Optimisation of in silico derived 2-aminobenzimidazole hits as unprecedented selective kappa opioid receptor agonists

Article abstract of DOI:10.1016/j.bmcl.2014.12.064

Kappa opioid receptor (KOR) is an important mediator of pain signaling and it is targeted for the treatment of various pains. Pharmacophore based mining of databases led to the identification of 2-aminobenzimidazole derivative as KOR agonists with selectivity over the other opioid receptors DOR and MOR. A short SAR exploration with the objective of identifying more polar and hence less brain penetrant agonists is described herewith. Modeling studies of the recently published structures of KOR, DOR and MOR are used to explain the receptor selectivity. The synthesis, biological evaluation and SAR of novel benzimidazole derivatives as KOR agonists are described. The in vivo proof of principle for anti-nociceptive effect with a lead compound from this series is exemplified.

Full text of DOI:10.1016/j.bmcl.2014.12.064

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Optimisation of in silico derived 2-aminobenzimidazole hits
as unprecedented selective kappa opioid receptor agonists q
Pradip K. Sasmal a, , , C. Vamsee Krishna a, S. Sudheerkumar Adabala a, M. Roshaiah a,
⇑
Khaji Abdul Rawoof a, Emima Thadi a, K. Pavan Sukumar a, Srisailam Cheera a,
Chandrasekhar Abbineni a, K. V. L. Narasimha Rao a, A. Prasanthi a, Kamal Nijhawan a,
Mahaboobi Jaleel a, Lakshmi Ramachandran Iyer a, T. Krishna Chaitanya a, Nirbhay Kumar Tiwari a,
N. Lavanya Krishna a, Vijay Potluri a, Ish Khanna a, Thomas M. Frimurer b,c, Michael Lückmann c,
Øystein Rist b, Lisbeth Elster b, Thomas Högberg b, ,à
⇑
a Discovery Research, Dr. Reddy’s Laboratories Ltd, Bollaram Road, Miyapur, Hyderabad 500049, India
b 7TM Pharma A/S, Fremtidsvej 3, DK-2970 Hørsholm, Denmark
c NNF, Center for Protein Research and NNF Center for Basic Metabolic Research, University of Copenhagen, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
Kappa opioid receptor (KOR) is an important mediator of pain signaling and it is targeted for the treat-
ment of various pains. Pharmacophore based mining of databases led to the identification of 2-amino-
benzimidazole derivative as KOR agonists with selectivity over the other opioid receptors DOR and
MOR. A short SAR exploration with the objective of identifying more polar and hence less brain penetrant
agonists is described herewith. Modeling studies of the recently published structures of KOR, DOR and
MOR are used to explain the receptor selectivity. The synthesis, biological evaluation and SAR of novel
benzimidazole derivatives as KOR agonists are described. The in vivo proof of principle for anti-nocicep-
tive effect with a lead compound from this series is exemplified.
Received 3 September 2014
Revised 15 December 2014
Accepted 19 December 2014
Available online xxxx
Keywords:
Kappa opioid receptor (KOR)
Benzimidazoles
KOR agonists
Ó 2014 Elsevier Ltd. All rights reserved.
Anti-nociception
The kappa (KOR), delta (DOR) and mu (MOR) opioid receptors
are closely related G-protein coupled receptors responsible for
analgesic and other pharmacological effects exhibited by opioid-
type drugs and endogenous opioid peptides. All three receptors
appear to be present in the central and peripheral nervous system
of many organisms including humans.1,2 Although activation of all
three receptor subtypes is known to produce antinociception, the
majority of opioid drugs that are currently in clinical use as analge-
sic agents (e.g., morphine and fentanyl) are MOR agonists.3 How-
ever, MOR stimulation is also responsible for the spectrum of
unwanted side effects associated with opioids including respira-
tory depression, dependence liability, and inhibition of gastrointes-
tinal motility.4 It has been established that KOR agonists are
capable of producing analgesia without the side effects common
to morphine and other classical opioids.5 KOR agonists have the
potential for treatment of incisional pain, inflammatory pain, pru-
ritus, burn injury pain,6 neuropathic pain,7 visceral pain including
dysmenorrhea or gastrointestinal pain,8 and rheumatoid arthritis.9
The great potential of KOR agonists stimulated research which led
to identification of KOR ligands with great structural diversity.10–12
Dynorphin A (1) is an endogenous ligand for KOR whereas the
naturally occurring diterpenoid salvinorin A (2) is a unique exam-
ple of non-nitrogenous KOR agonists (Fig. 1). Many promising
small molecule KOR agonists have been identified at the discovery
stage, but clinical studies of the first generation centrally acting
KOR agonists (viz., spiradoline, bremazocine and enadoline)
showed CNS liabilities such as sedation and dysphoria.13 Further
efforts towards peripherally restricted KOR agonist as second gen-
eration compounds also led to limited success.
