Novel TIPP (H-Tyr-Tic-Phe-Phe-OH) analogues displaying a wide range of efficacies at the δ opioid receptor. Discovery of two highly potent and selective δ opioid agonists

Article abstract of DOI:10.1016/j.bmcl.2012.01.063

Analogues of the δ opioid antagonist peptide TIPP (H-Tyr-Tic-Phe-Phe-OH; Tic = 1,2,3,4-tetrahydroisoquinoline3-carboxylic acid) containing various 4′-[N-(alkyl or aralkyl)carboxamido]phenylalanine analogues in place of Tyr¹ were synthesized. Th

Full text of DOI:10.1016/j.bmcl.2012.01.063

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1899–1902
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel TIPP (H-Tyr-Tic-Phe-Phe-OH) analogues displaying a wide range
of efficacies at the d opioid receptor. Discovery of two highly potent
and selective d opioid agonists
Irena Berezowska a, Carole Lemieux a, Nga N. Chung a, Jinguo Ding a, , Peter W. Schiller a,b,
⇑
a Laboratory of Chemical Biology and Peptide Research, Clinical Research Institute of Montreal, 110 Pine Avenue West, Montreal, QC, Canada H2W 1R7
b Department of Pharmacology, Université de Montréal, Montreal, QC, Canada H3C 3J7
a r t i c l e i n f o
a b s t r a c t
Article history:
Analogues of the d opioid antagonist peptide TIPP (H-Tyr-Tic-Phe-Phe-OH; Tic = 1,2,3,4-tetrahydro-
isoquinoline3-carboxylic acid) containing various 40-[N-(alkyl or aralkyl)carboxamido]phenylalanine
analogues in place of Tyr1 were synthesized. The compounds showed subnanomolar or low nanomolar
d opioid receptor binding affinity and various efficacy at the d receptor (antagonism, partial agonism, full
Received 21 December 2011
Revised 15 January 2012
Accepted 17 January 2012
Available online 28 January 2012
agonism) in the [35S]GTP S binding assay. Two analogues, [1-Ncp1]TIPP (1-Ncp = 40-[N-(2-(naphthalene-
c
1-yl)ethyl)carboxamido]phenylalanine) and [2-Ncp1]TIPP (2-Ncp = 40-[N-(2-(naphthalene-2-yl)ethyl)car-
boxamido]phenylalanine), were identified as potent and selective d opioid agonists.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Amino acid synthesis
Peptide synthesis
Opioid peptides
d Opioid agonists
d Partial opioid agonists
d Opioid antagonist
Selective d opioid receptor agonists are of interest both as
pharmacological tools and as potential therapeutic agents (for a
review, see Ref. 1). While they have low analgesic efficacy in acute
pain models, their potential for the treatment of inflammatory and
neuropathic pain has been clearly demonstrated.2,3 Furthermore,
there is evidence to indicate that d opioid agonists produce anxio-
lytic and antidepressant effects4 and that they may be useful as
neuroprotective agents as well as for the treatment of addictive dis-
orders.1 Both peptide and non-peptide d opioid agonists have been
reviewed.1,5 Examples of highly selective peptide d opioid agonists
The tetrapeptide TIPP (H-Tyr-Tic-Phe-Phe-OH) is a highly selec-
tive d opioid antagonist.9 Recently, we prepared a TIPP analogue
containing 40-[N-((40-phenyl)phenethyl)carboxamido]phenylala-
nine (Bcp, Fig. 1) in place of Tyr1, [Bcp1]TIPP, which displayed high
d receptor binding affinity and d agonist activity in the MVD assay.10
Here we describe analogues of TIPP in which the Tyr1 residue was
replaced by p-carboxamidophenylalanine (Cpa, 1) and its deriva-
tives containing various substituents at the p-carboxamido group,
including 40-[N-(hexyl)carboxamido]phenylalanine (Hcp, 2), 40-[N-
(decyl)carboxamido]phenylalanine (Dcap, 3), 40-[N-((40-cyclohexyl)
phenethyl)carboxamido]phenylalanine (Cpcp, 5), 40-[N-(2-(naph-
thalene-1-yl)ethyl)carboxamido]phenylalanine (1-Ncp, 6), 40-[N-
(2-naphthalene-2-yl)ethyl)carboxamido]phenylalanine (2-Ncp, 7),
and 40-N-(2-(indole-3-yl)ethyl)carboxamido]phenylalanine (Tcp, 8)
(Fig. 1). The effects of these various substitutions on binding affinity
are the cyclic opioid peptide analogues H-Tyr-c[
Pen]Phe-OH6 and H-Tyr-c[ -Pen]OH (JOM-13)7, and the
-Cys-Phe-
linear tripeptide UFP-512.8
D-Pen-Gly-Phe(pF)-
D
D
and efficacy at the d and
binding- and functional assays.
