+

WO1997033590A1 - A method of treating disorders related to cytokines in mammals - Google Patents

A method of treating disorders related to cytokines in mammals Download PDF

Info

Publication number
WO1997033590A1
WO1997033590A1 PCT/DK1997/000107 DK9700107W WO9733590A1 WO 1997033590 A1 WO1997033590 A1 WO 1997033590A1 DK 9700107 W DK9700107 W DK 9700107W WO 9733590 A1 WO9733590 A1 WO 9733590A1
Authority
WO
WIPO (PCT)
Prior art keywords
alkyl
adenosine
phenyl
mmol
deoxy
Prior art date
Application number
PCT/DK1997/000107
Other languages
French (fr)
Inventor
Lars Knutsen
Uffe Bang Olsen
Andrew Neil Bowler
Original Assignee
Novo Nordisk A/S
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novo Nordisk A/S filed Critical Novo Nordisk A/S
Priority to AU20224/97A priority Critical patent/AU2022497A/en
Publication of WO1997033590A1 publication Critical patent/WO1997033590A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • 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

Definitions

  • the present invention relates to a method of treating type I or type LI diabetes, multiple schlerosis, stroke, osteoporosis, septic shock and menstrual complications.
  • the invention furthermore relates to new purine derivatives having affinity for i.a. the A 3 subtype of adenosine receptors and acting as cytokine inhibitors.
  • Adenosine receptors represent a subclass (Pi) of the group of purine nucleotide and nucleoside receptors known as purinoreceptors. This subclass has been further classified into distinct receptor types which are now known as A t , A ⁇ , Aa, and A 3 .
  • Adenosine A3 receptors Pharmacological Properties, Species Differences and Receptor Functions. ⁇ PS 1994, 75, 298-306; Jacobson, K.A.; Kim, H.A.; Siddiqi, S.M.; Olah, M.E.; Stiles, G.L.; von Lubitz, D..K.J.E.; A3 Adenosine Receptors: Design of Selective Ligands and Therapeutic Prope ⁇ s. Drugs of the Future 1995, 20, 689-699; Collis, M.G.; Hourani, S.M.O.; Adenosine Receptor Subtypes, ⁇ PS 1993, 360-366].
  • cytokine production Cerri, M.A; Beltran-Nunez, A.; Bernasconi, S.; Dejana, E.; Bassi, L.; Bazzoni, G. Inhibition of Cytokine Production and Endothelial Expression of Adhesion Antigens by 5'- Methylthioadenosine. Eur. J. Pharmacol. 1993, 232, 291-294).
  • adenosine agonists R-PLA, NEC A, CPCA, CGS 21680, 2-chloroadenosine and CHA have all been shown to have an inhibitory effect on Tumour Necrosis Factor (TNF) production (Le Vraux, V.; Chen, Y.L.; Masson, M.; De Sousa, M.; Giroud, J.P.; Florentin, I.; Chauvelot-Moachon, L. Inhibition of Human Monocyte TNF production by Adenosine Receptor Agonists. Life Sci.
  • TNF Tumour Necrosis Factor
  • Circulatory Shock 1995, 44, 97-103 as has their therapeutic potential (Giroud, J.P.; Lian Chen, Y.; Le Vraux, V.; Chauvelot-Moachon, L. Therapeutic Aspects of Adenosine in Relation to its anti-TNF properties. Bull. Acad Natl. Med (Paris) 1995, 179, 79 -101).
  • TNF- ⁇ levels are increased in obese rodents (Yamakawa, T , Tanaka, S-I , Yamakawa, Y , Kiuchi, Y , Isoda, F , Kawamoto, S, Okuda, K and Sekihara, H Augmented Production of Tumor Necrosis Factor- ⁇ Production in Obese Mice Clin.
  • adenosine and adenosine agonists, acting via A 2 receptors can be of benefit in for example sepdc shock or ischaemia-reperfusion injury, where cytokine production by mononuclear phagocytes can be inhibited by tiiese agents (Bouma, M G , Stad, R.K., van den Wildenberg, A.J.M. and Buurman, W Differential Regulatory Effects of Adenosine on Cytokine Release by Activated Human Monocytes J.
  • TNF- ⁇ inhibitors have application in disorders which involve an inflammatory response, but this cytokine has multiple inflammatory, metabolic and immunological activities (Jirillo, E. Pellegrino, N.M. and Antonaci, S. Role of Tumor Necrosis Factor- ⁇ in Physiological and Pathological Conditions. Med Sci. Res., 1995, 23, 75-79).
  • TNF inhibitors in the prevention of neuronal damage following cerebral ischaemia (Firestein, G.Z., Liu, T and Barone, F.C. Cytokines, Inflammation, and Brain Injury: Role of Tumor Necrosis Factor- ⁇ . Cerebrovascular and Brain Metabolism Reviews 1994, 6, 341-360).
  • Examples with hydrogen at the purine 2-position include N-aminoadenosine, N-[(N-methyl-N- phenyl)amino]adenosine, N-hydroxyadenosine, N-methoxyadenosine and N-benzyloxyadenosine (Kusachi, S., Thompson, R.D. Bugni, .J., Yamada, N. and Olsson, R.A. Dog Coronary Artery Adenosine Receptor: Structure of the //-Alkyl Subregion. J.
  • N-ethoxyadenosine (Fujii, TRON Wu, C.C., Itaya, T., Moro, S. and Saito, T. Purines.
  • XI The Synthesis of N-Alkoxyadenosines and Their 2',3'-O-Isopropylidene Derivatives. Chem. Pharm. Bull., 1973, 21, 1676 - 1682); (Fuji, T., W, C.C. and Itaya, T. Purines.
  • Examples of adenosine derivatives with oxygen or nitrogen atoms bonded to the 6-amino substituent, containing an additional purine 2-substituent are 2-amino-Jv ' -hydroxyadenosine (Kikugawa, K, Iizuka, K., Higuchi, Y, Hirayama, H. and Ichino, M. Platelet Aggregation Inhibitors. 2. Inhibition of Platelet Aggregation by 5'-,2-,6-, and 8-substituted Adenosines. J. Med Chem., 1972, 15, 387 - 390); 2-amino-iV-aminoadenosine (Saneyoshi, M. and Terashima, K.
  • (lR,4S)-9-[4-(DimethylthexylsUyloxymethyl)cyclopent-2-enyl]-6-methoxyamino-9H-purine-2- amine is featured as an intermediate in the synthesis of (-)-carbovir (Exall A.M., Jones, M.F., Mo, C-L., Myer, P.L., Paternoster, I.L., Singh, H, Storer, R, Weingarten, G.G., Williamson, C, Brodie, A.C., Cook, J., Lake, D.E., Meerholz, C.A., Tumbull, P.J. and Highcock, KM.
  • (-)-carbovir Exall A.M., Jones, M.F., Mo, C-L., Myer, P.L., Paternoster, I.L., Singh, H, Storer, R, Weingarten, G.G., Williamson, C, Brodie, A.C.
  • Pfizer Inc. claim a range of heterocycles, including some purine derivatives, as CRF antagonists.
  • ribose moiety in adenosine is chemically modified, and many of those known have poor affinity for the adenosine receptor (Taylor, M.D., Moos, W.H., Hamilton, H.W. Szotek, D.S. PAtt, W.C. Badger, E.W. Bristol, J.W. Bruns, KF. Hef ⁇ ier, T.G. Mertz, T.E. Ribose-Modified Adenosine Analogues as Adenosine Receptor Agonists. J. Med Chem., 1986, 29, 346-353).
  • adenosine A 3 receptor agonists are 5'-modified adenosine derivatives (Jacobson, K.A.; Kim, H.A.; Siddiqi, S.M.; Olah, M.E.; Stiles, G.L.; von Lubitz, D..K.J.E.; A 3 Adenosine Receptors: Design of Selective Ligands and Therapeutic Propects. Drugs of the Future 1995, 20, 689-699; Baraldi, P.G., Cacciari, B., Spalluto, G. Ji, X-d, Olah, M.E.
  • EP-A-181,128 and EP-A-181,129 disclose 5'-deoxy adenosine derivatives containing 5'- hydrogen, 5'-halogen and 5'-methylthio, which are claimed to have desirable anti-inflammatory, analgesic as well as CNS and antihypertensive properties respectively.
  • EP-A-232,813 discloses N-substituted adenosines including a larger range of 5'-modified compounds.
  • WO 88/03147 5'-substituted adenosine derivatives with selectivity for the adenosine A 2 receptor are disclosed.
  • compounds acting as A3 ligands are found to be potent inhibitors of cytokine production, especially TNF- ⁇ production, and to be useful in the treatment of type I and type U diabetes, multiple schlerosis, stroke, osteoporosis, septic shock and menstrual complications.
  • present invention relates to a method of treating a mammal suffering from type I or type U diabetes, multiple schlerosis, stroke, osteoporosis, septic shock or menstrual complications, preferably type I or type II diabetes, more preferably type II diabetes, which method comprises administering to a mammal in need of such treatment an effective amount of an adenosine A 3 ligand.
  • the present invention is furthermore concerned with a method of treating disorders related to cytokines in mammals comprising administering to a mammal in need thereof an effective amount of a compound of the general formula (I), or a pharmaceutically acceptable salt thereof: -R,
  • A is hydroxymethyl, methyl, chloromethyl, bromomethyl, fluoromethyl, cyanomethyl, aminomethyl, vinyl, methylthiomethyl or methoxymethyl;
  • Ri is selected from the groups consisting of
  • n is 1 to 3 and where the group (a) may be optionally substituted with one or two groups, Cw-alkenyl, C -6-alkynyl, phenoxy, phenylsulphonyl, phenylthio, hydroxy, phenyl, C w -alkoxy or d-e-alkoxy- Ci- ⁇ -alkyl, phenylthioalkyl or
  • Y is O, S or NZ, where Z is H, C ⁇ -6-alkyl or phenyl, and where the group (b) may be optionally substituted with C ⁇ - 6 -alkyI, C 2- 6-alkenyl, C 2 -6-alkynyl, phenoxy, phenyl, C ⁇ -alkoxy or
  • R 1 is -NR 2 R 3 or -YR 4 , wherein Y is oxygen;
  • R 2 is C,-s-alkyl;
  • X represents phenyl
  • A is hydroxymethyl, methyl, chloromethyl, bromomethyl, fluoromethyl, cyanomethyl, aminomethyl, vinyl, methylthiomethyl or methoxymethyl; is selected from the groups consisting of
  • n is 1 to 3 and where the group (a) may be optionally substituted with one or two Ci- ⁇ -alkyl groups, C M -alkenyl, C 2-6 -alkynyl, phenoxy, phenylsulphonyl, phenylthio, hydroxy, phenyl, Ci- ⁇ -alkoxy or phenylthioalkyl or
  • R' is -NR 2 R 3 or -YR 4 , wherein Y is oxygen; R 2 is Ci- ⁇ -alkyi;
  • R 3 is phenyl or Cw-alkyl which may be substituted by phenyl or phenoxy;
  • R 4 is straight-chain d-e-alkyl, branched C 3 - 8 -alkyl, C 2-g -alkenyl or C3-g-cycloalkyl, which may be substituted by phenyl or phenoxy which in turn may be substituted with nitro, halogen or amine.
  • the present invention is furthermore concerned with new compounds of the above formula (I) or pharmaceutically acceptable salts thereof, wherein
  • X represents hydrogen, halogen, amino, perhalomethyi, cyano, Ci- ⁇ -alkyl, C ⁇ -alkoxy, C «- alkylthio, or phenyl;
  • A is hydroxymethyl, methyl, chloromethyl, bromomethyl, fluoromethyl, cyanomethyl, aminomethyl, vinyl, methylthiomethyl or methoxymethyl;
  • Ri is selected from the groups consisting of
  • n is 1 to 3 and where the group (a) may be optionally substituted with one or two Ci- ⁇ -alkyl groups, C 2 ⁇ -alkenyl, C 2 -s-alkynyl, phenoxy, phenylsulphonyl, phenylthio, hydroxy, phenyl, Cw-alkoxy or C ⁇ -6-alkoxy- Ci- ⁇ -alkyl, phenylthioalkyl or
  • Y is O, S or NZ, where Z is H C ⁇ -alkyl or phenyl, and where the group (b) may be optionally substituted with C ⁇ -alkyl, C 2-6 -alkenyl, C M -alkynyl, phenoxy, phenyl, d- ⁇ -aikoxy or C ⁇ -6-alkoxy-Cw-alkyl, or R 1 is -NR 2 R 3 or -YR 4 , wherein Y is oxygen;
  • R 2 is C ⁇ -6-alkyl
  • R 3 is phenyl or which may be substituted by phenyl or phenoxy;
  • R 4 is branched C 3-g -alkyl or C 2 .g-alkenyl, which may be substituted by phenyl or phenoxy which in turn may be substituted with nitro, halogen or amine.
  • the compounds of formula (I) have affinity for the A 3 subtype of adenosine receptors, modulate cyclic AMP and act as cytokine inhibitors. Moreover, these compounds are found to be useful as drugs in the treatment of disorders where damaging effects of cytokines are observed in humans.
  • the compounds according to the invention possess desirable pharmacological properties which can be ascribed to cytokine modulation. For example they inhibit TNF- ⁇ release, indicated by lowering of plasma TNF- ⁇ following LPS challenge in rats.
  • the adenosine receptor subtype A3 has been expressed in a human embryonic kidney cell line (HEK 293) and shown to be negatively coupled to adenylate cyclase via a pertussis toxin sensitive G-protein.
  • HEK 293 human embryonic kidney cell line
  • Test procedure & data analysis Test compounds are dissolved in DMSO, ethanol or water and further diluted in assay buffer. The final concentrations of solvents should be less than 2%. Test solutions should be in the range of pH 6.5 - 7.5. IC50 values were calculated from dose-response curves (3 points minimum) by a non-linear regression analysis using the GraphPad Prism program (GraphPad Software, USA). The results expressed in nM.
  • test compound is dissolved in dimethyl sulphoxide (DMSO) at 8 mg mL and is diluted with Cremophor in 5% saline (0.9% aqueous NaCl) to 160, 16 and 1.6 ⁇ g/mL. 25 ⁇ L is added to each tube, and 350 mL heparinised (50 lE/mL) rat blood, 25 ⁇ L lipopolysaccharide (LPS) 1.6 mg/mL in saline are introduced, i.e. the concentrations of the test compound is 10, 1 and 0.1 mg/ ⁇ L respectively.
  • the samples are shaken carefully and are placed in a water bath for 5 h. at 37°C.
  • the samples are centrifuged for 10 min. at 3000 ⁇ m at 4°C.
  • the plasma is removed by pipette in plastic tubes and is frozen. TNF- ⁇ levels are determined using a Genzyme ELISA kit.
  • the compounds of the invention together with a conventional adjuvant, carrier or diluent, and if desired in the form of a pharmaceutically acceptable acid addition salt thereof, may be placed into the form of pharmaceutical compositions and unit dosages thereof, and in such form may be employed as solids, such as tablets of filled capsules, or liquids, such as solutions, suspensions, emulsions, elixirs, or capsules filled with the same, all for oral use, in the form of suppositories for rectal administration; or in the form of sterile injectable solutions for parenteral use (including subcutaneous administration and infusion).
  • Such pharmaceutical compositions and unit dosage forms thereof may comprise conventional ingredients in conventional proportions, with or without additional active compounds or principles, and such unit dosage forms may contain any suitable effective amount of the adenosine receptor agonist commensurate with the intended daily dosage range to be employed.
  • the compounds of this invention can thus be used for the formulation of pharmaceutical preparation, e.g. for oral and parenteral administration to mammals including humans, in accordance with conventional methods of galenic pharmacy.
  • excipients are such pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral or enteral application which do not deleteriously react with the active compounds.
  • Such carriers are water, salt solutions, alcohols, polyethylene glycols, polyhyroxyethoxylated castor oil, gelatine, lactose amylose, magnesium stearate, talc, silicic acid, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxy- methylcellulose and polyvinylpyrrolidone.
  • the pharmaceutical preparations can be sterilised and mixed, if desired, with auxiliary agents, emulsifiers, salt for influencing osmotic pressure, buffers and/or colouring substances and the like, which do not deleteriously react with the active compounds.
  • injectable solutions or suspensions preferably aqueous solutions with the active compound dissolved in polyhydroxylated castor oil.
  • Ampoules are convenient unit dosage forms.
  • the dosage of the compounds according to this invention is 0.1-300 mg/day, preferably 10-100 mg/day, when administered to patients, e.g. humans, as a drug.
  • a typical tablet which may be prepared by conventional tabletting techniques contains:
  • the compounds of the invention may accordingly be administered to a subject, e.g., a living animal body, including a human, in need of adenosine receptor agonist, and if desired in the form of a pharmaceutically acceptable acid addition salt thereof (such as the hydrobromide, hydrochloride, or sulphate), in any event prepared in the usual or conventional manner, e.g., evaporation to dryness of the free base in solution together with the acid), ordinarily concurrently, simultaneously, or together with a pharmaceutically acceptable carrier or diluent, especially and preferably in the form of a pharmaceutical composition thereof, whether by oral, rectal, or parenteral (including subcutaneous) route, in an effective amount of adenosine receptor agonist, and in any event an amount which is effective for the treatment diseases related to cytokines, owing to their adenosine receptor agonist activity.
  • a pharmaceutically acceptable acid addition salt thereof such as the hydrobromide, hydrochloride, or sulphate
  • Suitable dosage ranges are 1-200 milligrams daily, 10-100 milligrams daily, and especially 30-70 milligrams daily, depending as usual upon the exact mode of administration, form in which administered, the indication toward which the administration is directed, the subject involved and the body weight of the subject involved, and the preference and experience of the physician or veterinarian in charge.
  • TLC thin layer chromatography
  • THF tetrahydrofuran
  • TFA trifluoracetic acid
  • mp melting point. Where melting points are given, these are uncon n ected.
  • the structures of the compounds are confirmed by assignment of NMR spectra (from which representative peaks are quoted) and by microanalysis where appropriate.
  • Compounds used as starting materials are either known compounds or compounds which can be prepared by methods known per se. Column chromatography was carried out on Merck silica gel 60 (Art 9385).
  • HPLC was carried out on a Waters or Merck chromatograph with a multiwavelength detector and a reversed phase C18 column (250 x 4 mm, 5mm, lOOA; eluent flow rate 1 mL min at 35°C). Retention times are given in minutes. Examples
  • the title compound was prepared according to the method described in Example 1 by reacting 1- methyl- 1-phenylhydrazine (1.29 g, 11 mmol) with 9-(2 , ,3 , ,5 , -tri-O-benzoyl-b-D-ribofuranosyl)- -2,6-dichloro-9H-purine (1.58 g, 2.5 mmol) and debenzoylating the purified product using potassium carbonate in methanol to provide the title 2-Chloro-N-[(N-methyl-N- phenyl)amino]adenosine (0.70 g, 69%) (after column chromatography) as a colorless foam, TLC r f 0.50 [SiO 2 , THF], l H NMR (DMSO-d*) d 3.23 (3H, 2s, CH 3 ), 3.50 - 3.72 (2H, br, -CH r ), 5.06 (IH, br, 5'-OH),
  • This example was prepared by a method similar to that described in Example 1. 9-(2 , ,3 , ,5'-Tri-O-benzoyl- ⁇ -D-ribofuranosyl)-2,6-dichloro-9H-purine (2.0 g, 3.2 mmol), O- henylmethyl)hydroxylamine hydrochloride (0.77 g, 4.8 mmol) and N,N-diisopropylamine (1.03 g, 8.0 mmol) were dissolved in dioxan (50 mL). The reaction mixture was heated at reflux for 20h, filtered and evaporated in vacuo.
  • N-Hydroxyphthalimide (15.0 g, 92 mmol) and sodium acetate (7.5 g, 92 mmol) were stirred in dimethylsulfoxide (70 mL) at ambient temperature for 3h.
  • 2-Chloroethylbenzene (12.5 g, 89 mmol) was added and the reaction mixture was heated at reflux for 2 h. After cooling and standing overnight the reaction mixture was filtered and the filtrate was poured onto ice/water (300 mL). The mixture was extracted with dichloromethane (4 x 100 mL), the combined extracts were dried (MgSO 4 ) and evaporated in vacuo to a residue.
  • N-(2-Phenylethoxy)phthalimide (11.3 g, 42 mmol) was dissolved in hot 96% ethanol (100 mL). Hydrazine hydrate (2.5 g, 50 mmol) was introduced and the reaction mixture was heated at reflux with mechanical stirring for 1.5h. The reaction mixture was stored at 4°C for 72 h, filtered and the filtrate was evaporated in vacuo. The crude white residue was suspended in toluene and stored at 4°C for 16h and filtered. The filtrate was treated with a solution of chlorotrimethyl- silane (4.34 g) in toluene (200 mL) with methanol (1.05 g) present and the title compound precipitated.
  • the title compound was prepared according to the method described in Example 4 by reacting 0-methylhydroxylamine (0.20 g, 2.0 mmol) with 9-(2',3',5'-tri-O-benzoyl- ⁇ -D-riboft ⁇ ranosyl)- -2,6-dichloro-9H-purine (1.27 g, 2.0 mmol) and debenzoylating the purified product using methanolic ammonia to provide the title 2-chloro-N-methoxyadenosine (0.40 g, 45%) (after column chromatography) as a colorless foam which became crystalline on trituration with dichloromethane, providing 0.20 g of a white solid, mp 123 - 125°C.
  • the title compound was prepared according the method described in example 6 by reacting 0- ethylhydroxylamine hydrochloride (0.305 g, 3.1 mmol) with 9-(2 , ,3 , ,5'-tri-O-acetyl- ⁇ -D-ribo- furanosyl)-2,6-dichloro-9H-purine (1.79 g, 4.0 mmol), followed by deacylation of the purified product using sodium methoxide in methanol to provide the title 2-chloro-N-ethoxyadenosine
  • the title compound was prepared according to the method described in Example 6 by reacting O-tert-butylhydroxylamine hydrochloride (0.603 g, 4.8 mmol) with 9-(2',3',5 , -tri-O-acetyl- ⁇ -D- ribofuranosyl)-2,6-dichloro-9H-purine (1.79 g, 4.0 mmol), followed by deacylation of the purified product using sodium methoxide in methanol to provide the title 2-chIoro-N-ethoxyadenosine
  • the title compound was prepared according to the method described in Example 4 by reacting 1,1-dimethylhydrazine (0.79 g, 13.1 mmol) with 9-(2,3,5-tri-0-acetyl- ⁇ -D-ribofuranosyi)-2- amino-6-chloro-9H-purine (4.0 g, 9.35 mmol) and debenzoylating the purified product using methanolic ammonia to provide the title 2-ammo-N-(dimethylamino)adenosine (after column chromatography) as an amorphous foam (0.56 g, 45%), 1H NMR (DMSO-ds) d 2.54 (6H, s, 2 x -CH 3 ), 3.50 - 3.57 (IH, m, H-5' a ), 3.61 - 3.68 (Hi m, H-5' b ), 3.93 (Hi q, H-4'), 4.12 (IH, q, H-3'), 4.51 (IH q
  • the title compound was prepared by reacting 1 -methyl- l-(2-phenoxyethyl)hydrazine (prepared by the general method described in Example 1) (0.80 g, 4 mmol) and 9-(2,3,5-tri-O-benzoyl- ⁇ - D-ribofuranosyl)-2,6dichloro-9H-purine (2.53 g, 4 mmol) and triethylamine (1.11 mL, 8 mmol) in dioxan (25 mL), followed by debenzoylation of the purified product using methanolic ammonia to provide the title 2-chloro-N-(N-methyl-N-(2-phenoxyethyl)amino)adenosine (0.84 g, 47%) (after column chromatography) obtained as a solid, mp 166-8°C, ⁇ NMR (DMSO-ds) d 2.70 (3H, s, -CH 3 ), 3.22 (2H, br t, -
  • O-Methylhydroxylamine hydrochloride (0.20 g, 2.4 mmol) was reacted with 9-(2',3',5'-tri- O-acetyl- ⁇ -D-ribofuranosyl)-6-chloro-2-methyl-9H-purine (0.85 g, 2.0 mmol) (see Example 20) in dioxan (40 mL) in the presence of triethylamine (0.52 g, 5.0 mmol). The reaction mbcture was heated at 90°C in a sealed vessel for 70h, before being filtered and evaporated.
  • N-methoxy-2-phenyladenosine O-Methylhydroxylamine hydrochloride (10.2 g, 12.2 mmol) was reacted with 9-(2',3',5'-tri- O-acetyl- ⁇ -D-ribofuranosyl)-6-chloro-2-phenyl-9H-purine (0.60 g, 1.2 mmol) in dioxan (30 mL) in the presence of d ⁇ sopropylethylamine (1.70 g, 13.1 mmol). The reaction mixture was heated at 100°C in a sealed vessel for 18h, and at 60°C for 50h before being filtered and evaporated.
  • reaction mixture was concentrated in vacuo and the crude product was purified by flash chromatography eluting with dichloromethane and 10% ammonia in ethanol (95:5) to give 5'-deoxy-2,5'-dichloro-2',3 , -O-(l-methyl- ethylidene)-N-(l-piperidinyl)adenosine (0.10 g, 48%) as a foam.
  • This compound was prepared by the general method described in Example 24.
  • 2-Chloro-N-(4-phenoxy-l-piperidinyl)adenosine [WO 93/08206 (Novo Nordisk A/S)] (0.5 g, 1.05 mmol) was subjected to the chlorination conditions described above, providing the title 5'- deoxy-2,5'-dichloro-N-(4-phenoxy-l-piperidinyl)adenosine which precipitated on treatment with dichloromethane following trituration with ether. Drying in vacuo provided a solid (0.28 g, 56%), m.p.
  • This compound was prepared by the method described in Example 24.
  • 2-Chloro-N-(4-phenyl- thio-l-piperidinyl)adenosine [WO 93/08206 (Novo Nordisk A/S)] (0.5 g, 1 mmol) was subjected to the reartion conditions described above, providing the title 5'-deoxy-2,5'-dichloro-N-(3-phenyl- thio-l-piperidinyl)adenosine (mixture of diastereoisomers) as a foam (0.48 g, 94%) following flash chromatography on silica gel.
  • reaction mixture was evaporated to a residue and purified by flash chromatography on silica gel, eluting with a mixture of dichloromethane and ethanol (9:1) to provide 5'-deoxy-2,5'-dicWoro-N-(4-phenylsulfinyl-l-piperidinyl)adenosine (0.1 g, 53%) as a foam.
  • EtOH requires C, 42.1; H, 4.9; N, 19.6. Found: C, 41.6; H, 5.2; N, 19.0%.
  • Methyl 2,3-O-(l-mrthylethyUdene)-5-O-(p-toluenesulfonyl)-D-ribofuranoside (28 7 g, 80 mmol) was dissolved in dry acetonitrile (100 mL).
  • Tetra- «-butylammonium fluoride (100 mL, 1 0M in THF) was added dropwise and the reaction mixture was heated at 80°C for 72 h After cooling to room temperature, the mbcture was diluted with dichloromethane (200 mL), washed with water (3 x 50 mL) and dried (MgSO 4 ) Evaporation provided a residue which was purified by flash chromatography eluting with a mixture of ethyl acetate/w-heptane (1/3) to give methyl 5-deoxy-5-fluoro-2,3-O-(l-methylethylidene)- ⁇ -D-ribofuranoside (13 6 g, 82%) as a clear oil, 'H NMR (CDC1 3 ) d 1 34 (3H, s, CH 3 ), 1 50 (3H, s, CH 3 ), 3 35 (3H, s, -OCH 3 ), 4 29 - 448 (3H,
  • Methyl 5-deoxy-5-fluoro-2,3-O-(l-methylethylidene)-D-ribofuranoside (5.0 g, 24 mmol) was treated with sulfuric acid (0.02M, 40 mL) and heated at reflux for 4 h. The reaction mixture was cooled, neutralized with barium carbonate to pH 7, filtered and evaporated to an oil. The oil was dried by coevaporation with ethanol, and the residue was dissolved in dichloromethane (50 mL).
  • This compound was prepared by general method A, described in more detail in Example 39 by reacting O-benzylhydroxylamine hydrochloride (0.80 g, 5.0 mmol) with 9-[(2',3'-di-O-acetyl-5'- deoxy-5'-fluoro-D-ribofuranosyl)]-2,6-dichloro-9H-purine (1.0 g, 2.5 mmol) as described above.
  • the product was purified by flash chromatography eluting with a mixture of dichloromethane and 10% ammonia in ethanol (98:2) giving the intermediate 2',3'-di-0-acetyI-2-chloro- 5'-deoxy-5'-fluoro-N-benzyloxyadenosine (0.2 g, 16%).
  • Deacetylation was performed in methanolic ammonia to afford 2-chloro-5'-deoxy-5'-fluoro-iV-benzyloxyadenosine as a foam (0.11 g, 89%) after flash chromatography eluting with dichloromethane and 10% ammonia in ethanol (95:5).
  • 5-O-Methyl-2,3-O-(l-methylethylidene)-D-ribofuranoside (3.0 g, 14 mmol) was dissolved in a mixture of sulfuric acid (0.02M, 100 mL) and ethanol (50 mL) and heated at 80°C for 6 h and stirred for 20 h at 20°C.
  • the reaction mbcture was neutralised with aqueous sodium bicarbonate and concentrated in vacuo.
  • the residual oil was dried and acetylated in a mixture of dichloromethane (100 mL), acetic anhydride (8.5 g, 83 mmol) and triethylamine (16.7 g, 165 mmol) at 20°C for 20 h.
  • the reartion mixture was washed with hydrochloric acid (1M, 50 mL) and water (50 mL).
  • the organic phase was dried (MgSO ) and concentrated to an oil before being purified by flash chromatography.
  • Acetic acid (74.5 mL, 1.3 mol), acetic anhydride (173.8 mL, 1840 mmol) and sulfuric acid (1.7 mL, 32 mmol) were mixed together and methyl 2,3-O-dibenzoyl-5-cyano-5-deoxy- ⁇ -D- ribofuranoside (13.01 g, 35 mmol) was added.
  • the reartion mixture was stirred for 20 h at ambient temperature before sodium acetate (37 g, 450 mmol) was introduced. After 30 min.
  • Methyl 5-deoxy-2,3-O-(l-methylethylidene)-D-ribofuranoside (prepared by reduction of methyl 2,3-O-(l-methylethylidene)-5'-O-(p-toluenesulphonyl)-D-ribofi ⁇ ranoside using lithium aluminium hydride) (4.36 g, 23.2 mmol) was dissolved in methanol (120 mL) and Amberlyst resin (Ff form, 19 g) was introduced. The mbcture was stirred at 80°C for 60 h and filtered. The filter pad was washed with methanol and the filtrate was evaporated to an oily residue.
  • Triphenylmethylphosphonium bromide (26.79 g, 75 mmol) was suspended in THF (200 mL) and n-butyllithium (1.7M in hexanes) (42 mL, 71.2 mmol) was introduced. After stirring for 2 h, methyl 5-deoxy-5-oxo-2,3-O-(l-methylethylidene)-D-ribofi ⁇ ranoside (prepared by oxidation of 1- O-methyl-2,3-O-(l-methylethylidene)-D-ribofuranoside using the method described in Ranganathan, R.S., Jones, G.H.
  • Methyl 5-deoxy-2,3-O-(l-methylethylidene)-5-methylene-D-ribofuranoside (5.65 g, 28.2 mmol) was dissolved in methanol (250 mL) and Amberlyst resin (H * form, 30 g) was introduced. The mixture was stirred at ambient temperature for 40 h and was filtered. The filter pad was washed with methanol and the filtrate was evaporated to an oily residue.
  • 2,3-Di-O-benzoyl-2-chloro-5'-deoxy-5'-methylene-N-cyclopentyladenosine prepared by reartion of 9-(5'-deoxy-2',3'-di-O-benzoyl-5'-methylene- ⁇ -D-ribofi ⁇ ranosyl)-2,6-dichloro-9H-purine (see Example 48) with cyciopentylamine) (0.30 g, 0.52 mmol) was dissolved in methanolic ammonia (10 mL) and stirred at ambient temperature for 18 h.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

