Design, synthesis and antiproliferative evaluation of fluorenone analogs with DNA topoisomerase I inhibitory properties.

A series of 2,7-diamidofluorenones were designed, synthesized, and screened by SRB assay. Some synthesized compounds exhibited antitumor activities in submicromolar range. Ten compounds (3a, 3b, 3c, 3g, 3j, 3l, 4a, 4h, 4i, and 4j) were also selected by NCI screening system and 3c (GI50=1.66 µM) appeared to be the most active agent of this series. Furthermore, 3c attenuated topoisomerase I-mediated DNA relaxation at low micromolar concentrations. These results indicated that fluorenones have potential to be further developed into anticancer drugs.

Development of sustained-release matrix tablets of BKP-01-041 (tilorone derivative) containing Hypromellose.

The objective of this research was to develop and evaluate sustained-release matrix tablets of BKP-01-041 (tilorone derivative) based on Hypromellose (hydroxypropyl methylcellulose, HPMC) as the matrix forming polymer. The sustained-release tablets were prepared by the wet granulation method. The influence of HPMC viscosity and ratios on drug release was investigated in vitro. Dissolution of the tablets developed with 26% HPMC K4 M/K100 M (1:2) (w/w) content showed a better drug release profile than the other batches tested in 12 h. Drug release from the optimal formulation was analyzed using release kinetics equations. The release kinetics parameters were determined and the value of the exponent (n) representing the apparent drug release mechanism determined from the Peppas equation was about 0.726. These results suggest that the drug release mechanism was non-Fickian (0.45 < n < 0.89), and drug release was dependent on both drug diffusion and polymer erosion.

[Effect of amyxine, loramyxine and their composites with yeast RNA on spontaneous and induced mutagenesis in Salmonella typhymurium].

It was found out that amyxine, loramyxine and their composites with yeast DNA do not increase reliably the frequency of spontaneous mutations in test-strain S. typhymurium TA 98. The studied substances do not possess the antimutagen activity in respect of mutations, induced by potassium bichromate in S. typhymurium TA 98 and do not increase their quantity.

alpha7 nicotinic acetylcholine receptor agonist properties of tilorone and related tricyclic analogues.

BACKGROUND AND PURPOSE: The alpha7 nicotinic acetylcholine receptor (nAChR) has attracted considerable interest as a target for cognitive enhancement in schizophrenia and Alzheimer's Disease. However, most recently described alpha7 agonists are derived from the quinuclidine structural class. Alternatively, the present study identifies tilorone as a novel alpha7-selective agonist and characterizes analogues developed from this lead. EXPERIMENTAL APPROACH: Activity and selectivity were determined from rat brain alpha7 and alpha4beta2 nAChR binding, recombinant nAChR activation, and native alpha7 nAChR mediated stimulation of ERK1/2 phosphorylation in PC12 cells. KEY RESULTS: Tilorone bound alpha7 nAChR (IC(50) 110 nM) with high selectivity relative to alpha4beta2 (IC(50) 70 000 nM), activated human alpha7 nAChR with an EC(50) value of 2.5 microM and maximal response of 67% relative to acetylcholine, and showed little agonist effect at human alpha3beta4 or alpha4beta2 nAChRs. However, the rat alpha7 nAChR maximal response was only 34%. Lead optimization led to 2-(5-methyl-hexahydro-pyrrolo[3,4-c]pyrrol-2-yl)-xanthen-9-one (A-844606) with improved binding (alpha7 IC(50) 11 nM, alpha4beta2 IC(50)>30 000 nM) and activity at both human and rat alpha7 nAChR (EC(50)s 1.4 and 2.2 microM and apparent efficacies 61 and 63%, respectively). These compounds also activated native alpha7 nAChR, stimulating ERK1/2 phosphorylation in PC12 cells. CONCLUSIONS AND IMPLICATIONS: Tilorone, known as an interferon inducer, is a selective alpha7 nAChR agonist, suggesting utility of the fluorenone pharmacophore for the development of alpha7 nAChR selective agonists. Whether alpha7 stimulation mediates interferon induction, or whether interferon induction may influence the potential anti-inflammatory properties of alpha7 nAChR agonists remains to be elucidated.

