A Dual Inhibitor of DYRK1A and GSK3β for β-Cell Proliferation: Aminopyrazine Derivative GNF4877

Article abstract of DOI:10.1002/cmdc.202000183

Loss of β-cell mass and function can lead to insufficient insulin levels and ultimately to hyperglycemia and diabetes mellitus. The mainstream treatment approach involves regulation of insulin levels; however, approaches intended to increase β-cell mass are less developed. Promoting β-cell proliferation with low-molecular-weight inhibitors of dual-specificity tyrosine-regulated kinase 1A (DYRK1A) offers the potential to treat diabetes with oral therapies by restoring β-cell mass, insulin content and glycemic control. GNF4877, a potent dual inhibitor of DYRK1A and glycogen synthase kinase 3β (GSK3β) was previously reported to induce primary human β-cell proliferation in vitro and in vivo. Herein, we describe the lead optimization that lead to the identification of GNF4877 from an aminopyrazine hit identified in a phenotypic high-throughput screening campaign measuring β-cell proliferation.

Full text of DOI:10.1002/cmdc.202000183

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A Dual Inhibitor of DYRK1A and GSK3β for β-Cell
Proliferation: Aminopyrazine Derivative GNF4877
Yahu A. Liu,*[a] Qihui Jin,[a] Qiang Ding,[a] Xueshi Hao,[a] Tingting Mo,[a] Shanshan Yan,[a]
Yefen Zou,[a] Zhihong Huang,[a] Xiaoyue Zhang,[a] Wenqi Gao,[a] Tom Y.-H. Wu,[a] Chun Li,[a]
Badry Bursalaya,[a] Michael Di Donato,[a] You-Qing Zhang,[a] Lisa Deaton,[a] Weijun Shen,[a]
Brandon Taylor,[a] Anwesh Kamireddy,[a] George Harb,[a] Jing Li,[a] Yong Jia,[a]
Andrew M. Schumacher,[a] Bryan Laffitte,[a] Richard Glynne,[a] Shifeng Pan,[a]
Peter McNamara,[a] Valentina Molteni,[a] and Jon Loren*[a]
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Loss of β-cell mass and function can lead to insufficient insulin
levels and ultimately to hyperglycemia and diabetes mellitus.
The mainstream treatment approach involves regulation of
insulin levels; however, approaches intended to increase β-cell
mass are less developed. Promoting β-cell proliferation with
low-molecular-weight inhibitors of dual-specificity tyrosine-
regulated kinase 1A (DYRK1A) offers the potential to treat
diabetes with oral therapies by restoring β-cell mass, insulin
content and glycemic control. GNF4877, a potent dual inhibitor
of DYRK1A and glycogen synthase kinase 3β (GSK3β) was
previously reported to induce primary human β-cell prolifer-
ation in vitro and in vivo. Herein, we describe the lead
optimization that lead to the identification of GNF4877 from an
aminopyrazine hit identified in a phenotypic high-throughput
screening campaign measuring β-cell proliferation.
Introduction
human islets in NOD-SCID mice. It was elucidated that
harmine’s inhibition of dual-specificity tyrosine-regulated kinase
1A (DYRK1A) contributes to β-cell proliferation through the
attenuation of nuclear factor phosphorylation in activated T
cells (NFAT).[8–12] Besides harmine, numerous DYRK1A inhibitor
scaffolds have been reported (Figure 1);[13–34] including 5-
iodotubercidin (5-IT),[16] INDY,[17] and selective DYRK1A inhibitor
GNF2133,[18] which have shown to promote human β-cell
proliferation.
β-Cell proliferation could further be enhanced by combining
DYRK1A inhibitors with different mechanisms of action. Stewart
and co-workers recently demonstrated that the combination of
a glucagon-like peptide-1 receptor (GLP-1R) agonist with a
More than 400 million people worldwide are living with
diabetes which results from insufficient mass and/or impaired
function of pancreatic insulin-producing β-cells.[1–3] Progress
towards the pharmacological driven expansion of functional β-
cells faces multiple challenges, such as limited understanding of
the signaling pathways relevant for human β-cell growth and
the extremely low levels of proliferation capacity of adult
human β-cells, even after mutagenic stimulation.[4] Despite
these challenges, there has been steady progress identifying
molecules that could potentially increase proliferation of human
β-cells.[4] For example, circulating factors derived from hepato-
cytes were shown to promote proliferation of β-cells.[5]
SerpinB1, a conserved protease inhibitor, enhanced β-cell
proliferation in zebrafish, mice and humans.[6] Diarylamide
compound WS6 induced human β-cell proliferation in a
dispersed islet proliferation assay and in intact human islet
cultures.[7] Harmine and its derivatives were reported to induce
human β-cell proliferation both in vitro and in transplanted
DYRK1A inhibitor could induce
a synergistic increase in
replication and numbers of human β-cells.[35] In Shen et al., we
reported aminopyrazine derivative 1 (GNF4877, Figure 1), a dual
inhibitor of DYRK1A and GSK3β. This compound showed that
inhibition of both DYRK1A and glycogen synthase kinase 3β
(GSK3β) can have a synergistic effect enhancing β-cell prolifer-
ation, compared with DYRK1A inhibition alone.[36] We found
that human islets treated with 1 retain functionality both
in vitro and in vivo after transplantation into diabetic mice.[36]
Oral dosing of 1 in diabetic mice induced β-cell proliferation,
increased β-cell mass and insulin content and improved
glycemic control.[36] In this report, we describe the lead
optimization efforts which led to the discovery of 1.
[a] Dr. Y. A. Liu, Dr. Q. Jin, Q. Ding, Dr. X. Hao, T. Mo, S. Yan, Dr. Y. Zou,
Dr. Z. Huang, X. Zhang, W. Gao, Dr. T. Y.-H. Wu, C. Li, Dr. B. Bursalaya,
Dr. M. Di Donato, Dr. Y.-Q. Zhang, L. Deaton, Dr. W. Shen, Dr. B. Taylor,
A. Kamireddy, Dr. G. Harb, Dr. J. Li, Dr. Y. Jia, Dr. A. M. Schumacher,
Dr. B. Laffitte, Dr. R. Glynne, Dr. S. Pan, Dr. P. McNamara, Dr. V. Molteni,
Dr. J. Loren
Genomics Institute of the Novartis Research Foundation (GNF)
10675 John Jay Hopkins Drive
San Diego, CA 92121 (USA)
E-mail: yliu2@gnf.org
jloren@gnf.org
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.202000183
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Figure 1. Selected small-molecule DYRK1A inhibitors (reported literature IC50).[12–18,36]
Results and Discussion
then subjected to Suzuki coupling conditions with the corre-
sponding boronic acid (or boronic ester) to yield 7. Alterna-
tively, using method B, Suzuki coupling of aryl bromide 5 with
the corresponding boronic acid (or boronic ester) afforded
compound 8, which was subjected to amide bond formation
with aminopyridine 4 to yield 7. Hydrolysis of methyl or ethyl
ester afforded the final aminopyrazine products (1, 10–33).
