Software-assisted automated detection and identification of "unknown" analogues of benzodiazepines in liquid chromatography mass spectrometry analysis.

RATIONALE: Benzodiazepines (BZDs) construct a large group of psychoactive drugs acting as depressants of the central nervous system (CNS) and used in medicine as sedatives and anxiolytic and antiepileptic agents. The illicit use of these materials is a worldwide problem, and for many years, part of the benzodiazepines have been abused as rape drugs. For example, flunitrazepam (Rohypnol) is most commonly linked by media reports to drug-facilitated sexual assaults, more commonly referred to as "date rape." Furthermore, there are growing concerns for other misuses of these drugs. Over the last few years, there was an increase in the number, type, and availability of new psychoactive substances (NPS) belonging to the benzodiazepine group, challenging standard forensic labs to fully identify the chemical structure of new, unknown benzodiazepines. METHODS: This work demonstrates a new application of the automated tool for the detection and identification of benzodiazepine analogues using high-resolution-accurate-mass LC-MS analysis, followed by "Compound Discoverer" (CD) software data processing, to automatically detect various benzodiazepine analogues by picking peaks and compare them to in silico calculated modifications made on a predefined basic backbone. Subsequently, a complete structural elucidation for the proposed molecular formula is obtained by MS/MS data analysis of the suspected component. RESULTS: This method was found to be useful for the automated detection and putative identification of a series of nine "unknown" benzodiazepine analogues, at concentrations in the low ng/mL range. CONCLUSIONS: We hereby present a general demonstration of this powerful tool for the forensic community in the detection and identification of hazardous unknown compounds.

Functional effects of synthetic cannabinoids versus Δ9 -THC in mice on body temperature, nociceptive threshold, anxiety, cognition, locomotor/exploratory parameters and depression.

Synthetic cannabinoids are psychoactive substances designed to mimic the euphorigenic effects of the natural cannabis. Novel unregulated compounds appear once older compounds become illegal. It has been previously reported that synthetic cannabinoids are different than Δ9 -tetrahydrocannabinol (Δ9 -THC) as they have chemical structures unrelated to Δ9 -THC, different metabolism and, often, greater toxicity. This study aimed to investigate the effects of three novel synthetic cannabinoids and pure Δ9 -THC on body temperature, nociceptive threshold, anxiety, memory function, locomotor and exploratory parameters, and depression. We performed a battery of behavioural and motor tests starting 50 minutes post i.p. injection of each drug to adult ICR mice. The synthetic cannabinoids that were used are AB-FUBINACA, AB-CHMINACA and PB-22. All synthetic cannabinoids and Δ9 -THC caused hypothermia, but only Δ9 -THC induced a clear antinociceptive effect. All synthetic cannabinoids and Δ9 -THC caused decreased anxiety levels, spatial memory deficits and decreased exploratory behaviour as measured in the elevated plus maze, Y-maze and staircase paradigm, respectively. However, all synthetic cannabinoids but not Δ9 -THC demonstrated decreased locomotor activity in the staircase test. Moreover, only AB-FUBINACA and Δ9 -THC affected the gait balance and grip strength of the mice as was assessed by the latency time to fall from a rod. In the forced swimming test, PB-22 caused elevated depression-like behaviour while AB-FUBINACA induced a reversed effect. These results suggest varied effects among different synthetic cannabinoids and Δ9 -THC. Further studies are needed to characterize the overall effects and differences between these synthetic cannabinoids and Δ9 -THC.

Carbamoylphosphonate matrix metalloproteinase inhibitors 6: cis-2-aminocyclohexylcarbamoylphosphonic acid, a novel orally active antimetastatic matrix metalloproteinase-2 selective inhibitor--synthesis and pharmacodynamic and pharmacokinetic analysis.

cis-2-Aminocyclohexylcarbamoylphosphonic acid ( cis-ACCP) was evaluated in vitro and in two in vivo cancer metastasis models. It reduced metastasis formation in mice by approximately 90% when administered by a repetitive once daily dosing regimen of 50 mg/kg via oral or intraperitoneal routes and was nontoxic up to 500 mg/kg, following intraperitoneal administration daily for two weeks. Pharmacokinetic investigation of cis-ACCP in rats revealed distribution restricted into the extracellular fluid, which is the site of action for the antimetastatic activity and rapid elimination ( t 1/2 approximately 19 min) from blood. Sustained and prolonged absorption ( t 1/2 approximately 126 min) occurred via paracellular mechanism along the small and large intestine with overall bioavailability of 0.3%. The in vivo concentrations of cis-ACCP in the blood in rats was above the minimal concentration for antimetastatic/MMP-inhibitory activity, thus explaining the prolonged action following once daily administration. Finally, 84% of the intravenously administered cis-ACCP to rats was excreted intact in the urine.

Carbamoylphosphonate matrix metalloproteinase inhibitors 3: in vivo evaluation of cyclopentylcarbamoylphosphonic acid in experimental metastasis and angiogenesis.

