Transformation of a κ-opioid receptor antagonist to a κ-agonist by transfer of a guanidinium group from the 5′- to 6′-position of naltrindole

Article abstract of DOI:10.1021/jm010095v

The importance of the indole scaffold of GNTI 3 in directing its address (5′-guanidinium group) to associate with the Glu297 residue of the κ-opioid receptor was investigated by the synthesis and biological evaluation of its 4′- (4a), 6′- (4b), and 7′- (4

Full text of DOI:10.1021/jm010095v

J . Med. Chem. 2001, 44, 2073-2079
2073
Articles
Tr a n sfor m a tion of a K-Op ioid Recep tor An ta gon ist to a K-Agon ist by Tr a n sfer of
a Gu a n id in iu m Gr ou p fr om th e 5′- to 6′-P osition of Na ltr in d ole
Shiv Kumar Sharma, Robert M. J ones, Thomas G. Metzger, David M. Ferguson, and Philip S. Portoghese*
Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota 55455
Received March 3, 2001
The importance of the indole scaffold of GNTI 3 in directing its address (5′-guanidinium group)
to associate with the Glu297 residue of the κ-opioid receptor was investigated by the synthesis
and biological evaluation of its 4′- (4a ), 6′- (4b), and 7′- (4c) regioisomers. The finding that
only the 5′-regioisomer (GNTI) possessed potent κ-opioid antagonist activity and high affinity
at κ-receptors illustrates the importance of the 5′-position in orienting the guanidinium group
to the proper recognition locus (Glu 297) for potent κ-antagonist activity. The discovery that
the 6′-regioisomer of GNTI was a potent κ-agonist, together with the results of site-directed
mutagenesis studies that are consistent with association between the 6′-guanidinium group
and Glu297, suggest that the transition from an inactive to an active state of the κ-receptor
involves a conformational change of TM6. We propose that association of the 6′-guanidinium
group of 4b with Glu297 promotes axial rotational motion of transmembrane helix VI which
leads to receptor activation via a conformational change of inner loop 3.
In tr od u ction
nonconserved acidic residue (Glu297)6 located at the top
of the transmembrane spanning helix-VI (TM6) of the
κ-receptor.6 Moreover, it has also been shown that
incorporation of a glutamate residue into the corre-
sponding position of the µ-receptor leads to high affinity
binding of norBNI to the mutant µ-receptor, which
illustrates the importance of this glutamate residue.7
In light of the structural requirements of norBNI, 5′-
guanidinonaltrindole 3 (GNTI) was synthesized as it has
been reported to be a highly potent and selective κ-opioid
receptor antagonist.7a-c The design of GNTI utilized the
indole moiety of the δ-selective antagonist, naltrindole8
(NTI, 2), as a rigid scaffold9 for the attachment of a
The κ-opioid receptor is one of the three major opioid
receptors that are found in the central nervous system
and in the periphery.1 The precise roles of κ-receptors
have not yet been established, but it appears that
κ-selective endogenous opioid peptides (e.g., dynorphin
A) function both as neuro- and immunomodulators. A
large number of nonpeptide κ-agonists have been de-
veloped as promising potential analgesics, and some of
them have found wide use as pharmacological tools in
opioid research.2 In particular, norbinaltorphimine (norB-
NI 1), a bivalent ligand, which contains two naltrexone-
derived pharmacophores, is the only nonpeptide ligand
that is highly selective and widely used as a κ-opioid
receptor antagonist.3 The structure-activity relation-
ship of norBNI 1 has been extensively investigated4 and
it has been recently demonstrated that the basic group
(N17′) in the second pharmacophore of norBNI acts as
an “address”5 to confer selectivity by interacting with a
guanidinium group at the 5′-position to permit interac-
tion with Glu297 of the κ-opioid receptor. This position
orients the guanidinium group to a location similar to
that of the N17′ basic group of norBNI. It has been
established that the cationic 5′-guanidinium group
functions as an “address” to confer selectivity through
interaction with the anionic Glu297 residue of the
κ-receptor.7c
* To whom correspondence should be addressed: Prof. Philip S.
Portoghese, University of Minnesota, 308 Harvard Street SE, 8-111
WDH, Minneapolis, MN 55455. Phone: 612-624-9174. Fax: 612-626-
6891. E-mail: porto001@maroon.tc.umn.edu.
In the present study, we have investigated the
interaction of the 4′-, 6′-, and 7′-regioisomers (4a -c) of
10.1021/jm010095v CCC: $20.00 © 2001 American Chemical Society
Published on Web 05/24/2001

2074 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13
Sharma et al.
Sch em e 1. Synthetic Route to Guanidinonaltrindole Regioisomersa
a
Reagents: (i) (BocNH)(BocNd)CSMe, HgCl2, CH2Cl2, NEt3, rt, 18-24 h; (ii) TFA, CH2Cl2, N2, rt, 36 h; (iii) (CBZ-NH)(CBZ-Nd)CSMe,
HgCl2, CH2Cl2, NEt3, rt, 2 h or (CBZ-NH)(CBZ-Nd)C-NHSO2CF3, CH2Cl2, NEt3, rt, 4 days; (iv) Pd/C, H2, MeOH-dil. HCl, 65 psi, 4 h.
GNTI with opioid receptors in order to evaluate the
relationship between the position of the guanidinium
group and its interaction with Glu297 of the κ-receptor.
