Identification of 3-substituted N-benzhydryl-nortropane analogs as nociceptin receptor ligands for the management of cough and anxiety

Article abstract of DOI:10.1016/j.bmcl.2009.03.057

A series of nortropane analogs based on previously reported compound 1 have been synthesized and shown to bind to the nociceptin receptor with high affinity. The synthesis and structure-activity relationships around the C-3 nortropane substitution are des

Full text of DOI:10.1016/j.bmcl.2009.03.057

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2482–2486
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of 3-substituted N-benzhydryl-nortropane analogs as nociceptin
receptor ligands for the management of cough and anxiety
a
a
a
a
b
Shu-Wei Yang a, , Ginny Ho , Deen Tulshian , William J. Greenlee , Zheng Tan , Hongtao Zhang ,
April Smith-Torhan b, Ahmad Fawzi b, John Anthes b, Sherry Lu b, Geoffrey Varty b,
Xiomara Fernandez b, Robbie L. McLeod b, John Hey b
*
a Department of Chemical Research—CV & CNS, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
b Neurobiology Department of Biological Research, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of nortropane analogs based on previously reported compound 1 have been synthesized and
shown to bind to the nociceptin receptor with high affinity. The synthesis and structure–activity relation-
ships around the C-3 nortropane substitution are described. From the SAR study and hPXR screening
effort, compound 15 was identified to possess potent oral antitussive and anxiolytic-like activities in
the guinea pig models.
Received 28 January 2009
Revised 10 March 2009
Accepted 12 March 2009
Available online 18 March 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Nociceptin
Antitussive
Anxiolytic
NOP
Genetic and pharmacological studies indicate that NOP modulates
the anxiety response.5,6 It was found that local (ICV) administration
of nociceptin neuropeptide or IP administration of NOP agonist
Ro64-6198 led to anxiolytic-like activity in rats.6,7 Recently, NOP
agonist SCH 221510, a nortropane analog structurally distinct from
nociceptin and Ro64-6198, was disclosed to possess the oral anxio-
lytic-like activity in various animal models including the rat condi-
tioned lick suppression (CLS) model and the separation-induced
guinea pig pup vocalization (GPPV) model.8 The reported data for
SCH 221510 suggested that NOP agonists have the potential to be
developed as a novel class of anxiolytic agents.
The nociceptin receptor (NOP, ORL-1), first discovered in 1994,
displays low binding affinity for the classical opioid ligands, albeit
its sequence has ꢀ50% homology to those of the opioid receptor
family
l, j
, and d (or MOP, KOP, and DOP, respectively).1 NOP is
widely distributed throughout the brain and spinal cord and thus
is expected to participate in various physiological processes. Fol-
lowing the discovery of NOP, there has been remarkable advance
toward understanding its pharmacological significance. The NOP
endogenous ligand (nociceptin)2 does not interact with the other
opioid receptors, and has been reported to mediate various physi-
ological processes, for instance, pain, cough, anxiety, cognition,
feeding, sleep, substance abuse and urinary incontinence.3 Thus,
selective NOP agonists or antagonists might have clinical potential
for the treatment of related diseases.
In our nociceptin agonist program, we have reported structure–
activity relationships (SAR) based on the 4-hydroxy-4-phenyl
piperidinyl scaffold.9 Further SAR development was focused on
the conformationally-restricted nortropane scaffold. Compound 1
was identified previously as a potent NOP agonist with potent
in vivo antitussive activity in the guinea pig model from a series
of 3-axial-aminomethyl-N-benzhydryl-nortropane analogs.10 In
this paper, we describe our continuing efforts to explore SAR at
the C-3 position to modulate the binding affinity and selectivity
profile (e.g., human pregnane X receptor (hPXR)). Synthesis and
SAR of a new 3-axial-substituted-N-benzhydryl-nortropane series
including carboxylamide, amine, and carbamate series are dis-
closed along with our strategy to identify potent dual antitussive
and axiolytic agents.
