The Rise of Synaptic Density PET Imaging.

Many neurological disorders are related to synaptic loss or pathologies. Before the boom of positrons emission tomography (PET) imaging of synapses, synaptic quantification could only be achieved in vitro on brain samples after autopsy or surgical resections. Until the mid-2010s, electron microscopy and immunohistochemical labelling of synaptic proteins were the gold-standard methods for such analyses. Over the last decade, several PET radiotracers for the synaptic vesicle 2A protein have been developed to achieve in vivo synapses visualization and quantification. Different strategies were used, namely radiolabelling with either 11C or 18F, preclinical development in rodent and non-human primates, and binding quantification with different kinetic modelling methods. This review provides an overview of these PET tracers and underlines their perspectives and limitations by focusing on radiochemical aspects, as well as preclinical proof-of-concept and the main clinical outcomes described so far.

Robust design of some selective matrix metalloproteinase-2 inhibitors over matrix metalloproteinase-9 through in silico/fragment-based lead identification and de novo lead modification: Syntheses and biological assays.

Broad range of selectivity possesses serious limitation for the development of matrix metalloproteinase-2 (MMP-2) inhibitors for clinical purposes. To develop potent and selective MMP-2 inhibitors, initially multiple molecular modeling techniques were adopted for robust design. Predictive and validated regression models (2D and 3D QSAR and ligand-based pharmacophore mapping studies) were utilized for estimating the potency whereas classification models (Bayesian and recursive partitioning analyses) were used for determining the selectivity of MMP-2 inhibitors over MMP-9. Bayesian model fingerprints were used to design selective lead molecule which was modified using structure-based de novo technique. A series of designed molecules were prepared and screened initially for inhibitions of MMP-2 and MMP-9, respectively, as these are designed followed by other MMPs to observe the broader selectivity. The best active MMP-2 inhibitor had IC50 value of 24nM whereas the best selective inhibitor (IC50=51nM) showed at least 4 times selectivity to MMP-2 against all tested MMPs. Active derivatives were non-cytotoxic against human lung carcinoma cell line-A549. At non-cytotoxic concentrations, these inhibitors reduced intracellular MMP-2 expression up to 78% and also exhibited satisfactory anti-migration and anti-invasive properties against A549 cells. Some of these active compounds may be used as adjuvant therapeutic agents in lung cancer after detailed study.

SAR studies on hydropentalene derivatives--Important core units of biologically active tetramic acid macrolactams and ptychanolides.

Structurally diverse bicyclo[3.3.0]octanes were prepared and tested for their biological activity. Both the antiproliferative activity and the results of phenotypic characterization varied with the substitution patterns. Two derivatives displayed high inhibitory (IC50 ≤3µM) activity against the L-929 cell line, but differed in their mode of action. A cluster analysis with impedance profiling data showed the two compounds in relationship to microtubule interfering compounds. In PtK2 cells treated with both derivatives a perturbing effect on the microtubular network was observed, whereas the actin cytoskeleton in incubated PtK2 cells was disturbed only by one compound. The effects on tubulin and actin polymerization could be confirmed by in vitro polymerization experiments.

Reversal of nucleophilicity of enamides in water: control of cyclization pathways by reaction media for the orthogonal synthesis of dihydropyridinone and pyrrolidinone Clausena alkaloids.

A highly efficient, metal-free, and divergent method for the synthesis of 3,4-dihydropyridin-2-one and pyrrolidin-2-one Clausena alkaloids and their analogs is reported. While the oxirane-containing enamides underwent the TFA-mediated 6-endo-enamide-epoxide cyclization reaction in Bu(t)OH to produce homoclausenamides, an unprecedented nucleophilic reaction occurred at the alpha-carbon of enamides in water to yield 5-endo-enamide-epoxide cyclization products in excellent yields. The reversal of nucleophilicity of enamides in intramolecular cyclization is discussed in terms of steric and electronic effects of the oxirane-containing enamides.

Design, synthesis, and anticonvulsant activity of N-phenylamino derivatives of 3,3-dialkyl-pyrrolidine-2,5-diones and hexahydro-isoindole-1,3-diones.

