Design and synthesis of a library of tertiary amides: Evaluation as mimetics of the melanocortins' active core

Article abstract of DOI:10.1016/j.bmc.2007.06.003

Two hundred and ten tertiary amides were prepared on solid phase. Diamines were coupled to activated carboxylated Wang polymer, and the polymeric substituted benzyloxycarbonyl protected diamines obtained were reacted with aldehydes or ketones in trimethyl orthoformate giving resin attached Schiff bases. Coupled resins were then reduced to secondary amines by sodium cyanoborohydride in 4% acetic acid/trimethyl orthoformate, followed by acylation with the carboxylic acid in the presence of PyBroP and diisopropylethylamine. Cleavage of tertiary amides from the resin was made by trifluoroacetic acid in the presence of scavengers (mainly 1,2-ethanedithiol). When indole derivatives were prepared, parallel alkylation with the linker fragment occurred, giving derivatives of 2-(4-hydroxybenzyl)-indole as side products. Solution synthesis or mixed liquid/solid phase preparation of title substances proved to be advantageous in cases when the above method did not give acceptable results. According to this approach an efficient formation of Schiff bases was achieved in the presence of TiCl₄. Substances were isolated by reversed phase chromatography; in some cases isomers were additionally separated by chiral chromatography on Chirobiotic T. When tested on human recombinant melanocortin receptors all the tertiary amides showed some binding affinities; for the highest affinity compounds the K_is reached 400 nM on MC₁, 2 μM on MC₃ and 1 μM on MC₄ and MC₅ receptors. cAMP assays of some of the title compounds showed that the tertiary amides are melanocortin receptor antagonists on the four MC receptor subtypes.

Full text of DOI:10.1016/j.bmc.2007.06.003

Bioorganic & Medicinal Chemistry 15 (2007) 5787–5810
Design and synthesis of a library of tertiary amides: Evaluation as
mimetics of the melanocortins’ active core
Felikss Mutulis, Jana Kreicberga,ꢀ Sviatlana Yahorava, Ilze Mutule,ꢁ
Larisa Borisova-Jan, Aleh Yahorau, Ruta Muceniece,§ Sandra Azena,
Santa Veiksina,– Ramona Petrovska and Jarl E. S. Wikberg*
Department of Pharmaceutical Biosciences, Division of Pharmacology, Uppsala Biomedical center,
Uppsala University, PO Box 591, S-751 24 Uppsala, Sweden
Received 12 September 2006; accepted 5 June 2007
Available online 8 June 2007
Abstract—Two hundred and ten tertiary amides were prepared on solid phase. Diamines were coupled to activated carboxylated
Wang polymer, and the polymeric substituted benzyloxycarbonyl protected diamines obtained were reacted with aldehydes or
ketones in trimethyl orthoformate giving resin attached Schiff bases. Coupled resins were then reduced to secondary amines by
sodium cyanoborohydride in 4% acetic acid/trimethyl orthoformate, followed by acylation with the carboxylic acid in the presence
of PyBroP and diisopropylethylamine. Cleavage of tertiary amides from the resin was made by trifluoroacetic acid in the presence of
scavengers (mainly 1,2-ethanedithiol). When indole derivatives were prepared, parallel alkylation with the linker fragment occurred,
giving derivatives of 2-(4-hydroxybenzyl)-indole as side products. Solution synthesis or mixed liquid/solid phase preparation of title
substances proved to be advantageous in cases when the above method did not give acceptable results. According to this approach
an efficient formation of Schiff bases was achieved in the presence of TiCl4. Substances were isolated by reversed phase chromatog-
raphy; in some cases isomers were additionally separated by chiral chromatography on Chirobiotic T. When tested on human
recombinant melanocortin receptors all the tertiary amides showed some binding affinities; for the highest affinity compounds
the Kis reached 400 nM on MC1, 2 lM on MC3 and 1 lM on MC4 and MC5 receptors. cAMP assays of some of the title compounds
showed that the tertiary amides are melanocortin receptor antagonists on the four MC receptor subtypes.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
belong to the 7TM family of G-protein-coupled recep-
tors8,9 and their subtypes show both differing distribu-
tions and functions in the body. The MC1R is present
in melanocytes,10 in the CNS11 and in cells of the
immune system.12 This receptor is known to be in-
volved in the regulation of skin and fur pigmentation,13
and in the regulation of the immune system.14 The
MC2R, which is responsive to ACTH only, but not
the MSHs, controls the production of adrenal glucocor-
ticoids.3,15 The MC3, MC4 and MC5Rs are expressed in
the brain; the MC3 and MC5Rs also in the periph-
ery.6,16–18 These receptors have many multifaceted
actions, e.g., the MC4R regulates feeding behaviour17
and is considered a prime novel target for drugs aimed
to the control of body weight.19 The MC3 receptor may
have a role in controlling sexual behaviours, while the
MC5 receptor may control exocrine gland functions.1
Besides melanocortins the agouti and AgRP proteins
are endogenous ligands for the melanocortin receptors.
These small proteins are shown to act as inverse
agonists.20
Melanocortins are natural peptides formed by post-
translational cleavage of pro-opiomelanocortin and
include melanocyte stimulating hormones (a-, b-, c-
MSH) and adrenocorticotropin.1 Melanocortins interact
with five subtypes of melanocortin receptors MC1–5R,
identified by molecular cloning.2–7 The MC receptors
Keywords: Melanocortin receptor ligands; Tertiary amides; Solid phase
organic synthesis; Indole derivatives.
*
Corresponding author. Tel.: +46 18 4714238; fax: +46 18 559718;
e-mail: Jarl.Wikberg@farmbio.uu.se
ꢀ
Present address: Faculty of Material Science and Applied Chemistry,
Riga Technical University, Azenes 14/24, LV-1048 Riga, Latvia.
Present address: Department of Organic Chemistry Latvian Institute
of Organic Synthesis, Aizkraukles iela 21, LV-1006 Riga, Latvia.
Present address: Department of Pharmacy, Faculty of Medicine,
University of Latvia, Sarlotes iela la, LV-1001 Riga, Latvia.
Present address: Institute of Organic and Bioorganic Chemistry,
Tartu University, Jakobi 2, 51014 Tartu, Estonia.
ꢁ
§
–
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.06.003

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F. Mutulis et al. / Bioorg. Med. Chem. 15 (2007) 5787–5810
Melanocortins are linear peptides with about 11–27 ami-
no acid residues that share a conserved core of between
three and six amino acid residues the most essential
solely through the pharmacophoric benzene, guanidi-
nium and indole groups in the side chains of the phenyl-
alanine, arginine and tryptophan residues, while the
peptide backbone and the parts of the side-chains closer
to the backbone do not participate in the binding. Such
a view can be found, e.g., in a study devoted to docking
of ligands to a hypothetical melanocortin-1 receptor
structure.43 If this view is true, a tempting perspective
emerges to simplify a structure in an extreme way by
removing the peptide backbone and connecting the
remaining pharmacophoric groups through a minimal
scaffold. Optimization would then merely be to place
the pharmacophoric groups in a correct position by
changing distances between them and introducing spa-
tial constraints.
1
structure being Phe-Arg-Trp.21 L-Phenylalanine residue
of the active core can be exchanged to the D-isomer,
or to D-2-naphthylalanine and still retaining or even
increasing the peptides’ activity on the MSH responsive
MCRs.21–23 By replacing the Phe7 residue with its D-iso-
mer and Met4 with Nle in a-MSH, a very potent MC
receptor agonist, NDP–MSH, was obtained.22 A num-
ber of quite potent cyclic analogues of MSH (both of
disulfide and lactam types) have also been prepared
(see e.g., Refs. 23 and 24). Moreover, by combining se-
quences obtained from phage display library screening
with parts of the sequence of a-MSH we found some
highly active MC1R selective peptides.25
On the other hand, the most essential fragment of the
agouti and AgRP is the tripeptide Arg-Phe-Phe,44 which
is closely related to the melanocortin sequence Phe-Arg-
Trp. One may assume that the pharmacophoric groups
in these fragments are spatially oriented similarly and
determine the activity for both the melanocortins and
agouti/AgRP, despite the other dissimilarities in se-
quence. Incorporation of the pharmacophoric groups
of these tripeptides in compact organic structures might
thus lead to MC receptor active compounds. We here
developed this idea by trying to combine the three phar-
macophoric groups with an extremely simple scaffold,
based on tertiary amides.
To obtain substances suited for medical use there is an
interest to non-peptides imitating melanocortins. Em-
piric search and lead optimization led in some cases to
highly active and selective organic compounds, such as
the MC4R agonist THIQ26 and derivatives thereof,27,28
the MC4R antagonist MCL0129,29 and some MC5R
antagonistic phenylguanidines.30 To obtain ligands for
melanocortin receptors, an alternative possibility is the
incorporation of the most important structural features
of the melanocortins into non-peptide compounds.
Early studies led to the assumption that a feature of the
‘biologically active’ conformation of melanocortins is a
b-turn of their core region.31 Later investigators applied
NMR and energy calculations on cyclic melanocortin-
related peptides with reduced flexibility, and showed
that turn structures (b-turn for L-phenylalanine deriva-
tives and reversed turn for D-phenylalanine compounds)
indeed predominate in the core region.32–34 In one at-
tempt the presumed bioactive b-turn conformation was
mimicked in nine-membered cyclic non-peptides. The
scaffolds were connected with three side-chains contain-
ing, respectively, an indole moiety, benzene or naphtha-
lene structures and an amino or guanidine group.35
Recently, also tri-substituted tetrahydropyrans were
used as scaffolds to bring such pharmacophoric groups
together.36 Small peptoids are attractive as substituents
of peptides as well. G. Heizmann et al.37 synthesized a
library of tripeptoids, which included side chains resem-
bling those of the active core of a-MSH, Phe-Arg-Trp,
and identified moderately active ligands. Our efforts in
this direction concerning N-alkylated dipeptide amide
peptoids and related structures led also to structures
with moderate MC receptor activity.38 Moreover,
Thompson et al.39 synthesized peptoids that mimicked
the AgRP tripeptide and succeeded to obtain MC4R
antagonists. Urea derivatives, which included pharma-
cophoric groups from melanocortin tripeptide or their
equivalents, were also synthesized on solid phase.40
Our studies on the preparation of products of reductive
amination41 and N-alkylaminoacids and derivatives42
resulted in substances showing moderate binding activ-
ity on melanocortin receptors.
2. Results
2.1. Structures
The general structure of the tertiary amides is shown in
Figure 1A. Three parts of the molecule are connected
through a centrally located amide linkage. This conju-
gated scaffold creates some rigidity in the centre of
the molecule resulting in formation of two rotamers
(Fig. 1B). The existence of such rotamers is well-known.45
They were previously identified also by us in a study
devoted to peptoids.38 Here, rotamers were detected by
NMR, where doubling of signals was observed in many
cases (see Section 5).
It turned out that the tertiary amides were relatively easy
to synthesize by a solid or liquid phase process allowing
According to one contemporary idea, the active core
Phe-Arg-Trp of the melanocortins binds to the receptor
Figure 1. (A) General structure of tertiary amides. (B) Stabilized forms
(rotamers).

