Synthesis and biological activities of turkesterone 11-acyl derivatives

Laurence Dinan¹, Pauline Bourne¹, Pensri Whiting¹, Ada Tsitsekli¹, Ziyadilla Saatov², Tarlochan S. Dhadialla^3§, Robert E. Hormann^3¶, René Lafont⁴ and Josep Coll⁵

¹Department of Biological Sciences. University of Exeter, Hatherly Laboratories, Prince of Wales Road, Exeter, Devon, EX4 4PS, U.K.
²Institute of the Chemistry of Plant Substances, 700170 Tashkent, Uzbekistan
³Rohm and Haas Co., Research Laboratories, 727 Norristown Road, Spring House, PA 19477-0949, U.S.A
⁴Laboratoire d'Endocrinologie Moléculaire et Evolution, Université Pierre et Marie Curie, 7 Quai St. Bernard, 75252 Paris 05, France
⁵Dep. Química Orgànica Biològica, Institut d'Investigacions Químiques i Ambientals de Barcelona "Josep Pascual Vila", C.S.I.C., Jordi Girona 18, 08034 Barcelona, Spain
^§Current address: Dow AgroSciences L.L.C., Biochemistry, 9330 Zionsville Road, Indianapolis, IN 46268, U.S.A.
^¶Current address: RheoGene L.L.C., P.O. Box 949, Spring House, Pennsylvania 19477-0904, U.S.A.
L.N.Dinan@exeter.ac.uk

Received 10 November 2002
Accepted 11 February 2003
Published 24 February 2003

Cite this paper as:
Dinan L., Bourne P., Whiting P., Tsitsekli A., Saatov Z., Dhadialla T.S., Hormann R.E., Lafont R., Coll J. 2003. Synthesis and biological activities of turkesterone 11-acyl derivatives 11 pp. Journal of Insect Science, 3.6. Available online: insectscience.org/3.6

Keywords

steroid, ecdysteroid, 20-hydroxecdysone, QSAR, steroid hormone receptor

Abbreviations

20E	20-hydroxyecdysone
CoMFA	comparative molecular field analysis
DCM	dichloromethane
DMF	dimethylformamide
DMP	2,2-dimethoxypropane
4D-QSAR	4-dimensional quantitative structure-activity relationship
EcR	ecdysteroid receptor
EcRE	ecdysteroid response element
HPLC	high-performance liquid chromatography
LBD	ligand-binding domain
NMR	nuclear magnetic resonance
ponA	ponasterone A
QSAR	quantitative structure-activity relationship
RXR	retinoid X receptor
SAR	structure-activity relationship
SPE	solid-phase extraction
THF	tetrahydrofuran
TLC	thin-layer chromatography
p-TsOH	para-toluenesulphonic acid
USP	ultraspiracle
UV-VIS	ultraviolet-visible

Introduction
Materials and Methods
Results and Discussion
Conclusion
Acknowledgements
References

Turkesterone is a phytoecdysteroid possessing an 11-hydroxyl group. It is an analogue of the insect steroid hormone 20-hydroxyecdysone. Previous ecdysteroid QSAR and molecular modelling studies predicted that the cavity of the ligand-binding domain of the ecdysteroid receptor would possess space in the vicinity of C-11/C-12 of the ecdysteroid. We report the regioselective synthesis of a series of turkesterone 11-acyl derivatives in order to explore this possibility. The structures of the analogues have been unambiguously determined by spectroscopic means (NMR and low-resolution mass spectrometry). Purity was verified by HPLC. Biological activities have been determined in Drosophila melanogaster B_II cell-based bioassay for ecdysteroid agonists and in an in vitro radioligand-displacement assay using bacterially expressed D. melanogaster EcR/USP receptor proteins. The 11-acyl derivatives do retain a significant amount of biological activity relative to the parent ecdysteroid. Further, although activity initially drops with the extension of the acyl chain length (C₂ to C₄), it then increases (C₆ to C₁₀), before decreasing again (C₁₄ and C₂₀). The implications of these findings for the interaction of ecdysteroids with the ecdysteroid receptor and potential applications in the generation of affinity-labelled and fluorescently-tagged ecdysteroids are discussed.

Abstract
Materials and Methods
Results and Discussion
Conclusion
Acknowledgements
References

Ecdysteroids are the steroid hormones of arthropods and possibly of other invertebrate phyla too. They also occur in certain plant species, where they are known as phytoecdysteroids and are believed to contribute to the deterrence of invertebrate predators (Dinan, 2001). In insects, they regulate moulting and metamorphosis and have been implicated in the regulation of reproduction, diapause etc. (Koolman, 1989). Most actions of ecdysteroids are mediated by intracellular receptor complexes, which regulate gene expression in a tissue- and development-specific manner. In Drosophila melanogaster, the receptor complex consists of the ecdysteroid receptor (EcR) protein, of which there are 3 isoforms (Koelle et al., 1991; Talbot et al., 1993), and the Ultraspiracle (Usp) protein, which is a homologue of the vertebrate retinoid X receptor (RXR) protein (Oro et al., 1990). Both EcR and Usp are members of the nuclear receptor superfamily (Laudet, 1997). EcR binds ligand with high affinity only when it is complexed to Usp (Yao et al., 1993). It is not known for certain if there is a natural ligand for Usp, although it has been suggested that juvenile hormone might be a possible candidate (Jones and Jones, 2000). Owing to the critical role that the ecdysteroid system plays in regulating insect development, there is considerable interest in characterising ligand/receptor/EcRE interactions from different insect Orders and to exploit the differences to develop Order-specific pest control agents. The selectivity of the bisacylhydrazines, non-steroidal agonists, is a good example of what can be achieved (Dhadialla et al., 1998). Further, because the ecdysteroid receptor is specific to invertebrates and ecdysteroids appear to be non-toxic to vertebrates and plants (Sláma and Lafont, 1995), there is growing interest in the use of ecdysteroid systems in transgenic gene-switch systems in vertebrate or plant cells to generate medically or agronomically effective systems (Fussenegger, 2001; Lafont and Dinan, 2003).

