(4-Piperidin-1-yl)phenyl amides: Potent and selective human β3 agonists

Article abstract of DOI:10.1021/jm000544b

In search of potent and selective human β₃ agonists as potential drugs for the treatment of human obesity and type II diabetes, a series of (4-piperidin-1-yl)phenyl amides was prepared and evaluated for their biological activity on the human β<

Full text of DOI:10.1021/jm000544b

1456
J . Med. Chem. 2001, 44, 1456-1466
(4-P ip er id in -1-yl)p h en yl Am id es: P oten t a n d Selective Hu m a n â3 Agon ists
Baihua Hu,*,† J ohn Ellingboe,† Stella Han,‡ Elwood Largis,‡ Ruth Mulvey,‡ Alexander Oliphant,†
Fuk-Wah Sum,† and J eff Tillett‡
Chemical Sciences and Cardiovascular/ Metabolic Diseases Research, Wyeth-Ayerst Research, Pearl River, New York 10965
Received December 21, 2000
In search of potent and selective human â3 agonists as potential drugs for the treatment of
human obesity and type II diabetes, a series of (4-piperidin-1-yl)phenyl amides was prepared
and evaluated for their biological activity on the human â3-adrenergic receptor. The leucine
derivative 26e and the reverse amide 33b were found to be the two most potent and selective
compounds in this study. With EC50 values of 0.008 and 0.009 µM, respectively, at the â3
receptor, nearly completely abolished intrinsic activity at either the â1 or â2 receptor, and
significant thermogenesis effects on human â3-adrenergic receptor transgenic mice, 26e and
33b are among the most potent and selective human â3 agonists known to date.
In tr od u ction
that desired metabolic effects could be achieved by â3-
AR agonists. This encouraged us to continue the â3-AR
agonists discovery program. In a previous study, (4-
piperidin-1-yl)phenyl sulfonamide 2 was found to be a
potent (â3-AR: EC50 ) 0.004 µM, IA ) 1.0) and selective
(â1-AR: EC50 ) 2.0 µM; â2-AR: EC50 ) 5.0 µM) human
â3-AR agonist, but it is a partial agonist at both â1 (IA
) 0.51) and â2 (IA ) 0.45) receptors.9 In this paper we
will describe the synthesis and structure-activity study
(SAR) of a variety of (4-piperidin-yl)phenyl amides,
leading to the discovery of a leucine derivative 26e and
a reverse amide 33b, which are among the most potent
and selective human â3-AR agonists reported to date.
The â3-adrenergic receptor (â3-AR), located on the
surface of adipocytes, has been shown to mediate
various pharmacological and physiological effects such
as lipolysis in white adipocyte tissue (WAT) and ther-
mogenesis in brown adipocyte tissue (BAT).1 Conse-
quently, a number of laboratories are engaged in
developing potent and selective â3-AR agonists for the
treatment of diverse human disease states, such as
obesity and type II diabetes. Early â3-AR agonists,
which were developed based on the rodent â3-AR, are
represented by CL 316243,2 BRL 37344,3 and CGP
12177A.4 These compounds have shown antiobesity
effects, such as mobilization of fat from WAT depots
(lipolysis), increased BAT-mediated thermogenesis, and
increased fat oxidation in rodents. In addition to anti-
obesity effects, they exhibit potent antidiabetic effects
(such as an increase in insulin secretion and improve-
ment in insulin-mediated glucose uptake) in rodent
model type II diabetes.5 However, human clinical trials
with these early â3-AR agonists have been disappoint-
ing due to a lack of selectivity or potency. Subsequent
cloning and expression of the human and rat â3-ARs
indicated a significant difference between the two.4 This
led to the recognition that a cloned human receptor
assay would offer major advantages over rodent models
for the identification of future â3-AR agonists.
Second-generation â3-AR agonists currently under
preclinical development are represented by tetrahy-
droisoquinoline 1,6 SB 226552,7 L 755507,8a and L
770644.8b,c These highly potent and selective compounds
were evaluated in Chinese hamster ovary (CHO) cells
expressing the cloned human â3-AR.
Despite the difference between the human and rat â3-
AR, a recently reported study1a demonstrated that
treatment of lean volunteers with CL 316243, which was
developed in our laboratories based on rodent models,
did induce lipolysis and fat oxidation and increased
insulin sensitivity. These new findings clearly suggest
Ch em istr y
The (4-piperidin-1-yl)phenyl amides and related com-
pounds were readily prepared by utilizing reductive
amination of piperidones with arylethanolamine 6.9 The
starting piperidone 5 was prepared by NaOH hydrolysis
of the ester 310 followed by HCl-catalyzed hydrolysis of
the resulting ketal 4. Reductive amination of piperidone
5 with arylethanolamine 6 gave the desired product 7
(Scheme 1).
Synthesis of the homologous phenylacetic acid 11 was
achieved as illustrated in Scheme 2. The acetophenone
810 was directly converted to the methyl arylacetate 9
by an oxidative rearrangement using thallium(III)
nitrate (TTN) in acidic methanol.11 Ester and ketal
hydrolysis followed by reductive amination, as described
above, gave the desired analogue 11.
Phenylpropionic acid 17 was prepared as outlined in
Scheme 3. The aldehyde 1210 was subjected a Horner-
Emmons reaction in the presence of triethylamine to
give the phenylacrylate 14. Basic ester hydrolysis fol-
lowed by acidic ketal hydrolysis and catalytic hydroge-
nation, as previously described in Scheme 1, produced
the phenylpropionic acid derivative 17.
Scheme 4 depicts the synthesis of benzyloxyacetic acid
20. The aldehyde 12 was reduced to the benzyl alcohol
18 by NaBH4. Alkylation of the benzyl alcohol with
iodoacetate sodium salt followed by ketal hydrolysis
gave the piperidone 19, which was further converted
into the final product 20 by reductive amination.
* To whom correspondence should be addressed. Tel: 845-732-5773.
Fax: 845-732-5561. E-mail: hub@war.wyeth.com.
† Chemical Sciences.
‡ Cardiovascular/Metabolic Diseases Research.
10.1021/jm000544b CCC: $20.00 © 2001 American Chemical Society
Published on Web 03/27/2001

(4-Piperidin-1-yl)phenyl Amides
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9 1457
Ch a r t 1
Sch em e 1. Synthesis of Benzoic Acid 7a
a
(a) NaOH, MeOH/THF (1:1), 99%; (b) HCl, 88%; (c) H2, Pd/C, MeOH, AcOH, 53%.
Sch em e 2. Synthesis of Phenylacetic Acid 11a
a
(a) TTN, perchloric acid, MeOH; (b) i. HCl, ii. NH4OH, 17% over steps a and b; (c) 6, H2, Pd/C, MeOH, 5%.
When the ketal 2110 was subjected to hydrolysis in
the presence of concentrated HCl, the cyano group of
21 was also hydrolyzed to generate the amide 22, which
gave the amide 23 after reductive amination with 6.
The amino acid derivatives 26a -g were prepared
according to Scheme 6. Coupling of the benzoic acid 5
with amino acid esters using EDC as the coupling agent
gave the piperidone 24. The reductive amination of 24

1458 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9
Sch em e 3. Synthesis of 17a
Hu et al.
a
(a) Et3N, LiBr, THF, 81%; (b) NaOH, 100%; (c) HCl, 90%; (d) 6, H2, Pd/C, MeOH, 94%.
Sch em e 4. Synthesis of 20a
a
(a) NaBH4, MeOH, 100%; (b) i. ICH2CO2Na, NaH, THF, ii. HCl, 26% over steps i and ii; (c) 6, H2, Pd/C, MeOH, 45%.
Sch em e 5. Synthesis of 23a
a
(a) HCl, 82%; (b) 6, H2, Pd/C, MeOH, 54%.
with 6 was carried out with NaBH(OAc)3 in DMF. The
target carboxylic acids were prepared by basic hydroly-
sis of the intermediate alkyl esters 25a ,c-f or via
catalytic hydrogenation of the benzyl esters 24b,g with
6. The N-butylglycine derivative 29 was prepared by a
similar route as shown in Scheme 7.
The reverse amide 33 was conveniently prepared as
outlined in Scheme 8. Acylation of the aniline 309 with
ethyl 3-chloro-3-oxopropionate gave the corresponding
amide 31. Reductive amination followed by basic ester
hydrolysis, as previously described in Scheme 6, pro-
duced the reverse amide derivative 33.
hamster ovary (CHO) cells expressing the cloned human
â3-, â2-, and â1-AR receptors, and the results are
summarized in Table 1. As a reference, the parent
benzoic acid 7 was tested for activity and selectivity at
the human â3 receptor and was found to be a modestly
potent â3-AR full agonist (EC50 ) 0.22 µM, IA ) 1.20)
with greater than 200-fold selectivity versus both the
â2- and â1-ARs. The homologous analogue 11 showed
similar activity at the â3 receptor with EC50 ) 0.27 µM
and IA ) 0.86. Increasing the tether length between the
carboxylic acid and the right-hand-side phenyl (phenyl-
propionic acid 17 and benzoxylacetic acid 20) resulted
in a moderate increased (3-6-fold) activity at the â3
receptor, but selectivity versus the â1 or â2 receptor was
decreased. Furthermore, both showed significant ago-
nist activity at both the â2- and â1-AR receptors (IA )
0.56-0.92).
Biologica l Resu lts a n d Discu ssion
The (4-piperidin-1-yl)phenyl amides and related com-
pounds were tested for their in vitro activity13 in
stimulating an increase in cAMP levels in Chinese

(4-Piperidin-1-yl)phenyl Amides
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9 1459
Sch em e 6. Synthesis of Amino Acid Derivatives 26a -g
Sch em e 7. Synthesis of N-Butylglycine Derivative 29a
a
(a) N-Butylglycine ethyl ester,12 EDC, NMM, CH2Cl2; (b) 6, NaBH(OAc)3, DMF, 10% over steps a and b; (c) NaOH, 60%.
