Minli Zhang

Discovery of a potent respiratory syncytial virus RNA polymerase inhibitor.

Targeting viral polymerases has been a proven and attractive strategy for antiviral drug discovery. Herein we describe our effort in improving the antiviral activity and physical properties of a series of benzothienoazepine compounds as respiratory syncytial virus (RSV) RNA polymerase inhibitors. The antiviral activity and spectrum of this class was significantly improved by exploring the amino substitution of the pyridine ring, resulting in the discovery of the most potent RSV A polymerase inhibitors reported to date.

Synthesis and SAR of aminothiazole fused benzazepines as selective dopamine D2 partial agonists.

Dopamine (D(2)) partial agonists (D2PAs) have been regarded as a potential treatment for schizophrenia patients with expected better side effect profiles than currently marketed antipsychotics. Herein we report the synthesis and SAR of a series of aminothiazole fused benzazepines as selective D(2) partial agonists. These compounds have good selectivity, CNS drug-like properties and tunable D(2) partial agonism. One of the key compounds, 8h, has good in vitro/in vivo ADME characteristics, and is active in a rat amphetamine-induced locomotor activity model.

Identification of the sulfone functionality in protonated analytes via ion/molecule reactions in a linear quadrupole ion trap mass spectrometer.

A tandem mass spectrometric method is presented for the rapid identification of drug metabolites that contain the sulfone functional group. This method is based on a gas-phase ion/molecule reaction of protonated sulfone analytes with trimethyl borate (TMB) that yields a diagnostic product ion, adduct-Me2O, at high reaction efficiency. A variety of compounds with different functional groups, such as sulfoxides, hydroxylamines, N-oxides, anilines, phenol, an aliphatic amine, and an aliphatic alcohol, were examined to probe the selectivity of this reaction. Except for protonated sulfones, most of the protonated compounds react very slowly or not at all with TMB. Most importantly, none of them give the adduct-Me2O product. A mechanism that explains the observed selectivity is proposed for the diagnostic reaction and is supported by quantum chemical calculations. The reaction was tested with the anti-inflammatory drug sulindac and its metabolite, sulindac sulfone, which were readily distinguished. The presence of other functionalities in addition to sulfone was found not to influence the diagnostic reactivity.

Identification of N-Oxide and Sulfoxide Functionalities in Protonated Drug Metabolites by Using Ion–Molecule Reactions Followed by Collisionally Activated Dissociation in a Linear Quadrupole Ion Trap Mass Spectrometer

The in vivo oxidation of sulfur and nitrogen atoms in many drugs into sulfoxide and N-oxide functionalities is a common biotransformation process. Unfortunately, the unambiguous identification of these metabolites can be challenging. In the present study, ion-molecule reactions of tris(dimethylamino)borane followed by collisionally activated dissociation (CAD) in an ion trap mass spectrometer are demonstrated to allow the identification of N-oxide and sulfoxide functionalities in protonated polyfunctional drug metabolites. Only ions with N-oxide or sulfoxide functionality formed diagnostic adducts that had lost dimethyl amine (DMA). This was demonstrated even for an analyte that contains a substantially more basic functionality than the functional group of interest. CAD of the diagnostic product ions (M) resulted mainly in type A (M - DMA) and B fragment ions (M - HO-B(N(CH3)2)2) for N-oxides, but sulfoxides also formed diagnostic C ions (M - O═BN(CH3)2), thus allowing differentiation of the functionalities. Some protonated analytes yielded abundant TDMAB adducts that had lost two DMA molecules instead of just one. This provides information on the environment of the N-oxide and sulfoxide functionalities. Quantum chemical calculations were performed to explore the mechanisms of the above-mentioned reactions. The method can be implemented on HPLC for real drug analysis.

Mass spectrometric identification of the N‐monosubstituted N‐hydroxylamino functionality in protonated analytes via ion/molecule reactions in tandem mass spectrometry

RATIONALE N-Monosubstituted hydroxylamines correspond to an important class of metabolites for many bioactive molecules. In this study, a tandem mass spectrometric method based on ion/molecule reactions was developed for the identification of compounds with the N-monosubstituted hydroxylamino functionality. METHODS The diagnostic ion/molecule reaction occurs between protonated analytes with 2-methoxypropene (MOP) inside a linear quadrupole ion trap mass spectrometer. RESULTS Most protonated compounds with N-monosubstituted and disubstituted hydroxylamino and oxime functional groups react with MOP via proton transfer and formation of a stable adduct in a linear quadrupole ion trap mass spectrometer. However, only protonated compounds with N-monosubstituted hydroxylamino groups form the characteristic MOP adduct-MeOH product. Possible mechanisms of this reaction are discussed. CONCLUSIONS A method based on functional group-selective ion/molecule reactions in a linear quadrupole ion trap mass spectrometer has been demonstrated to allow the identification of protonated compounds with the N-monosubstituted hydroxylamino functionality. Only N-monosubstituted hydroxylamines react with MOP via formation of an adduct that has eliminated methanol.

