Analogues of (3 R)-7-hydroxy- N -[(1 S)-1-{[(3 R,4 R)-4-(3-hydroxyphenyl)- 3,4-dimethyl-1-piperidinyl]methyl}-2-methylpropyl)-1,2,3,4-tetrahydro-3- isoquinolinecarboxamide (JDTic). Synthesis and in vitro and in vivo opioid receptor antagonist activity

Article abstract of DOI:10.1021/jm1004978

The synthesis of compounds 6, 7a,b, 8a,b, 9a,b, and 10a,b where the amino -NH- group of JDTic (3) was replaced with an aromatic - CH-, CH₂, O, S, or SO group was accomplished and used to further characterize the SAR of the compound 3 class of κ opioid receptor antagonists. All of the compounds showed subnanomolar to low nanomolar K_e values at the κ opioid receptor. The most potent compound was 7a, where the amino -NH- group of 3 was replaced by a methylene (-CH₂-) group. This compound had a K _e = 0.18 nM and was 37- and 248-fold selective for the κ relative to the μ and δ opioid receptors, respectively. Similar to compound 3, compound 7a antagonized selective κ agonist U50,488-induced diuresis after sc administration in rats. In contrast to 3, where κ antagonist activity lasted for three weeks, compound 7a did not show any κ antagonist activity after one week.

Full text of DOI:10.1021/jm1004978

5290 J. Med. Chem. 2010, 53, 5290–5301
DOI: 10.1021/jm1004978
Analogues of (3R)-7-Hydroxy-N-[(1S)-1-{[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-
piperidinyl]methyl}-2-methylpropyl)-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide (JDTic).
Synthesis and in Vitro and in Vivo Opioid Receptor Antagonist Activity
†
†
†
†
†
‡
ꢀ
Scott P. Runyon, Lawrence E. Brieaddy, S. Wayne Mascarella, James B. Thomas, Hernan A. Navarro, James L. Howard,
Gerald T. Pollard,‡ and F. Ivy Carroll*,†
†Organic and Medicinal Chemistry, Research Triangle Institute, Research Triangle Park, North Carolina 27709, and
‡Howard Associates, LLC, Research Triangle Park, North Carolina 27709
Received April 22, 2010
The synthesis of compounds 6, 7a,b, 8a,b, 9a,b, and 10a,b where the amino -NH- group of JDTic (3)
was replaced with an aromatic dCH-, CH2, O, S, or SO group was accomplished and used to further
characterize the SAR of the compound 3 class of κ opioid receptor antagonists. All of the compounds
showed subnanomolar to low nanomolar Ke values at the κ opioid receptor. The most potent compound
was 7a, where the amino -NH- group of 3 was replaced by a methylene (-CH2-) group. This
compound had a Ke=0.18 nM and was 37- and 248-fold selective for the κ relative to the μ and δ opioid
receptors, respectively. Similar to compound 3, compound 7a antagonized selective κ agonist U50,488-
induced diuresis after sc administration in rats. In contrast to 3, where κ antagonist activity lasted for
three weeks, compound 7a did not show any κ antagonist activity after one week.
Introduction
10a,b, where the amino -NH- group was replaced with an
aromatic dCH-, CH2, O, S, or SO group, were synthesized
and evaluated for their ability to antagonize μ, δ, and κ opioid
agonists in a [35S]GTPγS in vitro antagonist efficacy test. The
stereochemical assignments for the position R to the carbonyl
group for compounds 7a,b, 8a,b, and 9a,b are arbitrary. The
isomer with the lowest Ke value for the κ opioid receptor in
each pair was given the a designation. Compounds 10a,b are
the two possible sulfoxide diastereoisomers derived from 9a.
Analogue 7a, with a Ke = 0.18 nM at the κ opioid receptor
and 37- and 248-fold selectivity for the κ receptor relative to
the μ and δ opioid receptors, was the most potent and selective
analogue tested. The potency and duration of action of 7a to
antagonize κ agonist U50,488-induced diuresis in rats was
compared to that of 3.
Kappa opioid receptor selective antagonists are of consid-
erable interest as potential pharmacotherapies for addiction
(cocaine, opiate, alcohol, nicotine, and possibly others),1-7
depression,1,8-10 anxiety disorders,11 obesity,12-14 and psychosis
disorders.15 Opioid antagonists with varying degrees of receptor
potency and selectivity have been developed for the κ opioid
receptor. The first highly selective and potent κ opioid receptor
antagonist was norBNI (1).16,17 Continued studies identified 50-
GNTI (2) as a more potent and selective κopioid antagonist.18-20
In 2001, JDTic (3), an N-substituted trans-(3R,4R)-dimethyl-
4-(3-hydroxyphenyl)piperidine analogue, was developed as the
first highly potent and selective κopioid receptor antagonist from
this class of opioid antagonists.21 More recently, the dynorphin
analogues zyklophin and arodyn (4 and 5, respectively) were
developed as selective κ opioid receptor antagonists.22,23
SARa studies previously conducted on 3 identified the
70-phenolic and (S)-20-isopropyl groups as features important
for the κ selectivity and suggested that the nitrogen of the
hydroxy-D-Tic group might also be uniquely required for the
κ selectivity.24-26 To gain additional informationas to the role
that the amine nitrogen of the hydroxy-D-Tic group plays in
determining the κ opioid in vitro antagonist efficacy and
selectivity, the JDTic analogues 6, 7a,b, and 8a,b, 9a,b, and
*To whom correspondence should be addressed. Phone: 919-541-
6679. Fax: 919-541-8868. E-mail: fic@rti.org.
a Abbreviations: GPCRs, G-protein-coupled receptors; cDNAs, cDNA;
SAR, structure-activity relationship; [35S]GTPγS, sulfur-35 guanosine-50-
O-(3-thio)triphosphate; DAMGO, [D-Ala2,MePhe4,Gly-ol5]enkephalin;
DPDPE, [D-Pen2,D-Pen5]enkephalin; U69,593, (5R,7R,8β)-(-)-N-methyl-
N-[7-(1-pyrrolidinyl)-1-oxaspiro[4,5]dec-8-yl]benzeneacetamide; CHO,
Chinese hamster ovary; GDP, guanosine diphosphate; BOP, benzotri-
azole-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate;
HBTU, O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium hexafluoro-
phosphate; HMPA, hexamethylphosphoramide; LOA, lithium diisopro-
pylamide; Tic, tetrahydroisoquinolinecarboxylic acid; SAR, structure-
activity relationship.
Chemistry
Analogues 6, 7a,b, 8a,b, 9a,b, and 10a,b of 3 were synthe-
sized by the methods depicted in Schemes 1-3. Coupling of
r
pubs.acs.org/jmc
Published on Web 06/22/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5291
6-hydroxy-2-naphthoic acid (11) with 3-{1-[(2S)-amino-3-
methylbutyl]-(3R,4R)-dimethyl-4-piperidinyl}phenol (12)27
using benzotriazole-1-yloxy-tris(dimethylamino)phosphonium
hexafluorophorphole (BOP) in tetrahydrofuran containing
triethylamine at 25 °C gave the desired analogue 6 (Scheme 1).
Our initial plan for the synthesis of 7a and 7b was to start with
(þ)- and (-)-13. Since all attempts to resolve (()-13 directly
into its optical isomers were unsuccessful, we used the strategy
outlined in Scheme 2 to prepare (þ)- and (-)-13, which were
then converted to 7a and 7b. This strategy was based on a
report from Fox et al.,28 who showed that the tetrahydro-
2H-indeno[1,2d]-2-ones serve as a readily cleavable chiral
auxiliary suitable for large-scale normal phase separation of
isomers. Thus, treatment of (3aR-cis)-3,3a,8,8a-tetrahydro-
2H-indeno[1,2d]oxazol-2-one (14) with ethyl lithium provided
the lithium salt, which was acetylated with the acid chloride
prepared by treating (()-1329 with thionyl chloride in toluene
to give 15a and 15b, which were separated by standard silica
chromatography. Hydrolysis of 15b using lithium hydroxide
in tetrahydrofuran containing 30% hydrogen peroxide yielded
(þ)-13.30 Coupling of (þ)-13 to 1227 using BOP in tetrahydro-
furan containing triethylamine at 25 °C provided 16a. Treat-
ment of 16a with boron tribromide in methylene chloride at
-78 °C afforded 7a. Subjecting 15a to the same set of reaction
conditions provided (-)-13, 15b, and 7b.
On the basis of the ease of separating 7a,b using chiral
auxiliary 14, a similar synthetic strategy was applied to the
synthesis of 9a,b and 10a,b. In this case, the synthesis of the
7-methoxy-isothiochroman-3-carboxylic acid [(()-20] had to
be developed. Scheme 3 outlines the synthesis used to prepare
(þ)- and (-)-20 and subsequent conversion to 9a and 9b.
Condensation of 7-methoxy-isothiochroman-4-one (17)31
with methyl cyanoformate using lithium diisopropylamide
(LDA) as the base in tetrahydrofuran containing hexamethyl-
phosphoramide (HMPA) gives keto ester (18).32 Reduction
of 18 using triethylsilane in trifluoroacetic acid effected re-
moval of the 4-keto group to give 19.33 Hydrolysis of 19 using
potassium hydroxide in methanol provided (()-20. The
lithium salt of 14 was acylated with the acid chloride prepared
by treating (()-20 with oxalyl chloride in methylene chloride
to give 21a and 21b, which were separated by normal phase
silica gel chromatography. Hydrolysis of 21a using lithium
hydroxide in aqueous tetrahydrofuran afforded (þ)-20. It
should be noted that hydrolysis of the auxiliary was equally
effective without the presence of peroxide in the basic media.
Coupling of (þ)-20 with 1227 using BOP in tetrahydrofuran
containing triethylamine at 25 °C provided 22a. Treatment of
22a with boron tribromide in methylene chloride at -78 °C
afforded 9a. Subjecting 21b to a set of reaction conditions
Scheme 1a
a Reagents and conditions: (a) BOP, TEA, THF, 25 °C.
Scheme 2a
a Reagents and conditions: (a) thionyl chloride, toluene; (b) EtLi, (3aR-cis)-3,3a,8,8a-tetrahydro-2-hindeno[1,2d]oxazol-2-one (14); (c) 30% H2O2,
LiOH, 3:1 THF/H2O; (d) 12, BOP, TEA, THF 25 °C; (e) BBr3, CH2Cl2 -78 °C.

5292 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Runyon et al.
Scheme 3a
analogous to those used for 21a provided (-)-20, 22b, and 9b.
