Discovery of a spiroindane based compound as a potent, selective, orally bioavailable melanocortin subtype-4 receptor agonist

Article abstract of DOI:10.1016/j.bmcl.2010.02.058

We report the design, synthesis and properties of spiroindane based compound 1, a potent, selective, orally bioavailable, non-peptide melanocortin subtype-4 receptor agonist. Compound 1 shows excellent erectogenic activity in the rodent models.

Full text of DOI:10.1016/j.bmcl.2010.02.058

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2106–2110
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of a spiroindane based compound as a potent, selective, orally
bioavailable melanocortin subtype-4 receptor agonist
a
a
a
a
a
Shuwen He a, , Zhixiong Ye , Peter H. Dobbelaar , Iyassu K. Sebhat , Liangqin Guo , Jian Liu ,
Tianying Jian a, Yingjie Lai a, Christopher L. Franklin a, Raman K. Bakshi a, James P. Dellureficio a,
Qingmei Hong a, Nancy N. Tsou a, Richard G. Ball a, Doreen E. Cashen b, William J. Martin b,
David H. Weinberg c, Tanya MacNeil c, Rui Tang c, Constantin Tamvakopoulos d, Qianping Peng d,
Randy R. Miller d, Ralph A. Stearns d, Howard Y. Chen c, Airu S. Chen c, Alison M. Strack b, Tung M. Fong c,
D. Euan MacIntyre b, Matthew J. Wyvratt Jr. a, Ravi P. Nargund a
*
a Department of Medicinal Chemistry, Merck Research Laboratories, Rahway, NJ 07065-0900, USA
b Department of Pharmacology, Merck Research Laboratories, Rahway, NJ 07065-0900, USA
c Department of Obesity Research, Merck Research Laboratories, Rahway, NJ 07065-0900, USA
d Department of Drug Metabolism and Pharmacokinetics, Merck Research Laboratories, Rahway, NJ 07065-0900, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the design, synthesis and properties of spiroindane based compound 1, a potent, selective,
orally bioavailable, non-peptide melanocortin subtype-4 receptor agonist. Compound 1 shows excellent
erectogenic activity in the rodent models.
Received 11 January 2010
Revised 12 February 2010
Accepted 16 February 2010
Available online 19 February 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Melanocortin subtype-4 receptor
MC4R
Agonist
Spiroindane
Erectile dysfunction
The melanocortin receptors, a family of seven-transmembrane
G-protein coupled receptors (GPCR’s), consist of five sub-types
(MC1R-MC5R) identified to date. Interactions of the melanocortin
receptors with the endogenous ligands, namely the melanocortins
and corticotropins, elicit a wide range of biological effects, includ-
ing feeding behavior and body-weight homeostasis, skin pigmen-
tation, steroid production, sexual behaviors, and exocrine gland
secretion.1
The melanocortin subtype-4 receptor (MC4R), primarily ex-
pressed in the brain, has been established as a crucial molecular
component of the complex homeostatic regulation in mammals.
Furthermore, MC4R has been implicated in male and female sexual
dysfunction.2 There have been intensive research efforts on identi-
fying small molecule ligands as well as peptide ligands for MC4R as
potential therapeutics for obesity and sexual dysfunction.3
Strong validation utilizing MC4R agonism as a potential mode of
action for the treatment of sexual dysfunction comes from the data
generating around MT-II and bremelanotide (formerly known as
PT-141) ( Fig. 1).4 MT-II and bremelanotide are cyclic peptidic
pan-melanocortin receptor agonists, with activity on MC1R,
NHAc
H
N
H
N
O
O
O
O
O
H
N
HN
HN
X
H
HN
O
N
N
O
H
N
N
H
O
HN
HN
NH2
X= NH2: MT-II
X= OH: Bremelanotide (PT-141)
* Corresponding author. Tel.: +1 732 594 0881; fax: +1 732 594 3007.
E-mail address: shuwen_he@merck.com (S. He).
Figure 1. MT-II and bremelanotide.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.02.058