Gas-phase ion-molecule reactions for the identification of the sulfone functionality in protonated analytes in a linear quadrupole ion trap mass spectrometer.

RATIONALE The oxidation of sulfur atoms is an important biotransformation pathway for many sulfur-containing drugs. In order to rapidly identify the sulfone functionality in drug metabolites, a tandem mass spectrometric method based on ion-molecule reactions was developed. METHODS A phosphorus-containing reagent, trimethyl phosphite (TMP), was allowed to react with protonated analytes with various functionalities in a linear quadrupole ion trap mass spectrometer. The reaction products and reaction efficiencies were measured. RESULTS Only protonated sulfone model compounds were found to react with TMP to form a characteristic [TMP adduct-MeOH] product ion. All other protonated compounds investigated, with functionalities such as sulfoxide, N-oxide, hydroxylamino, keto, carboxylic acid, and aliphatic and aromatic amino, only react with TMP via proton transfer and/or addition. The specificity of the reaction was further demonstrated by using a sulfoxide-containing anti-inflammatory drug, sulindac, as well as its metabolite sulindac sulfone. CONCLUSIONS A method based on functional group-selective ion-molecule reactions in a linear quadrupole ion trap mass spectrometer has been demonstrated for the identification of the sulfone functionality in protonated analytes. A characteristic [TMP adduct-MeOH] product ion was only formed for the protonated sulfone analytes. The applicability of the TMP reagent in identifying sulfone functionalities in drug metabolites was also demonstrated. Copyright

Identification of 2-Aminothiazolobenzazepine Metabolites in Human, Rat, Dog, and Monkey Microsomes by Ion-Molecule Reactions in Linear Quadrupole Ion Trap Mass Spectrometry

2-Aminothiazolobenzazepine (2-ATBA), 7-[(1-methyl-1H-pyrazol-4-yl)methyl]-6,7,8,9-tetrahydro-5H-[1,3]thiazolo[4,5-h][3]benzazepin-2-amine, is a D2 partial agonist that has demonstrated antipsychotic effects in a rodent in vivo efficacy model. The metabolite profile showed that 2-ATBA is mainly metabolized by oxidation. However, identification of the oxidation site(s) in the 2-aminothiazole group presents a challenge for the traditional metabolite identification methods such as liquid chromatography/mass spectrometry and NMR due to the lack of unique tandem mass spectrometry fragmentation patterns for ions with the 2-aminothiazole group oxidized at different sites and the lack of stability for purification or reference standard synthesis. We describe the characterization of the oxidized heteroatoms of the 2-aminothiazole group via gas-phase ion-molecule reactions (GPIMR) in a modified linear quadrupole ion trap mass spectrometer. The GPIMR reagents used were dimethyl disulfide, tert-butyl peroxide, and tri(dimethylamino)borane. Each reagent was introduced into the ion trap through the helium line and was allowed to react with the protonated metabolites. The ionic ion-molecule reaction products and their fragmentation profiles were compared with the profiles of the ionic ion-molecule reaction products of protonated reference compounds that had specific heteroatom functionalities. The oxidized 2-aminothiazole metabolite of 2-ATBA showed a similar GPIMR profile to that of the reference compounds with a tertiary N-oxide functionality and distinct from the profiles of the reference compounds with N-aryl hydroxylamine, nitroso, or pyridine N-oxide functionalities. This study demonstrates the feasibility of fingerprinting the chemical nature of oxidized nitrogen functional groups via GPIMR profiling for metabolite structure elucidation.

Identification of Carboxylate, Phosphate, and Phenoxide Functionalities in Deprotonated Molecules Related to Drug Metabolites via Ion–Molecule Reactions with water and Diethylhydroxyborane

