Oxindole derivatives as orally active potent growth hormone secretagogues

Article abstract of DOI:10.1021/jm0103763

A series of substituted oxindole derivatives was synthesized and evaluated for growth hormone (GH) releasing activity using cultured rat pituitary cells. (+)-6-Carbamoyl-3-(2-chlorophenyl)-(2-diethylaminoethyl)- 4-trifluoromethyloxindole (SM-130686, 37S) was found to have potent activity (EC₅₀ = 3.0 nM), while the other enantiomer 37R had reduced activity. The absolute configuration of 37S was confirmed by X-ray crystallographic analysis. Compound 37S showed a good pharmacokinetic profile in rats with 28% oral bioavailability at 10 mg/kg and excellent in vivo activity as evidenced by a significant weight gain after 4 days of oral administration at 10 mg/kg twice a day. Compound 37S displaced the binding of ³⁵S-MK-677 to human GHS-R with an IC₅₀ value of 1.2 ± 0.2 nM.

Full text of DOI:10.1021/jm0103763

J . Med. Chem. 2001, 44, 4641-4649
4641
Oxin d ole Der iva tives a s Or a lly Active P oten t Gr ow th Hor m on e Secr eta gogu es
Teruhisa Tokunaga,‡ W. Ewan Hume,‡ Takashi Umezome,‡ Kazuhiko Okazaki,‡ Yasuyuki Ueki,‡
Kazuo Kumagai,‡ Shinji Hourai,† J un Nagamine,‡ Hitoshi Seki,‡ Mutsuo Taiji,‡ Hiroshi Noguchi,‡ and
Ryu Nagata*,‡
Research Division, Sumitomo Pharmaceuticals Co., Ltd, 1-98 Kasugadenaka 3-chome, Konohana-ku,
Osaka 554-0022, J apan, and Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd,
4-2-1 Takatsukasa, Takarazuka, Hyogo 665-8555, J apan
Received August 7, 2001
A series of substituted oxindole derivatives was synthesized and evaluated for growth hormone
(GH) releasing activity using cultured rat pituitary cells. (+)-6-Carbamoyl-3-(2-chlorophenyl)-
(2-diethylaminoethyl)-4-trifluoromethyloxindole (SM-130686, 37S) was found to have potent
activity (EC50 ) 3.0 nM), while the other enantiomer 37R had reduced activity. The absolute
configuration of 37S was confirmed by X-ray crystallographic analysis. Compound 37S showed
a good pharmacokinetic profile in rats with 28% oral bioavailability at 10 mg/kg and excellent
in vivo activity as evidenced by a significant weight gain after 4 days of oral administration at
10 mg/kg twice a day. Compound 37S displaced the binding of 35S-MK-677 to human GHS-R
with an IC50 value of 1.2 ( 0.2 nM.
In tr od u ction
specificity not only in animal models11,12 but also in
human subjects,13 its peptidyl nature limited its clinical
use.14
Growth hormone (GH), a 191-amino acid hormone,
has important roles such as promotion of growth and
acceleration of metabolic processes such as lipolysis and
protein synthesis.1 GH is released episodically from the
pituitary gland, and its release is regulated by two
hypothalamic hormones, growth hormone releasing
hormone (GHRH), and somatostatin. GHRH stimulates
GH release, whereas somatostatin inhibits GH release.1
Recombinant human growth hormone (rGH) has been
used for the treatment of GH deficiency in children over
the past several decades.2 In addition, it has shown
beneficial effects in the treatment of patients with
burns, bone fractures, and Turner’s syndrome.1,3,4 rGH
also has a potential in reversing several effects of aging
in the elderly.5 However, the use of rGH has limitations
such as its high cost, and it must be administered by
injection due to its lack of oral efficacy. Therefore, there
have been many efforts to identify orally active GH
releasing agents.
Encouraged by the result that a small molecule such
as GHRP-6 could stimulate the release of endogenous
GH, many research groups have initiated studies for
identifying peptidomimetic and nonpeptidic GHSs with
improved pharmacokinetic properties. So far, several
small molecule GHSs have been reported (Chart 1).15,16
Among them, L-692,429 and MK-677 are noted as
extensively studied GHSs. In 1993, a nonpeptidyl ben-
zolactam compound L-692,429 (EC50 ) 60 nM) was
reported from Merck’s group.17 Although L-692,429
became the first nonpeptidyl GHS to be evaluated in
humans,18 its oral bioavailability in dog was less than
5%.19 The spiropiperidine compound MK-677 was re-
ported as the first orally active GHS in 1995.20 MK-677
had strong activity in vitro (EC50 ) 1.3 nM) and elevated
GH levels in beagles after oral doses as low as 0.125
mg/kg.20 Furthermore, in 1996, the same group also
succeeded in cloning the GHS receptor (GHS-R).21 The
GHS-R was found to be a G-protein coupled receptor
and expressed in the pituitary gland and in other
areas.21,22 Thus, it is now clear that synthetic GHSs
release GH through the GHS-R.
These reports stimulated us to search for a new GHS
by means of a GH releasing assay using cultured rat
pituitary cells.23 Herein, we report the preparation and
biological activities of the nonpeptidyl oxindole GHS 37S
(SM-130686) and related analogues.24 These oxindole
compounds are orally active and have a novel structure
totally different from other GHSs reported so far.
A recent major advance in this field was the discovery
of an endogenous ligand for the GHS-R. In 1999,
Kangawa et al. isolated a novel acylated peptide,
designated as ghrelin, from the stomach, which had
strong affinity to the GHS-R and stimulated GH release
after intravenous administration to rats.25 This discov-
In 1977, Bowers and co-workers reported that series
of peptides derived from Met-enkephalin released GH
from the pituitary gland6 and later discovered that these
growth hormone releasing peptides (GHRPs) stimulated
the secretion of GH via a mechanism distinct from
GHRH.7 This discovery opened the possibility that these
relatively small peptides, so-called GH secretagogues
(GHSs),8 might be alternatives to rGH. After continuous
investigations in Bowers’ group,9 hexapeptide GHRP-6
(His-D-Trp-Ala-Trp-D-Phe-Lys-NH2) was identified as a
potent GHS (EC50 ) 10 nM) in 1984.10,11 Although
GHRP-6 was proved to have excellent activity and
* To whom correspondence should be addressed. Tel: 81-6-6466-
5193. Fax: 81-6-6466-5483. E-mail: rnagata@sumitomopharm.co.jp.
‡ Sumitomo Pharmaceuticals Co., Ltd.
