Discovery and Characterization of 1H-Pyrazol-5-yl-2-phenylacetamides as Novel, Non-Urea-Containing GIRK1/2 Potassium Channel Activators

Article abstract of DOI:10.1021/acschemneuro.7b00217

The G protein-gated inwardly-rectifying potassium channels (GIRK, K_ir3) are a family of inward-rectifying potassium channels, and there is significant evidence supporting the roles of GIRKs in a number of physiological processes and as potential targets for numerous indications. Previously reported urea containing molecules as GIRK1/2 preferring activators have had significant pharmacokinetic (PK) liabilities. Here we report a novel series of 1H-pyrazolo-5-yl-2-phenylacetamides in an effort to improve upon the PK properties. This series of compounds display nanomolar potency as GIRK1/2 activators with improved brain distribution (rodent K_p > 0.6).

Full text of DOI:10.1021/acschemneuro.7b00217

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Letter
Discovery and characterization of 1H-pyrazol-5-yl-2-phenylacetamides
as novel, non-urea containing GIRK1/2 potassium channel activators
Joshua M. Wieting, Anish K. Vadukoot, Swagat H. Sharma, Kristopher K. Abney, Thomas M. Bridges,
J. Scott Daniels, Ryan D. Morrison, Kevin Wickman, C. David Weaver, and Corey R. Hopkins
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00217 • Publication Date (Web): 11 Jul 2017
Downloaded from http://pubs.acs.org on July 11, 2017
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Discovery and characterization of 1H-pyrazol-5-
yl-2-phenylacetamides as novel, non-urea
containing GIRK1/2 potassium channel
activators
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Joshua M. Wieting†, Anish K. Vadukoot‡, Swagat Sharma‡, Kristopher K. Abney∫,
Thomas M. Bridges∞, J. Scott Daniels∮, Ryan D. Morrison∮, Kevin Wickman∇, C. David
Weaver§,∆, and Corey R. Hopkins‡,*
†Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University, Nashville, TN 37232,
‡Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center,
Omaha, NE 68198, ∫Neuroscience and Pharmacology, Meharry Medical College, Nashville, TN 37208,
∞Vanderbilt Center for Neuroscience Drug Discovery, Nashville, TN 37232, ∮Sano Informed Prescribing,
∇
Franklin, TN 37067, Department of Pharmacology, University of Minnesota, Minneapolis, MN 55455,
§Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232,
∆Vanderbilt Institute of Chemical Biology, Nashville, TN 37232
Abstract: The G protein regulated inwardly rectifying potassium channels (GIRK, Kir3)
are a family of inwardꢀrectifying potassium channels, and there is significant evidence
supporting the roles of GIRKs in a number of physiological processes and as potential
targets for numerous indications. Previously reported urea containing molecules as
GIRK1/2 preferring activators have had significant pharmacokinetic liabilities. Here we
report a novel series of 1Hꢀpyrazoloꢀ5ꢀylꢀ2ꢀphenylacetamides in an effort to improve
upon the PK properties. This series of compounds display nanomolar potency as
GIRK1/2 activators with improved brain distribution (rodent Kp > 0.6).
KEYWORDS: Kir3, GIRK, activator, thallium flux, pharmacokinetics
The G proteinꢀgated regulated inwardly rectifying potassium channels (GIRK,
Kir3) are a family of inwardꢀrectifying potassium channels and are key effectors in Gꢀ
proteinꢀcoupled receptor cellular signaling pathways.1 GIRK channels are tetrameric
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complexes formed by homoꢀ and heteroassembly among four related subunits (GIRK1ꢀ
4 or Kir3.1ꢀ3.4), which are encoded by the genes KCNJ3, KCNJ6, KCNJ9, and KCNJ5,
respectively.2, 3 The GIRK1/2 channel subtype is the most common within the brain,
while other subunit combinations have more limited brain distribution; the GIRK1/4
subtype is primarily found in the heart atria, where it plays a key role in regulating heart
rate and has been a potential target for the treatment of atrial fibrillation.4 There is
significant evidence supporting the roles of GIRK channels in a number of physiological
processes and as potential targets for numerous indications, such as pain perception,
epilepsy, reward/addiction and anxiety.1, 5ꢀ7 Unfortunately, due to a dearth of selective
pharmacological tool compounds, the roles of GIRK channels as potential therapeutic
targets are not well understood. Herein, we report on a new class of GIRK1/2 channel
activators with improved pharmacokinetic properties over the previously reported ureaꢀ
class of activators.
