Design of 4-Oxo-1-aryl-1,4-dihydroquinoline-3-carboxamides as Selective Negative Allosteric Modulators of Metabotropic Glutamate Receptor Subtype 2

Article abstract of DOI:10.1021/acs.jmedchem.5b01371

Both orthosteric and allosteric antagonists of the group II metabotropic glutamate receptors (mGlus) have been used to establish a link between mGlu_2/3 inhibition and a variety of CNS diseases and disorders. Though these tools typically have good selectivity for mGlu_2/3 versus the remaining six members of the mGlu family, compounds that are selective for only one of the individual group II mGlus have proved elusive. Herein we report on the discovery of a potent and highly selective mGlu₂ negative allosteric modulator 58 (VU6001192) from a series of 4-oxo-1-aryl-1,4-dihydroquinoline-3-carboxamides. The concept for the design of this series centered on morphing a quinoline series recently disclosed in the patent literature into a chemotype previously used for the preparation of muscarinic acetylcholine receptor subtype 1 positive allosteric modulators. Compound 58 exhibits a favorable profile and will be a useful tool for understanding the biological implications of selective inhibition of mGlu₂ in the CNS.

Full text of DOI:10.1021/acs.jmedchem.5b01371

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Article
Design of 4-oxo-1-aryl-1,4-dihydroquinoline-3-carboxamides as selective
negative allosteric modulators of metabotropic glutamate receptor subtype 2
Andrew S Felts, Alice L Rodriguez, Katrina A Smith, Julie L. Engers, Ryan D. Morrison,
Frank W. Byers, Anna L. Blobaum, Charles W. Locuson, Sichen Chang, Daryl F. Venable,
Colleen M Niswender, J. Scott Daniels, P. Jeffrey Conn, Craig W Lindsley, and Kyle A Emmitte
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01371 • Publication Date (Web): 02 Nov 2015
Downloaded from http://pubs.acs.org on November 3, 2015
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Design of 4ꢀoxoꢀ1ꢀarylꢀ1,4ꢀdihydroquinolineꢀ3ꢀ
carboxamides as selective negative allosteric
modulators of metabotropic glutamate receptor
subtype 2
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†
†
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†
†,║
Andrew S. Felts, Alice L. Rodriguez, Katrina A. Smith, Julie L. Engers, Ryan D. Morrison,
†
†
†,§
†
Frank W. Byers, Anna L. Blobaum, Charles W. Locuson, Sichen Chang, Daryl F.
†
,
┴
†
†,║
†
†,‡
Venable, Colleen M. Niswender, J. Scott Daniels, P. Jeffrey Conn, Craig W. Lindsley,
,†,‡,#
and Kyle A. Emmitte*
†
Vanderbilt Center for Neuroscience Drug Discovery, Department of Pharmacology, Vanderbilt
University Medical Center, Nashville, Tennessee 37232
‡
Department of Chemistry, Vanderbilt University, Nashville, TN 37232
KEYWORDS. Metabotropic glutamate receptor subtype 2 (mGlu ), negative allosteric modulator
2
(NAM), major depressive disorder (MDD), treatmentꢀresistant depression (TRD), Alzheimer’s
disease
ABSTRACT. Both orthosteric and allosteric antagonists of the group II metabotropic glutamate
receptors (mGlus) have been used to establish a link between mGlu inhibition and a variety of
2
/3
CNS diseases and disorders. Though these tools typically have good selectivity for mGlu2/3
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versus the remaining six members of the mGlu family, compounds that are selective for only one
of the individual group II mGlus have proved elusive. Herein we report on the discovery of a
potent and highly selective mGlu negative allosteric modulator 58 (VU6001192) from a series
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of 4ꢀoxoꢀ1ꢀarylꢀ1,4ꢀdihydroquinolineꢀ3ꢀcarboxamides. The concept for the design of this series
centered on morphing a quinoline series recently disclosed in the patent literature into a
chemotype previously used for the preparation of muscarinic acetylcholine receptor subtype 1
positive allosteric modulators. Compound 58 exhibits a favorable profile and will be a useful tool
for understanding the biological implications of selective inhibition of mGlu in the CNS.
2
INTRODUCTION
Glutamate (Lꢀglutamic acid) is the major excitatory neurotransmitter in the mammalian
central nervous system (CNS) and exerts its effects through both ionotropic and metabotropic
glutamate receptors (mGlus). The mGlus belong to family C of the Gꢀproteinꢀcoupled receptors
(GPCRs) and are characterized by a seven transmembrane (7TM) αꢀhelical domain connected
via a cysteine richꢀregion to a large biꢀlobed extracellular aminoꢀterminal domain. The
orthosteric binding site is found within this aminoꢀterminal domain for each of the eight
members of the mGlu family. The mGlus are further categorized into three groups according to
their homology, preferred signal transduction mechanisms, and pharmacology. The group I
mGlus (mGlu and mGlu ) are primarily located postꢀsynaptically in neurons and coupled via G
1
5
q
to the activation of phospholipase C, which leads to the elevation of intracellular calcium and
activation of protein kinase C (PKC). On the other hand, group II mGlus (mGlu and mGlu ) and
2
3
group III mGlus (mGlu , mGlu , mGlu and mGlu ) are primarily located preꢀsynaptically and
4
6
7
8
1ꢀ3
are coupled via G to the inhibition of adenylyl cyclase activity. The expression of the group
i/o
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II mGlus is wide throughout the CNS; moreover, both are found in brain regions associated with
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,5
emotional states such as the amygdala, hippocampus, and prefrontal cortex.
With multiple compounds having advanced into clinical trials in schizophrenic patients, the
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design of selective and drugꢀlike positive allosteric modulators (PAMs) of mGlu is significantly
2
more advanced than complementary research directed toward selective negative allosteric
6
modulators (NAMs) of the same receptor. Still, the literature contains multiple examples of
highly optimized orthosteric antagonists and NAMs of the group II mGlus. Though these
compounds typically possess good levels of selectivity against the other members of the mGlu
7
family, they lack appreciable selectivity between mGlu and mGlu . Consequently, much has
2
3
been learned regarding the potential utility of group II mGlu inhibition through the use of these
tools in animal models of various CNS disorders. The bulk of such studies have employed the
8
9
two orthosteric antagonists 1 (LY341495) and 2 (MGS0039) (Figure 1). Specifically, potential
therapeutic applications of group II mGlu antagonists have been established in obsessiveꢀ
1
0,11
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compulsive disorder (OCD),
anxiety,
cognition,
and Alzheimer’s disease.
Additionally, substantial work with these compounds has been directed toward establishing a
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,10,12,17–22
role for mGlu2/3 antagonists as novel antidepressants.
Perhaps most intriguing are
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3
studies demonstrating efficacy in animal models of treatmentꢀresistant depression (TRD) and
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anhedonia.
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Figure 1. mGlu2/3 orthosteric antagonist tools 1 and 2, mGlu2/3 NAM tools 3 and 4, Roche
mGlu2/3 NAM clinical compound 5, firstꢀgeneration selective mGlu NAMs 6 and 7, and mGlu3
3
NAM in vivo tool 8.
Reports of in vivo studies with mGlu NAMs are less prevalent; yet, two related compounds
2/3
2
5
from a series of 4ꢀarylꢀ1,3ꢀdihydroꢀ2Hꢀbenzo[b][1,4]diazepinꢀ2ꢀones, 3 (RO4491533) and 4
2
6,27
25,28
(RO4432717)
(Figure 1), are worth noting. Studies in rodent models of depression
with these tools have been disclosed and show similar results as those observed
with mGlu orthosteric antagonists. Additionally, another structurally distinct mGlu NAM, 5
and
27,29,30
cognition
2/3
2/3
3
1
(
decoglurant, RO4995819) from Roche (Figure 1), advanced into a phase II trial in patients
32
with major depressive disorder (MDD) (NCT01457677). Thus, the evidence for a therapeutic
application with mGlu2/3 antagonists is compelling; however, further elucidation is required
regarding the individual importance of mGlu and mGlu in these various disorders. As such, we
2
3
have been pursuing the design of selective antagonists of each receptor for use as in vivo tools.
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Our initial success came in the design of selective mGlu NAMs from a series of 1,2ꢀ
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diphenylethyne compounds represented by tool compounds 6 (VU0463597, ML289) and 7
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4,35
(VU0469942, ML337)
(Figure 1). More recently we reported on another mGlu NAM, 8
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(
VU0650786), that is a superior in vivo tool and has demonstrated efficacy in rodent models of
36
anxiety/OCD and depression. Having selective mGlu NAMs from multiple chemotypes in
3
hand, we sought strategies for the design of selective mGlu NAMs for the purpose of thoroughly
2
evaluating the therapeutic potential of each individual target. Our first successful execution of
such a strategy is described in this manuscript.
