Aliphatic propargylamines: potent, selective, irreversible monoamine oxidase B inhibitors.

A series of aliphatic propargylamine derivatives has been synthesized. Some of them possess highly potent, irreversible, selective, inhibitory activity toward monoamine oxidase B (MAO-B). The potency of the inhibitors is related to chain length and substitution of a hydrogen on the terminal carbon of the aliphatic chain. MAO inhibitory activity as assessed in vitro increased as the aliphatic carbon chain length increased. Substitution of a hydrogen by hydroxyl, carboxyl, or carbethoxyl groups at the aliphatic chain terminal or replacement of the methyl group on the nitrogen atom by an ethyl group considerably reduced the inhibitory activity. Stereospecific effects were observed with the R-(-)-enantiomer being 20-fold more active than the S-(+)-enantiomer. Inhibitors with relatively short carbon chain lengths (i.e. four to six carbons) were found to be more potent than those with longer chains in inhibiting brain MAO-B activity in vivo especially after oral administration. Chronic administration of low doses of the aliphatic propargylamines caused a slight cumulative inhibition of MAO-A activity in the mouse brain. These MAO-B inhibitors appear to be nontoxic, and they do not possess an amphetamine-like moiety in their structure as is the case for deprenyl. We expect that these aliphatic propargylamines may be useful in the treatment in certain neuropsychiatric disorders.     

Nitric Oxide Synthase Inhibition by Pentacycloundecane Conjugates of Aminoguanidine and Tryptamine

This paper describes the synthesis and in‐vitro activity of pentacycloundecane‐conjugated aminoguanidine and tryptamine analogues on nitric oxide synthase (NOS) using rat brain homogenate. Both aminoguanidine and tryptamine‐derived NOS inhibitors show selectivity towards the inducible and neuronal isoforms of the NOS enzyme, but are weak inhibitors and complete inhibition of the enzyme occurs only at high millimolar concentrations. In view of the increased NOS inactivation observed with alkyl substitution of these structures, the present study aimed to evaluate the effect of the pentacycloundecane cage moiety as an alkyl substituent on the in vitro NOS inhibition of aminoguanidine and tryptamine compounds. Comparison of the IC₅₀ values of aminoguanidine (IC₅₀ = 2.306×10^–3 M) and 8‐imino‐N‐guanidino‐pentacyclo‐undecane 2 (IC₅₀ = 8.803×10^–5 M) revealed a more than 26‐fold increase in potency. The ability of tryptamine to inhibit NOS activity was also markedly improved by the various pentacycloundecane substituents. The compounds, 3‐hydroxy‐4‐[3‐(2‐aminoethyl)indole]‐azahexacyclo[5.4.1.0^2,6.0^3,10.0^5,9.0^8,11]dodecane 4 and 8‐[3‐(2‐aminoethyl) indole]‐pentacyclo[5.4.0^2,6.0^3,10.0^5,9]undecane 7 showed the best activity of the tryptamine analogues with a more than 3‐fold increase in nitric oxide synthase inhibition. The results confirmed that the pentacycloundecane structure substantially enhanced the NOS inhibitory potency as observed for the six new NOS inhibitors.     

Isotope labeled ‘HEA/HEE’ moiety in the synthesis of labeled HIV‐protease inhibitors—Part II

[²H₅]‐Amprenavir and [²H ₅]‐saquinavir have been prepared from a common labeled precursor (1S, 2S)‐(1‐oxiranyl‐2‐[²H₅]phenylethyl)‐carbamic acid tert‐butyl ester, 1 . Both of these compounds are in the ‘HEA’ class of HIV protease inhibitors. [²H₅]‐Indinavir, a representative of the ‘HEE’ group of protease inhibitors, has also been synthesized. In the case of indinavir, 1S‐(2,2‐dimethyl‐8, 8a‐dihydro‐3aH‐indeno‐[1,2‐d]‐oxazol‐3R‐yl)‐2‐oxiranylmethyl‐3‐[²H₅]phenylpropan‐1‐one, 11 , provided the [phenyl‐²H₅]‐HEE core structure for synthesis of the desired labeled compound. Copyright © 2005 John Wiley & Sons, Ltd.     

