Continued optimization of the MLPCN probe ML071 into highly potent agonists of the hM1 muscarinic acetylcholine receptor

Article abstract of DOI:10.1016/j.bmcl.2012.03.088

This Letter describes the continued optimization of the MLPCN probe molecule ML071. After introducing numerous cyclic constraints and novel substitutions throughout the parent structure, we produced a number of more highly potent agonists of the M₁ mACh receptor. While many novel agonists demonstrated a promising ability to increase soluble APPα release, further characterization indicated they may be functioning as bitopic agonists. These results and the implications of a bitopic mode of action are presented.

Full text of DOI:10.1016/j.bmcl.2012.03.088

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3467–3472
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Continued optimization of the MLPCN probe ML071 into highly potent
agonists of the hM1 muscarinic acetylcholine receptor
Bruce J. Melancon a,c,d, Rocco D. Gogliotti a,c,d, James C. Tarr a,c,d, Sam A. Saleh a,c,d, Brian A. Chauder a,c,d
,
Evan P. Lebois a,c,d, Hyekyung P. Cho a,c,d, Thomas J. Utley a,c,d, Douglas J. Sheffler a,c
,
Thomas M. Bridges a,c,d, Ryan D. Morrison a,c,d, J. Scott Daniels a,c,d, Colleen M. Niswender a,c,d
,
P. Jeffrey Conn a,c,d, Craig W. Lindsley a,b,c,d, Michael R. Wood a,b,c,d,
⇑
a Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232, USA
b Department of Chemistry, Vanderbilt University, Nashville, TN 37232, USA
c Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center, Nashville, TN 37232, USA
d Vanderbilt Specialized Chemistry Center for Probe Development (MLPCN), Nashville, TN 37232, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
This Letter describes the continued optimization of the MLPCN probe molecule ML071. After introducing
numerous cyclic constraints and novel substitutions throughout the parent structure, we produced a
number of more highly potent agonists of the M1 mACh receptor. While many novel agonists demon-
Received 2 March 2012
Revised 13 March 2012
Accepted 22 March 2012
Available online 29 March 2012
strated a promising ability to increase soluble APP
a release, further characterization indicated they
may be functioning as bitopic agonists. These results and the implications of a bitopic mode of action
are presented.
Keywords:
Ó 2012 Elsevier Ltd. All rights reserved.
Muscarinic acetylcholine receptor 1
M1
Allosteric
Agonist
Bitopic
ML071
VU0364572
Continued efforts to develop potent and highly subtype-selec-
tive ligands for each of the five (M1–5) muscarinic acetylcholine
receptors (mAChRs) remains a challenging, but potentially highly
rewarding endeavor. Tremendous clinical promise has been shown
for muscarinic activators in the treatment of Alzheimer’s disease
and schizophrenia with the moderately selective muscarinic ago-
nist xanomeline.1,2 In the absence of truly subtype-selective activa-
tors, it has not been definitively established which mAChRs are
primarily responsible for the positive outcomes observed with
xanomeline. While a spectrum of mAChR-knock out (KO) mice
have strongly implicated the M1 and M4 receptors in mediating
beneficial CNS effects,3 the study of selective mAChR ligands in
non-mutant species and under various disease states remains a
worthy goal. The ability to develop selective mAChR agonists is
complicated by the highly conserved orthosteric binding site
shared across all five receptor subtypes. As a result, classic orthos-
teric agonists, such as xanomeline, do not display the desired levels
of receptor subtype selectivity. Recently, the focus of novel mAChR
activators has moved away from the orthosteric site in favor of
allosteric sites.4 These allosteric sites have the potential to be less
conserved in their amino acid sequences, across the M1–5 receptors,
thereby giving rise to much higher levels of receptor subtype selec-
tivity. Allosteric ligands can function as purely allosteric agonists,
activating the receptor independent of acetylcholine (ACh), posi-
tive allosteric modulators (PAMs), which enhance the activity of
endogenous ACh, or as a combination of these two modes (an
ago-PAM). Considerable progress has been made in the develop-
ment of highly selective M1 PAMs,5–7 M4 PAMs,8 M5 PAMs,9 and
M1 allosteric agonists.10 Most recently, we have described the
Me
O
Me
O
H
N
N
N
H
N
H
N
N
CO2Et
CO2Et
ML071
(VU0357017)
hM1 EC50 = 1.6 µM (42% AChmax
VU0364572
hM1 EC50 = 0.89 µM (71% AChmax)
)
⇑
Corresponding author.