AbstractTandem mass spectrometry based on ion–molecule reactions has emerged as a powerful tool for structural elucidation of ionized analytes. However, most currently used reagents were designed to react with protonated analytes, making them suboptimal for acidic analytes that are preferentially detected in negative ion mode. In this work we demonstrate that the phenoxide, carboxylate, and phosphate functionalities can be identified in deprotonated molecules by use of a combination of two reagents, diethylmethoxyborane (DEMB) and water. A novel reagent introduction setup that allowed DEMB and water to be separately introduced into the ion trap region of the mass spectrometer was developed to facilitate fundamental studies of this reaction. A new reagent, diethylhydroxyborane (DEHB), was generated inside the ion trap by hydrolysis of DEMB on introduction of water. Most carboxylates and phenoxides formed a DEHB adduct, followed by addition of one water molecule and subsequent ethane elimination (DEHB adduct +H2O − CH3CH3) as the major product ion. Phenoxides with a hydroxy group adjacent to the deprotonation site and phosphates formed a DEHB adduct, followed by ethane elimination (DEHB adduct − CH3CH3). Deprotonated molecules with strong intramolecular hydrogen bonds or without the aforementioned functionalities, including sulfates, were unreactive toward DEHB/H2O. Reaction mechanisms were explored via isotope labeling experiments and quantum chemical calculations. The mass spectrometry method allowed the differentiation of phenoxide-, carboxylate-, phosphate-, and sulfate-containing analytes. Finally, it was successfully coupled with high-performance liquid chromatography for the analysis of a mixture containing hymecromone, a biliary spasm drug, and its three possible metabolites. Graphical Abstractᅟ

Identification of Protonated Sulfone and Aromatic Carboxylic Acid Functionalities in Organic Molecules by Using Ion–Molecule Reactions Followed by Collisionally Activated Dissociation in a Linear Quadrupole Ion Trap Mass Spectrometer

Gas-phase reactivity of protonated model compounds with different functional groups toward trimethoxymethylsilane (TMMS) was studied to explore the utility of this reagent in mass spectrometric identification of specific functionalities for potentially rapid characterization of drug metabolites. Only protonated analytes with a carboxylic acid, a sulfone, or a sulfonamide functionality formed diagnostic adducts that had lost a methanol molecule upon reactions with TMMS. Collisionally activated dissociation (CAD) of these methanol-eliminated adduct ions (MS3 experiments) produced characteristic fragment ions of m/z 75, 105, and 123 for sulfones, while an additional methanol elimination was observed for carboxylic acids and sulfonamides. CAD of latter products (MS4 experiments) resulted in elimination of diagnostic neutral molecules CO2 (44 Da) and C2H6O2Si (90 Da) for aromatic carboxylic acids. Both aliphatic carboxylic acids and sulfonamides yield several fragment ions in these MS4 experiments that are different from those observed for sulfones or aromatic carboxylic acids. Potential energy surfaces were calculated (at the M06-2X/6-311++G(d,p) level of theory) to explore the mechanisms of various reactions. In summary, sulfones and aromatic carboxylic acids can be differentiated from each other and also from sulfonamides and aliphatic carboxylic acids based on reactions with TMMS and one or two CAD experiments. Aliphatic carboxylic acids and sulfonamides could not be differentiated from each other.

Contribution of Artifacts to N-Methylated Piperazine Cyanide-Adduct Formation in vitro From N-Alkyl Piperazine Analogs

In the liver microsome cyanide (CN)-trapping assays, piperazine-containing compounds formed significant N-methyl piperazine CN adducts. Two pathways for the N-methyl piperazine CN adduct formation were proposed: 1) The α-carbon in the N-methyl piperazine is oxidized to form a reactive iminium ion that can react with cyanide ion; 2) N-dealkylation occurs followed by condensation with formaldehyde and dehydration to produce N-methylenepiperazine iminium ion, which then reacts with cyanide ion to form the N-methyl CN adduct. The CN adduct from the second pathway was believed to be an artifact or metabonate. In the present study, a group of 4′-N-alkyl piperazines and 4′-N-[13C]methyl–labeled piperazines were used to determine which pathway was predominant. Following microsomal incubations in the presence of cyanide ions, a significant percentage of 4′-N-[13C]methyl group in the CN adduct was replaced by an unlabeled natural methyl group, suggesting that the second pathway was predominant. For 4′-N-alkyl piperazine, the level of 4′-N-methyl piperazine CN adduct formation was limited by the extent of prior 4′-N-dealkylation. In a separate study, when 4′-NH-piperaziens were incubated with potassium cyanide and [13C]-labeled formaldehyde, 4′-N-[13C]methyl piperazine CN-adduct was formed without NADPH or liver microsome suggesting a direct Mannich reaction is involved. However, when [13C]-labeled methanol or potassium carbonate was used as the one-carbon donor, 4′-N-[13C]methyl piperazine CN adduct was not detected without liver microsome or NADPH present. The biologic and toxicological implications of bioactivation via the second pathway necessitate further investigation because these one-carbon donors for the formation of reactive iminium ions could be endogenous and readily available in vivo.