Syntheses of [3H2]T0901317 and a labeled structural isomer, and characterization of the dispersed labeled compounds via 19F NMR

Article abstract of DOI:10.1002/jlcr.3138

The synthesis and characterization of [³H₂]T0901317 and a structural isomer are described. The structural assignments of the closely related labeled compounds were primarily accomplished via ¹⁹F NMR analyses of the corresponding ethanolic compound dispersions. The synthesis and characterization of [³H₂]T0901317 and a structural isomer are described. The structural assignments of the closely related labeled compounds were primarily accomplished via ¹⁹F NMR analyses of the corresponding ethanolic compound dispersions. Copyright

Full text of DOI:10.1002/jlcr.3138

Note
Received 28 August 2013,
Revised 25 September 2013,
Accepted 11 October 2013
Published online 26 November 2013 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3138
3
Syntheses of [ H2]T0901317 and a labeled
structural isomer, and characterization of the
19
dispersed labeled compounds via F NMR
a
a
c
*
Benjamin P. Fauber, Adam R. Johnson, Stephen Bowerman,
Brenda Burton,b Adam Colebrook,c Annerley Flynn,c Gareth Harrold,c
Steve Huhn,a Gary Jones,c Peter Lockey,b Maxine Norman,b
Olivier René,a and Harvey Wonga
The synthesis and characterization of [3H2]T0901317 and a structural isomer are described. The structural assignments of the
closely related labeled compounds were primarily accomplished via 19F NMR analyses of the corresponding ethanolic
compound dispersions.
Keywords: T0901317; tritium; 19F NMR; LXR; PXR; RORγ; RORc
otherwise specified, all reagents and solvents were used as is from
commercial sources.
Introduction
The benzenesulfonamide compound T0901317 (Figure 1,
Compound 1) was initially disclosed by a team at Tularik (now
Amgen) as a potent liver X receptor (LXR) agonist.1 [3H]
T0901317 (specific activity = 27 Ci/mmol, Amersham) has since
been used to probe the binding of small-molecule ligands to
the nuclear receptors LXR,2 pregnane X receptor,3 and retinoic
acid receptor-related orphan receptor gamma.4 Although [3H]
T0901317 has been reported as an assay tool, to our knowledge,
the synthesis and characterization of this radioligand have not
been disclosed.
The scintillation proximity assay (SPA) format was used to detect
binding of the synthesized radioligands 7 and 8 to the nuclear receptor
LXRα. Incubations were carried out using 50 nM N-terminal GST-tagged
LXRα ligand binding domain (Life Technologies, PV4657) and 25 nM
radioligand. Yttrium silicate polylysine-coated SPA beads (PerkinElmer,
RPNQ0010) were used at 0.5 mg/well in 96-well format and a reaction
volume of 50 μL. The incubation buffer was as described,5 except we
used a pH of 7.4. Total binding (TB) was determined as the signal in
the presence of 50 nM LXRα receptor and 25 nM radioligand. Nonspecific
binding was determined in incubations of 50 nM LXRα, 25 nM
radioligand, and 3 μM unlabeled T0901317 (Sigma-Aldrich, T2320). All
It is well recognized that increasing the specific activity of a SPA binding mixtures were incubated at room temperature, and the
SPA signal (cpm) was measured after 10, 30, 60, 120, 180, and 240 min
using a MicroBeta plate reader (PerkinElmer). Specific binding (SB) was
calculated by subtracting the signal for nonspecific binding from the
signal for TB, while the percent SB (%SB) was calculated using the
equation: %SB = [(SB/TB) × 100].
radioligand can improve the signal-to-noise ratio when the
ligand is used in a radiometric assay. With such a consideration
in mind, we sought to synthesize [3H2]T0901317 as a radioligand
for use in nuclear receptor radiometric binding assays.
