Preparation and Use of a General Solid-Phase Intermediate to Biomimetic Scaffolds and Peptide Condensations.

The Distributed Drug Discovery (D3) program develops simple, powerful, and reproducible procedures to enable the distributed synthesis of large numbers of potential drugs for neglected diseases. The synthetic protocols are solid-phase based and inspired by published work. One promising article reported that many biomimetic molecules based on diverse scaffolds with three or more sites of variable substitution can be synthesized in one or two steps from a common key aldehyde intermediate. This intermediate was prepared by the ozonolysis of a precursor functionalized at two variable sites, restricting their presence in the subsequently formed scaffolds to ozone compatible functional groups. To broaden the scope of the groups available at one of these variable sites, we developed a synthetic route to an alternative, orthogonally protected key intermediate that allows the incorporation of ozone sensitive groups after the ozonolysis step. The utility of this orthogonally protected intermediate is demonstrated in the synthesis of several representative biomimetic scaffolds containing ozonolytically labile functional groups. It is compatible with traditional Fmoc peptide chemistry, permitting it to incorporate peptide fragments for use in fragment condensations with peptides containing cysteine at the N-terminus. Overall yields for its synthesis and utilization (as many as 13 steps) indicate good conversions at each step.

Effect of single agent high-dose methotrexate-related acute kidney injury on length of hospitalization and relative dose intensity in adult patients with central nervous system lymphoma.

Purpose Grade ≥3 adverse effects prolong hospitalization and reduce chemotherapy dose intensity. The purpose of this study was to evaluate the rate and severity of high-dose methotrexate-related acute kidney injury and analyze its effect on hospital length of stay and relative chemotherapy dose intensity. Methods This was a retrospective cohort analysis. Patients receiving ≥1 dose of high-dose methotrexate were analyzed for acute kidney injury and length of stay. Patients receiving ≥6 cycles of induction therapy were included in the analysis of relative chemotherapy dose intensity. Chi squared analysis was used to determine the differences between dichotomous data; Student's t-test for parametric data and Mann-Whitney U test for non-parametric data for continuous variables. Statistical analyses were performed with IBM SPSS Statistics (version 21). Results Twenty-six patients and 194 treatment encounters were identified. Thirteen patients were evaluated for relative chemotherapy dose intensity. Grade ≥3 acute kidney injury occurred in four patients (15% of patients; 2% of encounters). There were no grade 5 adverse events. Mean length of stay for encounters with grade ≥3 acute kidney injury was almost three times longer than for those with ≤ grade 2 acute kidney injury (p = 0.041). Mean relative chemotherapy dose intensity was reduced approximately in half for patients experiencing grade ≥3 acute kidney injury (p < 0.01). The most common adverse events were hypokalemia and nausea. Proton pump inhibitors were the most frequently co-administered medications with the potential to affect high-dose methotrexate pharmacokinetics. Conclusion At our cancer program, the rate of grade ≥3 acute kidney injury with high-dose methotrexate is similar to that reported by others. Grade ≥3 acute kidney injury following high-dose methotrexate administration significantly prolonged length of stay and reduced relative chemotherapy dose intensity.

Solid-Phase Synthesis of Amine/Carboxyl Substituted Prolines and Proline Homologues: Scope and Limitations.

A solid-phase procedure is used to synthesize racemic peptidomimetics based on the fundamental peptide unit. The peptidomimetics are constructed around proline or proline homologues variably substituted at the amine and carbonyl sites. The procedure expands the diversity of substituted peptidomimetic molecules available to the Distributed Drug Discovery (D3) project. Using a BAL-based solid-phase synthetic sequence the proline or proline homologue subunit is both constructed and incorporated into the peptidomimetic by an α-alkylation, hydrolysis and intramolecular cyclization sequence. Further transformations on solid-phase provide access to a variety of piperazine derivatives representing a class of molecules known to exhibit central nervous system activity. The procedure works well with proline cores, but with larger six- and seven-membered ring homologues the nature of the carboxylic acid acylating the cyclic amine can lead to side reactions and result in poor overall yields.

