Anti-juvenile hormone activity of ethyl 4-[(7-substituted 1,4-benzodioxan-6-yl)methyl]benzoates and their effect on the juvenile hormone titer in the hemolymph of the silkworm, Bombyx mori

Abstract

A series of ethyl 4-[(7-substituted 1,4-benzodioxan-6-yl)methyl]benzoates was synthesized and evaluated for their anti-juvenile hormone (anti-JH) activities to induce precocious metamorphosis in silkworm (Bombyx mori) larvae. The introduction of bulky alkyloxy substituents on the 7-position on the benzodioxan ring significantly increased activity. Ethyl 4-[(7-benzyloxy-1,4-benzodioxan-6-yl)methyl]benzoate (4c) showed the most potent activity among the test compounds, and its median-effective dose (ED₅₀) value was 41 ng/larva. The JH I, II, and III concentrations in the hemolymph of the 3rd instar larvae treated with compound 4c were determined by ultra-high-performance liquid chromatography/mass spectrometry (UHPLC/MS) after using a simple purification method. Compound 4c clearly decreased the JH I and II titers of 3rd instar larvae within 24 hr after treatment, and prevented JH I spike usually found immediately after 4th instar molting.

Introduction

A juvenile hormone (JH) is a sesquiterpenoid hormone that regulates metamorphosis, diapause, reproduction, and various other physiological processes in insects.¹⁾ During the larval stages, the JH controls ecdysis and metamorphosis in cooperation with the molting hormone (ecdysteroid). A high JH titer must be present in the hemolymph of immature larvae to suppress the action of the ecdysteroid and maintain larval characteristics. At the adult stage, the JH controls ovary development in females by promoting vitellogenin synthesis in some insects.²⁾ Therefore, the compounds that specifically inhibit the action of the JH (anti-JH agents) could be potential insect growth regulators (IGRs). Moreover, Methoprene-tolerant (Met), a transcription factor belonging to the bHLH-PAS family, was recently identified as a JH receptor.³⁾ Met bound JH with a nanomolar affinity and mediated the expression of Krüppel-homolog 1 (Kr-h1), a JH-inducible transcription factor.^4–6) Precocious metamorphosis was induced by injecting Met dsRNA into the flour beetle Tribolium castaneum.⁷⁾ Thus, Met plays a critical role in JH action during insect development, but the exact JH mode of action in insect development and reproduction remains unclear. Anti-JH agents, especially JH antagonists, could be used to elucidate the mechanism underlying JH signaling in various insects.

Several anti-JH agents have been found. Ethyl 4-[2-(t-butylcarbonyloxy)butoxy]benzoate (ETB) is known to possess both anti-JH and JH-like activities in the tobacco hornworm, Manduca sexta, and the silkworm, Bombyx mori, suggesting that it acts as a partial JH antagonist (Fig. 1).⁸⁾ We have also reported that (S)-ethyl 4-[2-benzylhexyloxy]benzoate (KF-13S), structurally derived from ETB, strongly induced precocious metamorphosis, a clear phenotype of JH deficiency in the silkworm, and its activity was completely counteracted by the application of methoprene, a JH agonist.⁹⁾ KF-13S was found to suppress the expression of many JH biosynthetic enzymes in the corpora allata of the silkworm.¹⁰⁾ However, the anti-JH activity of KF-13S was drastically decreased at high doses because it began to show JH-like activity. We attempted to design a new scaffold of anti-JH agents and found that ethyl 4-[(6-methoxy-2,2-dimethyl-2H-chromen-7-yl)methoxy]benzoate (KF-38), which was designed on the basis of the structure of retinoid analogs, exhibited anti-JH activity and weak JH-like activity.¹¹⁾ In a previous report, ethyl 4-[(7-methoxy-1,4-benzodioxan-6-yl)carbonyl]benzoate (KM-03) and its derivatives were prepared, but they showed no or less insecticidal activity against the western flower thrips, Frankliniella occidentalis.¹²⁾ In this study, further research on the structure–activity relationship of KM-03 was conducted. We have found that ethyl 4-[(7-substituted 1,4-benzodioxan-6-yl)methyl]benzoates showed precocious metamorphosis-inducing activity against silkworm larvae. Furthermore, to investigate their effect on JH titers in hemolymph, the JH quantification method by ultra-high-performance liquid chromatography/mass spectrometry (UHPLC/MS) was developed. JH I, II, and III titers in the hemolymph of silkworms treated with ethyl 4-[(7-benzyloxy-1,4-benzodioxan-6-y)methyl]benzoate (4c), which showed the most potent activity, were measured using this method.

