Harned Harki NF-kB ACS MCL SI Final Galley Stage 10May2012 ACS Med Che… · VII. Protocol for...

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SUPPORTING INFORMATION

Bicyclic Cyclohexenones as Inhibitors of NF-κB Signaling

Joseph K. Hexum,† Rodolfo Tello-Aburto,‡ Nicholas B. Struntz,† Andrew M. Harned‡,*and Daniel A. Harki†,*

†University of Minnesota, Department of Medicinal Chemistry, 717 Delaware St SE, Minneapolis, MN,

55414, USA ‡University of Minnesota, Department of Chemistry, 207 Pleasant St. SE, Minneapolis, MN, 55455, USA

Table of Contents for Supporting Information

Page

I. Chemical Synthesis: Materials & Methods S2 II. Chemical Synthesis: Experimental Procedures S2

III. Supplemental Figure 1. Initial screening of 4-9 for NF-κB inhibitory activity S7

IV. Supplemental Figure 2. Cytotoxicity of 10, 11, and 12 in the NF-κB Reporter Assay S7

V. Supplemental Figure 3. Western blot analysis of p65 and histone H2B proteins S8 VI. Preparation of stock solutions of compounds 4-12 S8 VII. Protocol for mammalian cell culture S8

VIII. Protocol for NF-κB reporter assay S9

IX. Cell culture cytotoxicity assays S9 X. Cell fractionation and western blotting S10 XI. Compound treatment for ELISA and PCR S11 XII. IL-8 Enzyme-Linked Immunosorbent Assays (ELISA) S11 XIII. mRNA isolation and purification S12 XIV. Reverse Transcription (RT) and Polymerase Chain Reaction (PCR) assays S12 XV. HPLC and NMR Michael acceptor assays S14 XVI. NMR Spectra S15 XVII. Chiral HPLC Chromatograms for 11 and 11* S27

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I. Chemical Synthesis: Materials and Methods

Compounds 4-9 were prepared as previously described.1 Iodobenzene diacetate (PIDA) and

bis(trifluoroacetoxy)iodobenzene (PIFA) were purchased from Acros. All other compounds were

purchased from commercial sources and used as received, unless otherwise specified.

Thin-layer chromatography (TLC) was performed using plates pre-coated with silica gel XHL w/UV254

(250 mm) or alumina w/UV purchased from Sorbent Technologies and visualized by UV light, KMnO4,

or anisaldehyde stains, followed by heating. ICN Silica gel (particle size 32-63 µm) was used for flash

chromatography.

1H and 13C NMR spectra were recorded on a Varian Inova 300 (operating at 300 MHz and 75 MHz

respectively) or Varian Inova 500 (operating at 500 MHz and 125 MHz respectively), and are reported

relative to residual solvent peak (δ 7.26 and δ 77.2 for 1H and 13C in CDCl3, δ7.16 and δ 128.0 for 1H

and 13C in C6D6, respectively). Data for 1H NMR spectra are reported as follows: chemical shift (δ ppm)

(multiplicity, coupling constant (Hz), integration). Spectra obtained are described using the following

abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, hept = heptuplet, m = multiplet. IR spectra

were recorded on a MIDAC Corporation M Series spectrometer and samples were prepared by

evaporation from CHCl3 or CH2Cl2. High resolution mass spectra were obtained from the University of

Minnesota Mass Spectrometry Facility. Chiral HPLC analysis was performed with an Agilent 1200

Series HPLC equipped with a diode array detector and 6-position column changer. Analysis of 11 and

11* was conducted with a Chiralcel OD column (4.6 mm x 25 cm) obtained from Daicel Chemical

Industries, Ltd with visualization at 225 nm.

II. Chemical Synthesis: Experimental Procedures

OH

Me

HO

PIDA, 0 °C

O

Me O

4-methyl-4-(prop-2-yn-1-yloxy)cyclohexa-2,5-dienone (S1). A solution of p-cresol (540.7 mg, 5

mmol) in propargyl alcohol (10 mL) was cooled to 0 ºC and treated with PIDA (1.61 g, 5 mmol). The

reaction mixture was stirred at the same temperature for about 30 min before concentrating in vacuo.

(1) Tello-Aburto, R; Harned, A. M. Org. Lett. 2009, 11, 3998–4000.

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Residual product was purified using 9:1 hexanes-EtOAc to give 381.9 mg of the title compound in 47%

yield.