Nociceptin has been shown to display antitussive activity in the
guinea pig model through peripheral (IV) or central (ICV) adminis-
tration.4 Thus, selective NOP agonists provide a novel therapeutic
approach for the management of cough.
NHAc
Cl
NOP Ki: 20 nM
N
Cl
γ
NOP (GTP S) EC50: 269 nM
Antitussive activity:
ED50: 0.19 mg/kg at 2 h
1
The synthetic route to the C-3-
rized in Scheme 1. The commercially available tropinone (2) was
a-substituted nitrile is summa-
* Corresponding author. Tel.: +1 908 740 7291; fax: +1 908 740 7164.
E-mail address: shu-wei.yang@spcorp.com (S.-W. Yang).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.057

S.-W. Yang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2482–2486
2483
de-methylated and subsequently alkylated using previously estab-
lished procedure to afford the ketone intermediate (3).9,10 Trans-
formation of to the nitrile and subsequent nucleophilic
addition of a benzyl or pyridyl group through the less hindered
CN
R
OH
R
Cl
Cl
a, b
c, d
4
N
Cl
N
Cl
3
a
11
12
face was achieved using the corresponding benzyl halides or pyri-
dyl halides under the basic condition to give 4.10 The C-3 stereo-
chemistry was confirmed with the NOE experiments of the
hydrolyzed products (5). The NOE correlations were observed be-
tween the amide protons and the protons on the ethylene bridge
in the NOESY experiment, confirming the b position of the amide
group. The detail NMR and high-resolution mass data of one exam-
ple of 5 are presented in the reference.11
R = Bn
CONH2
Cl
N
e
R
Cl
13
Scheme 4. Reagents and conditions: (a) DIBAL in toluene, 0 °C; (b) NaBH4, MeOH,
0 °C; (c) MsCl, Et3N, DCM, 0 °C; (d) KCN, 18-crown-6, DMF, 110 °C; (e) concd H2SO4,
neat, rt.
Further transformation of the C-3 nitrile (4) to the carboxyla-
mide, carbamate, and amino functional groups is detailed in
Scheme 2. Thus, the nitriles (4) were hydrolyzed to the carboxyla-
mides (5) with concd H2SO4, and then rearranged through Hof-
mann rearrangement to afford the corresponding methyl
carbamates (6) using bis(acetoxy)iodobenzene and KOH/MeOH.
The 3-amino analogs (7) were obtained by removal of the –CO2Me
group with TMSI in dichloromethane and then in MeOH under
reflux.
aldehyde intermediate using DIBAL, and further reduced to the
alcohol (11) with NaBH4. Mesylation of the alcohol and the subse-
quent replacement with a cyano group gave nitrile 12. Finally, the
nitrile (12) was hydrolyzed to the carboxylamide (13) under the
same acidic condition described above.
The N-substituted analogs were synthesized as described in
Scheme 3. Acetylation and methylation of 7 gave 8 and 9,
respectively.
To prepare the amide homolog (13), a five-step synthesis was
designed as shown in Scheme 4. The nitrile 4 was reduced to the
Target compounds were tested for their affinity at the cloned
human nociceptin receptor expressed in CHO cell membranes by
measuring their ability to compete with [125I][Tyr14]nociceptin.
The opioid receptor binding assays were performed with CHO cell
membranes expressing the human opioid receptors using [3H]-
diprenorphine as the radioligand. The functional activities of se-
lected compounds were evaluated as their ability to enhance the
binding of [35S]GTP
cS in the presence of GDP, using membranes
Cl
O
CN
3
isolated from CHO cells transfected with the human nociceptin
gene. Since most of the nortropane analogs identified previously
showed good selectivity over DOP and KOP,10 only selectivity over
MOP will be presented. To limit the number of the compounds for
further evaluation, the compounds with the NOP Ki less than
15 nM and selectivity (NOP vs MOP) higher than ꢀ10-fold were
selected.