Unexpected hydrolytic instability of N-acylated amino acid amides and peptides.

Remote amide bonds in simple N-acyl amino acid amide or peptide derivatives 1 can be surprisingly unstable hydrolytically, affording, in solution, variable amounts of 3 under mild acidic conditions, such as trifluoroacetic acid/water mixtures at room temperature. This observation has important implications for the synthesis of this class of compounds, which includes N-terminal-acylated peptides. We describe the factors contributing to this instability and how to predict and control it. The instability is a function of the remote acyl group, R(2)CO, four bonds away from the site of hydrolysis. Electron-rich acyl R(2) groups accelerate this reaction. In the case of acyl groups derived from substituted aromatic carboxylic acids, the acceleration is predictable from the substituent's Hammett σ value. N-Acyl dipeptides are also hydrolyzed under typical cleavage conditions. This suggests that unwanted peptide truncation may occur during synthesis or prolonged standing in solution when dipeptides or longer peptides are acylated on the N-terminus with electron-rich aromatic groups. When amide hydrolysis is an undesired secondary reaction, as can be the case in the trifluoroacetic acid-catalyzed cleavage of amino acid amide or peptide derivatives 1 from solid-phase resins, conditions are provided to minimize that hydrolysis.

Pseudohyperkalemia in a patient with chronic lymphoblastic leukemia and tumor lysis syndrome.

PURPOSE: Recognition of pseudohyperkalemia is essential to prevent medical mismanagement of erroneous hyperkalemia. The purpose of this case is to describe pseudohyperkalemia attributed to malignant leucocytosis in a patient with chronic lymphoblastic leukemia and tumor lysis syndrome. Methods for determination of pseudohyperkalemia are discussed. SUMMARY: A 75-year-old male with progressive chronic lymphoblastic leukemia was hospitalized for medical evaluation and chemotherapy administration. Notable laboratory findings included white blood cell count of 479 × 10(3) cells/µL (4.00 × 10(3) cells/µL-10.80 × 10(3) cells/µL) with 95% lymphocytes (20%-50%) and 5% blasts (zero) present in the differential, serum potassium 9.8 mM/L (3.4 mM/L-5.0 mM/L), uric acid of 11.8 mg/dL (3.5 mg/dL-8.0 mg/dL), serum creatinine 1.47 mg/dL (0.60 mg/dL-1.30 mg/dL), and lactate dehydrogenase of 2529 IU/L (100 IU/L-220 IU/L). The patient was anemic (Hb 7.6 g/dL (14.0 g/dL-18.0 g/dL)) and thrombocytopenic (17 × 10(3) platelets/µL (140 × 10(3) platelets/µL-400 × 10(3) platelets/µL)). There were no electrocardiographic findings indicating systemic hyperkalemia. Repeat analysis of the blood potassium level using a heparinized tube assayed immediately after specimen collection demonstrated a plasma potassium level 4.1 mM/L. Subsequent analysis of specimens using similar methodology demonstrated potassium results within the normal limits despite continued laboratory evidence of pseudohyperkalemia. Based on the patient's conscious and interactive condition, ECG findings, and normal plasma potassium level following immediate analysis, the diagnosis of pseudohyperkalemia was made. Laboratory findings of pseudohyperkalemia persisted throughout the period of leukocytosis. CONCLUSION: This case describes pseudohyperkalemia attributed to malignant leucocytosis in a patient with chronic lymphoblastic leukemia (CLL). Practitioners should consider pseudohyperkalemia as the underlying cause of elevated potassium levels in patients with malignant leucocytosis who do not have signs or symptoms of systemic hyperkalemia.

Solid-phase synthetic route to multiple derivatives of a fundamental peptide unit.

