Modeling directed design and biological evaluation of quinazolinones as non-peptidic growth hormone secretagogues

Article abstract of DOI:10.1016/S0960-894X(99)00584-3

Quinazolinone derivatives were synthesized and evaluated as non-peptidic growth hormone secretagogues. Modeling guided design of quinazolinone compound 21 led to a potency enhancement of greater than 200-fold compared to human growth hormone secretagogue affinity of a screening lead 4.

Full text of DOI:10.1016/S0960-894X(99)00584-3

Bioorganic & Medicinal Chemistry Letters 10 (2000) 5±8
Modeling Directed Design and Biological Evaluation
of Quinazolinones as Non-Peptidic Growth Hormone
Secretagogues
Zhixiong Ye, a,Ã Yingduo Gao, b,Ã Raman K. Bakshi, a Meng-Hsin Chen, a
Susan P. Rohrer, c Scott D. Feighner, c Sheng-Shung Pong, c Andrew D. Howard, c
Allan Blake, c Elizabeth T. Birzin, c Louis Locco, c Rupa M. Parmar, c
Wanda W.-S. Chan, c James M. Schae€er, c Roy G. Smith, c
Arthur A. Patchett a and Ravi P. Nargund a
aDepartment of Medicinal Chemistry, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
bDepartment of Molecular Systems, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
cDepartment of Biochemistry and Physiology, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
Received 6 August 1999; accepted 28 September 1999
AbstractÐQuinazolinone derivatives were synthesized and evaluated as non-peptidic growth hormone secretagogues. Modeling
guided design of quinazolinone compound 21 led to a potency enhancement of greater than 200-fold compared to human growth
hormone secretagogue anity of a screening lead 4. # 1999 Elsevier Science Ltd. All rights reserved.
Introduction
receptor agonists that evolved from a directed screening
lead 4 (hGHSr IC50=3.5 mM, Fig. 2).
Growth hormone (GH) secretagogues have received
considerable attention in the last several years based on
promising applications of recombinant GH in animals
and in humans. Potential therapeutic indications for
GH secretagogues in human health include idiopathic
GH de®ciency states in children and adults, stimulation
of anabolic processes in the elderly and supportive
therapy in catabolic wasting conditions including AIDS
and osteoporosis.1,2 After extensive studies of the
growth hormone releasing peptide GHRP-6 (1, hGHSr3
IC50=6 nM, rat pit.4 EC50=10 nM, Fig. 1)5 had estab-
lished its GH releasing ecacy in animals and in man,
e€orts were made in a number of laboratories to iden-
tify peptidomimetic GH secretagogues as potential
alternatives to GH replacement therapy.6 Several of GH
secretagogue peptidominetics entered clinical studies
including MK-0677 (2, hGHSr IC50=0.3 nM, rat pit.
EC50=1.3 nM)7 and CP-424,391 (3, rat pit. EC50=3
nM).8 In this paper we present the design, synthesis and
biological activities of quinazolinone-based compounds
as a new class of non-peptidic human GH secretagogue
Chemistry
The quinazolinones were prepared in a straightforward
fashion as outlined in Scheme 1. The cyclization of the
2-amino-5-bromobenzoic acid 5 with 2-naphthoyl chlo-
ride 6 was accomplished by heating the mixture with
DMAP and triethyl amine in DMF to yield the qui-
noxazolone intermediate 7. After addition of glycine
ethyl ester 8, heating was continued to ensure depletion
of the quinoxazolone 7 and formation of the quiazolin-
one ester 9. Base-catalyzed hydrolysis of the ester 9
a€orded the key quinazolinone acid 10 followed by a
standard peptide coupling with mono-Boc protected
diamines to furnish bromo-quinazolinones 11 and 12.
The bromo compounds were then either treated with
HCl to yield the quinazolinones 13 and 14 or converted
to the corresponding phenyl analogues 15 and 16 under
standard Suzuki coupling conditions [PhB(OH)2,
Pd(PPh3)4 and Na2CO3] followed by removal of the Boc
group. Similarly, the phenethyl analogues were synthe-
sized from the cyclization of 5 and 3-phenylpropionyl
chloride (17) and, by following the same route, phe-
nethyl analogues 18±21 were prepared (Scheme 2).
ÃCorresponding author. Tel.: +1-732-594-5644; fax: +1-732-594-
3007.
0960-894X/00/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(99)00584-3

