Identification 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 as a novel potent and selective opioid κ receptor antagonist

Article abstract of DOI:10.1021/jm030094y

(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) was identified as a potent and selective κ opioid receptor antagonist. Structure-activity relationship (SAR) studies on JDTic analogues revealed that the 3R,4R stereochemistry of the 3,4-dimethyl-4-(3-hydroxy-phenyl)piperidine core structure, the 3R attachment of the 7-hydroxy-1,2,3,4-tetrahydroisoquinoline group, and the 1S configuration of the 2-methylpropyl (isopropyl) group were all important to its κ potency and selectivity. The results suggest that, like other κ opioid antagonists such as nor-BNI and GNTI, JDTic requires a second basic amino group to express potent and selective κ antagonist activity in the [³⁵S] GTPγS functional assay. However, unlike previously reported κ antagonists, JDTic also requires a second phenol group in rigid proximity to this second basic amino group. The potent and selective κ antagonist properties of JDTic can be rationalized using the "message-address" concept wherein the (3R,4R)-3,4-dimethyl-4-(hydroxyphenyl)piperidinyl group represents the message, and the basic amino and phenol group in the N substituent constitutes the address. It is interesting to note the structural commonality (an amino and phenol groups) in both the message and address components of JDTic. The unique structural features of JDTic will make this compound highly useful in further characterization of the κ receptor.

Full text of DOI:10.1021/jm030094y

J . Med. Chem. 2003, 46, 3127-3137
3127
Id en tifica tion of (3R)-7-Hyd r oxy-N-((1S)-1-{[(3R,4R)-4-(3-h yd r oxyp h en yl)-
3,4-d im eth yl-1-p ip er id in yl]m eth yl}-2-m eth ylp r op yl)-1,2,3,4-tetr a h yd r o-
3-isoqu in olin eca r boxa m id e a s a Novel P oten t a n d Selective Op ioid K Recep tor
An ta gon ist
J ames B. Thomas,† Robert N. Atkinson,† N. Ariane Vinson,† J ennifer L. Catanzaro,† Carin L. Perretta,†
Scott E. Fix,† S. Wayne Mascarella,† Richard B. Rothman,‡ Heng Xu,‡ Christina M. Dersch,‡ Buddy E. Cantrell,§
Dennis M. Zimmerman,§ and F. Ivy Carroll*,†
Chemistry and Life Sciences, Research Triangle Institute, 3040 Cornwallis Road,
Research Triangle Park, North Carolina 27709, Clinical Psychopharmacology Section, Intramural Research Program,
National Institute on Drug Abuse, National Institutes of Health, 5500 Nathan Shock Drive, Baltimore, Maryland 21224, and
Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285
Received February 26, 2003
(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 (J DTic) was identified as a potent
and selective κ opioid receptor antagonist. Structure-activity relationship (SAR) studies on
J DTic analogues revealed that the 3R,4R stereochemistry of the 3,4-dimethyl-4-(3-hydroxy-
phenyl)piperidine core structure, the 3R attachment of the 7-hydroxy-1,2,3,4-tetrahydroiso-
quinoline group, and the 1S configuration of the 2-methylpropyl (isopropyl) group were all
important to its κ potency and selectivity. The results suggest that, like other κ opioid
antagonists such as nor-BNI and GNTI, J DTic requires a second basic amino group to express
potent and selective κ antagonist activity in the [35S]GTPγS functional assay. However, unlike
previously reported κ antagonists, J DTic also requires a second phenol group in rigid proximity
to this second basic amino group. The potent and selective κ antagonist properties of J DTic
can be rationalized using the “message-address” concept wherein the (3R,4R)-3,4-dimethyl-
4-(hydroxyphenyl)piperidinyl group represents the message, and the basic amino and phenol
group in the N substituent constitutes the address. It is interesting to note the structural
commonality (an amino and phenol groups) in both the message and address components of
J DTic. The unique structural features of J DTic will make this compound highly useful in further
characterization of the κ receptor.
In tr od u ction
established that opioid receptors belong to the super-
family of G-protein-coupled receptors (GPCRs).4-11
Opium, the exudate of the opium poppy (Papaver
somniferum), has been used for beneficial medicinal
purposes for thousands of years. For an equally long
time however, mankind has known the hardship as-
sociated with abuse of this powerfully addictive sub-
stance. Early in the past century, attempts to harness
the better nature of this natural product led to the
development of heroin (1, Chart 1), the diacetyl deriva-
tive of the principle constituent of opium, morphine (2).
Ironically, heroin is much more addictive than opium
and has since become the most abused opiate worldwide.
To counter this problem, a scientific campaign was
mounted early in the 20th century to probe the funda-
mentals of addiction and the addiction process.1,2 The
wealth of knowledge accumulated since its inception is
enormous and includes examples of milestone discover-
ies commensurate with its breadth from the original
demonstration of an endogenous opioid receptor3 to the
more recent cloning of the opioid receptor. With distinct
cDNAs encoding µ, δ, and κ receptors for animals and
humans identified, sequenced, and cloned, it is now well
An integral part of the effort to characterize the opioid
receptor has been the discovery of antagonists for the
opioid receptors. In addition, it has long been thought
that antagonists for the opioid receptors might provide
treatment medications for those suffering from opiate
abuse, especially those antagonists that are selective for
particular receptor subtypes. One of the more useful
compounds for studying the opioid receptor has been the
κ selective opioid antagonist nor-BNI (3), which is de-
rived from the nonselective opioid antagonist naltrexone
(4).12 We recently reported the discovery 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 (5, J DTic) as a
potent and selective antagonist for the κ opioid recep-
tor.13 Compound 5 arose via modification of the R group
in N-substituted trans-3,4-dimethyl-4-(3-hydroxyphe-
nyl)piperidines (6) first to give the analogue 7, which
was optimized by additional structural modification to
give the κ selective 5.13,14 Preliminary results from some
of these studies have been reported.13 In this article,
we report the results of an SAR investigation of J DTic
and present additional results on the in vitro pharma-
cological characterization of J DTic.
* To whom correspondence should be addressed. Phone: 919-541-
6679. Fax: 919-541-8868. E-mail: fic@rti.org.
† Research Triangle Institute.
‡ National Institute on Drug Abuse.
§ Eli Lilly and Company.
10.1021/jm030094y CCC: $25.00 © 2003 American Chemical Society
Published on Web 06/11/2003

3128 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14
Thomas et al.
Ch a r t 1
provided 3-methyltetrahydropyridine 21, which was
alkylated with dimethyl sulfate following deprotonation
with n-butyllithium. N-demethylation using phenyl
chloroformate followed by deprotection of the phenol
group with HBr in acetic acid gave (3S,4S)-dimethyl-
4-(3-hydroxyphenyl)piperidine (18). Acylation of 18 with
Boc-L-valine followed by reduction with the borane
dimethyl sulfide complex and deprotection with TFA
gave diamine 22. The completion of the synthesis of 17
from this point followed the same route employed in the
preparation of 5 from 8.
