The effects of C-terminal modifications on the opioid activity of [N-benzylTyr1]dynorphin A-(1-11) analogues

Article abstract of DOI:10.1021/jm900715m

Structural modifications affecting the efficacy of analogues of the endogenous opioid peptide dynorphin (Dyn) A have focused on the N-terminal "message" sequence based on the "message-address" concept. To test the hypothesis that changes in the C-terminal

Full text of DOI:10.1021/jm900715m

6814 J. Med. Chem. 2009, 52, 6814–6821
DOI: 10.1021/jm900715m
The Effects of C-Terminal Modifications on the Opioid Activity of [N-BenzylTyr1]Dynorphin
A-(1-11) Analogues
Kshitij A. Patkar,† Thomas F. Murray,‡ and Jane V. Aldrich*,†
†Department of Medicinal Chemistry, University of Kansas, Lawrence, Kansas 66045, and ‡Department of Pharmacology, Creighton University
School of Medicine, Omaha, Nebraska 68178
Received May 26, 2009
Structural modifications affecting the efficacy of analogues of the endogenous opioid peptide dynorphin
(Dyn) A have focused on the N-terminal “message” sequence based on the “message-address” concept. To
test the hypothesis that changes in the C-terminal “address” domain could affect efficacy, modified amino
acids and cyclic constraints were incorporated into this region of the partial agonist [N-benzylTyr1]Dyn
A-(1-11). Modifications in the C-terminal domain of [N-benzylTyr1]Dyn A-(1-11)NH2 resulted in
increased κ opioid receptor (KOR) affinity for all of the linear analogues but did not affect the efficacy of
these peptides at KOR. Cyclization between positions 5 and 8 yielded [N-benzylTyr1,cyclo(D-Asp5,
Dap8)]Dyn A-(1-11)NH2 (zyklophin, 13) (J. Med. Chem. 2005, 48, 4500-4503) with high selectivity for
KOR. In contrast to the linear peptides, this peptide exhibits negligible efficacy in the adenylyl cyclase (AC)
assay and is a KOR antagonist. These data are consistent with our hypothesis that appropriate
modifications in the “address” domain of Dyn A analogues may affect efficacy.
Introduction
the treatment of depression,11-13 anxiety,14,15 and opiate
and cocaine addiction.12,16,17 The KOR selective peptide
antagonist arodyn18 designed in our laboratory suppresses
stress-induced reinstatement of cocaine seeking behavior
in mice,17 suggesting its potential use in the development
of peptide-based therapeutics for the treatment of cocaine
addiction.
The heptadecapeptide dynorphin (Dyn) A (Figure 1) is an
endogenous agonist for KOR.19 Studies of Dyn A and its
fragments by Chavkin and Goldstein led them to propose the
“message-address” concept in which the four N-terminal
amino acids in Dyn A are the “message” sequence responsible
for opioid agonist activity and the C-terminal domain is the
“address” that enhances the affinity of this peptide for
KOR.20 The entire C-terminal sequence is not required for
KOR interaction, however, and the first 11 to 13 residues of
Dyn A account for essentially all of the opioid activity of the
longer peptide.20
Weareinterested inexploringthe structuralfeatures of Dyn
A that affect efficacy and result in peptide antagonists for
KOR. Structural modifications in the N-terminus of Dyn A
can alter efficacy as well as potency and selectivity. Alkylation
of the N-terminus can influence both KOR affinity and
efficacy. WhiletheN,N-diallylTyr1 analoguesof[D-Pro10]Dyn
A-(1-11) and Dyn A-(1-13) exhibit antagonist activity at
KOR and MOR21-23 (but with decreased binding affinity and
selectivity for KOR compared to the parent peptides without
the N-terminal modification), the monoalkylated derivatives
have high KOR affinity and selectivity.23,24 The N-allyl- and
N-cyclopropylmethyl analogues are potent agonists in the
guinea pig ileum (GPI) assay, while the N-benzyl analogue
exhibits weak agonist activity in the GPI assay23,24 and is a
partial agonist in the adenylyl cyclase (AC) assay using cloned
KOR expressed on Chinese hamster ovary (CHO) cells.25
Other modifications in the N-terminal sequence of Dyn
Narcotic analgesics that target μ opioid receptors (MORa)
