EP1907407A1 - Improvements to analogous compounds of 6-thioguanosine triphosphate, their use in medical fields and processes for their preparation - Google Patents

Improvements to analogous compounds of 6-thioguanosine triphosphate, their use in medical fields and processes for their preparation

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Publication number
EP1907407A1
EP1907407A1 EP06766084A EP06766084A EP1907407A1 EP 1907407 A1 EP1907407 A1 EP 1907407A1 EP 06766084 A EP06766084 A EP 06766084A EP 06766084 A EP06766084 A EP 06766084A EP 1907407 A1 EP1907407 A1 EP 1907407A1
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European Patent Office
Prior art keywords
thioguanosine
compound
group
thio
general formula
Prior art date
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EP06766084A
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German (de)
French (fr)
Inventor
Giancarlo Naccari
Sergio Baroni
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Nogra Pharma Ltd
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Giuliani International Ltd
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Priority claimed from IT000391A external-priority patent/ITRM20050391A1/en
Priority claimed from IE2006/0448A external-priority patent/IE84917B1/en
Application filed by Giuliani International Ltd filed Critical Giuliani International Ltd
Publication of EP1907407A1 publication Critical patent/EP1907407A1/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • 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 analogous compounds of 6-thioguanosine triphosphate, their use in medical field and process for their preparation.
  • the invention refers to the therapeutic use of analogous compounds of 6- thioguanosine triphosphate for example as immunosuppressant for the prevention of rejection of organ transplants and of post-transplant nephropathy and in the treatment of pathologies in which immune system is involved, such as, for instance, inflammatory chronic intestinal diseases, such as Crohn's disease, ulcerous rectocolitis, indeterminate colitis, or of autoimmune enteropathy, active chronic hepatitis, rheumatoid arthritis, Still's disease, systemic lupus erythematous, acquired haemolytic anaemia, idiopathic thrombocytopenia, polyarthritis nodosa, vasculitis, polyangitis, polymyositis, myasthenia gravis, sarcoidosis, lipoid nephritis, multiple sclerosis, dermatomyositis, pemphigus vulgaris, primary biliary cirrhosis, primary
  • the cells involved in the inflammatory immune response are able to survive at the inflammatory site, however, after completion of such response, the majority of cells must "die” to maintain the homeostasis of organism (Boise, 1995). Since the uncontrolled lymphocyte proliferation may cause the development of inflammatory chronic pathologies, the immune system controls the depletion of activated lymphocytes by a process named apoptosis (programmed cell death). This would assume a particular importance for the immune system of the mucosa, since the apoptosis resistance of lamina intestinal cells can lead to a chronic inflammatory response at the intestinal level (Tiede, 2003).
  • the activation of the mucosal immune system plays a key role in the pathogenesis of Crohn's disease.
  • pro-inflammatory cytokines produced by T lymphocytes and macrophages in particular interleukine-6 (IL-6) and interleukine-12 (IL-12), may cause T lymphocytes resistance against apoptosis, which in its turn provokes an intestinal accumulation of lymphocytes and establishes a long-lasting disease (Tiede, 2003).
  • the lymphocytes activation starts with two signals: the specific binding of antigens to the TCR (T cell receptor) and a second co-stimulatory signal represented by transmembrane proteins, such as CD28 (Maltzman, 2003). It has been shown that co-stimulation with CD28 enhances in vitro survival of activated T lymphocytes; in fact, CD28 induces an enhanced production of interleukine-2 (IL-2) acting as extrinsic factor for T lymphocytes survival, and the intrinsic ability of T lymphocytes to be resistant against apoptosis (Boise, 1995 bis). This occurs since CD28 action is associated with the expression of an anti-apoptotic gene, named bcl-x L gene (Khoshnan, 2000; Noel, 1996).
  • bcl-x L gene an anti-apoptotic gene
  • -CD28 acts through its cytoplasmaic portion with a complex of "adaptor” proteins and with a molecule, named Vav (Frauwirth, 2002);
  • -Vav acts as guanosine nucleotide exchange factor (GEF) for another molecule named Rac1 (Frauwirth, 2002);
  • NF- ⁇ B is not anymore retained in an inactive form in the cytosol but is able to translocate to nucleus where induces bcl-x L expression (Khoshnan, 2000);
  • activated Rad stimulates the activation of a protein belonging to the STAT family (Signal Transducers and Activators of Transcription), that is STAT-3, thus inducing its translocation to the nucleus and the corresponding expression of STAT-3 dependent genes (Faruqi, 2001).
  • STAT-3 induces bcl-x L expression thus contributing to the resistance against apoptosis and to the accumulation of T lymphocytes in the inflamed mucosa during the course of Crohn's disease (Mudter, 2003).
  • the study of intestinal T lymphocytes has pointed out that STAT-3 is steadily activated in patients with Crohn's disease, but not in healthy traditionsies (Lovato 2003).
  • RhoA and Cdc42 belongs to the Rho family which is a superfamily of small G protein characterized in that they are able to bind guanosine nucleotides and to regulate many cellular responses. They cycle between an inactive state, when bound to GDP, to an active state with GTP in place of GDP. This reaction is sustained upon guanosine nucleotide exchange factors named GEFs (like Vav).
  • GEFs guanosine nucleotide exchange factors
  • the binding with GTP induces a conformational change, which allows Rad and other GTPases to bind to their effectors.
  • GAPs GTPase-activating protein
  • Rad as in general all GTPase belonging to the Rho family, plays an important role in the mitogenesis processes, proliferation, and invasivity, since it stimulates alterations of the gene expression, in the present case of the gene bci-x L , modulating the activity of transcription factors, such as, in the present case, NF- ⁇ B and STAT-3 (Van Aelst, 1997).
  • Azathioprine is regarded as "gold standard" of the immunosuppressive therapy of Crohn's disease, also if the mechanism of action of such active principle is still unknown.
  • the inhibition of the purine nucleotide biosynthesis with suppression of DNA and RNA synthesis and downregulation of T and B lymphocytes function (Tiede 2003), is assumed to be the main therapeutic mechanism of azathioprine.
  • 6-thioguanosine triphosphate (6-thioGTP) which represents the real functional metabolite of the drug.
  • 6-thioGPT binds directly to
  • azathioprine by modulating Rad activity switches an anti- apoptotic co-stimulatory signal, mediated by CD28, into a pro-apoptotic signal.
  • This new mechanism of action is able to explain the well-known "delay" in the therapeutic effect of azathioprine, which needs a long treatment time to elicit a clinical response in such a way that benefits and clinical response were not observed earlier than 4 months of therapy.
  • This is due to the fact that 6-thioGPT has 20-fold less affinity to Rad in comparison with GTP, which is normally bound to Rad. Therefore, the azathioprine treatment needs the simultaneous and prolonged administration with high steroids doses, having remarkable side effects such as osteoporosis, diabetes, cataract.
  • the dashed bond in the sugar moiety can be either single or double and wherein R 1 , R 2 , R 3 , R 4 or R 5 , equal or different between each other, have general formula - ⁇ Int) m -Ter, wherein m is between 0 and 12 and lnt and Terare Internal and Terminal building blocks, wherein lnt is selected from the group consisting of
  • X represents either carbon or nitrogen atom within aromatic ring
  • Y represents either oxygen or sulphur atom
  • an additional group Q, group Qi or groups Qi are selected from the group consisting of -OH, -COOH, -N(CH 3 ) 2 , -N(CH 2 -CH 3 ) 2 , -CO-CH 3 , -CO-O-CH 3 , -0-CH 3 , -S-CH 31 -SO 2 -CH 3 , , -CN, -NO 2 or -Halogen elements.
  • R 5 may be
  • n is between O and 5, or oxygen or phosphorus is partially or completely replaced by nitrogen, sulphur, methylene groups or their derivatives.
  • TER may be selected from the group consisting of:
  • sugar moiety of compounds of formula (I) can be selected from the group consisting of the following sugar moieties or sugar-like moieties:
  • n 1, 2 or 3
  • m is between O and 5
  • lnt is selected from the group consisting of
  • X represents either carbon or nitrogen atom within aromatic ring
  • Y represents either oxygen or sulphur atom
  • an additional group Q or groups Q/ are selected from the group consisting of -CH 3 , -C(CH 3 ) 3l -OH, -COOH, -CO-CH 3 , -CO-O-CH 3 , -0-CH 3 , -S-CH 31 -SO 2 -CH 3 , -N(CHs) 2 , -N(CH 2 -CHs) 2 , -CN, -NO 2 or -Halogen elements.
  • Ter is selected from the group consisting of
  • the compounds of formula (I) are the compounds described below:
  • R-i, R 2 , R 3 , R 4 or R 5 equal or different between each other, have general formula - (Int) m -Ter, wherein m is between O and 12 and lnt and fer are Internal and Terminal building blocks, wherein lnt is selected from the consisting of
  • an additional group Q, group Qi or groups Qi are selected from the group consisting of -OH, -COOH, -N(CH 3 ) 2 , -N(CH 2 -CH 3 ) 2 or -Halogen elements
  • sugar moiety of compounds of formula (I) can be selected from the group consisting of the following sugar moieties or sugar-like moieties:
  • Q is selected from -OH (FAM) or -N(CH 3 ) 2 (TAMRA).
  • the compounds of formula (I), (Ia) and (II) are the compounds described below:
  • Threonine-2',3'-EDA-6-Thio-GTP ID: 05B-3
  • compositions comprising at least one of the compounds of the above mentioned formula (I) as active principle and one or more pharmaceutically acceptable co-adjuvants or excipients, that are known to those skilled in the art and currently in use in the pharmaceutical technology.
  • kidney, heart, lung, pancreas, liver transplantation kidney, heart, lung, pancreas, liver transplantation
  • post- transplant nephropathy in the treatment of pathologies in which immune system is involved, such as, for instance, inflammatory chronic intestinal diseases, such as Crohn's disease, ulcerous rectocolitis, indeterminate colitis, or auto-immune enteropathy, active chronic hepatitis, rheumatoid arthritis, Still's disease, systemic lupus erythematous, acquired haemolytic anaemia, idiopathic thrombocytopenia, polyarthritis nodosa, vasculitis, polyangitis, polymyositis, myasthenia gravis, sarcoidosis, lipoid nephritis, multiple sclerosis, dermatomyositis, pemphigus vulgaris, primary biliary cirrhosis, primary sclerosing cholangitis, recurrent multi
  • the present invention further relates to the use of labelled compounds of formula (I), particularly with R 3 or R 4 selected from
  • the present invention refers to a process for the preparation of compounds of formula (I), (Ia) and (II), wherein the introduction of the -NH-R group at the 2 position of guanosine ring comprises the following steps:
  • Figure 1 shows the scheme of the mechanism of action of the apoptosis inhibition and of the action of azathioprine.
  • FIG. 5 Induction of apoptosis.
  • Buffy stands for "buffy coat” which is the fraction of blood obtainable by centrifugation and containing leukocytes and platelets. In this case buffy coats were used to isolate monocytes for the experiments.
  • Buffy 1 vom 08/03/05 means "Buffy coat 1 of march 8th, 2005”.
  • V1 and V3 have been tested 4 times in four independent experiments, while V2 and V5 were tested twice in two separate experiments. For instance: V1 was tested twice on 08/03/2005 (where 08/03/2004 is written this is in error and should read 08/03/2005) and twice on 22/04/2005
  • Figure 6 Results of biological activity of compounds compiled in Figure 5.