Oxidation of 9a using 3-chloroperoxybenzoic acid in methylene
chloride provided sulfoxide diastereomers 10a and 10b that
were separated using normal phase silica gel chromatography.
The synthesis of compounds 8a and 8b was more complex
than that of 7a,b and 9a,b due to the requirement of phenolic
protecting groups in the presence of a benzylic ether (see
Scheme 4). Cyclization of 3-methoxybenzyloxyacetic acid (23)
using oxalyl chloride followed by tin(IV) chloride in chloro-
benzene yielded chromanone 24.34 Demethylation of 24 to
the phenol 25 was accomplished using sodium ethanethiolate
in dimethylformamide.35 The phenol group in 25 was repro-
tected as the methoxymethyl ether 26 using chloromethyl-
methyl ether and diisopropylethylamine in methylene chloride.
Condensation of 26 with methyl cyanoformate using lithium
diisopropylethylamine as the base in tetrahydrofuran contain-
ing hexamethylphosphoramide (HMPA) afforded the keto
ester 27.32 Treatment of 27 with triethylsilane in trifluoroacetic
acid afforded both removal of the 4-keto group and the meth-
oxylmethyl protecting group to give 28.33 Hydrolysis of 28
using lithium hydroxide in a water, tetrahydrofuran, and
ethanol mixture provided (()-29. Coupling of (()-29 to 12
using BOP in tetrahydrofuran containing triethylamine at
25 °C yielded a mixture of (þ)-8 that were separated using
reverse phase HPLC to provide 8a,b.
Pharmacology
Compounds 6, 7a,b, 8a,b, 9a,b, and 10a,b were first
evaluated at 10 μM for intrinsic activity in the [35S]GTPγS
binding assay at all three opioid receptors. As none of these
compounds displayed measurable intrinsic activity at this
concentration, they and the reference compounds1 and 3 were
evaluated for antagonist efficacy and selectivity at the opioid
receptors. These data were obtained by monitoring the
ability of test compounds to inhibit stimulated [35S]GTPγS
binding produced by the selective agonists DAMGO (μ),
DPDPE (δ), or U69,593 (κ) using cloned human opioid
receptors expressed in CHO cells.36 Agonist dose-response
curves were run in the presence or absence of a single
concentration of test compound. The Ke values were calcu-
lated using the formula: Ke = [L]/DR - 1, where [L] is the
concentration of test compound and DR is the ratio of
agonist EC50 value in the presence or absence of test com-
pound, respectively. At least two different concentrations
of test compound were used to calculate the Ke, and the
concentrations were chosen such that the agonist EC50
exhibited at least a 4-fold shift to the right and there was a
clear upper asymptote to the agonist þ compound concen-
tration-response curve. The Ke values along with those for
the reference compound 1 are shown in Table 1.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5293
Scheme 4a
a Reagents and conditions: (a) oxalyl chloride, CH2Cl2; (b) SnCl4, chlorobenzene, 0 °C; (c) NaSEt, DMF, reflux; (d) MOMCl, i-Pr2EtN; (e) methyl
cyanoformate, LDA HMPA, THF; (f) TFA, Et2SiH; (g) LiOH, THF/MeOH/H2O, (1:1:1) 0 °C; (h) 12, BOP, TEA, THF, 25 °C; (i) preparative HPLC.
Table 1. Comparison of Inhibition of Agonist-Stimulated [35S]GTPγS Binding in Cloned Human μ, δ, and κ Opioid Receptors for 6, 7a,b, 8a,b, 9a,b,
and 10a,b to 3 and 1a,b
compd
μ, DAMGO Ke (nM)
δ, DPDPE Ke (nM)
κ, U69,593 Ke (nM)
μ/κ
δ/κ
1
26.7 ( 7c
25.1 ( 3.5c
2.53 ( 0.34
6.67 ( 1.2
11.2 ( 2.4
7.4 ( 2.6
29 ( 8c
76.4 ( 2.7c
219 ( 41
44.8 ( 7.7
205 ( 59
197 ( 64
134 ( 26
395 ( 122
182 ( 48
208 ( 55
61 ( 11
0.05 ( 0.02c
0.02 ( 0.01c
3.93 ( 1.2
0.18 ( 0.03
1.37 ( 0.36
0.71 ( 0.16
5.7 ( 1.6
0.99 ( 0.25
1.58 ( 0.07
2.7 ( 2.2
520
1255
580
3830
56
3
6
0.7
37
8.2
10
0.9
7a
7b
8a
8b
9a
9b
10a
10b
248
150
278
24
5.2 ( 1.6
5.57 ( 1.27
13.4 ( 2.9
7.9 ( 2.2
1.8 ( 0.3
6
399
115
77
8.5
2.9
3.3
0.55 ( 0.12
111
a Data represent means ( SE from at least three independent experiments. b The average percent stimulation and agonist EC50 values for the μ, κ, and
δ [35S]GTPγS binding assays were 200% and 120 nM, 220% and 380 nM, and 50% and 6 nM, respectively. Ke values were calculated from experiments
where the antagonist produced at least a 4-fold shift in the agonist ED50
Program (OTDP) were 3.41, 79.3, and 0.01 nM for the μ, δ, and κ receptors, respectively (ref 24).
.
c The Ke values for 3 supplied by the NIDA Opioid Treatment Discovery
Compound 7a was evaluated for its ability to antagonize
U50,488-induced diuresis in male Sprague-Dawley rats
(Charles River Laboratories, Raleigh, NC).
0.18 to 5.7 nM for κ, 1.8 to 13.4 nM for μ, and 44.8 to 395 nM
for δ and thus are potent κ and μ opioid receptor antagonists.
In the cases of 7, 8, 9, and 10, one isomer was more potent than
the other isomer. The differences in potencies were 7.6-, 8-,
1.6-, and 4.9-fold for 7a,b, 8a,b, 9a,b, and 10a,b, respectively.
The Ke values at the κ opioid receptor for the more potent 7a,
8a, and 9a are 0.18, 0.77, and 0.99 nM, respectively. The
diastereomeric sulfoxides 10a and 10b prepared from 9a had
Ke values of 2.7 and 0.55 nM at the κ opioid receptor, res-
pectively. The more potent isomers 7a,8a,9a,and10b are 9-, 36-,
50-, and 28-times less potent than 3 as a κ opioid antagonist.
This data shows that replacing the amino -NH- group of 3
with a -CH2- was better tolerated at the κopioid receptor than
replacement by an -O- or -S-. Compound 7a with Ke values
of 6.67, 44.8, and 0.18 nM at the μ, δ, andκ opioid receptors was
the most potent and selective κ opioid receptor antagonist of
the compounds evaluated. The low antagonist efficacy of com-
pounds 6, 7a,b, 8a,b, 9a,b, and 10a,b relative to 3 strongly
suggests that the -NH group of 3 is critical to its high in vitro
antagonist efficacy at the κ opioid receptor.
Results and Discussion
Compounds 6, 7a,b, 8a,b, 9a,b, and 10a,b were evaluated
for their μ, δ, and κ agonist or antagonist activity in the
[35S]GTPγS antagonist efficacy binding assay. The data are
shown in Table 1 compared to 1 and 3. None of the com-
pounds had any μ, δ, or κ intrinsic activity at 10 μM. As
expected, all compounds were antagonists of the DAMGO
(μ), DPDPE (δ), or U69,593 (κ) stimulated binding. Similar to
1 and 3, the compounds were more potent at the μ and κ than
at the δ opioid receptor. Compounds 1 and 3 with Ke values of
0.05 and 0.02 nM, respectively, are highly potent antagonists
for the κ receptor. In addition, both compounds are highly
selective for the κ relative to the μ and δ opioid receptors. The
compounds 6, 7a,b, 8a,b, 9a,b, and 10a,b, where the amino
-NH- groups have been replaced by an aromatic dCH-,
-CH2-, O, S, or SO group, showed Ke values ranging from

5294 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Runyon et al.
With the exception of 8b, all of the compounds were less
potent at the μ opioid receptor than the κ opioid receptor.
However, with the exception of 7a, which had a 37-fold
selectivity for κ over μ, the potencies of the μ and κ opioid
receptor were similar. The Ke values at the μ opioid receptor
ranged from 1.8 to 11.2 nM. The sulfoxide 10b with a Ke =
1.8 nM was the most potent μ opioid antagonist. All of the
compounds had relatively low antagonist efficacies for the δ
opioid receptor. Compound 7a with a Ke =44.8 shows the
highest potency at the δ opioid receptor.
Structure-activity relationship (SAR) studies of 1 and 2
and analogues led Portoghese to propose that the 170-amino
group of 1 and the 50-guanidinyl amino moiety of 2 as the
primary structural features leading to the κ opioid selectivity
of these two compounds (see Metcalf and Coop37 and
Aldrich38 for reviews of the SAR studies). Portoghese and
co-workers explained the κ selectivity of 1 and 2 using the
message-addressconceptdeveloped by Schwyzer39 for peptide
hormones. In this concept, molecular features common to a
series of compounds recognized by a family of receptors are
defined as the message. The “address” portion of this concept
is a specific structural feature of the ligand that affords
subtype selectivity through interaction with a site or specific
residue(s) unique to a receptor subtype. In the case of 1 and 2,
Portoghese used molecular modeling studies40 coupled with
site-directed mutagenesis to provide additional evidence that
the 170-amino group of 1 and the 50-guanidinium amino group
of 2 serve as the address by forming an ion pair with Glu297 at
the top of the TM6 in the κ opioid receptor.19,41-44
To determine if 3 could be interacting with the κ opioid
receptor in a way similar to that proposed for 2 and 1,
structures of 3 and 2 were constructed and compared. Low-
energy conformations of 3 which provide an overlay of the
primary pharmacophoric groups of 3 and 2 (as represented by
the centroidof the phenolicrings of 3 and 2 and thecentroid of
the Tic aromatic ring of 3 and the guanidinium group of 2)
were identified via conformational analysis (Figure 1).
Although the geometry and dimensions of 3 conformations
do not permit an atom-for-atom overlay of the 7-hydroxy-D-
Tic-group hydrogen-bonding substituents of 3 with the gua-
nidinium nitrogens of 2, both the 7-hydroxy-D-Tic secondary
amine NH group and phenol oxygen are projected into the
same general regionand presumablycould participate insome
type of interactions in the κ-address locus proposed for 1 and 2
of the κ-opioid receptor (Figure 1).
