Discovery of BNC375, a Potent, Selective, and Orally Available Type i Positive Allosteric Modulator of α7 nAChRs

Article abstract of DOI:10.1021/acsmedchemlett.9b00001

Positive allosteric modulators (PAMs) of α7 nAChRs can have different properties with respect to their effects on channel kinetics. Type I PAMs amplify peak channel response to acetylcholine but do not appear to influence channel desensitization kinetics, whereas Type II PAMs both increase channel response and delay receptor desensitization. Both Type I and Type II PAMs are reported in literature, but there are limited reports describing their structure-kinetic profile relationships. Here, we report a novel class of compounds with either Type I or Type II behavior that can be tuned by the relative stereochemistry around the central cyclopropyl ring: for example, (R,R)-13 (BNC375) and its analogues with RR stereochemistry around the central cyclopropyl ring are Type I PAMs, whereas compounds in the same series with SS stereochemistry (e.g., (S,S)-13) are Type II PAMs as measured using patch-clamp electrophysiology. Further fine control over the kinetics has been achieved by changing the substitutions on the aniline ring: generally the substitution of aniline with strong electron withdrawing groups reduces the Type II character of these compounds. Our structure-activity optimization efforts have led to the discovery of BNC375, a small molecule with good CNS-drug like properties and clinical candidate potential.

Full text of DOI:10.1021/acsmedchemlett.9b00001

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Letter
Discovery of BNC375, a Potent, Selective, and Orally
Available Type I Positive Allosteric Modulator of #7 nAChRs.
Andrew J. Harvey, Thomas D. Avery, Laurent Schaeffer, Christophe Joseph, Belinda C. Huff, Rajinder
Singh, Christophe Morice, Bruno Giethlen, Anton A. Grishin, Carolyn J. Coles, Peter Kolesik, Stéphanie
Wagner, Emile Andriambeloson, Bertrand Huyard, Etienne Poiraud, Dharam Paul, and Susan M. O’Connor
ACS Med. Chem. Lett., Just Accepted Manuscript • Publication Date (Web): 25 Mar 2019
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Page 1 of 8
ACS Medicinal Chemistry Letters
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Discovery of BNC375, a Potent, Selective, and Orally Available Type
I Positive Allosteric Modulator of α7 nAChRs.
Andrew J. Harvey#, Thomas D. Avery#, Laurent Schaeffer$, Christophe Joseph$, Belinda C. Huff#,
Rajinder Singh#, Christophe Morice$, Bruno Giethlen$, Anton A. Grishin#, Carolyn J. Coles#, Peter
Kolesik#, Stéphanie Wagner†, Emile Andriambeloson†, Bertrand Huyard†, Etienne Poiraud†, Dharam
Paul*#, Susan M. O’Connor#
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# Bionomics Limited, 31 Dalgleish Street, Thebarton, SA-5031, Australia
† Neurofit, 850 Boulevard Sébastien Brant, Bioparc 1, Parc d'Innovation, 67400 Illkirch, France
$ Prestwick Chemicals, 220 Boulevard Gonthier d'Andernach, Parc d'Innovation, 67400, Illkirch, France
Alpha 7 nicotinic acetylcholine receptor, positive allosteric modulators, memory, T-maze, Attention
ABSTRACT: Positive allosteric modulators (PAMs) of α7 nAChRs can have different properties with respect to their effects on
channel kinetics. Type I PAMs amplify
peak channel response to acetycholine
but do not appear to influence channel
desensitization kinetics, whereas Type II
PAMs both increase channel response
and delay receptor desensitization. Both
Type I and Type II PAMs are reported in
literature, but there are limited reports
describing their structure-kinetic profile
relationships. Here we report a novel
class of compounds with either Type I or
Type II behavior, that can be tuned by the
relative stereochemistry around the
central cyclopropyl ring: for example,
(R,R)-13 (BNC375) and its analogues
with RR stereochemistry around the
OMe
1s
H
N
p A
(R,R)-13 (BNC375)
O
3 0 0
S
(R,R)-13
H2N
O
Cl
(BNC375)
Type I
OMe
H
(S,S)-13
N
O
1s
S
H2N
(S,S)-13
O
Cl
Type II
central cyclopropyl ring are Type I PAMs, whereas compounds in the same series with SS stereochemistry (e.g. (S,S)-13) are Type
II PAMs as measured using patch-clamp electrophysiology. Further fine control over the kinetics has been achieved by changing the
substitutions on the aniline ring: generally the substitution of aniline with strong electron withdrawing groups reduces the Type II
character of these compounds. Our structure-activity optimization efforts has led to the discovery of BNC375, a small molecule with
good CNS-drug like properties and clinical candidate potential.