have been used extensively in the clinical management of
pain,1 but their use is complicated by serious side effects.2
Thus there has been considerable interest in the development
of ligands for κ opioid receptors (KOR) as potential thera-
peutic agents for pain and other disorders.3 KOR agonists can
produce analgesia with less addiction liability and respiratory
depression than MOR agonists,4,5 and compounds with KOR
agonist activity are used clinically (e.g., pentazocine). How-
ever, KOR agonists can also cause dysphoria in humans.6 In
addition to the centrally mediated analgesia, KOR agonists
can produce antinociception through peripheral KOR7 and
peripherally selective KOR agonists are in clinical trials as
analgesics.8 Moreover, KOR agonists also exhibit anti-in-
flammatory activity.9 Other potentially useful therapeutic
effects of KOR agonists include anticonvulsant and neuro-
protective effects and their ability to down-regulate HIV-1
expression in human microglial and CD4 cells (see ref 10
for a review). KOR antagonists, initially used only as phar-
macological tools, also have potential therapeutic uses in
*To whom the correspondence should be addressed. Phone: (785) 864
2287. Fax: (785) 864 5326. E-mail: jaldrich@ku.edu.
a Abbreviations: AC, adenylyl cyclase; Boc, tert-butyloxycarbonyl;
CHO, Chinese hamster ovary; Dap, 2,3-diaminopropionic acid; DOR, δ
opioid receptor; DMF, N,N-dimethylformamide; Dyn, dynorphin; EL,
extracellular loop; Fmoc, 9-fluorenylmethoxycarbonyl; GPI, guinea pig
ileum; HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate; HOAt, 1-hydroxy-7-azabenzotriazole; KOR, κ
opioid receptor; MOR, μ opioid receptor; Mtr, 4-methoxy-2,3,6-tri-
methylbenzenesulfonyl; Mtt, 4-methyltrityl; NMM, N-methylmorpho-
line; NMM, N-methylmorholine; PAC, peptide acid linker; PAL,
peptide amide linker; Pbf, 2,2,4,6,7-pentamethyl-dihydrobenzofurane-
5-sufonyl; PEG-PS, poly(ethylene glycol)-polystyrene; Pip, 2-phenyl-
isopropyl; PyAOP, 7-azabenzotriazol-1-yloxytris(pyrrolidino)phos-
phonium hexafluorophosphate; TFA, trifluoroacetic acid; Tis,
triisospropylsilane; Trt, trityl.
r
pubs.acs.org/jmc
Published on Web 10/06/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6815
Figure 1. Dynorphin A.
A-(1-11) analogues that result in antagonist activity at KOR
are Pro in position 3,26 and the removal of the basic R-amine
of Tyr1 in analogues such as dynantin.27 Other analogues with
antagonist activity, namely JVA-901,28 arodyn,18 and
cycloN,5[Trp3,Trp4,Glu5]Dyn A-(1-11)NH2,29 prepared in
our laboratory, also lack a basic N-terminus.
Reports in the literature examining modifications in the
C-terminal or “address” sequence of Dyn A suggest that in
selected cases such modifications may affect the efficacy of
these analogues at KOR. While analogues of [Phe1]Dyn
A-(1-11)NH2 with D-Ala8 and D-Pro10 substitutions retain
similar KOR affinity (<50% difference), they exhibit large
differences in potency in the GPI30(almost a 50-fold decrease
fortheD-Ala8 analogue compared to the parent peptide). Such
discrepancies in affinity and potency suggest a possible role of
the C-terminus in the efficiency of receptor activation. A
variety of cyclic analogues of Dyn A, cyclized either with a
disulfide or lactam linkage, have been prepared to restrict
conformational freedom and study possible bioactive con-
formations (see refs 3 and 31 for reviews). Interestingly, some
of these analogues also display discrepancies between KOR
affinity and potency in the GPI. Cyclic analogues with
disulfide linkages between residues 5 and 11 ([5,11]), which
may stabilize a reverse turn in the C-terminal region of Dyn
A-(1-13)NH2, were reported to possess high KOR affinity in
brain homogenate but low potency in the GPI assay, which
the authors suggested could be due to different interactions
with central vs peripheral KOR.32 Similarly, a series of lactam
bridged [5,8] cyclic analogues of Dyn A-(1-13)NH2 with varying
ring sizes prepared in our laboratory exhibit much lower potency
in the GPI than anticipated based on their KOR binding affinity.33
Also, while the [5,8] cyclic analogues exhibit similar binding
affinities for KOR, they varied substantially in potency in the GPI
(IC50 = 530 - >5000 nM). Interestingly, a computational model
suggests a possible role for the hydrophobic residues Leu5 and Ile8
in the “address” sequence of Dyn A-(1-10) in KOR activation.34
Molecular simulation of Dyn A-(1-10) bound to KOR suggested
that these two residues interact with hydrophobic residues in
extracellular loop (EL) 2 of KOR.34 On the basis of this model,
it was proposed that these hydrophobic interactions may play a
role in the efficacy and/or affinity of Dyn A for this receptor. In
contrast, [6,9] cyclic analogues exhibit similar KOR affinities and
are potent agonists in the GPI (IC50 = 7-46 nM).33
On the basis of these results, we hypothesized that
C-terminal residues and/or the conformation of the C-term-
inal domain of Dyn A, while not a primary determinant, could
affect the efficacy of Dyn A analogues. To explore the possible
roles of the C-terminus in efficacy, we introduced a series of
modifications into [N-benzylTyr1]Dyn A-(1-11) (Figure 2).
This peptide was chosen because [N-benzylTyr1,D-Pro10]Dyn
A-(1-11) exhibits partial agonist activity in the AC assay
(71% relative to Dyn A-(1-13)NH2),25 and therefore we
could readily detect changes in efficacy. We anticipated that
selected changes in the C-terminal domain of the peptide
[N-benzylTyr1]Dyn A-(1-11) could alter the efficacy of
these analogues at KOR. Because the basic residues in the
C-terminal sequence of Dyn A were thought to be important
in KOR interaction,20 the modifications were mainly carried
out in the nonbasic residues in the “address”. On the basis of
the results with [Phe1]Dyn A-(1-11)NH2 analogues discussed
Figure 2. Dyn A-(1-11) and its analogues. Modifications in pep-
tides 4-16 were made in the “address” sequence (italicized in 3).