E-mail address: michael.r.wood@vanderbilt.edu (M.R. Wood).
Figure 1. The MLPCN probe molecule ML071 and the next-generation highly
selective, orally bioavailable, and CNS penetrant agonist VU0364572.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.03.088

3468
B. J. Melancon et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3467–3472
Me
O
O
development and characterization of VU0364572 (Fig. 1) starting
from the MLPCN probe ML071.11,12 Concurrently, we pursued an
alternative optimization of ML071 through the introduction of
numerous diverse constraints and now detail those efforts in this
Letter.
H
N
NCO2Et
F
NCO2Et
N
H
N
H
N
H
Me
1
ML071
hM1 EC50 = 1600 nM (35% AChmax
)
hM1 EC50 = 470 nM (40% AChmax
)
H
Continued optimization of ML071 built on the highly modular
and versatile nature of the synthesis routes utilized previously.
Briefly, all analogs appearing in subsequent tables and figures were
prepared according to the general routes appearing in Scheme 1,
employing routine methodology as required for elaborating pro-
tecting groups into libraries of targets. Starting materials were
either commercially available or prepared according to literature
procedures. Route A depicts the reductive amination step involved
when the Eastern piperidine-like portion of the final target arises
from a ketone building block. Route B depicts the corresponding
reaction between an aldehyde and the Eastern region already con-
taining a primary or secondary amine.
O
Me
O
H
H
N
N
CO2Me
NCO2Et
N
H
N
H
H
2
3
hM1 EC50 = 6200 nM (18% AChmax
)
hM1 EC50 = 4100 nM (60% AChmax
)
NCO2Et
NCO2Et
O
O
N
N
N
H
N
H
4
5
hM1 EC50 = 3200 nM (13% AChmax
)
hM1 EC50 = 1200 nM (13% AChmax)
Figure 2. Novel, bicyclic analogs of ML071.
Given the demonstrated benefits associated with the introduc-
tion of the central ring present in VU0364572 (Fig. 1),11 we first ex-
plored other modes of limiting molecular flexibility (Fig. 2). For the
compounds appearing in Figure 2, ML071 and 1 were prepared via
route A (Scheme 1), while the remaining analogs (2–5) were pre-
pared according to route B. Assay descriptions and experimental
protocols for the functional muscarinic assays have been described
previously.12 Compound 1 shows that a potency enhancement of
roughly threefold could be achieved by introducing the tropane
scaffold into the Eastern region. This was not surprising since these
types of tropane structures are commonly associated with musca-
rinic ligands (e.g., atropine and scopolamine). However, this was an
inseparable mixture of endo- and exo-isomers (roughly 70:30 endo/
exo based on proton NMR). Although, the four other compounds in
Figure 2 were less active than 1, the diazadecalins 4 and 5, were
viewed as an instructive pair of isomers. Relative to 1, the trans-
fused bicycle 4 has its central nitrogen, and the point of attachment
to the Western half of the molecule, displayed in an equatorial
fashion which might mimic the minor exo-isomer of compound
1. Conversely, the cis-fused bicycle 5 has its central nitrogen posi-
tioned in an axial sense, thereby mimicking the major, endo-isomer
component of 1. While the potencies of 4 and 5 are not drastically
different, it was encouraging to see that the axial nitrogen point of
attachment in 5, which more closely mimics the endo-tropane of 1,
was preferred over the equatorial sense of attachment in 4. This
implied that the endo-isomer component of 1 was likely the major
contributor to its improved activity, and that further efforts to
improve potency should focus on the production of endo-tropane
isomers.
Table 1
Structures and activities of tropane analogs 6a–i
NCO2Et
R
O
*
N
N
H
n
6a-i (>90:10 endo:exo)
a
b
Compd
R
⁄
n
hMl EC50
(l
M)
AChmax (%)
6a
6b
6c
6d
6e
6f
6g
6h
6i
Me
H
Me
H
Me
H
Me
H
(S)
(S)
(R)
(R)
(S)
(S)
(R)
(R)
—
2
2
2
2
1
1
1
1
0
2.1
>10
0.059
0.84
>10
1.2
52
12
75
75
22
39
55
62
34
1.2
0.32
2.2
H
a
Average of at least three independent determinations performed in triplicate.
