Tetrazole based amides as growth hormone secretagogues

Article abstract of DOI:10.1016/j.bmcl.2008.03.059

A novel series of N1 substituted tetrazole amides were prepared and showed to be potent growth hormone (GH) secretagogues. Among them, hydroxyl containing analog 31 displayed excellent in vivo activity by increasing plasma GH 10-fold in an anesthetized IV

Full text of DOI:10.1016/j.bmcl.2008.03.059

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Bioorganic & Medicinal Chemistry Letters 18 (2008) 2536–2539
Tetrazole based amides as growth hormone secretagogues
James J. Li,a,* Haixia Wang,a Jun Li,a Fucheng Qu,a Stephen G. Swartz,a
a
a
a
a,
*
´
´
Andres S. Hernandez, Scott A. Biller, Jeffrey A. Robl, Joseph A. Tino,
Dorothy Slusarchyk,b Ramakrishna Seethala,b Paul Sleph,b Mujing Yan,b
Gary Grover,b Neil Flynn,b Brian J. Murphyb and David Gordonb
aDiscovery Chemistry, Bristol-Myers Squibb Research and Development, PO Box 5400, Princeton, NJ 08543-5400, USA
bMetabolic Disease Research, Bristol-Myers Squibb Research and Development, PO Box 5400, Princeton, NJ 08543-5400, USA
Received 1 February 2008; revised 17 March 2008; accepted 18 March 2008
Available online 22 March 2008
Abstract—A novel series of N1 substituted tetrazole amides were prepared and showed to be potent growth hormone (GH) secret-
agogues. Among them, hydroxyl containing analog 31 displayed excellent in vivo activity by increasing plasma GH 10-fold in an
anesthetized IV rat model.
Ó 2008 Elsevier Ltd. All rights reserved.
H
Human growth hormone (GH) replacement therapy has
shown promising results for the prevention of muscle
functional decline in the elderly in a number of prelimin-
ary studies.1 However, longer term use is associated with
multiple side effects such as edema, arthralgia, carpal
tunnel syndrome, and hyperglycemia due to the need
to administer superphysiological doses of GH in order
to maintain an efficacious level in the systemic circula-
tion.2 Orally active GH secretagogues (GHS), on the
other hand, have been shown to enhance the amplitude
of GH during endogenous pulsatile release and are be-
lieved to more closely mimic human physiological condi-
tion.3 Therefore, treatment with an oral GHS agent
should offer a more optimal approach for restoring pul-
satile GH secretion in the elderly population.4 Several
classes of GHS agonists such as 1 (MK-677),5 2 (CP-
42491),6 and 3 (LY-444711)7 have advanced to the clin-
ics (Fig. 1). More recently, we have reported a novel tet-
razole based GHS 4 (BMS-317180) as our first clinical
candidate. Tetrazole 4 was shown to be orally active
and efficacious in promoting GH release in a preclinical
beagle dog model.8 As part of our efforts to further
delineate structural features critical for activity within
this series, SAR exploration of various amide and/or
reverse amide linkers, in place of the corresponding car-
H
N
N
BnO
NH2
BnO
NH2
O
O
O
O
N
N
O
N
N
N
Me
SO2Me
1 (MK-677)
2 (CP-424391)
H
N
Ph
NH2
BnO
H
O
O
N
NH2
HN
N
O
H
N
O
N
N
N
N
OH
N
Me
N
O
O
MeO
3 (LY-444711)
4 (BMS-317180)
Figure 1. Representative examples of GH secretagogues.
bamate in 4, was undertaken. Herein is a summary of
these SAR studies which led us to the identification of
a novel series of N1 substituted tetrazole amides as po-
tent GHS agonists.
Keywords: GHS, growth hormone secretagogues; Tetrazole amide.
*
The preparations of tetrazole amides were straightfor-
ward from readily available starting materials and are
Corresponding authors. Tel.: +1 609 818 7124 (J.J.L.); e-mail
addresses: James.Li@bms.com; joseph.tino@bms.com
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.03.059

