Nonpeptide analogues of dynorphin A(1-8): Design, synthesis, and pharmacological evaluation of κ-selective agonists

Article abstract of DOI:10.1021/jm990356p

Two novel series of κ opioid receptor agonist analogues of MPCB-GRRI and MPCB-RRI, hybrid ligands ofMPCB ((-)-cis-N-(2-phenyl-2-carbomethoxy)cyclopropylmethyl-N-normetazo cine) and of the C-terminal fragments of dynorphin A(1-8), have been synthesized. The critical functional groups of the peptide fragments of hybrid compounds were maintained, and the binding affinities and selectivities for compounds 1-40 to μ, δ, and κ opioid receptors were analyzed. Compounds 15 and 16, MPCB-Gly-Leu-NH-(CH₂)(n)-NH-C( = NH)-C₄H₉ (n = 5, 6), displayed high affinity and selectivity for κ opioid receptors (K(i)(κ) = 6.7 and 5.3 nM, K(i)(μ)/K(i)(κ) = 375 and 408, and K(i)(δ)/K(i)(κ) = 408 and 424, respectively). Since κ agonists may also cause psychotomimetic effects by interaction with σ sites, binding assays to σ₁ sites were performed where compounds 15 and 16 showed negligible affinity (K(i) > 10 000). Compounds 15 and 16 were further characterized in vivo and showed potent antinociceptive activity in mouse abdominal constriction tests (ED₅₀ = 0.88 and 1.1 mg/kg, respectively), fully prevented by nor-BNI. Thus, these novel analogues open an exciting avenue for the design of peptidomimetics of dynorphin A(1-8).

Full text of DOI:10.1021/jm990356p

2992
J . Med. Chem. 2000, 43, 2992-3004
Articles
Non p ep tid e An a logu es of Dyn or p h in A(1-8): Design , Syn th esis, a n d
P h a r m a cologica l Eva lu a tion of K-Selective Agon ists
Giuseppe Ronsisvalle,*,† Lorella Pasquinucci,† Valeria Pittala`,† Agostino Marrazzo,† Orazio Prezzavento,†
Rosanna Di Toro,‡ Barbara Falcucci,‡ and Santi Spampinato‡
Department of Pharmaceutical Sciences, University of Catania, Viale Andrea Doria, 6 - Citta` Universitaria,
95125 Catania, Italy, and Department of Pharmacology, University of Bologna, Via Irnerio, 48, 40126 Bologna, Italy
Received J uly 12, 1999
Two novel series of κ opioid receptor agonist analogues of MPCB-GRRI and MPCB-RRI, hybrid
ligands of MPCB ((-)-cis-N-(2-phenyl-2-carbomethoxy)cyclopropylmethyl-N-normetazocine) and
of the C-terminal fragments of dynorphin A(1-8), have been synthesized. The critical functional
groups of the peptide fragments of hybrid compounds were maintained, and the binding
affinities and selectivities for compounds 1-40 to µ, δ, and κ opioid receptors were analyzed.
Compounds 15 and 16, MPCB-Gly-Leu-NH-(CH2)n-NH-C(dNH)-C4H9 (n ) 5, 6), displayed high
affinity and selectivity for κ opioid receptors (Kiκ ) 6.7 and 5.3 nM, Kiµ/Kiκ ) 375 and 408, and
Kiδ/Kiκ ) 408 and 424, respectively). Since κ agonists may also cause psychotomimetic effects
by interaction with σ sites, binding assays to σ1 sites were performed where compounds 15
and 16 showed negligible affinity (Ki > 10 000). Compounds 15 and 16 were further
characterized in vivo and showed potent antinociceptive activity in mouse abdominal constric-
tion tests (ED50 ) 0.88 and 1.1 mg/kg, respectively), fully prevented by nor-BNI. Thus, these
novel analogues open an exciting avenue for the design of peptidomimetics of dynorphin A(1-
8).
In tr od u ction
of different opioid receptor types have suggested that
the second extracellular loop region (EL2) plays an
important role in the selective interaction of dynorphin
A with the κ opioid receptor.13,14 More specifically, the
interaction between EL2 and the C-terminal region of
dynorphin A may be relevant in receptor activation. In
fact studies on the mechanism of activation of cloned κ
opioid receptors have demonstrated the necessity of
simultaneous interaction with the two binding domains
of the κ opioid receptor.15 This event implies that the
peptidomimetic design of dynorphin A should require
both the essential chemical fragments of the C-terminal
region and the “message” sequence. Recently reported
molecular simulations of dynorphin A(1-10) binding to
EL2 does not exclude an interaction of dynorphin A with
the κ opioid receptor in a conformation similar to that
of an R-helical secondary structure as proposed by
Tessmer and Kallick.16,17
Opioid analgesics exert their actions through three
types of opioid receptors: µ, κ, and δ. Research in the
area of opioid receptors has focused on the development
of selective ligands, aimed to clarify their anatomical
distributions and functions.1 Over the past two decades,
a large number of novel κ-selective agonists have been
developed. In fact, κ agonists are potentially safer as
analgesic agents, without exhibiting many of the clini-
cally limiting side effects that characterize morphine.2
Recently, pharmacological studies which report activa-
tion of κ opioid followed by µ antagonist effects have
provided new interest for the development of κ-selective
ligands, opening a possible new therapeutic strategy in
pain treatment.3-5
The κ opioid receptor was first cloned in 1993, and
the analysis of its amino acid sequence identified this
receptor to be a member of the G-protein-coupled
receptor (GPCR) superfamily.6-9 Dynorphin A (H-Tyr-
Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-
Asp-Asn-Gln-OH) was then postulated to be the endo-
genous ligand for the κ opioid receptor.10 Dynorphin A
and its shorter homologues, dynorphin A(1-8) and
dynorphin A(1-13), are reported to have high affinity
for both natural and cloned κ opioid receptors.11,12
Studies on chimeric receptors composed of fragments
Structure-activity relationship (SAR) studies on pep-
tide and nonpeptide κ-selective ligands enabled us to
design and synthesize MPCB and its p-chlorophenyl
analogue (CCB), the first (-)-normetazocine agonist
derivatives that bind to κ opioid receptors with high
affinity and specificity (Figure 1).18,19 The cyclopropyl-
methyl-normetazocine (CPM) nucleus represents a scaf-
fold to support a phenolic ring, a basic nitrogen, and a
phenyl ring in a fixed and an appropriate conformation,
which then mimics the recognition process of the N-
terminal fragment of dynorphin A (Figure 2A). To
confirm this hypothesis, we later synthesized a series
* To whom correspondence should be addressed. Phone/Fax: +39
(95) 33 67 22. E-mail: ggrmedch@mbox.unict.it.
† University of Catania.
‡ University of Bologna.
10.1021/jm990356p CCC: $19.00 © 2000 American Chemical Society
Published on Web 07/20/2000

Nonpeptide Analogues of Dynorphin A(1-8)
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16 2993
diaminopropane,26 N-(tert-butoxycarbonyl)-1,4-diamino-
butane, N-(tert-butoxycarbonyl)-1,5-diaminopentane,
N-(tert-butoxycarbonyl)-1,6-diaminohexane,27 ethyl pro-
pionimidate hydrochloride, ethyl butyrimidate hydro-
chloride, ethyl valerimidate hydrochloride,28 butyra-
mide, and valeramide.29 cis-(-)-(2R,6R,11R)-N-Normeta-
zocine was separated from a racemic mixture as re-
ported by Brine et al.30 Alkylation of cis-(-)-(2R,6R,-
11R)-N-normetazocine with methyl (1S,2R)-2-(chloro-
methyl)-1-phenylcyclopropanecarboxylate31 afforded
methyl 1(S)-phenyl-2(R)-[(8-hydroxy-6(R),11(R)-dimethyl-
1,2,5,6-tetrahydro-4H-2(R),6-methanobenzazocin-3-yl)-
methyl]cyclopropanecarboxylate (MPCB) that was hy-
drolyzed to the corresponding 1(S)-phenyl-2(R)-[(8-
hydroxy-6(R),11(R)-dimethyl-1,2,5,6-tetrahydro-4H-
2(R),6-methanobenzazocin-3-yl)methyl]cyclopropane-
carboxylic acid.18 2(R)-[2-(Carbobenzoxy)aminoacety-
lamino]-4-methylpentanoic acid p-nitrophenyl ester was
synthesized following the same procedure to synthesize
2(R)-(carbobenzoxy)amino-4-methylpentanoic acid p-nitro-
phenyl ester25 (analytical data are available as Sup-
porting Information).
The Cbz-Gly-Leu-NH-(CH2)n-NH-Boc compounds 41-
44 and the Cbz-Leu-NH-(CH2)n-NH-Boc compounds 45-
48 were prepared by coupling mono-Boc-diamines with
p-nitrophenyl active esters Cbz-Gly-Leu-O-pNP and
Cbz-Leu-O-pNP, respectively. The Cbz-R-amino protect-
ing group was removed by hydrogenolysis to obtain the
compounds H-Gly-Leu-NH-(CH2)n-NH-Boc 49-52 and
the compounds H-Leu-NH-(CH2)n-NH-Boc 53-56. The
coupling reagents DCC and HOBt were employed to
synthesize compounds 57-60 and 61-64 by reacting
1(S)-phenyl-2(R)-[(8-hydroxy-6(R),11(R)-dimethyl-1,2,5,6-
tetrahydro-4H-2(R),6-methanobenzazocin-3-yl)methyl]-
cyclopropanecarboxylic acid with compounds 49-52 and
53-56, respectively. Treatment of Boc-protected com-
pounds with 90% TFA in the presence of anisole
provided the final compounds 1-4 and 21-24. Com-
pounds 1-4 and 21-24, with the terminal primary
amino group, were transformed to N-terminal ethyla-
midino derivatives 5-8 and 25-28 by reaction with
ethyl propionimidate hydrochloride in EtOH and DIEA
at pH 10.32-34 Similarly, N-terminal propylamidino
derivatives 9-12 and 29-32 were obtained with ethyl
butyrimidate hydrochloride and N-terminal butylami-
dino derivatives 13-16 and 33-36 with ethyl valerimi-
date hydrochloride. Finally, N-terminal guanidino de-
rivatives 17-20 and 37-40 were synthesized by reacting
compounds 1-4 and 21-24 with 3,5-dimethylpyrazole-
1-carboxamidine nitrate in EtOH and DIEA at pH 10.35
Thin-layer chromatography (TLC) and analytical
HPLC assessed the purity of the final compounds 1-40.
Molecular weights were confirmed by FAB-MS (Table
1).
F igu r e 1. MPCB,18 CCB,19 and hybrid compounds.20
of hybrid ligands of MPCB and CCB, in which the
heterocyclic nucleus was linked to various C-terminal
fragments of dynorphin A(1-8) (Figure 1). In particular,
MPCB-GRRI and MPCB-RRI exhibited high affinities
to both cloned and native κ opioid receptors, further
supporting the hypothesis that MPCB and CCB are
appropriate mimetics of the N-terminal fragment of
dynorphin A (Figure 2B).20
To further develop conformationally constrained pep-
tidomimetics of dynorphin A(1-8), we herein report the
design and synthesis of two novel series of analogues
of MPCB-GRRI and MPCB-RRI with nonpeptide re-
placement at the C-terminus (Figure 3). Only highly
flexible analogues were synthesized. The use of a flexible
spacer chain might make impossible a rigorous inter-
pretation of the results with respect to the stereochem-
ical requirements of the pharmacophore; however, it will
permit the alignment of critical functional groups with
the receptor. A systematic evaluation of the distance
among recognition features could in fact identify the
functionalities capable of mimicking relevant groups in
the C-terminal region of dynorphin A. In addition, a
qualitative analysis of the observed activity trends can
provide a basis to improve the design of the heterocyclic
scaffold for the dynorphin A pharmacophores. We
synthesized two series of compounds in which a spacer,
constituted by Leu or Gly-Leu, was linked to the MPCB
nucleus in order to mimic the amino acid fragments R
and GR of the hybrid compounds MPCB-RRI and
MPCB-GRRI. The amino acid residue Leu was chosen
to replace the Arg6 residue because it can be considered
an analogue lacking the positive charge on the side
chain. Previous structure-affinity studies of dynorphin
A have determined that only the charged residue Arg7
and the hydrophobic residue Ile8 are necessary for κ
affinity and selectivity.21-23 The amino acid residue Arg7
was replaced with a linear diamine, where the spacer
chain length ranged from 3 to 6 methylene units to
represent the distance between the pharmacophore and
the charged nitrogen of Arg7. Different basic groups
were considered to explore the influence of basicity and
optimal charge distribution. Thus we synthesized de-
rivatives with a guanidine group and with a substituted
amidine with lipophilic R groups of increasing hydro-
phobicity to mimic the side chain of the Ile8 residue.
Resu lts
Binding affinities of the synthesized compounds in
comparison with that of standard compounds (MPCB,
U50,488, and dynorphin A(1-8)) are summarized in
Tables 2 and 3.
The series with the dipeptide spacer Gly-Leu exhib-
ited the best values for κ binding affinity (Table 2). In
this series, compounds 1-4, without N-substitution on
the diamine, displayed a positive trend from compound
Ch em istr y
All of the compounds 1-40 listed in Figure 3 were
synthesized according to Scheme 1. The following com-
pounds were synthesized by published procedures: 2(R)-
[2-(carbobenzoxy)aminoacetylamino]-4-methylpentano-
ic acid,24 2(R)-(carbobenzoxy)amino-4-methylpentanoic
acid p-nitrophenyl ester,25 N-(tert-butoxycarbonyl)-1,3-