  • Figure 7 list of further compounds of the invention, as well as providing key to some of the shorthand used in identifying some of the molecules.lt will be noted that some compounds have different enantiomeric forms, and the representations in Figure 7 may show an alternative enantiomeric form than that discussed in the text.
  • EXAMPLE 1 Process for the preparation of 2-substituted-6-thio-guanosine nucleotides.
  • lnosine-2 ' ,3 ' ,5 ' -triacetate (4g, 10.2mmol) was dissolved in 100 ml dichloromethane and the solution was treated with 1 ,4 ml (12.2 mmol) 2-methoxyethoxymethylchloride at O 0 C in the presence of diisopropylethylamine as a supportive base. After 1 hour the reaction was quenched with water. The solution was stirred for 30 min and chloroform was added afterwards. The aqueous layer was extracted with chloroform and the combined and washed organic layers were concentrated to dryness.
  • Aqueous ammonia solution (28%, 20 ml) was added to a solution of 2',3',5'-Tri-O-acetyl-1-[(2- methoxyethoxy)methyl] inosine (3,5 g, 7.3 mmol) in 50 ml methanol.
  • the reaction mixture was stirred for 1 hour at room temperature and concentrated, yielding the deprotected nucleoside (2,44 g, 6.9 mmol, 95%). This product was used in the next step without further purification.
  • the nucleoside (2,44 g, 6.9 mmol) was refluxed with 50 ml aqueous sodium hydroxide (0.2 M) for 1 hour, cooled to room temperature, neutralized with HCI (6 M) and evaporated to dryness. The residue was dissolved in ethanol, filtered from insoluble material and concentrated to dryness. Purification of the crude product by column chromatography (silica gel, CHCI 3 :MeOH, 3:1) gave 1 ,32 g (5.11 mmol, 70%) of 5-Amino-1- ⁇ -ribofuranosylimidazole-4-carboxamide.
  • the 5-Amino-1- ⁇ -ribofuranosylimidazole-4-carboxamide (1,3 g, 5 mmol) was dissolved in pyridine and 18ml (15 mmol) phenyl-isothiocyanate were added slowly.
  • the reaction mixture was refluxed for 2 hours under argon atmosphere.
  • the solution was cooled to room temperature, the precipitate collected by filtration and washed with diethyl ether.
  • the obtained pyridinium salt of the product was dissolved in aqueous sodium hydroxide (15%, 40 ml). The solution was heated 30 minutes at 60 0 C and concentrated under vacuum. Methanol was then added and the solution was kept in the refrigerator overnight.
  • nucleoside (220 mg, 0.575 mmol) in 20 ml dry pyridine was stirred with 1ml (lOmmol) acetic anhydride at room temperature for 15 hours under argon atmosphere. The solvent was removed under vacuum and the residue dissolved in a mixture of CHCI 3 (10 ml) and CH 3 OH (2 ml). This solution was loaded on silica gel and eluted with CHCI 3 / CH 3 OH (5:1) to give 284,7 mg (0.52 mmol, 90%) of fully protected nucleoside.
  • EXAMPLE 2 Process for the preparation of analogous of ribose-modified 6-thio-guanosine- triphosphate.
  • 6-thio-guanosine (1 g, 3.34 mmol) was dissolved in 6 ml trimethylphosphate.
  • the solution was cooled to O 0 C and 1,3 ml of Lutidine were added. After 10 minutes, 0,4 ml (4.4 mmol) phosphorous oxychloride was carefully added to the solution. After 1 hour the excess of POCI 3 was removed under vacuum within ten minutes.
  • the N-hydroxy-succinimide ester of TAMRA (1 mg, 2 ⁇ mol) was dissolved in 200 ⁇ l of dry dimethylformamide and added to a solution of 1 mg (3 ⁇ mol) of 273'-EDA-6-Thio-Guanosine- Triphosphate in 500 ⁇ l of 10OmM sodium borate buffer (pH 8.5) at room temperature. After 2 hours, the reaction mixture was quenched with methanol. The TAMRA labelled product was obtained in 70% yield (2,4 mg, 1.4 ⁇ mol) after workup of the reaction mixture by reversed phase HPLC.
  • nucleoside 10 (0.1 g, 0.22 mmol) in 5 ml NH 3 /H 2 O (30%) was stirred for 90 minutes at 80 0 C. The solvent was removed in vacuo and the residue was dissolved in water (2 ml). N-2- (6"-Aminohexyl)-6-Thioguanosine (11) (0.80 g, 2.0 mmol, 91%) was obtained after purification of the crude product by column chromatography (silica gel RP-18, linear gradient from 100% water to 100% methanol).
  • N-2-(6"-Aminohexyl)-6-Thioguanosine (11) (0.091 g, 0.23 mmol) was dissolved in 1 ml DMF and the resulting solution was treated with 0.34 g (0.23 mmol) Triazole-1-carboxamidine hydrochloride at room temperature in the presence of 39 ⁇ l (0.23 mmol) N-Ethyl- diisopropylamine acting, as a supportive base. After 2 hours, the reaction mixture was evaporated to dryness.
  • nucleoside 9 (0.3 g, 0.6 mmol) was dissolved in 3 ml trimethylphosphate. The solution was cooled to 0 0 C and treated with 0.3 ml of Lutidine.
  • N-2-(6"-Aminohexyl)-6-Thioguanosine-5'-monophosphate (13) (0.053 g, 0.11 mmol) was dissolved in a mixture of 0.8 ml water and 0.5 ml DMF. The solution was subsequently treated with 0.16 g (0.11 mmol) Triazole-1-carboxamidine hydrochloride at room temperature in the presence of 18 ⁇ l (0.11 mmol) N-Ethyl-diisopropylamine, acting as a supportive base. After 16 hours, the reaction mixture was concentrated to dryness.
  • the N-hydroxy-succinimide ester of Boc/ffiu protected Aspartate (2.8 mg, 7 ⁇ mol) was dissolved in 200 ⁇ l of dry DMF and added to a solution of 1 (5 mg, 7 ⁇ mol) in 500 ⁇ l of 100 mM sodium borate buffer (pH 8.5) at room temperature. After 16 hours, the reaction mixture was quenched with methanol. The Aspartate derivative 4 was obtained in 70% yield (3.6 mg, 4.9 ⁇ mol) after workup of the reaction mixture by reversed phase HPLC.
  • the N-hydroxy-succinimide ester of Boc/fBu-protected Glutamate (2.9 mg, 7 ⁇ mol) was dissolved in 200 ⁇ l of dry DMF and added to a solution of 1 (5 mg, 7 ⁇ mol) in 500 ⁇ l of 100 mM sodium borate buffer (pH 8.5) at room temperature. After 16 hours, the reaction mixture was quenched with methanol. The Glutamate labeled product 5 was obtained in 70% yield (3.7 mg, 4.9 ⁇ mol) after workup of the reaction mixture by reversed phase HPLC.
  • the N-hydroxy-succinimide ester of Boc-protected Threonine (2.3 mg, 7 ⁇ mol) was dissolved in 200 ⁇ l of dry DMF and added to a solution of 1 (5 mg, 7 ⁇ mol) in 500 ⁇ l of 100 mM sodium borate buffer (pH 8.5) at room temperature. After 16 hours, the reaction mixture was quenched with methanol. The Threonine derivative 6 was obtained in 70% yield (3.4 mg, 4.9 ⁇ mol) after workup of the reaction mixture by reversed phase HPLC.
  • EXAMPLE 5 Analysis of the ability of 5 new synthesized 6-Thio-GTP-derivatives to induce apoptosis in human CD4* T Lymphocytes.
  • V3 and V4 were not soluble in water, therefore V3 was reconstituted with ethanol and V4 was reconstituted with methanol.
  • PBMC Human peripheral blood mononuclear cells from 4 buffycoats were isolated using Ficoll-Hypaque gradients. PBMC were further purified using CD4 monoclonal antibodies attached to immunomagnetic microbeads according to the protocol provided by the manufacturer (Miltenyi Biotec). T lymphocytes were stimulated in complete RPMI-1640 medium (RPMI-1640 + 10% FCS + 100 U/ml Penicillin/ Streptomycin + 3mM L-Glutamin) for 3, 4 or 5 days with coated antibodies to CD3 (0,04 ⁇ g/ml) and soluble CD28 antibodies (PharMingen; 1 Dg/ ml) plus IL-2 (R & D Systems, Wiesbaden, Germany; 40 U/ml).
  • apoptotic cells were detected by staining with annexin V and propidium iodide using the Annexin V FITC Apoptosis Detection Kit I (PharMingen).
  • Annexin V FITC Apoptosis Detection Kit I PharMingen
  • T cells were washed twice in PBS, and the pellet was resuspended in annexin V binding buffer (PharMingen) at a concentration of 10 6 cells per milliliter.
  • Annexin V FITC and propidium iodide were added (5 ⁇ l of each per 10 5 cells). Samples were gently mixed and incubated for 15 minutes at room temperature in the dark before FACS analysis.
  • Annexin-positive, propidium iodide-negative cells black bars present the rate of early apoptotic cells.
  • Annexin-positive, propidium iodide-positive cells white bars present late apoptotic or necrotic cells.
  • V1 and V5 were able to induce apoptosis in CD3/CD28 costimulated T Lymphocytes.
  • V2, V3 and V4 were not able to induce apoptosis. Comparing V1 and V5 mediated induction of apoptosis with azathioprine or 6-mercaptopurine mediated induction of apoptosis, V1 and even V5 mediated effects seemed to be more pronounced and appeared earlier ( Figures 2-4).
  • the dry tributylammonium salt of 6-Thio-GTP 1 (0.2mmol) was treated with 200 mg carbonyl- diimidazole in 4 ml dimethylformamide. The resulting mixture was stirred for 6 hours at 0° C, brought to room temperature and, subsequently, 80 ⁇ l of methanol were added. After 10 minutes, 2 mmol of the appropriate methyleno-amine and 2 ml of triethylamine were also added to the reaction mixture. The solution was stirred overnight at room temperature and the solvent was then removed under reduced pressure. The residue was taken up in 30 ml water and the mixture was adjusted to pH 1, in order to decompose the resulting intermediate phosphoramidate at the triphosphate moiety. After 20 minutes, the solution was adjusted to pH 7.5, the precipitate filtered off and the solvent was removed in vacuo. The resulting crude product was purified by ion exchange chromatography and subsequently by reversed phase HPLC.
  • the dry tributylammonium salt of compound 1 (0.2mmol) was treated with 200 mg carbonyl- diimidazole in 4 ml dimethylformamide. The resulting mixture was stirred for 6 hours at 0° C, brought to room temperature and, subsequently, 80 ⁇ l methanol were added. After 10 minutes, 2 mmol of the appropriate amine and 2 ml of 1 M potassium hexamethyldisilazide (KHMDS) in THF were carefully added to the solution. The solution was stirred at room temperature for 1 hour and the solvent was then removed under reduced pressure. The residue was taken up in 30 ml water and the mixture was adjusted to pH 1 , in order to decompose the resulting intermediate phosphoramidate at the triphosphate moiety. After 20 minutes, the solution was adjusted to pH 7.5, the precipitate filtered off and the solvent was removed in vacuo. The resulting crude product was purified by ion exchange chromatography and subsequently by reversed phase HPLC.
  • Example 8 In relation to figures 5 and 6, an overview of at least three experimental data sets on apoptosis is shown below. Here, some of the D compounds were able to induce apoptosis. BO was the strongest candidate drug for apoptosis induction. Two issues should be considered in these results.
