carba -analogues of fentanyl are opioid receptor agonists

Article abstract of DOI:10.1021/jm9019068

There is evidence to indicate that the Asp residue in the third transmembrane helix (TMH) of opioid receptors forms a salt bridge with the positively charged nitrogen of endogenous and exogenous opioid ligands. To further examine the role of this electrostatic interaction in receptor binding and activation, we synthesized carba -analogues of a published fentanyl analogue containing a 3-(guanidinomethyl)-benzyl group in place of the phenyl moiety attached to the ethylamido group (C. Dardonville et al., Bioorg. Med. Chem. 2006, 14, 6570-6580 (1)), in which the piperidine ring nitrogen was replaced with a carbon. As expected, the resulting cis and trans isomers (8a and 8b) showed reduced μ and - opioid receptor binding affinities as compared to 1 but, surprisingly, retained opioid full agonist activity with about half the potency of leucine-enkephalin in the guinea pig ileum assay. In conjunction with performed receptor docking studies, these results indicate that the electrostatic interaction of the protonated nitrogen in the piperidine ring of fentanyl analogues with the Asp residue in the third TMH is not a conditio sine qua non for opioid receptor activation.

Full text of DOI:10.1021/jm9019068

J. Med. Chem. 2010, 53, 2875–2881 2875
DOI: 10.1021/jm9019068
“Carba”-Analogues of Fentanyl are Opioid Receptor Agonists‡
Grazyna Weltrowska,† Nga N. Chung,† Carole Lemieux,† Jianxin Guo, Yixin Lu,*,^ Brian C. Wilkes,† and
Peter W. Schiller*,†,§
†Laboratory of Chemical Biology and Peptide Research, Clinical Research Institute of Montreal, 110 Pine Avenue West, Montreal,
Quebec, Canada H2W 1R7, §Deꢀpartement de Pharmacologie, Universiteꢀ de Montreꢀal, Montreal, Quebec, Canada H3C 3J7, Center for Drug
Discovery, Northeastern University, 360 Huntington Avenue, Boston, Massachusetts 02115, and ^Department of Chemistry and Medicinal
Chemistry Program, Office of Life Sciences, National University of Singapore, 3 Science Drive 3, Singapore
Received December 23, 2009
There is evidence to indicate that the Asp residue in the third transmembrane helix (TMH) of opioid
receptors forms a salt bridge with the positively charged nitrogen of endogenous and exogenous opioid
ligands. To further examine the role of this electrostatic interaction in receptor binding and activation,
we synthesized “carba”-analogues of a published fentanyl analogue containing a 3-(guanidinomethyl)-
benzyl group in place of the phenyl moiety attached to the ethylamido group (C. Dardonville et al.,
Bioorg. Med. Chem. 2006, 14, 6570-6580 (1)), in which the piperidine ring nitrogen was replaced with a
carbon. As expected, the resulting cis and trans isomers (8a and 8b) showed reduced μ and κ opioid
receptor binding affinities as compared to 1 but, surprisingly, retained opioid full agonist activity with
about half the potency of leucine-enkephalin in the guinea pig ileum assay. In conjunction with
performed receptor docking studies, these results indicate that the electrostatic interaction of the
protonated nitrogen in the piperidine ring of fentanyl analogues with the Asp residue in the third TMH is
not a conditio sine qua non for opioid receptor activation.
Introduction
affinity.3-5 Recently, a cyclic enkephalin analogue containing
Dhp in the place of Tyr obtained via ring closing metathesis
has been described.6 This peptide also lacks a positively
charged nitrogen and represents the first example of a neutral
compound with δ opioid agonist activity.
N-Formylnormorphine is an example of a nonpeptide
opioid compound lacking a positive charge. This compound
retained significant μ opioid receptor binding affinity, but was
unable to activate the μ opioid receptor, as demonstrated
using the [35S]GTPγS binding assay.7 More recently, N-for-
mylnormorphine was shown to be a μ and κ opioid antagonist
with similar binding affinity for the two receptors (Kiμ = 420
nM; Kiκ = 363 nM) (G. Weltrowska et al., manuscript in
preparation).
In the μ opioid agonist fentanyl, the positively charged
nitrogen is contained in the piperidine ring of this compound.
In an effort to develop bifunctional μ opioid receptor/I2-
imidazoline binding site ligands, a fentanyl analogue contain-
ing a 3-(guanidinomethyl)-benzyl group in place of the phenyl
moiety attached to the ethylamido group was prepared
(Figure 1, compound 1).8 This compound showed no affinity
for the I2-imidazoline binding site but very high μ opioid
receptor binding affinity (Kiμ = 0.0098 nM), about 300-fold
more potent than fentanyl under the assay conditions used.
This compound was not examined for binding affinity at δ
and κ receptors and was not characterized in a functional
opioid activity assay. To examine the role of the positively
charged nitrogen of fentanyl compounds in the interaction
with opioid receptors, we prepared “carba”-analogues of
compound 1, in which the piperidine nitrogen is replaced with
a carbon atom. Both the cis and the trans isomer were
obtained and pharmacologically characterized (Figure 1,
The positively charged nitrogen in nonpeptide opiates and
in opioid peptides with agonist or antagonist activity has been
thought until recently to be an absolute requirement for
binding to opioid receptors, presumably through formation
of a salt bridge with the side chain of the Asp residue in the
third transmembrane helix (TMH)a of opioid receptors.1 A
cyclic β-casomorphin-derived opioid peptide, CHO-Dmt-
c[D-Orn-2-Nal-Pro-Gly], lacking a positively charged nitro-
gen, was the first example of a neutral opioid compound with
significant μ and δ opioid receptor binding affinity and μ and
δ antagonist activity.2 Subsequently, it was reported that
replacement of the Tyr1 residue in cyclic opioid peptide
analogues with 3-(2,6-dimethyl-4-hydroxyphenyl)propanoic
acid (Dhp), (2S)-2-methyl-3-(2,6-dimethyl-4-hydroxyphenyl)-
propanoic acid[(2S)-Mdp], or (3S)-3-methyl-3-(2,6-dimethyl-
4-hydroxyphenyl)propanoic acid [(3S)-Mdp] resulted in
opioid antagonists lacking a positively charged N-terminal
amino group and showing very high opioid receptor binding
‡Dedicated to the memory of Ralph F. Hirschmann.
