Synthesis and SAR of potent and orally bioavailable tert-butylpyrrolidine archetype derived melanocortin subtype-4 receptor modulators

Article abstract of DOI:10.1016/j.bmcl.2008.04.049

Discovery of a series of tert-butyl pyrrolidine derived, potent and orally bioavailable melanocortin receptor subtype-4 (MC4R) selective modulators is disclosed.

Full text of DOI:10.1016/j.bmcl.2008.04.049

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 3242–3247
Synthesis and SAR of potent and orally bioavailable
tert-butylpyrrolidine archetype derived melanocortin
subtype-4 receptor modulators
Liangqin Guo,a,* Zhixiong Ye,a Feroze Ujjainwalla,a Heather L. Sings,a
Iyassu K. Sebhat,a John Huber,a David H. Weinberg,b Rui Tang,b Tanya MacNeil,b
Constantin Tamvakopoulos,a Qianping Peng,a Euan MacIntyre,b Lex H. T. van der Ploeg,b
Mark T. Goulet,a Matthew J. Wyvratta and Ravi P. Nargunda
aDepartment of Medicinal Chemistry, Merck Research Laboratories, 126 E. Lincoln Avenue,
PO Box 2000, RY 50G-332, Rahway, NJ 07065, USA
bDepartment of Obesity and Metabolic Research, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
Received 11 March 2008; revised 15 April 2008; accepted 21 April 2008
Available online 25 April 2008
Abstract—Discovery of a series of tert-butyl pyrrolidine derived, potent and orally bioavailable melanocortin receptor subtype-4
(MC4R) selective modulators is disclosed.
ꢀ 2008 Elsevier Ltd. All rights reserved.
The melanocortin receptors are known as a family of
five seven-transmembrane G protein-coupled receptors.
All the melanocortin receptor subtypes are activated
by their endogenous ligands (the corticotropins and mel-
anocortins), which are derived from the cleavage of pro-
opiomelanocortin (POMC), to mediate a variety of
physiological functions. Of these five subtypes, the mel-
anocortin-4 receptor (MC4R) has been clearly linked to
the regulation of energy homeostasis and feeding behav-
ior, as well as sexual functions.1 Since small molecules
are more likely to be brain penetrable and orally bio-
available than peptide ligands, significant amount of ef-
fort has been spent in drug discovery to develop potent
and selective non-peptide MC4R agonists for the poten-
tial treatment of human obesity.2 A recent report from
Merck research laboratories described the design and
pharmacology of tert-butylpyrrolidine archetype de-
rived MC4R agonist 1 (Fig. 1).3 Here, we report the dis-
covery of a series of interesting MC4R modulators by
modifying the 2,4-difluorophenyl part in 2B,4 a close
analog of 1.
The general synthetic procedures for synthesizing 1-tert-
butyl-4-arylpyrrolidine-3-carboxylic acid part structures
and the MC4R modulators are summarized in Scheme 1.
Preparation of racemic trans methyl 1-tert-butyl-4-aryl-
pyrrolidine-3-carboxylate (3A) as key intermediates in
this study involved [3 + 2] cycloaddition of trans methyl
arylacrylate and bis(trimethylsilylmethyl)-tert-butyl-
amine in the presence of silver fluoride. The methyl ary-
acrylates were synthesized either from esterification of
the corresponding cinnamic acids or from Wittig reac-
tions of methyl(triphenylphosphoranylidene) acetate
F
O
O
F
HN
R
N
N
Cl
1
R = Me
Keywords: G protein-coupled receptor; Melanocortin subtype 4 rece-
ptor agonist; Obesity; Oral bioavailability.
2B R = Et
*
Figure 1. MC4R modulators 1 and 2B.
