Herkinorin analogues with differential β-arrestin-2 interactions

Article abstract of DOI:10.1021/jm701162g

Salvinorin A is a psychoactive natural product that has been found to be a potent and selective κ opioid receptor agonist in vitro and in vivo. The activity of salvinorin A is unusual compared to other opioids such as morphine in that it mediates potent κ

Full text of DOI:10.1021/jm701162g

J. Med. Chem. 2008, 51, 2421–2431
2421
Herkinorin Analogues with Differential ꢀ-Arrestin-2 Interactions
Kevin Tidgewell,† Chad E. Groer,‡ Wayne W. Harding,† Anthony Lozama,† Matthew Schmidt,† Alfred Marquam,†,§
Jessica Hiemstra,† John S. Partilla,4 Christina M. Dersch,4 Richard B. Rothman,4 Laura M. Bohn,‡ and
Thomas E. Prisinzano*,†,§
DiVision of Medicinal and Natural Products Chemistry, The UniVersity of Iowa, Iowa City, Iowa 52242, Departments of Pharmacology and
Psychiatry, The Ohio State UniVersity College of Medicine, Columbus, Ohio 43210, Department of Medicinal Chemistry, The UniVersity of
Kansas, Lawrence, Kansas 66045, and Clinical Psychopharmacology Section, IRP, NIDA, NIH, DHHS, Baltimore, Maryland 21224
ReceiVed September 17, 2007
Salvinorin A is a psychoactive natural product that has been found to be a potent and selective κ opioid
receptor agonist in vitro and in vivo. The activity of salvinorin A is unusual compared to other opioids such
as morphine in that it mediates potent κ opioid receptor signaling yet leads to less receptor downregulation
than observed with other κ agonists. Our initial chemical modifications of salvinorin A have yielded one
analogue, herkinorin (1c), with high affinity at the µOR. We recently reported that 1c does not promote the
recruitment of ꢀ-arrestin-2 to the µOR or receptor internalization. Here we describe three new derivatives
of 1c (3c, 3f, and 3i) with similar properties and one, benzamide 7b, that promotes recruitment of ꢀ-arrestin-2
to the µOR and receptor internalization. When the important role µ opioid receptor regulation plays in
determining physiological responsiveness to opioid narcotics is considered, µ opioids derived from salvinorin
A may offer a unique template for the development of functionally selective µ opioid receptor–ligands with
the ability to produce analgesia while limiting adverse side effects.
Introduction
Increasing evidence indicates that chemically distinct ligands
can elicit different receptor regulation pathways.1 For example,
the opioids morphine, methadone, and fentanyl each promote
µ opioid receptor (µOR) coupling to G proteins, but they differ
in their ability to direct receptor trafficking.2,3 This may be due
to differences in agonist-induced receptor conformations, result-
ing in different degrees of phosphorylation, arrestin recruitment,
and vesicular trafficking. Such differences in µOR regulation
and trafficking may be physiologically relevant as mice lacking
ꢀ-arrestin-2 display enhanced antinociception, decreased toler-
ance, and greatly diminished side effects (constipation and
respiratory depression) following morphine treatment.4–7 There-
fore, an opioid agonist conferring nonconventional receptor
conformations may yield novel analgesics with reduced potential
to produce unwanted side effects.
Currently, there are no selective pharmaceutical or biochemi-
cal inhibitors of G-protein couple receptor (GPCR) desensitiza-
tion nor are there specific inhibitors of the GRKs or ꢀ-arrestins.
A therapeutic approach in which ꢀ-arrestins or GRKs were
individually inhibited might produce unwanted alterations of
the function of other GPCRs. Furthermore, because arrestins
regulate >1000 different GPCRs,8,9 it will be exceedingly
difficult to produce receptor-selective effects using this approach.
An alternate approach would be to selectively target µOR
regulation by designing ligands that confer µOR conformations
that allow for signaling yet disrupt receptor regulation.
Figure 1. Structures of salvinorin A (1a), salvinorin B (1b), herkinorin
(1c), and morphine (2).
family native to Oaxaca, Mexico.10,11 S. diVinorum has been
used as a vision-inducing plant by the Mazatec Indians in their
divination rituals for centuries.12 Previous studies have shown
that 1a is a potent and selective κ opioid receptor agonist in
vitro and in vivo.13–20 Interestingly, 1a activates κ opioid
receptor signaling with less receptor internalization than ob-
served with other κ agonists.21 These studies suggest that the
κOR conformation induced by 1a binding is conducive to G-
protein mediated signal transduction but resistant to internaliza-
tion-mediated regulation. Recent biochemical and site-directed
mutagenesis studies indicate that 1a has a unique binding epitope
at κORs.22–24 These findings support a novel mode by which
subtype selectivity for GPCR ligands is induced by a change
in the topology of conserved residues within a common binding
pocket.23,24
Salvinorin A (1a, Figure 1) is a neoclerodane diterpene
isolated from SalVia diVinorum, a member of the Lamiaceae
Our initial chemical modifications of 1a yielded several
ligands, some agonists and some antagonists at µ, δ, or κ
ORs.25–27 In particular, herkinorin (1c) was identified as the
first non-nitrogenous µ opioid receptor agonist and does not
lead to receptor internalization under any conditions tested but,
more interestingly, it does not promote the recruitment of
ꢀ-arrestin-2 to the µOR.28 As part of our ongoing program to
develop analgesics with reduced propensity to induce tolerance
and dependence, we synthesized several analogues of 1c. These
* To whom correspondence should be addressed. Thomas E. Prisinzano,
Associate Professor, 1251 Wescoe Hall Drive, 4035 Malott Hall, Lawrence,
Kansas 66045-7582. Telephone: (785) 864-3267. Fax: (785) 785-5326.
E-mail: prisinza@ku.edu.
†
The University of Iowa.
The Ohio State University College of Medicine.
The University of Kansas.
IRP, NIDA, NIH, DHHS.
‡
§
4
10.1021/jm701162g CCC: $40.75
2008 American Chemical Society
Published on Web 04/02/2008

2422 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8
Tidgewell et al.
Scheme 1a
a Reagents and conditions: (a) appropriate acid chloride, DMAP, NEt3, CH2Cl2; (b) appropriate acid, EDCI, HOBT, CH2Cl2.
analogues were prepared to further elucidate the role of structure
on µOR affinity, activity, and regulatory pathways.
compared to 1c. Given the clear effects of ring substitution, we
sought to probe additional modifications of the benzene ring.
The addition of a 2-methoxy group (3d) decreased affinity
for µ receptors over 130-fold compared to 1c (Ki ) 1640 vs 12
nM). Introduction of a methoxy group in the 3-position of the
benzene ring (3e) also decreased affinity for µORs and κORs
compared to 1c (Ki ) 30 vs 12 nM and Ki ) 550 vs 90 nM,
respectively). This modification, however, increased affinity 55-
fold for µORs compared to 3d (Ki ) 30 vs 1640 nM) and
improved selectivity for µORs over κORs compared to 1c (µ/κ
) 0.05 vs µ/κ ) 0.13). The presence of a 4-methoxy group
(3f) leads to an approximately 6-fold decrease in affinity (Ki )
70 vs 12 nM) and similar selectivity (µ/κ ) 0.12 vs µ/κ ) 0.13)
for µORs compared to 1c. This observation and our previous
finding that 3c has equal affinity when compared to 1c30 suggest
that an electron-withdrawing group in the 4-position is more
favorable for µOR affinity.
The introduction of a 2-nitro group (3g) decreased affinity
for µORs over 600-fold compared to 1c (Ki ) 7550 vs 12 nM).
This modification was better tolerated at κORs where only a
10-fold loss in affinity was observed (Ki ) 900 vs 90 nM). This
result, coupled with those observed for 3a, 3d, and 3g, would
indicate that factors other than electronics are likely involved
in the binding of 2-position analogues. Substitution of a 3-nitro
group (3h) abolished affinity at µORs (Ki > 10000) and
decreased affinity approximately 10-fold at κORs compared to
1c (Ki ) 800 vs 90 nM). Finally, a 4-nitro group (3i) was also
explored. This modification decreased affinity over 20-fold for
µORs and over 6-fold for κORs compared to 1c (Ki ) 260 vs
12 nM and Ki ) 570 vs 90 nM, respectively). This result,
coupled with those observed for 3c and 3f, would indicate that
factors other than the strength of the electron withdrawing group
are likely involved in the binding of 4-position analogues.
We then sought to further explore the size requirements for
the aromatic substituent. First, we annulated an additional
benzene ring onto the 2 and 3 positions (3j).31 This modification
resulted in a roughly 1000-fold loss of affinity at µOR compared
to 1c (Ki > 10000 vs 12 nM). This change, however, was better
tolerated at κORs with roughly a 5-fold loss in affinity compared
to 1c (Ki ) 410 vs 90 nM). This is interesting given the
observation that replacement of the acetoxy group in 1 with an
Chemistry
We synthesized compounds 3d–3o, 7a, 7b, 8a, 8b, 9a, and
9b, as described in Schemes 1 and 2. Diterpene 1a was isolated
from S. diVinorum and then converted to salvinorin B (1b), as
described previously.29 The reaction of 1b with the appropriate
acid halide or acid under basic conditions afforded compounds
3d–3o.30,31 Alternately, the reaction of 1b with CBr4 and PPh3
afforded a mixture of 432 (59%) and its C2 epimer (14%).
