Gamma-lactams-A novel scaffold for highly potent and selective α7 nicotinic acetylcholine receptor agonists

Article abstract of DOI:10.1016/j.bmcl.2009.01.073

A novel class of α7 nicotinic acetylcholine receptor (nAChR) agonists has been discovered through high-throughput screening. The cis γ-lactam scaffold has been optimized to reveal highly potent and selective α7 nAChR agonists with in vitro activity and selectivity and with good brain penetration in mice.

Full text of DOI:10.1016/j.bmcl.2009.01.073

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1287–1291
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Gamma-lactams—A novel scaffold for highly potent and selective
nicotinic acetylcholine receptor agonists
a7
a
b
Albert Enz a, Dominik Feuerbach a, Mathias U. Frederiksen b, , Conrad Gentsch , Konstanze Hurth ,
*
Werner Müller b, Joachim Nozulak b, Bernard L. Roy b,
*
a Novartis Institutes for Biomedical Research, Neuroscience Disease Area, 4002 Basel, Switzerland
b Novartis Institutes for Biomedical Research, Global Discovery Chemistry-Neuroscience, Postfach, 4002 Basel, BS, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel class of
throughput screening. The cis
7 nAChR agonists with in vitro activity and selectivity and with good brain penetration in mice.
Ó 2009 Elsevier Ltd. All rights reserved.
a
7 nicotinic acetylcholine receptor (nAChR) agonists has been discovered through high-
Received 17 December 2008
Revised 22 January 2009
Accepted 23 January 2009
Available online 7 February 2009
c
-lactam scaffold has been optimized to reveal highly potent and selective
a
Keywords:
c
-Lactams
a7
Nicotinic
nAChR
Nicotinic acetylcholine receptors (nAChRs) belong to a super-
family of ligand gated ion channels that include the receptors for
GABA, glycine and serotonin (5-HT3). For centrally expressed nAC-
pyridine in anabasein-derived scaffolds 2 or a bicyclic amine in 3–
5, most prominently quinuclidine as in 3 and 4) connected to a
hydrophobic element by a linker (e.g. carbamate, ether or amide)
(Fig. 1).5–7
In view of the populated chemical space around these general
scaffolds we sought to find alternative templates. Herein we de-
hRs several genes have been cloned to date including nine
units ( 2– 10) and three b subunits (b2–b4). Among the various
polymeric constellations that these subunits may adopt, the
nAChR can form functional homopentamers that bind -bungaro-
7 sub-
a sub-
a
a
a7
a
scribe the discovery of a series of
c-lactams as
a
7 nAChR agonists
toxin with high affinity. Recent findings indicate that the
a
unit may also assemble with other subunits.1
A substantial body of evidence suggests that the
involved in the pathophysiology of several human diseases. Cen-
trally, agonism of the 7 nAChR has been discussed as a therapeu-
a7 nAChR are
OMe
a
N
tic option for a number of neurological disorders such as; age
OMe
N
associated memory impairment (AAMI),2 Alzheimer’s disease and
N
H
schizophrenia.3 Additionally, peripherally
pressed in macrophages and stimulation of the
the release of inflammatory cytokines (e.g. TNF-
HMGB-1.4 Thus, the clinical use of agonists of the
a
7
nAChRs are ex-
7 nAChR inhibits
, IL-1) as well as
7 nAChR could
a viable strategy against a multitude of human
HN
O
a
a
N
O
a
N
GTS-21
1
2
3
represent
disorders.1
Nicotine
AR-R17779
N
O
Recently several
of which have progressed to the clinic. The common pharmaco-
phore of nicotine and 7 nAChR agonists usually consists of a
a7 nAChR agonists have been described, some
N
O
F
a
O
N
H
N
H
H
strong base (e.g. pyrolidine in nicotine 1, piperidine or tetrahydro-
N
O
4
5
JN403
PHA-709829
* Corresponding authors. Tel.: +41 0 61 3246502; fax: +41 0 61 6962455 (M.U.F.).
E-mail address: mathias.frederiksen@novartis.com (M.U. Frederiksen).
Figure 1. Representative
a
7 nAChR agonists.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.01.073

1288
A. Enz et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1287–1291
Table 2
that display high potency, selectivity and interesting in vivo phar-
macokinetic properties.