Standard deviations ranging from 5% to 18% of the value reported.
b
When hM1 EC50 is ‘>10’, the reported AChmax equals an average of the %ACh
obtained when the compound was tested at the highest concentration (30
Standard deviations ranging from 2% to 11% of the value reported.
lM).
NCO2Et
O
H
H
N
NCO2Et
O
One straight forward method for increasing the proportion of
the endo-isomer relative to the exo-isomer when preparing analogs
of 1 via route A (Scheme 1), would be to perform the reductive
amination step with secondary amines, thereby relying on the in-
creased steric interactions to provide more of the desired endo-iso-
mer. When the analogs appearing in Table 1 were prepared, an
increased preference for the endo-isomer was observed with
Ph
N
H
NH
Ph
N
H
H
7
8
hM1 EC50 = 0.11 µM (34% AChmax
)
hM1 EC50 = 0.97 µM (49% AChmax)
Figure 3. Comparison of the individually prepared endo- (7) and exo- (8) tropane
isomers.
Route A:
O
(COR)PG
(COR)PG
N
N
N
H
(COR)PG
NH
O
a
a
N
H
+
N
N
N
PG
PG
(CO2Et)
(CO2Et)
Route B:
HN
N
H
(COR)PG
N
H
+
N
PG
PG
H
(CO2Et)
(CO2Et)
Scheme 1. General methods for the production of novel mAChR agonists. Reagents: (a) NaHB(OAc)3, DCE or DCM, HOAc (0–4 equiv).

B. J. Melancon et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3467–3472
3469
Table 2
endo:exo-isomer ratios now heavily favoring the endo-isomer. In
the context of a central piperidine (Table 1, n = 2), this provided
the corresponding tropane analogs of VU0364572. The preference
for the (R)-stereochemistry, as seen previously for VU0364572,11
was maintained and was most pronounced for the n = 2 analogs
(compare 6a with 6c). Although the (S)-isomers 6a and 6b appear
to display hM1 activity, this could also result from contamination
with the (R)-isomer arising from the commercially available 95%
ee starting material. As the ring size decreased (n = 1) this stereo-
chemical preference for the (R)-isomer decreased beyond what
could be explained by trace impurities, while the high endo:exo
ratios were maintained. The achiral azetidine analog 6i possessed
a similar potency and efficacy as ML071.
Structures and activities of exo-substituted tropane analogs 8, 9a–j
O
R
H
N
N
Ph
N
H
H
8, 9a-j
a
b
Compd
R
hM1 EC50
(
lM)
AChmax (%)
8
–CO2Et
0.97
>10
2.0
—
49
21
40
—
—
23
7
—
—
—
—
9a
9b
9c
9d
Be
9f
9g
9h
91
9j
–CO2Me
–CO2allyl
–(CO)Et
–(CO)Pr
–(CO)i-Pr
–CO2Bn
–CO2Ph
—
5.4
>10
—
Compound 6c represented one of our most potent hM1 ago-
nists prepared to date and when administered to rats exhibited
moderate PK properties. A standard rat IV (1 mg/kg)/PO (10 mg/
kg) study found that 6c possessed a high CL of 189 mL/min/kg,
–(CO)NMe2
–SO2Me
–SO2Et
—
—
—
a
Average of at least three independent determinations performed in triplicate. ‘—’
indicates an inactive compound (<5% AChmax) up to the highest concentration tested
(30 M). Standard deviations ranging from 10% to 15% of the value reported.
When hMl EC50 is ‘>10’, the reported AChmax equals an average of the %ACh
but with a Vss of 11.8 L/kg and a t of 46 min. Additionally, 6c
½
was orally bioavailable with a %F of 70 (AUCIV = 92.2 ngh/mL,
AUCPO = 639 ngh/mL). Unfortunately, compound 6c was found to
be lacking in its muscarinic subtype selectivity profile, along with
6d. Table 5 (vide infra) shows that while both 6c and 6d were
l
b
obtained when the compound was tested at the highest concentration (30
Standard deviations ranging from 3% to 21% of the value reported.
lM).
