Design and synthesis of tetrazole-based growth hormone secretagogue: The SAR studies of the O-benzyl serine side chain

Article abstract of DOI:10.1016/j.bmcl.2008.02.021

The structure-activity relationship of the O-benzyl serine side chain was investigated based on the tetrazole-based growth hormone secretagogue BMS-317180 (2). The ortho position of the benzyl moiety was found to be favorable for introduction of substituents. A series of ortho-substituted compounds were synthesized with improved in-vitro and in-vivo activity. Among them, the biphenyl compound 2p shows twofold improvement in potency compared to its parent compound BMS-317180 (2).

Full text of DOI:10.1016/j.bmcl.2008.02.021

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1825–1829
Design and synthesis of tetrazole-based growth hormone
secretagogue: The SAR studies of the O-benzyl serine side chain
Jun Li,a,* Stephanie Y. Chen,a Shiwei Tao,a Haixia Wang,a James J. Li,a Steve Swartz,a
Christa Musial,a Andres A. Hernandez,a Neil Flynn,b Brian J. Murphy,b Blake Beehler,b
Kenneth E. Dickinson,b Leah Giupponi,b Gary Grover,b Ramakrishna Seethala,b
Paul Sleph,b Dorothy Slusarchyk,b Mujing Yan,b William G. Humphreys,c
Hongjian Zhang,c William R. Ewing,a Jeffrey A. Robl,a
David Gordonb and Joseph A. Tinoa,*
aDiscovery Chemistry, Bristol Myers Squibb, Princeton, NJ 08543, USA
bMetabolic Disease Biology, Bristol Myers Squibb, Princeton, NJ 08543, USA
cPharmaceutical Candidate Optimization, Bristol Myers Squibb, Princeton, NJ 08543, USA
Received 4 January 2008; revised 7 February 2008; accepted 8 February 2008
Available online 13 February 2008
Abstract—The structure–activity relationship of the O-benzyl serine side chain was investigated based on the tetrazole-based growth
hormone secretagogue BMS-317180 (2). The ortho position of the benzyl moiety was found to be favorable for introduction of sub-
stituents. A series of ortho-substituted compounds were synthesized with improved in-vitro and in-vivo activity. Among them, the
biphenyl compound 2p shows twofold improvement in potency compared to its parent compound BMS-317180 (2).
Ó 2008 Elsevier Ltd. All rights reserved.
Since Bowers discovered a novel mechanism for releas-
ing endogenous growth hormone (GH) from the pitui-
tary in 1982, numerous efforts to uncover an orally
active growth-hormone secretagogue (GHS) have been
reported.1 The initial focus was on small growth hor-
mone-releasing peptides (GHRPs), including hexarelin,
that produced a GH response from the pituitary in a
physiologically pulsatile pattern in animals and hu-
mans.1,2 A group from Merck disclosed a G-protein
coupled receptor found in the pituitary and the hypo-
thalamus was cloned and identified as the growth hor-
mone secretagogue receptor (GHSR), which is distinct
from the growth hormone releasing hormone (GHRH)
receptor.2c Subsequently, small non-peptidic GHS
agents including L-692429 and MK-0677 (1) were re-
ported.3 MK-0677 is the first potent GH secretagogue
with excellent oral bioavailability in dogs.3b Subse-
quently, more small molecule GHSs have been reported
by pharmaceutical companies.3,4 The natural ligand
ghrelin for the GHS receptor was also discovered.7
Recently, we disclosed a potent, orally active GHS ago-
nist, BMS-317180, which was based on a tetrazole core
and was advanced into clinical evaluation.5 To further
improve the potency and pharmacological properties
of BMS-317180, extensive SAR studies were conducted.
Herein we report the SAR study around the left-hand O-
benzyl serine side chain of BMS-317180 (2) (Fig. 1).
Our initial approach focused on halogen-substituted
benzyl analogues of 2. The synthesis of 2, 4-difluro-ben-
zyl-substituted BMS-317180 (2a) is exemplified in
O
H
O
H
N
N
O
NH2
O
NH2
O
N
N
N
N
N
O
NSO2CH3
NH(CH2)4OH
O
Keyword: Growth hormone secretagogue.
*
Figure 1. Small-molecule growth-hormone secretagogues: MK-677
and BMS-317180.
Corresponding authors. Tel.: +1 609 818 7123; fax: +1 609 818 6810
(J.L.); e-mail addresses: jun.li@bms.com; joseph.tino@bms.com
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.02.021