Figure 1. Alignment of a local-energy minimum conformation of 3
(gray) and 2 (green) with heteroatoms indicated by CPK-colored
spheres (blue for nitrogen and red for oxygen). Possible shared-
pharmacophore regions are labeled “A” for the opioid address
region, “M” for the opioid message region, and “P” for a polar
interaction site adjacent to the protonated nitrogens.
compound 3 showed dose-related antagonism of urine output
relative to vehicle (Figure 2A). At week 1 (no further dosing
with 3), 3 continued to show dose-related antagonism of
U50,488-induced diuresis. In keeping with our previous find-
ingswith3,45 the levelof antagonism atweek1 was the same as
or greater than at week 0.
Compounds 6, 7a,b, 8a,b, 9a,b, and 10a,b all have a phenol
group in a location similar to the 7-hydroxy-D-Tic phenol
group of 3 but lack the -NH group present in 3. Therefore,
the lower κ opioid antagonist efficacy of each to these
compounds seems due largely to the absence of the -NH
group present in 3. Previous studies have shown that the
7-hydroxy-D-Tic phenol oxygen is essential for the high κ
opioid receptor efficacy of 3.25,26 Thus, these results suggest
that the highly potent κ opioid receptor efficacy of 3 is due to
strong interactions at the κ opioid receptor by the 7-hydroxy-
D-Tic secondary amine -NH group and the phenol oxygen as
suggested by the molecular modeling studies (Figure 1). How-
ever, these results do not rule out the possibility that 3 is
interacting with the κ opioid receptor in a way different from
that of 1 and 2.
At week 0 (first 5 h after dosing sc), 7a showed dose-related
antagonism of U50,488-induced diuresis but was less potent
than 3, which is consistent with its 9-fold lower in vitro
antagonist potency at the κ opioid receptor compared to 3 in
the [35S]GTPγS in vitro antagonist efficacy test (Figure 2B).
Repeated measures ANOVA (StatView v5; SAS Institute Inc.,
Cary, NC) with factors of Dose and Week revealed a margin-
ally significant effect of Dose (F(3,12) = 2.74; P < 0.1) but a
significant Dose ꢀ Week interaction (F(1,12) = 106.8; P <
0.0001). Examining these data within Week indicated the
ability of 7a to antagonize U50,488-mediated diuresis was dose
dependent and confined to Week 0. The effect of individual
doses of 7a on urine output was compared to U50,488 using
Dunnett’s test. At Week 0, ANOVA indicated there was a signi-
ficant overall effect of Dose (F(3,12) = 7.53; P < 0.005) to lower
urine output, with the 10 and 30 mg/kg doses causing signifi-
cant antagonism of the diuretic effects of U50,488. However, at
Week 1 (no further dosing with 7a), antagonism of U50,488
could no longer be detected (ANOVA; F(1,12) =0.69; NS).
To gain information concerning the in vivo activity, the
effect of 7a to antagonize κ agonist U50,488-induced diuresis
was compared to that of 3. The results from the study are
summarized in Figure 2. At week 0 (first 5 h after dosing sc),

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5295
Figure 2. Comparison of 7a to 3 on U50,488-induced diuresis in rats. (A) Antagonism of U50,488-induced urine output in various sc doses
(mg/kg) of 3. (B) Antagonism of U50,488-induced urine output by various sc doses (mg/kg) of 7a, and compound 3 and 7a were only
administered once at week 0.
Thus, the activity of 7a lasted less than 1 week, which contrasts
sharply with the activity of 3 as well as other κ opioid receptor
antagonists such as 1, which can persist for up to 3 weeks.45 The
shorter duration of action of 7a compared to 3 and 1 showed
that selective, high affinity κ opioid receptor antagonists can be
identified with shorter durations of activity.
Elemental analysis was performed by Atlantic Microlab, Inc.,
Norcross, GA.
HPLC-gradesolvents werepurchasedfromBurdick& Jackson
(Muskegon, MI). Preparative HPLC was carried out utilizing a
Varian Prostar HPLC system (Walnut Creek, CA) equipped with
Prostar 210 pumps, Prostar 701 fraction collector, and a Prostar
335 photodiode array detector (PDA), with data collected and
analyzed using Galaxie Chromatography Workstation software
(version 1.9.3.2). Analytical HPLC was carried out utilizing a
Varian Prostar HPLC system (Walnut Creek, CA) equipped with
Prostar 210 pumps and a Prostar 330 photodiode array detector
(PDA), with data collected and analyzed using Star Chromatog-
raphy Workstation software (version 6.41). For preparative
HPLC, a Gemini-NX C18 (5 μM; 250 mm ꢀ 21.2 mm) column
was used with a 19 mL/min flow rate, while for analytical HPLC,
a Gemini-NX C18 (5 μm; 250 mm ꢀ 4.6 mm) column was used
with a 1 mL/min flow rate (both from Phenomenex, Torrance,
CA). For analytical HPLC, a MetaTherm HPLC column tem-
perature controller (Varian) maintained the column at 30 °C.
6-Hydroxynaphthalene-2-carboxylic Acid-{1-[4-(3-hydroxy-
phenyl)-(3R,4R)-trans-dimethyl-piperidinylmethyl]-(2S)-methyl-
propyl}amide Hydrochloride (6). 6-Hydroxy-2-naphthoic acid
(100 mg, 0.53 mmol) was added to a solution of 1227 (154 mg,
0.53 mmol) and BOP reagent (235 mg, 0.53 mmol) in THF
(40 mL) and was allowed to stir under nitrogen for 15 min.
Triethylamine (1.18 g, 0.012 mol) in THF (10 mL) was added,
and the reaction mixture was allowed to stir at room tempera-
ture for 3 h. NaHCO3 (50 mL) and Et2O (50 mL) were added to
the reaction mixture, and the organic layer was separated,
washed with brine, dried, (Na2SO4), and concentrated under
reduced pressure to afford 0.32 g of an amorphous solid. The
solid was purified using medium pressure silica gel chromato-
graphy, eluting with EtOAc-CMA80 (1:1) to afford 0.095 g of a
white amorphous solid. 1H NMR (CDCl3) δ 8.20 (s, 1H), 7.75
(m, 2H), 7.62 (d, J = 9.0 Hz, 1H), 7.10 (m, 4H), 6.71 (m, 2H),
6.60 (d, 6.0 Hz, 1H), 4.42 (m, 1H), 2.32-2.92 (bm, 7H), 2.25 (bt,
1H), 2.05 (m, 1H), 1.92 (bs, 1H), 1.57 (bd, 1H), 1.27 (s, 3H), 1.06
(dd, J = 4.8, 2.1 Hz, 6H), 0.55 (d, J = 6.9 Hz, 3H). The resulting
solid was dissolved in CH2Cl2-MeOH (1:1) and acidified with
1 M ethereal HCl. The mixture was concentrated in vacuo, then
dried to yield 0.065 g (23%) of 6 as a white solid: mp 207-
Conclusions
In summary, replacement of the amino -NH- group of 3
with an aromatic dCH-, -CH2-, O, or S provided com-
pounds with reduced κ opioid receptor in vitro antagonist
potency and reduced κ selectivity relative to the μ and δ opioid
receptors. These results confirm our earlier results showing
that the amino -NH- group of 3 is highly important to its
high κ opioid receptor in vitro antagonist potency as well as κ
selectivity. However, molecular modeling studies show that
the amino -NH- group of 3 and the amino group in GNTI
are not likely to be interacting with the κ opioid receptor in
the same way. Interestingly, compound 7a, where the amino
-NH- of 3 has been replaced by a methylene (-CH2-)
group, showed subnanomolar κ potency and reasonable
selectivity for the κ relative to the μ and δ opioid receptors.
Compound 7a antagonized selective κ agonist U50,488-
induced diuresis with a potency that parallels its κ efficacy in
the [35S]GTPγS antagonist assay. In contrast to 3, whose
activity as an antagonist of U50,488-induced diuresis lasted
for 3 weeks, the activity of 7a lasted less than 1 week.
Experimental Section
1H NMR spectra were determined on a Bruker 300 spectro-
meter using tetramethylsilane as an internal standard. Mass
spectral data were obtained using a Finnegan LCQ electrospray
mass spectrometer in positive ion mode at atmospheric pressure.
Silica gel 60 (230-400 mesh) was used for column chromato-
graphy. All reactions were followed by thin-layer chromatography
using Whatman silica gel 60 TLC plates and were visualized by
UV. Optical rotations were measured on an Auto Pol III polari-
meter. All solvents were reagent grade. HCl in dry diethyl ether
was purchased from Aldrich Chemical Co. and used while fresh
before discoloration. CMA-80 is a mixture of 80% chloroform,
18% methanol, and 2% concentrated ammonium hydroxide.
CMA-80 is a mixture of 80% methylene chloride, 18% methanol,
and 2% concentrated ammonium hydroxide. Purity of com-
pounds (>95%) was established by elemental analysis except
for 8a and 8b. HPLC analysis was used to establish their purity.
211 °C. Anal. (C29H37ClN2O3 2H2O) C,H, N.
3
6-Hydroxy-1,2,3,4-tetrahydro-naphthalene-2(þ)-carboxylic
Acid-{1-[4-(3-hydroxyphenyl)-(3R,4R)-trans-dimethyl-piperidinyl-
methyl]-(2S)-methylpropyl}amide (7a) Hydrochloride. A 1.0 M
solution of BBr3 (8.2 mL. 8.2 mmol) in CH2Cl2 was added at
-78 °C under N2 to 16b (0.39 g, 0.82 mmol) in CH2Cl2 (25 mL).
The dark-brown solution was allowed to stir at -78 °C for 0.5 h

5296 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Runyon et al.
and allowed to warm to room temperature. A saturated solution
of NaHCO3 (50 mL) was cautiously added, and the biphasic
mixture was extracted with EtOAc (3 ꢀ 100 mL). The organic
extracts were combined, dried (MgSO4), and concentrated under
reduced pressure to provide a brown oil. The oil was purified using
medium pressure column chromatography on silica using CHCl3-
MeOH-NH4OH (8:1.8:0.2) as the eluent to provide 0.30 g (77%)
of 7a as a colorless oil. The hydrochloride salt was prepared by
adding a 1.0 M solution of HCl in Et2O to the free base in MeOH.
The solution was concentrated under reduced pressure and
the resulting solid recrystallized from EtOH-Et2O to provide
for 15 min. Chromatograms were observed at 220 nm, and the
samples were dissolved in MeOH.
