The 1,4-benzodiazepine-2,5-dione small molecule template results in melanocortin receptor agonists with nanomolar potencies

Article abstract of DOI:10.1021/jm701303z

The melanocortin system consists of five seven-transmembrane spanning G-protein coupled receptors (MC1-5) that are stimulated by endogenous agonists and antagonized by the only two known endogenous antagonists of GPCRs, agouti and agouti-related protein (

Full text of DOI:10.1021/jm701303z

J. Med. Chem. 2008, 51, 1423–1431
1423
The 1,4-Benzodiazepine-2,5-dione Small Molecule Template Results in Melanocortin Receptor
Agonists with Nanomolar Potencies
Christine G. Joseph,† Krista R. Wilson,† Michael S. Wood, Nicholas B. Sorenson, Dong V. Phan, Zhimin Xiang,
Rachel M. Witek, and Carrie Haskell-Luevano*
Department of Medicinal Chemistry, College of Pharmacy, UniVersity of Florida, GainesVille, Florida 32610
ReceiVed October 16, 2007
The melanocortin system consists of five seven-transmembrane spanning G-protein coupled receptors
(MC1-5) that are stimulated by endogenous agonists and antagonized by the only two known endogenous
antagonists of GPCRs, agouti and agouti-related protein (AGRP). These receptors have been associated
with many physiological functions, including the involvement of the MC4R in feeding behavior and energy
homeostasis, making this system an attractive target for the treatment of obesity. Small-molecule mimetics
of endogenous ligands may result in the development of compounds with properties more suitable for use
as therapeutic agents. The research presented herein involves the synthesis and analysis of 12 melanocortin
receptor agonists using the 1,4-benzodiazepine-2,5-dione template and is the first report of these derivatives
as melanocortin receptor agonists. Structure–activity relationship studies using this privileged structure
template has resulted in molecules with molecular weights around 400 that possess nanomolar agonist potency
at the melanocortin receptors examined in this study.
Introduction
Themelanocortinsystemconsistsoffivereceptors(MC1-5),1–7
which are members of the superfamily of G-protein coupled
receptors (GPCRs).a These receptors are seven transmembrane-
spanning receptors, which upon agonist binding stimulate the
cAMP signal transduction pathway. The melanocortin receptors
are stimulated by the endogenous agonists R-MSH, ꢀ-MSH, γ1-
MSH, γ2-MSH, and ACTH-derived from the proopiomelano-
cortin (POMC) gene transcript. The melanocortin receptors are
antagonized by the only two known endogenous antagonists of
GPCRs, agouti8,9 and agouti-related protein (AGRP).10,11 The
MC1R is expressed in melanocytes and is involved in the
production of melanin, the pigment responsible for skin and
coat coloration.2,12–14 The MC2R is expressed in the adrenal
cortex where it mediates the production of glucocorticoids and
aldosterone.2 The MC3R is expressed in the brain, heart,
gastrointestinal tract, and placenta4,15 and has been shown to
be involved in energy homeostasis, though the mechanism of
action remains to be clarified.16,17 The MC4R is expressed in
the brain6,18 and testis and is involved in energy and weight
homeostasis,19 feeding behavior,20 and sexual function.21,22 The
MC4R knockout mice exhibit hyperphagia, hyperinsulinemia,
hyperglycemia, and increased linear growth when compared
with their wild-type littermates.19 It is hypothesized that potent
Figure 1. Structures of (a) 1,4-benzodiazepin-2-ones and (b) 1,4-
benzodiazepin-2,5-diones.
agonists selective for the MC4R may be used as therapeutic
agents to treat obesity. The MC5R is expressed in peripheral
tissues and is involved in the secretion of lipids from exocrine
glands in mice.7,23
Information obtained from peptide-related SAR studies may
be used for the design of peptidomimetic and small-molecule
templates that are potent and melanocortin receptor selective.
Peptides are not ideally suited for use as drugs due to their large
molecular weight, high rate of first-pass metabolism in the
stomach and small intestine, and extreme sensitivity to changes
in pH or temperature; therefore it is desirable to obtain a small-
molecule drug. In the design of small molecules, the information
obtained from the peptide SAR studies is used to create
conformationally restrained peptides or small molecules. Further
studies are done to optimize the molecule in regards to potency,
selectivity, and toxicity. Small-molecule libraries have become
an important part of the drug discovery process24–26 with
particular emphasis placed upon the preparation and evaluation
of libraries based upon privileged structures. These structures
display a number of different functionalities that result in a
number of potent and specific drugs or candidates for different
therapeutic targets. The 1,4-benzodiazepines are an important
class of privileged templates, and numerous derivatives have
been identified that have selective activities against a diverse
array of biological targets. A subset of the 1,4-benzodiazepines,
the 1,4-benzodiazepine-2,5-diones (Figure 1b), are the focus of
this work. The 1,4-benzodiazepin-2,5-diones (Figure 1b) are
perhaps the next most studied benzodiazepine framework after
the 1,4-benzodiazepin-2-ones (Figure 1a). 1,4-Benzodiazepin-
2,5-diones have been reported to possess anticonvulsant, anxi-
* To whom correspondence should be addressed. Phone: (352)846-2722.
Fax: (352) 392-8182. E-mail: Carrie@cop.ufl.edu.
†
These are equally contributing first authors.
a ACTH, adrenocorticotropic hormone; AGRP, agouti-related protein;
BAL, backbone amide linker resin; cAMP, cyclic 5′-adenosine monophos-
phate; DCM, dichloromethane; DMF, N,N-dimethylformamide; DMS,
dimethylsulfide; DNPH, dinitrophenylhydrazine; EDC, 1-[3-(dimethylami-
no)propyl]-1′-ethylcarbodiimide hydrochloride; GPCR, G-protein coupled
receptor; MC1R, melanocortin-1 receptor; MC2R, melanocortin-2 receptor;
MC3R, melanocortin-3 receptor; MC4R, melanocortin-4 receptor; MC5R,
melanocortin-5 receptor; MCR, melanocortin receptor; MeOH, methanol;
MSH, melanocyte stimulating hormone; NaBH(OAc)3, sodium triacetoxy-
borohydride; nBuLi, N-butyllithium; NMP, N-methyl pyrollidinone; POMC,
proopiomelanocortin; SAR, structure-activity relationship; SEM, standard
error of the mean; THF, tetrahydrofuran; TFA, trifluoroacetic acid; R-MSH,
R-melanocyte stimulating hormone; ꢀ-MSH, ꢀ-melanocyte stimulating
hormone; γ-MSH, γ-melanocyte stimulating hormone.
10.1021/jm701303z CCC: $40.75
2008 American Chemical Society
Published on Web 02/14/2008

1424 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Joseph et al.
Scheme 1a
a Reaction conditions: (a) NaBH(OAC)3, DMF/1% AcOH, rt, 2 h; (b) EDC in NMP, rt, overnight; (c) rt, 30 h, under argon; (d) rt, until pH ) 5; (e)
TFA/Me2S/H2O (90:5:5), rt, 50 h.