† Sumitomo Chemical Co., Ltd.
10.1021/jm0103763 CCC: $20.00 © 2001 American Chemical Society
Published on Web 11/16/2001

4642 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26
Ch a r t 1. Structures of Small Molecule GHSs
Tokunaga et al.
Sch em e 1a
Sch em e 2a
a
Reagents and conditions: (a) 1. NH2OH‚HCl, CCl3CHO‚H2O,
Na2SO4, HCl, H2O, reflux, 2. H2SO4, 80 °C, 41% (2 steps); (b) NaH,
Cl(CH2)2NEt2‚HCl, DMF, 60 °C, 38%; (c) 2-naphthylMgBr, THF,
rt, 56%.
a
Reagents and conditions: (a) 1. NH2OH‚HCl, CCl3CHO‚H2O,
Na2SO4, HCl, H2O, reflux, 2. H2SO4, 80 °C, 64% (2 steps); (b) NaH,
Cl(CH2)2NEt2‚HCl, DMF, 60 °C, 38% for 3, 15% for 4; (c) 2-naph-
thylMgBr, THF, rt, 48% for 5, 62% for 6.
Sch em e 3a
ery could unveil as yet unidentified new therapeutic
uses of SM-130686 as well as other GHSs.
Ch em istr y
Compounds 5 and 6 were synthesized as shown in
Scheme 1. The key intermediary isatins were prepared
according to the Sandmeyer procedure. Reaction of
m-bromoaniline with chloral hydrate in the presence of
hydroxylamine followed by heating in concentrated
sulfuric acid gave an inseparable regioisomeric mixture
of 2a and 2b in a 2.5:1 ratio in 64% yield. N-Alkylation
of the mixture with 2-diethylaminoethyl chloride, using
sodium hydride as a base, in DMF afforded 3 and 4 in
38% and 15% yield, respectively, which were easily
separated by silica gel column chromatography. Addi-
tion of 2-naphthylmagnesium bromide to either 3 or 4
afforded 4-bromooxindole 5 or 6-bromooxindole 6 in 48%
and 62% yield, respectively.
a
Reagents and conditions: (a) NaH, Cl(CH2)2NEt2‚HCl, DMF,
60 °C, 31% for 13, 64% for 14; (b) 2-naphthylMgBr, THF, rt, 56%
for 15, 42% for 16.
able 5-bromoisatin 11 and isatin 12, respectively, ac-
cording to Schemes 2 and 3.
Scheme 4 depicts the preparation of 20 and 21. An
inseparable mixture of N-alkylated oxindoles 19a and
19b were obtained in a 3:1 ratio from a 3:1 mixture of
17a and 17b26 by a method similar to the synthesis of
5. Sonogashira coupling27 of a mixture of 19a and 19b
with propargyl alcohol in the presence of a catalytic
In a similar way, regioisomeric 7-bromooxindole 10,
5-bromooxindole 15, and unsubstituted oxindole 16 were
synthesized from o-bromoaniline 7, commercially avail-

Oxindole Derivatives as Growth Hormone Secretagogues
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26 4643
Sch em e 4a
a
Reagents and conditions: (a) NaH, Cl(CH2)2NEt2‚HCl, DMF, 60 °C, 49%; (b) 2-naphthylMgBr, THF, rt, 79%. (c) propargyl alcohol,
Pd(PPh3)2Cl2, CuI, Et3N, THF, 50 °C, 44% for 20, 16% for 21.
Sch em e 5a
electron deficient nature of the aromatic ring. Regio-
specific iodination of 3-trifluoromethylnitrobenzene 22
was accomplished by a modification of Olah’s method29
using N-iodosuccimide in concentrated sulfuric acid to
provide 23 in 21% yield, which was then reduced to 24
by treatment with SnCl2 in refluxing ethanol in 80%
yield. Addition of sulfuryl chloride, ethyl methylthio-
acetate, and triethylamine successively to 24 at -78 °C
in dichloromethane gave a mixture of 3-methylthio-
oxindoles 25a and 25b, which was then oxidized by
cupric chloride and cupric oxide to generate the insepa-
rable isatins 26a and 26b in a 1:1 ratio in 81% overall
yield. After N-alkylation of the mixture of 26a and 26b,
the desired 6-iodo-4-trifluoromethylisatin 27 and the
other regioisomer, 4-iodo-6-trifluoromethylisatin, were
isolated in 27% and 29% yield, respectively, by silica
gel column chromatography. Addition of 2-naphthyl-
magnesium bromide or 2-chlorophenylmagnesium bro-
mide to 27 provided oxindole 28 or 29, respectively.
These oxindoles served as versatile intermediates for
derivatives with a substituent at the C-6 position. To
examine the stereochemical requirement at the C3
position, optical resolution of 29 into 29R and 29S using
preparative HPLC on a chiral stationary column was
performed.
Scheme 6 shows the preparation of C-6 substituted
derivatives. Compounds 30-33 were obtained using
Sonogashira coupling of 28 or 29 with the corresponding
alkynes. In the synthesis of 30, subsequent deprotection
of the silyl group was performed by treatment with
aqueous hydrogen fluoride in acetonitrile.
a
Reagents and conditions: (a) NIS, H2SO4, 50 °C, 21%; (b)
SnCl2‚H2O, EtOH, reflux 80%; (c) 1. SO2Cl2, MeSCH2CO2Et, Et3N,
CH2Cl2 -78 °C then rt, 2. 1 N HCl, Et2O, rt, 86% (2 steps); (d)
CuCl2, CuO, acetone, rt, 77%; (e) NaH, Cl(CH2)2NEt2‚HCl, DMF,
60 °C, 27%; (f) for 28, 2-naphthylMgBr, THF, rt, 78%; for 29,
2-chlorophenylMgBr, toluene, Et2O, rt, 55%; (g) optical resolution,
Chiralpak OD.