Fig. 1. Structures of ETB, KF-13, KF-38 and ethyl 4-[(7-substituted 1,4-benzodioxan-6-yl)methyl]benzoates.

Materials and Methods

The scheme for synthesizing ethyl 4-[(7-substituted 1,4-benzodioxan-6-yl)methyl]benzoates is outlined in Fig. 2.

Fig. 2. Synthetic scheme for the preparation of benzodioxan derivatives. (a) Methyl 4-chlorocarbonylbenzoate, AlCl₃, dichloromethane; (b) 2 M NaOH, methanol; (c) ethanol, concentrated H₂SO₄; (d) BBr₃, dichloromethane; (e) H₂, Pd/C, acetic acid, methanol; (f) substituted benzyl chloride or benzoyl chloride, K₂CO₃, DMF.

1. Instrumental analysis and chemicals

¹H NMR spectra were determined by using a JNM-AL 400 NMR spectrometer (JEOL, Japan). All samples were prepared in deuteriochloroform containing 0.03% (v/v) tetramethylsilane (TMS). Chemical shifts (δ values) are given in ppm relative to TMS and the J values are given in Hz. Spin multiplicities are expressed as follows: s (singlet), d (doublet), t (triplet), q (quartet), dd (double doublet), and m (multiplet). High-resolution mass spectrometry (HRMS) analyses were performed with a SYNAPT G2 mass spectrometer (Waters, UK) using the positive electrospray ionization (ESI) mode.

Methyl 4-[(7-methoxy-1,4-benzodioxan-6-yl)carbonyl]benzoate (1)

Methyl 4-chlorocarbonylbenzoate (1.5 g, 7.5 mmol) and AlCl₃ (1.0 g, 7.5 mmol) were added to a solution of 6-methoxy-1,4-benzodioxan (1.0 g, 6.3 mmol) in 10 mL of dichloromethane at 0°C. After stirring for 12 hr at room temperature, the mixture was quenched with 2 M HCl at 0°C and filtered. The product was extracted with dichloromethane, and the extract was washed with 2 M NaOH and brine, dried over Na₂SO₄, and concentrated. The residue was purified by column chromatography on silica gel by eluting with CH₂Cl₂–MeOH (100 : 1) to give 0.70 g (34%) of 1 as a colorless crystal. ¹H NMR (CDCl₃) δ: 3.59 (3H, s, OCH₃), 3.95 (3H, s, OCH₃), 4.24–4.34 (4H, m, OCH₂CH₂O), 6.50 (1H, s, phenyl), 7.05 (1H, s, phenyl), 7.80 (2H, d, J=8.3 Hz, phenyl), 8.08 (2H, d, J=8.8 Hz, phenyl).

Ethyl 4-[(7-methoxy-1,4-benzodioxan-6-yl)carbonyl]benzoate (2)

A solution of 1 (0.70 g, 2.1 mmol) in 10 mL of methanol and 5 mL of 1 M NaOH was refluxed for 12 hr. The product was extracted with ethyl acetate, and the ethyl acetate solution was washed with 1 N HCl and brine, dried over Na₂SO₄, and concentrated. The residue was collected by filtration using hexane to give 0.40 g of crude product as a white solid. A solution of crude product in 20 mL of ethanol containing a few drops of concentrated H₂SO₄ was refluxed for 15 hr. The product was extracted with ethyl acetate, and the ethyl acetate solution was washed with 2 M NaOH and brine, dried over Na₂SO₄, and concentrated. The residue was purified by column chromatography on silica gel by eluting with hexane–ethyl acetate (3 : 1) to give 0.75 g (97% in two steps) of 2 as a colorless crystal. ¹H NMR (CDCl₃) δ: 1.41 (3H, t, J=7.3 Hz, CH₃), 3.59 (3H, s, OCH₃), 4.24–4.34 (4H, m, OCH₂CH₂O), 4.41 (2H, q, J=7.3 Hz, OCH₂), 6.50 (1H, s, phenyl), 7.05 (1H, s, phenyl), 7.80 (2H, d, J=8.3 Hz, phenyl), 8.08 (2H, d, J=8.3 Hz, phenyl).

