Synthesis and biological evaluation of new opioid agonist and neurokinin-1 antagonist bivalent ligands

Article abstract of DOI:10.1016/j.bmc.2011.08.027

Newly designed bivalent ligands - opioid agonist/NK1-antagonists have been synthesized. The synthesis of new starting materials - carboxy-derivatives of Fentanyl (1a-1c) was developed. These products have been transformed to 'isoimidium perchlorates' (2a-

Full text of DOI:10.1016/j.bmc.2011.08.027

Bioorganic & Medicinal Chemistry 19 (2011) 6135–6142
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of new opioid agonist
and neurokinin-1 antagonist bivalent ligands
Ruben Vardanyan a, , Vlad K. Kumirov a, Gary S. Nichol a, Peg Davis b, Erika Liktor-Busa b, David Rankin b,
⇑
Eva Varga b, Todd Vanderah b, Frank Porreca b, Josephine Lai b, Victor J. Hruby a
a Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85719, USA
b Department of Pharmacology, University of Arizona Health Sciences Center, Tucson, AZ 85724, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Newly designed bivalent ligands—opioid agonist/NK1-antagonists have been synthesized. The synthesis of
new starting materials—carboxy-derivatives of Fentanyl (1a–1c) was developed. These products have been
transformed to ‘isoimidium perchlorates’ (2a–c). The new isoimidium perchlorates have been successfully
Received 11 April 2011
Revised 8 August 2011
Accepted 11 August 2011
Available online 24 August 2011
implemented in nucleophilic addition reactions, with
give the target compounds—amides (3a–c). Perchlorates (2a–c) successfully undergo reactions with other
nucleophiles such as alcohols, amines or hydrazines. The obtained compound 3b exhibited -opioid agonist
L-tryptophan 3,5-bis(trifluoromethyl)benzyl ester to
l
Keywords:
Analgesic
Bivalent ligands
activity and NK1-antagonist activity and may serve as a useful lead compound for the further design of a
new series of opioid agonist/NK1-antagonist compounds.
Published by Elsevier Ltd.
l-Opioids
NK1 antagonist
Fentanyl
1. Introduction
particularly to bind to opioid and NK1 receptors, and to produce
antinociception is becoming increasingly attractive.10
Opioid analgesics are the mainstay for treatment of moderate to
severe pain. Although opioids are still the drugs of choice for the
treatment of severe pain, they are less effective in the chronic pain
states and generally are accompanied by undesired secondary ef-
fects. On the other hand the tachykinin peptide family members,
such as Substance P, are known to play an important role as a
transmitter of pain signals and the actions of tachykinins are med-
iated by the neurokinin-1 (NK1) receptor. Impressive results of at-
tempts to combine action of two pharmacophores—opioid agonist
and NK1 antagonist have been successfully demonstrated in our
laboratory.1–9
Compounds that contain two pharmacophores or a single
pharmacophore and a non-pharmacophore recognition unit linked
through a connecting spacer or without it have been termed
‘bivalent ligands’.11,12 Over the past few decades, bivalent ligands
have been developed for a variety of G protein-coupled receptor
targets, including opioids.13 With the aim to improve the
pharmacological profile of potential analgesics while minimizing
common opioid-induced side-effects, dimers of monovalent ‘parent’
opioids have been prepared and include: peptide dimers, mixed
peptide-non-peptide bivalent ligands and dual non-peptide dimers.
Structures, using an opioid pharmacophore in combination with a
non-opioid pharmacophore, have also been prepared.14
In these earlier studies novel bivalent peptides with opioid ago-
nist and NK1 antagonist activities were designed and synthesized.
The synthesized peptides (Fig. 1) have excellent agonist activity for
l-Opioid receptors (MORs) are G-protein-coupled receptors
(GPCRs) that mediate the physiological effects of endogenous opioid
neuropeptides and synthetic opioid drugs. MORs are coexpressed
with NK1 receptors in several regions of the central nervous system
(CNS) that control opioid dependence and reward.15 Therefore, the
principal aim of this article is to design and synthesize bivalent
both l- and d-opioid receptors and exhibit high antagonist activity
for NK1 receptors both in vitro and in vivo. These results indicate
that the rational design of bifunctional ligands with opioid agonist
and NK1 antagonist activities could be accomplished and might
provide a tool for treatment of chronic pain states. Bivalent ligands
are likely to yield new drugs with improved properties. The use of
novel single molecules possessing multiple analgesic targets,
non-peptide compounds which combine l-opioid receptor agonist
and NK1 receptor antagonist pharmacophores in one molecule.
Novel small molecules represent variations of combinations of
structures of the opioid agonist Fentanyl and NK1 antagonist phar-
macophore L732138 and are presented in Figure 2. Ionic pair com-
pounds in which both cation and anion constituents theoretically
could play equal roles and thus could represent a new generation
of drugs.
⇑
Corresponding author. Tel.: +1 520 621 86 17; fax: +1 520 621 84 07.
E-mail address: vardanyr@email.arizona.edu (R. Vardanyan).
0968-0896/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmc.2011.08.027

6136
R. Vardanyan et al. / Bioorg. Med. Chem. 19 (2011) 6135–6142
NK1 Pharmacophore
piperidine ring H(2,6-ax) appear as 12 Hz triplets at d 2.80 (1a), 2.79
(1b), 3.01 (1c). H(2,6-eq) appear as 12 Hz doubletsat d 3.42 (1a), 3.41
(1b), 3.56 (1c). 2-Phenethyl methylene protons are enantiotopic and
appear at d 3.00 and 2.90 (1a), 2.99 and 2.89 (1b), 3.15 and 2.95 (1c).
