First-Time Disclosure of CVN424, a Potent and Selective GPR6 Inverse Agonist for the Treatment of Parkinson’s Disease: Discovery, Pharmacological Validation, and Identification of a Clinical Candidate

Article abstract of DOI:10.1021/acs.jmedchem.0c02081

Parkinson’s disease (PD) is a chronic and progressive movement disorder with the urgent unmet need for efficient symptomatic therapies with fewer side effects. GPR6 is an orphan G-protein coupled receptor (GPCR) with highly restricted expression in dopami

Full text of DOI:10.1021/acs.jmedchem.0c02081

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Drug Annotation
First-Time Disclosure of CVN424, a Potent and Selective GPR6
Inverse Agonist for the Treatment of Parkinson’s Disease: Discovery,
Pharmacological Validation, and Identification of a Clinical
Candidate
Huikai Sun,* Holger Monenschein,* Hans H. Schiffer, Holly A. Reichard, Shota Kikuchi, Maria Hopkins,
Todd K. Macklin, Stephen Hitchcock, Mark Adams, Jason Green, Jason Brown, Sean T. Murphy,
Nidhi Kaushal, Deanna R. Collia, Steve Moore, William J. Ray, Mark Beresford Lewis Carlton,
and Nicola L. Brice
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ABSTRACT: Parkinson’s disease (PD) is a chronic and progressive movement disorder with the urgent unmet need for efficient
symptomatic therapies with fewer side effects. GPR6 is an orphan G-protein coupled receptor (GPCR) with highly restricted
expression in dopamine receptor D2-type medium spiny neurons (MSNs) of the indirect pathway, a striatal brain circuit which
shows aberrant hyperactivity in PD patients. Potent and selective GPR6 inverse agonists (IAG) were developed starting from a low-
potency screening hit (EC50 = 43 μM). Herein, we describe the multiple parameter optimization that led to the discovery of multiple
nanomolar potent and selective GPR6 IAG, including our clinical compound CVN424. GPR6 IAG reversed haloperidol-induced
catalepsy in rats and restored mobility in the bilateral 6-OHDA-lesioned rat PD model demonstrating that inhibition of GPR6
activity in vivo normalizes activity in basal ganglia circuitry and motor behavior. CVN424 is currently in clinical development to treat
motor symptoms in Parkinson’s disease.
Replacing DA with levodopa (L-DOPA), a prodrug of DA, is
still the gold standard treatment to manage motor symptoms
of PD.7,8 However, long-term use can result in significant
dyskinesia and thus substantially decreases the quality of life of
patients.9,10 Other dopaminergic therapies with clinical
effectiveness have been developed: catechol-O-methyltransfer-
ase (COMT) inhibitors,11 monoamine oxidase-B (MAO)
inhibitors,12 and dopamine receptor agonists,13 they but are
associated with various side effects.14 Multiple non-dopami-
nergic therapies for PD have been developed,15 including the
adenosine A2A antagonist istradefylline (KW6002), which
achieved approval in the United States and Japan as an
INTRODUCTION AND BIOLOGICAL RATIONALE
■
Parkinson’s disease (PD) is a chronic and progressive
movement disorder.1−3 Neurodegeneration and loss of
dopamine-producing neurons in the substantia nigra pars
compacta is one of the hallmarks of PD pathology and leads to
dopamine (DA) depletion in the striatum.4,5 On the basis of
the classical model of basal ganglia function, dopamine release
into the striatum fine-tunes basal ganglia output by modulating
two opposing motor control circuits in the brainthe direct
(striatonigral) and the indirect (striatopallidal) pathways.6 In
the striatum, direct pathway medium spiny neurons expressing
dopamine D1 receptors (D1-type MSN) are intermingled with
indirect pathway MSN expressing dopamine D2 receptors
(D2-type MSN). Loss of DA in PD is believed to cause
hypoactivity in the direct and hyperactivity in the indirect
pathways, which produces a net increase of inhibitory
GABAergic output from the basal ganglia and suppresses
movement in PD patients.6
Received: December 1, 2020
https://doi.org/10.1021/acs.jmedchem.0c02081
© XXXX American Chemical Society
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A

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Drug Annotation
Table 1. Major SAR on the Right-Hand Side (RHS) Bicyclic Ring System
a
TR-FRET cAMP inverse agonist assay using a human GPR6 CHO-K1 cell line (Materials and Methods); EC50 values are the means of at least
b
c
two individual experiments with each concentration tested in triplicate. Mixture of two regioisomers (2:1). Efficacy (Ymax) is normalized to the
efficacy of a reference compound, which was validated to inhibit the apparent constitutive activity of GPR6 receptors expressed in cAMP TR-FRET
assays (Supporting Information).
adjunctive therapy to L-DOPA.16 Deep brain stimulation
(DBS) in the globus pallidus or subthalamic nucleus of the
indirect pathway is FDA approved and significantly improves
the motor symptoms of PD.1,17 Importantly, because of the
localized placement of the DBS probes, it avoids modulating
the direct pathway known to be a major driver of L-DOPA-
induced dyskinesia (LID). Therefore, development of a drug
that selectively targets the indirect pathway in PD could avoid
invasive DBS procedures and provide therapeutic benefit
without increased risk of drug-induced dyskinesia.
type MSN.25,26 The phenotype of GPR6 receptor knock out
mice support a role of GPR6 receptors in basal ganglia
function and its potential use as a PD drug target. They display
a two-fold increased phosphorylation of Thr43 of the
DARPP32 protein in the striatum, which is a key regulator
of DA signaling. They also display increased locomotor activity
and a reduced abnormal induced movement (AIM) in a
dyskinesia PD rodent model.23
Herein we report the discovery of the first potent and
selective GPR6 inverse agonists and their pharmacological
validation in preclinical PD behavioral animal models.
GPR6 is a highly constitutively active orphan G-protein
coupled receptor,18−20 with highly restricted expression in the
indirect pathway D2-type MSNs in the striatum21 and minimal
expression in the direct pathway D1-type MSNs, other brain
regions, and peripheral tissues. GPR6 receptors efficiently
couple to Gs proteins in heterologous expression systems and
constitutively stimulate adenylate cyclase activity and cyclic
adenosine monophosphate (cAMP) accumulation in cells in
vitro.18−20 cAMP levels are reduced in striatal tissue derived
from GPR6 knock out mice, which is consistent with GPR6 Gs
protein coupling and cAMP signaling in vivo.22,23 In contrast to
GPR6, dopamine D2 receptors expressed in the indirect
pathway D2-type MSN are Gi protein coupled and inhibit
cAMP signaling.24 We hypothesize that inhibition of GPR6
and D2 receptors coexpressed in the indirect pathway D2-type
MSN antagonize each other with respect to cAMP modulation.
Consequently, targeting GPR6 with antagonists or inverse
agonists could be a novel non-dopaminergic treatment to
improve motor symptoms in PD through selective modulation
of the indirect pathway by compensating for the lack of DA
acting on D2 receptors. In addition, pharmacological inhibition
of GPR6 is expected to have only a low risk for drug-induced
dyskinesia because of the restricted expression in striatal D2-
RESULTS AND DISCUSSION
■
A GPR6-selective high throughput screen (HTS) campaign
was run to identify novel GPR6 inverse agonists as starting
points for medicinal chemistry efforts. In total, 360 000
compounds were screened for inverse agonist activity in a
GPR6-selective, cell-based time-resolved fluorescence reso-
nance energy transfer (TR-FRET) cAMP assay using a stable
and inducible CHO-K1-based cell line expressing human
GPR6 receptor (see Materials and Methods). Only one very
low-potency hit, compound 1 (Table 1), was identified in the
screen and validated in a secondary full concentration−
response repeat assay (EC50 = 43 μM). Compound 1 did not
modulate the baseline cAMP level or forskolin-stimulated
cAMP levels in the uninduced CHO-K1-GPR6 cell line, which
did not express GPR6 (data not shown). Furthermore,
compound 1 did not modulate cAMP signaling in a
cannabinoid receptor CB1-specific counterscreening assay
(data not shown). Structure−activity relationship (SAR)
optimization was initiated with the aim to improve the
potency and druglike properties of compound 1 despite the
fact that it contains a [1,2,5]-oxadiazole-containing bicyclic
B
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Drug Annotation
Table 2. Optimization on the Pyrido[3,4-b]pyrazine Scaffold to Balance Selectivity and ADMET Properties
a
EC50
selectivity over GPR3
(fold)
selectivity over GPR12
(fold)
P450 (2D6)
pIC50
hERG binding % @10
b
analogue
R1
R2
R3
(nM)
uM
cLogP
3c
3d
3e
3f
3g
3h
4a
4b
4c
4d
4e
5-Cl
5-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
c-Pr
c-Pr
c-Pr
c-Pr
i-Pr
i-Pr
c-Pr
i-Pr
i-Pr
c-Pr
i-Pr
H
H
H
Me
H
Me
Me
Me
CN
Me
Me
29
151
39
29
21
51
29
16
55
95
61
638×
15×
3.8
3.2
3.2
3.3
3.4
3.6
3.4
3.7
3.8
3.4
3.7
6.9
7.3
5.4
40
40
23
21×
14×
9×
80×
78×
14×
19×
105×
27×
13×
105×
47×
5×
5.4
5.2
5.2
13
75
75
19×
13×
6×
19×
19×
5.2
5.4
71
a
TR-FRET cAMP inverse agonist assay using a CHO-K1-based cell line; EC50 values are the means of two or more individual experiments with
b
each done in triplicate. cLogP values are calculated with ChemAxon.
aromatic ring, which is a structural feature frequently found in
pan-assay interference compounds (PAINS) that often show
up as false positives in HTS.27
provided the benefit of improved physicochemical properties,
such as lower lipophilicity (cLogP), and higher lipophilic
ligand efficiency (LLE), which are known to correlate with
improved metabolic stability and solubility.
Accordingly, our initial focus was to explore less problematic
bicyclic ring systems with more vectors to explore for SAR and
to remove the structural alert at the onset of the program.
Fortunately, our first attempt to replace the five-membered
oxadiazole ring with an unsubstituted phenyl ring was tolerated
and even provided a modest improvement in potency (3-fold;
EC50 = 14 μM; 2a in Table 1). This new quinoxaline scaffold
served as the basis for further systematic SAR exploration.
While it was found that other small alkyl amines were also
accommodated as cyclopropyl replacements, the cyclopropyl-
amine was superior in terms of both potency and
physicochemical properties (data not shown) in this early
SAR. Next, introduction of small alkyl or phenyl groups on any
carbon of the piperazine ring was not tolerated (data not
shown). Modification of the benzyl group with one or two
substituents showed a more significant impact on the potency
as demonstrated by the initial compound 2b bearing a 2,5-
dichlorophenyl ring (EC50 = 2.1 μM) and providing a seven-
fold improvement in potency compared to 2a, likely as a result
of increased lipophilic interactions of the protein−ligand
complex.
In order to further lower lipophilicity of 3a and 3b (cLogP >
4.5), we conducted an extensive SAR investigation on the
more potent pyrido[3,4-b]pyrazine scaffold, focused on the
western side of the molecule with the goal to replace the two
very hydrophobic chlorine atoms. Gratifyingly, we found that
replacement of the 2-chloro group on that phenyl ring with a
fluorine atom was tolerated (3c in Table 2). In contrast,
replacing both Cl atoms with F atoms at the 2 and 5 position
decreased the potency by five-fold (3d in Table 2). Upon
further investigation into the optimal substitution pattern on
the benzyl moiety, we found that moving the 5-F atom to the 4
position (compound 3e) was much preferred and provided
potency below 50 nM. In addition, the lipophilicity of 3e
(cLogP = 3.2) was now within our acceptable range.
Additional pharmacological and physiochemical properties
of compound 3e are depicted in Table 3. In addition to the
Table 3. Pharmacological and Physicochemical Properties
of Tool Compound 3e
property
value
However, the most significant functional potency improve-
ment during the early phase of the project resulted from the
modification of the quinoxaline phenyl ring. Introduction of a
hydrophobic methyl group (2c) reduced the potency by eight-
fold. Although the introduction of an electron-withdrawing F
atom (2e) or the electron-donating OMe group (2d) was
better tolerated, no sub-micromolar potency breakthrough was
realized. In contrast, a strong electron-withdrawing group like
CN (2f) or dimethyl amide (2g) was found to significantly
enhance the potency to the 100 nM range. Incorporation of a
nitrogen atom into the phenyl ring at either the 6 or 7 position
(3a and 3b) also boosted potency (50 nM), with no
regiochemical preference observed between these two
positions. In addition, this introduction of a ring nitrogen
binding affinity Ki (nM)
functional EC50 (nM)
LLC-PK1-MDR1 A to B (nm/s)
P-gp efflux ratio
cLogP
CNS MPO
7
39
160
0.5
3.2
4.8
cAMP functional potency on GPR6 receptor, 3e possesses
strong binding affinity: Ki = 7.0 nM (in vitro competition
binding assay using a tritium-labeled tracer compound; see
Materials and Methods, and Table 3). Compound 3e also
exhibited high passive permeability and did not behave as a P-
gp substrate as indicated by the low efflux ratio (ER) in the
C
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Drug Annotation
LLC-PK1-MDR1 cell monolayer assay. On the basis of these in
vitro pharmacology properties in combination with acceptable
subcutaneous rodent PK (absorption and central uptakedata
not shown), we were enabled to explore for the first time the
effect of GPR6 inhibition on rodent behavior in pharmacody-
namic PD models.