Activation of alpha 7 nicotinic acetylcholine receptors (α7
nAChRs) improves cognitive function both in humans1 and
rodents1,2, and thus represents a promising therapeutic approach
for cognitive impairment in Alzheimer’s disease3 and
schizophrenia3,4. The activity of α7 nAChR can be enhanced
through either orthosteric agonists or positive allosteric
modulators (PAMs). In contrast to α7 agonists, α7 PAMs do not
activate α7 nAChRs by themselves and work by amplification
of responses induced by endogenous agonists, hence preserving
spatiotemporal signaling patterns. Furthermore, α7 nAChR
PAMs may allow for greater selectivity to be achieved as
compared to orthosteric agonists, providing for a potentially
cleaner side effect profile1,4. Based on the functional property
of modulation, α7 PAMs can be broadly classified into two
categories: Type I and Type II5. Type I α7 PAMs, such as AVL-
32886 (1), NS17387 (2) and Lu AF588018 (3, Figure 1), increase
channel responses to acetylcholine without affecting receptor
desensitization, whereas Type II α7 PAMs, such as TQS9 (4),
RO512694610 (5), JNJ-193094211 (6), PNU-12059612 (7) and
A-86774413 (8), not only increase channel response but also
delay receptor desensitization. The PAMs reported in the
literature are structurally quite diverse and small structural
changes have been reported to produce profound effects on the
pharmacological profile. For example, in the work reported by
Gill-Thind14, the cis-cis-diastereomers were all classified as
allosteric agonists or Type II PAMs. In contrast, all of the cis-
trans-diastereomers were Type I PAMs,
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Page 2 of 8
OMe
N
OMe
Cl
Cl
H
H
N
Cl
O
N
1
H
=
H
N
N
CF3
O
O
O
Lu AF58801
2
Cl
S
OH
9
11
10
H2N
O
N
H
O
O
NH
3
3
4
5
6
7
8
9
=
410%
P3
OH
710%
660%
2
(NS-1738)
O
O
O
Cl
N
H
N
1
Figure 2. Key compounds from the hit identification phase.
(AVL-3288)
OMe
O
NH2
HN
O
N
H
intraperitoneal (ip) doses of 3 mg/kg and 30 mg/kg. However,
compounds 10 and 11 were found to be inactive in T-maze after
oral doses of 3 and 30 mg/kg and compound 9 was not tested
orally. In order to achieve oral efficacy, further modification of
these hits to find a lead compound was carried out.
S
O
O
O
Cl
S
7
PNU-120596
H2N
O
Cl
H
H
O
5
RO5126946
S
NH
HO
N
F
F
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
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N
F
F
O
H2N
N
N
S
Table 1. Profile of hit compounds 9, 10 and 11
H
O
Cl
4
Compound
9
10
11
(TQS)
8
A-867744
6
JNJ-1930942
Figure 1. Representative examples of reported α7 nAChRs PAMS.
Primary Screening P3 (%)
Secondary Screening P3 (%)
Secondary Screening AUC/P3
logD7.4
660
420
5.6
3.7
710
360
5.6
4.2
410
NT
NT
NT
negative allosteric modulators or silent allosteric modulators.
Here we report a new class of compounds that can be tuned
either to a Type I or Type II α7 PAM, depending upon
the stereochemistry around the central cyclopropyl ring. Our
synthetic methods have allowed us to access both enantiomers
from the trans-racemates, and the structure-activity
optimization of these enantiomers has led to the identification
of a potential candidate for clinical development.
Sol6.5 (µg/mL)
3.1-6.3 1.6-3.1 NT
Primary in vivo Assay: ip
mouse T-maze
3, 30
NT
3, 30
NS
NT
NS
po
During the early days of the program, the knowledge about the
structural requirements for α7 PAM activity was ambiguous
because literature examples covered many diverse structural
classes containing two terminal rings joined through linear, as
well as more constrained cyclic linkers. Some examples
contained one ring decorated with a highly polar sulfamyl group
whereas others lacked such polar substituents. Therefore, the
lack of understanding in this area persuaded us to design a small
focused library of compounds belonging to different chemical
classes to discover novel α7 PAMs with identifiable structural
features crucial for biological activity. The primary in vitro
screening assay was performed by electrophysiology using the
Patchliner® (Nanion), an automatic planar patch clamp
instrument. The potentiation of an EC20 acetylcholine (ACh)
response by 3µM of each PAM was determined. Secondary
screening and more detailed characterisation of promising
compounds was performed with conventional manual patch-
clamp recordings using a fast-application system (Dynaflow®,
Cellectricon, Sweden). All experiments were performed in
stable cell lines expressing human or rat 7 nAChR in rat
GH4C1 cells. The percent change in peak current produced by
3 µM of test compound plus acetylcholine versus acetylcholine
alone was called P3 (percent potentiation at 3 µM). In vivo
efficacy was measured using the mouse T-maze Continuous
Alternation Task (T-maze) as the primary model and was
reported as the doses that significantly reversed the
scopolamine induced impairment of spontaneous alternation
(for details see SI: In vitro and in vivo evaluation).
