Discovery and optimization of a novel, selective and brain penetrant M 1 positive allosteric modulator (PAM): The development of ML169, an MLPCN probe

Article abstract of DOI:10.1016/j.bmcl.2010.12.015

This Letter describes a chemical lead optimization campaign directed at VU0108370, a weak M₁ PAM hit with a novel chemical scaffold from a functional HTS screen within the MLPCN. An iterative parallel synthesis approach rapidly established SAR

Full text of DOI:10.1016/j.bmcl.2010.12.015

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2697–2701
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and optimization of a novel, selective and brain penetrant M1
positive allosteric modulator (PAM): The development of ML169,
an MLPCN probe
Paul R. Reid e, , Thomas M. Bridges a,d, , Douglas J. Sheffler a,c, Hyekyung P. Cho a,d, L. Michelle Lewis e,
Emily Days e, J. Scott Daniels a,c,d, Carrie K. Jones a,c,d,f, Colleen M. Niswender a,c,d, C. David Weaver a,d,e
,
P. Jeffrey Conn a,c,d, Craig W. Lindsley a,b,c,d,e, Michael R. Wood a,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 Program in Drug Discovery, Nashville, TN 37232, USA
d Vanderbilt Specialized Chemistry Center(MLPCN), Nashville, TN 37232, USA
e Vanderbilt Institute of Chemical Biology/Chemical Synthesis Core, Nashville, TN 37232, USA
f U.S. Department of Veterans Affairs, Tennessee Valley Healthcare System, Nashville, TN 37212, 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 a chemical lead optimization campaign directed at VU0108370, a weak M1 PAM hit
with a novel chemical scaffold from a functional HTS screen within the MLPCN. An iterative parallel
synthesis approach rapidly established SAR for this series and afforded VU0405652 (ML169), a potent,
selective and brain penetrant M1 PAM with an in vitro profile comparable to the prototypical M1 PAM,
BQCA, but with an improved brain to plasma ratio.
Received 29 October 2010
Revised 29 November 2010
Accepted 2 December 2010
Available online 9 December 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Muscarinic
Acetylcholine
Positive allosteric modulator (PAM)
ML169
BQCA
Alzheimer’s disease
MLPCN
The muscarinic acetylcholine receptors (mAChRs) are members
of thefamily A G-protein-coupled receptors (GPCRs)and includefive
subtypes denoted M1–M5. All five of the mAChRs are known to play
critical roles in multiple basic physiological processes and represent
attractive therapeutic targets for a number of peripheral and CNS
pathologies.1–3 Within the mAChRs, a major challenge has been a
lack of subtype selective ligands to study the specific contribution
of discrete mAChRs in various disease states.3,4 To address this lim-
itation, we have focused on targeting allosteric sites on mAChRs as a
means to develop subtype selective small molecules, both allosteric
agonists and positive allosteric modulators (PAMs).5–9 Moreover,
the emerging phenomenon of ligand-biased signaling requires the
development of diverse chemical scaffolds of M1 ligands to success-
fully dissect of the roles of M1 activation through multiple, discrete
ligand-biased signaling pathways.10,11
As members of the Molecular Libraries Production Center
Network (MLPCN),12 we performed a real-time cell-based cal-
cium-mobilization assay employing a rat M1/CHO cell line (Z0 aver-
aged 0.7) and screened a 63,656 member MLPCN compound library
following a triple-add protocol to simultaneously identify M1
antagonists, agonists (both orthosteric and allosteric) and positive
allosteric modulators (PAMs). This screen proved to be a major
success providing viable leads that were optimized into potent
and highly selective M1 ligands (Fig. 1): an M1 antagonist (1,
VU0255035, ML012),13 an M1 allosteric agonist (2, VU0357017,
ML071),14 and both an M1 PAM (3, VU0366369, ML137)15 and the
first M5 PAM (4, VU0238429, ML129)16 derived from a pan-
M1,M3,M5-PAM (5, VU0119498).17,18 However, the brain penetra-
tion (brainAUC/plasmaAUC = 0.1) and efficacy (60% ACh Max) of 3
were poor, as was the brain penetration of the prototypical M1
PAM, BQCA (6, brainAUC/plasmaAUC = 0.1)19–21; therefore, M1 PAM
ligands with improved physicochemical properties for in vivo stud-
ies and novel scaffolds to address ligand-biased signaling are re-
quired. In this Letter, we describe the development of VU0405652
⇑
Corresponding author.
