Spirocyclic replacements for the isatin in the highly selective, muscarinic M1 PAM ML137: The continued optimization of an MLPCN probe molecule

Article abstract of DOI:10.1016/j.bmcl.2013.01.017

This Letter describes the further optimization of an MLPCN probe molecule (ML137) through the introduction of 5- and 6-membered spirocycles in place of the isatin ketone. Interestingly divergent structure-activity relationships, when compared to earlier M

Full text of DOI:10.1016/j.bmcl.2013.01.017

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1860–1864
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Spirocyclic replacements for the isatin in the highly selective,
muscarinic M1 PAM ML137: The continued optimization of an MLPCN
probe molecule
Michael S. Poslusney a,c,d, Bruce J. Melancon a,c,d, Patrick R. Gentry a,d, Douglas J. Sheffler 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 further optimization of an MLPCN probe molecule (ML137) through the intro-
duction of 5- and 6-membered spirocycles in place of the isatin ketone. Interestingly divergent struc-
ture–activity relationships, when compared to earlier M1 PAMs, are presented. These novel spirocycles
possess improved efficacy relative to ML137, while also maintaining high selectivity for the human
and rat muscarinic M1 receptor subtype.
Received 20 November 2012
Revised 19 December 2012
Accepted 2 January 2013
Available online 11 January 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Muscarinic acetylcholine receptor 1
M1
Spirocyclic
Positive allosteric modulator (PAM)
ML137
VU0413162
The tremendous potential for truly selective muscarinic acetyl-
choline (ACh) ligands in a variety of disease states is well appreci-
ated; in reality, however, this potential has been difficult to achieve
due to a historical reliance on targeting the highly conserved
orthosteric ACh binding site shared by the five muscarinic ACh
receptor subtypes (mAChRs, M1 through M5).1 As such, many of
our current beliefs pertaining to the potential utility of muscarinic
ligands in central nervous system (CNS) disorders has come from
the phenotypes of M1–5 knock out (KO) mice,2 CNS receptor local-
ization and the promising clinical outcomes observed with the
muscarinic agonist xanomeline in both Alzheimer’s disease and
schizophrenia patients.3,4 Unfortunately, xanomeline’s limited
muscarinic subtype selectivity has given rise to unacceptable levels
of adverse events consistent with peripheral activation of the M3
receptor. Although switching focus to allosteric agonists is a cred-
ible tactic for circumventing the selectivity issues associated with
engaging the orthosteric site, a fair number of allosteric agonists
have recently been shown to bind in a bitopic sense to the mAChRs,
bringing the orthosteric site issues back into unwelcome consider-
ation.5,6 Another tactic for achieving high mAChR subtype selectiv-
ity has been the development of positive allosteric modulators
(PAMs), compounds with selectivity profiles that benefit from
binding solely at sites distinct from the ACh binding site where
amino acid homology between the muscarinic receptors has the
potential to be reduced. This approach has been validated through
the development of numerous, highly selective PAMs for M1,7 M4,8
and M5.9
Recently, we described our initial success at replacing the isatin
portion of ML137 and were able to arrive at a variety of novel
structures partially summarized by compounds
1 and 2 in
Figure 1.10 Reduction or modification of the ketone carbonyl in
ML137 served as the starting point for these benzo-fused bicyclic
replacements. Alternatively, it might be envisioned that this
carbonyl could be partially hydrated when water is available
(in vitro/in vivo) or even preferentially present as the hydrate
when bound to the allosteric site on the hM1 receptor. This idea
formed the rationale for the preparation of the spirocyclic ketal 3
(Fig. 1) as a discrete and stable entity. When tested against the hu-
man M1 (hM1) receptor for functional activity, 3 provided a hM1
PAM EC50 of 2.0
lM with an AChmax response of 62%, showing rea-
⇑
Corresponding author.
E-mail address: michael.r.wood@vanderbilt.edu (M.R. Wood).
sonable potency and efficacy, respectively. Although not as potent
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.017

M. S. Poslusney et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1860–1864
1861
R1
spirocycles at this location. Some of these spirocycles were avail-
able from commercial sources and were alkylated with the appro-
priate benzyl chloride as described previously.10 However, the
majority of subsequent analogs were prepared according to
Schemes 1–3.