Significant dose
(3, 30) (3, 30)
(mg/kg)
NT: Not tested; NS: Not significant
As shown in Figure 2, the hit molecules 9-11 have a modular
structure, composed of three distinctive regions: RHS ring, LHS
ring and central linker. So, we planned to explore SAR for these
regions (a representative set of synthesized compounds is
shown in Figure 3). As found in the primary hits, the sulfamyl
group on the para position of LHS phenyl ring and 2-alkoxy-5-
halo group on RHS aniline ring were essential features for in
vitro α7 PAM activity. The central linker was modified in a
variety of ways and we found that compounds containing the
secondary amides with
a 3,3-dialkyl substituted 1,2-
cyclopropyl (12a, 12b) and 3,4-tetrahydrofuryl (12m) moieties
between the carbonyl carbon and LHS phenyl ring exhibited
good in-vitro activity. Removal of dimethyl group from
cyclopropyl (12c) or its replacement with
R1= SO2NH2, SO2NHOH,
3-hydroxy-isoxazol-3-yl,
H, Ph, X, CF3, NHSO2Me,
SO2Me, SO2NHCH2CF3,
R3
R2
Cl
R2
OEt
H
Cl
Cl
H
N
H
Me
H
OMe
O
N
N
O
S
N
N
R1
NH
,
N
12
O
SO2NH2
R3
Y
Y
N
N
H
N
H
N
O
f
N
O
O
j
k
X
l
O
O
O
N
Y
H
Nine hits were identified during the primary screen of a small
focused library. Three of them (9, 10 and 11; Figure 2) were of
particular interest15, exhibiting P3 values of 660, 710 and 410
%, respectively. The activity of 9 and 10 was confirmed in the
secondary screening assay where they exhibited P3 values of
420% and 360%, respectively (Table 1).
H
N
N
O
g X = O
h X = CH2
a
b
c
d
e
Et
m
cyclohexyl
n
O
O
O
H
H
H
N
N
Cl
Br
H
N
N
i
o
p
O
Figure 3. Representative examples exploring the hit to lead SAR
(green: active; orange: weakly active and red: inactive)
The hits 9 & 10 significantly reversed scopolamine induced
impairment of spontaneous alternation in the mouse T-maze at
2
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corresponding amines 13 and 14 and observed a significant
Table 2. Profile of lead compound 13
1
2
3
4
5
6
7
8
enhancement in α7 nAChR potentiation with P3 values of
7900%, 9890%, respectively. In contrast, the corresponding
amine of compound 11 was found to be inactive. The racemic
compound 13 was selected for further evaluation due to its
lower lipophilicity and the easy accessibility of its key
intermediate. Compound 13 showed in vivo activity (T-maze)
at the doses of 3 and 10 mg/kg when administered orally. To
confirm the allosteric nature of compound 13, it was evaluated
in the manual patch clamp assay in the absence of acetylcholine
and failed to evoke currents in stable cell lines expressing 7
channels. However, 13 exhibited typical allosteric behavior by
potentiating acetylcholine-evoked currents in a concentration
dependent manner. In the manual patch clamp, 13 showed an
EC50 of 25 nM and peak current potentiation Pmax of 650%
relative to the peak current evoked by ACh at an EC20
concentration. Overall, racemate 13 exhibited a good lead
profile (Table 2) and had a modular structure that enabled
further lead optimization. The main aim of lead optimization
was to improve the ADME properties of the compound. We
focused on decreasing lipophilicity to improve the solubility
and clearance while maintaining the potency. The initial
attempts to reduce lipophilicity began with removal of the
dimethyl group on the central cyclopropyl ring16. The obtained
des-dimethylcyclopropyl analogue had reduced activity. Also,
the replacement of the 1,2-cyclopropyl ring with more polar
3,4-tetrahydrofuryl ring reduced the activity. Synthetic attempts
OMe
Cl
H
N
O
S
H2N
13
O
Primary Screening P3 (%)
7900
650
25
Secondary Screening Pmax (%)
Secondary Screening EC50 (nM)
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
pH 2.0
pH 6.5
1.6-3.1
>100
0.64
Solubility (g/ml)
Microsomal
(EH)§
stability human
mouse
0.82
Primary in vivo Mouse T-maze
Oral significant dose (mg/kg)
3, 10
§ Method to calculate the predicted in vivo hepatic extraction
ratio (EH) is described in SI
dihalo (12d, 12e) rendered the compared to the amide
molecules 12m and 12n, was seen in the corresponding amines
12o and 12p, respectively. Hence, we compounds inactive.