Structural differences from peptide 5 are highlighted in bold in
subsequent analogues.
above, we chose D-Ala8 and D-Pro10 as modifications to
incorporate into the C-terminal domain. To retain the im-
portant Arg side chain but introduce a modification that
could alter peptide backbone conformation, NR-MeArg was
also incorporated in position 7. On the basis of the lactam
bridged cyclic Dyn A analogues cyclo[D-Asp5,Dap8]- and
cyclo[D-Asp6,Dap9]Dyn A-(1-13)NH2 synthesized pre-
viously in our laboratory,33 we also examined cyclic con-
straints between the fifth and eighth and between the
sixth and ninth positions in the C-terminal domain to evaluate
their effect on efficacy at KOR. These analogues were eval-
uated for their opioid receptor affinity in radioligand binding
assays and for opioid activity in the AC assay using cloned
KOR stably expressed on CHO cells.25 Among these, the [5,8]
cyclic analogue [N-benzylTyr1,cyclo(D-Asp5,Dap8)]Dyn A-
(1-11)NH2 (which we have named zyklophin) exhibits neg-
ligible efficacy at KOR and acts an antagonist in the AC assay
at KOR, as we reported in an earlier communication.35 Here
we describe the synthesis and report the pharmacological
profiles of the full series of C-terminally modified [N-benzyl-
Tyr1]Dyn A-(1-11) analogues.
Chemistry
The peptides were synthesized using the Fmoc (9-fluor-
enylmethoxycarbonyl) solid phase synthetic strategy
(Scheme 1). N-Benzyltyrosine was synthesized separately in
solution and then coupled to the peptide sequences. Selective
monobenzylation of tyrosine tert-butyl ester was carried
out by reductive amination with benzaldehyde using
sodium cyanoborohydride in an acidic solution, followed by
deprotection of the tert-butyl ester using trifluoroacetic acid
(TFA) to give N-benzyltyrosine. The Dyn A-(2-11) se-
quences were assembled on a poly(ethylene glycol)-polystyr-
ene (PEG-PS) resin containing the peptide amide linker [PAL,

6816 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Patkar et al.
Scheme 1. Solid Phase Synthesis of Linear [N-benzylTyr1]Dyn A-(1-11) Analogues (1-12)
5-(4-aminomethyl-3,5-dimethoxyphenoxy)valeric acid linker]
for the amides or the peptide acid linker [PAC, (hydroxy-
methyl)phenoxyacetic acid linker] for the acid derivatives 1, 3,
and 4, using 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl-
uronium hexafluorophosphate (HBTU) and N-methylmor-
pholine (NMM) in N,N-dimethylformamide (DMF) to cou-
ple Fmoc-amino acids to the growing peptide chain.28 The
side chain protecting groups used were Pbf (2,2,4,6,7-penta-
methyl-dihydrobenzofurane-5-sufonyl) and Mtr (4-methoxy-
2,3,6-trimethylbenzenesulfonyl) for the side chains of Arg and
NR-Me-Arg, respectively, and Boc (tert-butyloxycarbonyl) for
Lys. The coupling of Fmoc-Arg(Pbf) at position 6 to NR-Me-
Arg(Mtr) at position 7 and the coupling of N-benzyltyrosine to
the resin-bound Dyn A-(2-11) sequences were carried out using
7-azabenzotriazol-1-yloxytris(pyrrolidino)phosphonium hexa-
fluorophosphate (PyAOP),36 1-hydroxy-7-azabenzotriazole
(HOAt), and N,N-diisopropylethylamine (DIEA) in DMF.
The cyclic peptides were assembled (Scheme 2) similar to
the linear analogues except that Fmoc-D-Asp(Pip) (Pip=2-
phenylisopropyl) and Fmoc-Dap(Mtt) (Dap=2,3-diamino-
propionic acid and Mtt = 4-methyltrityl) were used in the
positions involved in cyclization. The hyperacid-labile Pip37
and Mtt38 protecting groups could be removed by dilute (3%)
TFA in the presence of the Boc and other tert-butyl type
side chain protecting groups that require stronger acidic
conditions for deprotection. The cyclizations between the
side chain carboxylic acid of D-Asp and the side chain free
amine of Dap were carried out using PyAOP, HOAt,
and DIEA in DMF. The cyclization between D-Asp in posi-
tion 6 and Dap in position 9, however, did not go to comple-
tion after 48 h, even using 6 equiv of the coupling reagents, so
the remaining unreacted amine was acetylated with N-acet-
ylimidazole and DIEA in DMF (Scheme 2). After cyclization,
the remaining amino acids were then coupled to complete the
peptide sequences. For the synthesis of the acetylated linear
analogues 15 and 16, Fmoc-D-Asn(Trt) (Trt = trityl) and
Fmoc-Dap(Mtt) were used; following deprotection of the
Scheme 2. Solid Phase Synthesis of Cyclic Peptide 14
Mtt group with dilute TFA, the free amine of Dap was
acetylated using N-acetylimidazole in DMF.