The data reveal that movement of the guanidinium
group from the 5′- to the 6′-position changes the activity
from a κ-antagonist 3 to a κ-agonist 4b, and that Glu297
of the κ-receptor may be involved in this transition. The
significance of these results is discussed in terms of the
conformational change that may occur in receptor
activation.
reported7c tedious chromatographic separation has been
developed. 5′-Amino-NTI 5b was allowed to react with
3.3 equiv of di-N′,N′′-carboxybenzyloxy-N′′′-trifluoro-
methanesulfonylguanidine14 in the presence of triethyl-
amine in CH2Cl2 for 4 days to give bis(benzyloxycar-
bonyl)- protected intermediate 7. This intermediate 7
was also prepared by an alternate route that involved
the reaction of 5′-amino-NTI 5b with 1,3-bis(benzyloxy-
carbonyl)-2-methyl-2-thiopseudourea using a HgCl2-
assisted coupling reaction.13 Deprotection of inter-
mediate 7 was carried out by catalytic hydrogenation
(10% Pd/C) to afford 3‚HCl. The overall yield from 5b
by either of the modified routes was 65-70%.
Ch em istr y
The synthetic route to the target compounds 4a -c is
outlined in Scheme 1. The 5′- and 7′-nitronaltrindole
intermediates were prepared as reported previously.8,10
Fisher-indole cyclization11 of naltrexone with 3-nitro-
phenylhydrazine yielded a mixture of regioisomers con-
sisting of the 4′-nitro (minor) and 6′-nitro (major) deriv-
atives of naltrindole. Separation of the regioisomers was
accomplished by repeated column chromatography.
Raney nickel-catalyzed reduction12 of the nitro func-
tion in all of the regioisomers using hydrazine hy-
drate in ethanol afforded the corresponding amino deriv-
atives 5a -d in 70-86% yields.10 Mercury(II) chloride-
assisted coupling13 of the regioisomers 5a ,c,d with
1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudo-
urea in dry CH2Cl2 yielded the Boc-protected guanidi-
nonaltrindole 6a -c (major product, 78-81%) and a
small amount (2.6-3.8%) of the esterified side product
6d -f. Trifluoroacetic acid (TFA) deprotection of the Boc
groups in intermediates 6a -c afforded regioisomers of
GNTI 4a -c as bis-TFA salts in good yield after reverse-
phase HPLC.
Biologica l Resu lts
Sm ooth Mu scle P r ep a r a tion s. The in vitro phar-
macological data for GNTI 3 and its regioisomers 4a -
c, as well as the standard κ-antagonist, norBNI 1, are
listed in Table 1. All of the compounds were tested on
the electrically stimulated guinea pig ileal longitudinal
muscle (GPI)15 and the mouse vas deferens (MVD)16
preparations for their agonist and antagonist activities
as previously described.17 The compounds were incu-
bated with the aforementioned preparations 15 min
prior to testing with standard selective agonists. Mor-
phine (M), (-)-ethylketazocine (EK), and [D-Ala2,D-Leu5]-
enkephalin (DADLE) were employed as µ-, κ-, and
δ-selective agonists, respectively. Morphine and EK
were used in the GPI, and DADLE was employed in the
MVD studies. Concentration-response curves were
obtained in the absence (control) and presence of the
antagonist in the same preparation. Antagonist potency
is expressed as an IC50 ratio (IC50 of the agonist in the
presence of antagonist divided by the control IC50 of the
A new procedure to synthesize GNTI 3 as its dihy-
drochloride salt in multigram quantities without the

κ-Antagonist to κ-Agonist Transformation
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13 2075
Ta ble 1. Opioid Antagonist Potency of GNTI Regioisomers in Smooth Muscle Preparations
EK (κ)a,c
IC50 ratiod
DADLE (δ)b,c
M (µ)a,c
IC50 ratiod
regioisomers
Ke (nM)
Ke (nM)
IC50 ratiod
Ke (nM)
1 (norBNI)
3 (GNTI)
4a
4b
4c
0.41
0.16
80
g
100
49.8 ( 7.8 (10)e
139 ( 33 (11)e
2.3 (2)
10.6
115
263
f
10.4 ( 2.9 (3)
1.9 ( 0.5 (3)
1.4 (2)
0.2 (2)
105 ( 30 (4)
12.5
30.3
f
g
238
2.6 ( 0.6 (12)e
4.3 ( 0.7 (5)
0.34 (2)
5′
4′
6′
7′
g
g
2.0 (2)
0.96
1.4 ( 0.9 (3)
a
b
Determined in the guinea pig ileum (GPI) preparation. Determined in the mouse vas deferens (MVD) preparation. c All antagonists
d
were tested at a concentration of 100 nM unless otherwise noted. Values are expressed mean ( standard error of the mean with the
number of determinations in parentheses. The agonists employed were ethylketazocine (EK), morphine (M), and [D-Ala2-D-leu5]enkephalin
(DADLE). e Tested at 20 nM. f Not calculated because IC50 ratio was <1. Not determined due to full agonist activity.
g
Ta ble 2. Binding Affinity of GNTI Regioisomers to Cloned
Opioid Receptors
Ki (nM)a
regioisomer
κ
δ
µ
3 (GNTI)
5′
4′
6′
7′
0.14 ( 0.03 24.8 ( 11.3 99.7 ( 8.7
4a
4b
4c
>1000
1.15 ( 0.39 20.3 ( 6.7
69.1 ( 25
>1000
>1000
81.8 ( 20.7
2.75 ( 0.48 181 ( 20
a
The Ki values were determined in competition binding using
[3H]diprenorphine in transiently expressed rat HEK-293 cells and
analyzed by whole cell binding. Ki values were derived from the
Cheng-Prusoff equation, Ki ) IC50/(1 + [L]/Kd), where IC50 is the
concentration of competing ligand producing 50% inhibition of the
specific binding of radioligand [L] and having dissociation constant
Kd. The values are the mean ( SE of three experiments in
duplicate.
