Structure-activity relationships of piperazinebenzylamines as potent and selective agonists of the human melanocortin-4 receptor

Article abstract of DOI:10.1016/j.bmcl.2004.06.059

SAR studies on a series of piperazinebenzenes directed toward the human melanocortin-4 receptor resulted in potent MC4R agonists. Replacement of the triazole moiety of an initial lead 4 by a basic nitrogen baring a lipophilic side-chain increased the binding affinities of these compounds. Analogs bearing an additional hetero-atom in the side-chain possessed good agonist potency. Thus, 11h had a K_i of 11 nM, and 13g exhibited an EC₅₀ of 3.8 nM and a K_i of 6.4 nM.

Full text of DOI:10.1016/j.bmcl.2004.06.059

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4417–4423
Structure–activity relationships of piperazinebenzylamines
as potent and selective agonists of the human
melanocortin-4 receptor
Joseph Pontillo,a Joseph A. Tran,a Melissa Arellano,a Beth A. Fleck,b Rajesh Huntley,b
Dragan Marinkovic,a Marion Lanier,a Jodie Nelson,a Jessica Parker,a John Saunders,a
Fabio C. Tucci,a Wanlong Jiang,a Caroline W. Chen,a Nicole S. White,a
Alan C. Fosterc and Chen Chena,
*
aDepartment of Medicinal Chemistry, Neurocrine Biosciences, Inc., 10555 Science Center Drive, San Diego, CA92121, USA
bDepartment of Pharmacology, Neurocrine Biosciences, Inc., 10555 Science Center Drive, San Diego, CA92121, USA
cDepartment of Neuroscience, Neurocrine Biosciences, Inc., 10555 Science Center Drive, San Diego, CA92121, USA
Received 24 March 2004; revised 18 June 2004; accepted 18 June 2004
Abstract—SAR studies on a series of piperazinebenzenes directed toward the human melanocortin-4 receptor resulted in potent
MC4R agonists. Replacement of the triazole moiety of an initial lead 4 by a basic nitrogen baring a lipophilic side-chain increased
the binding affinities of these compounds. Analogs bearing an additional hetero-atom in the side-chain possessed good agonist
potency. Thus, 11h had a Ki of 11nM, and 13g exhibited an EC50 of 3.8nM and a Ki of 6.4nM.
Ó 2004 Published by Elsevier Ltd.
1. Introduction
energy homeostasis, selective MC4R agonists and antag-
onists may be useful in human diseases such as obesity
and cachexia.3 Small molecule MC4R agonists such as
1–6 (Fig. 1) have been disclosed from several laborato-
ries.5–10 Compound 1, which has a (R)-Tic-(R)-(4-
Cl)Phe-piperidine, is the first potent small molecule
MC4R agonist reported (Fig. 1). It is designed and op-
timized based on the HFRW core of the melanotropin
peptides by mimicking the Arg-Trp residues with a priv-
ileged structure for GPCRs.4 1 is also highly selective
over other subtypes of the melanocortin receptors
MC1R, MC3R, and MC5R.5 Piperidine di-peptide com-
pounds represented by 2 are reported as potent MC1R
agonists (EC50 =0.19nM, 98% intrinsic activity), how-
ever, 2 also possesses high agonist potency at the
MC4R (EC50 =2.9nM, 95% intrinsic activity).6 This ser-
ies of compounds have been further derived by replace-
ment of the tetrahydroisoqinoline of 1 with a b-alanine
moiety (compound 3, EC50 =40nM, 98% intrinsic activ-
ity).7 An early report from our laboratory has disclosed
a series of piperazinebenzenes bearing a sulfonamide or
a triazole functionality as MC4R-selective agonists
(compound 4, EC50 =80nM, 100% intrinsic activity).8
Very recently, two independent publications, which
Five subtypes of melanocortin receptors, MC1-5R, have
been identified and cloned, and they belong to the Class
A G-protein-coupled receptor (GPCR) superfamily.1
The MC1R regulates skin pigmentation and the immune
system. The MC2R (ACTH receptor) controls steroid
production. The MC3R and MC4R are involved in
the regulation of central sexual behavior and the control
of feeding behavior, and the MC5R has a role for
regulating exocrine gland secretion.2 The melanocortic
peptides derived from a single prohormore, proopiome-
lanocortin (POMC), are the natural ligands for the mel-
anocortin receptors. They consist of the melanotropins
a-MSH, b-MSH, and c-MSH, and the adrenocortico-
tropin ACTH. All these peptides have a His-Phe-Arg-
Trp (HFRW) motif, which is crucial for activation of
the melanocortin receptors. Because of the importance
of the MC4R in feeding behavior, metabolism and
Keywords: Melanocortin; MC4R; Agonist.