Scheme 1 shows the alkylation of commercially available Boc-
protected spirocyclic amines 4 with benzylic bromides 5 employ-
ing potassium carbonate in acetonitrile to provide aryl bromides
6. Compound 6 can then undergo Suzuki couplings with a variety
of boronic acids/esters, such as 7, to form the N-Boc protected biar-
yl compounds 8. The N-Boc protecting group can then be removed
with a strong acid (TFA or anhydrous HCl) to give the secondary
amine 9. Amine 9 was an invaluable intermediate poised to engage
in a wide variety of well-precedented reactions with a near limit-
less number of electrophiles.
Scheme 2 depicts the construction of an isomeric spiropyrroli-
dine analog, which was not commercially available, starting from
the known, non-selective M1,3,5 PAM VU0119498.9a Condensation
of this isatin with tert-butyl sulfonamide promoted by titanium
(IV) tetraisopropoxide in DCM was followed, in a separate step,
by the addition of Grignard 10 to yield 11. In this manner, both
the nitrogen and the carbon chain required to form the spiropyrr-
olidine were efficiently introduced. Removal of both protecting
groups from 11 with HCl in dioxane gave the aminoaldehyde 12
R2
O
Remove or
G
N
O
O
replace the
ketone
N
N
O
R1 = R2 = H,
ML137
G = CH2, O, S
R
1 = OH, R2 = CF3
F
N Me
N
1a,b
2a-c
+ H2O - H2O
O
hM1 EC50 = 2.0 µM
AChmax = 62 2 %
pEC50 = 5.70 0.11
O
HO
OH
∴
O
N
F
O
N
3
N Me
N
Figure 1. Prior replacements for the isatin in ML137 and a rationale for the
preparation of the spirocyclic ketal 3.
(EC50) as its parent ML137 (hM1 EC50 = 0.60
pEC50 = 6.22 0.02),11 compound 3 produced improved efficacy
(%AChmax) at the highest concentration tested of 30 M. This novel
spirocycle was the impetus for exploring a wider range of diverse
lM, AChmax = 45 3%,
l
NBoc
NBoc
NBoc
B(OR)2
n
n
n
a
b
+
O
O
O
N
N
N
H
N
N
R3
n = 1 or 2
4
6
7
8
X
Br
X
Br
X
N R3
+
N
c
R(C=O)H
R(C=O)R'
R(C=O)Cl
NH
Br
5
n
Tables 1-3
O
N
RSO2Cl
R-I
R-OTf
ArBr
9
X
N R3
N
Scheme 1. Reagent and conditions: (a) K2CO3, MeCN; (b) PdCl2(dppf)ÁDCM, Cs2CO3, THF/H2O, 160 °C, 10 min; (c) TFA or HCl, 0 °C to rt.
t-Bu
S
O
O
O
NH
N
H2N
O
H
O
a
c
O
O
O
O
N
N
BrMg
O
b,
10
11
12
Br
Br
Br
VU0119498
d
Me
O
Me
O
B
N
NH
Me
O
f
e
Me
Me
18c
+
N
O
N
N
N
Me
14
13
15
Br
Br
Scheme 2. Reagents and conditions: (a) tert-butyl sulfinamide, Ti(OiPr)4, 0 °C to rt, DCM, 84% yield; (b) THF, À78 °C to rt, 72% yield; (c) HCl in dioxane (4 M), 0 °C to rt; (d)
NaHB(OAc)3, DCE, rt, 58% yield, 2 steps; (e) (CH2O)n, CO2H2, H2O, 100 °C, 2 h, 70% yield; (f) PdCl2(dppf)ÁDCM, Cs2CO3, THF/H2O, 160 °C, 10 min, 40% yield.

1862
M. S. Poslusney et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1860–1864
Table 1
TMS
Structures and activities of analogs 3, 18a–h showing spirocyclic alternatives to the
O
isatin core
O
O
a
b
c
X
O
O
O
18b
Me
N
N
N
N
N
16
17
VU0119498
Br
Br
Br
a
a,b
a
Compd
X
hM1 EC50
M)
AChmax
(%)
pEC50
(l
Scheme 3. Reagents and conditions: (a) allyltrimethylsilane, SnCl4, DCM, 0 °C, 5 h,
60% yield; (b) TBAF, THF, 60 °C, 18 h, 28% yield; (c) 15, PdCl2(dppf)ÁDCM, Cs2CO3,
THF/H2O, 160 °C, 10 min, 75% yield.
