Discovery of AMG 925, a FLT3 and CDK4 dual kinase inhibitor with preferential affinity for the activated state of FLT3

Article abstract of DOI:10.1021/jm500118j

We describe the structural optimization of a lead compound 1 that exhibits dual inhibitory activities against FLT3 and CDK4. A series of pyrido[4′,3′:4,5]pyrrolo[2,3-d]pyrimidine derivatives was synthesized, and SAR analysis, using cell-based assays, led to the discovery of 28 (AMG 925), a potent and orally bioavailable dual inhibitor of CDK4 and FLT3, including many FLT3 mutants reported to date. Compound 28 inhibits the proliferation of a panel of human tumor cell lines including Colo205 (Rb ⁺) and U937 (FLT3^WT) and induced cell death in MOLM13 (FLT3^ITD) and even in MOLM13 (FLT3^{ITD, D835Y}), which exhibits resistance to a number of FLT3 inhibitors currently under clinical development. At well-tolerated doses, compound 28 leads to significant growth inhibition of MOLM13 xenografts in nude mice, and the activity correlates with inhibition of STAT5 and Rb phosphorylation.

Full text of DOI:10.1021/jm500118j

Article
pubs.acs.org/jmc
Discovery of AMG 925, a FLT3 and CDK4 Dual Kinase Inhibitor with
Preferential Affinity for the Activated State of FLT3
Zhihong Li,*,† Xianghong Wang,† John Eksterowicz,† Michael W. Gribble, Jr.,† Grace Q. Alba,§
Merrill Ayres,† Timothy J. Carlson,∥ Ada Chen,† Xiaoqi Chen,† Robert Cho,∥ Richard V. Connors,†
Michael DeGraffenreid,† Jeffrey T. Deignan,† Jason Duquette,† Pingchen Fan,† Benjamin Fisher,†
Jiasheng Fu,† Justin N. Huard,⊥ Jacob Kaizerman,† Kathleen S. Keegan,⊥ Cong Li,‡ Kexue Li,†
Yunxiao Li,† Lingming Liang,‡ Wen Liu,† Sarah E. Lively,† Mei-Chu Lo,† Ji Ma,∥ Dustin L. McMinn,†
Jeffrey T. Mihalic,† Kriti Modi,† Rachel Ngo,† Kanaka Pattabiraman,† Derek E. Piper,†
Christophe Queva,⊥ Mark L. Ragains,§ Julia Suchomel,† Steve Thibault,† Nigel Walker,† Xiaodong Wang,†
Zhulun Wang,† Malgorzata Wanska,† Paul M. Wehn,† Margaret F. Weidner,⊥ Alex J. Zhang,†
Xiaoning Zhao,† Alexander Kamb,# Dineli Wickramasinghe,‡ Kang Dai,‡ Lawrence R. McGee,†
and Julio C. Medina†
Departments of †Therapeutic Discovery, ‡Oncology Research, §Pharmaceutics, and ∥Pharmacokinetics and Drug Metabolism, Amgen
Inc., 1120 Veterans Boulevard, South San Francisco, California 94080, United States
⊥Therapeutic Innovation Unit, Amgen Inc., 1201 Amgen Court West, Seattle, Washington 98119, United States
#Discovery Research, Amgen Inc., One Amgen Center Drive, Thousand Oaks, Callifornia 91320, United States
S
* Supporting Information
ABSTRACT: We describe the structural optimization of a lead compound 1
that exhibits dual inhibitory activities against FLT3 and CDK4. A series of
pyrido[4′,3′:4,5]pyrrolo[2,3-d]pyrimidine derivatives was synthesized, and SAR
analysis, using cell-based assays, led to the discovery of 28 (AMG 925), a potent
and orally bioavailable dual inhibitor of CDK4 and FLT3, including many FLT3
mutants reported to date. Compound 28 inhibits the proliferation of a panel of
human tumor cell lines including Colo205 (Rb+) and U937 (FLT3WT) and
induced cell death in MOLM13 (FLT3ITD) and even in MOLM13
(FLT3ITD, D835Y), which exhibits resistance to a number of FLT3 inhibitors
currently under clinical development. At well-tolerated doses, compound 28
leads to significant growth inhibition of MOLM13 xenografts in nude mice, and
the activity correlates with inhibition of STAT5 and Rb phosphorylation.
Despite extensive efforts toward developing targeted agents
for treatment of AML,7 a critical unmet need remains. FLT3
tyrosine kinase inhibitors used as monotherapies for AML have
effected reductions in peripheral blood and bone marrow blasts
in only a minority of AML patients, and these effects have
usually been transient. This may be due to selection of drug-
resistant mutants which are the primary resistance mechanism
observed in the clinic,8 increases in FLT3 expression during
treatment, or activation of other signaling pathways.9
Cyclin-dependent kinase 4 (CDK4), which is downstream of
FLT310 and other growth signaling pathways, is also up-
regulated in AML by overexpression of cyclin D through
activation of tyrosine kinase growth signaling pathways and loss
of p15 inhibitory function.11 Misregulated CDKs cause
uncontrolled proliferation as well as genomic and chromosomal
INTRODUCTION
■
Acute myeloid leukemia (AML) is a grievous disease for which
there is significant unmet medical need. There are 14600 cases
diagnosed each year, with an estimated 10400 deaths in the
U.S. alone.1 The standard of care for AML is poorly tolerated
among elderly patients. Durable responses are also lacking, and
rapid relapse is common. FMS-like tyrosine kinase 3 (FLT3)
belongs to the receptor tyrosine kinase class III family and is
expressed at high levels in most clinical samples from AML and
B-precursor acute lymphoblastic leukemia (ALL) patients.2−4
Activating mutations in FLT3, mostly internal tandem
duplication (ITD), are detected in approximately 30% of
AML patients and are associated with poor prognosis.5 The
important role that FLT3 plays in the survival and proliferation
of AML blast through the STAT5, RAS-ERK, and phosphati-
dylinositide 3 kinase (PI3K) signaling pathways, as well as its
mutation and overexpression in large numbers of AML patients,
make FLT3 a particularly attractive target for AML therapy.6
Received: January 22, 2014
Published: March 18, 2014
© 2014 American Chemical Society
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instability.12 In addition, synergistic activity has been observed
in preclinical studies when FLT3 and CDK4 were inhibited in
AML cell lines.10
These findings together suggest the potential for a FLT3/
CDK4 dual inhibitor to provide significant benefit in the
treatment of AML, including improving durability of clinical
response by simultaneously modulating two synergistic and
commonly dysregulated targets.
selectivity, and poor oral bioavailability. For structure−activity
relationship (SAR) analysis, FLT3 and CDK family kinases
assays were used as a primary screen to monitor on/off target
activities, with the hope that the selectivity within the CDK
family may be reflective of broader kinase selectivity. The
selectivity over CDK1 is necessary for reducing toxicity and
confounding in vivo analysis.11,15 Using biochemical assays to
optimize against 500 kinases was deemed impractical, therefore
cell-based FLT3 and CDK4 specific assays were used for
screening inhibitors. Three tumor cell lines, Colo205,
MOLM13, and MDA-MB436, were chosen. Colo205 is a
human colorectal cancer cell line whose viability depends on
the activation of the cyclin D-CDK4-Rb pathway. MOLM13 is
a human AML cell line whose viability depends on a
constitutively activated FLT3 kinase. MDA-MB436 is a
human breast cancer cell line whose viability is independent
of FLT3 and CDK4 and was thus included to monitor for off-
target effects. Compounds were anticipated to be selective if
they had high potency against MOLM13 and Colo205 cells but
low activity against MDA-MB436 cell line.
RESULTS AND DISCUSSION
Previously, we reported the discovery of substituted pyrido-
[4′,3′:4,5]pyrrolo[2,3-d]pyrimidine 1 (Figure 1) as a novel
■
While the CYP3A4 inhibitory activity of the lead 1 stems
from the fused pyridine ring of the tricyclic core, our earlier
SAR studies13 had established that this feature was critical for
maintaining good cellular potency. Our strategy for attenuating
the CYP3A4 inhibition, therefore sought not to eliminate this
feature but rather to modulate its ability to coordinate heme
iron through introduction of either sterically demanding or
electron-withdrawing substituents ortho to the pyridine nitro-
gen (Table 1). The applicability of this strategy quickly proved
limited, however, as only small substituents, such as chloro or
methyl, were tolerated by the kinases. While these changes
effectively eliminated the CYP3A4 inhibition, they diminished
either cellular or enzymatic potency and in some cases incurred
potential cardiovascular liabilities by markedly increasing
affinity for the hERG ion channel.
Figure 1. Lead compound 1.
CDK4/6 inhibitor.13 During kinase profiling it was found that
compound 1 also inhibits FLT3, yet possesses promising
selectivity against other kinases. This finding can be rationalized
on the basis of the high degree of similarity between the
binding domains of CDK4/6 (co-crystal structure of CDK6
with 1 in Figure 2a) and APO FLT3 (modeled with 1 in Figure
Thus, substitution with fluoro or chloro maintained
enzymatic potency and selectivity but resulted in reduced
cellular potencies and unacceptably high affinities for hERG (2
and 3, hERG IC50 = 0.72, 2.0 μM, respectively). Additionally,
these halopyridine analogues suffered from increased clearances
in rat (from 0.98 L/h/kg for 1 to 2.1 L/h/kg for 2 and 1.7 L/h/
kg for 3). Substitution with methyl and hydroxyl groups (4 and
5) maintained cellular potency against MOLM13 but
significantly decreased the CDK4-related activity against
Colo205 cells. Substitution with a cyano group (6) resulted
in decreased potency for both CDK4 and FLT3. Replacement
of the pyridine ring with a fused pyridazine (7) likewise
eliminated CYP3A4 inhibition at the expense of potency and
pharmacokinetic properties (rat CL = 5.6 L/h/kg). The
pyridine-modification strategy for attenuating CYP3A4 inhib-
ition was deemed at this point to be very challenging. One after
another, new analogues reinforced the view of the unsub-
stituted pyridine of 1 as critical for potency and physicochem-
ical and pharmacokinetic properties.
As we explored additional SAR of the cycloalkane moiety, we
noticed an opportunity for attenuating CYP3A4 inhibition
through remote influences on the heme iron coordination
sphere. The cyclopentane ring of 1 occupies a region of the
kinases known as the sugar pocket, where the ribose moiety of
ATP normally binds. The sugar pockets of CDK4 and FLT3
are formed by both polar and nonpolar residues, with the more
deeply buried region of the pocket being hydrophobic in
character and surrounded by a solvent exposed, polar rim
Figure 2. Binding of compound 1 in CDK6 and FLT3. (a) Co-crystal
structure of compound 1 in CDK6 at 2.9 Å resolution (PDB code
4P41). (b) Compound 1 with homology model of FLT3.14 The key
active site residues are highlighted in stick representation.
2b). The active sites of the two enzymes differ by only a few
residues, and the binding mode of 1 obtained by modeling with
FLT3 very closely resembles that observed in the co-crystal
structure with CDK6.
In both structures, the fused pyridine moiety of the inhibitor
engages the phenylalanine gatekeeper residue in an edge-to-face
interaction, while the aminopyrimidine ring system participates
in two hydrogen bonding interactions with residues in the
hinge loop. In both structures, the cyclopentyl group sits in a
small hydrophobic cleft between a leucine floor residue and
small side chain alanine (CDK4) or cysteine (FLT3). Taking
advantage of these shared structural features and binding
interactions, we sought to develop dual kinase inhibitors of
FLT3/CDK4 for the treatment of AML.