The peptides were cleaved from the resin using either
reagent B39 or modified reagent K40 (when NR-MeArg(Mtr)
was present in the peptide sequence). The crude peptides were
purified to g98% by preparative reversed phase HPLC; they
were analyzed for purity by reversed phase HPLC and their
molecular weights confirmed by ESI-MS (see Supporting
Information).

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6817
Table 1. Opioid Receptor Binding Affinities of Dyn A-(1-11)OH Analogues
Ki (nM) ( SEM
Ki ratio
peptide
KOR
MOR
DOR
KOR/MOR/DOR
1 Dyn A-(1-11)
2 Dyn A-(1-11)NH2
1.11 ( 0.36
0.57 ( 0.01
14.8 ( 4.5
15.5 ( 1.0
8.17 ( 2.16
3.50 ( 0.42
1.85 ( 0.52
98.1 ( 9.8
159 ( 30
8.18 ( 0.09
6.18 ( 1.01
345 ( 12
970 ( 131
474 ( 47
1/3/7
1/3/11
1/7/23
1/10/63
1/10/58
3 [N-benzylTyr1]Dyn A-(1-11)OH
4 [N-benzylTyr1,D-Pro10]Dyn A-(1-11)OH
5 [N-benzylTyr1]Dyn A-(1-11)NH2
84.9 ( 16.5
Analogues of [N-benzylTyr1]Dyn A-(1-11)NH2, 5
6 [NR-MeArg7]
7 [D-Ala8]
3.99 ( 1.09
4.64 ( 0.91
4.56 ( 0.42
3.11 ( 0.64
2.17 ( 0.22
3.10 ( 0.98
4.40 ( 0.75
30.3 ( 1.9
245 ( 37
49.9 ( 9.7
51.2 ( 3.0
45.1 ( 4.9
44.8 ( 7.3
36.5 ( 3.6
46.1 ( 8.6
69.8 ( 0.7
5880 ( 1420
1190 ( 270
1660 ( 440
1500 ( 310
345 ( 31
372 ( 25
148 ( 5
458 ( 130
120 ( 24
337 ( 54
665 ( 174
1/13/86
1/11/80
1/10/32
1/14/147
1/17/55
1/15/109
1/16/151
1/194/>330
1/5/15
8 [D-Pro10
]
9 [NR-MeArg 7,D-Ala8]
10 [NR-MeArg 7,D-Pro10
]
11 [D-Ala8,D-Pro10
]
12 [NR-MeArg 7,D-Ala8,D-Pro10
]
13a cyclo[D-Asp5,Dap8] (zyklophin)
14 cyclo[D-Asp6,Dap9]
>10,000
3660 ( 660
>10,000
4640 ( 340
15a [D-Asn5,Dap(Ac)8]
16 [D-Asn6,Dap(Ac)9]
66.9 ( 5.9
2840 ( 300
1/25/149
2/1/3
a From ref 35.
Figure 4. Efficacies of [N-benzylTyr1]Dyn A-(1-11) analogues as
inhibitors of adenylyl cyclase (AC) in CHO cells expressing KOR.
Percent AC inhibition relative to Dyn A-(1-13)NH2 (100%).
Figure 3. KOR affinities of [N-benzylTyr1]Dyn A-(1-11) Analo-
gues.
Results and Discussion
position 10 did not change the KOR affinity for analogue 4
but decreased affinity for DOR, thus improving KOR vs
DOR selectivity. Interestingly, converting the C-terminal acid
in analogues 3 and 4 to an amide (peptides 5 and 8, re-
spectively) increased KOR affinity slightly (2- to 3-fold).
EL2 of KOR, which has been implicated in Dyn A affinity
and selectivity for KOR,34,41 contains a unique cluster of
acidic residues that is not present in human and rodent
MOR or DOR.42,43 An increase in the KOR affinity upon
amidation may be due to the neutralization of unfavorable
interactions between the C-terminal acid with the acidic
residues on the EL2 of KOR. Conversion of the acid 3 to
the amide 5 also increased selectivity for KOR over DOR
3-fold. In contrast, the corresponding conversion of the acid
in the D-Pro10 peptide 4 to give the amide (analogue 8) in-
creased DOR affinity 6-fold, thus decreasing KOR vs DOR
selectivity.
In the linear analogues 6-12 modifications that could
affect the C-terminal conformation were well tolerated by
KOR, with these analogues exhibiting 2- to 4-fold higher
KOR affinitythanthe parent peptide 5 (Table1 and Figure3).
The introduction of a single modification in the C-terminus of
[N-benzylTyr1]Dyn A-(1-11)NH2 (analogues 6-8) resulted
in similar small increases (about 2-fold) in both KOR and
MOR affinities regardless of the substitution introduced.
