Design, synthesis, and biological activities of cyclic lactam peptide analogues of dynorphin A(1-11)-NH2

Article abstract of DOI:10.1021/jm950369c

We previously have reported four possible binding conformations of dynorphin A (Dyn A) for the central κ opioid receptors, induced by the address sequence, using a molecular mechanics energy minimization approach. The lowest energy conformation was found to exhibit an α-helical conformation in the cyclized address sequence. It was suggested that an α- helical conformation in the cyclized address sequence or a helical conformation induced by the conformational characteristics of the message sequence may be important for binding potency and κ opioid receptor selectivity. Side chain to side chain lactam bridges between the i and i + 4 positions have been shown to stabilize α-helical conformations. Thus, a series of cyclic lactam analogues of dynorphin A(1-11)-NH₂ have been designed, synthesized and evaluated by the guinea pig brain (GPB) binding assay and guinea pig ileum (GPI) bioassay to evaluate the conformational analysis prediction and, further, to investigate the conformational requirements for high potency and selectivity for κ opioid receptors. Positions 2-6, 3-7, and 5-9 were chosen as the sites for incorporating cyclic conformational constraints. Cyclization between D-Asp² and Lys⁶ in c[D- Asp²,Lys⁶]Dyn A(1-11)-NH₂ led to an analogue with pronounced potency and selectivity enhancement for the μ opioid receptor, whereas cyclization between D-Asp³ and Lys⁷ in c[D-Asp³,Lys⁷]Dyn A(1-11)-NH₂ led to a potent ligand (IC₅₀ 4.9 nM) with κ receptor selectivity. The other analogues in the series proved to be less selective. The biological results led to the suggestion that the binding conformation for the κ receptor may have structural requirements that are distinct from those of μ and δ receptors. Interestingly, analogues with a D-Asp at position 2, 3, or 9 were found to be more potent for the κ receptor than analogues with an L-Asp at the same positions. It is suggested that the incorporation of D-Asp into position 2, 3, or 9 of Dyn A(1-11)-NH₂ may have stereochemical and conformational effects on the nearby amino acids which can help discriminate the preference between κ, μ, and δ receptors.

Full text of DOI:10.1021/jm950369c

1
136
J . Med. Chem. 1996, 39, 1136-1141
Design , Syn th esis, a n d Biologica l Activities of Cyclic La cta m P ep tid e An a logu es
of Dyn or p h in A(1-11)-NH 1
2
†
†
‡
‡
‡
‡
Feng-Di T. Lung, Nathan Collins, Dagmar Stropova, Peg Davis, Henry I. Yamamura, Frank Porreca, and
Victor J . Hruby*,
†
Departments of Chemistry and Pharmacology, University of Arizona, Tucson, Arizona 85721
X
Received May 19, 1995
We previously have reported four possible binding conformations of dynorphin A (Dyn A) for
the central κ opioid receptors, induced by the address sequence, using a molecular mechanics
energy minimization approach. The lowest energy conformation was found to exhibit an
R-helical conformation in the cyclized address sequence. It was suggested that an R-helical
conformation in the cyclized address sequence or a helical conformation induced by the
conformational characteristics of the message sequence may be important for binding potency
and κ opioid receptor selectivity. Side chain to side chain lactam bridges between the i and i
+
4 positions have been shown to stabilize R-helical conformations. Thus, a series of cyclic
lactam analogues of dynorphin A(1-11)-NH have been designed, synthesized and evaluated
2
by the guinea pig brain (GPB) binding assay and guinea pig ileum (GPI) bioassay to evaluate
the conformational analysis prediction and, further, to investigate the conformational require-
ments for high potency and selectivity for κ opioid receptors. Positions 2-6, 3-7, and 5-9
were chosen as the sites for incorporating cyclic conformational constraints. Cyclization between
2
6
2
6
D-Asp and Lys in c[D-Asp ,Lys ]Dyn A(1-11)-NH
2
led to an analogue with pronounced potency
3
and selectivity enhancement for the µ opioid receptor, whereas cyclization between D-Asp and
Lys in c[D-Asp ,Lys ]Dyn A(1-11)-NH led to a potent ligand (IC50 4.9 nM) with κ receptor
2
7
3
7
selectivity. The other analogues in the series proved to be less selective. The biological results
led to the suggestion that the binding conformation for the κ receptor may have structural
requirements that are distinct from those of µ and δ receptors. Interestingly, analogues with
a D-Asp at position 2, 3, or 9 were found to be more potent for the κ receptor than analogues
with an L-Asp at the same positions. It is suggested that the incorporation of D-Asp into position
2
, 3, or 9 of Dyn A(1-11)-NH
2
may have stereochemical and conformational effects on the
nearby amino acids which can help discriminate the preference between κ, µ, and δ receptors.