Lead optimization was conducted to address three main
issues associated with 1: CYP3A4 inhibition, suboptimal
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a
Table 1. Modification of the Fused Pyridine Ring
Y
1 CH
2 CF
3 CCl
4 CCH3
5 COH
6 CCN
7 N
CDK4/D1 IC50 (μM)
FLT3 IC50 (μM)
0.003
0.014
3.00
0.025
0.005
1.76
90
0.003
0.011
7.56
0.052
0.021
2.61
<10
0.004
0.005
3.30
0.063
0.009
2.10
<10
0.020
0.022
2.26
0.28
0.005
3.42
<10
0.005
0.017
10.0
0.12
0.005
2.10
18
0.027
0.046
30.0
0.051
0.095
4.78
0.051
ND
CDK1/B IC50 (μM)
Colo205 IC50 (μM)
MOLM13 IC50 (μM)
MDA-MB436 IC50 (μM)
CYP3A4 INH%@3 μM
CYP3A4 IC50 (μM)
Rat CL (L/h/kg)
0.35
0.062
1.38
7.05
<10
ND
0.55
0.99
>10
>10
>10
>10
2.1
1.7
ND
ND
ND
5.6
a
Values were the means of at least three determinations; standard deviation was 30%. ND: not determined.
(Figure 3). These amphipathic pockets can accommodate a
variety of cyclic moieties, including rings substituted with either
the trans-isomer was responsible for the decreased affinity for
CYP3A4. Subsequently, the trans-isomer (12) was prepared in
stereochemically pure form and found to be free of this liability
(CYP3A4 IC50 >10 μM). Docking studies16 of a series of
inhibitors in the active site of CYP3A4 supported the
hypothesis that ligation of the heme iron could be disfavored
by a remote steric interaction with a sterically bulky
cycloalkane. While the studies revealed favorable binding
modes associated with coordination of the heme iron by the
pyridine moiety of 1, steric interactions developed between the
cycloalkane and key residues of the CYP active site when the
cycloalkane was appropriately substituted, and these destabiliz-
ing interactions grew more pronounced as the substituent was
made longer and bulkier. Compound 14 illustrates that this
effect does not derive from nonspecific lipophilicity but requires
the specific steric demand available in compounds 12 and 13.
As the trans-4-methycyclohexyl group abolished CYP3A4
inhibition without diminishing FLT3 or CDK4 potency or
inducing any other known liabilities, this group was chosen for
further analogues.
Having overcome one of the three major challenges
associated with this series by optimizing the tricyclic core and
its cycloalkyl substituent, we next sought to improve
bioavailability and FLT3 potency through appropriate mod-
ifications of the distal amine function (i.e., the piperazine ring
of 1) and the heteroarene ring linking it to the tricyclic core. In
the co-crystal structure, the piperazine ring sits in a channel of
the kinase that opens toward solvent and can accommodate a
wide array of polar groups. We therefore anticipated that
optimization of the physicochemical properties of the dual
inhibitors by variation of the pyridinylpiperazine could be
especially fruitful. Our initial work focused on developing a
series that retained a basic amine because this feature was
known to contribute favorably to selectivity within the CDK
family and to solubility. The piperazine basic nitrogen of 1,
which is protonated under physiological conditions, rests above
a threonine residue in the X-ray co-crystal structure with CDK6
(Thr-102 in CDK4) and engages this residue in a hydrogen-
bond interaction (Figure 2), whereas the corresponding lysine-
Figure 3. CDK6/compound 1 co-crystal structure highlighting ATP
binding site with P-loop surface removed for clarity (PDB code 4P41).
polar or nonpolar groups. We also considered the comple-
mentary possibility that replacing the cyclopentyl ring with a
polar surrogate could impact CYP3A4 inhibition by changing
the physicochemical properties of the molecule. Extensive
exploration of this region was performed, and only selected
examples are discussed here (Table 2).
As evidenced by compound 8, an acyclic moiety containing a
polar group can significantly reduce CYP3A4 inhibition;
however, in most cases this modification resulted in a more
than 10-fold loss of the binding affinity to the kinase target.
However, the hydroxyl substituted cyclohexane (9) did not
significantly reduce CYP3A4 inhibition and additionally
suffered losses of cellular potency and selectivity relative to 1.
Likewise, bulkier aliphatic rings, like cyclohexyl (10) and
cycloheptyl (data not shown), did not modulate CYP3A4
inhibitory activity either. However, addition of a methyl group
at the 4-position of the cyclohexyl moiety led to a significant
reduction in CYP3A4 inhibition (11). Entry 11 corresponds to
a mixture of cis and trans diastereomers (1/1 ratio), and based
on data collected from previous analogues, we predicted that
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a
Table 2. Optimization of the Sugar Pocket Binding Moiety
a
Values were the means of at least three determinations; standard deviation was 30%. ND: not determined.
a
Table 3. Optimization of the Heteroaryl Linker
a
b
Values were the means of at least three determinations; standard deviation was 30%. Formulation: 1.5% sodium acetate/pH4. ND: not
determined.
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89 of CDK1 should interact unfavorably with the basic amine
function.
Extending the basic nitrogen farther into the solvent channel
by making it exocyclic (17) (Table 3) substantially increased
cellular and FLT3 potency but diminished selectivity for CDK4
over CDK1 compared to 12. Selectivity for CDK4 was restored
when the primary amine of 17 was bis-methylated (18) while
maintaining similar potency across the biochemical and cellular
assays.
In parallel with these studies, we also explored the effects of
atom substitution within the heteroarene linker (15, 16, 19,
22−25) (Tables 3 and 4) as well as installation of an additional
substituent ortho to the pyridine nitrogen (20, 21, 27, 28).
Transposing the pyridine nitrogen markedly improved potency
against FLT3 but severely eroded selectivity within the CDK
family (12 versus 15). Replacing the pyridine with a more polar
pyridazine (16, 19, 22−25) did not affect potency or selectivity
but did favorably impact a number of important pharmacoki-
netic parameters such as solubility and bioavailability.
Unexpectedly, heteroatom substitution ortho to the pyridine
nitrogen gave remarkable increases in FLT3 potencies
(picomolar IC50s for 20, 21) and dramatic improvements in
oral bioavailability while maintaining acceptable selectivity
among the CDKs. Unfortunately, these compounds were not
tolerated in mice at doses that led to 100% tumor growth
inhibition (TGI) in xenograft studies.
Convergence of the heteroarene and distal amine-modifica-
tion strategies resulted in compound 19 (CDK4 IC50 = 2 nM;
FLT3 IC50 = 4 nM; MOLM13 IC50 = 9 nM; Colo205 IC50 = 11
nM; >1000-fold selectivity for CDK4 over CDK1; >200-fold
cellular off/on targets ratio; pRb IC50 = 23 nM and pSTAT5
IC50 = 52 nM in MOLM13 cell line, respectively), and we were
pleased to find that this molecule exhibited good-to-moderate
pharmacokinetics across the three species studied (rat, mouse,
and dog). A dose-dependent decrease in Rb phosphorylation
(pRb) was observed in a MOLM13 mouse xenograft PD study
with 19 starting at the 37.5 mg/kg dose (Figure 4b). In
addition, a dose-dependent increase in drug concentration in
plasma was observed. However, drug exposures in mouse
neither led to a dose-dependent reduction in STAT5
phosphorylation (pSTAT5) nor a sufficient decrease in
pSTAT5 even at the 150 mg/kg dose (Figure 4a). This may
have been due to limited drug exposure levels in the mouse
plasma (unbound Cmax = 0.024 μM at 150 mg/kg dose, much
lower than cellular IC50 of pSTAT5 in MOLM13 of 52 nM).
Reasoning that further optimization of the physicochemical
properties would be required for achieving efficacious drug
exposures, and knowing that the charged anime group of our
inhibitors was associated with reduced phospholipid membrane
permeability and intestinal absorption and with increased efflux
rates, we next turned our attention toward less basic polar
surrogates for the amine moiety of 19. Representative nonbasic
analogues are shown in Figures 5 and 6 and discussed in detail
below.
Figure 4. STAT5 and Rb phosphorylation in MOLM13 tumors
treated with 19. MOLM13 tumor bearing mice were dosed by oral
administration of 19 at 37.5, 75, or 150 mg/kg, and tumors were
harvested at 3 and 24 h after treatment. Tumors lysates were prepared
and the level of pSTAT5 (at 3 h post dose, (a)) or pRb (at 24 h post
dose, (b)) was determined. Unbound plasma levels of 19 (μM) for the
same time points are shown. Data are presented as mean SEM, n = 3
except for vehicle group (n = 6).
Figure 5. (a) Experimentally measured pKa of compound 19; (b)
R4N+ and Cl− ion pair may be stabilized by hydrogen bonds.
h at the 2 mg/kg oral dose). Unfortunately, further develop-
ment of this series was precluded by high binding affinities for
hERG (IC50 = 0.92 μM) and moderate-to-high clearance in the
dog.
Ultimately, we sought to replace the basic amine with an
effectively nonbasic polar surrogate (e.g., 23−28). Glycola-
mides, sulfones, and various spiro-lactams all resulted in
dramatically improved pharmacokinetic properties, particularly
increased drug exposures in high-dose mouse experiments
(Figure 6) at the expense of modest reductions in potency
(Table 4).
First, we considered that ethanolamine derivatives, exempli-
fied by 22, might benefit from several advantages, including
reductions in basicity compared to 19 (pKa = 9.2) through
either inductive or hydrogen-bonding effects as well as possible
stabilization of the ion pairs required for efficient passive
diffusion of the protonated structure (Figure 5).17 Propanol-
amine 22 indeed gave much higher drug exposure in the rat
(total Cmax = 0.28 μM and AUC = 3.24 μM·h at the 2 mg/kg
oral dose) than 19 (total Cmax = 0.13 μM and AUC = 1.57 μM·
Among these analogues, compound 28 (AMG 925) emerged
as the most suitable candidate for clinical development by
virtue of its excellent pharmacokinetic properties (in rat, CL =
0.39 L/h/kg, F% = 75), well-balanced inhibitory activities on
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Figure 6. Drug exposure levels vs molecular basicity (C3h: total drug concentration at 3 h post 150 mg/kg oral dose in mouse PK studies).
a
Table 4. Optimization of the Heteroaryl Linker
a
b
Values were the means of at least three determinations; standard deviation was 30%. Formulation: 1.5% sodium acetate/pH4.
Table 5. Binding Affinity of 28 to Wild-Type and Mutant FLT3
N841I
FLT3WT
0.004
FLT3ITD
0.004
FLT3D835Y
0.001
FLT3D835H
0.001
FLT3K663Q
0.004
FLT3
0.004
Kd (μM)
FLT3 and CDK4 with good selectivity against the Kinome Scan
panel18 (Supporting Information Tables 1 and 2), potent
antiproliferation efficacy against MOLM13 cells, and superior
safety profile and in vitro ADME properties (data not shown).
With a suitable candidate now in hand, we evaluated 28
against a panel of activated variants of FLT3. Activating
mutations of kinases are commonly observed in cancer and can
be selected by treatment with type II inhibitors, promoting the
development of cancer that is more difficult to treat and leading
to an acute need for agents that efficaciously target these
mutations.8 Gratifyingly, 28 exhibited excellent binding
affinities (1−4 nM) for all of the FLT3 mutants available in
the KINOMEscan (data in Table 5). This high affinity for
mutated forms of FLT3, particularly those mutants known to
confer pronounced resistance to FLT3-inhibition monotherapy,
make compound 28 very attractive for clinical development.