Table 3
Structures and activities of exo-substituted tropane analogs 10a–r
H
NCO2Et
R
N
H
N
H
10a-r
a
b
a
b
Compd
R
hM1 EC50
(lM)
AChmax (%)
Compd
R
hM1 EC50
2.5
(l
M)
AChmax (%)
Me
O
Me
O
N
10a
1.1
47
10j
39
Me
O
Cl
O
O
10b
10c
10d
10e
2.3
—
37
—
10k
10l
0.57
>10
2.0
53
11
44
—
N
CF3
O
O
O
O
N
O
Cl
0.44
3.6
63
33
10m
10n
N
O
O
MeO
F
—
N
H
O
10f
1.9
43
54
10o
10p
—
—
6
NH
F
O
O
O
O
10g
0.83
>10
N
F
F
F
10h
10i
0.81
1.5
50
34
10q
10r
1.0
—
33
—
S
O
O
S
O
MeO
a
Average of at least three independent determinations performed in triplicate. ‘—’ indicates an inactive compound (<5% AChmax) up to the highest concentration tested
M). Standard deviations ranging from 2% to 12% of the value reported.
When hM1 EC50 ‘>10’, the reported AChmax equals an average of the %ACh obtained when the compound was tested at the highest concentration (30
(30
l
b
lM). Standard
deviations ranging from 1% to 22% of the value reported.

3470
B. J. Melancon et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3467–3472
potent hM1 agonists, they showed activity at various hM2–5
receptors.13 Although 6c was about 100-fold selective over hM2,
this level of selectivity and the appearance of some level of ago-
nist activity across all muscarinic subtypes was not desirable.
Whether the emergence of hM2–5 activity resulted from structural
modifications that moved these agonists back into the orthosteric
binding site at the expense of the high-affinity allosteric site on
hM1 (bitopic agonists) or if these agonists are still allosteric
ligands at hM1 but not allosteric with respect to their binding
at hM2–5 has not yet been determined.
To enable a more rigorous comparison of endo- versus exo-
tropane isomers, each primary amine was individually prepared
according to literature procedures14 and utilized via synthesis
route B (Scheme 1) to prepare pure endo-analog 7 and pure exo-
analog 8 (Fig. 3). As surmised for the isomer components of 1,
the endo-isomer 7 was more potent than the exo-isomer 8. Inter-
estingly, the less potent isomer 8 displayed a superior efficacy
(%AChmax = 49%), compared to the more potent isomer 7. When
we determined the M1–5 receptor subtype selectivity for these
two compounds, the more potent endo-isomer 7 displayed antago-
nist activity across hM2–5, while the exo-isomer appeared com-
pletely selective (Table 5). This sort of agonist/antagonist profile
seen with 7 is similar to the profile seen with the M1 allosteric ago-
nist TBPB10b (Table 5, compound 12). As for the high apparent
selectivity demonstrated by 8, it remained to be seen if further
optimization with respect to hM1 potency would engender activity
at the other muscarinic subtypes. Ultimately, we decided to move
away from tropane analogs, where the point of attachment was
displayed in an axial fashion, and focus on tropane analogs which
position the central nitrogen in an equatorial manner, which might
be envisioned as better approximating the lowest energy confor-
mation of ML071.
mate, although this substitution resulted in a twofold decrease in
potency. The propyl- and butylamides (9c and 9d, respectively)
were not tolerated and speak to the importance of having a carba-
mate at this location. Ureas (like 9h) and sulfonamides (9i and 9j)
were similarly unproductive. Clearly the ethyl carbamate stood out
as the premier moiety at this location.
Concurrently we explored SAR around the Western end of com-
pound 8 (Table 3). Among the substituted benzamides (10a–h),
meta-substitution with a chlorine atom (10d) provided the only
clear improvement over 8. Various urea termini were tolerated at
this location; however, only 10k produced an increase in hM1 po-
tency. The two nitrogen-containing heterocycles 10o and 10p were
not tolerated, whereas, the thiophene analog 10q was equipotent
with compound 8. A sulfonamide analog 10r was relatively inac-
tive, as were a handful of other sulfonamides at this location (data
and structures not shown), but showed a trend toward hM1 antag-
onist activity. Although potencies were not improved to very low
nanomolar levels with the analogs 10, we continued to explore
alternate tropane scaffolds that positioned the nitrogen in an equa-
torial manner.