Haloperidol, a potent dopamine receptor D2 antagonist,
activates striatal D2-type MSN of the indirect pathway, causing
catalepsy in rats. GPR6 IAG 3e dose-dependently (0.1−30
mg/kg sc) reversed haloperidol-induced catalepsy (Figure 1a).
This result suggests that inhibition of GPR6 reduces activity of
the indirect pathway, which is expected to reduce inhibition of
movement.
reverse haloperidol-induced catalepsy in GPR6 knock out
mice, confirming that GPR6 receptor inhibition is mediating
the efficacy of GPR6 IAG 3e to reverse catalepsy in this PD
model. As expected, the adenosine receptor A2A antagonist
KW6002 was able to reverse haloperidol-induced catalepsy in
both wild-type and GPR6 knock out mice (Figure 1b).
Together, these results support the concept that GPR6 IAG
reduce activity of the indirect pathway and might have
potential as a therapeutic to treat motor symptoms of
Parkinson’s disease.
With the results from the catalepsy experiment, our
confidence in the mechanism of action as described earlier
had increased significantly. Therefore, we initiated lead
optimization of compound 3e by posing the question: What
properties of the tool compound need to be improved in order
to develop the candidate suitable for clinical development?
This analysis revealed numerous areas to focus the lead
optimization efforts on, and most of them could be addressed
by applying established medicinal chemistry strategies:
GPR6 knock out mice show similar sensitivity for
haloperidol to induce catalepsy than wild-type mice (Figure
1b). However, compound 3e (30 mg/kg sc) was unable to
• strong cytochrome P450 inhibition (mostly isoform
2D6): likely a result of the exposed eastern N atom
• poor metabolic stability in human microsomes and
hepatocytes leading to unacceptable human PK
predictions
• putative risk for time-dependent clearance inhibition
resulting from the cyclopropyl amine28,29
• insufficient selectivity over the closest neighbors GPR3
and GPR12, two G-protein coupled receptors in the
same GPCR subfamily as GPR630−32
With the above mitigation strategies, we embarked on a few
“quick wins”. Strong inhibition of one cytochrome P450
isoform (2D6) from 3e (pIC50 = 7.3) was diminished through
the strategic introduction of a methyl group on the carbon
adjacent to the pyridyl nitrogen (3f), minimally impacting
cLogP. Unfortunately, this modification also led to the further
erosion of the already poor selectivity ratios over GPR3 and
GPR12. Our initial goal was to keep a minimal 30-fold
selectivity, ideally higher, but with the very poor selectivity
observed from many of our pyridopyrazine analogues
especially against GPR3, this fused bicyclic scaffold was
deprioritized. Moreover, “quick win” attempt number two
failed for the same reasonthe introduction of an isopropyl-
amine group (compound 3g and 3h, Table 2) to replace the
cyclopropyl amine (a potential structural alert) also afforded a
loss of selectivity revealed by pairwise comparison with the
corresponding cyclopropylamine (compound 3e).
As these initial tactics had not proven successful, we
switched our focus toward improving metabolic stability. A
metabolite identification (Met ID) study revealed the major
route of oxidative metabolism takes place at the benzyl-
piperazine subunit via benzylic oxidation and subsequent N-
debenzylation (data not shown). Among many structural
iterations, it was found that a phenolic-piperidine replacement
was tolerated and maintained potency, and importantly, this
structural change appeared to be feasible to reduce oxidative
metabolism as demonstrated by comparing human microsomal
turnover rates of selected pairs such as 3f and 4a (74 mL/min/
kg over 45 mL/min/kg, respectively). The improved metabolic
stability of compounds containing the phenolic-piperidine vs
the benzyl-piperazine was consistent throughout the program.
Interestingly, a slight improvement in the selectivity profiles
versus GPR3 and GPR12 was also observed in pairwise
Figure 1. Studies on compound 3e in a haloperidol-induced catalepsy
model. (a) Compound 3e reverses haloperidol-induced catalepsy in
male rats. Haloperidol-induced catalepsy was measured 120 min after
dosing Sprague-Dawley rats (n = 9−10 per group) at the same time
with haloperidol (1 mg/kg, ip) and either vehicle (10% DMSO, 0.5%
methylcellulose) or compound 3e (0.1, 1, 10, and 30 mg/kg sc) or
positive control compound KW6002 (0.6 mg/kg ip; adenosine A2A
antagonist) treatments. (b) Compound 3e reverses catalepsy in wild-
type (WT) but not in male GPR6 knock out (KO) mice. Haloperidol-
induced catalepsy was measured in wild-type (C57BL/6J) and GPR6
knock out mice 120 min after dosing animals at the same time (n = 6
per group) with haloperidol (1 mg/kg, ip) and vehicle (10% DMSO,
0.5% methylcellulose) or after dosing at the same time with
haloperidol and either compound 3e (30 mg/kg sc) or positive
control compound KW6002 (0.3 mg/kg ip; adenosine A2A
antagonist). Applied statistics: one-way ANOVA using Dunnett’s
post hoc tests, significant effect of genotype difference or treatment as
indicated: *p < 0.05 and **p < 0.01.
D
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Table 4. Saturation of the Right-Hand Side (RHS) Pyridine Ring Leading to the Lead Compound 6h
EC
selectivity over GPR3 selectivity over GPR12
P450 (2D6)
pIC50
hERG binding % @
50a
c
analogue
X-R
CH2
CH2
Y-R
R1
(nM)
(fold)
(fold)
10 μM
cLogP
5a
5b
5c
6a
6b
6c
6d
6e
6f
-NCOMe
-NCOEt
-NSO2Me
CH2
CH2
-NH
-NMe
-NSO2Me
NCOOMe i-Pr
-NCOMe
-NCOMe
i-Pr
i-Pr
i-Pr
i-Pr
c-Pr
i-Pr
i-Pr
i-Pr
27
30
27
96
34
100
89
65
158
43
34
25×
20×
7×
30×
16×
8×
5.4
11
2.3
3.0
1.8
2.3
2.0
2.7
3.1
1.8
3.0
2.0
2.3
<5.0
b
CH2
5.7
-NCOMe
-NCOMe
CH2
CH2
CH2
CH2
CH2
CH2
15×
40×
1.6×
21×
15×
190×
52×
110×
15×
120×
16×
69×
26×
34×
272×
97×
4.5
<5.2
5.4
<5.0
<5.0
5.1
<5.0
5.4
30
58
41
36
10
81
69
6g
6h
c-Pr
i-Pr
a
TR-FRET cAMP inverse agonist assay using a human GPR6 CHO-K1 cell line (Materials and Methods); EC50 values are the means of at least
b
c
two individual experiments with each concentration tested in triplicate. Over P450 isoform 1A2. cLogP values are calculated with ChemAxon.
comparisons for the phenolic-piperidines (4a and 3f, and 4b
and 3h; Table 2). Expanding this SAR briefly to explore
additional potential selectivity gains, the pyridyl nitrogen was
moved from position 6 to 7 (4d and 4a, and 4e and 4b), which
did not afford a change in selectivity, unfortunately. The
introduction of the electron-withdrawing group CN (4c) at the
carbon adjacent to the pyridyl nitrogen decreased the
selectivity versus both GPR3 and GPR12.
The SAR described in Table 2 seemed to be moderately
promising; however, another concerning liability was intro-
duced and had to be carefully monitored. As shown in Table 2,
hERG binding affinity was significantly increased for
compounds (4a,b and 4e).
After only limited success was achieved thus far, we explored
more incisive modifications on the bicyclic aromatic ring
system. We hypothesized that lowering lipophilicity and
increasing fraction sp3 of the rather flat and highly aromatic
system could be beneficial to addressing some key areas for
optimization on a more overall compound quality and
balanced property basis, decreasing promiscuity and increasing
chances of clinical success.33,34 Accordingly, compounds 5a−c
and 6a−i with saturation of the RHS pyridine ring were
synthesized. We anticipated that removal of the exposed N
atom and the increased three-dimensional character would lead
to decreased CYP2D6 inhibition, decreased hERG activity, and
improved selectivity over GPR3 and GPR12. The results are
summarized in Table 4.
After saturation of the aromatic bicyclic ring system in
combination with the LHS piperazine ring motif, the newly
generated basic nitrogen was masked as either an amide or a
sulfonamide (5a−5c, Table 4). Encouragingly, all compounds
exhibited potency levels in the desired range and, indeed,
demonstrated lower hERG binding in many compounds as
hypothesized earlier (5a). Moreover, 5a showed a 2−3-fold
improvement in selectivity over both GPR3 and GPR12.
However, sterically more demanding N-masking groups were
detrimental to the selectivity.
piperidine subunit was combined with the new partially
saturated RHS core, leading to a number of notable
improvements. To our surprise, 6b (R1 = cyclopropyl)
exhibited much improved selectivity, 40-fold over GPR3 and
120-fold over GPR12. Although the selectivity trend of the
cyclopropylamine over isopropylamine was evidenced again by
comparing 6b with 6a, it was difficult to manifest a clear
selectivity strategy lacking a more reliable SAR trend. With this
observation, we attempted a similar idea as described for the
aromatic analogues from Table 3 and also synthesized 6g and
6h as the regioisomers of 6a and 6b, respectively. In contrast
to the aromatic analogues, a significant selectivity improvement
was achieved. Both 6g and 6h showed selectivity values for
both GPR3 and GPR12 well above our criteria of 30 fold. Our
investigation on the RHS saturated ring was expanded, but
none of these attempts were successful. These efforts either led
to the loss of potency, like 6c (unmasked nitrogen), 6d
(methylated nitrogen), and 6f (N masked as a carbamate), or
were not tolerable to the selectivity, like 6e (masked as
sulfonamides). After a careful review of all relevant data, we
realized that compound 6h was the most promising of all late-
stage lead optimization contenders, eliminating all the
concerns that were identified with compound 3e. Compound
6h was selected as the project’s first advanced lead and in vivo
tool compound. A snapshot of the relevant data contributing to
the selection process of compound 6h is depicted in Tables 5
and 6. Summarizing a few key features, 6h exhibited 16 nM
binding affinity in competitive radioligand binding assays,
similar to its functional potency in cell-based cAMP TR-FRET
assays (EC50 = 34 nM). LLC-PK1-MDR1 efflux assays
indicated a high brain permeability, and robust metabolic
stability was observed in human hepatocyte (hHep) stability
assays. A follow-up hERG functional assay gave IC50 = 6.2 μM,
consistent with its hERG binding affinity.
As a result of the identification of 6h, the project objectives
were revised, leading to a parallel three-prong approach:
• Objective 1: initiate the development of a quantitative
receptor occupancy (RO) assay, either in vivo or ex vivo
The next important breakthrough in the project was
achieved when the newly developed western phenolic-
E
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As shown in Figure 2a, the selected cold tracer molecule RL-
338 achieved baseline enrichment of roughly nine-fold in
Table 5. Summary of Other Key Properties for Lead
Compound 6h
property
6h
binding affinity Ki (nM)
16
functional EC50 (nM)
34
selectivity over GPR3 (fold)
selectivity over GPR12 (fold)
logD
LLE (logEC50-clogP)
LLC-PK1-MDR1 A to B (nm/s)
P-gp efflux ratio
HLM, Clint (mL/min/kg)
HHep, Clint (mL/min/kg)/fu (%)
hERG screen (Cerep) functional IC50
dog CV (CorDynamics) no findings@
dog Cmax (total/free) at 30 mpk
110×
97×
4.5
5.0
160
0.7
45
5.7/16%
6.2 μM
30 mg/kg p.o.
11.0 μM/11 nM
• Objective 2: initiate all required in vivo tolerability,
pharmacology, and pharmacokinetic studies with 6h and
explore the feasibility of generating a candidate selection
package
• Objective 3: continue the lead optimization to mitigate
for a potential failure of 6h
Executing on the newly devised research plan, objective 1
started with the search for a suitable tool tracer compound to
measure RO in vivo. First, we focused on developing an
isotope (label)-free LC-MS/MS-based in vivo RO method for
GPR6 receptors in rodents, based on previously reported
successful strategies.35 The use of LC-MS/MS techniques to
quantify levels of unlabeled tracer compound in discrete brain
regions expressing the target of interest offers an attractive
alternative to assess real in vivo RO, circumventing the need to
identify and generate radiolabeled tracer, reducing associated
costs and increasing turnaround time. First, we applied
compound selection criteria (e.g., high potency, high
selectivity, rapid brain uptake, and rapid wash out),36 dosed
animals intravenously (i.v.), dissected brain regions, and
screened for brain penetrant compounds that demonstrated
strong enrichment in the striatum compared to the cerebellum,
using LC-MS/MS-based quantification methods. A suitable
GPR6 tracer compound is expected to show a several fold
enrichment in the striatum due to the high expression of GPR6
in striatal D2-type MSN, compared to nonspecific binding in
the cerebellum, a brain region that lacks significant expression
of GPR6 receptors. The time course of tracer enrichment and
knowledge of the lowest tracer dose that achieved largest fold
enrichment are additional criteria to be considered for tracer
selection and validation. Most importantly, after successful
optimization and assay development, the enrichment of a
suitable tracer can be blocked by predosing the test compound
at therapeutically relevant doses, which allows quantification of
receptor occupancy for the test compound.
Figure 2. In vivo label-free LC-MS/MS-based GPR6 receptor
occupancy (RO) of lead compound 6h in male Sprague-Dawley
rats. (a) GPR6 IAG tracer compound RL-338 (0.04 μg/kg, i.v.)
accumulates nine-fold in the rat striatum (STR), a brain region
expressing a high level of GPR6, in comparison to the cerebellum
(CB), a brain region without GPR6 expression (90 min post dosing).
