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Transforming growth factor β (TGF- β ) ligands activate a signaling cascade with multiple cell context dependent outcomes. Disruption or disturbance leads to variant clinical disorders. To develop strategies for disease intervention, delineation of the pathway in further detail is required. Current theoretical models of this pathway describe production and degradation of signal mediating proteins and signal transduction from the cell surface into the nucleus, whereas feedback loops have not exhaustively been included. In this study we present a mathematical model to determine the relevance of feedback regulators (Arkadia, Smad7, Smurf1, Smurf2, SnoN and Ski) on TGF- β target gene expression and the potential to initiate stable oscillations within a realistic parameter space. We employed massive sampling of the parameters space to pinpoint crucial players for potential oscillations as well as transcriptional product levels. We identified Smad7 and Smurf2 with the highest impact on the dynamics. Based on these findings, we conducted preliminary time course experiments.

Supplemental digital content is available in the text., Background Elevated liver enzymes are frequently observed in renal transplant recipients and warrant further exploration. In immunosuppressed patients, hepatitis E virus (HEV) infection may cause chronic hepatitis, cirrhosis, and extrahepatic manifestations such as renal injury. Methods We performed a retrospective cross-sectional study investigating the prevalence, clinical correlates, and outcome of chronic HEV infection in a cohort of renal transplant recipients with elevated liver enzymes. Results Over a period of 30 months, 140 of 1469 renal transplant recipients had elevated liver enzymes, of which serum samples from 98 patients were available to determine HEV status. Seventeen patients were detected with HEV infection, of which 16 developed chronic HEV infection, while 1 patient controlled viremia (prevalence of chronic infection of 16.3%, with a minimum prevalence of 1.1% in the whole cohort). Increased liver stiffness was indicated by an average FibroScan result of 11.2 kPa in these patients. All 16 patients with chronic HEV infection were treated with ribavirin for a mean duration of 3 months. Five patients developed a viral rebound and received a second treatment course, of which 2 controlled HEV replication. Six months after the end of therapy, HEV clearance was achieved in 81.3% of the patients. One patient developed ribavirin resistance. Hemolytic anemia after ribavirin treatment was frequent, requiring blood transfusion in 3 patients. Four patients developed de novo glomerulonephritis, of which 2 were possibly associated with HEV infection. Conclusions This retrospective study showed that prevalence of chronic HEV infection was high in our renal transplant patient cohort and was associated with significant liver impairment and the occurrence of renal injury. Ribavirin treatment was effective and should be initiated early to avoid complications, but the risk of severe hemolytic anemia makes strict monitoring essential.

There is significant clinical interest in primers which are specific for fungi and do not hybridize to DNA of other eukaryotes or prokaryotes. Such primers would allow specific amplification of fungal DNA from human tissue samples containing fungi. Fungal identification to the species level could follow by direct sequencing or restriction analysis. Several previously described primer systems cross-react with DNA of plants and animals. We have designed a primer system that amplifies a fragment of the gene coding for the small ribosomal subunit 18S rRNA. Database searches and sequence analyses were performed using the HUSAR (Heidelberg Unix Sequence Analysis Resources) computer system at the German Cancer Research Centre, Heidelberg, Germany. Primers TR1 (5'-GTTTCTAGGACCGCCGTA) and TR2 (5'-CTCAAACTTCCATCGACTTG) bind to sequences which are homologous within the fungi, but differ from corresponding DNA fragments of plants and animals. The amplified fragment is 581 base pairs in length and contains variable, and therefore species-specific, regions. The DNA of Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton verrucosum, Trichophyton terrestre, Microsporum canis, Microsporum gypseum and Epidermophyton floccosum and of several yeast species was amplified by the primers, but not the DNA from 42 normal human skin samples. Furthermore, other DNA preparations from plants and animals, including those from radish, cabbage, wheat and mouse, did not show amplification reactions.

Selective and potent P2Y(11) receptor antagonists have yet to be developed, thus impeding an evaluation of this G protein-coupled receptor mainly expressed on immune cells. Taking suramin with moderate inhibitory potency as a template, 18 ureas with variations of the methyl groups of suramin and their precursors were functionally tested at P2Y(11), P2Y(1), and P2Y(2) receptors. Fluorine substitution of the methyl groups of suramin led to the first nanomolar P2Y(11) antagonist (8f, NF157, pK(i): 7.35). For selectivity, 8f was also tested at various P2X receptors. 8f displayed selectivity for P2Y(11) over P2Y(1) (>650-fold), P2Y(2) (>650-fold), P2X(2) (3-fold), P2X(3) (8-fold), P2X(4) (>22-fold), and P2X(7) (>67-fold) but no selectivity over P2X(1). QSAR studies confirm that residues with favored resonance and size parameters in the aromatic linker region can indeed lead to an increased potency as is the case for 8f. A symmetric structure linking two anionic clusters seems to be required for bioactivity. 8f may be helpful for studies evaluating the physiological role of P2Y(11) receptors.