*To whom correspondence should be addressed. Phone: þ1-514-987-
5576. Fax: þ1-514-987-5513. E-mail: schillp@ircm.qc.ca (P.W.S.).
Phone: þ65-6516-1569. Fax: þ65-6779-1691. E-mail: chmlyx@nus.edu.sg.
a Abbreviations: DAMGO, H-Tyr-D-Ala-Gly-NRMePhe-Gly-ol; Dhp,
3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid; DMAP, 4-(dimethy-
lamino)-pyridine; Dmt, 20,60-dimethyltyrosine; DSLET, H-Tyr-D-Ser-
Gly-Phe-Leu-Thr-OH; GPI, guinea pig ileum; LENK, [Leu5]enkephalin;
(2S)-Mdp, (2S)-2-methyl-3-(2,6-dimethyl-4-hydroxyphenyl)propanoic
acid; (3S)-Mdp, (3S)-3-methyl-3-(2,6-dimethyl-4-hydroxyphenyl)propa-
noic acid; MVD, mouse vas deferens; 2-Nal, 3-(2-naphthyl)-L-alanine;
TEA, triethylamine; TFA, trifluoroacetic acid; THF, tetrahydrofuran;
TMH, transmembrane helix; U69,593, (5R,7R,8β-(;)-N-methyl-N-[7-
(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]benzeneacetamide.
r
2010 American Chemical Society
Published on Web 03/10/2010
pubs.acs.org/jmc

2876 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Weltrowska et al.
compounds 8a and 8b). Compound 1 was chosen as parent
structure because the presence of the guanidino group in-
creases water solubility as compared to a carba analogue of
fentanyl itself which likely would be poorly soluble in water.
To assess the possible contribution of the guanidino group to
receptor binding, an analogue of 1 lacking the guanidino
group (compound 10) was also synthesized and pharmaco-
logically characterized.
gave amine 7. Reaction of 7 with the Boc-protected thiourea
reagent and subsequent Boc-deprotection afforded the target
compounds 8a and 8b as the trifluoroacetate salts. Com-
pounds 6, 7, and 8 were obtained as mixtures of cis and trans
isomers, which could be separated by preparative HPLC. In
the case of 8, the stereochemical assignment was made on the
basis of the results of homonuclear decoupling NMR experi-
ments. The faster-eluting component was identified as the cis
isomer (8a) and the slower-eluting one was the trans isomer
(8b). Compound 10 was prepared by reductive amination of
1-phenylethyl-4-piperidone with 3-methylbenzylamine, yield-
ing 9, followed by acylation with propionic anhydride
(Scheme 2).
Chemistry
The “carba”-analogues 8a and 8b were synthesized accord-
ing to Scheme 1. 4-Phenylethylcyclohexanone 5 was prepared
by following a synthetic route described in the literature.9 The
Wittig reagent was obtained from phosphonium salt 2 with
NaH/DMSO and was condensed with 4,4-(ethylenedioxy)-
cyclohexanone to form a mixture of benzylmethylene 3a and
phenylmethylene 3b. Catalytic hydrogenation of the mixture
of 3a and 3b yielded phenylethyl derivative 4. Removal of
the ketal blocking group from 4 with HCl/THF afforded
4-phenylethylcyclohexanone 5. Cyclohexanone 5 was then
used to prepare the target compounds 8a and 8b by following
synthetic steps described for the preparation of fentanyl
analogue 1.8 Reductive amination of 5 with mono-Boc-
protected m-xylenediamine provided compound 6. Acylation
of 6 with propionic anhydride followed by Boc-deprotection
Results and Discussion
In the guinea pig ileum (GPI) assay, the carba analogues 8a
and 8b both showed full agonist activity with about half the
potency of [Leu5]enkephalin (LENK) (Table 1). Compounds
8a and 8b were about 25-fold less potent agonists than the
piperidine nitrogen-containing parent 1, which was character-
ized as a potent agonist in this assay system (IC50 = 22.4 nM).
The agonist effects of 8a, 8b, and 1 in the GPI assay were
naloxone-reversible with Ke (naloxone) values ranging from 2
to 13 nM, indicating that they were mediated by opioid
Scheme 2a
a
˚
Reagents and conditions: (a) 3-methylbenzylamine, NaBH3CN, 3 A
molecular sieves, 1% AcOH/MeOH, rt, 5 h, 50%; (b) (CH3CH2CO)2O,
DMAP, pyridine, dry CH2Cl2, rt, 20 h, 73%.
Figure 1. Structural formulas of fentanyl and fentanyl analogues 1,
8a, 8b, and 10.