Compound 6h (0.03, 0.3,3 and 30 mg/kg, p.o.) dose-dependently
reduced striatal enrichment of tracer RL-338 compared to vehicle
(Tween-80, 0.5% methylcellulose) 90 min post dosingan in vivo
measure of isotope-free GPR6 receptor occupancy. (b) Correlation of
compound 6h plasma exposure and striatal receptor occupancy using
in vivo label-free RO method (compound 6h Occ50 (plasma) = 2.8
ng/mL). Data are mean
s.e.m. (n = 5 per group) after 90 min
postinjection of 6h; *P < 0.05, ****P < 0.0001 vs vehicle.
mouse striatum over background tissue (cerebellum) where
GPR6 is not present. Striatal enrichment of compound RL-338
was absent in GPR6 knock out mice, confirming the high
specificity of RL-338 for GPR6 receptor binding in vivo (data
not shown). Rewardingly, predosing 6h (0.03, 0.3, 3, 30 mg/
kg; p.o.) achieved a clear and dose-dependent blockage of the
striatal enrichment of tracer RL-338, confirming that 6h
efficiently occupies GPR6 receptors in vivo (Figure 2,b). To
Table 6. Pharmacokinetic Properties of Lead Compound 6h in Male Sprague-Dawley Rats
route
i.v.
dose (mg/kg)
1
subject (n = 3)
AUC (0 − τ) (h*ng/mL)
AUC (0 − ∞) (h*ng/mL)
CL (mL/min/kg)
Vdss (L/kg)
term t1/2 (h)
mean
SD
339
53
341
53
49.7
7.9
F
3.68
0.37
1.12
0.12
route
p.o.
dose (mg/kg)
subject (n = 3)
Cmax (ng/mL)
Tmax (h)
2
mean
SD
62.5
9.9
1.00
0.87
0.50
0.09
F
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Drug Annotation
the best of our knowledge, this is the first time that the
successful application of the isotope-free LC-MS/MS RO
method is reported for an orphan GPCR. A plasma exposure of
6h (2.8 ng/mL; 6.1 nM) was required to achieve 50% maximal
GPR6 receptor occupancy (Occ50). To further study
compound 6h receptor occupancy, we additionally developed
an ex vivo GPR6 receptor occupancy assay, using a tritium-
labeled form of our tracer compound RL-338 (Supporting
Information). After oral dosing, compound 6h dose-depend-
ently achieved ex vivo-based RO in rats with a plasma Occ50
of 3.4 ng/mL (7.5 nM), which is similar to the Occ50
determined using the LC-MS/MS-based RO method (data not
shown). Both methods together confirmed that compound 6h
efficiently achieved RO in vivo.
Executing on objective 2, compound 6h did not show any
signals in our internal collection of in vitro safety assessments
including mutagenicity. To derisk the moderate hERG signal, a
cardiovascular study in dogs was conducted, and no adverse
effects (AEs) were detected at a dose of 30 mpk. Likewise, 14-
day toxicological studies in rat and dog did not unveil any
findings at the highest doses of 300 mpk and 30 mpk in rats
and dogs, respectively, despite achieving very high total plasma.
To further understand the efficacy of GPR6 inhibition to
treat the motor symptoms of PD, we focused on testing our
lead compound 6h in the bilateral 6-OHDA-lesion PD model
that exhibits the loss of dopaminergic neurons in the substantia
nigra and dopamine depletion in the striatum, reflecting one of
the pathological hallmarks of Parkinson’s disease. Rats with
bilateral 6-OHDA lesions develop loss of mobility (Figure 3)
and are used as preclinical behavioral in vivo models to study
compounds for efficacy to treat the motor symptoms in PD.37
and the 6-OHDA-lesion PD model, respectively, provide first
preclinical evidence that efficient GPR6 inhibition in vivo
could represent a novel mechanism to treat motor symptoms
in Parkinson’s disease. A manuscript expanding the study of
GPR6 biology and the pharmacological characterization of
compound 6h is in preparation (unpublished).
The commitment to objective 3 led to the discovery of
compound 6i, which has many of the favorable druglike
properties of 6h but with some advantages such as lower
lipophilicity and higher in vivo toxicity margins. Therefore, 6i
was selected for candidate development over 6h. Compound
6i, now named CVN424, is currently in phase 2 clinical
development to treat motor symptoms in PD by Cerevance,
Inc. A manuscript describing the preclinical in vitro and in vivo
pharmacological characterization of CVN424 (6i) is in press.38
A selection of key data is summarized in Table 7 for
compound 6i.
Table 7. Summary of Key in Vitro ADMET and
Pharmacological Properties of Lead Compound 6i
(CVN424)
property
6i
binding affinity Ki (nM)
functional EC50 (nM)
9.4
38
selectivity over GPR3 (fold)
selectivity over GPR12 (fold)
logD
265×
68×
3.6
LLE (logEC50-cLogP)
5.8
LLC-PK1-MDR1 A to B (nm/s)
P-gp efflux ratio
170
0.7
HLM, Clint (mL/min/kg)
HHep, Clint (mL/min/kg)/fu (%)
hERG Screen (Cerep) functional IC50
62
48/51%
3.5 μM
Figure 3. Compound 6h shows anti-Parkinsonian efficacy in the
bilateral 6-OHDA-lesioned rat model of Parkinson’s disease. 6-
OHDA-lesioned female animals are dosed with vehicle (0.5%
methylcellulose) or compound 6h (1 mg/kg, p.o.). Animals are
placed in automated activity monitors for 3 h post dosing to assess
mobility. The pen field activity of unperturbed study animals before
the introduction of a bilateral 6-OHDA lesion is shown as control.
Collected data are mean s.e.m. (n = 10 per group) of total counts
over 3 h. *P < 0.05 vs vehicle; repeated measures one-way ANOVA
followed by Newman-Keuls test.
CONCLUSION
■
Pharmacological modulation of the indirect pathway in
Parkinson’s disease is a novel therapeutic strategy to reduce
motor deficits in PD, without the risk of developing drug-
induced dyskinesia. GPR6 is an orphan G-protein coupled
receptor, with highly restricted expression in dopamine
receptor D2-type medium spiny neurons (MSNs) of the
indirect pathway, which triggered our interest to explore GPR6
as a novel non-dopaminergic drug target for PD. We started
our SAR optimization campaign with a weak HTS hit 1 (EC50
= 43 μM) and were able to achieve a 1000-fold boost in
functional potency by introduction of electron density
reducing heteroatom features on the bicyclic aromatic ring
system. Other major issues, including P450 enzyme inhibition,
poor selectivity over GPR3 and GPR12, and high lipophilicity
were simultaneously overcome by the partial saturation of the
RHS bicyclic aromatic ring system. We identified multiple
GPR6 IAG compounds suitable for pharmacological validation
Oral dosage (1 mg/kg) of compound 6h restored mobility
of 6-OHDA-lesioned animals to presurgery level (Figure 3).
This result is consistent with the proposed mechanism of
action: inhibition of GPR6 receptors normalizes dopamine-like
cAMP signaling in dopamine D2 type MSNs and reduces
hyperactivity in the indirect pathway in the striatum. The
results of our initial in vivo pharmacology studies character-
izing selective GPR6 IAG 3e and 6h in the catalepsy model
G
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Drug Annotation
a
Scheme 1. Synthesis of Compounds 2a−g
a
Reagents and conditions: (a) NaBH(OAc)3, DCE, rt; (b) HCl, dioxane, rt; (c) (i) cyclopropylamine, DIPEA, DCM, 0 °C; (ii) 1-(3-
methylbenzyl)piperazine, DIPEA, rt. (d) (i) cyclopropylamine, dioxane, 80 °C; (ii) 1-(3-methylbenzyl)piperazine or 8a, dioxane, 150 °C; for 2a−e
and 2g; (e) (i) 8a, DIPEA DCM, rt; (ii) cyclopropylamine, DIPEA, dioxane, 100 °C, for 2f; (f) (i) 2g, KOH, MeOH, rt; (ii) Me2NH, HATU,
DIPEA, DMF.
a
Scheme 2. Synthesis of Compounds 3a−h and 4a−f
a
Reagents and conditions: (a) 8a or 4-(2,4-difluorophenoxy)piperidine, DIPEA, DCM, 0 °C; (b) cyclopropylamine or isopropylamine, DIPEA,
dioxane, 110 °C in a sealed tube; (c) cyclopropylamine or isopropylamine, DIPEA dioxane or DCM, 0 °C; (d) 8a, 8b, 8c, 8d, or 4-(2,4-
difluorophenoxy)piperidine, DIPEA, dioxane, 80 °C; (e) 4f, 1,1′-bis(diphenylphosphino) ferrocene, Pd2(dba)3, Zn(CN)2, NMP, 120 °C.
of our therapeutic hypothesis in behavioral PD models. Tool
compound 3e reversed haloperidol-induced catalepsy, and
early lead compound 6h demonstrated anti-parkinsonian
efficacy in the bilateral 6-OHDA-lesioned PD model, providing
for the first-time experimental evidence that pharmacological
inhibition of GPR6 in vivo could be a feasible non-
dopaminergic therapeutic strategy to reduce motor deficits in
human PD patient. Compound 6i(CVN424) has successfully
progressed through clinical phase I safety testing and is
currently in clinical phase II trials to validate the therapeutic
efficacy to treat motor symptoms in PD.
SYNTHESIS
■
Some common amine intermediates were prepared via a
reaction sequence of reductive amination and subsequent Boc
deprotection with HCl as shown in Scheme 1. The initial hit 1
was resynthesized by sequentially reacting 9 with cyclopropyl-
H
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Drug Annotation
a
Scheme 3. Synthesis of Compounds 5a−c
a
Reagents and conditions: (a) (a) 8d, DIPEA, DCM, 0 °C; (b) isopropylamine, DIPEA, dioxane, 100 °C in a sealed tube; (c) Ac2O, H2, 10% Pd/
C, acetone/dioxane, 45 °C; (d) NaOH, MeOH, 65 °C; (e) propionic anhydride or MeSO2Cl, DIPEA, DCM, 0 °C.
a
Scheme 4. Synthesis of Compounds 6a−i
a
Reagents and conditions: (a) (i). BnBr, MeCN, 80 °C; (ii). NaBH(OAc)3, DCM, rt; (iii). H2, Pd/C, MeOH, rt; (b) 18a or 18b, Ac2O, DIPEA,
DCM, 0 °C to rt; (c) 19a or 6c, MeSO2Cl or Ac2O or MeO2CCl, DIPEA, DCM, 0 °C to rt.
amine and then 1-(3-methylbenzyl)piperazine (commercially
available) at 0 °C in a one-pot reaction.
By following the same directing effect as the ester group, 2f (7-
CN) was prepared as the only isomer by reversing the reaction
order of the two SNAr reactions, starting with 8a first.
Intermediates 10 with different substituents (R2 = H, 7-Me,
7-OMe, 7-F and 6-CO2Me) underwent one SNAr reaction with
cyclopropylamine, followed by a second SNAr reaction with 1-
(3-methylbenzyl)piperazine or 8a to afford compounds 2a−e
and 2g. The different electron-withdrawing or -donating effect
of the substituents on the phenyl ring resulted in different
regioselectivities during the first SNAr reaction. 2C (7-Me) was
obtained as a mixture of two regioisomers (2:1 ratio). In
contrast, 2e (7-F) was separated as one single isomer, but it is
likely that the other regioisomer was lost during the
purification process. 2d with a strong electron-donating
methoxy group and 2g with a strong electron-withdrawing
ester group were afforded as one single observed isomer;
however, the directing effect of these two substituents for the
regioselectivity is opposite during the first SNAr reaction. Ester
2g was hydrolyzed to the corresponding acid, which then
reacted with dimethylamine to provide amide compound 2h.
Compounds 3a−h and 4a−f were prepared as shown in
Scheme 2, in which the presence of the pyridine nitrogen
(11a−c) also played a critical directing effect during the first
SNAr reaction for the synthesis of 12a−e and 13a−b when the
reaction temperature and addition rate of the corresponding
amine were carefully controlled. The second SNAr reaction
with corresponding amines gave rise to 3a−h, 4a−b, and 4d−f,
respectively. Palladium-catalyzed cyanation with Zn(CN)2
based on substrate 4f afforded final compound 4c (10).
Intermediate 14 was constructed with both LHS amines via
the same reaction sequence as 3a, but the second SNAr reaction
with isopropylamine was significantly facilitated by adding KF
as a promoter. The pyridine ring in the bicyclic ring system was
then saturated after activation with acetic anhydride and in situ
hydrogenation catalyzed by palladium to afford intermediate
5a (11), which was readily hydrolyzed to the corresponding
I
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Drug Annotation
Step 2: A solution of 3-chloro-N-cyclopropylquinoxalin-2-amine
(30 mg, 0.137 mmol), 1-(3-methylbenzyl)piperazine (39.0 mg, 0.205
mmol), and N-ethyl-N-isopropylpropan-2-amine (0.072 mL, 0.410
mmol) in dioxane (0.25 mL) was heated at 150 °C overnight. HPLC
purification afforded the title compound 2a (30% yield) as a white
solid. 1H NMR (400 MHz, CDCl3) δ 7.71 (br t, J = 8.7 Hz, 2H), 7.42
(br t, J = 7.5 Hz, 1H), 7.35−7.28 (m, 1H), 7.24−7.18 (m, 1H), 7.18−
7.11 (m, 2H), 7.07 (br d, J = 7.1 Hz, 1H), 5.51 (br s, 1H), 3.55 (s,
2H), 3.22 (br s, 4H), 2.91 (dt, J = 3.0, 6.6 Hz, 1H), 2.61 (br s, 4H),
2.35 (s, 3H), 0.95−0.84 (m, 2H), 0.61−0.52 (m, 2H); ESI-MS calcd
for C23H27N5 [M + H]+ = 373.23, found: 374.1.
secondary amine 15 by treating it with sodium hydroxide in
MeOH at 65 °C. The resulting amine was reacted with either
propionic anhydride or methanesulfonyl chloride to provide 5b
and 5c, respectively.
