B-973, a Novel α7 nAChR Ago-PAM: Racemic and Asymmetric Synthesis, Electrophysiological Studies, and in Vivo Evaluation

Article abstract of DOI:10.1021/acsmedchemlett.8b00407

We report here the total synthesis of B-973 (five steps), a recently identified α7 nAChR ago-PAM, its enantiomeric resolution, and its electrophysiological characterization in Xenopus oocytes to identify (-)-B-973B as the bioactive enantiomer. The asymmetric synthesis of B-973B was accomplished in 99% ee, and X-ray crystallography studies revealed its absolute "S" stereochemistry. B-973B was effective in attenuating pain behavior and decreasing paw edema (formalin test), and its analgesic effects were mediated through α7 nAChR.

Full text of DOI:10.1021/acsmedchemlett.8b00407

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Letter
B-973 — A Novel #7 nAChR Ago-PAM : Racemic and Asymmetric
Synthesis, Electrophysiological Studies and in vivo Evaluation.
Sumanta Garai, Krishnamohan S. Raja, Roger L. Papke,
Jeffrey R. Deschamps, M. Imad Damaj, and Ganesh A Thakur
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00407 • Publication Date (Web): 11 Oct 2018
Downloaded from http://pubs.acs.org on October 14, 2018
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B-973 — A Novel α7 nAChR Ago-PAM: Racemic and Asymmetric
Synthesis, Electrophysiological Studies and in vivo Evaluation
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†
†
§
⁋
Sumanta Garai , Krishnamohan S Raja , Roger L. Papke Jeffrey R. Deschamps , M. Imad
,
‡
,†
Damaj and Ganesh A. Thakur*
†
Department of Pharmaceutical Sciences, Bouvé College of Health Science, Northeastern University, 140 The Fenway, 360
Huntington Avenue, Boston, Massachusetts, 02115, United States
§Department of Pharmacology and Therapeutics, University of Florida, Gainesville, Florida, USA
⁋
Naval Research Laboratory, Code 6930, 4555 Overlook Avenue, Washington, D.C. 20375, United States.
‡Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia, USA
ABSTRACT: We report here the total synthesis of B-973 (5 steps), a recently identified 7 nAChR ago-PAM, its enantiomeric
resolution and its electrophysiological characterization in Xenopus oocytes to identify (-)-B-973B as the bioactive enantiomer. The
asymmetric synthesis of B-973B was accomplished in 99% ee, and X-ray crystallography studies revealed its absolute “S”
stereochemistry. B-973B was effective in attenuating pain behavior and decreasing paw edema (formalin test), and its analgesic
effects were mediated through 7 nAChR. Keywords: Alpha7 Nicotinic Acetylcholine Receptors, Ligand-gated Ion Channels,
Positive Allosteric Modulators, Ago-PAMs, Neuropathic Pain, Asymmetric Synthesis, Electrophysiological Characterization
their utility as clinical candidates.2,4,5 PAMs of the 7 nAChR
provide an alternative targeting approach that facilitates natural
signaling by endogenous ACh and thus is differentiated from
Nicotinic acetylcholine receptors (nAChRs) are members of the
Cys-loop superfamily of cationic ligand-gated ion channels and
are involved in the physiological responses to the
neurotransmitter, acetylcholine (ACh). These receptors are
distributed throughout the central and peripheral nervous
1
-3
8
partial agonists.
In addition to PAMs, allosteric agonists as well as silent
systems
and
play
important
roles
in
several
6
agonists of 7 nAChR have been discovered. However, despite
(patho)physiological processes. Compounds that selectively
all of these efforts, only limited success has been achieved
clinically with galantamine (Figure 1). JNJ-39393406 is an 7
PAM that is currently undergoing phase II clinical trials for
target the homopentameric 7 nAChR subtype are being
pursued as potential pharmacotherapy agents for treating
cognitive dysfunctions in schizophrenia and Alzheimer’s
disease, as well as for treating neuropathic and inflammatory
pain. This type of targeting is safe and is not associated with
9
,10
depressive disorders and smoking cessation.
