Compounds that activate the mouse melanocortin-1 receptor identified by screening a small molecule library based upon the β-turn

Article abstract of DOI:10.1021/jm990190s

A library of 951 compounds based upon the β-turn motif were examined for their ability to stimulate the melanocortin-1 receptor. From this screening process, we have identified two compounds possessing low micromolar agonist activity at the mMC1R. The compound EL1 with racemic Nal(2') in the i + 1 position, DPro in the i + 2 position, and Trp in the i + 3 position possesses an EC₅₀ of 42.5 ± 6.9 μM. Compound EL2 with Trp in the i + 1 position, DLys in the i + 2 position, and Phe in the i + 3 position possesses an EC₅₀ value of 63.4 ± 26.9 μM. The results of the library screening process are consistent with a hypothesis dating back to the 1980s proposing that a β-turn conformation involving the melanocortin 'Phe-Arg-Trp' core amino acids provides the key recognition element. Additionally, these compounds represent the first nonpeptidic heterocyclic molecules reported to date that are able to activate the MC1R, a melanocyte receptor involved in skin pigmentation and animal coat coloration.

Full text of DOI:10.1021/jm990190s

4
380
J . Med. Chem. 1999, 42, 4380-4387
Com p ou n d s Th a t Activa te th e Mou se Mela n ocor tin -1 Recep tor Id en tified by
Scr een in g a Sm a ll Molecu le Libr a r y Ba sed u p on th e â-Tu r n
Carrie Haskell-Luevano,‡ Åsa Rosenquist, Andrew Souers, Kathy C. Khong, J onathon A. Ellman,* and
,∇
§
§
‡
,§
Roger D. Cone*,‡
Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201, and Department of Chemistry,
University of California at Berkeley, Berkeley, California 94720
Received April 20, 1999
A library of 951 compounds based upon the â-turn motif were examined for their ability to
stimulate the melanocortin-1 receptor. From this screening process, we have identified two
compounds possessing low micromolar agonist activity at the mMC1R. The compound EL1
with racemic Nal(2′) in the i + 1 position, DPro in the i + 2 position, and Trp in the i + 3
position possesses an EC50 of 42.5 ( 6.9 µM. Compound EL2 with Trp in the i + 1 position,
DLys in the i + 2 position, and Phe in the i + 3 position possesses an EC50 value of 63.4 ( 26.9
µM. The results of the library screening process are consistent with a hypothesis dating back
to the 1980s proposing that a â-turn conformation involving the melanocortin “Phe-Arg-Trp”
core amino acids provides the key recognition element. Additionally, these compounds represent
the first nonpeptidic heterocyclic molecules reported to date that are able to activate the MC1R,
a melanocyte receptor involved in skin pigmentation and animal coat coloration.
7
17
In tr od u ction
DPhe -Arg-Trp-Gly-Lys-Pro-Val-NH2, was initially de-
veloped for investigations of the melanocortin system.
Subsequently, a cyclic peptide referred to as MTII,
The melanocortin peptides include R-, â-, γ-melano-
cyte stimulating hormones (MSH) and adrenocorticotro-
pin (ACTH). These hormones are derived by posttrans-
lational processing of the pro-opiomelanocortin (POMC)
preprohormone and all possess a central His-Phe-Arg-
Trp sequence (Table 1). This His-Phe-Arg-Trp consensus
sequence is also referred to as the “message” sequence
for melanocortin hormones due to its conservation in
these peptides. This family of peptides mediates a
number of diverse physiological responses, including
4
7
Ac-Nle -c[Asp-His-DPhe -Arg-Trp-Lys]-NH2, was re-
ported.
gained from these peptides resulted in the generation
of the hypothesis that the bioactive conformation of the
MSH peptides involves a â-turn. Subsequent SAR
studies established that the core “His-Phe-Arg-Trp”
tetrapeptide and “Phe-Arg-Trp” tripeptide were active
in generating a physiological response in the frog and
1
8,19
The substructural and biological information
1
2
0-22
lizard skin bioassays.