Cl
a
O
b
c
N
N
R'
N
Cl
d
Cl
Cl
Cl
Br
2
4
3
R' = substituted 2-, 3-, or
4-pyridinyl, or benzyl
Scheme 1. Reagents and conditions: (a) a-chloroethyl chloroformate, DCE, reflux;
The C-3 2-pyridyl carboxylamide 14 showed potent NOP bind-
ing affinity with a Ki of 4 nM. Thus a series of the substituted pyr-
idyl analogs were prepared. Introduction of a small substituent
(e.g., F, Cl, Me, or OMe) at the 5 position of the pyridine ring slightly
reduced NOP affinity (15–19, Ki between 6 and 22 nM, Table 1).
Substitutions of the polar groups (OH or NH2), cyano, or relatively
large groups (e.g., Br, CF3 or CONH2) at the 5-position of the pyri-
dine ring were found to decrease the NOP binding affinity signifi-
cantly (17, 20–25). The binding selectivity of this series of
compounds for the NOP receptor versus MOP is around 4–17-fold.
The promising result of the 5-fluoro analog (15) triggered addi-
tional studies on the fluoro-substituted pyridyl analogs. Moving
the fluoro atom from the 5-position to the 6-position retained
NOP affinity and improved selectivity over MOP (26, 35-fold). Most
of the di-fluoro compounds (27–31) showed slightly reduced NOP
affinity (Ki 9–19 nM), and were selected for further hPXR evalua-
tion. A dramatic loss of NOP affinity was observed while carboxylic
acid was introduced to the 4-position of the pyridine ring (32). In
general the polar or large substitutions were not preferred at the
4- or 5-position of the 2-pyridine ring (Table 2).
then MeOH, reflux; (b) 2, K2CO3, CH3CN, reflux; (c) KO–tBu, tosylmethyl isocyanide,
DME, À40 °C to rt; (d) NaHMDS, RX, THF, À78 °C to rt.
O
O
NH2
R
HN
O
Cl
Cl
a
b
4
N
R
N
Cl
Cl
5
6
R = Bn, or 2-pyridyl
NH2
Cl
c
N
R
Cl
7
Scheme 2. Reagents and conditions: (a) concd H2SO4, neat, rt; (b) PhI(OAc)2, KOH,
MeOH, 0 °C to rt; (c) TMSI, DCM, reflux, then MeOH reflux.
To further explore the C-3 axial substitution, the C-3 nitrogen
directly-attached analogs were prepared. The binding affinity data
of these analogs are listed in Table 3. The C-3 unsubstituted amino
analogs 33 and 34 exhibited potent NOP affinity (Ki 10 nM; 7 nM,
respectively) and decent selectivity over MOP (32-fold). The acetyl
analogs (35 and 36) of 33 and 34 retained NOP affinity (Ki 6 and
10 nM) and increased selectivity over MOP to 52- and 60-fold,
respectively. The methyl carbamates (37–39) displayed similar
NOP affinity (Ki 10–12 nM) and improved selectivity (74–80-fold)
over MOP. However, the N,N-di-methyl substitution resulted in
NHAc
NH2
R
N
Cl
Cl
Cl
b
a
N
Cl
R
N
Cl
N
Cl
R
8
7
9
Scheme 3. Reagents and conditions: (a) Ac2O, pyridine, rt; (b) HCHO, HCO2H, rt;
NaBH(OAc)3, DCE, rt.

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S.-W. Yang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2482–2486
Table 1
Table 4
SAR of the C-3 substituted 2-pyridyl analogs
SAR of the C-3 benzyl substituted analogs
R1
Cl
NH2
N
O
Cl
N
N
Cl
Cl
R
R2
Compds
R1
R2
NOP Ki (nM)
MOP Ki (nM)
Compds
R
NOP Ki (nM)
MOP Ki (nM)
41
42
43
44
45
46
CONH2
CONH2
CH2CONH2
CH2CONH2
NH2
F
Cl
F
OH
Cl
Cl
3
10
3
17
36
56
85
14
15
16
17
18
19
20
21
22
23
24
25
H
F
Cl
Br
4
7
52
284
190
nd
447
nd
123
143
153
26
133
205
nd
481
nd
1315
nd
15
29
6
22
31
72
75
122
401
>1000
Me
NHCO2Me
OMe
NH2
OH
nd: not determined.
