piperidines and methoxyamine

The incubation of CPAQOP (1-[(2R)-2-[[4-[3-chloro-4-(2-pyridyloxy)anilino]quinazolin-5-yl]oxymethyl]-1-piperidyl]-2-hydroxy) with human liver microsomes generated several metabolites that highlighted the hydroxyacetamide side chain was a major site of metabolism for the molecule. The metabolites were derived predominantly from oxidative biotransformations; however, two unexpected products were detected by liquid chromatography/ultraviolet/mass spectrometry (LC/UV/MS) and identified as methanol adducts. This observation prompted further LC/MS investigations into their formation.. Three separate incubations of CPAQOP were conducted in human liver microsomes; Naïve, fortified with methoxyamine and fortified with glutathione. Separation was achieved via ultra-high-performance liquid chromatography with either methanol or acetonitrile gradients containing formic acid. MS analysis was conducted by electrospray ionisation LTQ Orbitrap mass spectrometry acquiring accurate mass full scan, data-dependent MS. No methanol adducts were detected by MS when acetonitrile was used in the mobile phase instead of methanol, verifying that a metabolite was reacting with methanol on column. Although this reactive metabolite could not be isolated or structurally characterised by LC/MS directly, product ion spectra of the methanol adducts confirmed addition of methanol on the hydroxyacetamide side chain. Additional experiments using methoxyamine showed the disappearance of the two methanol adducts and appearance of a methoxyamine adduct, confirming the presence of an aldhyde. Product ion spectra of the methoxyamine adduct confirmed addition of methoxyamine to the hydroxyacetamide side chain.. The proposed bioactivation of CPAQOP occurred via the reactive aldehyde intermediate, which readily reacted with methanol in the mobile phase to form a pair of isomeric hemiacetal methanol adducts. In acidified methanol the equilibrium favoured the methanol adduct and in acidified acetonitrile it favoured the hydrate; therefore, the reactive aldehyde metabolite was not detected and could not be structurally characterised directly. Copyright © 2016 John Wiley & Sons, Ltd.

Topics: Aldehydes; Chromatography, High Pressure Liquid; Glutathione; Humans; Hydroxylamines; Mass Spectrometry; Methanol; Microsomes, Liver; Piperidines

The synthesis of (±)-lasubine II has been achieved through a three-component allylation capitalizing on the unique properties of N-methoxyamines. This reaction enabled the installation of all the carbon atoms of lasubine II in a single operation. The N-methoxy group was efficiently used for the subsequent nitrone formation. A single-step cyclization of isoxazolidines or N-methoxyamines to form functionalized piperidine rings was also developed.

Topics: Cyclization; Hydroxylamines; Lagerstroemia; Piperidines; Quinolizines; Stereoisomerism

Indoleamine 2,3-dioxygenase-1 (IDO) is an immune regulatory enzyme expressed by most human tumors. IDO levels in tumor cells correlate with increased metastasis and poor patient outcome and IDO is linked to tumor cell resistance to immunotherapy, radiation therapy, and chemotherapy. Knowledge of tumor cell-autonomous effects of IDO, independent of its well-known role in regulating and suppressing anti-tumor immune responses, is limited. Clonal populations of A549 human lung adenocarcinoma cells stably transfected with anti-IDO shRNA or scrambled control shRNA were used to study IDO effects on drug sensitivity and resistance. IFNγ was used to induce IDO in those cells. We show, for the first time, that IDO mediates human tumor cell resistance to the candidate anticancer drugs FK866 (an NAD+ inhibitor), methoxyamine (MX, a base excision repair [BER] inhibitor) and approved anticancer drugs pemetrexed (a folate anti-metabolite) and gemcitabine (a nucleoside analogue), and combined treatment with pemetrexed and MX, in the absence of immune cells. Concurrent knockdown of IDO and thymidylate synthase (TS, a key rate-limiting enzyme in DNA synthesis and repair) sensitizes human lung cancer cells to pemetrexed and 5FUdR to a greater degree than knockdown of either target alone. We conclude that BER in IDO-expressing A549 cells plays a major role in mediating resistance to a range of approved and candidate anticancer drugs. IDO inhibitors are undergoing clinical trials primarily to improve antitumor immune responses. We show that targeting IDO alone or in combination with TS is a potentially valuable therapeutic strategy for cancer treatment, independent of immune activity and in combination with conventional chemotherapy.