IR (neat) 3236, 2925, 2863, 2122, 1669, 1632, 1449, 1380, 1078, 1042, 862, 711 cm-1 1H NMR (300 MHz, CDCl3) δ6.82 (d, J = 10.2 Hz, 2 H), 6.29 (d, J = 10.2 Hz, 2 H), 3.97 (d, J = 2.5 Hz, 2

H), 2.46 (t, J = 2.5 Hz, 1 H), 1.47 (s, 3 H) 13C NMR (75 MHz, CDCl3, DEPT) δ185.0 (C), 150.7 (CH × 2), 120.6 (CH × 2), 80.3 (C), 73.4 (C), 53.7

(CH2), 26.4 (CH3)

HRMS (ESI+) 185.0573 calc for C10H10O2Na found 185.0612

4-methyl-4-((3-phenylprop-2-yn-1-yl)oxy)cyclohexa-2,5-dienone (S2). A mixture of alkyne S1 (290.6

mg, 1.79 mmol) and iodobenzene (0.22 mL, 1.97 mmol) was dissolved in 10 mL of freshly distilled Et3N.

The solution was added to a flask containing CuI (1.7 mg, 0.0089 mmol) and Pd(Ph3P)2Cl2 (12.5 mg,

0.017 mmol) kept under N2. The reaction mixture was stirred at room temperature for 12 h before

diluting with 50 mL of Et2O. The solution was washed with 10% HCl solution (20 mL), then with water

(20 mL) and brine (20 mL), dried over MgSO4, filtered and concentrated. Crude product was purified by

flash column chromatography (FCC) using 5:1 hexanes-EtOAc to give 301.3 mg of the title compound

in 70% yield.

IR (neat) 3048, 2979, 2930, 2854, 1668, 1630, 1493, 1443, 1382, 1078, 1040, 863, 759, 695 cm-1 1H NMR (300 MHz, CDCl3) δ7.38–7.44 (m, 2 H), 7.27–7.33 (m, 3 H), 6.89 (d, J = 10.2 Hz, 2 H), 6.33 (d,

J = 10.2 Hz, 2 H), 4.22 (s, 2 H), 1.50 (s, 3 H) 13C NMR (75 MHz, CDCl3, DEPT) δ185.1 (C), 151.0 (CH × 2), 131.7 (CH × 2), 130.4 (CH × 2), 128.7

(CH), 128.3 (CH × 2), 122.4 (C), 86.8 (C), 85.8 (C), 73.2 (C), 54.6 (CH2), 26.4 (CH3)


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(Z)-(7α-methyl-5-oxo-4,5-dihydrobenzofuran-3(2H,3αH,7αH)-ylidene)(phenyl)methyl acetate (10).

A mixture of alkyne S2 (100 mg, 0.41 mmol), Pd(OAc)2 (4.7 mg, 0.02 mmol) and 2,2ʼ-bipyridine (6.55

mg, 0.041 mmol) was placed under N2 and dissolved in 4 mL of dry AcOH. The mixture was placed in a

preheated bath at 80 ºC and stirred overnight under N2. After 13 h the mixture was allowed to cool to

room temperature and concentrated in vacuo. Crude product was purified by FCC using 5:1 to 3:1

hexanes-EtOAc to give 108.5 mg of the title compound in 86% yield.

IR (neat) 3063, 3036, 2973, 2930, 2853, 1761, 1690, 1373, 1207, 1050, 782, 714 cm-1 1H NMR (300 MHz, CDCl3) δ7.28–7.40 (m, 5 H), 6.50 (dd, J = 10.2, 1.0 Hz, 1 H), 5.96 (d, J = 10.2 Hz, 1

H), 4.46 (d, J = 13.8 Hz, 1 H), 4.31 (dd, J = 13.8, 2.3 Hz, 1 H), 3.35 (bs, 1 H), 2.33 (dd, J = 16.7, 5.1 Hz,

1 H), 2.26 (dd, J = 16.7, 4.2 Hz, 1 H), 2.10 (s, 3 H), 1.52 (s, 3 H) 13C NMR (75 MHz, CDCl3, DEPT) δ 196.6 (C), 168.5 (C), 150.2 (CH × 2), 140.5 (C), 134.1 (C), 130.5

(C), 130.0 (CH), 129.3 (CH × 2), 128.7 (CH), 128.1 (CH × 2),81.2 (C), 68.6 (CH2),45.7 (CH), 36.2 (CH2),

23.3 (CH3), 20.8 (CH3)