RESULTS AND DISCUSSION
Scaffold Design. In our search for new scaffolds suitable for the design of selective mGlu2
NAMs, we were intrigued by a set of quinolineꢀ2ꢀcarboxamide compounds 9 developed at Merck
7
,37
and disclosed in the patent literature (Figure 2).
activity presented in this application showed substantial tolerance for functional diversity at the
ꢀposition with a variety of linkers connected to a number of unsaturated and saturated ring
A survey of the functional mGlu NAM
2
7
systems (A). The 4ꢀposition demonstrated a preference for aryl and heteroaryl rings (B), and a
primary amide was preferred over a nitrile at the 2ꢀposition. We prepared an exemplar compound
3
5
1
0 and tested it in our own cellꢀbased functional assays for mGlu and mGlu . These
2 3
fluorescenceꢀbased assays measure calcium mobilization induced by receptor activation in a cell
line stably expressing either rat mGlu or rat mGlu along with the promiscuous Gꢀprotein G
α15
2
3
and are capable of detecting agonists, PAMs, and NAMs. Compound 10 exhibited potent NAM
activity at mGlu and no evidence of mGlu activity up to the highest concentration tested (30
2
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µM). The quinolineꢀ2ꢀcarboxamide mGlu NAMs were reminiscent of another series of allosteric
2
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modulators developed at Merck, the 4ꢀoxoꢀ1,4ꢀdihydroquinolineꢀ3ꢀcarboxylic acid muscarinic
3
8–40
acetylcholine receptor subtype 1 (M ) PAMs 11.
Our hypothesis was that a new mGlu2
1
NAM scaffold 12 might be obtained within a 4ꢀoxoꢀ1,4ꢀdihydroquinoline series by appending
appropriately linked groups (A) at the 6ꢀposition and installing Nꢀaryl rings (B) at the 1ꢀposition
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in the context of a primary amide at the 3ꢀposition. In addition, the extensive M PAM SAR
1
already developed in this chemotype indicated that such changes would not be favorable for that
target.
Figure 2. Merck quinolineꢀ2ꢀcarboxamide mGlu NAM scaffold 9 and representative compound
2
1
0; Merck 4ꢀoxoꢀ1,4ꢀdihydroquinolineꢀ3ꢀcarboxylic acid M PAM scaffold 11; Proposed 4ꢀoxoꢀ
1
1
ꢀarylꢀ1,4ꢀdihydroquinolineꢀ3ꢀcarboxamide mGlu NAM scaffold 12.
2
Synthesis of Compounds. It was envisioned that a number of interesting analogs could be
prepared from versatile 6ꢀbromo intermediate 15 (Scheme 1). The synthesis began with
commercially available acid 13. Treatment of 13 with two equivalents of butyl lithium followed
by addition of 5ꢀbromoꢀ2ꢀfluoroꢀbenzoyl chloride provided βꢀketoester 14. Reaction of 14 with
N,Nꢀdimethylformamide dimethyl acetal followed by a suitable aryl amine under microwave
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heating afforded the desired intermediate 15. Compound 15 could be converted to primary amide
16 through heating in ammonia in methanol under microwave irradiation to give 16. Where
possible, 16 was used as a common intermediate; however, certain transformations proved
incompatible with the primary amide functional group and necessitated the use of ester 15 with
subsequent conversion to the primary amide at a later stage. Reaction of 16 with commercially
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1
available aryl alcohols (R OH) in the presence of copper (I) iodide and dimethylglycine provided
aryl ether analogs 17–19 (Table 1). For synthesis of ethers 23–29 (Table 1), conversion of
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bromide 15 to alcohol 20 was accomplished with a palladium catalyzed hydroxylation. Acid 20
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was converted to methyl ester 21 via a Fischer esterification. A Mitsunobu coupling with
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commercial alcohols (R OH) was employed for installation of the various 6ꢀsubstituted ethers to
afford 22. Conversion of the ester moieties to the corresponding primary amides to yield 23–29
was carried out as described previously. Finally, the synthesis of amine analogs 31–42 (Table 2)
was accomplished by first reacting bromide 15 with commercially available amines in a
4
3
BuchwaldꢀHartwig amination reaction to yield 30. Conversion of 30 to analogs 31–42 was
carried out as described previously.
a
Scheme 1. Synthesis of 6ꢀheteroatom linked analogs
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a
Reagents and conditions: (a) nꢀBuLi, 2,2’ꢀbipyridyl, –30 ºC to –5 ºC, then 5ꢀbromoꢀ2ꢀfluoroꢀ
benzoyl chloride, –78 ºC to –30 ºC, 67%; (b) N,Nꢀdimethylformamide dimethyl acetal, DMF,
microwave, 120 ºC, 15 min, then ArNH , microwave, 150 ºC, 20 min, 60–98%; (c) 7N NH in
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MeOH, microwave, 150 ºC, 60 min, 29–99%; (d) R OH, CuI, Cs CO , Me NCH CO H,
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microwave, 150 ºC, 15 min, 26–56% (e) KOH, Pd (dba) , tꢀBuXphos, dioxane, H O, microwave,
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t
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50 ºC, 15 min, 99%; (f) MeOH, con. H SO , reflux, 74%; (g) R OH, PPh , D BAD, THF, 40–
2 4 3
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9
8%; (h) HNR R , Pd (dba) , Xantphos, Cs CO , PhMe, 110 ºC, 8–54%.
2
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3
In addition to the 6ꢀheteroatom linked analogs, 6ꢀcarbon linked compounds were prepared
from intermediates 15 and 16 (Scheme 2). Methyleneꢀlinked tertiary amine analogs 45–66
(Tables 3 and 4) were accessed through bromide 16, which was first converted to vinyl
44
intermediate 43 via a Suzuki coupling with potassium vinyltrifluoroborate. Dihydroxylation of
the olefin and subsequent in situ periodate cleavage of the resultant diol gave aldehyde 44.
Analogs 45–66 were then prepared through reductive aminations with 44 and commercially
2
3
available secondary amines (HNR R ). For preparation of methyleneoxy linked analogs 70–78
(Table 5), bromide 15 was converted to aldehyde 67 via an analogous vinylation,
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dihydroxylation, and periodate cleavage as described above. Sodium borohydride reduction of 67
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gave primary alcohol 68, which was reacted in a Mitsunobu coupling with commercial alcohols
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R OH) to give ether intermediate 69. Conversion of the ester moieties to the corresponding
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primary amides to yield 70–78 was carried out as described previously. Ethylene linked analogs
81–91 (Table 6 and Table 7) were also prepared from bromide 15 through initial preparation of
alkynes 79. Two methods were employed for preparation of these alkyne intermediates 79, each
4
5
relying on Sonogashira couplings with bromide 15. A coupling with 15 and a terminal alkyne
6
(R CCH) gave 79 directly. Alternatively, a coupling with trimethylsilylacetylene followed by
fluoride mediated silyl cleavage gave a 6ꢀalkyne intermediate that was coupled to an aryl
6
bromide (R Br) to afford 79. A palladium catalyzed hydrogenation of the alkyne moiety
provided 80, which was reacted with ammonia as described previously to yield the target
compounds 81–91.
a
Scheme 2. Synthesis of 6ꢀcarbon linked analogs
a
Reagents and conditions: (a) H CCHBF K, Pd(dppf)·CH Cl , NEt , nꢀpropanol, 100 ºC, 75–
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3
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100%; (b) OsO , NMO, acetone, H O, then NaIO , 91–99%; (c) HNR R , NaBH(OAc) , AcOH,
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CH Cl , 7–81%; (d) NaBH , EtOH, 0 ºC, 36–57%; (e) R OH, PPh , D BAD, THF, 14–98%; (f)
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N NH in MeOH, microwave, 150 ºC, 15 min, 10–94%; (g) R CCH, PdCl (PPh ) , CuI, NEt ,
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microwave, 150 ºC, 15 min, 83%; (i) TBAF, THF, 70%; (j) R Br, PdCl (PPh ) , CuI, NEt ,
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DMF, microwave, 150 ºC, 15 min, 21–47%; (k) 10% Pd/C, MeOH, H (1 atm), 63–99%.
2
In the case of the aforementioned 4ꢀoxoꢀ1,4ꢀdihydroquinolineꢀ3ꢀcarboxylic acid M PAM
1
scaffold, Merck has also shown that a 4Hꢀquinolizinꢀ4ꢀone functioned as an effective bioisostere
46–48
for the core of the chemotype.
