Discovery of a potent, orally bioavailable β3 adrenergic receptor agonist, (R)-N-[4-[2-[[2-Hydroxy-2-(3-pyridinyl)ethyl]amino]ethyl]phenyl]-4-[4-[4-(tri fluoromethyl)phenyl]thiazol-2-yl]benzenesulfonamide

Article abstract of DOI:10.1021/jm000286i

As part of our investigation into the development of orally bioavailable β₃ adrenergic receptor agonists, we have identified a series of pyridylethanolamine analogues possessing a substituted thiazole benzenesulfonamide pharmacophore that are potent human β₃ agonists with excellent selectivity against other human β receptor subtypes. Several of these compounds also exhibited an improved pharmacokinetic profile in dogs. For example, thiazole sulfonamide 2e (R = 4-F₃C-C₆H₄) is a potent full β₃ agonist (EC₅₀ = 3.6 nM, 94% activation) with >600-fold selectivity over the human β₁ and β₂ receptors, which also displays good oral bioavailability in several mammalian species, as well as an extended duration of action.

Full text of DOI:10.1021/jm000286i

3832
J . Med. Chem. 2000, 43, 3832-3836
Expedited Articles
Discover y of a P oten t, Or a lly Bioa va ila ble â3 Ad r en er gic Recep tor Agon ist,
(R)-N-[4-[2-[[2-Hyd r oxy-2-(3-p yr id in yl)eth yl]a m in o]eth yl]p h en yl]-4-[4-[4-
(tr iflu or om eth yl)p h en yl]th ia zol-2-yl]ben zen esu lfon a m id e
Robert J . Mathvink,* J . Samuel Tolman, Dawn Chitty, Mari R. Candelore, Margaret A. Cascieri,
Lawrence F. Colwell, J r., Liping Deng, William P. Feeney, Michael J . Forrest, Gary J . Hom, D. Euan MacIntyre,
Randall R. Miller, Ralph A. Stearns, Laurie Tota, Matthew J . Wyvratt, Michael H. Fisher, and Ann E. Weber
Merck Research Laboratories, Rahway, New J ersey 07065
Received J une 29, 2000
As part of our investigation into the development of orally bioavailable â3 adrenergic receptor
agonists, we have identified a series of pyridylethanolamine analogues possessing a substituted
thiazole benzenesulfonamide pharmacophore that are potent human â3 agonists with excellent
selectivity against other human â receptor subtypes. Several of these compounds also exhibited
an improved pharmacokinetic profile in dogs. For example, thiazole sulfonamide 2e (R ) 4-F3C-
C6H4) is a potent full â3 agonist (EC50 ) 3.6 nM, 94% activation) with >600-fold selectivity
over the human â1 and â2 receptors, which also displays good oral bioavailability in several
mammalian species, as well as an extended duration of action.
In tr od u ction
Obesity is becoming an increasingly common disorder
in the United States and other parts of the world, and
is closely associated with the development of type II
diabetes, coronary heart disease, and hypertension.
Accordingly, the potential for therapeutic intervention
has received considerable attention in the past several
years, and researchers have described a number of
different approaches for treating obesity.1 One potential
therapeutic approach to altering body fat composition
is the enhancement of energy expenditure by increasing
metabolic rate. The recognition that stimulation of â3
adrenergic receptors (ARs) on the surface of adipocytes
evokes lipolysis and activation and upregulation of the
uncoupling protein UCP1, which leads to a net increase
in energy utilization,2 has led to considerable efforts to
identify selective agonists of the human â3 adrenergic
receptor.3 A recent report from our labs described the
in vitro activity and pharmacokinetic properties of the
pyridylethanolamine â3 agonist 1,4 a tetrazolone ben-
zenesulfonamide analogue which exhibited 27% oral
bioavailability in both dogs and rats. Concurrent studies
in our labs have examined a number of alternative
replacements for the tetrazolone nucleus (including
oxazole,5 oxadiazole,6 triazole,7 pyridyl,8 and pyrimidyl8),
but none have been found to possess the required
combination of potency, selectivity, and oral bioavail-
ability for a clinical drug candidate.
human â3 AR. However, these compounds exhibited poor
oral bioavailability in dogs and rats. Subsequent modi-
fications resulted in the identification of a related series
of substituted thiazole benzenesulfonamides 2 which
also display excellent in vitro agonist activity and
selectivity.10 In contrast to the SAR observed in several
other series of pyridylethanolamine â3 agonists,4,9,11 it
was subsequently found that several aryl-substituted
thiazoles 2 exhibited better potency, selectivity, and oral
bioavailability than the alkyl-substituted analogues.