*
Corresponding author. Tel.: +1-858-658-7630; fax: +1-858-658-
7619; e-mail: cchen@neurocrine.com
0960-894X/$ - see front matter Ó 2004 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2004.06.059

4418
J. Pontillo et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4417–4423
Cl
Cl
Cl
N
N
O
N
N
N
N
N
NH
O
O
O
N
N
N
N
H
N
H
N
H
N
O
HN
O
HN
O
2
3
O
1
N
O
N
Cl
Cl
Cl
N
N
N
N
N
O
O
N
O
N
N
N
N
H
N
H
N
H
O
NH
O
NH
O
6
4
5
Figure 1. Small molecule MC4 agonists.
reveal the triazole of 4 can be replaced by an amide
(compound 5, EC50 =15nM, 100% intrinsic activity)9
or a dimethylamino group (compound 6, EC50 =7.0nM,
intrinsic activity >80%),10 further elaborate the struc-
ture–activity relationships of this piperazine series as
MC4R agonists. Here we report our continued efforts
on the optimization of these piperazinebenzenes toward
potent and selective agonists of the human MC4 recep-
tor.
of 9a with a various primary or secondary amines, fol-
lowed by deprotection of the Boc-group, resulted in
the desired products 11 in 60–80% yields.11
The synthesis of the piperazine-a-methylbenzylamine
compounds 13a–i started from 20-fluoroacetophenone
7b as showed in Scheme 2. Thus, condensation of 7b
with mono-Boc-protected piperazine gave the corre-
sponding piperazineacetophenone 8b similarly as de-
scribed above. Next we used a sequential peptide
coupling reaction using the conditions with minimal
possibility of racemerization of the two R-amino acids.
After deprotection of 8b, the resulting free amine was
coupled with (R)-N-Boc-(4-Cl)Phe-OH in the presence
of EDC and HOBt in DCM to afford the amide 12,
which was deprotected with TFA and subjected to a sec-
ond coupling reaction with (R)-N-Boc-Tic-OH to give
the dipeptidic derivative 9b. Reductive amination of
9b were accomplished with various amines using a
standard protocol, followed by deprotecting the Boc-
moiety of the Tic-group, to give the desired products
2. Chemistry
The synthesis of the piperazinebenzylamines 11a–u
started from 2-fluorobenzaldehyde 7a (Scheme 1). Thus,
condensation of 7a with mono-Boc-protected piperazine
in DMF at 120–160°C gave the piperazinebenzaldehyde
8a. After deprotection of 8a, a coupling reaction of
the free amine with (R)-N-Boc-Tic-(R)-(4-Cl)Phe-OH
(10)10 in the presence of HOBt and EDC in DCM af-
forded the dipeptidic derivative 9a. Reductive amination
Cl
CHO
N
CHO
b,c
CHO
F
a
HN
+
N
O
NBoc
NBoc
N
N
H
O
N
7a
8a
Boc
9a
R1
R2
Cl
Cl
N
N
d
O
O
HO
N
N
H
Boc
N
H
O
N
O
HN
11
10
Scheme 1. Reagents and conditions: (a) DMF/K2CO3/D, 80%; (b) 50% TFA/CH2Cl2, quant.; (c) 10/EDC/HOBt/Et3N, 90%; (d) R1R2NH/Na-
BH(OAc)3; then TFA/CH2Cl2, 60–80%.

J. Pontillo et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4417–4423
4419
Cl
O
N
O
N
O
F
b
a
HN
+
NBoc
NBoc
N
NHBoc
O
8b
7b
12
R1
R2
Cl
Cl
N
N
O
N
c
d
O
O
N
N
O
N
H
N
H
O
HN
N
Boc
13
9b
Scheme 2. Reagents and conditions: (a) DMF/D, 85%; (b) TFA/CH2Cl2, then (R)-Boc-(4-Cl)Phe-OH/EDC/HOBt/Et3N, 90%; (c) TFA/CH2Cl2, then
(R)-Boc-Tic-OH/EDC/HOBt/Et3N, 85%; (d) R1R2NH/NaBH(OAc)3, then 50% TFA/CH2Cl2, 25–90%.
