Potent achiral agonists of the ghrelin (growth hormone secretagogue) receptor. Part I: Lead identification

Article abstract of DOI:10.1016/j.bmcl.2007.09.067

High throughput screening combined with efficient datamining and parallel synthesis led to the discovery of a novel series of indolines showing potent in vitro ghrelin receptor agonist activity and acceleration of gastric emptying in rats.

Full text of DOI:10.1016/j.bmcl.2007.09.067

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6584–6587
Potent achiral agonists of the ghrelin (growth hormone
secretagogue) receptor. Part I: Lead identification
Tom D. Heightman,a,* Jackie S. Scott,a Mark Longley,a Vincent Bordas,a David K. Dean,a
Richard Elliott,a Gail Hutley,a Jason Witherington,a Lee Abberley,a Barry Passingham,a
Manuela Berlanga,b Maite de los Frailes,b Alan Wise,b Ben Powney,b Alison Muir,b
Fiona McKay,b Sharon Butler,b Kim Winborn,b Christopher Gardner,c
Jill Darton,c Colin Campbellc and Gareth Sangerc
aDepartment of Medicinal Chemistry & DMPK, Neurology & GI Centre of Excellence for Drug Discovery, GlaxoSmithKline,
New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK
bMolecular Discovery Research, Department of Screening and Compound Profiling, GlaxoSmithKline,
New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK
cDepartment of Biology, Neurology & GI Centre of Excellence for Drug Discovery, GlaxoSmithKline,
New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK
Received 20 August 2007; revised 18 September 2007; accepted 20 September 2007
Available online 25 September 2007
Abstract—High throughput screening combined with efficient datamining and parallel synthesis led to the discovery of a novel series
of indolines showing potent in vitro ghrelin receptor agonist activity and acceleration of gastric emptying in rats.
Ó 2007 Elsevier Ltd. All rights reserved.
Ghrelin, a 28-amino acid gastric hormone, exhibits a
wide range of biological activities via its cognate G-pro-
tein coupled receptor, previously known as the growth
hormone secretagogue receptor. In humans as well as
in rodents, ghrelin stimulates pituitary growth hormone
secretion1 and in addition increases food intake and
body weight gain and regulates energy balance.2,3 Ghre-
lin exerts powerful effects on the gastrointestinal tract,
increasing gastric emptying4 and defecation,5 as well as
having the potential to reduce emesis.6 Hence, agents
which mimic the actions of ghrelin have potential in dis-
orders requiring increased nutritional intake, such as
cancer-associated cachexia and post-operative ileus,
and requiring increased gastric emptying, such as func-
tional dyspepsia.7 A number of first generation ghrelin
receptor agonists have undergone extensive clinical
evaluation in humans. To date these have shown disap-
pointing results in the treatment of diseases such as age-
related frailty8 but the potential for ghrelin receptor
agonists in other indications remains to be studied.
The discovery and development of ghrelin receptor ago-
nists has focussed on peptide-derived privileged frag-
ment approaches, which have led to relatively high
molecular weight, synthetically complex molecules such
as MK-0677,9 and CP-424391.10 More recently a series
of partial agonists represented by oxindole SM-130686
has been discovered using diversity screening.11 Here,
we report the discovery of a novel series of low molecu-
lar weight achiral ghrelin receptor agonists, employing
high throughput screening using a recombinant func-
tional assay, and early optimization by parallel
synthesis.
A high throughput screen of the GSK compound collec-
tion using FLIPR technology delivered a moderately
potent and tractable hit series represented by the indo-
line amide 1 (Table 1), originally prepared as 5-HT1B
receptor antagonists.12 Analogues of 1 were selected
for further screening with the goal of generating diver-
gent SAR data, taking into account the previous obser-
vation that arylpiperazines bearing an arylsulfonamide
Keywords: Ghrelin receptor agonist; Growth hormone secretagogue.
*
Corresponding author. Tel.: +44 1279 622031; fax: +44 1279
627779; e-mail: Tom.D.Heightman@gsk.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.09.067

