Novel series of highly potent non-peptide growth hormone secretagogues with improved bioavailability

Article abstract of DOI:10.3987/COM-05-S(T)73

The discovery and the SAR of acylproline derivatives as highly potent growth hormone secretagogues (GHSs) with good oral bioavailability are described. One representative compound, N-[3-(2,2-dimethylpropylamino)-2-hydroxypropyl]-2(R)-[1-(2,2-dimethylpropionyl)pyrrolidine-2(S)- carbonylamino]-3-naphthalen-2-ylpropionamide (4e), showed potent GHS activity (ED₅₀=1 nM) and good oral bioavailability (BA=33.2%). Moreover, the optically pure N-[3-(2,2-dimethylpropylamino)-2(S)-hydroypropyl]-2(R)-[1-(2,2-dimethylpropionyl)pyrrolidine-2(S)-carbonylamino]-3-naphthalen-2- ylpropionamide ((2S)-4e) showed a good metabolic stability against in vitro clearance (human liver microsome) with potent GHS activity.

Full text of DOI:10.3987/COM-05-S(T)73

HETEROCYCLES, Vol. 67, No. 2, 2006
561
HETEROCYCLES, Vol. 67, No. 2, 2006, pp. 561 - 566. © The Japan Institute of Heterocyclic Chemistry
Received, 18th August, 2005, Accepted, 10th November, 2005, Published online, 11th November, 2005. COM-05-S(T)73
NOVEL SERIES OF HIGHLY POTENT NON-PEPTIDE GROWTH
HORMONE SECRETAGOGUES WITH IMPROVED BIOAVAILABILITY
Hirohide Ishige,* Nobuo Ishiyama, Mitsuo Mimura, Mitsuo Hayashida,
Tadashi Okuno, Kiyoharu Ukai, Takeshi Kiyofuji, Yasuo Yoneda, Shinji
Tauchi, Akinori Aoyama, and Kiyoshi Inoguchi
General Research Laboratories, Kaken Pharmaceutical Co., Ltd., 14, Shinomiya,
Minamikawara-cho, Yamashina-ku, Kyoto 607-8042, Japan
Abstract – The discovery and the SAR of acylproline derivatives as highly potent
growth hormone secretagogues (GHSs) with good oral bioavailability are
described. One representative compound, N-[3-(2,2-dimethylpropylamino)-2-
hydroxypropyl]-2(R)-[1-(2,2-dimethylpropionyl)pyrrolidine-2(S)-carbonylamino]-
3
-naphthalen-2-ylpropionamide (4e), showed potent GHS activity (ED50=1 nM)
and good oral bioavailability (BA=33.2%). Moreover, the optically pure
N-[3-(2,2-dimethylpropylamino)-2(S)-hydroxypropyl]-2(R)-[1-(2,2-dimethylpro-
pionyl)pyrrolidine-2(S)-carbonylamino]-3-naphthalen-2-ylpropionamide
(
(2S)-4e) showed a good metabolic stability against in vitro clearance (human liver
microsome) with potent GHS activity.
INTRODUCTION
Since the discovery of the peptidyl growth hormone secretagogue, named GHRP-6, by Bowers and
1,2
Momany et al. in 1984, GHSs have received considerable attention with the hope of using them as a
3,4
replacement for the parenteral GH.
In addition to the stimulation of growth, GH has been known to play a
number of important roles in the metabolic processes, e.g., the stimulation of protein synthesis and the
5
mobilization of free fatty acids. Furthermore, the discoveries of the GHS receptors and its endogenous
6
ligand, Ghrelin has spured the research activity on orally active small molecular GHSs for their potential
use such as an appestat.
We have already created novel benzothiazepin derivatives represented as the compound (1) (Figure 1) with a
nanomolar GHS activity using a 3D pharmacophore technique and the site-dependent fragment QSAR
7
analysis, but most of these derivatives turned out to have a low bioavailability (BA<0.4%). We have also

