Discovery of naphthyl-fused 5-membered lactams as a new class of M 1 positive allosteric modulators

Article abstract of DOI:10.1021/ml500055h

Selective activation of the M₁ muscarinic receptor via positive allosteric modulation represents an original approach to treat the cognitive decline in patients with Alzheimer's disease. A series of naphthyl-fused 5-membered lactams were identified as a new class of M₁ positive allosteric modulators and were found to possess good potency and in vivo efficacy.

Full text of DOI:10.1021/ml500055h

Letter
pubs.acs.org/acsmedchemlett
Discovery of Naphthyl-Fused 5‑Membered Lactams as a New Class of
M1 Positive Allosteric Modulators
Zhi-Qiang Yang,†,* Youheng Shu,† Lei Ma,‡ Marion Wittmann,‡ William J. Ray,‡ Matthew A. Seager,‡
Kenneth A. Koeplinger,§ Charles D. Thompson,§ George D. Hartman,† Mark T. Bilodeau,†
and Scott D. Kuduk†
‡
§
†Departments of Medicinal Chemistry, Alzheimer’s Research, and Drug Metabolism, Merck Research Laboratories, Sumneytown
Pike, West Point, Pennsylvania 19486, United States
S
* Supporting Information
ABSTRACT: Selective activation of the M1 muscarinic receptor via positive allosteric modulation
represents an original approach to treat the cognitive decline in patients with Alzheimer’s disease. A series
of naphthyl-fused 5-membered lactams were identified as a new class of M1 positive allosteric modulators
and were found to possess good potency and in vivo efficacy.
KEYWORDS: M1, muscarinic, positive allosteric modulators, Alzheimer’s disease, acetylcholine
lzheimer’s disease (AD) is the most common neuro-
degenerative disease representing one of the largest unmet
glycoprotein (P-gp), which serves as the major efflux
transporter of xenobiotics at the blood−brain barrier.20 Amides
were identified that dialed out the P-gp efflux and led to M1
PAMs with good brain penetration and free drug concen-
tration.19 However, it was hoped that the 1S,2S-2-hydroxy
cyclohexyl group present in 3, which provided excellent
potency, could be utilized in a scaffold distinct from the
quinolizidinone amide. Herein we report the discovery of a
novel class of M1 allosteric modulators derived from a naphthyl
lactam core (4) that maintain the high potency without being
substrates for P-gp.
The preparation of the analogues of naphthyl lactam 4 is
shown in Scheme 1. Starting from 1-hydroxy-2-naphthoic acid
methyl ester 5, triflate (6) formation is followed by Stille cross-
coupling to provide 1-methyl derivative 7. Selective bromina-
tion at the C-4 position with bromine provided compound 8. A
second bromination at the C-1 methyl group with NBS
provided dibromo intermediate 9. Condensation with amines
afforded lactam 10. The bromo group at C-4 was then
transformed to an aldehyde via the standard two-step protocol:
vinylation via Suzuki followed by ozonolysis. To complete the
synthesis, reductive amination with previously reported
piperidine 1217 led to final product 4.
A
medical needs in human heath today. One of the hallmarks of
AD is the progressive degeneration of cholinergic neurons in
the basal forebrain leading to cognitive decline.1 Acetylcholine,
the key neurotransmitter in cholinergic neurons, targets both
nicotinic and muscarinic receptors. Muscarinic receptors are G-
protein coupled receptors (GPCRs) widely expressed in the
central nervous system (CNS). There are five subtypes in the
muscarinic family, designed M1−M5,2,3 of which M1 receptor is
the most abundantly expressed in the hippocampus, cortex,4
and striatum, suggesting a prominent role in memory and
cognition.
As a result, significant interest has been placed on developing
selective M1 agonists in order to minimize adverse gastro-
intestinal events associated with activation of the other
muscarinic subtypes.5 One approach to achieve selectivity is
to target allosteric sites on M1 that are less conserved than the
orthosteric site.6−8 To this end, we have previously reported
several highly promising selective allosteric positive modulators
of M1, derived from the original HTS lead, quinolone
carboxylic acid 1 (BQCA).9−14 Efforts to improve the potency
and brain penetration led to structural modification in the form
of a quinolizidinone ring system, such as 2 (PQCA).15−17 More
recent efforts focused on identification of replacements for the
carboxylic acid to enhance CNS exposure and avoid clearance
of the parent via the glucuronidation pathway.18 Accordingly,
quinolizidinone carboxamide 3 was identified and provided
significant improvement with respect to potency while
maintaining excellent selectivity over other muscarinic
receptors.19 However, compound 3 was found to be a substrate
for the multidrug resistant (MDR) efflux transporter P-
Compound potencies were determined in the presence of an
EC20 concentration of acetylcholine at human M1 expressing
CHO cells using calcium mobilization readout on a FLIPR384
fluorometric imaging plate reader. A number of analogues with
Received: February 7, 2014
Accepted: February 20, 2014
Published: February 20, 2014
© 2014 American Chemical Society
604
dx.doi.org/10.1021/ml500055h | ACS Med. Chem. Lett. 2014, 5, 604−608

ACS Medicinal Chemistry Letters
Letter
Table 1. SAR on N-Substitution of Lactams
a
Values represent the numerical average of at least two experiments.