Scheme 2a
positions R2 and R3. This compound is a high micromolar full
agonist at the mMC1R but does not exhibit any stimulatory
activity at the other three receptors examined at up to 100 µM
concentrations. Compounds 2 and 3 are substituted with a H at
a Reaction conditions: (a)-78 °C, 30 min, under argon; (b) DMF, -78
°C, 15 min.
position R1 and a benzyl group at R2 and differ in their
substitution at the R3 position. The R3 propyl guanidine moiety
in compound 2 resulted in a molecule that is equipotent (within
the 3-fold inherent experimental error) to compound 1 at the
mMC1R, but does not contain any agonist activity at the other
melanocortin receptors examined. Compound 3, containing
a methyl indole moiety at the R3 position, exhibited only a 70%
maximal stimulatory response at the mMC1R as compared with
the forskolin control at up to 100 µM concentrations and was
unable to stimulate the MC3-5 receptors. Compound 4 contains
9-methyl at R1, benzyl at R2, and 1-benzyl-1H-indol-3-yl-
methyl at the R3 position. The 1-benzyl-1H-indol-3-yl-methyl
moiety at the R3 position in 4 was derived from over alkylation
with benzyl bromide and alkylation of the 1-nitrogen of the
indole ring, and this compound possessed micromolar full
agonist activity at the mMC1R and was devoid of stimulatory
activity at the MC3-5Rs.
Compound 5, containing an 8-chloro substitution at R1, a
benzyl at R2, and a propyl guanidine group at R3, resulted in
full micromolar agonist activity at the mMC1, mMC3, and
mMC5 receptors. Substitution of the R3 guanidinyl group with
the 4-amino butyl moiety in compound 6 resulted in a full
nanomolar agonist activity at all four receptors examined.
Compounds 7-9 all contain an 8-chloro at the R1 position and
a napthylene-2-yl-methyl group in the R2 position, with
differential substitution at R3. Comparison of compounds 6 and
8 resulted in decreased potency of 8 by the addition of the
naphtyl moiety at the R2 position versus the benzyl moiety of
6. This difference may be attributed to the bulkier nature of the
naphtyl versus benzyl ring that perhaps modifies ligand–receptor
interactions or modified putative π-π stacking interactions
between the ligand and receptor. Compound 7, containing a
napthyl side chain at R2 and a benzyl group at R3, resulted in
a micromolar agonist at the mMC1R, mMC3R, and mMC5R
but was only able to stimulate the mMC4R to 70% maximal
activity observed for the forskolin control at 100 µM concentra-
tions. Compound 8, differing from compound 7 by the presence
of the 4-aminobutyl moiety at the R3 position, is equipotent
olytic, and antitumor properties, as well as being cholecystokinin
receptor (CCK), opiate receptor, and platelet glycoprotein IIb-
IIIa antagonists,27–29 as well as having herbicidal properties.30
In addition, the 1,4-benzodiazepine-2,5-dione core appears in
a number of natural products.31–33 We hypothesized that small-
molecule 1,4-benzodiazepine-2,5-diones may be created that
mimic the side chains of the Phe-Arg-Trp melanocortin agonist
core tetrapeptide resulting in potent and full agonist activity at
the melanocortin receptors. In order to achieve these goals, a
number of the synthetic schemes for the solid-phase organic
synthesis of benzodiazepines were evaluated.29,30,34–42 The
optimized synthesis approach that we utilized is shown in
Schemes 1 and 2.24,27,30,35 It begins with the attachment of the
R-amino ester to the solid support, followed by acylation with
an anthranilic acid, base-catalyzed lactamization, alkylation, and
cleavage. The sequence relies on the incorporation of three
different components, anthranilic acids (R1), alkylating agents
(R2), and R-amino esters (R3), available commercially with
appropriate side-chain protection. Synthesis with this scheme
also provided high yield and relatively clean crude products.
Twelve compounds were synthesized, analytically characterized,
and pharmacologically characterized for functional agonist
activity at the melanocortin receptors (MC1R, and MC3-5Rs).
Results
Analytical characterization and agonist functional data for
the 1,4-benzodiazepine-2,5-dione molecules (Figure 2) synthe-
sized in this study are summarized in Tables 1 and 2,
respectively. All functional agonist data are expressed as
nanomolar EC50 values with the associated standard error of
the mean (SEM) derived from at least three independent
experiments. Compounds that did not result in agonist activity
(>100 000 in Table 2) were not competitive antagonists at any
of the melanocortin receptors examined herein. Compound 1 is
substituted with a H at position R1, and benzyl groups at

Nanomolar Melanocortin Receptor Agonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1425
Figure 2. Structures of the ligands designed, synthesized, and pharmacologically tested in this study.
Table 1. Analytical Data of 1,4-Benzodiazepine-2,5-dione Analoguesa
HPLC retention HPLC retention
time (min)
(system 1)
time (min)
(system 2)
m/z
m/z
peptide
R1
R2
R3
% purity (M, calcd) (M + 1, expt)
1
2
3
4
5
6
7
8
H
H
H
benzyl
benzyl
benzyl
benzyl
propyl guanidine
1H-indol-3-yl-methyl
1-benzyl-1H-indol-3-yl-methyl
propyl guanidine
21.5
12.7
21.2
27.2
15.3
14.9
26.5
17.6
31.1
25.5
18.5
20.4
28.3
18.4
34.1
34.3
22.3
22.3
41.9b
26.0
41.4b
40.7b
26.2
33.2b
>98
>99
>95
>96
>99
>99
>99
>97
>97
>98
>98
>99
356.4
365.4
395.5
499.6
399.9
371.9
440.9
421.9
480.0
432.5
308.4
322.4
357.2
366.4
396.0
500.4
401.1
372.2
441.9
422.5
481.5
433.1
309.5
323.1
9-methyl benzyl
8-chloro benzyl
8-chloro benzyl
8-chloro naphthalen-2-yl-methyl benzyl
8-chloro naphthalen-2-yl-methyl 4-aminobutyl
8-chloro naphthalene-2-yl-methyl 1H-indol-3-yl-methyl
H
H
H
4-aminobutyl
9
10
11
12
biphenyl-2-yl-methyl
propyl
butyl
benzyl
benzyl
benzyl
a HPLC system 1 (10% acetonitrile in 0.1% trifluoroacetic acid/water and a gradient to 90% acetonitrile over 35 min) or solvent system 2 (10% methanol
in 0.1% trifluoroacetic acid/water and a gradient to 90% methanol over 35 min). b Indicates solvent system 2 that in which a gradient to 90% methanol over
45 min was used. An analytical Vydac C18 column (Vydac 218TP104) was used with a flow rate of 1.5 mL/min. The peptide purity was determined by
HPLC at a wavelength of 214 nm.
with compound 7 at the mMC1R yet was only able to generate
weak stimulatory responses at the mMC3R, MC4R, and
mMC5R at 100 µM concentrations. Compound 9, containing
the 1H-indol-3-yl-methyl moiety at the R3 position instead of
the benzyl group in compound 7, was devoid of any stimulatory
activity at any of the receptors at concentrations up to 100 µM.
Compounds 10-12 contain a H at the R1 position, a benzyl
group at the R3 position, and are differentially modified at the
R2 position. Compound 10, containing a biphenyl-2-yl-methyl
moiety at the R2 position, is a nanomolar agonist at the mMC1R,
is 18-fold mMC1R versus mMC3R selective, is ca. 9-fold
mMC1R versus mMC4R selective, is a micromolar agonist at
the mMC3R and the mMC4R, and is only able to stimulate a
60% maximal forskolin response at the mMC5R. When the R2
position contains the propyl moiety in compound 11, micromolar
full agonist activity is observed at the mMC1R, mMC4R, and
mMC5R with only an 80% maximal forskolin response at the
mMC3R. When the R2 position contains the butyl group in
compound 12, only a 70% maximal forskolin stimulation
response is observed at the mMC1R and no stimulatory response
is observed at any of the other receptors.
