Discovery of a novel, potent and selective human β3- adrenergic receptor agonist

Article abstract of DOI:10.1016/j.bmcl.2004.11.001

Design and structure-activity relationships of a novel β₃- adrenergic receptor agonist are described. The discovery of a novel, potent and selective β₃-adrenergic receptor (AR) agonist is described. SAR studies demonstrated the struc

Full text of DOI:10.1016/j.bmcl.2004.11.001

Bioorganic & Medicinal Chemistry Letters 15 (2005) 251–254
Discovery of a novel, potent and selective human b3-adrenergic
receptor agonist
Yutaka Nakajima,a,* Hitoshi Hamashima,a Ken-Ichi Washizuka,a
Yasuyo Tomishima,a Hiroaki Ohtake,a Emiko Imamura,b Toshiko Miura,b
Hiroshi Kayakiria and Masayuki Katoa
aMedicinal Chemistry Research Laboratories, Fujisawa Pharmaceutical Co. Ltd, 1-6, Kashima 2-Chome, Yodogawa-Ku,
Osaka 532-8514, Japan
bMedicinal Biology Research Laboratories, Fujisawa Pharmaceutical Co. Ltd, 1-6, Kashima 2-Chome, Yodogawa-Ku,
Osaka 532-8514, Japan
Received 24 September 2004; revised 1 November 2004; accepted 1 November 2004
Available online 18 November 2004
Abstract—The discovery of a novel, potent and selective b3-adrenergic receptor (AR) agonist is described. SAR studies demon-
strated the structural requirements for activity and selectivity. Compound 1c, which showed good b3-ARactivity and selectivity,
was identified and pharmacokinetics were investigated.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
b2-ARmay induce severe side effects such as enhance-
ment of heart rate and tracheal relaxation, respectively.
In our laboratory and in others, the search for novel,
potent human b3-ARagonists has been ongoing for a
number of years.9
The b3-adrenergic receptor (AR), which is present on the
surface of adipocytes, plays a significant role in mediat-
ing lipolysis in white adipocyte tissue and thermogenesis
in brown adipocyte tissue.1,2 It has been reported that
stimulation of b3-ARinduces a variety of pharmacologi-
cal effects such as increase of fat oxidation, enhance-
ment of energy expenditure and improvement in
insulin-mediated glucose uptake in rodent models and
thus b3-ARagonists have been developed as therapeutic
candidates for obesity and type II diabetes.3 Recent
studies indicated that in addition to adipocytes, the b3-
ARis also distributed in gall bladder, gastrointestinal
tract and prostate,4 therefore new therapeutic applica-
tions of b3-ARagonists in treatment of gastrointestinal
and urinary disease have been studied.5–7 Early b3 agon-
ists, which had been developed using rodent models
showed insufficient effects in clinical trials due to weak
agonistic activity for the human b3 receptor in spite of
high potency for rodent receptors.8 Thus, potent b3-
ARagonists against the human receptor are required.
Furthermore, b3-ARselectivity over b1-ARand b2-AR
is also important, because stimulation of b1-ARand
2. Design
Our design concept is outlined in Figure 1. We plan-
ned to introduce a hydroxymethyl group into the core
OH
H
HO
HO
N
isoproterenol
part A
OH
part B
part C
R2
H
N
R1
O
OH
N
H
N
H
Keyword: b3-Adrenergic receptor agonist.
Corresponding author. Tel.: +81 6 6390 1384; fax: +81 6 6304
5435; e-mail: yutaka_nakajima@po.fujisawa.co.jp
1
*
Figure 1. Design of b3 agonists.
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.11.001

