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Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-.

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Identification of Small Molecule Inhibitors that Suppress Cytokine-Induced Apoptosis in Human Pancreatic Islet Cells

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Received: ; Last Update: May 26, 2011.

Type 1 diabetes is caused by autoimmune destruction of insulin-producing beta cells in the pancreas. In this process, beta-cell apoptosis involves multiple signaling cascades stimulated by interleukin-1β (IL-1β), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α). These pathways result in decreased pancreatic beta-cell numbers that lead to the disease phenotype. Most of the compounds described in the literature protect cells from a single facet of cytokine treatment but do not provide wide-ranging protection from apoptosis nor do they restore insulin secretion. The goal of this project was to identify and to optimize small molecules that can prevent cytokine-induced pancreatic beta-cell apoptosis. To achieve this goal, we completed a screen of 339,000 compounds in rat INS-1E insulinoma cells treated with IL-1β, IFN-γ, and TNF-α. As a result, we identified MLS003179189, a member of a novel diversity-oriented synthesis (DOS) library with stereochemical diversity and complexity akin to naturally occurring small molecules. MLS003179189 contains 3 stereocenters and was the only stereoisomer (out of eight possible stereoisomers) to show activity in the primary assay for cell viability. About 50 analogs of the active stereoisomer were synthesized and tested, leading to a superior probe candidate (ML187). In studies with dissociated human primary pancreatic islets, (ML187) improved cell viability, decreased caspase activation, and improved insulin production. These data suggest that a consistent mechanism of action exists in both rat and human cells and that the probe (ML187) is a first-in-class probe for Type I diabetes that both protects against destruction of beta cells and restores function.

Assigned Assay Grant No.: DP2 DK083048

Screening Center Name & PI: Broad Institute Probe Development Center, Stuart Schreiber

Chemistry Center Name & PI: Broad Institute Probe Development Center, Stuart Schreiber

Assay Submitter & Institution: Bridget Wagner, Broad Institute, Chemical Biology Program

PubChem Summary Bioassay Identifier (AID): 435007

Probe Structure and Characteristics

CID/ML No.Target NameTarget IC50 (μM) [SID, AID]Anti-target NameAnti-target IC50 (μM) [SID, AID]Fold selectiveSecondary Assay 1 IC50 (μM) [SID, AID]Secondary Assay 2 IC50 (μM) [SID, AID]Secondary Assay 3 IC50 (μM) [SID, AID]
46907798/ML187Cytokine-induced beta cell apoptosis3.5 [SID-99376568, 488844]Beta-cell apoptosis in the absence of cytokinesNo activity up to 25 μM [99376568,488864]>10XCytokine-induced beta-cell apoptosis counter screen 2.2 [99376568, 4888936]Suppresses caspase-3 activation in primary human islets 6.7 [99376568, 488945]Restores glucose-stimulated insulin secretion in rat INS-1E cells 4.8 [99376568, 488951]

Recommendations for scientific use of the probe

The goal of this project is to identify and to optimize small molecules that can prevent cytokine-induced pancreatic beta-cell apoptosis. To achieve this goal, we have completed a pilot screen of 19,826 compounds and a primary screen of 319,000 compounds in rat INS-1E insulinoma cells treated with a cocktail of IL-1β, IFN-γ, and TNF-α. As a result, we have identified a probe compound (ML187) capable of increasing cellular ATP, decreasing caspase activation, and restoring glucose-stimulated insulin secretion (GSIS) in INS-1E cells in the presence of this cytokine cocktail. Importantly, this compound has exhibited activity in dissociated primary human islet cells, suggesting a consistency in the mechanism of action between species tested. This is a first-in-class probe with potential to be developed into an in vivo therapeutic agent to alleviate the beta-cell apoptosis that occurs during the development of type 1 diabetes. Although there are small molecules that exhibit some of the attributes listed above, no compound described in the literature contains all of the features in our new probe (ML187) disclosed in this report.

There are three main uses of a molecular probe discovered by this screening process:

  1. To uncover the molecular pathways by which cytokine-induced beta-cell apoptosis occurs, helping us to understand the process of type 1 diabetes development.
  2. To understand the points at which this process can be disrupted.
  3. To ascertain how closely the rodent cell system models the development of human type 1 diabetes.

The probe (ML187) generated from this project will be useful to the larger diabetes research community who focus on the role of the immune system and cytokine-induced beta cell injury. It also will be useful to immunologists who use diabetes as a model of autoimmune disease. For example, many laboratories are focused on the role of thymocytes in early type I diabetes (1). This probe (ML187) will help to parse out the roles of other immune cells that may play a role in the early phase of disease and will help to identify avenues for therapeutic intervention.

1. Introduction

Type 1 diabetes is caused by autoimmune destruction of insulin-producing beta cells in the pancreas. Human beta-cell apoptosis in this process involves a complex set of signaling cascades initiated by interleukin-1β (IL-1β), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α). IL-1β and TNF-α induce nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) expression, while downstream activation of gene expression is thought to occur through nitric oxide (NO) signaling, which both increases the endoplasmic reticulum stress-response pathway and decreases beta-cell function. These effects of cytokines are beta cell-specific, and we aim to find small-molecule suppressors that would have little to no effect on other cell types in the pancreas. We sought to build a physiologically relevant in vitro model of the development of type 1 diabetes. Therefore, we decided to use the rat INS-1E beta-cell line (2), which is sensitive to the cytokine cocktail in all assays performed. Further, it is an excellent model for studying both apoptosis and GSIS. The primary assay measured general cellular viability after 48 hours of cytokine treatment and served as a surrogate for programmed cell death. Despite the greater fold changes induced by cytokines in some of the secondary assays, we chose to use CellTiter-Glo (CTG) as the primary assay for reasons of cost, ease-of-performance, and data quality (e.g., the assay had the lowest coefficient of variation). Next, we utilized a panel of secondary assays that addressed the different outcomes of cytokine-induced apoptosis, such as activation of NO signaling, activation of caspases, breakdown of mitochondrial function, and loss of insulin production. A successful probe is one that increases cellular ATP, reduces caspase activity, reduces nitrite production, increases mitochondrial membrane potential, and restores insulin secretion compared to cells treated with cytokines. In addition, the probe must show activity in primary human pancreatic islets in the caspase and GSIS assays.

Small molecules that increase beta-cell survival in the presence of cytokines could be of potential clinical benefit to early-stage type 1 diabetic patients. Previous studies have described either anti-inflammatory or antioxidant small molecules that have general protective effects in beta cells in the presence of cytokines (see Figure 1). For example, sulforaphane induces the expression of multiple antioxidant proteins (3) while resveratrol (4,5), the flavonoids (6), and an extract of the Nardostachys jatamansi herb (7) have anti-inflammatory effects due to interaction with the NF-κB pathway. The known anti-inflammatory and immunosuppressant dexamethasone can increase beta-cell proliferation in rat islets at high doses (8); however, it does not restore GSIS. The histone deacetylase (HDAC) inhibitors suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA) can prevent some aspects of cytokine-induced beta-cell death (9), presumably by decreasing NF-κB transactivation, but lack specificity. The kinase inhibitors alsterpaullone (9) and Ro 31-8220 (10) can prevent beta-cell apoptosis at low concentrations, but act as GSK-3β inhibitors and are only partially protective. Some commercially available pyrazoles were identified in our pilot screening as inhibitors of cytokine-induced beta-cell apoptosis. These compounds increased ATP by 90% but only partially restored GSIS (10). In addition, these pyrazoles contain a chemically reactive thioester bond that may compromise activity. Most of the compounds described in the literature protect cells from a single facet of cytokine treatment but do not provide wide-ranging protection from apoptosis and restore insulin secretion. These studies also suggest that multiple mechanisms are involved in cytokine-induced apoptosis. Some of these compounds appear to be somewhat toxic to these cells, as evidenced by a decrease in ATP in the absence of cytokine treatment. Therefore, we seek to identify more potent and selective compounds that can suppress the effects of cytokines without cytotoxicity.

Figure 1. Structures of Compounds That Have at Least Some Protective Effects in Beta Cells.

Figure 1

Structures of Compounds That Have at Least Some Protective Effects in Beta Cells. General classes include kinase inhibitors, polyphenols such as flavonoids and resveratrol, pyrazoles, HDAC inhibitors, general antioxidants, and anti-inflammatory compounds (more...)

Only SAHA and alsterpaullone are currently available in the MLSMR.

2. Materials and Methods

Materials and Reagents

INS-1E cells (generously provided by C. Wollheim and P. Maechler, University of Geneva) are a rat insulinoma cell line that models certain aspects of pancreatic beta cell biology (2). This particular subclone maintains the ability to secrete insulin upon treatment with higher concentrations of glucose.

All compounds were dissolved in DMSO and were pinned into plates in a 0.1 μl-volume with the exception of the 96-well plate studies, where 0.24 μl of compound solution was added per well. For every assay plate, multiple DMSO and positive control wells were included. For dose-response studies in INS-1E cells, compounds were measured at a range of concentrations from 0.2 μM to 25 μM at 2-fold dilutions. For primary islet studies, the maximal dose of compound was 20 μM.

2.1. Assays

A summary listing of completed assays and corresponding PubChem AID numbers is provided in Appendix A (Table A1). Refer to Appendix B for the detailed assay protocols.

Table A1. Summary of Completed Assays and AIDs.

Table A1

Summary of Completed Assays and AIDs.

Cell Culture and Reagents

INS-1E cells were maintained in phenol red free RPMI-1640 (Invitrogen) containing 11 mM glucose, 10% fetal bovine serum (FBS), 10 mM HEPES, 50 μM 2-mercaptoethanol, 1 mM sodium pyruvate, cultivated at 37 °C with 5% CO2 in a humidified atmosphere, with fluid changes every 3 days and splitting every 7 days. Recombinant rat IL-1β, recombinant IFN-γ, and recombinant mouse TNF-α were purchased from R&D Systems.

2.1.1. Primary Caspase-3 Activity Assay (AID 488848)

Apoptosis itself is determined by measurement of caspase-3 activity using the commercially available luminescent Caspase-Glo 3/7 assay (Promega). The primary assay measures cell viability via ATP levels as a surrogate for apoptosis but does not measure apoptosis directly. A more selective assay is to measure the activity of a direct mediator of apoptosis, such as caspase-3. Our cytokine cocktail induced the activity of this assay 4- to 5-fold.

Procedures

INS-1E cells were seeded at 8,000 cells per well in white, opaque, 384-well plates and treated as described above. After treatment with cytokines and compounds for 48 hours, 20 μl Caspase-Glo 3/7 reagent was added. Luminescence was measured after 2 hours of incubation using an EnVision plate reader (Perkin-Elmer).

2.1.2. Counterscreen Measurement of Cellular ATP (Cellular Viability; AID Nos. 488910, 88844)

The primary assay was of cellular ATP levels, measured by the commercial kit CellTiter-Glo (Promega). This assay informed us of viability and energy status after compound treatment, which was decreased by about 50% after cytokine treatment.

Procedures

INS-1E cells were plated at 8,000 cells per well in 30 μl plating media (phenol red-free RPMI-1640 (Invitrogen) containing 11 mM glucose, 5% FBS, 10 mM HEPES, 50 μM 2-mercaptoethanol, 1 mM sodium pyruvate) into opaque, white, 384-well tissue culture plates. On the following day, the cells were treated with 10 μl of cytokine cocktail (10 ng/ml IL-1β, 50 ng/ml IFN-γ, and 25 ng/ml TNF-α) in plating media and pinned with compounds. After 48 hours, the combination of cytokines led to apoptotic cell death. The level of cell death was inferred by detecting a decrease in cellular ATP.

2.1.3. Secondary Measurement of Cellular Nitrite Production (AID Nos. 488866, 488868, 488870)

Levels of nitrite accumulated in the cell-culture media were measured colorimetrically using the Griess reagent (a commercially available mixture of naphthylenediamine dihydrochloride and sulfanilamide). IL-1β is known to induce gene expression of nitric oxide synthase (iNOS), an effect that is potentiated by IFN-γ. The subsequent formation of NO drives cell death by both necrosis and apoptosis. In this case, cytokine treatment increased NO in the media approximately 3-fold.