Experimental
2,4-dibromo-N-(4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-
2-yl)phenyl)benzenesulfonamide (4)
Reaction mixtures were analyzed for consumption of starting material on
an Agilent 1100 series LCMS using
a linear gradient of 0.1%
2-(4-aminophenyl)-1,1,1,3,3,3-hexafluoro-propan-2-ol (2) (1.5g, 5.8mmol),
2,6-lutidine (1.0mL, 8.7mmol), acetone (29 mL), and 2,4-dibromoben-
zenesulfonyl chloride (3) (2.1 g, 6.4mmol) were combined and stirred at
trifluoroacetic acid in water and acetonitrile mobile phases and a Waters
X-bridge C18 column (2.5 μm, 3 × 30 mm). 1H, 3H, and 19F NMR spectra
were recorded in DMSO-d6 solutions using a Bruker AV-400 spectrometer
or a Bruker AV-300 spectrometer with tetramethylsilane, or residual
solvent, as an internal reference for the 1H NMR spectra and
trifluoroacetic acid as an internal reference for the 19F NMR spectra
(À76.6 ppm). The 3H NMR spectrum was referenced to the corresponding
1H NMR proton frequency of the para-hydrogen on the sulfonamide
aromatic ring. High-resolution mass spectra were obtained on a Kratos
Concept IIH using positive or negative electrospray ionization. Flash column
aGenentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA
bArgenta, Units 7-9 Spire Green Centre, Flex Meadow, Harlow, Essex CM19 5TR, UK
cQuotient Bioresearch, The Old Glassworks, Nettlefold Road, Cardiff, Wales, CF24
5JQ, UK
chromatography purification was performed using
Teledyne ISCO instrument with SiliCycle SiliaSepHP (15–45 μm mesh size)
or ISCO RediSepRf Gold (20–40 μm mesh size) silica gel cartridges. Unless
a CombiFlashRf
*Correspondence to: Benjamin P. Fauber, Department of Discovery Chemistry,
Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
E-mail: fauber.benjamin@gene.com
J. Label Compd. Radiopharm 2014, 57 57–60
Copyright © 2013 John Wiley & Sons, Ltd.

B. P. Fauber et al.
was charged with 10% palladium on carbon (15 mg), and the resultant
black mixture was stirred for 3 h at 23°C under an atmosphere of tritium
gas (2 Ci, 1 atm). The reaction was then filtered, and the labile tritium
was removed under consecutive rotary evaporations from methanol
(3 × 5 mL). The crude material was purified by preparatory HPLC
(Hichrom Ltd. Ultrasphere C18 ODS 25 × 1 cm column, 5 μm particle size)
using a 60–100% gradient of water/acetonitrile (3 mL/min flow rate)
over a 60 min period (230 nm UV detection). The relevant fractions were
pooled and dried via rotary evaporation to provide the title compound
(7). The resulting product was re-constituted in ethanol (1 mCi/mL).
The radiochemical purity (95.0%) was determined by analytical HPLC
Figure 1. Structure of T0901317.
23°C. The reaction was heated at reflux for 36 h. The reaction was (Vydac Genesis C8 150 × 4.6 mm column, 5 μm particle size) using a
cooled to 23°C and quenched with EtOAc (30 mL) and 10% (w/v) 40–90% gradient of 20 mM aqueous ammonium acetate and acetonitrile
aqueous KH2PO4 (2× 30mL). The organic fraction was washed with brine (1 mL/min flow rate) over a 15 min period, and held for an additional
(30 mL), dried over Na2SO4, filtered, and concentrated under reduced 5 min (254 nm UV detection). Specific activity was determined by mass
pressure. The crude material was triturated with heptane, and the spectroscopy (54 Ci/mmol, ESI (m/z): [M-H]À calcd for C17H9T2F9NO3S,
resulting solid was subjected to column chromatography (SiO2, 10–30% 484.3; found, 484.0). 19F NMR (376 MHz, DMSO-d6): δ À72.2 (s, 6F),
EtOAc in heptane) to yield the title compound (4) as a white solid (2.58 g, -74.1 (m, 3F).