A dataset on 145 chemicals tested in alternative assays for skin sensitization undergoing prevalidation

Skin sensitization is a key endpoint for cosmetic ingredients, with a forthcoming ban for animal testing in Europe. Four alternative tests have so far been submitted to ECVAM prevalidation: (i) MUSST and (ii) h‐Clat assess surface markers on dendritic cell lines, (iii) the direct peptide reactivity assay (DPRA) measures reactivity with model peptides and (iv) the KeratinoSens^TM assay which is based on detection of Nrf2‐induced luciferase. It is anticipated that only an integrated testing strategy (ITS) based on a battery of tests might give a full replacement providing also a sensitization potency assessment, but this concept should be tested with a data‐driven analysis. Here we report a database on 145 chemicals reporting the quantitative endpoints measured in a U937‐ test, the DPRA and KeratinoSens^TM . It can serve to develop data‐driven ITS approaches as we show in a parallel paper and provides a view as to the current ability to predict with in vitro tests as we are entering 2013. It may also serve as reference database when benchmarking new molecules with in vitro based read‐across and find use as a reference database when evaluating new tests. The tests and combinations thereof were evaluated for predictivity, and overall a similar predictivity was found as before on three‐fold smaller datasets. Analysis of the dose–response parameters of the individual tests indicates a correlation to sensitization potency. Detailed analysis of chemicals false‐negative and false‐positive in two tests helped to define limitations in the tests but also in the database derived from animal studies. Copyright © 2013 John Wiley & Sons, Ltd.     

Tryptophan replacement in the nociceptin/orphanin FQ receptor ligand Ac‐RYYRWK‐NH2

Abstract: In the present study we describe the in vitro pharmacological characterization of the nociceptin/orphanin FQ (N/OFQ) receptor (NOP) ligand Ac‐RYYRWK‐NH₂ and the synthesis and biological evaluation of 13 Trp⁵ substituted Ac‐RYYRWK‐NH₂ analogs. Results indicate that Ac‐RYYRWK‐NH₂ behaves as a highly potent and selective partial agonist at the NOP receptors and that the whole indole moiety of the Trp⁵ side chain is not required, being a phenyl‐ethyl side chain already sufficient for maintaining high potency.     

Synthesis andanti‐aggregatory activity of linear retro‐inverso RGD peptides

Abstract: Six retro‐inverso tri‐ and tetrapeptide analogues of RGD were prepared and their anti‐aggregatory activity was determined by platelet aggregation tests in comparison with the corresponding parent peptides. An efficient method for the introduction of a malonyl‐aspartic residue into a peptide chain is described for the first time. A 2–3‐fold decrease in potency or total loss of bioactivity was observed with the new peptides; structure–activity relationships are discussed.     

New inhibitors of human renin that contain novel Leu-Val replacements. Examination of the P1 site

Stereoselective syntheses of several nonpeptide sulfidoethanol fragments that function as Leu10-Val11 (P1-P1') scissile bond replacements in human angiotensinogen are presented. These fragments are prepared from a variety of amino acids with formal P1 side chains varying in size and lipophilicity by converting them to their corresponding N-protected aminoalkyl epoxide 5 followed by ring opening with isopropyl mercaptan. The coupling of these fragments to either Boc-Phe-Ala-OH or Boc-Phe-His-OH produces inhibitors of human renin, 6 and 7, respectively, which are compared to a series of dipeptide-aldehyde inhibitors, 4, by molecular modeling and biochemical methods. Qualitatively, histidine-containing (P2) inhibitors 7 possess greater inhibitory potency than their corresponding alanine (P2) analogues 6, which are more potent than the corresponding aldehydic inhibitors from series 4. Within a given series, inhibitors with the cyclohexylmethyl P1 side chain are more potent than the benzyl analogues, which in turn are more potent than cyclohexyl or isobutyl derivatives. Inhibitors with parger P1 side chains (e.g. adamantylmethyl and benzhydryl) are much less active. The inhibitory potency of these compounds against human renin is discussed in terms of specific interactions with the enzyme.     

