Study of Mono and Di-O-caffeoylquinic Acid Isomers in Acmella oleracea Extracts by HPLC-MS/MS and Application of Linear Equation of Deconvolution Analysis Algorithm for Their Characterization.

Chlorogenic acids, the esters of caffeic and quinic acids, are the main phenolic acids detected in Acmella oleracea extracts and have gained increasing interest in recent years due to their important biological activities. Given their structural similarity and instability, the correct analysis and identification of these compounds in plants is challenging. This study aimed to propose a simple and rapid determination of the A. oleracea caffeoylquinic isomers, applying an HPLC-MS/MS method supported by a mathematical algorithm (Linear Equation of Deconvolution Analysis (LEDA)). The three mono- and the three di-caffeoylquinic acids in roots of Acmella plants were studied by an ion trap MS analyzer. A separation by a conventional chromatographic method was firstly performed and an MS/MS characterization by energetic dimension of collision-induced dissociation mechanism was carried out. The analyses were then replicated using a short HPLC column and a fast elution gradient (ten minutes). Each acquired MS/MS data were processed by LEDA algorithm which allowed to assign a relative abundance in the reference ion signal to each isomer present. Quantitative results showed no significant differences between the two chromatographic systems proposed, proving that the use of LEDA algorithm allowed the distinction of the six isomers in a quarter of the time.

Isomers Recognition in HPLC-MS/MS Analysis of Human Plasma Samples by Using an Ion Trap Supported by a Linear Equations-Based Algorithm.

The tandem mass spectrometry (MS/MS) approach employing an ion trap mass analyzer (IT) was evaluated in isomers recognition. The proposed approach consists of sole, simple, and rapid liquid chromatographic separation (HPLC) without requiring resolution between the analytes. Then, the MS/MS properties were optimized to solve the signal assignment using post-processing data elaboration (LEDA). The IT-MS/MS experiment uses the same site, helium as collision gas, and different time steps to modify the applied conditions on the studied ions. Nevertheless, helium cannot ensure the quick energization of the precursor ion due to its small cross-section. Then, different combinations between excitation amplitude (ExA) and excitation time (ExT) were tested to achieve the activation of the fragmentation channels and the formation of the MS/MS spectrum. Usually, the IT-MS/MS acquisition cycle is longer for other multistage instruments, decreasing the frequency of sample data collection and influencing the chromatographic profile. To solve these problems, two time segments were set up, and the elution conditions were optimized with a compromise between peaks distinction and run time reduction. The developed HPLC-MS/MS method was checked and applied to analyze a series of human plasma samples spiked with an equimolar mixture of pair of isomers.

Corrigendum: Characterization of the "gut microbiota-immunity axis" and microbial lipid metabolites in atrophic and potential celiac disease.

[This corrects the article DOI: 10.3389/fmicb.2022.886008.].

Tandem mass spectrometry approaches for recognition of isomeric compounds mixtures.

The present review aims to collect the published literature pertaining the recognition of isobaric compounds (isomers or stereoisomers) using the features of tandem mass spectrometry (MS) experiments without any chromatographic separation or chemical modification (derivatization or isotopic enrichment) of the analytes. MS/MS methods possess high selectivity, wide dynamic range and high throughput capabilities. Generally, tandem MS has limited capability for distinguishing isomers that fragment similarly. However, some MS/MS methods have been developed and positively applied to isomers discrimination. Among the literature on this topic, the applications that fit on the review subject can be summarized as follow: (1) chiral discrimination by the kinetic method, (2) the use energy-resolved tandem mass spectra and the survival yield (SY) representation, (3) the kinetics evaluation of the ion-molecule interaction and (4) the postprocessing mathematical algorithm to resolve the isomers in MS/MS signal.

Simultaneous Degradation Study of Isomers in Human Plasma by HPLC-MS/MS and Application of LEDA Algorithm for Their Characterization.

This paper proposes a tandem mass spectrometry (MS/MS) approach in isomer recognition by playing in the "energetic dimension" of the experiment. The chromatographic set up (HPLC) was tuned to minimize the run time, without requiring high efficiency or resolution between the isomers. Then, the MS/MS properties were explored to solve the signal assignment by performing a series of energy resolved experiments in order to optimize the parameters, and by applying an interesting post-processing data elaboration tool (LEDA). The reliability of the new approach was evaluated, determining the accuracy and precision of the quantitative results through analysis of the isomer mixture solutions. Next, the proposed method was applied in a chemical stability study of human plasma samples through the simultaneous addition of a pair of isomers. In the studied case, only one of the isomers suffered of enzymatic hydrolysis; therefore, the influence of the stable isomer on the degradation rate of the other was verified. In order to monitor this process correctly, it must be possible to distinguish each isomer present in the sample, quantify it, and plot its degradation profile. The reported results demonstrated the effectiveness of the LEDA algorithm in separating the isomers, without chromatographic resolution, and monitoring their behavior in human plasma samples.