Discussion
The concept of heterocyclic small molecule melanocortin
agonists based on a ꢀ-turn motif has been reported as early as
1999 and were identified as micromolar mMC1R agonists.43
Since the association between the MC4R subtype and human
obesity and energy homeostasis was recognized, the MC4R has
been a target for the design of potent and receptor-selective
small-molecule agonists.44–47 Some of the small-molecule
templates possessing nanomolar agonist potency at the mMC4R

1426 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Joseph et al.
Table 2. Functional Activity of 1,4-Benzodiazepine-2,5-dione Analogues at the Mouse Melanocortin Receptorsa
agonist EC50 (nM)
peptide
structure
mMC1R
mMC3R
mMC4R
mMC5R
R-MSH
Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2
0.7 ( 0.23
2.14 ( 0.46
2.56 ( 0.29
2.0 ( 0.20
structure
agonist EC50 (nM)
peptide
R1
R2
R3
mMC1R
mMC3R
mMC4R
>100000
>100000
>100000
>100000
mMC5R
1
2
3
4
5
6
7
8
H
H
H
benzyl
benzyl
benzyl
benzyl
32850 ( 8200
16450(6000
70% @ 100 µM >100000
>100000
>100000
>100000
propyl guanidine
1H-indol-3-yl-methyl
1-benzyl-1H-indol-3-yl-methyl 21600 ( 6900 >100000
propyl guanidine
4-aminobutyl
>100000
>100000
>100000
9-methyl benzyl
8-chloro benzyl
8-chloro benzyl
8-chloro naphthalen-2-yl-methyl benzyl
8-chloro naphthalen-2-yl-methyl 4-aminobutyl
8-chloro naphthalen-2-yl-methyl 1H-indol-3-yl-methyl
1800 ( 100
48 ( 23
4700 ( 1270
3400 ( 700
>100000
230 ( 0.27
6900 ( 2600
70% @ 100 µM >100000
30650 ( 23000 60% @ 100 µM 17000 ( 4500
320 ( 80
23300 ( 2500
240 ( 75
70% @ 100 µM 4350 ( 1700
87 ( 38
30% @ 100 µM 30% @ 100 µM 65% @ 10 µM
9
>100000
4160 ( 70
>100000
1970 ( 1230
>100000
60% @ 100 µM
10
11
12
H
H
H
biphenyl-2-yl-methyl
benzyl
benzyl
benzyl
propyl
butyl
80% @ 100 µM 35000 ( 16000 3267 ( 1010
>100000
>100000
a Indicated errors represent the standard error of the mean determined from at least four independent experiments. >100000 indicates that no agonist
activity was observed at up to 100 µM. Agonists that possessed some stimulatory response at 100 µM concentrations are indicated as percent response
relative to the maximal response observed for the forskolin control.
contain similar features such as a para(chloro)-substituted phenyl
ring and a heterocyclic scaffold.46
at all four receptors. Studies of π-π stacking in peptides have
suggested that the addition of an electron-withdrawing group
in the para position on a phenyl ring increases activity.63–65
Addition of chlorine creates an electron-deficient ring, which
interacts favorably with the relatively electron-rich ring of
tyrosine.66 Additional studies have shown that in proteins, a
T-shaped conformation is favored in π-stacking, resulting in a
greater decrease in free energy than the sandwich conformation.67,68
Based on this information, it may be proposed that the
halogenated ring of the benzodiazepine template may interact
with the tyrosine of the receptor in a T-shaped conformation to
create a favorable π-stacking charge-transfer relationship, result-
ing in increased agonist activity.
The most interesting compound discovered in this study is
compound 6, which is a low nanomolar agonist at all four
melanocortin receptors (Table 2). This compound has a chlorine
in position R1, a benzyl group in position R2, and a 4-ami-
nobutyl group in position R3. It is interesting to note that when
a propyl guanidine was substituted at position R3 (compound
5), the resulting compound exhibited a significant decrease in
agonist activity at all melanocortin receptors. It appears,
therefore, that the decreased cationic character of the 4-ami-
nobutyl group compared with the guanidine provides optimal
intermolecular interactions with the electrostatic binding pocket
of the melanocortin receptors.
The benzodiazepine template has been widely studied as a
scaffold for nonpeptide drugs. Derivatives have been identified
that have properties as anxiolytics, cholecystokinin receptor
antagonists,48 κ-opioid receptor agonists,49 oxytocin receptor
antagonists,50,51 endothelin antagonists,52 HIV Tat antagonists,53
and reverse transcriptase inhibitors,54 among others. Because
many targets are members of the G-protein coupled receptor
superfamily (i.e., cholecystokinin, κ-opioid, oxytocin, and
endothelin receptors), we hypothesized that benzodiazepine
derivatives could be designed as melanocortin ligands. The 1,4-
benzodiazepine-2,5-dione molecules synthesized in this study
was designed to interact with proposed binding sites in the
melanocortin receptors. Consistent with melanocortin in Vitro
receptor mutagenesis studies,14,55–57 homology molecular mod-
eling of the mMC1R58 and the MC4R59–62 suggest the presence
of hydrophobic and electrostatic domains within the putative
binding pocket that are important for melanocortin receptor
agonist molecular recognition and stimulation. Thus, using this
information and the design strategy of mimicking the melano-
cortin agonist Phe-Arg-Trp side chains, we designed the ligands
reported herein. Based on the results of this study, we speculate
that the p(Cl)-phenyl ring of the benzodiazepine and an aryl
side chain putatively interact with the hydrophobic receptor
domains and that a cationic component interacts with the anionic
receptor binding domain.
When a naphthalene group was inserted at position R2
(compounds 7–9), the compounds showed decreased agonist
activity at all receptors. This is consistent with previous SAR
studies on peptide agonists, which showed that when the DPhe
of the tetrapeptide His-DPhe-Arg-Trp was replaced with a bulky
aromatic group such as DNal(2′), the peptide was converted
into an antagonist at the mMC3R and the mMC4R.69 The most
significant example of this is the mMC3-4R antagonist
SHU9119, which exhibits partial agonist activity and potent
antagonist activity at the mMC3R and mMC4R.69 When a
The compounds synthesized in this study have provided
information about the mMC1R and mMC4R small-molecule
pharmacophore. There were several compounds synthesized that
only showed agonist activity at the mMC1R. These compounds
all had either a hydrogen or a methyl group in the R1 position,
a benzyl group in the R2 position, and either an aromatic or a
basic group in the R3 position. Interestingly, the addition of
chlorine at the R1 position converts the molecule into an agonist

Nanomolar Melanocortin Receptor Agonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1427
DCM (3 × 20 mL), MeOH (2 × 20 mL), and DCM (3 × 20 mL).
