1H−15N HMBC NMR as a tool for rapid identification of isomeric azaindoles: The case of 5F‐MDMB‐P7AICA

The high frequency of the synthetic cannabinoid receptor agonists (SCRAs) emergence renders this group of new psychoactive compounds particularly demanding in terms of detection, identification, and responding. Without the available reference material, one of the specific problems is differentiation and structure elucidation of constitutional isomers. Herein, we report a simple and efficient flow chart diagram applicable for a rapid nuclear magnetic resonance (NMR) identification and differentiation between azaindoles, 4‐, 5‐, 6‐, and 7‐azaindole, which is a common structural motif of synthetic cannabinoids. The flow chart diagram is based on ¹H NMR and ¹H–¹⁵N NMR spectra, and to prove the concept, it has been tested on 5F‐MDMB‐P7AICA ( 1 ). Spectral and analytical data including standard 1D and 2D NMR spectra, gas chromatography?mass spectrometry (GC?MS), Fourier transform infrared?attenuated total reflectant (FTIR?ATR), Raman, melting point, and combustion analysis are provided for compound 1 .     

Fast and precise quantitative analysis of metabolic mixtures by 2D H INADEQUATE NMR

Quantitative analysis of metabolic mixtures by ¹H 1D NMR offers a limited potential for precise quantification of biomarkers, due to strong overlap between the peaks. Two-dimensional spectroscopy is a powerful tool to unambiguously and simultaneously measure a larger number of metabolite contributions. However, it is still rarely used for quantification, first because quantitative analysis by 2D NMR requires a calibration procedure due to the multi-impulsional nature of 2D NMR experiments, and above all because of the prohibitive experiment duration that is necessary to obtain such a calibration curve. In this work, we develop and evaluate a 2D ¹H INADEQUATE protocol for a fast determination of metabolite concentrations in complex mixtures. The 2D pulse sequence is carefully optimized and evaluated in terms of precision and linearity. Quantitative ¹H INADEQUATE 2D spectra of metabolic mixtures are obtained in 7 min with a repeatability better than 2% for metabolite concentrations as small as 100 μM and an excellent linearity. The method described in this work allows a fast and precise quantification of metabolic mixtures, and it forms a promising tool for metabonomic studies.     

Solution conformation of Leu27 hGRF(1-32)NH2 and its deamidation products by 2D NMR

The solution structure and helical content of a human growth hormone releasing factor analog, Leu²⁷ hGRF(1–32)NH₂ (hGRF), and its deamidation products Asp⁸ Leu²⁷ hGRF(l-32)NH₂ and isoAsp⁸ Leu²⁷ hGRF(1-32)NH₂, were determined by CD and 2D NMR. Chemical-shift assignments of ¹H NMR resonances were made from DQFCOSY, HOHAHA and NOESY spectra, and qualitative secondary structure was determined from NOESY spectra. 2D NMR studies in aqueous MeOH showed the Asn⁸, Asp⁸ and isoAsp⁸ hGRF analogs to have significant α-helical character. However, the β-linked isoAsp⁸ analog did not retain helical structure in the N-terminal region, most likely because of disruption of the hydrogen bonding pattern upon substitution of the extra methylene into the peptide backbone. The helical content, as determined by CD, was ~ 12% in 0% MeOH for all three peptides, and 77, 72 and 69% in 80% MeOH for the Asn⁸, Asp⁸ and isoAsp⁸ hGRF analogs, respectively. However, 2D NMR solution structure data indicated a decrease in helicity in the N-terminal region for the isoAsp⁸ analog when compared with the other two analogs. In the Asn⁸ and Asp⁸ hGRF analogs, the helix began at Asp³ or Ala⁴, while the isoAsp⁸ analog helix was disrupted until Arg. The higher helicity value for the Asn⁸ peptide over the isoAsp⁸ analog may be associated with reported biological activity, where the in vitro activity decreased from 100 to 4 and < 1% for Asn⁸, Asp⁸ and isoAsp⁸ hGRF, respectively.     