In recent years, we have conducted an extensive SAR for ecdysteroids in a homogeneous bioassay system, based on the ecdysteroid-responsive l(2)mbn (B_II) permanent cell line of Drosophila melanogaster0 (Clément et al., 1993). The response of these cells reflects the affinity of the EcR/USP complex for the ecdysteroid analogue and can be measured in a simple microplate-based assay. In conjunction with molecular modelling methods (CoMFA and 4D-QSAR; Dinan et al., 1999; Ravi et al., 2001), SAR of an extensive range of pure ecdysteroid analogues has provided testable predictions for ecdysteroid interaction with the LBD and generated the first pharmacophore hypothesis for ecdysteroid binding. Amongst the predictions to come out of the CoMFA/4D-QSAR analyses was that a region capable of accommodating steric bulk was present around the location of C-11/C-12 of the ecdysteroid molecule. We have tested this hypothesis by generating a series of 11a-acyl derivatives of the phytoecdysteroid turkesterone (Figure 1: structure 1) and determining their biological activities relative to the parent ecdysteroid.

Abstract
Introduction
Results and Discussion
Conclusion
Acknowledgements
References

Chemicals

Turkesterone (1) was isolated from Ajuga turkestanica (Usmanov et al., 1975). 20-Hydroxyecdysone (6) was supplied by Dr. V. Volodin (Institute of Biology, Russian Academy of Sciences). Muristerone A (9) was purchased from Simes spa, Italy. Other ecdysteroids were generous gifts from other researchers (see acknowledgements in Dinan et al., 1999). Acyl anhydrides were purchased from Sigma/Aldrich (www.sigmaaldrich.com). Solvents were analytical or HPLC-grade and used without further purification, with the exception of pyridine, which was dried over sodium hydroxide pellets for at least 24 h before use. All other chemicals were obtained from Sigma-Aldrich Co. (Poole, Dorset, U.K.) or Lancaster Synthesis (www.Lancastersynthesis.com).

NMR

¹H and ¹³C nuclear magnetic spectroscopy was performed on a Varian Unity 300 spectrometer (at CSIC, Barcelona) or on a Bruker (www.bruker.com) DMX500 MHz spectrometer (NMR Service, University of Barcelona). Samples were dissolved in the indicated solvent. Chemical shifts are given in ppm and the coupling constants and widths at half height (_1/2) are given in Hz. Spectral assignments were achieved by comparison to published data where available (Lafont et al., 2002).

Mass spectrometry

CIMS and FABMS spectra were recorded at the Ecole Normale Supérieure (Paris, France) on a Jeol (www.jeol.com) MS 700 spectrometer equipped with a direct inlet probe. CIMS spectra were recorded in chemical ionisation/desorption mode using ammonia as the reagent gas.

UV spectrophotometry

Ultra-violet spectra of ecdysteroids were determined in methanol using a Shimadzu (www.shimadzu.com) UV-2401 PC UV-VIS dual-beam spectrophotometer and quartz cells. Extinction coefficients were obtained from Lafont et al. (2002).

Thin-layer chromatography

TLC was performed on Merck 60 F₂₅₄ pre-coated 10 x 10 cm plates (BDH/VWR www.chromatography.co.uk), with CHCl₃/EtOH (9:1 v/v) for development and visualisation by fluorescence quenching under short-wavelength UV light.

Solid-phase extraction

Reversed-phase (C₁₈; 0.5g) Sep-Pak cartridges (Waters/Millipore, www.waters.com) were activated with methanol (5 ml), followed by water (10 ml). The sample was applied in 10% aq. MeOH and then the column was sequentially eluted with 5 ml portions of 10%, 25% and 100% aq. MeOH.

HPLC

Two HPLC systems were employed. The analytical system consisted of a Gilson (www.gilson.com) model 811 HPLC coupled with a Gilson 160 diode-array detector and controlled by Gilson Unipoint program. The semi-preparative system consisted of two Gilson model 303 pumps, a model 802c manometric module, a mixing unit, a Holochrome variable wavelength detector and Rheodyne 7125 injector block (rheodyne.com). Solvent composition and flow was controlled by an Apple IIe computer with Gilson Gradient Manager program. Elution profiles were monitored at 242 nm. Three types of column were used: i) Spherisorb reversed-phase octadecyl (ODS2) column (analytical: 150 mm x 4.6 mm i.d., 5 µm particle size; semi-preparative: 250 mm x 10 mm i.d. 5 µm particle size), ii) normal-phase Zorbax SIL column (analytical: 250 mm x 4.6 mm i.d. 5 µm particle size), iii) normal-phase Apex II DIOL column (analytical: 250 mm x 4.6 mm i.d., 5 µm particle size). Columns were purchased from Jones Chromatography (www.jones-chrom.co.uk).