2994 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16
Ronsisvalle et al.
F igu r e 2. Superposition of (A) MPCB or CCB (red) and (B) hybrid compound MPCB-GRRI (red) with dynorphin A(1-8) (blue).
ylene spacer chain, respectively, and a butylamidino-
modified N-terminus.
Similar observations can be made for compounds 21-
40 which possess a Leu residue spacer, where their Kiκ
values were notably higher than the corresponding
values of compounds 1-20 (Table 3).
The binding affinity data of compounds 1-20 and
21-40 are also shown in Figure 4. It is evident that
there is a parallel trend of the κ opioid binding affinity
values of the two series of compounds. Their binding
affinity ratios for homologous compounds range from 1.3
to 15.
The series of compounds 1-20 shows higher values
of µ/κ and δ/κ selectivity than the series 21-40. In
particular, for compounds 13-16, with the butylamidine
modified N-terminus, the Kiµ/Kiκ ratio ranges from 192
to 408 and the Kiδ/Kiκ ratio ranges from 275 to 424.
Selectivity ratios of compounds 1-20 are represented
in Figure 5. Compounds 13-16, exhibiting the most
potent values for κ opioid binding affinity, are also the
compounds showing the greatest selectivity, mainly as
a consequence of their higher κ affinity.
F igu r e 3. Structures of novel synthesized compounds 1-40.
1 (trimethylene spacer chain) to compound 4 (hexam-
ethylene spacer chain) for κ binding affinities. The Kiκ
values ranged from 282.4 to 79.6 nM. Similar analysis
can be made for the other groups of compounds with
N-terminal substitution. Particularly, in the compounds
with alkylamidino substituents, lengthening of the
alkyldiamine chain produced an increase in the κ opioid
binding affinity. Compounds 5-8, with the N-terminal
ethylamidino substituent, have Kiκ values ranging from
256.1 to 46.9 nM, compounds 9-12 (N-terminal propy-
lamidino substituent) between 80.8 and 15.4 nM, and
compounds 13-16 (N-terminal butylamidino substitu-
ent) between 18.0 and 5.3 nM. Compounds 17-20, with
the N-terminal guanidino substituent, have Kiκ values
ranging between 72.5 and 35.5 nM. N-Substitution of
the diamine side chain with alkylamidino or guanidino
substituents in the series of compounds also enhances
κ binding affinities. Moreover, compounds with alkyl-
amidino substituents exhibit parallel increases in bind-
ing affinities with increases in lipophilic character of
the R group. Thus, the greatest κ opioid affinity occurred
in compounds 13-16 with the N-terminal butylamidino
substituent. In conclusion, among the synthesized com-
pounds, the greatest κ opioid affinity is shown by
compounds 15 and 16 (Kiκ ) 6.7 and 5.3 nM, respec-
tively) that possess a pentamethylene and a hexameth-
Since κ agonists may also interact with σ sites,
binding assays for σ1 sites were performed.36 However,
compounds 15 and 16 showed negligible affinity (Ki >
10 000).
Compounds 15, 16, and U50,488 (a selective κ opioid
receptor agonist; obtained from RBI, Natick, MA) were
effective in raising the nociceptive threshold in the
mouse abdominal constriction test after subcutaneous
administration. The nociceptive effect was fully antago-
nized by the selective κ opioid receptor antagonist nor-
BNI (Table 4).
Compounds 15, 16, and U50,488 produced sedation
(locomotor incapacitation), determined by visual obser-
vation and by the ability of mice to maintain their
position at higher doses in the rotarod test than of those
producing a full antinociceptive effect (Table 4). The
ratio of the sedative dose (MPE50) to the antinociceptive
dose (MPE50) was approximately 10. Moreover, com-
pounds 15 and 16 did not cause any stereotyped
behavior or ataxia in the mouse in doses up to 40 mg/
kg, as evaluated adopting the scoring scale described
by Tanaka et al. (data not shown).37

Nonpeptide Analogues of Dynorphin A(1-8)
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16 2995
Sch em e 1a
a
Reagents: (a) DMF; (b) H2, 10% Pd/C, CH3OH/H2O; (c) 1 N NaOH; (d) DCC, HOBt, DMF; (e) 90% TFA, anisole; (f) R-C(dNH)-O-
C2H5‚HCl, DIEA, EtOH (R ) C2H5, C3H7, C4H9); (g) 1-amidino-3,5-dimethylpyrazole‚HNO3, DIEA, EtOH.
Discu ssion
occur in compounds with the pentamethylene and
hexamethylene spacer chains. Nevertheless, the percep-
tion of optimal cation placement is difficult to infer
because it may be overshadowed by the effect of
increased hydrophobicity. It should also be noted that
the lipophilicity of these linear chains might favor the
interaction with the receptor. N-Substitution of the
diamine side chain with alkylamidino or guanidino
substituents is optimal with regard to κ binding affinity,
reflecting the possibility for a high basic fragment to
efficiently dock to κ opioid receptors. In fact, compounds
3 and 4 and the relative alkylamidino or guanidino
derivatives display binding affinity ratios ranging from
3.6 to 36.7 (pentamethylene spanner chain) and from
1.7 to 15.0 (hexamethylene spanner chain). It is evident
that the lipophilic character of the R group of alkyla-
midino substituents increases both κ binding affinity
and selectivity. In fact, butylamidino compounds 15 and
16 display 7.1 and 6.7 times higher κ binding affinity
than the guanidino compounds 19 and 20, respectively.
The introduction of the Gly-Leu spacer in compounds
1-20 results in an increase in κ binding affinity and
selectivity, as compared to compounds 21-40 which lack
the Gly residue. These data present evidence that the
Gly-Leu spacer allows for a better alignment of the
cation-functionalized peptide fragments designed to
mimic the “address” segment of the κ-selective dynor-
phins with the κ receptor.
Comparison of the binding profiles of compounds
1-20 with that of the hybrid compounds MPCB-GRRI
and MPCB-RRI suggests that the presence of only one
mimetic structure of the two arginine residues is suf-
ficient to maintain binding affinity and selectivity for
the κ receptor. This result may be well-correlated to
previous SAR studies that have identified Arg7 as the
critical basic residue in dynorphin A(1-8).21,22
For Arg-mimicking structures, the length of the linear
diamine is important for the binding properties of
compounds 1-20 of which the highest affinity values

2996 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16
Ta ble 1. Analytical Properties of Compounds 1-40
Ronsisvalle et al.
TLC Rf valuesa
HPLCb (tR)
no.
X
n
M
R
I
II
1 (min)
2 (min)
FAB-MS
1
2
3
4
5
6
7
8
9
Gly-Leu
Gly-Leu
Gly-Leu
Gly-Leu
Gly-Leu
Gly-Leu
Gly-Leu
Gly-Leu
Gly-Leu
Gly-Leu
Gly-Leu
Gly-Leu
Gly-Leu
Gly-Leu
Gly-Leu
Gly-Leu
Gly-Leu
Gly-Leu
Gly-Leu
Gly-Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
Leu
3
4
5
6
3
4
5
6
3
4
5
6
3
4
5
6
3
4
5
6
3
4
5
6
3
4
5
6
3
4
5
6
3
4
5
6
3
4
5
6
nil
nil
nil
nil
H
H
H
H
0.35
0.36
0.37
0.38
0.40
0.41
0.42
0.43
0.43
0.44
0.45
0.46
0.46
0.47
0.48
0.49
0.38
0.39
0.40
0.41
0.43
0.44
0.45
0.46
0.46
0.47
0.48
0.49
0.47
0.48
0.50
0.52
0.50
0.51
0.52
0.53
0.46
0.47
0.48
0.50
0.60
0.59
0.58
0.57
0.56
0.55
0.54
0.53
0.54
0.53
0.52
0.51
0.51
0.50
0.49
0.48
0.58
0.57
0.56
0.55
0.57
0.56
0.55
0.54
0.54
0.53
0.52
0.51
0.51
0.50
0.49
0.48
0.49
0.48
0.47
0.46
0.55
0.54
0.52
0.50
2.30
2.40
2.45
2.50
2.40
2.50
2.70
2.85
2.60
2.70
2.80
2.95
2.85
3.00
3.10
3.20
2.45
2.50
2.55
2.70
2.55
2.60
2.70
2.90
2.85
2.90
3.00
3.20
2.90
3.00
3.30
3.70
3.30
3.60
3.70
3.90
2.80
2.85
2.90
3.15
2.94
2.97
3.00
3.12
3.15
3.35
3.48
3.59
3.38
3.47
3.57
3.80
3.49
3.67
3.88
3.93
3.04
3.18
3.30
3.47
3.53
3.67
3.88
3.96
3.62
3.80
4.00
4.20
3.98
4.07
4.20
4.43
4.21
4.30
4.50
4.64
3.62
3.75
3.88
4.15
618 [M + H]+
632 [M + H]+
646 [M + H]+
660 [M + H]+
673 [M + H]+
687 [M + H]+
701 [M + H]+
715 [M + H]+
687 [M + H]+
701 [M + H]+
715 [M + H]+
729 [M + H]+
701 [M + H]+
715 [M + H]+
729 [M + H]+
743 [M + H]+
660 [M + H]+
674 [M + H]+
688 [M + H]+
702 [M + H]+
561 [M + H]+
575 [M + H]+
589 [M + H]+
603 [M + H]+
616 [M + H]+
630 [M + H]+
644 [M + H]+
658 [M + H]+
630 [M + H]+
644 [M + H]+
658 [M + H]+
672 [M + H]+
644 [M + H]+
658 [M + H]+
672 [M + H]+
686 [M + H]+
603 [M + H]+
617 [M + H]+
631 [M + H]+
645 [M + H]+
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
nil
C2H5
C2H5
C2H5
C2H5
C3H7
C3H7
C3H7
C3H7
C4H9
C4H9
C4H9
C4H9
NH2
NH2
NH2
NH2
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
nil
nil
nil
H
H
H
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C2H5
C2H5
C2H5
C2H5
C3H7
C3H7
C3H7
C3H7
C4H9
C4H9
C4H9
C4H9
NH2
NH2
NH2
NH2
Leu
Leu
Leu
a
b
Solvent systems: I, 1-butanol/water/acetic acid (45:45:10); II, acetonitrile/water/TFA (50:50:2). 1 and 2 refer to the HPLC systems
as described in the Experimental Section.
Moreover, compound 16 also show µ/κ and δ/κ selectivity
ratios 4.3 and 5.0 times higher than the corresponding
guanidino compound 20.
doses. These effects are fully inhibited by the selective
κ opioid receptor antagonist nor-BNI.39
Contreras et al.40 have observed that several benzo-
morphan derivatives cause stereotyped behavior and
ataxia in rodents by interacting with binding sites other
than opioid receptors. Compounds 15 and 16 do not
cause these effects in the mouse; therefore, it is unlikely
that they may bind to σ or phencyclidine sites to induce
psychotomimetic effects.
Compounds 15 and 16 show higher affinity and
similar selectivity with respect to the hybrid compounds
MPCB-RRI and MPCB-GRRI and also to dynorphin
A(1-8). Compounds 15 and 16 also have affinity and
selectivity comparable to that of the standard compound
U50,488. A representative superposition of compound
16 and the previously reported peptidic analogue MPCB-
GRRI is presented in Figure 6.
In conclusion, compounds 15 and 16 have κ binding
affinities higher than dynorphin A(1-8) and κ selectivity
higher than dynorphin A(1-17). Moreover, all the
compounds derived from nonpeptide ligands MPCB and
CCB,18,19 i.e., hybrid compounds MPCB-GRRI and
MPCB-RRI20 and compounds 15 and 16, show µ/κ and
δ/κ selectivity ratios higher than those shown by κ
endogenous ligands (µ/κ and δ/κ values higher than 42.4
and higher than 407.8 with respect to 10.9 and 1.9 for
dynorphin A(1-8) and 3.38 and 6.8 for dynorphin A(1-
Compounds 15 and 16 also show very low affinity for
σ1 binding sites. Therefore, contrary to other benzomor-
phan derivatives,36,38 compounds 15 and 16 are capable
of recognizing κ opioid receptors from σ1 binding sites.
Compounds 15 and 16 display typical in vivo profiles
as that of the κ opioid receptor agonist U50,488. In fact,
both compounds effectively increase the nociceptive
threshold and exhibit locomotor impairment at higher

