CYP51; fungicidal

Prothioconazole is a new triazolinthione fungicide used in agriculture. We have used Candida albicans CYP51 (CaCYP51) to investigate the in vitro activity of prothioconazole and to consider the use of such compounds in the medical arena. Treatment of C. albicans cells with prothioconazole, prothioconazole-desthio, and voriconazole resulted in CYP51 inhibition, as evidenced by the accumulation of 14α-methylated sterol substrates (lanosterol and eburicol) and the depletion of ergosterol. We then compared the inhibitor binding properties of prothioconazole, prothioconazole-desthio, and voriconazole with CaCYP51. We observed that prothioconazole-desthio and voriconazole bind noncompetitively to CaCYP51 in the expected manner of azole antifungals (with type II inhibitors binding to heme as the sixth ligand), while prothioconazole binds competitively and does not exhibit classic inhibitor binding spectra. Inhibition of CaCYP51 activity in a cell-free assay demonstrated that prothioconazole-desthio is active, whereas prothioconazole does not inhibit CYP51 activity. Extracts from C. albicans grown in the presence of prothioconazole were found to contain prothioconazole-desthio. We conclude that the antifungal action of prothioconazole can be attributed to prothioconazole-desthio [1].

Antifungal binding determinations. [1]
The chemical structures of the azoles used in this study are shown in Fig. 1. Binding of azole to CaCYP51 was performed as previously described. A stock 2 mg · ml−1 solution of Prothioconazole, a 0.2 mg · ml−1 solution of prothioconazole-desthio, and a 0.1 mg · ml−1 solution of voriconazole were prepared in DMF. Azoles were progressively titrated against 5 μM CaCYP51 in 0.1 M Tris-HCl (pH 8.1) and 25% (wt/vol) glycerol, with the difference spectra between 500 and 350 nm determined after each addition. Azole binding determinations were performed in triplicate for each compound. Binding saturation curves were constructed from ΔApeak-trough against the azole concentration. A rearrangement of the Morrison equation was used to determine the dissociation constant (Kd) values when ligand binding was “tight.” Tight binding is observed when the Kd for azole is similar to or lower than the concentration of CYP51 present. The Michaelis-Menten equation was used when the ligand binding was not tight. The Kd values reported are the mean values from three replicates along with the associated standard deviations.

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Substrate binding studies. [1]
A 0.1% (wt/vol) aqueous solution of lanosterol in 0.5% (vol/vol) Tween 80 was prepared as previously described. Lanosterol was progressively titrated against 10 μM CaCYP51 in the sample cuvette with equivalent amounts of 0.5% (vol/vol) Tween 80 added to the P450-containing reference cuvette. The absorbance difference spectrum between 500 and 350 nm was determined after each incremental addition of lanosterol, and binding saturation curves were constructed from the ΔA385–419, including corrections for changes in sample volume. The substrate binding constant (Ks) was determined by nonlinear regression (Levenberg-Marquardt algorithm) using the Michaelis-Menten equation. Ks values reported for lanosterol are the mean values from three replicates along with the associated standard deviations. The spin-state change of CaCYP51 was calculated from the ΔA385–419 value using an extinction coefficient of 118 mM−1 · cm−1 derived for the type I difference spectrum of CYP164A2 which was modulated from 100% low spin to nearly 100% high spin by physicochemical means.

Lanosterol binding difference spectra were determined with 10 μM CaCYP51 in the presence and absence of 4 μM voriconazole, 100 μM Prothioconazole, and 4 μM prothioconazole-desthio. Negative-control determinations were made in the presence of 1.25% (vol/vol) DMF. Determinations were performed in triplicate, and Lineweaver-Burk plots were constructed from resultant CaCYP51 substrate binding spectra.

