Pyrrolidines as potent functional agonists of the human melanocortin-4 receptor

Article abstract of DOI:10.1016/j.bmcl.2007.06.088

A series of pyrrolidine derivatives were synthesized and characterized as potent agonists of the human melanocortin-4 receptor. For example, 28c had a K_i of 13 nM in binding affinity and EC₅₀ of 6.9 nM in agonist potency with an intr

Full text of DOI:10.1016/j.bmcl.2007.06.088

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5165–5170
Pyrrolidines as potent functional agonists
of the human melanocortin-4 receptor
Joe A. Tran,a Caroline W. Chen,a Wanlong Jiang,a Fabio C. Tucci,a, Beth A. Fleck,b
Dragan Marinkovic,a Melissa Arellanoa and Chen Chena,*
aDepartment of Medicinal Chemistry, Neurocrine Biosciences, Inc., 12790 El Camino Real, San Diego, CA 92130, USA
bDepartment of Pharmacology, Neurocrine Biosciences, Inc., 12790 El Camino Real, San Diego, CA 92130, USA
Received 4 June 2007; revised 22 June 2007; accepted 28 June 2007
Available online 4 July 2007
Abstract—A series of pyrrolidine derivatives were synthesized and characterized as potent agonists of the human melanocortin-4
receptor. For example, 28c had a Ki of 13 nM in binding affinity and EC50 of 6.9 nM in agonist potency with an intrinsic activity
of 100% of the endogenous ligand a-MSH.
Ó 2007 Elsevier Ltd. All rights reserved.
The melanocortin-4 receptor (MC4R) is a member of
the G-protein-coupled receptor superfamily, and plays
an important role in regulating feeding behavior and
other biological functions.1 Therefore, MC4R agonists
have been extensively studied in an effort to discover
small molecules for the treatment of obesity.2 Several
MC4R agonists from different chemical classes have
been reported.3 For example, compound 1 (Fig. 1),4
characterized as a potent and selective MC4R agonist,
has demonstrated efficacy in obesity models in rodents.5
A series of phenylpiperazines 2–5 have been reported by
several research groups. Richardson and coworkers
have reported that compound 2 (EC50 = 71 nM) is mod-
agonist than the sulfonamide 3 (EC50 = 380 nM) in our
assay.7 Recently, we have discovered that benzylamine
with a small alkyl group at the benzylic position in-
creases the binding affinity of a series of antagonists
such as 8.10
Ujjainwalla has recently reported that a series of pyrroli-
dines are potent and selective MC4R agonists.11 For
example, compound 9 has an EC50 of 2 nM in a MC4R
functional assay and an IC50 of 14 nM in a binding assay.
One advantage of this compound is that it is less peptide-
like than 1. Here we report our exploratory work on the
combination of the pyrrolidine moiety in 9 with the
piperazinbenzylamine functionality in our MC4R antag-
onist template 8 to synthesize potent MC4R agonists.
erately active, while its imidazole derivative
4
(EC50 = 14 nM) possesses potent agonist activity at
hMC4R.6 The methanesulfonamide 3 as an MC4R ago-
nist has been reported by Dyck,7 Richardson,6 and
Fotsch and coworkers.8 In addition, a N,N-dimethylam-
inomethyl analog 5 (Ki = 60 nM, EC50 = 7 nM) exhibits
good binding affinity and agonist potency.6 We have
also found that an additional amino group at this site
improves the interaction of this series of compounds
The trans-N-isopropylpyrrolidine carboxylic amides
14–19 were synthesized by the reaction of trans-1-tert-
butoxycarbonyl-4-(4-chlorophenyl)pyrrolidine-3-car-
boxylic acid 11 with a variety of phenylpiperazines
12a– f under coupling conditions, followed by deprotec-
tion of the Boc-group of 13 and reductive alkylation with
acetone as shown in Scheme 1. For compounds 17–19,12
an HCl/MeOH treatment was required to remove the
tert-butylsulfinyl group before purification (Scheme 1).
with the receptor.9 For example, compound
6
(Ki = 6.4 nM, EC50 = 3.8 nM) is a 100-fold more potent
Keywords: Pyrrolidine; Melanocortin-4 receptor; Agonist; Synthesis.
*
Reduction of benzonitrile 15 with sodium borohydride
catalyzed with nickel chloride afforded the benzylamine
20a, which was converted to the corresponding
methanesulfonamide 20b. Reductive alkylation of 20a
with isobutyraldehyde provided the secondary amine
Corresponding author. Tel.: +1 858 617 7600; fax: +1 858 617
7967; e-mail: cchen@neurocrine.com
Present address: Department of Medicinal Chemistry, Tanabe
Research Laboratories, USA, Inc., 4540 Towne Centre Ct., San
Diego, CA 92121, USA.
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.06.088