The activity of 28 in various tumor cell lines demonstrated its
ability to block FLT3 and CDK4. Compound 28 showed
potent and broad antiproliferation activities against AML cell
lines independent of the FLT3 mutation status while
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maintaining simultaneous inhibition of CDK4. Compound 28
inhibited pSTAT5 in MOLM13 and pRb in Colo205 with IC50s
of 0.005 and 0.023 μM, respectively, indicating that the
observed efficacy in vitro was consistent with FLT3 and CDK4
inhibition (Table 6). In contrast, sorafenib,19 a multikinase
respectively, we modeled the binding modes of the two
compounds to FLT3, the conformation of the activation loop
(DFG-in, DFG-out),21 and key FLT3 activation mutations and
how they affect the activation loop conformation. The
activation loop in a kinase is inherently flexible and capable
of multiple conformations. For this analysis, we focused on two
representative conformations, DFG-in as the active conforma-
tion and DFG-out as the inactive conformation. Previously
published binding data suggest that sorafenib is a type II kinase
inhibitor that preferentially binds to the inactive DFG-out
kinase conformation.22 In this sorafenib−FLT3 model, the
phenolic ring of sorafenib would form a perpendicular
aromatic−aromatic interaction with F830 in the DFG motif
(Figure 7c), which is one of the key interactions with the
Table 6. The Antiproliferation Activity of 28 in Comparison
a
with a CDK4/6 and FLT3 Inhibitor
28
palbociclib
sorafenib
FLT3 IC50 (μM)
0.001
0.003
0.005
0.023
0.019
0.052
0.023
0.055
3.48
0.002
>3
0.004
>3
CDK4 IC50 (μM)
pSTAT5 IC50 (μM)
pRb IC50 (μM)
0.002
>3
0.011
0.096
0.14
MOLM13 IC50 (μM)
U937 IC50 (μM)
MOLM13SR IC50 (μM)
0.005
>3
0.096
0.036
>3
Colo205 IC50 (μM)
>3
a
The antiproliferation activity was determined by 14C-thymindine
incorporation DNA synthesis assay; MOLM13SR: (FLT3ITD+D835Y
)
clone was isolated by culturing MOLM13 in the presence of 1 nM
Sorafenib. Resistance cells were isolated in about 6 weeks, and the cells
can rapidly adapt to >1 μM of sorafenib in culture (about 2 weeks
from 1 nM to 1 μM).
inhibitor with FLT3 activity, showed potent inhibitory activity
toward the FLT3ITD mutant MOLM13 cell line but almost no
activity against U937, a FLT3 wild-type AML cell line. In
contrast to palbociclib,19 a CDK4/6 inhibitor, which arrests Rb
positive tumor cells including MOLM13 in G1 stage, 28
induced apoptosis in FLT3ITD AML cell lines and the
FLT3ITD,D835Y mutant AML cell line, the latter being a mutant
strain cultivated to exhibit marked resistance to sorafenib by
continuously treating MOLM13(FLT3ITD) cells with gradually
increased concentration of sorafenib (from 1 to 1000 nM) over
the course of several weeks (Table 7). Analogous experiments
Figure 7. Computational models of binding modes and activation loop
conformations of inactive DFG-out and active DFG-in activation loop
conformations.14,23 (a) DFG-out kinase with the activiation loop
(green) in the inactive conformation stabilized by intramolecular
hydrogen bonds. (b) DFG-in kinase with the activation loop (green)
in the active DFG-in conformation. (c) DFG-out kinase with sorafenib
model showing type 2 binding mode. (d) DFG-in kinase with 28
model showing type I binding mode.
Table 7. Compound 28 Induced Apoptosis in FLT3 Mutant
a
AML Cells
28
palbociclib sorafenib control
U937 (FLT3WT
MOLM13 (FLT3ITD
MOLM13SR (FLT3ITD+D835Y
)
23.68
82.30
70.86
9.09
16.95
8.35
8.76
84.60
5.16
7.14
5.44
7.57
)
protein. This interaction would not be possible in the active
DFG-in kinase conformation. In this inactive, DFG-out
conformation, FLT3 native residues D835 and Y842 stabilize
this conformation of the activation loop by forming hydrogen
bonds with the main chain amide group S838 and side chain
D811, respectively (Figure 7a). Thus, replacement of either
residue might destabilize the inactive conformation of the
activation loop, which would then be expected to hinder the
binding of sorafenib.
In contrast, 28 is a type I kinase inhibitor and preferentially
binds to the active DFG-in conformations (Figure 7d) and
maintains activity against those activation loop single point
mutations.
The pharmacokinetic profiles of 28 were investigated in rat,
dog, and cynomolgus monkey (Table 8). Plasma elimination
half-lives following intravenous dosing range from 1.7 h in dog
to 5.6 h in monkey. Steady state volumes of distribution values
(Vss) were fairly constant across species, with values ranging
from 1.8 L/kg in rat to 3.8 L/kg in cynomolgus monkey. The
plasma clearance in rat was low relative to hepatic blood flow,
while it was moderate to high in dog and moderate in
cynomolgus monkey. In all species, the compound showed
)
a
MOLM13, MOLM13SR, and U937 cells were treated with 28,
palbocilib, and sorafenib at 1 μM for 48 h, individually. The treated
cells were stained with Annexin V/Sytox Green and analyzed by flow
cytometry for apoptosis. Data represent percentage of apoptotic cells
in total cells analyzed.
conducted with the concentration of 28 ranging as high as 10
nM over treatment durations as long as 4 months failed to
produce appreciable resistance to 28 (IC50 = 0.028 μM), and
sequencing analysis showed that no additional FLT3 mutations
had been produced under these conditions.20 These results are
consistent with the hypothesis that dual inhibition of FLT3 and
CDK4 make it more challenging for AML cells to develop
resistance to FLT3 inhibition than in the case of FLT3-
inhibition monotherapy. In aggregate, these data indicate that
the FLT3/CDK4 dual inhibitor 28 has broader activity, and is
potentially more effective in treating AML, than the current
therapeutic agents.
To understand the structural properties that result in the
differential activities against FLT3 mutants of sorafenib and 28,
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Table 8. Pharmacokinetic Parameters of 28 in Rat, Dog, and
Cynomolgus Monkey
species
CL (L/h/kg)
Vss (L/kg)
T1/2,z (h)
F (%)
a
rat
0.39
1.41
1.42
1.8
2.6
3.2
5.5
4.1
6.6
75
ca. 100
44
b,c
dog
b,c
monkey
a
Vehicle used in iv and po studies in rat: water/acetate/tween at pH4,
b
96/3/1 (v/v/v). Vehicle used in iv studies in dog and monkey: 40%
PG aqueous solution at pH2. Vehicle used in po studies in dog and
c
monkey: 20% captisol aqueous solution.
moderate to high oral bioavailability following gavage
administration in solution formulation. This preclinical
pharmacokinetic profile was the basis for predicting, using in
vitro−in vivo extrapolation methods, human oral clearance of
0.21 L/h/kg and a terminal phase half-life of 3.3 h, a profile
consistent with twice-daily dosing.
The combination of potent tumor cell growth inhibition in
vitro and high exposure led to significant antitumor effects in
vivo. NCR nude mice bearing MOLM13 tumors were treated
with 28 orally at doses of 25, 50, 75, and 150 mg/kg once daily
for 8 days. Dose-dependent inhibition of tumor growth was
observed with 100% tumor growth inhibition (TGI) at 150
mg/kg dose (Figure 8). The calculated ED50 was 22 mg/kg,
Figure 9. Compound 28 inhibited STAT5 (a) and Rb (b)
phosphorylation in MOLM13 subcutaneous tumors. MOLM13
tumor bearing mice were dosed by oral administration of 28 at 50,
75, or 150 mg/kg, and tumors were harvested at 3, 6, and 24 h after
treatment. Tumors lysates were prepared, and the level of pSTAT5
(upper panel) or pRb (lower panel) was determined.24 Unbound
plasma levels of 28 (μM) same time points are shown. Data are
presented as mean SEM, n = 3 except for vehicle group (n = 6).
Figure 8. In vivo tumor growth inhibition for 28. NCR nude mice
bearing established human MOLM13 AML xenografts were dosed
orally with either vehicle or 28 at 25, 50, 75, and 150 mg/kg (day 14
TGI = 100%, p < 0.0001) once daily from day 5 to day 13.
and the corresponding AUC was determined to be 28 μM·h
from terminal PK analyzed from plasma harvested after the final
dose (Supporting Information Figure 1). Body weight losses
were within an acceptable range (10%) throughout the study
(Supporting Information Figure 2), and 28 was well tolerated.
In addition, the phosphorylation of STAT5, which is an
important downstream signaling protein of FLT3 signaling, and
the phosphorylation of Rb, a downstream protein of the CDK4
pathway, were also significantly reduced in MOLM13 tumor
tissues obtained from the 28 treated group compared with the
vehicle group (Figure 9), indicating that the observed efficacy
in vivo was consistent with FLT3 and CDK4 signaling
inhibition.
Figure 10. Numbering convention used for the pyrido[4′,3′:4,5]-
pyrrolo[2,3-d]pyrimidine core.26
the C4a−C4b heterobiaryl bond through either Negishi or
Suzuki cross-coupling (Scheme 1). Early variants of this
strategy generated bromopyrimidine Suzuki substrates 31a−e
in a two-step substitution sequence beginning with primary
aliphatic amines of interest and commercially available 5-
bromo-2,4-dichloropyrimdine 29. Variation of the amine
substituent at this juncture enabled broad characterization of
SAR trends associated with the cycloalkane sugar-pocket group
displayed at N9 of the tricyclic core (Table 2, 8−14).25 The
N9−C8a bond establishing the fused pyrrole ring of the core
was formed by intramolecular Hartwig−Buchwald amination of
heterobiaryl cross-coupling products 32a−e, with subsequent
installation of the heteroarene substituent at N1′ by
intermolecular amination of haloheteroarenes 34. Piperazine
SYNTHETIC CHEMISTRY
■
The general approach to the pyrido[4′,3′:4,5]pyrrolo[2,3-
d]pyrimidine series of dual inhibitors (Figure 10) fashioned
the key C−N bonds using nucleophilic aromatic substitution
and Hartwig−Buchwald amination sequences and established
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Scheme 1. First-Generation Synthetic Approach to the
Pyrido[4′,3′:4,5]pyrrolo[2,3-d]pyrimidine Dual Inhibitors
Scheme 2. Synthesis of ortho-Substituted Pyridine Analogues
via Pyridone 38
a
a
a
Reagents and conditions: (a) 31a, PdCl2(PPh3)2, Na2CO3, 1,4-
a
dioxane, 120 °C; (b) Pd2dba3, XantPhos, t-BuONa, 1,4-dioxane, 150
°C; (c) HCl, 1,4-dioxane/MeOH, 80 °C; (d) POCl3, 100 °C; (e)
Pd2dba3, XantPhos, t-BuONa, 1,4-dioxane, 80 °C; (f) TFA/CH2Cl2;
(g) Tf2NPh, NaH, DMF; (h) Fe(acac)3, MeMgBr, THF/NMP; (i)
34a, Pd2dba3, XantPhos, t-BuONa, 1,4-dioxane, 120 °C; (j) TFA/
CH2Cl2; (k) 34a, Pd2dba3, XantPhos, t-BuONa, 1,4-dioxane, 120 °C;
(l) TFA/CH2Cl2.
Reagents and conditions: (a) RNH2, DIPEA, reflux; (b) NH4OH, i-
PrOH, 120 °C; (c) PdCl2(PPh3)2, Na2CO3, 1,4-dioxane, 120−150 °C;
(d) Pd2dba3, XantPhos, t-BuONa, 1,4-dioxane, 150 °C; (e) Pd2dba3,
Xantphos, t-BuONa, 1,4-dioxane, 120 °C; (f) TFA/CH2Cl2.
derivatives (e.g., lead compound 1) were liberated upon acidic
cleavage of the t-butylcarbamoyl group, and alkylation or
acylation of the resulting basic nitrogen function enabled the
preparation of an array of higher analogues (e.g., 23).
