Structure-Guided Discovery of Potent and Selective DYRK1A Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.1c00023

The kinase DYRK1A is an attractive target for drug discovery programs due to its implication in multiple diseases. Through a fragment screen, we identified a simple biaryl compound that is bound to the DYRK1A ATP site with very high efficiency, although with limited selectivity. Structure-guided optimization cycles enabled us to convert this fragment hit into potent and selective DYRK1A inhibitors. Exploiting the structural differences in DYRK1A and its close homologue DYRK2, we were able to fine-tune the selectivity of our inhibitors. Our best compounds potently inhibited DYRK1A in the cell culture and in vivo and demonstrated drug-like properties. The inhibition of DYRK1A in vivo translated into dose-dependent tumor growth inhibition in a model of ovarian carcinoma.

Full text of DOI:10.1021/acs.jmedchem.1c00023

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Article
Structure-Guided Discovery of Potent and Selective DYRK1A
Inhibitors
Csaba Weber, Melinda Sipos, Attila Paczal, Balazs Balint, Vilibald Kun, Nicolas Foloppe, Pawel Dokurno,
Andrew J. Massey, David Lee Walmsley, Roderick E. Hubbard, James Murray, Karen Benwell,
Thomas Edmonds, Didier Demarles, Alain Bruno, Mike Burbridge, Francisco Cruzalegui,
and Andras Kotschy*
Cite This: J. Med. Chem. 2021, 64, 6745−6764
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Supporting Information
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ABSTRACT: The kinase DYRK1A is an attractive target for drug discovery programs due to its implication in multiple diseases.
Through a fragment screen, we identified a simple biaryl compound that is bound to the DYRK1A ATP site with very high efficiency,
although with limited selectivity. Structure-guided optimization cycles enabled us to convert this fragment hit into potent and
selective DYRK1A inhibitors. Exploiting the structural differences in DYRK1A and its close homologue DYRK2, we were able to
fine-tune the selectivity of our inhibitors. Our best compounds potently inhibited DYRK1A in the cell culture and in vivo and
demonstrated drug-like properties. The inhibition of DYRK1A in vivo translated into dose-dependent tumor growth inhibition in a
model of ovarian carcinoma.
eliminated,11 the identification of novel selective DYRK1A
INTRODUCTION
■
inhibitor chemotypes with drug-like properties remains highly
DYRK1A (dual-specificity tyrosine-phosphorylation-regulated
kinase 1A) belongs to the CMGC family of protein kinases and
desirable.12
is essential for maintaining the normal development and
RESULTS AND DISCUSSION
function of the brain, phosphorylating several proteins and
thereby regulating their functions.1 DYRK1A has also been
implicated in the pathogenesis of one of the most frequent
genetic disorders, Down’s syndrome,2 and recent evidence has
also implicated DYRK1A in the pathology of neurodegener-
ative disorders such as Alzheimer’s disease3 and Parkinson’s
disease.4 DYRK1A is also involved in the regenerative
pathways relevant to human insulin-producing pancreatic β-
cells,5 and its implication in tumorigenesis and cell survival has
also been reported.6 The natural product harmine was
identified as a selective and potent inhibitor of DYRK1A.7 In
spite of the inherent liabilities of harmine, including the potent
inhibition of MAO-A linked with hallucinogenic effects8 and
poor metabolic stability, the most potent DYRK1A inhibitors
reported so far are typically based on this scaffold,9 although
some new chemotypes were also identified recently.10
Although the MAO-A inhibition of β-carbolines could be
■
The accompanying paper describes the screening of a fragment
collection by NMR spectroscopy followed by orthogonal
validation to identify a number of fragments that bind to
DYRK1A with high ligand efficiency (LE), including 1 with a
LE of 0.59. The crystal structure of 1 in complex with the
DYRK1A kinase domain (hereafter termed Dyrk1a)
(PDB:7A4W), reported in the preceding article, showed
(Figure 1) that compound 1 completely filled the buried
Received: January 7, 2021
Published: May 12, 2021
https://doi.org/10.1021/acs.jmedchem.1c00023
© 2021 American Chemical Society
J. Med. Chem. 2021, 64, 6745−6764
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Figure 1. Structural characterization of fragment 1 bound to Dyrk1a, as determined by X-ray crystallography (PDB: 7A4W). (A) Overall view of
the structure, with 1 shown in green (space filling) and the ATP site of Dyrk1a as the molecular surface. (B) Overlay of Dyrk1a-bound harmine
(orange, PDB 3ANR) onto 1 (green) highlighting the hinge region in magenta and salt-bridge region in blue. (C) Specific interactions between 1
and Dyrk1a showing selected hydrogen atoms (added with software MOE13). Black dotted lines depict hydrogen bonds.
part of this site with a combination of apolar and polar
contacts. In particular, hydrogen bonds between the hinge
region of the protein (Leu241) and the methoxy group of 1, as
well as between the aminopyrimidine and the side chain of
Lys188 and Glu203 (a.k.a. “salt-bridge region”, also involving
Asp307, Figure 1C). The observed interactions were highly
reminiscent of those seen in harmine’s bound structure (PDB:
3ANR, Figure 1B). Because harmine (2) is known to be an
efficient and selective inhibitor of DYRK1A, 1 was selected as
the starting point for fragment elaboration. In addition, from
the outset, molecular modeling highlighted the distinctive
hinge conformation of Dyrk1a at the backbone carbonyl of
Leu241 and how it could be used to design ligand selectivity
for DYRK1A over a number of other kinases, via direct
contacts with the backbone carbonyl of Leu241. The methoxy
of 1 offered such contact. How this structural insight was used
to gain selectivity over other kinases is illustrated below. It is
important to note at this point that all DYRK1A inhibitors
exhibit a very similar effect on DYRK1B (for details, see Table
S1 in the Supporting Information) due to the very high
homology of these two proteins in their kinase domain.
The first set of compounds (3−6) were aimed at
establishing the minimum necessary elements for efficient
binding. In agreement with the X-ray structure, chemical
modifications that conserved the hydrogen acceptor moieties
in the correct geometric arrangements led only to small
variations in the affinity. Changing the diaminopyrimidine
moiety to aminopyridine (3) led to a minor improvement,
while modification to methylpyridine (4) slightly decreased the
affinity (Table 1). The X-ray structure of 3 in complex with
Dyrk1a (Figure 2A) confirmed the conservation of the binding
mode of 1; however, 3 binds with dual occupancy, its amine
being 50% buried toward Glu203 or 50% flipped toward the
solvent. Thus, neither orientation of the amine is over-
whelmingly preferred. In the buried orientation, the replace-
ment of this amine with methyl removes the hydrogen bond to
Glu203, consistent with the decreased affinity of 4.
It has been reported that chlorine attached to an aromatic
ring can interact efficiently with the hinge region of Dyrk1a.14
Indeed, the change of the hinge binder methoxy group to
chlorine with the methoxy group moved vicinal to chlorine (5)
led only to a minor deterioration. The X-ray structure of the
5−Dyrk1a complex (Figure 1B) confirmed that the hinge
binding occurs via the chlorine, accommodated by the above-
mentioned unusual orientation of the hinge carbonyl of
Leu241. The X-ray data for 5 cannot exclude two orientations
for its amine; however, signs of this equilibrium were weaker
with 5 than with 3; thus, the amino group of 5 was
conservatively modeled with single occupancy pointing toward
the protein. Removing the electron-donating substituents from
both aromatic rings (6) on the other hand led to a significant
drop in the affinity.
The overlaid X-ray structures of 1 and 5 (Figure 2B)
suggested that cyclization linking the attachment points of the
chlorine and methoxy should be tolerated. The cyclization of
the methoxy group to a benzofuran through a single (7) or
double bond (8) led to a marked increase in its affinity (Table
2), consistent with conformational restriction compared to 1.
The replacement of the pyrimidine ring by pyridine (9,10)
further improved binding to DYRK1A. This might be
attributed to the increased electron density of the pyridine
nitrogen which hydrogen bonds Lys188. The benzofuran
derivatives (8 and 10) and their dihydro analogues (7 and 9,
respectively) showed similar affinity. Moving from benzofuran
10 to the benzothiophene analogue (11) resulted in a marked
decrease of affinity, reflecting the poorer acceptor properties of
sulfur. The introduction of nitrogen into the five-membered
ring (benzoxazole 12) slightly decreased the affinity while the
same manipulation on the benzene ring (furo[3,2-b]pyridine
13) was well tolerated. In the next set of compounds, the hinge
binder oxygen was deliberately replaced by nitrogen because
nitrogen tends to be the better hydrogen bond acceptor in
oxazoles.15 Indeed, oxazole 14 showed a 2-fold improvement
over its analogue 12. The replacement of the oxygen of 14 by
sulfur was beneficial; the thiazole derivative 15 registering a
single-digit nanomolar affinity. Unfortunately, the sulfur of 15
blocked further growth at this position, but the corresponding
imidazoles do not have this limitation. The imidazole ring (16)
was about as efficient as the respective oxazole (14), while the
Table 1. Dyrk1a Inhibition with Our Starting Fragment (1),
Harmine (2), and Early Fragment-Derived Compounds (3−
6)
a
no
R1
R2
R3
DYRK1A IC50 (nM)
1
2
3
4
5
6
158
32
126
670
218
>10,000
MeO
MeO
Cl
NH2
Me
NH2
Me
H
H
OMe
H
Cl
a
Measured in the DYRK1A FRET assay.
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Figure 2. X-ray structures of Dyrk1a complexes of compounds derived by the early elaboration of fragment 1. Only selected structural elements and
hydrogen atoms are shown. Hydrogen bonds of interest are depicted with black dotted lines. (A) 3 (PDB: 7AJ2) showing double occupancy, with
its amine buried (green) or pointing toward the solvent (magenta). (B) Overlay of 1 (green) and 5 (orange, PDB: 7AJ4). (C) Overlay of 10
(orange sticks, PDB: 7AJ5) and 25 (green ball and stick, PDB: 7AJ8). The amine of 25 points toward Glu203. (D) 16 (PDB: 7AJ7) with the
imidazole methyl substituent offering a vector for growth toward the glycine loop (blue) and the opening of the ATP binding site including Asn244
and Asn292. (E) 28 (PDB: 7AJA) highlighting the desolvation of polar groups (red arrow) and a water molecule trapped between 28 and Glu203.
(F) 32 (PDB: 7AJM). It is unclear if the desolvation of the cationic amine by Phe170 is compensated by cation−π interactions.
Table 2. DYRK1A Inhibition of Diaminopyridines and Pyrimidines Carrying a Bicyclic Hinge Binder Motif (7−24)
a
no
X
Y
Z
bond
DYRKA IC50 (nM)
7
8
9
N
N
CH
CH
S
O
O
N
N
N
N
CH
N
single
double
single
22
36
16
14
137
22
10
10
1.0
14
13
17
15
2.0
19
11
42
10
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
double
CH
N
CH
O
CH
CH
N
CH
CH
CH
N
S
N-Me
N-Me
O
CO
single
double
a
Measured in the DYRK1A FRET assay.
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introduction of an additional nitrogen atom, resulting in the
imidazo[4,5-b]pyridine derivative 17 was tolerated. The X-ray
structure of the complexes with Dyrk1a of 10 (Figure 2C) and
16 (Figure 2D) confirmed that the cyclization as well as the
swap of the hinge binder atom closely maintain the initial
binding mode.
Table 3. DYRK1A Inhibition of Pyridine-Substituted
Benzofurans (25−32)
We also assessed the effect of introducing nitrogen into the
bridgehead position. The imidazo[1,2-a]pyridine (18)-,
[1,2,4]triazolo[2,3-a]pyridine (19)-, and pyrazolo[1,5-a]-
pyrimidine (20)-substituted diaminopyridine derivatives all
showed high affinities towards DYRK1A in the same range as
the other bicyclic compounds. Finally, we tested if further
variation in the moiety contacting the hinge could be sterically
accommodated by expanding the five-membered ring to six-
membered. The benzodioxane derivative 22 matched the
affinity of its dihydrobenzofuran analogue (9). Changing the
acceptor moiety to the less electron-rich chromenone analogue
(23) led to a perceptible drop of affinity due probably to
electronic effects, while the quinoline derivative 24 was as
efficient as benzodioxane 22. In summary, we took advantage
of the distinctive hinge of Dyrk1a to develop a large variety of
bicyclic hinge binder motifs, which were well accommodated
and offer a broad range of options. This variety was facilitated
because there is no constraint to the hydrogen bond of the
hinge backbone carbonyl of Leu241 in Dyrk1a, given its
comparatively unusual orientation. The main requirement was
an efficient heteroatom to accept a hydrogen bond from the
hinge amide nitrogen of Leu241. These observations were
important for the progression of the project because the
chemical space of ATP-competitive kinase inhibitors is quite
crowded, and the structural diversity of the identified bicyclic
inhibitors helped escape from areas that were already covered
by prior art.
The bound structure of 10 (Figure 2C) is consistent with its
particularly highly efficient binding (LE 0.63) because the
above-described hinge binding was combined with additional
interactions with the salt-bridge region. The hydrogen bond
between the pyridine nitrogen and Lys188 was conserved from
fragment 1 to more elaborated molecules. The amino group on
the pyridine point toward the protein hydrogen bond
glutamate-203, while the amino group that points toward the
solvent is in the proximity of Asp307. Thus, this region
involves multiple polar interactions, including water, combined
with protein sidechain shifts when comparing X-ray structures.
It was therefore challenging to establish precise structure−
activity relationships for the salt-bridge region because small
changes in the geometry of polar contacts have large effects on
the resulting energies. However, to address the contribution of
these interactions, we undertook the systematic variation of the
pyridine ring substitution pattern (Table 3), using 10 as a
reference for comparisons. The omission of one of the amino
groups (25) led to a minor decrease in affinity (IC50 increased
to 19 nM from 14 nM) while removing the second amino
group (26) resulted in a marked loss of affinity (320 nM).
Thus, at least one of the two amino groups strengthens
binding. Indeed, in the absence of both amines, there would be
either a trapped water molecule or a void between the
compound and Glu203. This interpretation is supported by the
Dyrk1a bound X-ray structure of 25 (Figure 2C), where the
amino group points toward the protein, hydrogen-bonding
Glu203. The polar nature of the interactions involving the
amines was confirmed when the replacement of one amine by a
methyl group (27) dropped the affinity to 77 nM. Deleterious
a
no
R1
R2
DYRK1A IC50 (nM)
25
26
27
28
29
30
31
32
NH2
H
NH2
H
H
CH3
H
NH2
19
320
77
3560
17
96
189
168
piperazinyl
NHCH3
MeOCH2CH2−
2-(3′-thienyl)ethyl
CH3NHCH2CO−
a
Measured in the DYRK1A FRET assay.
effects were also observed upon the alkylation of one of the
amines, although simple methylation in 29 maintained a
binding affinity similar to that of 10 and 25. Larger alkyl
substituents are exposed to the solvent but still have to form
specific interactions with the protein surface. The piperazinyl
analogue 28 showed a marked drop in affinity and ligand
efficiency, which may reflect desolvation because the X-ray
structure of its bound complex suggests that the piperazine ring
is in contact with the side chain of Asn292 but without forming
a hydrogen bond (Figure 2E). In addition, the deletion of both
plain amino groups in 28 resulted in a water molecule trapped
between 28 and glutamate-203, which is likely to be
entropically unfavorable.
Thus, the next set of compounds kept one unsubstituted
amine and explored the substitution of the second amino
group of the diaminopyridine moiety (29−32). A simple
methylation (29) resulted in a minor decrease of affinity that
translated to a drop of ligand efficiency to 0.59. Representative
examples for the introduction of a polar acceptor (30) or an
electron-rich aromatic group (31) led to a 10- to 100-fold drop
of affinity. Finally, the acylation of the amino group converting
it to a different H-bond donor (32) was also detrimental
despite the conservation of the hydrogen bond with the salt
bridge, as well as the formation of water-mediated hydrogen
bonds with Asn292 and Asp307 (Figure 2F).
At this stage of the program, small molecule 10 was our lead
DYRK1A inhibitor. It was not only bound to DYRK1A
potently and efficiently (IC50 = 14 nM, LE = 0.63) but was also
translated into the potent inhibition of DYRK1A autophos-
phorylation on Ser520 with an IC50 of 500 nM (Table 4). cis-
Autophosphorylation of DYRK1A on Ser520 results in protein
14-3-3β stimulating the catalytic activity of the kinase and
serves as a useful biomarker of intracellular DYRK1A
inhibition.16 However, there were some issues with this
compound’s selectivity. For example, compound 10 was also
a high affinity binder to the undesired target CDK9 (Table 4),
not a surprise considering the structural similarity between the
ATP-binding sites of Dyrk1a and CDK9 (Figure 3A). As
suggested by modeling analysis, a minor structural modifica-
tion, the introduction of a methyl group into the 2-position of
the benzofuran (33) drastically decreased the affinity for
CDK9 while 33 remained a 41 nM binder of DYRK1A and
maintained some cellular activity. This selectivity can be
rationalized by overlaying the Dyrk1a-bound X-ray structures
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b]pyridine ring system, which seemed superior with respect to
synthetic tractability, metabolic stability, and freedom to
operate. The methylated analogue 35 showed high affinity
toward DYRK1A while being over 100-fold less potent on
CDK9. This compound was also quite potent in the cellular
autophosphorylation inhibition assay registering at 250 nM.
Further synthetic modifications, such as the replacement of the
methyl group in the 1-position by a butyl group (36) was well
tolerated. This latter compound also showed a considerable
improvement in the cellular assay. Thus, to assess if a similar
increase of the apolar character at the 2-position of the five-
membered ring can be accommodated by the Dyrk1a hinge, we
prepared and tested the 2-ethyl (37) and 2-butyl (38)
analogues. These compounds maintained high affinity for
DYRK1A, high selectivity versus CDK9, and a potent
inhibition of DYRK1A autophosphorylation in the cellular
setting, consolidating our choice of the imidazopyridine core
for future optimization. We obtained the X-ray structure of 38
in its Dyrk1a bound complex (Figure 3C). Comparing it with
the structure of the 33-Dyrk1a complex shows that the cores
are closely overlaid, and the flexible butyl chain can avoid
clashing into the Dyrk1a hinge region. Such a substituent is
largely solvent exposed, which might be used to modulate
physicochemical properties but offers fewer opportunities for
further potency gains than elaboration at position 1 of the five-
membered ring.
Having a series of potent and selective DYRK1A inhibitors
in hand, subsequent optimization focused on the improvement
of the cellular activity of the compounds. Compound 36
suggested that growing the substituent in the 1-position while
maintaining a methyl group in position-2 could result in more
potent compounds in cells (100 vs 250 nM for 35). The
addition of a small branched alkyl group, such as tert-butyl
(39), cyclopropyl (40), or cyclopentyl (41), maintained the
on-target affinity (7−11 nM) and showed cellular activities in
the 80−228 nM range (Table 5). The selectivity versus CDK9
has dropped for 41 compared to the other compounds. The
introduction of a polar end group on a flexible chain (42) was
also well-tolerated affinity-wise but the cellular activity
decreased to 280 nM. The lipophilic cyclohexylethyl end
group (43) restored activity but again with a drop in CDK9
selectivity. The introduction of the less flexible 4-fluorobenzyl
substituent (44) led to a marked deterioration of the on-target
affinity along with a sharp decrease of cellular activity. When
Table 4. DYRK1A, CDK9 Inhibition, and Cellular
Inhibition of DYRK1A Autophosphorylation of Compounds
10,33−38
DYRK1A IC50
CDK9 IC50 pSer520 IC50 U2OS
a
b
c
no
R1
R2
(nM)
(nM)
(nM)
10
33
34
35
36
37
38
H
Me
Cl
Me
Me
Et
14
34
76
15
2.0
11
8.0
41
>10,000
5430
1257
1244
500
1800
NA
250
100
153
128
Me
Bu
Bu
Bu
>10,000
>3000
Bu
a
b
Measured in the DYRK1A FRET assay. Measured in the CDK9
c
FRET assay. Measured in the inhibition of the DYRK1A pSer520
autophosphorylation assay in U2OS cells.
of 10 and 33, with an X-ray structure of human CDK9 (PDB
entry 3BLH).17 The crystal structure 3BLH was solved at
sufficient crystallographic resolution (2.48 Å) and its unbound
ATP site presents the intrinsically preferred conformation of its
backbone hinge, which is consistent with other X-ray structures
of CDK9 (e.g., 3BLQ, 3BLR, and 4BCF). Figure 3B shows the
hinge region of the aligned X-ray structures with Dyrk1a in
gray and CDK9 in magenta. The two kinases differ in the
orientation of the backbone carbonyl groups of hinge residues
Leu241 (Dyrk1a) and Cys106 (CDK9), which confer the
desired selectivity to our inhibitors. The methyl substituent of
the benzofuran ring of 33 (green structure) clashes sterically
with the carbonyl oxygen of CDK9 (short C···O distance),
while the carbonyl of Dyrk1a avoids that clash due to its
orientation being somewhat perpendicular to the added
methyl. To further test this interpretation, we also prepared
34, the 2-chloro-analogue of 10. Although the replacement of
hydrogen with a bulkier chlorine substituent led to some
decrease of affinity toward DYRK1A, this compound still
maintained a 50-fold selectivity against CDK9.
