A novel synthetic route to 1-oxo-5-hydroxytryptamine, the benzofuran analogue of serotonin, has been developed. The new synthesis proceeds via the [3+2] cycloaddition of p-benzoquinone and 2,3-dihydrofuran, followed by a Lewis acid-catalyzed isomerization. This molecule proves to be a competent agonist (equipotent to serotonin) of the 5-HT_3 receptor, demonstrating that the indolic proton of serotonin is not essential to its activation of the receptor

Bower, Kiowa S.

Dougherty, Dennis A.

Kedrowski, Sean M. A.

Caltech Authors - Main

1
Supporting Information for:
1-Oxo-5-hydroxytryptamine: A Surprisingly Potent 
Agonist of the 5-HT3 (Serotonin) Receptor
Sean M. A. Kedrowski, Kiowa S. Bower, and Dennis A. Dougherty
Division of Chemistry and Chemical Engineering, California Institute of Technology, 
Pasadena, California 91125
e-mail: dadougherty@caltech.edu
Received Date (will be automatically inserted after manuscript is accepted)
2
Table of Contents
Topic Page
Chemical Synthesis
Molecular Biology and Electrophysiology
NMR Data
References
3
5
6
10
3
Chemistry—General.
All reactions were performed under Argon using solvents that were dried and purified according 
to the method of Grubbs.1 All flasks and vials were oven dried at 122°C and cooled in a 
desiccator box containing anhydrous calcium sulfate. Commercial reagents were purified 
according to the methods compiled by Armengo and Perrin.2 Hydrogen chloride gas was 
generated in situ by dropwise addition of concentrated HCl into stirred concentrated H2SO4.  
Reactions were monitored by thin-layer chromatography on Merck Siligel 60-F254. Compounds 
were visualized with UV lamp (254 nm) and stained with ceric ammonium molybdate solution.  
Column chromatography was carried out in accordance with the methods of Still3 using EMD-
Merck silica gel 60, 230-400 mesh ASTM or Aldrich Brockmann I alumina, 150 mesh (for acid 
sensitive compounds such as pyrroline). 1H and 13C NMR spectra were acquired on a Varian 
Mercury 300 MHz spectrometer. High-resolution mass spectrometry was performed on a JEOL 
JMS-600H HRMS using an Electrospray Ion Source.
Chemistry—Synthesis.
(6). Boron trifluoride-diethyl etherate (0.6 mL, 5 mmol) was added to a mixture of acetonitrile 
(10 mL) and dichloromethane (30 mL) in a 100 mL roundbottomed flask and cooled to -78 °C.  
A solution of p-benzoquinone (540 mg, 5 mmol) and 2,3-dihydrofuran (0.6 mL, 7.5 mmol) in 
acetonitrile (10 ml) was then added to the stirred BF3 solution at -78 °C by syringe pump over 2 
h.  After 5 total hours, additional 2,3-dihydrofuran (0.6 mL, 7.5 mmol) was added dropwise over 
10 minutes.  After 8 total hours, The reaction temperature was allowed to slowly rise to room 
temperature over the following hour, at which point methanol (10 mL) and 1 N HCl (10 mL) 
were added. The product was extracted with 4 x 30 mL dichloromethane, washed with 1 x 30 mL 
