A new HPLC method with multiple detection systems for impurity analysis and discrimination of natural versus synthetic cannabidiol.

Cannabidiol (CBD) is the main non-psychoactive phytocannabinoid derived from Cannabis sativa L. It is now an active pharmaceutical ingredient (API), given its usage in treating some types of pediatric epilepsy. For this reason, this compound requires a deep characterization in terms of purity and origin. Previous research work has shown two impurities in CBD samples from hemp inflorescences, namely, cannabidivarin (CBDV) and cannabidibutol (CBDB), while abnormal-cannabidiol (abn-CBD) has been described as the primary by-product that is generated from CBD synthesis. Both natural and synthetic CBD samples exhibit the presence of Δ9-tetrahydrocannabinol (Δ9-THC) and Δ8-THC. This study aimed to develop a new analytical method based on high-performance liquid chromatography (HPLC) with different detection systems to study the purity of CBD and to define its origin based on the impurity profile. In addition to the above-mentioned cannabinoids, other compounds, such as cannabigerovarin (CBGV), cannabigerol (CBG), cannabichromevarin (CBCV), and cannabichromene (CBC), were examined as potential discriminating impurities. Qualitative and quantitative analyses were carried out by UHPLC-HRMS and HPLC-UV/Vis, respectively. Principal component analysis was applied for statistical exploration. Natural CBD samples exhibited purities ranging between 97.5 and 99.7%, while synthetic samples were generally pure, except for three initially labeled as synthetic, revealing natural-derived impurities. To further confirm the origin of CBD samples, the presence of other two minor impurities, namely cannabidihexol (CBDH) and cannabidiphorol (CBDP), was assessed as unequivocal for a natural origin. Finally, an enantioselective HPLC analysis was carried out and the results confirmed the presence of the (-)-trans enantiomer in all CBD samples. In conclusion, the HPLC method developed represents a reliable tool for detecting CBD impurities, thus providing a clear discrimination of the compound origin.

Structure-guided optimization of 3-hydroxybenzoisoxazole derivatives as inhibitors of Aldo-keto reductase 1C3 (AKR1C3) to target prostate cancer.

AKR1C3 is an enzyme that is overexpressed in several types of radiotherapy- and chemotherapy-resistant cancers. Despite AKR1C3 is a validated target for drug development, no inhibitor has been approved for clinical use. In this manuscript, we describe our study of a new series of potent AKR1C3-targeting 3-hydroxybenzoisoxazole based inhibitors that display high selectivity over the AKR1C2 isoform and low micromolar activity in inhibiting 22Rv1 prostate cancer cell proliferation. In silico studies suggested proper substituents to increase compound potency and provided with a mechanistic explanation that could clarify their different activity, later confirmed by X-ray crystallography. Both the in-silico studies and the crystallographic data highlight the importance of 90° rotation around the single bond of the biphenyl group, in ensuring that the inhibitor can adopt the optimal binding mode within the active pocket. The p-biphenyls that bear the meta-methoxy, and the ortho- and meta-trifluoromethyl substituents (in compounds 6a, 6e and 6f respectively) proved to be the best contributors to cellular potency as they provided the best IC50 values in series (2.3, 2.0 and 2.4 µM respectively) and showed no toxicity towards human MRC-5 cells. Co-treatment with scalar dilutions of either compound 6 or 6e and the clinically used drug abiraterone led to a significant reduction in cell proliferation, and thus confirmed that treatment with both CYP171A1-and AKR1C3-targeting compounds possess the potential to intervene in key steps in the steroidogenic pathway. Taken together, the novel compounds display desirable biochemical potency and cellular target inhibition as well as good in-vitro ADME properties, which highlight their potential for further preclinical studies.

The discovery of aryl-2-nitroethyl triamino pyrimidines as anti-Trypanosoma brucei agents.

Pteridine reductase 1 (PTR1) is a catalytic protein belonging to the folate metabolic pathway in Trypanosmatidic parasites. PTR1 is a known target for the medicinal chemistry development of antiparasitic agents against Trypanosomiasis and Leishmaniasis. In previous studies, new nitro derivatives were elaborated as PTR1 inhibitors. The compounds showing a diamino-pyrimidine core structure were previously developed but they showed limited efficacy. Therefore, a new class of phenyl-, heteroaryl- and benzyloxy-nitro derivatives based on the 2-nitroethyl-2,4,6-triaminopyrimidine scaffold were designed and tested. The compounds were assayed for their ability to inhibit T. brucei and L. major PTR1 enzymes and for their antiparasitic activity towards T. brucei and L. infantum parasites. To understand the structure-activity relationships of the compounds against TbPTR1, the X-ray crystallographic structure of the 2,4,6-triaminopyrimidine (TAP) was obtained and molecular modelling studies were performed. As a next step, only the most effective compounds against T. brucei were then tested against the amastigote cellular stage of T. cruzi, searching for a broad-spectrum antiprotozoal agent. An early ADME-Tox profile evaluation was performed. The early toxicity profile of this class of compounds was investigated by measuring their inhibition of hERG and five cytochrome P450 isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4), cytotoxicity towards A549 cells and mitochondrial toxicity. Pharmacokinetic studies (SNAP-PK) were performed on selected compounds using hydroxypropyl-ß-cyclodextrins (50 % w/v) to preliminarily study their plasma concentration when administered per os at a dose of 20 mg/kg. Compound 1p, showed the best pharmacodynamic and pharmacokinetic properties, can be considered a good candidate for further bioavailability and efficacy studies.

Structure-Based Optimization of 1,2,4-Triazole-3-Thione Derivatives: Improving Inhibition of NDM-/VIM-Type Metallo-ß-Lactamases and Synergistic Activity on Resistant Bacteria.

The worldwide emergence and dissemination of Gram-negative bacteria expressing metallo-ß-lactamases (MBLs) menace the efficacy of all ß-lactam antibiotics, including carbapenems, a last-line treatment usually restricted to severe pneumonia and urinary tract infections. Nonetheless, no MBL inhibitor is yet available in therapy. We previously identified a series of 1,2,4-triazole-3-thione derivatives acting as micromolar inhibitors of MBLs in vitro, but devoid of synergistic activity in microbiological assays. Here, via a multidisciplinary approach, including molecular modelling, synthesis, enzymology, microbiology, and X-ray crystallography, we optimized this series of compounds and identified low micromolar inhibitors active against clinically relevant MBLs (NDM-1- and VIM-type). The best inhibitors increased, to a certain extent, the susceptibility of NDM-1- and VIM-4-producing clinical isolates to meropenem. X-ray structures of three selected inhibitors in complex with NDM-1 elucidated molecular recognition at the base of potency improvement, confirmed in silico predicted orientation, and will guide further development steps.