Ana Conejo-García

Homochiral Drugs: A Demanding Tendency of the Pharmaceutical Industry

The issue of drug chirality is now a major theme in the design and development of new drugs, underpinned by a new understanding of the role of molecular recognition in many pharmacologically relevant events. In general, three methods are utilized for the production of a chiral drug: the chiral pool, separation of racemates, and asymmetric synthesis. Although the use of chiral drugs predates modern medicine, only since the 1980s has there been a significant increase in the development of chiral pharmaceutical drugs. An important commercial reason is that as patents on racemic drugs expire, pharmaceutical companies have the opportunity to extend patent coverage through development of the chiral switch enantiomers with desired bioactivity. Stimulated by the new policy statements issued by the regulatory agencies, the pharmaceutical industry has systematically begun to develop chiral drugs in enantiometrically enriched pure forms. This new trend has caused a tremendous change in the industrial small- and large-scale production to enantiomerically pure drugs, leading to the revisiting and updating of old technologies, and to the development of new methodologies of their large-scale preparation (as the use of stereoselective syntheses and biocatalyzed reactions). The final decision whether a given chiral drug will be marketed in an enantiomerically pure form, or as a racemic mixture of both enantiomers, will be made weighing all the medical, financial and social proficiencies of one or other form. The kinetic, pharmacological and toxicological properties of individual enantiomers need to be characterized, independently of a final decision.

Novel Substituted Quinazolines for Potent EGFR Tyrosine Kinase Inhibitors

The type I receptor tyrosine kinases (RTKs) are involved in various aspects of cell growth, survival, and differentiation. Among the known RTKs, the epidermal growth factor receptor (EGFR) and ErbB-2 (HER-2) are two widely studied proteins that are prototypic members of the ErbB family which also includes ErbB-3 (Her-3) and ErbB-4 (Her-4). Overexpression of ErbB-2 and EGFR has been associated with aggressive disease and poor patient prognosis in a range of human tumour types (e.g. breast, lung, ovarian, prostate, and squamous carcinoma of head and neck). Disruption of signal transduction of these kinases has been shown to have an antiproliferative effect. Various approaches have been developed to target the ErbB signalling pathways including monoclonal antibodies (trastuzumab/Herceptin™ and cetuximab/Erbitux™) directed against the receptor, and synthetic tyrosine kinase inhibitors (gefitinib/Iressa™ and erlotinib/Tarceva™). Since many tumours overexpress ErbB receptors, simultaneous targeting of multiple ErbB receptors therefore becomes a promising approach to cancer treatment. Lapatinib (Tykerb™), a potent dual EGFR/ErbB-2 inhibitor, was approved for the treatment of ErbB-2-positive breast cancer. Despite years of intensive research on EGFR inhibitors, there is a surprising dearth of chemically distinct small inhibitors with a high degree of selectivity. There is also a need for new scaffolds due to the recent finding of EGFR mutations which render the kinase resistant to gefinitib and erlotinib. The structures under study will be quinazolines with different substituents. The structure-activity relationships and biological evaluation of compounds published during the last four years will be reviewed herein.

New (RS)-benzoxazepin-purines with antitumour activity: The chiral switch from (RS)-2,6-dichloro-9-[1-(p-nitrobenzenesulfonyl)-1,2,3,5-tetrahydro-4,1-benzoxazepin-3-yl]-9H-purine.

Completing an SAR study, a series of (RS)-6-substituted-7- or 9-(1,2,3,5-tetrahydro-4,1-benzoxazepine-3-yl)-7H or 9H-purines has been prepared under microwave-assisted conditions. Their antiproliferative activities on MCF-7 and MDA-MB-231 cancerous cell lines are presented, being the majority of the IC(50) values below 1μM. The most active compound (RS)-2,6-dichloro-9-[1-(p-nitrobenzenesulfonyl)-1,2,3,5-tetrahydro-4,1-benzoxazepin-3-yl]-9H-purine (14) presents an IC(50) of 0.166μM against the human cancerous cell line MDA-MB-231. Compound 14 was the most selective against the human breast adenocarcinoma MCF-7 and MDA-MB-231 cancer cell lines (Therapeutic Indexes, TIs=5.1 and 11.0, respectively) in relation to the normal one MCF-10A. (RS)-14 was resolved into its enantiomers. Both enantiomers are equally potent, but more potent than the corresponding racemic mixture. (R)-14 induces apoptosis against MCF-7 up to 52.50% of cell population after 48h, being more potent than the clinical-used drug paclitaxel (43%). (RS)-14 induces no acute toxicity in mice after two weeks of treatment.