Scheme 7 depicts the synthesis of C-6 carbamoyl
compounds. Compounds 28 and 29 were treated with
zinc cyanide in the presence of a catalytic amount of
tetrakis(triphenylphosphine)palladium in DMF at 80 °C
followed by hydrolysis of the resulting nitriles 34 and
35 with potassium hydroxide in anhydrous tert-butyl
alcohol at 50 °C30 to give 36 and 37, respectively.
Similarly, both enantiomers of 37, (+)-37S and (-)-37R,
were obtained from (+)-29S and (-)-29R, respectively.
The absolute configuration at the C-3 position of (+)-
amount of dichlorobistriphenylphosphine palladium and
copper iodide in a mixed solvent of triethylamine and
THF gave separable oxindoles 20 and 21 in 44% and
16% yield, respectively.
Scheme 5 illustrates the synthesis of a series of 6-iodo-
4-trifluoromethyloxindoles. For the synthesis of 3-iodo-
5-trifluoromethylaniline 24, we utilized the Gasmann
reaction,28 since the cyclization of 24 using the Sand-
meyer procedure appeared to be difficult owing to the

4644 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26
Tokunaga et al.
Sch em e 6a
a
Reagents and conditions: (a) acetylene derivatives (1-tert-
butyldimethylsilyloxy-2-propyne for 30, 4-morpholinocarbonyl-1-
butyne for 31 and 33, 4-carbamoyl-1-butyne for 32), Pd(PPh3)2Cl2,
CuI, Et3N, THF, 50 °C; (b) aq HF, CH3CN, rt; (c) HCl, dioxane,
rt, 66% for 30 (3 steps), 56% for 31 (2 steps), 48% for 32 (2 steps),
61% for 33 (2 steps).
Sch em e 7a
F igu r e 1. X-ray structure of 35S
Ta ble 1. Growth Hormone Releasing Activities of the Oxindole
Derivativesa
a
Reagents and conditions: (a) Zn(CN)2, Pd(PPh3)4, DMF, 60
°C, 80% for 34, 64% for 35, 60% for 35R, 71% for 35S; (b) 1.
powdered KOH, tert-butyl alcohol, 50 °C, 2. HCl, dioxane rt, 95%
for 36 (2 steps), quant. for 37 (2 steps), quant. for 37R (2 steps),
quant. for 37S (2 steps).
a
The growth hormone releasing activities were measured in
b
rat pituitary cells. The values were means of more than two
independent experiments and were normalized for MK-677 (1.3
nM).
37S was confirmed to be S by X-ray crystallographic
analysis of nitrile compound (+)-35S as shown in Figure
oxindoles (5, 10, 15). 5- or 7-Bromooxindoles (15, 10)
and 16 resulted in reduced potency, whereas 4-bro-
mooxindole 5 increased activity (EC50 ) 77 nM), com-
pared to 6. This indicated that both the C-4 and C-6
positions could be further modified. The major goals of
the modifications were not only to increase the activity
but also to reduce the lipophilicity, since the lead
compound 6 had a relatively high clogP value (5.12),
which would be unfavorable for an oral bioavailability.31
A versatile intermediate for introducing a hydrophilic
substituent into either the C-4 or C-6 position would
be the corresponding iodoisatin derivatives, which could
readily be functionalized using Pd catalyzed coupling
reactions. Although such iodo-derivatives are generally
not easily accessible, we fortunately had a mixture of
6-chloro-4-iodoisatin 17a and 4-chloro-6-iodoisatin 17b26
in hand. First of all, we prepared propargyl alcohol
derivatives 20 and 21 using the mixture of 17a and 17b.
Compound 20 showed only weak activity (EC50 > 1000
1.
Resu lts a n d Discu ssion
Str u ctu r e-Activity Rela tion sh ip s. In the course
of our study aimed at generating an orally active GH
secretagogue, we found that an oxindole derivative in
our chemical library showed activity in the in vitro GH
releasing assay using rat pituitary cells.23 After initial
modifications of that derivative, we found that 6-bromo-
1-diethylaminoethyl-3-(2-naphthyl)oxindole 6 exhibited
reproducible activity with an EC50 of 380 nM. Since the
chemical structure of 6 is totally different from any
other known GHSs, we were encouraged to synthesize
various derivatives of 6 and evaluated them for their
GH releasing activity, the results of which are sum-
marized in Table 1.
In the beginning of the study, we prepared unsubsti-
tuted oxindole 16 and the mono bromo substituted

Oxindole Derivatives as Growth Hormone Secretagogues
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26 4645
Ta ble 2. Growth Hormone Releasing Activities of the Oxindole
Ta ble 3. Growth Hormone Releasing Activities of the Oxindole
Derivativesa
Derivativesa
a
The growth hormone releasing activities were measured in
b
rat pituitary cells. The values were means of more than two
independent experiments and were normalized for MK-677 (1.3
nM).
a
The growth hormone releasing activities were measured in
b
rat pituitary cells. The values were normalized for MK-677 (1.3
nM). c Unnormalized value.
nM), whereas compound 21 retained the activity (EC50
) 310 nM), indicating that introduction of a hydrophilic
group into the C6 region could be tolerated. Thus,
4-chloro-6-iodoisatin could serve as a key intermediate
for the structure and activity relationship (SAR) study.
However, we found that 4-chloro-6-iodoisatin and even
4-chloro-6-iodooxindole were difficult to separate from
the regioisomeric by simple column chromatography, so
that it could not be used for that purpose. Next, we
prepared 1-diethylaminoethyl-6-iodo-4-trifluoromethyli-
satin 27. Fortunately, this was easily separated from
the isomer 1-diethylaminoethyl-4-iodo-6-trifluorometh-
ylisatin, and more importantly, compound 30 showed
comparable activity (EC50 ) 310 nM) to 21. These
results encouraged us to investigate 6-substituted-4-
trifluoromethyloxindole derivatives using 1-diethylami-
noethyl-6-iodo-4-trifluoromethylisatin 27 as an inter-
mediate.