13 in 25–80% yields.11 These compounds were presuma-
bly mixtures of diastereoisomers although we were not
able to see separation in the purity characterization by
HPLC on a C-18 column.
proved agonist potency of these analogs. For example,
11j with a 3-hydroxypropyl side-chain had a Ki of
120nM and an EC50 of 36nM. While the binding affinity
of this compound was only 3-fold better than that of
11a, its agonist potency improved about 8-fold.
3. Results and discussions
The successful improvement in both binding affinity and
agonist potency over the parent primary amine 11a by
introduction of a side-chain at the basic nitrogen
prompted us to examine N,N-dialkylated derivatives
(11m–u, Table 1). The diethylamino derivative 11m
had a Ki value of 52nM, which was 7-fold better than
11a, and an EC50 of 240nM.14 Similarly, other tertiary
amine analogs 11n–r had Ki values ranged from 35nM
(11r) to 84nM (11p). Once again, compounds bearing
a polar group at the side-chain (11n, 11o, and 11q)
exhibited improved agonist potency. For example, 11o
had a Ki of 49nM and an EC50 of 51nM. Among the
cyclic amines (11s–u), 11s possessed a Ki value of
24nM, however, its EC50 value was only 146nM. In
comparison, the cis-2,5-dimethylpiperazine 11u exhib-
ited similar binding affinity (Ki =19nM) to 11s, however,
it had an EC50 value of 18nM, which was about 8-fold
better than 11s. Although these tertiary amines 11m–u
had in general better binding affinities than 11a, the
agonist potency (EC50 values) of these compounds
exhibited little improvement other than compounds with
a side-chain bearing a hetero-atom.
The competition binding experiments were performed
using HEK293 cells stably transfected with the human
melanocortin-4 receptor as described before,12 using
[
125I]-NDP-MSH as the radiolabeled ligand in the bind-
ing assay. The agonist activities of these compounds
were tested for their stimulation of cAMP in CHO cells
stably expressed the human melanocortin-4 receptor and
a-MSH was used as a standard.13 The Ki and EC50 val-
ues reported in Tables 1 and 2 are the average of at least
three independent measurements.
The primary benzylamine (11a) showed modest binding
affinity (Ki =380nM) and agonist potency (EC50
=
274nM) at the human MC4R, which were comparable
to that of the triazole 4 (Ki =270nM, EC50 =110nM).
Incorporation of a small aliphatic side chain such as cy-
clopropyl and cyclopentyl group on the benzylamine
nitrogen of 11a resulted in a 4–6-fold increase in binding
affinity (11b and 11c). The 2-thiophenylethyl analog 11h
had a Ki of 11nM, which was a 35-fold increase over
11a. These results suggest that an additional aromatic
ring is favored for high receptor binding. For fluorine-
substituted phenethyl side-chain (11e–g), the 2- or
3-position seemed to be favored and the binding affini-
ties of 11e and 11f were over 5-fold better than that of
the corresponding 4-fluoro-analog 11g. However, while
a small lipophlic side-chain at the basic nitrogen of
11a enhanced the binding affinity, it had little effect on
the agonist potency of these compounds (EC50 164–
910nM). Furthermore, the intrinsic activities (maximal
levels of cAMP stimulation) of 11e and 11f (47% and
37%, respectively) were somewhat reduced.
