Classifications

• • 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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