Optimization of privileged structures for selective and potent melanocortin subtype-4 receptor ligands

Article abstract of DOI:10.1016/j.bmcl.2010.06.038

Design, syntheses and structure-activity relationships of N-acetylated piperazine privileged structures containing MC4R agonist compounds were described. The most potent derivatives were low nM MC4R selective full agonists. Several compounds from the series had modest pharmacokinetic properties.

Full text of DOI:10.1016/j.bmcl.2010.06.038

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4483–4486
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Optimization of privileged structures for selective and potent melanocortin
subtype-4 receptor ligands
a
a
a
a
a
Qingmei Hong a, , Raman K. Bakshi , James Dellureficio , Shuwen He , Zhixiong Ye , Peter H. Dobbelaar ,
Iyassu K. Sebhat a, Liangqin Guo a, Jian Liu a, Tianying Jian a, Rui Tang b, Rubana N. Kalyani b, Tanya MacNeil b,
Aurawan Vongs b, Charles I. Rosenblum b, David H. Weinberg b, Qingping Peng c,
*
Constantin Tamvakopoulos c, Randy R. Miller c, Ralph A. Stearns c, Doreen Cashen d, Willian J. Martin d,
Airu S. Chen b, Joseph M. Metzger d, Howard Y. Chen b, Allison M. Strack b, Tung M. Fong b, Euan Maclntyre d,
Lex H. T. Van der Ploeg b, Matthew J. Wyvratt a, Ravi P. Nargund a
a Department of Medicinal Chemistry, Merck Research Laboratories, Rahway, NJ 07065-0900, USA
b Department of Obesity Research Pharmacology, Merck Research Laboratories, Rahway, NJ 07065-0900, USA
c Department of Drug Metabolism, Merck Research Laboratories, Rahway, NJ 07065-0900, USA
d Department of Pharmacology, 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:
Design, syntheses and structure–activity relationships of N-acetylated piperazine privileged structures
containing MC4R agonist compounds were described. The most potent derivatives were low nM MC4R
selective full agonists. Several compounds from the series had modest pharmacokinetic properties.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 25 April 2010
Revised 4 June 2010
Accepted 7 June 2010
Available online 10 June 2010
Keywords:
MC4R
Melanocortin
Agonist
Privileged structures
N-acetylated piperazine
Sulfonamide piperazine
The melanocortin receptors are a family of seven-transmem-
brane G-protein-coupled receptors. Five different subtypes of mel-
anocortin receptors (MC1R–MC5R) have been identified and
cloned. They interact with their endogenous ligands (the cortico-
tropins and melanocortins) to regulate diverse physiological func-
tions such as skin pigmentation, steroidogenesis, energy
metabolism, feeding and erectile activity, and exocrine secretion.1
The melanocortin subtype-4 receptor (MC4R) is primarily ex-
pressed in the brain, and is involved in the regulation of energy
balance including food intake, and is also associated with sexual
behavior.2 The pharmacological link between MC4R and feeding
behavior comes from the studies with both agonists and the antag-
onists.3 Treatment with MC4R antagonist SHU9119 and agouti pro-
tein (an MC1R and MC4R antagonist) increase food intake and body
weight in rodents.4 The non-selective peptide melanocortin ago-
nist MTII reduces food intake and body weight in rodents.4b Studies
of MC4R knock-out mice, which exhibit obesity, further confirm
the relation between MC4R and feeding regulation.5 These findings
have attracted many medicinal chemists and biologists to seek
potent MC4R agonists for the treatment of obesity.
In the past several years, small-molecule MC4 receptor agonists,
both based on peptides and non-peptides, have been reported in
the literature.6 Earlier work from our laboratories and other re-
search groups identified small-molecule MC4R agonists 1–5
(Fig. 1).7 Both compounds 1 and 2 show significant reduction of
food intake and body weight, and stimulation of erectile response
in rodents.7a,b Compound 3 has good binding and functional MC4R
activity and selectivity against the other MCR subtypes.7c Com-
pound 4 also has good MC4R binding affinity.7d Compound 5 is a
potent and selective MC4R agonist and is effective in rodent
food-intake models.7e
Compared with the dipeptide analogs,7a,b non-peptide com-
pounds had a better PK profile.7e–g We envisioned that a hybrid
structure of t-butyl pyrrolidine acid (from 5) and piperazine privi-
leged structures (from 4) might improve potency while maintaining
* Corresponding author.
E-mail address: qingmei_hong@merck.com (Q. Hong).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.038