Most of the
drug candidates that were pursued as 7 nAChR (partial)
agonists or PAMs, however, failed in Phase II clinical trials for
cognitive disorders due to insufficient efficacy. Recently, a
unique class of compounds with dual modes of action —
allosteric agonism and positive allosteric modulation —have
been discovered and are referred to as “ago-PAMs” (e.g.,
4-7
opioids-like side effects such as addiction liability. Attempts
to target 7 nAChR has mainly focused on two approaches:
partial agonism via the ACh binding site, and positive allosteric
modulation (PAM) at sites topographically distinct from ACh
binding. Although 7 nAChR-selective and potent (partial)
agonists have been developed and studied (pre)clinically, the
rapid desensitization of 7 nAChR agents in response to high
agonist concentration, together with the possibility of
endogenous tone disruption, has led to some concerns regarding
11-14
GAT107, Figure 1).
Ago-PAMs of 7 nAChR activate receptors through site
distinct from orthosteric sites and positively modulate
orthosteric cholinergic signaling while preserving the
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Page 2 of 5
spatiotemporal features of synaptic transmission.13,14 Such
modulators that act through dual
producing gram-scale quantities of B-973 and is depicted in
Scheme 1.
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HO
N
H
R
N
N
Cl
O
O
N
N
F
F
O
Br
N
Br
N
O
S
O
N
O
Br
N
N
N
H
N
N
N
S
N
2 3
K CO , DMF
O
+
HN
N
N
N
H
H
15 min, 82%
O
9
7
N
8
H3CO
(-)-Galantamine (1)
JNJ-39393406 (2)
PNU-120596 (3)
O
N
N
Conc. HCl
,
Pd(PPh3)2Cl2,
SO2NH2
Sn(Bu)3
N
N
N
SO2NH2
O
THF, rt, 3 hrs
2% (over two steps)
CuI, CH3CN, reflux
overnight
8
R
S
10
0
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HN
O
N
N
S
HN
F
F
N
N
N
N
O
N
N
N
N
H
NH4OAc, NaCNBH4
*
N
N
N
N
N
N
H2N
EtOH, MW, 2 min
96%
multigram scale from 7 to 12
B-973 (6)
undefined sterochemistry
Br
11
12
TQS (4)
racemic
GAT107 (5)
O
O
N
N
F
R
Py, DMAP
DCM, rt,
F
F
*
N
N
N
12
H
F
N
2
hrs, 54%
SOCl2,
DCM, reflux,
R=OH,13
6
, (±)-B-973
R=Cl, 14
The synthesis begins with the formation of tricyclic bromo
pyrazine derivative 9 by mono-substitution of dibromo pyrazine
7 with piperazine 8 in the presence of K CO in DMF in 92%
Figure 1: Representative selective positive allosteric modulators of
7 nAChR: an approved drug (1), phase II clinical candidate (2)

2
3
and pre-clinical candidates (3–6).
yield. To install a keto functionality on 9, we examined several
modes of action are expected to be more efficacious and
potentially address the limitations observed in partial agonists
and PAMs in clinical trials. Indeed, we recently demonstrated
that one such dual modulator, GAT107, from the
tetrahydroquinoline (THQ) class of compounds is an orally
bioavailable and efficacious analgesic for treating inflammatory
Scheme 1. Racemic synthesis of B-973
reaction conditions involving BuLi-mediated lithiation
followed by quenching with appropriate electrophiles. Our
initial efforts were plagued by the formation of a range of
undesired products, which was presumably due to the
sensitivity of the pyrazine ring to these conditions. We further
attempted installing the ketone moiety under mild Pd-catalyzed
1
5
and neuropathic pain in animal models.
Recently, Bristol Myers Squibb reported HTS screening of a
6
diverse small molecule library (>10 molecules) using a
17
cross-coupling conditions. Stille cross coupling of compound
9 with tributyl (1-ethoxyvinyl)tin and Pd(PPh Cl in the
fluorescence-based assay configured to detect 7 nAChR
16
3
)
2
2
PAMs. Active compounds identified from the screen included
presence of CuI yielded the enol ether 10. The crude product
was hydrolyzed with aq. HCl to give the desired ketone
compound 11 in 82% yield over two steps. Thus, after obtaining
the key keto intermediate 11 in multi-gram quantities, we
a series of piperazine-containing molecules that displayed
robust 7 nAChR PAM activity. One compound from this
series, B-973 (Figure 1), was unique in that it was shown to be
a potent ago-PAM. Although this important work led to the
identification of a novel scaffold with 7 nAChR ago-PAM
activity, it left several questions unanswered, thus limiting
further drug optimization and developmental efforts on this
scaffold. Based on the information provided, it is not clear if B-
carried out its conversion to an imine with NH
4
OAc, followed
by in situ reduction with NaCNBH to yield racemic amine 12
4
in 96% yield. The conversion of acid 13 to the corresponding
acid chloride 14, followed by its coupling with the amine
fragment 12 in the presence of pyridine and catalytic DMAP
yielded the racemic B-973, 6 in 54% yield (Scheme 1).