A method of treating disorders related to cytokines such as TNFα in mammals. The disorder is an autoimmune disorder, inflammation, arthritis, type I or type II diabetes, multiple sclerosis, stroke, osteoporosis, septic shock or menstrual complications.

Description

A method of treating disorders related to cvtokines in mammals
Field of the invention
The present invention relates to a method of treating type I or type LI diabetes, multiple schlerosis, stroke, osteoporosis, septic shock and menstrual complications. The invention furthermore relates to new purine derivatives having affinity for i.a. the A3 subtype of adenosine receptors and acting as cytokine inhibitors.
Background of the Invention
Adenosine receptors represent a subclass (Pi) of the group of purine nucleotide and nucleoside receptors known as purinoreceptors. This subclass has been further classified into distinct receptor types which are now known as At, A^, Aa, and A3.
Extensive research has been carried out in a quest to identify selective ligands at these receptors [see, for example, Fredholm, B.B.; Abbracchio, M.P.; Burnstock, G.; Daly, J.W.; Harden, T.K.; Jacobson, K.A.; Williams, M. Nomenclature and Classification of Purinoceptors. Pharmacol. Rev. 1994, 46, 143-156; Van Galen, P.J.M.; Stiles, G.L.; Michaels, G. Jacobson, K.A. Adenosine Ai and A2 Receptors: Structure Activity Relationships. Med Chem. Rev. 1992, 12, 423-471; Linden, J. Cloned Adenosine A3 receptors: Pharmacological Properties, Species Differences and Receptor Functions. ΗPS 1994, 75, 298-306; Jacobson, K.A.; Kim, H.A.; Siddiqi, S.M.; Olah, M.E.; Stiles, G.L.; von Lubitz, D..K.J.E.; A3 Adenosine Receptors: Design of Selective Ligands and Therapeutic Propeαs. Drugs of the Future 1995, 20, 689-699; Collis, M.G.; Hourani, S.M.O.; Adenosine Receptor Subtypes, ΗPS 1993, 360-366].
Several purine derivatives are known to modulate the release and or action of cytokines. For example the naturally occurring 5'-methylthioadenosine has been shown to inhibit cytokine production (Cerri, M.A; Beltran-Nunez, A.; Bernasconi, S.; Dejana, E.; Bassi, L.; Bazzoni, G. Inhibition of Cytokine Production and Endothelial Expression of Adhesion Antigens by 5'- Methylthioadenosine. Eur. J. Pharmacol. 1993, 232, 291-294).
Furthermore, the adenosine agonists R-PLA, NEC A, CPCA, CGS 21680, 2-chloroadenosine and CHA as well as the adenosine uptake inhibitor dipyridamole have all been shown to have an inhibitory effect on Tumour Necrosis Factor (TNF) production (Le Vraux, V.; Chen, Y.L.; Masson, M.; De Sousa, M.; Giroud, J.P.; Florentin, I.; Chauvelot-Moachon, L. Inhibition of Human Monocyte TNF production by Adenosine Receptor Agonists. Life Sci. 1993, 52, 1917 - 1924) and these workers conclude that it is the A adenosine receptor which is involved in this aspect of TNF inhibition. The post-receptor event is presumably upregulation of adenylate cyclase. In a separate study, the specific adenosine Ai agonists 2-chloroadenosine and CCPA as well as the A2 agonist CPCA have been identified as inhibitors of lipopolysaccharide-stimulated TNF-α production, with the A2 agonist CPCA being 1000-fold more potent than CCPA, giving further evidence of the probable involvement of the adenosine A2 receptor. These findings have been confirmed by others (Prabhakar, U.; Brooks, D P. Lipschlitz, D.; Esser, K.M. Inhibition of LPS-induced TNFa Production in Human Monocytes by Adenosine (A2) Receptor Selective Agonists. Int. J. Immunopharmac. 1995, 17, 221-224). This effect of adenosine receptor agonists on TNF biosynthesis have been reviewed recently (Lee, J.C.; Prabhakar, U.; Griswold, D.E.; Dunnington, D.; Young, P.K; Badger, A. Low-Molecular-Weight TNF Biosynthesis Inhibitors: Strategies and Prospectives. Circulatory Shock 1995, 44, 97-103) as has their therapeutic potential (Giroud, J.P.; Lian Chen, Y.; Le Vraux, V.; Chauvelot-Moachon, L. Therapeutic Aspects of Adenosine in Relation to its anti-TNF properties. Bull. Acad Natl. Med (Paris) 1995, 179, 79 -101).
Disease states involving TNF-α
The suggestion has been made that TNF-α inhibitors are useful in the treatment of diabetes (Argiles, J M , Lopez-Soriano, J and Lopez-Soπano, F J Cytokmes and Diabetes- The Final Step Involvement of TNF-α in both Type I and Type II Diabetes Mellitus Harm. Metab. Res , 1994, 26, 447 - 449)
It is now clear that TNF-α levels are increased in obese rodents (Yamakawa, T , Tanaka, S-I , Yamakawa, Y , Kiuchi, Y , Isoda, F , Kawamoto, S, Okuda, K and Sekihara, H Augmented Production of Tumor Necrosis Factor-α Production in Obese Mice Clin. Immunol, and Immunopath , 1995, 75, 51-56) A clinical study of the expression pattern of TNF-α in adipose tissue of obese and normal premenopausal women has been carried out (Hotamisligil, G S , Amer, P Caro, J F , Atkinson, R L and Spielgelman, B Increased Adipose Tissue Expression of Tumor Necrosis Factor-α in Human Obesity and Insulin Resistance J. Clin. Invest , 1995, 95, 2409 - 2415) Obese individuals express 2 5-fold more TNF-α mRNA in fat tissue relative to lean controls, and there was a correlation to the level of hypeπnsulinemia, suggesting a role for TNF-α in the pathogenesis of obesity related insulin resistance The topic of TNF-α in insulin resistance has been reviewed by the same group (Hotamisligil, G S and Spielgelman, B Perspectives in Diabetes Tumor Necrosis Factor-α A Key Component of the Obesity-Diabetes Link Diabetes, 1994, 43, 1271 - 1278)
Some investigators suggest that adenosine and adenosine agonists, acting via A2 receptors can be of benefit in for example sepdc shock or ischaemia-reperfusion injury, where cytokine production by mononuclear phagocytes can be inhibited by tiiese agents (Bouma, M G , Stad, R.K., van den Wildenberg, A.J.M. and Buurman, W Differential Regulatory Effects of Adenosine on Cytokine Release by Activated Human Monocytes J. Immunol, 1994, 153, 4159 - 4168) These effects by A2 receptor agonists have also been demonstrated on human polymoφhonuclear leukocytes (Thiel, M. and Chouker, A Acting Via A2 Receptors, Adenosine Inhibits the Production of Tumor Necrosis Factor-α of Endotoxin-stimulated Human Polymoφhonuclear Leucocytes J. Lab Clm. Med. 1995, 275 - 282)
A massive release of TNF-α in the host produces severe damage to a range of tissues It is therefore clear that TNF-α inhibitors have application in disorders which involve an inflammatory response, but this cytokine has multiple inflammatory, metabolic and immunological activities (Jirillo, E. Pellegrino, N.M. and Antonaci, S. Role of Tumor Necrosis Factor-α in Physiological and Pathological Conditions. Med Sci. Res., 1995, 23, 75-79).
Many patented TNF-α inhibitors such as rolipram, pentoxyfylline and denbufylline are phosphodiesterase (PDE) inhibitors and exert their effects on TNF-α via control of cAMP (Davidsen, S.K. and Summers, J.B. Inhibitors of TNFa Synthesis. Exp. Opin. Ther. Patents 1995, 5, 1087 - 1100). Evidence is also available for some synergism between the effects of rolipram and adenosine in reduction of T primed neutrophil oxidative activity, thereby offering protection against inflammatory tissue damage (Sullivan, G., Caφer, H.T. and Mandell, G.L. Int. J. Immunopharmac. 1995, 17, 793-803).
There is also good evidence for TNF inhibitors in the prevention of neuronal damage following cerebral ischaemia (Firestein, G.Z., Liu, T and Barone, F.C. Cytokines, Inflammation, and Brain Injury: Role of Tumor Necrosis Factor-α. Cerebrovascular and Brain Metabolism Reviews 1994, 6, 341-360).
Prior art
Examples of adenosine derivatives in the chemical literature with the heteroatoms, oxygen or nitrogen bonded directly to the 6-amino substituent are summarised below.
Examples with hydrogen at the purine 2-position include N-aminoadenosine, N-[(N-methyl-N- phenyl)amino]adenosine, N-hydroxyadenosine, N-methoxyadenosine and N-benzyloxyadenosine (Kusachi, S., Thompson, R.D. Bugni, .J., Yamada, N. and Olsson, R.A. Dog Coronary Artery Adenosine Receptor: Structure of the //-Alkyl Subregion. J. Med Chem., 1985, 28, 1636 - 1643); N-ethoxyadenosine (Fujii, T„ Wu, C.C., Itaya, T., Moro, S. and Saito, T. Purines. XI. The Synthesis of N-Alkoxyadenosines and Their 2',3'-O-Isopropylidene Derivatives. Chem. Pharm. Bull., 1973, 21, 1676 - 1682); (Fuji, T., W, C.C. and Itaya, T. Purines. XH Catalytic Hydrogenolysis of Alkoxyarrtinopurines and Related Derivatives, ibid., 1973, 21, 1835 -1838); N-(methylamino)adenosine and N-[(N-hydroxy-N-methyl)amino]adenosine (Giner-Sorolla, A_, OTBryant, S.A., Nanos, C, Dollinger Ml, Bendich, A and Burchenal, J.H. The Synthesis and Biological Properties of Hydroxylaminopurines and Related Derivatives. J. Med Chem., 1968, 77, 521 - 523).
Examples of adenosine derivatives with oxygen or nitrogen atoms bonded to the 6-amino substituent, containing an additional purine 2-substituent are 2-amino-Jv'-hydroxyadenosine (Kikugawa, K, Iizuka, K., Higuchi, Y, Hirayama, H. and Ichino, M. Platelet Aggregation Inhibitors. 2. Inhibition of Platelet Aggregation by 5'-,2-,6-, and 8-substituted Adenosines. J. Med Chem., 1972, 15, 387 - 390); 2-amino-iV-aminoadenosine (Saneyoshi, M. and Terashima, K. Synthetic Nucleosides and Nucleotides. VII. A Direct Replacement of 6-Thiol Group of 6- Thioinosine and 6-Thioguanosine with Hydrazine Hydrate. Chem. Pharm. Bull, 1969, 17, 2373 - 2376); 2-amino-N-methoxyadenosine (Chem. Pharm. Bull, 1975, 23, 464 - 466); (Ueda, T, Miura, K. and Kasai, T., Synthesis of 6-Thioguanosine and 2,6-diarninopurine Nucleosides and Nucleotides from Adenosine Counteφarts via a facile Rearrangement in the Base Portion (Nucleosides and Nucleotides XLX). Chem. Pharm. Bull., 1978, 26, 2122 - 2127); 2-chloro-/V- hydroxyadenosine (Cristalli, G., Sauro, V., Eleuteri, A., Grifantini, M., Volpini, R., Lupidi, G, Capolongo, L. and Pesenti, E. Purine and 1-Deazapurine Ribonucleosides and Deoxyribonucleosides: Synthesis and Biological Activity. J. Med Chem., 1991, 34, 2226 - 2230); (LTzerman, A.P. von Frijtag Drabbe Kunzel, IK., Vittori, S. and Cristalli, G. Purine- Substituted Adenosine Derivatives with Small Λ^-Substituents as Adenosine Receptor Agonists. Nucleosides and Nucleotides, 1994, 13, 2267 - 2281): 2-fluoro-/V-hydroxyadenosine and 2- fluoro-TV-aminoadenosine (Montgomery, J.A. and Hewson, K. 2-Fluoropurine Ribonucleosides. J. Med Chem., 1970, 13, 427 - 430) and 2-fluoro-N-methoxyadenosine (Giner-Sorolla, A and Burchenal, J.H., Substituted Hydroxylaminopurines and Related Derivatives. Synthesis and Screening Tests. J. Med Chem., 1971, 14, 816 - 819). (lR,4S)-9-[4-(DimethylthexylsUyloxymethyl)cyclopent-2-enyl]-6-methoxyamino-9H-purine-2- amine is featured as an intermediate in the synthesis of (-)-carbovir (Exall A.M., Jones, M.F., Mo, C-L., Myer, P.L., Paternoster, I.L., Singh, H, Storer, R, Weingarten, G.G., Williamson, C, Brodie, A.C., Cook, J., Lake, D.E., Meerholz, C.A., Tumbull, P.J. and Highcock, KM. Synthesis from (-)-Aristeromycin and X-Ray structure of (-)-Carbovir. J. Chem. Soc. Perkin Trans. I, 1991, 2467 - 2477). A related purin-2-amine is featured as an intermediate in the synthesis of the antiheφetic agent carbocyclic 9-(2,-deoxy-2'-fluoro-b-D- arabinofuranosyl)guanine (Borthwick, A.D., Biggadike, K., Holman, S. and Mo, C-L., Enantiospecific Synthesis of the Potent Antiheφetic Carbocyclic 9-(2'-deoxy-2'-fluoro-b-D- arabinofuranosyl)guanine (+)-C-APG from Aristeromycin. Tetrahedron Lett., 1990, 31, 767- 770).
Some of the biological effects of the compounds contained within this application on the central nervous system have been published (Knutsen, L.J.S., Lau, J., Eskesen, K., Sheardown, M.J., Thomsen, C, Weis, J.U., Judge, M.E. and Klitgaard, H. Anticonvulsant Actions of Novel and Reference Adenosine Agonists. In Adenosine and Adenine Nucleotides: From Molecular Biology to Integrative Physiology, Belardinelli, L. and Pelleg, A, Eds.; Kluwer: Boston, MA; 1995, 479-487. See also Knutsen, L.J.S., Lau, J., Sheardown, M.J., Thomsen, C; The Synthesis and Biochemical Evaluation of New Ai Selective Adenosine Receptor Agonists Containing a 6-Hydrazinopurine Moiety; BioMed Chem. Lett., 1993, 3, 2661-2666).
In the above scientific articles, no mention is made of any pharmacological effects of the compounds concerned which can be ascribed to cytokine modulation.
The recent patents within novel purinergic agents has been reviewed (Bhagwhat, S.S.; Williams, M. Recent Progress in Modulators of Purinergic Activity. Exp. Opin. Ther. Patents 1995, 5, 547 - 558), as have patents covering TNF inhibitors (Davidsen, S.K. and Summers, J.B. Inhibitors of TNFa Synthesis. Exp. Opin. Ther. Patents 1995, 5, 1087 - 1100). Marion Merrell Dow Inc. has claimed some cyclopentyl substituted adenines which appear to exert their effects by control of TNF-α (US 5,244,896). A follow-up application by Merrell Dow Pharmaceuticals Inc. (WO 95/03304) disclosed some novel bicyclic 9-bicyclic nucleosides useful as selective inhibitors of proinflammatory cytokines.
Merck & Co. Inc. has filed a patent application claiming a method for inhibiting TNF-α production comprising contacting the Aa, subtype of the adenosine receptor with an adenosine agonist (GB 2289218-A).
Some other purine derivatives which are TNF inhibitors, presumably owing to their activity as PDE inhibitors, have been reviewed (This Years Drug News. Cardiovascular Drugs. Treatment of Septic Shock 1994 pp 185 - 189).
In WO 9533750-A1, Pfizer Inc. claim a range of heterocycles, including some purine derivatives, as CRF antagonists.
In US Patent 3,819,613, substituted adenosine analogues with hydrazone derivatives on the 6-amino function are disclosed as hypotensive agents. In GB 1,351,501, adenosine and 2-amino- adenosine derivatives having a -NH-R2 group joined to the 6-amino function are disclosed as coronary dilators and platelet aggregation inhibitors. In EP A 152,944, a series of 2-, 6- and 8-substituted adenosine derivatives are described having activity as anti-allergy agents. In EP 402,752A, derivatives of adenosine unsubstituted in the 2-position are described which have a substituted heteroaromatic 1-pyrrolyl moiety attached to the 6-amino group.
There are only relatively few examples where the ribose moiety in adenosine is chemically modified, and many of those known have poor affinity for the adenosine receptor (Taylor, M.D., Moos, W.H., Hamilton, H.W. Szotek, D.S. PAtt, W.C. Badger, E.W. Bristol, J.W. Bruns, KF. Hefδier, T.G. Mertz, T.E. Ribose-Modified Adenosine Analogues as Adenosine Receptor Agonists. J. Med Chem., 1986, 29, 346-353).
However, minor modifications at 3'- and 5'- appear to be allowed and amongst these the 5 - chloro-5'-deoxy adenosines show particularly good receptor affinity (Trivedi, B.K., Bridges, A.J., Patt, W.C. Priebe, S.R., Bruns, KF. TV'-Bicycloalkyladenosines with Unusually High Potency and Selectivity for the Adenosine Ai Receptor J. Med Chem., 1989, 32, 8-11). Other scientific articles also describe 5 '-modifications of adenosine derivatives (Olsson, R.A. Kusachi, S., Thompson, KD., Ukena, D., Padgett, W. and Daly, J.W. ^-Substituted N-Alkyladenosine-5'- uronamides: Bifunctional Ligands Having Recognition Groups for AI and A2 Adenosine Receptors. J. Med Chem., 1986, 29, 1683-1689).
Many of the new adenosine A3 receptor agonists are 5'-modified adenosine derivatives (Jacobson, K.A.; Kim, H.A.; Siddiqi, S.M.; Olah, M.E.; Stiles, G.L.; von Lubitz, D..K.J.E.; A3 Adenosine Receptors: Design of Selective Ligands and Therapeutic Propects. Drugs of the Future 1995, 20, 689-699; Baraldi, P.G., Cacciari, B., Spalluto, G. Ji, X-d, Olah, M.E. Stiles, G., Dionsiotti, S., Zocchi, C, Ongini, E. and Jacobson, K.A Novel Λ^-(Substituted-phenylcarbamoyl)adenosine-5'- uronamides as Potent Agonists for A3 Receptors. J. Med Chem., 1986, 39, 802-806).
EP-A-181,128 and EP-A-181,129 disclose 5'-deoxy adenosine derivatives containing 5'- hydrogen, 5'-halogen and 5'-methylthio, which are claimed to have desirable anti-inflammatory, analgesic as well as CNS and antihypertensive properties respectively.
EP-A-232,813 discloses N-substituted adenosines including a larger range of 5'-modified compounds. In WO 88/03147 5'-substituted adenosine derivatives with selectivity for the adenosine A2 receptor are disclosed.
In US 4,962,194 methods for preparing 5', N^-disubstituted adenosine derivatives are revealed. GB 1,101,108 discloses 5', N^-disubstituted adenosine analogues which possess cardiovascular activity. US Patent No. 3,910,885 reveals 4-alkoxy and 4-haloalkoxy nucleosides. PCT publication WO 94/06348 discloses a number of pyrrolo[3,4-d]pyrimidine structures which are formally isosteric with adenosine and which are modified at the sugar 5'-position. US Patent No. 5,308,837 covers the use of 5'-amine substituted adenosine analogues as immunosupressants.
In EP Publication No. 0 423 777 A2 a method for treating gastrointestinal motility disorders using N(6) (substituted aminoalkyl) adenosine derivatives is disclosed. EP Publication No. 0 490 818 AI describes a new use of 2'-O-methyl adenosine derivatives for a range of ailments including neurodegenerative disorders.
In WO 93/23417 (corresponds to US patent No. 5,430,027) and WO 95/07921 adenosine derivatives having central nervous system (CNS) properties are disclosed.
Description of the invention
According to the present invention, compounds acting as A3 ligands are found to be potent inhibitors of cytokine production, especially TNF-α production, and to be useful in the treatment of type I and type U diabetes, multiple schlerosis, stroke, osteoporosis, septic shock and menstrual complications.
Thus, present invention relates to a method of treating a mammal suffering from type I or type U diabetes, multiple schlerosis, stroke, osteoporosis, septic shock or menstrual complications, preferably type I or type II diabetes, more preferably type II diabetes, which method comprises administering to a mammal in need of such treatment an effective amount of an adenosine A3 ligand.
The present invention is furthermore concerned with a method of treating disorders related to cytokines in mammals comprising administering to a mammal in need thereof an effective amount of a compound of the general formula (I), or a pharmaceutically acceptable salt thereof: -R,
X N A
HO OH
wherein
X represents hydrogen, halogen, amino, perhalomethyl, cyano, Cι-5-alkyl,
Figure imgf000012_0001
C«- alkylthio, Ci-β-alkylamino or phenyl;
A is hydroxymethyl, methyl, chloromethyl, bromomethyl, fluoromethyl, cyanomethyl, aminomethyl, vinyl, methylthiomethyl or methoxymethyl;
Ri is selected from the groups consisting of
Figure imgf000012_0002
wherein Q is nitrogen or carbon, n is 1 to 3 and where the group (a) may be optionally substituted with one or two
Figure imgf000012_0003
groups, Cw-alkenyl, C -6-alkynyl, phenoxy, phenylsulphonyl, phenylthio, hydroxy, phenyl, Cw-alkoxy or d-e-alkoxy- Ci-β-alkyl, phenylthioalkyl or
Figure imgf000013_0001
wherein Y is O, S or NZ, where Z is H, Cι-6-alkyl or phenyl, and where the group (b) may be optionally substituted with Cι-6-alkyI, C2-6-alkenyl, C2-6-alkynyl, phenoxy, phenyl, C^-alkoxy or
Figure imgf000013_0002
R1 is -NR2R3 or -YR4, wherein Y is oxygen; R2 is C,-s-alkyl;
R3 is phenyl or
Figure imgf000013_0003
which may be substituted by phenyl or phenoxy;
R4 is straight-chain
Figure imgf000013_0004
branched C3.8-a.kyl, C2-g-alkenyl or C3-8-cycloalkyl, which may be substituted by phenyl or phenoxy which in turn may be substituted with nitro, halogen or amine.
A preferred method of treating the above-mentioned diseases comprises administering to a subject in need thereof a compound of the above general formula (I) wherein R1 is -OR4, wherein R4 is straight-chain Cι-5-alkyl, branched C3-8-alkyI, C2-8-alkenyl or C3-8-cycloalkyl, which may be substituted by phenyl or phenoxy which in turn may be substituted with nitro, halogen or amine.
The present invention is furthermore concerned with new compounds of the above formula (I) or pharmaceutically acceptable salts thereoζ wherein
X represents phenyl;
A is hydroxymethyl, methyl, chloromethyl, bromomethyl, fluoromethyl, cyanomethyl, aminomethyl, vinyl, methylthiomethyl or methoxymethyl; is selected from the groups consisting of