Table 2 summarizes the GH releasing activity of the
6-substituted series. First, we utilized Sonogashira
coupling to introduce various hydrophilic groups through
a triple bond linkage into the oxindole ring. Amide
derivatives 31 (EC50 ) 30 nM) and 32 (EC50 ) 30 nM)
showed enhanced activities compared to 30 and pro-
vided a 10-fold increase in potencies. Next, we synthe-
sized carbamoyl compound 36, since we calculated that
a carbamoyl group attached directly to the oxindole ring
would reduce the clogP value. For example, 31 had a
clogP value with 6.03, whereas the clogP value of 36 is
4.47. Gratifyingly, 36 also displayed the most potent
activity (EC50 ) 18 nM) among the 6-substituted
derivatives tested.
The naphthyl group is relatively lipophilic and we
replaced the naphthyl ring with other less lipophilic
aromatic rings. Among several aromatic rings, 2-chlo-
rophenyl was found to be optimal in terms of potency
and reducing the clogP value. We found that 2-chlo-
rophenyl derivative 33 exhibited more potent GH re-
leasing activity (EC50 ) 3.7 nM, clogP ) 5.40) than 31
as shown in Table 3. Similarly to the 2-naphthyl series,
6-carbamoyl compound 37 showed the strong activity
(EC50 ) 3.5 nM, clogP ) 3.83). Each enantiomer of
6-carbamoyl compound 37 was prepared and (+)-isomer
37S was found to retain the potency (EC50 ) 3.0 nM).
F igu r e 2. Effect of SM-130686 on the body weight gain. SM-
130686 was orally administered twice a day every day for 4
days in normal female rats. Data are represented as means (
SEM (n ) 6). *P < 0.025; *P < 0.005; **P < 0.01, Shirley-
Williams test (vs 0 mg/kg treatment group).
On the other hand, (-)-isomer 37R showed reduced
potency (EC50 ) 210 nM). The absolute configuration
of the more active isomer was revealed to be “S” as
determined by the X-ray crystallographic analysis of
precursor 35S.
In Vivo Effica cy a n d P h a r m a cok in etics. Com-
pound 37S was selected and was evaluated for in vivo
activity. Administration of 37S orally increased the
weight in a dose dependent manner after 4 days in rats.
When 37S was administered at 3, 10, and 30 mg/kg
twice a day, the body weight gains were 6.9, 8.9, and
12.3 g, respectively, whereas the distilled water admin-
istered group showed a weight gain of 3.5 g (Figure 2).
In addition to the significant effect on the weight gain,
37S was found to show excellent profile in continuous
studies. The body weight gain in the 37S administered

4646 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26
Tokunaga et al.
g, 153 mmol), 3-bromoaniline (1, 8.00 mL, 73.5 mmol) in a
mixture of H2O (75 mL) and 36% HCl (6.5 mL, 78.0 mmol),
and a solution of hydroxylamine hydrochloride (19.0 g, 273
mmol) in H2O (85 mL) with vigorous stirring. After the
addition was completed, the reaction mixture was heated at
reflux for 10 min and allowed to cool to room temperature.
The precipitate formed was collected by filtration, washed with
H2O, and then dried in vacuo to yield the crude isonitrosoac-
etanilide. This product was added portion-wise to rapidly
stirred concentrated sulfuric acid (250 mL) at a rate to keep
the reaction temperature between 50 and 70 °C. The resulting
solution was stirred at 80 °C for 10 min and allowed to cool to
room temperature. The cooled mixture was poured carefully
onto crushed ice (ca. 900 g). The mixture was allowed to stand
for 1 h. The orange precipitate was collected by filtration,
washed with H2O, and then dried to yield a 2.5:1 mixture of
2a and 2b (10.6 g, 64%). 2a : 1H NMR (270 MHz, CDCl3) δ
11.17 (brs, 1 H), 7.46 (dd, 1 H, J ) 8.0, 8.0 Hz), 7.22 (dd, 1 H,
J ) 0.7, 8.0 Hz), 6.89 (dd, 1 H, J ) 0.7, 8.0 Hz). 2b: 1H NMR
(270 MHz, CDCl3) δ 11.10 (brs, 1 H), 7.47 (d, 1 H, J ) 7.9 Hz),
7.26 (dd, 1 H, J ) 1.7, 7.9 Hz), 7.08 (d, 1 H, J ) 1.7 Hz). MS
m/z 225 (M+).
group was almost equivalent to the fat free mass gain.
After administration of 37S at 10 mg/kg orally twice a
day for 9 days in rats, there were significant increases
in both the body weight and the lean body mass (19.5
( 2.1 and 18.0 ( 7.5 g, respectively).32 This result
indicates that 37S promotes growth by accelerating
anabolic effects through GH secretion and is intriguing
because endogenous GHS ghrelin was reported to
increase the fat mass gain.33
In a series of in vitro studies, 37S was proven to have
the same mechanism of action in terms of GH release
as the other GHSs. It displaced the binding of [35S]MK-
67734 to human GHS-R21 expressed in CHO cells in a
dose-dependent manner with an IC50 value of 1.2 ( 0.2
nM. The in vitro specificity of 37S was evaluated in over
50 receptor and enzyme assays including adrenergic,
angiotensin, bradykinin, cholecystokinin, dopamine,
GABA, histamine, muscarinic, nicotinic acetylcholine,
opiate, serotonin somatostatin, and vasopressin recep-
tors, and no significant binding was observed at 1 µM.
Compound 37S was evaluated in a rat pharmacoki-
netic study. After an oral dose of 10 mg/kg to rats, Cmax
was 274 ng/mL with a Tmax of 0.4 h and the bioavail-
ability was 28%. When intravenously administered to
rats (5 mg/kg), Cl, Vd, and t1/2 were 119 mL/min/kg, 9.9
L/kg and 1.5 h, respectively.
