Indolyl and dihydroindolyl N-glycinamides as potent and in vivo active NPY5 antagonists

Article abstract of DOI:10.1016/j.bmcl.2012.01.117

A novel series of indolyl and dihydroindolyl glycinamides were identified as potent NPY5 antagonists with in vivo activity from screen hit 1. The dihydroindolyl glycinamide 10a significantly inhibits NPY5 agonist induced feeding at a dose of 0.1 mg/kg. The indolyl glycinamide 12c also inhibits NPY5 agonist induced feeding at a dose of 1 mg/kg. Both compounds 10a and 12c represent potential tools for further investigation into the biology of the NPY5 receptor.

Full text of DOI:10.1016/j.bmcl.2012.01.117

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2167–2171
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Indolyl and dihydroindolyl N-glycinamides as potent and in vivo active
NPY5 antagonists
Lingyun Wu a, , Kai Lu a, Mathivanan Packiarajan a, Vrej Jubian a, Gamini Chandrasena a,
⇑
Toni C. Wolinsky b, Mary W. Walker b
a Chemical & Pharmacokinetic Sciences, 215 College Road, Paramus, NJ 07652, USA
b Synaptic Transmission Disease Biology Unit, Lundbeck Research USA, 215 College Road, Paramus, NJ 07652, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of indolyl and dihydroindolyl glycinamides were identified as potent NPY5 antagonists
with in vivo activity from screen hit 1. The dihydroindolyl glycinamide 10a significantly inhibits NPY5
agonist induced feeding at a dose of 0.1 mg/kg. The indolyl glycinamide 12c also inhibits NPY5 agonist
induced feeding at a dose of 1 mg/kg. Both compounds 10a and 12c represent potential tools for further
investigation into the biology of the NPY5 receptor.
Received 19 December 2011
Revised 27 January 2012
Accepted 30 January 2012
Available online 4 February 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Indolyl glycinamide
Dihydroindolyl glycinamide
PGP efflux
Brain penetration
Neuropeptide Y (NPY) is a 36 amino acid neuropeptide widely
expressed in the central and peripheral nervous systems.1 It has
been shown that NPY influences a diverse array of biological func-
tions including blood pressure; food intake; circadian rhythms;
and stress sensitivity.2–4 NPY exerts its effects through the interac-
tion with specific receptors of the G-protein coupled receptor
(GPCR) superfamily. Five NPY receptor subtypes have been identi-
fied to date (Y1, Y2, Y4, Y5, and Y6).5 The NPY5 receptor is well
known for its role in appetite regulation, and there are multiple
studies supporting the potential utility of an NPY5 antagonist in
the treatment of obesity.6–8 Indeed, two compounds, MK0557
and Velneperit, from Merck and Shionogi, respectively, have ad-
vanced to human clinical trials. Both compounds induced statisti-
cally significant weight loss in obese subjects.9,10 Based on its
expression in the limbic system, we hypothesized that the NPY5
receptor might also modulate stress sensitivity.11 In order to better
understand the function of the NPY5 receptor, selective agonists
and antagonists are required. A diverse array of chemotypes having
NPY5 antagonist activity have been reported.12
and rat microsomal preparations. Unfortunately, these compounds
suffered from low brain penetration. Our strategy to improve brain
exposure by manipulating potential H-bonding interactions and
logD led to the discovery of diamine 5, a potent and brain pene-
trant NPY5 antagonist. A parallel effort to improve the brain pene-
tration of the glycinamides focused on further ring contraction of
the N-heterocycles and is reported herein. Here we describe the
discovery of the novel indolyl and dihydroindolyl glycinamides
as potent NPY5 antagonists with in vivo activity.