1826
J. Li et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1825–1829
Scheme 1. Direct alkylation of Boc-D-serine by 2,4-difl-
uro-benzyl bromide gave the desired benzylated serine 4.
The chirality of 4 was retained by ensuring no excess
NaH during the reaction. The completion of the synthe-
sis of 2a followed our previously reported procedures.5
Coupling of 4 with aminoethanol, followed by acylation
of the crude alcohol gave the desired acetate 5. The for-
mation of tetrazole was carried out by treatment of 5
with two equivalents of triphenylphosphine, diethyl azo-
dicarboxylate, and azidotrimethylsilane.6 After stirring
at room temperature for 48 h, the tetrazole 6 was iso-
lated in 55% yield. A three-step sequence of Boc-depro-
tection of 6, followed by amino-acid coupling of amine
with N-Boc-methylalanine, and subsequent hydrolysis
of the acetate by lithium hydroxide in THF yielded the
alcohol 7. Alcohol 7 was then treated with 4-nitrophenyl
chloroformate in the presence of pyridine to give the
carbonate 8 in 50% yield from 6. Carbonate 8 was re-
acted with 4-amino-butanol to give the Boc-protected
carbamate in 90% yield. Deprotection of the Boc group
using 4 N HCl in dioxane gave the HCl salt of 2a. The
overall yield ranged between 5% and 9% starting from
Boc-D-serine.
An alternative route was developed for multiple analog
synthesis as shown in Scheme 2. Hydrolysis of acetate 9
with lithium hydroxide followed by protection with tert-
butyldimethylsilyl resulted in the silyl ether 10. Hydro-
genation of 10 afforded a key intermediate, alcohol 11.
Alkylation of 11 with a substituted benzyl bromide or
chloride generated 12 without loss of chirality by using
no more than two equivalents of sodium hydride.
Further elaboration to the final compounds proceeded
as described in Scheme 1.
F
F
NHBoc
c
NHBoc
OH
NHBoc
OH
O
b
a
O
HO
O
F
N
H
F
O
O
OAc
5
4
F
F
O
H
N
NHBoc
d
e
O
O
NHBoc
OH
N
N
N
N
F
F
N
N
OAc
N
N
7
6
F
O
H
N
F
O
H
N
O
NHBoc
O
f
O
N
N
NH2
F
N
O
N
N
N
F
N
O
NH(CH2)4OH
N
O
O
NO2
8
2a
Scheme 1. Reagents and conditions: (a) i—2,4-difluoro-benzyl bromide, NaH, DMF; (b) i—NMM, iso-butyl-chloroformate, À40 °C, ii—2-amino-
ethanol, À40 °C, iii—Ac2O, pyridine; (c) TMSN3, DEAD, Ph3P, rt, 48 h; (d) i—HCl/dioxane, rt, ii—HOAT, EDAC, N-Boc-methylalanine, CH2Cl2,
overnight, iii—1 N LiOH; (e) 4-nitrophenyl chloroformate, pyridine, CH2Cl2; (f) i—4-amino-butanol, ii—HCl/dioxane.
NHBoc
NHBoc
NHBoc
HO
O
O
a
b
N
N
N
N
N
N
N
N
N
OTBDMS
OTBDMS
OAc
N
N
N
10
11
9
CN
O
CN
H
N
NHBoc
c
d
O
O
NH2
N
N
N
N
N
N
O
OTBDMS
NH(CH2)4OH
N
N
O
12
2k
Scheme 2. Reagents: (a) i—4 N LiOH, THF, ii—TBDMSCl, imidazole, DMF; (b) 5% Pt(OH)2/C (cat), AcOH (cat), H2 (60 psi), MeOH; (c) 2-cyno-
benzyl bromide, NaH, DMF; (d) See Scheme 1, step c–d.