80% yield). 1H NMR (400MHz, DMSO-d6): δ 11.08 (s, 1H), 8.57 (s, 1H), 8.13
(d, J = 4 Hz, 1H), 8.03 (d, J = 12Hz, 1H), 7.81 (dd, J = 8, 4 Hz, 1H), 7.54 (d,
J = 8 Hz, 2H), 7.20 (d, J = 8 Hz, 2H); 19F NMR (282 MHz, DMSO-d6): δ À75.5
2,4-ditritium-N-(4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-
(s, 6F). MS ESI (m/z): [M-H]- calcd for C15H8Br2F6NO3S, 556.1; found, 556
2-yl)phenyl)-N-(2,2,2-trifluoroethyl)benzenesulfonamide (8)
and 558 (dibromo pattern).
2,4-dibromo-N-(4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl)-N-
(2,2,2-trifluoroethyl)benzenesulfonamide (6) (6mg, 9.4μmol) was dissolved
in 1% (w/v) methanolic potassium hydroxide (1mL). The reaction vessel
was charged with 10% palladium on carbon (15mg), and the resultant
black mixture was stirred for 3h at 23°C under an atmosphere of tritium
gas (2Ci, 1 atm). The reaction was then filtered, and the labile tritium
was removed under consecutive rotary evaporations from methanol
(3× 5 mL). The crude material was purified by preparatory HPLC
(Hichrom Ltd. Ultrasphere C18 ODS 25 × 1cm column, 5μm particle size)
using a 30– 90% gradient of water/acetonitrile (3mL/min flow rate) over
a 60 min period (254nm UV detection). The relevant fractions were
pooled and dried via rotary evaporation to provide the title compound
(8). The resulting product was re-constituted in ethanol (1mCi/mL).
The radiochemical purity (99.9%) was determined by analytical HPLC
(Vydac Genesis C8 150 × 4.6mm column, 5μm particle size) using a
40– 90% gradient of 20mM aqueous ammonium acetate and acetonitrile
(1mL/min flow rate) over a 15 min period, and held for an additional
5 min (254 nm UV detection). Compound 8 also co-chromatographed with
compound 1 under the previously mentioned analytical HPLC conditions.
Specific activity was determined by mass spectroscopy (48 Ci/mmol, ESI
(m/z): [M-H]À calcd for C17H9T2F9NO3S, 484.3; found, 484.0). 3H NMR
(426MHz, DMSO-d6): δ 7.78 (s, 1T), 7.76 (s, 1T); 19F NMR (376 MHz,
DMSO-d6): δ À71.0 (m, 3F), À75.4 (s, 6F).
2,4-dibromo-N-(4-(1,1,1,3,3,3-hexafluoro-2-(2,2,2-
trifluoroethoxy)propan-2-yl)phenyl)benzenesulfonamide (5)
2,4-dibromo-N-(4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl)
benzenesulfonamide (4) (1.3 g, 2.33 mmol), 1,1,1-trifluoro-2-iodoethane
(2.2 g, 10.4 mmol), and K2CO3 (635 mg, 4.6 mmol) were combined in
DMF (15 mL) and stirred at 100°C for 18h. The reaction mixture was
cooled to 23°C and poured into water (100 mL). The mixture was
extracted with EtOAc (3 × 100 mL). The organic fractions were combined
and washed with brine (250 mL), dried over anhydrous Na2SO4, filtered,
and concentrated under reduced pressure. The residue was purified by
column chromatography (SiO2, 15% EtOAc in petroleum ether) to afford
the title compound (5) as a light yellow solid (135 mg, 10% yield). 1H
NMR (400 MHz, DMSO-d6): δ 11.28 (s, 1H), 8.15 (d, J = 2 Hz, 1H), 8.08 (d,
J = 9 Hz, 1H), 7.85 (dd, J = 9, 2 Hz, 1H), 7.47 (d, J = 9 Hz, 2H), 7.27 (d,
J = 9 Hz, 2H), 4.24 (q, J = 8 Hz, 2H); 19F NMR (282 MHz, DMSO-d6): δ -72.1
(s, 6F), À74.2 (m, 3F). MS ESI (m/z): [M-H]À calcd for C17H9Br2F9NO3S,
638.1; found, 638 and 640 (dibromo pattern).