brine, dried on magnesium sulfate, and reduced iv. The residue was filtered through a silica plug 
with ethyl acetate, then purified by flash chromatography on silica (25% EtOAc in hexanes). The 
product fraction could be purified further by recrystallization from 3 mL toluene to yield flaky, 
white crystals; 160 mg (18%) 6. 1H NMR (300 MHz, CDCl3) δ 6.59-6.70 (m, 3H), 6.27 (d, 1H, J
= 6 Hz), 4.66 (s, 1H, OH), 4.05 (t, 1H, J = 8 Hz), 3.95 (t, 1H, J = 7 Hz), 3.58-3.64 (m, 1H), 2.22-
2.29 (m, 1H), 2.03 (dd, 1H, J = 12, 4.5 Hz); 13C NMR (75 MHz, CDCl3) δ 153.6, 150.5, 128.9, 
115.5, 112.1, 111.3, 109.6, 67.5, 47.2, 33.6; HRMS (EI+) calculated for [C10H10O3] ([M]
+) 
178.0630, found 178.0638.  The characterization data matched reported values for this 
compound synthesized using the Corey oxazaborolidine catalyst.4
(8).  6 (89 mg, 0.50 mmol) was measured into a 2-dram vial, and acetonitrile (5 mL) was added 
to dissolve it.  Boron trifluoride-diethyl etherate (140 µL, 1.1 mmol) was then added.  The vial 
was then shaken at 37°C for 22 h.  Aqueous HCl (4 mL, 1 N) was then added, and the product 
was extracted with 5 x 8 mL dichloromethane, dried on sodium sulfate, and reduced iv.  The 
residue was then purified by flash chromatography on silica (50% EtOAc in hexanes) to yield 
74.1 mg (83%) 8. 1H NMR (300 MHz, acetone-d6) δ 8.03 (s, 1H), 7.58 (s, 1H), 7.27 (d, 1H, J = 9 
Hz), 7.01 (d, 1H, J = 3 Hz), 6.81 (dd, 1H, J = 9, 3 Hz), 3.99 (s, 1H), 3.82 (t, 2H, J = 6 Hz), 2.82 
(t, 2H, J = 6 Hz); 13C NMR (75 MHz, acetone-d6) δ 153.9, 150.0, 143.8, 129.8, 118.0, 113.5, 
112.0, 104.9, 61.6, 27.5; HRMS (EI+) calculated for [C10H10O3] ([M]
+) 178.0630, found 
178.0628.
4
(10). 8 (193 mg, 1.1 mmol), triphenylphosphine (341 mg, 1.3 mmol), and N-Boc-o-
nitrobenzenesulfonamide5 (393 mg, 1.3 mmol) were measured into a 2-dram vial.  THF (5 mL) 
was added.  Diisopropyl azodicarboxylate (256 µL, 1.3 mmol) was added to the stirred solution 
by a syringe over a 10 minute period.  After an additional 20 minutes stirring at room 
temperature, the reaction was reduced iv, and the residue was triturated with toluene (5 mL) to 
give 372.7 mg of a white solid.  Additional product could be recovered by flash chromatography 
of the filtrate on silica (30% EtOAc in hexanes), followed by trituration of the product fraction to 
give a total yield of 483.0 mg (96 %) 10. 1H NMR. (300 MHz, acetone-d6) δ 8.28-8.31 (m, 1H), 
7.95-8.02 (m, 3H), 7.70 (s, 1H), 7.32 (d, 1H, J = 9 Hz), 7.14 (d, 1H, J = 2 Hz), 6.86 (dd, 1H, J = 
9, 2 Hz), 4.03 (t, 2H, 7 Hz), 3.08 (t, 2H, 7 Hz), 1.27 (s, 9H); 13C NMR (75 MHz, acetone-d6) δ
153.7, 150.4, 150.0, 143.6, 135.3, 133.0, 132.6, 132.5, 128.9, 124.9, 116.6, 113.4, 111.7, 104.5, 
85.0, 47.2, 27.1, 21.6; HRMS (EI+) calculated for [C21H22N2O8S] ([M]
+) 462.1097, found 
462.1100.