7-Hydroxy-3,4-dihydro-1H-isochromene-3-carboxylic Acid-
{1-[4-(3-hydroxyphenyl)-(3R,4R)-trans-dimethyl-piperidinyl-
methyl]-(2S)-methylpropyl}amide (8a, peak 1). [R]22D þ108.6 °C,
(c = 0.11, MeOH). 1H NMR (CDCl3) δ 0.71 (d, J = 6 Hz, 3H),
0.93 (m, 6H), 1.28 (m, 4H), 1.56 (d, J = 12 Hz, 1H), 1.87 (m, 1H),
1.95 (m, 1H), 2.26 (ddd, 1H), 2.41-2.55 (m, 4H), 2.64-2.80 (m,
2H), 2.90-2.96 (dd, J = 3, 18 Hz, 1H), 3.99 (m, 1H), 4.14 (dd,
J = 6, 12 Hz, 1H), 4.84 (d, 1H), 4.87 (d, J = 12 Hz, 1H), 6.45 (s,
1H), 6.58 (m, 2H), 6.71 (m, 2H), 6.91 (d, J = 9 Hz, 1H), 7.07 (dd,
J = 9, 9 Hz, 1H). HRMS m/z 467.2908 (M þ H)þ, predicted
467.2910.
7a HCl as white plates: mp 189-191 °C; [R]22D þ113.7 °C (c =
3
0.18, MeOH). 1H NMR (CD3OH) δ 0.74 (d, J = 6.78 Hz, 3 H),
0.90 (d, J = 6.78 Hz, 3 H), 0.93 (d, J = 6.78 Hz, 3 H), 1.27(s, 3 H),
1.55 (d, J = 12.81 Hz, 1 H), 1.68-1.89 (m, 2 H), 1.95 (m, 2 H),
2.36-2.81 (m, 12H), 4.02 (ddd, J = 9.61, 5.09, 4.90 Hz, 1 H), 6.50
(d, J = 2.26 Hz, 1 H), 6.57 (ddd, J = 15.26, 8.10, 2.26 Hz, 2 H),
6.70-6.80 (m, 2 H), 6.85 (d, J=8.29Hz, 1H), 7.10(t, J=8.10Hz,
7-Hydroxy-3,4-dihydro-1H-isochromene-3-carboxylic Acid-
{1-[4-(3-hydroxyphenyl)-(3R,4R)-trans-dimethyl-piperidinyl-
methyl]-(2S)-methylpropyl}amide (8b, peak 2). [R]22 -16.3 °C
D
(c = 0.08, MeOH). 1H NMR (CDCl3) δ 0.71 (d, J = 6 Hz, 3H),
0.93 (m, 6H), 1.28 (m, 4H), 1.56 (d, J = 12 Hz, 1H), 1.87 (m, 1H),
1.95 (m, 1H), 2.26 (ddd, 1H), 2.41-2.55 (m, 4H), 2.64-2.80 (m,
2H), 2.90-2.96 (dd, J = 3, 18 Hz, 1H), 3.99 (m, 1H), 4.14 (dd,
J = 6, 12 Hz, 1H), 4.84 (d, 1H), 4.87 (d, J = 12 Hz, 1H), 6.45 (s,
1H), 6.58 (m, 2H), 6.71 (m, 2H), 6.91 (d, J = 9 Hz, 1H), 7.07 (dd,
J = 9, 9 Hz, 1H). HRMS m/z 467.2905 (M þ H)þ, predicted
467.2910.
1 H), 7.81 (br s, 1 H). Anal. (C29H41ClN2O3 0.75H2O) C, H, N.
3
6-Hydroxy-1,2,3,4-tetrahydro-naphthalene-2(-)-carboxylic
Acid-{1-[4-(3-hydroxyphenyl)-(3R,4R)-trans-dimethyl-piperidinyl-
methyl]-(2S)-methylpropyl}amide (7b) Hydrochloride. A 1.0 M
solution of BBr3 (8.2 mL, 8.2 mmol) in CH2Cl2 was added at
-78 °C under N2 to 16a (0.70 g, 1.45 mmol) in CH2Cl2 (50 mL).
The dark-brown solution was allowed to stir at -78 °C for 0.5 h
and allowed to warm to room temperature. A saturated solution
of NaHCO3 (100 mL) was cautiously added, and the biphasic
mixture was extracted with EtOAc (3 ꢀ 150 mL). The organic
extracts were combined, dried (MgSO4), and concentrated under
reduced pressure to provide a brown oil. The oil was purified using
medium pressure column chromatography on using silica CHCl3-
MeOH-NH4OH (8:1.8:0.2) as the eluent to provide 0.057 g (83%)
of 7b as a colorless oil. The hydrochloride salt was prepared by
adding a 1.0 M solution of HCl in Et2O to 7b in MeOH. The
solution was concentrated under reduced pressure and recrystal-
7-Hydroxyisothiochroman-3(þ)-carboxylic Acid-{1-[4-(3-hydro-
xyphenyl)-(3R,4R)-trans-dimethyl-piperidinylmethyl]-(2S)-methyl-
propyl}amide (9a). A 1.0 M solution of BBr3 (9.1 mL, 9.1 mmol) in
CH2Cl2 was added at -78 °C under N2 to 22a (0.45 g, 0.91 mmol)
in CH2Cl2 (100 mL). The dark-brown solution was allowed to stir
at -78 °C for 0.5 h and allowed to warm to 0 °C for 2 h. A
saturated solution of NaHCO3 (100 mL) was cautiously added,
and the biphasic mixture was extracted with EtOAc (3 ꢀ 150 mL).
The organic extracts were combined, dried (MgSO4), and con-
centrated under reduced pressure to provide a brown oil. The oil
was purified using medium pressure column chromatography on
silica gel using CHCl3-MeOH-NH4OH (8:1.8:0.2) as an eluent
to provide a 0.43 g (98%) of 9a tan semisolid. The solid was
recrystallized from acetone-petroleum ether to afford 9a as white
needles:mp 133-135 °C; [R]22D þ108.7 °C, (c =0.20, MeOH). 1H
NMR (CD3OD) δ 0.69-0.74 (m, 9H), 0.89-0.98 (m, 1H), 1.28 (s,
3H), 1.52-1.59 (d, J = 12.9 Hz, 1H), 1.64-1.68 (m, 1H), 1.94-
1.96 (m, 1H), 2.17-2.47 (m, 4H), 2.58-2.62 (d, J = 11.3 Hz, 1H),
2.72-2.75 (d, J = 11.3 Hz, 1H), 2.89-2.96 (dd, J = 5.3, 15 Hz,
1H), 3.11-3.18 (dd, J = 7.54, 14.3 Hz, 1H), 3.62-3.77 (m, 3H),
3.83-3.90 (m, 1H), 6.54-6.65 (m, 3H), 6.71-6.76 (m, 2H),
6.91-6.94 (d, J = 8.2 Hz, 1H), 7.06-7.11 (t, J = 7.9 Hz, 1H).
lized from EtOH-Et2O to provide7b HCl as tan cubes: mp 193-
3
195 °C;[R]22D þ 67.0°C(c = 0.22, MeOH). 1H NMR (CD3OD) δ
0.76 (d, J = 7.32 Hz, 3 H), 0.91 (d, J = 6.84 Hz, 3 H), 0.95 (d, J =
6.84 Hz, 3 H), 1.27-1.30 (s, 3 H), 1.57 (d, J = 11.23 Hz, 1 H),
1.75-1.86 (m, 2 H), 1.95-2.03 (m, 2 H), 2.29 (td, J = 12.57,
4.15 Hz, 1 H), 2.34-2.41 (m, 1 H), 2.42-2.87 (m, 10 H), 4.02 (dt,
J = 9.77, 4.88 Hz, 1 H), 6.49 (m, 1 H), 6.52 (dd, J = 8.30, 2.44 Hz,
1 H), 6.58 (dd, J = 7.81, 1.95 Hz, 1 H), 6.74 (m, 1 H), 6.77 (d,
J = 7.81 Hz, 1 H), 6.82 (d, J = 8.30 Hz, 1 H), 7.10 (t, J = 8.06 Hz,
1H). Anal. (C29H41ClN2O3 1.5H2O) C, H, N.
3
7-Hydroxy-3,4-dihydro-1H-isochromene-3-carboxylic Acid-
{1-[4-(3-hydroxyphenyl)-(3R,4R)-trans-dimethyl-piperidinyl-
methyl]-(2S)-methylpropyl}amide (8a,b). 7-Hydroxy-3,4-dihydro-
1H-isochromene-3-carboxylic acid [(()-29, 66 mg, 0.34 mmol]
was added to a solution of 1227 (98 mg, 0.34 mmol) and BOP
reagent (150 mg, 0.34 mmol) in THF (20 mL) and was allowed to
stir under nitrogen for 15 min. Triethylamine (69 mg, 0.68 mmol)
in THF (10 mL) was added, and the reaction was allowed to stir at
room temperature for 3 h. To the reaction mixture were added
NaHCO3 (30 mL) and Et2O (30 mL), and the organic layer was
separated, washed with brine, dried, (Na2SO4), and concentrated
under reduced pressure to afford an oil. The oil was purified by
medium pressure silica gel chromatography, eluting with CHCl3-
CMA80 (1:1) to afford 0.129 g of a pale-yellow oil as a mixture of
8a and 8b. The diastereomers were purified using preparative
HPLC. A sample of 41 mg of 8a,b in DMSO (200 μL) was purified
via reversed phase HPLC using an isocratic MeOH-H2O (0.1%
DEA) solvent system at 19 mL/min for 35 min, and 19 mL
fractions were collected. Fractions were combined to obtain two
pools. The first pool contained 21.6 mg of 100% pure compound
8a (the first compound to elute), and the second pool contained
16 mg of 8b, the second peak determined to be 87% pure. The
second peak was repurified to afford 9.2 mg of 8b at 98.7% purity.
The purity of isolates was determined via analytical HPLC using
an isocratic solvent system at MeOH-H2O (70:30) (0.1% DEA)
Anal. (C28H38N2O3S 0.25H2O) C, H, N.