S. He et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2106–2110
2107
MC3R and MC4R. Bremelanotide is a metabolite of MT-II, which
was originally studied as a potential tanning drug targeting
MC1R. Unexpectedly, MT-II demonstrates strong erectile responses
with accompanying skin tanning in men. Recently, preliminary
clinical trials show efficacy for bremelanotide as a treatment for
both male and female sexual dysfunction. However, bremelanotide
is not expected to have good exposure when administered orally
due to its peptide structure. Furthermore, it is a pan-melanocortin
receptor agonist, with significant activity on MC1R, which can
cause skin tanning effect.
tive, orally bioavailable MC4R agonist, with excellent erectile activ-
ity augmentation in rodent models (Fig. 2).
The design of compound 1 starts with compound 5, following
our previous SAR studies in this series (Fig. 3).6 We envision a cycli-
zation to form 6 containing a spiroindane privileged structure,
which has been shown to improve pharmacokinetic properties
during the work on a growth hormone secretagogue program.7
The substitution pattern on the phenyl ring in the spiroindane moi-
ety comes from a SAR study of a related series.8 The appendage is
extended to introduce the gem-dimethyl substituents to satisfy the
presumed steric requirement.9
Synthetic effort of compounds 1–4 focused on the preparation
of the spiroindane privileged structures in two stages. Stage 1 con-
structed the spiroindanone core structure. Stage 2 concentrated on
converting the ketone functionality to the appending acetamide
moiety.
Previously, we disclosed THIQ and MB243, selective MC4R ago-
nists, displaying proerectile activity as well as food-intake reduc-
tion effect in rodent models.5 In this Letter, we describe our
continued efforts culminating in the discovery of 1, a potent, selec-
Construction of the spiroindanone core structure began with a
Knoevenagel condensation of Boc protected 4-piperidone with
ethyl cyanoacetate (Scheme 1). Cuprate addition on unsaturated
cyanoester 7 set up the quarternary carbon center in the core of
the structure. Decarboethoxylation followed by hydrolysis of the
nitrile and protection of the piperidine nitrogen gave acid 9. Intra-
molecular Friedel–Crafts reaction afforded the spiroindanone 10.
With spiroindanone 10 in place, the rest of the synthesis in-
stalled the acetamide appendage (Scheme 2). Horner–Emmons
reaction on the ketone 10 gave the unsaturated nitrile as a mixture
of geometric isomers. Reduction with magnesium in MeOH fur-
nished the saturated nitrile 11 as a diastereomeric mixture.10
Methylation gave the gem-dimethyl nitrile, which underwent
hydrolysis to form the acid 12. Curtis rearrangement on acid 12
gave the isocyanate intermediate, which was trapped with benzyl
alcohol to give the Cbz protected amine 13. Hydrogenolysis fol-
lowed by acetylation of the amine provided the acetamide 14.
Chlorination on the phenyl ring with N-chlorosuccinimide gave
the privileged structure 15, with chlorination occurring primarily
at the required position.11 The racemic privileged structure was re-
solved on chiral HPLC to give separated enantiomers (15a and
15b). The absolute steric configuration (S) of 15b was established
by single-crystal X-ray diffraction analysis (Fig. 4).11 Coupling of
Boc de-protected 15a and 15b with the enantiomers of the pyrrol-
idine acids (16a and 16b), respectively, provided compounds 1–4.
Compounds 1–4 were initially evaluated in two in vitro assays:
MCR binding assays and MCR functional assays. Binding assay was
performed to assess the competitive binding of test compounds
N
HN
HN
HN
HN
O
O
O
N
N
N
N
NH
N
O
O
Cl
F
THIQ
MB243
N
F
O
N
F
Cl
NHAc
1
versus [129I]-NDP-
a-MSH. The functional assay was conducted by
Figure 2. MC4R selective agonists.
N
N
N
F
F
F
*
*
O
O
O
34
N
N
N
F
F
F
Cl
NHAc
*
NHAc
NHAc
Cl
Cl
1:
2:
3:
4:
R
,
trans(3
trans(3
trans(3
trans(3
S
, 4
, 4
, 4
, 4
R)
5
6
S
,
S
R)
R
,
,
R
R
S
)
)
S
S
Figure 3. Design of MC4R agonists.