Sch em e 8. Synthesis of Reverse Amide Derivative 33a
a
(a) Ethyl 3-chloro-3-oxopropionate, Et3N, CH2Cl2; (b) 6, NaBH(OAc)3, DMF; (c) NaOH.
While the amide derivative 23 showed improved (11-
fold) potency as compared to 7 at the human â3 receptor,
with EC50 ) 0.02 µM and 112% intrinsic activity, it was
not very selective versus the â1 receptor (IA ) 0.69). A
variety of amino acid derivatives were then examined.
To our delight, glycine derivative 26a proved to be a
potent agonist of the human â3 receptor (EC50 ) 0.06
µM, IA ) 0.95) with dramatically decreased intrinsic
activity at both the â2- and â1-ARs (IA ) 5% and 4%,
respectively). The corresponding ester 25a showed a
moderate increased (4-fold) activity at the â3 receptor,
but it was less selective versus the â2 (IA ) 0.36) and
â1 receptors (IA ) 0.25). This result is consistent with
a previous finding14 that carboxylic acids are usually
more selective than the corresponding esters.
Effects of alkylation of the amide NH on â3 agonist
activity/selectivity were then examined. N-Butyl amide
29 showed EC50 ) 0.036 µM. Only a minor improvement
(1.7-fold as compared to 26a ) in its agonist activity was
observed. However, it had significant partial agonist

1460 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9
Hu et al.
Ta ble 1. In Vitro Activity for Benzoic Acid Derivatives
EC50, µM (IA)a
Ta ble 2. Thermogenesis and â1-AR/â2-AR Binding Data
bindingb Ki, µM
compd
â3-AR
â2-AR
(0.02)b
â1-AR
compd
thermogenesis (%)a
â1-AR
â2-AR
L 75550716 0.001 (0.96)b
0.33 (0.30)b
48.7 ( 20 (0.6)
nd
9.8 ( 0.6 (0.56)
1.6 ( 0.1 (0.75)
1.2 ( 0.4 (0.69)
4.5 ( 0.3 (0.25)
(0.05)
CGP 12177A
vehiclec
L 755507
CL 316243
26a
0.00025
0.0001
7
11
17
20
0.22 (1.2)b
0.27 ( 0.05 (0.86)
>50 (0.19)
0 ( 10
28 ( 5
ndd
ndc
0.57
0.16
79.2
11.6
3.6
3.4
0.48
>100
0.039 ( 0.019 (1.1) 4.7 ( 1.2 (0.69)
0.066 ( 0.019 (0.69) 6.0 ( 0.1 (0.92)
0.020 ( 0.004 (1.12) (0.27)
>100
>100
>100
>100
>100
>100
51 ( 817
nd
23
26d
26e
25a
26a
26b
26c
26d
26e
26f
26g
29
0.015 (0.99)b
0.06 (0.95)b
3.2 ( 0.9 (0.36)
(0.04)
nd
(0.02)
53 ( 8
nd
35 ( 12
26f
0.26 (0.94)b
nd
(0.0)
33b
0.098 (1.01)b
0.026 (1.1)b
a
>10 (0.27)
>10 (0.31)
Compounds were tested for increased thermogenesis (%) using
â3 human transgenic mice at 10 mg/kg ip. Compounds were
considered active if they were able to produce a statistically
significant 15% increase in thermogenesis in â3 transgenic mice.
b Binding potency is reported as Ki, the binding inhibition constant,
determined by inhibition of 125I-iodocyanopindolol. Results are
given as the mean of two or three independent experiments.
cVehicle is 0.5% methylcellulose:0.1% Tween-80 and injected 0.2
mL/mouse ip. dnd ) not determined.
0.008 (0.96)b
0.004 (1.13)b
0.92 ( 0.16 (0.88)
(0.08)b
(0.06)b
1.5 (0.41)b
nd
43.7 (0.65)b
nd
0.036 ( 0.011 (1.1) 10.5( 0.28 (0.59) >1 (0.29)
33a
33b
0.13 (1.0)b
(0.09)
(0.1)
0.009 (1.1)b
(0.06)b
(0.04)b
a
â-AR agonistic activities were assessed by measurement of
cAMP accumulation levels in CHO cells expressing human â-ARs.
The intrinsic activities (IA) were given as a fraction of the maximal
stimulation with isoproterenol. All results are based on a single
dose-response determination ( SE, unless otherwise indicated.
Number of doses: n ) 7 for â2 and â1 dose-response curves; n )
reverse amide series. The parent amide 33a was a
rather modest â3 agonist (EC50 ) 0.13 µM), but N-butyl
derivative 33b exhibited increased activity (EC50
0.009 µM, IA ) 1.10). A good selectivity profile was seen
with both 33a and 33b.
)
b
14 for â3 dose-response curves. Results are given as the mean
of two or three independent dose-response determinations. c nd
) not determined.
Compounds with low cAMP functional activity in â1-
and â2-ARs may exhibited strong antagonist activity
which may cause unwanted side effects.1 Several of the
more selective compounds and three known selective â3
agonists (CGP 12177A, L 755507, and CL 316243) were
evaluated in â1- and â2-AR binding assays, and the
results are shown in Table 2. In contrast to the known
antagonist CGP 12177A, which had very strong binding
affinities for â2 and â1-ARs, glycine derivative 26a , three
branched amino acid analogues 26d -f, and the reverse
amide 33b all had a binding inhibition constant (Ki)
greater than 100 µM at the â1 receptor site and Ki )
11.6, 3.6, 3.4, 0.48, and >100 µM, respectively, at the
â2 receptor site. These results suggested that 26a , 26d -
f, and 33b had very weak binding affinities to â2- and
â1-ARs, thus further confirming their selectivity for â3-
AR versus â1- and â2-ARs.
Three of the most interesting compounds and the
standard L 755507 were examined in vivo for their
thermogenesis effect,15 and the results are included in
Table 2. L 755507 has been shown to be active when
given iv to monkeys resulting in increased UCP-1 mRNA
in BAT.8a When given ip to human â3 transgenic mice
at a dose of 10 mg/kg, L 755507 increased thermogenesis
by 28%. This confirmed that our â3 transgenic mice
model is giving a result that is comparable to this
reference compound when it was given to nonhuman
primates. Under our assay conditions, all three new
compounds (25a ,17 26e, 33b) produced a major effect
(more than 35% increase) in thermogensis.
activity at the â2 receptor (IA ) 0.59) and modest partial
agonist activity at the â1 receptor (IA ) 0.29).
More amino acid derivatives were then evaluated.
Lengthening the carboxylic acid tether by one carbon
atom (â-alanine derivative 26b) resulted in a moderate
decrease (4.3-fold) in â3-AR activity. Proline derivative
26c, a conformationally constrained analogue of 26a ,
was also less active (EC50 ) 0.098 µM at the â3-AR) as
compared to the glycine analogue 26a . While introduc-
tion of an isopropyl group at the R-position of glycine
led to a branched amino acid derivative (valine 26d ),
which had a minor increased â3 activity (2.3-fold) as
compared to the glycine analogue 26a , introduction of
an isobutyl group resulted in a leucine analogue 26e
which proved to be about 8-fold more potent than glycine
26a at the â3 receptor. Furthermore, agonist 26e
exhibited a remarkable selectivity profile with IA ) 8%
for the â2-AR and 6% for the â1-AR. This suggests that
the spatial arrangement of the alkyl substituent and
the carboxylic acid is important for maintaining â3
agonist activity and selectivity as the isomeric N-butyl
analogue 29 was about 5-fold less potent at the â3
receptor and much less selective versus either the â1-
or â2-AR. Thus, with EC50 ) 0.008 µM and full agonist
activity at the â3 receptor, and nearly completely
abolished intrinsic activity at either the â1- or â2-AR,
to our knowledge 26e is among the most potent and
selective human â3-AR agonists reported to date.
Replacement of the isobutyl substituent with the
larger benzyl group (phenylalanine derivative 26f)
resulted in a minor increase (2-fold) in â3-AR agonist
activity. However, it was less selective than 26e.
Introduction of another carboxylic acid substituent
(glutamic acid 26g) diminished the â3-AR activity (EC50
) 0.92 µM). All of these results suggest that the
combination of a lipophilic alkyl group with the correct
spatial orientation and a polar carboxylic acid is impor-
tant for activity and selectivity for the â3-AR. This same
combination/activity pattern was also observed with the
Con clu sion s
In this study the synthesis and SAR within a series
of (4-piperidin-1-yl)phenyl amide based â3 agonists has
been discussed. With EC50 ) 0.008 and 0.009 µM,
respectively, at the human â3 receptor, nearly com-
pletely abolished intrinsic activity at either the â1 or â2
receptor, the leucine derivative 26e and the reverse
amide 33b are the two most potent and selective
compounds in this study. These compounds also showed

(4-Piperidin-1-yl)phenyl Amides
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9 1461
1
Method A, 5, off-white solid, 53% yield: mp >85 °C dec; H
good thermogenesis effects in human â3-AR transgenic
mice. On the basis of their desirable in vitro and in vivo
properties, 26e and 33b were chosen for further biologi-
cal evaluations.