Recep tor Bin d in g. Receptor binding was deter-
mined in triplicate using [3H]diprenorphine on HEK-
293 cells that were transiently transfected with plas-
mids encoding rat κ-, rat µ-, or mouse δ-opioid receptors
(Table 2). GNTI 3 possessed greater than 2 orders of
magnitude higher affinity for the κ-receptor relative to
µ- and δ-receptors. Consistent with the smooth muscle
data, substitution at the 4′-position (4a ) eliminated
binding to all three receptor types. Like GNTI, the 6′-
regioisomer 4b was κ-selective but possessed one-tenth
the affinity of GNTI for κ-receptors. The results are
inconsistent with the κ-agonist selectivity of 4b observed
in the GPI. The δ-selectivity of regioisomer 4c also
corresponded well with the functional data. Binding to
µ-receptors was uniformly low for all regioisomers.
In view of the finding6,7a that Glu297 at the top of
TM6 is important for high κ-antagonist affinity, the
binding of the 5′-, 6′-, and 7′- regioisomers (3, 4b, 4c) to
mutant κ-receptor was determined (Table 3). It is
noteworthy that the 5′- and 6′-regioisomers displayed
a significant decrease in affinity for the Glu297Ala
mutant while the binding of the 7′-regioisomer was
virtually unchanged.
F igu r e 1. Concentration-response curves of 4b and mor-
phine in the guinea pig ileal preparation.
agonist in the same preparation) and as a Ke value
derived from the relationship, Ke ) [antagonist]/(IC50
ratio - 1).
The data presented in Table 1 clearly illustrate that
the 5′-regioisomer GNTI 3 is a highly potent and
selective κ-opioid receptor antagonist as reported
previously.7a,b In this regard, GNTI was approximately
4-fold more potent than norBNI 1, with δ/κ and µ/κ
selectivity ratios that are 718 and 188, respectively. The
4′-regioisomer 4a was virtually inactive at all opioid
receptor types. While the 7′-regioisomer 4c was inactive
at µ- and κ-receptors, it was a potent δ-opioid receptor
antagonist. This is inconsistent with the previously
reported8 steric tolerance for δ-antagonist activity when
the 7′-position of naltrindole is substituted. Surprisingly,
the 6′-regioisomer 4b was found to be an agonist with
a 51-fold greater potency relative to that of morphine
(Figure 1) in the GPI assay. The finding that the agonist
effect of 4b was only partially reversed by naltrexone
(500 nM) suggested that it was not a µ-selective agonist.
However, the κ-selective antagonist, norBNI (20 nM),
completely reversed the agonist effect. In the MVD
assay, 4b was inactive as an agonist at 1 µM and did
not antagonize DADLE (IC50 ratio ) 0.2). These results
strongly suggest that 4b is a κ-selective agonist.
Discu ssion
The importance of Glu297, located at the top of
transmembrane helix VI (TM6), for the antagonist

2076 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13
Sharma et al.
Ta ble 3. Effect on Binding to the κ-Opioid Receptor upon
Mutational Exchange of Glu297
Ki (nM)a
regioisomer
wild-type κ
κ (Glu297Ala)
3 (GNTI)
4b
4c
5′
6′
7′
0.21 ( 0.05
1.13 ( 0.56
32.7 ( 5.1
3.93 ( 0.58
12.3 ( 2.5
47.3 ( 6.7
a
The Ki values were determined in competition binding using
[3H]diprenorphine in transiently expressed rat HEK-293 cells and
analyzed by whole cell binding.
activity of norBNI 1 and GNTI 3 has been reported
previously.7a,c The results of the present study on the
GNTI regioisomers 4a -c clearly support the proposal
that the indole moiety functions as a rigid scaffold for
directing the guanidinium group to this nonconserved
glutamate residue (Glu297). In this regard, it is likely
that the 4′-, 6′-, and 7′-regioisomers (4a -c) exhibit
greatly reduced binding relative to the 5′-regioisomer,
GNTI 3, in part as a consequence of the less favorable
alignment of the guanidinium group with the glutamate
carboxylate group (Glu297) of the κ-receptor.
F igu r e 2. Depiction of the interaction of the GNTI 3 (green)
with Glu297 of the κ-opioid receptor. A counterclockwise
rotation of helix 6 (curved arrow) is required to enable the
interaction of the glutamate side chain (red) with the 6′-
guanidinium group (magenta). Transmembrane helix back-
bones are shown as ribbons in yellow.
Although GNTI 3 and its regioisomers bind uniformly
poorly to µ-receptors, this is not the case for δ-receptors,
where 4b and 4c have moderate affinity. These data
are inconsistent with the reported structure-activity
relationship8 of the parent ligand, naltrindole 2, which
is known to tolerate substitution in the order 7′ > 6′ >
5′ . 4′ for δ-receptor antagonist potency. Accordingly,
it is not surprising that the 7′-regioisomer 4c has the
greatest affinity for δ-receptors.
Significantly, the 6′-regioisomer 4b functions as a
potent κ-opioid receptor agonist in the guinea pig ileum
preparation. This represents a remarkable transition
from κ-antagonist (3) to a κ-agonist (4b), simply by
moving the guanidinium group from the 5′- to 6′-
position. The finding that the mutant (Glu297Ala)
κ-receptor exhibited reduced affinity for GNTI 3 and its
6′-regioisomer 4b suggests that the guanidinium group
of each of these ligands interacts with Glu297 (Table
3). Thus, one possible explanation for the antagonist-
agonist transition may involve stabilization of the
receptor via salt bridge formation between the carboxy-
late group of Glu297 and the 5′- or the 6′-guanidinium
group in either the inactive or active state of the
receptor, respectively.
Glu297 (Figure 2). Evidence for a counterclockwise
rotation of TM6 has been reported to be a consequence
of activation of the â2-adrenergic receptor.23-27
Con clu sion s
The critical nature of the position of attachment of
the guanidinium group to the indole moiety in naltrin-
dole 2 illustrates the principle of a rigid scaffold in
directing an “address” to a specific subsite (Glu297) on
the κ-opioid receptor. The remarkable transition from
potent κ-antagonist (3) to potent κ-agonist (4b) upon
moving the guanidinium group from the 5′- to 6′-position
may be due to salt bridge-induced stabilization of the
receptor in an inactive state with the former and an
active state with the latter. If both 3 and 4b interact
with Glu297 on TM6, a possible explanation for this
phenomenon may involve differential axial rotational
motion of TM6 by the 6′-guanidinium group to promote
a conformational change of inner loop 3, which is
involved in G protein coupling and activation.