However, addition of the PPh3 in two portions afforded almost
exclusively the ꢀ isomer. This method results in higher yields
than previously described methods using SOBr2.32 The reaction
of 4 with sodium azide in DMF was unsuccessful. However, if
the reaction was conducted in a mixture of acetic acid and
DMF,33 azide 532 was formed in 86% yield, a higher yield than
previously described.32 Interestingly, when the C2 epimer of 4
was subjected to identical conditions, azide 5 was also formed.
Reduction of 5 using Zn metal and NH4Cl34 afforded amine
6
35 in 36% yield. Staudinger reduction (PPh3, H2O)36 of 5 was
also attempted but led mainly to decomposition of starting
material.35 The treatment of amine 6 with acetic anhydride or
benzoyl chloride under basic conditions and in the presence of
a catalytic amount of DMAP afforded amides 7a32,35 and 7b,
respectively. The reaction of amine 6 with methanesulfonyl
chloride or benzenesulfonyl chloride using similar conditions
afforded 8a and 8b. Finally, the reaction of the potassium salt
of thioacetic acid or thiobenzoic acid with 4 gave 9a32,37 and
9b, respectively.
Results
Newly synthesized compounds 3d–3o, 7b, 8a, 8b, and 9b
were then evaluated for affinity at opioid receptors using
methodology previously described (Table 1).38 These analogues
were prepared to give insight as to the nature of the high affinity
and selectivity of 1a and 1c for κ and µ receptors, respectively.
Recently, we investigated the effects of the addition of a bromo
group to 1c (i.e., 3a–3c).30 It was found that substitution of the
bromo group in the 4-position (3c) retained high affinity at µ
receptors. This modification also increased µ/κ selectivity

Herkinorin Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8 2423
Scheme 2a
a Reagents and conditions: (a) CBr4, PPh3, CH2Cl2; (b) NaN3, DMF, AcOH; (c) Zn, NH4Cl, MeOH, CH2Cl2; (d) appropriate acid chloride or anhydride,
DMAP, NEt3, CH2Cl2; (e) appropriate sulfonyl chloride, DMAP, CH2Cl2; (f) RCOSK, CH3CN.
Table 1. Binding Affinities of Salvinorin A Analogues at Opioid
compared to 1c (Ki ) 10 vs 12 nM). However, it retained
Receptors Using [125I]IOXY as Radioligand39,40
selectivity for µORs over κORs. Previously, we showed that
Ki ( SD, nM
selectivity
µ/κ
1.9 ( 0.2 >526
bioisosteric replacement of the benzene ring with a 2-thiophene
(3m) retained affinity at µORs.30 We were curious if the point
of attachment might play a role in its affinity. To probe this,
we synthesized the corresponding 3-thiophene (3n). Compound
3n had similar affinity to 1c for µORs and κORs (Ki ) 10 vs
12 nM and Ki ) 80 vs 90 nM, respectively), indicating that the
point of attachment on the thiophene ring does not play a role
in µOR binding. This change, however, increases affinity 3-fold
for κORs (Ki ) 80 vs 260 nM). Finally, we sought to further
confirm the role of the aromatic moiety in the selectivity of 1c.
To address this, we prepared cyclohexyl analogue (3o). As
expected, 3o had reduced affinity for µORs and κORs compared
to 1c (Ki ) 1030 vs 12 nM and Ki ) 2010 vs 90 nM,
respectively). This change also decreased selectivity for µORs
over κORs (µ/κ ) 0.5 vs µ/κ ) 0.13).
While our studies were in progress, several groups reported
the effects of bioisosteric replacement of the 2-acetoxy group
in 1a with an acetamido group (7a).32,35 Consistent with those
reports, we found this change resulted in a loss in affinity at κ
receptors (Ki ) 30 vs 1.9 nM). However, 7a was found to have
affinity for µORs (Ki ) 4180 nM). Given our previous finding
that introduction of a benzene ring increases µ affinity,25 we
synthesized benzamide 7b. As expected, introduction of the
benzene ring resulted in a decreased affinity at κ receptors and
increased affinity at µ receptors. To our delight, 7b has 4-fold
higher affinity than 1c (Ki ) 3.1 vs 12 nM) and is more selective
for µ receptors over κ receptors (κ/µ ) 0.0004 vs κ/µ ) 0.13.
To further explore these developments, we synthesized sulfona-
mides 8a and 8b.
cmpd
µ
δ
κ
δ/κ
1aa
1b
1ca
3ac
3bc
3cc
3d
3e
>1000b
>10000
12 ( 1
110 ( 1
110 ( 1
10 ( 1
1640 ( 90
30 ( 2
70 ( 4
5790 ( 980
>10000
1170 ( 60
>10,000
>10000
1410 ( 80
>10000
1140 ( 60
1860 ( 140
3050
>35
12
280 ( 20
90 ( 2
>35
0.13
90 ( 7
1.2
1.6
0.01
7
0.05
0.12
8
>100
>100
2
70 ( 7
740 ( 40
230 ( 20
550 ( 30
540 ( 40
900 ( 50
800 ( 50
570 ( 40
410 ( 40
5490 ( 640
70 ( 2
>43
2
3
3f
3g
3 h
3i
7550 ( 970 >10000
>11
>12
>10000
260 ( 210
>10000
180 ( 20
10 ( 1
>10000
>10000
>10000
>10000
580 ( 30
1380 ( 130
690 ( 30
>10000
>12
13
>24
0.45
3j
3k
3l
>24
>2
8
0.03
0.14
0.04
0.16
0.5
13
0.0004
3mc 10 ( 2
260 ( 20
80 ( 3
2010 ( 110
30 ( 2
7430 ( 880
260 ( 30
1400 ( 110
5.7 ( 0.4
1410 ( 80
5
9
>5
3n
3o
7a
7b
8a
8b
9a
9b
10 ( 1
1030 ( 80
4180 ( 310 >10000
>330
3.1 ( 0.4
>10000
>10000
810 ( 30
>10000
>10000
0.11
>38
>38
>7
>7
700
1.4
4370 ( 310 3990 ( 290
290 ( 70 1930 ( 70
767
0.21
a Data from ref 25. b Partial inhibitor. c Data from ref 30. All results are
n ) 3.
1-naphthoate abolishes affinity for κORs (Ki > 10000 nM).31
This difference is likely due to the different radioligands used
([3H]bremazocine vs [125I]IOXY) or the possibility of misiden-
tification because these compounds were not rigorously evalu-
ated for purity.31 Annulation of the benzene ring into the 3 and
4 positions (3k) reduced affinity at µORs approximately 10-
fold compared to 1c (Ki ) 180 vs 12 nM). This modification
also decreased affinity for κORs greater than 50-fold (Ki ) 5490
vs 90 nM). This suggests that a ꢀ,γ-annulated system increases
selectivity for µORs over κORs. To probe this, we prepared
2-benzofuran 3l as an analogue that possesses a ꢀ,γ-annulated
system. Somewhat surprisingly, 3l had equal affinity at µORs
Previously, we showed that the addition of a methanesulfonyl
group retained high affinity and activity at κORs.25 The
replacement of the acetamido group with a methanesulfony-
lamino group (8a) decreased affinity approximately 9-fold for
κORs compared to 7a (Ki ) 260 vs 30 nM). This change also
abolished affinity for µORs (Ki > 10000 nM). The addition of
a benzene ring to 8a (8b) decreased affinity approximately 5-fold
for κORs (Ki ) 1400 vs 260 nM). The loss of affinity at κORs

2424 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8
Tidgewell et al.