Corresponding author. E-mail: liangqin_guo@merck.com
0960-894X/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.04.049

L. Guo et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3242–3247
3243
O
b, c
Ar
OH
ArCHO
ArCO2H
a
d
O
N
e
Si
Ar
OMe
Si
N
Ar
+
I
NH2
Si
O
f
OMe
(3A)
H
N
g
N
O
Ar
N
H
N
O
Ar
N
(3B)
Cl
i
4A - 14A
4B - 14B
O
h
OH
O
N
H
Cl
4 -20
Scheme 1. Reagents and conditions: (a) MeOH, SOCl2, reflux, 2 h; (b) BH3, THF, rt, overnight; (c) NMO, TPAP; (d) Ph3P@CHCO2Me, THF; (e)
K2CO3, CH3CN, 80 ꢁC, overnight; (f) AgF, CH3CN, 1 h; (g) 3 N HCl, 85 ꢁC, 24 h; (h) HOAt, HATU, DIEA, CH2Cl2; (i) Chiral HPLC resolution:
ChiralCel OD column; mobile phase = 10% IPA/heptane; flow rate = 9 ml/min.
with the corresponding aryl aldehydes. The aryl alde-
hydes were either commercially available or made from
N
N
aryl carboxylic acids by borane reduction followed by
TPAP oxidation. Bis(trimethylsilylmethyl)-tert-butyl-
amine was formed from reaction of (iodomethyl)tri-
methyl silane with tert-butylamine in the presence of
potassium carbonate in acetonitrile. Acid hydrolysis of
the ester functionality of 3A provided the corresponding
racemic trans-carboxylic acids, which were then coupled
with 4-chlorophenyl piperidine part structure 3B4,5 to
give the final compounds as a mixture of two diastereo-
mers. While only marginal separations were achieved
with racemic trans-t-butyl pyrrolidine methyl esters un-
der a variety of conditions, most diastereomers were sep-
arated well with chiral HPLC using ChiralCel OD
column (2 cm · 25 cm) and 10% isopropanol in heptane
as mobile phase.
N
Ar
O
Ar
O
a, b, c
+
Ar
O
O
O
N
N
OH
O
O
Ph
Ph
d
d
N
N
Ar
Ar
O
O
OH
OH
e
For scaled up synthesis of key compounds for in vivo
studies, Evans auxiliary method was applied to resolve
the racemic tert-butyl pyrrolidine carboxylic acids as
shown in Scheme 2. The products were then confirmed
with the authentic samples on chiral HPLC.
3B
4A, 7A & 12A
3B
e
6B, 7B, 8B & 14B
Scheme 2. Reagents and conditions: (a) trimethyl acetyl chloride,
DIEA, THF, À10 ꢁC, 2 h; (b) (R)-(+)-4-benzyl-2-oxazolidinone, LiCl,
À10 ꢁC to rt, overnight; (c) MPLC separation conditions: 20% EtOAc/
hexane to 80% EtOAc/hexane gradient; (d) LiOH, THF, H2O, rt,
overnight; (e) DIEA, HOAt, HATU, CH2Cl2.
Compound 2B, which was discovered through an earlier
SAR effort, showed modest binding affinity and good
functional activity as a full MC4R agonist (Table 1).4
It was our intention to further improve the MC4R bind-
ing and functional activity. One of our approaches was
replacing the 2,4-difluorophenyl part in compound 2B
with aromatics bearing various functional groups. The
preliminary SAR studies were carried out with com-
pounds as a mixture of two diastereomers, and the assay
results are summarized in Table 2.