A sample of the resin was removed and tested for the absence of
a secondary amine (chloranil test) and the presence of a tertiary
amine (bromophenol blue test). To an oven-dried round-bottomed
flask was added a magnetic stir bar and acetanilide (24 equiv, 7.2
mmol) followed by 10 mL of THF. The flask was purged with
argon gas and cooled to -78 °C in a dry ice/acetone bath. A hexane
solution of n-BuLi (20 equiv, 6.0 mmol) was added dropwise over
10 min followed by rapid stirring for 30 min at -78 °C (Scheme
2). After 30 min, 10 mL of DMF was added to homogenize the
solution, and the solution was stirred gently for 15 min. The flask
was then allowed to slowly come to rt. The acylated resin was
transferred to a round-bottomed flask and purged with argon gas.
The lithium salt was transferred to the flask containing the resin
and mixed gently for 30 h at rt. Following cyclization, the pH of
the solution was tested (pH ≈ 13–14) and then the alkylating agent
(40 equiv, 12.0 mmol) was added, and the solution was stirred at
rt until the pH of the solution was around 5. The resin was then
transferred back to a reaction vessel and washed with DMF (7 ×
20 mL), DCM (7 × 20 mL), MeOH (3 × 20 mL), and DCM (3 ×
20 mL). The resin was air-dried and then cleaved with a cleavage
cocktail of TFA/Me2S/H2O (90:5:5) for 50 h. Resin was removed
by filtration, and the filtrate was evaporated and lyophilized.
1,3-Dibenzyl-3,4-dihydro-1H-benzo[e][1,4]diazepine-2,5-di-
one (1). BAL resin (333 mg, 0.9 mmol/g) was mixed with sodium
triacetoxyborohydride [NaBH(OAc)3] (636 mg, 3.0 mmol) and
L-Phe-OMe·HCl (647 mg, 3.0 mmol) in 1% acetic acid/DMF for
2 h and washed with DMF, DCM, and MeOH. The resin containing
the amino ester was then treated with EDC (690 mg, 3.6 mmol) in
NMP and 2-aminobenzoic acid (411 mg, 3.0 mmol) overnight and
washed as before. A solution of acetanilide (973 mg, 7.2 mmol)
and N-butyllithium (252 µL, 3.0 mmol) in THF was stirred for 30
min, and then 15 mL of DMF was added; this solution was then
added to the acylated resin and stirred under argon for 30 h. Benzyl
bromide (1.4 mL, 12 mmol) was added following lactamization,
and the solution was stirred until pH ) 5. The resin was then
washed and dried in a desiccator. The compound was cleaved from
the resin using 90% TFA, 5% dimethylsulfide, and 5% H2O for
50 h. Semipreparative HPLC on an RP-HPLC C18 bonded silica
column (flow rate ) 5.0 mL/min, Vydac 218TP1010, 1.0 cm × 25
cm) using a gradient of 46–56% acetonitrile in 0.1% TFA/H2O over
10 min and collection from 8.0 to 8.7 min gave a compound that
was >95% pure. M + 1 ) 357.2 by MALDI-TOF mass spectrom-
etry. 1H NMR (600 MHz, DMSO-d6) δ 8.77 (d, J ) 1.83 Hz, 1H),
7.56 (d, J ) 2.0 Hz, 1H), 7.53–7.51 (m, 1H), 7.47 (d, J ) 2.3 Hz,
1H), 7.32 (d, J ) 2.0 Hz, 2H), 7.26–7.22 (m, 5H), 7.19–7.17 (m,
2H), 7.08 (d, J ) 2.0 Hz, 2H), 5.34 (d, J ) 4.3 Hz, 1H), 4.98 (d,
J ) 4.3 Hz, 1H), 4.03 (dt, J ) 2.3, 1.7 Hz, 1H), 4.19 (dd, J ) 1.5,
4.0 Hz, 1H), 2.96 (dd, J ) 4.0, 2.5 Hz, 1H).
N-[3-(1-Benzyl-2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]-
diazepin-3-yl)-propyl]-guanidine (2). BAL resin (333 mg, 0.9
mmol/g) was mixed with sodium triacetoxyborohydride [NaB-
H(OAc)3] (636 mg, 3.0 mmol) and L-Arg(Pbf)-OMe·HCl (1321
mg, 3.0 mmol) in 1% acetic acid/DMF for 2 h and washed with
DMF, DCM, and MeOH. The resin containing the amino ester was
then treated with EDC (690 mg, 3.6 mmol) in NMP and 2-ami-
nobenzoic acid (411 mg, 3.0 mmol) overnight and washed as before.
A solution of acetanilide (973 mg, 7.2 mmol) and N-butyllithium
(252 µL, 3.0 mmol) in THF was stirred for 30 min, and then 15
mL of DMF was added; this solution was then added to the acylated
resin and stirred under argon for 30 h. Benzyl bromide (1.4 mL,
12 mmol) was added following lactamization, and the solution was
stirred until pH ) 5. The resin was then washed and dried in a
desiccator. The compound was cleaved from the resin using 90%
TFA, 5% dimethylsulfide, and 5% H2O for 50 h. Semipreparative
HPLC on an RP-HPLC C18 bonded silica column (flow rate )
5.0 mL/min, Vydac 218TP1010, 1.0 cm × 25 cm) using a gradient
of 23–33% acetonitrile in 0.1% TFA/H2O over 10 min and
collection from 7.8 to 9.0 min gave a compound that was >95%
pure. M + 1 ) 366.4 by MALDI-TOF mass spectrometry. 1H NMR
(400 MHz, DMSO-d6) δ 8.68 (d, J ) 3.5 Hz, 1H), 7.31 (dd, J )
biphenyl was inserted at position R2 (compound 10), the
compound was a low nanomolar agonist at the mMC1R and a
low micromolar agonist at the mMC3-4R. This suggests that
there may be an additional interaction with the greater negative
potential on the face of the conjugated π system of napthylene
that is not present with the nonconjugated rings of the biphenyl
group that results in loss of agonist activity.
It should also be observed that an aromatic group in the R2
position provides for increased agonist activity. When this
position contained a propyl (compound 11) or butyl (compound
12) group, the agonist activity at all four receptors was
decreased. It is also interesting to note that the shorter propyl
chain in compound 11 was significantly more potent that the
butyl chain in compound 12 at all receptors.
Conclusions
This research has resulted in the identification and first report
of several 1,4-benzodiazepine-2,5-dione derivatives that exhibit
nanomolar agonist activity at the mouse melanocortin receptors.
These compounds are easy to synthesize due to the use of solid-
phase organic synthesis and may be readily purified allowing
for the design of a large number of compounds. When
substituted with building blocks that mimic the active sequence
of peptide melanocortin ligands, this template has the potential
to create compounds with increasing potency that may be used
in the future as drugs to treat obesity.
Experimental Design and Methods
Synthesis was performed in a manual reaction vessel (Peptides
International, Louisville, KY); mixing was accomplished with
nitrogen. All reagents used were ACS grade or better and used
without further purification. BAL resin (Backbone amide linker
resin) was purchased from Advanced ChemTech (Louisville, KY).
Dichloromethane, acetic acid, N-methyl pyrollidinone, acetonitrile,
acetone, and methanol were purchased from Fisher (Fair Lawn,
NJ). N,N-Dimethylformamide was purchased from Burdick and
Jackson (McGaw Park, IL). Sodium triacetoxyborohydride, 1-[3-
(dimethylamino)propyl]-1′-ethylcarbodiimide hydrochloride, ac-
etanilide, N-butyllithium (10 M in hexanes), benzyl bromide,
2-(bromomethyl)-napthalene, 2-phenylbenzylbromide, dimethyl-
sulfide, 1-iodopropane, 1-iodobutane, tetrahydrofuran, and trifluo-
roacetic acid were purchased from Sigma-Aldrich (St. Louis, MO).