Interestingly, the modest gain in activity, reduced the amide
functionality in the most active compounds 9 and 10 to their
Table 3. Representative examples for SAR on the RHS modifications
R1
R1
H
N
N
N
O
=
RING
RING
X
R2
R2
R3
R3
D
C
A
B
X
RING
R1
R2
H
H
H
F
R3
Cl
H
P3 (%)
SEM (n)
3560 (2)
660 (3)
3870 (3)
4280 (3)
900 (3)
2060 (4)
1190 (3)
660 (2)
550 (4)
400 (4)
100 (3)
260 (6)
12 (2)
13
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
A
A
A
A
A
A
A
A
B
B
A
A
A
A
A
B
C
D
OMe
H
7900
2570
12310
8660
9230
8170
3360
2440
2420
3000
130
14
15
16
17
18
19
20
21
22
32
33
58
66
67
69
72
74
Me
Me
Me
OMe
OCHF2
OCF3
OMe
H
Cl
H
F
Cl
CF3
H
H
F
H
H
H
H
H
H
F
H
F
SO2NHNH2
SO2NHOH
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
OMe
OMe
OMe
H
Cl
Cl
720
SO2NH2
<100
<100
460
morpholino
H
16 (3)
H
H
H
-
morpholino
12 (4)
Me
-
H
-
130
50 (2)
<100
<100
8 (2)
-
-
-
30 (6)
3
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to replace LHS p-sulfamoylphenyl ring with p-
in subsequent synthetic steps. The synthetic procedures and the
analytical data of the representative compounds are presented
in the supplementary information.
1
2
3
4
5
6
7
8
sulfamoylpyridyl proved unsuccessful. Replacement of p-
sulfamyl group on LHS phenyl ring with more polar groups
such as p-sulfonohydrazide (32) and N-hydroxysulfonamide
(33) rendered the analogs inactive or very weakly active.
Additionally, we attempted to replace the p-sulfamyl group
with comparable or slightly more lithophilic groups such as N-
alkylsulfonamides, saturated heterocycles, heteroaryl and aryl,
to identify a more active LHS that could be combined later with
a polar RHS to balance the lipophilicity of the molecule.
However, all the attempts proved unsuccessful. The only
change leading to the retention of activity was attachment of the
methanesulfonamide group through the nitrogen to the aryl ring
rather than the sulfur atom. However, the original lead
compound 13 with a p-sulfamyl group was still the most active
analog, indicating that the sulfamyl group was a critical feature
of potent α7 PAMs in the series (see the data in SI: SAR
analysis tables). Having learned that the central part and the
LHS phenyl ring could tolerate only limited modifications, we
then sought to explore the RHS of the molecule and synthesized
a set of 32 analogs (see the SI: SAR analysis tables).
Representative compounds and their PAM activities are shown
in Table 3. Some analogs with lower lipophilicity were
achieved by replacing the RHS 5-chloro-2-methoxyphenyl ring
in 13 with 2-methoxy-5-tert-butyl-3-oxazolyl (72), 2-methyl-
3-pyridyl (69), 2-methoxy-3-pyridyl (21) 5-fluoro-3-pyridyl
(22) or hexyl (74). These changes were found to be either
inactive or relatively less active. Substitution on the phenyl with
polar groups such as sulfamyl (58) or morpholino (66 or 67)
also reduced the activity. In general, decreasing lipholicity to
improve solubility and clearance failed to give an active
compound. Efforts devoted to explore the alternatives for
metabolically labile –OCH3 and Cl groups, and to protect the
para-position on the RHS ring provided some active
compounds (19, 26, 27, 28). This effort revealed that a 2,4- or
2,5-disubstitution pattern on the RHS phenyl ring retained good
levels of activity.
Scheme 1. Synthesis of enantiopure building block 23
O
O
O
O
LiN
Bn
O
O
OH
N
N
+
O
O
Bn
O
c
Bn
24
25
23
a, b
(mixture of
9
trans-enantiomers)
c
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
OH
OH
O
(R,R)-23
O
(S,S)-23
Reagents and conditions: a) SOCl2, DCM ; b) (S)-4-benzyl-2-oxazolidinone, nBuLi, THF;
c) H2O/THF, H2O2, LiOH
Both trans enantiomers of compound 13 were found to be active
in the primary screening assay at 3 µM. On Patchliner®, the
peak current potentiation by the enantiomer (R,R)-13 was
1160% which was considerably lower than the parent trans
racemic mixture, however the peak potentiation of other
enantiomer, (S,S)-13 was found to be 11840%, which was
consistent with the parent trans racemic mixture. The shape of
the current trace of (R,R)-13 was sharp and resembled a Type I
PAM, whereas (S,S)-13 was like a Type II PAM (Patchliner®
current traces are shown in Table 4). It appeared that the R,R
enantiomer was only moderately active compared to the parent
racemic mixture and much less active than the S,S-enantiomer.