Exploration of hydrophobic moiety
In a recent screening campaign a small cluster of cis gamma-lac-
tams emerged as a structurally distinct hit class represented by 6
and 7 (Fig. 2).8
O
N
R3
N
R2
R1
These hits were found to be a7 nAChR agonists with pEC50 val-
ues of 5.9 (6) and 6.1 (7) in a Ca-influx assay.9 Both compounds
also showed good relative efficacy in comparison to the known
agonists nicotine (1), GTS-21 (2) and AR-R17779 (3) as well as
Compounda
R1
R2
R3
pEC50
Emax (%)
b
a
7 nAChR.9 Intrigued
10
6
11
7
12
13
14
15
16
OMe
H
H
Cl
H
H
H
H
H
H
OMe
H
H
Cl
H
H
H
H
H
H
OMe
H
H
Cl
Br
Me
Cy
5.9
5.9
5.9
6.1
6.0
6.0
6.2
6.2
6.5
107
128
124
89
102
110
89
(+)-epibatidine which is a full agonist at the
by the discovery of a novel 7 agonist scaffold of good potency, hit-
a
to-lead optimization was initiated. We started with the exploration
of the piperidine moiety which presumably mimics the basic nitro-
gen of nicotine (Table 1).
Replacing the piperidine in 6 by a smaller pyrolidine led to loss
of potency (e.g. 8, Table 1). Expanding the ring in 7 to a seven-
membered ring (9) also led to lower potency.
114
60
Further efforts focused on improving potency by modifying the
aromatic portion of the scaffold. A positional scan of the meta-meth-
oxy or ortho-chloro substituent in the original hits (e.g. 6, 10, 11, and
7, 12, 13, Table 2) had little effect on potency. However replacing the
para position with a bromide (14) or methyl (15) led to a small but
significant improvement. Introduction of a more lipophilic group,
for example a cyclohexyl (16) or cyclopentyl (17) moiety, led to im-
17
H
H
6.5
28
18
19
20
H
H
H
H
H
H
Ph
2-F-Ph
2-Cl-Ph
5.9
6.5
6.9
61
47
62
a
Compounds tested as diastereomerically pure cis racemates.
Agonist-activity in GH3 cells recombinantly expressing human
b
a7
nAChR.
Compounds measured in duplicate. EC50 values generated from individual 8-point
concentration response curves.9
Cl
O
O
O
N
N
proved potency albeit at a detriment to efficacy. Continuing in this
vein the central aromatic ring was substituted with a phenyl (18)
which proved inferior. However the potency could be resurrected
and even further improved by placing an ortho-fluoro (19) or
ortho-chloro substituent (20) providing the most potent agonist of
this series. Presumably the ortho substituent provides the ideal biar-
yl conformation to fill the lipophilic pocket.
N
N
6
7
pEC50 = 5.9
Emax = 128
pEC50 = 6.1
Emax = 89
Figure 2. Cis c-lactam HTS hits 6 and 7.
We hypothesized that the central phenyl served as a hydropho-
bic linker positioning the lactam and the distal hydrophobic moi-
ety in optimal distance for potency. Thus we sought to explore
by modifying the linker in order to investigate the effect it would
have on potency. Having attained good potency with 20, we also
Table 1
Initial hit exploration of
c
-lactam hits 6 and 7
O
started to monitor selectivity against the a3b4 nAChR sub-type.
N
The replacement of the phenyl linker by a thiazole led to a signif-
icant improvement in potency and efficacy and an improvement in
selectivity (21, Table 3). Other heterocyclic linkers such as ‘naked’
or substituted thiophene (22 and 23) or imidazoles (24 and 25)
were less potent. The breakthrough came by removing the cyclic
element altogether and linking the c-lactam and the hydrophobic
moiety with an alkyne which led to a potent, efficacious and selec-
tive lead (26).
The desirable profile of the alkyne led us to further examine this
linker. It was found that the distal ortho-chloro phenyl moiety,
which in 20 had proven decisive to obtain potency, could be ex-
changed with an ortho-methyl (27, Table 4) or even an unsubstitut-
ed phenyl (28) to yield potent and efficacious compounds. Potency
could be improved by replacing the phenyl with a N-methyl indole
(29), albeit without improving selectivity. To our satisfaction the
selectivity could be further improved by replacing the phenyl with
benzofuran or dihydrobenzofuran (30 and 31) yielding highly po-
R3
R1
R2
Compounda
R1
R2
R3
pEC50
Emax
b
1
2
3
—
—
—
—
—
—
—
—
—
5.8
5.7
6.4
7.4
100
71
104
100
—
—
—
(+)-Epibatidine
6
8
7
MeO
MeO
H
H
5.9
5.1
6.1
5.7
128
80
N
H
N
Cl
Cl
89
N
N
tent and selective
The alkynes 28–31 were profiled further in our ligand gated ion
channel panel, which included the 3b4, 4b2, and 1b1 d nico-
a7 agonists.
a
a
a
c
9
H
82
tinic sub-types, as well as the 5-hydroxytryptamine 3 (5-HT3)
receptors (Table 5).
a
Compounds tested as diastereomerically pure cis racemates.