In tr od u ction
for κ opioid receptors.7
The discovery of endogenous opioid peptides in the
970s has greatly accelerated research in opioid chem-
Dyn A: H-Tyr-Gly-Gly-Phe-Leu-ArgArg-Ile-Arg-
Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln-OH
2
1
istry and biology. Evidence accumulated has led to the
suggestion of multiple opioid receptors.3 At present, it
is generally accepted that there are at least three
different opioid receptors, namely µ (mu), δ (delta), and
Structure-activity relationships for the dynorphin
8
peptides have been reviewed. It was suggested that
7
11
13
the basic residues Arg , Lys , and Lys were important
for high selectivity and/or potency, but the deletion of
4
κ (kappa). Studies of the physiological and pharma-
residues 14-17 or 12-17 of dynorphin A did not
cological roles of these receptors require highly potent
and receptor-selective ligands for µ, δ, and κ receptors.
Research in the development of potent and selective
peptide ligands has led to the development of peptides
with high receptor selectivities for the µ and δ opioid
7,9,10
significantly affect its selectivity or potency.
Thus,
we have used the truncated derivative Dyn A(1-11)-
NH2 as a starting point to design analogues with high
potency and selectivity for the κ receptor.
In solution, Dyn A exhibits an extended and/or
random coil structure as indicated by several spectro-
5
receptors. The potential of targeting the κ opioid
receptor as an effector for analgesia with the putative
11,12
11,13
14
15
scopic methods (FT-IR,
NMR,
CD, Raman,
6
endogenous κ opioid ligand dynorphin A (Dyn A) has
16
and fluorescence energy transfer ). In anisotropic
environments such as membranes or phase inter-
faces, some R-helical or â-sheet structure has been
not been explored to date in great detail. Therefore, it
was necessary to design and synthesize highly selective
ligands for the κ receptor, especially in relation to the
endogenous dynorphins. Dynorphin A is a 17 amino
acid peptide that consists of a N-terminal message
sequence (Tyr-Gly-Gly-Phe) and an address sequence
in the more C-terminal region which imparts selectivity
17
18
observed for dynorphin and some of its analogues.
Conformational analyses examined in our laboratory
also have suggested that an R-helical conformation in
the cyclized address sequence or a helical structure
induced in the message sequence is important for κ
opioid receptor binding potency and selectivity.19 A H
NMR study of Dyn A(1-17)-NH2 indicated that residues
1
*
Author to whom reprint requests should be addressed at the
Department of Chemistry.
3
9
from Gly to Arg form a helical conformation when
†
Department of Chemistry.
Department of Pharmacology.
Abstract published in Advance ACS Abstracts, February 1, 1996.
2
0
‡
bound to lipid micelles. This supports our suggestion
that a helical structure in the message sequence of Dyn
X
0
022-2623/96/1839-1136$12.00/0 © 1996 American Chemical Society

Analogues of Dynorphin A(1-11)-NH
2
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5 1137
Ta ble 1. Opioid Receptor Binding Affinities and Selectivities of Various Dyn A Analogues in Guinea Pig Brain Homogenate
IC50 (nM)a
selectivity
analogues of
Dyn A(1-11)-NH2
κ
µ
δ
µ/κ
δ/κ
Dyn A(1-11)-NH2 (1)
0.58 ( 0.03
9.9 ( 2.0
2980 ( 614
3.4 ( 2.1
26 ( 3
910 ( 62
12 ( 5
1710 ( 256
130 ( 12
150 ( 7
50 ( 1
560 ( 29
1.15 ( 0.02
1800 ( 327
870 ( 251
3290 ( 809
1970 ( 328
17.1
29.8
0.03
30
64
1.2
1.8
1.3
0.05
7.0
14
45.0
9.1
0.11
68
27
1.5
2
,
6
c[Asp Lys ] (2)
100 ( 42
2
6
c[D-Asp ,Lys ] (3)
110 ( 26
25 ( 5
4.9 ( 0.6
100 ( 42
6.6 ( 2.5
220 ( 27
1.5 ( 0.4
3
7
c[Asp ,Lys ] (4)
740 ( 97
3
7
c[D-Asp ,Lys ] (5)
310 ( 40
9
c[Lys5,Asp ] (6)
c[Lys ,D-Asp ] (7)
Asp ,Lys ] (8)
120 ( 25
5
9
12 ( 4
7.6
2.5
2
6
[
[
[
[
[
[
290 ( 70
2
6
D-Asp ,Lys ] (9)
0.08 ( 0.04
1320 ( 421
660 ( 202
400 ( 110
555 ( 165
0.77
9.5
19
94
110
3
7
Asp ,Lys ] (10)
190 ( 46
47 ( 15
35 ( 9
3
7
D-Asp ,Lys ] (11)
5
9
Lys ,Asp ] (12)
Lys ,D-Asp ] (13)
12
32
5
9
17.5 ( 6.4
a
3
3
3
2
4
5
The radioligands used were [ H]U-69,593 (κ receptor), [ H]DAMGO (µ receptor) and [ H]c[D-Pen ,p-Cl-Phe ,D-Pen ]enkephalin (δ
receptor).