Simultaneous quantification of tiloronoxim and tilorone in human urine by liquid chromatography-tandem mass spectrometry.

A simple, sensitive and specific HPLC method with tandem mass spectrometry (HPLC/MS/MS) detection has been developed and validated for the simultaneous quantification of tiloronoxim and its major active metabolite, tilorone, in human urine. The analytes, together with metoprolol, which was employed as an internal standard (IS), were extracted with a mixture solvent of chloroform/ethyl ether (1/2, v/v). The chromatographic separation was performed on a narrow-bore reversed phase HPLC column with a gradient mobile phase of methanol/water containing 15 mM ammonium bicarbonate (pH 10.5). The API 3,000 mass spectrometer was equipped with a TurboIonSpray interface and was operated on positive-ion, multiple reaction-monitoring (MRM) mode. The mass transitions monitored were m/z 426.3-->100.0, m/z 411.3-->100.0 and m/z 268.3-->116.1 for tiloronoxim, tilorone and the IS, respectively. The assay exhibited a linear dynamic range of 1-100 ng/ml for both tiloronoxim and tilorone based on the analysis of 0.2 ml aliquots of urine. The lower limit of quantification was 1 ng/ml for both compounds. Acceptable precision and accuracies were obtained for concentrations over the standard curve ranges. Run time of 8 min for each injection made it possible to analyze a high throughput of urine samples. The assay has been successfully used to analyze human urine samples from healthy volunteers.

[Interferonogenic activity of amixin analogs and diphenyl derivatives].

Properties of interferon productivity of several inducers of interferon, analogs of amixin, have been studied in vivo. It has been shown, that under the influence of injected agents the content of endogenic interferon in the blood serum of laboratory animals increased and their non-fractionated cells of the peripheral blood (splenocites and macrophages) synthesized alpha- and gamma-interferon.

Inhibition of deoxynucleotide-polymerizing enzyme activities of human leukemia lymphoblasts and simian sarcoma virus by tilorone and thirteen of its analogs.

Tilorone, which is 2,7-bis[2-(diethylamino)ethoxy]-9H-fluoren-9-one dihydrochloride, and 13 of its analogs inhibited human cellular DNA polymerases alpha and beta assayed with activated DNA as template and also cellular DNA polymerase gamma and DNA polymerase from simian sarcoma virus assayed with poly(A) (dT)12-18 as template. Terminal deoxynucleotidyltransferase (TdT), which has no template requirement, was not inhibited by any of the 14 compounds when d(A)12-18 or d(G)12-18 was used as initiator. Three compounds did not inhibit TdT assayed with activated DNA as initiator, but 11 compounds did, and these 11 compounds were generally less inhibitory to TdT than to the other DNA polymerases. The three compounds that did not inhibit TdT assayed with activated DNA but did inhibit the other DNA polymerases will be useful in the characterization of TdT activity. Modifications of the polycyclic ring structure of tilorone and the kinds of substituent groups attached to the ring structures influenced the degree of inhibition of all enzymes.

Inhibition of purified DNA polymerase of RNA tumor viruses by fluoranthene derivatives and analogues of tilorone hydrochloride.

