Discovery of highly potent and selective biphenylacylsulfonamide-based β3-adrenergic receptor agonists and molecular modeling based on the solved X-ray structure of the β2-adrenergic receptor: Part 6

Article abstract of DOI:10.1016/j.bmcl.2009.06.083

As an extension of research, we have investigated modification of left-hand side (LHS) of biphenyl analogues containing an acylsulfonamide moiety in the development of potent and selective human β₃-adrenergic receptor (AR) agonists. Result of s

Full text of DOI:10.1016/j.bmcl.2009.06.083

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4679–4683
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of highly potent and selective biphenylacylsulfonamide-based
b3-adrenergic receptor agonists and molecular modeling based on the solved
X-ray structure of the b2-adrenergic receptor: Part 6
a
a
a
a
a
Kouji Hattori a, , Masaya Orita , Susumu Toda , Masashi Imanishi , Shinji Itou , Yutaka Nakajima ,
Daisuke Tanabe a, Kenichi Washizuka a, Takanobu Araki a, Minoru Sakurai a, Shigeo Matsui b,
Emiko Imamura b, Koji Ueshima c, Takao Yamamoto b, Nobuhiro Yamamoto b, Hirofumi Ishikawa b,
Keiko Nakano b, Naoko Unami b, Kaori Hamada b, Yasuhiro Matsumura d, Fujiko Takamura d
*
a Chemistry Research Laboratories, Astellas Pharma Inc., 21, Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585, Japan
b Pharmacology Research Laboratories, Astellas Pharma Inc., 21, Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585, Japan
c Applied Pharmacology Research Laboratories, Astellas Pharma Inc., 21, Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585, Japan
d Analysis & Pharmacokinetic Research Laboratories, Astellas Pharma Inc., 21, Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
As an extension of research, we have investigated modification of left-hand side (LHS) of biphenyl ana-
logues containing an acylsulfonamide moiety in the development of potent and selective human b3-
adrenergic receptor (AR) agonists. Result of structure–activity relationships (SAR) and cassette-dosing
evaluation in dogs showed that the hydroxynorephedrine analogue 16 had an excellent balance of
in vitro and in vivo potency with pharmacokinetic profiles. In addition, to facilitate structure-based drug
design (SBDD), we also have performed a docking study of biphenyl analogues based on the X-ray struc-
ture of the b2-adrenergic receptor.
Received 11 April 2009
Revised 3 June 2009
Accepted 20 June 2009
Available online 25 June 2009
Ó 2009 Elsevier Ltd. All rights reserved.
We previously discovered a series of biphenyl analogues con-
taining a carboxylic acid moiety on the right-hand side (RHS),
which exhibited good b3-adrenergic receptor (AR) activity, selec-
tivity, and bioavailability in Figure 1.1 We then focused our atten-
tion on a phenylethanolaminotetraline (PEAT) skeleton2 and on
substituting an acylsulfonamide moiety for the carboxylic acid on
the RHS for protecting against metabolism via acylglucuronide
conjugation.3 Building on this research regarding acylsulfonamide
analogues, we explored the replacement of left-hand side (LHS)
moieties to improve potency and bioavailability.
Since the discovery of b3-AR agonist by the Beecham group two
decades ago, the majority of LHS moieties has featured a 3-chloro-
phenyl group attached directly to the amino alcohol framework.4
Over the years, a range of successful modifications has been re-
ported in Figure 2.5 This structure also contributes extensively to
agonistic activity due to its mimicking of an adrenalin/noradrena-
lin unit. Here, we conducted a structure–activity relationship (SAR)
study on modifications to the LHS region of acylsulfonamide series
as well as reported on the pharmacokinetics (PK). Further, to iden-
tify successful drug candidates with an excellent balance of mark-
edly high potency and selectivity, we evaluated the results of a
docking study based on b3-AR and our biphenyl analogues as a
structure-based drug design (SBDD) approach.