Synthesis of 11-acyl derivatives of turkesterone

Conversion of turkesterone (1) to 2,3-isopropylidene turkesterone 20,22-phenylboronate (12)

The procedure was modified from Guédin-Vuong et al. (1985). A solution of turkesterone (10 mg; 20.8 µmol) and phenylboronic acid (2.7 mg, 22.1 µmol; 1.1 eq) in dry DMF (500 µL) was stirred at room temperature for two hours. Dry acetone (3.5 ml), dry DMP (200 µL) and fused p-TsOH (1 mg; 10.4 µmol; 0.5 eq) were then added and the reaction was stirred for a further 3 hours. Acetone and DMP were evaporated under nitrogen and 2 ml ethyl acetate was added to the remaining DMF. This mixture was partitioned three times with saturated aqueous sodium chloride solution leaving the 2,3-isopropylidene turkesterone 20,22-phenylboronate in the ethyl acetate phase. The product was assessed for purity by TLC (CHCl₃/EtOH 9:1, v/v, Rf 12: 0.52; cf Rf 1: 0.04) and used without further purification or assessment of yield. This reaction was repeated many times with acyl derivatization at C-11 occurring as a third step without complete purification of the intermediates.

Conversion of 2,3-isopropylidene turkesterone 20,22-phenylboronate (12) to 2,3-isopropylidene turkesterone (10)

To a solution of 2,3-isopropylidene turkesterone 20,22-phenylboronate (12: synthesised from 10 mg turkesterone 1) in a (9:1) mixture of THF/H₂O (2 ml) was added H₂O₂ (30 %; 1 ml), which had been previously adjusted to pH 7 with 0.1M NaOH. This mixture was stirred at room temperature for 2 hours. The reaction was quenched by the addition of H₂O (2 ml) and the THF was removed under reduced pressure. The residue was purified through a 0.5 g C₁₈ reversed-phase Sep-Pak cartridge and assessed on TLC (CHCl₃/EtOH, 9:1, v/v; Rf 10: 0.24, cf Rf 12: 0.52). The product could be used directly in acetylation reactions without further purification.

Conversion of 2,3-isopropylidene turkesterone 20,22-phenylboronate (12) to 11-acyl turkesterone (14)

i) turkesterone 11-acetate (14a)

To a solution of 2,3-isopropylidene turkesterone 20,22-phenylboronate (12: synthesised from 5 mg turkesterone [1]) in dry pyridine (800 µL) at room temperature was added acetic anhydride (400 µL). The reaction was stirred at room temperature for 1 hour and quenched with the addition of H₂O (20 ml). The reaction mixture was pre-purified on a SPE cartridge and the 100% MeOH fraction was dried under nitrogen. The residue was dissolved in dioxan (700 µL) and aqueous HCl (0.1M, 700 µL) was added dropwise. The reaction mixture was stirred at room temperature for 4 hours and diluted with H₂O (20 ml). The product was pre-purified on a SPE cartridge and analysed by HPLC using an ODS2 analytical column with detection at 242 and 300 nm (30 %-100 % MeOH linear gradient over 30 minutes; 1 ml/min; Rt (12) = 24.4 min, Rt (14a) = 8.3 min). The product was purified on an ODS2 semi-preparative column (MeOH/H₂O [45:55 v/v], 2 ml/min) and yielded turkesterone 11-acetate (14a: 1.61 mg; 3.0 µmol; 30% yield).

CIMS: 556 ([M+NH₄]⁺, 18%), 538 ([M]⁺, 3%), 521 ([M+H-H₂O]⁺, 10%), 503 ([M+H-2H₂O]⁺, 8%), 479 ([M+H-AcOH]⁺, 100%), 461 ([M+H-AcOH-H₂O]⁺, 34%), 443 ([M+H-AcOH-2H₂O]⁺, 13%), 425 (6%), 345 (9%)

¹H and ¹³C NMR: see Tables 3 and 4.

ii) turkesterone 11-propionate (14b)

To a solution of 2,3-isopropylidene turkesterone 20,22-phenylboronate (12: synthesised from 5 mg turkesterone [1]) in dry pyridine (800 µL) at room temperature was added propionic anhydride (400 µL). The reaction was stirred at room temperature for 2 hours and quenched with H₂O (20 ml). After SPE purification, the 100% MeOH fraction was dried and the residue dissolved in dioxan (2 ml) and aqueous HCl (0.1M, 1 ml). The reaction mixture was stirred at room temperature for 4 hours and diluted with H₂O (20 ml). The product was pre-purified on a SPE cartridge and analysed by HPLC using an ODS2 analytical column with detection at 242 and 300 nm (30 %-100 % MeOH linear gradient over 30 minutes; 1 ml/min; Rt (12) = 24.4 min, Rt (14b) = 11.2 min). The product was purified on an ODS2 semi-preparative column (MeOH/H₂O [45:55 v/v], 2 ml/min) and yielded turkesterone 11-propionate (14b: 924 µg; 1.7 µmol; 17 % yield). The product was identified by ¹H NMR and mass spectrometry.

CIMS: 570 ([M+NH₄]⁺, 39%), 552 ([M]⁺, 5%), 496 ([M+H-C₃H₅O]⁺, 90%), 479 ([M+H-C₃H₆O₂]⁺, 100%), 461 ([M+H-C₃H₆O₂-H₂O]⁺, 4%), 443 ([M+H-C₃H₆O₂-2H₂O]⁺, 4%), 425 (2%), 380 (2%), 345 (1%)

¹H NMR: see Table 3.

iii) turkesterone 11-butanoate (14c)