Pharmacological Development of Target-Specific Delocalized Lipophilic Cation-Functionalized Carboranes for Cancer Therapy

Purpose

Tumor cell heterogeneity and microenvironment represent major hindering factors in the clinical setting toward achieving the desired selectivity and specificity to malignant tissues for molecularly targeted cancer therapeutics. In this study, the cellular and molecular evaluation of several delocalized lipophilic cation (DLC)-functionalized carborane compounds as innovative anticancer agents is presented.

Methods

The anticancer potential assessment of the DLC-carboranes was performed in established normal (MRC-5, Vero), cancer (U-87 MG, HSC-3) and primary glioblastoma cancer stem (EGFR^pos, EGFR^neg) cultures. Moreover, the molecular mechanism of action underlying their pharmacological response is also analyzed.

Results

The pharmacological anticancer profile of DLC-functionalized carboranes is characterized by: a) a marked in vitro selectivity, due to lower concentration range needed (ca. 10 fold) to exert their cell growth-arrest effect on U-87 MG and HSC-3, as compared with that on MRC-5 and Vero; b) a similar selective growth inhibition behavior towards EGFR^pos and EGFR^neg cultures (>10 fold difference in potency) without, however, the activation of apoptosis in cultures; c) notably, in marked contrast to cancer cells, normal cells are capable of recapitulating their full proliferation potential following exposure to DLC-carboranes; and, d) such pharmacological effects of DLC-carboranes has been unveiled to be elicited at the molecular level through activation of the p53/p21 axis.

Conclusions

Overall, the data presented in this work indicates the potential of the DLC-functionalized carboranes to act as new selective anticancer therapeutics that may be used autonomously or in therapies involving radiation with thermal neutrons. Importantly, such bifunctional capacity may be beneficial in cancer therapy.

     

Structure-activity studies of flavonoids as inhibitors of hyaluronidase

The order of decreasing potency for five most potent flavonoids as inhibitors of hyaluronidase was found to be: condensed tannin less than luteolin less than apigenin less than kaempferol less than silybin. Kinetic studies of these inhibitors showed that their mode of inhibition was competitive. Aglycones were stronger inhibitors than their corresponding glycosides. The following flavonoid structure conferred potent inhibitory effect: a double bond between carbons 2 and 3; unsubstituted hydroxyl groups at positions 5, 7 and 4' and a ketone group at position 4.     

Pharmacokinetics and in vivo potency of soluble epoxide hydrolase inhibitors in cynomolgus monkeys

BACKGROUND AND PURPOSE

Soluble epoxide hydrolase inhibitors (sEHIs) possess anti-inflammatory, antiatherosclerotic, antihypertensive and analgesic properties. The pharmacokinetics (PK) and pharmacodynamics in terms of inhibitory potency of sEHIs were assessed in non-human primates (NHPs). Development of a sEHI for use in NHPs will facilitate investigations on the role of sEH in numerous chronic inflammatory conditions.

EXPERIMENTAL APPROACH

PK parameters of 11 sEHIs in cynomolgus monkeys were determined after oral dosing with 0.3 mg·kg⁻¹. Their physical properties and inhibitory potency in hepatic cytosol of cynomolgus monkeys were examined. Dose-dependent effects of the two inhibitors 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) and the related acetyl piperidine derivative, 1-trifluoromethoxyphenyl-3-(1-acetylpiperidin-4-yl) urea (TPAU), on natural blood eicosanoids, were determined.

KEY RESULTS

Among the inhibitors tested, TPPU and two 4-(cyclohexyloxy) benzoic acid urea sEHIs displayed high plasma concentrations (>10 × IC₅₀), when dosed orally at 0.3 mg·kg⁻¹. Although the 4-(cyclohexyloxy) benzoic acid ureas were more potent against monkey sEH than piperidyl ureas (TPAU and TPPU), the latter compounds showed higher plasma concentrations and more drug-like properties. The C_max increased with dose from 0.3 to 3 mg·kg⁻¹ for TPPU and from 0.1 to 3 mg·kg⁻¹ for TPAU, although it was not linear over this range of doses. As an indication of target engagement, ratios of linoleate epoxides to diols increased with TPPU administration.