Procedures

NS-1E cells were seeded at 8,000 cells per well into clear, 384-well tissue-culture plates and treated as described above. After treatment with cytokine and compounds for 48 hours, 10 μl of modified Griess reagent (1:1 mixture of 1% sulfanilamide in 30% acetic acid and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride in 60% acetic acid; Sigma) was added to each well. After 5 minutes of incubation at room temperature, the absorbance was measured at 540 nm using an EnVision plate reader (Perkin-Elmer).

2.1.4. Secondary Measurement of Glucose-Stimulated Insulin Secretion (GSIS; 488959)

We measured glucose-stimulated insulin secretion (GSIS), the gold standard for beta-cell function. The insulin stimulatory index can be measured by ELISA, after 1-hour incubation with “high glucose” (2 mM) in the experimental buffer, in comparison with “low glucose” (15 mM). The rat INS-1E cell line and beta cells treated with cytokines lose their insulin secretory response to glucose; small molecules that can promote beta-cell survival should restore insulin secretion.

Procedures

INS-1E cells were seeded in 96-well tissue-culture plates at 20,000 cells per well and incubated for 48 hours in 100 μl of fresh RPMI containing 1% FBS and the cytokine cocktail, in the presence or absence of compounds. The cells were washed once with and incubated for 2 hours in KRBH (135 mM NaCl, 3.6 mM KCl, 5 mM NaHCO3, 0.5 mM NaH2PO4, 0.5 mM MgCl2, 1.5 mM CaCl2, 10 mM HEPES, pH 7.4, 0.1% BSA) without glucose. The cells were subsequently incubated with KRBH containing 2 mM or 15 mM glucose for 1 hour. Next, 5 μl of the supernatant was taken for measurement of released insulin. Insulin was measured with a rat insulin ELISA kit (ALPCO).

2.1.5. Mitochondrial Membrane Potential (MMP) in INS-1E Cells (488867)

Cytokine-mediated beta-cell apoptosis has been reported to cause a loss of the mitochondrial membrane potential (MMP). JC-1 is a dye commonly used to measure MMP, and cytokine treatment decreases MMP by about 50%. JC-1 is a cationic dye that exhibits a membrane potential-dependent accumulation in mitochondria. The dye exists as a monomer at low concentrations yielding green fluorescence similar to fluorescein. At higher concentrations, the dye forms J-aggregates that exhibit a broad excitation spectrum and an emission maximum at approximately 590 nm (i.e., red fluorescence). In apoptotic cells, JC-1 remains in the cytoplasm as the green monomer and the amount of red fluorescence decreases. Mitochondrial membrane depolarization is indicated by a decrease in the red to green fluorescence intensity ratio.

Procedures

INS-1E cells were seeded into Aurora IQ-EB black, 384-well, optical-bottom plates at 8,000 cells per well using 30 μl per well. After overnight incubation, 10 μl RPMI-1640 containing 5% FBS and a combination of cytokines (10 ng/ml IL-1β, 50 ng/ml IFN-γ, 25 ng/ml TNF-α) was added to every well. Next, 0.1 μl of each compound or DMSO was added to each well. After incubation for 48 hours, medium was removed and 20 μl per well of 3.25 μM JC-1 in phenol-red media was added. The plates were incubated at 37 °C for 2 hours. The cells were gently washed three times with 50 μl per well of 1X PBS (with Ca2+ and Mg2+). Fluorescence was measured with an EnVision plate reader (Perkin-Elmer) at the rhodamine spectra (excitation/emission 530 nm/580 nm) followed by fluorescein (excitation/emission 485nm/530nm). The ratio of rhodamine to fluorescein intensity was determined and represents the degree of mitochondrial membrane potential.

2.1.6. Counterscreen Cytokine-free CellTiter-Glo Assay in INS-1E Cells (488864)

We also performed a counterscreen assay to test compounds for induction of ATP production in the absence of cytokines. INS-1E cells were treated with compounds at various doses for 48 hours in the absence of cytokines. While perhaps interesting in another context, compounds that elevate ATP levels in INS-1E cells under basal conditions are not suitable as probes in this assay. Further, compounds that suppress cytokine-induced beta-cell apoptosis, but are toxic in their own right, are similarly unattractive.

Procedures

INS-1E cells were plated at 8,000 cells per well in 30 μl plating media (phenol red free RPMI 1640 (Invitrogen) containing 11 mM glucose, 5% FBS, 10 mM HEPES, 50 μM 2-mercaptoethanol, 1 mM sodium pyruvate) into opaque, white, 384-well, tissue culture plates. On the following day, 10 μl of plating media was added per well and treated with compounds for 48 hours. The level of cellular ATP was measured with CellTiter-Glo assay reagent (Promega).

2.1.7. Caspase Activity in Primary Human Pancreatic Islet Cells (488945)

Human islets were obtained through the Islet Cell Resource Consortium (http://icr.coh.org/) and through the National Disease Research Interchange (http://www.ndriresource.org/). The purity and viability of human islets are reported to be 70–93% and 70–98%, respectively.

Procedures

Islets were washed with PBS and incubated in CMRL medium supplemented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. Islets were gently dissociated into a cell suspension by incubating with Accutase (37 °C, 10 min), and seeded in 384-well plates containing extracellular matrix secreted by the HTB-9 human bladder carcinoma cell line (adapted from Beattie et al.) (11). Islets were exposed to a range of cytokine concentrations for 6 days, with media changed after 3 days. The cells were processed for caspase-3 activity with the CaspaseGlo 3/7 assay (Promega). Activity was measured with the EnVision plate reader (Perkin-Elmer).

2.1.8. GSIS in Primary Human Pancreatic Islet Cells (488951)

The best indication of physiological relevance in cell culture is to recapitulate our results in human beta cells. Therefore, we tested the effects of promising screening positives in primary human pancreatic islets. We have confirmed that dissociated human islet cells are sensitive to a 6-day cytokine treatment. These results suggest that it is possible to use primary human islets for follow-up studies of promising compounds. Human islets were obtained through the Islet Cell Resource Consortium (http://icr.coh.org/) and through the National Disease Research Interchange (http://www.ndriresource.org/). The purity and viability of human islets are reported to be 70–93% and 70–98%, respectively.

Procedures

Islets were washed with PBS and incubated in CMRL medium supplemented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. Islets were gently dissociated into a cell suspension by incubating with Accutase (37 °C, 10 min), and seeded in 384-well plates containing extracellular matrix secreted by the HTB-9 human bladder carcinoma cell line (adapted from Beattie et al.) (11). Islets were exposed to a range of cytokine concentrations for 6 days, with media changed after 3 days. The culture supernatants were collected for insulin production. In this case, islets were treated as in the GSIS ELISA for INS-1E cells (see Section 2.1.4) and insulin was measured with a commercial ELISA kit (ALPCO).

2.2. Probe Chemical Characterization

After preparation as described in Section 2.3, the probe (ML187, SID-99376568) was analyzed by UPLC, 1H NMR and 13C NMR spectroscopy, and high-resolution mass spectrometry (HRMS (ESI) calcd for C35H38N4O8S [M + H]+: 675.2483, found: 675.2491). The data obtained from NMR and mass spectroscopy were consistent with the structure of the probe, and UPLC indicated an isolated purity of greater than 99%. The stereochemistry was confirmed by X-ray crystallography of an intermediate (12). Solubility, plasma protein binding (PPB), and plasma stability were analyzed (Figure 2). The associated spectroscopic data is provided in Appendix C.

Figure 2. Stability of the Probe (ML187) in PBS at 23°C.

Figure 2

Stability of the Probe (ML187) in PBS at 23°C. Amount of the probe (ML187) in 0.2% DMSO in PBS buffer over time (A); Amount of the probe at varying concentrations of DMSO in PBS (B); Amount of the probe in plasma after 5 hours (C). Note: The probe (more...)

The stability of the probe (ML187) was measured in phosphate buffered saline (PBS) (Figure 2A). Although the graph suggests that only a small percentage of the probe (ML187) remains after 2 hours, this anomalous result is likely due to the low solubility of the compound, as the PBS solubility of the probe (ML187) is <0.5 μM.

To support this theory, DMSO was used to dilute the sample that was tested in the PBS stability assay, and it was found that as more DMSO was added the signal for the product also increased (Figure 2B). This finding indicates that the additional DMSO is solubilizing the precipitate, and the compound is actually stable at 72 hours. Of note, the compound is stable in the plasma stability assay, which is also analyzed in PBS buffer (Figure 2C).

The probe (ML187, MLS003179193) and four additional analogs MLS003179190 (CID44489675), MLS003179192 (CID44492462), MLS003179189(CID44500012), and MLS003179191(CID46907806) (Figure 3) were submitted to the MLSMR collection were characterized and analyzed by the same methods.

Figure 3. Structures of Compounds Submitted to the MLSMR Library.

Figure 3

Structures of Compounds Submitted to the MLSMR Library.

2.3. Probe Preparation

The probe (ML187) was prepared by solid-phase synthesis (see Scheme 1) with Synphase L-Series lanterns (12). The synthesis is amenable to diversification and has been used to create large libraries of molecules (12). Specifically, approximately 8,000 compounds with this substructure were synthesized and tested in this screen. Full experimental details are provided in this section.

Scheme 1. Synthesis of Probe (ML187).

Scheme 1

Synthesis of Probe (ML187).

The synthesis of probe (ML187) began with the anti-aldol reaction of the ephedrine-based starting material 1. Enolization of 1 was carried out using freshly prepared dicyclohexylboron triflate and further reaction of the presumed boron enolate with aldehyde 2 provided the aldol product. Hydrolysis with sodium hydroxide/hydrogen peroxide followed by TBS protection of the alcohol yielded compound 3 in good yield. Coupling of acid 3 with amine 4 followed by reduction of the amide gave 5. This 5-step sequence is robust and has been successfully applied on large scale to provide up to 150 grams of amine 5 and all of its stereoisomers with just one flash chromatography purification.

Acylation of the secondary amine gave linear precursor 6. Cyclization through intramolecular SNAr reaction of 6 with CsF provided compound 7. Reduction of the nitro group and protection of the resulting aniline as the Fmoc carbamate gave 8. Removal of the Boc carbamate followed by subsequent protection as the allyl carbamate and removal of the PMB ether formed 9. Exchanging the protecting group from Boc to Alloc was necessary to allow compatibility with the silyl linker used in the subsequent solid phase transformations.

Loading of compound 9 onto Synphase lanterns followed by removal of the Fmoc protecting group gave 10. Compound 10 can then be diversified by reaction with a variety of isocyanates, sulfonyl chlorides, or acid chlorides. To generate the probe (ML187), reaction of the aniline with naphthyl isocyanate provided 11, and removal of the Alloc protecting group gave free amine 12. Free amine 12 allows for an additional position of diversification via reaction with a number of sulfonyl chlorides, isocyanates, and aldehydes. En route to the probe, 12 was capped with 2,3-dihydrobenzo[b][1,4]dioxine-6-sulfonyl chloride. Cleavage of the desired product from the lantern provided the probe (ML187).

Using this solid-phase diversification platform, a library of 8,000 compounds was made from all eight stereoisomers of 9 in highly enantioenriched form.

Chemistry Experimental Methods

General details. All reagents and solvents were purchased from commercial vendors and used as received, or synthesized according to the footnoted references. All oxygen and/or moisture sensitive reactions were carried out under nitgrogen (N2) atmosphere in glassware that had been flame-dried under vacuum (approximately 0.5 mm Hg) and purged with N2 prior to use.

NMR spectra were recorded on a Bruker 300 (300 MHz 1H, 75 MHz 13C) or Varian UNITY INOVA 500 (500 MHz 1H, 125 MHz 13C) spectrometer. Proton and carbon chemical shifts are reported in ppm (δ) referenced to the NMR solvent. Data are reported as follows: chemical shifts, multiplicity (br = broad, s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet; coupling constant(s) in Hz;). Unless otherwise indicated, NMR data were collected at 25 °C. Flash chromatography was performed using 40–60 μm Silica Gel (60 Å mesh) on a Teledyne Isco Combiflash Rf. Tandem Liquid Chromotography/Mass Spectrometry (LC/MS) was performed on a Waters 2795 separations module and 3100 mass detector. Analytical thin layer chromatography (TLC) was performed on EM Reagent 0.25 mm silica gel 60-F plates. Visualization was accomplished with UV light and aqueous potassium permanganate (KMnO4) stain followed by heating. High-resolution mass spectra were obtained at the MIT Mass Spectrometry Facility (Bruker Daltonics APEXIV 4.7 Tesla Fourier Transform Ion Cyclotron Resonance Mass Spectrometer).