Table 1 shows the result of several substituted derivatives to
the LHS with the reference compound, isoproterenol (ISP) as a non-
selective b-AR agonist. PK properties of select compounds were
evaluated by cassette-dosing assay in dogs.6 In a previous article,
we reported that leading compounds 1, 2, 3, and 6 showed potent
b3-AR activity, good selectivity, and good PK.3 We initially exam-
ined the effects of introduction at the
a-position of the secondary
amino group. Addition of an -methyl group (4) resulted in
a
improvement of b3-AR and b1-AR activity relative to the parent
compound (3), which was similar trend to for the previous series.1b
In contrast, addition of a hydroxymethyl group (5) resulted in a
two-fold decrease in b3-AR activity but improvement in
bioavailability.
We then focused on substitution of the phenyl/pyridyl ring on
the LHS. Although introduction of an amino group to the pyridine
ring (8) greatly increased b3-AR activity, Cmax and AUC levels were
fairly reduced compared with 6 in the cassette-dosing assay.
Abbreviations: b-AR, b-adrenergic receptors; SAR, structure–activity relation-
ship; SBDD, structure-based drug design; PK, pharmacokinetic; LHS, left-hand side;
RHS, right-hand side; PEAT, phenylethanolaminotetraline; cAMP, cyclic adenosine
monophosphate; ISP, isoproterenol; CHO, chinese hamster ovary; IVP, intravesical
pressure; PB, protein binding.
* Corresponding author. Tel.: +81 29 863 7179; fax: +81 29 852 5387.
E-mail address: kouji.hattori@jp.astellas.com (K. Hattori).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.06.083

4680
K. Hattori et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4679–4683
OH
H
N
OH
H
Ar
O
CO2H
N
R
Ar
O
OH
R
OH
H
N
Ar
A: Biphenyl Series
B: Biphenylether Series
R
O
CO2H
OH
H
HN
SO2
OH
H
N
O
CO2H
N
Ar
Ar
R
C: Biphenylacylsulfonamide Series
R
E: PEAT-Biphenylether Series
D: PEAT-Biphenyl Series
Figure 1. Design and discovery strategy for biphenyl analogues.
Acetylamino analogue 9 resulted in a substantial decrease in b3-AR
activity relative to 8. SAR results for the 3- and 4-aniline analogues
(10–13) were similar of those seen in phenyl analogues (1 and 2).
Addition of a 4-hydroxy-3-methylsulfonamide unit to the LHS (14),
as explored by the BMS group,7 resulted in greatly improved activ-
ity relative to the original compound 1. Compound 15 with a 3-hy-
droxy group also exhibited an improvement in potency of over
10-fold compared with 1. However, both compounds 14 and 15 re-
sulted in the fairly decreased the Cmax and AUC levels by oral
administration. A series of hydroxynorephedrine studied by the
Kissei group in Figure 2.8 We also demonstrated for our previous
series that the represented structure (19) provided potent activity
and good selectivity for b1-AR, but low bioavailability (F = 22%)
shown in Table 1.1b Of particular note is the fact that compound
16 showed good potency and selectivity while maintaining a good
PK profile (F = 56%; t1/2 = 6.7 h), indicating improved metabolic sta-
bility over 14, 15, and 19.9 Unfortunately, the more lipophilic ana-
logue with a cyclohexyl group on the RHS (17) showed lower
activity and bioavailability than the compound with an iso-propyl
group (16).
We further examined the inhibitory effect of a carbachol-in-
duced increase in intravesical pressure in an anesthetized dog
overactive bladder model.1 Table 2 shows in vitro potency for hu-
man and dog b3-AR activity in CHO cell lines and in vivo inhibition
data. The hydroxynorephedrine analog 16 showed improved activ-
ity (in vivo ED50 = 5.9 lg/mL) compared to compound 4 and 6 of a
previous series in spite of similar in vitro dog b3-AR activity and
dog protein binding (PB).
