Complex antibiotics based on natural products are almost invariably prepared by semisynthesis, or chemical transformation of the isolated natural products. This approach greatly limits the range of accessible structures that might be studied as new antibiotic candidates. Here we report a short and enantioselective synthetic route to a diverse range of 6-deoxytetracycline antibiotics. The common feature of this class is a scaffold of four linearly fused rings, labeled A through D. We targeted not a single compound but a group of structures with the D ring as a site of structural variability. A late-stage, diastereoselective C-ring construction was used to couple structurally varied D-ring precursors with an AB precursor containing much of the essential functionality for binding to the bacterial ribosome. Five derivatives were synthesized from benzoic acid in yields ranging from 5 to 7% over 14 to 15 steps, and a sixth, (-)-doxycycline, was synthesized in 8.3% yield over 18 steps.

We present a series of small molecule drug discovery case studies where computational methods were prospectively employed to impact Roche research projects, with the aim of highlighting those methods that provide real added value. Our brief accounts encompass a broad range of methods and techniques applied to a variety of enzymes and receptors. Most of these are based on judicious application of knowledge about molecular conformations and interactions: filling of lipophilic pockets to gain affinity or selectivity, addition of polar substituents, scaffold hopping, transfer of SAR, conformation analysis, and molecular overlays. A case study of sequence-driven focused screening is presented to illustrate how appropriate preprocessing of information enables effective exploitation of prior knowledge. We conclude that qualitative statements enabling chemists to focus on promising regions of chemical space are often more impactful than quantitative prediction.

Identification of novel antibiotics remains a major challenge for drug discovery. The present study explores use of phenotypic readouts beyond classical antibacterial growth inhibition adopting a combined multiparametric high content screening and genomic approach. Deployment of the semi-automated bacterial phenotypic fingerprint (BPF) profiling platform in conjunction with a machine learning-powered dataset analysis, effectively allowed us to narrow down, compare and predict compound mode of action (MoA). The method identifies weak antibacterial hits allowing full exploitation of low potency hits frequently discovered by routine antibacterial screening. We demonstrate that BPF classification tool can be successfully used to guide chemical structure activity relationship optimization, enabling antibiotic development and that this approach can be fruitfully applied across species. The BPF classification tool could be potentially applied in primary screening, effectively enabling identification of novel antibacterial compound hits and differentiating their MoA, hence widening the known antibacterial chemical space of existing pharmaceutical compound libraries. More generally, beyond the specific objective of the present work, the proposed approach could be profitably applied to a broader range of diseases amenable to phenotypic drug discovery.

Tetracyclines and tetracycline analogs are prepared by a convergent, single-step Michael-Claisen condensation of the AB precursors 1 or 2 with D-ring precursors of wide structural variability, followed by removal of protective groups (typically in two steps). A number of procedural variants of the key C-ring-forming reaction are illustrated in multiple examples. These include stepwise deprotonation of a D-ring precursor followed by addition of 1 or 2, in situ deprotonation of a D-ring precursor in mixture with 1 or 2, and in situ lithium-halogen exchange of a benzylic bromide D-ring precursor in the presence of 1 or 2, followed by warming. The AB plus D strategy for tetracycline synthesis by C-ring construction is shown to be robust across a range of different carbocyclic and heterocyclic D-ring precursors, proceeding reliably and with a high degree of stereochemical control. Evidence suggests that Michael addition of the benzylic anion derived from a given D-ring precursor to enones 1 or 2 is quite rapid at −78 °C, while Claisen cyclization of the enolate produced is ratedetermining, typically occurring upon warming to 0 °C. The AB plus D coupling strategy is also shown to be useful for the construction of tetracycline precursors that are diversifiable by latter-stage transformations, subsequent to cyclization to form the C ring. Results of antibacterial assays and preliminary data obtained from a murine septicemia model show that many of the novel tetracyclines synthesized have potent antibiotic activities, both in bacterial cell culture and in vivo. The platform for tetracycline synthesis described gives access to a broad range of molecules that would be inaccessible by semi-synthetic methods (presently the only means of tetracycline production), and provides a powerful engine for the discovery and, perhaps, development of new tetracycline antibiotics.