CN
CF3
CONH2
CO2Me
pound 43 with an additional carbon extension on the C-3 carbo-
xylamide retained potent NOP affinity (Ki 3 nM) and enhanced
selectivity over MOP (ꢀ63-fold), compared to 41. Replacement of
the fluoro with a polar hydroxyl group (44) at the para position re-
sulted in a sixfold decrease in the NOP binding affinity. Replace-
ment of the C-3 carboxylamide with an amino (45) or methyl
carbamate (46) group led to the diminished NOP binding affinity
(Ki 36 and 56 nM, respectively).
The 3- and 4-pyridyl analogs (47–49) were evaluated for NOP
and MOP affinities. The 3-pyridyl analogs (47 and 49) retained
NOP affinity (Table 5) and selectivity over MOP, whereas the 4-pyr-
idyl analog (48) showed a 20-fold decrease in NOP affinity, com-
pared to 2-pyridyl 14. The racemic C-3 piperidinyl carboxylamide
(50), prepared from reduction of 14 with PtO2 and H2, showed a
fourfold decrease in NOP affinity, when compared to 14.
nd: not determined.
Table 2
SAR of the C-3 substituted 2-pyridyl analogs (continued)
NH2
N
O
Cl
6
N
Cl
3
5
R1,R2
Compds
R1, R2
NOP Ki (nM)
MOP Ki (nM)
26
27
28
29
30
31
32
6-F
10
15
16
47
9
19
348
468
nd
nd
74
4-F, 6-F
3-F, 6-F
3-F, 5-F
4-F, 5-F
4-CH2F, 5-F
4-CO2H, 5-F
The functional activity (EC50) was evaluated for selected com-
pounds using the GTPcS assay. The data listed in Table 6 showed
the NOP and MOP functional activities (EC50) and percent stimula-
tion at certain concentration of compounds 15–17, 35, 37, 41, and
46. The data demonstrated their full NOP agonist activity with the
EC50s ranging from 11 to 87 nM and >75% stimulation at the high
367
nd
>1000
nd: not determined.
concentration in the NOP GTPcS assay. Most of the compounds
(15, 35, 37, 41, and 46) showed medium to high selectivity over
MOP, except compounds 16 and 17 with selectivity equal or less
than 10-fold over MOP.
Table 3
SAR of the C-3 2-pyridyl C-3 amino analogs
R1
N
N
Cl
Table 5
N
Cl
SAR of the additional C-3 heterocyclic analogs
R2
CONH2
Q
Cl
Compds
R1
R2
NOP Ki (nM)
MOP Ki (nM)
N
33
34
35
36
37
38
39
40
H
H
Ac
Ac
CO2Me
CO2Me
CO2Me
Me, Me
H
F
H
F
H
F
Cl
H
10
7
6
10
10
12
12
42
343
209
312
601
741
887
961
840
Cl
Compds
Q
NOP Ki (nM)
MOP Ki (nM)
N
N
47
3
71
48
49
79
13
265
46
the diminished NOP affinity (40, 42 nM). Overall, the amino, N–Ac
and N–CO2Me groups were all tolerated at the C-3 axial position for
NOP affinity with improved selectivity over MOP, compared to the
carboxylamide series (Table 1).