Table 2. Results from [35S]GTP-γ-S Functional Assay Carried out in
CHO Cells Containing DNA for Human µ and κ Receptors39,40
µ EC50 ( SD,
κ EC50 ( SD,
a
a
cmpd
nM
µ Emax ( SD
nM
κ Emax ( SD
1a
1c
3c
3e
3f
3i
3l
3m
3n
7a
7b
NTb
NTb
40 ( 10
1320 ( 150
NTb
3590 ( 550
2610 ( 470
NTb
120 ( 2
140 ( 2
NTb
500 ( 140
4890 ( 980
1670 ( 250
830 ( 100
1370 ( 230
1680 ( 250
1150 ( 250
690 ( 60
NTb
130 ( 4
108 ( 8c
72 ( 3c
94 ( 3c
46 ( 2c
104 ( 5c
95 ( 3c
108 ( 3c
NTb
97 ( 2c
106 ( 5c
NTb
1120 ( 170
109 ( 5c
NTb
NTb
840 ( 210
120 ( 20
NTb
95 ( 8c
108 ( 3c
NTb
360 ( 60
134 ( 5
35 ( 2
100 ( 4
(-)-U50,488 4840 ( 890
DAMGO
30 ( 6
100 ( 4
40 ( 4
NTb
NTb
a Emax is % compound that stimulates binding compared to DAMGO
(10 µM) at µ and (-)-U50,488 (500 nM) at κ receptors, respectively; b Not
tested. c P < 0.05 when compared to the Emax of 1c at µ and κ receptors
(Student’s t-test).
may be due to the increased ionizability of a sulfonamide
compared to a sulfonyl ester. Recently, it has been shown that
a tertiary amide has higher affinity for κORs than a secondary
amide.35 This data would seem to confirm this observation, as
well as, our previous finding that sulfonyl esters of 1a are not
binding in an identical manner to alkyl esters.30
Finally, we probed the replacement of the 2-acetoxy group
with a 2-acetylthio group. As seen previously,37 this change
resulted in a slight reduction in affinity at κ receptors (Ki ) 5.7
vs 1.9 nM). However, 9a was found to have low affinity for
µORs (Ki ) 4370 nM). The addition of a benzene ring to 9a
(9b) lead to an increase in affinity at µORs (Ki ) 290 vs 4370
nM). However, this change lead to a 24-fold decrease in affinity
compared to 1c, indicating an ester or amide linkage is
preferential for binding at µORs.
To test the hypothesis that µ opioids derived from 1a have
functional activity at opioid receptors, several analogues were
then evaluated in a [35S]GTP-γ-S assay (Table 2).39,40 The
introduction of a 4-bromo substituent (3c) resulted in an
approximately 10-fold decrease in activity compared to 1c (EC50
) 4890 vs 500 nM). This modification also reduced the efficacy
compared to 1c (Emax ) 108 vs 130), but 3c is just as efficacious
as DAMGO (Emax ) 108 vs 100). The presence of a 3-methoxy
group (3e) resulted in an approximately 3-fold loss in activity
at µORs compared to 1c (EC50 ) 1670 vs 500 nM). A similar
effect was seen at κORs. Interestingly, 3e is not as efficacious
as 1c (Emax ) 72 vs 130) and appears to be a partial agonist
when compared to DAMGO (Emax ) 72 vs 100). A 4-methoxy
group (3f) had similar activity compared to 1c (EC50 ) 830 vs
500 nM). However, 3f is not as efficacious at µORs as 1c (Emax
) 94 vs 130) but is approximately as efficacious as DAMGO
(Emax ) 94 vs 100). A 4-nitro group (3i) decreased activity at
µORs approximately 3-fold compared to 1c (EC50 ) 1370 vs
500 nM). This change, however, resulted in a large decrease in
efficacy compared to 1c and DAMGO (Emax ) 46 vs 130 and
Emax ) 46 vs 100).
Figure 2. Agonist-induced ꢀ-arrestin-2-GFP translocation. HEK-293
cells transfected with MOR1 and ꢀarr2-GFP and with or without GRK2
overexpression were treated with the indicated drugs. Representative
cells of at least three independent experiments are shown in which
several cells were imaged. (A) DAMGO induces robust translocation
of ꢀarr2-GFP (puncta; arrows) to the plasma membrane. Morphine,
however, can only induce translocation when GRK2 is overexpressed.
Ester 1c is unable to induce robust ꢀarr2-GFP translocation to the
plasma membrane even in the presence of GRK2 overexpression. (B)
Amide 7b is the only herkinorin derivative that induces ꢀarr2-GFP
translocation in the absence or presence of GRK2 overexpression.
replacement of the benzene ring in 1c with a 2-thiophene (3m)
reduced activity and efficacy at µORs compared to 1c (EC50
1150 vs 500 nM and Emax ) 95 vs 130). Substitution of a
3-thiophene (3n) had little effect on activity at µORs (EC50
)
)
690 nM vs 500 nM) and decreased efficacy (Emax ) 108 vs
130). Compound 3n, however, is roughly as efficacious as
DAMGO (Emax ) 108 vs 100) at µORs.
Replacement of the 2-acetoxy group in 1a with a 2-acetamido
group (7a) resulted in a 3-fold loss in activity at κORs compared
to 1a (EC50 ) 120 vs 40 nM). This change, however, had little
effect on efficacy (Emax ) 108 vs 120). Replacement of the
2-benzoyloxy group in 1c with a 2-benzoylamino group (7b)
resulted in a slight increase in activity and no change in efficacy
(EC50 ) 360 vs 500 nM and Emax ) 134 vs 130).
To better understand the role of drug structure on µOR
regulation pathways, we examined the ability of 3c, 3f, 3i, and
7b to induce ꢀ-arrestin-2-GFP translocation HEK-293 cells
(Figure 2). The effects of DAMGO, morphine, and 1c are shown
for comparison.3,28 DAMGO induces robust translocation of
ꢀarr2-GFP to the plasma membrane. Morphine, however, can
only induce translocation when GRK2 is overexpressed. Com-
pounds 3c, 3f, and 3i, like 1c, are unable to induce robust ꢀarr2-
GFP translocation to the plasma membrane even in the presence
of GRK2 overexpression. Amide 7b induces robust ꢀarr2-GFP
translocation under both conditions.
Substitution of the benzene ring in 1c with a 2-benzofuran
(3l) resulted in an approximately 3-fold loss in activity and
decreased efficacy at µORs compared to 1c (EC50 ) 1680 vs
500 nM and Emax ) 104 vs 130). However, 3l is still a full
agonist when compared to DAMGO (Emax ) 104 vs 100).
Benzofuran 3l had similar activity at κORs compared to 1c (EC50
) 1120 vs 1320 nM). Strikingly, 3l was less efficacious as an
agonist at κORs compared to 1a (Emax ) 109 vs 140), but more
efficacious than U50,488H (Emax ) 109 vs 100). Bioisosteric
To further support the conclusion that µ opioids derived from
1a have altered receptor regulation, we examined the ability of
3c, 3f, 3i, and 7b to induce µOR-YFP internalization in HEK-
293 cells (Figure 3). The effects of DAMGO, morphine, and
1c are shown for comparison.3,28 DAMGO induces robust
internalization of µOR-YFP. Morphine, however, can only
induce µOR-YFP internalization when GRK2 is overexpressed.
Unlike DAMGO and morphine and similar to 1c, 3c, 3f, and 3i
are unable to induce robust µOR-YFP internalization even in

Herkinorin Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8 2425
most potent neoclerodane µ agonist described to date. However,
this change promotes ꢀ-arrestin translocation and receptor
internalization in HEK-293 cells. The discovery of two com-
pounds with nearly identical chemical structure and similar
binding affinity and efficacies that elicit differential signaling
at the cellular level would suggest that not only receptor
conformation but also ligand structure contribute to signaling
events. Future studies of the effect of chemical alterations of
1c on the activation of cellular pathways may serve as a basis
for the development of compounds that can selectively activate
or block ꢀ-arrestin-receptor interactions may determine specific
physiological responses.
The differences in affinity and receptor regulation between
1c and 7b are interesting. One potential explanation is that these
two molecules, while very similar in structure are not binding
in an identical manner at the µOR. This type of phenomenon
has been seen previously with other opioids.41 Another explana-
tion is that the benzene rings in 1c and 7b may have different
orientations relative to the A ring of the salvinorin core. X-ray
crystallographic studies26,30 indicate that the benzene ring in
1c is out of the plane of the A ring of the salvinorin core.
Preliminary molecular modeling indicates that the benzene ring
in 7b is in the plane of the A ring. This orientation of 7b may
be responsible for the increased affinity and activity at µORs
compared to 1c. However, the out of the plane orientation of
the benzene ring in 1c and esters 3c, 3f, and 3i may be required
for the lack of the ꢀ-arrestin translocation and receptor
internalization. Conformationally constrained analogues will
need to be prepared to further delineate the role of the benzene
ring on affinity, activity, and receptor regulation pathways.
Figure 3. Agonist-induced MOR1-YFP internalization. HEK-293 cells
stably transfected with MOR1-YFP were treated with the indicated
drugs with or without GRK2 overexpression. Representative cells of
at least three independent experiments are shown in which several cells
were imaged. (A) DAMGO induces robust internalization of MOR1-
YFP (vesicles; arrows). Morphine, however, can only induce robust
MOR1-YFP internalization when GRK2 is overexpressed. Ester 1c is
unable to induce robust MOR1-YFP internalization even in the presence
of GRK2 overexpression. (B) Amide 7b is the only herkinorin derivative
that can induce MOR1-YFP internalization, even in the in the presence
of GRK2 overexpression.
An alternate explanation for the differences seen in affinity
and receptor regulation is that ester 1c hydrolyzes too rapidly
in media to cause internalization and other chronic effects.