T. D. Heightman et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6584–6587
6585
F
F
Cl
CF3
OH
O
O
N
O
N
O
O
N
N
NH2
N
H2N
S
CF3
N
H
N
NH
O
O
O
O
O
O
N
NH2
NEt2
MK-0677
CP-424391
SM-130686
Table 1. Potency and selectivity of HTS hits14
Table 2. GHSR potency and efficacy for sulfonamide and piperazine
analogues14
Me
Me
R2
O
N
R1
R
O
N
S
N
N
Me
N
N
OMe
OMe
Compound
R
GHSR pEC50 5HT1B
(IA)a-FLIPR
Compound
R1
R2
GHSR
pEC50 (IA)a
pKi-binding
EtO
Cl
O
1
6.8 (0.7)
7.9
2
Me
6.9 (0.7)
Br
O
O
Br
S
2
6.9 (0.7)
5.7
3
Me
H
H
H
H
H
H
H
7.4 (0.8)
9.3 (1.0)
7.5 (0.8)
8.3 (0.9)
9.7 (1.1)
7.7 (0.9)
9.8 (0.9)
8.6 (0.9)
a IA refers to intrinsic activity compared to human ghrelin.
4
moiety in a meta-orientation show reduced 5-HT1 recep-
tor affinity.13 This led to the discovery of the sulfonyl
indoline 2 which retains the promising activity of the
amide and shows a 100-fold reduction in affinity for
5-HT1B receptors (Table 1).
5
I
Cl
6
Synthetic studies were initiated to explore SAR in the
sulfonamide series. Replacement of the 3-bromophenyl
group in 2 with a 2-naphthyl group to give 3 led to a
moderate increase in potency and efficacy (Table 2).
The 2,6-dimethylpiperazinyl analogue 4 of this com-
pound showed subnanomolar potency and full efficacy.
As a result, the 2,6-dimethylpiperazinyl indoline was
used for subsequent sulfonyl group modifications.
7
N
8
N
9
S
N
Replacement of the 2-naphthyl group with more flexible
substituted benzyl or phenethyl groups led to a moder-
ate drop in potency (compounds 5 and 6, respectively).
Extension to a biaryl system uncovered more marked
SAR: the 4-linked 2-pyridylphenyl sulfonamide 7 shows
over 100-fold higher potency compared to the 3-linked
isomer 8. In the biaryl system, the sulfonyl-bearing phe-
nyl group could be replaced by a thiophene with little
effect on potency and efficacy (compounds 9 and 10).
10
S
O
N
a IA refers to intrinsic activity compared to human ghrelin.
mers 11 or 12 was observed; however, after introduction
of a third methyl substituent (compounds 14 and 15), a
preference for the S-configuration at the 6-position of
the piperazine was observed. The geminal dimethylpi-
perazine 16 shows somewhat reduced potency compared
to either of the monomethyl enantiomers 11 or 12.
Retaining the highly potent 2-pyridylthienyl sulfonyl
group of compound 9, systematic removal of the methyl
groups in the dimethylpiperazine moiety led to incre-
mental loss in potency (11–13, Table 3). No significant
preference for either of the 2-methylpiperazine enantio-
The thienyl analogue 9 was further characterized in a
number of in vitro pharmacology assays. The