CONCLUSION AND IMPLICATIONS

Our data indicate that TPPU is suitable for investigating sEH biology and the role of epoxide-containing lipids in modulating inflammatory diseases in NHPs.     

2‐Prenylated m‐dimethoxybenzenes as potent inhibitors of 15‐lipo‐oxygenase: inhibitory mechanism and SAR studies

15‐lipo‐oxygenases are one of the iron‐containing proteins capable of performing peroxidation of unsaturated fatty acids in animals and plants. The critical role of enzymes in the formation of inflammations, sensitivities, and some cancers has been demonstrated in mammals. The importance of enzymes has led to the development of mechanistic studies, product analysis, and synthesis of inhibitors. In this study, a series of allyl and prenyl dimethoxybenzenes were synthesized and their inhibitory potency against soybean 15‐Lipo‐oxygenase (L1; EC 1,13,11,12) was determined. Among the synthetic compounds, 2,6‐dimethoxy‐1‐isopentenyl‐4‐methylbenzene, 2,6‐dimethoxy‐1‐geranyl‐4‐methylbenzene, and 2,6‐dimethoxy‐1‐farnesyl‐4‐methylbenzene showed the most potent inhibitory activity with IC₅₀ values of 7.6, 5.3, and 0.52 μm , respectively. For some of the compounds, SAR studies showed acceptable relationship between inhibitory potency and enzyme–ligand interactions. Radical scavenging assessment results apart from the SAR studies indicate that electronic properties are the major factors for lipo‐oxygenase inhibition potency of the mentioned compounds. Based on the theoretical studies, it was suggested that CH…O intramolecular hydrogen bond between ortho‐methoxy oxygen and methine hydrogen atoms is one of the major factors in the stability of 2,6‐dimethoxyallyl(or prenyl)benzenes radical via the planarity fixation between phenyl and allyl (or prenyl) pi orbitals.     

Modified melanocortin tetrapeptide Ac‐His‐dPhe‐Arg‐Trp‐NH2 at the arginine side chain with ureas and thioureas

Abstract: The Ac‐His‐d Phe‐Arg‐Trp‐NH₂ tetrapeptide is a nonselective melanocortin agonist and replacement of Arg in the tetrapeptide with acidic, basic or neutral amino acids results in reduced potency at the melanocortin receptor (MCR) isoforms (MC1R and MC3–5R). To determine the importance of the positive charge and the guanidine moiety for melanocortin activity, a series of urea‐ and thiourea‐substituted tetrapeptides were designed. Replacement of Arg with Lys or ornithine reduced agonist activity at the mouse mMC1 and mMC3–5 receptors, thus supporting the hypothesis that the guanidine moiety is important for receptor potency, particularly at the MC3–5 receptors. The Arg side chain‐modified tetrapeptides examined in this study include substituted phenyl, naphthyl, and aliphatic urea and thiourea residues using a Lys side‐chain template. These ligands elicit full‐agonist pharmacology at the mouse MCRs examined in this study.     

Design,Synthesis, Biological Evaluation,and Docking Studies of (S)‐Phenylalanine Derivatives with a 2‐Cyanopyrrolidine Moiety as Potent Dipeptidyl Peptidase 4 Inhibitors

A novel series of (S)‐phenylalanine derivatives with a 2‐cyanopyrrolidine moiety were designed and synthesized through a rational drug design strategy. Biological evaluation revealed that most tested compounds were potent dipeptidyl peptidase 4 (DPP‐4) inhibitors; among them, the cyclopropyl‐substituted phenylalanine derivative 11h displayed the most potent DPP‐4 inhibitory activity with an IC₅₀ value of 0.247  μ m . In addition, molecular docking analysis of the representative compounds 11h , 11k , and 15a were performed, which not only revealed the impact of binding modes on DPP‐4 inhibitory activity but also provided additional methodological values for design and optimization.     

