Structure-guided evolution of potent and selective CHK1 inhibitors through scaffold morphing

Article abstract of DOI:10.1021/jm2007326

Pyrazolopyridine inhibitors with low micromolar potency for CHK1 and good selectivity against CHK2 were previously identified by fragment-based screening. The optimization of the pyrazolopyridines to a series of potent and CHK1-selective isoquinolines demonstrates how fragment-growing and scaffold morphing strategies arising from a structure-based understanding of CHK1 inhibitor binding can be combined to successfully progress fragment-derived hit matter to compounds with activity in vivo. The challenges of improving CHK1 potency and selectivity, addressing synthetic tractability, and achieving novelty in the crowded kinase inhibitor chemical space were tackled by multiple scaffold morphing steps, which progressed through tricyclic pyrimido[2,3-b] azaindoles to N-(pyrazin-2-yl)pyrimidin-4-amines and ultimately to imidazo[4,5-c]pyridines and isoquinolines. A potent and highly selective isoquinoline CHK1 inhibitor (SAR-020106) was identified, which potentiated the efficacies of irinotecan and gemcitabine in SW620 human colon carcinoma xenografts in nude mice. (Figure presented)

Full text of DOI:10.1021/jm2007326

Article
pubs.acs.org/jmc
Structure-Guided Evolution of Potent and Selective CHK1 Inhibitors
through Scaffold Morphing
John C. Reader,*,§,⊥ Thomas P. Matthews,†,⊥ Suki Klair,§ Kwai-Ming J. Cheung,† Jane Scanlon,§
Nicolas Proisy,† Glynn Addison,§ John Ellard,§ Nelly Piton,§ Suzanne Taylor,§ Michael Cherry,§
Martin Fisher,§ Kathy Boxall,† Samantha Burns,† Michael I. Walton,† Isaac M. Westwood,†,‡
Angela Hayes,† Paul Eve,† Melanie Valenti,† Alexis de Haven Brandon,† Gary Box,†
Rob L. M. van Montfort,†,‡ David H. Williams,§ G. Wynne Aherne,† Florence I. Raynaud,†
Suzanne A. Eccles,† Michelle D. Garrett,† and Ian Collins*,†
†Cancer Research UK Cancer Therapeutics Unit and ‡Division of Structural Biology, The Institute of Cancer Research, 15 Cotswold
Road, Sutton, Surrey SM2 5NG, U.K.
§Sareum Ltd., 2a Langford Arch, London Road, Cambridge CB22 3FX, U.K.
S
* Supporting Information
ABSTRACT: Pyrazolopyridine inhibitors with low micromolar potency
for CHK1 and good selectivity against CHK2 were previously identified
by fragment-based screening. The optimization of the pyrazolopyridines
to a series of potent and CHK1-selective isoquinolines demonstrates how
fragment-growing and scaffold morphing strategies arising from a
structure-based understanding of CHK1 inhibitor binding can be
combined to successfully progress fragment-derived hit matter to
compounds with activity in vivo. The challenges of improving CHK1
potency and selectivity, addressing synthetic tractability, and achieving novelty in the crowded kinase inhibitor chemical space
were tackled by multiple scaffold morphing steps, which progressed through tricyclic pyrimido[2,3-b]azaindoles to N-(pyrazin-2-
yl)pyrimidin-4-amines and ultimately to imidazo[4,5-c]pyridines and isoquinolines. A potent and highly selective isoquinoline
CHK1 inhibitor (SAR-020106) was identified, which potentiated the efficacies of irinotecan and gemcitabine in SW620 human
colon carcinoma xenografts in nude mice.
by siRNA and several small molecule inhibitors, including the
lead compound resulting from the studies reported herein,17
has confirmed this in preclinical studies.18−23
INTRODUCTION
■
The DNA damage response network ensures the fidelity of
DNA replication and controls the repair of damage arising
during cellular replication or from exogenous agents such as
genotoxic drugs. Checkpoint kinase 1 (CHK1) is a serine/
threonine kinase occupying a central position in this complex
network of cell regulatory and DNA repair mechanisms.1−3
CHK1 is predominantly activated through phosphorylation on
amino acid residues Ser317 and Ser345 by the upstream kinase
Ataxia Telangiectasia and Rad3 Related (ATR) in response to
single strand breaks in DNA,4,5 and it undergoes autophos-
phorylation on Ser296.6,7 G1/S, S, or G2/M cell cycle
checkpoints are activated in response to genotoxic antitumor
drugs to provide an opportunity for repair of damaged DNA or
to activate apoptotic pathways.8 CHK1 is involved in the S-
phase checkpoint and stabilizing replication forks, and in the
G2/M checkpoint through regulating the stability of the
CDC25 phosphatases which control cell cycle progression by
regulation of CDK1.9−12 Human cancers frequently have
functional defects in the tumor suppressor p53, with
consequent loss of G1/S checkpoint control13,14 and greater
reliance on S and G2/M checkpoints. Thus, S or G2/M
checkpoint inhibitors will selectively sensitize p53 deficient
cancer cells to DNA damaging agents.1,8,15,16 CHK1 inhibition
A range of adenosine 5′-triphosphate (ATP) competitive
CHK1 inhibitors have been reported, with varying selectivity
for the target enzyme over other kinases.23−26 Inhibitor
development has been assisted by structural biology studies
of the enzyme, in particular crystallographic analysis of inhibitor
binding to the protein.27−30 As well as ATP-competitive
inhibitors, allosteric modulators of CHK1 have been identi-
fied.31 Some ATP-competitive CHK1 inhibitors have reached
early clinical trials in combination with DNA-damaging agents,
although the therapeutic outcomes remain to be deter-
mined.2,15,23,32
RESULTS AND DISCUSSION
■
Previously we have shown how a fragment-based screening
strategy identified several low molecular weight, ligand efficient
templates which were developed into early stage CHK1
inhibitors.33,34 The purine template hit 1 was initially
Received: June 8, 2011
Published: November 23, 2011
© 2011 American Chemical Society
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elaborated to the pyrazolopyridine lead 2.33 Here we describe
how structure-based design led, through several scaffold
morphing steps, to the evolution of 2 into a potent and
selective CHK1 inhibitor (R)-3 (SAR-020106,17 Figure 1).
environment for the binding of water molecules or polar ligand
functionality. Thus, successful targeting of the interior pocket in
CHK1 with polar functionality is predicted to lead to selectivity
against CHK2.
Among several analogues prepared,38 the trisubstituted
pyrazolopyridine analogue 4 retained the potency of 2 (Table
1) and was shown by X-ray crystallography to hydrogen bond
to a water in the interior pocket through the nitrile group
(Figure 2B). However, the crystal structure also indicated that
the ligand was unable to simultaneously position all three
substituents optimally in the ATP-binding site due to steric
congestion. The 2-(aminomethyl)morpholine and ethyl ester
were both twisted into potentially undesirable conformations
compared to 2, with the ester oriented orthogonal to the
bicycle. The ligand attained only equivalent potency to 2
despite the addition of extra interacting functionality, reflected
in the reduced ligand efficiency. We attempted to resolve the
overcrowding by reverting to a pyrrolo[2,3-d]pyrimidine core33
and removing the pendant ethoxycarbonyl group. Substitution
with the 3-(3-cyanophenyl) group resulted in compound 5,
having slightly reduced CHK1 potency relative to 4, while the
3-(3-(hydroxymethyl)phenyl) analogue 6 gave a 3-fold increase
in activity. However, the selectivities for CHK1 against CHK2
were reduced for both 5 and 6 compared to 2.
Figure 1. Structures of the template hit 1, early stage lead 2, and (R)-3
(SAR-020106). Features conserved in the elaboration of the template
hit and early stage lead into a potent and selective CHK1 inhibitor are
highlighted in blue and red.
The crystal structure of 2 bound to CHK1 confirmed that
the ligand occupied the ATP pocket and interacted with the
hinge region of the kinase. The predicted hydrogen bonds were
observed between N1 and N7 of the pyrazolo[3,4-b]pyridine
with the backbone amides of Glu85 and Cys87, respectively
(Figure 2A). The 2-(aminomethyl)morpholine group was
located in the ribose pocket, though no specific polar
interactions were observed, while the ethyl ester was directed
out onto the solvent exposed surface. The area around the
gatekeeper and interior pocket was unexplored, and our initial
synthetic effort concentrated on probing this area. CHK1
contains a polar amino acid (Asn59; see Figure 2A) in the
interior pocket beyond the gatekeeper residue, which differ-
entiates the enzyme from many other protein kinases where a
hydrophobic amino acid is located at the equivalent
position.27,29 As a result, the interior pocket of CHK1, defined
by Asn59, Glu55, and the Lys38-Asp148 salt bridge, is often
observed in crystal structures to contain between 1 and 3
protein bound water molecules (see Figure 2A−E). Ligand
interactions with these protein bound water molecules have
been shown to be important in conferring both CHK1 potency
and selectivity against other kinases.27,29
Examination of the crystal structure of 2 suggested that a
pendant phenyl at the 3-position of the pyrazolo[3,4-b]pyridine
could provide a suitable vector toward the interior pocket.
Alternatively, fusing a third ring to the core to form a tricycle
could also provide a platform for substitution to contact the
protein-bound water molecules. Accordingly, sets of analogues
exploring these hypotheses were prepared and evaluated. We
focused on substitution with functional groups having the
potential to form hydrogen bonding interactions with the
bound water molecules or the polar residues comprising the
Asp148-Lys38 salt bridge in CHK1. Inhibition data for selected
compounds tested against CHK1 are described in Table 1. In
addition, we investigated the ability of the compounds to
inhibit the functionally distinct checkpoint kinase 2 (CHK2).35
Selectivity for inhibition of CHK1 over CHK2 is desirable,
since simultaneous ablation of both CHK1 and CHK2 by RNAi
has been shown to be inferior to selective depletion of CHK1
alone in overriding the S-phase checkpoint.36 The interior
pocket of CHK2 contains a hydrophobic leucine residue at the
equivalent site to Asn59 in CHK1, providing a less favorable
The crystal structure of 6 (Figure 2C) showed that although
steric crowding around the core was reduced, the 3-
hydroxymethyl substituent had adopted an alternative
orientation along a vector into the ribose pocket and away
from the interior pocket. The selectivity of compound 2 for
CHK1 over CHK2 indicates that, in common with many ATP-
competitive inhibitors binding to active kinase conformations,39
some specificity for inhibition can be obtained through
interactions with the selectivity (or solvent-exposed) surface
at the entrance to the ATP-binding cleft. The amino acid
residues comprising this region vary between kinases and thus
alter the properties of the surface. In CHK1 the surface includes
contributions from Tyr86, Cys87, and Ser88 (see Figure 2F),
while in CHK2 the equivalent residues are leucine, methionine,
and glutamate, respectively. In compound 6 the absence of the
pendant ethoxycarbonyl group and the failure of the
hydroxymethyl group to engage with the water-filled pocket
removed the potential contributions to CHK1 selectivity.