Protein-tyrosine phosphatases (PTPs) are important signaling enzymes that have emerged within the last decade as a new class of drug targets. It has previously been shown that suramin is a potent, reversible, and competitive inhibitor of PTP1B and Yersinia PTP (YopH). We therefore screened 45 suramin analogs against a panel of seven PTPs, including PTP1B, YopH, CD45, Cdc25A, VHR, PTPalpha, and LAR, to identify compounds with improved potency and specificity. Of the 45 compounds, we found 11 to have inhibitory potency comparable or significantly improved relative to suramin. We also found suramin to be a potent inhibitor (IC(50) = 1.5 microm) of Cdc25A, a phosphatase that mediates cell cycle progression and a potential target for cancer therapy. In addition we also found three other compounds, NF201, NF336, and NF339, to be potent (IC(50) < 5 microm) and specific (at least 20-30-fold specificity with respect to the other human PTPs tested) inhibitors of Cdc25A. Significantly, we found two potent and specific inhibitors, NF250 and NF290, for YopH, the phosphatase that is an essential virulence factor for bubonic plague. Two of the compounds tested, NF504 and NF506, had significantly improved potency as PTP inhibitors for all phosphatases tested except for LAR and PTPalpha. Surprisingly, we found that a significant number of these compounds activated the receptor-like phosphatases, PTPalpha and LAR. In further characterizing this activation phenomenon, we reveal a novel role for the membrane-distal cytoplasmic PTP domain (D2) of PTPalpha: the direct intramolecular regulation of the activity of the membrane-proximal cytoplasmic PTP domain (D1). Binding of certain of these compounds to PTPalpha disrupts D1-D2 basal state contacts and allows new contacts to occur between D1 and D2, which activates D1 by as much as 12-14-fold when these contacts are optimized.

Extracellular adenine and uracil 5'-nucleotides are important signalling molecules that exert a great variety of effects in numerous tissues and cell types through the activation of P2 receptors. In the past eight years, an extended series of P2 receptors (P2X(17), ionotropic subunits; P2Y(1,2,4,6,11,12), metabotropic receptors) has been cloned from vertebrate tissues. In this rapidly expanding field, one of the main current challenges is to relate the cloned P2 receptor subtypes to the diverse physiological responses mediated by the pharmacological phenotypes of native P2 receptors. Unfortunately, subtype-selective P2 ligands, especially potent and selective antagonists, have been only slowly forthcoming, and this acts as a considerable impediment to progress. However, a number of new P2 receptor antagonists have recently been described which to some degree are more potent and more selective than earlier antagonists like suramin or pyridoxal-5'-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). This work moves us closer to the ideal goal of classifying the recombinant and native P2 receptor subtypes on the basis of antagonist profiles. This review begins with a brief account of the current status of P2 receptors and their ligands. It then focuses on structure-activity relationships of PPADS and suramin analogues and will finish with a brief discussion of some related therapeutic possibilities.

Intracellular microelectrodes were used to record the transmembrane potential and excitatory junction potentials (e.j.p.s) produced by sympathetic nerve stimulation (1 Hz) in smooth muscle cells of the guinea-pig isolated vas deferens. The symmetrical 3′-urea of 8-(benzamido)naphthalene-1,3,5-trisulphonic acid (NF023) produced a concentration-dependent inhibition of e.j.p. magnitude (IC50=4.8×10−6 M), but had no effect on the resting membrane potential of the smooth muscle cells. Pyridoxal-5-phosphate (P-5-P) also depressed e.j.p. magnitude in a concentration-dependent manner, but was less potent than NF023 (IC50=2.2×10−5 M). At 10−4 M and above P-5-P significantly depolarized the smooth muscle cells. The nucleoside triphosphatase inhibitor 6-N,N-diethyl-D-β,γ-dibromomethyleneATP (ARL 67156) (5×10−5 M) significantly increased e.j.p. amplitude. ARL 67156 (10−4 M) further increased e.j.p. amplitude such that they often reached threshold for initiation of action potentials, causing muscle contraction and expulsion of the recording electrode. After reduction of e.j.p.s by NF023 or P-5-P (both 10−5 M), subsequent co-addition of ARL 67156 (10−4 M) significantly increased their magnitude. The overflow of endogenous ATP evoked by field stimulation of sympathetic nerves (8 Hz, 1 min) was measured by HPLC and flurometric detection. ARL 67156 (10−4 M) enhanced ATP overflow by almost 700% compared to control. We conclude that for electrophysiological studies NF023 is preferable to other P2X receptor antagonists such as pyridoxalphosphate -6-azophenyl-2′,4′-disulphonic acid (PPADS), suramin or P-5-P. Furthermore, breakdown of endogenous ATP by nucleoside triphosphatases is an important modulator of purinergic neurotransmission in the guinea-pig vas deferens. British Journal of Pharmacology (2000) 129, 1089–1094; doi:10.1038/sj.bjp.0703163

Suramin analogs are polyanionic naphthylureas structurally related to suramin, an antitumor agent with a narrow therapeutic window. The angiostatic activities of suramin and 16 suramin analogs were investigated using an easily quantifiable in vitro angiogenesis system. In addition, the antiproliferative activities of the analogs were studied in four different human tumor cell lines and in porcine aortic endothelial cells. The suramin analogs encompassed two main structural variations, i.e. their molecular size, and the number and substitution pattern of the sulfonate groups. Some suramin analogs with a reduced number of sulfonate groups (NF062, NF289 and NF326) showed significant dose-dependent angiostatic and also antiproliferative activities. The disulfonate NF062 was superior to suramin in inhibiting HT29 and T47D tumor cells while demonstrating a similar angiostatic potential as suramin. Therefore, the sulfonate groups in the para position of the amino groups of the naphthyl residues of suramin seem to be of special importance. The very small disulfonates (NF108, NF109, NF499, NF500 and NF241) and the asymmetric compound NF520, one half of the suramin molecule, are inactive. Therefore, a minimal molecule size seems to be essential for the biological activity. Suramin is a rather rigid molecule. The highly flexible analogs (NF527, NF528 and NF529) are inactive. This indicates that the molecular rigidity is important for the biological activity.