1-oxa-5-hydroxytryptamine hydrochloride (2).  10 (445 mg, 0.96 mmol) was measured into a 
25 mL round-bottomed flask.  DMF (9.6 mL), followed by lithium hydroxide monohydrate (162 
mg, 3.85 mmol) and mercaptoacetic acid (134 µL, 1.93 mmol) were then added.  After 6 h room 
temperature stirring, the reaction was poured onto 60 mL aqueous sodium bicarbonate, and the 
product was extracted with 5 x 20mL EtOAc, washed with 1 x 20 mL brine, reduced iv, and 
filtered through a silica plug (30% EtOAc in hexanes) to yield 247 mg, which was transferred 
into a 50 mL 3-neck flask appended with an HCl purge.  Chloroform (20 mL) and methanol (2 
mL) were added, and HCl was purged through the stirring solution for 30 minutes.  The reaction 
was then reduced iv, and the residue was redissolved in acetone (5 mL).  This acetone solution 
was then added dropwise to a stirring beaker of ether (20 mL); the resultant white crystals were 
then isolated by filtration to yield 130 mg (64%, 2 steps) 1-oxa-5-hydroxytryptamine 
hydrochloride. 1H NMR (300 MHz, DMSO-d6) δ 9.31 (s, 1H, OH), 8.13 (s, 3H, NH), 7.78 (s, 
1H), 7.34 (d, 1H, J = 8 Hz), 6.95 (d, 1H, J = 2 Hz), 6.78 (dd, 1H, J = 9, 2 Hz), 3.06 (t, 2H, J = 8 
Hz), 2.91 (t, 2H, J = 8 Hz); 13C NMR (75 MHz, DMSO-d6) δ 154.0, 149.4, 144.1, 128.7, 116.1, 
113.9, 112.3, 104.7, 38.7, 22.0; HRMS (EI+) calculated for [C10H11NO2] ([M]
+) 177.0790, found 
177.0794.
5
Molecular Biology and Electrophysiology
The measurement of EC50 and efficacy values for 5-HT, 1-OT and 5-FT was carried out as 
previously described.6  Briefly, the 5-HT3 receptor was expressed in Xenopus laevis oocytes by 
manual injection of the corresponding mRNA.  Following 1-3 days incubation at 18 °C, 
transmembrane current in response to drug application was measured using a two-electrode 
whole-cell voltage clamp.  From these data a dose-response curve was created for each drug and 
was fit to the Hill equation.
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6
NMR Data
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O O
HO
6
7
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O
HO
8
OH
8
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O
HO
10
N
S
O
O
NO2
O
O
9
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O
HO
NH3 Cl
2
10
References
(1) Pangborn, A. B.; Giardello, M. A.; Grubbs, R. H.; Rosen, R. K.; Timmers, F. J. 
Organometallics 1996, 15, 1518-1520.
(2) Hallmann, G.; Hagele, K. Annalen Der Chemie-Justus Liebig 1963, 662, 147-&.
(3) Still, W. C.; Kahn, M.; Mitra, a. Journal of Organic Chemistry 1978, 43, 2923-2925.
(4) Zhou, G.; Corey, E. J. Journal of the American Chemical Society 2005, 127, 11958-
11959.
(5) Kan, T.; Fukuyama, T. Chemical Communications 2004, 353-359.
(6) Beene, D. L.; Price, K. L.; Lester, H. A.; Dougherty, D. A.; Lummis, S. C. R. Journal of 
Neuroscience 2004, 24, 9097-9104.


