Gas‐phase fragmentation study of a novel series of synthetic 2‐oxo‐2H‐benzopyrano[2,3‐d]pyrimidine derivatives using electrospray ionization tandem mass spectroscopy measured with a quadrupole orthogonal time‐of‐flight hybrid instrument

The fragmentation patterns of a series of six novel synthesized benzopyranopyrimidine derivatives 1–6, possessing the same 2‐oxo‐2H‐benzopyrano[2,3‐d]pyrimidine backbone structure, were investigated by electrospray ionization mass spectrometry (ESI‐MS) and tandem mass spectrometry (MS/MS) techniques using a quadrupole orthogonal time‐of‐flight (QqToF)‐hybrid instrument. The series of six pure benzopyranopyrimidine compounds contained three constitutional isobaric isomers (compounds 4–6). A simple methodology, based on the use of ESI (positive ion mode) and increasing the declustering potential in the atmospheric pressure/vacuum interface resulting in collision‐induced dissociation (CID), was used to enhance the formation of the product ions. In general, the novel synthetic benzopyranopyrimidine derivatives 1–6 afforded exact accurate masses for the protonated molecules. This led to the confirmation of both molecular masses and chemical structures of the studied compounds. The breakdown routes of the protonated molecules were rationalized by conducting low‐energy CID‐MS/MS analyses. It was shown that the MS/MS fragmentation routes for the protonated molecules 1 and 2 were similar, and that the MS/MS fragmentations of the constitutional isobaric protonated molecules 5 and 6 were identical. It was also shown that the gas‐phase CID fragmentations of 5 and 6 were different from that of their constitutional isomer 4. Finally, the ESI‐MS and CID‐MS/MS analyses of the protonated molecules that were obtained from the monodeuterated benzopyranopyrimidine derivatives 1–6 confirmed the values obtained for the exact masses, the precise structural assignments of all product ions and all the pathways described in the proposed CID fragmentations. Copyright © 2008 John Wiley & Sons, Ltd.     

Reactivity of imidazolidin-4-one derivatives of primaquine: implications for prodrug design

In contrast to peptide-based imidazolidin-4-ones, those synthesized from N-(α-aminoacyl) derivatives of the antimalarial drug, primaquine and ketones are unexpectedly stable in pH 7.4 at 37 °C. The kinetics of hydrolysis of primaquine-based imidazolidin-4-ones were investigated in the pH range 0.3-13.5 at 60 °C. The hydrolysis to the parent α-aminoacylprimaquine is characterized by sigmoidal-shaped pH-rate profiles, reflecting the spontaneous decomposition of both unionized and protonated (at N-1) forms of the imidazolidin-4-one. The kinetically determined pK_a values are ca. 3.6-4.0, i.e., 4 pK_a units lower than those of amino acid amides, thus implying that hydrolysis of imidazolidin-4-ones at pH 7.4 involves the unionized form. Reactivity of this form decreases with the steric crowding of the amino acid α-substituent. In contrast, the rate constant for the spontaneous decomposition of the unionized form increases sharply for imidazolidin-4-ones derived from cyclic ketones, an observation that can be explained by the I-strain (internal strain) effect. These results are consistent with a mechanism of hydrolysis involving an S_N1-type unimolecular cleavage of the imidazolidin-4-one C2-N3 bond with departure of an amide-leaving group. The mechanism for the decomposition of the protonated imidazolidin-4-one is likely to involve an amide-carbonyl oxygen protonated species, followed by the C2-N3 bond scission, as supported by computational studies. The results herein presented suggest that imidazolidin-4-ones derived from simple N-alkyl α-aminoamides are too stable and therefore, may be useful as slow drug release prodrugs.     

α-Ureidoalkylation of thiosemicarbazide and aminoguanidine

azo[4,5-e][1,2,4]triazin-6-ones, 4,5-bis(3-thiosemicarbazido(guanidinoamino))imidazolidin-2-ones, and 1,3-dialkyl-4-(guanidinoimino)imidazolidin-2-ones by α-ureidoalkylation of thiosemicarbazide or aminoguanidine were found. A novel conglomerate in the series of imidazolidin-2-one derivatives was detected: 4,5-bis(guanidinoamino)-1,3-dimethylimid-azolidin-2-one dihydrochloride dihydrate. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 5, pp. 836–843, May, 2006.     

