Isatin replacements applied to the highly selective, muscarinic M 1 PAM ML137: Continued optimization of an MLPCN probe molecule

Article abstract of DOI:10.1016/j.bmcl.2012.11.092

This Letter describes the continued optimization of an MLPCN probe molecule (ML137) with a focused effort on the replacement/modification of the isatin moiety present in this highly selective M₁ PAM. A diverse range of structures were validated as viable replacements for the isatin, many of which engendered sizeable improvements in their ability to enhance the potency and efficacy of acetylcholine when compared to ML137. Muscarinic receptor subtype selectivity for the M₁ receptor was also maintained.

Full text of DOI:10.1016/j.bmcl.2012.11.092

Bioorganic & Medicinal Chemistry Letters 23 (2013) 412–416
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Isatin replacements applied to the highly selective, muscarinic M1 PAM
ML137: Continued optimization of an MLPCN probe molecule
Bruce J. Melancon a,c,d, Michael S. Poslusney a,c,d, Patrick R. Gentry a,d, James C. Tarr a,c,d
Douglas J. Sheffler a,c, Margrith E. Mattmann a,c, Thomas M. Bridges a,c,d, Thomas J. Utley 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 an MLPCN probe molecule (ML137) with a focused
effort on the replacement/modification of the isatin moiety present in this highly selective M1 PAM. A
diverse range of structures were validated as viable replacements for the isatin, many of which engen-
dered sizeable improvements in their ability to enhance the potency and efficacy of acetylcholine when
compared to ML137. Muscarinic receptor subtype selectivity for the M1 receptor was also maintained.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 11 October 2012
Revised 12 November 2012
Accepted 20 November 2012
Available online 1 December 2012
Keywords:
Muscarinic acetylcholine receptor 1
M1
Allosteric
Positive allosteric modulator (PAM)
ML137
VU0448350
Acetylcholine (ACh) is a critical neurotransmitter influencing a
broad range of physiological processes both inside and outside of
the central nervous system by binding to both nicotinic (nAChR)
and muscarinic acetylcholine receptors (mAChR). The challenges
associated with developing highly selective mAChR ligands inter-
acting at the orthosteric binding site of ACh are most significantly
impacted by the high level of receptor homology displayed across
the five mAChR subtypes (M1 through M5).1 Despite this seemingly
insurmountable roadblock towards the production of truly selec-
tive muscarinic agonists, a great deal of information has been
learned from M1–5 KO mice2 and the testing of less-than-selective
muscarinic agonists in preclinical and clinical experiments. The
clinical promise for dual, or potentially individual, M1/M4 mAChR
activators in treating aspects of both Alzheimer’s disease and
schizophrenia has been demonstrated with the orthosteric agonist
xanomeline;3,4 however, this clinical candidate’s limited selectivity
(particularly against M3) ultimately caused its withdrawal from
further clinical trials irrespective of the encouraging positive
outcomes. A new and exciting chapter in the search for selective/
specific mAChR activators commenced with the identification of
novel positive allosteric modulators (PAMs) and allosteric agonists
through the use of functional high throughput screening (HTS) as-
says.5 These next-generation ligands have the novel benefit of
interacting at sites removed from the orthosteric binding site,
where the potential for selective interactions with just one of the
five mAChR is greatly enhanced. This revitalized interest in selec-
tively activating the mAChRs has been very successful, providing
a number of M1 PAMs (BQCA,6 ML137,7 and ML1698), M4 PAMs
(ML108,9 ML173,10 ML253,11 and ML29312
) and M5 PAMs
(ML129,13 and ML17214). Here we describe the continued struc-
tural exploration around the M1 PAM, MLPCN probe molecule
ML137.
Figure 1 shows the development of two M5 PAMs and one M1
PAM from a common HTS lead, VU0119498, all of which share
the isatin core in common. Although VU0119498 displayed PAM
activity at the M1, M3, and M5 mAChRs,13 specific structural mod-
ifications resulted in highly preferring (ML129) or completely
selective (ML172) M5 PAMs and a highly selective M1 PAM
(ML137). The ability to dial in muscarinic receptor subtype selec-
tivity was facilitated by the wide variety of commercially available
⇑
Corresponding author.