2108
S. He et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2106–2110
CO2t-Bu
N
CO2t-Bu
N
O
N CO2t-Bu
a
b, c
CN
EtO2C
CN
EtO2C
CN
8
d, e
7
CO2t-Bu
N
CO2
N
t-Bu
f, g, h, i
CO2H
9
10
O
Scheme 1. Reagents and conditions: (a) NH4Cl, AcOH, C6H6; (b) p-tolylmagnesium bromide, CuCN, THF; (c) LiCl, H2O, DMSO; (d) concd HCl; (e) (Boc)2O, NaOH (aq.), dioxane;
(f) (COCl)2, CH2Cl2, DMF (cat.); (g) HCl, dioxane; (h) AlCl3; (i) (Boc)2O/Et3N/CH2Cl2.
measuring the accumulated cAMP in CHO cells expressing the
human receptors.12
Compound 1 was further evaluated on human, rat, and mouse
melanocortin receptors for in vitro binding and functional assays.
It had excellent selectivity against other sub-types in human
receptors. It also has single digit nanomolar potency on rat and hu-
man MC4R with slight loss of potency in mouse MC4R (Table 3).
Compound 1 was then tested in pharmacokinetics study. It
showed an outstanding pharmacokinetic profile with excellent
Compounds 1 and 2 were significantly more potent than
compounds 3 and 4 in both binding and functional assays on
MC4R (Tables 1 and 2). In functional assay, compound 1 had better
MC4R selectivity against MC1R than compound 2 (Table 2). There-
fore, compound 1 was selected for further evaluation.
CO2t-Bu
N
CO2t-Bu
N
CO2t-Bu
N
a, b
c, d, e
CN
CO2H
O
10
11
12
f, g
CO2t-Bu
N
CO2t-Bu
N
CO2t-Bu
N
j, k
h, i
NHAc
Cl
NHAc
NHCbz
15a (R)
15b (S)
14
13
l
N
O
*
16a trans (3
S
, 4
R
)
N
*
3
16b trans (3
R
, 4
S
)
4
HO
F
F
*
*
O
3 4
N
F
F
1:
2:
3:
4:
R
,
trans(3
trans(3
trans(3
trans(3
S
,4
,4
,4
,4
R
)
Cl
S
,
S
R
)
*
R
,
R
S
)
NHAc
S
,
R
S
)
Scheme 2. Reagents and conditions: (a) (EtO)2POCH(CH3)CN, NaH, THF, reflux; (b) Mg, MeOH; (c) LDA, MeI, HMPA, THF; (d) concd HCl, reflux; (e) (Boc)2O, NaOH, dioxane; (f)
DPPA, Et3N, toluene, reflux; (g) BnOH, reflux; (h) H2, Pd/C, EtOH; (i) AcCl, Et3N, CH2Cl2; (j) N-chlorosuccinimide, DMF, 50 °C; (k) Chiral AD column, EtOH–Heptane; (l) 16a or
16b, HATU, HOAt, NMM, CH2Cl2.

S. He et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2106–2110
2109
Table 3
Binding affinity and functional activity of 1 at human, rat, and mouse melanocortin
receptors
Receptor Bindinga IC50 (nM) cAMPb EC50 (nM) Activationc at 10
lM (%)
hMC1R
hMC3R
hMC4R
hMC5R
rMC3R
rMC4R
mMC4R
1938
890
13.0
83.9
1020
351
18
71
105
9
97
126
197
4.9
997
2.0
22
ndd
a
b
c
Displacement of [125I]-NDP-
Concentration of compound at 50% maximum cAMP accumulation.
Percentage of cAMP accumulation at 10
Data is not available.
a
-MSH receptors expressed in CHO cells.
lM compound relative to a-MSH.
d
bioavailability, low clearance, and high plasma drug exposure,
especially in dog and monkey (Table 4).
Compound 1 was evaluated in rodent in vivo obesity models. It
showed mechanism based acute food-intake reduction in DIO
mice.13 Dose-dependent food-intake and body-weight reduction
were observed in a 14-day food-intake study in DIO rats when
compound 1 was administered orally at 10 and 20 mpk.14
Finally, in vivo studies were performed to evaluate the erecto-
genic activity of compound 1 using a conscious rat model in which
the occurrence of spontaneous erections was quantified in the ab-
sence of an external stimulus.15 Compound 1 elicited 1.0 0.27–
1.5 0.19 erections at doses of 0.001–3.0 mg/kg, iv, compared to
vehicle with 0.5 0.16 erections. Even more significantly 75–
100% of compound 1 treated rats presented at least one erection
during the observation period versus 28% for the vehicle treated
rats. The number of erections produced by compound 1 at its max-
imally effective dose (0.1 mg/kg) compared favorably to the maxi-
mal number of erections produced by bremelanotide (PT-141)
(1.3 0.42; 1 lg/kg, iv) (Fig. 5).
Table 4
Pharmacokinetic data for 1
PK parameter
Rata
Dogb
Monkeyc
F (%)
33
12
5.1
4.5
0.8
71
53
Figure 4. Structure of 15b by single-crystal X-ray diffraction.
Cl (mL minÀ1 kgÀ1
)
3.5
3.5
11.2
6.1
9.6
3.5
3.9
1.5
Vdss (L kgÀ1
t1/2 (h)
AUCn (lM h/mpk)
)
Table 1
a
Binding affinity and selectivity of compounds 1–4 for the human melanocortin
subtype-4 receptor
Compound dosed in Sprague-Dawley rats as a solution in EtOH/PEG400/water
(10:40:50) at 1 mg/kg, iv and 4 mg/kg, po.
b
Compound dosed in beagles as a solution in EtOH/PEG400/water (10:40:50) at
a
Compounds
MC4R IC50 (nM)
0.5 mg/kg, iv and in 0.5% methylcellulose in water at 1.0 mg/kg, po.
c
1
2
3
4
13.0
4.3
89.0
39.7
Compound dosed in rhesus as a solution in EtOH/PEG400/water (10:40:50) at
0.5 mg/kg, iv and in 0.5% methylcellulose in water at 2 mg/kg po.
a
Displacement of [125I]-NDP-
a
-MSH from human receptors expressed in CHO
83%
92%
100%
2
1
0
cells.
88% 75%
75%
Table 2
28%
Functional activity of compounds 1–4 at human melanocortin receptors
EC50 (nM) (% max)b
a
Compounds
MC4R
MC1R
Vehicle, IV
PT-141
1 ug/kg, i.v.
0.001 0.01
0.1
1.0
3.0
1
2
3
4
4.9 [105]
3.2 [97]
94.0 [107]
37.2 [111]
1020 [18]
228 [39]
[15]
Compound 1 (mg/kg, i.v.)
Figure 5. Penile erections in conscious rat observation model following treatment
with vehicle (n = 28), PT-141 (n = 6) or compound 1 (n = 8/group). Compound 1
dose-dependently increased the number of penile erections comparable to PT-141.
Bar graph represents mean S.E.M., values depict percent incidence of responder.
620 [41]
a
Concentration of compound at 50% maximum cAMP accumulation.
Percentage of cAMP accumulation at 10 lM compound relative to a-MSH.
b