Figure imgf000014_0001
wherein Q is nitrogen or carbon, n is 1 to 3 and where the group (a) may be optionally substituted with one or two Ci-β-alkyl groups, CM-alkenyl, C2-6-alkynyl, phenoxy, phenylsulphonyl, phenylthio, hydroxy, phenyl, Ci-δ-alkoxy or
Figure imgf000014_0002
phenylthioalkyl or
Figure imgf000014_0003
wherein Y is O, S or NZ, where Z is H,
Figure imgf000014_0004
or phenyl, and where the group (b) may be optionally substituted with Ci-β-alkyl, C2-s-alkeny-, C2-5-alkynyl, phenoxy, phenyl, Ci-s-alkoxy or
Figure imgf000014_0005
R' is -NR2R3 or -YR4, wherein Y is oxygen; R2 is Ci-β-alkyi;
R3 is phenyl or Cw-alkyl which may be substituted by phenyl or phenoxy;
R4 is straight-chain d-e-alkyl, branched C3-8-alkyl, C2-g-alkenyl or C3-g-cycloalkyl, which may be substituted by phenyl or phenoxy which in turn may be substituted with nitro, halogen or amine. The present invention is furthermore concerned with new compounds of the above formula (I) or pharmaceutically acceptable salts thereof, wherein
X represents hydrogen, halogen, amino, perhalomethyi, cyano, Ci-β-alkyl, C^-alkoxy, C«- alkylthio,
Figure imgf000015_0001
or phenyl;
A is hydroxymethyl, methyl, chloromethyl, bromomethyl, fluoromethyl, cyanomethyl, aminomethyl, vinyl, methylthiomethyl or methoxymethyl;
Ri is selected from the groups consisting of
Figure imgf000015_0002
wherein Q is nitrogen or carbon, n is 1 to 3 and where the group (a) may be optionally substituted with one or two Ci-β-alkyl groups, C2^-alkenyl, C2-s-alkynyl, phenoxy, phenylsulphonyl, phenylthio, hydroxy, phenyl, Cw-alkoxy or Cι-6-alkoxy- Ci-β -alkyl, phenylthioalkyl or
Figure imgf000015_0003
wherein Y is O, S or NZ, where Z is H C^-alkyl or phenyl, and where the group (b) may be optionally substituted with C^-alkyl, C2-6-alkenyl, CM-alkynyl, phenoxy, phenyl, d-β-aikoxy or Cι-6-alkoxy-Cw-alkyl, or R1 is -NR2R3 or -YR4, wherein Y is oxygen;
R2 is Cι-6-alkyl;
R3 is phenyl or
Figure imgf000016_0001
which may be substituted by phenyl or phenoxy;
R4 is branched C3-g-alkyl or C2.g-alkenyl, which may be substituted by phenyl or phenoxy which in turn may be substituted with nitro, halogen or amine.
Various salts of compounds of formula (I) can be prepared which can be considered physiologically acceptable. These include addition salts derived from inorganic or organic acids, for example, acetates, fiimarates, glutarates, glutaconates, lactates, maleates, methanesulphonates, phosphates, salicylates, succinates, sulphates, sulphamates, tartrates and paratoluenesulphonates. In some cases, solvates of either the free nucleosides or the acid addition salts can be isolated and these solvates may, for example, be hydrates or alcoholates.
It has been found that the compounds of formula (I) have affinity for the A3 subtype of adenosine receptors, modulate cyclic AMP and act as cytokine inhibitors. Moreover, these compounds are found to be useful as drugs in the treatment of disorders where damaging effects of cytokines are observed in humans.
Evaluation of these compounds in established animal models has indicated that the compounds according to the invention possess desirable pharmacological properties which can be ascribed to cytokine modulation. For example they inhibit TNF-α release, indicated by lowering of plasma TNF-α following LPS challenge in rats.
Evaluation of vitro binding to adenosine Ai receptors
An assay for the human adenosine A3 receptor is described in Jacobson, M. Cloning and Expression of Human Adenosine Receptor Subtypes. In Adenosine and Adenine Nucleotides: From Molecular Biology to Integrative Physiology, Belardinelli, L. and Pelleg, A., Eds.; Kluwer: Boston, MA; 1995, pp 5-13.
\ n-AB-MECA binding to adenosine A3 receptors
The adenosine receptor subtype A3 has been expressed in a human embryonic kidney cell line (HEK 293) and shown to be negatively coupled to adenylate cyclase via a pertussis toxin sensitive G-protein. When the A3 receptor is expressed in CHO cells, it has been shown that [1 5I]-AB-MECA binds with a high affinity to this receptor A3 (K; = 1.5 nM) (Linden, J. Trends Pharmacol. Sci. 1994, 15, 298).
Method
HEK 293 cells were cultured in Dulbecco's modified Eagles media supplemented with 10% fetal bovine serum, 2 MM glutamine, 0.1 mg/mL streptomycin, 0.1 mg/mL penicillin and 0.8 mg mL G418 in an incubator at 37°C (95% air, 5% CO2). HEK 293 cells were seeded in 24-well plates two days before the experiment so that they were 70-80% confluent on the day of the assay. Cells were washed twice with 1 mL of assay buffer (the composition was: 118 mM NaCl, 25 MM NaHCO3, 4.7 mM KCI, 1.2 mM KH2PO4, 1.2 mM CaCl2, 3.8 mM MgCl2, 1.2 mM MgSO4, 11 mM D-glucose and 10 mM HEPES, pH 7.4). The cells were subsequently incubated for 15 min at 37°C in assay buffer supplemented with 2 U/mL adenosine deaminase. Test compounds or buffer were added to the cells before [125I]-AB- MECA and incubated for 45 min. at 37°C in a final volume of 500mL. The cells were washed quickly with 2 x 2 mL ice-cold assay buffer, solubilised with 1 mL of NaOH (2 M) and transferred to g-counting vials with 0.5 mL water.
Test procedure & data analysis Test compounds are dissolved in DMSO, ethanol or water and further diluted in assay buffer. The final concentrations of solvents should be less than 2%. Test solutions should be in the range of pH 6.5 - 7.5. IC50 values were calculated from dose-response curves (3 points minimum) by a non-linear regression analysis using the GraphPad Prism program (GraphPad Software, USA). The results expressed in nM.
In vitro assay of TNF-α
The test compound is dissolved in dimethyl sulphoxide (DMSO) at 8 mg mL and is diluted with Cremophor in 5% saline (0.9% aqueous NaCl) to 160, 16 and 1.6 μg/mL. 25 μL is added to each tube, and 350 mL heparinised (50 lE/mL) rat blood, 25 μL lipopolysaccharide (LPS) 1.6 mg/mL in saline are introduced, i.e. the concentrations of the test compound is 10, 1 and 0.1 mg/μL respectively. The samples are shaken carefully and are placed in a water bath for 5 h. at 37°C. The samples are centrifuged for 10 min. at 3000 φm at 4°C. The plasma is removed by pipette in plastic tubes and is frozen. TNF-α levels are determined using a Genzyme ELISA kit.
Biological activity of selected examples in vitro
Example Inhibition of LPS-induced rat brain
Ratio TNF-α number
K, (nM) A3 A^A, inhibition rat whole blood IC50 (μM)
8 10.4 1.5 3.0
34 26.3 6.5 0.9
43 4.6 22 3.0
50 20.2 61 0.9
Λ/-benzyl 41 58 3.0 MECA Pharmaceutical compositions
The compounds of the invention, together with a conventional adjuvant, carrier or diluent, and if desired in the form of a pharmaceutically acceptable acid addition salt thereof, may be placed into the form of pharmaceutical compositions and unit dosages thereof, and in such form may be employed as solids, such as tablets of filled capsules, or liquids, such as solutions, suspensions, emulsions, elixirs, or capsules filled with the same, all for oral use, in the form of suppositories for rectal administration; or in the form of sterile injectable solutions for parenteral use (including subcutaneous administration and infusion). Such pharmaceutical compositions and unit dosage forms thereof may comprise conventional ingredients in conventional proportions, with or without additional active compounds or principles, and such unit dosage forms may contain any suitable effective amount of the adenosine receptor agonist commensurate with the intended daily dosage range to be employed. Tablets containing ten (10) milligrams of active ingredient or, more broadly, ten (10) to hundred (100) milligrams, per tablet, are accordingly suitable representative unit dosage forms.
The compounds of this invention can thus be used for the formulation of pharmaceutical preparation, e.g. for oral and parenteral administration to mammals including humans, in accordance with conventional methods of galenic pharmacy.
Conventional excipients are such pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral or enteral application which do not deleteriously react with the active compounds.
Examples of such carriers are water, salt solutions, alcohols, polyethylene glycols, polyhyroxyethoxylated castor oil, gelatine, lactose amylose, magnesium stearate, talc, silicic acid, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxy- methylcellulose and polyvinylpyrrolidone. The pharmaceutical preparations can be sterilised and mixed, if desired, with auxiliary agents, emulsifiers, salt for influencing osmotic pressure, buffers and/or colouring substances and the like, which do not deleteriously react with the active compounds.
For parenteral application, particularly suitable are injectable solutions or suspensions, preferably aqueous solutions with the active compound dissolved in polyhydroxylated castor oil.
Ampoules are convenient unit dosage forms.
Tablets, dragees, or capsules having talc and/or carbohydrate carrier or binder or the like, the carrier preferably being lactose and/or corn starch and/or potato starch, are particularly suitable for oral application. A syrup, elixir or the like can be used in cases where a sweetened vehicle can be employed.
Generally, the compounds of this invention are dispensed in unit form comprising 0.05-100 g in a pharmaceutically acceptable carrier per unit dosage.
The dosage of the compounds according to this invention is 0.1-300 mg/day, preferably 10-100 mg/day, when administered to patients, e.g. humans, as a drug.
A typical tablet which may be prepared by conventional tabletting techniques contains:
Active compound 5.0 mg Lactosum 67.0 mg Ph.Eur.
Avicel™ 31.4 mg
Amberlite™IRP 88 1.0 mg
Magnesϋ stearas 0.25 mg Ph.Eur. Owing to activity against diabetes, multiple schlerosis, stroke, osteoporosis, septic shock and menstrual complications the compounds of the invention are extremely useful in the treatment of related symptoms in mammals, when administered in an amount effective for agonist activity of compounds of the invention. The compounds of the invention may accordingly be administered to a subject, e.g., a living animal body, including a human, in need of adenosine receptor agonist, and if desired in the form of a pharmaceutically acceptable acid addition salt thereof (such as the hydrobromide, hydrochloride, or sulphate), in any event prepared in the usual or conventional manner, e.g., evaporation to dryness of the free base in solution together with the acid), ordinarily concurrently, simultaneously, or together with a pharmaceutically acceptable carrier or diluent, especially and preferably in the form of a pharmaceutical composition thereof, whether by oral, rectal, or parenteral (including subcutaneous) route, in an effective amount of adenosine receptor agonist, and in any event an amount which is effective for the treatment diseases related to cytokines, owing to their adenosine receptor agonist activity. Suitable dosage ranges are 1-200 milligrams daily, 10-100 milligrams daily, and especially 30-70 milligrams daily, depending as usual upon the exact mode of administration, form in which administered, the indication toward which the administration is directed, the subject involved and the body weight of the subject involved, and the preference and experience of the physician or veterinarian in charge.
The preparation of compounds of the invention is further illustrated in the following examples.
Hereinafter, TLC is thin layer chromatography, THF is tetrahydrofuran, TFA is trifluoracetic acid and mp is melting point. Where melting points are given, these are unconnected. The structures of the compounds are confirmed by assignment of NMR spectra (from which representative peaks are quoted) and by microanalysis where appropriate. Compounds used as starting materials are either known compounds or compounds which can be prepared by methods known per se. Column chromatography was carried out on Merck silica gel 60 (Art 9385). HPLC was carried out on a Waters or Merck chromatograph with a multiwavelength detector and a reversed phase C18 column (250 x 4 mm, 5mm, lOOA; eluent flow rate 1 mL min at 35°C). Retention times are given in minutes. Examples
EXAMPLE 1
2-CMoro-N-(N-methyl-N-(2-phenylethvπamino adenosine.
N-Methyl-2-phenylethylamine hydrochloride.
Phenylacetaldehyde (24.0 g, 0.20 mol), a 33% solution of methylamine in ethanol (42.35 g, 0.41 mol) and methylamine hydrochloride (10.13 g, 14.8 mmol) were dissolved in methanol (200 mL).
Sodium cyanoborohydride (3.77 g, 60 mmol) was introduced and the reaction mixture was stirred at room temperature for 18 h. The reaction mixture was evaporated in vacuo to ca. 50 mL, concentrated hydrochloric acid (40 mL) was added and once the exotherm had subsided the reaction mixture was stirred for 2 h. Water (200 mL) was introduced followed by potassium hydroxide (27g, 0.48 mol) and the cooled solution was extracted with dichloromethane (7 x 100 mL). The combined dichloromethane extracts were extracted with 2 N hydrochloric acid solution (200 mL) and 0.2 N hydrochloric acid solution (200 mL). The combined acidic extracts were washed with dichloromethane (2 x 50 mL) and the aqueous phase was basified with 4 N sodium hydroxide solution before being extracted with dichloromethane (2 x 100 mL). The combined extracts were dried (MgSO4) and evaporated to a residue (9.2 g) which was dissolved in toluene (200 mL) with methanol (5 mL) present. Chlorotrimethylsilane (8.63 mL) dissolved in toluene (300 mL) was added and the hydrochloride salt of N-methyl-2-phenylethylamine preci¬ pitated. After cooling the suspension, the solid was collected by filtration and dried in vacuo; 1H NMR (DMSO-dβ) d 2.54 (3H, s, -CHj), 2.92-3.07 (4H, m, -CH2CH2-), 7.20 -7.40 (5H, m, Ar¬ il).
N-Methyl-N-rritroso-2-phenylethylamine A sample of N-methyl-2-phenylethylamine hydrochloride (6.4 g, 37.3 mmol) was dissolved in water (18 mL), ethanol (11 mL) and 2 N hydrochloric acid ( 4 mL) were introduced and UIQ solution was heated to 70°C. A solution of sodium nitrite (2.6g, 37.7 mmol) in water (11 mL) was added dropwise to the reaction mixture over 30 min. During the addition the reaction mixture was acidified with 2 N hydrochloric acid (4 mL). Cooling was followed by extraction with n-heptane (4 x 100 mL); the combined extracts were dried (MgSO4) and evaporated in vacuo to provide the N-methyl-N-nitroso-2-phenylethylamine as an oil (5.0 g, 81%) (a ca. 65:35 mixture of apparent geometric isomers), *H NMR (DMSO-ds)d 2.98 (3H, s, -CH3), 3.05 (2H, t, PhCH2-), 4.37 (2H, t, -CH2-N-), 7.13-7.36 (5H, m, Ar-H) (major isomer); 2.78 (2H, t, PhCH2-), 2.98 (3H, s, -CH3), 3.77 (2H, t, -CH2-N-), 7.13-7.36 (5H, m, Ar-H) (minor isomer).
1 -Methyl- 1 -(2-phenylethyl)hydrazine.
The above nitrosamine (5.0 g, 30.4 mmol) was dissolved in dry THF (100 mL) and a 1 M solution of lithium aluminium hydride in THF was added dropwise. On heating to 55°C reaction commenced, the mixture was heated at reflux for 30 min. and cooled to room temperature. The reaction mixture was kept cool using a 23 °C water bath and water (50 mL) was carefully introduced dropwise with rapid stirring, followed by IN sodium hydroxide solution (4 mL). The precipitate was removed by filtration and the filtrate was extacted with dichloromethane (3 x 50 mL). The combined extracts were dried (MgSO ) and evaporated to a residue (4.08 g) which 35was dissolved in toluene (100 mL) with methanol (2 mL) present. Chlorotrimethylsilane (3.46 mL) dissolved in toluene (200 mL) was introduced and the hydrochloride salt of 1 -methyl- 1 -(2- phenylethyl)hydrazine was separated as a gum, Η NMR (CDC13) d 2.44 (3H, s, -CH3), 2.56 (2H, t, -CH2), 2.76 (2H, t, -CH2), 3.36 (2H, br s, -NH2), 7.13 - 7.32 (5H, m, Ar-H).
9-(2,,3',5,-tri-O-Benzoyl-β-D-ribofuranosyl)-2,6-dichloro-9H-purine
2,6-dichloro-9H-purine (5.8 g, 30.7 mmol) and 1-O-acetyl-tri-O-benzoyl-β-D-ribofuranose (16.26 g, 32.2 mmol) were thoroughly mixed (as powdery solids) and fused together at 145-150°C under oil pump vacuum. The resultant oily mixture was stirred gently for 0.75 h (during which time the acetic acid by-product evaporated) and cooled to ca. 50°C before being dissolved in dichloromethane (100 mL) with stirring. This solution was applied directly to a column of silica gel (6 x 22 cm) and eluted initially with cyclohexane/dichloromethane (1/1), then with dichloromethane and finally with cyclohexane/ethyl acetate (1/1) to provide 9-(tri-O-benz- oyl-β-D-ribofuranosyl)-2,6-dichloro-9H-purine (16.6 g, 87%) as a colorless foam, TLC rf 0.50 [SiO , cyclohexane/ ethyl acetate (1/1)]. Η NMR (DMSO-de) d 4.72 (1H, dd, H-5',), 4.88 (1H, q, H-4'), 4.93 (1H, dd, H-5'b), 6.15 (2H, m, H-2' & H-3'), 6.50 (1H, d, H-l'), 7.34 - 7.65 (9H, m, m- & p-ArH), 7.90 - 8.13 (6H, m, o-ArH), 8.28 (1H, s, H-8). (This method of preparation is a modification of that described by Imai, K-i. et ai, Chemical and Pharmaceutical Bulletin, 1966, 14, 1377-1381, but without the use of a catalyst).
2, \5'-Tri-O-berιzoyl-2-chloro-N-[N-methyl-N-(2-phenylethyl)- amino]adenosine
The above hydrochloride salt of 1 -methyl- l-(2-phenylethyl)hydrazine (0.67 g, 1.05 mmol), 9-(2,,3,,5,-tri-O-benzoyl-β-D-ribofuranosyl)-2,6-dichloro-9H-purine (1.90 g, 3 mmol) and triethylamine (1.24 mL, 9 mmol) were dissolved in 1,4-dioxan (20 mL). The solution was stirred at ambient temperature for 20 h. The cooled reaction mixture was evaporated to a gum and purified by flash chromatography on silica gel. Elution with cyclohexane/ethyl acetate (4/1) initially and then with a 1/1 mixture of these solvents provided 2,'3l,51-tri-O-benzoyl-2-chloro-N- (N-methyl-N-(2-phenylethyl)amino)adenosine (1.28 g, 60%) as a foam, Η NMR (DMSO-de)d 2.63 (3H, s, -CH3), 4.66 (1H, dd, H-5'.), 4.77 (1H, dd, H-5'b), 4.86 (1H, q, H-4'), 6.23 (1H, t, H-3'), 6.36 (1H, t, H-2'), 6.54 (1H, d, H-l'), 7.11 - 7.98 (20H, m, Ar-H), 8.44 (1H, s, H-8), 9.40 (1H, br s, N-H).
2, 5'-Tri-O-benzoyl-2-cWoro-N-(N-methyl-N-(2-phenylethyl)amino)- adenosine (1.24 g, 1.74 mmol) was dissolved in methanolic ammonia (50 mL) and allowed to stand at ambient temperature for 20 h. The reaction mixture was evaporated and the residue was purified by flash chromatography on silica gel. Elution with dichloromethane/methanol (19/1) initially, increasing the polarity of the eluent to dichloromethane/methanol (9/1) provided the title compound (0.57 g, 78%) as semi-solid foam, TLC rf 0.25 [SiO2, dichloromethane/ethanol/25% aqueous ammonia solution (60/10/1)], Η NMR (DMSO-dβ) d 2.65 (3H, s, -CH3), 3.53 - 3.60 (IH, m, H-5'.), 3.63 - 3.70 (IH, m, H-5',), 4.05 (IH, q, H-4'), 4.15 (IH, q, H-3'), 4.55 (IH, q, H-2'), 5.08 (IH, t, 5'- OH), 5.22, 5.50 (2H, 2d, 2'-and 3'-OH), 5.56 (IH, d, H-l'), 7.11 - 7.28 (5H, m, Ar-H), 8.42 (IH, s, H-8), 9.33 (lH, br s, N-H).
Figure imgf000025_0001
0.6 H2O requires C, 51.2 ; H, 5.5 ; N, 18.9. Found: C, 51.3 ; H 5.4 ; N 18.7%.
EXAMPLE 2
2-Chioro-N-[(N-methyl-N-phenvDamino]adenosine
The title compound was prepared according to the method described in Example 1 by reacting 1- methyl- 1-phenylhydrazine (1.29 g, 11 mmol) with 9-(2,,3,,5,-tri-O-benzoyl-b-D-ribofuranosyl)- -2,6-dichloro-9H-purine (1.58 g, 2.5 mmol) and debenzoylating the purified product using potassium carbonate in methanol to provide the title 2-Chloro-N-[(N-methyl-N- phenyl)amino]adenosine (0.70 g, 69%) (after column chromatography) as a colorless foam, TLC rf 0.50 [SiO2, THF], lH NMR (DMSO-d*) d 3.23 (3H, 2s, CH3), 3.50 - 3.72 (2H, br, -CHr), 5.06 (IH, br, 5'-OH), 5.24, 5.52 (2H, 2d, 2'- and 3'-OH), 5.86 (IH, br, H-l'), 6.70 - 6.84 (3H, m, Ar-H), 7.15 - 7.24 (2H, t, Ar-H).
Cι7Hι9CIN6θ4. 0.66 H2O requires C, 48.7; H, 4.9; N, 20.0. Found: C, 49.1; H 4.9; N 19.6%
EXAMPLE 3
2-Chloro-/V-fΛ^N-dimethylamino adenosine
The title compound was prepared according to the method described in Example 1 by reacting 1,1-dimethylhydrazine (0.15 g, 2.52 mmol) with 9-(2,,3',5'-tri-O-benzoyl-β-D-ribofuranosyl)- -2,6-dichloro-9H-purine (1.45 g, 2.29 mmol) and debenzoylating the purified product using methanolic ammonia to provide the title 2-chloro-N-(N^V-dimethylamino)adenosine (0.140 g, 29%) (after column chromatography) as a colorless foam, lH NMR (DMSO-de) d 2.58 (6H, s, 2 x -CH3), 3.52 - 3.57 (IH, m, H-5',), 3.62-3.68 (IH, m, H-5'b), 3.93 (IH, q, H-4'), 4.11 (IH, q, H-3'), 4.50 (IH, q, H-2'), 5.06 (IH, t, 5'-0H), 5.22, 5.48 (2H, 2d, 2'-and 3'-OH), 5.82 (IH, d, H-l'), 8.38 (IH, s, H-8), 9.34 (IH, br s, N-H).