Next, we confirmed the PAM activity by performing full dose
responses using manual electrophysiology. Similar to the
primary screening results, the enantiopure (R,R)-13 and (S,S)-
13 exhibited dramatically different kinetics of desensitization
on 7 nAChRs. Like the prototypical Type I PAM, AVL-3288
(1), (R,R)-13 significantly potentiated the acetylcholine signal
without changing the rapid receptor desensitization. This was
reflected in the AUC/Pmax value of 1.7. In contrast, (S,S)-13, not
only potentiated the signal but also significantly delayed the
desensitization (AUC/Pmax = 25). This kinetic profile resembled
that of PNU-120596 (7), a typical Type II PAM. This profound
difference in channel modulation was further investigated by
comparing the activity profile of enantiomers of related active
compounds. The enantiomeric pairs of a range of analogues of
13 (Table 4) are shown in Table 4 with P3 values and current
traces measured at 3 µM using Patchliner®. In general, the S,S-
enantiomers had a greater effect on receptor desensitization (i.e.
more Type II in nature) than their R,R-enantiomeric partners.
Interestingly, it was possible to tune the compounds toward
more Type I-like PAM activity by changing the substituents on
the RHS aryl ring, as indicated by their current traces (Table 4).
The electron withdrawing substituents on the RHS ring directed
the enantiomers more towards Type I PAM character. The
greatest impact in shifting the S,S-enantiomer towards Type I
activity appeared to come from the nature of the ortho
substituents, where electron-withdrawing substituents (CF3 and
OCF2H) pushed the kinetics more towards Type I, however
electron-donating substituents (methoxy and methyl) yielded
compounds that had a greater effect on delaying desensitization
(Type II). These results suggest that the electron density on the
aniline ring may have a pivotal role in tuning desensitization
All the compounds with chiral centers were prepared using
racemic building blocks and were a mixture of more than one
diastereoisomer or enantiomer. Compound 13 and related active
analogs (shown in Table 3) that featured a cyclopropyl ring in
the linker were prepared from trans building block 23 and were
a mixture of two trans enantiomers with R,R stereochemistry in
one and S,S in the other, around the cyclopropane ring. In order
to profile both enantiomers, we prepared an enantiopure
building block by developing a method to separate the trans
enantiomeric pair of acid 23 (Scheme 1). The racemic mixture
of acid 23 was reacted with a chiral auxiliary (S)-4-isopropyl-
2-oxazolidinone to prepare the mixture of two diastereoisomers,
24 and 25. The resulting diastereoisomers were separated by
repeated flash column chromatography.
The absolute stereochemistry of 24, the first eluting
diastereoisomer, was determined by X-ray crystallography to be
R,R,S (cyclopropyl chiral centers were found to be R,R) and the
stereochemistry of the other, 25 was assigned to be S,S,S. These
two diastereoisomers were then separately hydrolyzed to
corresponding enantiopure trans acids (R,R)-23 and (S,S)-23.
The enantiopure acids were used to produce the enantiopure
final compounds and the enantiomeric-excess of trans
enantiomers of compound 13 was measured by analytical
HPLC and found to be >98%, demonstrating no racemization
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kinetics. The discovery of different kinetics of the trans-
enantiomers provided an opportunity to make a
enantiomer. The in vivo efficacy of (R,R)-13 and (S,S)-13 was
tested in mouse T-maze model at
1
2
3
4
5
6
7
8
9
Table 5. Profiling of enantiomers of compound 13
Table 4. Effect of RHS substituents on kinetics of 7
nAChR modulation
Compounds
(R,R)-13
(S,S)-13
H
N
Primary Screening (r7 GH4C1 planar patch clamp)
Ar
*
*
O
P3 (%)
1160
3.1
11840
57
S
H2N
O
AUC/P3
No.
-Ar
Current Trace^
P3 (%)
Secondary screen: r7 GH4C1 manual patch clamp
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
EC50
1.9 µM
1570%
1.7
0.063 µM
1630%
25
R,R
S,S
OMe
Cl
13
Pmax
AUC/Pmax
Physicochemistry & in vitro ADME
1160%
470%
460%
420%
11840%
17580%
17430%
16
26
27
Solubility at pH 6.5
Solubility at pH 2.0
logD7.4
1.6-3.1 µg/mL
>100 µg/mL
4.2
F
Papp (x10-6 cm/s) 11
CACO-2
14
Microsomal
stability (EH)
h
0.84
0.79
0.59
0.71
F
m
Rat PK
F
Clp (mL/min/kg)
Vss (L/kg)
BA (%)
35
43
F
8930%
1.8
62
5.1
77
OCF2H
19
28
F
AUC0-inf /Dose
(h*µM)/mg
1.2
1.0
170%
410%
8170%
2860%
CF3
Plasma half-life (hr)
1.2
3.9
a range of 0.003-10.0 mg/kg dose (Table 6). The compounds
were formulated in saline based vehicle containing 25%
cremephor ELP and were administered orally. The significant
difference between the group means were assessed by one-way
analysis of variance (ANOVA) followed by Fisher’s Protected
Least Significant Difference using GraphPad Prism 7.0
(GraphPad Software Inc., San Diego, CA). A p value <0.05 was
considered statistically significant. (R,R)-13 exhibited the MED
of 0.03 mg/kg and full reversal of the scopolamine-induced
impairment was achieved at 1.0 mg/kg. (S,S)-13 was found to
be less potent with MED of 0.3 mg/kg and full reversal of the
scopolamine-induced impairment was achieved at 3.0 mg/kg.