Biologica l Resu lts
The binding affinities of the test compounds for the
µ, δ, and κ opioid receptors listed in Table 1 were
determined using previously reported competitive bind-
ing assays.13,14,16 [3H]DAMGO, [3H]DADLE, and [3H]-
U69,593 radioligands were used to label the µ, δ, and κ
receptors, respectively. The tissues used in the binding
assays included rat brain (µ and δ receptors) and guinea
pig brain (κ receptors). Measures of functional antago-
nism were obtained by monitoring the ability of test
compounds to inhibit stimulation of [35S]GTPγS binding
produced by the selective agonists (D-Ala2,MePhe4,Gly-
ol5)enkephalin (DAMGO, µ receptor), (+)-4-[(RR)-R-
(2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxy-
benzyl]-N,N-diethylbenzamide (SNC-80, δ receptor), and
5R,7R,8â-(-)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro-
[4,5]dec-8-yl]benzeneacetamide (U69,593, κ receptor) in
guinea pig caudate membranes (Table 2).13,14,16 The
binding affinities of selected compounds for the µ, δ, and
κ receptors using [3H]DAMGO, [3H]Cl-DPDPE, and [3H]-
U69,593, respectively (Table 3), and the ability to inhibit
[35S]GTPγS binding stimulated by DAMGO (µ), DPDPE
(δ), and U69,593 (κ), using cloned human opioid recep-
tors transfected into CHO cells, were also determined
(Table 4).17,18
Ch em istr y
In this study to develop a potent and κ-selective opioid
receptor antagonist, we synthesized and evaluated
J DTic (5) and the J DTic analogues 9-17. J DTic (5),
J LTic (9), the desphenolic analogue 10, and the tyrosine
analogues 11 and 12 were all prepared from the com-
mon intermediate 814 (Scheme 1). Coupling of Boc-D-7-
hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid,
Boc-L-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-car-
boxylic acid, and Boc-D-1,2,3,4-tetrahydroisoquinoline-
3-carboxylic acid with 8 using benzotriazol-1-yloxy-
tris(dimethylamino)phosphonium hexafluorophosphate
(BOP) in THF, followed by removal of the Boc protecting
group with trifluoroacetic acid in methylene chloride,
yielded 5, 9, and 10, respectively. The tyrosine ana-
logues 11 and 12 were synthesized by coupling Boc-D-
tyrosine and Boc-L-tyrosine to 8 using BOP followed by
removal of the Boc protecting group with trifluoroacetic
acid in methylene chloride. The reduced and N-substi-
tuted analogues 13-16 were all synthesized from 5. The
desired products were obtained via carbonyl reduction
with borane dimethyl sulfide (13), methylation using
sodium triacetoxy borohydride, and formaldehyle (14),
and acylation using BOP and acetic acid (15) or N,N-
dimethylglycine (16) (Scheme 2).
Resu lts a n d Discu ssion
In our studies of novel opioid agonists and antago-
nists,16,19 we commonly utilized radioligand binding
assays as a primary screen. Once compounds possessing
desirable biological properties, i.e., enhanced affinity or
selectivity for a particular receptor subtype, were iden-
tified, the selected compounds were evaluated in the in
vitro [35S]GTPγS functional assay to reveal their ef-
ficacy, potency, and selectivity. This progression scheme
proved to be reliable when dealing with µ and δ receptor
ligands, where a good correlation was observed between
a compound’s receptor affinity in binding studies and
its potency in functional assays. However, this scheme
proved to be less useful in our studies directed toward
the 3,4-dimethyl-4-(3-hydroxyphenyl)piperidine-based κ
opioid receptor antagonists. The data from the radioli-
gand binding assays did not accurately predict those
compounds that would be most selective for the κ over
the µ and δ receptors in the in vitro efficacy assays. In
fact, some compounds exhibited an inverse behavior
between κ selectivity in binding relative to selectivity
in functional assays. For example, we reported that
compound 7 with radioligand binding Ki values of 171,
>3400, and 3.84 nM at the µ, δ, and κ receptors,
respectively, and µ/κ and µ/δ selectivity ratios of 45 and
The 3S,4S diastereomer (17) was synthesized from
(3S,4S)-dimethyl-4-(3-hydroxyphenyl)piperidine (18),
which was in turn prepared according to the method of
Werner and co-workers starting from 1,3-dimethyl-4-
piperidone (19) as depicted in Scheme 3.15 Thus, 19 was
treated with 3-isopropoxyphenyllithium followed by
formation of the ethyl carbonate and recrystallization
of the (+)-diethyl ditoluyltartrate salt to give optically
pure carbonate 20. Heating 20 in refluxing Decalin

Isoquinolinecarboxamide
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14 3129
Sch em e 1a
a
Reagents: all coupling reactions used BOP and Et3N in THF; (a) Boc-D-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid;
(b) Boc-L-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; (c) Boc-D-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; (d) Boc-
D-tyrosine; (e) Boc-L-tyrosine; (f) TFA, CH2Cl2.
Sch em e 2a
a
Reagents: (a) BH3‚SMe2, THF; (b) NaBH(OAc)3, 37% aqueous CH2O; (c) BOP, TEA, CH3CO2H; (d) BOP, TEA, (CH3)2NCH2CO2H.
Sch em e 3a
>885 were superior to 5 (J DTic), which possessed Ki
values of 3.73, 301, and 0.32 nM at the µ, δ, and κ
receptors and µ/κ and µ/δ ratios of 12 and 940, respec-
tively (Table 1). In fact, compound 7 possesses a µ/κ Ki
selectivity ratio comparable to that of nor-BNI and a
far superior δ/κ Ki ratio and, thus, is selective for the κ
opioid receptor over the µ or δ receptor. In contrast, 5
shows very little κ versus µ selectivity relative to 7 or
nor-BNI in the radioligand binding assays. The principle
reason for this lack of κ selectivity is the greater affinity
of 5 for the µ receptor relative to 7 and nor-BNI. The
overall result is that 5 possesses low κ versus µ selec-
tively in the radioligand binding test. In contrast, 5
shows a 16-fold improvement in its κ receptor Ke value
in the [35S]GTPγS assay relative to the radioligand
binding assay (0.02 versus 0.32 nM). Since the Ke values
a
Reagents: all coupling reactions used BOP and Et3N in THF,
for 5 in the µ and δ assays do not increase substantially,
and all deprotections used TFA in CH2Cl2; (a) 3-isopropoxyphen-
the shift to higher potency for 5 in the κ receptor func-
yllithium, THF; (b) EtOCOCl, EtOAc; (c) (+)-DTTA; (d) Decalin
reflux; (e) n-BuLi, (CH3)2SO4; (f) C6H5OCOCl; (g) 48% HBr, HOAc;
tional assay results in greater than 100-fold µ versus κ
selectivity and a remarkable >15000-fold selectivity for
the δ versus κ receptor. Similar to J DTic (5), nor-BNI
(3) shows a 28-fold increase in its Ke value for the κ re-
ceptor relative to its Ki value in the radioligand binding
assay and only a 4- and 8-fold increase at the µ and δ
(h) Boc-L-valine; (i) BH3‚SMe2, THF; (j) TFA; (k) Boc-D-7-hydroxy-
1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid.
receptors. Overall, this translates into a significant in-
crease in µ versus κ and δ versus κ selectivity in [35S]-

3130 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14
Thomas et al.
Ta ble 1. Radioligand Binding Results at the µ, δ, and κ Opioid Receptors Using Opioid Receptors Obtained from Brain Tissue (µ and
δ of Rat, κ of Guinea Pig)
Ki ( SD, nM
compd
µ [3H]DAMGOa
δ [3H]DADLEb
κ [3H]U69,593c
µ/κ
δ/κ
79
Nor-BNI, 3
J DTic, 5
65 ( 5.6d
3.73 ( 0.17d
171 ( 15d
596 ( 29
775 ( 75
208 ( 14
633 ( 34
107 ( 11
37 ( 1.4
764 ( 66
164 ( 10
138 ( 8
86 ( 7.2d
301 ( 50d
>3400
1.09 ( 0.14d
0.32 ( 0.05d
3.84 ( 0.26d
9.8 ( 1.6
2.1 ( 0.17
20.4 ( 2
16 ( 1.1
0.63 ( 0.05
0.053 ( 0.003
146 ( 15
15.5 ( 1.2
17.5 ( 2.5
60
12
45
61
369
10
940
>885
>500
>2333
>247
>315
>7777
11,622
>34
7
9
>4900
10
11
12
13
14
15
16
17
>4900
>5051
>5051
40
>4900
170
700
5
11
7.8
616 ( 59
>4900
>3400
>219
144 ( 15
8.2
a
b
[3H]DAMGO [(D-Ala2,MePhe4,Gly-ol5)enkephalin]. Tritiated ligand selective for µ opioid receptor. [3H]DADLE [(D-Ala2,D-
Leu5)enkephalin]. Tritiated ligand selective for δ opioid receptor. c [3H]U69,593 {[3H](5R,7R,8â)-(-)-N-methyl-N-[7-(1-pyrrolidinyl)-1-
oxaspiro[4,5]dec-8-yl]benzeneacetamide}. Tritiated ligand selective for κ opioid receptor. Data taken from ref 13.