Further studies examining
skin pigmentation and animal coat coloration, energy
homeostasis, and adrenocortical steroidogenesis.1-5 These
stereochemically modified tetra- and tripeptides estab-
lished that Ac-His-DPhe-Arg-Trp-NH2 bound and acti-
vated the human melanocortin receptor subtypes hMC1R
and hMC4R at sub micromolar concentrations.23 Ad-
ditionally, tripeptides Ac-DPhe-Arg-Trp-NH2 and Ac-
DPhe-Arg-DTrp-NH2 were determined to bind and stimu-
late the hMC4R subtype only at 10 µM concentrations.23
These data, in conjunction with the involvement of the
MC4R subtype in energy homeostasis, prompted this
study which involves the screening of a library of small
molecules based upon the â-turn. The general synthesis
hormones and neuropeptides are known to mediate their
responses via interactions with the G-protein coupled
melanocortin receptors and stimulate the adenylyl
cyclase second messenger signal transduction cascade.
Five melanocortin receptor subtypes have been iden-
tified to date in humans and a variety of animal
6
-11
species.
The MC1R subtype is involved in skin
pigmentation1 and animal coat coloration. The MC2R
2
13
responds only to ACTH and is involved in adrenocortical
steroidogenesis. The MC3R is present in a variety of
6
2
4-26
sequence as reported previously
, and the building blocks used to prepare the library
are shown in Figure 1.
is shown in Scheme
tissues including the brain, gut, and testes,8 but its
physiological role remains to be determined. The MC4R
participates in energy homeostasis.4 Finally, the MC5R
has been demonstrated to be highly expressed in
exocrine glands and plays a role in these glandular
,11
1
,5
Resu lts
1
4
The 951 compounds derived from a small molecule
library consisting of 5544 compounds were evaluated
for agonist activity at the melanocortin-1 receptor
subtype (data for all the compounds provided in Sup-
porting Information). The 951-compound subset of the
small molecule library was selected based upon peptides
containing the putative melanocortin pharmacophore
side chain moieties identified from previous studies to
be important for molecular recognition and receptor
functions.
Structure-activity studies on the melanocortin pep-
tides dates back to the 1960s.1
5,16
A linear tridecapeptide
4
known as NDP-MSH, Ac-Ser-Tyr-Ser-Nle -Glu-His-
*
To whom reprint requests should be addressed.
Oregon Health Sciences University.
University of California at Berkeley.
‡
§
∇
Present address: Department of Medicinal Chemistry, University
of Florida, P.O. Box 100485, Gainesville, FL 32610-0485. E-mail:
Carrie@cop.ufl.edu.
2
2,23
stimulation.
These compounds were generated using
1
0.1021/jm990190s CCC: $18.00 © 1999 American Chemical Society
Published on Web 09/23/1999

Compounds That Activate Melanocortin-1 Receptor
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21 4381
Ta ble 1. Primary Sequence of POMC-Derived Melanocortin Peptides with the Core “His-Phe-Arg-Trp” Sequence Emphasizeda
name
sequence
ACTH (1-39)
R-MSH
NH2-SYSMEHF RWGKPVGKKRRPVKVYPNGAEDESAEAFPLEF-OH
Ac-SYSMEHF RWGKPV-NH2
NH2-AEKKDEGPYRMEHF RWGSPPKE-OH
NH2-YVMGHF RWDRF-OH
â-MSH
γ-MSH
a
Sequences of human MCs are provided.
Sch em e 1. Synthetic Route for the Preparation of Heterocyclic â-Turn Mimetics 1
parallel synthesis techniques,24-26 and screened in
parallel at concentrations of 10 µM using a 96-well
colorimetric bioassay.27 Figures 2 and 3 summarize the
bioassay data from the two 96-well plates containing
the lead compounds (10 µM), both of which possessed
reproducible agonist activity at the mMC1R. The plate
possessing the Phe side chain in the i + 3 position
include determining the minimally active peptide frag-
ment, incorporating conformational restraints (global
and local), and inverting amino acid chirality. For our
screening efforts we selected a biased small molecule
library based upon the â-turn as a result of prior
2
0-23
classical studies of melanocortins.
First, these stud-
ies implicated the Phe-Arg-Trp side chains as important
for melanocortin agonist activity. Substitution of the
(Figure 3) contained compounds that had activities
7
above the basal activity of the system. Figure 4 il-
lustrates the three compounds that demonstrated the
greatest reproducible agonist activity at the mouse
MC1R subtype at concentrations of 10 µM.