As for the preparation of 6a−i (Scheme 4), it also requires a
saturation process on the pyridine ring. A two-step reaction
sequence (benzylation followed by reduction with sodium
triacetoxyborohydride) was developed to saturate the pyridine
ring in this case (12−13). This optimized two-step procedure
proved to be a more practical and scalable process in
comparison to the one-pot reaction as described in Scheme
3. The subsequent debenzylation under hydrogenation
conditions afforded common secondary amine intermediates
6c, 19a, 18a−b, with no purification needed over three steps.
Final compounds 6a−i were generated from the respective
secondary amines using methylation, acylation, or sulfonation
reaction conditions.
Using similar reaction conditions as described above, and utilizing
intermediates prepared as described above or commercially available
piperazines, the following compounds were synthesized, and purified
by either HPLC or silica gel column chromatography:
N-Cyclopropyl-3-(4-(2,5-dichlorobenzyl)piperazin-1-yl)-
1
quinoxalin-2-amine (2b). H NMR (400 MHz, CDCl3) δ ppm 7.72
(t, J = 8.2 Hz, 2H), 7.54 (d, J = 2.2 Hz, 1H), 7.43 (t, J = 7.4 Hz, 1H),
7.32 (br t, J = 7.4 Hz, 1H), 7.27 (d, J = 8.4 Hz, 1H), 7.16 (dd, J = 2.3,
8.5 Hz, 1H), 5.52 (br s, 1H), 3.66 (s, 2H), 3.25 (br s, 4H), 2.92 (br
dd, J = 3.3, 6.6 Hz, 1H), 2.69 (br s, 4H), 0.93−0.88 (m, 2H), 0.64−
0.53 (m, 2H); ESI-MS calcd for C22H23Cl2N5 [M + H]+ = 427.13,
found: 428.0.
N-Cyclopropyl-3-(4-(2,5-dichlorobenzyl)piperazin-1-yl)-7-meth-
ylquinoxalin-2-amine (2c). The titled compound was obtained as a
mixture of two regioisomers (2:1 ratio). 1H NMR (400 MHz,
CD3OD) δ ppm 0.87−0.98 (m, 2H), 1.11 (ddt, J = 6.96, 3.45, 1.82,
1.82 Hz, 2H), 2.37−2.46 (m, 3H), 2.65−2.85 (m, 4H), 2.92 (ddt, J =
10.79, 7.22, 3.54, 3.54 Hz, 1H), 3.21−3.29 (m, 4H), 3.73 (s, 2H),
7.17−7.24 (m, 1H), 7.30 (dd, J = 8.53, 2.76 Hz, 1H), 7.37−7.42 (m,
1H), 7.50−7.62 (m, 2H), 7.66−7.81 (m, 1H); ESI-MS calcd for
C23H25Cl2N5 [M + H]+ = 441.2, found: 442.0.
N-Cyclopropyl-3-(4-(2,5-dichlorobenzyl)piperazin-1-yl)-7-me-
thoxyquinoxalin-2-amine (2d). 1H NMR (400 MHz, CD3OD) δ
ppm 0.81−0.90 (m, 2H), 1.04−1.13 (m, 2H), 2.88 (dt, J = 7.09, 3.36
Hz, 1H), 3.58 (br d, J = 2.76 Hz, 8H), 3.94 (s, 3H), 4.56 (s, 2H), 7.15
(dd, J = 9.03, 2.51 Hz, 1H), 7.34 (d, J = 2.76 Hz, 1H), 7.53−7.58 (m,
1H), 7.59−7.63 (m, 1H), 7.72 (d, J = 9.03 Hz, 1H), 7.78 (d, J = 2.26
Hz, 1H); ESI-MS calcd for C23H25Cl2N5O [M + H]+ = 457.1, found:
458.0.
MATERIALS AND METHODS
■
General Methods. All reagents and solvents were purchased from
commercial suppliers and used without purification unless otherwise
noted. All reactions were performed at ambient temperature and in
anhydrous solvents unless otherwise noted. Flash chromatography
was performed on ISCO CombiFlash with prepacked silica gel
columns and eluted with ethyl acetate in heptane. Preparative HPLC
was performed on a Shimadzu LC-8A (UV/vis: SPD-20A; Software:
LCSolution) with a Phenomenex Gemini C18, 5 μm, ID 30 × 100
mm column using either acid (0.1% TFA) or base (0.1% NH4OH)
modified MeCN/H2O gradients. 1H NMR spectra were recorded on a
500 or 400 MHz Bruker instrument. Chemical shifts are expressed in
parts per million (ppm) δ relative to residual solvent as an internal
reference. The mass spectra were obtained using liquid chromatog-
raphy mass spectrometry (LC-MS) on an Agilent 1260 Infinity
instrument using electrospray ionization (ESI). All test compounds
showed >95% purity as determined by LC-MS. All animal
experiments performed in the manuscript were conducted in
compliance with institutional guidelines.
Synthesis of N-Cyclopropyl-6-(4-(3-methylbenzyl)piperazin-1-
yl)-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-amine (1). To a solution of
5,6-dichloro-[1,2,5]oxadiazolo[3,4-b]pyrazine (200 mg, 1.05 mmol,
1.0 equiv) in DCM (5.0 mL) were added DIPEA (547.2 μL, 3.14
mmol, 3.0 equiv) and cyclopropanamine (59.79 mg, 1.05 mmol, 72.56
μL, 1 equiv). The mixture was stirred at 0 °C for 2 h. 1-(m-
Tolylmethyl)piperazine (241.6 mg, 1.26 mmol, 1.2 equiv) was then
added, and stirring was continued for 3 h at 20 °C. The mixture was
concentrated under reduced pressure, and the resulting residue was
purified by prep-HPLC (column: Boston Prime C18 150 × 30 mm ×
5 μm; mobile phase: [water (0.05% NH3H2O + 10 mM NH4HCO3)-
ACN]; B%: 55−85%,7 min) to give N-cyclopropyl-5-[4-(m-
tolylmethyl)piperazin-1-yl]-[1,2,5] oxadiazolo[3,4-b]pyrazin-6-amine
(100.7 mg, 25.8% yield) as a white solid. 1H NMR (400 MHz,
CDCl3) δ ppm 7.21 (t, J = 7.5 Hz, 1H), 7.14−7.06 (m, 3H), 5.88 (br
s, 1H), 3.53 (s, 2H), 3.47−3.36 (m, 4H), 2.98 (dt, J = 3.4, 7.1 Hz,
1H), 2.61 (br d, J = 4.4 Hz, 4H), 2.34 (s, 3H), 1.04−0.95 (m, 2H),
N-Cyclopropyl-3-(4-(2,5-dichlorobenzyl)piperazin-1-yl)-7-fluoro-
quinoxalin-2-amine (2e). 1H NMR (400 MHz, CD3OD) δ ppm
0.61−0.68 (m, 2H), 0.90−0.99 (m, 2H), 2.78 (br s, 4H), 2.86 (tt, J =
7.03, 3.64 Hz, 1H), 3.23−3.31 (m, 4H), 3.74 (s, 2H), 7.10 (td, J =
8.66, 2.76 Hz, 1H), 7.24 (dd, J = 8.53, 2.51 Hz, 1H), 7.35 (d, J = 8.53
Hz, 1H), 7.39 (dd, J = 10.29, 2.76 Hz, 1H), 7.56 (d, J = 2.51 Hz, 1H),
7.70 (dd, J = 8.91, 5.90 Hz, 1H); ESI-MS calcd for C22H22Cl2FN5 [M
+ H]+ = 445.1, found: 446.0.
Synthesis of 2-(Cyclopropylamino)-3-(4-(2,5-dichlorobenzyl)-
piperazin-1-yl)-N,N-dimethylquinoxaline-6-carboxamide (2h).
Step 1: To a solution of methyl 2-(cyclopropylamino)-3-(4-(2,5-
dichlorobenzyl)piperazin-1-yl)quinoxaline-6-carboxylate (2g, pre-
pared by following the procedures for the synthesis of 2a) (100
mg, 0.206 mmol, 1.0 equiv) in a mixture of dioxane (3.0 mL) and
methanol (1.0 mL) was added an aqueous solution of KOH (2 N,
0.185 mL, 0.370 mmol, 1.8 equiv). The resulting reaction mixture was
stirred at rt overnight and then neutralized by the addition of HCl
(1.0 N, 0.37 mL). Concentration under reduced pressure gave 2-
(cyclopropylamino)-3-(4-(2,5-dichlorobenzyl)piperazin-1-yl)-
quinoxaline-6-carboxylic acid as a white solid (78% purity), which was
used directly in the next step without further purification.
Step 2: A solution of 2-(cyclopropylamino)-3-(4-(2,5-
dichlorobenzyl)piperazin-1-yl)quinoxaline-6-carboxylic acid (45 mg,
0.095 mmol, 1.0 equiv) and HATU (36.2 mg, 0.095 mmol, 1.0 equiv)
in DMF (1.5 mL) was stirred at rt for 5 min, and then DIPEA (36.9
mg, 0.286 mmol, 3.0 equiv) and dimethylamine hydrochloride (11.7
mg, 0.143 mmol, 1.5 equiv) were added. The resulting solution was
stirred at rt for 3 h. The solvent was removed under reduced pressure,
and then the residue was purified by silica gel column chromatog-
raphy (1−5% MeOH in DCM) to give the titled compound 2h as an
0.69−0.57 (m, 2H); ESI-MS calcd for C19H23N7O [M + H]+
=
365.20; found 366.1.
Synthesis of N-Cyclopropyl-3-(4-(3-methylbenzyl)piperazin-1-
yl)quinoxalin-2-amine (2a). Step 1: A solution of 2,3-dichloroqui-
noxaline (400 mg, 2.010 mmol), cyclopropanamine (149 mg, 2.61
mmol), and DIPEA (526 μL, 3.01 mmol) in dioxane (2871 μL) was
heated at 80 °C for 2 days. The solvent was removed under reduced
pressure, and the residue obtained was purified by silica gel column
chromatography (10−40% EtOAc/hexanes) that afforded 3-chloro-
N-cyclopropylquinoxalin-2-amine as a light yellow solid. 1H NMR
(400 MHz, CDCl3) δ ppm 7.79 (br d, J = 7.9 Hz, 2H), 7.58 (br t, J =
7.6 Hz, 1H), 7.39 (br t, J = 7.7 Hz, 1H), 5.72 (br s, 1H), 3.01−2.91
(m, 1H), 0.93 (br d, J = 6.8 Hz, 2H); ESI-MS calcd for C11H10ClN3
[M + H]+ = 219.06; found: 219.9.
J
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Drug Annotation
1
off-white solid (29 mg, 59% yield). H NMR (400 MHz, CD3OD) δ
7.20 (m, 1H), 7.62 (s, 1H), 8.64 (s, 1H); ESI-MS calcd for
C22H25F2N5O [M + H]+ = 413.2, found: 414.4.
ppm 0.89−0.96 (m, 2H), 1.06−1.13 (m, 2H), 2.78 (br d, J = 2.76 Hz,
4H), 2.91−2.99 (m, 1H), 3.03 (br s, 3H), 3.12 (br s, 3H), 3.22−3.28
(m, 4H), 3.71−3.77 (m, 2H), 7.21−7.29 (m, 1H), 7.31 (d, J = 2.01
Hz, 1H), 7.34−7.38 (m, 1H), 7.38−7.43 (m, 1H), 7.55 (d, J = 2.51
Hz, 1H), 7.66−7.75 (m, 1H); ESI-MS calcd for C25H28Cl2FN6O [M
+ H]+ = 498.1, found: 499.1.
Synthesis of 3-(Cyclopropylamino)-2-(4-(2,5-dichlorobenzyl)-
piperazin-1-yl)quinoxaline-6-carbonitrile (2f). Step 1: To a solution
of 2,3-dichloroquinoxaline-6-carbonitrile (400 mg, 1.785 mmol, 1.0
equiv) and 1-(2,5-dichlorobenzyl)piperazine hydrogen chloride (8a)
(601 mg, 2.14 mmol, 1.2 equiv) in DCM (30 mL) was added DIPEA
(0.780 mL, 4.46 mmol, 2.5 equiv) at 20 °C, and the resulting reaction
mixture was stirred for 6 h. Water was added, and the reaction was
extracted with DCM. The combined organic layers were concen-
trated, and the residue was purified by silica gel column
chromatography (PE/EtOAc = 50:1) to afford 3-chloro-2-(4-(2,5-
dichlorobenzyl)piperazin-1-yl)quinoxaline-6-carbonitrile (657 mg,
85% yield) as a yellow solid.