C12H17ClN6O4. 0.5 EtOH. 0.5 H20 requires C, 41.5 ; H, 5.6; N, 22.3. Found: C, 41.5; H, 5.5; N, 22.1%.
EXAMPLE 4
2-Chloro-N-(phenylmethoxy')adenosine.
2',3,,5'-Tri-O-benzoyl-2-chloro-N-(phenylmethoxy)adenosine
This example was prepared by a method similar to that described in Example 1. 9-(2,,3,,5'-Tri-O-benzoyl-β-D-ribofuranosyl)-2,6-dichloro-9H-purine (2.0 g, 3.2 mmol), O- henylmethyl)hydroxylamine hydrochloride (0.77 g, 4.8 mmol) and N,N-diisopropylamine (1.03 g, 8.0 mmol) were dissolved in dioxan (50 mL). The reaction mixture was heated at reflux for 20h, filtered and evaporated in vacuo. The crude product was coevaporated with dichloromethane and crystallised from a mixture of dichloromethane and methanol to provide the title compound (1.0 g, 43%), as white crystals, mp 115 - 119°C, 1H NMR (CDC13) d 3.52 - 3.70 (2H, m, H-5'a and H-5'b) 3.95 (IH, d, H-4'), 4.15 (IH, dd, H-3'), 4.52 (IH, dd, H-2'), 5.00 (2H, s, CH2), 5.88(1H, d, H-l'), 8.52 (IH, s, H-8).
C38H29ClN5O8 requires C, 63.4; H, 4.1; N, 9.7; Cl, 4.9. Found C, 63.5; H, 4.3; N, 9.5; Cl, 5.2%. 2',3',5'-Tri-O-benzoyl-2-chloro-N-(phenylmethoxy)adenosine (0.80g, 1.1 mmol) was suspended in methanolic ammonia (50 mL). The reaction mixture was stirred at room temperature for 72h. The solvent was evaporated in vacuo and the crude product was purified by flash chromato¬ graphy (2 x 40cm) eluting with dichloromethane/ethanol/aqueous ammonia solution (90/10/1) to give the title 2-chloro-N-(phenylmethoxy)adenosine as a foam (0.40 g, 89%), Η NMR (DMSO- dβ) d 3.52 - 3.70 (2H, m, H-5'a and H-5'b), 3.95 (IH, d, H-4*), 4.15 (IH, dd, H-3'), 4.52 (IH, dd, H-2'), 5.00 (2H, s, -CH2-), 5.88 (IH, d, H-l'), 8.52 (IH, s, H-8). HPLC retention time 13.02 min (gradient elution over 30min; 20-80% acetonitrile 0.1% TFA in water; 99.6% purity/ 254nm).
EXAMPLE 5
N-f(4-Nitrophenyl)methoxyladenosine
The title compound was prepared by reacting O-[(4-nitrophenyl)methyl]hydroxylamine hydro¬ chloride (1.43 g, 7 mmol) with 6-chloropurine riboside (i.e. 9-β-D-ribofuranosyl-6-chloro-9H- purine) (1.0 g, 3.5 mmol) in DMF (40 mL) at 110°C for 2 h with dϋsopropylethylamine (1.80 g, 14 mmol) present. The reaction mixture was evaporated and to the resultant residue was added saturated sodium bicarbonate solution (20 mL) and water (20 mL). Methanol was gradually added until the residue dissolved, and the solid which gradually precipitated was removed by filtration. The filtrate was concentrated to a residue which was purified by flash chromatography on silica gel. Elution with ethyl acetate/ ethanol (30/1) initially, followed by a (8/1) mixture of these solvents provided a solid which was recrystallised from ethanol. The first crop of material was discarded, but the second crop was confirmed as the title compound (70 mg, 7%) mp 115- 117°C. TLC x{ 0.20 [SiO2, ethyl acetate/ methanol (9/1)]; 1H NMR (DMSO-d«) d 3.46 - 3.57 (2H, m, H-5', and H-5'b), 3.90 (IH, q, H-4'), 4.08 (IH, q, H-3'), 4.43 (IH, q, H-2'), 5.07 (IH, t, 5'-OH), 5.44, 5.74 (2H, 2d, 2* and 3'-OH), 5.82 (IH, d, H-l'), 7.67 (2H, d, Ar-H), 8.08 (lh, s, H- 8), 8.22 (2H, d, Ar-H), 9.34 (IH, br s, N-H). Cι7H,8N6O7. 0.75 H20 requires C, 47.3 ; H, 4.6 ; N, 19.45. Found: C, 48.8; H, 4.55; N, 19.45%.
EXAMPLE 6
2-Chloro-/V*-(2-phenylethoxy adenosine.
N-(2-Phenylethoxy)phthalimide
N-Hydroxyphthalimide (15.0 g, 92 mmol) and sodium acetate (7.5 g, 92 mmol) were stirred in dimethylsulfoxide (70 mL) at ambient temperature for 3h. 2-Chloroethylbenzene (12.5 g, 89 mmol) was added and the reaction mixture was heated at reflux for 2 h. After cooling and standing overnight the reaction mixture was filtered and the filtrate was poured onto ice/water (300 mL). The mixture was extracted with dichloromethane (4 x 100 mL), the combined extracts were dried (MgSO4) and evaporated in vacuo to a residue. Purification by flash chromatography on a silica gel column (3 x 40 cm) eluting with heptane ethyl acetate (1/1) afforded the title compound as a colorless foam (15.7 g, 33%); 1H NMR (DMSO-ds) d 3.05 (2H, t, -OCH2-), 4.39 (2H, t, -CH2Ph), 7.20-7.35 (5H, m, Ar-H), 7.85 (4H, s, Ar-H).
O-(2-PhenylethyI)hydroxylamine hydrochloride.
N-(2-Phenylethoxy)phthalimide (11.3 g, 42 mmol) was dissolved in hot 96% ethanol (100 mL). Hydrazine hydrate (2.5 g, 50 mmol) was introduced and the reaction mixture was heated at reflux with mechanical stirring for 1.5h. The reaction mixture was stored at 4°C for 72 h, filtered and the filtrate was evaporated in vacuo. The crude white residue was suspended in toluene and stored at 4°C for 16h and filtered. The filtrate was treated with a solution of chlorotrimethyl- silane (4.34 g) in toluene (200 mL) with methanol (1.05 g) present and the title compound precipitated. The suspension was allowed to cool and the product was collected and dried in vacuo, giving a white hygroscopic solid product (5.84g, 84%). 'H NMR (DMSO-dβ) d 2.95 (2H, t, -OCH2-), 4.25 (2H, t, -CH2Ph), 7.20 - 7.35 (5H, m, Ar-H).
2,,3',5'-Tri-O-benzoyl-2-chloro-N-(2-phenylethoxy)adenosine.
9-(2',3,,5,-Tri-O-benzoyl-β-D-ribofuranosyl)-2,6-dichloro-9H-purine (2.0 g, 3.2 mmol), O-(2-phenylethyl)hydroxylamine hydrochloride (0.70 g, 4.0 mmol) and N,N-dϋsopropylamine (0.95 g, 7.4 mmol) were dissolved in 1,4-dioxan (40 mL) and heated at reflux for 3 days. After cooling the reaction mixture was diluted with dichloromethane (50 mL) and washed with water (2 x 30 mL). The organic phase was dried (MgSΟ ). Flash chromatography on a silica gel column (2 x 40 cm), eluting with heptane/ethyl acetate (1/1) gave the desired product as a foam (1.54 g, 66%), Η NMR (DMSO-ds) d 3.05 (2H, t, -OCH2-), 4.19 (2H, t, -CH2Ph), 4.70 (IH, dd, H-5'a), 4.80 (IH, dd, H-5'b), 4.90 (IH, dd, H-4'), 5.22 (IH, t, H-3'), 6.39 (IH, t, H-2'), 6.59 (Hi d, H-l'), 8.51 (lH, s, H-8).
Figure imgf000029_0001
requires C, 63.8; H, 4.4; N, 9.5. Found C, 63.7; H, 4.5; N, 9.4%.
2')3l,5,-Tri-O-benzoyl-2-chJoro-N-(2-phenylethoxy)adenosine (1.54 g, 2.0 mmol) was suspended in a solution of methanolic ammonia and stirred at room temperature for 2 days. The reaction mixture was concentrated in vacuo and the crude product was purified by flash chromatography (2 x 40 cm) eluting with dichloromethane/ethanol aqueous ammonia solution (90/10/1), providing the product as a foam (0.48 g, 57%), Η NMR (DMSO-de) d 3.00 (2H, t, -CH2O-), 3.55 - 3.70 (2H, m, H-5', and H-5'b), 3.99 (IH, d, H-4'), 4.15 - 4.20 (3H, m, -CH2Ph and H-3'), 4.52 (IH, dd, H-2'), 5.88 (IH, d, H-l*), 8.51 (IH, s, H-8). HPLC retention time 13.64 min (gradient elution over 30min; 20-80% acetonitrile/0.1% trifluoroacetic acid in water; 99.0% purity/ 254nm).
CISHMCINJOS. 0.75 H2O requires C, 49.7; H, 5.0; N, 16.1. Found C, 49.9; H, 4.9; N, 15.8%. EXAMPLE 7
2-Chloro-N-cyclopentyloxyadenosine
The title compound was prepared according to the method described in Example 6 by reacting O-cyclopentylhydroxylamine (prepared by the overall procedure described in example 5) (0.78 g, 5.67 mmol) with 9-(2',3',5'-tri-O-benzoyl-β-D-ribofuranosyl)-2,6-dichloro-9H-purine (3.0 g, 4.74 mmol), followed by debenzoylation of the purified product using methanolic ammonia to provide the title 2-chloro-JV-cyclopentoxyadenosine (0.11 g) (after column chromatography) as a solid, mp 116-119°C. Η NMR (DMSO-d*) d 1.50 - 1.80 (8H, m, -CH2CH2CH2CH2-), 3.52 - 3.58 (IH, m, H-5',), 3.63 - 3.70 (Hi m, H-5'b), 3.94 (Hi q, H-4'), 4.13 (IH, q, H-3'), 4.51 (IH, q, H-2'), 4.57 (IH, br m, -OCH-), 5.06 (IH, t, 5'-0H), 5.22, 5.50 (2H, 2d, 2'-and I'-OH), 5.86 (IH, d, H-l'), 8.46 (IH, s, H-8), 11.40 (Hi s, N-H)
EXAMPLE 8
2-Chloro-N-methoxyadenosine.
The title compound was prepared according to the method described in Example 4 by reacting 0-methylhydroxylamine (0.20 g, 2.0 mmol) with 9-(2',3',5'-tri-O-benzoyl-β-D-riboftιranosyl)- -2,6-dichloro-9H-purine (1.27 g, 2.0 mmol) and debenzoylating the purified product using methanolic ammonia to provide the title 2-chloro-N-methoxyadenosine (0.40 g, 45%) (after column chromatography) as a colorless foam which became crystalline on trituration with dichloromethane, providing 0.20 g of a white solid, mp 123 - 125°C. Η NMR (DMSO-ds) d 3.52 -3.59 (IH, m, H-5',), 3.63 - 3.70 (Hi m, H-5'b), 3.78 (3H, s, -OCH3), 3.96 (IH, q, H-4'), 4.14 (IH, q, H-3'), 4.52 (IH q, H-2'), 5.06 (IH t, 5'-0H), 5.22, 5.51 (2H, 2d, 2'-and 3'-OH), 5.87 (Hi d, H-l*), 8.50 (Hi s, H-8), 11.60 (IH, s, N-H). CΠHMCINSOS. 1.33 H2O requires C, 37.1 ; H 4.7 ; N, 19.7. Found: C, 37.4; H, 4.4; N, 19.3%.
EXAMPLE 9
2-Chloro-N-ethoxyadenosine
The title compound was prepared according the method described in example 6 by reacting 0- ethylhydroxylamine hydrochloride (0.305 g, 3.1 mmol) with 9-(2,,3,,5'-tri-O-acetyl-β-D-ribo- furanosyl)-2,6-dichloro-9H-purine (1.79 g, 4.0 mmol), followed by deacylation of the purified product using sodium methoxide in methanol to provide the title 2-chloro-N-ethoxyadenosine
(0.161 g, 21%) (after column chromatography) as a foam, Η NMR (DMSO-de) d 1.25 (3H t, -
CH2CH3), 3.52 - 3.58 (Hi m, H-5'_), 3.63 - 3.70 (IH, m, H-5'b), 3.94 (IH, q, H-4'), 4.00 (2H, q,
-CILCHj), 4.13 (IH q, H-3'), 4.51 (IH, q, H-2'), 5.04 (IH, t, 5'-0H), 5.22, 5.49 (2H, 2d, 2'- and 3'-OH), 5.86 (IH d, H-l'), 8.48 (IH, s, H-8), 11.45 (IH, s, N-H).
EXAMPLE 10
2-Chloro-N-( 1 -propyloxytedenosine
The title compound was prepared according to the method described in Example 6 by reacting O-(l-propyl)hydroxylamine hydrochloride with 9-(2',3',5'-tri-O-acetyl-β-D-ribofuranosyl)- -2,6-dichloro-9H-purine, followed by deacylation of the purified product using sodium methoxide in methanol to provide the title 2-chloro-JV-(l-propyloxy)adenosine (after column chromatography) as a foam. EXAMPLE 1 1
2-Chloro-jV-f 2-methyl- 1 -propyloxy)adenosine
The title compound was prepared according to the method described in Example 6 by reacting isobutoxyamine hydrochloride (0.603 g, 4.8 mmol) with 9-(2',3',5'-tri-O-acetyl-β-D-ribo- furanosyl)-2,6-dichloro-9H-purine (1.79 g, 4.0 mmol), followed by deacylation of the purified product using sodium methoxide in methanol to provide the title 2-chloro-N-ethoxyadenosine (0.31 g, 42%) (after column chromatography) as a foam, H NMR (DMSO-de) d 0.96 (6H, 2t, - CH(CH3)2), 2.0 (IH, h, -CH2CH(CH3>.), 3.52 - 3.58 (IH, m, H-5',), 3.63 - 3.70 (IH, m, H-5'b), 3.73 (2H, d, -CHzCH CHj),), 3.95 (IH, q, H-4'), 4.14 (Hi q, H-3'), 4.50 (IH, q, H-2'), 5.04 (IH, t, 5'-0H), 5.20, 5.48 (2H, 2d, 2'- and 3'-OH), 5.85 (Hi d, H-l'), 8.47 (IH, s, H-8), 11.48 (Ir s, N-H).
EXAMPLE 12
2-Chloro-N-(3-propenyloxV)adenosine
The title compound was prepared according to the method described in Example 6 by reacting O-allylhydroxylamine hydrochloride (0.526 g, 4.8 mmol) with 9-(2,,3',5'-tri-O-acetyl-β-D-ribo- furanosyl)-2,6-dichloro-9H-purine (1.79 g, 4.0 mmol), followed by deacylation of the purified product using sodium methoxide in methanol to provide the title 2-chloro-N-ethoxyadenosine (0.39 g, 60%) (after column chromatography) as a foam, 1H NMR (DMSO-de) d 3.53 - 3.59 (IH, m, H-5',), 3.62 - 3.69 (IH, m, H-5* b), 3.95 (IH, q, H-4'), 4.04 (IH, q, H-3'), 4.48 (2H, d, - CH2CH=CH2), 4.51 (Hi q, H-2'), 5.06 (Hi t, 5'-0H), 5.22, 5.48 (2H, 2d, 2'- and 3'-OH), 5.26 (Hi dd, -CH2CH=CH), 5.37 (IH, dd, -CH2CH=CH), 5.85 (IH, d, H-l'), 6.04 (Hi qt, - CH2CH=CH2), 8.48 (IH, s, H-8), 11.50 (IH s, N-H). EXAMPLE 13
2-Crύoro-N-( 1.1 -dimethylethoxy)adenosine
The title compound was prepared according to the method described in Example 6 by reacting O-tert-butylhydroxylamine hydrochloride (0.603 g, 4.8 mmol) with 9-(2',3',5,-tri-O-acetyl-β-D- ribofuranosyl)-2,6-dichloro-9H-purine (1.79 g, 4.0 mmol), followed by deacylation of the purified product using sodium methoxide in methanol to provide the title 2-chIoro-N-ethoxyadenosine
(0.296 g, 56%) (after column chromatography) as a foam, lH NMR (DMSO-ds) d .1.27 (9H, m,
-C(CH3)3), 3.52 - 3.58 (Hi m, H-5',), 3.62 - 3.69 (IH, m, H-5'b), 3.94 (IH q, H-4'), 4.14 (IH, q, H-3'), 4.52 (IH, q, H-2'), 5.05 (IH, t, 5'-0H), 5.22, 5.50 (2H, 2d, 2'- and 3'-OH), 5.85 (IH, d,
H-l'), 8.47 (IH, s, H-8), 11.01 (IH, s, N-H).
EXAMPLE 14
2-Chloro-N-f2-(phenoxy ethoxy1adenosine
The title compound was prepared by reacting O-[2-(phenoxy)ethyl]hydroxylamine (prepared by the procedure described in Example 6) (0.94 g, 5.0 mmol) with 9-(2',3',5'-tri-O-benzoyl-β-D- ribofuranosyl)-2,6-dichloro-9H-purine (2.0 g, 3.16 mmol), followed by debenzoylation of the purified product using methanolic ammonia to provide the title 2-chloro-N-[2- (phenoxy)ethoxy]adenosine (0.44 g, 32%) (after column chromatography) as a colorless foam, 1HNMR (DMSO-de) d 3.52 - 3.59 (IH m, H-5' , 3.63 - 3.70 (IH, m, H-5'„), 3.94 (Hi q, H-4'), 4.11 (Hi q, H-3'), 4.32 (4H, dt, -OCH2CH2O-), 4.50 (IH, q, H-2'), 5.06 (IH, t, S'-OH), 5.22, 5.50 (2H, 2d, 2'-and 3'-OH), 5.86 (IH, d, H-l1), 6.92 - 6.97 (3H, m, Ar-H), 7.26 - 7.33 (2H, dd, Ar-H), 8.51 (IH, s, H-8), 11.64 (IH, br, N-H). CιgH20ClN5O4. 0 66 H20 requires C, 48.1 ; H 4.8 ; N, 15.6. Found. C, 48.1; H, 4.7; N, 15.4%.
EXAMPLE 15
2-Chloro-N-f 1.2.3,4-tetrahvdronaphth- 1 -yloxy)adenosine.
The title compound was prepared according to the method described in Examples 4 and 5 by reacting O-[l,2,3,4-tetrahydronaphth-l-yl)hydroxylamine (prepared by the procedure described in example 6) (0.35 g, 2.0 mmol) with 9-(2',3,,5'-tri-O-benzoyl-β-D-ribofuranosyl)-2,6-dichloro-
-9H-purine (1.0 g, 1.58 mmol) and debenzoyiating the purified product using methanolic ammonia to provide the title 2-chloro-N-(l,2,3,4-tetrahydronaphth-l-yloxy)adenosine (0.10 g,
15%) (after column chromatography) as a colorless foam (a mixture of diastereoisomers), 1H NMR (DMSO-ds) d 1.65 - 2.85 (6H, m, -CH2CH2CH2-), 3.53 -3.61 (IH, m, H-5',), 3.64 - 3.70
(IH, m, H-5'b), 3.94 (IH, br d, H-4'), 4.16 (IH, br d, H-3'), 4.53 (IH, q, H-2'), 5.07 (2H, m, 5'-
0H and -OCH-), 5.26, 5.53 (2H, 2d, 2'-and 3'-OH), 5.88 (IH, d, H-l'), 7.13 - 7.30 (3H, m, Ar-
H), 7.88 - 7.96 (Hi m, Ar-H), 8.52 (IH, s, H-8), 11.60 (Hi br s, N-H). HPLC retention time
20.71 and 20.95 min (gradient elution over 25 min.; 25-45% acetonitrile/ 0.1M
EXAMPLE 16
2JV-Dimethoxyadenosine.
2-Chloro-N-methoxyadenosine (Example 8) (0.20 g, 0.60 mmol) was added to a mixture of sodium hydride (40 % oil dispersion) (0.12 g, 3.0 mmol) methanol (0.24 mL) and DMF (5 mL) which had been stirred under nitrogen at room temperature for 1 h. The reaction mixture was heated at 80°C for 16 h, cooled and evaporated. Flash chromatography provided the title 2,N-dimethoxyadenosine, still however containing some starting 2-chloro-N-methoxyadenosine. 1H NMR (DMSO-ds)d 3.52 -3.59 (IH m, H-5',), 3.63 - 3.70 (IH, m, H-5'b), 3.77 (3H, s, - OCH3), 3.97 (IH, q, H-4'), 4.14 (IH, q, H-3'), 4.60 (IH, q, H-2'), 5.09 (IH, t, 5'-0H), 5.18, 5.44 (2H, 2d, 2'-and 3'-OH), 5.83 (IH, d, H-l'), 8.24 (IH, s, H-8), 11.04 (Hi s, N-H) (desired product only quoted).
EXAMPLE 17
2-Methylthio-/V-|'2-(phenoxy ethoxy1 adenosine.
9-(2',3 5'-tri-O-acetyl-β-D-ribofuranosyl)-6-chloro-2-methylthio-9H-purine
(0.50 g, 1.1 mmol) (prepared using the procedure described in US 5,432,164), O-[2-
(phenoxy)ethoxy]hydroxylamine hydrochloride (0.41 g, 2.16 mmol) (see Example 9) and triethylamine (0.45 g, 4.4 mmol) were stirred at 100°C in dioxan (20 mL) for 6 h, and at ca. 95°C for 65 h. The product, after column chromatography, was deprotected using methanolic ammonia, to provide the title 2-methylthio-N-[2-(phenoxy)ethoxy]adenosine (0.09 g, 18%), (af¬ ter column chromatography) as a colorless foam, [tic Rf 0.26 (SiO2, dichloromethane/methanol (9/1)], 1H NMR (DMSO-ds) d 2.51 (3H s, -SCH3), 3.52 - 3.60 (Hi m, H-5'a), 3.62 - 3.69 (IH, m, H-5'b), 3.94 (Hi q, H-4'), 4.16 (IH, q, H-3'), 4.30 (4H, br d, -OCH2CH2O-), 4.61 (IH, q, H- 2'), 5.04 (IH, t, 5'-0H), 5.23, 5.50 (2H, 2d, 2'-and 3'-OH), 5.90 (IH, d, H-l'), 6.92 - 7.01 (3H m, Ar-H), 7.32 (2H, dd, Ar-H), 8.36 (Hi s, H-8), 11.20 (IH, s, N-H).
EXAMPLE 18
2-Anτino-N-fdimethylamino)adenosine.
The title compound was prepared according to the method described in Example 4 by reacting 1,1-dimethylhydrazine (0.79 g, 13.1 mmol) with 9-(2,3,5-tri-0-acetyl-β-D-ribofuranosyi)-2- amino-6-chloro-9H-purine (4.0 g, 9.35 mmol) and debenzoylating the purified product using methanolic ammonia to provide the title 2-ammo-N-(dimethylamino)adenosine (after column chromatography) as an amorphous foam (0.56 g, 45%), 1H NMR (DMSO-ds) d 2.54 (6H, s, 2 x -CH3), 3.50 - 3.57 (IH, m, H-5'a), 3.61 - 3.68 (Hi m, H-5'b), 3.93 (Hi q, H-4'), 4.12 (IH, q, H-3'), 4.51 (IH q, H-2'), 5.12, 5.38 (2H 2d, 2'-and 3'-OH), 5.42 (IH, t, 5'-0H), 5.76 (IH, d, H-l'), 5.95 (2H s, -NH2), 7.93 (IH, s, H-8), 8.20 (IH, s, N-H).
EXAMPLE 19
2-Chloro-N-f/V-methyl-N-('2-phenoxyethvπamino]adenosine
The title compound was prepared by reacting 1 -methyl- l-(2-phenoxyethyl)hydrazine (prepared by the general method described in Example 1) (0.80 g, 4 mmol) and 9-(2,3,5-tri-O-benzoyl-β- D-ribofuranosyl)-2,6dichloro-9H-purine (2.53 g, 4 mmol) and triethylamine (1.11 mL, 8 mmol) in dioxan (25 mL), followed by debenzoylation of the purified product using methanolic ammonia to provide the title 2-chloro-N-(N-methyl-N-(2-phenoxyethyl)amino)adenosine (0.84 g, 47%) (after column chromatography) obtained as a solid, mp 166-8°C, Η NMR (DMSO-ds) d 2.70 (3H, s, -CH3), 3.22 (2H, br t, -CH2-), 3.51 - 3.58 (IH, m, H-5',), 3.62 - 3.69 (IH, m, H-5',), 3.95 (IH, q, H-4'), 4.05 - 4.15 (3H, m, -CH2- and H-3'), 4.50 (IH, q, H-2 5.06 (IH, t, 5'-0H), 5.21, 5.49 (2H, 2d, 2'-and 3'-OH), 5.81 (IH, d, H-l'), 6.79 - 7.25 (5H, , Ar-H), 8.41 (IH, s, H-8), 9.41 (Hi br s, N-H).
EXAMPLE 20
N-Cyclopeπtoxy-2-methyladenosine O-Cyclopentymydroxylamine hydrochloride (0.52 g, 3.75 mmol) was reacted with 9-(2',3',5,-tri- O-acetyl-β-D-ribofuranosyl)-6-chloro-2-methyl-9H-purine (1.07 g, 2.5 mmol) [prepared from 2-methylinosine (Journal of Organic Chemistry, 1967, 32, 3258 - 3260) by standard acylation and chlorination steps] in dioxan (40 mL) in the presence of triethylamine (0.63 g, 6.25 mmol). The reartion mixture was heated at 100°C in a sealed vessel for 70h, before being filtered and evaporated. The product (after purification by chromatography) was debenzoylated using methanolic ammonia to provide the title N-cyclopentoxy-2-methyladenosine (after column chromatography) as a foam, Η NMR (DMSO-ds) d 1.47 - 1.93 (8H, m,
Figure imgf000037_0001
2.32 (3H, s, -CH3), 4.59 (IH, br m, -O-CH-), 5.88 (IH, d, H-l').
EXAMPLE 21
N-methoxy^-methyladenosine
O-Methylhydroxylamine hydrochloride (0.20 g, 2.4 mmol) was reacted with 9-(2',3',5'-tri- O-acetyl-β-D-ribofuranosyl)-6-chloro-2-methyl-9H-purine (0.85 g, 2.0 mmol) (see Example 20) in dioxan (40 mL) in the presence of triethylamine (0.52 g, 5.0 mmol). The reaction mbcture was heated at 90°C in a sealed vessel for 70h, before being filtered and evaporated. The product (after purification by chromatography) was debenzoylated using methanolic ammonia to provide the tide compound (after column chromatography) as a foam, Η NMR (DMSΟ-ds) d 2.30 (3H, s, 2-CH3), 3.48 - 3.55 (IH, m, H-5'_), 3.60 - 3.67 (IH, m, H-5'b), 3.77 (3H, s, -OCH3), 3.92 (IH, q, H-4'), 4.08 (IH, q, H-3'), 4.45 (IH, q, H-2'), 5.22 (Hi dt, 5'-0H), 5.17, 5.40 (2H, 2d, 2*- and 3'-OH), 5.71 (IH, d, H-l*), 7.99 (IH, s, H-8), 10.85 (IH, s, N-H).
EXAMPLE 22
N-methoxy-2-phenyladenosine O-Methylhydroxylamine hydrochloride (10.2 g, 12.2 mmol) was reacted with 9-(2',3',5'-tri- O-acetyl-β-D-ribofuranosyl)-6-chloro-2-phenyl-9H-purine (0.60 g, 1.2 mmol) in dioxan (30 mL) in the presence of dϋsopropylethylamine (1.70 g, 13.1 mmol). The reaction mixture was heated at 100°C in a sealed vessel for 18h, and at 60°C for 50h before being filtered and evaporated. The product (after purification by chromatography) was debenzoylated using methanolic ammonia to provide the title N-methoxy-2-phenyladenosine (0.085 g, 19%) (after column chromatography) as a foam, Η NMR (DMSO-ds) d 3.55 - 3.62 (IH, m, H-5',), 3.66 - 3.73 (IH, m, H-5'b), 3.87 (3H, s, -OCH3) 3.97 (IH, q, H-4'), 4.23 (Hi q, H-3'), 4.72 (IH, q, H- 2'), 5.02 (IH, dt, 5'-0H), 5.26, 5.52 (2H, 2d, 2'- and 3'-OH), 6.05 (IH, d, H-l'), 7.44 - 7.52 (3H, m, Ar-H), 8.38 (2H, dd Ar-H), 8.49 (IH, s, H-8), 11.04 (IH, s, N-H).