Two compounds were then individually resynthesized,
purified, and characterized (see Experimental Section).
These two compounds, EL1 (rac-Nal(2′) i + 1, DPro i +
melanocortin Phe with Gly or Ala in the 6-9 and 7-9
tetrapeptides (R-MSH numbering nomenclature) re-
2
2
sulted in peptides that were inactive. Substitution of
8
the melanocortin Arg by Lys, Gln, Ala, and Gly resulted
2
2
in tetrapeptides possessing 2-65 µM EC50 values,
9
whereas substitution of Trp by Gly or Ala resulted in
inactive tri- and tetrapeptides (unpublished results, C.
Haskell-Luevano, M. E. Hadley, and V. J . Hruby).
Second, based upon biological characterization of NDP-
2
, Trp i + 3) and EL2 (Trp i + 1, DLys i + 2, Phe i + 3)
were tested in triplicate and in at least two independent
experiments (10
-
4
-10
17
4
10
M to 10
M) at the mMC1R,
MSH, the design and evaluation of c[Cys , Cys ]-R-
2
8
1,18
mMC3R, mMC4R, and mMC5R subtypes (Figure 5).
EL1 possessed an EC50 value of 42.5 ( 6.9 µM, and EL2
possessed an EC50 value of 63.4 ( 26.9 µM at the
mMC1R subtype. As illustrated in Figure 5, these two
compounds did not generate an agonist response at the
mMC3R and mMC4R subtypes. Interestingly, EL1
possessed some agonist activity at the mMC5R.
MSH, and extensive model building,
Sawyer, Ha-
dley, and Hruby proposed that the bioactive conformation
of R-MSH involves a â-turn structure involving the Glu-
His-Phe-Arg-Trp region. This hypothesis was further
supported by the enhanced potency observed for the
1
8,19
cyclic lactam analogue MTII.
Conformational analy-
sis of MTII, R-MSH, and NDP-MSH by nuclear mag-
netic resonance (NMR) techniques provided further
evidence that a â-turn is the bioactive conformation of
Discu ssion
2
9,30
melanocortin peptides.
Rational design approaches toward the generation of
small molecules that mimic peptide-receptor interac-
tions have been pursued for decades. The use of peptide
hormones as therapeutic agents has several shortcom-
ings including poor enzymatic stability, bioavailability,
and receptor specificity. Classic approaches in pursuit
of generating small molecule agonists and antagonists
We have screened a library of compounds based upon
the â-turn motif, with the library members biased to
include side chains present in the putative â-turn region
of R-MSH, against the melanocortin-1 receptor. These
studies have led to the identification of the first het-
erocyclic small molecules that are able to stimulate the

4
382 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21
Haskell-Luevano et al.
F igu r e 1. Side chain building blocks used in the generation of the â-turn library examined at the melanocortin receptor subtypes.
8
mouse melanocortin-1 receptor subtype. Furthermore,
these findings identify a combinatorial chemistry scaf-
fold upon which further modifications can be examined
to increase ligand affinities to the mMC1R.
activities of R-MSH analogues with Arg replacements
8
clearly implicate the importance of Arg in melanocortin
recognition and receptor activation. Interestingly, al-
though EL1 lacks any potential electrostatic/hydrophilic
functionality that corresponds to the Arg side chain,
8
Because the identified compounds, EL1 and EL2, are
agonists at the mMC1R, it is possible that a similar
mode of receptor interactions and activation are com-
mon to these molecules and the pharmacophore ele-
ments of the melanocortin peptides. Previously reported
homology molecular modeling of the MC1R31 suggested
specific receptor side chain residue interactions with the
ligand DPhe-Arg-Trp. As shown in Figure 6, the model
proposes two hydrophobic pockets and an electrostatic
pocket for accommodating binding of the peptidic ligand.