Steroid-like compounds in Chinese medicines promote blood circulation via inhibition of Na^＋/K^＋- ATPase

Aim:

To examine if steroid-like compounds found in many Chinese medicinal products conventionally used for the promotion of blood circulation may act as active components via the same molecular mechanism triggered by cardiac glycosides, such as ouabain.

Methods:

The inhibitory potency of ouabain and the identified steroid-like compounds on Na⁺/K⁺-ATPase activity was examined and compared. Molecular modeling was exhibited for the docking of these compounds to Na⁺/K⁺-ATPase.

Results:

All the examined steroid-like compounds displayed more or less inhibition on Na⁺/K⁺-ATPase, with bufalin (structurally almost equivalent to ouabain) exhibiting significantly higher inhibitory potency than the others. In the pentacyclic triterpenoids examined, ursolic acid and oleanolic acid were moderate inhibitors of Na⁺/K⁺-ATPase, and their inhibitory potency was comparable to that of ginsenoside Rh2. The relatively high inhibitory potency of ursolic acid or oleanolic acid was due to the formation of a hydrogen bond between its carboxyl group and the Ile322 residue in the deep cavity close to two K⁺ binding sites of Na⁺/K⁺-ATPase. Moreover, the drastic difference observed in the inhibitory potency of ouabain, bufalin, ginsenoside Rh2, and pentacyclic triterpenoids is ascribed mainly to the number of hydrogen bonds and partially to the strength of hydrophobic interaction between the compounds and residues around the deep cavity of Na⁺/K⁺-ATPase.

Conclusion:

Steroid-like compounds seem to contribute to therapeutic effects of many cardioactive Chinese medicinal products. Chinese herbs, such as Prunella vulgaris L, rich in ursolic acid, oleanolic acid and their glycoside derivatives may be adequate sources for cardiac therapy via effective inhibition on Na⁺/K⁺-ATPase.     

1,2,3,4‐Tetrahydroisoquinolines as inhibitors of HIV‐1 integrase and human LEDGF/p75 interaction

Alkaloids are a class of organic compounds with a wide range of biological properties, including anti‐HIV activity. The 1,2,3,4‐tetrahydroisoquinoline is a ubiquitous structural motif of many alkaloids. Using a short and an efficient route for synthesis, a series of 1,2,3,4‐tetrahydroisoquinolines/isoquinolines was developed. These compounds have been analysed for their ability to inhibit an important interaction between HIV‐1 integrase enzyme (IN) and human LEDGF/p75 protein (p75) which assists in the viral integration into the active genes. A lead compound 6d is found to inhibit the LEDGF/p75‐IN interaction in vitro with an IC₅₀ of ~10 μm . Molecular docking analysis of the isoquinoline 6d reveals its interactions with the LEDGF/p75‐binding residues of IN. Based on an order of addition experiment, the binding of 6d or LEDGF/p75 to IN is shown to be mutually exclusive. Also, the activity of 6d in vitro is found to be unaffected by the presence of a non‐specific DNA. As reported earlier for the inhibitors of LEDGF/p75‐IN interaction, 6d exhibits a potent inhibition of both the early and late stages of HIV‐1 replication. Compound 6d differing from the known inhibitors in the chemical moieties and interactions with CCD could potentially be explored further for developing small molecule inhibitors of LEDGF/p75‐IN interaction having a higher potency.     

Effects of short-chain alcohols and norepinephrine on brain (Na+, K+)ATPase activity

(Na⁺, K⁺)ATPase activity in synaptic membranes from whole brains of mice was inhibited by a series of short-chain aliphatic alcohols (ethanol through pentanol). The relationship of inhibitory potency to alcohol chain length and to alcohol membrane: water partition coefficient suggested that the inhibitory effect of the alcohols does not depend totally on their interaction with neuronal membrane lipids. Although partitioning into the membranes is important for this inhibitory effect, a direct interaction of alcohol with the enzyme protein may also be involved in the inhibition. Norepinephrine did not significantly potentiate inhibition of (Na⁺, K⁺)ATPase activity by low concentrations of ethanol in preparations of either mouse or rat brain. Thus, under our conditions, ethanol, at levels which can be reached in vivo, only slightly inhibited enzyme activity, and the possible importance of this inhibition in mediating the in vivo acute or chronic effects of ethanol on the CNS remains open to question.     