The compounds described in this Letter were synthesized fol-
lowing the methodology as described in Scheme 1. The amine
intermediate (18) was obtained from the commercially available
amino acid (16) following the procedure previously reported.13
H
N
H
N
R
N
N
H
N
H
N
S
X
O
O
N
O
O
S
S
O O
O O
1
2: X = S; 3: X = O; 4: X = C
We recently reported the identification of benzothiazepinone 1
(Fig. 1) and our lead optimization efforts to overcome its clearance
and PK liabilities.13 In these studies, replacement of the benzo-
thiazepinone ring system with various six-membered N-heteroaryl
glycinamides, for example, 2, 3 and 4, was shown to improve the
potency at the NPY5 receptor as well as the stability in both human
F
F
F
H
N
N
H
O
N
S
O O
5
⇑
Corresponding author. Tel.: +1 201 350 0362; fax: +1 201 261 0623.
E-mail address: wuly2000@yahoo.com (L. Wu).
Figure 1. Lead optimization of NPY5 antagonist 1.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.117

2168
L. Wu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2167–2171
H2N
The dihydroindole carboxylic acids (22) were prepared from
HOOC
HOOC
b,c
dihydroindole intermediates (21) which were either purchased
from commercial source or made by condensation of hydrazines
(19) with the appropriate aldehydes (20) under acidic conditions
followed by reduction with sodium triacetoxyborohydride. Alkyl-
a
NH
NH
S
NH2
S
O O
O O
16
17
18
O
ation with a-bromomethyl acetate (for 10) or b-bromomethyl ace-
n
H
N
H
O
OH
N
f,g
R1
R2
N
d,e
tate (for 11) followed by hydrolysis gave the carboxylic acids (22).
Coupling of carboxylic acids (22) with the amine 18 generated the
dihydroindolyl amides ( 10, 11). The synthesis of other glycina-
mides or close analogs ( 8, 9, 12, 13) involves similar synthetic
methodology to dihydroindolyl glycinamdies (10–11) synthesis.
Starting with the commercially available materials (23), the carbon
linked analogs (14 and 15) were obtained in moderate yields.
The six-membered N-heteroaryl glycinamides (3, 4, 6, 7) were
reported in a previous publication to be potent NPY5 antagonists
suffering from low brain penetration.13 Further contraction of the
six-membered heterocycles to the five-membered compounds
8–10 maintained potent NPY5 antagonist activity (Table 1).
Pharmacokinetic studies revealed that while benzoxazolinone
and benzothiazolinone derivatives 8 and 9 had poor brain penetra-
tion, the dihydroindole 10 showed a marked improvement in brain
penetration.
NH2
+
R
R
H
R
R1
R1
R2
R2
19
20
21
22
H
N
n
h
R
N
10:
11: n = 2
n = 1
O
NH
R2
S
R1
O O
10,11
O
H
H
N
n
OH
n
N
R
N
Y
f,g
h
N
X
R
X
R
X
NH
O
Y
Y
S
O O
8, 9, 12, 13
Based on the above mentioned results, the dihydroindolyl
glycinamide (10a) was singled out as the most promising lead
compound with sub-nanomolar binding affinity, good plasma
exposure, and acceptable brain penetration.
12: X = Y = CH, n = 1
13: X = Y = CH, n = 2
8
:
X = CO, Y = S, n = 1
9: X = CO, Y = O, n = 1
O
The structure–activity relationship on the dihydroindolyl por-
tion was explored next (Table 2). The methyl substitution at C-4
position (10b) has lower NPY5 affinity and similar clearance and
exposure level as chlorine analog (10a). The fluorine substitution
at C-4 (10c) improved the in vitro clearance, at the expense of low-
er NPY5 affinity, and plasma and brain exposure. The di-substi-
tuted fluorine analog (10d) did not show any improvement in
the in vitro clearance. The gem dimethyl compound 10e also main-
tains potency at the NPY5 receptor, but suffers from low micro-
somal stability and poor plasma and brain exposure. The b-
amino acid linker (11) was also investigated. The longer linker
H
N
OH
14: R = H
15: R = Me
h
NH
O
N
N
R3
S
R3
O O
23
14, 15
Scheme 1. Reagents and conditions: (a) iPrSO2Cl, NaOH, H2O, 5 °C or rt 30–50%; (b)
DPPA, NEt3, BnOH/toluene, reflux, 80–90%; (c) Pd/C, H2, EtOH, 95%; (d) TFA, DCM,
40 °C; (e) NaBH(OAc)3, DCE, 50% for two steps; (f) for n = 1:
a-bromo methyl
acetate, TBAB, KOH, THF; for n = 2: b-bromo methyl propionate, K2CO3, DMF,
100 °C; (g) LiOH, MeOH, H2O, 30–50% for two steps; (h) 20, EDCI, DMAP, DCM, 10–
50%.