The only notable difference in the affinities of these three
The [N-benzylTyr1] Dyn A-(1-11) analogues were evalu-
ated for KOR, MOR, and δ opioid receptor (DOR) affinity in
radioligand binding assays (Table 1 and Figure 3) and for
efficacy in the AC assay using cloned rat KOR stably ex-
pressed on CHO cells (Figure 4). N-Benzylation of Dyn
A-(1-11) and [D-Pro10]Dyn A-(1-11) resulted in peptides 3
and 4, respectively, with Ki values of 15 nM for KOR using
[3H]diprenorphine as the radioligand. These results are com-
parable to those we reported previously for 4 (Ki = 18 nM).25
In contrast to other N-alkyl Dyn A analogues, the KOR
affinity determined for the [N-benzylTyr1,D-Pro10]Dyn
A-(1-11) (4) depends on the radioligand used,25 and the 14-
fold decrease upon N-benzylation observed here was not
found when the KOR affinity of 4 was determined using
[3H]bremazocine in guinea pig cerebellum.23,24 N-Benzylation
of Dyn A-(1-11) (1) and Dyn A-(1-11)NH2 (2) resulted in
analogues
3 and 5, respectively, that exhibit 2- to
3-fold increases in KOR vs MOR selectivity and 3- to 5-fold
increases in KOR vs DOR selectivity compared to 1 and 2
(Table 1) due to greater decreases in the affinities for MOR
and DOR compared to those for KOR.
We initially prepared both [N-benzylTyr1]Dyn A-(1-11)
acid and amide derivatives (3 and 5, respectively) for compar-
ison to [N-benzylTyr1,D-Pro10]Dyn A-(1-11) (4), which we
described previously.23-25 Compared to peptide 3, D-Pro in

6818 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Patkar et al.
analogues was the 3-fold increase in DOR affinity as a result
of incorporating D-Pro (analogue 8). Introduction of an
additional modification in the C-terminus yielded analogues
9-11 with similar to slightly (up to 2-fold) higher KOR
affinity than the monosubstituted analogues 6-8; analogue
12 with all three modifications exhibits KOR affinity similar
to the monosubstituted derivatives 6-8 (Table 1 and
Figure 3).
The linear analogues 15 and 16 were designed to evaluate
the effect of the substitutions that were used to carry out the
cyclizations between positions 5 and 8 or 6 and 9, respec-
tively, on affinity and efficacy. To avoid effects due to the
introduction of charged groups in the side chains, Asn and
Dap(Ac) were introduced into these positions. Substitution of
Asn and Dap(Ac) in positions 5 and 8, respectively, of 5
resulted in an 8-fold decrease in KOR affinity and a much
larger decrease in MOR affinity compared to analogue 5. As
reported earlier,35 the [5,8] cyclization resulted in increases in
KOR affinity and KOR vs MOR selectivity compared to the
linear analogue 15. The linear peptide 16 with substitutions in
positions 6 and 9 exhibits extremely low affinity for all three
opioid receptors (Table 1). However, cyclization between
these positions led to an 11-fold gain in KOR affinity for
analogue 14 compared to 16, while the MOR affinity for 14
remained similar to the corresponding linear analogue 16.
Thus the conformation induced by the cyclic constraint does
not appear to be responsible for the low KOR affinity of 14.
These analogues were evaluated for their efficacy in the AC
assay usingclonedratKOR stablyexpressed on CHO cells. At
concentrations of 10 μM the linear peptides generally exhibit
partial to full agonist activity at KOR (Figure 4), similar to
that originally reported for peptide 3 (71% maximum inhibi-
tion of AC relative to Dyn A-(1-13)NH2).25 The linear
analogues with different C-terminal modifications (3-12)
showed very similar maximum inhibition of AC (70-90%
relative to Dyn A-(1-13)NH2) regardless of the type of
substitution or number of substitutions. Thus, these changes
in the C-terminal domain (NR-MeArg7, D-Ala8, D-Pro10, and/
or C-terminal amidation) of linear [N-benzylTyr1]Dyn
A-(1-11) analogues had little effect on the efficacy of the
analogues at cloned KOR in the AC assay. In contrast, the
cyclic analogue zyklophin (13) and the corresponding linear
analogue 15 show minimal efficacy in the AC assay, and as
reported previously, zyklophin exhibits antagonist activity
(KB=84 nM) at KOR in this assay. The linear analogues 14
and 16 were not tested for efficacy due to their low KOR
affinities.
These modifications affected MOR and DOR affinities
differently (Table 1). The MOR affinities of the linear ana-
logues 6-12 were similar to one another, regardless of the
substitutions, and generally about 2-fold higher than the
parent peptide 5. Because these changes in MOR affinity
generally paralleled those for KOR, the selectivity for KOR
over MOR varied by only 2-fold among these analogues. The
changes in DOR affinity, however, were dependent on the
type of substitution. While the NR-MeArg7 and D-Ala8 mod-
ifications improved selectivity for KOR over DOR slightly, D-
Pro10 increased DOR affinity in the C-terminal amide series,
thereby reducing KOR vs DOR selectivity. In peptides 9-12
containing substitutions in addition toD-Pro10, D-Ala8 but not
NR-MeArg7 alone decreased DOR affinity and restored se-
lectivity for KOR over DOR. Thus, analogue 11 containing D-
Ala8 retained KOR vs DOR selectivity, while the NR-MeArg7
analogue 10 exhibited higher DOR affinity and therefore
lower KOR selectivity. The addition of D-Ala8 to
NR-MeArg7 and D-Pro10, resulting in analogue 12, restored
the high KOR vs DOR selectivity. Thus, substitution of D-Ala
in position 8 appears to be important for maintaining high
selectivity for KOR over DOR in these analogues.