F. Mutulis et al. / Bioorg. Med. Chem. 15 (2007) 5787–5810
5789
us to apply a high diversity of reagents of three types,
namely diamines, aldehydes or ketones and carboxylic
acids. A large set of compounds was thereby possible
to prepare. We thus introduced the three pharmaco-
phoric groups of melanocortins, presumed to be the
most essential, namely an amino or guanidino group,
a benzene or naphthalene function, and an indole struc-
ture (or the equivalents thereof). An advantage of our
approach was that the distances between the pharmaco-
phores could be easily varied. Moreover, very compact
structures, not covered by turn mimetics, could be
prepared.
(3-aminocyclohexyl)ureido-trans-cyclohexan-4-yl; 24,
trans-4-aminocyclohexyl; 25, (4-aminomethyl-cyclohexyl)
methyl; 26, (trans-4-aminocyclohexyl)ureido-cyclohex-
an-3-yl; 27, (3-aminocyclohexyl)ureido-cyclohexan-3-yl;
28, 3-aminocyclohexyl; 29, (3-aminomethyl-cyclohexyl)
methyl.
Number codes for the aldehyde or ketone parts (A) were
as follows: 0, 2-naphthylmethyl; 1, 3,4-dimethoxybenzyl;
2, 3-ethoxy-4-methoxybenzyl; 3, 2-fluorobenzyl; 4,
4-carbmethoxybenzyl, 5, 4-methylthiobenzyl; 6, 3-meth-
ylbenzyl; 7, 2-methylbenzyl; 8, 4-methylbenzyl; 9,
1-(benzothien-3-yl)ethyl; 10, 2-bromobenzyl; 11, 2-phenyl-
ethyl; 12, 3-(2-methoxyphenyl)allyl; 13, 2-pyridylmethyl;
14, 3-bromo-4-methoxybenzyl; 15, 3-phenylpropyl; 16,
3-(2-methoxy-4-hydroxyphenyl)allyl; 17, 1-(2-methyl-5-
phenylthien-2-yl)ethyl; 18, 2-bromo-4,5-dimethoxy-
benzyl; 19, 4-pyridylmethyl; 20, 2-chlorobenzyl; 21,
2-chloro-4,5-dimethoxybenzyl; 22, 2-fluoro-6-chlorob-
enzyl; 23, 4-cyanobenzyl; 24, 2,6-dichlorobenzyl; 25,
2,6-difluorobenzyl; 26, 2-adamantyl; 27, 1-methylethyl; 28,
2-bromoallyl; 29, 1-(2-naphthyl)ethyl; 30, (2-methylth-
ien-6-yl)methyl; 31, 1-(4-trifluoromethylphenyl)ethyl;
32, cycloheptyl; 33, 3,4-methylenedioxybenzyl; 34,
3-(2-naphthyl)allyl; 35, 3-phenylallyl; 36, 2-chloro-3-
phenylallyl; 37, 2-methyl-3-phenylallyl; 38, 1,2,3,4-tetra-
hydronaphthalene-2-yl; 39, 4-diethylaminobenzyl; 40,
benzyl; 41, 2-methyl-3-phenylpropyl; 42, spiro[5.5]und-
ecan-3-yl; 43, spiro[5.5]undec-8-en-3-yl.
2.1.1. Coding of compounds. Compounds were coded by
a
three-number code referring to the structure
D(A)@NAC(O)AC (Fig. 1). The numbers in the code
denote, respectively, the diamine (D), aldehyde or
ketone (A) and carboxylic acid [C(O)AC] parts listed
below. Moreover, for the cases that the three-number-
codes are followed by one or two numbers within paren-
theses the first of the numbers within parentheses
denotes that the substance is obtained from the corre-
sponding fraction of a reversed phase chromatography.
The second number within the parentheses denotes the
fraction number of a preparative chiral chromatogra-
phy. Codes marked with letter ‘a’ at the end correspond
to 4-hydroxybenzyl derivatives (Table 1).
The number codes for the diamine part (D) were as
follows: 1, pentyl; 2, 2-aminoethyl; 3, 3-aminopropyl;
4, 4-aminobutyl; 5, 5-aminopentyl; 6, 6-aminohexyl; 7,
7-aminoheptyl; 8, 8-aminooctyl; 9, 2-(3-aminopropyl)-
2-ethyl-hexyl and/or 4-(aminomethyl)-4-ethyl-octyl;
10a, 4-(4-amino-cyclohexylmethyl)-cyclohexyl, mixture
of isomers from commercial diamine; 10b, 4-(4-amino-
cyclohexylmethyl)-cyclohexyl, mixture of isomers ob-
tained from commercial diamine after partial separation
of its trans, trans form; 10c, trans,trans-4-(4-amino-cyclo-
hexylmethyl)-cyclohexyl; 10d, 4-(4-amino-cyclohexylm-
ethyl)-cyclohexyl, mixture of isomers from commercial
tritylpolymer attached diamine; 10e, cis-4-(trans-4-ami-
no-cyclohexylmethyl)-cyclohexyl; 11, 2-guanidinoethyl;
12, 3-guanidinopropyl; 13, 4-guanidinobutyl; 14a, 4-
(4-guanidino-cyclohexylmethyl)-cyclohexyl, mixture of
isomers obtained from commercial bis-(4-amin-
ocyclohexyl)-methane after partial separation of its trans,
trans form; 14b, 4-(4-guanidino-cyclohexylmethyl)-
cyclohexyl, mixture of isomers obtained from
commercial tritylpolymer attached diamine; 15, 3-ami-
nomethyl-benzyl; 16, 4-aminomethyl-benzyl; 17, trans-
2-aminocyclohexyl; 18, 5-amino-2-methyl-pentyl and/or
5-amino-4-methyl-pentyl, mixture of isomers obtained
from commercial diamine; 19, 6-amino-2,4,4-trimethyl-
hexyl and/or 6-amino-2,2,4-trimethyl-hexyl and/or
6-amino-3,3,5-trimethyl-hexyl and/or 6-amino-3,5,5-trim-
ethylhexyl mixture of isomers obtained from commercial
diamine; 20a, 4-(4-amino-3-methyl-cyclohexylmethyl)-2-
methyl-cyclohexyl, mixture of isomers obtained from
commercial diamine; 20b, 4-(4-amino-3-methyl-cyclo-
hexylmethyl)-2-methyl-cyclohexyl, mixture of isomers
obtained from fractionated commercial diamine; 21,
3-[4-(3-aminopropyl)-piperazine-1-yl]-propyl; 22, (trans-
4-aminocyclohexyl)ureido-trans-cyclohexan-4-yl; 23,
Number codes for the carboxylic acid parts (CAC@O)
were as follows: 0, indole-3-carbonyl; 1, indole-3-acetyl;
2, 3-(2-naphthyl)-D-alanyl; 3, 3-(2-naphthyl)-L-alanyl;
4, 4-nitro-D-phenylalanyl; 5, indole-3-propanoyl; 6,
3-(2-pyridyl)-D-alanyl; 7, 3-(4-biphenyl)-D-alanyl; 8,
indole-3-butanoyl; 9, 1-(4-chlorobenzoyl)-2-methyl-5-
methoxyindole-3-acetyl; 10, D-tryptophanyl; 11, indole-
3-glyoxylyl; 12, L-histidyl; 13, 2-naphthylacetyl; 14,
2-naphthoxyacetyl; 15, trans-3-indoleacryloyl; 16, 2-thi-
ophenebutanoyl; 17, L-tryptophanyl; 18, 5-benzyloxyin-
dole-2-carbonyl; 19, 5-hydroxyindole-2-carbonyl; 20,
4-chloro-D-phenylalanyl.
A list of all end-products prepared herein is given in
Table 1.
2.2. Chemistry
2.2.1. Preparation of reagents. We used in one case 3-(2-
naphthyl)-2-propenal (2), which is not commercially
available. We synthesized it in a simple and efficient
way from 2-naphthaldehyde (1) and acetaldehyde
(Scheme 1).
The use of commercial mixtures of diamines and/or
unsymmetrical diamines led to the formation of mix-
tures of isomers already at the first step of the solid
phase synthesis.
As we were interested to obtain information on the
activity of individual compounds, diamine fractionation
experiments were done before introducing them into the
reactions, in some cases. Thus, in order to improve the

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F. Mutulis et al. / Bioorg. Med. Chem. 15 (2007) 5787–5810
Table 1. Preparation procedures of tertiary amides and their binding
affinities to melanocortin receptorsa
Table 1 (continued)
Substance
P.p.b
(yield, %)
Ki (lM)
Substance
P.p.b
(yield, %)
Ki (lM)
MC3R MC4R
MC1R MC3R MC4R MC5R
MC1R
MC5R
5–35–8
5–36–8
5–36–15
5–37–8
5–37–15
5–39–8
5–40–2
5–40–3
5–40–4
5–40–6
5–40–7
5–40–8
5–40–8a
5–40–10
5–40–17
5–43–14
6–36–8
6–37–19
6–40–8
6–40–8a
6–41–19
7–36–8
7–40–8
8–35–5
9–36–8
9–37–12
9–40–8
9–40–8a
A(20)
A(18)
A(26)
A(8)
3.0
0.77
2.7
3.5
2.7
6.8
5.0
10
36
16
2.2
28
1–39–8
2–0–0
2–0–1
H(16)
A(13)
A(24)
J(21)
C(23)
C(7)
72
289
134
68
78
66
408
27
5.4
27
2.3
59
3.6
36
5.4
8.7
17
140
ndc
26
18
20
22
4.7
99
2–15–5
2–17–8
2–29–1
2–30–9
2–35–5
2–36–8
2–38–1
2–38–13
2–40–8
2–42–8
2–42–14
2–42–20
3–0–1
21
127
210
11
A(5)
ndc
33
7.5
4.4
2.3
17
36
19
A(24)
D(23)
D(26)
D(27)
D(21)
D(24)
A(28)
A(3)
25
13
34
24
11
41
39
C(27)
A(13)
A(8)
35
51
4.0
106
5.8
42
4.4
13
ndc
29
21
34
37
162
nbd
18
96
102
nbd.
20
5.6
4.0
1.6
22
65
16
nbd
3.9
3.2
2.0
32
nbd
14
C(28)
C(33)
A(8)
29
49
27
21
25
15
47
6.4
5.0
87
4.6
5.1
159
54
ndc
12
J(22)
J(10)
J(25)
A(20)
A(26)
A(27)
C(23)
A(2)
3.2
6.0
3.7
20
2.7
2.1
1.7
63
10
D(23)
D(28)
J(22)
106
43
4.9
2.4
161
19
3.4
3.2
12
34
44
7.4
2.4
3.2
13
3.7
26
5.9
3.8
11
8.0
21
A(24)
A(10)
A(18)
A(2)
3–0–8
3–0–13
3–9–8
3.7
5.9
7.4
21
26
12
23
27
43
20
16
267
55
3.4
3.7
9.8
7.4
3.8
17
3.8
2.1
31
104
30
9.8
164
32
2.3
2.9
5.0
4.0
1.2
2.5
5.7
2.1
2.1
0.51
3.2
1.9
2.9
2.1
2.4
1.0
2.1
4.3
3.2
17
36
46
3–11–8
3–15–1
3–35–0
3–35–1
3–35–5
3–35–11
3–35–13
3–36–8
3–36–11
3–37–0
3–37–1
3–37–11
3–37–16
3–38–0
3–38–1
3–38–5
3–38–15
3–38–18
3–40–8
4–0–0
33
13
A(12)
A(15)
A(13)
A(21)
A(14)
D(12)
A(17)
A(2)
A(31)
A(29)
A(19)
A(27)
A(6)
5.0
17
5.7
172
21
42
28
29
23
26
12
3.2
40
6.4
21
nbd
11
24
65
17
81
131
23
113
15
391
7.5
3.2
40
1.6
2.5
6.1
1.0
0.85
9.3
1.8
6.8
4.8
2.4
3.7
6.0
26
5.0
6.4
7.0
6.8
7.4
67
4.9
10
A(15)
A(28)
A(23)
A(24)
A(22)
A(29)
A(24)
C(27)
C(26)
I(14)
33
18
11
31
4.7
8.6
4.1
3.7
2.4
2.1
2.1
3.7
1.5
2.2
2.7
4.3
4.5
3.0
3.4
2.3
4.1
2.1
1.9
4.4
4.9
4.1
5.6
4.8
2.5
6.4
8.0
3.2
1.3
7.0
5.6
2.8
14
31
7.4
24
4.3
0.93
89
10a–36–8(1) A, K(5)
7.1
22
47
10b–1–8(2)
10b–1–8a
10b–2–8(2)
10b–2–8a
10b–3–8(2)
10b–3–8a
10b–4–8(2)
10b–4–8a
10b–5–8(2)
10b–5–8a
10b–6–8(2)
10b–6–8a
10b–7–8(2)
10b–7–8a
10b–8–8(2)
10b–8–8a
A, K(13)
A(6)
8.3
11
44
23
16
9.1
63
6.2
14
58
91
A, K(10)
A(3)
9.5
12
26
20
131
37
ndc
48
74
33
23
A, K(10)
A(3)
7.2
36
25
55
14
7.0
11
ndc
12
A, K(10)
A(3)
9.6
6.7
85
I(27)
I(38)
46
542
45
21
16
681
25
A, K(13)
A(4)
249
201
22
A(10)
A(28)
A(26)
A(15)
C(26)
C(4)
17
8.9
12
1.7
15
19
63
6.4
111
158
10
18
16
A, K(9)
A(2)
1.3
2.9
1.2
4.7
1.3
4.7
3.6
1.5
1.6
4.6
2.9
2.6
2.9
3.3
2.0
0.94
2.5
6.3
3.2
6.6
1.3
1.9
0.63
2.1
4–0–1
78
16
15
4–15–5
4–26–9
4–27–0
4–27–0a
4–35–5
4–36–8
4–37–9
4–40–8
4–40–8a
5–0–1
15
38
19
13
35
A, K(4)
A(2)
121
349
220
147
80
3.1
106
90
4.4
10
112
106
67
222
165
18
279
353
10
A, K(10)
A(3)
C(0.2)
A(10)
A(17)
A(16)
A(9)
2.2
9.1
7.5
5.0
49
8.3
2.4
1.8
12
10b–10–8(2) A, K(8)
10b–12–8(1) A, K(7)
10b–12–8(2) A, K(10)
32
30
5.4
7.1
17
85
56
17
31
38
16
20
10b–13–8
10b–14–8(2) A, K(8)
10b–14–8a A(2)
A(29)
39
A(1)
7.7
1.5
14
6.2
13
4.6
5.2
7.3
34
9.3
132
37
278
23
A(28)
A(23)
A(24)
A(6)
50
58
5–0–5
5–0–8
62
12
10b–16–8(1) A, K(7)
10b–16–8(2) A, K(9)
10b–18–8(1) A, K(9)
7.6
11
5.1
12
20
47
51
14
5–15–1
5–15–8
5–26–8
5–29–1
5–31–1
5–32–1
5–33–8
5–34–8
5–35–5
5–35–5a
77
7.9
19
266
18
ndc
A(6)
18
61
25
ndc
14
10b–18–8a
A(4)
94
28
C(14)
C(18)
C(25)
C(42)
A(26)
A(9)
6.1
4.5
2.6
6.0
5.5
2.5
3.7
3.3
46
10b–19–8(1) A, K(7)
10b–19–8(2) A, K(9)
10b–20–8(2) A, K(4)
13
12
9.4
16
8.0
11
172
22
96
5.2
97
14
38
16
12
35
23
10b–20–8a
A(3)
26
17
10b–21–8(2) A, K(8)
10b–21–8(3) A, K(9)
1.9
1.5
1.9
2.3
4.3
8.9
11
5.4
27
8.4
8.1
7.1
nd
8.0
6.7
25
A(22)
A(2)
10b–21–8a A(2)
10b–22–8(2) A, K(4)
8.3
24
27
5.2

