Javier A. Ramírez

Antiviral effect of brassinosteroids against herpes virus and arenaviruses.

A natural brassinosteroid and a series of synthetic derivatives were found to be good inhibitors of herpes simplex virus type 1 (HSV-1) and arenavirus replication in cell culture. The synthetic compounds tested were analogues of the 24(S) ethylbrassinone. Compounds (22R,23R,24S)-2α, 3α,5α,22,23-pentahydroxy-stigmastan-6-one and (22R,23R,24S)-3β-bromo-5α,22,23-trihydroxy stigmastan-6-one were cytotoxic at concentrations of 20–40 µM. (22S,23S,24S)-2α,3α,22,23-tetrahydroxy-5α,stigmastan-6-one, (22R,23R,24S)-3β-acetoxy-22,23-dihydroxy-5α-cholestan-6-one, (22S,23S,24S)-3β-bromo-22,23-dihydroxy-5α-cholestan-6-one and (22S,23S,24S)-3β-bromo-5α,22,23-trihydroxy-stigmastan-6-one were the most active of the series against HSV-1, with selectivity index (SI) values (CC50/EC50) ranging from 10.6 to 16.5. The majority of the compounds were potent inhibitors of arenaviruses, (22S,23S,24S)-3β-bromo-5α,22,23-trihydroxy-stigmastan-6-one being the most active, with SI values of 307.8 and 692.5 for Tacaribe and Junin viruses, respectively. The antiviral activity of brassinosteroid derivatives was not because of direct inactivation; time-of-addition experiments suggested that a late step in HSV-1 multiplication was affected, whereas arenaviruses remained susceptible to the compounds throughout the replicative cycle.

In vitro and in vivo antiherpetic activity of three new synthetic brassinosteroid analogues

Brassinosteroids are a novel group of steroids that appear to be ubiquitous in plants and are essential for normal plant growth and development. It has been previously reported that brassinosteroid analogues exert an antiviral activity against herpes simplex virus type 1 (HSV-1) and arenaviruses. In the present study, we report the chemical synthesis of compounds (22S,23S)-3beta-bromo-5alpha,22,23-trihydroxystigmastan-6-one (2), (22S,23S)-5alpha-fluoro-3beta-22,23-trihydroxystigmastan-6-one (3), (22S,23S)-3beta,5alpha,22,23-tetrahydroxy-stigmastan-6-one (4) as well as their antiherpetic activity both in a human conjunctive cell line (IOBA-NHC) and in the murine herpetic stromal keratitis (HSK) experimental model. All compounds prevented HSV-1 multiplication in NHC cells in a dose dependent manner when added after infection with no cytotoxicity. Administration of compounds 2, 3, and 4 to the eyes of mice at 1, 2, and 3 days post-infection delayed and reduced the incidence of HSK, consisting mainly of inflammation, vascularization, and necrosis, compared to untreated, infected mice. However, viral titers of eye washes showed no differences among samples from treated and untreated mice. Since the decrease in the percentage of mice with ocular lesions occurred 5 days after treatment had ended, we suggest that brassinosteroids 2, 3, and 4 did not exert a direct antiviral effect in vivo, but rather may play a role in immune-mediated stromal inflammation, which would explain the improvement of the clinical signs of HSK observed.

Synthesis and bioactivity evaluation of brassinosteroid analogs

Four new analogs of 28-homocastasterone have been synthesized and completely characterized for the first time from stigmasterol. (22R, 23R,24S)-3beta-acetoxy-22,23-dihydroxy-5alpha-stigmastan+ ++-6-one (17), (22R,23R,24S)-3beta-bromo-22,23-dihydroxy-5alpha-stigmast an-6-one (18), (22R,23R,24S)-3beta-acetoxy-5,22, 23-trihydroxy-5alpha-stigmastan-6-one (20), and (22R,23R, 24S)-3beta-bromo-5,22,23-trihydroxy-5alpha-stigmastan-6-one (21), were obtained through a synthetic route based on regioselective Delta(5) epoxidation. Compounds 17 and 18, bearing a 5alphaH moiety, were prepared through a reductive opening of the 5beta,6beta epoxy precursor, and compounds 20 and 21, analogs with a 5alphaOH moiety were obtained by hydrolytic opening of a mixture of 5alpha,6alpha and 5beta,6beta epoxy precursors. Known compounds 19 and 22 were also obtained following the described synthetic routes, respectively. The new compounds were tested with the traditional auxin-like bioassay for brassinosteroids with 19 and 22 as standards. All compounds were comparatively evaluated for their inhibitory effect on the replication of DNA (HSV-1) virus.

Effects on Bioactivity due to C-5 Heteroatom Substituents on Synthetic 28-Homobrassinosteroid Analogs

Abstract Five new 28-homobrassinosteroids have been synthesized, namely, (22 R ,23 R )-5-fluoro-3α,22,23-trihydroxy-5α-stigmastan-6-one, (22 R ,23 R )-5-fluoro-3β,22,23-trihydroxy-5α-stigmastan-6-one, (22 R ,23 R )-5-fluoro-2α,3α,22,23-tetrahydroxy-5α-stigmastan-6-one, (22 R ,23 R )-3α,5,22,23-tetrahydroxy-5α-stigmastan-6-one and (22 R ,23 R )-3β,5,22,23-tetrahydroxy-5α-stigmastan-6-one. Their bioactivities were evaluated by the rice lamina inclination test. C-5α Fluorinated analogs showed excellent in vitro bioactivity, also revealed at low doses, while C-5α hydroxylated analogs resulted in an important decrease in bioactivity. Previously given explanations to justify the decreasing effect due to C-5α electronegative groups should be revised.