In the present study on the development of new anticonvulsants, the library of differently substituted N-phenylamino pyrrolidine-2,5-dione and hexahydro-isoindole-1,3-dione derivatives was synthesized. The anticonvulsant activity of all the compounds was evaluated using the maximal electroshock (MES) and pentylenetetrazole (scPTZ) screens, which are the most widely employed seizure models for early identification of candidate anticonvulsants. Their neurotoxicity was determined applying the rotorod test. The pharmacological results revealed that the majority of compounds were effective in electrical (MES) and/or pentylenetetrazole induced seizure (scPTZ) models. The quantitative in vivo anticonvulsant evaluation of N-phenylamino-3,3-dimethyl-pyrrolidine-2,5-dione (15), conducted at the time of peak pharmacodynamic activity (TPE), showed the MES ED(50) value of 69.89mg/kg in rats. The median toxic dose (TD(50)) was 500mg/kg, providing compound 15 with a protective index (TD(50)/ED(50)) of 7.15 in the MES test.

2-[3-[2-[(2S)-2-Cyano-1-pyrrolidinyl]-2-oxoethylamino]-3-methyl-1-oxobutyl]- 1,2,3,4-tetrahydroisoquinoline: a potent, selective, and orally bioavailable dipeptide-derived inhibitor of dipeptidyl peptidase IV.

Dipeptidyl peptidase IV (DPP-IV) inhibitors are expected to become a new type of antidiabetic drugs. Most known DPP-IV inhibitors often resemble the dipeptide cleavage products, with a proline mimic at the P1 site. As off-target inhibitions of DPP8 and/or DPP9 have shown profound toxicities in the in vivo studies, it is important to develop selective DPP-IV inhibitors for clinical usage. To achieve this, a new class of 2-[3-[[2-[(2S)-2-cyano-1-pyrrolidinyl]-2-oxoethyl]amino]-1-oxopropyl]-based DPP-IV inhibitors was synthesized. SAR studies resulted in a number of DPP-IV inhibitors, having IC(50) values of <50 nM with excellent selectivity over both DPP8 (IC(50) > 100 microM) and DPP-II (IC(50) > 30 microM). Compound 21a suppressed the blood glucose elevation after an oral glucose challenge in Wistar rats and also inhibited plasma DPP-IV activity for up to 4 h in BALB/c mice. The results show that compound 21a possesses in vitro and in vivo activities comparable to those of NVP-LAF237 (4), which is in clinical development.

A novel class of achiral seco-analogs of CC-1065 and the duocarmycins: design, synthesis, DNA binding, and anticancer properties.

The synthesis, DNA binding properties, and in vitro and in vivo anticancer activity of fifteen achiral seco-cyclopropylindoline (or achiral seco-CI) analogs (5a-o) of CC-1065 and the duocarmycins are described. The achiral seco-CI analogs contain a 4-hydroxyphenethyl halide moiety that is attached to a wide range of indole, benzimidazole, pyrrole, and pyridyl-containing noncovalent binding components. The 4-hydroxyphenethyl halide moiety represents the simplest mimic of the seco-cyclopropylpyrroloindoline (seco-CPI) pharmacophore found in the natural products, and it lacks a chiral center. The sequence and minor groove specificity of the achiral compounds was ascertained using a Taq DNA polymerase stop assay and a thermal induced DNA cleavage experiment using either a fragment of pBR322 or pUC18 plasmid DNA. For example, seco-CI-InBf (5a) and seco-CI-TMI (5c) demonstrated specificity for AT-rich sequences, particularly by reacting with the underlined adenine-N3 position of 5'-AAAAA(865)-3'. This is also the sequence that CC-1065 and adozelesin prefer to alkylate. The achiral seco-CI compounds were subjected to cytotoxicity studies against several human (K562, LS174T, PC3, and MCF-7) and murine cancer cell lines (L1210 and P815). Following continuous drug exposure, the achiral compounds were found to be cytotoxic, with IC(50) values in the muM range. Interestingly, the carbamate protected compound 5p was significantly less cytotoxic than agent 5c, supporting the hypothesis that loss of HCl and formation of a spiro[2,5]cyclopropylcyclohexadienone intermediate is necessary for biological activity. The achiral seco-CI compounds 5a and 5c were submitted to the National Cancer Institute for further cytotoxicity screening against a panel of 60 different human cancer cell lines. Both compounds showed significant activity, particularly against several solid tumor cell lines. Flow cytometry studies of P815 cells that were incubated with compound 5c at its IC(50) concentration for 24h showed induction of apoptosis in a large percentage of cells. Compounds 5a and 5c were selected by the NCI for an in vivo anticancer hollow-fiber test, and received composite scores of 18 and 22, respectively. These two compounds were subsequently evaluated for in vivo anticancer activity against the growth of a human advanced stage SC UACC-257 melanoma in skid mice. At a dose of 134 mg/kg administered IP, compound 5c gave a T/C value of 40% (for day 51), and the median number of days of doubling tumor growth was 27.7, versus 15.8 for untreated animals. For compound 5a, at 200mg/kg, the T/C was 58% and the median number of days of doubling tumor growth was 20.0 versus 8.7 for untreated animals. At these doses no toxicity or weight loss was observed for either compound. Furthermore, compound 5c was not toxic to murine bone marrow cell growth in culture, at a dose that was toxic for the previously reported seco-CBI (cyclopropylbenzoindoline)-TMI (4).