l opioid receptors were determined in
Abbreviations: Boc, tert-butyloxycarbonyl; Cl-HOBt, 6-chloro-1-hydroxybenzo-
triazole; DAMGO, H-Tyr- -Ala-Gly-Phe(NMe)-Gly-ol; DIC, 1,3-diisopropylcarbodii-
mide; DIPEA, N,N-diisopropylethylamine; DPDPE, H-Tyr-c[ -Pen-Gly-Phe-D-Pen]OH;
DSLET, H-Tyr- -Ser-Gly-Phe-Leu-Thr-OH; ES-MS, electrospray mass spectrometry;
GPI, guinea pig ileum; HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate; HPLC, high performance liquid chromatography; MVD, mouse
vas deferens, Pen, penicillamine; Tic, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic
D
For the preparation of the various amino acids, Boc-Phe(40-
COOH)-OMe was synthesized by activating the hydroxyl group of
Boc-Tyr-OMe as the triflate, followed by carbonylation with carbon
monoxide, as described.11 Boc-Phe-(40-CONH2)-OMe (Boc-Cpa, 1)12
was prepared by reacting Boc-Phe(40-COOH)-OMe with ammonium
chloride using HBTU as coupling agent, followed by hydrolysis with
2 N NaOH. The other Boc-protected amino acids ( 2, 3, 5–9)12
were synthesized by reacting Boc-Phe(40-COOH)-OMe with the
appropriate alkyl- or aralkylamine using HBTU as coupling agent,
D
D
acid; TIPP, H-Tyr-Tic-Phe-Phe-OH; U69,593, (5a,7a,8b-(ꢀ)-N-methyl-N-[7-(1pyrroli-
dinyl)-1-oxaspiro[4.5]dec-8-yl]benzeneacetamide.
⇑
Corresponding author. Tel.: +1 514 987 5576; fax: +1 514 987 5513.
E-mail address: schillp@ircm.qc.ca (P.W. Schiller).
Present address: Shanghai No. 1 Biochemical & Pharmaceutical Co. Ltd, 1317
Jianchuan Road, Shanghai 200240, China.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.063

1900
I. Berezowska et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1899–1902
Figure 1. Structures of Phe(40-CONHX) amino acids.
followed by hydrolysis with 2 N NaOH. All amines were commer-
cially available, except for 2-((40-cyclohexyl)phenyl)ethylamine
(5a)12 required for the synthesis of Cpcp-OH. The latter amine was
prepared by catalytic hydrogenation of 2-(40-biphenyl)ethylamine
in EtOH using palladium on carbon as catalyst with hydrogen
pressure of 140 psig during 45 h at 95 °C. 2-((40-cyclo-
hexyl)phenyl)ethylamine was obtained as the predominant product
(45%) with unreacted 2-(40-biphenyl)ethylamine (35%) and 2-((40-
phenyl)cyclohexyl)ethylamine (20%) also present. This mixture
was used in the synthesis of Boc-Cpcp-OH which was isolated from
the reaction product by reversed-phase HPLC.