As such, we decided to prepare analogs with this core to
evaluate its potential as an mGlu NAM scaffold as well (Scheme 3). The synthesis began with
2
lithiation of 5ꢀhydroxyꢀ2ꢀmethylpyridine and subsequent in situ reaction with commercially
available diethyl 2ꢀ(ethoxymethylene)malonate 92 to give 7ꢀhydroxyꢀ4ꢀoxoꢀ4Hꢀquinolizine 93.
The alcohol was protected as its methoxymethyl ether 94, which was then selectively brominated
at the 1ꢀposition to afford 95. A Suzuki coupling with 4ꢀfluorophenylboronic acid provided
intermediate 96. Acidic cleavage of the protecting group and simple filtration of the precipitated
product gave 97, which served as the key intermediate for the synthesis of analogs. Ether
4
2
compounds 99–102 (Table 8) were prepared via Mitsunobu coupling with commercial alcohols
1
(
R OH) to yield 98, which was converted to primary amides 99–102 as described above.
Intermediate 97 was also converted to the corresponding triflate 103, which was subjected to an
analogous vinylation, dihydroxylation, and periodate cleavage as described previously to afford
aldehyde 104. Finally, conversion of 104 to amines 105 and ultimately final compounds 106–108
followed methods outlined herein above.
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a
Scheme 3. Synthesis of 4Hꢀquinolizinꢀ4ꢀone analogs
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
Reagents and conditions: (a) 5ꢀhydroxyꢀ2ꢀmethylpyridine, nꢀBuLi, THF, –78 ºC to –30 ºC,
5
7%; (b) CH OCH Cl, DIEA, CH Cl , 0 ºC to r.t., 96%; (c) NBS, CHCl , 0 ºC to r.t., 96%; (d) 4ꢀ
3
2
2
2
3
fluorophenylboronic acid, Pd(dppf)·CH Cl , 1M aq. Na CO , DME, 90 ºC, 94%; (e) pTSA·H O,
2
2
2
3
2
1
t
EtOH, DCE, 80 ºC, 66%; (f) R OH, PPh , D BAD, THF, 0 ºC to 45 ºC, 38–82%; (g) 7N NH in
3
3
MeOH, microwave, 150 ºC, 2.0–3.0 h, 26–90%; (h) PhN(SO CF ) , NEt , CH Cl , 0 ºC, 96%; (i)
2
3 2
3
2
2
H CCHBF K, Pd(dppf)·CH Cl , NEt , nꢀpropanol, 90 ºC, 96%; (j) OsO , NMO, THF, H O, then
2
3
2
2
3
4
2
4
5
NaIO , 70%; (k) HNR R , NaBH(OAc) , AcOH, CH Cl , 27–67%.
4
3
2
2
mGlu NAM Activity and Preliminary DMPK SAR. As new analogs were evaluated for
2
potency in our functional mGlu assay, interesting compounds were further assessed in our
2
frontline in vitro drug metabolism and pharmacokinetics (DMPK) assays. Specifically, metabolic
stability was determined by measuring the intrinsic clearance of the compound when incubated
49
with rat liver microsomes (RLM). The intrinsic clearance obtained was used to calculate a
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8
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
predicted hepatic clearance, and compounds were binned accordingly into low (< ⅓ hepatic
blood flow), moderate (⅓ to ⅔ hepatic blood flow) and high (> ⅔ hepatic blood flow) groups.
5
0
The extent to which the compounds were bound to rat plasma was also measured. We also
calculated the lipophilicity of new analogs and attempted to assess the efficiency of the structural
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
1
modifications being tested. Much of the SAR work was conducted in the context of a 4ꢀ
fluorophenyl ring at the 1ꢀposition of the scaffold as this was a group with good potency in the
quinoline series, and it was likely to be somewhat metabolically stable. As expected, the mGlu2
NAM SAR with the new 4ꢀoxoꢀ1,4ꢀdihydroquinoline ether analogs showed a good deal of
tolerance at the 6ꢀposition (Table 1). The ethers with directly linked heteroaryl rings (17–19)
were among the least active in this set; however, in vitro DMPK was benchmarked. The fraction
unbound in rat plasma with 17 was 0.083, and the predicted hepatic clearance was moderate. The
3
remaining analogs 23–29 possessed a single sp hybridized carbon between the 6ꢀposition ether
oxygen and the heteroaryl ring (A). This feature generally improved mGlu NAM activity. The
2
methyl groups of analogs 24 and 25 both provided small boosts of potency relative to
unsubstituted 3ꢀpyridyl ring 23. Analogous 4ꢀpyridyl methyl analogs 27 and 28 were
approximately twoꢀfold more potent than unsubstituted comparator 26. Pyrimidine analog 29
was less potent than 3ꢀpyridyl analog 24; however installation of this nitrogen atom reduced
lipophilicity, and the ligandꢀlipophilicity efficiency (LLE) of 29 indicated that this 2ꢀ
methylpyrimidinꢀ5ꢀyl functional group was worthy of continued evaluation in other analogs. The
compounds examined (24, 25, 27, 28) in our in vitro DMPK assays again had fraction unbound
in rat plasma similar to 17, but only 24 had moderate predicted hepatic clearance with the
remaining analogs having CLhep values near liver blood flow.
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9
Table 1. mGlu NAM and in vitro DMPK results with 6ꢀsubstituted ethers
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
2
mGlu
2
pIC50
mGlu
2
IaC50
% Glu Max
(± SEM)
c
d
rat
plasma f
rat CLhep
f
No.
A
R
R
a
a,b
cLogP
LLE
e
u
(± SEM)
(nM)
850
1280
887
895
515
386
895
403
450
723
(mL/min/kg)
1
1
7
8
I
I
H
F
H
H
6.07 ± 0.09
5.89 ± 0.07
6.05 ± 0.12
6.05 ± 0.07
6.29 ± 0.10
6.41 ± 0.05
6.05 ± 0.07
6.39 ± 0.09
6.35 ± 0.06
6.14 ± 0.06
1.84 ± 0.47
0.87 ± 0.15
1.20 ± 0.29
1.63 ± 0.33
1.55 ± 0.39
1.31 ± 0.45
1.63 ± 0.33
1.09 ± 0.16
1.07 ± 0.13
1.30 ± 0.58
2.80
2.90
3.31
2.92
3.11
3.42
2.92
3.11
3.42
2.73
3.27
2.99
2.74
3.13
3.18
2.99
3.13
3.28
2.93
3.41
0.083
—
40.6
—
19
I
H
F
—
—
2
2
2
3
4
5
II
H
H
—
—
II
Me
H
H
0.056
0.072
—
43.0
64.2
—
II
Me
H
26
III
III
III
IV
H
2
2
2
7
8
9
Me
H
H
0.091
0.061
—
58.0
63.6
—
Me
—
—
a
b
c
d
e
f
2
Calcium mobilization mGlu assay; values are average of n ≥ 3
Amplitude of response in the presence of 30 µM test compound as a percentage of maximal response (100 µM glutamate); average of n ≥ 3
Calculated using Dotmatics Elemental (www.dotmatics.com/products/elemental/)
LLE (ligandꢀlipophilicity efficiency) = pIC50 – cLogP
f
u
= fraction unbound
Predicted hepatic clearance based on intrinsic clearance (CLint) in rat liver microsomes
4
ꢀOxoꢀ1,4ꢀdihydroquinoline amine analogs further illustrated the tolerance for variation at
the 6ꢀposition (Table 2). Additionally, many of these analogs exhibited superior mGlu NAM
2
potency compared to the ether analogs discussed above. Simple alkylation of the nitrogen linker
generally had minimal impact on mGlu NAM activity as evidenced by comparing secondary
2
amine analogs 31 and 34 to their tertiary amine comparators 32, 35, and 36; however, in each
case, the tertiary amine analogs exhibited a higher predicted hepatic clearance, possibly due to Nꢀ
dealkylation. In the case of analogs with the ring (A) directly attached to the nitrogen linker, the
3
ꢀpyridyl ring (32) exhibited superior mGlu NAM activity compared to the 4ꢀpyridyl ring (33).
2
On the other hand, the difference in potency was minimal when a methylene (35 and 37) or
ethylene spacer (40 and 41) was inserted between the nitrogen atom and the ring (A). Combining
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the methylene spacer with the 2ꢀmethylpyrimidinꢀ5ꢀyl ring (38) provided the most potent
compound in this set. The fraction unbound was considerably higher with 38 relative to other
similar analogs (35 and 37), and the predicted hepatic clearance, though still high, was less than
the majority of the other analogs in this set. Though analogs 39 and 42 were not among the most
potent amine analogs, these derivatives demonstrated that saturated heteroaryl rings were also
tolerated at the 6ꢀposition of the chemotype.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. mGlu NAM and in vitro DMPK results with 6ꢀsubstituted amines
2
mGlu
(± SEM)
2
pIC50
mGlu
2
IaC50
% Glu Max
(± SEM)
c
d
rat
plasma f
rat CLhep
f
No.