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J. A. Tran et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5165–5170
O
O
O
HN
HN
HN
HN
HN
HN
N
N
N
N
N
O
O
O
N
R'
2: R' = MeO
3: R' = MeSO2NH
N
HN
Cl
Cl
Cl
N
1
6
NH
F
4: R' = Imidazol-1-ylCH2
5: R' = Me2NCH2
Cl
N
F3C
N
N
N
N
F
O
O
NH
NH2
N
O
R"
Cl
O
Cl
N
O
O
7: R" = Me
8: R" = iBu
F
9
10
N
Figure 1. Small molecules as hMC4R ligands.
O
O
O
O
O
N
N
N
b
a
N
N
N
N
HO
X
X
O
O
Cl
Cl
Cl
14-15, 17-19
13a-f
11 (trans)
F
F3C
N
N
N
N
R
NH
NH
NH
NH
NH
S
O
NH
S
O
NH
S
O
12a: R = MeO
12b: R = CN
12c: R = CHO
12d
12e
12f
Scheme 1. Reagents and conditions: (a) HBTU/DIEA/DMF/rt, 4 h, ꢀ80%; (b) i—TFA/CH2Cl2/rt, 1 h; ii—Me2CO/NaBH(OAc)3/AcOH/
dichloroethane/rt, 8 h; for 17–19: iii—HCl/MeOH/rt, 1 h.
24a, which was coupled with b-alanine to give the amide
25 (Scheme 2).
treatment to remove the Boc-group, with acetone affor-
ded the benzylalcohol 21, which was converted to the
corresponding mesylate, followed by nucleophilic
replacement with various imidazoles, triazoles, and pyr-
azoles to give compounds 22. Oxidation of 13c with
The benzaldehyde 13c was derivatized as described in
Scheme 3. Reductive alkylation of 13c, after a TFA
N
N
c
a
15
N
N
N
N
O
O
HN
R'
N
R"
Cl
Cl
20a: R' = H
20b: R = MeSO2
24a: R" = H
25: R" = COCH2CH2NH2
b
d
Scheme 2. Reagents and conditions: (a) NiCl2/NaBH4/EtOH/60 °C, 24 h; (b) MeSO2Cl/Et3N/CH2Cl2/rt, 1 h; (c) Me2CHCHO/NaBH(OAc)3/
CH2Cl2/rt, 8 h; (d) i—N-Boc-b-Ala-OH/EDC/HOBt/Et3N/CH2Cl2/rt, 2 h; ii—TFA/CH2Cl2/rt, 1 h.

J. A. Tran et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5165–5170
5167
N
N
b
a
N
N
13c
N
N
O
O
O
HO
Cl
Cl
21
16
c
d
N
N
N
N
N
N
O
O
R1
N
Y
23: R2 = alkyl
24: R2 = H
Cl
R2
Cl
22
Scheme 3. Reagents and conditions: (a) i—TFA/CH2Cl2, rt, 1 h; ii—Me2CO/NaBH(OAc)3/CH2Cl2/rt, 8 h; (b) PCC/CH2Cl2/rt, 3 h, 55%;
(c) i—MeSO2Cl/Et3N/CH2Cl2/rt, 3 h; ii—YH/NaI/DMF/80 °C, 16 h; (d) R1R2NH/NaBH(OAc)3/Cl CH2CH2Cl/rt, 16 h.
PCC gave the benzaldehyde 16, which was subjected to
reductive amination with secondary or primary amines
to afford the tertiary or secondary amines 23 and 24,
respectively (Scheme 3).
variety of Boc-protected amino acids under peptide
coupling conditions provided the corresponding amides
27, 28, and 29, respectively, after Boc-deprotection
(Scheme 4). A sulfonamide 30 was also obtained by
the reaction of 19 with methanesulfonyl chloride. All
final compounds were tested in a binding assay using
membranes from HEK293 cells transfected with the
hMC4R and [125I]-NDP-MSH as the radioligand, and
the results are listed in Tables 1 and 2. The functional
Reductive alkylation of 17 with excess of formaldehyde
afforded the N,N-dimethylamine 26. Acetylation of 17
with acetic anhydride in the presence of DMAP gave
the acetamide 27a. Coupling reactions of 17–19 with a
N
Z
N
a
N
N
N
N
O
O
NH2
N
Cl
Cl
17: Z = 4-Me
18: Z = 6-F
26
b
N
Z
N
N
NH
O
N
O
R3
F3C
N
N
O
NH
Cl
R3
Cl
O
27: Z = 4-Me
28: Z = 6-F
29
b
N
N
F3C
F3C
N
c
N
N
N
O
O
NH2
NH
S
Cl
Cl
O
O
19
30
Scheme 4. Reagents and conditions: (a) CH2O/NaBH(OAc)3/CH2Cl2/rt, 2 h, 50%; (b) R3COOH/EDC/HOBt/i-Pr2NEt/rt, 16 h; (c) MeSO2Cl/Et3N/
CH2Cl2/rt, 2 h.