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Q. Hong et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4483–4486
structure improved the MC4R potency. Initially, we decided to
HN
HN
make the cyclohexyl-based amine privileged compounds.
The synthesis of the cyclohexyl-based amine privileged struc-
ture compounds, 15–18, is shown in Scheme 1. Cyclohexanecarbol-
dehyde 8 underwent potassium tert-butoxide catalyzed nitro-aldol
condensation with nitromethane to afford nitro alcohol 9. Dehy-
dration with trifluoroacetic anhydride and triethylamine generated
nitroalkene 10. Reaction of 10 with Boc-piperazine in CH2Cl2 gave
nitro compound 11, which was reduced using NaBH4 in the pres-
ence of NiCl2 to generate amine 12. Reductive amination of com-
pound 12 with acetone to give amine 13, which was followed by
deprotection with HCl and coupled with t-butyl pyrrolidine acid
7 to generate 15. Alkylation of intermediate 12 by reductive amina-
tion with isobutylaldehyde or reaction with appropriate bromide
in DMF in the presence of potassium carbonate, followed by depro-
tection and coupling sequence gave compounds 16–18. Com-
pounds 15–18 are the mixtures of diastereomers since compound
12 is racemic.
NH
O
N
NH
O
O
O
Cl
Cl
N
N
N
H
N
N
N
O
2
1
HN
HN
NH
O
NH
O
O
O
Cl
Cl
N
N
N
O
O
N
N
3
4
The compounds (15–18) with piperazine amine privileged
structure were evaluated in a competitive binding assay and func-
tional assay (Table 1). The binding assay was performed to assess
F
F
N
the competitive binding of test compounds versus [129I]-NDP-
a-
O
MSH. The functional assay was conducted by measuring the accu-
mulated cAMP in CHO cells expressing the human receptors.8
As shown in Table 1, compounds (15–18) have modest MC4R
binding and functional activity. The smaller alkyl group shows en-
hanced MC4R binding and functional activity. For example, the iso-
propyl amine compound 15 shows good functional activity in this
series (IC50 = 310 nM, EC50 = 240 nM).
N
O
N
H
Cl
In order to further improve the MC4R binding and functional
activity, we prepared the isopropyl amide and sulfonamide privi-
leged compounds.
5
Figure 1. MC4R agonists.
a good PK profile. This Letter will discuss the discovery of novel
piperazine derivatives 6 as MC4R agonists based on this design
(Fig. 2). Wewillfocusonthemodificationoftheprivilegedstructures
while keeping the t-butyl-4-(2,4-difluorophenyl)pyrrolidine-3-
carboxylate.
OH
CHO
a
NO2
NO2
b
8
9
10
Earlier’s work in our group showed that cyclohexyl ring (R in
structure 6) which had replaced a phenyl ring in the privileged
Boc
Boc
Boc
N
N
N
d
N
c
e
N
N
H
N
NO2
NH2
F
F
N
13
11
12
O
f, g
h, f, g
N
F
F
F
F
F
F
O
F
N
F
N
N
N
H
O
O
O
N
N
N
N
N
Cl
5
N
N
O
OH
Acid 7
R
N
H
N
R1
N
HN
R
NH
O
R
15
O
16-18
Cl
6
N
N
16 R = CH2CH(CH3)2
17 R = CH3CH2CH2
18 R = CH3CH2
N
Scheme 1. Reagent and conditions: (a) CH3NO2, t-BuOK, t-BuOH, THF, 0 °C, 2 h, rt
16 h; (b) TFAA, Et3N, CH2Cl2, À10 °C, 1 h; (c) Boc-piperizane, CH2Cl2, rt 16 h; (d)
NiCl2, NaBH4, CH3OH, 0 °C, 2 h; (e) CH3COCH3, AcOH, NaBH(OAc)3, CH2Cl2, rt 16 h;
(f) HCl, dioxane, rt 1 h; (g) EDC, HOBt, CH2Cl2, NMM, acid 7, rt 16 h; (h)
(CH3)2CHCHO, AcOH, NaBH(OAc)3, CH2Cl2, rt 4 h; or RBr, K2CO3, DMF, rt 16 h.
4
Figure 2. Design of MC4R agonists.

