Modesto de Candia

Novel factor Xa inhibitors: a patent review

Importance of the field. New oral anticoagulants with favorable safety profiles and fixed doses are required for the management of thromboembolism and stroke prevention in patients with atrial fibrillation. Among them, fXa inhibitors (the so-called xabans) are attractive options that can overcome limitations (e.g., bleeding) of the current oral antithrombotic therapy. The rational design of small-molecule direct fXa inhibitors, whose importance is testified by the growing number of publications and patents recently registered, has been fully supported by the X-ray crystallography of enzyme–ligand complexes. Areas covered in this review. Pubmed, SciFinder® Scholar, ISI web of knowledgeSM, http://ep.espacenet.com/ and Google websites were used as the main sources for literature retrieving, and > 100 patents filed between 2006 and April 2009, reviewed and discussed herein, highlight the variety among the P1 and P4 moieties on suitable scaffolds. What the reader will gain. The replacement of the benzamidine P1 moiety, which characterizes the first generation, with less basic bioisosteric or nonpolar neutral P1 groups led to the disclosure of numerous fXa inhibitors with high potency, selectivity and oral bioavailability. Novel selective fXa inhibitors with stable pharmacokinetics, better therapeutic windows and ease-of-use than the existing anticoagulants are currently under advanced stage clinical trials. Take-home message. Available data from Phase II and Phase III studies reflect the drive towards fXa inhibitors as potentially more effective and safer antithrombotic drugs. Their development is expected to address two major needs for anticoagulation, namely safety and ease-of-use, and to significantly affect the anticoagulant market.

Inhibition of 6-hydroxydopamine-induced oxidative damage by 4,5-dihydro-3H-2-benzazepine N-oxides

A number of new analogs of 3,3-dimethyl-4,5-dihydro-3H-2-benzazepine 2-oxide, structurally related to the nitrone spin trap alpha-phenyl-N-tert-butylnitrone (PBN), were synthesized and evaluated for their activity in vitro as protectants against oxidative stress induced in rat brain mitochondria by 6-hydroxydopamine (6-OHDA), a neurotoxin producing experimental model of Parkinsons disease (PD). As assessed by a fluorimetric assay, all 2-benzazepine-based nitrones were shown to decrease hydroxyl radicals (OH) generated during 6-OHDA autoxidation. The inhibition effects on the OH formation shown by the 5-gem-dimethyl derivatives, 2-4 times higher than those of the corresponding 5-methyl derivatives, were attributed to the flattening effect of the 5-gem-dimethyl group on the azepine ring, which should enhance nitrone reactivity and/or increase stability of the radical adducts. In contrast, owing to steric hindrance, a methyl group to C-1 diminishes the OH-scavenging activity of the nitrone group. All the assayed compounds were more potent than PBN as inhibitors of 6-OHDA-induced lipid peroxidation (LPO) and protein carbonylation (PCO), taken as an indicator of mitochondrial protein oxidative damage. The most promising antioxidant (compound 11), bearing 5-gem-dimethyl and spiro C-3 cyclohexyl groups, highlighted in this study as the best features, inhibited LPO and PCO with IC50 values of 20 and 48 microM, respectively, showing a potency improvement over PBN of two order magnitude. Both LPO and PCO inhibition potency data were found primarily related to the OH-scavenging activities, whereas lipophilicity plays a role in improving the LPO (but not PCO) inhibition, as a statistically valuable two-parameter equation proved.

Protein crystallography and fragment-based drug design

Crystallography is a major tool for structure-driven drug design, as it allows knowledge of the 3D structure of protein targets and protein-ligand complexes. However, the route for crystal structure determination involves many steps, some of which may hamper its high-throughput use. Recent efforts have produced significant advances in experimental and computational tools and protocols. They include automatic crystallization tools, faster data collection devices, more efficient phasing methods and improved ligand-fitting procedures. The timescales of drug-discovery processes have been also reduced by using a fragment-based screening approach. Herein, the achievements in protein crystallography over the last 5 years are reviewed, and advantages and disadvantages of the fragment-based approaches to drug discovery that make use of x-ray crystallography as a primary screening method are examined. In particular, in some detail, five recent case studies pertaining to the development of new hits or leads in relevant therapeutic areas, such as cancer, immune response, inflammation, metabolic syndrome and neurology are described.

