Experimental and Theoretical Analysis of the Thiol-Promoted Fragmentation of 2-Halo-3-tosyl-oxanorbornadienes.

2-Halo-3-tosyl-oxanorbornadienes are able to accept two thiol molecules through an initial nucleophilic substitution, giving isolable oxabicyclic thiovinyl sulfones that, subsequently, can react with a second thiol molecule via thio-Michael addition. The resulting oxanorbornenic thioketals undergo retro-Diels-Alder (rDA) fragmentation to release a furan derivative and a ketene S,S-acetal. The substitution pattern of the oxanorbornadienic skeleton influences the rate of the rDA through electronic and steric factors examined by quantum mechanical calculations.

Transferring Substituents from Alkynes to Furans and Pyrroles through Heteronorbornadienes as Intermediates: Synthesis of ß-Substituted Pyrroles/Furans.

The use of 7-oxa/azanorbornadienes as synthetic intermediates for the preparation of 3/4-substituted (ß-substituted) furans/pyrroles is presented. The method lies in the inverse electron demand Diels-Alder (iEDDA) cycloaddition between a substituted heteronorbornadiene and an electron-poor tetrazine followed by spontaneous fragmentation of the resulting cycloadduct via two retro-Diels-Alder (rDA) reactions affording a ß-substituted furan/pyrrole. The scope of this tandem iEDDA/rDA/rDA reaction was explored in the preparation of 29 heterocycles. A one-pot procedure starting directly from the alkyne precursors of the heteronorbornadiene intermediates is also described.

Synthesis and structure-activity relationship of new nicotinamide phosphoribosyltransferase inhibitors with antitumor activity on solid and haematological cancer.

Cancer cells are highly dependent on Nicotinamide phosphoribosyltransferase (NAMPT) activity for proliferation, therefore NAMPT represents an interesting target for the development of anti-cancer drugs. Several compounds, such as FK866 and CHS828, were identified as potent NAMPT inhibitors with strong anti-cancer activity, although none of them reached the late stages of clinical trials. We present herein the preparation of three libraries of new inhibitors containing (pyridin-3-yl)triazole, (pyridin-3-yl)thiourea and (pyridin-3/4-yl)cyanoguanidine as cap/connecting unit and a furyl group at the tail position of the compound. Antiproliferative activity in vitro was evaluated on a panel of solid and haematological cancer cell lines and most of the synthesized compounds showed nanomolar or sub-nanomolar cytotoxic activity in MiaPaCa-2 (pancreatic cancer), ML2 (acute myeloid leukemia), JRKT (acute lymphobalistic leukemia), NMLW (Burkitt lymphoma), RPMI8226 (multiple myeloma) and NB4 (acute myeloid leukemia), with lower IC50 values than those reported for FK866. Notably, compounds 35a, 39a and 47 showed cytotoxic activity against ML2 with IC50 = 18, 46 and 49 pM, and IC50 towards MiaPaCa-2 of 0.005, 0.455 and 2.81 nM, respectively. Moreover, their role on the NAD+ synthetic pathway was demonstrated by the NAMPT inhibition assay. Finally, the intracellular NAD+ depletion was confirmed in vitro to induced ROS accumulation that cause a time-dependent mitochondrial membrane depolarization, leading to ATP loss and cell death.

Studies on the Regioselective Rearrangement of Azanorbornanic Aminyl Radicals into 2,8-Diazabicyclo[3.2.1]oct-2-ene Systems.

Aminyl radicals are nitrogen-centered radicals of interest in synthetic strategies involving C-N bond formation due to their high reactivity. These intermediate radicals are generated by the reaction of an organic azide with tributyltin hydride (Bu3SnH) in the presence of substoichiometric amounts of azobisisobutyronitrile (AIBN). In this work, we report the regioselective rearrangement of azanorbornanic ([2.2.1]azabicyclic) aminyl radicals into 2,8-diazabicyclo[3.2.1]oct-2-ene systems. With the aim to establish the structural requirements for this ring expansion, we have studied the effect of different bridgehead atoms of the [2.2.1]bicyclic system and the presence of an alkyl substituent at C4. Attempts to perform this ring expansion on a monocyclic analogue have been also explored to evaluate the influence of the bicyclic skeleton on the rearrangement. A detailed mechanistic proposal supported by computational studies is reported.

