Ecdysone Analogues Research Articles

New lead discovery of insect growth regulators based on the scaffold hopping strategy

Insect growth regulators (IGRs), which can interrupt or inhibit pest life cycles, are low-toxicity pesticides widely used in integrated pest management (IPM). Ecdysone analogues and chitinase inhibitors are familiar IGRs that have attracted considerable attention because of their unique modes of action and low toxicity to non-target organisms. To find new and highly effective candidate IGRs with novel mechanisms, D-08 (N-(4-(tert-butyl)phenyl)-2-phenyl-2,4,5,6,7,8-hexahydrocyclohepta[c]pyrazole-5-carboxamide) was chosen as a lead compound, and a series of novel heptacyclic pyrazolamide derivatives were designed and synthesized using the scaffold hopping strategy. The bioassay showed that III-27 (N-(2-methylphenethyl)-1-phenyl-1,4,5,6,7,8-hexahydrocyclohepta[c]pyrazole-5-carboxamide) had excellent activity against Plutella xylostella. Protein verification and molecular docking indicated that III-27 could act on both the ecdysone receptor (EcR) and Ostrinia furnacalis chitinase (Of ChtI) and is a promising new lead IGRs. The interaction mechanism of III-27 with EcR and Of ChtI was then studied by molecular docking. These results provide important guidance for the study of new dual-target IGRs.

Design, Synthesis, and Biological Activity of Novel Heptacyclic Pyrazolamide Derivatives: A New Candidate of Dual-Target Insect Growth Regulators.

Insect growth regulators (IGRs) can cause abnormal growth and development in insects, resulting in incomplete metamorphosis or even death of the larvae. Ecdysone receptor (EcR) and chitinase in insects play indispensable roles in the molting process. Ecdysone analogues and chitinase inhibitors are considered as potential IGRs. In order to find new and highly effective IGR candidates, based on the structure-activity relationship and molecular docking results of the active compound 6i (3-(tert-butyl)-N-(4-(tert-butyl)phenyl)-1-phenyl-1H-pyrazole-5-carboxamide) discovered in our previous work, we changed the t-butyl group on the pyrazole ring into heptacycle to enhance the hydrophobicity. Consequently, a series of novel heptacyclic pyrazolamide derivatives were designed and synthesized. The bioassay results demonstrated that some compounds showed obvious insecticidal activity. Especially, D-27 (N-(4-(tert-butyl)phenyl)-2-phenyl-2,4,5,6,7,8-hexahydrocyclohepta[c]pyrazole-5-carboxamide) showed good activities against Plutella xylostella (LC50, 51.50 mg·L-1) and Mythimna separata (100% mortality at 2.5 mg·L-1). Furthermore, protein validation indicated that D-27 acts not only on the EcR but also on chitinase Of ChtI. Molecular docking and molecular dynamics simulation explained the vital factors in the interaction between D-27 and receptors. D-27 may be a new lead candidate with a dual target in which Of ChtI shall be the main one. This work created a new starting point for discovering a novel type of IGRs.

Skin UV-protective effect of Microsorum grossum extract on human dermal fibroblast

Microsorum grosum (Polypodiaceae family), locally called ”metuapua'a”, are two of the most frequently fern species used in Polynesian traditional medicine. Fronds or rizhomes of this species are common ingredient of popular medicine recipes to cure various ailments. M. grosum frond and rizhome extracts contains phytoecdysteroids as main bioactive components such as ecdysone analogues, known to have many interesting biological activities and so considered as adaptogenic compounds. Skin UV-protective effect and mode of action of M. grosum extract was investigated using three ways: a transcriptomic study with c-DNA array for gene expression modulation, a stress induced premature senescence (SIPS) test on human dermal fibroblasts and cellular response experiment by activation of protein p53. The total extract of M. grosum up-regulates both heme oxygenase 1 and ferritin in human fibroblasts of the m-RNAs, enzymes which protect cells from oxidative stress (through the liberation of biliverdin, itself quickly converted into the antioxidant bilirubin) and which exerts several activities like the photoimmunoprotection of skin by UVA through the liberation of CO. The present work also report that premature senescence of human skin induced by repeated UV irradiations can be prevented by an ecdysteroid fraction of M. grosum. The activation of the protein p53 by ecdysone content of M. grosum is also indicative of a protective effect provided by cellular response including damage DNA repair processes. It seems therefore that extracts of M. grosum could protect skin against oxidative stresses and they could be used to formulate innovative cosmetic products.