Despite (S,S)-13 being more potent in vitro, the in vivo potency
was lower and thus higher in vitro potency did not offer any
clear advantage in this case.
F
^ All current traces are plotted on same time scale (x-axis) but
peak current amplitude (y-axis) is not drawn to scale
comparison between the efficacy and safety of Type I and II
PAMs. The in vitro ADME and pharmacokinetics properties of
the enantiomers of compound 13 (Table 5), showed that both
(R,R)-13 and (S,S)-13 exhibited similar permeability in in vitro
CACO-2 assay. In a human liver microsome stability assay,
compound (S,S)-13 was slightly more stable (EH= 0.59) than
compound (R,R)-13 (EH = 0.84), whereas the stability in mouse
liver microsomes was very similar for both enantiomers (EH =
0.79 and 0.71 for (R,R)-13 and (S,S)-13, respectively). These
results were further supported by comparable in vivo plasma
clearance and bioavailability in rats. Single dose IV (bolus, 4.0
mg/kg) PK in male Sprague Dawley rats of each compound,
formulated as 1.1 mg/mL in saline-based vehicle containing
0.1M hydroxypropyl-β-cyclodextrin and 10% (v/v) DMSO,
showed moderate plasma clearance and good exposure with
dose normalized AUC of 1.2 and 1.0 h*µM, respectively. The
oral bioavailability (BA) of these enantiomers in rat was found
to be 62% for the R,R-enantiomer and 77% for the S,S-
Considering that a Type I PAM with an EC50 ≈ 1 M can
achieve comparable or even better in vivo efficacy to a Type II
PAM with an EC50 < 100 nM, we compared the in vivo efficacy
of modulators exhibiting similar potentiation profiles, but lower
in vitro microsomal clearance. Compounds (R,R)-19, (S,S)-19,
(R,R)-26, (R,R)-27, (S,S)-27, (R,R)-28 and (S,S)-28 were
chosen because they all showed improved intrinsic clearance in
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ACS Medicinal Chemistry Letters
mouse and human microsomes relative to (R,R)-13 and (S,S)-
Page 6 of 8
metabolizing enzymes such as CYP3A4, CYP2C9, CYP2D6,
CYP1A2 and CYP2C19 (IC50 >10 μM). It is worth mentioning
that, although some of the related analogs have similar or
improved solubility and in vitro microsomal clearance, the
overall combination of properties of (R,R)-13 (BNC 375),
including percent potentiation of the acetylcholine response,
oral bioavailability, half-life and brain exposure, has provided a
molecule with efficacy across a broad range of oral doses in
vivo, that warrants further evaluation as a potential clinical
candidate.
1
2
3
4
5
6
7
8
13 (data not shown).
Table 6. In vivo efficacy of selected compound in mouse T-
maze
Comp.
Dose
(mg/kg)
Significance
Reversion
(%)
n
of reversal*
ns
0.003
0.03
0.3
-9
10
10
10
10
10
10
9
***
***
***
***
ns
51
62
78
78
-3
(R,R)-13
9
Supporting Information
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13
14
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16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
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56
57
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60
1.0
The Supporting Information is available free of charge on the ACS
Publications website.
Synthetic procedures and analytical characterization data for key
compounds, detailed SAR analysis tables, biological assay
protocols and crystallography experimental ( PDF).
Author Information:
3.0
0.003
0.03
0.3
ns
11
65
90
28
8
(S,S)-13
***
***
ns
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
9
3.0
Corresponding Author
1.0
*E-mail: dpaul@bionomics.com.au
ORCID Dharam Paul: 0000-0001-9540-9455
(R,R)-19
(S,S)-19
(R,R)-26
(R,R)-27
(S,S)-27
(R,R)-28
(S,S)-28
3.0
ns
10.0
1.0
**
41
46
16
51
-9
Author Contributions: The manuscript was written by D.P. with
editing support from S.O.C., A.J.H., T.D.A, C.M. and R.S. All
authors have given approval to the final version of the
manuscript.