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Previous reports of GIRK1/2 channel activators from our laboratory started from
a highꢀthroughput screen of the molecular libraries small molecule repository (MLSMR)
compound collection – part of the Molecular Libraries Screening Center Network
(MLSCN).8 This screen provided the ureaꢀcontaining compounds, which led to the
discovery of 1 (Figure 1).9, 10 The urea scaffold has been a rich source of a variety of
GIRK1/2 channel activators, as well as ‘molecular switches’ leading to the mode of
pharmacology converting from activator to inhibitor as well as compounds with varying
selectivity profiles.11, 12 Although the urea scaffold has led to the identification of
compounds with in vivo activity in an antiepileptic model in mice, these compounds
suffer from significant pharmacokinetic liabilities, namely poor brain penetration as well
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as only modest selectivity against the GIRK1/4 channel subtype.9, 10 In addition to the
urea scaffold, an alternative amideꢀcontaining scaffold was reported that resulted from
the same MLSCN HTS that yielded the urea scaffold.13 A medicinal chemistry effort
focused on the amide scaffold led to the discovery of 2; however, this scaffold was
significantly less potent as a GIRK1/2 channel activator, as compared to 1, and it was
equipotent as an activator against the GIRK1/4 channel.13 Mining this previous data led
us to postulate that it may be possible to merge these two scaffolds – utilizing the
pyrazole ‘privileged scaffold’ from the urea scaffold with the phenylacetamide portion of
2 to develop novel amide compounds that may allow for improved brain penetration as
well as improved selectivity for GIRK1/4 (selectivity criteria of >10ꢀ30ꢀfold). The results
of this effort are presented below.
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Figure 1. Structures of the previously reported GIRK1/2 activators and the merged
pyrazolꢀ5ꢀylphenylacetamides.
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RESULTS AND DISCUSSION
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The synthesis of the pyrazolꢀ5ꢀylphenylacetamide analogs is outlined in Scheme
1. The appropriately substituted hydrazine, 3, was reacted with 3ꢀaminobutꢀ2ꢀenenitrile,
4, under acidic conditions to generate the substituted aminopyrazoles, 5 in modest
yield.14 The final compounds (7 – 10) were obtained by PyClU amide coupling of the
acid15, 6, with the newly formed (or commercially available) aminopyrazoles, 5.
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Scheme 1. Synthesis of the pyrazolꢀ5ꢀylphenylacetamide analogs.
The initial SAR library focused on maintaining the substituent on the pyrazole
constant (benzyl) and evaluating a variety of acetamides to test whether these were
viable replacements of the urea moiety (Table 1). The initial 3,4ꢀdifluoro comparator,
7a, was tested and was shown to lose significant activity compared to the urea analog,
1 (EC50 = 1.97 ꢀM vs. EC50 = 0.073 ꢀM). Replacing the 3,4ꢀdifluorophenyl with a 3ꢀ
chloroꢀ4ꢀfluorophenyl, 7b, produced an ~4ꢀfold increase in potency (EC50 = 0.478 ꢀM);
which is still ~5ꢀfold less potent than the urea analog, but did improve upon the potency
of the original amide scaffold (2, EC50 = 1.1 ꢀM). However, 7b, provided a slight
improvement in the selectivity for GIRK1/2 versus GIRK1/4 channel subtypes (~4ꢀfold
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selectivity vs. ~2ꢀfold selectivity for 1). Unfortunately, other modifications of the phenyl
portion did not lead to an improvement over 7b. The unsubstituted phenyl derivative
(7c, EC50 = >10 ꢀM) lost significant activity and we were able to regain some of the
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activity with other substituents (4ꢀbromoꢀ3ꢀfluoro, 7d, EC50 = 1.35 ꢀM; naphthyl, 7f, EC50
= 1.05 ꢀM; 3,4ꢀdichloro, 7g, EC50 = 1.45 ꢀM), but none of these compounds produced a
molecule as active as the urea analog, 1.
Table 1. SAR evaluation of the initial pyrazolꢀ5ꢀylphenylacetamides.