2,4-dibromo-N-(4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-
2-yl)phenyl)-N-(2,2,2-trifluoroethyl)benzenesulfonamide (6)
2,4-dibromo-N-(4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl)
benzenesulfonamide (4) (249mg, 0.45mmol), K2CO3 (68mg, 0.49 mmol),
and acetonitrile (1.5mL) were combined and stirred at 23°C. 2,2,2-
trifluoroethyl trifluoromethanesulfonate (109mg, 0.47 mmol) was added,
and the reaction was stirred at reflux for 16h. The reaction was quenched
with saturated aqueous NH4Cl (50 mL) and extracted with dichloromethane
(3× 50 mL). The organic fractions were combined, rinsed with brine (50 mL),
dried over anhydrous MgSO4, filtered, and concentrated under reduced
pressure. The resulting residue was purified by column chromatography
(SiO2, 0–50% EtOAc in heptane) to afford the title compound (6) as a
white solid (95mg, 33% yield). 1H NMR (400 MHz, DMSO-d6): δ 8.78
(s, 1H), 8.17 (s, 1H), 7.80–7.59 (m, 4H), 7.48 (d, J = 9 Hz, 2H), 4.85 (q,
J = 9 Hz, 2H); 19F NMR (282 MHz, DMSO-d6) δ -À71.2 (t, JHF = 9 Hz, 3F),
À75.5 (s, 6F). MS ESI (m/z): [M-H]À calcd for C17H9Br2F9NO3S, 638.1;
found, 638 and 640 (dibromo pattern).
Results and discussion
Synthesis of the target molecule commenced with sulfonamide
formation between the commercially available 2-(4-amino-
phenyl)-1,1,1,3,3,3-hexafluoropropan-2-ol (2) and sulfonyl chloride
(3) under basic conditions (Scheme 1). The resulting sulfonamide
(4) was subjected to alkylation conditions similar to those
described by Li and co-workers for the synthesis of related tertiary
sulfonamide analogs.6 Only one alkylated product was identified in
our reaction mixture. Based on the literature precedent from Li and
co-workers, we presumed that the alkylation would occur on the
sulfonamide nitrogen to provide dibromo-intermediate 6. After
extensive analysis of the alkylation product described in the
succeeding texts, we found that the alkylation had instead
occurred on the oxygen of the hexafluoro-2-isopropanol moiety
to provide compound 5. The O-alkylation result was most apparent
when compound 5 was subjected to metal-catalyzed tritium-
halogen exchange conditions under a tritium gas atmosphere
2,4-ditritium-N-(4-(1,1,1,3,3,3-hexafluoro-2-(2,2,2-trifluoro-
ethoxy)propan-2-yl)phenyl)benzenesulfonamide (7)
2,4-dibromo-N-(4-(1,1,1,3,3,3-hexafluoro-2-(2,2,2-trifluoroethoxy)propan-
2-yl)phenyl)benzenesulfonamide (5) (7.7 mg, 12.1 μmol) was dissolved in
1% (w/v) methanolic potassium hydroxide (1 mL). The reaction vessel (Scheme 2). The positive and negative ion mass spectrometry data
www.jlcr.org
Copyright © 2013 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2014, 57 57–60

B. P. Fauber et al.
Scheme 1. Syntheses of dibromo-intermediates 5 and 6.
Scheme 2. Synthesis of labeled ligand 7.
of the reaction product matched that of the desired compound,
A more effective alkylation strategy of compound 4 was
but the HPLC retention time of the labeled product did not match envisioned in which the N-alkyl product would predominate to
that of T0901317 1 (Cayman Chemicals). At that point, we were provide compound 6 (Scheme 1). A comparison of the reported
uncertain of the labeled product’s chemical structure. To rule out relative pKa values of the N―H and O―H hydrogen atoms found
the risk of differing chromatographic retention times due to in the functional group substructures of compound 4 revealed
isotopic fractionation,7 and to further characterize the labeled that they had similar values (pKa ~ 11).12,13 Thus, discriminating
product, we explored other analytical methods to gather structural between the two heteroatoms during the alkylation step could
information.
not be achieved on the basis of pKa alone.