Peptides were synthesized by the solid-phase method using a Mer-
rifield resin with Boc-protection and DIC/Cl-HOBt or HBTU/DIPEA as
coupling agents, and were cleaved from the resin by HF/anisole treat-
ment. The crude products were purified by preparative reversed-phase
HPLC and their purity (>98%) and structural identity were established
by TLC, analytical HPLC and ES-MS.13
Cpa was also a good surrogate for Tyr1 in opioid agonist pep-
tides.16,17 The Hcp1-analogue (11) displayed about 2.5-fold higher
d receptor binding affinity than 10 and retained high d versus
l
receptor selectivity. Peptide 11 was a d full agonist in the MVD as-
say with subnanomolar potency and a weak partial agonist in the
GPI assay. It showed d partial agonist activity in the [35S]GTP
cS
binding assay (e = 0.486) with an ED50 of 0.103 nM. In comparison
with 11, the [Dcap1]analogue (12) displayed somewhat diminished
d receptor binding affinity and lower d versus
l selectivity in the
binding assays, fourfold lower d agonist potency in the MVD assay,
and slightly decreased d partial agonist activity (ED50 = 0.239 nM)
with comparable efficacy (e = 0.527) in the [35S]GTP
cS binding
assay.
As previously reported,10 [Bcp1]TIPP (13) was a potent d full
agonist in the MVD assay; however, it was now characterized as
a d partial agonist (e = 0.678) in the [35S]GTP
c
S binding assay with
an overall in vitro activity profile similar to that of 12. The results
obtained with peptides 11–13 in the [35S]GTP
S binding assay
Binding affinities (Ki-values) for
l
and d opioid receptors were
c
determined by displacing, respectively, [3H]DAMGO and [3H]DSLET
indicate that they all behave as d partial agonists with regard to
G protein activation. The fact that these compounds show d full
agonist behavior in the MVD assay could be due to the presence
of spare d receptors in the vas preparation or to a different signal
transduction mechanism.
from rat brain membrane binding sites and
j receptor binding
affinities were measured by displacement of [3H]U69,593 from
guinea pig brain membrane binding sites, as described.14 None of
the compounds showed significant
j receptor binding affinity
(Kji >10
l
M). Opioid agonist potencies (IC50s) or antagonist activi-
The Cpcp1-analogue (14) showed diminished d versus
l recep-
ties (Ke-values) were determined in the mouse vas deferens (MVD)
tor binding selectivity and somewhat reduced d full agonist po-
assay (d receptor-representative) and in the guinea pig ileum (GPI)
tency in the MVD assay. It was essentially a d full agonist
assay using previously described protocols.14 Furthermore,
d
(e = 0.933) with an ED50 of 1.69 nM in the [35S]GTP
cS binding as-
receptor agonist potencies and efficacies were determined in the
say. [1-Ncp1]TIPP (15) (1-NIPP) was a potent and selective d opioid
agonist with an IC50 of 0.533 nM in the MVD assay and with d full
[
35S]GTP
cS binding assay using HEK293 cells stably expressing
the human d opioid receptor15 (efficacies were indicated relative
to the efficacy of DPDPE [e = 1.0]).
agonist properties (e = 0.894) in the [35S]GTP
cS binding assay. Sim-
ilarly, [2-Ncp1]TIPP (16) (2-NIPP) displayed high d receptor binding
affinity (Kdi = 1.01 nM), high d agonist potency in the MVD assay
(IC50 = 0.950 nM) and d full agonist potency (ED50 = 0.475 nM,
In comparison with the TIPP parent peptide, [Cpa1]TIPP (10)
showed comparable d and
sus selectivity (Table 1), and about threefold lower d antagonist
activity in the MVD assay (Table 2). In the [35S]GTP
l receptor binding affinities and d ver-
l
e = 0.989) in the [35S]GTP
c
S binding assay. In a direct comparison
with the standard d opioid agonist DPDPE, 1-NIPP and 2-NIPP show
5–8 fold higher d receptor binding affinity, comparable d versus
c
S binding assay
both TIPP and [Cpa1]TIPP behaved as d neutral antagonists (e = 0,
Table 3). These data indicate that replacement of Tyr1 in TIPP with
Cpa had little effect on the binding affinity and efficacy at the d
receptor and are in agreement with earlier results indicating that
l
l
l
receptor selectivity (K /(Kdi = 250–520 versus (K /(Kid = 336 for
i
i
DPDPE), 2–4-fold higher d agonist potency in the MVD assay and
7–14-fold higher d agonist potency in the [35S]GTP
c
S binding as-
say. Finally, replacement of the naphthyl moiety in 15 with an
indole moiety resulted in a compound, [Tcp1]TIPP (17) with some-
what diminished d agonist potency in the MVD assay and reduced
d partial agonist activity in the [35S]GTP
cS binding assay.