Scheme 3. Negishi Coupling Approach to Substituted Fused-
a
Pyridine Derivatives 2 and 6
Our discovery efforts ultimately led us toward extensive
characterization of SAR trends associated with the fused
pyridine ring of the tricyclic core, with particular concentration
in studying the effects of substitution at C8. We found it most
expedient to prepare many of the substituted pyridine
analogues via interconversions between pyridone derivatives
(Scheme 2). The parent pyridone core 38 could be readily
obtained from methoxypyridine 37 and thus from commercially
available methoxypyridylboronic acid 35. Certain analogues
more easily accessed when the synthesis design was modified to
employ a milder construction of the heterobiaryl C4a−C4b
bond by Negishi coupling of iodopyrimidines 44a,b (Scheme
3) with suitable arylzinc precursors. ortho-Fluoropyridine 2
could thus be prepared by directed lithiation/transmetalation of
iodobromofluoropyridine 45 (Scheme 3). ortho-Cyano deriva-
tives (e.g., 6) were in turn generated from a common
fluoropyridine intermediate 47 by the action of tetraethylam-
monium cyanide in hot DMF (Scheme 3). Synthesis of
pyridazine derivative 7 also proceeded by way of Negishi
chemistry (Scheme 4), with formation of the central ring via
nucleophilic displacement.
a
Reagent and conditions: (a) cyclopentylamine or trans-Me-cyclo-
hexylamine, 1,4-dioxane or BuOH, 100 °C; (b) NIS, DMF or AcOH,
80 °C; (c) i-PrMgCl, ZnCl2; (d) 44a, Pd(PPh3)4, THF, reflux; (e) X-
phos, Cs2CO3, 1,4-dioxane, 120 °C; (f) 34a, Pd2dba3, XantPhos, t-
BuONa, 1,4-dioxane, 120 °C; (g) TFA/CH2Cl2; (h) NEt4CN, DMF,
150 °C; (i) 34a, Pd2dba3, XantPhos, t-BuONa, 1,4-dioxane, 120 °C;
(j) TFA/CH2Cl2.
Surveying the effects of varying the cycloalkane and fused-
pyridine binding motifs led to identification of 33d as the
optimal scaffold, and our escalating demand for this material
necessitated the development of a highly efficient and scaleable
route from cost-effective precursors. Applying the Negishi
methodology developed above, we were gratified to discover
that the readily available feedstock 3-fluoropyridine 54
underwent high-yielding and facile cross-coupling with 44b
upon directed lithiation by LiTMP at cryogenic temperature
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a
Scheme 4. Negishi Route to Fused Pyridazine Derivative 7
Scheme 6. Synthesis and Coupling of Haloheteroarene
a
Amination Substrates
a
Reagents and conditions: (a) LiTMP, ZnCl2; (b) 44a, Pd(PPh3)4,
THF, reflux; (c) NaH, THF, 150 °C; (d) PPTS, 180 °C; (e) PhNTf2,
TEA, DMAP; (f) Pd(OAc)2, dppf, TEA, HCO2H, DMF; (g) 34a,
Pd2dba3, XantPhos, t-BuONa, 1,4-dioxane, 120 °C; (h) TFA/CH2Cl2.
followed by transmetalation to zinc bromide (Scheme 5).27,28
Intramolecular displacement of fluoride by the pendant
a
Reagents and conditions: (a) Pd2dba3, XantPhos, t-BuONa or
Cs2CO3, 1,4-dioxane or PhMe, 90−120 °C (for 58a, 58c−d), DIPEA,
1,4-dioxane, 80 °C (for 58b,e); (b) 33d, Pd2dba3, XantPhos, t-BuONa
or Cs2CO3, 1,4-dioxane or PhMe, 100−120 °C; (c) TFA/CH2Cl2.
Scheme 5. Process Synthesis of Optimized Tricyclic Core
33d
a
Scheme 7. Modular Approach to 4-Aminopiperidine
Derivatives 17 and 22 via Late-Stage Reductive Amination
a
a
Reagents and conditions: (a) LiTMP, ZnBr2, −78 °C; (b) 44b,
Pd(PPh3)4, THF, reflux; (c) LiHMDS, NMP, 90 °C.
secondary amine function in the presence of LiHMDS
completed the synthesis of 33d. The optimized sequence
enabled rapid preparation of kilogram quantities of this critical
intermediate.
Exploring SAR trends associated with the N1′ pyridyl
substituent and the piperazine ring (more generally, the distal
solubilizing group) situated at its 5 position required us to
prepare a wide array of haloheteroarene Hartwig−Buchwald
substrates (58a−d, Scheme 6); this was generally accomplished
by arylating the corresponding secondary amines (e.g., 57).
Studies directed at optimizing substituents on the basic
nitrogen in the 4-aminopiperidine series effected late-stage
diversification by reductive amination of advanced ketones
60a,b (illustrated for 17 and 22, Scheme 7).
Derivatives featuring nonbasic distal solubilizing moieties
often required the development of more involved, target-
specific methodologies. Syntheses of representative compounds
from the most important classes are outlined in Schemes 8−10.
The halopyridazine precursor 64 to aliphatic sulfone 24 was
prepared from primary alcohol 61 by a straightforward
amination/mesylation/SN2 methylsulfanation/oxidation se-
quence (Scheme 8).
a
Reagents and conditions: (a) 33d, Pd2dba3, XantPhos, t-BuONa, 1,4-
dioxane; (b) 6N HCl (aq); (c) NaBH3(CN), NH4OAc, MeOH, 60
°C; (d) NaBH(OAc)3, H2CO (aq), CH2Cl2.
Finally, the requisite amination substrate for the synthesis of
28 could be prepared by Boc protection of commercially
available chlorotetrahydronaphthyridine 70 (Scheme 10).
Hartwig−Buchwald coupling of 71 with 33d and subsequent
Boc deprotection both occurred in virtually quantitative yields
under optimized conditions. Acetoxyacetylation and acetate
saponification completed the synthesis, giving 28 in high overall
yield (97% from 70).
Arylation of the spirocyclic piperidines 65 and 68 gave the
corresponding amidation substrates 66, 69a, and 69b used to
prepare compounds 25−27 (Scheme 9).
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Scheme 8. Synthesis of the Precursor to Aliphatic Sulfone
24
CONCLUSION
■
a
From lead compound 1, a combination of structure-based drug
design, traditional, and novel medicinal chemistry approaches
led to the discovery of 28, a FLT3/CDK4 dual inhibitor with
preferential affinity for the activated state of FLT3. Compound
28 shows potent in vitro activities against CDK4 and FLT3,
including several key FLT3 mutants, and displays potent
antitumor efficacy mediated by inhibition of FLT3 and Rb
phosphorylation in vivo. Compound 28 possesses excellent
cross-species pharmacokinetic properties in multiple preclinical
models. Compound 28 may improve the durability of clinical
response in the treatment of AML by simultaneously
modulating two key targets.
a
Reagents and conditions: (a) 56b, 90 °C; (b) MsCl, TEA; (c)
NaSMe, DMF; (d) oxone, MeOH; (e) 33d, Pd2dba3, XantPhos, t-
BuONa, 1,4-dioxane, 120 °C.
EXPERIMENTAL SECTION
■
Scheme 9. Synthesis of the Precursor to Spirocyclic Sulfone
and Spirocyclic Lactams
General Chemistry. All reactions were conducted under an inert
gas atmosphere (nitrogen or argon) using a Teflon-coated magnetic
stirbar at the temperature indicated. Commercial reagents and
anhydrous solvents were used without further purification. Removal
of solvents was conducted by using a rotary evaporator, and residual
solvent was removed from nonvolatile compounds using a vacuum
manifold maintained at approximately 1 Torr. All yields reported are
isolated yields. Preparative reversed-phase high pressure liquid
chromatography (RP-HPLC) was performed using an Agilent 1100
series HPLC and Phenomenex Gemini C18 column (5 μm, 100 mm ×
30 mm i.d.), eluting with a binary solvent system A and B using a
gradient elusion [A, H2O with 0.1% trifluoroacetic acid (TFA); B,
CH3CN with 0.1% TFA] with UV detection at 220 nm. All bioassayed
compounds were purified to ≥95% purity as determined by a Agilent
1100 series HPLC with UV detection at 220 nm using the following
method: Zorbax SB-C8 column (3.5 μm, 150 mm × 4.6 mm i.d.);
mobile phase, A = H2O with 0.1% TFA, B = CH3CN with 0.1% TFA;
gradient, 5−95% B (0.0−15.0 min); flow rate, 1.5 mL/min. Low-
resolution mass spectral (MS) data were determined on an Agilent
1100 series LCMS with UV detection at 254 nm and a low-resolution
electrospray mode (ESI). High-resolution mass spectra (HRMS) were
obtained on an Agilent 6510 Q-TOF MS with an Agilent 1200 LC on
the front end. 1H NMR spectra were obtained on a Bruker Avance III
500 (500 MHz) or Bruker Avance II 400 (400 MHz) spectrometer.
Chemical shifts (δ) are reported in parts per million (ppm) relative to
residual undeuterated solvent as an internal reference. The following
abbreviations were used to explain the multiplicities: s = single, d =
doublet, t = triplet, q = quartet, dd = doublet of doublets, dt = doublet
of triplets, m = multiplet, br = broad.
a
a
Reagents and conditions: (a) 56b, DIPEA, 1,4-dioxane, 90 °C; (b)
33d, Pd2dba3, XantPhos, t-BuONa, 1,4-dioxane, 150 °C; (c) HCl, 1,4-
dioxane; (d) 56a or 56d, XantPhos, Pd2dba3, t-BuONa, PhMe; (e)
33d, Pd2dba3, Xantphos, t-BuONa, 1,4-dioxane, 150 °C.
a
Scheme 10. Synthesis of 28
5-Bromo-2-chloro-N-cyclopentylpyrimidin-4-amine (30a). To a
solution of 5-bromo-2,4-dichloropyrimidine (45.6 g, 200 mmol) in
dioxane (400 mL) was added N-cyclopentylamine (20.4 g, 240 mmol)
at room temperature. The mixture thus obtained was stirred at room
temperature for 6 h. The reaction mixture was then diluted with ethyl
acetate, washed with brine, and dried over MgSO4. The solvent was
evaporated to give compound 30a as light-yellow solid (56 g, 100%
1
yield), which was used in next step without further purification. H
NMR (500 MHz, DMSO-d6) δ ppm 8.23 (1H, s), 7.37 (1H, d, J = 7.3
Hz), 4.31 (1H, m), 1.92 (2H, m), 1.71 (2H, m), 1.53−1.59 (4H, m).
MS (ESI) m/z: 276.0 [M + H]+.
5-Bromo-N4-cyclopentylpyrimidine-2,4-diamine (31a). A solution
of 30a (45 g, 200 mmol) in 28% NH4OH/2-propanol (1/1, 400 mL)
was heated at 120 °C in sealed tube for 22 h. The product was
extracted with dichloromethane, and the organic layers were washed
with brine and dried. The solvent was evaporated, and the residue was
purified by flash chromatography on silica gel eluting with 25% ethyl
acetate in hexanes to give compound 31a as white solid (34 g, 66%
1
yield). H NMR (500 MHz, DMSO-d6) δ ppm 7.77 (1 H, s), 6.19 (2
H, br s), 6.12 (1 H, d, J = 7.3 Hz), 4.33 (1 H, m), 1.90 (2 H, m), 1.69
(2 H, m), 1.49−1.55 (4 H, m). MS (ESI) m/z: 257.0 [M + H]+.
