New substituted piperazines as ligands for melanocortin receptors. Correlation to the X-ray structure of "THIQ"

Article abstract of DOI:10.1021/jm0311285

A series of piperazine analogues of the melanocortin 4 receptor (MC4R) specific small-molecule agonist "THIQ" was synthesized and characterized structurally and pharmacologically. First, several THIQ imitations lacking the triazole moiety were prepared. Syntheses included acylation of 4-phenylpiperazine or 4-cyclohexylpiperazine. In two cases the tertiary amine function was replaced by the corresponding N-oxide. To obtain more complex structures, a 4-substituted piperazine ring was formed by alkylation of the primary amino group of cyclohexane-derived amino alcohols with N,N-bis(2-chloroethyl)benzylamine. The hydroxylic group of the intermediate was first activated with methanesulfonyl chloride, and the sulfonic ester formed in situ was introduced into the reaction with the sodium salt of 1,2,4-triazole. In one case (i.e., preparation of 23c) introduction of the 1,2,4-triazole moiety was performed at a carbon of the cyclohexane ring. In addition, this intermediate contained a piperazine moiety connected via its nitrogen atom to a cyclohexane ring carbon neighboring the reaction center. As established in NMR and X-ray investigations herein, this substitution proceeded with retention of the initial trans configuration of 1,2-disubstituted cyclohexane. To obtain pure enantiomers of 23c, its precursor 21c was subjected to chiral chromatography on a Chirobiotic V column. The derivatives (R,R)-21cand (S,S)-21c obtained were introduced into further syntheses steps, giving (R,R)-23c and (S,S)-23c, respectively. Melanocortin MC_1,3-5 receptor binding studies showed that all tested piperazine derivatives were active. Several compounds showed clear selectivity for MC₄R, with submicromolar affinities being obtained. Among them, one substance, (R,R)-23c, displayed a biphasic curve in displacement of [¹²⁵I]NDP-MSH on MC₄R [K(i)_high = 1 nM and K(i)_low = 260 nM]. This biphasic competition curve was similarly biphasic to the competition curve obtained herein using THIQ. An X-ray study performed on crystals of the THIQ sulfate salt revealed two closely related conformations, which resemble the shape of the letter "Y", where piperidine and 4-chlorophenyl groups are situated close to each other, but the 1,2,3,4-tetrahydroisoquinoline residue is remote, the triazole function being highly exposed to the environment. The crystals of the dinitrate salt of (R,R)-23c showed a different conformation, where parts of the molecule are spread out almost symmetrically around the central section. Molecular modeling, based on the THIQ crystal structure and the functional similarity of THIQ and (R,R)-23c, allowed us to suggest a possible "bioactive" conformation of (R,R)-23c that is similar to the crystal conformation of THIQ.

Full text of DOI:10.1021/jm0311285

J . Med. Chem. 2004, 47, 4613-4626
4613
New Su bstitu ted P ip er a zin es a s Liga n d s for Mela n ocor tin Recep tor s.
Cor r ela tion to th e X-r a y Str u ctu r e of “THIQ”
Felikss Mutulis,† Sviatlana Yahorava,† Ilze Mutule,† Aleh Yahorau,† Edvards Liepinsh,‡ Sergei Kopantshuk,†,§
Santa Veiksina,† Kaspars Tars,# Sergey Belyakov,| Anatoly Mishnev,| Ago Rinken,§ and J arl E. S. Wikberg*,†
Department of Pharmaceutical Biosciences, Division of Pharmacology, Uppsala University, Box 591, Biomedicum,
SE-751 24 Uppsala, Sweden, Department of Medical Biochemistry and Biophysics, Karolinska Institutet,
S-171 77 Stockholm, Sweden, Institute of Organic and Bioorganic Chemistry, Tartu University,
J akobi 2, 51014 Tartu, Estonia, Department of Cell and Molecular Biology, Uppsala University, Box 596,
SE-751 24 Uppsala, Sweden, and Latvian Institute of Organic Synthesis, LV-1006, Riga, Latvia
Received December 8, 2003
A series of piperazine analogues of the melanocortin 4 receptor (MC4R) specific small-molecule
agonist “THIQ” was synthesized and characterized structurally and pharmacologically. First,
several THIQ imitations lacking the triazole moiety were prepared. Syntheses included acylation
of 4-phenylpiperazine or 4-cyclohexylpiperazine. In two cases the tertiary amine function was
replaced by the corresponding N-oxide. To obtain more complex structures, a 4-substituted
piperazine ring was formed by alkylation of the primary amino group of cyclohexane-derived
amino alcohols with N,N-bis(2-chloroethyl)benzylamine. The hydroxylic group of the intermedi-
ate was first activated with methanesulfonyl chloride, and the sulfonic ester formed in situ
was introduced into the reaction with the sodium salt of 1,2,4-triazole. In one case (i.e.,
preparation of 23c) introduction of the 1,2,4-triazole moiety was performed at a carbon of the
cyclohexane ring. In addition, this intermediate contained a piperazine moiety connected via
its nitrogen atom to a cyclohexane ring carbon neighboring the reaction center. As established
in NMR and X-ray investigations herein, this substitution proceeded with retention of the initial
trans configuration of 1,2-disubstituted cyclohexane. To obtain pure enantiomers of 23c, its
precursor 21c was subjected to chiral chromatography on a Chirobiotic V column. The
derivatives (R,R)-21cand (S,S)-21c obtained were introduced into further syntheses steps, giving
(R,R)-23c and (S,S)-23c, respectively. Melanocortin MC1,3-5 receptor binding studies showed
that all tested piperazine derivatives were active. Several compounds showed clear selectivity
for MC4R, with submicromolar affinities being obtained. Among them, one substance, (R,R)-
23c, displayed a biphasic curve in displacement of [125I]NDP-MSH on MC4R [K(i)high ) 1 nM
and K(i)low ) 260 nM]. This biphasic competition curve was similarly biphasic to the competition
curve obtained herein using THIQ. An X-ray study performed on crystals of the THIQ sulfate
salt revealed two closely related conformations, which resemble the shape of the letter “Y”,
where piperidine and 4-chlorophenyl groups are situated close to each other, but the 1,2,3,4-
tetrahydroisoquinoline residue is remote, the triazole function being highly exposed to the
environment. The crystals of the dinitrate salt of (R,R)-23c showed a different conformation,
where parts of the molecule are spread out almost symmetrically around the central section.
Molecular modeling, based on the THIQ crystal structure and the functional similarity of THIQ
and (R,R)-23c, allowed us to suggest a possible “bioactive” conformation of (R,R)-23c that is
similar to the crystal conformation of THIQ.
In tr od u ction
mediated control of steroid production.4 The MC3R is
present in the hypothalamus and is there involved in
energy homeostasis regulation.5 The MC4R is expressed
in the brain and other tissues, and it is known to control
feeding. In addition, this receptor (as well as presumably
the MC3R receptor) regulates sexual behavior.3,6 MC5R
is found in the skin among other places and appears to
regulate functions of exocrine glands.7
Melanocortin receptors belong to the family of hep-
tahelical transmembrane G-protein-coupled receptors.1
Up to now five subtypes of melanocortin receptors
(MC1-5R) have been cloned.2 These receptors are in-
volved in multiple physiological processes. For example,
the MC1R is expressed in the skin where it plays a major
role in skin and hair pigmentation, and it has a role in
controlling the immune system.3 The MC2R is found in
the adrenal gland where it is responsible for the ACTH
Agonists and antagonists of melanocortin receptors
have a potential for treatment of several diseases and
health disorders such as overweight, sexual dysfunc-
tions, and conditions related to inflammation.8 MC4R
agonists reduce appetite and decrease body weight,
while antagonists of the receptor has the opposite effect,
where long-term treatment causes a dramatic increase
in body weight.3,9 The natural agonists for the melano-
* To whom correspondence should be addressed. Phone: +46-18-
4714238. Fax: +46-18-559718. E-mail: J arl.Wikberg@farmbio.uu.se.
† Division of Pharmacology, Uppsala University.
‡ Karolinska Institutet.
§ Tartu University.
#
Department of Cell and Molecular Biology, Uppsala University.
| Latvian Institute of Organic Synthesis.
10.1021/jm0311285 CCC: $27.50 © 2004 American Chemical Society
Published on Web 08/03/2004

4614 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Mutulis et al.
receptors.8,18-21 Among them, “THIQ” (Figure 1) exhibits
outstanding specificity for the MC4 receptor and a high
agonist activity.22 Publishing of its structural formula
and activity data in the year 2000 was followed by an
extensive exploration of related structures by Merck and
several other drug development companies, which is
reflected in patents (see refs 8, 18, and 19). However,
up to now only few academic studies were devoted to
this theme.23-26
In the present study we have synthesized some new
piperazine derivatives related to THIQ and investigated
their molecular structures and receptor binding proper-
ties. For comparison, we resynthesized also THIQ22 for
use in parallel investigations.
F igu r e 1. THIQ.
cortin receptors are the melanocortins. These peptides
include adrenocorticotropic (ACTH) and R-, â-, and
γ-melanocyte stimulating hormones (R-, â-, and γ-MSH),
all which are formed by post-translational cleavages of
the precursor protein proopiomelanocortin (POMC).10 In
addition, the activity of the melanocortin receptors
is regulated by endogenous antagonists, namely, the
agouti protein and agouti-related peptide (AgRP).11
Investigations of melanocortins include preparation
of many peptide analogues.3 Among them, substances
displaying valuable properties such as antagonism, high
activity, and selectivity were discovered.12-16 However,
in most cases peptides are not suited for therapeutic
application because they usually do not pass the blood-
brain barrier and are rapidly metabolized in blood and
tissues.17 Accordingly there is a demand for non-peptide
ligands with low molecular weight showing high affinity
and selectivity for the melanocortin receptors. Many
drug development companies exert considerable efforts
to create MC4R-directed drugs for treatment of obesity
or anorexia (for reviews see refs 8, 18, and 19 ). A large
number of low molecular weight compounds were re-
ported to show a binding activity on melanocortin
Ch em istr y
To prepare some piperazine derivatives related to
THIQ that lacked the triazole function, we started with
Boc-4-chloro-D-phenylalanine 1 (Scheme 1). Reaction
with 4-nitrobenzyl bromide27 gave the corresponding
4-nitrobenzyl ester 2. Elimination of the Boc group then
followed. The amino acid derivative 3 formed was
coupled28 with Boc-D-1,2,3,4-tetrahydroisoquinoline-3-
carboxylic acid to obtain the protected dipeptide 4.
Further, alkaline hydrolysis27 of the 4-nitrobenzyl ester
function led to dipeptide acid 5, which was reacted with
1-phenylpiperazine 6a or 1-cyclohexylpiperazine 6b,
giving amides 7a and 7b. Elimination of the Boc group
converted them to compounds 8a and 8b, bearing a free
amino group.
To prepare N-oxides, Boc-protected amide 7a or 7b
were oxidized by hydrogen peroxide.29 Protected N-oxide
9a or 9b formed. Deprotection with trifluoroacetic acid
provided the desired N-oxides 10a and 10b.
For preparation of more complex end products, di-
ethanolamine 11 was treated with benzyl bromide
(Scheme 2), as described in the literature.30 The N-
Sch em e 1a
a
Reagents and conditions: (a) 4-nitrobenzylbromide, TEA, EtOAc, reflux, 7 h; (b) 4 M HCl/dioxane, AcOH, room temp, 1 h; (c) Boc-
D-Tic-OH, HATU, DIEA, DMF, room temp, 2 h; (d) 2 N aqueous NaOH, MeOH, room temp, 1.5 h; (e) HATU, DIEA, DMF, room temp, 2
h; (f) 30% aqueous H2O2, room temp, 1 h, then 60 °C, 2 h; (g) TFA/CH2Cl2 (2:1), room temp, 1 h.