Herein we detail the preparation and biological profiles
of a series of thiazole sulfonamides possessing fluori-
nated aromatic substituents, culminating in the discov-
ery of 2e (R ) 4-F3C-C6H4), a potent and selective
pyridylethanolamine â3 agonist with an improved phar-
macokinetic profile.
Ch em istr y
We recently reported9 the discovery of a class of
pyridylethanolamine â3 AR agonists possessing a sub-
stituted indoline-5-sulfonamide pharmacophore which
exhibited exceptional potency and selectivity for the
Thiazole benzenesulfonamides 2a -h were prepared
from aniline intermediate 312 by the divergent route
illustrated in Scheme 1. Coupling of 3 with 4-cyanoben-
zenesulfonyl chloride, followed by treatment with hy-
drogen sulfide, afforded the thioamide 4, which was
condensed with the requisite R-halo ketones to afford
* To whom correspondence should be addressed. Tel: 732-594-1504.
Fax: 732-594-5790. E-mail: robert_mathvink@merck.com.
10.1021/jm000286i CCC: $19.00 © 2000 American Chemical Society
Published on Web 09/29/2000

Potent, Orally Bioavailable â3 AR Agonist
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21 3833
Sch em e 1. Synthesis of Thiazole Sulfonamides 2a
binding affinities to membranes prepared from CHO
cells expressing the cloned human â1 and â2 ARs were
determined, and these values permit an assessment of
the selectivity of such compounds for the human â3
receptor. The results are shown in Table 1 and indicate
that all of these fluorinated aryl-substituted thiazoles
behave as full agonists of the human â3 AR (82-100%
activation relative to the full agonist isoproterenol) and
were much less efficacious at the human â1 and â2
receptors. Several of the more potent analogues (2a ,e,h )
were also evaluated for â3 binding affinity (â3 IC50) and
were titrated in the â1 and â2 functional assays for direct
comparison with â3 functional efficacies (â3 EC50).
Four of the most potent aryl-substituted thiazoles 2
were evaluated in vivo for their ability to evoke hyper-
glycerolemia in beagle dogs. Administration (10 mg/kg
po) of 2a ,d ,e,h to fasted dogs produced elevated glycerol
levels, and drug levels and pharmacokinetic properties
of these analogues were determined (Table 2). While
2a ,d ,e exhibited comparable oral bioavailability in dogs,
that of trifluoromethoxy-substituted analogue 2h was
signficantly higher. More importantly, both 2e,h dis-
played substantially longer half-lives than the di- and
trifluorophenyl analogues 2a ,d .
a
Reagents: (a) 4-CNC6H4SO2Cl, pyridine, CH2Cl2; (b) H2S,
Et3N, pyridine, 25 °C; (c) ArCOCH2Cl, ethanol, reflux; (d) trifluo-
roacetic acid, CH2Cl2, 25 °C.
the thiazole intermediates. Removal of the BOC protect-
ing group afforded the final products 2.
Resu lts a n d Discu ssion
The extended duration of action observed for the
4-substituted arylthiazole analogues 2e,h led us to
further examine the in vivo efficacy of these compounds
in other animal models. The oral bioavailabilities of 2e,h
in rats (dosing 10 mg/kg po, 3 mg/kg iv) were determined
to be 17% and 7%, respectively. Both compounds
displayed a plasma half-life of greater than 8 h in rats.
When administered to anesthetized rhesus monkeys as
a series of sequential rising dose intravenous infusions,
2e,h produced dose-dependent increases in serum glyc-
erol accompanied by tachycardia, with ED50 values for
hyperglycerolemia of 0.26 and 0.14 mg/kg, respectively.