A new antibacterial compound from Luo Han Kuo fruit extract (Siraitia grosvenori)

Luo Han Kuo fruit (Siraitia grosvenori Swingle) has been used in China for centuries as a sweetening agent, and also used to treat sore throat and cough. In our recent study, a new bioactive compound, (2R,3S,4S)-2,3-trans-3,4-cis-5,3′-bimethoxy-7-(trans-2-propenal)-3,4-flavandiol (1), named siraitiflavandiol was obtained. The structure has been determined on the basis of spectroscopic studies including 1D and 2D NMR (¹H, ¹³C NMR, ¹H–¹H COSY, HSQC, HMBC, and NOESY), CD, EI-MS, and HR-EI-MS spectra. The new compound was evaluated in vitro for its inhibitory ability against the growth of oral bacterial species Streptococcus mutans, Porphyromonas gingivalis, and yeast Candida albicans. The minimum inhibitory concentrations were 6, 24, and 6 μg/ml, respectively.     

Seimisochromenes A and B: two new dihydroisochromenes from the endophytic fungus,Seimatosporium sp.

Two new dihydroisochromenes, named seimisochromenes A and B (1 and 2), were isolated from an endophytic fungus, Seimatosporium sp. The structures of seimisochromenes A and B have been determined from 1D (¹H and ¹³C NMR spectra) and 2D (COSY, HMQC, HMBC, and NOESY) NMR experiments.     

Solution structure of the cytoplasmic domain of the human immunodeficiency virus type 1 encoded virus protein U (Vpu)

The HIV-1-specific Vpu protein is an 81 amino acid class I integral membrane phosphoprotein that induces degradation of the virus receptor CD4 in the endoplasmic reticulum and enhances the release of virus particles from infected cells. Vpu is of amphipathic nature and consists of a hydrophobic N-terminal membrane anchor proximal to a polar C-terminal cytoplasmic domain. In our recent work, focussed on the structural analysis of the cytoplasmic tail, we established an α-helix-flexible-α-helix-turn model. Now we present the experimental solution structure of the Vpu cytoplasmic domain which has been elucidated in aqueous 50% trifluoroethanol solution by 2D ¹H NMR spectroscopy, and restrained molecular dynamics and energy minimization calculations. Under these conditions the peptide, Vpu^32-81, is predominantly monomeric and adopts a well defined helix-interconnection-helix-turn conformation, in which the four regions are bounded by residues 37-51, 52-56, 57-72 and 73-78. The presence of the cis isomer of Pro-75 manifests itself as a doubling of cross peaks of neighbouring residues in the 2D spectra. A related variant peptide, Vpum^32-81, in which the Vpu-phosphoacceptor sites Ser⁵² and Ser⁵⁶ were exchanged for Asn, adopts a very similar structure and, taken together, provides evidence that the second helix and the turn form a comparatively rigid region. Both helices are amphipathic in character, but show different charge distributions. In general the cytoplasmic region is N-terminally positively charged, passes through a region of alternating charges in helix 1 and then becomes negatively charged. The flexibility of the interconnection permits orientational freedom of the two helices. The motif found here is the first experimentally refined solution structure of the cytoplasmic domain of Vpu, and it is conceivable that these α-helices are important for a previously defined physical interaction with an α-helical Vpu-responsive element located within the cytoplasmic tail of CD4. © Munksgaard 1996.     

Establishing the carbon skeleton of pharmaceutical agents using HSQC-ADEQUATE spectra

Two-dimensional NMR methods are the cornerstone of modern structure elucidation methods. When the ensemble of 1D and 2D NMR experiments normally employed for structure assignment fails, investigators typically resort to successively more complex 2D NMR experiments for structure determination and/or spectral assignment. Unsymmetrical Indirect Covariance (UIC) NMR data processing methods provide a convenient and highly efficient means of accessing the connectivity information embodied in more complex experiments such as HSQC-TOCSY spectra. Using Unsymmetrical Indirect Covariance (UIC) or General Indirect Covariance (GIC) processing to mathematically combine multiplicity-edited GHSQC and 1,1-ADEQUATE 2D NMR spectra affords an HSQC-ADEQUATE spectrum that offers a new method for establishing the carbon skeleton of a molecule. The application of this technique is demonstrated for a novel cyclin-dependant kinase inhibitor, Dinaciclib™ (SCH 727965).     

