Absorption, Distribution and Excretion
Forty-two groups of Sprague Dawley rats (maximum of five males and females per group) were orally administered (14)Ct-butyl-, (14)CA-cyclo-, or (14)CB-cyclo-methoxyfennozine at doses of 10 or 1000 mg/kg. Some treatment groups received an appropriate amount of unlabeled or (13)C-labeled methoxyfennozine mixed with (14)C-methoxyfennozine. Three types of experiments were performed: (1) determining excretion, distribution, and mass balance 120 hours after administration; (2) determining plasma concentrations (Cmax and 1/2 Cmax); and (3) determining tissue distribution of (14)C at Cmax and 1/2 Cmax. Most of (14)C was excreted within the first 24 hours after administration, with 58-77% of the administered dose recovered in feces and 4-9% recovered in urine on days 0-1. The location of carbon labeling did not significantly alter the excretion pattern. Approximately 0.07–0.23% of (14)C remained in tissues, and 0.03–0.11% of (14)C was recovered from day 0–5 post-administration as (14)C-CO2 and volatile organic compounds. All three (14)C-labeled (14)C-methoxyphenylhydrazines reached peak plasma concentrations 15–30 minutes post-administration. The highest tissue concentrations of (14)C were observed in the liver. (14)C residues were rapidly cleared from all organs of the rats. Based on the recovery of ¹⁴C from bile, urine, tissues, and carcass, 62–70% of the administered dose was systemically absorbed.
The distribution of radioactivity in tissues was investigated after a single administration of A-ring-labeled or tert-butyl-labeled methoxyfenozide via gavage (at C_max, 1/2 C_max, and 5 days post-administration, at doses of 10 or 1000 mg/kg body weight, respectively); the distribution of radioactivity in tissues was also investigated after a single administration of B-ring-labeled methoxyfenozide (5 days post-administration, at a dose of 10 mg/kg body weight). Furthermore, tissue distribution after administration of A-ring-labeled methoxyfenozide at a dose of 10 mg/kg body weight, pulsed (5 days post-administration), and repeated administration (0.25 hours after the last administration, approximately at C_max) was also studied. Similar results were observed in all experiments. The absorbed radioactive material was widely distributed, with the highest concentrations in the liver 0.5–2 hours post-administration (the higher concentrations in the gastrointestinal tract were mainly attributed to unabsorbed material). …The drug was widely eliminated from the body; the highest percentage of radioactive material in the liver was observed 5 days after a single oral dose of 10 mg/kg body weight, but this was only <0.1% of the administered dose. This study investigated the bile excretion of radiolabeled substances following a single oral dose of 10 mg/kg ((14)CA-cyclic)methoxyfenoxazine in cannulated rats. Bile excretion was rapid; within 12 hours, 22% of the administered dose was excreted in female rats and 50% in male rats. Overall, within 72 hours, 38% of the radiolabeled substances in female rats and 64% in male rats were excreted via bile. Significant inter-individual differences were observed in cannulated female rats (bile absorption rate 13-55% and urine absorption rate 5-43% within 72 hours), but the total absorption (absorption rate in bile, urine, carcass, and tissues) was similar in all four female rats (56-67%). Considering the bile component, the oral absorption rate of methoxyfenozide at a dose of 10 mg/kg body weight was 60-70% in both male and female rats. In a study conforming to US EPA guidelines and Good Laboratory Practice (GLP), the in vivo transdermal absorption of methoxyfenozide was tested in either an aqueous flowing liquid (RH-112,485 2F) or a wettable powder (RH-112,485 280WP). All administered methoxyfenozide rings were uniformly labeled with 14C. Given the limited degree of cleavage observed in oral metabolism studies, these results are acceptable. To provide data on concentrated products and exposure to diluents, four male Cr1:CD BR rats were randomly assigned to four groups and administered 100 μL of radiolabeled methoxyfenoxazine aqueous solution at three different concentrations (0.025%, 0.25%, or 2.5% w/v) to a shaved area of approximately 10 cm² for 1, 10, or 24 hours. Systemic absorption of methoxyfenoxazine was defined as the radiolabel detected in the carcass, urine (including urine funnel and cage flushing fluid), feces, and whole blood. For RH-112,485 2F, the mean total radiolabel recovery across all groups ranged from 98% to 114%. After animals were exposed to (14)C-labeled RH-112,485 2F formulation (dissolved in water) at concentrations of 2.5%, 0.25%, or 0.025% (w/v) for 1, 10, or 24 hours, a small amount of radiolabeled material (<1-4%) was systematically absorbed. For RH-112,485 280 WP, three animals were excluded from subsequent analyses due to low recovery. The overall mean recovery of radiolabeled material across all groups ranged from 85% to 110%. After animals were exposed to (14)C-labeled RH-112,485 280 WP formulation at concentrations of 2.5%, 0.25%, or 0.025% (w/v) for 1, 10, or 24 hours, <1-2% of the radiolabeled material was systematically absorbed. Results were similar for both formulations. During the 10-hour and 24-hour exposure periods, the amount of systemically absorbed radiolabeled material did not increase linearly, indicating that most of the radiolabeled material remaining in or on the skin after washing was tightly bound and could not be absorbed by the system. This study shows that the absorption rate of methoxyphenylhydrazine after skin contact preparation or dilution is very low (<4%). The low skin absorption rate may be attributed to its extremely low solubility in water (3.3 mg/L at 20°C).