The maximum extent of hyperglycerolemia elicited by
both compounds was equivalent to that produced by the
infusion of a maximally effective dose (100 µg/kg) of
Compounds 2a -h were evaluated for in vitro func-
tional efficacy in stimulating increases in cAMP in
Chinese hamster ovary (CHO) cells expressing the
cloned human â3 receptor. The activity of an agonist at
the â3 AR is best described by its ability to stimulate
adenylyl cyclase in a functional assay, since this method
measures the affinity of a compound for the high-
affinity, G-protein-coupled state of the receptor. As a
result, this assay accurately predicts the lipolytic po-
tential of compounds in native adipocytes.13 However,
because the compounds under study generally have very
low efficacy at â1 and â2 receptors, the activities of these
compounds for the â1 and â2 ARs are better described
by their binding affinities for these receptors. Thus,
Ta ble 1. Comparison of â3 AR Agonist Activity and â1 and â2 Binding Affinity of Thiazole Sulfonamides 2
â3 â1
EC50, nM (% act)a binding IC50, nMb EC50, nM (% act)a binding IC50, nMb EC50, nM (% act)a binding IC50, nMb
â2
compd
Ar
2a
2b
2c
2d
2e
2f
3,4-F2C6H3
2,3-F2C6H3
2,4-F2C6H3
3,4,5-F3C6H2
4-F3C-C6H4
3-F3C-C6H4
2-F3C-C6H4
7.1 ( 4.7 (92)
7.6 ( 3.3 (99)
19 ( 12 (87)
6.3 ( 4.4 (100)
3.6 ( 2.2 (94)
6.9 ( 3.7 (100)
53 ( 42 (82)
20 ( 8
nd
nd
nd
46 ( 29
nd
2600 (20)c
1700 ( 580
2600 ( 540
2500 ( 1600
2000 ( 1400
2300 ( 1000
1800 ( 410
1900 ( 110
8400 ( 4900
1800 (12)c
1700 ( 1200
760 ( 100
(6)d
(13)d
(7)d
(20)d
(3)d
(12)d
2600 ( 470
5000 ( 4200
2300 ( 700
980 ( 320
4800 ( 2600 (24)
2400 ( 950 (25)
(17)d
(22)d
2g
2h
nd
30 ( 20
nd
nd
1800 ( 480
1200 ( 1000
4-F3CO-C6H4 4.3 ( 1.6 (93)
2700 ( 1500 (24)
1400 ( 140(18)
a
Adenylyl cyclase activation given as % of the maximum stimulation with isoproterenol. Results are given as the mean ( SD from
b
dose-response curves of at least two experiments (n g 2), unless otherwise indicated. Receptor binding assays were carried out with
membranes prepared from CHO cells expressing the cloned human receptor in the presence of [125I]iodocyanopindolol. Results are given
as the mean ( SD from at least two experiments (n g 2), unless otherwise indicated. nd ) not determined. c n ) 1. Single point data,
d
% activation at 10 µM.
Ta ble 2. Pharmacokinetic Properties of Compounds 2a ,d ,e,h in Dogs
comd
avg AUCiva (µg‚min/mL)
avg AUCpob (µg‚min/mL)
Clp (mL/min/kg)
Vdss (L/kg)
t1/2 (h)
F (%)
2a
2d
2e
2h
103 ( 1
148 ( 5
116 ( 20
212 ( 23
124 ( 21
134 ( 76
136 ( 40
377 ( 57
29.3 ( 0.3
20.5 ( 0.5
26.8 ( 4.6
14.3 ( 5.2
19.0 ( 1.8
14.1 ( 0.4
31.3 ( 5.9
12.8 ( 2.0
7.5 ( 0.6
8.0 ( 0.4
13.5 ( 0.3
10.3 ( 0.5
36 ( 6.2
27 ( 14
38 ( 3.0
53 ( 2.3
a
Dogs (n ) 2) were dosed intravenously at 3 mg/kg test compound dissolved in vehicle (PEG400/EtOH/0.9% saline, 60/20/20 v/v/v);
b
plasma drug levels were determined by LC/MS/MS. Dogs (n ) 2) were dosed orally at 10 mg/kg test compound dissolved in 0.5%
methylcellulose/0.02% sodium lauryl sulfate containing 2 equiv of HCl; plasma drug levels were determined by LC/MS/MS.

3834 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21
Mathvink et al.
and the residue was purified by flash chromatography on silica
gel (8% methanol in dichloromethane eluant), affording 9.31
g of 4 as a bright yellow foam: 1H NMR (CD3OD) δ 8.45 (m,
2H), 7.87 (d, J ) 8.5 Hz, 2H), 7.80 (m, 1H), 7.70 (m, 2H), 7.01
(overlapping s, 4H and m, 1H), 4.84 (m, 1H), 3.15-3.45 (m,
4H), 2.7 (m, 2H), 1.30 (s, 9H).
isoproterenol, indicating that both 2e,h act as full
agonists for hyperglycerolemia in the rhesus monkey.