Table 1
Receptor binding affinities of TIPP analogues
In conclusion, all compounds described here displayed high d opi-
oid receptor binding affinity, d antagonist or d full agonist activity in
the MVD assay and various efficacy, ranging from d antagonism to d
l
a
l
Kdi (nM)
Ki =Kdi
a
Compound
Ki (nM)
10
11
12
13
14
15
16
17
H-Cpa-Tic-Phe-Phe-OH
0.978 0.195
0.369 0.028
1.54 0.25
0.605 0.058
2.72 0.40
1.65 0.31
1.01 0.10
1.84 0.28
1.22 0.07
8.39 0.70
2190 170
507 17
174 40
87.7 8.0
47.6 17.3
850 190
249 32
3600 1250
1720 50
2820 0.7
2240
1370
113
145
17.5
515
partial agonism to d full agonism, in the [35S]GTP
cS binding assay.
H-Hcp-Tic-Phe-Phe-OH
H-Dcap-Tic-Phe-Phe-OH
H-Bcp-Tic-Phe-Phe-OHb
H-Cpcp-Tic-Phe-Phe-OH
H-1-Ncp-Tic-Phe-Phe-OH
H-2-Ncp-Tic-Phe-Phe-OH
H-Tcp-Tic-Phe-Phe-OH
H-Tyr-Tic-Phe-Phe-OH
DPDPE
Two of the compounds, 1-NIPP and 2-NIPP, turned out to be potent
d full agonists in both functional assays. The d partial agonist vs. d full
agonist behavior of the compounds in G protein activation can be
explained with two different receptor activation models. Assuming
that the receptor exists in only two functional conformational states,
inactive and active, the partial agonists may simply induce a smaller
fraction of receptors to undergo the transition to the active state than
do the full agonists. The more likely explanation is that the partial
247
1960
1410
336
a
Mean of 3 determinations SEM. Data taken from Ref. 10.

I. Berezowska et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1899–1902
1901
Table 2
Opioid activities of TIPP analogues in the MVD and GPI assaysa
Compound
MVD
GPI
l
c
Kde (nM)
b
IC50 (nM)
IC50 (nM)
Ke (nM)
6030 1600
10
11
12
13
14
15
16
17
H-Cpa-Tic-Phe-Phe-OH
H-Hcp-Tic-Phe-Phe-OH
H-Dcap-Tic-Phe-Phe-OH
H-Bcp-Tic-Phe-Phe-OHe
H-Cpcp-Tic-Phe-Phe-OH
H-1-Ncp-Tic-Phe-Phe-OH
H-2-Ncp-Tic-Phe-Phe-OH
H-Tcp-Tic-Phe-Phe-OH
H-Tyr-Tic-Phe-Phe-OH
DPDPE
18.3 2.1
d
d
0.392 0.051
1.54 0.25
3.42 0.36
6.20 0.40
0.533 0.047
0.950 0.142
10.1 1.4
875 171 (IC30
730 109 (IC30
223 37 (IC40
23.4 5.1 (IC35
)
)
d
)
d
)
l
Inactive at 10
M
P.A.d
734 17
P.A.d
4.80 0.20
> 10000 (Inactive)
6340 410
2.02 1.6
a
b
c
Mean of 3 determinations SEM.
Determined against DPDPE.
Determined against TAPP.
Partial agonist.
d
e
Data taken from Ref. 10.