Interassay variability was 30% (IP, nM), unless otherwise noted.
potency with an M1 IP value of 290 nM, approximately 5-fold
less potent than 4d. Similar potency was observed with 4-
tetrahydropyran lactam 4b (M1 IP = 280 nM). However, the
corresponding 3-pyran (4c) was ∼5-fold weaker. It is worth
noting that this SAR pattern at the R1 position was consistent
with previously reported quinolizidinone amides.19 Moreover, it
is important to recognize that this new naphthyl fused lactam
still provides potent M1 PAMs such that cyclization of the
amide in 3 into a lactam is tolerated and that the
quinolizidinone can indeed be replaced by the naphthalene
ring system. This shows that the carbonyl moiety present in
previous quinolone and quinolizidinone M1 PAMs is not
required for activity. As a novel class of M1 PAM, selected
lactams were profiled in functional assays at other muscarinic
subtypes and showed no activity at M2, M3, or M4 receptors,
indicating that lactams maintain selectivity for M1.
Figure 1. Evolution of M1 PAM.
a
Scheme 1
Having identified the naphthyl lactam as a potent new
structural class, it was important to verify if it showed an
advantage over amide 3 with respect to reduced P-gp efflux.
Plasma protein binding was also determined using the
equilibrium dialysis method in the presence of rat and human
serum. The most potent lactam 4d did indeed display
significantly reduced efflux and was a borderline substrate for
human and rat P-gp with efflux ratios (ERs) of 3.2 and 4.3,
respectively (Table 2). This is a significant improvement over
the analogous quinolizidinone amide 3, with efflux ratios (ERs)
of 11 and 21, respectively (Figure 1). Compound 4d also
displayed excellent passive-permeability and good unbound
fraction in plasma (5% in rat and 8% in human).
In order to further identify non-P-gp substrates, an SAR
campaign was initiated on the amine motif. When the 2-pyridyl
at R3 was replaced with other pyridines (4g and 4h) or less
basic diazines (4i−k), the M1 potency was maintained but the
P-gp ERs increased. Next, a methyl group was placed at key
positions on the pyridine ring. The ortho-methyl analogue 4l
was found not to be a human P-gp substrate, while the rat P-gp
ER was also improved. The para-methyl compound 4m also
showed similar advance on human P-gp but not on rat.
a
Reagents and conditions: (a) Tf2O, Py. −5 °C; (b) SnMe4, LiCl,
Pd(PPh3)2Cl2, DMF, 110 °C; (c) Br2, HOAc, 90 °C; (d) NBS,
(BzO)2, CCl4, 90 °C; (e) R1NH2, THF; (f) vinylBF3K, Pd2(dba)3,
PCy3, K3PO4, dioxane, 140 °C; (g) O3, CH2Cl2, −78 °C, then resin-
bound PPh3; (h) NaBH(OAc)3, CH2Cl2.
varied substituents on the lactam nitrogen were synthesized as
described in Scheme 1. Representative examples are shown in
Table 1. Not surprisingly, compound 4d bearing the
aforementioned 1S,2S-2-hydroxy cyclohexyl group off the
lactam is the most potent compound among various R1 groups
explored and displayed an M1 IP value of 15 nM. This specific
stereochemistry was critical for potency as the associated
1R,2R-trans isomer 4e and cis isomer 4f were both significantly
less active. ortho-Fluoro phenyl lactam 4a gave only moderate
605
dx.doi.org/10.1021/ml500055h | ACS Med. Chem. Lett. 2014, 5, 604−608

ACS Medicinal Chemistry Letters
Letter
Table 2. M1 FLIPR, Protein Binding, and Pgp Data for Selected Compounds
a
b
Values represent the numerical average of at least two experiments. Interassay variability was 30% (IP, nM), unless otherwise noted. Passive
c
permeability (10−6 cm/s). MDR1 directional transport ratio (B to A)/(A to B). Values represent the average of three experiments, and interassay
variability was 20%.