Furthermore, the doses of 2e,h which produced a 15%
elevation in heart rate (ED15HR) under the same experi-
mental conditions were determined to be 10 and 2.5 mg/
kg, respectively. Trifluoromethyl analogue 2e thus
exhibits a significantly better therapeutic index for
Gen er a l P r oced u r e for P r ep a r a tion of Th ia zole Ben -
zen esu lfon a m id es (2): (R)-N-[4-[2-[[2-Hyd r oxy-2-(3-p yr -
id in yl)eth yl]a m in o]eth yl]p h en yl]-4-[4-(3,4-d iflu or op h en -
yl)th ia zol-2-yl]ben zen esu lfon a m id e (2a ). A mixture of 62
mg (0.111 mmol) of 4 and 24 mg (0.126 mmol) of 2-chloro-3′,4′-
difluoroacetophenone in absolute ethanol (1 mL) was warmed
at reflux for 4 h. The cooled reaction mixture was concentrated
under reduced pressure, and the residue was dissolved in 1
mL of dichloromethane and 1 mL of trifluoroacetic acid (TFA).
After stirring for 1 h at ambient temperature, the solution was
concentrated under reduced pressure. Residual TFA was
removed by azeotropic distillation with dichloromethane, and
the residue was purified by flash chromatography (9:1 dichlo-
romethane/10% NH4OH in methanol eluant), affording 2a (48
mg) as a light yellow foam: 1H NMR (CD3OD) δ 8.49 (d, J )
2.1 Hz, 1H), 8.40 (dd, J ) 5.0 and 1.5 Hz, 1H), 8.09 (d, J ) 8.6
Hz, 2H), 7.94 (s, 1H), 7.91 (m, 1H), 7.75-7.85 (overlapping d,
J ) 8.6 Hz, 2H and m, 2H), 7.25-7.38 (m, 2H), 7.09 (d, J )
8.6 Hz, 2H), 7.04 (d, J ) 8.6 Hz, 2H), 4.80 (dd, J ) 7.3 and 5.7
Hz, 1H), 2.90-2.70 (m, 6H); EIMS m/z 593 (M+ + H). Anal.
(C30H26F2N4O3S2) C, H, N.
hyperglycerolemia over cardiovascular effects (ED50gly
/
ED15HR) than the trifluoromethoxy analogue 2h . In
addition, compound 2e is >100-fold selective for â3 AR
agonist activity when tested against a panel of receptors
and ion channels, except for the human dopamine D2
and D3 receptors, where the selectivity is 61- and 26-
fold, respectively. Since the D2 and D3 receptors are
located within the CNS, and 2e displays poor brain
penetration, this level of cross-reactivity is not likely to
be biologically significant.
Con clu sion s
The introduction of a range of fluorinated aryl sub-
stituents onto the thiazole ring of the 3-pyridylethanol-
amine benzenesulfonamide 2 results in a number of
exceptionally potent and selective â3 agonists, several
of which exhibit improved pharmacokinetic profiles. The
4-(trifluoromethyl)phenyl analogue 2e, in particular, is
a potent (EC50 3.6 nM) full agonist which displays good
oral bioavailability in both dogs (%F ) 38) and rats
(%F ) 17), as well as an exceptionally long half-life
(t1/2 > 8h) in all species tested. On the basis of these
data and favorable safety assessment, compound 2e was
selected for phase I clinical studies, the results of which
will be reported elsewhere.
(R)-N-[4-[2-[[2-Hydr oxy-2-(3-pyr idin yl)eth yl]am in o]eth -
yl]p h en yl]-4-[4-(2,3-d iflu or op h en yl)th ia zol-2-yl]ben zen e-
su lfon a m id e (2b). Prepared by general procedure outlined
for the preparation of 2a : 1H NMR (CD3OD) δ 8.49 (d, J )
1.9 Hz, 1H), 8.40 (dd, J ) 4.9 and 1.6 Hz, 1H), 8.12 (d, J ) 8.4
Hz, 2H), 8.03 (m, 1H), 8.01 (d, J ) 2.2 Hz, 1H), 7.82 (d, J )
8.4 Hz, 2H), 7.78 (m, 1H), 7.36 (dd, J ) 7.9 and 5.0 Hz, 1H),
7.25 (m, 2H), 7.09 (d, J ) 8.5 Hz, 2H), 7.04 (d, J ) 8.5 Hz,
2H), 4.78 (m, 1H), 2.90-2.70 (m, 6H); EIMS m/z 593 (M+
H). Anal. (C30H26F2N4O3S2) C, H, N.