O
O
O
O
O
3
F
2.0
>10
>10
>10
3.2
—
62
43
43
53
65
2
2
3
4
3
5.70 0.11
O
(alternatively in a protonated/cyclized version, not shown) which
readily underwent reductive cyclization to form the spiropyrroli-
dine 13. The secondary amine of 13 was reductively methylated
with formaldehyde in an aqueous mixture of formic acid at ele-
vated temperatures to give the tertiary amine 14. Suzuki coupling
with 15 provided the fully elaborated spiropyrrolidine 18c (see Ta-
ble 1 for structure). The analogous spirotetrahydrofuran was pre-
pared starting from the same isatin (Scheme 3). Tin (IV) chloride
promoted the formal dipolar cycloaddition of allyltrimethylsilane
to the ketone in VU0119498 giving the spirocycle 16 in a single
step and in good yield (60%).12 Heating 16 with TBAF in THF
yielded the unadorned spirotetrahydrofuran 17, which was effi-
ciently converted to the desired biaryl 18b under our standard Su-
zuki coupling conditions.
Table 1 presents a survey of spirocycles and their functional
PAM activity at hM1. Compounds 18a and 18f showed an interest-
ing divergence in the SAR between these spirocycles and our ear-
lier series of non-spiro isatin replacement analogs in that
deletion of the amide carbonyl (18a) or deletion of the phenyl ring
(18f) did not abolish PAM activity, as was previously observed.10
Although an EC50 could not be determined for these two com-
pounds, a measureable positive allosteric modulation of ACh’s
activity was observed (AChmax >20%) and presumably indicates
beneficial interactions between the receptor and portions of the
new spirocycle ring.13 Relative to 3, the tetrahydrofuran 18b and
N-methyl pyrrolidine 18c lost potency but retained a measurable
level of hM1 PAM efficacy. This loss in potency is most likely not
related to the absence of a fluorine on the phenyl ring (vide infra,
compare 19d with 20i from Tables 2 and 3, respectively). The iso-
meric N-methyl spiropyrrolidine 18d displayed measurable po-
N
N
N
N
N
N
18a
18b
18c
18d
18e
18f
F
H
H
F
O
NMe
O
NMe
O
5.49 0.10
NH
O
F
NMe
F
>10
28
49
3
4
O
N
NMe
18g
F
F
3.8
5.42 0.06
O
N
N
tency with a hM1 EC50 of 3.2
18e lacked any PAM activity at concentrations up to 30
the N-methyl spiropiperidine 18g was about twofold less potent
than the lead spirocycle 3, for this structure, removal of the N-
methyl group did not abolish all the PAM activity for 18h.
l
M. However, the desmethyl analog
l
M. While
NH
18h
>10
29
2
O
Because compound 18d showed measurable potency, with the
potential for a twofold potency increase upon chiral resolution,
and was readily available for rapid analog preparation, this spiro-
pyrrolidine was chosen for the additional modifications shown in
Table 2. HPLC chiral column chromatography resolution of 18d
provided the individual enantiomers (>95% ee) 19a and 19b which
surprisingly shared the identical potency and only slightly differ-
ent efficacy, favoring enantiomer 19a. A series of N-cycloalkyl
groups were explored (19c–f), showing the cyclopentyl ring to be
the preferred size, which exhibited an equivalent potency to the
N-methyl version (18d). The increase in alkyl group size from
R = Me to R = c-pentyl may help to explain why there was now
an enantioselective preference for one enantiomer (19g) over the
other (19h), both with unknown absolute configurations. Given
the range of activities seen with the various cycloalkyl groups, it
was surprising to see such a large decline in activity upon going
to the isopropyl analog 19i. The N-ethyl analog 19j shared the
same potency as the N-methyl analog, but as fluorines were intro-
duced onto the ethyl terminus, potency decreased (19k–m).