*
3.0
ns
10.0
1.0
**
Present address:
A.J.H.: UniQuest Pty Ltd, Level 7, General Purpose South
Building, Staff House Road, Brisbane, QLD-4072, Australia
R.S.: Department of Medicinal Chemistry, Monash Institute of
Pharmaceutical Sciences, 381 Royal Parade, Parkville VIC-
3052, Australia
A.G.: Bigtincan, Level 20, 320 Pitt Street Sydney NSW 2000
Australia
ns
3.0
ns
0
10.0
1.0
ns
10
8
ns
3.0
ns
31
15
-13
-15
-5
10.0
1.0
ns
ns
T.D.A: Centre for Nanoscale BioPhotonics, The University of
Adelaide, North Terrace, Adelaide, SA-5000, Australia
3.0
ns
10.0
1.0
ns
Note: The authors declare the following competing financial
interest(s): The authors are employees of Bionomics and its
subsidiaries (Prestwick and Neurofit) and Bionomics has a
commercial interest in positive allosteric modulation of nicotine
acetylcholine receptors.
ns
-18
38
-9
3.0
ns
10
10
10
9
10.0
1.0
ns
*
40
39
79
Acknowledgement: The authors gratefully thank Ian Bell, Jason
Uslaner and Xiaohai Wang of MSD for their review and
constructive comments, and Ben Harvey of Bionomics for
providing the publication quality Patchliner® current trace
figures.
3.0
*
10.0
***
10
* ns: not significant; * p <0.05; ** p <0.01; *** p <0.001
Upon evaluation in the mouse T-maze at 1, 3 and 10 mg/kg
doses, these compounds were found to be less potent than
(R,R)-13 and (S,S)-13 (Table 6). Only (S,S)-28exhibited
comparable efficacy but at higher dose of 10 mg/kg. The lower
in vivo potency/efficacy of these compounds may have been due
to other DMPK properties, which were not fully profiled for
these compounds. Based upon its overall profile and superior
potency in the mouse T-maze assay, we selected (R,R)-13 for
further evaluation and profiling. In addition to being highly
potent in vivo, (R,R)-13 possessed an excellent safety profile:
no change in the spatial and temporal response of the receptor
to endogenous ligand ACh, no activity on other related channels
(data not disclosed) and no inhibition of major drug
Abbreviations: PAM, Positive allosteric modulator; nAChRs,
nicotinic acetylcholine receptors; ACh, acetylcholine; SAR,
structure-activity relationship; ADME, absorption, distribution,
metabolism and excretion; EH, hepatic extraction ratio; BA,
bioavailability;
DMPK,
drug
metabolism
and
pharmacokinetics; CYP, cytochrome P450;
(1) Lewis, A.S.; Schalkwyk, G.I.; Bloch, M.H. Alpha-7
nicotinic agonists for cognitive deficits in neuropsychiatric
disorders: A translational meta-analysis of rodent and
human studies. Prog. Neuropsychopharmacol. Biol.
Psychiatry. 2017, 75, 45–53.
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ACS Medicinal Chemistry Letters
(2) Potasiewicz, A.; Nikiforuk, A.; Hołuj, M.; Popik, P.
novel positive allosteric modulator of the alpha7 neuronal
nicotinic acetylcholine receptor: in vitro and in vivo
characterization". J. Neurosci. 2005, 25 (17): 4396–4405
1
2
3
4
5
6
7
8
Stimulation of nicotinic acetylcholine alpha7 receptors
rescue schizophrenia-like cognitive impairments in rats. J.
Psychopharmacol. 2017, 31(2), 260-271.
(13) Malysz, J.; Grønlien, J. H.; Anderson, D. J.; Håkerud, M.;
Thorin-Hagene, K.; Ween, H.; Wetterstrand, C.; Briggs, C.
A.; Faghih, R.; Bunnelle, W. H.; Gopalakrishnan, M. In
vitro pharmacological characterization of a novel allosteric
modulator of α7 neuronal acetylcholine receptor, 4-(5-(4-
chlorophenyl)-2-methyl-3-propionyl-1H-pyrrol-1-
yl)benzenesulfonamide (A-867744), exhibiting unique
pharmacological profile. J. Pharmacol. Exp. 2009, 330 (1),
257-267.
(3) Balázs Lendvaia, B.; Kassaia, F.; Szájli, Á.; Némethya, Z.
α7 Nicotinic acetylcholine receptors and their role in
cognition. Brain Res. Bull. 2013, 93, 86–96
(4) Yang, T.; Xiaoa, T.; Sun, Q.; Wang, K. The current
agonists and positive allosteric modulators of α7 nAChR
for CNS indications in clinical trials. Acta Pharm. Sin. B.
2017, 7(6), 611-622.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(5) Uteshev, V.V. The therapeutic promise of positive
allosteric modulation of nicotinic receptors. Eur. J.
Pharmacol. 2014, 727, 181–185.
(14) Gill-Thind, J.K.; Dhankher, Persis.; D’Oyley,J.M.;
Sheppard, T.D.; Millar, N.S. Structurally similar allosteric
modulators of α7 nicotinic acetylcholine receptors exhibit
five distinct pharmacological effects. J. Biol. Chem. 2015,
290(6), 3552-3562.