NMR (DMSO-d6) δ 1.20-1.40 (m, 2 H), 1.80-1.95 (m, 2 H),
2.60-3.00 (m, 5 H), 2.92 (s, 3 H), 3.75-3.85 (m, 2 H), 4.53
(dd, J ) 8.3, 4.0 Hz, 1 H), 6.82 (d, J ) 8.3 Hz, 1 H), 6.92 (d, J
) 8.9 Hz, 2 H), 7.01 (dd, J ) 8.3, 2.0 Hz, 1 H), 7.19 (d, J ) 2.0
Hz, 1 H), 7.73 (d, J ) 8.9 Hz, 2 H); MS (ES) m/z 449.9 (MH+);
HRMS calcd for C21H26N3O6S (M - H)- 448.1549, found
448.1541. Anal. (C21H27N3O6S‚2H2O‚0.5MeOH) C, H, N.
Exp er im en ta l Section
Gen er a l. Melting points were determined in open capillary
tubes on a Meltemp melting point apparatus and are uncor-
rected. 1H NMR spectra were determined with a Bruker DPX-
300 spectrometer at 300 MHz. Chemical shifts δ are expressed
in parts per million relative to the internal standard tetram-
ethylsilane and J values (coupling constant) in Hz. Electro-
spray (ES) mass spectra were recorded in positive or negative
mode on a Micromass Platform spectrometer. Electron impact
and high-resolution mass spectra were obtained on a Finnigan
MAT-90 spectrometer. Combustion analyses were obtained
using a Perkin-Elmer Series II 2400 CHNS/O analyzer.
Chromatographic purifications were performed by flash chro-
matography using Baker 40-µm silica gel. Thin-layer chroma-
tography (TLC) was performed on Analtech silica gel GHLF
250 M prescored plates.
4-(1,4-Dioxa -8-a za sp ir o[4.5]d ec-8-yl)ben zoic Acid (4).
A mixture of 4-(1,4-dioxa-8-azaspiro[4.5]dec-8-yl)benzoic acid
ethyl ester10 (14 g, 48.1 mmol) in MeOH/THF (1:1, 100 mL)
and NaOH (2 N, 150 mL) was refluxed for 2 h. After cooling
to room temperature, the solution was acidified with acetic
acid and the solid which was formed was collected and dried
to give the title compound as a white solid (12.5 g, 99%): mp
170-172 °C; 1H NMR (CDCl3) δ 1.67 (t, J ) 5.6 Hz, 4 H), 3.43
(t, J ) 5.6 Hz, 4 H), 3.92 (s, 4 H), 6.96 (d, J ) 9.0 Hz, 2 H),
7.75 (d, J ) 9.0 Hz, 2 H); MS (ES) m/z 264.4 (MH+); HRMS
calcd for C14H17NO4(M+) 263.1158, found 263.1160. Anal.
(C14H17NO4) C, H, N.
4-(4-Oxop ip er id in -1-yl)b en zoic Acid (5). Compound 4
(12.5 g, 47.5 mmol) was treated with concentrated HCl (500
mL) at room temperature. After 15 h, ∼28% NH4OH was
added dropwise and the precipitate was collected by filtration
and dried over P2O5 to give the title compound as a white solid
(9.2 g, 88%): mp 209-211 °C; 1H NMR (CDCl3) δ 2.46 (t, J )
6.1 Hz, 4 H), 3.74 (t, J ) 6.1 Hz, 4 H), 7.02 (d, J ) 9.0 Hz, 2
H), 7.80 (d, J ) 9.0 Hz, 2 H); MS (ES) m/z 219.8 (MH+); HRMS
calcd for C12H14NO3 (MH+) 220.0974, found 220.0947. Anal.
(C12H13NO3) C, H, N.
Gen er a l P r oced u r es for P r ep a r a tion of Com p ou n d s
7, 11, 17, 20, 23, 25a -f, 26a -f, 33a ,b. Meth od A. A mixture
of the piperidone (as specified, 1 equiv), acetic acid (1-5 equiv)
and N-[5-((1R)-2-amino-1-hydroxyethyl)-2-hydroxyphenyl]-
methanesulfonamide (6)9 (1 equiv) in EtOH (0.01-0.5 M) was
hydrogenated in the presence of 10% Pd/C (5-15 wt %) under
H2 (5-20 psi) for overnight. The catalyst was then removed
by filtering through a short pad of silica gel. The filtrate was
concentrated and purified by preparative TLC, silica gel
chromatography using 0-10% MeOH/CH2Cl2 as eluent or
recrystallization from MeOH/CH2Cl2.
[4-(4-Oxop ip er id in -1-yl)p h en yl]a cetic Acid (10). 1-[4-
(1,4-Dioxa-8-azaspiro[4.5]dec-8-yl)phenyl]ethanone10(2.61 g, 10
mmol) was added to a solution of thallium trinitrate trihy-
drate11 (4.44 g, 10 mmol) in MeOH (40 mL) and CH2Cl2 (20
mL) containing perchloric acid (70%, 10 mL). After 5 h at room
temperature, the precipitated thallium(I) nitrate was removed
by filtration and the filtrate was diluted with water (100 mL).
The organic phase was separated, the aqueous layer was
extracted with CH2Cl2, and the combined organic phases were
washed with water, dried over Na2SO4 and concentrated. The
residue was dissolved in concentrated HCl (250 mL) at room
temperature. After 3 h the solution pH was adjusted to 5 with
∼28% NH4OH. The aqueous layer was extracted with CH2-
Cl2, and the combined organic phases were washed with water,
dried over Na2SO4 and concentrated. The product was purified
by chromatography on silica gel with EtOAc/hexanes as eluant
to give the title compound as a pale yellowish solid (0.4 g,
17%): 1H NMR (DMSO-d6) δ 2.42 (t, J ) 6.0 Hz, 4 H), 3.41 (s,
2 H), 3.56 (t, J ) 6.0 Hz, 4 H), 6.98 (d, J ) 8.7 Hz, 2 H), 7.12
(d, J ) 8.7 Hz, 2 H), 12.20 (s, 1 H); MS (ES) m/z 234.3 (MH+);
HRMS calcd for C13H15NO3 (M+) 233.1052, found 233.1045.
Anal. (C13H15NO3) C, H, N.
(4-{4-[(2R)-2-Hydr oxy-2-(4-h ydr oxy-3-m eth an esu lfon yl-
a m in op h en yl)et h yla m in o]p ip er id in -1-yl}p h en yl)a cet ic
Acid (11). Method A, 10, pale yellowish solid, 5% yield: mp
>140 °C dec; 1H NMR (DMSO-d6) δ 1.25-1.40 (m, 2 H), 1.75-
1.95 (m, 2 H), 2.50-3.60 (m, 7 H), 2.88 (s, 3 H), 3.16 (s, 2 H),
4.47 (dd, J ) 8.0, 4.1 Hz, 1 H), 6.78 (d, J ) 8.2 Hz, 1 H), 6.84
(d, J ) 8.3 Hz, 2 H), 6.95 (dd, J ) 8.2, 1.7 Hz, 1 H), 7.05 (d, J
) 8.3 Hz, 2 H), 7.16 (d, J ) 1.7 Hz, 1 H); MS (ES) m/z 464.1
(MH+); HRMS calcd for C22H28N3O6S (M - H)- 462.1704, found
462.1696. Anal. (C22H29N3O6S‚2H2O‚0.15AcOH) C, H, N.
(E)-3-[4-(1,4-Dioxa-8-azaspir o[4.5]dec-8-yl)ph en yl]acr yl-
ic Acid E t h yl E st er (14). To a solution of LiBr (0.53 g, 6
mmol) in THF (5 mL) was added triethyl phosphonoacetate
(1.14 g, 5 mmol), followed by Et3N (0.51 g, 5 mmol). After
stirring for 10 min, a solution of 4-(1,4-dioxa-8-azaspiro[4.5]-
dec-8-yl)benzaldehyde10 (1.24 g, 5 mmol) in THF (2 mL) was
added, and the mixture was stirred at room temperature for
20 h. Additional triethyl phosphonoacetate (0.57 g, 2.5 mmol)
and Et3N (0.25 g, 2.5 mmol) were added, and stirring was
continued for 3 days. The mixture was evaporated, and the
residue stirred with 20 mL of water and 5 mL of 1 N HCl.
The aqueous suspension was filtered, and the precipitate was
washed with water and dried in vacuo to give 1.28 g (81%) of
a light yellow solid: mp 115-116 °C; 1H NMR (CDCl3) δ 1.33
(t, 3 H), 1.81 (dd, 4 H), 3.47 (dd, 4 H), 4.00 (s, 4 H), 4.25 (q, 2
H), 6.26 (d, 1 H), 6.89 (d, 2 H), 7.42 (d, 2 H), 7.62 (d, 1 H); MS
(ES) m/z 318.0 (MH+); HRMS calcd for C18H24NO4 (MH+)
318.1700, found 318.1699. Anal. (C18H23NO4) C, H, N.
Meth od B. The piperidone (as specified, 1 equiv) and 6 (1
equiv) were mixed in DMF (0.05-0.3M) and then treated with
NaBH(OAc)3 (1.5-5 equiv) and acetic acid (1.5-5 equiv). After
stirring at room temperature under a N2 atmosphere for 1-24
h the mixture was quenched with 1 N NaOH and then poured
into a saturated aqueous NaHCO3. The precipitate which
formed was collected and purified by preparative TLC or silica
gel chromatography using 0-10% MeOH/CH2Cl2 as eluent.
Meth od C. To a stirred solution of the alkyl ester (as
specified, 1 equiv) in distilled water (0.01-0.5 M) was added
1 N NaOH (1-10 equiv). The reaction was stirred at room
temperature for 2-24 h. The reaction mixture was made acidic
(pH 6) with glacial acetic acid, and the solid was collected and
dried over P2O5. The product was purified (if needed) by
preparative TLC, silica gel chromatography using 0-10%
MeOH/CH2Cl2 as eluent or recrystallization from MeOH/
CH2Cl2.