A is biologically important. Side chain to side chain
lactam cyclizations between the side chains of aspartic
acid and lysine at positions i and i + 4 have been shown
to stabilize R-helical peptides.21 Very recently we
reported several lead compounds with modifications
involving the 3 position of Dyn A and suggested that
position 3 in the message sequence of Dyn A is a
promising site for further modifications.22 We recently
and δ vs κ receptors. The linear Dyn A(1-11)-NH2 (1)
was chosen as the standard for comparison with the
synthetic Dyn A analogues.
In the GPB binding assay, it was found that Dyn A
analogues with a D-residue at position 2, 3, or 9
(analogue 5, 7, 9, 11, or 13) are generally more potent
for the κ receptor than the corresponding analogues with
an L-residue at the same position (4, 6, 8, 10, and 12).
3
7
reported in preliminary studies that c[D-Asp ,Lys ]Dyn
2
6
The cyclic c[Asp ,Lys ]Dyn A(1-11)-NH2 analogue (2)
displayed poor affinities for κ, µ, and δ receptors (IC50
are 100, 2980, and 910 nM, respectively), but slightly
increased selectivity for κ vs µ receptors (IC50 ratio is
3
7
A(1-11)-NH2 and c[Asp ,Lys ]Dyn A(1-11)-NH2 were
the first message-cyclized ligands to retain the κ recep-
2
3
tor selectivity.
To further analyze these conforma-
tional suggestions, to evaluate the importance of global
constraints, and to investigate further the conforma-
tional requirements for high potency and selectivity for
the κ opioid receptor, cyclic [i, i + 4] lactams of Dyn A
analogues have been designed, synthesized, and evalu-
2
9.2). Interestingly, the related cyclic analogue c[D-
2
6
Asp , Lys ]Dyn A(1-11)-NH2 (3) displayed similar af-
finity for the κ receptor (IC50 value is 110 nM), but
greatly improved affinities for µ and δ receptors (IC50
values are 3.4 and 12 nM, respectively), leading to a
ligand that preferentially interacts with µ and δ recep-
1
4
ated for their biological activities. Since Tyr and Phe
were reported to be critical for opioid agonist activity
and potency, positions 2-6, 3-7, and 5-9 were chosen
as the sites for incorporation of a conformational
constraint to determine whether the helical conforma-
tion is important in the address or message sequence
of Dyn A. We report here the results of this compre-
hensive study.
3
7
tors. The cyclic analogue c[Asp ,Lys ]Dyn A(1-11)-NH2
4) displayed moderate affinity for the κ receptor (IC50
(
is 25 nM) and greatly decreased affinities for µ and δ
receptors (IC50 values are 740 and 1710 nM, respec-
tively), leading to increased selectivities for κ vs µ and
κ vs δ receptors (IC50 ratios are 30 and 69). On the other
3
7
hand, the corresponding cyclic analogue c[D-Asp ,Lys ]-
Dyn A(1-11)-NH2 (5) displayed high affinity for the κ
receptor (IC50 value is 4.9 nM) and also is the most
selective for κ vs µ receptors (IC50 ratio is 64) of all
analogues reported here. To our knowledge, analogues
4 and 5 are the only message-cyclized peptide ligands
that retain good κ opioid receptor selectivity. These
interesting biological results provide new insights into
the requirements for high affinity and selectivity for the
κ receptor. These biological results and the preliminary
results of NMR studies of analogues 4 and 5 suggest
that the conformational changes induced in the message
Resu lts a n d Discu ssion
All Dyn A analogues were synthesized as C-terminal
carboxamide analogues (to impart stability to exopep-
2
4
25,26
tidase ) by solid phase methods,
purified by RP-
HPLC, and characterized by FAB mass spectrometry
and amino acid analysis. The purity of the synthesized
peptides was assessed by TLC (single spot in four
different solvent systems, ninhydrin detection) and
HPLC (one single peak, UV detection at 280 nm and
2
25 nm, using two independent gradients) (see the
Experimental Section).
3
sequence of Dyn A may be stabilized by a D-Asp and
demonstrate for the first time that a basic residue in
position 7 is not needed for good κ receptor binding and
selectivity in dynorphin analogues. On the other hand,
Bin d in g Assa y
The peptides were evaluated for opioid receptor
affinities at κ, µ, and δ receptors by measuring the
inhibition of binding of [ H]U-69,593 (N-methyl-N-[7-
2
2
3
the structural effects of L-Asp vs D-Asp and L-Asp vs
3
3
D-Asp clearly play an important role in discriminating
(
1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]benzo[b]furan-4-
binding preference for κ, µ, and δ receptors. For the
3
2
4
5
acetamide), [ H]DAMGO ([D-Ala ,MePhe ,Glyol ]en-
5,9 cyclized analogues we placed a Lys in position 5
3
2
4
5
5
kephalin), and [ H]c[D-Pen ,p-Cl-Phe ,D-Pen ]enkephalin,
respectively, to opioid receptors in plasma membrane
homogenates of the guinea pig brain (GPB) (Table 1).
The selectivities are defined as the IC50 ratios of µ vs κ
which has a side chain more homologous to Leu , and
because our previous work had shown that a D-amino
acid in position 5 was not consistent with high κ receptor
2
7
5
9
potency.