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CYP51 reconstitution assays and IC50 determinations. [1]
The CYP51 enzyme reconstitution system previously described containing 2.5 μM CaCYP51 and 10 μM truncated yeast cytochrome P450 reductase was used. The reaction was terminated by the addition of 2 ml 15% (wt/vol) KOH in ethanol followed by incubation at 85°C for 90 min. IC50 determinations were performed by adding various concentrations of voriconazole, Prothioconazole, and prothioconazole-desthio in 5 μl of DMF prior to incubation at 37°C and the addition of β-NADPH-Na4. Sterol substrates and products were extracted and analyzed by GC/MS as described below.

Antifungal treatment of cells. [1]
C. albicans cells were grown overnight in RPMI 1640 l-glutamine. A starting concentration of 1 × 103 cells/ml was used to inoculate RPMI medium containing 4 μg · ml−1 Prothioconazole, 4 μg · ml−1 prothioconazole-desthio, or 1 μg · ml−1 voriconazole (all with a final concentration of 1% DMSO) and a control containing 1% DMSO (untreated). Cultures were incubated at 37°C and 200 rpm overnight and cells harvested and washed twice with deionized water prior to sterol extraction.

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Solid-phase extraction of antifungals from Candida albicans cells and growth media. [1]
A 100-μl aliquot of 1 × 107 cells/ml from overnight cultures of C. albicans (ATCC SC5314) was subcultured into 250 ml RPMI l-glutamine and yeast extract-peptone-dextrose (YPD) broth containing 8 μg · ml−1 Prothioconazole with a final concentration of 1% (vol/vol) DMSO (and controls without any prothioconazole) and incubated at 37°C and 200 rpm for 24 h. Cells were harvested and washed three times with deionized water before sonication (60-s bursts and 60-s rests for 10 min) in 99:1 methanol:acetic acid. Samples were then dried in a vacuum centrifuge and resuspended in 20% (vol/vol) methanol. Sep-Pak cartridges (Vac 3 cc, 200 mg) were prepared by washing with 6 ml of methanol and subsequently 6 ml of 20% (vol/vol) methanol. Extracts were each applied to a cartridge which was then washed with 4 sequential 1-ml volumes of 40%, 60%, 80%, and 100% (vol/vol) methanol. The eluent was collected for each wash.

Extracts from the media used for the growth of C. albicans with prothioconazole, control media containing Prothioconazole and no cells, and sterile deionized water with prothioconazole and no cells were prepared in a manner similar to that described for the cell extractions. Methanol was added to samples to reach a final concentration of 20% (vol/vol) (standards were diluted in 20% [vol/vol] methanol) prior to solid-phase extraction.

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Identification of Prothioconazole and prothioconazole-desthio. [1]
Samples were analyzed by ESI-MS and -MS/MS on a Q-Tof Ultima spectrometer in positive-ion mode. Samples were electrosprayed from nano-ESI metal-coated capillaries, and collision-induced dissociation was achieved with argon gas. The following instrument parameters were used: capillary voltage, 1.8 kV; collision voltage, 8 V for MS (25 V for MS/MS); m/z range, 50 to 1,000; scan time, 2.4 s/scan; MCP detector voltage, 2,100 V. The instrument was calibrated immediately prior to use with 1 pmol/μl Glu-1-Fibrinopeptide B. The data obtained were processed using the MassLynx V4.1 software package.

Prothioconazole and prothioconazole-desthio were identified by their measured mass and by their fragmentation patterns obtained through MS/MS. Both prothioconazole and prothioconazole-desthio standards were found to be most abundant in the 80% (vol/vol) methanol eluent; therefore, analysis of extractions was performed by comparing the 80% (vol/vol) methanol eluents of all samples.