Chemistry
The aminopyrazine analogues in this report, except for com-
pound 9 (Table 1) whose synthesis was previously described,[37]
were synthesized through the general reaction sequence out-
lined in Scheme 1. The synthesis began with an aromatic
nucleophilic substitution reaction between 4-chloro-3-nitropyr-
idine and a cyclohexylamine compound 2 with R being either
methyl or ethyl to give the nitropyridine 3, which was
subsequently reduced to the aminopyridine building block 4 by
catalytic hydrogenation. The synthesis of intermediate 7 was
achieved by permutating between amide and Suzuki coupling
conditions with 3-amino-6-bromopyrazine-2-carboxylic acid (5)
and aminopyridine 4. By using method A, amide bond
formation between 4 and 5 afforded compound 6, which was
Lead optimization
A safe orally administered small-molecule drug that can
selectively expand functional β-cell mass would be a highly
desirable option for diabetic patients over routine insulin
injections. The aim of the medicinal chemistry effort described
here was to discover potent orally bioavailable small-molecule
DYRK1A and GSK3β dual inhibitors that can proliferate human
°
°
Scheme 1. Synthesis of the aminopyrazine analogues. a) K2CO3, DMF, 25 C, 4 h; b) Pd/C (10%), H2 (balloon), MeOH, 25 C, 16 h; c) Pd/C (10%), H2 (50 psi),
°
°
°
°
EtOAc, 25 C, 3 h; d) 4, HATU, DMF, 25 C, 2 h; e) 4, EDC HCl, HOAt, DMF, 25 C, 18 h; f) ArB(OH)2 or ArBpin, Pd(dppf)Cl2, Et3N, CH3CN, 90 C, 4 h; g) ArB(OH)2 or
°
°
°
ArBpin, Pd(PPh3)4, Na2CO3, dioxane/H2O, 90 C, 3 h; h) LiOH (aqueous, 1.0 M), EtOH, 25 C, 16 h; i) LiOH (aqueous, 1.0 M), THF/MeOH, 25 C, 4–16 h.
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put screening (HTS) campaign measuring β-cell proliferation by
using growth-arrested reversibly immortalized mouse β-cell line
R7T1 (EC50 =0.018 μM).[7] Compound 9 was later demonstrated
to be a dual inhibitor of DYRK1A and GSK3β.[36] Further profiling
however showed that compound 9 was unable to proliferate
primary rat β-cells. In contrast, the carboxylic acid 10, albeit less
potent in the R7T1 assay (EC50 =0.33 μM), showed moderate
proliferative activity in rat islet β-cell proliferation assay (EC50 =
3.5 μM). We conjectured that the lack of primary islet activity
observed in 9 could be attributed to its basic amino group and
a related propensity to sink in the lysosome.[38] Therefore,
further optimization was focused on carboxylic acids. The
enzymatic DYRK1A activity was used as the primary driver for
lead optimization, while retaining activity on both DYRK1A and
GSK3β as basic criteria for selecting compounds for further
profiling in a rat islet proliferation assay.
Docking of 10 into the ATP binding pocket of DYRK1A using
an in-house co-crystal structure[18] showed that the pyrazine
core forms two hydrogen bonds in the hinge area, one between
the pyrazine nitrogen and backbone NH of L241 (hidden from
view), and the other between the pyrazinylamino group and
the backbone carbonyl oxygen of E239 (Figure 2). Additionally,
10 forms H-bonds with the side chain of K188 via the pyridine
nitrogen and with N244 via the carboxyl group. The interaction
with K188 proved to be critical, as moving (11–13) or removing
(14) the pyridyl nitrogen resulted in partial or complete loss of
enzymatic DYRK1A inhibitory activity (Table 1).
We further evaluated the structure-activity relationship
(SAR) by replacing the piperidine-4-carboxylic acid ring (Ta-
ble 1). The ring size of the cyclic amine had a significant impact
on the activity on DYRK1A, where four- and five-membered
rings decreased activity substantially (15, 16 vs 10). Introducing
a fluorine atom at the α position relative to the carboxylic acid
caused a sixfold reduction in potency (17 vs 10). Moving the
carboxylic acid from the 4-position to the 3-position of the
piperidine ring maintained the activity, but only when the
stereogenic center is in the R configuration (19 vs 10), whereas
the S enantiomer (18) was less potent. Replacing the piperidine
ring in 19 with a piperazine ring caused a ninefold decrease in
inhibition (20 vs 19). Homologating the carboxylic acid by one
carbon at the 3-position of the piperidine ring resulted in more
than a 35-fold decrease in inhibition independently of stereo-
chemistry (21 and 22 vs 19).
Table 1. Structure-activity relationship (SAR) of aminopyrazine derivatives
on enzymatic DYRK1A and GSK3β inhibition.
1
2
3
4
5
6
7
8
9
DYRK1A
IC50
GSK3β
IC50
[a]
[a]
Compound
R
[μM]
[μM]
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9
0.039
0.003
10
11
12
13
14
0.100
0.113
4.51
0.040
0.088
3.747
0.288
0.280
22.68
>50
15
16
17
25.02
>50
25.83
1.478
0.085
0.583
18
19
20
0.427
0.068
0.606
0.019
0.006
0.088
21
22
2.426
2.493
0.039
0.033
[a] Obtained from three or more independent experiments. ATP
concentration=1.0 mM.
β-cells. Our work started from the aminopyrazine derivative 9,
which was identified from a 850,000 compound high-through-
Figure 2. Molecular docking model of 10 in the DYRK1A crystal structure
(PDB ID: 6UIP).
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To potentially fill the unoccupied space surrounding the
Table 2. The effects of phenyl ring substitution on enzymatic DYRK1A and
GSK3β inhibition.
1
2
3
4
5
6
7
8
9
phenyl ring at the 6-position of the pyrazine core (Figure 2), we
set out to explore whether substituted aromatic groups
(designated R’) could increase potency on DYRK1A (Table 2).
Introduction of methyl and fluoro substituents at the ortho and
para position resulted in compounds two to three times less
potent than the unsubstituted analogue (23, 24, 27, 28 vs 10)
whereas other slightly larger substituents led to about a tenfold
loss in activity (25, 26, 29 vs 10). Modeling studies suggested
that larger substituents in the ortho position bring the phenyl
out of plane and this possibly accounts for the difference in
activity between ortho-substituted compounds (23, 24 vs 26).
The lower potency of para-substituted compounds with larger
groups (29 vs 27, 28) can be rationalized by a possible clash
with Y243. Interestingly, substituents at the meta position
yielded compounds with improved potency (30, 31 vs 10), as
they likely avoided twisting of the ring out of plane and
prevented negative interactions with Y243. Further inhibitory
potency improvement was observed when combining a small
substitution (F) at the ortho position and a larger substitution
(trifluoromethoxy) at the meta position onto the phenyl ring
especially when combined with the (R)-piperidine-3-carboxylic
Compound R’
R
DYRK1A GSK3β
[a] [a]
IC50
IC50
[μM]
[μM]
10
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23
24
0.243
0.093
0.330
1.071
1.120
0.06
25
26
0.469
0.063
acid at
R (32, 33). Finally, replacement of the meta-
trifluoromethoxy substitution with an isopropoxy group led to
compound (1) with single-digit nanomolar potency. Docking of
27
28
29
0.187
0.281
1.023
0.041
0.067
0.08
1
into the DYRK1A crystal structure revealed that the
isopropoxy substituent fits into the hydrophobic pocket created
by residues K175, I165, M240, and Y243, which may explain the
increased inhibitory potency (Figure 3).
Compared with 10, aminopyrazine 1 demonstrated a 14-
fold improvement in activity for DYRK1A and 2.5-fold for GSK3β
and was selected for further profiling in vitro and in vivo.
Consistent with the increased biochemical potency on both
DYRK1A and GSK3β, compound 1 showed 23-fold increase in
potency in the rat islet β-cell proliferation assay compared to
compound 10 (Table 3). Importantly, 1 also demonstrated good
proliferative activity in the human islet β-cell assay. Compound
1 showed acceptable physicochemical and in vitro ADME
properties, with desirable PSA (�140) and clogP (<5), good
permeability, moderate metabolic stability and no major CYP
inhibition or hERG liabilities. Solubility of 1, however, was low,
as measured by high throughput thermodynamic solubility. On
a broad kinase panel, compound 1 showed comparable
inhibition for SYK and modest inhibitions on ROS, CDK2 and
Axl, as previously reported.[36] In addition, 1 has preferential
inhibitory activity for both DYRK1A and DYRK1B subtypes,
compared to DYRK2.[39]
30
31
0.074
0.097
0.147
0.106
32
33
0.075
0.055
0.800
0.085
1
0.006
0.016
In vivo studies
[a] Obtained from three or more independent experiments. ATP
concentration=1.0 mM
In in vivo pharmacokinetic (PK) studies in mice dosed intra-
venously (IV), compound
1 displayed moderate to high
clearance (CL) at approximately 74% of mouse hepatic blood
flow, moderate volume of distribution (Vss) and short half-life
(t1/2; Table 4). Following a single 10 mg/kg oral (PO) admin-
istration, 1 showed low to moderate oral bioavailability (F%).
