CYP3A4; CYP24A1; ergosterol synthesis; active metabolite of Ketoconazole
Cytochrome P450 enzymes, particularly CYP3A4, and fungal lanosterol 14α-demethylase (CYP51). Deacylketoconazole, like ketoconazole, inhibits fungal lanosterol 14α-demethylase, an enzyme essential for ergosterol biosynthesis in fungi. However, deacylketoconazole has reduced potency compared to ketoconazole. The compound also inhibits human cytochrome P450 enzymes, contributing to drug-drug interaction potential.

Deacylated ketoconazole (R-39519) also affect the growth of Staphylococcus aureus, miconazole being 12.5 and 14 times, respectively, more active than R-39519 and ketoconazole.[2]
Deacylketoconazole has reduced antifungal activity compared to ketoconazole. In vitro susceptibility testing against various fungal strains shows that deacylketoconazole is less potent than the parent compound. The compound also inhibits human CYP3A4 and other cytochrome P450 enzymes, though with reduced potency compared to ketoconazole. Its inhibitory effects on human enzymes contribute to the pharmacokinetic profile of ketoconazole.

Deacylketoconazole was 15- to 50-fold more active against Plasmodium falciparum than was ketoconazole, based on [(3)H]hypoxanthine uptake and quantitative parasite counts. In contrast, there were no significant differences between these drugs in their activity against clinical isolates of Candida spp.[1]
Deacylketoconazole is a major metabolite of ketoconazole found in plasma after oral administration. Its in vivo activity is limited compared to ketoconazole. The compound contributes to the overall pharmacokinetic profile of ketoconazole and may contribute to its drug-drug interaction potential through inhibition of CYP3A4. However, deacylketoconazole is not administered as a therapeutic agent.

Ketoconazole (KC), an antifungal agent, rarely causes severe liver injury when orally administered. It has been reported that KC is mainly hydrolyzed to N-deacetyl ketoconazole (DAK), followed by the N-hydroxylation of DAK by flavin-containing monooxygenase (FMO). Although the metabolism of KC has been considered to be associated with hepatotoxicity, the responsible enzyme(s) remain unknown. The purpose of this study was to identify the responsible enzyme(s) for KC hydrolysis in humans and to clarify their relevance to KC-induced toxicity. Kinetic analysis and inhibition studies using human liver microsomes (HLM) and recombinant enzymes revealed that human arylacetamide deacetylase (AADAC) is responsible for KC hydrolysis to form DAK, and confirmed that FMO3 is the enzyme responsible for DAK N-hydroxylation. In HLM, the clearance of KC hydrolysis occurred to the same extent as DAK N-hydroxylation, which indicates that both processes are not rate-limiting pathways. Cytotoxicity of KC and DAK was evaluated using HepaRG cells and human primary hepatocytes. Treatment of HepaRG cells with DAK for 24h showed cytotoxicity in a dose-dependent manner, whereas treatment with KC did not show due to the low expression of AADAC. Overexpression of AADAC in HepaRG cells with an adenovirus expression system elicited the cytotoxicity of KC. Cytotoxicity of KC in human primary hepatocytes was attenuated by diisopropylfluorophosphate, an AADAC inhibitor. In conclusion, the present study demonstrated that human AADAC hydrolyzes KC to trigger hepatocellular toxicity.[3]
In vitro enzyme inhibition assays for Deacylketoconazole typically involve measuring inhibition of fungal CYP51 or human CYP3A4. The assay uses recombinant enzymes or microsomal preparations and appropriate substrates (e.g., testosterone for CYP3A4). The formation of metabolites is measured by HPLC or LC-MS/MS. IC50 values are calculated from dose-response curves. Antifungal susceptibility testing is performed using broth microdilution methods according to CLSI guidelines.

Cellular assays for Deacylketoconazole are performed using fungal cultures to assess antifungal activity. Minimum inhibitory concentrations (MICs) are determined using broth microdilution or agar dilution methods. Human cell lines (e.g., HepG2) may be used to assess cytotoxicity and CYP3A4 inhibition. The compound's effects on cellular sterol biosynthesis can be assessed by analyzing sterol profiles.

Deacylketoconazole is not administered to animals as a therapeutic agent. The compound is studied in the context of ketoconazole pharmacokinetics. In animal studies, ketoconazole is administered and plasma levels of deacylketoconazole are measured to assess metabolism. The metabolite's contribution to the overall pharmacological and toxicological profile of ketoconazole is evaluated in these studies.

Deacylketoconazole has a molecular weight of approximately 489.39 g/mol and molecular formula C24H26Cl2N4O3. The compound is soluble in organic solvents such as DMSO and should be stored under standard laboratory conditions. It is typically analyzed by HPLC or LC-MS/MS in pharmacokinetic studies. The compound is stable under appropriate storage conditions.

Deacylketoconazole, as a metabolite of ketoconazole, shares some of the toxicological properties of the parent compound. Ketoconazole is known to have hepatotoxicity and drug-drug interaction potential. Deacylketoconazole may contribute to these effects through CYP inhibition. Standard laboratory safety precautions should be followed when handling the compound.