Exp er im en ta l Section
Gen er a l. Ma t er ia ls. Naltrexone was obtained from
Mallinckrodt & Co. All reactions were carried out under an
inert atmosphere of nitrogen. Triethylamine (NEt3) was dis-
tilled from KOH, while dichloromethane and acetonitrile were
distilled from calcium hydride prior to their use. Dry dimethy-
formamide (DMF) was obtained by storing reagent grade
material over 3 Å sieves for at least 24 h. All other chemicals
were either HPLC or reagent grade and used without further
purification. Thin-layer chromatography (TLC) was performed
on analytical Uniplate silica gel GF plates (250 µm by 2.5 ×
20 cm2), and preparative thin-layer chromatography was
performed on 1.0 or 0.5 mm silica gel plates purchased from
Analtech. Gravity and low pressure column chromatography
were performed over silica gel (200-400 mesh, 60 Å, Aldrich)
as the stationary phase under N2. The reverse phase high
pressure liquid chromatography (HPLC) was performed with
Beckman model 110A pumps, a Beckman Analytical Optical
Unit (fixed wavelength UV), and a Hewlett-Packard HP 3390A
integrating recorder. Purification was performed with a Ranin
Dynamax macro HPLC column (C18, 5 µm, 1 × 28 cm2) or an
Alltech Altima C18 column (10 µm, 250 × 22 mm2). Chro-
matographic elution solvent systems are reported as vol:vol
Interaction with the Glu297 residue on TM6 may
have special relevance to the receptor function, as this
helix is contiguous with an inner loop 3 (IL3) which is
the major domain involved in G protein activation.
Conformational differences in TM6 may therefore be a
key feature that distinguishes an inactive state from
an agonist state of the receptor. Such a conformational
change could involve rotation and/or tilting of TM6,
which could promote a conformational change of IL3.
Conceivably, interaction of 3 or 4b with Glu297 may
therefore lead to different conformational transitions of
TM6 due to the regioisomeric relationship of the 5′- or
6′-guanidinium group.
Our structure-activity results are consistent with
recent studies that implicate movement of TM6 in the
activation of rhodopsin and the â2 adrenergic recep-
tor.18-24 Transfer of the guanidinium group from the 5′-
to the 6′-position could result in a counterclockwise
rotation of the TM6 in order to facilitate interaction with

κ-Antagonist to κ-Agonist Transformation
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13 2077
ratios. Infrared (IR) spectra were recorded on a Perkin-Elmer
PE-281 spectrophotometer or a Nicolet 5DXC FT-IR spectrom-
eter as potassium bromide (KBr) disks. Low resolution (LRMS)
and high resolution (HRMS) mass spectra were obtained on a
Finnigan 4000 or VG-707EHF spectrometer by the Chemistry
Mass Spectrometry Laboratory at the Department of Chem-
istry, University of Minnesota. All 1H and 13C spectra were
recorded on a Varian 300 MHz or a GE 300 MHz spectrometer,
and the chemical shifts are reported as δ values with units of
parts-per-million (ppm). 1H NMR spectra are referenced to
tetramethylsilane (TMS) at 0.00 ppm as an internal standard,
and 13C NMR spectra are referenced to either CDCl3 (77.00
ppm) or DMSO-d6 (40.00 ppm) and are recorded at room
temperature (20 ( 1 °C). All recorded spectra are for the free
base unless otherwise stated. Elemental analyses were per-
formed by the M-H-W Laboratory in Phoenix, AZ, and are
within (0.4% of theoretical values. Melting points were
determined in open capillary tubes on a Thomas-Hoover
melting point apparatus and are uncorrected. All of the tested
regioisomers of GNTI (3, 4a -c) were purified by silica gel flash
chromatography (CH2Cl2-MeOH-NH4OH, 78:20:2), then by
silica gel preparative TLC (CH2Cl2-MeOH-NH4OH, 78:20:
2), and finally by reverse-phase HPLC (CH3CN-H2O, 65:35
containing 0.1% of TFA) to afford as their TFA salts.
Tr a n sien t Tr a n sfection . HEK-293 in DMEM (Gibco, BRL)
supplemented with 10% bovine calf serum (Hyclone) and 1%
penicillin/streptomycin (Gibco, BRL) were maintained at 37
°C and in 5% CO2. Cells were seeded at 16% for 24 h prior to
transfection. Fresh media was added 2 h prior to transfection.
Cells were transfected with plasmid DNA (20 µg/100 mm plate)
of either wild-type or mutant receptor cDNA using the calcium
phosphate precipitation method.28 Media was changed 5 h after
transfection. Transfected cells were harvested 48-72 h after
tranfection for binding studies.
Recep tor Bin d in g Assa ys. Sixty to 72 h after transfection,
HEK cells were washed three times with 25 mM HEPES buffer
(pH 7.4) and were resuspended with 8-12 mL of 25 mM
HEPES/100 mm plate. Saturation binding assays were per-
formed in triplicate. Nonselective binding was determined
using 10 µM naltrexone. Assays were incubated at room
temperature for 90 min in a total binding volume of 0.5 mL
and were terminated by filtration through a Whatman GF/B
filter that had been presoaked in 0.25% poly(ethyleneimine)
immediately prior to filtration. Filters were washed three
times with 4 mL of ice-cold 25 mM HEPES buffer, and
scintillation counting was performed with a Beckman 3801 LS
scintillation counter. Protein concentrations were determined
by the method of Bradford.29 Raw binding data was analyzed
with RADLIG and LIGAND (G. A. McPherson, Biosoft, Cam-
bridge, U.K.). Inhibition constants (Ki) were determined from
IC50 values with the Cheng-Prusoff equation.30
Site-Dir ected Mu ta gen esis. Rat κ-opioid receptor cDNA
was subcloned into pcDNA3 (Invitrogen). Point mutation
Glu297Ala was introduced in the opioid receptor genes by
polymerase chain reaction (QuikChange Site Directed Mu-
tagenesis Kit, Stratogene). Primers were designed to incorpo-
rate an Eco47 III restriction site that was used for screening
of mutant DNA. Mutations were then confirmed by DNA
sequencing.