Application of LEDA algorithm for the recognition of P-glycoprotein and Carbonic Anhydrase hybrid inhibitors and evaluation of their plasma stability by HPLC-MS/MS analysis.

Design and synthesis of new candidate drugs produces a large number of compounds that must be qualified and tested to evaluate their characteristics and potential applications. Therefore, many studies will be scheduled and, consequently, it will be necessary to arrange specific, reliable, fast and relatively cheap analytic methods to support this research. The manuscript proposes a new approach in the HPLC-MS/MS analysis by using a sole chromatographic set up, tuned to minimize the run time, without requiring high efficiency or resolution between the analytes. The chromatographic column was used only to avoid or limit the interference of sample matrix towards the analyte ionization process (matrix-effects). Then, the MS/MS properties were explored to solve the signal assignment, by performing a series of energy resolved experiments to optimize the parameters and applying an interesting post-processing data elaboration tool (LEDA). The reliability of the new approach was evaluated in a chemical stability study in PBS and human plasma samples of a series of isomeric compounds P-glycoprotein/Carbonic Anhydrase (P-gp/CA) hybrid inhibitors. The obtained results demonstrated the effectiveness (reliability 97%-100%) of the LEDA algorithm to recognize and to separate the possible isomers present in the samples. The obtained matrix-effects values (ME 96%-106%) established that the chromatographic set up (short column and fast elution gradient) was proper to avoid the matrix interferences, while recovery values (RE 88%-108%) indicate a suitable sample preparation, despite only a protein precipitation was carried out. The quantitative performances of proposed HPLC-MS/MS methods showed an accuracy ranging between 92% and 108% and a precision lower than 13% that allows to be confident on the determination of new P-gp/CA hybrid inhibitors in the degradation study. Therefore, the general procedure proposed was found adequate to study a series of isomeric compounds without their chromatographic separation but only by applying and developing the MS/MS features.

A method for assessing plasma free fatty acids from C2 to C18 and its application for the early detection of colorectal cancer.

The targeted analysis of free fatty acids (FFAs) is attracting interest since several years with a plenty of studies. However, most of them are devoted to the solely determination of the short-chain fatty acids (SCFAs) arising from the symbiotic gut microbiota metabolism. Recently, the FFAs analysis highlighted changes in the plasma levels of octanoic and decanoic acids (medium-chain fatty acids or MCFAs) may be associated to gastrointestinal diseases, including colorectal cancer (CRC). Then, the simultaneous quantification of both SCFAs and MCFAs could be useful to put in evidence the interconnection between microbiota and metabolic alterations during hosts' disease. To this aim, it was developed an isotopic dilution gas-chromatography coupled mass spectrometry (ID/GC-MS) method for the targeted analysis of both linear and branched FFAs (SCFAs, MCFAs, and LCFAs) in human plasma samples as specific markers for both microbiota and host metabolic alterations. In order to minimize sample manipulation procedures, an efficient, sensible and low time-consuming procedure is presented, which relies in a simple liquid-liquid extraction before the determination of underivatized free acids (FFAs) by Single Ion Monitoring (SIM) acquisition. The reached detection limits (LODs) were less than 100 µg L-1 for most of analytes, except for acetic, hexadecanoic and octadecanoic acids that showed a LOD > 1 mg L-1. Methods accuracy and precision, obtained by the analysis of the FFAs mixtures showed accuracy values between 84% and 100% and precision (RSD %) between 0.1% and 12.4% at the concentration levels tested. The proposed ID/GC-MS method was applied in a case study to evaluate the FFAs as specific markers for both microbiota and host alterations in CRC patients. Obtained results highlight the advantage of present method for its rapidity, simplicity, and robustness.

Intestinal microbiota profiles in a genetic model of colon tumorigenesis correlates with colon cancer biomarkers.