Synthesis and Biological Evaluation of Direct Thrombin Inhibitors Bearing 4‑(Piperidin-1-yl)pyridine at the P1 Position with Potent Anticoagulant Activity

The design and synthesis of a new class of nonpeptide direct thrombin inhibitors, built on the structure of 1-(pyridin-4-yl)piperidine-4-carboxamide, are described. Starting from a strongly basic 1-amidinopiperidine derivative (6) showing poor thrombin (fIIa) and factor Xa (fXa) inhibition activities, anti-fIIa activity and artificial membrane permeability were considerably improved by optimizing the basic P1 and the X-substituted phenyl P4 binding moieties. Structure-activity relationship studies, usefully complemented with molecular modeling results, led us to identify compound 13b, which showed excellent fIIa inhibition (Ki = 6 nM), weak anti-Xa activity (Ki = 5.64 μM), and remarkable selectivity over other serine proteases (e.g., trypsin). Compound 13b showed in vitro anticoagulant activity in the low micromolar range and significant membrane permeability. In mice (ex vivo), 13b demonstrated anticoagulant effects at 2 h after oral dosing (100 mg·kg(-1)), with a significant 43% prolongation of the activated partial thromboplastin time (aPTT), over controls (P < 0.05).

Biarylmethoxy isonipecotanilides as potent and selective inhibitors of blood coagulation factor Xa.

New chloro-substituted biarylmethoxyphenyl piperidine-4-carboxamides were synthesized and assayed in vitro as inhibitors of the blood coagulation enzymes factor Xa (fXa) and thrombin. An investigation of effects of the amidine and isopropyl groups attached at the piperidine nitrogen and 5-(halogenoaryl)isoxazol-3-yl groups as biaryl substituents led us to identify new compounds which proved to be selective fXa inhibitors, with inhibition constants in the low nanomolar range. The most potent compound 21e, that incorporates 2-Cl-thiophen-5-yl group as the P1 motif and 1-isopropylpiperidine P4 group, inhibited fXa with K(i) value of 0.3nM and very high selectivity over thrombin and some other tested serine proteases, achieving moderate levels of anticoagulant activity in the low micromolar range, as assessed by the prothrombin time clotting assay (PT(2)=3.30μM). Based on reliable docking simulations, molecular modeling provided a rationale for interpreting structure-activity relationships. The predicted binding modes highlighted the structural requirements for addressing the subsites S1 and S4 of the fXa enzyme.

2-Benzazepine Nitrones Protect Dopaminergic Neurons against 6-Hydroxydopamine-Induced Oxidative Toxicity

A number of C‐3 spirocyclic 2‐benzazepine analogs of α‐phenyl‐N‐tert‐butyl nitrone (PBN) were synthesized and tested for their activity in protecting rat brain mitochondria and dopaminergic (DA) neurons against 6‐hydroxydopamine (6‐OHDA), a toxin inducing destruction of the DA nigro‐striatal pathway in rodent models of Parkinsons disease. The newly synthesized nitrone derivatives were firstly investigated for their activity in decreasing the level of hydroxyl radicals generated during 6‐OHDA oxidation, and inhibit lipid peroxidation (TBARS assay) and protein carbonyl content (PCC) in rat brain mitochondria. Most of the studied 2‐benzazepine nitrones showed inhibitory potencies in both TBARS and PCC assays at least two magnitude orders higher than that of PBN. The data obtained usefully complemented the known structure–activity relationships. In particular, 5 and 10, bearing C‐3 spiro cyclopentyl and tetrahydropyranyl moieties, respectively, at 8 µM concentration proved to be significantly more effective than PBN in protecting cultured DA neurons exposed to 6‐OHDA, which alone causes about 45% cell loss in 24 h. In addition, we found that 5 inhibited butyrylcholinesterase with an IC50 value of 16.8 µM, which would enhance its potential as neuroprotective agent in Alzheimers neurodegeneration. These findings extend the utility of benzazepine‐based PBN analogs in the treatment of age‐related free radical‐mediated disorders.

Investigation on the antiplatelet activity of pyrrolo[3,2‐c]pyridine‐containing compounds

A series of 4,5,6,7‐tetrahydro‐1H‐pyrrolo[3,2‐c]pyridines (THPPs), mostly C(2)‐substituted derivatives, and some 2, 3, 4, 5‐tetrahydro‐1H‐pyrido[4, 3‐b]indoles (THPIs) were synthesized and tested in‐vitro for their ability to inhibit aggregation of human platelet‐rich plasma (PRP) induced by adenosine 5′‐diphosphate (ADP) and adrenaline (epinephrine). 5‐Benzyl THPP (3), 2‐(benzylamino)methyl THPP (5f) and 2‐ethyl THPI (6) moderately and dose‐dependently inhibited platelet aggregation induced by adrenaline and, to a lesser extent, by ADP. These compounds inhibited the second phase of the PRP aggregation triggered by adrenaline, which largely depends upon thromboxane A2 production and ADP release. In the adrenaline‐stimulated aggregation, the THPI derivative 6 was found to be nearly equipotent with aspirin, their IC50 values (concentration effecting 50% inhibition of aggregation) being 90 and 60 μM, respectively. A relation between activity and calculated octanol‐water partition coefficient suggested that a log P value around 2.5 should be the optimal lipophilicity value for the activity of THPP‐containing compounds.