0.5) to give two products. Major product 6a (410 mg, 79%):
mp 255 °C (dec); 1H NMR (DMSO-d6) δ 11.57 (s, 1H, NH),
11.33 (s, 1H, NH), 10.00 (s, 1H, NH), 8.93 (s, 1H, Ar-OH),
7.17-7.11 (m, 2H, ArH), 7.09-6.97 (m, 1H, ArH), 6.46 (m, 2H,
ArH), 5.46 (s, 1H, 5-H), 4.70 (b, 1H, 14-OH), 3.24-3.02 (m,
1H), 2.84-2.79 (m, 1H), 2.64-2.59 (m, 2H), 2.45-2.38 (m, 1H),
2.36 (m, 2H), 2.27-2.25 (m, 2H), 2.16-2.12 (m, 1H), 1.49 (s,
t
t
9H, Bu), 1.27 (s, 9H, Bu), 0.99 (m, 1H), 0.45 (m, 2H), 0.09
(m, 2H); HRMS (FAB) m/z 672.3386 (M + H)+, C37H45N5O7
requires 671.3397. Minor product 6d (26 mg, 3.8%): mp >280
°C; 1H NMR (DMSO-d6) δ 11.46 (s, 1H, NH), 11.22 (s, 1H, NH),
10.70 (s, 1H, NH), 9.83 (s, 1H, NH), 7.36 (s, 1H, ArH), 7.25 (d,
1H, J ) 8.1 Hz, ArH), 7.16 (d, 1H, J ) 8.1 Hz, ArH), 6.70 (d,
1H, J ) 8.1 Hz, ArH), 6.66 (d, 1H, J ) 8.4 Hz, ArH), 5.61 (s,
1H, 5-H), 4.76 (b, 1H, 14-OH), 3.14 (d, 1H, J ) 18.9 Hz), 2.81-
2.63 (m, 5H), 2.39-2.46 (m, 3H), 2.12 (m, 1H), 1.53 (d, 1H, J
t
t
) 13.2 Hz), 1.47 (s, 9H, Bu), 1.38 (s, 9H, Bu), 1.31 (s, 9H,
tBu), 1.25 (s, 9H, Bu), 0.84 (m, 1H), 0.47 (m, 2H), 0.12 (m,
t
2H); HRMS (FAB) m/z 914.4717 (M + H)+, C48H63N7O11
requires 913.4585.
6′-N′-(N′′,N′′′-Bis(ter t-b u t oxyca r b on yl)gu a n id in o-17-
(cyclop r op ylm et h yl)-6,7-d id eh yd r o-4,5r-ep oxy-3,14-h y-
d r oxyin d olo[2′,3′:6,7]m or p h in ia n (6b). A mixture of 5c (216
mg, 0.5 mmol), HgCl2 (250 mg, 0.83 mmol), 1,3-bis(tert-
butoxycarbonyl)-2-methyl-2-thiopseudourea (200 mg, 0.7 mmol)
in freshly dried CH2Cl2 containing few drops of NEt3 was
allowed to stir for 18 h under a sealed N2 atmosphere at room
temperature. After work up of the reaction according to the
above procedure for 6a , it gave two products which were
separated by column chromatography (CH2Cl2-MeOH-NH4-
OH, 94.5:5.0:0.5); Major product 6b (260 mg, 78%): mp 270
1
°C (dec); H NMR (DMSO-d6): δ 11.43 (s, 1H, NH), 11.20 (s,
1H, NH), 10.00 (s, 1H, NH), 8.88 (s, 1H, Ar-OH), 7.69 (s, 1H,
ArH), 7.25 (d, 1H, J ) 8.7 Hz, ArH), 6.90 (d, 1H, J ) 8.7 Hz,
ArH), 6.47 (d, 1H, J ) 8.1 Hz, ArH), 6.42 (d, 1H, J ) 8.1 Hz,
ArH), 5.44 (s, 1H, 5-H), 4.70 (b, 1H, 14-OH), 3.24-3.07 (m,
2H), 3.02 (m, 1H), 2.60 (m, 2H), 2.48-2.08 (m, 5H), 1.53 (m,
t
t
1H), 1.48 (s, 9H, Bu), 1.38 (s, 9H, Bu), 0.80 (m, 1H), 0.45 (m,
2H), 0.12 (m, 2H);13C NMR (DMSO-d6): δ 153.35, 152.81,
143.56, 140.31, 136.98, 131.64, 131.44, 130.87, 124.73, 124.24,
118.73, 117.37, 114.99, 110.56, 106.23, 84.34, 72.66, 62.11,
59.11, 47.74, 43.80, 39.15, 31.79, 29.18, 28.40, 23.14, 9.71, 4.35,
3.97. HRMS (FAB) m/z 672.3405 (M + H)+, C37H45N5O7
requires 671.3397. Minor product 6e (12 mg, 2.6%): mp >280
°C; 1H NMR (DMSO-d6): δ 11.44 (s, 1H, NH), 11.28 (s, 1H,
NH), 10.70 (s, 1H, NH), 10.02 (s, 1H, NH), 7.37 (s, 1H, ArH),
7.27 (d, 1H, J ) 7.8 Hz, ArH), 6.89 (d, 1H, J ) 8.1 Hz, ArH),
6.72 (d, 1H, J ) 8.4 Hz, ArH), 6.68 (d, 1H, J ) 8.1 Hz, ArH),
5.60 (s, 1H, 5-H), 4.76 (b, 1H, 14-OH), 3.12 (d, 1H, J ) 18.1
Hz), 2.78-2.56 (m, 3H), 2.48-2.36 (m, 5H), 2.16-2.03 (m, 1H),
t
t
1.52 (m, 1H), 1.49 (s, 9H, Bu), 1.36 (s, 18H, Bu), 1.26 (s, 9H,
tBu), 0.86 (m, 1H), 0.47 (m, 2H), 0.12 (m, 2H); HRMS (FAB)
m/z 914.4670 (M + H)+, C48H63N7O11 requires 913.4585.