EXAMPLE 23
5'-Deoxy-2.5'-dichloro-N-(l-piperidinyπadenosine
5'-Deoxy-2,5'-dichloro-2',3,-O-l-methylethylidene)-N-(l-piperidinyl)- adenosine
2-Chloro-2',3'-O-(l-methylethylidene)-N-(l-piperidinyl)adenosine [prepared by protection of 2-Chloro-N-(l-piperidinyl)adenosine (Knutsen, L.J.S., Lau, J., Sheardown, M.J., Thomsen, C; Bioorganic and Medicinal Chemistry Letters, 1993, 5, 2661-2666)] (0.28 g, 0.47 mmol), triphenylphosphine (0.31 g, 1.18 mmol) and tetrachloromethane (0.18 g, 1.18 mmol) was stirred in dry dimethylformamide (10 mL) at 20°C for 48 h. The reaction mixture was concentrated in vacuo and the crude product was purified by flash chromatography eluting with dichloromethane and 10% ammonia in ethanol (95:5) to give 5'-deoxy-2,5'-dichloro-2',3,-O-(l-methyl- ethylidene)-N-(l-piperidinyl)adenosine (0.10 g, 48%) as a foam. Η-NMR (400 MHz, DMSO-ds) d 1.34 (3H, s, -CH3), 1.36 (2H, m, piperidine C-H), 1.62 (4H, m, piperidine C-H), 2.82 (4H, br, piperidine C-H), 3.78, 3.88 (2H, ABX, H-5'a and H-5'b), 4.35 (IH, ddd, H-4'), 5.02 (IH, dd, H-3'), 5.39 (IH dd, H-2'), 6.20 (IH, d, H-l'), 8.36 (Hi s, H-8). HPLC retention time 21.45 min. (gradient elution over 30 min.; 20-80% acetonitrile/0.1% TFA in water).
Deprotection of 5'-deoxy-2,5'-dichloro-2',3'-O-(l-methylethylidene)-N-(l-piperidinyl)adenosine (0.11 g, 0.25 mmol) was performed by dissolving the compound in a mixture of ethanol (5 mL) and sulfuric acid (0.2M, 5 mL) and stirring the mixture for 72 h. at room temperature. The reaction mixture was neutralized with aqueous sodium bicarbonate and extracted with dichloro¬ methane (3 x 50 mL). The organic phase was dried (MgSO4) and evaporated in vacuo. The product was purified by flash chromatography on silica gel, eluting with a mbcture of dichloromethane and 10% ammonia solution in ethanol (9:1), to provide the title 5'deoxy- 2,5'-dichloro-N-(l-piperidinyI)adenosine (0.1 g, 99%) as a foam, Η-NMR (400 MHz, DMSO-ds) d 1.35 (2H, br, piperidine C-H), 1.62 (4H, br, piperidine C-H), 2.80 (4H br, piperidine C-H), 3.82, 3.93 (IH, ABX, H-5'a and H-5'b), 4.10 (Hi dd, H-4'), 4.18 (IH, dd, H-3'), 4.63 (IH, dd, H-2'), 5.88 (IH, d, H-l'), 8.39 (IH, s, H-8). HPLC retention time 10.95 min. (gradient elution over 30 min.; 20-80% acetonitrile/0.1% TFA in water, 99% purity at 250 nm).
EXAMPLE 24
("S1-5'-Deoxy-2.5'-dichloro-N- 2-(methylmethoxyVl-pyrrolidinyl]adenosine
(S)-2-ChIoro-N-[2-(methylmethoxy)-l-pyrrolidinyl]adenosine [WO 93/08206 (Novo Nordisk A/S), Knutsen, L.J.S., Lau, J., Sheardown, M.J., Thomsen, C; Bioorganic and Medicinal Chemistry Letters, 1993, 5, 2661-2666] (0.35 g, 0.8 mmol) was dissolved in acetonitrile (5 mL) and cooled on an ice-water bath. Under a nitrogen atmosphere, thionyl chloride (0.34 g, 0.2 mL, 2.4 mmol) (see Borchardt, R.T., Huber, J.A. and Wu, Y.S., Journal of Organic Chemistry, 1976, 41, 565-567) was added and a precipitate appeared which dissolved over the following 15 min. Pyridine (0.13 mL, 0.13 g, 1.6 mmol) was introduced gradually, the reaction mixture became yellow in color and was allowed to reach room temperature gradually. After stirring the mixture overnight, ice was added and the reaction mixture was neutralised to pH 7 with aqueous sodium bicarbonate prior to extraction with ethyl acetate (2 x 10 mL). The combined extracts were dried (MgSO ) and evaporated to provide the intermediate 2,3-O-sulphinyl derivative (0.35 g), to which was added methanol (5 mL), water (1 mL) and 25% aqueous ammonia solution (0.25 mL) and this mixture was stirred for 16 h. The solution was evaporated in vacuo, and the resultant residue was purified by flash chromatography on silica gel eluting with a mixture of dichloromethane and 10% ammonia in ethanol (17:3), to provide the title (S)-5'-deoxy-2,5,-di- chloro-N-[2-(methylmethoxy)-l-pyrrolidinyl]adenosine (0.12 g, 34%) as a foam, 1H-NMR (400MHz, DMSO-ds) d 1.50 - 1.60 (IH br, pyrrolidine C-H), 1.78 (2H, br q, pyrrolidine C-H), 1.92 - 2.03 (IH, br, pyrrolidine C-H), 3.85, 3.95 (2H, ABX, H-5'. and H-5'b), 4.09 (Hi br dd, H-4'), 4.17 (IH, dd, H-3'), 4.67 (Hi dd H-2'), 5.49, 5.62 (2H, 2d, 2'- and 3'-OH), 5.87 (IH, d, H-l'), 8.38 (IH s, H-8), 9.34 (IH, br s, -NH). HPLC retention time 9.65 min. (gradient elution over 30 min.; 20-80% acetonitrile/0.1% TFA in water, 99% purity at 250 πm).
CISHΏCIJNSO,,. 0.25 H2O requires C, 43.9; H, 5.2; N, 19.2. Found: C, 43.7; H, 5.4; N, 19.4%.
EXAMPLE 25
5'-Deoxy-2.5'-dichloro-N-(4-phenoxy- 1 -piperidinvDadenosine
This compound was prepared by the general method described in Example 24. 2-Chloro-N-(4-phenoxy-l-piperidinyl)adenosine [WO 93/08206 (Novo Nordisk A/S)] (0.5 g, 1.05 mmol) was subjected to the chlorination conditions described above, providing the title 5'- deoxy-2,5'-dichloro-N-(4-phenoxy-l-piperidinyl)adenosine which precipitated on treatment with dichloromethane following trituration with ether. Drying in vacuo provided a solid (0.28 g, 56%), m.p. 165-170°C, Η-NMR (400MHz, DMSO-ds) d 1.74 - 1.84 (2H, br, piperidine C-H), 1.99 - 2.08 (2H, br, piperidine C-H), 2.80 - 2.90 (2H, br, piperidine C-H), 3.04 - 3.12 (2H, br, piperidine C-H), 3.85, 3.94 (2H, ABX, H-5*. and H-5'b), 4.09 (Hi q, H-4'), 4.18 (IH, q, H-3'), 4.44 (IH, br, PhO-C-H) 4.66 (IH, dd, H-2'), 5.50, 5.63 (2H, 2d, 2'- and 3'-OH), 5.89 (IH d, H-l'), 6.94 (IH, t, Ar-H), 6.99 (2H, d, Ar-H), 7.30 (2H, t, Ar-H), 8.40 (IH, s, H-8). HPLC retention time 19.15 min. (gradient elution over 30 min.; 20-80% acetonitrile 0.1% TFA in water, 99.9% purity at 250 nm).
EXAMPLE 26
5'-Deoxy-2.5'-dichloro-N-(3-methoxy-l-piperidinvDadenosine
This compound was prepared by the general method described in Example 24. 2-Chloro-N— (3-methoxy-l-piperidinyl)adenosine (prepared by O-methylation of N-tertbutyloxycarbonyI-3- hydroxypiperidine, followed by use of the N-amination technique described in Overberger, C.G. and Herin, L.P. Journal of Organic Chemistry, 1962, 27, 417, and further reaction of the resultant hydrazine as described in Knutsen, L.J.S., Lau, J., Sheardown, M.J., Thomsen, C; Bioorganic and Medicinal Chemistry Letters, 1993, 3, 2661-2666) (0.1 g, 0.24 mmol) was subjected to the chlorination conditions described in Example 2, providing the title 5'-deoxy-2,5'-di- chioro-N-(3-methoxy-l-piperidinyl)adenosine (mixture of diastereoisomers) (0.07 g, 67%), following column chromatography, as a solid, m.p. 200 - 202°C. 'H-NMR (400MHz, DMSO-ds) d 3.86, 3.94 (2H, ABX, H-5'a and H-5'b), 4.11 (IH, q, H-4'), 4.19 (IH, q, H-3'), 4.65 (Hi dd H- -2'), 5.49, 5.62 (2H, 2d, 2'- and 3'-OH), 5.88 (IH, d, H-l'), 8.39 (Hi s, H-8). HPLC retention time 17.08 min. (gradient elution over 30 min.; 20-80% acetonitrile/0.1% TFA in water, 98.7% purity at 250 nm).
EXAMPLE 27
5'-Deoxy-2.5'-dichloro-/V-(4-phenylthio- 1 -piperidinyDadenosine This compound was prepared by the method described in Example 24. 2-Chloro— N-(4-ρhenylthio-l-piperidinyl)adenosine [WO 93/08206 (Novo Nordisk A/S)] (5.49 g, 11.1 mmol) was subjected to the reartion conditions described above, providing the title 5'- deoxy-2,5'-dichloro-N-(4-phenylthio-l-piperidinyl)adenosine which precipitated from die aqueous methanolic ammonia. Recrystallization provided a solid (3.93 g, 69%), m.p. 154 - 157°C, 1H-NMR (400MHz, DMSO-ds) d 1.74 - 1.84 (2H, br, piperidine C-H), 1.95 - 2.05 (2H br, piperidine C-H), 2.80 - 2.90 (IH, br, piperidine C-H), 3.04 - 3.12 (2H, br, piperidine C-H),' 3.84, 3.93 (2H, ABX, H-5*. and H-5'b), 4.10 (IH, q, H-4'), 4.17 (IH, q, H-3'), 4.64 (IH, dd, H- 2'), 5.48, 5.62 (2H, 2d, 2'- and 3'-OH), 5.87 (IH, d, H-l'), 7.26 (IH, t, Ar-H), 7.35 (2H, t, Ar-H), 7.42 (2H, d, Ar-H), 8.38 (IH, s, H-8), 9.49 (IH, s, N-H). HPLC retention time 22.19 min. (gradient elution over 30 min.; 20-80% acetonitrile/0.1% TFA in water, 100% purity at 250 nm).
EXAMPLE 28
5'-Deoxy-2.5'-dichloro-N-O-phenylthio-l-piperidinv0adenosine
This compound was prepared by the method described in Example 24. 2-Chloro-N-(4-phenyl- thio-l-piperidinyl)adenosine [WO 93/08206 (Novo Nordisk A/S)] (0.5 g, 1 mmol) was subjected to the reartion conditions described above, providing the title 5'-deoxy-2,5'-dichloro-N-(3-phenyl- thio-l-piperidinyl)adenosine (mixture of diastereoisomers) as a foam (0.48 g, 94%) following flash chromatography on silica gel. Η-NMR (400MHz, DMSO-ds) d 3.84, 3.92 (2H, ABX, H-5'. and H-5'b), 4.10 (IH q, H-4'), 4.17 (IH, q, H-3'), 4.64 (IH, dd, H-2'), 5.49, 5.62 (2H, 2d, 2'- and 3'-OH), 5.87 (Hi d, H-l'), 7.22 (IH, t, Ar-H), 7.31 (2H, t, Ar-H), 7.43 (2H, d, Ar-H), 8.39 (IH, s, H-8). HPLC retention time 17.08 min. (gradient elution over 30 min.; 20-80% acetonitrile/0.1% TFA in water, 98.7% purity at 250 nm). EXAMPLE 29
5'-Deoxy-2.5'-dichloro-N-(4-phenylsulfinyl- 1 -piperidinvDadenosine
2',3'-Di-O-acetyl-5,-deoxy-2,5,-dichloro-N-(4-phenylthio-l-piperidinyl)adenosine (prepared by acetylation of Example 5) (2.95 g, 5 mmol) was dissolved in dry dichloromethane (100 mL). "Oxone" (7.7 g, 2.5 equiv.) and wet clay (Hirano, M., Tomaru, J. and Morimoto, T. Bull. Chem. Soc. Japan, 1991, 64, 3752-3754) (5.95 g) were introduced with vigourous stirring. Both the phenylsulphinyl product (Rf = 0.21) and the phenylsulphonyl product (Rf = 0.45) were apparent by TLC [SiOi etiiyl acetate/methanol (9:1)] and after 0.5 h. reartion time the reartion mixture was filtered. The filtrate was washed with water (2 x 100 mL), dried (MgSO ) and evaporated. The residue was purified by flash chromatography on silica gel eluting with a mixture of ethyl acetate and heptane (1 :1), then with ethyl acetate, and finally with a mixture of ethyl acetate and methanol (19:1), to provide 2',3'-di-O-acetyl-5'-deoxy-2,5,-dichloro-N- (4-phenylsulfinyl-l-piperidinyl)adenosine (0.18 g, 6%) and 2',3'-di-O-acetyl-5'-deoxy-2,5'-di- chloro-N-(4-phenylsulphonyl-l-piperidinyl)adenosine (1.2 g, 39 %) as foams.
5'-Deoxy-2,5'-dichloro-N-(4~phenylsulfinyl- 1 -piperidinyl)adenosine
2,,3-Di-O-acetyl-5'-deoxy-2,5'-dichloro-N-(4-phenylsulfinyl-l-piperidinyl)adenosine (0.22 g, 0.36 mmol) was dissolved in methanol (10 mL) and methanolic ammonia (1 mL) was introduced. After 1 h. at room temperature, the reaction mixture was evaporated to a residue and purified by flash chromatography on silica gel, eluting with a mixture of dichloromethane and ethanol (9:1) to provide 5'-deoxy-2,5'-dicWoro-N-(4-phenylsulfinyl-l-piperidinyl)adenosine (0.1 g, 53%) as a foam. Η-NMR (400MHz, DMSO-ds) d 3.84, 3.93 (2H, ABX, H-5'a and H-5'b), 4.10 (IH q, H-4'), 4.17 (Hi q, H-3'), 4.63 (Hi dd, H-2'), 5.49, 5.62 (2H, 2d, 2'- and 3'-OH), 5.87 (Hi d, H-l'), 7.53 - 7.66 (5H, m, Ar-H), 8.38 (IH, s, H-8). HPLC retention time 14.24 min. (gradient elution over 30 min.; 20-80% acetonitrile/0.1% TFA in water, 95.4% purity at 250 nm). EXAMPLE 30
5'-Deoxy-2.5'-dichloro-N-(4-phenylsulfonyl-l-piperidinyl)adenosine
2',3-Di-O-acetyl-5'-deoxy-2,5'-dichloro-N-(4-phenylsulfonyl-l-piperidinyl) adenosine (generated during the preparation of Example 29) (1.2 g, 1.9 mmol) was dissolved in methanol (90 mL) and methanolic ammonia (10 mL) was introduced. After 0.5 h. at room temperature, the reartion mixture was evaporated to a residue and purified by flash chromatography on silica gel, eluting with a mixture of dichloromethane and ethanol (9:1) to provide 5'-deoxy-2,5'-dichloro-N~ (4-phenylsulphonyl-l-piperidinyl)adenosine (0.82 g, 79%) as a foam. 1H-ΝMR (400MHz, DMSΟ-ds) d 3.84, 3.93 (2H, ABX, H-5'a and H-5'b), 4.09 (IH, dt, H-4'), 4.16 (IH, ps t, H-3'), 4.62 (IH, dd, H-2'), 5.49, 5.62 (2H, 2d, 2'- and 3'-OH), 5.87 (Hi d, H-l'), 7.71 (2H, t, Ar-H), 7.80 (Hi t, Ar-H), 7.90 (Hi d, Ar-H), 8.39 (IH, s, H-8). HPLC retention time 13.78 min. (gradient elution over 30 min.; 20-80% acetonitrile/0.1% TFA in water, 96.9% purity at 250 nm).
EXAMPLE 31
5'-Deoxy-2.5'-dichloro-N-(4-phenyl- 1 -piperidinvOadenosine
2-Chloro-N-(4-phenyl-l-piperidinyl)adenosine [WO 93/08206 (Novo Nordisk A/S)] (0.3 g, 0.65 mmol) was subjected to the chlorination conditions described in Example 24, providing the title 5'-deoxy-2,5'-dichloro-N-(4phenyl-l-piperidinyl)adenosine as a foam (0.28 g, 90%), 'H-NMR (400MHz, DMSO-d«) d 3.86, 3.95 (2H, ABX, H-5'a and H-5'b), 4.11 (Hi q, H-41), 4.21 (IH, q, H-3'), 4.66 (IH, dd, H-2'), 5.49, 5.63 (2H 2d, 2'- and 3'-OH), 5.89 (IH, d, H-l'), 7.20 (IH, dt, Ph-C-H), 7.31 (5H, d, Ar-H), 8.40 (Hi s, H-8), 9.45 (IH, s, N-H). HPLC retention time 20.92 min. (gradient elution over 30 min.; 20-80% acetonitrile/0.1% TFA in water, 99.75% purity at 250 nm). EXAMPLE 32
5'-Deoxy-2.5'-dichloro-N-π-moφholinyl)adenosine
This compound was prepared by the method described in Example 23. 2-Chloro-N— (l-morpholinyl)adenosine [WO 93/08206 (Novo Nordisk A S)] (1.0 g, 2.6 mmol) was subjerted to the chlorination conditions described in Example 24, providing the tide 5'- deoxy-2,5'-dichIoro-N-(l-moφholinyl)adenosine as a foam (0.78 g, 74%), Η-NMR (400MHz, DMSO-ds) d 2.38 (4H, br, morpholine C-H), 3.71 (4H, br, moφholine C-H), 3.85, 3.94 (2H, ABX, H-5'a and H-5'b), 4.10 (IH q, H-4'), 4.18 (IH, q, H-3'), 4.64 (IH, q, H-2'), 5.50, 5.63 (Hi 2d 2'-and 3'-OH), 5.88 (IH d, H-l'), 8.41 (IH, s, H-8), 9.50 (IH, s, NH). HPLC retention time 7.82 min. (gradient elution over 30 min.; 20-80% acetonitrile/0.1% TFA in water, 97.8% purity at 250 nm).
Figure imgf000045_0001
EtOH requires C, 42.1; H, 4.9; N, 19.6. Found: C, 41.6; H, 5.2; N, 19.0%.
EXAMPLE 33
5'-Deoxy-2.5'-dichloro-N-fdimethylamino)adenosine
This compound was prepared by the method described in Example 23. 2-Crύoτo-N- (dimethylamino)adenosine [WO 93/23417 (Novo Nordisk A S)] (0.62 g, 1.8 mmol) was subjerted to the chlorination conditions described in Example 24, providing the title 5'- deoxy-2,5,-dichloro-N-(dimethylamino)adenosine as a solid (0.23. g, 41%) after column chromatography, m.p. 188-189°C, Η-NMR (400MHz, DMSO-ds) d 2.59 (6H, s, Nrø}.), 3.85, 3.94 (2H, ABX, H-5'. and H-5'b), 4.10 (Hi q, H^'), 4.18 (IH, q, H-3'), 4.66 (Hi q, H-2'), 5.50, 5.62 (IH, 2d, 2'-and 3'-OH), 5.88 (IH d, H-l'), 8.38 (Hi s, H-8), 9.39 (IH, br, NH). Ci2H,6Cl2N6O3.0.25 H2O. 0.25 EtOH requires C, 39.6; H, 4.8; N, 22.2. Found: C, 39.5; H 4.5; N, 22.3%.
EXAMPLE 34
5'-Deoxy-2.5'-dichloro-N-methoxyadenosine
This compound was prepared by the method described in Example 23. 2-Chloro-N-- (methoxy)adenosine [WO 93/23417 (Novo Nordisk A/S)] (0.8 g, 2.4 mmol) was subjerted to the chlorination conditions described in Example 24, providing the title 5'- deoxy-2,5'-dichloro-JV-methoxyadenosine as a foam (0.34 g, 40%), Η-NMR (400MHz, DMSO-ds) d 3.78 (3H, s, -OCH3), 3.85, 3.94 (2H ABX, H-5'a and H-5'b), 4.10 (IH, q, H-4'), 4.19 (Hi q, H-3'), 4.66 (IH, q, H-2'), 5.51, 5.65 (IH, 2d, 2'-and 3'-OH), 5.90 (IH, d, H-l'), 8.45 (Hi s, H-8), 11.59 (IH, s, NH). HPLC retention time 6.99 min. (gradient elution over 30 min.; 20-80% acetonitrile/0.1% TFA in water, 96.5% purity at 250 nm).
EXAMPLE 35
iV-Cvclopentoxy-5'-deoxy-2.5'-dichloroadenosine
This compound was prepared by the method described in more detail in Example 23. N-Cyclo- ρentoxy-2-chloroadenosine [WO 93/23417 (Novo Nordisk A/S)] (1.0 g, 2.6 mmol) was subjected to die chlorination conditions described in Example 24, providing the title N-cyclopentoxy-5'-deoxy-2,5'-dichloroadenosine as a foam (0.82 g, 78%), Η-NMR (400MHz, DMSO-ds) d 1.49 - 1.92 (8H, 3m, cyclopentyl C-H), 3.86, 3.94 (2H, ABX, H-5'a and H-5'b), 4.11 (Hi q, H-4'), 4.20 (IH, q, H-3'), 4.59 (IH, m, -OC-H), 4.67 (IH, q, H-2'), 5.51, 5.64 (IH 2d, 2'-and 3'-OH), 5.90 (IH, d, H-l'), 8.44 (IH, s, H-8), 11.44 (IH s, NH). HPLC retention time 12.2 min. (gradient elution over 30 min.; 20-80% acetonitrile/0.1% TFA in water, 97.4% purity at 250 nm).
C,5HI9Cl2N5θ4.0.5 EtOH requires C, 45.0; H, 5.2; N, 16.4. Found: C, 45.2; H, 5.1, N, 16.2%.
EXAMPLE 36
2-Bromo-5'-chloro-5'-deoxy-N-f 1 -piperidinyPadenosine
This compound was prepared by the method described in Example 23. 2-Bromo-N— (l-piperidinyl)adenosine [WO 93/23417 (Novo Nordisk A S)] (0.06 g, 0.14 mmol) was subjected to the chlorination conditions described in Example 24, providing the title 2- bromo-5'-chloro-5,-deoxy-Λr-(l-piperidinyl)adenosine as a foam (0.016 g, 26%), lH-NMR (400MHz, DMSO-ds) d 1.37 (2H br, piperidine C-H), 1.64 (4H, br, piperidine C-H), 2.80 (4H, br, piperidine C-H), 3.85, 3.93 (2H, ABX, H-5*. and H-5'b), 4.09 (IH, q, H-4'), 4.18 (IH q, H-3'), 4.65 (IH, q, H-2'), 5.48, 5.62 (IH, 2d, 2'-and 3'-OH), 5.87 (IH, d, H-l'), 8.33 (IH, s, H-8), 9.36 (IH, s, NH). HPLC retention time 9.65 min. (gradient elution over 30 min.; 20-80% acetonitrile/0.1% TFA in water, 94% purity at 250 nm).
EXAMPLE 37
2-Anιmo-5'-chloro-5'-deoxy-N-f 4-phenylthio- 1 -piperidinvDadenosine
This compound was prepared by the method described in Example 24. 2-Amino-N- -(4-phenylthio-l-piperidinyl)adenosine [prepared by reaction of 9-(2',3,,5'-tri-O-acetyl-β-D-ribo- furanosyl)-2,6-dichloro-9H-purine (Knutsen, L.J.S., Lau, J., Sheardown, M.J., Thomsen, C; Bioorganic and Medicinal Chemistry Letters, 1993, 3, 2661-2666) with l-amino-4- phenyldiiopiperidine] (0.47 g, 1.0 mmol) was subjerted to the chlorination conditions described above. Column chromatography on silica gel, eluting initially with a mixture of heptane and ethyl acetate (7:3), increasing polarity to pure ethyl acetate provided the title 2-amino-5'-chloro-5'- deoxy-N-(4-phenylthio-l-piperidinyl)adenosine (0.1 g, 20%) as a foam. 1H-NMR (400MHz, DMSO-ds) d 1.65 (2H, dq, piperidine C-H), 1.91 - 2.01 (2H, br, piperidine C-H), 2.75 (2H, br t, piperidine C-H), 2.98 - 3.07 (2H, br, piperidine C-H), 3.82, 3.92 (2H, ABX, H-5'a and H-5*b), 4.04 (Hi dt, H-4'), 4.15 (IH, q, H-3'), 4.64 (IH, dd, H-2'), 5.36, 5.52 (2H, 2d, 2'- and 3'-OH), 5.77 (IH, d, H-l'), 5.96 (Hi br s, -NH2), 7.27 (IH, t, Ar-H), 7.37 (2H, t, Ar-H), 7.42 (2H d, Ar-H), 7.91, 8.18 (2H, 2s, H-8, N-H).
EXAMPLE 38
5'-Chloro-5'-deoxy-2-methylthio-N-( 1 -piperidinvDadenosine
2-Methylthio-N-(l-piperidinyl)adenosine [WO 93/23417 (Novo Nordisk A/S)] (0.15 g, 0.38 mmol) was subjerted to the chlorination conditions described in Example 24, providing 5,-chloro-5'-deoxy-2-methylthio-N-(l-piperidinyl)adenosine as a solid (0.07 g, 45%) mp 213 - 215°C. 'H-NMR (400MHz, DMSO-ds) d 1.37 (2H, br, piperidine C-H), 1.62 (4H, br q, piperi¬ dine C-H), 2.48 (3H, s, -SCH3), 2.81 (4H, br, piperidine C-H), 3.83, 3.93 (2H, ABX, H-5'a and H-5'b), 4.08 (IH, q, H-4'), 4.23 (IH, q, H-3'), 4.75 (IH, q, H-2'), 5.47, 5.59 (IH, 2d, 2'-and 3'- OH), 5.88 (IH, d, H-l'), 8.22 (Hi s, H-8), 8.85 (Hi s, NH). HPLC retention time 9.15 min. (gradient elution over 30 min.; 20-80% acetonitrile/0.1% TFA in water, 96.4% purity at 250 nm).
EXAMPLE 39
5'-Bromo-2-chloro-5'-deoxy-/V-( 1 -piperidinviladenosine 2-Chloro-N-(l-piperidιnyl)adenosine [WO 93/08206 (Novo Nordisk A/S)] (3 08 g, 8 mmol) was subjerted to the same reartion conditions described m Example 24, except that thionyl bromide was substituted for thionyl chloride The procedure provided the desired 5'-bromo-2-chloro-5'-deoxy-N-(l-piperidinyl)adenosine as a foam (0 19 g, 9%) after column chromatography, Η-NMR (400MHz, DMSO-ds) d 1 37 (2H, br, piperidine C-H), 1 62 (4H, m, piperidine C-H), 2.80 (4H, br, piperidine C-H), 3 72, 3.82 (2H, ABX, H-5'a and H-5'b), 4 10 (IH, dt, H-4'), 4 17 (IH, dt, H-3'), 4 68 (IH, q, H-2'), 5 50, 5.62 (IH, 2d, 2'-and 3'-OH), 5.88 (Hi d, H-l'), 8 38 (Hi s, H-8), 936 (IH, br, NH) HPLC retention time 1 1 06 mm (gradient elution over 30 min., 20-80% acetonitrile/0 1% TFA in water, 964% purity at 250 nm)
CuH2oNsBrClθ3 1.3 H2O requires C, 38 2, H 8, N, 17.8 Found: C, 38 7, H, 4 7, N, 173%
EXAMPLE 40
2-Chloro-5'-deoxy-5'-fluoro-N-C 1 -piperidinvDadenosine
Methyl 5-Deoxy-5-fluoro 2,3-0-(l-methylethyudene)-D-ribofuranoside