Genetics and in vitro mutagenesis of the MC1R have
clearly implicated the receptor residues Glu92, Asp115,
and Asp119 (mouse numbering nomenclature) in trans-
membrane (TM) domains 2 and 3 as being particularly
important for endogenous melanocortin ligand binding
and receptor activation.13,32 These studies and functional
this molecule possesses low micromolar EC50 values at
the mMC1R. These finding are consistent with previous
reports on activation of frog skin MC1R with a series
of tri- and tetrapeptides, Ac-DPhe-Xaa-DTrp-NH2 and
Ac-His-DPhe-Xaa-DTrp-NH2, where Xaa is Lys, Gln, Ala,
or Gly. The tetrapeptides possess low micromolar EC50
values, and the tripeptides possess EC50 values in the
range of 100 µM. These studies suggest that spatial
orientation of the hydrophobic Phe and Trp side chains
play a critical role in receptor activation. Figure 7
illustrates putative EL1 interactions with the mMC1R
side chains based upon the assumption that the ligand
receptor interactions mimic the melanocortin peptide-
receptor hydrophobic interactions. Notably, although
EL2 possesses a DLys side chain at the i + 2 position

Compounds That Activate Melanocortin-1 Receptor
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21 4383
F igu r e 2. Summary of the â-turn library plate containing the EL1 compound evaluated at concentrations of 10 µM in the
â-galactosidase bioassay at the mMC1R.
F igu r e 3. Summary of the â-turn library plate containing the EL2 compound evaluated at concentrations of 10 µM in the
â-galactosidase bioassay at the mMC1R.
8
that could substitute for Arg -mMC1R interactions,
slightly poorer agonist activity is observed (Figure 5).
In addition, the library members that incorporated D
or L Arg in place of Pro in EL1 and D or L Arg in place
of Lys in EL2 were not as potent as either EL1 or EL2
generations of non-peptide heterocyclic agonists at the
MC1R. (During the preparation of this manuscript,
reports of MC1R peptoids and oligomeric R-MSH ana-
3
3,34
logues were reported.
)
Exp er im en ta l Section
(
Figures 3 and 4). Detailed SAR of EL1 and EL2 along
with mutagenesis studies of mMC1R will be required
in order to evaluate the validity of the proposed binding
model.
Gen er a l Meth od s a n d Rea gen ts. All materials, unless
otherwise noted, were obtained from commercial suppliers and
used without further purification. When used as a reaction
solvent CH
distilled under N
mediately prior to use. Deoxygenation of solvents and reaction
mixtures was achieved by N
2
Cl
2
was distilled from CaH
2
; THF and dioxane were
2
from sodium benzophenone ketyl, all im-
Con clu sion s
By screening a small molecule library based upon the
â-turn, we have identified, to our knowledge, the first
reported non-peptide heterocyclic compounds which
activate the mouse melanocortin-1 receptor subtype.
These results support the hypothesis put forth a decade
ago that the melanocortin peptide possesses a â-turn
conformation involving the melanocortin Phe-Arg-Trp
core amino acids as a key recognition element. The
compounds reported herein are the first leads for future
2
or Ar bubbling for 15-20 min.
N-Fmoc-protected amino acids and side-chain-protected amino
acids were purchased from Calbiochem-Novabiochem Corp.
(San Diego, CA) or Bachem Bioscience Inc. (King of Prussia,
PA). R-Bromo acids were prepared from the corresponding
side-chain-protected amino acids in one step in optically
3
5
enriched form according to the previously reported procedure.
Chromatography was carried out using Merck 60 230-240
mesh silica gel. Compounds were visualized either by ultra-
violet light, by cobalt nitrate/ammonium molybdate staining,

4
384 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21
Haskell-Luevano et al.
resin was dried in vacuo. Complete disappearance of support-
bound amine was confirmed by a negative bromophenol blue
test.
Disu lfid e F or m a tion for Ba ck bon e Cou p lin g. Thioester
resin 2 (49 g) was solvated in 500 mL of THF under an inert
atmosphere. Sodium (3.2 g, 140 mmol) was dissolved in 170
mL of methanol and slowly added to the solvated resin with
mechanical stirring. The reaction slurry was stirred for 1 h,
quenched by addition of 20 mL (350 mmol) of acetic acid. After
being stirred for 5 min, the resin was filtered and rinsed with
2
.5% acetic acid in THF, methanol, THF, methanol, THF,
methanol (2×), and dry CH Cl (2×). 2-Benzothiazolyl 2-meth-
anesulfonoxyethyl disulfide (20.5 g, 63.7 mmol) dissolved in
00 mL of dry CH Cl was added to the deprotected resin. The
2
2
3
2
2
mixture was mechanically stirred under an inert atmosphere.