Substituted Piperazines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4615
Sch em e 4. Relative Stereochemistrya
Sch em e 2a
a
Reagents and conditions: (a) benzyl bromide, K2CO3, toluene,
60-70 °C, 1 h; (b) SOCl2, CHCl3, reflux, 4 h.
Sch em e 3. Relative Stereochemistrya
a
Reagents and conditions: (a) LiAlH4, THF under Ar atmo-
sphere, 70-80 °C, 28 h; (b) Boc2O, TEA/MeOH (1:9), room temp,
4 h; (c) 4 M HCl/dioxane, room temp, 1 h.
protected diol 12 formed. Reaction with SOCl2 converted
it to the dichloride 13.30 To obtain the two reactants 15a
and 15b for reaction with 13, cis-2-aminocyclohexane-
carboxylic acid (14a ) or 1-aminocyclohexanecarboxylic
acid (14b) was reduced with LiAlH4 (Scheme 3).31,32
However, it was difficult to isolate alcohols 15a ,b from
the mixture formed. To enable chromatography on silica
gel, the reaction products were converted to Boc deriva-
tives 16a ,b.33 After purification by flash chromatogra-
phy the Boc group was removed to afford pure alcohol
15a or 15b. Two other alcohols 15c and 15d were
commercial available. In the next step (Scheme 4), the
cyclic alcohols 15a -d were introduced into reaction with
dichloride 13 (preparation detailed above). This led to
the creation of piperazine derivatives 17a -d .34,35 The
next stage was the introduction of the triazole moiety.
The hydroxyl group was first activated with methane-
sulfonyl chloride, and the sulfonic acid derivative formed
in situ was reacted with the sodium salt of 1,2,4-
triazole.22,36 Triazole derivatives 18a -d were obtained.
In one case (preparation of 18c), the introduction of the
triazole moiety proceeded at an asymmetrical carbon
atom included in the cyclohexane ring. We found only
one example in the literature where a similar reaction
had been investigated earlier. There it was found that
upon reaction of 2,3-disubstituted oxirane (substitu-
ents: halogen and tertiary butyl group) with sodium
triazolide only the trans triazole introduction product
was obtained.37 In the present study the orientation of
substituents was determined by a more insightful NMR
analysis of the end product of the whole synthesis of
23c (trans configuration was established; a full account
of this study is given below). Further, it could be inferred
that already in the 1,2-disubstituted cyclohexane 18c
and its further synthesis products the piperazine and
triazole moieties were in trans relation to each other.
a
Reagents and conditions: (a) 13, NaHCO3, EtOH, reflux, 3-12
h; (b) (1) MeSO2Cl, CH2Cl2, TEA, 0 °C to room temp, 2 h; (2)
sodium salt of 1,2,4 triazole, DMF, under Ar atmosphere, 80-100
°C, 12 h; (c) HCOONH4, 10% Pd/C, 1 N HCl, MeOH, reflux, 1.5 h.
Thus, the reaction where the triazole moiety was
introduced using the secondary alcohol 17c proceeded
with retention of the initial relative configuration (trans
for the starting material 2-aminocyclohexanol, 15c). We
assume that the reaction mechanism includes two
successive SN2 steps. First, the adjacent piperazine
nitrogen forms a transient aziridinium ion by back-side
attack on the mesylate. The meso-aziridinium ion is
then attacked by the triazolide from the far side, leading
to overall retention of stereochemistry.38
Removal of the N-benzyl group by transfer hydroge-
nation39 afforded diamines 19a -d . They were subjected
to stepwise peptide synthesis in solution (Scheme 5).
First, the Boc derivative of 4-chloro-D-phenylalanine was
attached, giving intermediates 20a -d . After deprotec-
tion (products 21a -d ), coupling with Boc-D-1,2,3,4-
tetrahydroisoquinoline-3-carboxylic acid followed. Pro-
tected derivatives 22a -d were obtained. Removal of the
Boc group furnished raw end products 23a -d , which
were purified by HPLC.
Several other compounds obtained in the present
study included an asymmetrical center in their cyclo-
hexane ring, namely, 23a and its precursors (15a -22a ).
However, there is no doubt that the relative cis config-
uration, which was present in the starting amino acid
(14a ), was retained in the end product 23a because its
multistep preparation process did not touch the asym-
metrical carbon atoms (Schemes 3-5).

4616 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Mutulis et al.
Sch em e 5a
us to estimate the coupling constant between the above
protons. 3J HH was found to be about 11.3 Hz, something
which again corresponds to the interaction between
protons in axial-axial positions.44 Consequently, both
substituents at cyclohexane are equatorial. Such a
relation between cyclohexane 1,2-substituents presumes
that they are in a trans relative configuration. Analysis
was problematic, however, because signal doubling was
observed in most cases. This could be explained as the
presence of equal amounts of two trans isomers at
cyclohexane asymmetric centers: R,R and S,S.
Other NMR observations for the 23c molecule (DMSO-
d6 solution) indicated that the aliphatic ring of the
tetrahydroisoquinoline ring system mainly exists in a
half-chair conformation, where the carbon atoms of the
CH2 groups are situated in the same plane as the
benzene ring, whereas the asymmetrical carbon atom
and charged nitrogen atom are each located in opposite
sides of this plane. (This could be reasoned from data
concerning the spin-spin coupling constants and the
nuclear Overhauser effect in the aliphatic cycle, includ-
ing those for the disubstituted ammonium ion.) Besides,
the ROESY spectra showed several types of exchange
peaks for piperazine protons and the whole analysis was
complicated because of broadness of the signals. This
can be explained both by a conversion process between
several chair conformations of this ring and by a slow
rotation around the amide bond attached to the pipera-
zine cycle occurring at the rates intermediate to the
NMR time scale.
a
Reagents and conditions: (a) Boc amino acid, HATU, DIEA,
DMF, room temp, 2 h; (b) TFA/CH2Cl2 (2:1), room temp, 1 h.
After separation of isomers (described below), an
additional NMR study, which again involved a wide
application of two-dimensional approaches, allowed us
to make a full assignment of most 1H and 13C NMR
spectral signals for both forms of 23c [(R,R)-23cand
(S,S)-23c] and their intermediates (R,R)-21c and (S,S)-
21c. Data for this are provided in Supporting Informa-
tion.
F igu r e 2. Combined picture of a ROESY (above the diagonal)
and double-quantum filtered COSY (below the diagonal) NMR
spectra for 23c in DMSO-d6. Presence (in COSY spectrum) or
absence (in ROESY spectrum) of a cross-peak reflecting
interaction between protons at the cyclohexane asymmetrical
carbon atoms is shown by circles drawn with a broken line.
Sep a r a tion of Isom er s
According to our NMR data, substance 23c had been
initially prepared as an equimolar mixture of R,R and
S,S isomers in relation to its trans 1,2-substituents at
the cyclohexane ring. In our binding experiments on the
MC4R, this mixture gave a strongly biphasic competition
binding curve, showing one apparent high-affinity bind-
ing component with about 1 nM affinity and one
apparent low-affinity component with more than 200-
fold lower affinity (similarly as did THIQ), and we
decided that 23c should deserve further studies using
its individual isomers.
We investigated both 23c and those intermediates of
its synthesis that included disubstituted cyclohexane,
trying to separate the isomers. Because 23c and some
of its precursors (20c, 21c, 22c) contained one or two
fixed asymmetric centers (included in the residues of
4-chloro-D-phenylalanine and D-1,2,3,4-tetrahydroiso-
quinoline-3-carboxylic acid) in addition to the above
cyclohexane centers, their separation by conventional
chromatography was in principle possible. However, no
double peaks were observed in our experiments on
reversed-phase HPLC columns. Crystallization of salts
to obtain individual diastereomers was unsuccessful too
(we prepared salts of 19c with L-tartaric acid, dibenzoyl-
NMR In vestiga tion s
The aim of the investigations was to establish the
relative configuration of the carbon atom in cyclohexane
position 2, which could be affected by the nucleophilic
reaction introducing the triazole moiety. 23c was in-
vestigated by NMR at 600 MHz in DMSO, 28 °C: proton
spectra, 60 ms mixing time TOCSY,40 250 ms mixing
time ROESY,41 HSQC and HMBC (13C-1H correla-
tion).42,43 Protons, which are attached to carbon atoms
of the cyclohexane ring (positions 1 and 2) were detected
at resonances 2.77 and 4.36 ppm, respectively. They
showed a cross-peak in a double-quantum-filtered COSY
spectrum (500 MHz, in deuterium oxide, pH 5.5) but
did not exhibit any cross-peak in the ROESY spectrum
(Figure 2). In other words, the COSY spectrum showed
the presence of a spin-spin coupling between protons
at the cyclohexane asymmetrical centers, but from the
ROESY data there was no sign of a nuclear Overhauser
effect between them. This reflects the diaxial location
of these protons. Other spectra, which were recorded
in deuterium oxide (500 MHz, pH 4.4, 25 °C), allowed

Substituted Piperazines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4617
with the parent compound, but still it retained about a
2-fold selectivity for the MC4R. From these data, we can
make the conclusion that the benzene derivatives of our
THIQ-related piperazines, and particularly the N-
oxides, are less compatible with efficient MC4R binding.
Therefore, in the subsequent studies we concentrated
on alicyclic cyclohexane and cyclopentane derivatives
introducing additionally a 1,2,4-triazole moiety (which
is present also in THIQ, Figure 1).
The first substance of the more complex type, 23a ,
contains two substituents at neighboring carbon atoms
in the cyclohexane ring (Schemes 4 and 5). 23a showed
a submicromolar affinity on the MC4R and low micro-
molar affinities on MC1R and MC3R. Thus, the affinities
for 23a on these receptors were improved when com-
pared with the more simple cyclohexane derivative 8b
described above. In contrast, the affinity of 23a for the
MC5R turned out to be 2-fold lower compared with the
simpler compound 8b.
F igu r e 3. Analytical separation of 21c isomers on Chirobiotic
V 4.6 mm × 250 mm column: (eluent) dioxane-MeOH-
AcOH-NEt3-H2O (50:50:0.04:0.01:0.03); detection at 220 nm;
flow rate of 1 mL/min.
D-tartaric acid, and di-p-toluoyl-D-tartaric acid and tried
their fractional crystallization, but no separation of
isomers was observed).
The problem was solved using chiral HPLC (analyti-
cal and preparative) on a Chirobiotic V (Advanced
Separation Technologies Inc.) stationary phase. A some-
what less satisfactory separation was obtained also on
a Chirobiotic T column. These phases are obtained
covalently bonding the natural glycopeptides vancomy-
cin (18 chiral centers) or teicoplanin (20 chiral centers)
to silica gel.45 Data on our trials with Chirobiotic V and
Chirobiotic T columns are included in Supporting
Information. After some optimization in analytical scale
experiments with intermediate 21c, a division close to
baseline separation was achieved (Figure 3). On a
semipreparative Chirobiotic V column using a mobile
phase containing dioxane-methanol-acetic acid-tri-
ethylamine (50:50:0.02:0.01), we thereafter succeeded
with a multimilligram scale separation. Both products
obtained, (R,R)-21c (faster moving on chiral HPLC) and
(S,S)-21c (a slower moving isomer), were introduced into
the two remaining synthesis steps (Scheme 6) to give
individual diastereomers (end products (R,R)-23c and
(S,S)-23c) as ditrifluoroacetates. The absolute stereo-
chemistry of them was established later when we
obtained the X-ray structure for (R,R)-23c dinitrate
(described below).
23b, similar to THIQ (Figure 1), contains a carbon
atom with no hydrogens attached to it, which is con-
nected to the 1,2,4-triazol-1-ylmethyl group. Two sub-
stituents are attached to the same carbon atom, which
is also part of the cyclohexane ring, and there are no
asymmetric centers in this part of the molecule (Schemes
4, 5). 23b displayed an affinity pattern that was very
similar to that of 23a except that the affinity to MC4R
was diminished by a factor of 2.5.
23c, which has a rigid trans triazole substitution at
the carbon atom neighboring the carbon where the
piperazine is attached to the cyclohexane ring, showed
obvious MC4R selectivity. Moreover, the curve for
displacement of [125I]NDP-MSH binding was heteroge-
neous, which can be interpreted as the interaction with
two apparent binding sites (e.g., representing stabilized
receptor conformations). When the heterogeneous bind-
ing curves were analyzed using law-of-mass-action
modeling under the assumption that two discrete MC4R
binding sites were present, the apparent low-affinity site
appeared to show a submicromolar affinity, while the
high affinity binding site appeared to show a low
nanomolar affinity for 23c. In parallel experiments
using THIQ, we obtained a similar heterogeneous curve
(Figure 4). Thus, the binding patterns of THIQ and 23c
were closely similar, indicating that their “biologically
active” structures should be similar. However, 23c was
a mixture of two isomers. These isomers were obtained
as individual substances, (R,R)-23c and (S,S)-23c (see
“Separation of Isomers”). Binding experiments showed
that these isomers exhibit different properties; (R,R)-
23c was more active than (S,S)-23c on the MC3, MC4,
and MC5Rs, but the activity toward MC1R was the same
for both isomers. However, the most striking difference
between the isomers was the absence of any apparent
high- and low-affinity binding sites for (S,S)-23c on the
MC4R. On the MC4R, (S,S)-23c exhibited a classical
monophasic competition binding curve. The heterogene-
ity of binding to the MC4R, which was observed for the
mixture of isomers 23c, showed up again in the binding
experiments using (R,R)-23c (Figure 4).
Recep tor Bin d in g Stu d ies
The synthesized compounds were investigated for
their ability to displace the specific binding of [125I]NDP-
MSH to recombinant human melanocortin receptors
MC1,3-5Rs in Sf9 cell membranes.46,47 All substances
displayed some binding affinity (Table 1). By comparison
of data for 8a and 8b, which differ only by the presence
of an aromatic (benzene) or aliphatic (cyclohexane) ring,
it can be seen that the benzene-related 8a displays an
approximately equal affinity for all four MC receptors,
the Ki values in the range 13-30 µM with about a 2-fold
lower affinity for MC4R than that for the three other
receptors. The cyclohexane derivative 8b on the other
hand showed the same or higher affinity for all receptors
compared with 8a , on the MC1R the affinities being
roughly the same as 8a , on the MC3 and MC5Rs being
twice as high, and on the MC4R being 20-fold as high
as 8a . Thus, 8b displayed some selectivity for the MC4R.
When 8a was converted into the N-oxide (substance
10a ), the affinities decreased about 2- to 5-fold. As in
8a , some loss of affinity was seen for the MC4R. 10b,
which is the N-oxide derived from 8b, behaved like 10a ,
showing a 12- to 41-fold lower affinity when compared
The next synthesis product, 23d , contains a cyclopen-
tane moiety and a triazole group attached to the rest of
the molecule through a methylene group. We found that
the MC4R selectivity, which had been observed for the