E-mail address: michael.r.wood@vanderbilt.edu (M.R. Wood).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.12.015

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P. R. Reid et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2697–2701
O
N
N
O
S
O
N
HN
O
N
O
O
S
O
N
NH
HN
N
F
N
NMe
N
O
3, VU0366369
1, VU0255035
2, VU0357017
ML012
ML071
ML137
O
N
O
O
O
OH
F3CO
O
O
N
N
Br
OMe
OMe
6
, BQCA
4
5, VU0119498
, VU0238429
ML129
Figure 1. Structures of selective M1 and M5 MLPCN probes developed from hits from a triple-add functional M1 HTS MLPCN screen (1–5) and BQCA (6).
(ML169),22 a highly selective M1 PAM MLPCN probe, with a novel
chemical scaffold and improved brain penetration.
Perusal of the HTS data, which also yielded the non-selective hit
5, identified a second weak M1 PAM hit 7, VU0108370, with an EC50
Reduce to sulfoxide/sulfide
O
N
O
O
O
Me
S
N
H
N
of ꢀ13
lM. Confirmation of 7 from fresh powder and counter-
screening against M2–M5 increased our enthusiasm for this highly
Vary substitution
on the phenyl ring
Truncate or replace
Substitute
M1 mAChR selective hit (Fig. 2); however, the CRC did not plateau,
suggesting the M1 EC50 was actually >13 lM. Despite the weak po-
Cl
tency, the confirmation of a novel M1 PAM scaffold with high M1
selectivity initiated a lead optimization campaign to improve M1
potency while maintaining the high M2–M5 selectivity.
Figure 3. Initial optimization strategy for VU0108370, 7.
Our initial optimization strategy is outlined in Figure 3, and as
SAR with allosteric ligands is often shallow, we employed an iter-
ative parallel synthesis approach, along with targeted syntheses for
structures encompassing more speculative modifications. At-
tempted modifications of the Eastern oxazole-amide, although
not extensive, met with no success, returning only compounds
with undetectable activity. In a straightforward attempt to reduce
molecular weight the benzyl group attached to the indole nitrogen
O
O
N
O
a
S
b
Me
N
+
HS
OH
H2N
OMe
N
H
8
9
10
O
O
O
N
O
O
S
O
c
Me
Me
S
N
N
H
was omitted, but met with a similar lack of success (EC50 >10
as did the sulfide and sulfoxide congeners.
lM)
H
N
H
HN
Thus, we planned to hold the northern portion of 7 constant,
11
12
and survey diversity on the southern benzyl moiety employing a
O
O
O
N
O
S
Me
N
H
d
N
R
13
Scheme 1. Reagents and conditions: (a) (i) indole, I2, KI, MeOH, H2O; (ii) 2 M LiOH,
THF (38%); (b) PyClu, DCE, 110 °C, 20 min, mw (71%); (c) Oxone, MeOH, H2O (88%);
NaH, DMF, BnX (50–90%).
library synthesis approach. As shown in Scheme 1, the key library
scaffold 12 was readily prepared in three steps from methyl thio-
glycolate 8.
A PyClu-mediated microwave-assisted coupling
between 9 and 10 provided 11 in 71% yield, which was then oxi-
dized to the corresponding sulfone 12 with Oxone in 88% yield.
Figure 2. Concentration response curves (CRCs) for M1–M5 for HTS hit VU0108370.
M1 EC50 ꢀ13
lM (does not plateau) and M2–M5 EC50 >30 lM.