Discovery of 4-substituted pyrrolidone butanamides as new agents with significant antiepileptic activity.

(S)-alpha-ethyl-2-oxopyrrolidine acetamide 2 (levetiracetam, Keppra, UCB S.A.), a structural analogue of piracetam, has recently been approved as an add-on treatment of refractory partial onset seizures in adults. This drug appears to combine significant efficacy and high tolerability due to a unique mechanism of action. The latter relates to a brain-specific binding site for 2 (LBS for levetiracetam binding site) that probably plays a major role in its antiepileptic properties. Using this novel molecular target, we initiated a drug-discovery program searching for ligands with significant affinity to LBS with the aim to characterize their therapeutic potential in epilepsy and other central nervous system diseases. We systematically investigated the various positions of the pyrrolidone acetamide scaffold. We found that (i) the carboxamide moiety on 2 is essential for affinity; (ii) among 100 different side chains, the preferred substitution alpha to the carboxamide is an ethyl group with the (S)-configuration; (iii) the 2-oxopyrrolidine ring is preferred over piperidine analogues or acyclic compounds; (iv) substitution of positions 3 or 5 of the lactam ring decreases the LBS affinity; and (v) 4-substitution of the lactam ring by small hydrophobic groups improves the in vitro and in vivo potency. Six interesting candidates substituted in the 4-position have been shown to be more potent antiseizure agents in vivo than 2. Further pharmacological studies from our group led to the selection of (2S)-2-[(4R)-2-oxo-4-propylpyrrolidin-1-yl]butanamide 83alpha (ucb 34714) as the most interesting candidate. It is approximately 10 times more potent than 2 as an antiseizure agent in audiogenic seizure-prone mice. A clinical phase I program has been successfully concluded and 83alpha will commence several phase II trials during 2003.

Highly efficient sequence-specific DNA interstrand cross-linking by pyrrole/imidazole CPI conjugates.

We have developed a novel type of DNA interstrand cross-linking agent by synthesizing dimers of a pyrrole (Py)/imidazole (Im)-diamide-CPI conjugate, ImPyLDu86 (1), connected using seven different linkers. The tetramethylene linker compound, 7b, efficiently produces DNA interstrand cross-links at the nine-base-pair sequence, 5'-PyGGC(T/A)GCCPu-3', only in the presence of a partner triamide, ImImPy. For efficient cross-linking by 7b with ImImPy, one A.T base pair between two recognition sites was required to accommodate the linker region. Elimination of the A.T base pair and insertion of an additional A.T base pair and substitution with a G.C base pair significantly reduced the degree of cross-linking. The sequence specificity of the interstrand cross-linking by 7b was also examined in the presence of various triamides. The presence of ImImIm slightly reduced the formation of a cross-linked product compared to ImImPy. The mismatch partners, ImPyPy and PyImPy, did not produce an interstrand cross-link product with 7b, whereas ImPyPy and PyImPy induced efficient alkylation at their matching site with 7b. The interstrand cross-linking abilities of 7b were further examined using denaturing polyacrylamide gel electrophoresis with 5'-Texas Red-labeled 400- and 67-bp DNA fragments. The sequencing gel analysis of the 400-bp DNA fragment with ImImPy demonstrated that 7b alkylates several sites on the top and bottom strands, including one interstrand cross-linking match site, 5'-PyGGC(T/A)GCCPu-3'. To obtain direct evidence of interstrand cross-linkages on longer DNA fragments, a simple method using biotin-labeled complementary strands was developed, which produced a band corresponding to the interstrand cross-linked site on both top and bottom strands. Densitometric analysis indicated that the contribution of the interstrand cross-link in the observed alkylation bands was approximately 40%. This compound efficiently cross-linked both strands at the target sequence. The present system consisted of a 1:2 complex of the alkylating agent and its partner ImImPy and caused an interstrand cross-linking in a sequence-specific fashion according to the base-pair recognition rule of Py-Im polyamides.