2-Amino-4-chlorobenzoic acid and 2-amino-3-methylbenzoic acid
were purchased from Acros Organics (Morris Plains, NJ). Arginine-
(Pbf) methyl ester, and lysine-(Boc) methyl ester were purchased
from Bachem (Torrence, CA). Phenylalanine methyl ester was
purchased from Advanced Chemtech (Louisville, KY). Tryptophan
methyl ester was purchased from Fluka (St. Louis, MO). 2-Ami-
nobenzoic acid was purchased from Alfa Aesar (Ward Hill, MA).
Ligand Synthesis. Synthesis was performed on solid phase using
the method described in Scheme 1. Bal resin was swollen in DCM
for 1 h and tested for the presence of an aldehyde using the DNPH
method. To a reaction vessel containing 0.3 mmol of BAL resin
was added NaBH(OAc)3 (10 equiv, 3.0 mmol) dissolved in 1%
AcOH/DMF, generating a white suspension. The suspension was
mixed until the mixture became clear. Methyl ester (10 equiv, 3.0
mmol) was then added, and the solution was stirred for 3–4 h. The
resin was filtered and washed with DMF (3 × 20 mL), DCM (3 ×
20 mL), MeOH (2 × 20 mL), and DCM (3 × 20 mL). A sample
of the resin was removed and tested for the presence of a secondary
amine (chloranil test) and the absence of an aldehyde (DNPH test
for aldehydes). The resin was dissolved in N-methyl pyrollidinone
(NMP) followed by the addition of 1-[3-(dimethylamino)propyl]-
1′-ethylcarbodiimide hydrochloride (EDC). After the EDC was
completely dissolved, the 2-aminobenzoic acid was added slowly
to minimize side reaction that may occur with an unprotected amine,
and the reaction was mixed with nitrogen overnight. Resin was
filtered and washed with NMP (2 × 20 mL), DMF (3 × 20 mL),

1428 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Joseph et al.
5.0, 1.0 Hz, 1H), 7.54–7.50 (m, 1H), 7.44 (d, J ) 4.75 Hz),
7.29–7.17 (m, 4H), 7.07 (d, J ) 4.25 Hz, 2H), 5.29 (d, J ) 10 Hz,
1H), 4.98 (d, J ) 10.0 Hz, 1H), 3.80–3.78 (m, 1H), 3.32–3.30 (m,
2H), 3.29 (s, 2H), 3.07–3.05 (m, 2H), 1.86–1.78 (m, 1H), 1.70–1.62
(m, 1H), 1.68–1.62 (m, 1H), 1.58–1.45 (m, 2H).
and the solution was stirred until pH ) 5. The resin was then
washed and dried in a desiccator. The compound was cleaved from
the resin using 90% TFA, 5% dimethylsulfide, and 5% H2O for
50 h. Semipreparative HPLC on an RP-HPLC C18 bonded silica
column (flow rate ) 5.0 mL/min, Vydac 218TP1010, 1.0 cm × 25
cm) using a gradient of 28–38% acetonitrile in 0.1% TFA/H2O over
10 min and collected from 6.9 to 7.5 min gave a compound that
was >95% pure. M + 1 ) 401.1 by MALDI-TOF mass spectrom-
etry. 1H NMR (400 MHz, DMSO-d6) δ 8.77 (d, J ) 3.25 Hz, 1H),
7.63 (d, J ) 5.25 Hz, 1H), 7.58 (d, J ) 1.25 Hz, 1H), 7.34 (dd, J
) 5.5, 1.25 Hz, 1H), 7.26–7.22 (m, 2H), 7.20–7.16 (m, 1H), 7.06
(d, J ) 4.25 Hz, 2H), 5.34 (d, J ) 10.25 Hz, 1H), 4.99 (d, J )
10.0 Hz, 1H), 3.10–3.03 (m, 2H), 1.88 (s, 5H), 1.84–1.78 (m, 1H),
1.71–1.59 (m, 1H), 1.58–1.42 (m, 2H).
1-Benzyl-3-(1H-indol-3-ylmethyl)-3,4-dihydro-1H-benzo[e][1,4]-
diazepine-2,5-dione (3). BAL resin (333 mg, 0.9 mmol/g) was
mixed with sodium triacetoxyborohydride [NaBH(OAc)3] (636 mg,
3.0 mmol) and L-Trp-OMe·HCl (764 mg, 3.0 mmol) in 1% acetic
acid/DMF for 2 h and washed with DMF, DCM, and MeOH. The
resin containing the amino ester was then treated with EDC (690
mg, 3.6 mmol) in NMP and 2-aminobenzoic acid (411 mg, 3.0
mmol) overnight and washed as before. A solution of acetanilide
(973 mg, 7.2 mmol) and N-butyllithium (252 µL, 3.0 mmol) in
THF was stirred for 30 min, and then 15 mL of DMF was added;
this solution was then added to the acylated resin and stirred under
argon for 30 h. Benzyl bromide (1.4 mL, 12 mmol) was added
following lactamization, and the solution was stirred until pH ) 5.
The resin was then washed and dried in a desiccator. The compound
was cleaved from the resin using 90% TFA, 5% dimethylsulfide,
and 5% H2O for 50 h. Semipreparative HPLC on an RP-HPLC
C18 bonded silica column (flow rate ) 5.0 mL/min, Vydac
218TP1010, 1.0 cm × 25 cm) using a gradient of 43–53%
acetonitrile in 0.1% TFA/H2O over 10 min and collection from
6.7 to 7.0 min gave a compound that was >95% pure. M + 1 )
3-(4-Aminobutyl)-1-benzyl-8-chloro-3,4-dihydro-1H-
benzo[e][1,4]diazepine-2,5-dione (6). BAL resin (333 mg, 0.9
mmol/g) was mixed with sodium triacetoxyborohydride [NaBH
(OAc)3] (636 mg, 3.0 mmol) and L-Lys(Boc)-OMe·HCl (1187 mg,
3.0 mmol) in 1% acetic acid/DMF for 2 h and washed with DMF,
DCM, and MeOH. The resin containing the amino ester was then
treated with EDC (690 mg, 3.6 mmol) in NMP and 2-amino-4-
chlorobenzoic acid (515 mg, 3.0 mmol) overnight and washed as
before. A solution of acetanilide (973 mg, 7.2 mmol) and N-
butyllithium (252 µL, 3.0 mmol) in THF was stirred for 30 min
and then 15 mL of DMF was added; this solution was then added
to the acylated resin and stirred under argon for 30 h. Benzyl
bromide (1.4 mL, 12 mmol) was added following lactamization,
and the solution was stirred until pH ) 5. The resin was then
washed and dried in a desiccator. The compound was cleaved from
the resin using 90% TFA, 5% dimethylsulfide, and 5% H2O for
50 h. Semipreparative HPLC on an RP-HPLC C18 bonded silica
column (flow rate ) 5.0 mL/min, Vydac 218TP1010, 1.0 cm × 25
cm) using a gradient of 26–36% acetonitrile in 0.1% TFA/H2O over
10 min and collected from 7.8 to 8.5 min gave a compound that
was >95% pure. M + 1 ) 372.2 by MALDI-TOF mass spectrom-
etry. 1H NMR (400 MHz, DMSO-d6) δ 8.75 (d, J ) 3.75 Hz, 1H),
7.65–7.61 (m, 2H), 7.36 (dd, J ) 5.25, 1.0 Hz, 1H), 7.28–7.18 (m,
3H), 7.09 (d, J ) 4.5 Hz, 2H), 5.39 (d, J ) 10.0 Hz, 1H), 5.00 (d,
J ) 10.0 Hz, 1H), 3.82–3.77 (m, 1H), 2.76 (t, J ) 3.65 Hz, 2H),
1.84–1.66 (m, 2H), 1.57–1.48 (m, 2H), 1.38–1.28 (m, 4H).