Amide 7b would be expected to be more stable in serum, as
recently shown for 7a.42 Additional stability studies of 1c and
7b will be necessary to further investigate the role of metabolism
in the differences seen in receptor regulation pathways. How-
ever, 1b, the likely metabolism product of ester 1c, has no
affinity for µORs (Ki > 10000 nM),43 and after 30 min, 1c still
produces a 3.5-fold increase in ERK phosphorylation, demon-
strating a persisting agonistic activity.28 Moreover, cells treated
with DAMGO will internalize the µOR in approximately 10–15
min, therefore, the compound, which is still active at 30 min in
the ERK activation assay, should be sufficiently potent to induce
internalization. Furthermore, chronic treatment of 1c produces
desensitization in cells, suggesting that it is active long enough
to induce some yet undescribed mode of receptor desensitiza-
tion.44
the presence of GRK2 overexpression. However, 7b induces
robust µOR-YFP internalization in HEK-293 cells under both
conditions.
To further assess agonist activity in parallel with the current
studies, we used the phosphorylation of the downstream MAP
kinases (ERK1/ERK2) as an indicator of receptor activation.
Compounds 3c, 3f, 3i, and 7b were examined for their ability
to activate ERK in hµOR-CHO cells. In Figure 4, 3c, 3f, 3i,
and 7b are similar to DAMGO in that they are able to induce
a µOR1-mediated, dose-dependent increase in ERK phospho-
rylation that is blocked by naloxone.28
Taken together, these data indicate that 3c, 3f, and 3i are
able to induce receptor conformations that are able to activate
both G protein coupling and MAP kinase activation pathways,
yet have unique properties compared to the morphine or
DAMGO bound µOR rendering the receptor resistant to
ꢀ-arrestin interactions or internalization. Amide 7b appears to
induce receptor conformations that are different than other
derivatives of 1c and produces effects similar to other opioids
such as DAMGO.
The molecular basis for the unique signaling properties of
1c is not clear at this time. A likely explanation is that they are
the result of a unique binding mode at the µOR relative to other
opioids. Most nonpeptide opioid ligands, which contain a basic
nitrogen atom, interact with aspartate 147 in TM III.45 Given
the structure of 1c, this interaction is unlikely. This explanation
is further supported by recent studies, indicating that 1a utilizes
unique residues in binding to κORs.22–24 Ester 1c and related
analogues may have a similar mode of binding at µORs. The
exact nature of the interaction of 1c with the µOR will have to
be confirmed through site-directed mutagenesis and/or affinity
labeling experiments.
Discussion
Our results indicate that the structure–activity relationships
for affinity and activity at µ opioid receptors are not identical
to those for receptor regulation. Addition of substituents to the
aromatic ring of 1c results in agonists and partial agonists at
µORs and similar receptor regulation to 1c. These changes do
not affect the unique receptor regulation properties of 1c.
Analogues 3c, 3f, 3i are unable to induce robust ꢀarr2-GFP
translocation and µOR-YFP internalization even in the presence
of GRK2 overexpression in HEK-293 cells. Replacement of the
ester linkage in 1c with an amide linkage (7b) increases affinity
at µORs compared to 1c. Amide 7b has been identified as the
With regards to chemical structure, 1a and 1c have an
interesting structural motif for GPCR ligands. The neoclerodane
nucleus is not considered to be a priVileged structure, which is
defined as a selected substructure that is able to provide high-

2426 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8
Tidgewell et al.
Figure 4. Herkinorin and its four derivatives induce dose-dependent, MOR1-mediated ERK activation. CHO cells stably expressing the human
MOR1 were treated with the indicated drugs for 10 min. Top: Representative concentration–response data of 1c, 3c, 3f, 3i, and 7b are shown.
Experiments were performed at least three times in triplicate. Bottom: Densitometric analysis of two experiments done in triplicate compare efficacy
of 1c, 3c, 3f, 3i, and 7b. Bar graph shows means and SEM for the densitometric analysis (Student’s t-test p < 0.0001 vs vehicle for all treatments;
* p < 0.05 vs 3i (100 nM) for all other treatments) Representative immunoblots of a single experiment are shown.
affinity ligands for more than one type of receptor.46,47 However,
natural products, such as 1a, can be viewed as a population of
privileged structures selected by evolutionary pressures to
interact with a wide variety of proteins and other biological
targets for specific purposes.48 Finding additional molecules that
have unique receptor regulation pathways for GPCRs may
require examining additional natural products or natural product-
like libraries.49
The life cycle of a GPCR is to reside at the cell surface and,
upon activation, become phosphorylated, desensitized, internal-
ized, and then either degraded or recycled. While internalized,
the GPCR may also take part in activating signaling cascades.50,51
Usual drug discovery efforts for GPCRs are to develop agonists,
antagonists, or inverse agonists for the GPCR of interest. In
our case, this is the µ opioid receptor. Our results illustrate a
novel drug discovery strategy that seeks to develop a series of
compounds that retain signaling properties at a GPCR but avoid
typical regulation pathways. This has a clear impact on the
development of novel opioids with reduced side effects and
GPCR drug discovery because this finding illustrates the ability
of selecting or designing novel agents that differentially activate
regulation pathways of a single receptor. This has the potential
to optimize therapeutic action in vivo by alleviating unwanted
side effects.
Experimental Section
General Methods. Unless otherwise indicated, all reagents were
purchased from commercial suppliers and are used without further
purification. All melting points were determined on a Thomas-
1
Hoover capillary melting apparatus and are uncorrected. The H
NMR spectra were recorded at 300 MHz on a Bruker Avance-300
spectrometer using CDCl3 as solvent, δ values in ppm (TMS as
internal standard), and J (Hz) assignments of 1H resonance coupling.
Thin-layer chromatography (TLC) was performed on 0.25 mm
plates Analtech GHLF silica gel plates using n-hexanes/EtOAc, 1:1
as the solvent system. Spots on TLC visualized with vanillin/H2SO4
in ethanol. Column chromatography was performed with silica gel
(32–63 µ particle size) from Bodman Industries (Atlanta, GA).
Analytical HPLC was carried out on an Agilent 1100 Series

Herkinorin Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8 2427
Capillary HPLC system with diode array detection at 254.8 nm on
an Agilent Eclipse XDB-C18 column (4.6 × 150 mm, 5 µm) with
isocratic elution in 70% CH3CN/30% H2O at a flow rate of 5.0
mL/min.
General Procedure A. A solution of 1b (1 equiv), appropriate
acid chloride (1–3 equiv), NEt3 (3 equiv), and a catalytic amount
of DMAP in CH2Cl2 was stirred at room temperature. Absolute
MeOH was added and the solvent was removed under reduced
pressure. CH2Cl2 was added to the residue and the solution was
washed with 10% HCl (3 × 20 mL) and saturated NaCl (3 × 20
mL) and dried (Na2SO4). Removal of the solvent under reduced
pressure afforded a crude solvent that was purified by column
chromatography (eluent: n-hexanes/EtOAc) to yield the desired
compound.
(s, 3H), 1.64 (m, 3H), 1.83 (dd, J ) 2.7, 11.7 Hz, 1H), 2.15 (m,
2H), 2.27 (s, 1H), 2.40 (m, 2H), 2.55 (dd, J ) 5.4, 12.3 Hz, 1H),
2.83 (dd, J ) 3.6, 13.2 Hz, 1H), 3.75 (s, 3H), 5.42 (dd, J ) 7.5,
12.6 Hz, 1H), 5.54 (dd, J ) 5.1, 11.4 Hz, 1H), 6.41 (dd, J ) 0.9,
1.8 Hz, 1H), 7.42 (dd, J ) 1.5, 1.8 Hz, 1H), 7.45 (dd, J ) 0.9, 1.5
Hz, 1H), 7.69 (td, J ) 1.8, 7.8 Hz, 1H), 7.74 (td, J ) 1.5, 7.5 Hz,
1H), 7.92 (dd, J ) 1.8, 7.8 Hz, 1H), 8.00 (dd, J ) 1.8, 7.5 Hz,
1H). Anal. (C28H29NO10 ·0.25H2O): C, H, N.
2S,4aR,6aR,7R,9S,10aS,10bR)-9-(3-Nitrobenzoyloxy)-2-(3-
furanyl)-dodecahydro-6a,10b-dimethyl-4,10-dioxo-2H-naph-
tho[2,1-c]pyran-7-carboxylic Acid Methyl Ester (3 h). Compound
3h was synthesized from 1b using general procedure A and
3-nitrobenzoyl chloride to afford 0.110 g (80%) as a white solid,
mp 148–150 °C. 1H NMR (300 MHz, CDCl3): δ 1.19 (s, 3H), 1.46
(s, 3H), 1.64 (m, 3H), 1.85 (dd, J ) 2.7, 9.9 Hz, 1H), 2.15 (m,
2H), 2.28 (s, 1H), 2.52 (m, 3H), 2.85 (dd, J ) 5.1, 11.7 Hz, 1H),
3.76 (s, 3H), 5.43 (dd, J ) 8.4, 11.7 Hz, 1H), 5.53 (dd, J ) 5.1,
11.7 Hz, 1H), 6.39 (dd, J ) 0.9, 1.8 Hz, 1H), 7.41 (m, 2H), 7.69
(t, J ) 8.1, 8.1 Hz, 1H), 8.41 (dt, J ) 1.5, 1.5, 7.5 Hz, 1H), 8.46
(ddd, J ) 0.9, 2.4, 8.1 Hz, 1H), 8.91 (t, J ) 2.1, 2.1 Hz, 1H).