P. R. Reid et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2697–2701
2699
Table 1
Table 2
Structures and activities of analogs 13
Structures and activities of analogs 14
O
O
N
O
O
N
O
O
O
O
R
Me
Me
S
S
N
H
N
H
N
N
R
14
13
Compound Ar/Het
M1EC50
3.21
(l
M)a %Ach Maxa
Compound
R
M1EC50
(
l
M)a
%Ach Maxa
N
N
7
2-Cl
3-Cl
4-Cl
2-OMe
3-OMe
4-OMe
3-F
3-Br
3-CF3
3-CN
3,5-diBr
3,4-diCl
4-CF3
4-OCF3
2-F
9.71
5.82
>10
>10
6.54
>10
5.22
3.79
>10
>10
>10
>10
>10
>10
>10
>10
>10
83
96
—
—
79
—
96
91
—
—
—
—
—
—
—
—
—
14a
14b
102
90
—
13a
13b
13c
13d
13e
13f
13g
13h
13i
13j
13k
13l
13m
13n
13o
13p
Me
N
NH
N
2.19
14c
>10
N
s-Bu
14d
14e
>10
>10
—
—
N
N
N
N
Cl
N
N
N
14f
14g
14h
>10
>10
>10
—
—
—
O
2,4-diF
2-Br-4-F
NH
a
Average of at least three independent determinations.
An 18-membered library of analogs 13 was then prepared by treat-
ment with NaH and 18 diverse benzyl halides.
a
Average of at least three independent determinations.
As shown in Table 1, SAR, as with many allosteric ligands, was
shallow affording only five active compounds from the eighteen
synthesized. Upon resynthesis, HTS hit 7 (a 2-Cl congener) showed
an EC50 of 9.7 lM with 83% ACh Max; however, the CRC once again
did not plateau. The other four actives, all with substituents in the
tive (c Log P = 3.1) to 13g. Additional steric bulk on the pyrazole, as
in the sec-butyl derivative 14c, led to an inactive analog. Phenyl
derivative 14d and basic amino pyridine analogs 14e–14h, were
3-position, did afford sigmoidal CRCs with up to 96% ACh Max and
inactive (M1 EC50s >10 lM).
EC50s in the 3.8–6.5
lM range. The most potent analog was 13g, a
To further the development of this novel series of M1 PAMs we
focused on three of the more potent analogs (13a, 13g and 14b)
and applied a fine-tuning process of introducing fluorine atoms
at various locations to provide analogs 15 (Table 3), an approach
we found successful for multiple allosteric ligands. Across the ser-
ies, substitution at the 4-position was uniformly not tolerated
(15a–c), consistent with the SAR appearing in Table 1. Bis-fluorina-
tion of the indole ring eroded activity in the context of the bromine
analog 15d but conversely augmented the activity of the pyrazole
congener 15e. This type of subtle/confounding SAR was similarly
observed with respect to fluorination at the R2 position in analogs
15f–h. While the presence of a fluorine at R2 was neutral or slightly
beneficial in the context of the chlorine analog (15f), its presence
resulted in clearly decreased activity for both the bromine and pyr-
azole analogs, 15g and 15h. Lastly, the introduction of a single fluo-
rine at the R6 position could either be moderately detrimental in
the context of pyrazole 15i or decidedly beneficial with respect
to bromine analog 15j, where an almost 3-fold improvement in
potency was observed. In this manner, both VU0405652 (15j)
and the related difluorindole analog VU0405645 (15e) were chosen
for further evaluation.
3-Br derivative (EC50 = 3.8
l
M, 91% ACh Max) which provided a
significant increase in potency, but at the cost of physiochemical
properties (c Log P >4 and poor solubility). Replacement of the ben-
zyl moiety with a pyridyl analog also led to an inactive compound
(data not shown). However, this first generation library indicated
that substitution at the 3-position of the benzyl moiety was pre-
ferred. This result then prompted us to employ 13g as a starting
material for a small Suzuki coupling library (Scheme 2) to replace
the lipohilic bromide with various aryl and heteroaryl moieties to
introduce basicity and/or polarity.
As shown in Table 2, SAR was again shallow, but we identified
pyrazole as a preferred heterocyclic replacement for the bromide.
N-Me pyrazole 14a possessed an M1 EC50 of 3.2
Max), while the unsubstituted pyrazole congener 14b was essen-
tially equipotent (M1 EC50 = 2.2 M, 90% ACh Max). Moreover, both
pyrazole congeners lowered c Log P a full order of magnitude rela-
lM (102% ACh
l
O
O
N
O
N
O
O O
S
O
O
Me
Me
S
N
H
N
H
a
N
N
Both 15e and 15j were selective (Fig. 4A) for M1 (>30 lM vs.