4618 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Sch em e 6. Absolute Stereochemistrya
Mutulis et al.
a
Reagents and conditions: (a) Boc amino acid, HATU, DIEA, DMF, room temp, 2 h; (b) TFA/CH2Cl2 (2:1), room temp, 1 h.
cyclohexane derivatives, had disappeared for this com-
pound. However, 23d was more active on the MC1R
compared to all other compounds tested in the present
study because it showed a submicromolar affinity for
this receptor. For the MC4R, its affinity was 6-fold lower
compared to that for (R,R)-23c (low affinity site), and
the affinities for the MC3 and MC5Rs were 2-fold lower.
were obtained from a crystal in the form of a thin plate
with linear dimensions 0.05 mm × 0.17 mm × 0.45 mm.
(Further experimental details, fractional atomic coor-
dinates, and crystal data are presented in Supporting
Information).
From the X-ray study it was found that the structure
of the crystals corresponded to the formula 2C33H41
-
ClN6O2‚H2SO4‚6H2O; i.e., two protonated THIQ mol-
ecules form a complex with one sulfate ion and six water
molecules. Both the crystal conformations of THIQ differ
insignificantly from each other; some difference could
be seen for the location of the triazole rings. These
structures resemble the shape of the letter Y, where the
4-chlorophenyl group and cyclohexylpiperidine are close
to each other and the D-Tic residue is remote. The
triazole function turns away from the rest of the
molecule and is highly exposed to the environment
(Figure 5).
X-r a y An a lysis
THIQ was synthesized according to the patent de-
scription.22 Its trifluoroacetate salt was dissolved in
methanol and passed through a small column filled with
Dowex 1 × 4 in OH- form. The solution of the base
obtained was diluted with water (20% of volume),
sulfuric acid (0.6 molar equiv) was added, and the
mixture was evaporated to dryness in vacuo. The
residue was dissolved in a small volume of hot water.
When the mixture was slowly cooled to room tempera-
ture, crystals formed. The largest of these crystals was
sampled together with the mother liquid, using a glass
capillary. The mother liquor was then removed with a
small piece of filter paper. The crystal in the capillary
was subjected to X-ray diffraction. However, it turned
out that the crystals were unstable when exposed to air.
An initial trial experiment resulted in a model for the
structure that could not be refined to a reliable R factor.
To resolve this, different glues were tried for coating
the crystals. It turned out that the best results were
obtained using nitrocellulose glue, which resulted in
practically no intensity decay during the course of 12 h
of X-ray exposure. The final successful measurements
(R,R)-23c was synthesized as described above. Its
ditrifluoroacetate salt was dissolved in methanol and
passed through a small column filled with Dowex 1 × 4
in OH- form. The solution of the base obtained was
evaporated. To the oily residue formed, a solution of
nitric acid (2 molar equiv) was added in a small volume
of water. After shaking the mixture on a Vortex, a
homogeneous solution formed, from which after a short
standing crystals precipitated.
Crystals of (R,R)-23c showed sufficient stability in air,
and therefore, no special measures to protect them were
required. To perform X-ray structure analysis, es-
sentially the same instrumentation and experimental

Substituted Piperazines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4619
Ta ble 1. Affinity of Synthesized Substances to Recombinant
Human Melanocortin Receptors in Membrane Preparations, Ki
(in µM Unless Otherwise Noted)
F igu r e 5. ORTEP image of two molecules of THIQ in
elementary crystallographic unit.
F igu r e 6. ORTEP image of (R,R)-23c conformation in crys-
tals.
a
R ) H-D-Tic-D-Phe(4-Cl)-; 23a structure is shown in terms of
relative stereochemistry.
The crystallographic data for the structure analysis
of THIQ and (R,R)-23c have been deposited at the
Cambridge Crystallographic Data Centre.
Molecu la r Mod elin g
A THIQ tertiary structure, which is very similar (if
not identical) to those that we found in crystals, was
presented in a previous publication25 without any
information about its origin. According to the authors
of this study, this conformation correlated with a
calculated low-energy structure of the highly active
cyclic melanocortins related peptide MT-II. It was
proposed that it represents a “biologically active” con-
formation interacting with the MC4R. We have utilized
the same assumption in our present work.
F igu r e 4. Displacement of receptor-bound [125I]NDP-MSH by
THIQ (experimental points shown as circles), (R,R)-23c
(squares), and (S,S)-23c (triangles). Experiments were made
on MC4R containing membranes.
methods as described for THIQ were used. The crystal
structure was solved by use of direct methods and
refined by full-matrix least squares on F2. All non-
hydrogen atoms were refined anisotropically. All hy-
drogen atoms were located by difference Fourier syn-
thesis. The X-ray study showed that the structure of
the crystals corresponded to the formula C31H38ClN7O2‚
2HNO3‚3H2O. The single conformation of (R,R)-23c,
which was found in the crystals, is shown in Figure 6.
These data allowed us to establish the absolute stereo-
chemistry of the molecule, which was R,R in relation
to the substituents at the cyclohexane ring. Accordingly,
the corresponding configuration of the other isomer
(S,S)-23c is S,S (Scheme 6). More experimental facts
are presented in Supporting Information.
Taking into account that both isomers of compound
23c (Scheme 6) showed an increased affinity and
selectivity for the MC4R and that these are character-
istic features of THIQ, we suggested that their “biologi-
cally active” structure is in general similar to that of
THIQ and the differences should be located in the
differing part. To investigate this further, we first
introduced fractional atomic coordinates obtained from
the X-ray structure of THIQ (form 1 from two forms
found in crystals; see Supporting Information) into the
CRYSIN program, which is included in molecular
modeling software package SYBYL.48 Application of the
“Connect” command led to an image of the tertiary
structure of the molecule. This was then saved as a mol2
file to enable its modification following molecular mod-

4620 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Mutulis et al.
protonated charged nitrogen atom, which is present in
the piperazine ring, is not favorable for interactions with
the receptor. This disubstituted ammonium ion is
shielded in 23c by the rigidly connected triazole so that
the ion is not able to contact the receptor. In 23b the
triazole is connected through a methylene group and
has more conformational freedom. Therefore, upon
interaction with the receptor the disubstituted am-
monium ion might interfere negatively with the binding
process. This explanation could be expanded also to 23d
and 23a , which have structures similar to 23c but
reduced activity and selectivity for the MC4R.
The importance for separation of the piperazine
substituted ammonium ion from the receptor is sup-
ported by the fact that other recently disclosed MC4R
agonists related to THIQ contain piperazine with a
bulky substituent close to the ammonium nitrogen.18,49
A steric hindrance here could be an important factor to
maintain high MC4R activity.
F igu r e 7. Modeling on SYBYL: (A) Proposed “bioactive”
conformation of (R,R)-23c; (B) proposed “bioactive” conforma-
tion of (S,S)-23c; (C) overlaid proposed “bioactive” conforma-
tions of THIQ and (R,R)-23c.
eling in SYBYL. Further, the part of the THIQ molecule
that is identical to that of (R,R)-23c and (S,S)-23c
molecules (residues of 4-chloro-D-phenylalanine and
D-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid and
nitrogen atom of piperidine (THIQ) or piperazine (23c)
residues) was defined as an “aggregate” to exclude
change of its tertiary structure during the following
modeling. The image of THIQ was then modified using
the “Sketch molecule” option, changing its differing part
in relation to the isomers of 23c in order to obtain their
approximate tertiary structure. To suggest a minimum
energy conformer for the investigated molecules, the
imaginary structures of (R,R)-23c and (S,S)-23c, whose
conformational freedom was limited by the “aggregates”,
were introduced into the SYBYL program MAXIMIN.
As a result, conformations of both 23c forms were
generated (Figure 7A,B). If we look at the parts of the
molecules not included in the “aggregate”, it can be seen
that only the conformation of (R,R)-23c (Figure 7A) is
very similar to that of the X-ray structure of THIQ
(Figure 5). The generated conformation of (S,S)-23c
(Figure 7B) differs substantially because the triazole
moiety is here turned to the rest of the molecule. To
obtain a better comparison with the X-ray structure of
the THIQ form 1 found in crystals, its structure was
overlaid with the conformation of (R,R)-23c created by
MAXIMIN using the SYBYL “Fit atoms” function. From
the overlaid image (Figure 7C), the following features
could be noted. The position of the piperazine of (R,R)-
23c differs insignificantly from the position of the
piperidine of THIQ. However, the cyclohexane ring of
(R,R)-23c is turned away from its place in THIQ and
the same can be said about the triazole residue. Both
in THIQ and (R,R)-23c the triazole is exposed to the
environment, i.e., turned away from the rest of the
molecule. Upon measurement of the distances between
centers of the corresponding cycles in both molecules
(SYBYL options “Centroid” and “Measure”), the follow-
ing results were obtained: distance piperidine-pipera-
zine 0.54 Å, distance cyclohexane-cyclohexane 3.82 Å,
distance triazole-triazole 3.27 Å. Such deviations could,
at least partly, explain the reduced activity of (R,R)-
23c compared with THIQ.
Con clu sion
Comparison of the binding data for THIQ-related
piperazine derivatives, which do not include a triazole
moiety, indicated the importance of the cyclohexane ring
for attaining MC4R selectivity over other MC receptors.
N-Oxides were considerably less active than the corre-
sponding amines. The cyclopentane derivative 23d
exhibited preference for the MC1R and not for the MC4R.
Introduction of a triazole moiety improved the affinity
of all compounds for all melanocortin receptors evalu-
ated, the affinities reaching a low micromolar to sub-
micromolar level. Among the compounds studied, only
THIQ and (R,R)-23c displayed an MC4R-selective,
heterogeneous binding profile in competition with [125I]-
NDP-MSH. According to the two binding sites interpre-
tation, they showed submicromolar affinity for the
apparent low-affinity site and a nanomolar or subna-
nomolar affinity for the apparent high-affinity site.
In line with a previous study by others,25 the crystal
conformation of THIQ may be looked upon as a “bioac-
tive” one. By adoption of this assumption, a model for
explaining the different activity patterns of the 23c
isomers was developed. According to this model, an
important feature for a “bioactive” form of (R,R)-23c is
the location of its triazole function so that it is turned
away from the rest of the molecule, similarly as in
THIQ, whereas for (S,S)-23c it is turned toward the rest
of the molecule, thus not being exposed to the receptor.
According to our interpretation, this is the reason (S,S)-
23c does not delineate the apparent high- and low-
affinity binding sites of THIQ and (R,R)-23c.
Exp er im en ta l Section
Ma ter ia ls a n d Meth od s. Reagents were used without
purification. Unless otherwise noted, they were obtained from
Aldrich or Fluka. DMF, DIEA, and HATU were supplied from
Applied Biosystems. Boc-D-Tic-OH was a product of Neosys-
tem. Most of 1H NMR spectra were recorded on a J EOL J NM-
EX270 spectrometer, using DMSO-d6 (2.50 ppm downfield
from TMS) as an internal standard. In a particular study the
Bruker DMX-600 and Bruker DRX-500 instruments were
used. Exact molecular masses were determined on Micromass
Q-Tof2 mass spectrometer equipped with an electrospray ion
source. LC/MS was performed on a Perkin-Elmer instrument
PE SCIEX API 150EX with a Turboionspray ion source and a
Dr. Maisch Reprosil-Pur C18-AQ, 5 µm, 150 mm × 3 mm
Performing similar modeling for 23b and comparing
the structure obtained with that of THIQ gave very
similar data as above in terms of positions of rings and
distances between them. However, 23b displayed uni-
form and considerably lower affinity on the MC4R
compared with 23c (i.e., no high- and low-affinity sites
involved). This could be explained if we assume that the