5-(3-Chloropyridin-4-yl)-N4-cyclopentylpyrimidine-2,4-diamine
(32a). To a solution of 31a (2.57 g, 10.0 mmol) in dioxane (75 mL)
were added 3-chrolopyridine-4-boronic acid (4.72 g, 10.0 mmol),
a
Reagents and conditions: (a) (Boc)2O, DIPEA, DCM; (b) 33d,
Pd2dba3, XantPhos, t-BuONa, 1,4-dioxane, 100 °C; (c) HCl, MeOH/
H2O; NaOH; (d) DIPEA, CHCl3; (e) MeONa, DCM/MeOH.
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PdCl2(PPh3)2 (702 mg, 1.0 mmol), and sodium carbonate (3.82 g, 36
mmol, in 36 mL of water). The mixture thus obtained was purged with
N2 for 10 min and heated at 120 °C in a sealed tube for 22 h. The
reaction mixture was diluted with water, and the product was extracted
with chloroform. The organic layers were dried over MgSO4 and
concentrated. The residue was purified by flash chromatography on
silica gel, eluting with 2.5% methanol in dichloromethane to give
compound 32a as white solid (2.31g, 80% yield). 1H NMR (500 MHz,
DMSO-d6) δ ppm 8.64 (1 H, s), 8.50 (1 H, d, J = 4.9 Hz), 7.51 (1 H,
s), 7.34 (1 H, d, J = 4.9 Hz), 6.21 (2 H, br s), 5.97 (1 H, d, J = 7.4 Hz),
4.43 (1 H, p, J = 7.4 Hz), 1.84 (2 H, m), 1.62 (2 H, m), 1.40−1.49 (4
H, m). MS (ESI) m/z: 290.0 [M + H]+.
9-Cyclopentyl-9H-pyrido[4′,3′:4,5]pyrrolo[2,3-d]pyrimidin-2-
amine (33a). To a solution of 32a (2.3 g, 7.9 mmol) in 1,4-dioxane
(60 mL) were added Pd2dba3 (368 mg, 0.4 mmol), XantPhos (696
mg, 1.2 mmol), and sodium t-butoxide (1.15 g, 12 mmol). The
mixture thus obtained was heated at 150 °C under microwave
irradiation for 3 h. The reaction mixture was passed through a short
pack silica gel column and concentrated. The residue was purified by
flash chromatography on silica gel eluting with 2% methanol in
dichloromethane to give compound 33a as white solid (1.71 g, 85%
yield). 1H NMR (500 MHz, DMSO-d6) δ ppm 9.05 (1 H, s), 8.87 (1
H, br s), 8.37 (1 H, d, J = 4.9 Hz), 7.93 (1 H, d, J = 5.1 Hz), 6.97 (2 H,
br s), 5.33 (1 H, quin, J = 8.6 Hz), 2.27 (2 H, m), 2.04 (4 H, m), 1.76
(2 H, m). HRMS (ESI) m/z: calculated for [M + H]+ 254.1400, found
254.1406.
in vacuo. The residue was subjected to flash column chromatography
on silica gel eluting with 1% methanol in DCM to give compound 44a
as a light-yellow solid (39 g, 73% yield). 1H NMR (500 MHz, DMSO-
d6) δ ppm 7.89 (1 H, s), 6.18 (2 H, s), 5.63 (1 H, d, J = 7.6 Hz), 4.30
(1 H, p, J = 7.4 Hz), 1.84−1.96 (2 H, m), 1.61−1.74 (2 H, m), 1.42−
1.58 (4 H, m). MS (ESI) m/z: 305.0 [M + H]+.
5-Iodo-N4-((1r,4r)-4-methylcyclohexyl)pyridine-2,4-diamine
(44b). 4-Chloropyrimidine-2-amine (1000 g, 7.72 mol, 1.0 equiv),
trans-4-methylcyclohexylamine hydrochloride (1500 g, 10.03 mol, 1.3
equiv) and TEA (3.23 L, 23.2 mol, 3.0 equiv) were mixed together in
n-butanol (8 L). The reaction mixture was heated at reflux for 36 h and
monitored using LCMS. Upon completion, the reaction mixture was
cooled to room temperature, diluted with water (8 L), and extracted
with ethyl acetate (2 × 10 L). The organic layers were combined, dried
over Na2SO4, and concentrated under reduced pressure to give N4-
((1r,4r)-4-methylcyclohexyl)pyridine-2,4-diamine (43b) (1770 g),
which was used in the next step without further purification.
Compound 43b (1770 g, 8.58 mol, 1.0 equiv) was dissolved in
anhydrous DMF (8 L). To this solution under N2 atmosphere at 10
°C was added iodosuccinmide (1.93 g, 8.58 mol, 1.0 equiv) in portions
over 10 min. Upon completion of the addition, the reaction mixture
was stirred at room temperature for 2 h. The reaction was monitored
using LCMS. Upon completion, the reaction mixture was cooled using
an ice bath, quenched with saturated aqueous sodium carbonate (5 L),
and extracted with ethyl acetate (2 × 15 L). The combined organic
extracts were washed with saturated aqueous sodium carbonate (2 × 5
L), water (3 × 2 L), dried over Na2SO4, and concentrated under
reduced pressure. The residue was purified using column chromatog-
raphy on silica gel eluting with 25−40% ethyl acetate in hexanes to
provide compound 44b (1470 g, 57% over two steps). 1H NMR (400
MHz, DMSO-d6) δ ppm 7.86 (1H, s), 6.16 (2H, br s), 5.48 (1H, d, J =
8.1 Hz), 3.85 (1H, m), 1.78 (2H, d, J = 12.3 Hz), 1.66 (2H, d, J = 12.3
Hz), 1.41−1.27 (3H, m), 0.98 (1H, dd, J = 12.9, 2.7 Hz), 0.85 (3H, d,
J = 7.2 Hz). MS m/z: 333.0 [M + H]+.
4-(4-Chloro-pyrid-2-yl)-piperidine-1-carboxylic Acid t-Butyl Ester
(34a). To a solution of 5-bromo-2-chloropyridine (11.54 g, 60 mmol)
in toluene (300 mL) were added t-butyl 1-piperazinecarboxylate
(11.18 g, 60 mmol), sodium t-butoxide (8.64 g, 90 mmol), Pd2(dba)3
(1.10 g, 1.20 mmol), and XantPhos (2.08 g, 3.6 mmol). The mixture
was evacuated and purged with argon (3 cycles), then heated at 100
°C for 5 h. After the reaction went to completion, the mixture was
cooled to room temperature, diluted with ethyl acetate (1200 mL),
and washed with water (300 mL). The organic solution was
concentrated under pressure, and the residue was purified by flash
chromatography on silica gel eluting with 17% ethyl acetate in hexane
5-(3-Bromo-2-fluoro-4-pyridinyl)-N4-cyclopentyl-2,4-pyrimidine-
diamine (46). In a dry 3 neck 2 L flask equipped with a dry addition
funnel, thermometer, and stir bar was added 3-bromo-2-fluoro-4-
iodopyridine (83.4 g, 276 mmol) followed by 300 mL of anhydrous
THF under an atmosphere of nitrogen. The solution was cooled to
−70 °C in a 2-propanol/dry ice bath. A solution of isopropylmagne-
sium chloride 2.0 M in diethyl ether (145 mL, 290 mmol) was added
dropwise over a period of 30 min. The solution was then stirred for 30
min before zinc(II) chloride (0.5 M in THF, 276 mL, 138 mmol) was
cannulated in. The solution was warmed to room temperature and
stirred for 1 h. The addition funnel was replaced with a reflux
condenser and 44a (28.00 g, 92.1 mmol) was added, followed by
Pd(PPh3)4 (5.32 g, 4.60 mmol). The solution was heated overnight at
a gentle reflux. After concentrating the solution to 1/10th of the
volume under vacuum, it was cooled in an ice bath. To this was added
100 mL of cold saturated NH4Cl, followed by 500 mL of water. Then
500 mL of 12% 2-propanol/DCM was then added and the solution
was stirred at room temperature for 1 h before being filtered through
filter paper. The filter cake was washed in succession with water,
DCM, and 12% 2-propanol/DCM. The filtrate was partitioned in a
separation funnel, and the aqueous layer was washed with 12% 2-
propanol/DCM. The organics were dried over MgSO4 and then
concentrated under vacuum. The residue obtained was partially
dissolved in DCM then sonicated and filtered to give compound 46
(26.5 g, 82% yield) as a light-yellow solid. 1H NMR (500 MHz,
DMSO-d6) δ ppm 8.18 (1 H, d, J = 4.9 Hz), 7.52 (1 H, s), 7.26 (1 H,
dd, J = 4.9, 0.7 Hz), 6.34 (2 H, s), 6.12 (1 H, d, J = 7.6 Hz), 4.42 (1 H,
p, J = 7.4 Hz), 1.76−1.96 (2 H, m), 1.55−1.68 (2 H, m), 1.31−1.53 (4
H, m)). MS (ESI) m/z: 352.0 [M + H]+.
1
to give compound 34a as a light-yellow solid (14.6 g, 82% yield). H
NMR (400 MHz, CDCl3) δ ppm 8.02 (1 H, s), 7.19−7.18 (2 H, m),
3.59 (4 H, dd, J = 12 Hz, J = 4 Hz), 3.14 (4 H, dd, J = 12 Hz, J = 4
Hz), 1.49 (9 H, s). MS (ESI) m/z: 298.1 [M + H]+.
9-Cyclopentyl-N-(5-(1-piperazinyl)-2-pyridinyl)-9H-pyrido-
[4′,3′:4,5]pyrrolo[2,3-d]pyrimidin-2-amine (1). To a solution of 33a
(1.6 g, 6.3 mmol) in dioxane (60 mL) were added 34a (2.3 g, 7.6
mmol), Pd2dba3 (293 mg, 0.32 mmol), XantPhos (370 mg, 0.64
mmol), and sodium t-butoxide (908 mg, 9.45 mmol). The mixture
thus obtained was heated at 150 °C under microwave irradiation for 1
h. The reaction mixture was passed through a short pack silica gel
column and concentrated to give t-butyl 4-(6-((9-cyclopentyl-9H-
pyrido[4′,3′:4,5]pyrrolo[2,3-d]pyrimidin-2-yl)amino)-3-pyridinyl)-1-
piperazinecarboxylate as a yellow solid (3.28 g), which was then
treated with TFA/DCM (60 mL, 1:1) at room temperature for 30
min. The reaction mixture was concentrated, and the residue was
purified by chromatography on silica gel eluting with CH2Cl2/MeOH/
NH4OH (200:10:1) to give compound (1) as an off-white solid (2.60
1
g, 98% yield). H NMR (400 MHz, DMSO-d6) δ ppm 9.86 (1 H, s),
9.28 (1 H, s), 9.01 (1 H, s), 8.68 (1 H, br s), 8.45 (1 H, d, J = 4 Hz),
8.23 (1 H, d, J = 8 Hz), 8.10 (1 H, d, J = 4 Hz), 8.04 (1 H, d, J = 8
Hz), 7.54 (1 H, dd, J = 8 Hz, J = 4 Hz), 5.43 (1 H, quin, J = 8 Hz),
3.35 (4 H, m), 3.26 (4 H, m), 2.40 (2 H, m), 2.03−2.10 (4 H, m), 1.78
(2 H, m). HRMS (ESI) m/z: calculated for [M + H]+ 415.2354, found
415.2350.