E-mail address: michael.r.wood@vanderbilt.edu (M.R. Wood).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.092

B. J. Melancon et al. / Bioorg. Med. Chem. Lett. 23 (2013) 412–416
413
O
O
O
O
hM1 EC50 >10 µM
AChmax = 41 3 %
hM1 EC50 = 2.4 µM
AChmax = 64 2 %
pEC50 = 5.62 0.07
S
O
N
F
N
F
O
O
N
N
N
Me
N Me
Br
F
N
N
VU0119498
(M1, 3, 5 PAM)
N
N
Me
13
14
ML137 (M1 PAM)
Collection of inactive surrogates: (no statistically significant PAM activity at 30 µM)
N
H
N
Ph
O
N
O
Me
F3CO
F3CO
O
O
N
N
N
O
N
O
O
N
N
15
16
17
19
20
21
22
N
N
OMe
OMe
O
O
N
N
ML129 (M5 PAM)
ML172 (M5 PAM)
N
18
Figure 1. HTS lead VU0119498 parlayed into selective M1 and M5 PAMs.
Figure 2. Rapid foray into isatin replacements while holding the benzyl pyrazole
constant.
substituted isatins and the efficiency of analog preparation, clearly
demonstrating that this scaffold can be very advantageous.
However, it was also appreciated that the isatin motif has an estab-
lished history of appearing in a wide range of medicinal chemistry
targets across the full spectrum of potential clinical indications.15
Additionally, HTS hits containing the isatin core are so frequent
that the isatin scaffold has earned a position of notoriety in some
measures of HTS promiscuity.16 As such, it seemed reasonable to
explore replacements, or modifications, to the isatin core in the
context of ML129, ML137, and ML172.
Our first efforts focused on a large and extensive library which
thoroughly and rapidly explored small modifications to the isatin
core employing commercially available benzyl, biphenyl and biaryl
ethers similar to those found in ML129 and ML172. As is often the
case with steep allosteric SAR,5 this effort proved unfruitful (data
and structures not shown). We then hypothesized that a more fo-
cused effort directed solely at replacing the isatin moiety, while
retaining the exact biaryl from ML137, could be more productive.
These analogs and the requisite phenylpyrazoles were prepared
according to the general synthesis route appearing in Scheme 1.
Readily available 4-bromobenzyl alcohols 1 undergo Suzuki
reactions with pyrazole boronic acids/esters 2 employing standard
conditions and provided the biaryl benzyl alcohols 3. Ghosez’ re-
agent (1-chloro-N,N,2-trimethylprop-1-en-1-amine) in DCM
smoothly converted the benzylic alcohol into the corresponding
benzyl chloride 4. With ample quantities of 4 in place, isatin or a
variety of isatin replacements could be alkylated, employing a
range of bases, to give analogs 5 and the majority of structures
appearing in Figure 2 and Table 1. Alternative routes towards the
preparation of these initial analogs can be seen in Schemes 2 and 3.
Scheme 2 depicts the alkylation of isatin with a 4-bromobenzyl
bromide 6 using a carbonate base in acetonitrile to give N-
benzylated isatins 7. Next, a trifluoromethyl group was introduced
at the ketone carbon employing the conditions of Shreeve, and
co-workers,17 to yield the aryl bromides 8. A Suzuki reaction anal-
ogous to the first step in Scheme 1 could then provide analogs 9, as
well as a diverse range of alternative biaryls, simply by changing
the boronate in the final step.
Scheme 3 shows the route used in the preparation of sultam 13
(See Fig. 2 for structure), which was necessary due to a lack of reac-
tivity and selectivity (N vs C benzylation) associated with the de-
sired parent sultam. Starting from commercially available
benzylic amine 11 and sulfonyl chloride 10, the linear sulfonamide
12 was prepared in 93% yield. The nitrogen-aryl bond was then
formed using Cu(I) catalysis in degassed toluene to give the desired
sultam 13, in 59% yield.
Figure 2 shows the functional activity for two active analogs
along with a host of unsuccessful isatin replacements (inactive at
human M1 (hM1) up to a concentration of 30 lM). Although not
as potent (EC50) as their parent ML137 (hM1 EC50 = 0.60 lM,
AChmax = 45 3%, pEC50 = 6.22 0.02),18 both 13 and 14 produced
comparable efficacy (% AChmax) at the highest concentration tested
of 30 lM and, more importantly, no longer contained the isatin
core. Indolinone 14 appeared to be more promising than sultam
13 from activity and synthesis standpoints and so was explored
further. While reduction of the isatin ketone was gratifyingly toler-
ated, attachment of the biaryl at the resultant methylene (as in 15)
or further reduction of the amide carbonyl to the indoline 16 was
not tolerated. The overall planarity of the indolinone core appeared
to be important for PAM activity since scission of the 5-membered
lactam to give the N-acetyl amide 17 provided an inactive
compound. However, annulation of a second aryl ring, as in 18,
proved detrimental to activity as well, despite maintaining a planar
system. The remaining analogs in Figure 2 (19–22) attempted to
mimic various heteroatom interactions potentially occurring with
ML137 or 14, but all retained no hM1 PAM activity.