Nonpeptide Analogues of Dynorphin A(1-8)
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16 2997
Ta ble 2. Binding Affinities and Selectivities of Compounds 1-20 toward κ, µ, and δ Opioid Receptors
Ki ( SEM (nM)a,b
selectivity ratio
no.
n
M
R
κ
µ
δ
µ/κ
δ/κ
MPCBc
240 ( 39
54.3 ( 8.5
78.4 ( 6.4
282.4 ( 19.7
269.6 ( 13.5
246.0 ( 12.3
79.6 ( 5.6
256.1 ( 20.5
175.6 ( 8.8
68.4 ( 4.1
46.9 ( 3.3
80.8 ( 4.8
67.8 ( 4.7
65.3 ( 3.3
15.4 ( 0.9
18.0 ( 1.1
10.2 ( 0.6
6.7 ( 0.4
>25000
>25000
ncd
42.36
58.67
57.0
49.1
44.4
101.4
42.5
42.6
70.5
89.8
132.8
100.1
110.1
124.9
192.1
325.3
375.0
408.0
127.5
91.7
81.1
94.3
142.9
10.9
ncd
MPCB-GRRIe
2300 ( 356
4600 ( 817
>25000
ncd
MPCB-RRIe
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
>25000
ncd
3
4
5
6
3
4
5
6
3
4
5
6
3
4
5
6
3
4
5
6
nil
nil
nil
nil
H
H
H
H
16100 ( 805.0
13230 ( 661.5
10920 ( 567.8
8070 ( 468.1
10890 ( 620.7
7480 ( 433.8
4820 ( 250.6
4210 ( 214.7
10730 ( 579.4
6780 ( 372.9
7190 ( 359.5
2270 ( 120.3
3460 ( 197.2
3310 ( 172.1
2530 ( 136.6
2150 ( 111.8
9240 ( 517.4
6160 ( 334.9
3850 ( 204.0
3350 ( 170.8
716 ( 37.9
10610 ( 604.7
10450 ( 543.4
9580 ( 507.7
6050 ( 326.7
10370 ( 591.1
9700 ( 514.1
5580 ( 284.6
5060 ( 268.2
9540 ( 534.2
7280 ( 393.1
5210 ( 270.9
2430 ( 136.0
4950 ( 267.3
3660 ( 204.9
2750 ( 145.7
2230 ( 113.7
3720 ( 200.8
3500 ( 185.5
3480 ( 117.5
2990 ( 155.5
8100 ( 162.0
240.7 ( 12.5
37.6
38.7
38.9
76.0
40.5
55.2
81.6
107.9
118.1
107.4
79.8
157.9
275.0
359.2
407.8
423.8
51.4
52.1
73.1
84.2
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C2H5
C2H5
C2H5
C2H5
C3H7
C3H7
C3H7
C3H7
C4H9
C4H9
C4H9
C4H9
NH2
16
17
18
19
5.3 ( 0.3
72.5 ( 5.1
67.2 ( 4.0
47.5 ( 2.4
35.5 ( 2.1
5.01 ( 0.3
123.8 ( 6.2
NH2
NH2
NH2
20
U50,488
Dyn A(1-8)
1616
1.9
1350 ( 71.5
a
Values are the mean of three separate experiments each carried out in duplicate. b Ki values were obtained as [3H]U69,593 displacement
for κ receptor and [3H]diprenorphine displacement for µ and δ receptors. c Ronsisvalle et al.18 nc ) not calculated. e Ronsisvalle et al.20
d
Ta ble 3. Binding Affinities and Selectivities of Compounds 21-40 toward κ, µ, and δ Opioid Receptors
Ki ( SEM (nM)a,b
selectivity ratio
µ/κ δ/κ
no.
n
M
R
κ
µ
δ
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
3
4
5
6
3
4
5
6
3
4
5
6
3
4
5
6
3
4
5
6
nil
nil
nil
nil
H
H
H
H
367.3 ( 22.0
382.1 ( 19.7
276.5 ( 14.6
242.1 ( 14.7
254.1 ( 15.2
191.5 ( 10.9
149.0 ( 10.4
116.5 ( 6.5
237.6 ( 16.1
203.5 ( 10.3
155.8 ( 8.72
121.9 ( 6.94
138.9 ( 8.6
116.1 ( 6.6
98.1 ( 5.5
4220 ( 223.6
3400 ( 193.8
3250 ( 169.0
3080 ( 308.0
4980 ( 278.8
2200 ( 118.8
2210 ( 112.7
2940 ( 149.9
3570 ( 192.7
2280 ( 120.8
2000 ( 108.0
1900 ( 108.3
2230 ( 124.8
2130 ( 112.8
2380 ( 128.5
2260 ( 117.5
2430 ( 131.2
2950 ( 156.3
2790 ( 159.0
2630 ( 136.7
ndc
ndc
ndc
11.5
8.9
ndc
ndc
ndc
11.8
12.7
19.6
11.5
14.8
25.3
15.0
11.2
12.8
15.6
16.0
18.3
24.3
27.9
8.7
3420 ( 181.2
14.1
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C(dNH)
C2H5
C2H5
C2H5
C2H5
C3H7
C3H7
C3H7
C3H7
C4H9
C4H9
C4H9
C4H9
NH2
ndc
ndc
ndc
ndc
ndc
ndc
3550 ( 191.7
30.5
ndc
ndc
ndc
ndc
ndc
ndc
3460 ( 179.9
28.4
ndc
ndc
ndc
ndc
3590 ( 186.6
3260 ( 177.3
ndc
36.6
40.2
81.0 ( 4.7
279.7 ( 16.5
249.5 ( 13.2
216.1 ( 12.3
138.9 ( 7.3
ndc
NH2
NH2
NH2
ndc
11.8
12.9
18.9
ndc
ndc
ndc
2830 ( 155.6
20.3
a
Values are the mean of three separate experiments each carried out in duplicate. b Ki values were obtained as [3H]U69,593 displacement
for κ receptor and [3H]diprenorphine displacement for µ and δ receptors. c nd ) not determined.
17),20 respectively). These results further support the
hypothesis that the κ-selective nonpeptide ligands MPCB
and CCB already have all the structural and conforma-
tional requirements permitting a κ-selective interaction.
The attachment of a moiety mimicking the C-terminal
fragment of dynorphin A(1-8) does not reduce κ binding
affinity and maintains high κ preference, thus support-
ing the idea that these molecules might interact with κ
opioid receptors possibly occupying binding sites very
close to that of endogenous ligands. Compounds 15 and

2998 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16
Ronsisvalle et al.
F igu r e 4. κ opioid binding affinities of compounds 1-20 (dotted tonality) and compounds 21-40 (gray tonality).
F igu r e 5. Selectivity ratios Kiµ/Kiκ (dotted tonality) and Kiδ/Kiκ (gray tonality) of compounds 1-20.
Ta ble 4. Antinociceptive and Sedative Activity of Compounds 15, 16, and U50,488 in the Mouse
ED50 (95% confidence limits), mg/kg sc
rotarod value MPE50
/
compound
antinociceptive activity
rotarod value
antinocicpetive activity MPE50
15
0.88 (0.5-1.3)
a
9.2 (4.5-13.8)
a
10.45
15 + nor-BNI (10 mg/kg; administered
10 min before agonist)
16
1.1 (0.6-1.5)
11.9 (7.0-18.7)
10.81
10.78
16 + nor-BNI (10 mg/kg; administered
a
a
10 min before agonist)
U50,488
0.76 (0.4-1.1)
a
8.2 (3.7-11.4)
a
U50,488 + nor-BNI (10 mg/kg; administered
10 min before agonist)
a
Full antagonism of the pharmacological effect produced by the ED50 dose of the agonist.
determined by FAB-MS on a Finnigan MHT 90 mass spec-
trometer. Analytical RP-HPLC was performed on a Waters
model 600E, operating at a flow rate of 1.5 mL/min using a
Lichrosorb RP-18 10 µm column, and monitoring at 280 and
220 nm with a Knauer UV/VIS filter photomer. The HPLC
solvent systems were (1) an isocratic system of 80% A and 20%
B, where solvent A is 10% water in acetonitrile with 0.1% TFA
and solvent B is 10% acetonitrile in water with 0.1% TFA, and
(2) a linear gradient system of 30% A and 70% B to 0% A and
100% B over 10 min, where solvent A is water with 0.1% TFA
and solvent B is acetonitrile with 0.1% TFA. Melting points
were determined on a Bu¨chi 530 capillary apparatus and are
uncorrected. Analytical thin-layer chromatography (TLC) was
performed on precoated silica gel 60 RP-18 F254 plates (Merck)
and precoated silica gel F254 plates (Merck). Merck silica gel
16, therefore, allowing to identify the functionalities
relevant in the C-terminal region, provide insights for
future design of peptidomimetics of dynorphin A.
Exp er im en ta l Section
Reagents were purchased from Aldrich Chemical Co. unless
otherwise indicated. NMR spectra were recorded on a Varian
Inova-200 spectrometer, using tetramethylsilane (TMS) as an
internal standard. Infrared spectra were recorded on 1600
FTIR Perkin-Elmer instruments and are consistent with the
assigned structures. Elemental analyses were measured on an
elemental analyzer (model 1106, Carlo Erba) and were within
0.4% of the theoretical values. Mass spectra (EI) were recorded
on a Kratos 2S RFA spectrometer using a Tektroniks 4205
computer. Molecular weights of the final products were