Image ml187fu2

Preparation of 4-(tert-Butoxycarbonyl(methyl)amino)-3-(tert-butyldimethylsilyloxy)-2-methylbutanoic acid, 1: An oven-dried, 3-liter, 3-neck, round-bottom flask was equipped with a mechanical stirrer and temperature probe. Under a positive flow of N2, the vessel was charged with (1R,2S)-1-phenyl-2-(N,2,4,6-tetramethylphenylsulfonamido)propyl propionate (109 g, 270 mmol) and methylene chloride (CH2Cl2; 932 ml). The reaction was cooled to −78 °C with constant agitation at which point triethylamine (Et3N 112 ml, 810 mmol) was added dropwise, maintaining an internal reaction temperature no greater than −65 °C (approximately 10 minutes). After 15 minutes of additional stirring, a solution of dicyclohexylboron triflate (1.0 M in CH2Cl2, 176 g, 540 mmol) was added dropwise, maintaining an internal reaction temperature below −67 °C (approximately 30 minutes). The resulting yellow enolate reaction solution was stirred at −78 °C for an additional 2 hours. At this point t-butyl methyl(2-oxoethyl) carbamate (68.1 ml, 405 mmol) was added dropwise at a rate so as to maintain an internal temperature below −67 °C (approximately 15 minutes). The reaction mixture was stirred at −78 °C for 2 hours and then was allowed to warm to 0 °C for 1 hour. The reaction was quenched by addition of methyl alcohol (MeOH; 1.09 L) and pH 7 buffer (130 ml). Then, aqueous hydrogen peroxide (35% by wt, 130 ml, 1.5 mol) was slowly added such that the internal reaction temperature did not exceed 20 °C. After the addition was complete, the mixture was allowed to warm to ambient temperature where it was stirred for 1 hour. The resultant slurry was then concentrated in vacuo, and the residue was partitioned between water (550 ml) and CH2Cl2 (500 ml). The aqueous layer was extracted with CH2Cl2 (4 × 300 ml), and the combined organic layers were washed with water (100 ml) followed by brine (100 ml). The organic layer was dried over anhydrous magnesium sulfate (MgSO4), and the solvent was removed in vacuo to yield 1-phenyl-2-(N,2,4,6-tetramethylphenylsulfonamido) propyl 4-(tert-butoxycarbonyl(methyl)amino)-3-hydroxy-2-methylbutanoate (156 g, 270 mmol) as a yellow oil, which was deemed sufficiently pure and carried forward without any further purification. A small sample was purified on silica gel for full characterization.

(R,S,S,R)-(+)-11b: [α]D20 +6.42 (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3, 60 °C) d 7.18 (q, J = 5, 3H), 7.26 (d, J = 5, 1H), 7.18 (d, J = 5, 2H), 4.68 (m, 1H), 4.21 (t, J = 10, 1H), 4.15 (dd, J = 10, 5, 1H), 4.12 (m, 1H), 3.80 (m, 1H), 3.25 (dd, J = 15, 5, 2H), 2.91 (s, 3H), 2.79 (dd, J = 10, 5, 1H), 1,45 (s, 9H), 1.30 (d, J = 10, 3H); 13C NMR (125 MHz, CDCl3, 60 °C) d 176.1, 153.0, 135.4, 129.4, 129.0, 128.9, 127.3, 80.0, 71.3, 70.0, 66.3, 58.1, 55.2, 53.7, 52.8, 41.4, 41.2, 36.0, 28.5, 28.4, 27.5, 25.9, 18.3, 11.9; HRMS (ESI) calcd for C21H30N2O6Na [M + Na]+: 429.1996. Found: 429.2000.

A 3-liter, 3-neck, round-bottom flask was equipped with an overhead stirrer, an internal temperature probe and an addition funnel. The vessel was charged with the crude 1-phenyl-2-(N,2,4,6-tetramethylphenylsulfonamido)propyl-4-(tert-butoxycarbonyl-(methyl)amino)-3-hydroxy-2-methylbutanoate (156.0 g, 270 mmol, 1.0 equiv) and 1:1 t-BuOH/MeOH (1700 ml) was added and cooled 0 °C. A solution of H2O2 (30% in water, 166 ml, 1623 mmol, 6.0 equiv) was added via addition funnel, followed by aqueous NaOH (1.0 M, 811 ml, 811 mmol, 3.0 equiv), which was added at such a rate that the internal temperature did not rise above 10 °C. The mixture was warmed to room temperature slowly and allowed to stir overnight. In the morning, organic solvents were removed in vacuo. The resulting aqueous layer was diluted with water (300 ml) and extracted with EtOAc (4 × 200 ml) to remove the auxiliary. The resulting aqueous layer was then acidified with 6 M HCl to pH 4 and extracted with EtOAc (5 × 250 ml). This second set of EtOAc extracts were combined, dried (MgSO4), filtered, and concentrated, which provided the pure product 12b as a yellow oil (66.0 g, 99%). While the crude product was carried on to the next step without further purification, an analytical sample was obtained by chromatography on silica gel.

(S,R)-(+)-12b: [α]D20 +2.1 (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3, 60 °C) d 4.07 (dt, J = 10, 5, 1H), 3.41 (br s, 1H), 3.22 (d, J = 15, 1H), 2.91 (s, 3H), 2.62 (ddd, J = 16, 10, 5, 1H), 1,45 (s, 9H), 1.25 (d, J = 10, 3H). 13C NMR (125 MHz, CDCl3, 60 °C) d 181.0, 157.9, 81.0, 72.6, 53.4, 43.8, 36.2, 28.7, 15.0. HRMS (ESI) calcd for C11H21NO5Na [M + Na]+: 270.1312. Found: 270.1312.

An oven-dried, 5-liter, 3-neck, round-bottom flask equipped with a magnetic stirrer and internal temperature probe was charged with 4-(tert-butoxycarbonyl(methyl)amino)-3-hydroxy-2-methylbutanoic acid (58.7 g, 237 mmol, 1.0 equiv), CH2Cl2 (0.1 M) and 2,6-lutidine (91 ml, 783 mmol, 3.3 equiv) under a positive flow of N2. The reaction mixture was cooled to −78 °C and tert-butyldimethylsilyl trifluoromethanesulfonate (TBSOTf) (120 ml, 522 mmol, 2.2 equiv) was added dropwise over 20 min. The reaction mixture was stirred at −78 °C for 1 hour, quenched with aqueous saturated sodium bicarbonate (NaHCO3,) the layers were separated, and the aqueous layer was extracted with diethyl ether. The combined organic extracts were washed with saturated ammonium chloride (NH4Cl) and brine, dried (MgSO4), filtered, and the solvent evaporated. The residue was dissolved in MeOH and THF and cooled to 0 °C. An aqueous solution of potassium carbonate (K2CO3; 1.5–2.2 equiv) was added, and the mixture was stirred at 0 °C for 1 hour. The reaction mixture was concentrated to remove volatiles, and the remaining aqueous layer was extracted with EtOAc. The organic layer was washed with 1 N HCl, then dried over MgSO4, filtered, and concentrated in vacuo to yield the crude product as a clear oil, which was coevaporated with toluene to remove excess TBSOH and dried to provide the TBS ether 1 as a clear oil.

[α]D20 −3.2 (c 0.6, CHCl3). 1H NMR (500 MHz, CDCl3, 60 °C) d 4.15 (br s, 1H), 3.35 (dd, J = 14.5, 6.0, 1H), 3.23 (br s, 1H), 2.88 (s, 3H), 2.65 (br s, 1H), 1.44 (s, 9H), 1.21 (d, J = 7.0, 1H), 0.89 (s, 9H), 0.09 (s, 6H); 13C NMR (125 MHz, CDCl3, 60 °C) d 176.8, 156.3, 80.3, 72.4, 52.9, 43.8, 36.7, 28.7, 26.5, 25.9, 24.2, 18.2, −4.4; HRMS (ESI) calcd for C17H25NO5SiNa [M + Na]+: 384.2177. Found: 384.2167.

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Preparation of tert-Butyl 2-(tert-butyldimethylsilyloxy)-4-(1-(4-methoxybenzyloxy)propan-2-ylamino)-3-methylbutyl(methyl)carbamate, 5. An oven-dried, 3-liter, 3-neck, round-bottom flask was equipped with an overhead stirrer, addition funnel, and a temperature probe. Under a positive flow of N2, the vessel was charged with 4-(tert-butoxycarbonyl(methyl)amino)-3-(tert-butyldimethylsilyloxy)-2-methylbutanoic acid 1 (1.0 equiv) dissolved in CH2Cl2 (80% of total solvent, final concentration of 1 = 0.2 M), followed by benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP;1.0 equiv), and diisopropyl ethylamine (DIPEA; 3.0 equiv) The resulting mixture was cooled in an ice bath before 1-(4-methoxybenzyloxy)propan-2-amine 2 (1.1–1.2 equiv) was added as a solution in CH2Cl2 (remaining 20% of total solvent) by addition funnel. The rate of addition was controlled so as to maintain an internal temperature between 3–5 °C. When addition was complete, the mixture was warmed to ambient temperature and allowed to stir for 15 hours. The reaction was quenched with water, and extracted with CH2Cl2. The combined organic extracts were dried over MgSO4, filtered, and concentrated. The yellow oil was taken up in Et2O, and the phosphoramide byproducts were removed via filtration. The solvent was removed in vacuo, and the crude product was isolated. Flash chromatography on silica gel (4:1 Hexanes/EtOAc to 7:3 Hexanes/EtOAc) gave the product 13 as a colorless oil.Following the general reaction protocol (−) −1a (131 g, 362 mmol, 1.0 equiv) was reacted with PyBOP (189 g, 362 mmol, 1.0 equiv), DIPEA (190 ml, 1087 mmol, 3.0 equiv) and (S)-alaninol (+)−2 (78 g, 399 mmol, 1.1 equiv) in CH2Cl2 (1812 ml), which provided pure product (156 g, 80%) as a clear oil.

[α]D20 −39.9 (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3, 55 °C) δ 7.21 (d, J = 8.5, 2H), 6.85 (d, J = 8.5, 2H), 6.41 (br s, 1H, NH), 4.45 (d, J = 11.7, 1H), 4.41 (d, J = 11.7, 1H), 4.20-4.14 (m, 1H), 4.13-4.04 (m, 1H), 3.78 (s, 3H), 3.47 (dd, J = 3.6, 14.3, 1H), 3.39 (d, J = 4.3, 2H), 3.00-2.85 (m, 1H), 2.86 (s, 3H), 2.38 (dq, J = 3.8, 7.2, 1H), 1.45 (s, 9H), 1.19 (d, J = 6.7, 3H), 1.12 (d, J = 7.2, 3H), 0.89 (s, 9H), 0.07 (s, 3H), 0.05 (s, 3H). 13C NMR (125 MHz, CDCl3, 55 °C) δ 172.3 (br), 159.4, 155.8 (br), 130.5, 129.2 (2C), 113.9 (2C), 79.5 (br), 73.5 (br), 72.93, 72.85, 55.2, 51.8, 44.87, 44.85, 36.7 (br), 28.5 (3C), 25.9 (3C), 17.89, 17.88, 12.6, −4.7, −4.8. HRMS (ESI) calcd for C28H50N2NaO6Si [M + Na]+: 561.3330. Found: 561.3313.