Methyl 2,3-O-(l-mrthylethyUdene)-5-O-(p-toluenesulfonyl)-D-ribofuranoside (28 7 g, 80 mmol) was dissolved in dry acetonitrile (100 mL). Tetra-«-butylammonium fluoride (100 mL, 1 0M in THF) was added dropwise and the reaction mixture was heated at 80°C for 72 h After cooling to room temperature, the mbcture was diluted with dichloromethane (200 mL), washed with water (3 x 50 mL) and dried (MgSO4) Evaporation provided a residue which was purified by flash chromatography eluting with a mixture of ethyl acetate/w-heptane (1/3) to give methyl 5-deoxy-5-fluoro-2,3-O-(l-methylethylidene)-β-D-ribofuranoside (13 6 g, 82%) as a clear oil, 'H NMR (CDC13) d 1 34 (3H, s, CH3), 1 50 (3H, s, CH3), 3 35 (3H, s, -OCH3), 4 29 - 448 (3H, m, H-4, H-5a and H-5„), 460 (IH, d, H-3), 4 70 (Hi d, H-2), 4 99 (IH, d, H-l) 1 ,2,3 -Tri-O-acetyl-5-deoxy-5-fluoro-D-ribofuranose
Methyl 5-deoxy-5-fluoro-2,3-O-(l-methylethylidene)-D-ribofuranoside (5.0 g, 24 mmol) was treated with sulfuric acid (0.02M, 40 mL) and heated at reflux for 4 h. The reaction mixture was cooled, neutralized with barium carbonate to pH 7, filtered and evaporated to an oil. The oil was dried by coevaporation with ethanol, and the residue was dissolved in dichloromethane (50 mL).
Acetic anhydride (25 mL) and pyridine (25 mL) were introduced, and the reartion mixture was stirred for 16 h before being poured onto ice (100 mL). The cool suspension was extracted with dichloromethane (3 x 100 mL), and the combined extracts was washed with 2N hydrochloric acid solution (50 mL) and aqueous sodium bicarbonate solution (50 mL). The organic phase was dried (MgSO4), evaporated in vacuo and the residue was purified by flash chromatography, eluting with a mixture of dichloromethane and 10% ammonia in ethanol (97:3) to provide l,2,3-tri-O-acetyl-5-deoxy-5-fluoro-D-ribofuranose (5.3 g, 79%), which crystallized on standing. Recrystallization from absolute ethanol provided analytically pure material, m.p. 98 - 101°C, 1H
NMR (CDC13) d 2.09 (3H, s, -ΟCΟCH3), 2.10 (3H, s, -OCOCH3), 2.14 (3H, s, -OCOCH3),
4.34 (IH, ddd, H-4), 4.49 (IH, ddd, H-5a), 4.52 (IH, ddd, H-5b), 5.36 (IH, d, H-3), 5.46 (IH, dd, H-2), 6.17 (lH, s, H-l).
C,ιHι5FO7 requires C, 47.5; H 5.4. Found: C, 47.7; H, 5.6%.
9-[(2',3,-Di-O-acetyl-5'-deoxy-5,-fluoro-D-ribofuranosyl)]-2,6-dichloro-9H-purine
A mixture of the above l,2,3-tri-0-acetyl-5-deoxy-5-fluoro-D-ribofuranose (5.0 g, 18 mmol) and 2,6-dichloropurine (3.4 g, 18 mmol) was heated to 160°C. A catalytic amount of sulfuric acid (one drop) was added at which point a homogeneuos melt was obtained. The fusion was continued at 160°C under oil pump vacuum for 0.5 h. After cooling, the reaction mixture was dissolved in chloroform (200 mL) and washed with aqueous sodium bicarbonate (3 x 50 mL) and water (50 mL). The organic phase was dried (MgSO ) and evaporated in vacuo before purification by flash chromatography. Elution with a mixture of dichloromethane and 10% ammonia in ethanol (98:2) afforded an anomeric mbcture of 9-[(2',3'-di-O-acetyl-5'-deoxy-5*- fluoro-D-ribofuranosyl)]-2,6-dichloro-9H-purine (4.5 g, 90%) as a gum, lH NMR (DMSΟ-ds)d (a-anomer) 1.84 (3H, s, -OCOCHj), 2.00 (3H s, -OCOCH3), 4.71 (2H, dd, H-5'a, H-Sti), 5.98 (IH, ddd, H-4*), 5.48 (IH, t, H-3), 5.68 (IH, t, H-2'), 6.73 (IH d, H-l'), 8.92 (Hi s, H-8); (β-anomer) 2.05 (3H, s, -OCOCH3), 2.11 (3H, s, -COCH3), 4.50 (IH, ddd, H-4), 4.77 (2H, dd, H-5'a and H-5b'), 5.61 (Hi t, H-3), 5.89 (IH, t, H-2'), 6.33 (IH, d, H-l), 8.92 (IH, s, H-8).
2',3 '-Di-O-acetyl-2-chloro-51-deoxy-5'-fluoro-N-( 1 -piperidinyl)adenosine
An a b mixture of 9-[(2',3'-di-O-acetyl-5'-deoxy-5'-fluoro-D-ribofuranosyl])-2,6-dichloro-9H- purine (1.24 g, 3.0 mmol), N-N-dϋsopropylethyl amine (0.79 g, 6.1 mmol) and 1-aminopiperidine (0.60 g, 6.0 mmol) were stirred in dioxan (20 mL) for 4 h. The reartion mixture was diluted with dichloromethane (200 mL) and washed with water (2 x 50 mL). After drying over (MgSO4) the organic phase was evaporated in vacuo and the residue was purified by flash chromatography. Elution with dichloromethane and 10% ammonia in ethanol (98:2) afforded the b-anomer of 2',3'- di-O-acetyl-2-chloro-5'-deoxy-5-fluoro-N-(l-piperidinyl)adenosine (0.40 g, 28%) as a foam. HPLC retention time 14.8 min. (gradient elution over 30 min.; 20-80% acetonitrile/0.1% TFA in water).
The above 2',3,-di-O-acetyl-2-chloro-5'-deoxy-5,-fluoro-N-(l-piperidinyl)adenosine (0.38 g, 0.8 mmol) was dissolved in methanolic ammonia (15 mL) and stirred for 1 h. The reaction mixture was evaporated in vacuo and the resultant residue was purified by flash chromatography eluting with dichloromethane and 10% ammonia in ethanol (95:5) to afford 2-chloro-5'-deoxy-5'-fluoro- N-(l-piperidinyl)adenosine (1.8 g, 86%) as a white solid, m.p. 199-201°C. 1H-NMR (400MHz, DMSΟ-ds) d 1.35 (2H, br, piperidine C-H), 1.52 (4H br, piperidine C-H), 2.80 (4H, br, piperidine C-H), 4.10 (IH, ddd, H-4'), 4.20 (IH, br, H-3'), 4.51 (Hi br, H-2'), 4.65 (2H, dd, H-5'a and H-5'b), 5.88 (IH, d, H-l'), 8.28 (Hi s, H-8). HPLC retention time 7.15 min. (gradient elution over 30 min.; 20-80% acetonitrile/0.1% TFA in water). Ci5H2oClFN6O3, 0.75H2O requires C, 45.2; H, 5.4; N, 21.1. Found: C, 45.2; H, 5.2; N, 20.8%.
EXAMPLE 41
2-Chloro-5'-deoxy-5'-fluoro-N-benzyloxyadenosine
This compound was prepared by general method A, described in more detail in Example 39 by reacting O-benzylhydroxylamine hydrochloride (0.80 g, 5.0 mmol) with 9-[(2',3'-di-O-acetyl-5'- deoxy-5'-fluoro-D-ribofuranosyl)]-2,6-dichloro-9H-purine (1.0 g, 2.5 mmol) as described above. The product was purified by flash chromatography eluting with a mixture of dichloromethane and 10% ammonia in ethanol (98:2) giving the intermediate 2',3'-di-0-acetyI-2-chloro- 5'-deoxy-5'-fluoro-N-benzyloxyadenosine (0.2 g, 16%). Deacetylation was performed in methanolic ammonia to afford 2-chloro-5'-deoxy-5'-fluoro-iV-benzyloxyadenosine as a foam (0.11 g, 89%) after flash chromatography eluting with dichloromethane and 10% ammonia in ethanol (95:5). 1H-NMR (400MHz, DMSO-ds) d 4.12 (IH m, H-4'), 4.20 (Hi m, H-3"), 4.53 (IH, m, H-2'), 4.65 (2H, dd, H-5'a and H-5'b), 5.00 (2H, s, -CH2-), 5.48, 5.60 (2H, 2d, 2'-and 3'-OH) 5.91 (IH, d, H-l'), 7.30 -7.55 (5H, m, Ar-H), 8.43 (IH, s, H-8), 11.65 (IH, s, N-H). HPLC retention time 14.60 min. (gradient elution over 30 min.; 20-80% acetonitrile/0.1% TFA in water, 96% purity at 250 nm).
EXAMPLE 42
fS)-2-Chloro-5'-O-methyl-N-(2-(methylmethoxyVl-pyrrolidinvπadenosine
Methyl 5-O-methyl-2,3 -O-( 1 -methylethylidene)-D-ribofuranoside.
Metiiyl 2,3-O-(l-methylethylidene)-D-ribofuranoside (13.3 g, 60 mmol), 2,6-di-t-butyl-4-methyl- pyridine (20.0 g, 100 mmol) and methyl trifluoromethylsulfonate (16.0 g, 100 mmol) were dis- solved in dry dichloromethane (150 mL) placed in a closed reactor and heated to 80°C. After cooling, the reaction mixture was poured onto ice (150 mL). After standing, the product was extracted into dichloromethane (2 x 100 mL) and the combined extracts were dried (MgSO4) and evaporated in vacuo. The residue was purified by flash chromatography eluting with a mixture of cyclohexane and ethyl acetate (3:1) to afford methyl 5-O-methyI-2,3-O-(l-methylethylidene)-β- D-ribofuranoside (8.0 g, 61%) as an oil. Η-NMR (400MHz, CDC13) d 1.30 (3H, s, -CH3), 1.50 (3H, s, -CH3), 3.32 (3H, s, -OCH3), 3.39 (3H, s, -OCH3), 3.35 - 3.45 (2H, m, H-5a and H-5b), 4.30 (IH, t, H-4), 4.57 (IH, d, H-3), 4.65 (IH, d, H-2), 4.97 (IH, s, H-l).
l,2,3-Tri-O-acetyi-5-O-methyl-D-ribofuranose.
5-O-Methyl-2,3-O-(l-methylethylidene)-D-ribofuranoside (3.0 g, 14 mmol) was dissolved in a mixture of sulfuric acid (0.02M, 100 mL) and ethanol (50 mL) and heated at 80°C for 6 h and stirred for 20 h at 20°C. The reaction mbcture was neutralised with aqueous sodium bicarbonate and concentrated in vacuo. The residual oil was dried and acetylated in a mixture of dichloromethane (100 mL), acetic anhydride (8.5 g, 83 mmol) and triethylamine (16.7 g, 165 mmol) at 20°C for 20 h. The reartion mixture was washed with hydrochloric acid (1M, 50 mL) and water (50 mL). The organic phase was dried (MgSO ) and concentrated to an oil before being purified by flash chromatography. Elution with a mixture of cyclohexane and ethyl acetate (6:4) provided l,2,3-tri-O-acetyl-5-O-methyl-β-D-ribofuranose (2.5g, 62%) as an oil.
9-(2,,3'-Di-O-acetyl-5,-O-methyl-β-D-ribofuranosyl)-2,6-dichloro-9H-purine
5-O-Methyl-l,2,3-tri-O-acetyl-D-ribofuranose (5.0 g, 17 mmol) and 2,6-dichloropurine (3.3 g, 17 mmol) were thoroughly mixed. A catalytic amount of p-toluene sulfonic acid (50 mg) was added and the reartion mixture was heated to 140°C at which point a homogeneous melt was obtained. The fusion was continued at 140°C under oil pump vacuum for 0.5 h. The reaction mixture was dissolved in chloroform (200 mL) and washed with aqueous sodium bicarbonate (3 x 50 mL) and water (2 x 50 mL). The organic phase was dried (MgSO4), evaporated in vacuo and purified by flash chromatography eluting with a mixture of n-heptane and ethyl acetate (1:1) to provide - 9-(2',3'-di-O-acetyl-5,-O-methyl-β-D-ribofiιranosyl)-2,6-dichloro-9H-purine (1.0 g, 14%) as an oil which crystallized from diethyl ether. A mixture of a/β-anomers (1.2 g, 17%) was also isolated, with mp 59 - 61°C. Η-NMR (400MHz,CDCl3) d 2.06 (3H, s, -OCOCH3), 3.49 (3H, s, OCH3), 3.67, 3.72 (2H, ABX, H-5'a and H-5'b), 4.39 (IH d, H-4'), 5.58 (IH, d, H-3'), 5.75 (IH t, H-2'), 6.38 (IH, d, H-l'), 8.56 (IH, s, H-8).
Cι5HιsCl2N4O6 requires C, 43.0; H, 3.9; N, 13.4. Found C, 43.1, H 3.9, N, 13.2%.
(S)-2,,3,-Di-O-acetyl-2-chloro-5'-O-methyl-N-[2-(methylmethoxy)-l-pyrrolidinyl]adenosine.
9-(2',3'-Di-O-acetyl-5'-O-methyl-β-D-ribofuranosyl)-2,6-dichloro-9H-purine (1.3 g, 3.1 mmol), (S)-N-amino-2-(medιoxymethyl)pyrrolidine (0.81 g, 6.2 mmol) and triethylamine (0.63 g, 6.2 mmol) were dissolved in dioxan. After stirring for 20 h the reaction mixture was diluted with dichloromethane (150 mL) and washed with water (2 x 75 mL). The organic phase was dried (MgSΟ ) and concentrated in vacuo. The resultant residue was purified by flash chromatography eluting with a mixture of dichloromethane and 10% ammonia in ethanol (97:3) to afford (S)- 2',3'-di-O-acetyl-2-chloro-5'-O-methyl-N-[2-(methyl-methoxy)-l-pyrrolidin-yl] adenosine (0.28 g, 18%) as a foam. 1H-NMR (400MHz, CDC13) d 1.70 - 2.10 (4H, m, pyrrolidine C-H), 2.05 (3H, s, -ΟCΟCH3), 2.18 (3H, s, -OCOCH3), 2.85 (IH, m, pyrrolidine C-H), 3.10 (Hi br, pyrrolidine C-H), 3.22 (3H, s, -OCH3), 3.35 - 3.70 (8H, m, H-5'a and H-5't, -OCH3, pyrrolidine, -CH2-), 4.32 (IH, s, H-4'), 5.55 (IH, d, H-3'), 5.75 (IH, t, H-3'), 6.32 (IH, d, H-l'), 8.17 (Hi s, H-8). HPLC retention time 15.57 min. (gradient elution over 25 min.; 20-80% acetonitriie/0.1% TFA in water).
The above (S)-2',3'-di-O-acetyl-2-chloro-5'-O-methyl-N-[2-(methylmethoxy)- 1 -pyrrolidinyl]- adenosine (0.26 g, 0.52 mmol) was treated with methanolic ammonia for 1.5 h at room temperature. The crude product was purified by flash chromatography eluting with dichloromethane and 10% ammonia in ethanol (9:1) to give (S)- 2-chloro-5'-O-methyl-N-(2-(methylmethoxy)-l-pyrrolidinyl)adenosine (0.16 g, 72%) as a foam. 'H-NMR (400MHz, DMSO-ds) d 1.55 (IH m, pyrrolidine C-H), 1.75 (2H m, pyrrolidine C-H), 1.97 (IH, m, pyrrolidine C-H), 3.51, 3.59 (Hi ABX, H-5'a and H-5'b), 4.02 (IH, dd, H-4'), 4.10 (Hi dd, H-3'X 4.52 (Hi dd, H-2'), 5.82 (IH, d, H-l'), 8.32 (IH, s, H-8). HPLC retention time 7.67 min. (gradient elution over 25 min.; 20-80% acetonitrile/0.1% TFA in water).
Cι7H23ClNsθ5. 0.5H2O requires C, 46.3; H 5.8; N, 18.5. Found C, 46.7; H, 6.1; N, 18.6%.
EXAMPLE 43
2-chloro-N-methoxy-5'-O-methyladenosine
2',3'-Di-O-acetyl-2-chloro-iV-methoxy-5'-O-methyladenosine
9-(2,,3,-Di-O-acetyl-5,-O-methyl-β-D-ribofiιranosyI)-2,6-dichloro-9H-purine (0.364g, 0.87 mmol), O-methylhydroxyamine hydrochloride (0.087g; 1.04 mmol) and diisopropylethylamine (0.34 mL, 2.0 mmol) were dissolved in 1,4-dioxan (7.0 mL). The mixture was heated to 50°C under nitrogen for 17 hours. After this time, further methoxylamine hydrochloride (0.087g; 1.04mmol) and dϋsopropylethylamine (0.340 mL; 2.0mmol) were added and the reartion mixture was heated at 50°C for a further 48 h. The solvent was evaporated and the residue partitioned between ethyl acetate (20 mL) and water (10 mL). The aqueous phase was separated and die organic phase was washed with water (10 mL) and brine (10 mL) before being dried (Na SO4). Evaporation of solvent afforded a foam (0.33 g) which was purified by flash chromatography, eluting with ethyl acetate/heptane (3 :2), affording a white foam (0.211 g, 57%).
'H-NMR (200MHz; CDC13) d 2.05 (3H, s, -OCOCH3), 2.18 (3H, s, -OCOCH3), 3.48 (3H, s, -
OCH3), 3.68 (2H, ABX, H-5'. and H-5'b), 4.00 (3H, s, -N(6)-OCH3), 4.35 (Hi dd, H-4'), 5.56
(IH, dd, H-3'), 5.76 (IH, dd, H-2'), 6.35 (IH, d, H-l'), 8.28 (IH, s, H-8) and 9.25 (IH, s, exch.
NH). 2',3'-Di-O-acetyl-2-chloro-N-methoxy-5'-O-methyladenosine (0.20 g, 0.47 mmol) was dissolved in dry methanol (5 mL) and treated with a methanolic solution of sodium methoxide (3.5 mL, 0.047 mmol). The solution was stirred at room temperature for 24 h. After this time, the pH was adjusted from 9 to 5 using acetic acid (0.02 mL). Solvents were evaporated to afford a pale yellow oil (0.17g) which was purified by flash chromatography. Elution with dichloromethane/ethanol (14: 1) gave a white foam (0.115 g, 72%), Η-NMR (400MHz; DMSO- ds) d 3.34 (3H, s, -OCH3), 3.58 (2H, ddd, H-5'a and H-5'b), 3.78 (3H, s, -N-OCH3), 4.05 (IH, dd, H^'), 4.12 (IH dd, H-3'), 4.52 (Hi dd, H-2'), 5.33 (IH, d, OH) 5.55 (IH d, OH) 5.76 (IH, d, H-l'), 8.40 (IH, s, H-8), 11.55 (IH, s, NH). HPLC retention time 10.98 min. (gradient elution over 30 min.; 10-80% acetonitrile/0.1%TF A in water). MS (El): m/e 347 and 345 [M*], 314 [(M-OMe)
EXAMPLE 44
2-Chloro-5'-deoxy-5'-methylthio-N-( 1 -piperidinvDadenosine
2-Chloro-2,,3'-O-(l-methylethylidene)-Λr-(l-piperidinyl) adenosine tosylate salt
2-Chloro-N-(l-piperidinyl)adenosine (1.5 g, 3.9 mmol), 2,2-dimethoxypropane (0.9 g, 8.6 mmol) and 4-toluenesulfonic acid monohydrate (1.6 g, 18.6 mmol) was stirred in acetone (25 mL) for 72 h. Further 2,2-dimethoxypropane (0.9 g, 8.6 mmol) was added. After a further 24 h the tosylate salt of 2-chloro-2',3'-O-(l-methylethylidene)-N-(l-piperidinyl)adenosine (1.78 g, 76%) was collerted by filtration, m.p. 169-170°C, Η-NMR (400MHz, DMSO-ds) d 1.35 (3H, s, -CH3), 1.46 (2H, br, piperidine C-H), 1.55 (3H s, -CH3), 1.75 (4H, m, piperidine -CH), 3.10 (4H br, piperidine C-H), 3.55 (2H, ABX, H-5.' and H-5'b), 4.29 (Hi m, H-4'), 4.95 (IH, dd, H-3'), 5.30 (IH, dd, H-2'), 6.15 (Hi d, H-l'), 8.72 (IH, s, H-8). HPLC retention time 14.87 min. (gradient elution over 30 min.; 20-80% acetonitrile/0.1% TFA in water). C25H33ClN6O7S requires C, 50.3; H, 5.6; N, 14.1. Found C, 50.7; H, 5.8; N, 13.7%.
2-Chloro-5'-deoxy-5'-methylthio-2',3'-O-(l-methylethylidene)-N-(l-piperidinyl)adenosine
2-Chloro-2',3'-O-(l-methylethylidene)-N-(l-piperidinyl) adenosine tosylate (0.5 g, 0.84 mmol), tributylphosphine (1.7 g, 8.4 mmol) and dimethyldisulfide (0.4 g, 4.20 mmol) were stirred in dry dimethylformamide (5 mL) under nitrogen for 7 days. The reaction mixture was poured into ice (50 mL) and after standing for 1 h was extracted with dichloromethane (3 x 25 mL). The organic phase was dried (MgSO ) and evaporated in vacuo. The crude product was purified by flash chromatography eluting with a mixture of dichloromethane and 10% ammonia in ethanol (95:5) to give 2-chloro-5,-deoxy-5'-methylthio-2',3'-O-(l-methylethylidene)-N-(l-piperidinyl)adenosine (0.12 g, 31%) as a foam. Η-NMR (400MHz, CDC13) d 1.40 (3H s, -CH3), 1.45 (2H, m, piperidine C-H), 1.65 (3H, s, -CH3), 1.70 - 1.85 (4H, m, piperidine C-H), 2.15 (3H, s, -SCH3), 2.85 (4H, m, piperidine C-H), 4.02, 4.38 (2H AB H-5'a and H-5'b), 5.05 (IH, dd, H-4'), 5.38 (IH, dd, H-3'), 6.05 (IH, d, H-2'), 6.50 (IH, br, H-l'), 7.83 (IH, s, H-8).
2-Chloro-5,-deoxy-5'-methylthio-N-( 1 -piperidinyl)adenosine
2-Chloro-5'-deoxy-5'-methylthio-2',3'-O-(l-methylethylidene)-N-(l-piperidinyl)adenosine (0.10 g, 0.22 mmol) was reacted in a mixture of water (2.5 mL) and ethanol (2.5 mL) containing sulfuric acid (0.1 mL) for 10 h at 60°C. The reartion mixture was diluted with dichloromethane (100 mL) and washed with aqueous sodium bicarbonate (2 x 25 mL) followed by water (25 mL). The organic phase was dried (MgSO4) and concentrated in vacuo. The crude product was purified by flash chromatography eluting with a mixture of dichloromethane and 10% ammonia in ethanol (95:5) to provide 2-chloro-5'deoxy-5'-methylthio-N-(l-piperidinyl)adenosine (0.75 g, 82%) as a foam, 'H-NMR (400MHz, DMSO-ds) d 1.39 (2H br, piperidine C-H), 1.62 (4H m, piperidine C-H), 2.05 (3H, s, -SCH3), 2.75 - 2.80 (8H, m, H-5'a, H-5'b, piperidine C-H), 4.01 (Hi m, H-41), 4.10 (Hi m, H-3'), 4.65 (IH, br, H-2'), 6.82 (Hi d, H-l'), 8.39 (IH s, H-8). HPLC retention time 9.90 min. (gradient elution over 30 min.; 20-80% acetonitrile/0.1% TFA in water, purity 96% at 250 nm).
EXAMPLE 45
2-Chloro-5'-cyano-5'-deoxy-N-(l-piperidinyl adenosine
Methyl 2,3-O-( 1 -methylethylidene)-5-O-(4-nitrobenzenesulfonyl)-β-D-ribofiιranoside
Methyl 2,3-O-(l-methylethyϋdene)-β-D-ribofuranose (37 g, 180 mmol) and triethylamine (54.6 g, 540 mmol) were dissolved in dry dichloromethane (100 mL). 4-Nitrobenzenesulfonyl chloride (40.0 g, 180 mmol) was added dropwise at 0°C over 0.5 h. After stirring for 20 h, the reaction mixture was diluted with dichloromethane (1000 mL) and washed with aqueous ammonium chloride (2 x 250 mL) and water (250 mL). After drying (MgSΟ4) the organic phase was evapor¬ ated to dryness in vacuo. Recrystallization from ethyl acetate gave methyl 2,3-O-(l-methylethyl- idene)-5-O-(4-nitrobenzenesulfonyl)-β-D-ribofuranoside as a white solid (54.8 g, 85%), m.p. 97-98°C, Η-NMR (400MHz, CDC13) d 1.38 (3H, s, -CH3), 1.45 (3H, s, -CH3), 3.26 (3H, s, - ΟCH3), 4.13 (2H, ABX, H-5. and H-5b), 4.32 (Hi t, H-4), 4.53 (Hi d H-3), 4.61 (Hi d, H-2), 4.95 (IH, s, H-l), 8.12 (2H, d, Ar-H), 8.40 (2H, d, Ar-H).
Methyl 5-cyano-5-deoxy-2,3-O-(l-methylethylidene)-β-D-ribofuranoside.
Methyl 2,3-O-(l-methylethylidene)-5-O-(4-nitrobenzenesulfonyl)-β-D-ribofuranoside (45.3 g, 120 m mol) was added over 1.5 h to a suspension of sodium cyanide (6.8 g, 140 mmol) in dry dimethylformamide (1000 mL). The reartion mixture was heated to 50°C for 3 h before being poured onto ice (500 mL). This mixture was extracted with dichloromethane (3 x 500 mL), the combined extracts were dried (MgSO ) and concentrated in vacuo. The residue was distilled under vacuum to give methyl 5-cyano-5-deoxy-2,3-O-(l-methylethylidene)-β-D-ribofuranoside as an oil (6.3 g, 25%), bp 110-115°C/0.6 mm Hg. Η-NMR (400MHz, CDC13) d 1.30 (3H, s, - CH3), 1.48 (3H, s, -CH3), 2.62, 2.70 (2H, ABX, H-5a and H-5b), 3.40 (3H, s, -OCH3), 4.45 (IH, s, H-4), 4.61 (IH, d, H-3), 4.63 (IH, d, H-2), 5.00 (Hi s, H-l).
Methyl 2,3-di-O-benzoyl-5-cyano-5-deoxy-β-D-ribofuranoside
Methyl 5-cyano-5-deoxy-2,3-O-(l-methylethylidene)-β-D-ribofuranoside (18.7 g, 87 mmol) and Amberlyst (H* form, 84 g) were mixed and heated at reflux for 24 h. The reartion mixture was filtered and evaporated to a residue which was dissolved in dichloromethane (200 mL), which was washed with water (300 mL). The separated water phase was extracted with ethyl acetate (7 x 200 mL), combined with the earlier organic phase and dried (MgSΟ ). Evaporation provided the intermediate methyl 5-cyano-5-deoxy-β-D-ribofuranoside (6.85 g) which was dissolved in dichloromethane (200 mL). Benzoyl chloride (24 g, 170 mmol) and triethlamine (34 g, 340 mmol) were introduced and the reartion mixture was stirred for 20 h. at ambient temperature before being washed with 1 N hydrochloric acid solution (2 x 85 mL) and saturated sodium bicarbonate solution. The organic phase was dried (MgSO4) and evaporated to a residue which was purified by flash chromatography on silica gel. Elution with a mixture of heptane and ethyl acetate (39:1), increasing polarity to a 9:1 mixture of these solvents provided methyl 2,3-O- dibenzoyl-5-cyano-5-deoxy-β-D-ribofuranoside (13.01 g, 40%), Η-NMR (400MHz, CDCI3) d 3.04, 3.20 (2H, ABX, H-5. and H-5„), 3.43 (3H, s, -ΟCH3), 4.67 (IH, d, H-4), 5.25 (IH, s, H-l), 5.50 - 5.57 (2H, m, H-3 and H-4), 7.4 - 7.95 (10H, 6m, Ar-H).
l-O-Acetyl-2,3-di-O-benzoyl-5-cyano-5-deoxy-D-ribofuranose