Table 3
35S]GTP
cS binding assay of TIPP analogues
6. Hruby, V. J.; Bartosz-Bechowski, H.; Davis, P.; Slaninova, J.; Zalewska, T.;
Stropova, D.; Porreca, F.; Yamamura, H. I. J. Med. Chem. 1997, 40, 3957.
7. Mosberg, H. I.; Omnaas, J. R.; Medzihradsky, F.; Smith, G. B. Life Sci. 1988, 1013,
43.
[
a
Compound
ED50 (nM)
ea
8. Vergura, R.; Balboni, G.; Spagnolo, G.; Gavioli, E.; Lambert, D. G.; McDonald, J.;
Trapella, C.; Lazarus, L. H.; Regoli, D.; Guerrini, R.; Salvadori, S.; Caló, G. Peptides
2008, 29, 93.
9. Schilller, P. W.; Nguyen, T. M.-D.; Weltrowska, G.; Wilkes, B. C.; Marsden, B. J.;
Lemieux, C.; Chung, N. N. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 11871.
10. Berezowska, I.; Chung, N. N.; Lemieux, C.; Wilkes, B. C.; Schiller, P. W. J. Med.
Chem. 2009, 52, 6941.
10
11
12
13
14
15
16
17
H-Cpa-Tic-Phe-Phe-OH
Antagonist
0.103 0.012
0.239 0.023
0.413 0.015
1.69 0.25
1.03 0.05
0.475 0.064
7.08 2.19
Antagonist
6.81 0.68
0
H-Hcp-Tic-Phe-Phe-OH
H-Dcap-Tic-Phe-Phe-OH
H-Bcp-Tic-Phe-Phe-OH
H-Cpcp-Tic-Phe-Phe-OH
H-1-Ncp-Tic-Phe-Phe-OH
H-2-Ncp-Tic-Phe-Phe-OH
H-Tcp-Tic-Phe-Phe-OH
H-Tyr-Tic-Phe-Phe-OH
DPDPE
0.486 0.060
0.527 0.080
0.678 0.076
0.933 0.026
0.894 0.009
0.989 0.44
0.643 0.051
0
11. Wang, W.; Obeyesekere, N. U.; McMurray, J. S. Tetrahedron Lett. 1996, 37, 6661.
12. All new Boc-protected amino acids were fully characterized by optical rotation
measurements, melting point determinations, 1H and 13C NMR spectra and
HRMS:
1
Compound 1. a2D0 + 15.7° (c 1, MeOH); mp 240–242 °C; 1H NMR (500 MHz,
CD3OD) d 7.88–7.79 (d, 2H, J = 8.0 Hz), 7.40–7.32 (d, 2H, J = 7.8 Hz), 4.46–4.36
(m, 1H), 3.27–3.21 (m, 1H), 3.02–2.95 (m, 1H), 1.41–1.37 (s, 8H), 1.36–1.34 (s,
1H); 13C NMR (125 MHz, CD3OD) d 173.9, 171.1, 156.6, 142.0, 132.1, 129.4,
127.6, 79.4, 54.8, 37.3, 27.5; HRMS (EI) m/e calcd for C15H20N2NaO5 [M+Na]+
331.1270, found 331.1023.
a
Mean of 3–6 determinations SEM.
agonists induce a receptor conformation distinct from that induced
by the full agonists. It has been convincingly demonstrated that
b2-adrenergic receptor partial agonists and full agonists induce
distinct receptor conformations.18,19 In the case of the d opioid partial
agonists and full agonists described here, distinct d receptor confor-
mations could be induced through diverse interactions of the various
large lipophilic substituents at the 1-position residue of these
peptides with hydrophobic residues of an accessory receptor binding
site. Because these structurally flexible peptides contain 4–6
aromatic rings they may have unique ability to selectively induce
or stabilize a number of distinct receptor conformations through
diverse hydrophobic interactions with the numerous aromatic and
aliphatic residues present in the receptor binding site.10 For this
reason, they need to be further examined in various assay systems
for possible functional selectivity.