Synthesis of N-Cyclopropyl-3-(4-(2,5-dichlorobenzyl)piperazin-
1-yl)pyrido[3,4-b]pyrazin-2-amine (3b). A solution of 3-chloro-N-
cyclopropyl-pyrido[3,4-b]pyrazin-2-amine (12a) (50 mg, 172.2 μmol,
1.0 equiv), 1-[(2,5-dichlorophenyl)methyl]piperazine (42.2 mg, 172.2
μmol, 1.0 equiv), and DIPEA (90.0 μL, 516.6 μmol, 3.0 equiv) in
dioxane (3.0 mL) was stirred at 80 °C for 24 h. The reaction mixture
was concentrated under reduced pressure and then purified by prep-
HPLC (column: Phenomenex Gemini-NX 150 × 30 mm × 5 μm;
mobile phase: [water (0.05% NH3H2O)-ACN]; B%: 54−84%,7 min)
to give N-cyclopropyl-3-[4-[(2,5-dichlorophenyl)methyl]piperazin-1-
yl]pyrido[3,4-b]pyrazin-2-amine (17.4 mg, 23.5% yield) as a white
solid. 1H NMR (400 MHz, CDCl3) δ ppm = 8.98 (s, 1H), 8.46 (d, J =
5.5 Hz, 1H), 7.57−7.46 (m, 2H), 7.28 (d, J = 8.4 Hz, 1H), 7.17 (dd, J
= 2.4, 8.6 Hz, 1H), 5.80 (br s, 1H), 3.66 (s, 2H), 3.33−3.19 (m, 4H),
2.96 (dt, J = 3.2, 6.9 Hz, 1H), 2.70 (br s, 4H), 1.01−0.89 (m, 2H),
0.66−0.56 (m, 2H); ESI-MS calcd for C21H22Cl2N6 [M + H]+
428.13, found: 429.0.
=
Step 2: A solution of 3-chloro-2-(4-(2,5-dichlorobenzyl)piperazin-
1-yl)quinoxaline-6-carbonitrile (30 mg, 0.069 mmol, 1.0 equiv),
cyclopropanamine (12 mg, 0.208, 3.0 equiv), and DIPEA (0.030 mL,
0.173 mmol, 2.5 equiv) in dioxane (2.0 mL) in a sealed vial was
heated at 100 °C overnight. HPLC purification under acidic
conditions afforded the title compound 2f (21 mg, 66%) as a yellow
solid. 1H NMR (400 MHz, methanol-d4) δ ppm 0.67−0.73 (m, 2H),
0.88−0.97 (m, 2H), 2.88 (tt, J = 7.15, 3.64 Hz, 1H), 3.60 (br s, 8H),
4.58 (s, 2H), 7.55−7.57 (m, 1H), 7.58 (d, J = 1.00 Hz, 1H), 7.60−
7.64 (m, 1H), 7.77 (s, 1H), 7.78 (d, J = 5.77 Hz, 1H), 8.04 (d, J =
1.76 Hz, 1H); ESI-MS calcd for C23H22Cl2N6 [M + H]+ = 452.1,
found: 453.1.
Synthesis of N-Cyclopropyl-2-(4-(2,5-dichlorobenzyl)piperazin-
1-yl)pyrido[3,4-b]pyrazin-3-amine (3a). To a solution of 3-chloro-2-
[4-[(2,5-dichlorophenyl)methyl]piperazin-1-yl]pyrido [3,4-b]-
pyrazine (13a) (70 mg, 171.3 μmol, 1.0 equiv) and cyclopropanamine
(9.8 mg, 171.3 μmol, 1.0 equiv) in dioxane (3.0 mL) was added
DIPEA (89.5 μL, 513.8 μmol, 3.0 eq). The resulting mixture was
stirred in a 30 mL sealed tube at 100 °C for 30 h. The reaction
mixture was evaporated to dryness and purified by prep-HPLC
(column: Phenomenex Gemini-NX150 × 30 mm × 5 μm; mobile
phase: [water (0.05% NH3H2O)-ACN]; B%: 60−90%,7 min) to give
N-cyclopropyl-2-[4-[(2,5-dichlorophenyl)methyl]piperazin-1-yl]-
pyrido[3,4-b]pyrazin-3-amine (7.6 mg, 10.2% yield) as a yellow solid.
1H NMR (400 MHz, CDCl3) δ 9.02 (s, 1H), 8.38 (br d, J = 6.0 Hz,
Using similar reaction conditions as described above and utilizing
intermediates prepared as described above or commercially available
piperazines, the following compounds were synthesized and purified
by either HPLC or silica gel column chromatography:
3-(4-(5-Chloro-2-fluorobenzyl)piperazin-1-yl)-N-
cyclopropylpyrido[3,4-b]pyrazin-2-amine (3c). The title compound
was prepared in a manner similar to 3b using 1-(5-chloro-2-
fluorobenzyl)piperazine hydrogen chloride and 12a at 80 °C. 1H
NMR (400 MHz, CD3OD) δ ppm 0.78−0.86 (m, 2H), 0.93−1.02
(m, 2H), 3.12 (tt, J = 7.40, 3.76 Hz, 1H), 3.45 (br t, J = 4.77 Hz, 4H),
3.74 (br s, 4H), 4.37 (s, 2H), 7.30 (t, J = 9.16 Hz, 1H), 7.56 (ddd, J =
8.78, 4.52, 2.76 Hz, 1H), 7.65 (dd, J = 6.27, 2.76 Hz, 1H), 7.86 (d, J =
6.78 Hz, 1H), 8.41 (dd, J = 6.53, 1.00 Hz, 1H), 8.98 (s, 1H); ESI-MS
calcd for C21H22ClFN6 [M + H]+ = 412.2, found: 413.3.
N-Cyclopropyl-3-(4-(2,5-difluorobenzyl)piperazin-1-yl)pyrido-
[3,4-b]pyrazin-2-amine (3d). The title compound was prepared in a
manner similar to 3b using 1-(2,5-difluorobenzyl)piperazine hydrogen
1
chloride and 12a at 80 °C. H NMR (400 MHz, CD3OD) δ ppm
0.76−0.86 (m, 2H), 0.93−1.02 (m, 2H), 3.08−3.17 (m, 1H), 3.38 (br
d, J = 4.02 Hz, 4H) 3.71 (br s, 4H), 4.30 (s, 2H), 7.24−7.32 (m, 2H),
7.37 (ddd, J = 7.28, 5.27, 1.76 Hz, 1H), 7.85 (d, J = 6.53 Hz, 1H),
8.40 (dd, J = 6.52, 0.75 Hz, 1H), 8.96 (s, 1H); ESI-MS calcd for
C21H22F2N6 [M + H]+ = 396.2, found: 397.4.
N-Cyclopropyl-3-(4-(2,4-difluorobenzyl)piperazin-1-yl)pyrido-
[3,4-b]pyrazin-2-amine (3e). The title compound was prepared in a
manner similar to 3b using 1-(2,4-difluorobenzyl)piperazine hydrogen
chloride and 12a at 80 °C (90% yield). 1H NMR (500 MHz, CDCl3)
δ ppm 0.61−0.65 (m, 2 H), 0.94−0.99 (m, 2H), 2.67 (br. s., 4H),
2.93−3.00 (m, 1H), 3.26 (t, J = 4.64 Hz, 4H), 3.65 (s, 2H), 6.79−
6.85 (m, 1H), 6.86−6.92 (m, 1H), 7.37−7.44 (m, 1H), 7.54 (d, J =
5.37 Hz, 1H), 8.47 (d, J = 5.86 Hz, 1H), 8.98 (s, 1H); ESI-MS calcd
for C21H22F2N6 [M + H]+ = 396.2, found: 397.9.
N-Cyclopropyl-3-(4-(2,4-difluorobenzyl)piperazin-1-yl)-7-
methylpyrido[3,4-b]pyrazin-2-amine (3f). The title compound was
prepared in a manner similar to 3b using 1-(2,4-difluorobenzyl)-
piperazine hydrogen chloride and 12c at 80 °C and obtained as a
white solid (22% yield). 1H NMR (500 MHz, CD3OD) δ ppm 0.77−
0.86 (m, 2H), 0.94−2.04 (m, 2H), 2.75 (S, 3H), 3.13 (tt, J = 7.44,
3.78 Hz, 1H), 3.54 (br, s., 8H), 4.48 (s, 2H), 7.12−7.25 (m, 2H),
7.66 (td, J = 8.54, 6.35 Hz, 1H), 7.71 (s, 1H), 8.88 (s, 1H); ESI-MS
calcd for C22H24F2N6 [M + H]+ = 410.2, found: 411.0.
3-(4-(2,4-Difluorobenzyl)piperazin-1-yl)-N-isopropylpyrido[3,4-
b]pyrazin-2-amine (3g). The title compound was prepared in a
manner similar to 3b using 1-(2,4-difluorobenzyl)piperazine hydrogen
chloride and 12b at 80 °C and obtained as a gray solid (82% yield).
1H NMR (500 MHz, CD3OD) δ ppm 1.32 (d, J = 6.35 Hz, 6H), 2.74
1H), 7.61 (d, J = 5.7 Hz, 1H), 7.54 (br s, 1H), 7.31−7.27 (m, 1H),
7.21−7.17 (m, 1H), 5.51 (br s, 1H), 3.69 (s, 2H), 3.51 (br s, 4H),
2.93 (br dd, J = 3.6, 6.7 Hz, 1H), 2.72 (br s, 4H), 1.02−0.91 (m, 2H),
0.68−0.58 (m, 2H); ESI-MS calcd for for C21H22Cl2N6 [M + H]+ =
428.13, found: 429.1.
Using similar reaction conditions as described above and utilizing
intermediates prepared as described above or commercially available
piperazines, the following compounds were synthesized and purified
by either HPLC or silica gel column chromatography:
N-Cyclopropyl-2-(4-(2,4-difluorophenoxy)piperidin-1-yl)-7-
methylpyrido[3,4-b]pyrazin-3-amine (4d). The title compound was
prepared in a manner similar to 3a using 10 equiv of cyclopropan-
amine and 13b at 110 °C to give a dark yellow semisolid (23.8%
1
yield). H NMR (400 MHz, CD3OD) δ ppm 0.70−0.74 (m, 2H),
0.88−0.90 (m, 2H), 1.93 (dt, J = 6.83, 3.42 Hz, 2 H) 2.06−2.15 (m, 2
H), 2.71 (s, 3H), 2.90 (dt, J = 7.44, 3.36 Hz, 1H), 3.77−3.80 (m,
2H), 3.99−4.02 (m, 2H), 4.57−4.58 (m, 1H), 6.87−6.89 (m, 1H),
6.99−7.01 (m, 1H), 7.18−7.19 (m, 1H), 7.65 (s, 1H), 8.73 (s, 1H);
ESI-MS calcd for C22H23F2N5O [M + H]+ = 411.2, found: 412.4.
2-(4-(2,4-Difluorophenoxy)piperidin-1-yl)-N-isopropyl-7-
methylpyrido[3,4-b]pyrazin-3-amine (4e). The title compound was
prepared in a manner similar to 3a using 10 equiv of propan-2-amine
and 13b at 110 °C to give a yellow solid (15.6% yield). 1H NMR (400
MHz, CD3OD) δ ppm 1.32 (d, J = 6.35 Hz, 6H), 1.93−1.96 (m, 2H),
2.09−2.11 (m, 2H), 2.70 (s, 3H), 3.83−3.84 (m, 2H), 4.05−4.06 (m
2H), 4.42−4.44 (m, 1H), 6.89 (m, 1H), 7.00−7.02 (m, 1H), 7.19−
(t, J = 4.64 Hz, 4H), 3.36 (br. s., 4H), 3.68 (s, 2H), 4.45 (dt, J =
13.06, 6.41 Hz, 1H), 6.90−7.03 (m, 2H), 7.41−7.56 (m, 2H), 8.29
(d, J = 5.37 Hz, 1H), 8.75 (s, 1H); ESI-MS calcd for C21H24F2N6 [M
+ H]+ = 398.2, found: 399.3.
K
https://doi.org/10.1021/acs.jmedchem.0c02081
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Drug Annotation
3-(4-(2,4-Difluorobenzyl)piperazin-1-yl)-N-isopropyl-7-
methylpyrido[3,4-b]pyrazin-2-amine (3h). The title compound was
prepared in a manner similar to 3b using 1-(2,4-difluorobenzyl)-
piperazine hydrogen chloride and 12d at 80 °C and obtained as a
2H), 4.00−4.15 (m, 1H), 4.40 (s, 0.8H), 4.43 (s, 1.2H), 5.35 (d, J =
7.8 Hz, 0.4H), 5.38 (d, J = 8.3 Hz, 0.6H) 7.08 (td, J = 8.3, 2.4 Hz,
1H), 7.21 (td, J = 10.0, 2.4 Hz, 1H), 7.44−7.51 (m, 1H); ESI-MS
calcd for C23H30F2N6O [M + H]+ = 444.2, found: 445.0.