d
Ta ble 2. Inhibition by Antagonists of [35S]GTPγS Binding in
picted, the selectivity ratios obtained from binding data
for 5 and 16 suggest that these ligands would possess
Guinea Pig Caudate Stimulated by the Opioid Receptor
Subtype-Selective Agonists, DAMGO (µ), SNC80 (δ), and
U69,593 (κ)
low selectivity for the κ receptor versus the µ receptor
in the functional assay, but as is clear from the [35S]-
Ke ( SD, nM
GTPγS functional assay selectivity ratios, this is not the
compd µ DAMGOa δ SNC-80b
κ U69,593c
µ/κ
δ/κ
268
>15000
case. Indeed, in the more relevant functional assay,
these compounds were found to be among the most
potent and selective κ antagonists ever identified. At the
opposite end of the spectrum, the binding assay data
for compounds 10 and 14 indicate that these compounds
should possess extraordinary selectivity in the func-
tional assay, yet as was the case for 7, this stands in
contrast to the actual selectivity found in the functional
assay. Nevertheless, it is clear that the development of
J DTic required input from both series of data. Radio-
ligand binding data provided the novel lead structure
7, and the [35S]GTPγS assay, which is more correlated
with in vivo functional assays, led to the identification
of J DTic as a potent and κ-selective opioid antagonist.
On the basis of the higher correlation of [35S]GTPγS
data with in vivo functional data, the SAR analysis of
the J DTic analogues is limited to the data obtained in
the functional assays.
The data obtained for the test compounds in the [35S]-
GTPγS functional assay using guinea pig caudate
membranes are presented in Table 2. By means of
comparison, the SAR for compound 5 and that of the
original lead compound 7 guided the design of additional
compounds to reveal those structural features under-
pinning the dramatic change in biological activity
observed between these two compounds. The addition
of a methyleneamino bridge to compound 7, giving
compound 5, has a tremendous impact on conforma-
tional flexibility, overall basicity, and solubility; yet it
allows 5 to still meet all five of Lipinski’s requirements
for a compound to possess druglike properties.20 For
example, from a comparison of the data for 5 and 7 in
Table 2, the overall result to antagonist activity of this
structural change is expressed as a 3-fold increase of
potency in the µ receptor assay (Ke ) 7.25 versus 2.16
nM) and a 235-fold increase in potency at the κ receptor
(Ke ) 4.75 versus 0.02 nM).
3
5
7
16.7 ( 1.5d 10.2 ( 1.0d 0.038 ( 0.005d 440
2.16 ( 0.75d >300d 0.02 ( 0.002d 108
7.25 ( 0.52 450 ( 74.1 4.7 ( 0.56
1.5
2.6
5.9
0.3
0.55
64
34
0.9
181
10.7
96
78
12.8
7.5
15
9
11 ( 0.8
68.6 ( 6.5
12 ( 0.8
16.5 ( 2
12.8 ( 1.4
12.7 ( 1.4
17.4 ( 1.2
29 ( 2
327 ( 35
147 ( 11
334 ( 41
452 ( 35
>300
4.2 ( 0.20
11.5 ( 1
44.6 ( 6
30 ( 2.9
0.20 ( 0.03
0.37 ( 0.04
19.6 ( 1.6
0.16 ( 0.03
16.7 ( 1.9
10
11
12
13
14
15
16
17
>1500
810
15
3925
>18
>300
>300
628 ( 50
>300
178 ( 17
a
DAMGO [(D-Ala2,MePhe4,Gly-ol5)enkephalin]. Agonist selec-
b
tive for µ opioid receptor. SNC-80 ([(+)-4-[(RR)-R-(2S,5R)-4-allyl-
2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenz-
amide). Agonist selective for δ opioid receptor. c U69,593 [(5R,7R,8â)-
(-)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4,5]dec-8-yl]benze-
d
neacetamide]. Agonist selective for κ opioid receptor. Taken from
ref 13.
GTPγS assay relative to that observed in the binding
assay.
As already mentioned, compound 7 also shows sig-
nificant changes in behavior between these two assays,
but the changes do not parallel those found for J DTic
(5) and nor-BNI (3). Instead, compound 7 shows no µ
versus κ selectivity in the [35S]GTPγS assay compared
with its 45-fold selectivity seen in the radioligand
binding assay, an effect driven primarily by its 25-fold
increase in µ receptor Ke. Overall, compound 7 is not
selective in [35S]GTPγS assay and does not compare
favorably with J DTic (5) and nor-BNI (3). Similar
differences in the radioligand and [35S]GTPγS assays
seen for compounds 3, 5, and 7 were also noted for other
compounds assayed (Tables 1 and 2). Figure 1 shows a
comparison of the µ versus κ selectivity ratios from the
radioligand binding and [35S]GTPγS functional assays,
as listed in Tables 1 and 2, respectively, for a relevant
set of N-substituted 3,4-dimethyl-4-(3-hydroxyphenyl)-
piperidine-based antagonists. In this representation, the
nature of this phenomenon of a lack of correlation
between the radioligand and [35S]GTPγS assays is
readily appreciated with compounds 5, 16, 10, and 14,
which illustrate the two observable extremes. As de-
The presence of pendant amino groups in the nal-
trexone-based κ antagonists such as nor-BNI (3) has
been shown to be essential to its selectivity for the κ
receptor versus the µ or δ opioid receptors.21 Thus,

Isoquinolinecarboxamide
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14 3131
Ta ble 3. Radioligand Binding Results for Test Compound Using Human Cloned µ, δ, and κ Receptors
Ki ( SD, nM
compd
µ [3H]DAMGO
δ [3H]Cl-DPDPE
κ [3H]U69,593
µ/κ
105
206
2.3
15
2.1
δ/κ
nor-BNI, 3a
21 ( 5.0
5.7 ( 0.9
0.2 ( 0.05
29
389
72
500
46
GNTI, 23b
36.9 ( 2.3
0.96 ( 0.01
33.1 ( 0.89
3.80 ( 0.30
8.86 ( 0.69
13.2 ( 1.16
70.0 ( 0.3
29.6 ( 11.9
1090 ( 174
83.6 ( 8.09
118 ( 6.66
175 ( 25.5
0.18 ( 0.10
0.41 ( 0.10
2.18 ( 0.12
1.82 ( 0.20
1.01 ( 0.17
16.2 ( 2.58
J DTic, 5
10
13
14
16
8.8
0.81
117
11
a
b
Taken from ref 24. Taken from ref 23.
Ta ble 4. Inhibition of Agonist Stimulated [35S]GTPγS Binding by J DTic and J DTic Analogues in Cloned Human µ, δ, and κ Opioid
Receptors
µ, DAMGOa
Ke (nM)
δ, DPDPEb
Ke (nM)
κ, U69,593c
Ke (nM)
0.04
compd
pA2
pA2
pA2
µ/κ
δ/κ
nor-BNI,3d
19 ( 1.8
4.4 ( 0.38
475
115
GNTI, 23e
8.49 ( 0.09
7.81 ( 0.06
f
10.40 ( 0.10
J DTic, 5
3.41 ( 0.36
8.38 ( 0.30
14.3 ( 2.27
8.92 ( 1.23
21.2 ( 1.48
79.3 ( 9.3
ND
0.01 ( 0.00
2.95 ( 0.42
0.14 ( 0.02
0.10 ( 0.02
1.10 ( 0.09
341
2.8
102
89
19
7930
10
13
14
16
427 ( 109
120 ( 11
299 ( 54
3100
1200
272
a
b
DAMGO [(D-Ala2,MePhe4,Gly-ol5)enkephalin] (selective for µ opioid receptor). DPDPE cyclo[D-Pen2,D-Pen5]enkephalin (selective for
δ opioid receptor). c U69,593 {(5R,7R,8â)-(-)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4,5]dec-8-yl]benzeneacetamide} (agonist selective
for κ opioid receptor). pA2 values taken from ref 24; no Ke values were given in the reference. e Data taken from ref 23. f The pA2 value
d
could not be determined because this compound is a noncompetitive antagonist at the δ opioid site.