To a solution of 2,3-isopropylidene turkesterone 20,22-phenylboronate (12: synthesised from 10 mg turkesterone [1]) in dry pyridine (800 µL) at room temperature was added butanoic anhydride (400 µL). The reaction was stirred at room temperature for 6 hours and then heated to 50°C for 3 hours, after which time the reaction appeared to be complete by TLC analysis. The mixture was diluted with H₂O (20 ml) and pre-purified on a SPE cartridge, leaving the sample in methanol. This was dried under nitrogen and the residue was dissolved in dioxan (2 ml) and aqueous HCl (0.1M, 1 ml). The reaction mixture was stirred at room temperature for 4 hours and diluted with H₂O (20 ml). The product was pre-purified on a SPE cartridge and analysed by HPLC using an ODS2 analytical column with detection at 242 and 300 nm (30% - 100% MeOH linear gradient over 30 minutes, 1 ml/min, Rt (12) = 24.4 min, Rt (14c) = 12.5 min). The product was purified on an ODS2 semi-preparative column (MeOH/H₂O [50:50 v/v], 2 ml/min) and yielded turkesterone 11-butanoate (14c: 1.8 mg; 3.1 mmol; 16 % yield). The product was identified by ¹H NMR and mass spectrometry.

CIMS: 584 ([M+NH₄]⁺, 100%), 566 ([M]⁺, 8%), 548 ([M-H₂O]⁺, 2%), 496 ([M+H-C₄H₇O]⁺, 58%), 479 ([M+H-C₄H₈O₂]⁺, 72%), 461 ([M+H-C₄H₈O₂-H₂O]⁺, 6%), 443 ([M+H-C₄H₈O₂-2H₂O]⁺, 3%), 344 (2 %), 246 (7%)

1H NMR: see Table 3.

iv) turkesterone 11-hexanoate (14d)

2,3-Isopropylidene turkesterone 20,22-phenylboronate (12: synthesised from 10 mg turkesterone 1) was dissolved in pyridine/benzene 6:5, v/v (4 ml) and hexanoic anhydride (335 µL) was added. After incubation at 50°C for 20 hours, the reaction mixture was reduced to dryness by rotary evaporation. The residue was dissolved in DCM and fractionated by silica gel chromatography. Aliquots of each fraction were analysed by TLC (CHCl₃/EtOH 9:1 v/v; Rf = 0.76). Fractions containing 2,3-isopropylidene turkesterone 20,22-phenylboronate 11-hexanoate were dried and redissolved in dioxan (2 ml) and aqueous HCl (0.1M, 1 ml). The reaction mixture was stirred at room temperature for 4 hours and diluted with H₂O (20 ml). The product was pre-purified on a SPE cartridge and analysed by HPLC using an ODS2 analytical column with detection at 242 and 300 nm (30%-100% MeOH linear gradient over 30 minutes; 1 ml/min; Rt (12) = 24.4 min, Rt (14d) = 17.17 min). The product was purified on an ODS2 semi-preparative column (MeOH/H₂O [60:40 v/v], 2 ml/min) and yielded turkesterone 11-hexanoate (14d: 5.72 mg; 9.6 mmol; 48% yield). The product was identified by ¹H and ¹³C NMR and mass spectrometry.

CIMS: 612 ([M+NH₄]⁺, 2%), 597 ([M+H]⁺, 2%), 579 ([M+H-H₂O]⁺, 5%), 561 ([M+H-2H₂O]⁺, 6%) 543 ([M+H-3H₂O]⁺, 21%), 496 ([M-C₆H₁₁O]⁺, 4%), 479 ([M+H-C₆H₁₂O₂]⁺, 62%), 461 ([M+H-C₆H₁₂O₂-H₂O]⁺, 71%), 443 ([M+H-C₆H₁₂O₂-2H₂O]⁺, 33%), 427 (27%), 362 (35%), 345 (54%), 299 (5%), 178 (11%), 160 (13%), 143 (13%)

¹H and ¹³C NMR: see Tables 3 and 4.

v) turkesterone 11-decanoate (14e)

The production of turkesterone 11a-decanoate was completed as for 14d, but using decanoic anhydride (474 mg). The product was pre-purified on a SPE cartridge and analysed by HPLC using an ODS2 analytical column with detection at 242 and 300 nm (30% - 100% MeOH linear gradient over 30 minutes; 1 ml/min; Rt (12) = 24.4 min, Rt (14e) = 24.4 min). The product was purified on an ODS2 semi-preparative column (MeOH/H₂O [60:40 v/v], 2 ml/min) and yielded turkesterone 11-decanoate (14e: 4.48 mg; 6.9 µmol; 34% yield). The product was identified by mass spectrometry.

CIMS: 669 ([M+NH₄]⁺, 1%), 651 ([M+H]⁺, 1%), 634 ([M+H-H₂O]⁺, 1.5%), 616 ([M+H-2H₂O]⁺, 1.5%), 479 ([M+H-C₁₀H₂₀O₂]⁺, 81%), 461 ([M+H-C₁₀H₂₀O₂-H₂O]⁺, 80%), 443 ([M+H-C₁₀H₂₀O₂-2H₂O]⁺, 57%), 425 (35%), 361 (32%), 345 (47%), 256 (14%), 190 (44%), 143 (28%)

vi) turkesterone 11-laurate (14f)

The production of turkesterone 11-laurate was completed as for 14d, but using lauric anhydride (555 mg). The product was pre-purified on a SPE cartridge and analysed by HPLC using an ODS2 analytical column with detection at 242 and 300 nm (30% - 100% MeOH linear gradient over 30 minutes; 1 ml/min; Rt (12) = 24.4 min, Rt (14f) = 26.8 min). The product was purified on an ODS2 semi-preparative column (MeOH/H₂O [60:40 v/v], 2 ml/min) and yielded turkesterone 11-laurate (14f: 1.47 mg; 2.2 µmol; 11% yield). The product was identified by ¹H and ¹³C NMR and mass spectrometry.