Anal. (C28H29NO10 ·0.5H2O) C, H, N.
2S,4aR,6aR,7R,9S,10aS,10bR)-9-(4-Nitrobenzoyloxy)-2-(3-
furanyl)-dodecahydro-6a,10b-dimethyl-4,10-dioxo-2H-naph-
tho[2,1-c]pyran-7-carboxylic Acid Methyl Ester (3i). Compound
3i was synthesized from 1b using general procedure A and
4-nitrobenzoyl chloride to afford 0.093 g (67%) as a white solid,
mp 195–200 °C. 1H NMR (300 MHz, CDCl3): δ 1.18 (s, 3H), 1.46
(s, 3H), 1.66 (m, 3H), 1.84 (dd, J ) 3.0, 9.9 Hz, 1H), 2.15 (m,
2H), 2.27 (s, 1H), 2.51 (m, 3H), 2.85 (dd, J ) 6.9, 15.9 Hz, 1H),
3.76 (s, 3H), 5.42 (dd, J ) 9.3, 10.8 Hz, 1H), 5.53 (dd, J ) 5.1,
11.7 Hz, 1H), 6.39 (dd, J ) 0.9, 1.8 Hz, 1H), 7.40 (dd, J ) 1.8,
1.8 Hz, 1H), 7.42 (dd, J ) 0.9, 1.8 Hz, 1H), 8.25 (dt, J ) 1.8, 2.1,
9.3 Hz, 2H), 8.31 (dt, J ) 1.8, 2.1, 9.0 Hz, 2H). Anal. (C28H29NO10)
C, H, N.
(2S,4aR,6aR,7R,9S,10aS,10bR)-9-(1-Naphthoyloxy)-2-(3-fura-
nyl)-dodecahydro-6a,10b-dimethyl-4,10-dioxo-2H-naphtho[2,1-
c]pyran-7-carboxylic Acid Methyl Ester (3j). Compound 3j was
synthesized from 1b using general procedure A and 1-naphthoyl
chloride to afford 0.044 g (63%) of 3j as a white solid, mp 155–160
°C. 1H NMR (CDCl3): δ 1.19 (s, 3H), 1.49 (s, 3H), 1.66 (m, 3H),
1.84 (dd, J ) 2.7, 9.9 Hz, 1H), 2.15 (m, 2H), 2.30 (s, 1H), 2.55
(m, 3H), 2.87 (dd, J ) 8.1, 8.4 Hz, 1H), 3.75 (s, 3H), 5.53 (m,
2H), 6.40 (dd, J ) 0.9, 1.8 Hz, 1H), 7.40 (dd, J ) 1.8, 1.8 Hz,
1H), 7.43 (d, J ) 0.9 Hz, 1H), 7.56 (m, 3H), 7.89 (dd, J ) 1.2, 7.8
Hz, 1H), 8.05 (d, J ) 8.1 Hz, 1H), 8.32 (dd, J ) 1.5, 7.5 Hz, 1H),
8.86 (d, J ) 8.7 Hz, 1H).
(2S,4aR,6aR,7R,9S,10aS,10bR)-9-(2-Naphthoyloxy)-2-(3-fura-
nyl)-dodecahydro-6a,10b-dimethyl-4,10-dioxo-2H-naphtho[2,1-
c]pyran-7-carboxylic Acid Methyl Ester (3k). Compound 3k was
synthesized from 1b using general procedure A and 2-naphthoyl
chloride to afford 0.020 g (29%) as a white solid, mp 155–160 °C.
1H NMR (300 MHz, CDCl3): δ 1.19 (s, 3H), 1.48 (s, 3H), 1.67
(m, 3H), 1.85 (dd, J ) 2.7, 9.9 Hz, 1H), 2.15 (m, 2H), 2.29 (s,
1H), 2.56 (m, 3H), 2.86 (dd, J ) 6.0, 10.8 Hz, 1H), 3.76 (s, 3H),
5.44 (m, 1H), 5.55 (dd, J ) 5.4, 12.0 Hz, 1H), 6.39 (dd, J ) 0.9,
1.8 Hz, 1H), 7.40 (dd, J ) 1.8, 3.0 Hz, 1H), 7.42 (dd, J ) 0.9, 1.8
Hz, 1H), 7.59 (m, 2H), 7.90 (m, 2H), 7.97 (d, J ) 8.1 Hz, 1H),
8.08 (dd, J ) 1.8, 10.2 Hz, 1H), 8.67 (m, 1H). HRMS (m/z): [M+]
calcd for C32H32O8Cs, 677.1152; found, 677.1150. HPLC tR ) 7.38
min; purity ) 98.22%.
Benzofuran-2-carboxylic Acid (2S,4aR,6aR,7R,9S,10aS,10bR)-
7-Carbomethoxy-2-(3-furanyl)dodecahydro-6a,10b-dimethyl-
4,10-dioxo-2H-naphtho[2,1-c]pyran-9-yl Ester (3l). A solution
of 1b (0.10 g, 0.26 mmol), benzofuran 2-carboxylic acid (0.08 mg,
0.51 mmol), HOBT (0.07 g, 0.51 mmol), and EDCI (0.120 g, 0.64
mmol) in CH2Cl2 (20 mL) was stirred at room temperature for 4 d.
The mixture was washed with 2 N HCl (3 × 15 mL), saturated
NaHCO3 (3 × 15 mL), and H2O (3 × 15 mL) and dried (Na2SO4).
Removal of the solvent under reduced pressure afforded a crude
product that was purified by column chromatography (eluent:
n-hexanes/EtOAc, 1:1) to afford 0.05 g of 1b and 0.016 g (22%)
(2S,4aR,6aR,7R,9S,10aS,10bR)-9-(4-Bromobenzoyloxy)-2-(3-
furanyl)-dodecahydro-6a,10b-dimethyl-4,10-dioxo-2H-naphtho-
[2,1-c]pyran-7-carboxylic Acid Methyl Ester (3c). Compound 3c
was synthesized from 1b using general procedure A and 4-bro-
mobenzoyl chloride to afford 0.083 g (57%) as a white solid, mp
190–192 °C. 1H NMR (CDCl3): δ 1.17 (s, 3H), 1.46 (s, 3H), 1.65
(m, 3H), 1.83 (dd, J ) 3.3, 9.9 Hz, 1H), 2.10 (dd, J ) 2.7, 11.4
Hz, 1H), 2.17 (s, 1H), 2.20 (m, 1H), 2.50 (m, 3H), 2.83 (dd, J )
11.1, 11.7 Hz, 1H), 3.75 (s, 3H), 5.38 (dd, J ) 9.9, 10.2 Hz, 1H),
5.52 (dd, J ) 5.1, 11.7 Hz, 1H), 6.38 (dd, J ) 0.9, 1.8 Hz, 1H),
7.41 (m, 2H), 7.61 (m, 2H), 7.94 (m, 2H).
(2S,4aR,6aR,7R,9S,10aS,10bR)-9-(2-Methoxybenzoyloxy)-2-
(3-furanyl)-dodecahydro-6a,10b-dimethyl-4,10-dioxo-2H-naph-
tho[2,1-c]pyran-7-carboxylic Acid Methyl Ester (3d). Compound
3d was synthesized from 1b using general procedure A and
2-anisoyl chloride to afford 0.010 g (14%) as a white solid, mp
105–107 °C. 1H NMR (300 MHz, CDCl3): δ 1.16 (s, 3H), 1.47 (s,
3H), 1.65 (m, 3H), 1.84 (m, 1H), 2.14 (m, 2H), 2.27 (s, 1H), 2.43
(m, 2H), 2.55 (dd, J ) 5.1, 13.2 Hz, 1H), 2.83 (dd, J ) 8.4, 8.7
Hz, 1H), 3.74 (s, 3H), 3.90 (s, 3H), 5.38 (dd, J ) 9.9, 9.9 Hz, 1H),
5.52 (dd, J ) 5.4, 11.7 Hz, 1H), 6.38 (s, 1H), 7.00 (dd, J ) 7.5,
8.1 Hz, 2H), 7.40 (m, 2H), 7.51 (ddd, J ) 1.8, 7.5, 8.1 Hz, 1H),
7.95 (d, J ) 7.5 Hz, 1H). HRMS (m/z): [M+] calcd for C29H32O9,
525.2125; found, 525.2117. HPLC tR ) 4.43 min; purity ) 97.76%.