1
white solid (97% yield). H NMR (500 MHz, CD3OD) δ ppm 1.35
Synthesis of 2-(4-(2,4-Difluorobenzyl)piperazin-1-yl)-N-isoprop-
yl-5,6,7,8-tetrahydropyrido[3,4-b]pyrazin-3-amine (15). To a sol-
ution of 1-(2-(4-(2,4-difluorobenzyl)piperazin-1-yl)-3-(isopropylami-
no)-7,8-dihydropyrido[3,4-b]pyrazin-6(5H)-yl)ethanone (5a) (0.367
g, 0.826 mmol) in MeOH (4 mL) was added NaOH, 15% solution
(2.201 g, 8.26 mmol) at 23 °C. The reaction mixture was stirred at 65
°C for 16 h, cooled to 23 °C, and neutralized with 1 N HCl (9.5 mL)
to furnish a suspension. The crude mixture was concentrated via
rotary evaporation, and stirred overnight. The resulting solid was
filtered, rinsed with water, and dried in vacuo to provide the title
compound 15 (253 mg, 76%) as a yellow foam. 1H NMR (500 MHz,
DMSO-d6) δ ppm 1.15 (d, J = 6.8 Hz, 6H), 2.42 (br s, 1H), 2.51−
2.61 (m, 6H), 2.89−3.00 (m, 6H), 3.58 (s, 2H), 3.63 (s, 2H), 4.00−
4.10 (m, 1H), 5.15 (d, J = 8.3 Hz, 1H), 7.04−7.11 (m, 1H), 7.21 (td,
J = 10.0, 2.4 Hz, 1H), 7.44−7.52 (m, 1H); ESI-MS calcd for
C21H28F2N6 [M + H]+ = 402.2, found: 403.0.
(d, J = 6.83 Hz, 6H), 2.73 (s, 4H), 3.57 (br. s., 6H), 4.48 (s, 2H), 4.66
(quin, J = 6.59 Hz, 1H), 7.11−7.24 (m, 2H), 7.61 (s, 1H), 7.68 (td, J
= 8.54, 6.35 Hz, 1H), 8.83 (s, 1H); ESI-MS calcd for C22H26F2N6 [M
+ H]+ = 412.2, found: 413.00.
N-Cyclopropyl-3-(4-(2,4-difluorophenoxy)piperidin-1-yl)-7-
methylpyrido[3,4-b]pyrazin-2-amine (4a). The title compound was
prepared in a manner similar to 3b using 4-(2,4-difluorophenoxy)-
piperidine hydrogen chloride and 12c at 80 °C and obtained as a light
yellow solid (35% yield). 1H NMR (500 MHz, CD3OD) δ ppm
1.59−1.64 (m, 2H), 1.64−1.70 (m, 2H), 2.55−2.74 (m, 2H), 2.82−
2.98 (m, 2H), 3.45 (s, 3H), 3.82−3.96 (m, 1H), 4.04−4.17 (m, 3H),
4.48 (br. s., 2H), 5.44 (dt, J = 7.57, 4.03 Hz, 1H), 7.77−7.91 (m, 1H),
8.03−8.22 (m, 2H), 8.47 (s, 1H), 9.27 (d, J = 4.39 Hz, 1H), 9.78 (s,
1H); ESI-MS calcd for C22H23F2N5O [M + H]+ = 411.2, found:
412.9.
3-(4-(2,4-Difluorophenoxy)piperidin-1-yl)-N-isopropyl-7-
methylpyrido[3,4-b]pyrazin-2-amine (4b). The title compound was
prepared in a manner similar to 3b using 4-(2,4-difluorophenoxy)-
piperidine hydrogen chloride and 12d at 60 °C and obtained as an
Synthesis of 1-(2-(4-(2,4-Difluorobenzyl)piperazin-1-yl)-3-(iso-
propylamino)-7,8-dihydropyrido[3,4-b]pyrazin-6(5H)-yl)propan-1-
one (5b). To a solution of 2-(4-(2,4-difluorobenzyl)piperazin-1-yl)-N-
isopropyl-5,6,7,8-tetrahydropyrido[3,4-b]pyrazin-3-amine 15 (50 mg,
0.124 mmol) and DIPEA (0.032 mL, 0.186 mmol) in DCM (1 mL)
was added propionic anhydride (0.019 mL, 0.149 mmol) at 0 °C. The
reaction mixture was stirred at 0 °C for 1 h, then warmed to 23 °C,
and concentrated via rotary evaporation. The resulting crude material
was partitioned between EtOAc and water. The organic phase was
washed with brine, dried over Na2SO4, filtered, rinsed with EtOAc,
and concentrated via rotary evaporation. The crude material was
dissolved in EtOAc and MeOH, adsorbed on silica gel (0.75 g), and
purified via medium pressure silica gel column chromatography using
a 20% to 100% gradient of EtOAc in heptane. The pure fractions were
combined, concentrated via rotary evaporation, and dried in vacuo to
provide the title compound 5b (48 mg, 84% yield) as an off-white
1
off-white solid (43% yield). H NMR (500 MHz, CD3OD) δ ppm
1.35 (d, J = 6.35 Hz, 6H), 1.99 (tdd, J = 10.25, 10.25, 7.32, 3.42 Hz,
2H), 2.12−2.22 (m, 2H), 2.71 (s, 3H), 3.45 (ddd, J = 13.06, 7.69,
3.66 Hz, 2H), 3.74−3.83 (m, 2H), 4.58 (tt, J = 6.83, 3.42 Hz, 1H),
4.64 (quin, J = 6.71 Hz, 1H), 6.90 (dddd, J = 9.34, 7.87, 3.05, 1.71
Hz, 1H), 7.00 (ddd, J = 11.35, 8.66, 2.93 Hz, 1H), 7.20 (td, J = 9.28,
5.37 Hz, 1H), 7.56 (s, 1H), 8.73 (s, 1H); ESI-MS calcd for
C22H25F2N5O [M + H]+ = 413.2, found: 414.0.
Synthesis of 3-(4-(2,4-Difluorobenzyl)piperazin-1-yl)-2-
(isopropylamino)pyrido[3,4-b]pyrazine-7-carbonitrile (4c). A vial
containing
a mixture of 7-bromo-N-isopropyl-3-(4-(2,4-
difluorobenzyl)piperazin-1-yl)pyrido[3,4-b]pyrazin-2-amine (4g, pre-
pared in a manner similar to 3b using 4-(2,4-difluorophenoxy)-
piperidine hydrogen chloride and 12e) (17 mg, 0.035 mmol, 1.0
equiv), Pd2(dba)3 (1.581 mg, 1.726 μmol, 0.05 equiv), dicyanozinc
(2.433 mg, 0.021 mmol, 0.6 equiv) and 1,1′-bis(diphenylphosphino)
ferrocene (dppf) (1.914 mg, 3.4 μmol, 0.1 equiv) in NMP (345 μL)
was evacuated and filled with N2 three times then heated at 120 °C
for 2 h. The crude reaction mixture was filtered through a Millipore
filter, and purified by HPLC to afford the title compound 4c as a
1
foam. H NMR (500 MHz, DMSO-d6, mixture of rotamers) δ ppm
0.94−1.05 (m, 3H), 1.16−1.18 (m, 6H), 2.41 (quin, J = 7.8 Hz, 2H),
2.52−2.65 (m, 5H), 2.69 (t, J = 5.6 Hz, 1H), 2.98 (br s, 3H), 3.58 (s,
2H), 3.70 (t, J = 5.9 Hz, 1.1H), 3.73 (t, J = 5.9 Hz, 0.9H), 4.03 (q, J =
7.2 Hz, 2H), 4.06−4.15 (m, 1H), 4.42 (br s, 2H), 5.34 (d, J = 7.8 Hz,
0.6H), 5.37 (d, J = 8.3 Hz, 0.4H), 7.08 (td, J = 8.5, 2.4 Hz, 1H), 7.21
(td, J = 9.9, 2.7 Hz, 1H), 7.44−7.52 (m, 1 H); ESI-MS calcd for
C24H32F2N6O [M + H]+ = 458.2, found: 459.0.
1
white solid. H NMR (400 MHz, CD3OD) δ ppm 1.32 (d, J = 6.53
Synthesis of 2-(4-(2,4-Difluorobenzyl)piperazin-1-yl)-N-isoprop-
yl-6-(methylsulfonyl)-5,6,7,8-tetrahydropyrido[3,4-b]pyrazin-3-
amine (5c). To a solution of 2-(4-(2,4-difluorobenzyl)piperazin-1-yl)-
N-isopropyl-5,6,7,8-tetrahydropyrido[3,4-b]pyrazin-3-amine (50 mg,
0.124 mmol) and DIPEA (0.032 mL, 0.186 mmol) in DCM (1 mL)
was added methanesulfonyl chloride (10.6 μL, 0.137 mmol) at 0 °C.
The reaction mixture was stirred at 0 °C for 1 h and then
concentrated via rotary evaporation. The resulting crude material was
partitioned between EtOAc (2 mL) and water (1 mL). The organic
phase was washed with brine, dried over Na2SO4, filtered, rinsed with
EtOAc, and concentrated via rotary evaporation. The crude material
was dissolved in EtOAc and MeOH, adsorbed on silica gel (0.75 g),
and purified via medium pressure silica gel column chromatography
using a gradient eluant of 50% to 100% EtOAc in heptane. The pure
fractions were combined, concentrated via rotary evaporation, and
dried in vacuo to provide the title compound 5c (50.6 mg, 85%) as an
Hz, 6H), 1.29−1.29 (m, 1H), 1.95−2.07 (m, 2H), 2.11−2.24 (m,
2H), 3.35−3.44 (m, 2H), 3.68−3.79 (m, 2H), 4.41−4.51 (m, 1H),
4.55 (td, J = 7.15, 3.51 Hz, 1H), 6.83−6.92 (m, 1H), 6.99 (ddd, J =
11.32, 8.50, 3.01 Hz, 1H), 7.19 (td, J = 9.19, 5.46 Hz, 1H), 7.87 (s,
1H), 8.76 (s, 1H); ESI-MS calcd for C22H23F2N7 [M + H]+ = 423.2,
found: 424.4.
Synthesis of 1-(2-(4-(2,4-Difluorobenzyl)piperazin-1-yl)-3-(iso-
propylamino)-7,8-dihydropyrido[3,4-b]pyrazin-6(5H)-yl)ethan-1-
one (5a). To a pressure reactor charged with a red-orange solution of
2-(4-(2,4-difluorobenzyl)piperazin-1-yl)-N-isopropylpyrido[3,4-b]-
pyrazin-3-amine (14) (0.5 g, 1.255 mmol) in acetone (25 mL) and
dioxane (25 mL) was added palladium, 10 wt % on activated carbon
(0.053 g, 0.502 mmol) as a slurry in dioxane (3 mL) under nitrogen.
Next, acetic anhydride (1.179 mL, 12.55 mmol) was added at 23 °C.
The mixture was stirred under hydrogen at 45 °C and 310 kPa for 9.5
h. The reaction mixture was filtered through Celite, rinsed with
EtOAc, and concentrated via rotary evaporation. The crude material
was dissolved in toluene (5 mL) and purified via medium pressure
chromatography using a 10% to 100% gradient eluant of EtOAc in
heptane on an NH column. The pure fractions were combined,
concentrated via rotary evaporation, and dried in vacuo to provide the
1
off-white solid. H NMR (500 MHz, DMSO-d6) δ ppm 1.12−1.19
(m, 6H), 2.53−2.61 (m, 4H), 2.74 (t, J = 5.9 Hz, 2H), 2.97 (s, 3H),
3.00 (d, J = 7.3 Hz, 4H), 3.44−3.48 (m, 1H), 3.59 (s, 2H), 4.02−4.10
(m, 1H), 4.16 (s, 2H), 5.41 (d, J = 7.8 Hz, 1H), 7.08 (td, J = 8.4, 2.2
Hz, 1H), 7.22 (td, J = 9.9, 2.7 Hz, 1H), 7.44−7.54 (m, 1H); ESI-MS
calcd for C22H30F2N6O2S [M + H]+ = 480.2, found: 480.9.
1
title compound 5a (341 mg, 61.1%) as an off-white foam. H NMR
Substrates 16a,b were prepared under the reaction conditions
similar to 3b (Scheme 2) through double SNAr reactions on 11a with
isopropylamine (or cyclopropylamine) and 4-(2,4-difluorophenoxy)-
piperidine sequentially. Substrates 17a,b were prepared under the
(500 MHz, DMSO-d6, mixture of rotamers) δ ppm 1.14−1.19 (m,
6H), 2.08 (s, 1.2H), 2.09 (s, 1.8H), 2.52−2.65 (m, 5H), 2.70 (t, J =
5.9 Hz, 1H), 2.98 (br s, 4H), 3.58 (s, 2H), 3.70 (dt, J = 12.0, 5.7 Hz,
L
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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Drug Annotation
reaction conditions similar to 3a through double SNAr reactions on
11a with 4-(2,4-difluorophenoxy)piperidine and isopropylamine (or
cyclopropylamine) sequentially.
CDCl3) δ ppm 0.66−0.75 (m, 2 H) 1.03−1.13 (m, 2 H) 1.94−2.03
(m, 2 H) 2.08−2.15 (m, 2 H) 2.23−2.28 (m, 3 H) 2.96−3.14 (m, 5
H) 3.38−3.47 (m, 2 H) 3.75−3.83 (m, 1 H) 3.90−3.97 (m, 1 H)
4.38−4.45 (m, 1 H) 4.51−4.56 (m, 1 H) 4.66−4.70 (m, 1 H) 6.78−
6.85 (m, 1 H) 6.86−6.92 (m, 1 H) 6.97−7.04 (m, 1 H); ESI-MS
calcd for C23H27F2N5O2 [M + H]+ = 443.2, found: 444.4.