The cyclic analogue c[Lys ,Asp ]Dyn A(1-

1
138 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5
1)-NH2 (6) was found to be not very potent at any of
Lung et al.
Ta ble 2. Bioassays with the Smooth-Muscle Tissue of the
1
Guinea Pig Ileum
the opioid receptors and was nonselective for the κ
receptor (Table 1). Since the L-configuration in position
IC50 (nM)
analogues of
Dyn A(1-11)-NH2
2
7
5
has been reported to be important for κ affinity, the
fold-shifta
GPI
related cyclic analogue 7 with a D-amino acid in position
i + 4 and L-amino acid in position i was synthesized,
although this linkage would be expected to be shorter
Dyn A(1-11)-NH2 (1)
1.07 ( 0.31
16000 ( 1300
610 ( 23
ns
nt
17
nt
nt
nt
nt
nt
4.5
nt
nt
nt
nt
2
6
c[Asp ,Lys ] (2)
2
6
c[D-Asp ,Lys ] (3)
2
8
3
7
than an idealized helix would require. Nonetheless,
c[Asp ,Lys ] (4)
1500 ( 86
3
7
5
9
c[D-Asp ,Lys ] (5)
600 ( 51
9300 ( 95
the cyclic analogue c[Lys ,D-Asp ]Dyn A(1-11)-NH2 (7)
exhibited good affinity (6.6 nM) for the κ receptor, but
decreased selectivity for the κ receptor. We suggest that
further modifications in the message sequence of cyclic
Dyn A analogues may lead to novel peptides with high
potency and selectivity. Of all the synthesized ana-
5
9
c[Lys ,Asp ] (6)
c[Lys ,D-Asp ] (7)
Asp ,Lys ] (8)
[D-Asp ,Lys ] (9)
Asp ,Lys ] (10)
D-Asp ,Lys ] (11)
Lys ,Asp ] (12)
Lys ,D-Asp ] (13)
5
9
2500 ( 180
490 ( 74
2
6
[
2
6
14.2 ( 1.8
3
7
[
[
[
[
13300 ( 182
4900 ( 288
3170 ( 510
4160 ( 423
3
7
5
9
2
6
logues, the linear [D-Asp ,Lys ]Dyn A(1-11)-NH2 (9)
was found to be the most potent for central κ, µ, and δ
receptors (IC50 values are 1.5, 0.08, and 1.15 nM,
respectively). However, this linear analogue preferen-
tially interacted with µ and δ receptors. The high
affinities for all three receptors may be due to increased
enzymatic stability and/or to stabilization of the helical
conformation. Of particular note is the very high
affinity of 9 at the µ opioid receptor. It would appear
that an acidic amino acid in the 2 position can lead to
very strong binding to µ receptors that deserves further
examination in other opioid ligands. The structural
5
9
a
nt, not tested; ns, no significant shift observed with 1000 nM
CTAP used as a µ antagonist.
Ta ble 3. Central (GPB) vs Peripheral (GPI) Nervous Systems
Selectivities at the κ Opioid Receptors of Various Dyn A
Analogues
ratio
ratio
of IC50
analogues of
Dyn A(1-11)-NH2
of IC50
analogues of
GPI/GPB Dyn A(1-11)-NH2 GPI/GPB
2
6
Dyn A(1-11)-NH2 (1)
1.8
[Asp ,Lys ] (8)
2.2
9.5
2
6
2
6
a
c[Asp ,Lys ] (2)
160
[D-Asp ,Lys ] (9)
2
6
5.6a
3
7
c[D-Asp ,Lys ] (3)
[Asp ,Lys ] (10)
72
110
91
2
effects of D-Asp may be responsible for the preference
c[Asp3,Lys7] (4)
61
120
91
[D-Asp3,Lys7] (11)
3
7
5
9
for µ and δ receptors for this analogue. The other
c[D-Asp ,Lys ] (5)
[Lys ,Asp ] (12)
5
9
5
9
2
c[Lys ,Asp ] (6)
[Lys ,D-Asp ] (13)
240
message sequence-modified linear analogues, [Asp ,-
5
9
c[Lys ,D-Asp ] (7)
380
6
3
7
Lys ]Dyn A(1-11)-NH2 (8), [Asp ,Lys ]Dyn A(1-11)-
a
3
7
Not really κ receptor selective.
NH2 (10), and [D-Asp ,Lys ]Dyn A(1-11)-NH2 (11), all
displayed decreased affinities for opioid receptors and
decreased selectivities for κ vs µ and κ vs δ receptors. It
is interesting to note that, with the exception for
analogue 9, the message-cyclized analogues generally
display somewhat higher affinity for the κ receptor than
the related linear analogues (2 vs 8, 4 vs 10, and 5 vs
peripheral κ receptor systems is defined as the IC50 ratio
of the GPB and GPI (Table 3).