Agonist-activity in GH3 cells recombinantly expressing human
Overall the alkynes displayed excellent selectivity against the
nAChR sub-types as well as the 5-HT3 receptor (all > 100-fold).
Especially noteworthy is the selectivity against the 5-HT3 receptor
b
a
7
nAChR.
Compounds measured in duplicate. EC50 values generated from individual 8-point
concentration response curves.9

A. Enz et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1287–1291
1289
Table 3
Table 5
Exploration of linker
Selectivity profile of alkynes 28–31 compared to JN403 (4)
Compounda
a
pEC50
7
a
3b4 pIC50
a
4b2 pIC50
a1b1cd pIC50 5-HT3 pIC50
Cl
O
(ratio)c
(ratio)c
(ratio)c
(ratio)c
b
N
R
4
28
29
30
31
7.0
8.0
8.1
8.1
8.1
4.7 (200)
5.9 (126)
6.0 (111)
6.1 (123)
6.0 (154)
4.6 (251)
5.1 (820)
5.7 (232)
5.3 (660)
5.0 (1263)
3.9 (1259)
4.9 (1299)
5.4 (431)
4.0 (16982)
5.2 (773)
4.7 (200)
N
3.6 (26368)
4.6 (3221)
4.0 (16982)
4.3 (5389)
Compounda
R
pEC50
6.9
Emax (%)
Selectivity
3
a7/a
3b4c
b
a
Compounds tested as pure enantiomers.
Agonist-activity in GH3 cells recombinantly expressing human
20
62
b
a7 nAChR.
Compounds measured in duplicate. EC50 values generated from individual 8-point
concentration response curves.9
Antagonistic activity in cells expressing the different nAChR10 and 5-HT3
c
5
N
21
22
23
24
25
7.6
7.5
6.2
5.9
6.2
7.6
89
79
81
61
47
89
14
26
nd
nd
nd
52
receptors.
S
S
when compared to the quinuclidene derivative 4. When tested
against human recombinant muscarinic receptors the compounds
also displayed IC50 > 10 lM on all sub-types (M1–M5).
In vitro cardiovascular safety profiling was also undertaken,
with the compounds submitted for patch-clamp hERG K+ channel
assay. In general the compounds display reasonable cardiosafety,
for example, alkyne 28, having an IC50 of 6.85 lM. Patch clamp
experiments at the cardiac sodium channel were also performed,
and alkyne 28 was found to display a 33% inhibition at a concentra-
tion of 30 lM. Finally to investigate the safety window in an
ex vivo whole heart assay, we submitted alkyne 28 to the ‘‘Lange-
ndorff” isolated rabbit heart assay, and found to have no QT-inter-
val prolongation with doses up to 10 lM.
The ADME and pharmacokinetic data were also assessed in vitro
and in vivo (Table 6).
Me
S
N
N
H
N
N
Me
26
The phenyl-substituted alkyne 28 showed rapid clearance and
short half-lives in the mouse liver microsome preparations
(ER = 90%, t1/2 = 6.6). The benzofuran 30 showed improved in vitro
metabolic stability, displaying an ER and t1/2 of 67% and 12.5 in
MLM and 68% and 25.8 in HLM, respectively. The rapid in vitro clear-
ance of 28 was reflected in the low plasma levels when administered
orally to mice. In spite of the low plasma levels the compound
showed excellent brain exposure and no retention. The improved
microsomal stability of the benzofuran 30, translated into improved
in vivo pharmacokinetic properties. High brain and plasma levels
were observed at one and four hours following administration. As
was the case with phenyl-substituted alkyne 28 the brain/plasma
ratios at both time points indicated good brain penetration.