M2–M5) and both compounds demonstrated impressive left-ward
shifts of the ACh CRC (98-fold and 49-fold, respectively) in fold-
shift assays at 30 lM (Fig. 4B), values comparable to BQCA. How-
ever, a combination of calculated properties (TPSA, Hacc, etc. . .),
ancillary pharmacology and in vivo PK suggested 15j was the more
optimal MLPCN probe molecule compared to 15e.
Br
13g
Ar(Het)
14
Scheme 2. Reagents and conditions: (a) Ar-B(OH)2 or Het-B(OH)2, 10 mol % Pd(t-
Bu)2, 1.0 M aq Cs2CO3, THF, mw, 120 °C (65–90%).

2700
P. R. Reid et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2697–2701
Table 3
Structures and activities of analogs 15
R1
O
O
N
O
R1
O
Me
S
N
H
N
R2
N
NH
for G, A =
G
R6
R4
15
R4
R1
R2
R6
M1EC50 (lM)
%Ach Maxa
a
Compound
G
15a
15b
15c
15d
15e
15f
15g
15h
15i
A
H
H
H
F
H
H
H
H
H
F
F
F
H
H
F
F
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
>10
>10
>10
5.37
1.85
5.10
—
—
—
103
102
56
—
103
96
84
Br
Cl
Br
A
Cl
Br
A
F
H
H
H
H
H
>10
Figure 5. M1 PAM VU04505652 (ML169) potentiates the CCh-mediated non-
amyloidogenic APPs release in TREx293-hM1 cells.
4.40
3.07
1.38
a
A
Br
15j
F
importantly, 15j was selective versus the biogenic amines (D2,
H-HT2B, etc. . .) and displayed no orthosteric binding at M1–M5.
Based on the exciting in vitro profile, we then evaluated 15j for
brain penetration in rat. A 10 mg/kg IP dose of 15j afforded a brai-
a
Average of at least three independent determinations.
nAUC/plasmaAUC of 0.32 at 1 h, providing an improvement over both
3 (ML137) and BQCA with brainAUC/plasmaAUC of ꢀ0.1. Based on
this profile, 15j (VU0405652) was declared an MLPCN probe,
ML169.
As we have previously demonstrated with both an M1 PAM
(BQCA)20 and M1 allosteric agonists (2, ML071 and TBPB),23
ML169 also shifted APP processing towards a non-amyloidogenic
pathway.24 As shown in Figure 5 10
l
M carbachol (CCh) affords a
significant increase in soluble APP (APPs ), while 100 nM CCh pro-
vides a modest increase. VU0405652 (ML169) at a dose of 2 M has
no effect, but in combination with low dose CCh (100 nM), ML169
potentiates the CCh-mediated non-amyloidogenic APPs release to
the same degree as 10 M CCh. These data once again suggest that
a
l
a
l
selective activation of M1 may have a disease modifying role in Alz-
heimer’s disease.14,20
In summary, we have developed a potent, selective and brain
penetrant M1 PAM, ML169, based on a novel indole scaffold from
an MLPCN functional HTS. Further in vivo evaluation of this probe
is underway and results from preclinical models of Alzheimer’s dis-
ease and schizophrenia will be reported in due course. ML169 is an
MLPCN probe and freely available upon request.
Acknowledgments
The authors thank the MLPCN (1U54 MH084659), NIMH (1RO1
MH082867), NIH and the Alzheimer’s Association (IIRG-07-57131)
for support of our Program in the development of subtype selective
allosteric ligands of mAChRs.
References and notes
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8. Bridges, T. M.; LeBois, E. P.; Hopkins, C. R.; Wood, M. R.; Jones, J. K.; Conn, P. J.;
Lindsley, C. W. Drug News Perspect. 2010, 23, 229.
Figure 4. (A) CRC for 15j, VU0405652 at M1–M5 (15e, VU0405645 looked similar);
(B) ACh fold-shift experiment at 30
(98-fold).