OH

Ph

HO

0 °C

O

Ph O

Me

Me

1. (Me3Si)2NH, µwave2. PIFA

1-(but-2-yn-1-yloxy)-[1,1'-biphenyl]-4(1H)-one (S3) In a microwave vial, 4-phenylphenol2 (80.3 mg,

0.46 mmol) was treated with (Me3Si)2NH (2 mL). The vial was sealed, and the reaction mixture was

heated to 125 ºC for 10 min. before concentrating in vacuo. Crude TMS-protected phenol was dissolved

in 1 mL of 2-butyn-1-ol and the solution cooled to 0 ºC. PIFA (198.2 mg, 0.46 mmol) was added in one

portion and the cooling bath was removed. The mixture was stirred at room temperature for 2 hrs

before an additional portion of PIFA (30 mg) was added. Stirring continued for 30 min. at which point

(2) Freundlich, J. S.; Landis, H. E. Tetrahedron Lett. 2006, 47, 4275–4279

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the reaction was complete by TLC. Concentration in vacuo afforded an oil that was purified by FCC (9:1

hexanes-EtOAc) to give 49.2 mg of the title compound in 44% yield.

IR (neat) 3036, 2359, 2336, 1665, 1624, 1447, 1276, 1169, 1052, 922, 753, 693 cm-1 1H NMR (300 MHz, CDCl3) δ7.40–7.58 (m, 2 H), 7.28–7.40 (m, 3 H), 6.87 (d, J = 10.2 Hz, 2 H), 6.38 (d,

J = 10.2 Hz, 2 H), 4.19 (q, J = 2.3 Hz, 2 H), 1.86 (t, J = 2.3 Hz, 3 H) 13C NMR (75 MHz, CDCl3, DEPT) δ185.6 (C), 150.0 (CH × 2), 137.8 (C), 129.9 (CH × 2), 128.9 (CH ×

2), 128.5 (CH), 125.9 (CH × 2), 83.5 (C), 76.8 (C), 75.9 (C), 54.2 (CH2),3.8 (CH3)


(Z)-1-((3αR,7αR)-5-oxo-7α-phenyl-4,5-dihydrobenzofuran-3(2H,3αH,7αH)-ylidene)ethyl acetate

(11*). A mixture of alkyne S3 (26.1 mg, 0.1 mmol), (-)-Iso-PINDY ligand3 (3.77 mg, 0.01 mmol) and

Pd(OAc)2 (1.23 mg, 0.0054 mmol) was placed under N2 and dissolved in 0.5 mL of dry AcOH. The

reaction mixture was placed in a preheated oil bath at 80 ºC and stirred overnight under N2. After ~16 h

the mixture was allowed to cool to room temperature and then was concentrated in vacuo. Residual

product was purified by FCC using 5:1 hexanes-EtOAc to give the title compound in 70% yield. Chiral

stationary phase HPLC analysis (Chiralcel OD, 90:10 hexanes-isopropyl alcohol, 1 mL/min. l = 225 nm,

RTmajor = 12.8 min, RTminor = 17.5 min) showed 62% enantiomeric excess (ee). Racemic 11 was

prepared in a similar fashion using 2ʼ2-bipyridine as ligand.


H), 4.52 (bd, J = 13.1 Hz, 1 H), 4.36 (dquint, J = 13.1, 2.1 Hz, 1 H), 3.36 (bt, J = 5.1 Hz, 1 H), 2.82 (dd,

J = 16.5, 5.6 Hz, 1 H), 2.68 (dd, J = 16.5, 5.5 Hz, 1 H), 2.10 (s, 3 H), 1.88 (q, J = 1.7 Hz, 3 H)

(3) Malkov, A.V.; Pernazza, D.; Bell, M.; Bella, M.; Masa, A.; Teplý, F.; Meghani, P; Kočovský, P. J. Org. Chem. 2003, 68, 4727–4742

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13C NMR (75 MHz, CDCl3, DEPT) δ197.4 (C), 168.4 (C), 140.6 (C), 148.1 (CH), 138.9 (C), 130.5(CH),

129.0 (CH× 2), 128.5(CH), 127.5 (C), 125.5 (CH× 2), 84.4 (C),68.4 (CH2), 47.4(CH),37.7 (CH2),20.8

(CH3), 17.2 (CH3)


cat. Pd/CH2 (1 atm)