One possible approach to restoring selectivity was to replace
the morpholine substituent with a less bulky, unbranched
alkylamine and reintroduce the 5-substituent to the pyrazolo-
[3,4-b]pyridine core. Less bulky alkylamine 4-substituents
would be anticipated to relieve the crowding seen in 4 and
permit both the 3- and 5-substituents to be present in favorable
conformations for binding to CHK1. However, the 2-
(aminomethyl)morpholine or similar cyclic 4-substituents on
the 3-aryl substituted bicyclic cores were important in
establishing novelty to provide patentable compounds in an
area which has been intensively exemplified as kinase inhibitor
scaffolds.40−46 Instead, we maintained the morpholine sub-
stituent but elaborated the bicyclic core by the addition of a
fused ring.
Initial compounds prepared from a commercially available
pyrimidoindole tricyclic core (7) gave compounds with similar
CHK1 potencies and ligand efficiencies to 2. No preference was
seen for the enantiomers of the 2-(aminomethyl)morpholine
group, (R)-7 and (S)-7. The requirement for the inclusion of a
suitable H-bond donor or acceptor capable of interacting with
the protein-bound water molecules led to a more involved
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Figure 2. Crystal structures of 2 (panel A, PDB 2ym3), 4 (panel B, PDB 2ym4), 6 (panel C, PDB 2ym5), 8 (panel D, PDB 2ym6), 20 (panel E,
PDB 2ym7), and (R)-3 (panel F, PDB 2ym8) bound to the CHK1 kinase domain. Red spheres represent water molecules modeled in the
refinement. Dashed gray lines represent hydrogen bonds. Panel G: Plan view of the overlay of CHK1 inhibitors 2 (cyan), 8 (gray), 20 (blue), and
(R)-3 (gold) relative to the hinge region of CHK1 (cartoon).
synthetic effort, resulting in both the nitrile substituted
analogue 9 and the introduction of a nitrogen into the fused
ring to give the 9H-pyrimido[2,3-b]azaindole 8. While addition
of the nitrile group 9 led to a drop in LE, the more compact 8
gave equivalent potency and LE to 2 and restored some of the
selectivity over CHK2. X-ray crystallography confirmed that the
azaindole nitrogen effectively contacted the network of bound
waters in the interior pocket of CHK1, providing a rationale for
the recovery of the selectivity in the absence of a substituent
directed toward Ser88 and the selectivity surface (Figure 2D).
Importantly, the pyrimido[2,3-b]azaindole core of 8 provided
the opportunity to introduce a more varied range of alkylamino
substituents to replace the 2-(aminomethyl)morpholine while
still generating novel inhibitors.47 The influence of the
alkylamino group was probed with an array of analogues,
including 10−13, resulting in the identification of the 4-
(aminomethyl)piperidine 13 as a more potent and selective
alternative. However, the rigid planar nature of the tricyclic
scaffold and synthetic challenges to further elaboration
prompted us to consider another scaffold morphing step and
to disconnect the middle ring of the tricycle. This was
envisaged to introduce a degree of flexibility into the core
hinge-binding pharmacophore, potentially allowing the ligand
to adopt a better conformation for simultaneous interaction
with the hinge region and the water-filled pocket. An efficient
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Table 1. Inhibition of CHK1 and CHK2 by Bicyclic and Tricyclic Compounds
a
a
b
c
CHK1 Inhibition IC50 (μM)
CHK2 Inhibition IC50 (μM)
Selectivity (CHK2/CHK1)
50
LE (kcal mol−1 non-H atom−1
)
2
1.0 (0.86, 1.2)
1.5 (1.3, 1.8)
3.2 (2.2, 4.2)
0.43 (0.26, 0.60)
3.2 (2.4, 3.9)
3.0 (2.9, 3.2)
2.3 (1.55, 3.0)
50 (26, 74)
0.38
0.27
0.31
0.36
0.37
0.37
0.38
0.40
0.33
0.47
0.45
0.43
0.43
d
4
n.d.
5
28 (13, 43)
9
8
e
6
3.4 (±1.16)
rac-7
(R)-7
(S)-7
8
12 (11, 13)
13 (11, 16)
3.5 (2.3, 4.6)
19 (16, 23)
10 (9.3, 11)
23 (22, 24)
11 (8.1, 14)
16 (10, 21)
12 (6.8, 16)
4
4
2
e
0.88 (±0.21)
22
3
9
3.7 (3.2, 4.2)
1.7 (1.7, 1.8)
1.7 (1.7, 1.8)
0.67 (0.53, 0.80)
10
11
12
13
14
6
24
e
0.29 (±0.02)
41
a
IC50 determined in a dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA).33 Mean of two independent determinations; individual
b
values in parentheses. Standard inhibitor staurosporine gave CHK1 IC50 = 2.1 (±1.8) nM and CHK2 IC50 = 27 (±8) nM. Ratio of IC50 values
(CHK2/CHK1). Ligand efficiency (LE) calculated using LE = [−1.4 log10(IC50 (M))]/(number of heavy atoms).37 Not determined. Mean
c
d
e
(±SD) of at least three independent determinations.
synthesis of a small array of compounds allowed this variation
to be quickly assessed (Table 2).
relationship is in contrast with that demonstrated for (6-
cyanopyrazin-2-yl)urea CHK1 inhibitors,48,49 where replace-
ment of a pendant amine with nonbasic polar groups was
tolerated. Thus, although occupying similar space within the
ATP-binding site, as shown by the crystallographic data, and
interacting with some similar points on the CHK1 protein, the
two series did not have identical structure−activity relation-
ships.
With limited points of substitution that would enable us to
probe the solvent-exposed selectivity surface, we sought to
exchange the pyrimidine for a pyridine. Consistent with the
structure−activity relationships discussed above, the reintro-
duction of the ester 5-substituent in 23 increased CHK1 affinity
by 4-fold (23 vs 20) and CHK1 vs CHK2 selectivity to
approximately 300-fold. The 4,5-disubstituted pyridine scaffold
presented one opportunity for optimization of the inhibitors,
which will be reported separately. We also pursued a further
scaffold morphing step, seeking to combine the 4- and 5-
substituents into a fused ring to give imidazo[4,5-c]pyridines
and isoquinolines. This gave the micromolar inhibitors 24 and
27 (Table 3), with encouraging ligand efficiencies (>0.3 kcal
mol−1 heavy atom−1) for the unsubstituted scaffolds.
We explored the reintroduction of basic amine substituents
to mimic the substitution of 20 and 23, originating either from
the imidazole nitrogen of the imidazo[4,5-c]pyridines or
through tethering via an ether linkage to the pyrazine portion
of the scaffolds.27 As shown by the representative example 25,
the analogues with the side chain originating from the
imidazo[4,5-c]pyridines had substantially decreased affinity
(Table 3). Moving the basic amine substituent to the pyrazine
in 26 and 28 gave much improved activity against CHK1 with
good selectivity over CHK2. While both imidazo[4,5-c]-
The potency of simple N-(pyridin-3-yl)pyrimidin-4-amines
(see representative example 14) suffered, presumably as the
steric clash between the pyrimidine 5-H and pyridine 4′-H
would force the core to adopt too extreme a nonplanar
conformation. By switching the pyridine to a pyrazine ring, this
was overcome, and the first “ring opened” adaptation, N-
(pyrazin-2-yl)pyrimidin-4-amine (15), showed some activity,
though disappointingly 75-fold less than that of the parent
compound, 13. The incorporation of pyrazine substitution into
CHK1 inhibitors has been reported previously for a series of
pyrazin-2-yl ureas,48,49 where the additional nitrogen atom in
the pyrazine ring was shown by crystallographic studies to be
important in restraining the urea moiety in the bioactive
conformation.27 Comparisons with these CHK1 inhibitors
suggested that appending a nitrile in the 2-position of the
pyrazine could be beneficial through achieving additional
interactions with the protein in the region of the water-filled
pocket. Comparing 15 with 16, the addition of the nitrile
increased the CHK1 potency 15-fold. Optimization of the
diamine substituent proved fruitful, as 4-(aminomethyl)-
piperidine gave excellent CHK1 potency while retaining
selectivity over CHK2 when incorporated into the N-
(pyrazin-2-yl)pyrimidin-4-amine 20.
Crystallographic data explained the increased activity and
selectivity of the 2-cyanopyrazines, as there was a clear
interaction of the nitrile with the conserved Lys38 and the
water network filling the interior pocket (see Figure 2E).
Interestingly, a sharp loss of activity was seen when the terminal
amine (19−20) was replaced with structurally similar but
nonbasic polar groups (21−22). This structure−activity
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to Lys38, and the dimethylamine nestled in the ribose pocket.
The branched methyl on the side chain fitted closely to Val23
while the isoquinoline chloro substituent was close to the
specificity surface without projecting too far toward solvent.
The distance from the chlorine to the carbonyl oxygen of Ser88
measured in the 2.1 Å resolution crystal structure was 3.44 Å,
greater than the sum of the Cl and O van der Waals radii. The
potency of (rac)-3 was only approximately twice that of the
des-chloro analogue 34, suggesting that an oxygen−halogen
interaction was not strongly contributing to the binding energy
of (R)-3. However, comparison of 34 and (rac)-3 showed that
selectivity against CHK2 was improved from 120-fold to
greater than 3000-fold by the addition of the 8-chloro
substituent.