Dyn A(1-11)-NH2 (1) showed an IC50 value of 1.07
nM in the GPI bioassay. It has been shown that this
activity is due only to a peripheral κ receptor rather
than the µ receptor because the highly µ selective
antagonist ligand CTAP has no antagonist effect on
the activity of 1 in the GPI assay (Table 2). All the
analogues synthesized are less potent than Dyn A(1-
1
1). This indicates that the conformational change
induced in the message sequence may be important for
maintaining high affinity for the κ receptor, but not for
µ and δ receptors, although cyclic analogue 5 also is
more potent than linear analogue 11 at the µ and δ
30
1
1)-NH2 (1), but display increased selectivities for the
5
9
receptor as well. The linear analogue [Lys ,Asp ]Dyn
A(1-11)-NH2 (12) displayed moderate affinity for the κ
receptor (IC50 value is 35 nM) and enhanced selectivities
for κ vs δ receptors (IC50 ratio is 94). Interestingly, the
central vs periphery κ receptors (ratios range from 2.2
to 380, measured as the IC50 ratios of the GPB and GPI).
Except for analogue 13, analogues with a D-residue at
position 2, 3, or 9 are more potent and selective than
analogues with an L-residue at the same position. The
5
9
linear analogue [Lys ,D-Asp ]Dyn A(1-11)-NH2 (13)
exhibited moderate affinity for the κ receptor (IC50 value
is 17.5 nM) and enhanced selectivities for κ vs µ (ΙC50
ratio is 32) and κ vs δ receptors (IC50 ratio is 112). These
results indicate that the stereochemical and electro-
chemical properties produced by the replacement of an
3
7
cyclic analogues c[D-Asp ,Lys ]Dyn A(1-11)-NH2 (5)
exhibited good selectivity for the central vs peripheral
κ receptors (IC50 ratio is 122) and was found to be the
most potent in the periphery (IC50 value is 600 nM) of
2
6
the cyclic analogues. The cyclic analogue c[Asp ,Lys ]-
Dyn A(1-11)-NH2 (2) also exhibited good selectivity for
the central vs peripheral κ receptors (IC50 ratio is 160).
These results suggest that the incorporation of confor-
mational constraints into the message sequence may
produce or stabilize a conformation which is important
for selectivity for central vs peripheral κ receptors. The
Arg with an L-Asp9 or a D-Asp residue and by the
replacement of an Leu with an Lys5 (12 and 13,
respectively), though reducing affinity for the κ receptor,
generally have an even greater effect on µ and δ receptor
bindings. Interestingly, except for 6 at the κ receptor,
cyclization of these analogues leads to increased affini-
ties at κ, µ, and δ receptors.
9
9
5
5
9
cyclic analogue c[Lys ,D-Asp ]Dyn A1-11NH2 (7) showed
the highest selectivity (IC50 value is 380) of all analogues
Bioa ssa y
3
7
synthesized. Linear analogues [D-Asp ,Lys ]Dyn A(1-
5
9
The opioid activities of these peptides also were
measured by their ability to inhibit the electrically
evoked contraction of the GPI29 (guinea pig ileum)
11)-NH2 (11) and [Lys ,D-Asp ]Dyn A(1-11)-NH2 (13)
exhibited poor potency for the peripheral κ receptor, but
high selectivities for central vs peripheral κ receptors.
(IC50 ratios are 110 and 240, respectively).
(Table 2). The selectivity between the central and

Analogues of Dynorphin A(1-11)-NH
2
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5 1139
Ta ble 4. Analytical Properties of Various Dyn A Analogues
TLC Rf valuesa
K′ HPLCb
analogues of
Dyn A(1-11)-NH2
I
II
III
IV
1
2
FAB-MSc
Dyn A(1-11)-NH2 (1)
0.65
0.66
0.69
0.71
0.74
0.61
0.63
0.53
0.58
0.65
0.58
0.49
0.49
0.20
0.24
0.29
0.37
0.41
0.25
0.28
0.26
0.22
0.17
0.39
0.30
0.16
0.80
0.74
0.73
0.93
0.92
0.91
0.64
0.79
0.73
0.68
0.58
0.71
0.66
0.65
0.77
0.77
0.82
0.80
0.73
0.77
0.72
0.74
0.78
0.77
0.69
0.65
3.4
5.0
1362 [M]+
2
6
+
+
+
+
+
+
+
+
+
+
+
+
c[Asp ,Lys ] (2)
3.72
2.91
3.51
2.51
2.44
2.39
2.50
2.53
2.58
2.60
1.88
2.59
3.19
2.76
2.90
2.32
3.29
2.63
2.25
3.22
2.64
2.50
1.64
1.56
1374 [M + H]
1374 [M + H]
1374 [M + H]
1374 [M + H]
1318 [M + H]
1318 [M + H]
1392 [M + H]
1392 [M + H]
1392 [M + H]
1392 [M + H]
1336 [M + H]
1336 [M + H]
2
6
c[D-Asp ,Lys ] (3)
3
7
c[Asp ,Lys ] (4)
3
7
c[D-Asp ,Lys ] (5)
5
9
[
Lys ,Asp ] (6)
c[Lys ,D-Asp ] (7)
Asp ,Lys ] (8)
5
9
2
6
[
[
[
[
[
[
2
6
D-Asp ,Lys ] (9)
3
7
Asp ,Lys ] (10)
3
7
D-Asp ,Lys ] (11)
5
9
Lys ,Asp ] (12)
Lys ,D-Asp ] (13)
5
9
a
Solvent systems: I, 1-butanol/pyridine/acetic acid/water (15/10/3/8); II, 1-butanol/acetic acid/water (4/1/5); III, 2-propanol/concentrated
b
ammonium hydroxide/water (3/10/10); IV, 1-butanol/pyridine/acetic acid/water (6/6/1/5). 1 and 2 refer to the HPLC gradients as described
in the Experimental Section. Fast atom bombardment mass spectroscopy.