A
X
R
a
a,b
cLogP
LLE
e
u
(nM)
328
318
874
341
201
159
201
147
535
296
376
816
(mL/min/kg)
48.2
60.4
—
3
3
3
1
2
3
I
I
—
—
H
Me
Me
H
6.48 ± 0.12
6.50 ± 0.37
6.06 ± 0.06
6.47 ± 0.10
6.70 ± 0.03
6.80 ± 0.01
6.70 ± 0.09
6.83 ± 0.09
6.27 ± 0.05
6.53 ± 0.12
6.42 ± 0.11
6.09 ± 0.07
2.11 ± 0.42
–0.65 ± 1.98
1.16 ± 0.55
1.38 ± 0.58
2.39 ± 0.13
2.36 ± 0.14
1.76 ± 0.74
1.86 ± 0.26
1.15 ± 0.47
1.98 ± 0.67
2.01 ± 0.40
1.13 ± 0.18
2.57
2.95
2.95
2.61
2.92
3.33
2.92
2.74
2.87
2.71
2.71
1.56
3.91
3.55
3.11
3.86
3.78
3.47
3.78
4.09
3.40
3.82
3.71
4.53
0.084
0.061
—
II
I
—
34
CH
CH
CH
CH
CH
CH
2
2
2
2
2
2
0.137
0.082
0.067
0.089
0.340
—
51.3
67.6
68.2
67.0
47.1
—
3
3
3
5
6
7
I
Me
Et
I
II
III
IV
I
Me
Me
H
38
3
4
4
9
0
1
CH
CH
CH
2
2
2
CH
CH
CH
2
2
2
H
0.037
0.172
—
63.8
60.5
—
II
V
H
42
H
a
b
c
d
e
f
2
Calcium mobilization mGlu assay; values are average of n ≥ 3
Amplitude of response in the presence of 30 µM test compound as a percentage of maximal response (100 µM glutamate); average of n ≥ 3
Calculated using Dotmatics Elemental (www.dotmatics.com/products/elemental/)
LLE (ligandꢀlipophilicity efficiency) = pIC50 – cLogP
u
f = fraction unbound
Predicted hepatic clearance based on intrinsic clearance (CLint) in rat liver microsomes
Having observed that aromatic rings were not required at the 6ꢀposition, we were interested
to evaluate the numerous methylene amine analogs prepared at that position. Several of these
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analogs were simple tertiary amines without additional heteroatoms in the ring system (Table 3).
The mGlu NAM activity observed with these compounds was generally more dependent on
2
minor structural changes than had been observed with previous compounds. For example,
difluorocyclobutylamine 45 was approximately 5ꢀfold more potent than cyclopentylamine 46,
and difluropyrrolidine 48 was approximately 8ꢀfold more potent than difluoroazaspiroheptane
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
7. Likewise, though unsubstituted piperidine 49 was only a weak mGlu NAM, inhibiting the
2
glutamate response only at the highest concentration (30 µM), further substitution of the ring
with a variety of moieties enhanced potency (50–54). Three analogs (45, 48, and 54) were
evaluated in our in vitro DMPK assays, and while the protein binding results were encouraging
with more than ten percent unbound in each case, predicted hepatic clearance remained high
(>48 mL/min/kg).
Table 3. mGlu NAM and in vitro DMPK results with 6ꢀsubstituted methylene amines
2
1
2
mGlu
2
pIC50
mGlu
(nM)
2
IaC50
% Glu Max
(± SEM)
c
d
rat
plasma f
rat CLhep
f
No.
45
A
R
R
a
a,b
cLogP
LLE
e
u
(± SEM)
(mL/min/kg)
I
—
—
—
—
H
—
—
—
—
H
6.40 ± 0.01
5.66 ± 0.09
5.75 ± 0.06
6.67 ± 0.09
396
1.16 ± 0.18
0.43 ± 0.75
1.44 ± 0.30
1.10 ± 0.12
37.2 ± 9.7
3.86
4.09
3.39
3.32
3.55
4.21
2.94
3.08
2.34
3.77
2.54
1.57
2.36
3.35
< 1.45
2.00
3.29
2.69
3.71
3.02
0.138
—
60.9
—
4
4
4
6
7
8
II
2170
1770
214
III
IV
V
V
V
V
V
V
—
—
0.156
—
56.5
—
g
49
< 5.0
> 10,000
618
5
5
5
0
1
2
H
CF
3
6.21 ± 0.09
6.23 ± 0.07
5.77 ± 0.11
6.05 ± 0.09
6.79 ± 0.19
1.11 ± 0.28
0.90 ± 0.33
1.03 ± 0.26
1.36 ± 0.26
1.59 ± 0.27
—
—
H
CN
587
—
—
H
OMe
1720
886
—
—
53
H
SO
2
Me
—
—
5
4
F
F
161
0.109
48.6
a
b
c
d
2
Calcium mobilization mGlu assay; values are average of n ≥ 3
Amplitude of response in the presence of 30 µM test compound as a percentage of maximal response (100 µM glutamate); average of n ≥ 3
Calculated using Dotmatics Elemental (www.dotmatics.com/products/elemental/)
LLE (ligandꢀlipophilicity efficiency) = pIC50 – cLogP
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
e
f
u
f = fraction unbound
Predicted hepatic clearance based on intrinsic clearance (CLint) in rat liver microsomes
Weak activity; concentrationꢀresponse curve (CRC) does not plateau
g
In addition to the simple tertiary amines highlighted above, we also prepared a number of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
analogs with heterocyclic amines (Table 4). Substituted morpholine analogs 55–58 were potent
mGlu NAMs with the dimethyl substituted analogs 57 and 58 offering potency superior to
2
monomethyl analogs 55 and 56. Particularly encouraging was analog 58, which was predicted to
be a low–moderate clearance compound in rats and was approximately 30% unbound in rat
plasma. On the other hand, thiomorpholine 59 exhibited high clearance in vitro, and
thiomorpholine 1,1ꢀdioxide 60 showed reduced potency. We also prepared several analogs with
sevenꢀmembered rings (63–66). Though most of these medium ringꢀcontaining analogs were
moderate to weak mGlu NAMs, 1,4ꢀthiazepane 65 was quite potent. Unfortunately, 65 was
2
highly cleared in vitro; however, oxidation of the sulfur atom was a likely metabolic softꢀspot as
clearance with 1,4ꢀthiazepane 1,1ꢀdioxide 66 was substantially reduced.
Table 4. mGlu NAM and in vitro DMPK results with 6ꢀsubstituted methylene amines (cont.)
2
1
2
mGlu
2
pIC50
mGlu
2
IaC50
% Glu Max
(± SEM)
c
d
rat plasma
rat CLhep
f
No.