Substituted Piperazines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4621
HPLC column using a gradient formed from water and
acetonitrile with 5 mM ammonium acetate additive. Small-
scale preparative HPLC was carried out on an LKB system
consisting of a 2150 HPLC pump, 2152 LC controller, a 2151
variable-wavelength monitor, and a Vydac RP C18 column (10
mm × 250 mm, 90 Å, 201HS1010), with the eluent being an
appropriate concentration of MeCN in water + 0.1% TFA, a
flow rate of 5 mL/min, and detection at 280 nm. TLC was
performed using Merck silica gel 60 F 254 glass plates; flash
chromatography was performed using Merck Silica gel (70-
230 mesh). Evaporations of solvents were made on a vacuum
rotary evaporator at 30 °C and 20 mbar. Freeze-drying was
carried out at 0.01 bar on a Lyovac GT2 freeze-dryer (Finn-
Aqua) equipped with a Trivac D4B (Leybold Vacuum) vacuum
pump and a liquid nitrogen trap.
N-(ter t-Bu toxycar bon yl)-4-ch lor o-D-ph en ylalan in e 4-Ni-
tr oben zyl Ester (2). To a solution of Boc-D-Phe(4-Cl)-OH
(0.99 g, 3.30 mmol) in EtOAc (50 mL), 4-nitrobenzyl bromide
(0.75 g, 3.47 mmol) and TEA (0.69 mL, 4.95 mmol) were added.
The resulting mixture was refluxed with stirring for 7 h. After
the mixture was cooled to room temperature, the precipitate
formed was filtered off. The filtrate was diluted with EtOAc
to 200 mL and washed successively with aqueous 10% KHSO4
(3 × 50 mL), saturated aqueous NaHCO3 (2 × 20 mL), and
water (2 × 50 mL). The organic layer was dried over MgSO4
and filtered, and the filtrate was evaporated. Product 1 was
obtained as a white solid (yield 1.28 g, 89%). 1H NMR (270
MHz, DMSO-d6): δ 8.20 (2H, m), 7.54 (2H, m), 7.47 (1H, d, J
) 7.6 Hz), 7.29 (4H, m), 5.24 (2H, s), 4.25 (1H, m), 2.95 (2H,
m), 1.31 (9H, s). MS calculated for C21H23ClN2O6: 434.1, found
435.1 (M + H)+. Rf ) 0.90 (CHCl3-MeOH, 7:1).
1-[(N-ter t-Bu toxyca r bon yl-D-1,2,3,4-tetr a h yd r oisoqu in -
olin e-3-ca r bon yl)-4-ch lor o-D-p h en yla la n yl]-4-p h en ylp ip -
er a zin e (7a ). To a solution of 5 (0.15 g, 0.33 mmol) in DMF
(1.5 mL) were added 1-phenylpiperazine (50 µL, 0.33 mmol),
HATU (0.14 g, 0.36 mmol), and DIEA (0.17 mL, 0.99 mmol).
The reaction mixture was stirred for 2 h at room temperature,
diluted with CH2Cl2 to 50 mL, and washed with aqueous 10%
KHSO4 (2 × 10 mL), saturated aqueous NaHCO3 (2 × 10 mL),
and water (2 × 5 mL), dried over MgSO4, and evaporated to
afford 7a as a somewhat yellow oil (0.10 g, quantitative yield).
MS calculated for C34H39ClN4O4: 602.3, found 603.3 (M + H+).
1-[(N-ter t-Bu toxyca r bon yl-D-1,2,3,4-tetr a h yd r oisoqu in -
olin e-3-ca r b on yl)-4-ch lor o-D-p h en yla la n yl]-4-cycloh ex-
ylp ip er a zin e (7b) was prepared from 5 (0.114 g, 0.25 mmol),
1-cyclohexylpiperazine (42 mg, 0.25 mmol), HATU (0.104 g,
0.27 mmol), and DIEA (0.13 mL, 0.75 mmol) as described above
for 7a . 7b was obtained as a clear oil (0.15 g, quantitative
yield). MS calculated for C34H45ClN4O4: 608.3, found 609.3 (M
+ H+). Rf ) 0.56 (CHCl3-MeOH, 7:1).
1-[(D-1,2,3,4-Tetr ah ydr oisoqu in olin e-3-car bon yl)-4-ch lo-
r o-D-p h en yla la n yl]-4-p h en ylp ip er a zin e Dih yd r och lor id e
(8a ). To a solution of 7a (90 mg, 0.15 mmol) in MeOH (1 mL)
was added 4 M HCl in dioxane (0.38 mL), and the reaction
mixture was stirred for 1.5 h at room temperature and
evaporated. The residue was dissolved in MeOH (1 mL) and
treated with dry ether. The crude 8a was precipitated as a
white-yellow powder (80 mg), which was purified by HPLC
(eluent, 19% MeCN; k′ ) 0.47) followed by freeze-drying. A
1
light powder formed. Yield 30 mg (36%). H NMR (270 MHz,
DMSO-d6): δ 9.87 (1H, m), 9.50 (1H, m), 9.13 (1H, d, J ) 7.6
Hz), 8.18 (2H, m), 7.46 (2H, m), 7.25 (9H, m), 5.60 (1H, br),
5.02 (1H, m), 4.20 (3H, m), 3.65 (4H, m), 3.00 (8H, m). HRMS
(M + H+): 503.2236, C29H32ClN4O2 requires 503.2214. Rf )
0.70 (n-BuOH-AcOH-H2O, 4:1:1).
4-Ch lor o-D-p h en yla la n in e 4-Nitr oben zyl Ester Hyd r o-
ch lor id e (3). To a solution of 2 (1.00 g, 2.30 mmol) in AcOH
(2 mL) 4 M HCl was added in dioxane (4 mL), and the mixture
was stirred for 1 h at room temperature. The resulting solution
was evaporated and the residue was triturated with dry ether
to give product 3 as a white solid (yield 0.85 g, 96%). 1H NMR
(270 MHz, DMSO-d6): δ 8.70 (3H, br s), 8.19 (2H, m), 8.49
(2H, m), 7.29 (4H, m), 5.28 (2H, s), 4.42 (1H, m), 3.15 (2H, m).
HRMS (M + H+): 335.0784, C16H16ClN2O4 requires 335.0789.
Rf ) 0.54 (CHCl3-MeOH, 7:1).
1-[(D-1,2,3,4-Tetr ah ydr oisoqu in olin e-3-car bon yl)-4-ch lo-
r o-D-p h en yla la n yl]-4-cycloh exylp ip er a zin e Ditr iflu or o-
a ceta te (8b). To a solution of 7b (75 mg, 0.125 mmol) in
CH2Cl2 (1 mL) was added TFA (2 mL), and the resulting
solution was allowed to stand for 1 h at room temperature.
The reaction mixture was evaporated to dryness, and the
residue was triturated with a dry ether and hexane mixture
to furnish crude 8b as a fine white powder (70 mg), which was
purified by HPLC (eluent, 13% MeCN; k′ ) 0.67) followed by
(N-ter t-Bu toxyca r bon yl-D-1,2,3,4-tetr a h yd r oisoqu in o-
lin e-3-ca r bon yl)-4-ch lor o-D-p h en yla la n in e 4-Nitr oben zyl
Ester (4). To a solution of Boc-D-Tic-OH (0.46 g, 1.65 mmol)
in DMF (3 mL) were added 3 (0.65 g, 1.65 mmol), HATU (0.69
g, 1.81 mmol), and DIEA (0.85 mL, 4.94 mmol). The reaction
mixture was stirred for 2 h at room temperature, diluted with
CH2Cl2 to 100 mL, and washed with aqueous 10% KHSO4 (2
× 25 mL), saturated aqueous NaHCO3 (2 × 25 mL), and water
(2 × 25 mL), dried over MgSO4, filtered, and evaporated to
1
freeze-drying. A light powder formed. Yield 37 mg (40%). H
NMR (270 MHz, DMSO-d6): δ 10.10 and 9.93 (1H, 2 br s),
9.58 (1H, br s), 9.34 (1H, br s), 9.01 (1H, m), 7.30 (8H, m),
5.00 (1H, m), 4.30 (5H, m), 3.30 (6H, m), 2.90 (4H, m), 2.20
(1H, m), 1.85 (4H, m), 1.60 (1H, m), 1.20 (5H, m). HRMS (M
+ H+): 509.2697, C29H38ClN4O2 requires 509.2683. Rf ) 0.28
(n-BuOH-AcOH-H2O, 4:1:1).
1
1-[(N-ter t-Bu toxyca r bon yl-D-1,2,3,4-tetr a h yd r oisoqu in -
o lin e -3-c a r b o n y l)-4-c h lo r o -D -p h e n y la la n y l]-4-o x y -4-
p h en ylp ip er a zin e (9a ). A solution of 7a (90 mg, 0.15 mmol)
in 30% H2O2-AcOH (2 mL) was allowed to stand for 1 h at
room temperature and for 2 h at 60 °C. The reaction mixture
was evaporated to afford 9a (82 mg, 88%). MS calculated for
afford product 4 (yield 0.97 g, 99%) as a fine white power. H
NMR (270 MHz, DMSO-d6): δ 8.39 (1H, d, J ) 7.6 Hz), 8.17
(2H, m), 7.48 (2H, m), 7.15 (8H, m), 5.19, 5.12 (2H, 2s), 4.50
(4H, m), 3.00 (4H, m), 1.20 (9H, m). MS calculated for C31H32
-
ClN3O7: 593.2, found 594.2 (M + H+). Rf ) 0.80 (CHCl3-
MeOH, 7:1).
C
34H39ClN4O5: 618.3, found 619.3 (M + H+).
(N-ter t-Bu toxyca r bon yl-D-1,2,3,4-tetr a h yd r oisoqu in o-
lin e-3-ca r bon yl)-4-ch lor o-D-p h en yla la n in e (5). To a stirred
suspension of 4 (0.15 g, 0.25 mmol) in MeOH (2 mL), aqueous
2 N NaOH (131 µL, 0.26 mmol) was added, and the mixture
was stirred for 1 h at room temperature. Reaction progress
was monitored using TLC and LC/MS. After 1 h, 31 µL (0.06
mmol) of aqueous 2 N NaOH was additionally added, and the
reaction mixture was stirred for 30 min more and then
evaporated. The residue was partitioned between EtOAc (50
mL) and aqueous 10% KHSO4 (25 mL), and the aqueous layer
was extracted with EtOAc (2 × 25 mL). The combined organic
extracts were washed with water (2 × 25 mL), dried over
MgSO4, filtered, and evaporated to give 5 (0.11 g, quantitative
1-[(N-ter t-Bu toxyca r bon yl-D-1,2,3,4-tetr a h yd r oisoqu in -
olin e-3-ca r b on yl)-4-ch lor o-D-p h en yla la n yl]-4-oxy-4-cy-
cloh exylp ip er a zin e (9b) was synthesized from 7b as de-
scribed for the preparation of 9a . Yield 92%. MS calculated
for C34H43ClN4O5: 624.3, found 625.3 (M + H+). Rf ) 0.41
(CHCl3-MeOH, 7:1).
1-[(D-1,2,3,4-Tetr ah ydr oisoqu in olin e-3-car bon yl)-4-ch lo-
r o-D-p h en yla la n yl]-4-oxy-4-p h en ylp ip er a zin e Ditr iflu o-
r oa ceta te (10a ). 9a (82 mg, 0.13 mmol) was dissolved in
CH2Cl2 (1 mL) and treated with TFA (2 mL). The resulting
solution was allowed to stand for 1 h at room temperature.
The reaction mixture was evaporated, and the residue was
triturated with a dry ether and hexane mixture to furnish
crude 10a (76 mg), which was purified by HPLC (eluent, 14%
MeCN; k′ ) 0.67) followed by freeze-drying. A light powder
1
yield) as a somewhat yellow oil. H NMR (270 MHz, DMSO-
d6): δ 12.80 (1H, br s), 8.00 (1H, m), 7.15 (8H, m), 4.40 (4H,
m), 2.90 (4H, m), 1.30 (9H, m). MS calculated for C24H27
-
ClN2O5: 458.2, found 459.2 (M + H+). Rf ) 0.22 (CHCl3-
formed. Yield 36 mg (36%). H NMR (270 MHz, DMSO-d6): δ
1
MeOH, 7:1).
9.50 (1H, br s), 9.31 (1H, br s), 9.17 and 9.08 (1H, 2d, J ) 8.6