GIRK1/2
(ꢀM ± SEMa;
% ± SEMb)
GIRK1.4
(ꢀM ± SEMa;
% ± SEMb)
Cmpd
1
R
0.073 ± 0.007;
96.0 ± 1.1
0.150 ± 0.021;
94.0 ± 1.5
1.97 ± 0.33;
74.7 ± 7.8
0.478 ± 0.077;
94.5 ± 5.5
>10;
3.98 ± 0.19;
46.0 ± 4.0
2.0 ± 0.8;
100 ± 0
>10;
7a
7b
7c
>25
>15
1.35 ± 0.17;
36.3 ± 4.9
2.75 ± 0.74;
17.0 ± 0.7
7d
7e
inactive
inactive
1.05 ± 0.14;
32 ± 1%
1.45 ± 0.35;
62.0 ± 5.5%
>6.9;
1.32 ± 0.22;
12.0 ± 0.3%
2.01 ± 0.41;
35.0 ± 3.0%
>10;
7f
7g
7h
7i
>56%
>42%
inactive
inactive
a
Potency values were obtained from triplicate determinations;
values are average of n = 3. b Reported efficacy values shown are
obtained from triplicate determinations; values are average of n =
3 and are normalized to a standard compound, 1.
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The next round of SAR evaluation was focused on the pyrazole Nꢀsubstituent
(Table 2). For this evaluation, we chose to hold the 3ꢀchloroꢀ4ꢀfluorophenylacetamide
constant in order to obtain a frame of reference for potency comparisons. Replacing the
benzyl group with cyclohexyl, 8a, proved to be a productive change as the potency
increased ~3ꢀfold over the Nꢀbenzylpyrazole analog (8a, EC50 = 0.165 ꢀM vs. 7b, EC50
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= 0.478 ꢀM). The potency was maintained after the insertion of a flexible methylene
linker to form the cyclohexylmethyl substituent, 8b (EC50 = 0.135 ꢀM). These changes,
although offering an improvement in GIRK1/2 channel potency, did not dramatically
change the selectivity profile of the compounds. Eliminating the cyclohexyl ring and
replacing with the secꢀbutyl group, 8c, led to a loss of activity (EC50 = 0.536 ꢀM);
however, it was similar in potency to the original benzyl analog. Interestingly, addition
of a heteroatom to moieties in this region of the molecule led to significant reduction in
activity. Namely, addition of a nitrogen to the benzyl moiety (i.e., 4ꢀpyridylmethyl, 8d,
EC50 >10 ꢀM) led to an ~20ꢀfold reduction compared to the benzyl counterpart; and
addition of an oxygen to the cyclohexyl analog (i.e., 4ꢀpyran, 8e, EC50 = 3.07 ꢀM) also
led to an ~20ꢀfold reduction in activity. Evaluation of additional alkyl (branched or
unbranched, 8g-l) led to the identification of other submicromolar compounds (8j, EC50
= 0.840 ꢀM; 8k, EC50 = 0.483 ꢀM; 8l, EC50 = 0.684 ꢀM), signifying tolerance for flexible
groups. However, none of these compounds improved upon the potency or selectivity
compared to the cyclohexyl compounds. In addition, cycloalkyl groups were also
investigated both directly attached to the pyrazole and those with a methylene spacer
group. Contracting the ring system from a 6ꢀmembered ring to 3ꢀ, 4ꢀ or 5ꢀmembered
rings was deleterious to the activity, with only the cyclopentyl group showing
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midnanomolar activity (8p, EC50 = 0.518 ꢀM). Lastly, direct branched alkyl groups (tꢀ
Bu, 8o, inactive; iꢀPr, 8q, EC50 = 1.06 ꢀM) were either inactive or showed significant
loss of activity.
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Table 2. Evaluation of the pyrazole Nꢀsubstituent.