A
R
R
X
a
a,b
cLogP
LLE
e
(± SEM)
(nM)
341
551
119
207
420
870
1110
612
f
u
(mL/min/kg)
5
5
I
Me
H
H
Me
Me
—
—
—
—
—
—
—
—
—
S
6.47 ± 0.16
6.26 ± 0.16
6.93 ± 0.09
6.69 ± 0.04
6.38 ± 0.09
6.06 ± 0.08
5.95 ± 0.06
6.21 ± 0.07
0.98 ± 0.24
1.33 ± 0.15
2.14 ± 0.17
1.48 ± 0.29
1.52 ± 0.63
1.33 ± 0.38
1.15 ± 0.32
1.10 ± 0.07
2.59
2.59
2.99
3.10
2.76
1.35
2.09
2.90
3.88
3.67
3.94
3.59
3.62
4.71
3.86
3.31
—
—
—
—
56
I
5
5
5
7
8
9
I
Me
—
—
—
—
—
0.16
0.306
0.205
—
51.2
24.5
59.6
—
II
III
III
III
III
60
SO
2
6
6
1
2
NMe
NCH CF
—
—
2
3
—
—
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6
6
6
3
4
5
IV
IV
IV
IV
—
—
—
—
—
—
—
—
CH
2
5.68 ± 0.10
2080
> 10,000
138
0.55 ± 0.21
24.2 ± 3.6
2.19 ± 0.24
1.24 ± 0.04
4.00
2.54
3.21
1.80
1.68
< 2.46
3.65
—
—
—
—
g
O
< 5.0
S
6.86 ± 0.10
6.38 ± 0.06
0.074
0.253
63.8
23.7
66
SO
2
415
4.58
a
b
c
d
e
f
2
Calcium mobilization mGlu assay; values are average of n ≥ 3
Amplitude of response in the presence of 30 µM test compound as a percentage of maximal response (100 µM glutamate); average of n ≥ 3
Calculated using Dotmatics Elemental (www.dotmatics.com/products/elemental/)
LLE (ligandꢀlipophilicity efficiency) = pIC50 – cLogP
u
f = fraction unbound
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Predicted hepatic clearance based on intrinsic clearance (CLint) in rat liver microsomes
Weak activity; CRC does not plateau
g
Turning our attention to the 6ꢀaryloxymethyl ether analogs 70–78 uncovered several
additional compounds with good mGlu NAM potency (Table 5). Several 3ꢀpyridyl derivatives
2
(70–74) were prepared, and 6ꢀmethyl derivative 72 and 6ꢀchloro derivative 73 exhibited good
potency. Interestingly, a trifluoromethyl group (74) did not function as an adequate alternative at
this position. The pyridyl derivatives (75–77) demonstrated more modest differences in mGlu2
NAM activity, and in this case the trifluoromethyl (77) was only slightly less potent than its
corresponding methyl comparator (76). Fraction unbound with these pyridyl analogs was in line
with other similar analogs (see Table 1), and predicted clearance ranged from moderate (72 and
7
6) to high (73 and 75). Once again, we installed a 2ꢀmethylpyrimidinꢀ5ꢀyl ring (78) and
observed positive results. Specifically, not only was 78 a potent mGlu NAM, it exhibited more
2
than 10% fraction unbound in rat plasma and a low predicted clearance in rat liver microsomes.
Table 5. mGlu NAM and in vitro DMPK results with 6ꢀaryloxymethyl ethers
2
1
2
mGlu
2
pIC50
mGlu
(nM)
2
IaC50
% Glu Max
c
d
rat plasma
rat CLhep
f
No.
70
A
R
R
a
a,b
cLogP
LLE
e
u
(± SEM)
(± SEM)
f
(mL/min/kg)
I
I
F
H
F
6.13 ± 0.06
6.35 ± 0.10
746
0.31 ± 0.30
1.13 ± 0.38
3.42
3.42
2.71
2.93
—
—
—
—
7
1
H
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
7
7
7
2
3
4
I
I
H
H
H
F
Me
Cl
6.61 ± 0.11
6.71 ± 0.10
5.88 ± 0.04
6.64 ± 0.10
6.45 ± 0.09
6.31 ± 0.06
6.56 ± 0.08
247
193
1330
228
351
486
277
1.62 ± 0.23
1.70 ± 0.12
1.23 ± 0.02
1.83 ± 0.20
1.90 ± 0.32
1.70 ± 0.14
1.71 ± 0.20
3.11
3.94
3.83
3.02
3.11
3.83
2.73
3.50
2.77
2.05
3.62
3.34
2.48
3.83
0.087
0.033
—
43.9
52.1
—
I
CF
3
75
II
II
II
III
H
0.063
0.050
—
59.8
37.2
—
7
7
7
6
7
8
H
H
—
Me
CF
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
—
0.109
22.4
a
b
c
d
e
f
2
Calcium mobilization mGlu assay; values are average of n ≥ 3
Amplitude of response in the presence of 30 µM test compound as a percentage of maximal response (100 µM glutamate); average of n ≥ 3
Calculated using Dotmatics Elemental (www.dotmatics.com/products/elemental/)
LLE (ligandꢀlipophilicity efficiency) = pIC50 – cLogP
= fraction unbound
f
u
Predicted hepatic clearance based on intrinsic clearance (CLint) in rat liver microsomes
Finally, examination of 6ꢀethylene linked analogs 81–87 yielded a range of results (Table 6).
Unsubstituted phenyl analog 81 demonstrated weak mGlu NAM activity; however, modification
2
of the aromatic ring (A) to pyridine (82 and 84) improved potency approximately 15ꢀfold.
Unfortunately, both 82 and 84 were highly cleared in vitro. Substitution of 4ꢀpyridyl analog 82
with a trifluoromethyl group (83) modestly enhanced potency but without reducing clearance.
Substitution of 3ꢀpyridyl analog 84 with trifluoromethyl (85) and fluorine (86) was unfavorable
for mGlu NAM activity. Again the 2ꢀmethylpyrimidinꢀ5ꢀyl ring (87) proved an attractive
2
moiety, having demonstrated the most potent activity and highest fraction unbound in this set of
analogs. Also, although predicted hepatic clearance for 87 remained on the high end, it was
improved relative to other analogs in this class (82–84).
Table 6. mGlu NAM and in vitro DMPK results with 6ꢀethylene linked analogs
2
1
2
mGlu
2
pIC50
mGlu
(nM)
2
IaC50
% Glu Max
c
d
rat plasma
rat CLhep
f
No.
A
R
R
a
a,b
cLogP
LLE
e
u
(± SEM)
(± SEM)
f
(mL/min/kg)
8
1
I
—
—
5.16 ± 0.09
6890
2.07 ± 1.38
4.65
0.21
—
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8
9
8
8
8
2
3
4
II
II
H
—
—
H
6.33 ± 0.09
6.52 ± 0.09
6.33 ± 0.08
5.76 ± 0.03
5.93 ± 0.04
6.67 ± 0.10
471
304
1.88 ± 0.38
0.88 ± 0.06
1.90 ± 0.11
1.22 ± 0.14
0.80 ± 0.34
1.52 ± 0.26
3.34
4.25
3.34
4.25
3.44
3.16
2.99
2.27
2.99
1.51
2.49
3.51
0.057
0.039
0.062
—
66.7
68.4
64.1
—
CF
3
III
III
III
IV
H
466
85
CF
3
H
1720
1170
215
8
8
6
7
H
F
—
—
—
—
0.157
46.9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
b
c
d
e
f
2
Calcium mobilization mGlu assay; values are average of n ≥ 3
Amplitude of response in the presence of 30 µM test compound as a percentage of maximal response (100 µM glutamate); average of n ≥ 3
Calculated using Dotmatics Elemental (www.dotmatics.com/products/elemental/)
LLE (ligandꢀlipophilicity efficiency) = pIC50 – cLogP
f
u
= fraction unbound
Predicted hepatic clearance based on intrinsic clearance (CLint) in rat liver microsomes
Having developed substantial SAR at the 6ꢀposition of the chemotype, we wanted to conduct
limited exploration of another area as well. We chose ethylene linked analog 87 as a useful
comparator given its overall profile. As such, additional analogs of 87 with alternative aromatic
rings (B) to the 4ꢀfluorophenyl ring were prepared and tested (Table 7). Replacement of the 4ꢀ
fluoro group (87) with a 4ꢀmethoxy group (88) improved mGlu NAM potency slightly, but the
2
predicted hepatic clearance of 88 remained high. Since methoxy groups increase electron density
on the ring, fluorinated analogs 89 and 90 were prepared; however, these modifications failed to
improve metabolic stability. It was encouraging to see that the 4ꢀfluorophenyl ring could be
replaced altogether with a 3ꢀmethylisothiazolꢀ5ꢀyl ring (91). Analog 91 demonstrated a 2ꢀfold
drop in potency relative to 87; yet, this modification was considered efficient by the LLE
quotient as it was a less lipophilic compound. Of note, 91 had an increased fraction unbound and
a marginally and perhaps insignificantly lower predicted hepatic clearance than 87. Continued
exploration of this region (B) of the scaffold is clearly worthwhile; however, at this point, we
decided to more thoroughly profile some of the promising analogs discovered thus far to evaluate
the full potential of the 4ꢀoxoꢀ1ꢀarylꢀ1,4ꢀdihydroquinolineꢀ3ꢀcarboxamides as a lead series for
the discovery of drugꢀlike mGlu NAMs.
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2
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 7. mGlu NAM and in vitro DMPK results with modified 1ꢀposition analogs
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
mGlu
2
pIC50
mGlu
(nM)
2
IaC50
% Glu Max
(± SEM)
c
d
e
u
rat CLhep
f
No.