  • Negative induction of apoptosis means that there were more apoptotic cells in the untreated group than in the treated group. This phenomenon may appear from time to time and may be explained by a kind of statistical variance.
  • 6-Thio-GTP should be a positive control to induce apoptosis in T cells.
  • the positive control did not work very well. This might be explained by the fact that the experiments are often preformed with primary T cells, which are freshly isolated from blood of different donors. It is well known, that some people are not sensitive for azathioprine therapy. In this way, T cells of some donors might be resistant against 6-Thio-GTP induced apoptosis. In any case, however, BO and some of the D compunds were able to induce apoptosis suggesting that they are candidate drugs.
  • the Caspase-Glo 3/7 Assay (PromegaTM) was used. This assay is based on a caspase dependent luminescent signal. Protocol: CD4 + T cells were isolated from human blood by magnetic beads (Dynal). T lymphocytes were stimulated in complete RPMI-1640 medium (RPMI-1640 + 10% FCS + 100 U/ml Penicillin/ Streptomycin + 3mM L-Glutamin) for 3 days with coated antibodies to CD3 (0,04 ⁇ g/ml) and soluble CD28 antibodies (PharMingenTM; 1 ⁇ g/ ml) plus IL-2 (R & D SystemsTM, Wiesbaden, Germany; 40 U/ml) in 96-well plates.
  • RPMI-1640 + 10% FCS + 100 U/ml Penicillin/ Streptomycin + 3mM L-Glutamin coated antibodies to CD3 (0,04 ⁇ g/ml) and soluble CD28 antibodies (PharMingenTM; 1 ⁇ g/ ml
  • CD28 costimulation can promote T cell survival by enhancing the expression of bcl-x L . Immunity 1995; 3: 87-98.

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Abstract

The invention relates to analogous compounds of 6-thioguanosine triphosphate of general formula (I). A compound of the general formula (I); wherein the dashed bond in the sugar moiety can be either single or double and wherein R1 , R2, R3, R4 or R5, equal or different between each other, have general formula -(Int)m-Ter, wherein m is between 0 and 12 and lnt and Ter are Internal and Terminal building blocks, wherein lnt is selected from the group consisting of formula (II); and Ter is selected from the group consisting of formula (III). And wherein X represents either carbon or nitrogen atom within aromatic ring, Y represents either oxygen or sulphur atom and an additional group Q, group Qi or groups Qi (Qi indicates that the group or several groups may be bound to any unsaturated moiety of the ring) are selected from the group consisting of -OH, -COOH, -N(CH3)2, -N(CH2-CH3)2| -CO-CH3, -CO-O-CH3, -O-CH3, -S-CH3,-SO2-CH3, -CN, -NO2 or -Halogen elements, and wherein R5 may be formula (IV) and metal and ammonium salts thereof, wherein n is between O and 5, or oxygen or phosphorus is partially or completely replaced by nitrogen, sulphur, methyleno groups or their derivatives. The invention also concerns the uses of the above mentioned compounds in medical field and the process for their preparation.

Description

IMPROVEMENTS TO ANALOGOUS COMPOUNDS OF 6-THIOGUANOSINE TRIPHOSPHATE, THEIR USE IN MEDICAL FIELDS AND PROCESSES FOR THEIR PREPARATION.
The present invention relates to analogous compounds of 6-thioguanosine triphosphate, their use in medical field and process for their preparation.
Particularly, the invention refers to the therapeutic use of analogous compounds of 6- thioguanosine triphosphate for example as immunosuppressant for the prevention of rejection of organ transplants and of post-transplant nephropathy and in the treatment of pathologies in which immune system is involved, such as, for instance, inflammatory chronic intestinal diseases, such as Crohn's disease, ulcerous rectocolitis, indeterminate colitis, or of autoimmune enteropathy, active chronic hepatitis, rheumatoid arthritis, Still's disease, systemic lupus erythematous, acquired haemolytic anaemia, idiopathic thrombocytopenia, polyarthritis nodosa, vasculitis, polyangitis, polymyositis, myasthenia gravis, sarcoidosis, lipoid nephritis, multiple sclerosis, dermatomyositis, pemphigus vulgaris, primary biliary cirrhosis, primary sclerosing cholangitis, recurrent multiform erythema, chronic actinic dermatitis, gangrenous hypoderm, ptyriasis rubra, Wegener's granulomatosis, cutaneous vasculitis, atopic dermatitis, psoriasis, pimply pemphigoid and, in general, in the immunosuppressive treatment in addition to radiotherapy, corticosteroids and other cytotoxic agents. The latter also involves immunosuppressive therapy after organ transplantation (e.g. kidney, heart, lung, pancreas and liver transplantation).
The cells involved in the inflammatory immune response are able to survive at the inflammatory site, however, after completion of such response, the majority of cells must "die" to maintain the homeostasis of organism (Boise, 1995). Since the uncontrolled lymphocyte proliferation may cause the development of inflammatory chronic pathologies, the immune system controls the depletion of activated lymphocytes by a process named apoptosis (programmed cell death). This would assume a particular importance for the immune system of the mucosa, since the apoptosis resistance of lamina propria cells can lead to a chronic inflammatory response at the intestinal level (Tiede, 2003).
The activation of the mucosal immune system plays a key role in the pathogenesis of Crohn's disease. Particularly, pro-inflammatory cytokines produced by T lymphocytes and macrophages, in particular interleukine-6 (IL-6) and interleukine-12 (IL-12), may cause T lymphocytes resistance against apoptosis, which in its turn provokes an intestinal accumulation of lymphocytes and establishes a long-lasting disease (Tiede, 2003).
The lymphocytes activation starts with two signals: the specific binding of antigens to the TCR (T cell receptor) and a second co-stimulatory signal represented by transmembrane proteins, such as CD28 (Maltzman, 2003). It has been shown that co-stimulation with CD28 enhances in vitro survival of activated T lymphocytes; in fact, CD28 induces an enhanced production of interleukine-2 (IL-2) acting as extrinsic factor for T lymphocytes survival, and the intrinsic ability of T lymphocytes to be resistant against apoptosis (Boise, 1995 bis). This occurs since CD28 action is associated with the expression of an anti-apoptotic gene, named bcl-xL gene (Khoshnan, 2000; Noel, 1996).
The steps through which an inhibition of apoptosis takes place, will be synthesized as follows, as shown in Figure 1 :
-CD28 acts through its cytoplasmaic portion with a complex of "adaptor" proteins and with a molecule, named Vav (Frauwirth, 2002);
-Vav acts as guanosine nucleotide exchange factor (GEF) for another molecule named Rac1 (Frauwirth, 2002);
-Rac1 , a small GTPase, in such a way switches between an inactive state bound to GDP and an active state bound to GTP (Frauwirth, 2002);
-Activated Rad , in its turn, leads to the activation of kinases (IKK) that phosphorylate the NF-κB inhibitory proteins (like l-κB alfa) (Marinari, 2002) through MEK phosphorylation;
-Thus, NF-κB is not anymore retained in an inactive form in the cytosol but is able to translocate to nucleus where induces bcl-xL expression (Khoshnan, 2000);
-Further, activated Rad stimulates the activation of a protein belonging to the STAT family (Signal Transducers and Activators of Transcription), that is STAT-3, thus inducing its translocation to the nucleus and the corresponding expression of STAT-3 dependent genes (Faruqi, 2001). In particular, STAT-3 induces bcl-xL expression thus contributing to the resistance against apoptosis and to the accumulation of T lymphocytes in the inflamed mucosa during the course of Crohn's disease (Mudter, 2003). On the other hand, the study of intestinal T lymphocytes has pointed out that STAT-3 is steadily activated in patients with Crohn's disease, but not in healthy voluntaries (Lovato 2003).
Rad , together with RhoA and Cdc42, belongs to the Rho family which is a superfamily of small G protein characterized in that they are able to bind guanosine nucleotides and to regulate many cellular responses. They cycle between an inactive state, when bound to GDP, to an active state with GTP in place of GDP. This reaction is sustained upon guanosine nucleotide exchange factors named GEFs (like Vav). The binding with GTP induces a conformational change, which allows Rad and other GTPases to bind to their effectors. The action of other proteins called GTPase-activating protein (GAPs) stimulates the innate GTPase activity of these small G proteins and causes them to turn back to their GDP bound inactive state. Rad , as in general all GTPase belonging to the Rho family, plays an important role in the mitogenesis processes, proliferation, and invasivity, since it stimulates alterations of the gene expression, in the present case of the gene bci-xL, modulating the activity of transcription factors, such as, in the present case, NF-κB and STAT-3 (Van Aelst, 1997).
Azathioprine is regarded as "gold standard" of the immunosuppressive therapy of Crohn's disease, also if the mechanism of action of such active principle is still unknown. However, the inhibition of the purine nucleotide biosynthesis with suppression of DNA and RNA synthesis and downregulation of T and B lymphocytes function (Tiede 2003), is assumed to be the main therapeutic mechanism of azathioprine.
Recently, a new mechanism of action of azathioprine acting at the T lymphocytes level has been shown. After the evidence that azathioprine induces in vitro apoptosis of activated T lymphocytes and that treatment with azathioprine causes apoptosis of both circulating and lamina propria T lymphocytes of IBD patients, the specific molecular mechanisms were pointed out (Tiede 2003).
The key point is represented by the metabolite 6-thioguanosine triphosphate (6-thioGTP) which represents the real functional metabolite of the drug. Specifically, 6-thioGPT binds directly to
Rac1 in place of GTP, thus blocking its activation. Such a block is highly specific for Rad , as other GTPases belonging to the same family are not inhibited by 6-thioGTP, and this specificity suggests that the block would be correlated to the structure of Rad protein. The fact that an accumulation of the Rad guanosine nucleotide exchange factor vav was observed, is consistent with a compensatory mechanism to achieve Rad activation.
The block of the activation of Rad would result in the block of NF-κB and STAT-3, normally induced by Rad itself, and thus in the block of the bcl-xL gene expression, detected both at the mRNA and protein levels. Thus, azathioprine, by modulating Rad activity switches an anti- apoptotic co-stimulatory signal, mediated by CD28, into a pro-apoptotic signal.
This new mechanism of action is able to explain the well-known "delay" in the therapeutic effect of azathioprine, which needs a long treatment time to elicit a clinical response in such a way that benefits and clinical response were not observed earlier than 4 months of therapy. This is due to the fact that 6-thioGPT has 20-fold less affinity to Rad in comparison with GTP, which is normally bound to Rad. Therefore, the azathioprine treatment needs the simultaneous and prolonged administration with high steroids doses, having remarkable side effects such as osteoporosis, diabetes, cataract.
In the light of the above it would be desirable to have at disposal new immunosuppressive drugs eliciting a faster and more efficacious therapeutic response in comparison with the already known compounds. According to the present invention, a new class of 6-thioGTP analogous drugs able to inhibit Rad and characterized by greater affinity to Rad , an higher suppression of Rac1 activity and, thus having a greater immunosuppressive power and action in comparison with the delayed effect of azathioprine treatment and a best therapeutic effect, was prepared.