The title compound is one of a large number of compounds related to the drug suramin which have been found to have anti-HIV properties. However, its toxicity to uninfected cells is high. We crystallized the compound, determined its structure, and investigated the molecular basis for its high toxicity. The crystals are monoclinic, C2/c, a = 28.614(1), b = 10.031(0), c = 24.051(1) Å, β = 98.36(2)°, Z = 8, convergence at R = 0.066 for 4264 reflections with I > 2σ(I). The oxygen atoms of one of the sulfonate groups are disordered, as are several water molecules. Stereochemical comparisons with a segment of B-DNA have yielded a plausible explanation for the molecule's toxicity. Key words: crystal structure, cellular toxicity, stereochemistry, NF-033.

The synthesis of suramin analogues bearing a 2-phenyl-benzimidazole moiety is described. Aminoarene sulfonic acids 2a-e are acylated with 3,4-dinitrobenzoyl chloride 3 yielding the amides 4a-e which are hydrogenated to the corresponding diamines 5a-e. These are treated with 3-nitrobenzaldehyde, yielding the azomethines 7a-e and their isomers 8a-e and 9a-e. Key step in the synthesis of the target compounds 12a-e is the oxidation of the azomethines with oxygen to the benzimidazoles 10a-e. These are hydrogenated to the amines 11a-e reacting with phosgene to yield the symmetric ureas 12a-e. Results of the anti-HIV, cytostatic, and antiangiogenic screening are presented.

The purpose of this study was to test the ability of 70 polyanionic analogues of suramin to inhibit angiogenesis. The ID50, the dose that produced 50% inhibition of angiogenesis, was determined for suramin and each of the analogues by measuring the ability of various amounts to inhibit angiogenesis in vivo in the chick egg chorioallantoic membrane (CAM) assay. Of the 70 analogues, 11 had antiangiogenic activities similar to suramin and an additional 7 were significantly more potent than suramin. All seven of these analogues were from the naphthalenetrisulfonic acid group and contained large urea groups. The benzene sulfonic and disulfonic acid analogues were less active inhibitors of angiogenesis than the naphthalenetrisulfonic acid analogues. Replacement of the naphthalenetrisulfonic acid groups by aliphatic carboxylic acids or benzoic acid gave analogues with very little antiangiogenic activity. In subsequent experiments, the antiproliferative activity of selected analogues on basic FGF (bFGF)-stimulated growth of immortalized human microvascular endothelial cells in vitro was determined. Analogues that inhibited angiogenesis to a greater extent than suramin in the CAM assay generally showed a greater antiproliferative effect on bFGF-induced growth of human microvascular endothelial cells. These results suggest that some of the polyanionic analogues may be potent therapeutic agents for cancers and angiogenesis-dependent diseases.

1. The effects of NF023, the symmetrical 3'-urea of 8-(benzamido)naphthalene-1,3,5-trisulphonic acid), and its parent compound suramin were investigated on vasoconstrictor responses to alpha, beta-methylene ATP in rabbit isolated saphenous artery and vasodilator responses to ATP in noradrenaline-precontracted rabbit isolated thoracic aorta. 2. In rabbit isolated saphenous artery, alpha, beta-methylene ATP-induced vasoconstrictor responses via P2X-receptors were concentration-dependently and competitively antagonised by NF023 (30-300 microM; pA2 = 5.69 +/- 0.04). Suramin (100-1000 microM) also competitively blocked vasoconstrictor responses to alpha, beta-methylene ATP, albeit with lower potency (pA2 = 4.79 +/- 0.05). In contrast, NF023 (100 microM) did not significantly affect contractile responses to noradrenaline or histamine in the saphenous artery. 3. In noradrenaline-precontracted rabbit isolated thoracic aorta preparations, ATP (3-3000 microM) concentration-dependently induced relaxations via endothelium-dependent or smooth muscle P2Y-receptor subtypes. NF023 (30-300 microM) failed to block relaxant responses to ATP at endothelium-dependent P2Y-receptors, whereas suramin (100-1000 microM) did antagonise endothelium-dependent vasodilator responses to ATP. Neither NF023 (100 microM) nor suramin (300 microM) influenced vasorelaxant responses to ATP via endothelium-independent P2Y-receptors. 4. In conclusion, this study outlines the selectivity of NF023 as an effective P2X-receptor antagonist in rabbit isolated blood vessels without affecting endothelium-dependent or endothelium-independent P2Y-receptor subtypes, adrenoceptors or histamine receptors.

A high-performance liquid chromatography (HPLC) method for the determination of suramin, its precursors and analogues in a aqueous solutions and in plasma samples with advantages compared to earlier methods is described. Due to the method's high sensitivity (detection limit of suramin in plasma samples: 7 ng/ml; in aqueous solutions: 5 ng/ml) and selectivity suramin t R : 7.05 min, precursor amine 2 t R : 4.68 min ), it is possible to analyze degradation products, impurities and possible metabolites of suramin besides suramin. Tetrabutylammonium hydrogensulfate (TBAHS) (5 mM) is used as ion-pairing reagent in a mixture of 36% methanol and 0.02 M phosphate buffer pH 6.5 is used as the mobile phase. After sample injection, a linear gradient from 36 to 62.9% methanol is run. A C8 stationary phase (100 × 2.1 mm I.D.) is used and ultraviolet (UV) detection at 238 nm is applied. Plasma extraction is performed with tetrabutylammonium bromide (pH 8.0) and acetonitrile. This procedure allows the determination of suraminn and its precursor amine 2 in the range of 0.05–400 μg/ml with high precision [relative standard deviation of peak areas at 0.05 μg/ml: 2.10% (n = 5)] and nearly complete recovery (>96.5%). Because of the high flexibility of the chromatographic system and subsequently the universality of the method, the analysis of a broad range of suramin analogues is possible. The result of the purity check of two suramin analogues is given.