Compared with F, Larger halogen atoms (Cl and Br) at
para position resulted in improvement in binding and

3244
L. Guo et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3242–3247
Table 1. Binding affinity and functional activity of 2B and its
diastereomer 2A at the human MC4R6
Table 2. Binding affinity and functional activity of diastereomeric
compounds at the human MC4R6
F
O
O
Ar
N
HN
N
O
O
F
HN
**
N
*
Cl
N
Compounda Ar
MC4R MC4R cAMP
Binding EC50 (nM)
IC50 (nM) (% Max)
Cl
( ,
Compound
)
MC4R Binding
IC50 (nM)
MC4R cAMP
EC50 (nM) [% Max]
* **
Cl
2A
2B
(R,S)
(S,R)
640
70
565 [83%]
17.9 [108%]
4
11.4
184.9 (29%)
Cl
Cl
functional activity (6 and 7), and the larger the halogen,
the greater the improvement in IC50 and EC50. How-
ever, introducing a larger group, such as Cl, at ortho po-
sition decreased the activation (5). Replacing 2, 4-
difluorophenyl with 2, 4-dichlorophenyl resulted in
improvement in binding affinity but significant loss in
functional activity (4). Introducing OCH3 (8) or
OCF3(13) to the para position also provided modest
improvement in binding and functional activities. CF3
group at the para position improved binding affinity
but decreased MC4R activation (9 and 12). Installing
one more F at 5 position resulted in loss in binding affin-
ity and functional activity (11). Incorporating 2-naph-
thyl group (14) showed modest binding affinity and
much lowered activation at MC4R. Functional groups
like NH2, NMe2 and NHAc at the para position all re-
sulted in great loss in binding and functional activities
(17, 19, and 18), suggested that the receptor does not fa-
vor basicity or polarity at this position.
5
6
157.7
10.1
342.9 (77%)
12.7(117%)
F
F
F
Cl
7
4.3
4.7 (92%)
Br
F
8
14.1
26.7
45.1
6 (98%)
190.7 (66%)
256.4 (45%)
O
F
9
After preliminary SAR studies with the mixture prod-
ucts, we then separated the two diastereomers of inter-
esting compounds by means of chiral HPLC. The
assay results of single compounds are summarized in
Table 3. Generally the fast eluting isomers (A series)
on ChiralCel OD column in isopropanol/heptane system
are less potent in functional assay than the slow eluting
isomers (B series). With 2,4-dichlorophenyl part, the fast
eluting isomer (4A) showed higher MC4R affinity
(IC50 = 2.8 nM), but showed no or little activation at
MC4R. Its slow eluting isomer (4B) showed modest
MC4R agonism. With 2-F-4-Cl, 2-F-4-Br or 2-F-4-
OMe phenyl parts, all three slow eluting isomers (6B,
7B, and 8B) showed very strong MC4R potency with
high activation of the receptor, nearly 3-fold improve-
ment in EC50 and 5- to 10-fold improvement in IC50
compared with compound 2B. Compounds incorporat-
ing 2-F-4-CF3 phenyl or 4-CF3 phenyl part (9A vs
12A; 9B vs 12B) showed minimal difference in terms of
MC4R potency, but compounds with 2-F-4-CF3 phenyl
part showed slightly better selectivity, suggesting that F
group at ortho position is not crucial for MC4R recep-
tor binding and functional activities. Replacing 2,4-
difluorophenyl part with 2-naphthyl resulted in a mod-
est agonist with partial activation (14B). Both 9A and
CF3
10
F
F
11
180.5
320.4 (92%)
F
12
13
17.2
22.9
67.1 (37%)
177 (88%)
CF3
OCF3
14
119.8
17% at 10 lM

L. Guo et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3242–3247
3245
Table 3. Binding affinity and functional activity of single compounds
at the human MC4R6
Table 2. (continued)
Compounda Ar
MC4R MC4R cAMP
Binding EC50 (nM)
IC50 (nM) (% Max)
O
O
Ar
HN
**
N
*
N
15
74.6
99.4
198.3 (59%)
Cl
Ar
Compound
a
* **
MC4R
binding
IC50 (nM)
MC4R cAMP
EC50 (nM)
[% Max]
16
17
261.4 (80%)
1363 (69%)
( ,
)
NO2
NH2
Cl
Cl
1274
4A (R,S)
4B (S,R)
3.3
17.9
1% at 10 lM
155.6 (48%)
Cl
18
19
20
10,780
27% at 10 lM
6129 (38%)
NHAc
N
5A (R,S)
5B (S,R)
82.2
216.2
10% at 10 lM
214.5 (84%)
686.1
F
F
6A (R,S)
6B (S,R)
13.23
6.2
110.8 (45%)
2.8 (97%)
O
O
257.9
200.4 (94%)
Cl
a All compounds contain two trans isomers at 3,4 positions on the
pyrrolidine ring.
