ars.els-cdn.com · web viewwere evaluated by colorometric assay against a549 (ccl-185) (human small...

Supplementary Material

The Total Syntheses of JBIR-94 and Two Synthetic Analogs and Their Cytotoxicities Against A549 (CCL-185) Human Small Lung Cancer Cells

By Cathy L. Mangum, Mica B. Munford, Alyssa B. Sam, Sandra K. Young, Jeremy T. Beales, Yagya Prasad Subedi, Chad D. Mangum, Tanner J. Allen, Miranda S.

Liddell, Andrew I. Merrell, Diana I. Saavedra, Becky J. Williams, Nicole Evans, Joseph L. Beales, and Michael A. Christiansen*

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Table of Contents

Contents Page Number

1. Experimental Details S31.1. General Materials and Methods S31.2. Cytotoxicity Assay S31.3. Synthetic Procedures and Characterization Data S5

1.3.1. Syntheses of Molecules in Scheme 1 S51.3.2. Syntheses of Molecules in Scheme 2 S81.3.3. Syntheses of Molecules in Scheme 3 S91.3.4. Syntheses of Molecules in Scheme 4 S121.3.5. Syntheses of Molecules in Scheme 5 S161.3.6. Spectra S19

2. References S57

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1. Experimental Details

1.1. General Materials and Methods

Air and water sensitive reactions were performed in oven-dried glassware and under nitrogen

atmosphere. Air and moisture sensitive reagents were introduced via dry syringe. When used as

a reaction solvent, methylene chloride (CH2Cl2) was pre-dried by distillation from CaSO4

according to Purification of Laboratory Chemicals, 5th ed.; Butterworth-Heinemann: Burlington,

MA, USA, 2003; p 198. ACS-grade methanol, ethanol, N,N-dimethylformamide, tetrahydrofuran,

and triethylamine were purchased from Fisher Scientific and dried overnight with molecular

sieves (4Å, 4 to 8 mesh) before use. Flash chromatography was carried out using 230 x 400

mesh silica gel. Analytical thin-layer chromatography (TLC) was performed using silica gel 60

F254, 0.255 mm pre-coated TLC plates, purchased from EMD Millipore Chemicals. TLC plates

were visualized using UV254 and a cerium molybdate stain with charring, as described below. In-

class 1H-NMR spectra were obtained by undergraduates using a 60 MHz NMR spectrometer,

with TMS (0.0 ppm) or chloroform (7.27 ppm) as an internal reference. High-field 1H-NMR

spectra, provided below, were obtained with JEOL (300 MHz) or Bruker (500 MHz)

spectrometers using TMS (0.0 ppm) as an internal reference. Signals are reported as m

(multiplet), s (singlet), d (doublet), t (triplet), q (quartet), bs (broad singlet), dd (doublet of

doublets), or dd (doublet of doublets); the coupling constants are ported in hertz (Hz). 13C-NMR

(75 or 125 MHz) were acquired with chloroform (77.2 ppm) as the internal standard. High-

Resolution Mass Spectral (HRMS) data were obtained using an Agilent multi-mode source

mass spectrometer. GCMS data were obtained using a Shimadzu QP2010 Ultra Gas

Chromatograph Mass Spectrometer. Melting points were obtained with a MEL-TEMP® capillary

melting point apparatus. IR spectra were obtained using a Nicolet 6700 FT-IR spectrometer.

Equipment and procedural details for each experiment, with accompanying spectra, are

thoroughly outlined below.

1.2. Cytotoxicity Assay

The cytotoxicities of JBIR-94 and analogs 3-4 were evaluated by colorometric assay against

A549 (CCL-185) (human small lung cancer) cells using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide) dye. For this, A549 (CCL-185) cells were grown in Dulbecco’s

modified eagle’s medium, supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and

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100 µM streptomycin at 37 oC with 5% CO2 atmosphere. Two-hundred microliters (200 µL) of

cell suspension in growth medium with 5000 cells was seeded to the wells of a 96-well cell

culture plate and incubated for 24 hours. The medium was removed, and the compounds at

different concentrations (0.01, 0.1, 1.0, 10, and 100 µM) in growth medium were added to the

cells, including a control containing growth medium only and a separate control containing 1%

of 100X triton in medium. Cells were then incubated for an additional 48 hours. Twenty

microliters of MTT dye (5 mg/mL stock) was then added to each cells-containing well of a 96-

well plate and incubated for five more hours. The medium was removed, and the cells were

washed with 100 µL of 1x PBS (pH = 7.4). The reduced MTT dye in the cells was dissolved in

100 µL of DMSO, and absorption was measured at 570 nm and 650 nm. The experiment was

performed in triplicate of triplicate.

The cytotoxicity of the tested compounds at different concentrations and IC50 values are

presented in Figure 1. The results show that compound 4 has relative cell viability around 55%

at 100 μM, whereas JBIR-94 and analog 3 are relatively more toxic, with cell viability aroud

60%, even at 10 µM concentration. Moreover, JBIR-94 and compounds 3-4 have IC50 values of

52.88 ± 11.69 µM, 78.92 ± 8.92 µM, and >100 µM, respectively, showing mild toxicity to human

lung cells.

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Figure 1. Gradient cytotoxicities of JBIR-94 and analogs 3-4 towards A549 (CCL-185) cells.