F. Mutulis et al. / Bioorg. Med. Chem. 15 (2007) 5787–5810
5791
Table 1 (continued)
Table 1 (continued)
Substance
P.p.b
(yield, %)
Ki (lM)
Substance
P.p.b
(yield, %)
Ki (lM)
MC1R MC3R MC4R MC5R
MC1R MC3R MC4R MC5R
10b–22–8a
A(3)
1.1
1.6
1.5
1.4
2.5
1.6
1.8
2.1
1.2
1.4
1.0
0.81
0.96
2.3
1.4
0.61
1.5
1.9
1.5
0.41
0.96
1.9
1.9
1.3
4.2
4.5
1.4
3.1
2.7
0.42
2.3
2.9
23
12
1.9
2.0
1.6
2.4
2.6
5.3
3.0
3.0
3.2
5.5
3.3
3.8
2.0
5.2
5.6
6.0
3.7
4.7
5.9
3.4
4.4
3.5
5.0
4.0
4.8
12
15
20a–40–8(1) A, K(4)
20a–40–8(2) A, K(6)
20b–40–8(1) A, K(3)
20b–40–8(2) A, K(10)
20b–40–8(3) A, K(4)
20b–40–8(4) A, K(2)
0.69
0.87
1.6
1.7
1.3
1.3
4.6
6.4
11
4.3
22
62
6.6
12
31
78
52
99
31
10
50
33
27
59
45
57
25
29
30
25
24
1.2
3.2
3.7
3.7
3.8
2.1
9.2
16
1.7
2.2
42
10b–23–8(2)
10b–23–8(3)
10b–23–8a
A, K(10)
A, K(9)
A(2)
6.5
3.9
21
20
25
6.9
5.6
5.0
6.6
22
10b–24–8(2)
10b–24–8a
A, K(4)
A(1)
25
27
7.2
22
10b–25–8(2)
10b–25–8a
A, K(8)
A(1)
52
65
83
21
21–36–8
21–40–8
22–40–8
23–40–8
24–36–8
24–40–8
25–36–8
25–40–8
26–40–8
27–36–8
27–40–8
27–40–8a
28–36–8
28–40–8
29–36–8
29–40–8
A(16)
A(14)
E(27)
E(23)
A(18)
A(15)
A(16)
A(17)
E(25)
E(19)
E(6)
10b–28–8(1)
10b–28–8(1,1)
10b–28–8(1,2)
10b–28–8(3)
10b–36–8(1)
10b–36–8(1,1)
10b–36–8(1,2)
10b–36–8(2)
10b–36–14
A, K(6)
A, K, L(2)
A, K, L(2)
A, K(7)
A, K(9)
A, K, L(2)
A, K, L(3)
A, K(8)
A(17)
19
29
4.9
9.4
7.8
5.5
8.7
5.4
4.9
3.2
5.5
ndc
ndc
ndc
ndc
6.7
ndc
ndc
12
25
11
176
52
1.8
2.8
4.3
1.6
2.5
2.7
3.1
2.2
2.9
2.8
3.3
1.5
2.7
21
27
3.2
8.4
2.5
4.4
7.3
18
10
40
17
33
11
18
31
21
37
16
23
5.3
6.5
2.1
5.7
3.7
3.6
49
23
10b–36–14(1,1) A, K, L(2)
10b–36–14(1,2) A, K, L(3)
10b–36–14(2,1) A, K, L(2)
10b–36–14(2,2) A, K, L(2)
ndc
ndc
ndc
ndc
22
E(1)
A(16)
A(24)
A(13)
A(28)
10
18
9.1
8.7
10b–36–14(3)
A, K(10)
10b–36–14(3,1) A, K, L(3)
10b–36–14(3,2) A, K, L(3)
ndc
ndc
12
a All values represent means of at least two experiments, and difference
between measurements for the same sample did not exceed 30%.
b Preparation procedure, see Section 5.
c Not determined.
d Not binding up to concentration 1 mM.
10b-36-18
10b-38–8
10b–40–8(1)
10c–36–8
10d–35–8
10d–40–8
10e–18–8
10e–18–8a
11-0–1
A(27)
C(28)
A, K(7)
A(25)
B(8)
278
15
44
2.6
6.8
9.2
8.3
7.8
7.4
179
149
179
150
16
105
8.4
7.7
18
45
16
B(12)
F(6)
F(16)
9.4
8.1
5.4
57
6.9
9.1
15
A(24), G(50) 19
A(21), G(49) 14
A(29), G(55) 8.3
A(20), G(54) 35
A(27), G(52) 12
A(26), G(42) 1.9
A(26), G(60) 17
A(21), G(49) 12
A(24), G(40) 3.2
11–0–5
11–0–8
65
17
204
228
53
8.3
28
Scheme 1. Reagents and conditions: (a) CH3CHO, KOH, MeOH, 0–
5 ꢁC, 2 h; (b) (CH3CO)2O, 100 ꢁC, 30 min, (c) HCl, H2O, 100 ꢁC, 20
min.
12–0–1
12–0–5
13
12–0–8
13–0–1
7.5
137
50
6.0
182
116
96
6.1
16
13–0–5
13–0–8
14a–36–8(1)
21
11
homogeneity of 4,40-diaminodicyclohexylmethane deriv-
atives, the commercial product was crystallized from
isooctane.46 Most of the trans, trans isomer separated
as crystals; after evaporation of solvent a mixture en-
riched with cis, trans and cis, cis forms was obtained
from the mother liquor. Both pure trans, trans isomer
and the enriched mixture were introduced in the tertiary
amide synthesis.
134
13
A(7), G(55),
K(30)
2.7
3.4
27
14a–36–8(2)
A(10), G(43), 5.4
K(45)
9.6
4.0
24
14a–40–8
14b–35–8
15–0–8
A(25), G(46) 1.5
A(20), G(49) 3.5
19
23
17
45
40
44
62
43
17
34
18
62
19
10
16
54
31
37
64
13
3.4
38
2.6
37
B(13)
B(22)
1.5
1.9
4.0
2.6
12
7.7
33
6.4
32
15–40–8
16–0–8
B(26)
B(27)
11
17
More complicated operations were applied to introduce
the pure cis, trans isomer of the above diamine (4,
Scheme 2),47 whose amino groups are not equivalent,
in a selective way into the tripartide structure. In other
words, our goal was that the central carbonyl group of
the end product should be attached exclusively to, e.g.,
cis amino group of the diamine. First, prolonged reac-
tion of trans,trans-4,40-diaminodicyclohexylmethane (3)
and its cis,trans isomer (4) with trifluoroacetic anhydride
in trifluoroacetic acid solution gave the model sub-
16–35–8
17–35–8
7.3
116
3.0
14
9.7
555
22
B(28)
A(24)
18–36–8
18–37–17
18–40–8
1.1
8.0
6.8
1.8
3.9
2.8
2.2
1.0
5.0
3.2
2.6
5.6
2.2
D(19)
A(22)
4.4
9.2
5.4
64
6.7
3.7
6.6
14
18–40–8(1)
19–36–8
A, K(8)
A(16)
19–37–10(1)
19–37–10(2)
19–40–8
D, K(8)
D, K(11)
A(26)
2.1
1.2
5.2
20
4.8
5.2
4.5
4.7
2.7
7.7
3.1
19–40–8(1)
19–40–8(2)
19–40–8(3)
20a–36–8(1)
20a–36–8(2)
A, K(7)
A, K(13)
A, K(6)
A, K(5)
A, K(12)
stances trans,trans-bis-(4-trifluoroacetylamino-cyclo-
hexyl)-methane (5) and its cis,trans isomer (6) (Scheme
25
14
1
2). The H NMR spectra of both of these diacylated
substances (Fig. 2A and B) in DMSO-d6 solution
9.1
4.8

5792
F. Mutulis et al. / Bioorg. Med. Chem. 15 (2007) 5787–5810
Scheme 3. Reagents and condition: (a) 40% (CF3CO)2O in
CF3COOH, 40 ꢁC, 48 h.
Partial trifluoroacetylation of 4 was performed using tri-
fluoroacetic anhydride in trifluoroacetic acid solution
using a short reaction time. All three expected prod-
ucts—two monosubstituted (7 and 8; Scheme 3) and
one disubstituted (6, Scheme 2)—are formed. Reaction
at the cis-amino group proceeded slightly easier, result-
ing in a mixture of trifluoroacetate salts of products 8
and 7 in the proportion 3:2. Further, the trifluoroacetate
ion was exchanged with tosylate and several recrystalli-
zations from methanol–acetonitrile allowed us to isolate
the main isomer—N-[cis-4-(trans-4-amino-cyclohexyl-
methyl)-cyclohexyl]trifluoroacetamide (8) in pure form.
Scheme 2. Reagents and condition: (a) 50% (CF3CO)2O in
CF3COOH, 40 ꢁC, 7 days.
showed a doublet from the NHCO proton at 9.25 ppm,
J = 7.6 or 7.9 Hz, and a multiplet from the neighbouring
CH proton at 3.54 ppm. These almost identical signals
could only be related to the trifluoroacetylated trans
amino group present in both compounds. On the other
hand, only the 1H NMR spectrum of cis,trans-bis-(4-tri-
fluoroacetylamino-cyclohexyl)-methane (6, Fig. 2B) dis-
played a doublet from the NHCO proton at 9.09 ppm,
J = 7.3 Hz, and a multiplet from the neighbouring CH
proton at 3.72 ppm. These signals could be related only
to the trifluoroacetylated cis-amino group, uniquely
present in 6. The same signals could be observed also
in the NMR spectra of monoacylated products
(Fig. 2C and D), which allowed us to identify these sub-
stances, also in their mixtures.
To fractionate 4,40-methylene-bis-(2-methylcyclohexyl-
amine), the commercial substance was kept at +5 ꢁC
causing crystallization. A small part of product re-
mained liquid. This was used for synthesis of a tripartide
(20b–40–8).
2.2.2. Solid phase synthesis. The main process for prepa-
ration of compounds on solid phase is illustrated in
Scheme 4 for the preparation of compounds 4–40–8
and 4–40–8a.
The process started with activated carboxylated poly-
mer—4-nitrophenyl carbonate Wang resin (9) or its imi-
dazolecarbonyl analogue. It was reacted with excess
diamine in DMF yielding diamine, which was partially
protected with the polymer attached substituted benzyl-
oxycarbonyl group (10). In some cases we used commer-
cially available diamines attached to trityl resin
(Novabiochem).
The polymer attached partially protected diamine (10)
was further introduced into reaction48 with aldehydes
or ketones dissolved in trimethyl orthoformate in order
to obtain Schiff base (11). Aldehydes reacted better,
whereas the reactions with ketones were generally far
from complete. When diamines had been attached to
the trityl resin, ketones did not react at all. In order to
get better yields with ketones the reactions were carried
out for several days at 30 ꢁC in the presence of acetic
acid (10%). Attempts to form Schiff bases from polymer
attached derivatives of cyclohexylamine in reaction with
ketones essentially failed, obviously due to steric hin-
drance. Use of trityl polymer and branched diamines
also gave lower yields if sterically hindered structures
were to be synthesized.
Figure 2. NMR spectra of N-trifluoroacetylated 4, 40-diam-
inodicyclohexylmethane in DMSO-d6. (A) trans,trans-Bis-(4-
trifluoroacetylamino-cyclohexyl)-methane (5). (B) cis,trans-Bis-(4-tri-
fluoroacetylamino-cyclohexyl)-methane (6). (C) Mixture of trifluoroac-
etate salts of N-[trans-4-(cis-4-amino-cyclohexylmethyl)-cyclohexyl]
trifluoroacetamide (7) and N-[cis-4-(trans-4-amino-cyclohexylmethyl)-
cyclohexyl] trifluoroacetamide (8). (D) N-[cis-4-(trans-4-Amino-cyclo-
hexylmethyl)-cyclohexyl] trifluoroacetamide (8) 4-toluenesulfonate salt.
Schiff bases formed were then further reduced with so-
dium cyanoborohydride in 4% acetic acid/trimethyl

F. Mutulis et al. / Bioorg. Med. Chem. 15 (2007) 5787–5810
5793
Scheme 4. Reagents and conditions: (a) tetramethylenediamine, DMF, rt, 24 h; (b) benzaldehyde, trimethyl orthoformate, rt, 20 h; (c) NaCNBH3,
4% AcOH in trimethyl orthoformate, rt, 5 min; (d) diisopropylethylamine, CH2Cl2, rt, 10 min; (e) 3-indolebutyric acid, PyBroP, diisopropyleth-
ylamine, CH2Cl2, rt, 20 h; (f) trifluoroacetic acid–1,2-ethanedithiol–triisopropylsilane–water (10:2:0.25:0.25), rt, 1 h.
orthoformate,49 which gave polymer attached secondary
amines (12). We found that yields increased dramatically
if we added the solvent–acetic acid mixture rapidly to a
dry resin that had been carefully mixed with solid
sodium cyanoborohydride using a spatula. Obviously
the intensive and rapid reduction avoided the accumula-
tion of intermediates that otherwise resulted in double
alkylations. Slow reactions gave mainly side products.50
molecular masses were increased by 106 Da. As shown
in our detailed NMR investigation,53 these substances
could be identified as 4-hydroxybenzyl derivatives, where
an aralkyl group formed by fragmentation of the linker is
introduced at position 2 of the indole residue (like in 4–
40–8a, Scheme 4). Yields of these side products reached
up to 25% of the yield of the main product, even when
the cleavage from the polymer was performed in presence
of 1,2-ethanedithiol. Cleaving without this scavanger
gave alkylated derivatives as the main products.
4-Hydroxybenzyl derivatives were investigated as poten-
tial melanocortin receptor ligands as well.
Acetate ions, which could have caused acetylation at the
next step, were removed from 12 by treatment of resin
with diisopropylethylamine. The polymeric secondary
amine (12) was then introduced into reaction with car-
boxylic acid in methylene chloride (a small amount of
DMF was added if necessary, in order to dissolve the
carboxylic acid) in the presence of PyBroP51 and diiso-
propylethylamine forming resin bound tertiary amide
(13). The acylation usually proceeded well. Even the ste-
rically hindered indomethacin could be introduced as
the carboxylic component using this procedure.
After cleavage raw end-products were isolated by filtra-
tion, partial evaporation of filtrates and precipitation
with a mixture of dry ether and hexane. Along with
abovementioned 4-hydroxybenzyl derivatives other side
products were often present. Examples of such side
products are shown in Figure 3. Thus, 14 was the result
of an incomplete Schiff base formation during the syn-
thesis of 4–26–9; 6–37–19 formed by cleavage of benzyl
ether in the carboxylic acid part during the final depro-
tection step in the attempt to synthesize 6–37–18; 6–41–
19 formed during the synthesis of the cinnamaldehyde
derivative 6–37–18 due to double bond reduction. For-
tunately, a-halogenated cinnamaldehyde derivatives
were stable to double bond reduction in our process
and were preferred for the purpose of the present inves-
tigations. One more side product, namely an unidenti-
fied dimer, turned out to be the main outcome in the
preparation of tertiary amides derived from trans-3-
indoleacrylic acid.
Next, 13 was treated with trifluoroacetic acid in combi-
nation with 1,2-ethanedithiol (20%),52 triisopropylsilane
and water, which gave tertiary amides like 4–40–8.
Cleavage of the tertiary amides coupled to trityl resin
was accomplished by using a mixture of TFA–1,2-ethan-
edithiol–methylene chloride (1:1:20).
Preliminary identification of substances present in the
raw products was made by LC/MS. On preparing indole
derivatives on Wang resin (a typical situation in this
study), we observed formation of side products whose

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F. Mutulis et al. / Bioorg. Med. Chem. 15 (2007) 5787–5810
Figure 3. Side products of tertiary amide synthesis corresponding to the following processes: 14, incomplete Schiff base formation; 6–37–19,
fragmentation at trifluoroacetic acid treatment, 3–15–1, reduction of C@C bond.
When amino acids were brought in as carboxylic com-
ponents, they were introduced as Fmoc-derivatives
(preparation of 18–37–17 is shown on Scheme 5, as an
example). Solid phase synthesis then included an addi-
tional step, namely elimination of the Fmoc group from
polymeric intermediate 14 using piperidine solution in
DMF, forming intermediate 15.
amino group 20. The following synthesis was performed
in the usual way yielding tertiary amides 10e–18–8 and
10e–18–8a.
2.2.3. Solution synthesis. Some of the tertiary amides
were subjected to guanidation in DMF using 1H-pyra-
zole 1-carboxamidine,55 which gave guanidinoderiva-
tives (like 12–0–8, Scheme 8). The reaction was carried
out in a concentrated DMF solution. This allowed us
to apply the reaction mixture (after dilution with water)
directly to the preparative reversed phase HPLC.
A particular case constituted the preparation of tertiary
amides containing two diamine residues connected
through a carbonyl group (Scheme 6 shows synthesis
of one of them, 27–40–8, as a typical example). First,
activated carboxylated polymer reacted with one of the
diamines. The polymeric product 16 formed was then
introduced into reaction with 4-nitrophenylchlorofor-
mate yielding activated intermediate 17. The latter re-
acted with the second diamine giving the polymeric
urea derivative 18, which was then introduced into
reductive amination, acylation and cleavage steps in
the usual way.
Tertiary amide 1–39–8 was prepared in solution
(Scheme 9). The Schiff base from 4-diethylaminobenzal-
dehyde (21) and pentylamine was formed first. Subse-
quent
reduction
with
NaCNBH3
gave
the
corresponding secondary amine (22). Acylation with 3-
indolebutyric acid and purification by HPLC finished
the synthesis.
Solution synthesis proved to be very useful when ke-
tones needed to be introduced into the preparation pro-
cess of the tertiary amides. Thus, to prepare 3–38–5
(Scheme 10), b-tetralone (23) (dissolved in CH2Cl2)
was reacted with 1,3-propylenediamine in the presence
of TiCl4.56 Without isolating the Schiff base formed,
NaCNBH3 was added, and the diamine (24) formed
was separated by extractions. The CH2Cl2 solution
Another specific case was the introduction of a
cis,trans-4,40-diaminodicyclohexylmethane residue in a
regioselective way (Scheme 7). N-[cis-4-(trans-4-amino-
cyclohexylmethyl)-cyclohexyl] trifluoroacetamide (8)
was reacted with 4-nitrophenylcarbonate Wang resin
(9). Polymeric diamine derivative 19 formed was depro-
tected using NaBH4,54 giving a product carrying a free
Scheme 5. Reagents and conditions: (a) 20% piperidine in DMF, rt, 10 min; (b) trifluoroacetic acid–1,2-ethanedithiol–triisopropylsilane–water
(10:2:0.25:0.25), rt, 1 h.