An oven-dried, 2-liter, 1-necked, round-bottom flask was equipped with a magnetic stirrer. Under a positive flow of N2, the flask was charged with tert-butyl 2-(tert-butyldimethylsilyloxy)-4-(1-(4-methoxybenzyloxy)propan-2-ylamino)-3-methyl-4-oxobutyl(methyl)carbamate 13 (1.0 equiv) and anhydrous THF (final concentration 0.1 M). Borane dimethylsulfide complex (BH3.DMS;5.0 equiv) was added dropwise via syringe. Afterwards, the reaction mixture was heated at 65 °C for 5 hours. After cooling to ambient temperature, excess hydride was quenched by the careful addition of MeOH. The mixture was concentrated under reduced pressure to afford a colorless oil, which was then co-evaporated with MeOH three times to remove excess B(OMe)3. The oil was then re-dissolved in MeOH and 10% aqueous potassium sodium tartrate (2:3 ratio, final concentration 0.067 M). The resulting slurry was heated at reflux for 12 hours. The volatiles were removed under reduced pressure, and the aqueous layer was extracted three times with ethyl acetate. The combined organic layers were washed once with brine, dried over magnesium sulfate, filtered and concentrated to provide the desired amine 3 as a colorless oil.

[α]D20 −10.65 (c 2.46, CHCl3). 1H NMR (500 MHz, CDCl3, 60 °C) δ 7.19 (d, J = 8.5 Hz, 2H), 6.82 (d, J = 8.5 Hz, 2H), 4.40 (s, 2H), 3.89 (m, 1H), 3.74 (s, 3H), 3.29 (m, 4H), 2.98 (dd, J = 13.5, 7.5 Hz, 1H), 2.85 (m, 4H), 2.63 (m, 1H), 2.41 (dd, J = 11, 9 Hz, 1H), 1.72 (m, 1H), 1.41 (s, 9H), 0.98 (d, J = 6.5 Hz, 1H), 0.92 (d, J = 6.5 Hz, 1H), 0.86 (s, 9H), 0.02 (s, 3H), 0.00 (s, 3H). 13C NMR (125 MHz, CDCl3, 60 °C) δ 159.3, 155.7, 130.8, 129.1, 113.9, 79.1, 74.6, 73.3, 72.8, 55.2, 52.5, 52.0, 49.4, 38.7, 36.4, 28.6, 26.0, 18.0, 17.6, 13.7, −4.5. HRMS (ESI) calcd for C28H53N2O5Si [M + H]+: 525.3718. Found: 525.3698.

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Preparation of tert-Butyl-((2R,3R)-2-((tert-butyldimethylsilyl)oxy)-4-(2-fluoro-N-((S)-1-((4-methoxybenzyl)oxy)propan-2-yl)-3-nitrobenzamido)-3-methylbutyl)(methyl)carbamate, 4. To a stirred solution of 2 (21 g, 40.0 mmol, 1 equiv) and 2-fluoro-3-nitrobenzoyl chloride (20.36 g, 100 mmol, 2.5 equiv) in CH2Cl2 (120 ml) at 0 °C was added NEt3 (27.7 ml, 200 mmol, 5 equiv). The reaction was allowed to warm to room temperature as it stirred, and no starting material remained after 1.5 hours. Water (H2O;50 ml) was added to the reaction, and it was extracted with CH2Cl2 (2 x 100 ml). The combined organic portion was dried over MgSO4, filtered, and concentrated. The crude material was purified by silica gel chromatography in EtOAc/hexanes (10% →30%) to give the product.

[α]20D −49.5 (c 1.0, CHCl3); 1H NMR (DMSO-d6, 500 MHz, 150 °C) δ 8.12 (dd, J = 7.7, 7.7, 1H), 7.64 (dd, J = 6.6, 6.6, 1H), 7.45 (dd, J = 7.9, 7.9, 1H), 7.21 (d, J = 8.4, 2H), 6.90 (d, J = 8.4, 2H), 4.41 (s, 2H), 3.95-3.83 (m, 2H), 3.78 (s, 3H), 3.54-3.45 (m, 1H), 3.40-3.24 (m, 4H), 3.16-3.08 (m, 1H), 2.84 (s, 3H), 2.15-2.06 (m, 1H), 1.43 (s, 9H), 1.25 (br s, 3H), 0.94 (br s, 3H), 0.85 (s, 9H), 0.04 (s, 3H), 0.02 (s, 3H); 13C NMR (DMSO-d6, 125 MHz, 150 °C) δ 164.2, 158.5, 154.5, 149.7, 137.1, 133.5, 129.7, 128.2 (2C), 128.0, 125.3, 124.6, 113.3 (2C), 78.1, 72.4 71.5, 70.4, 54.6, 54.0, 51.0, 36.3 (br), 34.8, 27.5 (3C), 25.0 (3C), 16.9, 14.7, 12.5, −5.4, −5.6 (one carbon absent); HRMS (ESI) calc’d for C35H55FN3O8Si [M + H]+: 692.3737, found: 692.3764.

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Preparation of tert-Butyl (((2R,3R)-5-((S)-1-((4-methoxybenzyl)oxy)propan-2-yl)-3-methyl-10-nitro-6-oxo-3,4,5,6-tetrahydro-2H-benzo[b][1,5]oxazocin-2-yl)methyl)(methyl)carbamate, 5. To a stirred solution of 4 (24.5 g, 35.4 mmol, 1 equiv) in DMF (708 ml) was added CsF (10.76 g, 70.8 mmol (2 equiv). The resulting suspension was heated to 85 °C for 5 hours. The solvent was then removed under reduced pressure, and the crude solid was dissolved in EtOAc (250 ml), washed with H2O (1 × 100 ml) and brine (1 × 100 ml), dried over Na2SO4, filtered, and concentrated. The product was used in the next reaction without purification.

[α]20D −52.1 (c 1.0, CHCl3); 1H NMR (DMSO-d6, 500 MHz, 150 °C) δ 7.91 (d, J = 8.0, 1H), 7.64 (d, J = 8.0, 1H), 7.39 (dd, J = 8.0, 8.0, 1H), 7.27 (d, J = 8.4, 2H), 6.91 (d, J = 8.4, 2H), 4.52 (d, J = 11.8, 1H), 4.48 (d, J = 11.8, 1H), 4.35-4.28 (m, 1H), 3.96-3.91 (m, 1H), 3.86 (dd, J = 7.3, 9.8, 1H), 3.79 (s, 3H), 3.66 (dd, J = 5.8, 10.2, 1H), 3.63 (dd, J = 5.0, 15.0, 1H), 3.50 (dd, J = 2.0, 15.0, 1H), 3.38 (dd, J = 10.0, 16.0, 1H), 3.15 (br d, J = 16.0, 1H), 2.89 (s, 3H), 2.18-2.12 (m, 1H), 1.47 (s, 9H), 1.34 (d, J = 6.7, 3H), 0.87 (d, J = 6.8, 3H); 13C NMR (DMSO-d6, 125 MHz, 150 °C) δ 165.0, 158.5, 154.5, 146.6, 143.0, 133.7, 132.2, 130.0, 128.0, 125.4, 124.3, 113.3, 88.5, 78.2, 71.4, 71.0, 54.6, 52.4, 51.5, 50.4, 35.2, 34.4, 27.4, 15.2, 13.9; HRMS (ESI) calcd for C29H39N3NaO8 [M + Na]+: 580.2629, found: 580.2614.

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Preparation of tert-Butyl(((2R,3R)-10-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-((S)-1-((4-methoxybenzyl)oxy)propan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-benzo[b][1,5]oxazocin-2-yl)methyl)(methyl)carbamate, 6. A mixture of compound 5 (7.51 g, 13.47 mmol, 1 equiv) and Pd/C (1.43g, 1.347 mmol, 0.1 equiv) in EtOH (500 ml) was stirred under H2 at 40 °C. No starting material remained after 2 hours, and the reaction was cooled, filtered through Celite, and concentrated. The crude material was used in the next reaction without purification. The product (0.852 g, 1.614 mmol) was taken up in 10% aqueous NaHCO3 (10 ml) in dioxane (60 ml), and a solution of fluorenylmethyloxycarbonyl chloride (FMOC-Cl 2.088 g, 8.07 mmol, 5 equiv) in dioxane (5 ml) at 0 °C was added via syringe. The resulting cloudy solution was allowed to stir for 15 minutes at 0 °C and then room temperature overnight. The solution became clear as it warmed to room temperature. The reaction was quenched with sat. NH4Cl solution and extracted with EtOAc (2 × 100 ml). The combined organic portion was dried over MgSO4, filtered, and concentrated. The crude material was purified by silica gel chromatography (EtOAc/hexanes gradient).

[α]20D −71.7 (c 1.00, CHCl3); 1H (DMSO-d6, 500 MHz, 130 °C) δ 7.84 (d, J = 7.5, 2H), 7.80 (d, J = 7.5, 2H), 7.41 (t, J = 7.5, 2H), 7.35 (t, J = 7.5, 2H), 7.29 (d, J = 8, 2H), 6.91 (d, J = 8.5, 2H), 6.89 (t, J = 7.5, 1H), 6.84 (d, J = 7.5, 1H), 6.56 (d, J = 7, 1H), 4.61 (s, 2 H), 4.49 (dd, J = 12.0, 2 H), 4.40 (dd, J = 7.0, 6.5, 1 H), 3.80 (m, 4H), 3.65-3.62 (m, 3 H), 3.38 (dd, J = 10.5, 1H), 3.33 (dd, J = 10.0, 1H), 3.03 (d, J = 16, 1H), 2.97 (s, 3H), 2.82-2.81 (m, 1H), 1.99 (m, 1H), 1.46 (s, 9H), 1.29 (d, J = 6.5, 3H), 0.80 (d, J = 6.5, 3H) ppm; 13C (DMSO-d6, 500 MHz, 130 °C, as a mixture of rotomers) δ 169.1, 159.9, 156.1, 143.7, 142.2, 140.9, 140.4, 138.4, 132.6, 131.5, 129.5, 129.4, 127.8, 125.4, 121.8, 120.4, 118.0, 116.6, 114.8, 109.2, 84.4, 80.0, 72.8, 72.7, 72.0, 60.2, 56.0, 54.1 54.0, 52.7, 52.6, 51.4, 51.2, 37.2, 36.9, 28.9, 17.0, 15.4; C44H51N3NaO8: 772.3568 [M+Na]+ found: 772.3593.

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Preparation of Allyl (((2R,3R)-10-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-((S)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-benzo[b][1,5]oxazocin-2-yl)methyl)(methyl)carbamate, 7. To a stirred solution of 6 in CH2Cl2 (200 ml, 0.1 M) was added 2,6-lutidine (9.94 ml, 85 mmol, 4.0 equiv), and TBSOTf (14.7 ml, 64 mmol, 3.0 equiv). The mixture was stirred for 2 hours and then quenched with saturated NH4Cl solution and extracted with EtOAc. The combined organic extracts were dried over MgSO4, filtered, and concentrated. The resulting oil was dissolved in CH2Cl2 (120 ml) and cooled to −78 °C before triethylamine (12.5 ml, 90 mmol, 5.0 equiv) and allyl chloroformate (1.78 ml, 16.7 mmol, 1.0 equiv) were added. After 10 minutes, the reaction was quenched with saturated NH4Cl and extracted with CH2Cl2. The combined organic extracts were dried over MgSO4, filtered, and concentrated. The crude material was purified by silica gel chromatography (EtOAc/hexanes 30% →50%) to give the product (12.2 g, 78%).

The product (12.2 g, 16.6 mmol, 1.0 equiv) was then dissolved in CH2Cl2 (200 ml) and pH 7 buffer (15 ml). The mixture was cooled to 0 °C and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ;5.66 g, 24.9 mmol, 1.5 equiv) was added. The mixture was stirred for 10 minutes at 0 °C and then 1 hour at room temperature before quenching with H2O. The product was then extracted with CH2Cl2, and the combined organic portion was washed with saturated NaHCO3 before activated carbon was added. The mixture was then filtered through Celite, and the Celite was washed several times with hot CH2Cl2. The filtrate was concentrated, and the crude material was purified by silica gel chromatography (0% →5% MeOH/CH2Cl2) to give the product (10.0 g, 98%).