In anticipation of the future challenges, we decided to move
away from the benzofuran core and focus on the imidazo[4,5-
Figure 3. Structural basis for the design of compounds selective for DYRK1A over CDK9. Black dotted lines denote hydrogen bonds. (A)
Alignment the ATP-binding sites of Dyrk1a bound to 10 (green) and unliganded CDK9 (PDB: 3BLH). Residues within 5 Å of 10 are shown for
both proteins; differing residue pairs (Dyrk1a residues labeled) are in red and same residue types are in gray. (B) Overlay of CDK9 (PDB: 3BLH)
onto Dyrk1a bound to 10 (gold sticks) or 33 (green ball and sticks, PDB: 7AJS). Only the hinge backbone of Dyrk1a (grey) and CDK9 (magenta)
is shown. The red oval highlights the clash between the methyl group of 33 and the hinge backbone carbonyl Cys106 in CDK9. (C) Overlay of
Dyrk1a bound to 33 (green sticks) and 38 (brown ball and sticks, PDB: 7AJV). The butyl substituent at position 2 crosses the path of the protein
chain (gray ribbon) into the solvent.
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Table 5. DYRK1A, DYRK2, and CDK9 Inhibition and
Cellular Inhibition of DYRK1A Autophosphorylation of
Compounds 39−50
DYRK1A DYRK2
CDK9
pSer520
IC
IC
IC
IC50 U2OS
50a
50b
50c
d
no
R
(nM)
(nM)
(nM)
(nM)
39
40
41
42
43
44
45
46
47
48
49
50
t-Bu
cyclopropyl
cyclopentyl
MeO(CH2)3−
cyclohexylethyl
4-fluorobenzyl
2-(2′-thienyl)ethyl
2-PhO-ethyl
CF2HCH2−
CF3CH2−
7.0
11
13
6.0
3.0
1.0
5.0
9.0
134
6.0
9.0
4.0
6.0
10
1837
2149
314
3027
470
4575
1952
1019
5692
5700
1407
8835
228
90
80
280
110
3000
226
35
34
28
47
1650
6.0
4.0
32
3.0
1.0
3.0
2.0
6.0
1.0
Figure 4. Use of X-ray structures for the design of Dyrk1a inhibitors
selective over Dyrk2. Black and green dotted lines depict hydrogen
bonds and van der Waals contacts, respectively. (A) Overlay of 50
bound to Dyrk1a (green, PDB: 7AKL) and Dyrk2 (magenta, PDB:
7AKF). (B) Overlay of 46 (green, PDB: 7AJW) and 53 (orange,
PDB: 7AK2) bound to Dyrk1a. (C) Overlay of 56 (green, PDB:
7AKB) and 58 (orange, PDB: 7AKE) bound to Dyrk1a. (D) Overlay
of 58 bound to Dyrk1a (green) and Dyrk2 (magenta, PDB: 7AKH).
The glycine loop is shown in blue ribbons.
CF3(CH2)3−
21
b
a
Measured in the DYRK1A FRET assay. Measured in the DYRK2
c
d
FRET assay. Measured in the CDK9 FRET assay. Measured in the
inhibition of the DYRK1A pSer520 autophosphorylation assay in
U2OS cells.
we increased the flexibility by removing the aromatic end
group by 2 (45) or 3 atoms (46), we generated highly potent
DYRK1A inhibitors with affinities at 3.0 and 1.0 nM,
respectively. The cellular activity of 45 was in the usual
range at 226 nM, while 46 showed the highest activity
registered so far at 35 nM. We have also synthesized some
fluoroalkylated analogues of 35. The difluoroethyl compound
(47) showed very high affinity and cellular activity (3.0 and 34
nM, respectively) that was even further improved by the
trifluoroethyl substitution. This compound (48) registered at
2.0 and 28 nM in the affinity and cellular assays, respectively.
Finally, the addition of two more methylene groups into the
linker (49) led to a slight drop in affinity and activity compared
to 48 (6.0 vs 2.0 nM affinity, and 47 vs 28 nM activity). The
cellular target hitting of 40 and 48 was also confirmed in a
nanoBRET assay (for details see Supporting Information).
We systematically determined the affinity of our DYRK1A
inhibitors 39−49 toward DYRK2. It was not clear if the
downstream effects of DYRK2 inhibition are antagonistic or
synergistic with the biological consequences of DYRK1A
inhibition, so generating compounds of different selectivities
could be useful to probe the biology of these targets. Before
addressing DYRK2 selectivity, the compounds showed high
affinity for DYRK2 (Table 5), consistent with the DYRK2
sequence and X-ray structures being very similar to those of
Dyrk1a in the ATP site, as demonstrated by the X-ray
structures of Dyrk1a and the kinase domain of DYRK2
(hereafter Dyrk2) complexed with a benzofuran derivative 50
(Figure 4A). Within the ATP site, Val222, Met240 (hinge),
and Val306 in Dyrk1a are changed to Ile285, Leu303, and
Ile367 in Dyrk2 (isoform 1 residue numbering), respectively
(Figure 4A). Despite two valines in the Dyrk1a ATP site being
changed to bulkier isoleucines in Dyrk2, compound 50 and the
isosteric imidazo[4,5-b]pyridine ring systems, are accommo-
dated unhindered in Dyrk2. The corresponding two additional
methyl groups in Dyrk2 do not occlude the binding volume of
the imidazo-pyridine or benzofuran cores, instead maybe
increasing favorable van der Waals contacts between the
compound core and Dyrk2. Thus, gaining selectivity over
DYRK2 with the present chemotype was challenging.
However, compounds with an aromatic end group (44−46)
linked to the imidazole position 1 seemed to have a mild
preference for DYRK1A over DYRK2, in particular 46 (9.0 vs
1.0 nM). An overlay of X-ray structures of Dyrk2 and 46
complexed to Dyrk1a (PDB: 7AJW) gave some insights into
this emerging selectivity (Figure 4B). The 2-PhO-ethyl side
chain of 46 sits alongside the imidazo-pyridine core, which
largely removes this apolar substituent from water. This
orientation of the side chain is also stabilized by van der Waals
contacts with the compound core and the protein, in particular
regarding the end phenyl. One edge of this phenyl is tucked
under the glycine loop (residues 165−173 in Dyrk1a), while
the opposite edge donates a C-H hydrogen bond to Asn292
(Figure 4B) and, importantly, contacts the side chain of Val306
(Ile367 in Dyrk2). This shape complementarity between the
phenyl of 46 and Dyrk1a would be disrupted when facing the
additional methyl of Ile367 in Dyrk2 (Figure 4B), likely
contributing to the poorer affinity for DYRK2.
Based on this insight, it was hypothesized that rigidifying the
linker may help confine the phenyl to its Dyrk1a-bound
orientation, reinforcing a clash with Ile367 in Dyrk2, leading to
increased selectivity. Thus, a methyl substituent was
introduced in the middle of the linker chain, affording the
enantiomers R-methyl (51) and S-methyl (52). In DYRK1A,
both compounds showed the same affinity of 8.0 nM while
their DYRK2 affinity differed by a factor of 2 (46 vs 22 nM)
(Table 6). The absolute configuration of the stereocenter in 51
(and 54 below) was elucidated by the X-ray structure of its
Dyrk1a complex. Overlaying the bound structures of 46 and 51
(PDB: 7AJY) showed that the aromatic rings occupy the same
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structures of 46 and 53 is shown in Figure 4B. Compared to
46, the potency of 53 with respect to the different targets
dropped, possibly due to the desolvation of pyridine. Indeed,
the pyridine of Dyrk1a-bound 53 and 54 adopts essentially the
same position and interactions as those of the phenyl of 46 and
51, contacting the (∼4 Å) Val306 targeted for selectivity. This
is consistent with the affinities of 53 and 54 for DYRK1A being
equivalent to that of 51, ∼7 nM. The selectivity against
DYRK2 was between 6- and 17-fold for 53−55, consistent
with the approach targeting the Val306/Ile367 pair. Interest-
ingly, the affinity toward DYRK2 degraded more with the
methylated linkers of 54 (116 nM) and 55 (117 nM) than
with the plain linker of 53 (34 nM), maybe the reduced linker
flexibility lessens the ability to adapt to DYRK2. However, the
present linkers remain flexible with several torsions able to
adjust the conformation when binding to DYRK2. Increased
selectivity would require substituents which cannot reorientate
to alleviate a clash with Ile367. Overall, there was evidence that
a substituent reaching the Val306/Ile367 pair can impart some
DYRK1A/DYRK2 selectivity.
Building on the above approach, a natural progression was to
increase the size of the aryl moiety reaching the Val306/Ile367
pair, to increase the steric repulsion with Ile367. The X-ray
structures of 53 and 54 suggested that a small apolar
substituent at position 6 of the pyridine may strengthen the
contacts with the glycine loop, further anchoring the pyridine
in a position that clashes with Ile367 in Dyrk2. Thus, we
introduced a halogen into the 6-position of the pyridine ring of
53. The addition of a bromine (56) or chlorine (57) was well
tolerated by DYRK1A giving 2.0 nM-ar or 3.0 nM-ar
inhibitors, respectively. Reassuringly the cellular activities of
56 and 57 were among the best observed at 32 and 26 nM,
respectively. The X-ray structure of 56 confirmed that its
bromine is nested under the glycine loop, with favorable apolar
Table 6. DYRK1A, DYRK2, and CDK9 Inhibition and
Cellular Inhibition of DYRK1A Autophosphorylation by
Compounds 51−58
pSer520
DYRK1A DYRK2 CDK9
IC50
IC
IC
IC
50c
U2OS
(nM)
50a
50b
d
no
X
R1
R2
(nM)
(nM)
(nM)
51
52
53
54
55
56
57
58
CH
CH
N
N
N
N
N
N
R-Me
S-Me
H
S-Me
R-Me
H
H
H
H
H
H
Br
Cl
Br
8.0
8.0
6.0
7.0
16
2.0
3.0
5.0
46
22
34
116
117
52
>3000
>3000
881
>3000
>3000
289
111
73
89
131
>300
32
H
R-Me
33
195
222
467
26
44
a
b
Measured in the DYRK1A FRET assay. Measured in the DYRK2
c
d
FRET assay. Measured in the CDK9 FRET assay. Measured in the
inhibition of the DYRK1A pSer520 autophosphorylation assay in
U2OS cells.
space in Dyrk1a despite an altered conformation of the
aliphatic linker. Unfortunately, both 51 and 52 lost in terms of
affinity and cellular activity when compared to 46. We also
prepared the pyridine analogues without (53) or with (54,55)
the methyl substituent in the linker, and X-ray structures of 53
and 54 were obtained (PDB: 7AKA). An overlay of the X-ray
Scheme 1. Synthetic Strategies Applied in the Preparation of Our DYRK1A Inhibitors
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contacts to Val173 and Phe170 of Dyrk1a (Figure 4C). The
bigger halogen substituent (56) decreased the affinity toward
DYRK2 (52 vs 34 nM) but not the chlorine (33 vs 34 nM).
Still, both compounds increased the DYRK1A/DYRK2
selectivity with respect to 53 (26-fold and 11-fold vs 5.7-
fold). Finally, we tested if the methyl substitution of the linker
would further add to the selectivity gained with the
halogenated pyridine. Compound 58, which combines these
two features showed a 39-fold selectivity, the highest observed
in this series, and both its affinity for DYRK1A at 5.0 nM and
its cellular activity at 44 nM were respectable. We also
obtained the X-ray structure of 58 with both Dyrk1a and
Dyrk2 (Figure 4D). With both proteins, the pyridine is
anchored to the glycine loop via the bromine, but the pyridine
is clearly shifted in Dyrk2 to avoid a clash with Ile367; this shift
in turn disrupts a number of contacts, including the C-H
hydrogen bond to the side chain of Asn353 in Dyrk2. Overall,
we obtained a collection of compounds with high affinity for
DYRK1A, good cellular potency, and covering a range of
selectivity toward Dyrk2.
The selectivity profile of 40 for the inhibition of other
protein kinases was characterized in a DiscoverX KINOMEs-
can assay which measured the percentage activity remaining for
each of 442 kinases (Table S1). From the kinases that showed
a dose-dependent inhibition between 10 nM and 1 μM of 40,
only two kinases were inhibited to less than 10% activity at the
lower dose, DYRK1A (9.2%) and DYRK2 (1.6%) and only 8
other kinases to less than 75% activity, most notably DYRK1B
(20%) CLK1 (48%), CLK2 (40%), CLK4 (52%), CSNK1G1
(69%), HIPK1 (72%), and RPS6KA4 (61%). We also
determined the affinity of 40 and 48 toward closely related
kinases in a binding assay. The measured t values for 40 were
0.76 nM for DYRK1B, 4.7 nM for CLK1, 5.7 nM for CLK2,
and 3.2 nM for CLK4. 48 bound to the same targets with
affinities of 0.12 nM (DYRK1B), 3.2 nM (CLK1), 2.2 nM
(CLK2), and 5.7 nM (CLK4), respectively. Leu244 in CLK1
adopts a similar conformation to the equivalent to Leu241 in
DYRK1A (see the Supporting Information in ref 19),
providing some explanation for the observed selectivity,
which is a feature seen for other DYRK1A inhibitors.10a It is
interesting to note that efficient inhibitors of CLK1 carrying a
similar combination of a bicyclic hinge binder and monocyclic
salt-bridge binder motif were reported recently.18 At 1 μM
compound, 37 kinases were inhibited to less than 50% activity.
It is also worth mentioning that none of the studied 37
receptor targets was affected more than 40% at 10 μM
concentration of 40. In the patch-clamp model, 40 induced a
52% decrease of hERG activity at 10 μM.
achieved in an amidine formation−cyclization sequence
(Scheme 1). The key intermediate tetra-substituted bipyridyl
compound was prepared via the formerly established Suzuki
approach. The R2 substituent was introduced in a conventional
acylation, while the activation of the amide function enabled us
to use primary amines to install the R1 group. The palladium-
catalyzed cyclization of the amidine and protecting group
removal completed the sequence to give 36−49, 51, and 52.
This synthetic route was used also to prepare the common
intermediate leading to 54−58. Aromatic nucleophilic
substitution with 2-halopyridines, followed by protecting
group removal furnished the desired selective DYRK1A
inhibitors. The developed synthetic routes were sufficiently
robust to furnish building blocks and final products on the
multigram scale needed for in vivo studies and detailed
characterization.
To evaluate the inhibition of DYRK1A in vivo, we selected
compound 40, which had favorable in vitro ADME properties
including the complete predicted oral absorption, moderate to
good microsomal stability, and a high unbound (free) fraction
in plasma (for details see Supporting Information). The in vivo
PK characterization of 40 showed a good in vitro: in vivo
correlation with high plasma clearance (226 mL/min/kg) and
an oral bioavailability of 39% at 3 mg/kg that showed an over
proportional increase with the dose (for details see Supporting
Information). To determine whether the in vitro functional
effect could be recapitulated in vivo, mice bearing RS4;11
tumors were treated with different doses of 40. Following the
oral administration of 3.125, 6.25, 12.5, or 25 mg/kg of 40, the
tumors were harvested after 2 or 6 h and the change in the
level of DYRK1A phosphorylated on Ser520 was determined
by the western blot. We observed a dose-dependent inhibition
of DYRK1A autophosphorylation at both timepoints with the
effect appearing more pronounced after 2 h (Table 7). It
Table 7. In Vivo Inhibition of DYRK1A
Autophosphorylation by Compounds 40, 46, and 48 (n = 3)
pDYRK1A (S520)
inhibition (%)
compound
xenograft
dose (mpk, p.o.)
2 h
6 h
40
40
40
40
46
48
40
40
40
40
40
RS4;11
RS4;11
RS4;11
RS4;11
RS4;11
RS4;11
A2780
A2780
A2780
A2780
A2780
3.125
6.25
12.5
25
6.25
6.25
4.687
9.375
18.75
37.5
75
51
77
87
87
59
93
60
60
82
82
80
38
25
84
91
25
95
0
10
80
95
92
In order to ensure the rapid generation of our DYRK1A
inhibitors, we needed to establish a collection of robust and
scalable synthetic routes that would ideally allow for the late-
stage introduction of the differentiating elements. Our early
DYRK1A inhibitors (3−6) were prepared from commercially
available building blocks through the formation of the biaryl
link in a Suzuki coupling. As we wanted to explore the bicyclic
hinge binder element and the substitution pattern on the
diaminopyridine moiety (Scheme 1, 7−35, 50), the Suzuki
coupling remained the key construction step but some of the
coupling reagents needed to be prepared, and in certain cases
the Suzuki coupling was also followed by the modification of
the pyridine ring’s substituents (Z = CH).
should also be emphasized that at the highest dose of 25 mg/
kg, the effect was maintained after 6 h. In a similar experiment,
we evaluated 46 and 48 at the 6.25 mg/kg dose. Both
compounds led to a sustained decrease of phosphorylated
DYRK1A levels with 46 being slightly less active than 40 giving
59 and 25% inhibition of DYRK1A phosphorylation at the 2
and 6 h time points compared to 77 and 25% observed for 40.
Compound 48, on the other hand, demonstrated a very strong
and sustained inhibition of pDYRK1A (93% after 2 h and 95%
after 6 h, Table 7) superior to that of 40 and 46, which might
After the identification of the imidazo[4,5-b]pyridine hinge
binder motif, the variation of the R1 and R2 substituents was
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Table 8. Effect of DYRK1A Inhibitors 40 and 48 on the Viability of Selected Cancer Cell Lines in 2D and 3D Cultures
2D viability (MTT assay) IC50 (nM)
3D viability (soft agar assay) IC50 (nM)
compound
pSer520 IC50 (nM)
A2780
SK-N-MC
C-33A
A2780
SK-N-MC
C-33A
40
48
90
28
1539
1099
958
1281
1022
1154
126
13
77
31
157
21
reflect a combination of its increased cellular activity and
stability.
We evaluated the antiproliferative activity of our DYRK1A
inhibitors 40 and 48 against a range of human cancer cell lines.
These cell lines exhibited little sensitivity to DYRK1A
inhibition in 2D culture. However, when growing in 3D
culture in soft agar as spheroids, their sensitivity to DYRK1A
inhibition increased considerably (for the detailed graphs see
the Supporting Information). In three cell lines (ovarian cancer
cell line A2780, neuroblastoma cell line SK-N-MC, and cervical
cancer cell line C33-A) in 2D culture, all compounds
demonstrated IC50s around 1 μM, with no differentiation
between the two compounds (Table 8). In 3D culture, a clear
difference in cellular activity between 40 and 48 was observed
with the latter compound being more potent against all cell
lines, and the observed cellular IC50s correlated nicely with the
cellular pDYRK1A inhibition activities observed in the U2OS
cell line (90 nM for 40 and 28 nM for 48). Although one can
hypothesize that the efficient inhibition of DYRK1B, CLK1,
CLK2, and CLK4 might play an active role in these biological
processes, the explanation of the different sensitivity of a given
cell line in the 2D and 3D culture requires further
experimentation.