Absorption, Distribution and Excretion
Following single oral low dose administration, the absorption of prothioconazole in male rats was approximately 94% for the triazole label. The absorption for the phenyl label was estimated to be approximately 90% at 48 hours based on extrapolation of the course of excretion for the triazole label at 48 hours. Plasma radioactivity time-course data showed that absorption following single oral low dose administration was rapid, with peak plasma concentrations occurring between 0.33 and 0.66 hours post administration in males and females. Peak plasma concentrations following single oral high dose administration occurred between 0.66 and 1.00 hours post administration in males and females. The absorption of the phenyl-labelled prothioconazole was slightly more rapid, with peak plasma concentrations occurring between 0.16 and 0.33 hours post administration of a single oral low dose in males, and at 0.16 hours post administration of a repeat oral low dose in males and females. Oscillations in the plasma time course were noted, indicating that the radioactivity was subjected to enterohepatic circulation. This effect was more prominent in the female rats. A slight delay in absorption compared to males was also noted in females.
Residual radioactivity in the rats 168 hours after a single oral low dose administration was low. For the triazole label, 1.5% of the administered dose was recovered in the tissues and carcass of males, and 0.4% was recovered in females. The highest tissue levels were found in liver, carcass and gastrointestinal tract. In all other tissues examined, residual radioactivity levels ranged from 0.0004-0.07%. For the phenyl label (administered to males only), 5.8% of the administered dose was recovered in the tissues and carcass. The highest tissue levels were found in the gastrointestinal tract, liver and carcass. In all other tissues examined, residual radioactivity levels ranged from 0.0001-0.05%. Residual radioactivity in the rats 48 hours after a repeat oral low dose administration was also low, with 3.8% of the administered dose recovered in the tissues and carcass of males, and 0.8% recovered in females. The highest tissue levels were found in liver, gastrointestinal tract and carcass. In all other tissues examined, residual radioactivity levels ranged from 0.0002-0.05%. Total body accumulation as well as liver accumulation was consistently higher in males. Residual radioactivity in the rats 168 hours after a single oral high dose administration was also low, with 0.11% of the administered dose recovered in the carcass and tissues of both males and females. Total body accumulation was approximately the same in males and females, with liver accumulation higher in the males.
The primary route of excretion for both labels and both sexes was via the feces. Following single oral low dose administration (triazole label), total recovery was approximately 94-95% of the administered dose for both sexes, with 10% (males) and 16% (females) of the administered dose eliminated in the urine, and 84% (males) and 78% (females) eliminated in the feces. In the phenyl-labelled group (males only), 5% of the administered dose was eliminated in the urine and 85% in the feces, for a total recovery of approximately 90%. In the bile duct-cannulated triazolelabel group (males only), approximately 90% of the administered dose was eliminated in the bile within 24-48 hours. In the phenyl-label group (males only), approximately 81% of the administered dose was eliminated in the bile after 24 hours and 93% after 48 hours.
In a whole body autoradiography distribution study, peak concentrations in males were noted 1 hour post-administration and continued to decline until sacrifice at 168 hours. In females, absorption was slightly delayed with peak concentrations in some tissues noted at 8 hours post-administration. The highest concentrations were noted in liver (up to 1.78 g/g in males and up to 0.97 ug/g in females), followed by kidney (renal medulla, up to 0.64 g/g), brown/perirenal fat (up to 0.36 ug/g), thyroid (up to 0.23 ug/g) and adrenal gland (up to 0.27 ug/g). All other tissues showed peak concentrations of <0.13 ug/g. Concentrations of radioactivity decreased rapidly from 24 to 168 hours post-administration, indicative of continued elimination from the tissues.