EtOAc

O

OMe

H

OAc

Ph

10

O

OMe

H

OAc

Ph

12

(Z)-(7α-methyl-5-oxohexahydrobenzofuran-3(2H)-ylidene)(phenyl)methyl acetate (12). Compound

10 (25.8 mg, 0.0865 mmol) was dissolved in 2 mL EtOAc and 10% Pd/C (11.0 mg, 0.0134 mmol, 15

mol%) was added. The reaction vessel was evacuated under gentle vacuum and backfilled with H2

(balloon) three times. The mixture was stirred at ambient temperature for 1.5 hours at which time TLC

(2x 3:1 hexane/EtOAc) indicated completed conversion. The mixture was filtered through a short plug of

silica gel and eluted with fresh EtOAc. The filtrate was concentrated in vacuo. The crude product was

purified by FCC using 4:1 hexanes-EtOAc to give 24.8 mg of the title compound in 95% yield as a

colorless oil.


H), 3.08 (dd, J = 8.0, 8.0 Hz, 1 H), 2.65–2.51 (m, 1 H), 2.37 (dd, J = 7.1, 15.1 Hz, 1 H), 2.32–2.09 (m, 3

H), 2.15 (s, 3 H), 2.05–1.91 (m, 1 H), 1.29 (s, 3 H) 13C NMR (75 MHz, CDCl3, DEPT) δ 211.0 (C), 168.4 (C), 139.8 (C), 134.5 (C), 132.7 (C), 128.8 (CH),

128.6 (CH), 127.2 (CH), 82.0 (C), 67.1 (CH2), 46.2 (CH), 41.5 (CH2), 35.6 (CH2), 33.6 (CH2), 25.2 (CH3),

20.7 (CH3)


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III. Supplemental Figure 1. Initial Screening of Compounds 4-9 for NF-κB Inhibitory Activity.

Assay was performed in A549 cells as described below. NI = non-induced control wells, I = cells

induced with TNF-α (15 ng/mL). A549 cells are induced with TNF-α (15 ng/mL) and treated with 4-9 at

a concentration of 50 μM. Notably, 4 was poorly soluble in this assay and significant amounts of

precipitate were noticed upon serial dilution of the DMSO stock into media.

IV. Supplemental Figure 2. Cytotoxicity of 10-12 in the NF-κB Reporter Assay. Cells were treated

with 10, 11, and 12 at 250 μM, 100 μM, and 50 μM concentrations and cell viability was assessed by

Alamar Blue staining.

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V. Supplemental Figure 3. Analysis of NF-κB Nuclear Translocation in CCRF-CEM cells and

Control Blots. (A) Western blot analysis of p65 and histone H2B proteins from nuclear lysate fractions

of compound-treated CCRF-CEM cells. Cells were dosed with either vehicle control (DMSO; NI and I

lanes) or analogues 10 or 11 (100 μM and 25 μM), followed by induction of the NF-κB pathway with

PMA. (B) Nuclear (N) and cytoplasmic (C) control blots for DU-145 cells. This blot shows that α-tubulin,

a cytoplasmic protein, was not found in the nuclear samples.

VI. Preparation of Stock Solutions of Compounds 4-12

Compound stock solutions were prepared in DMSO (100 mM or 200 mM concentrations) and stored at

-20° C when not in use. Compound purities were assessed frequently by analytical reverse-phase

HPLC analysis and fresh solutions were prepared as needed.

VII. Protocol for Mammalian Cell Culture

All cell lines were maintained in a humidified 5% CO2 environment at 37 °C. CCRF-CEM cells (ATCC,

CCL-119) were cultured in RPMI-1640 media (ATCC) supplemented with 10% fetal bovine serum (FBS,

Gibco), penicillin (100 I.U./mL), and streptomycin (100 μg/mL, ATCC) at a density of 2 × 105 - 2 × 106

cells/mL. A549/NF-κB-luc cells (Panomics, RC0002) were cultured in DMEM media (ATCC)

supplemented with 10% FBS (Gibco), penicillin (100 I.U./mL), streptomycin (100 μg/mL, ATCC), and

hygromycin (100 μg/mL, Roche). DU-145 cells (ATCC, HTB-81) were cultured in EMEM media (ATCC)

supplemented with 10% FBS (Gibco), penicillin (100 I.U./mL), and streptomycin (100 μg/mL, ATCC).