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HETEROCYCLES, Vol. 67, No. 2, 2006
2
learned that the addition of non-polar groups to the R position of benzothiazepin derivatives (2) could
bestow a better gastrointestinal absorption on this series, but at the same time, resulted in weaker in vitro
activities (EC50>100 nM). In order to resolve this incompatibility, we resumed our research to create a
8
new scaffold that would be able to harmonize the GHS activity with the oral bioavailability. In this
communication, we report new acylproline derivatives which have the acylprolinyl scaffold replacing the
benzothiazepin group, and discuss their GHS activity and in vivo behavior.
S
N
S
N
O
O
HCl
HCl
H
N
OH
H
N
OH H
N
NH2
R2
O
N
H
O
N
H
O
O
1
2
R1
R1
HCl
HCl
N
O
N
H
OH
H
OH H
N
O
O
N
NH2
N
R2
N
H
O
N
H
O
O
3
4
Figure 1.
RESULTS AND DISCUSSION
The general method for the synthesis of the acylproline derivatives as a mixture of two distereoisomers is
outlined in Scheme 1. The epoxide (6) was obtained from the phtalimide (5) with glycidol by the
8
Mitsunobu reaction in 75%. The cleavage of the epoxide (6) with sodium azide followed by the
Pd-catalyzed hydrogenation in the presence of conc. HCl produced the amino alcohol salt (8) in 42% from
6
. On the other hand, the -proline ethyl ester hydrochloride (9) was converted to the corresponding
L
amides with acetic acid, isobutyric acid, 2-ethylbutanoic acid or pivalic acid and then hydrolysis of the
ethyl ester gave acylprolines (10a-d) in good to excellent yields. The coupling of 10a-d with
D
-3-(2-naphtyl)alanine methyl ester and subsequent hydrolysis of the methyl ester produced the acid
11a-d) . The coupling of 11a-d with 8 produced compound (12a-d). The deprotection of 12a-d with
hydrazine hydrate gave the amine (13a-d). The resulting amine (13a-d) was then converted to the HCl salt
3a-d) with 4M HCl/AcOEt in good to excellent yields from 10a-d. The reductive amination of 13d with
(
(

HETEROCYCLES, Vol. 67, No. 2, 2006
563
isobutyl aldehyde or pivaloyl aldehyde followed by treatment of the resulting secondary amine with 4M
HCl/AcOEt provided the target compounds (4d,e) as the HCl salt in moderate yields from 10a-d.
O
O
O
O
i
ii
HCl
NH2
iii
NH
N
N3
N
N
O
OH
OH
O
O
O
O
5
6
7
8
R1
R1
O
R1
N
O
iv,v
N
vi,v
N
O
H
OH
HN
HCl
vii
O
N
N
O
O
N
H
COOEt
COOH
N
COOH
O
O
1
H
1
0a: R =Me
b: R =i-Pr
c: R =ethylpropyl
d: R =t-Bu
1
1
9
11a: R =Me
1
2a: R =Me
1
1
1
b: R =i-Pr
b: R =i-Pr
1
1
1
c: R =ethylpropyl
c: R =ethylpropyl
1
1
1
d: R =t-Bu
d: R =t-Bu
R1
N
HCl
NH2
OH
H
viii
O
N
O
N
H
1
O
3a: R =Me
b: R =i-Pr
c: R =ethylpropyl
d: R =t-Bu
ix
1
1
R1
N
1
OH
H
N
O
NH2
O
N
H
O
R1
1
HCl
x,ix
13a: R =Me
N
OH
1
H
H
b: R =i-Pr
O
N
N
1
O
N
R2
c: R =ethylpropyl
H
1
2
O
1
4
e: R =t-Bu, R =i-Bu
f: R =t-Bu, R =neo-Pentyl
d: R =t-Bu
1
2
Scheme 1. Conditions: (i) glycidol, PPh
3
, DEAD, THF; (ii) NaN
3
, NH
4
Cl, DMF, 80℃; (iii) 10% Pd-C,
Cl ; (v) 2M NaOH,
1
conc. HCl, EtOH, H
2
/2MPa; (iv) R COOH, HOBt, EDC, N-Methylmorpholine, CH
2
2
MeOH; (vi)
D-3-(2-Naphtyl)alanine methyl ester, HOBt, EDC, N-Methylmorpholine, DMF; (vii) 8, HOBt,
2
EDC, N-Methylmorpholine, DMF; (viii) NH
2
NH
2
-H
2
O, EtOH, reflux; (ix) 4M HCl/AcOEt, (x) R CHO,
NaBH CN, MeOH.
3
Table 1 shows the GHS activity and the oral bioavailability of the acylproline compounds. Several repeats
of the appropriate structural modifications followed by SAR analyses of this scaffold yielded compounds
with a strong GHS activity and eventually with a good oral bioavailability (Table 1). The SAR studies of
1
the acylproline derivatives revealed that when R was a small aliphatic group, the GHS activity was not
improved at all (4a:1000 nM). However the activity significantly increased when the α-branched aliphatic
1
1
1
alkyl groups were inserted in the R . The compounds (4b, R =isopropyl), (4, R =ethylpropyl) and (4d,
1
R =pivalyl) depicted in the Table 1 showed high GHS activities (4b:10 nM, 4c:1 nM, 4d:10 nM), but their