6
Z. Ye et al. / Bioorg. Med. Chem. Lett. 10 (2000) 5±8
Figure 1.
3-fold increase in binding anity of human GH secre-
tagogue receptor while altering the chain length from
®ve to six carbon atoms (14) provided a slight increase
in potency.9
Previous work on GH secretagogues has suggested that
the agonist pharmacophore of highly active GH secre-
tagogue is comprised of two aryl groups and a basic
amine.10 Site-directed mutagenesis studies of the human
GH secretagogue receptor indicated that the basic
amine group of 2 forms a salt bridge with the negatively
charged side-chain of E124 in TM3.11 We hypothesized
that the benzyl group of 2 may lie in a hydrophobic
pocket formed by the side-chains of M213 in TM5 and
H280 in TM6 and the spiroindane group p±p stacks
with the side-chain of W274 in TM6 (Fig. 3a).
Figure 2.
Results and Discussion
The initial screening lead compound 4 showed modest
anity toward the human GH secretagogue receptor.
Modi®cation of the amine side-chain was investigated
®rst. Removal of the ester group (13, Table 1) led to a
Scheme 1. (a) DMAP, Et3N, DMF; (b) Gly-OMe (8), heat; (c) NaOH, H2O, THF; (d) NH2(CH2)nNHBoc, HOBt, EDC, NMM; (e) HCl; (f)
PhB(OH)2, Pd(PPh3)4, Na2CO3.

Z. Ye et al. / Bioorg. Med. Chem. Lett. 10 (2000) 5±8
7
Using this hypothesis, the naphthyl quinazolinone 16
was docked12 in the receptor model with its positively
charged basic amine group interacting with E124 and
the phenyl group at C-6 p±p stacking with W274 (Fig.
3b). This docking hypothesis was supported by the
observation that substitution of the bromine at C-6 in
13 and 14 with a phenyl group (15 and 16) increased
potency about 4- to 5-fold. This model indicates that the
naphthyl group at the C-2 position of compound 16
may make unfavorable interactions with some of the
side-chains near the top of TM4 and TM6 and not stack
ideally in the hydrophobic pocket formed by M213 and
H280. As shown in Figure 3c, the replacement of the
rigid naphthyl group with a ¯exible aryl group should
improve its orthogonal stacking. The phenethyl analo-
gue (21) exempli®es this better ®t in the hydrophobic
Scheme 2. (a) DMAP, Et3N, DMF; (b) Gly-OMe (8), heat; (c) NaOH, H2O, THF; (d) NH2(CH2)nNHBoc, HOBt, EDC, NMM; (e) PhB(OH)2,
Pd(PPh3)4, Na2CO3; (f) HCl.
Table 1. SAR trends in the quinazolinone leads
No.
R1
R2
n
hGHSr IC50 (mM)
No.
R1
R2
n
hGHSr IC50 (mM)
13
14
15
16
BR
Br
5
6
5
6
1.4
18
19
20
21
Br
Br
5
6
5
6
0.34
0.96
0.34
0.20
0.067
0.048
0.016
Figure 3.