N4-Cyclopentyl-5-iodo-2,4-pyrimidinediamine (44a). To a stirred
solution of compound 43a (25.5 g, 143 mmol) in DMF (120 mL) was
added N-iodosuccinimide (32 g, 143 mmol) in two portions at rt. The
resulting mixture was stirred at rt for 2 h. Upon workup, the mixture
was poured into a mixture of ice and saturated aqueous sodium
carbonate with some sodium sulfite, then extracted with ethyl acetate
(2×). The combined organics were washed with saturated aqueous
sodium carbonate (3×), dried over sodium sulfate, and concentrated
9-Cyclopentyl-8-fluoro-9H-pyrido[4′,3′:4,5]pyrrolo[2,3-d]-
pyrimidin-2-amine (47). Compound 46 (0.143 g, 0.41 mmol), and
cesium carbonate (0.40 g, 1.2 mmol) were combined in anhydrous 1,4-
dioxane (5 mL). Nitrogen was briefly bubbled through the solution
before adding Pd2(dba)3 (0.037 g, 0.041 mmol) and XantPhos (0.047
g, 0.081 mmol). The solution was heated at 120 °C under microwave
irradiation for 1 h. The solution was purified with flash column
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chromatography on silica gel eluting with DCM/MeOH/NH4OH
(200:10:1) to give compound 47 (94 mg, 85% yield) as a light-
brownish solid. 1H NMR (500 MHz, DMSO-d6) δ ppm 9.10 (1 H, s),
7.94 (1 H, dd, J = 5.2, 1.5 Hz), 7.91 (1 H, dd, J = 5.1, 2.9 Hz), 7.10 (2
H, br s), 5.38−5.49 (1 H, m), 2.10−2.23 (2 H, m), 1.93−2.08 (4 H,
m), 1.64−1.79 (2 H, m). MS (ESI) m/z: 272.0 [M + H]+.
brine, dried, and concentrated . The residue was purified by flash
chromatography on silica gel, eluting with DCM/MeOH/NH4OH
(100:10:1) to give compound 40 as a yellow solid (0.102 g, 67%
yield). MS (ESI) m/z: 402.1 [M + H]+.
9-Cyclopentyl-8-methyl-9H-pyrido[4′,3′:4,5]pyrrolo[2,3-d]-
pyrimidin-2-amine (41). MeMgBr (3 M, in ether, 5.5 mmol, 1.8 mL)
was added dropwise to a stirred solution of 40 (1.0 g, 1.8 mmol) and
ferric acetylacetonate (1. 93 g, 5.5 mmol) in THF (20 mL) and NMP
(1 mL) under argon. The resulting mixture was stirred at room
temperature for 30 min. The reaction mixture was concentrated, and
the residue was purified by flash chromatography on silica gel eluting
with DCM/MeOH/NH4OH (200:10:1) to give compound 41 as a
light-yellow solid (260 mg, 54% yield). 1H NMR (400 MHz, CDCl3) δ
ppm 8.90 (1 H, s), 8.33 (1 H, d, J = 8.0 Hz), 7.64 (1 H, d, J = 8.0 Hz),
5.38 (1 H, quin, J = 8.6 Hz), 5.15 (2 H, br s), 3.01 (3 H, s), 2.56−2.86
(2 H, m), 1.96−2.23 (4 H, m), 1.67−1.89 (2 H, m). MS (ESI) m/z:
268.1 [M + H]+.
9-Cyclopentyl-8-methyl-N-(5-(1-piperazinyl)-2-pyridinyl)-9H-
pyrido[4′,3′:4,5]pyrrolo[2,3-d]pyrimidin-2-amine (4). Compound 4
was prepared according to the methods described in the synthesis of 1.
1H NMR (500 MHz, CD3OD) δ ppm 9.01 (1 H, s), 8.36 (1 H,s), 8.29
(1 H, d, J = 8.0 Hz), 8.01 (1 H, d, J = 4.0 Hz), 7.62 (1 H, d, J = 8.0
Hz), 7.28 (1 H, d, J = 4.0 Hz), 5.36 (1 H, m), 3.09 (4 H, m), 3.02 (4
H, m), 2.95 (3 H, s), 2.73−2.70 (2 H, m), 2.09−1.98 (4 H, m), 1.80 (2
H, m). HRMS (ESI) m/z: calculated for [M + H]+ 429.2510, found
429.2527.
8-Cyano-9-cyclopentyl-9H-pyrido[4′,3′:4,5]pyrrolo[2,3-d]-
pyrimidin-2-amine (48). A mixture of 47 (1.50 g, 5.53 mmol) and
tetraethylammonium cyanide (1.30 g, 8.29 mmol) in DMF (10 mL)
was heated at 150 °C under microwave irradiation for 2 h. The
reaction mixture was concentrated under reduced pressure and the
residue was purified by flash chromatography on silica gel, eluting with
DCM/MeOH/NH4OH (100:10:1) to give 48 as a tan solid (1.17 g,
76% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 9,17 (1 H, s), 8.49
(1 H, d, J = 5.1 Hz), 8.25 (1 H, d, J = 5.1 Hz), 7.24 (2 H, br s), 5.59 (1
H, m), 2.49−2.56 (2 H, m), 2.05−2.14 (4 H, m), 1.70−1.75 (2 H, m).
MS (ESI) m/z: 279.2 [M + H]+.
9-Cyclopentyl-2-((5-(1-piperazinyl)-2-pyridinyl)amino)-9H-
pyrido[4′,3′:4,5]-pyrrolo[2,3-d]pyrimidine-8-carbonitrile (6). Com-
pound 6 was prepared according to the methods described in the
synthesis of 1. 1H NMR (500 MHz, DMSO-d6) δ ppm 10.42 (1 H, br
s), 9.40−9.49 (1 H, m), 8.67−8.80 (2 H, m), 8.58−8.65 (1 H, m),
8.39−8.48 (1 H, m), 8.09−8.16 (1 H, m), 7.98−8.07 (1 H, m), 5.62−
5.73 (1 H, m), 3.34−3.44 (5 H, m), 3.25−3.34 (4 H, m), 2.64 (2 H,
m), 2.01−2.20 (4 H, m), 1.68−1.80 (2 H, m). HRMS (ESI) m/z:
calculated for [M + H]+ 440.2307, found 440.2302.
9-Cyclopentyl-8-fluoro-N-(5-(1-piperazinyl)-2-pyridinyl)-9H-
pyrido[4′,3′:4,5]pyrrolo[2,3-d]pyrimidin-2-amine (2). Compound 2
was prepared from 47 using chemistry similar to that described in the
1
synthesis of 1. H NMR (500 MHz, CD3OD) δ ppm 9.46 (1 H, s),
8.10−8.15 (3 H, m), 8.05 (1 H, d, J = 2.9 Hz), 7.64 (1 H, d, J = 9.5
Hz), 5.20 (1 H, m), 3.51−3.57 (4 H, m), 3.42−3.51 (4 H, m), 2.40−
2.50 (2 H, m), 2.10−2.25 (4 H, m), 1.80−1.90 (2H, m). HRMS (ESI)
m/z: calculated for [M + H]+ 433.2260, found 433.2258.
8-Chloro-9-cyclopentyl-9H-pyrido[4′,3′:4,5]pyrrolo[2,3-d]-
pyrimidin-2-amine (39). Compound 39 was prepared from 2-chloro-
3-fluoropyridine using chemistry similar to that described in the
synthesis of 47. 1H NMR (500 MHz, DMSO-d6) δ ppm 9.11 (1 H, s),
8.14 (1 H, d, J = 4.9 Hz), 7.98 (1 H, d, J = 4.9 Hz), 7.07 (2 H, br s),
5.90 (1 H, quin, J = 8.7 Hz), 2.44−2.49 (2 H, m), 2.04−2.13 (2 H, m),
1.95−2.03 (2 H, m), 1.63−1.73 (2 H, m). MS (ESI) m/z: 2.88.1 [M +
H]+.
8-Chloro-9-cyclopentyl-N-(5-(1-piperazinyl)-2-pyridinyl)-9H-
pyrido[4′,3′:4,5]-pyrrolo[2,3-d]pyrimidin-2-amine (3). Compound 3
was prepared using chemistry similar to that described in the synthesis
of 1. 1H NMR (500 MHz, DMSO-d6) δ ppm 10.56 (1 H, br s), 9.42 (1
H, s), 8.87 (2 H, br s), 8.28 (1 H, d, J = 4.9 Hz), 8.19 (1 H, d, J = 5.1
Hz), 8.09 (1 H, d, J = 2.9 Hz), 7.97 (1 H, d, J = 9.0 Hz), 7.75 (1 H, dd,
J = 9.3, 2.0 Hz), 5.96−6.07 (2 H, m, J = 9.2, 8.9, 8.8, 8.8 Hz), 3.37−
3.43 (4 H, m), 3.26−3.33 (4 H, m), 2.54−2.65 (2 H, m), 2.01−2.12 (4
H, m), 1.66−1.77 (2 H, m). HRMS (ESI) m/z: calculated for [M +
H]+ 449.1963, found 449.1963.
9-Cyclopentyl-8-methoxy-9H-pyrido[4′,3′:4,5]pyrrolo[2,3-d]-
pyrimidin-2-amine (37). Compound 37 was prepared from (3-chloro-
2-methoxypyridin-4-yl)boronic acid using chemistry similar to that
described in the synthesis of 33a. 1H NMR (400 MHz, CDCl3) δ ppm
8.87 (1 H, s) 7.98 (1 H, d, J = 5.3 Hz), 7.42 (1 H, d, J = 5.3 Hz), 5.70
(1 H, t, J = 9.0 Hz), 5.21 (2 H, br s), 4.13 (3 H, s), 2.33−2.47 (2 H,
m), 1.98−2.15 (4 H, m), 1.67−1.83 (2 H, m). MS (ESI) m/z: 284.0
[M + H]+.
2-Amino-9-cyclopentyl-7,9-dihydro-8H-pyrido[4′,3′:4,5]pyrrolo-
[2,3-d]pyrimidin-8-one (38). A solution of compound 37 (1.42 g, 5.0
mmol) was dissoved in conc HCl (10.4 mL, 125 mmol), and the
solution thus obtained was heated at 140 °C under microwave
irradiation for 1 h. The reaction mixture was neutrallized to pH 7−8
by addition of sodium carbonate, and the precepitate was collected by
filtration, washed with water, and dried under high vacuum to give 38
as a yellow solid (1.35 g, 100% yield) that was used in the next step
N4-Cyclopentyl-5-(3,6-dimethoxy-4-pyridazinyl)-2,4-pyrimidine-
diamine (50). A solution of nBuLi (2.5 M solution in hexanes) (7.5
mL, 19 mmol) was added to a cold (0 °C) solution of 2,2,6,6-
tetramethylpiperidine (3.4 mL, 20 mmol) in THF (24 mL). The
resulting mixture was allowed to stand at 0 °C for 30 min. The ice−
water bath was then replaced with a dry ice−2-propanol bath. A
precooled solution of 3,6-dimethoxypyridazine (2.40 g, 17 mmol) in
THF (40 mL) was added through a cannula over a period of 30 min.