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HETEROCYCLES, Vol. 67, No. 2, 2006
bioavailavilities were not as good as we expected (BA=3.6% and 0.6%, respectively). Based on these
results, we added a hydrophobic functional group at the amino moiety in order to increase the
hydrophobisity of the entire molecule for the enhancement of the gastrointestinal absorption through the
effect of the cell membrane permeability improvement. The amino moiety was the most influential
pharmacophare on the GHS activity and the improvised change at that part in the series of benzodiazepine
derivatives only caused a loss of the GHS activity. On the other hand, the acylproline derivatives tolerated
the modification at this sensitive part toward the hydrophobisity. As shown in Table 1, the t-butyl
1
2
substitution at R in the presence of the isobutyl or neopentyl group at R drastically improved the oral
bioavailability while the GHS activity remained intact (4e:BA=33.2%, 4f:BA=30.1%).
Table 1 GHS Activity and Oral Bioavailability of Acylproline Compounds
R1
HCl
N
O
H
N
OH H
O
N
R2
N
H
2
O
4
a
b
R1
R2
Compound
EC (nM)
Bioavailability(%)
5
0
4
a
Me
H
H
1
000
Me
4b
1
0
Me
Me
Me
4
c
H
H
1
10
1
3.6
0.6
Me
Me
Me
Me
Me
4
d
Me
Me
Me
Me
4
e
3
3
3.2
0.1
Me
Me
Me
Me
Me
4
f
10
a
EC50 values were measured with rat primary anterior pituitary cells.7,10
b
Rats, 10mg/kg, po.
These results naturally prompted us to further examine compound (4e) as to whether it would have a
suitable profile as a drug candidate. The in vitro clearance in the liver microsome of 4e was tested and the
results are shown in Table 2. The compound (4e) turned out to be quite stable against the rat liver

HETEROCYCLES, Vol. 67, No. 2, 2006
565
microsome, but relatively vulnerable to the human one. Furthermore, the optically pure compounds
1
1
12
13
(
(2S)-4e and (2R)-4e ) were synthesized and examined the GHS activity and the clearance in the liver
microsome of humans (Table 2). The GHS activity of (2S)-4e was 100-times better than that of (2R)-4e.
In addition, it should be noted that (2S)-4e was 2-fold more metabolically resistant to in vitro clearance
(
human liver microsome) than (2R)-4e. These results strongly suggested that the compound ((2S)-4e)
would have an excellent in vivo behavior, i.e., a high Cmax and a long half-life time in the human body.
Table 2 in vitro Clearance in Liver Microsome of Compound (4e)
Me
Me
Me
N
O
H
N
HCl
O
N
H
X
O
4e
a
CLint.(mL/min/mg)
Compound
X
EC (nM)
50
rat
human
OH H Me
N
1
1
0.00
0.11
4
e
Me
2
OH H Me
N
(
2S)-4e
2R)-4e
0.07
0.14
Me
2
S
OH H Me
N
(
100
Me
2R
a
EC50 values were measured with rat primary anterior pituitary cells.7,10
In conclusion, we discovered novel acylproline derivatives with a high a GHS activity and drastically
improved oral bioavailability. Moreover, we demonstrated that (2S)-4e had a more potent GHS activity
and a more favorable metabolic stability against in vitro clearance (human liver microsome) than (2R)-4e.
ACKNOWLEDGEMENTS
The authors thank Dr. Ping Huang at the Molecular Research Institute (Mountain View, CA) for the fruitful
discussions on the SAR results.
REFERENCES AND NOTES
1
2
3
.
.
.
F. A. Momany, C. Y. Bowers, G. A. Reynolds, and A. Hong, Endocrinology, 1984, 114, 1531.
C. Y. Bowers, F. A. Momany, G. A. Reynolds, and A. Hong, Endocrinology, 1984, 114, 1537.
Z. Laron, Drugs, 1995, 50, 595.