(2S,4aR,6aR,7R,9S,10aS,10bR)-9-(3-Methoxybenzoyloxy)-2-
(3-furanyl)-dodecahydro-6a,10b-dimethyl-4,10-dioxo-2H-naph-
tho[2,1-c]pyran-7-carboxylic Acid Methyl Ester (3e). Compound
3e was synthesized from 1b using general procedure A and
3-anisoyl chloride to afford 0.017 g (26%) as a white solid, mp
200–202 °C. 1H NMR (300 MHz, CDCl3): δ 1.17 (s, 3H), 1.46 (s,
3H), 1.65 (m, 3H), 1.82 (dd, J ) 2.4, 9.9 Hz, 1H), 2.14 (m, 2H),
2.27 (s, 1H), 2.46 (m, 2H), 2.54 (dd, J ) 5.4, 13.8 Hz, 1H), 2.84
(dd, J ) 6.3, 10.5 Hz, 1H), 3.75 (s, 3H), 3.86 (s, 3H), 5.39 (dd, J
) 9.6, 10.5 Hz, 1H), 5.51 (dd, J ) 5.1, 11.7 Hz, 1H), 6.39 (d, J )
0.9 Hz, 1H), 7.13 (ddd, J ) 0.9, 0.9, 7.1 Hz, 1H), 7.40 (m, 3H),
7.58 (dd, J ) 1.5, 2.4 Hz, 1H), 7.69 (dt, J ) 0.9, 0.9, 7.5 Hz, 1H).
HRMS (m/z): [M+] calcd for C29H32O9, 525.2125; found, 525.2140.
HPLC tR ) 5.14 min; purity ) 98.16%.
(2S,4aR,6aR,7R,9S,10aS,10bR)-9-(4-Methoxybenzoyloxy)-2-
(3-furanyl)-dodecahydro-6a,10b-dimethyl-4,10-dioxo-2H-naph-
tho[2,1-c]pyran-7-carboxylic Acid Methyl Ester (3f). Compound
3f was synthesized from 1b using general procedure A and 4-anisoyl
chloride to afford 0.083 g (60%) as a white solid, mp 185–187 °C.
1H NMR (300 MHz, CDCl3): δ 1.17 (s, 3H), 1.46 (s, 3H), 1.65
(m, 3H), 1.83 (dd, J ) 2.7, 11.7 Hz, 1H), 2.15 (m, 2H), 2.25 (s,
1H), 2.45 (m, 2H), 2.55 (dd, J ) 5.1, 13.2 Hz, 1H), 2.83 (dd, J )
7.8, 8.7 Hz, 1H), 3.74 (s, 3H), 3.87 (s, 3H), 5.37 (dd, J ) 9.6, 10.2
Hz, 1H), 5.52 (dd, J ) 5.1, 11.7 Hz, 1H), 6.38 (dd, J ) 0.9,
1.8 Hz, 1H), 6.93 (dt, J ) 2.1, 3.0, 8.7 Hz, 2H), 7.39 (dd, J ) 1.8,
1.8 Hz, 1H), 7.41 (dd, J ) 0.9, 1.5 Hz, 1H), 8.04 (dt, J ) 2.1, 3.0,
9.0 Hz, 2H). Anal. (C29H32O9): C, H.
2S,4aR,6aR,7R,9S,10aS,10bR)-9-(2-Nitrobenzoyloxy)-2-(3-
furanyl)-dodecahydro-6a,10b-dimethyl-4,10-dioxo-2H-naph-
tho[2,1-c]pyran-7-carboxylic Acid Methyl Ester (3g). Compound
3g was synthesized from 1b using general procedure A and
2-nitrobenzoyl chloride to afford 0.103 g (75%) as a white solid,
mp 144–146 °C. 1H NMR (300 MHz, CDCl3): δ 1.15 (s, 3H), 1.48

2428 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8
Tidgewell et al.
of 3l as a white solid, mp 226–227 °C. 1H NMR (300 MHz, CDCl3):
δ 1.19 (s, 3H), 1.48 (s, 3H), 1.64 (m, 3H), 1.84 (dd, J ) 2.8, 10.5
Hz, 1H), 2.19 (m, 2H), 2.28 (s, 1H), 2.60 (m, 3H), 2.86 (1H, dd,
J ) 5.25, 10.5 Hz, 1H), 3.77 (s, 3H), 5.48 (dd, J ) 9.3, 10.8 Hz,
1H), 5.57 (dd, J ) 5.1, 11.7 Hz, 1H), 6.40 (dd, J ) 0.9, 1.2 Hz,
1H), 7.36 (s, 1H), 7.43 (m, 2H), 7.52 (m, 1H), 7.66 (m, 2H), 7.73
(dd, J ) 0.6, 7.8 Hz, 1H). Anal. (C30H30O9): C, H.
Thiophene-3-carboxylic Acid (2S,4aR,6aR,7R,9S,10aS,10bR)-
7-Carbomethoxy-2-(3-furanyl)dodecahydro-6a,10b-dimethyl-
4,10-dioxo-2H-naphtho[2,1-c]pyran-9-yl Ester (3m). Compound
3m was synthesized from 1b using general procedure A and
3-thiophenoyl chloride to afford 0.056 g (44%) as a white solid,
mp 211–212 °C. 1H NMR (300 MHz, CDCl3): δ 1.18 (s, 3H), 1.47
(s, 3H), 1.69 (m, 3H), 1.82 (dd, J ) 2.7, 10.0 Hz, 1H), 2.18 (m,
3H), 2.27 (s, 1H), 2.42 (m, 2H), 2.52 (dd, J ) 5.1, 13.2 Hz, 1H),
2.84 (dd, J ) 7.5, 12.6 Hz, 1H), 3.76 (s, 3H), 5.39 (m, 1H), 5.56
(dd, J ) 5.1, 11.7 Hz, 1H), 7.36 (dd, J ) 3.0, 5.1 Hz, 1H), 7.42
(m, 2H), 7.57 (dd, J ) 1.0, 4.6 Hz, 1H), 8.22 (dd, J ) 0.6, 2.7 Hz,
1H). Anal. (C26H2808S): C, H.
to afford 0.08 g (86%) of 5 as a white solid, mp 200–203 °C (lit.32
179–181 °C; EtOAc/n-hexanes). 1H NMR spectra was in agreement
with that previously reported.32
(2S,4aR,6aR,7R,9S,10aS,10bR)-9-(Amino)-2-(3-furanyl)-
dodecahydro-6a,10b-dimethyl-4,10-dioxo-2H-naphtho[2,1-c]py-
ran-7-carboxylic Acid Methyl Ester (6). A mixture of 5 (0.21 g,
0.50 mmol), Zn dust (0.33 g, 5.0 mmol), and NH4Cl (0.27 g, 5.0
mmol) in a mixture of CH2Cl2/MeOH (1:4, 10 mL) was stirred at
room temperature for 3 h. The mixture was filtered and the filtrate
was concentrated to dryness under reduced pressure. NaOH (2 N,
30 mL) was added to the residue and the mixture was extracted
with CH2Cl2 (2 × 20 mL). The combined CH2Cl2 portion was
washed with H2O (30 mL) and dried (Na2SO4). Removal of the
solvent under reduced pressure afforded 0.07 g (36%) of 6 as an
1
orange solid, mp 237–240 °C (EtOAc/ n-hexanes). The H NMR
spectra was in agreement with that previously reported.35
(2S,4aR,6aR,7R,9S,10aS,10bR)-9-(Acetylamino)-2-(3-furanyl)-
dodecahydro-6a,10b-dimethyl-4,10-dioxo-2H-naphtho[2,1-c]py-
ran-7-carboxylic Acid Methyl Ester (7a). Compound 7a was
prepared from 6 using a method similar to that previously
described35 to afford 0.04 g (58%) as a white solid, mp 222–224
(2S,4aR,6aR,7R,9S,10aS,10bR)-9-(1-Cyclohexanecarbonyloxy)-
2-(3-furanyl)-dodecahydro-6a,10b-dimethyl-4,10-dioxo-2H-naph-
tho[2,1-c]pyran-7-carboxylic Acid Methyl Ester (3o). Compound
3o was synthesized from 1b using general procedure A and
cyclohexane carbonyl chloride to afford 0.091 g (71%) as a white
1
°C (lit.32 137–138 °C; EtOAc/n-hexanes). The H NMR spectra
was in agreement with that previously reported.35 Anal. (C23H29-
NO7): C, H, N.