Compound 2. a2D0 ꢀ 5.8° (c 1, DMSO); mp 152–153 °C; 1H NMR (500 MHz,
DMSO-d6) d 12.64–12.50 (br s, 1H), 8.38–8.32 (t, 1H, J = 5.5 Hz), 7.77–7.71 (d,
2H, J = 7.8 Hz), 7.34–7.29 (d, 2H, J = 8.0 Hz), 7.14–7.10 (d, 1H, J = 8.3 Hz), 4.15–
4.08 (m, 1H), 3.24–3.20 (m, 2H), 3.09–3.03 (m, 1H), 2.90–2.83 (m,1H), 1.54–
1.46 (m, 2H), 1.33–1.30 (s, 8H), 1.30–1.23 (m 7H), 0.89–0.84 (m, 3H); 13C NMR
(125 MHz, DMSO-d6) d 174.1, 166.5, 156.1, 141.9, 133.5, 129.6, 127.7, 78.8,
55.6, 39.8, 36.9, 31.7, 29.8, 28.8, 26.8, 22.8, 14.6; HRMS (EI) m/e calcd for
C
21H32O5N2Na [M+Na]+ 415.2209, found 415.2201.
Compound 3. a2D0 ꢀ 3.2° (c 1, DMSO); mp 136–137 °C; 1H NMR (500 MHz,
DMSO-d6) d 12.74–12.34 (br s, 1H), 8.36–8.32 (t, 1H, J 5.6 Hz), 7.77–7.72 (d, 2H,
J = 8.1 Hz), 7.34–7.28 (d, 2H, J = 8.1 Hz), 7.14–7.08 (d, 1H, J = 8.3 Hz), 4.15–4.08
(m, 1H), 3.26–3.19 (m, 2H), 3.09–3.03 (m, 1H), 2.90–2.83 (m, 1H), 1.54–1.46
(m, 2H), 1.34–1.30 (s, 8H), 1.29–1.21 (m, 15H), 0.88–0.83 (m, 3H); 13C NMR
(125 MHz, DMSO-d6) d 174.2, 166.5, 156.1, 141.9, 133.5, 129.6, 127.7, 78.8,
55.6, 39.8, 36.9, 32.0, 29.8, 29.7, 29.6, 29.5, 29.4, 28.8, 27.2, 22.8, 14.7; HRMS
(EI) m/e calcd for C25H40O5N2Na [M+Na]+ 471.2835, obsd 471.2823.
Compound 5. a2D0 + 12.2° (c 1, MeOH); mp 206–208 °C; 1H NMR (500 MHz,
DMSO-d6) d 12.75–12.50 (br s, 1H), 8.53–8.47 (t, 1H, J = 5.5 Hz), 7.77–7.72 (d,
2H, J = 8.0 Hz), 7.36–7.29 (d, 2H, J = 8.0 Hz),7.17–7.11 (m, 5H), 4.18–4.09
(m,1H), 3.48–3.41 (m, 2H), 3.10–3.04 (m, 1H), 2.91–2.85 (m, 1H), 2.81–2.73 (m,
2H), 2.49–2.40 (m, 1H), 1.80–1.66 (m, 6H), 1.40–1.34 (m, 4H), 1.33–1.30 (s,
8H), 1.27–1.24 (s, 1H); 13C NMR 125 MHz, DMSO-d6) d 175.4, 166.6, 146.0,
142.2, 137.6, 133.0, 129.6, 129.2, 127.7, 127.3, 78.8, 55.6, 45.0, 44.1, 36.9, 35.4,
34.7, 26.3, 25.5; HRMS (EI) m/e calcd for C29H38N2O5Na [M+Na]+ 517.2678,
obsd 517.3116.