J. J. Li et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2536–2539
2537
outlined in Scheme 1. The syntheses started with amide
coupling of N-Boc-O-benzyl-D-Serine (5) and amines 6
via conventional mixed anhydride activation protocol.
The formation of tetrazole intermediate 7 proceeded in
the presence of triphenylphosphine, diethyl azodicar-
boxylate (DEAD), and azidotrimethylsilane (TMSN3).9
The removal of Boc protecting group in 7 with strong
acids and subsequent reaction with Boc-Aib-OH gave
8a–c. Hydrolysis under basic condition provided acid 9
or 11; or the alcohol intermediate 10. Amide formation
from acid 9 or 11 via the activation of EDAC and HOAT
followed by deprotection of Boc group gave the desired
amides 12–36 or benzyl amides 40–43. For reverse amide
analogs, alcohol 10 was first converted to an azide inter-
mediate using a modified Mitsunobu condition, followed
by hydrogenation of the azide to provide the correspond-
ing amine. Once again, reverse amides 37–39 were pre-
pared in a similar manner as previously described using
a variety of acids. The last two steps of these sequences,
amide formation and de-Boc deprotection, were easily
amended to a parallel synthesis procedure using auto-
mated solid phase extraction purification and generally
afforded the final compounds in >95% purity.10
Simple amides with various terminal lipophilic groups
such as 12–17 exhibited modest potency in the range
of 26–70 nM (Table 1). The incorporation of more polar
functional groups at the distal position, such as a methyl
sulfone in 18, a sulfonamide in 19, a primary amide in
20, or a urea in 23 improved GHS potency to give com-
pounds with EC50 value less than 10 nM. The dramatic
effect of the terminal moiety on the in vitro potency was
evident by comparison to ester 21 or acid 22. These
modifications resulted in a significant loss of functional
potency (EC50 > 140 nM).
We have demonstrated earlier that the incorporation of
a distal hydroxyl group in the tetrazole carbamate series
(i.e., 4) significantly improved oral activity in our pre-
clinical beagle dog model.8 Consequently, a series of tet-
razole amides containing a terminal hydroxyl group
were evaluated. A longer length in general appeared to
be preferred as compounds 27–32 showed considerably
greater potency than shorter length analogs 25–26 (Ta-
ble 1). Interestingly, replacement with a phenolic hydro-
xyl moiety as in 33 provided
a
very potent
subnanomolar GHS agonist. However, further exten-
sion of the terminal hydroxyl group as in 35, or restric-
tion as in cyclohexanol 36, resulted in a substantial loss
of activity. For two of the most potent amides 31 and 33
(EC50 ꢀ 0.2 nM), the corresponding N-methylated ter-
tiary amides 32 and 34 were also generated, but an
approximate 10-fold loss of the functional potency was
observed in both cases.
A H4 glioma cell based GHS functional FLIPR assay
was developed for the in vitro evaluation of these
GHS agents.11,12 All the compounds reported herein
showed an intrinsic activity greater than 85% as com-
pared with endogenous Ghrelin (EC50 = 1.4 nM) and
therefore are considered as full agonist.13
Me Me
H
NHBoc
NHBoc
O
N
further elaboration
c, d
O
a, b
O
NHBoc
+
NH2G
N
N
CO2H
O
N
N
N
G
shown below
N
G
N
5
6
N
7
8a-c
Me Me
Me Me
H
H
N
N
e
f, c
O
NHBoc
OH
O
NH2
8a
O
O
N
N
N
N
N
N
NR1R2
CO2Me
G =
G =
G =
N
N
O
O
9
12-36
Me Me
Me Me
H
H
N
N
e
e
O
NHBoc
OH
g, h, i, c
O
NH2
8b
8c
O
O
H
N
N
N
N
N
OAc
N
N
N
R3
N
O
10
37-39
Me Me
Me Me
H
H
N
N
f, c
O
NHBoc
O
NH2
O
2
O
2
O
O
N
N
N
N
NHR4
CO2Me
OH
O
O
1
N
N
N
1
2
3
3
O
1
N
3
11
40-43
Scheme 1. Reagents and conditions: (a) NMM, i-BuOCOCl, 70–95%, THF; (b) TMSN3, DEAD, Ph3P, 50–85%, THF; (c) 15–20% TFA/CH2Cl2 or
4 N HCl in dioxane/methanol; (d) Boc-Aib-OH, EDAC, HOAT, 65–83%, CH2Cl2; (e) 1 N aq NaOH, 80–90%; (f) EDAC, HOAT, appropriate
amines, 30–90%, CH2Cl2; (g) PPh3, DEAD, DPPA, 95%, THF; (h) H2/5% Pd–C catalyst, 95%, MeOH; (i) EDAC, HOAT, appropriate acids,
30–90%, CH2Cl2.