q
DRL Publication No. 739.
⇑
Corresponding authors. Tel.: +91 40 4434 6865 (P.K.S.), +45 44 94 58 88 (T.H.).
E-mail addresses: pradipks@drreddys.com (P.K. Sasmal), thomas.hogberg@
leo-pharma.com (T. Högberg).
Asimadoline (3), a small molecule KOR agonist, is currently in
Phase III clinical trial for the treatment of patients with diarrhea-
predominant irritable bowel syndrome (Fig. 1).14 The tetra-peptide
CR-845 (4) has shown positive results in a phase II trial for post-
operative pain in women following hysterectomy. KOR agonists
Current address: Dr. Reddy’s Laboratories Ltd, Proprietary Products, Innovation
Plaza, Bachupally, Hyderabad 500090, India.
à
Current address: LEO Pharma, Chemical Research, Industriparken 55, DK-2750
Ballerup, Denmark.
http://dx.doi.org/10.1016/j.bmcl.2014.12.064
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Sasmal, P. K.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2014.12.064

2
P. K. Sasmal et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys
in-house) were selected using clustering based on 2D UNITY finger
O
prints to maximize the chemical diversity of the extracted set of
compounds. The compounds were screened in a KOR IP1 HTRF
assay (COS-7 cells transiently transfected with the human KOR
1
Dynorphin A ( )
H
O
O
H
H
receptor cDNA)23 at 10
lM concentration and 374 primary hits
N
AcO
O
showed >50% activation of KOR compared to control.24,25 The high
hit rate (>14.3%) demonstrated the robustness of the developed
pharmacophore model. These compounds were subjected to
in vitro dose response studies and counter screen against the clos-
est related receptors, that is, DOR and MOR using protocols18 anal-
ogous to KOR IP1. This exercise led to identification of 39
compounds from 12 distinct chemical classes having EC50 values
between 10 and 500 nM. Based on the chemical attractiveness,
excellent in vitro potency (39 nM) and selectivity over DOR (200-
fold) and MOR (1000-fold) one such compound 6 (Fig. 3) was cho-
sen for further SAR studies.
OH
O
O
OMe
O
HO
N
2
Salvinorine A ( )
5
Nalfurafine ( )
O
O
O
Ph
OH
N
Ph
N
Ph
O
H
Ph
H
N
Asimadoline (3)
N
N
H2N
N
H
COOH
NH2
O
O
To access the structural basis for ligand binding, we docked
compound 6 into the KOR, DOR and MOR structures.19–21,26 The
predicted binding pose of compound 6 (yellow sticks) in the KOR
structure are shown in Figure 2A and B. For comparison, subtype
selective ligands in complex with recently published crystal struc-
tures of KOR, MOR and DOR (Protein Data Bank codes 4DJH, 4DKL,
and 4EJ4) are shown in the Supplementary material. Analysis of
compound 6 binding to the KOR, in combination with SAR results,
suggest that Val1082.53 (Ala and Ala; II:13), Val1182.63 (Asn and
Lys; II:23), Met2265.38 (Leu and Thr; V:08), Ile2946.55 (Val and
Val; VI:20), and in particular Tyr3127.35 (Trp and Leu; VII:02) con-
tribute to the subtype selectivity of compound 6 (corresponding
amino acids in MOR and DOR are shown in parentheses followed
by the generic pocket numbering as indicated in bottom of
Fig. 2C). Thus, changes in the Val1182.63 side chain, where larger
hydrophilic residues, Asn2.63 and Lys2.63 are found in MOR and
DOR, respectively, are likely to introduce unfavorable contacts with
the benzimidazole moiety located between TM II, -III and -VII. The
remaining two hydrophobic side chains replacements, Val to Ala at
position 2.53 and Ile to Val at position 6.55, may cause a reduction
of the hydrophobic contact between compound 6 and the receptor.