Scheme 1a
a Reagents and conditions: (a) NaH, dry DMSO, 4,4-(ethylenedioxy)-cyclohexanone, rt, 20 h, 70%; (b) Pd/C, H2 (g), EtOAc/AcOH, 60 psi, rt, 2 h,
˚
100%; (c) 2 N HCl, THF, rt, 48 h, 82%; (d) tert-Butyl 3-(aminomethyl)benzylcarbamate, NaBH3CN, 3 A molecular sieves, 1% AcOH/MeOH, rt, 3 h,
71%; (e) (CH3CH2CO)2O, DMAP, pyridine, dry CH2Cl2, rt, 20 h, 95%; (f) 50% TFA/CH2Cl2, rt, 45 min, 63%; (g) N,N0-Boc-thiourea, HgCl2, Et3N, dry
CH2Cl2, rt, 3 h, 96%; (h) 50% TFA/CH2Cl2, rt, 1 h, 95%.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2877
Table 1. In Vitro Opioid Activity Profiles of Fentanyl Analoguesa
GPI
MVD
receptor binding
Ki ratio
μ/δ/κ
cpd
IC50 (nM)
IC50 (nM)
Kiμ (nM)
Kiδ (nM)
Kiκ (nM)
1
22.4 ( 3.7
521 ( 67
590 ( 85
114 ( 9
0.448 ( 0.079
300 ( 8
224 ( 13
96.0 ( 5.9
5.9 ( 1.4c
9.43 ( 2.07
43.5 ( 4.1
4110 ( 80
7800 ( 280
6820 ( 670
568 ( 159c
2.53 ( 0.35
0.536 ( 0.043
238 ( 41
162 ( 8
1850 ( 160
298 ( 40c
4570 ( 550
1/97/1.2
1/14/0.8
1/35/0.7
1/71/19
1/96/51
1/0.3/485
8a
8b
10
5820 ( 1040
4750 ( 790
610 ( 78
647 ( 77
Fentanyl
LENK
3.45 ( 0.45b
246 ( 39
9.45 ( 4.05b
11.4 ( 1.1
a Values represent means of 3-6 determinations ( SEM. b ref 14. c ref 15.
receptors. In the δ receptor-representative MVD assay, the
carba analogues 8a and 8b displayed about 10-fold lower
potency as compared to the potencies determined in the GPI
assay, similar to the 5-fold weaker potency seen with com-
pound 1 in this assay system.
3-(guanidinomethyl)-benzyl group (1), the phenylethyl group
assumes an almost fully extended conformation with the
phenyl ring interacting with the Tyr148, Val300, Phe237, and
Met151 side chain residues of the receptor. The piperidine
nitrogen forms a salt bridge with Asp147 in the third TMH
˚
Inthe opioid receptorbindingassays, compounds8aand 8b
showed marked binding affinities for μ opioid receptors
(Kiμ = 220-300 nM) and κ opioid receptors (160-230 nM)
(Table 1). Subnanomolar binding affinities were determined
for 1 at both μ and κ receptors. The discrepancy between the
μ receptor binding affinity of 1 (Kiμ = 0.448 nM) determined
in this study and the Kiμ of 0.0098 nM reported for this
compound in the literature8 is likely due to the different
μ receptor binding assays used in the two laboratories. The
δ opioid receptor binding affinities of 8a, 8b, and 1 were more
than 10-fold lower than their respective μ and κ receptor
binding affinities, and these three compounds showed similar
μ/δ/κ Ki ratios (Table 1). This result indicates that the carbon
replacement of the piperidine nitrogen in 1 did not have much
of an effect on the opioid receptor selectivity profile. Further-
more, the opioid receptor binding affinities obtained for 8a,
8b, and 1 indicate that the agonist effects produced by these
three compounds in the GPI assay are due to activation of
bothμ and κ receptors which arebothpresent inthe ileum. It is
also of interest to point out that the configuration at the
cyclohexane ring in the carba analogues does not have much
of an effect on the in vitro opioid activity profile, as both the
cis isomer (8a) and the trans isomer (8b) show very similar
agonist potencies and opioid receptor binding affinities and
similar opioid receptor selectivity profiles. Compound 10, an
analogue of 1 lacking the guanidino group, showed about
30-fold lower agonist potency than 1 in the GPI assay as well
as lower potency in the MVD assay. In the receptor binding
assays, compound 10 displayed about 150-200-fold lower μ
and δ receptor binding affinities in comparison with 1 and
3500-fold lower κ receptor affinity. These results indicate that
the guanidino group of 1 significantly contributes to the
receptor binding energy of this compound at all three recep-
tors and to the largest extent at the κ receptor. In comparison
with carba analogues 8a and 8b, compound 10 has similar
μ and δ receptor binding affinities but about 10-fold lower κ
receptor binding affinity. Interestingly, compound 10 has
about 15-fold lower μ receptor binding affinity than fentanyl
and about 180-fold lower μ agonist potency in the GPI assay
(Table 1).
(nitrogen-oxygen distance = 2.8 A), as is typically observed
with nitrogen-containing opioid receptor ligands. A second
salt bridge between the guanidino moiety of the ligand and the
side chain of Asp216 in the second extracellular loop close to
the interface between the cell membrane and the extracellular
domain is also observed. The aromatic ring of the
3-(guanidinomethyl)-benzyl moiety interacts with the side
chains of Phe313, Trp318, Thr218, and Leu219
.
The lowest-energy conformer of compound 8b, the trans
isomer of the “carba”-analogue of 1, with the phenylethyl and
N-(3-guanidinomethyl)benzylpropanamide moieties equator-
ial, shows a receptor binding mode which overall is quite
similar to that of its parent (1) (Figure 2). Again, the phenyl-
ethyl substituent is in an extended conformation and interacts
with the same receptor residues as the phenylethyl group of 1.
However, the distance between the carbon replacing the
nitrogen in the piperidine ring of 1 and the Asp147 oxygen is
˚
larger (5.2 A as compared to the corresponding distance of
˚
2.8 A in the case of 1) and the cyclohexane ring is displaced by
˚
about 1.2 A as compared to the piperidine ring in 1. The
guanidino group of 8b forms a salt bridge with the side chain
of Asp216 but from the opposite side as compared to the salt
bridge formation of the guanidino group of 1 with that same
residue. The phenyl ring of the 3-(guanidinomethyl)benzyl
moiety of 8b interacts with the same receptor moieties as in the
case of compound 1 (Phe313, Thr218, Trp318).