[α]20D −23.8 (c 1.0, CHCl3); 1H NMR (DMSO-d6, 500 MHz, 150 °C, as a 10:1 mixture of rotomers, only major rotomer reported) δ 8.04 (br s, 1H), 7.83 (d, J = 7.3, 2H), 7.66 (dd, J = 2.3, 7.3, 2H), 7.64 (dd, J = 2.3, 7.0, 1H), 7.40 (dd, J = 7.3, 7.3, 2H), 7.30 (dd, J = 7.5, 7.5, 2H), 7.15-7.11 (m, 2H), 5.93 (ddq, J = 5.4, 5.4, 10.7, 1H), 5.28 (d, J = 17.2, 1H), 5.17 (d, J = 10.5, 1H), 4.61-4.51 (m, 4H), 4.33 (dd, J = 6.4, 6.4, 1H), 4.23-4.13 (m, 2H), 3.79 (dd, J = 6.7, 10.5, 1H), 3.72-3.60 (m, 4H), 3.31 (dd, J = 10.0, 15.5, 1H), 3.11 (d, J = 15.5, 1H), 3.01 (s, 3H), 2.21-2.14 (m, 1H), 1.30 (d, J = 7.0, 3H), 0.83 (d, J = 6.7, 3H); 13C NMR (DMSO-d6, 125 MHz, 150 °C, as a mixture of rotomers) δ 167.7, 166.7, 155.7, 155.3, 153.0, 146.7, 143.1, 141.0, 140.2, 139.3, 139.0, 137.0, 132.7, 131.3, 131.2, 129.9, 128.0, 126.7, 126.3, 126.1, 125.8, 124.5, 124.1, 123.8, 123.6, 120.3, 119.1, 118.9, 116.7, 116.2, 116.1, 115.5, 107.5, 85.7, 82.9, 65.7, 64.8, 63.1, 63.0, 54.6, 54.2, 52.7, 51.6, 50.8, 50.5, 46.4, 35.7, 35.4, 34.8, 34.2, 15.4, 15.1, 13.5; HRMS (ESI) calcd for C35H40N3O7 [M + H]+: 614.2861, found: 614.2840.

General protocol A for cleavage of the compound from the lantern and its characterization for qualitative analysis. A portion of each product was used to assure that each reaction was complete. To complete this qualitative analysis and characterization, one segment of a lantern (1/4) was severed and placed in a polypropylene vial (or 96-well plate) and HF/pyridine (200 uL) was added to completely submerge the lantern. The mixture was allowed to sit at room temperature for 3 hours. The reaction was then quenched by slow addition of methoxytrimethylsilane (400 μl, >2 equiv) via pipette. After 15 minutes, the quenched solution was then removed from the vial and combined with additional MeOH washes of the lantern (2 × 200 μl). The product was dried under reduced pressure to yield a solid which was dissolved in DMSO and analyzed by UPLC/MS.

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Preparation of Allyl (((2R,3R)-10-amino-5-((S)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-benzo[b][1,5]oxazocin-2-yl)methyl)(methyl)carbamate, 8. Compound 7 (353 mg, 0.576 mmol) was rigorously dried by evaporating with benzene (3 × 5 ml) to azeotrope H2O. The resulting white solid was dried under reduced pressure overnight. SynPhase L-series alkyl tethered diisopropylarylsilane lanterns (32 Lanterns, approximately 15 μmol/lantern) were prepared for loading by washing with CH2Cl2 (3 × 20 min) and dried overnight under reduced pressure. The lanterns were then activated in an oven-dried vial by addition of 3% TfOH in CH2Cl2. The vial was shaken for 10 minutes, and the lanterns turned bright red. The liquid was removed, 2,6-lutidine was added, and the lanterns were shaken until the red color disappeared. A small amount of CH2Cl2 was added to the lanterns followed by 7 (353 mg, 0.576 mmol, 1.2 equiv) in CH2Cl2. Enough CH2Cl2 was added to cover the lanterns, and the reaction was shaken for 60 hours. The reaction solvent was then removed, and the lanterns were washed with CH2Cl2 (2 × 8 ml) and DMF (2 × 8 ml). The lanterns were shaken in 20% piperidine in DMF (8 ml) for 30 minutes to remove the Fmoc protecting group. The lanterns were then washed with DMF (2 × 8 ml), 3:1 THF/H2O (1 × 8 ml), 3:1 THF:isopropanol (1 × 8 ml), THF (1 × 8 ml), and CH2Cl2 (2 × 8 ml). General Protocol A was used to release the compound from the lantern for subsequent for characterization to ascertain that the reaction was complete. LRMS (M + H)+: 392.19.

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Preparation of Allyl (((2R,3R)-5-((S)-1-hydroxypropan-2-yl)-3-methyl-10-(3-(naphthalen-1-yl)ureido)-6-oxo-3,4,5,6-tetrahydro-2H-benzo[b][1,5]oxazocin-2-yl)methyl)(methyl)carbamate, 9. To 8 (26 3/4 lanterns, 0.401 mmol) in CH2Cl2 (8 ml) was added 1-isocyanatonaphthalene (1.357 g, 8.02 mmol, 20 equiv). The vial was sealed and shaken at room temperature for 36 hours. The reaction mixture was then removed and the lanterns were washed with CH2Cl2 (1 × 8 ml), DMF (2 × 8 ml), THF/H2O (3:1, 1 × 8 ml), THF/isopropanol (3:1, 1 × 8 ml), THF (1 × 8 ml), and CH2Cl2 (2 × 8 ml). General protocol A was used to release the compound from the lantern for subsequent characterization to ascertain that the reaction was complete. LRMS (M + H)+: 561.20.

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Preparation of 1-((2R,3R)-5-((S)-1-Hydroxypropan-2-yl)-3-methyl-2-((methylamino)methyl)-6-oxo-3,4,5,6-tetrahydro-2H-benzo[b][1,5]oxazocin-10-yl)-3-(naphthalen-1-yl)urea, 10. To 9 (26 1/2 lanterns, 0.383 mmol) in THF (8 ml) was added dimethylbarbituric acid (1.798 g, 11.51 mmol, 30 equiv) and Pd(PPh3)4 (444 mg, 0.384 mmol, 1.0 equiv). The vial was sealed and the reaction was allowed to shake at room temperature overnight. The reaction mixture was then removed, and the lanterns were washed with CH2Cl2 (2 × 8 ml) and DMF (5 × 8 ml). The lanterns were then shaken in DMF (8 ml) overnight. The solvent was removed and the lanterns were washed with DMF (2 × 8 ml), THF/H2O (3:1, 1 × 8 ml), THF/isopropanol (3:1, 1 × 8 ml), THF (1 × 8 ml), and CH2Cl2 (2 × 8 ml). General Protocol A was used to release the compound from the lantern for subsequent characterization to ascertain that the reaction was complete. LRMS (M + H)+: 477.22.

Image ml187fu11

Preparation of N-(((2R,3R)-5-((S)-1-Hydroxypropan-2-yl)-3-methyl-10-(3-(naphthalen-1-yl)ureido)-6-oxo-3,4,5,6-tetrahydro-2H-benzo[b][1,5]oxazocin-2-yl)methyl)-N-methyl-2,3-dihydrobenzo-[b][1,4]dioxine-6-sulfonamide, ML187, MLS003179193. To 10 (11 1/4 lanterns, 0.169 mmol) in CH2Cl2 (8 ml) was added 2,6-lutidine (490 uL, 4.22 mmol, 25 equiv) and 2,3-dihydrobenzo[b][1,4]dioxine-6-sulfonyl chloride (792 mg, 3.38 mmol, 20 equiv). The reaction was allowed to shake for 60 hours, and then the reaction mixture was removed. The lanterns were washed with CH2Cl2 (1 × 8 ml), DMF (2 × 8 ml), THF/H2O (3:1, 1 × 8 ml), THF/isopropanol (3:1, 1 × 8 ml), THF (1 × 8 ml), and CH2Cl2 (2 × 8 ml). General Protocol A was used to release the compound from the lantern for subsequent characterization to ascertain that the reaction was complete (LRMS (M + H)+: 675.21). The remaining lanterns were then cleaved according to General Protocol A to give the product as a white solid.

[α]20D −14.0 (c 1.0, CHCl3); 1H NMR (CDCl3, 500 MHz) δ 8.45 (d, J = 8.3, 1H), 8.23 - 8.07 (m, 3H), 7.84 (d, J = 7.7, 1H), 7.69 (dd, J = 7.8, 14.5, 2H), 7.57 - 7.41 (m, 3H), 7.26 (t, J = 10.1, 2H), 7.18 (t, J = 7.9, 1H), 7.11 (d, J = 6.6, 1H), 6.97 (d, J = 8.5, 1H), 4.28 (dd, J = 4.6, 16.9, 4H), 4.15 (s, 1H), 3.88 (d, J = 8.7, 1H), 3.83 - 3.68 (m, 2H), 3.58 (dd, J = 10.9, 15.6, 1H), 3.25 (s, 2H), 3.03 (d, J = 15.4, 1H), 2.94 (s, 3H), 2.07 (d, J = 7.1, 1H), 1.41 (d, J = 6.9, 3H), 0.89 (d, J = 6.7, 3H); 13C NMR (CDCl3, 125 MHz, 169.7, 153.8, 148.3, 143.9, 142.7, 134.3, 133.2, 132.4, 130.9, 129.1, 128.2, 126.9, 126.0, 125.9, 125.9, 125.5, 122.9, 122.6, 121.8, 121.7, 118.1, 117.4, 85.1, 77.2, 76.9, 76.7, 65.2, 64.5, 64.1, 56.1, 55.9, 51.8, 38.8, 35.1, 16.8, 14.4, −0.04; HRMS (ESI) calcd for C35H38N4O8S [M + H]+: 675.2483, found: 675.2491.

Image ml187fu12

Preparation of 2-Fluoro-N-(((2R,3R)-5-((S)-1-hydroxypropan-2-yl)-3-methyl-10-(3-(naphthalen-1-yl)ureido)-6-oxo-3,4,5,6-tetrahydro-2H-benzo[b][1,5]oxazocin-2-yl)methyl)-N-methylbenzenesulfonamide, MLS003179191. Compound MLS003179191 was synthesized in the same manner as the probe (ML187) above except o-fluoro sulfonyl chloride was used in place of 2,3-dihydrobenzo[b][1,4]dioxine-6-sulfonyl chloride in the final reaction to yield the product as a white solid.

1H NMR (300 MHz, CDCl3) δ 8.36 (d, J = 6.8, 1H), 8.15 (d, J = 17.9, 2H), 7.89 – 7.73 (m, 3H), 7.72 – 7.54 (m, 2H), 7.47 (dd, J = 6.6, 14.7, 3H), 7.33 – 7.06 (m, 4H), 4.12 (s, 1H), 3.82 (d, J = 32.5, 4H), 3.52 (s, 3H), 3.10 – 2.84 (m, 4H), 2.14 (s, 1H), 1.42 (d, J = 6.9, 3H), 0.89 (d, J = 6.7, 3H); 13C NMR (75 MHz, CDCl3) δ 169.6, 153.7, 143.3, 84.9, 65.2, 56.3, 55.3, 52.1, 40.9, 37.7, 35.0, 16.8, 14.4; HRMS (ESI) calcd for C33H35FN4O6S [M + H]+: 635.2334, found: 635.2316.

Image ml187fu13

Preparation of N-(((2R,3R)-5-((S)-1-Hydroxypropan-2-yl)-3-methyl-10-(3-(naphthalen-1-yl)ureido)-6-oxo-3,4,5,6-tetrahydro-2H-benzo[b][1,5]oxazocin-2-yl)methyl)-4-methoxy-N-methylbenzenesulfonamide, MLS003179190. Compound MLS003179190 was synthesized in the same manner as the probe (ML187) above except p-methoxy sulfonyl chloride was used in place of 2,3-dihydrobenzo[b][1,4]dioxine-6-sulfonyl chloride in the final reaction to yield the product as a white solid.