9
73 is a racemate or a single enantiomer. Neither its absolute
stereochemistry nor the activity of the other enantiomer are
known. Information on the chemical synthesis of this
compound to facilitate in vivo studies and library syntheses is
also not reported.
B-973 has one chiral center with two possible configuration. To
test whether the allosteric agonist and PAM activity was
attributed to a single enantiomer or was present in both
compounds, we first developed conditions for the enantiomeric
separation of B-973 (Scheme 2; see SI). Using chiral super fluid
chromatography (SFC), baseline separation of the B-973
enantiomers was accomplished in 99% ee.
Given the therapeutic potential of 7 nAChR ago-PAMs and
the excitement surrounding this structurally novel HTS, an
effort was made to pursue its synthesis and enantiomeric
separation and identify the stereochemical requirements for

7nAChR ago-PAM activity through electrophysiological
O
N
N
studies followed by in vivo evaluation of the active enantiomer.
We report an efficient and scalable synthesis of the racemate as
well as of the active enantiomer in 99% ee. Access to copious
amounts of the active enantiomer enabled its preclinical
evaluation for inflammatory pain (formalin test) in a mouse
model, where it was shown to be efficacious. (sentence deleted).
The five-step synthetic route reported here was optimized for
F
F
N
N
N
N
H
6, (±)-B-973
SFC Chiral seperation
O
N
N
O
N
N
F
F
N
N
+
F
F
N
N
N
N
N
H
H
N
15, (+)-B-973, Inactive
16, (-)-B-973, Active
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the subsequent 60 µM ACh response was greater (p < 0.05) for B-
973B than for GAT107. Additionally, the responses to racemic B-
73 were smaller (p < 0.05) than the response to the B-973B
enantiomer. These studies thus concluded that the ago-PAM
activity resided exclusively in (-)-B-973B, while (+)-B-973A was
inactive at 7 nAChR.
To understand the absolute stereochemical requirements for 7
nAChR ago-PAM activity, we attempted to crystallize both
enantiomers to obtain crystals suitable for X-ray studies. However,
even after several crystallization attempts, neither of the B-973
enantiomers could be crystallized in an acceptable form.
Furthermore, to facilitate structure-activity relationship (SAR)
studies, there was a need for easy access to the homochiral version
of amine 12 due to the observed high enantio-specificity for
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5
6
7
8
9
9
Scheme 2. Enantiomeric resolution of racemic B-973.
Both enantiomers were then tested in Xenopus oocytes
expressing human 7 nAChR with a two-electrode voltage
clamp method (Figure 2). Following the measurement of 60 µM
ACh control responses, the B-973 isomers were co-applied with
a control concentration at 10 or 100 µM. The average net-charge
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0

7nAChR activity. We therefore decided to develop an
enantioselective approach that would yield the active enantiomer in
high ee and allow derivatization of the chiral amine to expand the
possibility of obtaining crystals suitable for X-ray studies.
Among the various methods for the preparation of optically
1
8
active amines , we planned to synthesize chiral 12 via an
approach involving imine formation with chiral tert-
butanesulfinamide, followed by diastereoselective reduction
with the appropriate reducing agent as a key step.
Advantageously, both the enantiomers of tert-
butanesulfinamide are inexpensive and commercially available
in high optical purity. Moreover, condensation of tert-
butanesulfinamide and ketone gives stable ketimine under mild
reaction conditions, yields good diastereoselectivity during
reduction and allows easy removal of the N-tert-butanesulfinyl
in high yield.
1
9
Accordingly, the chiral N-tert-butanesulfinyl ketimine 17 was
synthesized from ketone 11 with R-(+)-2-methyl-2-propane
sulfonamide in the presence of Ti(OEt) in dichloromethane
4
under reflux conditions as a yellow solid in 89% yield. The
chiral N-tert-butanesulfinyl ketimine 17 was stereospecifically
responses (± SEM) of the oocytes (n ≥ 5) to the A and B
isomers, normalized to the initial ACh control responses from
the same cells, are shown in Figure 2A. While there were
negligible effects from the B-973A isomer, the B-973B isomer
greatly increased the responses compared to those evoked by
ACh alone.