Vero cells (ATCC, CCL-81) were cultured in EMEM media (ATCC) supplemented with 10% FBS,

penicillin (100 I.U./mL), and streptomycin (100 μg/mL). RWPE-1 cells (ATCC, CRL-11609), which are

S9

non-cancerous prostate epithelial cells, were cultured in Keratinocyte Serum-Free Medium (K-SFM;

Gibco) supplemented with human recombinant epidermal growth factor (EGF, 5 ng/mL, PeproTech),

bovine pituitary extract (BPE, 0.05 mg/mL, PeproTech), penicillin (100 I.U./mL, ATCC), and

streptomycin (100 μg/mL, ATCC).

VIII. Protocol for NF-κB Reporter Assay

A549/NF-κB-luc cells were seeded at a density of 5,000 cells/well in cell culture media (50 μL) in 96-

well white plates with clear bottoms (Costar) 24 h prior to treatment. Compounds were serially diluted

in pre-warmed media and dosed to cells (final volume/well = 100 μL; final DMSO concentration =

0.5%). Thirty minutes after treating the cells, NF-κB was induced by adding TNF-α (15 ng/mL final

concentration, delivered in PBS; Invitrogen) to the treated wells and the induced control wells. PBS

alone was delivered to the blank and non-induced wells. After 7 h, Bright-Glo luciferase reagent

(Promega) was added to each well (100 μL) and the plate was allowed to stand for two minutes.

Luminescence measurements were then obtained using an LJL BioSystems HT Analyst plate reader.

Background luminescence from reagents (no cell controls) was subtracted. Typical induction yields

were ~7-fold. Negligible decreases in cell viability (<15%) were observed with test compounds under

these conditions, as measured by colorimetric viability staining (Alamar Blue, Invitrogen). Figure S2

depicts the changes in cell viabilities following treatment with analogues 10, 11, and 12 in this system.

Each experiment was performed in biological triplicate (at minimum) with three technical replicates per

experiment. Mean activity values were obtained by averaging the mean values of the individual

biological replicates. Uncertainty in each % NF-κB activity value was calculated by propagating the

standard deviations from the individual biological replicates (the square root of the sum of squares of

the individual standard deviations). Statistical analyses were performed with Microsoft Excel and the

results were plotted with GraphPad Prism (v. 5.0).

IX. Cell Culture Cytotoxicity Assays

CCRF-CEM cells were seeded at a density of 10,000 cells/well in cell culture media (50 μL) in standard

96-well plates (Costar) 24 h prior to treatment. DU-145, Vero, and RWPE-1 cells were seeded at a

density of 5,000 cells/well in cell culture media (50 μL) in standard 96-well plates (Costar). Blank (no

cells) wells and control (vehicle control treated) wells were prepared with each experiment. Compounds

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were serially diluted in pre-warmed media and dosed to cells (final volume/well = 100 μL; final DMSO

concentration = 0.5%). Approximately 2 h before the end of the treatment period (48 h), Alamar Blue

(Invitrogen) cell viability reagent was added to each well (10 μL). This procedure yields a quantitative

measure of cell viability by evaluating the ability of metabolically active cells (which are proportional to

the number of living cells) to convert resazurin (non-fluorescent dye) to red-fluorescent resorufin.

Fluorescence data were obtained on either a Molecular Devices SpectraMax M2 plate reader or an LJL

BioSystems HT Analyst plate reader. Background fluorescence (no cell controls) was subtracted from

each well and cellular viability values following compound treatment were normalized to vehicle-only

treated wells (control wells only treated with aqueous DMSO, which were arbitrarily assigned 100%

viability). Individual IC50 curves were generated by fitting data to the sigmoidal (dose response) function

of varied slope in GraphPad Prism (v. 5.0) software. Only curve fits with r2 > 0.95 were deemed

sufficient. Each experiment was performed in biological triplicate and mean IC50 values were calculated

from the individual IC50 values obtained from each replicate. Standard deviation was calculated from the

individual IC50 values obtained for each biological replicate.

X. Cell Fractionation and Western Blotting

CCRF-CEM and DU-145 cells were plated at 4 x 106 cells/well in 6-well culture dishes 24 h before

dosing. Cells were then treated with compounds 10 and 11 for 16 h. The final volume of media/well

following compound dosing was 2.25 mL. CCRF-CEM cells were induced with phorbol 12-myristate 13-

acetate (PMA, 850 ng/mL, Fisher BioReagents) for the last 4 h of the 16 h dosing.4 DU-145 cells were

induced with TNF-α (117 ng/mL, Gibco) for the final 2 h of the 16 h dosing.