Compound 5a. A small quantity of 5a was purified by HPLC for analytical
characterization: mp 118–120 °C; 1H NMR (500 MHz, CD3OD) d 7.24–7.17 (m,
4), 3.19–3.13 (t, 2H, J = 7.5 Hz), 2.96–2.89 (t, 2H, J = 7.5 Hz), 2.56–2.46 (m, 1H),
1.91–1.74 (m, 5H), 1.51–1.39 (m, 5H), 1.38–1.26 (m, 2H); 13C NMR (125 MHz,
CD3OD) d 147.2, 133.9, 128.6, 128.5, 127.3, 127.2, 44.4, 40.8, 34.5, 33.0, 26.8,
26.1; HRMS (EI) m/e calcd for C14H22N [M+H]+ 204.1752, found 204.1747.
Compound 6. a2D0 ꢀ 15.5° (c 1, DMSO); mp 138–140 °C; 1H NMR (500 MHz,
DMSO-d6) d 12.84–12.38 (br s, 1H), 8.68–8.61 (t, 1H, J = 5.6 Hz), 8.31–8.25 (d,
1H, J = 8.3 Hz), 7.96–7.90 (d, 1H, J = 8.0 Hz), 7.82–7.79 (d, 1H, J = 8.3 Hz), 7.79–
7.73 (d, 2H, J = 8.3 Hz), 7.62–7.56 (m, 1H), 7.56–7.50 (m, 1H), 7.46–7.38 (m,
1H), 7.38–7.30 (d, 2H, J = 8.3 Hz), 7.17–7.10 (d, 1H, J = 8.5 Hz), 4.18–4.10 (m,
1H), 3.63–3.55 (m, 2H), 3.35–3.28 (m, 2H), 3.10–3.04 (m, 1H), 2.91–2.85 (m,
1H), 1.38–1.33 (s, 8H),1.28–1.25 (s 1H); 13C NMR (125 MHz, DMSO-d6) d 174.2,
166.8, 156.4, 142.1, 136.2, 134.2, 132.4, 129.6, 127.7, 127.6, 126.4, 124.5, 78.8,
Acknowledgments
This work was supported by Grants from the Canadian Insti-
tutes of Health Research (MOP-89716) and the U.S. National Insti-
tutes of Health (DA004443).
References and notes
1. Pradhan, A. A.; Befort, K.; Nozaki, C.; Gavériaux-Ruff, C.; Kieffer, B. L. Trends
Pharmacol. Sci. 2011, 32, 581.
2. Cahill, C. M.; Morinville, A.; Hoffert, C.; O’Donnell, D.; Beaudet, A. Pain 2003,
101, 199.
3. Holdridge, S. V.; Cahill, C. M. Eur. J. Pain 2007, 11, 685.
4. Saitoh, A.; Kimura, Y.; Suzuki, T.; Kawai, K.; Nagase, H.; Kamei, J. J. Pharmacol.
Sci. 2004, 95, 374.
5. Hruby, V. J.; Agnes, R. S. Biopolymers (Peptide Sci.) 1999, 51, 391.

1902
I. Berezowska et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1899–1902
55.8, 41.0, 36.5, 33.3, 28.8; HRMS (EI) m/e calcd for C27H30O5N2Na [M+Na]+
485.2052, found 485.2038.
13. Analytical data of peptides. TLC in the following systems (all v/v): (I) n-BuOH/
AcOH/H2O (4:1:1), (II) n-BuOH/pyridine/AcOH/H2O (15:10:3:12); analytical
reversed-phase HPLC performed on a Vydac column (10 ꢁ 250 mm) with a
linear gradient of 45–95% MeOH in 0.1% TFA over 50 min (K0 = (tr ꢀ tm)/tm); ES-
MS (m/e). 10. Rf 0.571 (I), Rf 0.768 (II); K0 1.72; ES-MS m/e 662. 11. Rf 0.457 (I), Rf
0.840 (II); K0 4.65; ES-MS m/e 746. 12. Rf 0.493 (I), Rf 0.852 (II); K0 6.17, ES-MS
m/e 802. 14. Rf 0.593 (I), Rf 0.841 (II); K0 4.97; ES-MS m/e 848. 15. Rf 0.709, Rf
0.860 (II); K0 4.86; ES-MS m/e 816. 16. Rf 0.463 (I), Rf 0.846 (II); K0 4.77; ES-MS
m/e 816. 17. Rf 0.743 (I), Rf 0.797 (II), K’ 3.74; ES-MS m/e 805.