GIRK1/2
(ꢀM ± SEM;
% ± SEM)
GIRK1.4
(ꢀM ± SEM;
% ± SEM)
Cmpd
8a
Structure
0.165 ± 0.012;
87.3 ± 0.9%
0.720 ± 0.213;
50.5 ± 5.5%
0.135 ± 0.024;
82.0 ± 3.5%
0.536 ± 0.093;
99.5 ± 3.5%
>10;
0.534 ± 0.285;
52.3 ± 7.4%
3.26 ± 0.44;
111 ± 4%
>10;
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8c
8d
>42%
>25%
3.07 ± 0.79;
86.0 ± 5.6%
>10;
>35%
8e
8f
5.78 ± 1.52;
66.8 ± 6.8%
>10;
>33%
5.58 ± 0.66;
77.0 ± 11.4%
2.63 ± 0.73;
97.3 ± 10.4%
1.40 ± 0.27;
89.7 ± 9.1%
0.840 ± 0.180;
82.3 ± 5.8%
0.483 ± 0.084;
57.3 ± 6.3%
0.684 ± 0.072;
69.5 ± 5.3%
>10;
>10;
>63%
8g
8h
8i
6.70 ± 1.68;
100.3 ± 10.0%
5.93 ± 1.36;
80.0 ± 4.6%
1.95 ± 0.52;
91.7 ± 8.4%
1.49 ± 0.31;
55.3 ± 2.5%
1.74 ± 0.13;
30.5 ± 8.5%
>10;
8j
8k
8l
8m
8n
8o
8p
>34%
1.52 ± 0.41;
102.0 ± 5.5%
>18%
5.40 ± 1.34;
97.7 ± 6.8%
inactive
inactive
0.518 ± 0.091;
87.0 ± 7.3%
1.06 ± 0.34;
101 ± 2%
2.15 ± 0.56;
80.3 ± 8.5%
8.50 ± 1.50;
67.5 ± 0.5%
8q
a Potency values were obtained from triplicate determinations;
values are average of n = 3. b Reported efficacy values shown
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are obtained from triplicate determinations; values are average
of n = 3 and are normalized to a standard compound, 1.
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In order to more fully explore the scope of the phenylacetamide substitution
tolerance as well as other urea replacements, we synthesized a number of alternative
analogs (Table 3). The branching of the methylene group of the phenylacetamide was
explored with both quaternary center (cyclopropyl, 9a) and (R)ꢀ and (S)ꢀmethyl
substituents (9b and 9c). Truncation of the phenylacetamide to the benzamide (9c) as
well as reverse amide analogs (9e and 9f), both with and without the methylene spacer,
and finally squaramide derivatives as complete replacements were all examined.
Unfortunately, none of these derivatives were active.
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Table 3.
SAR of substituted phenylacetamides, reverse amides and urea
replacements.
GIRK1/2
(ꢀM ± SEM;
% ± SEM)
GIRK1.4
(ꢀM ± SEM;
% ± SEM)
Cmpd
9a
Structure
inactive
inactive
inactive
inactive
inactive
inactive
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9c
inactive
inactive
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inactive
inactive
inactive
inactive
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inactive
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values are average of n = 3. Reported efficacy values shown
are obtained from triplicate determinations; values are average
of n = 3 and are normalized to a standard compound, 1.
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Having established from the previous SAR evaluations that the cyclohexylmethyl
and cyclohexyl substituents on the pyrazole were the most productive in terms of
activity, we next more deeply investigated the SAR around the acetamide portion of the
molecule (Table 4). As seen previously, the 3,4ꢀdifluorophenyl derivative, 10a, was an
active compound (EC50 = 0.149 ꢀM) and was a significant improvement over the initial
analog, 7a (EC50 = 1.97 ꢀM), and offered a slight improvement in selectivity (~5.7ꢀfold
selective over the GIRK1/4 subtype). Both the 2ꢀchloroꢀ4ꢀfluoro, 10b (EC50 = 0.343
ꢀM), and 4ꢀchloroꢀ2ꢀfluoro, 10c (EC50 = 0.382 ꢀM), derivatives were active, but were
less active than the 3,4ꢀsubstituted variants (8b or 10a). This disparity in activity is also
seen in the 2ꢀfluoroꢀ4ꢀtrifluormethoxy (10d, EC50 = 0.600 ꢀM) versus the 4ꢀfluoroꢀ3ꢀ
trifluoromethoxy analog (10g, EC50 = 0.245 ꢀM). Further carbon substitutions were also
tolerated with naphthalene (10f, EC50 = 0.405 ꢀM) and 2ꢀmethylphenyl acetamide (10i,
EC50 = 0.293 ꢀM) having subꢀ500 nM activity.
Replacing the phenyl group with a variety of heterocyclic analogs was not as
productive. The initial analog, 2ꢀchloropyridineꢀ4ꢀyl acetamide, 10j, retained most of the
activity (EC50 = 0.396 ꢀM); however, many of the other derivatives lost significant
activity or were inactive. These include additional pyridine analogs (10k-l) and other 5ꢀ
membered ring heteroaryl groups (10m-o) and indole derivatives (10p-q). Further
replacement with cycloalkyl groups (indane, 10r; difluorocyclopropyl, 10s) were also not
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productive analogs as they both lost activity. Lastly, a few analogs with the Nꢀ
pyrazolecyclohexyl group were also evaluated (10u-z). As was seen previously, the
cyclohexyl moiety produced compounds with similar range of potencies as the
cyclohexylmethyl derivatives, and it was not obvious from the potency data that the
cyclohexyl derivatives offered any advantages. However, the one analog where a
dramatic increase in potency was observed was the 6ꢀmethylpyridinꢀ3ꢀyl acetamide,
(10w, EC50 = 0.333 ꢀM vs. 10k, EC50 = >9.4 ꢀM). Although the potency improvement
was not obvious, a few cyclohexyl derivatives were more selective against GIRK1/4
(10w, 14ꢀfold selectivity; 10x, 12ꢀfold selectivity; 10z, inactive against GIRK1/4) than
their previous counterparts.
