Design, synthesis, and biological studies of efficient multivalent melanotropin ligands: Tools toward melanoma diagnosis and treatment

Article abstract of DOI:10.1021/jm2009937

To achieve early detection and specific cancer treatment, we propose the use of multivalent interactions in which a series of binding events leads to increased affinity and consequently to selectivity. Using melanotropin (MSH) ligands, our aim is to target melanoma cells which overexpress melanocortin receptors. In this study, we report the design and efficient synthesis of new trivalent ligands bearing MSH ligands. Evaluation of these multimers on a cell model engineered to overexpress melanocortin 4 receptors (MC4R) showed up to a 350-fold increase in binding compared to the monomer, resulting in a trivalent construct with nanomolar affinity starting from a micromolar affinity ligand. Cyclic adenosine monophosphate (cAMP) production was also investigated, leading to more insights into the effects of multivalent compounds on transduction mechanisms.

Full text of DOI:10.1021/jm2009937

ARTICLE
pubs.acs.org/jmc
Design, Synthesis, and Biological Studies of Efficient
Multivalent Melanotropin Ligands: Tools toward Melanoma
Diagnosis and Treatment
Nabila Brabez,†,‡,§,|| Ronald M. Lynch,^ Liping Xu,# Robert J. Gillies,# Gerard Chassaing,†,‡,§
Solange Lavielle,†,‡,§ and Victor J. Hruby*,||
†UPMC Paris06, UMR 7203, Laboratoire des BioMolꢀecules, Universitꢀe P. et M. Curie, 75005 Paris France
‡CNRS, UMR 7203, France
§ENS, UMR 7203, Dꢀepartement de Chimie, Ecole Normale Supꢀerieure, 75005 Paris France
University of Arizona, Department of Chemistry and Biochemistry, 1306 East University Boulevard, P.O. Box 210041, Tucson, Arizona
85721, United States
^University of Arizona, Department of Physiology, Tucson, Arizona 85721, United States
#Lee Moffitt Cancer Center, Tampa, Florida 33612, United States
ABSTRACT: To achieve early detection and specific cancer treatment, we propose
the use of multivalent interactions in which a series of binding events leads to
increased affinity and consequently to selectivity. Using melanotropin (MSH)
ligands, our aim is to target melanoma cells which overexpress melanocortin
receptors. In this study, we report the design and efficient synthesis of new trivalent
ligands bearing MSH ligands. Evaluation of these multimers on a cell model
engineered to overexpress melanocortin 4 receptors (MC4R) showed up to a
350-fold increase in binding compared to the monomer, resulting in a trivalent
construct with nanomolar affinity starting from a micromolar affinity ligand. Cyclic
adenosine monophosphate (cAMP) production was also investigated, leading to
more insights into the effects of multivalent compounds on transduction
mechanisms.
’ INTRODUCTION
selectivity to cancer cells targets. Previously, our group and
others have described the use of different templates to construct
multimers,15ꢀ24 resulting in relative enhancement, up to about
50-fold. Herein, we describe the synthesis of new dendritic based
multimers that can bear up to three melanotropin ligands and the
study of their affinity and activity toward a cancer cell model
readily available overexpressing MC4R.
Targeted therapy consists of drugs that will target specific
patterns (e.g., proteins) involved in tumor spread and growth.
Targeting tumor characteristics has the advantage of leading to
specific action on the cancer cells, leaving normal cells intact.1,2 G
protein coupled receptors (GPCRs) are intensively studied as
targets for many diseases because they are involved in cell
signaling pathways and have been shown to be up-regulated in
many cancers.3 Melanoma is the most threatening skin cancer:
once the metastatic stage is reached, the prognosis is poor
because the tumor is resistant to most cures. Therefore, early
diagnosis and specific treatment could increase the chances of
survival. The melanocortin 1 receptor (MC1R) is known to be
overexpressed4ꢀ6 in most forms of melanoma, which made an
important target7ꢀ10 because it can be targeted using a variety of
ligands including well-studied melanotropin ligands. The coop-
erative multivalency effect is involved in many biological
pathways11,12 and consists of the multiple and simultaneous
binding of ligands to multiple targets leading to an increased
affinity. Increases in specificity can also be provided by multi-
valency by a complex interaction between ligand affinity and
receptor number.13,14 Our strategy consists in seeking coopera-
tive effects using multimers to bind with a high affinity and
’ RESULTS
Design. To seek multivalent interactions, different criteria
have to be considered such as the target, the nature of the
multimer backbone, the linker length, the valency, and the
ligands to be used. Different kinds of scaffold have been used
in the literature such as peptides,23,24 polymers,9,19,20 and
dendrimers.21,22 Our aim is to use a tetravalent scaffold that will
be able to bear three peptides to seek a cooperative effect and one
tag that could be used for imaging or therapeutic purposes. In
2006, Chassaing et al. described the synthesis of a small tetra-
valent template,25 which appeared readily modifiable and hence
Received: July 25, 2011
Published: September 19, 2011
r
2011 American Chemical Society
7375
dx.doi.org/10.1021/jm2009937 J. Med. Chem. 2011, 54, 7375–7384
|

Journal of Medicinal Chemistry
ARTICLE
Figure 1. Sequences of Eu-NDP-α-MSH, Ac-MSH(4), protected peptides (A) and (B) and peptides (A) and (B).
fit our criteria. However, the distance between ligands is
important15ꢀ24 and the described template was too small to
“adjust” concomitantly three ligands in the binding sites of three
GPCRs. Thus, we modified the backbone of our scaffold on the
N-terminal with a Boc-Gly so that every added peptide of a
trivalent construct would be equidistant from the scaffold
quaternary carbon. An additional β-Ala spacer was placed on
the C-terminal of the scaffold as a small spacer to the potential tag
moiety. Previous studies have described the optimal length to
achieve cross-linking of GPCRs and a potential increase in
affinity as between 25 and 50 Å.23,24,26 The cooperative effect
being a thermodynamic process, it is known that a compromise
between linker properties such as flexibility and length are
one with on its N-terminal a lysine bearing a triple bond on the
side chain (peptide A) and one directly acylated on the N-
terminal by a triple bond containing moiety (peptide B). To be
able to have a strategic scheme that can be used for any other
applications, the lysine modification was appropriate when
modification within the peptide sequence is possible but not at
the N- or C-terminus.