Table 2. Inhibition of CHK1 and CHK2 by N-(Pyridin-3-yl)-
or N-(Pyrazin-2-yl)pyrimidin-4-amines
Selected compounds were investigated for activity in cellular
assays during optimization of CHK1 potency. Cytotoxicity was
measured with a sulforhodamine B (SRB) assay51 using the
human colon carcinoma cell line HT29. A cell based enzyme-
linked immunosorbent assay (ELISA) was developed to
measure G2 checkpoint abrogation by quantifying the level of
M-phase phosphoprotein 2 (MPM2) expression (a measure of
mitosis) in HT29 cells that passed through an etoposide-
induced G2 arrest into mitosis, where they were trapped using
nocodazole.17 The ratio of SRB cytoxicity to cellular ELISA
activity gave an activity index (AI), which we found to be a
useful indicator of selective inhibition of CHK1 in cells.17,48
As shown in Table 4 with selected examples of the scaffolds
described above, the cytotoxity was generally weak, with only
30 and (R)-3 giving GI50 values in the nanomolar range. The
potency in the G2 checkpoint abrogation assay in general
tracked the CHK1 activity. Isoquinolines 28, 30, and (R)-3,
three compounds with CHK1 IC50 <100 nM, abrogated the
etoposide-induced G2 checkpoint with IC50 < 200 nM.
Conversely, the imidazo[4,5-c]pyridines, e.g. 26, demonstrated
good potency in the enzyme assay but showed minimal activity
in both the G2 checkpoint abrogation and SRB assays. We
attributed this to poor permeability of the imidazo[4,5-
c]pyridine core scaffold when paired with the piperidine,
consistent with the high polar surface area calculated for 26.
Compounds abrogating the G2 checkpoint at nanomolar
concentrations while remaining relatively noncytotoxic (AI > 4)
were assessed further to gauge the enhanced efficacy when used
in combination with a cytotoxic drug. HT29 colon cancer cells
were exposed to a fixed concentration of SN38 (the active
metabolite of the DNA topoisomerase I inhibitor irinotecan52)
that inhibited growth by 50% relative to untreated controls, and
they were additionally exposed to increasing concentrations of
the CHK1 inhibitor in a 96 h SRB assay. The ability of the
CHK1 inhibitor to enhance the cytotoxicity of SN38 was
expressed as a potentiation index (PI), which was the ratio of
GI50 for the CHK1 inhibitor alone and the GI50 for the CHK1
inhibitor in combination with SN38.17 Potentiation was
observed for 27, 28, 29, and (R)-3, with all except 27 giving
a greater than 2-fold potentiation. The two analogues having
the most favorable properties overall were 28 and (R)-3, which
had GI50 values of 345 nM and 190 nM, respectively, in the
presence of SN38. These encouraging cellular assay data led to
further profiling of (R)-3.
a
IC50 determined in DELFIA.33 Mean of two independent
determinations; individual values in parentheses. Standard inhibitor
staurosporine gave CHK1 IC50 = 2.1 (±1.8) nM and CHK2 IC50 = 27
b
c
(±8) nM. Ratio of IC50 values (CHK2/CHK1). LE = [−1.4
log10(IC50 (M))]/(number of heavy atoms).37 Not determined.
d
e
f
Mean (±SD) of at least three independent determinations. Single
determination.
pyridines (e.g., 26) and isoquinolines (e.g., 28) were potent and
selective CHK1 inhibitors, the cellular activities of the
imidazo[4,5-c]pyridines were substantially lower than those of
the corresponding isoquinolines (see Table 4 and discussion
below), and thus, further optimization concentrated on the
latter heterocycle.
Several published urea-based CHK1 inhibitors49 direct a
chlorine substituent toward the selectivity surface. Overlays of
our isoquinoline scaffold with these ureas showed that a
chlorine in the 8-position could be similarly positioned and
have the potential to make a short oxygen−halogen interaction
with the carbonyl oxygen of Ser88.50 To explore this,
reoptimization of the basic amine substituent was conducted
on isoquinolines with and without 8-chloro substitution (Table
3). In the main, the chlorinated analogues retained CHK1
activity, and this set of compounds led us to the potent and
selective compound (rac)-3. The CHK1 enzyme inhibitory
activity of (rac)-3 could be attributed, on testing of the
enantiomeric pair, to the R-enantiomer, (R)-3.
The inhibitory activity of (R)-3 was assessed at 1 μM
concentration against a representative sample of 124 kinases
from across the human kinome, using radiometric assay
format56 at [ATP] ∼ Km,ATP for all the enzymes (Figure 3).
Only CHK1 and 6 other kinases out of 124 showed >80%
X-ray crystallography of (R)-3 in the CHK1 enzyme (Figure
2F) showed binding through hydrogen-bonding to Glu85 and
Cys87 in the hinge region, the hydrogen-bonding of the nitrile
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Table 3. Inhibition of CHK1 and CHK2 by Imidazo[4,5-c]pyridines and Isoquinolines
a
IC50 determined in DELFIA.33 Mean of two independent determinations; individual values in parentheses. Standard inhibitor staurosporine gave
CHK1 IC50 = 2.1 (±1.8) nM and CHK2 IC50 = 27 (±8) nM. LE = [−1.4 log10(IC50 (M))]/(number of heavy atoms).34 Not determined. Mean
(±SD) of at least three independent determinations. Single determination. Poor solubility gave variability for this analogue in this assay.
b
c
d
e
f
inhibition at 1 μM. A further 8 kinases showed some inhibition
(40−80%) at 1 μM, but the majority of kinases tested (109/
124) showed less than 40% inhibition at 1 μM concentration of
(R)-3, indicative of >100-fold selectivity. The kinase inhibitory
activities of (R)-3 were also determined in a smaller panel at
the higher concentration of 10 μM (see Supporting
Information). The compound also displayed good selectivity
at this higher concentration, where only CHK1 and 8 other
kinases out of 50 tested showed >80% inhibition.
Metabolic turnover of (R)-3 in mouse and rat liver
microsomes was high (80% and 92% metabolized at 30 min,
respectively), but the compound was appreciably more stable in
human liver microsomes (35% metabolized at 30 min). Good
permeability was seen for a monolayer of human intestinal
epithelial cells (CaCo-2; A to B Pe 1.43 × 10−6 cm s−1) with no
active efflux observed (ratio A → B/B → A 0.25).
(R)-3 was studied in vivo as previously described,17 including
pharmacodynamic and efficacy determinations in SW620
human colon carcinoma xenografts in nude mice.59 Although
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Table 4. G2 Checkpoint Abrogation and Potentiation of SN38 Cytotoxicity in Human Colon Cancer Cells by Selected
Compounds
CHK1 IC
SRB (HT29) GI50
Checkpoint abrogation (HT29) IC50
Potentiation of SN38 cytotoxicity (HT29)
a 50
b
c
d
e
e
(μM)
(μM)
(μM)
P.I.
ALogP
TPSA
f
f
2
1.0
64
18.5
−0.22
−0.44
1.0
106
93
f
f
8
0.88
0.29
0.40
0.12
1.9
62
13.5
f
f
f
13
19
20
24
26
27
28
29
30
(R)-3
17.5
f
3.1
84
24
n.d.
−0.21
125
111
103
124
74
76.5
n.d.
0.077
0.19
1.5
46 (±39, n = 3)
125 (120, 130)
26 (21, 30)
23 (±22, n = 3)
128 (120, 135)
>100, >100
0.17 (0.16, 0.17)
2.7 (2.0, 3.3)
0.049
1.2
1.3
3.3
2.6
2.2
0.017
0.74
0.022
0.013
1.2 (1.1, 1.2)
2.3
96
120 (120, 120)
0.53 (0.52, 0.53)
0.47 (±0.19, n = 11)
2.2
74
g
0.18, 0.008
2.9
96
0.055 (±0.019, n = 5)
3.1 (±1.6, n = 5)
3.9
87
a
b
IC50 determined in DELFIA. Mean of at least two independent determinations. SRB cytotoxicity assay; mean of two independent determinations
unless otherwise stated. G2 checkpoint abrogation assay.17 PI; ratio of SRB GI50 for CHK1 inhibitor alone to GI50 for the combination of CHK1
c
d
inhibitor and SN38 (see ref 17). ALogP53 and TPSA54 calculated with Pipeline Pilot (v7).55 Single determination. Poor aqueous solubility gave
e
f
g
variability for this analogue in this assay.
Figure 3. Selectivity profile of (R)-3 against a panel of 124 kinases measured at 1 μM concentration of the test compound with [ATP] ∼ Km,ATP for
each kinase. Kinase dendrogram58 reproduced courtesy of Cell Signaling Technology, Inc. (www.cellsignal.com).
having minimal oral bioavailability in mice (F = 5%),
distribution of (R)-3 following i.p. dosing (40 mg/kg) was
sufficient to inhibit CHK1 in the tumors, as shown by
inhibition of the irinotecan-induced CHK1 pS296 autophos-
phorylation (Figure 4A).17 At doses giving inhibition of CHK1
activity in vivo, the selective CHK1 inhibitor (R)-3 showed no
single agent activity in the SW620 xenograft model, and tumors
grew at similar rates to the vehicle-treated controls. When
dosed (i.p.) in combination with irinotecan, (R)-3 was
observed to potentiate the antitumor activity of the genotoxic
drug in the SW620 xenograft model. Tumor growth was
delayed when compared to the vehicle-treated control, CHK1
inhibitor alone, or cytotoxic drug alone. Similar data were
obtained for the combination of (R)-3 with gemcitabine57
(Figure 4B and see ref 17). These experiments demonstrated
that the in vitro cellular potency and potentiation of genotoxic
cytotoxicity by (R)-3 translated into in vivo biomarker
modulation and potentiation of genotoxic drug efficacy
expected for a selective CHK1 inhibitor.17,23
CONCLUSION
■
The evolution of pyrazolopyridine 2, a micromolar inhibitor of
CHK1, to the potent and selective isoquinoline inhibitor (R)-3
demonstrates how fragment-growing and scaffold morphing
strategies can be combined to successfully progress fragment-
derived hit matter to compounds with appropriate activity in
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Figure 4. (A) Status of pS296 CHK1 autophosphorylation in SW620 tumor xenografts following treatment with either irinotecan alone or in
combination with (R)-(3).17 Tumor bearing animals were administered either vehicle alone, irinotecan alone (25 or 50 mg/kg ip), or irinotecan
combined with a fixed dose of (R)-(3) (40 mg kg−1 ip) 1 h prior to irinotecan administration. Tumors were collected 6 or 24 h following treatment
(6 or 24 after administration of the first agent for combinations) and snap frozen. Tumor protein expression was characterized by Western blotting
using 50 μg of sample per lane. (B) Antitumor effects of combining (R)-3 with irinotecan in nude mice bearing SW620 xenograft tumors.17 Symbols:
(↑) dose administered; (■) vehicle alone; (shaded up triangles) irinotecan alone (12.5 mg/kg ip); (shaded down triangles) (R)-3 alone (40 mg/kg
ip); (⧫) irinotecan and (R)-3 combined ((R)-3 dosed 1 h prior to and 24 h after irinotecan). Values are mean ± SEM; n = 9−11.
vivo. By incorporating inhibitor selectivity against CHK2 as a
goal from the start of the medicinal chemistry, and guided by
determinants of CHK1 selectivity apparent from the structural
biology of the enzyme, the promising selectivity for CHK1 in
the early fragment-derived leads was maintained and improved.