Amino acids are Nature's combinatorial building blocks. When substituted on both the amino and carboxyl sides they become the basic scaffold present in all peptides and proteins. We report a solid-phase synthetic route to large combinatorial variations of this fundamental scaffold, extending the variety of substituted biomimetic molecules available to successfully implement the Distributed Drug Discovery (D3) project. In a single solid-phase sequence, compatible with basic amine substituents, three-point variation is performed at the amino acid a-carbon and the amino and carboxyl functionalities.

Multi-Institution Research and Education Collaboration Identifies New Antimicrobial Compounds.

New antibiotics are urgently needed to address increasing rates of multidrug resistant infections. Seventy-six diversely functionalized compounds, comprising five structural scaffolds, were synthesized and tested for their ability to inhibit microbial growth. Twenty-six compounds showed activity in the primary phenotypic screen at the Community for Open Antimicrobial Drug Discovery (CO-ADD). Follow-up testing of active molecules confirmed that two unnatural dipeptides inhibit the growth of Cryptococcus neoformans with a minimum inhibitory concentration (MIC) ≤ 8 µg/mL. Syntheses were carried out by undergraduate students at five schools implementing Distributed Drug Discovery (D3) programs. This report showcases that a collaborative research and educational process is a powerful approach to discover new molecules inhibiting microbial growth. Educational gains for students engaged in this project are highlighted in parallel to the research advances. Aspects of D3 that contribute to its success, including an emphasis on reproducibility of procedures, are discussed to underscore the power of this approach to solve important research problems and to inform other coupled chemical biology research and teaching endeavors.

Distributed Drug Discovery, Part 3: using D(3) methodology to synthesize analogs of an anti-melanoma compound.

For the successful implementation of Distributed Drug Discovery (D(3)) (outlined in the accompanying Perspective), students, in the course of their educational laboratories, must be able to reproducibly make new, high quality, molecules with potential for biological activity. This article reports the successful achievement of this goal. Using previously rehearsed alkylating agents, students in a second semester organic chemistry laboratory performed a solid-phase combinatorial chemistry experiment in which they made 38 new analogs of the most potent member of a class of antimelanoma compounds. All compounds were made in duplicate, purified by silica gel chromatography, and characterized by NMR and LC/MS. As a continuing part of the Distributed Drug Discovery program, a virtual D(3) catalog based on this work was then enumerated and is made freely available to the global scientific community.

Distributed Drug Discovery, Part 2: global rehearsal of alkylating agents for the synthesis of resin-bound unnatural amino acids and virtual D(3) catalog construction.

Distributed Drug Discovery (D(3)) proposes solving large drug discovery problems by breaking them into smaller units for processing at multiple sites. A key component of the synthetic and computational stages of D(3) is the global rehearsal of prospective reagents and their subsequent use in the creation of virtual catalogs of molecules accessible by simple, inexpensive combinatorial chemistry. The first section of this article documents the feasibility of the synthetic component of Distributed Drug Discovery. Twenty-four alkylating agents were rehearsed in the United States, Poland, Russia, and Spain, for their utility in the synthesis of resin-bound unnatural amino acids 1, key intermediates in many combinatorial chemistry procedures. This global reagent rehearsal, coupled to virtual library generation, increases the likelihood that any member of that virtual library can be made. It facilitates the realistic integration of worldwide virtual D(3) catalog computational analysis with synthesis. The second part of this article describes the creation of the first virtual D(3) catalog. It reports the enumeration of 24,416 acylated unnatural amino acids 5, assembled from lists of either rehearsed or well-precedented alkylating and acylating reagents, and describes how the resulting catalog can be freely accessed, searched, and downloaded by the scientific community.

On Planar Discrete Elastic Rod Models for the Locomotion of Soft Robots.