Based on single dose oral PK data, the regimen of 50 mg/kg
twice daily oral dosing in 0.5% w/v MC/0.5% w/v Tween 80
suspension was chosen for testing in a diabetes efficacy study
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trations of 1 in the efficacy study exceeded DYRK1A enzymatic
IC50 (6 nM) and was transiently above the GSK3β enzymatic IC50
(16 nM) for the dosing interval, as shown in Figure 4.
1
2
3
As reported by Shen et al., human islets treated with 1
in vitro demonstrated increases in DNA and ATP content, and
an increase in islet equivalent units compared with vehicle-
treated cultures.[36] In addition, human islets treated with 1
maintained insulin secretion capacity. Compound 1 stimulated
proliferation of functional primary human β-cells in vivo when a
suboptimal dose of human islets were transplanted under the
kidney capsule of immune-compromised streptozotocin (STZ)-
treated mice.[36] In a separate in vivo study using RIP-DTA mice,
1 demonstrated a significant increase in β-cell mass and
improved glycemic control.[36]
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Figure 3. Molecular docking model of 1 in the DYRK1A crystal structure (PDB
ID: 6UIP).
Table 3. Enzymatic and primary islet activity, ADME and safety profiling for
compounds 10 and 1.
Compound
10
1
Conclusions
Primary islet activity
rat islet EC50[a] [μM] (% efficacy[b]
)
3.5 (100)
0.76 (100)
0.15 (180)
0.54 (430)
In conclusion, the aminopyrazine scaffold was identified from a
phenotypic high-throughput screening campaign measuring β-
cell proliferation using mouse R7T1 β-cells.[7] Lead optimization
efforts starting from screening hit 9 led to the identification of a
potent dual DYRK1A and GSK3β inhibitor aminopyrazine 1
(GNF4877). SAR studies on the aminopyrazine scaffold were
driven by an enzymatic DYRK1A inhibition assay using a
structure-guided approach. The DYRK1A and GSK3β dual
inhibitor GNF4877 showed low-nM inhibition on both targets
and demonstrated unambiguous β-cell proliferation in vitro and
in vivo. As there is the possibility that long-term inhibition of
GSK3β might cause adverse effects,[18,40] GNF4877 was not
progressed beyond preclinical research. However, the work on
the aminopyrazine series laid a foundation for discovering
selective DYRK1A inhibitors to expand functional human β-cell
mass.[18,36]
human islet EC50[a] [μM] (% efficacy[b]
Cardiac ion channels
binding P4 [μM]18
QPatch S8 [μM]18
patch clamp Nav1.5 [μM][18]
ADME
)
>30
>30
n.d.[d]
n.d.[d]
n.d.[d]
>50
Microsomal stability (% remaining)[c]
human
43.0
69.0
61.0
39.0
33.3
20.64
rat
mouse
CYP450 inhibition [μM]
CYP2C9
CYP2D6
CYP3A4 I
CYP3A4 II
thermodynamic solubility [μM]
logPe PAMPA (pH 6.8)
mouse plasma protein binding
>25
>25
>25
>25
<2
8.6
20.3
>25
>25
14.1
À 4.7
99.5%
À 5.4
n.d.[d]
[a] Obtained from three or more independent experiments. [b] Prolifera-
tion % efficacy relative to the assay standard 10 set at 100%. [c] After
30min of incubation. Microsomes derived from pieces of endoplasmic
reticulum (ER) were used. [d] Not determined.
Table 4. Pharmacokinetic (PK) parameters for 1 in CD-1 mice.
(IV) [a]
(PO) [b]
dose [mgkgÀ 1
CL [mLminÀ 1 kgÀ 1
Vss [LkgÀ 1
AUC [hnM]
max [nM]
]
2
10
]
66.8
1.85
1013
2819
0.03
0.91
]
805
269
1.0
2.32
15.9
C
t
t
max [h]
1/2 [h]
F%
[a] Solution formulation (2.5 mg/mL in 50 mM Na2CO3 buffer, pH 9.3). [b]
Suspension formulation (1.0 mg/mL in 0.5% w/v MC/0.1% w/v Tween 80).
(rat insulin promoter and diphtheria toxin A (RIP-DTA) mice).
Compound 1-treated diabetic mice demonstrated a significant
increase in β-cell mass and improved glycemic control.[36] The
plasma exposure of 1 was determined on day 10. The exposure
was significantly higher in diabetic mice than predicted from
single dose PK study in normal CD-1 mice. The free concen-
Figure 4. Plasma exposure of 1 in RIP-DTA mice following 50 mg/kg twice
daily oral dosing in 0.5% w/v MC/0.5% w/v Tween 80 suspension
formulation at day 10. Mean�SD; n=3. Mouse plasma protein binding of 1
is 99.5%.
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aminopyridin-4-yl)piperidine-4-carboxylate (4a; 2.35 g, 10.0 mmol)
in DMF (50 mL) was added HATU (4.56 g, 12.0 mmol) and DIEA
Experimental Section
1
2
3
4
5
6
7
8
9
°
(5.23 mL, 30.0 mmol) to result in a mixture that was stirred at 25 C
Biology
for 2 h, poured into saturated Na2CO3 solution (400 mL) and
extracted with EtOAc (3×120 mL). The combined organic extracts
were washed with brine, dried over Na2SO4 and concentrated. The
residue was subjected to ISCO chromatography (0–5% MeOH/
CH2Cl2 eluent) to give 6a (3.13 g, 72%). 1H NMR ([D6]DMSO,
500 MHz): δ=10.05 (s, 1H, NH), 9.34 (s, 1H), 8.47 (s, 1H), 8.27 (d, J=
5.3 Hz, 1 H), 7.80 (br s, 2H, NH2), 7.17 (d, J=5.3 Hz, 1H), 3.64 (s, 3H),
3.13–3.18 (m, 2H), 2.76–2.81 (m, 2H), 2.53–2.61 (m, 1H), 1.92–2.03
(m, 4H). LRMS (ESI) m/z 435.3 [M+H]+.
All animal care and experimental procedures were approved by the
Institutional Animal Care and Use Committee (IACUC) of the
Genomics Institute of the Novartis Research Foundation (GNF) and
strictly followed the NIH guidelines for the humane treatment of
animals. R7T1 β-cells were obtained from Dr. S. Efrat (Tel Aviv
University) and expanded in growth medium.[36] Human islets were
obtained from Prodo Laboratories, Inc. (Irvine, CA, USA), University
of Minnesota and University of Miami, in accordance with internal
review board (IRB) ethical guidelines for the use of human tissue.[36]
Informed consent was obtained from the donors for studies using
human islets.
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Methyl 1-(3-(3-amino-6-phenylpyrazine-2-carboxamido)pyridin-4-yl)pi-
peridine-4-carboxylate (7a): A mixture of methyl 1-(3-(3-amino-6-
bromopyrazine-2-carboxamido)pyridin-4-yl)piperidine-4-carboxylate
(6a; 870.6 mg, 2.0 mmol), phenylboronic acid (292.6 mg, 2.4 mmol),
Pd(PPh3)4 (115.8 mg, 0.10 mmol) and Na2CO3 (635.9 mg, 6.0 mmol)
DYRK1A and GSK3β biochemical assays were used as previously
discribed.[18,36] A β-cell proliferation assay with growth-arrested
reversibly immortalized mouse β-cell line R7T1 was used as
previously discribed.[7,18,36] Rat and human primary β-cell prolifer-
ation assays were used as previously discribed.[7,18,36]
°
was heated at 90 C for 3 h, poured into water (200 mL), and
extracted with CH2Cl2 (3×40 mL). The combined extracts were
washed with brine, dried over Na2SO4, and concentrated to result in
a residue which was subjected to chromatography (0–5% MeOH/
1
CH2Cl2) to give 7a (66.2 mg, 77%). H NMR ([D6]DMSO, 500 MHz):
Chemistry
δ=10.38 (s, 1H, NH), 9.37 (s, 1H), 8.97 (s, 1H), 8.29 (d, J=5.3 Hz, 1H),
8.06–8.11 (m, 2H), 7.79 (br s, 2H, NH2), 7.38–7.47 (m, 3H), 7.21 (d, J=
5.3 Hz, 1H), 3.50 (s, 3H), 3.14–3.22 (m, 2H), 2.70–2.80 (m, 2H), 2.44–
2.49 (m, 1H), 1.92–2.00 (m, 2H), 1.70–1.83 (m, 2H); 13C NMR ([D6]
DMSO, 125 MHz): δ=173.87, 163.63, 154.39, 149.74, 145.94, 145.42,
141.23, 138.44, 135.38, 128.77, 128.29, 125.17, 122.89, 114.92, 51.40,
50.34, 39.31, 27.95. LRMS (ESI) m/z 433.2 [M+H]+.
