Species differences in metabolism of EPZ015666, an oxetane-containing protein arginine methyltransferase-5 (PRMT5) inhibitor

Abstract

1.?Metabolite profiling and identification studies were conducted to understand the cross-species differences in the metabolic clearance of EPZ015666, a first-in-class protein arginine methyltransferase-5 (PRMT5) inhibitor, with anti-proliferative effects in preclinical models of Mantle Cell Lymphoma. EPZ015666 exhibited low clearance in human, mouse and rat liver microsomes, in part by introduction of a 3-substituted oxetane ring on the molecule. In contrast, a higher clearance was observed in dog liver microsomes (DLM) that translated to a higher in vivo clearance in dog compared with rodent.

2.?Structure elucidation via high resolution, accurate mass LC-MSⁿ revealed that the prominent metabolites of EPZ015666 were present in hepatocytes from all species, with the highest turnover rate in dogs. M1 and M2 resulted from oxidative oxetane ring scission, whereas M3 resulted from loss of the oxetane ring via an N-dealkylation reaction.

3.?The formation of M1 and M2 in DLM was significantly abrogated in the presence of the specific CYP2D inhibitor, quinidine, and to a lesser extent by the CYP3A inhibitor, ketoconazole, corroborating data from human recombinant isozymes.

4.?Our data indicate a marked species difference in the metabolism of the PRMT5 inhibitor EPZ015666, with oxetane ring scission the predominant metabolic pathway in dog mediated largely by CYP2D.     

Mechanistic investigations into the species differences in pinometostat clearance: impact of binding to alpha-1-acid glycoprotein and permeability-limited hepatic uptake

1.?The plasma clearance of the first-in-class DOT1L inhibitor, EPZ-5676 (pinometostat), was shown to be markedly lower in human compared to the preclinical species, mouse, rat and dog.

2.?This led to vertical allometry where various interspecies scaling methods were applied to the data, with fold-errors between 4 and 13. We had previously reported the elimination and metabolic pathways of EPZ-5676 were similar across species. Therefore, the aim of this work was to explore the mechanistic basis for the species difference in clearance for EPZ-5676, focusing on other aspects of disposition.

3.?The protein binding of EPZ-5676 in human plasma demonstrated a non-linear relationship suggesting saturable binding at physiologically relevant concentrations. Saturation of protein binding was not observed in plasma from preclinical species. Kinetic determinations using purified serum albumin and alpha-1-acid glycoprotein (AAG) confirmed that EPZ-5676 is a high affinity ligand for AAG with a dissociation constant (K_d) of 0.24?μM.

4.?Permeability limited uptake was also considered since hepatocyte CL_int was much lower in human relative to preclinical species. Passive unbound CL_int for EPZ-5676 was estimated using a correlation analysis of logD and data previously reported on seven drugs in sandwich cultured human hepatocytes.

5.?Incorporation of AAG binding and permeability limited hepatic uptake into the well-stirred liver model gave rise to a predicted clearance for EPZ-5676 within 2-fold of the observed value of 1.4?mL min^?1?kg^?1. This analysis suggests that the marked species difference in EPZ-5676 clearance is driven by high affinity binding to human AAG as well as species-specific hepatic uptake invoking the role of transporters.     

Synthesis,anticancer activity,and molecular modeling of etodolac‐thioether derivatives as potent methionine aminopeptidase (type II) inhibitors

A series of (R,S)‐1‐{[5‐(substituted)sulfanyl‐4‐substituted‐4H‐1,2,4‐triazole‐3‐yl]methyl}‐1,8‐diethyl‐1,3,4,9‐tetrahydropyrano[3,4‐b]indoles ( 5a–v ) were designed and synthesized using a five‐step synthetic protocol that involves substituted benzyl chlorides and (R,S)‐5‐[(1,8‐diethyl‐1,3,4,9‐tetrahydropyrano[3,4‐b]indole‐1‐yl)methyl]‐4‐substituted‐2,4‐dihydro‐3H‐1,2,4‐triazole‐3‐thiones in the final step. The synthesized derivatives were evaluated for cytotoxicity and anticancer activity in vitro using the MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) colorimetric method against VERO, HEPG2 (human hepatocellular liver carcinoma), SKOV3 (ovarian carcinoma), MCF7 (human breast adenocarcinoma), PC3 and DU145 (prostate carcinoma) cells at 10^?5 M (10 μM) for 24 h. Compounds 5d and 5h showed the best biological potency against the SKOV3 cancer cell line (IC₅₀ = 7.22 and 5.10 μM, respectively) and did not display cytotoxicity toward VERO cells compared to etodolac. Compounds 5k , 5s , and 5v showed the most potent biological activity against the PC3 cancer cell line (IC₅₀ = 8.18, 3.10, and 4.00 μM, respectively) and did not display cytotoxicity. Moreover, these compounds were evaluated for caspase‐3, ‐9, and ‐8 protein expression and activation in the apoptosis pathway for 6, 12, and 24 h, which play a key role in the treatment of cancer. In this study, we also investigated the apoptotic mechanism and molecular modeling of compounds 5k and 5v on the methionine aminopeptidase (type II) enzyme active site in order to get insights into the binding mode and energy.