Although both methyl analogues showed similar potency and
reduced P-gp efflux compared to 4d, they were considerably
more lipophilic and showed very high plasma protein binding at
∼99%. When the 2-pyridine was replaced with a phenyl group,
compound 4n showed reduced P-gp efflux as expected, but at
the expense of a ∼5-fold loss of potency. The phenyl
606
dx.doi.org/10.1021/ml500055h | ACS Med. Chem. Lett. 2014, 5, 604−608

ACS Medicinal Chemistry Letters
Letter
Table 3. Brain and Plasma Distribution in Rats for Selected Compounds
a
Sprague−Dawley rats, concentration at 2 h postdose. Oral dose 10 mg/kg in 0.5% methocel. Interanimal variability was less than 20% for all values.
Determined using rat plasma protein binding from Table 2.
b
replacement also resulted in higher plasma protein binding for
4n. A similar SAR trend was observed when exchanging CN
group with F: compound 4o showed a loss of ∼4-fold of M1
potency but did have reduced P-gp efflux. In addition, the
methyl group could be introduced onto benzylic position of
lactam ring (R2). In this case, racemic product 4p maintained
potency and showed improved human P-gp efflux. Compound
4p also retained a reasonable unbound free fraction despite the
addition of the methyl group but was more lipophilic with a
logP of 3.3.
On the basis of the potency, P-gp profile, and free fraction
properties, compounds 4d, 4m, and 4p were selected as
candidates to determine the CNS exposure in rats (Table 3).
Plasma and cerebrospinal fluid (CSF) levels were measured
after 2 h following a 10 mg/kg oral dose. Compound 4d gave
significant plasma (5.2 uM) and CSF (134 nM) levels with a
CSF/Uplasma ratio of 0.51. Although compound 4p provided a
similar CSF/Uplasma ratio (0.52) compared to 4d, the absolute
CSF level (8.6 nM) was significantly lower. The other methyl
analogue 4m gave a moderately reduced CSF/Uplasma ratio
(0.3) as well as low absolute CSF level (4.7 nM). The high CSF
level of 4d was believed to be driven by higher unbound drug
concentration than the corresponding methyl analogues 4m
and 4p. Consistent with their increased protein binding, methyl
analogues 4m and 4p have higher measured logP values than
4d. All three compounds showed excellent solubility at pH 2
(existed as salt form) but poor solubility at pH 7 (neutral
form).
On the basis of the robust CSF levels of compound 4d,
further studies were performed to investigate the properties of
this new class of lactam-derived M1 modulators. Fold
potentiation with a fixed concentration of modulator 4d was
evaluated on the M1 dose response with acetylcholine as the
agonist. As shown in Figure 2, with increasing concentration of
compound 4d, a left shift was observed up to ∼40-fold at 1.8
μM in the acetylcholine dose−response curve, indicating that
4d is a potent positive allosteric modulator of human M1
receptor. It worth noting that PAM 4d displayed dose-
dependent partial agonism as indicated by an upward shift of
acetylcholine dose−response curves at the two highest
concentrations tested.
Figure 2. Fold potentiation of 4d. Mean values from four replicate
wells are plotted; data are representative of 12 independent
experiments.
Figure 3. Evaluation of lactam 4d in the mouse CFC model. Data are
representative of four experiments. *, Different from vehicle; #,
different from scopolamine + vehicle (P < 0.05, Dunnet test).
deficit at 1 mpk relative to mice treated with scopolamine
alone. The corresponding plasma level at 1 mpk was 1.5 μM. By
way of comparison, the previous lead, carboxylic acid 2 showed
significant reversal at ∼1 μM plasma levels. The result
demonstrated robust proof-of-action for the new series despite
the fact that lactam 4d is a rodent P-gp substrate.
Pharmacokinetics of lead compound 4d was also evaluated in
rat and dog (Table 4). Low clearance was observed in these two
species, further highlighting the potential to provide potent M1
PAMs with potential for good human pharmacokinetic
properties.
To evaluate the in vivo efficacy, PAM 4d was tested in a
mouse contextual fear conditioning (CFC) assay, which serves
as a model of episodic memory (Figure 3). In this study, mice
were treated with scopolamine, a nonselective muscarinic
antagonist, prior to exposure to a novel environment to impair
a new association. Mice dosed by intrapertitoneal injection with
4d exhibited a significant reversal of scopolamine-induced
Table 4. Pharmacokinetics of 4d in Rat and Dog
dose (mg/kg) route Cl (min/ml/kg) Vdss (L/kg) T1/2 (h)
rat
2
iv
iv
4.1
2.1
2.8
1.2
12
dog
0.125
6.7
607
dx.doi.org/10.1021/ml500055h | ACS Med. Chem. Lett. 2014, 5, 604−608

ACS Medicinal Chemistry Letters
Letter
(10) Yang, F. V.; Shipe, W. D.; Bunda, J. L.; Wisnoski, D. D.; Zhao,
Z.; Lindsley, C. W.; Ray, W. J.; Ma, L.; Wittmann, M.; Seager, M. W.;
Koeplinger, K.; Thompson, C. D.; Hartman, G. D. Parallel synthesis of
N-biaryl quinolone carboxylic acids as selective M1 positive allosteric
modulators. Bioorg. Med. Chem. Lett. 2010, 19, 531−535.