It is therefore an object of the present invention to provide a class of analogous compounds of 6-thioguanosine triphosphate of general formula (I):
wherein the dashed bond in the sugar moiety can be either single or double and wherein R1, R2, R3, R4 or R5, equal or different between each other, have general formula -{Int)m-Ter, wherein m is between 0 and 12 and lnt and Terare Internal and Terminal building blocks, wherein lnt is selected from the group consisting of
and Ter is selected from the group consisting of
wherein X represents either carbon or nitrogen atom within aromatic ring, Y represents either oxygen or sulphur atom and an additional group Q, group Qi or groups Qi (Qi indicates that the group or several groups may be bound to any unsaturated moiety of the ring) are selected from the group consisting of -OH, -COOH, -N(CH3)2, -N(CH2-CH3)2, -CO-CH3, -CO-O-CH3, -0-CH3, -S-CH31-SO2-CH3, , -CN, -NO2 or -Halogen elements.
Alternatively R5 may be
and metal and ammonium salts thereof, wherein n is between O and 5, or oxygen or phosphorus is partially or completely replaced by nitrogen, sulphur, methylene groups or their derivatives.
In some embodiments (particularly, but not limited to those where R5 is HO-[PO3Jn, and metal and ammonium salts thereof, wherein n is between O and 5, or oxygen or phosphorus is partially or completely replaced by nitrogen, sulphur, methylene goups or their derivatives) TER may be selected from the group consisting of:
Compounds of formula (I) can be labelled, particularly with R3 or R4 selected from
wherein Q is selected from -OH (FAM) or -N(CH3)2 (TAMRA). In addition the sugar moiety of compounds of formula (I) can be selected from the group consisting of the following sugar moieties or sugar-like moieties:
Compounds of the invention may be of the general formula (II) derived from general formula (Ia) and/or (I) where R1 is [-SH] (it is understood that the Guanosine moiety may undergo keto-enol tautomeric shifts and so give rise to [=S]), R2 is [-H], R5 is [-(PO3)n-OH] and one of R3 and R4 is [-OH] and the other of R3 and R4 is [-O-CO-NH-lntm-Ter] (both versions are provided in the same mixture, i.e., [-O-CO-NH -Intm-Ter]: will be the same, but a proportion of the molecules will have [-O-CO-NH- -Intm-Ter]: attached at R3 with R4 being OH, and another proportion of the molecules will have [-O-CO-NH-lntm-Ter]: attached at R4 with R3 being OH):
wherein n=1, 2 or 3, m is between O and 5, lnt is selected from the group consisting of
and Ter is selected from the consisting of
wherein X represents either carbon or nitrogen atom within aromatic ring, Y represents either oxygen or sulphur atom and an additional group Q or groups Q/ (/ indicating the position of any unsaturated moiety of the ring to which the group Q may be bound) are selected from the group consisting of -CH3, -C(CH3)3l -OH, -COOH, -CO-CH3, -CO-O-CH3, -0-CH3, -S-CH31-SO2-CH3, -N(CHs)2, -N(CH2-CHs)2, -CN, -NO2 or -Halogen elements.
In some embodiments of the invention of formula (I), (Ia) and (II), Ter is selected from the group consisting of
According to some embodiments of the present invention the compounds of formula (I) are the compounds described below:
wherein R-i, R2, R3, R4 or R5, equal or different between each other, have general formula - (Int)m-Ter, wherein m is between O and 12 and lnt and fer are Internal and Terminal building blocks, wherein lnt is selected from the consisting of
and Ter is selected from the consisting of
wherein an additional group Q, group Qi or groups Qi (Qi indicates that the group or several groups may be bound to any unsaturated moiety of the ring) are selected from the group consisting of -OH, -COOH, -N(CH3)2, -N(CH2-CH3)2 or -Halogen elements
In addition the sugar moiety of compounds of formula (I) can be selected from the group consisting of the following sugar moieties or sugar-like moieties:
Compounds of formula (I), (Ia) and (II) can be labelled, particularly with R3 or R4 selected from
wherein Q is selected from -OH (FAM) or -N(CH3)2 (TAMRA).
According to some embodiments of the present invention the compounds of formula (I), (Ia) and (II) are the compounds described below:
',3J-EDA-6-Thio-GTP, ID: 05B-0
FAM-2', 3'-EDA-6-Thio-GTP
TAMRA-2',3'-EDA-6-Thio-GTP
O
Aspartate-2',3'-EDA-6-Thio-GTP, ID: 05B-1
Glutamate-2',3'-EDA-6-Thio-GTP, ID: 05B-2
Threonine-2',3'-EDA-6-Thio-GTP, ID: 05B-3
Serine-2',3'-EDA-6-Thio-GTP
',3',5',O-Triacetyl-N-2-(Acetyl-6"-aminohexyl)-guanosine
',3'15'-Triacetyl-N-2-(6"-thioacetamide-hexyl)-6-Thioguanosine (V4) [TW1 107/7]
N-2-(6"-thioacetamide-hexyl)-6-Thioguanosine
N-2-(6"-Aminohexyl)-6-Thioguanosine, ID: 05A-0
N-2-(6"-guanidino-hexyl)-6-Thioguanosine, ID: 05A-1
N-2-(6"-Aminohexyl)-6-Thio-GMP
N-2-(6">guanidino-hexyl)-6-Thio-GMP, ID: 05A-2
N-2-(6"-Aminohexyl)-6-Thio-GTP
N-2-(6"-guanidino-hexyl)-6-Thio-GTP, ID: 05A-3
N-2-(6"-Aspartate-hexyl)-6-Thioguanosine
N-2-(6"-Glutamate-hexyl)-6-Thioguanosine
N-2-(6"-Threonine-hexyl)-6-Thioguanosine
N-2-(6"-Serine-hexyl)-6-Thioguanosine
N-2-(6"Αminobutyl)-6-Thio-Guanosine, ID: 05C-0
- N-2- (6"-guanidino-butyl)-6-Thioguanosine, ID: 05C-1
- N-2- (6"-guanidino-butyl)-6-Thio-GMP, ID: 05C-2
2- Λ/-2- (6"-guamdmo-butyl)-6-Thio-GTP, ID: 05C-3
N-2-(6"-Aspartate-butyl)-6-Thioguanosine
N-2-(6"-Glutamate-butyl)-6-Thioguanosine
N-2-(6"-Threonine-butyl)-6-Thioguanosine
N-2-(6"-Serine-butyl)-6-Thioguanosine
N-2-(6"-Aminopropyl)-6-Thioguanosine
N-2-(6"-guanidino-propyl)-6-Thioguanosine
N-2-(6"-Aspartate-propyl)-6-Thioguanosine
N-2-(6"-Glutamate-propyl)-6-Thioguanosine
N-2-(6"-Threonine-propyl)-6-Thioguanosine
N-2-(6"-Serine-propyl)-6-Thioguanosine
N-2-(6"-Amino-2-butene)-6-Thioguanosine
N-2-(6"-guanidino-2-butene)-6-Thioguanosine
N-2-(6"-Aspartate-2-butene)-6-Thioguanosine
N-2-(6"-Glutamate-2-butene)-6-Thioguanosine
N-2-(6"-Threonine-2-butene)-6-Thioguanosine
N-2-(6"-Serine-2-butene)-6-Thioguanosine
N-2-(6"-Amino-2-butyne)-6-Thioguanosine
N-2-(6"-guanidino-2-butyπe)-6-Thioguanosine
N-2-(6"-Aspartate-2-butyne)-6-Thioguanosine
N-2-(6"-Glutamate-2-butyne)-6-Thioguanosine
N-2-(6"-Threonine-2-butyne)-6-Thioguanosine
N-2-(6"-Serine-2-butyne)-6-Thioguanosine
N-2-(6"-Amino-2,4-hexadiyne)-6-Thioguanosine
N-2-(6"-guanidino-2,4-hexadiyne)-6-Thioguanosine
N-2-(6"-Aspartate-2,4-hexadiyne)-6-Thioguanosine
N-2-(6"-Glutamate-2,4-hexadiyne)-6-Thioguanosine
N-2-(6"-Threonine-2,4-hexadiyne)-6-Thioguanosine
N-2-(6"-Serine-2,4-hexadiyne)-6-Thioguanosine
Further compounds of the invention are disclosed, with reference titles indicated. 06D-1
06D-2
6D-3
D-10
D-11
D-12
06D-13
06D-14
06D-15
06D-16
6D-17
6D-18
6D-22
6D-23
6D-24
06D-25
06D-26
06D-27
06D-28
6D-29
6D-30
06D-31
6D-32
6D-33
Compounds according to the present invention, can be advantageously used in medical field; therefore another object of the present invention are pharmaceutical composition comprising at least one of the compounds of the above mentioned formula (I) as active principle and one or more pharmaceutically acceptable co-adjuvants or excipients, that are known to those skilled in the art and currently in use in the pharmaceutical technology.
It is an object of the invention to provide for compounds for the preparation of an immunosuppressive drug,. Its uses and therapeutical and medical uses thereof, for It is an object of the present invention to provide the compounds and compositions of the invention, and their uses, in the following methods of treatment, and/or therapy; the prevention of rejection of organ transplants (e.g. kidney, heart, lung, pancreas, liver transplantation) and of post- transplant nephropathy and in the treatment of pathologies in which immune system is involved, such as, for instance, inflammatory chronic intestinal diseases, such as Crohn's disease, ulcerous rectocolitis, indeterminate colitis, or auto-immune enteropathy, active chronic hepatitis, rheumatoid arthritis, Still's disease, systemic lupus erythematous, acquired haemolytic anaemia, idiopathic thrombocytopenia, polyarthritis nodosa, vasculitis, polyangitis, polymyositis, myasthenia gravis, sarcoidosis, lipoid nephritis, multiple sclerosis, dermatomyositis, pemphigus vulgaris, primary biliary cirrhosis, primary sclerosing cholangitis, recurrent multiform erythema, chronic actinic dermatitis, gangrenous hypoderm, ptyriasis rubra, Wegener's granulomatosis, cutaneous vasculitis, atopic dermatitis, psoriasis, pimply pemphigoid and, in general, in the immunosuppressive treatment in addition to radiotherapy, corticosteroids and other cytotoxic agents. In addition, the compounds according to the present invention can be advantageously used for the preparation of a medicament for the treatment of cancer.
The present invention further relates to the use of labelled compounds of formula (I), particularly with R3 or R4 selected from
wherein Q is selected from -OH (FAM) or -N(CH3)2 (TAMRA) as probes for the evaluation of the binding properties of the compounds of formula (I) by the Racl/Vav system. According to a further aspect, the present invention refers to a process for the preparation of compounds of formula (I), (Ia) and (II), wherein the introduction of the -NH-R group at the 2 position of guanosine ring comprises the following steps:
a) protection of the NH moiety of tri-O-acetyl-inosine;
b) oxidative guanosine ring-opening and O-deprotection;
c) guanosine ring-closing and introduction of a SH group at the 2 position of the guanosine ring through the use of CS2;
d) replacing the SH group at the 2 position with an amino-linker by using an excess of an aliphatic diamine. The process may further comprise an additional step e) of protection of ribose OH groups and of the primary amine group by acetylation, and, moreover, an additional step f) of thiolation of C=O groups through the use of Lawesson's reagent.
The present invention will be now described, for illustrative but not limitative purposes, according to its preferred embodiments, with particular reference to the Figures of the enclosed drawings.
Descripton of Drawings
Figure 1 shows the scheme of the mechanism of action of the apoptosis inhibition and of the action of azathioprine.