English

1-Oxo-5-hydroxytryptamine: A Surprisingly Potent Agonist of the 5-HT_3(Serotonin) Receptor

1Supporting Information for:
1-Oxo-5-hydroxytryptamine: A Surprisingly Potent 
Agonist of the 5-HT3 (Serotonin) Receptor
Sean M. A. Kedrowski, Kiowa S. Bower, and Dennis A. Dougherty
Division of Chemistry and Chemical Engineering, California Institute of Technology, 
Pasadena, California 91125
e-mail: dadougherty@caltech.edu
Received Date (will be automatically inserted after manuscript is accepted)
2Table of Contents
Topic Page
Chemical Synthesis
Molecular Biology and Electrophysiology
NMR Data
References
3
5
6
10
3Chemistry—General.
All reactions were performed under Argon using solvents that were dried and purified according 
to the method of Grubbs.1 All flasks and vials were oven dried at 122°C and cooled in a 
desiccator box containing anhydrous calcium sulfate. Commercial reagents were purified 
according to the methods compiled by Armengo and Perrin.2 Hydrogen chloride gas was 
generated in situ by dropwise addition of concentrated HCl into stirred concentrated H2SO4.  
Reactions were monitored by thin-layer chromatography on Merck Siligel 60-F254. Compounds 
were visualized with UV lamp (254 nm) and stained with ceric ammonium molybdate solution.  
Column chromatography was carried out in accordance with the methods of Still3 using EMD-
Merck silica gel 60, 230-400 mesh ASTM or Aldrich Brockmann I alumina, 150 mesh (for acid 
sensitive compounds such as pyrroline). 1H and 13C NMR spectra were acquired on a Varian 
Mercury 300 MHz spectrometer. High-resolution mass spectrometry was performed on a JEOL 
JMS-600H HRMS using an Electrospray Ion Source.
Chemistry—Synthesis.
(6). Boron trifluoride-diethyl etherate (0.6 mL, 5 mmol) was added to a mixture of acetonitrile 
(10 mL) and dichloromethane (30 mL) in a 100 mL roundbottomed flask and cooled to -78 °C.  
A solution of p-benzoquinone (540 mg, 5 mmol) and 2,3-dihydrofuran (0.6 mL, 7.5 mmol) in 
acetonitrile (10 ml) was then added to the stirred BF3 solution at -78 °C by syringe pump over 2 
h.  After 5 total hours, additional 2,3-dihydrofuran (0.6 mL, 7.5 mmol) was added dropwise over 
10 minutes.  After 8 total hours, The reaction temperature was allowed to slowly rise to room 
temperature over the following hour, at which point methanol (10 mL) and 1 N HCl (10 mL) 
were added. The product was extracted with 4 x 30 mL dichloromethane, washed with 1 x 30 mL 
brine, dried on magnesium sulfate, and reduced iv. The residue was filtered through a silica plug 
with ethyl acetate, then purified by flash chromatography on silica (25% EtOAc in hexanes). The 
product fraction could be purified further by recrystallization from 3 mL toluene to yield flaky, 
white crystals; 160 mg (18%) 6. 1H NMR (300 MHz, CDCl3)  6.59-6.70 (m, 3H), 6.27 (d, 1H, J
= 6 Hz), 4.66 (s, 1H, OH), 4.05 (t, 1H, J = 8 Hz), 3.95 (t, 1H, J = 7 Hz), 3.58-3.64 (m, 1H), 2.22-
2.29 (m, 1H), 2.03 (dd, 1H, J = 12, 4.5 Hz); 13C NMR (75 MHz, CDCl3)  153.6, 150.5, 128.9, 
115.5, 112.1, 111.3, 109.6, 67.5, 47.2, 33.6; HRMS (EI+) calculated for [C10H10O3] ([M]+) 
178.0630, found 178.0638.  The characterization data matched reported values for this 
compound synthesized using the Corey oxazaborolidine catalyst.4
(8).  6 (89 mg, 0.50 mmol) was measured into a 2-dram vial, and acetonitrile (5 mL) was added 
to dissolve it.  Boron trifluoride-diethyl etherate (140 µL, 1.1 mmol) was then added.  The vial 
was then shaken at 37°C for 22 h.  Aqueous HCl (4 mL, 1 N) was then added, and the product 
was extracted with 5 x 8 mL dichloromethane, dried on sodium sulfate, and reduced iv.  The 
residue was then purified by flash chromatography on silica (50% EtOAc in hexanes) to yield 
74.1 mg (83%) 8. 1H NMR (300 MHz, acetone-d6)  8.03 (s, 1H), 7.58 (s, 1H), 7.27 (d, 1H, J = 9 
Hz), 7.01 (d, 1H, J = 3 Hz), 6.81 (dd, 1H, J = 9, 3 Hz), 3.99 (s, 1H), 3.82 (t, 2H, J = 6 Hz), 2.82 
(t, 2H, J = 6 Hz); 13C NMR (75 MHz, acetone-d6)  153.9, 150.0, 143.8, 129.8, 118.0, 113.5, 
112.0, 104.9, 61.6, 27.5; HRMS (EI+) calculated for [C10H10O3] ([M]+) 178.0630, found 
178.0628.
4(10). 8 (193 mg, 1.1 mmol), triphenylphosphine (341 mg, 1.3 mmol), and N-Boc-o-