F igu r e 1. Comparison of the κ to µ ratio for radioligand binding and [35S]GTPγS assays.
addition of pendant amino groups to 7 might be expected
to have the same κ directing effect. However, the data
for compounds 11 and 12 in Table 2, both of which
possess pendant amino groups, clearly show that this
modification does not produce κ selective compounds.
In fact, at the µ and δ receptors there is little change in
behavior for these amine-bearing ligands relative to 7,
and with respect to κ receptor potency, compounds 11
and 12 were found to be less potent than 7. On the
whole, the net affect of this change provided a decrease
in κ antagonist activity and leads to the conclusion that
the amino group alone is insufficient to promote κ
receptor selectivity in the absence of a methylene bridge.
Viewed from a different perspective, one must consider
compounds 11 and 12 as analogues of compound 5,
which lack this methylene bridge. The 2230- and 1500-
fold losses in κ antagonist potency displayed by 11 and
12 relative to 5 clearly highlight the importance of this
conformationally constraining methylene bridge.
In a previous report, it was established that the
pendant phenol group in the structure of 7 is a require-
ment for its κ binding affinity.14 This suggested that the
corresponding phenol group in 5 might also be required
for the expression of its κ antagonist activity. The data
for compound 10 (Table 2), which differs from 5 by the
absence of this phenol group, support such an assertion.
In fact, this structural change impacts activity at all of
the opioid receptors. At the µ receptor, the loss of this
hydroxyl group provides a 32-fold decrease in potency
(Ke ) 68.6 nM for 10 versus 2.16 nM for 5), while at

3132 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14
Thomas et al.
the δ receptor the same change results in an improve-
ment in potency. The most dramatic change, however,
is seen in the κ receptor assay where 10 shows a 575-
fold loss of antagonist potency relative to 5. Clearly, the
phenol group plays an integral part in the action of this
compound at the κ receptor. More importantly, this is
the first example of a pendant phenol group showing
such a profound effect upon the potency of a κ opioid
receptor antagonist.
versus κ selectivity compared to that found in compound
5 (181-fold versus 108-fold, respectively).
In the initial investigation of the N-substituted 3,4-
dimethyl-4-(3-hydroxyphenyl)piperidine antagonists, it
was found that the relative position of the 3,4-dimethyl
substituents on the piperidine ring affected the overall
selectivity and potency but not the agonist versus
antagonist activity of the resulting compounds.22 To
establish a link with this historical information, the
diastereomer of 5, compound 17, which has a 3S,4S
versus the 3R,4R configuration for the 3,4-dimethyl
groups found in 5, was prepared and tested. A compari-
son of the data for compounds 5 and 17 in Table 2
clearly shows that the stereochemistry of these methyl
groups contributes substantially to the observed potency
and selectivity of compound 5. In the κ receptor assay,
the shift of the 3-methyl group from one side of the
piperidine ring to the other resulted in an 835-fold loss
of antagonist potency. On the whole, the behavior
observed for these two compounds is congruent with
historical precedent because the placement of the meth-
yl groups does impact overall activity but the magnitude
of the observed difference for these diastereomers is far
greater than any other pair yet studied in this series.
Importantly, however, while compound 17 was found
to lack the selectivity and potency of 5, it still retains
full antagonist character because it showed no agonist
properties up to 10 µM (data not shown). Taken to-
gether, this information suggests that the methyl group
in the piperidine 3-position of compound 5 could be
interacting directly with the κ receptor in a highly
specific manner or that the placement of the methyl
group in 17 is such that it makes the “κ critical”
conformation energetically unavailable.
Compound 9 was prepared to examine the importance
of the configuration of the carbon adjacent to the amino
group in 5. Opioid receptors have been shown to
distinguish between enantiomers as well as changes in
a single stereocenter. The data for compound 9 indicate
that the stereocenter of interest is also critical to κ
receptor potency and selectivity but has little affect on
µ or δ activity. Overall, 9 is not a selective κ compound,
and this change in behavior is driven primarily by the
210-fold loss of potency of this compound for the κ
receptor relative to 5. Taken together, it is clear that
this stereocenter is a key structural determinant of the
selectivity and potency displayed by compound 5.
Reduction of the amide carbonyl in compound 5,
giving 13, provided a means of probing the contribution
of this functional group to the overall activity of 5. The
data obtained from 13 indicated that the loss of this
group does not impact the potency or selectivity of the
resulting compound to the same degree as that seen in
other modifications. For example, compared to com-
pound 5, 13 shows only a 10-fold loss of potency for the
κ receptor, maintains about the same affinity for the δ
receptor, and loses about 4-fold activity in the µ receptor
assay. On the whole, the change in this functional group
had little overall impact on antagonist activity.
Compounds J DTic (5), 10, 13, 14, and 16 were also
evaluated in radioligand binding and [35S]GTPγS assays
using cloned human µ, δ, and κ opioid receptors trans-
fected into Chinese hamster ovary (CHO) cells and
compared to reported values for nor-BNI (3) and GNTI
(23).17,23,24 The results are listed in Tables 3 and 4. In
general, we observed a similar pattern of results in both
assay systems. Interestingly, some compounds were
more potent at cloned receptors than native receptors.
As noted above, there were at times striking differences
in potency between the binding Ki value and the
functional Ke value. For example, J DTic (5) and nor-
BNI (3) possessed Ke values of 0.01 and 0.04 nM,
respectively, at the κ receptor and showed a 40- and 20-
fold increase, respectively, in the [35S]GTPγS assay
relative to the radioligand binding assay. The Ki (ra-
dioligand binding) to Ke ([35S]GTPγS binding) ratio was
very small for both compounds at the µ and δ receptors.
This is represented graphically in Figure 2.
Compounds 14-16 were produced to examine the
requirements of basicity of the pendant amino group of
5 as well as to uncover a potential hydrogen-bonding
interaction or size constraints. Substitution of the amine
hydrogen for methyl to give compound 14 provided an
18-fold loss of potency at the κ receptor and a 6-fold
decrease in potency for the µ opioid receptor. The
activity in the δ assay remained unchanged. Together,
these shifts in potency combine to produce a less
selective κ antagonist, 34-fold for 14 versus 108-fold for
5, and suggest that a hydrogen-bonding interaction
could contribute to the activity of compound 5.
Acylation of 5 to give compound 15 effectively removes
the pendant basic center that has historically been a
requirement for κ antagonist selectivity. As the data
from Table 2 show, compound 15 fits the pattern
established for earlier κ compounds because removal of
its basic center affords a 980-fold loss of κ potency. To
demonstrate that the loss of activity was not the result
of a steric interaction between receptor and binding site,
compound 16 was prepared that incorporates the acyl
group from 15 but that also contains a basic amino
group. The addition of this basic center to 15 affords a
dramatic 122-fold increase in potency relative to 15 and
effectively restores most of the κ antagonist potency lost
by the acylation. Interestingly, this modification of 5
produces a significant decrease in µ antagonist potency
that, coupled with the subnanomolar activity of 16 at
the κ receptor, results in a compound with improved µ
Unlike J DTic, GNTI with Ki values of 36.9, 70, and
0.18 nM at the µ, δ, and κ receptor was selective for the
κ receptor in the radioligand binding assays. However,
the pA2 values of 8.49 and 10.40 for the µ and κ receptor,
respectively, in the [35S]GTPγS test are almost identical
to the pA2 values of 8.50 and 10.46 at the µ and κ
receptors found for J DTic. Since J DTic is a noncompeti-
tive antagonist at the δ receptor, a comparison of pA2
values was not possible. The 79.3 nM Ke values for
J DTic and 7.81 pA2 for GNTI show that neither com-
pound has significant potency at the δ receptor.