Faecal (FM) and colon mucosal associated microbiota (MAM) were studied in a model of colorectal cancer (CRC), the Apc-mutated Pirc rats, and in age-paired wt F344 rats. Principal Coordinates Analysis indicated that samples' distribution was driven by age, with samples of young rats (1 month old; without tumours) separated from older ones (11-month-old; bearing tumours). Diversity analysis showed significant differences between FM and MAM in older Pirc rats, and between MAM of both Pirc and wt rats and the tumour microbiota, enriched in Enterococcus, Escherichia/Shigella, Proteus and Bifidobacteriaceae. In young animals, Pirc FM was enriched in the genus Delftia, while wt FM was enriched in Lactobacillus and Streptococcus. Some CRC biomarkers and faecal short chain fatty acids (SCFAs) were also measured. Colon proliferation and DClK1 expression, a pro-survival mucosal marker, were higher in Pirc than in wt rats, while the mucin MUC2, was lower in Pirc rats. Branched SCFAs were higher in Pirc than in wt animals. By Spearman analysis CRC biomarkers correlated with FM (in both young and old rats) and with MAM (in young rats), suggesting a specific relationship between the gut microbiota profile and these functional mucosal parameters deserving further investigation.

Vaginal Lactobacilli and Vaginal Dysbiosis-Associated Bacteria Differently Affect Cervical Epithelial and Immune Homeostasis and Anti-Viral Defenses.

Persistent infection with High Risk-Human Papilloma Viruses (HR-HPVs) is a primary cause of cervical cancer worldwide. Vaginal-dysbiosis-associated bacteria were correlated with the persistence of HR-HPVs infection and with increased cancer risk. We obtained strains of the most represented bacterial species in vaginal microbiota and evaluated their effects on the survival of cervical epithelial cells and immune homeostasis. The contribution of each species to supporting the antiviral response was also studied. Epithelial cell viability was affected by culture supernatants of most vaginal-dysbiosis bacteria, whereas Lactobacillus gasseri or Lactobacillus jensenii resulted in the best stimulus to induce interferon-γ (IFN-γ) production by human mononuclear cells from peripheral blood (PBMCs). Although vaginal-dysbiosis-associated bacteria induced the IFN-γ production, they were also optimal stimuli to interleukin-17 (IL-17) production. A positive correlation between IL-17 and IFN-γ secretion was observed in cultures of PBMCs with all vaginal-dysbiosis-associated bacteria suggesting that the adaptive immune response induced by these strains is not dominated by TH1 differentiation with reduced availability of IFN-γ, cytokine most effective in supporting virus clearance. Based on these results, we suggest that a vaginal microbiota dominated by lactobacilli, especially by L. gasseri or L. jensenii, may be able to assist immune cells with clearing HPV infection, bypasses the viral escape and restores immune homeostasis.

The Gut Microbiota-Immunity Axis in ALS: A Role in Deciphering Disease Heterogeneity?

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder with an unknown etiology and no effective treatment, and is characterized by large phenotypic heterogeneity, including variable sites, ages of symptom onset and rates of disease progression. Increasing data support the role of the microbiota-immunity axis in the pathogenesis of neurodegenerative diseases. In the present study, we compared the inflammatory and microbiota profile of ALS patients with different clinical characteristics, with healthy family caregivers. Measuring a panel of 30 inflammatory cytokines in serum and fecal samples, we observed a distinct cytokine profile both at the systemic and intestinal level in patients compared to controls and even in patients with different clinical phenotypes and progression rates. The 16S targeted metagenome analysis revealed slight differences in patients compared to controls as well as in patients with slow progression, marked by the reduction of butyrate-producing bacteria and a decrease of the Firmicutes/Bacteroidetes ratio in ALS. Finally, the short chain fatty acid analysis did not show a different distribution among the groups. If confirmed in a larger number of patients, the inflammatory cytokine profile and the microbial composition could be appropriate biomarker candidates for deciphering ALS heterogeneity.

Free Fatty Acids Signature in Human Intestinal Disorders: Significant Association between Butyric Acid and Celiac Disease.

