Semi‐Synthetic Ecdysteroids as Gene‐Switch Actuators: Synthesis, Structure–Activity Relationships, and Prospective ADME Properties

Abstract

The ligand-inducible, ecdysteroid receptor (EcR) gene-expression system can add critical control features to protein expression in cell and gene therapy. However, potent natural ecdysteroids possess absorption, distribution, metabolism and excretion (ADME) properties that have not been optimised for use as gene-switch actuators in vivo. Herein we report the first systematic synthetic exploration of ecdysteroids toward modulation of gene-switch potency. Twenty-three semi-synthetic O-alkyl ecdysteroids were assayed in both a natural insect system (Drosophila B(II) cells) and engineered gene-switch systems in mammalian cells using Drosophila melanogaster, Choristoneura fumiferana, and Aedes aegypti EcRs. Gene-switch potency is maintained, or even enhanced, for ecdysteroids methylated at the 22-position in favourable cases. Furthermore, trends toward lower solubility, higher permeability, and higher blood-brain barrier penetration are supported by predicted ADME properties, calculated using the membrane-interaction (MI)-QSAR methodology. The structure-activity relationship (SAR) of alkylated ecdysteroids indicates that 22-OH is an H-bond acceptor, 25-OH is most likely an H-bond donor, and 2-OH and 3-OH are donors and/or acceptors in network with each other, and with the EcR. The strategy of alkylation points the way to improved ecdysteroidal actuators for switch-activated gene therapy.

HPLC and TLC characterisation of ecdysteroid alkyl ethers

“…For example, dacryhainansterone 22-O-methyl ether 23, a compound generated during methylation of PoA, shows a max = 299 nm. The observed max value results from the presence of the 14␣-hydroxy-7,9(11)-dien-6-one conjugated system, as confirmed by structural assignment by NMR [20]. We measured the extinction coefficient of 23 by recording its ultra-violet spectrum in methanol and found an ε-value at 299 nm = 14,190 L mol −1 cm −1 .…”

“…The solvent systems were a linear gradient of methanol/water from 30 to 100% in 25 min using C18-RP-HPLC (method A) or C6-RP-HPLC (method B); or the diol-NP-HPLC with a linear gradient of methanol/dichloromethane from 2 to 10% (method C) or 4 to 10% (method D) in 20 min. PoA mono-and dimethyl ether analogues (17)(18)(19)(20)(21)(22) showed slightly greater retention times than the corresponding 20E derivatives (Table 1), but with a generally similar sequence of elution, reflecting the chromatographic behaviour of the parent ecdysteroids [26,27]. Dacryhainansterone (or 9,11 -ponasterone A) 22-OMe 23 has an additional double bond between C-9 and C-11, which shifts the UV maximum ( max ) from 242 nm (for the typical ecdysteroid chromophore) to 299 nm (see below).…”

“…1) were unambiguously assigned by solution 1D-and 2D-NMR and high-resolution MS. Twenty-two (1-22) of them possess a typical ecdysteroid structure with a cis-A/B ring junction (5␤-H) and a 14␣-hydroxy-7-en-6-one chromophore. Methyl ether groups are present at one or more of the 2␤-, 3␤-, 14␣-, 22-hydroxyl groups of 20E (1)(2)(3)(4)(5)(6)(7)(8) or PoA (17)(18)(19)(20)(21)(22), as well as 25-OH in 20E (5). Other substituents attached to the 22-OH of 20E include C 2 -C 4 alkyl chains (4,(11)(12)(13), allyl (14), benzyl (15) or 2 -ethyloxiranyl (16) moieties.…”

“…PoA (25) and derivatives with the 2,3-and/or the 20,22-diol protected by acetonide groups (labelled a-c) were synthetically prepared from the parent ecdysteroids 20E and PoA, as previously described [20]. The structures and stereochemistry of 1-23 (summarised in Fig.…”

“…In order to overcome these drawbacks, we have recently reported novel semi-synthetic analogues of prototypical ecdysteroids, PoA and its 25-hydroxy analogue, 20-hydroxyecdysone (20E 24), in which the hydroxyl groups have been refashioned into more suitable pharmacophore elements by alkyl-capping [20]. The alkyl ether analogues appear to possess more favourable absorption, distribution, metabolism and excretion properties with respect to their parent ecdysteroids, while retaining gene-switch potency in favourable cases [20,21]. There is a demand for scalable syntheses of such analogues for the development of novel ecdysteroidregulated gene switches.…”