2
0-22
reduced by 9-BBN
as a single diastereomer (dr ≥ 99%; determined by H and
in THF at -78 °C to yield sulfinamide 18
1
13
C
NMR spectroscopy) in 96% yield The hydrolysis of the
sulfinamide group of 17 was accomplished using 6 M HCl in
methanol and gave chiral amine 19 in 86% yield. Finally, the
coupling of chiral amine 19 with acid chloride 14 yielded
optically pure 15 in 94% ee (Scheme 3). By comparing the
optical rotation of 15 with the enantiomers obtained from chiral
SFC, 15 was identified as the inactive enantiomer.
.
H2N
S
S
,
Ti(OEt)4
N
O
N
N
N
9
-BBN, THF
O
11
N
-78 0C to rt, 12 hrs
dr= > 99%, 96%
DCM, reflux, 36 hrs
95%
Figure 2: Electrophysiological characterization of the B-973
isomers on human 7 nAChR expressed in Xenopus oocytes.
N
17
S
N
N
Following measurement of the 60 µM ACh control responses, the
B-973 isomers were applied alone at a concentration of 100 µM,
followed by another application of 60 µM ACh. The average net-
charge responses (± SEM) of the oocytes (n = 6) to the A and B
isomers, normalized to the initial ACh control responses from the
same cells, are shown in Figure 2B. The B-973A isomer failed to
evoke a response, and the subsequent ACh controls were not
significantly different from the initial controls. In contrast, B-973B
evoked large responses, and the ACh responses greatly increased
after washout compared to initial controls. The normalized net-
charge responses of 7-expressing cells to 10 µM B-973B
compared to 10 µM of the previously characterized ago-PAM
HN
O
N
N
N
6
M HCl
N
N
N
N
H2N
MeOH, rt, 12 hrs
91%
N
19
18
O
N
N
1
4, Pyridine, DMAP
F
F
N
N
N
H
DCM, rt, 2 hrs
ee  94%, 57%
N
15, (+)-B-973A
1
2, 13
GAT107 (Figure 2C).
While the initial allosteric activation
produced by the two agents was similar, the primed potentiation of
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the antinociceptive effects of B-973B in the formalin test. As
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shown in Figure 3C, a significant effect of treatment was found
[F(3,15) = 33.05; P < 0.0001]. MLA given alone significantly
affected the nociceptive behavior (P = 0.77). MLA (10 mg/kg,
s.c.) totally blocked the antinociceptive effect of B-973B in both
phase I and II of the formalin test (P < 0.001).
Scheme 3. Asymmetric synthesis of (+)-B-973A.
To obtain the other enantiomer, N-tert-butanesulfinyl ketimine
17 was stereospecifically reduced with L-Selectride20-22 in
anhydrous THF at -78 C to yield 20 as a single diastereomer (dr
1
13
≥ 99%; determined by H and C NMR spectroscopy) in 92%
yield. An X-ray crystallographic study revealed the absolute
stereochemistry of 20 at C4 to be “S”. The hydrolysis of the
sulfinamide group of 20 using 6 M HCl gave the chiral amine
2
1 in 86% yield. The absolute “S” stereochemistry of the chiral
0
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4
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8
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0
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0
center C4 was further confirmed by a single-crystal X-ray study
of p-nitrobenzoyl derivative 22 of chiral amine 21 (See SI).
Finally, the coupling of 21 with acid chloride 14 yielded the
optically pure bioactive isomer 16, (S)-(-)-B-973B, in 56%
yield and with 99% optical purity (Scheme 4).
Figure 3: In vivo evaluation of the active enantiomer B-973B
in a mouse model of inflammatory pain, and establishment of
target specificity.