1-(2-Dieth yla m in oeth yl)-4-br om oisa tin (3) a n d 1-(2-
Dieth yla m in oeth yl)-6-br om oisa tin (4). To a solution of a
2.5:1 mixture of 2a and 2b (2.25 g, 9.95 mmol) in DMF (30
mL) was added sodium hydride (60% in oil, 960 mg, 24.0 mmol)
with stirring at 0 °C, followed by addition of 2-diethylamino-
ethyl chloride hydrochloride (2.05 g, 11.9 mmol). The mixture
was stirred at 60 °C for 2 h and cooled to room temperature.
Water was then added, and the mixture was extracted with a
mixture of toluene and ethyl acetate (1:1). The organic phase
was washed with brine, dried over magnesium sulfate, and
concentrated. The residue was purified by silica gel column
chromatography (AcOEt) to give 3 (1.22 g, 38%) and 4 (478
mg, 15%). 3: Rf 0.21 (AcOEt); LC-Ms m/z 327 (M+ + 3), 325
Con clu sion
We have successfully synthesized a new class of GH
secretagogue oxindole derivatives. Among them, 37S
(SM-130686) showed the same range of potencies as
MK-677 in vitro, although the structure was different
from that of MK-677. Compound 37S (SM-130686) also
increased the body weight gain after oral administration
to rats. Recently, an endogenous ligand for GHS-R,
ghrelin, was identified and showed strong GH releasing
activity.25 More recently, adenosine was discovered as
another endogenous ligand for GHS.35 Although Ca2+
concentration in the cells was elevated by adenosine
similarly to the case of ghrelin, it does not exhibit any
GH releasing property in pituitary cells.35 Thus the
complexity of the GHS-R has emerged, and unknown
roles of GHS-R by these natural ligands may soon be
elucidated. Further study indicated that 37S (SM-
130686) might be a partial agonist to GHS-R,32 and the
more detailed pharmacological evaluation of 37S and
its derivatives is now in progress in our laboratory.
1
(M+ + 1); H NMR (270 MHz, CDCl3) δ 7.44 (d, 1 H, J ) 7.7
Hz), 7.25 (dd, 1 H, J ) 1.5, 7.7 Hz), 7.17 (d, 1 H, J ) 1.5 Hz),
3.77 (t, 2 H, J ) 6.6 Hz), 2.69 (t, 2 H, J ) 6.6 Hz), 2.55 (q, 4
H, J ) 7.1 Hz), 0.97 (t, 6 H, J ) 7.1 Hz); HRMS calcd for
C
14H18BrN2O2 325.0552, found 325.0580. 4: Rf 0.42 (AcOEt);
mp 62-64 °C; LC-MS m/z 327 (M+ + 3), 325 (M+ + 1); 1H NMR
(270 MHz, CDCl3) δ 7.39 (dd, 1 H, J ) 7.8, 7.8 Hz), 7.23 (d, 1
H, J ) 7.8 Hz), 6.90 (d, 1 H, J ) 7.8 Hz), 3.80 (t, 2 H, J ) 6.8
Hz), 2.68 (t, 2 H, J ) 6.8 Hz), 2.56 (q, 4 H, J ) 7.1 Hz), 0.97
(t, 6 H, J ) 7.1 Hz); Anal. (C14H17BrN2O2): C, H, N.
4-Br om o-1-(2-d ieth yla m in oeth yl)-3-h yd r oxy-3-(2-n a p h -
th yl)oxin d ole (5). To a solution of 3 (205 mg, 0.631 mmol) in
anhydrous THF (3.0 mL) was added freshly prepared 1.0 N
2-naphthylmagnesium bromide in THF (1.2 mL, 1.20 mmol)
at room temperature. The reaction mixture was stirred
overnight. Saturated aqueous NaHCO3 was added, and the
mixture was extracted with ethyl acetate, and then washed
with brine. The organic layer was dried over magnesium
sulfate, and concentrated. The residue was purified by silica
gel column chromatography (CHCl3-MeOH, 60:1, then 30:1)
to give the title compound (134 mg, 48%): mp 158-162 °C;
Exp er im en ta l Section
1
LC-MS m/z 455 (M+ + 3), 453 (M+ + 1); H NMR (270 MHz,
Gen er a l Notes. Melting points were measured on either a
CDCl3) δ 8.00 (d, 1 H, J ) 1.6 Hz), 7.75 ∼ 7.85 (m, 3 H), 7.44
∼ 7.50 (m, 2 H), 7.34 (dd, 1 H, J ) 2.0, 8.6 Hz), 7.19 ∼ 7.30
(m, 2 H), 6.97 (dd, 1 H, J ) 1.0, 7.6 Hz), 3.61 ∼ 3.93 (m, 2 H),
2.72 (t, 2 H, J ) 6.9 Hz), 2.57 (q, 4 H, J ) 7.1 Hz), 0.97 (t, 6
H, J ) 7.1 Hz); HRMS calcd for C24H26BrN2O2 453.1178, found
453.1167.