Isoquinolinecarboxamide
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14 3133
series clearly illustrated that the second basic amino
group in these compounds is critical to their κ selectivity
and that the second phenolic hydroxyl group in nor-BNI
did not have to be present. The present SAR study of
J DTic (5) revealed that this structurally unique κ
antagonist also requires a second basic amino group to
express κ potency and in this sense behaves similarly
to previously identified naltrexone-based κ antagonists
such as nor-BNI (3). In the case of J DTic, the trans-
(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine could
be viewed as the “message” and some portion of the N
substituent as the “address”. If one assumes that this
“message-address” is as suggested, the J DTic studies
reveal that the κ potency is due to at least two structural
features in the N substituent “address” moiety. The first
involves the interaction of the N-H moiety of the
D-hydroxy Tic group, possibly via a charged interaction
with Glu297 in the κ receptor similar to the interaction
found for N′-17 group for nor-BNI and the guanidino
amino group in GNTI. Equally important is the interac-
tion of the phenol moiety in the D-hydroxy Tic group
with the κ receptor. This interaction is unique to J DTic
and opens the possibility that site-directed mutagenesis
studies might identify the complementary residues in
the κ receptor, leading to further characterization of the
κ receptor. The κ selectivity of J DTic is due to the
combination of high κ affinity resulting from these two
D-hydroxy Tic interactions plus possible steric hindrance
of these groups at the address-recognition locus present
in the µ and δ opioid receptors.
F igu r e 2. Comparison of ratios of radioligand binding and
GTPγS assays for J DTic (5) and nor-BNI (3) using cloned
human opioid receptors.
Ch a r t 2
Con clu sion s
The discovery of the potent and selective κ opioid
receptor antagonist (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-isoquinolinecar-
boxamide (JDTic, 5) represents a significant advancement
in the development of the trans-3,4-dimethyl-4-(3-hy-
droxyphenyl)piperidine class of opioid antagonist. To our
knowledge, J DTic is the only potent and selective κ
opioid receptor antagonist not derived from the opiate
class of compounds. On the basis of historical precedent,
we believe the potent and pure opioid antagonist activ-
ity results from the (3R,4R)-3,4-dimethyl-4-(3-hydroxy-
phenyl)piperidine core structure. An SAR study of J DTic
analogues suggests that the κ opioid receptor potency
and selectivity result from a combination of (a) the
isoquinoline amino group and 7-hydroxy group held in
a rigid orientation by the 1,2,3,4 tetrahydroisoquinoline
structure in its 3R attachment to the amide carboxyl,
(b) an S configuration of the 2-methylpropyl group in
the spacer between the piperidine ring and the D-
hydroxy Tic acyl group, and (c) the lack of a substituent
on the amide nitrogen. The unique structure of J DTic
provides an additional pharmacological tool to further
characterize the κ opioid receptor. Using site-directed
mutagenesis studies, Portoghese et al. have shown that
Glu297 in the κ receptor is critical to the κ selectivity
shown by nor-BNI and GNTI.28 It will be particularly
interesting to determine if Glu297 is also critical for the
κ selectivity of J DTic. In addition, since the 7-hydroxy
(phenolic) group is important to the κ selectivity of
J DTic, it will be of interest to see if site-directed
mutagenesis studies can identify the amino acid residue
in the κ receptor responsible for this interaction. It will
Portoghese has explained the selectivity of opioid
ligands in terms of the “message-address” concept of
Schwyzer,25 wherein the “message” component of the
ligand specifies primary receptor recognition and the
“address” portion confers selectivity by specific recogni-
tion of a particular receptor subsite.26 Portoghese pro-
vided an elegant demonstration of the “message-
address” concept by showing that the selectivity of
naltrindole (NTI, 24) could be changed from a δ- to a
κ-selective ligand by attaching a methyleneamidino
group to its 5′ position to give 5′-[(N2-butylamidino)-
methyl]naltrindole (25) (Chart 2).21 Further refinements
of this concept, targeting the distance between the
amino address group and the naltrexone message frag-
ment in 25, led to the more potent and selective κ
antagonist, C5′-guanidinylnaltrindole (GNTI, 23).27
Taken together, the studies in the naltrexone-derived

3134 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14
Thomas et al.
(2R)-Am in o-3-(4-h yd r oxyp h en yl)-N-{1-[4-(3-h yd r oxy-
p h en yl)-3R,4R-d im eth yl-p ip er id in -1-ylm eth yl]-2-m eth yl-
1-p r op yl}p r op a n a m id e (11). 11 was prepared according to
the general procedures from Boc-D-tyrosine and 3-[1-(2S-
amino-3-methylbutyl)-3R,4R-dimethyl-4-piperidinyl]phenol (8,
1 equiv).14 The impure product was purified by flash chroma-
tography using 67% (CHCl3/MeOH/NH4OH, 80:18:2) in CHCl3
be of great interest to determine if the potent and κ
opioid receptor properties of J DTic seen in the in vitro
binding and functional assay translate to in vivo tests.
Exp er im en ta l Section
1H NMR were determined on a Bruker WM-250 or a Bruker
300 spectrometer 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 all column chromatography. All reactions were followed by
thin-layer chromatography using Whatman silica gel 60 TLC
plates and were visualized by UV or by charring using 5%
phosphomolybdic acid in ethanol. All solvents were reagent
grade. Tetrahydrofuran and diethyl ether were dried over
sodium benzophenone ketyl and distilled prior to use. Meth-
ylene chloride and chloroform were distilled from calcium
hydride if used as reaction solvents. HCl in dry ethyl ether
was purchased from Aldrich Chemical Co. and used while fresh
before discoloration.
1
to afford 11 (99%) as a white foam. H NMR (CD3OD): δ 7.02
(m, 3H), 6.71 (m, 4H), 6.55 (d, 1H, J ) 7.8 Hz), 3.91 (m, 1H),
3.50 (t, 1H, J ) 7.1 Hz), 2.90-2.21 (m, 9H), 1.97 (br, 1H), 1.74
(m, 1H), 1.56 (d, 1H, J ) 12.6 Hz), 1.28 (s, 3H), 0.74 (m, 9H).
13C NMR (CD3OD): δ 176.8, 158.6, 157.7, 153.5, 131.8, 130.4,
129.9, 118.3, 116.7, 114.1, 113.6, 61.4, 58.3, 57.2, 52.7, 52.3,
41.9, 40.5, 39.8, 32.2, 32.1, 28.4, 20.0, 18.2, 17.0. Anal.
(C27H39N3O3) C, H, N.
(2S)-Am in o-3-(4-h yd r oxyp h en yl)-N-{1-[4-(3-h yd r oxy-
p h en yl)-3R,4R-d im eth ylp ip er id in -1-ylm eth yl]-2-m eth yl-
1-p r op yl}p r op a n a m id e (12). 12 was prepared according to
the general procedures from Boc-L-tyrosine and 3-[1-(2S-
amino-3-methylbutyl)-3R,4R-dimethyl-4-piperidinyl]phenol (8,
1 equiv).14 The crude was purified by flash chromatography
using 67% (CHCl3/MeOH/NH4OH, 80:18:2) in CHCl3 to afford
The [3H]DAMGO, DAMGO, and [3H][D-Ala2,D-Leu5]enkeph-
alin were obtained via the Research Technology Branch, NIDA,
and were prepared by Multiple Peptide Systems (San Diego,
CA). The [3H]U69,593 and [35S]GTPγS (SA ) 1250 Ci/mmol)
were obtained from DuPont New England Nuclear (Boston,
MA). U69,593 was obtained from Research Biochemicals
International (Natick, MA). Levallorphan was a generous gift
from Kenner C. Rice, Ph.D., NIDDK, NIH (Bethesda, MD).
GTPγS and GDP were obtained from Sigma Chemical Com-
pany (St. Louis, MO). Boc-D- and Boc-L-7-hydroxy-1,2,3,4-
tetrahydroisoquinoline-3-carboxylic acid were purchased from
CSPS Pharmaceuticals, Inc. (San Diego, CA). The sources of
other reagents are published.