Fragment-growing identified two productive vectors for
enhancing the potency and selectivity of the initial bicyclic
inhibitors: an extension to contact the water-filled pocket of
CHK1, best achieved with a tricyclic heteroaromatic frame-
work, and substitution to bind at the specificity surface of the
ATP site. The determination of multiple protein−ligand
structures during the optimization confirmed the introduction
of productive new binding interactions and allowed a
rationalization of the poor affinity of sterically crowded bicycles
which adopted energetically unfavorable conformations on
binding.
xenografts. At a pharmacodynamically active dose, the inhibitor
potentiated the antiproliferative efficacy of the genotoxic agents
irinotecan and gemcitabine toward human SW620 colon cancer
xenografts in nude mice, and minimal effects of the selective
CHK1 inhibitor alone were shown. While optimization of
pharmacokinetic properties is desirable to generate compounds
suitable for development, the isoquinoline (R)-3 is a useful in
vitro and in vivo chemical tool to investigate the effects of
selective CHK1 inhibition.
EXPERIMENTAL SECTION
■
Synthetic Chemistry. The synthetic procedures for the four
scaffolds and the analogues discussed in this paper (3−34) are
outlined in Schemes 1−4. The previously described pyrazolopyridine
a
Scheme 1.
Scaffold modification, or morphing, is a common strategy for
improvement and diversification of lead molecules derived from
screening approaches. In this case, breaking up the tricyclic
scaffold to a series of N-(pyrazin-2-yl)pyrimidin-4-amines,
followed by optimization of a basic amino-substituted side-
chain, maintained the hinge-binding hydrogen bonds and other
key interactions for CHK1 potency and selectivity, while
addressing challenges of synthetic tractability and crowded
intellectual property space encountered with the tricyclic and
bicyclic inhibitors, respectively. A further scaffold morphing
step was achieved through translocation of the basic amine side-
chain, allowing diversification of the hinge-binding group to
include a series of 2-amino-isoquinolines. This approach led to
the isoquinoline (R)-3 (SAR-020106), a potent inhibitor of
CHK1 with excellent selectivity with respect to CHK2 and a
broader sample of the kinome, representing one of the most
selective CHK1 inhibitors reported to date. Despite the
extensive scaffold modification, the isoquinoline (R)-3 (MW
= 382) retained the good ligand efficiency of the original
template hit, 6-(morpholin-4-yl)purine 1.
Cell-based assays for selective CHK1 inhibition, through
measuring the abrogation of an etoposide-induced G2
checkpoint, and for nonselective cytotoxicity were used to
confirm that the observed kinase selectivity translated into the
expected CHK1-selective pharmacology in cells. The pharma-
cokinetic properties of the optimized isoquinoline (R)-3 were
sufficient to demonstrate inhibition of CHK1 in vivo on i.p.
dosing. Pharmacodynamic biomarkers showing the activation of
CHK1 by genotoxic drugs were inhibited in human tumor
a
Reagents and conditions: (i) NBS, DMF, rt, 77%; (ii)
PdCl2(dppf)·CH2Cl2, 3-cyanophenylboronic acid, Na2CO3, DME/
H2O, 140 °C, 2 h, microwave, 48%; (iii) TFA, MeOH, 80 °C, 62%;
(iv) NBS, CH2Cl2, rt, 83%; (v) NaH, SEM-Cl, DMF, 0 °C to rt, 93%;
(vi) Boc-amine, TEA, n-BuOH, 100 °C, 68%; (vii) Pd(PPh3)4,
phenylboronic acid, Na2CO3, DME/H2O, 120 °C, 30 min, microwave,
49−59%; (viii) TBAF, ethane-1,2-diamine, DMF, 60 °C, 7 h and then
TFA, MeOH, 80 °C, 21%; (ix) TBAF, ethane-1,2-diamine, DMF, 60
°C, 7 h and then 4 M HCl−dioxane, MeOH, 24 h, rt, 17%.
3533 was brominated at the 3-position using NBS (Scheme 1). A
Suzuki−Miyaura coupling and deprotection of the amine gave the
pyrazolopyridine 4. For the pyrrolopyrimidine analogues commercially
available 4-chloro-7H-pyrrolo[2,3-d]pyrimidine 37 was first bromi-
nated followed by SEM protection of the pyrazole nitrogen to give 38.
Displacement of the chlorine substituent with Boc-protected 2-
(aminomethyl)morpholine at 100 °C in n-butanol gave an advanced
intermediate 39, which was elaborated using Suzuki−Miyaura
couplings. Dual deprotection using TBAF and ethylenediamine in
DMF followed by HCl in dioxane/MeOH gave 5 and 6.
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a
Scheme 2.
a
Reagents and conditions: (i) NaH, NCCH2CO2Et, DMF, 0 °C to rt and then 40 or 41, 2 h, rt, 48%; (ii) Zn/AcOH, 95 °C, 1 h 15 min, 88%; (iii)
formamide, ammonium formate, 170 °C, 18 h, 45%; (iv) POCl3, 75 °C, 18 h, 100%; (v) amine, TEA, DMF, 120 °C, 1 h, microwave and then 4 M
HCl, dioxane/MeOH, 2.5 h, 3−46%; (vi) amine, TEA, NMP, 140 °C, microwave and then MP-TsOH column, 29%.
a
azabenzimidazoles were effected by heating with acetic anhydride and
triethylorthoformate.
Scheme 3.
In the course of our investigations into the isoquinoline analogues,
several different isoquinoline reagents were coupled using a variety of
Buchwald−Hartwig conditions (Scheme 4 and Supporting Informa-
tion). The lengthy synthesis of 64 has been previously described,60
while 3-chloroisoquinoline 63 is commercially available and 8-
chloroisoquinolin-3-yl trifluoromethanesulfonate 65 was synthesized
in large quantities using the method reported by Ventura et al.61
Buchwald−Hartwig palladium mediated aminations of 60 and 62
with commercially available 2-amino-5-cyanopyrazine 66 followed by
deprotection of Boc or PMB protecting groups using TFA in CH2Cl2
at ambient temperature, or with neat TFA at 80 °C, respectively, gave
analogues 24 and 25. Similarly, palladium mediated aminations of 63
and 64 with 66 afforded analogues 27 and 29 directly.
The Buchwald−Hartwig coupling partners for the other isoquino-
lines and azabenzimidazoles were constructed from 2,6-dichloropyr-
azine.60 Substitution of one chlorine using NH3 followed by
bromination ortho to the remaining chlorine gave the precursor to
the desired 5-amino-3-chloropyrazine-2-carbonitrile 70. Cyanation was
achieved by heating with potassium cyanide in DMF with a crown
ether and Pd(PPh3)4 to complete the synthesis of 70. SNAr with
protected or tertiary aminoalkoxides gave a range of substituted
pyrazines 71−76 for Buchwald−Hartwig palladium mediated coupling
to the haloheterocycles 62−65. The single enantiomers of 1-
(dimethylamino)propan-2-ol, e.g. 68, required for the synthesis of
(R)-3 and (S)-3 were prepared by the careful addition of an aqueous
solution of dimethylamine to the appropriate, neat, enantiomerically
pure propylene oxide, e.g. 67 (Scheme 4). Palladium mediated
couplings between isoquinolines 63 or 65 with the relevant substituted
pyrazines 73, 74, 75, or 76 afforded products 33, 34, 3, (R)-3, or (S)-3
directly. The optical purities of (R)-3 and (S)-3 were determined to be
>95% ee by chiral HPLC analysis (see Supporting Information).
Where required, Boc or PMB protecting groups were removed
following coupling reactions using TFA in dichloromethane at ambient
temperature, or with neat TFA at 80 °C, respectively, to give
compounds 26, 28, and 30−32.
a
Reagents and conditions: (i) PdCl2(PPh3)2, LiHMDS, THF, 135 °C,
20 min, 19%−22%; or when X = CH(tBu3P)2Pd(0), NaOtBu, toluene,
80 °C, 2 h, 45%; (ii) amine, NMP, 145 °C, 20 min, (26−41%); (iii)
amine, NMP, 145 °C, 20 min, followed by TFA, CH2Cl2, 1 h, rt, or
HCl, dioxane, MP-TsOH column (3−63%).
(4-(9H-Pyrimido[4,5-b]indol-4-yl)-morpholin-2-yl)methanamines
7 were synthesized by reacting the racemic or single enantiomer33
Cbz-protected 2-(aminomethyl)morpholine with commercially avail-
able 4-chloro-9H-pyrimido[4,5-b]indole (48) and subsequent acid-
mediated removal of the Cbz protecting group (Scheme 2). The
pyrido[4′,3′:4,5]pyrrolo[2,3-d]pyrimidine and pyrimido[4,5-b]indole-
7-carbonitrile tricyclic cores 49 and 50 were constructed from 4-
chloro-5-nitropyridine (40) and 4-chloro-3-nitrobenzonitrile (41),
respectively (Scheme 2). Nucleophilic aromatic subsitution of the
starting material with the anion of ethylcyanoacetate and reduction of
the nitro group with zinc in acetic acid allowed an intramolecular
cyclization which provided the ethyl 2-amino-1H-indole-3-carboxylate
45 or its 6-aza analogue 44. Condensation with formamide completed
the tricyclic cores (46, 47), and chlorination with phosphorus
oxychloride installed the reactive chloride functionality of 49 and 50.
The diamines were introduced as described for the previous examples
to provide 7−13.
The N-(pyridin-2-yl)pyrimidine-4,6-diamines and N-(pyrazin-2-
yl)pyrimidine-4,6-diamines were assembled via a palladium-mediated
amination of 4,6-dichloropyrimidine (51) with 3-aminopyridine (52),
2-aminopyrazine (53), or 2-amino-5-cyanopyrazine (54) (Scheme 3).
The resulting intermediates 55−57 were derivatized by SNAr reactions
with amines followed by a deprotection step, where required, to yield
14−22.