F. Mutulis et al. / Bioorg. Med. Chem. 15 (2007) 5787–5810
5795
Scheme 6. Reagents and conditions: (a) 1,3-diaminocyclohexane, DMF, rt, 24 h; (b) 4-nitrophenylchloroformate, DIEA, DMF, rt, 24 h.
Scheme 7. Reagents and conditions: (a) DIEA, DMF, rt, 60 h; (b) NaBH4, n-BuOH, rt, 49 h; 50 ꢁC, 3 h.
containing 24 was treated with di-tert-butyl dicarbonate,
giving a solution containing the selectively protected
diamine 25. This compound was purified by silica gel
chromatography and precipitated as hydrochloride.
Further, acylation with 3-indolepropionic acid in the
presence of PyBroP was carried out. The product,

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F. Mutulis et al. / Bioorg. Med. Chem. 15 (2007) 5787–5810
Scheme 8. Reagents and condition: (a) 1H-pyrazole-1-carboxamidine hydrochloride, DIEA, DMF, rt, 24 h.
Scheme 9. Reagents and conditions: (a) pentylamine, trimethyl orthoformate, rt, 3 days; (b) NaCNBH3, 0–5 ꢁC, 2 h; (c) 3-indolebutyric acid,
PyBroP, diisopropylethylamine, CH2Cl2, rt, 1 h.
Scheme 10. Reagents and conditions: (a) 1,3-propylenediamine, TiCl4, CH2Cl2, Ar atmosphere, 0–20 ꢁC, 20 h; (b) NaCNBH3, MeOH, 0–20 ꢁC,
45 min; (c) NaOH, water; (d) di-tert-butyl dicarbonate, CH2Cl2, rt, 24 h; (e) 4M HCl/dioxane, ether; (f) 3-indolepropionic acid, PyBroP, DIEA,
CH2Cl2, rt, 24 h; (g) CF3COOHACH2Cl2 (1:8), rt, 15 min.
Boc-protected tertiary amide 26, was cleaved with
CF3COOHACH2Cl2 yielding the desired 3–38–5.
bond took also place, resulting in diamine 28. This com-
pound was further coupled with activated carboxylated
Wang polymer. The reaction proceeded at the primary
amino group forming intermediate 29. Finally, acylation
with 2-naphthoxyacetic acid and deprotection led to ter-
tiary amide 2–42–14. Similarly, using spiro[5.5]undec-
1,8-diene-3-one57 as a ketone, end-product 5–43–14
was obtained. Remarkably, double bond in position 8
(not conjugated with carbonyl group) was stable to
NaCNBH3 procedure.
A combination of solution and solid phase synthesis was
applied to prepare 2–42–14,
a
derivative of
spiro[5.5]undecane (Scheme 11). Like in the previous
example, spiro[5.5]undec-1-en-3-one57 (27) was intro-
duced into reaction with ethylenediamine in the presence
of TiCl4. During the following reduction of the Schiff
base formed, hydrogenation of carbonAcarbon double

F. Mutulis et al. / Bioorg. Med. Chem. 15 (2007) 5787–5810
5797
Scheme 11. Reagents and conditions: (a) ethylenediamine, TiCl4, CH2Cl2, Ar atmosphere, 0–20 ꢁC, 20 h; (b) NaCNBH3, MeOH, 0–20 ꢁC, 45 min;
(c) 4-nitrophenyl carbonate Wang resin, rt, 24 h.
2.2.4. HPLC procedures. All end-products were first
purified by semipreparative HPLC on reversed phase
in acetonitrile–water–trifluoroacetic acid (0.1%). Eluted
fractions containing individual substances or their iso-
mer mixtures were freeze-dried. In some cases, further
separations were performed on chiral columns. (We
tried both Chirobiotic T and Chirobiotic V and found
that the first one separated tertiary amides slightly bet-
ter.) An example for this is shown in Figure 4, for the
separation of isomers of the 4,40-methylene-bis-(2-meth-
ylcyclohexylamine) derivative 20b–40–8. Thus, separa-
tion of isomers could be achieved already at the
preparative reversed phase HPLC step by dividing the
eluate corresponding to main product peak into several
parts (Fig. 4A). The separation was verified by the Chi-
robiotic T column chromatography (Fig. 4B). Isomer-
ism of substances was confirmed from their identical
UV spectra of the chiral chromatography peaks [UV
spectral data could be collected directly online using
the HPLC photodiode array detector (Fig. 4C)], and
from the LC/MS data showing the same molecular ions
and fragmentation patterns. In selected cases Chirobiot-
ic V preparative separations of the tertiary amides were
performed in order to improve the isomeric homogene-
Figure 4. Chromatography of 20b–40–8. (A) Preparative HPLC run of
˚
ity of the substances before investigating them for their
activity on melanocortin receptors. The exact structures
of tertiary amides obtained by the use of commercial
mixtures of diamines or/and unsymmetrical diamines
followed by separation by chromatography were not
established.
a raw product (25 mg). Vydac RP C18 column (10 · 250 mm, 90 A,
201HS1010), eluent–30% MeCN in water + 0.1% TFA, flow rate 5 mL/
min, detection at 280 nM. Eluate fractions I–III were lyophilized to
give 1.8 mg 20b–40–8(1), 5.1 mg 20b–40–8(2) and 2.2 mg 20b–40–8(3)
correspondingly. (B) Analytical chiral HPLC of 20b–40–8(1) (I), 20b–
40–8(2) (II) and 20b–40–8(3) (III) on
a Chirobiotic T column
(4.6 · 250 mm). Eluent–MeOH–AcOH–triethylamine (100:0.1:0.1),
flow rate 1 mL/min, detection at 220 nm. Analysis time 20 min. (C)
UV spectra of 20b–40–8(2) components (obtained at chiral HPLC
using a photodiode array detector).
Overall yield of purified tertiary amides was not high.
However, quantitative determination of tertiary amides
in the raw products using HPLC showed that the actual
yields of them were about 1.5–2 times higher. Obviously,
substance losses occurred due to irreversible adsorption
on the reversed phase during semipreparative HPLC.
Nevertheless, the amounts of compounds obtained were
sufficient for their pharmacological characterizations.
2.3. Pharmacology
The 210 tertiary amide samples obtained were tested on
recombinant human MC receptors in radioligand

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F. Mutulis et al. / Bioorg. Med. Chem. 15 (2007) 5787–5810
Figure 5. Competition binding curves for 2–30–9 (negative logarithm of ligand concentration versus percent of receptor bound [125I]-NDP–MSH).
(A) MC1R, Ki 2575 nM. (B) MC3R, Ki 12019 nM. (C) MC4R, Ki 3443 nM. (D) MC5R, Ki 5649 nM.
binding assays and most of them bound to all the MC1,
MC3, MC4 and MC5 receptors evaluated in these tests
(Fig. 5 and Table 1). For the most active compounds
(structures shown in Fig. 6) the Kis reached 0.41 lM
for 10b–36–14(2,1) on the MC1 receptor, 1.7 lM for
2–42–20 on the MC3 receptor, 1.2 lM for 20a–40–8
(1) on the MC4 receptor, and 0.93 lM for 3–37–0 on
the MC5 receptor. The compounds also showed varying
selectivity patterns (Table 1). A few were uniquely selec-
tive for one of the receptor subtypes, namely the MC1R.
E.g., the binding affinities of 9–36–8 (Fig. 6), 2–38–13,
3–38–1, 8–35–5, 10d–40–8 and 15–40–8 for the MC1R
exceeded those for the MC3–5Rs by 10-fold or more.
The most MC1R selective was 9–36–8 (Fig. 6). It showed
45-fold selectivity or better. The most non-selective was
5–40–17, whose affinity was practically the same for all
the evaluated MC receptor subtypes.
expected increase in cAMP for all the MC receptor
assays.
In order to probe the possibility that the compounds are
antagonists we investigated their ability to block the in-
crease in the accumulation of cAMP caused by a-MSH.
Results are shown graphically on Figure 8. Thus, at a
concentration of 100 lM all the seven compounds
caused clear inhibition of the increase in the cAMP lev-
els caused by a-MSH; the latter being applied in concen-
trations up to 1 lM. The data are thus compatible with
the notion that the tertiary amides are antagonists at
melanocortin receptors.
3. Discussion
Our method for parallel synthesis of individual tertiary
amides on solid phase made it convenient to prepare a
large series of compounds. The raw product mixtures
obtained could be well separated on reversed phase
using acetonitrile–water–TFA as an eluent. The process
allowed us to isolate title substances as well as side prod-
ucts. Freeze drying of eluates resulted mainly in amor-
phous powders that were convenient to handle. We
found that the chiral phases Chirobiotic T and Chirobiotic
V, with a standard methanol–acetic acid–triethylamine
(100:0.1:0.1) eluent, were efficient to separate tertiary
amide isomers. The chiral selector system was useful
for preparative separations, as well.
In order to determine if tertiary amides are agonists or
antagonists on melanocortin receptors some of the title
compounds (structures shown in Fig. 7) were tested
for their ability to promote, respectively, block the accu-
mulation of cAMP in cells expressing different MC
receptor subtypes. The evaluated compounds had per-
se only negligible effects on cAMP in cells expressing
either of the MC1, MC3, MC4 and MC5-receptor sub-
types. [However, one of them (12–0–5) marginally stim-
ulated cAMP in MC1 and MC4 receptor expressing cells
at the highest concentration (100 lM) evaluated]. Con-
trol experiments where a-MSH was added showed the

F. Mutulis et al. / Bioorg. Med. Chem. 15 (2007) 5787–5810
5799
Figure 6. Selected tertiary amides’ structures. Affinity (in lM, for MC1R, MC3R, MC4R and MC5R correspondingly): 2–42–20 (3.7, 1.7, 2.4, 3.2), 3–
35–5 (21, 65, 81, 11), 3–37–0 (0.85, 24, 7.4, 0.93), 4–27–0 (106, 112, 222, 279), 5–15–8 (18, 61, not determined, 19), 5–26–8 (6.1, 25, 14, 46), 1–39–8 (72,
289, 78, 408), 5–39–8 (6.8, 33, 25, 34), 5–40–6 (not binding to all receptors), 9–36–8 (2.5, 131, 113, 391), 10b–1–8(2) (3.2, 22, 3.7, 8.3), 10b–12–8(2)
(1.6, 31, 4.1, 5.0), 10b–36–14 (1.5, 23, 3.7, 5.5), 16–35–8 (2.6, 44, 7.3, 9.7), 20a–40–8(1) (0.69, 4.3, 1.2, 1.7), 21–40–8 (6.4, 52, 16, 22).
From Table 1 it can be seen that despite considerable
structural differences most of the tertiary amides show
quite similar binding affinity for the MSH peptide bind-
ing MC receptors. Most of the compounds show a selec-
tivity pattern: MC1R > MC4R, MC5R > MC3R.
Looking at the structure–activity relationships we may
see that the diamine part can be varied widely and still
retaining MC receptor binding affinity. We thus intro-
duced all oligomethylenediamines having from two to
eight methylene groups, as well as diamines with

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F. Mutulis et al. / Bioorg. Med. Chem. 15 (2007) 5787–5810
Figure 7. Structures of tertiary amides tested for functional activity.
affinities. We also prepared some substances where
diamines containing a benzene ring were incorporated
[i.e., substances 15–0–8, 15–40–8, 16–0–8 and 16–35–8
(Fig. 6)]. All of these compounds exhibited relatively
good binding to the MC receptors. Only slightly reduced
affinities were found for the triple charged derivatives of
piperazine 21–36–8 and 21–40–8 (Fig. 6) obtained from
1,4-bis(3-aminopropyl)piperazine. Thus, even tertiary
amides with two additional positive charges are toler-
ated by the MC receptors.
In order to determine the optimal number of methylene
groups in oligomethylenediamine derivatives, we investi-
gated series of compounds of the X–40–8 type (Fig. 9),
where the number of methylene groups was varied be-
tween two and seven. For benzaldehyde derivatives of
the X–40–8 type we found that, at least for the MC1
and MC4Rs a shorter chain of between two and four
methylene groups was unfavourable. Moreover, in the
range of 4–7 methylene groups an alternating pattern
of affinities was observed as the compounds with odd
numbers of methylene groups (5 and 7) were more active
than compounds with even numbers (4 and 6). (This
alternating pattern was documented in repeated parallel
binding experiments, where the substances were applied
on the same assay plates). The finding might be ex-
plained in terms of a stretched conformation of the
Figure 8. Effect of a-MSH on cAMP level in natural melanoma cells
B16 (MC1R) and COS-1 cells transfected with MC3R, MC4R, MC5R
DNA in absence and in presence of tertiary amides (100 lM). White
bars, experiments performed at a-MSH concentration 0.1 nM. Light
grey bars, experiments performed at a-MSH concentration 10 nM.
Grey bars, experiments performed at a-MSH concentration 1 lM. 1,
no tertiary amide; 2, 2–0–1; 3, 3–0–1; 4, 4–0–1; 5, 5–0–8; 6, 5–35–5; 7,
12–0–5; 8, 13–0–5; C, cell control. Standard error of mean (SEM) is
shown in each bar.
branched chains, like the ones in compounds 9–36–8
(Fig. 6), 18–36–8 and 19–40–8. Besides, many deriva-
tives of 4,40-methylenebis(cyclohexylamine), like 10b–
36–14 (Fig. 6), and derivatives of 4,40-methylenebis(2-
methylcyclohexylamine), like 20b–40–8 (Fig. 4), were
prepared. Substances of this type showed relatively good
Figure 9. Affinity of compounds of type X–40–8 on MC1R and MC4R
as a function of the number of methylene groups.