The hydroxynorephedrine analogues were synthesized follow-
ing the procedure in Scheme 1. Intermediate 21 was prepared by
OH
H
N
OH
H
Cl
N
N
NH
O
O
S
O
CO2H
N
F3C
S
BRL-37344
L-796568
OH
OCHF2
H
N
OH
CO2H
MsHN
HO
H
N
O
H2N
N
OCHF2
BMS-194449
CP-331684
HO
OH
H
N
O
CO2H
KUL-7211
Figure 2. Representative LHS structure for b3-AR agonists.

K. Hattori et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4679–4683
4681
Table 1
SAR result of b3-AR activity and pharmacokinetic profilesa
OH
H
2
OH
H
N
N
3
R1
R4
R2 R3
O
O
HO
X
4
H
OH
N
19
S
O O
O
Compound
Human EC50 (nM)b
Pharmacokinetic parameters in dogsc,d
po (n = 3) iv (n = 3)
AUC0–24 h (ngꢀh/mL)
Fe (%)
R1
X
R2
R3
R4
b3
b2
b1
Cmax (ng/mL)
t1/2 (h)
CLtot (mL/min/kg)
1f
H
H
H
H
H
H
Cl
CH
CH
CH
CH
CH
N
N
N
N
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
O-iso-Pr
O-c-Hex
iso-Bu
3.1
NT
NT
NT
NT
NT
>10,000
NT
290
NT
NT
>1000
>1000
>1000
73
>1000
800
33.41.4
22.2 3.6
24.9 1.9
NT
59.1 3.2
34.5 3.8
NT
2.5 0.3
16.2 3.8
17.2 4.0
6.1 0.7
NT
17.5 1.0
1.1 0.2
2.9 1.3
23.2 8.2
5.3 0.8
NT
562 60.6
363 41.3
210.5 22.6
NT
653.8 36.5
384.6 57.7
NT
26.7 1.0
86.5 11.5
143.0 17.6
79.3 12.1
NT
212.5 14.4
4.4 0.9
7.3 0.5
117.4 15.8
39.7 3.2
NT
12.0 2.0
7.8 1.2
3.5 0.2
NT
5.0 0.28
5.5 0.4
NT
6.7 1.3
1.8 0.05
4.9 0.2
NT
2.3 0.1
3.0 0.1
5.3 0.8
NT
2.4 0.25
3.2 0.3
NT
34.9 2.5
8.9 0.77
5.1 0.6
NT
75
70
67
NT
95
81
NT
54
43
46
NT
NT
68
NT
NT
56
25
NT
22
2f
0.43
0.46
0.10
0.82
0.13
0.23
0.029
0.23
6.0
3f
4
Me
CH2OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
iso-Bu
5
O-c-Hex
O-c-Hex
O-c-Hex
O-c-Hex
O-c-Hex
O-iso-Pr
O-c-Hex
O-iso-Pr
O-c-Hex
O-iso-Pr
O-iso-Pr
O-iso-Pr
O-c-Hex
O-c-Hex
O-iso-Pr
0.97
6f
7
88
140
8f
4-NH2
4-NHAc
3-NH2
3-NH2
4-NH2
4-NH2
3-MSNH, 4-OH
3-OH
4-OH
4-OH
4-OMe
4-OH
9
>1000
>1000
>1000
>1000
>1000
150
130
290
110
>1000
500
10
11
12
13
14
15
16
17
18
19g
ISPh
1.2
5.5
NT
NT
NT
NT
NT
5.7 0.3
NT
NT
5.0 0.2
NT
0.50
0.26
0.044
0.16
0.35
28
NT
NT
NT
CH3
CH3
CH3
CH3
>10,000
>10,000
NT
NT
0.84
6.7 4.3
3.5 1.4
NT
7.9 1.4
11.0 2.1
NT
0.14
2.0
4.0 1.4
16.3 8.1
5.7 0.9
23.5 3.1
a
b
c
Detail experimental method was expressed in Ref. 1.
The results are shown the mean (n = 2–3).