To further optimize this series of compounds toward
better binding and agonist potency at the MC4R, we
hypothesized that, since a relatively long and flexible
alkylamino group, such as 2-thiophenylethyl, was used
in this series, restriction of the free rotation of this
side-chain could improve affinity if favorable low-energy
conformers could be found. One simple way to achieve
this objective is to introduce a methyl group at the benz-
ylic position, and therefore, to reduce the flexibility of
the side-chain. Among these a-methylbenzylamines,
the N-cyclopropyl derivative 13a had a Ki value of
23nM, about 4-fold better than the corresponding benz-
ylamino analog 11b (Ki =93nM), and its EC50 value
(174nM) was, however, only slightly better than that
of 11b. The a-methyl compound 13b was 6-fold better
Incorporation of a side-chain bearing an additional po-
lar group at the basic nitrogen of 11a had no significant
impact on the binding affinity for 11i–l, however, it im-

4420
J. Pontillo et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4417–4423
Table 1. SAR of N-alkylpiperazinebenzylamines at the hMC4 receptor
1
2
R
R
Cl
N
N
O
N
N
H
O
NH
11
Compd
R1NR2
hMC4a Ki (nM)b
cAMP Stimulationc
EC50 (nM)d
IA (%)e
100a
1
33 11
270 65
380 34
90 26
5.7a
N
N
N
4
110 61a
274 122
253 82
100a
11a
11b
NH2
74 19
85 22
HN
HN
11c
11d
66 20
53 16
164 190
274 205
89 11
91 24
O
HN
HN
11e
27
14
9
3
910 878
81 23
47 10
F
HN
HN
F
11f
460 160
487 125
11g
81 44
37 8
F
HN
HN
11h
11i
11j
11
8
357 312
91 28
105 22
S
127 26
120 35
69 7
NH2
HN
HN
OH
36 19
83 25
OH
11k
11l
490 125
102 11
90 31
77 13
75 20
O
NH
208 94
240 17
HN
11m
11n
52 18
55 10
55 8
N
OH
40
9
78 16
82 14
N
O
11o
11p
49 14
84 23
51 25
N
N
632 374
71 13
70 12
57 16
N
N
11q
11r
77 28
35 18
94 30
N
190 100
N

J. Pontillo et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4417–4423
4421
Table 1 (continued)
Compd
R1NR2
hMC4a Ki (nM)b
cAMP Stimulationc
EC50 (nM)d
IA (%)e
87 22
24
3
146 50
11s
11t
N
N
145 67
300 34
61 2
N
N
N
11u
19
4
18
3
64 16
NH
a Human melanocortin-4 receptor stably expressed in HEK 293 cells.
b
[
125I]-NDP-MSH used as radiolabeled ligand.
c Human melanocortin-4 receptor stably expressed in CHO cells.
d EC50 determined by compound concentration at 50% maximum cAMP release.
e Intrinsic activity, percentage of a-MSH-stimulated cAMP level.
Table 2. SAR of N-alkylpiperazine-a-methylbenzylamines at the hMC4 receptor
R2
R1
Cl
N
N
O
N
N
H
O
NH
13
hMC4a Ki (nM)b
Compd
R1NR2
cAMP Stimulationc
EC50 (nM)d
IA (%)e
13a
13b
23
11
3
2
174 80
72 28
75
1
HN
HN
76 13
80 20
13c
13d
13e
13f
13g
13h
12
33
24
13
2
8
6
3
138 76
O
HN
HN
2060 1550
577 111
50 23
52 17
F
HN
HN
S
R
NH
12
2
66 7
6.4 4.4
25 13
3.8 2.6f
34 15
84 15f
79 13
NH
H2N
NH2
HN
H2N
13i
13
3
15
6
93 30
HN
a Human melanocortin-4 receptor stably expressed in HEK 293 cells.
b
[
125I]-NDP-MSH as the radiolabeled ligand.
c Human melanocortin-4 receptor stably expressed in CHO cells.
d EC50 determined by compound concentration at 50% maximum cAMP release.
e Intrinsic activity, percentage of a-MSH-stimulated cAMP level.
f EC50 value of this compound at the hMC4R in HEK 293 cells was 4.7nM with IA of 100%.

4422
J. Pontillo et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4417–4423
Table 3. Binding affinities at the human MC subtypesa
the current series had improved binding affinity, but
not agonistic potency. Based on these facts and receptor
modeling,18 we speculated the Asp-122 was the acidic
residue, which interacts with the basic benzylamine.
Compound
Ki (nM)b
hMC1Rc
hMC3Rc
hMC4R
hMC5R
1
1500
2300
(24%)
(33%)
2300
1800
3600
2000
33
270
380
27
860
1200
2300
260
4
(36%)
(27%)
(42%)
6000
4. Conclusion
11a
11e
11h
13f
13g
11
13
240
590
A series of piperazinebenzylamines bearing the (R)-Tic-
(R)-(4-Cl)Phe dipeptide was synthesized and tested as
melanocortin-4 receptor ligands. Replacement of the
triazole of 4 by a basic amine, in combination with a
small lilophilic side-chain, improved the binding affinity
of this series of compounds. Structure–activity relation-
ship studies around the benzylamine revealed that an
additional basic nitrogen at the side-chain increased
agonistic potency. Thus, 11h exhibited a Ki value of
11nM, and 13g had an EC50 of 3.8nM (Ki =6.4nM).