F. Mutulis et al. / Bioorg. Med. Chem. 15 (2007) 5787–5810
5801
methylene groups in the diamine part, were slightly (2-
to 3-fold) better binders on the MC1 and MC4Rs than
the corresponding benzaldehyde derivatives 2–40–8, 3–
40–8 and 4–40–8 (Figs. 9 and 10). Apparently a larger
distance between the presumed pharmacophoric amino
and phenyl groups (i.e., introduction of chloroethylene
moiety) is preferred by these receptors.
These data supported the assumption that the benzene
ring in the aldehyde part and the amino group from
the diamine part are interacting with the receptors. In
order to investigate this further we prepared two deriv-
atives of 4-diethylaminobenzaldehyde: 5–39–8 and 1–
39–8 (Fig. 6). These compounds differ only by the pres-
ence or absence of the primary amino group. Indeed, 1–
39–8 showed a considerably reduced affinity for all the
MC receptors, compared to 5–39–8. Moreover, almost
all other tertiary amides obtained in the present study
showed higher activities than 1–39–8 (Table 1). Thus,
the positively charged amino or guanidino group is a
crucial function for the compounds’ binding to MC
receptors.
Figure 10. Geometry model explaining alternating affinity of com-
pounds of type X–40–8 on MC1R. (A,C) Reduced complexation of
positively charged ammonium group of tertiary amide with negatively
charged receptor group for compounds whose diamine part contains
four or six methylene groups (methylene chain turns ammonium group
away from the receptor). (B,D) Improved complexation of compounds
whose diamine part contains five or seven methylene groups. It is
assumed that the remaining identical part of substances (R) is kept in
position by other interactions with the receptor. Probably, methylene
groups from diamine parts are also involved in such interactions.
Guanidation of the amino group in tertiary amides did
not generally improve the affinities. Thus, comparison
of the affinity data for compounds 2–0–1, 3–0–1, 4–0–
1 (structures shown in Fig. 7), 3–0–8, 10d–35–8, 10b–
36–8 and 10b–40–8, with the corresponding data for
their guanidated analogues 11–0–1, 12–0–1, 13–0–1,
12–0–8 (Scheme 8), 14b–35–8, 14a–36–8 and 14a–40–8
(Table 1), revealed that guanidine derivatives exhibited
better affinity in 10 cases, while in 17 cases they dis-
played lower affinity. This was quite unexpected if we as-
sume that amino group of the tertiary amides mimics the
guanidino group of arginine from the active core of the
natural peptides.
The most common aldehyde components in our study
were benzaldehyde, b-naphthaldehyde or a-chlorocin-
namaldehyde. Changing the number of methylene
groups between the benzene ring in the aldehyde part
and the central amide nitrogen atom, as in compounds
3–40–8 (one methylene group), 3–11–8 (two methylene
groups) and 5–15–8 (Fig. 6; three methylene groups),
did not lead to any substantial change in binding affini-
ties (Table 1). However, as pointed out above, introduc-
tion of a double bond in the aldehyde part improved the
affinity slightly. For the series of substituted benzaldehyde
derivatives 10b–1–8 (Fig. 6; 3,4-dimethoxy), 10b–2–8
(3-ethoxy-4-methoxy), 10b–3–8 (2-fluoro), 10b–4–8
(4-carbmethoxy), 10b–5–8 (4-methylthio), 10b–6–8 (3-
methyl), 10b–7–8 (2-methyl), 10b–8–8 (4-methyl) and
10b–10–8 (2-bromo) no substantial differences in activity
were seen.
Figure 11. Affinity of compounds of type X–36–8, with varied number
of methylene groups, on MC1R and MC4R.
methylene chain, which determines position of the ami-
no group so that it is more favourable for binding to the
MC1R for compounds 5–40–8 and 7–40–8 (Fig. 10).
However, binding of these substances to the MC4R
did not reveal the same alternating pattern. Instead, a
higher activity was shown for substances containing be-
tween five and seven methylene groups in the diamine
residue, while the ones including 2–4 groups showed
lower activity (Fig. 9).
A similar study was made for derivatives of a-chlorocin-
namaldehyde of the X–36–8 type (Fig. 11). For this ser-
ies the optimal number of methylene groups turned out
to be five for both the MC1R and MC4R. Increasing the
chain length further to six and seven methylene groups
was unfavourable, which contrasted to the findings for
the benzaldehyde derivatives (Fig. 9). The results can
be explained on the basis that a too long distance be-
tween benzene ring and the amino group in 6–36–8
and 7–36–8 might not fit the receptor geometry well.
On the other hand, a-chlorocinnamaldehyde derivatives
2–36–8, 3–36–8 and 4–36–8, which contain two to four
Differently substituted cinnamaldehydes were also
incorporated into tertiary amides as follows: 2-methoxy-
cinnamaldehyde (10b–12–8, Figure 6), 2-methoxy-4-
hydroxycinnamaldehyde (10b–16–8), a-methylcinnamal-
dehyde (3–37–0, and others), a-chlorocinnamaldehyde
(2–36–8, and many others), a-bromocinnamaldehyde
(10b–28–8) and 3-(2-naphthyl)-2-propenal (5–34–8).
However, all the resulting tertiary amides exhibited

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F. Mutulis et al. / Bioorg. Med. Chem. 15 (2007) 5787–5810
similar affinities. Heteroaromatic derivatives obtained
from 2-pyridinaldehyde and 4-pyridinaldehyde 10b–
13–8 and 10b–19–8 and 5-methyl-2-thiophenecarboxal-
dehyde 2–30–9 (Fig. 5) also showed about the same
affinity (Table 1).
which contains a D-3-pyridinalanine residue, thus dem-
onstrated a total absence of activity. In other words,
introduction of a pyridine ring in the carboxylic acid
part completely destroyed the tertiary amide binding.
When other amino acids were used as the carboxylic
component the resulting compounds did not exhibit
any particular anomalies. E.g., naphthalene derivatives,
like 5–40–2, bound well to the MC receptors. The re-
duced affinity of derivatives of D- and L-tryptophan
when introduced as the carboxylic part in tertiary
amides (i.e., derivatives 5–40–10, 5–40–17 and 18–37–
17, Scheme 5) was surprising also as this part could be
considered as an imitation of the tryptophan in the nat-
ural melanocortin peptides. The highest activity from
this ‘amino acid’ series was shown by the D-biphenylala-
nine derivative 5–40–7.
Ketone derivatives like 2–29–1 (product of 2-acetonaph-
thone), 3–9–8 (product of 2-acetylbenzothiophene), 5–
26–8 (Fig. 6; product of 2-adamantanone), 3–38–5
(Scheme 10, product of b-tetralone), 2–17–8 (product
of 2-methyl-3-acetyl-5-phenyl-thiophene), 5–31–1 (prod-
uct of 4-trifluoromethyl-acetophenone) and 5–32–1
(product of cycloheptanone), which have reduced con-
formational freedom compared to aldehyde products,
did not show any significant difference in binding affinity
compared to aldehyde derivatives.
Derivatives of cyclic ketones, which contain bulky alicy-
clic residues instead of a benzene ring like in 5–32–1
(product of cycloheptanone) and 5–26–8 (Fig. 6,
product of 2-adamantanone), displayed quite good
activity in our tests with a preference for the MC1R.
Among the most active compounds, in particular for
MC3–5Rs, found were products of spiro[5.5]undec-1-
en-3-one: 2–42–8, 2–42–14 (Scheme 11) and 2–42–20
(Fig. 6), as well as spiro[5.5]undec-1,8-dien-3-one: 5–
43–14. The presence of an aromatic ring in the aldehyde
or ketone part is thus not obligatory for the MC recep-
tor binding as aliphatic groups interact with the receptor
as well. However, it turned out that the acetone deriva-
tive 4–27–0 (Fig. 6) was considerably less active com-
pared to most of the other investigated substances.
Probably, this compact compound is not able to fill
the receptor pocket efficiently and might therefore not
interact well with receptor.
Another observation was that affinities seem to increase
with increasing size and hydrophobicity of the tertiary
amide molecule. Thus, e.g., good MC receptor binding
was found for the bulky adamantane derivative 5–26–8
(Fig. 6), as well as for the even more bulky and hydro-
phobic adamantane/indomethacine derivative 4–26–9.
On the other hand, e.g., the small molecule derivative
of acetone and indole-3-carboxylic acid, 4–27–0
(Fig. 6), showed a markedly reduced binding for all
the four MC receptor subtypes evaluated herein.
Comparison of binding affinities of solid phase synthesis
side products, 4-hydroxybenzyl derivatives, with those
of the corresponding main products (Table 1) gave re-
sults as follows: In 33 cases the affinity of the main prod-
ucts was better, but in 34 cases the affinity was better for
the side product. For eight cases main and side products
showed essentially the same affinities. However, the dif-
ferences between the affinity values were quite small.
Thus, in general the introduction of the 4-hydroxybenzyl
group did not influence much the binding affinity of
the tertiary amides; thus this group does not interact
with the receptors and it also does not hinder the
binding.
In order to imitate the tryptophan residue present in the
natural MSH peptides’ active core, we introduced, in
most cases, indole derivatives in the tertiary amide tripar-
tides’ carboxylic acid part. For homologous 3-indole car-
boxylic acid derivatives changing the distance between
the indole moiety and the central nitrogen atom [cf sub-
stances 2–0–0 and 4–0–0 (no methylene groups in car-
boxylic part), 2–0–1, 3–0–1, 4–0–1 (structures shown in
Fig. 7), 5–0–1 (one methylene group in carboxylic acid
part), 5–0–5 (two methylene groups in carboxylic acid
part), 5–0–8 (three methylene groups in carboxylic acid
part)] leads to compounds with similar affinities. Excep-
tions were compounds 2–0–1, 3–0–1 and 5–0–5, which
showed a lower affinity on the MC1R. Similar affinities
were also found for the series 3–38–0, 3–38–1, 3–38–15
(Scheme 10) (b-tetralone derivatives) and 3–35–0, 3–
35–1, 3–35–5 (Fig. 6) (cinnamaldehyde derivatives).
Other indole derivatives investigated included 3-indolyl-
glyoxalic acid, 3-indolylacrylic acid, 5-benzyloxyindol-2-
carbonic acid, D- and L-tryptophan and indomethacine
residues. Besides indole derivatives, other carboxylic
acids such as 2-naphthoxyacetic acid, 4-(2-thienyl)butyric
acid and L-histidine could be used to prepare MC recep-
tor active compounds (Table 1).
In the present study, we evaluated seven tertiary amides,
12–0–5, 13–0–5, 2–0–1, 3–0–1, 4–0–1, 5–0–8 and 5–35–5
(structures shown in Fig. 7), for their agonist/antagonist
properties by assaying cAMP in MC1R, MC3R, MC4R
and MC5R expressing cells. As all of these compounds
were capable of inhibiting the increase in the levels of
cAMP induced by a-MSH (Fig. 8), it appears that our
tertiary amides are antagonists at melanocortin respon-
sive MC receptors [However, one of the compounds, 12–
0–5, induced a marginal activation of cAMP release at
the highest concentration (100 lM) applied.].
4. Conclusions
The tertiary amides reported herein were conveniently
prepared, allowing us to obtain a diverse library of pep-
tidomimetics. Remarkable enough almost all of the 210
tertiary amides samples obtained showed moderate
binding affinity to all of the MSH binding MC receptors
(MC1, MC3–5). Seven title compounds were tested for
Surprising data were obtained when we introduced
unnatural amino acids. Compound 5–40–6 (Fig. 6),

F. Mutulis et al. / Bioorg. Med. Chem. 15 (2007) 5787–5810
5803
their agonistic/antagonistic properties and the data
showed that they were antagonists on all the four
MSH binding MC receptors. The structure–affinity rela-
tionships derived from the data indicate that hydropho-
bic structures are advantageous. We found also that
bigger molecules are better binders than smaller ones.
These data are in agreement with our earlier modelling
results,8 which indicated that hydrophobic interactions
dominate the binding of ligands to melanocortin recep-
tors. From our data, the participation of the amino or
guanidino group in complexation of the peptidomimet-
ics with the receptor is also evident. Some optimization
of structures leading to increased affinity could be
obtained by systematically changing the number of
methylene groups in the compounds’ diamine part.
The data obtained for the present series of compounds
may find use in the further development of melanocortin
receptor subtype active compounds.
Solid phase synthesis was performed on a Nautilus
2400 synthesiser (Argonaut Technologies). Exact molec-
ular masses were determined on a Micromass Q-Tof2
mass spectrometer equipped with an electrospray ion
source. LC/MS was performed on a Perkin-Elmer PE
SCIEX API 150EX instrument with a Turboionspray
Ion Source (m/z obtained are labelled below as TISMS
data) and Dr. Maisch Reprosil-Pur C18-AQ, 5 l,
150 · 3 mm HPLC column using a gradient formed
from water and acetonitrile with 5 mM ammonium ace-
1
tate additive. H NMR spectra were recorded on a Jeol
JNM-EX270 spectrometer or a Varian Mercury-400
spectrometer, using DMSO-d6 (2.50 ppm downfield
from TMS) as an internal standard. Analytical and mi-
cro-preparative HPLC was performed on a Waters sys-
tem equipped with Millenium-32 Workstation, 2690
Separation Module, 996 Photodiode Array Detector
and Waters Fraction Collector. Semi-preparative HPLC
was carried out on a LKB system consisting of a 2150
HPLC Pump, 2152 LC Controller and 2151 Variable
Wavelength Monitor and Vydac RP C18 column
From our experience, it remains unclear whether really
high affinity and selectivity compounds are attainable
relying solely on the concept of the correct placement
of three pharmacophoric groups from melanocortins’
active core. However, taking into account the moderate
affinity of large number high diversity compounds,
which we obtained, it seems rather that the answer is
negative. In other words, more attachment points be-
tween ligand and receptor are necessary. Essential ele-
ments of binding can be, e.g., neighbouring histidine
residue and peptide bonds, present in the active core.
Such idea is supported by the high affinity reported for
slightly modified active core tetrapeptide sequences.58
˚
(10 · 250 mm, 90 A, 201HS1010), the eluent being an
appropriate concentration of MeCN in water + 0.1%
TFA, flow rate 5 mL/min, detection at 280 nM.
Freeze-drying was performed at 0.1 bar on a Lyovac
GT2 Freeze-Dryer (Finn-Aqua) equipped with a Busch
010-112 vacuum pump and a liquid nitrogen trap.
Tertiary amides were prepared according to type proce-
dures A–L; see also Table 1.
5.1.1. 3-(2-Naphthyl)-2-propenal (2). To 2-naphth-
aldehyde (3.12 g, 19 mmol) in a flask cooled in a salt-
ice bath acetaldehyde (24 mL) was added with stirring.
A clear solution formed, to which potassium hydroxide
(0.26 g, 4.6 mmol) dissolved in methanol (1.3 mL) was
added dropwise, keeping the temperature of the reaction
mixture at 0–5 ꢁC. Stirring was continued for 2 h. At the
end of the period the temperature was allowed to rise
until it reached room temperature. Acetic anhydride
(10 mL) was then added and the mixture was heated
to 100 ꢁC for 30 min. The reaction mixture was then
poured into a flask containing water (72 mL) and con-
centrated hydrochloric acid (9.6 mL) was added, heated
to 100 ꢁC and stirred for 20 min. The reaction mixture
was then cooled to 0 ꢁC and the precipitate formed
was filtered off, washed with water, dried under vacuum
in the presence of P2O5 and KOH, and crystallized from
Despite variable success in getting valuable properties,
simulations of the features of the melanocortins’ active
core continue to emerge in the newest scientific and pat-
ent literature. In particular, diketopiperazine template
was connected with 2-naphthylmethyl, biphenylmethyl
and tetramethyleneamino groups, thus imitating
Phe-Arg-Trp tripeptide.59 Similarly, dipeptide from
L-arginine and trans-3-benzyl-L-proline, capped with
1-naphthylacetyl and benzylamino groups, was pre-
pared.60 Remarkably, this substance exhibited a high
and selective MC4R affinity. Moreover, dipeptide from
THIQ26 was connected to a bicyclic template, the latter
was coupled also with trimethylene-guanidine and meth-
yleneoxy-2-naphthalene groups (associations with
arginine and tryptophan from melanocortins’ active
core).61 Analogous approach, using piperazine as the
central template and functions resembling natural pep-
tides and THIQ, was disclosed in another application,62
and utilization of diphenyloxide structure in related case
was investigated as well.63
1
heptane. Yield 2.2 g (64%). Mp 118–119 ꢁC. H NMR
(270 MHz, DMSO-d6, ppm): d 9.75 and 9.72 (1H, 2s,
CHO), 7.66–7.54 and 8.05–7.84 (8H, 2m, naphthyl),
6.99 (1H, dd, J = 16.0, 7.9 Hz, CHCHO). 2,4-Dini-
trophenylhydrazone: red crystals, mp 254–255 ꢁC.
TISMS: m/e 363.1 (M+H+).
5. Experimental
5.1. Chemistry
5.1.2. trans,trans-Bis-(4-trifluoroacetylamino-cyclohexyl)-
methane (5). trans,trans-Bis-(4-aminocyclohexyl)-meth-
ane47 (1.50 g, 7.13 mmol) was dissolved in trifluoroacetic
acid (10 mL) by stirring and cooling (0 ꢁC). Trifluoro-
acetic anhydride (10 mL, 70 mmol) was added and the
mixture stirred at 40 ꢁC for 7 days. The volatiles were re-
moved under vacuum, and the oily residue was dissolved
in acetonitrile (20 mL) and water (100 mL) was added.
Reagents were used without purification. Unless other-
wise noted, they were purchased from Aldrich, Fluka,
Merck or Lancaster. DMF was purchased from Per-
kin-Elmer Biosystems. Melting points are uncorrected.