Stoffwechselprodukte von Mikroorganismen. 139. Mitteilung. Synthesen in der Sideramin-Reihe: Rhodotorulasäure und Dimerumsäure

t-Butyl (E)-O-acetyl-Δ²-anhydromevalonate could be prepared. by a Reformatsky reaction of 4-acetoxybutan-2-one and t-butyl bromoacetate. Condensation of its activated derivatives with the diketopiperazine of N⁵-hydroxy-L -ornithine led to di-O-acetyl dimerumic acid, which could be transformed, by ammonolysis, to dimerumic acid, identical with the natural compound. The corresponding acetohydroxamic acid, prepared by acetylation of the diketopiperazine, was identical with natural rhodotorulic acid.     

Synthesis of imidazolidin-4-one and 1H-imidazo[2,1-a]isoindole-2,5(3H,9bH)-dione derivatives of primaquine: scope and limitations

The synthesis of imidazolidin-4-one derivatives of primaquine as potential antimalarial agents is described. The target compounds were synthesized in three steps: (i) condensation of (±)-primaquine with N^α-protected amino acids, (ii) removal of the N^α-protecting group, and (iii) reaction of the N-acylprimaquine with a carbonyl compound: acetone, three cyclic ketones and veratraldehyde. Using 2-formylbenzoic acid in the third step afforded 1H-imidazo[2,1-a]isoindole-2,5(3H,9bH)-diones. All products were isolated in good to excellent yields. Whereas imidazolidin-4-ones were formed as mixtures of all possible diastereomers in equal amounts, 1H-imidazo[2,1-a]isoindole-2,5(3H,9bH)-diones were produced in a stereoselective fashion. The compounds hydrolyse very slowly (t_1/2 5-30 d) in pH 7.4 buffer to release primaquine. These primaquine derivatives are being submitted to biological assays, and preliminary results of their antimalarial activity are quite encouraging.     

Novel route of the reaction of trifluoromethyl-containing N-methyl(4-ethoxyphenyl)imidazolidin-2-ones with urea

The reactions of perfluorobiacetyl with N-(4-ethoxyphenyl)- and N-methylurea afforded cis- and trans-isomers of 1-methyl-4,5-dihydroxy-4,5-bis(trifluoromethyl)imidazolidin-2-one and 1-(4-ethoxyphenyl)-4,5-dihydroxy-4,5-bis(trifluoromethyl)imidazolidin-2-one in a yield of ～60—75%. N-Alkyl(aryl)bis(trifluoromethyl)imidazooxazoles were obtained as unexpected products in the reaction of 1-alkyl(aryl)-4,5-dihydroxy-4,5-bis(trifluoromethyl)imidazolidin-2-ones with urea in dimethylacetamide. The reaction is accompanied by the rearrangement of imidazolidin-2-ones to N-alkyl(aryl)-5,5-bis(trifluoromethyl)hydantoins with CF₃ group migration from position 5 to position 4 of the starting heterocycle. A similar rearrangement is observed on boiling of the studied imidazolidin-2-ones in dimethylacetamide. The molecular structures of 3-(4-ethoxyphenyl)-5,5-bis(trifluoromethyl)imidazolidine-2,4-dione and 6-(4-ethoxyphenyl)-3a,6a-bis(trifluoromethyl)tetrahydroimidazo[4,5-d]oxazole-2,5-dione were studied by X-ray diffraction analysis.     

Polycyclic N-Heterocyclic compounds. 50. Synthesis and pharmacological evaluation of 2,3,6,7-Tetrahydrothieno[2,3-H]-imidazo[2,1-f][1,6]naphthyridines,3,4,7,8-Tetrahydro-2H-thieno-[2,3-h]pyrimido[2,1-f][1,6]naphthyridines and their precursor

Some novel 5-hydroxyalkylamino-1,2-dihydrothieno[2,3-h][1,6]naphthyridines were prepared by the reaction of 5-chloro-1,2-dihydrothieno[2,3-h][1,6]naphthyridine derivatives with some aminoalcohols in the presence of base. These derivatives were cyclized to the corresponding imidazo or pyrimido derivatives. The bronchodilatory activity of these compounds was evaluated on the basis of their relaxation activity to tracheal contraction induced by carbamylcholine chloride as a primary in vitro assays. Effect of some naphthyridines on carbamylcholine chloride-induced contractions of trachea in the presence or absence of milrinone or 4-(3-butoxy-4-methoxyphenyl)imidazolidin-2-one, which is inhibitor of phosphodiesterase III or IV, were also evaluated.     