After 18 h, Ellman’s free thiol test showed that no free thiol
remained on the resin. The resin was isolated by filtration and
rinsed with CH
overnight to give 3.
2
Cl
2
(2×) and MeOH (3×) and dried in vacuo
Am in e Disp la cem en ts (i + 3 Sid e Ch a in ). Each support-
bound secondary amine 4 was prepared by treating 4 g of resin
3
in a 50 mL round bottom flask with a 0.75 M solution of the
corresponding primary amine in 24 mL of NMP for 18 h at
5
0-55 °C. The resin was isolated by filtration, rinsed with
DMF (3×) and (CH
2
Cl
2
(2×), MeOH (2×)) (2×), and dried in
vacuo.
F igu r e 4. Structures of EL1, EL2, and the third compound
which were identified as possessing agonist activity at the
mMC1R.
Cou p lin g of F m oc Am in o Acid s (i + 2 Sid e Ch a in ) a n d
F m oc Clea va ge. Each secondary amine resin 4 was weighed
(mass depended on R , 62.5 mmol/cartridge) into 28 12 mL
i+3
filter cartridges with 70 mm hydrophobic polyethylene frits
{
Applied Separations (Allentown, PA), cat.# 2427)} for coupling
of the 28 Fmoc-amino acids. Solutions of each Fmoc amino acid
0.2 M) with HATU (0.2 M) and i-Pr EtN (0.4 M) in DMF were
or by a 0.3% ninhydrin solution in butanol/3% acetic acid.
Melting points were determined in open Pyrex capillaries and
are uncorrected. NMR spectra were recorded on Bruker AMX
(
2
prepared, and 2 mL of the coupling solution containing the
appropriate Fmoc amino acid was added to each filter car-
tridge. HOAt (0.2 M) was added to coupling solutions of Fmoc-
Arg and Fmoc-D-Arg to protect the guanidine side chain as
the HOAt salt.38 The filter cartridges were stoppered and
shaken for 12 h. After complete consumption of starting
material as confirmed by negative bromophenol blue test, the
3
00 (300 MHz), AMX 400 (400 MHz), AM 400 (400 MHz), or
1
3
AM 500 (500 MHz) machines. Proton-decoupled C spectra
were obtained at 101 or 126 MHz with the same instruments
with a line broadening of 1.5 Hz. Samples for NMR analysis
which contained rotamers were heated in order to cause
coalescence of the different rotameric peaks. NMR chemical
shifts are expressed in ppm downfield, relative to internal
solvent peaks. Coupling constants, J , are listed in hertz and
chemical shifts δ are reported in ppm. MALDI TOF mass
spectrometry was performed on a VoyagerTM BioSpectrometry
workstation from PerSeptive Biosystems (South San Francisco,
CA). Elemental analyses were performed by M-H-W Labora-
tories (Phoenix, AZ) or by the Microanalytical Laboratory at
the University of California at Berkeley.
resin was rinsed with DMF (2×), CH
and allowed to air-dry. Each resin batch was then treated with
mL of DMF containing 20% (v/v) piperidine for 30 min with
shaking. The resin was drained, rinsed with DMF (2×), CH Cl
2×), and MeOH (2×), and dried in vacuo.
Cou p lin g of r-Br om o Acid s (i + 1 Sid e Ch a in ). Filter
2
Cl
2
(2×), and MeOH (2×)
2
2
2
(
cartridges (152) of deprotected resin (62.5 mmol) were equili-
brated in 5 mL of a 17:13 comixture of 1,2-dichloroethane/DMF
for at least 10 min. Resin from each filter cartridge was then
transferred as an isopycnic slurry to each of 22 respective wells
of a high-throughput solid-phase synthesis block (∼200 mL of
slurry/well, 2.6 mmol/well). Each block was then fitted into a
deep-well (2 mL) microtiter plate (Beckmann, cat.# 267006)
with one glass bead present in each well. The coupling solution
corresponding to one of the 22 R-bromo acids (0.1 M), DICI
Syn t h esis of Am in e (i + 3 Sid e Ch a in ) Bu ild in g
3
6
Block s. 1-N-ter t-Bu toxyca r bon yl-1,5-d ia m in op en ta n e.
