Synthesis of [1-15N,2-13C]-labeled difloxacin

Abstract  

The synthesis of [1-¹⁵N,2-¹³C]-difloxacin, an arylfluoroquinolone antibacterial agent, is reported. As a crucial initial step, the starting materials ethyl 2,4,5-trifluorobenzoylacetate, [formyl-¹³C]-triethyl orthoformate, and [¹⁵N]-4-fluoroaniline were reacted to ethyl [¹⁵N,3-¹³C]-3-(4-fluoroanilino)-2-(2,4,5-trifluorobenzoyl)acrylate. After cyclization and ester cleavage, the resulting intermediate was reacted with 1-methylpiperazine to [1-¹⁵N,2-¹³C]-1-(4-fluorophenyl)-6-fluoro-7-(4-methyl-1-piperazinyl)-1,4-dihydro-4-oxoquinoline-3-carboxylate, i.e., [1-¹⁵N,2-¹³C]-difloxacin. The overall yield was 62% based on the non-labeled and 43% based on the labeled starting materials (both used in 1.4 molar excess). The product was identified by ¹H-, ¹³C-, and ¹⁵N-NMR spectroscopy and by cochromatography (TLC, HPLC) with an authentic reference; its purity (HPLC) was above 98%. Prior to synthesis of [1-¹⁵N,2-¹³C]-difloxacin, non-labeled difloxacin was synthesized in order to optimize procedures and to identify and characterize all intermediates.     

Biosynthesis of kinamycin D. incorporation of [1,2-13C] acetate and of [2-2H3,1-13C]acetate

Results from the incorporation of [1,2-¹³C₂]- and [2-²H₃,1-¹³C]acetates into kinamycin D. indicate the involvement of only unsymmetrical intermediates; the identity of some of these are suggested.     

Biosynthesis of psilotin from [2′,3′-13C2,1′-14C,4-3H]phenylalanine studied with 13C-NMR

The 6-phenyl-dihydro-α-pyrone moiety of psilotin is formed from [2′,3′-¹³C₂,1′-¹⁴C,-4-³H]phenylalanine in the plant $\text{Psilotum}$$\text{nudum}$ with retention of all the isotopes.     

13C13C spin spin coupling constants in azulenes

[4-¹³C]- Azulene and [4-¹³C]-4-methylazulene have been synthesized. The ¹³C¹³C spin coupling constants have been measured and interpreted in terms of πMO theory.     

Biosynthesis of the antibiotic verrucarin E use of [1-13C]-, [2-13C]-, [1,2-13C]- and [2-13C, 2-2H3]-acetates. Verrucarins and roridins, 37th communication [1]

The origin of the carbon skeleton of verrucarin E (1) from acetate as precursor is confirmed. Incorporation studies with [1,2-¹³C]-acetate have demonstrated that two acetoacetate units couple together as shown in pattern A (Scheme 2) and not as in B . Analysis of the deuterium distribution in both verrucarin E (1) isolated after the incorporation of [2-¹³C,2-²H₃]-acetate and in sodium acetate obtained after Kuhn-Roth oxidation of the metabolite demonstrated that C(7) is derived from the starter unit of one of the acetoacetate moieties. The deuterium exchange in verrucarin E (1) occurring during fermentation was investigated.     

Photolyse von 2-oxo-[2-13c]-1-diazocyclohexan. Ein beitrag zum oxiren-problem

The synthesis of 2 - oxo - [2 - ¹³C] -1 - diazocyclohexan (10) starting with K¹³CN is described. Photolysis of 10 in dioxan-water yields cyclopentancarboxylic acid containing all the label in the carboxy group, which has been proved by C-13 NMR spectroscopy and electron impact induced fragmentation. The absence of isotope scrambling in the photolytic ring contraction excludes an oxiren participation.     

Studies on the biosynthesis of terrecyclic acid A II confirmation of the cyclization mechanism and hydrogen shifts using [2-2H3]acetate and [213C2H3]acetate

Feeding experiments with [2-³H₃]acetate and [2-¹³C²H₃]acetate in $\text{Aspergillus terreus}$ Thom No. 14 indicated that the hydrogen at C-2 in terrecyclic acid A is incorporated without migration from the precursor acetic acid; the results favour our group's earlier speculation for the cyclization to the tricyclic skeleton in the biosynthetic scheme.     