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Metabolism / Metabolites
Prothioconazole was extensively metabolized in the rat following oral administration. Eighteen metabolites and the parent compound were identified in urine, feces and bile. The biotransformation of prothioconazole consisted of three major reaction types including desulfuration, oxidative hydroxylation of the phenyl moiety and glucuronic acid conjugation. Identification of the metabolites ranged from 26-63% of the administered dose. A higher percentage of metabolite isolation and identification could not be achieved due to difficulties in fecal extraction where 67-79% of the administered dose remained in non-extractable residues in the solids.
The major route of excretion for prothioconazole was in the feces, representing 22-53% of the administered dose. The parent compound prothioconazole was the most abundant in the feces (1-22% of the administered dose), followed by the prothioconazole-desthio metabolite (3-16% of the administered dose). All other fecal metabolites represented less than 7% of the administered dose. The 1,2,4-triazole metabolite was not detected in the feces. The major urinary metabolite was prothioconazole-S- or O-glucuronide (0.1-8% of the administered dose) and was preferentially excreted in females. The 1,2,4-triazole metabolite represented 0.8-2.3% of the administered dose following administration of single oral low and/or high doses. The remaining urinary metabolites accounted for 0-1.4% of the administered dose. Prothioconazole- S- or O-glucuronide was the most abundant metabolite in the bile, representing approximately 46% of the administered dose. This metabolite was excreted in females only in the urine, however, it was noted in the bile in males. The glucuronic acid metabolites in the bile represented 8-10% of the administered dose. Parent compound represented 3-5% of the administered dose in the bile. The 1,2,4-triazole metabolite was not detected in the bile.
In a metabolism study, the absorption, distribution, metabolism and excretion of prothioconazole-desthio were investigated. Absorption of the radioactive test material from the gastro-intestinal tract (GIT) commenced as early as 4 minutes following dosing. A maximum concentration of 0.052 ug/g was observed at 1.5 hours. The largest amount of radioactivity was observed in the liver and the GIT, likely due to long-lasting enterohepatic circulation. The remaining tissues contained levels of radioactivity of less than 1%. Greater than 90% of the administered radioactive dose was excreted in the bile and urine. Very little of the administered radioactivity was recovered in the expired carbon dioxide. The majority of the radioactive administered dose was excreted in the feces, with a minor portion being excreted in the urine. The skin contained a minute amount of the recovered radioactivity, while the carcass and GIT contained up to 4 and 2.25%, respectively. Excretion was not likely complete at 48 hours, as the total body radioactive residue was 5 to 6% of the administered dose at that time point. The elimination half-life was found to be 44.3 hours, and the mean residence time was 48.2 hours. These observations indicate that the process of redistribution of the radioactivity into the plasma before elimination was slow, as supported by the total clearance of 10.9 mL/min kg bw and the renal clearance of 1.4 mL/min kg bw. Following the intraduodenal administration of the radioactive dose in bile-cannulated animals, 84 to 85% of the administered dose was found in the bile after 24 and 48 hours, respectively. Excretion in urine in these animals accounted for almost 6% of the administered dose after 48 hours, while excretion in faeces accounted for 2% of the administered dose for the same time period. In the pooled bile sample, 18 radioactive HPLC peaks were observed accounting for 84.3% of the administered dose. Five compounds were isolated and identified, while the remaining 13 metabolites were not identified, accounting for 44.7% of the administered dose. /Prothioconazole-desthio/
In whole-body autoradiography experiments, the quick onset of absorption of the test material was demonstrated. Absorption was not complete after one hour. The autoradiograms also showed that the blood concentration was less than the concentration present in the fatty tissues, demonstrating the lipophilic nature of prothioconazole-desthio, and perhaps of its metabolites. The mucous membrane of the stomach walls were observed with radioactivity throughout the various observation periods, which was considered an indication of extrabiliary secretion of the absorbed radioactivity back into the stomach lumen. The muscle, heart, lung, brain, thyroid, and mineral portion of the bones showed minor concentrations of radioactivity. The testes had a radioactivity distribution pattern indicative of the blood circulation in the organ. Medium amounts of radioactivity were observed in some glandular organs, including the preputial gland and the adrenals. The gums were also observed with increased radioactivity, with unknown physiological significance. The distributions noted at one hour were fairly consistent for 48 hours, though declining due to excretion. The renal cortex contained radioactivity to a much greater extent than did the renal pelvis, indicating that the radioactivity was reabsorbed in the duodenum. As well, the radioactivity that is absorbed is likely not transformed into metabolites that are adequately polar to be eliminated by the kidney. This results in increased passage through the liver by the radioactive test material. /Prothioconazole-desthio/
For more Metabolism/Metabolites (Complete) data for Prothioconazole (6 total), please visit the HSDB record page.

