Dirk Leysen

Tibolone is not converted by human aromatase to 7α-methyl-17α-ethynylestradiol (7α-MEE):: Analyses with sensitive bioassays for estrogens and androgens and with LC-MSMS

To exclude that aromatization plays a role in the estrogenic activity of tibolone, we studied the effect tibolone and metabolites on the aromatization of androstenedione and the aromatization of tibolone and its metabolites to 7alpha-methyl-17alpha-ethynylestradiol (7alpha-MEE) by human recombinant aromatase. Testosterone (T), 17alpha-methyltestosterone (MT), 19-nortestosterone (Nan), 7alpha-methyl-19-nortestosterone (MENT) and norethisterone (NET) were used as reference compounds. Sensitive in vitro bioassays with steroid receptors were used to monitor the generation of product and the reduction of substrate. LC-MSMS without derivatization was used for structural confirmation. A 10 times excess of tibolone and its metabolites did not inhibit the conversion of androstenedione to estrone by human recombinant aromatase as determined by estradiol receptor assay whereas T, MT, Nan, and MENT inhibited the conversion for 75, 53, 85 and 67%, respectively. Tibolone, 3alpha- and 3beta-hydroxytibolone were not converted by human aromatase whereas the estrogenic activity formed with the Delta4-isomer suggests a conversion rate of 0.2% after 120 min incubation. In contrast T, MT, Nan, and MENT were completely converted to their A-ring aromates within 15 min while NET could not be aromatized. Aromatization of T, MT, Nan and MENT was confirmed with LC-MSMS. Structure/function analysis indicated that the 17alpha-ethynyl-group prevents aromatization of (19-nor)steroids while 7alpha-methyl substitution had no effect. Our results with the sensitive estradiol receptor assays show that in contrast to reference compounds tibolone and its metabolites are not aromatized.

A NEW CLEAVAGE STRATEGY FOR THE SOLID-PHASE SYNTHESIS OF SECONDARY AMINES

Abstract The clean and efficient cleavage of N-benzyl linked tertiary amines from a solid support (e.g. 4 ) by treatment with α-chloroethyl chloroformate (ACE-Cl) / methanol to yield secondary amines 7 is described. This allows the solid-phase synthetic transformation of the secondary amine 2 into 7 . When the Merrifield resin 1 is used the N-tethered amine-polymeric matrix ensemble is stable towards a wide variety of reaction conditions.

A comparison of the physicochemical and biological properties of mirtazapine and mianserin

Although the chemical structures of the antidepressants mirtazapine and mianserin are closely related there are considerable differences in their biological properties. To find an explanation of this, various physicochemical properties of mirtazapine and mianserin were measured or calculated.

AMA0076, a Novel, Locally Acting Rho Kinase Inhibitor, Potently Lowers Intraocular Pressure in New Zealand White Rabbits with Minimal Hyperemia

PURPOSEnTo determine whether ROCK inhibition for the treatment of glaucoma can be improved by using novel, locally acting Rho kinase (ROCK) inhibitors, such as AMA0076, that lower IOP without inducing hyperemia.nnnMETHODSnOn-target potency of AMA0076 was compared with other ROCK inhibitors (Y-27632 and Y-39983) and conversion of AMA0076 into its functionally inactive metabolite was evaluated in rabbit eye tissues. Human trabecular meshwork (HTM) cell morphology, actin filaments, and focal adhesion were studied in vitro after exposure to AMA0076. The effect of AMA0076 on IOP was investigated in normotensive rabbits and a new, acute hypertensive rabbit model. Intraocular pressure lowering efficacy of AMA0076 was compared with pharmacologic treatments. Hyperemia after single topical dosing of AMA0076 and Y-39983 was scored.nnnRESULTSnAMA0076 and Y-39983 showed similar on-target potency. AMA0076 was most stable in aqueous humor and converted into its metabolite in other eye tissues. Exposure of HTM cells to AMA0076 led to significant and reversible changes in cell shape and a decrease in actin stress fibers and focal adhesions. Both AMA0076 and Y-39983 provided an equivalent IOP control. Compared with latanoprost and bimatoprost, AMA0076 was more potent in preventing the IOP elevation in the acute hypertensive rabbit model. The degree of hyperemia was significantly lower in rabbits treated with AMA0076 then with Y-39983.nnnCONCLUSIONSnAMA0076 is a locally acting ROCK inhibitor that is able to induce altered cellular behavior of HTM cells. Administration of AMA0076 effectively reduces IOP in ocular normotensive and acute hypertensive rabbits without causing distinct hyperemia.

