Aspartic acid conjugates of 2-(3,4-dichlorophenyl)-N-methyl-N-[(1S)-1- (3-aminophenyl)-2-(1-pyrrolidinyl)ethyl]acetamide: κ Opioid receptor agonists with limited access to the central nervous system

Article abstract of DOI:10.1021/jm960459x

Aspartic acid conjugates of 2-(3,4-dichlorophenyl)-N-methyl-N-[(1S)-1- (3-aminophenyl)-2-(1-pyrrolidinyl)ethyl]acetamide (5) were synthesized and evaluated in mice for antinociceptive activity by intravenous and intracerebroventricular routes of administr

Full text of DOI:10.1021/jm960459x

4478
J . Med. Chem. 1996, 39, 4478-4482
Asp a r tic Acid Con ju ga tes of 2-(3,4-Dich lor op h en yl)-N-m eth yl-N-
[(1S)-1-(3-a m in op h en yl)-2-(1-p yr r olid in yl)eth yl]a ceta m id e: K Op ioid Recep tor
Agon ists w ith Lim ited Access to th e Cen tr a l Ner vou s System
An-Chih Chang,† Alan Cowan,‡ Akira E. Takemori,§ and Philip S. Portoghese*,†
Department of Medicinal Chemistry, College of Pharmacy, and Department of Pharmacology, Medical School, University of
Minnesota, Minneapolis, Minnesota 55455, and Department of Pharmacology, School of Medicine, Temple University,
Philadelphia, Pennsylvania 19140
Received J une 25, 1996X
Aspartic acid conjugates of 2-(3,4-dichlorophenyl)-N-methyl-N-[(1S)-1-(3-aminophenyl)-2-(1-
pyrrolidinyl)ethyl]acetamide (5) were synthesized and evaluated in mice for antinociceptive
activity by intravenous and intracerebroventricular routes of administration. The intravenously-
administered R-conjugate of L-Asp (2), its D-Asp diastereomer (3), and the â-conjugate of L-Asp
(4) were found to be 11-, 31-, and 40-fold, respectively, less effective than the parent ligand 1
(ICI 199,441) in producing central nervous system mediated antinociception in the mouse
abdominal stretch assay. In addition, iv-administered 2 and 3 were found to also produce
potent antinociception in the tonic phase of the mouse formalin assay, which is a model of
tonic rather than acute pain. This study suggests that the attachment of a zwitterionic moiety
to a position in the molecule that exhibits bulk tolerance is a viable strategy for the design of
peripherally-selective and peripherally-active opioids.
In tr od u ction
dichlorophenyl)-N-methyl-N-[(1S)-1-phenyl-2-(1-pyrrol-
idinyl)ethyl]acetamide (1, ICI 199,441).12,13
In view of the pain and suffering inflicted by chronic
inflammatory diseases such as rheumatoid arthritis,
there is an urgent need for new and improved methods
of treatment. Now there is considerable evidence
indicating that opioid ligands can produce opioid recep-
tor-mediated antinociception outside the central nervous
system (CNS).1-3 Furthermore, the peripheral analge-
sic effects of opioids are enhanced under inflammatory
conditions. However, the use of opioids for alleviating
inflammatory pain has been limited because of the
variety of CNS side effects produced by opioids. There
has been an interest in the preparation of peripherally-
acting opioid agonists that have limited or no access to
the CNS in an effort to reduce or eliminate these side
effects. Another potential application of peripherally-
acting opioids is the treatment of gastrointestinal motil-
ity disorders.4-6
Previous work in our laboratory has shown that
aspartic acid conjugates of opioid ligands naltrexamine
and oxymorphamine crossed the blood-brain barrier
(BBB) poorly, as indicated by very large iv/icv dose
ratios in mice.7 The poor CNS penetration of these
conjugates was attributed to the highly charged nature
of the zwitterionic group, which decreases the lipophi-
licity of the conjugates.