     

A novel five‐lipoxygenase activity protein inhibitor labeled with carbon‐14 and deuterium

2‐[4‐(3‐{(1R)‐1‐[4‐(2‐Aminopyrimidin‐5‐yl)phenyl]‐1‐cyclopropylethyl}‐1,2,4‐oxadiazol‐5‐yl)‐1H‐pyrazol‐1‐yl]‐N,N‐dimethylacetamide (1), is a novel and selective five‐lipoxygenase activity protein (FLAP) inhibitor with excellent pharmacokinetics properties. The availability of a key chiral intermediate allowed the synthesis of [¹⁴C]‐(1) in six radiochemical steps and in 47% overall radiochemical yield with a specific activity of 51 mCi/mmol using carbon‐14 zinc cyanide. 2‐Chloro‐N,N‐dimethyl‐²H₆‐acetamide was prepared and condensed with a penultimate intermediate to give [²H₆]‐(1) in very high yield and in more than 99% isotopic enrichment.     

The in vivo and in vitro phase I metabolism of FYL‐67, a novel oxazolidinone antibacterial drug,studied by LC‐MS/MS

In our previous study, FYL‐67, a novel linezolid analogue with the morpholinyl ring replaced by a 4‐(pyridin‐2‐yl)‐1H‐pyrazol‐1‐yl group, was demonstrated to own an excellent activity against Gram‐positive organisms,such as methicillin‐resistant Staphylococcus aureus (MRSA). However, metabolic biotransformation was not investigated. This study was performed to identify the phase I metabolites of FYL‐67 using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). The chemical structures were confirmed by comparison with corresponding chemical standards obtained internal. Primary elucidation of the metabolic pathway of FYL‐67 in vitro was performed using liver preparations (microsomes and hepatocytes) from rats and humans, and SD (Sprague Dawley, rat, rattus norvegicus) rats were used for the study of in vivo approach. To the end, two metabolites (M₁ and M₂) were detected after in vitro as well as in vivo experiments. Based on LC‐MS/MS analyses, the metabolites were demonstrated to be 5‐(aminomethyl)‐3‐(3‐fluoro‐4‐(4‐(pyridin‐2‐yl)‐1H‐pyrazol‐1‐yl)phenyl)oxazolidin‐2‐one (M₁) and 3‐(3‐fluoro‐4‐(4‐(pyridin‐2‐yl)‐1H‐pyrazol‐1‐yl)phenyl)‐5‐(hydroxymethyl)oxazolidin‐2‐one (M₂). Amide hydrolysis at acetyl group of FYL‐67 leading to the formation of M₁ was observed and suggested to play a major role in both in vivo and in vitro phase I metabolism of FYL‐67. M₁ was demonstrated to undergo a further oxidation to form M₂. In addition, the results indicated no species difference existing between rats and humans. The outcomes of our research can be utilized for the development and validation of the analytical method for the quantification of FYL‐67 as well as its metabolites in biological samples. Furthermore, it is helpful to conduct studies of pharmacodynamics and toxicodynamics. Copyright © 2015 John Wiley & Sons, Ltd.     

Synthesis of the tritiated isotopomers of enzastaurin and its N‐des‐pyridylmethyl metabolite for use in ADME studies

Enzastaurin (3‐(1‐methyl‐1H‐indol‐3‐yl)‐4‐[1‐[1‐(2‐pyridinylmethyl)‐4‐piperidinyl]‐1H‐indol‐3‐yl]‐1H‐pyrrole‐2,5‐dione, 1), an agent with potential utility in the treatment of solid tumors, is currently in phase II clinical trials. Enzastaurin undergoes metabolism in vitro and in vivo to several products of oxidative metabolism, the major one of which is 3‐(1‐methyl‐1H‐indol‐3‐yl)‐4‐(1‐piperidin‐4‐yl‐1H‐indol‐3‐yl)‐1H‐pyrrole‐2,5‐dione (2). In a model study, the attempted synthesis 1‐[²H] by reaction of 1 with deuterium gas in the presence of Ir[(COD)(Cy₃P)pyr]PF₆ (Crabtree's catalyst) was unsuccessful. Alternatively, it was decided to prepare tritiated 2 as both a final product and the starting material for the tritiation of 1. We have reported herein a route that was developed for use in the preparation of tritium‐labeled 2‐[³H] and its successful conversion to 1‐[³H]. Copyright © 2008 John Wiley & Sons, Ltd.     