Unblocking the Sink: Improved CID-Based Analysis of Phosphorylated Peptides by Enzymatic Removal of the Basic C-Terminal Residue

A one-step enzymatic reaction for improving the collision-induced dissociation (CID)-based tandem mass spectrometry (MS/MS) analysis of phosphorylated peptides in an ion trap is presented. Carboxypeptidase-B (CBP-B) was used to selectively remove C-terminal arginine or lysine residues from phosphorylated tryptic/Lys-C peptides prior to their MS/MS analysis by CID with a Paul-type ion trap. Removal of this basic C-terminal residue served to limit the extent of gas-phase neutral loss of phosphoric acid (H₃PO₄), favoring the formation of diagnostic b and y ions as determined by an increase in both the number and relative intensities of the sequence-specific product ions. Such differential fragmentation is particularly valuable when the H₃PO₄ elimination is so predominant that localizing the phosphorylation site on the peptide sequence is hindered. Improvement in the quality of tandem mass spectral data generated by CID upon CBP-B treatment resulted in greater confidence both in assignment of the phosphopeptide primary sequence and for pinpointing the site of phosphorylation. Higher Mascot ion scores were also generated, combined with lower expectation values and higher delta scores for improved confidence in site assignment; Ascore values also improved. These results are rationalized in accordance with the accepted mechanisms for the elimination of H₃PO₄ upon low energy CID and insights into the factors dictating the observed dissociation pathways are presented. We anticipate this approach will be of utility in the MS analysis of phosphorylated peptides, especially when alternative electron-driven fragmentation techniques are not available.

Molecular formula analysis of fragment ions by isotope‐selective collision‐induced dissociation tandem mass spectrometry of pharmacologically active compounds

The purpose of this work is to explore the mass fragment characterization of commonly used drugs through a novel approach, which involves isotope‐selective tandem mass spectrometry (MS/MS). Collision‐induced dissociation (CID) was performed with a low‐resolution linear ion trap mass spectrometer in positive electrospray ionization. Three pharmacologically active ingredients, i.e. omeprazole, meloxicam and brinzolamide, selected as model compounds in their own formulation, were investigated as a sodiated adduct [C₁₇H₁₉N₃O₃S + Na]⁺ (omeprazole) and as protonated adducts, [C₁₄H₁₃N₃O₄S₂ + H]⁺ and [C₁₂H₂₁N₃O₅S₃ + H]⁺, meloxicam and brinzolamide, respectively. Selecting a narrow window of ±0.5 m/z units, precursor ion fragmentation by CID‐MS/MS of isotopologues A + 0, A + 1 and A + 2 was found very useful to confirm the chemical formula of product ions, thus aiding the establishment of characteristic fragmentation pathways of all three examined compounds. The correctness of putative molecular formula of product ions was easily demonstrated by exploiting the isotope peak abundance ratios (i.e. I_F+0/I_F+1 and I_F+0/I_F+2) as simple constraints in low‐resolution MS instrumentations. Copyright © 2014 John Wiley & Sons, Ltd.     

Identification and fragmentation pathways of caffeine metabolites in urine samples via liquid chromatography with positive electrospray ionization coupled to a hybrid quadrupole linear ion trap (LTQ) and Fourier transform ion cyclotron resonance mass spectrometry and tandem mass spectrometry