1.3. Synthetic Procedures and Characterization Data

1.3.1.[1.2.1.] Syntheses of Molecules in Scheme 1

3-(4-hydroxy-3-methoxyphenyl)propanoic acid (6). Following literature procedure,1 trans-

ferulic acid (10.0 g, 51.50 mmol, 1.0 equivalent) was dissolved in H2O (515 mL, 0.1 M), and

methanol (515 mL, 0.1 M) in a 2.0 L round-bottom flask with a magnetic stir bar at room

temperature (RT). CAUTION (flammability hazard): 5% Pd/C (2.0 g, 0.2 wt/wt equivalents) was

then added carefully. The reaction vessel was topped with an H2 balloon, purged with H2 gas

(3x), and stirred at RT under H2 balloon pressure for 24 hours. Once the reaction was complete

(determined by TLC), the reaction slurry was concentrated and purified by column

chromatography (50% EtOAc/hexanes, followed by 100% EtOAc flush). This procedure yielded

10.12 grams of 6 (quantitative yield) as an off-white solid. Melting point was 89 – 91 °C. Data

are: TLC Rf = 0.47 (streak, 60% EtOAc/hexanes); 1H NMR (DMSO-d6, 300 MHz) δ 12.09 (bs,

1H), 8.85 (bs, 1H), 6.59 (m, 2H), 3.70 (s, 3H), 2.68 (t, J=6 Hz, 2H), 2.44 (t, J=6Hz, 2H); 13C NMR

(DMSO-d6, 75 MHz) δ 174.5, 147.9, 145.2, 132.2, 120.8, 115.8, 113.0, 56.0, 36.3, 30.6.

3-[4-(acetyloxy)-3-methoxyphenyl]propanoic acid (7a). Following literature procedure,2

molecule 6 (253 mg, 1.29 mmol, 1.0 equivalent) was dissolved in CH2Cl2 (7.5 mL, 0.17 M) in a

25.0 mL round-bottom flask with a magnetic stir bar at room temperature (RT). Triethylamine

(0.36 mL, 2.57 mmol, 2.0 equivalents) and 4-dimethylaminopyridine (DMAP, 4.4 mg, 0.036

mmol, 0.028 equivalents) were then added, and the flask was cooled to 0 °C in an ice bath. At

this point, acetic anhydride (Ac2O, 0.243 mL, 2.57 mmol, 2.0 equivalents) was added dropwise.

The reaction was then warmed to RT, stirred for 20 minutes, and diluted with CH 2Cl2 (20 mL)

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and H2O (20 mL). The suspension was now mixed and transferred to a separatory funnel, where

the layers were separated. The organic (CH2Cl2) layer was then washed with brine (10 mL),

dried over Na2SO4, concentrated, and purified by column chromatography (20%

EtOAc/hexanes). This procedure yielded 240 mg (78%) of 7a as a white powder. Melting point

was 92 – 93 °C. Data are: TLC Rf = 0.44 (streak, 50% EtOAc/hexanes); 1H NMR (DMSO-d6, 300

MHz) δ 12.02 (bs, 1H), 6.97-6.90 (m, 2H), 6.76 (d, J=4.5 Hz, 1H), 3.76 (s, 3H), 2.79 (t, J=9Hz,

2H), 2.50 (t, J=9Hz, 2H), 2.20 (s, 3H); 13C NMR (DMSO-d6, 75 MHz) δ 174.3, 169.2, 151.1,

140.4, 138.0, 123.0, 120.6, 113.4, 56.2, 35.8, 30.8, 20.9.

3-[4-(tert-butyldimethylsilyloxy)-3-methoxyphenyl]propanoic acid (7b). Diisopropyl-

ethylamine (6.66 mL, 38.23 mmol, 3.0 equivalents) and TBSCl (4.8 g, 31.86 mmol, 2.5

equivalents) were added sequentially and in single portions to a solution of molecule 6 (2.50 g,

12.74 mmol, 1.0 equivalent) suspended in CH2Cl2 (16.5 mL, 0.77 M) in a 50.0 mL round-bottom

flask with a magnetic stir bar at room temperature (RT). The flask was then stirred at RT for 14

hours. The reaction was afterward diluted with EtOAc (20 mL), mixed, and transferred to a

separatory funnel containing H2O (20 mL). More EtOAc was then added (~50 mL). The layers

were separated, and the organic (EtOAc) layer was washed with 1M aqueous HCl (10 mL) and

brine (10mL), dried over MgSO4, and concentrated to form the bis-silylated intermediate as a

yellow-orange oil.

At this point, the concentrated intermediate was dissolved in wet THF (16.5 mL, 0.77 M),

and solid K2CO3 (2.0 g, 1.14 equivalents) was added in a single portion. The resulting slurry was

then stirred vigorously at RT for 2 hours. The reaction mixture was then diluted with EtOAc (50

mL), mixed, and transferred to a separatory funnel, where it was further diluted with H2O (20

mL). The layers were separated, and the organic layer was then washed with HCl (20 mL) and

brine (20mL). The crude product was then concentrated in a pre-weighed RB flask, which was

afterward suspended in an oil bath at 60 °C and left under high vacuum to remove excess TBS–

OH. This procedure yielded 3.13 g (79%) of 7b as a dark-yellow solid. Melting point was 62 °C.

Data are: TLC Rf = 0.47 (streak, 50% EtOAc/hexanes); 1H NMR (CDCl3, 300 MHz) δ 10.89 (bs,

1H), 7.05-6.64 (m, 3H), 3.78 (s, 3H), 2.90 (t, J=9Hz, 2H), 2.66 (t, J=9Hz, 2H), 1.00 (s, 9H), 0.15

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(s, 6H); 13C NMR (CDCl3, 75 MHz) δ 179.7, 150.9, 143.6, 133.8, 120.9, 120.4, 112.5, 55.6, 36.6,

30.5, 25.8, 18.5, -4.53.