Ecto-nucleotidases are plasma membrane-bound enzymes that sequentially dephosphorylate extracellular nucleotides such as ATP. This breakdown of ATP and other nucleotides obscures the characterization and classification of P2 (nucleotide) receptors. We therefore studied suramin and several of its analogs, divalent cations and ATP gamma S for their ability to inhibit ecto-ATPase in human blood cells. Suramin itself and Ni2+ were the more potent, non-competitive inhibitors with micromolar affinity. ATP gamma S also displayed micromolar affinity and inhibited ecto-ATPase competitively. The data obtained with the divalent cations demonstrate that coordination of the phosphate chain but not the N7 of the adenine ring is required for the breakdown of ATP by ecto-ATPase. Divalent cations that coordinate both the phosphate chain and N7 inhibit ecto-ATPase in a non-competitive manner.

Binding of the radioligand [35S]adenosine 5'-O-(2-thiodiphosphate) (ADP beta 35S) to P2 gamma purinoceptors on turkey erythrocyte membranes was used to determine the affinity of suramin and various suramin congeners belonging to different structure classes (large urea, small urea, dibenzamides and benzamides) for these receptors. Suramin was shown to be a competitive antagonist with a Ki value of 7.3 +/- 2.2 microM. The simple benzamide compound XAMR0721 (8-(3,5-dinitrophenylene carbonylimino)-1,3,5-naphthalene trisulfonate, trisodium salt) displays a high affinity for the P2 gamma purinoceptor (Ki value of 19 +/- 6 microM). Similar to suramin, compound XAMR0721 is a competitive antagonist at P2 gamma purinoceptors. In contrast to suramin, which is a potent inhibitor of the ecto-nucleotidase activity in human blood cells (44 +/- 2% residual activity at 100 microM), compound XAMR0721 is hardly active in this assay (93 +/- 1% residual activity at 100 microM). So XAMR0721, the first competitive antagonist for P2 purinoceptors that is able to discriminate between P2 purinoceptor affinity and ecto-nucleotidase activity, is an interesting pharmacological tool for the characterization of P2 purinoceptor mediated effects.

The reverse transcriptase (RT) inhibition and the specificity of 15 aminonaphthalenesulfonic acid derivatives were examined with RT of a simian immunodeficiency virus derived from an African green monkey (SIVagmTYO-7). The two compounds with the strongest RT inhibition (NF415) or the highest specificity (NF345), together with suramin, were evaluated against polymerase alpha-primase complex from calf thymus. We have also compared the kinetics of inhibition of the viral and the cellular polymerase by these three compounds. While RT inhibition followed a mixed competitive and non-competitive mechanism, inhibition of the DNA polymerase alpha was competitive for suramin and non-competitive for NF415 and NF345. Certain structural characteristics appeared to be common for specific RT inhibitors.

A synthetic way for obtaining 6-amino-2,4-dimethylquinoline derivatives from 2,5-dichloraniline by successive ring closure, nitration, aromatic substitution at so activated quinoline ring, followed by reduction of the nitro group to amino group and alkylation of the last is described.

A synthetic way for obtaining 6-amino-2,4-dimethylquinoline derivatives from 2,5-dichloraniline by successive ring closure, nitration, aromatic substitution at so activated quinoline ring, followed by reduction of the nitro group to amino group and alkylation of the last is described.

Suramin is a polysulfonated naphthylurea currently investigated for the treatment of advanced malignancy. We have previously reported that suramin was a potent inducer of the differentiation of the human colic adenocarcinoma cell clone HT29-D4 (Fantini et al., J Biol Chem, 1989; 264: 10282-10286). In this report, we show that chronic suramin treatment of these cells is associated with a lysosomal storage disease. We have tested five suramin-related compounds for their ability to induce HT29-D4 differentiation and we looked at their action on the lysosomal apparatus. We conclude that one of the derivatives used is a more potent differentiation inducer than suramin, while it does not elicit any perturbation of the lysosomal system.

The dose-response curve for the vasopressor effect of alpha, beta-methylene ATP in pithed rats was influenced by four suramin-related drugs (each at 100 mumol/kg). The curve was shifted to the right by a factor of 8 by 'compound 3' and by a factor of 2 by two other derivatives, but was not affected by the fourth analogue. Compound 3 had no effect on the vasopressor response to noradrenaline or neuropeptide Y. In conclusion, compound 3 is a purine P2X receptor antagonist which is approximately as potent as suramin, and which, like suramin, does not exhibit antagonistic properties at alpha-adrenoceptors and neuropeptide y receptors.

The role of ATP-stimulated P2X1 receptors in human platelets is still unclear. They may act alone or in synergy with other pathways, such as P2Y1 or P2Y12 receptors, to accelerate and enhance calcium mobilisation, shape change and aggregation. To date very few pharmacological means of selectively inhibiting platelet P2X1 receptors have been described, although recent work has shown that suramin is a useful lead compound for the development of high-affinity P2X1 antagonists. We therefore investigated the effects of a series of bivalent and tetravalent suramin analogues on alphabeta meATP (P2X1 receptors)-induced or ADP (P2Y1 receptors)-induced intracellular calcium increases and shape change, as well as on ADP-induced aggregation (P2Y1P2Y12 receptors) in human platelets. Changes in intracellular calcium were measured using standard fluorescence techniques, while shape change and aggregation were determined by turbidimetry. The novel tetravalent compound NF864 (8,8',8'',8'''-(carbonylbis(imino-5,1,3-benzenetriyl-bis(carbonylimino)))tetrakis-naphthalene-1,3,5-trisulfonic acid-dodecasodium salt) proved to be the most potent platelet P2X1 antagonist reported to date, blocking alphabeta meATP-induced Ca2+ increases and shape change in a concentration-dependent manner, with a pA2 of 8.17 and 8.49, respectively. The ability to inhibit the platelet P2X1 receptor displayed the following order : NF864NF449or = NF110NF023 = MK-HU1 = suramin. A different antagonistic profile was observed for ADP-induced Ca2+ increases, shape change and aggregation; however, overall four compounds showed sufficient ability to selectively inhibit P2X1 responses, with the order NF110NF449or = NF864or = MK-HU1. Therefore, these compounds should prove useful tools for investigating the functional significance of platelet P2X1 receptors in thrombosis and haemostasis, NF864 being the most promising compound.