DU-145 cells were then trypsinized and pelleted, and CCRF-CEM cells were immediately pelleted (500

x g for 5 min, room temperature). The media was decanted and cell pellets were resuspended once in

ice-cold phosphate buffered saline (PBS, 1 mL) and pelleted again (500 x g for 5 min, 4 °C). Cell pellets

were subsequently lysed in Buffer 1 (400 μL; Buffer 1 components (to make 50 mL): 10 mM HEPES, 10

mM KCl, 1.5 mM MgCl2, 1 mM DTT, and one tablet of cOmplete (Roche) EDTA-free protease inhibitor

cocktail) and incubated on ice for 10 min. The cells were homogenized by passing them through a 28-

gauge syringe four times. Cell nuclei were pelleted by centrifugation at maximum speed (21,000 x g) for

(4) Cogswell, P.; Mayo, M.; Baldwin, A. J. Exp. Med. 1997, 185, 491-497

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5 min at 4 °C. The supernatants (cytosolic extracts) were collected in pre-chilled tubes and stored at

-80 °C until further use. The residual pellets were gently washed with PBS without pellet disruption (200

μL) and subsequently resuspended in Buffer 2 (400 μL; Buffer 2 components (to make 50 mL): 20 mM

HEPES, 600 mM KCl, 1.5 mM MgCl2, 1 mM DTT, 0.2 mM EDTA, 25% (v/v) glycerol, and one tablet of

cOmplete [Roche] EDTA-free protease inhibitor cocktail) to lyse the nuclei. Samples were incubated on

ice and vortexed on the highest setting for 15 sec every 10 min for a total of 40 min. The suspension

was centrifuged at maximum speed (21,000 x g) for 10 min at 4 °C. The supernatants (nuclear extracts)

were collected in pre-chilled tubes and stored at -80 °C until further use. The protein concentrations

were determined using the BCA protein assay kit (Pierce).

Samples of nuclear and cytosolic extracts (30 μg) were combined with NuPAGE 4X LDS sample buffer

and NuPAGE 10X sample reducing agent (Invitrogen) and put in heat block at 99 °C for 5 minutes.

Protein samples were electrophoresed on a gradient 4-20% SDS-PAGE gel (BioRad), then

electrotransferred to a polyvinylidene difluoride membrane (Immobilon). The membrane was then

blocked with Odyssey blocking buffer (LI-COR Biotech.) for 1 h. Proteins were detected by incubation

with primary antibodies for p65 (Active Motif, 39283), histone H2B (Active Motif, 61038), and α-tubulin

(Active Motif, 39528) in blocking buffer supplemented with 0.1% Tween 20 for 1 h. The membrane was

then briefly washed by gentle rocking in a solution of PBS (50 mL, 1 min, total 5x), and then incubated

with IRDye 800 anti-rabbit (LI-COR Biotech., 926-32211) and IRDye 680 anti-mouse conjugated

secondary antibodies (LI-COR Biotech., 926-68020) in blocking buffer supplemented 0.1% Tween 20

for 1 h. The membrane was again washed via gentle rocking in a solution of PBS (50 mL, 1 min, total

5x). The immunocomplexes were visualized using the Odyssey classic infrared imaging system (LI-

COR Biotech.).

XI. Compound Treatment for ELISA and PCR

DU-145 cells were seeded into 24-well plates at a cell density of 2 × 105 cells/well. The plate was dosed

with compounds 10-12 for 24 hours. The cells were induced with TNF-α (117 ng/mL) six hours after the

initial dosing, resulting in an 18 hour induction.

XII. IL-8 Enzyme-Linked Immunosorbent Assays

A human IL-8 ELISA kit (Thermo Scientific, EH2IL82) was used to quantitate secreted IL-8 protein

levels in NF-κB-induced DU-145 cells treated with 10-12. Following the dosing and induction of DU-145

S12

cells (described above), cell media (500 µL) was collected from each treated well and stored at -20 °C

until analysis. Immediately prior to analysis, samples were centrifuged at maximum speed (21,000 x g)

for 1 minute and then secreted IL-8 protein levels were measured for each sample according to vendor

instructions. The absorbance of each sample at 450 nm and 550 nm was collected following addition of

the substrate stop solution using a Molecular Devices SpectraMax M2 plate reader. The standard curve

was fitted using a 4-parameter logistic (4-PL) algorithm in GraphPad Prism (v. 5.0) software. Unknown

samples were plotted against the standard curve to yield IL-8 protein concentrations in pg/mL. Triplicate

biological replicates were performed and each bar represents at least 6 individual wells. The data

shown in Figure 4 is mean ± SEM, which was calculated from mean values of each of the three

biological replicates. Statistical analyses were performed in Microsoft excel and data was plotted using

GraphPad Prism (v. 5.0) software.