Figure 5. Tumor volume changes in mice xenografted with A2780
cells on treatment with 40. BALB/C nude mice bearing established
A2780 xenografted tumors were dosed BID with the indicated
amounts of 40 for 14 days (black triangles). Tumor volume was
determined as described in the Experimental section.
Table 9. Summary of In Vivo Responses in A2780-
Encouraged by these results, we grafted A2780 cells
subcutaneously into mice to evaluate levels of DYRK1A
phosphorylation on Ser520 by the western blot at 2 or 6 h
post-treatment. Analysis of the data revealed dose- and time-
dependent inhibitions of pDYRK1A similar to that observed in
the RS4;11 cell line in vivo. At the lowest dose of 4.7 mg/kg,
we observed a transient inhibition of pDYRK1A (S520) at 2 h
but with complete recovery by 6 h (Table 7). Increasing the
dose to 37.5 mg/kg led to the almost complete inhibition of
DYRK1A autophosphorylation at both 2 (82% inhibition) and
6 h (95% inhibition) that was retained but not increased upon
at 75 mg/kg. We measured the plasma and tumor
concentrations of 40 at the different doses and time points
and established that tumors were well exposed. At doses of
18.75 mg/kg and above, the tumor concentration of 40 was
greater than the measured cellular pDYRK1A (S520) IC50 for
at least 6 h (see Supporting Information), demonstrating a
clear correlation between tumor exposure and PD response.
Finally, we determined the antitumor activity of 40 in a
dose-dependent efficacy study in A2780-xenografted mice.
Animals were treated with different doses of 40 daily for 2
weeks. At the lower doses of 4.75−9.5 mg/kg, we observed no
effect on tumor growth (Figure 5 and Table 9). For the dose of
18.75 mg/kg, at which the PD response was more robust, we
observed some inhibition of tumor growth (TGI 55%, P <
0.01). Increasing the dose to 37.5 and 75 mg/kg increased the
antitumor activity of 40, resulting in an almost complete tumor
stasis while on treatment (TGI 63 and 92%, P < 0.001).
Increased toxicity was observed at the higher doses with a
mean body weight changes of −9.7 and −16.3% at 37.5 and 75
mg/kg doses, respectively (Figure S1). The 75 mg/kg dose was
also the maximal tolerated dose in female Balb/C nude mice.
The body weight changes were transient and recovered rapidly
a
Xenografted Mice Treated with 40
dose 40
T/C (%) @
D14
TGI (%) @
D14
MBWC (%) @
D14
(mg/kg)
P
0
4.75
9.5
18.75
37.5
75.0
+4.2
−1.5
−8.7
−10.7
−9.7
−16.3
91
95
63
52
24
28
28
55
63
92
ns
ns
<0.01
<0.001
<0.001
a
T/C, tumor/control; TGI, tumor growth inhibition; MBWC, mean-
body-weight change.
on the cessation of treatment. The antitumor effects of 40 on
A270 tumors appeared cytostatic as upon stopping dosing, the
tumors rapidly regrew with similar kinetics to the untreated
controls. These results suggest that DYRK1A inhibitors, at
least in this ovarian model, exert their antitumor activity
through inhibiting tumor growth and might not be efficient as
single agents to induce tumor regressions. Further work is
needed to understand if this effect on tumor growth might play
an effective role in combination therapy.
CONCLUSIONS
■
Starting from a fragment hit identified by NMR spectroscopy,
we conducted a structure-guided drug discovery program that
led to the identification of potent and selective DYRK1A
inhibitors. By exploiting the unique orientation of the
backbone CO group of Leu241 in the hinge region in
DYRK1A, we identified a reliable way to confer selectivity on
our inhibitors against the majority of other kinases by placing a
small substituent in the proximity of this moiety. We were also
able to fine-tune the selectivity of our DYRK1A inhibitors
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Compound 40 was formulated in PEG300/Ethanol/water (4/1/5,
v/v) and administered by oral gavage twice daily for 14 days. Tumor
size was measured twice weekly with electronic calipers and tumor
volume calculated according to the formula ((width × width) ×
length)/2. Body weight was measured daily for the first 14 days then
twice weekly thereafter. T/C (%) = (Median tumor volume of treated
group at D14/Median tumor volume of the control group at D14) ×
100. TGI (%) = 1 − ((T14/T0)/(CD14/C0))/1 − (C0/C14) × 100,
where T14 and T0 equals the median tumor volume of the treated
group at day 14 and day 1, respectively, and C14 and C0 equals the
median tumor volume of the control group at day 14 and day 1,
respectively.
For the assessment of the Ser520 phosphorylation of DYRK1A,
tumors were excised at 2 and 6 h post-treatment and lysed in RIPA
buffer containing protease and phosphatase inhibitor cocktails
(Roche). Western blotting for the phosphorylation of anti P-
(S520)DYRK1A (Rabbit Polyclonal, Eurogentec) was performed
using standard techniques. Blots were scanned using a Bio-Rad GS-
800 calibrated densitometer, quantitative analysis of western blots was
performed using TotalLab software (Amersham), and % inhibition
was calculated with respect to the control values.
General Synthetic Remarks. All reagents obtained from
commercial sources were used without further purification. Anhy-
drous solvents were obtained from commercial sources and used
without further drying.
The reactions were monitored using LCMS and GCMS instru-
ments. Analytical LC−MS: Agilent HP1200 LC with Agilent 6140
quadrupole MS, operating in positive or negative ion electrospray
ionization mode. Molecular weight scan range was 100−1350 m/z.
Parallel UV detection was done at 210 and 254 nm. Samples were
supplied as a 1 mM solution in MeCN or in THF/water (1:1) with 5
μL loop injection. LCMS analyses were performed on two
instruments, one of which was operated with basic, and the other
with acidic eluents.
against the closely related DYRK2 kinase by targeting the
sequence differences in the two kinase ATP pockets. Our
developed compounds also have the desired properties to
probe the in vitro and in vivo biology of DYRK1A inhibition.
They are potent inhibitors of DYRK1A in the cellular setting,
which was also recapitulated in vivo. Interestingly, we observed
a significant difference between the sensitivity of cancer cell
lines to our inhibitors in 2D and 3D models. The good in vivo
PK properties of our inhibitors enabled us to translate their in
vitro cell killing into tumor growth inhibition in the
xenografted mice model potentially opening the way to new
therapeutic strategies in oncology and Down’s syndrome.
EXPERIMENTAL SECTION
■
TR-FRET Assay. A TR-FRET assay was used to measure the
activity of the protein kinases DYRK1A, DYRK2, and CDK9. The
assay was performed on a TECAN Evo robotic platform. Compound
at 5% DMSO was added to reaction buffer (50 mM Hepes pH 7.4, 1
mM EGTA, 10 mM MgCl2, 2 mM DTT, and 0.01% Tween20)
containing 20 ng/μL DYRK1A kinase (Carna Biosciences #04-130),
100 μM Ulight-MBP substrate (PerkinElmer #TRF109L) and 10 μM
ATP co-substrate (Sigma-Aldrich #A2383) and incubated for 40 min
at room temperature. The kinase reaction was stopped by adding 10
mM EDTA (Sigma-Aldrich #E7889) and 1 nM Anti-phospho-MBP
antibody (PerkinElmer #TRF0201L) and incubated for 1 h minimum
at room temperature. Plates were read on a multimode reader
(PerkinElmer Envision 2400) with an excitation wavelength of 337
nm and emission wavelengths of 620 nm for the donor and 665 nm
for the acceptor. A similar format was used for other TR-FRET kinase
inhibition assays.
DYRK1A (pS520) Cell Assay. U2OS cells (ATCC) were
transiently transfected with a plasmid expressing full length wild-
type DYRK1A (pcDNA3.1). After 5 h, compounds were added and
cells were incubated for a further 5 h. Cells were lysed and equal
amounts of protein were separated by SDS-PAGE and transferred to
the PVDF membrane. Membranes were probed for pS520 DYRK1A
(custom rabbit-polyclonal antibody immunized with the synthetic
peptide SNSGRARpSDPTHQHR, Eurogentec; 1:500 in 5% BSA-
TBST), detected with anti-rabbit IgG-HRP (7074, CST; 1:3000), and
then quantified on an ECL camera (BioRad). Membranes were
subsequently stripped and reprobed with a total anti-DYRK1A
antibody (H0000-1859, Abnova; 1:1000 in 5% BSA-TBST) and
quantified as above.
Basic LCMS: Gemini-NX, 3 μm, C18, 50 mm × 3.00 mm i.d.
column at 23 °C, at a flow rate of 1 mL min−1 using 5 mM aq.
NH4HCO3 solution and MeCN as eluents.
Acidic LCMS: ZORBAX Eclipse XDB-C18, 1.8 μm, 50 mm × 4.6
mm i.d. column at 40 °C, at a flow rate of 1 mL min−1 using water and
MeCN as eluents, both containing 0.02 V/V % formic acid.
Combination gas chromatography and low-resolution mass
spectrometry were performed on an Agilent 6850 gas chromatograph
and Agilent 5975C mass spectrometer using 15 m × 0.25 mm column
with 0.25 μm HP-5MS coating and helium as the carrier gas. Ion
source: EI+, 70 eV, 230 °C, quadrupole: 150 °C, interface: 300 °C.
Flash chromatography was performed on a ISCO CombiFlash Rf
200i with pre-packed silica-gel cartridges (RediSepRf Gold High
Performance).
Preparative HPLC purifications were performed on a Gemini-NX
10 μm C18, 250 mm × 50 mm i.d. columns running at a flow rate of
118 mL min−1 with UV diode array detection (210−400 nm).
1H NMR and proton-decoupled 13C NMR measurements were
performed on Bruker Avance III 500 MHz and Bruker Avance III 400
MHz spectrometers, using DMSO-d6 or CDCl3 as the solvent. 1H and
13C NMR data are in the form of delta values, given in parts per
million (ppm), using the residual peak of the solvent as the internal
standard (DMSO-d6: 2.50 ppm (1H)/39.5 ppm (13C); CDCl3: 7.26
ppm (1H)/77.0 ppm (13C)). Splitting patterns are designated as: s
(singlet), d (doublet), t (triplet), q (quartet), sp (septet), m
(multiplet), br s (broad singlet), dd (doublet of doublets), td (triplet
of doublets), and qd (quartet of doublets). In some cases, two sets of
signals appear in the spectra due to hindered rotation.
2D and 3D Cell Viability Assays. For 2D viability assays, A2780,
C-33A, and SK-N-MC cells (ATCC) were seeded in 96-well plates
and supplemented 48 h later with serial dilutions of compounds. After
120 h incubation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazo-
lium bromide (MTT; Sigma) was added to each well. After 4 h, the
formazan metabolite was solubilized in DMSO and cell viability was
estimated by the measurement of optical density at 540 nm. For 3D
vitality assays, 2000 (A2780), 6000 (C-33A), or 25,000 (SK-N-MC)
cells were seeded in Agarose type VII (Sigma A-4018) and cultured 7,
11, or 14 days, respectively. Vital colonies were then stained overnight
with MTT. Colonies were counted using a light microscope and
image analysis software (AxioVision, Zeiss). IC50 values for the
inhibition of cell viability, migration, or colony formation were
calculated from dose−response curves (XLfit).
Animal Studies. Female BALB/C nu/nu mice were purchased
from Charles River Laboratories. Xenograft studies were undertaken
by Oncodesign S.A., Dijon, France in accordance with “Principe
HRMS were determined on a Shimadzu IT-TOF, ion source
temperature 200 °C, ESI +/−, ionization voltage: ( )4.5 kV. Mass
resolution min. 10,000.
́
́
d’ethique de l’experimentation animale, Directive no 86/609”, France.
Tumors were induced subcutaneously by injecting 1 × 107 of
RS4;11 or A2780 cells (ATCC) in 200 μL of RPMI 1640 into the
right flank of the female nu/nu mice and allowed to reach a target
volume of 150 mm3. On day 0 of the study, tumors were randomized
into treatment groups before compound administration (n = 8−10 for
efficacy studies and n = 3 for pharmacodynamic studies).
All obtained products had an LC purity above 96% that was
1
corroborated by their H and 13C NMR spectra unless specifically
mentioned otherwise.
Reagent Synthesis and General Procedures. tert-Butyl N-[6-
(tert-Butoxycarbonylamino)-4-(4,4,5,5-tetramethyl-1,3,2-dioxabor-
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olan-2-yl)-2-pyridyl]carbamate (R1). To 109.7 g of (4-bromo-6-tert-
butoxycarbonylamino-pyridin-2-yl)-carbamic acid tert-butyl ester19
(283 mmol), 107.7 g of bis(pinacolato)diboron (424 mmol), 0.29 g
of Pd(OAc)2 (1.27 mmol), 0.70 g of 1,1′-bis(diphenylphosphino)-
ferrocene (1.27 mmol), and 83.2 g of KOAc (848 mmol) were added
to 1100 mL of previously degassed 1,4-dioxane, and the mixture was
stirred at 80 °C under an argon atmosphere until no further
conversion was observed. Then, the reaction mixture was filtered; the
solid was washed with dioxane. 5.5 g of charcoal was added to the
filtrate, and it was stirred for 2 min at the reflux temperature. The
mixture was filtered, washed with warm 1,4-dioxane, and the volatiles
were evaporated under reduced pressure. The residue was crystallized
from tert-butyl-methyl-ether to give 92 g (75%) of R1 as a white
crystalline solid.
acetamide (25.7 mmol), 3.15 g of 2-aminoethanol (51.5 mmol), and 5
mL of N,N-diethylethanamine were stirred at 50 °C until no further
conversion was observed then the solvent was removed under reduced
pressure, then 150 mL of acetic acid was added, and the mixture was
heated under reflux conditions until full conversion was observed
(approximately 4 days). The reaction mixture was cooled down to
room temperature then 250 mL of water was added followed by the
extraction of the water phase with diethyl ether (3 × 250 mL) and
EtOAc (2 × 250 mL). The combined organic layers were extracted
with brine, then they were dried over MgSO4. Solvents were removed
in vacuum to give 5.3 g of 2-(5-bromo-2-methyl-imidazo[4,5-
b]pyridin-3-yl)ethanol as a crude product, which was used in the
next step without further purification.
1H NMR (400 MHz, DMSO-d6): δ ppm 7.87 (d, J = 8.2 Hz, 1H),
7.37 (d, J = 8.2 Hz, 1H), 4.95 (t, J = 5.5 Hz, 1H), 4.23 (t, J = 5.5 Hz,
2H), 3.71 (q, J = 5.5 Hz, 2H), 2.58 (s, 3H); HPLC-MS: (M + H)+:
256.0.
1H NMR (500 MHz, CDCl3-d6): δ ppm 8.16 (br s, 2H), 7.92 (s,
2H), 1.54 (s, 18H), 1.34 (s, 12H); 13C NMR (125 MHz, CDCl3-d6):
δ ppm 153.1, 150.9, 111.9, 84.7, 80.0, 49.2, 28.4, 27.3, 25.1.
tert-Butyl N-[6-(tert-Butoxycarbonylamino)-4-[6-chloro-5-(ace-
tylamino)-2-pyridyl]-2-pyridyl]carbamate (R2). Step A: N-(6-
Bromo-2-chloro-3-pyridyl)acetamide. To 31.4 g of 6-bromo-2-
chloro-pyridin-3-amine (151.3 mmol) was dissolved in 200 mL of
glacial acetic acid, then 15 mL of acetic anhydride (158.9 mmol) was
added to this solution dropwise, and the reaction mixture was stirred
at room temperature until no further conversion was observed. The
reaction mixture was concentrated under reduced pressure. The
residue was dissolved in EtOAc and the organic phase was washed
with 10% aqueous K2CO3 and brine. Following drying over Na2SO4
removal of the solvents under reduced pressure gave 35.5 g (94%) of
N-(6-bromo-2-chloro-3-pyridyl)acetamide.
Step C: tert-Butyl N-[6-(tert-Butoxycarbonylamino)-4-[3-(2-hy-
droxyethyl)-2-methyl-imidazo[4,5-b]pyridin-5-yl]-2-pyridyl]-
carbamate. To 5.32 g of 2-(5-bromo-2-methyl-imidazo[4,5-b]-
pyridin-3-yl)ethanol (20.9 mmol), 10.01 g of R1 (23.0 mmol),
0.469 g of Pd(OAc)2 (0.1 mmol), 1.50 g of PBuAd2 (4.18 mmol), and
13.31 g of K3PO4 (62.7 mmol) were suspended in 100 mL of DME,
and the mixture was stirred under nitrogen at 105 °C until no further
conversion was observed. 500 mL of water was added, then the
mixture was extracted with EtOAc (3 × 250 mL) and with brine. The
organic layer was dried over MgSO4, then volatiles were removed in
vacuum to give 10.1 g of tert-butyl N-[6-(tert-butoxycarbonylamino)-
4-[3-(2-hydroxyethyl)-2-methyl-imidazo[4,5-b]pyridin-5-yl]-2-
pyridyl]carbamate as the crude product, which was used in the next
1H NMR (500 MHz, DMSO-d6): δ ppm 9.75 (s, 1H), 8.16 (d, J =
8.4 Hz, 1H), 7.66 (d, J = 8.5 Hz, 1H), 2.12 (s, 3H); HPLC-MS: (M −
H)−: 247.0; 249.0.
1
steps without purification. H NMR (400 MHz, DMSO-d6): δ ppm
9.34 (s, 2H), 8.03 (m, 3H), 7.68 (d, J = 8.3 Hz, 1H), 4.98 (t, J = 5.4
Hz, 1H), 4.33 (t, J = 5.4 Hz, 2H), 3.81 (q, J = 5.4 Hz, 2H), 2.63 (s,
3H), 1.49 (s, 18H); HPLC-MS: (M + H)+: 485.2.
Step B: tert-Butyl N-[6-(tert-Butoxycarbonylamino)-4-[6-chloro-
5-(acetylamino)-2-pyridyl]-2-pyridyl]carbamate. To 13.1 g of N-(6-
bromo-2-chloro-3-pyridyl)acetamide (52.5 mmol), 24.0 g of R1 (55.1
mmol) and 33.4 g of K3PO4 (157.3 mmol) were dissolved in 250 mL
of 1,2-dimethoxyethane-water 4:1, then 0.304 g of tetrakis-
(triphenylphosphine)palladium(0) (0.26 mmol) was added, and the
mixture was heated under nitrogen at 90 °C until no further
conversion was observed. Then, the mixture was diluted with 250 mL
of water and extracted with EtOAc (3 × 400 mL). The organic layer
was washed with brine (1 × 800 mL), dried over MgSO4, the volatiles
were removed under reduced pressure, and the residue was
recrystallized from EtOAc to obtain 18.2 g (73%) of tert-butyl N-
[6-(tert-butoxycarbonylamino)-4-[6-chloro-5-(acetylamino)-2-pyrid-
yl]-2-pyridyl]carbamate.
tert-Butyl N-[6-(tert-Butoxycarbonylamino)-4-[3-(2-hydroxy-
propyl)-2-methyl-imidazo[4,5-b]pyridin-5-yl]-2-pyridyl]carbamate
(R4). Step A: 1-(5-Bromo-2-methyl-imidazo[4,5-b]pyridin-3-yl)-
propan-2-ol. The mixture of 12.0 g of N-(6-bromo-2-fluoro-3-
pyridyl)acetamide (51.5 mmol), 9.15 g of 1-aminopropan-2-ol (113.3
mmol), and 14 mL of triethylamine was stirred at 60 °C until no
further conversion was observed. The solvent and excess amine were
removed under reduced pressure.
The remained solid was dissolved in 300 mL of acetic acid and the
solution was heated at 130 °C until no further conversion was
observed. The solvent was removed under reduced pressure. The
residue was taken up the mixture of 200 mL of methanol and 40 mL
of water. 9.6 g of LiOH*H2O (230 mmol) was added and the mixture
was stirred at ambient temperature until no further conversion was
observed, then it was poured into water. The precipitate was filtered
off, washed with water, and dried to give 10.7 g of (77%) of 1-(5-
bromo-2-methyl-imidazo[4,5-b]pyridin-3-yl)propan-2-ol.