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Table 4. Further modification of phenylacetamide and Nꢀpyrazole substituents.
GIRK1/2
(ꢀM ± SEM;
% ± SEM)
0.149 ± 0.009;
71.3 ± 2.6%
GIRK1.4
(ꢀM ± SEM;
% ± SEM)
0.850 ± 0.475;
47.3 ± 7.5%
Cmpd
10a
R
R1
0.343 ± 0.127;
50.0 ± 4.6%
0.462 ± 0.019;
21.3 ± 3.3%
10b
10c
10d
10e
0.382 ± 0.165;
51.0 ± 2.3%
1.01 ± 0.05;
28.7 ± 4.7%
0.600 ± 0.201;
53.7 ± 2.9%
1.22 ± 0.03;
27.3 ± 3.7%
0.800 ± 0.130;
50.0 ± 5.0%
0.925 ± 0.220;
18.7 ± 2.8%
0.405 ± 0.105;
59.0 ± 5.6%
0.245 ± 0.063;
81.3 ± 4.4%
0.126 ± 0.013;
82.3 ± 3.5%
0.923 ± 0.162;
28.0 ± 5.3%
0.751 ± 0.142
65.3 ± 0.9%
0.295 ± 0.071;
75.0 ± 3.6%
10f
10g
10h
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0.293 ± 0.085;
44.0 ± 2.1%
0.930 ± 0.222;
29.7 ± 4.6%
10i
0.396 ± 0.029;
79.3 ± 2.0%
>9.4;
1.28 ± 0.02;
61.3 ± 4.7%
10j
10k
10l
inactive
inactive
>40%
>10;
>32%
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<10;
>56%
2.38 ± 0.77;
61.7 ± 3.4%
0.892 ± 0.107;
59.5 ± 0.5%
>10;
>21%
<10;
10m
10n
10o
>21%
2.59 ± 0.25;
28.0 ± 3.3%
inactive
inactive
inactive
10p
10q
inactive
inactive
inactive
inactive
10r
10s
10t
1.10 ± 0.04;
25.5 ± 0.5%
2.04 ± 0.74;
73 ± 21%
>10;
>79%
2.30 ± 0.02;
91.0 ± 7.3%
>10;
>60%
10u
10v
1.01 ± 0.30;
83 ± 12%
>10;
>54%
0.333 ± 0.023;
75 ± 0%
0.150 ± 0.020;
75.0 ± 2.5%
0.392 ± 0.072;
76.0 ± 7.5%
4.79 ± 2.78;
23.5 ± 4.5%
1.79 ± 1.07;
37.7 ± 1.9%
2.06 ± 0.42;
41.0 ± 2.5%
10w
10x
10y
0.505 ± 0.175;
50.0 ± 9.5%
inactive
10z
a
Potency values were obtained from triplicate determinations; values are
average of n = 3. b Reported efficacy values shown are obtained from triplicate
determinations; values are average of n = 3 and are normalized to a standard
compound, 1.
Having identified a number of unique 1Hꢀpyrazolꢀ5ꢀylꢀ2ꢀphenylacetamide
GIRK1/2 channel activators, we further profiled selected compounds in in vitro DMPK
assays to assess their human and mouse liver microsomal intrinsic clearance and
plasma protein binding. All but a few compounds evaluated displayed high intrinsic
clearance and were predicted to have high hepatic clearance in vivo (CLHEP > 75%
QH).16, 17 Both compounds 8e and 8f were predicted to have moderate clearance
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(~<50% QH) in both human and mouse. Interestingly, both of these compounds were
cycloheteroalkyl substituents (8e, 4ꢀtetrahydropyran; 8f, tetrahydrothiopheneꢀ1,1ꢀ
dioxide). Using equilibrium dialysis, the plasma protein binding of the selected
compounds was determined in human and mouse plasma.18 The results revealed that
the compounds had varying degrees of free fraction in human (Fu: 0.003 to 0.348) and
mouse (Fu: 0.010 to 0.423) plasma; however, a number of compounds were unstable in
mouse plasma. Interestingly, both 8e and 8f displayed the highest free fraction in
human and mouse, and coupled with the predicted clearance these compounds
possess the most attractive in vitro DMPK profile; unfortunately, these compounds are
not very active as GIRK1/2 channel activators. However, these substituents may inform
the next round of compound optimization. Further compounds for evaluation (10f–10y)
all exhibited high clearance and many of these compounds were unstable in mouse
plasma. Lastly, we evaluated a smaller set of selected compounds (7b, 8a, 8c) in a
mouse IP cassette study in order to assess their ability to cross the bloodꢀbrain barrier
(BBB).19, 20 All of the compounds were brain penetrant with Kp values > 0.5 – 0.9. This
represents a significant improvement over the previously reported urea scaffolds where
many of those analogs had low brain distribution (Kp < 0.2).