Identification of new FK866 analogues with potent anticancer activity against pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal diseases for which chemotherapy has not been very successful yet. FK866 ((E)-N-(4-(1-benzoylpiperidin-4-yl)butyl)-3-(pyridin-3-yl)acrylamide) is a well-known NAMPT (nicotinamide phosphoribosyltransferase) inhibitor with anti-cancer activities, but it failed in phase II clinical trials. We found that FK866 shows anti-proliferative activity in three PDAC cell lines, as well as in Jurkat T-cell leukemia cells. More than 50 FK866 analogues were synthesized that introduce substituents on the phenyl ring of the piperidine benzamide group of FK866 and exchange its buta-1,4-diyl tether for 1-oxyprop-3-yl, (E)-but-2-en-1,4-diyl and 2- and 3-carbon tethers. The pyridin-3-yl moiety of FK866 was exchanged for chlorinated and fluorinated analogues and for pyrazin-2-yl and pyridazin-4-yl groups. Several compounds showed low nanomolar or sub-nanomolar cell growth inhibitory activity. Our best cell anti-proliferative compounds were the 2,4,6-trimethoxybenzamide analogue of FK866 ((E)-N-(4-(1-(2,4,6-trimethoxybenzoyl)piperidin-4-yl)butyl)-3-(pyridin-3-yl)acrylamide) (9), the 2,6-dimethoxybenzamide (8) and 2-methoxybenzamide (4), which exhibited an IC50 of 0.16 nM, 0.004 nM and 0.08 nM toward PDAC cells, respectively.

Discovery of human hexosaminidase inhibitors by in situ screening of a library of mono- and divalent pyrrolidine iminosugars.

Two libraries of mono- and dimeric pyrrolidine iminosugars were synthesized by CuAAC and (thio)urea-bond-forming reactions from the respective azido/aminohexylpyrrolidine iminosugar precursors. The resulting monomeric and dimeric compounds were screened for inhibition of ß-N-acetylglucosaminidase from Jack beans, the plant ortholog of human lysosomal hexosaminidases. A selection of the best inhibitors of these libraries was then evaluated against human lysosomal ß-N-acetylhexosaminidase B (hHexB) and human nucleocytoplasmic ß-N-acetylglucosaminidase (hOGA). This evaluation identified a potent (nM) and selective monomeric inhibitor of hOGA (compound 7A) that showed a 6770-fold higher affinity for this enzyme than for hHexB. The corresponding dimeric derivative (compound 9D) further remarkably improved the selectivity in the inhibition of hOGA (2.7 × 104 times more selective for hOGA over hHexB) and the inhibition potency (by one order of magnitude). Docking studies were performed to explain the selectivity of inhibition observed in compound 7A.

Regioselectivity of the 1,3-Dipolar Cycloaddition of Organic Azides to 7-Heteronorbornadienes. Synthesis of ß-Substituted Furans/Pyrroles.

An efficient procedure for the preparation of ß-substituted furans/pyrroles is presented. The methodology is based on the use of 7-oxa/azanorbornadienes as dipolarophiles in 1,3-dipolar cycloaddition with benzyl azide. The triazoline cycloadduct thus formed spontaneously decomposes via a retro-Diels-Alder (rDA) reaction to afford a ß-substituted furan/pyrrole derivative and a stable triazole. The scope of this tandem 1,3-dipolar cycloaddition/rDA reaction was studied with thirteen 7-heteronorbornadienes. This study allowed a deep knowledge of the regioselectivity of the reaction, which can be tuned through the substituents of the heteronorbornadienic systems.

Exploring Multi-Subsite Binding Pockets in Proteins: DEEP-STD NMR Fingerprinting and Molecular Dynamics Unveil a Cryptic Subsite at the GM1 Binding Pocket of Cholera Toxin†B.