In the lowest-energy conformer of compound 8a, the cis
isomer of the “carba”-analogue of 1, the phenylethyl moiety is
axial and the N-(3-guanidinomethyl)benzylpropanamide
moiety is equatorial. The binding mode of compound 8a
differs somewhat from the binding modes of 8b and 1. The
phenylethyl group assumes a bent conformation and its
˚
aromatic ring is displaced by about 4 A relative to the
corresponding aromatic ring of docked 1, interacting with
Tyr148, Phe237, Val300, and Lys308. The cyclohexane ring is
˚
displaced by 3.6 A relative to the cyclohexane ring of the trans
isomer 8b, and the carbon replacing the nitrogen in the
147
˚
piperidine moiety of 1 is located 8.5 A away from the Asp
side chain oxygen. The guanidino group of 8a again forms a
salt bridge with the side chain of Asp216 and the aromatic ring
of its 3-(guanidinomethyl)-benzyl moiety interacts with the
side chains of the Tyr128, Thr315, and His319 residues of the
receptor.
Compound 10, the analogue of 1 lacking the guanidino
group, has a binding mode very similar to that of 1, with the
phenylethyl moietyin an extended conformation. To compare
the μ receptor binding mode of 10 with that of fentanyl, a
docking study with fentanyl was also performed (data not
Flexible docking studies of the fentanyl analogues were
performed using Mosberg’s model of the μ opioid receptor in
the activated state.10 The results indicated that in all cases
the phenylethyl group is accommodated in a binding pocket
formed by transmembrane helixes (TMHs) 3, 5, and 6,
but significant differences in the detailed binding modes
among the different ligands are observed (Figure 2). In
the case of the docked fentanyl analogue containing the

2878 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Weltrowska et al.
Figure 2. Receptor docking studies. Fentanyl analogues bound to the μ opoid receptor in the activated state: 1, 8a, 8b, 10.
shown). Earlier studies on the docking of fentanyl to the μ
receptor, using receptor models developed prior to the rho-
dopsin crystal structure determination have been reviewed.11
In agreement with two of these studies,12,13 the results of the
present docking study indicated an interaction of the posi-
tively charged piperidine nitrogen with Asp147 in the third
TMH and an orientation of the phenylethyl moiety down-
ward into a cavityformed by TMHs 3, 5, and 6, as described in
one of these studies,12 whereas in the other study, it was
located in a cavity formed by TMHs 2, 3, and 7.13 Further-
more, as in the case of one of these studies,13 the phenethyl
moiety assumed an extended conformation. The aromatic
rings of the phenylethyl group and of the 3-methylbenzyl
group in 10 interact with the same receptor residues as the
corresponding aromatic rings of 1, and the nitrogen in the
piperidine ring forms a salt bridge with Asp147. In a compar-
ison of docked fentanyl and docked compound 10, the
N-phenylethyl pipiperidine moiety of the two compounds shows
the same receptor binding mode. However, the aromatic rings of
the N-phenyl group in fentanyl and of the N-3-methylbenzyl
group in compound 10 occupy different receptor sites. This is in
part due to the presence of the methylene group between the
aromatic ring and the amide nitrogen in 10. Furthermore,
the torsion angle at the bond between the amide nitrogen and
the piperidine ring is quite different for fentanyl (Φ = 172ꢀ) and
compound 10 (Φ=11ꢀ). Consequently, the propanamide moiety
also has a different orientation in the two docked compounds.
The different receptor interactions of the N-propanamide sub-
stituents may explain the observed decrease in μ opioid agonist
potency of 10 as compared to fentanyl (Table 1).14,15
and 8b have 670- and 500-fold lower μ receptor binding
affinity than 1, corresponding to a loss in binding energy
(ΔG) of 3.95 and 3.71 kcal/mol, respectively. This reduction in
receptor binding energy is due to the inability of these
“carba”-analogues to engage in an electrostatic interaction
with Asp147. Such electrostatic interactions typically contri-
bute about 4 kcal/mol to the binding energy in ligand-protein
interactions.16 The analogous analysis of the κ receptor bind-
ing data of 8a and 8b as compared to 1 indicates a similar loss
in binding energy of the two “carba”-analogues (3.71 and 3.47
kcal/mol, respectively). This decrease in binding energy is
likely due to the elimination of the electrostatic interaction
with the Asp residue (Asp138) in the third TMH of the κ
receptor, which was observed in a study on the docking of 1 to
this receptor in the activated state (data not shown). The
performed flexible docking studies reveal overall similar μ
receptor binding modes for 8a, 8b, and 1. In all cases, an
electrostatic interaction between the guanidino group of these
three compounds and the Asp216 side chain of the receptor is
observed. The analogue of compound 1 lacking the guanidino
group (compound 10) shows a 214-fold decrease in μ receptor
binding affinity. This corresponds to a reduction in binding
energy of 3.26 kcal/mol, in agreement with the observation
that the guanidino group of 1 engages in an electrostatic bond
with Asp216 16
The agonist activity of “carba”-analogues 8a
.
and 8b can be explained with their overall similar binding
modes as compared to 1 and 10, and with their guanidino
group interacting with Asp216. The latter electrostatic inter-
action contributes to binding the active conformation of the
receptor and, thereby, compensates to some extent for the loss
of the salt bridge with Asp147 in the third TMH.