Note should be taken of the downfield shift observed for (1c) and not
for (1a) and (1b). H(4-ax) appears as a triple-triplet at d 4.70 (1a),
4.72 (1b), 4.74 (1c) and does not show a relative downfield shift.
The H(2,6-ax) signals appear as triplets and not quartets, which
probably is due to rapid exchange on the HN with the methanol-d4
solvent.
The acids obtained could not be converted into acid chlorides by
standard methods. A convenient method for creation of substituted
fentanyls was developed by us based on the idea of sporadically and
rarely reported ‘succinisoimidium perchlorates chemistry’ which
was ‘born’ over 100 years ago21 and was minimally developed by
the late 1970s.22–25 Now it has become revitalized, developed,
simplified and extended, including new isoimidium compounds—
H-Tyr-D-Ala-Gly-Phe-Xxx-Pro-Leu-Trp-O-3,5-Bzl(CF3)2
Opioid Pharmacophore
Figure 1. Protoptype peptide dimers.
The analogue approach was demonstrated in a patent16 for opi-
oid-NSAID (non-steroid anti-inflammatroy drug) ion pairs wherein
the opioid represents a cation and a NSAID represents an anion.
Under the conditions prescribed for their use, that ion pair com-
pounds exhibit poor or complete insolubility, but excellent chem-
ical stability in low pH environments, such as those found in the
stomach.
glutarisoimidium
lorates (Scheme 2, 2b,c). All three isoimidium perchlorates (2a–c)
were treated with -tryptophan 3,5-bis(trifluoromethyl)benzyl es-
and
3-oxaglutarisoimidium
perch-
2. Results and discussion
2.1. Chemistry
L
ter for creation of the main target of this paper—amides (3a–c).
The isoimidium perchlorate products obtained (2a–c) have been
also checked for their ability to participate in different nucleophilic
addition reactions. This method as developed could be implemented
for any other nucleopiles as alcohols (4a), amines (5b) or hydrazines
(6c), as well as to other amino acids or peptides. These results show
thattheisoimidiumperchlorates(2a–c)couldbesuccessfullyimple-
mented in reactions with other nucleophiles (Scheme 2).
The synthesis of one of the starting materials
a-N-Boc protected
3,5-bis(trifluoromethyl)benzyl ester of -tryptophan, has been
L
described.17,18 The large scale synthesis of functionalized
Fentanyls—carboxyfentanyl (1a), carboxymethylfentanyl (1b) and
fentanyl derivative (1c) starting from 1-phenethyl-N-phenylpiperi-
din-4-amine19 and appropriate acid anhydride was carefully
worked out by us (Scheme 1). The three compounds differ essen-
tially only in the construction of the side acid chain.
A ‘peptide chemistry’ methods with the carbodiimide activation
of carboxyl function with BOP/HOBt [benzotriazole-1-yl-oxy-
Crystal structures of all three compounds were obtained.20 Two
of the crystal structures (1b,c) share several structural similarities,
including the length of the chain, while the third (1a), with a shorter
chain, is quite different. In particular the structures of 1b and 1c are
solvated zwitterions featuring hydrogen bonding between adjacent
ions. Proton transfer occurs via these hydrogen bonds, and in most
cases is complete. Compound 1a does not form the same dimer motif
and has not formally formed a zwitterion. NMR data confirm the ob-
tained X-ray results. 1H NMR for the carboxylic acids suggests that a
zwitterion may exist on (1c), but not (1a) or (1b). The protons of the
tris(dimethylamino)-phosphonium
hexafluorophosphate]/
[1-hydroxybenzotriazole] or with EDAC/HOBt [1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide]/[1-hydroxybenzotriazole]
were also successfully implemented for creation of covalently
bonded Fentanyl carboxylic acids with tryptophan 3,5-bis(trifluo-
romethyl) benzyl ester with high yields.
The structures of isoimidium perchlorates (2a–c) produced were
confirmed by NMR methods using 1H–13C HSQC and DQF-COSY.
Existing earlier data on confirming the structures of isoimidium
CF3
NH
O
O
X
O
N
N
N
H
CF3
O
O
O
H
N
CF3
N
N
O
O
NH
CF3
Fentanyl (Opioid part)
L 73213 (NK-1 Antagonist part)
CF3
NH
O
O
_
+
X
O
N
O
H2N
CF3
O
X = -- ; -CH2 -; -O-
N
Figure 2. Synthetic approach.

R. Vardanyan et al. / Bioorg. Med. Chem. 19 (2011) 6135–6142
6137
O
O
O
OH
N
N
HN
HO
N
N
O
O
O
O
O
O
O
H+
H+
N
1b
1a
O
O
O
O
H+
O
O
O
HO
N
N
1c
Scheme 1. Synthesis of functionalized Fentanyls.
CF3
CF3
O
NH
NH
O
O
O
O
O
+
+
H3N
Cl-
+
O
X
O
X
X
CF3
N
N
N
H
CF3
N
N
OH
N
Ac2O
O
O
Et3N
HClO4
N
-
NK1 Antagonist
(L 73213 )
1a-c
2ClO4
Isoimidium Salts 2a-c
3a-c
Functionalized Fentanyls
C2H5OH
PhCH2NH2
NH2NH2
O
O
O
H
N
OC2H
5
NHNH2
N
N
N
N
O
O
O
N
N
4a
5a
6a
a - X = -;
b - X = CH2;
c - X = O;
Scheme 2. Synthesis of succinisoimidium salts and their use in nucleophilic addition reactions.
perchlorates was based only on IR data.22–25 Compound 2a exists in
two conformations on the NMR scale based on the HSQC data (Table
S1). The major conformation is listed first; the minor conformation
is listed second. The two forms were distinguished by HSQC using
relative 1H integration (1.0 major: 0.8 minor) and 13C peak heights.