(E)-3-[4-(1,4-Dioxa-8-azaspir o[4.5]dec-8-yl)ph en yl]acr yl-
ic Acid (15). Prepared from 14 according to the procedure for
1
4 as a light yellow solid in 100% yield: mp 221-222 °C; H
NMR (DMSO-d6) δ 1.67 (dd, 4 H), 3.40 (dd, 4 H), 3.91 (s, 4 H),
6.26 (d, 1 H), 6.96 (d, 2 H), 7.47 (d, 1 H), 7.50 (d, 2 H), 12.10
(br s, 1 H); MS (ES) m/z 290 (MH+); HRMS calcd for C16H20
-
NO4 (MH+) 290.1421, found 290.1421. Anal. (C16H19NO4)
C, H, N.
(E)-3-[4-(4-Oxop ip er id in -1-yl)p h en yl]a cr ylic Acid (16).
Prepared from 15 according to the procedure for 5 as a light
yellow solid in 90% yield: mp 215 °C; H NMR (DMSO-d6) δ
2.42 (t, 4 H), 3.71 (t, 4 H), 6.28 (d, 1 H), 7.01 (d, 2 H), 7.50 (d,
1 H), 7.55 (d, 2 H); MS (ES) m/z 246.3 (MH+); HRMS calcd for
C14H16NO3 (MH+) 246.1123, found 246.1127. Anal. (C14H15NO3‚
0.55H2O) C, H, N.
1
4-{4-[(2R)-2-Hyd r oxy-2-(4-h yd r oxy-3-m eth a n esu lfon yl-
am in oph en yl)eth ylam in o]piper idin -1-yl}ben zoic Acid (7).

1462 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9
Hu et al.
3-(4-{4-[(R)-2-H yd r oxy-2-(4-h yd r oxy-3-m et h a n esu lfo-
n yla m in op h e n yl)e t h yla m in o]p ip e r id in -1-yl}p h e n yl)-
p r op ion ic Acid (17). Method A, 16, off-white solid, 94%
Gen er a l P r oced u r e for P r ep a r a tion of Com p ou n d s
24a -f. A mixture of 4-(4-oxopiperidin-1-yl)benzoic acid (5) (1
equiv), 1-[3-(dimethylamino)propyl-3-ethylcarbodiimide hy-
drochloride (1.1-1.5 equiv) and the amine salt (as specified,
1-1.5 equiv) was stirred in CH2Cl2 (0.01-0.3 M). N-Methyl-
morpholine (2-5 equiv) was added dropwise and the mixture
was stirred for 2-24 h. The mixture was then washed with
0.05 N HCl and water. The resulting solution was dried with
MgSO4 and concentrated. The residue was purified by silica
gel chromatography using 0-10% MeOH/CH2Cl2 as eluant.
1
yield: mp 130-133 °C; H NMR (DMSO-d6) δ 1.30-1.46 (m,
2 H), 1.84-1.95 (m, 2 H), 2.37-2.50 (m, 4 H), 2.60-2.78 (m, 4
H), 2.92 (s, 3 H), 3.53-3.62 (m, 3 H), 4.46-4.55 (m, 1 H), 6.85
(d, 2 H), 6.92-7.10 (m, 4 H), 7.18 (dd, 1 H); MS (ES) m/z 478.3
(MH+); HRMS calcd for C23H32N3O6S (MH+) 478.2011, found
478.2013. Anal. (C23H31N3O6S‚2.5H2O) C, H, N.
[4-(1,4-Dioxa -8-a za sp ir o[4.5]d ec-8-yl)p h en yl]m et h a -
n ol (18). NaBH4 (3.78 g, 100 mmol) was added in four portions
to a stirred solution of 4-(1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-
benzaldehyde10 (4.94 g, 20 mmol) in MeOH /THF (1:1, 100 mL)
at 0 °C and the resulting solution was stirred at 0 °C for 30
min and at room temperature for 1 h. CH2Cl2 was added and
the solution was washed with water and dried over Na2SO4.
The solvents were removed and the residue solidified upon
standing (5.0 g, 100%): 1H NMR (CDCl3) δ 1.84 (t, J ) 5.7
Hz, 4 H), 3.33 (t, J ) 5.7 Hz, 4 H), 3.99 (s, 4 H), 4.59 (d, J )
5.8 Hz, 2 H), 6.93 (d, J ) 8.7 Hz, 2 H), 7.24 (d, J ) 8.7 Hz, 2
H); MS (ES) m/z 249.9 (MH+); HRMS calcd for C14H20NO3
(MH+) 250.1443, found 250.1434. Anal. (C14H19NO3‚0.18H2O)
C, H, N.
Eth yl {[4-(4-Oxo-1-p ip er id in yl)ben zoyl]a m in o}a ceta te
(24a ). Glycine ethyl ester hydrochloride, pale yellowish solid,
1
66% yield: mp 98-99 °C; H NMR (DMSO-d6) δ 1.20 (t, J )
7.1 Hz, 3 H), 2.43 (t, J ) 6.1 Hz, 4 H), 3.72 (t, J ) 6.1 Hz, 4
H), 3.95 (d, J ) 5.8 Hz, 2 H), 4.10 (q, J ) 7.1 Hz, 2 H), 7.04 (d,
J ) 8.9 Hz, 2 H), 7.77 (d, J ) 8.9 Hz, 2 H), 8.63 (t, J ) 5.8 Hz,
1 H); MS (ES) m/z 305.2 (MH+); HRMS calcd for C16H20N2O4
(M+) 304.1423, found 304.1422. Anal. (C16H20N2O4‚0.2H2O) C,
H, N.
3-[4-(4-Oxopiper idin -1-yl)ben zoylam in o]pr opion ic Acid
Ben zyl Ester (24b). â-Alanine benzyl ester p-tosylate, white
solid, 41% yield: mp 87-89 °C; 1H NMR (DMSO-d6) δ 2.42 (t,
J ) 6.0 Hz, 4 H), 2.63 (t, J ) 6.9 Hz, 2 H), 3.40-3.50 (m, 2 H),
3.71 (t, J ) 6.0 Hz, 4 H), 5.10 (s, 2 H), 6.74 (d, J ) 8.9 Hz, 2
H), 7.30-7.40 (m, 5 H), 7.73 (d, J ) 8.9 Hz, 2 H), 8.29 (t, J )
5.5 Hz, 1 H); MS (ES) m/z 381.3 (MH+); HRMS calcd for
C22H24N2O4 (M+) 380.1737, found 380.1755. Anal. (C22H24N2O4)
C, H, N.
[4-(4-Oxop ip er id in -1-yl)ben zyloxy]a cetic Acid (19). A
solution of 18 (3.49 g, 14 mmol) in THF (100 mL) was treated
with 60% NaH in mineral oil (0.50 g, 21 mmol) and heated to
a gentle reflux for 2 h. After cooling to room temperature,
iodoacetic acid sodium salt (2.91 g, 14 mmol) was added in
one portion. The resulting mixture was heated to a gentle
reflux for another 2 h. After cooling to room temperature the
reaction was quenched by careful addition of water and then
partitioned between water and CH2Cl2. The aqueous layer was
acidified with acetic acid and extracted with CH2Cl2. The
organic phase was dried over Na2SO4, and concentrated. The
residue was treated with concentrated HCl (150 mL) at room
temperature. After 3 h the solution pH was adjusted to 4 with
∼28% NH4OH. The aqueous layer was extracted with CH2-
Cl2, and the combined organic phases were washed with water,
dried over Na2SO4 and concentrated to give a gum (19) (0.95
g, 26%): 1H NMR (CDCl3) δ 2.55 (t, J ) 6.1 Hz, 4 H), 3.61 (t,
J ) 6.1 Hz, 4 H), 4.10 (s, 2 H), 4.56 (s, 2 H), 6.95 (d, J ) 8.6
Hz, 2 H), 7.29 (d, J ) 8.6 Hz, 2 H); MS (ES) m/z 264.3 (MH+).
Meth yl 1-[4-(4-Oxo-1-p ip er id in yl)ben zoyl]-L-p r olin a te
(24c). L-Proline methyl ester hydrochloride, pale yellowish
1
solid, 54% yield: mp 79-81 °C; H NMR (DMSO-d6) δ 1.70-
1.95 (m, 4 H), 2.20-2.40 (m, 1 H), 2.41 (t, J ) 6.0 Hz, 4 H),
3.32 (s, 3 H), 3.69 (t, J ) 6.0 Hz, 4 H), 4.35-4.50 (m, 2 H),
7.01 (d, J ) 8.7 Hz, 2 H), 7.49 (d, J ) 8.7 Hz, 2 H); MS (ES)
m/z 331.2 (MH+); HRMS calcd for C18H22N2O4 (M+) 330.1580,
found 330.1572. Anal. (C18H22N2O4‚0.2H2O) C, H, N.
E t h yl 1-[4-(4-Oxo-1-p ip er id in yl)b en zoyl]-L-va lin a t e
(24d ). L-Valine ethyl ester hydrochloride, pale yellowish solid,
1
72% yield: mp 51-52 °C; H NMR (DMSO-d6) δ 0.93 (d, J )
6.8 Hz, 3 H), 0.97 (d, J ) 6.8 Hz, 3 H), 1.20 (t, J ) 7.1 Hz, 3
H), 2.10-2.30 (m, 1 H), 2.43 (t, J ) 6.0 Hz, 4 H), 3.72 (t, J )
6.0 Hz, 4 H), 4.00-4.30 (m, 3 H), 4.46 (t, J ) 5.1 Hz, 1 H),
7.05 (d, J ) 8.9 Hz, 2 H), 7.82 (d, J ) 8.9 Hz, 2 H), 8.21 (d, J
) 7.9 Hz, 1 H); MS (ES) m/z 347.3 (MH+); HRMS calcd for
C19H26N2O4 (M+) 346.1892, found 346.1896. Anal. (C19H26N2O4‚
0.25H2O) C, H, N.