The results are shown the mean SE (n = 3).
Cassette assay data.
F = bioavailability.
See in Ref. 3.
d
e
f
g
h
See in Ref. 1b.
ISP = isoproterenol.
Table 2
Inhibitory effect on intraduodenal administration of 16 on increase in IVP (intravesical pressure), induced by carbachol in anesthetized dogsa
Compound
In vitrob
In vivoc
ED50 g/kg)
PBd (%)
Clog Pe
Human b3 EC50 (nM)
Dog b3 EC50 (nM)
(l
6f
0.13
0.029
0.16
1.4
1.2
1.0
16.2 4.6
14.3 5.2
5.9 1.2
83
76
90
3.92
3.59
3.86
8f
16
a
Details experimental are shown in Ref. 1
b
c
d
e
f
The results are shown as the mean (n = 3).
The results are shown as the mean SE (n = 3).
PB = dog protein binding.
Biobyte CLOGP ver 4.3.
See in Ref. 3.
coupling the optically pure (
a
-S, b-R)-40-hydroxynorepherine 208
the complex of b3-AR receptor bound to 8. The gray region indi-
cates amino acids are completely conserved among b-AR receptors,
while the magenta region contains of different amino acids, specif-
ically Leu97 (Ile for b1 and His for b2), Ala98 (Val for b1 and Ile for
b2), Leu329 (Val for b1 and Ile for b2) and Ala197 (Asp for b1 and b2),
which were expected to generate selectivity in our analogues. In
the RHS, the cyclohexyl moiety of 8 lies in the hydrophobic region
around Leu97 and Ala197.14 The acylsulfonamide part is not lo-
cated within hydrogen-bonds distance and protrudes into the sol-
vent. Conformational changes in the loop regions may result in
hydrogen-bonds between these two basic residues and the acyl-
with 4-bromophenylethylbromide, followed by protection with a
Boc group. Suzuki cross-coupling of the intermediate Boc-amine
21 with the corresponding boronic acids was performed, followed
by de-protection to provide the targets compounds 16 and 17 in a
previous method of the biphenyl analogues.3
Next, we studied a docking analysis of several biphenyl ana-
logues to explain the observed potency and selectivity based on a
molecular modeling of the b-AR receptor using data from the pre-
viously determined X-ray structure of the b2-AR receptor bound to
carazolol (accession code of the PDB: 2RH1).10,11 Figure 3 shows

4682
K. Hattori et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4679–4683
Br
1.
OH
Br
Boc
N
OH
iPr2NH / DMF
90ºC
NH2
BocO
Br
2. Boc2O, DMAP
/ CH3CN
HO
20
21
27%
OH
B
1.
OR
CONHSO2Me
HO
OH
H
N
PdCl2(dppf) (0.2eq), dppf (0.05eq)
2N Na2CO3 / DMF, 95ºC
OR
HO
2. 4N-HCl / AcOEt
40-55%
CONHSO2Me
16: R = iso-Pro
17: R = cyclo-Hex
Scheme 1. Synthesis route to hydroxynorephedrine analogues.
Figure 3. Compound 8 bound to the human b3 receptors. 8 is yellow with heteroatom being blue for nitrogen, red for oxygen, and green for sulfur. (A) The surface around the
conserved residues among b-AR receptor is shown grey and the surface around the non-conserved residues is shown magenta. (B) The conserved residues among b-AR
receptor is colored grey and the non-conserved residues is colored magenta. Yellow dashed lines indicate hydrogen bonds.
sulfonamide portiont.15 Our SAR results indicated that the acidic
portion is essential for b3-AR activity and selectivity.16 We also
confirmed docking study using biphenyl analogues (A), biphenyle-
ther analogues (B), biphenylacylsulfonamide analogues (C), and
aminotetraline analogues (D and E), suggesting that both the ter-
minal carboxylic acid portion in the RHS and arylethanolamine
unit in the LHS might occupy in the similar space as important
pharmacophores for b3-AR (in Fig. 4).