We previously demonstrated the antinociceptive and anti-
inflammatory efficacy of GAT107 towards pain in a series of
mouse models. Here, we tested whether the systemic
administration of B-973B would produce antinociception in
mouse formalin testing. As seen in Figure 3A, B-973B did
significantly reduce nociceptive behavior during phase I [F(3,28)
1
5
Acute B-973B mainly attenuated pain behavior in the second
phase of the formalin test, which is associated with the
development of inflammation and spinal dorsal horn
sensitization but was only effective in phase I at the highest dose
(immediately after formalin injection), which is mediated by C-
=
3.941, P = 0.018] at the highest dose of 10 mg/kg. Moreover,
2
3, 24
B-973B (1 – 10 mg/kg, i.p.)
dose-dependently
attenuated formalin-
induced paw licking
fiber activity.
Moreover, B-973B decreased formalin-
induced paw edema, which is consistent with the idea that it acts
on the inflammatory phase of the formalin test. Furthermore,
using a complementary pharmacological agent (i.e., the α7
nAChR antagonist, MLA), we confirmed that the
antinociceptive effects of B-973B in the formalin test were
mediated through α7 nAChRs.
S
HN
O
N
N
N
L-Selectride, THF
17
N
78 0C to rt, 12 hrs
4
-
dr=  99%, 85%
N
20
MeOH, rt,
6M HCl,
12 hrs, 86%
O
N
N
Thus, in this work, we have developed the first total, gram-scale
synthesis of B-973, a structurally novel 7 nAChR ago-PAM.
We also developed conditions for its enantiomeric separation
using chiral SFC to give both enantiomers in high enantiomeric
excess. The (-)-B-973B enantiomer retained all 7 nAChR ago-
PAM activity, while the (+)-B-973B was inactive in
electrophysiological assays. Asymmetric synthesis of (-)- B-
N
N
F
F
N
N
14, Pyridine, DMAP
DCM, rt, 2 hrs H2N
99% ee, 56%
N
H
N
N
4
N
N
16, (-)-(S)-B-973B
2
1
behavior in Phase II [F(3,28) = 13.07, P < 0.001] with 3 and 10
mg/kg doses, differing from the vehicle group.
9
73B was accomplished in high enantiomeric excess (99% ee).
When tested in vivo, B-973B was effective in attenuating pain
behavior, and decreased formalin-induced paw edema
(formalin test). Target specificity of B-973B for analgesic
activity was established using MLA. B-973B was as effective
as structurally distinct GAT107 in in vitro assays and in vivo
experiments, thus providing a suitable lead for future SAR
optimization.
Scheme 4. Asymmetric synthesis of (-)-B-973B.
In addition, B-973B significantly reduced paw edema [F(3,28)
=
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website at DOI:
Experimental details, structural characterization data, Chiral SFC
separation details, NMR spectra, X-ray study details and biological
studies are provided (PDF).
Accession Codes: CCDC 1842480 and 1842483 contains the
supplementary crystallographic data for this paper. These data can
2
1.05, P < 0.001; Fig 3B] in mice treated with 3 and 10 mg/kg
doses, differing from the vehicle group. We next explored the
possible role of α7 nAChRs as related to B-973B’s effect in the
formalin test. To study this, we tested whether the α7 nAChR
antagonist,
MLA, blocks
be
obtained
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receptors via an allosteric transmembrane site. Proc Natl Acad Sci U S
A 2011, 108, 5867.
12) Thakur, G. A.; Kulkarni, A. R.; Deschamps, J. R.; Papke, R. L.
Expeditious synthesis, enantiomeric resolution, and enantiomer
functional characterization of (4-(4-bromophenyl)-3a,4,5,9b-
tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide (4BP-TQS): an
allosteric agonist-positive allosteric modulator of α7 nicotinic
acetylcholine receptors. J. Med. Chem. 2013, 56, 8943.
Corresponding Author
g.thakur@northeastern.edu
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ACKNOWLEDGMENT
This work was supported by the National Institute of Health (NIH)
RO1-EY024717 to GAT and RO1-GM057481 to RLP, a grant
from a Pilot Project from the VCU Massey Cancer Center and R01-
CA206028 to MID. Oocyte experiments were conducted by Clare
Stokes and Alexander den Boef.
(13) Papke, R. L.; Horenstein, N. A.; Kulkarni, A. R.; Stokes, C.;
Corrie, L. W.; Maeng, C. Y.; Thakur, G. A. The activity of GAT107,
an allosteric activator and positive modulator of α7 nicotinic
acetylcholine receptors (nAChR), is regulated by aromatic amino acids
that span the subunit interface. J. Biol. Chem. 2014, 289, 4515.
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