3-[2-(Aminomethyl)-5-[(pyridin-4-yl)carbamoyl]phenyl] benzoates as soft ROCK inhibitors.

Clinical development of ROCK inhibitors has so far been limited by systemic or local ROCK-associated side effects. A soft drug approach, which involves predictable metabolic inactivation of an active compound to a nontoxic metabolite, could represent an attractive way to obtain ROCK inhibitors with improved tolerability. We herein report the design and synthesis of a new series of soft ROCK inhibitors structurally related to the ROCK inhibitor Y-27632. These inhibitors contain carboxylic ester moieties which allow inactivation by esterases. While the parent esters display strong activity in enzymatic (ROCK2) and cellular (MLC phosphorylation) assays, their corresponding carboxylic acid metabolites have negligible functional activity. Compound 32 combined strong efficacy (ROCK2 IC50=2.5 nM) with rapid inactivation in plasma (t1/2 <5). Compound 32 also demonstrated in vivo efficacy when evaluated as an IOP-lowering agent in ocular normotensive New-Zealand White rabbits, without ocular side effects.

The effect of AMA0428, a novel and potent ROCK inhibitor, in a model of neovascular age-related macular degeneration.

PURPOSEnRho kinase (ROCK) is associated with VEGF-driven angiogenesis, as well as with inflammation and fibrosis. Therefore, the effect of AMA0428, a novel ROCK inhibitor, was studied in these processes, which highly contribute to the pathogenesis of neovascular AMD.nnnMETHODSnThe effect of AMA0428 (0.5-5.0 μM) on human umbilical vein endothelial cells (HUVECs), human brain microvascular endothelial cells (HBMECs), and human brain microvascular pericytes (HBVPs) was determined using cell viability (WST-1), apoptosis (caspase 3/7), and migration (scratch and under-agarose) assays. The in vivo response was investigated using a laser-induced choroidal neovascularization (CNV) mouse model, in which intravitreal injections of AMA0428, murine anti-VEGF-R2 mAb (DC101), or placebo was given. Outcome was assessed by analysis of inflammation (CD45), angiogenesis (FITC-dextran), vessel leakage (Texas Red-conjugated Dextran and FITC-labeled lectin) and fibrosis (Sirius Red/Collagen I).nnnRESULTSnThe AMA0428 dose-dependently reduced proliferation and VEGF-induced migration of HUVEC and HBMEC (P < 0.05). No significant effect was seen on HBVP proliferation; however, migration and pericyte recruitment were enhanced (P < 0.05) by AMA0428 administration. There was no apoptosis induction. The AMA0428 significantly reduced CNV and vessel leakage 2 weeks after laser treatment, comparable to DC101. In addition, AMA0428 inhibited inflammation on day 5 by 42% (P < 0.05) and collagen deposition on day 30 by 43% (P < 0. 05), whereas DC101 had no effect on inflammation nor on fibrosis.nnnCONCLUSIONSnThe results suggest that targeting ROCK with AMA0428 not only reduces neoangiogenesis, but also blocks inflammation and fibrosis (contrary to VEGF suppression). These results point to a potential therapeutic benefit of ROCK inhibition in neovascular AMD.

Q) SAR study on the metabolic stability of steroidal androgens.