Insect moulting hormones. LIV. The synthesis and biological acitvity of some alkylated ecdysone analogues

A number of ecdysone analogues were prepared, with alkyl substituent groups projecting above and below the plane of the A-ring of the analogue (37), to gain an insight into the steric requirements for the binding of ecdysteroids to the hormone receptor. Moulting hormone activities of these analogues, determined with the Calliphora bioassay, revealed that the β-face is more sensitive to projecting alkyl groups than the α-face. It is concluded that binding of the hormone to the receptor probably takes place on both sides of the A-ring but that a close fit is more important on the β-face than the α-face.

Prothoracotropic action of ecdysone analogues in Bombyx mori

The prothoracotropic action of ecdysone analogues was examined, using the brainless, diapausing pupae of Bombyx mori with or without inclusion of prothoracic glands. The most effective of those hormones to stimulate prothoracic glands to secrete the moulting hormone was found to be cyasterone. The other analogues such as ponasterone A, ecdysterone, and inokosterone showed a lower activity with regard to prothoracotropic action. The female prothoracic glands were found to be more sensitive to the ecdysones than the male ones. The time lag from hormone injection to emergence indicated the dual actions of the injected ecdysones, directly on the target organs and indirectly on the prothoracic glands subsequent to secreting the moulting hormone.

Trace ahalysis of ecdysones by gas-liquid chromatography, radioimmunoassay and bioassay

A radioimmunoassay was compared to a gas-liquid Chromatographic assay and a bioassay as a method for quantifying ecdysone titers in arthropods. In addition a continuous-development thin layer Chromatographic technique was evaluated as a means for separating various ecdysone analogues. The carboxyuethoxyamine derivative of β-ecdysone was synthesized and characterized by several spectral methods. This derivative was conjugated to bovine serum albumin to afford a suitable antigen and injected into rabbits. The antisera produced shows a moderate affinity for β-ecdysone and cross-reacts with many ecdysone analogues. Ecdysones were also quantified directly as trlmethylsilyl ethers using a 63Ni electron capture detector in a gas-liquid Chromatographic system. Less than 50 pg of material could be detected per injection. Both of these techniques and the Mosca domeetica bioassay were used for the comparative analysis of a number of biological samples. The advantages and disadvantages of the various methods are discussed.

Effects of ecdysone analogues on development and metabolic activity of wing disks of the fleshfly, Sarcophaga peregrina, in vitro

Effects of ecdysone analogues on development and metabolic activities of Sarcophaga wing disks were studied in cultures. Development of disks was induced by ecdysterone, ponasterone A, and cyasterone in vitro, whereas rubrosterone was quite inactive in inducing development. As well as morphogenetic effects, a proper concentration (3 × 10 −5 M to 3 × 10 −7 M) was required to induce the incorporation of tritiated uridine, thymidine, and leucine into RNA, DNA, and protein, respectively. Higher concentration of the hormone was more favourable to development of disks and enhancement of RNA synthesis. However, the hormone at concentration higher than 2 × 10 −9 M seemed to be rather toxic to both development and metabolic activity.

Cuticle deposition in imaginal disks of three species of Lepidoptera: Effects of ecdysones in vitro

We report the effects of three ecdysone analogues in the presence and absence of fat body on the wing imaginal disks of Cadra cautella, Paramyelois transitella, and Plodia interpunctella cultured in vitro in a modified Grace's medium. Alpha-ecdysone, unlike beta-ecdysone, did not stimulate cuticle deposition in Plodia disks at low doses and was only slightly effective at high doses. Also, the response of wing disks of Cadra and Paramyelois to beta-ecdysone and alpha ecdysone with and without fat body was similar to that of Plodia. Combinations of alpha-ecdysone and beta-ecdysone were more effective than beta-ecdysone alone in Plodia disks, but a synthetic analogue, 22-iso-ecdysone, had no effect on cuticle deposition when it was used alone or with beta-ecdysone in the presence or absence of fat body. Thus, the results with alpha-ecdysone and beta-ecdysone were not non-specific steroidal effects.