Synthesis and anticancer activity of (RS)-9-(2,3-dihydro-1,4-benzoxaheteroin-2-ylmethyl)-9H-purines.

Herein are reported the synthesis and anticancer activity against the human breast cancer cell line MCF-7 of a series of substituted (RS)-9-(2,3-dihydro-1,4-benzoxathiin-2-ylmethyl)-9H-purine derivatives and (RS)-9-(2,3-dihydro-1,4-benzodioxin-2-ylmethyl)-9H-purine derivatives. When the Mitsunobu reaction was carried out between (RS)-2,3-dihydro-1,4-benzoxathiin-3-methanol and the heterocyclic bases 6-chloro-, 2,6-dichloro, and 6-bromo-purines under microwave-assisted conditions, a formal 1,4-sulfur migration takes place through two consecutive oxyranium and episulfonium rings, giving rise to the corresponding (RS)-9-(2,3-dihydro-1,4-benzodioxin-3-ylmethyl)-9H-purine derivatives, previously reported by us. The most active compound (RS)-2,6-dichloro-9-(2,3-dihydro-1,4-benzoxathiin-2-ylmethyl)-9H-purine shows an IC(50) = 2.75 ± 0.02 μM. When the cancerous cells were treated with this compound, a significant increase of apoptotic cells (70.08 ± 0.33%) was obtained in relation to the control ones. The induction of the G(2)/M cell cycle arrest and apoptosis by the three most active compounds is associated with increased phosphorylation of eIF2α in human breast cancer cells.

Homodimeric bis-quaternary heterocyclic ammonium salts as potent acetyl- and butyrylcholinesterase inhibitors: a systematic investigation of the influence of linker and cationic heads over affinity and selectivity.

A molecular library of quaternary ammonium salts (QASs), mainly composed of symmetrical bis-quaternary heterocyclic bromides exhibiting choline kinase (ChoK) inhibitory activity, were evaluated for their ability to inhibit acetyl- and butyrylcholinesterase (AChE and BChE, respectively). The molecular framework of QASs consisted of two positively charged heteroaromatic (pyridinium or quinolinium) or sterically hindered aliphatic (quinuclidinium) nitrogen rings kept at an appropriate distance by lipophilic rigid or semirigid linkers. Many homodimeric QASs showed AChE and BChE inhibitory potency in the nanomolar range along with a low enzymatic selectivity. Computational studies on AChE, BChE, and ChoK allowed identification of the key molecular determinants for high affinity and selectivity over either one of the three enzymes and guided the design of a hybrid bis-QAS (56) exhibiting the highest AChE affinity (IC(50) = 15 nM) and selectivity over BChE and ChoK (SI = 50 and 562, respectively) and a promising pharmacological potential in myasthenia gravis and neuromuscular blockade.

The Mechanism of Allosteric Coupling in Choline Kinase α1 Revealed by the Action of a Rationally Designed Inhibitor

Applying a CHOK hold: Combined experimental and computational studies of the binding mode of a rationally designed inhibitor of the dimeric choline kinase α1 (CHOKα1) explain the molecular mechanism of negative cooperativity (see scheme) and how the monomers are connected. The results give insight into how the symmetry of the dimer can be partially conserved despite a lack of conservation in the static crystal structures.

Lysophosphatidylcholine Regulates Sexual Stage Differentiation in the Human Malaria Parasite Plasmodium falciparum

Summary Transmission represents a population bottleneck in the Plasmodium life cycle and a key intervention target of ongoing efforts to eradicate malaria. Sexual differentiation is essential for this process, as only sexual parasites, called gametocytes, are infective to the mosquito vector. Gametocyte production rates vary depending on environmental conditions, but external stimuli remain obscure. Here, we show that the host-derived lipid lysophosphatidylcholine (LysoPC) controls P. falciparum cell fate by repressing parasite sexual differentiation. We demonstrate that exogenous LysoPC drives biosynthesis of the essential membrane component phosphatidylcholine. LysoPC restriction induces a compensatory response, linking parasite metabolism to the activation of sexual-stage-specific transcription and gametocyte formation. Our results reveal that malaria parasites can sense and process host-derived physiological signals to regulate differentiation. These data close a critical knowledge gap in parasite biology and introduce a major component of the sexual differentiation pathway in Plasmodium that may provide new approaches for blocking malaria transmission.