4622 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Mutulis et al.
and 8.3 Hz), 8.10 (2H, m), 7.67 (2H, m), 7.30 (9H, m), 5.20
(1H, m), 4.70-3.80 (9H, m), 3.50 (3H, m), 3.00 (3H, m). HRMS
(M + H+): 519.2143, C29H32ClN4O3 requires 519.2163. Rf )
0.30 (n-BuOH-AcOH-H2O, 4:1:1).
evaporated. EtOAc (50 mL) was added to the residue, and the
mixture was extracted with saturated aqueous NaHCO3 (30
mL). The organic layer was washed with brine (2 × 15 mL),
dried over MgSO4, filtered, and evaporated to give the crude
product as a white oil. It was purified by crystallization from
petroleum ether to afford 17a (yield 0.70 g, 69%). 1H NMR
(270 MHz, DMSO-d6): δ 7.42 (5H, m), 4.42 (1H, m), 4.00 (2H,
m), 3.75 (1H, m), 3.70-2.80 (8H, m), 2.40 (1H, m), 2.00-1.00
(10H, m). MS calculated for C18H28N2O: 288.2, found 289.2
(M + H+). Rf ) 0.43 (CHCl3-MeOH, 7:1).
1-[(D-1,2,3,4-Tetr ah ydr oisoqu in olin e-3-car bon yl)-4-ch lo-
r o-D-p h en yla la n yl]-4-oxy-4-cycloh exylp ip er a zin e Ditr i-
flu or oa ceta te (10b). 10b was synthesized from 9b as de-
scribed for the preparation of 10a . HPLC eluent, 16% MeCN;
1
k′ ) 2.47. Yield 52%. H NMR (270 MHz, DMSO-d6): δ 9.62
(1H, br, s), 9.37 (1H, br, s), 9.04 and 9.05 (1H, 2d, J ) 6.9 and
7.6 Hz), 7.30 (8H, m), 5.05 (1H, m), 4.30 (5H, m), 3.65 (5H,
m), 3.30 (3H, m), 2.96 (3H, m), 2.17 (2H, m), 1.87 (2H, m),
1.70-1.00 (6H, m). HRMS (M + H+): 525.2617, C29H38ClN4O3
requires 525.2632. Rf ) 0.33 (n-BuOH-AcOH-H2O, 4:1:1).
1-[ci s-2-(t er t -B y t o x y c a r b o n y la m in o )c y c lo h e x y l]-
m eth a n ol (16a ). To a stirred suspension of lithium aluminum
hydride (0.40 g, 10.50 mmol) in anhydrous THF (25 mL) was
added cis-2-amino-1-cyclohexanecarboxylic acid (0.50 g, 3.49
mmol). The reaction mixture was stirred under Ar atmosphere
for 28 h at 70-80 °C, then cooled to 0 °C and quenched with
saturated aqueous K2CO3 (25 mL). The resulting white emul-
sion was diluted with EtOAc (50 mL) and filtered through a
short pad of Celite, which was afterward rinsed with EtOAc
(25 mL). The filtrate was evaporated, 1-butanol (50 mL) was
added, and the organic layer formed was washed with brine
(3 × 10 mL), dried over Na2SO4, and evaporated to afford a
white solid. MS analysis of the product showed that it
contained alcohol 15a . MS calculated for C7H15NO: 129.1,
found 130.1 (M + H+).
1-Ben zyl-4-(1-h yd r oxym et h ylcycloh exyl)p ip er a zin e
(17b) was synthesized from 15b as described for the prepara-
tion of 17a . Yield 25%. 1H NMR (270 MHz, DMSO-d6): δ 7.26
(5H, m), 4.23 (1H, m), 3.65-3.1 (5H, m), 2.60 (4H, m), 2.28
(3H, m), 1.70-1.00 (10H, m). MS calculated for C18H28N2O:
288.2, found 289.2 (M + H+). Rf ) 0.47 (CHCl3-MeOH, 7:1).
1-Ben zyl-4-(tr a n s-2-h ydr oxycycloh exyl)piper azin e (17c)
was synthesized from trans-2-aminocyclohexanol hydrochlo-
ride as described for the preparation of 17a . Yield 69%. 1H
NMR (270 MHz, DMSO-d6): δ 7.40 (5H, m), 3.97 (1H, m),
3.80-2.60 (11H, m), 1.90 (2H, m), 1.65 (2H, m), 1.20 (5H, m).
MS calculated for C17H26N2O: 274.2, found 275.2 (M + H+).
Rf ) 0.43 (CHCl3-MeOH, 7:1).
1-Ben zyl-4-(1-h yd r oxym et h ylcyclop en t yl)p ip er a zin e
(17d ) was synthesized from 1-amino-1-cyclopentanemethanol
1
as described for the preparation of 17a . Yield 74%. H NMR
(270 MHz, DMSO-d6): δ 7.27 (5H, m), 4.44 (1H, br s), 4.07
(1H, m), 3.61 (1H, m), 3.35 (2H, m), 2.66 (4H, m), 2.32 (4H,
m), 1.50 (8H, m). MS calculated for C17H25N2O: 274.2, found
275.2 (M + H+). Rf ) 0.27 (CHCl3-MeOH, 7:1).
The above product was dissolved in 10% TEA solution in
MeOH (30 mL), di-tert-butyl dicarbonate (0.76 g, 3.49 mmol)
was added, and the reaction mixture was stirred for 4 h at
room temperature. The mixture was evaporated, the residue
was dissolved in EtOAc (70 mL) and successively washed with
saturated aqueous NaHCO3 (2 × 10 mL), water (2 × 10 mL),
and brine (1 × 10 mL), dried over Na2SO4, and filtered, and
the filtrate was evaporated to afford a clear colorless oil (0.53
g). The product was purified on a silica gel column (2 cm × 20
cm) eluting it with a petroleum ether and ether mixture
(gradually changing their volume proportions from 4:1 to 5:3).
Eluate fractions containing the desired product were pooled
and evaporated to furnish 16a as a clear colorless oil (0.254 g,
32% for two steps). 1H NMR (270 MHz, DMSO-d6): δ 6.67 (1H,
d, J ) 8.6 Hz), 3.69 (1H, m), 3.32-3.17 (2H, m), 1.50 (4H, m),
1.37 (9H, s), 1.40-1.15 (6H, m). MS calculated for C12H23NO3:
229.2, found 230.2 (M + H+). Rf ) 0.67 (CHCl3-MeOH, 7:1).
1-(cis-2-Am in ocycloh exyl)m et h a n ol H yd r och lor id e
(15a ). 16a (0.218 g, 0.952 mmol) was dissolved in a 4 M
solution of HCl in dioxane (4.76 mL, 19.04 mmol), allowed to
stand for 1 h at room temperature, and evaporated. The
residue was dried in vacuo in the presence of KOH and treated
with dry ether to furnish the product 15a as white oil (0.157
1-Ben zyl-4-[cis-2-(1,2,4-tr ia zol-1-ylm eth yl)cycloh exyl]-
p ip er a zin e (18a ). To a cooled solution of 17a (0.27 g, 0.937
mmol) and TEA (0.26 mL, 1.87 mmol) in CH2Cl2 (10 mL) at 0
°C was added dropwise with stirring a solution of methane-
sulfonyl chloride (0.145 mL, 1.87 mmol) in CH2Cl2 (5 mL). The
reaction mixture was allowed to warm to room temperature
and was stirred for 2 h more. The volatiles were removed under
reduced pressure, and the residue containing 1-benzyl-4-[cis-
(2- methanesulfonyloxymethyl)cyclohexyl]piperazine was used
in the next step without any purification as clear-yellow oil.
MS calculated for C19H29N2O3S: 366.2, found 367.2 (M + H+).
To a solution of the above-mentioned oil in DMF (2 mL) was
added the sodium salt of 1,2,4-triazole (0.257 g, 2.81 mmol).
The resulting suspension was heated for 12 h at 80-100 °C
and cooled to room temperature. The reaction mixture was
poured into water (15 mL) and extracted with EtOAc (2 × 20
mL). The combined organic extracts were washed with brine
(2 × 10 mL), dried over NaSO4, and filtered, and the filtrate
was evaporated, affording a yellow oil (0.444 g). The product
was purified on a silica gel chromatography column (2 cm ×
25 cm) and eluted with a hexanes-EtOAc-MeOH mixture
(gradually changing their volume proportions from 1:1:0 to 1:6:
1). Eluate fractions containing the desired product were pooled
and evaporated to furnish 18a as a clear colorless oil. Yield
1
g, quantitative yield). H NMR (270 MHz, DMSO-d6): δ 7.86
(3H, br s), 4.86 (1H, br s), 3.40 (2H, m), 1.90-1.20 (10H, m).
HRMS (M + H+): 130.1237, C7H16NO requires 130.1232. Rf
) 0.57 (n-BuOH-AcOH-H2O, 4:1:1).
1
36 mg (11% for two steps). H NMR (270 MHz, DMSO-d6): δ
8.50 (1H, s), 7.92 (1H, s), 7.28 (5H, m), 4.23 (2H, d, J ) 7.3
Hz), 3.42 (2H, s), 3.33 (8H, m), 2.36 (2H, m), 2.07 (1H, m),
1.78 (2H, m), 1.50-1.00 (5H, m). MS calculated for C20H29N5:
339.2, found 340.2 (M + H+). Rf ) 0.36 (CHCl3-MeOH, 7:1).
1-Ben zyl-4-[1-(1,2,4-tr ia zol-1-ylm eth yl)cycloh exyl]p ip -
er a zin e (18b) was synthesized from 17b as described for the
preparation of 18a . Yield 19% (oil). 1H NMR (270 MHz, DMSO-
d6): δ 8.49 (1H, s), 7.97 (1H, s), 7.46 (5H, m), 4.29 (2H, s),
4.16 (2H, s), 3.21 (2H, m), 2.96 (6H, m), 1.74 (2H, m), 1.60-
1.00 (8H, m). MS calculated for C20H29N5: 339.2, found 340.2
(M + H+). Rf ) 0.41 (CHCl3-MeOH, 7:1).
1 -[ 1 -( t e r t -B y t o x y c a r b o n y l a m i n o ) c y c l o h e x y l ] -
m eth a n ol (16b) was synthesized from 1-aminocyclohexane-
carboxylic acid as described for the preparation of 16a . Yield
68%. 1H NMR (270 MHz, DMSO-d6): δ 6.03 (1H, br s), 4.54
(1H, br s), 3.34 (2H, s), 1.89 (2H, dm, J ) 12.2 Hz), 1.50-1.10
(8H, m), 1.35 (9H, s). MS calculated for C12H23NO3: 229.2,
found 230.2 (M + H+). Rf ) 0.69 (CHCl3-MeOH, 7:1).
1-(1-Am in ocycloh exyl)m eth a n ol Hyd r och lor id e (15b).
15b was synthesized from 16b as described for the preparation
of 15a . Yield 80%. 1H NMR (270 MHz, DMSO-d6): δ 7.80 (3H,
br s), 5.41 (1H, br s), 3.45 (2H, s), 1.80-1.10 (10H, m). HRMS
(M + H+): 130.1235, C7H16NO requires 130.1232. Rf ) 0.45
(n-BuOH-AcOH-H2O, 4:1:1).
1-Ben zyl-4-[tr a n s-2-(1,2,4-tr ia zol-1-yl)cycloh exyl]p ip -
er a zin e (18c) was synthesized from 17c as described for the
preparation of 18a . Yield 23% (oil). 1H NMR (270 MHz, DMSO-
d6): δ 8.41 (1H, s), 7.88 (1H, s), 7.25 (5H, m), 4.32 (1H, m),
3.34 (3H, m), 2.74 (1H, m), 2.62 (2H, m), 2.50 (1H, m), 2.13
(4H, m), 1.89 (2H, m), 1.74 (2H, m), 1.30 (4H, m). MS
calculated for C19H26N5: 325.2, found 326.2 (M + H+). Rf )
0.65 (CHCl3-MeOH, 7:1).
1-Be n zyl-4-(cis-2-h yd r oxym e t h ylcycloh e xyl)p ip e r -
a zin e (17a ). To a solution of 1330 (0.251 g, 0.937 mmol) in
EtOH (25 mL) were added 15a (0.155 g, 0.937 mmol) and
NaHCO3 (0.283 g, 3.373 mmol). The reaction mixture was
refluxed for 5 h, then cooled to room temperature and