Altered circulating levels of free fatty acids (FFAs), namely short chain fatty acids (SCFAs), medium chain fatty acids (MCFAs), and long chain fatty acids (LCFAs), are associated with metabolic, gastrointestinal, and malignant diseases. Hence, we compared the serum FFA profile of patients with celiac disease (CD), adenomatous polyposis (AP), and colorectal cancer (CRC) to healthy controls (HC). We enrolled 44 patients (19 CRC, 9 AP, 16 CD) and 16 HC. We performed a quantitative FFA evaluation with the gas chromatography-mass spectrometry method (GC-MS), and we performed Dirichlet-multinomial regression in order to highlight disease-specific FFA signature. HC showed a different composition of FFAs than CRC, AP, and CD patients. Furthermore, the partial least squares discriminant analysis (PLS-DA) confirmed perfect overlap between the CRC and AP patients and separation of HC from the diseased groups. The Dirichlet-multinomial regression identified only strong positive association between CD and butyric acid. Moreover, CD patients showed significant interactions with age, BMI, and gender. In addition, among patients with the same age and BMI, being male compared to being female implies a decrease of the CD effect on the (log) prevalence of butyric acid in FFA composition. Our data support GC-MS as a suitable method for the concurrent analysis of circulating SCFAs, MCFAs, and LCFAs in different gastrointestinal diseases. Furthermore, and notably, we suggest for the first time that butyric acid could represent a potential biomarker for CD screening.

Susceptibility of cosmeceutical peptides to proteases activity: Development of dermal stability test by LC-MS/MS analysis.

Recently, several peptides are used as active ingredients in topical cosmetic formulations, few information are available on their dermal stability against proteases. In this study, it was developed a simple and reliable assay to evaluate the stability of cosmeceutical peptides in skin homogenates. The quantification of studied peptides was performed by liquid chromatography coupled with a triple quadrupole mass spectrometer operating in tandem mass spectrometry mode (LC-MS/MS) and the conditions were tuned through energy resolved MS/MS (ERMS) experiments. The sample preparation procedure was carried out on rat skin homogenates by employing pal-KTTKS (reference peptide and the parameters that may affect the assay results were evaluated, including substrate concentration, dilution of skin homogenate, protein concentration and batch-to-batch variation of the homogenate. The optimized conditions were applied to check the degradation profile of pal-KTTKS in human skin samples and the obtained results were compared. Finally, the degradation profiles of SA1-III and pamSA1-III, recently described as cosmeceutical peptides, in human skin homogenate were evaluated. The results showed that proposed peptides are stable toward proteases for up to 8 h of incubation. Thanks to this characteristic, these peptides can be considered very interesting candidates as active ingredients for creams intended for a daily application.

Determination of coeluted isomers in wine samples by application of MS/MS deconvolution analysis.

Two organic acids isomers, 3-isopropylmalic acid (3-IPMA) and 2-isopropylmalic acid (2-IPMA), were identified and quantified in wine samples by using an LC-MS/MS method without any chromatographic separation, but processing the MS/MS data with a recently developed deconvolution algorithm (LEDA: linear equations deconvolution analysis), thus decreasing the time necessary for the process. In particular, the LEDA tool processes the MS/MS signals and assigns the relative concentrations (abundances) of the isomers in the sample, at the mg L-1 level. The efficiency of MS/MS signal assignment was improved by introducing five linear equations to define the LEDA matrix. Then, as a novel approach, an overdetermined system of linear equations was applied for the deconvolution of isomers. The use of LEDA to identify and quantify the isomers in wine samples, together with the choice of a short LC column and a fast elution gradient, simplifies the process and shortens the time needed. Furthermore, it was evaluated the quantitative determination of the IPMA isomers by using the calibration curve provided by the precursor ion MRM transition data. The calculated values of accuracy (recovery between 82.6% and 99.8%) and precision (RSD between 0.4% and 4.0%) confirm the validity of this quantitative approach and the ability of LEDA to establish the correct percentage of the MS/MS signal for each isomer. Finally, to compare the conventional LC-MS/MS method and our proposed method of LC-MS/MS coupled with LEDA post-processing elaboration, a series of real wine samples were analysed by both methods, and the results were compared.

Influence of a 3-month low-calorie Mediterranean diet compared to the vegetarian diet on human gut microbiota and SCFA: the CARDIVEG Study.

PURPOSE: We evaluated the effect of low-calorie mediterranean (MD) and vegetarian (VD) diets on gut microbiome (GM) composition and short-chain-fatty acids (SCFA) production. METHODS: We performed next generation sequencing (NGS) of 16S rRNA and SCFA analysis on fecal samples of 23 overweight omnivores (16 F; 7 M) with low-to-moderate cardiovascular risk. They were randomly assigned to a VD or MD, each lasting 3 months, with a crossover study design. RESULTS: Dietary interventions did not produce significant diversity in the GM composition at higher ranks (family and above), neither between nor within MD and VD, but they did it at genus level. MD significantly changed the abundance of Enterorhabdus, Lachnoclostridium and Parabacteroides, while VD significantly affected the abundance of Anaerostipes, Streptococcus, Clostridium sensu stricto, and Odoribacter. Comparison of the mean variation of each SCFA between MD and VD showed an opposite and statistically significant trend for propionic acid (+ 10% vs - 28%, respectively, p = 0.034). In addition, variations of SCFA were negatively correlated with changes of some inflammatory cytokines such as VEGF, MCP-1, IL-17, IP-10 and IL-12, only after MD. Finally, correlation analyses showed a potential relationship-modulated by the two diets-between changes of genera and changes of clinical and biochemical parameters. CONCLUSIONS: A short-term dietary intervention with MD or VD does not induce major change in the GM, suggesting that a diet should last longer than 3 months for scratching the microbial resilience. Changes in SCFA production support their role in modulating the inflammatory response, thus mediating the anti-inflammatory and protective properties of MD.