1H NMR (300 MHz, CDCl3) δ 8.44 (d, J = 8.0, 1H), 8.17 (dd, J = 7.5, 18.1, 2H), 7.85 (d, J = 7.0, 1H), 7.69 (d, J = 9.0, 3H), 7.57 – 7.41 (m, 3H), 7.13 (dd, J = 7.2, 16.3, 2H), 6.98 (d, J = 8.8, 2H), 4.15 (s, 1H), 3.81 (d, J = 21.9, 7H), 3.55 (d, J = 10.7, 1H), 3.24 (s, 2H), 3.03 (d, J = 15.3, 1H), 2.92 (s, 3H), 2.06 (s, 1H), 1.40 (d, J = 6.9, 3H), 0.88 (d, J = 6.7, 3H); 13C NMR (75 MHz, CDCl3) δ 169.7, 163.8, 153.9, 142.8, 114.7, 85.2, 65.2, 56.1, 55.9, 55.7, 51.9, 38.7, 35.2, 16.8, 14.4; HRMS (ESI) calcd for C34H38N4O7S [M + H]+: 647.2534, found: 647.2532.

Image ml187fu14

Preparation of 4-Chloro-N-(((2R,3R)-5-((S)-1-hydroxypropan-2-yl)-3-methyl-10-(3-(naphthalen-1-yl)ureido)-6-oxo-3,4,5,6-tetrahydro-2H-benzo[b][1,5]oxazocin-2-yl)methyl)-N-methylbenzenesulfonamide, MLS003179192. Compound MLS003179192 was synthesized in the same manner as the probe (ML187) above except p-chloro sulfonyl chloride was used in place of 2,3-dihydrobenzo[b][1,4]dioxine-6-sulfonyl chloride in the final reaction to yield the product as a white solid.

1H NMR (300 MHz, CDCl3) δ 8.35 (s, 1H), 8.14 (s, 2H), 7.96 (s, 1H), 7.84 (s, 1H), 7.70 (d, J = 8.4, 3H), 7.59 – 7.42 (m, 4H), 7.14 (d, J = 14.9, 2H), 4.28 – 4.07 (m, 1H), 3.79 (d, J = 27.7, 5H), 3.07 (s, 2H), 2.91 (s, 2H), 2.23 – 2.02 (m, 1H), 1.38 (d, J = 6.8, 3H), 1.25 (s, 3H), 0.87 (d, J = 6.6, 3H). 13C NMR (75 MHz, CDCl3) δ 169.7, 153.7, 143.2, 140.4, 84.9, 65.1, 55.8, 55.4, 51.5, 41.0, 38.3, 34.9, 29.7, 16.8, 14.4; HRMS (ESI) calcd for C33H35ClN4O6S [M + H]+: 651.2039, found: 651.2050.

Image ml187fu15

Preparation of N-(((2R,3R)-5-((S)-1-hydroxypropan-2-yl)-3-methyl-6-oxo-10-(3-phenylureido)-3,4,5,6-tetrahydro-2H-benzo[b][1,5]oxazocin-2-yl)methyl)-4-methoxy-N-methylbenzenesulfonamide, MLS003179189. Compound MLS003179189 was synthesized in the same manner as the probe (ML187) above except phenyl isocyanate was used in place of 1-isocyanatonaphthalene in the reaction with the aniline, and p-methoxy sulfonyl chloride was used in place of 2,3-dihydrobenzo[b][1,4]dioxine-6-sulfonyl chloride in the final reaction to yield the product as a white solid.

1H NMR (500 MHz, CDCl3) δ 8.50 (s, 1H), 8.03 (s, 2H), 7.82 (d, J = 8.5, 2H), 7.68 (s, 2H), 7.35 (s, 2H), 7.24 (s, 1H), 7.10 (d, J = 8.1, 4H), 4.20 – 4.07 (m, 1H), 3.93 (s, 4H), 3.76 (s, 2H), 3.68 – 3.54 (m, 2H), 3.08 – 2.99 (m, 1H), 2.94 (s, 3H), 2.07 – 1.93 (m, 1H), 1.44 (d, J = 6.8, 3H), 0.88 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 169.6, 163.9, 152.6, 142.5, 139.1, 85.1, 65.3, 56.3, 55.8, 51.7, 35.4, 16.8, 14.4; HRMS (ESI) calcd for C30H36N4O7S [M + H]+: 597.2377, found: 597.2392.

3. Results

Probe attributes

Biological CharacteristicsPrior Art: SAHADesired Probe
Target Activity (IC50/EC50 nM; not available)Complete protection from cytokine induced cell death at 1 μMIC50 < 10 μM in the primary cell-based assay
Selectivity: ATP Levels without cytokinesToxic at 2 μMNontoxic at 10X IC50
Selectivity: Caspase-3 activationNot activeIC50 < 10 μM
Biological Mode of Action: Measurement of glucose stimulation in rat INS-1E cellsNot activeIC50< 5 μM
Biological Mode of Action: Apoptosis and insulin secretion in primary human islet cellsNot testedDose-dependent response observed
Cellular Toxicity (nontoxic @ level/not-applicable)Toxic at 2 μMNontoxic to cells at 10X the effective IC50

Compound Summary in PubChem

IUPAC Chemical NameN-(((2R,3R)-5-((S)-1-hydroxypropan-2-yl)-3-methyl-10-(3-(naphthalen-1-yl)ureido)-6-oxo-3,4,5,6-tetrahydro-2H-benzo[b][1,5]oxazocin-2-yl)methyl)-N-methyl-2,3-dihydrobenzo[b][1,4]dioxine-6-sulfonamide
PubChem CID46907798
Molecular Weight674.76
Molecular FormulaC35H38N4O8S
AlogP3.57
H-Bond Donor3
H-Bond Acceptor8
Rotatable Bond Count7
Exact Mass674.2410
Topological Polar Surface Area146.74

In the primary screen, cell viability was measured using CTG to measure cellular ATP levels in the INS-1E cells. Towards this goal, we completed a pilot screen of 19,000 compounds and 319,000 compounds for the MLPCN primary screen. For the MLPCN hits, we have performed dose-response studies using the primary screening assay to retest 2,351 compounds that showed greater than 74% suppression of cytokine-induced cell death at 7.5 μM. 1,959 compounds were confirmed at dose, of which 529 showed EC50 values ≤ 10 μM. Analysis of the MLPCN hit compounds is in progress.

For the pilot screen hits, each have been profiled in dose through a panel of secondary assays to assess: 1) apoptosis, 2) nitrite levels, 3) mitochondrial membrane potential, and 4) GSIS. The probe (ML187), an active compound identified during the pilot phase, is the most mature lead and has shown activity in all secondary assays in INS-1E cells. The only assay in which the probe (ML187) was not active was the ATP assay in the absence of cytokines (488864), suggesting that the compound does not elevate cellular ATP levels in a nonspecific manner. In addition to acting as a potent suppressor of cytokine-induced beta-cell apoptosis in INS-1E cells, the probe (ML187) reduced cytokine-induced caspase-3 activity (Figure 7A; 488945) and restored GSIS to nearly normal levels in primary human islets (Figure 7B; 488951).

Figure 7. Dose-dependent Activity of the Probe (ML187) in Primary Human Pancreatic Islets.

Figure 7

Dose-dependent Activity of the Probe (ML187) in Primary Human Pancreatic Islets. Primary human pancreatic islets were dissociated, plated, and treated with cytokines for 6 days in the presence of cytokines and compounds. Caspase activity was reduced in (more...)

Figure 4. Dose-response Curves for the Initial Hit (MLS003179189).

Figure 4Dose-response Curves for the Initial Hit (MLS003179189)

Compounds were used over a range of concentrations up to 25 μM. Dose curves were generated with Genedata Condeseo and show percent activity (normalized) for the individual doses. CellTiter-Glo (488844), EC50=2.7 μM (A); Caspase-Glo (488848), IC50=9.84 μM (B); nitrite production (488868) (C) and mitochondrial membrane potential (488867) (D). ○= replicate 1, △=replicate 2, greyed out symbols=masked wells

3.1. Summary of Screening Results

Figure 5 displays the critical path for probe development.

Figure 5. Critical Path for Probe Development.

Figure 5

Critical Path for Probe Development.

In the primary high-throughput screen (HTS), compounds were called active if they increased cellular ATP levels, indicated by an increase in luminescence (Figure 5). The positive control, SAHA, increased ATP levels at 5 μM, and was used to normalize data. Compounds with >74% activity compared to SAHA were considered hits and chosen for confirmation in dose studies (488844). For the primary screen and other assays, negative-control (NC) wells and positive-control (PC) wells were included on every plate. The raw signals of the plate wells were normalized using the ‘Stimulators Minus Neutral Controls’ method in Genedata Assay Analyzer (v7.0.3). The median raw signal of the intra-plate NC wells was set to a normalized activity value of 0, while the median raw signal of the intra-plate PC wells was set to a normalized activity value of 100. Experimental wells were scaled to this range, resulting in an activity score representing the percent change in signal relative to the intra-plate controls. We used the mean of the replicate percent activities as the final ‘Pubchem Activity Score’.

The ‘Pubchem Activity Outcome’ class was assigned as described below, based on an activity threshold of 75%:

  • Activity_Outcome = 1 (inactive), less than half of the replicates fell outside the threshold.
  • Activity_Outcome = 2 (active), all of the replicates fell outside the threshold, OR at least half of the replicates fell outside the threshold AND the ‘Pubchem Activity Score’ fell outside the threshold.
  • Activity_Outcome = 3 (inconclusive), at least half of the replicates fell outside the threshold AND the ‘Pubchem Activity Score did not fall outside the threshold.

3.2. Dose-Response Curves for Probe

3.3. Scaffold/Moiety Chemical Liabilities

The probe (ML187) has no functional groups with known chemical reactivity that may lead to instability.

3.4. SAR Tables

Before screening the MLPCN compound collection, a pilot screen was carried out to survey the Broad Institute compound collection. This collection included 18,560 diverse compounds synthesized by our Diversity-Oriented Synthesis (DOS) chemistry group. Compound MLS003179190 (Table 1, entry 1), which had been synthesized as a member of a DOS library, was identified as a hit in the primary screen, and was active in all the appropriate secondary assays. It had an IC50 value of 2.71 μM in the CTG assay (449756), it decreased caspase levels with an EC50 value of 9.84 μM in the caspase-glo 3/7 assay (488936), and showed inhibition in the nitrite production assay (488866) with an EC50 of 15.4 μM. This very promising DOS hit was chosen for follow-up chemistry.

Table 1. Stereochemical SAR Analysis of Nine Synthetic Compounds for Target.

Table 1

Stereochemical SAR Analysis of Nine Synthetic Compounds for Target.

Since all of the stereoisomers of this hit (Table 1, entries 2–7), as well as a number of compounds with various substituents at both the aniline and amine sites, were included in this library, a great deal of SAR could be obtained directly from the primary screen. Only one of the eight stereoisomers was active in the primary screen (Figure 8). The stereochemistry of the substituents within the ring must be in the 2R,3R configuration for the compound to be active. In contrast, the stereochemistry of the aliphatic alcohol external to the ring can be either 5R or 5S in most cases (Table 1, entries 8 and 9). Both the urea and the sulfonamide are necessary for activity, as nitrogens capped with other functional groups (e.g., amide) were inactive.

Figure 8. Stereochemical SAR Heat Map of the HTS Hit Compound (MLS003179190).

Figure 8

Stereochemical SAR Heat Map of the HTS Hit Compound (MLS003179190). Only one of the eight stereoisomers of the hit compound (MLS003179190) was active in the primary screen.

After the optimal stereochemistry was determined, about 50 analogs with different substituents at the aniline and sulfonamide positions were readily synthesized, leading to a superior analog. The probe compound (Table 2, entry 1) was active in all assays, with an EC50 value of 3.5 μM in the CTG assay. It had an EC50 value of 4.8 μM in the GSIS assay and was completely inactive in the CTG assay in the absence of cytokines (488864), demonstrating good selectivity for cytokine-induced apoptosis. Other analogs with 3- or 4- substituted sulfonamides exhibited micromolar potency.

Table 2. SAR Analysis of 22 Synthetic Compounds for Target.

Table 2

SAR Analysis of 22 Synthetic Compounds for Target.