6-Ch lor o-1-(2-d iet h yla m in oet h yl)-3-h yd r oxy-4-(3-h y-
dr oxy-1-pr opyn yl)-3-(2-n aph th yl)oxin dole (20) an d 4-Ch lo-
r o-1-(2-dieth ylam in oeth yl)-3-h ydr oxy-6-(3-h ydr oxy-1-pr o-
p yn yl)-3-(2-n a p h t h yl)oxin d ole (21). To a mixture of 19a
and 19b (198 mg, 0.370 mmol) and dichlorobis(triphenylphos-
phine)palladium (II) (33.8 mg, 0.0482 mmol) in a mixed solvent
of triethylamine (1.0 mL) and tetrahydrofuran (3.0 mL) were
added propargyl alcohol (0.04 mL, 0.687 mmol) and copper(I)
iodide (8.4 mg, 0.0441 mmol). The mixture was stirred at 50
°C for 3 h. After cooling, water was added and the mixture
was extracted with ethyl acetate. The organic phase was dried
Thomas-Hoover or a Yanaco melting point apparatus and were
uncorrected. H NMR spectra were recorded on a J EOL GX-
1
270 or Bruker AVANCE 400 spectrometers using tetrameth-
ylsilane as an internal standard. LC mass spectra were
obtained on a PE SCIEX API 150EX spectrometer. Elemental
analyses, low-resolution mass spectra, and high-resolution
mass spectra were obtained from Sumitomo Analytical Center,
Inc. Thin-layer chromatography and flash column chromatog-
raphy was performed on silica gel glass-backed plates (5719,
Merck & Co.) and silica gel 60 (230-400 or 70-230 mesh,
Merck & Co.), respectively. Optical rotations were measured
on a J ASCO DIP-370. Optical purity was determined by HPLC
using Hitachi L 6000 pump and L 4000 UV detector with a
chiral column (Chiralpak AD, Daicel).
4-Br om oisa tin (2a ) a n d 6-Br om oisa tin (2b). To a solu-
tion of trichloroacetaldehyde monohydrate (15.8 g, 95.5 mmol)
in H2O (200 mL) were successively added sodium sulfate (21.8

Oxindole Derivatives as Growth Hormone Secretagogues
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26 4647
over magnesium sulfate and concentrated. The residue was
purified by silica gel column chromatography (CHCl3-MeOH,
20:1) to give 20 (75.0 mg, 44%) and 21 (27.3 mg, 16%). 20: Rf
26b (46.6 g, 77%): MS m/z 341 (M+); 1H NMR (270 MHz,
DMSO-d6) δ (11.34 (s), 11.26 (s), 1 H), (7.77 (s), 7.68 (s), 1 H),
(7.55 (s), 7.08 (s), 1 H); HRMS calcd for C9H3F3INO2 340.9161,
found 340.9164.
1
0.18 (CHCl3-MeOH, 10:1); MS m/z 462 (M+); H NMR (270
MHz, CDCl3) δ 8.04 (d, 1 H, J ) 3.3 Hz), 7.74 ∼ 7.82 (m, 3 H),
7.44 ∼ 7.48 (m, 2 H), 7.35 (dd, 1 H, J ) 1.8, 8.7 Hz), 7.06 (d,
1 H, J ) 2.0 Hz), 6.98 (d, 1 H, J ) 2.0 Hz), 4.15 (s, 2 H), 3.74
∼ 3.90 (m, 2 H), 2.68 ∼ 2.82 (m, 2 H), 2.62 (q, 4 H, J ) 7.1
Hz), 0.99 (t, 6 H, J ) 7.1 Hz). For elemental analysis, HCl
salt of 20 was prepared by treating 20 with 4 N HCl in dioxane
followed by concentration to dryness: Anal. (C27H28Cl2N2O3‚
1/2H2O‚1/2C4H8O2): C, H, N. 21: Rf 0.11 (CHCl3-MeOH, 10:
1); mp 172-173 °C; MS m/z 462 (M+); 1H NMR (270 MHz,
CDCl3) δ 7.99 (d, 1 H, J ) 1.7 Hz), 7.75 ∼ 7.84 (m, 3 H), 7.44
∼ 7.50 (m, 2 H), 7.36 (dd, 1 H, J ) 1.8, 8.7 Hz), 7.11 (d, 1 H,
J ) 1.2 Hz), 6.96 (d, 1 H, J ) 1.2 Hz), 4.50 (s, 2 H), 3.83 (t, 2
H, J ) 6.3 Hz), 2.71 ∼ 2.85 (m, 2 H), 2.65 (q, 4 H, J ) 7.2 Hz),
0.99 (t, 6 H, J ) 7.2 Hz); Anal. (C27H27ClN2O3‚1/3CH4O): C,
H, N.
1-(2-Die t h yla m in oe t h yl)-6-iod o-4-t r iflu or om e t h yl-
isa tin (27). The title compound was prepared from a 1:1
mixture of 26a and 26b by the method described in the
preparation of 3. Purification was carried out by silica gel
column chromatography (hexane-ethyl acetate, 3:1, then
1:1): Rf 0.13 (hexane-AcOEt, 1:1); mp 108-111 °C dec; MS
m/z 440 (M+); 1H NMR (270 MHz, CDCl3) δ 7.71 (s, 1 H), 7.68
(s, 1 H), 3.80 (t, 2 H, J ) 6.1 Hz), 2.69 (t, 2 H, J ) 6.1 Hz),
2.55 (q, 4 H, J ) 7.1 Hz), 0.97 (t, 6 H, J ) 7.1 Hz); Anal.
(C15H16F3lN2O2): C, H, N.
3-(2-Ch lor oph en yl)-1-(2-dieth ylam in oeth yl)-3-h ydr oxy-
6-iod o-4-tr iflu or om eth yloxin d ole (29). To a solution of 27
(4.96 g, 11.3 mmol) in a mixed solvent of diethyl ether (81 mL)
and toluene (27 mL) was added dropwise freshly prepared 0.64
N 2-chlorophenylmagnesium bromide in ether (18.0 mL, 11.5
mol) at room temperature. The mixture was stirred for 20 min.