Utilizing synthesis route A (Scheme 1), and an alternate tropa-
none wherein the bridgehead was moved away from the ethyl car-
bamate, we easily obtained the analogs appearing in Table 4.
Interestingly, the reductive amination step which sets the endo/
exo-connectivity in 11 now afforded only the endo-product as a re-
sult of the bridgehead being closer to the reaction center thereby
enhancing its steric influence. Furthermore, since the bridgehead
had moved to the opposite side of the piperidine ring (compare
10 with 11) we could now directly obtain the desired equatorial
orientation of the central nitrogen.
This change in tropane scaffold afforded an almost uniform in-
crease in potency for analogs 11 compared to the corresponding
tropanes 10. Only a handful of analogs were prepared, but even
with this small sampling of R-groups, hM1 potencies were now
in the low nanomolar range for compounds 11a and 11c. Interest-
ingly, when central piperidines analogous to compounds 6a–d
were introduced into this alternate tropane scaffold, only weakly
potent agonists or wholly inactive compounds were obtained (data
and structures not shown).
Viewing compound 8 as a new starting point for medicinal
chemistry optimization, we began by exploring replacements for
the ethyl carbamate terminus (R-groups, Table 2). The structure-
activity relationship (SAR) in this region was remarkably steep.
The methyl carbamate 9a lost greater than 10-fold potency, but
remained an agonist. The only minor modification which appeared
to be somewhat tolerated was the introduction of an allyl carba-
Table 4
Structures and activities of endo-substituted tropane analogs 11a–j
O
H
N
NCO2Et
R
N
H
H
11a-j
a
b
a
b
Compd
R
hM1 EC50
0.092
(l
M)
AChmax (%)
Compd
R
hM1 EC50
1.0
(l
M)
AChmax (%)
11a
53
11f
37
9
Me
N
11b
0.17
51
11g
0.69
N
Cl
11c
11d
0.047
0.24
52
47
11h
11i
3.6
28
11
MeO
F
N
>10
O
11e
0.20
53
11j
0.17
54
S
F
a
Average of at least three independent determinations performed in triplicate. Standard deviations ranging from 3% to 23% of the value reported.
When hM1 EC50 is ‘>10’, the reported AChmax equals an average of the %ACh obtained when the compound was tested at the highest concentration (30
b
lM). Standard
deviations ranging from 2% to 10% of the value reported.

B. J. Melancon et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3467–3472
3471
Table 5
hM1–5 selectivity profile for selected hM1 agonists from an assay of calcium mobilization
Compd hM1
EC50 or
%AChc hM2
EC50 or
%AChc hM3
modea IC50
EC50 or
%AChc hM4
modea IC50
EC50 or
%AChc hM5
modea IC50
(lM)
EC50 or
%AChc
modea IC50
(
l
M)
modea IC50
(l
M)
(l
M)
(
l
M)
b
b
b
b
b
ML071
6c
6d
7
8
11a
11c
12
E
E
E
E
E
E
E
E
E
2.6
0.017
0.35
0.21
1.2
0.018
0.096
0.27
34
Inactive
1.8
>10
Inactive
>10
Inactive
>10
Inactive
>10
Inactive
2.2
Inactive
>10
Inactive
3.1
Inactive
>10
>10
Inactive
3.7
>10
4.9
Inactive
2.8
4.2
4.6
1.0
59
65
37
57
46
58
40
81
E
E
I
66
27
15
E
I
36
13
45
E
I
28
2
E
E
I
62
21
2
>10
Inactive
Inactive
Inactive
2.7
I
I
I
I
I
48
40
3
E
E
I
31
22
3
I
I
1
30
I
E
0
19
0.70
>10
13
0.041
>10
5.9
26
E
28
a
Mode of action in the calcium flux assay: E-represented an agonist response, I-represented an antagonist response and a blank represented a lack of clearly discernible
activity at the receptor subtype up to the top dose of 30 M. Experiments were single determinations conducted in triplicate, with routine error in the 1–25% range.