Benzyl 3-[4-(benzyloxy)-3-methoxyphenyl]propanoate. Following literature procedure,3

molecule 6 (2.40 g, 12.23 mmol, 1.0 equivalent) and K2CO3 (6.71 g, 48.55 mmol, 3.97

equivalents) were dissolved in N,N-dimethylformamide (DMF, 11.9 mL, 1.03 M) in a 250 mL

round-bottom flask with a magnetic stir bar at room temperature (RT). CAUTION (caustic smell

hazard): Benzyl bromide (9.5 mL, 79.73 mmol, 6.52 equivalents) was then added dropwise at

RT. The flask was now warmed to 80 °C and heated for 4.0 hours. Once complete (TLC), the

mixture was cooled to RT and quenched with H2O (20 mL). The vessel was then cooled to 0 °C

in an ice bath, and the mixture was gradually brought to pH <4.0 by slow addition of 1N HCl. At

this point, the mixture was transferred to a separatory funnel, where the layers were mixed and

separated. The aqueous layer was then extracted with EtOAc (3×20 mL). The combined organic

layers were washed with saturated aqueous NaHCO3 (20 mL), brine (20 mL), dried over

Na2SO4, concentrated, and purified by column chromatography (20% EtOAc/hexanes). This

procedure yielded 4.73 g (quantitative yield) of the ester product as a light-yellow oil. When

stored in a freezer overnight at –20 °C, the compound converted to an off-white solid with a

melting point of 35.5 – 37 °C. Data are: TLC R f = 0.36 (20% EtOAc/hexanes); 1H NMR (CDCl3,

500 MHz) δ 7.46 (d, J=2.5Hz, 2H), 7.40-7.33 (m, 8H), 6.83-6.77 (m, 2H), 6.68 (d, J=2.5Hz, 1H),

5.15 (s, 2H), 5.14 (s, 2H), 3.87 (s, 3H), 2.94 (t, J=5Hz, 2H), 2.69 (t, J=5Hz, 2H); 13C NMR

(CDCl3, 125 MHz) δ 172.8, 149.6, 146.7, 137.4, 135.9, 133.7, 128.6, 128.5, 128.2, 128.2, 127.8,

127.3, 120.2, 114.3, 112.2, 71.2, 66.3, 56.0, 36.1, 30.6.

3-[4-(benzyloxy)-3-methoxyphenyl]propanoic acid (7c). Benzyl 3-[4-(benzyloxy)-3-

methoxyphenyl]propanoate (1.40 g, 3.72 mmol, 1.0 equivalent) was dissolved in H2O (6.9 mL,

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0.54 M) and methanol (6.9 mL, 0.54 M) in a 250 mL round-bottom flask with a magnetic stir bar

at room temperature (RT). Potassium hydroxide (396 mg, 7.07 mmol, 1.9 equivalents) and THF

(1.78 mL, 2.7 M) were then added. The reaction was then warmed to 60 °C and stirred for 4.0

hours. Once complete (TLC), the mixture was cooled to RT and quenched with H2O (20 mL).

The vessel was then cooled to 0 °C in an ice path, and the mixture was gradually brought to pH

<2.0 by slow addition of 1N HCl. At this point, the mixture was transferred to a separatory

funnel, where the layers were mixed and separated. The aqueous layer was then extracted with

EtOAc (5×40 mL), dried over Na2SO4, concentrated, and purified by column chromatography

(25% EtOAc/hexanes, followed by column flush with 100% methanol). This procedure yielded

905 mg (85%) of compound 7c as a light-yellow oil. When stored in a freezer overnight at –20

°C, the compound converts to a white solid. Melting point was 98.9 – 100 °C. Data are: TLC R f

= 0.08 (streak, 20% EtOAc/hexanes); 1H NMR (DMSO-d6, 500 MHz) δ 12.27 (bs, 1H), 7.44-7.31

(m, 5H), 6.92-6.87 (m, 2H), 6.70 (d, J=2.5Hz, 1H), 5.03 (s, 2H), 3.76, (s, 3H), 2.76 (t, J=5Hz,

2H), 2.50 (t, J=5Hz, 2H); 13C NMR (DMSO-d6, 125 MHz) δ 174.5, 149.5, 146.5, 137.8, 134.5,

128.8, 128.2, 128.2, 120.4, 114.2, 113.0, 70.5, 56.0, 36.2, 30.6; HRMS found 287.1311 [M+H]+,

calcd 286.12 for [C17H18O4].


1-{[3-(4-acetoxy-3-methoxyphenyl)propanoyl]oxy}pyrrolidine-2,5-dione (11a). Molecule 7a (200 mg, 0.84 mmol, 1.0 equivalent) and N-hydroxysuccinimide (96.6 mg, 0.84 mmol, 1.0

equivalent) were dissolved in THF (2.8 mL, 0.3 M) in a 25 mL round-bottom flask with a

magnetic stir bar at room temperature (RT) under N2 atmosphere. The reaction mixture was

then cooled to 0 °C and stirred until it turned clear. At this point, pre-dissolved DCC (175 mg,

0.85 mmol, 1.01 equivalents) in THF (0.85 mL, 0.3 M) was added in five to six portions, via

syringe. The reaction was then stirred at 0 °C for four hours. The reaction mixture was now

poured through a sintered glass filter, and the mother liqueur was concentrated in a pre-

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weighed RB flask. This procedure yielded 244 mg (87%) of compound 11a as an impure, off-

white solid. Data are: TLC Rf = 0.08 (20% EtOAc/hexanes). The final product was too impure to

clearly evaluate by NMR spectroscopy.