The observation that the “carba”-analogues of 1, com-
pounds 8a and 8b, behave as full opioid agonists in the GPI
assay with about half the potency of leucine-enkephalin
represents a novel and important finding. Compounds 8a
Conclusions
Compound 1 was confirmed as a ligand with subnano-
molar μ receptor binding affinity and, furthermore, it was

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2879
determined that this fentanyl analogue also has high κ recep-
tor binding affinity as well as high agonist potency in the GPI
assay. The most important finding of this study is the novel
observation that an electrostatic interaction between the
positively charged nitrogen of fentanyl compounds and the
Asp147 residue of the μ opioid receptor is not a conditio sine
qua non for μ opioid receptor activation. To the best of our
knowledge, this has not been demonstrated before for opioid
receptor ligands or for other GPCRs having a conserved Asp
residue in the third TMH and interacting with endogenous
ligands containing an amine moiety. Previously, it has been
shown that elimination of the N-terminal amino group or its
replacement with a methyl group in opioid peptides contain-
ing an N-terminal 20,60-dimethyltyrosine (Dmt) residue led
to neutral compounds showing high opioid antagonist
activity.3-5 Similarly, N-formylnormorphine, which also
lacks a positively charged nitrogen, also turned out to be an
opioid antagonist (G. Weltrowska et al., manuscript in pre-
paration). Taken together, these results indicate that the
elimination of the positively charged nitrogen in opioid
compounds may have divergent effects on the efficacy, de-
pending on the receptor binding interactions of other moieties
present in the molecule. The nonnitrogenous salvinorin A
analogue herkinorin, which also lacks a positive charge, has
been reported to be a μ opioid agonist with an EC50 of 500 nM
in a functional in vitro assay.17 However, there is evidence to
indicate that salvinorin A and its analogues have a receptor
binding mode totally different from the binding modes of all
classical, nitrogen-containing opioid agonists.18
stirred for 20 h. The mixture was then poured over ice and
extracted with petroleum ether. The organic layers were com-
bined, dried over MgSO4, and evaporated. Flash chromatogra-
phy on silica gel with hexane-EtOAc (6:1, v/v) afforded 3 as a
mixture of 8-(2-phenylethylidene)-1,4-dioxaspiro[4.5]decane
(3a) and 8-styryl-1,4-dioxaspiro[4.5]decane (3b) in the form of
a colorless oil (4.5 g, 70%) . The mixture of 3a and 3b was used
without separation in the subsequent hydrogenation step.
8-Phenylethyl-1,4-dioxaspiro[4.5]decane (4). An argon-
purged reaction vessel was charged with 3a and 3b (4.5 g, 18
mmol), 10% Pd/C (0.5 g) in 75 mL EtOAc and AcOH (10 μL).
The reaction vessel was then pressurized to 60 psi with hydrogen
and the mixture was stirred for 2 h at room temperature. After
filtration, the solvent was evaporated to yield 4 as a colorless oil
1
(4.5 g, 100%). H NMR (500 MHz, CDCl3) δ 7.18-7.35 (m,
5H), 3.95 (s, 4H), 2.63-2.66 (t, 2H, J = 8.0 Hz), 1.28-1.82 (m,
11H). 13C NMR (125 MHz, CDCl3) δ 142.7, 128.5, 125.7, 108.5,
64.4, 38.2, 36.5, 33.1, 29.5, 28.9. HRMS (EI) m/e calcd for
C16H23O2 [MþH]þ 247.1698, obsd 247.1691.
4-Phenylethylcyclohexanone (5). 4.5 g (18 mmol) of 4 in a
mixture of 50 mL THF and 25 mL 2 N HCl was stirred for 48 h at
room temperature. After evaporation of the solvent, the product
was partitioned between EtOAc and H2O. Evaporation of the
organic layer afforded 5 (3.0 g, 82%) as a colorless oil. 1H NMR
(500 MHz, CDCl3) δ 7.18-7.38 (m, 5H), 2.68-2.71 (t, 2H, J =
8.0 Hz) 1.42-2.45 (m, 11H). 13C NMR (125 MHz, CDCl3) δ
211.8, 142.7, 128.5, 128.2, 125.7, 41.0, 38.3, 36.5, 31.2, 28.1.
HRMS (EI) m/e calcd for C14H19O [MþH]þ 203.1436, obsd
203.1431.
tert-Butyl-3-((4-phenylethylcyclohexylamino)methyl) Benzyl-
carbamate (6). NaBH3CN (942 mg, 15 mmol) was added to a
solution of mono-Boc-protected m-xylenediamine8 (2.8 g, 12
mmol), 4-phenylethylcyclohexanone (5) (2.02 g, 10 mmol) and
˚
Experimental Section
3 A molecular sieves in 1% AcOH/MeOH dry, and the mixture
was stirred for 3 h at room temperature. After filtration on a pad
of Celite, the organic solvent was evaporated and 6 was obtained
as a 50%/50% mixture of the cis and trans isomers, as deter-
mined by integration of the isomeric proton peaks at 2.94 and
3.08 ppm in the 1H NMR spectrum. Purification by flash
chromatography on silica gel with CH2Cl2/7N NH3 in MeOH
(97:3) afforded 6 (3.080 g, 71% [3 g of cis/trans mixture, 40 mg of
isomer 1 and 40 mg of isomer 2]). Isomer 1: 1H NMR (500 MHz,
CDCl3) δ 8.45 (s, 1H), 7.08-7.25 (m, 9H), 5.12 (s, 1H), 4.12 (s,
2H), 4.08 (s, 2H), 3.08 (br s, 1H). 2.60 (t, 2H, J = 8.0 Hz),
1.42-1.9 (m, 11H), 1.4 (s, 9H). 13C NMR (125 MHz, CDCl3) δ
156.2, 142.6, 137.4, 136.2, 128.5, 128.3, 128.1, 127.7, 125.9, 80.0,
56.3, 52.5, 45.4, 38.3, 36.7, 32.1, 28.4. HRMS (EI) m/e calcd for
General Methods. Reactions were monitored by ascending
TLC using precoated plates (silica gel 60 F254, 250 μm, Merck
Darmstadt, Germany). Flash chromatography was performed
with silica gel 60 columns (particle size 230-400 μm). 1H and 13
C
NMR spectra were recorded on a Varian INOVA 500 MHz
spectrometer and referenced with respect to the residual signals
of the solvent. The following abbreviations were used in report-
ing spectra: s = singlet, d = doublet, t = triplet, m = multiplet.