J. Li et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1825–1829
1827
Table 1. In-vitro potency of O-benzyl serine side chain analogs8
Table 1 (continued)
a
EC50 (nM)
O
Compound
R
H
N
R
O
2n
1.66
NH2
N
N
N
O
NH(CH2)4OH
N
O
2o
0.34
a
EC50 (nM)
Compound
R
Ghrelin
2
1.41
1.92
Bn
N
F
2p
2q
1.18
1.04
2a
0.42
F
O
N
Cl
2b
2c
2d
0.45
1.6
Cl
a Values are means of three experiments.
The major objective of this work was to study the
substituent effects on the benzyl ring. Halogen-related
substituents were initially chosen to study the posi-
tional effect of benzyl substitution as shown in Table
1. Small substituents such as fluorine at both para
and ortho positions (2a) improved the potency over
compound 2. The ortho-chlorine or ortho-trifluoro-
methyl-substituted benzyl analogs (2b, 2f) were also
found to improve functional in-vitro activity 3- to 4-
fold when compared to 2. The same substituents at
the meta position (2c, 2g, and 2i) had little effect on
the potency. In contrast, a para-substituted trifluorom-
ethyloxy (2j) resulted in a significant loss in potency
when compare to the meta-substituted analog 2i. The
potency was also significantly reduced when both meta
positions of the benzyl were occupied by either chlo-
rine or trifluoromethyl groups (2e, 2h). However, the
2,3-di-chloro-substituted phenyl (2d) still maintained
the excellent functional activity. These initial results
suggested that the ortho-position may be a favorable
site for further SAR study of the substituent effects
on the benzyl ring. For this purpose, several non-hal-
ogen functional groups with various stereo and elec-
tronic properties were introduced. All groups
including both electron-withdrawing group such as cy-
ano (2k), and electron-donating groups such as methyl
(2l) or methoxy (2m), gave similar results with good to
excellent in vitro potency. This result indicated that
the electronic effect of the substituents on the benzyl
ring had minimal or negligible consequences on
potency. Furthermore, removing the aromaticity by
using cyclohexane as the replacement for the phenyl
ring resulted in 2n showed in-vitro potency similar
to the parent compound 2. This result suggested that
the aromatic ring may not be essential to maintain
the potency.
Cl
Cl
Cl
Cl
0.61
2e
2f
6.75
0.72
1.79
CF3
F3C
F3C
2g
2h
2i
2j
34
CF3
F3CO
3.1
33.2
F3CO
CN
2k
1.26
CH3
2l
0.51
1.11
OCH3
We next turned our attention to aromatic substitutions
at the ortho-position (Scheme 3). Addition of the second
aromatic ring was achieved via Suzuki coupling of aryl
2m

1828
J. Li et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1825–1829
Br
O
H
O
H
N
N
O
NHBoc
OH
a
O
NHBoc
OH
N
N
N
N
N
N
N
N
13
14
Scheme 3. Reagent and condition: (a) PhB(OH)2, Pd (0) (cat), toluene, 100 °C.
the target site(s) and/or more favorable pharmaco-
dynamic properties. Several of the analogs in Table 2
were studied in a rat PK screen at a 10 mg/kg oral dose.
However, all compounds tested, including the most po-
tent in vitro compound 2o, and the most active in vivo
compound 2q, gave low plasma exposures. Since the
in vitro activity was determined on human cell-based as-
say, a species difference might be another reason for this
unexpected in vivo activity in the rat model. The discon-
nection of the in vitro and in vivo activities for some
compounds in certain animal models was also reported
by other research groups.10
Table 2. In vivo activity of the compounds in the acute anesthetized
rat model at 1.74 lmol/kg
Compound
Increase GHa at 1.74 lmol/kg
(% of vehicle)
Respondersb
2
750 ( 112)
1081 ( 130)
920 ( 257)
1441 ( 332)
1654 ( 117)
836 ( 192)
5237 ( 1233)
5/5
5/5
5/5
5/5
5/5
5/5
5/5
2a
2k
2l
2n
2o
2q
a Values are means of five experiments, standard deviation is given in
parentheses.
b A rat with more than 200% GH increase over vehicle control is
considered as a responder.
In summary, the SAR of the benzyl moiety of BMS-
317180 (2) has been described. The results suggest that
the ortho position of the benzyl moiety is the preferred
position for substitution. A hydrophobic aromatic
group at the ortho position of the phenyl was found
to greatly increase the functional activity at the GHS
receptor. Some revealed analogs also improved the
in vivo activity in the rat model when compared to par-
ent compound 2. The initial efforts to replace the phenyl
ring also resulted in the discovery of the non-aromatic
functional group cyclohexane as an alternative for the
aromatic benzene ring.
boronic acids with the ortho-bromo-phenyl intermediate
13, which was prepared similarly according to Scheme 1.
The completion of the synthesis from 14 proceeded as
described in Scheme 1. The biphenyl analog 2p was
found to be the most potent compound within the series
in this in-vitro assay. However, polar aromatic substitu-
ents such as pyridine or dimethyl-isoxazole (2p, 2q), only
slightly improved in-vitro potency over compound 2.
This phenomenon suggested a deeper hydrophobic bind-
ing pocket might be located in this region of the GHS
receptor.9
Acknowledgment
Several compounds were chosen for evaluation in an
acute anesthetized rat model measuring the increased
GH levels as the efficacious response. The compounds
We thank Dr. Robert Zahler for his advice and sugges-
tions for the manuscript.
were administered intravenously at
a
dose of
1.74 lmol/kg. After 15 min, blood samples were col-
lected and the plasma isolated and analyzed for rat
growth hormone (GH) via radioimmunoassay.12 Data
are expressed as the percentage of increase GH release
compared with vehicle control animals (Table 2).
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