Synthesis,molecular docking,and pharmacological evaluation of N‐(2‐(3,5‐dimethoxyphenyl)benzoxazole‐5‐yl)benzamide derivatives as selective COX‐2 inhibitors and anti‐inflammatory agents

A series of N‐(2‐(3,5‐dimethoxyphenyl)benzoxazole‐5‐yl)benzamide derivatives ( 3am ) was synthesized and evaluated for their in vitro inhibitory activity against COX‐1 and COX‐2. The compounds with considerable in vitro activity (IC₅₀ < 1 μM) were evaluated in vivo for their anti‐inflammatory potential by the carrageenan‐induced rat paw edema method. Out of 13 newly synthesized compounds, 3a , 3b , 3d , 3g , 3j , and 3k were found to be the most potent COX‐2 inhibitors in the in vitro enzymatic assay, with IC₅₀ values in the range of 0.06–0.71 μM. The in vivo anti‐inflammatory activity of these six compounds ( 3a , 3b , 3d , 3g , 3j , and 3k ) was assessed by the carrageenan‐induced rat paw edema method. Compounds 3d (84.09%), 3g (79.54%), and 3a (70.45%) demonstrated significant anti‐inflammatory activity compared to the standard drug ibuprofen (65.90%) and were also found to be safer than ibuprofen, by ulcerogenic studies. A docking study was done using the crystal structure of human COX‐2, to understand the binding mechanism of these inhibitors to the active site of COX‐2.

     

Syntheses,calcium channel modulation effects,and nitric oxide release studies of O2‐alkyl‐1‐(pyrrolidin‐1‐yl) diazen‐1‐ium‐1,2‐diolate 4‐aryl(heteroaryl)‐1,4‐dihydro‐2,6‐dimethyl‐3‐nitropyridine‐5‐carboxylates

A group of racemic 4‐aryl(heteroary)‐1,4‐dihydro‐2,6‐dimethyl‐3‐nitropyridine‐5‐carboxy‐lates possessing a potential nitric oxide donor C‐5 O²‐alkyl‐1‐(pyrrolidin‐1‐yl)diazen‐1‐ium‐1,2‐diolate ester [alkyl=(CH₂)_n, n=1–4] substituent were synthesized using a modified Hantzsch reaction. Compounds having a C‐4 2‐trifluoromethylphenyl ( 16 ), 2‐pyridyl ( 17 ), or benzofurazan‐4‐yl ( 20 ) substituent generally exhibited more potent smooth‐muscle calcium channel antagonist activity (IC₅₀ values in the 0.55 to 38.6 μM range) than related analogs having a C‐4 3‐pyridyl ( 18 ), or 4‐pyridyl ( 19 ) substituent with IC₅₀ values > 29.91 μM, relative to the reference drug nifedipine (IC₅₀=0.0143 μM). The point of attachment of C‐4 isomeric pyridyl substituents was a determinant of antagonist activity where the relative potency profile was 2‐pyridyl > 3‐pyridyl and 4‐pyridyl. Subgroups of compounds 16a–d , 17a–d , and 20a–d having alkyl spacer groups of variable chain length [–CO₂(CH₂)_nO–, n=1–4] exhibited small differences in calcium channel antagonist potency. Replacement of the ester “methyl” moiety of Bay K 8644 by an O²‐alkyl‐1‐(pyrrolidin‐1‐yl)diazen‐1‐ium‐1,2‐diolate group provided the Bay K 8644 group of analogs 16a‐d that retained the desired cardiac positive inotropic effect. The most potent compound in this group, O²‐ethyl‐1‐(pyrrolidin‐1‐yl)diazen‐1‐ium‐1,2‐diolate 1,4‐dihydro‐2,6‐dimethyl‐3‐nitro‐4‐(2‐trifluoromethylphenyl)pyridine‐5‐carboxylate ( 16b , EC₅₀=0.096 μM) is about eightfold more potent positive inotrope (cardiac calcium channel agonist) than the reference compound Bay K 8644 (EC₅₀=0.77 μM). A similar replacement of the ester “isopropyl” group in the C‐4 benzofurazan‐4‐yl group of compounds by an O²‐alkyl‐1‐(pyrrolidin‐1‐yl)diazen‐1‐ium‐1,2‐diolate ester substituent provided compounds 20 (n=1 and 4) that were approximately equipotent cardiac positive inotropes with the parent reference compound PN 202‐791 ( 3 , EC₅₀=9.40 μM). The O²‐alkyl‐1‐(pyrrolidin‐1‐yl)diazen‐1‐ium‐1,2‐diolate ester moiety present in 1,4‐dihydropyridine calcium channel modulating compounds 16–20 is not a suitable ?NO donor moiety because the percent nitric oxide released upon in vitro incubation with either l ‐cysteine, rat serum, or pig liver esterase was less than 1%. Drug Dev. Res. 60:204–216, 2003. © 2003 Wiley‐Liss, Inc.     