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J. A. Tran et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5165–5170
Table 1. Binding affinity of phenylpiperazines at hMC4R
activity of selected compounds was tested using a cAMP
assay as previously described,13 and the results are listed
in Table 2.
N
The 2-methoxy compound 14 displayed a moderate
binding affinity (Ki = 520 nM), while the 2-cyano analog
15 and 2-carboxaldehyde 16 possessed lower affinity. In
comparison, the 2-methoxyphenylpiperazine with Tic-
(4-Cl)Phe dipeptide 2 (Fig. 1) has been reported by
Richardson to have a Ki of 1100 nM.6 The 2-amino-
methyl compound 20a displayed similar binding affinity
to its methanesulfonamide analog 20b (Ki = 1700 nM),
which was less potent than its dipeptide counterpart
(3, Ki = 210 nM reported, 250 nM in our assay).6
However, the imidazole 22a (Ki = 220 nM) possessed a
Ki value similar to its dipeptide analog 4 (Ki = 110 nM,
reported). The variation of this functional group (tria-
zole and pyrazole) resulted in compounds with similar
affinity (22b–g, Ki = 280–580 nM). In our functional
assay, 22a only exhibited partial agonism with moderate
potency (EC50 = 320 nM, IA = 52%). In comparison,
the dipeptide 4 (EC50 = 14 nM) is a full agonist with
high potency as reported.6 Similarly, the N,N-dimethy-
laminomethyl 23a (Ki = 1200 nM) was also much less
potent than its dipeptide analog 5 (Ki = 60 nM, re-
ported),6 and this trend was further confirmed by 23b
and cyclic amines, such as pyrrolidine 23e and piperidine
23f (Ki’s 34, 24, and 27 nM, and EC50’s 14, 39, and
37 nM, respectively, have been reported for the dipep-
tide analogs of 23b, 23e, and 23f).6 The current series
of compounds possessed low binding affinity and no
clear structure–activity relationship at this site of the
molecule (Table 1), which is somewhat different from
the Tic-(4-Cl)Phe compounds 2 and analogs.
N
N
O
W
Cl
14-16, 20-25
Compound
W
Ki (nM)
14
15
MeO
CN
520
4800
1150
1100
1700
220a
280
16
CHO
NH2CH2
MeSO2NHCH2
20a
20b
22a
22b
22c
22d
22e
22f
22g
23a
23b
23c
23d
23e
23f
23g
23h
23i
24a
24b
24c
24d
24e
24f
24g
24h
25
Imidazol-1-yl-CH2
2-Methylimidazol-1-yl-CH2
5-Methylimidazol-1-yl-CH2
1,2,4-Triazol-4-yl-CH2
1,2,4-Triazol-1-yl-CH2
1,2,3-Triazol-1-yl-CH2
Pyrazol-1-yl-CH2
250
580
320
420
490
Me2NCH2
Et2NCH2
1200
1200
1100
320
(Me2CHCH2)2NCH2
MeOCH2CH2N(Me)CH2
Pyrrolidin-yl-CH2
550
950
Piperidin-1-yl-CH2
Piperazin-1-yl-CH2
360
840
92b
4-Methylpiperazin-1-yl-CH2
4-Aminopiperidin-1-yl-CH2
Me2CHCH2NHCH2
NH2CH2CH2NHCH2
MeNHCH2CH2NHCH2
Azetidin-3-yl-NHCH2
Azetidin-3-yl-N(Me)CH2
Piperidin-4-yl-CH2
510
230
270
520
240
190
Pyridin-3-yl-NHCH2
2-NH2C6H4NHCH2
Me2CHCH2N(COCH2CH2NH2)CH2
1600
1400
450
Previously we have found from the SAR study of the
Tic-(4-Cl)Phe dipeptide series that an additional amine
on the benzylamine side chain increases potency. For
example, compound 6 possesses a Ki of 6.4 nM and an
EC50 of 3.8 nM. We tried a set of diamines on the cur-
rent series (23h–i and 24b–h), and the most potent binder
a This compound exhibited partial agonist activity in a functional assay
(EC50 = 320, IA = 52%).
b Partial agonist (EC50 = 390, IA = 40%).
Table 2. Binding affinity and agonist potency of phenylpiperazines at hMC4Ra
F
F3C
Ar =
N
NH2
NH2
NH2
NH
26
19
17
27
18
28
N
F
Ar
F3C
F3C
N
N
O
NH
NH
NH
S
O
R3
R3
Cl
R3
O
17-19, 26-30
R3
O
30
O
29
O
Compound
Ki (nM)
EC50 (nM)
Emax (%)
17
18
19
26
12
21
26
13
105
270
99
68
94
44
1100
b