l
b
When the mode of action was that of an agonist (E) value is an EC50, when an antagonist (I) value is an IC50 and when an acceptable curve fit could not be generated, but a
clear mode of action could be determined, ‘>10’ is entered.
c
When the mode of action was that of an agonist %ACh represents the maximal activity observed as a percentage of the maximum response generated from 80% of a
saturating dose of ACh. When the mode of action was that of an antagonist %ACh represents the remaining activity observed at the highest dose (30
maximum response generated from 80% of a saturating dose of ACh. When the IC50 or EC50 is ‘>10’, the reported %ACh equals an average of the %ACh obtained when the
compound was tested at the highest concentration (30 M).
lM) as a percentage of the
l
increase in M1 potency brings into focus a potential weakness
within this series of hM1 bitopic agonists. Although the highly selec-
tive interaction with the M1 receptor may be allosteric at low
concentrations due to a high-affinity allosteric site, the bitopic
nature of these agonists may result in orthosteric interactions at
higher concentrations or at the M2–5 receptors as potency is im-
proved at the M1 receptor. As such, a potentially more tractable ap-
proach toward the selective activation of each individual
muscarinic subtype may be realized with subtype selective PAMs.
Detailed in vitro pharmacological studies around this group of
bitopic17 partial agonists are in progress and will be reported
shortly.18
In summary, we have expanded the SAR surrounding ML071,
resulting in the development of highly potent hM1 partial agonists
containing two different tropane scaffolds. However, as potency
was increased within each of the three series, off-target activity
at the other muscarinic receptors surfaced. This may be related
to the bitopic nature of these weak partial agonists.
Acknowledgments
Figure 4. Ability of hM1 partial agonists to stimulate nonamyloidogenic sAPP
a
release in TREx293-hM1 cells. Agonists were tested at
presented were from three independent experiments.
2 lM and the results
The authors thank the NIH and NIMH for support of our M1
program (1RO1MH082867-01) and TJU (5T32MH065215-09).
Vanderbilt University is a Specialized Chemistry Center within
the MLPCN (U54MH084659), and ML071 is an MLPCN probe, freely
available upon request.19
With another series of highly potent hM1 partial agonists in
hand, the paramount question of receptor subtype selectivity
was quickly explored. Unfortunately, as shown in Table 5, both
compounds displayed off target activity at subsets of the hM3–5
receptors. Compound 11a had mixed activity as an antagonist at
hM3 and hM4, but functioned as a partial agonist at hM5. Similarly,
11c was an antagonist at hM4, but a partial agonist at hM5. We
have observed this mixed profile with other hM1 allosteric agonists
in our functional calcium mobilization assays, in particular
AC260584 (compound 13).15
References and notes
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Calligaro, D. O.; Shipley, L. A.; Buelke-Sam, J. L.; Bodick, N. C.; Farde, L.;
Sheardown, M. J.; Olesen, P. H.; Hansen, K. T.; Suzdak, P. D.; Swedberg, M. D. B.;
Sauerberg, P.; Mitch, C. H. Drug Dev. Res. 1997, 40, 158.
2. Shekhar, A.; Potter, W. Z.; Lightfoot, J.; Lienemann, J.; Dube, S.; Mallinckrodt, C.;
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Having optimized hM1 potency in three related series of tro-
pane partial agonists, we tested select compounds for their ability
to enhance the release of soluble APPa (sAPPa
),16 as has been dem-
onstrated with previous M1 agonists11 and PAMs.7 Gratifyingly, all
five of the tropane agonists tested (6c, 6d, 7, 8 and 11a, Fig. 4) were
able to stimulate the release of sAPP
a
in TREx293-hM1 cells to the
M ML071. These
same extent as 10 M carbachol (CCh) and 2
l
l
experiments continue to support the belief that activation of M1
may have the potential to be disease modifying for the treatment
of Alzheimer’s disease.
