This review describes simple and useful concepts for predicting and tuning the pK(a) values of basic amine centers, a crucial step in the optimization of physical and ADME properties of many lead structures in drug-discovery research. The article starts with a case study of tricyclic thrombin inhibitors featuring a tertiary amine center with pK(a) values that can be tuned over a wide range, from the usual value of around 10 to below 2 by (remote) neighboring functionalities commonly encountered in medicinal chemistry. Next, the changes in pK(a) of acyclic and cyclic amines upon substitution by fluorine, oxygen, nitrogen, and sulfur functionalities, as well as carbonyl and carboxyl derivatives are systematically analyzed, leading to the derivation of simple rules for pK(a) prediction. Electronic and stereoelectronic effects in cyclic amines are discussed, and the emerging computational methods for pK(a) predictions are briefly surveyed. The rules for tuning amine basicities should not only be of interest in drug-discovery research, but also to the development of new crop-protection agents, new amine ligands for organometallic complexes, and in particular, to the growing field of amine-based organocatalysis.

In our molecular recognition studies, aimed at quantifying the energetics of individual protein-ligand interactions, [1] we became interested in exploring cation-p interactions [2,3] in the D-pocket of thrombin, a central serine protease in the blood coagulation cascade. The bottom of this hydrophobic pocket is lined by the indole residue of Trp 215 (Figure 1), an aromatic amino acid side chain frequently involved in cationp interactions in biological systems.[4] To probe this interaction, we prepared the tricyclic inhibitors [5] (AE )-1 and (AE )-2, predicted by computer modeling [6] to position a quaternary ammonium ion and an uncharged tert-butyl group above the indole ring of Trp 215.The synthesis of (AE )-1 started with the 1,3-dipolar cycloaddition between maleimide 3, aldehyde 4, and l-proline (5) to give (AE )-6, which was transformed into amidinium salt (AE )-7 using a Pinner-reaction (Scheme 1, for full experimen-

Using intravital fluorescence microscopy in the ears of hairless mice, we determined skin microvascular adaptations during the process of aging from juvenile to adult and senescent life (6-78 wk). Despite an increase of ear area within the first 36 wk, the number and branching pattern of both arteriolar and venular microvessels remained constant during the whole life period. Both arterioles and venules exhibited an increase in length, diameter, and intervascular distance up to the age of 36 wk. With the increase of the size of the ears, the observation that cutaneous capillary density remained unchanged implied new capillary formation. During aging to 78 wk, capillary density in the ears was reduced to approximately 40%. Functional analysis revealed an appropriate hyperemic response to a 2-min period of ischemia during late juvenile and adult life, which, however, was markedly reduced during senescence. Thus, except for capillaries, there is no indication for age-related new vessel formation. The process of aging from adult to senescent life does not cause any significant remodeling but is associated with a decrease of nutritive perfusion and a functional impairment to respond to stimuli such as ischemia.

Im Verlauf unserer Studien zur molekularen Erkennung mit dem Ziel einer Quantifizierung des energetischen Beitrages einzelner Protein-Ligand-Wechselwirkungen [1] richtete sich unser Interesse auf die Erforschung von Kation-p-Wechselwirkungen [2,3] in der D-Tasche von Thrombin, einer zentralen Serin-Protease in der Blutgerinnungskaskade. Den Boden dieser hydrophoben Tasche bildet der Indol-Rest von Trp 215 (Abbildung 1), einer aromatischen Aminosäure-Seitenkette, die in biologischen Systemen häufig Kation-p-Wechselwirkungen eingeht.[4] Zur Erkundung dieser Wechselwirkung wurden die tricyclischen Inhibitoren (AE )-1 und (AE )-2 synthetisiert, [5] die entsprechend der Voraussage eines Computer-Modells [6] beim Binden im aktiven Zentrum von Thrombin ein quartäres Ammonium-Ion bzw. einen ungeladenen tert-Butyl-Rest oberhalb des Indol-Ringes von Trp 215 positionieren.