β-D-glucosyl conjugates of highly potent inhibitors of blood coagulation factor Xa bearing 2-chorothiophene as a P1 motif.

We synthesized a novel O‐glucoside of the recently reported potent factor Xa (fXa) inhibitor 1, which bears a 5‐chlorothien‐2‐yl moiety and 1‐isopropylpiperidine as fragments that bind the S1 and S4 enzyme pockets, respectively. A β‐D‐glucosyl unit was conjugated through an ether‐linked C3‐alkyl spacer to the central phenyl ring of 1. The synthesized β‐D‐glucose‐based compound 16 achieved picomolar inhibitory potency against human fXa (Ki=60 pM) and high selectivity over thrombin and other serine proteases. In addition to the chlorothienyl S1 binder, a large gain in ΔG resulted from the addition of protonated 1‐isopropylpiperidine (ΔΔG=29.7–30.5 kJ mol−1), which should bind to the aromatic S4 pocket through efficient cation–π and CH⋅⋅⋅π interactions. Instead, the C3‐alkyl‐linked glucose fragment, which is likely directed toward the solvent outside the enzyme binding site, improves ΔG by an average of 2.9–3.8 kJ mol−1. Compound 16 showed sub‐micromolar in vitro anticoagulant activity, as assessed by prothrombin time (PT) and activated thromboplastin time (aPTT) clotting assays in pooled human plasma (PT2 and aPTT2 equal to 0.135 and 0.389 μM, respectively). Although compound 16 was 1.4‐fold less active than parent compound 1 in the ex vivo anticoagulant assay in mice, it showed a significant (1.6‐fold) prolongation of PT relative to controls (P<0.05) 60 min after oral dosing (75 mg kg−1).

Oxazepam-dopamine conjugates increase dopamine delivery into striatum of intact rats

The neurotransmitter dopamine (DA) was covalently linked to oxazepam (OXA), a well-known positive allosteric modulator of γ-aminobutyric acid type-A (GABAA) receptor, through a carbamate linkage (4) or a succinic spacer (6). These conjugates were synthesized with the aim of improving the delivery of DA into the brain and enhancing GABAergic transmission, which may be useful for the long-term treatment of Parkinson disease (PD). Structure-based permeability properties, in vitro stability, and blood-brain barrier (BBB) permeability studies led to identify the OXA-DA carbamate conjugate 4a as the compound better combining sufficient stability and ability to cross BBB. Finally, in vivo microdialysis experiments in freely moving rats demonstrated that 4a (20 mg/kg, i.p.) significantly increases extracellular DA levels into striatum, with a peak (more than 15-fold increase over the baseline) at about 80 min after a single administration. The stability and delivery data proved that 4a may be a promising candidate for further pharmacological studies in animal models of PD.

Pharmaceutical development of novel lactate-based 6-fluoro-l-DOPA formulations.

Abstract 6‐[18F]fluoro‐l‐dihydroxyphenylalanine (18F–DOPA) is a diagnostic positron emission tomography (PET) agent, which has been used for decades in imaging the loss of dopaminergic neurons in Parkinsons disease, and more recently to detect, stage and restage neuroendocrine tumors (NETs) and to search for recurrence of viable glioma tissue. The commercially available 18F–DOPA PET radiopharmaceutical for diagnostic use in European Union countries, is formulated in an aqueous solution of acetic acid (1.05 mg/mL) and has the disadvantages that, immediately before injection, the pH must be adjusted to 4.0–5.0 by the addition of a sterile solution of sodium bicarbonate (84 mg/mL) causing a light and transient burning sensation at the injection site. To overcome these drawbacks, preformulation studies were accomplished to confirm that F‐DOPA degradation was affected by pH. Hence, two formulations of F‐DOPA, namely ND1 and ND2, were prepared maintaining the pH = 5.0 using 1 mM l‐(+)‐lactate buffer, excluding oxygen, and incorporating in the formula the chelating agent Na2EDTA (1 mM). F‐DOPA oxygen exposure, the presence of free metal cations in formulation and high pH values seem to promote F‐DOPA degradation. The resulting formulations proved to guarantee the chemical stability of F‐DOPA in solution at pH 5.0, value also compatible with the direct infusion. In vitro cell viability tests on mouse skeletal muscle fibers, renal tsa201 and neuronal SH‐SY5Y cell lines, and in vivo studies in rats reported elsewhere, showed cell tolerability to the new F‐DOPA formulations providing the basis for their further in vivo evaluation.