3-Benzyl-8-chloro-1-naphthalen-2-ylmethyl-3,4-dihydro-1H-
benzo[e][1,4]diazepine-2,5-dione (7). BAL resin (333 mg, 0.9
mmol/g) was mixed with sodium triacetoxyborohydride [NaBH
(OAc)3] (636 mg, 3.0 mmol) and L-Phe-OMe·HCl (647 mg, 3.0
mmol) in 1% acetic acid/DMF for 2 h and washed with DMF,
DCM, and MeOH. The resin containing the amino ester was then
treated with EDC (690 mg, 3.6 mmol) in NMP and 2-amino-4-
chlorobenzoic acid (515 mg, 3.0 mmol) overnight and washed as
before. A solution of acetanilide (973 mg, 7.2 mmol) and N-
butyllithium (252 µL, 3.0 mmol) in THF was stirred for 30 min,
and then 15 mL of DMF was added; this solution was then added
to the acylated resin and stirred under argon for 30 h. 2-(Bromom-
ethyl)-napthalene (2.7 g, 12 mmol) was added following lactam-
ization, and the solution was stirred until pH ) 5. The resin was
then washed and dried in a desiccator. The compound was cleaved
from the resin using 90% TFA, 5% dimethylsulfide, and 5% H2O
for 50 h. Semipreparative HPLC on an RP-HPLC C18 bonded silica
column (flow rate ) 5.0 mL/min, Vydac 218TP1010, 1.0 cm × 25
cm) using a gradient of 55–65% acetonitrile in 0.1% TFA/H2O over
10 min and collection from 7.5 to 8.2 min gave a compound that
was >95% pure. M + 1 ) 441.9 by MALDI-TOF mass spectrom-
etry. 1H NMR (400 MHz, DMSO-d6) δ 8.92 (d, J ) 4.0 Hz, 1H),
7.86–7.83 (m, 1H), 7.81 (d, J ) 5.5 Hz, 1H), 7.75–7.72 (m, 1H),
7.68 (d, J ) 1.3 Hz, 1H), 7.58 (s, 1H), 7.55 (d, J ) 5.3 Hz, 1H),
7.51–7.45 (m, 2H), 7.36–7.34 (m, 2H), 7.31–7.24 (m, 3H),
7.23–7.18 (m, 2H), 5.56 (d, J ) 10.0 Hz, 1H), 5.15 (d, J ) 10.0
Hz, 1H), 4.19–4.14 (m, 1H), 3.19 (dd, J ) 3.5, 8.8 Hz, 1H), 2.98
(dd, J ) 8.8, 5.5 Hz, 1H).
1
396.0 by MALDI-TOF mass spectrometry. H NMR (400 MHz,
DMSO-d6) δ 10.83 (d, J ) 1.0 Hz, 1H), 8.70 (d,J ) 4.0 Hz, 1H),
7.52 (d,J ) 1.0 Hz 1H), 7.50–7.44 (m, 3H), 7.29 (d, J ) 5.3 Hz,
2H), 7.23–7.16 (m, 4H), 7.08–7.06 (m, 2H), 7.00 (t, J ) 4.5 Hz,
1H), 6.87 (t, J ) 4.5 Hz, 1H), 5.34 (d, J ) 10 Hz, 1H), 4.95 (d, J
) 10 Hz, 1H), 4.00–3.95 (m, 1H), 3.07 (dd, J ) 5.5, 9.5 Hz, 2H).
1-Benzyl-3-(1-benzyl-1H-indol-3-ylmethyl)-9-methyl-3,4-dihy-
dro-1H-benzo[e][1,4]diazepine-2,5-dione (4). BAL resin (333 mg,
0.9 mmol/g) was mixed with sodium triacetoxyborohydride [NaB-
H(OAc)3] (636 mg, 3.0 mmol) and L-Trp-OMe·HCl (764 mg, 3.0
mmol) in 1% acetic acid/DMF for 2 h and washed with DMF,
DCM, and MeOH. The resin containing the amino ester was then
treated with EDC (690 mg, 3.6 mmol) in NMP and 2-amino-3-
methylbenzoic acid (453 mg, 3.0 mmol) overnight and washed as
before. A solution of acetanilide (973 mg, 7.2 mmol) and N-
butyllithium (252 µL, 3.0 mmol) in THF was stirred for 30 min,
and then 15 mL of DMF was added; this solution was then added
to the acylated resin and stirred under argon for 30 h. Benzyl
bromide (1.4 mL, 12 mmol) was added following lactamization,
and the solution was stirred until pH ) 5. The resin was then
washed and dried in a desiccator. The compound was cleaved from
the resin using 90% TFA, 5% dimethylsulfide, and 5% H2O for
50 h. Semipreparative HPLC on an RP-HPLC C18 bonded silica
column (flow rate ) 5.0 mL/min, Vydac 218TP1010, 1.0 cm × 25
cm) using a gradient of 52–67% acetonitrile in 0.1% TFA/H2O over
10 min and collection from 9.7 to 10.4 min gave a compound that
was >95% pure. M + 1 ) 500.4 by MALDI-TOF mass spectrom-
etry. 1H NMR (400 MHz, DMSO-d6) δ 8.62 (d, J ) 3.8 Hz, 1H),
7.51 (d, J ) 5.5 Hz, 1H), 7.46 (d, J ) 5.3 Hz, 1H), 7.35 (d, J )
4.8 Hz, 2H), 7.30–7.17 (m, 8H), 7.12–7.10 (m, 2H), 7.05–7.01 (m,
3H), 6.92 (t, J ) 4.5 Hz, 1H), 5.40 (d, J ) 9.4 Hz, 1H), 5.33 (s,
2H), 4.28 (d, J ) 9.4 Hz, 1H), 3.86–3.85 (m, 1H), 3.00 (dd, J )
5.3, 9.5 Hz, 2H), 2.40 (s, 3H).