Synthetic inhibitors of endopeptidase EC 3.4.24.15: potency and stability in vitro and in vivo.

1. The role of the metalloendopeptidase EC 3.4.24.15 (EP 24.15) in peptide metabolism in vivo is unknown, in part reflecting the lack of a stable enzyme inhibitor. The most commonly used inhibitor, N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate (cFP-AAY-pAB, Ki = 16 nM), although selective in vitro, is rapidly degraded in the circulation to cFP-Ala-Ala, an angiotensin converting enzyme (ACE) inhibitor. This metabolite is thought to be generated by neutral endopeptidase (NEP; EC 3.4.24.11), as the Ala-Tyr bond of cFP-AAY-pAB is cleaved by NEP in vitro. In the present study, we have examined the role of NEP in the metabolism of cFP-AAY-pAB in vivo, and have tested a series of inhibitor analogues, substituted at the second alanine, for both potency and stability relative to the parent compound. 2. Analogues were screened for inhibition of fluorescent substrate cleavage by recombinant rat testes EP 24.15. D-Ala or Asp substitution abolished inhibitory activity, while Val-, Ser- and Leu-substituted analogues retained activity, albeit at a reduced potency. A relative potency order of Ala (1) > Val (0.3) > Ser (0.16) > Leu (0.06) was observed. Resistance to cleavage by NEP was assessed by incubation of the analogues with rabbit kidney membranes. The parent compound was readily degraded, but the analogues were twice (Ser) and greater than 10 fold (Leu and Val) more resistant to cleavage. 3. Metabolism of cFP-AAY-pAB and the Val-substituted analogue was also examined in conscious rabbits. A bolus injection of cFP-AAY-pAB (5 mg kg-1, i.v.) significantly reduced the blood pressure response to angiotensin I, indicating ACE inhibition. Pretreatment with NEP inhibitors, SCH 39370 or phosphoramidon, slowed the loss of cFP-AAY-pAB from the plasma, but did not prevent inhibition of ACE. Injection of 1 mg kg-1 inhibitor resulted in plasma concentrations at 10 s of 23.5 microM (cFP-AAY-pAB) and 18.0 microM (cFP-AVY-pAB), which fell 100 fold over 5 min. Co-injection of 125I-labelled inhibitor revealed that 80-85% of the radioactivity had disappeared from the circulation within 5 min, and h.p.l.c. analysis demonstrated that only 25-30% of the radiolabel remained as intact inhibitor at this time. Both analogues were cleared from the circulation at the same rate, and both inhibitors blunted the pressor response to angiotensin I, indicative of ACE inhibition. 4. These results suggest that both NEP and other clearance/degradation mechanisms severely limit the usefulness of peptide-based inhibitors such as cFP-AAY-pAB. To examine further EP 24.15 function in vivo, more stable inhibitors, preferably non-peptide, must be developed, for which these peptide-based inhibitors may serve as useful molecular templates.     

Pharmacophore Identification and Validation Study of CK2 Inhibitors Using CoMFA/CoMSIA