+
(R)-N-[4-[2-[[2-Hydr oxy-2-(3-pyr idin yl)eth yl]am in o]eth -
yl]p h en yl]-4-[4-(2,4-d iflu or op h en yl)th ia zol-2-yl]ben zen e-
su lfon a m id e (2c). Prepared by general procedure outlined
for the preparation of 2a : 1H NMR (CD3OD) δ 8.48 (d, J )
2.2 Hz, 1H), 8.39 (dd, J ) 4.9 and 1.6 Hz, 1H), 8.27 (m, 1H),
8.10 (d, J ) 8.6 Hz, 2H), 7.86 (d, J ) 2.4 Hz, 1H), 7.81 (d, J )
8.6 Hz, 2H), 7.77 (m, 1H), 7.34 (dd, J ) 7.9 and 5.0 Hz, 1H),
7.05 (m, 6H), 4.77 (m, 1H), 2.90-2.70 (m, 6H); EIMS m/z 593
(M+ + H). Anal. (C30H26F2N4O3S2) C, H, N.
(R)-N-[4-[2-[[2-H yd r oxy-2-(3-p yr id in yl)et h yl]a m in o]-
et h yl]p h en yl]-4-[4-(3,4,5-t r iflu or op h en yl)t h ia zol-2-yl]-
ben zen esu lfon a m id e (2d ). Prepared by general procedure
outlined for the preparation of 2a : 1H NMR (CD3OD) δ 8.50
(d, J ) 2.0 Hz, 1H), 8.42 (dd, J ) 4.9 and 1.6 Hz, 1H), 8.07 (d,
J ) 8.6 Hz, 2H), 8.01 (s, 1H), 7.81 (d, J ) 8.6 Hz, 2H), 7.76
(m, 3H), 7.37 (dd, J ) 7.8 and 4.7 Hz, 1H), 7.11 (d, J ) 8.6 Hz,
2H), 7.06 (d, J ) 8.6 Hz, 2H), 4.80 (m, 1H), 2.90-2.70 (m, 6H);
EIMS m/z 611 (M+ + H). Anal. (C30H25F3N4O3S2) C, H, N.
(R)-N-[4-[2-[[2-Hydr oxy-2-(3-pyr idin yl)eth yl]am in o]eth -
yl]p h en yl]-4-[4-[4-(tr iflu or om eth yl)p h en yl]th ia zol-2-yl]-
ben zen esu lfon a m id e (2e). Prepared by general procedure
outlined for the preparation of 2a : 1H NMR (CD3OD) δ 8.49
(d, J ) 2.1 Hz, 1H), 8.40 (dd, J ) 5.0 and 1.5 Hz, 1H), 8.18 (d,
J ) 7.9 Hz, 2H), 8.12 (d, J ) 8.6 Hz, 2H), 8.09 (s, 1H), 7.82 (d,
J ) 8.6 Hz, 2H), 7.77 (m, 1H), 7.71 (d, J ) 8.6 Hz, 2H), 7.35
(dd, J ) 7.9 and 5.0 Hz, 1H), 7.09 (d, J ) 8.6 Hz, 2H), 7.04 (d,
J ) 8.6 Hz, 2H),), 4.80 (dd, J ) 7.3 and 5.7 Hz, 1H), 2.90-
2.70 (m, 6H); EIMS m/z 625 (M+ + H). Anal. (C31H27F3N4O3S2)
C, H, N.