Table 1
SAR of benzothiazepine and benzothiazepinone glycinamides
L
L
L
H
N
O
O
N
O
N
O
N
L =
O
NH
O O
Cl
Cl
Cl
Cl
Cl
S
3
8
4
6
L
L
L
L
N
N
S
N
N
O
O
Cl
O
9
7
10a
Compound
hY5, Kia (nM)
LE
hCLint (L/min)b,14
rCLint (mL/min)c,14
Brain (nM)16
Plasma (nM)16
1
3
4
6
7
8
9
10a
27.0
0.3
3.9
0.9
0.8
0.9
0.32
0.44
0.41
0.43
0.43
0.43
0.39
0.46
35.0
0.5
0.8
2.9
2.8
1.0
0.5
3.6
230.0
15.0
40.0
35.0
36.0
12.0
1.9
0
33
17
43
88
11
19
304
2
480
877
428
498
870
1638
5607
10.0
0.5
49.0
a
Binding affinity was determined using a [125I]PYY radioligand binding assay to membranes from cells expressing human NPY5 receptor.11 Data shown are an average of
two or more assays.
b
Maximum human liver blood flow = 1.5 L/min.15
c
Maximum rat liver blood flow = 20 mL/min.15

L. Wu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2167–2171
2169
Table 2
SAR of dihydroindolyl glycinamides
3
2
H
N
4
[ ]n
R
10: n = 1;
11:
N
H
N
5
;
n = 2
O
R1
S
R2
O O
Compound
R
R1,R2
hY5, Ki (nM)a
hCLint (L/min)14
rCLint (mL/min)14
Brain (nM) 10 mpk16
Plasma (nM) 10 mpk16
10a
10b
10c
10d
10e
11
4-Cl
4-Me
4-F
4,5-DiF
4-Cl
H
H,H
H,H
H,H
H,H
Me,Me
H,H
0.5
3.5
4.0
1.5
1.1
1.8
3.6
3.1
1.4
4.5
14.0
5.8
49.0
54.0
21.0
39.0
205.0
7.1
304
477
117
105
50
5607
6142
2676
1515
417
7
24
a
Binding affinity was determined using a [125I]PYY radioligand binding assay to membranes from cells expressing human NPY5 receptor.11 Data shown are an average of
two or more assays.
around the indole ring (12b, 12c, 12d, 12e) did not further improve
the NPY5 affinity. The C-6 substituted analog was evaluated for
metabolic stability and PK, and was found to be inferior compared
to 10a. It seems that the C-5 substitution is optimal for both po-
tency and metabolic stability. Replacing the chlorine with fluorine
(12f vs 12c; 12g vs 12d) or cyano (12i) reduced the NPY5 affinity
while the in vitro clearance was shown to be improved. The C-5
and C-6 di-substituted fluorine analog (12h) did not improve the
metabolic stability. Methyl substitution on the C-2 position re-
duced the potency and exposure. The isomeric indole (14) had re-
duced NPY5 potency and brain exposure. Replacement of the
indole NH with a methyl group (15) improved the NPY5 affinity,
but resulted in decreased plasma exposure.
Table 3
MDR-MDCK assay data of compound 10a
Papp (Â10À6 cm/s)
Efflux ratio
26
A–B
2.31
B–A
60.1
had little effect on NPY5 affinity, but was found to be detrimental
to in vitro microsomal stability perhaps due to a reverse Michael
addition mechanism.