In the cyclic peptides there was a marked difference in
KOR affinity depending upon the position of the cyclic
constraint. As reported previously,35 the [5,8] cyclic peptide
zyklophin (13) shows approximately a 4-fold decrease in
KOR affinity (Ki = 30 nM) compared to the linear peptide
5. Cyclization between residues 6 and 9 (cyclic analogue 14)
led to a much larger decrease in KOR affinity (Ki = 245 nM)
compared to the linear analogue 5. Zyklophin and 14 both
show drastic decreases in MOR and DOR affinities compared
to the linear peptide 5. The decreases in MOR and DOR
affinities were larger as a result of the [5,8] cyclization than due
to the [6,9] cyclization. Thus the [5,8] cyclic analogue zyklo-
phin shows much higher affinity for KOR and much lower
affinities for MOR and DOR compared to the [6,9] cyclic
analogue 14, resulting in much greater KOR selectivity
(Table 1). The affinities for all three opioid receptors for
zyklophin (13) and analogue 14 with N-benzylation of
Tyr1 are lower than those for the unbenzylated cyclo[D-
Asp5,Dap8]- (17) and cyclo[D-Asp6,Dap9]Dyn A-(1-13)NH2
(18).33 For the [5,8] cyclic derivative zyklophin the KOR
affinity is 4-fold lower than that of 17 (Ki = 8 nM), but for
the [6,9] cyclic derivative 14 the KOR affinity is 100-fold
lower than that of 18 (Ki = 2.6 nM), suggesting that the steric
bulk of the N-benzyl group may have shifted 14 in the KOR
binding site relative to 18, leading to the large decrease in
affinity. Interestingly, zyklophin shows much higher KOR
selectivity over MOR compared to analogue 17 (Ki ratio
(KOR/MOR/DOR) = 1/9/412)33 that does not possess the
benzyl group on its N-terminal amine. Only a modest increase
in selectivity for KOR over MOR and DOR upon N-benzyla-
tion of Tyr1 was observed for the cyclic analogue cyclo[D-
Asp2,Dap5]Dyn A-(1-11)NH2 (Ki = 3.8 nM, Ki ratio (KOR/
MOR/DOR) = 1/5/56 for the N-benzylTyr1 derivative vs Ki
ratio = 1/1.1/11 for the unbenzylated analogue).44
Conclusions
Various C-terminal modified analogues of [N-benzyl-
Tyr1]Dyn A-(1-11) were synthesized to evaluate the effect
of these modifications on opioid receptor affinity and efficacy.
The effects on opioid receptor affinity varied depending upon
the type of substitution and the receptor type. Interestingly,
introducing a C-terminal amide functional group appeared to
enhance KOR affinity slightly. While N-benzylation substan-
tially decreased KOR affinity in the linear analogues, incor-
poration of additional modifications in the C-terminus
increased KOR affinity by 2- to 4-fold in peptides 6-12
(Table 1 and Figure 3) such that the highest affinity
N-benzylated analogue had relatively high KOR affinity
(Ki=2.2 nM for peptide 10). Generally, the effects on MOR
affinity were similar so that the selectivity of 6-12 for KOR
over MOR was within 2-fold that of 5. The effects of the
substituents on DOR affinity were more variable.
The most drastic changes in opioid receptor affinities
occurred with the [5,8] and [6,9] cyclic analogues. Modifica-
tions in positions 5 and 8 were tolerated by KOR but not by
MOR or DOR, leading to a large increase in KOR selectivity
for zyklophin (13). The KOR affinity of zyklophin is only
4-fold lower compared to the cyclic peptide 17 without an
N-benzyl group. In contrast, cyclization between positions 6

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6819
and 9 led to a very large decrease in KOR affinity. Peptide 18
without an N-benzyl group exhibits nanomolar affinity for
KOR (Ki = 2.6 nM), suggesting that N-terminal alkylation
shifts the peptide in the binding site, resulting in the lower
KOR affinities of 14 and 16.
There was a marked difference in the effects of the mod-
ifications in the linear analogues 3-12 vs peptides 13 and 15
on efficacy in the AC assay. The linear analogues 3-12 all
exhibit similar efficacies regardless of the modifications in
positions 7, 8, and/or 10 that could affect peptide conforma-
tion. In contrast, the modifications in positions 5 and 8 in
peptides 13 and 15 eliminated efficacy at KOR. These results
are consistent with our hypothesis that appropriate modifica-
tions in the C-terminal domain can alter the efficacy of Dyn A
analogues at KOR. To further elucidate the role of the
conformational constraint and stereochemistry on efficacy,
we are preparing a variety of analogues of zyklophin.
Because of their target specificity and high potency, opioid
peptides and their analogues could be useful lead compounds
forthe development oftherapeutic agents. Thetherapeutic use
of peptide-based drugs has been limited because of their low
oral bioavailability. However, alternative routes of adminis-
tration such as inhalation45 are being explored that could
increase the acceptance of agents that are not taken orally.