Bioisosteric Development of Multitarget Nonsteroidal Anti-Inflammatory Drug-Carbonic Anhydrases Inhibitor Hybrids for the Management of Rheumatoid Arthritis.

Multitarget nonsteroidal anti-inflammatory drug (NSAID)-carbonic anhydrase inhibitor (CAI) agents for the management of rheumatoid arthritis are reported. The evidence of the plasma stability of the amide-linked hybrids previously reported prompted us to investigate their pain-relieving mechanism of action. A bioisosteric amide to ester substitution yielded a series of derivatives showing potent target CAs inhibition and to undergo cleavage in rat or human plasma depending on the NSAID portion. A selection of derivatives were assayed in vitro to indirectly evaluate their effect on COX-1 and COX-2. MD simulations demonstrated that the entire hybrids are also able to efficiently bind the COX active site. In a rat model of RA, the most promising derivative (5c) showed major antihyperalgesic action compared with the equimolar coadministration of the single agents. The gathered data provided new insights on the action mechanism of these multitarget compounds, which induce markedly improved pain relief compared with the parent NSAIDs.

Evaluation and comparison of short chain fatty acids composition in gut diseases.

BACKGROUND: An altered (dysbiosis) and unhealthy status of the gut microbiota is usually responsible for a reduction of short chain fatty acids (SCFAs) concentration. SCFAs obtained from the carbohydrate fermentation processes are crucial in maintaining gut homeostasis and their determination in stool samples could provide a faster, reliable and cheaper method to highlight the presence of an intestinal dysbiosis and a biomarker for various gut diseases. We hypothesize that different intestinal diseases, such as celiac disease (CD), adenomatous polyposis (AP) and colorectal cancer (CRC) could display a particular fecal SCFAs' signature. AIM: To compare the fecal SCFAs' profiles of CD, AP, CRC patients and healthy controls, using the same analytical method. METHODS: In this cross-sectional study, we defined and compared the SCFAs' concentration in fecal samples of 9 AP, 16 CD, 19 CRC patients and 16 healthy controls (HC). The SCFAs' analysis were performed using a gas-chromatography coupled with mass spectrometry method. Data analysis was carried out using Wilcoxon rank-sum test to assess pairwise differences of SCFAs' profiles, partial least squares-discriminate analysis (PLS-DA) to determine the status membership based on distinct SCFAs' profiles, and Dirichlet regression to determine factors influencing concentration levels of SCFAs. RESULTS: We have not observed any difference in the SCFAs' amount and composition between CD and healthy control. On the contrary, the total amount of SCFAs was significantly lower in CRC patients compared to HC (P = 0.044) and CD (P = 0.005). Moreover, the SCFAs' percentage composition was different in CRC and AP compared to HC. In detail, HC displayed higher percentage of acetic acid (P value = 1.3 × 10-6) and a lower amount of butyric (P value = 0.02192), isobutyric (P value = 7.4 × 10-5), isovaleric (P value = 0.00012) and valeric (P value = 0.00014) acids compared to CRC patients. AP showed a lower abundance of acetic acid (P value = 0.00062) and higher percentages of propionic (P value = 0.00433) and isovaleric (P value = 0.00433) acids compared to HC. Moreover, AP showed higher levels of propionic acid (P value = 0.03251) and a lower level of isobutyric acid (P value = 0.00427) in comparison to CRC. The PLS-DA model demonstrated a significant separation of CRC and AP groups from HC, although some degree of overlap was observed between CRC and AP. CONCLUSION: Analysis of fecal SCFAs shows the potential to provide a non-invasive means of diagnosis to detect patients with CRC and AP, while CD patients cannot be discriminated from healthy subjects.