1
solid, mp 104–107 °C. H NMR (300 MHz, CDCl3): δ 1.12 (s,
(2S,4aR,6aR,7R,9S,10aS,10bR)-9-(Benzoylamino)-2-(3-fura-
nyl)-dodecahydro-6a,10b-dimethyl-4,10-dioxo-2H-naphtho[2,1-
c]pyran-7-carboxylic Acid Methyl Ester (7b). A solution of 6
(0.10 g, 0.26 mmol), benzoyl chloride (0.11 g, 0.78 mmol), and
DMAP (0.08 g, 0.78 mmol) in CH2Cl2 (20 mL) was stirred at room
temperature for 2 h. Absolute MeOH (15 mL) was added and the
solvent was removed under reduced pressure. CH2Cl2 (25 mL) was
added to the residue, and the solution was washed with 10% HCl
(2 × 20 mL), H2O (3 × 20 mL), and saturated NaCl (3 × 20 mL)
and dried (Na2SO4). Removal of the solvent under reduced pressure
afforded 0.09 g (67%) of 7b as a white crystalline solid, mp
3H), 1.28 (m, 4H), 1.51 (m, 2H), 1.60 (m, 4H), 1.79 (m, 3H), 1.94
(m, 1H), 2.02 (m, 1H), 2.08 (m, 1H), 2.16 (m, 1H), 2.19 (s, 1H),
2.29 (dd, J ) 8.7, 9.8 Hz, 2H), 2.42 (tt, J ) 3.6, 11.3 Hz, 1H),
2.51 (dd, J ) 5.1, 13.5 Hz, 1H), 2.76 (dd, J ) 7.5, 9.3 Hz, 1H),
3.73 (s, 3H), 5.14 (dd, J ) 9.8, 10.4 Hz, 1H), 5.52 (dd, J ) 5.3,
11.6 Hz, 1H), 6.38 (dd, J ) 0.8, 1.7 Hz, 1H), 7.32 (dd, J ) 1.4,
1.4 Hz, 1H), 7.42 (m, 1H). Anal. (C28H36O8 ·0.5H2O): C, H.
(2S,4aR,6aR,7R,9R,10aS,10bR)-9-(Bromo)-2-(3-furanyl)-
dodecahydro-6a,10b-dimethyl-4,10-dioxo-2H-naphtho[2,1-c]py-
ran-7-carboxylic Acid Methyl Ester (4a) and (2S,4aR,6aR,7R,
9S,10aS,10bR)-9-(Bromo)-2-(3-furanyl)-dodecahydro-6a,10b-di-
methyl-4,10-dioxo-2H-naphtho[2,1-c]pyran-7-carboxylic Acid
Methyl Ester (4b). A mixture of salvinorin B (1b)29 (0.15 g, 0.38
mmol), triphenylphosphine (0.21 g, 0.80 mmol), and carbon
tetrabromide (0.15 g, 0.45 mmol) in CH2Cl2 (30 mL) was stirred
at room temperature overnight. TLC indicated that starting material
was still present after 16 h, thus additional triphenylphosphine (0.11
g, 0.42 mmol) and carbon tetrabromide (0.07 g, 0.21 mmol) were
added and the mixture was stirred for an additional 3 h. The solvent
was removed under reduced pressure affording a crude residue.
The residue was purified by column chromatography (eluent: 30%
EtOAc/ n-hexanes) to afford 0.10 g (59%) of 4a as a white solid,
mp 170–173 °C (lit.32 156–158 °C). 1H NMR (300 MHz, CDCl3):
δ 1.15 (s, 3H), 1.48 (s, 3H), 1.60 (m, 3H), 1.81 (dd, J ) 2.7, 9.9
Hz, 1H), 1.95 (dd, J ) 13.2, 26.1 Hz, 1H), 2.1 (m, 2H), 2.27 (s,
1H), 2.47 (dd, J ) 4.8, 13.2 Hz, 1H), 2.66 (m, 1H), 2.80 (dd, J )
3.3, 13.2 Hz, 1H), 3.70 (s, 3H), 3.89 (d, J ) 2.4 Hz, 2H), 4.45 (m,
1H), 5.55 (dd, J ) 4.8, 11.7 Hz, 1H), 6.38 (dd, J ) 0.9, 1.5 Hz,
1H), 7.4 (m, 2H).
1
155–157 °C (EtOAc/n-hexanes). H NMR (300 MHz, CDCl3): δ
1.44 (s, 3H), 1.50 (s, 3H), 1.63 (m, 3H), 1.82 (dd, J ) 2.1, 10.5
Hz, 1H), 2.0 (m, 1H), 2.12 (dd, J ) 2.7, 8.4 Hz, 1H), 2.17 (m,
1H), 2.32 (s, 1H), 2.48 (dd, J ) 5.4, 13.2 Hz, 1H), 2.79 (dd, J )
3.3, 6.9 Hz, 1H), 2.87 (dd, J ) 2.7, 13.5 Hz, 1H), 3.71 (s, 3H),
4.69 (m, 1H), 5.55 (dd, J ) 5.1, 11.4 Hz, 1H), 6.37 (dd, J ) 0.9,
1.8 Hz, 1H), 7.1 (d, J ) 6.0 Hz, 1H), 7.39 (t, J ) 1.8 Hz, 1H),
7.41 (dd, J ) 0.9, 1.8 Hz, 1H), 7.46 (m, 1H), 7.53 (tt, J ) 1.5, 2.7,
7.2 Hz, 1H), 7.80 (t, J ) 2.4 Hz, 1H), 7.82 (t, J ) 1.2 Hz, 1H).
Anal. (C28H31NO7 ·0.5H2O): C, H, N.
(2S,4aR,6aR,7R,9S,10aS,10bR)-9-(Methanesulfonylamino)-2-
(3-furanyl)-dodecahydro-6a,10b-dimethyl-4,10-dioxo-2H-naph-
tho[2,1-c]pyran-7-carboxylic Acid Methyl Ester (8a). A solution
of 6 (0.10 g, 0.26 mmol), methanesulfonyl chloride (0.08 mL, 1.03
mmol), NEt3 (0.04 mL, 0.28 mmol), and a catalytic amount of
DMAP in CH2Cl2 (50 mL) was stirred at room temperature for
2 h. The mixture was washed with 2 N HCl (30 mL), 2 N NaOH
(30 mL), and H2O (30 mL) and dried (Na2SO4). Removal of the
solvent under reduced pressure afforded a crude solid. The crude
solid was purified by column chromatography (eluent: 2% MeOH/
CH2Cl2) to afford 0.7 g (56%) of 8a as a white crystalline solid,
mp 262–265 °C (EtOAc/n-hexanes). 1H NMR (300 MHz, CDCl3):
δ 1.09 (s, 3H), 1.46 (s, 3H), 1.60 (m, 3H), 1.79 (dd, J ) 2.7, 9.6
Hz, 1H), 2.07 (m, 2H), 2.18 (m, 1H), 2.21 (s, 1H), 2.50 (m, 2H),
2.75 (dd, J ) 3.6, 13.2 Hz, 1H), 2.99 (s, 3H), 3.72 (s, 3H), 4.15
(m, 1H), 5.34 (d, J ) 5.4 Hz, 1H), 5.55 (dd, J ) 5.1, 11.4 Hz,
1H), 6.38 (dd, J ) 0.9, 1.2 Hz, 1H), 7.41 (dd, J ) 1.5, 1.8 Hz,
1H), 7.43 (dd, J ) 0.9, 1.5 Hz, 1H). Anal. (C22H29NO8S): C, H, N.
(2S,4aR,6aR,7R,9S,10aS,10bR)-9-(Benzenesulfonylamino)-2-
(3-furanyl)-dodecahydro-6a,10b-dimethyl-4,10-dioxo-2H-naph-
tho[2,1-c]pyran-7-carboxylic Acid Methyl Ester (8b). A solution
of 6 (0.08 g, 0.21 mmol), benzenesulfonyl chloride (0.07 g, 0.42
mmol), triethylamine (0.06 g, 0.63 mmol), and a catalytic amount
of DMAP in CH2Cl2 (40 mL) was stirred at room temperature for
18 h. Absolute MeOH was then added and the solution was washed
with 10% HCl (3 × 25 mL) and saturated NaCl (2 × 25 mL),
A more polar spot was isolated to afford 0.02 g (14%) of 4b as
1
an oil. H NMR (300 MHz, CDCl3): δ 1.14 (s, 3H), 1.48 (s, 3H),
1.52 – 1.73 (m, 4H), 1.80 (dd, J ) 3.0, 9.6 Hz, 1H), 2.08 (dd, J )
3.0, 11.4 Hz, 1H), 2.18 (m, 1H), 2.24 (s, 1H), 2.57 (dd, J ) 5.1,
13.2 Hz, 1H), 2.63–2.70 (m, 2H), 2.76 (dd, J ) 3.3, 12.9 Hz, 1H),
3.73 (s, 3H), 4.60 (dd, J ) 7.8, 12.3 Hz, 1H), 5.56 (dd, J ) 5.1,
11.7 Hz, 1H), 6.38 (dd, J ) 0.9, 0.9 Hz, 1H), 7.41 (m, 1H).
(2S,4aR,6aR,7R,9S,10aS,10bR)-9-(Azido)-2-(3-furanyl)-
dodecahydro-6a,10b-dimethyl-4,10-dioxo-2H-naphtho[2,1-c]py-
ran-7-carboxylic Acid Methyl Ester (5). A solution of 4a (0.10
g, 0.22 mmol), sodium azide (0.05 g, 0.77 mmol), and glacial acetic
acid in DMF (3 mL) was stirred at room temperature for 4 h. H2O
(30 mL) was added and the mixture was extracted with EtOAc (20
mL). The EtOAc solution was washed with H2O (2 × 20 mL) and
saturated NaCl (20 mL) and dried (Na2SO4). Removal of the solvent
under reduced pressure afforded a crude solid. The crude solid was
purified by column chromatography (eluent: 30% EtOAc/n-hexanes)

Herkinorin Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 8 2429
dried (Na2SO4), filtered, and concentrated under reduced pressure
to yield a crude solid. The solid was purified by flash column
chromatography (eluent: n-hexanes/EtOAc, 1:1). Removal of the
solvent under reduced pressure gave 0.11 g (97%) of 8b as a white
indicated, naloxone was included during serum-starvation and drug
treatment. After washing with PBS on ice, cells were collected in
lysis buffer (20 mM Tris HCl pH 8.0, 150 mM NaCl, 2 mM EDTA,
0.1% SDS, 1% NP-40, 0.25% dioxycholate, 1 mM sodium
orthovanedate, 1 mM PMSF, 1 mM NaF, and protease inhibitor
cocktail (Roche)) and centrifuged at 20000 × g for 30 min.