There has been a limited number of reports of
κ-selective agonists with restricted access to the CNS.8-11
Because the coupling of a zwitterionic group to naltrex-
amine has been shown to greatly reduce its ability to
produce CNS-mediated effects,7 we sought to produce
peripherally-selective κ opioid receptor agonists by the
introduction of zwitterionic groups on the central phenyl
group of the potent and selective κ agonist 2-(3,4-
Ch em istr y
Aspartic acid conjugates 2-4 were prepared via DCC/
HOBT coupling of 2-(3,4-dichlorophenyl)-N-methyl-N-
[(1S)-1-(3-aminophenyl)-2-(1-pyrrolidinyl)ethyl]acetam-
ide14,15 (5) and the suitably protected aspartic acid
(Scheme 1). The protected conjugates 6-8 (not char-
acterized) were deprotected by treatment with a 1:1
mixture of 2 N HCl and glacial acetic acid to remove
the Boc group, followed by Pd/C-catalyzed hydrogenoly-
sis of the benzyl ester.
Resu lts
Sm oot h Mu scle P r ep a r a t ion s. Compounds 2-4
were tested on the electrically stimulated guinea pig
ileal longitudinal muscle16 (GPI) and mouse vas defer-
ens17 (MVD) preparations as described previously.18 All
three conjugates were found to be potent agonists in
* To whom correspondence should be addressed.
† Department of Medicinal Chemistry.
‡ Department of Pharmacology, Temple University.
§ Department of Pharmacology, University of Minnesota.
X Abstract published in Advance ACS Abstracts, September 15, 1996.
S0022-2623(96)00459-1 CCC: $12.00 © 1996 American Chemical Society

κ Opioid Receptor Agonists
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 22 4479
Sch em e 1
Ta ble 1. Agonist Potencies in Smooth Muscle Preparations
IC50 (nM) ( SEMa
Ta ble 3. Antinociceptive Potencies of 1-4 in Mice
ED50 (nmol/kg)
dose ratio
iv/icv
compd
GPI
MVD
compd assay
icva
iv
writhing 11.8 (11.7-11.9) 16.5 (10.8-23.8)
writhing 6.8 (4.8-9.2) 105 (90-120)
1
2
3
4
0.27 ( 0.09
0.84 ( 0.30
4.59 ( 1.11
0.55 ( 0.16
2.50 ( 1.65
2.49 ( 0.64
4.06 ( 1.46
6.36 ( 1.44
1
2
1.4
15.7
2.5
tail-flick 335 (265-412) 850 (340-2070)
formalin ND
10 (4-16)
2015 (1850-2180)
3
writhing 46 (31-72)
tail-flick no effect foundb
formalin ND
43.6
a
Values are arithmetic means of three experiments.
1050 (510-1590)
628 (360-960)
Ta ble 2. Opioid Receptor Binding Affinities
Ki (nM)a
4
writhing 11 (8-15)
56.5
10.2
tail-flick 502 (460-542) 5120 (3940-6660)
a
compd
κ
µ
δ
Dose is converted from units of nmol/mouse to nmol/kg by
employing the average weight of 25 g/mouse. The icv potencies in
nmol/mouse were the following: 1 (0.3), 2 (0.17 in writhing, 8.37
in tail-flick), 3 (1.15), 4 (0.28 in writhing, 12.54 in tail-flick). No
effect was found at icv doses of up to 160 nmol/mouse or 6.4 µmol/
kg.
2
3
0.34 ( 0.23
1.20 ( 0.43
462 ( 80
280 ( 84
1072 ( 157
573 ( 33
b
a
Values are arithmetic means of three experiments.
smooth muscle preparations (Table 1). While they have
similar potencies in MVD, the D-aspartic acid conjugate
3 is significantly less potent than 2 and 4 in GPI.
Bin d in g. The opioid receptor binding affinity and
selectivity of diastereomers 2 and 3 were determined
by competition with radioligands in guinea pig brain
membranes employing a modification of the method of
Werling et al.19 Binding to κ receptors was evaluated
with 1 nM [3H]-(5R,7R,8â)-(-)-N-methyl-N-1-pyrroli-
dinyl-1-oxaspiro[4.5]dec-8-ylbenzeneacetamide20 ([3H]-
U69,593), to µ receptors with 2 nM [3H][D-Ala2,MePhe4,-
Gly-ol6]enkephalin21 ([3H]DAMGO), and to δ receptors
with 5 nM [3H][D-Pen2,D-Pen5]enkephalin22 ([3H]DP-
DPE). Both 2 and 3 were found to bind κ opioid
receptors with high affinity and selectivity (Table 2).
In Vivo Stu d ies. In order to determine their pe-
ripheral selectivity, compounds 1-4 were evaluated for
in vivo activity using either the mouse tail-flick23 or the
mouse abdominal stretch24 assay via both iv and icv
routes. The analgesic effects of opioid agonists in these
models of acute pain are believed to be mediated
through opioid receptors in the CNS. The fact that the
compounds are more potent in the mouse abdominal
stretch assay also suggests the involvement of opioid
receptors, inasmuch as this assay is known to be more
sensitive to κ agonists.25 In evaluating agonist selectiv-
ity, nor-BNI,26 naltrindole27 (NTI), and â-funaltrexam-
ine28 (â-FNA) were employed as κ-, δ-, and µ-selective
antagonists, respectively. Intravenously administered
2 and 3 were also evaluated for analgesic activity in the
second (tonic) phase of the mouse formalin assay, which
is a model for tonic inflammatory pain.29 Attempts to
determine the icv potencies of these compounds in the
mouse formalin assay were unsuccessful because of
interference from either ether or halothane anesthesia.
When tested in the abdominal stretch assay, the
derivatives 2-4 had iv/icv dose ratios of 11, 31, and 40-
fold, respectively, greater than the iv/icv dose ratio of 1
(Table 3). In addition, the ability of the iv-administered
L-aspartic acid conjugate 2 to produce CNS effects was
enhanced 3-fold relative to that of the corresponding
D-aspartic acid conjugate 3. A similar, but less pro-
nounced, reduction in the abilities of intravenously
administered 2 and 4 to produce CNS-mediated effects
was also observed in the mouse tail-flick assay (Table
3). Interestingly, the D-aspartyl conjugate 3 was inac-
tive at icv doses of up to 160 nmol/mouse (6.4 µmol/kg)
in the tail-flick assay. In the mouse formalin assay, iv-
administered 2 and 3 were found to have ED50 doses of
10 and 1050 nmol/kg, respectively.
The antinociceptive effects of compounds 2 and 4 in
the mouse abdominal stretch assay were found to be
κ-selective, as indicated by the fact that the κ-selective
antagonist nor-BNI26 significantly increased the ED50
values of 2 and 4, while the µ and δ antagonists,
â-FNA28 and NTI,27 were less effective or ineffective in
this regard (Table 4). Both 2 and 4 also exhibited weak
agonist activity at the µ receptors. Surprisingly, the
ED50 of 3 was not affected significantly by any of the
three opioid receptor selective antagonists.

4480 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 22
Chang et al.
Ta ble 4. Receptor Selectivity of Antinociception in the Mouse Abdominal Stretch Assay
ED50 ratioa
antagonist
selectivity
1
2
3
4
nor-BNIb
â-FNAc
NTId
κ
µ
δ
2.31 (1.6-3.3)
1.72 (1.00-2.86)
1.19 (0.55-2.77)
5.00 (3.13-8.33)
2.86 (1.69-4.76)
0.92 (0.56-1.49)
1.69 (0.83-2.94)
1.75 (0.88-3.23)
0.55 (0.28-0.96)
7.14 (5.00-11.11)
2.56 (1.67-4.00)
1.01 (0.66-1.56)
a
The ED50 of the agonist (2-4 were given icv; 1 was given sc) in the antagonist-treated mice divided by the control ED50 (determined
in the absence of antagonist); numbers in parentheses are 95% confidence levels. 12.25 µmol/kg, sc, 2 h after administration. c 20.37
µmol/kg, sc, 24 h after administration. 44.44 µmol/kg, sc, 30 min after administration.
b
d
Ta ble 5. Peak Times for Antinociception of 2-4
writhing (min) tail-flick (min)
less likely in view of the short time intervals involved
in the in vivo testing.
A comparison of their iv potencies shows that com-
pounds 2 and 3 are 10- and 2-fold, respectively, more
potent in the mouse formalin assay than in the writhing
assay. This may indicate that both 2 and 3, especially
2, are more effective analgesics against tonic pain than
acute pain. In view of the well-documented evidence
indicating that peripheral opioid receptor mediated
antinociception is manifested during inflammation,1-3
however, the greater potencies of 2 and 3 in the mouse
formalin assay may indicate that a greater portion of
the antinociception produced by 2 and 3 in the formalin
assay is mediated by localized peripheral opioid recep-
tors at the site of inflammation. Assuming the iv
administration of 2 results in the same CNS penetration
in both formalin and writhing assays, the amount of
CNS-mediated analgesia by 2 should be the same in
both assays. The greater potency of iv-administered 2
in the formalin assay may therefore reflect increased
contribution to the overall antinociception by peripheral
opioid receptors.
While both 2 and 3 exhibited high affinity and
selectivity binding to κ opioid receptors in competitive
binding assays against [3H]U69,593, their in vivo activi-
ties are significantly different. This may be due to
different pharmacokinetic properties that may result
from their diastereomeric relationship. Alternatively,
the low potency and low ED50 ratios of 3 suggest that 3
may be producing its antinociceptive effect via a non-
opioid mechanism of action or by interacting with a κ
subtype that is not effectively antagonized by nor-BNI.
The unexpected finding that the peak time of anti-
nociception of 4 by the icv route in the mouse abdominal
stretch assay was later than that when administered
iv is significant. The faster onset of action suggests the
possibility of a peripheral site of action for 4 at the high
doses required for iv administration.
compd
icv
iv
icv
30
iv
2
3
4
10
10
20
10
10
no effect was founda
30b
10c
30
30
a
No effect was found for icv doses of up to 160 nmol/mouse or
b
6.4 µmol/kg. Antinociceptive activity evaluated at 10, 20, 30, and
60 min after administration. c Antinociceptive activity evaluated
at 10, 20, and 40 min after administration.
While 2 and 3 have identical peak times (10 min) in
the abdominal stretch assay via iv and icv routes, the
antinociceptive effect of 4 administered iv peaked
significantly earlier relative to icv administration (Table
5).
Discu ssion
Previous work in our laboratory has shown that
aspartate conjugates of opioid ligands naltrexamine and
oxymorphamine crossed the BBB poorly, as indicated
by very large iv/icv ED50 dose ratios in mice.7 Peripher-
ally administered aspartic acid conjugates 2-4 were
also found to have reduced antinociceptive potencies,
but the dose ratios were smaller than those of the
opiates. This was indicated by much smaller iv/icv ED50
dose ratios in the mouse abdominal stretch and tail-
flick assays. In this regard, the iv-administered aspar-
tate conjugates 2-4 were 11-, 31-, and 40-fold, respec-
tively, less effective than the parent compound 1 in
producing CNS-mediated antinociception in the mouse
abdominal stretch assay.
There is a possibility that amino acid conjugates may
be transported across the BBB. As a matter of fact,
γ-glutamyl transpeptidase (γ-GT), which is present in
the BBB, facilitates CNS uptake of polar substances by
conjugation with L-Glu through the γ-carboxyl group.30,31
Furthermore, transporters specific for one of three
classes of R-amino acids (acidic,32 basic,33,34 and neu-
tral35) are also present in the BBB, and competition for
transport occurs among both natural and unnatural
amino acids of the same class.36,37 In addition to broad
substrate specificity,38 the neutral amino acid (NAA)
transporter has varying degrees of stereoselectivity in
its transport of the L and D isomers of different amino
acids.39 There are examples of small unnatural amino
acids or amino acid conjugates being transported by the
NAA transporter.40-43
In conclusion, the present study has been successful
in the development of κ agonists that have reduced
access into the CNS. In addition to retaining κ receptor
affinity and selectivity, the aspartate conjugates 2-4
were found to be significantly less effective than the
parent compound 1 in producing CNS-mediated opioid
antinociception after peripheral administration, and
they may be useful as potential therapeutic agents and
as pharmacologic tools in studying the peripheral opioid
receptors. Furthermore, the actions of peripherally-
administered 2-4, though limited, hint of the possible
involvement of an amino acid transporter system in
their CNS penetration.
The antinociceptive action of iv administered aspar-
tate conjugates 2-4 may in part be facilitated by
transport across the BBB. Alternately, the relative ease
with which iv-administered 2-4 produce CNS-mediated
effects may be a result of hydrolysis of the conjugates
to yield the parent amine 5, which would easily pen-
etrate the BBB. However, this latter possibility appears
Exp er im en ta l Section
All NMR spectra were recorded on a GE 300 MHz spec-
trometer at room temperature. Optical rotations were mea-
sured using a Rudolph Research Autopol III automatic pola-

κ Opioid Receptor Agonists
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 22 4481
rimeter. Melting points were determined using a Thomas
Hoover capillary melting point apparatus and are uncorrected.
Elemental analyses were conducted by M-H-W Laboratories
in Phoenix, AZ. Mass spectra were recorded by the Chemistry
Mass Spec Labs at the University of Minnesota’s Chemistry
Department. The diprotected aspartic acid derivatives were
purchased from Sigma Chemical Co., St Louis, MO 63178.
2-(3,4-Dich lor op h en yl)-N-m eth yl-N-{(1S)-1-[3-N-(S)-a s-
p a r t yla m in o]p h e n yl]-2-(1-p yr r olid in yl)e t h yl}a ce t a m -
id e (2). To an ice-cold mixture of N-t-Boc-L-aspartic acid-â
benzyl ester (0.3541 g, 1.095 mmol) and 1-hydroxybenzotria-
zole (HOBT) (0.1495 g, 1.106 mmol) in dry CH2Cl2 (20 mL)
was added with stirring under N2 a solution of N,N′-dicyclo-
hexylcarbodiimide (DCC) (0.2331 g, 1.130 mmol) in dry CH2-
Cl2 (10 mL). After 1 h of stirring at 0 °C, a solution of 514,15
(0.2228 g, 0.5483 mmol) in dry CH2Cl2 (6 mL) was added, and
the reaction mixture was stirred at 25 °C under N2 for 23 h
before it was filtered. The filtrate was then washed with
saturated NaHCO3 before it was dried (Na2SO4), filtered, and
evaporated under reduced pressure. Flash column chroma-
tography with CHCl3:2% NH3:2% MeOH yielded 0.3315 g
(85%) of the protected intermediate, which was further purified
on HPLC using CHCl3:2% NH3:1% MeOH. After the protected
intermediate (0.0905 g, 0.1272 mmol) was stirred in 1 mL of
2 N HCl, 1 mL of AcOH, and 1 drop of anisole at 25 °C for 20
min, 2 mL of MeOH and 10% Pd/C (about 15 mg) were added,
and the mixture was hydrogenated at 25 °C using a hydrogen
balloon. After 1 h, more Pd/C (about 10 mg) was added, and
20 min later the mixture was filtered through Celite and
evaporated in vacuo. The clear film which remained was
converted to a white solid by addition of iPrOH, and evapora-
g, 5.855 mmol), HOBT (0.8070 g, 5.972 mmol), and DCC
(1.2441 g, 6.030 mmol) in dry CH2Cl2 (31 mL). The procedure
and reaction conditions are similar to those employed for the
preparation of 2, but more than 2 equiv of the aspartic acid
derivative, DCC, and HOBT were needed to drive the reaction
to completion. After 17 h, the reaction was worked up in a
manner similar to that of 2, and purification by flash column
chromatography with CH2Cl2:2% NH3:3% MeOH yielded 0.3
g (72%) of the protected intermediate. After removal of the
Boc group by treatment with 2 N HCl (3 mL), AcOH (3 mL),
and 3 drops of anisole at 25 °C for 105 min, the benzyl ester
protected intermediate was worked up as the Boc deprotection
of 3 and purified by flash column chromatography with CH2-
Cl2:2% NH3:5% MeOH. The benzyl ester was converted to the
2HCl salt with Et2O‚HCl and cleaved by hydrogenation at 39
psi with 25 mg of 10% Pd/C in MeOH (8 mL) for 1 h, followed
by the workup analogous to the benzyl ester deprotection of 3
to yield 4‚2HCl (0.0714 g, 20.5%): mp 185 °C dec; [R]25D +147°
(c ) 0.10, MeOH); 1H NMR (DMSO-d6) δ 1.937 (br s, 4H, CH2-
CH2), 2.795 (s, 3H, NCH3), 2.967-4.170 (complex, 11H, 5 CH2
and 1 CH), 6.06 (m, 1H, CH), 6.965-7.611 (complex, 7H,
aromatic), 8.4 (br s, exchangeable proton), 10.5 (s, exchange-
able proton), 10.9 (br s, exchangeable, proton); MS (FAB) m/ z
521.2. Anal. (C25H30N4O4Cl2‚2HCl) C, H, N, Cl.
Ack n ow led gm en t . We thank Veronika Doty,
Michael Powers, J oan Naeseth, and Idalia Sanchez for
in vitro and in vivo testing of the compounds. We also
thank Dr. Diane DeHaven-Hudkins for pleasant and
insightful discussions of the results.
tion to dryness in vacuo yielded 2‚2HCl (50.6 mg, 66.9%): mp
1
170 °C dec; [R]25 +113° (c ) 0.2, MeOH); H NMR (DMSO-
D
d6) δ 1.97 (br s, 4H, CH2CH2), δ 2.82 (s, 3H, NCH3), 2.85-4.23
(complex, 11H, 5 CH2 and 1 CH), 6.10 (m, 1H, CH), 7.04-7.67
(complex, 7H, aromatic), 8.3 (s, exchangeable proton), 8.2-
8.8 (br s, exchangeable proton), 10.6-10.8 (br s, exchangeable
proton); MS (FAB) m/ z 521.3. Anal. (C25H30N4O4Cl2‚2HCl)
C, H, N, Cl.
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(0.1012 g, 30.6%): mp 200 °C dec; [R]25 +103.9° (c ) 0.23,
D
MeOH); 1H NMR (DMSO-d6) δ 1.94 (br s, 4H, CH2CH2), 2.808
(s, 3H, NCH3), 2.77-4.24 (complex, 11H, 5 CH2 and 1 CH),
6.076 (m, 1H, CH), 7.014-7.658 (complex, 7H, aromatic), 8.6
(br s, exchangeable proton), δ10.9 (br s, exchangeable proton);
MS (FAB) m/ z 521.2. Anal. (C25H30N4O4Cl2‚2HCl) C, H, N,
Cl.
2-(3,4-Dich lor op h en yl)-N-m et h yl-N-{(1S)-1-[N-(S)-a s-
p a r t yla m in o]p h e n yl]-2-(1-p yr r olid in yl)e t h yl}a ce t a m -
id e (4). Compound 4 was prepared from 514,15 (0.2379 g,
0.5855 mmol), N-t-Boc-L-aspartic acid R-benzyl ester (1.8933

4482 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 22
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