FABMS: 701 ([M+Na]⁺, 11%), 661 ([M+H-H₂O]⁺, 6%), 643 ([M+H-2H₂O]⁺, 9%), 625 ([M+H-3H₂O]⁺, 6%), 565(11%), 479 ([M+H-C₁₂H₂₄O₂]⁺, 82%), 461 ([M+H-C₁₂H₂₄O₂-H₂O]⁺, 16%), 443 ([M+H-C₁₂H₂₄O₂-2H₂O]⁺, 16%), 425 ([M+H-C₁₂H₂₄O₂-3H₂O]⁺, 18%), 391 (70%), 369 (36%).

¹H and ¹³C NMR: see tables 3 and 4

vii) turkesterone 11-myristate (14g)

The synthesis of turkesterone 11-myristate was completed as for 14d, but using myristic anhydride (635 mg). The deprotection reactions were diluted with H₂O as before, but this brought about precipitation of the products. The sample was centrifuged and the supernatant removed. The pellet was redissolved in methanol and analysed by HPLC using an ODS2 analytical column with detection at 242 and 300 nm (80 %-100 % MeOH linear gradient over 20 minutes; 1 ml/min; Rt (14g) = 9.1 min). The product was purified on an ODS2 semi-preparative column (MeOH/H₂O, 90:10 v/v, 2 ml/min) and yielded turkesterone 11-myristate (14g: 1.3 mg; 1.9 µmol; 9 % yield). The product was identified by ¹H NMR and mass spectrometry.

FABMS: 730 ([M+Na]⁺, 15%), 689 ([M+H-H₂O]⁺, 14%), 672 ([M+H-2H₂O]⁺, 24%), 654 ([M+H-3H₂O]⁺, 17%), 493 (9%), 479 ([M+H-C₁₄H₂₈O₂]⁺, 100%), 461 ([M+H-C₁₄H₂₈O₂-H₂O]⁺, 66%), 443 ([M+H-C₁₄H₂₈O₂-2H₂O]⁺, 52%), 425 ([M+H-C₁₄H₂₈O₂-3H₂O]⁺, 55%), 391 (62%), 362 (30%), 345 (40%), 327 (32%), 309 (96%), 299 (67%), 281 (55%), 228 (92%), 211 (30%)

¹H NMR: see Table 3.

viii) turkesterone 11-arachidate (14h)

The synthesis of turkesterone 11-arachidate was completed as for 14g, but using arachidic anhydride (880 mg). The deprotection reactions were diluted with H₂O, but this brought about precipitation of the products. The sample was centrifuged and the supernatant removed. The pellet was redissolved in methanol and analysed by HPLC using an ODS2 analytical column with detection at 242 and 300 nm (80 %-100 % MeOH linear gradient over 20 minutes; 1 ml/min; Rt (14h) = 19.2 min). The product was purified on an ODS2 semi-preparative column (MeOH/H₂O, 95:5 v/v, 2 ml/min) and yielded turkesterone 11-arachidate (14h: 2.6 mg; 3.3 µmol; 16% yield). The product was identified by ¹H NMR and mass spectrometry.

CIMS: 809 ([M+NH₄]⁺, 7%), 790 ([M]⁺, 8%), 479 ([M+H-C₂₀H₄₀O₂]⁺, 55%), 461 ([M+H-C₂₀H₄₀O₂-H₂O]⁺, 55%), 443 ([M+H-C₂₀H₄₀O₂-2H₂O]+, 34%), 428 (34%), 363 (47%), 346 (65%), 331 (34%), 283 (15%), 141 (36%).

¹H NMR: see Table 3.

Synthesis of turkesterone acetates

Conversion of turkesterone (1) to turkesterone 2-monoacetate (2)

To a solution of turkesterone (1: 5 mg; 10.4 µmol) in dry pyridine (400 µL) at room temperature was added acetic anhydride (200 µL; 2.08 mmol; 200 eq.). The reaction was stirred at room temperature for 1 hour and quenched with H₂O (20 ml). The residue was pre-purified on a SPE cartridge and analysed by HPLC using an ODS2 analytical column with detection at 242 and 300 nm (30%-100% MeOH linear gradient over 30 minutes, 1 ml/min; Rt (1) = 6.3 min, Rt (2) = 8.4 min]. The product was purified on an ODS2 semi-preparative column (MeOH/H₂O [45:55 v/v], 2 ml/min) and yielded turkesterone 2-acetate (2: 613 µg; 1.1 µmol; 12%). The product was identified by mass spectrometry.

CIMS: 557 ([M+NH₄]⁺, 62%), 539 ([M+H]⁺, 32 %), 522 ([M+H-H₂O]⁺, 61%), 486 ([M+H-3H₂O]⁺, 65%), 462 (40%), 444 (33%), 439 (74%), 423 (84%), 405 (47%), 362 (32%), 344 (28%), 328 (16%), 178 (18%), 160 (18%).

Conversion of turkesterone (1) to turkesterone 2,11-diacetate (4)

To a solution of turkesterone (1: 10 mg; 20.8 µmol) in dry pyridine (800 µL) at room temperature was added acetic anhydride (400µL; 4.16 mmol; 200 eq.). The reaction was stirred at room temperature for 1 hour and quenched with the addition of H₂O (20 ml). The residue was pre-purified on a SPE cartridge and analysed by HPLC using an ODS2 analytical column with detection at 242 and 300 nm (30%-100% MeOH linear gradient over 30 minutes; 1 ml/min; Rt (1) = 6.3 min, Rt (4) = 11.3 min). The product was purified on an ODS2 semi-preparative column (MeOH/H₂O [45:55 v/v], 2 ml/min) and yielded turkesterone 2,11-diacetate (4: 5.6 mg; 9.7 µmol; 48% yield). The product was identified by ¹H and ¹³C NMR and mass spectrometry.