Antioxidant-Conjugated 1,2,4-Triazolo[4,3-a]pyrazin-3-one Derivatives: Highly Potent and Selective Human A2A Adenosine Receptor Antagonists Possessing Protective Efficacy in Neuropathic Pain.

New 8-amino-6-aryl-1,2,4-triazolo[4,3-a]pyrazin-3-ones were designed to obtain dual antioxidant-human A2A adenosine receptor (hA2A AR) antagonists. Two sets of compounds were synthesized, the first featuring phenol rings at the 6-position, the second bearing the lipoyl and 4-hydroxy-3,5-di-tertbut-benzoyl residues appended by different linkers on the 6-phenyl ring. Several new triazolopyrazines (1-21) were potent and selective hA2A AR antagonists (Ki = 0.17-54.5 nM). Compounds 11, 15, and 21, featuring antioxidant moieties, and compound 12, lacking the antioxidant functionality, reduced oxaliplatin-induced toxicity in microglia cells, the most active being the lipoyl-derivative 15 and the (4-hydroxy-3,5-di-tert-butyl)benzoyl-analogue 21 which were effective in reducing the oxygen free radical level. The lipoyl-derivative 15 was also able to revert oxaliplatin-induced neuropathy in the mouse. In vivo efficacy of 15 makes it a promising neuroprotective agent in oxidative stress-related diseases.

Synthesis and Evaluation of Carbonic Anhydrase Inhibitors with Carbon Monoxide Releasing Properties for the Management of Rheumatoid Arthritis.

Carbon monoxide (CO) is a gas endogenously produced in humans, reported to exhibit anti-inflammatory and cytoprotective effects at low concentration. In this context, CO releasing molecules (CORMs) are attracting enormous interest. Herein, we report a series of small-molecule hybrids consisting of a carbonic anhydrase (CA; EC 4.2.1.1) inhibitor linked to a CORM tail section (CAI-CORMs). All compounds were screened in vitro for their inhibition activity against the human (h) CA I, II, IV, IX, and XII isoforms. On selected CAI-CORM hybrids, the CO releasing properties were evaluated, along with their pain-relieving effect, in a model of rheumatoid arthritis. One CAI-CORM hybrid (5b) induced a higher pain-relieving effect compared to the one exerted by the single administration of CAI (5a) and CORM (15b) fragments, shedding light on the possibility to enhance the pain relief effect of CA inhibitors inserting a CO releasing moiety on the same molecular scaffold.

Modifications on the Amino-3,5-dicyanopyridine Core To Obtain Multifaceted Adenosine Receptor Ligands with Antineuropathic Activity.

A new series of amino-3,5-dicyanopyridines (1-31) was synthesized and biologically evaluated in order to further investigate the potential of this scaffold to obtain adenosine receptor (AR) ligands. In general, the modifications performed have led to compounds having high to good human (h) A1AR affinity and an inverse agonist profile. While most of the compounds are hA1AR-selective, some derivatives behave as mixed hA1AR inverse agonists/A2A and A2B AR antagonists. The latter compounds (9-12) showed that they reduce oxaliplatin-induced neuropathic pain by a mechanism involving the alpha7 subtype of nAchRs, similar to the nonselective AR antagonist caffeine, taken as the reference compound. Along with the pharmacological evaluation, chemical stability of methyl 3-(((6-amino-3,5-dicyano-4-(furan-2-yl)pyridin-2-yl)sulfanyl)methyl)benzoate 10 was assessed in plasma matrices (rat and human), and molecular modeling studies were carried out to better rationalize the available structure-activity relationships.

Benzensulfonamides bearing spyrohydantoin moieties act as potent inhibitors of human carbonic anhydrases II and VII and show neuropathic pain attenuating effects.

Carbonic Anhydrases have been recently validated as novel therapeutic targets in neuropathic pain. In this study, we combine the anticonvulsant propriety of spyrohydantoin and the CA inhibitor moiety of benzenesulfonamide to synthesize a novel series of spyrohydantoin bearing sulfonamides with strong activity against hCA II and VII. These isoforms are present in the nervous system and largely expressed both at the central as well as at peripheral level and can be modulated for pain relief. The crystal structures of hCA II in complex with selected compounds 5a-c demonstrate the importance of the tail in the binding modes within the isoform. Finally, in vivo, in an animal model of oxaliplatin induced neuropathy, compounds with organoselenium tails (8b-c) showed potent neuropathic pain attenuating effects. Taken together, these data strongly suggest the translational utility of these inhibitors as novel pain relievers.