Gen er a l Am id e Cou p lin g P r oced u r e. The appropriate
Boc-protected carboxylic acid derivative (1.2 equiv) was added
to a solution of the appropriate amine intermediate (1 equiv)
in dry THF (0.5 mL/mg) followed by BOP reagent (1.2 equiv)
and triethylamine (5 equiv). The reaction mixture was stirred
for 2 h at room temperature, and then ether was added (2 mL/
mL THF) and the mixture was washed with saturated
NaHCO3 and then water. The organic layer was collected and
dried over magnesium sulfate, and the solvent was removed
under reduced pressure.
Gen er a l Boc-Dep r otection P r oced u r e. The material
obtained from the coupling reaction was dissolved in dry
CH2Cl2 (50 mL/g of Boc intermediate) and cooled to -20 °C
whereupon TFA (27 mL/g of Boc intermediate) was added
dropwise. The reaction flask was left in a MeOH/ice bath for
10 min and then was allowed to warm to room temperature.
The solvent was removed under reduced pressure, and the
residue diluted with CH2Cl2. To this was added saturated
NaHCO3. The organic layer was separated, and the solvent
was removed under reduced pressure. The material obtained
from the coupling reactions was purified using flash column
chromatography, if necessary, and was then converted to its
HCl salt by dissolving in methanol at 0 °C followed by addition
of 1 equiv of 1 N HCl in ethyl ether.
(3R)-7-Hydr oxy-N-((1S)-1-{[(3R,4R)-4-(3-h ydr oxyph en yl)-
3,4-d im e t h yl-1-p ip e r id in yl]m e t h yl}-2-m e t h ylp r op yl)-
1,2,3,4-tetr a h yd r o-3-isoqu in olin eca r boxa m id e (5). 5 was
prepared according to the general procedures starting from
Boc-D-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid
and 3-[1-(2S-amino-3-methylbutyl)-3R,4R-dimethyl-4-piperidi-
nyl]phenol. The impure product was purified by silica gel
preparative plate chromatography (50% (CHCl3/MeOH/NH4-
OH, 80:18:2) in CHCl3 to give 5 as a white solid that was
converted to its HCl salt via the general procedures. 1H NMR
(CD3OD): δ 7.11 (t, 1H, J ) 7.9 Hz), 6.92 (d, 1H, J ) 8.3 Hz),
6.74 (m, 2H), 6.59 (m, 2H), 6.50 (m, 1H), 4.03 (m, 1H), 3.94 (d,
2H, J ) 5.9 Hz), 3.54 (dd, 1H, J ) 4.8, 10.2 Hz), 2.94 (dd, 1H,
J ) 4.7, 15.7 Hz), 2.80 (m, 2H), 2.67-2.37 (m, 5H), 2.27 (dt,
1H, J ) 4.2, 12.6 Hz), 1.99-1.85 (m, 2H), 1.57 (d, 1H, J )
12.7 Hz), 1.30 (s, 3H), 0.95 (m, 6H), 0.74 (d, 3H, J ) 6.7 Hz).
Anal. (C28H39N3O3‚2HCl‚H2O) C, H, N.
1
12 (94%) as a white foam. H NMR (CD3OD): δ 6.94 (m, 3H),
6.60 (m, 4H), 6.45 (d, 1H, J ) 8.0 Hz), 3.80 (m, 1H), 3.36 (m,
1H), 2.81 (m, 1H), 2.66 (m, 1H), 2.57-2.07 (m, 7H), 1.84 (br,
1H), 1.73 (m, 1H), 1.43 (d, 1H, J ) 12.7 Hz), 1.16 (s, 3H), 0.76
(m, 6H), 0.6 (d, 3H, J ) 6.9 Hz). 13C NMR (CD3OD): δ 176.3,
158.1, 157.3, 153.2, 131.4, 130.0, 129.4, 117.9, 116.3, 113.7,
113.1, 61.0, 57.8, 56.8, 52.4, 51.7, 41.6, 40.1, 39.3, 31.7, 28.0,
19.7, 17.8, 16.6. Anal. (C27H39N3O3) C, H, N.
(3R)-N-((1S)-1-{[(3R,4R)-4-(3-H yd r oxyp h en yl)-3,4-d i-
m et h yl-1-p ip er id in yl]m et h yl}-2-m et h ylp r op yl)-1,2,3,4-
tetr a h yd r o-3-isoqu in olin eca r boxa m id e (10). 10 was pre-
pared according to the general procedures from Boc-D-1,2,3,4-
tetrahydroisoquinoline-3-carboxylic acid and 3-[1-(2S-ami-
no-3-methylbutyl)-3R,4R-dimethyl-4-piperidinyl]phenol (8, 1
equiv).14 The crude product was purified by silica preparative
plate chromatography 50% (CHCl3/MeOH/NH4OH, 80:18:2) in
1
CHCl3, yielding 10 as a white foam. H NMR (CDCl3) 7.22 (d,
J ) 8.8 Hz, 1H), 7.05-7.13 (m, 3H), 6.98-7.01 (m, 1H), 6.79
(s, 1H), 6.73 (d, J ) 7.8 Hz, 1H), 6.65 (dd, J ) 7.9, 1.8 Hz,
1H), 4.05-4.14 (m, 1H), 3.99 (s, 2H), 3.56 (dd, J ) 10.7, 4.9
Hz, 1H), 3.18 (dd, J ) 16.5, 4.8 Hz, 1H), 2.67-2.82 (m, 3H),
2.31-2.54 (m, 4H), 2.18-2.25 (m, 1H), 1.86-1.96 (m, 2H), 1.52
(d, J ) 12.9 Hz, 1H), 1.25 (s, 3H), 0.92 (t, J ) 7.6 Hz, 6H),
0.67 (d, J ) 6.9 Hz, 3H). Anal. (C28H39N3O2) C, H, N.
(3S)-7-Hydr oxy-N-((1S)-1-{[(3R,4R)-4-(3-h ydr oxyph en yl)-
3,4-d im e t h yl-1-p ip e r id in yl]m e t h yl}-2-m e t h ylp r op yl)-
1,2,3,4-tetr a h yd r o-3-isoqu in olin eca r boxa m id e (9). 9 was
prepared according to the general procedures starting from
Boc-L-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid
and 3-[1-(2S-amino-3-methylbutyl)-3R,4R-dimethyl-4-piperidi-
nyl]phenol (8, 1 equiv).14 The crude product was purified using
silica gel column chromatography gradient elution starting
with neat CHCl3 and moving to 50% (CHCl3/MeOH/NH4OH,
80:18:2) in CHCl3 to give 9 as an off-white solid. 1H NMR (CD3-
OD): δ 7.09 (t, J ) 7.8 Hz, 1H), 6.87 (d, J ) 4.1 Hz, 1H), 6.77
(s, 1H), 6.74 (d, J ) 0.8 Hz, 1H), 6.58 (dd, J ) 8.3, 2.3 Hz,
2H), 6.48 (d, J ) 2.3 Hz, 1h), 4.01-3.95 (m, 1H), 3.90 (s, 2H),
3.51-3.48 (m, 1H), 3.35 (s, 3H), 2.94-2.21 (m, 9H), 1.96 (d, J
) 13 Hz, 1H), 1.28 (s, 3H), 0.89 (t, 7.2 Hz, 6H), 0.74 (d, J )