Scaffold Synthesis. The synthesis of scaffold (7) is reported
in Scheme 1. Compound (1) was synthesized as described in the
literature by Chassaing et al.25 Coupling of (1) with Boc-glycine
was achieved using O-(benzotriazol-1-yl)-N,N,N0,N0-tetra-
methyluronium hexafluorophosphate (HCTU) in dimethylfor-
mamide (DMF) in the presence of diisopropylamine (DIEA) to
afford the coupling product, which was then hydrolyzed into (3)
with a 2N solution of lithium hydroxide in tetrahydrofuran
(THF). Compound (3) was coupled to β-alanine methyl ester
(β-Ala-OMe) using (benzotriazol-1-yloxy)-tris-dimethylamino-
phosphonium hexafluorophosphate (BOP) and DIEA in DMF,
and the resulting product (4) was saponified to yield (5),
following the procedure described above, but at 0 ꢀC. Reduction
of cyano groups into amines was done in a Parr apparatus under
50 psi of hydrogen with PtO2 in a mixture of methanol and
chloroform to afford (6), which was further treated with di-tert-
butyl dicarbonate (Boc2O) in a mixture of water and acetonitrile
in the presence of triethylamine to generate (7). This scaffold
(7) was used for the synthesis of the various trivalent ligands
necessary. We have previously described PEGO-(Pro-Gly)3ꢀ6
-
PEGO as a more functional semiflexible spacer, compared to
fully flexible PEGO, moderately flexible β-Ala, and the more rigid
(Pro-Gly) as linkers.21,22
In the current work, we explore the multivalent cooperative
effect of a low affinity melanotropin ligand pharmacophore,
MSH(4): His-DPhe-Arg-Trp-CONH2 (Figure 1). This peptide
was chosen as a model because it binds with micromolar affinity
to both receptors MC1R and the MC4R. It can also be modi-
fied on its N-terminal without affecting the binding affinity
and activity. Furthermore, the relatively low affinity provided a
larger potential dynamic range with which to measure affinity
enhancements.13 We designed two different ligands (Figure 1):
7376
dx.doi.org/10.1021/jm2009937 |J. Med. Chem. 2011, 54, 7375–7384

Journal of Medicinal Chemistry
ARTICLE
Scheme 1. Scaffold Synthesisa
a Reagents and conditions: (a) Boc-Gly-OH, HCTU, DIEA, DMF, rt, 92%; (b) LiOH (2N), THF, rt, 95%; (c) β-Ala-OMe, BOP, DMF, DIEA, rt, 85%;
(d) LiOH (2N), THF:MeOH, 0 ꢀC, 90%; (e) PtO2, H2, 50 psi, MeOH:CHCl3, rt, 80%; (f) Boc2O, DIEA, H2O:MeCN, rt, 80%; (g) Fmoc-OSu, H2O:
MeCN, NaHCO3, 0 ꢀC to rt, 65%.
Figure 2. Structures of monovalent, bivalent, and trivalent ligands.
7377
dx.doi.org/10.1021/jm2009937 |J. Med. Chem. 2011, 54, 7375–7384

Journal of Medicinal Chemistry
ARTICLE
Scheme 2. Assembly of the Trivalent Construct on Solid Supporta
a Reagents and conditions: (a) 7, HCTU, DIEA, DMF; (b) 100% TFA; (c) 20% DIEA in DCM; (d) Boc-linker-OH, HCTU, DIEA, DMF; (e)
N3CH2CO2H, HBTU, DIEA, DMF; (f) CuI, ascorbic acid, DIEA, peptide (A) or (B), (cf. Figure 2 for the sequences), DMF:2,6-lutidine; (g) HF.
Table 1. Analytical Data of Synthesized Multimers
Table 2. Influence of Linker Length on Affinitya
mass spectrumb
estimated length
linker between ligands (Å)b IC50 (nM) to Ac-MSH(4)
relative potency
compd
HPLCc, tR
Ac-MSH(4)
NB300
NB299
NB298
NB302
NB301
NB297
NB342
NB341
4600 ( 790
compda
linker
ion
calcd
obsd
(min)
(Gly-Pro)3
(Gly-Pro)2
(Gly-Pro)1
(β-Ala)2
β-Ala
60 ( 20
52 ( 20
45 ( 20
48 ( 10
43 ( 10
35 ( 10
35 ( 5
620 ( 51
410 ( 70
220 ( 59
120 ( 10
88 ( 12
71 ( 5.3
73 ( 3.5
14 ( 1.5
7
11
Ac-MSH(4)
NB297
NB298
NB299
NB300
NB301
NB302
NB341
NB342
NB397
NB399
(M + 1)+ 686.3482 686.6389
(M + 5)5+ 650.1385 650.1376
(Gly-Pro)1 (M + 6)6+ 647.4978 647.4982
(Gly-Pro)2 (M + 6)6+ 724.5349 724.5359
(Gly-Pro)3 (M + 6)6+ 801.5720 801.5709
9.75
12.37
12.97
12.90
12.86
12.90
12.83
12.94
12.90
11.58
12.62
no linker
21
38
52
no linker
β-Ala
64
β-Ala
(M + 6)6+ 577.4685 577.4683
(M + 6)6+ 612.9871 612.9861
(M + 5)5+ 548.0752 548.0752
(M + 5)5+ 590.6974 590.6969
(M + 2)2+ 689.3679 689.3666
(M + 4)4+ 578.8050 578.8053
63
(β-Ala)2
no linker
β-Ala
no linker
24 ( 5
330
a Competition experiments were carried out using time resolved fluor-
escence.31 Multimers were competed against Eu-NDP-α-MSH. The
IC50 values are the average of 4 experiments each done in quadruplicate.
b Linker length was estimated from an energy-minimized structure using
MacroModel.
No linker
No linker
a NB297 to NB302 were synthesized using peptide (A) and NB341 to
NB399 were synthesized using peptide (B). NB397 is the monovalent
ligand and NB399 is the bivalent ligand. b Maldi-TOF or ESI-MS.
c Peptides were eluted with a linear gradient from 0 to 100% acetonitrile
containing 0.1%TFA in 30 min at 1 mL/min. The purity of all peptides
determined by HPLC was g95%.
solid support using a Sieber amide resin following the standard
Fmoc strategy synthesis procedure. These peptides were func-
tionalized on the N-terminal with a triple bond either by
acylation with pentynoic acid or by coupling a lysine modified
on its side chain by pentynoic acid. Cleavage of the resins, a very
critical to keep the protections on the side chains, was success-
fully processed using 1% trifluoroacetic acid (TFA) in dichlor-
omethane (DCM) to give the protected peptides (A) and (B)
(Figure 1) with 85% yields. These crude protected peptides (A)
and (B) were used without purification for the coupling to the
different scaffolds.
(Figure 2). Another scaffold with orthogonal protections was
synthesized starting from compound (6) in order to selectively
produce either the monovalent ligand or the divalent one
(Figure 2). This orthogonally protected scaffold (8) was ob-
tained after Fmoc protection of (6) using Fmoc-OSu in a mixture
of water and acetonitrile in the presence of NaHCO3 (Scheme 1).
Azide Moiety and Protected Peptide Synthesis. The azido
acetic acid was prepared with good yields by substitution of
sodium azide on the corresponding bromoacetic acid in water.
Adequately protected MSH(4) analogues were synthesized on
Assembly of Multivalent Constructs. The synthesis of the
final trivalent constructs, described in Scheme 2, was achieved on
MBHA resin using regular Boc strategy. The scaffold (7) plus the
amino acids composing the linkers were successively attached on
7378
dx.doi.org/10.1021/jm2009937 |J. Med. Chem. 2011, 54, 7375–7384

Journal of Medicinal Chemistry
ARTICLE
Table 3. Influence of Valency on Affinitya
compd
IC50 (nm)
relative potency to NB397
NB397ꢀ1xMSH(4)
NB399ꢀ2xMSH(4)
NB341ꢀ3xMSH(4)
4900 ( 760
310 ( 73
14 ( 1.5
16
350
a Competition experiments were carried out using time resolved fluor-
escence.31 Multimers were competed against Eu-NDP-α-MSH. The
IC50 values are the average of 4 experiments each done in quadruplicate.