The azabenzimidazoles 60 and 62 were prepared by first stirring 2-
bromo-4-chloro-5-nitropyridine (58) at rt with tert-butyl 4-
(aminomethyl)piperidine-1-carboxylate or 4-methoxybenzylamine fol-
lowed by a reduction of the nitro group with tin(II) chloride dihydrate
to give the diamine 59 or 61 (Scheme 4). Cyclizations to the
General Synthetic Chemistry. Reactions were carried out under
N2. Organic solutions were dried over MgSO4 or Na2SO4. Starting
materials and solvents were purchased from commercial suppliers and
were used without further purification. Microwave reactions were
carried out using Biotage Initiator 60 or CEM microwave reactors.
Flash silica chromatography was performed using Merck silica gel 60
(0.025−0.04 mm). Ion exchange chromatography was performed
using Isolute Flash SCX-II (acidic) or Flash NH2 (basic) resin
cartridges. 1H NMR spectra were recorded on a Bruker AMX500
instrument at 500 MHz or Bruker Avance instrument at 400 MHz
using internal deuterium locks. Chemical shifts (δ) are reported
relative to TMS (δ = 0) and/or referenced to the solvent in which they
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a
UV detector, and ionization was by positive or negative ion
electrospray. The molecular weight scan range was 80−1000 amu.]
All tested compounds gave >95% purity as determined by these
methods. All purified synthetic intermediates gave >95% purity as
determined by these methods except where indicated in the text. High-
resolution mass spectra were measured using the Agilent TOF system
described above.
Scheme 4.
Ethyl 3-Bromo-4-(2-((tert-butoxycarbonyl)aminomethyl)-
morpholino)-1H-pyrazolo[3,4-b]pyridine-5-carboxylate (36). N-
Bromosuccinimide (0.044 g, 0.25 mmol) was added in portions at rt
to a solution of ethyl 4-(2-((tert-butoxycarbonylamino)methyl)-
morpholino)-1H-pyrazolo[3,4-b]pyridine-5-carboxylate33 (35; 0.083
g, 0.20 mmol) in DMF (2 mL). After stirring for 2 h, saturated
brine was added and the precipitate was collected by filtration, washed
with water, and dried, to yield 36 as a pale yellow powder (0.076 g,
1
77%). H NMR (500 MHz, d4-MeOD) δ 1.40−1.46 (9H + 3H, m),
3.05−3.13 (1H, m), 3.13−3.29 (3H, m), 3.39−3.48 (1H, m), 3.55−
3.62 (1H, m), 3.95−4.00 (2H, m), 4.02−4.07 (1H, m), 4.44 (2H, q, J
= 7.5 Hz), 8.53 (1H, s); LC-MS (LCT, 6 min) Rt 4.83 min; m/z (ESI)
484, 486 [MH+].
Ethyl 4-(2-(Aminomethyl)morpholino)-3-(3-cyanophenyl)-1H-
pyrazolo[3,4-b]pyridine-5-carboxylate (4). A mixture of 36 (14
mg, 0.029 mmol), PdCl2(dppf)·CH2Cl2 (18 mg, 10 mol %), 3-
cyanophenylboronic acid (9 mg, 0.06 mmol), and Na2CO3 (8 mg,
0.075 mmol) in DME (2 mL) and water (0.5 mL) was heated to 140
°C in a microwave reactor for 2 h. The mixture was partitioned
between brine (10 mL) and EtOAc (2 × 8 mL). The combined
organic layers were washed with brine (10 mL) and water (10 mL),
dried, filtered, and concentrated. Preparative TLC, eluting with 1:2
hexane/EtOAc, gave ethyl 4-(2-((tert-butoxycarbonylamino)methyl)-
morpholino)-3-(3-cyanophenyl)-1H-pyrazolo[3,4-b]pyridine-5-car-
a
Reagents and conditions: (i) 4-(aminomethyl)piperidine-1-carbox-
1
ylate, TEA, MeCN, 1.5 h, rt, 93%; (ii) SnCl2·2H2O, EtOH, 70 °C, 2 h,
88%; (iii) (EtO)3CH, Ac2O, 100 °C, 18 h, 40%−100%; (iv) 4-
methoxybenzylamine, TEA, MeCN, 1.5 h, rt, 77%; (v) aryl halide
(ArCl for 24, 25, 29; ArI for 27), Pd(OAc)2, (±)-BINAP, NaOtBu,
DMF/Tol, 150 °C, 30 min, microwave and then MP-TsOH column,
3−41% (11% for 25 including Boc deprotection); (vi) NHMe2 (40%
in H2O), 0−20 °C, 2 h 40%; (vii) aq NH3 (28%), 100 °C, o/n, 91%;
(viii) NBS, CH2Cl2, 0 °C, 1 h, 42%; (ix) CuI, 18-crown-6, Pd(PPh3)4,
KCN, DMF, reflux, 3 h, 82%; (x) NaH (60% in oil), dioxane,
aminoalcohol, 100 °C, o/n, 14−32%; (xi) Pd(OAc)2, (±)-BINAP,
NatBu, DMF/toluene, microwave, 140−150 °C, 30 min, or Pd2(dba)3,
Xantphos, Cs2CO3, toluene, microwave, 140 °C, 45 min (2−45%);
(xii) Pd(OAc)2, (±)-BINAP, NatBu, DMF/Tol, microwave, 140−
150 °C, 30 min, or Pd2(dba)3, Xantphos, Cs2CO3, toluene, microwave,
140 °C, 45 min followed by TFA, 80 °C, 30 min or TFA, CH2Cl2, rt,
1 h MP-TsOH column (2−26%).
boxylate as a light yellow oil (7 mg, 48%). H NMR (500 MHz, d4-
MeOD) δ 1.44 (3H, t, J = 7.5 Hz), 1.46 (9H, s), 2.86−3.05 (4H, m),
3.06−3.26 (4H, m), 3.57−3.65 (1H, m), 4.45 (2H, q, J = 7.5 Hz), 7.75
(1H, dd, J = 7.5, 7.5 Hz), 7.87 (1H, d, J = 7.5 Hz), 8.01 (1H, d, J = 7.5
Hz), 8.08 (1H, s), 8.55 (1H, s); LC-MS (LCT, 6 min) Rt 4.74 min; m/
z (ESI) 507 [MH+]. Ethyl 4-(2-((tert-butoxycarbonylamino)methyl)-
morpholino)-3-(3-cyanophenyl)-1H-pyrazolo[3,4-b]pyridine-5-car-
boxylate (2 mg, 0.004 mmol) was dissolved in MeOH (2 mL), and
TFA (1 mL) was added. After being refluxed at 80 °C for 16 h, the
solvents were evaporated and the residue was purified on SCX-II acidic
resin (2 g), eluting with MeOH and then 2 M NH3/MeOH. The basic
fractions were combined and concentrated. The crude oil was purified
by preparative TLC, eluting with EtOAc, to give 4 as a yellow oil (1
mg, 62%). 1H NMR (500 MHz, d4-MeOD) δ 1.43 (3H, t, J = 7.5 Hz),
2.46−2.51 (2H, m), 2.85−2.91 (1H, m), 2.97−3.05 (2H, m), 3.08−
3.25 (3H, m), 3.62−3.67 (1H, m), 4.44 (2H, q, J = 7.5 Hz), 7.75 (1H,
dd, J = 7.5, 7.5 Hz), 7.90 (1H, d, J = 7.5 Hz), 8.00 (1H, d, J = 7.5 Hz),
8.08 (1H, s), 8.56 (1H, s); LC-MS (LCT, 6 min) Rt 2.56 min; m/z
(ESI) 407 [MH+]. HRMS (ESI) m/z calcd for C21H23N6O3 (M + H)
407.1826, found 407.1831.
were measured. Combined HPLC-MS analyses were recorded using
either
(1) (LCT) a Waters Alliance 2795 separations module and Waters/
Micromass LCT mass detector with electrospray ionization (+ve or
−ve ion mode as indicated) with HPLC performed using Supelco
DISCOVERY C18, 50 mm × 4.6 mm or 30 mm × 4.6 mm i.d.
columns or an Agilent 6210 TOF HPLC-MS with a Phenomenex
Gemini 3 μm C18 (3 cm × 4.6 mm i.d.) column [Both were run at a
temperature of 22 °C with gradient elution of 10−90% MeOH/0.1%
aqueous formic acid at a flow rate of 1 mL/min and a run time of 3.5
or 6 min as indicated. UV detection was at 254 nm, and ionization was
by positive or negative ion electrospray. The molecular weight scan
range was 50−1000 amu.]
(2) (ZQ) a Micromass ZQ mass spectrometer/Waters Alliance
2795 HT HPLC with a Phenomenex Gemini 5 μm, C18, 30 mm × 4.6
mm i.d. column or a Waters X-Bridge C18, 2.5 μM, 3.0 × 30 mm
column. [Both were run at a temperature of 35 °C with a gradient
elution of 5−95% [(0.1% ammonia in acetonitrile)/(0.1% ammonia,
5% acetonitrile, and 0.063% ammonium formate in water)] at a flow
rate of 2 mL/min and a run time of 4 or 7 min as indicated. UV
detection was at 220−400 nm using a Waters 996 photodiode array
5-Bromo-4-chloro-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-
pyrrolo[2,3-d]pyrimidine (38). A suspension of 4-chloro-7H-pyrrolo-
[2,3-d]pyrimidine (37; 0.49 g, 3.2 mmol) and N-bromosuccinimide
(0.68 g, 3.8 mmol) in dry CH2Cl2 (20 mL) was stirred at rt for 2.5 h.
The suspension was diluted with MeOH and evaporated onto silica.
The crude product was purified using flash column chromatography,
eluting with 2:1 hexanes/EtOAc, to yield 5-bromo-4-chloro-7H-
pyrrolo[2,3-d]pyrimidine as an off-white solid (0.613 g, 2.64 mmol,
1
83%). H NMR (500 MHz, d4-MeOD) δ 7.64 (1H, s), 8.56 (1H, s);
LC-MS (LCT, 6 min) Rt 3.34 min; m/z (ESI) 236, 234, 232 [MH+].
To a solution of 5-bromo-4-chloro-7H-pyrrolo[2,3-d]pyrimidine (1.03
g, 4.43 mmol) in DMF (12 mL) at 0 °C was added NaH (0.21 g, 60%
suspension in oil, 5.05 mmol). After the suspension was stirred at this
temperature for 15 min, SEM-Cl (0.89 g, 5.34 mmol) in DMF (2 mL)
was added. The resulting suspension was then stirred at 0 °C for 20
min and at rt for 1 h. Water (30 mL) was added; the precipitate was
collected by filtration, washed with water (2 × 30 mL), and dried in
vacuo. Compound 38 was obtained as light pink crystals (1.5 g, 93%).