The largest differences in chemical shifts between the two confor-
mations occur around the C@N group. The difference in chemical
shift decreases proportionally with the distance from the C@N
group. Compounds 2b and 2c, which are both six-membered rings,
exist in one conformation on the NMR scale based on the HSQC data
(Tables S2 and S3). Isoimidium perchlorates 2a–c are all protonated
at the N(1) position due to a large 13 Hz quartet splitting of the
H(2,6-ax) signal and a broad singlet at 6.8 ppm corresponding to
the H(1N).
The next part of our investigation was devoted to creation of io-
nic pair compounds (7a–c) Scheme 3 based on the newly synthe-
sized acids (1a–c) and
L-tryptophan 3,5-bis(trifluoromethyl)
benzyl ester. The method we developed for synthesis is very sim-
ple. The potassium salts of acids (1a–c) obtained from the corre-
sponding acids and potassium hydroxide are simply mixed with

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R. Vardanyan et al. / Bioorg. Med. Chem. 19 (2011) 6135–6142
CF3
NH
+
O
O
O
O
O
_
+
H2N
H3N
Cl-
CF3
X
X
O
O
N
N
OK
N
N
O
CF3
O
+
NH
-KCl
CF3
1a- c (K)
NK1 Antagonist
(L 73213)
7a-c
Functionalized Fentanyls
Scheme 3. Synthesis of ionic bifunctional opioid/NK1 antagonist compounds.
Figure 3. Simulated spectrum (top) and actual phenethyl methylenene multiplets in 7b (bottom) as an example. Arrows point to small outer peaks of the multiplets to show
consistency between simulation and experimental.
Table 1
L
-tryptophan 3,5-bis (trifluoromethyl)benzyl ester hydrochloride
Ki values of the bivalent ligands and the parent compounds
followed by removal of the resulting KCl (Scheme 3).
1H–13C HSQC and DQF-COSY were used to confirm the six
compounds (3a–c) and (7a–c) with covalent and ionic links. NMR
for covalent compounds showed two different conformers or dia-
stereomers present in solution—1a (1.0:0.7), 1b (1.0:0.9), 1c
(1.0:0.7). The variation in chemical shift (1H and 13C) happens
mostly in the Trp-O-3,5-Bzl(CF3)2 moiety, with Fentanyl chemical
shifts appearing as broadened averages. This may be caused by
the chiral center on the Trp-O-3,5-Bzl(CF3)2 moiety or slow
rotation of the aromatic rings. It is worth noting that H(4-ax) in
Fentanyl appears at two different chemical shifts, unlike other
Fentanyl protons. Ionic compounds only show a difference in
chemical shifts in the benzyl moiety, appearing in a 1:1 ratio.
The covalent link can be identified in 1H NMR spectra by the amide
doublet at 6–7 ppm. The ionic link can be easily seen in (7c) with a
prominent ammonium peak of the tryptophan at 5.4 ppm, which is
weakly observed in (7a) and (7c).
Compd
Ki [3H]DAMGO (nM)
Ki [3H]Substance P (nM)
3a
3b
3c
7a
7b
7c
130
120
400
>10,000
3900
1300
13 1.2
6.8 0.5
31 1.2
21 4.7
44 2.3
23 9.8
and spin simulation using MestreNova. Instead of two triplets,
there often appear as two multiplets (m⁄) that correspond to the
trans conformation of the phenethyl group observed, which is con-
sistent with X-ray structures. There may be hindered rotation of
the phenethyl group that cause those multiplets, which would ap-
pear as two triplets due to motional averaging (3JHH 6–8 Hz). Sim-
0
ulation parameters (Fig. 3): 600 MHz, 1.5 Hz linewidth, dA,A = 3.75,
Most of the compounds showed enantiotopic (AA0BB0) methy-
lene groups in the phenethyl moiety, confirmed by 1H–13C HSQC
dB,B = 2.63, JAA = 2JBB = 12 Hz (geminal), JAB = 3JA B = 11 Hz (anti),
2
3
0
0
0
0
0
3
JAB = 3JA B = 5 Hz (gauche).