[1]. Pfaller MA, et al., Activity of ketoconazole and its deacyl derivative against Plasmodium falciparum and Candida isolates. Antimicrob Agents Chemother. 1982 Nov;22(5):917-9.
[2]. Bossche H V, et al., Molecular basis for the antimycotic and antibacterial activity of N‐substituted imidazoles and triazoles: The inhibition of isoprenoid biosynthesis. Pesticide science, 1984, 15(2): 188-198.
[3]. Fukami T, et al., Human arylacetamide deacetylase hydrolyzes ketoconazole to trigger hepatocellular toxicity. Biochem Pharmacol. 2016 Sep 15;116:153-61.

Antifungal N-substituted imidazoles and triazoles, such as imidazolid, ketoconazole, and itraconazole, selectively interfere with the 14α-demethylase system (which depends on cytochrome P-450) in fungal cells (e.g., Candida albicans and Penicillium italicum) at low concentrations (≥0.01 nM). This leads to reduced ergosterol availability and promotes the accumulation of 14α-methylsterols (e.g., lanosterol). For example, cholesterol synthesis is much less sensitive to ketoconazole in rat subcellular fractions of the liver, intact fibroblasts, and in vivo in rat liver. Imidazole derivatives imidazolid, miconazole, ketoconazole, and perconazole, and triazole derivatives propiconazole, teconazole, and itraconazole all affect cytochrome P-450 in Saccharomyces cerevisiae and rat liver microsomal fractions. The lower sensitivity of rat liver microsomes to cytochrome P-450 is consistent with the lower sensitivity to cholesterol synthesis. Using monolayered vesicles composed of phosphatidylcholine, phosphatidylethanolamine, and diphosphatidylcholine, multilayered vesicles composed of dipalmitoylphosphatidylcholine, and intact Saccharomyces cerevisiae, studies have shown that replacing ergosterol with lanosterol leads to altered membrane function. It is speculated that the selective interaction of azole derivatives with yeast microsomal cytochrome P-450 results in the accumulation of 14α-methylsterol, subsequently causing altered cell membrane permeability and leakage. The observed growth inhibition may stem from these alterations. Miconazole, ketoconazole, and deacylated ketoconazole (R-39519) also affected the growth of Staphylococcus aureus, with miconazole exhibiting 12.5-fold and 14-fold greater activity than R-39519 and ketoconazole, respectively. The stronger antibacterial activity of miconazole is consistent with its stronger inhibitory effect on the biosynthesis of C-55 isopreneol and vitamin K. Phosphorylated derivatives of C-55 isoprene alcohol play important functional roles in the biosynthesis of bacterial cell walls and membrane polymers, while menadione-type vitamin K plays a role in electron transport in Gram-positive bacteria. Reduced synthesis of these important compounds may contribute to the antibacterial activity of miconazole. [2]
Deacylketoconazole is a major metabolite of the antifungal drug ketoconazole, formed by deacetylation. It retains some of the pharmacological activity of ketoconazole but is generally less potent as an antifungal agent. Deacylketoconazole inhibits fungal lanosterol 14α-demethylase and human cytochrome P450 enzymes, particularly CYP3A4, contributing to the drug-drug interaction potential of ketoconazole. The compound is primarily of interest in pharmacokinetic and metabolism studies of ketoconazole. Deacylketoconazole is for research use only and is not used as a therapeutic agent.

C24H26CL2N4O3

489.397

488.138

C, 58.90; H, 5.36; Cl, 14.49; N, 11.45; O, 9.81

67914-61-8

12854716

White to off-white solid powder

4.34

1

6

7

33

621

2

c1cc(ccc1N2CCNCC2)OC[C@H]3CO[C@](O3)(Cn4ccnc4)c5ccc(cc5Cl)Cl

LOUXSEJZCPKWAX-URXFXBBRSA-N

InChI=1S/C24H26Cl2N4O3/c25-18-1-6-22(23(26)13-18)24(16-29-10-7-28-17-29)32-15-21(33-24)14-31-20-4-2-19(3-5-20)30-11-8-27-9-12-30/h1-7,10,13,17,21,27H,8-9,11-12,14-16H2/t21-,24-/m0/s1

1-[4-[[(2R,4S)-2-(2,4-dichlorophenyl)-2-(imidazol-1-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]piperazine

Deacyl ketoconazole; 67914-61-8; Deacetyl Ketoconazole; Deacylketoconazole; N-Deacetylketoconazole; P7P4A1FD7Z; CIS-1-[4-[[2-(2,4-DICHLOROPHENYL)-2-(1H-IMIDAZOL-1-YLMETHYL)-1,3-DIOXOLAN-4-YL]METHOXY]PHENYL]PIPERAZINE; rel-1-(4-(((2R,4S)-2-((1H-Imidazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl)methoxy)phenyl)piperazine; Piperazine, 1-(4-((2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-dioxolan-4-yl)methoxy)phenyl)-, cis-; Deacylketoconazole

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.)