F
7A (R,S)
7B (S,R)
4.1
4
13.4 (33%)
2.1 (100%)
Br
12A showed strong MC4R binding affinity with very low
activation.
F
8A (R,S)
8B (S,R)
37.2
11.2
244.7 (34%)
4.6 (100%)
The complete MCR activity profiles and oral bioavail-
ability for potent MC4R modulators are shown below
in Table 4.
O
F
9A (R,S)
9B (S,R)
9.8
22.7
5% at 10 lM
88.8 (99%)
These data indicate that 6B shows great MC4 receptor
subtype selectivity with high activation, while 7B and
8B only show modest MC4R selectivity. Compound
6B is over 100-fold functionally selective towards human
MC4R and shows excellent selectivity versus human
MC1b, MC3R, and MC5R. Following oral dosing to
rats at 4 mpk, good PK profiles were observed with both
6B (AUCN = 0.72 lM h, CLp = 11.4 ml/min/kg) and 7B
(AUCN = 1.18 lM h, CLp = 7.3 ml/min/kg), incorpo-
rating 2-F-4-Cl and 2-F-4-Br phenyl parts, respectively.
However, compound incorporating 2-F-4-methoxy phe-
nyl part (8B) showed poor PK profile with relatively
high clearance (CLp = 66.4 ml/min/kg).
CF3
12A (R,S)
12B (S,R)
7.8
28.1
1% at 10 lM
67.2 (90%)
CF3
14A (R,S)
14B (S,R)
528
1922 (57%)
120.5 (47%)
76.2
F
F
11A (R,S)
11B (S,R)
101.1
127.6
3702 (50%)
130.3 (93%)
The more detailed MCR activity profiles of 7A and 14B
(Table 4) showed that both compounds are modest
MC4R agonists with weak binding and functional activ-
ities at other receptors. While 7A showed decent oral
bioavailability with good drug level (AUCN =
1.2 lM h) and fairly low plasma clearance (CLp = 9.12
ml/min/kg), compound 14B, incorporating 2-naphthyl
part, showed only modest oral bioavailability with a rel-
atively high clearance rate (CLp = 37.6 ml/min/kg).
F
a The stereochemistry was assigned based on the analogy of 2B.7
The complete MCR activity profiles of 4A and 12A (Ta-
ble 4) indicate that both compounds are potent and
selective MC4R modulators with very low activation
on all the human melanocortin receptors. Compound

3246
L. Guo et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3242–3247
Table 4. MCR activity profiles and oral bioavailability data of selected
MC4R modulators
References and notes
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P. Curr. Top. Med. Chem. 2007, 7, 1121; (b) Nargund, R.
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Pharm. Des. 2003, 9, 627; Van der (d) Ploeg, L. H. T.;
Howard, A. D.; Nargund, R. P.; Austin, C. P.; Guan, X.;
Drisko, J.; Cashen, D.; Sebhat, I.; Pattchett, A. A.;
Figueroa, D. J.; DiLella, A. G.; Connolly, D. H.;
Weinberg, D. H.; Tan, C. T.; Palyha, O. C.; Pong, S.;
MacNeil, T.; Rosenblum, C.; Vongs, A.; Tang, R.; Yu, H.;
Sailer, A. W.; Fong, T. M.; Huang, C.; Tota, M.; Chang,
R. S.; Sterns, R.; Tamvakopoulos, T.; Christ, G.; Drazen,
D. L.; Spar, B. D.; Nelson, R. J.; Maclntyre, D. E. Proc.
Natl. Acad. Sci. U.S.A. 2002, 99, 11381; (e) Eberle, A. N.