CIMS: 598 ([M+NH₄]⁺, 8%), 580 ([M]⁺, 6%), 563 ([M+H-H₂O]⁺, 10%), 545 ([M+H-2H₂O]⁺, 31%), 538 ([M-Ac]⁺, 12%), 485 ([M+H-Ac-3H₂O]⁺, 48%), 480 ([M-2Ac-H₂O]⁺, 28%), 464 (70%), 420 (38%), 404 (82%), 387 (100%), 344 (24%), 325 (16%), 128 (65%)

¹H and ¹³C NMR: see Tables 1 and 2.

Conversion of 2,3-isopropylidene turkesterone (10) to turkesterone 11,22-diacetate (5)

To a solution of 2,3-isopropylidene turkesterone (10: synthesised from 10 mg turkesterone [1]) in dry pyridine (800 µL) at room temperature was added acetic anhydride (400 µL). The reaction was stirred at room temperature for 3 hours and quenched with H₂O (20 ml). The reaction mixture was pre-purified on a SPE cartridge and the 100% MeOH fraction dried under nitrogen. The residue was dissolved in dioxan (2 ml) and aqueous HCl (0.1M, 1 ml). The reaction mixture was stirred at room temperature for 4 hours and was diluted by the addition of H₂O (20 ml). The product was pre-purified on a SPE cartridge and analysed by HPLC using an ODS2 analytical column with detection at 242 and 300 nm (30% - 100% MeOH linear gradient over 30 minutes, 1 ml/min, Rt (10) = 12.5 min, Rt (5) = 11.2 min). The product was purified on an ODS2 semi-preparative column (MeOH/H₂O [45:55 v/v], 2 ml/min) and yielded turkesterone 11,22-diacetate (5: 5.4 mg, 9.3 mmol, 46% yield). The product was identified by ¹H and ¹³C NMR and mass spectrometry.

CIMS: 598 ([M+NH₄]⁺, 27%), 563 ([M+H-H₂O]⁺, 8%), 545 ([M+H-2H₂O]⁺, 5%), 538 ([M-Ac]⁺, 5%), 521 ([M+H-Ac-H₂O]⁺, 100%), 503 ([M+H-Ac-2H₂O]⁺, 40%), 485 ([M+H-Ac-3H₂O]⁺, 22%), 461 (18%), 443 (35%), 425 (41%), 344 (19%), 176 (20%), 136 (52%), 128 (25%).

¹H and ¹³C NMR: see Tables 1 and 2.

Purity of test compounds

All compounds for bioassay were purified by RP-HPLC using water/methanol mixtures. Purity of samples for bioassay was >98%.

B_II bioassay

The microplate-based BII bioassay, based on the ecdysteroid-responsive D. melanogaster l(2)mbn permanent cell line, was performed as described previously (Clément et al., 1993).

Competitive radioligand binding assay with in vitro-expressed receptor complexes

Bacterially-expressed DmEcR and DmUSP proteins were used in a competitive binding assay with [24,25,26,27-³H]ponasterone A (0.2 nM; 150 Ci/mmol; ARC Inc.) as previously described (Dinan et al., 2001). K_i values were calculated from IC₅₀ values by means of the Cheng-Prusoff equation (K_i = IC₅₀/{1 + [radiolabelled ligand]/K_d}; Cheng and Prusoff, 1973), with the K_d for [³H]ponA being 0.15 nM.

Extraction of receptor complexes from B_II cells and cell-free receptor assays

Ecdysteroid receptor complexes were extracted from B_II cells by sonication as described previously (Dinan, 1985). After centrifugation (20,000 x g for 30 min), aliquots of the supernatant were incubated with [³H]ponA (0.2 nM; 183 Ci/mmol) and known concentrations of the test compounds. Specifically-bound radiolabel was determined by dextran-coated charcoal assay (Dinan, 1985). K_d values were determined by the LIGAND program.

Abstract
Introduction
Materials and Methods
Conclusion
Acknowledgements
References

Regioselective synthesis of 11-acyl derivatives of turkesterone

Owing to the presence of several reactive secondary hydroxyl groups in turkesterone, it was necessary to protect the diols at C-2/C-3 and C-20/C-22 in order to selectively acylate the 11-hydroxyl. The acylation sequence of the hydroxyls in 20-hydroxyecdysone has been investigated previously (Galbraith and Horn, 1969) and found to be: 2>22>3>>25. Acetylation experiments comparing turkesterone and 20-hydroxyecdysone indicated that the sequence for turkesterone was 2>11>22>3>>25 (data not shown). Figure 2 demonstrates the protection strategy employed. The 20,22-phenylboronate was first produced, followed by the 2,3-acetonide derivative. The 11-hydroxyl group could then be derivatised with the appropriate acyl anhydride. Conditions for this reaction had to be modified to take account of the solid state and lower solubility of the higher acyl anhydrides relative to their lower homologues. The diol-protecting groups were readily removed with dilute acid. Overall yields for the 11-acyl derivatives were variable. The synthesis of turkesterone 11-benzoate was attempted, but the product proved unstable, degrading rapidly back to turkesterone.