N-[3-(1-Benzyl-8-chloro-2,5-dioxo-2,3,4,5-tetrahydro-1H-
benzo[e][1,4]diazepin-3-yl)-propyl]-guanidine (5). BAL resin (333
mg, 0.9 mmol/g) was mixed with sodium triacetoxyborohydride
[NaBH(OAc)3] (636 mg, 3.0 mmol) and L-Arg(Pbf)-OMe·HCl
(1321 mg, 3.0 mmol) in 1% acetic acid/DMF for 2 h and washed
with DMF, DCM, and MeOH. The resin containing the amino ester
was then treated with EDC (690 mg, 3.6 mmol) in NMP and
2-amino-4-chlorobenzoic acid (515 mg, 3.0 mmol) overnight and
washed as before. A solution of acetanilide (973 mg, 7.2 mmol)
and N-butyllithium (252 µL, 3.0 mmol) in THF was stirred for 30
min and then 15 mL of DMF was added; this solution was then
added to the acylated resin and stirred under argon for 30 h. Benzyl
bromide (1.4 mL, 12 mmol) was added following lactamization,
3-(4-Aminobutyl)-8-chloro-1-naphthalen-2-ylmethyl-3,4-dihy-
dro-1H-benzo[e][1,4]diazepine-2,5-dione (8). BAL resin (333 mg,
0.9 mmol/g) was mixed with sodium triacetoxyborohydride [NaBH

Nanomolar Melanocortin Receptor Agonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1429
(OAc)3] (636 mg, 3.0 mmol) and L-Lys(Boc)-OMe·HCl (890 mg,
3.0 mmol) in 1% acetic acid/DMF for 2 h and washed with DMF,
DCM, and MeOH. The resin containing the amino ester was then
treated with EDC (690 mg, 3.6 mmol) in NMP and 2-amino-4-
chlorobenzoic acid (515 mg, 3.0 mmol) overnight and washed as
before. A solution of acetanilide (973 mg, 7.2 mmol) and N-
butyllithium (252 µL, 3.0 mmol) in THF was stirred for 30 min,
and then 15 mL of DMF was added; this solution was then added
to the acylated resin and stirred under argon for 30 h. 2-(Bromom-
ethyl)-napthalene (2.7 g, 12 mmol) was added following lactam-
ization, and the solution was stirred until pH ) 5. The resin was
then washed and dried in a desiccator. The compound was cleaved
from the resin using 90% TFA, 5% dimethylsulfide, and 5% H2O
for 50 h. Semipreparative HPLC on an RP-HPLC C18 bonded silica
column (flow rate ) 5.0 mL/min, Vydac 218TP1010, 1.0 cm × 25
cm) using a gradient of 35–45% acetonitrile in 0.1% TFA/H2O over
10 min and collection from 6.0 to 6.8 min gave a compound that
was >95% pure. M + 1 ) 422.5 by MALDI-TOF mass spectro-
metry. 1H NMR (400 MHz, DMSO-d6) δ 8.83–8.81 (m, 1H),
7.87–7.85 (m, 1H), 7.82 (d, J ) 5.25 Hz, 1H), 7.78–7.75 (m, 1H),
7.70 (d, J ) 0.75 Hz, 1H), 7.64–7.61 (m, 1H), 7.52–7.46 (m, 1H),
7.34 (dd, J ) 5.25, 1.25 Hz, 1H), 7.21 (dd, J ) 5.0, 1.0 Hz, 1H),
5.58 (d, J ) 10.25 Hz, 1H), 5.15 (d, J ) 10.0 Hz, 1H), 2.70–2.67
(m, 2H), 1.90 (s, 2H), 1.86–1.80 (m, 1H), 1.77–1.71 (m, 1H),
1.47–1.34 (m, 4H).
acetonitrile in 0.1% TFA/H2O over 10 min and collection from
7.1 to 7.9 min gave a compound that was >95% pure. M + 1 )
1
433.1 by MALDI-TOF mass spectrometry. H NMR (400 MHz,
DMSO- d6) δ 8.79 (d, J ) 4.0 Hz, 1H), 7.56 (dd, J ) 1.0, 2.25
Hz, 1H), 7.48- 7.37 (m, 4H), 7.31–7.22 (m, 9H), 7.20–7.15 (m,
2H), 7.09–7.04 (m, 2H), 5.16 (d, J ) 10.0 Hz, 1H), 4.89 (d, J )
10.0 Hz, 1H), 4.02–3.97 (m, 1H), 3.14 (dd, J ) 3.5, 8.8 Hz, 1H),
2.92 (d, J ) 8.8, 5.25 Hz, 1H).
3-Benzyl-1-propyl-3,4-dihydro-1H-benzo[e][1,4]diazepine-2,5-
dione (11). BAL resin (333 mg, 0.9 mmol/g) was mixed with
sodium triacetoxyborohydride [NaBH(OAc)3] (636 mg, 3.0 mmol)
and L-Phe-OMe·HCl (647 mg, 3.0 mmol) in 1% acetic acid/DMF
for 2 h and washed with DMF, DCM, and MeOH. The resin
containing the amino ester was then treated with EDC (690 mg,
3.6 mmol) in NMP and 2-aminobenzoic acid (411 mg, 3.0 mmol)
overnight and washed as before. A solution of acetanilide (973 mg,
7.2 mmol) and N-butyllithium (252 µL, 3.0 mmol) in THF was
stirred for 30 min, and then 15 mL of DMF was added; this solution
was then added to the acylated resin and stirred under argon for
30 h. 1-Iodopropane (1.2 mL, 12 mmol) was added following
lactamization, and the solution was stirred until pH ) 5. The resin
was then washed and dried in a desiccator. The compound was
cleaved from the resin using 90% TFA, 5% dimethylsulfide, and
5% H2O for 50 h. Semipreparative HPLC on an RP-HPLC C18
bonded silica column (flow rate ) 5.0 mL/min, Vydac 218TP1010,
1.0 cm × 25 cm) using a gradient of 40–50% acetonitrile in 0.1%
TFA/H2O over 10 min and collection from 6.4 to 7.1 min gave a
compound that was >95% pure. M + 1 ) 309.5 by MALDI-TOF
8-Chloro-3-(1H-indol-3-ylmethyl)-1-naphthalen-2-ylmethyl-
3,4-dihydro-1H-benzo[e][1,4]diazepine-2,5-dione (9). BAL resin
(333 mg, 0.9 mmol/g) was mixed with sodium triacetoxyborohy-
dride [NaBH(OAc)3] (636 mg, 3.0 mmol) and L-Trp-OMe·HCl (764
mg, 3.0 mmol) in 1% acetic acid/DMF for 2 h and washed with
DMF, DCM, and MeOH. The resin containing the amino ester was
then treated with EDC (690 mg, 3.6 mmol) in NMP and 2-amino-
4-chlorobenzoic acid (515 mg, 3.0 mmol) overnight and washed
as before. A solution of acetanilide (973 mg, 7.2 mmol) and
N-butyllithium (252 µL, 3.0 mmol) in THF was stirred for 30 min,
and then 15 mL of DMF was added; this solution was then added
to the acylated resin and stirred under argon for 30 h. 2-(Bromo-
methyl)-napthalene (2.7 g, 12 mmol) was added following lacta-
mization, and the solution was stirred until pH ) 5. The resin was
then washed and dried in a desiccator. The compound was cleaved
from the resin using 90% TFA, 5% dimethylsulfide, and 5% H2O
for 50 h. Semipreparative HPLC on an RP-HPLC C18 bonded silica
column (flow rate ) 5.0 mL/min, Vydac 218TP1010, 1.0 cm × 25
cm) using a gradient of 52–62% acetonitrile in 0.1% TFA/H2O over
10 min and collected from 8.0 to 8.6 min gave a compound that
was >95% pure. M + 1 ) 481.5 by MALDI-TOF mass spectrom-
1
mass spectrometry. H NMR (400 MHz, DMSO- d6) δ 8.73 (d, J
) 4.0 Hz, 1H), 7.61–7.57 (m, 2H), 7.49 (d, J ) 5.25 Hz, 1H),
7.33–7.28 (m, 3H), 7.23 (t, J ) 4.5 Hz, 2H), 7.18–7.14 (m, 1H),
4.23 (dt, J ) 5.0, 8.5 Hz, 1H), 3.89–3.84 (m, 1H), 3.61 (dt, J )
5.0, 7.0 Hz, 2H), 3.13 (dd, J ) 3.25, 8.75 Hz, 1H), 2.91 (dd, J )
8.75, 5.5 Hz, 1H), 1.47–1.38 (m, 1H), 1.33–1.24 (m, 1H), 0.70 (t,
J ) 4.5 Hz, 3H).