3
7-Hydroxyisothiochroman-3(-)-carboxylic Acid-{1-[4-(3-hydro-
xyphenyl)-(3R,4R)-trans-dimethyl-piperidinylmethyl]-(2S)-methyl-
propyl}amide Hydrochloride (9b). A 1.0 M solution of BBr3
(9.0 mL, 9.0 mmol) in CH2Cl2 was added at -78 °C under N2 to
22b (0.44 g, 0.90 mmol) in CH2Cl2 (100 mL). The dark-brown
solution was allowed to stir at -78 °C for 0.5 h and allowed to
warm to 0 °C for 2 h. A saturated solution of NaHCO3 (100 mL)
was cautiously added, and the biphasic mixture was extracted
with EtOAc (3 ꢀ 150 mL). The organic extracts were combined,
dried (MgSO4), and concentrated under reduced pressure to
provide a brown oil. The oil was purified using medium pressure
column chromatography on silica gel using CHCl3-MeOH-
NH4OH (8:1.8:0.2) as an eluent to provide 0.40 g (93%) of 9b as a
tan semisolid. 1H NMR (CD3OD) δ 0.71-0.74 (d, J = 6.8 Hz,
3H), 0.83-0.85 (d, J = 6.8 Hz, 3H), 0.88-0.90 (d, J = 6.8 Hz,
3H), 1.06-1.13 (m, 1H), 1.26 (s, 3H), 1.50-1.54 (d, J = 12.4 Hz,
1H), 1.79-1.94 (m, 2H), 2.15-2.39 (m, 4H), 2.48 (brs, 1H),
2.71-2.75 (d, J = 11 Hz, 1H), 2.97-3.10 (m, 2H), 3.58-3.78
(m, 3H), 3.85-3.91 (m, 1H), 6.57-6.60 (d, J = 7.9 Hz, 1H), 6.63
(m, 2H), 6.73 (m, 2H), 6.95-6.98 (d, J = 8.2 Hz, 1H), 7.06-7.12
(t, J = 7.9 Hz, 1H). The hydrochloride salt was prepared by
adding a 1.0 M solution of HCl in Et2O to 9b in MeOH. The solu-
tion was concentrated under reduced pressure and recrystallized

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5297
from EtOH-Et2O to provide 9b HCl as white cubes: mp
and for 2 h at room temperature. A 1.5 N solution of Na2SO3
(10 mL) was added in a dropwise manner, and the biphasic
solution was basified (pH ≈ 10) with saturated sodium bicar-
bonate solution. The solution was extracted (2 ꢀ 50 mL) with
EtOAc, made acidic to pH 3 with HCl (10 M solution), and
extracted (3 ꢀ 100 mL) with CH2Cl2. The organic extracts were
combined, dried (MgSO4), and concentrated under reduced
pressure to provide a white solid. The solid was recrystallized
from EtOAc-petroleum ether to provide 0.102 g (90%) of (-)-
13 as white needles: mp 121-122 °C; [R]22D -56.9 °C (c = 0.25,
CHCl3). 1H NMR (CDCl3) δ 1.87-1.90 (m, 1H), 2.20-2.25 (m,
1H), 2.74-2.98 (m, 5H), 3.77 (s, 3H), 6.63 (s, 1H), 6.68-6.72
(dd, J = 2.7, 8.4 Hz, 1H), 7.0-7.03 (d, J = 8.4 Hz, 1H).
(3aR-cis)-3-(6-Methoxy-1,2,3,4-tetrahydronaphthalene-2(þ
and -)-carbonyl)-3,3a,8,8a-tetrahydro-2H-indeno[1,2-d]oxazol-2-
one (15a,b). A 2.0 M solution of thionyl chloride (7.25 mL, 14.3
mmol) in CH2Cl2 was added to a solution of (()-1329 (0.29 g, 1.43
mmol) in toluene (20 mL). The solution was heated at reflux for
8 h, cooledtoroom temperature, andconcentrated under reduced
pressure to provide 6-methoxy-1,2,3,4-tetrahydronaphthalene-2-
carbonyl chloride as a tan solid. In a separate flask, a 0.50 M
solution of ethyl lithium (3.0 mL, 1.50 mL) in benzene-yclohexane
(90:10) was added to a solution of (3aR-cis)-3,3a,8,8a-tetra-
hydro-2H-indeno[1,2-d]oxazol-2-one (0.25 g, 1.43 mmol) in
THF (20 mL) at 0 °C under N2. The suspension was allowed
to stir at 0 °C for 0.5 h and was cooled to -78 °C. A solution of
6-methoxy-1,2,3,4-tetrahydronaphthalene-2-carbonyl chloride
(0.29 g, 1.43 mmol) in THF (10 mL) was then added in a dropwise
manner to the -78 °C slurry. The resulting slurry was allowed to
warm to room temperature over 2 h, and water (100 mL) was
added. The suspension was extracted with CH2Cl2 (3 ꢀ 100 mL).
The organic extracts were combined, dried (MgSO4), and con-
centrated under reduced pressure to provide a mixture of 15a
and 15b as a tan solid. The two compounds were separated using
silica gel medium pressure column chromatography petroleum
ether-Et2O (70:30) to provide each of the diastereomers in
approximately 50% theoretical yield. The yield improves with
additional chromatography. The less polar spot was later identi-
fied as the (þ) isomer while the more polar was (-).
3
224-227 °C (191-194 °C softens), [R]22 þ62.1 °C, (c = 0.38,
D
MeOH). Anal. C, H, N.
7-Hydroxy-2-oxo-2-isothiochroman-3(þ)-carboxylic Acid-
{1-[4-(3-hydroxyphenyl)-(3R,4R)-trans-dimethyl-piperidinylmethyl]-
(2S)-methylpropyl}amide (10a,b) Resorcylate. 3-Chloroperoxyben-
zoic acid (0.94 g, 0.41 mmol) was added to an ice-cold solution of 9a
(0.20 g, 0.41 mmol) in CH2Cl2 (20 mL). The solution was allowed
to stir at 0 °C for 30 min and was quenched by the addition of
saturated sodium bicarbonate. The slurry was extracted with
CH2Cl2 (3 ꢀ 30 mL), dried (MgSO4), and concentrated under
reduced pressure to afford a mixture of diastereomers as a pale-
yellow oil. The diastereomers were separated by silica gel chro-
matography using CHCl3-MeOH-NH4OH (8:1.8:0.2) as the
eluent to provide 10a (first to elute, 28 mg, 14%) and 10b (second
to elute, 21 mg, 10%) as pale-yellow oils. Additional product was
collected as a mixture of diastereomers.
7-Hydroxy-2-oxo-2-isothiochroman-3(þ)-carboxylic Acid-
{1-[4-(3-hydroxyphenyl)-(3R,4R)-trans-dimethyl-piperidinylmethyl]-
(2S)-methylpropyl}amide (10a) Resorcylate. The first spot to elute
off the column was converted to the resorcylate salt by dissolving
3,5-dihydroxybenzoic acid (1.1 equiv) in acetone and adding the
amine in acetone. The solution was concentrated, and the solid was
recrystallized from EtOAc and hexane to provide 17 mg of 10a as a
white solid: mp 170-173 °C; [R]22D þ4.7 °C, (c = 0.11, MeOH). 1H
NMR (CD3OD) δ 0.80 (d, J = 6.8 Hz, 3H), 1.01 (m, 6H),
1.27-1.40 (m, 4H), 1.72 (d, 1H), 1.85 (m, 1H), 2.13 (m, 1H), 2.41
(m, 1H), 2.79-2.86 (m, 3H), 3.0-3.08 (m, 3H), 3.18-3.23 (m, 1H),
3.62 (dd, J = 6, 9 Hz, 1H), 4.10-4.16 (m, 3H), 6.38 (dd, 1H),
6.59-6.79 (m, 5H), 6.91 (dd, 1H), 6.96-6.99 (d, J = 9 Hz, 1H),
7.10-7.15 (m, 2H). Anal. (C35H44N2O8S 1.5H2O) C, H, N.
3
7-Hydroxy-2-oxo-2-isothiochroman-3(þ)-carboxylic acid-
{1-[4-(3-hydroxyphenyl)-(3R,4R)-trans-dimethyl-piperidinylmethyl]-
(2S)-methylpropyl}amide (10b) Resorcylate. The second spot to
elute off the column was converted to the resorcylate salt by
dissolving 3,5-dihydroxybenzoic acid (1.1 equiv) in acetone
and adding the amine in acetone. The solution was concen-
trated, and the solid was recrystallized from EtOAc and hexane
to provide 12 mg of 10b resorcylate as a white solid: mp
1
186-189 °C; [R]22 þ102.0 °C, (c = 0.15, MeOH). H NMR
D
(3aR-cis)-3-(6-Methoxy-1,2,3,4-tetrahydronaphthalene-2(þ)-
carbonyl)-3,3a,8,8a-tetrahydro-2H-indeno[1,2-d]oxazol-2-one (15a).
The solid was recrystallized from EtOAc-petroleum ether to
provide 0.12 g (46%) of 15a as a white solid: mp 168-169 °C. 1H
NMR (CDCl3) δ 1.80-1.85 (m, 1H), 2.10-2.21 (m, 1H), 2.71-
3.13 (m, 4H), 3.38 (d, J = 3.3 Hz, 2H), 3.76 (s, 3H), 3.84 (m, 1H),
5.27 (m, 1H), 5.96-5.99 (d, J = 9 Hz, 1H), 6.62 (s, 1H), 6.70-6.71
(dd, J = 2.4, 8.1 Hz, 1H), 6.99-7.04 (dd, J = 3.6, 8.4 Hz, 1H),
7.24-7.32 (m, 3H), 7.57-7.60 (d, J = 7.5 Hz, 1H).
(CD3OD) δ 0.82 (dd, J = 6, 3 Hz, 3H), 0.97 (m, 6H), 1.26 (dd,
J = 4, 12 Hz 1H), 1.36 (s, 3H), 1.72-1.76 (d, 8 Hz, 1H), 1.90 (m,
1H), 2.18 (m, 1H), 2.46 (m, 1H), 2.91-3.32 (m, 6H), 3.51 (m,
1H), 3.72-3.87 (m, 1H), 4.0-4.33 (m, 3H), 6.37 (dd, J = 6,
6 Hz, 1H), 6.60-6.78 (m, 5H), 6.91 (d, J = 3 Hz, 2H), 7.01-
7.15 (m, 2H). Anal. (C35H44N2O8S 1.25H2O) C, H, N.
3
2(þ)-6-Methoxy-1,2,3,4-tetrahydronaphthalene-2-carboxylic
Acid [(þ)-13]. A 30% solution of hydrogen peroxide (6.96 mmol,
0.24 mL) in H2O was added at 0 °C to a solution of 15a (0.42 g,
1.16 mmol) in 3:1 THF-H2O (25 mL). Lithium hydroxide hydrate
(0.098 g, 2.32 mmol) was added to the solution in portions. The
suspension was allowed to stir for 0.5 h at 0 °C and for 2 h at room
temperature. A 1.5 N solution of Na2SO3 (15 mL) was added in a
dropwise manner, and the biphasic solution was basified (pH ≈ 9)
with saturated sodium bicarbonate solution. The solution was
extracted (2 ꢀ 50 mL) with EtOAc, made acidic to pH 3 with HCl
(10 M solution), and extracted (3ꢀ100 mL) with CH2Cl2. The
organic extracts were combined, dried (MgSO4), and concentrated
under reduced pressure to provide a white solid. The solid was re-
crystallized from EtOAc-petroleum ether to provide 0.22 g (92%)
of (þ)-13 as white needles: mp 129-130 °C; [R]22D þ57.27 °C, (c =
0.22, CHCl3). 1H NMR (CDCl3) δ 1.87-1.90 (m, 1H), 2.20-2.25
(m, 1H), 2.74-2.98 (m, 5H), 3.77 (s, 3H), 6.63 (s, 1H), 6.68-6.72
(dd, J = 2.7, 8.4 Hz, 1H), 7.0-7.03 (d, J = 8.4 Hz, 1H).