(4-{4-[(2R)-2-Hydr oxy-2-(4-h ydr oxy-3-m eth an esu lfon yl-
a m in op h e n yl)e t h yla m in o]p ip e r id in -1-yl}b e n zyloxy)-
a cetic Acid (20). Method A, 19, off-white solid, 45% yield:
1
mp >145 °C dec; H NMR (DMSO-d6) δ 1.30-1.50 (m, 2 H),
1.80-2.00 (m, 2 H), 2.60-2.80 (m, 5 H), 2.90 (s, 3 H), 3.50-
3.70 (m, 2 H), 3.65 (s, 2 H), 4.52 (s, 2 H), 4.54 (dd, J ) 8.3, 3.8
Hz, 1 H), 6.75-6.90 (m, 3 H), 7.00 (dd, J ) 8.3, 2.0 Hz, 1 H),
7.02-7.20 (m, 3 H); MS (ES) m/z 494.2 (MH+); HRMS calcd
for C23H30N3O7S (M - H)- 492.1810, found 492.1804. Anal.
(C23H31N3O7S‚H2O‚CH2Cl2) C, H, N.
E t h yl 1-[4-(4-Oxo-1-p ip er id in yl)b en zoyl]-L-leu cin a t e
(24e). L-Leucine ethyl ester hydrochloride, white flakes, 76%
1
yield: mp 75-77 °C; H NMR (CDCl3) δ 0.99 (d, J ) 6.2 Hz,
6 H), 1.30 (t, J ) 7.1 Hz, 3 H), 1.60-1.80 (m, 3 H), 2.56 (t, J
) 6.0 Hz, 4 H), 3.72 (t, J ) 6.0 Hz, 4 H), 4.22 (q, J ) 7.1 Hz,
2 H), 4.80-4.90 (m, 1 H), 6.42 (d, J ) 8.2 Hz, 1 H), 6.94 (d, J
) 9.0 Hz, 2 H), 7.77 (d, J ) 9.0 Hz, 2 H); MS (ES) m/z 361.3
(MH+); HRMS calcd for C20H28N2O4 (M+) 360.2049, found
360.2081. Anal. (C20H28N2O4) C, H, N.
4-(4-Oxo-1-p ip er id in -1-yl)ben za m id e (22). A suspension
of 4-(1,4-dioxa-8-azaspiro[4.5]dec-8-yl)benzonitrile (15.0 g, 61.5
mmol)10 in 150 mL of concentrated HCl was stirred at room
temperature overnight. The solution was neutralized with 5
N NaOH and the solid that formed was collected to give the
title compound (11.0 g, 82%) as a white solid: mp 186-188
Met h yl 1-[4-(4-Oxo-1-p ip er id in yl)b en zoyl]-L-p h en yl-
a la n in a te (24f). L-Phenylalanine methyl ester hydrochloride,
1
pale yellowish solid, 75% yield: mp 30 °C; H NMR (DMSO-
1
°C; H NMR (CDCl3) δ 2.58 (t, J ) 6.1 Hz, 4 H), 3.74 (t, J )
d6) δ 2.41 (t, J ) 6.0 Hz, 4 H), 3.05-3.20 (m, 2 H), 3.71 (t, J )
6.0 Hz, 4 H), 4.55-4.65 (m, 1 H), 6.97 (d, J ) 8.9 Hz, 2 H),
7.10-7.45 (m, 5 H), 7.73 (d, J ) 8.9 Hz, 2 H), 8.52 (d, J ) 7.8
Hz, 1 H); MS (ES) m/z 381.3 (MH+); HRMS calcd for C22H25N2O4
(MH+) 381.1809, found 381.1807. Anal. (C22H24N2O4‚0.4H2O)
C, H, N.
6.1 Hz, 4 H), 5.76 (brs, 2 H), 6.94 (d, J ) 9.0 Hz, 2 H), 7.74 (d,
J ) 9.0 Hz, 2 H); MS (ES) m/z 218.8 (MH+); HRMS calcd for
C12H14N2O2 (M+) 218.1055, found 218.1051. Anal. (C12H14N2O2)
C, H, N.
4-{4-[(2R)-2-Hyd r oxy-2-(4-h yd r oxy-3-m eth a n esu lfon yl-
am in oph en yl)eth ylam in o]piper idin -1-yl}ben zam ide (23).
Method A, 22, white solid, 54% yield: mp >75 °C dec; 1H NMR
(DMSO-d6) δ 1.2-1.40 (m, 2 H), 1.70-1.90 (m, 2 H), 2.55-
2.95 (m, 5 H), 2.97 (s, 3 H), 3.70-3.85 (m, 2 H), 4.48 (dd, J )
8.0, 4.3 Hz, 1 H), 6.81 (d, J ) 8.2 Hz, 1 H), 6.91 (d, J ) 9.0 Hz,
2 H), 6.99 (dd, J ) 8.2, 2.0 Hz, 1 H), 7.17 (d, J ) 2.0 Hz, 1 H),
7.67 (br s, 2 H), 7.72 (d, J ) 9.0 Hz, 2 H); MS (ES) m/z 448.9
(MH+); HRMS calcd for C21H29N4O5S (MH+) 449.1853, found
449.1853. Anal. (C21H28N4O5S‚0.8AcOH) C, H, N.
Diben zyl 1-[4-(4-Oxo-1-piper idin yl)ben zoyl]-L-glu tam ate
(24g). L-Glutamic acid dibenzyl ester p-tosylate, white solid,
45% yield: mp 64-66 °C; 1H NMR (DMSO-d6) δ 1.90-2.20
(m, 4 H), 2.42 (t, J ) 6.0 Hz, 4 H), 3.72 (t, J ) 6.0 Hz, 4 H),
4.45-4.55 (m, 1 H), 5.07 (s, 2 H), 5.13 (s, 2 H), 7.03 (d, J ) 9.0
Hz, 2 H), 7.25-7.40 (m, 8 H), 7.85 (d, J ) 9.0 Hz, 2 H), 8.49
(d, J ) 9.0 Hz, 2 H); MS (ES) m/z 529.1 (MH+); HRMS calcd
for C31H33N2O6 (MH+) 529.2339, found 529.2331. Anal.
(C31H32N2O6) C, H, N.

(4-Piperidin-1-yl)phenyl Amides
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9 1463
Eth yl [(4-{4-[((2R)-2-Hyd r oxy-2-{4-h yd r oxy-3-[(m eth yl-
sulfonyl)amino]phenyl}ethyl)amino]-1-piperidinyl}benzoyl)-
a m in o]a ceta te (25a ). Method B, 24a , white solid, 69%
yield: mp >140 °C dec; 1H NMR (DMSO-d6) δ 1.20 (t, J ) 7.1
Hz, 3 H), 1.20-1.35 (m, 2 H), 1.75-1.90 (m, 2 H), 2.50-2.90
(m, 5 H), 2.90 (s, 3 H), 3.70-3.85 (m, 2 H), 3.93 (d, J ) 5.8 Hz,
2 H), 4.10 (q, J ) 7.1 Hz, 2 H), 4.47 (dd, J ) 7.9, 4.4 Hz, 1 H),
6.79 (d, J ) 8.3 Hz, 1 H), 6.90-7.00 (m, 3 H), 7.16 (d, J ) 2.0
Hz, 1 H), 7.72 (d, J ) 8.9 Hz, 2 H), 8.58 (t, J ) 5.8 Hz, 1 H);
MS (ES) m/z 535.2 (MH+); HRMS calcd for C25H35N4O7S (MH+)
535.2221, found 535.2216. Anal. (C25H34N4O7S‚0.4CH2Cl2‚
1.2MeOH) C, H, N.
N-(4-{4-[((2R)-2-H yd r oxy-2-{4-h yd r oxy-3-[(m et h ylsu l-
fonyl)amino]phenyl}ethyl)amino]-1-piperidinyl}benzoyl)-â-
a la n in e (26b). Method A, 24b, white solid, 42% yield: mp
>170 °C dec; 1H NMR (DMSO-d6) δ 1.20-1.45 (m, 2 H), 1.80-
1.95 (m, 2 H), 2.43 (t, J ) 7.0 Hz, 2 H), 2.60-2.90 (m, 5 H),
2.90 (s, 3 H), 3.00-3.40 (m, 2 H), 3.70-3.85 (m, 2 H), 4.50
(dd, J ) 8.0, 4.2 Hz, 1 H), 6.82 (d, J ) 8.3 Hz, 1 H), 6.92 (d, J
) 8.9 Hz, 2 H), 7.01 (dd, J ) 8.3, 1.9 Hz, 1 H), 7.18 (d, J ) 1.9
Hz, 1 H), 7.68 (d, J ) 8.9 Hz, 2 H), 8.26 (t, J ) 5.2 Hz, 1 H);
MS (ES) m/z 521.2 (MH+); HRMS calcd for C24H33N4O7S (MH+)
521.2064, found 521.2056. Anal. (C24H32N4O7S‚MeOH) C, H,
N.
1-(4-{4-[((2R)-2-H yd r oxy-2-{4-h yd r oxy-3-[(m et h ylsu l-
fon yl)am in o]ph en yl}eth yl)am in o]-1-piper idin yl}ben zoyl)-
L-p r olin e (26c). Method C, 25c, white solid, 47% yield: mp
>220 °C dec; 1H NMR (DMSO-d6) δ 1.20-1.40 (m, 2 H), 1.75-
1.95 (m, 4 H), 2.50-2.90 (m, 7 H), 2.62 (s, 3 H), 3.50-4.00 (m,
5 H), 4.35-4.45 (m, 1 H), 6.60 (d, J ) 8.3 Hz, 1 H), 6.79 (d, J
) 8.7 Hz, 2 H), 6.90 (dd, J ) 8.2, 1.4 Hz, 1 H), 7.06 (d, J ) 1.4
Hz, 1 H), 7.37 (d, J ) 8.6 Hz, 2 H); MS (ES) m/z 545.6 (M -
H)-; HRMS calcd for C26H33N4O7S (M - H)- 545.2075, found
545.2064; HPLC purity 95.5% at 5.3 min (pH 3 phosphate/
ACN).