0
0

R. Vardanyan et al. / Bioorg. Med. Chem. 19 (2011) 6135–6142
6139
Table 2
Binding data values of compounds 3a–c and 7a–c at d- and l-opioid receptors
Compd
Delta opioid activity
MVD inhibition contraction MVD
antagonism at
Mu opioid activity
GPI inhibition of contraction height
at 1 M (or IC50 (nM))
NK1 activity
GPI
Concentration tested as
NK1 antagonist
NK1 antagonism
nM Ke SEM
height at 1
lM (%)
l
Antagonism at
1
lM
1
lM
3a
3b
3c
7a
7b
7c
14
13
30
4.3
19.8
17.1
None
None
None
None
None
None
410 42
55 12
30%
1.8%
19.5%
11%
—
—
None
None
None
None
1
l
M
240 39
21 4.3
480 12
210 42
500 130
490 68
100 nM
1
1
1
1
lM
lM
lM
lM
Table 3
Results for the MVD and GPI/LMMP testing of starting compounds 1a–c
Compd
MVD
(agonist activity at 1
GPI/LMMP
DPDPE antagonism at 1
lM in the MVD
PL-017 antagonism at 1 lM in the GPI/LMMP
l
M or nM IC50 SEM)
1a
1b
1c
10
10
10
l
l
l
M—6.9%
M—19.3%
M—10.4%
10
10
10
l
l
l
M—2%
M—10%
M—0%
None at 10
None at 10
None at 10
—
l
l
l
M
M
M
None at 10
None at 10
None at 10
—
lM
lM
lM
Fentanyl citrate
(n = 1) 7.2 nM
1.9 0.70
2.2. Pharmacology
exhibited the best agonist potency at the l-opioid receptor, indi-
cating that this compound may serve as a useful lead compound
for further design of new, improved bivalent opioid-agonist/NK1-
antagonist compounds. The other compounds in the series exhib-
ited low affinities for the hMOR in radioligand binding assays
The affinities of the bivalent compounds 3a–c and 7a–c to opi-
oid and NK1 receptors have been measured using competitive radi-
oligand binding assays in recombinant cell lines expressing human
l
or human NK1 receptors, respectively, as described previously.1–
and accordingly were weak agonists at the
tors in in vitro functional assays.
l- and d-opioid recep-
9 The functional characterization of the same compounds 3a–c and
7a–c, and the starting compounds 1a–c at d- and -opioid recep-
tors, has been carried out using guinea pig isolated ileum/longitu-
dinal muscle myenteric plexus (GPI/LMMP) as a source of opioid
l
3. Conclusions
l
and NK1 receptors and mouse vas deferens (MVD) as a source of d
opioid receptors as is described in the part 4.2 ‘Biological assays’.
The results are presented in Tables 1–3.
The overall conclusion is that the results obtained in these stud-
ies confirm that properties obtained for peptide molecules could be
successfully transferred for creation of novel small molecules and
particularly bivalent ligands by maintaining appropriate distance
between the two active pharmacophores. Probably we can take
into account data published concerning bivalent ligands containing
2.3. Discussion
The biological assays indicate that the synthesized starting
functionalized Fentanyl derivatives—(1a–c) themselves dramati-
cally lose analgesic activity in comparison with Fentanyl (Table
3), which probably can be explained with the appearance of the
‘bulky’ carboxyl group with negative charge in the Fentanyl struc-
ture, and which is more or less restored on distancing of the car-
boxyl group from the ethyl part of the propionyl group, (compare
(1a–c) as well as on transformation of a charged group back to
covalently bonded derivatives.
At the same time the behavior of the tryptophan 3,5-bis(trifluo-
romethyl) benzyl ester does not change so sharply on variations of
substituents on the nitrogen atom, even on its transformation to a
charged form in the ionic series. The obtained data suggests that
both ionic and covalently linked bivalent ligands compete with
high affinity for [3H] Substance P binding sites in recombinant
CHO cells expressing the human NK1 receptor. Bivalent ligands
with covalent linkage between the two pharmacophors exhibit re-
duced affinities, relative to the parent opioid (Fentanyl) to the rat
MOR and for [3H]DAMGO binding sites with very low affinities,
confirming that introduction of a bulky carboxyl group with nega-
l
-opioid agonist and CCK2 receptor antagonist pharmacophores
linked through spacers containing 16–22 atoms, which indicate
that for the pair of pharmacophores ( -opioid agonist and CCK2
l
receptor antagonist) spacers cannot be shorter than nine atoms.26
In the case of close fusion of two pharmacophores it is more
probable to get a molecule with new pharmacological properties,
unless these pharmacophores can overlap (share common struc-
tural features).27 The particular conclusion of this work is that
the place of possible conjugation for Fentanyl and any other known
pharmacophore molecule has been chosen correctly and the series
of novel hybrid covalently bonded Fentanyl/NK1 antagonist mole-
cules will be continued: a. by variations of the length and the nat-
ure of the linker between opioid and NK1 antagonist parts; and b.
what is conceptually the same, by elongation of the peptide chain
to synthesize Fent-Leu-Trp-O-3,5-Bzl(CF3)2, Fent-Pro-Leu-Trp-O-
3,5-Bzl(CF3)2 or Fent-Xxx-Pro-Leu-Trp-O-3,5-Bzl(CF3)2 chimeric
compounds according to previously obtained data.1–9
4. Experimental
4.1. Chemistry
tive charge into the Fentanyl pharmacophore may interfere with
opioid receptor recognition.
l
In functional assays, all compounds, where opioid and NK1
antagonist are bound covalently or by ionic bonds showed NK1
antagonism in vitro with no NK1 agonist activity up to 1 lM.
Compound 3b was the most potent NK1 antagonist by more than
10-fold in comparison with the others. The same compound 3b
4.1.1. General method for the synthesis of the 4-oxo-4-((1-
phenethylpiperidin-4-yl)(phenyl)amino) alcanoic acids (1a–c)
A mixture of (0.01) mol of [1-(2-phenyl)-ethyl-piperidin-4-yl]-
phenyl-amine, 0.011 mol of appropriate acid anhydride (succinic,

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R. Vardanyan et al. / Bioorg. Med. Chem. 19 (2011) 6135–6142
glutaric or diglycolic) and 2–3 drops of acetic acid in 30 mL of
dichloromethane was heated in a closed pressure proof flask on a
boiling water bath for 5 h and left for the night at room tempera-
ture. The solvent was evaporated; ether was added and on cooling
(ice) 0.022 mol of NaHCO3 as a 5% solution was added. The ether
layer was separated, and the water solution was added 50 mL of
chloroform. The mixture was neutralized on cooling and stirring
by 0.022 mol of acetic acid. The chloroform solution was dried on
MgSO4 and after evaporation of solvent the product was dissolved
in the minimum quantity of boiling ethyl acetate from which on
cooling crystals of the appropriate acid was separated with 75–
90% yield.