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
l
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 M).
b
l
Whether this trend in potency resulted from decreasingly basic
pyrrolidine nitrogens, an increase in steric size, or a combination
of both factors was not rigorously explored. Carbonate, acyl and
sulfonyl groups were also attached to this nitrogen (19n–r) but re-
sulted only in compounds with less functional activity than the
parent analog 18d. Interestingly, the N-phenyl pyrrolidine 19s
was prepared and found to be inactive at the hM1 receptor,
whereas the 2-pyrimidine analog 19t produced low micro-molar
activity. Table 2 serves to illustrate the type of shallow SAR fre-
quently observed with allosteric ligands, where even minor
changes often result in few gains, and more frequently, significant
losses in functional activity.

M. S. Poslusney et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1860–1864
1863
Table 2
Table 4
Structures and activities of spiropyrrolidine analogs 19a–t
ACh fold-shifts for selected hM1 PAMs
Compd
hM1 ACh fold-shifta
2.7 0.2
R
N
ML137
3
19a
19b
19g
19j
19k
19l
19t
*
27
21
3
2
O
7.2 0.2
28
N
2
8.3 0.8
13.5 0.7
5.9 0.5
F
N Me
17.4 0.9
N
a
Leftward shift of an ACh CRC in the presence of 30
lM compound relative to the
a
b
b,c
b
ACh CRC control. Values represent the mean standard error of the means of at
least three independent determinations.
Compd
⁄
hM1 EC50 (lM) AChmax (%) pEC50
19a
19b
19c
19d
19e
19f
19g
19h
19i
A
B
Me
Me
2.9
2.9
>10
3.1
5.2
7.5
1.6
>10
>10
3.2
4.1
>10
—
67
50
48
57
60
50
69
33
30
59
60
52
3
3
3
3
2
3
1
3
2
2
3
3
5.54 0.06
5.54 0.07
c-Butyl
c-Pentyl
c-Hexyl
c-Heptyl
c-Pentyl
c-Pentyl
i-Pr
5.51 0.02
5.29 0.06
5.13 0.05
5.80 0.03
A
B
19j
19k
19l
Et
5.49 0.03
5.39 0.02
CH2CH2F
CH2CHF2
CH2CF3
Boc
19m
19n
19o
19p
19q
19r
19s
19t
—
Ac
>10
41
45
50
41
2
3
4
1
(C@O) c-butyl 6.3
(C@O) c-hexyl 5.0
5.20 0.07
5.30 0.05
SO2Me
Ph
2-Pyrimidyl
>10
—
2.5
64
1
5.61 0.07
Figure 2. Fold-shift CRCs for the hM1 PAMs: ML137 (VU0366369-1),
(VU0413162-1), 19a (VU0450158-1), 19b (VU0450257-1).
3
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 showing no PAM activity up to the highest concentration tested (30 lM).
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
Having explored the spirocycle region of these new M1 PAMs,
we decided to re-examine the biaryl region in the context of the
N-cyclopentyl spiropyrrolidine 19d. The results appearing in Ta-
ble 3 indicated that the 3-position of the pyrazole could be substi-
c
(30 lM).
tuted with
a methyl group, leaving PAM activity virtually
unaffected (20a). However, if a larger and more electron withdraw-
ing trifluoromethyl group was introduced at the same location, all
PAM activity was absent. Methyl substitution at the 5-position
eliminated PAM activity (20c) and simultaneous methylations at
both the 3- and 5-positions provided an inactive compound
(20d). Although not shown in Table 3, compounds not methylated
on the N-1 position of the pyrazole were devoid of PAM activity.
Fluorination patterns on the central ring provided relatively flat
SAR. The slightly preferred location for a single fluorine atom
was that found in 20e. Of the difluorides tested, compound 20f
was equipotent with the best monofluoride, while the other difluo-
rides brought significantly reduced activity. Unexpectedly, com-
pound 20i demonstrated that an absence of fluorination on the
central phenyl was potentially superior with respect to potency.
This fluorine SAR is very different from the SAR elucidated during
the development of ML137,7b and exemplifies the potential pitfalls
associated with trying to transfer established SAR from one series
into even a closely related PAM series.
Even after a considerable amount of effort was invested to ex-
plore a great deal of structural diversity, no improvements in po-
tency were forthcoming relative to the lead compound ML137.
However, as shown in Table 4, encouraging improvements in effi-
cacy were realized in the arena of hM1 ACh fold-shift values. Our
initial foray into spirocycles, ketal 3, demonstrated an order of
magnitude increase in its ability to shift the ACh concentration-re-
sponse relative to ML137. Perhaps the most interesting pair of
compounds in Table 4 was the pair of enantiomers 19a and 19b.