2110
S. He et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2106–2110
Ploeg, L. H. T.; Patchett, A. A.; Nargund, R. P. J. Med. Chem. 2002, 45, 4589; (b)
In summary, we report the design, synthesis and evaluation of
Martin, W. J.; McGowan, E.; Cashen, D. E.; Gantert, L. T.; Drisko, J. E.; Hom, G. J.;
Nargund, R.; Sebhat, I.; Howard, A. D.; Van der Ploeg, L. H. T.; MacIntyre, D. E.
Eur. J. Pharmacol. 2002, 454, 71; (c) Van der Ploeg, L. H. T.; Martin, W. J.;
Howard, A. D.; Nargund, R. P.; Austin, C. P.; Guan, X.; Drisko, J.; Cashen, D.;
Sebhat, I.; Patchett, A. A.; Figueroa, D. J.; DiLella, A. G.; Connolly, B. M.;
Weinberg, D. H.; Tan, C. P.; Palyha, O. C.; Pong, S.-S.; MacNeil, T.; Rosenblum, C.;
Vongs, A.; Tang, R.; Yu, H.; Sailer, A. W.; Fong, T. M.; Huang, C.; Tota, M. R.;
Chang, R. S.; Stearns, R.; Tamvakopoulos, C.; Christ, G.; Drazen, D. L.; Spar, B. D.;
Nelson, R. J.; MacIntyre, D. E. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 11381; (d)
Cashen, D. E.; MacIntyre, D. E.; Martin, W. J. Br. J. Pharmacol. 2002, 136; (e)
Palucki, B. L.; Park, M. K.; Nargund, R. P.; Ye, Z.; Sebhat, I. K.; Pollard, P. G.;
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I.; Doss, G. A.; Miller, R. R.; Stearns, R. A.; Peng, Q.; Tamvakopoulos, C.;
McGowan, E.; Martin, W. J.; Metzger, J. M.; Shepherd, C. A.; Strack, A. M.;
MacIntyre, D. E.; Van der Ploeg, L. H. T.; Patchett, A. A. Bioorg. Med. Chem. Lett.
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MC4R agonists containing spiroindane substructure. This study
led to the discovery of compound 1, a potent, selective, bioavail-
able, non peptidic MC4R agonist. Compound 1 displayed excellent
proerectile activity comparable to the effect of bremelanotide (PT-
141).
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