Evaluation of building blocks at the aniline site indicated that compounds lacking the naphthyl urea substituent were generally less potent or inactive. For example, when the naphthyl urea was replaced by aryl sulfonamides or aryl amides, all activity was lost. In particular, a naphthyl amide was also completely inactive (Table 2, entry 21). The probe (ML187) exhibits low solubility in PBS (< 0.5 μM), so more soluble analogs were made, but potency was diminished (Table 2, entries 19 and 22).

Evaluation of building blocks at the amine site was also informative. Analogs with a urea or amine linker were inactive, whereas aryl sulfonamides were most active. Furthermore, para- and meta-substituted aryl sulfonamides (Table 2, entries 1, 8 and Table 2, entries 3, 4, 5) were much more active than ortho-substituted aryl sulfonamides (Table 2, entries 6, 9, 10).

The biological assay data and physical properties of these analogs are presented below in Table 1 and Table 2. Characterization data (1H NMR spectra and UPLC chromatograms) of these analogs are provided in Appendix C.

3.5. Cellular Activity

All assays used for this project were cell-based. The probe (ML187) and several analogs showed activity in all of the assays, suggesting that these compounds are cell-permeable. We tested the probe (ML187) and several analogs in INS-1E cells for 48 hours at concentrations up to 25 μM without cytokine treatment (488864). No significant alteration in ATP levels was observed at any of the doses tested, suggesting that there is little to no cytotoxicity. In studies outside the scope of the CPDP, the probe was tested in HepG2 cells, HeLa cells, and PANC-1 cells for general cytotoxicity using the CellTiter-Glo assay. The cells were exposed to up to 25 μM compound for 48 hours. The probe (ML187) exhibited no cytotoxicity in any of the cell lines tested (Table 3).

Table 3. Comparison of the Probe to Project Criteria.

Table 3

Comparison of the Probe to Project Criteria.

3.6. Profiling Assays

The probe (ML187) will be sent to Ricerca for evaluation of off-target binding to a broad panel of receptors, ion channels, and enzymes.

4. Discussion

The HTS campaign described in Section 3.1 of a library of complex compounds accessed by DOS identified a hit that had three chiral centers and two sites of diversity. The hit was part of an 8,000-membered library that contained numerous related analogs and all the individual stereoisomers. We screened all 8,000 compounds from this library in the primary assay and identified several key structure-activity relationships (SARs). We subsequently designed and synthesized about 50 analogs, leading to the identification of the probe (ML187).

4.1. Comparison to Existing Art and How the New Probe is an Improvement

The probe (ML187) is a substantial improvement over existing art. It is the first probe to come out of the beta-cell apoptosis project. The probe (ML187) was identified during a pilot screen performed at the Broad using a stereochemically diverse library of compounds. The probe (ML187) has met the probe criteria set for this project: It increases cellular ATP (i.e., cell viability), decreases caspase activity, reduces nitrite production, improves mitochondrial membrane potential, and restores GSIS.

Prior to the probe (ML187), other compounds were shown to increase ATP levels and/or reduce caspase activity, but not restore GSIS. A good performance in the nitrite and caspase assays correlates with restored insulin secretion, a hallmark for physiological beta-cell function (10). We found that a 6-day treatment of primary islets with the cytokine cocktail was necessary to induce caspase-3/7 activity by approximately 60%; this effect was abolished by co-incubation with the probe (ML187) (Figure 6). Importantly, the probe (ML187) was also able to restore GSIS in primary human islets to 75% of islets not treated with cytokines. This probe shows unprecedented characteristics and provides a unique tool for beta-cell biology and diabetes research (Figure 7).

Figure 6. Dose-dependent Activity of the Probe (ML187).

Figure 6

Dose-dependent Activity of the Probe (ML187). Compounds were used over a range of concentrations up to 25 μM. Dose curves were generated with Genedata Condeseo and show percent activity (normalized) for the individual doses. CellTiter-Glo measuring (more...)

4.2. Mechanism-of-Action Studies

To date, there have been no direct mechanism-of-action studies for the probe (ML187). Currently, we are taking a focused approach to further characterize this probe, with the goal of substantially improving its performance as a biological tool. The approach will integrate proteomic, genomic, and computational methods to address the mechanism of action of the probe (see Section 4.3).

4.3. Planned Future Studies

We have developed a probe (ML187) that increases beta-cell survival in the presence of pro-inflammatory cytokines. The probe will be used in a number of studies to determine mechanism of action (see Section 4.2). In addition, there will be a concerted effort to improve the potency and solubility of the probe (ML187) by our medicinal chemists. Following these advances, the probe will be tested for in vivo activity.

We have applied for two Extended Probe Characterization grants for the medicinal chemistry and mechanism-of-action studies. For the in vivo work, we have established collaborations with researchers who routinely study type 1 diabetes using rodent models. Together, these studies will significantly enhance the utility of the probe (ML187) and/or its derivatives as tool compounds for use in type 1 diabetes model systems and may ultimately result in a lead compound for clinical use in patients with early-stage type 1 diabetes.

4.3.1. Determination of Binding Partners

A phenotypic cell-based screening approach is extremely powerful for identifying novel compounds of interest when the exact mechanisms of cellular damage and repair are not fully understood. We have developed a robust, scalable method for confident identification of the protein targets of small molecules in their cellular context called stable isotope labeling by amino acids in cell culture (SILAC). SILAC-based target identification technology overcomes prior difficulties with affinity-based target ID methods (13). This technology is routinely applied at the Broad Institute to identify targets of a variety of small molecules with drug-like properties, including a kinase inhibitors, immunophilin modulators and others (13).

4.3.2. Gene-expression Studies

A gene-expression profiling of rat INS-1E cells in the absence or presence of the cytokine cocktail, and treated with or without the probe (ML187) will be performed. Tools developed at the Broad Institute, such as GenePattern and Gene Set Enrichment Analysis, will be applied to determine the comparative markers responsible for the greatest difference between the different states. A computational effort that facilitates target identification is to analyze small-molecule signatures that we call the Connectivity Map, or cmap (14). This tool uses gene-expression profiles in a systematic approach to enable the discovery of functional connections between diseases, genetic perturbations, and small-molecule perturbations.

4.3.3. RNAi Modifier Screen

Numerous genetic efforts to determine the target(s) of small molecules have used the principle of genetic dosage (for example, in yeast cells) (15,16). This approach is also attractive in mammalian cell culture. We proceed by the hypothesis that a reduction in the expression levels of the target(s) of the probe (ML187) will cause a similar phenotype in beta cells. In anticipation of the need to do large-scale RNAi screening, the Broad Institute created an RNAi Platform dedicated to developing genome-scale reagents for RNAi as well as the technologies necessary to apply RNAi systematically. We will perform a pooled RNAi screen to identify genes which, when knocked-down, synergize with a low dose of the probe (ML187) to suppress cytokine-induced beta-cell apoptosis.

4.3.4. Assay Performance Profiling

The Computational Chemical Biology group at the Broad Institute routinely analyzes profiles of compound activity drawn from historical screening data to generate hypotheses about mechanisms of action. We expect that compounds inhibiting the same proteins or biological pathways will have similar performance across various assays, and that using profiles built over multiple assays can help overcome problems associated with error rates in any given assay. We have used this approach to good effect in determining that a compound of unknown function acts as a kinase inhibitor in vitro and in cells (17).

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Appendix A. Assay Summary Table

Table A1. Summary of Completed Assays and AIDs

Appendix B. Detailed Assay Protocols

CellTiter-Glo assay in INS-1E Cells (488844)

Day 0

  1. Collect cells and generate single cell suspension by trypsinization.
  2. Seed 8,000 cells/well of INS-1E rat beta-cell line in 30 μl plating media (phenol red free RPMI-1640 (Invitrogen) containing 11 mM glucose, 10% fetal bovine serum (FBS), 10 mM HEPES, 50 μM 2-mercaptoethanol, 1 mM sodium pyruvate) using white, opaque, bar-coded, 384-well Corning 8867 plates.
  3. Incubate at 37 °C overnight.

Day 1

  1. Add 10 μL plating media with cytokine cocktail to each well using the Multidrop Combi (VWR).
  2. Pin-transfer 100 nl compounds immediately after the addition of cytokines. Positive control added by additional pin-transfer step.

Day 3

  1. After 48 hours, add 20 μl CellTiter-Glo reagent to plates.
  2. Agitate gently for 15 seconds to maximize cell lysis. Incubate 10 minutes.
  3. Use EnVision plate reader (Perkin-Elmer) to read plate luminescence with standard luminescence parameters.

Caspase Activity Assay with INS1-E Cells (488848)

Day 0

  1. Collect cells.
  2. Seed 8,000 cells/well of INS-1E rat beta-cell line in 30 μl (phenol red free RPMI-1640 (Invitrogen) containing 11 mM glucose, 10% fetal bovine serum, 10 mM HEPES, 50 μM 2-mercaptoethanol, 1 mM sodium pyruvate) using white-walled, bar-coded, 384-well plates (Corning 8867).
  3. Incubate at 37 °C overnight.

Day 1

  1. Add 10 μL medium with cytokine cocktail to each well using the Multidrop Combi (VWR) (cocktail contains 250 ml INS-1E media, 92.5 μl IFN-γ, 32.5 μl TNF-α and 20 μl IL-1-β).

Day 3

  1. Add 20 μl Caspase-Glo 3/7 reagent to each well.
  2. Agitate plate for 15 seconds to maximize cell lysis.
  3. Incubate 1 hour at room temperature.
  4. Use EnVision plate reader (Perkin-Elmer) to read plate luminescence with ultra-sensitive settings.

Cytokines: 10 ng/ml IL-1β (R&D Systems, 501-RL-050), 25 ng/ml TNF-α (R&D Systems, 410-MT-050), 50 ng/ml IFN-γ (R&D Systems)

Mitochondrial membrane potential (MMP) in INS-1E cells (488867)

Day 0

  1. Seed INS-1E cells into Aurora IQ-EB black 384-well optical-bottom plates at 8000 cells per well in 40 μl of phenol red free RPMI-1640 (Invitrogen) containing 11 mM glucose, 5% fetal bovine serum, 10 mM HEPES, 50 μM 2-mercaptoethanol, 1 mM sodium pyruvate with Multidrop Combi (Thermo).

Day 1

  1. After overnight incubation, remove the medium and add 40 μl RPMI containing 1% FBS and a combination of cytokines (10 ng/mL IL-1β, 50 ng/ml IFN-γ, 25 ng/ml TNF-α) every well.
  2. Using libraries of compounds dissolved in DMSO and a CyBi-Well pin-transfer robot (CyBio Corp.), pin transfer 100 nL of each compound to each of the wells.

Day 3

  1. After incubation for 48 hours, remove the medium.
  2. Dissolve JC-1 powder to 1 mM stock in 100% DMSO.
  3. Dilute the 1 mM JC-1 stock in phenol-red free media to a final concentration of 3.25 μM.
  4. Aspirate media from the plates, and add 20 μl per well of JC-1 containing-media.
  5. Incubate plates at 37°C for 2 hours.
  6. Wash cells three times with 50 μl per well of 1X PBS.
  7. Read fluorescence on the EnVision plate reader (Perkin-Elmer) at rhodamine channel (ex/em 530/580) followed by fluorescein channel (ex/em 485 nm/530 nm). Each time a new assay is read, run an optimization cycle on the instrument to ensure the appropriate reading levels of the assay.

Cytokine-free CellTiter-Glo assay in INS-1E Cells (488864)

Day 0

  1. Collect cells and generate single cell suspension by trypsinization.
  2. Seed 8,000 cells/well of INS-1E rat beta-cell line in 30 μl plating media (phenol red free RPMI-1640 (Invitrogen) containing 11 mM glucose, 5% fetal bovine serum, 10 mM HEPES, 50 μM 2-mercaptoethanol, 1 mM sodium pyruvate) using white, opaque, bar-coded, 384-well Corning 8867 plates;.
  3. Incubate at 37°C overnight.

Day 1

  1. Add 10 μL plating media to each well using the Multidrop Combi (VWR)
  2. Pin transfer compounds to plates right after the addition of cytokines in 100 nL volume.

Day 3

  1. After 48 hours, add 20 μl CellTiter-Glo reagent to plates.
  2. Agitate gently for 15 seconds to maximize cell lysis.
  3. Incubate 10 minutes.
  4. Use EnVision plate reader (Perkin Elmer) to read plate luminescence with standard luminescence parameters.