Synthesis and anticancer activity of (R,S)-9-(2,3-dihydro-1,4-benzoxathiin-3-ylmethyl)-9H-purines.

A series of eleven 2‐ and 6‐substituted (R,S)‐9‐(2,3‐dihydro‐1,4‐benzoxathiin‐3‐ylmethyl)‐9H‐purine derivatives was obtained by applying a standard Mitsunobu protocol that led to a six‐membered ring contraction from (R,S)‐3,4‐dihydro‐2H‐1,5‐benzoxathiepin‐3‐ol via an episulfonium intermediate. The signal ∼δ=151 ppm, which corresponds to the C4′ carbon atom, is unequivocal proof of the N9′ regioisomer. The potential of the target molecules as anticancer agents is reflected in their activity against the MCF‐7 cancer cell line. The most active compounds have IC50 values of (6.18±1.70) and (8.97±0.83) μM. The results indicate that the anticancer activity for the most active compounds is correlated with their capacity to induce apoptosis.

Design, synthesis and anticancer activity against the MCF-7 cell line of benzo-fused 1,4-dihetero seven- and six-membered tethered pyrimidines and purines.

Having previously reported the synthesis and anticancer activities of cyclic 5-fluorouracil (5-FU) O,N-acetalic compounds, the decision was made to change 5-FU for uracil (U), with the prospect of finding an antiproliferative agent endowed with a new mechanism of action. The use of a reverse transcription-PCR-based assay decreased cyclin D1 mRNA, suggesting that this cyclic U O,N-acetalic compound exerts its regulatory action on cyclin D1 at the level of transcription. Following the ongoing Anticancer Drug Programme we planned the synthesis of compounds bearing a natural pyrimidine base and also, the oxygen atom at position 1 of the seven-membered cycle was replaced by its isosteric sulfur atom, and its oxidized states. Next, the pyrimidine base was substituted for the purine one, with the objective of increasing both the lipophilicity and the structural diversity of the target molecules. If the previously described compounds were not prodrugs, it would not be necessary to maintain the O,N-acetalic characteristic. Therefore, molecules were designed in which both structural entities (such as the benzoheterocyclic ring and the purine base) were linked by a heteroatom-C-C-N bond. A series of (RS)-9-(2,3-dihydro-1,4-benzoxathiin-3-ylmethyl)-9H-purine derivatives was obtained and the anticancer activity for the most active compounds was correlated with their capability to induce apoptosis. Finally, completing a SAR study, a series of (RS)-6-substituted-7- or 9-(1,2,3,5-tetrahydro-4,1-benzoxazepine-3-yl)-7H- or 9H-purines was prepared. The studies by microarray technology showed that the main molecular targets of some of these compounds are pro-apoptotic genes with protein kinase activity such as GP132, ERN1 or RAC1, which prevent the metastatic progression.

Discovery of a new binding site on human choline kinase α1: design, synthesis, crystallographic studies, and biological evaluation of asymmetrical bispyridinium derivatives.

Human choline kinase α (CKα) is a validated drug target for the treatment of cancer. In recent years, a large number of CK inhibitors have been synthesized, and one of them is currently being evaluated in Phase I clinical trials as a treatment for solid tumors. Here we have evaluated a new series of asymmetrical biscationic CK inhibitors by means of enzymatic, crystallographic, and antitumor studies. We demonstrate that one of these structures adopts a completely new binding mode not observed before inducing the aperture of an adjacent binding site. This compound shows antiproliferative and apoptotic effects on cancer cells through activation of caspase-3. Therefore, this study not only provides fruitful insights into the design of more efficient compounds that may target different regions in CKα1 but also explains how these compounds induce apoptosis in cancer cells.