Ethyl 4-[(7-hydroxy-1,4-benzodioxan-6-yl)methyl]benzoate (3)

Boron tribromide, 1.0 M in dichloromethane (2.6 mL, 2.6 mmol), was added to a solution of 2 (0.75 g, 2.2 mmol) in 10 mL of dichloromethane at −7°C. The mixture was quenched with ice water after stirring for 30 min at the same temperature. The product was extracted with dichloromethane, and the extract was washed with water and brine, dried over Na₂SO₄, and concentrated to give 0.65 g of crude product as a white solid. A solution of crude product in 8.5 mL of methanol and 1.5 mL of acetic acid containing Pd/C (0.01 g) was stirred for 16 hr at room temperature under hydrogen gas. After filtration, the product was extracted with ethyl acetate. The ethyl acetate solution was washed with water and brine, dried over Na₂SO₄, and concentrated. The residue was purified by column chromatography on silica gel by eluting with hexane–ethyl acetate (3 : 1) to give 0.61 g (89% in two steps) of 3 as a colorless crystal. ¹H NMR (CDCl₃) δ: 1.38 (3H, t, J=7.3 Hz, CH₃), 3.92 (2H, s, CH₂), 4.18–4.21 (4H, m, OCH₂CH₂O), 4.36 (2H, q, J=7.3 Hz, OCH₂), 6.35 (1H, s, phenyl), 6.59 (1H, s, phenyl), 7.28 (2H, d, J=8.3 Hz, phenyl), 7.95 (2H, d, J=8.3 Hz, phenyl). HRMS (ESI) m/z ([M+Na]⁺): Calcd. for C₁₈H₁₈NaO₅: 337.1052; Found: 337.1064.

Ethyl 4-[(7-methoxy-1,4-benzodioxan-6-yl)methyl]benzoate (4a)

Methyl iodide (0.027 g, 0.19 mmol) and K₂CO₃ (0.026 g, 0.19 mmol) were added to a solution of 6 (0.040 g, 0.13 mmol) in 10 mL of DMF. After stirring for 15 hr at room temperature, the product was extracted with ethyl acetate. The ethyl acetate solution was washed with 2 M NaOH and brine, dried over Na₂SO₄, and concentrated. The residue was purified by column chromatography on silica gel by eluting with hexane–ethyl acetate (3 : 1) to give 0.038 g (89%) of 4a as a colorless crystal. ¹H NMR (CDCl3) δ : 1.37 (3H, t, J=7.3 Hz, CH₃), 3.71 (3H, s, OCH₃), 3.89 (2H, s, CH₂), 4.17–4.24 (4H, m, OCH₂CH₂O), 4.35 (2H, q, J=7.3 Hz, OCH₂), 6.41 (1H, s, phenyl), 6.57 (1H, s, phenyl), 7.25 (2H, d, J=8.3 Hz, phenyl), 7.93 (2H, d, J=8.3 Hz, phenyl). HRMS (ESI) m/z ([M+H]⁺): Calcd. for C₁₉H₂₁O₅: 329.1389; Found: 329.1400.

Compounds 4b–i were prepared in the same manner as described above using benzoyl chloride or the corresponding benzyl chloride.

Ethyl 4-[(7-benzoyloxy-1,4-benzodioxan-6-yl)methyl]benzoate (4b)

Yield: 86%, ¹H NMR (CDCl₃) δ: 1.38 (3H, t, J=7.3 Hz, CH₃), 3.85 (2H, s, CH₂), 4.25 (4H, s, OCH₂CH₂O), 4.35 (2H, q, J=7.3 Hz, OCH₂), 6.67 (1H, s, phenyl), 6.73 (1H, s, phenyl), 7.18 (2H, d, J=8.3 Hz, phenyl), 7.47 (2H, d, J=8.3 Hz, phenyl), 7.62 (1H, t, J=7.8 Hz, phenyl), 7.90 (2H, d, J=8.3 Hz, phenyl), 8.06 (2H, d, J=7.8 Hz, phenyl). HRMS (ESI) m/z ([M+Na]⁺): Calcd. C₂₅H₂₂NaO₆: 441.1314; Found: 441.1307.