Although, these tropane agonists were very attractive from an
M1 potency aspect, the loss of selectivity accompanied by their
8. Brady, A. E.; Jones, C. K.; Bridges, T. M.; Kennedy, J. P.; Thompson, A. D.; Heiman,
J. U.; Breininger, M. L.; Gentry, P. R.; Yin, H.; Jadhav, S. B.; Shirey, J. K.; Conn, P. J.;
Lindsley, C. W. J. Pharmacol. Exp. Ther. 2008, 327, 941.

3472
B. J. Melancon et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3467–3472
9. Bridges, T. M.; Marlo, J. E.; Niswender, C. M.; Jones, C. K.; Jadhav, S. B.; Gentry, P.
R.; Plumly, H. C.; Weaver, C. D.; Conn, P. J.; Lindsley, C. W. J. Med. Chem. 2009,
52, 3445.
receptor expression and/or receptor reserve levels between the experiments
used in the selectivity profiling experiments and those used for the rest of the
manuscript, as well as other forms of experimental variability.
14. Blackburn, C.; LaMarche, M. J.; Brown, J.; Che, J. L.; Cullis, C. A.; Lai, S.; Maguire,
M.; Marsailje, T.; Geddes, B.; Govek, E.; Kadambi, V.; Doherty, C.; Dayton, B.;
Brodjian, S.; Marsh, K. C.; Collins, C. A.; Kym, P. R. Bioorg. Med. Chem. Lett. 2006,
16, 2621.
15. Bradley, S. R.; Lameh, J.; Ohrmund, L.; Son, T.; Abhishek, B.; Nguyen, D.; Friberg,
M.; Burstein, E. S.; Spalding, T. A.; Ott, T. R.; Schiffer, H. H.; Tabatabaei, A.;
McFarland, K.; Davis, R. E.; Bonhaus, D. W. Neuropharmacology 2010, 58, 365.
16. See Ref. 7 for experimental details.
17. Bitopic refers to a ligand which can occupy two sites on a given receptor either
individually or concurrently, wherein one site is the orthosteric site and the
other is an allosteric site, which may or may not be adjacent to the orthosteric
site. See Refs. 12,18 for a more detailed description of the pharmacology.
18. Manuscripts in preparation.
10. (a) Lebois, E. P.; Bridges, T. M.; Dawson, E. S.; Kennedy, J. P.; Xiang, Z.; Jadhav, S.
B.; Yin, H.; Meiler, J.; Jones, C. K.; Conn, P. J.; Weaver, C. D.; Lindsley, C. W. ACS
Chem. Neurosci. 2010, 1, 104; (b) Jones, C. J.; Brady, A. E.; Davis, A. A.; Xiang, Z.;
Bubser, M.; Tantawy, M. N.; Kane, A. S.; Bridges, T. M.; Kennedy, J. P.; Bradley, S.
R.; Peterson, T. E.; Ansari, M. S.; Baldwin, R. M.; Kessler, R. M.; Deutch, A. Y.;
Lah, J. J.; Levey, A. I.; Lindsley, C. W.; Conn, P. J. J. Neurosci. 2008, 28, 10422.
11. Lebois, E. P.; Digby, G. J.; Sheffler, D. J.; Melancon, B. J.; Tarr, J. C.; Cho, H. P.;
Miller, N. R.; Morrison, R.; Bridges, T. M.; Xiang, Z.; Daniels, J. S.; Wood, M. R.;
Conn, P. J.; Lindsley, C. W. Bioorg. Med. Chem. Lett. 2011, 21, 6451.
12. Digby, G. J.; Noetzel, M. J.; Bubser, M.; Utley, T. J.; Walker, A. G.; Byun, N. E.;
Lebois, E. P.; Xiang, Z.; Sheffler, D. J.; Cho, H. P.; Davis, A. A.; Nemirovsky, N. E.;
Mennenga, S. E.; Camp, B. W.; Bimonte-Nelson, H. A.; Bode, J.; Italiano, K.;
Morrison, R.; Daniels, S.; Niswender, C. M.; Olive, M. F.; Lindsley, C. W.; Jones, C.
K.; Conn, P. J. J. Neurosci., in press.
19. For information on the MLPCN and information on how to request probe
compounds, such as ML071, see: http://mli.nih.gov/mli/mlpcn/.
13. Absolute potencies for hM1 appearing in Table 5 are different from the values
appearing in earlier tables/figures and may arise from slightly different