The resulting mixture was stirred at −70 °C for 1.5 h. A solution of
zinc(II) chloride (0.5 M in THF) (34 mL, 17 mmol) was added
through a syringe over a period of 15 min. The cold bath was removed,
and the resulting mixture was allowed to warm up to rt. A solution of
compound 44a (1.7 g, 5.7 mmol) and Pd2(PPh3)4 (0.33 g, 0.29 mmol)
in THF (20 mL) was added, and the resulting mixture was heated at
reflux for 18 h. Upon workup, the crude mixture was cooled in an ice−
water bath before being poured onto ice and saturated NH4Cl aqueous
solution and extracted with ethyl acetate (2×). The combined organics
were dried over sodium sulfate and concentrated in vacuo. The residue
was purified by flash chromatography on silica gel, eluting with DCM/
MeOH/NH4OH (200:10:1) to give compound 50 as an off-white
solid (1.2 g, 66% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 7.53
(1 H, s), 7.01 (1 H, s), 6.18−6.38 (3 H, m), 4.35−4.51 (1 H, m, J =
7.3, 7.3, 7.3, 7.3, 7.0 Hz), 3.95 (3 H, s), 3.91 (3 H, s), 1.77−1.90 (2 H,
1
without further purification. H NMR (400 MHz, DMSO-d6) δ ppm
11.38 (1 H, br s), 8.95 (1 H, s), 7.14 (1 H, d, J = 8.0 Hz), 6.87 (1 H, d,
J = 8.0 Hz), 6.75 (2 H, br s), 5.92 (1 H, quin, J = 9.0 Hz), 2.32−2.42
(2 H, m), 1.80−2.07 (4 H, m), 1.54−1.70 (2 H, m). MS (ESI) m/z:
270.1 [M + H]+.
9-Cyclopentyl-2-((5-(1-piperazinyl)-2-pyridinyl)amino)-7,9-dihy-
dro-8H-pyrido[4′,3′:4,5]-pyrrolo[2,3-d]pyrimidin-8-one (5). Com-
pound 5 was prepared from 38 using the methods described in the
1
synthesis of 1. H NMR (500 MHz, CD3OD) δ ppm 9.31 (1 H, s),
8.03 (1 H, s), 8.01 (1 H, d, J = 4.o Hz), 7.75 (1 H, d, J = 8.0 Hz), 7.35
(1 H, d, J = 4.0 Hz), 7.16 (1 H, d, J = 8.0 Hz), 6.05 (1 H, m), 3.52 (4
H, m), 3.47 (4 H, m), 2.50−2.60 (2 H, m), 2.09−2.25 (4 H, m), 1.55−
1.75 (2H, m). HRMS (ESI) m/z: calculated for [M + H]+ 431.2303,
found 431.2314.
2-Amino-9-cyclopentyl-9H-pyrido[4′,3′:4,5]pyrrolo[2,3-d]-
pyrimidin-8-yl Trifluoromethanesulfonate (40). Sodium hydride
(60% in oil, 16 mg) was added to a solution of compound 38 (0.10
g, 0.371 mmol) in DMF (2 mL) and stirred at 0 °C for 30 min, then
N-phenyl-bis-(trifluoromethanesulfonimide) (0.146 g, 0.408 mmol) in
DMF (1 mL) was added dropwise at 0 °C, and the resulting mixture
was stirred at 0 °C for 2 h. The reaction mixture was concentrated and
dissolved in DCM. The resulting organic solution was washed with
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m), 1.55−1.69 (2 H, m), 1.35−1.55 (4 H, m). MS (ESI) m/z: 317.2
[M + H]+.
m), 1.86 (1 H, m), 1.65 (2 H, m). HRMS (ESI) m/z: calculated for
[M + H]+ 445.2459, found 445.2458.
9-Cyclopentyl-3-methoxy-9H-pyrimido[5′,4′:4,5]pyrrolo[2,3-c]-
pyridazin-7-amine (51). To a 35 mL microwave reaction vessel was
added compound 50 (200 mg, 0.63 mmol) followed by THF (15 mL)
and sodium hydride (60% in mineral oil, 0.063 g, 1.6 mmol). The
resulting mixture was stirred at room temperature for 10 min to allow
gas release. The vessel was then subjected to microwave irradiation at
150 °C for an hour. The reaction mixture was concentrated, and the
residue was purified by flash chromatography on silica gel eluting with
2% methanol in DCM to give compound 51 as a light-yellow solid
(170 mg, 95% yield). 1H NMR (500 MHz, DMSO-d6) δ ppm 8.97 (1
H, s), 7.57 (1 H, s), 7.28 (2 H, s), 5.28 (1 H, quin, J = 8.7 Hz), 4.02 (3
H, s), 2.38−2.46 (2 H, m), 1.89−2.10 (4 H, m), 1.62−1.78 (2 H, m).
MS (ESI) m/z: 285.1 [M + H]+.
7-Amino-9-cyclopentyl-9H-pyrimido[5′,4′:4,5]pyrrolo[2,3-c]-
pyridazin-3-yl Trifluoromethanesulfonate (52). A mixture of
compound 51 (0.17 g, 0.60 mmol) and pyridine hydrochloride
(0.69 g, 6.0 mmol) was heated at 200 °C overnight. After cooling to
room temperature, the mixture was dissolved in THF (6 mL).
Triethylamine (1.3 mL, 9.0 mmol), N,N-dimethylpyridin-4-amine (7.3
mg, 0.060 mmol), and N-phenyltrifluoromethanesulfonimide (0.54 g,
1.5 mmol) were added at room temperature. The resulting mixture
was sonicated for 10 min and stirred for 2 h. The reaction mixture was
concentrated, and the residue was purified by flash chromatography on
silica gel eluting with 15−65% ethyl actate in hexane to give
compound 52 as an off-white solid (150 mg, 62% yield). MS (ESI) m/
z: 403.0 [M + H]+.
(9-Cyclohexyl-9H-pyrido[4′,3′,4,5]pyrrolo[2,3-d]pyrimidin-2-yl)-
(5-piperazin-1-yl-pyridin-2-yl)-amine (10). Compound 10 was
prepared using chemistry similar to that described in the synthesis
of 1. 1H NMR (500 MHz, DMSO-d6) δ ppm 9.56 (1 H, s), 9.47 (1 H,
s), 8.77 (1 H, d, J = 6.1 Hz), 8.65 (1 H, d, J = 6.1 Hz), 8.10 (1 H, dd, J
= 9.5, 2.7 Hz), 8.00 (1 H, d, J = 2.7 Hz), 7.54 (1 H, s), 7.42 (1 H, d, J
= 9.5 Hz), 4.77−4.84 (1 H, m), 3.50−3.54 (4 H, m), 3.43−3.48 (4 H,
m), 2.31−2.44 (2 H, m), 2.04−2.16 (4 H, m), 1.86−1.95 (1 H, m),
1.65−1.77 (2 H, m), 1.46−1.59 (1 H, m). HRMS (ESI) m/z:
calculated for [M + H]+ 429.2510, found 429.2507.
9-(4-Methylcyclohexyl)-N-(5-(1-piperazinyl)-2-pyridinyl)-9H-
pyrido[4′,3′:4,5]pyrrolo[2,3-d]pyrimidin-2-amine (11). Compound
11 was prepared according to the methods described in the synthesis
of 1. 1H NMR (500 MHz, CD3OD) δ ppm 9.19 (1 H, s), 8.97 (1H, s),
8.39 (1 H, d, J = 6.1 Hz), 8.27 (1 H,, d, J = 6.1 Hz), 8.05 (1 H, m),
7.53 (1 H, m), 3.20 (4 H, m), 3.07 (4 H, m), 2.80 (1 H, m), 2.60 (1 H,
m), 2.10 (1 H br s), 1.97 (3 H, m), 1.75 (3 H, m), 1.40 (1 H, m),
1.08−1.25 (3 H, m). HRMS (ESI) m/z: calculated for [M + H]+
443.2666, found 443.2666.
5-(3-Fluoropyridin-4-yl)-N4-((1r,4r)-4-methylcyclohexyl)-
pyrimidine-2,4-diamine (55). To a solution of 2,2′,6,6′-tetramethyl-
piperidine (997 mL, 5.87 mol, 3 equiv) in anhydrous THF (6 L) under
N2 atmosphere at 0 °C, was added n-BuLi (2.5 M in hexanes, 2350
mL, 5.87 mol, 3 equiv) via an addition funnel over 30 min. Upon
completion of the addition, the reaction mixture was stirred at 0 °C for
1 h. The reaction mixture was cooled to −74 °C, and a solution of 3-
fluoropyridine (561 g, 5.773 mol, 2.95 equiv) in anhydrous THF (500
mL) was added over 15 min, keeping the temperature below −63 °C.
Upon completion of the addition, the reaction mixture was stirred at
−74 °C for an additional 2 h. A solution of ZnBr2 (1422 g, 6.32 mol,
3.22 equiv) in anhydrous THF (3 L) was then added dropwise over 35
min, keeping the temperature below −60 °C. Upon completion of the
addition, the cold bath was removed and the reaction mixture was
allowed to warm to room temperature. Then 44b (650 g, 1.95 mol, 1.0
equiv) was added in one portion, followed by Pd(PPh3)4 (113 g, 97.8
mmol, 0.05 equiv). The reaction mixture was heated at reflux
overnight and monitored using LCMS. Upon completion, the reaction
mixture was cooled to room temperature, quenched with saturated
aqueous NaHCO3 (6 L), and extracted with ethyl acetate (2 × 10 L).
The organic extracts were washed with saturated NaHCO3 (2 × 2.5 L)
and brine (2.5 L) and were then concentrated under vacuum. The
residue was dissolved in 2N HCl (2.5 L) and washed with DCM (3 ×
1.25 L). The aqueous phase was adjusted to pH 10−12 by addition of
aqueous 4N NaOH and extracted with DCM (3 × 1.5 L). The organic
extracts were washed with water (2 × 1.25 L), dried, and concentrated
to give compound 55 (540 g, 92% yield). 1H NMR (300 MHz,
DMSO-d6) δ ppm 8.50 (1 H, d, J = 6.6 Hz), 8.37 (1 H, d, J = 4.8 Hz),
7.58 (1 H, s), 7.35 (1 H, dd, J = 6.6, 4.4 Hz), 6.24 (2 H, br s), 5.00 (1
H, d, J = 8.4 Hz), 3.96 (1 H, m), 1.74 (2 H, d, J = 11.7 Hz), 1.64 (2H,
d, J = 12.3 Hz), 1.30−1.18 (3H, m), 0.98 (1H, dd, J = 12.9, 2.7 Hz),
0.85 (3H, d, J = 7.2 Hz). MS m/z: 302.2 [M + H]+.
9-((1r,4r)-4-Methylcyclohexyl)-9H-pyrido[4′,3′:4,5]pyrrolo[2,3-d]-
pyrimidin-2-amine (33d). To a solution of 55 (854 g, 2.84 mol, 1.0
equiv) in anhydrous 1-methyl-2-pyrrolidinone (8 L) under N2
atmosphere at room temperature was added LiHMDS (1.0 M in
toluene, 8.5 L, 8.5 mol, 3.0 equiv) over 30 min. Upon completion of
the addition, the reaction mixture was heated at 90 °C overnight and
monitored using LCMS. Upon completion, the reaction mixture was
cooled to room temperature, quenched with ice-cold water (10 L), and
extracted with ethyl acetate (12 L). The organic phase was washed
with saturated aqueous NaHCO3 (2 × 4 L) and water (3 × 2 L). The
aqueous layers were combined and back-extracted with ethyl acetate (2
× 15 L). The organic layers were combined, dried over Na2SO4, and
concentrated under reduced pressure. The solid thus obtained was
suspended in DCM (2.5 L) and agitated using a rotary evaporator for
30 minutes. The solid was collected by filtration, washed with DCM,
and dried to afford compound 33d (400 g). The mother liquor was
purified by column chromatography (eluting with DCM/MeOH =
9-Cyclopentyl-9H-pyrimido[5′,4′:4,5]pyrrolo[2,3-c]pyridazin-7-
amine (53). To a 25 mL single-necked round-bottom flask were added
compound 52 (94 mg, 234 μmol), palladium diacetate (21 mg, 93
μmol), and 1,1′-bis(diphenylphosphino)ferrocene (130 mg, 234
μmol). The flask was subjected to three cycles of evacuation and
backfilling with N2. DMF (3 mL) was added under N2, followed by
triethylamine (715 μL, 5140 μmol) and formic acid (176 μL, 4.67
mmol). The resulting mixture was stirred at 65 °C for 1.5 h. The
mixture was poured into ice and saturated NaHCO3 aqueous solution,
then extracted with 10% i-PrOH/DCM (2×). The combined organics
were dried over sodium sulfate and concentrated in vacuo. The residue
was purified by flash chromatography on silica gel eluting with DCM/
MeOH/NH4OH (200:10:1) to give compound 53 as a reddish solid
1
(48 mg, 80% yield). H NMR (400 MHz, DMSO-d6) δ ppm 9.08 (1
H, s), 9.01 (1 H, d, J = 5.1 Hz), 8.07 (1 H, d, J = 5.1 Hz), 7.28 (2 H,
s), 5.42 (1 H, quin, J = 8.6 Hz), 2.40−2.49 (2 H, m), 1.92−2.14 (4 H,
m), 1.63−1.80 (2 H, m). MS (ESI) m/z: 255.1 [M + H]+.