1-{[3-(4-tert-butyldimethylsilyloxy-3-methoxyphenyl)propanoyl]oxy}pyrrolidine-2,5-dione (11b). Following the procedure described above for 11b, 100 mg of molecule 7b (0.322 mmol,

1.0 equivalent) yielded 174 g (132% crude yield) of compound 11b as an impure, off-white solid.

Data are: TLC Rf = 0.35 (20% EtOAc/hexanes, developed 2x). 1H NMR (1:1 CDCl3 : DMSO-d6

300 MHz) δ 7.16-7.01 (m, 4H), 6.76-6.61 (m, 3H), 3.76 (s, 3H), 2.96-2.81 (m, 10H), 2.29 (d,

J=1.5 Hz, 4H), 0.95 (s, 9H); 13C NMR (CDCl3, 300 MHz) δ 169.2, 132.7, 122.1, 121.0, 120.4,

112.3, 56.1, 55.5, 49.4, 33.8, 33.1, 25.8, 25.0, 20.7, -4.6.


3-[4-(benzyloxy)-3-methoxyphenyl]tert-butyl (4-propanamidobutyl)carbamate (13). Molecule 7c (1.40 g, 4.87 mmol, 1.0 equivalent) was dissolved in CH2Cl2 (13 mL, 0.37 M) in a

50 mL round-bottom flask with a magnetic stir bar at room temperature (RT) under N2

atmosphere. The reaction mixture was then cooled to 0 °C. At this point, commercial N-BOC-

1,4-butanediamine4 (1.03 mL, 5.36 mmol, 1.1 equivalents), EDCI5 (1.03 g, 5.36 mmol, 1.1

equivalents), and triethylamine (0.72 mL, 5.36 mmol, 1.1 equivalents) were added. The reaction

was then stirred overnight (~21 hours), during which time it gradually warmed to RT. The

reaction mixture was then diluted with H2O (20 mL) and transferred to a separatory funnel. The

layers were mixed and separated, and the aqueous layer was extracted with CH2Cl2 (5 × 25

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mL). The combined organic layer was then washed with brine (15 mL), dried over Na2SO4,

concentrated, and recrystallized overnight from warm 50% CH2Cl2/hexanes. Subsequent

vacuum filtration yielded 1.91 g (86%) of compound 13 as a white solid. Melting point was 116.4

°C. Data are: TLC Rf = 0.1 (60% EtOAc/hexanes); 1H NMR (DMSO-d6, 500 MHz) δ 7.43-7.25

(m, 5H), 6.88-6.82 (m, 2H), 6.62 (dd, J1=7.5 Hz, J2=5Hz, 1H), 5.04 (s, 2H), 4.88 (s, 3H), 3.82

(s, 3H), 3.32-3.29 (m, 2H), 3.15-3.06 (m, 3H), 2.98 (t, J=10Hz, 2H), 2.83 (t, J=10Hz, 2H), 2.42 (t,

J=10Hz, 3H), 1,41 (s, 9H), 1.37 (t, J=5 Hz, 2H); 13C NMR (CDCl3, 125 MHz) δ 166.6, 149.9,

142.5, 139.3, 130.3, 127.2, 120.9, 120.3, 120.2, 113.1, 107.4, 105.4, 71.3, 63.8, 36.1, 32.4,

31.4, 30.6, 24.0, 20.2, 19.7, 19.1; GCMS found 456 [M]+, calcd 456.26 for [C26H36N2O5].

N-(4-aminobutyl)-3-[4-(benzyloxy)-3-methoxyphenyl] propanamide (14). Carbamate 13 (100 mg, 0.22 mmol, 1.0 equivalent) was dissolved in toluene (6.3 mL, 0.035 M) in a 25 mL

round-bottom flask with a magnetic stir bar at room temperature (RT) under N2 atmosphere.

Trifluoroacetic acid (0.63 mL, 0.347 M) was then added, and the reaction was stirred at RT for

15-20 minutes. The reaction mixture was then stirred and diluted with enough 2M aqueous

NaOH to reach a pH of >10.0. The mixture was now transferred to a separatory funnel. The

layers were separated, and the aqueous layer was extracted exhaustively with CH2Cl2 (20 × 10

mL). The combined organic layer was then dried over Na2SO4 and concentrated in a pre-

weighed flask. This procedure yielded 64 mg (82%) of compound 14 as a heavy oil, which

crystallized to form a sticky, crystalline solid after being stored overnight at –20 °C. Melting point

undetected, due to the solid’s stickiness. Data are: TLC R f = 0.31 (5% methanol/CH2Cl2); 1H

NMR (DMSO-d6, 500 MHz) δ 7.41-7.24 (m, 5H), 6.87-6.82 (m, 2H), 6.68 (dd, J1=7.5 Hz,

J2=5Hz, 1H), 5.02 (s, 2H), 4.92 (s, 5H), 3.81 (s, 3H), 3.30-3.28 (m, 1H), 3.13 (t, J=10Hz, 2H),

2.87-2.80 (m, 2H), 2.46 (t, J=15Hz, 2H), 1.56-1.40 (m, 4H); 13C NMR (CDCl3, 125 MHz) δ

166.4, 142.1, 138.8, 129.8, 126.8, 120.4, 119.9, 112.7, 107.0, 104.5, 63.4, 47.5, 32.4, 31.4,

30.4, 30.1, 23.5, 18.3, 17.0; GCMS found 356 [M]+, calcd 356.21 for [C21H28N2O3].