(11) Kuduk, S. D.; Di Marco, C. N.; Cofre, V.; Pitts, D. R.; Ray, W. J.;
Ma, L.; Wittmann, M.; Seager, M.; Koeplinger, K. A.; Thompson, C.
D.; Hartman, G. D.; Bilodeau, M. T. Pyridine containing M1 positive
allosteric modulators with reduced plasma protein binding. Bioorg.
Med. Chem. Lett. 2010, 19, 657−660.
(12) Kuduk, S. D.; Di Marco, C. N.; Cofre, V.; Pitts, D. R.; Ray, W. J.;
Ma, L.; Wittmann, M.; Seager, M.; Koeplinger, K. A.; Thompson, C.
D.; Hartman, G. D.; Bilodeau, M. T. N-Heterocyclic Derived M1
Positive Allosteric Modulators. Bioorg. Med. Chem. Lett. 2010, 19,
1334−1337.
(13) Kuduk, S. D.; Di Marco, C. N.; Chang, R. K.; Ray, W. J.; Ma, L.;
Wittmann, M.; Seager, M.; Koeplinger, K. A.; Thompson, C. D.;
Hartman, G. D.; Bilodeau, M. T. Heterocyclic fused pyridone
carboxylic acid M1 positive allosteric modulators. Bioorg. Med. Chem.
Lett. 2010, 19, 2533−2537.
In summary, naphthyl-fused 5-membered lactams have
emerged as a new class of M1 positive allosteric modulators.
This naphthyl fused lactam is novel not only because it shows
that cyclization of the amide in 3 into a lactam is tolerated but
that the quinolizidinone can be replaced by the naphthalene
meaning that the carbonyl moiety present in previous
quinolone and quinolizidinone M1 PAMs is not required for
activity. The trans-1S,2S-2-hydroxy cyclohexyl group was found
to be the most potent group off the amide position, and
significant attenuation of P-gp efflux could be garnered.
Compound 4d demonstrated high CSF drug levels and good
efficacy in a mouse contextual fear model of episodic memory
despite being a rodent P-gp substrate. Further SAR study of
these lactams with respect to improving solubility at neutral pH
and reducing P-gp efflux is expected to provide optimized M1
PAMs and will be reported in due course.
ASSOCIATED CONTENT
* Supporting Information
■
(14) Kuduk, S. D.; DiPardo, R. M.; Beshore, D. C.; Ray, W. J.; Ma,
L.; Wittmann, M.; Seager, M.; Koeplinger, K. A.; Thompson, C. D.;
Hartman, G. D.; Bilodeau, M. T. Hydroxy cycloalkyl fused pyridone
carboxylic acid M1 positive allosteric modulators. Bioorg. Med. Chem.
Lett. 2010, 19, 2538−2541.
S
Representative assay and experimental procedures and data for
test compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
(15) Kuduk, S. D.; Chang, R. K.; Di Marco, C. N.; Ray, W. J.; Ma, L.;
Wittmann, M.; Seager, M.; Koeplinger, K. A.; Thompson, C. D.;
Hartman, G. D.; Bilodeau, M. T. Identification of quinolizidinone
carboxylic acids as CNS penetrant, selective M1 allosteric muscarinic
receptor modulators. ACS Med. Chem. Lett. 2010, 1, 263−267.
(16) Kuduk, S. D.; Chang, R. K.; Di Marco, C. N.; Ray, W. J.; Ma, L.;
Wittmann, M.; Seager, M.; Koeplinger, K. A.; Thompson, C. D.;
Hartman, G. D.; Bilodeau, M. T. Quinolizidinone carboxylic acid
selective M1 allosteric modulators: SAR in the piperidine series. Bioorg.
Med. Chem. Lett. 2011, 21, 1710−1713.
(17) Kuduk, S. D.; Chang, R. K.; Di Marco, C. N.; Pitts, D. R.;
Greshock, T. J.; Ma, L.; Wittmann, M.; Seager, M.; Koeplinger, K. A.;
Thompson, C. D.; Hartman, G. D.; Bilodeau, M. T.; Ray, W. J.