c
Con clu sion s
follows: (1) 10-90% acetonitrile over 40 min and (2) 10-50%
over 30 min. The flow rate was 2 mL/min for both cases. The
column used for analytical chromatography had dimensions
of 4.5 × 250 mm (Vydac, 10 µm particle size, C-18). HPLC on
a semipreparative scale was performed with a reverse phase
column (Vydac, 10 × 250 mm, 10 µm particle size, C-18)
employing the solvent system (1) with a flow rate of 5 mL/
min. Mass spectra (fast-atom bombardment, low-resolution
full scan, glycerol matrix) were performed by the Center for
Mass Spectrometry, University of Arizona, Tucson, AZ. Hy-
drolysis of the peptides was performed in 4 N methanesulfonic
acid (0.2% 3-(2-aminoethyl)indole) at 110 °C for 24 h, and
amino acids were analyzed with an automatic analyzer (Beck-
man Instruments, Model 7300). The results are reported in
Table 5.
The biological results provide some new insights for
the future design of conformationally constrained Dyn
A analogues. The biological results for the new ana-
logues demonstrate that the configuration, position, and
electrochemical properties of the residues that are
incorporated into the cyclic lactam analogues of Dyn A
have great effects on the potency and selectivity for the
κ receptor. It is found that analogues with a D-Asp
incorporated into position 2, 3, or 9 of Dyn A(1-11)-
NH2 are more potent at the central κ receptor than
analogues with an L-Asp incorporated into the same
3
7
positions. The cyclic analogues c[Asp ,Lys ]- and c[D-
3
7
R
Asp ,Lys ]Dyn A(1-11)-NH2 (4 and 5) which displayed
good affinity and increased selectivity for the κ receptor
appear to be lead compounds based on this study. These
results demonstrate that the incorporation of confor-
mational constraints involving residues at the 3 and 7
positions of Dyn A into lactam analogues of Dyn A have
important effects on the selectivity for κ vs µ and/or κ
vs δ receptors. It is suggested that cyclization through
these positions to give conformationally constrained
analogues will have useful effects for potency and
selectivity for κ receptors. To further investigate the
effect of ring size of this global conformation on potency
and selectivity for the κ receptor, the incorporation of
conformational constraints between positions 3 and 6,
Gen er a l Meth od for P ep tid e Syn th esis. The N -Boc
amino acids (4 equiv) were sequentially coupled to the growing
peptide chain using diisopropylcarbodiimide (DIC) and N-
hydroxybenzotriazole (HOBt) in N-methyl-2-pyrrolidone (NMP).
The coupling reaction time was 1 h. Side chain protecting
groups used were 2,4-dichlorobenzyloxycarbonyl or 9-fluore-
nylmethyloxycarbonyl (Fmoc) for Lys, tolylsulfonyl (Tos) for
Arg, 2,6-dichlorobenzyl (2,6-Cl Bzl) for Tyr, and fluorenyl-
2
methyl (Fm) for Asp. Trifluoroacetic acid (TFA, 50% in DCM)
R
was used to remove the N -Boc protecting group, and 20%
piperidine was used to remove the Fmoc side chain protecting
group from Lys and the OFm group from Asp. Diisopropyl-
ethylamine (DIEA) was used as a base, and dichloromethane
(DCM) and NMP were used as solvents for washing. The fully
protected peptide-resin was dried and separated into two
portions for synthesizing the linear and cyclic peptides. The
linear peptide-resin was produced by removing the Fmoc side
chain protecting group from Lys and the OFm group from Asp
3
and 8, or 3 and 9 of Dyn A(1-11)-NH2 may be a
rational approach.
R
and then deprotecting the last N -Boc group. The cyclic
Exp er im en ta l Section
peptides were cyclized on the resin by removing the Fmoc side
chain protecting group from Lys and the OFm group from Asp,
cyclizing to the lactam ring using 3 equiv of BOP reagent and
P ep tid e Syn th esis a n d P u r ifica tion . Peptides were
synthesized by the solid phase method utilizing a Boc/benzyl
strategy and a p-methylbenzhydrylamine (p-MBHA) resin
6
equiv of DIEA in NMP as coupling reagent, reacting for 7-15
R
h at room temperature, and then deprotecting the last N -
Boc group. Each peptide-resin was dried in vacuo, and the
peptide was then cleaved from the resin using liquid anhy-
drous hydrofluoric acid (HF) in the presence of cresol (10%
w/v) for 1 h at 0 °C. After removal of HF in vacuo, the residue
was washed with anhydrous ether and extracted with 30%
aqueous acetic acid. The acetic acid solution was lyophilized
to give a white residue. The crude peptide was then purified
by semipreparative reverse phase HPLC under the conditions
described above to yield a white powder after lyophilization.