Compounds were prepared following two general procedures
outlined below (Schemes 1 and 2). The diastereomerically pure race-
mates were prepared as summarized in Scheme 1. Various
aldehydes (e.g. 32, R = aryl, heteroaryl or alkyne) were converted
to methyl-imines (e.g. 33). From which the corresponding lactams
35 were generated by treating the imine with an organozinc reagent
a
Compounds tested as diastereomerically pure cis racemates.
Agonist-activity in GH3 cells recombinantly expressing human
b
a7 nAChR.
Compounds measured in duplicate. EC50 values generated from individual 8-point
concentration response curves.9
Antagonistic activity in cells expressing the different nAChR10 and 5-HT3
c
5
receptors.
Table 4
Optimization of alkyne linked
a
7 agonists
O
N
R
N
Compounda
R
pEC50
Emax (%)
Selectivity a7/a
3b4c
b
26
27
28
2-Cl-Ph
2-Me-Ph
Ph
7.6
7.8
8.0
82
83
104
52
104
126
derived in situ from bromide 34. The resulting
a,b-unsaturated
lactams were treated with piperidine and the major cis isomers
(6–17, 36, and 37) were isolated (typical cis/trans ratio 3/1) in good
overall yields. The aryl linked agonists 18–20, could be obtained by
Suzuki12 coupling of 14 with the corresponding aryl boronic acids.
The heteroaryl linked agonists 21–25 were obtained in a similar
fashion from the corresponding brominated heterocycle 36. Finally,
the alkyne linked derivatives were obtained by desilylation of 37 fol-
lowedbySonogashira13 coupling. In theexamples26–31, pureenan-
tiomers could be isolated after chromatography on chiral support.14
Additionally we developed an asymmetric route to access this
scaffold. This sequence is exemplified for compound 20 below in
Scheme 2. The route was based on the work of Villiéras15 and
Yamamoto.16 Aldehyde 38 was condensed with (S)-phenyl glycidol
to afford imine 39. The diastereoselective addition of an organozinc
reagent derived from bromide 34 in situ onto the corresponding
29
30
8.1
8.1
8.1
100
90
111
123
154
N
O
O
31
80
a
Compounds tested as pure enantiomers.
Agonist-activity in GH3 cells recombinantly expressing human
b
a7 nAChR.
Compounds measured in duplicate. EC50 values generated from individual 8-point
concentration response curves.9
Antagonistic activity in cells expressing the different nAChR10 and 5-HT3
c
5
receptors.

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A. Enz et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1287–1291
Table 6
ADME and pharmacokinetic data
Plasma protein binding (human/rat)a
MLM ER/t1/2
HLM ER/t1/2
Mouse in vivo pharmokokinetic datac
Brain/plasma levels 1 h (pmol/g) Brain/plasma levels 4 h (pmol/g)
b
b
Compound
LogP (o/w)
28
30
3.7
3.9
17%/31%
nd
90%/6.6
67%/12.5
nd
68%/25.8
239/18
6533/1001
64/4
3676/544
a
Plasma protein binding expressed as free fraction.
MLM and HLM, mouse liver microsomes or human liver microsomes, respectively, in vitro hepatic extraction ratios (ER) are reported as% compound depleted at
b
completion of experiment.
c
Mouse in vivo pharmacology: Mice (n = 6) were treated with 30
l
mol/kg po of compound. Plasma and brain concentrations were assessed at the time points indicated.11
For full protocols see Ref. 11.
Br
CO2Me
O
O
34
a
c
N
R
N
b
R
H
R
32
33
35
R = Aryl, heteroaryl
or alkynyl
O
O
O
N
N
N
N
N
N
R3
N
X
Br
Ar
R2
R1
Y
Y
TIPS
Z
6 - 17
36
37
d
d
e, f
O
O
O
N
N
N
N
N
X
Ar
Z
Ar
18 - 20
21 - 25
26 - 31
Scheme 1. General synthetic route for the preparation of
c-lactams. Reagents and conditions: (a) MeNH2 (33% in EtOH), 25 °C, 4 h (85–95%); (b) 34, Zn in THF, 25 °C, 20 min,
followed by addition of 33, then stirred for 3.5 h at 25 °C (70–90% over two steps); (c) piperidine, 2 h, 85 °C, (50–60%); (d) arylboronic acid, Pd(OAc)2, Na2CO3, PhMe, 12 h,
110 °C (40–65%); (e) TBAF, THF, 2 h, 25 °C (90%); (f) ArI, (PPh3)2PdCl2, CuI, THF/Et3 N, 2 h, 25 °C (40–60%).