XIII. mRNA Isolation and Purification

RNeasy Plus Micro Kits (Qiagen, 74034) and QIAshredders (Qiagen, 79654) were used to isolate and

purify mRNA from DU-145 cells. Following compound dosing, NF-κB induction, and harvesting of the

cell media (described above), cell monolayers were washed with PBS (1X) and then lysed according to

the method described in the RNeasy Plus Micro Kit (β-Mercaptoethanol supplement was included). The

samples were then homogenized using QIAshredders according to vendor instructions. mRNA was

isolated and purified using the RNease Plus Micro Kit and eluted in RNase-free water (14 μL). RNA

samples were immediately stored at -80 °C.

XIV. Reverse Transcription (RT) & Polymerase Chain Reaction (PCR) Assays

The RNA concentration and purity of each sample were determined using a NanoDrop instrument

(Thermo Scientific). RNA samples with A260/A280 ratios ≥ 1.9 were used in PCR assays. The

concentration of each mRNA sample within a biological experiment was normalized to the value

obtained from the sample with the lowest measured mRNA concentration. Reverse transcription of

mRNA into cDNA was carried out using the QuantiTect Reverse Transcription Kit (Qiagen, 205311).

Template mRNA was added to gDNA Wipeout Buffer (7X) and RNase-free water for a total volume of

14 μL (less than 1 μg of mRNA was added to each sample). Samples were then incubated at 42 °C for

2 minutes (Bio-Rad T100 thermal cycler) and then immediately placed on ice. Next, the entire contents

of the previous mix were added to 6 μL of reverse-transcription master mix, and mRNA was reverse

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transcripted to cDNA per vendor instructions. cDNA samples were stored at -20 °C immediately

following reverse transcription.

The cDNA samples were carried forward to the PCR assays. These assays utilized HotStarTaq DNA

Polymerase Kit (Qiagen, 203203) along with a dNTP Mix (Qiagen, 201900). Primers specific to either

IL-8 or GAPDH (control) were purchased for Integrated DNA Technologies. The following IL-8 and

GAPDH oligonucleotide primers were reported previously in the literature5,6 and were used in our study:

IL-8 Forward: 5ʼ – CTC TCT TGG CAG CCT TCC TGA TT – 3ʼ

IL-8 Reverse: 5ʼ – AAC TTC TCC ACA ACC CTC TGC AC – 3ʼ

GAPDH Forward: 5ʼ – GTA AAG TGG ATA TTG TTG CCA TCA – 3ʼ

GAPDH Reverse: 5ʼ – AAA TTC GTT GTC ATA CCA GGA AAT – 3ʼ

Primers (IL-8 and GAPDH) were added to separate HotStarTaq DNA Polymerase master mixes (no

extra Mg2+ was added) containing PCR Buffer, dNTP mix, HotStarTaq DNA Polymerase, and RNase-

free water. An aliquot (2 μL) of the template cDNA obtained from the previous reverse transcription

procedures was then added to the master mix in 0.200 mL PCR tubes for a total volume of 100 μL. A

total of 27 PCR cycles were performed (on a Bio-Rad T-100 thermal cycler) for both IL-8 and GAPDH

according to the following method:

1. Initial Activation: 15 minutes at 95 °C 2. Denaturation: 1 minute at 94 °C 3. Annealing: 1 minute at 54 °C 4. Extension: 1 minute at 72 °C 5. Final Extension: 10 minutes at 72 °C 6. Final Hold: 4 °C

Steps 2-4 were repeated 27 times (27 cycles).

(5) Cubitt, C. L.; Lausch, R. N.; Oakes, J. E., Invest. Ophthalmol. Vis. Sci., 1995, 36, 330-336

(6) Doyon, P.; Servant, M. J., J. Bio. Chem., 2010, 40, 30708-18

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An aliquot of the resulting PCR products (2 μL) was added to loading buffer (8 μL; recipe for loading

buffer: 10 mg of orange G, 10 mg of bromophenol blue sodium salt, and 10 mg of xylene cyanol FF in 6

mL of formamide). The samples were then electrophoresed on a 1% agarose gel made in 0.5% TBE

buffer (10X TBE components (to make 1 L): 108 g tris base, 55 g boric acid, and 7.4 g EDTA in distilled

and deionized water). The agarose gel was stained using SYBR Gold nucleic acid gel stain (Invitrogen)

and then visualized using the Typhoon FLA 7000 (GE Healthcare). Gels were analyzed using

ImageQuant TL software (GE, version 7.0).