NF449 [4,4',4",4"'-(carbonylbis(imino-5,1,3-benzenetriyl-bis(carbonylimino)))tetrakisbenzene-1,3-disulfonic acid-octasodiumsalt)] was recently described to inhibit recombinant rP2X(1) receptors (Naunyn Schmiedeberg's Arch. Pharmacol. 364 (2001) 285). The purpose of this study was to examine structure-activity-relationships at P2 receptors of a series of NF449 analogues. Thus, compounds containing various arylaminemono-, di-, or trisulfonic acids and a replacement of the central urea bridge were synthesized. NF449 displayed a pIC(50) at P2X(1) receptors (rat vas deferens) of 6.31 +/- 0.04 being at least 19-fold more potent at P2X(1) than at P2X(3), P2Y(1), P2Y(2), or P2Y(11). Any deletion or change of position of sulfonic acid groups or replacing the central urea bond by the bisamide of terephthalic acid reduced the potency at P2X(1) by at least 90%. All compounds were very weak antagonists at P2Y(2) or P2Y(11) receptors (pIC(50)4.5). In conclusion, NF449 remains the most potent and selective P2X(1) antagonist known. Potential lead compounds among the suramin class for P2X(3) (16d) and P2Y(1) (16a) receptors were identified.

FTY720 is highly effective in various models of transplantation and autoimmunity. In order to find drugs that act synergistically with a tolerance-inducing nondepleting anti-CD4 mAb we studied this combination in a strong DA to LEW kidney transplantation model. Rats were treated with 0.3 mg/kg of FTY720 for 14 days and anti-CD4 mAb RIB5/2, alone or in combination. After kidney transplantation serum creatinine and blood lymphocyte counts were monitored. Immunohistology, ELISPOT and TaqMan trade mark -PCR analysis of biopsies were performed. Short-term application of RIB5/2 but not FTY720 induced long-term survival of kidney transplants. Moreover, the combination of FTY720 + RIB5/2 prevented tolerance induction. In the combination group serum creatinine levels increased 1 week after cessation of therapy and all rats died from uremia within 72 days. Intragraft immunohistology, ELISPOT and real-time RT-PCR analysis at day 21 demonstrated an enhanced T-cell infiltration and activation but a diminished up-regulation of protective genes in the grafts from recipients receiving the combination therapy. In contrast, delayed application of FTY720 to RIB5/2-treated rats did not interact with RIB5/2-induced tolerance. In summary, FTY720 is powerful in preventing intragraft infiltration by naive T cells but this might also affect the early development of graft-protecting regulatory T cells and tolerance induction.

Antagonistic effects of the novel suramin analogue 4,4',4",4"'-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid (NF449) were studied on contractions of the rat vas deferens elicited by alpha,beta-methylene ATP (alphabetameATP; mediated by P2X1 receptors), contractions of the guinea-pig ileal longitudinal smooth muscle elicited by alphabetameATP (mediated by P2X3 receptors) or adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS; mediated by P2Y1 receptors), ATP-induced increases of [Ca2+]i in human embryonic kidney (HEK) 293 cells (mediated by P2Y2 receptors), inward currents evoked by ATP in follicle cell-free Xenopus laevis oocytes expressing rP2X1 or rP2X3 receptors and degradation of ATP by ecto-nucleotidases in folliculated Xenopus laevis oocytes. In addition, NF449 was examined for its P2 receptor specificity in rat vas deferens (alpha1A-adrenoceptors) and guinea-pig ileum (histamine H1 and muscarinic M3 receptors). At native (pIC50=7.15) and recombinant (pIC50=9.54) P2X1 receptors, NF449 was a highly potent antagonist. The P2X3 receptors present in guinea-pig ileum (pIC50=5.04) or expressed in oocytes (pIC50 approximately 5.6) were much less sensitive for NF449. It also was a very weak antagonist at P2Y1 receptors in guinea-pig ileum (pIC50=4.85) and P2Y2 receptors in HEK 293 cells (pIC50=3.86), and showed very low inhibitory potency on ecto-nucleotidases (pIC503.5). NF449 (100 microM) did not interact with alpha1A-adrenoceptors or histamine H1 and muscarinic M3 receptors. Thus, the antagonism by NF449 is highly specific for P2 receptors. In conclusion, the subnanomolar potency at rP2X1 receptors and the rank order of potency, P2X1P2X3P2Y1P2Y2ecto-nucleotidases, make NF449 unique among the P2 receptor antagonists reported to date. NF449 may fill the long-standing need for a P2X1-selective radioligand.