Figure 4. cAMP accumulation in hMC4R cells in response to multi-
valent ligands. cAMP was quantified using a chemiluminescent immu-
noassay described on the Experimental Section. cAMP is expressed as
the amount released from treated cells above that observed for
control cells.
NB397 and NB399, respectively (Figure 2) and compared their
affinities to the corresponding trivalent ligand (Table 3 and
Figure 3). As predicted, the affinity of the monovalent ligand
NB397, i.e. the ligand being directly attached to the scaffold, was
found similar to the affinity of acetylated MSH, Ac-MSH(4)
(IC50 about 4700 nM ( 200 nM). Surprisingly, the affinity of the
bivalent ligand, NB399 (IC50 310 nM), showed an order of
magnitude improvement in binding compared to the monova-
lent ligand and with the trivalent ligand, NB341, another addi-
tional order of magnitude increase in the affinity was observed,
leading to an IC50 of 14 nM. The fact that an order of magnitude
is reached per additional ligand provides evidence that three
receptors are involved in binding, which suggests a cooperative
mechanism of binding rather than a statistic effect.26
cAMP Assays. Melanotropin ligands are known to stimulate
cAMP when bound to melanocortin receptors such as the human
MC4R. cAMP measurements were made using the mono-, bi-,
and trivalent constructs (NB397, NB399, and NB341, re-
spectively) to investigate how these ligands combinations affect
cell signaling. cAMP production was activated by each of the
constructs, demonstrating that they all are agonists for the MC4R
receptor. The sensitivity for activation of cAMP production was
found to increase with the valency (Figure 4). To determine if the
efficacy of signaling was similar between the ligands, the relation-
ship between the number of ligands bound and the amount of
cAMP released was evaluated. Receptor occupancy was calcu-
lated at different percentages of the maximal cAMP response,
which was measured as the amount of cAMP produced (above
control) compared to that observed in response of 10 μM of
forskolin. As shown in Table 4, the number of receptors occupied
by each construct was within the same range, suggesting that the
bivalent and trivalent constructs activate the transduction me-
chanisms as a monovalent construct. It is also important to
remember that the bivalent NB399 and trivalent NB341 mol-
ecules showed orders of magnitude higher affinity, which sup-
ports the fact that two and three receptors are bound. However,
because the occupancy was similar at the same concentration of
cAMP for every construct, it provides evidence that the bivalent
and trivalent ligands activate the transduction mechanisms with
effectiveness similar to a monovalent construct.
Figure 3. Time-resolved fluorescence assay.31 Competitive binding
assay was performed by competing 10 nM of Eu-NDPꢀαꢀMSH with
various concentrations of multimers.
the resin until the desired sequence was reached. The azidoacetic
acid was finally coupled with equal amounts of coupling agent
and DIEA. The resin was dried and the protected peptides (A) or
(B) were then attached to the template via reaction of click
chemistry,27 which was achieved in the presence of copper
iodide, ascorbic acid, and DIEA in a mixture of DMF and 2,6-
lutidine either at room temperature or by using microwave
irradiation to accelerate the reaction rate. The monovalent and
bivalent constructs NB397 and NB399, respectively, were
synthesized, using the same strategy as above, starting from the
scaffold (8) and by removing first the proper orthogonal pro-
tecting group, depending on the desired compound, i.e. mono-
meric or dimeric ligands (Figure 2). Analytical data is reported
in Table 1.
Binding Assays. Ac-MSH(4) (Figure 1) has poor binding
affinity for the MC4R (IC50 4900 nM, Table 3). The linker
length effect on potency compared to Ac-MSH(4) was studied
using two kinds of linker within the range of 25ꢀ60 Å (Table 2).
A general trend can be observed: the shorter the linker, the better
the affinity. Also, we can observe a difference depending on the
type of linker used. When the semiflexible Gly-Pro linker is used,
the affinity was only increased from 8- to 20-fold compared to the
monomer (IC50 600 to 200 nM). However, the use of the more
flexible β-Ala linker showed an affinity enhancement from 40- to
70-fold depending on the length used. It is notable that the
trivalent compounds, NB297 and NB342, are made of ligands
(A) and (B), respectively, but the length from ligand to ligand is
the same. These two trivalent ligands show the same IC50 of
about 72 nM, which is consistent with the importance of the
length and the N-terminal modifiability of MSH(4). Although
analogues containing different linkers were synthesized, the
construct that showed the best affinity was the shortest one,
NB341, (24 ( 5 Å), which does not have any linker besides the
scaffold bonds.
’ DISCUSSION
To get more insights on the valence effect, we synthesized
and investigated monovalent and bivalent ligands of MSH(4),
We have been engaged in a program to design ligands
that might selectively target human cancers for diagnosis and
7379
dx.doi.org/10.1021/jm2009937 |J. Med. Chem. 2011, 54, 7375–7384

Journal of Medicinal Chemistry
ARTICLE
Table 4. Receptor Occupancy
compd
[RL] at 5% of forskolin response
[RL] at 10% of forskolin response
[RL] at 25% of forskolin response
NB397ꢀ1xMSH(4)
NB399ꢀ2xMSH(4)
NB341ꢀ3xMSH(4)
13000 ( 1000
12000 ( 1300
14000 ( 1000
60000 ( 4900
45000 ( 4100
75000 ( 5400
420000 ( 34000
400000 ( 22000
450000 ( 33000
Receptor occupancy was calculated for the concentration of each ligand required to activate cAMP increase at 5%, 10%, and 25% of the
maximum observed with forskolin (10 μM) treatment using the following established equation: [RL] = Bmax ꢁ [L]/[L] + Kd. The Kd
values used for this calculation were estimated from the competition binding assays (Figure 3). The numbers of receptors was assumed to
be constant at 7.5 ꢁ 105 receptors per cell, as previously measured for this cell line.32
therapeutic purposes. Multivalent interactions are part of numer-
ous biological systems and high-affinity highly selective com-
plexes may be formed starting from low-affinity ligands. In this
context, our aim was to: (i) design new tools for diagnosis and
treatment of melanoma cells using novel multivalent melano-
tropin trivalent molecules and (ii) investigate their binding and
activity properties. In the multivalent strategy, particular care
should be taken in the choice/design of the scaffold and the
linker, which should have the “right size” in order to avoid a
too large cost in entropy.11,12 Two types of linkers have been
investigated within the proposed ideal range for spanning ligands
for simultaneous binding (25ꢀ50 Å).23,26 “Bis-ornithine” repre-
sented a powerful scaffold to be explored for grafting ligands
because the χ1 and χ2 rotamers of this α,α0-amino acid side
chains are locked, with a minimal to maximal distance between
the two NH2 around 5ꢀ9 Å. The third amino function involved
in the grafting of the MSH(4) ligand is on a more flexible
position, as the three rotamers (Φ, Ψ, θ) of β-Ala might
theoretically be accessible. Altogether this construct appeared
as the best compromise for offering four anchoring points with a
minimal mobility, a critical point in this strategy. The synthetic
scheme was developed so diverse multivalent compounds can be
easily prepared with high yields. Competitive binding experi-
ments were performed by evaluating displacement of a potent
Eu-NDPꢀαꢀMSH ligand (Figure 1). Although β-Ala linkers
showed a 40- to 70-fold increase in affinity, the best results were
reached for multimers without any linker. The monovalent
compound attached to the β-Ala-“bis-ornithine” scaffold led to
a similar micromolar affinity as Ac-MSH(4), but increasing the
ligand valency increased the affinity from the monovalent to the
divalent and the trivalent constructs, as expected for successful
concomitant binding to multiple receptors. Selectivity of this
trivalent construct, which displayed a 350-fold increase in affinity
when compared to the monovalent ligand, will be ensured when a
high density of MSH receptors is reached as shown for many
melanoma cell lines.
of the ligand and/or at the basal level.28,29 Dimers of receptors
first identified on cells overexpressing receptors, probably as a
consequence of the high density of receptors, have been shown to
exist in tissues. However, very recently28,29 it has been demon-
strated that the dimers of GPCRs were in fact asymmetric and
that only one G protein in the complex presented the expected
orientation in the complex to be activated. Thus, we can
speculate that even though MSH divalent and trivalent con-
structs must recruit two to three receptors, respectively, as
ascertained by the gain in affinity, in the final ligandꢀreceptor
complex formed with G protein only one G protein can be
activated.