All commercial reagents and solvents were used without purifica-
1
tion. H and 13C NMR spectra were recorded on a Bruker 400 or
500 MHz spectrometer using solvent residual peak as the internal
standard. TLC analyses were carried out on aluminum sheets pre-
coated with silica gel 60 F254, and UV radiation was used for
detection. Flash column chromatography was performed on Tele-
dyne ISCO CombiFlash systems. The purity of the final compounds
was determined to be >95% by 1H NMR, LCMS and/or CHN
elemental analysis. LRMS (ESI) data were recorded using an Agilent
6520 Accurate-Mass Q-TOF LC–MS system with HPLC-Chip Cube
interface and an Agilent 1200 HPLC. Elemental analysis was
performed at Midwest Microlab (Indianapolis, IN 46250).
1-(3-(3-Amino-6-phenylpyrazine-2-carboxamido)pyridin-4-yl)piperi-
dine-4-carboxylic acid (10): To a suspension of methyl 1-(3-(3-amino-
6-phenylpyrazine-2-carboxamido)pyridin-4-yl)piperidine-4-carboxy-
late (7a; 432.5 mg, 1.0 mmol) in a mixed solvent of THF and MeOH
(2.5:1, v/v, 18 mL) was added LiOH (1.0 M, 2.0 mL, 2.0 mmol) to
°
result in a mixture which was stirred at 25 C for 16 h, and then
concentrated under reduced pressure. The residue was dissolved
into water (100 mL), washed with CH2Cl2 (15 mL), and then acidified
with an aqueous HCl solution (1.0 M, 2.0 mL, 2.0 mmol). The solid
precipitate was collected by filtration, and dried under reduced
Syntheses
Methyl 1-(3-nitropyridin-4-yl)piperidine-4-carboxylate (3a): A suspen-
sion of 4-chloro-3-nitropyridine (1; 3.17 g, 20.0 mmol), methyl
piperidine-4-carboxylate (2a; 2.86 g, 20.0 mmol) and K2CO3 (5.53 g,
1
pressure to afford 10 (410 mg, 98%). H NMR ([D6]DMSO, 500 MHz):
δ=10.46 (s, 1H), 8.98 (s, 1H), 8.68 (s, 1H), 8.34 (d, J=6.9 Hz, 1H),
8.18–8.21 (m, 2H), 7.67 (br s, 2H, NH2), 7.46–7.51 (m, 2H), 7.38–7.43
(m, 2H), 3.94–4.02 (m, 2H), 3.19–3.27 (m, 2H), 2.51–2.59 (m, 1H),
1.86–1.94 (m, 2H), 1.63–1.74 (m, 2H); 13C NMR ([D6]DMSO, 125 MHz):
δ=175.23, 165.00, 156.34, 154.37, 145.40, 138.90, 138.69, 137.90,
135.60, 128.80, 128.42, 125.57, 123.10, 122.80, 113.24, 48.24, 39.26,
27.71. LRMS (ESI) m/z 419.2 [M+H]+. Anal. calcd. (%) for C22H22N6O3:
C 63.15, H 5.30, N 20.08; found: C 62.83, H 5.28, N 19.97; purity
�99.6%.
°
40.0 mmol) in MeOH (100 mL) was stirred at 25 C for 4 h, and then
concentrated under reduced pressure, mixed with water (200 mL),
and extracted with CH2Cl2 (3×40 mL). The combined organic
extracts were washed with brine, dried over Na2SO4, and concen-
trated under reduced pressure. The residue was subjected to
column chromatography (hexanes/EtOAc 4:1 to 1:99) to afford 3a
(4.40 g, 83%). 1H NMR ([D6]DMSO, 500 MHz): δ=8.75 (s, 1H), 8.34 (d,
J=6.1 Hz, 1 H), 7.18 (d, J=6.1 Hz, 1 H), 3.63 (s, 3H), 3.36–3.40 (m,
2H), 3.05–3.10 (m, 2H), 2.62–2.70 (m, 1H), 1.88–1.95 (m, 2H), 1.62–
1.70 (m, 2H). LRMS (ESI) m/z 266.1 [M+H]+.
(R)-Ethyl 1-(3-nitropyridin-4-yl)piperidine-3-carboxylate (3b): To
a
suspension of 4-chloro-3-nitropyridine (12.68 g, 80.0 mmol) in
ethanol (100 mL) were added K2CO3 (11.06 g, 80.0 mmol.) and a
solution of (R)-ethyl piperidine-3-carboxylate (2b; 12.58 g,
Methyl 1-(3-aminopyridin-4-yl)piperidine-4-carboxylate (4a): To
a
solution of methyl 1-(3-nitropyridin-4-yl)piperidine-4-carboxylate
(3a; 4.24 g, 16.0 mmol) in MeOH (100 mL) was added Pd/C (10 wt.
% loading, 424 mg). The resultant mixture was stirred under H2
(balloon) for 16 h, and filtered through Celite. The filtrate was
°
20.0 mmol) in ethanol (20 mL) at 0 C. The resultant mixture was
°
stirred at 0 C for 4.5 h, poured into water (500 mL), and extracted
with CH2Cl2 (3×100 mL). The combined organic extracts were
washed with brine, dried over Na2SO4, and concentrated under
reduced pressure. The residue was subjected to column chromatog-
raphy (20–100% EtOAc/hexanes) to afford 3b as yellow oil (20.98 g,
1
evaporated to give 4a (3.43 g, 91%). H NMR ([D6]DMSO, 500 MHz):
δ=7.90 (s, 1H), 7.72 (d, J=5.2 Hz, 1 H), 6.75 (d, J=5.2 Hz, 1 H), 4.73
(br s, 2H, NH), 3.63 (s, 3H), 3.20–3.23 (m, 2H), 2.54–2.60 (m, 2H),
2.42–2.52 (m, 1H), 1.89–1.94 (m, 2H), 1.75–1.83 (m, 2H). LRMS (ESI)
m/z 236.3 [M+H]+.
93%). ee=98.5% (CHIRALPAK® ADÀ H column, CO2/MeOH 5% to
1
°
50% in 8 min, flow rate 2 mL/min, temperature 30 C). H NMR ([D6]
DMSO, 500 MHz): δ=8.74 (s, 1H), 8.34 (d, J=6.1 Hz, 1H), 7.19 (d, J=
6.1 Hz, 1H), 4.02 (q, J=7.2 Hz, 2H), 3.44–3.47 (m, 1H), 3.24–3.30 (m,
2H), 3.10–3.15 (m, 1H), 2.67–2.72 (m, 1H), 1.89–1.99 (m, 1H), 1.68–
1.76 (m, 2H), 1.53–1.60 (m, 1H), 1.24 (t, J=7.2 Hz, 3H); 13C NMR ([D6]
Methyl 1-(3-(3-amino-6-bromopyrazine-2-carboxamido)pyridin-4-yl)pi-
peridine-4-carboxylate (6a): To a suspension of 3-amino-6-bromo-
pyrazine-2-carboxylic acid (5; 2.18 g, 10.0 mmol) and methyl 1-(3-
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DMSO, 125 MHz): δ=172.31, 152.33, 148.97, 147.51, 135.73, 113.54,
60.11, 50.80, 49.49, 40.09, 25.78, 23.08, 13.87. LRMS (ESI) m/z 280.1
[M+H]+.