As a standard laboratory practice, labeled products were
Alteration of the sulfonamide 4 alkylation conditions to
stored as dispersals in ethanol to minimize radiation exposure include a solvent with greater ionizing-power,14 and a more
risks, increase long-term stability, and ease handling of small reactive electrophile,15 resulted in the alkylation of the
sample quantities.8 Although the ethanolic dispersal had the sulfonamide nitrogen and provided the dibromo-intermediate
previously mentioned benefits, it limited our capability to 6 (Scheme 1). This was the only alkylation product in the reaction
1
perform H NMR analysis of the dispersed labeled product and mixture. Analysis of the 19F NMR spectrum revealed that the
gather structural information. The amount of labeled product fluorine shifts of compound 6 were in close agreement with
was also too minuscule to acquire a 13C NMR spectrum in a those of T0901317 (1), providing additional confirmation for
reasonable timeframe, thus we turned our attention toward 19F the structure of compound 6 as drawn (Scheme 1).
NMR techniques. The high relative abundance of 19F and
Compound 6 was then subjected to a palladium on carbon
compatibility with the ethanolic sample media made 19F NMR suspension in methanolic potassium hydroxide under an
a suitable tool for the analysis of the dispersed radioligand.9 atmosphere of tritium gas to provide [3H2]T0901317 (Scheme 3,
The 19F NMR spectrum of the labeled product had very different Compound 8). Compound 8 was manipulated and stored as an
fluorine chemical shifts than those for compound 1, and thus ethanolic dispersal (1mCi/mL). The HPLC retention time and 19F
provided additional evidence for the labeled product bearing NMR spectrum of the labeled material (8) were nearly identical to
the structure of compound 7.10
that of T0901317 (1). Tritium NMR analysis indicated the presence
In addition to the comparative 19F NMR studies of compounds of two distinct aromatic tritium atoms on compound 8 in a 1:1
1 and 7, the connectivity of compound 5 (the precursor to ratio. The radiochemical purity of compound 8 was >99%, and
compound 7) was analyzed using a series of 2D NMR the specific activity was 48 Ci/mmol.
experiments.11 A 1H―15N HSQC NMR experiment on compound
Radioligands 7 and 8 were tested in SPA format to assess their
5 demonstrated the presence of an N―H bond on the ability to bind to LXRα (Table 1). Compound 7 showed low SB to
sulfonamide. A separate 1H―13C HMBC NMR experiment LXRα, with an average of 19% ( 14%) SB across all time points.
illustrated that the carbon bearing the bis(trifluoromethyl) On the other hand, [3H2]T0901317 (8) showed 2.6-fold higher
moiety was in close proximity to the methylene protons of the SB to LXRα than ligand 7, with an average over all time points
2,2,2-trifluoroethyl group. The outcomes of both 2D NMR of 50% ( 11%) SB. These SPA binding data were consistent with
experiments were consistent with the structure of compound 5 the 19F NMR characterization of ligand 8 being the desired [3H2]
as drawn (Scheme 1).
T0901317 product.
J. Label Compd. Radiopharm 2014, 57 57–60
Copyright © 2013 John Wiley & Sons, Ltd.
www.jlcr.org

B. P. Fauber et al.
Scheme 3. Synthesis of [3H2]T0901317 (8).
[3] N. Mitro, L. Vargas, R. Romeo, A. Koder, E. Saez, FEBS Lett. 2007, 581,
1721–1726.
Table 1. Specific binding of radioligands 7 and 8 to the
LXRα ligand binding domain as assessed in a scintillation
proximity assay.