Meth yl 1-(4-{4-[((2R)-2-Hydr oxy-2-{4-h ydr oxy-3-[(m eth yl-
sulfonyl)amino]phenyl}ethyl)amino]-1-piperidinyl}benzoyl)-
L-p r olin a te (25c). Method B, 24c, off-white solid, 58% yield:
mp >80 °C dec; 1H NMR (DMSO-d6) δ 1.20-1.40 (m, 2 H),
1.75-1.95 (m, 4 H), 2.15-2.30 (m, 2 H), 2.50-2.90 (m, 5 H),
2.90 (s, 3 H), 3.55-3.80 (m, 7 H), 4.40-4.55 (m, 2 H), 6.81 (d,
J ) 8.3 Hz, 1 H), 6.92 (d, J ) 8.7 Hz, 2 H), 6.98 (dd, J ) 8.3,
2.0 Hz, 1 H), 7.17 (d, J ) 2.0 Hz, 1 H), 7.43 (d, J ) 8.7 Hz, 2
H); MS (ES) m/z 561.3 (MH+); HRMS calcd for C27H37N4O7S
(MH+) 561.2377, found 561.2369. Anal. (C27H36N4O7S‚0.7CH2-
Cl2) C, H, N.
N-(4-{4-[((2R)-2-H yd r oxy-2-{4-h yd r oxy-3-[(m et h ylsu l-
fonyl)amino]phenyl}ethyl)amino]-1-piperidinyl}benzoyl)-L-
va lin e (26d ). Method C, 25d , pale yellowish solid, 27% yield:
mp >135 °C dec; 1H NMR (DMSO-d6) δ 0.90 (d, J ) 7.3 Hz, 3
H), 0.92 (d, J ) 7.3 Hz, 3 H), 1.45-1.55 (m, 2 H), 1.90-2.05
(m, 2 H), 2.05-2.20 (m, 1 H), 2.60-2.94 (m, 5 H), 2.94 (s, 3
H), 3.70-3.85 (m, 2 H), 4.11 (t, J ) 6.0 Hz, 1 H), 4.65-4.75
(m, 1 H), 6.86 (d, J ) 8.3 Hz, 1 H), 6.91 (d, J ) 8.9 Hz, 2 H),
7.06 (dd, J ) 8.3, 2.0 Hz, 1 H), 7.22 (d, J ) 2.0 Hz, 1 H), 7.65-
7.75 (m, 3 H); MS (ES) m/z 549.3 (MH+); HRMS calcd for
Eth yl N-(4-{4-[((2R)-2-Hydr oxy-2-{4-h ydr oxy-3-[(m eth yl-
sulfonyl)amino]phenyl}ethyl)amino]-1-piperidinyl}benzoyl)-
L-va lin a te (25d ). Method B, 24d , off-white solid, 59% yield:
1
mp >85 °C dec; H NMR (DMSO-d6) δ 0.92 (d, J ) 6.8 Hz, 3
H), 0.97 (d, J ) 6.8 Hz, 3 H), 1.17 (t, J ) 7.1 Hz, 3 H), 1.20-
1.40 (m, 2 H), 1.75-1.95 (m, 2 H), 2.05-2.20 (m, 1 H), 2.55-
2.90 (m, 5 H), 2.90 (s, 3 H), 3.70-3.85 (m, 2 H), 4.00-4.20 (m,
2 H), 4.22 (t, J ) 7.7 Hz, 1 H), 4.48 (dd, J ) 8.0, 4.4 Hz, 1 H),
6.80 (d, J ) 8.2 Hz, 1 H), 6.95 (d, J ) 8.9 Hz, 2 H), 6.98 (dd,
J ) 8.2, 2.0 Hz, 1 H), 7.17 (d, J ) 2.0 Hz, 1 H), 7.76 (d, J ) 8.9
Hz, 2 H), 8.17 (d, J ) 7.9 Hz, 1 H); MS (ES) m/z 577.2 (MH+);
HRMS calcd for C28H41N4O7S (MH+) 577.2690, found 577.2682.
Anal. (C28H40N4O7S‚2.1H2O) C, H, N.
C
26H35N4O7S (M - H)- 547.2231, found 547.2229. Anal.
(C26H36N4O7S‚2.4H2O) C, H, N.
N-(4-{4-[((2R)-2-H yd r oxy-2-{4-h yd r oxy-3-[(m et h ylsu l-
fon yl)am in o]ph en yl}eth yl)am in o]-1-piper idin yl}ben zoyl)-
L-leu cin e (26e). Method C, 25e, white solid, 45% yield: mp
>177 °C dec; 1H NMR (DMSO-d6) δ 0.87 (d, J ) 6.3 Hz, 3 H),
0.90 (d, J ) 6.3 Hz, 3 H), 1.35-1.75 (m, 5 H), 1.90-2.00 (m, 2
H), 2.60-2.90 (m, 5 H), 2.94 (s, 3 H), 3.65-3.80 (m, 2 H), 4.25-
4.35 (m, 1 H), 4.60-4.65 (m, 1 H), 6.85 (d, J ) 8.3 Hz, 1 H),
6.90 (d, J ) 8.9 Hz, 2 H), 7.05 (dd, J ) 8.3, 1.9 Hz, 1 H), 7.21
(d, J ) 1.9 Hz, 1 H), 7.73 (d, J ) 8.9 Hz, 2 H), 7.98 (d, J ) 7.7
Hz, 1 H); MS (ES) m/z 561.3 (M - H)-; HRMS calcd for
Eth yl N-(4-{4-[((2R)-2-Hydr oxy-2-{4-h ydr oxy-3-[(m eth yl-
sulfonyl)amino]phenyl}ethyl)amino]-1-piperidinyl}benzoyl)-
L-leu cin a te (25e). Method B, 24e, off-white solid, 78% yield:
1
mp >85 °C dec; H NMR (DMSO-d6) δ 0.86 (d, J ) 6.3 Hz, 3
H), 0.92 (d, J ) 6.3 Hz, 3 H), 1.18 (t, J ) 7.1 Hz, 3 H), 1.20-
1.40 (m, 2 H), 1.45-1.95 (m, 5 H), 2.55-2.92 (m, 5 H), 2.92 (s,
3 H), 3.70-3.85 (m, 2 H), 4.10 (q, J ) 7.1 Hz, 2 H), 4.40-4.55
(m, 2 H), 6.82 (d, J ) 8.3 Hz, 1 H), 6.94 (d, J ) 8.9 Hz, 2 H),
7.00 (dd, J ) 8.3, 2.0 Hz, 1 H), 7.18 (d, J ) 2.0 Hz, 1 H), 7.75
(d, J ) 8.9 Hz, 2 H), 8.33 (d, J ) 7.7 Hz, 1 H); MS (ES) m/z
591.3 (MH+); HRMS calcd for C29H43N4O7S (MH+) 591.2847,
found 591.2840. Anal. (C29H42N4O7S‚0.6CH2Cl2) C, H, N.
C
27H37N4O7S (M - H)- 561.2377, found 561.2381. Anal.
(C27H38N4O7S‚0.35CH2Cl2) C, H, N.
N-(4-{4-[((2R)-2-H yd r oxy-2-{4-h yd r oxy-3-[(m et h ylsu l-
fonyl)amino]phenyl}ethyl)amino]-1-piperidinyl}benzoyl)-L-
p h en yla la n in e (26f). Method C, 25f, pale yellowish solid, 74%
Meth ylN-(4-{4-[((2R)-2-h ydr oxy-2-{4-h ydr oxy-3-[(m eth yl-
sulfonyl)amino]phenyl}ethyl)amino]-1-piperidinyl}benzoyl)-
L-p h en yla la n in a te (25f). Method B, 24f, off-white solid, 59%
yield: mp >98 °C dec; 1H NMR (DMSO-d6) δ 1.20-1.40 (m, 2
H), 1.80-1.90 (m, 2 H), 2.05-2.90 (m, 5 H), 2.91 (s, 3 H), 3.00-
3.20 (m, 2 H), 3.70-3.80 (m, 2 H), 4.47 (dd, J ) 8.0, 4.3 Hz, 1
H), 4.50-4.65 (m, 1 H), 6.81 (d, J ) 8.2 Hz, 1 H), 6.92 (d, J )
8.9 Hz, 2 H), 7.00 (dd, J ) 8.2, 2.0 Hz, 1 H), 7.10-7.30 (m, 6
H), 7.67 (d, J ) 8.9 Hz, 2 H), 8.47 (d, J ) 7.8 Hz, 1 H); MS
(ES) m/z 611.2 (MH+); HRMS calcd for C31H39N4O7S (MH+)
611.2534, found 611.2525. Anal. (C31H38N4O7S‚0.85CH2Cl2)
C, H, N.
1
yield: mp >160 °C dec; H NMR (DMSO-d6) δ 1.40-1.50 (m,
2 H), 1.90-2.05 (m, 2 H), 2.50-3.50 (m, 7 H), 2.95 (s, 3 H),
3.69-3.75 (m, 2 H), 4.30-4.45 (m, 2 H), 4.70-4.75 (m, 1 H),
6.83 (d, J ) 8.8 Hz, 2 H), 6.88 (d, J ) 8.3 Hz, 1 H), 7.05-7.30
(m, 7 H), 7.63 (d, J ) 8.8 Hz, 2 H), 7.93 (br d, J ) 6.6 Hz, 1
H); MS (ES) m/z 597.1 (MH+); HRMS calcd for C30H35N4O7S
(M - H)- 595.2231, found 595.2232. Anal. (C30H36N4O7S‚
2.5H2O) C, H, N.
