J . Med. Chem. 2003, 46, 9-11
9
these compounds versus a potent agonist to the MCRs,
[Nle4,D-Phe7]MSH (NDP-MSH), will also be discussed.
Ch em istr y. The dipiperazines (7a -l) were prepared
following the general method described by Vejdelek10
and are summarized in Scheme 1. The R-bromopropio-
phenones 1a -d were reacted with a protected pipera-
zine to provide intermediates 2a -d . These intermedi-
ates were reduced to give alcohols 3a -d , which were
then converted to the corresponding chlorides 4a -d by
treatment with thionyl chloride. Treatment of 4a -d
with an N-substituted piperazine gave the dipiperazines
5a -g. Compounds 6a -g were obtained by deprotection
with potassium hydroxide in methanol. A Mannich
reaction was performed to convert 6a -g to the corre-
sponding dipiperazines 7a -l.
Str u ctu r e-Activity Rela tion sh ip of
(1-Ar yl-2-p ip er a zin yleth yl)p ip er a zin es:
An ta gon ists for th e AGRP /Mela n ocor tin
Recep tor Bin d in g
Premilla N. Arasasingham, Christopher Fotsch,
Xiaohu Ouyang, Mark H. Norman, Michael G. Kelly,
Kevin L. Stark, Bill Karbon, Clarence Hale,
J ames W. Baumgartner, Martha Zambrano,
J anet Cheetham, and Nuria A. Tamayo*
Departments of Small Molecule Drug Discovery and
Metabolic Disorders, Amgen Inc., One Amgen Center Drive,
Thousand Oaks, California 91320-1799
The synthesis of compounds 8-10 is described in
Scheme 2. Compounds 8 and 9 were prepared by treat-
ing the dipiperazine 6d with 1-bromo-3-phenylpropane
and â-chloropropiophenone, respectively. Compound 10
was prepared via Mannich reaction of 6b with R-tetra-
lone.
Received J une 10, 2002
Abstr a ct: Agouti-related protein (AGRP) is an endogenous
antagonist of the melanocortin action.1 In the hypothalamus,
melanocortin peptide agonists act as satiety-inducing factors
that mediate their action through the melanocortin-4 receptor
(MC4R) whereas AGRP is an opposing orexigenic agent. Novel
inhibitors of the AGRP/MC4 binding based on (piperazinyl-
ethyl)piperazines were prepared, and their structure-activity
relationship was established.
Resu lts a n d Discu ssion
All analogues were evaluated in vitro for their ability
to inhibit the binding of 125I-AGRP to the human MC4R.
The inhibition of the 125I-NDP-MSH/MC4R binding was
also measured as a secondary assay. The potency and
the selectivity for AGRP vs NDP-MSH binding are
reported in Table 1.
In tr od u ction . Obesity is the most common and costly
nutritional disorder in the industrialized world.2 It is a
chronic condition that leads to increased risk for dia-
betes and cardiovascular disease. A great deal of
evidence suggests that the central melanocortin system
may be a fundamental component in the regulation of
food intake and body weight.3 As part of our antiobesity
program, we investigated agouti-related protein (AGRP),
a 132 amino acid protein expressed in the feeding
centers of the brain (e.g., hypothalamus). When AGRP
binds to the melanocortin-4 (MC4) receptor, it stimu-
lates food intake.4 AGRP increases feeding in rodents
when administered icv,5 and mice that express AGRP
ectopically are hyperphagic and obese.4 On the other
hand, melanocortin peptides (e.g., R-MSH) decrease
feeding in rodents by agonizing the MC4 receptor.6
Inhibition of the interaction between AGRP and the
MC4R could lead to a new approach for the treatment
of obesity. However, finding a small-molecule antagonist
for the AGRP/MC4R interaction is particularly chal-
lenging for two reasons. First, the interaction between
AGRP and the MCRs is a protein/protein interaction
where contact may occur over a large area. A small
molecule may not be able to disrupt the interactions
between these two proteins.7 Second, it is not clear, as
Haskell-Luevano and co-workers8 suggest, that AGRP
and R-MSH share overlapping binding sites on the
MCRs. Recent reports have established that some
regions of the receptor that contribute to AGRP binding
are not responsible for binding to R-MSH.9 The present
report describes efforts in the screening, design, and
synthesis of AGRP/MC4 inhibitors. The selectivity of
Screening of our internal compound collection for
inhibitors of AGRP/MC4 resulted in the identification
of the N-phenyldipiperazine 7f. Although this compound
was a weak antagonist of AGRP, it was 10-fold better
inhibitor of the MSH/MC4 binding. To optimize the
potency and selectivity, we prepared several analogues
with different substituents at R1. Analogues containing
an alkyl group were much more potent and selective
(e.g., 7d ,e and g). Incorporation of an electron-with-
drawing group X at the meta and para positions of the
phenyl substituent increased the AGRP potency by more
than 10-fold, with para substitution being slightly
preferred (c.f. 7a to 7b-d ). On the other phenyl ring,
unsubstitution, para chloro and para and meta dichloro
substitutions were favored over para methoxy, para
trifluoromethyl, and the para nitro groups (see 7d ,h -
l). Analogues 8 and 9 exhibited no activity and minimal
selectivity, indicating that the R-substituted ketone was
required for activity. The cyclic analogue 10 was also
less active. All compounds were also tested in a 125I-
AGRP/MC3R binding assay where the IC50 values were
all >10µM. To determine if these compounds are truly
inhibitors of a protein/protein interaction, we evaluated
their ability to bind to AGRP in the absence of MC4R.
Binding to AGRP could also lead to inhibition of the
AGRP/MC4 interaction. The interaction of compound 7g
with des62 AGRP, a functionally active C terminal
domain truncation of the full-length protein, was stud-
ied via HSQC11 based NMR titration. A 100 µM solution
of 15N-labeled des62 AGRP was titrated from 500 µM
to 2.5 mM 7g, using 5 × 10 µL aliquots of 25 mM stock
solution prepared in DMSO-d6. The resulting HSQCs
* To whom correspondence should be addressed. Address: Depart-
ment of Small Molecule Drug Discovery, Amgen Inc., One Amgen
Center Drive, Thousand Oaks, CA 91320-1799. Phone: (805) 4478814.
Fax: 805-480-1337. E-mail: ntamayo@amgen.com.
10.1021/jm0255522 CCC: $25.00 © 2003 American Chemical Society
Published on Web 11/27/2002
Arasasingham, Premilla N.