2538
J. J. Li et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2536–2539
Table 1. In vitro potency of amides
Table 2. In vitro potency of reverse amides
Me Me
Me Me
H
H
N
O
N
NH2
O
NH2
O
O
H
N
N
N
NR1R2
N
N
R3
N
N
N
N
O
O
Compounda
R1
EC50 (nM)
Compound
R3
–(CH2)4OH
–(CH2)2SO2Me
EC50 (nM)
12
13
14
15
16
17
18
19
20
21
22
–(CH2)3CH3
–CH2CF3
–Ph
30
26
37
38
4.4
14.7
70
32
OH
–CH2Ph
–(CH2)2Ph
–(CH2)3Ph
39
0.64
23
33
–(CH2)2SO2Me
–(CH2)2SO2NH2
–(CH2)2CONH2
–(CH2)2CO2Et
–(CH2)2CO2H
4.1
1.4
7.6
140
204
Table 3. In vitro potency of benzyl amides
Me Me
H
N
O
NH2
O
2
O
O
N
N
NHR4
23
3.3
O
N
1
N
3
NH
N
24
25
26
27
28
–(CH2)2OH
–(CH2)3OH
–(CH2)4OH
–(CH2)5OH
–(CH2)6OH
40
27
Compound
Position
R4
EC50 (nM)
1.8
1.3
0.6
40
41
42
43
1,2
1,3
1,2
1,3
–(CH2)2OH
–(CH2)4OH
11
8.4
0.9
3.0
OH
29
30
0.7
0.2
O
S
S
OH
OH
To assess the in vivo efficacy of these novel tetrazole
amide based GHS compounds, analogs 19, 28, 31, 33,
and 37 were selected for evaluation in an anesthetized
IV rat model for their ability to stimulate GH release.14
All the compounds showed good activity in generating
4- to 10-fold increases in plasma GH upon a single
screening dose of 0.17 lmmole/kg, and were slightly
more potent than carbamate 4 in this model (Table 4).
Compound 31 afforded the greatest response and was
subsequently tested at lower doses (0.017 or
0.052 lmmole/kg). The observed response indicates that
analog 31 was the most potent in vivo analog of the ser-
ies. Unfortunately, in a subsequent PK evaluation in rat,
compound 31 was shown to have poor oral bioavailabil-
ity (<3%), precluding it for further evaluation.
31
0.2
O
O
32a
33
3.1
0.2
OH
34a
35
2.9
O
S
O
105
OH
36
521
1.4
OH
In summary, we have identified a novel series of tetra-
zole amides as exceptionally potent GHS agonists. The
Ghrelin
a R2 is hydrogen; R2 is methyl for compounds 32 and 34.
Table 4. In vivo activity in the IV anesthetized rat modela
Three reverse amides, generated based on SAR observa-
tions in the initial amide series (e.g., 18, 26, and 33),
were also evaluated (Table 2). Analogs 37–39 showed
approximately a two- to fourfold loss of potency as
compared to their amide analogs, limiting our interest
in further optimization in the reverse amide chemotype.
Finally, selected benzyl amides were assessed as part of
the linker evaluation. Similar to the results obtained in
the simple amide series, longer chain lengths were also
preferred (i.e., 42, EC50 of 0.9 nM vs 40, EC50 of
11 nM) but the difference in potency between 1,2 and
1,3 substitution scaffold was unremarkable in the two
pairs evaluated (Table 3).
Compound
EC50 (nM)
GH SEMb (%)
4
19
28
33
37
31
1.9
1.4
0.6
0.2
4.4
0.2
264 39
408 69
522 88
606 104
722 67
1290 119
956 120 (0.052 lmole/kg)
398 65 (0.017 lmole/kg)
a Five rats per group are treated with each compound.
b The values represent the increase of GH SEM (%) relative to
control after IV dosing of 0.17 lmole/kg of compound; lower doses
were also evaluated for compound 31.

J. J. Li et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2536–2539
2539
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incorporation of a terminal hydroxyl group in general
was preferred for both potent in vitro and in vivo activ-
ity. The most potent analog 31 (EC50 = 0.2 nM) showed
an increase in plasma GH levels of approximately 10-
fold in an anesthetized IV rat model.
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