8
Z. Ye et al. / Bioorg. Med. Chem. Lett. 10 (2000) 5±8
References and Notes
1. Rosen, T.; Johannsson, G.; Johannsson, J.-O.; Bengtsson,
B.-A. Horm. Res. 1995, 43, 93.
2. Strobl, J. S.; Thomas, M. J. Pharmacol. Rev. 1993, 46.
3. Human growth hormone secretagogues receptor binding
assayÐa ligand-displacement assay from cloned human GH
secretagogues receptor with [35S] MK-0677 as radiolabeled
ligand (ref 9).
4. Rat pituitary cell GH release assayÐtest compounds incu-
bated with cultured rat pituitary cells for 15 min at 37ꢀC; GH
release in culture medium measured using GH radio-
immunoassay (ref, Cheng. K.; Chan, W. W.-S.; Barreto, A.;
Convey, E. M.; Smith, R. G. Endocrinology 1989, 124, 2791).
5. Bowers, C. Y.; Mommany, F. A.; Reynolds, G. A.; Hong,
A. Endocrinol. 1984, 114, 1537.
Figure 4.
binding pocket and should, therefore, have enhanced
anity.
Based on this modeling hypothesis, the phenethyl com-
pounds 18±21 were synthesized and, as predicted, they
were 8- to 15-fold more potent than the naphthyl
analogues. The most active of this new class of human
GH secretagogues was compound 21 (Fig. 4), which is
200-fold more potent than compound 4.
6. For a summary please see: Nargund, R. P.; Patchett, A. A.;
Bach, M. A.; Murphy, M. G.; Smith, R. G. J. Med. Chem.
1998, 41, 1.
7. Patchett, A. A.; Nargund, R. P.; Tata, J. R.; Chen, M.-H.;
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A. PCT Patent Publication WO97/24369.
Conclusion
Compound 21 is the ®rst reported non-peptidic agonist
of the human GH secretagogue receptor. Introduction
of a phenyl group at the C-6 position of the quinazoli-
none core and extension of the amine side-chain
improved the binding activity of lead compound 4
against the GH secretagogue receptor. We found that in
all quinazolinone-type analogues, activities on the
human GH secretagogue receptor increased when a
phenethyl group was positioned at the C-2 position.
Molecular modeling proved to be an e€ective tool in
guiding the optimization of the screening lead 4. Qui-
nazolinones such as 21 provide a potent alternative to
the privileged structure based dipeptide design exempli-
®ed by MK-0677.
9. Similar improvements were observed earlier in imidazopyr-
idine and benzimidazole series and the results will be published
by Bakshi, R. K. and Chen, M.-C.
10. Howard, A. D.; Feighner, S. D.; Cully, D. F.; Arena, J. P.;
Liberator, P. A.; Rosenblum, C. I.; Hamelin, M.; Hreniuk, D.
L.; Palyha, O. C.; Anderson, J.; Paress, P. S.; Diaz, C.; Chou,
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Elbrecht, A.; Dashkevicz, M.; Heavens, R.; Righy, M.; Sir-
inathsinghji, D. J. S.; Dean, D. C.; Melillo, D. G.; Patchett, A.
A.; Nargund, R. P.; Grin, P. R.; DeMartino, J. A.; Gupta,
S. K.; Shae€er, J. M.; Smith, R. G.; Van der Ploeg, L. H. T.
Science 1996, 273, 974.
11. Feighner, S. D.; Howard, A. D.; Prendergast, K.; Palyha,
O. C.; Hreniuk, D. L.; Nargund, R. P.; Underwood, D.; Tata,
J. R.; Dean, D. C.; Tan, C.; McKee, K. K.; Woods, J. W.;
Patchett, A. A.; Smith, R. G.; Van der Ploeg, L. H. T. Mol.
Endocrinol 1998, 12, 137.
12. Modeling of compounds 2, 16 and 21 was carried out using
the MMFFs force ®eld (ref. Halgren T. A. J. Comput. Chem.
1999, 20, 720). Conformational generation and minimization
in accord with solution NMR structure of GHRP-6 (Baum,
M. W., Merck Research Laboratories) were performed and
low energy structures which ®t the constraints were used in the
docking studies.
Acknowledgements
The authors thank Dr. Kristine Prendergast for provid-
ing the human GH secretagogue receptor model, Ms.
Amy M. Bernick for performing MS analysis and Dr.
Laurie Costonguay for reviewing the manuscript.