(3aR-cis)-3-(6-Methoxy-1,2,3,4-tetrahydronaphthalene-2(-)-
carbonyl)-3,3a,8,8a-tetrahydro-2H-indeno[1,2-d]oxazol-2-one (15b).
The solid was recrystallized from EtOAc-petroleum ether to
provide 0.13 g (50%) of 15b as a white solid: mp 162-164 °C. 1H
NMR (CDCl3) δ 1.85-1.98 (m, 1H), 2.12-2.18 (m, 1H), 2.84-
2.95(m,4H),3.40-3.41 (d, J= 3.3 Hz, 2H), 3.77 (s, 3H), 3.85-3.95
(m, 1H), 5.28-5.33 (m, 1H), 5.97-5.99 (d, J = 6.9 Hz, 1H),
6.57-6.69 (m, 2H), 6.95-6.98 (d, J = 8.4 Hz, 1H), 7.26-7.42 (m,
3H), 7.60-7.62 (d, J = 7.5 Hz, 1H).
6-Methoxy-1,2,3,4-tetrahydro-naphthalene-2(þ)-carboxylic
Acid-{1-[4-(3-hydroxyphenyl)-(3R,4R)-trans-dimethyl-piperidinyl-
methyl]-(2S)-methylpropyl}amide (16a). 2(þ)-6-Methoxy-1,2,3,4-
tetrahydronaphthalene-2-carboxylic acid [(þ)-13, 0.22 g, 1.07
mmol] was added under N2 to a solution of BOP (0.47 g, 1.07
mmol), TEA (0.23 g, 2.35 mmol), and 1227 (0.31 g, 1.07 mmol) in
anhydrous THF(50mL). The solutionwas allowedtostiratroom
temperature for 6 h, and a saturated NaHCO3 solution (100 mL)
was added. The biphasic mixture was extracted with EtOAc (3 ꢀ
100 mL). The organic extracts were combined, dried (MgSO4),
and concentratedunder reduced pressuretoprovide anoil. The oil
was purified using medium pressure column chromatography on
silica gel using CHCl3-MeOH-NH4OH (9:0.8:0.2) at the eluent
2(-)-6-Methoxy-1,2,3,4-tetrahydronaphthalene-2-carboxylic
Acid [(-)-13]. A 30% solution of hydrogen peroxide (3.3 mmol,
0.11 mL) in H2O was added at 0 °C to a solution of 15b (0.20 g,
0.55 mmol) in THF-H2O (3:1) (15 mL). Lithium hydroxide
hydrate (0.046 g, 1.10 mmol) was added to the solution in
portions. The suspension was allowed to stir for 0.5 h at 0 °C

5298 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Runyon et al.
toprovide0.39g (77%) of16a as acolorlessoil. 1HNMR (CDCl3)
δppm 0.72 (d, J =6.78Hz, 3H), 0.82-0.96 (m,6 H), 1.26 (s, 3H),
1.55 (d, J = 12.43 Hz, 1 H), 1.78-2.07 (m, 4 H), 2.18-2.88 (m, 12
H), 3.73 (s, 3 H), 4.00-4.16 (m, 1 H), 6.05 (d, J = 7.54 Hz, 1 H),
6.57 (d, J = 2.64 Hz, 1 H), 6.62-6.77 (m, 3 H), 6.84 (m, 1 H), 6.93
(d, J = 8.67 Hz, 1 H), 7.11 (t, J = 7.91 Hz, 1 H).
The reaction was made basic (pH ≈ 9) with saturated sodium bi-
carbonate solution, and the solution was extracted with Et2O
(1 ꢀ 100 mL), made acidic to pH 3 with HCl (6 N solution), and
extracted with EtOAc (3 ꢀ 100 mL). The organic extracts were
combined, dried (MgSO4), and concentrated under reduced
pressure to provide 0.25 g (100%) of (þ)-20 as a white solid.
The solid was recrystallized from toluene-petroleum ether to
6-Methoxy-1,2,3,4-tetrahydro-naphthalene-2(-)-carboxylic
Acid-{1-[4-(3-hydroxyphenyl)-(3R,4R)-trans-dimethyl-piperidinyl-
methyl]-(2S)-methylpropyl}amide (16b). 2(-)-6-Methoxy-1,2,3,4-
tetrahydronaphthalene-2-carboxylic acid [(-)-13, 0.31 g, 1.48
mmol] was added under N2 to a solution of BOP (0.65 g, 1.48
mmol), TEA (0.33 g, 3.26 mmol), and 12 (0.43 g, 1.48 mmol) in
anhydrous THF(65mL). The solutionwas allowedtostirat room
temperature for 6 h, and a saturated NaHCO3 solution (100 mL)
was added. The biphasic mixture was extracted with EtOAc (3 ꢀ
100 mL). The organic extracts were combined, dried (MgSO4),
and concentrated under reduced pressure to provide an oil. The oil
was purified using medium pressure column chromatography on
silica gel using CHCl3-MeOH-NH4OH (9:0.8:0.2) as the eluent
toprovide0.70g (98%) of16b as acolorlessoil. 1HNMR (CDCl3)
δ ppm 0.66-0.78 (d, J = 6.9 Hz, 3 H), 0.83-0.97 (m, 6 H), 1.25 (s,
3 H), 1.53 (d, J = 12.43 Hz, 1 H), 1.78-2.10 (m, 4 H), 2.20-2.97
(m, 12 H), 3.73 (s, 3 H), 4.03 (m, 1 H), 6.03 (d, J = 7.54 Hz, 1 H),
6.57 (d, J = 2.26 Hz, 1 H), 6.61-6.75 (m, 3 H), 6.82 (m, 1H), 6.90
(d, J = 8.29 Hz, 1 H), 7.10 (t, J = 7.72 Hz, 1 H).
provide (þ)-20 as tan cubes: mp 117-118 °C; [R]22 þ98.3 °C
D
(c = 0.24, MeOH). 1H NMR (CD3OD) δ 2.97-3.01 (dd, J =
9.3, 15 Hz, 1H), 3.10-3.17 (dd, J = 5.1, 15.3, 1H), 3.63-3.88
(m, 6H), 6.75-6.77 (m, 2H), 7.08-7.11 (d, J = 8.2 Hz, 1H).
3(-)-7-Methoxy-isothiochroman-3-carboxylic Acid [(-)-20].
Lithium hydroxide hydrate (0.055 g, 1.32 mmol) was added at
0 °C to a solution of 21b (0.25 g, 0.66 mmol) in THF-H2O (3:1)
(15 mL). The suspension was allowed to stir for 0.5 h at 0 °C. The
reaction was made basic (pH ≈ 9) with saturated sodium bicar-
bonate solution, and the solution was extracted with Et2O (1 ꢀ
100 mL), made acidic to pH 3 with 6 N HCl, and extracted with
EtOAc (3 ꢀ 100 mL). The organic extracts were combined, dried
(MgSO4), and concentrated under reduced pressure to provide
0.14 g (92%) of (-)-20 as a white solid (0.136 g, 92%). The solid
was recrystallized from toluene-petroleum ether to provide
(-)-20 as pale-yellow needles: mp 121-122 °C; [R]22D -100.8 °C
(c = 0.26, MeOH). 1H NMR (CD3OD) δ 2.97-3.01 (dd, J =
9.3, 15 Hz, 1H), 3.10-3.17 (dd, J = 5.1, 15.3, 1H), 3.63-3.88
(m, 6H), 6.75-6.77 (m, 2H), 7.08-7.11 (d, J = 8.2 Hz, 1H).
(3aR-cis)-3-(7-Methoxy-isothiochroman-3(þ and -)-carbonyl)-
3,3a,8,8a-tetrahydro-2H-indeno[1,2-d]oxazol-2-one (21a,b). A
2.0 M solution of oxalyl chloride (3.57 mL, 7.14 mmol) in CH2Cl2
was added under N2 to a solution of (()-20 (0.80 g, 3.57 mmol)
and a drop of DMF in CH2Cl2 (100 mL). The solution was
allowed to stir at room temperature for 3 h and was concentrated
under reduced pressure to provide 7-methoxy-isothiochroman-3-
carbonyl chloride as a tan oil. 7-Methoxy-isothiochroman-3-
carbonyl chloride was used in the next step without further
purification. In a separate flask, a 0.50 M solution of ethyl lithium
(8.6 mL, 4.28 mmol) in benzene-cyclohexane 90:10 was added
to a solution of (3aR-cis)-3,3a,8,8a-tetrahydro-2H-indeno[1,2-d]-
oxazol-2-one (14, 0.75 g, 4.28 mmol) in THF (100 mL) at
0 °C under N2. The suspension was allowed to stir at 0 °C for
0.5 h and was then cooled to -78 °C. A solution of 7-methoxy-
isothiochroman-3-carbonyl chloride (0.86 g, 3.57 mmol) in THF
(10 mL) was added in a dropwise manner to the -78 °C slurry.
The resulting slurry was allowed to warm to room temperature
over 2 h, and water (150 mL) was then added. The suspension was
extracted with CH2Cl2 (3 ꢀ 150 mL). The organic extracts were
combined, dried (MgSO4), and concentrated under reduced
pressure to provide a mixture of 21a and 21b as a tan solid. The
mixture was separated by silica gel medium pressure column
chromatography using petroleum ether-Et2O (60:40) as the
eluent to provide each of the diastereomers in 62% [21a, (þ)-
isomer] and 37% [21b, (-)-isomer] theoretical yield. The yield
improves with additional chromatography. The higher Rf spot on
TLC was identified as the (þ)-isomer, while the more polar spot
was the (-)-isomer.