Combined use of NMR,distance geometry and MD calculations for the conformational analysis of opioid peptides of the type [D(L)-Cys2, D(L)-Cys5]enkephalin

The solution structures of a series of conformationally restricted pentapeptides with a sequence H-Tyr¹-Cys²-Gly³ Phe⁴-Cys⁵-OH cyclic (2-5) disulfide, where the cysteines possess either the D or L configuration, were examined by a combined approach including NMR measurements as well as MD calculations. It turned out that at least one low energy conformer of H-Tyr¹-Cys²-Gly³-Phe⁴-Cys⁵-OH cyclic (2-5) disulfide (DCDCE), as well as one conformer out of the group of calculated conformers for H-Tyr¹-D-Cys²-Gly³-Phe⁴-Cys⁵-OH cyclic (2-5) disulfide (DCLCE), satisfies the NMR data obtained in this study, whereas for the derivative H-Tyr^l-Cys²-Gly³-Phe⁴-Cys⁵-OH cyclic (2-5) disulfide, which contains solely L-Cys (LCLCE), there is no single structure compatible with the NMR data. © Munksgaard 1996.     

The Structure of Celiprolol

The crystal structure and nuclear magnetic resonance (NMR) spectra and assignments of celiprolol, N-[3-acetyl-4[3-[N-t-butylamino-2-hydroxypropoxy]phenyl]-N, N-diethylurea, are reported. Celiprolol crystallizes in the monoclinic space group, P2_l/a, with a = 9.081(2), b = 13.800(4), and c = 17.471(5) Å and = 95.04(2)°. Structure was solved by direct methods; structure refinement to R of 0.058. Intermolecular hydrogen-bonding in the crystal is discussed. The ¹H, ¹³C, and two-dimensional (2D) NMR spectra of the hydrochloride have been obtained and definitive signal assignments made.     

Two new dilactonized glycerol glycosides of the dual anticholinesterase active extract from Ocotea daphnifolia using bioguided fractionation and molecular docking studies

The dual inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) is considered as an important strategy for the treatment of Alzheimer's disease. In this study, we applied the bioguided fractionations of Ocotea daphinifolia ethyl acetate active extract to furnish a fraction with high inhibitory activity for AChE and BuChE (82% and 92%, respectively). High-performance liquid chromatography semipreparative purification of this fraction provided two new natural products: 1-β-D-galactopyranosyl-glycerol-2,3-heptanedionate, ( 1 ) whose complete chemical structural elucidation was made with spectrometric analysis (MS, 1D, and 2D NMR) and its minor derivative 1-β-D-gulopyranosyl-glycerol-2,3-heptanedionate; ( 2 ) which could be characterized by 2D ¹H-¹³C heteronuclear single-quantum correlation spectra analysis. Investigation of the intermolecular interactions with cholinesterases was carried out by molecular docking studies, and results suggested that both compounds are capable to interact with the catalytic site of both enzymes. Compounds 1 and 2 interact with residues of catalytic domains and the peripheral anionic binding site of AChE and BuChE. The results are comparable to those achieved with rivastigmine and galantamine. Thus, this study provides evidence for consideration of the glycosylglycerol from O. daphnifolia as new valuable dual cholinesterases inhibitor.     

A novel indole alkaloid from deep-sea sediment metagenomic clone-derived Escherichia coli fermentation broth

To explore secondary metabolites in deep-sea sediment metagenomic clone-derived Escherichia coli fermentation broth, different kinds of chromatography methods were used in the isolation procedures, while the structures of the isolated compounds were assigned based on the MS analysis and their ¹H and ¹³C NMR spectra including 2D NMR techniques such as COSY, HMQC, and HMBC experiments. As a result, a novel compound was isolated and characterized as N-{1-[4-(acetylamino)phenyl]-3-hydroxy-1-(1H-indol-3-yl)propan-2-yl}-2,2-dichloroacetamide (1). In addition, eight known compounds were also obtained. Fatty acid amide hydrolase and monoacylglycerol lipase were used to screen analgesic activity, and the new compound showed analgesic activity to some extent in pharmacological test.     

Structure elucidation of the designer drug N‐(1‐amino‐3,3‐dimethyl‐1‐oxobutan‐2‐yl)‐1‐(5‐fluoropentyl)‐3‐(4‐fluorophenyl)‐pyrazole‐5‐carboxamide and the relevance of predicted 13C NMR shifts – a case study