lM for VU040652 (49-fold) and VU0405645
In terms of ancillary pharmacology, the Lead Profile screen at
Ricerca, evaluating 68 GPCRs, ion channels and transporters in
radioligand binding assays, resulted in only two significant activi-
9. Lewis, J. A.; Lebois, E. P.; Lindsley, C. W. Curr. Opin. Chem. Biol. 2008, 12, 269.
10. Digby, G. J.; Conn, P. J.; Lindsley, C. W. Curr. Opin. Drug Disc. Dev. 2010, 13, 577.
ties (DAT, 83% at 10 lM and sodium channel site 2, 83% at 10 lM);

P. R. Reid et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2697–2701
2701
11. Marlo, J. E.; Niswender, C. M.; Luo, Q.; Brady, A. E.; Shirey, J. K.; Rodriguez, A. L.;
Bridges, T. M.; Williams, R.; Days, E.; Nalywajko, N. T.; Austin, C.; Williams, M.;
Xiang, Y.; Orton, D.; Brown, H. A.; Kim, K.; Lindsley, C. W.; Weaver, C. D.; Conn,
P. J. Mol. Pharm. 2009, 75(3), 577.
12. For information on the MLPCN and information on how to request probe
compounds, such as ML169, see: http://mli.nih.gov/mli/mlpcn/.
13. Sheffler, D. J.; Williams, R.; Bridges, T. M.; Lewis, L. M.; Xiang, Z.; Zheng, F.;
Kane, A. S.; Byum, N. E.; Jadhav, S.; Mock, M. M.; Zheng, F.; Lewis, L. M.; Jones, C.
K.; Niswender, C. M.; Weaver, C. D.; Conn, P. J.; Lindsley, C. W.; Conn, P. J. Mol.
Pharmacol. 2009, 76, 356.
removed in vacuo, the aqueous layer neutralized with 1.2 N HCl and extracted
with CH2Cl2. The organic layer was dried over magnesium sulfate and removed
in vacuo to produce an oily residue. Upon diluting the residue in
dichloromethane a reddish-brown solid formed which was filtered and dried
to yield compound 9 (2.00 g, 9.65 mmol, 38% yield over two steps, LCMS >98%).
Compound 10 (650 mg, 3.14 mmol), 3-amino-5-methyl-isoxazole (616 mg, 6.28
mmol), PyClU (2.00 g, 6.28 mmol), and DIEA (1.36 mL, 7.85 mmol) were added
to dichloroethane (25 mL) and microwave irradiated at 110 °C for 20 min. After
cooling, the solvent was removed in vacuo and the remaining residue purified on
a
silica gel column (0–70% ethyl acetate:hexanes over 33 min) to yield
14. 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.
15. Bridges, T. M.; Kennedy, J. P.; Cho, H. P.; Conn, P. J.; Lindsley, C. W. Bioorg. Med.
Chem. Lett. 2010, 20, 1972.
16. Bridges, T. M.; Marlo, J. E.; Niswender, C. M.; Jones, J. K.; Jadhav, S. B.; Gentry, P.
R.; Weaver, C. D.; Conn, P. J.; Lindsley, C. W. J. Med. Chem. 2009, 52, 3445.
17. Bridges, T. M.; Kennedy, J. P.; Cho, H. P.; Conn, P. J.; Lindsley, C. W. Bioorg. Med.
Chem. Lett. 2010, 20, 558.
18. Bridges, T. M.; Kennedy, J. P.; Hopkins, C. R.; Conn, P. J.; Lindsley, C. W. Bioorg.
Med. Chem. Lett. 2010, 20, 5617.
19. Ma, L.; Seager, M.; Wittman, M.; Bickel, N.; Burno, M.; Jones, K.; Graufelds, V.
K.; Xu, G.; Pearson, M.; McCampbell, A.; Gaspar, R.; Shughrue, P.; Danzinger, A.;
Regan, C.; Garson, S.; Doran, S.; Kreatsoulas, C.; Veng, L.; Lindsley, C. W.; Shipe,
W.; Kuduk, S.; Jacobson, M.; Sur, C.; Kinney, G.; Seabrook, G. R.; Ray, W. J. Proc.
Natl. Acad Sci. U.S.A. 2009, 106, 15950.
