Structure-activity relationship of linear peptide Bu-His-DPhe-Arg-Trp-Gly-NH2 at the human melanocortin-1 and -4 receptors: Histidine substitution

Article abstract of DOI:10.1016/S0960-894X(02)00830-2

Systematic substitution of His⁶ residue using non-selective hMC4R pentapeptide agonist (Bu-His⁶-DPhe⁷-Arg⁸-Trp⁹-Gly ¹⁰-NH₂) as the template led to the identification of Bu-Atc

Full text of DOI:10.1016/S0960-894X(02)00830-2

Bioorganic & Medicinal Chemistry Letters 13 (2003) 133–137
Structure–Activity Relationship of Linear Peptide Bu-His-DPhe-
Arg-Trp-Gly-NH2 at the Human Melanocortin-1 and
-4 Receptors: Histidine Substitution
Adrian Wai-Hing Cheung,* Waleed Danho, Joseph Swistok, Lida Qi, Grazyna Kurylko,
Karen Rowan, Mitch Yeon, Lucia Franco, Xin-Jie Chu, Li Chen and Keith Yagaloff
Roche Research Center, Hoffmann-La Roche Inc., Nutley, NJ 07110, USA
Received 10 July 2002; accepted 16 September 2002
Abstract—Systematic substitution of His6 residue using non-selective hMC4R pentapeptide agonist (Bu-His6-DPhe7-Arg8-Trp9-
Gly10-NH2) as the template led to the identification of Bu-Atc6(2-aminotetraline-2-carboxylic acid)-DPhe7-Arg8-Trp9-Gly10-NH2
which showed moderate selectivity towards hMC4R over hMC1R. Further SAR studies resulted in the discovery of Penta-5-
BrAtc6-DPhe7-Arg8-Trp9-Gly10-NH2 and Penta-5-Me2NAtc6-DPhe7-Arg8-Trp9-Gly10-NH2 which are potent hMC4R agonists and
are inactive in hMC1R, hMC3R and hMC5R agonist assays.
# 2002 Published by Elsevier Science Ltd.
In the last decade, five human melanocortin receptor
subtypes (hMC1R–hMC5R) have been cloned and
characterized.1 The melanocortin receptors belong to
the superfamily of G-protein coupled receptors
(GPCRs) mediating a wide range of physiological func-
tions: pigmentation (MC1R), glucocorticoid production
(MC2R), food intake and energy expenditure (MC3R
and MC4R) as well as exocrine gland function
(MC5R).1 Our laboratories are interested in the identi-
fication of potent and selective human melanocortin-4
receptor (hMC4R) agonists for the treatment of
obesity.2
reported our results in replacing Arg8 residue of penta-
peptide 1;3 this report summarizes our initial effort in
replacing the His6 residue of peptide 1.
All new peptides and NDP-MSH were synthesized on
solid phase from suitably protected amino acids using
standard Fmoc methodology.4 The crude peptides were
purified to homogeneity using reversed-phase HPLC
and characterized by fast atom bombardment mass
spectroscopy. Peptide a-MSH; amino acids Fmoc-
His(Trt)-OH, Fmoc-Ala-OH and racemic Fmoc-Atc-
OH were purchased from commercial sources.
As previously reported, our lead pentapeptide 1 (Bu-
His6-DPhe7-Arg8-Trp9-Gly10-NH2, a-MSH numbering)
is a potent hMC4R agonist (EC50=20 nM), selective
against hMC3R (no agonist activity at 50 mM) and
hMC5R (no agonist activity at 50 mM) but not selective
against hMC1R (EC50=10 nM).3 In an extensive struc-
ture–activity relationship (SAR) study of pentapeptide
1, we systematically replaced each of the five amino
acids of peptide 1 by other coding or non-coding amino
acids in an effort to dial out hMC1R activity and to
maintain or improve hMC4R activity. We previously
Synthesis of racemic Fmoc-protected substituted Atc
(2-aminotetraline-2-carboxylic acid) amino acids 5. Vari-
ous substituted b-tetralones 2 were converted into the
corresponding racemic hydantoins 3 by Bucherer–Bergs
reaction (Scheme 1).5 Basic hydrolysis of hydantoins 3
gave racemic amino acids 4, which were then Fmoc
protected under standard conditions to give amino acids
5.