(R)-N-[4-[2-[[2-Hydr oxy-2-(3-pyr idin yl)eth yl]am in o]eth -
yl]p h en yl]-4-[4-[3-(tr iflu or om eth yl)p h en yl]th ia zol-2-yl]-
ben zen esu lfon a m id e (2f). Prepared by general procedure
outlined for the preparation of 2a : 1H NMR (CD3OD) δ 8.49
(d, J ) 2.3 Hz, 1H), 8.39 (dd, J ) 4.9 and 1.6 Hz, 1H), 8.31 (s,
1H), 8.25 (d, J ) 6.5 Hz, 1H), 8.13 (d, J ) 8.5 Hz, 2H), 8.09 (s,
1H), 7.83 (d, J ) 8.5 Hz, 2H), 7.77 (m, 1H), 7.62 (m, 2H), 7.36
(dd, J ) 7.9 and 5.0 Hz, 1H), 7.09 (d, J ) 8.6 Hz, 2H), 7.05 (d,
Exp er im en ta l Section
Ch em istr y. Gen er a l Meth od s. Proton magnetic resonance
spectra were recorded on a Varian XL 400 spectrometer. Low-
resolution mass spectral analyses were obtained with a LKB
9000 mass spectrometer at an ionizing voltage of 70 eV. All
reagents, solvents and drying agents were obtained from
commercial sources and used without further purification
unless otherwise noted. Precoated plates (silica gel F254, 250
mM; Analtech, Inc., Newark, DE) were used for TLC. Flash
chromatography was carried out utilizing silica gel 60 (E.
Merck). Elemental analyses were performed by Robertson
Microlit Laboratories, Inc., Madison, NJ .
(R)-N-[4-[2-[N-(1,1-Dim eth yleth oxyca r bon yl)-N-[2-h y-
d r oxy-2-(3-p yr id in yl)eth yl]a m in o]eth yl]p h en yl]-4-(a m i-
n oth ioca r bon yl)ben zen esu lfon a m id e (4). To a solution of
6.5 g (18.2 mmol) of (R)-N-[2-(4-aminophenyl)ethyl]-2-hydroxy-
2-(3-pyridyl)ethylcarbamic acid 1,1-dimethylethyl ester (3)
(Fisher et. al. U.S. Patent 5,561,142, Oct 1, 1996) in 70 mL of
methylene chloride were added 2.1 mL of pyridine and 5.0 g
of 4-cyanobenzenesulfonyl chloride. The reaction mixture was
stirred at room temperature overnight. TLC (3:1 methylene
chloride:acetone) on silica gel indicated the formation of a
major fast moving (Rf 0.48) spot. Purification by flash chro-
matography (silica gel, 3:1 methylene chloride:acetone) gave
8.7 g of (R)-N-[4-[2-[N-(1,1-dimethylethoxycarbonyl)-N-[2-hy-
droxy-2-(3-pyridyl)ethyl]amino]ethyl]phenyl]-4-cyanobenzene-
sulfonamide as a white solid: 1H NMR (CDCl3) δ 8.53-8.44
(m, 2H), 7.78 (d, J ) 8.7 Hz, 2H), 7.72-7.68 (m, 1H), 7.67 (d,
J ) 8.7 Hz, 2H), 7.3-7.23 (m, 1H), 7.1-6.9 (m, 4H), 4.8 (m,
1H), 3.5-2.6 (m, 6H), 1.42 (s, 9H).
A steady stream of hydrogen sulfide was bubbled into a
solution of 10.2 g of the product from the previous step and
triethylamine (2.9 mL) in 100 mL of pyridine at 25 °C for 15
min. The green solution was stirred for 2.5 h, and then
nitrogen was bubbled through the solution for 30 min. The
reaction mixture was concentrated under reduced pressure,

Potent, Orally Bioavailable â3 AR Agonist
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21 3835
J ) 8.6 Hz, 2H), 4.80 (m, 1H), 2.90-2.70 (m, 6H); EIMS m/z
625 (M+ + H); ESI HRMS m/z 625.1558 (M + H) (C31H27F3-
N4O3S2 requires 625.1549). Anal. (C31H27F3N4O3S2) H, N; C:
calcd, 59.60; found, 59.00.
cate tubes and then evaporated to dryness. The residue was
reconstituted in 100 µL of 50% acetonitrile/water, vortexed,
and then sonicated in an ultrasonic agitator for 10 min. The
mixture was transferred to autosampler vials with low-volume
micro inserts. Sample analysis was performed by APCI-LC/
MS/MS in the positive ion mode using a PhaseSep SCN
(4.6- × 150-mm) column. A mobile phase containing 60%
acetonitrile/water, 20 mM ammonium acetate and 0.4% tri-
fluoroacetic acid was used at a flow rate of 1.0 mL/min at
ambient temperature. Data reduction was performed using
Sciex MacQuan software. Area under the curve (AUC) calcula-
tions were performed by the trapezoid method using Kaleida-
graph.