The temperature dependence of the isotope effects in the polymerization of [α-14C]styrene and [β-14C]styrene

Styrene has been copolymerized with [α-¹⁴C]styrene under radical conditions and with [β-¹⁴C]styrene under both radical and cationic conditions; radical copolymerizations were studied over the temperature range 40–110° and cationic copolymerizations over the range 30–90°. By relating the specific activities of the comonomer feed and of the copolymer, reactivity ratios were found. Arrhenius parameters have been determined for the isotope effects for the propagation steps in each copolymerization. They show that, with [α-¹⁴C]styrene, the difference in the energies of activation predominate in determining the magnitude of the isotope effect; in the case of [β-¹⁴C]styrene, the ratio of the pre-exponential factors is more important under both radical and cationic conditions.     

Synthesis of dl-[2-13C]leucine and it use in the preparation of [3-dl-[2-13C]leucine]oxytocin and [8-dl-[2-13C]leucine]oxytocin: Preparative separation of diastereoisomeric peptides by partition chromatography and high pressure liquid chromatography

dl-[2-¹³C]Leucine was prepared by condensing the sodium salt of ethyl acetamido-[2-¹³C]cyanoacetate with isobutylbromide in hexamethylphosphoroustriamide followed by acid hydrolysis. N-Boc-dl-[2-¹³C]Leucine was prepared and incorporated into [8-dl-[2-¹³C]leucine]oxytocin by total synthesis. The ¹³C-labeled hormone derivative [8-[2-¹³C]leucine]oxytocin was separated from its 8-position diastereoisomer by partition chromatography. The specifically ¹³C-labeled peptide hormone diastereoisomeric analog [3-dl-[2-¹³C]leucine]oxytocin also was prepared by solid phase peptide synthesis. No suitable solvent system for partition chromatography separation of the latter diastereoisomeric peptide mixture could be found. However an excellent preparative separation of the diastereoisomers could be obtained by reverse phase high pressure liquid chromatography on a partisil 10 M9 ODS column using the solvent system 0.05 M ammonium acetate (pH 4.0), acetonitrile (81:19, $\text{v}\text{v}$) to give pure [3-(2-¹³C]leucine]oxytocin and [3-D-(2-¹³C]leucine]oxytocin. An excellent separation of [8[2-¹³C]leucine]oxytocin and the corresponding 8-D-leucine diastereoisomer derivative could also be accomplished by high pressure liquid chromatography.     

Deuterium-induced 13C NMR isotope shifts for signal assignments and determination of deuteriation site in cyclododecanone

Deuterium-induced ¹³C isotope shifts were measured in the ¹³C {¹H} NMR spectra of monodeuteriated cyclododecanones formed upon oxidation of [1-²H₁] cyclododecanol with lead tetraacetate. These shifts, when used in conjunction with ¹³C NMR data of specifically labelled analogues, [2, 2, 12, 12-²H₄]- and [2-²H₁] cyclododecanone, enabled the ketone formed with lead tetraacetate to be identified as a mixture of [5-²H₁]- and [6-²H₁] isotopomers.     

Application of biosynthetic 13C-enrichment using [1-13C]-, [2-13C]- and [1,2-13C2]-acetate as precursor for 13C NMR spectral assignment of higher plant metabolites. The assignments of some bryonolic acid derivatives

The ¹³C NMR spectra of some derivatives of bryonolic acid (1) (D:C-friedoolean-8-en-3β-ol-29-oic acid) were assigned by means of ¹³C-enrichment, lanthanide-induced shifts (LIS) and comparison of chemical shift data between derivatives. The ¹³C-enriched species of 1, i.e., 1a, 1b and 1c were biosynthesized by Luffa cylindrica (Cucurbitaceae) callus fed with [1-¹³C]-, [2-¹³C]- or [1,2-¹³C₂]-acetate, respectively. Methyl acetylbryonolates 2, 2a, 2b and 2c, methyl bryonolates 3, 3a, 3b and 3c, methyl bryononates 4 and 4a, diacetyl-3β,29-diols (3,29-diacetyl-D:C-friedoolean-8-en-β,29-diol) 5, 5a, 5b and 5c, and 3-acetyl-3β,29-diols 6, 6a and 6b were prepared from 1, 1a, 1b and 1c, and their ¹³C NMR spectra were recorded. The ¹³C concentration of the ¹³C-enriched species was high enough to exhibit the satellite peaks clearly, and the analysed data were very useful for this study. Thus, total assignments for 2, 3, 4, 5 and 6 were established. It was found that conversion of the methoxycarbonyl group at C-29 into an acetoxymethyl group caused complex changes in the chemical shifts of the C, D- and E-ring carbons and those of the methyl carbons linked to these rings.     