5804
F. Mutulis et al. / Bioorg. Med. Chem. 15 (2007) 5787–5810
A crystalline precipitate formed, which was filtered off
and recrystallized from acetonitrile–water. Yield 2.32 g
(81%). Mp 216–218 ꢁC. H NMR (270 MHz, DMSO-
d6, ppm): d 9.26 (2H, d, J = 7.6 Hz, 2CONH), 3.54
(2H, m, 2CHNH), 1.71 (8H, m), 1.28 (6H, m), 0.95
(6H, m). HRMS (M+H+): 403.1823, C17H25F6N2O2 re-
quires 403.1820. Anal. Calcd for C17H24F6N2O2: C,
50.75; H, 6.01; N, 6.96. Found: C, 50.82; H, 6.07; N,
6.83.
acetonitrile (250 mL) was added and the mixture was
placed into a refrigerator. After 20 h the crystals formed
were filtered off and recrystallized (thrice) from methanol–
acetonitrile. Yield 0.77 g (38%). Mp >234 ꢁC. 1H
NMR (270 MHz, DMSO-d6, ppm): d 9.11 (1H, d,
J = 6.9 Hz, CONH, cis), 7.7 (3H, br, NH3þ), 7.47 and
7.11 (4H, AA0XX0 spin system, Tos), 3.73 (1H, m,
CHNHCO, cis), 2.88 (1H, m, CHNH3þ, trans), 2.28
(3H, s, CH3, Tos), 2.0–0.8 (m, 20H). HRMS (M+H+):
307.1984, C15H26F3N2O requires 307.1997. Anal. Calcd
for C22H33F3N2O4S: C, 55.21; H, 6.95; N, 5.85. Found:
C, 55.37; H, 7.00; N, 5.88.
1
5.1.3. cis,trans-Bis-(4-trifluoroacetylamino-cyclohexyl)-
methane (6). Compound (6) was prepared from cis,-
trans-bis-(4-aminocyclohexyl)-methane47 as described
for preparation of 5. Crystallization solvent was acetoni-
trile–water. Yield 83%. Mp 159–160 ꢁC. 1H NMR
(270 MHz, DMSO-d6, ppm): d 9.25 (1H, d, J = 7.9 Hz,
CONH, trans), 9.09 (1H, d, J = 7.3 Hz, CONH, cis),
3.72 (1H, m, CHNH, cis), 3.54 (1H, m, CHNH, trans),
1.72 (4H, m), 1.8–1.1 (14H, m), 0.93 (2H, m). HRMS
(M+H+): 403.1835, C17H25F6N2O2 requires 403.1820.
Anal. Calcd for C17H24F6N2O2: C, 50.75; H, 6.01; N,
6.96. Found: C, 50.71; H, 6.15; N, 6.77.
5.1.5. Procedure A. N-(4-Amino-butyl)-N-benzyl-4-(1H-
indol-3-yl)-butyramide (4–40–8) trifluoroacetate salt and
N-(4-Amino-butyl)-N-benzyl-4-[2-(4-hydroxy-benzyl)-1H-
indol-3-yl]-butyramide (4–40–8a) trifluoroacetate salt.
Wang resin (Bapeks, 0.64 g, 0.7 mmol) was suspended
in CH2Cl2 (5 mL), 4-nitrophenylchloroformate (0.42 g,
2.1 mmol) was added, and the mixture was cooled to
0 ꢁC. A solution of N-methylmorpholine (0.5 mL,
4.0 mmol) in CH2Cl2 (2 mL) was added in small por-
tions with shaking. The reaction mixture was allowed
to warm to room temperature and gently agitated for
4 h. The resin was filtered off, washed successively with
DMF (6· 4 mL), MeOH (3· 4 mL) and CH2Cl2 (2·
4 mL) and dried in a desiccator. The 4-nitrophenyl car-
bonate Wang resin obtained (0.84 g, 0.7 mmol) was
placed in a reaction vessel (size 15 mL), a solution of
1,4-diaminobutane (0.31 g, 3.5 mmol) in DMF (5 mL)
was added and agitated for 24 h at room temperature.
The mixture was then filtered, the resin washed with
DMF, MeOH and CH2Cl2 (each solvent 3· 5 mL) and
dried in vacuum. To the 1,4-diaminobutane resin ob-
tained a solution of benzaldehyde (0.37 g, 3.5 mmol) in
trimethyl orthoformate (5 mL) was added and agitated
for 20 h at room temperature. The mixture was then fil-
tered, the resin washed with CH2Cl2 (3· 5 mL) and dried
under vacuum. Sodium cyanoborohydride (220 mg,
3.5 mmol) was added, carefully mixed with the resin,
4% acetic acid in trimethyl orthoformate (6 mL) was
added in one portion and immediately intensely shaken
for 5 min. The mixture was thereafter filtered, the resin
washed with MeOH, water, MeOH again and CH2Cl2
(each solvent 3· 5 mL), and a 10% solution of diisopro-
pylethylamine in CH2Cl2 (6 mL) was added and agitated
for 10 min at room temperature. The mixture was then
filtered again and the resin washed with CH2Cl2
(6 mL). 3-Indolebutyric acid (213 mg, 1.05 mmol), PyB-
roP (489 mg, 1.05 mmol) and diisopropylethylamine
(0.37 mL, 2.1 mmol) in CH2Cl2 (6 mL) were added fol-
lowed by agitation for 20 h at room temperature. The
mixture was filtered, the resin washed with DMF,
MeOH and CH2Cl2 (each solvent 3· 5 mL) and dried
in vacuum. The resin was then treated with 1,2-ethane-
dithiol (1.2 mL) followed by addition of a freshly pre-
pared mixture of water (0.15 mL), triisopropylsilane
(0.15 mL) and trifluoroacetic acid (6 mL). The reaction
mixture was gently agitated at room temperature for
1 h, filtered and the resin on the filter was washed with
trifluoroacetic acid (3 mL). The combined filtrate was
evaporated under vacuum at room temperature and
the oily residue treated with dry ether-hexane (1:1,
5.1.4. N-[cis-4-(trans-4-Amino-cyclohexylmethyl)-cyclo-
hexyl]trifluoroacetamide (8) 4-toluenesulfonate salt. cis,-
trans-Bis-(4-aminocyclohexyl)-methane47 (4, 5.9 g, 28
mmol) was dissolved in trifluoroacetic acid (30 mL) at
stirring and cooling (0 ꢁC). Trifluoroacetic anhydride
(20 mL, 140 mmol) was added and the mixture stirred
at 40 ꢁC for 48 h. The volatiles were removed under vac-
uum, and the oily residue formed triturated with anhy-
drous ether (200 mL). The precipitate formed was
filtered off, washed with anhydrous ether, dried and
water (300 mL) added. The mixture was stirred at room
temperature for 1 h and then filtered. The filter cake was
crystallized from acetonitrile–water giving side prod-
uct—cis,trans-bis-(4-trifluoroacetyl-aminocyclohexyl)-
methane (6, 0.75 g, 6.7%). The aqueous filtrate was
evaporated to 100 mL volume, causing crystallization.
The suspension obtained was heated to boiling, and
the hot solution formed was slowly cooled to 4 ꢁC.
The crystals formed were filtered off and recrystallized
from 60 mL water. Yield of mixture of trifluoroacetate
salts of N-[trans-4-(cis-4-amino-cyclohexylmethyl)-
cyclohexyl]trifluoroacetamide (7) and N-[cis-4-(trans-4-
amino-cyclohexylmethyl)-cyclohexyl]trifluoroacetamide
(8) was 2.55 g (22%). 1H NMR (270 MHz, DMSO-
d6, ppm): d 9.30 (0.4H, d, J = 7.9 Hz, CONH, trans),
9.14 (0.6H, d, J = 6.9 Hz, CONH, cis), 7.93 (3H, br
s, NH3þ), 3.72 (0.6H, m, CHNHCO, cis), 3.52
(0.4H, m, CHNHCO, trans), 3.14þ (0.4H, m,
CHNH3þ, cis), 2.89 (0.6H, m, CHNH3 trans), 2.0–
0.8 (m, 20H).
1.8 g (4.28 mmol) of the above mixture was dissolved in
methanol (10 mL) and slowly passed through a column
(10 · 195 mm) filled with a slurry of Dowex 1 · 4 in.
OHꢀ form in methanol. The collected eluate was intro-
duced into this column again, and then again (repeating
four times). At the end the resin was washed with meth-
anol. The united eluate was evaporated to 15 mL vol-
ume, 4-toluenesulfonic acid hydrate (0.9 g, 4.7 mmol)
was added and the sample agitated until dissolution,

F. Mutulis et al. / Bioorg. Med. Chem. 15 (2007) 5787–5810
5805
50 ml). The suspension formed was placed in a refriger-
ator for 15 h, and the supernatant liquid was thereafter
removed by decantation. The material obtained was
dried under vacuum in presence of KOH and P2O5. This
raw product was further dissolved in 25% MeCN in
water, centrifuged and the clear solution applied in sev-
eral portions onto an HPLC semipreparative column;
eluent — 30% MeCN in water + 0.1% TFA. Eluate frac-
tions, containing pure putative 4–40–8 and 4–40–8a tri-
fluoroacetates (k0 0.56 and 1.56 correspondingly) were
separately pooled and lyophilized. White powders
formed. Yield of 4–40–8 trifluoroacetate: 32 mg
5.1.8. Procedure D. S-2-Amino-N-(5-amino-2-methyl-
pentyl)-3-(1H-indol-3-yl)-N-(2-methyl-3-phenyl-allyl)-pro-
pionamide and/or S-2-amino-N-(5-amino-4-methyl-pen-
tyl)-3-(1H-indol-3-yl)-N-(2-methyl-3-phenyl-allyl)-propi-
onamide (18-37-17, prepared as a mixture) ditrifluoracetate
salts. Synthesis was made mainly as described in Section
5.1.5 with the exception that an a-Fmoc protected ami-
no acid was applied. After addition of it the mixture was
filtered, the resin washed with DMF (3· 5 mL), 20%
piperidine in DMF (5 mL) was then added and the sam-
ple was agitated for 10 min. The mixture was filtered
again, the resin washed with DMF (5· 5 mL), MeOH
and CH2Cl2 (each solvent 3· 5 mL) and dried in vac-
uum. Cleavage from the polymer and the next proce-
dures followed. Yield 19%. 1H NMR (270 MHz,
DMSO-d6, ppm): d 11.12 and 11.08 (1H, 2s, NH indole),
8.28 (3H, br, COCHNH3þ), 7.79 (3H, br, CH NH þ),
7.56 (1H, m, CH indole), 7.42–7.15 (7H, m, CH benzene
and indole), 7.06 (2H, m, CH indole), 6.23 (1H, m,
CHCCH3), 4.35 (1H, m, COCHNH3þ), 3.49 (2H, m,
CH3CCH2), 3.20 (2H, m, CH3CHCH2), 2.72 (4H, m,
CH2-indole and CH2NH3þ), 1.64 (3H, s, CCH3), 1.63,
1.27 and 1.07 (5H, 3m, CH2CH2CHCH3), 0.77 (3H,
m, CHCH3). HRMS (M+H+): 433.2965, C27H37N4O re-
quires 433.2967.
1
(9.4%). H NMR (270 MHz, DMSO-d6, ppm): d 10.81
and 10.78 (1H, 2s, NH indole), 7.70 (3H, br, NH3þ),
7.46 (1H, m, indole), 7.35–6.86 (9H, m, indole and
benzene CH), 4.53 and 4.51 (2H, 2s, CH2-benzene),
3.32 (2H, m, CH2CH2NCO), 2.84-2.62 (4H, m,
2
3
þ
CH2NH3 and CH2-indole),2.45 (2H, m, CH2CO),
2.01–1.65 (6H, m, CH2CH2CO and CH2CH2CH2NCO).
HRMS (M+H+): 364.2377, C23H30N3O requires
364.2389. Yield of 4–40–8a trifluoroacetate: 4.4 mg
1
(1.0%). H NMR (270 MHz, DMSO-d6, ppm): d 10.81
and 10.78 (1H, 2s, NH indole), 9.19 (1H, br s, OH),
7.70 (3H, br, NH3þ), 7.48-6.86 (11H, m, indole and ben-
zene CH), 6.65 (2H, m, phenol CH), 4.53 (2H, m, CH2-
benzene), 3.88 (2H, m, CH2-phenol), þ3.35 (2H, m,
CH2NCO), 2.87-2.63 (4H, m, CH2NH3 and CH2-in-
dole),2.48 (2H, m, CH2CO), 2.05-1.66 (6H, m,
5.1.9. Procedure E. N-{3-[3-(3-Amino-cyclohexyl)-ureido]-
cyclohexyl}-N-benzyl-4-(1H-indol-3-yl)-butyramide (27-40-8)
trifluoroacetate salt and N-{3-[3-(3-Amino-cyclohexyl)-
ureido]-cyclohexyl}-N-benzyl-4-[2-(4-hydroxybenzyl)-1H-
indol-3-yl]-butyramide (27-40-8a) trifluoroacetate salt. To
a 4-nitrophenyl carbonate Wang resin (preparation de-
scribed in Section 5.1.5., 0.84 g, 0.7 mmol) placed into
a reaction vessel (size 15 mL), a solution of 1,3-diamino-
cyclohexane (0.40 g, 3.5 mmol) in DMF (5 mL) was
added and the mixture agitated for 24 h at room temper-
ature. The suspension was then filtered, and the resin
washed with DMF (4· 5 mL). To the wet resin 4-nitro-
phenylchloroformate (0.42 g, 2.1 mmol) and N,N-diiso-
propylethylamine (0.37 mL, 2.1 mmol) in DMF (5 mL)
were added and the mixture agitated for 24 h at room
temperature. The mixture was then filtered and the resin
washed with DMF (4· 5 mL). To the wet resin a solu-
tion of 1,3-diaminocyclohexane (0.40 g, 3.5 mmol) in
DMF (5 mL) was added and the suspension agitated
for 24 h at room temperature. The mixture was then fil-
tered, the resin washed with DMF, MeOH and CH2Cl2
(each solvent 3· 5 mL) and dried in vacuum. The subse-
quent operations (starting with addition of benzalde-
hyde) were made as described under Section 5.1.5.
CH2CH2CO
and
CH2CH2CH2NCO).
HRMS
(M+H+): 470.2823, C30H36N3O2 requires 470.2807.
5.1.6. Procedure B. N-[4-(4-Amino-cyclohexylmethyl)-
cyclohexyl]-N-benzyl-4-(1H-indol-3-yl)-butyramide (10d-
40-8) trifluoroacetate salt. Synthesis was made essentially
as described under 5.1.5 with the exception that
4,40-Diamino-dicyclohexylmethane trityl resin (Nova-
biochem) was used. For the cleavage of the synthesis
product from the polymer, a mixture of TFA-1,2-ethane
dithiol-methylene chloride (1:1:20) was applied for
30 min. Yield 12%. 1H NMR (270 MHz, DMSO-
d6, ppm): d 10.79 and 10.70 (1H, 2s, indole NH), 7.73
(3H, br, NH3þ), 7.52 and 7.44 (2H, 2d, J = 7.7 and
7.6 Hz, indole CH), 7.36–6.87 (8H, m, benzene and indole
CH), 4.47 and 4.44 (2H, 2s, CH2N), 4.40-4.15 (1H, m,
NCH), 3.2-0.5 (27H, m, aliphatic CH2, CH). HRMS
(M+H+): 486.3481, C32H44N3O requires 486.3484.
5.1.7. Procedure C. N-(2-Amino-ethyl)-2-(1H-indol-3-yl)-
N-(1-naphthalen-2-yl-ethyl)-acetamide (2-29-1) trifluoro-
acetate salt. Synthesis was made essentially as de-
scribed in Section 5.1.5, with the exception that
20-acetonaphthone was dissolved in 10% anhydrous
AcOH in trimethyl orthoformate, added to ethylene-
diamine resin and agitated for 20 h at 30 ꢁC. Yield
7.4%. 1H NMR (270 MHz, DMSO-d6, ppm): d 10.97
(1H, s, indole NH), 7.90-6.90 (15H, m, naphthalene
and indole CH, NH3þ), 5.98 and 5.60 (1H, 2q,
J = 6.9 and 6.4 Hz, CH-naphthyl), 4.07 and 4.05
(2H, 2s, COCH2), 3.55 and 3.47 (2H, þ2t,
J = 6.7 Hz, NCH2CH2), 3.35-3.15 (2H, m, CH2NH3 ),
1.60 and 1.49 (3H, 2d, J = 7.0 and 6.5 Hz, CH3).
HRMS (M+H+): 372.2074, C24H26N3O requires
372.2076.
1
Yield of 27–40–8 26.8 mg (6.0%). H NMR (270 MHz,
DMSO-d6, ppm): d 10.78 and 10.70 (1H, 2s, indole
NH), 7.74 (3H, br, NH3þ), 7.53 and 7.44(1H, 2d,
J = 8.2 and 7.9 Hz, CH indole), 7.36–6.88 (9H, m, ben-
zene and indole CH), 5.77 and 5.58 (2H, 2m,
NHCONH), 4.46 (2H, s, CH2-benzene), 3.84 (1H, m,
CH2N-CH cyclohexane-1), 3.37 (2H, m, 2CH-NH
cyclohexanes-3, 10), 3.01 (1H, m, CHNH3 cyclohex-
þ
ane-30), 2.74 and 2.58 (2H, 2t, J = 7.2 Hz, COCH2),
2.20 (2H, m, COCH2CH2CH2), 2.11-0.80 (18H, m,
COCH2CH2 and cyclohexanes). HRMS (M+H+):
530.3496, C32H44N5O2 requires 530.3495. Yield of
27–40–8a 7.4 mg (1.4%). 1H NMR (270 MHz,