Nonpeptide Analogues of Dynorphin A(1-8)
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16 2999
+ Leu-CγH), 3.14-3.06 (2H, m, Leu-NH-CH2), 3.26-3.19 (2H,
m, CH2-NH-Boc), 3.81 (1H, dd, Gly-CHa, J ) 5.4, 17.0 Hz),
3.93 (1H, dd, Gly-CHb, J ) 6.0, 17.0 Hz), 4.50-4.39 (1H, m,
Leu-CRH), 4.75 (1H, br s, Leu-NH), 5.11 (2H, s, Cbz-CH2), 5.85
(1H, br s, Gly-NRH), 6.59 (1H, br s, NH-Boc), 6.83 (1H, d, Leu-
NRH, J ) 8.0 Hz), 7.34-7.27 (m, 5H, Cbz-aromatic-H); 13C
NMR (CDCl3) δ 22.0, 22.8, 23.8, 24.7, 28.4, 28.8, 29.5, 39.2,
40.3, 40.8, 44.5, 51.8, 67.2, 79.1, 128.0, 128.2, 128.5, 136.1,
156.1, 156.8, 169.2, 171.7; MS m/z (EI) 506 [M]+. Anal.
(C26H42N4O6) C, H, N.
N-[6-(ter t-Bu t oxyca r b on yl)a m in oh exyl]-2(R)-[2-(ca r -
b ob en zoxy)a m in oa cet yla m in o]-4-m et h ylp en t a n a m id e
1
(44): 89% yield; mp 99-101 °C; H NMR (CDCl3) δ 0.89 (3H,
d, Leu-CH3, J ) 5.8 Hz), 0.91 (3H, d, Leu-CH3, J ) 5.8 Hz),
1.33-1.21 (8H, m, -(CH2)4-), 1.43 (9H, s, Boc-CH3), 1.77-1.40
(3H, m, Leu-CâH2 + Leu-CγH), 3.13-3.03 (2H, m, Leu-NH-
CH2), 3.24-3.15 (2H, m, CH2-NH-Boc), 3.81 (1H, dd, Gly-CHa,
J ) 5.4, 17.0 Hz), 3.93 (1H, dd, Gly-CHb, J ) 6.0, 17.0 Hz),
4.48-4.37 (1H, m, Leu-CRH), 4.65 (1H, br s, Leu-NH), 5.12
(2H, s, Cbz-CH2), 5.76 (1H, br s, Gly-NRH), 6.49 (1H, br s, NH-
Boc), 6.70 (1H, d, Leu-NRH, J ) 8.0 Hz), 7.34-7.28 (m, 5H,
Cbz-aromatic-H); 13C NMR (CDCl3) δ 22.0, 22.8, 24.7, 25.8,
28.4, 29.0, 29.7, 39.1, 40.0, 40.9, 44.6, 51.7, 67.2, 79.1, 128.0,
128.2, 128.5, 136.1, 156.1, 156.7, 169.1, 171.6; MS m/z (EI) 520
[M]+. Anal. (C27H44N4O6) C, H, N.
The following compounds 45-48 were prepared from 2(R)-
(carbobenzoxy)amino-4-methylpentanoic acid p-nitrophenyl
ester and N-(tert-butoxycarbonyl)-1,3-diaminopropane, N-(tert-
butoxycarbonyl)-1,4-diaminobutane, N-(tert-butoxycarbonyl)-
1,5-diaminopentane, and N-(tert-butoxycarbonyl)-1,6-diami-
nohexane, respectively, following the same methodology as
that described for compound 41.
F igu r e 6. Superposition of novel synthesized compound 16
(red) with MPCB-GRRI (blue).
60 (mesh 230-400) was used for flash column chromatogra-
phy. All compounds exhibited NMR, IR, and MS spectral data
consistent with those of the assigned structures.
N-[3-(ter t-Bu toxyca r bon yl)a m in op r op yl]-2(R)-[2-(ca r -
b ob en zoxy)a m in oa cet yla m in o]-4-m et h ylp en t a n a m id e
(41). To a solution of N-(tert-butoxycarbonyl)-1,3-diaminopro-
pane (575 mg, 3.3 mmol) in DMF (3 mL), cooled at 0 °C, was
added 2(R)-[2-(carbobenzoxy)aminoacetylamino]-4-methylpen-
tanoic acid p-nitrophenyl ester (1.33 g, 3 mmol) portionwise
over 1 h. The reaction mixture was then stirred for 5 h at room
temperature and the residue was dissolved in EtOAc and
washed consecutively with 5% citric acid, 5% NaHCO3 and
brine. The organic layer was separated and dried over anhy-
drous Na2SO4 and evaporated to dryness to give 1.19 g of the
pure desired compound 41 as a white solid: 83% yield; mp
113-115 °C; 1H NMR (CDCl3) δ 0.89 (3H, d, Leu-CH3, J ) 5.8
Hz), 0.91 (3H, d, Leu-CH3, J ) 5.8 Hz), 1.40 (9H, s, Boc-CH3),
1.75-1.49 (5H, m, CH2 + Leu-CâH2 + Leu-CγH), 3.16-3.06
(2H, m, Leu-NH-CH2), 3.29-3.20 (2H, m, CH2-NH-Boc), 3.82
(1H, dd, Gly-CHa, J ) 5.0, 17.0 Hz), 4.03 (1H, dd, Gly-CHb, J
) 6.2, 17.0 Hz), 4.53-4.41 (1H, m, Leu-CRH), 4.96 (1H, br s,
Leu-NH), 5.12 (2H, s, Cbz-CH2), 5.99 (1H, br s, Gly-NRH), 6.69
(1H, d, Leu-NRH, J ) 8.0 Hz), 7.11 (1H, br s, NH-Boc), 7.37-
7.30 (m, 5H, Cbz-aromatic-H); 13C NMR (CDCl3) δ 21.8, 22.9,
24.8, 28.4, 29.8, 35.5, 36.9, 40.8, 44.6, 51.8, 67.2, 79.5, 128.0,
128.2, 128.5, 136.1, 156.2, 156.7, 169.4, 171.9; MS m/z (EI) 478
[M]+. Anal. (C24H38N4O6) C, H, N.
The following compounds 42-44 were prepared from 2(R)-
[2-(carbobenzoxy)aminoacetylamino]-4-methylpentanoic acid
p-nitrophenyl ester and N-(tert-butoxycarbonyl)-1,4-diami-
nobutane, N-(tert-butoxycarbonyl)-1,5-diaminopentane, and
N-(tert-butoxycarbonyl)-1,6-diaminohexane, respectively, fol-
lowing the same methodology as that described for compound
41.
N-[4-(ter t-Bu t oxyca r b on yl)a m in ob u t yl]-2(R)-[2-(ca r -
b ob en zoxy)a m in oa cet yla m in o]-4-m et h ylp en t a n a m id e
(42): 80% yield; mp 112-114 °C; 1H NMR (CDCl3) δ 0.89 (3H,
d, Leu-CH3, J ) 5.8 Hz), 0.90 (3H, d, Leu-CH3, J ) 5.8 Hz),
1.42 (9H, s, Boc-CH3), 1.74-1.40 (7H, m, -(CH2)2- + Leu-CâH2
+ Leu-CγH), 3.13-3.06 (2H, m, Leu-NH-CH2), 3.29-3.21 (2H,
m, CH2-NH-Boc), 3.81 (1H, dd, Gly-CHa, J ) 5.4, 17.0 Hz),
3.92 (1H, dd, Gly-CHb, J ) 6.0, 17.0 Hz), 4.53-4.40 (1H, m,
Leu-CRH), 4.83 (1H, br s, Leu-NH), 5.11 (2H, s, Cbz-CH2), 5.90
(1H, br s, Gly-NRH), 6.82 (1H, br s, NH-Boc), 6.92 (1H, d, Leu-
NRH, J ) 8.0 Hz), 7.34-7.29 (m, 5H, Cbz-aromatic-H); 13C
NMR (CDCl3) δ 22.0, 22.8, 24.7, 26.3, 27.4, 28.4, 39.1, 40.1,
40.9, 44.5, 51.8, 67.2, 79.2, 128.0, 128.2, 128.5, 136.1, 156.2,
156.8, 169.3, 171.8; MS m/z (EI) 492 [M]+. Anal. (C25H40N4O6)
C, H, N.
N -[3-(t er t -Bu t oxyca r b on yl)a m in op r op yl]-2(R )-(ca r -
boben zoxy)a m in o-4-m eth ylp en ta n a m id e (45): 84% yield;
1
mp 118-119 °C; H NMR (CDCl3) δ 0.94 (6H, d, Leu-CH3, J
) 6.0 Hz), 1.43 (9H, s, Boc-CH3), 1.75-1.49 (5H, m, CH2
+
Leu-CâH2 + Leu-CγH), 3.16-3.04 (2H, m, Leu-NH-CH2), 3.33-
3.24 (2H, m, CH2-NH-Boc), 4.22-4.11 (1H, m, Leu-CRH), 4.95
(1H, br s, Leu-NH), 5.10 (2H, s, Cbz-CH2), 5.30 (1H, d, Leu-
NRH, J ) 7.8 Hz), 6.79 (1H, br s, NH-Boc), 7.37-7.29 (m, 5H,
Cbz-aromatic-H); 13C NMR (CDCl3) δ 21.9, 22.9, 24.7, 28.4,
30.0, 35.8, 36.8, 41.7, 53.7, 67.0, 79.3, 128.0, 128.1, 128.5, 136.2,
156.1, 156.5, 172.5; MS m/z (EI) 421 [M]+. Anal. (C22H35N3O5)
C, H, N.
N-[4-(ter t-Bu toxycar bon yl)am in obu tyl]-2(R)-(car boben -
zoxy)a m in o-4-m eth ylp en ta n a m id e (46): 88% yield; mp
117-119 °C; 1H NMR (CDCl3) δ 0.93 (6H, d, Leu-CH3, J ) 6.0
Hz), 1.43 (9H, s, Boc-CH3), 1.70-1.40 (7H, m, -(CH2)2- + Leu-
CâH2 + Leu-CγH), 3.16-3.06 (2H, m, Leu-NH-CH2), 3.29-3.20
(2H, m, CH2-NH-Boc), 4.21-4.10 (1H, m, Leu-CRH), 4.66 (1H,
br s, Leu-NH), 5.10 (2H, s, Cbz-CH2), 5.34 (1H, d, Leu-NRH, J
) 7.8 Hz), 6.40 (1H, br s, NH-Boc), 7.37-7.29 (m, 5H, Cbz-
aromatic-H); 13C NMR (CDCl3) δ 21.9, 22.9, 24.7, 26.3, 27.5,
28.4, 39.1, 39.9, 41.4, 53.6, 67.0, 79.2, 128.0, 128.2, 128.5, 136.1,
156.1, 156.2, 172.2; MS m/z (EI) 435 [M]+. Anal. (C23H37N3O5)
C, H, N.
N -[5-(t er t -Bu t oxyca r b on yl)a m in op e n t yl]-2(R )-(ca r -
boben zoxy)a m in o-4-m eth ylp en ta n a m id e (47): 89% yield;
1
mp 109-110 °C; H NMR (CDCl3) δ 0.93 (6H, d, Leu-CH3, J
) 6.0 Hz), 1.43 (9H, s, Boc-CH3), 1.70-1.25 (9H, m, -(CH2)3-
+ Leu-CâH2 + Leu-CγH), 3.14-3.05 (2H, m, Leu-NH-CH2),
3.25-3.16 (2H, m, CH2-NH-Boc), 4.20-4.09 (1H, m, Leu-CRH),
4.70 (1H, br s, Leu-NH), 5.10 (2H, s, Cbz-CH2), 5.30 (1H, d,
Leu-NRH, J ) 7.8 Hz), 6.24 (1H, br s, NH-Boc), 7.37-7.29 (m,
5H, Cbz-aromatic-H); 13C NMR (CDCl3) δ 21.9, 22.9, 23.8, 24.7,
28.4, 29.0, 29.4, 39.1, 40.3, 41.4, 53.6, 67.0, 79.1, 128.0, 128.2,
128.5, 136.1, 156.0, 156.3, 172.1; MS m/z (EI) 449 [M]+. Anal.
(C24H39N3O5) C, H, N.
N-[6-(ter t-Bu toxycar bon yl)am in oh exyl]-2(R)-(car boben -
zoxy)a m in o-4-m eth ylp en ta n a m id e (48): 85% yield; mp
108-111 °C; 1H NMR (CDCl3) δ 0.93 (6H, d, Leu-CH3, J ) 6.0
Hz), 1.33-1.25 (8H, m, -(CH2)4-), 1.43 (9H, s, Boc-CH3), 1.70-
1.25 (3H, m, Leu-CâH2 + Leu-CγH), 3.14-3.04 (2H, m, Leu-
NH-CH2), 3.26-3.17 (2H, m, CH2-NH-Boc), 4.19-4.09 (1H, m,
N-[5-(ter t-Bu toxyca r bon yl)a m in op en tyl]-2(R)-[2-(ca r -
b ob en zoxy)a m in oa cet yla m in o]-4-m et h ylp en t a n a m id e
(43): 85% yield; mp 112-113 °C; 1H NMR (CDCl3) δ 0.89 (3H,
d, Leu-CH3, J ) 5.8 Hz), 0.91 (3H, d, Leu-CH3, J ) 5.8 Hz),
1.42 (9H, s, Boc-CH3), 1.76-1.21 (9H, m, -(CH2)3- + Leu-CâH2