’ CONCLUSION
New multimeric ligands were designed for multivalent inter-
actions with MSH receptors. The optimal construct showed a
350-fold increase in the affinity vs the corresponding micromolar
affinity of the monovalent construct and Ac-MSH(4). Activity
studies confirmed that starting from a weak agonist the final
trivalent construct yielded an agonist with nanomolar potency.
The pharmacokinetic properties of this highly potent melano-
tropin trivalent ligand will be explored to ascertain the sensitivity
and selectivity to detect and target melanoma cells overexpres-
sing MC1R. From a more fundamental point of view, these
multimeric ligands should constitute powerful tools to explore
the transduction mechanisms of G protein coupled receptors.
’ EXPERIMENTAL SECTION
Chemicals and Materials. Reagents and solvents used were
reagent grade quality from commercial sources and used without further
purification unless noted otherwise. Purifications by flash chromatogra-
phy were performed on EMD chemicals silica gel 60 (0.04ꢀ0.063 mm).
p-Methylbenzhydrylamine resin (MBHA resin) (0.7 mmol/g) and
Sieber amide resin (0.5 mmol/g) were purchased from ChemPep, and
amino acids were purchased from Aapptec and Iris Biotech GmbH.
Other starting materials and chemicals were mostly purchased from
Sigma Aldrich and from Acros for platinium dioxide. NMR spectra were
recorded on Bruker ARX 250 or 500 MHz and are referenced in
ppm (δ), and coupling constants (J) are expressed in Hz. Melting
points were measured using a Kofler apparatus and are uncorrected.
Mass spectra were obtained with an ESI-mass spectrometer (Finnigan,
thermoelectron, lcq classic), and high-resolution fast atom bombard-
ment spectrometer (JEOL HX110 sector instrument) or by MALDI-
TOF (Voyager DE-Pro). HPLC was performed on a Hewlett-Packard
1100 series liquid chromatograph (Agilent Technologies) with a C8
column (SymmetryPrep, 0.78 cm ꢁ 30 cm) or C18 column (Vydac,
1.0 cm ꢁ 25 cm), separations were monitored at 230 and 280 nm, and
Bivalent and trivalent ligands showed orders of magnitude
increases in affinity, which supports the premise that when
bound, these ligands occupy two and three receptors, respec-
tively. Activity experiments established that starting from
AcMSH(4), which is a weak agonist of MSH receptors, cAMP
production increased with affinity of the multivalent ligand, the
trivalent construct exhibiting an EC50 value in the nanomolar
range close to its IC50 value. However, the occupancy is similar at
the same concentration of cAMP for every construct, even
though two or three receptors are involved in binding. This
suggests that the bivalent and trivalent ligands activate the trans-
duction mechanisms much as a monovalent ligand. G protein
coupled receptors are known to dimerize, either in the presence
7380
dx.doi.org/10.1021/jm2009937 |J. Med. Chem. 2011, 54, 7375–7384

Journal of Medicinal Chemistry
ARTICLE
final purity was determined as g95% using a C18 Vydac column
(0.46 cm ꢁ 25 cm).
(CH2), 30.4 (CH3), 33.6 (CH2), 36.6 (CH2), 54.5 (CH2), 57.4 (CH3),
62.22 (C), 80.9 (C), 119.7 (CN), 156.68 (CO), 170.3 (CO), 173.4
(CO), 176.57(CO). MS (HRMS) m/z calcd for C18H28N5O6 410.2034,
found 410.2040.
Methyl 2-(2-(tert-Butoxycarbonylamino-acetamido)-4-
cyano-2-(2-cyanoethyl-butanoate (2). To compound (1) (5.85 g,
30 mmol, 1 equiv) dissolved in 50 mL of dry DMF were added Boc-Gly
(37 mmol, 1.2 equiv), HCTU (30 mmol, 1 equiv), and DIEA (33 mmol,
1.1 equiv). After overnight stirring at room temperature under argon
atmosphere, the mixture was poured into a saturated solution of
ammonium hydrochloride. After extraction with EtOAc, the organic
phase was washed with 5% citric acid, satd NaCl, and 10% NaHCO3,
dried over MgSO4, and concentrated in vacuo leading to (2), which was
purified by silica gel chromatography EtOAc:Cy (6:4), crystallization
from EtOAc afforded (2) as a white solid, (9.8 g, 28 mmol, 92% yield); Rf
0.52 EtOAc:Cy (8:2); mp 102ꢀ103 ꢀC. 1H NMR (CDCl3) δ (ppm):
3.91 (s, 3H), 3.75 (m, 2H), 2.95 (m, 2H), 2.32 (m, 4H), 2.17 (m, 2H),
1.48 (s, 9H). 13C NMR (CDCl3) δ (ppm): 12.1 (CH2), 28.2 (CH2),
30.2 (CH3), 45.2 (CH2), 53.9 (CH3), 62.5 (C), 80.8 (C), 118.4 (CN),
156.2 (CO), 165.6 (CO), 171.7 (CO). MS (HRMS) m/z calcd for
C16H24N4O5Na 375.1644, found 375.1638.
4-(2-(tert-Butoxycarbonylamino-acetamido)-4-((2-carboxye-
thylcarbamoyl) heptane-1,7-diaminium Chloride (6). Compound
(5) (2 g, 4.8 mmol, 1 equiv) in 15 mL of MeOH:CHCl3 (5:0.15) was
reduced in the presence of PtO2 (0.228 g, 0.77 mmol, 0.16 equiv) for 2 h in a
Parr apparatus under 4 bar of H2. The suspension was filtered through Celite
and evaporated under vacuum to give (6) as a white powder (1.6 g, 3.9
mmol, 80% yield), which was used in the next step without further
purification because the NMR showed no detectable impurities; mp
1
116ꢀ117 ꢀC. H NMR (D2O) δ (ppm): 3.68 (s, 2H), 3.39 (m, 2H),
2.88 (m, 4H), 2.42 (m, 2H), 2.06 (m, 2H), 1.81 (m, 2H), 1.44 (m, 4H), 1.36
(s, 9H). 13C NMR (D2O) δ (ppm): 21.5 (CH2), 28.1 (CH3), 31.3 (CH2),
36.4 (CH2), 39.2 (CH2), 44.1 (CH2), 52.6 (CH2), 62.3 (C), 81.3 (C),
158.5 (CO), 171.2 (CO), 173.7 (CO), 175.8 (CO). MS (HRMS) m/z
calcd for C18H36N5O6 418.2660, found 418.2664.