Modeling soft robots that move on surfaces is challenging from a variety of perspectives. A recent formulation by Bergou et al. of a rod theory that exploits new developments in discrete differential geometry offers an attractive, numerically efficient avenue to help overcome some of these challenges. Their formulation is an example of a discrete elastic rod theory. In this article, we consider a planar version of Bergou et al.'s theory and, with the help of recent works on Lagrange's equations of motion for constrained systems of particles, show how it can be used to model soft robots that are composed of segments of soft material folded and bonded together. We then use our formulation to examine the dynamics of a caterpillar-inspired soft robot that is actuated using shape memory alloys and exploits stick-slip friction to achieve locomotion. After developing and implementing procedures to prescribe the parameters for components of the soft robot, we compare our calibrated model to the experimental behavior of the caterpillar-inspired soft robot.

Discovery of potent and selective inhibitors of the Escherichia coli M1-aminopeptidase via multicomponent solid-phase synthesis of tetrazole-peptidomimetics.

The Escherichia coli neutral M1-aminopeptidase (ePepN) is a novel target identified for the development of antimicrobials. Here we describe a solid-phase multicomponent approach which enabled the discovery of potent ePepN inhibitors. The on-resin protocol, developed in the frame of the Distributed Drug Discovery (D3) program, comprises the implementation of parallel Ugi-azide four-component reactions with resin-bound amino acids, thus leading to the rapid preparation of a focused library of tetrazole-peptidomimetics (TPMs) suitable for biological screening. By dose-response studies, three compounds were identified as potent and selective ePepN inhibitors, as little inhibitory effect was exhibited for the porcine ortholog aminopeptidase. The study allowed for the identification of the key structural features required for a high ePepN inhibitory activity. The most potent and selective inhibitor (TPM 11) showed a non-competitive inhibition profile of ePepN. We predicted that both diastereomers of compound TPM 11 bind to a site distinct from that occupied by the substrate. Theoretical models suggested that TPM 11 has an alternative inhibition mechanism that doesn't involve Zn coordination. On the other hand, the activity landscape analysis provided a rationale for our findings. Of note, compound TMP 2 showed in vitro antibacterial activity against Escherichia coli. Furthermore, none of the three identified inhibitors is a potent haemolytic agent, and only two compounds showed moderate cytotoxic activity toward the murine myeloma P3X63Ag cells. These results point to promising compounds for the future development of rationally designed TPMs as antibacterial agents.

Documenting and harnessing the biological potential of molecules in Distributed Drug Discovery (D3) virtual catalogs.

Virtual molecular catalogs have limited utility if member compounds are (i) difficult to synthesize or (ii) unlikely to have biological activity. The Distributed Drug Discovery (D3) program addresses the synthesis challenge by providing scientists with a free virtual D3 catalog of 73,024 easy-to-synthesize N-acyl unnatural α-amino acids, their methyl esters, and primary amides. The remaining challenge is to document and exploit the bioactivity potential of these compounds. In the current work, a search process is described that retrospectively identifies all virtual D3 compounds classified as bioactive hits in PubChem-cataloged experimental assays. The results provide insight into the broad range of drug-target classes amenable to inhibition and/or agonism by D3-accessible molecules. To encourage computer-aided drug discovery centered on these compounds, a publicly available virtual database of D3 molecules prepared for use with popular computer docking programs is also presented.

Solid-phase synthesis of constrained terminal and internal lactam peptidomimetics.

Lactams are key components of many peptidomimetic structures. Five- and six-membered lactam peptidomimetics with hydrogen or amino acid side chains at the alpha-position can be constructed from peptide precursors during a solid-phase synthesis. There is no significant racemization of remote stereocenters during synthesis.

A phase II trial of neoadjuvant gemcitabine, epirubicin, and docetaxel as primary treatment of patients with locally advanced or inflammatory breast cancer.