3000 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16
Ronsisvalle et al.
Leu-CRH), 4.60 (1H, br s, Leu-NH), 5.10 (2H, s, Cbz-CH2), 5.27
(1H, d, Leu-NRH, J ) 7.8 Hz), 6.24 (1H, br s, NH-Boc), 7.37-
7.29 (m, 5H, Cbz-aromatic-H); 13C NMR (CDCl3) δ 21.9, 22.9,
24.7, 25.9, 28.4, 29.2, 29.8, 39.1, 40.1, 41.4, 53.6, 67.0, 79.1,
128.0, 128.2, 128.5, 136.1, 156.0, 156.2, 172.0; MS m/z (EI) 463
[M]+. Anal. (C25H41N3O5) C, H, N.
0.93 (3H, d, Leu-CH3, J ) 6.0 Hz), 0.96 (3H, d, Leu-CH3, J )
6.0 Hz), 1.44 (9H, s, Boc-CH3), 1.75-1.35 (7H, m, -(CH2)2- +
Leu-CâH2 + Leu-CγH), 1.90 (2H, br s, NH2), 3.17-3.10 (2H,
m, Leu-NH-CH2), 3.30-3.24 (2H, m, CH2-NH-Boc), 3.43-3.38
(1H, m, Leu-CRH), 4.62 (1H, br s, Leu-NH), 7.41 (1H, br s, NH-
Boc); 13C NMR (CDCl3) δ 22.1, 23.2, 24.5, 26.2, 27.3, 28.4, 38.3,
39.5, 44.5, 53.0, 77.5, 155.9, 175.6; retention time (tR) (HPLC
system 1) 2.10 min.
N-[3-(ter t-Bu toxyca r bon yl)a m in op r op yl]-2(R)-(2-a m i-
n oa cetyla m in o)-4-m eth ylp en ta n a m id e (49). A mixture of
41 (850 mg, 1.77 mmol) and 10% Pd-C (212.5 mg) in MeOH
(15 mL) and water (0.75 mL) was allowed to stir under
hydrogen (1 atm) at room temperature for 45 min. The catalyst
was filtered and the filtrate was evaporated and dried to give
573.1 mg of the desired product 49 as a pale yellow oil: 94%
N-[5-(ter t-Bu toxyca r bon yl)a m in op en tyl]-2(R)-a m in o-
1
4-m eth ylp en ta n a m id e (55): 95% yield; H NMR (CDCl3) δ
0.94 (3H, d, Leu-CH3, J ) 6.0 Hz), 0.97 (3H, d, Leu-CH3, J )
6.0 Hz), 1.45 (9H, s, Boc-CH3), 1.75-1.25 (9H, m, -(CH2)3- +
Leu-CâH2 + Leu-CγH), 1.83 (2H, br s, NH2), 3.16-3.07 (2H,
m, Leu-NH-CH2), 3.30-3.20 (2H, m, CH2-NH-Boc), 3.43-3.38
(1H, m, Leu-CRH), 4.60 (1H, br s, Leu-NH), 7.34 (1H, br s, NH-
Boc); 13C NMR (CDCl3) δ 22.1, 23.3, 23.8, 24.3, 28.4, 28.9, 29.3,
38.3, 39.9, 44.4, 53.0, 77.6, 155.8, 175.5; retention time (tR)
(HPLC system 1) 2.20 min.
1
yield; H NMR (CDCl3) δ 0.92 (3H, d, Leu-CH3, J ) 5.6 Hz),
0.94 (3H, d, Leu-CH3, J ) 5.6 Hz), 1.43 (9H, s, Boc-CH3), 1.75-
1.41 (5H, m, CH2 + Leu-CâH2 + Leu-CγH), 2.18 (2H, br s, NH2),
3.16-3.05 (2H, m, Leu-NH-CH2), 3.27-3.20 (2H, m, CH2-NH-
Boc), 3.44 (2H, s, Gly-CH2), 4.48-4.40 (1H, m, Leu-CRH), 4.80
(1H, br s, Leu-NH), 6.83 (1H, br s, NH-Boc), 7.71 (1H, d, Leu-
NRH, J ) 8.0 Hz); 13C NMR (CDCl3) δ 22.1, 22.8, 24.7, 28.4,
29.7, 35.5, 36.8, 40.7, 44.5, 51.4, 79.2, 156.1, 172.1, 173.0;
retention time (tR) (HPLC system 1) 1.85 min.
N-[6-(ter t-Bu toxyca r bon yl)a m in oh exyl]-2(R)-a m in o-4-
m eth ylp en ta n a m id e (56): 95% yield; 1H NMR (CDCl3) δ
0.94 (3H, d, Leu-CH3, J ) 6.0 Hz), 0.97 (3H, d, Leu-CH3, J )
6.0 Hz), 1.35-1.20 (8H, m, -(CH2)4-), 1.44 (9H, s, Boc-CH3),
1.75-1.40 (3H, m, Leu-CâH2 + Leu-CγH), 1.80 (2H, br s, NH2),
3.14-3.05 (2H, m, Leu-NH-CH2), 3.29-3.18 (2H, m, CH2-NH-
Boc), 3.41-3.36 (1H, m, Leu-CRH), 4.58 (1H, br s, Leu-NH),
7.30 (1H, br s, NH-Boc); 13C NMR (CDCl3) δ 22.1, 23.3, 24.3,
25.8, 28.4, 29.2, 29.7, 38.0, 39.8, 44.4, 53.0, 77.6, 155.8, 175.4;
retention time (tR) (HPLC system 1) 2.30 min.
Compounds 50-52 were prepared from compounds 42-44,
respectively, following the same methodology as that described
for compound 49.
N-[4-(ter t-Bu t oxyca r b on yl)a m in ob u t yl]-2(R)-(2-a m i-
n oa cetyla m in o)-4-m eth ylp en ta n a m id e (50): 92% yield; 1H
NMR (CDCl3) δ 0.92 (3H, d, Leu-CH3, J ) 5.6 Hz), 0.94 (3H,
d, Leu-CH3, J ) 5.6 Hz), 1.44 (9H, s, Boc-CH3), 1.75-1.38 (7H,
m, -(CH2)2- + Leu-CâH2 + Leu-CγH), 2.16 (2H, br s, NH2),
3.16-3.09 (2H, m, Leu-NH-CH2), 3.26-3.22 (2H, m, CH2-NH-
Boc), 3.39 (2H, s, Gly-CH2), 4.43-4.40 (1H, m, Leu-CRH), 4.74
(1H, br s, Leu-NH), 6.79 (1H, br s, NH-Boc), 7.69 (1H, d, Leu-
NRH, J ) 8.0 Hz); 13C NMR (CDCl3) δ 22.1, 22.8, 24.7, 26.3,
27.3, 28.4, 38.9, 40.0, 40.6, 44.4, 51.4, 79.1, 156.1, 172.0, 172.9;
retention time (tR) (HPLC system 1) 1.90 min.
N-[5-(ter t-Bu toxyca r bon yl)a m in op en tyl]-2(R)-(2-a m i-
n oa cetyla m in o)-4-m eth ylp en ta n a m id e (51): 98% yield; 1H
NMR (CDCl3) δ 0.91 (3H, d, Leu-CH3, J ) 5.6 Hz), 0.94 (3H,
d, Leu-CH3, J ) 5.6 Hz), 1.44 (9H, s, Boc-CH3), 1.75-1.26 (9H,
m, -(CH2)3- + Leu-CâH2 + Leu-CγH), 2.13 (2H, br s, NH2),
3.14-3.04 (2H, m, Leu-NH-CH2), 3.27-3.17 (2H, m, CH2-NH-
Boc), 3.45 (2H, s, Gly-CH2), 4.47-4.36 (1H, m, Leu-CRH), 4.74
(1H, br s, Leu-NH), 6.71 (1H, br s, NH-Boc), 7.73 (1H, d, Leu-
NRH, J ) 8.0 Hz); 13C NMR (CDCl3) δ 22.1, 22.8, 23.8, 24.7,
28.4, 28.9, 29.5, 39.1, 40.3, 40.6, 44.4, 51.4, 79.0, 156.0, 171.9,
172.8; retention time (tR) (HPLC system 1) 1.95 min.
N-[6-(ter t-Bu t oxyca r b on yl)a m in oh exyl]-2(R)-(2-a m i-
n oa cetyla m in o)-4-m eth ylp en ta n a m id e (52): 95% yield; 1H
NMR (CDCl3) δ 0.91 (3H, d, Leu-CH3, J ) 5.6 Hz), 0.94 (3H,
d, Leu-CH3, J ) 5.6 Hz), 1.32-1.20 (8H, m, -(CH2)4-), 1.44 (9H,
s, Boc-CH3), 1.75-1.40 (3H, m, Leu-CâH2 + Leu-CγH), 2.08
(2H, br s, NH2), 3.13-3.04 (2H, m, Leu-NH-CH2), 3.26-3.16
(2H, m, CH2-NH-Boc), 3.40 (2H, s, Gly-CH2), 4.45-4.36 (1H,
m, Leu-CRH), 4.71 (1H, br s, Leu-NH), 6.65 (1H, br s, NH-
Boc), 7.70 (1H, d, Leu-NRH, J ) 8.0 Hz); 13C NMR (CDCl3) δ
22.1, 22.8, 24.7, 25.8, 28.4, 29.0, 29.6, 39.1, 40.2, 40.6, 44.3,
51.3, 79.0, 156.0, 171.8, 172.7; retention time (tR) (HPLC
system 1) 2.00 min.
N-{{1-[3-(ter t-Bu toxycar bon yl)am in opr opylcar bam oyl]-
3-m eth yl-1(R)-bu tylcar bam oyl}m eth yl}-1(S)-ph en yl-2(R)-
[(8-h yd r oxy-6(R),11(R)-d im et h yl-1,2,5,6-t et r a h yd r o-4H -
2(R),6-m et h a n ob en za zocin -3-yl)m et h yl]cyclop r op a n e-
ca r boxa m id e (57) a n d N-{[1-(3-Am in op r op ylca r ba m oyl)-
3-m eth yl-1(R)-bu tylca r ba m oyl]m eth yl}-1(S)-p h en yl-2(R)-
[(8-h yd r oxy-6(R),11(R)-d im et h yl-1,2,5,6-t et r a h yd r o-4H -
2(R),6-m et h a n ob en za zocin -3-yl)m et h yl]cyclop r op a n e-
ca r boxa m id e‚2TF A (1). To a solution of 1(S)-phenyl-2(R)-
[(8-hydroxy-6(R),11(R)-dimethyl-1,2,5,6-tetrahydro-4H-2(R),6-
methanobenzazocin-3-yl)methyl]cyclopropanecarboxylic acid
(563.8 mg, 1.44 mmol) and compound 49 (413.3 mg, 1.2 mmol)
in DMF (5 mL) was added HOBt (201.6 mg, 1.44 mmol). The
reaction mixture was cooled to 0 °C and DCC (297.1 mg, 1.44
mmol) was added. The reaction mixture was kept at 0-5 °C
for 48 h, then the DCU precipitated was filtered and the
filtrate was evaporated to dryness. The residue was dissolved
in EtOAc and washed with H2O, 5% NaHCO3 and brine. The
separated organic layer was dried over anhydrous Na2SO4 and
evaporated to dryness to give compound 57 as a yellow pale
solid, which was used without further purification. The tert-
butoxycarbonyl protecting group was removed by treating
crude compound 57 with aqueous 90% TFA (1:10 mL, w/v) and
anisole (1:100 mL, w/v). The mixture was stirred at room
temperature for 30-40 min, then the solvent was evaporated
in vacuo at 0 °C, and the residue was triturated with n-hexane/
Et2O (2:1). The resulting solid was purified by flash column
chromatography (LiChroprep RP-18, 40-63 µm, eluted with
acetonitrile/water/TFA, 30:70:0.25). The fractions containing
the desired product 1 were collected and lyophilized to constant
weight: 42% yield. Anal. (C36H51N5O4‚2CF3COOH‚3H2O) C,
H, N.
Compounds 53-56 were prepared from compounds 45-48,
respectively, following the same methodology as that described
for compound 49.
Compounds 2-4 were prepared from 1(S)-phenyl-2(R)-[(8-
hydroxy-6(R),11(R)-dimethyl-1,2,5,6-tetrahydro-4H-2(R),6-meth-
anobenzazocin-3-yl)methyl]cyclopropanecarboxylic acid and
compounds 50-52, respectively, following the same methodol-
ogy as that described for compound 1.
N-[3-(ter t-Bu toxyca r bon yl)a m in op r op yl]-2(R)-a m in o-
1
4-m eth ylp en ta n a m id e (53): 91% yield; H NMR (CDCl3) δ
0.93 (3H, d, Leu-CH3, J ) 6.0 Hz), 0.96 (3H, d, Leu-CH3, J )
6.0 Hz), 1.44 (9H, s, Boc-CH3), 1.75-1.44 (5H, m, CH2 + Leu-
CâH2 + Leu-CγH), 1.94 (2H, br s, NH2), 3.18-3.09 (2H, m, Leu-
NH-CH2), 3.32-3.25 (2H, m, CH2-NH-Boc), 3.44-3.35 (1H, m,
Leu-CRH), 4.66 (1H, br s, Leu-NH), 7.53 (1H, br s, NH-Boc);
13C NMR (CDCl3) δ 22.1, 23.2, 24.5, 28.4, 29.9, 35.6, 36.4, 44.6,
53.0, 77.5, 155.9, 175.8; retention time (tR) (HPLC system 1)
1.95 min.
N-{{1-[4-(ter t-Bu toxyca r bon yl)a m in obu tylca r ba m oyl]-
3-m eth yl-1(R)-bu tylcar bam oyl}m eth yl}-1(S)-ph en yl-2(R)-
[(8-h yd r oxy-6(R),11(R)-d im et h yl-1,2,5,6-t et r a h yd r o-4H -
2(R),6-m et h a n ob en za zocin -3-yl)m et h yl]cyclop r op a n e-
ca r boxa m id e (58) a n d N-{[1-(4-Am in obu tylca r ba m oyl)-
3-m e t h yl-1(R )-b u t ylca r b a m oyl]m e t h yl}-1(S )-p h e n yl-
2(R)-[(8-h yd r oxy-6(R),11(R)-d im eth yl-1,2,5,6-tetr a h yd r o-
4 H -2 ( R ) , 6 -m e t h a n o b e n z a z o c i n -3 -y l ) m e t h y l ] -
N-[4-(ter t-Bu toxyca r bon yl)a m in obu tyl]-2(R)-a m in o-4-
m eth ylp en ta n a m id e (54): 93% yield; 1H NMR (CDCl3) δ