Glucose Stimulated Insulin Secretion (GSIS) ELISA (AID# 488959)

Day 0

  1. Seed INS-1E cells in 100 μl of phenol red free RPMI-1640 (Invitrogen) containing 11 mM glucose, 5% fetal bovine serum, 10 mM HEPES, 50 μM 2-mercaptoethanol, 1 mM sodium pyruvate into 96-well plates at 20,000 cells per well

Day 1

  1. Remove media was removed and replace with 100 μl of phenol red free RPMI-1640 (Invitrogen) containing 11 mM glucose, 1% fetal bovine serum, 10 mM HEPES, 50 μM 2-mercaptoethanol, 1 mM sodium pyruvate containing the cytokine cocktail, in the presence or absence of compounds.

Day 3

  1. Wash cells and incubate for 2 hours in KRBH (135 mM NaCl, 3.6 mM KCl, 5 mM NaHCO3, 0.5 mM NaH2PO4, 0.5 mM MgCl2, 1.5 mM CaCl2, 10 mM HEPES, pH 7.4, 0.1% BSA) without glucose.
  2. Incubate cells were incubated with KRBH containing 2 or 15 mM glucose for 1 hour.
  3. Take the supernatant for measurement of released insulin.
  4. Measure insulin was measured with a rat insulin ELISA kit (Alpco).

Insulin ELISA Protocol

  1. Bring all reagents and microplate strips to room temperature prior to use.
  2. Gently mix all reagents before use. Perform a standard curve with each assay and with each microplate if more than one is run at a time.
  3. Run all standards, samples, and the control should be run in duplicate.

Reagent Preparation

Conjugate (11X): is diluted with 10 parts Conjugate Buffer. For example, to prepare enough Working Strength Conjugate for one complete microplate, dilute 0.9 ml of Conjugate (11X) with 9 ml of Conjugate Buffer. Working Strength Conjugate is stable for 4 weeks at 2–8°C.

Mammalian Insulin Control (High) is provided in a lyophilized form. Reconstitute the control with 0.6 ml of distilled water. Close the vial with the rubber stopper and cap, then gently swirl the vial and allow it to stand for 30 minutes prior to use. The contents should be in solution with no visible particulates. The reconstituted control is stable for 7 days stored at 2–8°C. If desired, the control can be aliquoted and stored at < −20°C for up to 6 months. The control should not be repeatedly frozen and thawed.

Wash Buffer (21X) is diluted with 20 parts distilled water. For example, to prepare Working Strength Wash Buffer, dilute 20 ml of Wash Buffer (21X) with 400 ml of distilled water. Working Strength Wash Buffer is stable for 4 weeks at room temperature (18–25 °C).

  1. Ensure that microplates are at room temperature prior to opening foil pouch. Designate enough microplate strips for the standards, desired number of samples, and control. Store the remaining strips at 2–8 °C in the tightly sealed foil pouch containing the desiccant.
  2. Pipette 5 μl of each standard, reconstituted control (see Reagent Preparation), or sample into its respective wells.
  3. Pipette 75 μl of Working Strength Conjugate (see Reagent Preparation) into each well.
  4. Incubate for 2 hours, shaking at 700–900 rpm on a horizontal microplate shaker at room temperature (18–25 °C).
  5. Wash the microplate 6 times with Working Strength Wash Buffer (see Reagent Preparation) with a microplate washer. After the final wash with the microplate washer, remove any residual Wash Buffer and bubbles from the wells by inverting and firmly tapping the microplate on absorbent paper towels.
  6. Pipette 100 μl of TMB Substrate into each well.
  7. Incubate for 15 minutes at room temperature (18–25 °C) on a horizontal microplate shaker (700–900 rpm).
  8. Pipette 100 μl of Stop Solution into each well. Gently shake the microplate to stop the reaction. Remove bubbles before reading with microplate reader.
  9. Place the microplate in a microplate reader capable of reading the absorbance at 450nm with a reference wavelength of 620–650 nm. The microplate should be analyzed within 30 minutes following the addition of Stop Solution.

Primary Human Pancreatic Beta Islet Assay (488959)

  1. Wash islets with PBS and incubate in CMRL medium supplemented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin.
  2. Gently dissociate islets into a cell suspension by incubating in Accutase (37 °C, 10 minutes), and seed in 384-well plates containing extracellular matrix secreted by the HTB-9 human bladder carcinoma cell line (adapted from Beattie et al.) (11)
  3. Expose islets to a range of cytokine concentrations for 6 days, with media and compound changed after 3 days.
  4. Process cells for caspase activity with the Caspase-Glo 3/7 assay (Promega). Add an equal volume of Caspase-Glo per well and incubate for 2 hours at room temperature in the dark.
  5. Measure luminescence-based activity with the EnVision plate reader (Perkin-Elmer). In addition, collect the culture supernatants for insulin production. In this case, islets are treated as in the GSIS ELISA for INS-1E cells (see above) with 2 hours in the KRBH buffer and 1 hour with glucose stimulation.
  6. Next, use 5 μl of culture supernatant from each well for ELISA studies.

Appendix C. NMR and LC Data of Probe and Analogs

1H NMR Spectrum of the Probe (ML187).

1H NMR Spectrum of the Probe (ML187)

13 C NMR Spectrum of the Probe (ML187).

13 C NMR Spectrum of the Probe (ML187)

UPLC/MS Chromatogram of the Probe (ML187), indicating >99% purity.

UPLC/MS Chromatogram of the Probe (ML187), indicating >99% purity

Spectroscopic Data for SAR Analogs

LC/MS of BRD-K64058329-001-01-8 / SID-85807046 / CID 44500012 / MLS003179189.

LC/MS of BRD-K64058329-001-01-8 / SID-85807046 / CID 44500012 / MLS003179189

1H NMR spectrum of BRD-K64058329-001-01-8 / SID-85807046 / CID 44500012 / MLS003179189.

1H NMR spectrum of BRD-K64058329-001-01-8 / SID-85807046 / CID 44500012 / MLS003179189

13C NMR spectrum of BRD-K64058329-001-01-8 / SID-85807046 / CID 44500012 / MLS003179189.

13C NMR spectrum of BRD-K64058329-001-01-8 / SID-85807046 / CID 44500012 / MLS003179189

LC/MS of BRD-K64610608-001-01-8 / SID-85796266 / CID4489675 / MLS003179189.

LC/MS of BRD-K64610608-001-01-8 / SID-85796266 / CID4489675 / MLS003179189

1H NMR spectrum of BRD-K64610608-001-01-8 / SID-85796266 / CID4489675 / MLS003179189.

1H NMR spectrum of BRD-K64610608-001-01-8 / SID-85796266 / CID4489675 / MLS003179189

13 C NMR spectrum of BRD-K64610608-001-01-8 / SID-85796266 / CID4489675 / MLS00317918.

13 C NMR spectrum of BRD-K64610608-001-01-8 / SID-85796266 / CID4489675 / MLS00317918

LC/MS of BRD-K68437527-001-01-0 / SID-85799159 / CID 44492462 / MLS003179192.

LC/MS of BRD-K68437527-001-01-0 / SID-85799159 / CID 44492462 / MLS003179192

1H NMR spectrum of BRD-K68437527-001-01-0 / SID-85799159 / CID 44492462 / MLS003179192.

1H NMR spectrum of BRD-K68437527-001-01-0 / SID-85799159 / CID 44492462 / MLS003179192

13C NMR spectrum of BRD-K68437527-001-01-0 / SID-85799159 / CID 44492462 / MLS003179192.

13C NMR spectrum of BRD-K68437527-001-01-0 / SID-85799159 / CID 44492462 / MLS003179192

LC/MS of BRD-K98896788-001-02-3 / SID-99368326 / CID 46907806.

LC/MS of BRD-K98896788-001-02-3 / SID-99368326 / CID 46907806

1H NMR spectrum of BRD-K98896788-001-02-3 / SID-99368326 / CID 46907806.

1H NMR spectrum of BRD-K98896788-001-02-3 / SID-99368326 / CID 46907806

13 C NMR spectrum of BRD-K98896788-001-02-3 / SID-99368326 / CID 46907806.

13 C NMR spectrum of BRD-K98896788-001-02-3 / SID-99368326 / CID 46907806

BRD-K39732687-001-01-6.

BRD-K39732687-001-01-6

BRD-K20197062-001-01-8.

BRD-K20197062-001-01-8

BRD-K04695623-001-01-3.

BRD-K04695623-001-01-3

BRD-K33116223-001-01-9.

BRD-K33116223-001-01-9

BRD-K44510578-001-01-1.

BRD-K44510578-001-01-1

BRD-K71138616-001-01-1.

BRD-K71138616-001-01-1

BRD-K53816294-001-01-3.

BRD-K53816294-001-01-3

BRD-K06311439-001-01-9.

BRD-K06311439-001-01-9

BRD-K87426499-001-02-2.

BRD-K87426499-001-02-2

BRD-K93441996-001-02-1.

BRD-K93441996-001-02-1

BRD-K36654975-001-02-4.

BRD-K36654975-001-02-4

BRD-K15672523-001-01-0.

BRD-K15672523-001-01-0

BRD-K80971603-001-02-7.

BRD-K80971603-001-02-7

BRD-K46337260-001-02-8.

BRD-K46337260-001-02-8

BRD-K00852784-001-01-9.

BRD-K00852784-001-01-9

BRD-K58134727-001-02-1.

BRD-K58134727-001-02-1

BRD-K47275644-001-01-8.

BRD-K47275644-001-01-8

BRD-K77989909-001-01-7.

BRD-K77989909-001-01-7

BRD-K58704510-001-01-4.

BRD-K58704510-001-01-4

BRD-K90648686-001-01-1.

BRD-K90648686-001-01-1

BRD-K10685739-001-01-7.

BRD-K10685739-001-01-7

BRD-K26687226-001-01-5.

BRD-K26687226-001-01-5

BRD-K46716321-001-02-0.

BRD-K46716321-001-02-0

BRD-K77229785-001-01-7.

BRD-K77229785-001-01-7

BRD-K09580233-001-01-4.

BRD-K09580233-001-01-4

BRD-K26687226-001-01-5.

BRD-K26687226-001-01-5

BRD-K39732687-001-01-6.

BRD-K39732687-001-01-6

BRD-K20197062-001-01-8.

BRD-K20197062-001-01-8

BRD-K04695623-001-01-3.

BRD-K04695623-001-01-3

BRD-K44510578-001-01-1.

BRD-K44510578-001-01-1

BRD-K71138616-001-01-1.

BRD-K71138616-001-01-1

BRD-K06311439-001-01-9.

BRD-K06311439-001-01-9

BRD-K09580233-001-01-4.

BRD-K09580233-001-01-4

BRD-K93441996-001-02-1.

BRD-K93441996-001-02-1

BRD-K15672523-001-01-0.

BRD-K15672523-001-01-0

BRD-K47275644-001-01-8.

BRD-K47275644-001-01-8

BRD-K77989909-001-01-7.

BRD-K77989909-001-01-7

BRD-K58704510-001-01-4.

BRD-K58704510-001-01-4

BRD-K90648686-001-01-1.

BRD-K90648686-001-01-1

BRD-K65064613-001-01-0.

BRD-K65064613-001-01-0

BRD-K10685739-001-01-7.

BRD-K10685739-001-01-7

BRD-K46716321-001-02-0.

BRD-K46716321-001-02-0

BRD-K77229785-001-01-7.

BRD-K77229785-001-01-7

Appendix D. Compounds Submitted to BioFocus

Table A2Probe and Analog Information

BRDSIDCIDP/AMLS IDML No.
BRD-K11540476-001-03-6SID-9937656846907798P003179193ML187
BRD-K64058329-001-01-8SID-8580704644500012A003179189NA
BRD-K64610608-001-01-8SID-8579626644489675A003179190NA
BRD-K68437527-001-01-0SID-8579915944492462A003179192NA
BRD-K98896788-001-02-3SID-9936832646907806A003179191NA

A = analog; NA= not applicable; P = probe

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