Ligand-based NMR techniques to study protein-ligand interactions are potent tools in drug design. Saturation transfer difference (STD) NMR spectroscopy stands out as one of the most versatile techniques, allowing screening of fragments libraries and providing structural information on binding modes. Recently, it has been shown that a multi-frequency STD NMR approach, differential epitope mapping (DEEP)-STD NMR, can provide additional information on the orientation of small ligands within the binding pocket. Here, the approach is extended to a so-called DEEP-STD NMR fingerprinting technique to explore the binding subsites of cholera toxin subunit B (CTB). To that aim, the synthesis of a set of new ligands is presented, which have been subject to a thorough study of their interactions with CTB by weak affinity chromatography (WAC) and NMR spectroscopy. Remarkably, the combination of DEEP-STD NMR fingerprinting and Hamiltonian replica exchange molecular dynamics has proved to be an excellent approach to explore the geometry, flexibility, and ligand occupancy of multi-subsite binding pockets. In the particular case of CTB, it allowed the existence of a hitherto unknown binding subsite adjacent to the GM1 binding pocket to be revealed, paving the way to the design of novel leads for inhibition of this relevant toxin.

Synthesis of multimeric pyrrolidine iminosugar inhibitors of human ß-glucocerebrosidase and α-galactosidase A: First example of a multivalent enzyme activity enhancer for Fabry disease.

The synthesis of a chemical library of multimeric pyrrolidine-based iminosugars by incorporation of three pairs of epimeric pyrrolidine-azides into different alkyne scaffolds via CuAAC is presented. The new multimers were evaluated as inhibitors of two important therapeutic enzymes, human α-galactosidase A (α-Gal A) and lysosomal ß-glucocerebrosidase (GCase). Structure-activity relationships were established focusing on the iminosugar inhitope, the valency of the dendron and the linker between the inhitope and the central scaffold. Remarkable is the result obtained in the inhibition of α-Gal A, where one of the nonavalent compounds showed potent inhibition (0.20 µM, competitive inhibition), being a 375-fold more potent inhibitor than the monovalent reference. The potential of the best α-Gal A inhibitors to act as pharmacological chaperones was analyzed by evaluating their ability to increase the activity of this enzyme in R301G fibroblasts from patients with Fabry disease, a genetic disorder related with a reduced activity of α-Gal A. The best enzyme activity enhancement was obtained for the same nonavalent compound, which increased 5.2-fold the activity of the misfolded enzyme at 2.5 µM, what constitutes the first example of a multivalent α-Gal A activity enhancer of potential interest in the treatment of Fabry disease.

Structural basis of the inhibition of GH1 ß-glucosidases by multivalent pyrrolidine iminosugars.

The synthesis of multivalent pyrrolidine iminosugars via CuAAC click reaction between different pyrrolidine-azide derivatives and tri- or hexavalent alkynyl scaffolds is reported. The new multimeric compounds, together with the monomeric reference, were evaluated as inhibitors against two homologous GH1 ß-glucosidases (BglA and BglB from Paenibacillus polymyxa). The multivalent inhibitors containing an aromatic moiety in the linker between the pyrrolidine and the scaffold inhibited the octameric BglA (µM range) but did not show affinity against the monomeric BglB, despite the similarity between the active site of both enzymes. A modest multivalent effect (rp/n = 12) was detected for the hexavalent inhibitor 12. Structural analysis of the complexes between the monomeric and the trimeric iminosugar inhibitors (4 and 10) and BglA showed the insertion of the inhibitors at the active site of BglA, confirming a competitive mode of inhibition as indicated by enzyme kinetics. Additionally, structural comparison of the BglA/4 complex with the reported BglB/2F-glucose complex illustrates the key determinants responsible for the inhibitory effect and explains the reasons of the inhibition of BglA and the no inhibition of BglB. Potential inhibition of other ß-glucosidases with therapeutic relevance is discussed under the light of these observations.

Exploring substituent diversity on pyrrolidine-aryltriazole iminosugars: Structural basis of ß-glucocerebrosidase inhibition.