Ethyl 4-[(7-benzyloxy-1,4-benzodioxan-6-yl)methyl]benzoate (4c)

Yield: 92%, ¹H NMR (CDCl₃) δ: 1.38 (3H, t, J=7.3 Hz, CH₃), 3.94 (2H, s, CH₂), 4.18–4.23 (4H, m, OCH₂CH₂O), 4.36 (2H, q, J=7.3 Hz, OCH₂), 4.93 (1H, s, OCH₂), 6.48 (1H, s, phenyl), 6.62 (1H, s, phenyl), 7.10–7.36 (7H, m, phenyl), 7.92 (2H, d, J=8.3 Hz, phenyl). HRMS (ESI) m/z ([M+Na]⁺): Calcd. C₂₅H₂₄NaO₅: 427.1521; Found: 427.1517.

Ethyl 4-{[7-(2-methylbenzyloxy)-1,4-benzodioxan-6-y]methyl}benzoate (4d)

Yield: 91%, ¹H NMR (CDCl₃) δ: 1.37 (3H, t, J=7.3 Hz, CH₃), 2.25 (3H, s, CH₃), 3.91 (2H, s, CH₂), 4.18–4.24 (4H, m, OCH₂CH₂O), 4.35 (2H, q, J=7.3 Hz, OCH₂), 4.90 (2H, s, OCH₂), 6.52 (1H, s, phenyl), 6.61 (1H, s, phenyl), 7.17–7.29 (6H, m, phenyl), 7.89 (2H, d, J=8.3 Hz, phenyl). HRMS (ESI) m/z ([M+Na]⁺): Calcd. C₂₆H₂₆NaO₅: 441.1678; Found: 441.1674.

Ethyl 4-{[7-(3-methylbenzyloxy)-1,4-benzodioxan-6-y]methyl}benzoate (4e)

Yield: 69%, ¹H NMR (CDCl₃) δ: 1.37 (3H, t, J=7.3 Hz, CH₃), 2.31 (3H, s, CH₃), 3.94 (2H, s, CH₂), 4.18–4.23 (4H, m, OCH₂CH₂O), 4.35 (2H, q, J=7.3 Hz, OCH₂), 4.88 (2H, s, OCH₂), 6.47 (1H, s, phenyl), 6.63 (1H, s, phenyl), 7.03–7.11 (3H, m, phenyl), 7.21 (1H, d, J=7.3 Hz, phenyl), 7.24 (2H, d, J=8.3 Hz, phenyl), 7.92 (2H, d, J=8.3 Hz, phenyl). HRMS(ESI) m/z ([M+Na]⁺): Calcd. C₂₆H₂₆NaO₅: 441.1678; Found: 441.1674.

Ethyl 4-{[7-(4-methylbenzyloxy)-1,4-benzodioxan-6-y]methyl}benzoate (4f)

Yield: 83%, ¹H NMR (CDCl₃) δ: 1.38 (3H, t, J=7.3 Hz, CH₃), 2.35 (3H, s, CH₃), 3.92 (2H, s, CH₂), 4.18–4.22 (4H, m, OCH₂CH₂O), 4.36 (2H, q, J=7.3 Hz, OCH₂), 4.89 (2H, s, OCH₂), 6.48 (1H, s, phenyl), 6.61 (1H, s, phenyl), 7.14 (4H, s, phenyl), 7.23 (2H, d, J=8.3 Hz, phenyl), 7.91 (2H, d, J=8.3 Hz, phenyl). HRMS(ESI) m/z ([M+Na]⁺): Calcd. C₂₆H₂₆NaO₅: 441.1678; Found: 441.1674.