Figure 2 shows the apoptosis induction in human CD4+ T Lymphocytes by V1=BMB20= EDA-6- Thio-GTP, V2= TWI 35/1= N-2-(6"-Aminohexyl)-guanosine, V3 = TWI 71/2 = 2',3',5',o-Triacetyl-
N-2-(Acetyl-6"-aminohexyl)-guanosine, V4 = TWI 107/7 = 2',3',5'-Triacetyl -N-2-(6"- thioacetamide-hexyl)-6-Thioguanosine, V5 = BMB = TAM RA-E DA-6-Thio-GTPafter 3 days.
Figure 3 shows the apoptosis induction in human CD4+ T Lymphocytes by V1=BMB20= EDA-6- Thio-GTP, V2= TWI 35/1= N-2-(6"-Aminohexyl)-guanosine, V3 = TWI 71/2 = 2',3',5\o-Triacetyl- N-2-(Acetyl-6"-aminohexyl)-guanosine, V4 = TWI 107/7 = 2',3',5'-Triacetyl -N-2-(6"- thioacetamide-hexyl)-6-Thioguanosine, V5 = BMB = TAMRA-EDA-6-Thio-GTPafter 4 days.
Figure 4 shows the apoptosis induction in human CD4+ T Lymphocytes by V1=BMB20= EDA-6- Thio-GTP, V2= TWI 35/1= N-2-(6"-Aminohexyl)-guanosine, V3 = TWI 71/2 = 2'r3',5',o-Triacetyl- N-2-(Acetyl-6"-aminohexyl)-guanosine, V4 = TWI 107/7 = 2',3\5'-Triacetyl -N-2-(6"- thioacetamide-hexyl)-6-Thioguanosine, V5 = BMB = TAMRA-EDA-6-Thio-GTPafter 5 days.
Figure 5: Induction of apoptosis. Buffy stands for "buffy coat" which is the fraction of blood obtainable by centrifugation and containing leukocytes and platelets. In this case buffy coats were used to isolate monocytes for the experiments. "Buffy 1 vom 08/03/05" means "Buffy coat 1 of march 8th, 2005". V1 and V3 have been tested 4 times in four independent experiments, while V2 and V5 were tested twice in two separate experiments. For instance: V1 was tested twice on 08/03/2005 (where 08/03/2004 is written this is in error and should read 08/03/2005) and twice on 22/04/2005
Figure 6: Results of biological activity of compounds compiled in Figure 5.
Figure 7: list of further compounds of the invention, as well as providing key to some of the shorthand used in identifying some of the molecules.lt will be noted that some compounds have different enantiomeric forms, and the representations in Figure 7 may show an alternative enantiomeric form than that discussed in the text.
Figure 8: Caspase-graph: Luminescence value of untreated cells was defined as 100% Caspase activity. Due to their specific luminescence value the caspase activity of treated cells were calculated accordingly. Induction of Caspase activityx [%]=Caspase activityx[%] - Caspase activityuπtreated[%]- Means ± SEM (Standart Error of the Mean= Standard deviation/radical(n)) of three different experiments are presented. Summary of this graph: 6-thio- GTP was able to induce caspase activity (as positive control). 05B-0 was also very effective. 05B-1 was less effective than 05B-0 in agreement with the previous data on AnnexinV/PI- staining. Concerning Group-D derivatives 06D-13, 06D-14 and to a lesser extent 06D-22 were promising.
EXAMPLE 1 : Process for the preparation of 2-substituted-6-thio-guanosine nucleotides.
Preparation of 2',3',5'-Tri-O-acetyl-1-[(2-methoxyethoxy)methyl] inosine
(Kohyoma et al., 2003)
lnosine-2',3',5'-triacetate (4g, 10.2mmol) was dissolved in 100 ml dichloromethane and the solution was treated with 1 ,4 ml (12.2 mmol) 2-methoxyethoxymethylchloride at O0C in the presence of diisopropylethylamine as a supportive base. After 1 hour the reaction was quenched with water. The solution was stirred for 30 min and chloroform was added afterwards. The aqueous layer was extracted with chloroform and the combined and washed organic layers were concentrated to dryness. The reaction yielded 3.93 g (8.2 mmol, 80%) of 2', 3',5'-Tn-O- acetyl-1-[(2-methoxyethoxy)methyl] inosine after chromatographic purification (silica gel, EtOAc - MeOH, 50:1).
Preparation of 5-Amino-1-β-ribofuranosylimidazole-4-carboxamide (Kohyoma et al., 2003)
Aqueous ammonia solution (28%, 20 ml) was added to a solution of 2',3',5'-Tri-O-acetyl-1-[(2- methoxyethoxy)methyl] inosine (3,5 g, 7.3 mmol) in 50 ml methanol. The reaction mixture was stirred for 1 hour at room temperature and concentrated, yielding the deprotected nucleoside (2,44 g, 6.9 mmol, 95%). This product was used in the next step without further purification.
The nucleoside (2,44 g, 6.9 mmol) was refluxed with 50 ml aqueous sodium hydroxide (0.2 M) for 1 hour, cooled to room temperature, neutralized with HCI (6 M) and evaporated to dryness. The residue was dissolved in ethanol, filtered from insoluble material and concentrated to dryness. Purification of the crude product by column chromatography (silica gel, CHCI3:MeOH, 3:1) gave 1 ,32 g (5.11 mmol, 70%) of 5-Amino-1-β-ribofuranosylimidazole-4-carboxamide.
Preparation of 2-Mercaptoinosine (Imai et al., 1971)
The 5-Amino-1-β-ribofuranosylimidazole-4-carboxamide (1,3 g, 5 mmol) was dissolved in pyridine and 18ml (15 mmol) phenyl-isothiocyanate were added slowly. The reaction mixture was refluxed for 2 hours under argon atmosphere. The solution was cooled to room temperature, the precipitate collected by filtration and washed with diethyl ether. The obtained pyridinium salt of the product was dissolved in aqueous sodium hydroxide (15%, 40 ml). The solution was heated 30 minutes at 600C and concentrated under vacuum. Methanol was then added and the solution was kept in the refrigerator overnight. After 20 hours the precipitated colourless prisms were collected by filtration to give 1,2 g (80%) 2-Mercaptoinosine. According to an alternative method of preparation, compound-Amino-1-β- ribofuranosylimidazole-4-carboxamid (1 ,3 g, 5 mmol) was added to a solution of sodium hydroxide (1 g, 25 mmol) in 20ml of methanol at 300C. Carbon disulfide (1 ,9 g, 25 mmol) was added, and the solution was heated in an autoclave at 18O0C for 3 hours. The mixture was cooled to room temperature, the precipitate was filtered off, washed with cold methanol and recrystallized from water to yield 1,1 g (3.8 mmol, 75%) 2-Mercaptoinosine.
Preparation of N-2-(6"-aminohexyl)-Guanosine
2-Mercaptoinosine (1 g, 3 mmol) was dissolved in 100 ml water and cooled to O0C. A clear solution was obtained after ultrasonic irradiation. Hydrogen peroxide (1 ml, 9 mmol) was added within 20 min under vigorous stirring. After 1 hour of stirring at O0C, HPLC chromatography indicated that the starting compound has been completely oxidized to the sodium salt of lnosine-2-sulfonic acid. Without further purification, the resulting solution was treated with an excess of 1 ,6-diaminohexane (20 g, 200 mmol). The mixture was refluxed for 2.5 hours at 1550C. The excess of 1 ,6-diaminohexane was removed by vacuum distillation to give an orange residue. N-2-(6"-Aminohexyl)-guanosine (0,85 g, 2.2 mmol, 50%) was obtained after purification of the crude product by column chromatography (silica gel RP-18, linear gradient from 100% water to 100% methanol). Preparation of 2',3',5'-triacetyl-N-2-(6"-acetamide-hexyl)- guanosine (Ostermann et al.,1999)
A solution of nucleoside (220 mg, 0.575 mmol) in 20 ml dry pyridine was stirred with 1ml (lOmmol) acetic anhydride at room temperature for 15 hours under argon atmosphere. The solvent was removed under vacuum and the residue dissolved in a mixture of CHCI3 (10 ml) and CH3OH (2 ml). This solution was loaded on silica gel and eluted with CHCI3 / CH3OH (5:1) to give 284,7 mg (0.52 mmol, 90%) of fully protected nucleoside.
Preparation of 2',3\5'-triacetyl-N-2-(6"-thioacetamido-hexyl)-6-thioguanosine
Dioxane (200ml) was added to the fully protected nucleoside (5,03 g, 9,13 mmol). After addition of 8 g (19.8 mmol) of Lawesson's reagent the suspension was vigorously stirred for 2 hours at 8O0C. The initially opaque reaction mixture became clear after 10 minutes. The solution was cooled at room temperature and the solvent was evaporated by vacuum distillation. Purification of the raw product by column chromatography (silica gel, CHCI3), resulted in 2',3',5'-triacetyl-N- 2-(6"-thioacetamido-hexyl)-6-thioguanosine (2,92 g, 5,02mmol) in 55% yield.
EXAMPLE 2: Process for the preparation of analogous of ribose-modified 6-thio-guanosine- triphosphate.
Preparation of 6-Thio-Guanosine-Triphosphate (Ludwig, 1981)
Under argon atmosphere, 6-thio-guanosine (1 g, 3.34 mmol) was dissolved in 6 ml trimethylphosphate. The solution was cooled to O0C and 1,3 ml of Lutidine were added. After 10 minutes, 0,4 ml (4.4 mmol) phosphorous oxychloride was carefully added to the solution. After 1 hour the excess of POCI3 was removed under vacuum within ten minutes.
The solution of the initially formed intermediate dichlorophosphate was then treated with a solution of tri-n-butyl ammonium pyrophosphate (17 ml, 100 mM) in dimethylformamide. After 2 minutes, the reaction was quenched by adding 100 ml of 0.25 M triethylammonium bicarbonate buffer. Purification by ion exchange chromatography gave 6-Thio-Guanosine-Triphosphate (0,5 g, 1 mmol, 30%).
Preparation of 273'-EDA-6-Thio-Guanosine-triphosphate
The dry tributylammonium salt of compound (0.5 mmol) was treated with 500 mg carbonyldiimidazole in 25 ml dimethylformamide. The resulting mixture was stirred for 6 hours at 0° C, brought to room temperature and 0,2 ml methanol and subsequently 0,3 ml ethylene diamine were added.
The resulting precipitate was centrifuged down and dissolved in water. The solution was adjusted to pH 2, in order to decompose the resulting intermediate phosphoramidate at the triphosphate moiety. After 18 hours, pH was adjusted to 7.5 and the solvent was subsequently removed under reduced pressure. Purification of the crude product by ion exchange chromatography gave 116 mg (0.4 mmol, 80%) 2V3'-EDA-6-Thio-Guanosine-triphosphate.
Preparation of TAMRA-2V3'-EDA-6-Thio-Guanosine-Triphosphate
The N-hydroxy-succinimide ester of TAMRA (1 mg, 2 μmol) was dissolved in 200μl of dry dimethylformamide and added to a solution of 1 mg (3 μmol) of 273'-EDA-6-Thio-Guanosine- Triphosphate in 500μl of 10OmM sodium borate buffer (pH 8.5) at room temperature. After 2 hours, the reaction mixture was quenched with methanol. The TAMRA labelled product was obtained in 70% yield (2,4 mg, 1.4 μmol) after workup of the reaction mixture by reversed phase HPLC.