At concentrations of 7 times 10(-6) to 7 times 10(-5) M, derivatives consisting of the polycylic ring structures fluoranthene, fluorenone, fluorene, anthraquinone, xanthenone, and dibenzofuran with appropriate amine side chains inhibited by over 90% the purified RNA-directed DNA polymerase of avian myeloblastosis virus acting on poly(deoxyadenylate-deoxythymidylate) [poly(dA-dT)]. Of these, only the fluoranthene derivatives were strong inhibitors of the viral DNA polymerase directed by polyadenylate-oligodeoxythymidylate [poly(A)-(dT)12-18]. Low levels of fluoranthene derivatives (1 times 10(-5) M) also strongly inhibited polymerase with polyinosinate-oligodeoxycytidylate [poly(I)-(dC)12-18], activated calf thymus DNA, and viral 70S RNA as templates, but not with polycytidylate-oligodeoxyguanylate as template. A comparison of the activity of 11 fluoranthene derivatives with different side chains showed that the structure of the amine side chain influenced both the extent of antipolymerase activity with a given template and the relative inhibition with different synthetic DNA and RNA templates. The naturally occurring polyamines, spermine, spermidine, and putrescine, did not inhibit the activity of the viral DNA polymerase. Studies on the mechanism of action indicated that the synthetic derivatives inhibited polymerase activity by binding to the template and not to the enzyme: 1) inhibition by fluoranthene derivatives was overcome by the addition of excess template including poly(dA-dT), poly(A)-(dT)12-18, poly(I)-(dC)12-18, viral 70S RNA, and activated calf thymus DNA; 2) the degree of inhibition by fluoranthene derivatives was unaffected by the addition of the creased viral DNA polymerase; 3) with the same template, Escherichia coli DNA-directed RNA polymerase and the viral RNA-directed DNA polymerase were inhibited to about the same extent; and 4) the derivatives formed a complex with DNA, poly(I), and poly(A) that was stable to exclusion chromatography on Sephadex G-100. Several derivatives also had biologic activity, since they blocked the ability of the murine sarcoma virus to transform cells.

New synthetic routes to tilorone dihydrochloride and some of its analogues.

New synthetic routes to the orally active, interferon-inducing antiviral agent tilorone dihydrochloride, 2,7-bis-[(diethylamino)ethoxy]fluoren-9-one dihydrochloride (1a), were developed. The routes involved the preparation and solvolysis of tetrazonium fluoroborate salts of 2,7-diaminofluoren-9-one. Nonplanar (1b), 9-sulfone (1c), and fluorene (1d) analogues of tilorone dihydrochloride were also prepared. Compounds 1b and 1c were evaluated for interferon induction.

Drug-induced glycosaminoglycan storage: dose-dependent changes in the pattern of accumulated glycosaminoglycans in cultured bovine and human fibroblasts.

The present study determines the amounts and patterns of glycosaminoglycans stored in cultured corneal fibroblasts after treatment with tilorone and three related compounds. The compounds have immunomodulatory properties and have been shown to impair the lysosomal degradation of glycosaminoglycans as a side effect. This side effect has been described as drug-induced mucopolysaccharidosis because the induced lysosomal storage of glycosaminoglycans leads to cellular lesions resembling those in patients with inherited mucopolysaccharidosis. In the present study, the dose-dependency of glycosaminoglycan storage was analyzed after treatment (96 hr) of bovine corneal fibroblasts. The investigated drug concentrations ranged from low concentrations inducing cytological lesions typical of drug-induced mucopolysaccharidosis to high concentrations at the borderline of cytotoxicity. The intracellular amounts of dermatan sulfate, heparan suflate, and chondroitin sulfate were quantified by densitometric scanning of Alcian Blue-stained bands after electrophoresis. All investigated compounds induced a predominant dermatan sulfate storage (3-4-fold accumulation) at low drug concentrations. With rising drug concentrations, a shift of the pattern of stored glycosaminoglycans was observed, characterized by the additional accumulation of heparan sulfate (up to 5-fold of control levels). In cultured human fibroblasts, tilorone also caused a marked dermatan sulfate storage, reaching maximum values at 5 microM and marked heparan sulfate storage at 20 microM. The present data provide evidence: (a) that selective dermatan sulfate accumulation is a characteristic feature of drug-induced glycosaminoglycan storage in cultured bovine and human fibroblasts, if these cells are treated with low concentrations (< or = 5 microM), that are likely to reflect the situation in vivo; and (b) that additional heparan sulfate storage is induced in vitro only by treatment with high concentrations that induce nonspecific cellular lesions.

Enhanced resistance to experimental systemic candidiasis in tilorone-treated mice.

Candida albicans is an increasingly important opportunistic fungal pathogen in immunocompromised patients. Natural killer (NK) cells constitute an important immune effector mechanism and are involved in the response to different pathological disorders. We wished to determine if this immune mechanism is involved in the specific response to C. albicans. Tilorone hydrochloride and related compounds have been described to display antiviral and antitumoral activity, as well as to enhance NK cell activity. In this study, we show the antimicrobial activity of different tilorone analogues and the enhanced resistance of tilorone-treated mice in experimental systemic candidiasis. We also present data suggesting that there is a correlation between NK cell activation and the resistance to experimental systemic candidiasis. Thus, it seems that the immunosurveillance of metastatic spread and the infection by C. albicans share some immune effector mechanisms, in particular activation of NK cells.