B
a
a,b
cLogP
LLE
rat plasma f
(± SEM)
(mL/min/kg)
8
8
8
7
8
9
I
6.67 ± 0.10
215
1.52 ± 0.26
3.16
3.02
3.12
3.12
2.66
3.51
3.85
3.46
3.58
3.72
0.157
0.098
0.085
0.159
0.220
46.9
g
g
g
II
6.87
136
1.61
53.4
III
IV
V
6.58 ± 0.09
6.70 ± 0.13
6.38 ± 0.05
266
198
414
1.30 ± 0.21
1.52 ± 0.38
1.20 ± 0.50
53.6
90
60.5
9
1
41.9
a
b
c
d
e
f
2
Calcium mobilization mGlu assay; values are average of n ≥ 3
Amplitude of response in the presence of 30 µM test compound as a percentage of maximal response (100 µM glutamate); average of n ≥ 3
Calculated using Dotmatics Elemental (www.dotmatics.com/products/elemental/)
LLE (ligandꢀlipophilicity efficiency) = pIC50 – cLogP
u
f = fraction unbound
Predicted hepatic clearance based on intrinsic clearance (CLint) in rat liver microsomes
Value is average of n=2
g
Prior to discussing the results of extended profiling of select 4ꢀoxoꢀ1ꢀarylꢀ1,4ꢀ
dihydroquinolines, it is worth briefly discussing the results obtained with the 4Hꢀquinolizinꢀ4ꢀ
one analogs (Table 8). Whether in the case of the ether analogs (99–102) or methylene amine
analogs (106–108), mGlu NAM potency was consistently weak. In fact, when compared to their
2
analogous compounds in the 4ꢀoxoꢀ1ꢀarylꢀ1,4ꢀdihydroquinoline series, these analogs were
notably less potent in each case. These results are important because it illustrates that these two
cores are not uniformly interchangeable. Moreover, it provides another example of the subtleties
5
2–54
of SAR often seen in the design of allosteric modulators of class C GPCRs.
Table 8. mGlu NAM results with 4Hꢀquinolizinꢀ4ꢀone analogs
2
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7
8
9
mGlu
2
pIC50
mGlu
(nM)
2
IaC50
% Glu Max
(± SEM)
c
d
e
fold decrease
in potency
No.
A
X
Y
a
a,b
cLogP
LLE
comparator
f
(± SEM)
9
9
I
I
O
O
C–H
N
5.40 ± 0.02
3980
2.67 ± 0.26
19.4 ± 9.3
2.83
2.45
2.63
3.54
2.65
2.77
2.87
2.57
< 2.55
2.64
24
29
26
—
58
65
48
5.8
> 13
6.0
—
g
1
00
< 5.0
> 10,000
5360
101
II
O
H
5.27 ± 0.07
5.34 ± 0.01
0.71 ± 1.86
2.19 ± 0.69
3.95 ± 1.84
1.04 ± 2.27
0.93 ± 2.27
1
1
1
02
06
07
II
O
CF
3
4610
1.80
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
g
III
IV
V
CH
CH
CH
2
2
2
—
—
—
< 5.0
> 10,000
6620
< 2.35
2.41
> 48
48
5.18 ± 0.13
5.36 ± 0.21
108
4400
2.49
21
a
b
c
d
e
f
2
Calcium mobilization mGlu assay; values are average of n ≥ 3
Amplitude of response in the presence of 30 µM test compound as a percentage of maximal response (100 µM glutamate); average of n ≥ 3
Calculated using Dotmatics Elemental (www.dotmatics.com/products/elemental/)
LLE (ligandꢀlipophilicity efficiency) = pIC50 – cLogP
Direct comparator from 4ꢀoxoꢀ1,4ꢀdihydroquinoline series
Fold decrease in potency relative to direct comparator from 4ꢀoxoꢀ1,4ꢀdihydroquinoline series
Weak activity; CRC does not plateau
g
Extended Characterization of Selected Analogs. In choosing the initial compounds for
more in depth evaluation, we sought molecules with both promising mGlu NAM potency and in
2
vitro DMPK profiles while also desiring some structural diversity at the 6ꢀposition substituent.
As such, we selected ether 17, amine 34, methylene amines 54 and 58, and ethylene linked
analog 87 for further profiling (Table 9). Since the potential therapeutic applications for an
mGlu NAM are in the area of CNS disorders, bloodꢀbrain barrier (BBB) penetration seemed a
2
logical next step for evaluation in this new series. It should be noted that the fraction unbound in
rat plasma ranged from 0.083 to 0.306 with these compounds; thus, consideration of unbound
fraction alongside potency and CNS exposure is required to fully evaluate these compounds.
Toward this end, we employed rat cassette pharmacokinetics (PK) tissue distribution studies
5
5
using intravenous (IV) dosing and single time point analysis. Such an approach has repeatedly
proven a rapid and costꢀeffective mechanism for preliminary assessment of BBB penetration. In
addition to the already measured protein binding in rat plasma, the protein binding of these
compounds in rat brain homogenates was also assessed. Unfortunately, the observed brain to
plasma ratio (K ) for each compound was low, ranging from 0.04 (34) to 0.36 (54). Calculation
p
of the unbound brain to unbound plasma ratio (Kp,uu) gave values that were all below 0.25,
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
56
indicating possible transporter effects. Thus, analogs 58 and 87 were selected for permeability
studies in MadinꢀDarby canine kidney (MDCK) cells transfected with the human MDR1 gene to
5
7
assess potential Pꢀglycoprotein (Pꢀgp) mediated efflux. Both compounds demonstrated
substantial efflux with ratios of 52 and 44, respectively. While it was disappointing to learn that
this scaffold appeared to suffer from Pꢀgpꢀmediated efflux, the absolute CNS concentrations
observed with 58 and its other properties raised the possibility that it might still be a valuable
tool.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 9. Rat IV cassette and MDR1ꢀMDCK permeability results
b
rat IV tissue distribution results
Permeability in MDR1-MDCK cells
mGlu
2
IC50
rat plasma
rat brain
plasma conc.
brain conc.
(nM)
d
e
A–B Papp
(10 cm/sec)
B–A Papp
(10 cm/sec)
Efflux
Ratio
No.
a
a
c
c
K
p
K
p,uu
-6
-6
(
nM)
850
341
161
207
215
f
u
f
u
(nM)
17
34
0.083
0.137
0.109
0.306
0.157
0.087
0.095
0.068
0.191
0.160
55.8
83.0
60.5
56.9
122
9.2
3.0
0.16
0.04
0.36
0.32
0.05
0.17
0.02
0.22
0.23
0.05
—
—
—
—
—
—
—
52
44
5
5
8
4
8
7
21.6
18.0
5.6
—
—
1.54
1.58
79.7
69.4
a
f
u
= fraction unbound
n = 2; dose = 0.2 mg/kg per compound; solution in 8% EtOH, 30% PEG 400, 62% DMSO (2 mg/mL total)
5 minutes post dose
= total brain to total plasma ratio
p,uu = unbound brain (brain f · total brain) to unbound plasma (plasma f
b
c
d
e
1
K
K
p
u
u
· total plasma) ratio
Further profiling of compound 58 (VU6001192) began with determination of its full
selectivity versus other members of the mGlu family. Selectivity versus fellow group II receptor
subtype, mGlu , was particularly critical to assess. Gratifyingly, we evaluated the selectivity of
3
5
8 versus rat mGlu using 10 point concentrationꢀresponse curve (CRC) analysis in the presence
3
of an EC concentration of glutamate, and 58 was inactive up to the highest concentration tested
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9
(
30 µM). For evaluation of selectivity versus other members of the mGlu family, the effect of 10
5
8,59
µM 58 on the orthosteric agonist CRC was measured in foldꢀshift experiments.
Fortunately,
no activity at the other mGlus was noted in these assays. Because the genesis for the 4ꢀoxoꢀ1ꢀ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
arylꢀ1,4ꢀdihydroquinoline chemotype as a mGlu NAM scaffold was inspired in part by a known
2
6
0
M PAM scaffold, we also evaluated 58 in our human M functional assay and observed no
1
1
activity up to the highest concentration tested (30 µM). Ancillary pharmacology was evaluated
through screening 58 at 10 µM in a commercially available radioligand binding assay panel of 68
6
1
clinically relevant GPCRs, ion channels, kinases, and transporters, and no significant responses
62
were noted.