In addition, these compounds displayed high MC4R-
selectivity.
2100
1500
6.4
270
a Human melanocortin receptors stably expressed in HEK 293 cells.
b
[
125I]-NDP-MSH as the radiolabled ligand.
c Data in parenthesis are percentage of inhibition at 10lM concen-
tration.
than 11b in binding affinity, but only 2-fold in agonist
potency (Table 2). This approach did not work well
for 11h (Ki =11nM, EC50 =357nM), and the a-methyl-
ated analog 13e had a Ki value of 24nM and an EC50
value of 577nM. However, incorporation of an addi-
tional basic nitrogen at the side-chain improved agonist
potency of these analogs. Thus, 13f with a 3R-pyrroli-
dineamino group had a Ki of 13nM and an EC50 of
12nM. The agonist potency 13f was much better than
13b (Ki =11nM and EC50 =72nM), which had a cyclo-
pentyl group instead. The most potent MC4R agonist
of this series was 13g, which had an EC50 of 3.8nM
(Ki =6.4nM). The other two diamine analogs 13h
(EC50 =34nM) and 13i (EC50 =15nM) also exhibited
good binding and agonist potency.
References and notes
1. Karnik, S. S.; Gogonea, C.; Patil, S.; Saad, Y.; Takezako,
T. Trends Endocrinol. Metab. 2003, 14, 431.
2. (a) Wikberg, J. E. S.; Muceniece, R.; Mandrika, I.; Prusis,
P.; Post, C.; Skottner, A. Pharmacol. Res. 2000, 42, 393;
(b) Wikberg, J. E. S. Eur. J. Pharmacol. 1999, 375, 295; (c)
Wikberg, J. E. S. Exp. Opin. Ther. Pat. 2001, 11, 61.
3. Goodfellow, V.; Saunders, J. Curr. Topic Med. Chem.
2003, 3, 855.
4. Patchett, A. A.; Nargund, R. P. Ann. Rep. Med. Chem.
2000, 35, 289.
Selected compounds from this series were also tested
and found to be selective in binding to the MC4R over
the other melanocortin receptors. For example, 11h
showed Ki values of 6000, 1800, and 240nM, respec-
tively, at the human melanocortin-1, -3, and -5 recep-
tors, which were over 20-fold selective (Table 3). The
potent MC4R agonist 13g possessed Ki values of 1500,
2000, 6.4, and 270nM, respectively, at the MC1R,
MC3R, MC4R, and MC5R. Thus, 13g exhibited more
than 300-fold selectivity in binding over the MC3R,
which is the relevant receptor in control of feeding be-
havior. 11e and 13f were also proved to be selective
and the data are summarized in Table 3.
5. 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.;
Stearns, R. A.; Miller, R. R.; Tamvakopoulos, C.; Strack,
A. M.; McGowan, E.; Cashen, D. E.; Drisko, J. E.; Hom,
G. J.; Howard, A. D.; MacIntyre, D. E.; van der Ploeg, L.
H. T.; Patchett, A. A.; Nargund, R. P. J. Med. Chem.
2002, 45, 4589.
6. Herpin, T. F.; Yu, G.; Carlson, K. E.; Morton, G. C.; Wu,
X.; Kang, L.; Tuerdi, H.; Khanna, A.; Tokarski, J. S.;
Lawrence, R. M.; Macor, J. E. J. Med. Chem. 2003, 46,
1123.
7. Ruel, R.; Herpin, T. F.; Iben, L.; Luo, G.; Martel, A.;
Mason, H.; Mattson, G.; Poirier, B.; Ruediger, E. H.;
Shi, D.; Thibault, C.; Yu, G.; Zimanyi, I. A.; Poindexter,
G. S.; Macor, J. E. Bioorg. Med. Chem. Lett. 2003, 13,
4341.