(6) Ng, H.J.; Whittemore, E.R.; Tran, M.B.; Hogenkamp, D.J.;
Broide, R.S.; Johnstone, T.B.; Zheng, L.; Stevens, K.E.;
Gee, K.W. Nootropic α7 nicotinic receptor allosteric
modulator derived from GABAA receptor modulators.
Proc. Natl. Acad. Sci. U.S.A. 2007, 104(19), 8059-8064
(15) Harvey, A.; Fluck, A.; Giethlen, B.; Paul, D.; Schaeffer, L.
Positive allosteric modulators of the alpha 7 nicotine
acetylcholine receptors and uses thereof. WO 2012103583.
(7) Timmermann, D. B.; Grønlien, J. H.; Kohlhaas, K. L.;
Nielsen, E. Ø.; Dam, E.; Jørgensen, T. D.; Ahring, P. K.;
Peters, D.; Holst, D.; Chrsitensen, J. K.; Malysz, J.;
Briggs, C. A.; Gopalakrishnan, M.; Olsen, G. M. An
allosteric modulator of the α7 nicotinic acetylcholine
receptor possessing cognition-enhancing properties in
vivo. J. Pharmacol. Exp. Ther. 2007, 323(1), 294-307.
(16) Harvey, A..; Avery, T.; Paul, D.; Ripper, J.; Huff, B.;
Singh, R.; Schaeffer, L.; Joseph, C.; Morice, C.; Giethlen,
B. α7 Nicotine acetylcholine receptor modulators and uses
thereof. WO 2014019023.
(8) Eskildsen, J.; Redrobe, J.P.; Sams, A.G.; Dekermendjian,
K.; Laursen, M.; Boll, J.B.; Papke, R.L.; Bundgaard, C.;
Frederiksen, K,; Bastlund, J.F. Discovery and optimization
of Lu AF58801, a novel, selective and brain penetrant
positive allosteric modulator of alpha-7 nicotinic
acetylcholine receptors: attenuation of subchronic
phencyclidine (PCP)-induced cognitive deficits in rats
following oral administration. Bioorg. Med. Chem. Lett.
2014, 24(1), 288-93
(9) Grønlien, J.H.; Håkerud, M.; Ween, H.; Thorin-Hagene.
K.; Briggs, C.A.; Gopalakrishnan, M.; Malysz, J. Distinct
profiles of 7 nAChR positive allosteric modulation
revealed by structurally diverse chemotypes. Mol.
Pharmacol. 2007,72,715–724.
(10) Sahdeo, S.; Wallace, T.; Hirakawa, R.; Knoflach, F.;
Bertrand, D.; Maag, H.; Misner, D.; Tombaugh, G.C.;
Santarelli, L.; Brameld, K.; Milla, M.E.; Button, D.C.
Characterization of RO5126946, a novel α7 nicotinic
acetylcholine receptor-positive allosteric modulator. J.
Pharmacol. Exp. Ther. 2014, 350(2),455-68
(11) Dinklo, T.; Shaban, H.; Thuring, J.W.; Lavreysen, H.;
Stevens, K.E.; Zheng, L.; Mackie, C.; Grantham, C.;
Vandenberk, I.; Meulders, G.; Peeters, L.; Verachtert, H.;
Prins, E.D.; Lesage A. S. J. Characterization of 2-[[4-
fluoro-3-(trifluoromethyl)phenyl]amino]-4-(4-pyridinyl)-
5-thiazolemethanol (JNJ-1930942),
a novel positive
allosteric modulator of the α7 nicotinic acetylcholine
receptor. J. Pharmacol. Exp. Ther. 2010, 336(2),560-74
(12) Hurst, R.; Hajós, M.; Raggenbass, M.; Wall, T.; Higdon,
N.; Lawson, J.; Rutherford-Root, K.; Berkenpas, M.;
Hoffmann, W.; Piotrowski, D.; Groppi, V. E.; Allaman, G.;
Ogier, R.; Bertrand, S.; Bertrand, D.; Arneric, S. P. "A
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Discovery of BNC375, a Potent, Selective, and Orally Available Type
I Positive Allosteric Modulator of α7 nAChRs.
Andrew J. Harvey#, Thomas D. Avery#, Laurent Schaeffer$, Christophe Joseph$, Belinda C. Huff#, Rajinder Singh#, Christophe
Morice$, Bruno Giethlen$, Anton A. Grishin#, Carolyn J. Coles#, Peter Kolesik#, Stéphanie Wagner†, Emile Andriambeloson†,
Bertrand Huyard†, Etienne Poiraud†, Dharam Paul*#, Susan M. O’Connor#
ACh
Type I Positive Allosteric
Modulator of
nAChRs

OMe
H
N
ACh + (R,R)-13 (BNC375)
O
S
H2N
(R,R)-13
(BNC375)
O
Cl
1s
EC50 = 1.9 M
Emax = 1570%
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