Substituted Piperazines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4623
1-Ben zyl-4-[1-(1,2,4-tr ia zol-1-ylm eth yl)cyclop en tyl]p i-
p er a zin e (18d ) was synthesized from 17d as described for
the preparation of 18a . Yield 21%. 1H NMR (270 MHz, DMSO-
d6): δ 8.61 (1H, s), 7.97 (1H, s), 7.46 (5H, m), 4.24 (2H, s),
3.40 (2H, m), 3.33 (4H, m), 2.28 (4H, m), 1.70-1.30 (8H, m).
MS calculated for C19H27N5: 325.2, found 326.2 (M + H+). Rf
) 0.45 (CHCl3-MeOH, 7:1).
20a . Yield 82% (oil). MS calculated for C26H37ClN6O3: 516.3,
found 517.3 (M + H+). Rf ) 0.63 (CHCl3-MeOH, 7:1).
1-[N-(ter t-Bu toxyca r bon yl)-4-ch lor o-D-p h en yla la n yl]-
4-[1-(1,2,4-tr iazol-1-ylm eth yl)cyclopen tyl]piper azin e (20d)
was synthesized from 19d as described for the preparation of
20a . Yield 81% (oil). MS calculated for C26H37ClN6O3: 516.3,
found 517.3 (M + H+). Rf ) 0.51 (CHCl3-MeOH, 7:1).
4-[cis-2-(1,2,4-Tr ia zol-1-ylm et h yl)cycloh exyl]p ip er a -
zin e Dih yd r och lor id e (19a ). To a solution of 18a (32 mg,
0.094 mmol) in MeOH (3 mL) were added 10% Pd/C (55 mg)
and ammonium formate (0.16 g, 2.51 mmol). The mixture was
heated under reflux for 1.5 h. After cooling to room temper-
ature, the mixture was filtered and the filtrate was evaporated.
The residue was dissolved in water (1.0 mL) and purified by
HPLC (detection at 205 nm; eluent, water + 0.1% CF3COOH).
Eluate fractions containing the presumed product of interest
were pooled and freeze-dried. The oil formed was dissolved in
MeOH (1.0 mL), and the solution slowly passed through a
column (5 mm × 10 mm) packed with Dowex 1 × 4 in Cl-
form. The column was rinsed with methanol, and the united
eluate evaporated. Yield 20 mg (66%, amorphous solid). 1H
NMR (270 MHz, DMSO-d6): δ 11.91 (1H, br s), 9.70 and 9.46
(2H, 2br s), 8.72 (1H, m), 8.06 (1H, m), 4.02 (2H, m), 3.69-
2.68 (8H, m), 1.95-1.50 (10H, m). HRMS (M + H+): 250.2030,
1-(4-Ch lor o-D-p h en yla la n yl-4-[cis-2-(1,2,4-tr ia zol-1-yl-
m eth yl)cycloh exyl]p ip er a zin e Ditr iflu or oa ceta te (21a ).
To a solution of 20a (21.0 mg, 0.0396 mmol) in CH2Cl2 (1 mL),
TFA (2 mL) was added, and the resulting mixture was stirred
for 1 h at room temperature. The volatiles were removed in
vacuo, and the residue was triturated with a mixture of dry
ether and hexane to furnish the product 21a as a fine white
powder (19.0 mg, 73%). 1H NMR (270 MHz, DMSO-d6): δ 8.53
(1H, m), 8.39 (1H, m), 8.21 (3H, m), 8.01 (1H, s), 7.42 (2H, m),
7.26 (2H, m), 4.67 (1H, m), 4.25 (2H, m), 3.44 (2H, m), 3.44-
2.85 (8H, m), 1.95-1.65 (2H, m), 1.40 (8H, m). HRMS (M +
H+): 431.2318, C22H32ClN6O requires 431.2326. Rf ) 0.24 (n-
BuOH-AcOH-H2O, 4:1:1).
1-(4-Ch lor o-D-p h e n yla la n yl)-4-[1-(1,2,4-t r ia zol-1-yl-
m eth yl)cycloh exyl]p ip er a zin e Ditr iflu or oa ceta te (21b).
21b was synthesized from 20b as described for the preparation
of 21a . Yield 79% (white powder). 1H NMR (270 MHz, DMSO-
d6): δ 8.57 (1H, br s), 8.23 (4H, m), 8.04 (1H, br s), 7.42 (2H,
m), 7.25 (2H, m), 4.66 (1H, m), 4.15 (4H, m), 3.08-2.75 (8H,
C
13H24N5 requires 250.2031. Rf ) 0.06 (n-BuOH-AcOH-H2O,
4:1:1).
4-[1-(1,2,4-Tr iazol-1-ylm eth yl)cycloh exyl]piper azin e Di-
m), 1.78-1.16 (10H, m). HRMS (M + H+): 431.2301, C22H32
-
h yd r och lor id e (19b). 19b was synthesized from 18b as
described for the preparation of 19a . The HPLC eluent
contained 1.6% MeCN. Yield 72% (amorphous solid). 1H NMR
(270 MHz, DMSO-d6): δ 9.30 (1H, m), 8.50 (1H, s), 8.51 (2H,
m), 7.98 (H, s), 4.16 (2H, m), 3.00 (4H, m), 2.71 (4H, m), 1.77
(2H, m), 1.45 (5H, m), 1.18 (3H, m). HRMS (M + H+):
250.2028, C13H24N5 requires 250.2031. Rf ) 0.16 (n-BuOH-
AcOH-H2O, 4:1:1).
ClN6O requires 431.2326. Rf ) 0.30 (n-BuOH-AcOH-H2O,
4:1:1).
1-(4-Ch lor o-D-p h en yla la n yl)-4-[tr a n s-2-(1,2,4-tr ia zol-1-
yl)cycloh exyl]p ip er a zin e Ditr iflu or oa ceta te (21c). 21c
was synthesized from 20c as described for the preparation of
21a . Yield 72% (white powder). 1H NMR (270 MHz, DMSO-
d6): δ 8.48 (1H, s), 8.14 (4H, m), 7.92 (1H, s), 7.39 (2H, m),
7.19 (2H, m), 4.59 (1H, m), 4.38 (1H, m), 3.51 (2H, m), 3.45-
3.15 (4H, m), 3.15-2.76 (4H, m), 2.26-1.08 (9H, m). HRMS
(M + H+): 417.2173, C21H30ClN6O requires 417.2169. Rf ) 0.22
(n-BuOH-AcOH-H2O, 4:1:1).
4-[tr a n s-2-(1,2,4-Tr ia zol-1-yl)cycloh exyl]p ip er a zin e Di-
h yd r och lor id e (19c). 19c was synthesized from 18c as
described for the preparation of 19a . HPLC eluent, water +
1
0.1% CF3COOH. Yield 35% (amorphous solid). H NMR (270
1-(4-Ch lor o-D-p h en yla la n yl)-4-[R,R-2-(1,2,4-tr ia zol-1-yl-
)cycloh exyl]p ip er a zin e Dih yd r och lor id e [(R,R)-21c] a n d
1-(4-Ch lor o-D-p h en yla la n yl)-4-[S,S-2-(1,2,4-t r ia zol-1-yl)-
cycloh exyl]p ip er a zin e Dih yd r och lor id e [(S,S)-21c]. 21c
(50.6 mg) was dissolved in 8.5 mL of methanol. This solution
was introduced in 150 µL portions into a 11034 Chirobiotic V
250 mm × 10 mm HPLC column (Advanced Separation
Technologies Inc.). Eluent, dioxane-methanol-acetic acid-
triethylamine (50:50:0.02:0.01); flow rate 5 mL/min; UV
monitoring at 220 nm. At each run, three eluate fractions were
collected. The first fraction contained pure (R,R)-21c, the
second contained a mixture of (R,R)-21c and (S,S)-21c, and
the third contained pure (S,S)-21c. When all of the initial
mixture was passed through the column, the corresponding
eluate fractions were separately pooled in three flasks and
evaporated. The residues were dried at 0.01 bar for 24 h, and
then each of them was dissolved in 50 mL of water and freeze-
dried for 2 days. Each of the products obtained were dissolved
in water (2.0 mL) and the solutions separately and slowly
passed through one of three columns (5 mm × 10 mm) with
Dowex 1 × 4 in Cl- form. Columns were rinsed with water,
and the correspondingly united eluates were freeze-dried. Each
of three residues was dissolved in 1.5 mL of water and freeze-
dried again. Three samples of fluffy white powders were
obtained. The yield of (R,R)-21c was 12.4 mg (32%), ee )
95.5%, and the yield of (S,S)-21c was 12.7 mg (33%), ee )
92.6%. (Optical purities, ee, were determined from data of
analytical chiral HPLC on Chirobiotic V.) The intermediate
fraction gave 9.8 mg (25%) of a regenerated isomer mixture.
MHz, DMSO-d6): δ 9.45 (1H, m), 8.67 (1H, s), 8.62 (2H, m),
8.03 (1H, s), 4.42 (1H, m), 3.08 (3H, m), 2.82 (5H, m), 2.40
(2H, m), 2.03-1.68 (4H, m), 1.30 (3H, m). HRMS (M + H+):
236.1876, C12H22N5 requires 236.1875. Rf ) 0.13 (n-BuOH-
AcOH-H2O, 4:1:1).
4-[1-(1,2,4-Tr ia zol-1-ylm eth yl)cyclop en tyl]p ip er a zin e
Dih yd r och lor id e (19d ). 19d was synthesized from 18d as
described for the preparation of 19a . The HPLC eluent
contained 1.6% MeCN. Yield 47% (amorphous solid). 1H NMR
(270 MHz, DMSO-d6): δ 9.31 (1H, m), 8.94 (1H, s), 8.41 (2H,
m), 8.19 (1H, s), 4.50 (2H, m), 2.88 (4H, m), 2.53 (4H, m), 1.92
(3H, m), 1.75-1.44 (5H, m). HRMS (M + H+): 236.1876,
C
12H22N5 requires 236.1875. Rf ) 0.14 (n-BuOH-AcOH-H2O,
4:1:1).
1-(N-ter t-Bu toxyca r bon yl-4-ch lor o-D-p h en yla la n yl)-4-
[cis-2-(1,2,4-t r ia zol-1-yl-m et h yl)cycloh exyl]p ip er a zin e
(20a ). To a solution of 19a (17.0 mg, 0.0528 mmol) in DMF
(200 µL) were added Boc-D-Phe(4-Cl)-OH (15.8 mg, 0.0528
mmol), HATU (22.1 mg, 0.0581 mmol), and DIEA (27.1 µL,
0.158 mmol). The reaction mixture was stirred for 2 h at room
temperature, then diluted with EtOAc to 20 mL and washed
with aqueous 10% KHSO4 (2 × 5 mL), saturated aqueous
NaHCO3 (2 × 5 mL), water (2 × 5 mL), and brine (1 × 5 mL),
dried over Na2SO4, filtered, and evaporated to afford 20a as a
clear oil (21.0 mg, 75%). MS calculated for C27H39ClN6O3:
530.3, found 531.3 (M + H+). Rf ) 0.44 (CHCl3-MeOH, 7:1).
1-[N-(ter t-Bu toxyca r bon yl)-4-ch lor o-D-p h en yla la n yl]-
4-[1-(1,2,4-tr ia zol-1-ylm eth yl)cycloh exyl]p ip er a zin e (20b)
was synthesized from 19b as described for the preparation of
1-(4-Ch lor o-D-p h e n yla la n yl)-4-[1-(1,2,4-t r ia zol-1-yl-
m eth yl)cyclop en tyl]p ip er a zin e Ditr iflu or oa ceta te (21d ).
21d was synthesized from 20d as described for the preparation
of 21a . Yield 59% (white powder). 1H NMR (270 MHz, DMSO-
d6): δ 8.62 (1H, s), 8.38 (1H, m), 8.13 (3H, m), 7.98 (1H, s),
7.39 (2H, m), 7.21 (2H, m), 4.57 (1H, m), 4.35 (2H, m), 3.69
(2H, m), 3.15-2.85 (4H, m), 2.30 (4H, m), 2.02-1.45 (m, 8H).
20a . Yield, quantitative (oil). MS calculated for
C27H39-
ClN6O3: 530.3, found 531.3 (M + H+). Rf ) 0.40 (CHCl3-
MeOH, 7:1).
1-[N-(ter t-Bu toxyca r bon yl)-4-ch lor o-D-p h en yla la n yl]-
4-[tr a n s-2-(1,2,4-tr ia zol-1-yl)cycloh exyl]p ip er a zin e (20c)
was synthesized from 19c as described for the preparation of