Since the observed brain to plasma ratio was consistently low
for this series, we suspected that efflux transporters, like p-glyco-
protein (Pgp),17 may contribute to the observed low brain expo-
sure. The permeability rates of lead dihydroindolyl glycinamide
(10a) were measured in a Pgp over-expressing mdr-MDCK cell
monolayer to determine the extent of Pgp efflux. The results (Table
3) confirmed that dihydroindolyl glycinamide (10a) is a strong Pgp
substrate with an efflux ratio of 26.
To address the high clearance issue, a metabolite ID study of
compound 10a was conducted and the major metabolite was iden-
tified as the indolyl analog 12c. The exposure of indole (12c) was
measured in a PK study, and the results are shown in Table 4. Both
the brain and plasma levels of the metabolite were higher than the
parent with slightly improved brain/plasma ratio compared to the
parent.
Encouraged by the observed exposure results, the metabolite
12c was synthesized and profiled (Table 5). The indolyl analog
showed similar NPY5 affinity verses the dihydroindolyl glycinam-
ide (10a) whereas the in vitro clearance seems to be improved.
However the overall plasma and brain exposure levels at 1 h fol-
lowing 10 mpk PO dose did not show any improvement compared
to dihydroindolyl glycinamide 10a. The indolyl glycinamide (12c)
plasma levels were much lower when compared to the dihydroind-
olyl glycinamide (10a) at a similar oral dose (Table 4) suggesting
poor oral absorption of indolyl glycinamide. Close analogs of 12c
were synthesized to investigate the SAR. Moving the chlorine
Compounds 10a and 12c were selected for further investigation.
Compound 10a has 154
99.1% human protein binding. In comparison, the compound 12c
exhibits similar solubility (160 M), low LogD7.4 (1.2), and slightly
lM solubility at pH 7.4, 3.0 LogD7.4, and
l
higher human protein binding (99.6%). The off target activities of
both compounds were determined in an in-house screening panel
of 18 GPCRs as well a broad cross reactivity panel and the data
were shown to be devoid of any activity that could have contrib-
uted to the in vivo efficacy shown below. The compound 10a
exhibited strong CYP3A4 and weak CYP2C8 inhibition of 0.7
IC50 and 8.2 IC50, respectively, and was inactive against
CYP1A2, CYP2C19, and CYP2D6 (IC50 >40 M). In comparison, the
lM
lM
l
indolyl analog 12c is inactive against all tested CYP isoforms.
The rat pharmacokinetic properties were evaluated for com-
pounds 10a and 12c as outlined in Table 6. Both compounds
showed acceptable PK profiles with good oral bioavailability and
were deemed suitable for in vivo efficacy studies.
Both dihydroindolyl 10a and indolyl glycinamide 12c were
evaluated for their ability to antagonize NPY5 agonist (cPP) in-
duced feeding and the results are shown in Figures 2 and 3. Signif-
icant inhibition of cPP induced feeding was observed by
dihydroindolyl glycinamide 10a pretreated with a 0.1, 1, and
10 mg/kg PO dose (1 h before intracerebroventricular infusion of
Y5 selective peptide agonist cPP at 0.6 nmol in normal saline).11
Table 4
Metabolite identification and the metabolite (12c) exposure data of 10a in rat brain and plasma
H
N
Cl
H
N
Cl
N
N
H
N
H
N
O
O
S
S
O O
O O
10a
12c
10 mpk, PO, 1 h
Plasma, 10a (nM)
Brain, 10a (nM)
Plasma, 12c (nM)
Brain, 12c (nM)
5467
295
8009
641

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L. Wu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2167–2171
Table 5
SAR of indolyl glycinamides 12, 14, and 15
5
4
6
7
3
H
6
R
5
H
N
N
R
N
H
N
7
H
N
4
O
N
2
O
S
2
S
R'
O O
O O
12
R' = H, 14; R' = Me, 15
Compound
R
hY5, Kia (nM)
hCLint (L/min)14
rCLint (mL/min)14
Brain (nM)16
Plasma (nM)16
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
14
H
1.5
1.7
0.3
1.0
0.9
2.2
1.2
0.9
5.6
8.9
12.0
2.0
1.0
—
1.9
2.6
—
27.0
—
11.0
37.0
—
11.0
—
28.0
8.7
81
—
54
134
—
460
—
751
157
—
4-Cl
5-Cl
6-Cl
7-Cl
5-F
0.6
—
100
—
1296
—
6-F
5,6-2F
5-CN
2-Me
H
2.5
0.4
1.1
0.5
1.2
328
43
49
54
17
2084
769
242
2557
296
49.0
3.4
22.0
15
H
a
Binding affinity was determined using a [125I]PYY radioligand binding assay to membranes from cells expressing human NPY5 receptor.11 Data shown are an average of
two or more assays.