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Table 5. In vitro and in vivo DMPK properties of selected compounds.
Plasma Protein
Binding (Fu)b
GIRK1/2
Intrinsic Clearance (mL/min/kg)
a
a
Cmpd
7b
8a
8b
8c
8e
8f
8h
10f
(ꢀM ± SEM)
0.478 ± 0.077
0.165 ± 0.012
0.135 ± 0.024
0.536 ± 0.093
3.07 ± 0.79
5.78 ± 1.52
2.63 ± 0.73
0.405 ± 0.105
hCLINT
ND
hCLHEP
ND
mCLINT
690
mCLHEP
79.6
Human
ND
Mouse
0.071
0.136
*
246
19.3
20.7
18.5
11.1
8.50
14.6
20.8
622
78.6
88.7
83.0
43.9
15.0
79.2
88.7
0.009
0.003
0.016
0.211
0.348
0.054
0.003
1390
156
23.6
14.3
47.7
2080
>5930
1070
85.7
18.0
657
0.172
0.423
0.362
0.171
*
>5930
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0.245 ± 0.063
0.126 ± 0.013
2.04 ± 0.74
0.333 ± 0.023
0.150 ± 0.020
0.392 ± 0.072
1900
648
>2770
139
1020
92.5
20.7
20.3
20.8
18.2
20.6
17.1
>5930
2970
>5930
990
>5930
2079
88.7
87.4
88.7
82.5
88.7
86.3
0.002
0.020
0.003
0.021
0.013
0.029
0.010
0.071
*
0.125
*
10g
10h
10t
10w
10x
10y
*
Mouse Cassette (IP, 0.25 mg/kg, 0.25 h)
c
Plasma (ng/mL)
Brain (ng/g)
63.4
Kp
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87.6
190
0.61
0.87
0.55
7b
8a
8c
75.5
104
aPredicted hepatic clearance based on intrinsic clearance in mouse and human liver microsomes using the
well-stirred organ CL model (binding terms excluded). bFu = fraction unbound. Kp = total brain:plasma
c
ratio. *unstable in mouse plasma.
CONCLUSION
In conclusion, we have identified a novel series of 1Hꢀpyrazolꢀ5ꢀylꢀ2ꢀ
phenylacetamides as brainꢀpenetrant GIRK1/2 channel activators. The genesis of this
series of compounds originated from a merging of two previously disclosed scaffolds
that contained a urea moiety and an amide moiety. SAR around the pyrazole identified
the cyclohexyl and cyclohexylmethyl groups as the optimal substituents. The acetamide
portion allowed for more structural diversity, incorporating substituted phenyl and
heteroaryl groups culminating in number of compounds with EC50s between 100 and
200 nM. An improvement in the channel subtype selectivity profile was also seen for
selected compounds; however, a universal improvement has not been realized at this
point. Lastly, a number of compounds evaluated displayed moderateꢀhigh brain
distribution (Kp = >0.5), which signifies an improvement over the previous GIRK1/2
channel activators that have been reported. Further in vivo evaluation of the lead
molecules in models of anxiety is onꢀgoing and will be reported in due course.