Substituent effects in 13C NMR spectra of 6-endo substituted 9-thiabicyclo[3.3.1]non-2-enes

A number of new 6-endo-X-9-thiabicyclo[3.3.1]non-2-enes have been synthesized (X = cyano, carbimino-methoxy, carbomethoxy, carboxylic acid, aminomethyl, tosylmethyl and methyl). The ¹³C NMR spectre of six of these compounds were measured along with those of several known analogues with X = chloro, hydroxy, hydrogen and deuterium. Assignments were carried out with the aid of hetero (¹³C-¹H) and homo (¹H-¹H) nuclear decoupling techniques. For the compounds with X = H and X = Cl our assignments differ from those published previously and hence result in different values of the substituent-induced chemical shifts (SIS-values) in the 9-thiabicyclo[3.3.1] non-2-ene skeleton.Some of the existing theories regarding SIS-values in multiple substituted cyclic compounds are reviewed and reconsidered in view of the new evidence presented here. Mutual repolarization of the C-hetero atom bonds is proposed as an important factor. In the systems under study in this paper an electron donation of the sulfur atom to the endo C₆-X group is postulated in order to explain the SIS-values on saturated C atoms. The signals of the unsaturated carbons are assigned assuming that electric field effects play a major role.     

Zum Mechanismus der α-Alkinon-Cyclisierung: Synthese und Thermolyse von 1-(1-Methylcyclopentyl)[3-13C]prop-2-inon

On the Mechanism of the α-Alkynone Cyclization: Synthesis and Thermolysis of 1-(1-Methylcyclopentyl)[3-¹³C]prop-2-ynone The relative migratory aptitude of two acetylenic substituents in the α-alkynone cyclization, a thermal conversion of α-acetylenic ketones A to 2-cyclopentenones C , was investigated by isotope-labeling experiments. The α-alkynone [β-¹³C]- 1 , specifically labeled with ¹³C at the β-acetylenic C-atom C(3), was synthesized by an intramolecular Witting reaction (230–300°) of the diacylmethylidenephosphorane [¹³C]- 7. The latter resulted from acylation of methylidenetriphenylphosphorane with the acid chloride 4 to yield the acylmethylidenephosphorane 5 , which in turn was formylated with acetic [¹³C]formic anhydride ([¹³C]- 6. ) Upon thermolysis of [β-¹³C]- 1 , its label at C(β) was transferred almost exclusively to C(β) of the 2-cyclopentenone moiety in the resulting cyclization product [¹³C]- 2. We conclude that there is a distinct preference for hydrogen migration in the acetylene → alkylidene carbene isomerization (A → B) which precedes the cyclization step (B → C). No evidence was found for a fast reversibility of this isomerization (A ? B) involving both acetylenic substituents.     

An Efficient Synthesis of Optically Active [4-13C] Labelled Quorum Sensing Signal Autoinducer-2

A new synthetic route for the quorum sensing signal Autoinducer-2 (AI-2) is described and used for the preparation of [4-¹³C]-AI-2 starting from [1-¹³C]-bromoacetic acid. The key step in this process was the enantioselective reduction of an intermediate ketone. This synthesis provides, selectively, both enantiomers of the labelled or unlabelled parent compound, (R) or (S)-4,5-dihydroxypentane-2,3-dione (DPD) and was used for an improved synthesis of [1-¹³C]-AI-2.     