1H NMR (500 MHz, DMSO-d6): δ ppm 9.80 (s, 1H), 9.51 (s, 2H),
8.40 (d, 1H), 8.00 (s, 2H), 7.92 (d, J = 8.4 Hz, 1H), 2.18 (s, 3H),
1.49 (s, 18H). 13C NMR (125 MHz, DMSO-d6): δ ppm 169.7, 153.2,
152.1, 150.1, 147.8, 142.6, 134.7, 132.9, 120.9, 104.4, 80.2, 28.5, 24.1.
tert-Butyl N-[6-(tert-Butoxycarbonylamino)-4-[3-(2-hydroxyeth-
yl)-2-methyl-imidazo[4,5-b]pyridin-5-yl]-2-pyridyl]carbamate (R3).
Step A: N-(6-Bromo-2-fluoro-3-pyridyl)acetamide. To 17.0 g of 6-
bromo-2-fluoro-pyridin-3-amine (89.0 mmol) was dissolved in 100
mL of acetic acid, then 8.82 mL of acetic anhydride (93.5 mmol) was
added, and the reaction mixture was stirred at room temperature until
no further conversion was detected. The reaction mixture was
concentrated under reduced pressure, the remaining solid was
dissolved in DCM. The DCM solution was extracted with 10%
potassium carbonate solution then with brine. The organic layer was
dried over MgSO4 then the solvent was removed under reduced
pressure to give 18.2 g (88%) of N-(6-bromo-2-fluoro-3-pyridyl)-
acetamide.
1H NMR (400 MHz, DMSO-d6): δ ppm 7.87 (d, J = 8.1 Hz, 1H),
7.37 (d, J = 8.1 Hz, 1H), 4.97 (d, J = 4.3 Hz, 1H), 4.19−3.94 (m,
3H), 2.57 (s, 3H), 1.21 (d, J = 5.9 Hz, 3H); HPLC-MS: (M + H)+:
270.0.
Step B: tert-Butyl N-[6-(tert-Butoxycarbonylamino)-4-[3-(2-hy-
droxypropyl)-2-methyl-imidazo[4,5-b]pyridin-5-yl]-2-pyridyl]-
carbamate. To 5.38 g of 1-(5-bromo-2-methyl-imidazo[4,5-b]-
pyridin-3-yl)propan-2-ol (20 mmol), 8.70 g of R1 (20 mmol),
0.449 g of palladium acetate (2.0 mmol), 1.43 g of triphenylphosphine
(4.0 mmol), and 12.70 g of K3PO4 the (60 mmol) were suspended in
133 mL of DME, and the mixture was stirred under argon at 110 °C
until no further conversion was observed. The volatiles were removed
under reduced pressure, the remained solid was dissolved in 700 mL
of EtOAc which was extracted with 3 × 300 mL of water. The organic
phase was washed with brine then it was dried over MgSO4. The
evaporation of EtOAc under reduced pressure gave the crude product,
which was purified by flash chromatography using DCM and
methanolic ammonia as eluents. 4.9 g (49%) of tert-butyl N-[6-
(tert-butoxycarbonylamino)-4-[3-(2-hydroxypropyl)-2-methyl-
1H NMR (400 MHz, DMSO-d6): δ ppm 10.04 (s, 1H), 8.35−8.52
(m, 1H), 7.58 (d, J = 8.25 Hz, 1H), 2.11 (s, 3H); 13C NMR (100
MHz, DMSO-d6): δ ppm 169.9, 152.5, 135.7, 129.4, 126.5, 121.9,
24.0; HRMS (TOF, ESI) m/z: calcd for C7H6N2OFBr [M + H]+:
232.9720; found, 232.9729.
Step B: 2-(5-Bromo-2-methyl-imidazo[4,5-b]pyridin-3-yl)-
ethanol. The mixture of 6.00 g of N-(6-bromo-2-fluoro-3-pyridyl)-
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n
imidazo[4,5-b]pyridin-5-yl]-2-pyridyl]carbamate was obtained as a
white solid.
palladium acetate and 10 mol % of BuPAd2 were added, and the
mixture was heated at 100 °C under nitrogen in a microwave reactor
until no further conversion was observed. Celite was added to the
reaction mixture and the volatiles were evaporated under reduced
pressure. The solid residue was purified by flash chromatography on
silica gel using DCM and methanolic ammonia as eluents.
1H NMR (500 MHz, DMSO-d6): δ ppm 9.41 (s, 2H), 8.10 (s, 2H),
8.03 (d, J = 8.3 Hz, 1H), 7.70 (d, J = 8.3 Hz, 1H), 5.03 (d, J = 4.9 Hz,
1H), 4.28 (dd, J = 13.7, 3.5 Hz, 1H), 4.18 (m, 1H), 4.10 (dd, J = 13.6,
7.8 Hz, 1H), 2.64 (s, 3H), 1.50 (s, 18H), 1.19 (d, J = 6.2 Hz, 3H);
HRMS (TOF, ESI) m/z: calcd for C25H35N6O5 [M + H]+: 499.2591;
found, 499.2646.
Examples in the Paper. 4-(4-Methoxyphenyl)pyrimidin-2,6-
diamine (1). Prepared following published procedures.20
4-(4-Methoxyphenyl)pyridin-2-amine (3). To 0.173 g of 4-
bromopyridine-2-amine (1.0 mmol), 0.304 g of (4-methoxyphenyl)-
boronic acid (2.0 mmol). and 0.420 g of K2CO3 (3.0 mmol) in 15 mL
of 1,2-dimethoxyethane and 3.75 mL of water was added 0.116 g of
Pd(PPh3)4 (0.1 mmol) and heated at 100 °C under nitrogen in a
microwave reactor until no further conversion was observed. The
crude product was purified by flash chromatography on silica gel using
heptane and EtOAc as eluents, to give 0.121 g (60%) of 3.
1H NMR (500 MHz, DMSO-d6): δ ppm 7.92 (d, J = 5.5 Hz, 1H),
7.59 (m, 2H), 7.03 (m, 2H), 6.76 (dd, J = 5.5, 1.6 Hz, 1H), 6.67 (d, J
= 1.0 Hz, 1H), 5.97 (s, 2H), 3.8 (s, 3H); 13C NMR (125 MHz,
DMSO-d6): δ ppm 160.7, 160.3, 148.4, 148.3, 130.9, 128.1, 114.9,
110.2, 104.8, 55.7; HRMS (TOF, ESI) m/z: calcd for C12H13N2O [M
+ H]+: 201.1022; found, 201.1022.
General Procedure ISuzuki Coupling and Deprotection.
Step A. 1 equiv of the appropriate halide derivative, 1−1.2 equiv of R1
and 3 equiv of K2CO3 were dissolved in 1,2-dimethoxyethane-water
7:1 (8 mL/mmol), then 0.05 equiv of palladium acetate and 0.1 equiv
of nBuPAd2 were added, and the mixture was heated at 100 °C under
nitrogen in a microwave reactor until no further conversion was
observed. Celite was added to the reaction mixture and the volatiles
were evaporated under reduced pressure. The solid residue was
purified by flash chromatography on silica gel using DCM and
methanolic ammonia as eluents.
Step B. The product obtained in step A was stirred in a mixture of
DCM (5 mL/mmol) and TFA (5 mL/mmol) until no further
conversion was observed. The volatiles were evaporated under
reduced pressure, the solid residue was dissolved in the ammonia
solution (7 N in methanol, 20 mL/mmol), and the volatiles were
evaporated under reduced pressure again. The crude product was
purified by preparative reversed phase chromatography using 5 mM
aqueous NH4HCO3 solution and MeCN as eluents.
General Procedure IIAmidine Formation, Ring Closure,
and Deprotection. Step A. 1.0 equiv of the appropriate amide and
5.0 equiv of 2,6-lutidine were dissolved in dry DCM (10 mL/mmol of
the amide). The DCM solution was cooled to 0 °C under nitrogen
and DCM solution of 5.0 equiv of nonafluorobutanesulfonic
anhydride (1.5 M) was added dropwise. The reaction mixture was
allowed to warm up to room temperature over 1 h then 5 equiv of the
appropriate amine was added in one portion, and the mixture was
stirred until no further conversion was observed. The DCM mixture
was washed with water, dried over Na2SO4, concentrated under
reduced pressure, and purified by flash chromatography using DCM
and methanolic ammonia as eluents to give the amidine intermediate.
Step B. To 1 equiv of the amidine intermediate from step A was
dissolved in DME (6.5 mL/mmol). 0.2 equiv of Pd(OAc)2, 0.4 equiv
of PBuAd2, and 2 equiv of K3PO4 were added, and the reaction
mixture was stirred under nitrogen at 115 °C in a microwave reactor
until no further conversion was observed. The reaction mixture was
concentrated under reduced pressure and purified by flash
chromatography using DCM and methanolic ammonia as eluents to
yield the appropriate Boc-protected example.
4-(4-Methoxyphenyl)-2-methyl-pyridine (4). To 0.172 g of 4-
bromo-2-methyl-pyridine (1.0 mmol), 0.304 g of (4-methoxyphenyl)-
boronic acid (2.0 mmol), and 0.420 g of K2CO3 (3.0 mmol) in 20 mL
of 1,2-dimethoxyethane and 5 mL of water 0.230 g of Pd(PPh3)4 (0.2
mmol) was added, and the mixture was heated at 100 °C under
nitrogen in a microwave reactor until no further conversion was
observed. The crude product was purified by flash chromatography on
silica gel using heptane and EtOAc as eluents, to give 0.150 g (75%)
of 4.
1H NMR (500 MHz, DMSO-d6): δ ppm 8.43 (d, J = 5.4 Hz, 1H),
7.74 (d, J = 9.0 Hz, 2H), 7.52 (s, 1H), 7.44 (d, J = 5.3 Hz, 1H), 7.05
(d, J = 8.6 Hz, 2H), 3.81 (s, 3H), 2.50 (s, 3H); 13C NMR (125 MHz,
DMSO-d6): δ ppm 160.6, 158.9, 149.9, 147.2, 129.9, 128.5, 120.2,
118.2, 115.0, 55.7, 24.6; HRMS (TOF, ESI) m/z: calcd for
C13H14NO [M + H]+ 200.1070; found, 200.1069.
4-(4-Chloro-3-methoxy-phenyl)pyridin-2-amine (5). To 0.173 g
of 4-bromopyridine-2-amine (1.0 mmol), 0.224 g of (4-chloro-3-
methoxy-phenyl)boronic acid (1.2 mmol), and 0.420 g of K2CO3 (3.0
mmol) in 15 mL of 1,2-dimethoxyethane and 3.75 mL of water 0.116
g of Pd(PPh3)4 (0.1 mmol) was added, and the mixture was heated at
100 °C under nitrogen in a microwave reactor until no further
conversion was observed. The crude product was purified by
preparative HPLC (C18) using H2O (0.1% TFA) and MeCN as
eluents, to give 0.104 g (44%) of 5.
1H NMR (500 MHz, DMSO-d6): δ ppm 7.98 (d, J = 5.5 Hz, 1H),
7.52 (d, J = 8.0 Hz, 1H), 7.33 (d, J = 1.9 Hz, 1H), 7.21 (dd, J = 8.2,
2.1 Hz, 1H), 6.83 (dd, J = 5.4, 1.6 Hz, 1H), 6.72 (d, J = 0.9 Hz, 1H),
6.05 (br, 2H), 3.94 (s, 3H); 13C NMR (125 MHz, DMSO-d6): δ ppm
160.7, 155.2, 148.7, 147.9, 139.3, 130.8, 122.0, 119.9, 111.2, 110.6,
105.7, 56.6; HRMS (TOF, ESI) m/z: calcd for C12H12N2OCl [M +
H]+ 235.0633; found, 235.0614.
Step C. Starting from the product of step B, the final product was
obtained following the deprotection protocol described in General
Procedure I Step B.
General Procedure IIINucleophilic Substitution and
Deprotection. Step A. To the solution of 1 equiv of R3 or R4 in
dry DMF (4.0 mL/mmol) under nitrogen 3 equiv of sodium hydride
was added, and the resulting mixture was stirred at 0 °C for 15 min.
Following the addition of 2 equiv of the appropriate aryl halide, the
mixture was stirred at 50 °C for 5 h. If the formation of the expected
product was not observed by HPLC-MS at this point, the reaction
temperature was raised to 120 °C and stirring continued until no
further conversion was observed. After cooling water was added to the
reaction mixture and the aqueous phase was extracted with EtOAc.
The combined organic phases were washed with brine, dried over
MgSO4, and solvent was removed under reduced pressure. The crude
product was purified by flash chromatography using DCM and
MeOH as eluents to give the Boc-protected example.
Step B. Starting from the product of step A, the final product was
obtained following the deprotection protocol described in General
Procedure I Step B.
General Procedure IVSuzuki Coupling. 1 equiv of 4-
bromopyridine-2,6-diamine, 1−1.5 equiv of the appropriate boronic
acid or ester, and 3 equiv of K2CO3 were dissolved in 1,2-
dimethoxyethane−water 4:1 (5 mL/mmol). Then, 5 mol % of
4-(4-Chlorophenyl)-2-methyl-pyridine (6). To 0.250 g of 4-
bromo-2-methyl-pyridine (1.45 mmol), 0.272 g of (4-chlorophenyl)-
boronic acid (1.74 mmol), and 0.600 g of K2CO3 (4.35 mmol) in 10
mL of 1,2-dimethoxyethane and 2.5 mL of water 0.116 g of
Pd(PPh3)4 (0.10 mmol) was added, and the mixture was heated at
100 °C under nitrogen in a microwave reactor until no further
conversion was observed. The crude product was purified by flash
chromatography on silica gel using heptane and EtOAc as eluents, to
give 0.157 g (53%) of 6.
1H NMR (400 MHz, DMSO-d6): δ ppm 8.50 (d, J = 5.2 Hz, 1H),
7.82 (d, J = 8.7 Hz, 2H), 7.59 (d, J = 1.2 Hz, 1H), 7.57 (d, J = 8.7 Hz,
2H), 7.50 (dd, J = 5.1, 1.9 Hz, 1H), 2.53 (s, 3H); 13C NMR (100
MHz, DMSO-d6): δ ppm 159.2, 150.1, 146.4, 136.7, 134.5, 129.6,
129.1, 120.8, 118.7, 24.6; HRMS (TOF, ESI) m/z: calcd for
C12H11NCl [M + H]+ 204.0575; found, 204.0585.
6-(2,3-Dihydro-benzofuran-5-yl)-pyrimidine-2,4-diamine (7). To
0.145 g of 6-chloropyrimidine-2,4-diamine (1.0 mmol), 0.270 g of
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benzofuran-5-ylboronic acid (1.1 mmol), and 0.414 g of K2CO3 (3.0
mmol) in 5 mL of 1,2-dimethoxyethane and 1 mL of water 0.072 g of
nBuPAd2 (0.2 mmol) and 0.022 g of Pd(OAc)2 (0.1 mmol) was
ylboronic acid (2.0 mmol), 0.115 g of tetrakis(triphenylphosphine)-
palladium(0) (0.1 mmol), and 0.414 g of K2CO3 (3.0 mmol) were
suspended in the mixture of 7.5 mL of DME and 1.8 mL of water, and
the mixture was stirred under nitrogen at 100 °C in a microwave
reactor until no further conversion was observed. Solvents were
removed under reduced pressure, and the crude product was purified
by flash chromatography using DCM and methanolic ammonia as
eluents to give 0.100 g (44%) of 12 was obtained.
added, and the mixture was heated at 130 °C under nitrogen in a
microwave reactor until no further conversion was observed. The
crude product was purified by preparative HPLC (C18) using H2O
(0.1% TFA) and MeCN as eluents, to give 0.075 g (33%) of 7.
1H NMR (500 MHz, DMSO-d6): δ ppm 7.79 (d, J = 1.1 Hz, 1H),
7.66 (dd, J = 8.3, 1.7 Hz, 1H), 6.79 (d, J = 8.4 Hz, 1H), 6.23 (s, 2H),
6.11 (s, 1H), 5.86 (s, 2H), 4.57 (t, J = 8.7 Hz, 2H), 3.21 (t, J = 8.7 Hz,
2H) ppm; 13C NMR (125 MHz, DMSO-d6): δ ppm 165.6, 164.0,
162.6, 161.4, 131.2, 128.0, 126.8, 123.6, 109.0, 89.8, 71.8, 29.3;
HRMS (TOF, ESI) m/z: calcd for C12H13N4O [M + H]+ 229.1014;
found, 229.1087.
1H NMR (500 MHz, DMSO-d6): δ ppm 8.79 (s, 1H), 7.87 (d, J =
1.7 Hz, 1H), 7.82 (d, J = 8.5 Hz, 1H), 7.58 (dd, J = 8.5, 1.7 Hz, 1H),
5.94 (s, 2H), 5.49 (s, 4H); 13C NMR (125 MHz, DMSO-d6): δ ppm
159.9, 155.4, 150.0, 149.8, 140.7, 137.5, 124.7, 117.9, 111.8, 94.2;
HRMS (TOF, ESI) m/z: calcd for C12H10N4O [M + H]+ 227.0927;
found, 227.0915.
6-Benzofuran-5-yl-pyrimidine-2,4-diamine Trifluoroacetic Acid
(1:1) Salt (8). To 0.145 g of 6-chloropyrimidine-2,4-diamine (1.0
mmol), 0.244 g of 2,3-dihydrobenzofuran-5-ylboronic acid (1.0
mmol), and 0.415 g of K2CO3 (3.0 mmol) in 15 mL of 1,2-
dimethoxyethane and 3.75 mL of water 0.116 g of Pd(PPh3)4 (0.1
mmol) was added, and the mixture was heated at 100 °C under
nitrogen in a microwave reactor until no further conversion was
observed. The crude product was purified by preparative HPLC
(C18) using H2O (0.1% TFA) and MeCN as eluents, to give 0.128 g
(57%) of 8.
4-Furo[3,2-b]pyridin-5-yl-pyridine-2,6-diamine (13). Starting
from 0.522 g of R1 (1.2 mmol) and 0.154 g of 5-chloro-furo[3,2-
b]pyridine (1.0 mmol) following general procedure I, 0.096 g (42%)
of 13 was obtained.
1H NMR (500 MHz, DMSO-d6): δ ppm 8.35 (d, J = 2.3 Hz, 1H),
8.09 (dd, J = 8.7, 1.0 Hz, 1H), 7.67 (d, J = 8.7 Hz, 1H), 7.17 (dd, J =
2.3, 1.0 Hz, 1H), 6.35 (s, 2H), 5.51 (s, 4H); 13C NMR (125 MHz,
DMSO-d6): δ ppm 159.9, 153.0, 151.5, 148.9, 147.5, 147.3, 119.6,
116.8, 108.4, 93.8; HRMS (TOF, ESI) m/z: calcd for C12H11N4O [M
+ H]+ 227.0850; found, 227.0923.
1H NMR (500 MHz, DMSO-d6): δ ppm 12.24 (br s, 1H), 8.17 (br
s, 2H), 8.17 (d, J = 2.2 Hz, 1H), 8.08 (d, J = 1.8 Hz, 1H), 7.85 (dm,
1H), 7.67 (dd, J = 8.7, 1.8 Hz, 1H), 7.51 (vbr s, 2H), 7.13 (dd, J =
2.2, 0.9 Hz, 1H), 6.34 (s, 1H); 13C NMR (125 MHz, DMSO-d6): δ
ppm 165.6, 156.7, 156.3, 148.5, 128.4, 126.5, 123.7, 120.8, 112.8,
107.6, 94.6; HRMS (IT-TOF, ESI) m/z: calcd for C12H11N4O [M +
H]+ 227.0927; found, 227.0917.
4-(2,3-Dihydro-benzofuran-5-yl)-pyridine-2,6-diamine (9). Start-
ing from 0.100 g of 4-bromopyridine-2,6-diamine (0.53 mmol) and
0.196 g 2-(2,3-dihydrobenzofuran-5-yl)-4,4,5,5-tetramethyl-1,3,2-di-
oxaborolane (0.79 mmol) as the appropriate boronic ester, following
General procedure IV, 0.100 g (83%) of 9 was obtained.
4-Benzooxazol-6-yl-pyridine-2,6-diamine (14). Starting from
0.188 g of 4-bromopyridine-2,6-diamine (1.0 mmol) and 0.245 g of
6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3-benzoxazole (1.0
mmol) following general procedure IV, 0.078 g (34%) of 14 was
obtained.