DQF-COSY investigations of the obtained compounds are summa-
rized in the Supplementary data.
4.1.3. Methods for the synthesis of the amides composed of L-
tryptophane 3,5-bis(trifluoromethyl) benzyl esters and carboxy
Fentanyls (3a–c)
A. General method for the addition of isoimidium perchlorates to
nucleophiles. To the stirred solution of 0.5 mmol of the 3,5-bis(tri-
fluoromethyl)benzyl ester of tryptophan hydrochloride dissolved
in 5 mL CHCl3 at 0 °C was added dropwise triethylamine (2.2 mmol
(4.4 equiv) dissolved in 5 mL CHCl3. After 15 min. solution of
0.5 mmol of one of isoimidium perchlorates (2a–c) dissolved in
5 mL CHCl3/1 mL CH3CN mixture was added dropwise to a solution
of nucleophile. The mixture was left for a night, cooled (ice bath)
and was washed consecutively with 1.0 mmol NaHCO3 5% solution
of, 5% acetic acid and water. The organic layer was dried over anhy-
drous MgSO4 and concentrated under reduced pressure. The resid-
ual oil were finally purified by flash chromatography on SiO2
column using 4:1 CHCl3/MeOH solvent system, which gave com-
pounds (3a–c).
4.1.1.1. 4-Oxo-4-((1-phenethylpiperidin-4-yl)(phenyl)amino) bu
tanoic acid 1a. Crystalline solid (89.7%), mp 119–120 °C, 1H NMR
(600 MHz, methanol-d4) d 7.50 (t, J = 7.3 Hz, 2H), 7.48–7.43 (m,
1H), 7.32–7.25 (m, 4H), 7.25–7.18 (m, 3H), 4.70 (tt, J = 12.0,
3.5 Hz, 1H), 3.42 (d, J = 12.2 Hz, 2H), 3.00 (m, 2H), 2.90 (dd,
J = 10.5, 6.1 Hz, 2H), 2.80 (t, J = 12.0 Hz, 2H), 2.42 (t, J = 6.8 Hz,
2H), 2.18 (t, J = 6.8 Hz, 2H), 2.00 (d, J = 12.2 Hz, 2H), 1.62 (dq,
J = 3.3, 13.1 Hz, 2H). 13C NMR (150 MHz, methanol-d4) d 172.77,
138.08, 137.48, 130.16, 129.40, 128.65, 128.31, 126.44, 58.09,
51.92, 50.76, 30.85, 30.47, 30.25, 28.14. EI-MS: m/z 381; HRMS
calcd for C23H29N2O3: 381.2178; found (ESI, [M+H]+): 381.2183.
B. General coupling method. To the stirred mixture of one of the
amino acids (1a–c), HOBt (1.1 equiv), BOP (1.1 equiv) and 3,5-
bis(trifluoromethyl)benzyl
ester
tryptophan hydrochloride
(1.1 equiv) dissolved in CHCl3 (1.5 mL/mmol) at 0 °C was added
dropwise triethylamine (4.4 equiv). After stirring for 30 min at
0 °C, the mixture warmed up to room temperature and stirred
for an additional 3–5 h until the disappearance of starting compo-
nents (monitored by TLC). The reaction mixture was cooled (ice
bath) and was washed consecutively with 5% NaHCO3 solution
(three times), 5% acetic acid (two times) and water. The organic
layer was dried over anhydrous MgSO4 and concentrated under re-
duced pressure. The residual oil represents pure compounds (3a–c)
solidified at room temperature. Yields of compounds obtained by
both methods A and B are comparable and are around 80%.
1H–13C HSQC and DQF-COSY investigations of obtained compounds
are summarized in the Supplementary data.
4.1.1.2. 5-Oxo-5-((1-phenethylpiperidin-4-yl)(phenyl)amino) pe
ntanoic acid 1b. Crystalline solid (84.9%), mp 104–105 °C, 1H
NMR (600 MHz, methanol-d4) d 7.49 (t, J = 7.3 Hz, 2H), 7.45 (d,
J = 7.2 Hz, 1H), 7.29 (t, J = 7.2 Hz, 2H), 7.24– 7.19 (m, 5H), 4.72 (tt,
J = 12.0, 3.6 Hz, 1H), 3.42 (d, J = 12.3 Hz, 2H), 2.99 (m, 2H), 2.89
(m, 2H), 2.79 (t, J = 11.9 Hz, 2H), 2.10 (t, J = 7.3 Hz, 2H), 2.05–1.95
(m, 4H), 1.79 (dd, J = 14.7, 7.3 Hz, 2H), 1.61 (dd, J = 12.8, 3.2 Hz,
2H). 13C NMR (150 MHz, methanol-d4) d 173.27, 138.07, 137.56,
130.05, 129.44, 128.66, 128.32, 126.43, 58.11, 51.87, 50.78, 34.52,
33.99, 30.89, 28.19, 21.05. EI-MS: m/z 395; HRMS calcd for
C
24H31N2O3: 395.2325; found (ESI, [M+H]+): 395.2323.