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N,N'-(butane-1,4-diyl)-bis-{3-[4-(benzyloxy)-3-methoxyphenyl]}dipropanamide (9c). Molecule 7c (211 mg, 0.74 mmol, 1.0 equivalent) was dissolved in CH2Cl2 (4.4 mL, 0.167 M) in

a 25 mL round-bottom flask with a magnetic stir bar at room temperature (RT) under N2

atmosphere. The reaction mixture was then cooled to 0 °C. At this point, molecule 14 (290 mg,

0.81 mmol, 1.1 equivalents) that was pre-dissolved in CH2Cl2 (4.4 mL, 0.167 M, 1.0 equivalent)

was added, followed by EDCI5 (156 mg, 0.81 mmol, 1.1 equivalents) and triethylamine (0.11

mL, 0.81 mmol, 1.1 equivalents). The reaction was then stirred overnight (~21 hours), during

which time it gradually warmed to RT. The reaction mixture was now diluted with H2O (20 mL)

and transferred to a separatory funnel. The layers were separated, and the aqueous layer was

extracted with CH2Cl2 (5 × 25 mL). The combined organic layer was now washed with brine (15

mL), dried over MgSO4, concentrated, and purified by column chromatography (50%

EtOAc/hexanes, followed by 100% MeOH flush). This procedure yielded 382 mg (83%) of

compound 9c as a white solid. Melting point was 168 – 172 °C. Data are: TLC Rf = 0.16 (streak,

50% EtOAc/hexanes); 1H NMR (CDCl3, 500 MHz) δ 7.44-7.29 (m, 10H), 6.81-6.74 (m, 4H), 6.66

(dd, J1=10 Hz, J2=5Hz, 2H), 5.12 (s, 4H), 4.13-4.07 (m, 4H), 3.87 (s, 6H), 3.39 (t, J=10Hz, 2H),

2.88 (t, J=10Hz, 4H), 2.63-2.56 (m, 4H), 1.92-1.73 (m, 4H); 13C NMR (CDCl3, 125 MHz) δ 173.0,

149.7, 146.8, 137.4, 133.8, 128.6, 127.9, 127.3, 120.2, 114.4, 112.3, 71.2, 63.5, 56.1, 36.1,

33.1, 30.7, 29.3, 27.4; GCMS found 624 [M]+, calcd 624.32 for [C38H44N2O6].

JBIR-94 (1). Molecule 9c (30 mg, 0.048 mmol, 1.0 equivalent) was dissolved in ethanol (0.48

mL, 0.1 M) in a 25 mL round-bottom flask with a magnetic stir bar at room temperature (RT).

The reaction vessel was then topped with an H2 balloon, purged with H2 gas (3x), and stirred at

RT under H2 balloon pressure for 30 minutes. The reaction slurry was next filtered through

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florisil using EtOAc and methanol, and the product was concentrated. This procedure yielded 18

mg (84%) of JBIR-94 as a chalky-white solid. Melting point was 178 – 180 °C. Data are: 1H

NMR (DMSO-d6, 300 MHz) δ 6.83 (d, J=3Hz, 2H), 6.75 (d, J=9Hz, 2H), 6.65 (dd, J1=4.5 Hz,

J2=3Hz, 2H), 3.82 (s, 6H), 3.09 (d, J=3Hz, 4H), 2.79 (t, J=9Hz, 4H), 2.60, (t, J=3Hz, 2H), 2.40 (t,

J=9 Hz, 4H), 1.41 (bs, 4H); 13C NMR (DMSO-d6, 75 MHz) δ 172.7, 148.7, 146.1, 133.4, 121.6,

116.6, 113.7, 56.8, 39.5, 38.9, 32.2, 28.0; 1H NMR (1:1 CDCl3 : CD3OD, 300 MHz) δ 6.74 (d,

J=4.5Hz, 2H), 6.69 (d, J=1.5 Hz, 2H), 6.62 (dd, J1=4.5 Hz, J2=3Hz, 2H), 3.82 (s, 6H), 3.04 (t,

J=6Hz, 4H), 2.83 (t, J=6Hz, 4H), 2.43 (t, J=6 Hz, 4H), 1.22-1.18 (m, 4H); 13C NMR (1:1 CDCl3 :

CD3OD, 75 MHz) δ 175.0, 148.3, 145.4, 133.6, 122.1, 116.1, 113.0, 57.1, 40.3, 39.7, 32.8, 27.5.


Benzyl 3-[4-(benzyloxy)phenyl]propanoate (15b). Commercial molecule 15a6 (2.0 g, 12.04

mmol, 1.0 equivalent) and K2CO3 (6.6 g, 47.78 mmol, 3.97 equivalents) were dissolved in N,N-

dimethylformamide (DMF, 11.7 mL, 1.03 M) in a 250 mL round-bottom flask with a magnetic stir

bar at room temperature (RT). CAUTION (caustic smell hazard): Benzyl bromide (9.4 mL, 78.47

mmol, 6.52 equivalents) was then added dropwise at RT. The flask was then warmed to 80 °C

and heated for 4.0 hours. Once complete (TLC), the mixture was cooled to RT and quenched

with H2O (20 mL). The vessel was then cooled to 0 °C in an ice bath, and the mixture was

gradually brought to pH <4.0 by slow addition of 1N HCl. At this point, the suspension was

transferred to a separatory funnel, where the layers were mixed and separated. The aqueous

layer was then extracted with EtOAc (3×20 mL). The combined organic layers were washed

with saturated aqueous NaHCO3 (20 mL), brine (20 mL), dried over Na2SO4, concentrated, and

purified by column chromatography (8% EtOAc/hexanes). This procedure yielded 3.82 g (91%)

of the ester product as a light-yellow oil. Data are: TLC Rf = 0.40 (10% EtOAc/hexanes); 1H

NMR (CDCl3, 500 MHz) δ 7.37-7.34 (m, 10H), 7.14 (d, J=5Hz, 2H), 6.93 (d, J=5Hz, 2H), 5.15 (s,

2H), 5.07 (s, 2H), 2.96 (t, J=10Hz, 2H), 2.69 (t, J=10 Hz, 2H); 13C NMR (CDCl3, 125 MHz) δ

172.8, 157.4, 137.2, 136.0, 132.8, 129.3, 128.6, 128.5, 128.3, 128.2, 127.9, 127.5, 114.9, 70.1,

66.3, 36.2, 30.2; GCMS found 346 [M]+, calcd 346.16 for [C23H22O3].