N-(4-{4-[((2R)-2-H yd r oxy-2-{4-h yd r oxy-3-[(m et h ylsu l-
fon yl)am in o]ph en yl}eth yl)am in o]piper idin -1-yl}ben zoyl)-
L-glu ta m ic Acid (26g). Method A, 24g, white solid, 42%
1
yield: mp >230 °C dec; H NMR (DMSO-d6) δ 1.40-1.60 (m,
[(4-{4-[((2R)-2-H yd r oxy-2-{4-h yd r oxy-3-[(m et h ylsu l-
fon yl)am in o]ph en yl}eth yl)am in o]-1-piper idin yl}ben zoyl)-
a m in o]a cetic Acid (26a ). Method C, 25a , white solid, 92%
2 H), 1.90-2.10 (m, 4 H), 2.31 (t, J ) 7.5 Hz, 2 H), 2.65-3.20
(m, 7 H), 3.00 (s, 3 H), 3.85-4.00 (m, 2 H), 4.25-4.35 (m, 1
H), 4.65-4.80 (m, 1 H), 6.88 (d, J ) 8.4 Hz, 1 H), 6.95 (d, J )
8.7 Hz, 2 H), 7.08 (dd, J ) 8.4, 1.2 Hz, 1 H), 7.25 (d, J ) 1.8
Hz, 1 H), 7.74 (d, J ) 8.7 Hz, 2 H), 8.14 (d, J ) 7.2 Hz, 1 H);
MS (ES) m/z 579.1 (MH+); HRMS calcd for C26H35N4O9S (MH+)
579.1973, found 579.1955. Anal. (C26H34N4O9S‚CH2Cl2)
C, H, N.
1
yield: mp >85 °C dec; H NMR (DMSO-d6) δ 1.20-1.40 (m,
2 H), 1.75-1.90 (m, 2 H), 2.55-2.90 (m, 5 H), 2.80 (s, 3 H),
3.49 (d, J ) 4.5 Hz, 2 H), 3.70-3.80 (m, 2 H), 4.43 (dd, J )
7.9, 4.4 Hz, 1 H), 6.70 (d, J ) 7.4 Hz, 1 H), 6.81 (dd, J ) 7.4,
2.0 Hz, 1 H), 6.92 (d, J ) 8.9 Hz, 2 H), 7.12 (d, J ) 2.0 Hz, 1
H), 7.54 (t, J ) 4.5 Hz, 1 H), 7.64 (d, J ) 8.9 Hz, 2 H); MS
(ES) m/z 507.2 (MH+); HRMS calcd for C23H31N4O7S (MH+)
507.1908, found 507.1912. Anal. (C23H30N4O7S‚3H2O‚0.7CH2-
Cl2) C, H, N.
[Bu t yl(4-{4-[(2R)-2-h yd r oxy-2-(4-h yd r oxy-3-m et h a n e-
su lfon ylam in oph en yl)eth ylam in o]piper idin -1-yl}ben zoyl)-
a m in o]a cetic Acid Eth yl Ester (28). A mixture of 5 (0.30
g, 1.37 mmol), 1-[3-(dimethylamino)propyl-3-ethylcarbodiimide

1464 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9
Hu et al.
hydrochloride (0.53 g, 2.74 mmol) and N-butylglycine ethyl
ester12 (0.44 g, 2.7 mmol) was stirred in CH2Cl2 (70 mL).
N-Methylmorpholine (0.28 g, 2.74 mmol) was added dropwise
and the mixture was stirred overnight. The mixture was then
washed with 0.05 N HCl and water. The resulting solution
was dried with MgSO4 and concentrated to give a gum (27).
The gum was dissolved in DMF (15 mL) and then treated with
6 (0.25 g, 1 mmol), NaBH(OAc)3 (0.85 g, 4 mmol) and acetic
acid (0.3 mL). After stirring at room temperature under a N2
atmosphere for 2 h, the mixture was poured into a saturated
aqueous NaHCO3. The aqueous layer was extracted with
n-butanol and the concentrated gum was purified by silica gel
chromatography (MeOH/CH2Cl2) to give the title compound
as a white solid (60 mg, 10%): mp >60 °C dec; 1H NMR
(DMSO-d6) δ 0.70-1.60 (m, 12 H), 1.75-1.95 (m, 2 H), 2.50-
3.50 (m, 7 H), 2.91 (s, 3 H), 3.60-3.75 (m, 2 H), 4.00-4.15 (m,
4 H), 4.47 (dd, J ) 8.0, 4.3 Hz, 1 H), 6.81 (d, J ) 8.5 Hz, 1 H),
6.90-7.00 (m, 3 H), 7.05-7.25 (m, 3 H); MS (ES) m/z 591.3
(MH+); HRMS calcd for C29H43N4O7S (MH+) 591.2847, found
591.2840.
1 H); MS (ES) m/z 591.2 (MH+); HRMS calcd for C29H42N4O7S-
(M+) 590.2774, found 590.2847. Anal. (C29H42N4O7S‚H2O)
C, H, N.
3-(4-{4-[((2R)-2-H yd r oxy-2-{4-h yd r oxy-3-[(m et h ylsu l-
fonyl)amino]phenyl}ethyl)amino]-1-piperidinyl}anilino)-3-
oxop r op a n oic Acid (33a ). Method C, 32a , pale gray solid,
59% yield: mp >170 °C dec; 1H NMR (DMSO-d6) δ 1.30-1.45
(m, 2 H), 1.80-1.95 (m, 2 H), 2.50-2.80 (m, 5 H), 2.90 (s, 2
H), 2.91 (s, 3 H), 3.45-3.60 (m, 2 H), 4.48-4.53 (m, 1 H), 6.80-
6.90 (m, 3 H), 7.00 (d, J ) 8.3 Hz, 1 H), 7.18 (d, J ) 1.6 Hz, 1
H), 7.38 (d, J ) 9.0 Hz, 2 H), 11.50-11.60 (m, 1 H); MS (ES)
m/z 505.3 (M - H)-; HRMS calcd for C23H31N4O7S (MH+)
507.1908, found 507.1895. Anal. (C23H30N4O7S‚CH2Cl2‚AcOH)
C, H, N.
3-(Butyl-4-{4-[((2R)-2-hydroxy-2-{4-hydroxy-3-[(methylsul-
fonyl)amino]phenyl}ethyl)amino]-1-piperidinyl}anilino)-3-
oxop r op a n oic Acid (33b). Method C, 32b, tan solid, 19%:
1
mp >200 °C dec; H NMR (DMSO-d6) δ 1.20-1.50 (m, 6 H),
1.70-1.95 (m, 2 H), 2.60-2.80 (m, 5 H), 2.83 (s, 2 H), 2.91 (s,
3 H), 3.45-3.70 (m, 4 H), 4.55-4.65 (m, 1 H), 6.81 (d, J ) 8.0
Hz, 1 H), 6.90 (d, J ) 8.7 Hz, 2 H), 7.09 (d, J ) 8.7 Hz, 2 H),
7.07 (s, 1 H); MS (ES) m/z 563.2 (MH+); HRMS calcd for
[Bu t yl(4-{4-[(2R)-2-h yd r oxy-2-(4-h yd r oxy-3-m et h a n e-
su lfon ylam in oph en yl)eth ylam in o]piper idin -1-yl}ben zoyl)-
a m in o]a cetic Acid (29). Method C, 28, pale yellowish solid,
C27H39N4O7S (MH+) 563.2461, found 563.2526. Anal. (C27H38
-
1
N4O7S‚0.4CH2Cl2‚0.7AcOH) C, H, N.
60% yield: mp >95 °C dec; H NMR (DMSO-d6) δ 0.70-1.70
(m, 9 H), 1.90-2.10 (m, 2 H), 2.50-3.50 (m, 7 H), 2.94 (s, 3
H), 3.60-3.85 (m, 4 H), 4.75-4.90 (m, 1 H), 6.80-7.40 (m, 8
H); MS (ES) m/z 561.5 (M - H)-; HRMS calcd for C27H37N4O7S
(M - H)- 561.2377, found 561.2388. Anal. (C27H38N4O7S‚2H2O)
C, H, N.
P h a r m a cologica l Stu d ies. In Vitr o F u n ction a l Assa ys.
CHO cells expressing either human â1-, â2-, or â3-AR subtypes
were used as previously described.13 Clones expressing receptor
levels of 70-110 fmol/mg protein were used in the assays. CHO
cells were grown in 24-well tissue culture plates in Dulbecco’s
modified Eagle media (DMEM) with 10% fetal bovine serum,
MEM nonessential amino acids, penicillin-streptompycin and
geneticin. On the day of assay, growth medium was replaced
with preincubation media (DMEM; Gibco, #1199-065) and
incubated for 30 min at 37 °C. Preincubation medium was
replaced with 0.2 mL treatment medium containing DMEM
media containing 250 µM IBMX (isobutyl-1-methylxantine)
plus 1 mM ascorbic acid with test compound dissolved in
DMSO. Test compounds were tested over a concentration
range of 10-9 to 10-5 M for â3-AR cells and 10-8 to 10-4 M for
â1- and â2 -AR transfected cells. Isoproterenol (10-5 M) was
used as an internal standard for comparison of activity. Cells
were incubated at 37 °C on a rocker for 30 min with the â3-
AR cells and 15 min for â1- and â2-AR cells. Incubation was
stopped with the addition of 0.2 N HCl and neutralized with
2.5 N NaOH. The plates, containing the cells and neutralized
media, were stored at -20 °C until ready to assay for cAMP
using the SPA assay kit (Amersham). Data collected from the
SPA assay was analyzed as a percent of the maximal isopro-
terenol response at 10-5 M. Activity curves were plotted using
the SAS statistical and graphics software. EC50 values were
generated for each compound and the maximal response
developed for each compound was compared to the maximal
response of isoproternol at 10-5 M from the following for-
mula: intrinsic activity (IA) ) (% activity of compound)/(%
activity of isoproterenol).