4624 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Mutulis et al.
HRMS (M + H+): 417.2184, C21H30ClN6O requires 417.2169.
Rf ) 0.25 (n-BuOH-AcOH-H2O, 4:1:1).
(8H, m), 2.20-1.60 (6H, m), 1.26 (3H, m). HRMS (M + H+):
576.2830, C31H39ClN7O2 requires 576.2854. Rf ) 0.52 (n-
BuOH-AcOH-H2O, 4:1:1).
1-[N-(ter t-Bu toxycar bon yl)-D-1,2,3,4-tetr ah ydr oisoqu in -
olin e-3-ca r bon yl-4-ch lor o-D-p h en yla la n yl]-4-[cis-2-(1,2,4-
tr ia zol-1-ylm eth yl)cycloh exyl]p ip er a zin e (22a ) was syn-
thesized from 21a (17.0 mg, 0.0258 mmol), Boc-D-Tic-OH (7.2
mg, 0.0258 mmol), HATU (10.8 mg, 0.0284 mmol), and DIEA
(13.2 mL, 0.0774 mmol) as described for the preparation of
20a . The title compound was obtained as a clear oil (17.8 mg,
quantitative yield). MS calculated for C37H48ClN7O4: 689.3,
found 690.3 (M + H+). Rf ) 0.40 (CHCl3-MeOH, 7:1).
1-[N-(ter t-Bu toxycar bon yl)-D-1,2,3,4-tetr ah ydr oisoqu in -
olin e-3-ca r bon yl-4-ch lor o-D-p h en yla la n yl]-4-[1-(1,2,4-tr i-
a zol-1-ylm eth yl)cycloh exyl]p ip er a zin e (22b) was synthe-
sized from 21b as described for the preparation of 22a . Yield,
quantitative (oil). MS calculated for C37H48ClN7O4: 689.3,
found 690.3 (M + H+). Rf ) 0.39 (CHCl3-MeOH, 7:1).
1-[N-(ter t-Bu toxycar bon yl)-D-1,2,3,4-tetr ah ydr oisoqu in -
olin e-3-car bon yl-4-ch lor o-D-ph en ylalan yl]-4-[tr a n s-2-(1,2,4-
tr ia zol-1-yl)cycloh exyl]p ip er a zin e (22c) was synthesized
from 21c as described for the preparation of 22a . Yield,
quantitative (oil). MS calculated for C36H46ClN7O4: 675.3,
found 676.3 (M + H+). Rf ) 0.60 (CHCl3-MeOH, 7:1).
1-[N-(ter t-Bu toxycar bon yl)-D-1,2,3,4-tetr ah ydr oisoqu in -
olin e-3-ca r bon yl-4-ch lor o-D-p h en yla la n yl]-4-[R,R-2-(1,2,4-
tr ia zol-1-yl)cycloh exyl]p ip er a zin e [(R,R)-22c] was syn-
thesized from (R,R)-21c as described for the preparation of
1-(D-1,2,3,4-Tetr ah ydr oisoqu in olin e-3-car bon yl-4-ch lor o-
D-p h en yla la n yl)-4-[R,R-2-(1,2,4-t r ia zol-1-yl)cycloh exyl]-
p ip er a zin e Ditr iflu or oa ceta te [(R,R)-23c]. (R,R)-23c was
synthesized from (R,R)-22c as described for the preparation
of 21a . The HPLC eluent contained 9.6% MeCN, k′ ) 0.87.
Yield 67% (white powder). HRMS (M + H+): 576.2834, C31H39
-
ClN7O2 requires 576.2854. Rf ) 0.52 (n-BuOH-AcOH-H2O,
4:1:1).
1-(D-1,2,3,4-Tetr ah ydr oisoqu in olin e-3-car bon yl-4-ch lor o-
D-p h en yla la n yl)-4-[S,S-2-(1,2,4-t r ia zol-1-yl)cycloh exyl]-
p ip er a zin e Ditr iflu or oa ceta te [(S,S)-23c]. (S,S)-23c was
synthesized from (S,S)-22c as described for the preparation
of 21a . The HPLC eluent contained 9.6% MeCN, k′ ) 0.87.
Yield 43% (white powder). HRMS (M + H+): 576.2838, C31H39
-
ClN7O2 requires 576.2854. Rf ) 0.52 (n-BuOH-AcOH-H2O,
4:1:1).
1-(D-1,2,3,4-Tetr ah ydr oisoqu in olin e-3-car bon yl-4-ch lor o-
D-p h en yla la n yl)-4-[1-(1,2,4-tr ia zol-1-ylm eth yl)cyclop en -
tyl]p ip er a zin e Ditr iflu or oa ceta te (23d ). 23d was synthe-
sized from 22d as described for the preparation of 21a . The
HPLC eluent contained 9.6% MeCN, k′ ) 1.00. Yield 17%
(white powder). 1H NMR (270 MHz, DMSO-d6): δ 9.42 (2H,
m), 9.33 (1H, m), 8.92 (1H, d, J ) 7.9), 8.62 (1H, s), 7.97 (1H,
s), 7.26 (8H, m), 4.92 (1H, m), 4.42-4.02 (5H, m), 3.70-3.15
(4H, m), 3.00-2.69 (4H, m), 2.30 (4H, m), 2.03-1.47 (8H, m).
HRMS (M + H+): 576.2865, C31H39ClN7O2 requires 576.2854.
Rf ) 0.41 (n-BuOH-AcOH-H2O, 4:1:1).
22a . Yield, quantitative (oil). MS calculated for
C36H46-
ClN7O4: 675.3, found 676.3 (M + H+). Rf ) 0.60 (CHCl3-
MeOH, 7:1).
1-[N-(ter t-Bu toxycar bon yl)-D-1,2,3,4-tetr ah ydr oisoqu in -
olin e-3-ca r bon yl-4-ch lor o-D-p h en yla la n yl]-4-[S,S-2-(1,2,4-
tr ia zol-1-yl)cycloh exyl]p ip er a zin e [(S,S)-22c] was syn-
thesized from (S,S)-21c as described for the preparation of 22a .
Yield, quantitative (oil). MS calculated for C36H46ClN7O4:
675.3, found 676.3 (M + H+). Rf ) 0.60 (CHCl3-MeOH, 7:1).
1-[N-(ter t-Bu toxycar bon yl)-D-1,2,3,4-tetr ah ydr oisoqu in -
olin e-3-ca r bon yl-4-ch lor o-D-p h en yla la n yl]-4-[1-(1,2,4-tr i-
a zol-1-ylm eth yl)cyclop en tyl]p ip er a zin e (22d ) was syn-
thesized from 21d as described for the preparation of 22a .
Yield 92% (oil). MS calculated for C36H46ClN7O4: 675.3, found
676.3 (M + H+). Rf ) 0.34 (CHCl3-MeOH, 7:1).
1-(D-1,2,3,4-Tetr ah ydr oisoqu in olin e-3-car bon yl-4-ch lor o-
D-p h en yla la n yl)-4-[cis-2-(1,2,4-t r ia zol-1-ylm et h yl)cyclo-
h exyl]p ip er a zin e Ditr iflu or oa ceta te (23a ). 23a was syn-
thesized from 22a as described for the preparation of 21a . The
crystalline crude product obtained was purified by HPLC (the
eluent contained 8% MeCN, k′ ) 4.00). Freeze-drying of eluate
fractions afforded the pure product as a white powder. Yield
10%. 1H NMR (270 MHz, DMSO-d6): δ 9.47 (2H, 2m), 9.32
(1H, m), 8.95 (1H, d, J ) 7.6 Hz), 8.57 (1H, s), 7.98 (1H, s),
7.40-7.12 (8H, m), 5.00 (1H, m), 4.60-4.03 (5H, m), 3.85-
3.16 (8H, m), 3.00-2.65 (4H, m), 1.90-1.05 (10H, m). HRMS
(M + H+): 590.3034, C32H41ClN7O2 requires 590.3010. Rf )
0.17 (n-BuOH-AcOH-H2O, 4:1:1).
Mela n ocor tin Recep tor Bin d in g Stu d ies. Cell Cu l-
tu r es. Sf9 cells were grown in 50-100 mL Sf-900 II medium
(Gibco-BRL) at 27 °C in small spinner bottles (250 mL) as
described.47 Recombinant viruses for human MC1, MC3, MC4,
and MC5R were added to the cell culture ((2-3) × 106 cells/
mL), and the incubation continued for an additional 72 h before
harvest.
Mem br a n e P r ep a r a tion s. Cells were centrifuged at 800g
for 5 min and Dounce homogenized (5 times by 10 stokes with
30 s intervals) in ice-cold 50 mL/(5 × 108 cells) in 20 mM Na-
HEPES, 0.1 mM phenylmethylsulfonyl fluoride, 0.25 mM
benzamidine, 1 µg/mL leupeptin, 1 µg/mL aprotinin, 1 µg/mL
soybean trypsin inhibitor, pH 7.4. The homogenate was
centrifuged at 700g for 5 min at 4 °C, and the pellet was then
homogenized and centrifuged again. The combined superna-
tants were collected, sedimented at 70000g for 60 min at 4
°C, washed once in new buffer, and recentrifuged. The final
pellet was resuspended in homogenization buffer at a protein
concentration of 1-3 mg of protein/mL, and aliquots were
stored at -80 °C. Protein was determined using the Bradford
method46 with bovine serum albumin as a standard.
Ra d ioliga n d Bin d in g Assa ys. Assays were performed by
incubating membranes (10 µg of protein/100 µL) in 20 mM
K-Hepes (pH 7.4), 5 mM NaCl, 1 mM CaCl2, 0.5 mM MgCl2,
and 0.5 mg/mL BSA with appropriate concentrations of [125I]-
NDP-MSH ([125I-Tyr2,Nle4,D-Phe7]R-MSH) and competitive
nonlabeled ligands. Incubations were for 3 h at 25 °C and were
terminated by rapid filtration through 0.3% polyethyleneimine
and 1 mg/mL BSA pretreated GF/B glass-fiber filters (What-
man International, Ltd., Madistone, U.K.) using a Brandell
cell harvester and three washes of 5 mL of ice-cold 50 mM
Tris-HCl, pH 7.4. Nonspecific binding was determined by using
3 µM NDP-MSH ([Nle4,D-Phe7]R-MSH). All binding data were
analyzed by nonlinear least-squares regression analysis using
the commercial program GraphPad PRISM 3.0 (GraphPad
Software, San Diego, CA), and the statistical analysis was
carried out with the StatView 4.5 package (Abacus Concepts,
Berkeley, CA) for Macintosh.
1-(D-1,2,3,4-Tetr ah ydr oisoqu in olin e-3-car bon yl-4-ch lor o-
D-p h en yla la n yl)-4-[1-(1,2,4-tr ia zol-1-ylm eth yl)cycloh exyl]-
p ip er a zin e Ditr iflu or oa ceta te (23b). 23b was synthesized
from 22b as described for the preparation of 21a . HPLC eluent
contained 9.6% MeCN, k′ ) 1.87. Yield 18% (white powder).
1H NMR (270 MHz, DMSO-d6): δ 9.50 (2H, H), 9.38 (1H, m),
8.98 (1H, d, J ) 7.6 Hz), 8.48 (1H, br s), 7.95 (1H, br s), 7.45-
7.20 (8H, m), 4.99 (1H, m), 4.48-3.20 (13H, m), 3.10-2.75 (4H,
m), 1.85-0.98 (10H, m). HRMS (M + H+): 590.2999, C32H41
-
ClN7O2 requires 590.3010. Rf ) 0.37 (n-BuOH-AcOH-H2O,
4:1:1).
1-(D-1,2,3,4-Tetr ah ydr oisoqu in olin e-3-car bon yl-4-ch lor o-
D-p h en yla la n yl)-4-[tr a n s-2-(1,2,4-tr ia zol-1-yl)cycloh exyl]-
p ip er a zin e Ditr iflu or oa ceta te (23c). 23c was synthesized
from 22c as described for the preparation of 21a . The HPLC
eluent contained 9.6% MeCN, k′ ) 0.87. Yield 41% (white
Meth od s a n d Con d ition s for Molecu la r Mod eln g. By
use of the subprogram MAXIMIN, default parameters were
introduced with following exceptions: charges, Gasteiger-
Marsili;50 PBC, minimum. Some other parameters were the
following: method, Powell; initial optimization, simplex; ter-
mination gradient, 0.05 kcal/mol; max iterations, 2000; force
1
powder). H NMR (270 MHz, DMSO-d6): δ 9.48 (2H, m), 9.37
(1H, m), 8.94 (1H, m), 8.47 (1H, s), 7.90 (1H, s), 7.39-7.15 (8H,
m), 4.93 (1H, m), 4.44-4.05 (6H, m), 3.30 (2H, m), 3.20-2.70