[4] N. Kumar, B. Lyda, M. R. Chang, J. L. Lauer, L. A. Solt, T. P. Burris,
T. M. Kamenecka, P. R. Griffin, ACS Chem. Biol. 2012, 7, 672–677.
[5] B. A. Janowski, M. J. Grogan, S. A. Jones, G. B. Wisely, S. A. Kliewer,
E. J. Corey, D. J. Mangelsdorf, Proc. Natl. Acad. Sci. U. S. A. 1999,
96, 266–271.
Compound
LXRα (%) specific binding
7
8
19 ( 14%)
50 ( 11%)
[6] L. Li, J. Liu, L. Zhu, S. Cutler, H. Hasegawa, B. Shan, J. C. Medina,
Bioorg. Med. Chem. Lett. 2006, 16, 1638–1642.
[7] C. N. Filer, J. Labelled Comp. Radiopharm. 1999, 42, 169–197.
[8] R. Voges, J. R. Heys, T. Moenius, Introduction. Preparation of
Compounds Labeled with Tritium and Carbon-14, 1st ed., John Wiley
& Sons, Ltd., West Sussex, UK, 2009, pp 6–15.
[9] W. R. Dolbier, An overview of fluorine NMR. Guide to Fluorine NMR
for Organic Chemists, 1st ed., John Wiley & Sons, Inc., Hoboken, NJ,
2009, pp 9–34.
[10] NMR spectra for the T0901317 samples supplied by Cayman
Chemicals and Sigma-Aldrich were identical: 1H NMR (400 MHz,
DMSO-d6): δ 8.81 (s, 1H), 7.74 (t, J = 6 Hz, 1H), 7.67-7.56 (m, 6H),
7.29 (d, J = 8 Hz, 2H), 4.63 (q, J = 8 Hz, 2H); 19F NMR (282 MHz,
DMSO-d6): δ -71.5 (t, JHF = 14 Hz, 3F), -75.4 (s, 6F).
[11] S. Berger, S. Braun, 2D NMR spectroscopy with field gradients. 200
and More NMR Experiments: A Practical Course, 2nd ed., Wiley-VCH
Verlag GmbH & Co. KGaA, Weinheim, Germany, 2004, pp 525–612.
[12] S. M. Ivanova, B. G. Nolan, Y. Kobayashi, S. M. Miller, O. P. Anderson,
S. H. Strauss, Chem. Eur. J. 2001, 7, 503–510.
[13] F. G. Bordwell, D. Algrim, J. Org. Chem. 1976, 41, 2507–2508.
[14] J. March, Aliphatic nucleophilic substitution. Advanced Organic
Chemistry: Reactions Mechanism and Structure, 4th ed., John Wiley
& Sons, Inc., New York, NY, 1992, pp 357–362.
Acknowledgements
We thank Joe Pease for helpful discussions regarding the
analytical data.
Conflict of Interest
The authors did not report any conflict of interest.
References
[1] J. R. Schultz, H. Tu, A. Luk, J. J. Repa, J. C. Medina, L. Li, S. Schwendner,
S. Wang, M. Thoolen, D. J. Mangelsdorf, K. D. Lustig, B. Shan, Genes
Dev. 2000, 14, 2831–2838.
[2] B. Hu, M. Collini, R. Unwalla, C. Miller, R. Singhaus, E. Quinet, D. Savio,
A. Halpern, M. Basso, J. Keith, V. Clerin, L. Chen, C. Resmini, Q.-Y. Liu,
I. Feingold, C. Huselton, F. Azam, M. Farnegardh, C. Enroth, T. Bonn,
A. Goos-Nilsson, A. Wilhelmsson, P. Nambi, J. Wrobel, J. Med. Chem.
2006, 49, 6151–6154.
[15] J. March, Aliphatic nucleophilic substitution. Advanced Organic
Chemistry: Reactions Mechanism and Structure, 4th ed., John Wiley
& Sons, Inc., New York, NY, 1992, pp 352–354.
www.jlcr.org
Copyright © 2013 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2014, 57 57–60