The N-ethylpyrrolidine moiety reaches deep into the orthosteric
ligand binding pocket to form hydrophobic interaction with the
conserved Trp2876.48 side chain (the ‘rotamer toggle switch’),27
possibly playing a critical role in the pharmacological properties
of this ligand. The carboxyl side chain of Asp1383.32 III:08 are
simultaneously engaged in charge–charge interactions with the
positively charged N-ethylpyrrolidine moiety and hydrogen bond
interaction with the central amide linker. Additionally, changing
Tyr3127.35 to the Trp7.35 and Leu7.35 residues found in MOR and
DOR, respectively, are likely to result in the loss of an important
polar interaction with the central amide linker in compound 6, a
predicted key interaction for the chemical series. Lastly, the p-
methyl benzamide binds in a predominantly hydrophobic pocket
between TM III, -V and VI.
A further aspect of our studies was the identification of more
polar compounds (lower logP and higher polar surface area
(PSA)) with retained potency in an effort to identify compounds
with lower propensity to pass the blood–brain barrier (BBB) and
thereby identify compounds with a reduced central action. We
have previously used this strategy to develop CB1 antagonists with
such properties.28,29 Compound 6 is a good starting point with a
relatively low lipophilicity (chromatographically determined
logD28 of 1.9, calculated values logP of 3.8 and logD of 2.8) albeit
with a too low PSA of 50 Å2.
The synthesis of compounds was fairly straight forward as men-
tioned in Scheme 1. SNAr reaction of compounds I with amines II
followed by reduction of nitro to amine and cyanogen bromide
mediated cyclization led to the formation of compounds III.
Coupling of various acids with III afforded final compounds.
Ph
4
CR-845 ( )
NH2
Figure 1. KOR agonists with structural diversity.
also have demonstrated in experimental and clinical studies anti-
pruritic effects in antihistamine-resistant and antihistamine-sensi-
tive pruritus in animals and humans.15 Nalfurafin (5), a compound
from the morphinan class, has been approved in Japan for the
treatment of uremic pruritus in people undergoing hemodialysis.15
Considering the wide potential of KOR agonism the search for an
efficacious and safe KOR agonist is still in demand. We describe
herein the identification of an unprecedented chemotype as KOR
agonist by pharmacophore-based in silico screening, a brief SAR
exploration and in vivo efficacy with a prototype compound.
We have previously described a physicogenetic methodology of
identifying binding pocket-related GPCRs and associated ligands to
construct pharmacophore models to mine commercial and in-
house data bases to extract ligands for the GPCR of interest.13 In
the case of the kappa opioid receptor we identified receptors which
are related also from a conventional phylogenetic relationship, that
is, in particular the other opioid receptors and monoaminergic
receptors. The challenge lies in the construction of a search query
specific enough for the KOR, which was based on the construction
of a homology model, build from the recently disclosed adrenergic
b2-receptor, to refine the pharmacophore model with guidance
from interactions of ligands to the binding pocket-related GPCRs
as described previously.16 According to our physicogenetic classifi-
cation system,16 and the Schwartz numbering system17 the minor
amino acid variations between the receptors in TM II:13, V:05 and
VI:20 belong to the same groups, whereas the variations in TM
II:23, V:08, VI:23 and VII:02 in the binding pockets are distinctly
different and offer opportunities to design in receptor selectivity
(Fig. 2C). Today the situation would have been quite different with
access to crystalline structures of all the opioid receptors with
bound ligands which allows for identification of minimal differ-
ences towards DOR and MOR.19–21 A more detailed analysis based
on the actual crystal structures will follow (vide infra). As a second
step we utilized known ligands for the MOR and DOR in the con-
struction of discriminative filters.