Nonpeptide Analogues of Dynorphin A(1-8)
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16 3001
cyclop r op a n eca r b oxa m id e‚2TF A (2): 45% yield. Anal.
(C37H53N5O4‚2CF3COOH‚3H2O) C, H, N.
trituration with diethyl ether to give 18.65 mg of the pure
desired compound 5 as a white solid with a 64% yield.
Compounds 6-8 were prepared from compounds 2-4,
respectively, following the same methodology as that described
for compound 5.
N-{[1-(4-Am id in oeth ylbu tylca r ba m oyl)-3-m eth yl-1(R)-
bu t ylca r ba m oyl]m et h yl}-1(S)-p h en yl-2(R)-[(8-h yd r oxy-
6(R),11(R)-d im et h yl-1,2,5,6-t et r a h yd r o-4H -2(R),6-m et h -
a n ob en za zocin -3-yl)m et h yl]cyclop r op a n eca r b oxa m id e
(6): 62% yield.
N-{[1-(5-Am idin oeth ylpen tylcar bam oyl)-3-m eth yl-1(R)-
b u t ylca r b a m oyl]m et h yl}-1(S)-p h en yl-2(R)-[(8-h yd r oxy-
6(R),11(R)-d im et h yl-1,2,5,6-t et r a h yd r o-4H -2(R),6-m et h -
a n ob en za zocin -3-yl)m et h yl]cyclop r op a n eca r b oxa m id e
(7): 61% yield.
N-{[1-(6-Am id in oeth ylh exylca r ba m oyl)-3-m eth yl-1(R)-
bu t ylca r ba m oyl]m et h yl}-1(S)-p h en yl-2(R)-[(8-h yd r oxy-
6(R),11(R)-d im et h yl-1,2,5,6-t et r a h yd r o-4H -2(R),6-m et h -
a n ob en za zocin -3-yl)m et h yl]cyclop r op a n eca r b oxa m id e
(8): 64% yield.
N-{{1-[5-(ter t-Bu toxycar bon yl)am in open tylcar bam oyl]-
3-m eth yl-1(R)-bu tylcar bam oyl}m eth yl}-1(S)-ph en yl-2(R)-
[(8-h yd r oxy-6(R),11(R)-d im et h yl-1,2,5,6-t et r a h yd r o-4H -
2(R),6-m et h a n ob en za zocin -3-yl)m et h yl]cyclop r op a n e-
ca r boxa m id e (59) a n d N-{[1-(5-Am in op en tylca r ba m oyl)-
3-m eth yl-1(R)-bu tylca r ba m oyl]m eth yl}-1(S)-p h en yl-2(R)-
[(8-h yd r oxy-6(R),11(R)-d im et h yl-1,2,5,6-t et r a h yd r o-4H -
2(R),6-m et h a n ob en za zocin -3-yl)m et h yl]cyclop r op a n e-
ca r boxa m id e‚2TF A (3): 40% yield. Anal. (C38H55N5O4‚2CF3-
COOH‚3H2O) C, H, N.
N-{{1-[6-(ter t-Bu toxyca r bon yl)a m in oh exylca r ba m oyl]-
3-m eth yl-1(R)-bu tylcar bam oyl}m eth yl}-1(S)-ph en yl-2(R)-
[(8-h yd r oxy-6(R),11(R)-d im et h yl-1,2,5,6-t et r a h yd r o-4H -
2(R),6-m et h a n ob en za zocin -3-yl)m et h yl]cyclop r op a n e-
ca r boxa m id e (60) a n d N-{[1-(6-Am in oh exylca r ba m oyl)-
3-m e t h yl-1(R )-b u t ylca r b a m oyl]m e t h yl}-1(S )-p h e n yl-
2(R)-[(8-h yd r oxy-6(R),11(R)-d im eth yl-1,2,5,6-tetr a h yd r o-
4H -2(R ),6-m e t h a n o b e n z a z o c i n -3-y l)m e t h y l]c y c lo -
p r op a n eca r b oxa m id e‚2TF A (4):
50% yield. Anal.
(C39H57N5O4‚2CF3COOH‚3H2O) C, H, N.
Compounds 25-28 were prepared from compounds 21-24,
respectively, following the same methodology as that described
for compound 5.
Compounds 21-24 were prepared from 1(S)-phenyl-2(R)-
[(8-hydroxy-6(R),11(R)-dimethyl-1,2,5,6-tetrahydro-4H-2(R),6-
methanobenzazocin-3-yl)methyl]cyclopropanecarboxylic acid
and compounds 53-56, respectively, following the same
methodology as that described for compound 1.
N-{1-[3-(ter t-Bu toxycar bon yl)am in opr opylcar bam oyl]-
3-m eth yl-1(R)-bu tyl}-1(S)-p h en yl-2(R)-[(8-h yd r oxy-6(R),-
11(R)-dim eth yl-1,2,5,6-tetr ah ydr o-4H-2(R),6-m eth an oben -
za zocin -3-yl)m eth yl]cyclop r op a n eca r boxa m id e (61) a n d
N-[1-(3-Am in op r op ylca r b a m oyl)-3-m et h yl-1(R)-b u t yl]-
1(S)-p h en yl-2(R)-[(8-h yd r oxy-6(R),11(R)-d im eth yl-1,2,5,6-
tetr a h yd r o-4H-2(R),6-m eth a n oben za zocin -3-yl)m eth yl]-
cyclop r op a n eca r boxa m id e‚2TF A (21): 43% yield. Anal.
(C34H48N4O3‚2CF3COOH‚3H2O) C, H, N.
N-{1-[4-(ter t-Bu toxyca r bon yl)a m in obu tylca r ba m oyl]-
3-m eth yl-1(R)-bu tyl}-1(S)-p h en yl-2(R)-[(8-h yd r oxy-6(R),-
11(R)-dim eth yl-1,2,5,6-tetr ah ydr o-4H-2(R),6-m eth an oben -
za zocin -3-yl)m eth yl]cyclop r op a n eca r boxa m id e (62) a n d
N-[1-(4-Am in obu tylcar bam oyl)-3-m eth yl-1(R)-bu tyl]-1(S)-
p h en yl-2(R)-[(8-h yd r oxy-6(R),11(R)-d im eth yl-1,2,5,6-tet-
r a h yd r o-4H -2(R ),6-m e t h a n ob e n za zocin -3-yl)m e t h yl]-
cyclop r op a n eca r boxa m id e‚2TF A (22): 47% yield. Anal.
(C35H50N4O3‚2CF3COOH‚3H2O) C, H, N.
N-{1-[5-(ter t-Bu toxyca r bon yl)a m in op en tylca r ba m oyl]-
3-m eth yl-1(R)-bu tyl}-1(S)-p h en yl-2(R)-[(8-h yd r oxy-6(R),-
11(R)-dim eth yl-1,2,5,6-tetr ah ydr o-4H-2(R),6-m eth an oben -
za zocin -3-yl)m eth yl]cyclop r op a n eca r boxa m id e (63) a n d
N-[1-(5-Am in op en t ylca r b a m oyl)-3-m et h yl-1(R)-b u t yl]-
1(S)-p h en yl-2(R)-[(8-h yd r oxy-6(R),11(R)-d im eth yl-1,2,5,6-
tetr a h yd r o-4H-2(R),6-m eth a n oben za zocin -3-yl)m eth yl]-
cyclop r op a n eca r boxa m id e‚2TF A (23): 44% yield. Anal.
(C36H52N4O3‚2CF3COOH‚3H2O) C, H, N.
N-{1-[6-(ter t-Bu toxyca r bon yl)a m in oh exylca r ba m oyl]-
3-m eth yl-1(R)-bu tyl}-1(S)-p h en yl-2(R)-[(8-h yd r oxy-6(R),-
11(R)-dim eth yl-1,2,5,6-tetr ah ydr o-4H-2(R),6-m eth an oben -
za zocin -3-yl)m eth yl]cyclop r op a n eca r boxa m id e (64) a n d
N-[1-(6-Am in oh exylcar bam oyl)-3-m eth yl-1(R)-bu tyl]-1(S)-
p h en yl-2(R)-[(8-h yd r oxy-6(R),11(R)-d im eth yl-1,2,5,6-tet-
r a h yd r o-4H -2(R ),6-m e t h a n ob e n za zocin -3-yl)m e t h yl]-
cyclop r op a n eca r boxa m id e‚2TF A (24): 49% yield. Anal.
(C37H54N4O3‚2CF3COOH‚3H2O) C, H, N.
N-{[1-(3-Am idin oeth ylpr opylcar bam oyl)-3-m eth yl-1(R)-
bu t ylca r ba m oyl]m et h yl}-1(S)-p h en yl-2(R)-[(8-h yd r oxy-
6(R),11(R)-d im et h yl-1,2,5,6-t et r a h yd r o-4H -2(R),6-m et h -
an oben zazocin -3-yl)m eth yl]cyclopr opan ecar boxam ide (5).
To a solution of compound 1 (30 mg, 0.0354 mmol) in EtOH
(0.5 mL) was added ethyl propionimidate hydrochloride (5.85
mg, 0.0425 mmol) and the pH was adjusted to 10 by adding
DIEA. The reaction mixture was stirred for 24 h at room
temperature and then evaporated to dryness. The crude
compound was purified by flash column chromatography (silica
gel, eluted with CHCl3/MeOH/NH4OH, 9:1:0.1), followed by
N-[1-(3-Am id in oeth ylp r op ylca r ba m oyl)-3-m eth yl-1(R)-
bu tyl]-1(S)-p h en yl-2(R)-[(8-h yd r oxy-6(R),11(R)-d im eth yl-
1,2,5,6-t et r a h yd r o-4H -2(R),6-m et h a n ob en za zocin -3-yl)-
m eth yl]cyclop r op a n eca r boxa m id e (25): 47% yield.
N-[1-(4-Am id in oeth ylbu tylca r ba m oyl)-3-m eth yl-1(R)-
bu tyl]-1(S)-p h en yl-2(R)-[(8-h yd r oxy-6(R),11(R)-d im eth yl-
1,2,5,6-t et r a h yd r o-4H -2(R),6-m et h a n ob en za zocin -3-yl)-
m eth yl]cyclop r op a n eca r boxa m id e (26): 53% yield.
N-[1-(5-Am id in oeth ylp en tylca r ba m oyl)-3-m eth yl-1(R)-
bu tyl]-1(S)-p h en yl-2(R)-[(8-h yd r oxy-6(R),11(R)-d im eth yl-
1,2,5,6-t et r a h yd r o-4H -2(R),6-m et h a n ob en za zocin -3-yl)-
m eth yl]cyclop r op a n eca r boxa m id e (27): 56% yield.
N-[1-(6-Am id in oeth ylh exylca r ba m oyl)-3-m eth yl-1(R)-
bu tyl]-1(S)-p h en yl-2(R)-[(8-h yd r oxy-6(R),11(R)-d im eth yl-
1,2,5,6-t et r a h yd r o-4H -2(R),6-m et h a n ob en za zocin -3-yl)-
m eth yl]cyclop r op a n eca r boxa m id e (28): 74% yield.
N-{[1-(3-Am id in op r op ylp r op ylca r b a m oyl)-3-m et h yl-
1(R )-b u t y lc a r b a m o y l]m e t h y l}-1(S )-p h e n y l-2(R )-[(8-
h ydr oxy-6(R),11(R)-dim eth yl-1,2,5,6-tetr ah ydr o-4H-2(R),6-
m e t h a n o b e n z a z o c i n -3 -y l )m e t h y l ]c y c l o p r o p a n e -
ca r boxa m id e (9). To a solution of compound 1 (30 mg, 0.0354
mmol) in EtOH (0.5 mL) was added ethyl butyrimidate
hydrochloride (6.44 mg, 0.0425 mmol) and the pH was adjusted
to 10 by adding DIEA. The reaction mixture was stirred for
24 h at room temperature and then evaporated to dryness.
Purification by flash column chromatography (silica gel, eluted
with CHCl3/MeOH/NH4OH, 9:1:0.1), followed by trituration
with diethyl ether, gave 17.8 mg of the pure desired compound
9 as a white solid with a 60% yield.
Compounds 10-12 were prepared from compounds 2-4,
respectively, following the same methodology as that described
for compound 9.
N-{[1-(4-Am idin opr opylbu tylcar bam oyl)-3-m eth yl-1(R)-
b u t ylca r b a m oyl]m et h yl}-1(S)-p h en yl-2(R)-[(8-h yd r oxy-
6(R),11(R)-d im et h yl-1,2,5,6-t et r a h yd r o-4H -2(R),6-m et h -
a n ob en za zocin -3-yl)m et h yl]cyclop r op a n eca r b oxa m id e
(10): 63% yield.
N-{[1-(5-Am id in op r op ylp en t ylca r b a m oyl)-3-m et h yl-
1(R )-b u t y lc a r b a m o y l]m e t h y l}-1(S )-p h e n y l-2(R )-[(8-
h ydr oxy-6(R),11(R)-dim eth yl-1,2,5,6-tetr ah ydr o-4H-2(R),6-
m e t h a n o b e n z a z o c i n -3 -y l )m e t h y l ]c y c l o p r o p a n e -
ca r boxa m id e (11): 70% yield.
N-{[1-(6-Am idin opr opylh exylcar bam oyl)-3-m eth yl-1(R)-
bu t ylca r ba m oyl]m et h yl}-1(S)-p h en yl-2(R)-[(8-h yd r oxy-
6(R),11(R)-d im et h yl-1,2,5,6-t et r a h yd r o-4H -2(R),6-m et h -
a n ob en za zocin -3-yl)m et h yl]cyclop r op a n eca r b oxa m id e
(12): 70% yield.
Compounds 29-32 were prepared from compounds 21-24,
respectively, following the same methodology as that described
for compound 9.