Discovery of a selective allosteric M1 receptor modulator with suitable
development properties based on a quinolizidinone carboxylic acid
scaffold. J. Med. Chem. 2011, 54, 4773−4780.
AUTHOR INFORMATION
Corresponding Author
*(Z.-Q.Y.) Tel: 732-594-5765. Fax: 732-594-9490. E-mail:
zhiqiang_yang@merck.com.
■
Notes
The authors declare no competing financial interest.
REFERENCES
■
(1) Geula, C. Abnormalities of neural circuitry in Alzheimer’s disease:
hippocampus and cortical innervation. Neurology 1998, 51, 518−529.
(2) Bonner, T. I. The molecular basis of muscarinic acetylcholine
receptor diversity. Trends Neurosci. 1989, 12, 148−151.
(3) Bonner, T. I. Subtypes of muscarinic acetylcholine receptors.
Trends Pharmacol. Sci. 1989, 11−15.
(18) For examples of noncarboxylic positive allosteric M1
modulators, see Shirey, J. K.; Brady, A. E.; Jones, P. J.; Davis, A. A.;
Bridges, T. M.; Kennedy, J. P.; Jadhav, S. B.; Menon, U. N.; Xiang, Z.;
Watson, M. L.; Christian, E. P.; Doherty, J. J.; Quirk, M. C.; Snyder, D.
H.; Lah, J. J.; Nicolle, M. M.; Lindsley, C. W.; Conn, P. J. A selective
allosteric potentiator of the M1 muscarinic acetylcholine receptor
increases activity of medial prefrontal cortical neurons and restores
impairments in reversal learning. J. Neurosci. 2009, 45, 14271−14286.
(19) Kuduk, S. D.; Chang, R. K.; Greshock, T. J.; Ray, W. J.; Ma, L.;
Wittmann, M.; Seager, M.; Koeplinger, K. A.; Thompson, C. D.;
Hartman, G. D.; Bilodeau, M. T. Identification of amides as carboxylic
acid surrogates for quinolizilinone-based M1 positive allosteric
modulators. ACS Med. Chem. Lett. 2012, 3, 1070−1074.
(4) Levey, A. I. Muscarinic acetylcholine receptor expression in
memory circuits: Implications for treatment of Alzheimer disease. Proc.
Natl. Acad. Sci. 1996, 93, 13451−13546.
(5) Conn, P. J.; Christopulos, A.; Lindsley, C. W. Allosteric
modulators of GPCRs: a novel approach for the treatment of CNS
disorders. Nat. Rev. Drug Discovery 2009, 8, 41−54.
(6) For an example of an allosteric activator of the M1 receptor, see
Jones, C. K.; Brady, A. E.; Davis, A. A.; Xiang, Z.; Bubser, M.; Noor-
Wantawy, M.; 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. Novel
selective allosteric activator of the M1 muscarinic acetylcholine
receptor regulates amyloid processing and produces antipsychotic-
like activity in rats. J. Neurosci. 2008, 41, 10422−10433.
(20) Hitchcock, S. A. Structural modifications that alter the P-
glycopprotein efflux properties of compounds. J. Med. Chem. 2012, 55,
4877−4895.
(7) Kuduk, S. D.; Beshore, D. C. Novel M1 allosteric ligands: a patent
review. Exp. Opin. Ther. Pat. 2012, 22, 1385−1398.
(8) Davie, B. J.; Christopoulos, A.; Scammells, P. J. Development of
M1 mAChR allosteric and bitopic ligands: therapeutics for the
treatment of cognitive deficits. ACS Chem. Neurosci. 2013, 4, 1026−
1048.
(9) Ma, L.; Seager, M.; Wittmann, M.; Jacobsen, M.; Bickel, D.;
Burno, M.; Jones, K.; Kuzmick-Graufelds, V.; Xu, G.; Pearson, M.;
McCampbell, A.; Gaspar, R.; Shughrue, P.; Danziger, A.; Regan, C.;
Flick, R.; Garson, S.; Doran, S.; Kreatsoulas, C.; Veng, L.; Lindsley, C.;
Shipe, W.; Kuduk, S. D.; Sur, C.; Kinney, G.; Seabrook, G.; Ray, W. J.
Selective activation of the M1 muscarinic acetylcholine receptor
achieved by allosteric potentiation. Proc. Natl. Acad. Sci. U.S.A. 2009,
106, 15950−19555.
608
dx.doi.org/10.1021/ml500055h | ACS Med. Chem. Lett. 2014, 5, 604−608