Synthesis and anticonvulsant properties of 2,3,3a,4-tetrahydro-1H-pyrrolo[1,2-a]benzimidazol-1-one derivatives.

A number of novel 1H-pyrrolo[1,2-a]benzimidazol-1-one derivatives were prepared and their anticonvulsant properties evaluated. The new synthesized compounds proved to possess anticonvulsant effects depending on the nature of substituents at C-6, C-2, and C-3a positions of the polycyclic system. In particular, the 6-chloro-3a-(p-tolyl)-2,3,3a,4-tetrahydro-1H-pyrrolo[1,2-a]benzimidazol-1-one derivative (22) displayed potency fivefold higher than unsubstituted compound (13).

Synthesis of the optical isomers of 4-[1-(4-tert-butylphenyl)-2-oxo- pyrrolidine-4-yl]methyloxybenzoic acid (S-2) and their biological evaluation as antilipidemic agent.

The enantiomers of (+/-)-4-[1-(4-tert-butylphenyl)-2-oxo-pyrrolidine- 4-yl]methyloxybenzoic acid (S-2), a new antilipidemic agent having dual action on the plasma triglyceride (TG) and cholesterol (Cho) lowering effects, were prepared via separation by Chiralcel OJ column chromatography of their methyl ester and also by the same method as the described racemate's synthesis from optically active 1-(4-tert-butylphenyl)-2-oxo-pyrrolidine-4-carboxylic acid respectively. These optically active carboxylic acids were prepared by the resolution of diastereomeric N-[(S)-(-)-[4-methyl-(alpha-methyl)benzyl]]-1-(4-tert-butylphenyl)-2-oxo - pyrrolidine-4-carboxyamide using silica gel column chromatography, followed by deamination with N2O4. The absolute configurations for the enantiomers of S-2 were indirectly determined using X-ray analysis of the 4-bromo-2-fluorobenzamide of the (+)-4-[1-(4-tert-butylphenyl)-2- oxo-pyrrolidine-4-yl]-methyloxybenzoic acid. S-2 and its enantiomers showed an essentially equipotent activity on the fatty acid- and sterol-biosynthesis inhibition in vitro. On the other hand, in the in vivo activity, (S)-(+)-4-[1-(4-tert-butylphenyl)-2-oxo-pyrrolidine- 4-yl]methyloxybenzoic acid (S-2E) was superior in the lowering abilities of the plasma TG and phospholipid(PL) and was chosen as a candidate for a novel antilipidemic agent. The difference in the in vivo activity among S-2 and its enantiomers was explained from the pharmacokinetics after administration p.o.

Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. 4. Incorporation of P1 lactam moieties as L-glutamine replacements.

The structure-based design, chemical synthesis, and biological evaluation of various human rhinovirus (HRV) 3C protease (3CP) inhibitors which incorporate P1 lactam moieties in lieu of an L-glutamine residue are described. These compounds are comprised of a tripeptidyl or peptidomimetic binding determinant and an ethyl propenoate Michael acceptor moiety which forms an irreversible covalent adduct with the active site cysteine residue of the 3C enzyme. The P1-lactam-containing inhibitors display significantly increased 3CP inhibition activity along with improved antirhinoviral properties relative to corresponding L-glutamine-derived molecules. In addition, several lactam-containing compounds exhibit excellent selectivity for HRV 3CP over several other serine and cysteine proteases and are not appreciably degraded by a variety of biological agents. One of the most potent inhibitors (AG7088, mean antirhinoviral EC90 approximately 0.10 microM, n = 46 serotypes) is shown to warrant additional preclinical development to explore its potential for use as an antirhinoviral agent.

Synthesis and anticonvulsant properties of 2,3,3a,4-tetrahydro-1H-pyrrolo[1,2-a]benzimidazol-1-ones.

A series of 2,3,3a,4-tetrahydro-1H-pyrrolo[1,2-a]benzimidazol-1-ones were synthesized and evaluated for anticonvulsant activity in DBA/2 mice against sound-induced seizures and in rats against maximal electroshock-induced seizures. Most of the derivatives showed an anticonvulsant effect better than that of valproate, a commonly used anticonvulsant drug. Compound 3 possessed an anticonvulsant activity comparable to that of diphenylhydantoin in both tests and was selected for further studies. Structure-activity relationships are discussed.