Substituted Piperazines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4625
field, Tripos;51 NB cutoff, 12.0; dielectric function, constant;
dielectric constant, 80.
(21) Mutulis, F.; Mutule, I.; Lapins, M.; Wikberg, J . E. S. Reductive
Amination Products Containing Naphthalene and Indole Moi-
eties Bind to Melanocortin Receptors. Bioorg. Med. Chem. Lett.
2002, 12, 1035-1038.
(22) Bakshi, R. K.; Barakat, K. J .; Nargund, R. P.; Palucki, B. L.;
Patchett, A. A.; Sebhat, I.; Ye, Z.; Van Der Ploeg, L. H. T. World
Patent Application WO 0074679 A1, December 14, 2000; Chem.
Abstr. 2001, 134, 42445.
Ack n ow led gm en t. This work was supported by the
Swedish VR (Grant 04X-05957) and Estonian SF (Grant
4603).
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectra
and assignments of 1H and 13C signals for (R,R)-21c, (S,S)-
21c, (R,R)-23c, and (S,S)-23c, chiral HPLC data, experimental
methods for X-ray structure analysis, crystal data and struc-
ture refinements for THIQ sulfate and (R,R)-23c dinitrate,
fractional atomic coordinates and U(iso) for THIQ sulfate, and
fractional atomic coordinates and U(eq) for (R,R)-23c dinitrate.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(23) Martin, W. J .; McGowan, E.; Cashen, D. E.; Gantert, L. T.;
Drisko, J . E.; Hom, G. J .; Nargund, R.; Sebhat, I.; Howard, A.
D.; Van der Ploeg, L. H. T.; MacIntyre, D. E. Activation of
Melanocortin MC4 Receptors Increases Erectile Activity in Rats
Ex Copula. Eur. J . Pharmacol. 2002, 454, 71-79.
(24) Van der Ploeg, L. H. T.; Martin, W. J .; Howard, A. D.; Nargund,
R. P.; Austin, C. P.; Guan, X.; Drisko, J .; Cashen, D.; Sebhat, I.;
Patchett, A. A.; Figueroa, D. J .; DiLella, A. G.; Connolly, B. M.;
Weinberg, D. H.; Tan, C. P.; Palyha, O. C.; Pong, S.-S.; MacNeil,
T.; Rosenblum, C.; Vongs, A.; Tang, R.; Yu, H.; Sailer, A. W.;
Fong, T. M.; Huang, C.; Tota, M. R.; Chang, R. S.; Stearns, R.;
Tamvakopoulos, C.; Christ, G.; Drazen, D. L.; Spar, B. D.;
Nelson, R. J .; MacIntyre, D. E. A Role for the Melanocortin 4
Receptor in Sexual Function. Proc. Natl. Acad. Sci. U.S.A. 2002,
99, 11381-11386.
(25) Sebhat, I. K.; Martin, W. J .; Ye, Z.; Barakat, K.; Mosley, R. T.;
J ohnston, D. B. R.; Bakshi, R.; Palucki, B.; Weinberg, D. H.;
MacNeil, T.; Kalyani, R. N.; Tang, R.; Stearns, R. A.; Miller, R.
R.; Tamvakopoulos, C.; Strack, A. M.; McGowan, E.; Cashen,
D. E.; Drisko, J . E.; Hom, G. J .; Howard, A. D.; MacIntyre, D.
E.; van der Ploeg, L. H. T.; Patchett, A. A.; Nargund, R. P. Design
and Pharmacology of N-[(3R)-1,2,3,4-Tetrahydroisoquinolinium-
3-ylcarbonyl]-(1R)-1-(4-chlorobenzyl)-2-[4-cyclohexyl-4-(1H-1,2,4-
triazol-1-ylmethyl)piperidin-1-yl]-2-oxoethylamine (1), a Potent,
Selective, Melanocortin Subtype-4 Receptor Agonist. J . Med.
Chem. 2002, 45, 4589-4593.
(26) Mutulis, F.; Yahorava, S.; Mutule, I.; Yahorau, A.; Kopanchuk,
S.; Veiksina, S.; Rinken, A.; Wikberg, J . E. S. A Non-Peptide
Radioiodinated High Affinity Melanocortin-4 Receptor Ligand.
J . Labelled Compd. Radiopharm. 2003, 46, 1007-1017.
(27) Schwarz, H.; Arakawa, K. The Use of p-Nitrobenzyl Esters in
Peptide Synthesis. J . Am. Chem. Soc. 1959, 81, 5691-5695.
(28) Albericio, F.; Bofill, J . M.; El-Faham, A.; Kates, S. A. Use of
Onium Salt-Based Coupling Reagents in Peptide Synthesis. J .
Org. Chem. 1998, 63, 9678-9683.
(29) Pruesse, W.; Amschler, H.; Schoetensack, W. German Patent
2638184, March 10, 1977; Chem. Abstr. 1977, 87, 6022.
(30) Szarvasi, E.; Festal, D.; Grand, M.; Depin, J . C.; Chabert, J .
Spiro[piperidine-4,6′-thiazolo[3,2-a]pyrimidines]. Thymoanalep-
tics and Blood Platelet Aggregation Inhibitors. Eur. J . Med.
Chem. 1976, 11, 115-24.
(31) Hsiao, Y.; Hegedus, L. S. Synthesis of Optically Active Imida-
zolines, Azapenams, Dioxocyclams, and Bis-dioxocyclams. J . Org.
Chem. 1997, 62, 3586-3591.
(32) Veeresa, G.; Datta, A. Stereoselective Synthesis of the Antifungal
Antibiotic (+)-Preussin. Tetrahedron 1998, 54, 15673-15678.
(33) Pozdnev, V. F. Acylation of Amino Acids by tert-Alkyl Pyrocar-
bonates. Khim. Prir. Soedin. 1971, 7, 384-385; Chem. Abstr.
1971, 75, 110568.
(34) Natsuka, K.; Nakamura, H.; Uno, H.; Umemoto, S. 1-Substituted
4-(1,2-diphenylethyl)piperazine Derivatives and Their Analgesic
Activities. J . Med. Chem. 1975, 18, 1240-1244.
Refer en ces
(1) Haskell-Luevano, C.; Hadley, M. E. The Melanocortin Receptors.
Drug News Perspect. 1999, 12, 197-205.
(2) Wikberg, J . E. S. Melanocortin Receptors: Perspectives for Novel
Drugs. Eur. J . Pharmacol. 1999, 375, 295-310.
(3) Wikberg, J . E. S.; Muceniece, R.; Mandrika, I.; Prusis, P.; Post,
C.; Skottner, A. New aspects on the melanocortins and their
receptors. Pharmacol. Res. 2000, 42, 393-420.
(4) Penhoat, A.; Naville, D.; Begeot, M. The Adrenocorticotropic
Hormone Receptor. Curr. Opin. Endocrinol. Diabetes 2001, 8,
112-117.
(5) Kesterson, R. A. The Melanocortin-3 Receptor. In Melanocortin
Receptors; Cone, R. D., Ed.; Humana Press Inc.: Totowa, NJ ,
2000; pp 385-403.
(6) Cone, R. D. The Melanocortin-4 Receptor. In Melanocortin
Receptors; Cone, R. D., Ed.; Humana Press Inc.: Totowa, NJ ,
2000; pp 405-447.
(7) Chen, W. The Melanocortin-5 Receptor. In Melanocortin Recep-
tors; Cone, R. D., Ed.; Humana Press Inc.: Totowa, NJ , 2000;
pp 449-472.
(8) Wikberg, J . E. S. Melanocortin Receptors: New Opportunities
in Drug Discovery. Expert Opin. Ther. Pat. 2001, 11, 61-76.
(9) MacNeil, D. J .; Howard, A. D.; Guan, X.; Fong, T. M.; Nargund,
R. P.; Bednarek, M. A.; Goulet, M. T.; Weinberg, D. H.; Strack,
A. M.; Marsh, D. J .; Chen, H. Y.; Shen, C.-P.; Chen, A. S.;
Rosenblum, C. I.; MacNeil, T.; Tota, M.; MacIntyre, E. D.; Van
der Ploeg, L. H. T. The Role of Melanocortins in Body Weight
Regulation: Opportunities for the Treatment of Obesity. Eur.
J . Pharmacol. 2002, 440, 141-157.
(10) Tatro, J . B. Receptor Biology of the Melanocortins, a Family of
Neuroimmunomodulatory Peptides. NeuroImmunoModulation
1996, 3, 259-284.
(11) Wilkison, W. O. Regulation of the Melanocortin Receptors by
Agouti. In Melanocortin Receptors; Cone, R. D., Ed.; Humana
Press Inc.: Totowa, NJ , 2000; pp 475-490.
(12) Szardenings, M.; Muceniece, R.; Mutule, I.; Mutulis, F.; Wikberg,
J . E. S. New Highly Specific Agonistic Peptides for Human
Melanocortin MC1 Receptor. Peptides 2000, 21, 239-243.
(13) Schioth, H. B.; Kask, A.; Mutulis, F.; Muceniece, R.; Mutule, I.;
Mutule, I.; Mandrika, I.; Wikberg, J . E. S. Novel Selective
Melanocortin 4 Receptor Antagonist Induces Food Intake after
Peripheral Administration. Biochem. Biophys. Res. Commun.
2003, 301, 399-405.
(14) Grieco, P.; Lavecchia, A.; Cai, M.; Trivedi, D.; Weinberg, D.;
MacNeil, T.; Van der Ploeg, L. H. T.; Hruby, V. J . Structure-
Activity Studies of the Melanocortin Peptides: Discovery of
Potent and Selective Affinity Antagonists for the hMC3 and
hMC4 Receptors. J . Med. Chem. 2002, 45, 5287-5294.
(15) Bednarek, M. A.; MacNeil, T.; Kalyani, R. N.; Tang, R.; Van der
Ploeg, L. H. T.; Weinberg, D. H. Selective, High Affinity Peptide
Antagonists of R-Melanotropin Action at Human Melanocortin
Receptor 4: Their Synthesis and Biological Evaluation in Vitro.
J . Med. Chem. 2001, 44, 3665-3672.
(35) Natsuka, K.; Nakamura, H.; Nishikawa, Y.; Negoro, T.; Uno,
H.; Nishimura, H. Synthesis and Structure-Activity Relation-
ships of 1-Substituted 4-(1,2-diphenylethyl)piperazine Deriva-
tives Having Narcotic Agonist and Antagonist Activity. J . Med.
Chem. 1987, 30, 1779-1787.
(36) Kaulen, J . The Enantiomers of the Azole Fungicide HWG-1608.
Asymmetric Synthesis of a 2-Hydroxyethylazole Fungicide.
Angew. Chem. 1989, 101, 507-508.
(37) Gasteiger, J .; Kaufmann, K. Diastereospecific Synthesis of
Fungicidal Threo- and Erythro-R-hydroxy Aminals. Tetrahedron
Lett. 1985, 26, 4341-4344.
(38) Sekar, G.; Nishiyama, H. Nonenzymatic kinetic resolution of
â-amino alcohols: chiral BINAP mediated SN2 displacement of
hydroxy groups by halogens through formation of an aziridinium
ion intermediate. Chem. Commun. 2001, 1314-1315.
(39) Diouf, O.; Depreux, P.; Chavatte, P.; Poupaert, J . H. Synthesis
and Preliminary Pharmacological Results on New Naphthalene
Derivatives as 5-HT4 Receptor Ligands. Eur. J . Med. Chem.
2000, 35, 699-706.
(40) Otting, G.; Wuethrich, K. Pre-TOCSY, a New Experiment for
Obtaining Complete Two-Dimensional Proton NMR Spectra of
Proteins in Aqueous Solution. J . Magn. Reson.1987, 75, 546-
549.
(16) Haskell-Luevano, C.; Holder, J . R.; Monck, E. K.; Bauzo, R. M.
Characterization of Melanocortin NDP-MSH Agonist Peptide
Fragments at the Mouse Central and Peripheral Melanocortin
Receptors. J . Med. Chem. 2001, 44, 2247-2252.
(17) Rocchi, R.; Gobbo, M. Peptide Science in the Years to Come.
Chim. Oggi 2002, 20, 26-29.
(18) Speake, J . D.; Bishop, M. J . Recent Advances in the Development
of Melanocortin-4 Receptor Agonists. Expert Opin. Ther. Pat.
2002, 12, 1631-1638.
(19) Andersson, P. M.; Boman, A.; Seifert, E.; Skottner, A.; Lundstedt,
T. Ligands to the Melanocortin Receptors. Expert Opin. Ther.
Pat. 2001, 11, 1583-1592.
(41) Bax, A.; Davis, D. G. Practical Aspects of Two-Dimensional
Transverse NOE Spectroscopy. J . Magn. Reson. 1985, 63, 207-
213.
(20) Mutulis, F.; Mutule, I.; Wikberg, J . E. S. N-Alkylaminoacids and
Their Derivatives Interact with Melanocortin Receptors. Bioorg.
Med. Chem. Lett. 2002, 12, 1039-1042.

4626 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Mutulis et al.
(42) Bodenhausen, G.; Ruben, D. J . Natural Abundance Nitrogen-
15 NMR by Enhanced Heteronuclear Spectroscopy. Chem. Phys.
Lett. 1980, 69, 185-189.
(43) Kessler, H.; Mronga, S.; Mueller, G.; Moroder, L.; Huber, R.
Conformational Analysis of a IgG1 Hinge Peptide Derivative in
Solution Determined by NMR Spectroscopy and Refined by
Restrained Molecular Dynamics Simulations. Biopolymers 1991,
31, 1189-1204.
(44) Brownstein, S.; Miller, R. Conformational Analysis of Disubsti-
tuted Cyclohexanes by Proton Resonance Spectroscopy. J . Org.
Chem. 1959, 24, 1886-1887.
(45) Armstrong, D. W.; Tang, Y.; Chen, S.; Zhou, Y.; Bagwill, C.;
Chen, J . R. Macrocyclic Antibiotics as a New Class of Chiral
Selectors for Liquid Chromatography. Anal. Chem. 1994, 66,
1473-1484.
(46) Bradford, M. M. A Rapid and Sensitive Method for the Quan-
titation of Microgram Quantities of Protein Utilizing the Prin-
ciple of Protein-Dye Binding. Anal. Biochem. 1976, 72, 248-
354.
(47) O’Reilly, D. R.; Miller, L. K.; Luckow, V. A. Baculovirus Expres-
sion Vectors: A Laboratory Manual; Freeman: New York, 1992.
(48) SYBYL Molecular Modeling Package, version 6.5; Tripos: St.
Louis, MO, 1998.
(49) Backer, R. T.; Briner, K.; Collado, C. I.; De Frutos-Garica, O.;
Doecke, C. W.; Fisher, M. J .; Garcia-Paredes, C.; Kuklish, S. L.;
Mancoso, V.; Martinelli, M. J .; Mateo, H. A. I.; Mullaney, J . T.;
Ornstein, P. L.; Wu, Z.; Xie, C. World Patent Application WO
0259095 A1, August 1, 2002; Chem. Abstr. 2002, 137, 140775.
(50) Gasteiger, J .; Marsili, M. Iterative Partial Equalization of Orbital
Electronegativity: A Rapid Access to Atomic Charges. Tetrahe-
dron 1980, 36, 3219-3222.
(51) Clark, M.; Cramer, R. D., III.; Van Opdenbosch, N. Validation
of the General Purpose Tripos 5.2 Force Field. J . Comput. Chem.
1989, 10, 982-1012.
J M0311285