Structural modifications to the endogenous peptides are
necessary to impart sufficient metabolic stability for systemic
administration and for the development of clinically useful
peptides. Incorporation of unnatural amino acids along with
conformational constraints, as are found in zyklophin, pro-
vides the improved enzymatic stability needed for in vivo
studies. Metabolically stable, highly potent, and KOR-selec-
tive peptide analogues can be used for a variety of central or
peripheral effects depending upon their penetration of the
blood-brain barrier.10 Dyn A-(1-11) analogues synthesized
in our laboratory have shown blood-brain barrier penetra-
tion in the in vitro bovine brain microvessel endothelial cell
(BBMEC) model.46 KOR selective peptide antagonists such
as zyklophin can serve as lead compounds for the develop-
ment of potential treatments for opiate and cocaine addiction.
Additional studies with this promising leadpeptideantagonist
are ongoing in our laboratories.47
Purification and Analysis of the Peptides. The crude peptides
were purified by preparative reversed phase HPLC (Rainin
HPXL HPLC system equipped with a Shimadzu SPD-
˚
10A detector) on a Vydac C18 column (10 μ, 300 A, 21
mm ꢀ 250 mm) equipped with a Vydac guard cartridge. For
purification, a linear gradient of 10-60% MeCN containing
0.1% TFA over 50 min, at a flow rate of 20 mL/min, was used.
The purification was monitored at 214 nm. The purity of the
final peptides was verified on a Vydac 218-TP column (5 μ,
˚
300 A, 4.6 mm ꢀ 250 mm) equipped with a Vydac guard
cartridge using a Beckman System Gold analytical HPLC,
consisting of a model 126 programmable solvent module and
a model 168 diode array detector module. Two systems, a linear
gradient of 10-60% solvent B (solvent A, aqueous 0.1% TFA,
and solvent B, MeCN containing 0.1% TFA) over 50 min, at a
flow rate of 1 mL/min (system 1), and a linear gradient of
5-80% solvent B (solvent A, aqueous 0.1% TFA, and solvent
B, MeOH containing 0.1% TFA) over 50 min, at a flow rate of
1.5 mL/min (system 2), were used for the analysis. The final
purity of all peptides by both analytical systems was generally
g98% (see Supporting Information).
Peptide Synthesis: General Procedure for Peptides 1-12, 15,
and 16. All of the peptides were synthesized by coupling a 4-fold
excess of Fmoc-protected amino acids (200 mM) on a Sym-
phony multiple peptide synthesizer (Rainin, Inc.) using DMF as
the solvent or a 3-fold excess of Fmoc-protected amino acids
using a 1:1 mixture of DCM and DMF on a manual multiple
peptide synthesizer (“CHOIR”) constructed in house.48 Pep-
tides 2, 5-12, 15, and 16 were assembled on a high load PAL-
PEG-PS resin (0.4 mmol/g, 200 mg), and peptides 13 and 14
were assembled on the low load PAL-PEG-PS resin (0.17 mmol/
g, 200 mg). Peptides 1, 3, and 4 were assembled on an Fmoc-Lys-
(Boc)-PAC-PEG-PS (0.19 mmol/g, 200 mg). The side chains of
the amino acids Lys, Arg, and NR-MeArg were protected by
Boc, Pbf, and Mtr groups, respectively. Following removal of
the Fmoc protecting group using 20% piperidine in DMF for
20 min, the amino acids were coupled using HBTU (equimolar
to the amino acid) and NMM (4-fold excess relative to the amino
acid) as the coupling reagents for 2 h unless otherwise noted. N-
Benzyltyrosine (1.5-3 equiv) was coupled to the resin-bound
peptides using PyAOP, HOAt, and DIEA (1:1:2 relative to the
amino acid) in DMF (2-3 mL). Prior to coupling, complete
dissolution of N-benzyltyrosine was achieved by warming the
solution to 90 °C, followed by cooling to about 50-60 °C and
then adding PyAOP, HOAt, and DIEA. Completion of the
coupling reactions was indicated by the qualitative ninhydrin
test and/or chloranil test.49 During the synthesis of the Dyn
A-(2-11) sequences, coupling of Fmoc-Arg(Pbf)OH (6-fold
excess) in position 6 to NR-MeArg(Mtr) at position 7 of the
Dyn A-(7-11) segment was carried out manually using PyAOP,
HOAt, and DIEA (1:1:2 relative to the amino acid) in a 1:1
mixture of DMF and DCM. The coupling was complete in
12-24 h, as indicated by the chloranil test.
For peptides 15 and 16, after coupling Fmoc-Dap(Mtt) at
position 8 or 9, respectively, the Mtt group was selectively
removed from the Dap side chain as follows: after swelling of
the peptide-resin for 5 min in DCM (5 mL), the resin was treated
with a mixture of 3% TFA and 5% TIS in DCM (3 ꢀ 10 min,
5 mL each). The resin was subsequently washed first with DCM
and then with DMF (5 ꢀ 10 mL each). The free amine of the Dap
side chain was then acetylated using N-acetylimidazole (10-fold
excess) and DIEA (2-fold excess) in DMF:DCM (3:1, 5 mL) for
1 h. The completion of the acylation was confirmed by the
qualitative ninhydrin test. Fmoc-D-Asn(Trt) was incorporated
in positions 5 or 6, respectively, for peptides 15 and 16. After
removal of the N-terminal Fmoc protecting group using 20%
piperidine in DMF, the rest of the amino acids were then
coupled as described in the general procedure to complete the
assembly of the peptides.