1H NMR (500 MHz, DMSO-d6): δ ppm 8.79 (s, 1H), 7.87 (d, J =
1.7 Hz, 1H), 7.85 (d, J = 8.3 Hz, 1H), 7.55 (dd, J = 8.3, 1.6 Hz, 1H),
5.95 (s, 2H), 5.52 (s, 4H); 13C NMR (125 MHz, DMSO-d6): δ ppm
159.9, 155.3, 150.4, 149.9, 140.1, 138.4, 123.7, 120.6, 109.2, 94.2;
HRMS (IT-TOF, ESI) m/z: calcd for C12H11N4O [M + H]+
227.0927; found, 227.0921.
4-Benzothiazol-6-yl-pyridine-2,6-diamine (15). Starting from
0.435 g of R1 (1.0 mmol) and 0.215 g of 6-bromo-1,3-benzothiazole
(1.0 mmol) following general procedure I, 0.067 g (28%) of 15 was
obtained.
1H NMR (500 MHz, DMSO-d6): δ ppm 7.38 (d, J = 1.4 Hz, 1H),
7.25 (dd, J = 8.2, 2.0 Hz, 1H), 6.80 (d, J = 8.2 Hz, 1H), 5.84 (s, 2H),
5.39 (s, 4H), 4.55 (t, J = 8.7 Hz, 2H), 3.21 (t, J = 8.7 Hz, 2H); 13C
NMR (125 MHz, DMSO-d6): δ ppm 160.4, 159.7, 150.3, 132.5,
128.4, 126.4, 123.4, 109.4, 93.4, 71.7, 29.4; HRMS (IT-TOF, ESI) m/
z: calcd for C13H14N3O [M + H]+ 228.1131; found, 228.1122.
4-(Benzofuran-5-yl)pyridine-2,6-diamine (10). 0.188 g of 4-
bromopyridine-2,6-diamine (1 mmol), 162 mg of benzofuran-5-
ylboronic acid (1.0 mmol), 0.115 g of tetrakis(triphenylphosphine)-
palladium(0) (0.1 mmol), and 0.350 g of K2CO3 (2.5 mmol) were
suspended in the mixture 7.5 mL of DME and 1.8 mL of water, the
mixture was stirred under nitrogen at 100 °C in a microwave reactor
until no further conversion was observed. The crude product was
purified by flash column chromatography using DCM and methanolic
ammonia as eluents to give 0.110 g (49%) of 10.
1H NMR (500 MHz, DMSO-d6): δ ppm 9.41 (s, 1H), 8.32 (d, J =
1.6 Hz, 1H), 8.13 (d, J = 8.5 Hz, 1H), 7.67 (dd, J = 8.5, 1.8 Hz, 1H),
5.97 (s, 2H), 5.52 (br s, 4H); 13C NMR (125 MHz, DMSO-d6): δ
ppm 159.9, 157.3, 153.4, 149.8, 137.8, 134.8, 125.3, 123.6, 120.5,
94.1; HRMS (TOF, ESI) m/z: calcd for C12H11N4S [M + H]+
243.0626; found, 243.0694.
4-(1-Methylbenzimidazol-5-yl)pyridine-2,6-diamine (16). 0.188 g
of 4-bromopyridine-2,6-diamine (1.0 mmol), 0.352 g of (3-
methylbenzimidazol-5-yl)boronic acid (2.0 mmol), 0.072 g of
CataCXium (0.2 mmol), 0.0224 g of palladium (II) acetate (0.1
mmol), and 0.414 g of K2CO3 (3.0 mmol) were suspended in the
mixture of 4 mL of DME and 2 mL of water, and the mixture was
stirred under nitrogen at 100 °C in a microwave reactor until no
further conversion was observed. The crude product was purified by
flash chromatography using DCM and methanolic ammonia as
eluents to give 0.078 g (33%) of 16.
1H NMR (500 MHz, DMSO-d6): δ ppm 8.03 (d, J = 2.3 Hz, 1H),
7.78 (dm, J = 1.9 Hz, 1H), 7.64 (dm, J = 8.6 Hz, 1H), 7.45 (dd, J =
8.6, 1.9 Hz, 1H), 7.01 (dd, J = 2.3, 0.9 Hz, 1H), 5.92 (s, 2H), 5.46 (s,
4H); 13C NMR (125 MHz, DMSO-d6): δ ppm 159.8, 154.7, 150.6,
147.1, 135.5, 128.1, 119.3, 111.9, 107.5, 94.1; HRMS (IT-TOF, ESI)
m/z: calcd for C13H12N3O [M + H]+ 226.0975; found, 226.0956.
4-Benzo[b]thiophen-5-yl-pyridine-2,6-diamine (11). Starting
from 0.100 g of 4-bromopyridine-2,6-diamine (0.53 mmol) and 277
mg of 2-(benzothiophen-5-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane
(1.07 mmol) as the appropriate boronic ester following general
procedure IV, 0.105 g (80%) of 11 was obtained.
1H NMR (500 MHz, DMSO-d6): δ ppm 8.21 (s, 1H), 7.70 (d, J =
1.5 Hz, 1H), 7.67 (d, J = 8.5 Hz, 1H), 7.38 (dd, J = 8.5 Hz, 1.5 Hz,
1H), 5.99 (s, 2H), 5.47 (s, 4H), 3.87 (s, 3H); 13C NMR (125 MHz,
DMSO-d6): δ ppm 159.7, 151.1, 145.9, 143.9, 135.4, 134.8, 120.7,
119.8, 108.3, 94.2, 31.2; HRMS (TOF, ESI) m/z: calcd for C13H13N5
[M + H]+ 240.1244; found, 240.1233.
1H NMR (500 MHz, DMSO-d6): δ ppm 8.05 (d, J = 8.4 Hz, 1H),
8.03 (d, J = 1.4 Hz, 1H), 7.80 (d, J = 5.4 Hz, 1H), 7.53 (d, J = 5.7 Hz,
1H), 7.51 (dd, J = 8.4, 1.6 Hz, 1H), 5.98 (s, 2H), 5.53 (s, 4H); 13C
NMR (125 MHz, DMSO-d6): δ ppm 159.7, 150.5, 140.5, 139.4,
136.6, 128.7, 124.8, 123.4, 123.2, 121.5, 94.0; HRMS (IT-TOF, ESI)
m/z: calcd for C13H12N3S [M + H]+ 242.0746; found, 242.0743.
4-(1,3-Benzoxazol-5-yl)pyridine-2,6-diamine (12). 0.188 g of 4-
bromopyridine-2,6-diamine (1.0 mmol), 0.490 g of 1,3-benzoxazol-5-
4-(3-Methyl-3H-imidazo[4,5-b]pyridin-5-yl)-pyridine-2,6-dia-
mine (17). Starting from 0.289 g R1 (0.9 mmol) and 0.159 g of 3-
methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)imidazo[4,5-b]-
pyridine (0.75 mmol) following general procedure I, 0.100 g (55%)
17 was obtained.
1H NMR (500 MHz, DMSO-d6): δ ppm 8.45 (s, 1H), 8.11 (d, J =
8.3 Hz, 1H), 7.62 (d, J = 8.3 Hz, 1H), 6.37 (s, 2H), 5.51 (s, 4H), 3.87
(s, 3H); 13C NMR (125 MHz, DMSO-d6): δ ppm 159.9, 150.9, 149.0,
6757
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147.6, 147.1, 135.1, 128.1, 115.4, 93.8, 29.9; HRMS (TOF, ESI) m/z:
calcd for C12H13N6 [M + H]+ 241.1122; found, 241.1195.
4-(Imidazo[1,2-a]pyridin-6-yl)-pyridine-2,6-diamine (18). Start-
ing from 0.435 g of R1 (1.0 mmol) and 0.197 g of 6-
bromoimidazo[1,2-a]pyridine (1.0 mmol) following general proce-
dure I, 0.028 g (12%) of 18 was obtained.
8.8 Hz, 1H), 7.91 (dd, J = 8.8, 2.2 Hz, 1H), 7.56 (dd, J = 8.4, 4.2 Hz,
1H), 6.05 (s, 2H), 5.55 (br s, 4H); 13C NMR (125 MHz DMSO-d6):
δ ppm 159.9, 151.2, 149.5, 148.0, 138.1, 136.8, 129.8, 128.4, 125.6,
125.5, 122.3, 94.1; HRMS (IT-TOF, ESI) m/z: calcd for C14H13N4
[M + H]+ 237.1135; found, 237.1143.
4-(Benzofuran-5-yl)pyridin-2-amine (25). Starting from 0.173 g of
4-bromopyridin-2-amine (1.0 mmol) and 0.193 g of benzofuran-5-
ylboronic acid (1.2 mmol) following general procedure IV, 0.091 g
(43%) of 25 was obtained.
1H NMR (400 MHz, DMSO-d6): δ ppm 8.85 (br s, 1H), 8.02 (br
s, 1H), 7.64 (d, J = 9.4 Hz, 1H), 7.62 (d, J = 1.2 Hz, 1H), 7.39 (dd, J
= 9.4, 1.8 Hz, 1H), 6.00 (br s, 4H), 5.98 (s, 2H); 13C NMR (100
MHz, DMSO-d6): δ ppm 157.6, 144.4, 134.1, 125.0, 124.3, 124.1,
117.3, 114.4, 93.3; HRMS (TOF, ESI) m/z: calcd for C12H12N5 [M +
H]+ 226.1021; found, 226.1094.
1H NMR (500 MHz, DMSO-d6): δ ppm 8.06 (d, J = 2.1 Hz, 1H),
7.96 (d, J = 5.3 Hz, 1H), 7.92 (d, J = 1.5 Hz, 1H), 7.69 (d, J = 8.7 Hz,
1H), 7.57 (dd, J = 8.6, 1.9 Hz, 1H), 7.03 (d, J = 2.0 Hz, 1H), 6.82
(dd, J = 5.4, 1.6 Hz, 1H), 6.74 (d, J = 0.9 Hz, 1H), 6.01 (br s, 2H);
13C NMR (125 MHz, DMSO-d6): δ ppm 160.7, 155.0, 149.2, 148.6,
4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)pyridine-2,6-diamine (19).
Starting from 0.130 g of R1 (0.30 mmol) and 0.060 g of 6-bromo
[1,2,4]triazolo [1,5-a] pyridine (0.30 mmol) following general
procedure I, 0.031 g (46%) of 19 was obtained.
147.4, 134.1, 128.4, 123.6, 119.8, 112.2, 110.9, 107.5, 105.8; HRMS
(IT-TOF, ESI) m/z: calcd for C13H11N2O [M + H]+ 211.0866;
found, 211.0864.
1H NMR (500 MHz, DMSO-d6): δ ppm 9.07 (dd, J = 2.24, 0.9 Hz,
1H), 8.54 (s, 1H), 7.91 (dm, 1H), 7.80 (dd, J = 9.1, 1.8 Hz, 1H), 5.96
(s, 2H), 5.57 (s, 4H); 13C NMR (125 MHz, DMSO-d6): δ ppm
160.0, 154.9, 150.0, 145.7, 129.9, 127.6, 126.4, 116.6, 93.7; HRMS
(TOF, ESI) m/z: calcd for C11H11N6 [M + H]+ 226.0967; found,
227.1033.
4-(Benzofuran-5-yl)pyridine (26). 0.400 g of 5-bromobenzofuran
(2.0 mmol), 0.458 g of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)pyridine (2.0 mmol), 0.072 g of CataCXium (0.2 mmol), 0.0224 g
of palladium (II) acetate (0.05 mmol), and 0.832 g of K2CO3 (6.0
mmol) were suspended in the mixture of 5 mL of DME and 2.5 mL of
water, and the mixture was stirred under nitrogen at 90 °C until no
further conversion was observed. The crude product was purified by
flash column chromatography using DCM and methanolic ammonia
as eluents to give 0.036 g (18%) of 4-(benzofuran-5-yl)pyridine 26.
1H NMR (500 MHz, DMSO-d6): δ ppm 8.63 (m, 2H), 8.11 (t, J =
1.3 Hz, 1H), 8.09 (d, J = 2.2 Hz, 1H), 7.75 (m, 4H), 7.05 (d, J = 2.2
Hz, 1H); 13C NMR (125 MHz, DMSO-d6): δ ppm 155.3, 150.6,
147.9, 147.6, 132.8, 128.6, 123.9, 122.0, 120.4, 112.5, 107.6; HRMS
(IT-TOF, ESI) m/z: calcd for C13H9NO [M + H]+ 196.0757; found,
196.0760.
4-(Pyrazolo[1,5-a]pyrimidin-5-yl)-pyridine-2,6-diamine (20).
Starting from 0.435 g of R1 (1.0 mmol) and 0.198 g of 5-
bromopyrazolo[1,5-a]pyrimidine (1.0 mmol) following general
procedure I, 0.044 g (19%) of 20 was obtained.
1H NMR (500 MHz, DMSO-d6): δ ppm 9.14 (dd, J = 7.4, 0.5 Hz,
1H), 8.24 (d, J = 2.3 Hz, 1H), 7.29 (d, J = 7.5 Hz, 1H), 6.74 (dd, J =
2.4, 0.6 Hz, 1H), 6.38 (s, 2H), 5.64 (br s, 4H); 13C NMR (125 MHz,
DMSO-d6): δ ppm 160.2, 155.4, 148.1, 146.4, 145.9, 136.6, 105.8,
96.9, 93.5; HRMS (TOF, ESI) m/z: calcd for C11H11N6 [M + H]+
226.0967; found, 227.1033.
4-(3-Methyl-benzo[c]isoxazol-5-yl)-pyridine-2,6-diamine (21).
Starting from 0.113 g of R1 (0.26 mmol) and 0.050 g of 5-bromo-
3-methyl-3aH-2,1-benzoxazole following general procedure I, 0.010 g
(16%) of 21 was obtained.
4-(Benzofuran-5-yl)-6-methyl-pyridin-2-amine (27). Step A: 4-
(Benzofuran-5-yl)-6-methyl-pyridin-2-ol. 1.40 g of 4-bromo-6-
methyl-pyridin-2-ol (7.45 mmol), 2.07 g of 2-(benzofuran-5-yl)-
4,4,5,5-tetramethyl-1,3,2-dioxaborolane (8.50 mmol), 0.501 g of
CataCXium (1.40 mmol), 0.157 g of palladium (II) acetate (0.70
mmol), and 3.08 g of K2CO3 (22.0 mmol) were suspended in the
mixture of 30 mL of DME and 15 mL of water. The mixture was
stirred under nitrogen at 100 °C in a microwave reactor until no
further conversion was observed. The precipitate was filtered off,
washed with water, and dried in vacuum to give 1.67 g of (quant.) of
4-(benzofuran-5-yl)-6-methyl-pyridin-2-ol.
1H NMR (500 MHz, DMSO-d6): δ ppm 7.85 (br s, 1H), 7.60 (d, J
= 9.4 Hz, 1H), 7.51 (dd, J = 9.3, 1.5 Hz, 1H), 5.93 (s, 2H), 5.49 (br s,
4H), 2.86 (s, 3H); 13C NMR (125 MHz, DMSO-d6): δ ppm 168.1,
159.9, 156.6, 149.4, 134.6, 131.7, 117.9, 116.0, 115.1, 93.6, 12.2;
HRMS (TOF, ESI) m/z: calcd for C13H13N4O [M + H]+ 241.1011;
found, 241.1080.
4-(2,3-Dihydro-1,4-benzodioxin-6-yl)pyridine-2,6-diamine (22).
Starting from 0.188 g of 4-bromopyridine-2,6-diamine (1.0 mmol)
and 0.216 g of 2,3-dihydro-1,4-benzodioxin-6-ylboronic acid (1.2
mmol) following general procedure IV, 0.165 g (68%) of 22 was
obtained.
1H NMR (500 MHz, DMSO-d6): δ ppm 11.6 (br s 1H), 8.06 (d, J
= 2.1 Hz, 1H), 7.97 (d, J = 1.7 Hz, 1H), 7.67 (d, J = 1.8 Hz, 1H), 7.61
(dd, J = 8.7, 1.85 Hz, 1H), 7.01 (dd, J = 2.2, 0.9 Hz, 1H), 6.45−6.35
(br m, 2H), 2.23 (s, 3H); 13C NMR (125 MHz, DMSO-d6): δ ppm
163.8, 155.2, 152.7, 147.5, 146.1, 133.2, 128.3, 123.7, 120.2, 113.1,
112.1, 107.5, 104.0, 19.2; HRMS (IT-TOF, ESI) m/z: calcd for
C14H11NO2 [M + H]+ 226.0863; found, 226.0852.
Step B: 4-(Benzofuran-5-yl)-2-chloro-6-methyl-pyridine. 1.67 g of
4-(benzofuran-5-yl)-6-methyl-pyridin-2-ol (7.45 mmol) was dissolved
in 5 mL of POCl3 under nitrogen. The reaction mixture was stirred at
140 °C until no further conversion was observed. Excess of POCl3
was distilled off, the remaining material was poured into ice water,
stirred, and the solid was filtered off and was washed with 0.1 M
NaHCO3 solution, then with water. The crude product was purified
by flash column chromatography using DCM as the eluent to give
0.600 g (36%) of 4-(benzofuran-5-yl)-2-chloro-6-methyl-pyridine.
1H NMR (500 MHz, DMSO-d6): δ ppm 8.13 (d, J = 1.4 Hz, 1H),
8.09 (d, J = 2.1 Hz, 1H), 7.75 (dd, J = 8.6 Hz, 1.8 Hz, 1H), 7.72 (dm,
J = 8.6 Hz, 1H), 7.65 (s, 1H), 7.65 (s, 1H), 7.03 (d, J = 2.0 Hz, 1H),
2.51 (s, 3H); 13C NMR (125 MHz, DMSO-d6): δ ppm 160.0, 155.5,
151.8, 150.7, 147.7, 131.6, 128.5, 124.4, 120.8, 120.7, 119.0, 112.4,
107.5, 24.1; HRMS (IT-TOF, ESI) m/z: calcd for C14H10NOCl [M +
H]+ 244.0524; found, 244.0530.
1H NMR (500 MHz, DMSO-d6): δ ppm 6.99 (dd, J = 8.2 Hz, 2.2
Hz, 1H), 6.97 (d, J = 2.2 Hz, 1H), 6.90 (d, J = 8.2 Hz, 1H), 5.84 (s,
2H), 5.39 (s, 4H), 4.26 (s, 4H); 13C NMR (125 MHz, DMSO-d6): δ
ppm 159.6, 149.6, 144.0, 133.3, 119.4, 117.7, 115.0, 93.4, 64.6;
HRMS (IT-TOF, ESI) m/z: calcd for C13H14N3O2 [M + H]+
244.1080; found, 244.1094.
6-(2,6-Diamino-4-pyridyl)chromen-4-one (23). Starting from
0.188 g of 4-bromopyridine-2,6-diamine (1.0 mmol) and 0.327 g of
6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)chromen-4-one (1.2
mmol) as the appropriate boronic ester and following general
procedure IV, 0.150 g (59%) of 23 was obtained.
1H NMR (500 MHz, DMSO-d6): δ ppm 8.33 (d, J = 6.0 Hz, 1H),
8.16 (d, J = 2.4 Hz, 1H), 7.94 (dd, J = 8.8, 2.4 Hz, 1H), 7.73 (d, J =
8.8 Hz, 1H), 6.39 (d, J = 6.0 Hz, 1H), 5.99 (s, 2H), 5.56 (s, 4H); 13C
NMR (125 MHz DMSO-d6): δ ppm 176.8, 160.1, 157.6, 156.3, 148.2,
137.1, 132.4, 124.8, 122.2, 119.7, 112.7; HRMS (IT-TOF, ESI) m/z:
calcd for C14H12N3O2 [M + H]+ 254.0924; found, 254.0932.
4-(6-Quinolyl)pyridine-2,6-diamine (24). Starting from 0.188 g of
4-bromopyridine-2,6-diamine (1.0 mmol) and 0.306 g of 6-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)quinoline (1.2 mmol) following
general procedure IV, 0.160 g 24 (68%) was obtained.