Table 3
Structure and activities of analogs 20a–i
N-c-pentyl
O
X
N
Z
Y
R5
N Me
N
R3
a
a,b
a
Compd
X
Y
Z
R3
R5
hM1 EC50
M)
AChmax
(%)
pEC50
(
l
20a
20b
20c
20d
20e
20f
20g
20h
20i
F
F
F
F
H
F
F
H
H
H
H
H
H
F
H
F
F
H
H
H
H
H
F
H
F
H
Me
CF3
H
H
H
Me
3.0
—
—
41
4
5.52 0.02
Me Me
—
H
H
H
H
H
H
H
H
H
H
2.0
2.6
>10
4.1
1.8
28
66
43
51
44
3
2
2
3
4
5.70 0.09
5.58 0.07
5.39 0.05
5.74 0.05
H
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).
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 M).
l
b
l

1864
M. S. Poslusney et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1860–1864
William K. Warren, Jr. Chair in Medicine (to CWL), for support of
our M1 program. Vanderbilt University is a Specialized Chemistry
Center within the MLPCN, and all ML# probes are freely available
upon request.14
References and notes
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Figure 3. Muscarinic selectivity for 3 (VU0413162-1).
Although both compounds share the same potency of 2.9 lM (hM1
6. Digby, G. J.; Utley, T. J.; Lamsal, A.; Sevel, C.; Sheffler, D. J.; Lebois, E. P.; Bridges,
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EC50), the enantiomer with a slightly better efficacy (19a: ACh-
max = 67% versus 19b: AChmax = 50%) is roughly 3-times better at
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graphically in Figure 2 by comparing 19a (open inverted triangles)
and 19b (open circles). Returning to Table 4, in the context of the
N-ethyl spiropyrrolidines 19j and 19k, the potency trend relative
to the number of fluorines present was not strictly recapitulated
in their abilities to produce an ACh fold-shift. For example,
although 19j was slightly more potent than 19k, with both sharing
the same AChmax, 19k elicited a higher fold-shift of the full ACh
concentration-response curve (CRC). For the final compound in Ta-
ble 4, 19t, it was encouraging to see that such a structurally diverse
analog could also show such robust efficacy.
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To round out the characterization of these novel spirocyclic M1
PAMs, we profiled 3 and 18d across all five human and all five rat
muscarinic acetylcholine receptors. Up to the highest concentra-
tions tested (30 lM) both compounds were completely selective
for the M1 receptors. A representative example of these results is
shown in Figure 3 for compound 3 (VU0413162-1) against the hu-
man receptors.
In summary, a number of isatin replacements containing vari-
ous spirocycles have been explored and these novel structures
have been confirmed as M1 PAMs. Although flat SAR was observed
with respect to potency, significant improvements in efficacy were
achieved for a range of structures. Very high receptor subtype
selectivity for 3 and 18d was observed across the spectrum of mus-
carinic subtypes (M2 through M5). Further optimization within any
of these scaffolds holds great promise for the development of
highly active and selective M1 PAMs and will be reported in due
course.
10. Melancon, B. J.; Poslusney, M. S.; Gentry, P. R.; Tarr, J. C.; Sheffler, D. J.;
Mattmann, M. E.; Bridges, T. M.; Utley, T. J.; Daniels, J. S.; Niswender, C. M.;
Conn, P. J.; Lindsley, C. W.; Wood, M. R. Bioorg. Med. Chem. Lett. 2013, 23, 412.
11. Although the M1 PAM EC50 has been previously reported as 0.83
%AChmax = 65% (Ref. 7b), the inherent variability associated with functional
assays supports that the most recent EC50 = 0.60 M for ML137 is equally valid
lM with a
l
and can serve as a baseline comparator between the two papers.
12. Nair, V.; Rajesh, C.; Dhanya, R.; Rath, N. P. Tetrahedron Lett. 2002, 43, 5349.
13. It is worth noting that intermediates 13, 14, 16 and 17 (Schemes 2 and 3),
which bear a close resemblance to the M1,3,5 PAM VU0119498, were found to
be inactive at hM1
Acknowledgments
14. 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/.
The authors thank the NIH (U54MH084659), NIMH
(1RO1MH082867) and William K. Warren, Jr., who funded the