EXAMPLE 3: Synthesis of 2-substituted 6-Thio-Guanosine nucleotides
Preparation of N-2-(6"-Thioacetamido-hexyl)-6-Thioguanosine (10)
A, 7 M solution of ammonia in methanol (70 ml) was added to the fully protected nucleoside 9 (1.15 g, 1.97 mmol). The solution was stirred at room temperature for 20 hours. The solvent was removed by distillation to give an off white residue. The crude N-2-(6"-thioacetamido-hexyl)-6- Thioguanosine (10) (0.85 g, 1.8 mmol, 95%) was used without further purification.
Preparation of N-2-(6"-Aminohexyl)-6-Thioguanosine (11)
10 11
A solution of nucleoside 10 (0.1 g, 0.22 mmol) in 5 ml NH3/H2O (30%) was stirred for 90 minutes at 800C. The solvent was removed in vacuo and the residue was dissolved in water (2 ml). N-2- (6"-Aminohexyl)-6-Thioguanosine (11) (0.80 g, 2.0 mmol, 91%) was obtained after purification of the crude product by column chromatography (silica gel RP-18, linear gradient from 100% water to 100% methanol).
Preparation of N-2-(6"-Guanidino-hexyl)-6-Thioguanosine (12)
11 12
N-2-(6"-Aminohexyl)-6-Thioguanosine (11) (0.091 g, 0.23 mmol) was dissolved in 1 ml DMF and the resulting solution was treated with 0.34 g (0.23 mmol) Triazole-1-carboxamidine hydrochloride at room temperature in the presence of 39 μl (0.23 mmol) N-Ethyl- diisopropylamine acting, as a supportive base. After 2 hours, the reaction mixture was evaporated to dryness. Purification of the crude product by reversed phase chromatography (silica gel RP 18, linear gradient from 100% H2O to 100% ACN) gave 0.92 g (0.21 mmol, 90%) of N-2-(6"-Guanidino-hexyl)-6-Thioguanosine (12).
Preparation of N-2-(6"-Aminohexyl)-6-Thioguanosine-5'-monophosphate (13)
13 Under argon atmosphere, nucleoside 9 (0.3 g, 0.6 mmol) was dissolved in 3 ml trimethylphosphate. The solution was cooled to 00C and treated with 0.3 ml of Lutidine.
After 10 minutes, 0.15 ml (1.1 mmol) phosphorous oxychloride was carefully added. After 1 hour, the excess of POCI3 was removed in vacuo within ten minutes.
The solution was quenched by adding 100 ml of 0.25 M (pH 7.5) triethylammonium bicarbonate buffer. Purification by ion exchange chromatography gave N-2-(6"-thioacetamido-hexyl)-6- Thioguanosine-5'-monophosphate. The product was dissolved in 5 ml NH3/H2O (30%) and stirred for 90 minutes at 8O0C. The solvent was subsequently removed in vacuo. N-2-(6"- Aminohexyl)-6-Thioguanosine-5'-monophosphate (13) (0.17 g, 0.36 mmol, 60%) was obtained after purification of the crude product by ion exchange chromatography.
Preparation of N-2-(6"-Guanidino-hexyI)-6-Thioguanosine-5'-monophosphate (14)
HCI
13 14
N-2-(6"-Aminohexyl)-6-Thioguanosine-5'-monophosphate (13) (0.053 g, 0.11 mmol) was dissolved in a mixture of 0.8 ml water and 0.5 ml DMF. The solution was subsequently treated with 0.16 g (0.11 mmol) Triazole-1-carboxamidine hydrochloride at room temperature in the presence of 18 μl (0.11 mmol) N-Ethyl-diisopropylamine, acting as a supportive base. After 16 hours, the reaction mixture was concentrated to dryness. Purification of the crude product by reversed phase chromatography (silica gel RP 18, linear gradient from 100% H2O to 100% ACN) gave 0.037 g (0.072 mmol, 65%) of N-2-(6"-Guanidino-hexyl)-6-Thioguanosine~5'- monophosphate (14).
EXAMPLE 4: Synthesis of Ribose-modified 6-Thio-GTP Analogs
Preparation of Aspartate-273'-EDA-6-Thio-Guanosine-Triphosphate (4)
The N-hydroxy-succinimide ester of Boc/ffiu protected Aspartate (2.8 mg, 7 μmol) was dissolved in 200 μl of dry DMF and added to a solution of 1 (5 mg, 7 μmol) in 500 μl of 100 mM sodium borate buffer (pH 8.5) at room temperature. After 16 hours, the reaction mixture was quenched with methanol. The Aspartate derivative 4 was obtained in 70% yield (3.6 mg, 4.9 μmol) after workup of the reaction mixture by reversed phase HPLC.
Preparation of Glutamate-273'-EDA-6-Thio-Guanosine-Triphosphate (5)
The N-hydroxy-succinimide ester of Boc/fBu-protected Glutamate (2.9 mg, 7 μmol) was dissolved in 200μl of dry DMF and added to a solution of 1 (5 mg, 7μmol) in 500 μl of 100 mM sodium borate buffer (pH 8.5) at room temperature. After 16 hours, the reaction mixture was quenched with methanol. The Glutamate labeled product 5 was obtained in 70% yield (3.7 mg, 4.9 μmol) after workup of the reaction mixture by reversed phase HPLC.
Preparation of Threonine-273'-EDA-6-Thio-Guanosine-Triphosphate (6)
1 6
The N-hydroxy-succinimide ester of Boc-protected Threonine (2.3 mg, 7 μmol) was dissolved in 200 μl of dry DMF and added to a solution of 1 (5 mg, 7 μmol) in 500 μl of 100 mM sodium borate buffer (pH 8.5) at room temperature. After 16 hours, the reaction mixture was quenched with methanol. The Threonine derivative 6 was obtained in 70% yield (3.4 mg, 4.9 μmol) after workup of the reaction mixture by reversed phase HPLC.
EXAMPLE 5: Analysis of the ability of 5 new synthesized 6-Thio-GTP-derivatives to induce apoptosis in human CD4* T Lymphocytes.
Used substances: • Azathioprine
• 6-Mercaptopurine
• V1= BMB20= EDA-6-Thio-GTP
• V2= TWI 35/1= N-2-(6"-Aminohexyl)-guanosine
• V3 = TWI 71/2 = 2',3',5',o-Triacetyl-N-2-(Acetyl-6"-aminohexyl)-guanosine
• V4 = TW1 107/7 = 2',3',5'-Triacetyl -N-2-(6"-thioacetamide-hexyl)-6-Thioguanosine
• V5 = BMB = TAMRA-EDA-6-Thio-GTP
V3 and V4 were not soluble in water, therefore V3 was reconstituted with ethanol and V4 was reconstituted with methanol.
Protocol:
Human peripheral blood mononuclear cells (PBMC) from 4 buffycoats were isolated using Ficoll-Hypaque gradients. PBMC were further purified using CD4 monoclonal antibodies attached to immunomagnetic microbeads according to the protocol provided by the manufacturer (Miltenyi Biotec). T lymphocytes were stimulated in complete RPMI-1640 medium (RPMI-1640 + 10% FCS + 100 U/ml Penicillin/ Streptomycin + 3mM L-Glutamin) for 3, 4 or 5 days with coated antibodies to CD3 (0,04 μg/ml) and soluble CD28 antibodies (PharMingen; 1 Dg/ ml) plus IL-2 (R & D Systems, Wiesbaden, Germany; 40 U/ml). Azathioprine, 6- Mercaptopurine , V1, V2, V3, V4 or V5 were added to the T cell cultures at day 0 at a final concentration of 5 μM. To determine induction of apoptosis in these T lymphocytes, cells were analyzed by FACS. For FACS analysis, apoptotic cells were detected by staining with annexin V and propidium iodide using the Annexin V FITC Apoptosis Detection Kit I (PharMingen). In brief, T cells were washed twice in PBS, and the pellet was resuspended in annexin V binding buffer (PharMingen) at a concentration of 106 cells per milliliter. Annexin V FITC and propidium iodide were added (5 μl of each per 105 cells). Samples were gently mixed and incubated for 15 minutes at room temperature in the dark before FACS analysis.
Results:
• Annexin-positive, propidium iodide-negative cells (black bars) present the rate of early apoptotic cells. Annexin-positive, propidium iodide-positive cells (white bars) present late apoptotic or necrotic cells.
• Induction of apoptosis = (Rate of apoptotic cells after indicated treatment) - (Rate of apoptosis of untreated cells) • V1 , V2 and V3 were tested in 4 independent experiments. V4 and V5 were tested in 2 independent experiments.
Conclusion:
Our first results showed, that V1 and V5 were able to induce apoptosis in CD3/CD28 costimulated T Lymphocytes. V2, V3 and V4 were not able to induce apoptosis. Comparing V1 and V5 mediated induction of apoptosis with azathioprine or 6-mercaptopurine mediated induction of apoptosis, V1 and even V5 mediated effects seemed to be more pronounced and appeared earlier (Figures 2-4).
Example 5
Preparation of 2'/3'-Methylenoaminocarbamate derivatives of 6-Thio-Guanosine- triphosphate
The dry tributylammonium salt of 6-Thio-GTP 1 (0.2mmol) was treated with 200 mg carbonyl- diimidazole in 4 ml dimethylformamide. The resulting mixture was stirred for 6 hours at 0° C, brought to room temperature and, subsequently, 80 μl of methanol were added. After 10 minutes, 2 mmol of the appropriate methyleno-amine and 2 ml of triethylamine were also added to the reaction mixture. The solution was stirred overnight at room temperature and the solvent was then removed under reduced pressure. The residue was taken up in 30 ml water and the mixture was adjusted to pH 1, in order to decompose the resulting intermediate phosphoramidate at the triphosphate moiety. After 20 minutes, the solution was adjusted to pH 7.5, the precipitate filtered off and the solvent was removed in vacuo. The resulting crude product was purified by ion exchange chromatography and subsequently by reversed phase HPLC.
Example 6 273'-Methylenoaminocarbamate-6-Thio-Guanosine-triphosphate derivative 2a
Reaction of 3-thienyl-methylamine (226 mg, 2 mmol) with 1 according to the general procedure yielded 2a (0.58 mmol, 29 %) after purification by ion exchange chromatography and subsequent reversed phase HPLC.
2V3'-Methylenoaminocarbamate-6-Thio-Guanosine-triphosphate derivative 2b
Reaction of (1 ,5-Dimethyl-1H-pyrazol-3-yl)methylamine (250 mg, 2 mmol) with 1 according to the general procedure yielded 2b (0.66 mmol, 33 %) after purification by ion exchange chromatography and subsequent reversed phase HPLC.
Example 7
Preparation of 273'-Carbamate derivatives of 6-Thio-Guanosine-triphosphate
The dry tributylammonium salt of compound 1 (0.2mmol) was treated with 200 mg carbonyl- diimidazole in 4 ml dimethylformamide. The resulting mixture was stirred for 6 hours at 0° C, brought to room temperature and, subsequently, 80 μl methanol were added. After 10 minutes, 2 mmol of the appropriate amine and 2 ml of 1 M potassium hexamethyldisilazide (KHMDS) in THF were carefully added to the solution. The solution was stirred at room temperature for 1 hour and the solvent was then removed under reduced pressure. The residue was taken up in 30 ml water and the mixture was adjusted to pH 1 , in order to decompose the resulting intermediate phosphoramidate at the triphosphate moiety. After 20 minutes, the solution was adjusted to pH 7.5, the precipitate filtered off and the solvent was removed in vacuo. The resulting crude product was purified by ion exchange chromatography and subsequently by reversed phase HPLC.