3002 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16
Ronsisvalle et al.
N-[1-(3-Am idin opr opylpr opylcar bam oyl)-3-m eth yl-1(R)-
bu tyl]-1(S)-p h en yl-2(R)-[(8-h yd r oxy-6(R),11(R)-d im eth yl-
1,2,5,6-t et r a h yd r o-4H -2(R),6-m et h a n ob en za zocin -3-yl)-
m eth yl]cyclop r op a n eca r boxa m id e (29): 60% yield.
N-[1-(4-Am id in op r op ylbu tylca r ba m oyl)-3-m eth yl-1(R)-
bu tyl]-1(S)-p h en yl-2(R)-[(8-h yd r oxy-6(R),11(R)-d im eth yl-
1,2,5,6-t et r a h yd r o-4H -2(R),6-m et h a n ob en za zocin -3-yl)-
m eth yl]cyclop r op a n eca r boxa m id e (30): 72% yield.
N-[1-(5-Am idin opr opylpen tylcar bam oyl)-3-m eth yl-1(R)-
bu tyl]-1(S)-p h en yl-2(R)-[(8-h yd r oxy-6(R),11(R)-d im eth yl-
1,2,5,6-t et r a h yd r o-4H -2(R),6-m et h a n ob en za zocin -3-yl)-
m eth yl]cyclop r op a n eca r boxa m id e (31): 53% yield.
N-[1-(6-Am id in op r op ylh exylca r ba m oyl)-3-m eth yl-1(R)-
bu tyl]-1(S)-p h en yl-2(R)-[(8-h yd r oxy-6(R),11(R)-d im eth yl-
1,2,5,6-t et r a h yd r o-4H -2(R),6-m et h a n ob en za zocin -3-yl)-
m eth yl]cyclop r op a n eca r boxa m id e (32): 65% yield.
N-{[1-(3-Am idin obu tylpr opylcar bam oyl)-3-m eth yl-1(R)-
bu t ylca r ba m oyl]m et h yl}-1(S)-p h en yl-2(R)-[(8-h yd r oxy-
6(R),11(R)-d im et h yl-1,2,5,6-t et r a h yd r o-4H -2(R),6-m et h -
a n ob en za zocin -3-yl)m et h yl]cyclop r op a n eca r b oxa m id e
(13). To a solution of compound 1 (30 mg, 0.0354 mmol) in
EtOH (0.5 mL) was added ethyl valerimidate hydrochloride
(7.04 mg, 0.0425 mmol) and the pH was adjusted to 10 by
adding DIEA. The reaction mixture was stirred for 24 h at
room temperature and then evaporated to dryness. Purification
by flash column chromatography (silica gel, eluted with CHCl3/
MeOH/NH4OH, 9:1:0.1), followed by trituration with diethyl
ether, gave 20.2 mg of the pure desired compound 13 as a
white solid with a 67% yield.
Compounds 14-16 were prepared from compounds 2-4,
respectively, following the same methodology as that described
for compound 13.
N-{[1-(4-Am id in obu tylbu tylca r ba m oyl)-3-m eth yl-1(R)-
bu t ylca r ba m oyl]m et h yl}-1(S)-p h en yl-2(R)-[(8-h yd r oxy-
6(R),11(R)-d im et h yl-1,2,5,6-t et r a h yd r o-4H -2(R),6-m et h -
a n ob en za zocin -3-yl)m et h yl]cyclop r op a n eca r b oxa m id e
(14): 50% yield.
N-{[1-(5-Am idin obu tylpen tylcar bam oyl)-3-m eth yl-1(R)-
bu t ylca r ba m oyl]m et h yl}-1(S)-p h en yl-2(R)-[(8-h yd r oxy-
6(R),11(R)-d im et h yl-1,2,5,6-t et r a h yd r o-4H -2(R),6-m et h -
a n ob en za zocin -3-yl)m et h yl]cyclop r op a n eca r b oxa m id e
(15): 59% yield. Anal. (C43H64N6O4‚7H2O) C, H, N.
N-{[1-(6-Am id in obu tylh exylca r ba m oyl)-3-m eth yl-1(R)-
bu t ylca r ba m oyl]m et h yl}-1(S)-p h en yl-2(R)-[(8-h yd r oxy-
6(R),11(R)-d im et h yl-1,2,5,6-t et r a h yd r o-4H -2(R),6-m et h -
a n ob en za zocin -3-yl)m et h yl]cyclop r op a n eca r b oxa m id e
(16): 61% yield. Anal. (C44H66N6O4‚5.5H2O) C, H, N.
Compounds 33-36 were prepared from compounds 21-24,
respectively, following the same methodology as that described
for compound 13.
N-[1-(3-Am id in obu tylp r op ylca r ba m oyl)-3-m eth yl-1(R)-
bu tyl]-1(S)-p h en yl-2(R)-[(8-h yd r oxy-6(R),11(R)-d im eth yl-
1,2,5,6-t et r a h yd r o-4H -2(R),6-m et h a n ob en za zocin -3-yl)-
m eth yl]cyclop r op a n eca r boxa m id e (33): 57% yield.
N-[1-(4-Am id in obu tylbu tylca r ba m oyl)-3-m eth yl-1(R)-
bu tyl]-1(S)-p h en yl-2(R)-[(8-h yd r oxy-6(R),11(R)-d im eth yl-
1,2,5,6-t et r a h yd r o-4H -2(R),6-m et h a n ob en za zocin -3-yl)-
m eth yl]cyclop r op a n eca r boxa m id e (34): 63% yield.
N-[1-(5-Am id in obu tylp en tylca r ba m oyl)-3-m eth yl-1(R)-
bu tyl]-1(S)-p h en yl-2(R)-[(8-h yd r oxy-6(R),11(R)-d im eth yl-
1,2,5,6-t et r a h yd r o-4H -2(R),6-m et h a n ob en za zocin -3-yl)-
m eth yl]cyclop r op a n eca r boxa m id e (35): 69% yield.
N-[1-(6-Am id in obu tylh exylca r ba m oyl)-3-m eth yl-1(R)-
bu tyl]-1(S)-p h en yl-2(R)-[(8-h yd r oxy-6(R),11(R)-d im eth yl-
1,2,5,6-t et r a h yd r o-4H -2(R),6-m et h a n ob en za zocin -3-yl)-
m eth yl]cyclop r op a n eca r boxa m id e (36): 72% yield.
N-{[1-(3-Gu a n id in op r op ylca r b a m oyl)-3-m et h yl-1(R)-
bu t ylca r ba m oyl]m et h yl}-1(S)-p h en yl-2(R)-[(8-h yd r oxy-
6(R),11(R)-d im et h yl-1,2,5,6-t et r a h yd r o-4H -2(R),6-m et h -
a n ob en za zocin -3-yl)m et h yl]cyclop r op a n eca r b oxa m id e
(17). To a solution of compound 1 (25 mg, 0.0295 mmol) in
EtOH (1 mL) was added 3,5-dimethylpyrazole-1-carboxamidine
nitrate (7.17 mg, 0.0354 mmol) and the pH was adjusted to
10 by adding DIEA. The reaction mixture was stirred for 48 h
at 60 °C, under N2 atmosphere, and then evaporated to
dryness. Purification by flash column chromatography (silica
gel, eluted with CHCl3/MeOH/NH4OH, 9:1:0.1), followed by
trituration with diethyl ether, gave 15.8 mg of the pure desired
compound 17 as a white solid with a 64% yield.
Compounds 18-20 were prepared from compounds 2-4,
respectively, following the same methodology as that described
for compound 17.
N-{[1-(4-Gu a n id in obu tylca r ba m oyl)-3-m eth yl-1(R)-bu -
tylcar bam oyl]m eth yl}-1(S)-ph en yl-2(R)-[(8-h ydr oxy-6(R),-
11(R)-dim eth yl-1,2,5,6-tetr ah ydr o-4H-2(R),6-m eth an oben -
zazocin -3-yl)m eth yl]cyclopr opan ecar boxam ide (18): 67%
yield.
N-{[1-(5-Gu a n id in op en t ylca r b a m oyl)-3-m et h yl-1(R)-
bu t ylca r ba m oyl]m et h yl}-1(S)-p h en yl-2(R)-[(8-h yd r oxy-
6(R),11(R)-d im et h yl-1,2,5,6-t et r a h yd r o-4H -2(R),6-m et h -
a n ob en za zocin -3-yl)m et h yl]cyclop r op a n eca r b oxa m id e
(19): 58% yield.
N-{[1-(6-Gu a n id in oh exylca r ba m oyl)-3-m eth yl-1(R)-bu -
tylcar bam oyl]m eth yl}-1(S)-ph en yl-2(R)-[(8-h ydr oxy-6(R),-
11(R)-dim eth yl-1,2,5,6-tetr ah ydr o-4H-2(R),6-m eth an oben -
zazocin -3-yl)m eth yl]cyclopr opan ecar boxam ide (20): 60%
yield.
Compounds 37-40 were prepared from compounds 21-24,
respectively, following the same methodology as that described
for compound 17.
N-[1-(3-Gu a n id in op r op ylca r ba m oyl)-3-m eth yl-1(R)-bu -
t yl]-1(S)-p h en yl-2(R)-[(8-h yd r oxy-6(R),11(R)-d im et h yl-
1,2,5,6-t et r a h yd r o-4H -2(R),6-m et h a n ob en za zocin -3-yl)-
m eth yl]cyclop r op a n eca r boxa m id e (37): 60% yield.
N-[1-(4-Gu a n id in obu tylca r ba m oyl)-3-m eth yl-1(R)-bu -
t yl]-1(S)-p h en yl-2(R)-[(8-h yd r oxy-6(R),11(R)-d im et h yl-
1,2,5,6-t et r a h yd r o-4H -2(R),6-m et h a n ob en za zocin -3-yl)-
m eth yl]cyclop r op a n eca r boxa m id e (38): 50% yield.
N-[1-(5-Gu a n id in op en tylca r ba m oyl)-3-m eth yl-1(R)-bu -
t yl]-1(S)-p h en yl-2(R)-[(8-h yd r oxy-6(R),11(R)-d im et h yl-
1,2,5,6-t et r a h yd r o-4H -2(R),6-m et h a n ob en za zocin -3-yl)-
m eth yl]cyclop r op a n eca r boxa m id e (39): 52% yield.
N-[1-(6-Gu a n id in oh exylca r ba m oyl)-3-m eth yl-1(R)-bu -
t yl]-1(S)-p h en yl-2(R)-[(8-h yd r oxy-6(R),11(R)-d im et h yl-
1,2,5,6-t et r a h yd r o-4H -2(R),6-m et h a n ob en za zocin -3-yl)-
m eth yl]cyclop r op a n eca r boxa m id e (40): 51% yield.
Ra d ioliga n d Bin d in g Assa ys. Bin d in g to K, µ, a n d δ
Op ioid Recep tor s. Male Hartley guinea pigs and Sprague-
Dawley rats were purchased from Charles River (Como, Italy).
Guinea pig cerebellum or rat brain membrane fractions were
prepared following a procedure reported by Bowen41 and the
protein content was evaluated.42 For radioligand binding
assays on κ receptors, aliquots of homogenate obtained from
guinea pig cerebella (1 mg protein/tube) were incubated in 50
mM Tris‚HCl (pH 7.4) containing 0.5 µg/mL aprotinin, 10 µg/
mL leupeptin, 200 µg/mL bacitracin at 25 °C for 60 min with
2 nM [3H]U69,593 (Amersham; 60 Ci/mmol, Kd ) 1.98 ( 0.04
nM, n ) 3). The concentration of the tested compounds ranged
from 10-11 to 10-5 M. Binding studies were performed in the
presence of [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO;
100 nM) and [D-Ala2,D-Ala5]-enkephalin (DADLE; 100 nM) to
eliminate the interaction with µ and δ receptors, respectively.
Nonspecific binding was determined by addition of U50,488
(Upjohn; 10 µM).
Binding to µ and δ sites was carried out on crude membrane
fractions obtained from the whole rat brain minus the cerebel-
lum as previously reported.18 For radioligand binding assays
to µ receptors, aliquots of homogenate (1 mg protein/tube) were
incubated in 50 mM Tris‚HCl (pH 7.4) containing 0.5 µg/mL
aprotinin, 10 µg/mL leupeptin, 200 µg/mL bacitracin at 25 °C
for 60 min with 1 nM [3H]diprenorphine (Amersham; 30 Ci/
mmol, Kd ) 0.22 ( 0.03 nM, n ) 5). The concentration of the
tested compounds ranged from 10-11 to 10-5 M. Binding studies
were performed in the presence of U50,488 (300 nM) and
DADLE (300 nM) which were added to saturate the κ and δ
receptors, respectively. Nonspecific binding was determined
by the addition of DAMGO (1 µM). For radioligand binding
assays to δ receptors, aliquots of homogenate (1 mg protein/