Test compounds for intravenous administration were pre-
pared as 10 mg/mL solutions in a vehicle consisting of poly-
(ethylene glycol) 400 (PEG400), ethanol and normal saline (60/
20/20 v/v/v). The test compound was dosed to animals via an
accessible peripheral vein in a dose volume of 0.3 mL/kg thus
providing a dose of 3 mg/kg. Blood samples were collected prior
to dosing and at 5, 15, and 30 min and 1, 2, 4, 6, 8, and 24 h
postdosing, for determination of plasma glycerol concentrations
and plasma drug concentrations. Oral bioavailability (F) was
calculated according to the following equation: F (%) )
(AUCoral × doseiv) ÷ (AUCiv × doseoral).
(R)-N-[4-[2-[[2-Hydr oxy-2-(3-pyr idin yl)eth yl]am in o]eth -
yl]p h en yl]-4-[4-[2-(tr iflu or om eth yl)p h en yl]th ia zol-2-yl]-
ben zen esu lfon a m id e (2g). Prepared by general procedure
outlined for the preparation of 2a : 1H NMR (CD3OD) δ 8.55
(d, J ) 2.1 Hz, 1H), 8.46 (dd, J ) 4.9 and 1.6 Hz, 1H), 8.08 (d,
J ) 8.6 Hz, 2H), 7.84 (m, 2H), 7.82 (d, J ) 8.6 Hz, 2H), 7.69
(m, 3H), 7.61 (m, 1H), 7.41 (m, 1H), 7.15 (d, J ) 8.5 Hz, 2H),
7.08 (d, J ) 8.5 Hz, 2H), 4.85 (m, 1H), 3.10-2.80 (m, 6H);
EIMS m/z 625 (M+ + H); ESI HRMS m/z 625.1565 (M + H)
(C31H27F3N4O3S2 requires 625.1549).
(R)-N-[4-[2-[[2-Hydr oxy-2-(3-pyr idin yl)eth yl]am in o]eth -
yl]p h en yl]-4-[4-[4-(tr iflu or om eth oxy)p h en yl]th ia zol-2-yl]-
ben zen esu lfon a m id e (2h ). Prepared by general procedure
outlined for the preparation of 2a : 1H NMR (CD3OD) δ 8.52
(d, J ) 2.0 Hz, 1H), 8.43 (dd, J ) 4.9 and 1.6 Hz, 1H), 8.12 (d,
J ) 8.5 Hz, 2H), 8.10 (d, J ) 8.6 Hz, 2H), 7.98 (s, 1H), 7.83
(overlapping d, J ) 8.5 Hz, 2H and m, 1H), 7.38 (dd, J ) 7.9
and 5.0 Hz, 1H), 7.33 (d, J ) 8.6 Hz, 2H), 7.12 (d, J ) 8.5 Hz,
2H), 7.07 (d, J ) 8.5 Hz, 2H), 4.85 (dd, J ) 8.8 and 4.2 Hz,
1H), 2.7-3.1 (m, 6H); EIMS m/z 641 (M+ + H). Anal.
(C31H27F3N4O4S2) C, H, N.
4. Measu r em en t of Lipolysis an d Hear t Rate in Rh esu s
Mon k eys. Due to the small signal-to-noise ratio in conscious
animals, this assay was run in anesthetized rhesus monkeys,
which effectively precludes oral dosing. Male lean rhesus
monkeys (4-7 kg body weight, age 3-5 years; n ) 2-4/group)
were fasted for 24 h and were lightly anesthetized with
ketamine (Fort Dodge Labs, Fort Dodge, IA; 10 mg/kg, im). A
22-gauge intraveneous catheter (Becton Dickinson & Co.,
Sandy, UT) was placed in a saphenous vein for the adminis-
tration of test compounds after which the animals were
administered Nembutal (Abbott Labs, North Chicago, IL; 25
mg/kg, iv). A 20-gauge angiocatheter, connected to a TNF-R
pressure transducer (Ohmeda Medical Device Systems, Madi-
son, WI), was placed in a femoral artery for monitoring blood
pressure. ECG leads were connected for the continuous
measurement of heart rate. Heart rate and blood pressure
were monitored for approximately 30 min until stable baseline
values were obtained, at which time animals were adminis-
tered a series of rising dose infusions (0.1 mL/min) of test
compound or an equivalent volume of vehicle over a 15-min
period. Infusion periods were separated by an interval of
approximately 20 s. Blood samples (2 mL) were collected from
the femoral artery 1 min prior to the initiation of infusions
and 14 min into each infusion period. Serum glycerol was
measured using an enzymatic colorimetric assay and serum
potassium was determined using an ion-specific electrode.