â1- a n d â2-AR Bin d in g Assa ys. CHO cells, permanently
transfected with either human â1- or â2-AR, were grown to 60-
75% of confluency and harvested using nonenzymatic buffers.
Intact CHO cells were incubated in 96-well plates using [125I]-
iodocyanopindolol (ICYP) as the radioligand. Assays were in
duplicate using 25 000 cells for â1-AR and 30 000 cells for â2-
AR incubations. PBS buffer, containing 0.1% BSA and 1 mM
l-ascorbic acid, was used to dilute the cells and ICYP. The â3-
AR agonists to be tested were dissolved in DMSO to a
concentration of 10 mM and then diluted to appropriate
concentrations in the PBS-BSA buffer. Nonspecific binding
was determined by addition of 2 µM D-propranol. The total
assay volume was 0.3 mL and was incubated at room tem-
perature for 45 min with gentle shaking (30 cycles/min). The
assay was terminated by filtering on Unifilter -96, GF/C
plates (Packard) using a Filtermate-196 cell harvester (Pack-
ard). The filters were dried, 30 µL of Microscint-20 added and
counted on a Topcount microplate scintillation counter (Pack-
ard). Duplicate values were averaged and counts for nonspe-
Eth yl 3-Oxo-3-[4-(4-oxo-1-p ip er id in yl)a n ilin o]p r op a n -
oa te (31a ). To a stirred solution, under N2 atmosphere, of 1-(4-
aminophenyl)piperidin-4-one (30a )9 (10.0 g, 38 mmol), and
Et3N (15.35 g, 150 mmol) in CH2Cl2 (100 mL) was added ethyl
3-chloro-3-oxopropionate (6.32 g, 42 mmol). The reaction was
stirred for 18 h. The reaction mixture was concentrated down
in vacuo and purified by flash silica gel chromatography
eluting with 1:1 EtOAc/hexanes to give an orange solid (0.86
g, 7%): 1H NMR (CDCl3) δ 1.32 (t, J ) 7.2 Hz, 3 H), 2.55 (t, J
) 6 Hz, 4 H), 3.46 (s, 2 H), 3.56 (t, J ) 6 Hz, 4 H), 4.22 (q, J
) 7.2 Hz, 2 H), 6.93 (d, J ) 9.0 Hz, 2 H), 7.47 (d, J ) 9.0 Hz,
2 H), 9.09 (s, 1 H); MS (ES) m/z 305.3 (MH+, 100%).
Eth yl 3-(4-{4-[((2R)-2-Hyd r oxy-2-{4-h yd r oxy-3-[(m eth -
ylsulfonyl)amino]phenyl}ethyl)amino]-1-piperidinyl}anilino)-3-
oxop r op a n oa te (32a ). Method B, 31a , yellowish solid, 36%
yield: mp >120 °C dec; 1H NMR (DMSO-d6) δ 1.19 (t, J ) 7.0
Hz, 3 H), 1.25-1.45 (m, 2 H), 1.75-1.95 (m, 2 H), 2.50-2.70
(m, 5 H), 2.92 (s, 3 H), 3.33 (s, 2 H), 3.45-3.60 (m, 2 H), 4.09
(q, J ) 7.0 Hz, 2 H), 4.48 (dd, J ) 8.1, 4.2 Hz, 1 H), 6.80 (d,
J ) 8.3 Hz, 1 H), 6.90 (d, J ) 9.0 Hz, 2 H), 7.00 (dd, J ) 8.3,
2.0 Hz, 1 H), 7.18 (d, J ) 2.0 Hz, 1 H), 7.39 (d, J ) 9.0 Hz,
2 H), 9.92 (s, 1 H); MS (ES) m/z 535.3 (MH+); HRMS calcd for
C
25H35N4O7S (MH+) 535.2221, found 535.2208. Anal. (C25H34
N4O7S‚1.5H2O) C, H, N.
-
Ethyl3-(Butyl-4-{4-[((2R)-2-hydroxy-2-{4-hydroxy-3-[(meth-
ylsulfonyl)amino]phenyl}ethyl)amino]-1-piperidinyl}anilino)-3-
oxop r op a n oa te (32b). To a stirred mixture of 1-[4-(buty-
lamino)phenyl]-4-piperidinone (30b;9 2.46 g, 10 mmol) in 30
mL of CH2Cl2, Et3N (1.81 mL, 13 mmol), and DMAP (catalytic
amount) was added ethyl 3-chloro-3-oxopropionate (2.18 g, 13
mmol) dropwise. The reaction was stirred for 18 h. The
reaction mixture was filtered, concentrated in vacuo, taken
up in methylene chloride, and washed with saturated sodium
bicarbonate two times and dried over sodium sulfate to give
ethyl 3-[butyl-4-(4-oxo-1-piperidinyl)anilino]-3-oxopropanoate
(31b) as a yellow oil (3.84 g, 68%): MS (ES) m/z 361.3 (MH+,
100%). The title compound was prepared from 31b and 6 as
described in method B to give 29% of the compound as a tan
solid: mp 172-174 °C; 1H NMR (DMSO-d6) δ 0.81 (t, J ) 7.0
Hz, 3 H), 1.11 (t, J ) 7.0 Hz, 3 H), 1.20-1.50 (m, 6 H), 1.80-
2.00 (m, 2 H), 2.65-2.90 (m, 5 H), 2.93 (s, 3 H), 3.09 (s, 2 H),
3.56 (t, J ) 6.8 Hz, 2 H), 3.65-3.80 (m, 2 H), 3.97 (q, J ) 7.0
Hz, 2 H), 4.55-4.65 (m, 1 H), 6.85 (d, J ) 8.3 Hz, 1 H), 6.96
(d, J ) 9.0 Hz, 2 H), 7.00-7.10 (m, 3 H), 7.20 (d, J ) 2.0 Hz,

(4-Piperidin-1-yl)phenyl Amides
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9 1465
1992, 44, 2333. (b) Hollenga, C.; Brouwer, F.; Zaagsma, J .
Differences in Functional Cyclic AMP Compartments Mediating
Lipolysis by Isoprenaline and BRL 37344 in Four Adipocyte
Types. Eur. J . Pharmacol. 1991, 200, 325-330.
cific binding subtracted. Data were analyzed for IC50 and Ki
values using GraphPad Prism software (GraphPad Software,
Inc., San Diego, CA).
Mea su r em en t of Th er m ogen esis in Tr a n sgen ic Mice.
Female FVB â3-AR transgenic mice15 were used to determine
in vivo activity. Compounds were tested for increased ther-
mogenesis using the Oxymax indirect calorimeter (Columbus
Instruments, Columbus, OH). Eight fed mice are weighed in
pairs and placed in 4 chambers, 2/chamber, for 3 h to obtain
baseline O2 and CO2 values. The relative gas content of each
chamber was sampled and recorded at 10-12-min intervals.
For each sample, energy expenditure values were calculated
by the Oxymax and expressed as kcal/h. After 3 h of baseline
measurement, the mice were removed, treated with drugs, and
replaced in the chambers. The â3 agonists were injected at
doses of 10 mg/kg (ip). Compounds in 10 mM or 10 mg/mL
DMSO solutions were suspended in 0.5% methylcellulose:0.1%
Tween-80 and injected (ip). Posttreatment kcal/h values were
taken between 40 min and 2.5 h after dosing. The 6-10 sample
sections of the pretreatment and posttreatment periods that
appeared to best represent stable resting thermogenesis were
selected. Each of these sample values was corrected for body
weight and used such that each pair of mice served as its own
baseline for both T-test and percent increase in thermogenesis
calculations. An ANOVA and a one-sided T-test (H1: post-
treatment > pretreatment) were performed using the SAS
software modified to down-weight extreme values. The mean
baseline value for each chamber was subtracted from the mean
posttreatment value for that chamber. This baseline-subtract-
ed value was divided by the mean baseline value and multi-
plied by 100 to obtain a percent increase in thermogenesis for
each chamber. The combined mean percent increase and the
standard error of the mean for each chamber were calculated
using Excel software. Compounds were considered active if
they were able to produce a statistically significant 15%
increase in thermogenesis in â3 transgenic mice.
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Ack n ow led gm en t. The authors acknowledge Dr.
Lee J ennings, Wyeth-Ayerst Research, for critical read-
ing of this manuscript, Dr. Youping Huang, Wyeth-
Ayerst Research, for biological data support, and the
members of the Wyeth-Ayerst Discovery Analytical
Chemistry group for analytical and spectral determina-
tions.
Su p p or tin g In for m a tion Ava ila ble: Elemental analysis
data for novel compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
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0.00043 ( 0.00031 µM, IA ) 0.52; â2 EC50 > 10 µM, IA ) 0.02;
â1 EC50 ) 0.58 ( 0.30 µM, IA ) 0.30).
(17) We occasionally submitted the esters instead of the acids for in
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than the acids. We anticipate that the ester 25a , a prodrug of
the acid, should have a similar activity in thermogenesis effect
to 26a . Presumably, the esters were rapidly hydrolyzed to the
corresponding acids upon injection. We have observed esters to
have similar in vivo activity as the corresponding acids. For
example, the methyl ester of 26e produced a 47 ( 10% thermo-
genesis effect which is comparable to that of the acid (53 ( 8%).
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(16) To compare the â3 activity and selectivity of our new compounds
with a known selective human â3 agonist, the in vitro activity
of L 755507, under our assay conditions, is incorporated into
the data set (Table 1, reported data8a for L 755507: â3 EC50
)
J M000544B