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Biological Half-Life
The elimination half-life was found to be 44.3 hours. ... /Prothioconazole-desthio/

Non-Human Toxicity Values
LD50 Rat (male, female) oral >/= 6200 mg/kg (Prothioconazole technical)
LC50 Rainbow trout 1.83 mg/L/96 hr
LC50 Rat inhalation > 4990 mg/cu m /Duration not specified/
LD50 Rat (male, female) dermal >/= 2000 mg/kg

[1]. Prothioconazole and prothioconazole-desthio activities against Candida albicans sterol 14-α-demethylase. Appl Environ Microbiol. 2013 Mar;79(5):1639-45.

2-[2-(1-chlorocyclopropyl)-3-(2-chlorophenyl)-2-hydroxypropyl]-1,2-dihydro-1,2,4-triazole-3-thione is a member of the class of triazoles that is 1,2,4-triazole-3-thione substituted at position 2 by a 2-(1-chlorocyclopropyl)-3-(2-chlorophenyl)-2-hydroxypropyl group. It is a member of monochlorobenzenes, a member of triazoles, a tertiary alcohol, a member of cyclopropanes and a thiocarbonyl compound.

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Mechanism of Action
Prothioconazole is a systemic demethylation inhibitor fungicide which belongs to the triazolinthione class of fungicides. ... It acts against susceptible fungi through the inhibition of demethylation at position 14 of lanosterol or 24-methylene dihydroano-sterol, both of which are precursors of sterols in fungi; i.e., it works through disruption of ergosterol biosynthesis (Ergosterol, a precursor to Vitamin D2, is an important component of fungal cell walls).

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The poor in vitro CaCYP51 inhibition properties of prothioconazole relative to prothioconazole-desthio stimulated further investigation of the mode of action. Prothioconazole-desthio was found to be present in both the cells and the media of C. albicans cultures treated with prothioconazole. Therefore, the antifungal effect seen during treatment with prothioconazole is due to the presence of the desthio product. Our results also showed that the desthio analog was present in both YPD media containing prothioconazole and RPMI media containing prothioconazole, but not in sterile deionized water containing prothioconazole after incubation at 37°C for 24 h. Therefore, prothioconazole is readily converted to the desthio form and accounts for the antifungal effect. It is conceivable that the high potency of prothioconazole as an agricultural fungicide is also enhanced due to intracellular metabolism of the relatively inactive prothioconazole in the pathogenic fungi to the highly active desthio form and in the host. Additional work on triazolinthiones may further the development of new antifungal compounds of this type for therapeutic use in the clinic, or as more effective compounds in crop protection, and stimulate consideration of the value of a profungicide or prodrug approach. [1]

C14H15CL2N3OS

344.25

343.031

C, 48.85; H, 4.39; Cl, 20.60; N, 12.21; O, 4.65; S, 9.31

178928-70-6

6451142

White to light yellow solid powder

1.5±0.1 g/cm3

486.7±55.0 °C at 760 mmHg

139.1-144.5°

248.2±31.5 °C

0.0±1.3 mmHg at 25°C

1.698

1.77

2

2

5

21

458

0

MNHVNIJQQRJYDH-UHFFFAOYSA-N

InChI=1S/C14H15Cl2N3OS/c15-11-4-2-1-3-10(11)7-14(20,13(16)5-6-13)8-19-12(21)17-9-18-19/h1-4,9,20H,5-8H2,(H,17,18,21)

2-[2-(1-chlorocyclopropyl)-3-(2-chlorophenyl)-2-hydroxypropyl]-1H-1,2,4-triazole-3-thione

Proline 480 SC Fungicide; JAU 6476; Prothioconazole; 178928-70-6; 3H-1,2,4-Triazole-3-thione, 2-[2-(1-chlorocyclopropyl)-3-(2-chlorophenyl)-2-hydroxypropyl]-1,2-dihydro-; JAU 6476; Prothioconazole [ISO:BSI]; 2-[2-(1-Chlorocyclopropyl)-3-(2-chlorophenyl)-2-hydroxypropyl]-1,2-dihydro-3H-1,2,4-triazole-3-thione; UNII-27B9FV58IY; PROLINE 480 SC Fungicide; Prothioconazole

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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