7-Methoxy-isothiochroman-4-one-3-carboxylic Acid Methyl
Ester (18). A 2.0 M solution of LDA in heptane-THF-
ethylbenzene (1.61 mL, 3.21 mmol) was added in a dropwise
manner to a solution of 7-methoxyisothiochroman-4-one (17)31
(0.50 g, 2.57 mmol) in THF (50 mL) at -78 °C under N2. After
30 min at -78 °C, HMPA (0.46 g, 2.57 mmol) and methyl
cyanoformate (0.27 g, 3.21 mmol) were added, and the yellow
solution was allowed to stir at -78 °C for 30 min. The solution
was then allowed to warm to room temperature, and a saturated
solution of NH4Cl (100 mL) was added. The slurry was ex-
tracted with EtOAc (3 ꢀ 75 mL), and the organic extracts were
combined, dried (MgSO4), and concentrated under reduced
pressure to provide a bright-yellow oil. The oil was purified on
silica gel medium pressure chromatography using petroleum
ether-EtOAc (9:1) as the eluent to provide 0.51 g (78%) of 18 as
1
a bright-yellow oil. H NMR (CDCl3) δ 3.73 (s, 2H), 3.83 (s,
3H), 3.85 (s, 3H), 6.67 (d, J = 3 Hz, 1H), 6.45 (dd, J = 3, 8.7 Hz,
1H), 7.80 (d, J = 8.7 Hz, 1H), 12.52 (s, 1H).
7-Methoxy-isothiochroman-3-carboxylic Acid Methyl Ester
(19). Triethylsilane (8.08 mmol, 0.94 g) was added to a solution
of 18 (0.51 g, 2.02 mmol) in trifluoroacetic acid (15 mL) at room
temperature under N2. The reaction mixture was allowed to stir
at room temperature for 2 h and was concentrated under
reduced pressure. The resulting oil was dissolved in EtOAc
(100 mL) and washed with a saturated NaHCO3 solution (3 ꢀ
75 mL). The organic extracts were combined, dried (MgSO4),
and concentrated to provide an oil. The oil was purified by silica
gel medium pressure chromatography using petroleum ether-
EtOAc (9:1) as the eluent to provide 0.34 g, (70%) of 19 as a pale-
yellow oil. 1H NMR (CDCl3) δ 3.14 (m, 2H), 3.58-3.63 (d, J =
15 Hz, 1H), 3.73-3.86 (m, 8H), 6.70 (d, J = 3 Hz, 1H), 6.75 (dd,
J = 3, 9 Hz, 1H), 7.10 (d, J = 9 Hz, 1H).
7-Methoxy-isothiochroman-3-carboxylic Acid [(()-20]. Pota-
ssium hydroxide (0.80 g, 14.3 mmol) was added to a solution of
19 (0.34 g, 1.43 mmol) in MeOH (50 mL). The solution was
heated at 60 °C for 2 h, cooled to room temperature, and diluted
with H2O (100 mL). The solution was made acidic with 6 N HCl
and extracted with EtOAc (3ꢀ100 mL). The organic extracts
were combined, dried (MgSO4), and concentrated to give 0.28 g
(88%) of (()-20 as a pale-yellow solid. The solid was used in the
next step without further purification.
(3aR)-cis)-3-(7-Methoxy-isothiochroman-3(þ)-carbonyl)-
3,3a,8,8a-tetrahydro-2H-indeno[1,2-d]oxazol-2-one (21a). The solid
was recrystallized from EtOAc-petroleum ether to provide 0.42 g
(62%) of 21a as a white solid: mp 146-147 °C. 1H NMR (CDCl3)
δ 3.09-3.16 (dd, J = 6, 15.6 Hz, 1H), 3.20-3.28 (dd, J = 7.2,
15.3 Hz, 1H), 3.38-3.39 (d, J = 3.6 Hz, 2H), 3.59-3.64 (d, J =
15Hz, 1H), 3.79(s, 3H), 3.85-3.90 (d, J = 15 Hz, 1H), 4.97-5.01
(m, 1H), 5.30-5.35 (m, 1H), 5.92-5.95 (d, J = 7 Hz, 1H),
6.71-6.72 (d, J = 2.7 Hz, 1H), 6.75-6.79 (dd, J = 2.4, 8.4 Hz,
1H), 7.06-7.09 (d, J=8.4 Hz, 1H), 7.23-7.38 (m, 3H), 7.58-
7.61 (d, J = 7.5 Hz, 1H).
3(þ)-7-Methoxy-isothiochroman-3-carboxylic Acid [(þ)-20].
Lithium hydroxide hydrate (0.093 g, 2.2 mmol) was added at
0 °C to a solution of 21a (0.42 g, 1.10 mmol) in THF-H2O (3:1)
(25 mL). The suspension was allowed to stir for 0.5 h at 0 °C.
(3aR-cis)-3-(7-Methoxy-isothiochroman-3(-)-carbonyl)-3,3a,8,8a-
tetrahydro-2H-indeno[1,2-d]oxazol-2-one (21b). The solid was recrys-
tallized from EtOAc-petroleum ether to provide 0.25 g (37%) of 21b
as a white solid: mp 176-178 °C. 1H NMR (CDCl3) δ 3.14-3.19

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5299
(dd, J = 6, 12.6 Hz, 1H), 3.24-3.29 (dd, J = 7.5, 15.3 Hz, 1H),
3.39-3.40 (d, J= 3.6 Hz, 2H), 3.48-3.55(d,J= 15 Hz, 1H), 3.79 (s,
3H), 3.83-3.88 (d, J = 15 Hz, 1H), 4.91-4.95 (m, 1H), 5.29-5.33
(m, 1H), 5.96-5.99 (d, J = 7 Hz, 1H), 6.71-6.72 (d, J = 2.7 Hz,
1H), 6.76-6.80 (dd, J = 2.4, 8.4 Hz, 1H), 7.10-7.12 (d, J = 8.4 Hz,
1H), 7.26-7.38 (m, 3H), 7.58-7.61 (d, J = 7.5 Hz, 1H).
DMF (320 mL), and EtSNa (7.8 g, 0.093 mol) was added. The
reaction mixture was heated at reflux for 3 h, H2O (450 mL) was
added, and the mixture was extracted with EtOAc (300 mL). The
aqueous layer was cooled, acidified to pH ≈ 4 with 1 M HCl,
and then extracted with EtOAc (3 ꢀ 300 mL). The combined
organics were washed with bleach (200 mL) and dried over
sodium sulfate, and the solvent was removed under reduced
pressure to afford crude product, which was purified by silica gel
medium pressure column chromatography using 10-50%
EtOAc in hexane as the eluent to give 7.2 g (71%) of 25 as a
7-Methoxy-isothiochroman-3(þ)-carboxylic Acid-{1-[4-(3-hy-
droxyphenyl)-(3R)-(4R)-trans-dimethyl-piperidinylmethyl]-(2S)-
methylpropyl}amide (22a). 3(þ)-7-Methoxyisothiochroman-3-
carboxylic acid [(þ)-20, (0.25 g, 1.12 mmol)] was added under
N2 to a solution of BOP (0.50 g, 1.12 mmol), TEA (0.23 g, 2.24
mmol), and 1227 (0.33 g, 1.12 mmol) in anhydrous THF (50 mL).
The solution was allowed to stir at room temperature for 6 h,
and a saturated NaHCO3 solution (100 mL) was added. The
biphasic mixture was extracted with EtOAc (3ꢀ100 mL). The
organic extracts were combined, dried (MgSO4), and concen-
trated under reduced pressure to provide an oil. The oil was
purified using medium pressure column chromatography using
CHCl3-MeOH-NH4OH (9:0.8:0.2) to provide 0.5 g (81%) of
22a as a pale-yellow semisolid. 1H NMR (CDCl3) δ 0.49-0.55
(m, 6H), 0.67-0.69 (d, J = 6 Hz, 3H), 1.24 (s, 3H), 1.48-1.52 (d,
J = 12 Hz, 1H), 1.62-1.70 (m, 1H), 1.86-1.88 (m, 1H), 2.14-
2.52 (m, 6H), 2.61-2.71 (m, 2H), 2.89-2.95 (dd, J = 5.1, 14.4
Hz, 1H), 3.31-3.38 (dd, J = 5.4, 14.4 Hz, 1H), 3.57-3.62 (d,
J = 13.8 Hz, 1H), 3.65-3.69 (d, J = 13.8 Hz, 1H), 3.74 (s, 3H),
3.84-3.87 (m, 1H), 6.70-6.72 (m, 3H), 6.84-6.89 (m, 2H),
7.03-7.12 (m, 2H).
1
colorless solid. H NMR (CD3OD) δ 7.85-7.80 (m, 1H), 6.77
(d, J = 6.4 Hz, 1 H), 6.6 (s, 1H), 4.76 (s, 3H), 4.22 (s, 2H). 13
C
NMR (CD3OD, 75 MHz) δ 195.5, 165.2, 147.0, 130.3, 123.3,
116.8, 111.7, 74.4, 69.2. LCMS (ESI) m/z 165.3 (M þ H)þ; 163.4
(M - H)þ.
7-(Methoxymethoxy)-1H-isochromen-4(3H)-one (26). 7-Hy-
droxy-1H-isochromen-4(3H)-one (25, 7.0 g, 0.43 mol) was dis-
solved in CH2Cl2 (350 mL), and the solution was cooled to 0 °C
under N2. i-Pr2EtN (8.3 g, 0.064 mol) was added, and then
MOMCl (5.15 g, 0.064 mol) was added slowly. The reaction
mixture was stirred for 8 h at room temperature, and saturated
NaHCO3 (200 mL) was added. The suspension was extracted
with EtOAc (3 ꢀ 250 mL), the combined organics were dried
over sodium sulfate, and the solvent was removed under reduced
pressure. The solid was purified by silica gel medium pressure
column chromatography using 10-40% EtOAc in hexane as the
1
eluent to give 7.8 g (88%) of 26 as a colorless solid. H NMR
(CDCl3) δ 8.02 (d, J = 8.6 Hz, 1H), 7.03 (d, J = 8.6 Hz, 1H), 6.84
(s, 1H), 5.24 (s, 2H), 4.85 (s, 2H), 4.33 (s, 2H), 3.49 (s, 3H). 13C
NMR (CDCl3, 75 MHz) δ 192.8, 161.8, 144.2, 128.9, 124.0, 116.1,
110.7, 94.1, 73.4, 68.1, 56.4. LCMS (APCI) m/z 209.3 (M þ H)þ.