Table 6
Pharmacokinetic properties of 10a and 12c in rats18
Fa (%)
CLb (L/h/kg)
CLp (L/h/kg)
T1/2 (h)
Tmax (h)
Cmax (ng/mL)
Vss (L/kg)
b
c
d
e
f
g
Compound
10a
12c
100
79
1.4
0.9
1.5
0.6
4.4
7.2
0.8
2.0
700h
480h
1.9
2.8
a
b
c
F (%) = absolute oral bioavailability.
CLp = blood clearance (L/h/kg).
CLp = plasma clearance (L/h/kg).
T1/2 = plasma half-life (h).
Tmax = time of maximal concentration.
Cmax = maximum plasma concentration.
Vss = volume of distribution at steady state (L/kg).
d
e
f
g
h
Compound limit of quantification (LOQ) in the plasma measurement was 2 ng/mL.
6
5
4
3
2
1
0
5
4
*
*
3
**
**
**
2
n = 14
n = 9
n = 7
n = 12
n = 7
n = 9
1
0
n = 9
n = 9
Vehicle
0.1mg/kg
1mg/kg
10 mg/kg
0.1mg/kg
1mg/kg
10 mg/kg
Vehicle
(20% CD)
(20% CD)
Figure 2. The effect of dose-dependent 10a on the cPP induced food intake.
Figure 3. The effect of dose-dependent 12c on the cPP induced food intake.
The corresponding brain and plasma levels were measured at each
dose in satellite animals (Table 7) to support the observed efficacy.
The indolyl glycinamides 12c inhibited the cPP induced feeding at
1 and 10 mg/kg PO dose with projected minimum effective dose
(MED) between 0.1 and 1 mg/kg. The corresponding brain and
plasma levels of 12c were also measured at each dose in satellite
animals as outlined in Table 7. The plasma and brain levels of
12c and 10a are similar to each other. The observed lower MED
of 10a compared to 12c could be attributed to increased efficacy
as a result of generating the active metabolite 12c upon adminis-
tration of 10a.
Table 7
Dose dependent brain and plasma levels at 1 h of 10a and 12c in cPP induced feeding
study
Dose (mg/
kg)
10a
Plasma (ng/
mL)
76
12c
Plasma (ng/
mL)
84
Brain (ng/
g)
Brain (ng/
g)
0.1
1.0
10
11
104
151
6
38
132
516
1703
613
1262

L. Wu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2167–2171
2171
Overstreet, D. H.; Gerald, C. P. G.; Craig, D. A. J. Pharmacol. Exp. Ther. 2009, 328,
900.
In summary, novel dihydroindolyl glycinamides (10a) and indo-
lyl glycinamides (12c) were identified through a systematic SAR
investigation as potent NPY5 antagonists. Dihydroindolyl glycin-
amide 10a significantly inhibits the cPP induced feeding with
<0.1 mg/kg MED dose. Likewise, the indolyl glycinamide (12c) also
inhibits the feeding at a MED dose between 0.1 and 1 mg/kg. Both
compounds represent interesting tools to further investigation into
the biology of the NPY5 receptor. The details of these analogs for
the treatment of various mood and anxiety disorders will be the
subject of future communications.