Q. Hong et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4483–4486
4485
Table 1
Table 2
Binding affinity and functional activity of compounds at human melanocortin
Binding affinity and functional activity of compounds at human melanocortin
subtypes 4 receptorsa
subtype-4 receptorsa
Compds MC4R bindingb IC50
(nM)
cAMPc EC50
(nM)
Activationd at 10
(%)
l
M
Compds MC4R bindingb IC50
(nM)
cAMPc EC50
(nM)
Activationd at 10
(%)
lM
15
16
17
18
310 30
1900 91e
200 21e
260 24
240 43
6700 3300e
710 170e
570 98
95
10
97
19A
19B
20A
20B
21A
21B
22A
22B
23A
23B
29
28
15
13
18
5
3
2
1
4
50 11
134
123
114
117
102
99
98
88
106
81
25
25
13
5
8
2
2
1
101
5.4
9.1
a
Values represent mean standard error. All data represent at least three
determinations except for where indicated.
64 20
35 4e
10 1e
Displacement of [125I]-NDP-
cells.
a
-MSH from human receptors expressed in CHO
b
130 66e
15 2.9e
18
62
3
8
6.4
19
1
3
c
d
e
Concentration of compound at 50% maximum cAMP accumulation.
Percentage of cAMP accumulation at 10
Values (n = 2) with standard error.
l
M compound relative to
a-MSH.
24A
24B
25A
25B
26A
26B
27A
27B
49
23
88
27
150 16
150 3.7
53 3.1e
46 12
4
5
4
1
29
9.7
4
1
101
113
117
100
89
94
88
102
47 12
19
6
180 87
180 81
19 2.5e
44 10
F
F
F
F
N
O
N
N
O
N
a
Values represent mean standard error. All data represent at least three
determinations except for where indicated.
b
Displacement of [125I]-NDP-
a
-MSH from human receptors expressed in CHO
cells.
N
R
N
N
R
N
c
d
e
Concentration of compound at 50% maximum cAMP accumulation.
Percentage of cAMP accumulation at 10 lM compound relative to a-MSH.
Values (n = 2) with standard error.
24A R = COCH3 d1
24B R = COCH3 d2
25A R = SO2CH3 d1
25B R = SO2CH3 d2
26A R = COCH3 d1
26B R = COCH3 d2
27A R = SO2CH3 d1
27B R = SO2CH3 d2
Table 3
Binding affinity and functional activity of selected compounds at human melanocor-
tin receptorsa
Figure 3. Structures of Compounds 24–27.
Compds Receptor Bindingb IC50
(nM)
cAMPc EC50
(nM)
Activationd at
10 M (%)
l
19B
20B
21A
23A
24B
25B
hMC1R
hMC3R
hMC4R
hMC5R
1800 380
6500 540
890 180
2000f
61
81
123
64
F
28
3
25
5
F
660f
2800f
Boc
N
hMC1R
hMC3R
hMC4R
hMC5R
1000 105
3600 186
540 150
1800 300
63
94
117
57
N
O
a, b, c
13
1
13
2
N
13
d, e, f
H
N
250f
>5000f
N
N
Racemic
hMC1R
hMC3R
hMC4R
hMC5R
1900 720
770 170
1200 450
90 19
58
108
102
68
R
N
13A e1
13B e2
18
4
5.4
2
130f
490 83
hMC1R
hMC3R
hMC4R
hMC5R
730 97
870 49
520 212
60
102
106
62
79
9
19A-23A
19B-23B
18
3
6.4
1
620f
1000 143
560 115e
880 215
hMC1R
hMC3R
hMC4R
hMC5R
3600 960
4700 1000
77
109
113
56
19A R = COCH3 d1
20A R = SO2CH3 d1
19B R = COCH3 d2
20B R = SO2CH3 d2
23
5
9.7
1
1100f
>5000f
hMC1R
hMC3R
hMC4R
hMC5R
5000 160
4900 350
>5000f
62
87
100
76
2500 0e
27
1
19
6
O
O
N
N
O
N
N
>5000e
d1
2800 270
R =
21B
22B
21A
R =
R =
d2
a
Values represent mean standard error. All data represent at least three
determinations except for where indicated.
O
N
N
N
Displacement of [125I]-NDP-
cells.
a
-MSH from human receptors expressed in CHO
d2
b
R =
22A
23A
d1
d1
N
O
O
c
d
e
f
Concentration of compound at 50% maximum cAMP accumulation.
23B R=
R=
Percentage of cAMP accumulation at 10
Values (n = 2) with standard error.
Values (n = 1).
lM compound relative to a-MSH.
d2
N
N
N
N
Scheme 2. Reagent and conditions: (a) NaHCO3, CH2Cl2, H2O, Cbz-succinimide, rt
16 h; (b) chiral HPLC resolution: AD column; mobile phase 40% IPA/heptane; (c)
Pd(OH)2, H2, HOAc, EtOH, rt 5 h; (d) (CH3CO)2O or CH3SO2Cl, DIEA, DMAP, CH2Cl2; or
RCOOH, EDC, HOBt, CH2Cl2; (e) HCl, dioxane; (f) EDC, HOBt, CH2Cl2, NMM, acid 7.
The single compounds 19A–23A and 19B–23B were prepared
from intermediate 13. In order to resolve the enantiomers, amine