252
Y. Nakajima et al. / Bioorg. Med. Chem. Lett. 15 (2005) 251–254
O
OH
R1
H
N
H
N
H2N
HO
a, b
d, e
4
R1
Boc
NO2
c
MeO
O
NO2
HO
NO2
2
3
5
OH
OH Boc
N
H
N
f, g, h
R1
O
R1
R2
HO
N
H
N
HO
NH2
H
6
1
Scheme 1. Reagents: (a) NaBH4, THF, MeOH; (b) HCl, dioxane; (c) 4, EtOH; (d) Boc2O, THF; (e) Fe, AcOH, EtOH, H2O; (f) BSA, 1-methyl-2-
pyrrolidinone; (g) R2–NCO, DIEA, 1-methyl-2-pyrrolidinone; (h) TFA or HCl.
Table 1. Effect of conversion of left-wing part on b3-ARactivity
scaffold (part B) to attempt to improve b3-ARselectivity,
water solubility and pharmacokinetics. The left-wing
(part A) represents the core pharmacophore of b3-AR
agonists, an aminoethanol moiety. The right-wing (part
C) is considered critical for b3-ARselectivity since iso-
proterenol is a non-selective b-ARagonist. We designed
compounds 1 based on a urea scaffold to facilitate rapid
synthesis and SARevaluation. This paper describes these
efforts and the discovery of a novel, potent analogue.
OH
H
N
R
O
HO
N
H
N
H
Compd
RHuman
b3 EC50 (nM)a
Cl
1a
74
3. Synthesis
1b
1c
22
HN
Our general synthetic approach to the compounds in
this letter is illustrated in Scheme 1. Boc-protected p-
nitrophenylalanine methyl ester 2 was reduced to the
alcohol with NaBH4. Removal of the Boc group under
acidic conditions gave amine 3. The first point of diver-
sity was introduced by coupling of 3 with various epox-
ides 4. The resulting amines 5 were protected as Boc and
the nitro group was reduced to afford key amines 6. The
second point of diversity was introduced by parallel cou-
pling of 6 with a variety of isocyanates. For efficient urea
formation, pretreatment of 6 with N,O-bis(trimethylsilyl)-
acetamide (BSA) to protect the hydroxyl groups,
addition of isocyanate and finally acidic deprotection
of the silyl and Boc protecting groups afford the desired
urea derivatives 1 in good yield.
O
O
0.5
Isoproterenol
0.97
a b3-ARagonistic activity was assessed by measuring cAMP accumu-
lation in CHO cell lines expressing cloned human b3-AR.
Next, we confirmed the importance of the hydroxy-
methyl group in the scaffold, as indicated in Table 2.
Compound 1d the stereoisomer of 1c, showed a 26-fold
decrease in b3-ARactivity. The stereochemistry of the
hydroxymethyl group contributed to enhancement of
b3-ARactivity. Compound 1e with no substituent, was
3-fold less potent in b3-ARactivity. Therefore, it is sug-
gested that the hydroxymethyl group may interact with
the b3-ARand the spatial configuration is important.
Furthermore b3-ARselectivity over b1-ARof these
compounds was evaluated. Compound 1c showed 40-
fold b1/b3 selectivity, whereas compound 1d and 1e re-
sulted in more than 10-fold increase in b1-ARactivity
and decrease in b1/b3 selectivity. Interestingly, com-
pound 1d and 1e showed b1-ARselectivity and non-
selectivity, respectively. These results indicate that the
presence and stereochemistry of the hydroxymethyl
group enhanced the b3-ARselectivity as well as the b3-
ARactivity. In addition, compound 1c was investigated
for b2/b3 selectivity and was shown to be inactive to-
wards the b2-AR.
4. Results and discussion
All compounds were evaluated for their ability to pro-
duce cAMP in Chinese hamster ovary (CHO) cell lines
expressing cloned human b3-AR. Selected compounds
were also evaluated for human b1 or b2-ARactivity
using a similar method.
We first aimed to determine whether a urea type struc-
ture would show potent b3-ARagonist activity, by
introducing representative left-wing structures to evalu-
ate potential. Table 1 shows these results. Using 3-chlo-
rophenyl, carbazoloxymethyl and phenoxymethyl as
representatives, it can be seen that urea compounds have
moderate to potent activity (1a, 1b and 1c). In particu-
lar, the phenoxymethyl compound 1c displayed potent
activity as compared to isoproterenol.