Characterization of cytochrome P450 enzymes and P‐glycoprotein involved in the metabolism and transport of a new anti‐angiogenic agent KR‐31831

The in vitro metabolism and the transport of a novel anti‐angiogenic agent KR‐31831, (2R,3R,4S)‐6‐amino‐4‐[N‐(4‐chlorophenyl)‐N‐(1H‐imidazol‐2‐ylmethyl)amino]‐3‐hydroxy‐2‐dimethyoxymethyl‐3,4‐dihydro‐2‐methyl‐2H‐1‐benzopyran were investigated. Liquid chromatography‐mass spectrometry and tandem mass spectrometry were used for qualitative and quantitative analysis. The bidirectional transport studies of KR‐31831 using Caco‐2 cell monolayers showed the efflux to be significantly higher than influx (29.1 × 10^?6 compared to 11.5 × 10^?6 cm/s). P‐glycoprotein inhibitors significantly increased the influx of KR‐31831 and decreased the efflux of KR‐31831. These data indicate that KR‐313831 is a substrate for an efflux pump, P‐glycoprotein. The incubations of KR‐31831 with human liver microsomes produced three metabolites, M1, M2, and M3. M1 and M2 were identified as N‐(4‐chlorophenyl)‐N‐(1H‐imidazol‐2‐ylmethyl)amine and (2R,3R,4S)‐6‐amino‐4‐[N‐(4‐chlorophenyl)‐N‐(1H‐imidazol‐2‐ylmethyl)amino]‐3‐hydroxy‐2‐hydroxymethyl‐3,4‐dihydro‐2‐methyl‐2H‐1‐benzopyran by comparison with the authentic standards. M3 was tentatively characterized as hydroxy‐KR‐31831. CYP3A4 was identified as the major enzyme responsible for KR‐31831 metabolism to a major metabolite M1 using the combination of correlation analysis, immuno‐inhibition, chemical inhibition in human liver microsomes, and metabolism by cDNA expressed CYP enzymes. There is the possibility of drug–drug interactions when prescribing KR‐31831 concomitantly with known inhibitors or inducers of CYP3A4 and P‐glycoprotein. KR‐31831 was found to inhibit potently the metabolism of CYP2D6 substrate, suggesting that coadministration of KR‐31831 with CYP2D6 substrates may have significant effects on the pharmacokinetics of CYP2D6 substrates. Drug Dev. Res. 66:40–49. 2006. © 2006 Wiley‐Liss, Inc.     

Synthesis and 11C‐labelling of (E,E)‐1‐(3′,4′‐dihydroxystyryl)‐4‐(3′‐methoxy‐4′‐hydroxystyryl) benzene for PET imaging of amyloid deposits†

Carboxylic acid derivatives of the amyloid‐binding dye Congo red do not enter the brain well and are thus unable to serve as in vivo amyloid‐imaging agents. A neutral amyloid probe, (E,E)‐1‐(3′,4′‐dihydroxystyryl)‐4‐(3′‐methoxy‐4′‐hydroxystyryl)benzene ( 3 ), devoid of any carboxylate groups has been designed and synthesized via a 12‐step reaction sequence with a total yield of 30%. The unsymmetric compound 3 has also been labelled with C‐11 via [¹¹C]methyl iodide ([¹¹C]CH₃I) methylation of a symmetric 4,4′‐dimesyl protected precursor followed by deprotection. Preliminary evaluation indicated that compound 3 selectively stained plaques and neurofibrillary tangles in post‐mortem AD brain, and exhibited good binding affinity (K_i=38±8 nM) for Aβ(1–40) fibrils in vitro. In vivo pharmacokinetic studies indicated that [¹¹C] 3 exhibited higher brain uptake than its carboxylic acid analogs and good clearance from normal control mouse brain. [¹¹C] 3 also exhibited specific in vivo binding to pancreatic amyloid deposits in the NOR‐beta transgenic mouse model. These results justify further investigation of 3 and similar derivatives as surrogate markers for in vivo quantitation of amyloid deposits. Copyright © 2002 John Wiley & Sons, Ltd.     