The synthesis of a library of pyrrolidine-aryltriazole hybrids through CuAAC between two epimeric dihydroxylated azidomethylpyrrolidines and differently substituted phenylacetylenes is reported. The evaluation of the new compounds as inhibitors of lysosomal ß-glucocerebrosidase showed the importance of the substitution pattern of the phenyl moiety in the inhibition. Crystallization and docking studies revealed key interactions of the pyrrolidine motif with aminoacid residues of the catalytic site while the aryltriazole moiety extended along a hydrophobic surface groove. Some of these compounds were able to increase the enzyme activity in Gaucher patient fibroblasts, acting as a new type of chemical chaperone for Gaucher disease.

Discovery of a Potent α-Galactosidase Inhibitor by in Situ Analysis of a Library of Pyrrolizidine-(Thio)urea Hybrid Molecules Generated via Click Chemistry.

The parallel synthesis of a 26-membered-library of aromatic/aliphatic-(thio)urea-linked pyrrolizidines followed by in situ biological evaluation toward α-galactosidases has been carried out. The combination of the (thio)urea-forming click reaction and the in situ screening is pioneer in the search for glycosidase inhibitors and has allowed the discovery of a potent coffee bean α-galactosidase inhibitor (IC50 = 0.37 µM, Ki = 0.12 µM) that has also showed inhibition against human lysosomal α-galactosidase (α-Gal A, IC50 = 5.3 µM, Ki = 4.2 µM).

Harnessing pyrrolidine iminosugars into dimeric structures for the rapid discovery of divalent glycosidase inhibitors.

The synthesis of three libraries (1a-l, 1a'-l' and 2a-l) of dimeric iminosugars through CuAAC reaction between three different alkynyl pyrrolidines and a set of diazides was carried out. The resulting crude dimers were screened in situ against two α-fucosidases (libraries 1a-l and 1a'-l') and one ß-galactosidase (2a-l). This method is pioneer in the search of divalent glycosidase inhibitors. It has allowed the rapid identification of dimer 1i as the best inhibitor of α-fucosidases from bovine kidney (Ki = 0.15 nM) and Homo sapiens (Ki = 60 nM), and dimer 2e as the best inhibitor of ß-galactosidase from bovine liver (Ki = 5.8 µM). In order to evaluate a possible divalent effect in the inhibition, the synthesis and biological analysis of the reference monomers were also performed. Divalent effect was only detected in the inhibition of bovine liver ß-galactosidase by dimer 2e.

Tuning of ß-glucosidase and α-galactosidase inhibition by generation and in situ screening of a library of pyrrolidine-triazole hybrid molecules.

The preliminary screening of two libraries of epimeric (pyrrolidin-2-yl)triazoles (14a-s and 22a-s), generated via click chemistry, allowed the rapid identification of four α-galactosidase (coffee beans) inhibitors (22b,k,p,r) and two ß-glucosidase (almond) inhibitors (14b,f) in the low µM range. The additional biological analysis of 14b,f towards ß-glucocerebrosidase (human lysosomal ß-glucosidase), as target enzyme for Gaucher disease, showed a good correlation with the inhibition results obtained for the plant (almond) enzyme. Surprisingly, although these compounds showed inhibition towards ß-glucocerebrosidase as acid hydrolase, they did not inhibit bovine liver ß-glucosidase as neutral hydrolase. In contrast to what was observed for ß-glucosidase inhibition, the coffee bean α-galactosidase inhibitors of the epimeric library (22b,k,p,r) only showed weak inhibition towards human lysosomal α-galactosidase.

Expanding the library of divalent fucosidase inhibitors with polyamino and triazole-benzyl bridged bispyrrolidines.

A small library of divalent fucosidase inhibitors containing pyrrolidine motifs and separated by polyamino and triazole-benzylated spacers was prepared and evaluated as α-fucosidase inhibitors. Although a weak multivalent effect was observed in polyamino derived dimers, useful structural information can be deduced about the length of the bridge, the number of nitrogen atoms present and the moieties close to the pyrrolidine. Within these investigations one of the best α-fucosidase inhibitors containing a pyrrolidine framework was obtained (18, Ki = 3.7 nM).