HO
H2N
Br
OH
CHO
CO2Me
Ph
Cl
N
Ph
34
H
Cl
Cl
Cl
a
b
38
HO
39
HO
Ph
O
CO2Me
Ph
NH
N
d
c
Cl
Cl
40
HN
41
O
O
N
N
e
42
Scheme 2. General synthetic route for the preparation of enantiomerically pure
20
c
-lactams. Reagents and conditions: (a) (S)-phenyl glycidol, CH2Cl2, 4 Å molecular sieves,
reflux, 3 h (95%); (b) 34, Zn in THF, 25 °C, 20 min, followed by addition of 37 then stirred for 3.5 h, 25 °C; (c) i—NaOH (1 M), THF, 24 h, 25 °C; ii—mesitylenesulfonyl chloride,
diethyl isopropyl amine, NBu4HSO4, 4.5 h, 25 °C (37% from 37); (d) i—SOCl2,THF, 45 min, reflux; ii—DBU, CH2Cl2, 24 h, 25 °C (79%); (e) i—KOtBu, MeI, THF, 45 min, 25 °C; ii—
piperidine, 3.5 h, 90 °C (60%).

A. Enz et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1287–1291
1291
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chiral imine afforded the methyl acrylate 40 as a single diastereo-
mer. This was directly transformed into the -lactam 41 after
c
hydrolysis of the ester and cyclization of the mixed anhydride gen-
erated in situ. The chiral auxiliary in 41 was removed by conver-
sion of the hydroxyl to the chloride followed by DBU assisted
elimination to the enamine and subsequent hydrolysis. Finally,
alkylation with MeI of 42 and conjugate addition of piperidine
afforded 20 in a separable cis/trans mixture (3:1). The exemplified
compound 20 was shown to have an ee > 99% by chiral
chromatography.13
In summary, we have discovered and optimized a series of cis
c-
7. Sheridan, R. P.; Nilakantan, R.; Dixon, J. S.; Venkataraghavan, R. J. Med. Chem.
1986, 29, 899.
lactams as 7 nAChR agonists that display excellent potency, sub-
a
type selectivity and favorable in vitro and in vivo pharmacokinetic
properties. Additionally, the scaffold displays good cardiosafety
profile. The scaffold was discovered through high-throughput
screening, and is devoid of the classical bicyclic amine, thus provid-
ing a valuable lead series for further development.
8. The cis diastereomer was 40- to 60-fold more potent to the corresponding trans
diastereomer (data not shown). Moreover the eutomer/distomer relationship
for 7 was ca. 3-fold with pEC50 values 6.34 versus 6.01, respectively. The
depicted (S,S)-configuration was later confirmed to be the eutomer by
enantioselective synthesis of 20 (vide infra Scheme 2). All stereochemistry is
therefore given as the (S,S)-configuration.
9. Feuerbach, D.; Lingenhöhl, K.; Dobbins, P.; Mosbacher, J.; Corbett, N.; Nozulak,
J.; Hoyer, D. Neuropharmacology 2005, 48, 215.
10. Michelmore, S.; Croskery, K.; Nozulak, J.; Hoyer, D.; Longato, R.; Weber, A.;
Bouhelal, R.; Feuerbach, D. Naunyn-Schmiedeberg’s. Arch. Pharmacol 2002, 366,
235.
11. Feuerbach, D.; Lingenhoehl, K.; Olpe, H.-R.; Vassout, A.; Gentsch, C.; Chaperon,
F.; Nozulak, J.; Enz, A.; Bilbe, G.; McAllister, K.; Hoyer, D. J. Neuropharm. 2009,
56, 254.
Acknowledgment
We wish to thank Dr. Eric Francotte for chiral separation
support.
12. Miyaura, N.; Suzuki, A. Chem. Rev. 1995, 95, 2457.
13. Tohda, Y.; Sonogashira, K.; Hagihara, N. Synthesis 1977, 777.
14. Chiral separation performed on Chiralpak OD or AH columns.
15. Dembélé, Y. A.; Belaud, C.; Hitchcock, P.; Villiéras, J. Tetrahedron: Asymm. 1992,
3, 351.
References and notes
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R.; Rodefer, J. S. Curr. Opin. Invest. Drugs 2008, 9, 47; b Mazurov, A.; Hauser, T.;
16. Yamamoto, Y.; Ito, W. Tetrahedron 1988, 44, 5415.