9,9-Bis(3-(tert-butoxycarbonylamino-propyl)-2,2-dimeth-
yl-4,7,10-trioxo-3-oxa-5,8,11-triazatetradecan-14-oic Acid (7).
To a solution of (6) (10.18 g, 16.5 mmol, 1 equiv) in 80 mL of H2O:
MeCN (1:1), Boc2O (50 mmol, 3 equiv), and triethylamine (6 equiv)
were added. The solution was stirred at room temperature for 24 h. The
pH was then decreased to 3 with 1N NaHSO4. The solution was
extracted with CH2Cl2. The organic phase was dried over MgSO4 and
then concentrated in vacuo to afford an oil, which was purified by silica
gel chromatography in EtOAc:Cy:MeOH:acetic acid (50:50:2.5:1) to
afford (7) as a white solid (8.2 g, 13.2 mmol, 80% yield). Rf 0.27 EtOAc:
Cy:MeOH:acetic acid (50:46:3:1); mp 140ꢀ141 ꢀC. 1H NMR
(CDCl3) δ (ppm): 3.72 (m, 2H), 3.51 (m, 2H), 2.99 (m, 4H), 2.57
(m, 2H), 2.39 (m, 2H), 1.65 (m, 2H), 1.42 (s, 9H), 1.39 (s, 18H), 1.38
(m, 2H). 13C NMR (CDCl3) δ (ppm): 21.6 (CH2), 24.6 (CH2), 28.4
(CH3), 32.5 (CH2), 34.1 (CH2), 40.4 (CH2), 44.9 (CH2), 63.7 (C),
79.6 (C), 156.7 (CO), 169.1 (CO), 172.7 (CO), 173.4 (CO). MS
(HRMS) m/z calcd for C28H51N5O10Na 640.3534, found 640.3528.
9,9-Bis(3-(9-fluorenylmethoxy-carbonyl-aminopropyl)-2,
2-dimethyl-4,7,10-trioxo-3-oxa-5,8,11-triazatetradecan-14-
oic Acid (8). To a solution, at 0 ꢀC, of (6) (1.7 g, 4 mmol, 1 equiv) in
20 mL of H2O:MeCN (1:1) is added NaHCO3 (1.36 g, 16 mmol, 4
equiv) and dropwise Fmoc-OSu (3 g, 9 mmol, 2.2 equiv) dissolved in
6 mL of MeCN. The temperature was then raised at room temperature,
and the solution was stirred at room temperature for 24 h. The pH was
then decreased to 3 with 1N NaHSO4, and the solution was extracted
with DCM. The organic phase was dried over MgSO4 and then
concentrated in vacuo to afford an oil, which was purified by silica gel
chromatography in EtOAc:Cy:MeOH:acetic acid (50:50:2.5:1) to af-
ford (8) as a white solid (2.2 g, 2.6 mmol, 65% yield). Rf 0.35 EtOAc:Cy:
MeOH:acetic acid (50:46:3:1); mp 159ꢀ160 ꢀC. 1H NMR (CDCl3) δ
(ppm): 7.75 (m, 4H), 7.58 (m, 4H), 7.39 (m, 4H), 7.31 (m, 4H), 4.35
(d, J 8 Hz, 4H), 4.19 (m, 2H), 3.75 (m, 2H), 3.53 (m, 2H), 3.13 (m, 4H),
2.59 (m, 2H), 2.47 (m, 2H), 1.64 (m, 2H), 1.44 (s, 9H), 1.40 (m, 2H),
1.27 (m, 2H). 13C NMR (CDCl3) δ (ppm): 24.4 (CH2), 28.6 (CH3),
32.8 (CH2), 33.7 (CH2), 36.21 (CH2), 40.86 (CH2), 44.85 (CH), 63.59
(CH2), 80.6 (C), 120.1 (CH-Ar), 125.2 (CH-Ar), 127.34 (CH-Ar),
129.15 (CH-Ar), 141.4 (CꢀAr), 143.8 (CꢀAr), 157.2 (CO), 172.5
(CO), 172.86 (CO), 174.76 (CO). MS (HRMS) m/z calcd for
C48H56N5O10 862.4022, found 862.4036.
2-(2-(tert-Butoxycarbonylamino-acetamido)-4-cyano-2-
(2-cyanoethyl) Butanoic Acid (3). A solution of (2) (8.8 g, 25
mmol, 1 equiv) in THF (100 mL) was treated by slow addition of
25 mL of LiOH 2N (51 mmol, 2 equiv). The solution was stirred for 1
h, and a solution of NaHSO4 (1N) was added until pH 3. The product
was extracted using EtOAc, and the organic phase was dried over
MgSO4 and concentrated in vacuo to afford (3) as a white solid (8 g,
24 mmol, 95% yield). Rf 0.38 DCM:MeOH:CH3COOH (8:2:0.1);
mp 74ꢀ75 ꢀC. 1H NMR (CDCl3) δ (ppm): 3.88 (m, 2H), 2.96 (m,
2H), 2.37 (t, J 8 Hz, 4H), 2.23 (m, 2H), 1.46 (s, 9H). 13C NMR
(CDCl3) δ (ppm): 12.2 (CH2), 28.4 (CH2), 30.1 (CH3), 45.3 (CH2),
53.9 (CH3), 64.5 (C), 80.8 (C), 118.4 (CN), 156.2 (CO), 158.2 (CO),
171.2 (CO). MS (HRMS) m/z calcd for C15H22N4O5Na 361.1488,
found 361.1482.
Methyl 9,9-Bis(2-cyanoethyl)-2,2-dimethyl-4,7,10-trioxo-
3-oxa-5,8,11-triazatetradecan-14-oate (4). To a solution of
(3) (4 g, 12 mmol, 1 equiv), β-Ala-OMe (14 mmol, 1.2 equiv), and
BOP (12 mmol, 1 equiv) in 24 mL of dry DMF was added DIEA (24
mmol, 2 equiv). After overnight stirring at room temperature under
argon atmosphere, the mixture was poured into a saturated solution of
ammonium hydrochloride. After extraction with EtOAc, the organic
phase was washed with 5% citric acid, satd NaCl, and 10% NaHCO3.
The organic phase was dried over MgSO4 and concentrated under
vacuum to yield compound (3) as an oil, which was purified by silica gel
chromatography, EtOAc:Cy (6:4), leading to an oil, which was crystal-
lized in EtOAc to afford (4) as white crystals (4.3 g, 10 mmol, 85%
yield); Rf 0.56 EtOAc:Cy (8:2); mp 138ꢀ139 ꢀC. 1H NMR (CDCl3) δ
(ppm): 3.75 (m, 2H), 3.71 (s, 3H), 3.57 (q, 2H), 3.01 (m, 2H), 2.61 (t,
2H), 2.27 (m, 4H), 1.92 (m, 2H), 1.46 (s, 9H). 13C NMR (CDCl3) δ
(ppm): 12.1 (CH2), 28.3 (CH2), 30.9 (CH3), 32.85 (CH2), 36.05
(CH2), 52.2 (CH2), 52.2 (CH3), 61.9 (C), 80.7 (C), 118.9 (CN),
160.63 (CO), 169.63 (CO), 169.76 (CO), 173.34 (CO). MS (HRMS)
m/z calcd for C19H29N5O6Na 446.2010, found 446.2017.