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3-[4-(benzyloxy)phenyl]propanoic acid (15c). Molecule 15b (1.05 g, 3.03 mmol, 1.0

equivalent) was dissolved in H2O (5.6 mL, 0.54 M) and methanol (5.6 mL, 0.54 M) in a 250 mL

round-bottom flask with a magnetic stir bar at room temperature (RT). Potassium hydroxide

(323 mg, 5.76 mmol, 1.9 equivalents) and THF (0.37 mL, 2.7 M) were then added. The reaction

was warmed to 60 °C and stirred for 4.0 hours. Once complete (TLC), the mixture was cooled to

RT and quenched with H2O (20 mL). The vessel was then cooled to 0 °C in an ice bath, and the

suspension was gradually brought to pH <2.0 by slow addition of 1N HCl. At this point, the

mixture was transferred to a separatory funnel, where the layers were shaken and separated.

The aqueous layer was then extracted with EtOAc (5×40 mL), dried over Na2SO4, concentrated,

and purified by column chromatography (20% EtOAc/hexanes, followed by column flush with

100% methanol). This procedure yielded 750 mg (97%) of compound 15c as a white, crystalline

solid. Melting point was 113 – 115 °C. Data are: TLC R f = 0.2 (streak, 20% EtOAc/hexanes); 1H

NMR (DMSO-d6, 500 MHz) δ 12.18 (bs, 1H), 7.45-7.31 (m, 5H), 7.13 (d, J=5Hz, 2H), 6.91 (d,

J=5Hz, 2H), 5.06 (s, 2H), 2.75 (d, J=10 Hz, 2H), 2.48 (d, J=10 Hz, 2H); 13C NMR (DMSO-d6, 125

MHz) δ 174.3, 157.1, 137.2, 133.5, 129.7, 128.9, 128.2, 128.1, 115.1, 69.6, 36.1, 30.0; HRMS

found 274.1501 [M+NH4]+, calcd 256.11 for [C16H16O3].

3-[4-(benzyloxy)phenyl]tert-butyl (4-propanamidobutyl)carbamate (16). Molecule 15c (2.50

g, 9.75 mmol, 1.0 equivalent) was dissolved in CH2Cl2 (14.4 mL, 0.676 M) in a 50 mL round-

bottom flask with a magnetic stir bar at room temperature (RT) under N2 atmosphere. The

reaction mixture was then cooled to 0 °C. At this point, commercial N-BOC-1,4-butanediamine4

(2.05 mL, 10.73 mmol, 1.1 equivalents), EDCI5 (2.05 g, 10.73 mmol, 1.1 equivalents), and

triethylamine (1.42 mL, 10.73 mmol, 1.1 equivalents) were added. The reaction was then stirred

overnight (~22 hours), during which time it gradually warmed to RT. The reaction mixture was

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then diluted with H2O (20 mL) and transferred to a separatory funnel. The layers were

separated, and the aqueous layer was extracted with CH2Cl2 (5 × 25 mL). The combined

organic layer was then washed with brine (15 mL), dried over Na2SO4, concentrated, and

recrystallized overnight from warm 50% CH2Cl2/hexanes. Subsequent vacuum filtration yielded

4.16 g (quantitative yield) of compound 16 as a white solid. Melting point was 129.5–131.6 °C.

Data are: TLC Rf = 0.1 (60% EtOAc/hexanes); 1H NMR (DMSO-d6, 500 MHz) δ 7.41-7.24 (m,

5H), 7.09 (d, J=7.5 Hz, 2H), 6.88 (d, J=7.5, Hz, 2H), 5.01 (s, 2H), 3.30-3.28 (m, 2H), 3.09 (t,

J=6Hz, 2H), 2.97 (t, J=6Hz, 2H), 2.82 (t, J=9Hz, 2H), 2.40 (t, J=9Hz, 2H), 1.40 (s, 9H), 1.37-1.35

(m, 2H); 13C NMR (DMSO-d6, 125 MHz) δ 175.9, 173.9, 157.4, 137.5, 133.1, 129.0, 128.1,

127.5, 127.2, 114.6, 78.5, 69.6, 39.6, 38.6, 37.9, 30.9, 27.5, 26.9, 26.3; GCMS found 426 [M]+,

calcd 426.25 for [C25H34N2O4].

N-(4-aminobutyl)-3-[4-(benzyloxy)phenyl] propanamide (17). Carbamate 16 (365 mg, 0.856

mmol, 1.0 equivalent) was dissolved in toluene (15.6 mL, 0.055 M) in a 25 mL round-bottom

flask with a magnetic stir bar at room temperature (RT) under N2 atmosphere. Trifluoroacetic

acid (1.56 mL, 0.55 M) was then added, and the reaction was stirred at RT for 15-20 minutes.

The reaction mixture was then stirred and diluted with enough 2M aqueous NaOH to bring the

pH to >10.0. The resulting mixture was then transferred to a separatory funnel. The layers were

separated, and the aqueous layer was extracted exhaustively with CH2Cl2 (20 × 10 mL). The

combined organic layer was then dried over Na2SO4 and concentrated in a pre-weighed flask.