served residues (Asp113, Asn312 and Tyr316) as observed in the
crystal structure of the carazolol-b2-AR receptor complex. In our
docking model, the amino group on the pyridyl ring of 8 and the
hydroxyl group on the phenyl ring of 16 are located near the oxy-
gen atom in the side-chain of Asn312, while both of Ser209 and
Ser212 are too far away for hydrogen-bonds to occur (>5 Å). Nev-
ertheless, Ser209 and Ser212 are expected to be the potential
The biphenyl portion on the RHS has interacts with the hydro-
phobic side-chain of Trp113, Phe198 and Leu329. In particular, the
side-chain of Phe198 was interacted hydrophobically with (R)-
methyl group of compound 4 to enhance activity (in Fig. 5) while
the (S)-methyl group protrudes into solvent, thus explaining our
SAR results.1b
In the LHS, the secondary amine and b-hydroxyl group are
strongly suggested to interact via hydrogen bonds with three con-
Figure 4. Overlay of biphenyl analogue: A (grey), biphenylether analogue: B (light-
blue), biphenylacylsulfonamide analogue: C (green), PEAT-biphenyl analogue: D
(white), and PEAT-biphenyleher analogue: E (orange).
Figure 5. Interaction of a-methyl group.

K. Hattori et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4679–4683
4683
4. Arch, J. R.; Ainsworth, A. T.; Cawthorne, M. A.; Piercy, V.; Sennitt, M. V.; Thod, V.
E.; Wilson, C.; Wilson, S. Nature 1984, 309, 163.
5. Sawa, M.; Harada, H. Curr. Med. Chem. 2006, 13, 25.
hydrogen-bonding partners, as agonist binding may cause the
structural changes in the binding site.17
In summary, we have identified potent and selective b3-AR ago-
nists in a novel acylsulfonamide series with modification to the
LHS to enhance potency and PK profiles. Among the biphenyl ana-
logues examined, compound 16 with hydroxynorephedrine exhib-
ited the most potent activity in vivo among our biphenyl series. We
also clarified molecular-level reasoning behind the observed selec-
tivity and potency by performing a docking study based on the X-
ray structure of b2-AR bound to carazolol, which provides for ra-
tional drug design of potent and selective b3 agonists.
6. A cassette-dosing assay by in vivo dog pharmacokinetic assay to generate the
desired compound with high oral bioavailability and long plasma half life,
because the phase 1 study of FK175 indicated that the pharmacokinetic profile
was similar in humans and dogs.
7. Washburn, N. W.; Sher, M. P.; Poss, M. K.; Girotra, N. R.; McCann, J. P.; Gavai, V.
A.; Mikkilineni, B. A.; Mathur, A.; Cheng, P.; Dejneka, C. T. Bioorg. Med. Chem.
Lett. 2001, 11, 3035.
8. Tanaka, N.; Tamai, T.; Mukaiyama, H.; Hirabayashi, A.; Muranaka, H.; Ishikawa,
T.; Kobayashi, J.; Akahane, S.; Akahane, M. J. Med. Chem. 2003, 46, 105.
9. Metabolic stability of 16 by in vitro clearance using liver microsomes has very
stable in all species.
10. (a) Cherezov, V.; Rosenbaum, D. M.; Hanson, M. A.; Rasmussen, S. G.; Thian, F.
S.; Kobilka, T. S.; Cho, I. H. J.; Kuhn, P.; Weis, W. I.; Kobilka, B. K.; Stevens, R. C.
Science 2007, 318, 1258; (b) Rosenbaum, D. M.; Cherezov, V.; Hanson, M. A.;
Rasmussen, S. G.; Thian, F. S.; Kobilka, T. S.; Choi, H. J.; Yao, X. J.; Weis, W. I.;
Stevens, R. C.; Kobilka, B. K. Science 2007, 318, 1266.