Preparative reversed-phase HPLC was performed on a Vydac
218-TP1022 column (22 ꢀ 250 mm) with a linear gradient of
50-70% MeOH in 0.1% TFA/H2O over 40 min at a flow rate of
12 mL/min. For analytical reversed-phase HPLC a Vydac 218-
TP54 column (5 ꢀ 250 mm) was used with a linear gradient of
50-70% MeOH in 0.1% TFA/H2O over 30 min at a flow rate of
1 mL/min. All final compounds were obtained in >95% purity,
as established by analytical HPLC. Molecular masses of com-
pounds were determined by electrospray mass spectrometry on
a hybrid Q-Tof mass spectrometer interfaced to a Mass Lynx
4.0 data system.
Syntheses of Compounds 8a, 8b, and 10. Phenylethyltriphe-
nylphosphonium Iodide (2). A solution of (2-iodoethyl)benzene
(10.4 mL, 72 mmol, 1.2 equiv) and 15.64 g (60 mmol) triphenyl-
phosphine in 80 mL xylene was heated at 90 ꢀC under argon for
24 h. After cooling in an ice bath, the formed precipitate was
collected, washed with Et2O, and dried in vacuo. Crystallization
from acetone-ethyl ether yielded 2 as white crystals (25.0 g,
83%); mp 130-132 ꢀC. 1H NMR (500 MHz, DMSO-d6) δ
7.77-7.95 (m, 15H), 7.24-7.35 (m, 5H), 3.92-3.98 (m, 2H),
2.86-2.91 (m, 2H). HRMS (EI) m/e calcd for C26H24P [M]þ
367.1610, obsd 367.1611.
1
C27H39N2O2 [MþH]þ 423.3012, obsd 423.3013. Isomer 2: H
NMR (500 MHz, CDCl3) δ 8.45 (s, 1H), 7.08-7.22 (m, 9H), 5.12
(br s, 1H), 4.12 (s, 1H), 4.05 (br s, 1H), 2.94 (br s, 1H), 2.60 (t, 2H,
J = 8.0 Hz), 2.10 (br, 2H), 1.98 (br, 2H), 1.25-1.4 (br m, 14H),
0.81 (br m, 2H). 13C NMR (125 MHz, CDCl3) δ 156.2, 142.6,
137.4, 136.2, 128.5, 128.3, 128.1, 127.7, 125.9, 80.0, 56.3, 52.5,
45.5, 38.3, 36.7, 32.1, 28.4. HRMS (EI) m/e calcd for
C27H39N2O2 [MþH]þ 423.3012, obsd 423.3018.
N-(3-(Aminomethyl)benzyl)-N-(4-phenylethylcyclohexyl)pro-
pionamide (7). Propionic anhydride (1 mL, 7.8 mmol) was added
at room temperature to a solution of amine 6 (mixture of cis/
trans isomers), DMAP (0.1 equiv), and pyridine (2.11 mL,
26 mmol) in dry CH2Cl2, and the mixture was stirred for 20 h
at room temperature. After evaporation of the organic solvent,
the oily residue was dissolved in EtOAc, extracted with a
saturated NH4Cl aqueous solution, brine, and water, and dried
under MgSO4 and concentrated. The crude product (2.5 g, 95%)
was then taken up in 50% TFA/CH2Cl2, and the solution was
stirred for 45 min at room temperature. The volatiles were
evaporated, and the resulting crude oil was triturated with dry
Et2O. Purification of the oily residue by flash chromatography
on silica gel with CH2Cl2/7N NH3 in MeOH (97:3) afforded 7 as
a mixture of cis and trans isomers (1.2 g, 63%). The mixture was
8-(2-Phenylethylidene)-1,4-dioxaspiro[4.5]decane (3). 1.2 g (30
mmol) NaH (60% oil dispersion) was stirred in 75 mL DMSO
(dry) at 60 ꢀC under argon for 1 h. After cooling of the solution
to room temperature, 12.8 g (26 mmol) of phosphonium salt 2
was added under stirring. 4,4-(Ethylenedioxy)-cyclohexanone
(4.06 g, 26 mmol) was then added and the reaction mixture was

2880 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Weltrowska et al.
used in the next synthetic step without separation of the isomers.
For the analytical characterization, the isomers were separated
by preparative HPLC of a 100 mg sample of the mixture. Isomer
1: 1H NMR (500 MHz, DMSO-d6) δ 8.26 (s, 2H), 7.25-7.49 (m,
9H), 4.58 (d, 2H, J = 23 Hz), 4.36 (br t, 0.5H), 4.05-4.11 (dd,
2H, J1 = 5 Hz, J2 = 17 Hz) 3.82 (br t, 0.5H), 3.16 (m, 0.5H), 2.82
(d, 0.5H, J = 5 Hz), 2.52 (m, 1H), 2.21 (m, 1H), 1.44-1.71 (m,
11H), 1.12 (t, 1.5H, J = 7 Hz), 1.0 (t, 1.5H, J = 7 Hz). 13C NMR
(125 MHz, DMSO-d6) δ 171.7, 142.4, 141.3, 136.2, 128.5, 128.3,
128.1, 127.7, 125.9, 125.2, 46.7, 46.2, 38.3, 36.7, 29.4, 28.8, 26.6,
10.3. HRMS (EI) m/e calcd for C25H35N2O [MþH]þ 379.2749,
obsd 379.2735. Isomer 2: 1H NMR (500 MHz, DMSO-d6) δ 8.22
(br s, 2H), 7.15-7.41 (m, 9H), 4.46 (d, 2H, J = 24 Hz), 4.31 (t,
0.5H, J = 12 Hz), 3.99 (m, 2H), 3.72 (t, 0.5H, J = 12 Hz), 3.05
(m, 0.5H), 2.75 (d þ m, 1.5H, J = 5 Hz), 2.47 (m, 3H), 2.13 (m,
1H), 1.75 (d, 2H, J = 12 Hz), 1.56 (t, 2H, J = 18 Hz), 0.92-1.48
(m, 8H). 13C NMR (125 MHz, DMSO-d6) δ 171.7, 142.4, 141.3,
136.2, 128.5, 128.3, 128.1, 127.7, 125.4, 125.2, 46.7, 46.2, 38.3,
36.7, 29.4, 28.8, 26.6, 10.3. HRMS (EI) m/e calcd for
C25H35N2O [MþH]þ 379.2749, obsd 379.2738.