Example 8 In relation to figures 5 and 6, an overview of at least three experimental data sets on apoptosis is shown below. Here, some of the D compounds were able to induce apoptosis. BO was the strongest candidate drug for apoptosis induction. Two issues should be considered in these results.
• Negative induction of apoptosis means that there were more apoptotic cells in the untreated group than in the treated group. This phenomenon may appear from time to time and may be explained by a kind of statistical variance.
• The drug 6-Thio-GTP on average was not able to induce apoptosis in these experiments. Generally 6-Thio-GTP should be a positive control to induce apoptosis in T cells. In these experiments the positive control did not work very well. This might be explained by the fact that the experiments are often preformed with primary T cells, which are freshly isolated from blood of different donors. It is well known, that some people are not sensitive for azathioprine therapy. In this way, T cells of some donors might be resistant against 6-Thio-GTP induced apoptosis. In any case, however, BO and some of the D compunds were able to induce apoptosis suggesting that they are candidate drugs.
In addition, an alternative method was performed for screening of group-D derivatives (figure 8). It was decided to analyse the activity of caspase-3/7 in T cells, which were treated with group-D derivatives. As compared to AnnexinV/PI staining this new method might have some advantages:
• It is an easier protocol. Therefore there are fewer possibilities for individual errors.
• Increased activity of caspase-3 is very specific for apoptosis. Therefore this method is very sensitive for the detection of apoptosis. There is no interfering influence of necrotic cells.
• The measurement is done in duplicate. In this way there is an internal control.
The Caspase-Glo 3/7 Assay (Promega™) was used. This assay is based on a caspase dependent luminescent signal. Protocol: CD4+ T cells were isolated from human blood by magnetic beads (Dynal). T lymphocytes were stimulated in complete RPMI-1640 medium (RPMI-1640 + 10% FCS + 100 U/ml Penicillin/ Streptomycin + 3mM L-Glutamin) for 3 days with coated antibodies to CD3 (0,04 μg/ml) and soluble CD28 antibodies (PharMingen™; 1 μg/ ml) plus IL-2 (R & D Systems™, Wiesbaden, Germany; 40 U/ml) in 96-well plates. Cells were treated with different group-D derivatives or were left untrated. At day three of culture Caspase- 3/7 Assay was performed. 25 μl of Capase-Glo 3/7 reagent was added to each well. Probes were mixed gently for 2 minutes and incubated at room temperature for 30 minutes. Finally 100 μl of each probe were transfered to a white walled 96-well luminometer plate and analysed in a plate-reading luminometer. The added reagent contains a specific substrate of caspase-3 and caspase-7. Cleavage of this substrate by activated caspase-3 results in luciferase reaction. Luminescence is proportional to the amount of caspase activity present (figure 8)
Example 8
2'/3'-Carbamate-6-Thio-Guanosine-triphosphate derivative 3a
3a
Reaction of 3-aminopyrimidine (190 mg, 2 mmol) with 1 according to the general procedure yielded 3a (0.076 mmol, 3.8 %) after purification by ion exchange chromatography and subsequent reversed phase HPLC.
Example 9
273'-Carbamate-6-Thio-Guanosine-trϊphosphate derivative 3b
3b
Reaction of 3-(tert.-Butyl)-1-methyl-1H-pyrazol-5-amine (306 mg, 2 mmol) with 1 according to the general procedure yielded 3b (0.11 mmol, 5.5 %) after purification by ion exchange chromatography and subsequent reversed phase HPLC.
BIBLIOGRAPHY
-Boise LH et al. Receptors that regulate T-cell susceptibility to apoptotic cell death. Ann N Y Acad Sci 1995; 766:70-80.
-Tiede I. et al. CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes. J Clin Invest 2003; 111: 1133-1145.
-Maltzman JS et al. Azathioprine: old drug, new action. J Clin Invest 2003; 111 : 1122-1124.
-Boise LH et al. CD28 costimulation can promote T cell survival by enhancing the expression of bcl-xL . Immunity 1995; 3: 87-98.
-Khoshnan A. et al. The NF-κB cascade is important in bcl-xL expression and for the anti- apoptotic effects of CD28 receptor in primary human CD4+ T lymphocytes. J Immunol 2000; 165: 1743-1754.
-Noel PJ et al. CD28 costimulation prevents cell death during primary T cell activation. J lmmmunol 1996; 157: 636-642.
-Frauwirth KA et al. Activation and inhibition of lymphocytes by costimulation. J Clin Invest 2002, 109: 295-299.
-Marinari B et al. Vav cooperates with CD28 to induce NF-κB activation via a pathway involving Rac1 an mitogen-activated kinase kinase 1. Eur J Immunol 2002; 32: 447-456.
-Faruqi TR et al. Rad mediates STAT-3 activation by autocrine IL-6. PNAS 2001 ; 98: 9014- 9019.
-Mudter J and Neurath MF. The role of signal transducers and activators of transcription in T inflammatory bowel diseases. IBD 2003; 9: 332-337.
-Lovato P et al. Constitutive STAT-3 activation in intestinal T cells from patients with Crohn's disease. J Biol Chem 2003; 278: 16777-16781.
-Van Aelst L et al. Rho GTPases and signaling networks. Genes & Development 1997; 11 : 2295-2322.
-Kohyoma et al.(2003) A facile synthesis of AICAR from inosine. Synthesis 17:2639.
-Imai et al. (1971) Synthesis of compounds related to inosine 5'-phosphate and their flavor enhancing activity. IV 2-substituted inosine %'-phosphates. Chem. Pharm. Bull. 19:576. -Ostermaπn et al (1999), New N-2-labelled fluorescent derivates of guanosine nucleotides and their interaction with GTP-binding proteins. Nucleosides & Nucleotides 18:245.
-Ludwig (1981) Acta Biochim. Acad. Sci.Hung. 16:131

Claims

Claims:
1. A compound of the general formula (I):
wherein the dashed bond in the sugar moiety can be either single or double and wherein R1 , R2, R3, R4 or R5, equal or different between each other, have general formula -{Int)m-Ter, wherein m is between 0 and 12 and lnt and Ter are /πternal and Terminal building blocks, wherein lnt is selected from the group consisting of
and Ter is selected from the group consisting of
and
And wherein X represents either carbon or nitrogen atom within aromatic ring, Y represents either oxygen or sulphur atom and an additional group Q1 group Qi or groups Qi (Qi indicates that the group or several groups may be bound to any unsaturated moiety of the ring) are selected from the group consisting of -OH, -COOH, -N(CH3)2, -N(CH2-CH3)2, -CO-CH3, -CO-O-CH3, -0-CH3, -S-CH31-SO2-CH3, , -CN, -NO2 or -Halogen elements, and wherein R5 may be
and metal and ammonium salts thereof, wherein n is between O and 5, or oxygen or phosphorus is partially or completely replaced by nitrogen, sulphur, methyleno groups or their derivatives.
2. A compound according to claim 1 lnt is selected from the consisting of
3. A compound according to claim 1or claim 2 wherein Ter is selected from the consisting of
and
4. A compound according to any preceding claim wherein , wherein said compounds are labelled.
5. A compound according to any preceding claim with the general formula (Ia):
wherein R1, R2, R3, R4 or R5, equal or different between each other, have general formula - (Int)m-Ter, wherein m is between 0 and 12 and lnt and Ter are Internal and Terminal building blocks, wherein lnt is selected from the consisting of
and Ter is selected from the consisting of
wherein an additional group Q, group Qi or groups Qi (Qi indicates that the group or several groups may be bound to any unsaturated moiety of the ring) are selected from the group consisting of -OH, -COOH, -N(CH3)2, -N(CH2-CH3)2 or -Halogen elements
6. A compound according to any one of the preceding claims, wherein sugar moiety of compounds of formula (I) are selected from the group consisting of the following sugar moieties or sugar-like moieties:
7. A compound according to any one of the preceding claims wherein R3 or R4 are selected from
and wherein Q is selected from -OH (FAM) or -N(CH3)2 (TAMRA)
8. A compound as claimed in any one of claims 1-6 with the following structure:
05B-0
9. A compound as claimed in any one of claims 1-6 with the following structure:
05B-1
10. A compound as claimed in any one of claims 1-6 with the following structure:
05B-2
11. A compound as claimed in any one of claims 1-6 with the following structure:
05B-3
12. A compound as claimed in any one of claims 1-6 with the following structure:
05C-0
13. A compound as claimed in any one of claims 1-6 with the following structure:
05C-1
14. A compound as claimed in any one of claims 1-6 with the following structure:
05C-2
15. A compound as claimed in any one of claims 1-6 with the following structure:
05C-3
16. A compound as claimed in any one of claims 1-6 with the following structure:
V4
17. A compound as claimed in any one of claims 1-6 with the following structure:
18. A compound as claimed in any one of claims 1-6 with the following structure:
V3
19 A compound according to any one of claims 1-5 of the general formula (II):
wherein n=1 , 2 or 3, m is between 0 and 5, Int is selected from the group consisting of
and Ter is selected from group the consisting of
wherein X represents either carbon or nitrogen atom within aromatic ring, Y represents either oxygen or sulphur atom and an additional group Q or groups Q/ (/' indicating the position of any unsaturated moiety of the ring to which the group Q may be bound) are selected from the group consisting of -CH3, -C(CHs)3, -OH, -COOH, -CO-CH3, -CO-O-CH3, -0-CH3, -S-CH31-SO2-CH3, -N(CH3)2, -N(CH2-CH3)Z, -CN, -NO2 or -Halogen elements.
20.A compound according to claim 19 wherein , Ter is selected from the group consisting of