Synthesis of 2-(4-(2,4-Difluorophenoxy)piperidin-1-yl)-N-iso-
propyl-5,6,7,8-tetrahydropyrido [3,4-b]pyrazin-3-amine (6c). Step
1: To a yellow-orange suspension of 2-(4-(2,4-difluorophenoxy)-
piperidin-1-yl)-N-isopropylpyrido[3,4-b]pyrazin-3-amine 17a (5.1 g,
12.77 mmol) in MeCN (63.8 mL) was added benzyl bromide (2.184
g, 12.77 mmol) over 1 min at rt. The mixture was stirred at 80 °C for
4 h to furnish a red-brown solution, cooled to 23 °C, and then
concentrated via rotary evaporation to provide 6-benzyl-2-(4-(2,4-
difluorophenoxy)piperidin-1-yl)-3-(isopropylamino)pyrido[3,4-b]
pyrazin-6-ium bromide (7.30 g) as a red-brown solid, which was used
directly in the next step without further purification.
Step 2: To a solution of 6-benzyl-2-(4-(2,4-difluorophenoxy)-
piperidin-1-yl)-3-(isopropylamino)pyrido[3,4-b]pyrazin-6-ium bro-
mide (7.2 g, 12.62 mmol) in DCM (126 mL) was added
NaBH(OAc)3 (16.05 g, 76 mmol) in one portion at rt. The resulting
solution was stirred at rt for 3 days. The solvent was removed under
reduced pressure, and EtOAc (200 mL) was added to redissolve the
residue. Saturated NaHCO3 (150 mL) was added, and the mixture
was vigorously stirred for 30 min. The organic layer was washed with
water (50 mL) and brine (50 mL), and dried with anhydrous Na2SO4
overnight. Removal of the solvent gave 6-benzyl-2-(4-(2,4-
difluorophenoxy)piperidin-1-yl)-N-isopropyl-5,6,7,8-tetrahydropyrido
[3,4-b]pyrazin-3-amine (6.43 g) as a yellow solid, which was used
without further purification. ESI-MS calcd for C28H33F2N5O [M +
H]+ = 493.3, found: 494.1.
Preparation of 2-(4-(2,4-Difluorophenoxy)piperidin-1-yl)-N-iso-
propyl-6-methyl-5,6,7,8-tetrahydropyrido[3,4-b]pyrazin-3-amine
(6d). Sodium triacetoxyhydroborate (9.9 mg, 0.047 mmol) was added
to a solution of DIPEA (8 μL, 0.047 mmol), 2-(4-(2,4-
difluorophenoxy)piperidin-1-yl)-N-isopropyl-5,6,7,8-
tetrahydropyrido[3,4-b]pyrazin-3-amine (6c) (12.1 mg, 0.023 mmol)
and formaldehyde (2 μL, 0.023 mmol) in MeOH (230 μL) at rt. After
30 min, the mixture was purified by HPLC to afford the title
1
compound 6d as a TFA salt (8.9 mg, 71.6%) as a yellow film. H
NMR (400 MHz, methanol-d4) δ ppm 1.24 (d, J = 6.3 Hz, 6H),
1.90−2.00 (m, 2H), 2.08−2.17 (m, 2H), 2.95−3.06 (m, 3H), 3.12 (br
s, 4H), 3.35−3.53 (m, 3H), 3.76 (br s, 1H), 4.15 (dt, J = 13.1, 6.5, 6.5
Hz, 1H), 4.19−4.38 (m, 2H), 4.48 (tt, J = 7.3, 3.7 Hz, 1H), 6.85−
6.92 (m, 1H), 6.99 (ddd, J = 11.4, 8.5, 3.0 Hz, 1H), 7.17 (td, J = 9.2,
5.6 Hz, 1H); ESI-MS calcd for C22H29F2N5O [M + H]+ = 417.2,
found: 418.3.
Preparation of 2-(4-(2,4-Difluorophenoxy)piperidin-1-yl)-N-iso-
propyl-6-(methylsulfonyl)-5,6,7,8-tetrahydropyrido[3,4-b]pyrazin-
3-amine (6e). In a 50 mL round-bottomed flask equipped with a stir
bar, a solution of 2-(4-(2,4-difluorophenoxy)piperidin-1-yl)-N-iso-
propyl-5,6,7,8-tetrahydropyrido[3,4-b]pyrazin-3-amine TFA salt (6c)
(735.3 mg, 1.421 mmol) in DCM (14.2 mL) at 0 °C was treated with
triethylamine (0.594 mL, 4.26 mmol), followed by addition of
methanesulfonyl chloride (0.221 mL, 2.84 mmol). The reaction
mixture was stirred at 0 °C for 1 h. The reaction mixture was
concentrated under reduced pressure. The residue was purified by
HPLC to give the title compound 6e as an off-white solid (229.6 mg,
33.6%). 1H NMR (500 MHz, DMSO-d6) δ ppm 1.18 (d, J = 6.3 Hz,
6H), 1.88 (m, 2H), 2.07 (m, 2H), 2.75 (t, J = 5.9 Hz, 2H), 2.89 (m,
2H), 2.97 (s, 3H), 3.29 (m, 2H), 3.45 (t, J = 5.9 Hz, 2H), 4.09 (m,
1H), 4.16 (s, 2H), 4.52 (tt, J = 8.1, 3.9 Hz, 1H), 5.57 (d, J = 8.3 Hz,
1H), 7.01 (m, 1H), 7.30 (m, 2H); ESI-MS calcd for C22H29F2N5O3S
[M + H]+ = 481.2, found: 481.90.
Step 3: A mixture of 6-benzyl-2-(4-(2,4-difluorophenoxy)piperidin-
1-yl)-N-isopropyl-5,6,7,8-tetrahydropyrido[3,4-b]pyrazin-3-amine
(6.43 g, 13.03 mmol) and 10% Pd/C (640 mg) in MeOH (86 mL)
under H2 in a balloon was stirred at rt overnight. Removal of the
solvent gave 2-(4-(2,4-difluorophenoxy)piperidin-1-yl)-N-isopropyl-
5,6,7,8-tetrahydropyrido [3,4-b]pyrazin-3-amine (6c) (4.4 g, 84%) as
1
a yellow oil. H NMR (400 MHz, CD3OD) δ ppm 1.24 (d, J = 6.4
Hz, 6H), 1.89−1.99 (m, 2H), 2.09−2.17 (m, 2H), 2.74 (t, J = 5.9 Hz,
2H), 2.95 (ddd, J = 12.3, 8.7, 3.4 Hz, 2H), 3.11 (t, J = 6.1 Hz, 2H),
3.82 (s, 2H), 4.14 (septet, J = 6.4 Hz, 1H), 4.46 (tt, J = 7.7, 3.5 Hz,
1H), 6.85−6.92 (m, 1H), 6.99 (ddd, J = 11.4, 8.4, 3.2 Hz, 1H), 7.18
(td, J = 9.3, 5.4 Hz, 1H); ESI-MS calcd for C21H27F2N5O [M + H]+ =
403.2, found: 404.0.
Compounds 18a, 18b, and 19a were prepared in a reaction
sequence similar to the synthesis of 6c, starting with 16a, 16b, and
17b, respectively.
Preparation of Methyl 2-(4-(2,4-difluorophenoxy)piperidin-1-yl)-
3-(isopropylamino)-7,8-dihydropyrido[3,4-b]pyrazine-6(5H)-car-
boxylate (6f). To a solution of 2-(4-(2,4-difluorophenoxy)piperidin-
1-yl)-N-isopropyl-5,6,7,8-tetrahydropyrido[3,4-b]pyrazin-3-amine
(6c) (45.6 mg, 0.113 mmol) and triethylamine (0.039 mL, 0.283
mmol) in DCM (0.75 mL) was added methyl carbonochloridate
(0.013 mL, 0.170 mmol) at 0 °C. The reaction mixture was stirred at
0 °C for 1 h, warmed to 23 °C, and concentrated under reduced
pressure. The resulting crude material was partitioned between EtOAc
and H2O. The organic phase was washed with brine, dried over
Na2SO4, filtered, rinsed with EtOAc, and concentrated under reduced
pressure. The crude material was purified by silica gel column
chromatography (2% to 80% gradient eluant of EtOAc in heptane) to
Preparation of 1-(3-(4-(2,4-Difluorophenoxy)piperidin-1-yl)-2-
(isopropylamino)-7,8-dihydropyrido[3,4-b]pyrazin-6(5H)-yl)-
ethanone (6a). Acetic anhydride (5 μL, 0.058 mmol) was added to a
solution of 3-(4-(2,4-difluorophenoxy)piperidin-1-yl)-N-isopropyl-
5,6,7,8-tetrahydropyrido[3,4-b]pyrazin-2-amine TFA salt (18a)
(15.1 mg, 0.029 mmol) and pyridine (7 μL, 0.088 mmol) in DCM
(290 μL) at rt. After being stirred for 1 h, the mixture was purified by
HPLC to afford the title compound 6a as its TFA salt (12.7 mg, 78%)
as a yellow film. 1H NMR (400 MHz, CD3OD, mixture of two
rotamers) δ ppm 1.33 (d, J = 6.6 Hz, 6H), 1.91−2.02 (m, 2H), 2.09−
2.18 (m, 2H), 2.19 (s, 1.4H), 2.21 (s, 1.6H), 2.77−2.83 (m, 0.9H),
2.88−2.94 (m, 1.1H), 3.12 (td, J = 8.5, 4.0 Hz, 2H), 3.43−3.51 (m,
2H), 3.80−3.85 (m, 1.1H), 3.86−3.91 (m, 0.9H), 4.10−4.18 (m,
1H), 4.47−4.54 (m, 1H), 4.55 (br s, 2H), 6.85−6.92 (m, 1H), 6.99
(ddd, J = 11.4, 8.6, 3.0 Hz, 1H), 7.18 (td, J = 9.2, 5.6 Hz, 1H); ESI-
MS calcd for C23H27F2N5O2 [M + H]+ = 445.2, found: 446.4.
Preparation of 1-(2-(Cyclopropylamino)-3-(4-(2,4-
difluorophenoxy)piperidin-1-yl)-7,8-dihydropyrido[3,4-b]pyrazin-
6(5H)-yl)ethenone (6b). To a solution of N-cyclopropyl-3-(4-(2,4-
difluorophenoxy)piperidin-1-yl)-5,6,7,8-tetrahydropyrido[3,4-b]-
pyrazin-2-amine (18b) (1.76 g, 4.38 mmol) in DCM (50 mL) at 0 °C
was added acetyl chloride (0.467 mL, 6.58 mmol) and DIPEA (2.291
mL, 13.15 mmol) dropwise. The resulting solution was stirred at rt for
10 min and then concentrated under reduced pressure. The residue
was purified by HPLC to give the title compound 6b (699 mg, 36%)
as a white solid. Melting point: 106.8 °C. 1H NMR (500 MHz,
1
provide the title compound 4i (48.7 mg, 93%) as a white foam. H
NMR (500 MHz, DMSO-d6) δ ppm 1.17−1.19 (m, 6H), 1.80−1.94
(m, 2H), 2.06 (ddd, J = 9.5, 5.9, 3.2 Hz, 2H), 2.64 (t, J = 5.9 Hz, 2H),
2.82−2.94 (m, 2H), 3.19−3.31 (m, 2H), 3.64 (s, 3H), 3.64−3.69 (m,
2H), 4.07−4.17 (m, 1H), 4.35 (br s, 2H), 4.51 (tt, J = 8.1, 3.9 Hz,
1H), 5.53 (d, J = 8.3 Hz, 1H), 7.01 (dddd, J = 9.3, 8.1, 3.2, 1.5 Hz,
1H), 7.26−7.35 (m, 2H); ESI-MS calcd for C23H29F2N5O3 [M + H]+
= 461.2, found: 461.9.
Preparation of 1-(3-(cyclopropylamino)-2-(4-(2,4-
difluorophenoxy)piperidin-1-yl)-7,8-dihydropyrido[3,4-b]pyrazin-
6(5H)-yl)ethanone (6g) is similar to the synthesis of compound 6a
starting with N-cyclopropyl-2-(4-(2,4-difluorophenoxy)piperidin-1-
yl)-5,6,7,8-tetrahydropyrido[3,4-b]pyrazin-3-amine (19a). 1H NMR
(500 MHz, CDCl3) δ ppm 0.46−0.56 (m, 2H) 0.78−0.87 (m, 2H)
1.87−1.96 (m, 2H) 2.05−2.14 (m, 2H) 2.18−2.22 (m, 3H) 2.72−
2.94 (m, 5H) 3.27−3.37 (m, 2H) 3.70−3.77 (m, 1H) 3.86−3.92 (m,
1H) 4.28−4.36 (m, 1H) 4.56 (s, 1H) 4.70 (s, 1H) 5.05−5.15 (m,
M
https://doi.org/10.1021/acs.jmedchem.0c02081
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Journal of Medicinal Chemistry
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Drug Annotation
1H) 6.76−6.82 (m, 1H) 6.84−6.90 (m, 1H) 6.96−7.03 (m, 1H);
ESI-MS calcd for C23H27F2N5O2 [M + H]+ = 443.2, found: 444.4.
2-(4-(2,4-Difluorophenoxy)piperidin-1-yl)-N-isopropyl-6-(methyl-
sulfonyl)-5,6,7,8-tetrahydropyrido[3,4-b]pyrazin-3-amine (6h) was
prepared in a manner similar to the synthesis of 6a starting with 6c
(50 mM Tris, pH 7.4, 50 mM NaCl, 6 mM MgCl2, fatty acid free 0.1%
BSA, proteinase inhibitor cocktail, Sigma USA) was added per well
(2.4 nM final assay concentration). Binding reactions were initiated
by adding 40 μL of total membranes obtained from cells expressing
human GPR6 receptors. Membranes were prepared in assay buffer
(50 mM Tris, pH 7.4, 50 mM NaCl, 6 mM MgCl2, fatty acid free 0.1%
BSA, 1× proteinase inhibitor cocktail, Sigma USA) and added per
well to 15 μg/well final assay concentration. Plates were sealed, mixed
for 30 s at 300 rpm, and incubated for 2 h at room temperature.
