Synthesis and activity evaluation of tilorone analogs as potential anticancer agents.

Tilorone is an interferon inducer with anticancer activity. Twenty-two novel tilorone analogs were synthesized by improvements of fluorenone skeleton, side chains and amino groups to screen new anticancer agents. In vitro evaluation showed that ten new compounds had better anticancer activities than tilorone. Among them, 2c (IC50 < 7 µM against cancer cell lines and IC50 > 35 µM against non-cancer cell lines) and 5d (IC50 < 10 µM against cancer cell lines and IC50 > 53 µM against non-cancer cell lines) exhibited the best anticancer activities and selectivities. Pharmacophore modeling of highly active compounds was carried out by Molecular Operating Environment (MOE) to generate a visualized model for compound design in future study.

Bone morphogenetic protein-inducer tilorone identified by high-throughput screening is antifibrotic in vivo.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with a poor prognosis and very few therapeutic options. On the molecular level, patients with IPF have increased amounts of the bone morphogenetic protein (BMP) inhibitor gremlin in their lungs, which results in decreased BMP signaling, and an increase in transforming growth factor-ß signaling. Based on these findings, we hypothesized that restoration of the impaired BMP signaling would offer a novel strategy for the prevention of fibrosis progression or for the treatment of pulmonary fibrosis. We used reporter cell lines and high-throughput screening of a chemical compound library as an approach to finding molecules that increase BMP signaling in lung epithelial cells, without increasing transforming growth factor-ß signaling. The most promising candidate drug was analyzed further by studying its effects on BMP target gene expression, Smad protein phosphorylation, and a mouse model of silica-induced pulmonary fibrosis. The most promising drug candidate, tilorone, induced BMP signaling in the reporter cells and increased the expression of BMP-7 and a BMP target gene, Id3, in lung epithelial A549 cells. In a mouse model of pulmonary fibrosis, tilorone decreased lung hydroxyproline content and the expression of collagen genes Col1A1 and Col3A1. Mice treated with tilorone showed markedly decreased histological changes, compared with untreated mice. These findings indicate that tilorone has biologically significant antifibrotic properties.

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.

Time course of the tilorone-induced lysosomal accumulation of sulphated glycosaminoglycans in cultured fibroblasts.

Dicationic amphiphilic drugs such as the immunomodulator tilorone [2,7-bis-[2-(diethylamino)ethoxy]fluoren-9-one] are accumulated in lysosomes and disturb the degradation of sulphated glycosaminoglycans (GAGs) thus leading to generalized lysosomal GAG storage (mainly dermatan sulphate) in vivo and in cultured cells. In the present study, the time course of the tilorone-induced GAG storage was determined in cultured bovine corneal fibroblasts by a radiochemical approach and by morphological examination. In contrast to the rapid lysosomal accumulation of the drug as reported previously, it took approximately 42 h to reach 50% of the GAG storage obtained after 96 h. This is thought to be due to the fact that the temporal development of storage of undigested GAGs depends on the natural delivery of GAGs towards the lysosomal apparatus.

Effect of antirheumatic agents on the mitogen response of arthritic rat spleen cells.

Splenic lymphoid cells from rats with adjuvant induced polyarthritis show a diminished response to the T-cell mitogens Con A and PHA. This report describes the in vitro effects of various antirheumatic agents on the mitogen induced proliferative response of normal (NSC) and arthritic (ASC) rat spleen cells. Indomethacin enhanced only the arthritic responses. Other PG synthesis inhibitors such as ibuprofen and naproxen enhanced the arthritic as well as the normal spleen cell proliferative responses. Gold sodium thiomalate augmented arthritic but not normal blastogenesis. Penicillamine significantly enhanced only the Con A response of arthritic cells at 10(7)M. Levamisole produced a significant increase in the PHA response of arthritic cells and the Con A NSC response. Chloroquine diphosphate enhanced the Con A response of normal cells at 10(-5)M and 10(-6)M; both chloroquine and tilorone suppressed blastogenesis of arthritic spleen cells at 10(-4)M and 10(-5)M. When classifying antirheumatic agents as stimulator or suppressors of immune function, one must account for their effects on arthritic as well as normal lymphoid cells at the predicted pharmacologic plasma levels.