Metamorphic shortening of interganglionic connectives of Galleria mellonella (Lepidoptera) in vitro: Stimulation by ecdysone analogues

In the absence of other organ systems, β-ecdysone (0·05 to 0·10 μg/ml culture medium) stimulates the shortening of interganglionic connectives of Galleria mellonella that occurs during metamorphosis. There is a direct relations-ship between the amount of β-ecdysone in the medium and the fraction of the sample shortening to at least half the initial length. β-Ecdysone is ∼ 140 × more active than α-ecdysone in eliciting the response. When β-ecdysone and other ecdysone analogues are assayed on this system at uniform dosages (10 μg/ml tissue culture medium), the order of effectiveness (percentage sample shortening to at least half the initial length) is: cyasterone > ponasterone-A = β-ecdysone > inokosterone > α-ecdysone. 22-Iso-α-ecdysone is ineffective in stimulating shortening.

Action of insect hormones at the cellular level: Morphological changes of a diploid cell line of Drosophila melanogaster, treated with ecdysone and several analogues in vitro

Cells of a diploid line established from embryos of Drosophila melanogaster, and maintained in the laboratory for more than 3 years, respond specifically to the synthetic moulting hormones α-ecdysone and β-ecdysone and to several ecdysone analogues. The cells are most sensitive to β-ecdysone and respond to doses as low as 0.006 μg/ml. β-Ecdysone was found to be 5 or 10 times more active than inokosterone and cyasterone, 500 times more active than α-ecdysone, and 5 000 times more than 22-iso-ecdysone. Hormone-treated cells display a series of morphological modifications which are identical for all the ecdysone analogues tested: 16 to 24 h after introduction of the hormone, the cells are seen to flatten or take a spindle shape. Then they tend to aggregate and become fibroblastic or very flattened, and the membrane shows an apparent expansion. The cells appear afterwards full of various inclusions. The rapidity of such an evolution is dose dependent. This morphological evolution requires the presence of foetal calf serum in the culture medium, and becomes irreversible when the cells have attained the fibroblastic stage, which coincides with an arrest of multiplication. Lines of ecdysone-resistant cells have been isolated.

Effects of α- and β-Ecdysone on in vitro Diploid Cell Multiplication in Drosophila melanogaster

THE moulting hormone of insects is sometimes referred to as a growth and differentiation hormone. The steroid ecdysone and ecdysone analogues were shown to promote differentiation both in vivo and in vitro1–3 but their action on cell multiplication is not certain4–10. I used a diploid cell line, established from Drosophila melanogaster embryos by Echalier and Ohanessian11,12, to assay insect hormones.

Effect of ecdysone and ecdysone analogues on two Balbiani rings of Acricotopus lucidus

The activity of two Balbiani rings (BR-3, BR-4) in the salivary gland of Acricotopus lucidus is controlled by ecdysone. This result agrees with our former conclusion worked out by indirect methods [22]. The injection of ecdysone and ecdysone analogues (ecdysterone, inokosterone) into the last instar larvae led to an inactivation of BR-3 and BR-4 identical to that observed during normal development. The assayed substances were compared in terms of their inactivation ability; they showed activity in the following order: ecdysone > ecdysterone > inokosterone. From a study of the response of BR-3 and BR-4 to different ecdysone concentrations a threshold dose in the range of 10 −12 to 10 −11 μg/mg of larval weight for BR-3 and BR-4 has been obtained.

Control of molting in mandibulate and chelicerate arthropods by ecdysones.

1. The ability of ecdysones to induce molting in arthropods other than insects was examined in representatives of both the mandibulate and chelicerate subphyla.2. All five ecdysones tested caused molting in the fresh-water crayfish, Procambarus. Their relative activities were: ecdysterone > inokosterone > α ecdysone > ponasterone A > cyasterone. Doses as low as 3 µg/g of ecdysterone caused 100% of all test crayfish to undergo apolysis and secrete a new cuticle within 14 days after injection, but only in a few cases did the animals shed their old cuticles spontaneously after experimental treatment. At higher doses the new cuticle was thinner than normal and had abnormal bristles. The stimulation of molting was specific for ecdysones and was not copied by a variety of ecdysone analogues or other steroids.3. Histological and autoradiographic studies revealed that ecdysterone at the levels used in these experiments caused molting in crayfish without DNA replication.4. Ecdysterone also caused molting in the ma...