7′-N′-(N′′,N′′′-Bis(ter t-b u t oxyca r b on yl)gu a n id in o-17-
(cyclop r op ylm et h yl)-6,7-d id eh yd r o-4,5r-ep oxy-3,14-h y-
d r oxyin d olo[2′,3′:6,7]m or p h in ia n (6c). A mixture of 5d (420
mg, 1 mmol), HgCl2 (430 mg, 1.44 mmol), 1,3-bis(tert-butoxy-
carbonyl)-2-methyl-2-thiopseudourea (380 mg, 1.3 mmol), and
a few drops of NEt3 in dried CH2Cl2 was allowed to stir for 18
h at room temperature under a sealed N2 atmosphere and
worked up according to the procedure for 6a . On subjecting
the mixture to column chromatography (CH2Cl2-MeOH-NH4-
OH 94.5:5.0:0.5), it gave major product 6c (530 mg, 81%): mp
4′-N′-(N′′,N′′′-Bis(ter t-b u t oxyca r b on yl)gu a n id in o-17-
(cyclop r op ylm et h yl)-6,7-d id eh yd r o-4,5r-ep oxy-3,14-h y-
d r oxyin d olo[2′,3′:6,7]m or p h in ia n (6a ). In a 250 mL flask
was stirred a mixture of 5a (325 mg, 0.75 mmol) and HgCl2
(400 mg, 1.33 mmol) in HPLC grade CH2Cl2 (50 mL) for few
minutes, and 1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thio-
pseudourea (320 mg, 1.1 mmol) was added, followed by few
drops of NEt3. The mixture was allowed to stir at room
temperature under a sealed N2 atmosphere. The progress of
the reaction was monitored by TLC. After completion the
reaction (24 h), mixture was filtered through Celite under
vacuum to remove mercuric sulfide, and the residue was
washed throughly with methanol. The combined filtrate was
concentrated to give a solid product which was subjected to
column chromatography (CH2Cl2-MeOH-NH4OH, 94.5:5.0:
1
230 °C (dec); H NMR (DMSO-d6) δ 11.64 (s, 1H, NH), 11.28
(s, 1H, NH), 9.67 (s, 1H, NH), 8.89 (s, 1H, Ar-OH), 7.26 (d,
1H, J ) 7.5 Hz, ArH), 6.97-6.89 (m, 2H, ArH), 6.50-6.43 (m,
2H, ArH), 5.70 (s, 1H, 5-H), 4.70 (b, 1H, 14-OH), 3.27 (m, 2H),
3.06 (d, 1H, J ) 18.3 Hz), 2.73-2.65 (m, 2H), 2.44-2.03 (m,
t
t
5H), 1.51 (s, 9H, Bu), 1.26 (s, 9H, Bu), 0.86 (m, 1H), 0.46 (m,
2H), 0.11 (m, 2H); 13C NMR (DMSO-d6) δ 155.93, 143.54,
140.48, 133.38, 131.28, 130.74, 128.24, 124.52, 121.09, 118.95,
118.82, 117.98, 117.36, 111.11, 84.25, 72.72, 62.11, 59.02,
47.77, 43.89, 31.80, 29.29, 28.69, 28.31, 23.21, 9.52, 4.42, 3.95;
HRMS (FAB) m/z 672.3406 (M + H)+, C37H45N5O7 requires

2078 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13
Sharma et al.
671.3397. Minor product 6f (34 mg, 3.7%): mp 165 °C (dec);
1H NMR (DMSO-d6) δ 11.63 (s, 1H, NH), 11.37 (s, 1H, NH),
10.70 (s, 1H, NH), 9.66 (s, 1H, NH), 7.26 (d, 1H, J ) 8.1 Hz,
ArH), 6.98 (d, 1H, J ) 6.60 Hz, ArH), 6.91 (d, 1H, J ) 7.5 Hz,
ArH), 6.73 (d, 1H, J ) 8.1 Hz, ArH), 6.66 (d, 1H, J ) 7.2 Hz,
ArH), 5.61 (s, 1H, 5-H), 4.70 (b, 1H, 14-OH), 3.44 (m, 1H), 3.39
(m, 1H), 3.14 (d, 1H, J ) 18.9 Hz), 2.85-2.68 (m, 3H), 2.45-
2.29 (m, 4H), 2.18-2.13 (m, 1H), 1.51 (s, 9H, tBu), 1.37 (s, 9H,
tBu), 1.32 (s, 9H, tBu), 1.30 (s, 9H, tBu), 0.88 (m, 1H), 0.49 (m,
2H), 0.12 (m, 2H); HRMS (FAB) m/z 914.4630 (M + H)+,
C48H63N7O11 requires 913.4585.
4′-Gu a n id in o-17-(cyclop r op ylm et h yl)-6,7-d id eh yd r o-
4,5r-ep oxy-3,14-h yd r oxyin d olo-[2′,3′:6,7]m or p h in ia n (4a ).