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HETEROCYCLES, Vol. 67, No. 2, 2006
4
.
.
P. Saenger, J. Clin. Endroclinol. Metab., 1996, 50, 595.
5
A. D. Howard, S. D. Feighner, D. F. Cully, J. P. Arena, P. A. Liberator, C. I. Rosenblum, M. Hamelin,
D. L. Hreniuk, O. C. Palyha, J. Anderson, P. S. Paress, C. Diaz, M. Chou, K. K. Liu, K. K. Mckee, S. S.
Pong, L. Y. Chaung, A. Elbrecht, M. Dashkevicz, R. Heavens, M. Rigby, D. J. S. Sirinathsinghji, D. C.
Dean, D. G. Melillo, A. A. Patchett, R. P. Nargund, P. R. Griffin, J. A. DeMartion, S. K. Gupta, J. M.
Schaeffer, R. G. Smith, and L. H. T. Van der Ploeg, Science, 1996, 273, 974.
6
.
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M. Kojima, H. Hosoda, Y. Date, M. Nakazato, H. Hatsuo, and K. Kangawa, Nature, 1999, 402, 656.
P. Huang, G. H. Loew, H. Funamizu, M. Mimura, N. Ishiyama, M. Hayashida, T. Okuno, O. Shimada,
A. Okuyama, S. Ikegami, J, Nakano, and K. Inoguchi, J. Med. Chem., 2001, 44, 4082.
N. Ishiyama, H. Ishige, M, Mimura, T. Okuno, K. Ukai, T. Kiyofuji, S. Tauchi, and K. Inoguchi, WO
7
8
.
0
048623, 2000.
9
1
1
.
O. Mitsunobu, Synthesis, 1981, 1.
0. D. Wu, C. Chen, K. Katoh, J. Zhang, and I. J. Clake, Endocrinol, 1994, 140, R9.
2
5
o
1
1. Data for (2S)-4e: [α]
D 6
-40.9 (c=1.0, MeOH) ; H-NMR (DMSO-d ; 270 MHz) δ 0.93 (6H, d, J=6.6
Hz), 1.12 (9H, s), 1.20-1.40 (1H, m), 1.50-1.85 (3H, m), 1.90-2.10 (1H, m), 2.60-2.80 (3H, m),
2
4
.85-3.05 (2H, m), 3.10-3.40 (3H, m), 3.45-3.70 (2H, m), 3.85-4.05 (1H, br s), 4.20-4.35 (1H, br s),
.45-4.65 (1H, m), 5.68 (1H, d, J=4.6 Hz), 7.35-7.55 (3H, m), 7.71 (1H, s), 7.75-7.90 (3H, m), 8.19
+
(
1H, t, J=5.6 Hz), 8.32 (1H, d, J=8.6 Hz), 8.40-8.70 (2H, br s) ; MS (FAB) m/z 525 (M + H) .
2
5
o
1
1
2. Data for (2R)-4e: [α]
D 6
-20.0 (c=0.1, MeOH) ; H-NMR (DMSO-d ; 270 MHz) δ 0.94 (6H, d, J=6.6
Hz), 1.12 (9H, s), 1.20-1.40 (1H, m), 1.60-1.90 (3H, m), 1.95-2.10 (1H, m), 2.65-2.85 (3H, m),
.90-3.20 (3H, m), 3.20-3.70 (4H, m), 3.85-4.00 (1H, m), 4.20-4.35 (1H, br s), 4.45-4.65 (1H, m), 5.72
2
(
1H, d, J=5.0 Hz), 7.35-7.55 (3H, m), 7.71 (1H, s), 7.75-7.90 (3H, m), 8.15-8.30 (1H, m), 8.40 (1H, d,
+
J=8.3 Hz), 8.50-8.70 (2H, br s) ; MS (FAB) m/z 525 (M + H) .
1
3. (2S)-4e and (2R)-4e were synthesized from the (S)-glycidol and (R)-glycidol, respectively.