N
Br
The C-3 benzyl analogs were synthesized to compare to the pyr-
idyl series. The SAR data of the C-3 benzyl analogs are listed in Ta-
ble 4. The para-flouro (41) or para-chloro (42) benzyl substitution
at the C-3 equatorial position was tolerated for NOP affinity (Ki 3
and 10 nM, respectively) and led to improved selectivity over
MOP (ꢀ28-fold), compared to the pyridyl analogs 15 and 16. Com-
H
N
50
17
474

S.-W. Yang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2482–2486
2485
Table 6
respectively, compared to the reference compound CDP (ED50
8.8 mg/kg). Compound 15 also displayed potent oral antitussive
activity in the capsaicin-induced guinea pig model with an ED50
of 0.02 mg/kg, at 2 h, compared to the reference compound codeine
(ED50 6.7 mg/kg). The acetamide analog 43 only displayed ꢀ38%
cough reduction at 0.3 mg/kg, and the amino analogs 33 and 34
did not show any antitussive activity at the low dose (0.3 mg/kg).
Compound 43 did not display anxiolytic-like activity in the GPPV
model at a dose of 3 mg/kg.
The functional activity of selected compounds
Compds GTP
c
(nM)
S NOP, EC50 GTP
c
S % Stim
GTP
(nM)
c
S MOP EC50 GTP
cS % Stim
(l
M)
(lM)
15
16
17
41
46
35
37
20
44
63
11
14
19
87
95 (10)
145 (10)
75 (10)
108 (10)
85 (5.5)
84 (10)
92 (10)
733
368
421
89 (5)
97 (10)
89 (10)
107 (10)
59 (5.5)
68 (10)
52 (10)
540
1563
1933
3777
NOP Ki: 7 nM
O
NH2
N
γ
NOP (GTP S) EC50: 20 nM
Cl
MOP Ki: 123 nM
γ
MOP (GTP S) EC50: 733 nM
N
DOP: Ki: 3347 nM
F
Cl
Selected compounds were evaluated for the potential of Cyp3A4
KOP: Ki: 1610 nM
induction using the hPXR reporter-gene assay.12 The induction le-
vel was compared to the positive control rifampicin (RIF), and the
ratio of 0.4 was set to be the screening cut-off to minimize the pos-
sibility of false-positive results. Compound 1, identified previously,
Antitussive activity:
ED50: 0.02 mg/kg at 2h
GPPV: ED50: 0.3 mg/kg
CLS: ED50: 1.7 mg/kg
15
From these SAR studies and screening efforts, compound 15 was
found to possess potent dual antitussive and anxiolytic-like activi-
ties with the best overall profile. It displayed high binding selectiv-
ity for the NOP receptor versus DOP and KOP, and reasonable
selectivity over MOP in the functional assay (ꢀ37-fold). Compound
15 also exhibited improved hPXR data (ratio 0.29 to RIF) compared
to the previously identified lead compound 1 (ratio 0.94 to RIF).
Compound 15 displayed stronger efficacy in the antitussive assay
(ED50 0.02 mg/kg) than that in the anxiolytic-like GPPV assay
(ED50 0.3 mg/kg). The antitussive/anxiolytic-like efficacy ratio was
ꢀ15.
showed a high hPXR ratio (0.94) to RIF at 1 lM (Table 7). From this
study it was found that substitution on the 5-pyridyl position (15–
19) reduced the hPXR liability, compared to the unsubstituted pyr-
idyl analog (14). In the benzyl series, most tested compounds in
Table 4 displayed some liability for hPXR, except 43 (ratio to RIF
0.35), indicating the
a-benzyl group (e.g., 41, 42) could lead to
higher hPXR ratio to RIF, compared to the pyridyl analogs (15,
16). In the C-3 nitrogen directly-attached series, compounds 35–
39 and 46 showed high hPXR ratio (0.98–1.97) to RIF. Therefore
the terminal acetamide and methyl carbamate groups were identi-
fied as strong inducers for Cyp3A4 through the hPXR reporter gene
pathway in the pyridyl series.
Selected compounds which met the above criteria were investi-
gated in vivo.4,8 The GPPV model was used as a screening tool.