6586
T. D. Heightman et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6584–6587
Table 3. GHSR potency and efficacy for piperazine analogues14
compound is inactive at the closest homologue, the
motilin receptor (GPR38), and showed >1000-fold
selectivity against over a hundred diverse receptor and
enzyme selectivity targets.
O
O
S
S
R
N
N
Compound 9 possesses promising physicochemical
properties, with a logD7.4 of 1.8, aqueous solubility
from solid of 168 lg/ml at pH 7.4, and a molecular
weight of 484 Da. Its metabolic stability in liver
microsomes is moderate (rat 2.6, human 4.5 ml/min/g
liver). After intravenous dosing in rats the compound
shows a promising half-life and relatively high volume
of distribution (Table 4); however, after oral dosing
exposure is low resulting in negligible oral
bioavailability.
OMe
Compound
R
GHSR pEC50 (IA)a
Me
9
9.8 (0.9)
NH
NH
N
Me
11
12
13
8.5 (1.0)
8.4 (1.0)
7.2 (0.8)
N
N
Me
Compound 9 shows significant acceleration of gastric
emptying after subcutaneous dosing in rats (Fig. 1) with
an ED50 of 0.1 mg/kg.
NH
Me
NH
The synthesis of 1,2,6-trimethylpiperazinyl indoline
amides analogous to 1 has been described previously.12
Sulfonamides 2 and 3 were prepared from 1,2,6-trimeth-
ylpiperazinyl indoline 17 using standard conditions
(Scheme 1).
N
Me
Me
14
15
16
9.1 (1.0)
8.0 (0.8)
7.8 (0.9)
NH
N
Me
Me
Me
The 2,6-dimethylpiperazinyl analogue 4 was prepared
similarly by sulfonylation of the 2,6-dimethylpiperazinyl
indoline 18 (Scheme 2).12 In order to allow efficient
array synthesis of sulfonamide analogues, conditions
using polystyryl-N-methylmorpholine were chosen. This
allowed simple parallel extraction of reaction products
using solid phase cation exchange cartridges, thereby
removing any minor byproducts arising from sulfonyla-
tion of the more hindered piperazine nitrogen. In this
way two 24-member SAR arrays were prepared.
NH
N
Me
Me
Me
NH
N
a IA refers to intrinsic activity compared to human ghrelin.
Synthesis of the alternatively substituted piperazine tem-
plates present in 11–16 was carried out using Pd-cataly-
sed amination of the corresponding 6-bromoindoline, as
described previously for the trimethylpiperazine.12
Table 4. In vivo DMPK parameters after 1 mg/kg iv dose
Cmax/lM
t1/2
Clb
64
Vss (L/Kg)
8.8
0.25
2.3
60
50
40
30
20
10
0
*
*
(Values are means +/- S.E.M.)
Saline 2ml/kg sc (n=12)
SB-791016-B 0.1mg/kg sc (n=6)
SB-791016-B 0.3mg/kg sc (n=5)
SB-791016-B 1mg/kg sc (n=11)
Ptt: 15min
* p<0.002 cf. Control
One-way ANOVA and Dunnett's test
Figure 1. Effect of compound 9 on gastric emptying in the conscious rat (subcutaneous dosing, 60 min test meal).15