5806
F. Mutulis et al. / Bioorg. Med. Chem. 15 (2007) 5787–5810
DMSO-d6, ppm): d 10.66 and 10.61 (1H, 2s, indole NH),
9.18 (1H, br s, OH), 7.72 (3H, br, NH3þ), 7.50–6.84
(11H, m, phenol, benzene and indole CH), 6.63 (2H,
m, phenol CH), 5.74 and 5.56 (2H, 2m, NHCONH),
4.44 (2H, m, CH2-benzene), 3.93 (2H, m, CH2-phenol),
3.80 (1H, m, CH2N-CH cyclohexane-1), 3.38 (2H, 2m,
5.1.11. Procedure G. N-(3-Guanidino-propyl)-4-(1H-
indol-3-yl)-N-naphthalen-2-yl-methyl-butyramide (12–0–8)
trifluoroacetate salt. Compound 3–0–8 trifluoroacetate
salt (56 mg, 0.11 mmol), 1H-pyrazole-1-carboxamidine
hydrochloride (18 mg, 0.12 mmol) and N,N-diisopropyl-
ethylamine (40 ll, 0.23 mmol) were dissolved in DMF
(0.2 mL) and allowed to stand at room temperature
for 24 h. Then 0.1% trifluoroacetic acid in water
(1 mL) was added, centrifuged and the clear solution
applied onto an HPLC semipreparative (10· 250 mm)
Vydac 219TP510 Diphenyl column, eluent—34% MeCN
in water + 0.1% TFA. Eluate fractions, containing pure
putative 12–0–8 trifluoroacetate, were pooled and
lyophilized. A white powder formed. Yield 25.5 mg
þ
2CH-NH cyclohexanes-3, 10), 3.01 (1H, m, CHNH3
cyclohexane-30), 2.75 and 2.59 (2H, 2m, COCH2), 2.15
(2H, m, COCH2CH2CH2), 2.00-0.80 (18H, m,
COCH2CH2 and cyclohexanes). HRMS (M+H+):
636.3912, C39H50N5O3 requires 636.3913.
5.1.10. Procedure F. N-[cis-4-(trans-4-Amino-cyclohexyl-
methyl)-cyclohexyl]-N-(2-bromo-4,5-dimethoxy-benzyl)-4-
(1H-indol-3-yl)-butyramide (10e–18–8) trifluoroacetate
salt and N-[cis-4-(trans-4-Amino-cyclohexylmethyl)-
cyclohexyl]-N-(2-bromo-4,5-dimethoxy-benzyl)-4-[2-
(4-hydroxy-benzyl)-1H-indol-3-yl]-butyramide (10e–18–
8a) trifluoroacetate salt. 4-Nitrophenyl carbonate Wang
resin (preparation described in 5.1.5., 0.42 g,
0.35 mmol) was placed in a reaction vessel (size
1
(42%). H NMR (270 MHz, DMSO-d6, ppm): d 10.78
and 10.73 (1H, 2s, indole NH), 8.0–6.9 (12H, m, indole,
naphthalene CH), 7.60-7.40 (5H, br, guanidinium+),
4.70 (2H, s, CH2-naphthalene), 3.44–3.22 (2H, m,
NCH2CH2), 3.16-3.01 (2H, m, CH2-guanidinium+),
2.8–2.6 (2H, m, CH2-indole), 2.5–2.4 (2H, m, COCH2),
2.05–1.80 (2H, m, COCH2CH2), 1.8–1.6 (2H, m,
NCH2CH2). HRMS (M+H+): 442.2622, C27H32N5O
requires 442.2607.
8 mL),
a solution of 8 (4-toluenesulfonate salt,
167 mg, 0.35 mmol) and N,N-diisopropylethylamine
(61 lL, 0.35 mmol) in DMF (2 mL) was added and
the mixture agitated for 60 h at room temperature.
The resin was filtered off, washed successively with
DMF (4· 3 mL), MeOH (3· 3 mL) and CH2Cl2 (2·
3 mL) and dried in a desiccator. To the dry resin,
NaBH4 (199 mg, 5.25 mmol) in 1-butanol (3 mL) was
added and the mixture agitated, first for 49 h at room
temperature, and then for 3 h at 50 ꢁC. The resin was
filtered off, washed successively with 1-butanol (4·
3 mL), DMF (4· 3 mL), MeOH (3· 3 mL) and
CH2Cl2 (2· 3 mL) and dried in a desiccator. The sub-
sequent operations (starting from the addition of 2-
bromo-4,5-dimethoxy-benzaldehyde) were made as de-
scribed for Procedure A, with the exception that 1,2-
ethanedithiol was excluded from the cleavage mixture.
Yield of 10e–18–8 14.8 mg (5.8%). 1H NMR
(270 MHz, DMSO-d6, ppm): d 10.74 and 10.65 (1H,
2s, indole NH), 7.68 (3H, br, NH3þ), 7.45 (1H, m, in-
dole CH), 7.26 (1H, m, indole CH), 7.14-6.83 (5H, m,
benzene and indole CH), 4.31 (2H, m, CH2N), 3.72
and 3.69 (3H, 2s, CH3), 3.57 and 3.56 (3H, 2s,
CH3), 3.43 (1H, m, N-CH cyclohexane), 3.10 and
2.85 (1H, 2m, N-CH cyclohexane), 2.72 and 2.55
(2H, 2m, COCH2), 2.19 (2H, m, COCH2CH2CH2),
1.85 (2H, m, COCH2CH2), 1.73–0.70 (20H, m, CH2-
cyclohexane and CH and CH2 cyclohexanes). HRMS
(M+H+): 624.2795, C34H47BrN3O3 requires 624.2801.
Yield of 10e–18–8a 48.2 mg (16%). 1H NMR
(270 MHz, DMSO-d6, ppm): d 10.70 (1H, m, indole
NH), 9.18 (1H, m, OH), 7.65 (3H, br, NH3þ), 7.25
(1H, m, indole CH), 7.16 (1H, m, indole CH), 7.10-
6.85 (6H, m, benzene, indole and phenol CH), 6.62
(2H, m, phenol CH), 4.31 (2H, m, CH2N), 4.02
(2H, m, CH2–phenol), 3.75 (3H, m, CH3), 3.57 (3H,
m, CH3), 3.43 (1H, m, N-CH cyclohexane), 3.15 and
2.85 (1H, 2m, N-CH cyclohexane), 2.74 and 2.54
(2H, 2m, COCH2), 2.23 (2H, m, COCH2CH2CH2),
1.85 (2H, m, COCH2CH2), 1.76-0.80 (20H, m, CH2-
cyclohexane and CH and CH2 cyclohexanes). HRMS
(M+H+): 730.3192, C41H53BrN3O4 requires 730.3219.
5.1.12. Procedure H. N-Pentyl-N-(4-diethylamino-benzyl)-
4-(1H-indol-3-yl)-butyramide (1-39-8) trifluoroacetate salt.
4-Diethylaminobenzaldehyde (1.77 g, 10 mmol) and
1-aminopentane (1.28 mL, 11 mmol) were dissolved in
anhydrous trimethyl orthoformate (25 mL) and allowed
to stand for 3 days at room temperature. The reaction
mixture was then cooled to 0 ꢁC and sodium cyanoboro-
hydride (2.51 g, 40 mmol) was added during stirring. The
mixture was allowed to warm to room temperature and
stirred for 2 h. The reaction mixture was then evapo-
rated, the oily residue dissolved in CH2Cl2 (50 mL),
washed with a saturated NaHCO3 water solution
(50 mL), and then with water (50 mL). The combined
water phases were extracted with CH2Cl2 (50 mL) and
discarded, the organic layer was then added to the previ-
ous organic phase, dried with Na2SO4 and filtered. The
filtrate was evaporated, the residue dried under vacuum
in presence of P2O5 and KOH. Light yellow oil formed.
Yield of pentyl-(4-diethylamino-benzyl)-amine (22) 2.5 g
1
(quantitative). H NMR (270 MHz, DMSO-d6, ppm): d
7.18 (2H, d, J = 8.9 Hz, benzene CH), 6.65 (2H, d,
J = 8.8 Hz, benzene CH), 6.60 (1H, m, NH), 3.81 (2H,
s, benzene-CH2), 3.36-3.23 [4H, m, N(CH2)2], 2.66 (2H,
m, NHCH2CH2), 1.52 (2H, m, NHCH2CH2), 1.28 (4H,
m, CH2CH2CH3), 1.06 [6H, t, J = 6.9 Hz, N(CH2CH3)2],
0.86 (3H, t, J = 6.8 Hz, CH2CH2CH3). HRMS (M+H+):
249.2340, C16H29N2 requires 249.2330.
Compound 22 (100 mg, 0.40 mmol), 3-indolebutyric acid
(98.5 mg, 0.485 mmol), PyBroP (226 mg, 0.485 mmol)
and diisopropylethylamine (0.25 mL, 1.49 mmol) were
dissolved in 1.0 mL CH2Cl2 and allowed to stand for 1 h
at room temperature. The reaction mixture was then
washed with a saturated aqueous solution of NaHCO3
(2· 1 mL) followed by water (1 mL). The combined water
phases were extracted with CH2Cl2 (2 mL), the organic
layer was added to the previous organic phase and dried
with Na2SO4. The mixture was then filtered, the filtrate
evaporated and the residue dried under vacuum in the

