Structure Guided Discovery of Novel Pan Metallo-ß-Lactamase Inhibitors with Improved Gram-Negative Bacterial Cell Penetration.

The use of ß-lactam (BL) and ß-lactamase inhibitor combination to overcome BL antibiotic resistance has been validated through clinically approved drug products. However, unmet medical needs still exist for the treatment of infections caused by Gram-negative (GN) bacteria expressing metallo-ß-lactamases. Previously, we reported our effort to discover pan inhibitors of three main families in this class: IMP, VIM, and NDM. Herein, we describe our work to improve the GN coverage spectrum in combination with imipenem and relebactam. This was achieved through structure- and property-based optimization to tackle the GN cell penetration and efflux challenges. A significant discovery was made that inhibition of both VIM alleles, VIM-1 and VIM-2, is essential for broad GN coverage, especially against VIM-producing P. aeruginosa. In addition, pharmacokinetics and nonclinical safety profiles were investigated for select compounds. Key findings from this drug discovery campaign laid the foundation for further lead optimization toward identification of preclinical candidates.

Rapid Evolution of a Fragment-like Molecule to Pan-Metallo-Beta-Lactamase Inhibitors: Initial Leads toward Clinical Candidates.

With the emergence and rapid spreading of NDM-1 and existence of clinically relevant VIM-1 and IMP-1, discovery of pan inhibitors targeting metallo-beta-lactamases (MBLs) became critical in our battle against bacterial infection. Concurrent with our fragment and high-throughput screenings, we performed a knowledge-based search of known metallo-beta-lactamase inhibitors (MBLIs) to identify starting points for early engagement of medicinal chemistry. A class of compounds exemplified by 11, discovered earlier as B. fragilis metallo-beta-lactamase inhibitors, was selected for in silico virtual screening. From these efforts, compound 12 was identified with activity against NDM-1 only. Initial exploration on metal binding design followed by structure-guided optimization led to the discovery of a series of compounds represented by 23 with a pan MBL inhibition profile. In in vivo studies, compound 23 in combination with imipenem (IPM) robustly lowered the bacterial burden in a murine infection model and became the lead for the invention of MBLI clinical candidates.

Discovery of MK-8153, a Potent and Selective ROMK Inhibitor and Novel Diuretic/Natriuretic.

A renal outer medullary potassium channel (ROMK, Kir1.1) is a putative drug target for a novel class of diuretics with potential for treating hypertension and heart failure. Our first disclosed clinical ROMK compound, 2 (MK-7145), demonstrated robust diuresis, natriuresis, and blood pressure lowering in preclinical models, with reduced urinary potassium excretion compared to the standard of care diuretics. However, 2 projected to a short human half-life (∼5 h) that could necessitate more frequent than once a day dosing. In addition, a short half-life would confer a high peak-to-trough ratio which could evoke an excessive peak diuretic effect, a common liability associated with loop diuretics such as furosemide. This report describes the discovery of a new ROMK inhibitor 22e (MK-8153), with a longer projected human half-life (∼14 h), which should lead to a reduced peak-to-trough ratio, potentially extrapolating to more extended and better tolerated diuretic effects.

Transfer learning for bearing performance degradation assessment based on deep hierarchical features.

Bearing design, production and complicated operating condition can lead to the scattered life cycle degradation distribution, which will bring a challenge of generalization for performance degradation assessment models. And it is costly and time-consuming to collect a large amount of labeled data for supervised diagnosis, especially when the task comes from a new operating condition. Thus in this paper, a novel bearing degradation assessment model is proposed based on transfer learning and deep hierarchical features extraction. The research of degradation assessment is transformed to the classification task of degradation pattern, which divides degradation process into the normal, slight fault, fault development and damage patterns. The hierarchical network with random weight parameters is introduced to extract the local sub-band characteristics of spectrum, in which the multiple alternately convolution and pooling layers without supervised fine-tuning are employed. Joint Geometrical and Statistical Alignment method is then utilized to obtain projected sharing feature space, and thus the knowledge of bearing degradation process is transferred to accomplish degradation pattern assessment under different operating conditions. Results of the experiments on bearing fault severity and degradation process show that the proposed method reduces the feature distribution divergence between the degradation processes and accomplishes bearing performance degradation assessment in different operating condition.

Improvement of hERG-ROMK index of spirocyclic ROMK inhibitors through scaffold optimization and incorporation of novel pharmacophores.