Among suramin analogues, the properties of P2 receptor subtype blockade and ecto-nucleotidase inhibition appear to be controlled by different structural parameters (Fig. 1 and 2, Table 1; Van Rhee et al., 1994; Beukers et al., 1995; Bultmann et al., 1996; Damer et al., 1998a, 1998b; and this study): the molecular size of the compounds, the position of the sulfonic acid residues in the naphthalene rings, the substitution pattern of the benzoyl moieties and the 3'- or 4'-aminobenzoyl-linkages of the phenyl rings "1" and "2". As a result, compounds with different receptor selectivity profiles were obtained. A maximum in potency at and selectivity for P2X1 receptors is reached in NF279, which is a specific P2 receptor antagonist and the compound with the highest P2X1 vs. P2Y receptor and ecto-nucleotidase selectivity presently available.

8,8'-(Carbonylbis(imino-4, 1 -phenylenecarbonylimino-4,1-phenylenecarbonylimino))bis(1,3, 5-naphthalenetrisulfonic acid) (NF279) antagonized P2X receptor-mediated contractions in rat vas deferens, evoked by alpha,beta-methylene ATP (10 microM; pIC50=5.71) without affecting responses mediated via alpha1A-adrenoceptors, adenosine A1 and A2B receptors, histamine H1, muscarinic M3 and nicotinic receptors. The low inhibitory potency of NF279 on P2Y receptors in guinea-pig taenia coli (pA2=4.10) and at ecto-nucleotidases in folliculated Xenopus laevis oocytes (IC50 > 100 microM) indicates that NF279 is a novel specific and selective P2X receptor antagonist.

Suramin acts as a G protein inhibitor because it inhibits the rate-limiting step in activation of the G α subunit, i.e., the exchange of GDP for GTP. Here, we have searched for analogues that are selective for G sα . Two compounds have been identified: NF449 (4,4′,4",4′"-[carbonyl-bis[imino-5,1,3-benzenetriyl bis-(carbonylimino)]]tetrakis-(benzene-1,3-disulfonate) and NF503 (4,4′-[carbonylbis[imino-3,1-phenylene-(2,5-benzimidazolylene)carbonylimino]]bis-benzenesulfonate). These compounds ( i ) suppress the association rate of guanosine 5′-[γ-thio]triphosphate ([ 35 S]GTP[γS]) binding to G sα-s but not to G iα-1 , ( ii ) inhibit stimulation of adenylyl cyclase activity in S49 cyc − membranes (deficient in endogenous G sα ) by exogenously added G sα-s , and ( iii ) block the coupling of β-adrenergic receptors to G s with half-maximum effects in the low micromolar range. In contrast to suramin, which is not selective, NF503 and NF449 disrupt the interaction of the A 1 -adenosine receptor with its cognate G proteins (G i /G o ) at concentrations that are >30-fold higher than those required for uncoupling of β-adrenergic receptor/G s tandems; similarly, the angiotensin II type-1 receptor (a prototypical G q -coupled receptor) is barely affected by the compounds. Thus, NF503 and NF449 fulfill essential criteria for G sα -selective antagonists. The observations demonstrate the feasibility of subtype-selective G protein inhibition.

Effects of suramin and five analogs or fragments of suramin were studied on contractions of the rat vas deferens elicited by alpha, beta-methylene ATP (alpha, beta-MeATP; mediated by P2X-purinoceptors), relaxations of the carbachol-precontracted guinea-pig taenia coli elicited by adenosine 5'-O-(2-thiodiphosphate) (ADP beta S; mediated by P2Y-purinoceptors), and the degradation of ATP by rat vas deferens tissue. One compound, NF023, differed from suramin by removal of two p-methylbenzamido groups, whereas another, BSt101, differed from NF023 by additional removal of the three sulphonate residues from one of the terminal naphthalene rings. The compounds all shifted the concentration-response curve of alpha, beta-MeATP in the rat vas deferens to the right and simultaneously increased the maximum of the curve. Where three concentrations were tested, the Arunlakshana-Schild regression was linear, and the slope did not differ from 1. The apparent Kd values were between 1 and 3672 microM. In the guinea-pig taenia coli, the compounds shifted the concentration-response curve of ADP beta S to the right in a parallel manner, but in the one case where three concentrations were tested, the slope of the Arunlakshana-Schild regression was lower than 1. Apparent Kd values were between 10 and 786 microM. The removal of ATP from the medium by vas deferens tissue was decreased only by suramin, NF023 and BSt101, with IC25% values between 170 and 590 microM.

The stability of an aqueous solution of suramin has been determined. The only degradation product detectable by HPLC was the amine precursor 2. The decomposition kinetics of suramin at different temperatures are shown. Because of first order kinetics the Arrhenius equation could not be used to evaluate the decomposition data. A good correlation was obtained between the reaction constants and the temperature (r = 0.9898). After 42 days at 37 degrees C, 2 % of suramin are hydrolysed. Possible implications of our results for the search for suramin metabolites in patients are discussed.