An exhaustive in silico search of available chemical vendor
libraries containing over 107 compounds complemented with a
GPCR-biased in-house library was conducted. The accepted com-
pounds were filtered using a neural network for druglikeness22 fol-
lowed by rejection of compounds displaying high similarity to DOR
and MOR ligands extracted from the Integrity data base with
potencies <1
lM (Tannimoto coefficient >0.8 using UNITY finger-
prints). Finally, 2616 compounds (45% commercial and 55%
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P. K. Sasmal et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
Figure 2. Extracellular view (A: top left) and side view (B: top right) of the predicted binding pose of compound 6 (yellow sticks) to the human KOR (green). Dark grey dashed
lines show polar interactions. The MOR and DOR structures (PDBID: 4DKL and 4EJ4) are shown in grey for comparison. Among many contacts, compound 6 interacts with—or
are in close proximity—to five residues (purple sticks) in the KOR binding pocket that differ in the MOR and DOR highlighted by purple rectangles in the pseudo-sequence
alignment (C: bottom) of binding pocket residues in the three opioid subtype receptors. The residues predicted to contribute to the subtype selectivity are: Val1082.53 (II:13),
Val1182.63 (II:23), Met2265.38 (V:08), Ile2946.55 (VI:20), and Tyr3127.35 (VII:02). The two GPCR numbering annotations by Schwartz17 and Ballesteros18 are shown for clarity as
described in detail in Ref. 16b.
The potency of the test compounds was determined using KOR
IP1 assay25 and the results are summarized in Tables 1 and 2. The
select compounds were counter screened using DOR and MOR
assays25 and the result is presented in Table 3. Initially we investi-
gated three compounds 7–9 (Fig. 3) to understand their impact on
SAR and selectivity. Compound 7 (the truncated version of 6 with-
out southern N-ethylpyrrolidine) was tested and found to be inac-
tive (Table 1). Extending the methylene spacer from two to three
carbons as in compound 8 led to significant decrease in potency.
Similarly replacing benzamide with phenylacetamide as in 9 also
led to decrease in potency. Based on these observations we decided
to explore SAR on the eastern part of the molecule. Initially we
scanned through various polar and non-polar substitutions on
benzamides and the results are summarized in Table 1. The haloge-
nated derivatives p-chloro (12) and m,p-dichloro (30) benzamides
displayed single digit agonist potency on KOR. The selectivity over
MOR and DOR is also excellent (>200) as exemplified with 30
(Table 3). In general ortho-substituents are inferior to other posi-
tions (11 vs 10, and 14 vs 13 and 15) unless in the case of o-
hydroxy (24), capable of forming intramolecular hydrogen bonds
that is reasonably accepted (cf. 10). The p-fluoro (10) is having
lipophilicity and potency equal to 6 but the somewhat less lipo-
philic m-cyano derivative 15 is displaying slightly reduced
potency. Both compounds showed reduced but still reasonable
selectivity over DOR and MOR (Table 2). However, most of the lar-
ger polar groups were not tolerated. Thus, carboxylic acid ester (16,
17), ketone (20, 21), mesylate (22), sulfonamide (25, 28), sulfone
(26) and pyrazole (29) derivatives suffered from significantly
reduced potencies (Table 1). In every instance acidic groups (18,
19, 23 and 27) which would assist in driving PSA and lipophilicity
further in the desired direction were detrimental to KOR potency.
O
O
N
N
N
N
NH
NH
6
8
O
N
N
N
NH
N
O
9
N
NH
N
N
H
7
Figure 3. Initial KOR agonist in silico screening hit 6 and related structures 7–9.
H2N
NO2
Z
N
N
a
b
9-17;
n
NH2
+
20-22;
24-35;
37-47
X
I
N
X
RCOOH
n
X = CH, N
Y = H, OH
n = 1, 2
Y
X = CH, Z= F
X = N, Z = Cl
III
N
II
Y
Scheme 1. Reagents and conditions: (a) (i) DMF, K2CO3, rt, 16 h, (ii) 10% Pd/C, H2,
MeOH, rt, 16 h, (iii) BrCN, EtOH, CH2Cl2, rt, 16 h; (b) EDCI, HOBt, DCM, rt, 16 h.
Compound 6 and 8 were prepared from 7 by N-alkylation.