Mucopolysaccharidosis and lipidosis in rats treated with tilorone analogues.

The experimental immunomodulatory agent tilorone was previously reported to induce generalized mucopolysaccharidosis (lysosomal storage of sulfated glycosaminoglycans) and lipidosis in rats. While lipidosis is a side effect common to many cationic amphiphilic compounds, none of them except tilorone has been known to cause mucopolysaccharidosis in intact animals. The purpose of the present histochemical and ultrastructural study was to examine whether or not mucopolysaccharidosis can be induced by tilorone analogues. Three analogues were selected and administered to rats in short-term and subchronic experiments, and liver, spleen, kidney, and cornea were examined. The analogues caused generalized cellular lesions which had the same histochemical and cytological characteristics and the same distribution as the lesions produced by tilorone. The results show that the ability to induce mucopolysaccharidosis is not a unique property of tilorone; this drug side effect should be taken into account when developing new drugs with molecular structures resembling that of tilorone.

N,N-diethylaminoalkoxy-4,7-phenanthrolines and 1,8-diazaflourene derivatives, a novel class of potential interferon inducers and antiviral agents: interactions with nucleic acids in vitro and cellular activities.

Tilorone aza-analogues, derivatives of 4,7-phenanthroline and 1,8-diazafluorene, were examined as DNA-complexing agents by spectral and electrophoretic methods. The binding process includes at least two types of interactions: electrostatic and, possibly, intercalation. Complex formation with the denatured DNA was also observed, but its nature remained unsolved. Binding and thermodynamic parameters were determined. All ligands studied showed weak antiviral activity and essentially no interferon induction when assayed in vitro and in vivo. It was concluded that interferon induction by tilorone may involve specific cell receptors or intermediaries.

Abnormal dendritic maturation of neurons under the influence of a Tilorone analogue (R 10.874).

Tilorone analogue (R 10.874) has a close affinity to the lysosomal compartment of cells and forms a non degradable carbohydrate-lipid-drug complex accumulated within digesting organelles. Resembling biochemical and structural changes are seen in hereditary mucopolysaccharidoses accompanied with abnormal dendritogenesis. On the other hand, developmental toxicity (TERRY et al. 1992), antiproliferative effects (ALGARRA et al. 1993) and interactions with DNA (GELLER et al. 1985) are generated by tilorone. Therefore it should be interesting to know whether the amphiphilic cationic compound is able to produce an abnormal dendritogenesis as in storage diseases or an impaired arborisation of dendrites and what could be the reason for the misdevelopment. We demonstrate that there was a fetal retardation in the development of dendritic network, even under influence of low dosis of the analogue R 10.874. The dendritic dismaturation was concomitant with an increased amount of fatty acids and a slightly disarranged metabolic pathway of gangliosides. The dendritic arborisation closed the gap of retarded development between intrauterine treated and untreated rats after 7 days of postnatal drug elimination. We suppose that a fetotoxic effect and not the lysosomopathy is responsible for the reduced dendritic network.

Derivatives of benzo(c)fluorene. VI. Antiviral and interferon-inducing activities of three benzo(c)fluorenone derivatives in mice.

The antiviral effect of 3 derivatives of benzo(c)fluorenone against encephalomyocarditis (EMC) and vaccinia viruses in mice was compared with that of tilorone hydrochloride (THCl). All 3 derivatives were effective against either virus following single-dose oral application as well as after 4-times repeated subcutaneous (s.c.) treatment, but following oral administration only the VUFB 14162 derivative exerted an antiviral effect corresponding to that of THCl. After s.c. application, however, VUFB 14162 derivative was less toxic than THCl. Two benzo(c)fluorenone derivatives, namely VUFB 14162 and 13431, induced in mouse sera the levels of interferon (IFN), which production kinetics and hyporeactivity phenomenon were comparable with those induced by THCl. Because no IFN was found following administration of the third VUFB 13371 derivative, its antiviral effect consisted probably in an other than IFN-inducing mechanism.