Methyl 7-(Methoxymethoxy)-4-oxo-3,4-dihydro-1H-isochro-
men-3-carboxylate (27). LDA (31 mL, 2.0 M in THF-heptane-
ethylbenzene, 62.0 mmol) was then added to a solution of 26
(6.6 g, 0.032 mol) in THF (400 mL) at -78 °C. The mixture was
stirred at -78 °C for 30 min, and HMPA (5.74 g, 5.6 mL, 0.032
mol) was added followed by methylcyanoformate (5.4 g, 0.064
mol). The reaction mixture was stirred at -78 °C for 2 h and
warmed to room temperature. The reaction mixture was quenched
with saturated NH4Cl (250 mL) and then extracted with EtOAc
(3 ꢀ 300 mL). The combined organics were dried over sodium
sulfate, and the solvent was removed under reduced pressure to
afford crude compound which was purified by silica gel medium
pressure column chromatography using 10-30% EtOAc in hex-
ane as eluent to give 4.35 g (52%) of 27 as a colorless solid. NMR
shows a mixture of ketone and enol tautomers. 1H NMR (CDCl3)
δ 8.02 (d, J = 8.7 Hz, 0.7 H), 7.61 (d, J = 8.5 Hz, 0.2H), 7.03 (d,
J = 8.7 Hz, 1H), 6.81 (s, 1H), 5.24 (s, 1H), 5.23 (s, 2H), 4.97-4.85
(m, 2H), 3.91 (s, 0.7H), 3.84 (s, 2.5H), 3.48 (s, 3H). 13C NMR
(CDCl3, 75 MHz) δ 187.1, 166.9, 162.3, 143.5, 129.6, 124.4, 116.5,
110.5, 94.2, 80.5, 65.9, 56.4, 52.8. LCMS (ESI) m/z267.1 (M þ H)þ.
Methyl 7-hydroxy-3,4-dihyro-1H-isochromene-3-carboxylate
(28). TFA (80 mL) was added to 27 (4.3 g, 0.016 mol) at 0 °C,
and Et3SiH (7.52 g, 0.065 mol) was added. The reaction mixture
was stirred at room temperature for 2 h and then was concentrated
under reduced pressure. Saturated NaHCO3 (100 mL) was added,
and the mixture was extracted with EtOAc (3 ꢀ 150 mL). The
combined organics were dried (Na2SO4), and the solvent was
removed under reduced pressuretoafford crude compound which
was purified by silica gel medium pressure column chromato-
graphy using 10-40% EtOAc in hexane as eluent to give 2.22 g
(66%) of 28 as a colorless solid. 1H NMR (CDCl3) δ 6.99 (d, J =
8.0 Hz, 1H), 6.70 (d, J = 8.1 Hz, 1H), 6.48 (s, 1H), 5.76 (s, 1H),
4.91 (d, J= 15.0 Hz, 1H), 4.78 (d, J= 15.0 Hz, 1H), 4.36 (t, J=7.0
Hz, 1H), 3.82 (s, 3H), 2.99 (d, J = 6.9 Hz, 2H). 13C NMR (CDCl3,
75 MHz) δ 171.9, 154.4, 134.8, 129.9, 123.3, 114.4, 110.7, 73.6, 67.8,
52.4, 30.1. LCMS (ESI) m/z 209.3 (M þ H)þ; 231.5 (M þ Na)þ.
7-Hydroxy-3,4-dihydro-1H-isochromene-3-carboxylic Acid
(29). Lithium hydroxide hydrate (45 mg, 1.08 mmol) was
added to a solution of 28 (0.100 g, 0.48 mmol) in a mixture
7-Methoxy-isothiochroman-3(-)-carboxylic Acid-{1-[4-(3-
hydroxyphenyl)-(3R,4R)-trans-dimethyl-piperidinylmethyl]-(2S)-
methylpropyl}amide (22b). 3(-)-7-Methoxy-isothiochroman-3-
carboxylic acid [(-)-20, 0.24 g, 1.07 mmol] was added under N2
to a solution of BOP (0.47 g, 1.07 mmol), TEA (0.21 g, 2.14
mmol), and 1227 (0.31 g, 1.07 mmol) in anhydrous THF (50 mL).
The solution was allowed to stir at room temperature for 6 h, and
a saturated NaHCO3 solution (100 mL) was added. The biphasic
mixture was extracted with EtOAc (3ꢀ100 mL). The organic
extracts were combined, dried (MgSO4), and concentrated under
reduced pressure to provide an oil. The oil was purified by silica
gel medium pressure column chromatography using CHCl3-
MeOH-NH4OH (9:0.8:0.2) as the eluent to provide 0.44 g
1
(84%) of 22b as a pale yellow semisolid. H NMR (CDCl3) δ
0.65-0.68 (d, J = 6.9 Hz, 3H), 0.77-0.79 (d, J = 4.2 Hz, 3H),
0.84-0.86 (d, J = 6.6 Hz, 3H), 1.27 (s, 3H), 1.47-1.51 (d, J =
12.3 Hz, 1H), 1.80-2.70 (m, 11H), 3.03-3.09 (dd, J = 5.4,
14.7 Hz, 1H), 3.17-3.24 (dd, J = 6.3, 14.4 Hz, 1H), 3.60-3.65
(d, J = 14.1 Hz, 1H), 3.67-3.72 (d, J = 14.1 Hz, 1H), 3.77 (s,
3H), 3.83-3.87 (m, 1H), 6.59-6.83 (m, 5H), 7.05-7.16 (m, 2H).
7-Methoxy-1H-isochromen-4(3H)-one (24). Oxalyl chloride
(92.5 mL, 2.0 M in CH2Cl2, 185 mmol) was added dropwise to
a solution of (3-methoxybenzyloxy)acetic acid (23)34 (23.2 g,
0.12 mol) in CH2Cl2 (850 mL) at 0 °C under N2, and a few drops
of DMF were added to initiate the reaction. The reaction mixture
was stirred for 2 h at room temperature and was concentrated to
give a brown oil. Chlorobenzene (500 mL) was then added, and
the mixture was allowed to stir using a mechanical stirrer. The
mixture was cooled to 0 °C in an ice bath, SnCl4 (29 mL, 246.8
mmol) was added slowly, and the reactionmixture was allowed to
stir at 0 °C under N2 for 1 h. The reaction mixture was quenched
with saturated NaHCO3 (150 mL) and H2O (150 mL) and then
extracted with CH2Cl2 (3 ꢀ 250 mL). The combined organics
were dried over NaSO4, and the solvent was removed under
reduced pressure to afford crude compound which was purified
by silica gel medium pressure column chromatography using
10-40% EtOAc in hexane to give 10.9 g (52%) of 24 as a white
solid. 1H NMR (CDCl3) δ 8.02 (d, J = 8.6 Hz, 1H), 6.92 (d, J =
7.9 Hz, 1H), 6.67 (s, 1H), 4.85 (s, 2H), 4.33 (s, 2H), 3.88 (s, 1H).
13C NMR (CDCl3, 75 MHz) δ 193.1, 164.6, 144.6, 129.4, 123.6,
114.5, 109.1, 73.7, 68.4, 56.0. LCMS (APCI) m/z (M þ H)þ 179.2.
7-Hydroxy-1H-isochromen-4(3H)-one (25). 7-Methoxy-1H-
isochromen-4(3H)-one (24, 11.0 g, 0.062 mol) was dissolved in

5300 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Runyon et al.
of THF-H2O-MeOH (1:1:1) (25 mL) at 0 °C. The solution
was allowed to stir for 1 h and warmed to room temperature.
A 1 N solution of HCl was added until the mixture was acidic
and was then extracted with CHCl3 (3 ꢀ 50 mL). The organic
extracts were combined, dried (MgSO4), and concentrated
under reduced pressure to provide 0.75 g (82%) of (()-29 as a
white solid. This solid was used in the next step without
further purification. 1H NMR (CD3OD) δ 2.96, (m, 3H),
4.32 (dd, J = 3, 9 Hz, 1H), 4.74 (m, 2H), 6.46 (s, 1H), 6.63
(d, J = 9 Hz, 1H), 6.95 (d, J = 9 Hz, 1H).
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Compounds 3 and 2 Alignment. Three-dimensional structures
of 3 and 2 were built using the Sybyl fragment library and
optimized using the MMFF94 force-field and charges. A sys-
tematic conformational search followed by a multifit flexible
structural alignment was performed using 2 as the rigid template
(excepting the guanidinium group of 2, which was allowed to
freely rotate during the multifit alignment). Six 2-3 atom pairs
and a spring constant of 20 kcal/mol were used to align 3 to the 2
template. The atom pairs were the 3 and 2 phenolic ring cen-
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atoms 14, 13, 15, 10, and 5 (morphine numbering). The centroid
of the Tic aromatic ring of 3 was paired with the centroid of the
guanidinium group of 2. After alignment, a final energy mini-
mization was performed using the MMFF94 force-field to allow
the structure of 3 to relax to a local minimum energy conformation.
This process was repeated for each of the 10 lowest energy con-
formations of 3 obtained from an initial systematic conformational
search in order to identify the lowest energy conformation of 3 that
could provide a close alignment to the 2 pharmacophore template.
Evaluation of 7a to Block U50,488-Induced Diuresis. Adult
male Sprague-Dawley rates (Charles River Laboratories,
Raleigh, NC) were used for these studies. The test compound
and U50,488 doses were prepared fresh in distilled deionized water
(vehicle) and administered (1 mL/kg body weight) via subcuta-
neous injection. Six groups of four rats were used to evaluate each
test compound: vehicle control, agonist control (10 mg/kg), test
compound 7a at 3, 10, or 30 mg/kg followed by agonist (10 mg/kg),
and compound 7a followed by vehicle (30 mg/kg). Each rat was
weighed prior to dosing. One rat from each group was dosed in
succession and the pattern repeated to distribute any effects of time
of day across all groups. After dosing, each rat was placed into a
metabolic chamber and urine output was collected hourly for 5 h.
Urine output for each collection period was calculated as (urine þ
collection tube weight)-collection tube tare weight. The effect of
test compound on total urine output was assessed by analysis of
variance with repeated measures (subject within Group) and
factors of Group and Time and their interaction, or one-way
ANOVA, where appropriate. A univariate ANOVA was run only
if a significant effect was observed following the multivariate
ANOVA. Significance was assumed at p < 0.05 for the individual
factors and p < 0.1 for their interaction.
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Acknowledgment. This research was supported by the
National Institute on Drug Abuse grant DA09045.
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Supporting Information Available: Elemental analysis data
for compounds 6, 7a,b, 9a,b, and 10a,b and HPLC traces for 8a
and 8b. This material is available free of charge via the Internet
at http://pubs.acs.org.
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Mascarella, S. W.; Vinson, N. A.; Xu, H.; Dersch, C. M.; Lu, Y.;
Cantrell, B. E.; Zimmerman, D. M.; Carroll, F. I. Identification of
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derivative to possess highly potent and selective opioid kappa
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