4.1.3.1. 3,5-Bis(trifluoromethyl)benzyl 3-(1H-indol-3-yl)-2-(4-
oxo-4-((1-phenethylpiperidin-4-yl)(phenyl)amino)butanami-
do) propanoate 3a. EI-MS: m/z 793; HRMS calcd for
4.1.1.3. 2-(2-Oxo-2-((1-phenethylpiperidin-4-yl)(phenyl)amino)
ethoxy) acetic acid 1c. Crystalline solid (75.7%), mp 143–144 °C,
1H NMR (600 MHz, methanol-d4) d 7.55–7.45 (m, 3H), 7.35–7.26
(m, 4H), 7.25–7.22 (m, 3H), 4.74 (tt, J = 12.1, 3.6 Hz, 1H), 3.86 (d,
J = 6.0 Hz, 4H), 3.56 (d, J = 12.3 Hz, 2H), 3.15 (dd, J = 10.3, 6.6 Hz,
2H), 3.01 (t, J = 12.2 Hz, 2H), 2.95 (dd, J = 10.3, 6.6 Hz, 2H), 2.09
(d, J = 12.7 Hz, 2H), 1.72 (dd, J = 12.8, 2.8 Hz, 2H). 13C NMR
(150 MHz, methanol-d4) d 174.52, 170.05, 136.78, 136.17, 129.90,
129.70, 129.28, 128.46, 128.33, 126.67, 69.40, 68.52, 57.60, 51.69,
50.81, 30.36, 27.41, 19.81. EI-MS: m/z 397; HRMS calcd for
C
43H43F6N4O4: 793.3183; found (ESI, [M+H]+): 793.3175.
4.1.3.2. 3,5-Bis(trifluoromethyl)benzyl 3-(1H-indol-3-yl)-2-(5-
oxo-5-((1-phenethylpiperidin-4-yl)(phenyl)amino)pentanami-
do) propanoate 3b. EI-MS: m/z 807; HRMS calcd for
C
44H45F6N4O4: 807.3340; found (ESI, [M+H]+): 807.3333.
4.1.3.3. 3,5-Bis(trifluoromethyl)benzyl 3-(1H-indol-3-yl)-2-(2-
(2-oxo-2-((1-phenethylpiperidin-4-yl)(phenyl)amino)ethoxy)
acetamido)propanoate 3c. EI-MS: m/z 809; HRMS calcd for
C
23H29N2O4: 397.2127; found (ESI, [M+H]+): 397.2123.
43H43F6N4O5: 809.3132; found (ESI, [M+H]+): 809.3128.
4.1.2. General method for the synthesis of the isoimidium
perchlorates (2a–c)
C
The stirred mixture of 0.5 mmol of one of the amino acids
(1a–c) in 1.5 mL of acetic anhydride was heated slightly until
the beginning of dissolution of the starting amino acids and the
heater was turned off (but remains under the flask). The stirring
was continued for 1 h. Then the mixture was cooled (ice bath)
and 0.15 mL of 70% HClO4 was added dropwise. The solution
obtained was allowed to come to room temperature overnight
and 5 mL of dry ether was added with stirring. After 15–20 min
the liquid was decanted from the precipitated perchlorate, and
the residue was washed with three 5 mL portions of ether. For
4.1.4. General method for the addition of nucleophiles to
isoimidium perchlorates 4a, 5a, 6a
To the stirred solution of 0.5 mmol nucleophile [C2H5OH,
PhCH2NH2, H2NNH2 (10-fold excess)] dissolved in 5 mL CHCl3 at
0 °C was added dropwise triethylamine (2.2 mmol, 4.4 equiv) dis-
solved in 5 mL CHCl3. After 15 min. solution of 0.5 mmol of isoim-
idium perchlorate (2a) dissolved in 5 mL CHCl3/1 mL CH3CN
mixture was added dropwise to a solution of nucleophile. The mix-
ture was left for a night, cooled (ice bath) and was washed consec-
utively with 1.0 mmol NaHCO3 5% solution of, 5% acetic acid and
water. The organic layer was dried over anhydrous MgSO4 and con-
centrated under reduced pressure. The residual oil were finally
purified by flash chromatography on SiO2 column using 4:1
CHCl3/MeOH solvent system, which gave compounds (4a, 5a, 6a).
analytical purposes the residue was dried overnight in
a
vacuum-desiccator over phosphorous pentoxide. For synthetic
purposes it was dissolved in 5 mL CHCl3/1 mL CH3CN mixture
and added to a solution of the nucleophile. 1H–13C HSQC and

R. Vardanyan et al. / Bioorg. Med. Chem. 19 (2011) 6135–6142
6141
4.1.4.1. Ethyl 4-oxo-4-((1-phenethylpiperidin-4-yl)(phenyl)a-
mino) butanoate 4a. Crystalline solid (75.7%), mp 145–146 °C,
1H NMR (600 MHz, CDCl3) d 7.57 (d, J = 7.5 Hz, 2H), 7.48–7.33 (m,
6H), 7.13 (d, J = 7.4 Hz, 2H), 4.68 (t, J = 11.5 Hz, 1H), 4.09 (q,
J = 7.1 Hz, 2H), 3.36 (br s, 2H), 2.76 (br s, 2H), 2.52 (t, J = 6.4 Hz,
2H), 2.20 (t, J = 6.3 Hz, 2H), 2.14–2.07 (m, 2H), 1.93 (d,
J = 13.1 Hz, 2H), 1.23 (t, J = 7.1 Hz, 3H). 13C NMR (151 MHz, CDCl3)
d 172.98, 171.85, 131.48, 130.18, 130.14, 130.03, 129.89, 129.77,
129.36, 129.29, 60.62, 51.60, 45.93, 30.00, 29.37, 27.59, 14.29. EI-
MS: m/z 409; HRMS calcd for C25H33N2O3: 409.2486; found (ESI,
[M+H]+): 409.2478.