9,9-Bis(2-cyanoethyl)-2,2-dimethyl-4,7,10-trioxo-3-oxa-5,
8,11-triazatetradecan-14-oic Acid (5). To a solution of (4) (6.38
g, 16 mmol, 1 equiv) in THF:MeOH (5:1) cooled at 4 ꢀC, LiOH (2N)
(20 mL, 40 mmol, 2.5 equiv) was added dropwise. After stirring 25 min at
4 ꢀC, the reaction was quenched by addition of water and decreasing the
pH to 3 with a 1N NaHSO4 solution. After extraction with EtOAc (5ꢁ),
the organic phases were assembled, dried over MgSO4, and concentrated
in vacuo to yield the pure compound (5) as a white solid (5.9 g, 14 mmol,
90% yield). Rf 0.45 DCM:MeOH:CH3COOH (8:2:0.1); mp
124ꢀ125 ꢀC. 1H NMR (MeOD) δ (ppm): 3.69 (s, 2H), 3.52 (t, J 3.5
Hz, 2H), 2.77 (m, 2H), 2.61 (t, J 2.6 Hz, 2H), 2.39 (m, 4H), 2.17 (m,
2H), 1.49 (s, 9H). 13C NMR (CDCl3) δ (ppm): 12.1 (CH2), 28.3
Azidoacetic Acid. To a solution of sodium azide (8.2 g, 126 mmol,
2 equiv) in 42 mL of water bromoacetic acid (10 g, 63 mmol, 1 equiv)
was slowly added. The solution was stirred at room temperature
overnight. The reaction was then quenched by dropwise addition of
35 mL of conc HCl (37%). After extraction with Et2O (4 ꢁ 50 mL), the
organic phase was dried over MgSO4 and concentrated in vacuo to
afford azidoacetic acid as an oil (59 mmol, 95% yield). 1H NMR
7381
dx.doi.org/10.1021/jm2009937 |J. Med. Chem. 2011, 54, 7375–7384

Journal of Medicinal Chemistry
ARTICLE
(CDCl3) δ (ppm): 10.16 (s, 1H), 3.97 (s, 2H). IR: 2108.45 cmꢀ1. MS
(GC) m/z calcd for C2H3N3O2 101.02, found 101.02.
determined as g95% by analytical HPLC (0ꢀ100% acetonitrile in
30 min) with a C18 column Vydac (0.46 cm ꢁ 25 cm).
Bivalent Construct: NB399. The scaffold (8) was coupled to the resin,
followed by Boc group removal and acetylation of the resulting free
amine. Fmoc groups were then removed, and the azido acetic acid was
coupled onto the two remaining amines to afford a bivalent construct
with two azides. The azido group was coupled to protected peptide (B)
via a click reaction. After HF cleavage, the bivalent construct NB399 was
obtained and purified by HPLC with a final purity g95% by analytical
HPLC (0ꢀ100% acetonitrile in 30 min). All reactions were performed
with the same protocols as described for the synthesis of the trivalent
constructs.
Monovalent Construct: NB397. The scaffold (8) was coupled to the
resin, followed by Boc group removal and coupling of the azido acetic
acid on the free amine. The azido group was then coupled to protected
peptide (B) via a click reaction. The Fmoc groups were finally removed,
and the resulting free amines were acetylated. After HF cleavage, the
monovalent construct NB397 was purified by HPLC with a final purity
g95% by analytical HPLC (0ꢀ100% acetonitrile in 30 min). All
reactions were performed with the same protocols as described for the
synthesis of the trivalent constructs.
Synthesis of the Protected Peptides (A), (B). The protected
peptides (A) and (B) were synthesized starting from 1 g of Sieber amide
resin (0.5 mmol/g) using Fmoc chemistry in Torviq fritted syringes. The
resin was swollen in DMF:DCM (1:1) for 1 h. The resin was first
deprotected using 25% piperidine in DMF (1 ꢁ 5 min and 1 ꢁ 20 min).
The amino acids were coupled to the resin with an elongation from the
C-terminal to the N-terminal with Fmoc-Trp(Boc)-OH, Fmoc-Arg-
(Pbf)-OH, Fmoc-D-Phe-OH, Fmoc-His(Trt), and either the modified
lysine bearing a triple bond or pentynoic acid. Coupling was achieved for
1 h using 3 equiv (relative to resin loading) of HBTU and HOBt and 6
equiv of DIEA. Coupling completion was confirmed using Kaiser test
(negative test: yellow beads). Fmoc deprotections were performed as
described above for the first deprotection step. After each step (coupling
and deprotection), the resin was washed using DMF (3 ꢁ 2 min) and
DCM (3 ꢁ 2 min) washings. Once the sequence was achieved, the
peptidyl resin was thoroughly washed with DMF, DCM, and finally
MeOH before drying under vacuum. The resin was swollen in dry DCM,
and cleavage was performed by incubating (15 ꢁ 2 min) 1 g of resin with
a solution of distilled DCM (10 mL) containing 1%TFA and then
pouring into 10% pyridine in methanol (30 mL). The solution was then
evaporated under reduced pressure, and the resulting oil was dissolved in
DCM (10 mL), which was washed with water to remove the trifluor-
oacetate pyridinium salt. The DCM solution was then transferred into a
centrifuge tube, and the protected peptide was precipitated with cold
diethyl ether. The precipitate was isolated by centrifugation. The peptide
was then triturated with ether and the pellet dried in the air to afford 85%
yield of crude peptide, which was used for the next step without
purification.
Cell Lines. HEK293 cells overexpressing the human MC4R were
used to assess the affinity and the activity at the hMC4R. The coding
region of the human MC4R gene was expressed in pcDNA3.1
(Invitrogen, V790ꢀ20). Hek293/hMC4R cells were grown in Dulbec-
co’s Modified Eagle Medium (DMEM) supplemented with 10% SCS
and 1% penicillinꢀstreptomycin and were kept under standard condi-
tions (37 ꢀC and 5% CO2).
Time Resolved Fluorescence Binding Assay. The HEK293
cells were seeded at 20000 cells per well into 96-well Costar 3603 plates
3 days before the experiment. Competition assays were performed in
quadruplicate unless noted otherwise using a fixed concentration of Eu-
NDPꢀαꢀMSH (10 nM, 50 μL per well) and 8ꢀ10 different concen-
trations of ligand. The day of the experiment, media was aspirated.