Synthesis and Structure‐Activity Relationship Studies of Pyrazole‐based Heterocycles as Antitumor Agents

Several 4‐cyano‐1,5‐diphenylpyrazoles attached to different heterocyclic ring systems at position 3 were synthesized starting from ethyl 4‐cyano‐1,5‐diphenyl‐1H‐pyrazole‐3‐carboxylate 1 . The newly synthesized compounds were tested in vivo for their anti‐estrogenic effects and evaluated in vitro for their cytotoxic properties against estrogen‐dependent tumors. 3‐(5‐Mercapto‐1,3,4‐oxadiazole‐2‐yl)‐1,5‐diphenyl‐1H‐pyrazole‐4‐carbonitrile 13 revealed the highest cytotoxic activity with a GI₅₀ value equal to 40 nM against the IGROVI ovarian tumor cell line. It also showed an anti‐estrogen activity 1.6 more effective than the reference drug, in addition to a high tolerable dose. 3‐(5‐(Methylthio)‐4‐phenyl‐4H‐1,2,4‐triazol‐3‐yl)‐1,5‐diphenyl‐1H‐pyrazole‐4‐carbonitrile 7 was found to have the highest anti‐estrogenic activity, while 1,5‐diphenyl‐3‐[5‐(phenylamino)‐1,3,4‐thiadiazol‐2‐yl]‐1H‐pyrazole‐4‐carbonitrile 11 showed the lowest activity. The oral LD₅₀ values revealed that most of the tested compounds are relatively nontoxic.     

Physiologically based pharmacokinetic modeling to predict complex drug–drug interactions: a case study of AZD2327 and its metabolite,competitive and time‐dependent CYP3A inhibitors

4‐{(R)‐(3‐Aminophenyl)[4‐(4‐fluorobenzyl)‐piperazin‐1‐yl]methyl}‐N,N‐diethylbenzamide (AZD2327) is a highly potent and selective agonist of the δ ‐opioid receptor. AZD2327 and N‐deethylated AZD2327 (M1) are substrates of cytochrome P450 3A (CYP3A4) and comprise a complex multiple inhibitory system that causes competitive and time‐dependent inhibition of CYP3A4. The aim of the current work was to develop a physiologically based pharmacokinetic (PBPK) model to predict quantitatively the magnitude of CYP3A4 mediated drug–drug interaction with midazolam as the substrate. Integrating in silico, in vitro and in vivo PK data, a PBPK model was successfully developed to simulate the clinical accumulation of AZD2327 and its primary metabolite. The inhibition of CYP3A4 by AZD2327, using midazolam as a probe drug, was reasonably predicted. The predicted maximum concentration (C_max) and area under the concentration–time curve (AUC) for midazolam were increased by 1.75 and 2.45‐fold, respectively, after multiple dosing of AZD2327, indicating no or low risk for clinically relevant drug–drug interactions (DDI). These results are in agreement with those obtained in a clinical trial with a 1.4 and 1.5‐fold increase in C_max and AUC of midazolam, respectively. In conclusion, this model simulated DDI with less than a two‐fold error, indicating that complex clinical DDI associated with multiple mechanisms, pathways and inhibitors (parent and metabolite) can be predicted using a well‐developed PBPK model. Copyright © 2015 John Wiley & Sons, Ltd.     