Liquid chromatography (LC) with positive ion electrospray ionization (ESI+) coupled to a hybrid quadrupole linear ion trap (LTQ) and Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) was employed for the simultaneous determination of caffeine and its metabolites in human urine within a single chromatographic run. LC/ESI‐FTICRMS led to the unambiguous determination of the molecular masses of the studied compounds without interference from other biomolecules. A systematic and comprehensive study of the mass spectral behaviour of caffeine and its fourteen metabolites by tandem mass spectrometry (MS/MS) was performed, through in‐source ion trap collision‐induced dissociation (CID) of the protonated molecules, [M+H]⁺. A retro‐Diels‐Alder (RDA) process along with ring‐contraction reactions were the major fragmentation pathways observed during CID. The base peak of xanthine precursors originates from the loss of methyl isocyanate (CH₃NCO, 57 Da) or isocyanic acid (HNCO, 43 Da), which in turn lose a CO unit. Also uric acid derivatives shared a RDA rearrangement as a common fragmentation process and a successive loss of CO₂ or CO. The uracil derivatives showed a loss of a ketene unit (CH₂CO, 42 Da) from the protonated molecule along with the loss of H₂O or CO. To assess the potential of the present method three established metabolite ratios to measure P450 CYP1A2, N‐acetyltransferase and xanthine oxidase activities were evaluated by a number of identified metabolites from healthy human urine samples after caffeine intake. Copyright © 2009 John Wiley & Sons, Ltd.     

Gas‐phase fragmentation study of biotin reagents using electrospray ionization tandem mass spectrometry on a quadrupole orthogonal time‐of‐flight hybrid instrument

In this study, we evaluated, by electrospray ionization mass spectrometry (ESI‐MS) and collision‐induced dissociation tandem mass spectrometry (CID‐MS/MS) using a quadrupole orthogonal time‐of‐flight (QqToF)‐MS/MS hybrid instrument, the gas‐phase fragmentations of some commercially available biotinyl reagents. The biotin reagents used were: psoralen‐BPE 1, p‐diazobenzoyl biocytin (DBB) 2, photoreactive biotin 3, biotinyl‐hexaethyleneglycol dimer 4, and the sulfo‐SBED 5. The results showed that, during ESI‐MS and CID‐MS/MS analyses, the biotin reagents followed a similar gas‐phase fragmentation pattern and the cleavages usually occurred at either end of the spacer arm of the biotin reagents. In general we have observed that the CID‐MS/MS fragmentation routes of the five precursor protonated molecules obtained from the biotin linkers 1–5 afforded a series of product ions formed essentially by similar routes. The genesis and the structural identities of all the product ions obtained from the biotin linkers 1–5 have been assigned. All the exact mass assignments of the protonated molecules and the product ions were verified by conducting separate CID‐MS/MS analysis of the deuterium‐labelled precursor ions. Copyright © 2009 John Wiley & Sons, Ltd.     

Energy-dependent collision-induced dissociation study of buprenorphine and its synthetic precursors

The collision-induced dissociation (CID) of protonated buprenorphine ([M+H](+) ) and four related compounds was studied by electrospray quadrupole/time-of-flight mass spectrometry (ESI-QTOF MS). The fragmentation pathways were investigated by using energy-dependent CID and pseudo-MS(3) (in-source CID combined with tandem mass spectrometry (MS/MS)) methods. The first steps of the fragmentation are the parallel losses of the substituents from the non-aromatic ring moieties. Depending on the applied collision energies, a large number of further fragment ions arising from the cross-ring cleavages of the core-ring structure were observed. Based on the experimental results, a generalized fragmentation scheme was developed for the five buprenorphine derivatives highlighting the differences for the alternatively substituted compounds. The collision-energy-dependent fragmentation profile of buprenorphine is visualized in a two-dimensional plot to aid its fingerprint identification.     

A new and efficient route to amino derivatives of [1,6]- and [2,7]naphthyridones

Reaction of N-substituted amides of 2-chloro- or 4-chloronicotinic acid with CH-acidic nitriles in the presence of a base provides a convenient access to amino derivatives of 1,6-naphthyrid-5(6H)-one, compounds of type 4 and 5 , or 2,7-naphthyrid-1(2H)-one 7.     