Synthesis of substituted ꢀ-tetralones. For 8-(7) and 6-(9)
substituted b-tetralones, Burckhalter–Campbell syn-
thesis starting from the corresponding ortho-(6) or para-
(8) substituted phenylacetic acids is preferred (Scheme
2).6 For R=bromine and chlorine, Burckhalter–Camp-
bell reaction on meta-substituted (10) phenylacetic acid
*Corresponding author. Fax: +1-973-235-7239; e-mail: adrian.
cheung@roche.com
0960-894X/03/$ - see front matter # 2002 Published by Elsevier Science Ltd.
PII: S0960-894X(02)00830-2

134
A. W.-H. Cheung et al. / Bioorg. Med. Chem. Lett. 13 (2003) 133–137
values reported in Table 4 are the average of at least two
separate experiments.
As shown in Table 1, the lead pentapeptide 1 (Bu-His-
DPhe-Arg-Trp-Gly-NH2) is a potent hMC4R agonist
(EC50=20 nM) but is not selective against hMC1R
(EC50=10 nM). For comparison purpose, known linear
peptide agonist NDP-MSH12 was determined in our
assays to have EC50 values of 0.5 nM (hMC1R) and 1
nM (hMC4R) while a-MSH showed EC50 values of 0.8
nM (hMC1R) and 25 nM (hMC4R). When His6 in
peptide 1 was replaced with Ala, the resulting peptide 19
showed a 9-fold drop (the standard error in our assays
is about 2-fold) in potency at hMC4R and a 23-fold
drop in potency at hMC1R, compared with peptide 1.
As reported by Yang et al.,13 the same His6 to Ala sub-
stitution using linear peptide NDP-MSH as the tem-
plate resulted in a 4-fold drop in agonist potency at
hMC4R.
Scheme 1. (a) KCN, (NH4)2CO3, ethanol, H2O, 80 ꢀC, 85–100% yield;
(b) Ba(OH)2.H2O, H2O, 120 ꢀC, 75–95% yield; (c) Fmoc-OSu,
CH3CN, H2O, Et3N, rt or Fmoc-Cl, 10% Na2CO3, dioxane, rt, 70–
95% yield.
gave a separable mixture of 5-(11) and 7-(12) substituted
b-tetralones.7
As the evolving structure–activity relationship (vide
infra) indicated that 5-substituted Atc gave more
hMC4R selective peptides, two regioselective protocols
of preparing 5-substituted b-tetralones (11) were
employed. For b-tetralones with chlorine, bromine,
methyl, ethyl and isopropyl substituents at the 5-posi-
tion (Scheme 3), the key step in the synthesis involved a
rhodium-catalyzed intramolecular Buchner reaction of
aryl diazo ketones (14),8 which in turn were prepared
from the corresponding ortho-substituted phenyl pro-
panoic acid (13).9
Encouraged by the modest potency of peptide 19, doz-
ens of analogues of the general structure Bu-X-DPhe7-
Arg8-Trp9-Gly10-NH2, in which X is a coding or non-
coding amino acid,14 were prepared and tested in both
hMC1R and hMC4R agonist assays. The great majority
of these His-substituted pentapeptides suffered a modest
to substantial drop in hMC4R efficacy (data not
shown). In addition, none of the above His-substituted
pentapeptides, except the ones containing Atc (2-ami-
notetraline-2-carboxylic acid), showed any significant
selectivity towards hMC4R over hMC1R (data not
shown). When racemic Atc (5, R¼H, Scheme 1) was
used in place of His6, two separable diastereomers 20
and 21 were obtained.15 Peptides 20 and 21 suffered a
147- and 15-fold drop in hMC4R agonist potency,
compared with peptide 1; more importantly however,
peptide 21 showed about 15-fold hMC4R selectivity
over hMC1R. Peptides 22 and 23, made by extending
the N-cap of peptides 20 and 21 from n-butanoyl (Bu-)
to n-pentanoyl (Penta-), showed slight improvement in
agonist potency at both hMC1R and hMC4R, com-
pared with peptides 20 and 21. Individual preparation
of peptides 22 and 23 using optically pure (l)- and
For b-tetralones with methoxy (15), ethoxy (16), iso-
propoxy (17) and dimethylamino (18) substituents at the
5-position, the appropriate 1,6-disubstituted naphtha-
lene was reduced using the method of Cornforth
(Scheme 4).10
Agonist assays were performed using HEK293 cells
transfected with hMC1R–hMC5R as reported in detail
elsewhere.4,11 The EC50 values reported in Tables 1–3
are the average of at least two separate experiments.