Di-tert-butyl dicarbonate (16 g, 73 mmol) in 300 mL of 9:1
dioxane/water was added to a solution of 1,5-diaminopentane
(15 g, 147 mmol) in 450 mL of 9:1 dioxane/water over a period
of 4 h. The solution was stirred at room temperature overnight
and concentrated, and the residue was taken up in 300 mL of
water. The precipitated N,N′-di-Boc-1,5-diaminopentane was
removed by filtration through a fritted glass funnel, and the
(
(
0.1 M), and HOAt (0.1 M) in DMF was added to each well
300 mL/well). After 12 h, the solvent was drained, and the
filtrate was extracted with CH
organic extracts were concentrated, and the crude product was
chromatographed over silica gel (80:20:4 CH Cl /methanol/40%
aqueous NH OH) and dried to provide 9.3 g (46 mmol, 63%)
2
Cl
2
(4 × 250 mL). The combined
resin in each high-throughput synthesis block was rinsed with
DMF (2×), CH Cl (2×), and 1:1 CH Cl /MeOH (2×) to provide
support-bound 5.
2
2
2
2
2
2
4
of a dense liquid which appears as a solid: boiling point 97-
Clea va ge a n d Cycliza tion . Each high-throughput syn-
-
1
37 1
9
2
(
8° at 2 × 10 Torr; H NMR (400 MHz, CDCl
3
) δ 1.28 (m,
thesis apparatus was fitted into a microtiter plate and
transferred into a glovebag under a N atmosphere, and TCEP
2
H), 1.36 (s, 9H), 1.37-1.46 (m, 4H), 2.65 (t, 2H, J ) 7.0), 2.81
1
3
br s, 2H), 2.98-3.08 (m, 2H), 4.78 (br s, 1H); C NMR (81
(6 mM) in ∼1 mL of oxygen-free 19:1 DMF/water was added
MHz, CDCl ) δ 23.9, 28.3, 29.7, 32.3, 40.3, 41.4, 78.8, 155.9.
3
to each well. The plates were transferred into a glovebag under
Libr a r y Syn th esis. Cou p lin g of th e SAMI Lin k er to
Su p p or t. Aminomethylated resin beads (85 g, 100-200 mesh,
purchased form California Bachem, Lot # ZL725, loading level
2
a N atmosphere within an oven maintained at 50 °C. After
24 h, the cleaved resin in each synthesis block was rinsed once
with 200 mL of DMF, and the synthesis blocks were removed
from the microtiter plates to provide the acyclic â-turn
mimetics in 1.2 mL of DMF.
0
2
.6 mequiv/g) were slowly stirred in a solution of S-acetyl
-mercapto-2-methyl propionic acid (22.7 g, 140 mmol), PyBOP
(
73 g, 140 mmol), HOBt-H
EtN (48.8 mL, 280 mL) in 650 mL of DMF for 12 h. The resin
was then isolated by filtration and rinsed with DMF (8×), CH
Cl Cl (3×), and methanol (2×). The
(4×), methanol (2×), CH
2
O (21.4 g, 140 mmol), and i-Pr
2
-
Guanidine resin (50-60 mg, 108-120 mmol, 100-200 mesh)
was then added to each well of each microtiter plate. The
2
-
microtiter plates were transferred to a glovebag under a N
atmosphere and allowed to remain for at least 24 h at room
2
2
2
2

Compounds That Activate Melanocortin-1 Receptor
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21 4385
F igu r e 5. Agonist pharmacological analysis of purified EL1 and EL2 compound activity at the melanocortin receptor subtypes.
concentrator. Methanol was added to each well followed by
reconcentration as before to provide a white residue for most
of the â-turn mimetics 1.
Libr a r y Ch a r a cter iza tion . The presence of compounds at
their specified locations within the library was confirmed by
mass spectral (MALDI TOF) analysis. Next, 110 of the
deprotected compounds containing the lysine side chain func-
tionality at one or more positions were submitted for analysis.