Although many drugs are based on single-substrate analogues, it is well appreciated that enzymes often require, in addition to a substrate, a cofactor for function. The rational design of inhibitors, where a substrate analogue and a cofactor analogue are covalently linked to form a bisubstrate inhibitor, may provide innovative new lead structures in medicinal chemistry that feature both enhanced binding affinity and binding-site selectivity. Some attempts have been made to construct bisubstrate inhibitors for kinases which target both the ATP and the substrate binding sites. [1,2] Other important targets are methyltransferases that depend on S-adenosylmethionine (SAM). The enzyme catechol-O-methyltransferase (COMT) catalyzes the methylation of biologically active catechols, such as l-dopa and dopamine, in the presence of SAM and Mg 2 ions. The addition of COMT inhibitors to levodopa reduces the extracerebral catabolism of levodopa and increases its elimination half-life, thus ensuring that a higher quantity of orally administered l-dopa reaches its target in the brain. [3] This situation produces a stronger and longer lasting therapeutic effect in patients with Parkinsons disease. Nitro-substituted catechols were found to be potent COMT inhibitors, and two derivatives (tolcapone (Tasmar) and entecapone (Comptan)) have been introduced into the marketplace. [3] Recently, we described the first effective bisubstrate inhibitor 1 for COMT (IC 50 2 mm; IC 50 concentration of inhibitor at which 50 % inhibition of the enzyme is observed) which was developed by rational design using the crystal structure of the quaternary complex between the enzyme, SAM, 3,5-dinitrocatechol, and a Mg 2 ion. [4±6]

Inhibition of the enzyme catechol-O-methyltransferase (COMT) is an important approach in the treatment of Parkinson's disease. A series of new potent bisubstrate inhibitors for COMT, resulting from X-ray structure-based design and featuring adenosine and catechol moieties have been synthesised. Biological results show a large dependence of binding affinity on inhibitor preorganisation and the length of the linker between nucleoside and catechol moieties. The most potent bisubstrate inhibitor for COMT has an IC50 value of 9 nM. It exhibits competitive kinetics for the SAM and mixed inhibition kinetics for the catechol binding site. Its bisubstrate binding mode was confirmed by X-ray structure analysis of the ternary complex formed by the inhibitor, COMT and a Mg2+ ion.

A novel class of nonpeptidic, active, and selective thrombin inhibitors has resulted from X‐ray‐structure‐based design and subsequent improvement of the initial lead molecules. These inhibitors possess a bi‐ or tricyclic central core structure with attached side chains to reach the three binding pockets (selectivity S1 pocket, distal D pocket, and proximal P pocket) present in the active site of the enzyme. The key step in the preparation of these compounds is the 1,3‐dipolar cycloaddition between an azomethine ylide, prepared in situ by the decarboxylative method from an aromatic aldehyde and an α‐amino acid, with an N‐substituted maleimide (e.g., see Schemes 1 and 2). All potent inhibitors contain an amidinium residue in the side chain for incorporation into the S1 pocket, which was introduced in the last step of the synthesis by a Pinner reaction. The compounds were tested in biological assays for activity against thrombin and the related serine protease trypsin. The first‐generation lead compounds (±)‐11 and (±)‐19 (Scheme 1) with a bicyclic central scaffold showed Ki values for thrombin inhibition of 18 μM and 0.67 μM, respectively. Conformationally more restricted second‐generation analogs (Scheme 2) were more active ((±)‐22i: Ki=90 nM (Table 1)); yet the selectivity for thrombin over trypsin remained weak. In the third‐generation compounds, a small lipophilic side chain for incorporation into the hydrophobic P pocket was introduced (Schemes 7 and 8). Since this pocket is present in thrombin but not in trypsin, an increase in binding affinity was accompanied by an increase in selectivity for thrombin over trypsin. The most selective inhibitor (Ki=13 nM, 760‐fold selectivity for thrombin over trypsin; Table 2) was (±)‐1 with an i‐Pr group for incorporation into the P pocket. Optical resolution of (±)‐1 (Scheme 9) provided (+)‐1 with a Ki value of 7 nM and a 740‐fold selectivity, whereas (−)‐1 was 800‐fold less active (Ki=5.6 μM, 21‐fold selectivity). The absolute configuration of the stronger‐binding enantiomer was assigned based on the X‐ray crystal structure of the complex formed between thrombin and this inhibitor. Compound (+)‐1 mimics the natural thrombin substrate, fibrinogen, which binds to the enzyme with the Ph group of a phenylalanine (piperonyl in (+)‐1) in the distal D pocket, with the i‐Pr group of a valine (i‐Pr in (+)‐1) in the proximal P pocket, and with a guanidinium side chain of an arginine residue (phenylamidinium group in (+)‐1) in the selectivity S1 pocket of thrombin. A series of analogs of (±)‐1 with the phenylamidinium group replaced by aromatic and aliphatic rings bearing OH or NH2 groups (Schemes 10 – 14) were not effectively bound by thrombin. A number of X‐ray crystal‐structure analyses of free inhibitors confirmed the high degree of preorganization of these compounds in the unbound state. Since all inhibitors prefer similar modes of association with thrombin, detailed information on the strength of individual intermolecular bonding interactions and their incremental con...

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