added at room temperature to a solution of amine 9 (0.9 g, 2.9
mmol), DMAP (30 mg, 0.1 equiv) and pyridine (1.17 mL, 14.5
mmol) in dry CH2Cl2. After stirring the reaction mixture for
20 h, the organic solvent was evaporated and the crude product
was dissolved in AcOEt. The solution was washed with sat.
NH4Cl aqueous solution, brine, and water, dried over MgSO4,
and concentrated. The resulting crude oil was purified by flash
chromatography on silica gel (CH2Cl2/MeOH/TEA, 95:5:0.2),
affording 10 as a colorless oil (0.8 g, 73%). 1H NMR (500 MHz,
CDCl3) δ 6.93-7.34 (m, 9H), 4.88 (m, 1H), 4.52 (s, 2H), 3.66 (d,
2H, J = 12 Hz), 3.16 (br, 2H), 3.00 (m, 2H), 2.77 (br, 2H), 2.37
(d, 2H, J = 8 Hz), 2.35 (s, 3H), 2.11 (dd, 2H, J = 11 and 36 Hz),
1.82 (d, 2H, J = 12 Hz), 1.14 (t, 3H, J = 8 Hz). 13C NMR (125
MHz, CDCl3) δ 175.8, 139.1, 137.6, 135.8, 129.3, 128.8, 128.5,
127.7, 126.4, 122.7, 58.6, 52.8, 49.0, 46.5, 45.0, 30.7, 27.2, 26.7,
21.6, 9.7. HRMS (EI) m/e calcd for C24H33N2O [MþH]þ
365.2593, obsd 365.2580.
In Vitro Bioassays and Receptor Binding Assays. The GPI19
and MVD20 bioassays were carried out as reported in detail
elsewhere.21,22 A dose-response curve was determined with
[Leu5]enkephalin as standard for each ileum and vas prepara-
tion, and IC50 values of the compounds being tested were
normalized according to a published procedure.23 Ke-values
for naloxone as antagonist were determined from the ratio of
IC50 values obtained with an agonist in the presence and absence
of a fixed naloxone concentration (20 nM).24 Opioid receptor
binding studies were performed as described in detail else-
where.21 Binding affinities for μ and δ opioid receptors were
determined by displacing, respectively, [3H]DAMGO (Multiple
Peptide Systems, San Diego, CA) and [3H]DSLET (Multiple
Peptide Systems) from rat brain membrane binding sites, and κ
opioid receptor affinities were measured by displacement of
[3H]U69,593 (Amersham) from guinea pig brain membrane
binding sites. Incubations were performed for 2 h at 0 ꢀC with
[3H]DAMGO, [3H]DSLET, and [3H]U69,593 at respective con-
centrations of 0.72, 0.78, and 0.80 nM. IC50 values were
determined from log-dose displacement curves, and Ki values
were calculated from the IC50 values by means of the equation of
Cheng and Prusoff,25 using values of 1.3, 2.6, and 2.9 nM for the
dissociation constants of [3H]DAMGO, [3H]DSLET, and
[3H]U69,593, respectively. The free energy change, ΔG, asso-
ciated with ligand-receptor interactions was calculated using
the equation ΔG = -2.303 RT log Ki, which under physiolo-
gical conditions (T = 310ꢀ K) is approximated (in kcal/mol) by
ΔG = -1.4 log Ki.
Theoretical Conformational Analyses and Receptor Docking
Studies. All calculations were performed using the SYBYL
software version 7.0 (Tripos Associates, St. Louis, MO). The
Tripos force field was used for energy calculations. A dielectric
constant of 78 was used for the conformational analysis of the
isolated ligands in vacuo. A stepwise approach was used to
determine the low-energy conformations of the fentanyl analo-
gues.26 First, the cyclohexane ring or piperidine ring was
retrieved from the fragment library. The piperidine ring was in
the charged (protonated) form. Functional groups were at-
tached to the six-membered ring in each of four different
arrangements: axial-axial, axial-equatorial, equatorial-axial,
and equatorial-equatorial, resulting in four different starting
structures for each compound studied. For each structure, a
systematic conformational grid search was performed to identi-
fy low-energy structures. Each exocyclic rotatable bond was
rotated in 30ꢀ increments over all space. Energies were calcu-
lated and the obtained conformers were grouped into low-
energy families. The lowest-energy member of each family was
minimized and the resulting conformations were ranked accord-
ing to energy. The lowest-energy conformations of all com-
pounds have the cyclohexane or piperidine ring in the chair
conformation. In the lowest-energy conformation of the cis
isomer of the “carba”-analogue (8a), the larger substituent on
the cyclohexane ring is equatorial and the smaller substituent is
N-(3-(Guanidinomethyl)benzyl)-N-(4-phenylethylcyclohexyl)-
propionamide TFA Salt (8). To a solution of 7 (500 mg, 1 mmol),
N,N0-di-(tert-butoxycarbonyl)-thiourea (327 mg, 1.2 mmol) and
TEA (0.68 mL) in dry CH2Cl2 (20 mL), HgCl2 (325 mg, 1.2
mmol) was added. After stirring under argon for 3 h at room
temperature, the reaction mixture was filtered and the organic
phase was washed with water, dried over MgSO4, and concen-