Ethyl 4-{[7-(4-methoxybenzyloxy)-1,4-benzodioxan-6-y]methyl}benzoate (4g)

Yield: 72%, ¹H NMR (CDCl₃) δ: 1.38 (3H, t, J=7.3 Hz, CH₃), 3.81, (3H, s, OCH₃), 3.90 (2H, s, CH₂), 4.18–4.23 (4H, m, OCH₂CH₂O), 4.36 (2H, q, J=7.3 Hz, OCH₂), 4.85 (2H, s, OCH₂), 6.48 (1H, s, phenyl), 6.61 (1H, s, phenyl), 6.86 (2H, d, J=8.8 Hz, phenyl), 7.18 (2H, d, J=8.8 Hz, phenyl), 7.22 (2H, d, J=8.3 Hz, phenyl), 7.91 (2H, d, J=8.3 Hz, phenyl). HRMS (ESI) m/z ([M+Na]⁺): Calcd. C₂₆H₂₆NaO₆: 457.1627; Found: 457.1629.

Ethyl 4-{[7-(4-chlorobenzyloxy)-1,4-benzodioxan-6-y]methyl}benzoate (4h)

Yield: 94%, ¹H NMR (CDCl₃) δ: 1.38 (3H, t, J=7.3 Hz, CH₃), 3.92 (2H, s, CH₂), 4.19–4.23 (4H, m, OCH₂CH₂O), 4.36 (2H, q, J=7.3 Hz, OCH₂), 4.88 (2H, s, OCH₂), 6.43 (1H, s, phenyl), 6.63 (1H, s, phenyl), 7.15 (2H, d, J=8.3 Hz, phenyl), 7.21 (2H, d, J=8.3 Hz, phenyl), 7.28 (2H, d, J=8.3 Hz, phenyl), 7.92 (2H, d, J=8.3 Hz, phenyl). HRMS (ESI) m/z ([M+Na]⁺): Calcd. C₂₅H₂₃ClNaO₅: 461.1132; Found: 461.1122.

Ethyl 4-{[7-(4-trifluoromethylbenzyloxy)-1,4-benzodioxan-6-yl]methyl}benzoate (4i)

Yield: 88%, ¹H NMR (CDCl₃) δ: 1.38 (3H, t, J=7.3 Hz, CH₃), 3.95 (2H, s, CH₂), 4.19–4.23 (4H, m, OCH₂CH₂O), 4.36 (2H, q, J=7.3 Hz, OCH₂), 4.97 (2H, s, OCH₂), 6.43 (1H, s, phenyl), 6.65 (1H, s, phenyl), 7.22 (2H, d, J=8.3 Hz, phenyl), 7.33 (2H, d, J=8.3 Hz, phenyl), 7.57 (2H, d, J=8.8 Hz, phenyl), 7.92 (2H, d, J=8.8 Hz, phenyl). HRMS (ESI) m/z ([M+Na]⁺): Calcd. C₂₆H₂₃F₃NaO₅: 495.1395; Found: 495.1386.

2. Biological evaluation

Bombyx mori (Shunrei×Shougetsu) larvae were reared on Silkmate 2S (Nippon-nosan Kogyo, Japan) under a photoperiodic regime of 12 hr of light and 12 hr of darkness at 25°C.¹¹⁾ Each test compound in acetone (2 µL) was topically applied to 24-hr-old 3rd instar larvae. Twenty larvae were used for each dose. The activity of the compounds was evaluated by the rate of induction of precocious metamorphosis.

3. Quantification of JH in hemolymph

3.1. Reagents

Purified JH I, II, and III and deuterium-labeled JH III (JH III-d₃) were obtained as described in our previous reports.^13,14) JH III-d₃ was dissolved in toluene (50 pg/µL) and stored at −30°C. All solvents used for extraction and cleanup were of residual pesticide analysis grade (Wako Pure Chemicals, Japan).

3.2. Purification of JHs from hemolymph and analytical conditions for UHPLC-MS

The extraction and clean-up procedure for determination of the JH in hemolymph was carried out according to previous reports.^14,15) Briefly, 10 µL of JH III-d₃ in toluene (50 pg/µL) and 100 µL of hemolymph were transferred into a glass tube. The JHs in the hemolymph were extracted with n-hexane. The crude extract was roughly purified by a small aluminum oxide column. The eluent was evaporated under nitrogen flow. The residue was dissolved in 15 µL of 80% acetonitrile in water containing 1 µM sodium acetate.