METHODS
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General. All NMR spectra were recorded on a 400 MHz AMX, AVꢀ400 or Avance III HD
500 MHz Bruker NMR spectrometer. 1H and 13C chemical shifts are reported in δ values
in ppm downfield with the deuterated solvent as the internal standard. Data are reported
as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, b =
broad, m = multiplet), integration, coupling constant (Hz). Low resolution mass spectra
were obtained on an Agilent 6120 or 6150 with ESI source. Method A: MS parameters
were as follows: fragmentor: 70, capillary voltage: 3000 V, nebulizer pressure: 30 psig,
drying gas flow: 13 L/min, drying gas temperature: 350 oC. Samples were introduced via
an Agilent 1290 UHPLC comprised of a G4220A binary pump, G4226A ALS, G1316C
TCC, and G4212A DAD with ULD flow cell. UV absorption was generally observed at
215 nm and 254 nm with a 4 nm bandwidth. Column: Waters Acquity BEH C18, 1.0 x 50
mm, 1.7 um. Gradient conditions: 5% to 95% CH3CN in H2O (0.1% TFA) over 1.4 min,
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o
hold at 95% CH3CN for 0.1 min, 0.5 mL/min, 55 C. Method B: MS parameters were as
follows: fragmentor: 100, capillary voltage: 3000 V, nebulizer pressure: 60 psig, drying
o
gas flow: 12 L/min, drying gas temperature: 350 C. Samples were introduced via an
Agilent 1260 HPLC comprised of a degasser, G7111A quaternary pump, G4237A ALS,
G7116A TCC, G1314 VWD with a ULD flow cell. UV absorption was generally observed
at 215 nm and 254 nm with a 4 nm bandwidth. Column: Poroshell 120, SBꢀC18, 4.6 x
75 mm, 2.7 mm. Gradient conditions: 5% to 80% CH3CN in H2O (0.1% TFA) from 0 – 4
min, 80 to 95% CH3CN in H2O (0.1% TFA) from 4 – 6 min, hold at 95% CH3CN from 6 –
o
8 min, 95% CH3CN for 0.1 min, 1.5 mL/min, 45 C. High resolution mass spectra were
obtained on a Waters Micromass® QꢀTOF with ESI source. Solvents for extraction,
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washing and chromatography were HPLC grade. All reagents were purchased from
commercial sources and were used without purification.
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Materials and Methods for in vitro Pharmacology
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Thallium flux assays were preformed essentially as previously described (Kaufmann et
al). Briefly, HEKꢀ293 cells expressing either GIRK1 and GIRK2 or GIRK1 and GIRK4
were cultured in αꢀMEM (Corning, Corning, NY) containing 10% (v/v) fetal bovine serum
(Thermo Fisher Scientific, Waltham, MA) plus 1x glutagro (Corning, Corning, NY)
(referred to hereafter as cell culture medium) at 37 Co in a humidified 5% CO2
atmosphere. Cells at ~90% confluence were dislodged from the tissue culture vessel
using TrypLE Express (Thermo Fisher Scientific, Waltham, MA) and plated at a density
of 20,000 cells/well in 20 ꢀL/well cell culture medium in 384ꢀwell, clearꢀbottom, blackꢀ
walled, BD PureCoat Amine plates (Corning, Corning, NY) and incubated over night at
37o C in a humidified 5% CO2 atmosphere. On the day of assay the medium was
removed from the plates and replaced with 20 ꢀL/well of a solution containing assay
buffer (Hanks Buffered Saline Solution (Thermo Fisher Scientific, Waltham, MA) plus 10
mM HEPES (Thermo Fisher Scientific, Waltham, MA )ꢀNaOH, pH 7.2), 1 ꢀM Thallos
(TEFlabs, Austin, TX), 0.5% DMSO and 0.036% Pluronic Fꢀ127 (SigmaꢀAldrich, St.
Louis, MO). Cell plates containing Thallos solution were incubated 1 h at room
temperature. Following incubation the Thallosꢀcontaining solution was replaced with 20
ꢀL/well assay buffer. The Thallosꢀloaded cell plates were transferred to a Panoptic
kinetic imaging plate reader (WaveFront Biosciences, Franklin, TN). Images acquired at
1 Hz, 480/40 nm excitation and 538/40 nm emission were collected for 10 s after which
time 20 ꢀL/well of assay buffer containing test compounds at 2ꢀfold over their final
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concentrations were added. Imaging continued for 4 min at which time 10 ꢀL/well of a
solution containing 125 mM NaHCO3, 1.8 mM CaSO4, 1 mM MgSO4, 5 mM glucose,
and 2 mM Tl2SO4, 10 mM HEPESꢀNaOH pH 7.2 was added and images were collected
for an additional 2 min. To quantify test compound effects on GIRK channel activity, the
initial slopes of the thalliumꢀevoked changes in fluorescence were fit to a 4ꢀparameter
logistic equation using the Excel (Microsoft, Redmond, WA) plugin XLfit (IDBS,
Guildford, UK) to obtain potency and efficacy values. Efficacies are relative to a
maximally effective concentration of 1, our most potent and effective activator which
shows low selectivity between GIRK1/2 and GIRK1/4 channel subtypes. Tenꢀpoint
concentration series from 30 ꢀM to 1.5 nM were generated using an Echo liquid handler
(Labcyte, San Jose, CA). Final DMSO concentration, 0.24% (v/v), in the assay was
constant across all compound concentrations. Unless otherwise indicated, all buffer
salts were obtained from SigmaꢀAldrich, St. Louis, MO.