The average yield for linear and cyclic peptides are 14% and
(
Advanced Chem Tech, Louisville, KY), as previously described
3
1
R
for dynorphin analogues. Side chain protected N -Boc amino
acids were purchased from Bachem (Torrance, CA), whereas
the other amino acids were synthesized by standard methods
in our laboratory. The analytical data for the purified peptides
are given in Tables 4 and 5.
Thin-layer chromatography of synthetic peptides was per-
formed on silica gel plates (0.25 mm, Analtech, Newark, DE)
with the solvent systems given in Table 4. Peptides were
detected with ninhydrin reagent. HPLC was carried out by
using of a binary pump (Perkin-Elmer LC 250) equipped with
an UV/vis detector (Perkin-Elmer LC 90 UV model) and
integrator (Perkin-Elmer LCI 100 model). The solvent system
used for analytical HPLC was a binary system, water contain-
ing 0.1% of TFA (pH 2.0) and acetonitrile as the organic
modifier, and solvent programs involved linear gradients as
1
1%, respectively.
The structures were corroborated by the results of the amino
acid analysis and mass spectrometry, and the purity of each
product was assessed by analytical HPLC and TLC (Tables 4
and 5).

1
140 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5
Lung et al.
Ta ble 5. Amino Acid Analysis of Various Dyn A Analogues
amino acidsa
Arg
analogues of
Dyn A(1-11)-NH2
Tyr
1.1
Gly
2.0
Phe
Leu
Ile
Pro
0.9
Lys
Asp
Dyn A(1-11)-NH2 (1)
1.1
(1)
1.1
(1)
1.02
(1)
1.01
(1)
1.03
(1)
1.1
(1)
1.0
(1)
1.01
(1)
1.03
(1)
1.01
(1)
1.03
(1)
1.1
(1)
1.1
(1)
0.97
(1)
1.01
(1)
0.96
(1)
2.8
(3)
2.02
(2)
2.01
(2)
2.01
(2)
2.04
(2)
1.9
(2)
1.95
(2)
2.0
(2)
2.04
(2)
1.9
(2)
2.04
(2)
1.9
(2)
1.1
1.0
(1)
1.98
(2)
1.95
(2)
1.97
(2)
2.0
(2)
1.9
(2)
2.0
(2)
2.0
(2)
2.0
(2)
1.95
(2)
2.0
(2)
1.97
(2)
(1)
(2)
1.0
(1)
0.9
(1)
1.01
(1)
0.98
(1)
2.0
(2)
2.1
(2)
0.98
(1)
0.96
(1)
0.98
(1)
0.98
(1)
2.0
(2)
2.1
(2)
(1)
0.8
(1)
0.9
(1)
0.90
(1)
0.90
(1)
1.0
(1)
1.1
(1)
0.90
(1)
0.95
(1)
0.90
(1)
0.90
(1)
1.1
(1)
1.0
(1)
(1)
0.98
(1)
0.98
(1)
0.99
(1)
0.99
(1)
0.99
(1)
0.99
(1)
0.99
(1)
0.99
(1)
0.99
(1)
0.99
(1)
1.0
(1)
1.0
(1)
2
6
c[Asp ,Lys ] (2)
0.9
1)
0.95
(1)
0.95
(1)
0.95
(1)
0.97
(1)
0.9
(1)
0.95
(1)
0.9
(1)
0.97
(1)
0.97
(1)
0.97
(1)
1.1
(1)
0.95
(1)
(
(
(
(
(
(
(
(
(
(
(
(
2
6
c[D-Asp ,Lys ] (3)
0.95
1)
0.95
1)
0.95
1)
1.1
1)
1.2
1)
0.95
1)
0.95
1)
1.1
1)
3
7
c[Asp ,Lys ] (4)
3
7
c[D-Asp ,Lys ] (5)
1.01
(1)
5
9
[
Lys ,Asp ] (6)
5
9
c[Lys ,D-Asp ] (7)
2
6
[
[
[
[
[
[
Asp ,Lys ] (8)
1.01
(1)
1.01
(1)
1.01
(1)
1.01
(1)
2
6
D-Asp ,Lys ] (9)
3
7
Asp ,Lys ] (10)
3
7
D-Asp ,Lys ] (11)
0.95
1)
1.1
1)
1.0
1)
5
9
Lys ,Asp ] (12)
5
9
Lys ,D-Asp ] (13)
1.0
(1)
2.0
(2)
2.0
(2)
a
Theoretical values in parentheses. L- or D-amino acid for Asp. Hydrolysis in 4 N methanesulfonic acid (0.2% 3-(2-aminomethyl)indole)
at 110 °C for 24 h.