This procedure yielded 218 mg (78%) of compound 14 as a white solid. Melting point was 107.6

– 109.8 °C. Data are: TLC Rf = 0.13 (100% EtOAc); 1H NMR (DMSO-d6, 500 MHz) δ 7.42-7.28

(m, 5H), 7.10 (d, J=7.5 Hz, 2H), 6.89 (d, J=5Hz, 2H), 5.03 (s, 2H), 3.30 (bs, 2H), 3.15 (t,

J=10Hz, 2H), 2.91-2.81 (m, 4H), 2.43 (t, J=10Hz, 2H), 1.61-1.42 (m, 4H); 13C NMR (DMSO-d6,

125 MHz) δ 174.1, 157.4, 137.5, 133.1, 129.1, 128.2, 127.5, 127.2, 114.6, 69.7, 38.9, 38.0,

37.9, 30.8, 26.0, 24.4; GCMS found 326 [M]+, calcd 326.20 for [C20H26N2O2].

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N,N'-(butane-1,4-diyl)-bis-{3-[4-(benzyloxy)phenyl]}dipropanamide (18). Molecule 15c (156

mg, 0.61 mmol, 1.0 equivalent) was dissolved in CH2Cl2 (3.7 mL, 0.167 M) in a 25 mL round-


reaction mixture was then cooled to 0 °C. At this point, molecule 17 (219 mg, 0.67 mmol, 1.1

equivalents) that was pre-dissolved in CH2Cl2 (3.7 mL, 0.167 M) was added, followed by EDCI5

(129 mg, 0.67 mmol, 1.1 equivalents) and triethylamine (94 μL, 0.67 mmol, 1.1 equivalents).

The reaction was then stirred overnight (~20 hours), during which time it gradually warmed to

RT. The reaction mixture was now diluted with H2O (20 mL) and transferred to a separatory

funnel. The layers were separated, and the aqueous layer was extracted with CH2Cl2 (5 × 25

mL). The combined organic layer was then washed with brine (15 mL), dried over MgSO4,

concentrated, and purified by column chromatography (50% EtOAc/hexanes, followed by 100%

MeOH flush). This procedure yielded 183 mg (53%) of compound 18 as a white solid. Melting

point was 182 – 187.6 °C. Data are: TLC Rf = 0.0 (baseline spot, 50% EtOAc/hexanes); 1H NMR

(CD3OD, 500 MHz) δ 7.44-7.31 (m, 10H), 7.09 (d, J=2.5 Hz, 4H), 6.91 (t, J=10 Hz, 4H), 5.05 (s,

4H), 3.05-2.99 (m, 8H), 2.78-2.71 (m, 5H), 2.43 (bs, 4H), 2.33-2.29 (m, 4H); 13C NMR (CD3OD,

125 MHz) δ 167.5, 149.5, 138.7, 131.2, 126.6, 122.7, 121.9, 120.9, 118.7, 63.3, 39.9, 36.8,

31.9, 29.2; GCMS found 564 [M]+, calcd 564.30 for [C36H40N2O4].

N,N'-(butane-1,4-diyl)-bis-{3-[4-(hydroxy)phenyl]}dipropanamide (3). Molecule 18 (34 mg,

0.059 mmol, 1.0 equivalent) was dissolved in ethanol (0.59 mL, 0.1 M) in a 25 mL round-bottom

flask with a magnetic stir bar at room temperature (RT). The reaction vessel was then topped

with an H2 balloon, purged with H2 gas (3x), and stirred at RT under H2 balloon pressure for 30

minutes. The reaction slurry was now filtered through florisil using copious amounts of EtOAc

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and methanol, and the product was concentrated. This procedure yielded 22 mg (97%) of

product 3 as a colorless solid. Melting undetermined, due to small product amount. Data are: 1H

NMR (CD3OD, 500 MHz) δ 7.01 (d, J=5 Hz, 4H), 6.79 (d, J=5Hz, 4H), 3.05-2.92 (m, 5H), 2.77-

2.74 (m, 4H), 2.33 (t, J=5Hz, 4H), 1.40 (t, J=5Hz, 2H); 13C NMR (CD3OD, 125 MHz) δ 165.3,

136.5, 120.4, 119.6, 118.7, 37.6, 34.6, 29.6, 27.0; HRMS found 384.2056 [M]+, calcd 384.20 for

[C22H28N2O4].


3-phenyl-tert-butyl (4-propanamidobutyl)carbamate (20). Hydrocinnamic acid 197 (400 mg,

2.66 mmol, 1.0 equivalent) was dissolved in CH2Cl2 (3.93 mL, 0.676 M) in a 25 mL round-


reaction mixture was then cooled to 0 °C. At this point, commercial N-BOC-1,4-butanediamine4

(0.56 mL, 2.93 mmol, 1.1 equivalents), EDCI5 (561 mg, 2.93 mmol, 1.1 equivalents), and

triethylamine (0.39 mL, 2.93 mmol, 1.1 equivalents) were added. The reaction was then stirred

overnight (~24 hours), during which time it gradually warmed to RT. The reaction mixture was

now diluted with H2O (20 mL) and transferred to a separatory funnel. The layers were

separated, and the aqueous layer was extracted with CH2Cl2 (5 × 25 mL). The combined

organic layer was then washed with brine (15 mL), dried over Na2SO4, concentrated, and

purified by column chromatography (50% EtOAc/hexanes, followed by 100% EtOAc and 100%

methanol flushes). This procedure yielded 850 mg (quantitative yield) of compound 20 as a

white solid. Melting point was 98.0 – 98.9 °C. Data are: TLC Rf = 0.2 (streak, 50%