nitrobenzenesulfonamide5 (393 mg, 1.3 mmol) were measured into a 2-dram vial.  THF (5 mL) 
was added.  Diisopropyl azodicarboxylate (256 µL, 1.3 mmol) was added to the stirred solution 
by a syringe over a 10 minute period.  After an additional 20 minutes stirring at room 
temperature, the reaction was reduced iv, and the residue was triturated with toluene (5 mL) to 
give 372.7 mg of a white solid.  Additional product could be recovered by flash chromatography 
of the filtrate on silica (30% EtOAc in hexanes), followed by trituration of the product fraction to 
give a total yield of 483.0 mg (96 %) 10. 1H NMR. (300 MHz, acetone-d6)  8.28-8.31 (m, 1H), 
7.95-8.02 (m, 3H), 7.70 (s, 1H), 7.32 (d, 1H, J = 9 Hz), 7.14 (d, 1H, J = 2 Hz), 6.86 (dd, 1H, J = 
9, 2 Hz), 4.03 (t, 2H, 7 Hz), 3.08 (t, 2H, 7 Hz), 1.27 (s, 9H); 13C NMR (75 MHz, acetone-d6) 
153.7, 150.4, 150.0, 143.6, 135.3, 133.0, 132.6, 132.5, 128.9, 124.9, 116.6, 113.4, 111.7, 104.5, 
85.0, 47.2, 27.1, 21.6; HRMS (EI+) calculated for [C21H22N2O8S] ([M]+) 462.1097, found 
462.1100.
1-oxa-5-hydroxytryptamine hydrochloride (2).  10 (445 mg, 0.96 mmol) was measured into a 
25 mL round-bottomed flask.  DMF (9.6 mL), followed by lithium hydroxide monohydrate (162 
mg, 3.85 mmol) and mercaptoacetic acid (134 µL, 1.93 mmol) were then added.  After 6 h room 
temperature stirring, the reaction was poured onto 60 mL aqueous sodium bicarbonate, and the 
product was extracted with 5 x 20mL EtOAc, washed with 1 x 20 mL brine, reduced iv, and 
filtered through a silica plug (30% EtOAc in hexanes) to yield 247 mg, which was transferred 
into a 50 mL 3-neck flask appended with an HCl purge.  Chloroform (20 mL) and methanol (2 
mL) were added, and HCl was purged through the stirring solution for 30 minutes.  The reaction 
was then reduced iv, and the residue was redissolved in acetone (5 mL).  This acetone solution 
was then added dropwise to a stirring beaker of ether (20 mL); the resultant white crystals were 
then isolated by filtration to yield 130 mg (64%, 2 steps) 1-oxa-5-hydroxytryptamine 
hydrochloride. 1H NMR (300 MHz, DMSO-d6)  9.31 (s, 1H, OH), 8.13 (s, 3H, NH), 7.78 (s, 
1H), 7.34 (d, 1H, J = 8 Hz), 6.95 (d, 1H, J = 2 Hz), 6.78 (dd, 1H, J = 9, 2 Hz), 3.06 (t, 2H, J = 8 
Hz), 2.91 (t, 2H, J = 8 Hz); 13C NMR (75 MHz, DMSO-d6)  154.0, 149.4, 144.1, 128.7, 116.1, 
113.9, 112.3, 104.7, 38.7, 22.0; HRMS (EI+) calculated for [C10H11NO2] ([M]+) 177.0790, found 
177.0794.
5Molecular Biology and Electrophysiology
The measurement of EC50 and efficacy values for 5-HT, 1-OT and 5-FT was carried out as 
previously described.6  Briefly, the 5-HT3 receptor was expressed in Xenopus laevis oocytes by 
manual injection of the corresponding mRNA.  Following 1-3 days incubation at 18 °C, 
transmembrane current in response to drug application was measured using a two-electrode 
whole-cell voltage clamp.  From these data a dose-response curve was created for each drug and 
was fit to the Hill equation.
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6NMR Data
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O O
HO
6
7QuickTime™ and a
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O
HO
8
OH
8QuickTime™ and a
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O
HO
10
N
S
O
O
NO2
O
O
9QuickTime™ and a
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are needed to see this picture.
O
HO
NH3 Cl
2
10
References
(1) Pangborn, A. B.; Giardello, M. A.; Grubbs, R. H.; Rosen, R. K.; Timmers, F. J. 
Organometallics 1996, 15, 1518-1520.
(2) Hallmann, G.; Hagele, K. Annalen Der Chemie-Justus Liebig 1963, 662, 147-&.
(3) Still, W. C.; Kahn, M.; Mitra, a. Journal of Organic Chemistry 1978, 43, 2923-2925.
(4) Zhou, G.; Corey, E. J. Journal of the American Chemical Society 2005, 127, 11958-
11959.
(5) Kan, T.; Fukuyama, T. Chemical Communications 2004, 353-359.
(6) Beene, D. L.; Price, K. L.; Lester, H. A.; Dougherty, D. A.; Lummis, S. C. R. Journal of 
Neuroscience 2004, 24, 9097-9104.


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1-Oxo-5-hydroxytryptamine: A Surprisingly Potent Agonist of the 5-HT_3(Serotonin) Receptor

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