Salt of 2-(2-oxo-2-((1-phenethylpiperidin-4-yl)(phenyl) amino)
ethoxy)acetic acid (1c) and 3,5-bis(trifluoromethyl) benzyl ester
tryptophan (7c)
4.2. Biological assays
4.2.1. Tissue bioassays
4.2.1.1. Isolated guinea pig ileum/longitudinal muscle with
myenteric plexus. Male Hartley guinea pigs under CO2 anesthesia
were sacrificed by decapitation and a non-terminal portion of the
ileum removed. The longitudinal muscle with myenteric plexus
(LMMP) was carefully separated from the circular muscle and cut
into strips as described previously.8 These tissues were tied to gold
chains with suture silk and mounted between platinum wire elec-
trodes in 20 mL organ baths at a tension of 1 g and bathed in oxy-
genated (95:5 O2/CO2) Kreb’s bicarbonate buffer at 37 °C, then
stimulated electrically (0.1 Hz, 0.4 ms duration) at supramaximal
voltage. Following an equilibration period, compounds were added
4.1.4.2.
N1-Benzyl-N4-(1-phenethylpiperidin-4-yl)-N4-phenyl-
succinamide 5a. Crystalline solid (75.7%), mp 134–135 °C, 1H
NMR (600 MHz, CDCl3) d 7.42–7.34 (m, 3H), 7.33–7.27 (m, 2H),
7.27–7.19 (m, 8H), 7.06 (dd, J = 7.9, 1.3 Hz, 2H), 6.52 (s, 1H), 4.57
(tt, J = 12.1, 3.8 Hz, 1H), 4.40 (d, J = 5.8 Hz, 2H), 3.43 (s, 2H), 2.86
(d, J = 11.6 Hz, 2H), 2.50–2.37 (t, J = 6.4 Hz, 2H), 2.24 (t, J = 6.4 Hz,
2H), 2.06 (t, J = 11.6 Hz, 2H), 1.69 (d, J = 12.0 Hz, 2H), 1.38 (qd,
J = 12.2, 3.3 Hz, 2H). 13C NMR (151 MHz, CDCl3) d 172.44, 171.80,
138.70, 138.42, 138.21, 130.40, 129.58, 129.22, 128.67, 128.63,
128.27, 127.74, 127.37, 127.13, 63.04, 53.06, 52.91, 43.54, 31.66,
cumulatively to the bath in volumes of 14–60 lL until maximum
inhibition was reached. A PL-017 dose–response curve was con-
structed to determine tissue integrity before analog testing.
31.21, 30.52. EI-MS: m/z 407; HRMS calcd for
C
30H36N3O2:
4.2.1.2. Mouse isolated vas deferens preparation. Male ICR mice
under CO2 anesthesia were sacrificed by cervical dislocation and the
vasadeferentiaremoved. Tissuesweretiedtogoldchainswithsuture
silk and mounted between platinum wire electrodes in 20 ml organ
baths at a tension of 0.5 g and bathed in oxygenated (95:5 O2/CO2)
magnesium free Kreb’s buffer at 37 °C, then stimulated electrically
(0.1 Hz, single pulses, 2.0 ms duration) at supramaximal voltage as
previously described.8 Following an equilibration period, com-
pounds were added to the bath cumulatively in volumes of 14–
470.2802; found (ESI, [M+H]+): 470.2797.
4.1.4.3. 4-Hydrazinyl-4-oxo-N-(1-phenethylpiperidin-4-yl)-N-
phenylbutanamide 6a. Crystalline solid (75.7%), mp 215–216 °C,
1H NMR (600 MHz, CDCl3) d 7.85 (s, 1H), 7.44–7.33 (m, 3H), 7.24
(t, J = 7.5 Hz, 2H), 7.18–7.09 (m, 5H), 4.61 (tt, J = 12.1, 3.9 Hz, 1H),
3.59 (br s, 2H), 2.99 (d, J = 11.7 Hz, 2H), 2.75–2.67 (m⁄, 2H), 2.59–
2.48 (m⁄, 2H), 2.37 (t, J = 6.5 Hz, 2H), 2.26 (t, J = 6.5 Hz, 2H), 2.13
(td, J = 11.9, 1.7 Hz, 2H), 1.79 (d, J = 12.0 Hz, 2H), 1.44 (qd,
J = 12.3, 3.8 Hz, 2H). 13C NMR (151 MHz, CDCl3) d 173.24, 171.56,
140.19, 138.22, 130.33, 129.51, 128.61, 128.60, 128.38, 126.04,
60.37, 53.02, 52.72, 33.75, 30.70, 30.44, 29.42. EI-MS: m/z 395;
HRMS calcd for C23H31N4O2: 395.5173; found (ESI, [M+H]+):
395.5167.m⁄—The ethylene group multiplets are enantiotopic
(AA’BB’). Spin simulation was used to model the multiplets with
60 lL until maximum inhibition was reached. A DPDPE dose–
response curve was constructed to determine tissue integrity before
analog testing.