References and notes
1. (a) Imanishi, M.; Tomishima, Y.; Itou, S.; Hamashima, H.; Nakajima, Y.; Sakurai,
M.; Washizuka, K.; Matsui, S.; Imamura, E.; Ueshima, K.; Yamamoto, T.;
Yamamoto, N.; Ishikawa, H.; Nakano, K.; Unami, N.; Hamada, K.; Matsumura,
Y.; Takamura, F.; Hattori, K. J. Med. Chem. 2008, 51, 1925; (b) Imanishi, M.; Itou,
S.; Washizuka, K.; Hamashima, H.; Nakajima, Y.; Araki, T.; Tomishima, Y.;
Sakurai, M.; Matsui, S.; Imamura, E.; Ueshima, K.; Yamamoto, T.; Yamamoto,
N.; Ishikawa, H.; Nakano, K.; Unami, N.; Hamada, K.; Matsumura, Y.; Takamura,
F.; Hattori, K. J. Med. Chem. 2008, 51, 4002; (c) Nakajima, Y.; Imanishi, M.; Itou,
S.; Hamashima, H.; Tomishima, Y.; Washizuka, K.; Sakurai, M.; Matsui, S.;
Imamura, E.; Ueshima, K.; Yamamoto, T.; Yamamoto, N.; Ishikawa, H.; Nakano,
K.; Unami, N.; Hamada, K.; Matsumura, Y.; Takamura, F.; Hattori, K. Bioorg. Med.
Chem. Lett. 2008, 18, 5037.
2. Imanishi, M.; Nakajima, Y.; Tomishima, Y.; Hamashima, H.; Washizuka, K.;
Sakurai, M.; Matsui, S.; Imamura, E.; Ueshima, K.; Yamamoto, T.; Yamamoto,
N.; Ishikawa, H.; Nakano, K.; Unami, N.; Hamada, K.; Matsumura, Y.; Takamura,
F.; Hattori, K. J. Med. Chem. 2008, 51, 4804.
3. Hattori, K.; Toda, S.; Imanishi, M.; Itou, S.; Nakajima, Y.; Washizuka, K.; Araki, T.;
Hamashima, H.; Tomishima, Y.; Sakurai, M.; Matsui, S.; Imamura, E.; Ueshima, K.;
Yamamoto, T.; Yamamoto, N.; Ishikawa, H.; Nakano, K.; Unami, N.; Hamada, K.;
Matsumura, Y.; Takamura, F.; Hattori, K. J. Med. Chem. 2009, 52, 3063.
11. Docking Study was performed by the program, MOE (CCG, Montreal, Canada)12
and the program, GOLD 3.1.1 (CCDC, Cambridge, UK).13
12. MOE revision 2007.0902; Chemical Computing Group: Montreal, 2007.
13. Jones, G.; Willett, P.; Glen, R. C. J. Mol. Biol. 1995, 245, 43.
14. It is not also conserved in b1-AR and b2-AR receptors, arising from the amino
acids’ differences (Leu97: Ile for b1 and His for b2, Ala197: Asp for b1 and b2).
15. In our model, His102 (Arg for b1 and Met for b2) in the first extracellular loop
and Arg194 (Lys for b1 and The for b2) in the second extracellular loop are
located near by terminal acylsulfonamide part of 8.
16. The BMS first highlighted an important role to carboxylate and other
negatively charged groups on the RHS. (a) Sher, P. M.; Mathur, A.; Fisher, L.
G.; Wu, G.; Skwish, S.; Michel, I. M.; Seiler, S. M.; Dickinson, K. E. Bioorg. Med.
Chem. Lett. 1997, 7, 1583; (b) Malamas, M. S.; Largis, E.; Gunawan, I.; Li, Z.;
Tillett, J.; Han, S. C.-H.; Mulvey, R. Med. Chem. Res. 2000, 10, 164; (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.
17. Rosenbaum et al. also reported similar discussion, using their proposed model
of isoproterenol–b1-AR receptor complex.10b