8. Dyck, B.; Parker, J.; Philips, T.; Carter, L.; Murphy, B.;
Summers, R.; Hermann, J.; Baker, T.; Cismowski, M.;
Saunders, J.; Goodfellow, V. Bioorg. Med. Chem. Lett.
2003, 13, 3793.
The increase of binding affinity of this series of com-
pounds over the triazole 4 could be contributed to a
strong charge–charge interaction between the basic benz-
ylamine of 11 and 13 (the calculated pKa value for 11h
was 9.0),15 instead of the weakly basic triazole moiety
of 4 (calculated pKa of 2.7),15 and an acidic residue of
the MC4 receptor. Mutagenesis studies16 of the MC4 re-
ceptor have demonstrated that two aspartic residues,
Asp-122 and Asp-126, are important for peptide ligand
binding and activation. Recently receptor modeling
based on the rhodopsin template17 shows these two res-
idues are located at the top part of the putative binding
pocket. The fact that the Asp122Ala MC4 mutant can
still be activated by peptide and small molecule agonists
such as 113 might suggest that the Asp-122 residue is not
critical for receptor function. In addition, many benzyl-
amine analogs with a small lipophilic side-chain from
9. Xi, N.; Hale, C.; Kelly, M. G.; Norman, M. H.; Stec, M.;
Xu, S.; Baumgartner, J. W.; Fotsch, C. Bioorg. Med.
Chem. Lett. 2004, 14, 377.
10. Richardson, T. I.; Ornstein, P. L.; Briner, K.; Fisher, M.
J.; Backer, R. T.; Biggers, C. K.; Clay, M. P.; Emmerson,
P. J.; Hertel, L. W.; Hsiung, H. M.; Husain, S.; Kahl, S.
D.; Lee, J. A.; Lindstrom, T. D.; Martinelli, M. J.; Mayer,
J. P.; Mullaney, J. T.; OÕBrien, T. P.; Pawlak, J. M.;
Revell, K. D.; Shah, J.; Zgombick, J. M.; Herr, R. J.;
Melekhov, A.; Sampson, P. B.; King, C.-H. R. J. Med.
Chem. 2004, 47, 744.
11. For details of the synthesis, see: Pontillo, J.; Marinkovic,
D.; Lanier, M.; Tran, J. A.; Arellano, M.; Parker, J.;

J. Pontillo et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4417–4423
4423
Nelson, J.; Chen, C.; Chen, C. W.; Jiang, W.; White, N.;
Tucci, F. WO 03094918, 2003.
13. Nickolls, S. A.; Cismowski, M. I.; Wang, X.; Wolff, M.;
Conlon, P. J.; Maki, R. A. J. Pharmacol. Exp. Ther. 2003,
304, 1217.
14. A Ki value of 34nM and an EC50 value of 14nM (IA
>80%) were reported for compound 11m. For detailed
assay conditions, see Ref. 10.
15. ACD/pKa, Advanced Chemistry Development Inc., 90
Adelaide Street West, Toronto, Ontario, M5H 3V9,
Canada.
12. MC4 receptor binding assay. Crude membranes were
prepared by differential centrifugation from HEK293
cells transfected with human MC4 receptor. These mem-
branes were incunated for 90min with a fixed concentra-
tion of [125I]-NDP-MSH and varying concentrations of
competing ligand. Reactions were terminated by rapid
vacuum filtration using
a
Packard 96-well cell
harvester over PEI (1%) soaked GF/C filter plates (Pack-
ard). Filter plates were then washed with 600mLPBS
(0.01% Triton-X100). Microscint scintillation fluid
(Packard) was added to each well before monitoring
16. (a) Haskell-Luevano, C.; Cone, R. D.; Monck, E. K.;
Wan, Y.-P. Biochemistry 2001, 40, 6164; (b) Yang, Y.;
Fong, T. M.; Dickison, C. J.; Mao, C.; Li, J.-Y.; Tota, M.
R.; Mosley, R.; Van der Ploeg, L. H. T.; Gantz, I.
Biochemistry 2000, 39, 14900.
[
125I]-NDP-MSH in a TopCount-NXT (Packard) micro-
plate scintillation counter. Binding data were analyzed by
GraphPad Prism software program. See Ref. 13 for
details.
17. Yang, X.; Wang, Z.; Dong, W.; Ling, L.; Yang, H.; Chen,
R. J. Protein Chem. 2003, 22, 335.
18. Unpublished results.