At least five distinct P2-purinoceptor subtypes have been characterized to date, based on the rank order of potency of several ATP analogues: P2X, P2Y, P2U, P2T and P2Z. However, the characterization of P2-purinoceptor subtypes is hampered by an unavailability of potent, highly selective, competitive antagonists. In the search for selective P2-purinoceptor antagonists, the structure-activity relationships for a series of analogues of pyridoxal-5-phosphate and suramin at P2-purinoceptor subtypes were investigated in our laboratories. Two of these compounds were the subject of a more detailed pharmacological characterization: pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) and the symmetrical 3′-urea of 8-(benzamido)naphthalene-1,3,5-trisulfonic acid (NF023). The results demonstrate that the two parent compounds, pyridoxal-5-phosphate and suramin, do not differentiate between P2X- and P2Y-purinoceptors. In contrast, PPADS and NF023 were found to be selective antagonists of P2X-purinoceptor-mediated responses in several smooth muscle preparations. In addition, PPADS and NF023 were shown to displace competitively the binding of [3H]α,β-methylene ATP to rabbit and rat bladder membranes, respectively, which indicates that these two compounds act directly on P2X-receptors. PPADS and NF023 were ineffective at P2U-purinoceptors in rat mesenteric arterial bed. P2T-purinoceptor-mediated platelet aggregation was only affected by PPADS in concentrations higher than 100 μM. Suramin and NF023 were inhibitors of ecto-ATPase activity in the same concentration range needed for P2-purinoceptor antagonism. In contrast, PPADS was only very weakly active in inhibiting ecto-ATPase activity. AT 100 μM, PPADS and NF023 did not interact with α1-adrenoceptors, adenosine A1- and A2-, histamine H1- and muscarinic M1-, M2- and M3-receptors. In conclusion, PPADS and NF023 are specific P2-purinoceptor antagonists showing a high selectivity for the P2X-subtype. These two compounds may prove to be useful starting points in the synthesis of novel, highly potent and selective antagonists at P2-purinoceptor subtypes.

Versuche 5.8-Dimethoxychinolin-2,4-dicarbonsaure 10c, deren Dichlorid 11c oder deren Ester 12c und 13c zu 2.4-bis-Hydroxymethyl-5.8-dimethoxychinolin 6c zu reduzieren waren nicht erfolgreich. Die Chinolin-2-bzw. 4-Mono-carbonsaureester 12d bzw. 12e liesen sich dagegen glatt zu den entsprechenden 2-bzw. 4-Hydroxymethylchinolinen 6d bzw. 6e reduzieren. Antimalarial 6-Aminoquinolines, X: Further Experiments on 2- and 4-Hydroxymethyl Derivatives Attempts to reduce 5,8-dimethoxyquinoline-2,4-dicarboxylic acid 10c, the dichloride 11c or the esters 12c and 13c to 2,4-bis(hydroxymethyl)-5,8-dimethoxyquinoline 6c were not successful. However, the esters 12d and 12c of the 2- and 4-monocarboxylic acids could be easily reduced to yield the 2- and 4-hydroxymethyl derivatives 6d and 6e.

Aus der Umsetzung des als aktive Methylenverbindung fungierenden Malonsaure-diathylesters (2a), Cyanessigsaure-athylesters (2b), Acetessigsaure-athylesters (2d) und Malonitrils (7) mit s-Triazin (1) in Gegenwart eines sekundaren Amins 5 gehen die Dehydro-C-Mannich-Basen 6a--6d und 8 hervor. Abweichend von diesem Reaktionsverhalten bildet sich jedoch aus 2a durch Einwirkung von 1 und Diathylamin (5c) das 5-Athoxycarbonyl-4-hydroxy-pyrimidin (10). Application of Aminomethynylating Three-Component Reaction to CH-Active Compounds Reaction of diethyl malonate (2a), ethyl cyanoacetate (2b), ethyl acetoacetate (2d), or malononitrile (7), which act as CH-active methylene compounds, with s-triazine (1) in the presence of a secondary amine 5 leads to the dehydro-C-Mannich-bases 6a--6d and 8. This course is not followed by the reaction of 2a with 1 and diethylamine (5c) which leads to 5-ethoxycarbonyl-4-hydroxypyrimidine (10).

Die Synthese der 2- und 4-Hydroxymethyl-Derivate li und 1m von 6-(4-Diaathylamino-1-methyl-butylamino)-5,8-dimethoxychinolin (1a) wird beschrieben. 1i und 1m werden als mogliche Metabolite der Methyl-derivate 1b bzw. 1d angesehen. 1i und 1m sind weniger toxisch und vergleichbar wirksam gegen P. vinckei wie 1b bzw. 1d. Antimalarial 6-Aminoquinolines, IX: 2- and 4-Hydroxymethyl Derivatives The 2-and 4-hydroxymethyl derivatives 1i and 1m of 6-(4-diethylamino-1-methylbutylamino)-5,8-dimethoxy-quinoline (1a) were synthesized. 1i and 1m are regarded as potential metabolites of the methyl derivatives 1b and 1d, Hydroxylation of 1i and 1m reduces the toxicity and has little influence on the plasmodicidal activity.

Zwei mono-Chlorderivate, 7a und 7b, und zwei di-Chlor-derivate, 7c und 7d, von 6-(4-Diathylamino-1-methyl-butylamino)-5,8-dimethoxychinolin (1a) wurden synthetisiert, um den Einflus von Chlorsubstituenten auf Malaria-Wirksamkeit und Toxizitat von 1a zu untersuchen. 7a–d besitzen nur eine geringe Wirksamkeit gegen Plasmodium vinckei (in der Maus). Antimalarial 6-Aminoquinolines, VI: 2-, 3- and 4-Chloro Derivatives of 6-(4-Diethylamino-1-methylbutylamino)-5.8-dimethoxyquinoline Two monochloro derivatives, 7a and 7b, and two dichloro derivatives, 7c and 7d, of 6-(4-diethylamino-1-methylbutylamino)-5.8-dimethoxyquinoline (1a) were synthesized with the aim of studying the influence of chloro substituents on the antimalarial activity and toxicity of 1a. 7a–d showed only slight activity against Plasmodium vinckei (in mice).