The extract samples were injected into an ACQUITY-Xevo TQ UHPLC/MS system (Waters, UK) equipped with a 2.1×100 mm Kinetex 2.6 µm C₁₈ reversed phase column (Phenomenex, USA). The linear gradient elution was programmed as follows: Eluent A: 50% methanol in water with 1 µM sodium acetate; Eluent B: 95% methanol in water with 1 µM sodium acetate; 0–0.5 min, 0% B; 0.5–8 min, 0–100% B; 8–9 min 100% B. The solvent flow rate was set at 0.3 mL/min. The MS spectra were acquired in the electrospray ionization (ESI) positive ion mode. Capillary and cone voltages were set at 2500 and 20 V, respectively. The ionization source was working at 150°C, and the desolvation gas temperature was set at 600°C. Selected ion masses (SIMs) for each JH were monitored. These were m/z 289.2, 292.2, 303.2, and 317.2 for JH III, JH III-d₃, JH II, and JH I, respectively.

Results and Discussion

1. Anti-JH activity of benzodioxan derivatives

The anti-JH activity of ethyl 4-[(7-substituted 1,4-benzodioxan-6-yl)methyl]benzoates was evaluated by precocious metamorphosis-inducing tests against 3rd instar larvae of B. mori (Table 1). Compound 4a, possessing a methoxy group at the 7-position of the benzodioxan ring, slightly induced precocious metamorphosis at 10 µg. When a benzyloxy group (4c) was introduced, anti-JH activity was drastically improved. Compound 4c showed a dose-dependent activity, in contrast to ETB and KF-13, and its ED₅₀ value was 41 ng/larva. The anti-JH activity of compound 4c was completely counteracted by the treatment of 1 µg of methoprene, a JH agonist. However, the introduction of hydroxyl (3) or benzoyloxy (4b) groups resulted in the disappearance of anti-JH activity even at high doses. These results indicated that a bulky alkyloxy group is important for high anti-JH activity. Various substituents were introduced into the benzene ring of the benzyl group of 4c to further investigate the effect these changes may have on anti-JH activity. Compound 4f, containing a 4-methylbenzyloxy group, showed slightly weak activity compared to 4c (ED₅₀=131 ng/larva). However, the displacement of the methyl group from the para to the ortho or meta positions led to a considerable decrease in anti-JH activity. The introduction of an electron-donating 4-methoxy group (4g) also showed weak activity at 1 µg. In contrast, the electron-withdrawing substituents, 4-chloro (4h) and 4-trifluoromethyl (4i) derivatives, showed no activity at the same dose.

Table 1. Precocious metamorphosis-inducing activities of ethyl 4-[(7-substituted 1,4-benzodioxan-6-yl)methyl]benzoates against 3rd instar silkworm larvae.

		Precocious metamorphosisa) (%)
No.	R	0.1	1	10	(µg/larva)
3	OH	N.T.	0	0
4a	OMe	0	0	15
4b	OBz	0	0	0
4c	OBn	78	92	100
4d	O(2-Me)Bn	0	0	0
4e	O(3-Me)Bn	0	5	10
4f	O(4-Me)Bn	39	92	90
4g	O(4-OMe)Bn	N.T.	15	67
4h	O(4-Cl)Bn	0	0	21
4i	O(4-CF3)Bn	0	0	16

^a) Values are the average of three experiments.

When 3rd instar larvae were treated with these compounds, precocious metamorphosis was always induced in the 4th instar. Interestingly, when 2nd instar larvae were treated with 10 µg of compound 4c, precocious metamorphosis also occurred in 4th instar. Daimon et al. reported that a JH-deficient mutant of the silkworm (mod strain), which is a null mutant in the JH epoxidase (CYP15C1) gene did not precociously metamorphose in the 1st or 2nd instar.¹⁶⁾ These results indicate that compound 4c was ineffective during the 2nd instar and showed anti-JH activity only after molting to the 3rd instar.