SAR in the previously described spirocyclic ROMK inhibitor series was further evolved from lead 4 by modification of the spirocyclic core and identification of novel right-side pharmacophores. In this process, it was discovered that the spiropyrrolidinone core with the carbonyl group α to the spirocenter was preferred for potent ROMK activity. Efforts aimed at decreasing hERG affinity within the series led to the discovery of multiple novel right-hand pharmacophores including 3-methoxythiadiazole, 2-methoxypyrimidine, and pyridazinone. The most promising candidate is pyridazinone analog 32 that showed an improved functional hERG/ROMK potency ratio and preclinical PK profile. In vivo evaluation of 32 demonstrated blood pressure lowering effects in the spontaneously hypertensive rat model.

Hyperbaric-Oxygen Therapy Improves Survival and Functional Outcome of Acute Severe Intracerebral Hemorrhage.

BACKGROUND: Prognosis of spontaneous intracerebral hemorrhage (ICH) remains poor worldwide. AIMS OF THE STUDY: To investigate the effect and optimal protocol for hyperbaric-oxygen therapy (HBOT), and reduce incidence of upper gastrointestinal bleeding (UGIB) in ICH. METHODS: This prospective, randomized, controlled trial included 565 patients with acute severe ICH. Participants were randomly assigned to a sham-control group (Group A) and four intervention groups: Groups B and C with 2.0 atmospheres absolute (ATA) pressure and HBOT exposure for 60 or 90 sessions, respectively; and Groups D and E with 1.5 ATA for 60 or 90 sessions, respectively. All patients received emergency craniotomy with hematoma evacuation. Outcome measures were modified Barthel Index (MBI) and modified Rankin Scale (mRS) scores, mortality rates at follow-up six months. UGIB rates were assessed as potential side effect. RESULTS: In four intervention groups, MBI and mRS scores were all significantly improved, and mortality rates were all significantly decreased compared with Group A (all p < 0.005). UGIB rates were 39.25, 60.00, 64.49, 36.79, and 34.26% in Groups A, B, C, D, and E, respectively. UGIB rates in Groups B and C were significantly increased compared with Groups A, D and E (all p < 0.005). None of UGIB were clinically significant. CONCLUSIONS: HBOT significantly improves survival and functional outcomes of ICH. HBOT at 1.5 and 2.0 ATA had the same beneficial effect. A pressure of 1.5 ATA and 60 HBOT exposures represents an optimal protocol for HBOT. Further studies are needed to optimize the protocol per specific patient.

(+)-Sorangicin A: evolution of a viable synthetic strategy.

An effective, asymmetric total synthesis of the antibiotic (+)-sorangicin A (1) has been achieved. Central to this venture was the development of first and second generation syntheses of the signature dioxabicyclo[3.2.1]octane core, the first featuring chemo- and stereoselective epoxide ring openings facilitated by a Co(2)(CO)(6)-alkyne complex, the second involving a KHMDS-promoted epoxide ring formation/opening cascade. Additional highlights include effective construction of the dihydro- and tetrahydropyran ring systems, respectively via a stereoselective conjugate addition/α-oxygenation protocol and a thioketalization/hydrostannane reduction sequence. Late-stage achievements entailed two Julia-Kociénski olefinations to unite three advanced fragments with high E-stereoselectivity, followed by a modified Stille protocol to introduce the Z,Z,E trienoate moiety, thereby completing the carbon skeleton. Mukaiyama macrolactonization, followed by carefully orchestrated Lewis and protic acid-promoted deprotections that suppressed isomerization and/or destruction of the sensitive (Z,Z,E)-trienoate linkage completed the first, and to date only, total synthesis of (+)-sorangicin A (1).

A case study from the chemistry core of the Pittsburgh Molecular Library Screening Center: the Polo-like kinase polo-box domain (Plk1-PBD).