Ligands were diluted in binding medium (DMEM, 1 mM 1,10-phenan-
troline, 200 mg/L bacitracin, 0.5 mg/L leupeptin, and 0.2% bovine
albumin serum (BSA)) and added to the cells that were incubated for 2 h
at 37 ꢀC. Following the incubation, cells were washed three times with
wash buffer (DMEM, 20 μM EDTA, 0.2% BSA, 0.01% Tween 20),
enhancement solution (Delfia, Perkin-Elmer) was added to the plates
(100 μL per well) and incubated for 30 min at 37 ꢀC. The plates were
read on a Wallac Victor instrument using the standard Eu(III) TRL
measurement (340 nm excitation, 400 s delay, and emission collection
for 400 s at 615 nm). Competitive binding data were analyzed with
GraphPad Prism software using nonlinear regression analysis and fitted
to a classic one-site-binding competition equation.
cAMP Biological Response Immunoassay. The HEK293 cells
were seeded at 20000 cells per well into 96-well Costar 3598 plates 3
days before the experiment and incubated at 37 ꢀC in 5% CO2. Assays
were performed in triplicate in 3 different experiments. Media was
aspirated and cells were preincubated with 1 mM 3-isobutyl-1-methyl-
xanthine (IBMX) in DMEM at 37 ꢀC for 10 min. Then 30 μL of
compounds and standards were added to the cells, eight concentrations
of ligand were tested (10ꢀ7 to 10ꢀ12), and plates were incubated for 15
min at 37 ꢀC. cAMP production was measured using a chemilumines-
cent immunoassay kit as described in detail in the user manual
(Invitrogen C10558). Briefly, cells were incubated in 60 μL of lysis
buffer (provided with the cAMP assay kit) for 25 min at 37 ꢀC and then
transferred to precoated 96-well plates. To these plates were added 30
μL of cAMP-AP (alkaline phosphatase conjugate) and 60 μL of anti-
cAMP antibody. The plates were incubated at room temperature for 1 h
and washed 5 times with wash buffer. Enhancement solution (CSPD
substrate/Saphire II enhencer) was added to the plates following by
incubation at room temperature in the dark for 30 min. Intensity of the
Assembly of the Multivalent Constructs. Multivalent con-
structs were synthesized on MBHA resin (20 mg, 0.7 mmol/g) using
Boc chemistry in Torviq fritted syringes. The resin was swollen in DCM
and then treated with 100% TFA (3 ꢁ 1 min), then rinsed with DCM
following another rinse with 20% DIEA in DCM. The amino acids were
coupled to the resin with an elongation from C-ter to N-ter.
Trivalent Constructs: NB297, NB298, NB299, NB300, NB301, NB302,
NB341, NB342: General Procedure. The scaffold (7) was first coupled to
the resin followed by the linkers either Boc-βAla-OH, Boc-Pro-OH, or
Boc-Gly-OH. Couplings were done using 3 equiv of HCTU and 6 equiv
of DIEA. Boc removal was achieved using 100% TFA (3 ꢁ 1 min)
followed by DCM wash and 20% DIEA in DCM wash. Completion was
monitored using Kaiser test. Azidoacetic acid was finally coupled using
HBTU (3 equiv) and DIEA (3 equiv). The reaction was monitored
using Kaiser test and a modified version of Kaiser test containing 5%
PPh3 to unveil the presence of the azido group (regular Kaiser test gives
yellow beads and the Kaiser test containing the phenyl phosphine gives
blue beads30). The resin was finally washed with DCM and MeOH and
dried under vacuum for click chemistry. The resin was used unswollen,
the “click reaction” was performed using per arm of template a 2-fold
excess of protected peptides in the presence of CuI (5-fold excess),
ascorbic acid (5-fold excess), and DIEA (7-fold excess) in a mixture of
DMF:2,6-lutidine (7:3), either at room temperature for 4 days or with
microwave activation at 250W, Tmax = 50 ꢀC for 60 ꢁ 5 s with liquid
nitrogen cooling between each cycle. The resin was then rinsed using
DMF (5 ꢁ 2 min), 0.5% of diethylthiocarbamate in DMF (2 ꢁ 10 min),
and finally DMF, DCM, and MeOH. The resin was dried under vacuum
and then transferred into a Teflon reactor, for 20 mg of resin, 30 μL of
anisole was added, and HF cleavage was processed for 2 h at 0 ꢀC. The
resin was first filtered then washed with ether. The multivalent con-
structs were washed off from the resin using 10% acetic acid in H2O,
transferred into a centrifuge tube for lyophilization to afford the crude
multivalent ligands, which were purified by HPLC. Final purity was
7382
dx.doi.org/10.1021/jm2009937 |J. Med. Chem. 2011, 54, 7375–7384

Journal of Medicinal Chemistry
ARTICLE
signal was measured using Wallac Victor instrument, and the concen-
trations of cAMP were calculated after the average value of the basal
condition was subtracted and based on a standard curve that was
acquired during the same assay.
hormone analog conjugate for melanoma targetting. Nucl. Med. Biol. 2008,
35, 91–99.
(11) Mammen, M.; Choi, S.; Whitesides, G. M. Polyvalent interac-
tions in biological systems: implications for design and use of multivalent
ligands and inhibitors. Angew. Chem. Int. Ed. 1998, 37, 2754–2794.
(12) Handl, H. L.; Vagner, J.; Han, H.; Mash, E.; Hruby, V. J.; Gillies,
R. J. Hitting multiple targets with multimeric ligands. Expert Opin. Ther.
Targets. 2004, 8, 565–586.
(13) Caplan, M. R.; Rosca, E. V. Targeting drugs to combinations of
receptors: a modeling analysis of potential specificity. Ann. Biomed. Eng.
2005, 33, 1113–1124.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: (520) 621-6332. Fax: (520) 621-8407. E-mail: hruby@
email.arizona.edu.
(14) Rosca, E. V.; Stukel, J. M.; Gillies, R. J.; Vagner, J.; Caplan, M. R.
Specificity and mobility of biomacromolecular, multivalent constructs
for cellular targeting. Biomacromolecules 2007, 8, 3830–3835.
(15) Vagner, J.; Handl, H. L.; Monguchi, Y.; Jana, U.; Begay, L. J.;
Mash, E. A.; Hruby, V. J.; Gillies, R. J. Rigid linkers for bioactive peptides.
Bioconjugate Chem. 2006, 17, 1545–1550.
(16) Lee, Y.; Sampson, N. S. Romping the cellular landscape: linear
scaffolds for molecular recognition. Curr. Opin. Struct. Biol. 2006, 16,
544–550.
(17) Arosio, D.; Vrasidas, I.; Valentini, P.; Liskamp, R. M. J; Pieters,
R. J.; Bernardi, A.. Synthesis and cholera toxin binding properties of
multivalent GM1 mimics. Org. Biomol. Chem. 2004, 2, 2113–2124.
(18) Vadas, O.; Harthley, O.; Rose, K. Characterization of new
multimeric erythropoietin receptors agonists. Peptide Sci. 2008, 90,
496–502.
(19) Kramer, R. H.; Karpen, J. W. Spanning binding sites on allosteric
proteins with polymer-linked ligand dimers. Lett. Nature 1998, 395,
710–713.
(20) Kanai, M.; Mortell, K. H.; Kiessling, L. H. Varying the size of
multivalent ligands: the dependence of concanavalin a binding on
neoglycopolymer length. J. Am. Chem. Soc. 1997, 119, 9931–9932.
(21) Fan, E.; Zhang, Z.; Minke, W. E.; Hou, Z.; Verlinde, C. L. M. J.;
Hol, W. G. H. High-affinity pentavalent ligands of E. coli heat-labile
enterotoxin by modular structure-based design. J. Am. Chem. Soc. 2000,
122, 2663–2664.