Step C: 4-(Benzofuran-5-yl)-6-methyl-pyridin-2-amine (27).
0.121 g of 4-(benzofuran-5-yl)-2-chloro-6-methyl-pyridine (0.5
mmol) and 0.126 g of diphenylmethaneimine (0.7 mmol) was
dissolved in toluene (15 mL) then 0.046 g of Pd2(dba)3 (0.05 mmol),
1H NMR (500 MHz, DMSO-d6): δ ppm 8.91 (dd, J = 4.2, 1.6 Hz,
1H), 8.44 (dm, J = 8.4 Hz, 1H), 8.14 (d, J = 1.8 Hz, 1H), 8.08 (d, J =
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0.031 g of BINAP (0.05 mmol), and 0.058 g of sodium tert-butylate
(0.6 mmol) were added. The reaction mixture was stirred in a
microwave reactor at 100 °C until no further conversion was
observed. The solvent was removed in vacuum, the remaining solid
was dissolved in the mixture of 5 mL of MeOH and 3 mL of 1 M
hydrochloric acid. The reaction mixture was stirred until no further
conversion was observed. The solvent was removed in vacuum then
the crude product was purified by flash chromatography using DCM
and methanolic ammonia as eluents to give 0.052 g of (48%) of (4-
(benzofuran-5-yl)-6-methyl-pyridin-2-amine.
give 0.130 g (46%) of N-[6-amino-4-(benzofuran-5-yl)-2-pyridyl]-3-
methoxy-propanamide as a white solid.
1H NMR (500 MHz, DMSO-d6): δ ppm 12.52 (s, 1H), 8.10 (d, J =
2.2 Hz, 1H), 7.97 (d, J = 1.6 Hz, 1H), 7.83−7.76 (m, 2H), 7.74 (d, J
= 8.6, 2H), 7.59 (dd, J = 8.6, 1.8 Hz, 1H), 7.44 (br s, 1H), 7.05 (dd, J
= 2.2, 0.8 Hz, 1H), 6.21 (d, J = 16.0 Hz, 2H), 5.86 (s, 2H), 3.43 (t, J =
6.0 Hz, 2H), 3.40−3.35 (m, 2H), 3.25 (s, 3H); 13C NMR (125 MHz,
DMSO-d6): δ ppm 170.4, 159.4, 155.0, 151.6, 151.1, 147.4, 134.6,
128.4, 123.6, 119.7, 112.2, 107.6, 101.7, 100.0, 68.6, 58.4, 37.1;
HRMS (TOF, ESI) m/z: calcd for C17H19N3O2 [M + H]+ 312.1342;
found, 312.1343.
1H NMR (500 MHz, DMSO-d6): δ ppm 8.04 (d, J = 2.17 Hz, 1H),
7.89 (d, J = 1.65 Hz, 1H), 7.67 (d, J = 8.65 Hz, 1H), 7.55 (dd, J =
8.65, 1.83 Hz, 1H), 7.02 (d, J = 2.17 Hz, 1H), 6.69 (br s, 1H), 6.53
(br s, 1H), 5.86 (b rs, 2H), 2.3 (s, 3H); 13C NMR (125 MHz,
DMSO-d6): δ ppm 160.3, 157.0, 154.9, 149.5, 147.3, 134.4, 128.2,
123.6, 119.7, 110.0, 112.1, 107.5, 102.9, 24.3; HRMS (IT-TOF, ESI)
m/z: calcd for C14H12N2O [M + H]+ 225.096; found, 225.1033.
1-[4-(Benzofuran-5-yl)-2-pyridyl]piperazine (28). 0.196 g of 6-
bromobenzofuran (1.00 mmol), 0.281 g of 1-[4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)-2-pyridyl]piperazine (0.75 mmol), 0.203 g of
tetrakis(triphenylphosphine)palladium(0) (0.20 mmol), and 0.400 g
of K2CO3 (3.00 mmol) were suspended in the mixture of 4.5 mL of
DME and 1.5 mL of water. The mixture was stirred under nitrogen at
100 °C in a microwave reactor until no further conversion was
observed. The solvent was removed in vacuum, and the remaining
mixture was purified by flash chromatography using DCM and
methanolic ammonia as eluents to give 0.030 g (11%) of 28.
1H NMR (500 MHz, DMSO-d6): δ ppm 8.15 (d, J = 5.2 Hz, 1H),
8.06 (d, J = 2.1 Hz, 1H), 8.04 (br s, 1H), 7.69 (s, 2H), 7.03 (br s,
1H), 7.02 (d, J = 2.1 Hz, 1H), 6.95 (dm, J = 5.2 Hz, 1H), 3.49 (m,
4H), 2.8 (m, 4H), 2.47 (br s, 1H); 13C NMR (125 MHz, DMSO-d6):
δ ppm 160.6, 155.1, 149.6, 148.5, 147.4, 134.2, 128.3, 124.0, 120.2,
112.1, 111.7, 107.5, 104.9, 46.4, 45.9; HRMS (IT-TOF, ESI) m/z:
calcd for C17H17N3O [M + H]+ 280.1444; found, 280.1427.
4-(Benzofuran-5-yl)-N6-methyl-pyridine-2,6-diamine (29). 0.500
g of 10 (2.22 mmol) was stirred in 1.7 mL of formic acid (4.44 mmol)
at 70 °C in the presence of 0.180 g of zinc oxide (2.22 mmol) until no
further conversion was observed. A mixture of the mono- and
diformylated products was formed in a 4:1 ratio. The reaction mixture
was evaporated directly onto Celite and purified by flash
chromatography using DCM and methanolic ammonia as eluents to
give 0.280 g of a mixture of the two products. The mixture was
dissolved in 5 mL of abs. THF, and the resulting solution was added
dropwise to the suspension of 0.156 g of LiAlH4 (4.12 mmol) in 15
mL of abs. THF. The resulting mixture was stirred until no further
conversion was observed. The excess of LiAlH4 was decomposed with
careful subsequent addition of 0.156 mL of H2O, 0.312 mL of 2 M
NaOH, and finally 0.312 mL of H2O. The inorganics were removed
by filtration, and the filtrate was evaporated onto Celite. The crude
product was purified by flash chromatography using DCM and
methanolic ammonia as eluents to give 0.050 g (30%) of 29 as a white
solid.
Step B: 4-Benzofuran-5-yl-N-(3-methoxy-propyl)-pyridine-2,6-
diamine Hydrochloride (30). The solution of the product of step A
in 5 mL of abs. THF was added to the suspension of 0.055 g of
LiAlH4 (1.44 mmol) in 35 mL of abs. THF. The resulting mixture was
stirred until no further conversion was observed. The excess of LiAlH4
was decomposed with careful subsequent addition of 0.055 mL of
H2O, 0.110 mL of 2 M NaOH, and finally 0.110 mL of H2O. The
inorganics were removed by filtration, and the filtrate was evaporated
onto Celite. The crude product was purified by flash chromatography
using DCM and methanolic ammonia as eluents. The product was
taken up in the solution of HCl in Et2O, then the precipitate was
collected by filtration to give 0.095 g (60%) of 30 as a white powder.
1H NMR (500 MHz, DMSO-d6): δ ppm 12.53 (s, 1H), 8.10 (d, J =
2.2 Hz, 1H), 7.98 (d, J = 1.7 Hz, 1H), 7.79 (br, 1H), 7.74 (dm, 1H),
7.59 (dd, J = 8.7, 1.8 Hz, 1H), 7.45 (br, 2H), 7.07 (dd, J = 2.2, 0.8 Hz,
1H), 6.23 (d, J = 0.9 Hz, 1H), 6.20 (d, J = 1.2 Hz, 1H), 3.43 (t, J = 6.1
Hz, 2H), 3.38 (m, 2H), 3.25 (s, 3H), 1.83 (m, 2H); 13C NMR (125
MHz, DMSO-d6): δ ppm 156.1, 155.7, 152.9, 151.5, 147.8, 132.7,
128.4, 123.8, 120.4, 112.4, 107.6, 93.3, 92.1, 69.6, 58.5, 39.4, 28.9;
HRMS (TOF, ESI) m/z: calcd for C17H19N3O2 [M + H]+ 298.1485;
found, 298.1558.
4-Benzofuran-5-yl-N-(2-thiophen-3-yl-ethyl)-pyridine-2,6-dia-
mine Hydrochloride (31). Step A: N-[6-Amino-4-(benzofuran-5-yl)-
2-pyridyl]-2-(3-thienyl)acetamide. The mixture of 0.139 g of (3-
thienyl)acetic acid (0.97 mmol), 10 mL of abs. THF, 0.090 mL of
(0.135 g, 0.97 mmol) oxalyl chloride, and a drop of DMF was stirred
at room temperature for 20 min, then it was cooled to −78 °C. Also,
added dropwise to the −78 °C solution of 0.200 g of 10 (0.88 mmol)
and 0.680 mL of TEA (4.42 mmol) in 40 mL of THF. The reaction
mixture was left to warm to room temperature and evaporated directly
on to Celite. The solid was purified by flash chromatography using
DIPE and EtOAc as eluents to give 0.209 g of (67%) of N-[6-amino-
4-(benzofuran-5-yl)-2-pyridyl]-2-(3-thienyl)acetamide as a white
solid.
Step B: 4-Benzofuran-5-yl-N-(2-thiophen-3-yl-ethyl)-pyridine-
2,6-diamine Hydrochloride (31). The solution of the product of
step A (0.137 g, 0.39 mmol) in 5 mL of abs. THF was added dropwise
to the suspension of 75 mg of LiAH4 (1.44 mmol) in 35 mL of THF.
The resulting mixture was stirred until no further conversion was
observed. The excess of LiAlH4 was decomposed with the careful
subsequent addition of 0.075 mL of H2O, 0.150 mL of 2 M NaOH,
and finally 0.150 mL of H2O. The inorganics were removed by
filtration, and the filtrate was evaporated onto Celite. The crude
product was purified by flash chromatography using DCM and
methanolic ammonia as eluents. The product was taken up in the
solution of HCl in Et2O, then the precipitate was collected by
filtration to give 0.075 g (62%) of 31 as a white powder.
1H NMR (500 MHz, DMSO-d6): δ ppm 8.03 (d, J = 2.2 Hz, 1H),
7.82 (d, J = 1.8 Hz, 1H), 7.65 (dm, J = 8.6 Hz, 1H), 7.49 (dd, J = 8.6,
1.8 Hz, 1H), 7.02 (dd, J = 2.2, 0.8 Hz, 1H), 6.03 (br q, 1 H), 5.94 (d,
J = 1.0 Hz, 1 H), 5.88 (d, J = 1.0 Hz, 1 H), 5.59 (br s, 2 H), 2.75 (d, 3
H); 13C NMR (125 MHz, DMSO-d6): δ ppm 159.7, 159.4, 154.8,
150.7, 147.2, 135.4, 128.1, 123.5, 119.4111.9, 107.5, 93.8, 92.8, 28.9;
HRMS (IT-TOF, ESI) m/z: calcd for C14H14N3O [M + H]+
240.1131; found, 240.1121.
4-Benzofuran-5-yl-N-(3-methoxy-propyl)-pyridine-2,6-diamine
Hydrochloride (30). Step A: N-[6-Amino-4-(benzofuran-5-yl)-2-
pyridyl]-3-methoxy-propanamide. The solution of 0.200 g of 10
(0.88 mmol) and 0.680 mL of TEA (4.42 mmol) in 40 mL of THF
was cooled to −78 °C. To the resulting mixture, 0.120 g of 3-
methoxypropanoyl chloride (0.97 mmol) dissolved in 10 mL of THF
was added dropwise. The reaction mixture was left to warm to room
temperature. It was evaporated directly onto Celite, and then it was
purified by flash chromatography using DIPE and EtOAc as eluents to
1H NMR (500 MHz, DMSO-d6): δ ppm 12.66 (br s, 1H), 8.10 (d,
J = 2.1 Hz, 1H), 7.97 (d, J = 1.7 Hz, 1H), 7.85 (br s, 1H), 7.74 (d, J =
8.6 Hz, 1H), 7.59 (dd, J = 8.6, 1.8 Hz, 1H), 7.52 (br s, 2H), 7.49 (dd,
J = 4.9, 3.0 Hz, 1H), 7.36 (br d, J = 2.3 Hz, 1H), 7.15 (dd, J = 4.9, 1.0
Hz, 1H), 7.07 (dd, J = 2.2, 0.8 Hz, 1H), 6.25 (s, 1H), 6.20 (s, 1H),
3.62 (m, 2H), 2.93 (t, J = 7.2 Hz, 2H); 13C NMR (125 MHz, DMSO-
d6): δ ppm 156.1, 155.7, 152.9, 151.4, 147.8, 139.4, 132.7, 129.1,
128.4, 126.5, 123.8, 122.4, 120.5, 112.4, 107.6, 93.4, 92.2, 42.9, 29.5;
HRMS (TOF, ESI) m/z: calcd for C19H17N3OS [M + H]+ 336.1101;
found, 336.1174.
N-[6-Amino-4-(benzofuran-5-yl)-2-pyridyl]-2-(methylamino)-
acetamide (32). The solution of 0.225 g 10 (1.0 mmol) and 0.278
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mL of TEA (2.0 mmol) in 8 mL of THF was cooled to −50 °C. 0.157
g of 2-bromoacetyl chloride (0.083 mL, 1.0 mmol) dissolved in 2 mL
of THF was added dropwise to the resulting mixture. After 10 min of
stirring, 5 mL of methyl amine (10.0 mmol, 2 M in THF) was added,
and the reaction mixture was left to warm up to room temperature,
where it was stirred until no further conversion was observed. The
reaction mixture was evaporated directly onto Celite, and then, it was
purified by flash chromatography using DCM and methanolic
ammonia as eluents to give 0.197 g (66%) of 32 as a white powder.
1H NMR (500 MHz, DMSO-d6): δ ppm 9.68 (br s, 1H), 8.06 (d, J
= 2.2 Hz, 1H), 7.86 (d, J = 1.8 Hz, 1 H), 7.70 (d, J = 8.4 Hz, 1 H),
7.61 (br s, 1 H), 7.52 (dd, J = 8.6, 1.8 Hz, 1 H), 7.06 (dd, J = 2.2, 0.9
Hz, 1 H), 6.47 (d, J = 1.4 Hz, 1 H), 6.01 (s, 2 H), 3.23 (s, 2 H), 2.93
(br s, 2 H), 2.33 (s, 3 H); 13C NMR (125 MHz, DMSO-d6): δ ppm
170.7, 159.7, 155.0, 151.4, 150.7, 147.4, 134.6, 128.4, 123.6, 119.7,
112.2, 107.6, 101.9, 99.1, 55.1, 36.5; HRMS (IT-TOF, ESI) m/z:
calcd for C16H17N4O2 [M + H]+ 297.1346; found, 297.1338.
4-(2-Methyl-benzofuran-5-yl)-pyridine-2,6-diamine Trifluoroace-
tic Acid (1:1) Salt (33). The mixture of 0.211 g of 5-bromo-2-
methylbenzofuran (1.0 mmol), 0.279 g of bis-4,4,5,5-tetramethyl-
1,3,2-dioxaborolane (1.1 mmol), 0.0225 g of palladium acetate (0.1
mmol), 0.072 g of PBuAd2 (0.2 mmol), and 0.553 g of K2CO3 (4.0
mmol) in 5 mL of DME and 1 mL of H2O was stirred under argon
for 30 min 0.170 g of 4-bromopyridin-2,6-diamine (0.90 mmol) was
added to the mixture, and the stirring was continued for 1 h. The
reaction mixture was evaporated directly onto Celite, and then, it was
purified by flash chromatography of a reverse phase silica column
using 0.1% TFA and MeCN as eluents to give 0.080 g (37%) of 33.
1H NMR (500 MHz, DMSO-d6): δ ppm 12.68 (br s, 1H), 7.77
(dm, 1H), 7.62 (dm, 1H), 7.26 (br s, 4H), 7.44 (dd, J = 8.5, 1.9 Hz,
1H), 6.68 (m, 1H), 6.18 (s, 2H), 2.47 (d, J = 0.8 Hz, 3H); 13C NMR
(125 MHz, DMSO-d6): δ ppm 157.5, 156.6, 155.5, 152.6, 132.3,
130.1, 122.5, 119.1, 111.7, 103.6, 93.5, 14.2; HRMS (TOF, ESI) m/z:
calcd for C14H14N3O [M + H]+ 240.1131; found, 240.1123.
4-(2-Chloro-benzofuran-5-yl)-pyridine-2,6-diamine Trifluoroace-
tic Acid (1:1.3) Salt (34). The mixture of 0.232 g of 5-bromo-2-
chlorobenzofuran (1.0 mmol), 0.279 g of bis-4,4,5,5-tetramethyl-
1,3,2-dioxaborolane (1.1 mmol), 0.0225 g of palladium acetate (0.1
mmol), 0.072 g of PBuAd2 (0.2 mmol), and 0.553 g of K2CO3 (4.0
mmol) in 5 mL of DME and 1 mL of H2O was stirred under argon
for 30 min. 0.170 g of 4-bromopyridin-2,6-diamine (0.90 mmol) was
added to the mixture and the stirring was continued for 1 h. The
reaction mixture was evaporated directly onto Celite, and then, it was
purified by flash chromatography of a reverse phase silica column
using 0.1% TFA and MeCN as eluents to give 0.019 g (7%) of 34.
1H NMR (500 MHz, DMSO-d6): δ ppm 12.33 (br s, 1H), 7.84
(dm, 1H), 7.75 (dm, 1H), 7.56 (dd, J = 8.6, 2.0 Hz, 1H), 7.23 (br s,
4H), 7.15 (d, J = 0.8 Hz, 1H), 6.16 (s, 2H); 13C NMR (125 MHz,
DMSO-d6): δ ppm 156.0, 154.8, 152.7, 141.9, 133.6, 129.1, 123.9,
119.5, 112.2, 104.7, 93.6; HRMS (IT-TOF, ESI) m/z: calcd for
C13H11N3OCl [M + H]+ 260.0585; found, 260.0587.
4-(2-Ethyl-3-butyl-3H-imidazo[4,5-b]pyridin-5-yl)pyridine-2,6-di-
amine (37). Step A: N-(6-Bromo-2-chloro-3-pyridyl)propanamide.
3.00 g of 6-bromo-2-chloro-pyridin-3-amine (14.5 mmol) and 2.20
mL of triethylamine (15.9 mmol) were dissolved in 60 mL of DCM.
The solution was cooled to 0 °C and 1.40 mL of propanoyl chloride
(15.9 mmol) was added dropwise over 30 min. On the completion of
the addition, the reaction mixture was allowed to warm up to room
temperature where it was stirred until no further conversion was
observed. The reaction mixture was diluted with EtOAc and washed
with water. The organic layer was washed with brine, dried over
MgSO4, filtered, and concentrated under reduced pressure. The
residue was purified by flash chromatography using DCM as the
eluent to give 3.55 g (93%) of N-(6-bromo-2-chloro-3-pyridyl)-
propanamide.
1H NMR (500 MHz, DMSO-d6): δ ppm 9.67 (s, 1H), 8.16 (d, J =
8.2 Hz, 1H), 7.64 (d, J = 8.4 Hz, 1H), 2.43 (q, J = 15.0, 7.5 Hz 2H),
1.08 (t, J = 7.6 Hz, 3H); HPLC-MS: (M − H)−: 261.0; 263.0.
Step B: tert-Butyl N-[6-(tert-butoxycarbonylamino)-4-[6-chloro-
5-(propanoylamino)-2-pyridyl]-2-pyridyl]carbamate. Starting from
3.53 g of N-(6-bromo-2-chloro-3-pyridyl)propanamide (13.4 mmol),
following step B of R2, 5.70 g of tert-butyl N-[6-(tert-butoxycarbo-
nylamino)-4-[6-chloro-5-(propanoylamino)-2-pyridyl]-2-pyridyl]-
carbamate (87%) was obtained.