1H NMR (500 MHz, d6-DMSO) δ 0.00 (9H, s), 0.90 (2H, t, J = 7.5
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Hz), 3.60 (2H, t, J = 7.5 Hz), 5.70 (2H, s), 7.25 (1H, s), 8.70 (1H, s);
LC-MS (LCT, 6 min) Rt 4.34 min; m/z (ESI) 366, 364, 362 [MH+].
tert-Butyl (4-(5-Bromo-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-
pyrrolo[2,3-d]pyrimidin-4-yl)morpholin-2-yl)methylcarbamate
(39). A solution of tert-butyl morpholin-2-ylmethylcarbamate (0.075
g, 0.35 mmol), 38 (0.12 g, 0.33 mmol), and Et3N (0.13 mL, 0.75
mmol) in n-BuOH (1.5 mL) was heated at 100 °C in a microwave
reactor for 1 h. The solution was concentrated onto silica gel. The
crude product was purified using flash column chromatography,
eluting with 2:1 hexanes/EtOAc, to yield 39 as a viscous oil (0.122 g,
fractions were combined. Preparative silica TLC, eluting with 1%
NH3−9% MeOH−90% CH2Cl2, gave rac-7 (0.007 g, 0.025 mmol,
46%) as a beige powder. 1H NMR (500 MHz, d4-MeOD) δ 2.75−2.80
(2H, m), 3.00−3.06 (1H, m), 3.35−3.40 (1H, m), 3.75−3.85 (1H, m),
3.90 (1H, dd, J = 10, 10 Hz), 4.10−4.13 (1H, m), 4.19 (1H, d, J = 13
Hz), 4.25 (1H, d, J = 13 Hz), 7.35 (1H, dd, J = 8.8 Hz), 7.47 (1H, dd,
J = 8.8 Hz), 7.57 (1H, d, J = 8 Hz), 7.79 (1H, d, J = 8 Hz), 8.46 (1H,
s); LC-MS (LCT, 6 min) Rt 1.98 min; m/z (ESI) 284 [MH+].
Ethyl 2-Cyano-2-(3-nitropyridin-4-yl)acetate (42). Ethylcyanoace-
tate (3.75 g, 38 mmol) in DMF (3 mL) was added dropwise at 0 °C to
a suspension of NaH (1.5 g, 60% in mineral oil, 38 mmol) in DMF (9
mL). The reaction mixture was stirred at rt for 30 min and then cooled
to 0 °C, and 3-nitro-4-chloropyridine (41; 2.63 g, 19 mmol, 1 equiv)
in DMF (3 mL) was added slowly. Two molar HCl (20 mL) and
EtOAc (20 mL) were added after 2 h stirring at rt, and the organic
layer was washed with water and brine, dried, and concentrated to give
a red-orange oil. The crude oil was triturated with EtOAc, and the
1
0.225 mmol, 68%). H NMR (500 MHz, CDCl3) δ −0.03 (9H, s),
0.93 (2H, t, J = 7 Hz), 1.27 (9H, s), 2.95 (1H, dd, J = 7, 7 Hz), 3.21−
3.23 (2H, m), 3.40−3.45 (1H, m), 3.56 (2H, t, J = 7 Hz), 3.85−3.90
(2H, m), 4.02−4.16 (2H, m), 4.90−4.95 (1H, m), 5.58 (2H, s), 7.28
(1H, s), 8.43 (1H, s); LC-MS (LCT, 6 min) Rt 4.30 min; m/z (ESI)
544, 542 [MH+].
3-(4-(2-(Aminomethyl)morpholino)-7H-pyrrolo[2,3-d]pyrimidin-
5-yl)benzonitrile (5). A mixture of 39 (44 mg, 0.08 mmol),
Pd(PPh3)4 (5 mg, 4 mol %), 3-cyanophenylboronic acid (24.5 mg,
0.22 mmol), and sodium carbonate (30 mg, 0.17 mmol) in DME (1.5
mL) and water (0.5 mL) was heated to 120 °C in a microwave reactor
for 30 min. The mixture was partitioned between brine (10 mL) and
EtOAc (2 × 8 mL). The combined organic layers were washed with
brine (10 mL) and water (10 mL), dried (Na2SO4), filtered, and
concentrated. Preparative TLC, eluting with EtOAc/n-hexane/1:1 (Rf
= 0.27), gave tert-butyl (4-(5-bromo-7-((2-(trimethylsilyl)ethoxy)-
methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)morpholin-2-yl)-
1
resultant solid was collected by filtration to give 42 (2.88 g, 48%). H
NMR (d6-DMSO) δ 1.18 (3H, t, J = 7.0 Hz), 4.06 (2H, q, J = 7.0 Hz),
7.58 (1H, br s), 7.88 (1H, dd, J = 1.0, 7.0 Hz), 8.70 (1H, s), 13.2 (1H,
br s); LC-MS (ZQ, 4 min) Rt 1.13 min; m/z (ES−) 190 [M − EtO],
234 [M − H].
2-Amino-1H-pyrrolo[2,3-c]pyridine-3-carboxylic Acid Ethyl Ester
(44). A solution of 42 (2.20 g, 9.35 mmol) in AcOH (20 mL) was
heated to 80 °C under nitrogen. Zinc dust (6.12 g, 94 mmol) in 500
mg portions was added, and then the reaction mixture was heated at
95 °C for 1 h 15 min. Upon cooling, the insoluble material was filtered
off and washed with fresh AcOH. The filtrate was concentrated, and
the residue was treated with saturated NaHCO3 to give a light brown
solid. This was filtered, washed with water, and dried to give 44 as a
light brown solid (1.69 g, 8.24 mmol, 88% yield). 1H NMR (d6-
DMSO) δ 1.32 (3H, t, J = 7.0 Hz), 4.23 (2H, q, J = 7.0 Hz), 7.0 (2H,
br s), 7.41 (1H, d, J = 4.5 Hz), 8.0 (1H, br), 8.31 (1H, br), 11.0 (1H, s,
br). LC-MS (TOF, 4 min), Rt = 1.70 min; m/z (ES+) 206.
1
methylcarbamate as a yellow oil (27 mg, 59%). H NMR (500 MHz,
CDCl3) δ 0.00 to −0.04 (9H, s), 0.95 (2H, t, J = 8.0 Hz), 1.45 (9H, s),
2.65−2.72 (1H, m), 2.82−2.98 (2H, m), 3.09−3.19 (1H, m), 3.35−
3.50 (2H, m), 3.61 (2H, t, J = 8.0 Hz), 3.62−3.78 (2H, m), 4.58 (1H,
m), 4.78 (1H, broad s), 5.67 (2H, s), 7.30 (1H, s), 7.51−7.53 (2H, m),
7.78 (1H, d, J = 7.5 Hz), 7.84 (1H, s), 8.55 (1H, s); LC-MS (LCT, 6
min) Rt 5.57 min; m/z (ESI) 565 [MH+]. A mixture of tert-butyl (4-
((5-(3-cyanophenyl)-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-
pyrrolo[2,3-d]pyrimidin-4-yl)morpholin-2-yl)methylcarbamate (37
mg, 0.066 mmol), 1.0 M TBAF/THF (0.55 mL, 0.55 mmol), and
ethane-1,2-diamine (20 μL) in DMF (1.5 mL) was stirred at 60 °C
under N2 for 16 h. It was diluted with brine (8 mL) and extracted with
EtOAc (2 × 10 mL). The combined organic layers were washed with
brine (10 mL) and water (10 mL), dried (Na2SO4), filtered, and
concentrated. The crude oil (12 mg) was purified by preparative TLC,
yielding 8 mg (LC-MS Rt 4.51 min; m/z (ESI) 435 [MH+]). It was
then dissolved in a mixture of MeOH (3 mL) and TFA (2 mL). The
solution was stirred at 80 °C for 12 h. The solvents were evaporated,
and the residue was purified on SCX-II acidic resin (1 g) eluting with
MeOH and then 2 M NH3−MeOH. The basic fractions were
9H-Pyrido[4′,3′:4,5]pyrrolo[2,3-d]pyrimidin-4-ol (46). A mixture
of 44 (1.80 g, 8.8 mmol) and ammonium formate (0.62 g, 9.8 mmol)
in formamide (10 mL) was heated at 170 °C for 18 h. One molar HCl
was added to the cooled reaction mixture, and the resulting suspension
was filtered to remove insolubles. The filtrate was then adjusted to pH
7 with saturated sodium bicarbonate solution. The resulting precipitate
was collected by filtration and dried to a constant weight in a vacuum
oven to give 46 (0.74 g, 45%). 1H NMR (d6-DMSO) δ 7.89 (1H, d, J
= 4.0), 8.26 (1H, s), 8.37 (1H, d, J = 4.0), 8.83 (1H, s), 12.5 (s, br);
LC-MS (ZQ, 4 min) Rt = 0.75 min; m/z (ES−) 185 [M − H].
4-Chloro-9H-pyrido[4′,3′:4,5]pyrrolo[2,3-d]pyrimidine (49). A
mixture of 46 (264 mg, 1.4 mmol) and Et3N (750 μL) in POCl3 (5
mL) was heated at 75 °C for 18 h. Toluene was added to the cooled
reaction mixture, and the solvents were then removed in vacuo to give
49 as a brown oil (290 mg, 100%) used directly in the subsequent
reaction. LC-MS (ZQ, 4 min) Rt 1.25 min; m/z (ES+) 205/207.
4-(2-(Aminomethyl)morpholino)-9H-pyrido[4′,3′:4,5]pyrrolo[2,3-
d]pyrimidine (8). A mixture of 49 (20 mg, 0.098 mmol), morpholin-
2-ylmethylcarbamic acid tert-butyl ester (32 mg, 0.147 mmol), and
Et3N (68 mL, 0.489 mmol) in NMP (1 mL) was heated in a
microwave reactor at 140 °C for 15 min. The reaction mixture was
diluted with MeOH and applied to a MP-TsOH cartridge which had
been preconditioned with MeOH. The cartridge was flushed with
MeOH and left for 20 min. The product was eluted using 2 M NH3 in
MeOH, affording a brown oil, which was purified using preparative
HPLC to give 4-(2-(aminomethyl)morpholino)-9H-pyrido[4′,3′:4,5]-
pyrrolo[2,3-d]pyrimidine (11 mg, 40%). 1H NMR (d4-MeOD) δ
3.10−3.04 (1H, m), 3.25−3.19 (2H, m), 3.58−3.51 (1H, m), 3.83 (1
H, td, J = 11.6, 2.5), 4.00−3.94 (1H, m), 4.14 (1H, dd, J = 11.6, 2.0),
4.32 (1H, d, J = 13.4), 4.46 (1H, d, J = 12.9), 7.78 (1H, d, J = 5.6),
8.43 (1H, d, J = 5.6), 8.46 (2H, br s), 8.54 (1H, s), 8.84 (1H, s); LC-
MS (ZQ, 7 min) Rt 1.78 min; m/z (ES+)285 [M + H]. HRMS (ESI)
m/z calcd for C14H17N6O (M + H) 285.1458, found 285.1457.