Anti-herpetic and anti-inflammatory activities of two new synthetic 22,23-dihydroxylated stigmastane derivatives

Stromal keratitis resulting from ocular infection with Herpes simplex virus type 1 (HSV-1) is a common cause of blindness. This report investigates the antiviral and anti-inflammatory properties of two new synthetic stigmastane analogs in the experimental model of HSV-1-induced ocular disease in mice. (22S,23S)-22,23-dihydroxystigmast-4-en-3-one (1) and (22S,23S)-22,23-dihydroxystigmasta-1,4-dien-3-one (2) exhibited anti-HSV-1 activity in vitro and ameliorated the signs of murine herpetic stromal keratitis (HSK), although none of the compounds showed antiviral activity in vivo. We discuss that the improvement of HSK could be due to an immunomodulatory effect of both compounds.

A new look into the reaction between ergosterol and singlet oxygen in vitro

The reaction of ergosterol (ERGO) with singlet oxygen in vitro was studied by using different combinations of the photosensitizers (i.e. rose Bengal and eosine) and solvents (i.e. pyridine, ethanol and methyl tert-butyl ether) and all the products obtained were isolated and fully characterized (mp, Rf, UV, 1H-NMR, 13C-NMR, EI-MS, ESI-MS and HR-MS). In pyridine. together with the expected (22E)-5alpha,8alpha-epidioxyergosta-6,22-dien-3beta-ol, EEP, the keto derivative (22E)-3beta-hydroxyergosta-5,8(9),22-trien-7-one. KE, was obtained. In ethanol the expected EEP was obtained together with (22E)-5alpha,8alpha-epidioxyergosta-6,9,22-trien-3beta-ol, EEP9(11), and (22E)-ergosta-6,9,22-triene-3beta,5alpha,8alpha-triol, DHOE, as main products and (22E)-ergosta-5,7,9,22-tetraen-3beta-ol, DHE, in trace amounts In methyl tert-butyl ether, a complex mixture of EEP, KE, DHOE, EEP9(11), DHE, together with (22E)-7alpha-hydroperoxyergosta-5.8(9),22-trien-3beta-ol, EHP, and (22E)-ergosta-5,8(9),22-triene-3beta, 7alpha-diol, EH, was obtained. The minor products were characterized and showed strong dependence on the reaction medium. The regioselective and stereoselective character of the singlet oxygen attack on the ERGO diene moiety is discussed in terms of ERGO HOMOs properties.

Plant and animal steroids a new hope to search for antiviral agents.

Scientific literature provides evidence about the use of steroids as an adjunct treatment to antiviral therapies. Immunomodulatory activity of some steroids would account for the recovery in patients with herpetic and other viral infections. However, in vitro studies have demonstrated a direct antiviral effect of this kind of molecules. In this review we discuss recent reports about the mechanism of antiviral action of steroids from animal and plant origin. Chemical structures of most active compounds are also provided.

Synthesis and bioactivity of natural and C-3 fluorinated biosynthetic precursors of 28-homobrassinolide

In this paper we describe the synthesis of two new fluorinated brassinosteroids: (22R,23R)-22,23-dihydroxy-3alpha-fluorostigmastan-6-one and (22R,23R)-22,23-dihydroxy-3beta-fluorostigmastan-6-one. Their bioactivities were evaluated in the rice lamina inclination test and compared with that of 28-homocastasterone, 28-homotyphasterol and 28-homoteasterone, possible biosynthetic precursors of 28-homobrassinolide. Results confirmed expected similarities between the biosynthesis of 24-ethylbrassinosteroids (named as the 28-homo series) and that described for 24-methylbrassinosteroids, and also indicated that these precursors might exhibit per se activities.

Synergistic in vitro Interactions between (22S,23S)-3β-Bromo-5α,22,23-Trihydroxystigmastan-6-one and Acyclovir or Foscarnet against Herpes simplex Virus Type 1

The replication of herpes simplex virus (HSV) type 1 in Vero cells is inhibited in the presence of (22S,23S)-3β-bromo-5α,22,23-trihydroxystigmastan-6-one (6b), a synthetic brassinosteroid derivative. Since a late step of virus multiplication is hindered by 6b, we performed studies of drug-drug combination with acyclovir (ACV) and foscarnet (FOS). It was determined that 6b would act synergistically with low concentrations of ACV and moderate concentrations of FOS against HSV. The best drug combination tested in this study resulted in an increase of 29.3 and 47.2% in antiviral activity for ACV (0.036 µM) and FOS (37.5 µM) in the presence of 14.8 and 6.9 µM of 6b, respectively.

Brassinosteroid-stimulated branch elongation in the marubakaido apple rootstock

Brassinosteroids (BRs) comprise a specific class of low-abundance plant steroids now recognized as a new class of phytohormones. In this paper, we demonstrate that a fluoro derivative of 28-homocastasterone (5F-HCTS) stimulates branch elongation in in vitro-grown shoots of Malus prunifolia, the marubakaido apple rootstock. In addition to that, we show that this BR-stimulated branch elongation is paralleled by an increase in ethylene release. However, either the presence of 1-amino-cyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene in higher plants, in the culture medium or an ethylene-enriched atmosphere resulted in inhibition of branch elongation, indicating that the stimulation of branch elongation observed for 5F-HCTS-treated shoots in this study was not, at least directly, related to the BR-induced enhancement in ethylene release rate. Besides its positive effect on the marubakaido shoot growth, i.e. branch elongation, the 5F-HCTS-driven enhancement of branch elongation found in this study is potentially useful to improve micropropagation techniques for other plant species as well, especially woody species, in which branch elongation is typically a constraint for efficient micropropagation.