4.2.1.3. Agonist and antagonist testing. Compounds were tested
as agonists by adding cumulatively to the bath until a full dose–
response curve was constructed or to a concentration of 1 lM.
good accuracy. Simulation parameters (Fig. 3): 600 MHz, 1.5 Hz
Compounds were tested as antagonists by adding to the bath
2 min before beginning the cumulative agonist dose-response
2
linewidth, dA,A = 3.0, dB,B = 3.3, JAA = 2JBB = 12 Hz (geminal),
0
0
0
0
3
3
JAB = 3JA B = 11 Hz (anti), JAB = 3JA B = 5 Hz (gauche). Trans-confor-
curves of the delta (DPDPE) or
P) agonists.
l (PL-017) opioid or NK1 (Substance
0
0
0
0
mation was taken from the X-ray structures.
4.1.5. General method for the synthesis of the ion pairs
composed of L-tryptophane 3,5-bis(trifluoromethyl)benzyl
esters and carboxyFentanyls (7a–c)
4.2.1.4. Analysis. Percentage inhibition was calculated using the
average tissue contraction height for 1 min preceding the addition
of the agonist divided by the contraction height 3 min after expo-
sure to the dose of agonist. IC50 values represent the mean of not
less than four tissues. IC50 and Emax estimates were determined
by computerized non-linear least-squares analysis (FlashCalc).
To the stirred solution of 0.5 mmol KOH in 2 mL of CH3OH on
stirring was added 0.5 mmol of one of amino acids (1a–c) and
the mixture was left for a night. Solution of 0.5 mmol of 3,5-bis(tri-
fluoromethyl)benzyl ester tryptophan hydrochloride dissolved in
1.5 mL of CH3OH was added carefully on cooling (ice bath) and stir-
ring to obtained mixture. Ice bath was removed and mixture was
allowed to come to room temperature. After one hour of stirring
CH3OH was evaporated, CHCl3 was added and KCl was washed
out with 3 ꢀ 5 mL of water. The CHCl3 was evaporated and traces
of water was azeotropically removed by benzene 3 ꢀ 10 mL.
Remaining residual oil represents pure compounds (7a–c) which
solidified at room temperature, with 70–80% yield. Data from
1H–13C HSQC and DQF-COSY investigations of obtained compounds
are summarized in the Supplementary data.
4.2.2. Radioligand binding analysis
CHO cells, stably transfected with the human neurokinin
receptor-1, were obtained from Dr. James Krause (University of
Washington Medical School, St. Louis, MI) and cultured as
described elsewhere.1–9 Upon 80–100% confluency, a crude mem-
brane preparation was prepared from the cells for [3H]-SP inhibi-
tion binding studies. The protein concentration was determined
by the method of Bradford and the membranes were stored at
ꢁ80 °C. On the day of the experiments, membranes were thawed
and diluted with working buffer (50 mM Tris-Mg buffer, pH: 7.4,
Saltof5-oxo-5-((1-phenethylpiperidin-4-yl)(phenyl)amino)pen-
tanoic acid (1b) and 3,5-bis(trifluoromethyl)benzyl ester tryptophan
(7a).
Saltof5-oxo-5-((1-phenethylpiperidin-4-yl)(phenyl)amino)pen-
tanoic acid (1b) and 3,5-bis(trifluoromethyl)benzyl ester tryptophan
(7b).
50
l
g/mL bacitracin, 30
l
M bestatin, 10
lM captopril, 100 lM
PMSF, 1 mg/mL BSA, 10–15
l
g/tube) and were co-incubated with
0.5 nM [3H]-SP (final concentration, 0.1 mCi/mL, 135 Ci/mmol, Per-
kin Elmer) and various concentrations (10ꢁ5 to 10ꢁ10 M) of unla-
beled ligands, in a final volume of 1 mL. Nonspecific binding was
measured by 10 lM unlabeled SP and subtracted from total

6142
R. Vardanyan et al. / Bioorg. Med. Chem. 19 (2011) 6135–6142
3. Yamamoto, T.; Nair, P.; Largent-Milnes, T.; Jacobsen, N. E.; Davis, P.; Ma, S.;
Navratilova, E.; Lai, J.; Yamamura, H. I.; Vanderah, T. W.; Porreca, F.; Hruby, V. J.
Pept. Sci. 2008, 45, 137.
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binding. The reaction mixtures were incubated in a shaking water
bath for 20 min at 25 °C and were filtered onto 0.3% PEI pre-soaked
Whatman GF/B glass fiber filter (Gaithersburg, MD). Each vial was
washed three times with cold saline. Filters were loaded into scin-
tillation vials and EcoLite scintillation cocktail (MP Inc.) was added
then the filter-bound radioactivities were measured by liquid scin-
tillation counter (Beckman LS 6000SC). The IC50 values for each
compound was determined from nonlinear regression analysis of
data points, which were then converted to inhibitory constants
(Ki, nM) using the Cheng–Prusoff equation (GraphPad Prism 4 soft-
ware, San Diego, CA). All experiments were carried out in duplicate
and are means of at least three independent experiments.
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compoundin 0.6 mL CDCl3 or CD3OD. All NMR spectrawere acquired
on a Bruker DRX-600 spectrometer equipped with 5-mm Narolac
triple-resonance single z-axis gradient probe at 25 °C with
XWinNMR. Spectra were processed with MestreNova software.
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Acknowledgment
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This work was supported from the U.S. Public National Institute
of Health DA 13449, DA 06284 and DA 06789.
19. Vardanyan, R.; Vijay, G.; Nichol, G. S.; Liu, L.; Kumarasinghe, I.; Davis, P.;
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.08.027.
References and notes
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