R1
R2
R1
R2
Y
B(OR)2
O
OH
X
N
O
a
H
+
N
c
N
N
X
N
R3
b
R3
Br
N
X
1
2
3
5
N
R3
, Y = OH
4, Y = Cl
N
Scheme 1. Reagents and conditions: (a) PdCl2(dppf)ꢀDCM, Cs2CO3, THF/H2O, 160 °C, 10 min; (b) Ghosez’ reagent (1-chloro-N,N,2-trimethylprop-1-en-1-amine), DCM, rt, 15 h;
(c) base (i.e. K2CO3, NaH, etc.), aprotic solvent (i.e. DCM, DMF, MeCN), 0 °C to rt.

414
B. J. Melancon et al. / Bioorg. Med. Chem. Lett. 23 (2013) 412–416
Table 1
Structure and activities of analogs 23a–r
R1
R2
*
O
N
X
N
R3
N
a
b
b,c
b
Compd
⁄
R1
R2
X
R3
hM1 EC50
(
lM)
AChmax (%)
pEC50
23a
23b
23c
23d
23e
23f
23g
23h
23i
23j
23k
23l
23m
23n
23o
23p
23q
23r
Me
NH2
NH2
NMe2
OH
OH
OH
OH
OH
OH
OH
OH
OH
OMe
F
H
H
F
F
F
H
F
F
F
F
F
F
F
F
F
F
F
F
Cl
F
Me
Me
Me
Me
Me
Me
Me
Me
Me
Et
n-pr
i-pr
H
Me
Me
Me
Me
Me
>10
1.3
>10
>10
>10
>10
4.8
3.3
—
3.3
—
—
—
—
53
50
28
48
49
59
51
57
2
3
2
2
3
2
3
1
5.90 0.04
Me
Me
Me
CH2F
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
F
5.32 0.07
5.48 0.07
A
B
37
2
5.48 0.07
>10
—
>10
4.0
25
2
F
OH
OH
CF3
CF3
29
66
2
2
5.40 0.05
a
Absolute stereochemistry is unknown, but each resolved enantiomer displayed an ee of >95% as determined by analytical chiral HPLC.
b
Values represent the mean standard error of the means of at least three independent determinations performed in triplicate. ‘—’ indicates an inactive compound
M).
When hM1 EC50 is reported as >10, this value represents the percent maximal ACh response when the test compound was present at its highest concentration (30
showing no PAM activity up to the highest concentration tested (30
l
c
lM).
O
O
HO
HO
CF3
CF3
O
O
O
O
a
b
c
N
N
N
N
H
Br
X
+
X
Br
X
Br
X
N
R3
N
Br
6
7
8
9
Scheme 2. Reagents and conditions: (a) K2CO3, MeCN, rt, overnight; (b) TMS-CF3, CsF, THF, rt, overnight; (c) 2, PdCl2(dppf)ꢀDCM, Cs2CO3, THF/H2O, 160 °C, 10 min.
derivatization of ML137 to provide the compounds appearing in
Table 1. The introduction of a methyl group onto the indolinone
methylene diminished hM1 PAM activity for 23a. However, the
introduction of an amino group at the same location, increased
the potency of 23b twofold over 14. Unfortunately, this primary
amino group instilled an undesirable level of chemical instability,
presumed to be air oxidation or self-condensation based on the
brightly colored byproducts observed. Attempts to block this deg-
radation with methyl groups provided only weakly active PAMs
(23c and 23d). Replacing the amino group with a hydroxyl group
provided the stable compound 23e, now with improved efficacy
relative to 23c. Fluorination of the methyl group in 23e was bene-
ficial, providing racemic 23g which displayed a measurable EC50 of
Br
Br
O
SO2Cl
+
S
O
10
HN
a
b
13
H2N
F
F
Me
N
N
N
Me
N
11
12
Scheme 3. Reagents and conditions: (a) NEt3, DCM, 30 min, 93% yield; (b) CuI(cat.),
K3PO4, DMEDA (N,N0-dimethylethylene-diamine), toluene, degassed with argon
(10 min), sealed, 110 °C for 3 h, 59% yield.