Supernatants were quantified using Bio-Rad Dc protein assay and
diluted to equal concentrations with 4 × XT sample buffer (Bio-
Rad; 62.5 mM Tris-HCl, pH 6.8, 25% glycerol, 2% SDS, 0.01%
bromophenol blue) with 5% ꢀ-mercaptoethanol and boiled at 95
°C for 3 min. Samples were subjected to SDS-PAGE and transferred
to PVDF membranes. Blots were first probed with an antibody
specific to total ERK1/2 (cell signaling; 1:1000). Blots were stripped
and reblotted for Phospho-ERK (Tyr204; Santa Cruz; 1:2000).
Bands were detected using secondary antibodies (Amersham;
antirabbit IgG 1:2000 and antimouse IgG 1:5000, respectively)
conjugated to horseradish peroxidase and Supersignal West Pico
Chemiluminescent Substrate (Pierce). Densitometric analysis was
performed on Kodak 1D imaging software. Phospho-ERK bands
were normalized to corresponding total ERK bands. Statistical
analysis was performed using GraphPad Prism software.
1
solid, mp 271–273 °C (EtOAc/n-hexanes). H NMR (300 MHz,
acetone-d6): δ 0.98 (s, 3H), 1.29 (s, 3H), 1.52 (m, 2H), 1.65 (m,
1H), 1.70 (ddd, J ) 3.0, 3.0, 12.6 Hz, 1H), 1.82 (ddd, J ) 1.8, 5.1,
13.5 Hz, 1H), 1.95 (ddd, J ) 6.3, 6.3, 10.2 Hz, 1H), 2.09 (d, J )
13.2 Hz, 1H), 2.22 (dd, J ) 2.7, 11.7 Hz, 1H), 2.29 (ddd, J ) 3.3,
6.9, 13.5 Hz, 1H), 2.62 (s, 1H), 2.96 (dd, J ) 3.5, 13.4 Hz, 1H),
3.66 (s, 3H), 4.19 (m, 1H), 5.47 (dd, J ) 5.4, 12.0 Hz, 1H), 6.53
(dd, J ) 0.9, 1.5 Hz, 1H), 6.69 (d, J ) 8.4 Hz, 1H), 7.38 (m, 1H),
7.40 (d, J ) 6.9 Hz, 1H), 7.44 (dd, J ) 2.1, 3.0 Hz, 1H), 7.60 (m,
2H), 7.80 (m, 2H). 13C NMR (acetone-d6): δ 15.7, 16.8, 19.4, 35.5,
36.4, 39.1, 43.4, 44.3, 51.5, 52.2, 54.8, 61.2, 64.7, 72.4, 110.0,
127.5, 128.2, 130.1, 133.5, 141.2, 142.4, 145.1, 171.7, 173.0, 205.3.
Anal. (C27H31NO8S·H2O): C, H, N.
(2S,4aR,6aR,7R,9S,10aS,10bR)-9-(Benzoylthio)-2-(3-furanyl)-
dodecahydro-6a,10b-dimethyl-4,10-dioxo-2H-naphtho[2,1-c]py-
ran-7-carboxylic Acid Methyl Ester (9b). A solution of 4 (0.10
g, 0.22 mmol) and the potassium salt of thiobenzoic acid (0.194 g,
1.10 mmol) was stirred in acetonitrile at room temperature for 3 h.
Solvent was removed under reduced pressure and it was then
redissolved in DCM (30 mL). The DCM solution was washed with
H2O (3 × 30 mL) and saturated NaCl (2 × 30 mL) and dried
(Na2SO4). Removal of solvent under reduced pressure affored 0.74 g
(66%) of 9b as a white solid, mp 212–215 °C. 1H NMR (300 MHz,
CDCl3): δ 1.16 (s, 3H), 1.47 (s, 3H), 1.63 (m, 3H), 1.80 (m, 1H),
2.12 (dd, 1H, J ) 1.8, 11.1 Hz), 2.17 (m, 1H), 2.36 (m, 1H), 2.39
(s, 1H), 2.49 (m, 1H), 2.57 (dd, 1H, J ) 5.1 13.5 Hz), 2.90 (dd,
1H, J ) 3.3, 12.9 Hz), 3.72 (s, 3H), 4.52 (dd, J ) 6.9, 13.2 Hz,
1H), 5.54 (dd, J ) 5.1, 11.4 Hz, 1H), 6.39 (d, J ) 0.9 Hz, 1H),
7.39 (dd, J ) 1.5, 1.8 Hz, 1H), 7.42 (m, 1H), 7.46 (m, 2H), 7.60
(m, 1H), 7.96 (m, 2H). HRMS (m/z): [M+] calcd for C28H30O7S,
511.1791; found, 511.1781. HPLC tR ) 6.34 min; purity ) 98.94%.
In Vitro Pharmacology. Cell culture, [35S]GTP-γ-S binding
assay, and [125I]IOXY binding assays proceeded as described
elsewhere.43,52,53 Recombinant CHO cells (hMOR-CHO, hDOR-
CHO, and hKOR-CHO) were produced by stable transfection with
the respective human opioid receptor cDNA and provided by Dr.
Larry Toll (SRI International, CA).
Statistics. Statistical analyses were performed using Prism
software (GraphPad Software), and the specific tests used are
presented in the figure legends.
Acknowledgment. This work described was supported by
Grant Numbers K01DA14600 (to L.M.B.), R01DA18860 (to
L.M.B.), R01DA018151 (to T.E.P.), and R01DA018151S1 (to
T.E.P.) from the National Institute on Drug Abuse. The content
is the sole responsibility of the authors and does not necessarily
represent the official views of the National Institute on Drug
Abuse or the National Institutes of Health. The authors also
thank support from AFPE (to K.T. and M.S.) and in part from
the Intramural Research Program of the NIH, NIDA, and
acknowledge the expert technical assistance of Mario Ayestas,
IRP, NIDA.
Supporting Information Available: HPLC analysis of com-
pounds 3d, 3e, 3k, and 9b. This material is available free of charge
via the Internet at http://pubs.acs.org.
ꢀ-Arrestin-2 Translocation. HEK-293 cells stably expressing
the µ opioid receptor (∼1000 fmol/mg membrane protein) were
transiently transfected with 2 µg of ꢀ-arrestin-2 tagged on the
C-terminus with Green Fluorescent Protein (ꢀarr2-GFP) and 1.5
µg G-protein receptor kinase 2 (GRK2). Experiments were also
done in HEK-293 cells transiently transfected with MOR1. After
incubation at 37 °C for 24 to 36 h, cells were serum-starved for 30
min. Basal ꢀarr2-GFP images were obtained, followed by drug
treatment for 10 min. Drugs included DAMGO (1 µM), morphine
(10 µM), and 1c and its derivatives (10 µM). Cells were monitored
each minute throughout the 10 min drug treatment. Representative
cells at 5 min are shown. Images were taken using an Olympus
Fluoview 300 confocal microscope and Olympus Fluoview imaging
software version 4.3.
MOR-YFP Internalization. HEK-293 cells stably expressing
MOR1 tagged with Yellow Fluorescent Protein at the C-terminus
(MOR1-YFP) were transiently transfected with GRK2. After
incubation at 37 °C for 24 to 36 h, cells were serum-starved for 30
min. Basal MOR1-YFP images were obtained, followed by drug
treatment for 2 h. Drugs included DAMGO (1 µM), morphine (10
µM), and 1c and its derivatives (10 µM). Cells were monitored
every 15 min throughout the 2 h drug treatment. In some
experiments, cells were left to incubate at 37 °C during the hour
treatment time, and this did not result in different internalization
profiles. Representative cells at 60 min are shown. Images were
taken using the Olympus 300 confocal microscope and Olympus
Fluoview imaging software version 4.3.
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