Experimental Section
Materials. The PAC-PEG-PS resin, PAL-PEG-PS resin,
HOBt, and DIEA were purchased from Applied Biosystems
(Foster City, CA). HBTU was purchased from EMD Bio-
sciences (San Diego, CA); HOAt and PyAOP were purchased
from Applied Biosystems. Standard Fmoc-protected amino
acids were purchased from Applied Biosystems, EMD Bio-
sciences, and Bachem Bioscience, Inc. (King of Prussia, PA).
Fmoc-NR-MeArG(Mtr), Fmoc-Dap(Mtt), and TyrOtBu were
purchased from EMD Biosciences. N-Benzyltyrosine was
synthesized as described in the Supporting Information. Piper-
idine, triisopropylsilane (TIS), and TFA were purchased from
Aldrich Chemical Co. (Milwaukee, WI). All solvents (AcOH,
MeCN HPLC grade, DMF, dichloromethane (DCM), and
MeOH (HPLC grade)) used for peptide synthesis or HPLC
analysis were obtained from either Burdick and Jackson, Inc.
(Muskegon, MI) or EM Sciences (Gibbstown, NJ). TFA for
HPLC analysis was purchased from Thermo-Fisher (Rockville,
IL). Molecular weights of the compounds were determined by
ESI-MS using a Thermoquest LCQ mass spectrometer
(University of Maryland Baltimore) and/or by fast atom bom-
bardment mass spectrometry (FAB-MS) on a Kratos MS50RF
mass spectrometer (Oregon State University).

6820 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Patkar et al.
The peptides were generally cleaved from the resin using
Reagent B39 (88.5% TFA containing 2.5% water, 2.5% phenol
and 5% TIS as scavengers) except for peptides containing
NR-MeArg. For these peptides containing the Mtr side chain
protecting group on NR-MeArg, 5% thioanisole and 2.5%
1,2-ethanedithiol in TFA40 were used along with 5% TIS and
2.5% phenol as scavengers. The peptides were filtered from the
resin, and the filtrates diluted with 10% acetic acid (about
10-15 mL) and extracted with diethyl ether (3 ꢀ 20 mL). The
ether extracts were back extracted with 10% acetic acid
(20 mL). The combined aqueous extracts were pooled and
lyophilized to give the crude peptides.
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Synthesis of Compounds 13 and 14. Peptide 14 was synthesized
using the same synthetic strategy as peptide 13 described pre-
viously.35 The cyclic peptides were assembled similar to the
linear analogues except that Fmoc-D-Asp(Pip) and Fmoc-
Dap(Mtt) were used in the positions involved in the cyclizations.
For 13, the amino acids from positions 5-11 and for 14, the
amino acids from positions 6-11 were coupled manually on
the “CHOIR” as described above using HBTU and NMM as
the coupling reagents. The Pip and Mtt groups were then
selectively deprotected with a mixture of 3% TFA and 5%
TIS in DCM (3 ꢀ 10 min). The cyclization was carried out by
mixing the resin with a mixture of PyAOP, HOAt, and DIEA
(3:3:6 relative to the resin substitution) dissolved in 1:1 DMF:
DCM (4 mL) twice for a total of 24 h for 13 and three times for a
total of 48 h for 14. Any remaining unreacted free amine groups
on Dap were acetylated as described above, and the rest of the
peptides were assembled as described in the general procedure.
Pharmacological Assays. Radioligand binding assays were
performed as previously described33 using cloned rat KOR and
MOR and mouse DOR stably expressed on CHO cells.
[3H]Diprenorphine (KD=0.45 nM), [3H]DAMGO ([D-Ala2,N-
MePhe4,glyol]enkephalin, KD) (KD = 0.49 nM), and [3H]-
DPDPE (cyclo[D-Pen2,D-Pen5]enkephalin, KD) (KD=1.76 nM)
were used as radioligands in assays for KOR, MOR, and DOR,
respectively. Results are mean ( SEM obtained from at least
three independent experiments.
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Antagonists in Models of Unlearned and Learned Fear in Rats.
J. Pharmacol. Exp. Ther. 2007, 323, 838–845.
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N.; Herzog, H.; Schwarzer, C. Prodynorphin-derived Peptides are
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Corticosterone. Neuropsychopharmacology 2009, 34, 775–785.
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Adenylyl cyclase assays were performed as previously de-
scribed25 using cloned rat KOR stably expressed on CHO cells.
Peptides were evaluated at 10 μM compared to reference agonist
Dyn A-(1-13)NH2 (100 nM) to determine efficacy. Results are
mean ( SEM obtained from at least three independent experi-
ments.
Acknowledgment. We thank Jennif Chandler and B.
Miranda Thomas in Dr. Thomas Murray’s laboratory for
performing the pharmacological assays. This research was
supported by grants R01 DA05195 and R01 DA018832 from
the National Institute on Drug Abuse.
Supporting Information Available: Details of the synthesis of
N-benzyltyrosine and analytical data for the peptides. This material
is available free of charge via the Internet at http://pubs.acs.org.
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A-(1-11) are Highly Selective for Kappa-Opioid Receptors.
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