Comparison of antiviral properties in mice of bis-pyrrolidinoacetamido-fluorenone (MLU-B75), bis-dipropylaminoacetamido-fluorenone (MLU-B76), and tilorone hydrochloride.

Like tilorone . HCl, its analogues, bis-pyrrolidinoacetamido-fluorenone (MLU-B75), and bis-dipropylaminoacetamido-fluorenone (MLU-B76), significantly protected mice against intraperitoneal challenge with a lethal dose of Mengo virus when administered prophylactically in a single oral dose of 250 mg per kg body weight. At lower doses the antiviral activity of MLU-B75 and MLU-B76 decreased more rapidly than that of tilorone. HCl. At the dose of 250 mg per kg the degree of antiviral protection and the titre of interferon induced by tilorone. HCl correlated well, but there was no correlation between the extent of protection and the detected amount of virus inhibitor induced by MLU-B76. It remains to be determined whether the antiviral protection elicited by the analogues of tilorone is mediated by interferon.

[Carbonyl derivatives of tilorone. Part 2: On the cancerostatic activity of carbonyl derivatives of tilorone (author's transl)]. / Carbonylderivate des Tilorons. 2. Mitteilung: Zur cancerostatischen Wirksamkeit von Carbonylderivaten des Tilorons.

A series of carbonyl derivatives of tilorone was synthetized by reaction with appropriate amino compounds, mainly hydrazines and hydrazides. The condensation products obtained were tested for cancerostatic activity against the murine leucaemia L 1210 and the Walker carcinosarcoma of the rat. Only three of the substances under investigation (1a, 5, 13) proved active against the Walker carcinosarcoma, one of which (5) being comparable to tilorone. No activity against L 1210 was observed, even tilorone exerted no effect. The reduction in activity against the Walker carcinosarcoma which resulted from the carbonyl substitution might be caused by a decrease in the ability to intercalate into DNA.

[N-quaternary derivatives of tilorone]. / N-quartäre Derivate des Tilorons.

Tilorone (free base) reacts with alkyl halides forming quaternary ammonium salts. Bis- as well as mono quaternary compounds (2 resp. 3) were synthesized. The tilorone-bis-methoiodide (2a) was converted to several carbonyl derivatives (4 and 5). All produced compounds did not show any cytostatic activity against the murine leukemias L 1210 and P 388 in vivo. Especially the bis-quaternary derivatives 2 were highly toxic in the mouse.

[Antiviral activity of amiksin incorporated into liposomes]. / Protivovirusnaia aktivnost' amiksina, zakliuchennogo v liposomy.

The results of a comparative study of the antiviral activity of amyxin (tilorone) without liposomes and incorporated into liposomes on the model of experimental enteral hepatitis of mice are presented. The use of amyxin in liposomes was shown to prolong interferon production and to provide significant 60% protection of the animals. The possible mechanism of increasing the antiviral effectiveness of amyxin upon its incorporation into liposomes, enteral administration and subsequent enteral infection of mice with hepatitis virus is discussed. It is assumed that use of amyxin in liposomes may increase its effectiveness in prevention of human hepatitis A.

[Effect of low-molecular interferon inducers in experimental hepatitis in mice]. / Effekt nizkomolekuliarnykh induktorov interferona pri éksperimental'nom gepatite myshei.

Among low molecular interferon inducers preparations were selected which were capable of inducing interferon (IFN) synthesis after oral administration and advantageous for further clinical use. The dynamics of interferon production was studied after oral administration of three national low molecular interferon inducers. The selected preparations: a synthetic inducer, amiksin, and 2 natural compounds (gossypol derivatives), kagocel-1 and PXL-6, stimulated high levels of interferon production (from 10,000 to 20,000 IU/ml) in the intestinal tract of the animals 4 hours after induction and protected the animals from hepatitis virus of mice (the Meshcherin strain) after oral administration 24 hours before infection (35-55%). Amiksin and PXL-6 produced significant protection (p less than 0.01 or 0.001)--40 or 50%, respectively, when administered 4 hours before virus infection.