J. A. Tran et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5165–5170
5169
Table 2 (continued)
Compound
R3
Ki (nM)
EC50 (nM)
Emax (%)
b
27a
27b
27c
28a
28b
28c
29a
29b
29c
29d
29e
29f
29g
29h
30
Me
CH2NMe2
47
35
100
102
114
70
7.1
4.7
11
170
16
CH2CH2NMe2
CH2NHMe
R-CH(Me)NH2
CH2CH2NH2
CH2NH2
16
4.4
13
9.0
6.9
44
100
115
63
8.9
7.7
17.3
4.9
47
CH2NHMe
CH2NMe2
67
52
99
R-CH(Me)NH2
S-CH(Me)NH2
CH2CH2NH2
CH2CH2NMe2
Azetidin-3-yl
31
210
103
104
95
8.7
10
4.6
91
6.6
450
76
80
18
87
96
a Data are average of two or more independent measurements. The affinity measurements for each compound differed by less than 3-fold, resulting in
an average coefficient of variance of 25% for the binding assay Ki values and 32% for the functional assay EC50 values.
b Not determined.
in this series was the 4-aminopiperidine 23g
(Ki = 94 nM), which possessed only moderate potency
and partial agonism (EC50 = 390 nM, IA = 40%).
29d (EC50 = 31 nM) was about 7-fold more potent than
its S-isomer 29e, demonstrating a stereo-effect and sug-
gesting specific interaction of this amino group with the
receptor. Finally, the methanesulfonamide 30 was less
potent in binding affinity than its parent 19, and this
result was similar to that from the pair of 20a and
20b, further demonstrating the importance of an amine
group in receptor binding.
We then focused our efforts on the benzylamines with a
small aliphatic group such as isopropyl or isobutyl
moiety which has been demonstrated to improve binding
affinity for a series of MC4R antagonists. For example,
compound 7 has a Ki of 490 nM in the binding assay,
while its isobutyl analog as an S-configure isomer 8 pos-
sesses a Ki of 74 nM, about 7-fold improvement. Thus,
compound 17 had a Ki of 12 nM in binding affinity, which
was almost 100-fold better than the simple benzylamine
20a (Ki = 1100 nM, Table 1).14 More importantly, 17
possessed good agonist activity (EC50 = 105 nM, IA =
99%). The 6-fluoro-analog 18 was somewhat less potent
compared to 17. Though the a-isobutyl compound 19
was less potent in the functional assay (Ki = 26 nM,
EC50 = 1100 nM), its binding affinity was much better
than the N-isobutylamine 24a (Ki = 510 nM).15
In conclusion, a series of pyrrolidines derived from piper-
azinebenzylamines were synthesized, and potent agonists
of the human melanocortin-4 receptor were identified.
While derivatives from the simple benzylamine 20a did
not yield a potent MC4R agonist, compounds with an
additional benzylic isopropyl or isobutyl group exhibited
improved agonist activity (17–19). Further increases in
potency were achieved by introducing an amino acid such
as b-alanine. Thus, 28c, 29f, and 29h possessed EC50 val-
ues of less than 10 nM.
We have also demonstrated that adding an amino acid
to the benzylic amine increases binding affinity for a
series of MC4R antagonists. For example, compound
10 possesses a Ki of 7.5 nM.16 Thus, while the N,N-
dimethylglycine 27b had a minimal effect on both bind-
ing affinity and agonist potency over its parent 17, the
N,N-dimethyl-b-alanine 27c (EC50 = 16 nM) greatly
improved its potency. In comparison, the acetyl deriva-
tive of 17 reduced its binding affinity (27a, Ki = 47 nM).
Like 27a (IA = 35%), N,N-dimethylation of 17 also re-
duced agonist efficacy (26. IA = 44%). The b-alanine
derivative of 18 increased its potency about 40-fold
(28c, EC50 = 6.9 nM), and sarcosine and D-alanine
derivatives (28a–b) were also potent MC4R agonists.
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