Acetic acid (74.5 mL, 1.3 mol), acetic anhydride (173.8 mL, 1840 mmol) and sulfuric acid (1.7 mL, 32 mmol) were mixed together and methyl 2,3-O-dibenzoyl-5-cyano-5-deoxy-β-D- ribofuranoside (13.01 g, 35 mmol) was added. The reartion mixture was stirred for 20 h at ambient temperature before sodium acetate (37 g, 450 mmol) was introduced. After 30 min. stirring the reartion mixture was filtered, the filter pad was washed with ethyl acetate (100 mL) and the filtrate was evaporated to a residue which was coevaporated with toluene (250 mL). The residue was dissolved in a mixture of ethyl acetate (250 mL) and water (250 mL). The ethyl acetate phase was washed with water (2 x 100 mL) and saturated brine (50 mL) before being dried (MgSO ) and evaporated. The residue was purified by flash chromatography on silica gel. Elution with a mbcture of hexane and ethyl acetate (9:1), increasing polarity to a 4:1 mixture of these solvents provided the title l-O-acetyl-2,3-di-O-benzoyl-5-cyano-5-deoxy-β-D- ribofuranose as a solid single isomer (2.85 g, 20%), mp 124-126°C, Η-NMR (400MHz, CDC13) d 2.22 (3 s, -OCOCH3), 2.92, 3.02 (2H ABX, H-5. and H-5b), 4.61 (IH, dt, H-4), 5.70 - 5.80 (2H, 2m, H-2 and H-3), 6.38 (IH s, H-l) and a mixture of isomers as a gum (7.5 g, 52%).
9-(2',3'-Di-O-benzoyl-5,-cyano-5,-deoxy-D-ribofiιranosyl)-2,6-dichloro-9H-purine
A mbcture of l-O-acetyl-2,3-di-O-benzoyl-5-cyano-5-deoxy-β-D-ribofuranose (2.8 g, 6.8 mmol) and 2,6-dichloro-9H-purine (1.36 g, 7.2 mmol) were heated at 145°C for 1.25 h in the presence of a catalytic amount of p-toluenesulphonic acid (0.025 g). The reaction mixture was dissolved in ethyl acetate (100 mL) and washed with aqueous sodium bicarbonate (100 mL) followed by saturated brine (100 mL). The organic phase was dried (MgSΟ4), and the solid residue was recrystallised from 2-propanol to provide 9-(2',3'-di-O-benzoyl-5'-cyano-5'-deoxy-β-D-ribo- furanosyl)-2,6-dichloro-9H-purine (3.3 g, 90%), m.p. 143-145°C, Η-NMR (400MHz, CDC13) d 3.15, 3.25 (2H, ABX, H-5'a and H-5'b), 4.74 (IH dt, H-4'), 5.95 (Hi t, H-3'), 6.12 (IH, t, H-2'), 6.42 (IH, d, H-l'), 7.36 - 8.05 (10H, 4m, Ar-H), 8.40 (Hi s, H-8).
2',3'-O-Benzoyl-2-chloro-5'cyano-5'-deoxy-N-(l-piperidinyl)adenosine
9-(2',3'-di-O-berιzoyl-5'-cyano-5'-deoxy-β-D-ribofiιranosyl)-2,6-dichloro-9H-purine (1.75 g, 3.25 mmol), triethylamine (0.9 mL, 6.5 mmol) and 1-aminopiperidine (0.7 mL, 6.5 mmol) were stirred in dioxan (20 mL) for 2 h. The reartion mbcture was evaporated and the residue dissolved in dichloromethane (10 mL) and purified by flash chromatography eluting with a mbcture of heptane and ethyl acetate (4.1), increasing polarity to a 1 :1 mixture of these solvents provided 2',3'-di-O-benzyl-2-chloro-5'-cyano-5'deoxy-N-(l-piperidinyl)adenosine (1.19 g, 61%) as a foam, Η-NMR (400MHz, DMSΟ-ds) d 3.15, 3.31 (2H ABX, H-5'a and H-5'b), 4.68 (IH, dd, H-4*), 5.99 (IH, t, H-3'), 6.11 (H, t , H-2'), 6.36 (IH, d, H-l'), 7.36 - 8.00 (lOH, m, Ar-H), 8.03 (IH, s, H-8). HPLC retention time 17.26 min. (gradient elution over 30 min.; 20-80% acetonitrile/0.1% TFA in water).
2-Chloro-5'-cyano-5'-deoxy-N-(l-piperidinyl)adenosine
2-Chloro-2',3'-O-benzoyl-5,-cyano-5'-deoxy-N-(l-piperidinyl)adenosine (1.5 g, 2.5 mmol) was dissolved in methanolic ammonia (30 mL) and stirred at ambient temperature for 3 h. Following evaporation, the crude product was purified by flash chromatography eluting with a mbcture of heptane and ethyl acetate, followed by ethyl acetate alone to provide 2-chloro-5'-cyano-
5'-deoxy-N-(l-piperidinyl)adenosine as a solid (0.6 g, 61%). Recrystallization from ethyl acetate provided an analytical sample (0.36 g, 37%), m.p. 192-193°C, Η-NMR (400MHz, DMSO-ds) d 1.38 (2H, br, piperidine C-H), 1.63 (4H, q, piperidine C-H), 2.81 (4H, br, piperidine C-H), 3.05
(2H, d, H-5'a and H-5'b), 4.12 (2H, m, H-4'and H-3'), 4.65 (Hi m, H-2'). 5.53, 5.67 (2H, 2d, 2'- and 3'-OH), 5.87 (IH, d, H-l'), 8.37 (IH, s, H-8). HPLC retention time 11.9 min. (gradient elution over 30 min; 25-45% acetonitrile/0.1% ammonium sulfate in water), purity 100% at 250 nm).
CsHzoCLN- requires C, 48.8; H, 5.1; N, 24.9. Found C, 48.9; H 5.3; N, 24.6%.
EXAMPLE 46
2-Chloro-5'-cvano-5'-deoxy-N-(4-phenylthio- 1 -piperidinyDadenosine
This compound was prepared by general metiiod A, described in more detail in Example 44 2-Chloro-2',3,-0-benzoyl-5'-cyano-5'-deoxy-Λr-(4-phenylthio- 1 -piperidinyl)adenosine [prepared from l-amino-6-phenylthiopiperidine (Knutsen, L.J.S., Lau, J., Sheardown, M.J., Thomsen, C; Bioorganic and Medicinal Chemistry Letters, 1993, 3, 2661-2666) and 9-(2',3'-di-O-benzoyl-5'-- cyano-5'-deoxy-β-D-ribofuranosyl)-2,6-dichloro-9H-purine as described in Example 24] (1.5 g, 2.5 mmol) in methanol (20 mL) was treated with methanolic ammonia (5 mL). The reaction mixture was stirred at ambient temperature for 0.75 h. Ethyl acetate (5 mL) was added to the residue on evaporation to provide 2-chloro-5'-cyano-5'-deoxy-N-(4-phenylthio-l-piper- idinyl)adenosine (0.11 g, 46%) as a solid, m.p. 159-161°C, Η-NMR (400MHz, DMSO-ds) d 1.68 (2H, br q, piperidine C-H), 1.98 (2H m, piperidine C-H), 2.78 (2H, br, piperidine C-H), 3.04 (2H, br d, H-5'a and H-5'b), 4.11 (2H, m, H-4'and H-3'), 4.63 (IH, m, H-2'), 5.54, 5.68 (2H, 2d, 2'-and 3'-OH), 5.88 (IH d, H-l'), 7.22 - 7.45 (5H, 3m, Ar-H), 8.38 (IH s, H-8), 9.49 (IH, s, N-H). HPLC retention time 20.34 min. (gradient elution over 30 min; 25-45% acetonitrile/0.1% ammonium sulfate in water), purity 96% at 250 nm).
C,sH20CIN7O3. 0.7 H2O. 0.1 EtOAc requires C, 51.4; H, 5.1; N, 18.7. Found C, 51.4; H, 4.9; N, 18.3%.
EXAMPLE 47
2-Chloro-5'-deoxy-Ar-( 1 -piperidinvOadenosine
Methyl 5-deoxy-2,3-di-O-benzoyl-D-ribofuranoside
Methyl 5-deoxy-2,3-O-(l-methylethylidene)-D-ribofuranoside (prepared by reduction of methyl 2,3-O-(l-methylethylidene)-5'-O-(p-toluenesulphonyl)-D-ribofiιranoside using lithium aluminium hydride) (4.36 g, 23.2 mmol) was dissolved in methanol (120 mL) and Amberlyst resin (Ff form, 19 g) was introduced. The mbcture was stirred at 80°C for 60 h and filtered. The filter pad was washed with methanol and the filtrate was evaporated to an oily residue. The residue was dissolved in dichloromethane and to this solution was added trietiiylamine (25.7 g, 185 mmol). Benzoyl chloride (13.08 g, 10.8 mL, 92.8 mmol) was added dropwise over 0.5 h and the reaction mbcture was stirred at ambient temperature for 40 h. The reartion mbcture was extracted with 0.5 M hydrochloric acid solution (2 x 50 mL) and sodium bicarbonate solution (30 mL) before being dried (MgSO4) and evaporated. The residue was purified by flash chromatography on silica gel eluting with a mixture of heptane and ethyl acetate (4:1), gradually increasing polarity a (1:1) mixture of these solvents, providing the title methyl 5-deoxy-2,3-di-O-benzoyl-D-ribofuranoside (6.11 g, 74%), Η-NMR (400MHz, CDClj) d 1.50 (3H, d, -CHCHj), 3.48 (3H, s, -COCHj), 4.48 (Hi q), 5.11 (IH, s), 5.46 (IH, t), 5.60 (IH, d), 7.25 - 8.19 (20 m, Ar-H).
9-(5'-Deoxy-2',3'-di-0-benzoyl-β-D-D-ribofuranosyl)-2,6-dichloro-9H-purine
l-O-Acetyl-2,3-di-O-benzoyl-5-deoxy-D-ribofuranose [prepared from the above methyl 5- deoxy-2,3-di-O-benzoyl-D-ribofuranoside by the method described in Lerner, L. Nucleic Acid Chemistry: Improved and New Synthetic Procedures, Methods and Techniques, Part Four. Townsend, L.B. and Tipson, R.S., Eds.; John Wiley and Sons, New York, 1991, pp 274 - 280] (1.02 g, 2.65 mmol) and 2,6-dichloro-9H-purine (0.48 g, 2.53 mmol) were mixed thoroughly and heated at 145°C under oilpump vacuum for 2 h. The cooled reartion mbcture was dissolved in dichloromethane (25 mL), evaporated, and coevaporated with toluene (2 x 50 mL). Purification of the residue by flash chromatography provided the title 9-(5'-deoxy-2',3'-di-O-benzoyl-β- D-ribofuranosyI)-2,6-dichloro-9H-purine (0.90 g, 58%) as a foam, Η-NMR (400MHz, DMSO-ds) d 1.67 (3H, d, -CHCH3), 4.64 (Hi dt, H-4'), 5.72 (Hi t, H-3'), 6.12 (IH, t, H-2'), 6.35 (IH, d , H-l'), 7.31 - 8.05 (10H, m, Ar-H), 8.31 (IH, s, H-8).
2,,3,-Di-O-benzoyl-2-chloro-5'-deoxy-N-(l-piperidinyl)adenosine
9-(5'-Deoxy-2',3,-di-O-benzoyl-β-D-ribofuranosyl)-2,6-dichloro-9H-purine (0.46 g, 0.75 mmol) was dissolved in dioxan (10 mL). 1-Arninopiperidine (0.06 mL, 0.90 mmol) and triethylamine (0.16 mL, 1.13 mmol) were added and the reaction mbcture was stirred at ambient temperature for 18 h before being evaporated. The residue was treated with water (50 mL) and ethyl acetate (100 mL). The organic phase was separated and washed with water (2 x 50 mL). The combined extracts were dried (MgSO4) and evaporated. The residue was purified by flash chromatography on silica gel eluting with a mixture of heptane and ethyl acetate (1 : 1) to provide the title 2,3-di-O- benzoyl-2-chloro-5'-deoxy-N-(l-piperidinyl)adenosine (0.30 g, 69%) as a foam, Η-NMR (400MHz, CDC13) 1.48 (2H, br, piperidine C-H), 1.80 (2H, m, piperidine C-H), 2.87 (2H, br, piperidine C-H), 4.57 (Hi dt, H-4'), 5.71 (IH, t, H-3'), 6.07 (IH, t, H-2'), 6.33 (IH, d , H-l'), 7.28 - 8.00 (1 Hi m, Ar-H and H-8).
2-Chloro-5'-deoxy- V-( 1 -piperidinyl)adenosine
2,3-Di-O-benzoyl-2-chloro-5'-deoxy-N-(l-piperidinyl)adenosine (0.30 g, 0.81 mmol) was dissolved in methanol (10 mL) and methanolic ammonia (5 mL) was introduced. The reartion mixture was stirred at ambient temperature for 18 h. and evaporated. The residue was purified by flash chromatography eluting with a mixture of dichloromethane and 10% ammonia in ethanol (95:5) to provide the title 2-chloro-5'-deoxy-N-(l-piperidinyl)adenosine (0.13 g, 43%) as a foam, Η-NMR (400MHz, DMSO-ds) d 1.21 - 1.43 (5H, m, piperidine C-H and CHCH3), 1.63 (4H, br q, piperidine C-H), 2.82 (4H, br m, piperidine C-H), 5.79 (Hi d, H-l'), 8.37 (IH, s, H-8), 9.32 (Hi s, N-H). HPLC retention time 6.82 min. (gradient elution over 30 min; 25-45% acetonitrile/0.1% ammonium sulfate in water).
EXAMPLE 48
2-Chloro-5'-deoxy-5'-methylene-N-( 1 -piperidinyPadenosine
Methyl 5-deoxy-2,3 -O-( 1 -methylethylidene)-5-methylene-D-ribofuranoside
Triphenylmethylphosphonium bromide (26.79 g, 75 mmol) was suspended in THF (200 mL) and n-butyllithium (1.7M in hexanes) (42 mL, 71.2 mmol) was introduced. After stirring for 2 h, methyl 5-deoxy-5-oxo-2,3-O-(l-methylethylidene)-D-ribofiιranoside (prepared by oxidation of 1- O-methyl-2,3-O-(l-methylethylidene)-D-ribofuranoside using the method described in Ranganathan, R.S., Jones, G.H. and Moffatt, J.G., Journal of Organic Chemistry, 1974, 39(3), 290-298) (5.06 g, 25 mmol) in THF (50 mL) was added dropwise. The reartion mixture was heated for 2 h at 50°C and cooled. A mbcture of water (10 mL) and THF (90 mL) were added carefully under a stream of nitrogen. Diethyl ether (250 mL) and water (250 mL) were introduced. The aqueous phase was washed with diethyl ether (250 mL) and the combined organic extracts were washed with saturated brine (150 mL) and dried (MgSO4). The residue on evaporation was purified by flash chromatography eluting with a mbcture of cyclohexane and ethyl acetate (19:1), increasing polarity to a mbcture of heptane and ethyl acetate (9:1) provided the desired methyl 5-deoxy-2,3-O-(l-methylethylidene)-5-methylene-D-ribofuranoside (3.6 g, 72%) as a gum, Η-NMR (400MHz, CDC13) d 1.32, 1.50 (6H 2s, C(CH3)2), 3.36 (3H, s, - COCH3), 4.64 (IH, s), 4.65 (Hi d), 5.00 (IH, s), 5.15 (IH, d), 5.26 (IH, d), 5.83 - 5.92 (Hi m).
Methyl 2,3-di-O-benzoyl-5-deoxy-5-methylene-D-ribofuranoside
Methyl 5-deoxy-2,3-O-(l-methylethylidene)-5-methylene-D-ribofuranoside (5.65 g, 28.2 mmol) was dissolved in methanol (250 mL) and Amberlyst resin (H* form, 30 g) was introduced. The mixture was stirred at ambient temperature for 40 h and was filtered. The filter pad was washed with methanol and the filtrate was evaporated to an oily residue. The residue was dissolved in dichloromethane and to this solution was added benzoyl chloride (8.47 g, 7.0 mL, 60 mmol) and triethylamine (6.49 g, 8.94 mL, 66 mmol) and the reaction mixture was stirred at ambient temperature for 18 h. The reaction mixture was extracted with 0.5 M hydrochloric acid solution (2 x 100 mL) and water (100 mL) before being dried (MgSO ) and evaporated. The residue was purified by flash chromatography on silica gel eluting with a mixture of heptane and ethyl acetate (29: 1), gradually increasing polarity to a (4: 1) mixture of these solvents, providing the title methyl 2,3-di-O-benzoyl-5-deoxy-5-methylene-D-ribofuranoside (1.66 g, 26%), Η-NMR (400MHz, CDCI3) d 3.50 (3H, s, -CΟCH3), 4.75 - 4.82 (Hi m), 5.16 (IH, s), 5.30 (Hi d), 5.45 (Hi d), 5.61 (IH, d), 5.94 - 6.09 (Hi m). 9-(5'-Deoxy-2,,3'-di-O-benzoyl-5'-methylene-β-D-ribofuranosyl)-2,6-dichloro-9H-purine
l-O-Acetyl-2,3-di-O-benzoyl-5-deoxy-5-methylene-D-ribofuranose [prepared from the above methyl 2,3-di-O-benzoyl-5-deoxy-(l-methylethylidene)-5-methylene-D-ribofuranoside by the method described in Lerner, L. Nucleic Acid Chemistry: Improved and New Synthetic Procedures, Methods and Techniques, Part Four. Townsend, L.B. and Tipson, R.S., Eds.; John Wiley and Sons, New York, 1991, pp 274 - 280] (4.2 g, 10.6 mmol) and 2,6-dichloro-9H-purine (2.0 g, 10.6 mmol) were suspended in dichloromethane (25 mL) and evaporated to a residue which was heated at 150°C under oilpump vacuum for 1.5 h. The cooled reartion mixture was dissolved in dichloromethane (25 mL), evaporated, and coevaporated with toluene (2 x 50 mL). Purification of the residue by flash chromatography eluting with a mixture of heptane and ethyl acetate (9: 1), increasing polarity to a mixture of heptane and ethyl acetate (4: 1) provided the title 9-(5,-deoxy-2',3'-di-0-berιzoyl-5'-methylene-β-D-ribofuranosyl)-2,6-dichloro-9H-purine (3.98 g, 71%) as a foam, Η-NMR (400MHz, DMSO-ds) d 4.98 (Hi d), 5.48 (IH, d), 5.58 (Hi d), 5.91 (IH, t), 6.16 (IH, t , H-2'X 6.22 (IH, m), 6.44 (IH, d, H-l'), 7.34 - 8.08 (10H, m, Ar-H), 8.34 (IH, s, H-8). HPLC retention time 15.51 min. (gradient elution over 30 min.; 20-80% acetonitrile/0.1% TFA in water).
2,,3'-Di-O-benzoyl-2-chloro-5'-deoxy-5'-methylene-N-( 1 -piperidinyPadenosine
9-(5'-Deoxy-2,,3'-di-O-benzoyl-5'-methylene-β-D-ribofuranosyl)-2,6-dichloro-9H-purine (0.3 g, 0.57 mmol) was dissolved in dioxan (20 mL). 1-Aminopiperidine (0.066 g, 0.63 mmol) and triediylamine (0.087 g, 0.12 mL, 0.86 mmol) were added and the reartion mbcture was stirred at ambient temperature for 40 h and evaporated. The residue was purified by flash chromatography on silica gel eluting with a mixture of heptane and ethyl acetate (4:1), gradually increasing polarity to a (1:1) mixture of these solvents, to afford the title 2,3-di-O-benzoyl- 2-chloro-5,-deoxy-5'-methylene-N-(l-piperidinyl)adenosine (0.28 g, 83%), Η-NMR (400MHz, CDC13) d 4.93 (IH, dt), 5.43 (IH, dd), 5.56 (IH d), 5.89 (IH, t), 6.10 (IH, t , H-2'), 6.21 (IH, m), 6.42 (IH, d, H-l'), 7.33 - 8.05 (10H, m, Ar-H).
2-Chloro-5'-deoxy-5'-methylene-Λr-( 1 -piperidinyl)adenosine
2,3-Di-O-benzoyl-2-chloro-51-deoxy-5'-methylene-iV-(l-piperidinyl)adenosine (0.28 g, 0.47 mmol) was dissolved in methanolic ammonia (10 mL) and stirred at ambient temperature for 18 h. The reartion mbcture was evaporated and purified by flash chromatography eluting with a mbcture of dichloromethane and 10% ammonia in ethanol (95:5) to provide the title 2-chloro-5'-deoxy-5'-methylene-N-(l-piperidinyl)adenosine (0.081 g, 45%) as a foam, Η-NMR (400MHz, DMSO-ds) d 1.30 - 1.45 (2H br m, piperidine C-H), 1.62 (4H, br q, piperidine C-H), 2.84 (4H, br, piperidine C-H), 4.07 (IH, dt, H-3'), 4.32 (Hi q, H-2'), 4.58 (IH, m, H-4'), 5.20 (IH, dd, C=C-H), 5.30 (Hi d, C=C-H), 5.40, 5.56 (2H, 2d, 2'-and 3'-OH), 5.86 (IH, d, H-l'), 6.07 (IH, m, C=C-H), 8.36 (IH, s, H-8), 9.34 (IH, s, N-H). HPLC retention time 9.35 min. (gradient elution over 30 min; 25-45% acetonitrile/0.1% ammonium sulfate in water), purity 99.5% at 250 nm).
EXAMPLE 49
2-Chloro-5'-deoxy-5'-methylene-N-(4-phenylthio-l-piperidinyPadenosine
2,,3'-Di-O-benzoyl-2-chloro-5'-deoxy-5'-methylene-N-(4-phenylthio-l-piperidinyl) adenosine, prepared as described in Example 48 from 9-(5'-deoxy-2,,3'-di-O-benzoyl-5'-methylene-β-D- ribofuranosyl)-2,6-dichloro-9H-purine (1.0 g, 1.9 mmol) and l-ammo-4-phenylthiopiperid e (0.44 g, 2.1 mmol), was dissolved in methanol (20 mL) and sat. methanolic ammonia (2.5 mL) was introduced. The reartion mixture was stirred at ambient temperature for 18 h, evaporated and purified by flash chromatography eluting with a mixture of dichlorometiiane and ethanol (50:1) to provide the title 2-chloro-5'-deoxy-5'-methylene-N-(4-phenylthio-l -piper¬ idinyPadenosine (0.27 g, 29%) as a foam, Η-NMR (400MHz, DMSO-ds) d 1.70 (2H, br q, piperidine C-H), 1.99 (2H, br d, piperidine C-H), 2.77 (2H, br m, piperidine C-H), 3.06 (2H br m, piperidine C-H), 4.07 (Hi q, H-3'), 4.32 (IH, dt, H-2'), 4.58 (IH, m, H-4'), 5.20 (IH, dd, C=C-H), 5.29 (IH, d, C=C-H), 5.39, 5.56 (2H, 2d, 2'-and 3'-OH), 5.86 (IH, d, H-l'), 6.07 (Hi m, C=C-H), 7.23 - 7.45 (5H 3 m, Ar-H), 8.36 (IH, s, H-8), 9.34 (IH, s, N-H). HPLC retention time 13.46 min. (gradient elution over 30 min; 25-45% acetonitrile/0.1% ammonium sulfate in water), purity 100% at 250 nm).
EXAMPLE 50
2-Chloro-5'-deoxy-N-methoxy-5'-methyleneadenosine
2',3'-Di-O-benzoyl-2-chloro-5'-deoxy-N-methoxy-5'-methyleneadenosine (0.2 g, 0.4 mmol), prepared by the method described in Example 48 from 9-(5'-deoxy-2',3'-di-O-benzoyl-5'- methylene-β-D-ribofuranosyl)-2,6-dichloro-9H-purine (0.5 g, 2.0 mmol) and O- methylhydroxlamine hydrochloride (0.167 g, 2.0 mmol), was treated with methanolic ammonia (10 mL) and stirred at ambient temperature for 18 h. The reaction mbcture was evaporated and purified by flash chromatography eluting with a mixture of dichloromethane and 10% ammonia in ethanol (19:1) to provide the tide 2-chloro-5'-deoxy-N-methoxy-5'-methyleneadenosine (0.03 g, 9%) as a foam, Η-NMR (400MHz, DMSO-ds) d 3.79 (3H, s, -CH3), 4.08 (IH, q, H-3'), 4.33 (Hi dt, H-2'X 4.61 (IH, m, H-4'), 5.20 (IH, dd, C=C-H), 5.31 (IH, d, C=C-H), 5.42, 5.59 (2H, 2d, 2'-and 3'-OH), 5.89 (IH, d, H-l'), 6.08 (Hi m, C=C-H), 8.44 (IH, s, H-8), 11.58 (IH, s, N- H). HPLC retention time 7.09 min. (gradient elution over 30 min; 25-45% acetonitrile/0.1% ammonium sulfate in water), purity 98.6% at 250 nm). EXAMPLE 51
2-Chloro-5'-deoxy-5'-methylene-N-( 1 -piperidinyPadenosine
2,3-Di-O-benzoyl-2-chloro-5'-deoxy-5'-methylene-N-cyclopentyladenosine (prepared by reartion of 9-(5'-deoxy-2',3'-di-O-benzoyl-5'-methylene-β-D-ribofiιranosyl)-2,6-dichloro-9H-purine (see Example 48) with cyciopentylamine) (0.30 g, 0.52 mmol) was dissolved in methanolic ammonia (10 mL) and stirred at ambient temperature for 18 h. The reaction mixture was evaporated and purified by flash chromatography eluting with a mixture of dichloromethane and 10% ammonia in ethanol (39: 1) to provide the title 2-chloro-5'-deoxy-5'-methylene-N-cyclopentyladenosine (0.051 g, 27%) as a foam, Η-NMR (400MHz, DMSO-ds) d 1.48 - 2.04 (8H, 3 br m, cyctopentyl C-H), 4.08 (IH, q, H-3'X 4.32 (IH, dt, H-2'), 4.42 (IH, m, -HN-C-H), 4.59 (IH m, H-4'), 5.19 (Hi dd, C=C-H), 5.30 (IH d, C=C-H), 5.40, 5.55 (2H, 2d, 2'-and 3'-OH), 5.85 (IH, d, H-l'), 6.08 (IH, m, C=C-H), 8.34 (IH, s & br s, H-8 and N-H).
EXAMPLE 52
5'-Deoxy-2.5'-dichloro-N-(4-phenylthio- 1 -piperidinyPadenosine
This compound was prepared by the method described in Example 24. 2-Chloro-N-(4-phenylthiocyclohexyl)adenosine (prepared from 4-hydroxycyclohexlamine by the general methods laid out in Knutsen, L.J.S., Lau, J., Sheardown, M.J., Thomsen, C; Bioorganic and Medicinal Chemistry Letters, 1993, 3, 2661-2666) (0.2 g, 0.44 mmol) was subjected to the reartion conditions described above, and the residue on evaporation was purified by flash chromatography on silica gel eluting with a mixture of heptane and ethyl acetate (4:1), gradually increasing polarity to a (19:1) mixture of ethyl acetate and methanol to afford the title 5'- deoxy-2,5'-dichloro-N-(4-phenylthiocyclohexyl)adenosine (0.08 g, 38%), Η-NMR (400MHz, CDC13) d 1.20 - 1.30 (2H, t, cyclohexyl C-H), 1.80 - 2.05 (6H, br m, cyclohexyl C-H), 3.75 - 3.84 (2H, m, H-5'a and H-5'b), 5.96 (Hi d, H-l'), 7.19 - 7.33 (3H, m, Ar-H), 7.41 (2H, d, Ar-H), 7.99 (Hi s, H-8), 8.29 (IH, s, N-H). HPLC retention time 9.1 min. (gradient elution over 30 min.; 20-80% acetonitrile/0.1% TFA in water, 100% purity at 250 nm).