Binding assays were performed using radiolabeled
NDP-MSH as reported in detail elsewhere.11 The IC50
Scheme 2. (a) (COCl)2, DMF, CH2Cl2, 0 ꢀC to rt; (b) AlCl3, ethene
gas, 0 ꢀC.
Scheme 4. (a) Ref 10(a); (b) K2CO3, EtI, DMF, 35 ꢀC, 65% yield; (c)
Na, ethanol, reflux; H2O, ethanol, p-TSA, reflux, 30–67% yield; (d)
Cs2CO3, BrCH(CH3)2, DMF, 40 ꢀC, 86% yield; (e) K2CO3, MeI, ace-
tone, reflux, 94% yield.
Scheme 3. (a) SOCl2, toluene, reflux or (COCl)2, DMF, CH2Cl2,
0 ꢀC to rt; (b) CH2N2, ether, 0 ꢀC to rt, 60–82% yield for two steps;
(c) Rh(OAc)2, CH2Cl2, reflux, 76–100% yield.

A. W.-H. Cheung et al. / Bioorg. Med. Chem. Lett. 13 (2003) 133–137
135
Table 1. Agonist activity of the His6 modified pentapeptides at the human melanocortin receptors
Peptide
Amino acid sequence
hMC4R
EC50 (nM)a
hMC1R
EC50 (nM)a
a-MSH
Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-
Gly-Lys-Pro-Val-NH2
25
1
0.8
NDP-MSH
Ac-Ser-Tyr-Ser-Nle-Glu-His-DPhe-Arg-
Trp-Gly-Lys-Pro-Val-NH2
0.5
b
1
19
20
Bu-His-DPhe-Arg-Trp-Gly-NH2
20
180
2940
10
230
5640
Bu-Ala-DPhe-Arg-Trp-Gly-NH2
Bu-Atc-DPhe-Arg-Trp-Gly-NH2
(1st isomer)c
21
22
23
24
25
26
27
28
29
30
Bu-Atc-DPhe-Arg-Trp-Gly-NH2
(2nd isomer)c
Penta-(d)Atc-DPhe-Arg-Trp-Gly-NH2
(1st isomer)
Penta-(l)Atc-DPhe-Arg-Trp-Gly-NH2
(2nd isomer)
Penta-5-BrAtc-DPhe-Arg-Trp-Gly-NH2
(1st isomer)
Penta-5-BrAtc-DPhe-Arg-Trp-Gly-NH2
(2nd isomer)
Penta-6-BrAtc-DPhe-Arg-Trp-Gly-NH2
(1st isomer)
Penta-6-BrAtc-DPhe-Arg-Trp-Gly-NH2
(2nd isomer)
Penta-7-BrAtc-DPhe-Arg-Trp-Gly-NH2
(mixture)d
290
1000
4380
1950
45
830
33
75% @ 50 mMe
50% @ 50 mMe
4630
35
50% @ 50 mMe
60% @ 50 mMe
40% @ 50 mMe
0% @ 50 mMe
0% @ 50 mMe
3380
0% @ 50 mMe
1710
Penta-8-BrAtc-DPhe-Arg-Trp-Gly-NH2
(1st isomer)
Penta-8-BrAtc-DPhe-Arg-Trp-Gly-NH2
(2nd isomer)
2910
aConcentration of peptide at 50% maximum cAMP accumulation or the % of cAMP accumulation (relative to NDP-MSH) observed at the highest
peptide concentration tested.
bBu stands for CH3CH2CH2C(¼O) and Penta stands for CH3CH2CH2CH2C(¼O).
c1st isomer and 2nd isomer refer to the order in which the two diastereomers eluted under our HPLC conditions.15
dTested as a 1:1 mixture of diastereomers.
eNot tested for antagonist activities.