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General Procedure for Amide Couplings. A one dram vial equipped with magnetic
stir bar and screw cap vial was charged with the appropriate phenyl acetic acid, 6,
derivative (0.10 mmol), chlorodipyrrolidinocarbenium hexafluorophosphate (PyClU) (33
mg, 0.10 mmol), DMF (0.3 mL), Hunig’s Base (0.1 mL, 0.57 mmol) and was stirred for
approximately 5 minutes. The appropriate 5ꢀAmino pyrazole, 5, (0.10 mmol) was added
and the reaction stirred until LCMS analysis indicated significant consumption of the
starting materials (10 minsꢀ18 hours). The crude reaction mixture was purified via massꢀ
directed acidic reverse phase preparative HPLC to yield the corresponding amide
products.
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2-(3-cyano-4-fluorophenyl)-N-(1-(cyclohexylmethyl)-3-methyl-1H-pyrazol-5-
yl)acetamide (10h): Following the general procedure above, compound (10h) was
obtained (25.0 mg, 69%).
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Analytical LCMS: RT = 4.135 min; >95% @ 254 nm; MS (ESI+) m/z 355.1 [M + H]+.
1H NMR (499 MHz, DMSOꢀd6) δ 9.97 (s, 1H), 7.86 (dd, J = 6.3, 2.2 Hz, 1H), 7.74 (ddd,
J = 8.0, 5.3, 2.3 Hz, 1H), 7.53 (t, J = 9.1 Hz, 1H), 5.96 (s, 1H), 3.74 (s, 2H), 3.66 (d, J =
7.2 Hz, 2H), 2.08 (s, 3H), 1.68 – 1.52 (m, 4H), 1.38 (d, J = 12.8 Hz, 2H), 1.07 (q, J =
9.7, 9.0 Hz, 3H), 0.84 – 0.69 (m, 2H). 13C NMR (126 MHz, DMSOꢀd6) δ 168.54, 145.98,
137.58, 137.50, 136.60, 134.65, 133.87, 133.85, 117.06, 116.91, 114.41, 100.34,
100.22, 98.95, 53.62, 41.12, 38.30, 30.32, 26.33, 25.61, 14.22. HRMS calc’d for
C20H23FN4O: 354.1856; found: 355.1934 [M + H]+.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website
at DOI:
Chemical synthesis of all analogs, in vitro Pharmacology procedures, in vitro PK
methods, in vivo PK methods (PDF).
AUTHOR INFORMATION
Corresponding Author
*Phone: +1ꢀ402ꢀ559ꢀ9729. Fax: +1ꢀ402ꢀ559ꢀ5643. Email: corey.hopkins@unmc.edu
Author Contributions
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C.R.H oversaw and designed the chemistry. J.M.W., A.K.V., and S.S. performed the
synthetic chemistry work. T.M.B., J.S.D., and R.D.M. designed and performed the drug
metabolism and pharmacokinetic in vitro experiments and in vivo studies. K.W. helped
with the manuscript and in vivo studies. C.D.W. designed and analyzed the in vitro
pharmacology experiments. K.K.A. performed and analyzed the in vitro pharmacology
experiments. C.R.H. wrote the manuscript with input from all authors.
Funding
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The work was supported by a NIH Grant 5R01MH107399 (C.R.H., C.D.W., and K.W.).
Notes
C.D.W. is the president of WaveFront Biosciences, the maker of the Panoptic kinetic
imaging plate reader used to perform the in vitro pharmacology experiments. C.D.W.
obtains royalties from the sale of Thallos thalliumꢀsensitive fluorescent dye used for the
in vitro pharmacology experiments. All other authors declare no competing financial
interest.
ACKNOWLEDGMENTS
The authors would like to thank Sichen Chang and Xiaoyan Zhan for their contributions
to the DMPK screening tier assays.
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