2. Analysis of JH titers in the silkworm hemolymph using UHPLC/MS

We previously reported a JH quantification method by LC/MS, in which JHs were derivatized to JH methoxyhydrines to remove interference from foreign ions.¹⁴⁾ However, the derivatization reaction and purification take a long time when this method is used. In this study, we developed a UHPLC/MS method with simpler preparation requirements. Figure 3 shows the chromatogram of a hemolymph extract from 2-d-old 3rd instar larvae. Each JH was detected with good sensitivity and separation when a methanol/water solution containing 1 µM sodium acetate was used as the mobile phase. The molecular ion peaks of JH III-d₃ (m/z 292.2), JH II (m/z 303.2) and JH I (m/z 317.2) were observed at 4.28, 5.02, and 5.62 min, respectively. JH III (m/z 289.2) could not be detected at the same retention time as JH III-d₃ (the limit of detection <15 pg). The JH I and JH II concentrations in the hemolymph sample could be calculated from the ratio of the peak area of JH III-d₃ when it was used as an internal standard, and they were found to be 1.67 and 1.15 ng/mL, respectively.

Fig. 3. Chromatogram of a hemolymph sample on day 2 of 3rd-instar larvae monitoring of JHs as sodium adducts. A, JH I (m/z 317.2); B, JH II (m/z 303.2); C, JH III (m/z 289.2); D, JH III-d₃ (m/z 292.2).

Under optimized conditions, JH I, II, and III in the hemolymph of silkworm larvae were quantified every 24 hr from 2-d-old 2nd instar larvae to just before pupation (Fig. 4A). There was a pronounced peak of JH I soon after ecdysis to the 3rd and 4th instars, and the JH I titer gradually decreased before the next ecdysis. The JH II titer changed very little through the 3rd and 4th larval stages. The developmental pattern of JH I during these stages was very similar to that obtained by LC/MS¹⁴⁾ and radioimmunoassay (RIA).¹⁷⁾ However, JH titers in the hemolymph of 5th instar larvae obtained by UHPLC/MS were completely different from those obtained when the RIA method was used. An apparent peak for JH I in the early stages of the 5th instar could not be detected. Furthermore, no increase in the JH titer in the prepupal stage was observed. Kayukawa et al. reported that the expression level of Kr-h1 was low in the epidermis of newly molted 5th instar larvae,⁶⁾ indicating that JH titers were low at the same period. On the other hand, Kr-h1 was strongly expressed in the prepupal stage. However, the expression of Kr-h1 was also observed in allatectomized larvae on day 1 of prepupal stage, suggesting that the expression of Kr-h1 was independent of the hemolymph JH titer in the prepupal stage.¹⁸⁾ These results indicated that JH titers obtained using the UHPLC/MS method were valid and reliable.

Fig. 4. (A) Developmental changes to JH concentration in hemolymph of Bombyx mori from the 2-day-old 2nd instar stage to just before pupation. Closed circle, JH I; open circle, JH II; open square, JH III. (B) Effects of compound 4c on JH I titers in hemolymph. Ten microgram of compound 4c in acetone (2 µL) was topically applied to 24 hr-old 3rd instar larvae, and then the concentration of JHs in the hemolymph was determined by UHPLC/MS every 24 hr. Closed circle, control larvae; open circle, compound 4c-treated larvae. E, larval ecdysis. Error bar means±S.D. (n=4).

The JH titers in the hemolymph of larvae treated with 10 µg of compound 4c were determined from just after treatment to before precocious pupation (Fig. 4B). The JH I titer was clearly decreased by the application of compound 4c within 24 hr. JH II and III were not detected over the same period (data not shown). Interestingly, a JH I peak in the 4th instar larvae was not detected, and the JH I concentration remained low until precocious pupation. This result suggested that compound 4c induced precocious metamorphosis by inducing the JH titer of normal 5th instar larvae during the 4th instar stage.

Ethyl 4-[(7-benzyloxybenzodioxan-6-yl)methyl]benzoate (4c) was shown to be a novel, potent anti-JH agent that induced precocious metamorphosis in silkworm larvae by decreasing the JH titers in hemolymph. However, the exact mode of action of compound 4c remains unclear because the decrease of JH titers were induced by various causes, such as the inhibition of JH biosynthetic enzymes, the suppression of these enzyme transcriptions, and the induction of the JH metabolic enzyme. Further investigation is necessary to identify the mode of action of compound 4c.

Acknowledgment

This work was supported by a grant-in-aid to E.K. for scientific research (no. 26870380) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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