5-(6-Chloropyrimidin-4-ylamino)pyrazine-2-carbonitrile (57). A
mixture of 4,6-dichloropyrimidine (51; 1.0 g, 6.7 mmol), 2-amino-5-
cyanopyrazine (54; 806 mg, 6.7 mmol), and bis(triphenylphosphine)-
1
combined and evaporated to give 5 as a yellow oil (4.5 mg, 21%). H
NMR (500 MHz, d4-MeOD) δ 2.52−2.60 (1H, m), 2.63−2.70 (1H,
m), 2.85−2.95 (2H, m), 3.41−3.51 (2H, m), 3.55−3.60 (1H, m), 3.74
(2H, d, J = 12 Hz), 7.48 (1H, s), 7.79−7.55 (2H, m), 7.88 (J = 7.7 Hz,
1H, d), 7.93 (1H, s), 8.39 (1H, s); LC-MS (LCT, 6 min) Rt 2.12 min;
m/z (ESI) 335 [MH+]. HRMS (ESI) m/z calcd for C18H19N6O (M +
H) 335.1620, found 335.1618.
C-[4-(9H-Pyrimido[4,5-b]indol-4-yl)-morpholin-2-yl]methylamine
(rac-7). A mixture of 4-chloro-9H-pyrimido[4,5-b]indole (47; 0.045
g, 0.221 mmol), morpholin-2-ylmethylcarbamic acid tert-butyl ester
(0.055 g, 0.25 mmol), and Et3N (0.10 mL, 0.75 mmol) in DMF (0.70
mL) was heated to 120 °C in a microwave reactor for 1 h. The cooled
solution was partitioned between water (20 mL) and EtOAc (20 mL).
The organic extract was dried (Na2SO4), filtered, and concentrated.
Flash column chromatography, eluting with EtOAc, gave [4-(9H-
pyrimido[4,5-b]indol-4-yl)morpholin-2-ylmethyl]carbamic acid tert-
butyl ester as a yellow solid (0.021 g, 0.0548 mmol, 25%). LC-MS
(LCT, 6 min) Rt 5.57 min; m/z (ESI) 384 [MH+]. A solution of [4-
(9H-pyrimido[4,5-b]indol-4-yl)morpholin-2-ylmethyl]carbamic acid
tert-butyl ester (0.021 g, 0.548 mmol) and 4 M HCl−dioxane (1
mL) in MeOH (5 mL) was stirred at rt for 2.5 h. The solution was
evaporated to dryness and purified by ion exchange on SCX-II acidic
resin (2 g), eluting with MeOH and then 2 M NH3−MeOH. The basic
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palladium(II) chloride (94 mg, 0.134 mmol) in dry THF (24 mL) was
degassed under a stream of nitrogen gas over 10 min with stirring. 1 M
LiHMDS in THF (7.38 mL, 7.4 mmol) was added, and the mixture
was heated at 135 °C for 20 min in a microwave reactor. The reaction
mixture was adsorbed onto silica and purified by flash chromatog-
raphy, eluting with 30% EtOAc in hexane, to give 57 (300 mg, 19%).
LC-MS (ZQ, 4 min) Rt = 1.64; min m/z (ES−) 231 [M − H].
5-(6-(Piperidin-4-ylmethylamino)pyrimidin-4-ylamino)pyrazine-
2-carbonitrile (20). A mixture of 57 (124 mg, 0.533 mmol), 4-
(aminomethyl)-1-N-Boc-piperidine (228 mg, 1.066 mmol), and Et3N
(150 μL, 1.07 mmol) in 1-methyl-2-pyrrolidinone (1 mL) was heated
at 145 °C for 15 min in a microwave reactor. The mixture was
concentrated in vacuo, and the residue was purified by preparative
HPLC. The purified solid was dissolved in CH2Cl2 (2 mL) and
trifluoroacetic acid (3 mL) and was stirred for 1 h at rt before being
applied to a MP-TsOH cartridge. After washing with MeOH, the pure
ylamino)pyrazine-2-carbonitrile (22.4 mg, 40%). 1H NMR (d6-DMSO,
400 MHz) δ 3.72 (3H, s), 5.41 (2H, s), 6.96 (2H, d, J = 8.8 Hz), 7.34
(2H, d, J = 8.8 Hz), 8.17 (1H, s), 8.49 (1H, s), 8.73−8.76 (2H, m),
8.77 (1H, d, J = 1.3 Hz), 10.84 (1H, br s). LC-MS (ZQ, 7 min) Rt =
2.32 min; m/z (ESI+) 358 (MH+), (ESI−) 356 (M − H). 5-(1-(4-
Methoxybenzyl)-1H-imidazo[4,5-c]pyridin-6-ylamino)pyrazine-2-car-
bonitrile (10.6 mg, 30 μmol) was treated with TFA at 80 °C over 30
min. Isolation by SPE on a MP-TsOH cartridge, eluting with 2M NH3
in MeOH, followed by concentration, gave 24 as a white solid (7.02
mg, 100%). 1H NMR (d6-DMSO, 400 MHz) δ 8.26 (1H, s), 8.35 (1H,
s), 8.75−8.77 (3H, m), 10.85 (1H, br s), 12.79 (1H, br s); LC-MS
(ZQ, 7 min) Rt = 1.36 min; m/z (ESI+) 238 (MH+), (ESI−) 236 (M −
H). HRMS (ESI) m/z calcd for C11H8N7 (M + H) 238.0836, found
238.0845.
(R)-1-(Dimethylamino)propan-2-ol (68). Dimethylamine 40% in
water (11.39 mL, 90 mmol) was slowly added to (R)-propylene oxide
(5.25 mL, 74.9 mmol) which had been cooled in an ice bath. This
solution was stirred at rt for 2 h before being extracted with CH2Cl2 (4
× 5 mL). The combined organic layers were dried over Na2SO4, and
the pure (R)-1-(dimethylamino)propan-2-ol (5.12 g, 49.6 mmol,
40.0% yield) was isolated as a clear oil using distillation under reduced
1
product was eluted using 7 M NH3 to give 20 (10 mg, 6.2%). H
NMR (d6-DMSO, 400 MHz): δ 1.30 (3H, m), 1.85 (4H, m), 2.71
(2H, m), 3.18 (2H, m), 3.26 (1H, br s), 7.14 (1H, br s), 8.21 (1H, s),
8.62 (s, 1H), 8.79 (1H, br s); LCMS (LCT, 4 min) Rt = 0.78 min m/z
(ES+) 311. HRMS (ESI) m/z calcd for C15H19N8 (M + H) 311.1742,
found 311.1727.
1
pressure (50 mbar). H NMR (CDCl3, 500 MHz) δ 1.12 (3H, d, J =
N4-(4-Methoxybenzyl)-6-bromopyridine-3,4-diamine (61). A sol-
ution of 4-methoxybenzylamine (0.756 g, 5.51 mmol) in acetonitrile
(2 mL) was added to a mixture of 2-bromo-4-chloro-5-nitropyridine
(53) (1.19 g, 5.01 mmol) and Et3N (0.768 mL, 5.51 mmol) in
acetonitrile (8 mL). After stirring for 1.5 h, the solution was diluted
with EtOAc (100 mL), and the resulting solution was then washed
successively with water and brine before being concentrated in vacuo
to a light brown oil which solidified on standing to give N-(4-
methoxybenzyl)-2-bromo-5-nitropyridin-4-amine (1.31 g, 3.87 mmol,
77%). 1H NMR (d6-DMSO, 400 MHz) δ 3.70 (3H, s), 4.60 (2H, d, J
= 6 Hz), 6.95 (2H, d, J = 9 Hz), 7.10 (1H, s), 7.35 (2H, d, J = 9 Hz),
8.80 (s, 1H), 9.00 (br t, 1H, J = 6 Hz). LC-MS (ZQ, 4 min) Rt = 2.13
min; m/z (ESI−) 336, 338 [M − H]. Tin(II) chloride dihydrate (4.37
g, 19.4 mmol) was added portionwise to N-(4-methoxybenzyl)-2-
bromo-5-nitropyridin-4-amine (1.31 g, 3.87 mmol) in absolute EtOH
(10 mL) at rt. The mixture was heated at 70 °C for 2 h before being
concentrated in vacuo. The residue was suspended in a mixture of
EtOAc and saturated sodium bicarbonate solution and filtered. The
insoluble solids were washed with EtOAc. The aqueous phase was re-
extracted with EtOAc, and the combined organic layers were washed
with brine, dried (Na2SO4), and concentrated to give 61 as a brown
6.0 Hz), 2.16−2.12 (1H, m), 2.25−2.21 (1H, m), 2.27 (6H, s), 3.40
(1H, br s), 3.82−3.76 (1H, m).
5-Amino-3-chloropyrazine-2-carbonitrile (70). 2,6-Dichloropyra-
zine (69; 2.89 g, 19.4 mmol) was stirred in aqueous NH3 (28%, 10
mL) and heated to 100 °C overnight in a sealed tube. The reaction
mixture was cooled, and the resultant precipitate was filtered.
Trituration with water and then ether gave 6-chloropyrazin-2-amine
1
as a white solid (2.28 g, 17.6 mmol, 91%). H NMR (d6-DMSO, 400
MHz) δ 6.9 (2H, br s), 7.70 (1H, d, J = 0.4 Hz), 7.80 (1H, d, J = 0.4
Hz); LC-MS (ZQ, 7 min) Rt = 1.05 min; m/z (ESI+) 130, 132 (MH+).