BACKGROUND: Three-drug regimens containing gemcitabine, an anthracycline, and a taxane produce response rates of 70%-90% in patients with metastatic breast cancer (MBC) although accompanied by considerable hematologic toxicity. We explored the combination of gemcitabine/epirubicin/docetaxel as neoadjuvant therapy. Docetaxel was administered weekly to decrease myelosuppression. PATIENTS AND METHODS: A total of 110 patients with locally advanced or inflammatory breast cancer received neoadjuvant gemcitabine 800 mg/m2 intravenously (I.V.) days 1 and 8, epirubicin 75 mg/m2 I.V. day 1, and docetaxel 30 mg/m2 I.V. days 1 and 8, repeated every 21 days for 4 cycles. Then patients had either mastectomy or breast conservation surgery, and pathologic treatment responses were assessed. After surgery, 4 cycles of adjuvant gemcitabine 1000 mg/m2 I.V. days 1 and 8 and docetaxel 35 mg/m2 I.V. days 1 and 8 were administered at 21-day intervals. After patients completed chemotherapy, locoregional radiation therapy and/or anti-estrogen therapy was administered per standard guidelines. RESULTS: Treatment with 4 cycles of neoadjuvant gemcitabine, epirubicin, and weekly docetaxel resulted in an objective response in 79 of 110 patients enrolled (72%; 95% CI, 63-80%). Twenty of 103 patients (19%) who had surgery had pathologic complete response (pCR). Moderate hematologic toxicity was evident during neoadjuvant therapy, with grade 3/4 neutropenia in 41% and febrile neutropenia in 11% of the patients. Protocol-specified dose modifications were required in 35% of the patients, and 58% of the patients used myeloid growth factors. CONCLUSION: The pCR rate of 19% achieved with gemcitabine, epirubicin, and weekly docetaxel confirms previous reports with similar 3-drug regimens. The use of a weekly schedule of docetaxel did not appear to reduce the incidence of grade 3/4 hematologic toxicity.

Solid-phase synthesis of amino- and carboxyl-functionalized unnatural alpha-amino acid amides.

A new solid-phase synthesis efficiently incorporates three different substituents (from R(1)-X, R(2)-CO(2)H, and R(3)-NH(2)) into a glycine-based peptidomimetic scaffold. The synthetic sequence is general and is typically accomplished in >50% overall isolated yield. Alkylating agents with a range of reactivities and normal and branched primary amines give good results. Utility was demonstrated by the synthesis of a series of protected phosphotyrosine mimetics.

Solid-phase synthesis of multiple classes of peptidomimetics from versatile resin-bound aldehyde intermediates.

A wide variety of highly substituted lactam containing peptidomimetic scaffolds are prepared by solid-phase synthesis from a single, versatile class of resin-bound aldehyde intermediates (1). These include monocyclics 3, bicyclics 4, tricyclics 5, and tetracyclics 6. The key intermediate 1 is readily synthesized from resin-bound natural or unnatural alpha-amino acids. The synthetic procedures permit the construction of a large diversity of substitution patterns for ready use in combinatorial chemistry. In every case, the release of final products from resin is by a cyclitive cleavage process. Since this depends on successful completion of multiple intermediate synthetic steps, the products are often quite pure, even though previous steps involve only a filtration workup. The mild conditions for many of these synthetic procedures offer the promise of using this chemistry in peptide fragment condensations to produce modified peptides, at either the N-terminus or C-terminus, or as individually assembled peptide segments with a wide variety of conformationally restricted peptidomimetic linkers at the point of juncture.

A solid-phase synthetic route to unnatural amino acids with diverse side-chain substitutions.

Reacting imine derivatives of resin-bound amino acids with alpha,omega-dihaloalkanes provides highly versatile intermediates to racemic alpha,alpha-disubstituted amino acids with a wide variety of side-chain functionality. Two strategies were developed to convert the intermediate omega-chloro or omega-bromo derivatives to the desired products. Together, they allow the creation of amino acids with diverse functionalities (omega-chlorides, nitriles, azides, acetates, thioacetates, thioethers, secondary and tertiary aliphatic amines, and anilines) placed at varying chain lengths (2-5) from the alpha-center of the amino acid.