6.9 Hz, 3H). Anal. (C28H39N3O3) C, H, N.
(3R)-3-{[((1S)-1-{[(3R,4R)-4-(3-Hyd r oxyp h en yl)-3,4-d i-
m et h yl-1-p ip er id in yl]m et h yl}-2-m et h ylp r op yl)a m in o]-
m eth yl}-1,2,3,4-tetr a h yd r o-7-isoqu in olin ol (13). A solu-
tion of 2 M BH3‚SMe2 in THF (0.495 mL, 0.99 mmol) was
added dropwise to a -20 °C solution of (3R)-7-hydroxy-
N-((1S)-1-{[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-pip-
eridinyl]methyl}-2-methylpropyl)-1,2,3,4-tetrahydro-3-isoquin-
olinecarboxamide (3, 46 mg, 0.099 mmol) in 5 mL of dry
THF.13 The reaction mixture was heated to reflux overnight
and was cooled to -20 °C, and 0.647 mL of methanol was
added. The contents were stirred at room temperature for 1
h, 1 M HCl in ether (0.142 mL, 0.142 mmol) was added, and

Isoquinolinecarboxamide
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14 3135
the mixture was stirred for 30 min. The solvent was then
removed under reduced pressure, the oil obtained was dis-
solved in 3:1 CH2Cl2/THF, and the pH was adjusted to 10 with
saturated NaHCO3. The organic layer was separated, and the
aqueous layer was extracted 5× with 3:1 CH2Cl2/THF. The
combined organic layers were dried over sodium sulfate, and
the solvent was removed under reduced pressure to give a
yellow foam. The crude product (0.03 g) was purified by silica
gel preparative plate chromatography using a solvent gradient,
starting with 25% (CHCl3/MeOH/NH4OH, 80:18:2) in CHCl3
dimethyl-4-(3-hydroxyphenyl)piperidine (18) used in the
synthesis of 17 was prepared according to the method of
Werner et al. with a single modification.15 The intermediate
tartrate salt (20) was recrystallized twice rather than once
from ethanol. The (3S,4S)-dimethyl-4-(3-hydroxyphenyl)pip-
eridine (18) thus obtained was found to be identical in all
respects to an authentic sample of 18. The 3-[1-(2S-amino-3-
methylbutyl)-3S,4S-dimethyl-4-piperidinyl]phenol (22) illus-
trated in Scheme 3 was prepared from 18 and Boc-L-valine
according to the method described for the synthesis of 8 in
Scheme 1.14 The impure product was purified via silica gel
flash chromatography using a gradient of 0-10% MeOH in
1
and then 40% and finally 65%. This afforded 6 mg of 13. H
NMR (CD3OD): δ 7.12 (t, J ) 5.1 Hz, 1H), 6.90 (d, J ) 4.1
Hz, 1H), 6.8 (d, J ) 3.9 Hz, 1H), 6.76 (d, J ) 1.1 Hz, 1h), 6.61
(dd, J ) 5.8, 2.2 Hz, 2H), 6.48 (d, J ) 1.2 Hz, 1H), 2.88-2.43
(m, 16H), 2.4-1.8 (m, 2H), 1.67 (d, J ) 13 Hz, 1H), 1.34 (s,
3H), 1.01 (d, J ) 6.9 Hz, 3H), 0.95 (d, J ) 6.9 Hz, 3H), 0.81 (d,
J ) 3.4 Hz, 3H). Anal. (C28H41N3O2) C, H, N.
1
CHCl3 to afford 22 as a yellow-white foam. H NMR (CDCl3):
δ 7.13 (t, J ) 7.9 Hz, 1H), 6.76 (d, J ) 7.9 Hz, 1H), 6.69 (s,
1H), 6.64 (dd, J ) 7.9 Hz, 1H), 4.25 (br s, 3H), 2.89 (d, J ) 5.1
Hz, 1H), 2.79 (dd, J ) 11.2, 2.5 Hz, 1H), 2.72-2.75 (m, 1H),
2.42 (d, J ) 11.2 Hz, 1H), 2.36 (dd, J ) 12.4, 2.9 Hz, 1H), 2.22
(d, J ) 8.4 Hz, 2H), 1.98 (d, J ) 6.5 Hz, 1H), 1.61-1.72 (m,
1H), 1.56 (d, J ) 9.7 Hz, 1H), 1.28 (s, 3H), 0.96 (d, 6.9 Hz,
3H), 0.93 (d, J ) 6.9 Hz, 3H), 0.79 (d, J ) 6.9 Hz, 3H). The
final product (17) was obtained according to the general
procedures starting from 22 and Boc-D-7-hydroxy-1,2,3,4-
tetrahydroisoquinoline-3-carboxylic acid. The product was
purified by silica gel preparative plate chromatography using
60% (CHCl3/MeOH/NH4OH, 80:18:2) in CHCl3 to give 17 as a
white solid. 1H NMR (CD3OD): δ 7.09 (t, J ) 7.9 Hz, 1H), 6.92
(d, J ) 8.3 Hz, 1H), 6.77 (s, 1H), 6.73 (d, J ) 2.3 Hz, 1H),
6.56-6.61 (m, 2H), 6.49 (d, J ) 2.3 Hz, 1H), 3.96-4.03 (m,
1H), 3.92 (d, J ) 6.5 Hz, 2H), 3.56 (dd, J ) 10.0, 4.9 Hz, 1H),
2.93 (dd, J ) 15.7, 4.9 Hz, 1H), 2.83 (d, J ) 9.9 Hz, 2H), 2.52-
2.66 (m, 2H), 2.18-2.48 (m, 5H), 1.86-1.97 (m, 2H), 1.53 (d,
J ) 12.5 Hz, 1H), 1.27 (s, 3H), 0.92 (t, J ) 6.6 Hz, 6H), 0.76
(d, J ) 7.0 Hz, 3H). Anal. (C28H39N3O3) C, H, N.
(3S)-7-Hydr oxy-N-((1S)-1-{[(3R,4R)-4-(3-h ydr oxyph en yl)-
3,4-d im et h yl-1-p ip er id in yl]m et h yl}-2-m et h ylp r op yl)-2-
m eth yl-1,2,3,4-tetr ah ydr o-3-isoqu in olin ecar boxam ide (14).
Formalin (0.02 mL, 0.215 mmol) was added to a stirring
solution of (3R)-7-hydroxy-N-((1S)-1-{[(3R,4R)-4-(3-hydroxy-
phenyl)-3,4-dimethyl-1-piperidinyl]methyl}-2-methylpropyl)-
1,2,3,4-tetrahydro-3-isoquinolinecarboxamide (3, 100 mg, 0.215
mmol) dissolved in 5 mL of dry DCE.13 To this mixture was
added Na(OAc)3BH (205 mg, 0.97 mmol). The reaction mixture
was stirred at room temperature for 1.5 h, and then the
reaction was quenched by the addition of saturated NaHCO3
until the bubbling subsided. The reaction mixture was ex-
tracted three times with a solution of 3:1 CH2Cl2/THF and the
residue was purified using silica gel preparative plate chro-
matography in 60% (CHCl3/MeOH/NH4OH, 80:18:2) in CHCl3
1
to give pure 14 as a white solid. H NMR (CD3OD): δ 7.09 (t,
J ) 3.8 Hz, 1H), 6.9 (d, J ) 4.5 Hz, 1H), 6.76 (s, 1H), 6.73 (d,
J ) 1.5 Hz, 1H), 6.57 (dd, J ) 9, 5.3 Hz, 2H), 6.51 (s, 1H),
3.98-3.83 (m, 3H), 3.80 (s, 2H), 3.47 (d, J ) 16 Hz, 1H), 3.31
(s, 1H), 3.13-2.44 (m, 11H), 2.37 (t, J ) 18 Hz, 1H), 1.27 (s,
3H), 0.90 (t, J ) 3 Hz, 6H), 0.71 (d, J ) 3 Hz, 3H). Anal.
(C29H41N3O3) C, H, N.
P h a r m a cologica l Meth od s. The biological methods fol-
lowed published procedures.14,29 The sources of reagents are
also descrbied in these papers. For [35S]GTPγS binding assays,
guinea pig caudate nuclei (from 10 brains) were homogenized
in 30 mL of ice-cold buffer containing 50 mM Tris, pH 7.4, 4
µg/mL leupeptin, 10 µg/mL bestatin, 100 µg/mL bacitracin, and
2 µg/mL chymostatin. The homogenate was centrifuged at
30000g and 4 °C for 10 min and then was washed by repeated
centrifugation and resuspension in buffer two times. After the
final wash, the pellets were resuspended in 50 mL of homog-
enization buffer. The suspension was divided into 1 mL ali-
quots and centrifuged. Pellets were stored at -80 °C until use.