The detailed structure elucidation process of the new cannabimimetic designer drug, N‐(1‐amino‐3,3‐dimethyl‐1‐oxobutan‐2‐yl)‐1‐(5‐fluoropentyl)‐3‐(4‐fluorophenyl)‐pyrazole‐5‐carboxamide, with a highly substituted pyrazole skeleton, using nuclear magnetic resonance (NMR) spectroscopic and mass spectrometric (MS) techniques is described. After a first analysis of the NMR spectra and comparison with 48 possible pyrazole and imidazole structures, a subset of six positional isomeric pyrazoles and six imidazoles remained conceivable. Four substituents of the heterocyclic skeleton were identified: a proton bound to a pyrazole ring carbon atom; a 5‐fluoropentyl group; a 4‐fluorophenyl substituent; and a carbamoyl group, which is N‐substituted with a methyl residue carrying a tert.‐butyl and a carbamoyl substituent. The 5‐fluoropentyl residue is situated at the nitrogen ring atom. Additional NMR experiments like the ¹H,¹³C HMBC were performed, but due to the small number of signals based on long‐range couplings, the comparison of predicted and observed ¹³C chemical shifts became necessary. The open access Internet shift prediction programs NMRDB, NMRSHIFTDB2, and CSEARCH were employed for the prediction of ¹³C shift values which allowed an efficient and unambiguous structure determination. For the identified N‐(1‐amino‐3,3‐dimethyl‐1‐oxobutan‐2‐yl)‐1‐(5‐fluoropentyl)‐3‐(4‐fluorophenyl)‐pyrazole‐5‐carboxamide, the best agreement between predicted ¹³C shifts and the observed chemical shifts and long‐range couplings for the pyrazole ring carbon atoms, with a standard error of about 2 ppm, was found with each of the predictions. For the comparison of measured and predicted chemical shifts model compounds with simple substituents proved helpful. The identified compound is a homologue of AZ‐037 which is offered by Internet suppliers. Copyright © 2015 John Wiley & Sons, Ltd.     

Role of tryptophan-14 in the interaction of dynorphin A(1-17) with micelles

Fluorescence spectroscopy has been used to examine the interaction between the opioid peptide dynorphin A(1-17) (dynorphin) and dodecylphosphocholine (DPC) micelles. Fluorescence emission spectra as a function of added lipid indicate insertion of the Trp¹⁴ side chain into the hydrophobic portion of the micelle, supporting NMR results from this laboratory. A model of interaction with micelles consistent with the fluorescence results and earlier NMR results is proposed. The critical micelle concentration in the presence of peptide was also determined, and is discussed in the context of relevance to both NMR spectroscopy and peptide-lipid interactions. © Munksgaard 1997.     

4-酰基-5-吡唑酮的二烃基锡配合物的合成、表征及其抗癌活性研究

目的　设计合成一系列新型有机锡化合物,通过药理筛选寻找具有抗肿瘤活性的药物并探讨其构效关系。方法　以4-酰基-5-吡唑酮为配体合成有机锡化合物,用元素分析、红外光谱、 ¹H,¹³C,¹¹⁹Sn NMR等方法对得到的化合物作结构表征并利用几种药理模型进行体外抗癌活性筛选。结果　合成了一系列R₂SnL₂ 型有机锡新配合物并确定其结构。结论　药理筛选结果表明,多个化合物(3～6,9～11)对HL-60, HCT-8, Bel-7402,BGC-823和KB癌细胞有效,显示了较强的抗癌活性。     

Studies of metabolism and disposition of potent human immunodeficiency virus (HIV) integrase inhibitors using 19F-NMR spectroscopy

¹⁹F-nuclear magnetic resonance (NMR) has been extensively used in a drug-discovery programme to support the selection of candidates for further development. Data on an early lead compound, N-(4-fluorobenzyl)-5-hydroxy-1-methyl-2-(4-methylmorpholin-3-yl)-6-oxo-1,6-dihydropyrimidine-4-carboxamide (compound A (+)), and MK-0518 (N-(4-fluorobenzyl)-5-hydroxy-1-methyl-2-(1-methyl-1-{[(5-methyl-1,3,4-oxadiazol-2-yl)carbonyl]amino}ethyl)-6-oxo-1,6-dihydropyrimidine-4-carboxamide), a potent inhibitor of this series currently in phase III clinical trials, are described. The metabolic fate and excretion balance of compound A (+) and MK-0518 were investigated in rats and dogs following intravenous and oral dosing using a combination of ¹⁹F-NMR-monitored enzyme hydrolysis and solid-phase extraction chromatography and NMR spectroscopy (SPEC-NMR). Dosing with the ³H-labelled compound A (+) enabled the comparison of standard radiochemical analysis with ¹⁹F-NMR spectroscopy to obtain quantitative metabolism and excretion data. Both compounds were eliminated mainly by metabolism. The major metabolite identified in rat urine and bile and in dog urine was the 5-O-glucuronide.     