1H NMR (500 MHz, DMSO-d6): δ ppm 9.68 (s, 1H), 9.45 (s, 2H),
8.40 (d, J = 8.5 Hz, 1H), 8.00 (s, 2H), 7.91 (d, J = 8.4 Hz, 1H), 2.48
(q, J = 13.0, 7.5 Hz 2H), 1.49 (s, 18H), 1.11 (t, J = 7.5 Hz, 3H); 13C
NMR (125 MHz, DMSO-d6): δ ppm 173.4, 153.1, 152.1, 150.0,
147.8, 142.6, 134.6, 132.9, 120.9, 104.4, 80.2, 29.5, 28.5, 9.9; HRMS
(TOF, ESI) m/z: calcd for C23H31N5O5Cl [M + H]+ 492.1935;
found, 492.2005.
Step C: 4-(3-Butyl-2-ethyl-3H-imidazo[4,5-b]pyridin-5-yl)-
pyridine-2,6-diamine (37). Starting from 0.49 g of tert-butyl N-[6-
(tert-butoxycarbonylamino)-4-[6-chloro-5-(propanoylamino)-2-pyrid-
yl]-2-pyridyl]carbamate (1.0 mmol) and 0.50 mL of butylamine (5.0
mmol) as the appropriate amine following general procedure II, 0.085
g (22%) of 37 was obtained.
1H NMR (500 MHz, DMSO-d6): δ 7.98 (d, J = 8.3 Hz, 1H), 7.55
(d, J = 8.3 Hz, 1H), 6.35 (s, 2H), 5.47 (br s, 4H), 4.27 (t, J = 7.2 Hz,
2H), 2.95 (q, J = 14.9, 7.3 Hz 2H), 1.79 (m, 2H), 1.37 (t, J = 7.4 Hz,
3H), 1.32 (m, 2H), 0.93 (t, J = 7.3 Hz, 3H); 13C NMR (125 MHz,
DMSO-d6): δ ppm 159.5, 158.5, 149.5, 148.4, 134.4, 126.5, 115.0,
93.6, 41.6, 31.7, 20.9, 19.9, 14.0, 11.5; HRMS (TOF, ESI) m/z: calcd
for C17H23N6 [M + H]+ 311.1906; found, 311.1982.
4-(2,3-Dibutyl-3H-imidazo[4,5-b]pyridin-5-yl)pyridine-2,6-dia-
mine (38). Step A: N-(6-Bromo-2-chloro-3-pyridyl)pentanamide.
To 3.00 g of 6-bromo-2-chloro-pyridin-3-amine (14.5 mmol) and 2.4
mL of triethylamine (17.4 mmol) were dissolved in 60 mL of DCM.
The solution was cooled to 0 °C and 2.1 mL of pentanoyl chloride
(17.4 mmol) was added dropwise over 30 min. On the completion of
the addition, the reaction mixture was allowed to warm up to room
temperature, where it was stirred until no further conversion was
observed. The reaction mixture was diluted with EtOAc and washed
with water. The organic layer was washed with brine, dried over
MgSO4, filtered, and concentrated under reduced pressure. The
residue was purified by flash chromatography using DCM as the
eluent to give 3.59 g (85%) of N-(6-bromo-2-chloro-3-pyridyl)-
pentanamide.
4-(2,3-Dimethyl-3H-imidazo[4,5-b]pyridin-5-yl)pyridine-2,6-dia-
mine (35). Starting from 0.500 g of R1 (1.15 mmol) and 0.190 g of 6-
chloro-2,3-dimethyl-imidazo[4,5-b]pyridine (1.05 mmol) following
general procedure I, 0.047 g (18%) of 35 was obtained.
1H NMR (500 MHz, DMSO-d6): δ ppm 7.96 (d, J = 8.3 Hz, 1H),
7.57 (d, J = 8.3 Hz, 1H), 6.40 (s, 2H), 5.63 (br s, 4H), 3.79 (s, 3H),
2.59 (s, 3H); 13C NMR (125 MHz, DMSO-d6): δ ppm 159.1, 155.3,
149.1, 148.6, 134.8, 126.4, 115.1, 93.7, 28.5, 14.5; HRMS (TOF, ESI)
m/z: calcd for C13H15N6 [M + H]+ 255.1289; found, 255.1361.
4-(2-Methyl-3-butyl-3H-imidazo[4,5-b]pyridin-5-yl)pyridine-2,6-
diamine (36). Starting from 0.143 g of R2 (0.3 mmol) and 0.219 g of
butylamine (0.297 mL, 3 mmol) following general procedure II, 0.019
g (21%) of 36 was obtained.
HPLC-MS: (M − H)− = 289.0; 291.0.
Step B: tert-Butyl N-[6-(tert-butoxycarbonylamino)-4-[6-chloro-
5-(pentanoylamino)-2-pyridyl]-2-pyridyl]carbamate. Starting from
N-(6-bromo-2-chloro-3-pyridyl)pentanamide following step B of R2,
4.35 g of tert-butyl N-[6-(tert-butoxycarbonylamino)-4-[6-chloro-5-
(pentanoylamino)-2-pyridyl]-2-pyridyl]carbamate (72%) was ob-
tained.
1H NMR (500 MHz, DMSO-d6): δ ppm 7.95 (d, J = 8.3 Hz, 1H),
7.55 (d, J = 8.3 Hz, 1H), 6.34 (s, 2H), 5.48 (br s, 4H), 4.27 (t, 2H),
2.6 (s, 3H), 1.79 (quint, 2H), 1.31 (m, 2H), 092 (t, 3H); 13C NMR
(125 MHz, DMSO-d6): δ ppm 159.8, 154.6, 149.6, 149.2, 148.4,
134.6, 126.5, 115.1, 93.7, 41.9, 31.6, 20.0, 14.7, 14.1; HRMS (TOF,
ESI) m/z: calcd for, C16H21N6 [M + H]+ 297.1769; found, 297.1842.
1H NMR (500 MHz, DMSO-d6): δ ppm 9.70 (s, 1H), 9.45 (s, 2H),
8.37 (d, J = 8.4 Hz, 1H), 8.00 (s, 2H), 7.91 (d, J = 8.3 Hz, 1H), 2.47
(t, J = 7.5 Hz, 2H), 1.6 (m, 2H), 1.49 (s, 18H), 1.36 (m, 2H), 0.91 (t,
J = 7.4 Hz, 3H); 13C NMR (125 MHz, DMSO-d6): δ ppm 172.7,
153.1, 152.1, 150.2, 147.8, 142.9, 134.8, 132.9, 120.9, 104.4, 80.2,
6760
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36.0, 28.5, 27.6, 22.2, 14.2; HRMS (TOF, ESI) m/z: calcd for
C25H35N5O5Cl [M + H]+ 520.2248; found, 520.2312.
26.1, 14.6; HRMS (TOF, ESI) m/z: calcd for C20H27N6 [M + H]+
351.2219; found, 351.2273.
4-[3-(4-Fluorobenzyl)-2-methyl-3H-imidazo[4,5-b]pyridin-5-yl]-
pyridine-2,6-diamine (44). Step A: 6-Chloro-N-[(4-fluorophenyl)-
methyl]-3-nitro-pyridin-2-amine. 0.94 g of 4-fluoro-benzylamine
(7.5 mmol) was added dropwise to the mixture of 1.0 g of 2,6-
dichloro-3-nitro-pyridine (5.39 mmol) and 1.86 g of K2CO3 (13.5
mmol) in 25 mL of dry DCE, and the reaction mixture was stirred at
ambient temperature until no further conversion was observed. The
reaction mixture was diluted with DCM and washed with water. The
organic layer was washed with brine, dried over MgSO4, filtered, and
concentrated under reduced pressure. The residue was purified by
flash chromatography using heptane and EtOAc as the eluent to give
1.10 g (74%) of 6-chloro-N-[(4-fluorophenyl)methyl]-3-nitro-pyr-
idin-2-amine.
Step C: 4-(2,3-Dibutyl-3H-imidazo[4,5-b]pyridin-5-yl)pyridine-
2,6-diamine (38). Starting from 0.52 g of tert-butyl N-[6-(tert-
butoxycarbonylamino)-4-[6-chloro-5-(pentanoylamino)-2-pyridyl]-2-
pyridyl]carbamate (1.0 mmol) and 0.5 mL of butylamine (5.0 mmol)
following general procedure II, 0.075 g (22%) of 38 was obtained.
1H NMR (500 MHz, DMSO-d6): δ ppm 7.97 (d, J = 8.3 Hz, 1H),
7.55 (d, J = 8.3 Hz, 1H), 6.34 (s, 2H), 5.48 (s, 4H), 4.27 (t, J = 7.1
Hz, 2H), 2.91 (t, J = 7.5 Hz, 2H), 1.82 (m, 2H), 1.79 (m, 2H), 1.44
(m, 2H), 1.31 (m, 2H), 0.95 (t, J = 7.3 Hz, 3H), 0.92 (t, J = 7.3 Hz,
3H); 13C NMR (125 MHz, DMSO-d6): δ ppm 159.8, 157.7, 149.7,
149.3, 148.4, 134.6, 126.6, 115.2, 93.7, 41.8, 31.8, 29.2, 27.1, 22.4,
20.0, 14.3, 14.1; HRMS (TOF, ESI) m/z: calcd for C19H28N6 [M +
H]+ 339.2219; found, 339.2289.
1H NMR (500 MHz, DMSO-d6): δ ppm 9.2 (s, 1H), 8.4 (d, J = 8.5
Hz, 1H), 7.4 (m, 2H), 7.13 (m, 2H), 6.79 (d, J = 8.5 Hz, 1H), 4.68 (s,
2H), 3.28 (d, J = 9.4 Hz, 3H); HPLC-MS: (M + H)+: 282.
Step B: 6-Chloro-N2-[(4-fluorophenyl)methyl]pyridine-2,3-dia-
mine. A mixture of 0.52 g of 6-chloro-N-[(4-fluorophenyl)methyl]-
3-nitro-pyridin-2-amine (1.85 mmol) and 0.040 g of Pt/C (0.19
mmol) in 30 mL of MeOH was stirred under 1 bar of hydrogen until
no further conversion was observed. The reaction mixture was filtered
through Celite and concentrated under reduced pressure to give 0.450
g (97%) of 6-chloro-N2-[(4-fluorophenyl)methyl]pyridine-2,3-dia-
mine, which was used without any further purification. HPLC-MS:
(M + H)+: 253.
Step C: 5-Chloro-3-[(4-fluorophenyl)methyl]-2-methyl-imidazo-
[4,5-b]pyridine. The mixture of 0.10 g of 6-chloro-N2-[(4-
fluorophenyl)methyl]pyridine-2,3-diamine (0.41 mmol) and 0.405
mL of 1 M HCl in Et2O (0.41 mmol) in 4 mL of acetic acid was
heated in a MW tube at 160 °C until no further conversion was
observed. After cooling to ambient temperature Celite was added to
the reaction mixture, and the volatiles were evaporated under reduced
pressure. The solid residue was purified by flash chromatography on
silica gel using DCM and MeOH as eluents to give 0.146 g (44%) of
5-chloro-3-[(4-fluorophenyl)methyl]-2-methyl-imidazo[4,5-b]-
pyridine.
4-(3-tert-Butyl-2-methyl-3H-imidazo[4,5-b]pyridin-5-yl)pyridine-
2,6-diamine (39). Starting from 0.30 g of R2 (0.63 mmol) and 0.33
mL of tert-butylamine (0.23 g, 3.2 mmol) following general procedure
II, 0.077 g (51%) of 39 was obtained.
1H NMR (500 MHz, DMSO-d6): δ ppm 7.90 (d, J = 8.3 Hz, 1H),
7.53 (d, J = 8.3 Hz, 1H), 6.32 (s, 1H), 5.49 (br s, 4H), 2.77 (s, 3H),
1.92 (s, 9H); 13C NMR (125 MHz, DMSO-d6): δ ppm 159.8, 154.5,
149.6, 149.5, 148.7, 134.8, 126.3, 114.7, 93.6, 59.8, 30.3, 20.6; HRMS
(TOF, ESI) m/z: Principal peak: 240.1125 [M + H-C4H8]+.
Fragment ion formula: C12H12N6 no molecular ion was detected
due to extensive fragmentation.
4-(3-Cyclopropyl-2-methyl-3H-imidazo[4,5-b]pyridin-5-yl)-
pyridine-2,6-diamine (40). Starting from 1.50 g of R2 (3.14 mmol)
and 1.1 mL of cyclopropylamine (0.896 g, 15.7 mmol) following
general procedure II, 0.205 g (23%) of 40 was obtained.
1H NMR (500 MHz, DMSO-d6): δ ppm 7.92 (d, J = 8.2 Hz, 1H),
7.53 (d, J = 8.3 Hz, 1H), 6.35 (s, 2H), 5.49 (br s, 4H), 3.36 (m, 1H),
2.64 (s, 3H), 1.26 (m, 2H), 1.20 (m, 2H); 13C NMR (125 MHz,
DMSO-d6): δ ppm 159.8, 156.9, 149.6, 149.3, 134.4, 126.5, 115.0,
93.7, 24.5, 15.5, 6.9; HRMS (TOF, ESI) m/z: calcd for C15H17N6 [M
+ H]+ 281.1436; found, 281.1518.
4-(3-Cyclopentyl-2-methyl-3H-imidazo[4,5-b]pyridin-5-yl)-
pyridine-2,6-diamine (41). Starting from 0.47 g of R1 (1.07 mmol)
and 0.23 g of 6-chloro-3-cyclopentyl-2-methyl-imidazo[4,5-b]pyridine
(0.98 mmol) following general procedure I, 0.106 g (35%) of 41 was
obtained.
1H NMR (500 MHz, DMSO-d6): δ ppm 8.04 (d, J = 8.2 Hz, 1H),
7.32 (d, J = 8.2 Hz, 1H), 7.25 (m, 2H), 7.18 (m, 2H), 5.45 (s, 2H),
2.51 (s, 3H); HRMS (TOF, ESI) m/z: calcd for C14H12N3FCl [M +
H]+: 276.0698; found, 276.0697.
Step D: 4-[3-(4′-Fluorobenzyl)-2-methyl-3H-imidazo[4,5-b]-
pyridin-5-yl]pyridine-2,6-diamine (44). Starting from 0.42 g of 4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-N2,N6-ditrityl-pyridine-
2,6-diamine (0.58 mmol) and 0.14 g of 5-chloro-3-[(4-fluorophenyl)-
methyl]-2-methylimidazo[4,5-b]pyridine (0.51 mmol) following
general procedure I, 0.064 g (36%) of 44 was obtained.
1H NMR (400 MHz, DMSO-d6): δ ppm 7.93 (d, J = 8.3 Hz, 1H),
7.53 (d, J = 8.3 Hz, 1H), 6.32 (s, 2H), 5.48 (br s, 4H), 4.91 (quint, J =
8.3 Hz, 1H), 2.62 (s, 3H), 2.42 (m, 2H), 2.11 (m, 2H), 2.08 (m, 2H),
1.72 (m, 2H); 13C NMR (100 MHz, DMSO-d6): δ ppm 159.8, 154.8,
149.4, 149.1, 148.1, 135.0, 126.5, 114.8, 93.7, 56.2, 30.6, 25.3, 15.4;
HRMS (TOF, ESI) m/z: calcd for C17H21N6 [M + H]+ 309.1749;
found, 309.1821.
4-[3-(3-Methoxypropyl)-2-methyl-3H-imidazo[4,5-b]pyridin-5-
yl]pyridine-2,6-diamine (42). Starting from 0.239 g of R2 (0.50
mmol) and 0.222 g of 3-methoxypropan-1-amine (0.255 mL, 2.5
mmol) following general procedure II, 0.047 g (31%) of 42 was
obtained.
1H NMR (500 MHz, DMSO-d6): δ ppm 8.01 (d, J = 8.3 Hz, 1H),
7.61 (d, J = 8.3 Hz, 1H), 7.34 (m, 2H), 7.19 (m, 2H), 6.36 (s, 2H),
5.52 (s, 2H), 5.49 (s, 4H), 2.52 (s, 3H); 13C NMR (125 MHz,
DMSO-d6): δ ppm 162.0, 159.9, 154.5, 150.0, 149.0, 148.3, 134.6,
133.5, 129.9, 126.9, 116.2, 115.5, 93.6, 44.4, 14.8; HRMS (TOF, ESI)
m/z: calcd for C19H18N6F [M + H]+ 348.1499; found, 348.1565.
4-{3-[2-(Furan-2-yl)ethyl]-2-methyl-3H-imidazo[4,5-b]pyridin-5-
yl}pyridine-2,6-diamine (45). Starting from 0.30 g of R2 (0.63 mmol)
and using 2-(2-furyl)ethanamine as the appropriate amine following
general procedure II, 0.025 g (12%) of 45 was obtained.
1H NMR (500 MHz, DMSO-d6): δ ppm 7.95 (d, J = 8.3 Hz, 1H),
7.55 (d, J = 8.3 Hz, 1H), 6.33 (s, 2H), 5.48 (br s, 4H), 4.31 (t, J = 7.0
Hz, 2H), 3.31 (t, J = 6.0 Hz, 2H), 3.22 (s, 3H), 2.59 (s, 3H), 2.07 (m,
2H); 13C NMR (125 MHz, DMSO-d6): δ ppm 159.8, 154.8, 149.7,
149.2, 148.4, 134.7, 126.5, 115.2, 93.7, 69.3, 58.3, 39.7, 29.3, 14.5;
HRMS (TOF, ESI) m/z: calcd for C16H21N6O [M + H]+ 313.1688;
found, 313.1761.
4-[3-(2-Cyclohexylethyl)-2-methyl-3H-imidazo[4,5-b]pyridin-5-
yl]pyridine-2,6-diamine (43). Starting from 0.30 g of R2 (0.63 mmol)
and 0.401 g of (2-aminoethyl)-cyclohexane (3.2 mmol) following
general procedure II, 0.053 g (24%) of 43 was obtained.
1H NMR (500 MHz, DMSO-d6): δ ppm 7.95 (d, J = 8.2 Hz, 1H),
7.55 (d, J = 8.2 Hz, 1H), 6.34 (s, 2H), 5.47 (br s, 4H), 4.29 (t, J = 7.4
Hz, 2H), 2.59 (s, 3H), 1.83 (d, J = 12.8 Hz, 2H), 1.67 (m, 5H), 1.23
(m, 6H); 13C NMR (125 MHz, DMSO-d6): δ 159.8, 154.5, 149.6,
149.2, 148.3, 134.6, 126.5, 115.1, 93.7, 40.0, 37.1, 35.2, 33.0, 26.5,
1H NMR (500 MHz, DMSO-d6): δ ppm 7.94 (d, J = 8.3 Hz, 1H),
7.55 (d, J = 8.3 Hz, 1H), 7.55 (dd, J = 2.0, 0.7 Hz, 1H), 6.34 (s, 2H),
6.33 (dd, J = 3.2, 1.9 Hz, 1H), 6.04 (d, J = 3.2 Hz, 1H), 5.49 (br s,
4H), 4.49 (t, J = 6.7 Hz, 2H), 3.20 (t, J = 6.7 Hz, 2H), 2.32 (s, 3H);
13C NMR (125 MHz, DMSO-d6): δ ppm 159.8, 154.7, 152.2, 149.8,
149.2, 148.1, 142.6, 134.7, 126.5, 115.3, 111.1, 107.6, 93.7, 41.4, 27.9,
14.0; HRMS (TOF, ESI) m/z: calcd for C18H19N6O [M + H]+
335.1545; found, 335.1618.
4-[2-Methyl-3-(2-phenoxyethyl)-3H-imidazo[4,5-b]pyridin-5-yl]-
pyridine-2,6-diamine (46). Starting from 0.60 g of R2 (1.26 mmol)
and using 2-phenoxyethanamine as the appropriate amine following
general procedure II, 0.108 g (30%) of 46 was obtained.
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1H NMR (500 MHz, DMSO-d6): δ ppm 7.97 (d, J = 8.3 Hz, 1H),
7.56 (d, J = 8.3 Hz, 1H), 7.25 (t, J = 7.9 Hz, 1H), 6.91 (m, 3H), 6.34
(s, 2H), 5.48 (br s, 4H), 4.66 (t, J = 5.4 Hz, 2H), 4.39 (t, J = 5.4 Hz,
2H), 2.67 (s, 3H); 13C NMR (125 MHz, DMSO-d6): δ ppm 159.8,
158.3, 155.4, 149.9, 149.2, 148.2, 134.7, 130.0, 126.7, 121.4, 115.5,
114.8, 93.8, 65.9, 42.0, 14.8; HRMS (TOF, ESI) m/z: calcd for
C20H21N6O [M + H]+: 361.1699; found, 361.1774.