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Metabolism/Metabolites
42 groups of Sprague Dawley rats (maximum of 5 males and females per group) were orally administered (14)Ct-butyl-, (14)CA-cyclo-, or (14)CB-cyclo-methoxyphenylhydrazine at doses of 10 or 1000 mg/kg. …(14)C-methoxyphenylhydrazine is extensively metabolized into 32 metabolites (26 of which have been identified), which were isolated from urine and feces; another 24 metabolites were found in bile and characterized. At dose levels of 10 mg/kg and 1000 mg/kg, the seven metabolites accounted for 59-69% and 42-56% of the administered dose, respectively. The parent compound accounted for 14-26% and 30-39% of the administered dose (14)C at dose levels of 10 mg/kg and 1000 mg/kg, respectively. The parent compound was found only in feces (not in urine or bile), accounting for 14-26% and 30-39% of the administered dose in the low-dose and high-dose groups, respectively, indicating that the low-dose group metabolized a higher proportion of the administered dose compared to the high-dose group. Seven metabolites (M10, M14, M16, M22, M24, M28, and M30) were detected in both feces and urine, with each metabolite exceeding 2% of the administered dose. The major metabolites were M14 (demethylated parent compound) and M24 (hydroxymethyl derivative). In all groups, the parent compound plus these seven metabolites accounted for 74-90% of the administered dose (in feces and urine). In each group, the total amount of the parent compound plus the identified metabolites accounted for ≥83% of the administered dose, indicating that the metabolic profile of methoxyphenylhydrazine in feces and urine was well-defined. Metabolites resulting from amide bridge cleavage accounted for less than 5% of the administered dose. The major metabolites in bile were two: M16 (A-ring glucuronide of M14) and M26 (A-ring glucuronide of M24). M16 was present in 13% of males and 18% of females, while M26 was present in 5% of males and 11% of females. All other metabolites accounted for <3% of the administered dose. The higher concentrations of M16 and M26 in bile compared to feces suggest that these two metabolites may have undergone subsequent hydrolysis. The primary metabolic pathway likely involves the demethylation of the A-ring methoxy group, yielding the corresponding phenolic compound (M14), which then combines with glucuronic acid to form M16. Hydroxylation of the B-ring methyl group is also an important metabolic pathway. Cleavage of methoxyphenylhydrazine to release the ring or tert-butyl group is only a minor metabolic pathway; the content of all cleavage metabolites (M06, M07, M13, M32-36) did not exceed 2% of the administered dose. However, in males, the proportion of cleavage products in urine is as high as approximately 50%. Based on the urinary metabolite pattern, there is evidence that males cleave a greater portion of the absorbed dose than females. Animals fed a diet containing methoxyfenozide for 14 consecutive days and given a single dose of (14C) methoxyfenozide by gavage showed increased concentrations of M22, M28, and M30, while the concentrations of M14 and M24 were lower than those of animals that received only a single dose of 10 mg/kg body weight (14C) methoxyfenozide.

Non-Human Toxicity Values
Rat inhalation LC50 >4.3 mg/L/4 hours
Rat dermal LD50 2000 mg/kg
Rat oral LD50 5000 mg/kg
Mouse oral LD50 5000 mg/kg

Methoxyfenozide is a carbamate compound with the structure hydrazine, in which the amino hydrogen is replaced by 3-methoxy-2-methylbenzoyl, 3,5-dimethylbenzoyl, and tert-butyl, respectively. It is an environmental pollutant, exogenous substance, and insecticide. It belongs to the carbamate class and the monomethoxybenzene class of compounds. Its function is similar to N'-benzoyl-N-(tert-butyl)benzoyl hydrazine. Methoxyfenozide has been reported to exist in Ganoderma lucidum, and relevant data are available. Methoxyfenozide is a diacylhydrazine insecticide that binds with extremely high affinity to the ecdysone receptor complex and acts as a potent agonist or analog of the insect ecdysone 20-hydroxyecdysone (20E). Methoxyfenozide has a high insecticidal effect on a variety of important caterpillar pests, including many species of lepidopteran insects such as the navel orange borer, peach fruit moth, leafroller, inchworm, armyworm, and citrus leafminer.

C22H28N2O3

368.4693

368.209

161050-58-4

Methoxyfenozide-d9;2469014-53-5

105010

White powder

1.1±0.1 g/cm3

530.3±60.0 °C at 760 mmHg

204-205ºC

274.5±32.9 °C

0.0±1.5 mmHg at 25°C

1.539

5.59

1

3

4

27

518

0

O=C(C1C([H])=C(C([H])([H])[H])C([H])=C(C([H])([H])[H])C=1[H])N(C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])N([H])C(C1C([H])=C([H])C([H])=C(C=1C([H])([H])[H])OC([H])([H])[H])=O

QCAWEPFNJXQPAN-UHFFFAOYSA-N

InChI=1S/C22H28N2O3/c1-14-11-15(2)13-17(12-14)21(26)24(22(4,5)6)23-20(25)18-9-8-10-19(27-7)16(18)3/h8-13H,1-7H3,(H,23,25)

N'-tert-butyl-N'-(3,5-dimethylbenzoyl)-3-methoxy-2-methylbenzohydrazide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~271.39 mM)

Solubility in Formulation 1: 2.5 mg/mL (6.78 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (6.78 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.78 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)