Reactions were filtered through filtermates (1450-421, filtermate A,
PerkinElmer, USA), and washed five times for 5 s with 4 °C cold 50
mM Tris, pH 7.4, 50 mM NaCl, 6 mM MgCl2, fatty acid free 0.1%
BSA using a Tomtec Harvester 96 instrument. Filters were dried in
the microwave, scintillator sheet (1450-411, PerkinElmer, USA)
melted on them and heat-sealed before CPM/wells were quantified in
a Microbeta Trilux instrument (PerkinElmer, USA). Before use,
filtermates were presoaked in 0.5% polyethylenimine solution for 3 h
with gentle shaking, followed by air drying overnight.
1
and acetic anhydride. H NMR (400 MHz, CD3OD, mixture of two
rotamers) δ ppm 1.23 (d, J = 6.6 Hz, 3.3H), 1.24 (d, J = 6.6 Hz,
2.7H), 1.93 (m, 2H), 2.11 (m, 2H), 2.18 (s, 1.3H), 2.20 (s, 1.7H),
2.71 (t, J = 6.1 Hz, 0.9H), 2.80 (t, J = 6.1 Hz, 1.1H), 2.95 (m, 2H),
3.34 (m, 2H), 3.79 (t, J = 5.9 Hz, 1.1H), 3.84 (t, J = 6.1 Hz, 0.9H),
4.15 (m, 1H), 4.44 (tt, J = 7.5, 3.7 Hz, 1H), 4.53 (s, 0.9H), 4.54 (s,
1.1H), 6.86 (m, 1H), 6.97 (ddd, J = 11.2, 8.5, 3.2 Hz, 1H), 7.16 (td, J
= 9.2, 5.6 Hz, 1H); ESI-MS calcd for C23H29F2N5O2 [M + H]+ =
445.2, found: 446.0.
Preparation of (R)-1-(2-(4-(2,4-difluorophenoxy)piperidin-1-yl)-3-
((tetrahydrofuran-3-yl)amino)-7,8-dihydropyrido[3,4-b]pyrazin-
6(5H)-yl)ethan-1-one (6i) is similar to the synthesis of compound 6a
starting with (R)-2-(4-(2,4-difluorophenoxy)piperidin-1-yl)-N-(tetra-
1
hydrofuran-3-yl)pyrido[3,4-b]pyrazin-3-amine (17c). H NMR (500
The same protocol was used for saturation binding assays to
MHz, DMSO-d6, mixture of two rotamers) δ ppm 1.83−2.00 (m,
3H), 2.06 (dd, J = 6.83, 3.42 Hz, 2H), 2.09 (d, J = 3.42 Hz, 3H),
2.13−2.23 (m, 1H), 2.60 (t, J = 5.86 Hz, 1H), 2.72 (t, J = 5.86 Hz,
1H), 2.84−2.95 (m, 2H), 3.29 (br. s., 1H), 3.56 (dt, J = 8.79, 5.37 Hz,
1H), 3.68−3.75 (m, 3H), 3.82−3.88 (m, 1H), 3.90 (ddd, J = 8.79,
6.10, 2.68 Hz, 1 H), 4.35−4.41 (m, 1 H), 4.42 (s, 1H), 4.45 (s, 1H),
4.51 (tt, J = 8.12, 3.84 Hz, 1H), 5.90 (dd, J = 13.42, 6.10 Hz, 1H),
7.01 (dddd, J = 9.28, 8.05, 2.93, 1.71 Hz, 1H), 7.23−7.35 (m, 2H);
SI-MS calcd for C24H29F2N5O3 [M + H]+ = 473.2, found: 474.3.
GPR6 cAMP TR-FRET Assay. Cell-based GPR6 cAMP TR-FRET
assays were used to quantify GPR6 receptor-dependent modulation of
the cAMP level in CHO-K1 cells. The unusually high level of
apparent constitutive activity of GPR6 in heterologous expression
systems provided the opportunity to measure inhibition of GPR6
without agonist stimulation. T-REx-CHO-K1 cells (Invitrogen, USA)
stably expressing tetracycline inducible human GPR6 were cultured in
growth media (F12K medium [Gibco, Life Technologies], containing
10% tetracycline free fetal bovine serum FBS [HyClone, Fisher
Scientific], 1% penicillin/streptomycin, and 200 μg/mL Hygromycin
B). To induce GPR6 expression, cells were dissociated and plated in
induction media (growth media plus 2.0 μg/mL doxycycline [Sigma
D9891]) at a density of 250−500 cells per well in half area white solid
bottom 96-well plates (Costar) and incubated (37 °C, 5% CO2) for
20 h. Following incubation, media was removed, and cells were
washed with 50 μL of Krebs Ringer’s buffer (Sigma K4002). Source
plates containing test compounds suspended in DMSO were diluted
in Krebs Ringer’s buffer containing 0.5% fatty acid free BSA. Cells
were incubated with compound for 45 min at 37 °C and 5% CO2,
followed by Eu-cAMP tracer for 10 min and ULight-anti-cAMP
antibody for 1 h at room temperature (PerkinElmer Lance Ultra
cAMP assay (TRF0264)). TR-FRET signal was detected on an
Envision plate reader (PerkinElmer). IC50 curves were generated with
a four-parameter logistic equation using GraphPad Prism 5.03.
GPR6 Radioligand Binding Assay. RL-338 was synthesized in
the chemistry laboratories of Envoy Therapeutics, Inc., FL. In vitro
pharmacological profiling showed that RL-338 is a potent and highly
selective GPR6 IAG, inhibiting GPR6 cAMP signaling in TR-FRET
assays with a EC50 = 16 nM and >100 fold selectivity against GPR3
and GPR12 (unpublished). RL-338 was radiolabeled with three
tritium atoms by the Quotient Bioresearch (Cardiff, UK) and
validated using standard in vitro pharmacology methods (Supporting
Information). To study the binding characteristics of GPR6 IAG
compounds, a competition binding assay using a filtration-based
format was developed featuring membranes prepared from T-REx-
CHO-GPR6 cells expressing human GPR6 cDNA driven by a
doxycycline inducible promoter. First, assay-ready 96-well plates
(651201, Greiner, USA) containing serial dilutions of test compounds
(1 μL of test ligand/well) were prepared in DMSO using liquid
handlers (5 μM, final assay top concentration). A total of 39 μL of
assay buffer was added, and plates were mixed on a plate shaker for 10
determine radioligand 3H-RL-338 affinity to GPR6. Different
3
concentrations of radioligand H-RL-338 ranging from 32 nM (high
concentration) to 0.015 nM (low concentration) with 1:2 dilution
steps were tested to generate saturation curves and obtain the
equilibrium dissociation constant Kd. Nonspecific background binding
of 3H-RL-338 was determined using 10 mM of an unlabeled
structurally diverse potent and selective GPR6 inverse agonists
(data not shown). IC50 and Kd values were calculated using nonlinear
regression analysis in Prism (GraphPad, USA). Saturation curves were
fitted to the Prism equation: one site - fit total and nonspecific binding
without constraints. Dose-response curves were fitted to the four-
parameter variable slope model without constraints in Prism to obtain
IC50’s. Ki values were calculated using the Cheng−Prusoff equation
(Cheng and Prusoff, 1973). CVN424 and other GPR6 IAG fully
3
displaced H-RL-338 from GPR6 and displayed binding properties
3
that were consistent with a single binding site. Radioligand H-RL-
338 demonstrated high affinity for human GPR6 receptors stably
expressed in T-REx-CHO-GPR6 cells (Kd
2.4 nM or 5 nM
=
dependent on synthesized radioligand batch) in saturation binding
assays.
Haloperidol-Induced Catalepsy. Haloperidol-induced catalepsy
in rodents is caused by blocking activity of striatal dopamine D2
receptors, resulting in elevated cAMP and overactivity of the indirect
pathway of the striatum and decreased movement. Compounds 3e
was tested for reversal of haloperidol-induced catalepsy in male wild-
type Sprague-Dawley rats (source: Harlan, Frederick (MD) USA),
male C57BL/6N (source: wild-type mice (source: Jackson Labo-
ratories, ME, USA) and GPR6 knock out mice using the bar test.22
The cataleptic state was induced by the subcutaneous administration
of the dopamine receptor antagonist haloperidol (0.3 mg/kg, sc rats
and 1 mg/kg i.p. mice), 90 min before testing the animals. Front paws
were placed on a horizontal metal bar raised 2’ (mice) or 6’ (rats)
above a Plexiglas platform and time was recorded for up to 60 s. The
test ended when the animal’s front paws returned to the platform or
after 60 s. The test was repeated three times, and the average of the
trials reported. Cataleptic animals treated with haloperidol will hold to
the bar without moving for several minutes. Efficacy of compound 3e
to reverse haloperidol-induced catalepsy was tested 120 min post
injection. The A2a antagonist KW6002 (Istradefylline) was dosed at
0.6 mg/kg i.p. as a positive control.
Bilateral 6-OHDA-Lesioned Parkinson’s Disease Rat Model.
Bilateral striatal 6-OHDA lesion in rats trigger degeneration of
dopaminergic neurons, striatal dopamine depletion and show motor
deficits, which mimics aspects of human PD pathology and
symptomology. Early lead compound 6h was tested for anti-
parkinsonian efficacy in this model, administered as monotherapy
(1 mg/kg, p.o.). Thirty minutes prior to surgery, animals (female
Sprague-Dawley rats, Charles River Laboratories, Wilmington, MA
USA) received pargyline (5 mg/kg; i.p.) and desipramine (10 mg/kg;
i.p.) to optimize subsequent 6-hydroxydopamine (6-OHDA)
availability and increase specificity for toxicity to dopaminergic
3
min. Next, 40 μL of radioligand H-RL-338 prepared in assay buffer
N
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Journal of Medicinal Chemistry
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Drug Annotation
neurons. Rats received bilateral injections of 6-OHDA (20 μg in 3 μL;
0.5 μL/min) into the striatum at coordinates AP: ML: DV, 1, 3, −5
mm relative to Bregma to introduce lesion. Open field activity of
animals was recorded pre- and 28 days postsurgery for 3 h. Bilateral
lesions induced a significant reduction in activity (40%) compared to
prelesion. In addition, there was a 32% reduction of dopamine
transporter expression in the striatum (not shown). Directly after
dosing compound 6h locomotor activity (defined as beam broken on
the lower level) was assessed in open field arenas equipped with
automatic activity monitors (Linton Instrumentation, U.K.).
Mark Beresford Lewis Carlton − Cerevance Ltd, Cambridge,
U.K.
Nicola L. Brice − Cerevance Ltd, Cambridge, U.K.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c02081
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
ASSOCIATED CONTENT
■
We thank the following individuals for their contributions: Ron
Chen, Padma Manam, Yang Song, and Paddi Ekhlassi for
analytical chemistry and formulation support, Ron Steigerwalt
for drug safety expertise, and our process chemistry colleagues
Wolfgang Notz, Chunrong Ma, and Dave Provencal.
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02081.
Materials and Methods; preparation of key intermedi-
ates; characterization of lead compounds: 1, 3e, and 6h;
original pharmacology data for compound 3e and lead
compound 6h; structure and validation data of radio-
ligand [3H]-RL338; reference compound for cAMP TR-
FRET assays: structure and in vitro pharmacology;
compound 3e in vitro pharmacology graphs (PDF)
ABBREVIATIONS USED
■
cAMP, cyclic adenosine monophosphate; DCM, dichloro-
methane; DMF, N,N-dimethylformamide; HPLC, high-per-
formance liquid chromatography; Hz, hertz; LC-MS, liquid
chromatography−mass spectrometry; MW, microwave; NMR,
nuclear magnetic resonance; rt, room temperature; TEA,
triethylamine; TFA, trifluoroacetic acid; THF, tetrahydrofuran
Molecular formula strings (CSV)
AUTHOR INFORMATION
■
REFERENCES
■
Corresponding Authors
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Huikai Sun − Takeda California, San Diego, California
92121, United States; orcid.org/0000-0002-4593-1306;
Phone: 619-930-8349; Email: Huikai.sun@takeda.com
Holger Monenschein − Takeda California, San Diego,
California 92121, United States; Email: holger.m@
takeda.com
Authors
Hans H. Schiffer − Takeda California, San Diego, California
92121, United States
Holly A. Reichard − Takeda California, San Diego, California
92121, United States
Shota Kikuchi − Takeda California, San Diego, California
92121, United States
Maria Hopkins − Takeda California, San Diego, California
92121, United States
Todd K. Macklin − Takeda California, San Diego, California
92121, United States
Stephen Hitchcock − Takeda California, San Diego,
California 92121, United States
Mark Adams − Takeda California, San Diego, California
92121, United States
Jason Green − Takeda California, San Diego, California
92121, United States
Jason Brown − Takeda California, San Diego, California
92121, United States
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Sean T. Murphy − Takeda California, San Diego, California
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Nidhi Kaushal − Takeda California, San Diego, California
92121, United States
Deanna R. Collia − Takeda California, San Diego, California
92121, United States
Steve Moore − Takeda California, San Diego, California
92121, United States
William J. Ray − Takeda California, San Diego, California
92121, United States
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