Metabolic stability is a key issue in the development of orally active androgens for Partial Androgen Deficiency in Aging Males (PADAM) and male contraception. Rates of metabolism in human hepatocyte suspensions provide useful information on the stability of compounds that undergo a first pass metabolism. We have derived a structure-pharmacokinetic relationship for a data set of 32 in-house steroidal androgens by means of the decision-trees technique. Volume, shape, number of rotatable bonds, and surface turned out to be the most important descriptors for classification. Only 2 of the 32 compounds were misclassified. The most stable compounds were classified in three leaf nodes on different branches of the tree, suggesting that higher metabolic stability can be achieved for the same substrate by different steric modifications. Further, it is generally assumed that the first step in cytochrome P450s oxidation reactions takes place by hydrogen abstraction to form a radical intermediate. An electronic model for hydrogen abstraction in steroidal androgens was, therefore, developed by means of ab initio calculations. Activation energies of steroid radical systems calculated as energy differences between the reactants equilibrium geometry energies and their corresponding transition states energies could be used to predict relative rates of metabolism to guide the design and redesign process of metabolically more stable steroidal androgens.

Structure–activity relationship study of human liver microsomes-catalyzed hydrolysis rate of ester prodrugs of MENT by comparative molecular field analysis (CoMFA)

A series of MENT esters (3-71) was designed, prepared and tested to study the structure-activity relationship (SAR) of the hydrolysis rate with human liver microsomes of these prodrugs. Compounds were obtained covering a wide range of metabolic stability. The results are useful for the proper selection of prodrugs for different pharmaceutical formulations to deliver the potent and prostate-sparing androgen MENT. The MENT esters can especially be administered for male hormone replacement therapy and male contraception. Comparative molecular field analysis (CoMFA) was applied to a dataset of 28 esters, for which ED50 values could be obtained. The CoMFA model where the electrostatic and H-bond molecular fields were combined turned out to be most predictive. Despite the limited size of the dataset, CoMFA can help to rationalize the SAR of the ester hydrolysis rate of ester prodrugs of MENT.

Novel Roflumilast analogs as soft PDE4 inhibitors

PDE4 inhibitors are of high interest for treatment of a wide range of inflammatory or autoimmune diseases. Their potential however has not yet been realized due to target-associated side effects, resulting in a low therapeutic window. We herein report the design, synthesis and evaluation of novel PDE4 inhibitors containing a γ-lactone structure. Such molecules are designed to undergo metabolic inactivation when entering circulation, thereby limiting systemic exposure and reducing the risk for side effects. The resulting inhibitors were highly active on both PDE4B1 and PDE4D2 and underwent rapid degradation in human plasma by paraoxonase 1. In contrast, their metabolites displayed markedly reduced permeability and/or on-target activity.

Chapter 1. Toward Third Generation Antidepressants

Publisher Summary In second generation anti-depressants, new tricyclics (TCAs), such as maprotiline and atypical agents, like mianserin, are used to reduce the cardiovascular and anti-cholinergic side effects of the first generation TCAs and monoamine oxidase inhibitors (MAOls), typified by amitriptyline and isocarboxazid. It continues with selective serotonin reuptake inhibitors (SSRls) and reversible inhibitors of monoamine oxidase A (RIMAs). Third generation agents retain the gains already made in minimizing side-effects and toxicity in overdosage. Chronic anti-depressant treatments produce adaptive hyporesponsitivity of the 5-HT 1A receptor–effector complex, as indicated by a decrease of hypothermic and neuroendocrine responses. Unravelling the functional alterations of components, of the intracellular signal transduction pathway, lead to new biochemical target mechanisms for future anti-depressants. Lithium is the most commonly used drug in bipolar disorders, for both acute mania and maintenance therapy. It is also effective in the acute treatment of depression, either alone or when used to augment mainline anti-depressant treatment, in refractory patients. In the enhancement of 5-HT transmission, lithium affects G protein-regulated phenomena, such as receptor-activated phosphatidyl-inositol and cyclic adenosine monophosphate (cAMP)-turnover, leading to altered functions of the inter-regulated cAMP-dependent protein kinase and protein kinase C. Lithium inhibits inositol monophosphatase, resulting in lowered cellular levels of ώ-inositol and reduced agonist-induced formation of inositol polyphosphate. Carbamazepine is now established as an alternative or adjunct to lithium.