Note: Capitulatin B,A new eudesmane derivative from Curculigo capitulata,and revised assignment of 13C NMR data of 6α,15α-epoxy-1β,4β-dihydroxyeudesmane

A new eudesmane derivative named capitulatin B (1) along with 6α,15α-epoxy-1β,4β-dihydroxyeudesmane (2) has been isolated from the rhizomes of Curculigo capitulata. The structure of compound 1 was established as 4α,6α-epoxy-1β-hydroxy-4β-methyleudesmane, and the ¹³C NMR data of compound 2 was reassigned on the basis of the spectral data, including 1D and 2D NMR (HMQC, HMBC, COSY, ROESY).     

Two new homoisoflavonoids from the fibrous roots of Ophiopogon japonicus (Thunb.) Ker-Gawl

Two new homoisoflavonoids ophiopogonone D (1) and ophiopogonanone G (2) were isolated from the fibrous roots of Ophiopogon japonicus. The structures of these two compounds were determined on the basis of spectroscopic means including HR-ESI-MS, 1D, and 2D NMR experiments. The cytotoxic activities of 1 and 2 against Hela and Hep2 cells are described.     

Synthesis of Chlorambucil-Tempol Adduct and its Delivery using Fluoroalkyl Double-Ended Poly (Ethylene Glycol) Micelles

In our pursuit to find potent anticancer drugs, we have covalently bonded free radical tempol to chlorambucil giving a chlorambucil-tempol (CT) adduct in which both of the anticancer active sites in tempol and chlorambucil were left intact. Analysis using NMR, Maldi-TOF, and EPR verified the designed chemical structure. Because the CT adduct is more hydrophobic than chlorambucil, its delivery also was investigated using fluoroalkyl double-ended poly (ethylene glycol) (R_f-PEG) micelles. Results from EPR spectra and¹⁹ F and¹ H NMR spin lattice relaxation times show that the R_f-PEG micelles are able to encapsulate CT into the R_f cores of the micelles.     

Jacaranone glycosides from Senecio scandens

Bioassay-guided fractionation of the ethanol extract of Senecio scandens led to the isolation of four new compounds 1–4. These compounds were obtained as tautomeric mixture of α/β epimers, but their structures were confirmed unambiguously by 1D and 2D NMR spectra and LC NMR technology. ¹H NMR spectra of pure 1α and 1β were furnished by HPLC NMR technology. Compounds 1–4 exhibited moderate cytotoxicities against five tumor cell lines.     

Didanosine Polymorphism in a Supercritical Antisolvent Process

Solid‐state properties of active ingredients are crucial in pharmaceutical development owing to their significant clinical and economical implications. In the present work we investigated the solid‐state properties and the solubility in water of didanosine, DDI, re‐crystallized from a dimethylsulfoxide solution using supercritical CO₂ as an antisolvent (SAS process) for comparison with the commercially available drug product. We also applied modern solid‐state NMR (SS NMR) techniques, namely 2D ¹H DQ CRAMPS (Combined Rotation And Multiple Pulse Spectroscopy) and ¹H–¹³C on‐ and off‐resonance CP (cross polarization) FSLG‐HETCOR experiments, known for providing reliable information about ¹H–¹H and ¹H–¹³C intra‐ and intermolecular proximities, in order to address polymorphism issues arising from the crystallization of a new form in the supercritical process. A new polymorph of didanosine was obtained from the supercritical antisolvent process and characterized by means of 1D and 2D multinuclear (¹H, ¹³C, ¹⁵N) SS NMR. The particle size of the new crystal phase was reduced by varying the antisolvent density through a pressure increase. The structural differences between the commercial product and the SAS re‐crystallized DDI are highlighted by X‐ray diffractometry and well described by solid‐state NMR. The carbon C6 ¹³C chemical shift suggests that both commercial and re‐crystallized didanosine samples are in the enol form. The analysis of homo‐ and heteronuclear proximities obtained by means of 2D NMR experiments shows that commercial and SAS re‐crystallized DDI possess very similar molecular conformation and hydrogen bond network, but different packing. The new polymorph proved to be a metastable form at ambient conditions, showing higher solubility in water and lower stability to mechanical stress. © 2009 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 99: 1855–1870, 2010