Identification of turkesterone derivatives

Turkesterone derivatives were identified by a combination of ¹H and ¹³C NMR and mass spectrometry. The major distinctive ¹H-NMR features amongst the compounds were the presence of one or more singlet absorptions at ca. 2.0 due to the acetyl groups for the acetates, and the shifts of ca. 1 ppm for the acylated oxymethine in acyl analogues. In the homogeneous series of 11-acyl analogues, significant differences were only observed in the number of signals in the ¹³C-NMR associated with the length of the acyl chain. Diagnostic absorptions are reported for all compounds and selected representatives were fully characterized by NMR. Minor differences were observed in the 11-O-acyl series and the expected shifts upon acylation of other positions.

¹H NMR for 11-acyl derivatives

The most noticeable change from the spectrum for turkesterone was the shift in the H-11 signal from 4.1 to 5.3. The signal for the acyl methyl (CH₃) was present at 0.8-0.9 and signals at 1.3-2.3 related to the acyl CH₂ groups.

¹³C NMR for 11-acyl derivatives

A small shift in the C-11 signal from 69.5 in turkesterone to 71-72 was apparent for all acyl derivatives. The acyl carbonyl (C=O) was identified by a signal at 171-173, the acyl methyl was identified by a signal at 14 and the acyl CH₂ groups were identified by a sequence of signals around 22-35.

¹H NMR for acetate derivatives

2ax-H and 3eq-H seen at 4.01 and 3.95, respectively, in turkesterone displayed downfield shifts to 5.1-5.3 and 4.1-4.2, respectively, upon derivatization at C-2. Similarly the peak for 11-H seen at 4.1 in turkesterone was seen at 5.3-5.6 in derivatives containing an acetate at C-11, and the signal for 22-H, seen at 3.44 in turkesterone, shifted to 4.8 upon derivatization at C-22. Acetyl (CH₃) signals were apparent at 2.0-2.1.

¹³C NMR for acetate derivatives

Only minor shifts were observed for signals corresponding to C-2, C-3, C-11 and C-22 for compounds derivatised at C-2, C-11 and/or C-22 (from 68.9, 68.6, 69.5 and 78.4 for turkesterone to 71.2, 65, 71 and 79, respectively, for derivatised compounds). The acetyl carbonyl (C=O) was the most obvious addition to the spectra at ca. 170 while the acetyl (CH₃) signals could be identified at ca. 21.

Biological activity of turkesterone 11-acyl derivatives

B_II bioassay

The biological activity of turkesterone in the B_II bioassay is ca. 100-fold lower than that of 20E (Table 5). It is notable that all the tested derivatives of turkesterone retain quantifiable biological activity in this bioassay, but their activities range from almost identical to over 1000-fold less than that of turkesterone. When one compares the activities of the various 11-acyl homologues, biological activity initially decreases with increasing acyl chain length (C₂ to C₄) with the acetate, propionate and butanoate being 5-, 10- and 50-fold less active than turkesterone. However, with intermediate length acyl groups (C₆ to C₁₄) the activity increases again, such that the activities of the 11-decanoate, -laurate and -myristate are similar to that of turkesterone. Activity is again lower with the C₂₂ arachidate ester (27.5-fold lower than turkesterone). These data indicate that the ligand binding pocket of EcR is capable of accepting steric bulk around C-11 of the ecdysteroidal ligand and that, further, interaction between the ligand and the binding pocket is enhanced with medium-chain length acyl groups (C₁₀ - C₁₄), relative to short- and long-chain acyl derivatives. Turkesterone 2-acetate has a biological activity similar to that of turkesterone 11-acetate, in accord with previous observations that small substituents at C-2 lower activity, but do not abolish it. The diacetates, 2,11- and 2,22-, have very much lower activities than turkesterone, but the 2,11-diacetate is more active than the 2,22-diacetate, again revealing that derivatisation at 11 is less detrimental than at C-22.

In vitro receptor assays

In addition to receptor affinity, a number of factors (e.g. penetration into the cells, sequestration, hydrolysis of ester-containing compounds) might affect potency in the B_II bioassay. Consequently, the ability of certain of the test compounds to displace [³H]ponA from in vitro-expressed DmEcR/DmUSP receptor complex was assessed (Table 5). Owing to limited quantities of the DmEcR and DmUSP proteins, it was not possible to assess all of the analogues. Comparison of the receptor assay with the B_II bioassay reveals a distinct difference between 20E and turkesterone. While 20E is more potent in the BII bioassay (EC₅₀ = 7.5 x 10^-9M) than in the receptor assay (K_i = 5.2 x 10^-8M), turkesterone is, conversely, ca. 9-fold less potent in the B_II bioassay (EC₅₀ = 8.0 x 10^-7M) than in the receptor assay (K_i = 9.0 x 10^-8M). This implies that other factors (entry, efflux, sequestration or metabolism) affect turkesterone and 20E differentially. It is known that the B_II cells do not metabolise 20E (Dinan, 1985). The differential activities in the two systems may relate to previous findings that turkesterone is a potent ecdysteroid in some insect systems, but not in others (Sláma et al., 1993). Alternatively, it may indicate that the in vitro preparation has the capacity to dehydrate 11-hydroxyl ecdysteroids to 7,9(11)-dien-6-ones (which are known to be biologically active) by a chemical reaction which occurs readily (Bourne et al., 2002).

However, there is good agreement between the K_i and EC₅₀ values for the 11-acyl derivatives of turkesterone and, as far as the somewhat restricted range of analogues assessed permits, the activity pattern in the receptor assay is similar to that in the B_II bioassay with the activity decreasing with increasing acyl length from C₂ to C₄ and then increasing again with C₆ and C₁₀ acyl derivatives. The C₂₀ derivative has a very low activity. There is also good agreement between the K_i and EC₅₀ values for turkesterone 2-acetate. Thus, although these data reveal inconsistencies for turkesterone and 20E, they support the notion that steric bulk can be accommodated around C-11 of the ecdysteroid within the ligand binding pocket of EcR.