Characterization of protostane triterpenoids in Alisma orientalis by ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry

A reliable and sensitive ultra‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry (UPLC/Q‐TOF‐MS) method has been optimized and established for analysis of protostane triterpenoids in a commonly used traditional Chinese herbal medicine Alisma orientalis (Sam.) Juzep. The separation of crude extract of A. orientalis was achieved on a Waters ACQUITY HSS T3 column (100 mm × 2.1 mm, 1.8 µm) eluting with 0.1% (v/v) formic acid/acetonitrile. A total of 20 protostane triterpenoids including 19 known compounds and a new one were well separated within 7 min. The collision‐induced dissociation (CID) tandem mass spectrometric (MS/MS) fragmentation patterns of protostane triterpenoids was firstly reported in this study. The hydrogen rearrangement at the C‐23‐OH leads to dissociation of the bond between C‐23 and C‐24 in the protostane triterpenoid skeleton during the CID process. This dissociation was the characteristic CID fragmentation pathway of this class of triterpenoids, and was useful for further differentiation of some positional isomers which contain an acetyl unit on the C‐23 or C‐24 position. The identities of isolated compounds were identified by comparing their retention times and CID fragmentation behaviors with those of reference standards or tentatively assigned by matching the empirical molecular formulae with those reported in the literature. It is concluded that this newly established UPLC/Q‐TOF‐MS method is a powerful approach for structural elucidation of protostane triterpenoids isolated from A. orientalis. Copyright © 2010 John Wiley & Sons, Ltd.     

A comparison of the peptide fragmentation obtained from a reflector matrix-assisted laser desorption-ionization time-of-flight and a tandem four sector mass spectrometer

The types, extent, and overall distribution of peptide fragmentation produced by matrix-assisted laser desorption-ionization-postsource decay (MALDI-PSD) on a reflector time-of-flight mass spectrometer were compared with those obtained from high and low energy collision-induced dissociation (CID) on a four-sector mass spectrometer and from liquid secondary ion mass spectrometry (LSIMS) ion source fragmentation and LSIMS metastable ion (MI) decomposition on a two-sector mass spectrometer. The model peptides studied had sequences and compositions that yielded predominantly either N- or C-terminal fragmentation from CID. For des-Arg¹ and des-Arg⁹ bradykinin (i.e., H-PPGFSPFR-OH and H-RP-PGFSPF-OH, respectively), the types of fragment ions and the extent to which each type is formed in both MALDI-PSD and low energy CID spectra are remarkably similar. This observation suggests that both methods deposit comparable internal energies (IE) into [M + H]⁺ precursor ions. The distribution of N-terminal, C-terminal, immonium, and internal fragmentation from MALDI-PSD spectra of des-Arg¹ and des-Arg⁹ bradykinin did not change dramatically with respect to the terminal arginine position, contrary to those from LSIMS MI decomposition, high and low energy CID spectra. This observation in combination with the prominent immonium, internal, and minus 17 fragment ion types in PSD indicates that the imparted IE from MALDI and the 14 µs of flight time may promote steady-state decomposition kinetics. Fragmentation distributions of MALDI-PSD spectra are also similar to those in LSIMS spectra. This implies that the distribution of protonation sites in [M + H]⁺ is comparable for both techniques.     

An isomer-specific high-energy collision-induced dissociation MS/MS database for forensic applications: a proof-of-concept on chemical warfare agent markers

Spectra database search has become the most popular technique for the identification of unknown chemicals, minimizing the need for authentic reference chemicals. In the present study, an isomer‐specific high‐energy collision‐induced dissociation (CID) MS/MS spectra database of 12 isomeric O‐hexyl methylphosphonic acids (degradation markers of nerve agents) was created. Phosphonate anions were produced by the electrospray ionization of phosphonic acids or negative‐ion chemical ionization of their fluorinated derivatives and were analysed in a hybrid magnetic‐sector–time‐of‐flight tandem mass spectrometer. A centre‐of‐mass energy (E_com) of 65 eV led to an optimal sequential carbon–carbon bond breakage, which was interpreted in terms of charge remote fragmentation. The proposed mechanism is discussed in comparison with the routinely used low‐energy CID MS/MS. Even‐mass (odd‐electron) charge remote fragmentation ion series were diagnostic of the O‐alkyl chain structure and can be used to interpret unknown spectra. Together with the odd‐mass ion series, they formed highly reproducible, isomer‐specific spectra that gave significantly higher database matches and probability factors (by 1.5 times) than did the EI MS spectra of the trimethylsilyl derivatives of the same isomers. In addition, ionization by negative‐ion chemical ionization and electrospray ionization resulted in similar spectra, which further highlights the general potential of the high‐energy CID MS/MS technique. Copyright © 2011 John Wiley & Sons, Ltd.     