Y. Nakajima et al. / Bioorg. Med. Chem. Lett. 15 (2005) 251–254
Table 2. Effect of hydroxymethyl group on b3-ARactivity and selectivity
253
OH
H
N
O
O
R2
R1
N
H
N
H
Compd
R1
R2
Human b3
Human b1
b1/b3 Selectivity
Human b2
b2/b3 Selectivity
EC50 (nM)a
EC50 (nM)a
EC50 (nM)a
1c
1d
CH2OH
H
0.5
13
20
40
>100
NT
NT
2.0
>200
—
—
H
H
CH2OH
H
3.0
3.6
0.2
2.3
1e
Isoproterenol
1.6
0.97
0.084
0.087
2.1
a b-ARagonistic activity was assessed by measuring cAMP accumulation in CHO cell lines expressing cloned human b-ARs.
Table 3 shows the effect of modification of the right part.
This moiety was expected to greatly affect the b3-AR
activity and selectivity. First, conversion of the terminal
phenyl ring to an alkyl group was examined. For b3-AR
activity, while a bulky group such as cyclohexyl group
was effective to maintain activity, the alkyl substituted
derivatives, 1f, 1g and 1h, were less potent than com-
pound 1c. This suggested that, in addition to bulkiness,
aromaticity in the right portion was significant for b3-
ARactivity. Next, we focused on introduction of sub-
stituents to the right terminal phenyl ring. The methoxy
substituents, 1i, 1j and 1k, maintained b3-ARactivity
compared to lead compound 1c, but increased b1-AR
activity and therefore lowered the b1/b3 selectivity of
these compounds. While the position of the methoxy
group affected b1-ARactivity and the b1/b3 selectivity
was increased in the order ortho > meta > para substitu-
ent, these analogues showed less than 10-fold b1/b3 selec-
tivity. Introduction of carboxylic acid (1l) and nitro
group (1m) also resulted in an increase in b1-ARactivity
and a decrease in b1/b3 selectivity. The effect of substi-
tuents appeared to influence b1/b3 selectivity and the
functional group on the right terminal phenyl ring was
suggested to contribute to interaction with the b1-AR.
The nonsubstituted phenyl analogue 1c was conse-
quently judged to be the most potent and selective.
The most potent and selective analogue 1c was investi-
gated in a pharmacokinetic (PK) study as shown in
Table 4. Oral bioavailability of compound 1c was low
in rats and moderate in dogs (F = 1.1% and 20.3%,
respectively). Values of clearance significantly differed
in rats and dogs. These results would be attributed to
species difference in metabolic stability. In dogs, how-
ever, compound 1c showed high AUC and long half-life,
indicating long duration of action.
Table 3. Effect of conversion of right-wing part on b3-ARactivity and
selectivity
OH
H
5. Summary
N
O
O
R
In summary, we have discovered a number of novel and
potent b3-ARagonists. A SARstudy revealed that the
hydroxymethyl and phenylurea groups were important
for b3-ARactivity and selectivity. Compound 1c was
identified as the most potent and selective in this series
of b3-ARagonists. In a PK study, compound 1c showed
prolonged plasma concentration and reasonable oral
bioavailability in dogs.
OH
N
H
N
H
Compd RHuman
b3
Human b1
b1/b3
EC50 (nM)a EC50 (nM)a Selectivity
1c
1f
Ph
c-Hex
0.5
2.1
8.3
4.9
1.1
0.8
2.5
20
15
51
27
12
40
7.1
1g
1h
1i
n-Pr
i-Pr
6.1
5.5
2-OMe–Ph
3-OMe–Ph
4-OMe–Ph
11
1j
4.0
5.0
0.9
0.9
0.9
1k
1l
2.2
1.3
0.9
Acknowledgements
3-Carboxy–Ph 1.5
3-NO2–Ph 1.0
1m
The authors would like to acknowledge Dr. David Barr-
ett for helpful discussions and critical reading of this
manuscript.
a b-ARagonistic activity was assessed by measuring cAMP accumu-
lation in CHO cell lines expressing cloned human b-ARs.
Table 4. Pharmacokinetic parameters of 1c after po and iv administration to rats and dogs
poa
iva
F (%)
Dose (mg/kg)
Cmax (ng/mL)
AUC0À24 (ngh/mL)
Dose (mg/kg)
t1/2 (h)
CLtot (mL/min/kg)
Rat
Dog
3.2
1.0
4.2
52.6
4.2
140.1
1.0
0.32
1.3
8.1
141.8
24.2
1.1
20.3
a n = 3.