4.8 lM. Although not routinely performed during these early
stages of structure–activity relationship (SAR) development, the
enantiomers of 23g were resolved employing HPLC chiral column
chromatography to demonstrate that all the hM1 PAM activity re-
sided in a single enantiomer of unknown absolute configuration
(compare 23h and 23i).
While full reduction of the isatin ketone to arrive at 14 appeared
to be the most tolerated modification so far, the decrease in po-
tency suggested that the introduction of various substituents to
this methylene might restore some of the lost activity. Numerous
small modifications were made to 14 based on the chemistries
appearing in Schemes 1 and 2, as well as through straightforward
With 23g showing promise as another viable replacement for
the isatin core, we explored alternatives to the N-methyl pyrazole.

B. J. Melancon et al. / Bioorg. Med. Chem. Lett. 23 (2013) 412–416
415
Table 2
Although a wide range of heterocycles could be efficiently intro-
Structures and activities of analogs 24a–k attached to the preferred biaryl
duced at the terminal location of the biaryl utilizing the chemistry
in Scheme 2, none was found to be superior to the pyrazole (data
and structures not shown for about 30 diverse alternative aryl
groups). Only the most minor of modifications was tolerated in
that the N-ethyl pyrazole 23j was found to be of comparable po-
tency, but with reduced efficacy. Alkyl groups larger than ethyl
(23k and 23l), or the non-alklyated pyrazole 23m resulted in a
complete loss of activity. Additional modifications to 23g appeared
detrimental towards hM1 PAM activity (23n–q) with the exception
of deleting the fluorine on the central phenyl ring providing 23r, a
compound of similar potency when compared to 23g.
Unfortunately, none of the analogs appearing in Table 1 dramat-
ically surpassed the potency of 14. However, the relative simplicity
of 14 inspired us to explore more benzo-fused bicycles which did
not contain a 5-membered ring as the point of attachment to the
biaryl southern region. In direct analogy to 14, expansion of the
5-membered lactam to a 6- or 7-membered version caused a drop
in activity (Table 2, 24a and 24b). Although 24a retained a measur-
F
Me
N
N
a
a,b
a
Compd
hM1EC50
(lM)
AChmax (%)
pEC50
24a
5.6
44
3
5.25 0.07
N
N
N
O
24b
—
O
24c
24d
>10
—
51
52
3
3
O
N
O
O
able hM1 EC50 of 5.6
lM, its unsaturated quinolinone analog 24c
possessed a hM1 EC50 >10
l
M, but with slightly improved efficacy.
24e
5.2
5.29 0.09
Deletion of the phenyl group from 24c to arrive at pyridone 24d re-
sulted in a loss of all PAM activity, indicating the importance of this
benzo-fused motif. Placing an oxygen adjacent to the aryl ring
(24e) resulted in a compound of similar potency to 24a, while
the carbamate and urea analogs 24f and 24g, respectively, main-
tained reasonable hM1 PAM efficacy but with EC50s greater than
N
O
O
O
24f
>10
>10
62
53
2
2
N
NH
24g
10 lM. The thio version of 24e, compound 24h, appears to have
N
S
O
improved potency relative to 24e; however, these two potency val-
ues are not statistically different as seen by comparing their exper-
imentally determined pEC50 values. Oxidized versions of 24h and
its carbonyl-reduced analog 24k all showed moderately reduced
activity (compounds 24i and 24j) or no activity at all (compound
24k). This complete lack of activity for 24k was not a surprise gi-
ven the results obtained for 16, and served to reiterate the impor-
tance of this carbonyl. This survey of expanded-ring analogs in
Table 2 barely scratches the surface of possible structures and a
more focused optimization of any slightly active structure could
prove highly productive.
