1
18
Y. Nakagawa et al. / Steroids 65 (2000) 117–123
tivity is caused by differences in the amino acid sequence
in the 20E-binding site of the ecdysteroid receptor (EcR)
among insect species. The amino–acid sequences of sev-
eral EcRs are known to demonstrate interspecies varia-
tion [13–18]. However, the binding modes between the
EcR and ecdysteroid analogs are unknown.
To gain insight into the molecular mechanism of nonste-
roidal ecdysteroid agonists, we quantitatively analyzed the
relationship between structure and activity [10,19–24] by
using quantitative structure-activity relationship (QSAR)
techniques such as the Hansch–Fujita approach [25] and
comparative molecular field analysis (CoMFA) [26]. Re-
cently, CoMFA was applied to the structure-activity rela-
tionship study of steroidal ecdysteroid agonists [27]. We
found that with increasing molecular hydrophobicity there
was both greater hormonal [21–23] and insecticidal activity
Ltd. (Osaka, Japan), and Nacalai Tesque Inc. (Kyoto,
Japan). Racemic 3-cyclohexene-1-carboxylic acid used to
synthesize compound 25 was purchased from Tokyo Ka-
1
4
sei Kogyo Co. Ltd. N-Acetyl-[1- C]glucosamine
1
4
([ C]GluNAc, 58.7 mCi/mmol) was purchased from
Amersham International plc (Buckinghamshire, UK).
The proportion of enantiomers of compound 25 was 1:1
by HPLC analysis with a Chiralcel OD-RH column (Dai-
cel Chemical Industries, Ltd., Tokyo, Japan). The inter-
mediates and final compounds were purified by either
recrystallization or column chromatography by using
Wakogel C-200 (Wako Pure Chemical Industries, Ltd.).
The structures and the purity of newly synthesized com-
1
pounds were confirmed by both [ H]NMR and elemental
analyses. The analytical values for C, H, and N agreed
1
with the calculated values within Ϯ 0.3%. [ H]NMR
[19,20] against lepidopterous rice stem borer Chilo suppres-
spectra were recorded on a Bruker AC-300 NMR spec-
salis Walker. A similar QSAR was obtained for the insec-
ticidal activity between rice stem borers and beet army
worms Spodoptera exigua [24], which both are lepidopter-
ous insects. However, the results were different from the
QSAR derived for the larvicidal activity against the co-
leopterous insect, the Colorado potato beetle [10]. The in-
secticidal activity against the two lepidopterous insects in-
creased with molecular hydrophobicity within a limited
range of log P (log P Ͻ 4.5), but the activity against potato
beetles varied in a bilinear manner with respect to log P. The
activity of mono–substituted compounds generally in-
creased with log P, except for several hydrophobic com-
pounds. However, activity decreased with log P for disub-
stituted and hydrophobic mono–substituted compounds
trometer at 300 MHz in deuteriochloroform (CDCl ) with
3
tetramethylsilane as the internal standard. Other com-
pounds are identical to those used in our previous studies
[20,23,28]. Melting points of all compounds were deter-
mined on a Yanako melting point apparatus (Yanagimoto
Seisakusho Co. Ltd., Kyoto, Japan) and were uncor-
rected. Melting points of final compounds are listed in
Table 1.
2.2. 3-Methoxy-2-methylbenzoic acid
Concentrated H SO (2.9 g, 29.8 mmol) was added
2
4
dropwise to 3-amino-2-methylbenzoic acid (3.0 g, 19.8
mmol) suspended in methanol (21.6 ml) in an ice bath.
After warming up the suspension to 55°C, 33.3% aqueous
NaNO2 (4.3 g, 89.3 mmol) was slowly added over a
period of 45 min. After letting the mixture stand for 1 h,
10 ml of 25% aqueous NaOH was slowly added over a
period of 2 h, then dimethylsulfate (5 g, 39.6 mmol) was
added over 45 min. Throughout the reaction the temper-
ature was kept between 55 and 65°C. After evaporating
the methanol, the residue was dissolved in ethyl acetate
(250 ml). The organic layer was washed with 1 M H SO
[10]. In an additional structure-activity study we found that
the alkyl side chain of 20-hydroxyecdysone corresponds to
the B-ring moiety in structure I [28]. By synthesizing and
assaying a number of alkanoyl analogs, this correspondence
was confirmed through the use of three-dimensional QSAR
[23]. The aim of this study was to obtain more precise
information on the structural factors that enhance hormonal
activity. We therefore quantitatively analyzed the hormonal
activity of various acylhydrazines, including alkanoyl ana-
logs, by using physicochemical parameters such as molec-
ular hydrophobicity and acyl moiety length.
2
4
and brine. After drying the organic layer over anhydrous
magnesium sulfate, the solvent was removed under re-
duced pressure. The residue was dissolved in a minimal
amount of hot methanol and poured into 1 M H SO (24
2
4
2
. Experimental
ml) to yield a solid material. The solid was collected by
filtration and manipulated from benzene to afford a
brown powder (1.76 g, yield 53.4%).
2
.1. Chemicals
Compounds 8, 13, 14, 16, 22–25 (Table 1) were newly
2.3. NЈ-t-Butyl-NЈ-(3,5-dimethylbenzoyl)-3-methoxy-2-
synthesized according to the conventional methods pre-
viously reported [10,19,20,23,24,28,29]. Methoxyfeno-
zide 15 was also synthesized according to the reported
method as shown in Fig. 1 [5]. Chemicals used for or-
ganic syntheses were obtained from Aldrich Chemical
Co. Inc. (Milwaukee, WI, USA), Tokyo Kasei Kogyo Co.
Ltd. (Tokyo, Japan), Wako Pure Chemical Industries,
methylbenzohydrazine (RH-2485; 15)
N-t-Butyl-N-3,5-dimethylbenzohydrazine (1.20 g, 5.5
mmol), prepared from 3,5-dimethylbenzoic acid and t-bu-
tylhydrazine according to conventional methods [23], was
suspended in anhydrous ether (9 ml). This ether solution
along with triethylamine (3 ml) was simultaneously added