In The melanocortin receptorsMelanocortin Receptors;
Cone, R. D., Ed.; Human Press, 2000; p 3; (f) Huszar,
D.; Lynch, C. A.; Fairchild-Huntress, V.; Dunmore, J. H.;
Fang, Q.; Berkemeier, L. R.; Gu, W.; Kesterson, R. A.;
Boston, B. A.; Cone, R. D.; Smith, F. J.; Campfield, L. A.;
Burn, P.; Lee, F. Cell 1997, 88, 131; (g) Gu, W.; Tu, Z.;
Kleyn, P. W.; Kissebah, A.; Duprat, L.; Lee, J.; Chin, W.;
Maruti, S.; Deng, N.; Fisher, S. L.; Franco, L. S.; Burn,
P.; Yagaloff, K. A.; Nathan, J.; Heymsfield, S.; Albu, J.;
Pi-Sunyer, F. X.; Allison, D. B. Diabetes 1999, 48, 635; (h)
Hinney, A.; Schmidt, A.; Nottebom, K.; Heibult, O.;
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Top. Med. Chem. 2007, 7, 1068; (b) Sebhat, I. K.; Lai, Y.;
Barakat, K.; Ye, Z.; Tang, R.; Kalyani, R. N.; Vongs, A.;
MacNeil, T.; Weinberg, D. H.; Cabello, M. A.; Maroto,
M.; Teran, A.; Fong, T. M.; Van der Ploeg, L. H. T.;
Patchett, A. A.; Nargund, R. P. Bioorg. Med. Chem. Lett.
2007, 17(20), 5720; (c) Ye, Z.; Guo, L.; Barakat, K. J.;
Pollard, P. G.; Palucki, B. L.; Sebhat, I. K.; Bakshi, R. K.;
Tang, R.; Kalyani, R. N.; Vongs, A.; Chen, A. S.; Chen, H.
Y.; Rosenblum, C. I.; MacNeil, T.; Weinberg, D. H.; Peng,
Q.; Tamvakopoulos, C.; Miller, R. R.; Stearns, R. A.;
Cashen, D. E.; Martin, W. J.; Metzger, J. M.; Strack, A.
M.; MacIntyre, D. E.; Van der Ploeg, L. H. T.; Patchett, A.
A.; Wyvratt, M. J.; Nargund, R. P. Bioorg. Med. Chem.
Lett. 2005, 15, 3501; (d) Ujjainwalla, F.; Warner, D.;
Snedden, C.; Grisson, R. D.; Walsh, T. F.; Wyvratt, M. J.;
Kalyani, R. N.; Mac Neil, T.; Tang, R.; Weinberg, D. H.;
Van der Ploeg, L.; Goulet, M. T. Bioorg. Med. Chem. Lett.
2005, 15, 4023; (e) Pan, K.; Scott, M. K.; Lee, D. H. S.;
Fitzpatrick, L. J.; Crooke, J. J.; Rivero, R. A.; Rosenthal,
D. I.; Vaidya, A. H.; Zhao, B.; Reitz, A. B. Bioorg. Med.
Chem. 2003, 11, 185; (f) Sebhat, I. K.; Martin, W. J.; Ye, Z.;
Barakat, K.; Mosley, R. T.; Johnston, D. B. R.; Bakshi, R.;
Palucki, B.; Weinberg, D. H.; MacNeil, T.; Kalyani, R. N.;
Tang, R.; Sterns, R. A.; Miller, R.; Tamvakopoulos, C.;
Strack, A.; McGowan, E.; Cashen, D. E.; Drisko, J. E.;
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Chem. 2002, 45, 4589.