Nonpeptide Analogues of Dynorphin A(1-8)
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16 3003
tube) were incubated in the same conditions with 1 nM [3H]-
diprenorphine (Kd ) 0.44 ( 0.03 nM, n ) 5). The concentration
of the tested compounds ranged from 10-11 to 10-5 M. Binding
studies were performed in the presence of U50,488 (300 nM)
and DAMGO (300 nM) which were added to saturate the κ
and µ receptors, respectively. Nonspecific binding was deter-
mined by the addition of DPDPE (1 µM).
The incubation was interrupted by rapid filtration through
Whatman GF/B glass filters which were presoaked in a 0.1%
poly(ethylenimine) solution for 1 h. Filters were washed twice
with 4 mL of ice-cold 50 mM Tris‚HCl solution.
(3) Pan, Z. Z. µ-Opposing actions of the κ-opioid receptors. Trends
Pharmacol. Sci. 1998, 19, 94-98.
(4) Portenoy, R. K.; Caraceni, A.; Cherny, N. I.; Goldblum, R.;
Ingham, J .; Inturrisi, C. E.; J ohnson, J . H.; Lapin, J .; Tiseo, P.
J .; Kreek, M. J . Dynorphin A(1-13) analgesia in opioid-treated
patients with chronic pain. Clin. Drug Invest. 1999, 17, 33-42.
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Bin d in g to σ1 Sites. σ1 binding assays were carried out on
guinea pig brain membranes prepared according to a method
reported by Matsumoto et al.43 Binding assays were performed
as described by DeHaven et al.44 Briefly, each tube contained
500 µg of membrane protein, 3 nM [3H]pentazocine (NEN Life
Science; 31.6 Ci/mmol, Kd ) 4.3 ( 0.8 nM, n ) 3). Nonspecific
binding was determined by the addition of haloperidol (10 µM).
The reaction was performed for 150 min at 37 °C and
terminated by filtration through Whatman GF/B glass filters
which were presoaked in a 0.5% poly(ethylenimine) solution.
Radioactivity on the filters was measured by a liquid
scintillation cocktail. Scatchard parameters and inhibition
constants were calculated using the EBDA-LIGAND pro-
gram,45 purchased from Elsevier/Biosoft.
An tin ociception . Male albino Swiss mice (20-25 g; Charles
River, Calco, Italy) were used. Test compounds were dissolved
in the vehicle (saline containing 0.5% carboxymethylcellulose,
w/v) and administered subcutaneously (sc) in a dose-volume
of 10 mL/kg (0.1-10 mg/kg) 30 min before testing. The opioid
antagonist nor-binaltorphimine (nor-BNI; RBI, Natik, MA)
was administered sc 10 min before the test compound at a 10
mg/kg dosage. Antinociception was measured by the acetyl-
choline-induced abdominal constriction test.19,46 Mice were
injected intraperitoneally with 0.25 mL of a solution of
acetylcholine iodine (0.75 mg/mL) and were then observed for
5 min during which the number of abdominal constrictions
was counted. The ED50 value was defined as the dosage that
reduced the mean number of constrictions to 50% of the
vehicle-treated mice.
(7) Minami, M.; Toya, T.; Katao, Y.; Maekawa, K.; Nakamura, S.;
Onogi, T.; Kaneko, S.; Satoh, M. Cloning and expression of a
cDNA for the rat kappa opioid receptor. FEBS Lett. 1993, 329,
291-295.
(8) Li, S.; Zhu, J .; Chen, C.; Chen, Y. W.; Deriel, J . K.; Ashby, B.;
Liu-Chen, L. Y. Molecular cloning and expression of a rat κ opioid
receptor. Biochem. J . 1993, 295, 629-633.
(9) Chen, Y.; Mestek, A.; Liu, J .; Yu, L. Molecular cloning of a rat
kappa opioid receptor reveals sequence similarities to the mu
and delta opioid receptors. Biochem. J . 1993, 295, 625-628.
(10) Chavkin, C.; J ames, I. F.; Goldstein, A. Dynorphin is a specific
endogenous ligand of the κ opioid receptor. Science 1982, 215,
413-415.
(11) Raynor, K.; Kong, H.; Chen, Y.; Yasuda, K.; Yu, L.; Bell, G. I.;
Reisine, T. Pharmacological characterization of the cloned κ-, δ-
and µ-opioid receptors. Mol. Pharmacol. 1994, 45, 330-334.
(12) Mansour, A.; Hoversten, M. T.; Taylor, L. P.; Watson S. J .; Akil,
H. The cloned µ, δ and κ receptors and their endogenous
ligands: Evidence for two opioid peptide recognition cores. Brain
Res. 1995, 700, 89-98.
(13) Wang, J . B.; J ohnson, P. S.; Wu, J . M.; Wang, W. F.; Uhl, G. R.
Human κ opiate receptor second extracellular loop elevates
dynorphin’s affinity for human µ/κ chimeras. J . Biol. Chem.
1994, 269, 25966-25969.
(14) Xue, J .-C.; Chen, C.; Zhu, J .; Kanapuli, S.; DeRiel, J . K.; Yu, L.;
Liu-Chen, L.-Y. Differential binding domains of peptide and non-
peptide ligands in the cloned rat κ opioid receptor. J . Biol. Chem.
1994, 269, 30195-30199.
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Akil, H. Controlling signaling with a specifically designed Gi-
coupled receptor. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 352-
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simulation of dynorphin A-(1-10) binding to extracellular loop
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Chem. 1997, 40, 3254-3262.
Beh a vior a l Stu d ies. Male albino Swiss mice (20-25 g)
were placed individually into clear acrylic cages and left for 1
h to acclimatize to the new environment. Test compounds were
dissolved in the vehicle and administered sc 30 min later. After
10 min, behavioral scores were recorded in 5-min intervals for
40 min. The scoring scale is modeled after by Tanaka et al;37
10 mice/group were used. The total score of the vehicle-treated
group was defined as 100% and ED50 values for treated mice
were determined using log-probit conversion of data.
(17) Tessmer, M. R.; Kallick, D. A. NMR and structural model of
dynorphin A (1-17) bound to dodecylphosphocholine micelles.
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Fronza, G.; Romagnoli, C.; Pistacchio, E.; Spampinato, S.; Ferri,
S. Non-peptide ligands for opioid receptors. Design of κ-specific
agonists. J . Med. Chem. 1993, 36, 1860-1865.
Rota r od Test. The test compounds were administered (as
previously described), and after 30 min, male albino Swiss mice
were placed individually on an accelerated rotarod (Ugo Basile,
Milan, Italy) with three opportunities to maintain balance on
the bar. Mice that failed to maintain balance in at least one
of three separate trials were considered impaired. The ED50
value was defined as the dosage that halved the latency of
the vehicle-treated mice for falling off the rotarod.
(19) Ronsisvalle, G.; Prezzavento, O.; Pasquinucci, L.; Marrazzo, A.;
Carboni, L.; Gomez-Vidal, J . A.; Carboni, L.; Spampinato, S. CCB
a novel specific κ-opioid agonist, which discriminates between
opioid and σ1 recognition sites. Life Sci. 1995, 57, 1487-1495.
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Pasquinucci, L.; Marrazzo, A.; Caccaguerra, S.; Spampinato, S.
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Ack n ow led gm en t. We thank Italian MURST and
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Su p p or tin g In for m a tion Ava ila ble: Elemental analyses
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