The utility of selective M1 PAMs as a therapeutic agent has not
been explored to the extent where it is definitively known whether
a high efficacy (high fold shift of an ACh concentration-response)
PAM or a low efficacy PAM is preferable under the various disease
states for which they might provide clinical efficacy. However,
ML137, with an ability to shift the ACh concentration-response
by just 2.7, rests firmly in the latter category. Therefore, it was
desirable to characterize tool compounds spanning a wide range
of fold-shift values. Although none of the hM1 PAM potencies pre-
sented here rivaled that of the lead compound ML137, improve-
ments with respect to efficacy were being realized and likely
contributed to the improved ACh fold-shift values appearing in
Table 3. Reduction of the isatin carbonyl (ML137 to 14) caused a
fourfold decrease in hM1 PAM EC50, but at the same time resulted
in a tenfold improvement in ACh fold-shift. As a group, the novel
compounds appearing in Table 3 all shared a reduced potency rel-
ative to ML137, while at the same time, displayed improved fold-
shifts ranging from 6.5 to 27.2. A graphical representation of four
hM1 PAMs, and the vehicle control for ACh, as part of a fold-shift
experiment is shown in Fig. 3. Compound 14 (VU0448350-1), with
the highest fold-shift, shifted the ACh concentration-response
curve (CRC) furthest to the left, indicating its superior ability to en-
hance the activity of ACh among this set of compounds. By way of
comparison, ML137 (VU0366369-1), which displays an order of
magnitude lower fold-shift value relative to 14, produces the first
concentration-response curve just to the left (open triangles) of
the vehicle control (ACh alone, open squares).
24h
24i
3.2
30
46
2
3
5.49 0.12
N
O
O
O
O
O
S
N
>10
O
S
24j
>10
—
36
2
N
S
24k
N
a
Values represent the mean standard error of the means of at least three
independent determinations performed in triplicate. ‘—’ indicates an inactive
compound showing no PAM activity up to the highest concentration tested (30 M).
l
b
When hM1 EC50 is reported as >10, this value represents the percent maximal
ACh response when the test compound was present at its highest concentration
(30 lM).
Table 3
ACh fold-shifts for selected hM1 PAMs
Compd
hM1 ACh fold-shifta
ML137
14
23g
23h
23e
23f
2.7 0.2
27.2 0.6
7.4 0.6
12
2
6.5 0.3
8.0 0.3
23r
15.2 0.5
a
Leftward shift of an ACh CRC in the presence of
30 lM compound relative to the ACh CRC control.
Values represent the mean standard error of the
means of at least three independent determinations.
Seemingly minor modifications to allosteric ligands can result
in large changes to both potency and receptor subtype selectivity

416
B. J. Melancon et al. / Bioorg. Med. Chem. Lett. 23 (2013) 412–416
receptor subtypes. The continued optimization of ML137 along
alternative lines will be reported shortly.
Acknowledgments
The authors thank the NIH (U54MH084659) and NIMH
(1RO1MH082867) for support of our M1 program. Vanderbilt Uni-
versity is a Specialized Chemistry Center within the MLPCN, and all
ML# probes are freely available upon request.20
References and notes
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Figure 3. Fold-shift CRCs for the hM1 PAMs: ML137 (VU0366369-1), 14
(VU0448350-1), 23g (VU0422337-1), and 23r (VU0449055-1).
4. Shekhar, A.; Potter, W. Z.; Lightfoot, J.; Lienemann, J.; Dube, S.; Mallinckrodt, C.;
Bymaster, F. P.; McKinzie, D. L.; Lelder, C. C. Am. J. Psychiatry 2008, 165, 1033.
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Christopoulos, A.; Conn, P. J.; Lindsley, C. W. J. Med. Chem. 2012, 55, 1445.
6. Ma, L.; Seager, M. A.; Wittmann, M.; Jacobson, M.; Bickel, D.; Burno, M.; Jones,
K.; Graufelds, V. K.; Xu, G.; Pearson, M.; McCampbell, A.; Gaspar, R.; Shughrue,
P.; Danziger, A.; Regan, C.; Flick, R.; Pascarella, D.; Garson, S.; Doran, S.;
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G.; Seabrook, G. R.; Ray, W. J. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 15950.
7. ML137 (a.k.a. VU0366369) Bridges, T. M.; Kennedy, J. P.; Noetzel, M. J.;
Breininger, M. L.; Gentry, P. R.; Conn, P. J.; Lindsley, C. W. Bioorg. Med. Chem.
Lett. 2010, 20, 1972.
8. ML169 (a.k.a VU0405652) Reid, P. R.; Bridges, T. M.; Sheffler, D. J.; Cho, H. P.;
Lewis, L. M.; Days, E.; Daniels, J. S.; Jones, C. K.; Niswender, C. M.; Weaver, C. D.;
Conn, P. J.; Lindsley, C. W.; Wood, M. R. Bioorg. Med. Chem. Lett. 2011, 21, 2697.