The Polo-like kinase (Plk) family comprises four cell cycle serine/threonine kinases, Plk1-4. Among these, Plk1 has been most thoroughly characterized; it contains a conserved kinase domain and a C-terminal docking site for S/T-phosphorylated proteins (polo-box domain, PBD). Polo-like kinases are deregulated in oncogenesis and therefore constitute a therapeutic target for cancer. A high throughput screening campaign was carried out by the Pittsburgh Molecular Library Screening Center (PMLSC), using a fluorescence polarization assay with recombinant Plk1-PBD to monitor the inhibition of binding of an optimal phosphopeptide substrate motif with recombinant Plk1-PBD. Screening of 97,090 small molecule library samples provided by the NIH Small Molecule Repository distributed by DPI Galapagos led to 11 confirmed hits. The Pittsburgh MLSCN Chemistry Core selected one of the structurally most tractable hits, SID 861574, for chemical hit-to-probe development. A broad chemistry program was initiated that developed new strategies for 6-amino- and 6-hydroxy uracil synthesis as well as acylanilides, and generated a total of 70 analogs. Out of 46 analogues tested, none, nor the resynthesized hit, showed affinity to Plk1-PBD in the follow up assays. In contrast, re-assays of the original screening materials displayed activities similar to the original HTS assay. We ultimately concluded that an impurity in the commercial material led to the positive screening artifact. This case study highlights our development of a synthesis of 6-position functionalized uracil analogs, but also illustrates the importance of careful quality and compound stability monitoring of screening collections.

Total synthesis of (+)-sorangicin A.

The final synthetic challenges associated with (+)-sorangicin A have been overcome, thus leading to the first total synthesis of this complex macrolide antibiotic. Highlights of the highly convergent synthesis include two Julia-Kociénski olefinations to unite three advanced fragments with high E-stereoselectivity. Critical to the final-stage success was the use of a carefully defined Stille coupling and a Mukaiyama macrolactonization as well as Lewis and protic acid-promoted deprotections carefully designed to suppress E/Z isomerization and/or destruction of the delicate (Z,Z,E)-trienoate linkage.

(+)-Rimocidin synthetic studies: construction of the C(1-27) aglycone skeleton.

Assembly of the C(1-27) macrocyclic skeleton of rimocidinolide, the aglycone of (+)-rimocidin (1), has been achieved in convergent fashion. Key features of the synthetic strategy entail application of multicomponent Type I Anion Relay Chemistry (ARC), in conjunction with the S(N)2/S(N)2' reaction manifolds of vinyl epoxides, both employing 2-substituted 1,3-dithianes to construct the C(1-19) carbon backbone. Yamaguchi union of a C(20-27) vinyl borate ester, possessing the all-trans triene, with an advanced C(1-19) vinyl iodide followed by macrocyclization via Suzuki-Miyaura cross-coupling completed construction of the C(1-27) rimocidinolide skeleton.

An efficient, second-generation synthesis of the signature dioxabicyclo[3.2.1]octane core of (+)-sorangicin A and elaboration of the (Z,Z,E)-triene acid system.

An efficient, second-generation synthesis of the signature dioxabicyclo[3.2.1]octane core of (+)-sorangicin A (1), in conjunction with an effective, stereocontrolled protocol to arrive at the requisite (Z,Z,E)-triene acid system has been developed. Highlights of the core construction entail a three-component union, a KHMDS-promoted epoxide ring formation-ring opening cascade, a Takai olefination, and a chemoselective Sharpless dihydroxylation. Assembly of the triene acid system was then achieved via Stille cross-coupling with the ethyl ester of (Z,Z)-5-tributylstannyl-2,4-pentadienoic acid, followed by mild hydrolysis preserving the triene configuration.

Pestalotiopsin a. Enantioselective construction of potential building blocks derived from antipodal cyclobutanol intermediates.

D-Glyceraldehyde acetonide has been used as the starting point for accessing the enantiomeric cyclobutanols 11 in optically pure condition. The dextrorotatory enantiomer has been transformed in five steps into the [3.2.0] bicyclic lactone 22. While the deoxygenation of 22 proved to be problematical, the uncyclized variant 25 underwent the Barton process smoothly. These findings guided the related conversion of (-)-11 into 34. Use was also made of ring-closing metathesis to bring about the conversion of (+)-11 into [4.2.0] bicyclic lactone building blocks. In general, all three pathways are efficient and offer the prospect of practical side-chain appendage for the purpose of installing the nine-membered ring of pestalotiopsin A (1).

Pestalotiopsin A. Side chain installation and exhaustive probing of olefin metathesis as a possible tool for elaborating the cyclononene ring.

A program directed toward an asymmetric synthesis of pestalotiopsin A is described. The routing begins with the dextrorotatory cyclobutanol 37, which is combined with the enantiomerically defined building blocks ent-15 and 16. These units are incorporated via stereocontrolled 1,2-nucleophilic addition and anti-aldol coupling, respectively. With these straightforward reactions accomplished, the sequel involved the introduction of terminal double bonds in anticipation of the fact that the (E)-cyclononene substructure could be realized by ring-closing metathesis. This central issue was evaluated with several diene substrates and catalysts, all to no avail. Cross-metathesis experiments involving 59 and 65 with the functionalized heptene 60 revealed a marked difference in the inability to engage interaction with the ruthenium catalyst. This awkwardness could not be skirted.