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Q. Hong et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4483–4486
Table 4
In summary, we report the design, synthesis and SAR and phar-
Pharmacokinetic data in rata
macokinetic of a new novel N-acetylated piperazine compounds as
MC4R agonists. SAR study of privileged structure led to the discov-
ery of potent, selective and orally bioavailable analogs with isopro-
pyl sulfonamide and amide piperazine as privileged structure for
MC4R agonists. Replacement of methyl group by heterocyclic
group in isopropyl amide piperazine privileged structures further
improved the MC4R binding and functional potency.
Compounds
19B
20B
22A
18
F (%)
12
42
6.8
2.2
0.10
21
25
6.0
2.7
0.26
26
31
3.8
1.8
0.24
22
7.3
19
34
1.3
Cl (mL minÀ1 kgÀ1
)
Vdss (L kgÀ1
t1/2 (h)
)
AUCN (l
M h/mpk)b
a
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.
References and notes
b
AUCN from oral dosing.
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13 was converted to Cbz-protected amine by treatment of Cbz-suc-
cinimide in the presence of sodium bicarbonate. The racemic com-
pound was resolved on chiral HPLC to give separated enantiomers
e1 and e2, followed by Cbz-deprotection gave 13A and 13B. The
first elution designated as e1 and second elution as e2. Acylation,
sulfonation or EDC coupling reaction of compounds 13A and 13B,
respectively, followed by Boc-deprotection with HCl and coupled
with t-butyl pyrrolidine acid 7 generated compounds 19A–23A
and 19B–23B.
In order to further explore the SAR of this series, we also re-
placed the cyclohexyl group with isobutyl and t-butyl group and
synthesis of compounds 24A–27A and 24B–27B (Fig. 3) using the
same methods for making cyclohexyl compounds 19A–19B and
20A–20B in Schemes 1 and 2.
The piperazine compounds (19A–27A and 19B–27B) were evalu-
ated in a competitivebindingassay and functionalassay (Table 2). As
illustrated in Table 2, converting amine compound 15 to acetyl
amideprivilegedcompound19BlargelyincreasedtheMC4R binding
potency (from 310 nM to 28 nM) and functional activity (from
240 nM to 25 nM). Replacing the acetyl amide privileged structure
with sulfonamide, compound 20B further improved the binding
(IC50 = 13 nM) and functional activity (EC50 = 13 nM). N-Isopropyl
heterocyclic amide (isopropyl pyridazine) and pyrazole privileged
structure compounds (21A and 23A) were more potent than isopro-
pyl sulfonamide compounds (20A–B) in functional assay. Further
SAR studies showed that the cyclohexyl group in the privileged
structure could be replaced with an isobutyl group (24–25) without
loss of potency. However, more hindered groups such as t-butyl
group compounds (26–27) reduced potency.
The MC4R binding affinity and functional activity at the human
receptor for several potent compounds is presented in Table 3. As
shown in Table 3 these compounds were at least 20-fold selective
in binding and functional activity versus the human MC1R and
MC5R except 21A. Most of these compounds were over 100-fold
selective in functional activity versus the human MC5R. Com-
pounds 21A and 23A showed the most potent MC3R activity,
EC50 <100 nM.
The pharmacokinetic (PK) properties of compounds 18, 19B,
20B and 22A were further evaluated in the rat (Table 4). These
compounds all had moderate bioavailability (12–26%), and most
had a half life of about 2 h, except 18 which had a long t1/2 of 34 h.
8. For a detailed description of the assay protocols, see: (a) Bednarek, M. A.;
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Med. Chem. 2001, 44, 3665; (b) Bednarek, M. A.; Siva, M. V.; Arison, B.; MacNeil,
T.; Kalyani, R. N.; Huang, R.-R. C.; Weinberg, D. H. Peptides 1999, 20, 401.