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.

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.

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.

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.

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.

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.

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.

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.

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 R_f. 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).

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.

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.

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]²⁰_D −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.

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]²⁰_D −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.

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]²⁰_D −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.

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]²⁰_D −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.

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.

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.

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.

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]²⁰_D −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.

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.

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.

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.

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.

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).

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.

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)

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.

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.

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.