(3R)-2-Acet yl-7-h yd r oxy-N-((1S)-1-{[(3R,4R)-4-(3-h y-
d r oxyp h e n yl)-3,4-d im e t h yl-1-p ip e r id in yl]m e t h yl}-2-
m eth ylp r op yl)-1,2,3,4-tetr a h yd r o-3-isoqu in olin eca r box-
a m id e (15). 15 was prepared according to the general proce-
dures starting from (3R)-7-hydroxy-N-((1S)-1-{[(3R,4R)-4-(3-
hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl}-2-methyl-
propyl)-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide (3)13 and
acetic acid. The crude product was purified using silica gel
preparative plate chromatography in 45% (CHCl3/MeOH/
[
35S]GTP γS Bin d in g Assa y. The [35S]GTPγS binding assay
proceeded as described elsewhere.14,29 Guinea pig caudate
membranes (10-20 µg protein in 300 µL of 50 mM Tris-HCl,
pH 7.4, with 1.67 mM DTT and 0.15% BSA) were added to
either polystyrene 96-well plates or 12 mm × 75 mm polysty-
rene test tubes, filled with 200 µL of a reaction buffer
containing 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 10 mM
MgCl2, 1 mM EDTA, 100 µM GDP, 0.1% BSA, 0.05-0.01 nM
[35S]GTPγS, and varying concentrations of drugs. The reaction
mixture was incubated for 3 h at 25 °C (equilibrium). The
reaction was terminated by the addition of 0.5 mL of ice-cold
Tris-HCl, pH 7.4 (4 °C), followed by rapid vacuum filtration
through Whatman GF/B filters previously soaked in ice-cold
Tris-HCl, pH 7.4 (4 °C). The filters were washed twice with
0.5 mL of ice-cold distilled H2O (4 °C). Bound radioactivity was
counted at an efficiency of 98% by liquid scintillation spec-
troscopy. Nonspecific binding was determined in the presence
of 10 µM GTPγS.
1
NH4OH, 80:18:2) in CHCl3. H NMR (CD3OD): δ 7.68 (t, J )
1.5 Hz, 1H), 7.10 (d, J ) 3 Hz, 1H), 6.99 (d, J ) 3 Hz, 2H),
6.69 (dd, J ) 13.5, 6 Hz, 2H), 6.61 (d, J ) 1.5 Hz, 1H), 4.73
(m, 1H), 4.63 (s, 2H), 3.36-2.56 (m, 8H), 2.46 (t, J ) 9 Hz,
1H), 2.31-1.49 (m, 9H), 1.29 (s, 3H), 0.73 (t, J ) 2.3 Hz, 6H),
0.68 (d, J ) 6 Hz, 3H). Anal. (C30H41N3O4) C, H, N.
(3R )-2-(N ,N -D im e t h y lg ly c y l)-7-h y d r o x y -N -((1S )-1-
{[(3R,4R)-4-(3-h ydr oxyph en yl)-3,4-dim eth yl-1-piper idin yl]-
m eth yl}-2-m eth ylp r op yl)-1,2,3,4-tetr a h yd r o-3-isoqu in o-
lin eca r boxa m id e (16). 16 was prepared according to the
general procedures starting from (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-isoquinolinecar-
boxamide (3) and N,N-dimethylglycine. The crude product was
purified by flash chromatography using 50% (CHCl3/MeOH/
NH4OH, 80:18:2) in CHCl3 to afford 16 as a white foam. 7.09
(t, J ) 7.91 Hz, 1H), 6.96 (d, J ) 8.29 Hz, 1H), 6.72 (m, 2H),
6.63 (m, 1H), 6.57 (m, 2H), 4.83-4.33 (m, 4H), 3.82 (m, 1H),
3.37 (m, 1H), 3.28 (m, 1H), 3.14 (m, 1H), 3.00 (m, 1H), 2.74-
2.17 (m, 12H), 1.91 (m, 1H), 1.69 (m, 1H), 1.51 (m, 1H), 1.24
(m, 3H), 0.78 (d, J ) 6.8 Hz, 3H), 0.69 (m, 6H). Anal.
(C32H46N4O4) C, H, N.
Op ioid Bin d in g Assa ys. Opioid binding assays proceeded
according to published procedures.14,30 The µ receptors were
labeled with [3H]DAMGO. Rat membranes for µ and δ receptor
binding assays were prepared each day using a partially
thawed frozen rat brain that was homogenized with a Polytron
in 10 mL/brain of ice-cold 10 mM Tris-HCl, pH 7.0. Membranes
were then centrifuged twice at 30000g for 10 min and
resuspended with ice-cold buffer following each centrifugation.
After the second centrifugation, the membranes were resus-
pended in 50 mM Tris-HCl, pH 7.4 (60 mL/brain), at 25 °C.
Incubations proceeded for 2 h at 25 °C in 50 mM Tris-HCl,
(3R)-7-Hydr oxy-N-((1S)-1-{[(3S,4S)-4-(3-h ydr oxyph en yl)-
3,4-d im e t h yl-1-p ip e r id in yl]m e t h yl}-2-m e t h ylp r op yl)-
1,2,3,4-t et r a h yd r o-3-isoq u in olin eca r b oxa m id e (3S,4S)-
Dim eth yl-4-(3-h ydr oxyph en yl)piper idin e (17). The (3S,4S)-

3136 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14
Thomas et al.
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pH 7.4, along with a protease inhibitor cocktail (PIC). The
nonspecific binding was determined using 20 µM of levallor-
phan. The δ binding sites were labeled using [3H]DADLE (2
nM) and rat brain membranes. Rat membranes were prepared
each day as described above. After the second centrifugation,
the membranes were resuspended in 50 mM Tris-HCl, pH 7.4
(50 mL/brain), at 25 °C. Incubations proceeded for 2 h at 25
°C in 50 mM Tris-HCl, pH 7.4, containing 100 mM choline
chloride, 3 mM MnCl2, 100 nM DAMGO to block binding to µ
sites, and PIC. Nonspecific binding was determined using 20
µM levallorphan. The κ binding sites were labeled using [3H]-
U69,593 (2 nM). Guinea pig brain membranes were prepared
each day using partially thawed guinea pig brain, which was
homogenized with a Polytron in 15 mL/brain of ice-cold 10
mM Tris-HCl, pH 7.0. The membranes were then centrifuged
twice at 30000g for 10 min and resuspended with ice-cold
buffer following each centrifugation. After the second centrifu-
gation, the membranes were resuspended in 50 mM Tris-HCl,
pH 7.4 (85 mL/brain), at 25 °C. Incubations proceeded for 2 h
at 25 °C in 50 mM Tris-HCl, pH 7.4, containing 1 µg/mL of
captopril and PIC. Nonspecific binding was determined using
1 µM U69,593. Each radioligand was displaced by 8-10
concentrations of test drug. Compounds were prepared as 1
mM solution with 10 mM Tris buffer (pH 7.4) containing 10%
DMSO before drug dilution. All drug dilutions were done in
10 mM Tris-HCl, pH 7.4, containing 1 mg/mL bovine serum
albumin. All washes were done with ice-cold 10 mM Tris-HCl,
pH 7.4. The IC50 and slope factor (N) were obtained by using
the program MLAB-PC (Civilized Software, Bethesda, MD).
Ki values were calculated according to the equation Ki ) IC50
(1 + [L]/Kd).
Da ta An a lysis. The data of the two separate experiments
(opioid binding assays) or three experiments ([35S]GTPγS
assay) were pooled and fit, using the nonlinear least-squares
curve-fitting language MLAB-PC (Civilized Software).
/
Ack n ow led gm en t. This research was supported by
the National Institute on Drug Abuse, Grant DA 09045.
We thank NIDA (OTDP, Division of Treatment Re-
search and Development) for obtaining the human
cloned functional assay data provided herein.
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