Compound 6a (400 mg, 0.6 mmol) was dissolved in HPLC
grade CH2Cl2, and the contents were cooled in an ice bath.
TFA (3.0 mL) was added in divided portions over a period of
10 min, and the flask was sealed under N2 atmosphere and
allowed to stir at room temperature. The reaction was moni-
tored by TLC, and after 36 h, CH2Cl2 and TFA were removed
with a stream of N2, leaving a residue which was subjected to
column chromatography (CH2Cl2-MeOH-NH4OH, 78:20:2) to
afford 4a along with CF3CO2-NH4+. Further purification was
accomplished by preparative TLC (CH2Cl2-MeOH-NH4OH,
78:20:2) to give 4a (211 mg, 75%); IR KBr disk υ (cm-1): 3400-
3150 (br), 1675 (s), 1507, 1463, 1432, 1332, 1202, 1134; 1H
NMR (DMSO-d6): δ 11.64 (s, 1H, NH), 9.84 (s, 1H, NH), 9.32
(s, 1H, NH), 8.92 (s, 1H, Ar-OH), 7.33-7.06 (m, 4H, ArH and
NH2), 6.78 (d, 1H, J ) 7.20 Hz, ArH), 6.59 (d, 1H, J ) 8.1 Hz,
ArH), 6.52 (d, 1H, J ) 8.1 Hz, ArH), 5.64 (s, 1H, 5-H), 4.11 (b,
1H, 14-OH), 3.44 (m, 1H), 3.12-2.99 (m, 4H), 2.87 (m, 1H),
2.70-2.55 (m, 4H), 1.76 (d, 1H, J ) 11.7 Hz), 1.07 (m, 1H),
0.64-0.56 (m, 2H), 0.37-0.44 (m, 2H); HRMS (FAB) m/z
472.2357 (M + H)+, C27H29N5O3 requires 471.2270). Anal.
(C27H29N5O3‚2TFA‚2H2O) C, H, N.
6′-Gu a n id in o-17-(cyclop r op ylm et h yl)-6,7-d id eh yd r o-
4,5r-epoxy-3,14-h yd r oxyin d olo-[2′,3′:6,7]m or p h in ia n (4b).
Compound 6b (500 mg, 0.75 mmol) was dissolved in a mixture
of TFA (3.0 mL) and dried CH2Cl2 (28 mL) and allowed to stir
under N2 atmosphere at room temperature for 36 h. The
reaction was worked up according to the procedure for 4a and
purified by preparative TLC to give 4b (260 mg, 74%) as a
free base: IR KBr disk υ (cm-1) 3450-3150 (br), 1683 (s), 1506,
1463, 1433, 1330, 1202, 1132; 1H NMR (DMSO-d6) δ 11.50 (s,
1H, NH), 9.96 (s, 1H, NH), 9.29 (s, 1H, NH), 8.95 (s, 1H, Ar-
OH), 7.36-7.09 (m, 3H, ArH and NH2), 6.77 (d, 1H, J ) 8.10
Hz, ArH), 6.59-6.52 (m, 2H, ArH), 6.39 (s, 1H), 5.67 (s, 1H,
5-H), 4.05 (b, 1H, 14-OH), 3.43-3.23 (m, 3H), 3.18-3.06 (m,
2H), 2.96-2.91 (m, 2H), 2.68-2.57 (m, 2H), 2.50 (m, 1H), 1.78
(d, 1H, J ) 11.7 Hz), 1.05 (m, 1H), 0.68 (m, 1H), 0.58 (m, 1H),
0.40 (m, 2H); HRMS (FAB) m/z 472.2356 (M + H)+, C27H29N5O3
requires 471.2270. Anal. (C27H29N5O3‚2TFA‚2H2O) C, H, N.
the mixture was diluted with CH2Cl2 (100 mL) and washed
with 2 M of sodium bisulfate, saturated NaHCO3, and brine.
After drying with MgSO4 and filtering, the solvent was
removed under reduced pressure, and the crude product was
subjected to flash column chromatography on silica gel (CH2-
Cl2-MeOH-NH4OH, 94.5:5.0:0.5) to give 1.41 g (76%) of 7.
Meth od B. 5′-Amino-NTI 5b (2.68 g, 6.24 mmol) was
dissolved in freshly distilled dry CH2Cl2, and 3.0 mL of
triethylamine was added. To this mixture was added HgCl2
(2.70 g, 10 mmol), followed by l,3-bis(benzyloxycarbonyl)-2-
methyl-2-pseudothiourea (2.64 g, 7.33 mmol) in portion. The
flask was sealed under N2 atmosphere and allowed to stir at
room temperature, and the progress of the reaction was
monitored by TLC (CH2Cl2-MeOH-NH4OH, 89:10:1). After
completion of the reaction (2 h), it was filtered through Celite
under vacuum to remove HgSO4, and the residue was washed
throughly with methanol. The combined filtrate was concen-
trated under reduced pressure to give a brownish material
which was subjected to column chromatography (CH2Cl2-
MeOH-NH4OH, 97.5:2.0:0.5) to give 7 (3.84 g, 78%): 1H NMR
(DMSO-d6) δ 11.35 (s, 1H, NH), 11.09 (s, 1H, NH), 9.83 (s,
1H, NH), 8.82 (s, 1H, 3-OH), 7.38 (s, 1H, Ar), 7.16-7.04 (m,
13H), 7.01 (m, 1H, ArH), 6.38-6.31 (m, 2H), 5.62 (s, 1H), 5.08
(b, 2H), 4.93 (b, 2H), 4.62 (b, 1H, 14-OH), 3.34-3.24 (m, 1H),
3.01 (d, 1H, J ) 19.2 Hz), 2.70-2.56 (m, 2H), 2.46-2.24 (m,
4H), 2.21 (m, 1H), 1.54 (d, 1H, J ) 11.1 Hz), 0.86 (m, 1H),
0.48 (m, 2H), 0.12 (m, 2H). 13C NMR (DMSO-d6) δ 153.78,
143.79, 143.59, 135.40, 131.77, 131.58, 129.10, 128.84, 128.73,
128.49, 127.31, 127.11, 126.73, 124.92, 119.30, 119.02, 117.67,
113.98, 112.04, 110.93, 84.53, 72.85, 63.66, 62.39, 59.30, 55.60,
48.04, 43.98, 31.83, 29.36, 23.31, 9.87, 4.54, 4.22; HRMS (FAB)
m/z 740.3093 (M + H)+, C43H41N5O7 requires 739.8355.