Ra d ioliga n d Bin d in g Assa y
two-site fits were made using the F-ratio test using a p value
of 0.05 as the cutoff for significance.33 Data best fitted by a
Membranes were prepared from whole brains taken from
3
4
one-site model was analyzed using the logistic equation. Data
obtained from at least three independent measurements in
adult male guinea pigs (200-400 g) obtained from SASCO
(Omaha, Nebraska). Following decapitation, the brain was
3
5
duplicate are presented as the arithmetic mean ( SEM. The
removed, dissected, and homogenized at 0 °C in 20 volumes
of 50 mM Tris-HCl (Sigma, St. Louis, MO) buffer adjusted to
pH 7.4 using a Teflon-glass homogenizer. The membrane
fraction obtained by centrifugation at 48 000g for 15 min at 4
results are not corrected for the actual peptide content.
In Vitr o GP I Bioa ssa y
°
C was resuspended in 20 volumes of fresh Tris-HCl buffer
and incubated at 25 °C for 30 min to dissociate any receptor
bound endogenous opioid peptides. The incubated homogenate
was centrifuged again as described and the final pellet
resuspended in 10 volumes of fresh Tris-HCl buffer. Radio-
ligand binding inhibition assay samples were prepared in an
assay buffer consisting of 50 mM Tris-HCl, 1.0 mg/mL bovine
serum albumin, 30 µM bestatin, 50 µg/mL bacitracin, 10 µM
captopril, and 0.1 mM phenylmethanesulfonyl fluoride, pH 7.4
Electrically induced smooth muscle contraction of strips of
guinea pig ileum longitudinal muscle-myenteric plexus was
used as bioassay.29 Tissues from male Hartley guinea pigs
weighing 250-500 g were prepared as described previously.36
The tissues were tied to gold chain with suture silk, suspended
in 20 mL baths containing 37 °C oxygenated (95% O
Krebs buffer (118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl
mM KH PO , 1.18 mM MgSO , 25 mM NaHCO , and 11.48
2
, 5% CO
2
)
2
, 1.19
2
4
4
3
(all from Sigma, St. Louis, MO, except bestatin that was
mM glucose), and allowed to equilibrate for 15 min. The
tissues were then stretched to optimal length previously
determined to be 1 g tension and again allowed to equilibrate
for 15 min. The tissues were stimulated transmurally between
platinum wire electrodes at 0.1 Hz, 0.4-ms pulses of supra-
maximal voltage. Drugs were added to the baths in 15-60
µL volumes. The agonists remained in contact with the tissue
for 3 min before the addition of the next cumulative dose, until
maximum inhibition was reached. Percent inhibition was
calculated by using the average contraction height for 1 min
preceding the addition of the agonist divided by the contraction
height 3 min after exposure to the dose of the agonist. To
further define the opioid selectivity of the agonist effect, the µ
selective antagonist CTAP was used at the concentration of
purchased from Peptides International, Louisville, KY). The
3
2
4
5
radioligand used were [ H]c[D-Pen ,p-Cl-Phe ,D-Pen ]enkeph-
alin3 (δ receptor) at a concentration of 0.75 nM, [ H]DAMGO
2
3
3
(
µ receptor) at a concentration of 1.0 nM, and [ H]U-69,593 (κ
receptor) at a concentration of 1.5 nM (all obtained from New
England Nuclear, Boston, MA). Peptides were dissolved in
the assay buffer prior to each experiment and added to
duplicate assay tubes at 10 different concentrations over an
8
00-fold concentration range. Control (total) binding was
measured in the absence of any inhibitor, while nonspecific
binding was measured in the presence of 10 µM naltrexone
(Sigma, St. Louis, MO). The final volume of the assay samples
was 1.0 mL, of which 10% consisted of the membrane prepara-
tion in 0.1 mL of Tris-HCl buffer. Incubation was performed
at 25 °C for 3 h after which the samples were filtered through
polyethylenimine (0.5% w/v, Sigma, St. Louis, MO)-treated
GF/B glass fiber filter strips (Brandel, Gaithersburg, MD). The
filters were washed three times with 4.0 mL of ice-cold 1 M
NaCl solution before transfer to scintillation vials. The filtrate
radioactivity was measured after adding 7-10 mL of cocktail
1
000 nM.37 IC50 values represent the mean of no less than
four tissues. IC50 estimates, relative potency estimates, and
their associated standard errors were determined by fitting
the mean data to the Hill equation by a computerized
nonlinear least-square method.35 The results are not corrected
for the actual peptide content.
(
EcoLiteTM (+), ICN Biomedicals, Inc) to each vial and
allowing the samples to equilibrate over 8 h at 4 °C.
Ack n ow led gm en t. This work was supported by a
grant from the National Institutes on Drug Abuse DA
04248.
Binding data was analyzed by nonlinear least-square re-
gression analysis using the program Inplot 4.03 (GraphPadTM,
San Diego, CA). Statistical comparisons between one- and

Analogues of Dynorphin A(1-11)-NH
2
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5 1141
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