Compound
Receptor
Binding
IC50
(nM)
cAMP
EC50
(nM)
% acti.
at 10
lMa
F%b
6B
hMC1b
hMC3
hMC4
hMC5
rMC4
1339
1492
6.2
905
249.9
2.7
57
92
97
98
106
24.3
129.4
—
372
0.9
7B
hMC1b
hMC3
hMC4
hMC5
rMC4
695
1050
4.0
273.6
97.4
2.1
57
88
100
88
94
24.7
3.8
65.3
1.7
101.6
0.5
8B
hMC1b
hMC3
hMC4
hMC5
rMC4
311.6
2958
11.2
205
—
156.7
4.6
—
91
100
101
—
166.7
—
—
14B
7A
hMC1b
hMC3
hMC4
hMC5
rMC4
6586
12,240
76.2
675
—
—
10
16
47
35
—
15.0
34.3
28.0
30.3
120.5
1233
—
—
hMC1b
hMC3
hMC4
hMC5
rMC4
173,500
2951
4.1
1065
917.5
13.4
455
33
38
33
30
44
280.3
1.5
10.3
12A
4A
hMC1b
hMC3
hMC4
hMC5
rMC4
16,370
3777
7.8
—
—
—
—
—
5
8
1
450
1.0
11
15
hMC1b
hMC3
hMC4
hMC5
rMC4
20,000
4293
3.3
—
—
3
5
—
1
245.4
0.5
1600
—
10
6
a Percentage of cAMP accumulation at 10 lM relative to a–MSH.
b Compounds were dosed in Sprague–Dawley rats as a solution in
EtOH/PEG/saline (1:4:5) at 1 mg/kg, iv (n = 2) and 4 mg/kg, po
(n = 3).
4A exhibits excellent selectivity against MC1bR (>6000-
fold) and MC3R (>1000-fold). Both compounds showed
good PK profiles in rats with good drug level and
low plasma clearance rate (4A, AUCN = 0.70 lM h;
CLp =11.4 ml/min/kg; 12A, AUCN = 1.9 lM h; CLp =
5.6 ml/min/kg).
3. Ujjainwalla, F. Presentation (MEDI-275), 230th ACS
National Meeting, Washington, DC, August 28–September
1, 2005.
4. Ujjainwalla, F et al., unpublished results (in preparation).
5. Intermediate 3B was synthesized in a similar way as
described in Ref. 3 for compound 1.
6. (a) MC4R binding IC50 was defined as the concentration
of compound that can inhibit binding of [125 I]NDP-a-
MSH by 50% from membranes prepared from CHO cells
expressing human MC4R. Agonist potency was deter-
In conclusion, we have described the synthesis, SAR and
pharmacokinetics of a new class of non-peptidyl mela-
nocortin subtype-4 receptor modulators derived from a
tert-butyl pyrrolidine core structure. A number of po-
tent, MC4R selective and orally bioavailable agonists
have been discovered. The in vivo efficacy studies in
terms of lowering food intake and body weight and
showing erectile activity for MC4R agonists will be dis-
cussed in future reports from this laboratory.

L. Guo et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3242–3247
3247
mined in cAMP release assays using CHO cells express-
ing the relevant receptors. MC4R cAMP EC50 was
defined as the inflection point of the cAMP dose–
response curve for any given compounds. Maxi percent-
age activation [% Max] is the percentage of cAMP
accumulation at 10 lM of compound relative to a-MSH;
For details about assay protocols see: (b) Bednarek, M.
A.; MacNeil, T.; Kalyani, R. N.; Tang, R.; Van der
Ploeg, L. H. T.; Weinberg, D. H. J. Med. Chem. 2001,
44, 3665; (c) Bednarek, M. A.; Silva, M. V.; Arison, B.;
MacNeil, T.; Kalyani, R. N.; Huang, R. R. C.; Wein-
berg, D. H. Peptides 1999, 20, 401.
7. The stereochemistry of 1-tert-butyl-4-aryl pyrrolidine-3-
carboxylic acid substructure in 2B is same as in 1 and
defined as shown in Ref. 3. The slow eluting isomers
discussed in this paper were assumed to have the same
stereochemistry as 2B, which is also the slow eluting isomer,
under the same chiral HPLC conditions.