Table 2. Agonist activity of pentapeptides containing various substituted Atc amino acids at the human melanocortin receptors
Peptide
Amino acid sequence
hMC4R
EC50 (nM)a
hMC1R
EC50 (nM)a
24
25
31
32
33
34
35
36
37
38
39
40
Penta-5-BrAtc-DPhe-Arg-Trp-Gly-NH2
(1st isomer)b,c
Penta-5-BrAtc-DPhe-Arg-Trp-Gly-NH2
(2nd isomer)c
Penta-5-ClAtc-DPhe-Arg-Trp-Gly-NH2
(1st isomer)
Penta-5-ClAtc-DPhe-Arg-Trp-Gly-NH2
(2nd isomer)
Penta-5-MeOAtc-DPhe-Arg-Trp-Gly-NH2
(mixture)d
Penta-5-EtOAtc-DPhe-Arg-Trp-Gly-NH2
(mixture)
Penta-5-iPrOAtc-DPhe-Arg-Trp-Gly-NH2
(mixture)
Penta-5-MeAtc-DPhe-Arg-Trp-Gly-NH2
(mixture)
Penta-5-EtAtc-DPhe-Arg-Trp-Gly-NH2
(mixture)
Penta-5-iPrAtc-DPhe-Arg-Trp-Gly-NH2
(mixture)
Penta-5-Me2NAtc-DPhe-Arg-Trp-Gly-NH2
(1st isomer)
Penta-5-Me2NAtc-DPhe-Arg-Trp-Gly-NH2
(2nd isomer)
33
35
75% @ 50 mMe
50% @ 50 mMe
600
130
90
1400
890
1480
990
120
130
35
680
1220
195
24
13
295
16
1850
940
2
aConcentration of peptide at 50% maximum cAMP accumulation or the % of cAMP accumulation (relative to NDP-MSH) observed at the highest
peptide concentration tested.
bPenta stands for CH3CH2CH2CH2C(¼O).
c1st isomer and 2nd isomer refer to the order in which the two diastereomers eluted under our HPLC conditions.15
dTested as a 1:1 mixture of diastereomers.
eNot tested for antagonist activities.

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A. W.-H. Cheung et al. / Bioorg. Med. Chem. Lett. 13 (2003) 133–137
(d)-Atc5 unambiguously confirmed that peptide 22 con-
tains (d)-Atc and peptide 23 contains (l)-Atc.
5-substituted Atc could yield more potent and/or more
selective hMC4R agonists. One of the more interesting
peptides discovered from this initial optimization
study is peptide 40, which is as potent as NDP-MSH
towards hMC4R (2 nM vs 1 nM, within the 2-fold
experimental error), >450-fold selective against
hMC1R and inactive in hMC3R and hMC5R agonist
assays (vide infra).
Peptide 23 is similar to a-MSH in hMC4R agonist
potency but it is significantly more selective towards
hMC4R compared with a-MSH. To further improve
the potency and selectivity of peptide 23 towards
hMC4R, substitutents were systematically introduced
into the four positions of the phenyl ring of Atc. Pep-
tides with 6-BrAtc (26 and 27), 7-BrAtc (28) and 8-
BrAtc (29 and 30) surprisingly were devoid of hMC4R
activity. Peptides containing 5-BrAtc, 24 and 25,
showed good hMC4R potency (33 and 35 nM) and were
unable to induce 100% hMC1R stimulation even at 50
mM concentration. Other 6-, 7-, or 8-substituted Atc
analogues investigated include 6-ClAtc, 7-MeOAtc and
8-HOAtc; none of the pentapeptides derived from these
amino acids is as potent or hMC4R selective as peptides
24 and 25 (data not shown).
The most hMC4R selective peptides described above
were tested in hMC3R and hMC5R agonist assays and
the results are shown in Table 3. As discussed pre-
viously, the lead pentapeptide 1 (Bu-His-DPhe-Arg-
Trp-Gly-NH2) was inactive in both hMC3R and
hMC5R agonist assays. This lack of agonist activity in
hMC3R and hMC5R is maintained when His6 of 1 was
replaced by 5-BrAtc (peptides 24 and 25) and
5-Me2NAtc (peptides 39 and 40). The above peptides
were also tested in hMC1R–hMC5R binding assays
(Table 4). In hMC4R, the binding affinities of the pep-
tides, within limits of experimental error, track with
their agonist activities. However, in hMC1R, the order
of binding affinity is 1>24, 25>39, 40 while the order
of agonist activity is 1>39, 40>24, 25. It is unclear why
peptides 24 and 25 are better binders but weaker ago-
nists than peptides 39 and 40 at hMC1R.
In an effort to optimize the substitution at the 5-posi-
tion of Atc, a series of halo- (chloro-), alkoxy- (meth-
oxy-, ethoxy-, isopropoxy-), alkyl- (methyl-, ethyl- and
isopropyl) and dimethylamino-Atc containing peptides
were prepared (peptides 31–40, Table 2). Although a
number of the above peptides showed equal or
improved hMC4R potency compared with 5-BrAtc
containing peptides 24 and 25, none of them was able to
dial out hMC1R activity as effectively as peptides 24
and 25. For peptides 31–40, the hMC1R potencies are
spread out over only 9-fold (195–1850 nM), while their
hMC4R potencies are spread out over 300-fold (2–600
nM), indicating a stronger influence of 5-substitution of
Atc on hMC4R than on hMC1R agonist activity. It is
reasonable to speculate that further fine-tuning of
In summary, our study shows16ꢁ18 that rigid non-basic
histidine surrogates such as Atc could lead to excellent
hMC4R selectivity in linear peptides. Pentapeptides
containing 5-BrAtc and 5-Me2NAtc are potent
hMC4R agonists and are inactive in hMC1R, hMC3R
and hMC5R agonist assays. Further modification of
Atc amino acid, incorporation of 5-substituted Atc
into cyclic peptides and in vivo studies using Atc-
containing linear/cyclic peptides would be reported in
due course.