A pyrazolylamine‐phosphonate monoester chelator for the fac‐[M(CO)3]+ core (M = Re, 99mTc): synthesis,coordination properties and biological assessment

Aiming to develop new strategies for the labeling of hydroxyl‐containing biomolecules with the organometallic core fac‐[^99mTc(CO)₃]⁺, we have prepared a new model bifunctional chelator, L4 (ethyl hydrogen (2‐{[2‐(3,5‐dimethyl‐1H‐pyrazol‐1‐yl)ethyl]amino}ethyl)phosphonate), combining a pyrazolyl‐amine chelating group and a monophosphonate ethyl ester function (–P(O)OHOEt). The phosphonate group allows metal stabilization, and, simultaneously, can be considered as a potential attachment site for a biomolecule. Reaction of L4 with the precursor [^99mTc(H₂O)₃(CO)₃]⁺ gave the model radiocomplex [^99mTc(CO)₃(k³‐L4)] ( 6a ). This radiocomplex was identified by comparing its chromatographic profile with that of the corresponding Re analog ( 6 ) under the same conditions, also prepared and fully characterized by the usual analytical techniques. Radiocomplex 6a is moderately lipophilic (log P_o/w = 1.07), presenting high stability in vitro without any measurable decomposition or ligand exchange, even in the presence of strong competing chelators such as histidine and cysteine (37°C, 24 h). Biodistribution studies of the complex in CD‐1 mice indicated a rapid blood clearance, and a rapid clearance from main organs, occurring primarily through the hepatobiliary pathway. Complex 6a presents also a high robustness in vivo, demonstrated by its resistance to metabolic degradation in blood, and intact excretion into the urine, after RP‐HPLC analysis of blood and urine samples. Copyright © 2007 John Wiley & Sons, Ltd.     

Species differences in metabolism of a new antiepileptic drug candidate,DSP‐0565 [2‐(2′‐fluoro[1,1′‐biphenyl]‐2‐yl)acetamide]

The metabolism and pharmacokinetics of DSP‐0565 [2‐(2′‐fluoro[1,1′‐biphenyl]‐2‐yl)acetamide], an antiepileptic drug candidate, was investigated in rats, dogs, and humans. In human hepatocytes, [¹⁴C]DSP‐0565 was primarily metabolized via amide bond hydrolysis to (2′‐fluoro[1,1′‐biphenyl]‐2‐yl)acetic acid (M8), while in rat and dog hepatocytes, it was primarily metabolized via both hydrolysis to M8 and hydroxylation at the benzene ring or the benzyl site to oxidized metabolites. After single oral administration of [¹⁴C]DSP‐0565 to rats and dogs, the major radioactivity fraction was recovered in the urine (71–72% of dose) with a much smaller fraction recovered in feces (23–25% of dose). As primary metabolites in their excreta, M8, oxidized metabolites, and glucuronide of DSP‐0565 were detected. The contribution of metabolic pathways was estimated from metabolite profiles in their excreta: the major metabolic pathway was oxidation (57–62%) and the next highest was the hydrolysis pathway (23–33%). These results suggest that there are marked species differences in the metabolic pathways of DSP‐0565 between humans and animals. Finally, DSP‐0565 human oral clearance (CL/F) was predicted using in vitro–in vivo extrapolation (IVIVE) with/without animal scaling factors (SF, in vivo intrinsic clearance/in vitro intrinsic clearance). The SF improved the underestimation of IVIVE (fold error = 0.22), but the prediction was overestimated (fold error = 2.4–3.3). In contrast, the use of SF for hydrolysis pathway was the most accurate for the prediction (fold error = 1.0–1.4). Our findings suggest that understanding of species differences in metabolic pathways between humans and animals is important for predicting human metabolic clearance when using animal SF.     

Identification and quantification of predominant metabolites of synthetic cannabinoid MAB‐CHMINACA in an authentic human urine specimen

An autopsy case in which the cause of death was judged as drug poisoning by two synthetic cannabinoids, including MAB‐CHMINACA, was investigated. Although unchanged MAB‐CHMINACA could be detected from solid tissues, blood and stomach contents in the case, the compound could not be detected from a urine specimen. We obtained six kinds of reference standards of MAB‐CHMINACA metabolites from a commercial source. The MAB‐CHMINACA metabolites from the urine specimen of the abuser were extracted using a QuEChERS method including dispersive solid‐phase extraction, and analyzed by liquid chromatography–tandem mass spectrometry with or without hydrolysis with β‐glucuronidase. Among the six MAB‐CHMINACA metabolites tested, two predominant metabolites could be identified and quantified in the urine specimen of the deceased. After hydrolysis with β‐glucuronidase, an increase of the two metabolites was not observed. The metabolites detected were a 4‐monohydroxycyclohexylmethyl metabolite M1 (N‐(1‐amino‐3,3‐dimethyl‐1‐oxobutan‐2‐yl)‐1‐((4‐hydroxycyclohexyl)methyl)‐1H–indazole‐3‐carboxamide) and a dihydroxyl (4‐hydroxycyclohexylmethyl and tert‐butylhydroxyl) metabolite M11 (N‐(1‐amino‐4‐hydroxy‐3,3‐dimethyl‐1‐oxobutan‐2‐yl)‐1‐((4‐hydroxycyclohexyl)methyl)‐1H–indazole‐3‐carboxamide). Their concentrations were 2.17 ± 0.15 and 10.2 ± 0.3 ng/mL (n = 3, each) for M1 and M11, respectively. Although there is one previous in vitro study showing the estimation of metabolism of MAB‐CHMINACA using human hepatocytes, this is the first report dealing with in vivo identification and quantification of MAB‐CHMINACA metabolites in an authentic human urine specimen.     