XV. HPLC and NMR Michael Acceptor Assays

A previously described NMR assay was performed to measure the reactivity of 10 towards thiols.7 In

brief, 10 (1.14 mg, 0.004 mmol) was dissolved in DMSO-d6 (50 μL) in a microcentrifuge tube. In a

separate tube, anhydrous dibasic sodium phosphate (3 mg) was added to D2O (450 μL). The PBS

solution (pH ~ 8) was added to the DMSO stock of 10 and vortexed. The solution was added to a

standard 5 mm NMR tube (Wilmad LabGlass) and the 1H NMR spectrum was recorded on a Varian 600

MHz NMR at 25 °C. Cysteamine (0.58 mg, 0.008 mmol, 2 mol equiv) was pre-weighed into a

microcentrifuge tube. The solution of 10 from the NMR tube was then added to the pre-weighed

cysteamine and mixed. This solution was then replaced in the NMR tube and additional spectra were

acquired at the time points shown.

For analysis of thiol reactivity of 10 by HPLC, the protocol described above was repeated, except the

PBS solution was prepared in distilled water. After 10 minutes, trifluoroacetic acid (50 μL, 80% v/v TFA

in distilled H2O) was added to the reaction mixture and the reaction products were separated using an

Agilent 1200 series instrument equipped with a diode array detector and a Zorbax SB-C18 column (4.6

x 150 mm, 3.5 μm, Agilent Technologies) at 23 ºC. The mobile phase comprised 0.1% TFA in water (0

to 10 min), followed by 0 to 85% 0.1% TFA in CH3CN (10 to 24 min) at a flow-rate of 1.0 mL/min. The

major peak (tR = 11.3 min) was collected and analyzed by negative ESI mass spectrometry confirming

formation of 13. Mass calculated for C20H25NO4S: 375.1504. Found [M – H]-: 374.1434.

(7) Avonto, C.; Taglialatela-Scafati, O.; Pollastro, F.; Minassi, A.; Di Marzo, V.; De Petrocellis, L.; Appendino, G., Angew. Chem. Int. Ed. Engl., 2011, 50, 467-471

S15

XVI. NMR Spectra

O

Me O

S1(CDCl3, 300 MHz)

S16

O

Me O

S1(CDCl3, 75 MHz)

S17

O

Me O

S2(CDCl3, 300 MHz)

S18

O

Me O

S2(CDCl3, 75 MHz)

S19

O

10(CDCl3, 300 MHz)

O OAc

Ph

Me

H

S20

O

10(CDCl3, 75 MHz)

O OAc

Ph

Me

H

S21

O

S3(CDCl3, 300 MHz)

O

Me

S22

O

S3(CDCl3, 75 MHz)

O

Me

S23

O

11(CDCl3, 300 MHz)

O OAc

MeH

Ph

S24

O

11(CDCl3, 75 MHz)

O OAc

MeH

Ph

S27

XVII. Chiral HPLC Chromatogram for 11 (top) and 11* (bottom)

Signal 1: DAD1 C, Sig=225,8 Ref=360,100 Peak RetTime Type Width Area Height Area # [min] [min] [mAU*s] [mAU] % ----|-------|----|-------|----------|----------|--------| 1 13.142 BB 0.4230 9017.05469 319.70926 49.9562 2 17.602 BV 0.5236 9032.87012 266.41418 50.0438 Totals : 1.80499e4 586.12344

Signal 1: DAD1 C, Sig=225,8 Ref=360,100 Peak RetTime Type Width Area Height Area # [min] [min] [mAU*s] [mAU] % ----|-------|----|-------|----------|----------|--------| 1 12.868 BB 0.4276 5.26892e4 1831.61230 81.0107 2 17.536 BV 0.5230 1.23506e4 364.79282 18.9893 Totals : 6.50398e4 2196.40512

OPh

H

O

OAc

Me

Harned Harki NF-kB ACS MCL SI Final Galley Stage 10May2012 ACS Med Che… · VII. Protocol for...

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