3-Benzyl-1-butyl-3,4-dihydro-1H-benzo[e][1,4]diazepine-2,5-
dione (12). BAL resin (333 mg, 0.9 mmol/g) was mixed with
sodium triacetoxyborohydride [NaBH(OAc)3] (636 mg, 3.0 mmol)
and L-Phe-OMe·HCl (647 mg, 3.0 mmol) in 1% acetic acid/DMF
for 2 h and washed with DMF, DCM, and MeOH. The resin
containing the amino ester was then treated with EDC (690 mg,
3.6 mmol) in NMP and 2-aminobenzoic acid (411 mg, 3.0 mmol)
overnight and washed as before. A solution of acetanilide (973 mg,
7.2 mmol) and N-butyllithium (252 µL, 3.0 mmol) in THF was
stirred for 30 min, and then 15 mL of DMF was added; this solution
was then added to the acylated resin and stirred under argon for
30 h. 1-Iodobutane (1.4 mL, 12 mmol) was added following
lactamization, and the solution was stirred until pH ) 5. The resin
was then washed and dried in a desiccator. The compound was
cleaved from the resin using 90% TFA, 5% dimethylsulfide, and
5% H2O for 50 h. Semipreparative HPLC on an RP-HPLC C18
bonded silica column (flow rate ) 5.0 mL/min, Vydac 218TP1010,
1.0 cm × 25 cm) using a gradient of 55–65% acetonitrile in 0.1%
TFA/H2O over 10 min and collection from 4.0 to 4.5 min gave a
compound that was >95% pure. M + 1 ) 323.1 by MALDI-TOF
1
etry. H NMR (400 MHz, DMSO- d6) δ 10.89 (s, 1H), 8.85 (d, J
) 4 Hz, 1H), 7.86–7.84 (m, 1H), 7.81 (d, J ) 5.25 Hz, 1H),
7.74–7.72 (m, 1H), 7.66 (d, J ) 1.25 Hz, 1H), 7.59–7.45 (m, 5H),
7.32 (d, J ) 4.75 Hz, 1H), 7.28–7.21 (m, 3H), 7.04 (t, J ) 4.5 Hz,
1H), 6.92 (t, J ) 5.0, 4.25 Hz, 1H), 5.59 (d, J ) 10 Hz, 1H), 5.15
(d, J ) 9.75 Hz, 1H), 4.14–4.09 (m, 1H), 3.12 (dd, J ) 5.50, 9.5
Hz, 2H).
3-Benzyl-1-biphenyl-2-ylmethyl-3,4-dihydro-1H-benzo[e][1,4]-
diazepine-2,5-dione (10). BAL resin (333 mg, 0.9 mmol/g) was
mixed with sodium triacetoxyborohydride [NaBH(OAc)3] (636 mg,
3.0 mmol) and L-Phe-OMe·HCl (647 mg, 3.0 mmol) in 1% acetic
acid/DMF for 2 h and washed with DMF, DCM, and MeOH. The
resin containing the amino ester was then treated with EDC (690
mg, 3.6 mmol) in NMP and 2-aminobenzoic acid (411 mg, 3.0
mmol) overnight and washed as before. A solution of acetanilide
(973 mg, 7.2 mmol) and N-butyllithium (252 µL, 3.0 mmol) in
THF was stirred for 30 min, and then 15 mL of DMF was added;
this solution was then added to the acylated resin and stirred under
argon for 30 h. 2-Phenylbenzylbromide (2.2 mL, 12 mmol) was
added following lactamization, and the solution was stirred until
pH ) 5. The resin was then washed and dried in a desiccator. The
compound was cleaved from the resin using 90% TFA, 5%
dimethylsulfide, and 5% H2O for 50 h. Semipreparative HPLC on
an RP-HPLC C18 bonded silica column (flow rate ) 5.0 mL/min,
Vydac 218TP1010, 1.0 cm × 25 cm) using a gradient of 55–65%
1
mass spectrometry. H NMR (400 MHz, DMSO- d6) δ 8.72 (d, J
) 4.25 Hz, 1H), 7.62–7.57 (m, 1H), 7.51 (d, J ) 5.0 Hz, 1H),
7.33–7.29 (m, 2H), 7.25–7.21 (m, 1H), 7.19–7.15 (m, 1H), 4.30
(dt, J ) 8.75, 4.5 Hz, 1H), 3.87–3.84 (m, 1H), 3.66–3.59 (m, 1H),
3.13 (dd, J ) 8.75, 3.50 Hz, 1H), 2.91 (dd, J ) 4.5, 5.75 Hz, 1H),
1.39–1.24 (m, 2H), 1.16–1.11 (m, 2H), 0.78 (t, J ) 4.5 Hz, 3H).
Ligand Purification and Analysis. Ligands were purified by
reverse phase high-performance liquid chromatography (RP-HPLC)
using a Shimadzu chromatography system with a photodiode array
detector and a semipreparative RP-HPLC C18 bonded silica column
(Vydac 218TP1010, 1.0 cm × 25 cm) and lyophilized. The purified
peptides were analyzed using RP-HPLC with an analytical Vydac
C18 column (Vydac 218TP104). Molecular mass was determined
by MALDI-TOF mass spectrometry (University of Florida protein
core facility). 1H NMR was performed on a 400 MHz Varian
1
instrument using DMSO-d6 as solvent and TMS as reference. H

1430 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Joseph et al.
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NMR of compound 2 was performed on a 600 MHz Bruker Avance
Console using DMSO-d6 as solvent and TMS as reference.
Melanocortin Receptor Agonist Pharmacology. Pharmacologi-
cal analysis was performed by ꢀ-galactosidase reporter gene assay.70
HEK-293 cells stably expressing the melanocortin receptors were
transiently transfected with CRE/ꢀ-galactosidase reporter gene.
Forty-eight hours post-transfection, the cells were stimulated with
100 µL of peptide (10-4 to 10-12 M) or forskolin (10-4 M) control
in assay medium (DMEM containing 0.1 mg/mL BSA and 0.1 mM
IBMX) for 6 h. To the cell lysate plates, 150 µL of substrate buffer
(60 mM sodium phosphate, 1 mM MgCl2, 10 mM KCl, 5 mM
ꢀ-mercaptoethanol, 2 mg/mL ONPG) was added to each well, and
the plates were incubated at 37 °C. The sample absorbance, OD405
,
was measured using a 96 well plate reader (Molecular Devices).
Data points were normalized both to the relative protein content
and to non-receptor-dependent forskolin stimulation.
Acknowledgment. This work was supported by NIH Grant
DK57080 and an American Diabetes Association Research
Award (C.H.L.). We also thank Mic E. Mouse for graciously
providing the inspiration for compound 1.
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