The spread of malignant tumor cells from a primary neoplasm to distant organs where they multiply and form new foci is the major cause of death from cancer. Despite the different modalities of cancer treatment, no effective curative therapy of metastatic lesions is available. To possess metastatic potential, a cell has to be able to invade the surrounding tissue, spread via lymphatics and/or the bloodstream, extravasate, and multiply at secondary sites. There is increasing evidence for a positive correlation between matrix metalloproteinase-2 (MMP-2) activity and tumor cell invasion. Agents blocking MMP-2 have been shown to prevent tumor cell invasion in vitro and in vivo. Inhibition of MMPs has, therefore, become the focus of considerable interest in connection with a variety of potential therapeutic applications. We have discovered a nontoxic MMP-2-selective inhibitor effective at nanomolar range on recombinant MMP. This compound, cyclopentylcarbamoylphosphonic acid, significantly inhibited cellular invasion and capillary formation in vitro. Further, i.p. or oral administration of the compound significantly reduced lung metastasis formation and s.c. tumor growth in a murine melanoma model. The effect of this novel compound on lung colonization, capillary formation, and s.c. tumor growth indicates that the compound might also be effective in treatment of primary tumor growth in reduction, or at least in prevention, of further tumor growth, thereby reducing the tumor burden of the patient by a nontoxic approach.

Carbamoylphosphonates, a new class of in vivo active matrix metalloproteinase inhibitors. 1. Alkyl- and cycloalkylcarbamoylphosphonic acids.

Matrix metalloproteinases (MMPs) are a family of over 20 zinc-dependent enzymes that hydrolyze connective tissue and are involved in a variety of diseases, which are associated with undesired tissue breakdown. This paper reports the synthesis, characterization, and biological evaluation of a novel class of MMP inhibitors based on the carbamoylphosphonic acid function. We report a series of 10 open chain N-alkylcarbamoylphosphonic acids (ranging from R = C(1) to C(6) groups), eight N-cycloalkylcarbamoylphosphonic acids (ranging from cyclopropyl to cyclooctyl rings), and four N,N-dialkylcarbamoylphosphonic acids. The compounds were evaluated in three in vitro models, which consisted of (a) the in vitro invasion across a reconstituted basement membrane, (b) determination of the IC(50) values on recombinant MMP-1, MMP-2 MMP-3, MMP-8, and MMP-9 enzymes, and (c) an in vitro capillary formation model, which is a model of angiogenesis. Several of the compounds were also tested in an in vivo murine melanoma model. The following general conclusions have been reached: Most compounds show selectivity for MMP-2 over the other MMP subtypes examined. Cycloalkylcarbamoylphosphonic acids are more potent than comparable open-chain alkyl compounds. Optimal activity against MMP-2 among the cycloalkyl derivatives was shown by N-cyclopentylcarbamoylphosphonic acid (3m). N,N-Dialkylcarbamoylphosphonic acids that were examined showed weak or no activity. The compounds examined showed toxic effects neither in vitro nor in vivo in the concentrations used. Carbamoylphosphonic acids are water soluble at physiological pH and are stable indefinitely.

Carbamoylphosphonate-based matrix metalloproteinase inhibitor metal complexes: solution studies and stability constants. Towards a zinc-selective binding group.

Overactive matrix metalloproteinases (MMPs) are associated with a variety of disease states. Therefore, their inhibition is a highly desirable goal. Yet, more than a decade of worldwide activity has not produced even one clinically useful inhibitor. Because of the crucial role of zinc in the activity of the enzyme, the design of inhibitors is usually based upon a so-called zinc binding group (ZBG). Yet, many of the hitherto synthesized potent inhibitors failed clinically, presumably because they bind stronger to metals other than zinc. We have developed in vivo potent inhibitors based on the carbamoylphosphonic group as a putative ZBG. In this paper we report stability constants for Ca(II), Mg(II), Zn(II) and Cu(II) complexes of two potent, in vivo active, MMP inhibitors, cyclopentylcarbamoylphosphonic acid (1) and 2-( N, N-dimethylamino)ethylcarbamoylphosphonic acid (2). Precipitation prevented the determination of stability constants for iron(III) complexes of1 and2. For comparison with carbamoylphosphonates1 and2, we synthesized 2-cyclohexyl-1,1-difluoroethylphosphonic acid (3), which does not inhibit MMP, and determined the stability constants of its complexes with Mg(II), Ca(II) and Zn(II). Comparison with the values obtained from the complexes of1 and2 with those from3 indicates participation of the C=O group in the metal binding of the former compounds. The complex stability orders for both1 and2 are Ca(II)8 the dimethylamino group of compound2 can also participate in the binding of the transition metals Cu and Zn. On the other hand, the amino group in carbamoylphosphonic acid2 lowers the stability of the complexes with metals favoring oxygen ligands (Ca, Mg and Fe) and increases the selectivity towards Zn. These results are helpful for rationalizing the results observed on our MMP inhibitors hitherto examined, and are expected to be useful for the design of new selective inhibitors.