Having established the excellent selectivity profile of 58, we progressed the compound to
6
3
additional and more definitive PK studies in both rats and mice (Table 10). In spite of its
predicted low–moderate clearance, a time course study using IV dosing showed 58 to be a high
clearance compound; however, the volume of distribution at steady state (V ) was high and the
SS
halfꢀlife was approximately 2 hours. Thus, intraperitoneal (IP) dosing was chosen as a route that
was both convenient for future use in behavioral models and had the likelihood of providing
superior exposure to oral dosing. An IP tissue distribution study in rats at 30 mg/kg gave K and
p
Kp,uu values similar to those observed previously; yet, we observed a brain concentration of 760
nM, which translates to an unbound brain concentration of 145 nM and is very near the
functional mGlu IC (207 nM). An analogous study in mice at the same dose gave similar
2
50
results with a brain concentration of 716 nM, which translates to an unbound brain concentration
of 184 nM. Finally, to verify the role of Pꢀgp in vivo, we repeated the study in mice with the
6
4
modification of preꢀtreating the animals with the known Pꢀgp inhibitor 109 (elacridar). The
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
impact of this modification was profound as the exposure of 58 in the brain was increased more
than 6ꢀfold without impacting the systemic exposure in plasma.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 10. IV PK and tissue distribution studies with compound 58
a
b
c,d
Protein Binding (f
rat plasma
u
)
Rat IV PK
Rat IP Tissue Distribution
0.306
0.191
0.315
0.257
dose
0.2 mg/kg
141 minutes
dose
30 mg/kg
3900 nM
760 nM
rat brain homogenates
mouse plasma
t
1/2
CLplasma
SS
plasma concentration
brain concentration
75.8 mL/min/kg
e
f
mouse brain homogenates
V
13.3 L/kg
K
p
K
p,uu
0.20
0.12
c,d
c,d,g
Mouse IP Tissue Distribution
Mouse IP Tissue Distribution + 109
dose
30 mg/kg
dose
30 mg/kg
plasma concentration
brain concentration
1820
716
plasma concentration
brain concentration
2160
4580
e
f
e
f
K
p
K
p,uu
0.39
0.32
K
p
K
p,uu
2.1
1.7
a
b
c
d
e
f
f
u
= fraction unbound
n = 2; solution in solution in 9% EtOH, 38% PEG 400, 53% DMSO (1 mg/mL)
n = 2; fine homogenous suspension in 10% Tween80 in H
5 minutes post dose of 58
= total brain to total plasma ratio
p,uu = unbound brain (brain f · total brain) to unbound plasma (plasma f
compound 109 dosed one hour prior to compound 58 at 20 mg/kg
2
O
1
K
K
p
u
u
· total plasma) ratio
g
CONCLUSION
A potent and highly selective mGlu NAM tool compound 58 was discovered through
2
scaffold hopping a series in the patent literature and recognizing the possibility that an
established M PAM series might function as a viable chemotype for new analog design. Diverse
1
functional groups were tolerated at the 6ꢀposition of the new chemotype, and limited work
established the potential for further modifications at the 1ꢀposition. While the utility of the
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compounds tested thus far is hampered by Pꢀgp mediated efflux that limits CNS exposure, the
overall profile of 58 remains interesting. The compound exhibits an unbound fraction of 25–30%
in rodent brain homogenates, and a dose of 30 mg/kg using IP dosing produces unbound brain
concentrations near the functional mGlu IC . Conceivably, higher doses could be employed in
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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58
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60
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50
order to reach pharmacologically relevant concentrations in the CNS. Perhaps more attractive is
the fact that preꢀtreatment with a commercially available Pꢀgp inhibitor boosts the unbound brain
exposure of 58 to more than 5ꢀfold the mGlu IC at the same dose. The study of 58 in
2
50
behavioral models relevant to mGlu inhibition is planned and will be the subject of future
2
communications.
EXPERIMENTAL SECTION
The synthesis of compound 58 and its associated intermediates is described below for
convenience. Synthetic details for other compounds can be found in the Supporting Information.
Ethyl 3-(5-bromo-2-fluorophenyl)-3-oxopropanoate (14). 3ꢀEthoxyꢀ3ꢀoxoꢀpropanoic acid 13
(2.16 mL, 18.3 mmol, 2.00 eq) was dissolved in THF (91 mL) in an oven dried roundꢀbottom
flask and 2,2’ꢀbipyridyl (8.00 mg, 0.0512 mmol, 0.0056 eq) was added as an indicator. The
reaction was cooled to –30 ºC and nꢀbutyllithium (1.6 M in hexanes) (29.0 mL, 45.6 mmol, 4.00
eq) was added dropwise over 20 minutes. Upon final addition the reaction turned red at which
point it was allowed to warm to –5 ºC. The reaction was allowed to stir at –5 ºC for 15 minutes,
during which time the red color began to dissipate. Enough nꢀbutyllithium was added to allow
the red color to persist. The reaction was then cooled to –78 ºC and 5ꢀbromoꢀ2ꢀfluoroꢀbenzoyl
chloride (2.17 g, 9.14 mmol, 1.00 eq) was added dropwise as a solution in THF (6.9 mL). The
reaction was allowed to stir at –78 ºC for 30 minutes and then allowed to warm to –30 ºC and
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stirred for an additional 30 minutes. The reaction was poured onto iceꢀcold 1N HCl (92 mL) and
the mixture was extracted with ethyl acetate (1x) and DCM (2x). The combined organics were
dried (MgSO ), filtered and concentrated in vacuo. Purification by flash chromatography on
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silica gel afforded 1.78 g (67%) of the title compound as an offꢀwhite solid. H NMR (400 MHz,
DMSOꢀd ): δ = 7.97 (dd, J = 6.5, 2.6 Hz, 1H), 7.91ꢀ7.86 (m, 1H), 7.40ꢀ7.34 (m, 1H), 4.13ꢀ4.07
6
1
3
(
m, 4H), 1.15 ppm (t, J = 7.1 Hz, 3H). C NMR (100 MHz, DMSOꢀd ): δ = 189.68 (d, J(C,F) =
6
3.2 Hz), 167.09, 160.26 (d, J(C,F) = 255 Hz), 138.09 (d, J(C,F) = 9.5 Hz), 132.49 (d, J(C,F) =
2.2 Hz), 126.16 (d, J(C,F) = 13.3 Hz), 119.51 (d, J(C,F) = 25.1 Hz), 116.64, 60.71, 48.81, 13.92
ppm. HRMS (ESI): calculated for C H BrFO [M]: 287.9797; found: 287.9794. LCMS R =
1
1
10
3
T
+
0
.989 min, ESꢀMS m/z = 289.0 [M+H] .
Ethyl 6-bromo-1-(4-fluorophenyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (15, where Ar
4-fluorophenyl). Compound 14 (2.87 g, 9.93 mmol, 1.00 eq) and N,Nꢀdimethylformamide
=
dimethyl acetal (1.87 mL, 14.9 mmol, 1.50 eq) were dissolved in DMF (33 mL) in a microwave
vial and heated in a microwave reactor at 120 ºC for 15 minutes. To this mixture was then added
4ꢀfluoroaniline (1.41 mL, 14.9 mmol, 1.50 eq) and the reaction was heated in a microwave
reactor at 150 ºC for 20 minutes. The reaction mixture was diluted with ethyl acetate and washed
with water (2x). The aqueous layers were backꢀextracted with ethyl acetate and the combined
organics were dried (MgSO ), filtered and concentrated in vacuo. Purification by flash
4
1
chromatography on silica gel afforded 3.79 g (98%) of the title compound as yellow solid. H
NMR (400 MHz, DMSOꢀd ) δ = 8.47 (s, 1H), 8.31 (d, J = 2.4 Hz, 1H), 7.81 (dd, J = 9.1, 2.4 Hz,
6
1H), 7.77ꢀ7.73 (m, 2H), 7.54ꢀ7.50 (m, 2H), 6.92 (d, J = 9.0 Hz, 1H), 4.20 (q, J = 7.1 Hz, 2H),
13
1.25 ppm (t, J = 7.1 Hz, 3H). C NMR (100 MHz, DMSOꢀd ): δ = 171.80, 163.90, 162.43 (d,
6
J(C,F) = 247.5 Hz), 149.02, 139.70, 136.38 (d, J(C,F) = 2.8 Hz), 135.37, 130.17 (d, J(C,F) = 9.0
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Hz), 128.83, 128.15, 120.73, 118.06, 117.26 (d, J(C,F) = 23.2 Hz), 110.88, 60.04, 14.21 ppm.
HRMS (ESI): calculated for C H BrFNO [M]: 389.0063; found: 389.0062. LCMS R = 0.934
18 13
3
T
+
min, ESꢀMS m/z = 390.2 [M+H] .
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6
-Bromo-1-(4-fluorophenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (16, where Ar = 4-
fluorophenyl). Ethyl 6ꢀbromoꢀ1ꢀ(4ꢀfluorophenyl)ꢀ4ꢀoxoꢀ1,4ꢀdihydroquinolineꢀ3ꢀcarboxylate
1.00 g, 2.56 mmol, 1.00 eq) was suspended in 7N ammonia in methanol (30 mL) in a
(
microwave vial and the reaction was heated in a microwave reactor at 150 ºC for 60 minutes.