12. Sato, N.; Ogino, Y.; Mashiko, S.; Ando, M. Expert Opin. Ther. Patents 2009, 19,
1401.
13. Wu, L.; Lu, K.; Desai, M.; Packiarajan, M.; Joshi, A.; Marzabadi, R. M.; Jubian, V.;
Andersen, K.; Chandrasena, G.; Boyle, J. N.; Walker, W. M. Bioorg. Med. Chem.
Lett. 2011, 21, 5573.
14. A 1 lM test compound in either rat or human (pooled) liver microsomes
(0.5 mg/mL) incubated at 37 °C for 0, 5, 15, 30, 60 min with NADPH
regenerating system. Following incubation, the acetonitrile quenched
reaction samples were centrifuged to afford the supernatant for LC/MS
analysis to determine the remained drug concentrations for T1/2 estimation.
The clearances were estimated according to: Obach, R. S.; Baxter, J. G.; Liston, T.
E.; Silber, B. M.; Jones, B. C.; Macintyre, F.; Rance, D. J.; Wastall, P. J. Pharmacol.
Exp. Ther. 1997, 283, 46.
Acknowledgments
15. Davies, B.; Morris, T. Pharm. Res. 1993, 10, 1093.
16. Femoral artery cannulated Sprague-Dawley male rats were purchased from
Taconic Laboratories. The average body weight was 300–350 g. Each
compound that was dissolved in an appropriate vehicle was orally
administered to three rats (N = 3) at a dose of 10 mg/kg. Blood samples were
collected at four time points: predose (0 h), 1 h, 2 h, and 4 h. The rats were then
sacrificed, and the brain tissues were collected and immediately stored at
À80 °C. Plasma samples were obtained by centrifuging the blood samples. To
increase bioanalysis throughput, an equal amount of N = 3 plasma or brain
samples at the same time point, which were collected from three different rats,
were pooled together. Each pooled rat brain sample was homogenized in an
aqueous solution. Protein precipitation of the pooled plasma or homogenized
brain sample afforded a supernatant that was analyzed by LC/MS/MS, an
Agilent 1100 HPLC (Agilent Technologies, Palo Alto, CA) and a TSQ Quantum
MS (ThermoFinnigan, San Jose, CA). Compound concentrations in the plasma
and brain matrices were quantified using ThermoFinnigan Xcalibur.
We thank Manuel Cajina, Asanthi Pieris, Martha Vallejo, and Chi
Zhang for providing bioanalysis, animal dosing (PK), metabolic sta-
bility, and physico-chemical data, respectively.
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concentrations were measured using an Agilent 1100 HPLC (Agilent
10. Okuno, T.; Takenaka, H.; Aoyama, Y.; Kanda, Y.; Yoshida, Y.; Okada, T.;
Hashizume, H.; Sakagami, M.; Nakatani, T.; Hattori, K.; Ichihashi, T.;
Yoshikawa, T.; Yukioka, H.; Hanasaki, K.; Kawanishi, Y. Abstr. Pap. Am. Chem.
Soc. 2010, 240, 284.
11. Walker, M. W.; Wolinsky, T. D.; Jubian, V.; Chandrasena, G.; Zhong, H.; Huang,
X.; Miller, S.; Hegde, L. G.; Marsteller, D. A.; Marzabadi, M. R.; Papp, M.;
Technologies, Palo Alto, CA) and
a TSQ Quantum MS spectrometry
(ThermoFinnigan, San Jose, CA) with quantitative analysis (LC/MS/MS)
performed using Xcalibur software. The PK parameters were determined
performing non-compartmental analysis of plasma concentration–time profile
using WinNonlin 5.2 Software (Pharsight, Cary, NC).