Compounds 18, 19, 23 and 36 were obtained by hydrolysis of the
corresponding esters.
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4
P. K. Sasmal et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Table 1
Table 2
Human KOR agonist potency of benzamide derivatives
Human KOR agonist potency of heteroaromatic derivatives
O
X
O
R
N
N
N
N
NH
NH
N
N
a
a
Compound
R
KOR EC50 (nM)
Compound
X
KOR EC50 (nM)
3b
6c
0.5
39
2000
878
1400
33
423
7
355
1225
72
1550
4250
>10,000
>10,000
537
970
478
>10,000
118
N
N
31
2050
7c
N
N
8c
32
33
34
35
36
37
70
9c
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
p-F
o-F
152
p-Cl
p-CN
o-CN
m-CN
p-COOMe
m-COOMe
p-COOH
m-COOH
p-COMe
m-COMe
m-OMs
S
S
1050
734
COMe
COOH
S
S
>10,000
22
S
N
38
39
40
41
122
8
m-OCH2COOH
p-F, o-OH
p-SO2NH2
H
N
2950
2400
>10,000
1550
1800
7
m-SO2Me
H
N
m-2-Tetrazolyl
m-SO2NH2, p-Cl
m-N-Pyrazolyl, p-Cl
m,p-diCl
1440
765
N
N
a
b
c
Values are mean of at least two experiments.
Refer Figure 1 for structure.
Refer Figure 3 for structures.
H
N
42
1
SMe
H
N
43
44
31
2
We then explored amides of a small set of heterocycles replac-
SO2Me
ing benzamides and the results are summarized in Table 2. Both
the pyridyl amides 32 and 33 showed moderate potency and good
selectivity (32, Table 3) whereas thiophenes (34 and 35) were less
tolerated. The corresponding carboxylic acid 36 was as expected
inactive. On the other hand the more polar pyrrole amide 39 with
logP of 2.7, logD of 1.8 and PSA of 66 (Table 3) was a very potent
agonist displaying 8 nM potency in the hKOR assay and several
100-fold selectivity over MOR and DOR (Table 3). Replacing pyrrole
NH with NMe (39 vs 41) resulted in a ꢀ100 fold drop in potency,
indicating a critical role of the N–H to stabilize a more coplanar
conformation. Surprisingly the pyrrole to pyrazole modification
as in 40 was not accommodated despite the tautomeric possibility
to resemble 39. When thiophene was replaced with the larger and
more lipophilic benzothiophene (logP 4.4/logD 3.5/PSA 50), a sig-
nificant improvement in KOR potency was observed (34 vs 37)
with a retained selectivity over the other opioid receptors. The cor-
responding benzothiazole 38 had reduced activity on all three opi-
H
N
45b
46b
47b
10
37
395
a
Values are mean of at least two experiments.
Refer Figure 4 for structures.
b
100) the later showed somewhat reduced potency KOR potency
and reduced opioid receptor selectivity (Tables 2 and 3). However,
compared to the benzamidesulfone 26 this is a higher potency than
expected once again indicating the positive conformational influ-
ence of the pyrrole system.
Another way of affecting the polarity is by manipulation of the
basic side chain, Thus, incorporation of a hydroxyl functionality on
the pyrrolidine as in 45 (Fig. 4) is completely tolerated as it dis-
played equipotency to corresponding compound 12. This deriva-
tive also showed 200-fold selectivity over the other two opioid
receptors and had improved polarity indexes (logP 3.1/logD 2.7/
PSA 70). Alternatively, replacement of benzimidazole (12) with
the slightly more polar imidazopyridine (46) led to little loss in
potency (Table 2). Hybridizing the structural features of 43 and
46 into 47 provided the most polar compound (logP 1.6/logD
1.0/PSA 113). It showed an additional 10-fold drop in potency to
KOR and the selectivity over DOR and MOR was significantly
reduced.
oid receptors (Table 3). Following
a similar pattern indole
derivative 44 displayed a higher KOR potency of 2 nM compared
to the plain pyrrole 39 but at the expense of a lower selectivity
(<100) over DOR and MOR (Table 2).