In vitro and in vivo metabolism of 14C-AZ11, a novel inhibitor of bacterial DNA gyrase/type II topoisomerase

Abstract

1.?(2R,4S,4aS)-11-Fluoro-2,4-dimethyl-8-((S)-4-methyl-2-oxooxazolidin-3-yl)-2,4,4a,6-tetrahydro-1H,1'H-spiro [isoxazolo[4,5-g][1,4]oxazino[4,3-a]quinoline-5,5′-pyrimidine]-2′,4′,6′(3′H)-trione (AZ11) is a novel mode-of-inhibition bacterial topoisomerase inhibitor that entered preclinical development for the treatment of Gram-positive bacteria infection.

2.?The in vitro biotransformation studies of AZ11 using mouse, rat, dog and human hepatocytes showed low-intrinsic clearance in all species attributed to microsomal metabolism.

3.?After a single intravenous administration of [¹⁴C]AZ11 in bile duct cannulated rats, the mean percentage of dose recovered in rat urine, bile and feces was approximately 18, 36 and 42%, respectively. Unchanged AZ11 recovered in rat urine and bile was less than 9% of the dose, indicating that AZ11 underwent extensive metabolism in rats.

4.?The most abundant in vivo metabolite detected in urine and bile was M1 formed via ring opening on the piperidine and morpholine rings accounting for 20% of the administered dose. The major fecal metabolite was M5, which accounted for approximately 32% of administered dose. M5 was not formed when AZ11 incubated with rat intestinal microsomes and cytosol but was formed when incubated with fresh rat feces, suggesting that unchanged AZ11 was directly excreted into gut lumen where M5 formed as an intestinal microflora-mediated product. This process could have significant impact on bioavailability or exposure of AZ11 in rat.     

1‐[(Imidazolidin‐2‐yl)imino]‐1H‐indoles as new hypotensive agents: synthesis and in vitro and in vivo biological studies

A series of 1‐[(imidazolidin‐2‐yl)imino]‐1H‐indole analogues of hypotensive α₂‐AR agonists, 1‐[(imidazolidin‐2‐yl)imino]‐1H‐indazoles, was synthesized and tested in vitro for their activities at α₁‐ and α₂‐adrenoceptors as well as imidazoline I₁ and I₂ receptors. The most active 1‐[(imidazolidin‐2‐yl)imino]‐1H‐indoles displayed high or moderate affinities for α₁‐ and α₂‐adrenoceptors and substantial selectivity for α₂‐adrenoceptors over imidazoline‐I₁ binding sites. The in vivo cardiovascular properties of indole derivatives 3 revealed that substitution at C‐7 position of the indole ring may result in compounds with high cardiovascular activity. Among them, 7‐fluoro congener 3g showed the most pronounced hypotensive and bradycardic activities in this experiment at a dose as low as 10 μg/kg i.v. Metabolic stability of the selected compounds of type 3 was determined using both in vitro and in silico approaches. The results indicated that these compounds are not vulnerable to rapid first‐phase oxidative metabolism.     

Synthesis of empagliflozin,a novel and selective sodium‐glucose co‐transporter‐2 inhibitor,labeled with carbon‐14 and carbon‐13

Empagliflozin, (2S,3R,4R,5S,6R)‐2‐[4‐chloro‐3‐[[4‐[(3S)‐oxolan‐3‐yl]oxyphenyl]methyl]phenyl]‐6‐(hydroxymethyl)oxane‐3,4,5‐triol was recently approved by the FDA for the treatment of chronic type 2 diabetes mellitus. Herein, we report the synthesis of carbon‐13 and carbon‐14 labeled empagliflozin. Carbon‐13 labeled empagliflozin was prepared in five steps and in 34% overall chemical yield starting from the commercially available α‐D‐glucose‐[¹³C₆]. For the radiosynthesis, the carbon‐14 atom was introduced in three different positions of the molecule. In the first synthesis, Carbon‐14 D‐(+)‐gluconic acid δ‐lactone was used to prepare specifically labeled empagliflozin in carbon‐1 of the sugar moiety in four steps and in 19% overall radiochemical yield. Carbon‐14 labeled empagliflozin with the radioactive atom in the benzylic position was obtained in eight steps and in 7% overall radiochemical yield. In the last synthesis carbon‐14 uniformly labeled phenol was used to give [¹⁴C]empagliflozin in eight steps and in 18% overall radiochemical yield. In all these radiosyntheses, the specific activities of the final compounds were higher than 53 mCi/mmol, and the radiochemical purities were above 98.5%.     