21. A compound as claimed in either of claims 19 or 20 wherein n is 1-3.
22. A compound as claimed in any of claims 19-21 wherein n is 3.
23. A compound as claimed in any of claims 19-21 wherein n is 1
24. A compound as claimed in any of claims 19-21 wherein n is 2.
25. A compound as claimed in any one of claims 19-22 with the following general formula:
06D-1
26. A compound as claimed in any one of claims 19-22 with the following general formula: 06D-3
27. A compound as claimed in any one of claims 19-22 with the following general formula:
06D-6
28. A compound as claimed in any one of claims 19-22 with the following general formula:
06D-12
29. A compound as claimed in any one of claims 19-22 with the following general formula: 06D-13
30. A compound as claimed in any one of claims 19-22 with the following general formula:
06D-14
31. A compound as claimed in any one of claims 19-22 with the following general formula:
06D-22
32. A compound as claimed in any one of claims 19-22 with the following general formula:
06D-130
33. A compound according to any one of claims 1-9, wherein said compounds are selected from the group consisting of 2',3'-EDA-6-Thio-GTP, FAM-2',3'-EDA-6-Thio-GTP, TAMRA-2',3'-EDA- 6-Thio-GTP, Aspartate-2'131-EDA-6-Thio-GTP, Glutamate-2',3'-EDA-6-Thio-GTP, Threonine- 2',3'-EDA-6-Thio-GTP, Serine-2',3'-EDA-6-Thio-GTP, 2',3',5',O-Triacetyl-N-2-(Acetyl-6"- aminohexyl)-guanosine, 2',3',5'-Triacetyl-N-2-(6"-thioacetamide-hexyl)-6-Thioguanosine, N-2- (6"-thioacetamide-hexyl)-6-Thioguanosine, N-2-(6"-Aminohexyl)-6-Thioguanosine, N-2-(6"- guanidino-hexyl)-6-Thioguanosine, N-2-(6"-Aminohexyl)-6-Thio-GMP, N-2-(6"-guanidino- hexyl)-6-Thio-GMP, N-2-(6"-Aminohexyl)-6-Thio-GTP, N-2-(6"-guanidino-hexyl)-6-Thio-GTP, N- 2-(6"-Aspartate-hexyl)-6-Thioguanosine, N-2-(6"-Glutamate-hexyl)-6-Thioguanosine, N-2-(6"- Threonine-hexyl)-6-Thioguanosine, N-2-(6"-Serine-hexyl)-6-Thioguanosine, N-2-(6"- Aminobutyl)-6-Thio-GTP, N-2-(6"-guanidino-butyl)-6-Thioguanosine, N-2-(6"-Aspartate-butyl)-6-
Thioguanosine, N-2-(6"-Glutamate-butyl)-6-Thioguanosine, N-2-(6"-Threonine-butyl)-6- Thioguanosine, N-2-(6"-Serine-butyl)-6-Thioguanosine, N-2-(6"-Aminopropyl)-6-Thioguanosine, N-2-(6"-guanidino-propyl)-6-Thioguanosine, N-2-(6"-Aspartate-propyl)-6-Thioguanosine, N-2- (6"-Glutamate-propyl)-6-Thioguanosine, N-2-(6"-Threonine-propyl)-6-Thioguanosine, N-2-(6"- Serine-propyl)-6-Thioguanosine, N-2-(6"-Amino-2-butene)-6-Thioguanosine, N-2-(6"-guanidino- 2-butene)-6-Thioguanosine, N-2-(6"-Aspartate-2-butene)-6-Thioguanosine, N-2-(6"-Glutamate- 2-butene)-6-Thioguanosine, N-2-(6"-Threonine-2-butene)-6-Thioguanosine, N-2-(6"-Serine-2- butene)-6-Thioguanosine, N-2-(6"-Amino-2-butyne)-6-Thioguanosine, N-2-(6"-guanidino-2- butyne)-6-Thioguanosine, N-2-(6"-Aspartate-2-butyne)-6-Thioguanosine, N-2-(6"-Glutamate-2- butyne)-6-Thioguanosine, N-2-(6"-Threonine-2-butyne)-6-Thioguanosine, N-2-(6"-Serine-2- butyne)-6-Thioguanosine, N-2-(6"-Amino-2,4-hexadiyne)-6-Thioguanosine, N-2-(6"-guanidino- 2,4-hexadiyne)-6-Thioguanosine, N-2-(6"-Aspartate-2,4-hexadiyne)-6-Thioguanosine, N-2-(6"- Glutamate-2,4-hexadiyne)-6-Thioguanosine, N-2-(6"-Threonine-2,4-hexadiyne)-6-
Thioguanosine, N-2-(6"-Serine-2,4-hexadiyne)-6-Thioguanosine.
34. A pharmaceutical composition comprising at least one of the compounds as claimed in any preceding claim as active principle and one or more pharmaceutically acceptable co-adjuvants or excipients.
35. Use of a compound as claimed in any one of claims 1-33 or a pharmaceutical composition as claimed in claim 34 for the preparation of an immunosuppressive medicament.
36. Use according to claim 35, wherein the immunosuppressive medicament is for the prevention of rejection of organ transplants or of post-transplant nephropathy.
37. Use of a compound as claimed in any one of claims 1-33 or a pharmaceutical composition as claimed in claim 34, for the preparation of a medicament for the treatment of pathologies in which immune system is involved.
38. Use of a compound as claimed in any one of claims 1-33 or a pharmaceutical composition as claimed in claim 34 for the preparation of a medicament for the treatment of one or more pathologies, wherein the pathologies are selected from the group consisting of inflammatory chronic intestinal diseases, auto-immune enteropathy, active chronic hepatitis, rheumatoid arthritis, Still's disease, systemic lupus erythematous, acquired haemolytic anaemia, idiopathic thrombocytopenia, polyarthritis nodosa, vasculitis, polyangitis, polymyositis, myasthenia gravis, sarcoidosis, lipoid nephritis, multiple sclerosis, dermatomyositis, pemphigus vulgaris, primary biliary cirrhosis, primary sclerosing cholangitis, recurrent multiform erythema, chronic actinic dermatitis, gangrenous hypoderm, ptyriasis rubra, Wegener's granulomatosis, cutaneous vasculitis, atopic dermatitis, psoriasis, pimply pemphigoid.
39. Use according to claim 38, wherein inflammatory chronic intestinal diseases are selected from the group consisting of Crohn's disease, ulcerous rectocolitis, indeterminate colitis.
40. Use according to anyone of the claims 35-39 in addition to radiotherapy, corticosteroids or cytotoxic agents.
41. Use of a compound as claimed in any one of claims 1-33 or a pharmaceutical composition as claimed in claim 34 for the preparation of a medicament for the treatment of cancer
42. Use of a compound as claimed in any one of claims 1-33 or a pharmaceutical composition as claimed in claim 34, as probes for the evaluation of the binding properties of the compounds of formula (I) by the Racl/Vav system.
43. Use of a compound as claimed in any one of claims 1-33 or a pharmaceutical composition as claimed in claim 34 in a method of therapy and/or treatment.
44. Use as claimed in claim 43 wherein the therapy and/or treatment is for one or more of the group comprising: rejection of organ transplants and of post-transplant nephropathy, pathologies in which immune system is involved, inflammatory chronic intestinal diseases, such as Crohn's disease, ulcerous rectocolitis, indeterminate colitis, or of auto-immune enteropathy, active chronic hepatitis, rheumatoid arthritis, Still's disease, systemic lupus erythematous, acquired haemolytic anaemia, idiopathic thrombocytopenia, polyarthritis nodosa, vasculitis, polyangitis, polymyositis, myasthenia gravis, sarcoidosis, lipoid nephritis, multiple sclerosis, dermatomyositis, pemphigus vulgaris, primary biliary cirrhosis, primary sclerosing cholangitis, recurrent multiform erythema, chronic actinic dermatitis, gangrenous hypoderm, ptyriasis rubra, Wegener's granulomatosis, cutaneous vasculitis, atopic dermatitis, psoriasis, pimply pemphigoid, the immunosuppressive therapy after organ transplantation including, kidney, heart, lung, pancreas and liver transplantation.
45. A process for the preparation of a compound as claimed in any one of claims 1-33 wherein the introduction of the -NH-R group at the 2 position of guanosine ring comprises the following steps:
a) protection of the NH moiety of tri-O-acetyl-inosine;
b) oxidative guanosine ring-opening and O-deprotection;
c) guanosine ring-closing and introduction of a SH group at the 2 position of the guanosine ring through the use of CS2;
d) replacing the SH group at the 2 position with an amino-linker by using an excess of an aliphatic diamine.
46. Process according to claim 45, comprising a further step e) of protection of ribose OH groups and of the primary amine group by acetylation.
47. Process according to claim 46, comprising a further step f) of thiolation of C=O groups through the use of Lawesson's reagent.
48. A compound substantially as herein described with reference to the accompanying formulae and figures
49. A use substantially as herein described with reference to the accompanying formulae and figures
50. A process substantially as herein described with reference to the accompanying formulae and figures.
51. A method of therapy and/or treatment substantially as herein described with reference to the accompanying formulae and figures
52. A pharmaceutical composition substantially as herein described with reference to the accompanying formulae and figures
Sped 794
EP06766084A 2005-07-22 2006-07-24 Improvements to analogous compounds of 6-thioguanosine triphosphate, their use in medical fields and processes for their preparation Withdrawn EP1907407A1 (en)

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IT000391A ITRM20050391A1 (en) 2005-07-22 2005-07-22 SIMPLE COMPOUNDS OF 6-THIOPHOUANOSINE TRIPOSPHATE, THEIR USE IN THE MEDICAL AREA AND PROCEDURE FOR THEIR PREPARATION.
IE2006/0448A IE84917B1 (en) 2006-06-14 Improvements to analogous compounds of 6-thioguanosine triphosphate, their use in medical fields and processes for their preparation
PCT/IE2006/000077 WO2007010515A1 (en) 2005-07-22 2006-07-24 Improvements to analogous compounds of 6-thioguanosine triphosphate, their use in medical fields and processes for their preparation

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Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1386585A (en) * 1970-11-16 1975-03-12 Wellcome Found Purine sugar derivatives
DE4418690A1 (en) * 1994-05-28 1996-01-11 Boehringer Mannheim Gmbh Nucleoside 5'-mono-phosphate ester(s) of glyceryl di:ether derivs.
ATE271063T1 (en) * 1996-10-16 2004-07-15 Icn Pharmaceuticals PURINE-L-NUCLEOSIDES, THEIR ANALOGUES AND USES

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
HIROKAZU MORISAWA ET AL: "A NEW METHOD FOR THE SYNTHESIS OF SOME 9-B-;-ARABINOFURANOSYLPURINES BY A COMBINATION OF CHEMICAL AND ENZYMATIC REACTIONS", TETRAHEDRON LETTERS, 1 January 1980 (1980-01-01), pages 479 - 482, XP055031796, Retrieved from the Internet <URL:http://ac.els-cdn.com/S0040403900714389/1-s2.0-S0040403900714389-main.pdf?_tid=825ab7887cf7e2eed1e5cbe973b144d1&acdnat=1341404645_7591a5fad96f44c98360afc1eccc8bbb> [retrieved on 20120704] *
J. MASSON GULLAND ET AL: "125. Constitution of the purine nucleosides. Part VII. Guanosine and guanine deoxyriboside", JOURNAL OF THE CHEMICAL SOCIETY (RESUMED), 1 January 1938 (1938-01-01), pages 692, XP055031853, ISSN: 0368-1769, DOI: 10.1039/jr9380000692 *
JOHN F. GERSTER ET AL: "Purine Nucleosides. X. The Synthesis of Certain Naturally Occurring 2-Substituted Amino-9-[beta]-D-ribofuranosylpurin-6(1H)-ones (N2-Substituted Guanosines)", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 87, no. 16, 20 August 1965 (1965-08-20), pages 3752 - 3759, XP055031791, ISSN: 0002-7863, DOI: 10.1021/ja01094a037 *
KARLHEINZ WÖRNER ET AL: "Synthesis and stability of GNRA-loop analogs", HELVETICA CHIMICA ACTA, 1 January 1999 (1999-01-01), pages 2094 - 2104, XP055031844 *
S. A. NARANG ET AL: "Studies on Polynucleotides. XLVI.1 The Synthesis of Hexanucleotides Containing the Repeating Trinucleotide Sequences Deoxycytidylyl-(3'->5')-deoxyadenylyl-(3'->5')-deoxyadenosine and Deoxyguanylyl-(3'->5')-deoxyadenylyl-(3'->5')- deoxyadenosine", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 87, no. 13, 5 July 1965 (1965-07-05), pages 2988 - 2995, XP055031792, ISSN: 0002-7863, DOI: 10.1021/ja01091a031 *
See also references of WO2007010515A1 *
UGO TRAVERSA ET AL: "Rat brain guanosine binding site", BIOORGANIC & MEDICINAL CHEMISTRY, vol. 11, no. 24, 1 December 2003 (2003-12-01), pages 5417 - 5425, XP055031787, ISSN: 0968-0896, DOI: 10.1016/j.bmc.2003.09.043 *

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