Since the receptor complex almost certainly consists of other protein components in addition to EcR and USP, which are unlikely to be present in the in vitro translated complexes, we include data (Table 6) on the comparison of the structure-activity relationships for the B_II bioassay and a cell-free receptor assay based on ecdysteroid receptor complexes extracted from B_II cells, where other endogenous transcription factors and binding proteins could be part of the extracted receptor complex. In general, there is reasonable agreement between the EC₅₀ and K_d values determined for most of the compounds. However, it is noticeable that the three 11-hydroxy compounds included here (ajugasterone C, muristerone A and turkesterone) all have very low K_d/EC₅₀ ratios, as seen above for turkesterone in the comparison of the B_II bioassay and in vitro-expressed DmEcR/DmUSP.

Implications

Enhanced activity of 25-deoxy compounds

With the D. melanogaster BII bioassay and in vitro DmEcR/DmUSP receptor assays, turkesterone shows considerably lower activity than 20E, although in other systems turkesterone is apparently more potent. Turkesterone was used in this study as a model compound, because it was the most readily available ecdysteroid possessing an 11-hydroxyl group. However, to exploit the potential of 11-derivatives it would be preferable to use higher potency analogues lacking the 25-hydroxyl group e.g. ajugasterone C (EC₅₀ = 3.0 x 10^-8M) or muristerone A (EC₅₀ = 2.2 x 10^-8M), which should generate 11-derivatives with ca. 100-fold greater activity than those listed in Table 5.

Future synthesis of 11-acyl derivatives

It would be desirable to synthesise a parallel series of 11-acyl derivatives in order to compare their activities to those of the 11-acyl analogues. Realisation of this goal will be complicated by the absence of any known 11-hydroxy ecdysteroids (Lafont et al., 2002) and the ready tendency of 11-hydroxy ecdysteroids to dehydrate, generating 7,9(11)-dien-6-ones, under a variety of chemical conditions (Canonica et al., 1977; Qui and Nie, 1983; Bourne et al., 2002), making sequential oxidation and reduction of the 11-hydroxyl group difficult.

Relationship to other steroid hormones

The availability of hydrophobic space in the ligand-binding pocket in the region of C-11 of the steroid appears to be a common feature of steroid hormone receptors. 11-Amidoalkoxyphenyl estradiols are potent antioestrogens (Nique et al., 1994). The hydrophobic pocket has been exploited to develop 11-substituted 19-norsteroids as fluorescent steroid derivatives for the glucocorticoid and progesterone receptors (Teutsch et al., 1994). The antiprogesterone RU486 (mifepristone), used as an abortifacient, is an 11-aryl-19-nor steroid (Rosen et al., 1995).

Application for synthesis of tagged ecdysteroids

The studies shown here reveal that there is considerable steric space in the ligand binding pocket of DmEcR extending out from C-11 of the ecdysteroid, as was predicted from the CoMFA models. This area would thus be a good candidate for the preparation of tagged ecdysteroids, for example the attachment of a fluorescent moiety for the generation of an alternative, non-radioactive receptor assay ligand. Currently, [³H]ponA is almost exclusively used as the ligand for receptor assays, but it is expensive and, owing to its very high specific activity (120 - 180 Ci/mmol), requires regular repurification. C-11 could also be a good location for an affinity labelling moiety as an alternative to the photoreactive endogenous 7-en-6-one (Schaltmann and Pongs, 1982) or an extended conjugated system (7,9[11]-dien-6-one; Bourne et al., 2002). For example, bromoacetate derivatives have proved effective electrophilic affinity agents for vitamin D binding protein (Swamy et al., 2000) and the human progesterone receptor (Holmes et al., 1981). Also, a successful photoaffinity label for human multidrug resistance protein has been based on the steroid agosterol A (Ren et al., 2001), which is structurally similar to ecdysteroids and possesses an 11-hydroxyl group, on to which the azidophenyl photoaffinity label is attached. Further, it has been found that insect EcR proteins when expressed in mammalian cells and partnered by RXR have altered ligand specificity and affinity, such that 20E, the endogenous hormone in insect systems, is virtually inactive. However, muristerone A, possessing an 11-hydroxyl group, is active, but micromolar concentrations are required. For gene switching it would be highly desirable to identify ecdysteroids with higher affinity than muristerone A. Production of 11-derivatives may enhance affinity by providing closer contact to the ligand binding pocket.

Abstract
Introduction
Materials and Methods
Results and Discussion
Acknowledgements
References

The prediction from CoMFA modelling of ecdysteroid QSAR data that the ligand binding domain of D. melanogaster EcR proteins possesses a hydrophobic pocket in the vicinity of C-11 of the steroid is substantiated by the biological activity of a series of 11-acyl derivatives of turkesterone. All the 11-acyl derivatives retain some biological activity and activity is higher with medium-chain acyl derivatives. The presence of a hydrophobic pocket in this region is a feature shared with several other steroid hormone receptors. The presence of this pocket may be exploited to generate fluorescently-tagged and affinity-labelled ecdysteroids that retain high biological activity.

Abstract
Introduction
Materials and Methods
Results and Discussion
Conclusion
References

This research was supported by a CASE award to PB from the BBSRC and Rohm and Haas Co (Spring House, PA, U.S.A.) and a studentship to AT from the EU-SCIENCE Programme.

Abstract
Introduction
Materials and Methods
Results and Discussion
Conclusion
Acknowledgements

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