Über die Hämolymphproteine von Pyrrhocoris apterus und über die Bindung von Ecdyson durch Hämolymphproteine

In Pyrrhocoris apterus the changes in total haemolymph protein concentration, the electrophoretic pattern, and the influence of hormonally active substances on these proteins have been investigated. The total protein concentration during the fifth larval instar shifts from 12 mg/ml to 115 mg/ml 5 days before the final ecdysis. In female adults the amount of protein in the haemolymph decreases during oöcyte growth. In male adults the concentration remains nearly constant. In short-day-induced diapause the amount of protein is doubled. With cellogel electrophoresis nine different bands are distinguishable. All bands show changes during development, and particularly three of them (Nos. 1, 8, 9) decrease during the moulting processes. One band (No. 9) is also found in immature eggs, and its haemolymph concentration is diminished during egg maturation. Two fractions (Nos. 4, 7) are increased during adult diapause. Farnesyl-methyl-ether and farnesenic-acid-methyl-ester dihydrochloride diminish fraction Nos. 8 and 9. Two ecdysone analogues induce changes in these fractions also and give rise to an increase in the amount of fractions 4 and 7. Ecdysone is reversibly bound to proteins of fraction 4. There are more than one kind of binding sites and two or more different association constants for these complexes. The prothoracic gland hormone is metabolized in the tissues, but not in the haemolymph of the bug. The ‘half-life time’ of injected ecdysone is about 80 min. One metabolite of the hormone has anionic characteristics and is chromatographically different from ecdysone. 3β-OH-5α-Cholestan-6-one influences the metabolism of ecdysone. Probably this substance acts as an inhibitor of the hormone inactivation system, perhaps due to an inductor for the degrading enzymes.

Differentiation of ommatidium-forming cells of Drosophila melanogaster in organ culture

Eye-antennal discs isolated from mature third-instar larvae (96 h after hatching at 25°C were cultivated in a chemically defined medium K-6′. In the presence of ring glands, ecdysone secreting organ, or ecdysone analogues of plant origin in the medium, a pronounced organization of ommatidium-forming cells in the eye disc portions was observed after 24 h of cultivation. Trypsin-dissociated eye-antennal disc cells formed in rotation culture cell aggregates in which prospective ommatidium-forming cells sorted out from other types of cells of eye-antennal discs. In the presence of ecdysone analogue, BUdR, actinomycin D and puromycin had no inhibitory effect on the differentiation of ommatidia in cultured eye-antennal discs. X-ray irradiation with 2000R demonstrated that there would be a differential sensitivity to X-ray in the differentiation of ommatidia at different stages of cultivation. Implications of these inhibitor effects of macromolecular syntheses and X-irradiation on the organization of ommatidium-forming cells are discussed.

Induction of metamorphosis by ecdysone analogues. Drosophila imaginal discs cultured in vivo.

The effects of molting hormone on the development of imaginal discs of Drosophila melanogaster were investigated. Imaginal leg discs from mature larvae were transplanted into the abdomens of fertilized adult female flies. The hosts were injected with a solution of a phytoecdysone—ecdysterone or cyasterone. The results showed: (1) Large doses of ecdysterone (over 1200 µg/g= 1.2 µg/fly) caused complete metamorphosis of the transplanted imaginal disc, including eversion of the disc, formation of a pupal cuticle, and formation of adult cuticle with bristles, hairs and sensilla arranged in normal orientation. (2) Ecdysterone was necessary as a sustained stimulus, and did not act merely as a trigger to metamorphosis. (3) Cyasterone was a more potent molting hormone than ecdysterone. The technique promises to simplify investigation of pattern formation and other developmental problems in Drosophila.

The structures of two ecdysone analogues, cheilanthones A and B, from the fern Cheilanthes tenuifolia

Cheilanthones A and B are shown to be 7,8-dihydroecdysone (II) and 25-deoxy-7,8-dihydroecdysone (V), respectively.