Saturated aqueous NaHCO3 was added, and the mixture was
extracted with ethyl acetate. The organic phase was washed
with saturated aqueous NaHCO3, dried over magnesium
sulfate, and concentrated. The residue was purified by silica
gel column chromatography (CHCl3-MeOH, 100:1, then 50:
1) to give the title compound (3.45 g, 55%): mp 160-162 °C;
3-Iod o-5-n itr oben zotr iflu or id e (23). To a mixture of
N-iodosuccimide (33.7 g, 150 mol) and concentrated sulfuric
acid (100 mL) was added dropwise a solution of 3-nitroben-
zotrifluoride (22, 13.3 mL, 100 mol) in concentrated sulfuric
acid (46 mL). After the addition was completed, the reaction
mixture was heated at 50 °C for 6 h and poured into crushed
ice. The mixture was extracted with ethyl acetate. The organic
phase was washed with aqueous Na2SO3, dried over magne-
sium sulfate, and concentrated. The residue was purified by
silica gel column chromatography (hexane-ethyl acetate, 1:0,
then 200:1) to give the title compound (6.65 g, 21%): MS m/z
1
MS m/z 552 (M+); H NMR (270 MHz, CDCl3) δ 8.09 (d, 1 H,
J ) 6.8 Hz), 7.61 (s, 1 H), 7.53 (s, 1 H), 7.41 (dd, 1 H, J ) 6.8,
6.8 Hz), 7.21 ∼ 7.32 (m, 2 H), 3.97 ∼ 4.11 (m, 1 H), 3.68 ∼
3.78 (m, 1 H), 2.55 ∼ 2.84 (m, 6 H), 1.03 (t, 6 H, J ) 7.1 Hz);
Anal. (C21H21ClF3lN2O2): C, H, N.
1
317 (M+); H NMR (270 MHz, CDCl3) δ 8.76 (s, 1 H), 8.47 (s,
(+)-3-(2-Ch lor op h en yl)-1-(2-d iet h yla m in oet h yl)-3-h y-
d r oxy-6-iod o-4-tr iflu or om eth yloxin d ole (29S) a n d (-)-3-
(2-Ch lor op h en yl)-1-(2-d iet h yla m in oet h yl)-3-h yd r oxy-6-
iod o-4-tr iflu or om eth yloxin d ole (29R). Each enantiomer of
racemic 29 was separated using preparative HPLC on a
Chiralpak OD with 6% 2-propanol/hexane as an eluent. The
enantiomeric excesses were determined by using HPLC on a
Chiralpak AD with 10% 2-propanol/hexane as an eluent at a
flow rate of 0.5 mL/min. The (+)-enantiomer eluted at the
retention time of 12.7 min, and the (-)-enantiomer eluted at
the retention time of 16.3 min. The spectral properties of the
title compounds were identical with those of 29. 29S: mp 63-
67 °C; [R]D +56.8 (c ) 0.26, MeOH); 89% ee. 29R: mp 61-64
°C; [R]D -60.0 (c ) 0.33, MeOH); 91% ee.
1 H), 8.28 (s, 1 H); HRMS calcd for C7H3F3INO2 316.9161,
found 316.9150.
3-Iod o-5-tr iflu or om eth yla n ilin e (24). To a solution of 23
(6.92 g, 21.8 mmol) in EtOH (150 mL) was added tin(II)
chloride hydrate (15.3 g, 67.8 mmol), and the solution was
heated at reflux for 1 h. After cooling to room temperature,
the mixture was poured into water, basified with NaHCO3,
and extracted with ethyl acetate. The organic phase was
washed with brine, dried over magnesium sulfate, and con-
centrated. The residue was purified by silica gel column
chromatography (hexane-ethyl acetate, 10:1) to give the title
compound (5.03 g, 80%): mp 37-38 °C; MS m/z 287 (M+); 1H
NMR (270 MHz, CDCl3) δ 7.29 (s, 1 H), 7.17 (s, 1 H), 6.83 (s,
1 H), 3.87 (brs, 2 H); HRMS calcd for C7H5F3IN 286.9419,
found 286.9438.
3-(2-Ch lor op h en yl)-6-cya n o-1-(2-d ieth yla m in oeth yl)-3-
h yd r oxy-4-tr iflu or om eth yloxin d ole (35). A mixture of 29
(2.97 g, 5.37 mmol), 60% zinc cyanide (926 mg, 4.73 mmol),
and tetrakis(triphenylphosphine)palladium (0) (630 mg, 0.545
mmol) in DMF (30 mL) was stirred at 60 °C for 3 h. After
cooling, saturated aqueous NaHCO3 was added, and the
mixture was extracted with a mixture of toluene and ethyl
acetate (1:1). The organic phase was washed with saturated
aqueous NaHCO3, dried over magnesium sulfate, and concen-
trated. The residue was purified by silica gel column chroma-
tography (chloroform-methanol, 80:1, then 50:1) to give the
6-Iod o-3-m eth ylth io-4-tr iflu or om eth yloxin d ole (25a )
an d 4-Iodo-3-m eth ylth io-6-tr iflu or om eth yloxin dole (25b).
To a solution of 24 (2.00 g, 6.98 mmol) in dichloromethane (35
mL) cooled to -78 °C was added sulfuryl chloride (0.68 mL,
8.36 mmol). The solution was stirred for 30 min. To the
mixture was then added ethyl methylthioacetate (1.17 mL,
9.09 mol), and stirring was continued for further 2 h at -78
°C. After triethylamine (2.43 mL, 17.4 mol) was added, the
mixture was allowed to warm to room temperature, treated
with an excess amount of 1 N HCl, and stirred overnight. The
organic phase was separated, dried over magnesium sulfate,
and concentrated. The residue was purified by silica gel column
chromatography (hexane-ethyl acetate, 4:1) to give a 1:1
1
title compound (1.56 g, 64%): mp 188-189 °C; H NMR (270
MHz, CDCl3) δ 8.07 (d, 1 H, J ) 7.6 Hz), 7.57 (s, 1 H), 7.40 ∼
7.47 (m, 2 H), 7.33 (ddd, 1 H, J ) 1.5, 7.6, 7.6 Hz), 7.24 (dd, 1
H, J ) 1.5, 7.9 Hz), 4.03 ∼ 4.13 (m, 1 H), 3.73 ∼ 3.83 (m, 1 H),
1
mixture of 25a and 25b (2.44 g, 86%): MS m/z 373 (M+); H
2.59 ∼ 2.78 (m, 6 H), 1.01 (t, 6 H, J ) 7.1 Hz); Anal. (C22H21
-
NMR (270 MHz, CDCl3) δ 8.88 (brs, 1 H), (7.72 (s), 7.65 (s), 1
H), (7.44 (s), 7.13 (s), 1 H), (4.33 (s), 4.14 (s), 1 H), (2.09 (s),
2.05 (s), 3 H); HRMS calcd for C10H7F3INOS 372.9245, found
372.9211.