Claims

Claims.
1. A method of treating a mammal suffering from type I or type U diabetes, multiple schlerosis, stroke, osteoporosis, septic shock or menstrual complications, preferably type I or type II diabetes, more preferably type LI diabetes, which method comprises administering to a mammal in need of such treatment an effective amount of an adenosine A3 receptor ligand.
2. A method according to claim l comprising administering a compound of the general formula (I), or a pharmaceutically acceptable salt thereof:
Figure imgf000071_0001
wherem
X represents hydrogen, halogen, amino, perhalomethyl, cyano, C^-alkyl, Ci-s-alkoxy, Cμ- alkylthio,
Figure imgf000071_0002
or phenyl;
A is hydroxymethyl, methyl, chlorometiiyl, bromomethyl, fluoromethyl, cyanomethyl, arninomethyl, vinyl, methylthiomethyl or methoxymethyl;
Ri is selected from the groups consisting of
Figure imgf000072_0001
wherein Q is nitrogen or carbon, n is 1 to 3 and where the group (a) may be optionally substituted with one or two Ci-s-alkyl groups, C2-s-alkenyl, C2-s-alkynyl, phenoxy, phenylsulphonyl, phenylthio, hydroxy, phenyl, Ci-β-alkoxy or
Figure imgf000072_0002
Cι-6 -alkyl, phenylthioalkyl or
Figure imgf000072_0003
wherein Y is O, S or NZ, where Z is H Ci-β-alkyl or phenyl, and where the group (b) may be optionally substituted with Ci-s-alkyl, C2^-alkenyl, C2^-alkynyl, phenoxy, phenyl, Ci-β-alkoxy or Cι-6-alkoxy-Cι-6-alkyl, or
R' is -NR2R3 or -YR4, wherein Y is oxygen;
R2 is Ci-s-alkyl;
R3 is phenyl or Ci-s-alkyl which may be substituted by phenyl or phenoxy;
R4 is straight-chain
Figure imgf000072_0004
C2.g-alkenyl or C3-g-cycloalkyl, which may be substituted by phenyl or phenoxy which in turn may be substituted with nitro, halogen or amine.
3. A method according to claim 1 or 2 comprising administering a compound of the general formula (H wherein R1 is -OR4, wherein R4 is straight-chain Ci-s-alkyl, branched C3^-alkyl, C2-g- alkenyl or C3.g-cycloalkyl, which may be substituted by phenyl or phenoxy which in turn may be substituted with nitro, halogen or amine.
4. A compound characterised by the general formula (I), or a pharmaceutically acceptable salt thereof:
^R1
HN
N
A
HO OH
wherein
X represents phenyl;
A is hydroxymethyl, metiiyi, chloromethyl, bromomethyl, fluoromethyl, cyanomethyl, aminomethyl, vinyl, methylthiomethyl or methoxymethyl;
Ri is selected from the groups consisting of
Figure imgf000073_0001
wherein Q is nitrogen or carbon, n is 1 to 3 and where the group (a) may be optionally substituted with one or two Ci-s-alkyl groups, C2^-alkenyl, C2-s-alkynyl, phenoxy, phenylsulphonyl, phenylthio, hydroxy, phenyl, Ci-s-alkoxy or Cι-6-alkoxy- Ci-s-alkyl, phenylthioalkyl or
Figure imgf000074_0001
wherein Y is O, S or NZ, where Z is H
Figure imgf000074_0002
or phenyl, and where the group (b) may be optionally substituted with Ci-s-alkyl, C2-β-alkenyl, C2-6-alkynyl, phenoxy, phenyl, Ci-s-alkoxy or Cι-6-alkoxy-Cι.6-alkyl, or
R' is -NR R3 or -YR4, wherein Y is oxygen; R2 is d-s-alkyl;
R3 is phenyl or Ci-s-alkyl which may be substituted by phenyl or phenoxy; R4 is straight-chain Ci-s-alkyl, branched C3.8-a.kyl, C2^-alkenyl or Cs-g-cycloalkyl, which may be substituted by phenyl or phenoxy which in turn may be substituted with nitro, halogen or amine.
5. A compound characterised by the general formula (I), or a pharmaceutically acceptable salt thereof:
Figure imgf000075_0001
wherein
X represents hydrogen, halogen, amino, perhalomethyl, cyano, Ct-β-alkyl, Ci-s-alkoxy, Cw alkylthio, Ci-s-alkylamino or phenyl;
A is hydroxymethyl, methyl, chloromethyl, bromomethyl, fluoromethyl, cyanomethyl, aminomethyl, vinyl, methylthiomethyl or methoxymethyl;
Ri is selected from the groups consisting of
Figure imgf000075_0002
wherein Q is nitrogen or carbon, n is 1 to 3 and where the group (a) may be optionally substituted with one or two Ci-β-alkyl groups, C2_s-alkenyl, C«-alkynyl, phenoxy, phenylsulphonyl, phenylthio, hydroxy, phenyl, -s-alkoxy or Ci-s-alkoxy- Ci-s-alkyl, phenylthioalkyl or
Figure imgf000076_0001
wherein Y is O, S or NZ, where Z is H Cι-5-alkyl or phenyl, and where the group (b) may be optionally substituted with Ci-s-alkyl, C -6-alkenyl, C2-6-alkynyl, phenoxy, phenyl, C^-alkoxy or Cι^-alkoxy-Cι-6-alkyl, or
R' is -NR2R3 or -YR4, wherein Y is oxygen; R2 is Cs-alkyl;
R3 is phenyl or Ci-β-alkyl which may be substituted by phenyl or phenoxy;
R4 is branched C3.g-a.kyl or C2-g-alkenyl, which may be substituted by phenyl or phenoxy which in turn may be substituted with nitro, halogen or amine.
7. A pharmaceutical composition comprising as artive component an adenosine A3 ligand and a pharmaceutically acceptable carrier or diluent.
8. A pharmaceutical composition according to claim 6 comprising a compound according to claim 4 or 5 and a pharmaceutically acceptable carrier or diluent.
9. The use of adenosine A3 ligands to modulate cytokines, preferably TNF-α, in the treatment of type I or type LI diabetes, multiple schlerosis, stroke, osteoporosis septic shock or menstrual complications, preferably type I or type II diabetes, more preferably type JJ diabetes.
10. The use of adenosine A3 ligands for the manufacture of a pharmaceutical composition for treating type I or type LI diabetes, multiple schlerosis, stroke, osteoporosis, septic shock or menstrual complications, preferably type I or type II diabetes, more preferably type LI diabetes.
11. The use of a compound according to claim 4 or 5 for the manufacture of a pharmaceutical composition for treating type I or type π diabetes, multiple schlerosis, stroke, osteoporosis, septic shock or menstrual complications, preferably type I or type LI diabetes, more preferably type LI diabetes.
PCT/DK1997/000107 1996-03-13 1997-03-12 A method of treating disorders related to cytokines in mammals WO1997033590A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU20224/97A AU2022497A (en) 1996-03-13 1997-03-12 A method of treating disorders related to cytokines in mammals

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DK0293/96 1996-03-13
DK29396 1996-03-13
DK59096 1996-05-21
DK0590/96 1996-05-21

Publications (1)

Publication Number Publication Date
WO1997033590A1 true WO1997033590A1 (en) 1997-09-18

Family

ID=26063710

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DK1997/000107 WO1997033590A1 (en) 1996-03-13 1997-03-12 A method of treating disorders related to cytokines in mammals

Country Status (2)

Country Link
AU (1) AU2022497A (en)
WO (1) WO1997033590A1 (en)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6544960B1 (en) * 1997-11-08 2003-04-08 Smithkline Beecham Corporation Chemical compounds
US7423144B2 (en) 2004-05-26 2008-09-09 Inotek Pharmaceuticals Corporation Purine Derivatives as adenosine A1 receptor agonists and methods of use thereof
US7732424B2 (en) 2005-11-30 2010-06-08 Inotek Pharmaceuticals Corporation Purine derivatives and methods of use thereof
US7863253B2 (en) 2004-09-20 2011-01-04 Inotek Pharmaceuticals Corporation Purine Derivatives and methods of use thereof
US9278991B2 (en) 2012-01-26 2016-03-08 Inotek Pharmaceuticals Corporation Anhydrous polymorphs of [(2R,3S,4R,5R)-5-(6-(cyclopentylamino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)} methyl nitrate and processes of preparation thereof
US9289383B2 (en) 2010-03-26 2016-03-22 Inotek Pharmaceuticals Corporation Method of reducing intraocular pressure in humans
US9370530B2 (en) 2010-01-11 2016-06-21 Inotek Pharmaceuticals Corporation Combination, kit and method of reducing intraocular pressure
US9522160B2 (en) 2013-03-15 2016-12-20 Inotek Pharmaceuticals Corporation Ophthalmic formulations

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0322242A2 (en) * 1987-12-23 1989-06-28 Glaxo Group Limited Adenosine derivatives
EP0472181A2 (en) * 1990-08-22 1992-02-26 Merrell Pharmaceuticals Inc. 5-Amine substituted adenosine analogs as immunosuppressants
EP0490818A1 (en) * 1990-12-07 1992-06-17 Sandoz Ltd. 6-cyclohexyl-2'-0-methyl-adenosine hydrate and uses thereof
WO1993008206A1 (en) * 1991-10-24 1993-04-29 Novo Nordisk A/S Novel 2,6-disubstituted purine derivatives
WO1993023417A1 (en) * 1992-05-14 1993-11-25 Novo Nordisk A/S Novel adenosine derivatives
WO1995003304A1 (en) * 1993-07-23 1995-02-02 Merrell Pharmaceuticals Inc. Novel 9-n-bicyclic nucleoside agents useful as selective inhibitors of proinflammatory cytokines
WO1995007921A1 (en) * 1993-09-17 1995-03-23 Novo Nordisk A/S Chemical compounds, their preparation and use
GB2289218A (en) * 1994-05-06 1995-11-15 Merck & Co Inc Inhibition of TNFalpha production with agonists of the A2b subtype of the adenosine receptor

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0322242A2 (en) * 1987-12-23 1989-06-28 Glaxo Group Limited Adenosine derivatives
EP0472181A2 (en) * 1990-08-22 1992-02-26 Merrell Pharmaceuticals Inc. 5-Amine substituted adenosine analogs as immunosuppressants
EP0490818A1 (en) * 1990-12-07 1992-06-17 Sandoz Ltd. 6-cyclohexyl-2'-0-methyl-adenosine hydrate and uses thereof
WO1993008206A1 (en) * 1991-10-24 1993-04-29 Novo Nordisk A/S Novel 2,6-disubstituted purine derivatives
WO1993023417A1 (en) * 1992-05-14 1993-11-25 Novo Nordisk A/S Novel adenosine derivatives
WO1995003304A1 (en) * 1993-07-23 1995-02-02 Merrell Pharmaceuticals Inc. Novel 9-n-bicyclic nucleoside agents useful as selective inhibitors of proinflammatory cytokines
WO1995007921A1 (en) * 1993-09-17 1995-03-23 Novo Nordisk A/S Chemical compounds, their preparation and use
GB2289218A (en) * 1994-05-06 1995-11-15 Merck & Co Inc Inhibition of TNFalpha production with agonists of the A2b subtype of the adenosine receptor

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CANADIAN JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY, Volume 72, No. 10, 1994, FRED D. ROMANO et al., "The Antiadrenergic Effect of Cyclopentyladenosine on Myocardial Contractility is Reduced in Vivo in Diabetic Rats", page 1245. *
JOURNAL OF IMMUNOLOGY, Volume 156, No. 9, 1996, FEREYDOUN G. SAJJADI et al., "Inhibition of TNF-alpha Expression by Adenosine". *
MOLECULAR AND CELLULAR BIOCHEMISTRY, Volume 144, 1995, LESLEY HESELTINE et al., "Adenosine Effects Upon Insulin Action on Lipolysis and Glucose Transport in Human Adipocytes", pages 147-151. *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6740644B2 (en) * 1997-11-08 2004-05-25 Smithkline Beecham Corporation Chemical compounds
US6544960B1 (en) * 1997-11-08 2003-04-08 Smithkline Beecham Corporation Chemical compounds
US8609833B2 (en) 2004-05-26 2013-12-17 Inotek Pharmaceuticals Corporation Purine derivatives as adenosine A1 receptor agonists and methods of use thereof
US7423144B2 (en) 2004-05-26 2008-09-09 Inotek Pharmaceuticals Corporation Purine Derivatives as adenosine A1 receptor agonists and methods of use thereof
US8183224B2 (en) 2004-05-26 2012-05-22 Inotek Pharmaceuticals Corporation Purine derivatives as adenosine A1 receptor agonists and methods of use thereof
US7863253B2 (en) 2004-09-20 2011-01-04 Inotek Pharmaceuticals Corporation Purine Derivatives and methods of use thereof
US8133880B2 (en) 2004-09-20 2012-03-13 Inotek Pharmaceuticals Corporation Purine derivatives and methods of use thereof
US7732424B2 (en) 2005-11-30 2010-06-08 Inotek Pharmaceuticals Corporation Purine derivatives and methods of use thereof
US9370530B2 (en) 2010-01-11 2016-06-21 Inotek Pharmaceuticals Corporation Combination, kit and method of reducing intraocular pressure
US9289383B2 (en) 2010-03-26 2016-03-22 Inotek Pharmaceuticals Corporation Method of reducing intraocular pressure in humans
US9278991B2 (en) 2012-01-26 2016-03-08 Inotek Pharmaceuticals Corporation Anhydrous polymorphs of [(2R,3S,4R,5R)-5-(6-(cyclopentylamino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)} methyl nitrate and processes of preparation thereof
US9718853B2 (en) 2012-01-26 2017-08-01 Inotek Pharmaceuticals Corporation Anhydrous polymorphs of [(2R,3S,4R,5R)-5-(6-(cyclopentylamino)-9H-purin-9-YL)-3,4-dihydroxytetrahydrofuran-2-YL)] methyl nitrate and processes of preparation thereof
US9522160B2 (en) 2013-03-15 2016-12-20 Inotek Pharmaceuticals Corporation Ophthalmic formulations

Also Published As

Publication number Publication date
AU2022497A (en) 1997-10-01

Similar Documents

Publication Publication Date Title
US5589467A (en) 2,5',N6-trisubstituted adenosine derivatives
AU784374C (en) Synthesis of 2'-deoxy-L-nucleosides
ES2236044T3 (en) 8-MODIFIED ADENOSINE N6-HETEROCICLIC DERIVATIVES.
EP1928475B1 (en) Antiviral phosphoramidates of 4'-c-azido-substituted pronucleotides
DE69933860T2 (en) 2'-FLUORONUKLEOSIDE
EP0496617B1 (en) Adenosine kinase inhibitors
EP0708781B1 (en) A 3 adenosine receptor agonists
SK47199A3 (en) Monocyclic l-nucleosides, analogs and uses thereof
KR20030061792A (en) Nucleoside derivatives
KR20080041296A (en) Modified nucleosides for the treatment of viral infections and abnormal cell proliferation
CA2476279A1 (en) Modified fluorinated nucleoside analogues
WO1998001459A1 (en) Novel n-alkoxyadenine derivatives acting as cytokine inhibitors
IL103513A (en) 2-N-disubstituted adenosine derivatives their preparation and pharmaceutical compositions containing them
WO1992012718A1 (en) Adenosine kinase inhibitors
WO1997033591A1 (en) A method of treating disorders related to cytokines in mammals
WO1997033590A1 (en) A method of treating disorders related to cytokines in mammals
WO1998016539A1 (en) Novel therapeutically active adenosine derivatives
EP1634888A2 (en) Synthesis of 2'-deoxy-L-nucleosides
IE920190A1 (en) Adenosine kinase inhibitors

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE HU IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TR TT UA UG UZ VN AM AZ BY KG KZ MD RU TJ TM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH KE LS MW SD SZ UG AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
NENP Non-entry into the national phase

Ref country code: JP

Ref document number: 97532209

Format of ref document f/p: F

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

NENP Non-entry into the national phase

Ref country code: CA

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载