Zwei Derivate von 5.8-Dimethoxy-2.4-dimethylchinolin mit einer 2-Diethyl-aminoethoxy-(3a) bzw. 3-diethyl-aminopropoxyseitenkette in 6-Stellung (3b) wurden synthetisiert, um zu uberprufen, ob in Verbindungen vom Typ 1 die Wirksamkeit gegen Plasmodien erhalten bleibt, wenn in der basischen Seitenkette der ringstandige Stickstoff durch Sauerstoff ersetzt wird. 3a und 3b zeigen keine Wirkung gegen P. vinckei. Potential Antimalarials, I:6-(Diethylaminoalkoxy)-5,8-dimethoxyquinolines Two derivatives of 5,8-dimethoxy-2,4-dimethylquinoline with a 2-diethylaminoethoxy (3a) and a 3-diethylaminopropoxy side chain (3b) at C-6 were synthesized with the aim to study the influence of the replacement of a nitrogen atom in the side chain of the antimalarial 6-aminoquinoline 1 by an oxygen atom on antimalarial activity. Compounds 3a and 3b are inactive against P. vinckei.

Die sieben moglichen 2-, 3- und 4-Mono-, Di- und Trimethylderivate 2b-h von 6-(4-Di-athylamino-1-methylbutylamino)-5,8-dimethoxychinolin (2a) wurden synthetisiert, um den Einflus von Methylsubstituenten im Pyridinring von 2a auf dessen Malariawirksamkeit und Toxizitat zu untersuchen. Antimalarial 6-Aminoquinolines, V. — 2-, 3-, and 4-Mono-, Di-, and Trimethyl Derivatives of 6-(4-Diethylamino-1-methylbutylamino)-5,8-dimethoxyquinoline The seven possible 2-, 3-, and 4-mono-, di-, and trimethyl derivatives 2b-h of 6-(4-diethyl-amino-1-methylbutylamino)quinoline (2a) were synthesised with the aim of studying the influence of methyl groups in the pyridine ring of 2a on antimalarial activity and toxicity.

Die Synthese der Titelverbindungen wurde versucht, ausgehend von 6-Aminochinolinen uber die 6-Diazoniumverbindung, ausgehend von 6-Bromchinolinen uber eine Grignardverbindung und durch selektive Etherspaltung von 5,6,8-Trialkoxychinolinen. Keiner der eingeschlagenen Wege fuhrte zum Erfolg. Attempted Synthesis of 5,8-Dimethoxy-6-hydroxyquinolines The synthesis of the title compounds was attempted starting from 6-aminoquinolines via diazonium compounds, starting from a 6-bromoquinoline via a Grignard compound or by selective cleavage of an ether group in a 5,6,8-trialkoxyquinoline. None of these ways was successful.

Neun in 2-, 3-, und/oder 4-Stellung unterschiedlich substituierte 8-Methoxy-chinolin-5,6-dione 3a-i werden durch Oxidation der entspr. 6-Amino-5,8-di-methoxy-chinoline 1a-i erhalten. Diese Oxidation verlauft nur glatt, wenn die 8-Stellung der 6-Aminochinoline substituiert ist. Aus 6-Amino-2,4-di-methyl-5-methoxychinolin (4) entsteht bei der Oxidation 2,4-Dimethyl-8-((2,4-dimethyl-5-methoxy-6-chinolyl)amino)chinolin-5,6-dion (5). 8-Methoxy-quinoline-5,6-diones, II Nine 8-methoxy-quinoline-5,6-diones with varying substituents in the 2-, 3-, and/or 4-position 3a-i were synthesized by oxidation of the corresponding 6-amino-5,8-dimethoxyquinolines 1a-i. By this oxidation quinones of type 3 are obtained only if the 8-position of the quinoline nucleus is substituted. Oxidation of 6-amino-2,4-dimethyl-5-methoxyquinoline (4) yields 2,4-dimethyl-8-((2,4-dimethyl-5-methoxy-6-quinolyl)amino)-quinoline-5,6-dione (5).

Zwolf in 6-Stellung basisch alkylierte 6-Amino-5.8-dimethoxy-chinaldine mit den Seiten-ketten (C2H5)2N(CH2)nNH (n = 2, 3; 15, 16) und H2N(CH2)nCHRNH(n = 19, R H, CH3; 21a–k) wurden synthetisiert, um den Einflus der basischen Seitenkette auf Malaria-Wirksamkeit und Toxizitat der 6-Amino-chinoline zu untersuchen. Antimalarial 6-Aminoquinolines, II. 6-[ω-Diethylaminoalkylamino]- and 6-[ω-Aminoalkyl-amino]-5,8-dimethoxyquinaldines Twelve derivatives of 6-amino-5,8-dimethoxyquinaldine with the basic side chains (C2H5)2N(CH2)nNH (n = 2, 3; 15, 16) and H2N(CH2)nCHRNH (n = 1 – 9, R H, CH3; 21a–k) in the 6-position are prepared with the aim of studying the influence of the basic side chain on malarial activity and toxicity of the 6-aminoquinolines.

Die spektrale Empfindlichkeitsverteilung eines Zeiss-Spektralfluorometers wird ermittelt. Die kauflichen Szintillatorsubstanzen POP, POPOP und Terphenyl werden als Vergleichssubstanzen in der Fluorometrie vorgeschlagen. Die UV-Absorptionsspektren und die korr. Fluoreszenzspektren von 2-Phenylthionaphthen-S-dioxyd und anderen ringgeschlossenen trans-Stilben-Derivaten sowie von ringgeschlossenen Diphenyl-Derivaten in Losung werden ermittelt; relative Quantenausbeuten der praktischen auseren Fluoreszenz werden angegeben. — Das 1.3-Dihydro-2H-1.3.2-benzodiazaborol fluoresziert stark, nicht dagegen die entsprechende Sauerstoffverbindung, o-Phenylen-phenyl-boronat.