Mass spectrometry of 2-substituted-1-keto-3-aryl-5H-imidazo-(1,5-a)-pyrroles derived from Schiff bases of N-terminal prolyl peptides

We have been able to extend the use of Schiff base derivatives in peptide sequencing to N-terminal prolyl peptides. Earlier studies from this laboratory revealed that certain aromatic Schiff bases of peptide esters gave electron-impact mass spectra with relatively intense molecular, sequence and internal fragment ions. We observed that the reaction of N-terminal prolyl peptide esters with 4-dimethylaminonaphthaldehyde, p-dimethylaminobenzaldehyde and 2-pyridinecarboxaldehyde gave cyclization products which were found to be 2-substituted-1-keto-3-aryl-5H-imidazo-[1,5-a]-pyrrole derivatives. The molecular ion and many of the expected cleavages were prominent in the mass spectra. Deuterium labeling at the α-carbon, amide nitrogen, or other exchangeable positions has been used in assigning the structure. It was also confirmed by the fragmentation pattern of the products derived by permethylation of the peptide derivative with tetramethylammonium hydroxide. Comparable cleavage patterns were seen among the N-terminal prolyl peptides examined. Proline amide gave the corresponding cyclized product. With the inclusion of N-terminal prolyl peptides in the list of peptides that we have examined, we may now prepare volatile derivatives of peptides containing any of the protein amino acids in two steps: esterification and treatment with the appropriate aromatic aldehyde.     

Dibutylphosphate (DBP) mediated synthesis of cyclic N,N′-disubstituted urea derivatives from amino esters: a comparative study

The N,N′-disubstituted urea derivatives such as amino acid hydantoins and dihydrouracil derivatives were prepared starting from natural and unnatural amino acid esters using dibutylphosphate (DBP). During the attempted synthesis of N-heterocycles with larger than six-membered rings containing the N,N′-disubstituted urea functionalities, three unexpected products namely squamolone, N-methyl pyrrolidine-2-one, and diketopiperazine were isolated.     

Structural studies of cyclic ureas: 1. Enthalpies of formation of imidazolidin-2-one and N,N′-trimethyleneurea

A thermophysical and thermochemical study has been carried out for crystalline imidazolidin-2-one and N,N′-trimethyleneurea [tetrahydropyrimidin-2(1H)-one]. The thermophysical study was made by differential scanning calorimetry, d.s.c., in the temperature intervals between T = 268 K and their respective melting temperatures. Several solid–solid transitions have been detected in imidazolidin-2-one. The standard (p^° = 0.1 MPa) molar enthalpies of formation, at T = 298.15 K, for crystalline imidazolidin-2-one and N,N′-trimethyleneurea [tetrahydropyrimidin-2(1H)-one], were determined using static-bomb combustion calorimetry. The standard molar enthalpies of sublimation, at T = 298.15 K, for the two compounds were derived from the variation of their vapour pressures, measured by the Knudsen effusion method, with the temperature. These two thermochemical parameters yielded the standard molar enthalpies of formation of the two cyclic urea compounds studied in the gaseous phase at T = 298.15 K. These values are discussed in terms of molecular structural contributions and interpreted on the bases of the “benzo-condensed effect” and of the ring strain of imidazolidin-2-one.     

Synthesis of piperidine analogs of 1-(3-chlorophenyl)piperazine,a well known serotonin ligand

Synthesis of arylpiperideines 3a-3d and arylpiperidines 4a-4d as analogs of a well known serotonin ligand 1-(3-chlorophenyl)piperazine is reported. Starting aryllithium derivatives were treated with 1-methyl-piperdin-4-one to provide the corresponding hydroxy derivatives 6a and 6b. N-Methylpiperideine derivatives 3a and 3c were obtained by their dehydration while the corresponding N-unsubstituted compounds 3b and 3d were prepared indirectly by a three-step procedure. Hydrogenation of piperideine 3a provided the corresponding piperidine derivative 4a which after demethylation yielded 4b. Similar 4-pyridyl derivatives 4c and 4d were prepared by a similar strategy via the corresponding methoxy derivatives.