T. D. Heightman et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6584–6587
6587
O
N
N
Cl
N
O
N
a)
b)
EtO
N
H
N
1
N
O
O
O
R
S
N
O
N
17
2: R=3-BrPh
3: R=2-Naphthyl
Scheme 1. Reagents: (a) RCO2H, EDC, HOBt, CH2Cl2; (b) RSO2Cl, Et3N, CH2Cl2.
9. Patchett, A. A.; Nargund, R. P.; Tata, J. R.; Chen, M.-H.;
Barkat, K. J.; Johnston, D. B. R.; Cheng, K.; Chan, W.
W.-S.; Butler, B.; Hickey, G.; Jacks, T.; Schleim, K.; Pong,
S.-S.; Chaung, L. Y.-P.; Chen, H. Y.; Frazier, E.; Leung,
K. H.; Chiu, S.-H. L.; Smith, R. G. Proc. Natl. Acad. Sci.
U.S.A. 1995, 92, 7001.
O
S
O
NH
NH
R1
a)
H
N
N
O
N
O
N
18
10. Pan, L. C.; Carpino, P. A.; Lefker, B. A.; Ragan, J. A.;
Toler, S. M.; Pettersen, J. C.; Nettleton, D. O.; Ng, O.;
Pirie, C. M.; Chidsey-Frink, K.; Lu, B.; Nickerson, D. F.;
Tess, D. A.; Mullins, M. A.; MacLean, D. B.; DaSilva-
Jardine, P. A.; Thompson, D. D. Endocrine 2001, 14, 121.
11. Tokunaga, T.; Hume, W. E.; Umezome, T.; Okazaki, K.;
Ueki, Y.; Kumagai, K.; Hourai, S.; Nagamine, J.; Seki,
H.; Taiji, M.; Noguchi, H.; Nagata, R. J. Med. Chem.
2001, 44, 4641.
12. Wyman, P. A.; Marshall, H. R.; Flynn, S. T.; King, R. J.;
Thompson, M.; Smith, P. W.; Hadley, M. S.; Price, G. W.;
Scott, C. M.; Dawson, L. A. Bioorg. Med. Chem. Lett.
2005, 15, 4708.
Scheme 2. Reagents: (a) R1SO2Cl, polystyryl-N-methylmorpholine,
DCM; SCX cartridge.
Conclusions. High throughput screening combined with
efficient parallel synthesis led to the discovery of a novel
achiral series of ghrelin receptor agonists with potential
as gastroprokinetic agents for the treatment of disorders
such as functional dyspepsia. Further optimization of
this series will be described in due course.
13. Bromidge, S. M.; Brown, A. M.; Clarke, S. E.; Dodgson,
K.; Gager, T.; Grassam, H. L.; Jeffrey, P. M.; Joiner, G.
F.; King, F. D.; Middlemiss, D. N.; Moss, S. F.; Newman,
H.; Riley, G.; Routledge, C.; Wyman, P. A. J. Med. Chem.
1999, 42, 202.
14. All novel compounds were characterized at 95% purity or
greater by 1H NMR and LC/MS. Ghrelin receptor agonist
BACMAM FLIPR assay was used to determine the
potency and efficacy of the test compounds. Media were
aspirated from cell plates using a cell washer (leaving 10 ll
of media). Cells are immediately loaded with loading
buffer (Tyrodes (Elga water + 145 mM NaCl + 5 mM
References and notes
1. Takaya, K.; Ariyasu, H.; Kanamoto, N.; Iwakura, H.;
Yoshimoto, A.; Harada, M.; Mori, K.; Komatsu, Y.;
Usui, T.; Shimatsu, A.; Ogawa, Y.; Hosoda, K.; Akamizu,
T.; Kojima, M.; Kangawa, K.; Nakao, K. J. Clin.
Endocrinol. Metab. 2000, 85, 4908.
2. Tschop, M.; Smiley, D. L.; Heiman, M. L. Nature 2000,
407, 908.
3. Nakazato, M.; Murakami, N.; Date, Y.; Kojima, M.;
Matsuo, H.; Kangawa, K.; Matsukura, S. Nature 2001,
409, 194.
KCl + 20 mM
Hepes + 10 mM
glucose + 1 mM
4. Rudd, J. A.; Ngan, M. P.; Wai, M. K.; King, A. G.;
Witherington, J.; Andrews, P. L. R.; Sanger, G. J.
Neurosci. Lett. 2006, 392, 79.
5. Shimizu, Y.; Chang, E. C.; Shafton, A. D.; Sanger, G. J.;
Witherington, J.; Furness, J. B. J. Physiol. 2006, 576.1,
329.
6. Murray, C. D. R.; Martin, N. M.; Patterson, M.; Taylor,
S. A.; Ghatei, M. A.; Kamm, M. A.; Johnston, C.; Bloom,
S. R.; Emmanuel, A. V. Gut 2005, 54, 1693.
7. Peeters, T. L. Curr. Opin. Pharmacol. 2006, 6, 553.
8. Carpino, P. Expert Opin. Ther. Patents 2002, 12, 1599.
MgCl2) + 1.5 mM CaCl2 + 0.714 mg/ml Probenicid (pre-
dissolved in 1 M NaOH) + 0.5 mM brilliant black +
2.5 lM Fluo 4 dye, and incubated at 37.5 °C for 1 h.
Ten microliter from compound plates is then added
immediately to cell plates using a FLIPR 3 calcium
imaging instrument. Fluorescence measurements are then
taken. Values given are means of at least three test
replicates.
15. Gastric emptying protocol based on procedure
described in Droppleman et al., J. Pharmacol. Methods,
1980, 4, 227.