The reaction was concentrated to afford 881 mg (95%) of the title compound as a brown solid
1
that was used without further purification. H NMR (400 MHz, DMSOꢀd ) δ = 9.08 (d, J = 4.0
6
Hz, 1H), 8.57 (s, 1H), 8.43 (d, J = 2.4 Hz, 1H), 7.85 (dd, J = 9.1, 2.4 Hz, 1H), 7.77ꢀ7.73 (m, 2H),
1
3
7.66 (d, J = 4.1 Hz, 1H), 7.56ꢀ7.50 (m, 2H), 7.02 ppm (d, J = 9.1 Hz, 1H). C NMR (100 MHz,
DMSOꢀd ): δ = 174.64, 164.83, 162.46 (d, J(C,F) = 247.6 Hz), 148.42, 139.85, 136.50 (d, J(C,F)
6
=
=
2.8 Hz), 135.66, 129.97 (d, J(C,F) = 9.3 Hz), 128.09, 128.07, 120.86, 118.19, 117.31 (d, J(C,F)
23.3 Hz), 112.07 ppm. HRMS (ESI): calculated for C H BrFN O [M]: 359.9910; found:
1
6
10
2
2
+
359.9909. LCMS R = 0.929 min, ESꢀMS m/z = 361.2 [M+H] .
T
1-(4-Fluorophenyl)-4-oxo-6-vinyl-1,4-dihydroquinoline-3-carboxamide (43, where Ar = 4-
fluorophenyl). To a solution of 6ꢀbromoꢀ1ꢀ(4ꢀfluorophenyl)ꢀ4ꢀoxoꢀ1,4ꢀdihydroquinolineꢀ3ꢀ
carboxamide (450 mg, 1.25 mmol, 1.0 eq), triethylamine (174 µL, 1.25 mmol, 1.0 eq), and
Pd(dppf)Cl ·CH Cl (18.2 mg, 0.025 mmol, 0.2 eq) in 1ꢀpropanol (8.3 mL) was added potassium
2
2
2
vinyltrifluoroborate (200 mg, 1.5 mmol, 1.2 eq). The mixture was purged with argon and stirred
at 100 ºC for 16 hours. The reaction was filtered through Celite® and washed very well with a
5%MeOH in DCM solution. The filtrate was concentrated in vacuo to give 385 mg (100%) of
1
the title compound, which was used without further purification. H NMR (400 MHz, DMSOꢀ
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d6): δ = 9.23 (d, J = 4.2 Hz, 1H), 8.55 (s, 1H), 8.36 (d, J = 1.6 Hz, 1H), 7.89 (dd, J = 1.8, 8.9 Hz,
1H), 7.79ꢀ7.75 (m, 2H), 7.64 (d, J = 4.2 Hz, 1H), 7.54 (t, J = 8.7 Hz, 2H), 7.03 (d, J = 8.8 Hz,
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H), 6.93 (q, J = 11, 6.6 Hz, 1H), 5.95 (d, J = 17.6 Hz, 1H), 5.39 ppm (d, J = 11 Hz, 1H). C
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NMR (100 MHz, DMSOꢀd ): δ = 175.79, 165.17, 162.36 (d, J(C,F) = 247.0 Hz), 147.77, 140.24,
6
136.74 (d, J(C,F) = 3.2 Hz), 135.38, 134.17, 130.1, 129.93 (d, J(C,F) = 9.0 Hz), 126.74, 123.72,
118.65, 117.35 (d, J(C,F) = 23.4 Hz), 115.9, 111.72 ppm. HRMS (ESI): calculated for
C H FN O [M]: 308.0961; found: 308.0964. LCMS R = 0.922 min, ESꢀMS m/z = 309.2
18
13
2
2
T
+
[M+H] .
1-(4-Fluorophenyl)-6-formyl-4-oxo-1,4-dihydroquinoline-3-carboxamide (44, where Ar = 4-
fluorophenyl). To a solution of 1ꢀ(4ꢀfluorophenyl)ꢀ4ꢀoxoꢀ6ꢀvinylꢀ1,4ꢀdihydroquinolineꢀ3ꢀ
carboxamide (385 mg, 1.25 mmol, 1.0 eq) in 3:1 acetone/water (8 mL) was added Nꢀoxideꢀ4ꢀ
methylmorpholine (220 mg, 1.87 mmol, 1.5 eq) and osmium tetroxide (6.3 mg, 0.025 mmol,
0
.02 eq). After the reaction stirred for one hour, sodium periodate (294 mg, 1.37 mmol, 1.1 eq)
was added. After another two hours, the reaction was diluted with EtOAc and washed well with a
0% NaS O solution. The organic layer was dried (MgSO ), filtered and concentrated in vacuo
1
2
3
4
1
to give 365 mg (94%) of the title compound that was used without further purification. H NMR
(
400 MHz, DMSOꢀd ): δ = 10.17 (s, 1H), 9.10 (d, J = 3.8 Hz, 1H), 8.93 (d, J = 1.8 Hz, 1H), 8.62
6
(s, 1H), 8.12 (dd, J = 1.8, 8.8 Hz, 1H), 7.82ꢀ7.78 (m, 2H), 7.74 (d, J = 3.8 Hz, 1H), 7.56 (t, J =
1
3
8.8 Hz, 2H), 7.22 ppm (d, J = 8.8 Hz, 1H).
C NMR (100 MHz, DMSOꢀd ): δ = 192.23,
6
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1
1
75.88, 164.68, 162.49 (d, J(C,F) = 247 Hz), 149.06, 144.23, 136.61 (d, J(C,F) = 3.2 Hz),
32.48, 130.98, 130.41, 130.0 (d, J(C,F) = 9.3 Hz), 126.54, 119.46, 117.37 (d, J(C,F) = 23.3 Hz),
12.78 ppm. HRMS (ESI) calculated for C H FN O [M]: 310.0754; found: 310.0757. LCMS
1
7
11
2
3
+
R = 0.732 min, ESꢀMS m/z = 311.2 [M+H] .
T
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-((cis-2,6-Dimethylmorpholino)methyl)-1-(4-fluorophenyl)-4-oxo-1,4-dihydroquinoline-3-
carboxamide (58). A solution of 1ꢀ(4ꢀfluorophenyl)ꢀ6ꢀformylꢀ4ꢀoxoꢀ1,4ꢀdihydroquinolineꢀ3ꢀ
carboxamide (580 mg, 1.87 mmol, 1.0 eq) in dichloromethane (1 mL), cisꢀ2,6ꢀ
dimethylmorpholine (461 µL, 3.74 mmol, 2.0 eq), and acetic acid (268 µL, 4.67 mmol, 2.5 eq)
was stirred for one hour. Sodium triacetoxyborohydride (594 mg, 2.80 mmol, 1.5 eq) was added.
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After 16 hours, the reaction was concentrated to dryness. Purification by reverse phase HPLC
1
afforded 620 mg (81%) of the title compound as a white solid. H NMR (400 MHz, DMSOꢀd ) δ
6
9.23 (d, J = 4.4 Hz, 1H), 8.55 (s, 1H), 8.26 (d, J = 1.4 Hz, 1H), 7.77ꢀ7.73 (m, 2H), 7.65 (dd, J =
1
.8, 8.7 Hz, 1H), 7.59 (d, J = 4.3 Hz, 1H), 7.52 (t, J = 8.7, 2H), 7.03 (d, J = 8.7, 1H), 3.57ꢀ3.52
1
3
(m, 4H), 2.65 (d, J = 10.7 Hz, 2H), 1.68 (t, J = 10.7 Hz, 2H), 1.01 ppm (d, J = 6.2 Hz, 6H). C
NMR (100 MHz, DMSOꢀd ): δ = 175.8, 165.26, 162.36 (d, J(C,F) = 247 Hz), 147.78, 139.9,
6
1
36.82 (d, J(C,F) = 3.1 Hz), 135.26, 133.91, 129.98 (d, J(C,F) = 8.9 Hz), 126.41, 125.85, 118.26,
17.22 (d, J(C,F) = 23.0 Hz), 111.59, 70.97, 61.23, 58.81, 18.96 ppm. HRMS (ESI) calculated
1
for C H FN O [M]: 409.1802; found: 409.1804. LCMS R = 0.644 min, ESꢀMS m/z = 410.3
2
3
24
3
3
T
+
[
M+H] .
ASSOCIATED CONTENT
Supporting Information. Experimental procedures and spectroscopic data for additional
compounds, detailed molecular pharmacology, DMPK, and behavioral methods, ancillary
1
13
pharmacology profile for compound 58, H and C spectra for compound 58 and its associated
intermediates
AUTHOR INFORMATION
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