Alkali metal counterion control of enolate protonation stereoselectivity.

[reaction: see text] Generation of the lithium salt of the norbornenol shown (M = H) followed by quenching with aqueous NH(4)Cl solution gives predominantly the beta-epimeric ketone 6. Similar production of the potassium alkoxide leads instead to the alpha-epimer (99:1). These results reveal the potential importance of alkali metal counterions as stereocontrol elements.

In pursuit of pestalotiopsin a via zirconocene-mediated ring contraction.

[reaction: see text] An asymmetric route from the epimeric beta-hydroxy esters 4 and 5 to the densely functionalized (+)-10 and (-)-10, respectively, is described. Either cyclobutanol can be made available as the predominant product. The levorotatory antipode has been transformed into the advanced intermediate 21 bearing side chains destined to become incorporated into the cyclononene ring of the title compound (1).

Highly stereoselective beta-anomeric glycosidation of a 2'-deoxy syn-5'-configured 4'-spironucleoside.

A direct enantioselective pathway that delivers exclusively the beta-anomer of a 4'-spironucleoside has been successfully developed. The key starting material is the enantiomerically pure dihydroxy lactone 19, which has proven amenable to conversion to glycal 22 via the chloro acetonide. This intermediate is then capped as the 3,5-O-(tetraisopropyldisiloxane-1,3-diyl) glycal. The latter can enter into N-iodosuccinimide-promoted glycosidation with persilylated thymine. Only the beta anomer is formed. Ensuing deiodination and desilylation proceed quantitatively to furnish the targeted, previously elusive anomer.

Stereoselective synthesis of beta-anomeric 4'-thiaspirocyclic ribonucleosides carrying the full complement of RNA-level hydroxyl substitution.

[reaction: see text] Stereoselective syntheses of a group of 4'-thiaspirocyclic ribonucleosides featuring both pyrimidine and purine classes and both possible configurations at C-5' are described. Use is made of the Pummerer reaction of substrates carrying an alpha-oriented 2,4-dimethoxybenzoyloxy substituent at C-2 in order to gain reliable stereocontrol via neighboring group participation. Irrespective of the S or R configuration of the pivotal sulfoxide intermediates, the nucleobase is captured from the beta-face. The competing process is formation of unsaturated sulfoxides, presumably via competing E2-type elimination. Although differences in reactivity between the two stereoisomeric series were noted, the common route has successfully given rise for the first time to desirable beta-anomeric sulfur-containing spiroribonucleosides with minimum formation of the alpha-anomers.

Stereoselective synthesis of conformationally constrained 2'-deoxy-4'-thia beta-anomeric spirocyclic nucleosides featuring either hydroxyl configuration at C5'.

An enantioselective approach to 2'-deoxy-4'-thia spirocyclic nucleosides featuring an alpha- or beta-hydroxyl substituent at C-5' of the carbocyclic ring is detailed. The starting point is the mandelate acetal 8. The overall strategy involves the stereocontrolled dihydroxylation of this dihydrothiophene, subsequent generation of the keto acetonide 12 followed by its Meerwein-Ponndorf-Verley reduction and beta-elimination, protection of the resulting dihydroxy thiaglycal, electrophilic glycosidation according to the Haraguchi protocol, reductive removal of the phenylseleno group, and end-game global deprotection. Acquisition of the alpha- and beta-5'-isomers is equally facile. Various 1D and 2D NMR techniques are used for assigning configuration.

C4'-spiroalkylated nucleosides having sulfur incorporated at the apex position.

Methodology based on the concept of thionium ion-initiated pinacolic ring expansion has been developed for accessing C4'-spirocyclic thionucleosides. The readily available racemic ketones 6 and 37 are conveniently resolved via their acetals with (R)-mandelic acid. Subsequent reactions beginning with utilization of the Pummerer rearrangement lend themselves to functionalization of the spirocyclic core and ultimately incorporation of the nucleosidic bases. Limitations to this strategy are pointed out. Acquisition of the alpha- and beta-isomers at C4' is equally facile. Absolute configurational assignments have been made possible by X-ray crystallography.