ClF3N3O2‚1/3H2O): C, H, N.
3-(2-Ch lor op h en yl)-6-ca r b a m oyl-1-(2-d iet h yla m in o-
eth yl)-3-h yd r oxy-4-tr iflu or om eth yloxin d ole (37). To a
solution of 35 (1.52 g, 3.36 mmol) in tert-butyl alcohol (70 mL),
warmed to 50 °C, was added powdered KOH (ca. 5.0 g). The
mixture was stirred at 50 °C for 1 h and passed through a
celite pad. The celite was washed with THF, and the filtrate
was concentrated. The residue was partitioned between water
and ethyl acetate, and the organic phase was separated. The
organic phase was dried over magnesium sulfate and concen-
trated, which was treated with 4 N HCl in dioxane followed
by concentration to dryness to give the title compound (1.66
g, 98%): mp 213-217 °C; MS m/z 469 (M+); 1H NMR (270
MHz, DMSO-d6) δ 10.41 (brs, 1 H), 8.33 (brs, 1 H), 8.15 (s, 1
6-Iod o-4-tr iflu or om eth ylisa tin (26a ) a n d 4-Iod o-6-tr i-
flu or om eth ylisa tin (26b). To a solution of a 1:1 mixture of
25a and 25b (66.5 g, 178 mmol) in acetone (730 mL) were
added copper(II) chloride (27.5 g, 205 mmol) and copper(II)
oxide (16.4 g, 205 mmol). The mixture was stirred at room
temperature for 3 h. To the resulting mixture were added 1 N
HCl and ethyl acetate. The organic phase was separated,
washed with 1 N HCl, and then brine, dried over magnesium
sulfate, and concentrated. The resulting solid was filtered and
washed with dichloromethane to give a 1:1 mixture of 26a and

4648 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26
Tokunaga et al.
H), 8.08 (d, 1 H, J ) 7.3 Hz), 7.84 (s, 1 H), 7.77 (brs, 1 H), 7.48
(ddd, 1 H, J ) 1.7, 7.6, 7.6 Hz), 7.11 ∼ 7.40 (m, 4 H), 4.16 ∼
4.36 (m, 2 H), 3.25 ∼ 3.51 (m, 6 H), 1.26 (t, 6 H, J ) 7.3 Hz);
Anal. (C22H24Cl2F3N3O3‚H2O): C, H, N.
with the binding buffer through a GF/B filter. The radioactivity
on the filters was counted in â-plate scintillation counter
(Amersham Pharmacia Biotech, England).
(+)-3-(2-Ch lor oph en yl)-6-cyan o-1-(2-dieth ylam in oeth yl)-
3-h yd r oxy-4-tr iflu or om eth yloxin d ole (35S). The title com-
pound was prepared from 29S by the method described in the
preparation of 35. The spectral properties of the title compound
were identical with those of 35. The enantiomeric excess was
determined by using HPLC on a Chiralpak AD with 10%
2-propanol/hexane as an eluent at a flow rate of 0.5 mL/min.
The title compound eluted at the retention time of 13.2 min:
mp 170-172 °C; [R]D +76.5 (c ) 0.31, MeOH); 90% ee.
(+)-3-(2-Ch lor op h en yl)-6-ca r b a m oyl-1-(2-d iet h yla m i-
n oeth yl)-3-h yd r oxy-4-tr iflu or om eth yloxin d ole (37S). The
title compound was prepared from 35S by the method de-
scribed in the preparation of 37. The spectral properties of the
title compound were identical with those of 37. The enantio-
meric excess was determined by using HPLC on a Chiralpak
AD with 10% 2-propanol/hexane as an eluent at a flow rate of
1.0 mL/min. The title compound eluted at the retention time
of 17.9 min: mp 168-172 °C; [R]D +81.4 (c ) 0.41, MeOH);
97% ee.
Ack n ow led gm en t. We are grateful to Ms. N. Oka-
hara-Mitsuhata and Ms. H. Ikeda for technical as-
sistance and to Dr. K. Yanagi for discussing concerning
X-ray crystallography.
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures and spectral data for 6, 8-10, 13-19, 28, 30-34, 35R,
36, and 37R, elemental analyses, and X-ray crystallographic
analysis. This material is available free of charge via the
Internet at http://pubs.acs.org.
Refer en ces
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a comprehensive review on the actions of GH and its
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pieces, and transferred to the isolation buffer (100 µL/lobe)
containing 0.8% collagenase and 0.2% DNase type I. The
mixture was incubated for less than 25 min at 37 °C. The cells
were collected and washed three times with the culture
medium; DMEM (Dulbecco’s modified Eagle’s medium) was
supplemented with 5% horse serum, 2.5% fetal calf serum, 1%
nonessential amino acids, 1% penicillin, and 1% streptomycin.
Cells were resuspended in the above medium and brought to
a density of 1.0 × 105 cell/mL. The cells (200 µL/well) were
seeded onto a 96 well-plate (Nunc, Denmark) and cultured for
5 days at 37 °C under 5% CO2.
Following the culture period the cells were washed once with
the stimulation buffer (the culture medium containing 25 mM
HEPES, pH ) 7.3) and then incubated for 90 min at 37 °C
under 5% CO2. This buffer was then replaced with the fresh
stimulation buffer containing test compound followed by
incubation for 15 min at 37 °C under 5% CO2. The medium
was collected and GH concentration measured.
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stimulation with the following scheme: GH content ) Emax
×
concentration of test compound/(EC50 + concentration of test
compound) + basal GH content.
In Vivo Stu d y: Effect of 37S (SM-130686) on th e Bod y
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10 a.m. and 4 p.m.) for 4 days (starting from day 0). The rats
were weighed every morning just before administration of the
compound.
GHS Bin d in g Activity. Crude membranes were isolated
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Oxindole Derivatives as Growth Hormone Secretagogues
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 26 4649
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