5′-Gu a n id in o-17-(cyclop r op ylm et h yl)-6,7-d id eh yd r o-
4,5r-ep oxy-3,14-h yd r oxyin d olo-[2′,3′:6,7]m or p h in ia n Hy-
d r och lor id e (3‚2HCl). Di-Cbz-protected GNTI 7 (820 mg,
1.10 mmol) was dissolved in MeOH (100 mL). To a Parr
hydrogenating bottle was added 100 mg of Pd/C (10% wt),
followed by anhydrous MeOH (care was taken in order to avoid
a possible fire; for a precaution, it is suggested to use a nitrogen
atmosphere in the bottle). Di-Cbz-protected GNTI solution was
added to the reaction bottle, stirred well, and added dilute HCl
dropwise. The reaction bottle was subjected to hydrogenation
at a pressure of 65 psi. After completion of the reaction (4 h),
it was left overnight at room temperature. The soluble part
was decanted, and the residue was stirred two times with 150
mL of hot MeOH (containing 5% of water) for a few minutes
and filtered off. The combined filtrate were concentrated to a
volume of ca. 5 mL under reduced pressure. Lyophilization
gave the dihydrochloride salt of 3 (520 mg, 90%). 3‚2HCl: IR
KBr disk υ (cm-1) 3400-3200 (br), 1675 (s), 1502, 1461, 1430,
1325, 1202, 1136; 1H NMR (DMSO-d6) δ 11.56 (s, 1H, NH),
9.92 (s, 1H, NH), 9.35 (s, 1H, NH), 9.00 (s, 1H, 3-OH), 7.40-
7.33 (m, 3H, NH2), 7.15 (s, 1H, ArH), 6.90 (d, 1H, J ) 8.10
Hz, ArH), 6.87 (d, 1H, J ) 5.7 Hz, ArH), 6.64-6.50 (m, 2H,
ArH), 5.68 (s, 1H, 5-H), 4.14 (d, 1H, J ) 5.7 Hz, 14-OH), 3.43
(m, 1H), 3.26 (m, 1H), 3.16-3.06 (m, 2H), 2.94-2.90 (m, 2H),
2.69-2.57 (m, 2H), 2.54-2.45 (m, 2H), 1.77 (d, 1H, J ) 15.7
Hz), 1.10 (m, 1H), 0.71-0.66 (m, 1H), 0.63-0.59 (m, 1H), 0.50-
0.47 (m, 1H), 0.43-0.39 (m, 1H); HRMS (FAB) m/z 472.2349
(M + H)+, C27H29N5O3 requires 471.2270.
7′-Gu a n id in o-17-(cyclop r op ylm et h yl)-6,7-d id eh yd r o-
4,5r-ep oxy-3,14-h yd r oxyin d olo-[2′,3′:6,7]m or p h in ia n (4c).
Intermediate 6c (530 mg, 0.78 mmol), TFA (3 mL), and CH2-
Cl2 (28 mL) were subjected to conditions similar to those
employed in the procedure for 4a to give 4c (251 mg, 67%) as
a free base: IR KBr disk υ (cm-1) 3450-3150 (br), 1676 (s),
1
1506, 1462, 1431, 1324, 1202, 1134; H NMR (DMSO-d6): δ
11.65 (s, 1H, NH), 9.88 (s, 1H, NH), 9.31 (s, 1H, NH), 8.96 (s,
1H, Ar-OH), 7.34-7.03 (m, 4H, ArH and NH2), 6.60 (d, 1H, J
) 8.10 Hz, ArH), 6.53 (d, 1H, J ) 8.7 Hz, ArH), 6.39 (s, 1H),
5.62 (s, 1H, 5-H), 4.07 (b, 1H, 14-OH), 3.43-3.23 (m, 4H),
3.18-3.06 (m, 2H), 2.98-2.90 (m, 2H), 2.67-2.52 (m, 2H), 1.78
(d, 1H, J ) 15.7 Hz), 1.05 (m, 1H), 0.68 (m, 1H), 0.59 (m, 1H),
0.41 (m, 2H); HRMS (FAB) m/z 472.2338 (M + H)+, C27H29N5O3
requires 471.2270. Anal. (C27H29N5O3‚2TFA‚3H2O) C, H, N.
Ack n ow led gm en t. This project was supported by
the National Institute on Drug Abuse. We thank Mike
Powers and Mary Lunzer for their excellent technical
assistance in biological testing.
5′-N ′-(N ′′,N ′′′-Bis(b e n zyloxyca r b on yl)gu a n id in o-17-
(cyclop r op ylm et h yl)-6,7-d id eh yd r o-4,5r-ep oxy-3,14-h y-
d r oxyin d olo[2′,3′:6,7]-m or p h in ia n (7). Meth od A. A mix-
ture of N,N′-dicarboxybenzyloxy-N′′-trifluoromethanesulfo-
nylguanidine (3.80 g, 8.27 mmol, 3.3 equiv), 5′-amino-NTI 5b
(1.06 g, 2.5 mmol), and triethylamine (1.5 mL) in dry CH2Cl2
(50 mL) was stirred at room temperature until all of 5b was
consumed (by TLC). After completion of the reaction (4 days),
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