Another modification of the pyrrole with a lower lipophilicity
than the indole 44 was the thiomethyl substituted pyrrole 42 (logP
3.4/logD 2.5/PSA70). The sulfide 42 showed a very high KOR ago-
nist potency of 1 nM and a moderate selectivity over DOR (10-fold)
and high selectivity over MOR (200-fold). At this stage we were
interested to introduce further polarity onto this scaffold. When
sulfide 42 was transformed to sulfone 43 (logP 2.1/logD 1.5/PSA
Please cite this article in press as: Sasmal, P. K.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2014.12.064

P. K. Sasmal et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
5
Table 3
Opioid receptor selectivity and physicochemical profile of selected compounds
Compound
KOR EC50 (nM)
DOR EC50 (nM)
MOR EC50 (nM)
logPa
logDa
tPSAa
M_NOa
3
6
0.5
39
33
7
72
7
70
22
122
8
1
31
2
10
37
395
8000
7800
1800
2000
5500
1700
100,000
5200
100,000
2800
12
72
82
1850
37,000
1150
60,000
39,000
2400
6100
7800
1800
100,000
3300
100,000
6800
255
3800
150
1750
52,500
34,500
3.58
3.75
3.79
4.10
3.29
4.67
2.87
4.40
3.72
2.66
3.36
2.12
3.96
3.10
3.26
1.55
3.42
2.83
2.90
3.10
2.45
3.70
2.05
3.47
2.95
1.83
2.45
1.46
3.19
2.70
2.41
1.00
43.8
50.2
50.2
50.2
73.9
50.2
63.1
50.2
63.1
65.9
69.9
100.1
66.0
70.4
63.1
113.0
4
5
5
5
6
5
6
5
6
6
6
8
6
6
6
9
10
12
15
30
32
37
38
39
42
43
44
45
46
47
a
S + logP, S + logD (pH 7.4), tPSA and number of nitrogen and oxygen were calculated with MedChem Designer(TM) version 2.0.0.34. Simulations Plus, Inc.
potential of this compound series by performing a few animal
experiments. The compound 42 showed good PK profile in mice
(Table 4) and was evaluated further to establish proof of concept
in an acetic acid induced writhing model. On oral administration
42 showed dose dependent anti-nociceptive effect at 1 and
10 mg/kg doses as shown in Figure 5.30,31
In summary, the in silico screening approach led us to discover
a benzimidazole derived novel series of potent KOR agonists which
displayed good selectivity over the other opioid receptors DOR and
MOR. Structural modifications helped to understand the initial SAR
and identify regions for incorporating polar motifs. A representa-
tive molecule 42 showed significant anti-nociceptive effect in ace-
tic acid induced mice writhing model. Further optimization of this
series using the SAR information obtained will be disclosed
elsewhere.
H
N
O
O
N
N
Cl
N
N
NH
NH
SO2Me
N
45
47
O
N
N
Cl
NH
N
N
N
46
OH
N
Figure 4. KOR agonists (45–47) with structural diversity.
Table 4
Acknowledgments
Pharmacokinetic profilea of compounds 3 and 42 in SAMb
Compound Oral dose (mg/kg) AUC0Àt (ng*h/ml) Cmax (ng/ml) t1/2 (h)
The authors thank the analytical chemists of Discovery
Research for their support to generate spectral data of all
compounds.
3
30
10
493
334
507
250
0.5
2
42
a
AUC: area under the curve—a measure of the exposure to the drug; Cmax: the
maximum concentration recorded.
Supplementary data
b
Swiss albino mice.
Supplementary data (synthesis and characterization data of
compounds and the details on the receptor models of ligands
bound to the KOR, DOR and MOR receptors are provided) associ-
ated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmcl.2014.12.064.
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Figure 5. Anti-nociceptive effect of 42 (1 mg/kg-B and 10 mg/kg-C) and asimad-
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As discussed above a number of highly potent KOR agonists
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6
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various concentrations and 1
Following an incubation of about 60 min at 37 °C, 10
10 l anti IP1-Cryptate (Cisbio) were added to each well. Plates were incubated
l
l was added to each well to stimulate cells.
l
l IP1-d2 (Cisbio) and
l
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Please cite this article in press as: Sasmal, P. K.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2014.12.064