(R)-1-(3-(3-Amino-6-(2-fluoro-5-isopropoxyphenyl)pyrazine-2-
1
2
3
4
5
6
7
8
9
carboxamido)pyridin-4-yl)piperidine-3-carboxylic acid (1): To a solu-
tion of ethyl (R)-1-(3-(3-amino-6-(2-fluoro-5-isopropoxyphenyl)
pyrazine-2-carboxamido)pyridin-4-yl)piperidine-3-carboxylate (7b;
156.7 mg, 0.3 mmol) in THF/MeOH (5:3, v/v, 8 mL) was added an
aqueous solution of LiOH (1.0 M, 0.6 mL, 0.6 mmol). The resulting
(R)-Ethyl 1-(3-aminopyridin-4-yl)piperidine-3-carboxylate (4b): To a
solution of (R)-ethyl 1-(3-nitropyridin-4-yl) piperidine-3-carboxylate
(3b; 12.11 g, 43.4 mmol) in EtOAc (100 mL) was added Pd/C (10 wt.
% loading, 1.21 g). The resultant mixture was shaken under H2 (50
psi) in a Parr hydrogenation flask for 3 h, and filtered through
Celite. The filtrate was evaporated to give 4b as brown oil (10.49 g,
97%). 1H NMR ([D6]DMSO, 500 MHz): δ=7.90 (s, 1H), 7.72 (d, J=
5.2 Hz, 1 H), 6.77 (d, J=5.2 Hz, 1 H), 4.92 (br s, 2H, NH2), 4.07 (q, J=
7.1 Hz, 2H), 2.97–3.10 (m, 2H), 2.86–2.94 (m, 1H), 2.74–2.78 (m, 1H),
2.64–2.72 (m, 1H), 1.81–1.89 (m, 1H), 1.61–1.77 (m, 3H), 1.19 (t, J=
7.1 Hz, 3H); 13C NMR ([D6]DMSO, 100 MHz): δ=173.33, 143.96,
138.74, 137.99, 136.50, 113.48, 60.07, 50.69, 49.58, 40.58, 25.46,
23.87, 14.03. LRMS (ESI) m/z 250.1 [M+H]+.
°
mixture was stirred at 25 C for 4 h and concentrated under reduced
pressure. The residue was dissolved in water (60 mL), washed with
CH2Cl2 (10 mL) and then acidified with an aqueous HCl solution
(1.0 M, 0.6 mL, 0.6 mmol). The solid precipitate was collected by
filtration and dried under reduced pressure to afford 1 (150.4 mg,
96%). ee=97.2% (CHIRALPAK® ADÀ H column, CO2/MeOH+0.5%
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
diethylamine, 5% to 50% in 8 min, flow rate 2 mL/min, temperature
1
°
30 C). H NMR ([D6]DMSO, 400 MHz): 10.30 (s, 1H, NH), 9.32 (s, 1H),
8.68 (d, J=2.1 Hz, 1H), 8.27 (d, J=5.3 Hz, 1H), 7.84 (br s, 2H, NH2),
7.41 (dd, J=6.5, 3.1 Hz, 1H), 7.25 (dd, J=10.6, 9.1 Hz, 1H), 7.18 (d,
J=5.3 Hz, 1H), 7.00–7.15 (m, 1H), 4.64 (septet, J=7.1 Hz, 1H), 3.26–
3.33 (m, 1H), 3.11–3.17 (m, 1H), 2.67–2.73 (m, 1H), 2.53–2.60 (m, 2H),
1.73–1.81 (m, 1H), 1.57–1.66 (m, 2H), 1.26–1.37 (m, 1H), 1.26 (d, J=
7.1 Hz, 6H); 13C NMR ([D6]DMSO, 100 MHz): δ=174.42, 163.38,
3-Amino-6-(2-fluoro-5-isopropoxyphenyl)pyrazine-2-carboxylic
acid
(8b): To a suspension of 3-amino-6-bromopyrazine-2-carboxylic
acid (5; 872.0 mg, 4.0 mmol), (2-fluoro-5-isopropoxyphenyl)boronic
acid (950.4 mg, 4.8 mmol), Pd(PPh3)4 (231.1 mg, 0.2 mmol) and
Na2CO3 (1.27 g, 12.0 mmol) in dioxane (16 mL) was added water
154.29, 153.97, 153.65 (d, JCF =238 Hz), 149.69, 147.87 (d, JCF
10.4 Hz), 145.97, 134.84 (d, JCF =3.2 Hz), 128.00, 124.52 (d, JCF
14.0 Hz), 123.71, 117.19, 116.82 (d, JCF =24.2 Hz), 115.72 (d, JCF
7.9 Hz), 114.89, 69.85, 52.97, 51.10, 41.48, 26.30, 24.61, 21.60. LRMS
(ESI) m/z 495.2 [M+H]+. Anal. calcd. (%) for C25H27FN6O4: C 60.72, H
5.50, N 16.99; found: C 60.60, H 5.46, N 16.88; purity �99.8%.
=
=
=
°
(4 mL) to result in a mixture which was heated at 90 C for 3 h,
concentrated. The residue was loaded on silica gel and subjected to
chromatography (CH2Cl2/MeOH/28%NH4OH, 40:20:1, v/v/v) to
afford the amonium salt of the product which was then dissolved
in water (160 mL), washed by EtOAc (2×20 mL) and adjusted to
pH 2 by adding concentrated HCl solution. The solid precipitated
3-Amino-N-(4-(4-aminopiperidin-1-yl)pyridin-3-yl)-6-phenylpyrazine-2-
1
carboxamide (9): H NMR (CD3OD, 400 MHz): δ=9.41 (s, 1H), 8.77 (s,
1
was collected by filtration to afford 8b (745.2 mg, 64%). H NMR
1H), 8.23 (d, J=5.4 Hz, 1H), 8.01–8.04 (dd, J=8.0, 1.3 Hz, 2H), 7.52–
7.58 (dd, J=8.0, 7.3 Hz, 2H), 7.42–7.49 (m, 1H), 7.21 (d, J=5.4 Hz,
1H), 3.20–3.27 (m, 2H), 2.78–2.81 (m, 2H), 2.60–2.68 (m, 1H), 1.82–
1.92 (m, 2H), 1.44–1.55 (m, 2H). LRMS (ESI) m/z 390.2 [M+H]+.
(CDCl3, 500 MHz): δ=8.80 (s, 1H), 7.26 (dd, J=6.3, 3.1 Hz, 1H), 7.10
(dd, J=10.5, 9.0 Hz, 1H), 6.92 (ddd, J=9.0, 4.0, 3.1 Hz, 1H), 4.54
(septet, J=6.0 Hz, 1H), 1.36 (d, J=6.0 Hz, 6H); 13C NMR ([D6]DMSO,
125 MHz): δ=167.60, 154.76 (JCF =3.1 Hz), 153.90, 153.78 (d, JCF
238 Hz), 147.45 (d, JCF =11.5 Hz), 135.44 (d, JCF =3.6 Hz), 124.53 (d,
CF =13.9 Hz), 123.15, 116.87 (d, JCF =24.7 Hz), 116.80 (d, JCF
=
1-(4-(3-Amino-6-phenylpyrazine-2-carboxamido)pyridin-3-yl)piperi-
dine-4-carboxylic acid (11): 1H NMR ([D6]DMSO, 400 MHz): δ=1.11 (s,
1H, NH), 9.05 (s, 1H), 8.65–8.71 (m, 2H), 8.52–8.60 (m, 1H), 8.10 (d,
J=7.2 Hz, 2H), 7.86 (br s, 2H, NH2), 7.48–7.52 (m, 2H), 7.39 (m, 1H),
3.09–3.16 (m, 2H), 2.86–2.89 (m, 2H), 2.38–2.46 (m, 1H), 1.97–2.05
(m, 2H), 1.77–1.86 (m, 2H). LRMS (ESI) m/z 419.2 [M+H]+.