Roflumilast analogs with improved metabolic stability,plasma protein binding,and pharmacokinetic profile

With the aim of studying their in vitro and in vivo pharmacokinetics, new chromatographic methods were developed for the determination of three new roflumilast synthetic analogs ( I?III ) as PDE‐4B inhibitors in rat liver S9 fraction, phosphate buffered saline, pH 7.4, and human and rat plasma. The developed high performance liquid chromatography?ultra violet (HPLC?UV) methods were performed on a Zorbax Eclipse C8 column and UV detection was carried out at 215 nm. The three compounds were tested for their metabolic stability and were found to be metabolically more stable than roflumilast especially the 2‐mercaptobenzothiazol‐6‐yl analog ( III ) which displayed an in vitro half‐life time (247.55 minutes) higher than that of roflumilast (12.29 minutes) and a low in vitro clearance of 5.67 mL/min./kg. Possible phase I metabolites were investigated using ultra‐performance liquid chromatography?tandem mass spectrometry (UPLC–MS/MS) showing hydroxylation of the unsubstituted benzothiazol‐2‐yl ( I ) and benzothiazole‐6‐yl ( II ) analogs and a cleaved benzothiazole metabolite of the 2‐mercaptobenzothiazol‐6‐yl analog ( III ). Plasma protein binding affinity was tested using equilibrium membrane dialysis method showing a very high percentage (more than 95%) of plasma protein binding of compounds I and II where compound III exhibited lower percentage (53.71%) demonstrating its accessibility for tissue distribution. Also, a UPLC–MS/MS method was developed using an Acquity UPLC BEH shield RP C18 column to be applied to an in vivo pharmacokinetic study in rats following a subcutaneous dose (1 mg/kg). Compounds I?III showed improved in vivo pharmacokinetic parameters especially compound III which displayed a half‐life 3‐fold greater than roflumilast (21 hours) and a C_max value of 113.958 ng/mL. Accordingly, this new chemical entity should be subjected to further investigation as it can be a good drug candidate for treating chronic obstructive pulmonary disease.     

Synthesis and preliminary characterization of radioiodinated benzofuran‐3‐yl‐(indol‐3‐yl)maleimide derivatives as potential SPECT imaging probes for the detection of glycogen synthase kinase‐3β (GSK‐3β) in the brain

We report on the synthesis and preliminary characterization of two radioiodinated benzofuran‐3‐yl‐(indol‐3‐yl)maleimides, 3‐(benzofuran‐3‐yl)‐4‐(5‐[¹²⁵I]iodo‐1‐methyl‐1H‐indol‐3‐yl)‐1H‐pyrrole‐2,5‐dione ([¹²⁵I]5), and 3‐(5‐[¹²⁵I]iodo‐1‐methyl‐1H‐indol‐3‐yl)‐4‐(6‐methoxybenzofuran‐3‐yl)‐1H‐pyrrole‐2,5‐dione ([¹²⁵I]6), as the first potential SPECT imaging probes targeting glycogen synthase kinase‐3β (GSK‐3β). In this study, we used ¹²⁵I as a surrogate of ¹²³I because of its ease of use. The radioiodinated ligands were prepared from the corresponding tributyltin precursors through an iododestannylation reaction using hydrogen peroxide as an oxidant with a radiochemical yield of 10–30%. In vitro binding experiments suggested that both compounds show high affinity for GSK‐3β at a level similar to a known GSK‐3β inhibitor. Biodistribution studies with normal mice revealed that the radioiodinated compounds display sufficient uptake into (1.8%ID/g at 10 min postinjection) and clearance from the brain (1.0%ID/g at 60 min postinjection). These preliminary results suggest that the further optimization of radioiodinated benzofuran‐3‐yl‐(indol‐3‐yl)maleimide derivatives may facilitate the development of clinically useful SPECT imaging probes for the in vivo detection of GSK‐3β.