Comparison of interferon-inducing activities and antiviral effects of tobacco mosaic virus, tilorone and sodium nucleinate.

Endogenous interferon was produced in animals in response to the administration of tobacco mosaic virus (TMV), tilorone and sodium nucleinate. The relationship between interferon production and the kind of inducer and the route of its administration was studied. TMV was completely innocuous for Macaca rhesus monkeys and mice and caused no untoward effects in humans upon peroral administration. TMV, tilorone and sodium nucleinate given per os exerted a marked protective effect in mice against tick-borne encephalitis, eastern and western equine encephalomyelitis and influenza virus infections.

Mutagenicity of cancer chemotherapeutic agents in the Salmonella/microsome test.

Seventeen cancer chemotherapeutic agents were tested for their ability to mutate Salmonella typhimurium tester strains in the Salmonella/microsome mutagenicity test. There was a high correlation between the mutagenicity and carcinogenicity of a given agent. Carcinogens positive in the test were Adriamycin, daunomycin, 1-propanol-3,3'-iminodimethanesulfonate, cyclophosphamide, isophosphamide, hycanthone, chlornaphazin, nitrogen mustard, uracil mustard, melphalan, and thio-tepa. Two carcinogesn, actinomycin D and bleomycin, were not detected as mutagens. The presumptive noncarcinogen, methotrexate, was negative in the test. Tilorone and 6-mercaptopurine, tentatively classified as noncarcinogens, were mutagenic. The carcinogenicity of cis-dichlorodiammineplatinum(II), which was positive in the test, has not been determined.

Evaluation of various analogues of tilorone hydrochloride against Venezuelan equine encephalitis virus in mice.

The antiviral activity of tilorone hydrochloride and three of its analogues (11,002, 11,567, and 11,877) was assessed by oral and intraperitoneal (i.p.) administration to Venezuelan equine encephalitis (VEE) virus-infected mice. Significant increases in the percentage of survival (P < 0.01) were apparent after oral administration of tilorone and analogue 11,877 at dosages of 250 and 500 mg/kg. Neither tilorone nor 11,877 increased percentage of survival when dosages of 31.25 to 500 mg/kg were given by the i.p. route. Orally administered analogue 11,002 was effective against 100 mouse intracranial median lethal doses (MICLD(50)) of VEE virus at doses at 250 to 1,000 mg/kg; doses of 31.25 to 250 mg/kg given i.p. were effective against 10 MICLD(50). Oral dosages of 250 to 1,000 mg of analogue 11,567 per kg were active against 100 MICLD(50) of virus. By the i.p. route, 250 mg of 11,567 per kg protected mice against 1,000 MICLD(50), and a dose of 125 mg/kg protected against 100 MICLD(50). Oral treatment of VEE infection with analogue 11,567 24 h after subcutaneous inoculation of VEE virus resulted in no significant increase in the percentage of survivors. All survivors of these studies were susceptible to rechallenge 21 days after the first inoculation of virus.

[Interferonogenic and antiviral activity of the tobacco mosaic virus, tilorone and sodium nucleinate]. / Interferonogennaia i protivovirusnaia aktivnost' virusa tabachnoi mozaiki, tilorona i nukleinata natriia

Production of endogenic interferon in animals in responce to administration of tobaco mozaic virus, tilorone and sodium nucleinate was shown. Dependence of interferon production on the type of the inductor and the route of its administration was studied. Absolute innocuiuty of the tobaco mozaic virus for monkeys (macaco-resus) and mice, as well as the absence of any side effects in humans treated with it perorally was shown. The tobaco mozaic virus, tilorone and sodium nucleinate used perorally in treatment of experimental infections of mice caused by the viruses of East and West encephalomyelitis, influenza and tick encephalitis had a pronounced protective effect.