J
=
6.7 Hz), 116.63 (d, JCF =2.9 Hz), 70.00, 21.78. LRMS (ESI) m/z 292.1
[M+H]+.
Ethyl
(R)-1-(3-(3-amino-6-(2-fluoro-5-isopropoxyphenyl)pyrazine-2-
carboxamido)pyridin-4-yl)piperidine-3-carboxylate (7b): To a mixture
of (R)-ethyl 1-(3-aminopyridin-4-yl) piperidine-3-carboxylate (4b;
565.9 mg, 2.27 mmol) and 3-amino-6-(2-fluoro-5-isopropoxyphenyl)
pyrazine-2-carboxylic acid (8b; 701.7 mg, 2.73 mmol) in DMF
(15 mL) was added EDCI (523.3 mg, 2.73 mmol) and HOAt
(371.5 mg, 2.73 mmol). After the resulting mixture was stirred at
1-(2-(3-Amino-6-phenylpyrazine-2-carboxamido)pyridin-3-yl)piperi-
dine-4-carboxylic acid (12): 1H NMR (CD3OD, 400 MHz): δ=8.90 (s,
1H), 8.16–8.23 (m, 2H), 8.02 (d, J=7.3 Hz, 2H), 7.49–7.53 (m, 3H),
7.37–7.44 (m, 1H), 3.17–3.25 (m, 2H), 2.86–2.95 (m, 2H), 2.46–2.52
(m, 1H), 2.08–2.18 (m, 2H), 1.91–2.03 (m, 2H). LRMS (ESI) m/z 419.2
[M+H]+.
°
25 C for 18 h, DMF was removed under reduced pressure, and the
residue was purified by reversed-phase HPLC [10–70% MeCN
(0.035% TFA)/water (0.05% TFA)]. After MeCN was removed from
the desired fractions under reduced pressure, the residue was
extracted with 5% MeOH in CH2Cl2 (3×30 mL). The combined
organic extracts were washed with a saturated aqueous NaHCO3
solution, dried over MgSO4 and evaporated to afford 7b (853.5 mg,
1-(3-(3-Amino-6-phenylpyrazine-2-carboxamido)pyridin-2-yl)piperi-
1
dine-4-carboxylic acid (13): H NMR ([D6]DMSO, 400 MHz): δ=10.53
(s, 1H, NH), 8.99 (s, 1H), 8.67 (dd, J=8.0, 1.6 Hz, 1H), 8.11–8.13 (m,
3H), 7.79 (br s, 2H, NH2), 7.47 (dd, J=7.9, 7.4 Hz, 2H), 7.38 (dd, J=
7.4, 6.4 Hz, 1H), 7.21 (dd, J=8.0, 4.8 Hz, 1H), 3.84–3.87 (m, 2H), 3.26–
3.32 (m, 2H), 2.36–2.43 (m, 1H), 2.10–2.28 (m, 2H), 1.96–2.03 (m, 2H).
LRMS (ESI) m/z 419.2 [M+H]+.
1
72%). H NMR ([D6]DMSO, 500 MHz): δ=10.27 (s, 1H, NH), 9.28 (s,
1H), 8.68 (d, J=2.5 Hz, 1H), 8.27 (d, J=5.3 Hz, 1H), 7.83 (br s, 2H,
NH2), 7.41 (dd, J=6.5, 3.2 Hz, 1H), 7.25 (dd, J=10.7, 9.0 Hz, 1H), 7.16
(d, J=5.3 Hz, 1H), 7.03 (m, 1H), 4.64 (septet, J=6.0 Hz, 1H), 3.88 (d,
J=7.1 Hz, 2H), 3.30–3.38 (m, 1H), 3.12–3.17 (m, 1H), 2.61–2.70 (m,
3H), 1.71–1.78 (m, 1H), 1.57–1.71 (m, 2H), 1.31–1.39 (m, 1H), 1.25 (d,
J=6.0 Hz, 6H), 1.00 (t, J=7.1 Hz, 3H); 13C NMR ([D6]DMSO, 125 MHz):
1-(2-(3-Amino-6-phenylpyrazine-2-carboxamido)phenyl)piperidine-4-
1
carboxylic acid (14): H NMR ([D6]DMSO, 400 MHz): δ=10.82 (s, 1H,
NH), 8.96 (s, 1H), 8.45 (dd, J=8.0, 1.6 Hz, 1H), 8.11 (dd, J=8.0,
1.3 Hz, 2H), 7.79 (br s, 2H, NH2), 7.49 (dd, J=7.8, 7.4 Hz, 2H), 7.37
(dd, J=7.4, 7.0 Hz, 1H), 7.33 (dd, J=7.8, 1.6 Hz, 1H), 7.11–7.21 (m,
2H), 2.95–3.04 (m, 2H), 2.70–2.80 (m, 2H), 2.33–2.42 (m, 1H), 1.95–
2.04 (m, 2H), 1.78–1.90 (m, 2H). LRMS (ESI) m/z 418.2 [M+H]+.
δ=172.28, 163.33, 154.26, 153.90 (d, JCF =1.1 Hz), 153.62 (d, JCF
238.3 Hz), 149.44, 147.81 (d, JCF =10.2 Hz), 146.00, 141.70, 134.79 (d,
CF =3.3 Hz), 127.77, 124.53 (d, JCF =14.2 Hz), 123.68, 117.20 (d, JCF
=
J
=
1-(3-(3-Amino-6-phenylpyrazine-2-carboxamido)pyridin-4-yl)azetidine-
2.4 Hz), 116.70 (d, JCF =24.2 Hz), 115.67 (d, JCF =8.0 Hz), 114.76,
69.75, 59,75, 52.70, 50.61, 41.32, 26.00, 24.38, 21.58, 13.68. LRMS
(ESI) m/z 523.3 [M+H]+.
3-carboxylic acid (15): LRMS (ESI) m/z 391.2 [M+H]+.
1-(3-(3-Amino-6-(pyridin-4-yl)pyrazine-2-carboxamido)pyridin-4-yl)pyr-
rolidine-3-carboxylic acid (16): LRMS (ESI) m/z 405.2 [M+H]+.
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1-(3-(3-Amino-6-phenylpyrazine-2-carboxamido)pyridin-4-yl)-4-fluoro-
piperidine-4-carboxylic acid (17): H NMR ([D6]DMSO, 400 MHz): δ=
10.45 (s, 1H, NH), 8.98 (s, 1H), 8.80 (br s, 1H), 8.36 (d, J=6.3 Hz, 1H),
8.18 (d, J=7.3 Hz, 2H), 7.71 (br s, 2H, NH2), 7.47 (dd, J=7.7, 7.3 Hz,
2H), 7.37–7.44 (m, 2H), 3.84–3.87 (m, 2H), 3.26–3.32 (m, 2H), 2.10–
2.28 (m, 2H), 1.96–2.03 (m, 2H). LRMS (ESI) m/z 437.2 [M+H]+.
1-(3-(3-Amino-6-(2-(trifluoromethyl)phenyl)pyrazine-2-carboxamido)
pyridin-4-yl)piperidine-4-carboxylic acid (26): 1H NMR (CD3OD,
400 MHz): δ=9.28 (s, 1H), 8.42 (s, 1H), 8.28 (d, J=6.0 Hz, 1H), 7.82
(d, J=7.6 Hz, 1H), 7.71 (dd, J=7.4, 7.3 Hz, 1H), 7.62 (dd, J=7.7,
7.6 Hz, 1H), 7.56 (J=7.6 Hz, 1H), 7.42 (d, J=6.8 Hz, 1H), 3.65–3.69
(m, 2H), 3.01–3.08 (m, 2H), 2.36–2.42 (m, 1H), 1.84–1.90 (m, 2H),
1.57–1.67 (m, 2H). LRMS (ESI) m/z 487.2 [M+H]+.