Rapid discovery of potent α-fucosidase inhibitors by in situ screening of a library of (pyrrolidin-2-yl)triazoles.

The synthesis of a small library of (pyrrolidin-2-yl)triazoles via copper catalysed cycloaddition of an alkynyl iminocyclopentitol and a set of commercial and synthetic azides has been achieved. The in situ screening for the activity towards α-fucosidase of the resulting triazoles has allowed the identification of one of the most potent and selective pyrrolidine derived inhibitors of this enzyme (Ki = 4 nM).

Synthesis, biological evaluation, WAC and NMR studies of S-galactosides and non-carbohydrate ligands of cholera toxin based on polyhydroxyalkylfuroate moieties.

The synthesis of several non-carbohydrate ligands of cholera toxin based on polyhydroxyalkylfuroate moieties is reported. Some of them have been linked to D-galactose through a stable and well-tolerated S-glycosidic bond. They represent a novel type of non-hydrolyzable bidentate ligand featuring galactose and polyhydroxyalkylfuroic esters as pharmacophoric residues, thus mimicking the GM1 ganglioside. The affinity of the new compounds towards cholera toxin was measured by weak affinity chromatography (WAC). The interaction of the best candidates with this toxin was also studied by saturation transfer difference NMR experiments, which allowed identification of the binding epitopes of the ligands interacting with the protein. Interestingly, the highest affinity was shown by non-carbohydrate mimics based on a polyhydroxyalkylfuroic ester structure.

Strain-promoted retro-Dieckmann-type condensation on [2.2.2]- and [2.2.1]bicyclic systems: a fragmentation reaction for the preparation of functionalized heterocycles and carbocycles.

The fragmentation reaction of differently functionalized [2.2.2]- and [2.2.1]bicyclic systems that leads to substituted five membered heterocycles and five/six membered carbocycles is broadly studied. This reaction is carried out through a retro-Dieckmann-type condensation on strained [2.2.1]bicyclic ß-ketosulfones and their counterparts ß-ketoesters under very mild catalytic acid or basic conditions and short reaction times. The same reaction is also achieved on [2.2.2]bicyclic ß-ketosulfones requiring harsher reaction conditions.

Three dimensional structure of a bacterial α-l-fucosidase with a 5-membered iminocyclitol inhibitor.

Fucosidases, enzymes that cleave fucose from the non-reducing end of a glycan, represent promising medicinal targets reflecting their roles in cancer metastasis, inflammation, host-parasite interactions and the lysosomal storage disorder fucosidosis. The X-ray crystal structures of Bacteroides thetaiotaomicron GH29 α-l-fucosidase (BtFuc2970) in a new crystal form (at a resolution of 1.59Å) and liganded with a 5-membered iminocyclitol inhibitor (1.73Å) are reported herein. The 5-membered iminocyclitol binds in a (3)E conformation, mimicking the proposed (3)H4 half chair transition-state of the enzyme catalysed reaction, and its Ki for BtFuc2970 was determined as 2µM. Structural analysis of fucosidase inhibition through 5-membered iminocyclitols will aid in the rational design of more potent fucosidase inhibitors for treatment of a range of medical conditions.

Exploiting the ring strain in bicyclo[2.2.1]heptane systems for the stereoselective preparation of highly functionalized cyclopentene, dihydrofuran, pyrroline, and pyrrolidine scaffolds.

The high strain of bicyclic systems drives retro-condensation reactions on bridgehead substituted bicyclo[2.2.1]hept-2-enes giving rise to orthogonally functionalized cyclopentene, 2,5-dihydrofuran, and 3-pyrroline scaffolds. Retro-Dieckman reactions were easily carried out on 3-tosyl-(7-carba/7-oxa/7-aza)bicyclo[2.2.1]hept-5-en-2-ones. Retro-aldol reactions of N-Boc-3-tosyl-7-azabicyclo[2.2.1]hept-5-en-2-ol and functionalized N-Boc-3-tosyl-7-azabicyclo[2.2.1]heptan-2-ols yield functionalized pyrrolidine scaffolds stereoselectively. The same reaction does not work with corresponding norbornene and 7-oxanorbornene derivatives.