Assessment of labelled products with different radioanalytical methods: study on 18F-fluorination reaction of 4-[18F]fluoro-N-[2-[1-(2-methoxyphenyl)-1-piperazinyl]ethyl-N-2-pyridinyl-benzamide (p-[18F]MPPF)

The serotonin receptor 5-HT_1A ligand 4-[¹⁸F]fluoro-N-[2-[1-(2-methoxyphenyl)-1-piperazinyl]ethyl-N-2-pyridinyl-benzamide (p-[¹⁸F]MPPF) was produced by a simplified method of Le Bars et al. Traditional oil bath heating was compared to microwave heating. Various radioanalytical methods, radio-Thin Layer Chromatography (TLC), High Pressure Liquid Chromatography (HPLC) and Mass Spectrometry (MS), were compared in the evaluation of the labelled product(s). The crude reaction mixture consisted of p-[¹⁸F]MPPF and 2–4 radioactive by-products eluting after the product fraction, and the reverse-phase HPLC method failed occasionally to separate p-[¹⁸F]MPPF from the radioactive by-product with close retention time. The heating method had no significant effect on the composition of labelled by-products. In LC-(ESI)-MS analysis of p-[¹⁸F]MPPF the labelled product was identified with m/z ratio of 435 ([M + H⁺]). The other HPLC fractions were measured to have following m/z ratios: (1) 327; 349; (675) (2) 402; 407/408; (791) and (3) 436, suggesting different kind of decomposition of the labelled product and/or the inactive precursor. The ion trap mass spectrometer was sufficient for the qualitative analysis of p-[¹⁸F]MPPF. However, differentiation of by-products arising from the decomposition of p-[¹⁸F]MPPF or from its precursor p-MPPNO₂ proved to be challenging.     

13C-NMR-spektren stereoisomerer 1,4-dialkylspiro[4.5]decan-derivate

The ¹³C NMR spectra of a series of 7-substituted 1,4-dialkylspiro[4.5]decanes and of suitable reference compounds are assigned and the derived substituent effects upon the chemical shifts are discussed. In particular, consideration of γ and δ-effects allows the differentiation between 1,4-cis- and 1,4-trans-compounds. Furthermore, the (relative) configuration of the chiral spiroatom C-5 is determined.     

Microwave spectra of tetrahydroselenophene-α13C, -β13C and molecular ring structure

Microwave spectra of isotopic species α-¹³C and β-¹³C of tetrahydroselenophene molecules have been investigated and rotational constants determined: A = 5608.98 Mc, B = 2819.532 Mc, C = 2022.624 Mc forα-¹³C isotopic species and A = 5695.94 Mc, B = 2770.714 Mc, C = 2009.166 Mc for β-¹³C isotopic species. The r_s-ring structure was found to be Se-C₂ = 1.963 Å, C₂-C₃ = 1.549 Å, C₃-C₄ = 1.527 Å, ∠C₅SeC₂ = 90° 44', ∠SeC₂C₃ = 104° 58', ∠C₂C₃C₄ = 106° 52', the angle of twist = 29° 44'.     

Craquage thermique de l'aniline 1-14C

Thermal cracking of aniline 1-¹⁴C at atmospheric pressure at 900°C and a contact time of 2.5 sec breakdown mechanisms of the formation of benzene with an elimination of nitrogen, and formation of heterocyclic compounds and condensation products has been studied. Most of the degradation compounds are produced by a mechanism similar to the primary mechanism for the degradation of phenol: elimination of HCN with formation of inactive C₅H₆. The formation of nitriles is not negligible.     

13C NMR spectroscopy of some 3- and 7-substituted bicyclo[3.3.1]nonanes

The ¹³C NMR spectra of a series of 3,7-substituted bicyclo[3.3.1]nonane derivatives are examined. Taken into account substituent influences, the ¹³C chemical shifts appear to be diagnostic for the conformation of the bicyclo[3.3.1]nonane derivative.     

Biosynthesis of the cytochalasans. Part III. C-NMR. of cytochalasin B (phomin) and cytochalasin D. Incorporation of [1-13C]- and [2-13C]-sodium acetate

Sequential single frequency decoupling and partially relaxed Fourier transform (PRFT) are used to assign the natural abundance ¹³C-NMR. spectra of cytochalasin B (phomin) ( 1 ) and cytochalasin D ( 2 ). Cultures of Phoma spec. S 298 were fed [2-¹³C]-sodium acetate, and the distribution of this precursor in cytochalasin B (phomin) ( 1 ) was determined by ¹³C-NMR. spectroscopy. Likewise, the labelling patterns in cytochalasin D (zygosporin A) ( 2 ) from Zygos-posium masonii could be identified after incorporation of [2-¹³C]-sodium acetate and [l-¹³C]-sodium acetate. The results confirm previous proposals for the biogenesis of the cytochalasans from phenylalanine, methionine, and a C₁₈, or C₁₆, polyketide part.