Protein kinase CK2, also known as casein kinase‐2, has been found to be involved in cell growth, proliferation and suppression of apoptosis, which is related to human cancers. The series of compounds were identified as casein kinase‐2 inhibitors and their inhibitory activities are a function of a variation of their structures. The current study deals with the pharmacophore identification and, accordingly, the three‐dimensional quantitative structure–activity relationship model development using Pharmacophore Alignment and Scoring Engine. Several hypotheses were developed for the molecular alignments. On the basis of statistical values, the best‐fitted model was identified and the same alignment was used for 3D‐QSAR using comparative molecular field analysis/comparative molecular similarity index analysis. Both the CoMFA (R ²_CV = 0.58, R ² = 0.82 and r ²_pred = 0.62) and the comparative molecular similarity index analysis (R ²_CV = 0.74, R ² = 0.98 and r ²_pred = 0.81) gave reasonable results. Besides pharmacophore‐based alignment, the maximum common substructure‐based alignment was also used for the comparative molecular field analysis and comparative molecular similarity index analysis. The pharmacophore‐based alignment was more prominent and it has provided important information for the modelling of potent inhibitors. The overall study implies that a highly positive and bulky group with H‐bond donating property is desirable around the nitrogen atom adjacent to the pyrrolidine ring.     

Metabolic patterns of JWH‐210, RCS‐4, and THC in pig urine elucidated using LC‐HR‐MS/MS: Do they reflect patterns in humans?

The knowledge of pharmacokinetic (PK) properties of synthetic cannabinoids (SCs) is important for interpretation of analytical results found for example in intoxicated individuals. In the absence of human data from controlled studies, animal models elucidating SC PK have to be established. Pigs providing large biofluid sample volumes were tested for prediction of human PK data. In this context, the metabolic fate of two model SCs, namely 4‐ethylnaphthalen‐1‐yl‐(1‐pentylindol‐3‐yl)methanone (JWH‐210) and 2‐(4‐methoxyphenyl)‐1‐(1‐pentyl‐indol‐3‐yl)methanone (RCS‐4), was elucidated in addition to Δ⁹‐tetrahydrocannabinol (THC). After intravenous administration of the compounds, hourly collected pig urine was analyzed by liquid chromatography‐high resolution mass spectrometry. The following pathways were observed: for JWH‐210, hydroxylation at the ethyl side chain or pentyl chain and combinations of them followed by glucuronidation; for RCS‐4, hydroxylation at the methoxyphenyl moiety or pentyl chain followed by glucuronidation as well as O ‐demethylation followed by glucuronidation or sulfation; for THC, THC glucuronidation, 11‐hydroxylation, followed by carboxylation and glucuronidation. For both SCs, parent compounds could not be detected in urine in contrast to THC. These results were consistent with those obtained from human hepatocyte and/or human case studies. Urinary markers for the consumption of JWH‐210 were the glucuronide of the N ‐hydroxypentyl metabolite (detectable for 3–4 h) and of RCS‐4 the glucuronides of the N ‐hydroxypentyl, hydroxy‐methoxyphenyl (detectable for at least 6 h), and the O ‐demethyl‐hydroxy metabolites (detectable for 4 h). Copyright © 2016 John Wiley & Sons, Ltd.     

Evaluation of Imidazole‐Based Compounds as Heme Oxygenase‐1 Inhibitors

Imidazole‐based compounds previously synthesized in our laboratory were selected and reconsidered as inhibitors of heme oxygenase‐1 obtained from the microsomal fractions of rat spleens. Most of tested compounds were good inhibitors with IC₅₀ values in the low micromolar range. Compounds were also assayed on membrane‐free full‐length recombinant human heme oxygenase‐1; all tested compounds were unable to interact with human heme oxygenase‐1 at 100 μm concentrations with the exception of compounds 11 and 13 that inhibited the enzyme of 54% and 20%, respectively. The binding of the most active compound 11 with heme or heme‐conjugated human heme oxygenase‐1 was also examined by spectral analyses. When heme was not conjugated to human heme oxygenase‐1, compound 11 caused changes in the heme spectrum only at concentration 50‐fold (100 μm ) higher than that required to inhibit rat heme oxygenase‐1; when heme was conjugated to human heme oxygenase‐1, compound 11 was able to form a heme‐compound 11 complex also at low micromolar concentrations. To obtain information on the binding mode of the tested compounds with enzyme, docking studies and pharmacophore analysis were performed. Template docking results were in agreement with experimental inhibition data and with a structure‐based pharmacophoric model. These data may be exploitable to design new OH‐1 inhibitors.