20. Shirey, J. K.; Brady, A. E.; Jones, P. J.; Davis, A. A.; Bridges, T. M.; Jadhav, S. B.;
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compound 11 (642 mg, 2.23 mmol, 71% yield, LCMS >98%). Compound 11
(502 mg, 1.77 mmol) was dissolved in 25 mL (9:1, methanol:water) and Oxone
(10.0 g, 17.7 mmol) was added. Stirring at ambient temperature continued
overnight. Water (20 mL) was added and the mixture extracted with ethyl
acetate (3 ꢁ 20 mL). The organics were combined, dried over magnesium
sulfate, and concentrated in vacuo to give an oily residue which was purified on
silica gel (0–50% ethyl acetate:hexanes over 19 min) to yield compound 12
(500 mg, 1.57 mmol, 88% yield, LCMS >98%). In
a 5 mL microwave vial,
compound 12 (55.0 mg, 0.174 mmol) was dissolved in DMF (3 mL) and cooled
to 0 °C. Sodium hydride (60% by weight, 14.0 mg, 0.348 mmol) was then added
in one portion and the reaction mixture vigorously stirred at 0 °C for 15 min. 4-
Bromo-2-bromomethyl-1-flouro-benzene (51.0 mg, 0.191 mmol) was added in
one portion and the reaction mixture was stirred while being allowed to warm
to ambient temperature over 3 h. The reaction mixture was quenched with
water (2 mL) and the solution was extracted with ethyl acetate (3 ꢁ 4 mL). The
combined organics were dried over magnesium sulfate, concentrated in vacuo to
give an oily residue which was purified on silica gel (0–70% ethyl
acetate:hexanes over 19 min) to yield ML169 (45 mg, 0.088 mmol, 51% yield).
LCMS >98% 214 nm, tR = 1.34 min, m/z = 506 ([79Br]m+1), 508 ([81Br]m+1). 1H
NMR (400 MHz, DMSO-d6) 11.27 (s, 1H), 8.23 (s, 1H), 7.82 (d, J = 8.0 Hz, 1H), 7.63
(d, J = 8.0 Hz, 1H), 7.56–7.58 (m, 1H), 7.47 (dd, J = 2.4 Hz, 6.4 Hz, 1H), 7.35–7.23
(m, 3H), 6.54 (s, 1H), 5.62 (s, 2H), 4.43 (s, 2H), 2.37 (s, 3H), HRMS found:
506.0184; calculated for C21H17BrFN3O4S: 506.0.
21. Yang, F. V.; Shipe, W. D.; Bunda, J. L.; Nolt, M. B.; Wisnoski, D. D.; Zhao, Z.;
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Hartman, G. D.; Lindsley, C. W. Bioorg. Med. Chem. Lett. 2010, 20, 531.
22. ML169,
2-((1-(5-bromo-2-fluorobenzyl)-1H-indol-3-yl)sulfonyl)-N-(5-
23. Jones, C. K.; Brady, A. E.; Davis, A. A.; Xiang, Z.; Bubser, M.; Tantawy, M. N.;
Kane, A.; Bridges, T. M.; Kennedy, J. P.; Bradley, S. R.; Peterson, T.; Baldwin, R.
M.; Kessler, R.; Deutch, A.; Lah, J. L.; Levey, A. I.; Lindsley, C. W.; Conn, P. J. J.
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methylisoxazol-3-yl)acetamide. To a solution of indole (3.00 g, 25.6 mmol)
and methyl thioglycolate 8 (2.40 mL, 25.6 mmol) in methanol:water (80 mL:
20 mL) was added iodine (6.50 g, 25.6 mmol) and potassium iodide (4.25 g,
25.6 mmol). The reaction mixture was stirred at ambient temperature for 60 h.
Methanol was removed in vacuo and the aqueous layer diluted with a saturated
solution of sodium bicarbonate and extracted with ethyl acetate. The organic
layer was dried over magnesium sulfate, evaporated in vacuo and the resulting
residue was purified on a silica gel column (0–100% ethyl acetate:hexanes over
33 min) to afford the ester as an oil (LCMS >98%). The ester was dissolved in a
mixture of tetrahydrofuran (20 mL) and 2.0 M aqueous LiOH (15 mL), then
stirred vigorously at ambient temperature for 30 min. Tetrahydrofuran was
24. APP processing. In order to test the effect of M1 PAM on M1-stimulated APPs
a
release, a human M1 overexpressing stable cell line was generated in TREx293
cells (Invitrogen). Cells were plated at 0.3 ꢁ 106 cells in 6-well plate 2 days
prior to experiments. Cells were pretreated with
2 lM VU0405652 or
dimethylsulfoxide (DMSO) for 15 min. Immediately, 100 nM or 10
lM
carbachol was added, and the medium was then conditioned for 1 h at 37 °C.
Western blot analysis of the endogenous APPs
performed as described.23
a in conditioned media was