Table 3. Agonist activity of pentapeptides containing various 5-sub-
stituted Atc amino acids at the human melanocortin receptors
Acknowledgements
Peptide
hMC1R
EC50 (nM)a
hMC3R
hMC4R
hMC5R
EC50 (nM)a EC50 (nM)a EC50 (nM)a
The authors are grateful to Roche Physical Chemistry
Department for spectroscopic measurements and inter-
pretations. We would also like to thank Dr. Ramakanth
Sarabu and Dr. Jefferson Tilley for critical reading of
the manuscript.
1
10
75% @ 50 mMb 0% @ 50 mMb
0% @ 50 mMb
20
33
35
16
2
0% @ 50 mMb
0% @ 50 mMb
32% @ 50 mMb
24% @ 50 mMb
0% @ 50 mMb
24
25
39
40
50% @ 50 mMb 0% @ 50 mMb
1850
940
0% @ 50 mMb
0% @ 50 mMb
aConcentration of peptide at 50% maximum cAMP accumulation or
the % of cAMP accumulation (relative to NDP-MSH) observed at the
highest peptide concentration tested.
References and Notes
bNot tested for antagonist activities.
1. Cone, R. D., Ed. The Melanocortin Receptors; Humana:
Totowa, 2000.
Table 4. Binding affinity of pentapeptides containing various 5-sub-
stituted Atc amino acids at the human melanocortin receptors
2. MacNeil, D. J.; Howard, A. D.; Guan, X.; Fong, T. M.;
Nargund, R. P.; Bednarek, M. A.; Goulet, M. T.; Weinberg,
D. H.; Strack, A. M.; Marsh, D. J.; Chen, H. Y.; Shen, C.-P.;
Chen, A. S.; Rosenblum, C. I.; MacNeil, T.; Tota, M.;
MacIntyre, E. D.; Van der Ploeg, L. H. T. Eur. J. Pharmacol.
2002, 440, 141.
3. Cheung, A. W.-H.; Danho, W.; Swistok, J.; Qi, L.; Kur-
ylko, G.; Franco, L.; Yagaloff, K.; Chen, L. Bioorg. Med.
Chem. Lett. 2002, 12, 2407.
Peptide
hMC1R
IC50 (nM)a
hMC3R
IC50 (nM)a
hMC4R
IC50 (nM)a
hMC5R
IC50 (nM)a
1
580
4900
5100
21,700
25,500
4000
150
200
105
150
25
13,300
24
25
39
40
Not determined
Not determined
Not determined
Not determined
Not determined
Not determined
Not determined
Not determined
4. (a) Chen, L.; Cheung, A. W.-H.; Chu, X.-J.; Danho, W.;
Swistok, J.; Yagaloff, K. A. WO 0174844, 2001; CAN
135:304143. (b) Chen, L.; Cheung, A. W.-H.; Chu, X.-J.;
aConcentration of peptide at 50% radiolabeled NDP-MSH displace-
ment.

A. W.-H. Cheung et al. / Bioorg. Med. Chem. Lett. 13 (2003) 133–137
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able using reversed-phase high performance liquid chromato-
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(10% buffer B to 60% buffer B) was carried out over 90 min at
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late, tri(o-tolyl)phosphine, Et3N, 100 ꢀC; Step 2: Mg, CH3OH,
10–15 ꢀC; Step 3: KOH, ethanol, water, reflux; overall yield
for three steps: 50% for 2-Et and 64% for 2-iPr].
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18. During the preparation of this manuscript, a report
appeared in which 17 histidine surrogates were introduced into
a Ac-His6-DPhe7-Arg8-Trp9-NH2 tetrapeptide template and
the resulting peptides were characterized in mouse melano-
cortin receptors. Holder, J. R.; Bauzo, R. M.; Xiang, Z.;
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14. Atc related non-coding amino acids used in our study
include the following: