Amino acid conjugates as κ opioid receptor agonists

Article abstract of DOI:10.1016/j.bmcl.2005.01.038

A novel series of kappa (κ) opioid receptor agonists were synthesized by incorporating the key structural features of known κ opioid agonists while replacing the aryl acetamide portion with substituted amino acid conjugates. Compounds 3j (K_i = 6.7 nM), 3k (K_i = 3.6 nM), 3l (K_i = 4.6 nM), 3m (K_i = 0.83 nM) and 3o (K_i = 2 nM) possessed potent affinities for the κ opioid receptor in vitro with reasonable selectivity over other opioid receptors.

Full text of DOI:10.1016/j.bmcl.2005.01.038

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1279–1282
Amino acid conjugates as j opioid receptor agonists
Virendra Kumar,* Deqi Guo, Jeffrey D. Daubert, Joel A. Cassel, Robert N. DeHaven,
Erik Mansson, Diane L. DeHaven-Hudkins and Alan L. Maycock
Adolor Corporation, 700 Pennsylvania Drive, Exton, PA 19341, USA
Received 7 December 2004; revised 14 January 2005; accepted 18 January 2005
Abstract—A novel series of kappa (j) opioid receptor agonists were synthesized by incorporating the key structural features of
known j opioid agonists while replacing the aryl acetamide portion with substituted amino acid conjugates. Compounds 3j
(Ki = 6.7 nM), 3k (Ki = 3.6 nM), 3l (Ki = 4.6 nM), 3m (Ki = 0.83 nM) and 3o (Ki = 2 nM) possessed potent affinities for the j opioid
receptor in vitro with reasonable selectivity over other opioid receptors.
Ó 2005 Elsevier Ltd. All rights reserved.
During the past decade efforts have been devoted to the
investigation of kappa (j) opioid receptor agonists as
analgesics lacking side effects such as sedation, dyspho-
ria and diuresis.1–3 A number of groups have taken
diversified approaches to achieve this goal, and the key
structural features or Ôprimary pharmacophoreÕ com-
mon to the aryl acetamide series of j opioid receptor
agonists that have emerged, are summarized in Figure
1.3–5 The amide and tertiary amine nitrogens are sepa-
rated by a two-carbon chain and acetamides are formed
from aryl acetic acids, illustrated by ICI 199441 (1).
Most of the efforts in the literature have focused on
modification of the substituents at these two-carbon
chains. The acetamide fragment of aryl acetamides was
not altered except for aryl and heteroaryl groups since
it was assumed that any substantial changes in this link-
age would reduce agonist activity at the j opioid recep-
tor.1,3,6 However, thiourea derivatives, bearing only the
diamine segment of the j opioid agonist Ôprimary phar-
macophoreÕ and represented by compound 2, not only
retain j opioid receptor activity but also selectivity over
other opioid receptors.6,7 This finding provided avenues
for the modification of the acetamide portion of j opi-
oid receptor agonists.
In a continuing effort of our research group to develop
new classes of j opioid receptor agonists that lack the
above mentioned central nervous system (CNS) liabili-
ties, we have synthesized an exploratory series of amino
acid conjugates of general structure 3 (Fig. 2). The ami-
no acid derivatives were selected to decrease the lipo-
philic character of centrally acting j opioid agonists
such as ICI 199441 (1), thus rendering compounds with
limited access to the CNS.8,9 These compounds were
prepared by incorporating the Ôprimary pharmacophoreÕ
Cl
O
O
N1
N
ArCH2
N2
Cl
N
CH3
aryl acetamide
diamine
1, ICI 199441
R2
N
O
O
S
R1
N1
N
N
X
N
H
N
CH3
ArCH2
N2
N
CH3
R3
Cl
O
2
3
a-p
Figure 1.
R1 = Alkoxy, Benzyloxy, or Aryl
2 = H, CH3
R
R3 = H, Phenyl, or Benzyl
X = H, OH
Keywords: Kappa opioid receptor agonists; Amino acid conjugates.
*
Corresponding author. Tel.: +1 484 595 1060; fax: +1 484 595
1573; e-mail: vkumar@adolor.com
Figure 2.
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.01.038

1280
V. Kumar et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1279–1282
common to known j opioid agonists but the aryl acet-
amide portion was replaced with substituted amino acid
conjugates. The initial goal of this research was to estab-
lish potent j opioid receptor affinity, followed by recep-
tor selectivity and an improvement in the peripheral
index in vivo as a means of limiting access to the
CNS.8,10 We describe here the syntheses and in vitro
binding affinities of these novel j opioid agonists.
diastereomer 1 (3b) bound to the j opioid receptor with
an affinity sixfold higher than the diastereomer 2 (3c)
with Ki values of 138 and 856 nM, respectively, indicat-
ing the chiral preference of the receptor in this part of
the molecule. The corresponding free amino compound
3a, as a mixture of diastereomers was, only half as active
in the binding assay with a Ki value of 258 nM. The dia-
stereomers of the amine 3a were not separated, but it
could be anticipated that one of the diastereomers of
the free amine would have a comparable binding po-
tency to 3b. Extension of the chain at R3 with a (S)-benzyl
group (3d, Ki = 150 nM) or substitution with a smaller
group at R1 (3e, Ki = 178 nM) did not lead to com-
pounds with improved j opioid receptor affinity.
Replacement of the benzyloxy group at R1 with a phenyl
group gave the benzoyl compound 3f, which showed a
modest improvement in binding affinity with a Ki value
of 99 nM.
The synthesis of amino acid conjugates utilized the pre-
viously reported diamine intermediates, (S)-411 and
(S,S)-5.12 A general synthetic methodology was devel-
oped for the preparation of these compounds and is
described in Scheme 1. Condensation of diamines with
N-carbobenzyloxy (Cbz)-protected amino acids gave
the intermediate 6. Removal of the Cbz protecting
group by catalytic hydrogenation followed by acylation
of the free amine (7) with aliphatic or aromatic acid
chlorides in the presence of HunigÕs base gave the
desired compounds, for example, compound 2j
(R1 = Ph, R2 = H, R3 = H and X = H). This method
was used to prepare other amino acid conjugates of
diamines 4 and 5 (Table 1).
It is evident that retaining a phenyl or benzyl group at
the R3 position of structure 3 would not allow for an in-
crease in j opioid receptor affinity, probably because of
the intolerance for sterically bulky groups by the recep-
tor in this position. To circumvent this problem, we pre-
pared the next series of compounds with no
substitutions at this position (R3 = H).
The new compounds were evaluated in vitro for opioid
receptor binding affinity. Determinations of affinities
for j, mu (l) and delta (d) opioid receptors were con-
ducted by displacement of bound [3H]diprenorphine
from membranes prepared from cells expressing the
cloned human opioid receptors. Methodological details
have been previously described in the literature.13,14
The selectivity for the j opioid receptor versus the l
and d opioid receptors was defined by the ratio of the
Ki values and the [35S]GTPcS functional binding assay15
was used to determine j agonist activity of the lead com-
pounds (Table 1).
Therefore, compound 3g containing a benzyloxy group
at the R1 position demonstrated an increased affinity
for the j opioid receptor (Ki = 84 nM) when compared
to compound 3b (Table 1). However, a significant
change in the j binding affinity was observed when the
benzyloxy group at R1 was replaced with a benzyl
group. The Ki value (16.3 nM) at the j opioid receptor
of the resulting compound, 3i, was 5- to 10-fold lower
than previous compounds. Shortening the chain further
to a phenyl group gave compound 3j with a Ki value of
6.7 nM for the j opioid receptor and 61- and 50-fold
selectivity, respectively, for j over l and d opioid
receptors.
Condensation of (R,S)-N-Cbz-phenylglycine with dia-
mine 4 resulted in two diastereomeric compounds 3b
and 3c, which were readily separated. The absolute ste-
reochemistry of these compounds was not assigned. The
Enhancement in the binding affinity of j opioid receptor
agonists has been reported with substitutions on the aro-
matic rings of aryl acetamides.1,3 Therefore, the replace-
ment of phenyl of 3j with 3,4-di-chlorophenyl (3k) and
benzofuroxan (3l) furnished compounds, which exhib-
ited potent j opioid receptor affinity with Ki values of
3.6 and 4.6 nM, respectively. It has been shown in the
literature that substitution of the pyrrolidine with
3-(S)-hydroxypyrrolidine often resulted in compounds
with improved j opioid receptor binding affinity,1,3 thus
compound 3m showed a fourfold higher affinity
(Ki = 0.83 nM) for the j opioid receptor than 3k. How-
ever, the j opioid receptor selectivity of 3m over the d
opioid receptor was somewhat compromised. A limited
number of compounds, 3j–o, were assessed in
[35S]GTPcS binding assay and were full agonists at the
j opioid receptor (Table 1).
DCC/HOBT
H
N
O
N
N
O
HN
CH3
N
CH3
Cbz-Gly-OH
84%
X
X
O
6
4, X = H
5, X = OH
O
Cl
H2, Pd/C
~100%
O
H2N
N
N
Hunig's Base
-20oC
X
CH3
7
H
O
N
N
N
X
A very limited structure–activity relationship was ex-
plored around the lead compound 3m. Substitution of
a methyl group at the R2 position of 3m resulted in com-
pound 3n, which had not only a 24-fold weaker affinity
O
CH3
3j (X = H)
Scheme 1.

V. Kumar et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1279–1282
Table 1. In vitro binding to cloned human opioid receptors
1281
R2
N
O
R1
N
X
N
CH3
R3
O
3
a-p
Compound
R1
—
R2
R3
X
Yielda Ki (nM)
Selectivity for
Selectivity for
[
35S]GTPcS
(%)
[3H]diprenorphine j over l (fold) j over d (fold) EC50 (nM)b
j (95% CI)
(R,S)
3a
H2
H
H
H
71
11
258 (104–674)
138 (27–690)
146
22
>100
27
ND
ND
3b
(Diastereomer 1)
(R or S)
(R or S)
CH2O
CH2O
CH2O
3c
(Diastereomer 2)
H
H
11
856 (401–1826)
4
>100
ND
CH2
3d
3e
3f
H
H
H
H
H
H
78
57
73
150 (86–259)
178 (83–382)
99 (39–252)
13
5
ND
ND
ND
(S)
(S)
(S)
OCH3
>100
>100
>100
13
3g
3i
H
H
H
H
H
H
H
H
H
H
H
H
84
44
68
59
84 (31–223)
16.3 (14–19)
6.7 (2.7–17)
3.6 (1.4–9.2)
11
2
ND
ND
CH2O
CH2
>100
61
49
50
24
3j
3.85
Cl
Cl
3k
34
2.25
4.37
0.76
41.42
1.98
ND
N
O
3l
H
H
H
H
H
H
H
58
4.6 (1.5–14)
0.83 (0.69–0.99)
20 (17–24)
55
11
11
1.5
25
+N
O-
Cl
3m
3n
3o
3p
H
OH 30
OH 75
OH 34
OH 48
160
>100
134
39
Cl
Cl
CH3
H
Cl
OCH3
2 (1.9–2.2)
H3CO
H
48 (39–57)
>100
567
ICI 199441 (1)
0.043 (0.027–0.21) 1241
ND = not determined.
a The compounds were isolated as either hydrochloride or methane sulfonic acid salts and characterized by MS, NMR, mp and elemental analyses.
b Geometric means computed from two to three separate determinations.
for the j opioid receptor but a further lowering of selec-
tivity with respect to the d opioid receptor. The j opioid
receptor affinity was also reduced when the 3,4-dichloro
groups of 3m were replaced with a methoxy in com-
pounds 3o and 3p. But, the 2-methoxy compound, 3o,
was 24 times more potent in receptor binding than the
corresponding 4-methoxy, 3p, indicating receptor sensi-
tivity to steric bulk rather than electronic effects.

1282
V. Kumar et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1279–1282
4. Vecchietti, V.; Giordani, A.; Giardina, G.; Colle, R.;
Clarke, G. D. J. Med. Chem. 1991, 34, 397.
In summary, we were successful in replacing the aryl
acetamide portion of j opioid receptor agonists, such
as ICI 199441 (1), with amino acid conjugates, resulting
in compounds with high affinity for the j opioid recep-
tor. Compounds 3j (Ki = 6.7 nM), 3k (Ki = 3.6 nM), 3l
(Ki = 4.6 nM), 3m (Ki = 0.83 nM) and 3o (Ki = 2 nM)
emerged as leads of this series and were full agonist at
the j opioid receptor. Even though most of the new
compounds were less potent in j opioid receptor binding
affinity and were less selective to other opioid receptors
when compared to ICI 199441 (1), these compounds
may have an advantage by limited access to the CNS
due to reduce lipophilicity. Further synthetic efforts will
be directed at improving the opioid receptor selectivity
and extending our observations to in vivo evaluation
of these compounds in various antinociceptive models
and assessing the CNS liabilities.
5. Soukara, S.; Maier, C. A.; Predoiu, U.; Ehret, A.;
Jackisch, R.; Wunsch, B. J. Med. Chem. 2001, 44, 2814.
6. Takeuchi, M.; Takayama, K.; Onda, K.; Motoie, H.;
Isomura, Y. W.O. 09303011, 1993; Chem. Abstr. 1993,
119, 139079.
7. Scopes, D. I. C. Drugs Future 1993, 18, 993; and Exp.
Opin. Invest. Drugs 1994, 3, 369.
8. Botros, S.; Lipkowaski, A. W.; Larson, D. A.; Stark, P.
A.; Takemori, A. E.; Portoghese, P. S. J. Med. Chem.
1989, 32, 2068.
9. Chang, A.-C.; Cowan, A.; Takemori, A. E.; Portoghese, P.
S. J. Med. Chem. 1996, 39, 4478.
10. Review of peripherally acting opioid agonists as novel
analgesic agents and additional references, see: DeHaven-
Hudkins, D. L.; Dolle, R. E. Curr. Pharm. Des. 2004, 10,
743.
11. Costello, G. F.; James, R.; Shaw, J. S.; Slater, A. M.;
Stutchbury, N. C. J. J. Med. Chem. 1991, 34, 181.
12. Gottschlich, R.; Ackermann, K. A.; Barber, A.; Bart-
osyzk, G. D.; Greiner, H. E. Bioorg. Med. Chem. Lett.
1994, 4, 677.
References and notes
1. (a) Barber, A.; Gottschlich, R. Exp. Opin. Invest. Drugs
1997, 6, 1351; (b) Szmuszkovicz, J. In Progress in Drug
Research; Jucker, E., Ed.; Birkhauser: Basel, 1999; Vol. 52,
pp 167–195; (c) Szmuszkovicz, J. In Progress in Drug
Research; Jucker, E., Ed.; Birkhauser: Basel, 1999; Vol. 53,
pp 1–51.
2. (a) Dionne, R. A.; Dobbins, K. R.; Hargreaves, K. M.
Clin. Pharmacol. Ther. 1991, 49, 183; (b) Pande, A. C.;
Pyke, R. E.; Greiner, M.; Wideman, G. L.; Benjamin, R.;
Pierce, M. W. Clin. Neuropharmacol. 1996, 19, 451.
3. Giardina, G.; Clarke, G. D.; Grugni, M.; Sbacchi, M.;
Vecchietti, V. Il Farmaco 1995, 50, 405.
13. DeHaven-Hudkins, D. L.; Cortes Burgos, L.; Cassel, J.
A.; Daubert, J. D.; DeHaven, R. N.; Mansson, E.;
Nagasaka, H.; Yu, G.; Yaksh, T. J. Pharmacol. Exp.
Ther. 1999, 289, 494.
14. DeHaven, R. N.; DeHaven-Hudkins, D. L. In Current
Protocols in Pharmacology; Enna, S. J., Williams, M.,
Ferkany, J. W., Kenakin, T., Porsolt, R. D., Sullivan, J.
P., Eds.; John Wiley and Sons: New York, 1998; pp 1.4.1–
1.4.12.
15. Schlechtingen, G.; DeHaven, R. N.; Daubert, J. F.;
Cassel, J. A.; Chung, N. N.; Schiller, P. W.; Taulane, J.
P.; Goodman, M. J. Med. Chem. 2003, 46, 2104.