Under these conditions, for isoproterenol the ED50 for hyper-
glycerolemia is 3 µg/kg and the ED50 for tachycardia is 0.2 µg/
kg.9
Biologica l Meth od s. 1. In Vitr o F u n ction a l Assa ys. The
human â3 receptor was obtained from Dr. J . Grannemann
(Wayne State University), and other receptors were cloned as
previously described.14,15 Human â1, â2 and â3 receptors were
expressed in mammalian cell lines for the primary screening
assays. CHO cells, stably transfected with the cloned â-adre-
nergic receptors, were harvested in enzyme-free dissociation
media (Specialty Media, Lavallette, NJ ) 3 days after plating.
Cells were counted and distributed in the assay tubes, after
being resuspended in ACC buffer (75 mM Tris, pH 7.4, 250
mM sucrose, 12.5 mM MgCl2, 1.5 mM EDTA), containing the
antioxidant sodium metabisulfite at a concentration of 0.2 mM
and a phosphodiesterase inhibitor (0.6 mM IBMX). The cAMP
production reaction was initiated by mixing cells with 20 µL
of a 6X stock of the ligand to be tested. Tubes were shaken at
275 rpm for 45 min at room temperature, and the reaction
was stopped by boiling the tubes for 3 min. The cAMP
produced in response to the ligand was measured in the lysate
by competing against [125I]cAMP for binding to a cAMP-
directed antibody using an automated RIA machine (ATTO-
FLO, Atto Instruments, Baltimore, MD). The cAMP level was
determined by comparison to a standard curve.
2. Bin d in g Assa ys. CHO cells expressing the cloned human
and rhesus â receptors were grown in selective media for 3
days and membranes prepared by hypotonic lysis in 1 mM
Tris, pH 7.2. Receptor binding assays were carried out in a
final volume of 250 µL containing 5-10 µg of membrane
protein, the radioligand [125I]cyanopindolol at a concentration
of 45 pM, and the compound of interest at various concentra-
tions. Binding reactions were carried out for 1 h at 23 °C and
terminated by filtration over GF/C filters using a 96-well cell
harvester from Inotech (Lansing, MI).
3. In Vivo Effica cy a n d Or a l Bioa va ila bility in Bea gle
Dogs. Test compounds for oral gavage were prepared as 10
mg/mL solutions in a vehicle consisting of methylcellulose
(0.5%) and sodium lauryl sulfate (0.2%) containing 2 equiv of
2 N HCl. The test compound was dosed by oral gavage to
animals in a dose volume of 1 mL/kg thus providing a dose of
10 mg/kg. Blood samples were collected prior to dosing and at
15 and 30 min and 1, 2, 6, 8, and 24 h postdosing, for
determination of plasma glycerol concentrations and, in some
cases, plasma drug concentrations. Glycerol levels were de-
termined on samples of cell-free plasma prepared by centrifu-
gation of the blood sample. Glycerol was assayed using a Sigma
triglyceride (GPO-TRINDER) assay kit. Plasma drug concen-
trations were determined by adding sodium carbonate (0.2 mL
of 0.1 M, pH ) 10) and ethyl acetate (4 mL) to 100 µL of plasma
sample. The mixture was shaken and then centrifuged. The
ethyl acetate layer was transferred to 13- × 100-mm borosili-
Ack n ow led gm en t. We thank Mr. Paul Cunning-
ham and Mr. Donald Hora, J r., for assistance with the
in vivo experiments, Ms. Amy Bernick for mass spectral
analyses, Mr. J oe Leone for preparation of intermediate
3, Dr. Gerard Kieczykowski for large-scale preparation
of 2e, and Prof. J ames G. Granneman (Wayne State
University) for supplying the cloned human â3 receptor.
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