Of the compounds analyzed, 95% (104/110) displayed the
1
desired mass spectral ions. H NMR was used to confirm the
structure, assess the purity, and determine the yield of a
selected set of compounds from the library. Thus, 26 com-
pounds were selected from the library to span a range of side
chain functionality at all three positions and to provide easily
interpretable spectra. An average of 1.4 ( 0.4 mmol of
compound was obtained per well as determined by comparison
of peak areas to that of an internal standard (p-xylene). The
analytical results from the library characterization are given
in the Supporting Information.
Compounds EL1 and EL2 (Figure 4), which showed inter-
esting activity in the preliminary screenings were prepared
on a 20 mg scale on solid support following the previously
F igu r e 6. Illustration of the putative ligand DPhe-Arg-Trp
amino acids interactions with the mouse MC1R.
described conditions in 65 and 74% total yield, respectively.
EL1: 1H NMR (MeOH-d
, 300 MHz) δ 1.10-1.28 (m, 2H),
4
1
3
6
8
.33-1.69 (m, 3H), 1.77-1.90 (m, 1H), 2.63-2.89 (m, 6H),
.07-3.37 (m, 5H), 3.62-3.79 (m, 2H), 4.62-4.74 (m, 1H),
.94-7.07 (m, 3H), 7.12-7.32 (m, 5H), 7.53 (d, 1H, J ) 7.87),
temperature. After the cyclization step, each microtiter plate
containing solutions of the cyclic â-turn mimetics and the
guanidine resin was positioned atop another microtiter plate
so that the corresponding wells of the lower plate were directly
beneath those of the upper plate. Syringe 18-gauge holes were
then punched into each well of the upper plate using a
microtiter-based punch apparatus. Filtration to remove the
guanidine resin was accomplished by draining the solution into
the new microtiter plate. To expedite the draining process the
plates were centrifuged. The wells of the upper plate contain-
ing the guanidine resin were rinsed with 400 mL of DMF and
then with 200 mL of DMF. Centrifugation was performed after
each rinse. After filtration and rinsing, the solvent containing
the â-turn mimetics was removed in vacuo on a microtiter plate
.75 (d, 1H, J ) 9.56). HRMS (FAB) m/z calcd for C27
35 4 2
H N O S
+
(MH ), 479.2481; found, 479.2482.
EL2: 1H NMR (DMSO-d
, 300 MHz) δ 2.42-2.95 (m, 4H),
6
2.97-3.45 (m, 2H), 3.48-3.90 (m, 4H), 4.54-4.67 (m, 1H),
4.81-5.18 (m, 2H), 6.12 (d, 0.5H, J ) 7.13), 6.36 (t, 0.5H, J )
7.21), 7.68-7.90 (m, 3H), 8.04-8.69 (m, 8H), 10.52 (b, 0.5H),
+
10.84 (b, 0.5H). HRMS (FAB) m/z calcd for C30
H
32
N
3
O
2
S (MH ),
498.2215; found, 498.2219.
Cell Cu ltu r e a n d Tr a n sfection . Briefly, HEK-293 cells
were maintained in Dulbecco’s modified Eagle’s medium with
10% fetal calf serum and seeded 1 day prior to transfection at

4
386 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21
Haskell-Luevano et al.
dye:water to the 10 µL cell lysate sample taken previously,
and the OD595 was measured on a 96-well plate reader
(Molecular Devices). Data points were normalized both to the
relative protein content and nonreceptor dependent forskolin
stimulation. Data analysis and EC50 values were determined
using nonlinear regression analysis with the PRISM program
(v2.0, GraphPad Inc.).
Ack n ow led gm en t. This work was supported by
grants from the U.S. Public Health Service, RO1-
AR42415 (R.D.C.), R01-53696 (J .E.), and DK09231
(C.H.L.). Carrie Haskell-Luevano is a recipient of a
Burroughs Wellcome fund Career Award in the Bio-
medical Sciences. Åsa Rosenquist thanks the Knut and
Alice Wallenberg Foundation for funding.
Su p p or tin g In for m a tion Ava ila ble: Characterization of
selected library members by low-resolution mass spectral
1
(
MALDI-TOF) analysis and H NMR is available. Additionally,
the data for plates indicating the exact side chain combinations
i + 1, i + 2, and i + 3) of the small molecules which were
(
pharmacological evaluated at the mMC1R are shown. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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