14. Berezowska, I.; Lemieux, C.; Chung, N. N.; Wilkes, B. C.; Schiller, P. W. Chem.
Biol. Drug Des. 2009, 74, 329.
Compound 7. a2D0 ꢀ 5.5° (c 1, DMSO); mp 226–227 °C; 1H NMR (500 MHz,
DMSO-d6) d 12.80–12.40 (br s, 1H), 8.56–8.50 (t, 1H, J = 5.6 Hz), 7.89–7.82 (m,
3H), 7.76–7.70 (m, 3H), 7.50–7.40 (m, 3H), 7.34–7.28 (d, 2H, J = 8.0 Hz), 7.16–
7.11 (d, 1H, J = 8.3 Hz), 4.15–4.08 (m, 1H), 3.62–3.56 (m, 2H), 3.09–3.04 (m,
1H), 3.03–2.98 (m, 2H), 2.89–2.82 (m, 1H), 1.33–1.28 (s, 8H), 1.27–1.22 (s, 1H);
13C NMR (125 MHz, DMSO-d6) d 174.1, 166.7, 156.1, 142.0, 137.9, 133.8, 133.4,
132.4, 129.6, 128.4, 128.2, 128.1, 128.0, 127.7, 127.4, 126.7, 126.0, 78.8, 55.5,
41.4, 36.9, 35.9, 28.8; HRMS (E/I) m/e calcd for
C
27H30N2O5Na [M+Na]+
485.2052, found 485.2038.
Compound 8. a2D0 ꢀ 5.8° (c 1, DMSO); mp 118–120 °C; 1H NMR (500 MHz,
DMSO-d6) d 12.77–12.58 (br s, 1H), 10.83–10.78 (s, 1H), 8.57–8.52 (t, 1H,
J = 5.6 Hz), 7.79–7.74 (d, 2H, J = 8.1 Hz), 7.62–7.58 (d, 1H, J = 7.8 Hz), 7.35–7.30
(m, 3H), 7.18–7.16 (s, 1H), 7.16–7.12 (d, 1H, J = 8.5 Hz); 7.09–7.04 (m, 1H),
7.00–6.96 (m, 1H), 4.15–4.08 (m, 1H), 3.56–3.49 (m, 2H), 3.10–3.04 (m, 1H),
2.97–2.90 (m, 2H), 2.90–2.84 (m, 1H), 1.33–1.30 (s, 8H), 1.28–1.25 (s, 1H); 13C
NMR (125 MHz, DMSO-d6) d 174.1, 166.6, 156.1, 142.0, 137.0, 133.5, 129.7,
128.0, 127.7, 123.2, 121.6, 118.9, 112.6, 78.8, 56.6, 45.0, 36.9, 28.8, 25.9; HRMS
(E/I) m/e calcd for C25H30O5N3 [M+H]+ 452.2185, found 452.2178.
15. Audet, N.; Paquin-Gobeil, M.; Landry-Paquet, O.; Schiller, P. W.; Piñeyro, G. J.
Biol. Chem. 2005, 280, 7808.
16. Dolle, R. E.; Machaut, M.; Martinez-Teipel, B.; Belanger, S.; Cassel, J. A.; Stabley,
G. J.; Graczyk, T. M.; DeHaven, R. N. Bioorg. Med. Chem. Lett. 2004, 14, 3545.
17. Weltrowska, G.; Lemieux, C.; Chung, N. N.; Schiller, P. W. J. Peptide Res. 2005,
65, 36.
18. Ghanouni, P.; Gryczynski, Z.; Steenhuis, J. J.; Lee, T. W.; Farrens, D. L.; Lakowicz,
J. R.; Kobilka, B. K. J. Biol. Chem. 2001, 276, 24433.
19. Swaminath, G.; Deupi, X.; Lee, T. W.; Zhu, W.; Thian, F. S.; Kobilka, T. S.;
Kobilka, B. J. Biol. Chem. 2005, 280, 22165.