Topics: Acrylamides; Animals; BRCA2 Protein; Cell Line, Tumor; Clone Cells; Deoxycytidine; DNA Repair; Down-Regulation; Drug Resistance, Neoplasm; Enzyme Induction; Floxuridine; Gemcitabine; Gene Knockdown Techniques; Humans; Hydroxylamines; Indoleamine-Pyrrole 2,3,-Dioxygenase; Mice, SCID; NAD; Pemetrexed; Piperidines; RNA, Small Interfering; Thymidylate Synthase; Xenograft Model Antitumor Assays

Glioblastoma multiforme (GBM) is a devastating brain tumor with poor prognosis and low median survival time. Standard treatment includes radiation and chemotherapy with the DNA alkylating agent temozolomide (TMZ). However, a large percentage of tumors are resistant to the cytotoxic effects of the TMZ-induced DNA lesion O(6)-methylguanine due to elevated expression of the repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) or a defect in the mismatch repair (MMR) pathway. Although a majority of the TMZ-induced lesions (N7-methylguanine and N3-methyladenine) are base excision repair (BER) substrates, these DNA lesions are also readily repaired. However, blocking BER can enhance response to TMZ and therefore the BER pathway has emerged as an attractive target for reversing TMZ resistance. Our lab has recently reported that inhibition of BER leads to the accumulation of repair intermediates that induce energy depletion-mediated cell death via hyperactivation of poly(ADP-ribose) polymerase. On the basis of our observation that TMZ-induced cell death via BER inhibition is dependent on the availability of nicotinamide adenine dinucleotide (NAD(+)), we have hypothesized that combined BER and NAD(+) biosynthesis inhibition will increase TMZ efficacy in glioblastoma cell lines greater than BER inhibition alone. Importantly, we find that the combination of BER and NAD(+) biosynthesis inhibition significantly sensitizes glioma cells with elevated expression of MGMT and those deficient in MMR, two genotypes normally associated with TMZ resistance. Dual targeting of these two interacting pathways (DNA repair and NAD(+) biosynthesis) may prove to be an effective treatment combination for patients with resistant and recurrent GBM.

Topics: Acrylamides; Adenosine Triphosphate; Antineoplastic Agents, Alkylating; Cell Line, Tumor; Cell Survival; Dacarbazine; DNA Glycosylases; DNA Repair; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Drug Synergism; Glioblastoma; Humans; Hydroxylamines; Immunoblotting; Methyl Methanesulfonate; NAD; Piperidines; Poly(ADP-ribose) Polymerases; RNA Interference; Temozolomide

In the present work, the characterization of the biotransformation and bioactivation pathways of the cannabinoid receptor 1 antagonist rimonabant (Acomplia) is described. Rimonabant was approved in Europe in 2006 for the treatment of obesity but was withdrawn in 2008 because of a significant drug-related risk of serious psychiatric disorders. The aim of the present work is to characterize the biotransformation and potential bioactivation pathways of rimonabant in vitro in human and rat liver microsomes. The observation of a major iminium ion metabolite led us to perform reactive metabolite trapping, covalent binding to proteins, and time-dependent inhibition of cytochrome P450 3A4 studies. The major biotransformation pathways were oxidative dehydrogenation of the piperidinyl ring to an iminium ion, hydroxylation of the 3 position of the piperidinyl ring, and cleavage of the amide linkage. In coincubations with potassium cyanide, three cyanide adducts were detected. A high level of covalent binding of rimonabant in human liver microsomes was observed (920 pmol equivalents/mg protein). In coincubations with potassium cyanide and methoxylamine, the covalent binding was reduced by approximately 40 and 30%, respectively, whereas GSH had no significant effect on covalent binding levels. Rimonabant was also found to inhibit cytochrome P450 3A4 irreversibly in a time-dependent manner. In view of these findings, it is noteworthy that, to date, no toxicity findings related to the formation of reactive metabolites from rimonabant have been reported.

Topics: Animals; Biotransformation; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Humans; Hydroxylamines; Microsomes, Liver; Oxidation-Reduction; Piperidines; Potassium Cyanide; Protein Binding; Pyrazoles; Rats; Receptor, Cannabinoid, CB1; Rimonabant