4-[3-(2,2-Difluoroethyl)-2-methyl-3H-imidazo[4,5-b]pyridin-5-
yl]pyridine-2,6-diamine (47). Starting from 0.50 g of R2 (1.05 mmol)
and using 2,2-difluoroethanamine as the appropriate amine following
general procedure II, 0.090 g (28%) of 47 was obtained.
Hz, 1H), 4.48 (dd, J = 14.5, 4.8 Hz, 1H), 2.65 (s, 3H), 1.35 (d, J = 6.1
Hz, 3H); 13C NMR (125 MHz, DMSO-d6): δ ppm 159.8, 157.5,
155.3, 149.9, 149.3, 148.5, 134.5, 130.1, 126.6, 121.3, 115.7, 115.5,
93.9, 72.2, 47.1, 18.2, 15.0; HRMS (TOF, ESI) m/z: calcd for
C21H23N6O [M + H]+ 375.1850; found, 375.1922; ee > 99.8%.
4-{2-Methyl-3-[2-(pyridin-2-yloxy)ethyl]-3H-imidazo[4,5-b]-
pyridin-5-yl}pyridine-2,6-diamine (53). Starting from 0.48 g of R3
(1.0 mmol) and using 2-fluoropyridine as the appropriate aryl halide
following general procedure III, 0.223 g (61%) of 53 was obtained.
1H NMR (500 MHz, DMSO-d6): δ ppm 11.95 (br s, 1H), 8.09
(dm, 1H), 7.95 (d, J = 8.3 Hz, 1H), 7.66 (m, 1H), 7.56 (d, J = 8.3 Hz,
1H), 6.95 (m, 1H), 6.75 (dm, 1H), 6.33 (s, 2H), 5.49 (s, 4H), 4.69
(m, 2H), 4.67 (m, 2H), 2.61 (s, 3H); 13C NMR (125 MHz, DMSO-
d6): δ ppm 163.0, 159.8, 155.2, 149.8, 149.2, 148.3, 147.2, 139.9,
134.7, 126.6, 117.9, 115.4, 111.2, 93.7, 63.7, 41.9, 14.7; HRMS (TOF,
ESI) m/z: calcd for C19H20N7O [M + H]+ 362.1651; found,
362.1731.
1H NMR (500 MHz, DMSO-d6): δ ppm 8.01 (d, J = 8.3 Hz, 1H),
7.60 (d, J = 8.3 Hz, 1H), 6.53 (tt, J = 54.6, 3.4 Hz, 1H), 6.34 (s, 2H),
5.49 (s, 4H), 4.78 (td, J = 15.5, 3.3 Hz, 2H), 2.62 (s, 3H); 13C NMR
(125 MHz, DMSO-d6): δ ppm 159.8, 155.0, 150.2, 149.0, 148.5,
134.5, 126.9, 115.8, 114.3, 93.7, 44.0, 14.6; HRMS (TOF, ESI) m/z:
calcd for C14H15N6F2 [M + H]+ 305.1321; found, 305.1318.
4-[2-Methyl-3-(2,2,2-trifluoroethyl)-3H-imidazo[4,5-b]pyridin-5-
yl]pyridine-2,6-diamine (48). Starting from 1.50 g of R2 (3.14 mmol)
and using 2,2,2-trifluoroethylamine as the appropriate amine following
general procedure II, 0.318 g (31%) of 48 was obtained.
4-{2-Methyl-3-[(2R)-2-(pyridin-2-yloxy)propyl]-3H-imidazo[4,5-
b]pyridin-5-yl}pyridine-2,6-diamine (54) and 4-{2-Methyl-3-[(2S)-2-
(pyridin-2-yloxy)propyl]-3H-imidazo[4,5-b]pyridin-5-yl}pyridine-
2,6-diamine (55). Starting from 0.41 g of R4 (1.5 mmol) and using 2-
fluoropyridine as the appropriate aryl halide following general
procedure III, a mixture of 54 and 55 was obtained. The enantiomers
were separated on CHIRALCEL OK column using 50:50 heptane/
EtOH +0.05% DEA as the eluent to give 0.085 g (15%) of 55 as the
first eluting enantiomer, and 0.082 g (15%) of 54 as the enantiomer
eluting second.
1H NMR (500 MHz, DMSO-d6): δ ppm 8.04 (d, J = 8.3 Hz, 1H),
7.63 (d, J = 8.3 Hz, 1H), 6.34 (s, 2H), 5.51 (br s, 4H), 5.24 (q, J = 9.3
Hz, 2H), 2.64 (s, 3H); 13C NMR (125 MHz, DMSO-d6): δ ppm
159.9, 154.9, 150.6, 148.8, 148.2, 134.4, 127.3, 124.4, 116.3, 93.7,
43.0, 14.5; HRMS (TOF, ESI) m/z: calcd for C14H14N6F3 [M + H]+
323.1165; found, 323.1238.
1
54: H NMR (500 MHz, DMSO-d6): δ ppm 7.97 (dm, 1H), 7.90
4-[2-Methyl-3-(4,4,4-trifluorobutyl)-3H-imidazo[4,5-b]pyridin-5-
yl]pyridine-2,6-diamine (49). Starting from 0.30 g of R2 and using
4,4,4-trifluorobutan-1-amine as the appropriate amine following
general procedure II, 0.077 g (35%) 49 was obtained.
(d, J = 8.2 Hz, 1H), 7.61 (m, 1H), 7.53 (d, J = 8.3 Hz, 1H), 6.87 (m,
1H), 6.69 (dm, 1H), 6.36 (s, 2H), 5.65 (m, 1H), 5.50 (br s, 4H), 4.53
(d, J = 5.9 Hz, 2H), 2.65 (s, 3H), 1.39 (d, J = 6.3 Hz, 3H); 13C NMR
(125 MHz, DMSO-d6): δ ppm 162.7, 159.8, 155.2, 149.7, 149.2,
148.5, 147.1, 139.8, 134.5, 126.5, 117.6, 115.3, 111.3, 93.7, 69.9, 46.8,
18.3, 14.9; HRMS (IT-TOF, ESI) m/z: calcd for C20H22N7O [M +
H]+ 376.1880; found, 376.1867; ee > 99.8%.
1H NMR (500 MHz, DMSO-d6): δ ppm 7.97 (d, J = 8.3 Hz, 1H),
7.56 (d, J = 8.3 Hz, 1H), 6.34 (s, 1H), 5.45 (br s, 4H), 4.35 (t, J = 7.4
Hz, 2H), 2.61 (s, 3H), 2.36 (m, 2H), 2.07 (quint, J = 7.4 Hz, 2H);
13C NMR (125 MHz, DMSO-d6): δ ppm 159.8, 154.7, 149.8, 149.2,
1
55: H NMR (500 MHz, DMSO-d6): δ ppm 7.97 (dm, 1H), 7.90
148.3, 134.7, 90.7, 127.9, 126.6, 115.4, 41.1, 30.6, 22.3, 14.6; HRMS
(TOF, ESI) m/z: calcd for C16H18N6F3 [M + H]+ 351.1467; found,
351.1533.
(d, J = 8.2 Hz, 1H), 7.61 (m, 1H), 7.53 (d, J = 8.3 Hz, 1H), 6.87 (m,
1H), 6.69 (dm, 1H), 6.36 (s, 2H), 5.65 (m, 1H), 5.50 (br, 4H), 4.53
(d, J = 5.9 Hz, 2H), 2.65 (s, 3H), 1.39 (d, J = 6.3 Hz, 3H); 13C NMR
(125 MHz, DMSO-d6): δ ppm 162.7, 159.8, 155.2, 149.7, 149.2,
148.5, 147.1, 139.8, 134.5, 126.5, 117.6, 115.3, 111.3, 93.7, 69.9, 46.8,
18.3, 14.9; HRMS (IT-TOF, ESI) m/z: calcd for C20H22N7O [M +
H]+ 376.1880; found, 376.1872; ee > 99.8%.
4-(3-{2-[(6-Bromopyridin-2-yl)oxy]ethyl}-2-methyl-3H-imidazo-
[4,5-b]pyridin-5-yl)pyridine-2,6-diamine (56). Starting from 0.30 g
of R3 (0.6 mmol) and using 2-fluoro-6-bromopyridine as the
appropriate aryl halide following general procedure III, 0.077 g
(18%) of 56 was obtained.
4-[2,3-Dimethyl-benzofuran-5-yl]pyridine-2,6-diamine (50).
Starting from 0.435 g of R1 (1.00 mmol) and using 0.337 g of 5-
bromo-2,3-dimethyl-benzofuran (1.50 mmol) as the appropriate
halide following general procedure I, 0.084 g (33%) of 50 was
obtained.
1H NMR (500 MHz, DMSO-d6): δ ppm 7.59 (d, J = 1.7 Hz, 1H),
7.47 (d, J = 8.5 Hz, 1H), 7.36 (dd, J = 8.5, 1.8 Hz, 1H), 5.95 (s, 2H),
5.44 (br s, 4H), 2.39 (br s, 3H), 2.16 (m, 3H); 13C NMR (125 MHz,
DMSO-d6): δ ppm 159.8, 153.7, 151.8, 150.8, 134.7, 130.8, 122.2,
116.7, 110.9, 110.2, 94.1, 12.1, 8.0; HRMS (TOF, ESI) m/z: calcd for
C15H16N3O [M + H]+ 254.1215; found, 254.1287.
1H NMR (500 MHz, DMSO-d6): δ ppm 7.94 (d, J = 8.3 Hz, 1H),
7.59(dd, J = 8.3, 7.4 Hz 1H), 7.55 (d, J = 8.3 Hz, 1H), 7.17(d, J = 7.4
Hz 1H), 6.78 (d, J = 8.3 Hz, 1H), 6.33 (s, 2H), 5.47 (s, 4H), 4.66 (m,
2H), 4.67 (m, 2H), 2.63s(3H); 13C NMR (125 MHz, DMSO-d6): δ
ppm 162.7, 159.8, 155, 1, 149.8, 149.2, 148.3, 142.5, 138.0, 134.6,
126.6, 121.4, 115.4, 110, 1, 64.6, 41.6, 14.7; HRMS (IT-TOF, ESI)
m/z: calcd for C19H19N7OBr [M + H]+ 440.0829; found, 440.0823.
4-(3-{2-[(6-Chloropyridin-2-yl)oxy]ethyl}-2-methyl-3H-imidazo-
[4,5-b]pyridin-5-yl)pyridine-2,6-diamine (57). Starting from 0.20 g
of R3 (0.4 mmol) and using 2-fluoro-6-chloropyridine as the
appropriate aryl halide following general procedure III, 0.028 g 57
(17%) was obtained.
4-{2-Methyl-3-[(2R)-2-phenoxypropyl]-3H-imidazo[4,5-b]-
pyridin-5-yl}pyridine-2,6-diamine (51) and 4-{2-Methyl-3-[(2S)-2-
phenoxypropyl]-3H-imidazo[4,5-b]pyridin-5-yl}pyridine-2,6-dia-
mine (52). Starting from 1.20 g of R2 (2.52 mmol) and using 2-
phenoxypropan-1-amine as the appropriate amine following general
procedure II, a mixture of 52 and 51 was obtained. The enantiomers
were separated on a CHIRALCEL OK column using MeOH+0.1%
DEA as an eluent to obtain 0.164 g (17%) of 51 as the first eluting
enantiomer and 0.166 g of (18%) 52 as the enantiomer eluting
second.
1
51: H NMR (500 MHz, DMSO-d6): δ ppm 7.94 (d, J = 8.3 Hz,
1H NMR (500 MHz, DMSO-d6): δ ppm 7.94 (d, J = 8.3 Hz, 1H),
7.69 (dd, J = 7.6, 0.5 Hz, 1H), 7.55 (d, J = 8.3 Hz, 1H), 7.04 (dd, J =
7.6, 0.5 Hz, 1H), 6.75 (dd, J = 8.3, 0.5 Hz, 1H), 6.33 (s, 2H), 5.47 (s,
4H), 4.67 (m, 4H), 2.63 (s, 3H); 13C NMR (125 MHz, DMSO-d6): δ
ppm 162.8, 159.8, 155.1, 149.8, 149.2, 148.3, 147.4, 142.7, 134.6,
126.6, 117.4, 115.4, 109.9, 93.7, 64.5, 41.8, 14.7; HRMS (IT-TOF,
ESI) m/z: calcd for C19H19N7ClO [M + H]+ 396.1334; found,
396.1331.
1H), 7.54 (d, J = 8.3 Hz, 1H), 7.20 (m, 2H), 6.87 (m, 2H), 6.86 (m,
1H), 6.35 (s, 2H), 5.49 (s, 4H), 4.97 (m, 1H), 4.52 (dd, J = 14.4, 7.1
Hz, 1H), 4.48 (dd, J = 14.5, 4.8 Hz, 1H), 2.65 (s, 3H); 13C NMR
(125 MHz, DMSO-d6): δ ppm 159.8, 157.5, 155.3, 149.9, 149.3,
148.5, 134.5, 130.1, 126.6, 121.3, 115.7, 115.5, 93.9, 72.2, 47.1, 18.2,
15.0; HRMS (TOF, ESI) m/z: calcd for C21H23N6O [M + H]+
375.1851; found, 375.1924; ee > 99.8%.
52: H NMR (500 MHz, DMSO-d6): δ ppm 7.94 (d, J = 8.3 Hz,
1H), 7.54 (d, J = 8.3 Hz, 1H), 7.20 (m, 2H), 6.87 (m, 2H), 6.86 (m,
1H), 6.35 (s, 2H), 5.49 (s, 4H), 4.97 (m, 1H), 4.52 (dd, J = 14.4, 7.1
1
4-(3-{(2R)-2-[(6-Bromopyridin-2-yl)oxy]propyl}-2-methyl-3H-
imidazo[4,5-b]pyridin-5-yl)pyridine-2,6-diamine (58). To 0.41 g of
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tert-butyl N-[6-(tert-butoxycarbonylamino)-4-[3-(2-hydroxypropyl)-
2-methyl-imidazo[4,5-b]pyridin-5-yl]-2-pyridyl]carbamate (0.8
mmol) was dissolved in 6 mL of DMF, then 0.098 g of sodium
hydride (2.5 mmol) was added portionwise, then the reaction mixture
was stirred at room temperature for 15 min followed by the addition
of 0.43 g of 2-bromo-6-fluoropyridine (2.5 mmol). The reaction
mixture was stirred at 50 °C until no further conversion was observed.
50 mL of EtOAc was added then the organic phase was washed with
water (3 × 25 mL) and brine (1 × 25 mL). The organic phase was
dried over MgSO4. The solvent was removed under reduced pressure
to give an intermediate, which was purified by flash column
chromatography using dichloromethane and methanolic ammonia as
eluents. The obtained solid was dissolved in 2 mL of DCM, 2 mL of
trifluoroacetic acid was added, and the mixture was stirred at room
temperature until no further conversion was observed. Volatiles were
removed under reduced pressure to give 0.17 g of racemic 4-(3-{2-
[(6-bromopyridin-2-yl)oxy]propyl}-2-methyl-3H-imidazo[4,5-b]-
pyridin-5-yl)pyridine-2,6-diamine as a crude product. The separation
of enantiomers by chiral HPLC on an AS-H column using heptane/
EtOH (60:40) containing 0.1% Et2NH gave 0.083 g (44%) of 58 as
the first eluting enantiomer.
Vilibald Kun − Servier Research Institute of Medicinal
Chemistry, H-1031 Budapest, Hungary
Nicolas Foloppe − Vernalis (R&D) Ltd., CB21 6GB
Cambridge, U.K.
Pawel Dokurno − Vernalis (R&D) Ltd., CB21 6GB
Cambridge, U.K.; orcid.org/0000-0002-7332-8889
Andrew J. Massey − Vernalis (R&D) Ltd., CB21 6GB
Cambridge, U.K.; orcid.org/0000-0002-0276-2573
David Lee Walmsley − Vernalis (R&D) Ltd., CB21 6GB
Cambridge, U.K.
Roderick E. Hubbard − Vernalis (R&D) Ltd., CB21 6GB
Cambridge, U.K.
James Murray − Vernalis (R&D) Ltd., CB21 6GB
Cambridge, U.K.; orcid.org/0000-0003-1007-8218
Karen Benwell − Vernalis (R&D) Ltd., CB21 6GB
Cambridge, U.K.
Thomas Edmonds − Institut de Recherches Servier, 78290
Croissy-sur-Seine, France
Didier Demarles − Technologie Servier, 45000 Orleans,
France
Alain Bruno − Institut de Recherches Servier, 78290 Croissy-
sur-Seine, France
Mike Burbridge − Institut de Recherches Servier, 78290
Croissy-sur-Seine, France
1H NMR (500 MHz, DMSO-d6): δ ppm 7.88 (d, J = 8.3 Hz, 1H),
7.52 (d, J = 8.3 Hz, 1H), 7.51 (t, J = 8.0 Hz, 1H), 7.07 (d, J = 7.5 Hz,
1H), 6.69 (d, J = 8.2 Hz, 1H), 6.36 (s, 2H), 5.60 (m, 1H), 5.48 (br s,
4H), 4.52 (d, J = 6.0 Hz, 2H), 2.65 (s, 3H), 1.41 (d, J = 6.3 Hz, 3H);
13C NMR (125 MHz, DMSO-d6): δ ppm 162.3, 159.8, 155.0, 149.7,
149.2, 148.5, 142.4, 137.8, 134.5, 126.5, 121.0, 115.3, 110.1, 93.8,
70.8, 46.7, 18.2, 14.9; HRMS (IT-TOF, ESI) m/z: calcd for
C20H21BrN7lO [M + H]+ 454.0985; found, 454.0970.
Francisco Cruzalegui − Institut de Recherches Servier, 78290
Croissy-sur-Seine, France
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00023
ASSOCIATED CONTENT
■
sı
* Supporting Information
Author Contributions
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00023.
The manuscript was written through contributions of all the
authors. All the authors have given approval to the final version
of the manuscript.
Description of data tables, structural determination
details, LC chromatograms, and H NMR spectra of
key compounds, (PDF)
1
Notes
The authors declare no competing financial interest.
Molecular formula strings (CSV)
ACKNOWLEDGMENTS
■
Accession Codes
The authors thank co-workers at the Analytical Division of the
Servier Research Institute of Medicinal Chemistry for
providing detailed chemical analysis of the compounds.
The X-ray structures mentioned in this paper have been
deposited in the PDB with the following codes. Dyrk1a: 3:
7AJ2, 5: 7AJ4, 10: 7AJ5, 16: 7AJ7; 25: 7AJ8, 28: 7AJA, 32:
7AJM, 33: 7AJS; 38: 7AJV, 46: 7AJW, 50: 7AKL; 51: 7AJY;
53: 7AK2, 54: 7AKA, 56: 7AKB, 58: 7AKE. Dyrk2: 50: 7AKF;
58: 7AKH. The authors will release the atomic coordinates and
experimental data upon article publication.
ABBREVIATIONS USED
■
DYRK, dual specificity tyrosine-regulated kinase; PDB, protein
databank; PK, pharmacokinetic; PD, pharmacodynamic; TR-
FRET, time-resolved fluorescence energy transfer; NMR,
nuclear magnetic resonance; LE, ligand efficiency; CDK9,
cyclin-dependent kinase 9; CLK, CDC-like kinase; hERG,
human ether-a-go-go related gene; HIPK2, Homeodomain-
interacting protein kinase 2; SCID, severe combined
immunodeficiency; po, per os; SAR, structure−activity
relationship
AUTHOR INFORMATION
Corresponding Author
■
Andras Kotschy − Servier Research Institute of Medicinal
Chemistry, H-1031 Budapest, Hungary; orcid.org/0000-
0002-7675-3864; Phone: +36 (1) 881-2000;
Email: andras.kotschy@servier.com; Fax: +36 (1) 881
2011
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