1
2
3
4
5
6
7
8
9
1
(S)-1-(3-(3-Amino-6-phenylpyrazine-2-carboxamido)pyridin-4-yl)piperi-
1
dine-3-carboxylic acid (18): H NMR ([D6]DMSO, 400 MHz): δ=10.49
1-(3-(3-Amino-6-(4-fluorophenyl)pyrazine-2-carboxamido)pyridin-4-yl)
1
(s, 1H, NH), 9.38 (s, 1H), 8.98 (s, 1H), 8.29 (d, J=5.3 Hz, 1H), 8.12 (dd,
J=8.5, 1.3 Hz, 2H), 7.78 (br s, 2H, NH2), 7.48–7.53 (m, 2H), 7.41–7.48
(m, 1H), 7.24 (J=5.3 Hz, 1H), 3.27–3.36 (m, 1H), 3.04–3.12 (m, 1H),
2.77–2.83 (m, 1H), 2.65–2.74 (m, 1H), 2.52–2.61 (m, 1H), 1.78–1.81
(m, 1H), 1.60–1.71 (m, 2H), 1.37–1.43 (m, H). LRMS (ESI) m/z 419.2
[M+H]+.
piperidine-4-carboxylic acid (27): H NMR (CD3OD, 400 MHz): δ=9.12
(s, 1H), 8.76 (s, 1H), 8.28 (dd, J=6.8, 1.9 Hz, 1H), 7.96 (dd, J=8.9,
8.6 Hz, 2H), 7.45 (d, J=6.8 Hz, 1H), 7.19 (dd, J=8.7, 8.6 Hz, 2H),
3.79–3.83 (m, 2H), 3.16–3.24 (m, 2H), 2.54–2.60 (m, 1H), 2.04–2.12
(m, 2H), 1.86–1.97 (m, 2H). LRMS (ESI) m/z 437.2 [M+H]+.
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19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
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1-(3-(3-Amino-6-(p-tolyl)pyrazine-2-carboxamido)pyridin-4-yl)piperi-
1
(R)-1-(3-(3-Amino-6-phenylpyrazine-2-carboxamido)pyridin-4-yl)piperi-
dine-3-carboxylic acid (19): 1H NMR ([D6]DMSO, 500 MHz) of the
sodium salt: δ 10.56 (s, 1H, NH), 9.40 (s, 1H), 8.99 (s, 1H), 8.27 (d, J=
5.3 Hz, 1H), 8.16 (dd, J=8.5, 1.3 Hz, 2H), 7.76 (br s, 2H, NH2), 7.51
(dd, J=7.9, 7.4 Hz, 2H), 7.39–7.45 (m, 1H), 7.19 (J=5.3 Hz, 1H), 3.32–
3.30 (m, 2H), 3.02–3.10 (m, 1H), 2.60–2.66 (m, 1H), 2.27–2.35 (m, 1H),
1.84–1.92 (m, 1H), 1.57–1.70 (m, 2H), 1.23–1.29 (m, 1H). LRMS (ESI)
m/z 419.2 [M+H]+.
dine-4-carboxylic acid (28): H NMR (CD3OD, 400 MHz): δ=9.08 (d,
J=0.9 Hz, 1H), 8.78 (s, 1H), 8.31 (dd, J=6.8, 1.1 Hz, 1H), 7.89 (d, J=
8.1 Hz, 2H), 7.45 (d, J=6.9 Hz, 1H), 7.29 (d, J=8.1 Hz, 2H), 3.82–3.88
(m, 2H), 3.16–3.22 (m, 2H), 2.54–2.60 (m, 1H), 2.39 (s, 3H), 2.02–2.09
(m, 2H), 1.84–1.95 (m, 2H). LRMS (ESI) m/z 433.2 [M+H]+.
1-(3-(3-Amino-6-(4-(trifluoromethyl)phenyl)pyrazine-2-carboxamido)
pyridin-4-yl)piperidine-4-carboxylic acid (29): 1H NMR (CD3OD,
400 MHz): δ=8.91 (s, 1H), 8.89 (d, J=1.0 Hz, 1H), 8.23–8.30 (m, 3H),
7.79 (d, J=8.2 Hz, 2H), 7.45 (d, J=6.9 Hz, 1H), 3.93–4.02 (m, 2H),
3.21–3.30 (m, 2H), 2.59–2.66 (m, 1H), 2.01–2.11 (m, 2H), 1.85–1.96
(m, 2H). LRMS (ESI) m/z 487.2 [M+H]+.
(R)-4-(3-(3-Amino-6-phenylpyrazine-2-carboxamido)pyridin-4-yl)piper-
1
azine-2-carboxylic acid (20): H NMR ([D6]DMSO, 400 MHz): δ=10.37
(s, 1H, NH), 9.26 (s, 1H), 8.99 (s, 1H), 8.30 (d, J=5.3 Hz, 1H), 8.14 (dd,
J=7.3, 1.1 Hz, 2H), 7.77 (br s, 2H, NH2), 7.52 (dd, J=7.8, 7.3 Hz, 2H),
7.40–7.47 (m, 1H), 7.23 (J=5.3 Hz, 1H), 3.14–3.51 (m, 4H), 2.86–2.97
(m, 2H), 2.77–2.82 (m, 1H). LRMS (ESI) m/z 420.2 [M+H]+.
1-(3-(3-Amino-6-(3-methoxyphenyl)pyrazine-2-carboxamido)pyridin-4-
1
yl)piperidine-4-carboxylic acid (30): H NMR (CD3OD, 400 MHz): δ=
9.08 (d, J=0.9 Hz, 1H), 8.81 (s, 1H), 8.30 (dd, J=6.9, 1.0 Hz, 1H),
7.53–7.60 (m, 2H), 7.48 (d, J=6.9 Hz, 1H), 7.39 (dd, J=8.1, 7.8 Hz,
1H), 6.97 (dd, J=7.8, 2.3 Hz, 1H), 3.81–3.85 (m, 2H), 3.18–3.24 (m,
2H), 2.56 (m, 1H), 2.01–2.10 (m, 2H), 1.85–1.96 (m, 2H). LRMS (ESI)
m/z 449.2 [M+H]+.
(R)-2-(1-(3-(3-Amino-6-phenylpyrazine-2-carboxamido)pyridin-4-yl)pi-
1
peridin-3-yl)acetic acid (21): H NMR ([D6]DMSO, 400 MHz): δ=10.50
(s, 1H, NH), 8.99 (s, 1H), 8.72 (s, 1H), 8.36 (d, J=6.9 Hz, 1H), 8.21 (dd,
J=7.3, 1.3 Hz, 2H), 7.69 (br s, 2H, NH2), 7.50 (dd, J=7.8, 7.3 Hz, 2H),
7.37–7.43 (m, 2H), 3.87–3.96 (m, 2H), 3.02–3.10 (m, 1H), 2.90–2.99
(m, 1H), 2.01–2.19 (m, 3H), 1.70–1.77 (m, 1H), 1.62–1.70 (m, 1H),
1.50–1.59 (m, 1H), 1.19–1.26 (m, 1H). LRMS (ESI) m/z 433.2 [M+H]+.
1-(3-(3-Amino-6-(m-tolyl)pyrazine-2-carboxamido)pyridin-4-yl)piperi-
1
dine-4-carboxylic acid (31): H NMR (CD3OD, 400 MHz): δ=9.03 (d,
J=1.0 Hz, 1H), 8.80 (s, 1H), 8.30 (dd, J=6.9, 1.2 Hz, 1H), 7.84 (s, 1H),
7.80 (d, J=7.9 Hz, 1H), 7.47 (d, J=6.9 Hz, 1H), 7.39 (dd, J=7.7,
7.6 Hz, 1H), 7.18–7.24 (m, 1H), 3.85–3.93 (m, 2H), 3.18–3.28 (m, 2H),
2.54–2.60 (m, 1H), 2.43 (s, 3H), 2.01–2.09 (m, 2H), 1.84–1.94 (m, 2H).