trated. The resulting crude oil (600 mg, 96%) was dissolved in
50% TFA/CH2Cl2 and the solution was stirred for 1 h at room
temperature. The volatiles were then evaporated and the crude
oily residue was triturated with dry Et2O. After evaporation of
Et2O, the TFA salt of 8 (mixture of cis and trans isomers) was
obtained as a very hygroscopic solid (400 mg, 95%). The cis and
trans isomers were separated by preparative HPLC and were
identified by homonuclear decoupling NMR experiments (see
Supporting Information). Isomer 1 (cis, 8a): 1H NMR (500
MHz, DMSO-d6) δ 7.94 (d, 1H, J = 27 Hz), 7.09-7.37 (m, 9H),
4.50 (d, 2H, J = 23 Hz), 4.32 (dd, 2H, J = 6 Hz), 4.26 (br t,
0.5H), 3.73 (br 0.5H), 2.75 (d, 1H, J = 5 Hz), 2.46 (m, 3H), 2.12
(m, 1H), 1.17-1.63 (m, 12H), 1.04 (t, 1.5H, J = 7 Hz), 0.92 (t,
1.5H, J = 7 Hz). 13C NMR (125 MHz, DMSO-d6) δ 171.7,
158.0, 142.6, 137.4, 128.5, 128.1, 127.7, 125.9, 53.3, 46.7, 44.9,
38.3, 36.7, 29.4, 29.1, 28.8, 26.6, 10.2. HRMS (EI) m/e calcd for
C26H37N4O [MþH]þ 421.2967, obsd 421.2955. Isomer 2 (trans,
8b): 1H NMR (500 MHz, DMSO-d6) δ 7.94 (d, 1H, J = 27 Hz),
7.08-7.37 (m, 9H), 4.46 (d, 2H, J = 26 Hz), 4.33-4.37 (dd,
2.5H, J = 6 Hz), 3.73 (br t, 0.5H), 2.74 (d, 1H, J = 5 Hz), 2.53
(t, 2H, J = 8 Hz), 2.40 (m, 1H), 2.12 (m, 1H), 1.75 (d, 2H, J = 12
Hz), 1.54 (m, 2H), 0.98-1.46 (m, 12H). 13C NMR (125 MHz,
DMSO-d6) δ 171.7, 158.0, 142.6, 137.4, 128.5, 128.1, 127.7,
125.0, 53.3, 46.7, 44.9, 38.3, 36.7, 29.4, 29.1, 28.8, 26.6, 10.2.
HRMS (EI) m/e calcd for C26H37N4O [MþH]þ 421.2967, obsd
421.2955.
N-(3-Methylbenzyl)-1-phenylethylpiperidine-4-amine (9).NaBH3-
CN (0.55 g, 8.9 mmol) was added to a solution of 3-methylbenzyl-
amine (0.88 mL, 7 mmol), 1-phenylethyl-4-piperidinone (1.2 g, 5.9
˚
mmol) and 3 A sieves in 1% AcOH/MeOH (dry). The mixture was
stirred at room temperature for 5 h, filtered over Celite, and washed
with dry MeOH. The combined organic filtrates were evaporated
and the crude product was purified by flash chromatography on
silica gel (CHCl3/MeOH, 9:1), affording 9 as a colorless oil (0.9 g,
50%). 1H NMR (500 MHz, CDCl3) δ 7.08-7.32 (m, 9H), 3.81 (s,
2H), 3.0 (d, 2H, J=14Hz), 2.83(m, 2H), 2.58(m, 4H), 2.37(s, 3H),
2.15 (t, 2H, J= 11Hz), 1.97(d, 2H,J =12Hz), 1.50(dd, 2H,J =4
and 11 Hz). 13C NMR (125 MHz, CDCl3) δ 140.5, 140.4, 129.1,
129.0, 128.9, 128.6, 128.5, 127.9, 126.3, 125.3, 60.7, 54.0, 51.0, 45.0,
38.9, 33.8, 32.5, 24.0, 21.6. HRMS (EI) m/e calcd for C21H29N2
[MþH]þ 309.2325, obsd 309.2319.
N-(3-Methylbenzyl)-N-(1-phenylethylpiperidine-4-yl)propio-
namide (10). Propionic anhydride (0.55 mL, 4.35 mmol) was

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2881
axial. The lowest-energy conformation of the trans isomer of the
“carba”-analogue (8b) has both substituents on the cyclohexane
ring in the equatorial position. In the lowest-energy conforma-
tions of compounds 1 and 10, both substituents on the piper-
idine ring are equatorial.
fentanyl active at the μ-opioid receptor and I2-imidazoline binding
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The model of the μ opioid receptor in the activated state,
constructed by Mosberg et al. by homology modeling based on
the crystal structure of rhodopsin10 (http://mosberglab.phar.
umich.edu/resources/), was used in the docking studies. A
dielectric constant of 1 was used for the receptor docking studies
as well as in all subsequent calculations involving receptor-
ligand complexes. Flexible docking of the lowest-energy con-
formation of each compound was performed using the software
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(11) Kane, B. E.; Svensson, B.; Ferguson, D. M. Molecular recognition
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€
program GLIDE (Schrodinger LLC). Each of the resulting
ligand-receptor complexes was minimized using the conjugate
gradient approach.27 Molecular dynamics simulations of 100 ps
at 300 K were performed in order to assess the stability of each
complex. In each case, no significant change in the complex
structure was observed during the simulation.
(15) Jagerovic, N.; Cano, C.; Elguero, J.; Goya, P.; Callado, L. F.;
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Acknowledgment. This work was financially supported by
the U.S. National Institutes of Health (Grant DA-004443)
and the Canadian Institutes of Health Research (Grant MOP-
89716).
Supporting Information Available: NMR data for stereo-
chemical assignment. This material is available free of charge
via the Internet at http://pubs.acs.org.
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