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A new route via intermediate pseudoenantiomers was developed to synthesize racemic and enantiomerically pure new non‐peptidic inhibitors of thrombin, a key serine protease in the blood‐coagulation cascade. These ligands feature a conformationally rigid tricyclic core and are decorated with substituents to fill the major binding pockets (distal (D), proximal (P), selectivity (S1), and oxyanion hole) at the thrombin active site (Fig. 1). The key step in the preparation of the new inhibitors is the 1,3‐dipolar cycloaddition between an optically active azomethine ylide, prepared in situ from L‐(4R)‐hydroxyproline and 4‐bromobenzaldehyde, and N‐piperonylmaleimide (Scheme 1). According to this protocol, tricyclic imide (compounds (±)‐15‐(±)‐18 and (+)‐21) and lactam (compound (+)‐2) inhibitors with OH or ether substituents at C(7) in the proline‐derived pyrrolidine ring were synthesized to specifically explore the binding features of the oxyanion hole (Schemes 2–4). Biological assays (Table) showed that the polar oxyanion hole in thrombin is not suitable for the accommodation of bulky substituents of low polarity, thereby confirming previous findings. In contrast, tricyclic lactam (+)‐2 (Ki=9 nM, Ki(trypsin)/Ki(thrombin)=1055) and tricyclic imide (+)‐21 (Ki=36 nM, Ki(trypsin)/Ki(thrombin)=50) with OH‐substituents at the (R)‐configured C(7)‐atom are among the most‐potent and most‐selective thrombin inhibitors in their respective classes, prepared today. While initial modeling predicted H‐bonding between the OH group at C(7) in (+)‐2 and (+)‐21 with the H2O molecule bound in the oxyanion hole (Fig. 2), the X‐ray crystal structure of the complex of (+)‐21 (Fig. 7, b) revealed a different interaction for this group. The propionate side chain of Glu192 undergoes a conformational change, thereby re‐orienting towards the OH group at C(7) under formation of a very short ionic H‐bond (OH⋅⋅⋅−OOC; d(O⋅⋅⋅O)=2.4 Å). The energetic contribution of this H‐bond, however, is negligible, due to its location on the surface of the protein and the unfavorable conformation of the H‐bonded propionate side chain.

A new route via intermediate pseudoenantiomers was developed to synthesize racemic and enantiomerically pure new non-peptidic inhibitors of thrombin, a key serine protease in the blood-coagulation cascade. These ligands feature a conformationally rigid tricyclic core and are decorated with substituents to fill the major binding pockets (distal (D), proximal (P), selectivity (S1), and oxyanion hole) at the thrombin active site (Fig. 1). The key step in the preparation of the new inhibitors is the 1,3-dipolar cycloaddition between an optically active azomethine ylide, prepared in situ from l-(4R)-hydroxyproline and 4-bromobenzaldehyde, and N-piperonylmaleimide (Scheme 1). According to this protocol, tricyclic imide (compounds (AE)-15-(AE)-18 and ()-21) and lactam (compound ()-2) inhibitors with OH or ether substituents at C(7) in the proline-derived pyrrolidine ring were synthesized to specifically explore the binding features of the oxyanion hole (Schemes 2 ± 4). Biological assays (Table) showed that the polar oxyanion hole in thrombin is not suitable for the accommodation of bulky substituents of low polarity, thereby confirming previous findings. In contrast, tricyclic lactam ()-2 (K i 9 nm, K i (trypsin)/K i (thrombin) 1055) and tricyclic imide ()-21 (K i 36 nm, K i (trypsin)/ K i (thrombin) 50) with OH-substituents at the (R)-configured C(7)-atom are among the most-potent and most-selective thrombin inhibitors in their respective classes, prepared today. While initial modeling predicted H-bonding between the OH group at C(7) in ()-2 and ()-21 with the H 2 O molecule bound in the oxyanion hole (Fig. 2), the X-ray crystal structure of the complex of ()-21 (Fig. 7, b) revealed a different interaction for this group. The propionate side chain of Glu192 undergoes a conformational change, thereby re-orienting towards the OH group at C(7) under formation of a very short ionic H-bond (OÀH ¥¥¥ À OOC; d(O ¥¥¥ O) 2.4 ä). The energetic contribution of this H-bond, however, is negligible, due to its location on the surface of the protein and the unfavorable conformation of the H-bonded propionate side chain.

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