LRMS (ESI) m/z 433.2 [M+H]+.
(S)-2-(1-(3-(3-Amino-6-phenylpyrazine-2-carboxamido)pyridin-4-yl)pi-
1
peridin-3-yl)acetic acid (22): H NMR ([D6]DMSO, 400 MHz): δ=10.50
(s, 1H, NH), 8.99 (s, 1H), 8.72 (s, 1H), 8.36 (d, J=6.9 Hz, 1H), 8.21 (dd,
J=7.3, 1.3 Hz, 2H), 7.69 (br s, 2H, NH2), 7.50 (dd, J=7.8, 7.3 Hz, 2H),
7.38–7.43 (m, 2H), 3.87–3.96 (m, 2H), 3.01–3.10 (m, 1H), 2.90–2.98
(m, 1H), 1.99–2.18 (m, 3H), 1.70–1.77 (m, 1H), 1.59–1.67 (m, 1H),
1.50–1.57 (m, 1H), 1.18–1.26 (m, 1H). LRMS (ESI) m/z 433.2 [M+H]+.
1-(3-(3-Amino-6-(2-fluoro-5-(trifluoromethoxy)phenyl)pyrazine-2-
carboxamido)pyridin-4-yl)piperidine-4-carboxylic acid (32): 1H NMR
(CD3OD, 400 MHz): δ=8.95 (d, J=1.0 Hz, 1H), 8.77 (d, J=2.0 Hz,
1H), 8.28 (d, J=6.9 Hz, 1H), 7.98 (d, J=6.3 Hz, 1H), 7.45 (d, J=
6.9 Hz, 1H), 7.37 (d, J=6.9 Hz 2H), 3.89–3.98 (m, 2H), 3.21–3.28 (m,
2H), 2.54–2.60 (m, 1H), 1.98–2.07 (m, 2H), 1.75–1.85 (m, 2H). LRMS
(ESI) m/z 503.2 [M+H]+.
1-(3-(3-Amino-6-(2-fluorophenyl)pyrazine-2-carboxamido)pyridin-4-yl)
1
piperidine-4-carboxylic acid (23): H NMR (CD3OD, 400 MHz): δ=9.17
(s, 1H), 8.71 (d, J=2.3 Hz, 1H), 8.28 (d, J=6.7 Hz, 1H), 7.91 (ddd, J=
7.8, 7.8, 1.8 Hz, 1H), 7.45 (d, J=6.8 Hz, 1H), 7.39–7.45 (m, 1H), 7.31
(ddd, J=7.6, 7.5, 1.1 Hz, 1H), 7.21 (ddd, J=11.4, 8.3, 0.9 Hz, 1H),
3.75–3.81 (m, 2H), 3.13–3.21 (m, 2H), 2.49–2.55 (m, 1H), 2.01–2.09
(m, 2H), 1.81–1.91 (m, 2H). LRMS (ESI) m/z 437.2 [M+H]+.
(R)-1-(3-(3-Amino-6-(2-fluoro-5-(trifluoromethoxy)phenyl)pyrazine-2-
1
carboxamido)pyridin-4-yl)piperidine-3-carboxylic acid (33): H NMR
([D6]DMSO, 400 MHz): δ=10.35 (s, 1H, NH), 9.18 (s, 1H), 8.73 (d, J=
2.4 Hz, 1H), 8.23 (d, J=5.4 Hz, 1H), 8.00 (dd, J=7.0, 2.4 Hz, 1H), 7.93
(br s, 2H, NH2), 7.48–7.54 (m, 2H), 7.09 (d, J=5.4 Hz, 1H), 3.21–3.28
(m, 1H), 3.11–3.17 (m, 1H), 2.52–2.59 (m, 2H), 2.00–2.07 (m, 1H),
1.71–1.78 (m, 1H), 1.43–1.62 (m, 2H), 1.19–1.25 (m, 1H). LRMS (ESI)
m/z 521.2 [M+H]+.
1-(3-(3-Amino-6-(o-tolyl)pyrazine-2-carboxamido)pyridin-4-yl)piperi-
dine-4-carboxylic acid (24): 1H NMR (CD3OD, 400 MHz): δ=9.23 (s,
1H), 8.43 (s, 1H), 8.28 (d, J=6.2 Hz, 1H), 7.44 (d, J=6.5 Hz, 1H), 7.39
(d, J=6.8 Hz, 1H), 7.26–7.35 (m, 3H), 3.66–3.71 (m, 2H), 3.09–3.16
(m, 2H), 2.43 (s, 1H), 2.39 (s, 3H), 1.90–1.98 (m, 2H), 1.68–1.78 (m,
2H). LRMS (ESI) m/z 433.2 [M+H]+.
1-(3-(3-Amino-6-(2-methoxyphenyl)pyrazine-2-carboxamido)pyridin-4-
1
yl)piperidine-4-carboxylic acid (25): H NMR (CD3OD, 400 MHz): δ=
Acknowledgements
9.14 (s, 1H), 8.79 (s, 1H), 8.29 (d, J=7.6 Hz, 1H), 7.76 (dd, J=7.6,
1.7 Hz, 1H), 7.47 (d, J=6.9 Hz, 1H), 7.40 (ddd, J=7.4, 6.6, 1.8 Hz, 1H),
7.12 (d, J=8.3 Hz, 1H), 7.08 (ddd, J=7.5, 7.5, 0.9 Hz, 1H), 3.90 (s,
3H), 3.79–3.84 (m, 2H), 3.15–3.22 (m, 2H), 2.49–2.55 (m, 1H), 1.98–
2.06 (m, 2H), 1.78–1.88 (m, 2H). LRMS (ESI) m/z 449.2 [M+H]+.
Funding for this work was provided by JDRF and GNF. We thank
the following individuals from GNF: Dr. David Jones for NMR
analysis; Lucas Westling, Michael Kwok, and Thomas Hollenbeck
ChemMedChem 2020, 15, 1–10
www.chemmedchem.org
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Keywords: aminopyrazines · diabetes · inhibitors · kinases ·
pancreatic beta-cell proliferation · synthases
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[39] In a LANCE TR-FRET assay at 1000 μM ATP (see ref. [18]) 1 had
comparable activity for both DYRK1A (IC50 =6 nM) and DYRK1B (IC50
=
16 nM). In a Caliper microfluidic-based assay 1 showed good selectivity
for DYRK1A (IC50 =0.99 nM) over DYRK2 (IC50 =500 nM).
[40] P. Cohen, M. Goedert, Drug Discovery Dev. 2004, 3, 479–487.
Manuscript received: March 18, 2020
Revised manuscript received: June 5, 2020
Version of record online: ■■■, ■■■■
ChemMedChem 2020, 15, 1–10
www.chemmedchem.org
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FULL PAPERS
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Getting more cells on site:
Dr. Y. A. Liu*, Dr. Q. Jin, Q. Ding, Dr. X.
Hao, T. Mo, S. Yan, Dr. Y. Zou, Dr. Z.
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Wu, C. Li, Dr. B. Bursalaya, Dr. M.
Di Donato, Dr. Y.-Q. Zhang, L. Deaton,
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Kamireddy, Dr. G. Harb, Dr. J. Li, Dr. Y.
Jia, Dr. A. M. Schumacher, Dr. B.
Laffitte, Dr. R. Glynne, Dr. S. Pan, Dr. P.
McNamara, Dr. V. Molteni, Dr. J.
Loren*
Promoting β-cell proliferation with
inhibitors of dual-specificity tyrosine-
regulated kinase 1A (DYRK1A) offers
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Lead optimization of an aminopyra-
zine hit led to the identification of
GNF4877, a potent dual inhibitor of
DYRK1A and glycogen synthase
kinase 3β (GSK3β).
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A Dual Inhibitor of DYRK1A and
GSK3β for β-Cell Proliferation: Ami-
nopyrazine Derivative GNF4877