F. Mutulis et al. / Bioorg. Med. Chem. 15 (2007) 5787–5810
5807
presence of P2O5 and KOH. The oil formed was further
dissolved in 30% acetonitrile in water + 0.1% TFA, centri-
fuged and the clear solution was divided in several por-
tions. Each portion was applied onto a semipreparative
HPLC column; the mobile phase consisting of 35%
MeCN in water + 0.1% TFA. Fractions from repeated
runs containing pure putative 1–39–8 were pooled and
lyophilized. A white powder formed. Yield 34.4 mg
(4H, m, CH2CH2CH2), 1.79 (4H, m, b -tetralone and
CH2CH2CH2), 1.38 (9H, s, Boc). HRMS (M+H+):
305.2227, C18H29N2O2 requires 305.2229. 100 mg
(0.29 mmol) of this product, 3-indolepropionic acid
(66 mg, 0.35 mmol), and PyBroP (163 mg, 0.35 mmol)
were dissolved in CH2Cl2 (3 mL), N,N-diisopropylethyl-
amine (0.17 mL, 1.0 mmol) added and allowed to stand
for 24 h. CH2Cl2 (3 mL) was added and solution was ex-
tracted with 10% aqueous KHSO4 (3· 5 mL), saturated
aqueous NaHCO3 (5· 5 mL) and water(3· 5 mL). The
organic solution was dried over MgSO4, filtered and
the filtrate evaporated. An oily residue, containing N-
(3-tert-butoxycarbonylaminopropyl)-N-(1,2,3,4-tetrahy-
dronaphthalene-2-yl)-3-(1H-indol-3-yl)-propionamide
1
(16%). H NMR (270 MHz, DMSO-d6, ppm): d 10.77
and 10.72 (1H, 2s, indole NH), 7.60–6.85 (9H, m, benzene
and indole CH), 7.39 (1H, br, NH+), 4.52 (2H, s, CH2-Ph),
3.52–3.36 [4H, m, +NH(CH2CH3)2], 3.28-3.08 (2H, m,
CONCH2CH2), 2.76–2.57 (2H, m, CH2-indole), 2.42
and 2.32 (2H, 2t, J = 7.2 Hz, COCH2), 1.98-1-76 (2H,
m, COCH2CH2), 1.51–1.28 (2H, m, NCH2CH2), 1.28–
1.05 (4H, m, CH3CH2CH2), 0.96 [6H, t, J = 6.9 Hz,
+NH(CH2CH3)2], 0.86–0.70 (3H, m, CH2CH2CH3).
HRMS (M+H+): 434.3156, C28H40N3O requires
434.3171.
1
(26) formed. H NMR (270 MHz, DMSO-d6): d 10.76
and 10.73 (1H, 2s, indole NH), 7.51 and 7.46 (1H, 2d,
J = 7.8 Hz, indole CH), 7.29 (1H, m, indole CH),
7.11–6.93 (7H, m, b-tetralone and indole CH), 6.89
and 6.80 (1H, 2m, NHBoc), 3.98 (1H, m, NCH b-tetr-
alone), 3.20 (2H, m, b-tetralone CH2), 2.98-2.79 (6H,
m, b-tetralone CH2, COCH2CH2 and CH2NH), 2.66
(4H, COCH2CH2), 1.76 and 1.63 (4H, 2m, b-tetralone
CH2 and CH2CH2CH2), 1.34 and 1.29 (9H, 2s, Boc).
TISMS: m/e 476.3 (M+H+). This product was dissolved
in a mixture CF3COOH–water–triisopropylsilane-1,2-
ethanedithiol (92.5:2.5:2.5:2.5; 2 mL), allowed to react
for 45 min at room temperature, the reaction mixture
was evaporated under vacuum at room temperature
and the oily residue treated with mixture dry ether–hex-
ane (1:1, 50 mL). A suspension formed, which was
placed in a refrigerator for 15 h whereafter the liquid
was removed by decantation. The material obtained
was dried under vacuum in the presence of KOH and
P2O5. This raw product was further dissolved in 20%
MeCN in water, centrifuged and the clear solution ap-
plied onto an HPLC semipreparative column, eluent—
22% MeCN in water + 0.1% TFA. Eluate fractions, con-
taining pure putative (3–38–5) trifluoroacetate, were
pooled and lyophilized. A white powder formed. Yield
71.5 mg (57% from 24, 14% summary). 1H NMR
(270 MHz, DMSO-d6): d 10.78 and 10.75 (1H, 2s, indole
NH), 7.69 (3H, br s, NH3þ), 7.53 and 7.51 (1H, 2d,
J = 8.0 Hz and 7.8 Hz, indole CH), 7.31 (1H, m, indole
CH), 7.16–6.87 (7H, m, b-tetralone and indole CH), 4.03
(1H, m, b-tetralone NCH), 3.27 (2H, m, b-tetralone
CH2), 2.93 (2H, m, b-tetralone CH2), 2.89–2.62 (8H,
m, COCH2CH2 and CH2CH2CH2), 1.77 and 1.65 (4H,
2m, b-tetralone CH2 and CH2CH2CH2). HRMS
(M+H+): 376.2394, C24H30N3O requires 376.2389.
5.1.13. Procedure I. N-(3-Aminopropyl)-N-(1,2,3,4-tetra-
hydronaphthalene-2-yl)-3-(1H-indol-3-yl)-propionamide
(3–38–5) trifluoroacetate salt. To a dry 250-mL three-
necked flask equipped with septum and argon inlet,
CH2Cl2 (100 mL), 1,3-propylenediamine (4.37 mL,
51.7 mmol) and b-tetralone (2.52 g, 17.2 mmol) were
added. The mixture was cooled to 0 ꢁC, and TiCl4
(0.95 mL, 8.6 mmol) was added using a syringe. The
reaction mixture was allowed to warm to room temper-
ature and stirred for 20 h, maintaining the argon atmo-
sphere. The mixture was thereafter again cooled to 0 ꢁC,
and a solution of NaCNBH3 (2.92 g, 46.5 mmol) in
MeOH (35 mL) was slowly added. The stirring was con-
tinued for 15 min at 0 ꢁC and 30 min at room tempera-
ture. Solution of NaOH (1.24 g, 31 mmol) in water
(18 mL) was then added portionwise. The organic phase
was separated, and the aqueous phase was extracted
with CH2Cl2 (50 mL). The combined organic solutions,
which contained N-(1,2,3,4-tetrahydronaphthalene-2-
yl)-1,3-propylenediamine [24, TISMS: m/e 205.1
(M+H+)], were directly used for the following synthesis.
Di-tert-butyl dicarbonate (3.76 g. 17.2 mmol) was
added, and the solution allowed to stand for 24 h at
room temperature. The mixture was then applied onto
a silica gel column (3.5 · 35 cm) and eluted with a
chromatographic system comprising CHCl3–MeOH–
NH4OH–petrol ether, gradually changing its volume
proportions from 30:2:1:90 to 9:1:1:0. Fractions,
containing pure N-tert-butoxycarbonyl-N0-(1,2,3,4-
tetrahydronaphthalene-2-yl)-1,3-propylenediamine [25,
TISMS: m/e 305.2 (M+H+)], were pooled, evaporated
and dried in desiccator over H2SO4 until constant
weight. The product was dissolved in anhydrous ether
(50 mL), and a 4 M HCl solution in dioxane was added
at stirring until pH reached 5 (wet indicator paper). A
brownish grey precipitate of N-tert-butoxycarbonyl-
N0-(1,2,3,4-tetrahydronaphthalene-2-yl)-1,3-propylen-
ediamine (25) hydrochloride formed. It was filtered off,
washed on filter with anhydrous ether and dried in des-
5.1.14. Procedure J. N-(2-Aminoethyl)-N-(spiro[5.5]und-
ecan-3-yl)-2-(naphthalen-2-yloxy)-acetamide
(2-42-14)
trifluoroacetate salt. To a dry 100-mL three-necked flask
equipped with septum and argon inlet, CH2Cl2 (50 mL),
ethylenediamine (2.49 mL, 29.3 mmol) and spiro[5.5]un-
dec-1-en-3-one57 (1.6 g, 9.8 mmol) were added. The mix-
ture was cooled to 0 ꢁC, and at stirring TiCl4 (0.54 mL,
4.9 mmol) added using a syringe. The reaction mixture
was allowed to warm to room temperature and stirred
for 20 h, maintaining the argon atmosphere. Then the
mixture was cooled to 0 ꢁC again, and a solution of
NaCNBH3 (1.23 g, 19.5 mmol) in MeOH (15 mL) was
slowly added. The stirring was continued for 15 min at
1
iccator over P2O5 and KOH. Yield 1.43 g (24%). H
NMR (270 MHz, DMSO-d6): d 9.08 (2H, m, NH2þ),
7.04 (4H, m, b-tetralone), 6.93 (1H, m, NHCO), 3.43
(1H, m, CHNH2þ), 3.02 (4H, m, b-tetralone), 2.85

5808
F. Mutulis et al. / Bioorg. Med. Chem. 15 (2007) 5787–5810
0 ꢁC, and 30 min at room temperature. A new portion of
NaCNBH3 in MeOH was added with stirring and the
temperature was changed as before. Then a saturated
aqueous solution of NaHCO3 (50 mL) was added with
stirring. The organic phase was separated, and the aque-
ous phase was extracted twice with CH2Cl2 (40 mL).
The combined organic solutions were dried over
Na2SO4, filtered, and the filtrate evaporated to afford
N-(spiro[5.5]undecan-3-yl)-ethylenediamine (27) as yel-
low oil. Yield 1.8 g (88%). 1H NMR (270 MHz,
DMSO-d6, ppm): d 3.36 (1H, m, NHCH spiro[5.5]und-
ecan-3-yl), 3.29 and 3.18 (2H, 2m, NHCH2), 2.97 and
2.80 (2H, 2m, CH2NH2), 1.84–0.88 (18H, m, CH2
spiro[5.5]undecan-3-yl). HRMS (M+H+): 211.2179,
C13H27N2 requires 211.2174. 0.31 g (1.5 mmol) of this
substance was dissolved in DMF (5 mL) and added to
4-nitrophenyl carbonate Wang resin (0.59 g, 0.50 mmol).
The mixture was agitated at room temperature for 24 h,
filtered, the resin washed with DMF (5· 5 mL), MeOH
and CH2Cl2 (each solvent 3· 5 mL), and dried in vac-
uum. Following operations (starting with addition of
carboxylic acid–2-naphthoxyacetic acid) were made as
described in Procedure A. Eluent for the semiprepara-
tive HPLC contained 32% MeCN. Yield 87 mg (11%
Each of the radioligand binding assays comprised a dilu-
tion series of12concentrations with duplicates with a51/2
order of magnitude of concentration range, and each
compound was analyzed with at least two repeats. In
brief, B16 mouse melanoma cells which express the
MC1 receptor naturally or Sf9 cells expressing either
one of the recombinant human MC3, MC4 or MC5
receptors were taken from frozen stocks, washed with
binding buffer (minimum essential medium with Earle’s
salts, pH 7.0, 0.2% bovine serum albumin, 1 mM 1,10-
phenanthroline, 0.5 mg per litre leupeptin and 200 mg
per litre bacitracin). The assays were performed in
96-well binding plates, to each well 50,000 cells were
dispensed, 0.5 nM (approximately 50,000 cpm) [125I]-
NDP–MSH and appropriate concentrations of tertiary
amide in 50 ll binding buffer. The plates were then incu-
bated for 1 h at 37 ꢁC whereafter the free radioligand was
separated from the bound radioligand by centrifugation
(3500 rpm for 5 min at 5 ꢁC). The cells were washed with
ice-cold binding buffer and then detached from the plates
with 0.1 N NaOH and the radioactivity counted in a
Wallac, Wizard automatic gamma counter and data
analyzed with the BindAid software, essentially as previ-
ously described.65
1
from 27, 10% summary). H NMR (270 MHz, CDCl3):
d 7.97 (3H, br s, NH3þ), 7.80–7.64 (3H, m, naphthalene),
7.47–7.27 (2H, m, naphthalene), 7.12 (2H, m, naphtha-
lene), 4.85 (2H, m, COCH2), 3.54 (1H, m, spiro[5.5]und-
ecan-3-yl NCH), 3.41 (1H, m, NCH2), 3.13 (2H, m,
CH2NH3þ), 1.72–0.9 (18H, m, spiro[5.5]undecan-3-yl
9CH2). HRMS (M+H+): 395.2709, C25H35N2O2 requires
395.2698.
5.2.2. cAMP assays. The ability of tertiary amides to
influence the levels of cAMP was, for the MC1 receptor,
assessed in B16 mouse melanoma cells which express the
MC1 receptor naturally, and for MC3, MC4 and MC5
receptors in COS-1 cells that had transiently been trans-
fected with the MC3, MC4 and MC5 receptor DNA,
using the procedures previously described.65 In brief,
cells were cultured in DMEM supplemented with heat-
inactivated 10% FBS at 37 ꢁC in humidified 5% CO2 cell
incubator. B16 cells were cultured until experiments
were performed. COS-1 cells were transfected when they
reached 50% confluence with the appropriate receptor
DNA by using Lipofectine reagent (Gibco-BRL)
according to manufacturers’ instruction. After 48 h
transfected COS-1 cells were used for cAMP determina-
tion. Cells were harvested and incubated for 20 min at
37 ꢁC in 0.05 mL DMEM without FBS containing
0.5 mM 3-isobutyl-1-methylxanthine and appropriate
concentrations of substances of interest. cAMP was then
extracted from cells by adding 4.4 M perchloric acid to
achieve a final concentration of 1.25 M. After 5 min
the samples were neutralized with 5 M KOH/1 M Tris
and centrifuged at 3000 rpm for 5 min at 20 ꢁC. After
centrifugation 50 ll of the supernatants or cAMP stan-
dards ranging from 0.2 to 250 nM were added to 96-well
microtitre plates. The cAMP concentration was mea-
sured by a bovine adrenal cAMP binding protein com-
petitive binding assay using [3H]-cAMP (specific
activity 24 Ci/mmol) as labelled ligand, essentially as de-
scribed.66 In brief, the sample, [3H]-cAMP (0.14 pmol,
approximately 11,000 cpm), cAMP standard and the
binding protein were incubated at 4 ꢁC for 150 min
whereafter the incubates were harvested by filtration
on Whatman GF/B filters using semiautomatic Brandel
cell harvester, Gaithersburg, MD, USA. Each filter was
rinsed with 10 mL ice-cold 50 mM TrisÆHCl, pH 7.4,
punched out and placed into scintillation vials with scin-
tillation fluid and counted.
5.1.15. Procedure K. Fractionation of mixtures of tertiary
amides on reversed phase. Raw syntheses product was
purified as usual with the exception that eluate portion,
which according to LC/MS analysis contained desired
mixture of isomers, was collected as several fractions,
which were numerated according to their order of com-
ing. They were then separately freeze-dried and used for
further investigations.
5.1.16. Procedure L. Fractionation of mixtures of tertiary
amides on chiral phase. Product (3–5 mg), obtained
according to Procedure K, was dissolved in MeOH–tri-
ethylamine–AcOH (100:0.1:0.1, 1.5 mL), and the solu-
tion divided in several portions. They were applied
onto a Chirobiotic V HPLC semipreparative (Astec,
10 · 250 mm) column equlibrated with the same solu-
tion, serving also as eluent. Flow rate 5 mL/min, detec-
tion at 220 nM. Eluate fractions corresponding to
observed UV peaks were separately pooled and repeat-
edly lyophilized (until a constant weight of the product
was reached). White powders formed. Fractions were
numerated according to their order of coming.
5.2. Pharmacology
5.2.1. Radioligand binding on recombinant human MC
receptor subtypes. Assays of tertiary amides binding to
human melanocortin receptors were performed using
(
125I–Tyr2, Nle4, D-Phe7)-a-MSH ([125I]-NDP–MSH)
radioligand binding essentially as described earlier.64

F. Mutulis et al. / Bioorg. Med. Chem. 15 (2007) 5787–5810
5809
20. Adan, R. A. H.; Nijenhuis, W. A. J.; Kas, M. J. H. Int.
Congr. Ser. 2003, 1249, 195–206.
Acknowledgments
21. Haskell-Luevano, C.; Sawyer, T. K.; Hendrata, S.; North,
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This work was supported by the Swedish MRC (04X-
05975). SviatlanaYahorava and Santa Veiksina were
supported by stipends from the Visby programme
administered by the Swedish Institute.
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Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.2007.
06.003.
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