254
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8. Weyer, C.; Tataranni, P. A.; Snitker, S.; Danforth, E., Jr.;
Ravussin, E. Diabetes 1998, 47, 1555.
References and notes
9. For recent studies, see: (a) Harada, H.; Hirokawa, Y.;
Suzuki, K.; Hiyama, Y.; Oue, M.; Kawashima, H.; Yosh-
ida, N.; Furutani, Y.; Kato, S. Bioorg. Med. Chem. Lett.
2003, 13, 1301; (b) Tanaka, N.; Tamai, T.; Mukaiyama, H.;
Hirabayashi, A.; Muranaka, H.; Ishikawa, T.; Kobayashi,
J.; Akahane, S.; Akahane, M. J. Med. Chem. 2003, 46, 105;
(c) Uehling, D. E.; Donaldson, K. H.; Deaton, D. N.;
Hyman, C. E.; Sugg, E. E.; Barrett, D. G.; Hughes, R. G.;
Reitter, B.; Adkison, K. K.; Lancaster, M. E.; Lee, F.;
Hart, R.; Paulik, M. A.; Sherman, B. W.; True, T.; Cowan,
C. J. Med. Chem. 2002, 45, 567; (d) Hu, B.; Ellingboe, J.;
Han, S.; Largis, E.; Mulvey, R.; Oliphant, A.; Sum, F.-W.;
Tillett, J. J. Med. Chem. 2001, 44, 1456; (e) Mathvink, R. J.;
Tolman, J. S.; Chitty, D.; Candelore, M. R.; Cascieri, M.
A., Jr.; Colwell, L. F., Jr.; Deng, L.; Feeney, W. P.; Forrest,
M. J.; Hom, G. J.; MacIntyre, D. E.; Miller, R. R.; Stearns,
R. A.; Tota, L.; Wyvratt, M. J.; Fisher, M. H.; Weber, A.
E. J. Med. Chem. 2000, 43, 3832.
1. Arch, J. R. S.; Ainsworth, A. T.; Cawthorne, M. A.; Piercy,
V.; Sennitt, M. V.; Thody, V. E.; Wilson, C.; Wilson, S.
Nature 1984, 309, 163.
2. Emorine, L. J.; Marullo, S.; Briend-Sutren, M.-M.; Patey,
G.; Tate, K.; Delavier-Klutchko, C.; Strosberg, A. D.
Science 1989, 245, 1118.
3. For recent reviews, see: (a) Hu, B.; Jennings, L. L. Prog.
Med. Chem. 2003, 41, 167; (b) de Souza, C. J.; Burkey, B. F.
Curr. Pharm. Des. 2001, 7, 1433; (c) Weyer, C.; de Souza, C.
J. Drug Dev. Res. 2000, 51, 80.
4. Strosberg, A. D. Annu. Rev. Pharmacol. Toxicol. 1997, 37,
421.
5. Rathi, S.; Kazerounian, S.; Banwait, K.; Schulz, S.;
Waldman, S. A.; Rattan, S. J. Pharmacol. Exp. Ther.
2003, 305, 615.
6. Bardou, M.; Dousset, B.; Deneux-Tharaux, C.; Smadja, C.;
Naline, E.; Chaput, J.-C.; Naveau, S.; Manara, L.; Croci,
T.; Advenier, C. Eur. J. Pharmacol. 1998, 353, 281.
7. Igawa, Y.; Yamazaki, Y.; Takeda, H.; Hayakawa, K.;
Akahane, M.; Ajisawa, Y.; Yoneyama, T.; Nishizawa,
O.; Andersson, K.-E. Br. J. Pharmacol. 1999, 126, 819.