EtOAc/hexanes); 1H NMR (CD3OD, 500 MHz) δ 7.27-7.11 (m, 5H), 3.10 (t, J=10 Hz, 2H), 3.01-

2.95 (m, 2H), 2.88 (t, J=15 Hz, 2H), 2.44 (t, J=15 Hz, 2H), 1.41 (s, 9H), 1.38-1.32 (m, 4H); 13C

NMR (CD3OD, 125 MHz) δ 173.8, 157.2, 140.8, 128.1, 125.9, 78.5, 39.8, 39.6, 38.7, 37.7, 31.7,

27.5, 26.9, 26.3; GCMS found 320 [M]+, calcd 320.21 for [C18H28N2O3].

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N-(4-aminobutyl)-3-phenyl propanamide (21). Carbamate 20 (428 mg, 1.34 mmol, 1.0

equivalent) was dissolved in toluene (24 mL, 0.055 M) in a 50 mL round-bottom flask with a

magnetic stir bar at room temperature (RT) under N2 atmosphere. Trifluoroacetic acid (2.43 mL,

0.55 M) was then added, and the reaction was stirred at RT for 15-20 minutes. The reaction

mixture was then stirred and diluted with enough 2M aqueous NaOH to bring the pH to >10.0.

The mixture was now transferred to a separatory funnel. The layers were separated, and the

aqueous layer was extracted exhaustively with CH2Cl2 (20 × 10 mL). The combined organic

layer was now dried over Na2SO4 and concentrated in a pre-weighed flask. This procedure

yielded 376 mg (127% crude yield) of impure compound 21 as a colorless solid, which was

taken on to the next step without purification. TLC R f = 0.41 (60% EtOAc/hexanes); 1H NMR

(CD3OD, 500 MHz) δ 7.26-7.11 (m, 5H), 3.14 (t, J=15Hz, 2H), 2.88 (t, J=10Hz, 4H), 2.46 (t,

J=10Hz, 2H), 1.61-1.42 (m, 4H); 13C NMR (CDCl3, 125 MHz) δ 174.0, 140.8, 128.2, 128.1,

125.9, 39.0, 38.1, 37.6, 31.6, 25.9, 24.4; HRMS found 220.1613 [M]+, calcd 220.16 for

[C13H20N2O].

N,N'-(butane-1,4-diyl)-bis-phenyldipropanamide (4). Hydrocinnamic acid 197 (108 mg, 0.722

mmol, 1.0 equivalent) was dissolved in CH2Cl2 (4.3 mL, 0.167 M) in a 25 mL round-bottom flask

with a magnetic stir bar at room temperature (RT) under N2 atmosphere. The reaction mixture

was then cooled to 0 °C. At this point, crude product 21 (175 mg, 0.79 mmol, 1.1 equivalents)

that was pre-dissolved in CH2Cl2 (4.3 mL, 0.167 M) was added, followed by EDCI5 (152 mg,

0.79 mmol, 1.1 equivalents) and triethylamine (110 μL, 0.79 mmol, 1.1 equivalents). The

reaction was then stirred overnight (~24 hours), during which time it gradually warmed to RT.

The reaction mixture was now diluted with H2O (20 mL) and transferred to a separatory funnel.

The layers were separated, and the aqueous layer was extracted with CH2Cl2 (5 × 25 mL). The

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combined organic layer was then washed with brine (15 mL), dried over Na2SO4, and

concentrated, with no further purification. This procedure yielded 198 mg (99% over two steps

from 20) of compound 4 as a white solid. Melting point was not obtained. Data are: TLC R f = 0.9

(60% EtOAc/hexanes); 1H NMR (CDCl3, 500 MHz) δ 7.28-7.13 (m, 5H), 3.06 (t, J=10Hz, 2H),

2.92-2.83 (m, 4H), 2.60 (t, J=10Hz, 2H), 2.44 (t, J=15Hz, 4H), 1.33-1.25 (m, 2H); 13C NMR

(CDCl3, 125 MHz) δ 173.8, 140.8, 128.11, 128.0, 125.9, 125.0, 38.6, 37.7, 31.7, 26.2; GCMS

found 352 [M]+, calcd 352.22 for [C22H28N2O2].

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1.3.6.[1.2.6.] Spectra

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2. References

1. Fumeaux, R.; Menozzi-Smarrito, C.; Stalmach, A.; Munari, C.; Kraehenbuehl, K.; Steiling, H.; Crozier,A.; Williamson, G.;

Barron, D. Org. Bioomolec. Chem. 2010, 8, 5199-5211.

2. Boschi, D.; Tron, G. C.; Lazzarato, L.; Chegaev, K.; Cena, C.; Di Stilo, A.; Giorgis, M.; Bertinaria, M.; Fruttero, R.; Gasco, A.

J. Med. Chem. 2006, 49, 2886-2897.

3. Leschot, A.; Tapia, R. A.; Eyzaguirre, J. Synth. Commun. 2002, 32, 3219-3223.4. N-BOC-1,4-butanediamine, 95%, available from Fischer Scientific, catalog #AC426190050.

5. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, available from Fischer Scientific, catalog #AC171440100.

6. 3-(4-Hydroxyphenyl)propionic acid, available from Fischer Scientific, catalog #AAA1456718.

7. Hydrocinnamic acid, available from Fischer Scientific, catalog #AC15910-1000.

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ars.els-cdn.com · web viewwere evaluated by colorometric assay against a549 (ccl-185) (human small...

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