6-Chloropyrazin-2-amine (2.50 g, 19.3 mmol) was stirred in CH2Cl2
(60 mL) and cooled to 0 °C. N-Bromosuccinimide (2.92 g, 16.4
mmol) was added slowly, and the reaction mixture was stirred at 0 °C
for 60 min. The reaction mixture was filtered through Celite and
concentrated to give a brown oil. Purification by flash chromatography,
eluting with 0−25% EtOAc−hexanes, gave 5-bromo-6-chloropyrazin-
1
2-amine as a yellow solid (1.69 g, 8.16 mmol, 42%). H NMR (d6-
DMSO, 400 MHz) δ 7.1 (2H, br s), 7.65 (1H, s); LC-MS (ZQ, 4 min)
Rt = 1.46 min; m/z (ESI−) 205 (M − H). A mixture of 5-bromo-6-
chloropyrazin-2-amine (1.00 g, 4.8 mmol), copper(I) iodide (914 mg,
4.8 mmol), 18-crown-6 (95 mg, 0.36 mmol), and tetrakis-
(triphenylphosphine)palladium (0) (83 mg, 0.072 mmol) was
suspended in dry DMF (20 mL), and a stream of nitrogen was
passed through it for 5 min. Potassium cyanide (312 mg, 4.8 mmol)
was added, and the mixture was stirred at rt for 30 min and then
refluxed at 200 °C for 3 h. The mixture was cooled and diluted with
EtOAc and absorbed onto silica gel (10 g). DMF was removed by
evaporation. The product was purified by flash chromatography,
eluting with 1:1 EtOAc−hexanes, to yield 70 as a yellow solid (607
1
solid (1.05 g, 3.41 mmol, 88%). H NMR (d6-DMSO, 400 MHz) δ
3.75 (3H, s), 4.30 (2H, d, J = 5.5 Hz), 4.85 (2H, br s), 6.35 (1H, br t,
5.5 Hz), 6.45 (1H, s), 6.90 (2H, d, J = 8.5 Hz), 7.30 (2H, d, J = 8.5
Hz), 7.40 (1H, s); LC-MS (ZQ, 4 min) Rt = 1.77 min; m/z (ESI−)
306, 308; (ESI+) 308, 310 [MH+].
1-(4-Methoxybenzyl)-6-bromo-1H-imidazo[4,5-c]pyridine (62).
Acetic anhydride (1.28 mL, 13.6 mmol) was added to a solution of
61 (1.05 g, 3.41 mmol) in triethylorthoformate (13 mL). The mixture
was heated at 100 °C for 18 h and then concentrated to give 62 as a
1
mg, 3.93 mmol, 82%). H NMR (d6-DMSO, 400 MHz) δ 7.87 (1H,
1
s), 8.1 (2H, br s); LC-MS (ZQ, 4 min) Rt = 1.20 min; m/z (ESI−) 153
(M − H).
brown oil (1.18 g, quantitative). H NMR (d6-DMSO, 400 MHz) δ
3.70 (3H, s), 5.45 (2H, s), 6.90 (2H, d, J = 9.0 Hz), 7.35 (2H, d, J = 9
Hz), 8.00 (1H, s), 8.60 (1H, s), 8.75 (1H, s). LC-MS (ZQ, 7 min) Rt =
2.27 min; m/z (ESI+) 318, 320 (MH+).
(R)-5-Amino-3-(1-(dimethylamino)propan-2-yloxy)pyrazine-2-
carbonitrile (74). (R)-1-(Dimethylamino)propan-2-ol (68; 0.667 g,
6.47 mmol) was added dropwise to a suspension of NaH 60% (0.388
g, 9.71 mmol) in dioxane (16.18 mL) and stirred for 30 min. 5-Amino-
3-chloropyrazine-2-carbonitrile (70; 1.000 g, 6.47 mmol) was added in
one portion and heated at 90 °C for 14 h. After cooling water (200
mL) was added and the solution was extracted with diethyl ether (4 ×
100 mL) and dried over MgSO4, the volatiles were removed under
vacuum. Column chromatography using a gradient of MeOH in
CH2Cl2 (+ 1% NH3) gave 74 (0.558 g, 2.52 mmol, 39.0% yield) as a
yellow solid. 1H NMR (500 MHz, CDCl3) δ 1.35 (3H, d, J = 6.5 Hz),
2.37 (6H, s), 2.52 (1H, dd, J = 4.0, 13.5 Hz), 2.76 (1H, dd, J = 7.5,
13.5 Hz), 5.31 (2H, br s), 5.42−5.35 (1H, m), 7.54 (1H, s). LC-MS
(TOF, 3.5 min) Rt = 0.80 min; m/z (ESI) 222 (M + H).
5-(1H-Imidazo[4,5-c]pyridin-6-ylamino)pyrazine-2-carbonitrile
(24). Palladium(II) acetate (3.5 mg, 16 μmol) was added to (±)-2,2″-
bis(diphenylphosphino)-1,1″-binaphthalene (59 mg, 94 μmol) in
DMF/toluene (1:2), and the resulting mixture was degassed under a
stream of nitrogen gas for 10 min. 2-Amino-5-cyanopyrazine (66; 19
mg, 0.16 mmol), sodium tert-butoxide (45 mg, 0.47 mmol), and 62
(50 mg, 0.16 mmol) were added, and the mixture was degassed for a
further 5 min before heating at 150 °C for 30 min in a microwave
reactor. The reaction mixture was partitioned between water and
CH2Cl2. The aqueous phase was extracted with CH2Cl2. The
combined organic layers were dried (Na2SO4) and concentrated.
The residue was dissolved in MeOH, passed through a PS-Thiol
column, and concentrated. The product was purified using preparative
HPLC to give 5-(1-(4-methoxybenzyl)-1H-imidazo[4,5-c]pyridin-6-
(R)-5-(8-Chloroisoquinolin-3-ylamino)-3-(1-(dimethylamino)-
propan-2-yloxy)pyrazine-2-carbonitrile ((R)-3). To four microwave
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vials was added Xantphos (66.8 mg, 0.116 mmol), Pd2(dba)3 (52.9
mg, 0.058 mmol), 8-chloroisoquinolin-3-yl trifluoromethanesulfonate
(180 mg, 0.578 mmol), 74 (128 mg, 0.578 mmol), and Cs2CO3 (376
mg, 1.155 mmol) followed by dry toluene (4 mL). The vials were
sealed, and dry nitrogen was bubbled through the stirred solution for 8
min. The reactions were irradiated at 130 °C for 45 min in a Biotage
microwave. Upon cooling, the mixtures were combined, diluted with
MeOH, and purified using an acidic ion exchange column which was
washed with MeOH before the basic components eluted with 2 M
NH3 in MeOH followed by CH2Cl2/2 M NH3 in MeOH. The residue
was purified by column chromatography eluting with a gradient of
MeOH in CH2Cl2 (+1% NH3) to give (R)-3 (301 mg, 0.786 mmol,
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1
45% yield) as a light yellow powder. H NMR (500 MHz, d6-DMSO)
δ 1.46 (3H, d, J = 6.5 Hz), 2.21 (6H, s), 2.54 (2H, dd, J = 5, 13 Hz),
2.66 (1H, dd, J = 7, 13 Hz), 5.54−5.46 (1H, m), 7.66 (1H, d, J = 7.5
Hz), 7.72 (1H, dd, J = 7.5, 8.0 Hz), 7.84 (1H, d, J = 8.0 Hz), 8.26 (1H,
s), 8.48 (1H, s), 9.40 (1H, s), 11.16 (1H, s). LC-MS (TOF, 3.5 min)
Rt = 2.58 min; m/z (ESI) 383 (M + H). HRMS (ESI) m/z calcd for
C19H20ClN6O (M + H) 383.1382, found 383.1366.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental details for the preparation and characterization of
compounds (rac)-3, (S)-3, 6, (R)-7, (S)-7, 9−19, 21, 22, and
25−34; experimental details for the determination of the
enantiopurity of (R)-3 and (S)-3; experimental details for
CHK1 and CHK2 enzyme inhibition assays; summary of crystal
structure determinations of CHK1 in complex with 2, 4, 6, 8,
20, and (R)-3; and kinase inhibitory profiles of (R)-3 at 1 μM
and 10 μM test concentrations. This material is available free of
charge via the Internet at http://pubs.acs.org.
Accession Codes
PDB ID Codes: 2ym3, 2ym4, 2ym5, 2ym6, 2ym7, 2ym8.
AUTHOR INFORMATION
Corresponding Author
■
*J.R.: Telephone, +44 (0)1223 497700; e-mail, john.reader@
sareum.co.uk. I.C.: Telephone, +44 (0)20 8722 4317; fax, +44
(0)20 8722 4126; e-mail, ian.collins@icr.ac.uk.
Author Contributions
⊥These authors contributed equally to this work.
ACKNOWLEDGMENTS
■
We thank Dr. A. Mirza, M. Richards, and Dr. M. Liu for
assistance in the spectroscopic characterization of test
compounds and Dr. M. Lamers for the preparation of CHK1
protein. This work was supported by Cancer Research UK
[CUK] grant numbers C309/A2187, C309/A8274, and C309/
A8365 and by The Institute of Cancer Research. We
acknowledge NHS funding to the NIHR Biomedical Research
Centre.
(19) Ganzinelli, M.; Carrassa, L.; Crippa, F.; Tavecchio, M.; Broggini,
M.; Damia, G. Checkpoint kinase 1 down-regulation by an inducible
small interfering RNA expression system sensitized in vivo tumors to
treatment with 5-fluorouracil. Clin. Cancer Res. 2008, 14, 5131−5141.
(20) Tse, A. N.; Rendahl, K. G.; Sheikh, T.; Cheema, H.; Aardalen,
K.; Embry, M.; Ma, S.; Moler, E. J.; Ni, Z. J.; Lopes de Menezes, D. E.;
Hibner, B.; Gesner, T. G.; Schwartz, G. K. CHIR-124, a novel potent
inhibitor of Chk1, potentiates the cytotoxicity of topoisomerase I
poisons in vitro and in vivo. Clin. Cancer Res. 2007, 13, 591−602.
(21) Xiao, Z.; Xue, J.; Sowin, T. J.; Zhang, H. Differential roles of
checkpoint kinase 1, checkpoint kinase 2, and mitogen-activated
protein kinase-activated protein kinase 2 in mediating DNA damage-
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ABBREVIATIONS USED
■
ATP, adenosine 5′-triphosphate; ATR, ataxia telangiectasia and
rad3 related; CHK1, checkpoint kinase 1; CHK2, checkpoint
kinase 2; DELFIA, dissociation-enhanced lanthanide fluores-
cent immunoassay; ELISA, enzyme-linked immunosorbent
assay; LE, ligand efficiency; MPM2, M-phase phosphoprotein
2; SRB, sulforhodamine B
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