9. ML108 (a.k.a. VU0152100) 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.
10. ML173 (a.k.a VU0359508, a.k.a compound 21n) Kennedy, J. P.; Bridges, T. M.;
Gentry, P. R.; Brogan, J. T.; Kane, A. S.; Jones, C. K.; Brady, A. E.; Shirey, J. K.;
Conn, P. J.; Lindsley, C. W. ChemMedChem 2009, 4, 1600.
Figure 4. Muscarinic selectivity for 14 (VU0448350-1).
11. ML253 (a.k.a VU0448088) Le, U.; Melancon, B. J.; Bridges, T. M.; Vinson, P. N.;
Utley, T. J.; Lamsal, A.; Rodriguez, A. L.; Venable, D.; Sheffler, D. J.; Jones, C. K.;
Blobaum, A. L.; Wood, M. R.; Daniels, J. S.; Conn, P. J.; Niswender, C. M.;
Lindsley, C. W.; Hopkins, C. R. Bioorg. Med. Chem. Lett. 2012. manuscript
accepted.
12. ML293 (a.k.a VU0409524) Salovich, J. M.; Vinson, P. N.; Sheffler, D. J.; Lamsal,
A.; Utley, T. J.; Blobaum, A. L.; Bridges, T. M.; Le, U.; Jones, C. K.; Wood, M. R.;
Daniels, J. S.; Conn, P. J.; Niswender, C. M.; Lindsley, C. W.; Hopkins, C. R. Bioorg.
Med. Chem. Lett. 2012, 22, 5084.
13. ML129 (a.k.a VU0238429, a.k.a. compound 42) Bridges, T. M.; Marlo, J. E.;
Niswender, C. M.; Jones, C. K.; Jadhav, S. B.; Gentry, P. R.; Plumley, H. C.;
Weaver, C. D.; Conn, P. J.; Lindsley, C. W. J. Med. Chem. 2009, 52, 3445.
14. ML172 (a.k.a. VU0400265, a.k.a compound 10) Bridges, T. M.; Kennedy, J. P.;
Cho, H. P.; Breininger, M. L.; Gentry, P. R.; Hopkins, C. R.; Conn, P. J.; Lindsley, C.
W. Bioorg. Med. Chem. Lett. 2010, 20, 558.
(off-target activity in general) and this observation has been
embodied within the concept of the ‘molecular switch’.5,19 With
this idea in mind, we chose to profile the highest efficacy com-
pound (14) across all five of the human muscarinic receptor sub-
types; encouragingly, we found little-to-no erosion of muscarinic
subtype selectivity (VU0448350-1, Fig. 4). Although it would ap-
pear from the graph that 14 displays very weak hM4 PAM activity,
examination of the raw calcium traces from the functional assay
indicated this was most likely an artifact resulting from an unex-
plained baseline increase at the two highest concentrations tested
(data not shown). Fundamentally, the identification of a selective
M1/M4 PAM would be a significant achievement in light of the po-
sitive clinical outcomes observed with xanomeline;3,4 unfortu-
nately, compound 14 does not represent a step along that path.
In summary, a number of bicyclic benzo-fused isatin replace-
ments have been identified which retain reasonable hM1 PAM
activity and may represent novel starting points for continued
optimization. Compound 14, which showed the largest improve-
ment in efficacy compared to ML137, was also demonstrated to
be very selective for the hM1 receptor over the other muscarinic
15. MacDonald, J. P.; Badillo, J. J.; Arevalo, G. E.; Silva-García, A.; Franz, A. K. ACS
Comb. Sci. 2012, 14, 285. and references therein.
16. Baell, J. B.; Holloway, G. A. J. Med. Chem. 2010, 53, 2719.
17. Singh, R. P.; Cao, G.; Kirchmeier, R. L.; Shreeve, J. M. J. Org. Chem. 1999, 64, 2873.
18. Although the M1 PAM EC50 has been previously reported as 0.83
AChmax = 65% (Ref. 7), the inherent variability associated with functional assays
supports that the most recent EC50 = 0.60 M for ML137 is equally valid and
lM with a %
l
can serve as a baseline comparator between the two papers.
19. Wood, M. R.; Hopkins, C. R.; Brogan, J. T.; Conn, P. J.; Lindsley, C. W.
Biochemistry 2011, 50, 2403.
20. For information on the MLPCN and information on how to request any of the
ML# probe compounds, such as ML137, see: http://mli.nih.gov/mli/mlpcn/.