Compounds 15 and 16 displayed an oral anxiolytic-like activity
with ED50s < 3 mg/kg in the GPPV screening assay. Further dose-re-
sponse studies determined both of their ED50 values as 0.3 mg/kg,
compared to the reference compound chlordiazepoxide (CDP)
(ED50 3.2 mg/kg). In the rat CLS model, 15 and 16 exhibited the
anxiolytic-like activity with ED50 values of 1.7 and 2.4 mg/kg,
In summary the C-3 2-pyridyl carboxylamide (14) displayed po-
tent NOP affinity. However, compound 14 showed high hPXR ratio
to RIF. Substitution at the 5-position of the pyridine reduced the
hPXR liability and led to the discovery of 15. The C-3 amine, acet-
amide, and methyl carbamate analogs in the pyridyl series (33–39)
showed good binding and selectivity profiles, whereas they suf-
fered from the hPXR-induction liability. The benzyl series with
the C-3 carboxylamide (41, 42) or acetamide (43) displayed good
binding profile, but again showed positive response in the hPXR as-
say. The 3-pyridyl analog 47 displayed good affinity profile, how-
ever, the hPXR ratio (0.40) was close to the cut-off value, and
this agonist was not further evaluated. In conclusion, among those
potent NOP agonists identified, compound 15 demonstrated the
best overall profile and superior in vivo activity. It exhibited potent
oral antitussive and anxiolytic-like activities in the animal models,
superior to the standard compounds codeine and chlodiazepoxide,
without having the hPXR liability.
Table 7
The hPXR data for selected compounds
Compd
hPXR ratioa (1
lM)
1
0.94
0.79
0.29
0.27
0.09
0.40
0.19
1.06
0.55
0.83
0.18
0.77
0.47
0.23
1.97
1.47
0.98
1.38
1.06
0.48
0.67
0.35
0.51
1.10
0.40
14
15
16
17
18
19
26
27
28
30
31
33
34
35
36
37
38
39
41
42
43
44
46
47
Acknowledgments
The authors wish to acknowledge Dr. Jianshe Kong, Dr. Jesse
Wong, and Mr. Meng Tao for preparation of intermediates; Dr.
Tze-Ming Chan and his group for structure confirmation of some
analogs and determination of the C-3 stereochemistry; Drs. Xiaoy-
ing Xu, Xiaoming Cui, and DMPK group for acquiring hPXR and
pharmacokinetic data.
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8. Varty, G. B.; Lu, S. X.; Morgan, C. A.; Cohen-Williams, M. E.; Hodgson, R. A.;
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11. Compound 15: 1H NMR (CDCl3): d 1.90 (dd, J = 14.4, 7.0 Hz, 2H), 2.09 (br s, 2H),
2.19 (dd, J = 13.7, 2.7 Hz, 2H), 2.77 (br d, J = 12.3 Hz, 2H), 3.08 (br s, 2H), 5.37
(br s, 1H, –NH), 5.42 (s, 1H), 6.15 (br s, 1H, –NH), 7.07 (td, J = 7.8, 1.8 Hz, 2H),
7.15 (t, J = 7.6 Hz, 2H), 7.26 (m, 4H), 7.69 (d, J = 7.7 Hz, 2H), 8.31 (d, J = 2.6 Hz,
1H). 13C NMR (CDCl3): d 25.9, 40.3, 48.8, 57.2, 59.4, 121.7 (J = 3.8 Hz), 123.3
(J = 18.2 Hz), 126.6, 127.9, 129.4, 130.1, 134.3, 136.2 (J = 22.8 Hz), 139.6, 158.0
(J = 253 Hz), 160.9, and 176.7. HRMS (ESI) calcd for C26H25Cl2N3OF, 484.1359
(M + H+); found, 484.1370.
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Metab. Dispos. 2002, 30, 795; (c) Cui, X.; Thomas, A.; Gerlach, V.; White, R. E.;
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