(22) Andre, S.; Pieters, R.; Vrasidas, I.; Kaltner, H.; Kuwabara, I.; Liu,
F.; Liskamp, R. M. J.; Gabius, H. Wedgelike glycodendrimers as
inhibitors of binding of mammalian galectins to glycoproteins, lactose
maxiclusters, and cell surface glycoconjugate. ChemBioChem 2001,
2, 822–830.
’ ACKNOWLEDGMENT
This research was supported in parts by grants from CNRS,
U.S. Public Health Service, National Institutes of Health, and
NCI. N. Brabez is a BDI recipient from CNRS. We are thankful
to James P. Cain and Yeon Sun Lee for their helpful discussions
and critical review of the manuscript and to Craig Weber, B. M.
Thanuja Jayasundera, and Kamya Ananthakrishnan for assistance
and discussions on the bioassays.
’ ABBREVIATIONS USED
cAMP, cyclic adenosine monophosphate; β-Ala, β-alanine, NH2-
CH2-CH2-COOH; Boc2O, di-tert-butyl-carbonate; BOP, (benzo-
triazol-1-yloxy)-tris-dimethylaminophosphonium hexafluoropho-
sphate; BSA, bovine serum albumin; Cy, cyclohexane; DCM, di-
chloromethane; DIEA, diisopropylethylamine; DMEM, Dulbecco’s
Modified Eagle Medium; DMF, dimethyformamide; EDTA, ethy-
lenediaminetetraacetic acid; Fmoc-OSu, N-(9-fluorenylmethoxycar-
bonyloxy) succinimide; GPCRs, G protein coupled receptors;
HBTU, O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium hexa-
fluorophosphate; HCTU, O-(6-chlorobenzotriazol-1-yl)-N,N,N0,
N0-tetramethyluronium hexafluorophosphate; MC1R, melanocortin
1 receptor; MC4R, melanocortin 4 receptor; MSH, melanotropin,
melanocyte stimulating hormone; MSH(4), His-DPhe-Arg-Trp-
CONH2;TFA, trifluoroacetic acid; THF, tetrahydrofuran
(23) Handl, H. L.; Sankaranarayanan, R.; Josan, J. S.; Vagner, J.;
Mash, E. A.; Gillies, R. J.; Hruby, V. J. Synthesis and evaluation of
bivalent NDP-α-MSH peptide ligands for binding to the human
melanocortin receptor 4 (hMC4R). Bioconjugate Chem. 2007, 18,
1101–1109.
(24) Vagner, J.; Xu, L.; Handl, H. L.; Josan, J. S.; Morse, D. L.; Mash,
E. A.; Gillies, R. J.; Hruby, V. J. Heterobivalent ligands crosslink multiple
cell-surface receptors: the human melanocortin-4 and δ-opioid recep-
tors. Angew. Chem., Int. Ed. 2008, 47, 1685–1688.
(25) Aussedat, B.; Chassaing, G.; Lavielle, G.; Burlina, F. Bis-
ornithine (2,2-bis(aminopropyl)glycine): a new tetravalent template
for assembling different functional peptides. Tetrahedron Lett. 2006, 47,
3723–3726.
(26) Josan, J. S.; Handl, H. L.; Sanbararyanan, R.; Xu, L.; Lynch,
R. M.; Vagner, J.; Mash, E. A.; Hruby, V. J.; Gillies, R. J. Specific targeting
by heterovalent ligands. Bioconjugate Chem. 2011, 22, 1270–1278.
(27) Zhang, Z.; Fan, E. Solid phase synthesis of peptidotriazoles with
multiple cycles of triazole formation. Tetrahedron Lett. 2006, 47, 665–669.
(28) Bulenger, S.; Marullo, S.; Bouvier, M. Emerging role of homo-
and heteromerization in G-protein-coupled receptor biosynthesis and
maturation. Trends Pharmacol. Sci. 2005, 26, 131–137.
(29) Pellisier, L. P.; Barthet, G.; Gaven, F.; Cassier, E.; Trinquet, E.;
Pin, J.; Marin, P.; Dumuis, A.; Bockaert, J.; Baneres, J.; Claeysen, J. G
protein activation by serotonin type 4 receptor dimers. J. Biol. Chem.
2011, 286, 9985–9996.
’ REFERENCES
(1) Finley, R. S. Overview of targeted therapies for cancer. Am.
J. Health-Syst. Pharm. 2003, 60, s5–s10.
(2) Wu, H.; De-Kuan, C.; Chia-Ting, H. Targeted therapy for
cancer. J. Cancer Mol. 2006, 2, 57–66.
(3) Dorsam, R. T.; Gutkind, R. J. G-protein-coupled receptors and
cancer. Nature Rev. 2007, 7, 79–94.
(4) Tatro, J. B.; Atkins, M.; Mier, J. M.; Hardason, S.; Wolfe, H.;
Smith, T.; Entwistle, M. L.; Reichlin, S. Melanotropin receptors demon-
strated in situ in human melanoma. J. Clin. Invest. 1990, 85, 1825–1832.
(5) Carlson, A. J.; Linette, G. P.; Aplin, A.; Ng, B.; Slominski, A.
Melanocyte receptors: clinical implications and therapeutic relevance.
Dermatol. Clin. 2007, 25, 541–557.
(6) Jiang, H.; Wortsman, J.; Matsuoka, L.; Granese, J.; Carlson, A. J.;
Mihm, M.; Slominski, A. Molecular spectrum of pigmented skin lesions;
from nevus to melanoma. Expert Rev. Dermatol. 2006, 1, 679–700.
(7) Ren, G.; Pan, Y.; Cheng, Z. Molecular probes for malignant
melanoma imaging. Curr. Pharm. Biotechnol. 2010, 11, 590–602.
(8) Sharma, S. D.; Granberry, M. E.; Jiang, J.; Leong, S. P. L.; Hadley,
M. E.; Hruby, V. J. Multivalent melanotropic peptide and fluorescent
macromolecular conjugates. Bioconjugate Chem. 1994, 5, 591–601.
(9) Sharma, S. D.; Jiang, J.; Hadley, M. E.; Bentley, D. L.; Hruby, V. J.
Melanotropic peptide-conjugated beads for microscopic visualization
and characterization. Proc. Nat. Acad. Sci. U.S.A. 1996, 93, 13715–13720.
(10) Raposinho, P. D.; Correia, J. D.G.; Alves, S.; Botelho, M. F.; Santos,
A. C.; Santos, I. A 99m Tc(CO)3-labeled pyrazolyl-a-melanocyte-stimulating
(30) Punna, S.; Finn, M. G. A convenient colorimetric test for
aliphatic azides. Synlett 2004, 1, 99–100.
7383
dx.doi.org/10.1021/jm2009937 |J. Med. Chem. 2011, 54, 7375–7384

Journal of Medicinal Chemistry
ARTICLE
(31) Handl, H. L.; Vagner, J.; Yamamura, H. I.; Hruby, V. J.; Gillies,
R. J. Anal. Biochem. 2004, 330, 242–250.
(32) Xu, L.; Vagner, J.; Josan, J. S.; Lynch, R. M.; Morse, D. L.;
Baggett, B.; Han, H.; Mash, E. A.; Hruby, V. J.; Gillies, R. J. Enhanced
targeting with heterobivalent ligands. Mol. Cancer Ther. 2009, 8,
2356–2365.
7384
dx.doi.org/10.1021/jm2009937 |J. Med. Chem. 2011, 54, 7375–7384