9-Cyclopentyl-N-(5-(1-piperazinyl)-2-pyridinyl)-9H-pyrimido-
[5′,4′:4,5]pyrrolo[2,3-c]pyridazin-7-amine (7). Compound 7 was
prepared using chemistry similar to that described in the synthesis
of 1. 1H NMR (400 MHz, CD3OD) δ ppm 9.47 (1 H, s), 9.23 (1 H, d,
J = 5.5 Hz), 8.49 (1 H, d, J = 5.5 Hz), 7.99−8.11 (2 H, m), 7.75 (1 H,
d, J = 9.4 Hz), 5.68 (1 H, quin, J = 8.5 Hz), 3.49−3.58 (4 H, m), 3.41−
3.49 (4 H, m), 2.50−2.71 (2 H, m), 2.12−2.29 (4 H, m), 1.76−1.94 (2
H, m). HRMS (ESI) m/z: calculated for [M + H]+ 416.2307, found
416.2310.
(S)-2-(2-((5-(Piperazin-1-yl)pyridin-2-yl)amino)-9H-pyrido-
[4′,3′:4,5]pyrrolo[2,3-d]pyrimidin-9-yl)propan-1-ol (8). Compound 8
was prepared using chemistry similar to that described in the synthesis
1
of 1. H NMR (400 MHz, CD3OD) δ ppm 9.66 (1 H, s), 9.40 (1 H,
s), 8.68 (1 H, d, J = 5.3 Hz), 8.64 (1 H, d, J = 5.3 Hz), 8.05−8.11 (3 H,
m), 7.77 (1 H, d, J = 9.8 Hz), 5.29 (1 H, m), 4.38 (1 H, dd, J = 12.0,
8.0 Hz), 4.03 (1 H, dd, J = 8.0, 4.0 Hz), 3.50−3.57 (4 H, m), 3.40−
3.47 (4 H, m), 1.79 (3 H, d, J = 8.0 Hz). HRMS (ESI) m/z: calculated
for [M + H]+ 405.2147, found 405.2127.
4-[2-(5-Piperazin-1-yl-pyridin-2-ylamino)-pyrido[4′,3′:4,5]-
pyrrolo[2,3-d]pyrimidin-9-yl]-trans-cyclohexanol (9). Compound 9
was prepared using chemistry similar to that described in the synthesis
1
of 1. H NMR (500 MHz, CD3OD) δ ppm 9.62 (1 H, s), 9.42 (1 H,
s), 8.64 (2 H, s), 8.11 (1 H, d, J = 2.5 Hz), 8.02 (1 H, d, J = 10.0 Hz),
7.87 (1 H, d, J = 10.0 Hz), 4.98 (1 H, m), 3.89 (1 H, m), 3.55 (4 H,
m), 3.47 (4 H, m), 2.77 (2 H, m), 2.21 (1 H, d, J = 15 Hz), 2.05 (2 H,
3443
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50:1) to afford, after triturating with DCM (750 mL), additional
compound 33d (277 g, total: 677 g, 84% yield). 1H NMR (400 MHz,
DMSO-d6) δ ppm 8.95 (1 H, s), 8.92 (1 H, s), 8.46 (1 H, d, J = 5.3
Hz), 7.78 (1 H, d, J = 5.3 Hz), 5.22 (2 H, br s), 4.72−4.88 (1 H, m),
2.45 (2 H, qd, J = 12.9, 4.0 Hz), 1.82−2.12 (4 H, m), 1.56−1.71 (1 H,
m), 1.21−1.42 (2 H, m), 1.02 (3 H, d, J = 6.5 Hz). HRMS (ESI) m/z:
calculated for [M + H]+ 282.1713, found 282.1709.
4.0 (4 H, s), 3.32−3.34 (4 H, m), 1.82−1.85 (4 H, m). MS (ESI) m/z:
255.2 [M + H]+.
1-(6-((9-((1r,4r)-4-Methylcyclohexyl)-9H-pyrido[4′,3′:4,5]pyrrolo-
[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperidin-4-one (60a). To
a solution of 33d (563 mg, 2.0 mmol) in 1,4-dioxane (20 mL) were
added 59a (611 mg, 2.40 mmol), Pd2dba3 (92 mg, 0.10 mmol),
XantPhos (175 mg, 0.30 mmol), and sodium t-butoxide (290 mg, 3.0
mmol). The reaction mixture was purged with argon, then heated at
150 °C under microwave irradiation for 2 h. The reaction mixture was
subjected to flash chromatography on silica gel to give N-(5-(1,4-
dioxa-8-azaspiro[4.5]decan-8-yl)pyridin-2-yl)-9-((1r,4r)-4-methylcy-
clohexyl)-9H-pyrido[4′,3′:4,5]pyrrolo[2,3-d]pyrimidin-2-amine as a
9-((1r,4r)-4-Methylcyclohexyl)-N-(5-(piperazin-1-yl)pyridin-2-yl)-
9H-pyrido[4′,3′:4,5]pyrrolo[2,3-d]pyrimidin-2-amine (12). Com-
pound 12 was prepared according to the methods described in the
1
synthesis of 1. H NMR (500 MHz, DMSO-d6) δ ppm 9.76 (1 H, s),
9.26 (1 H, s), 9.10 (1 H, s), 8.42 (1 H, d, J = 5.1 Hz), 8.20 (1 H, d, J =
9.0 Hz), 7.98−8.07 (2 H, m), 7.44 (1 H, dd, J = 9.0, 5.1 Hz), 4.80 (1
H, m), 3.04−3.06 (4 H, m), 2.06−2.08 (4 H, m), 2.55−2.61 (2 H, m),
1.80−1.93 (4 H, m), 1.66 (1 H, m), 1.21−1.29 (2 H, m), 1.01 (3 H, d,
J = 6.4 Hz). HRMS (ESI) m/z: calculated for [M + H]+ 443.2666,
found 443.2681.
1
yellow solid (1 g, 90% purity). H NMR (500 MHz, CDCl3) δ ppm
9.07 (1 H, s), 8.96 (1 H, s), 8.49 (1 H, d, J = 5.1 Hz), 8.42 (1 H, d, J =
9.0 Hz), 8.14 (1 H, br s), 8.09 (1 H, d, J = 3.2 Hz), 7.32 (1 H, d, J =
5.1 Hz), 7.40 (1 H, dd, J = 9.0, 3.2 Hz), 4.75 (1 H, m), 4.02 (4 H, s),
3.31−3.33 (4 H, m), 2.51−2.68 (2 H, m), 1.97−2.01 (4 H, m), 1.90−
1.92 (4 H, m), 1.87 (1 H, m), 1.18−1.41 (2 H, m), 1.07 (3 H, d, J =
6.6 Hz). MS (ESI) m/z: 500.2 [M + H]+]. The yellow solid was
dissoved in THF (10 mL) and 3 N HCl (10 mL), then stirred at 60 °C
overnight. To the reaction mixture was added 1 N NaOH (30 mL),
and the resulting mixture was extracted with DCM. The organic layer
was dried over MgSO4 and concentrated under reduced pressure to
give crude 60a as a yellow sold (1.1 g, 80% purity) that was used in the
next step without further purification. MS (ESI) m/z: 456.2 [M + H]+.
N-(5-(4-Aminopiperidin-1-yl)pyridin-2-yl)-9-((1r,4r)-4-methylcy-
clohexyl)-9H-pyrido[4′,3′:4,5]pyrrolo[2,3-d]pyrimidin-2-amine (17).
To a solution of 60a (285 mg, 80% purity, 0.5 mmol) in methanol (10
mL) were added molecular sieves (3 Å) (1.5 g) and amonium acetate
(385 mg, 5 mmol), and the mixture thus obtained was stirred at room
temperature for 20 min. To the reaction mixture was added sodium
cyanoborohydride (126 mg, 4 mmol), and the mixture was stirred at
60 °C for 2 h. The reaction mixture was filtered, and the filtrate was
concentrated under reduced pressure. The residue was purified by
flash chromatography on silica gel, eluting with DCM/MeOH/
9-(4,4-Dimethylcyclohexyl)-N-(5-(1-piperazinyl)-2-pyridinyl)-9H-
pyrido[4′,3′:4,5]-pyrrolo[2,3-d]pyrimidin-2-amine (13). Compound
13 was prepared according to the methods described in the synthesis
1
of 1. H NMR (400 MHz, DMSO-d6) δ ppm 10.35 (1 H, s), 9.52 (1
H, s), 9.35 (1 H, s), 8.62 (1 H, d, J = 5.9 Hz), 8.49 (1 H, d, J = 5.9
Hz), 8.15 (1 H, d, J = 2.9 Hz), 8.04 (1 H, d, J = 9.0 Hz), 7.51 (1 H, dd,
J = 9.0, 2.9 Hz), 4.66−4.79 (1 H, m), 3.34−3.43 (4 H, m), 3.30 (4 H,
br s), 2.62−2.77 (2 H, m), 1.73 (2 H, d, J = 11.3 Hz), 1.46−1.63 (4 H,
m), 1.11 (3 H, s), 1.02 (3 H, s). HRMS (ESI) m/z: calculated for [M
+ H]+ 457.2822, found 457.2806.
9-(3,3-Dimethylcyclohexyl)-N-(5-(piperazin-1-yl)pyridin-2-yl)-9H-
pyrido[4′,3′:4,5]pyrrolo[2,3-d]pyrimidin-2-amine (14). Compound
14 was prepared according to the methods described in the synthesis
1
of 1. H NMR (400 MHz, CD3OD) δ ppm 9.70 (1 H, s), 9.51 (1 H,
s), 8.75 (1 H, d, J = 5.3 Hz), 8.68 (1 H, d, J = 5.3 Hz), 8.17 (1 H, dd, J
= 9.8, 3.2 Hz), 8.06 (1 H, d, J = 3.2 Hz), 7.74 (1 H, d, J = 9.8 Hz), 5.21
(1 H, m), 3.52−3.57 (4 H, m), 3.44−3.47 (4 H, m), 2.45−2.55 (2 H,
m), 2.03 (1 H, m), 1.82−1.91 (2 H, m), 1.71 (1 H, m), 1.47−1.57 (2
H, m), 1.21 (3 H, s), 1.10 (3 H, s). HRMS (ESI) m/z: calculated for
[M + H]+ 457.2822, found 457.2829.
1
NH4OH to give 17 as a light-yellow solid (78 mg, 34% yield). H
NMR (500 MHz, DMSO-d6) δ ppm 9.73 (1 H, s), 9.25 (1 H, s), 9.10
(1 H, s), 8.42 (1 H, d, J = 5.1 Hz), 8.17 (1 H, d, J = 9.0 Hz), 8.01−8.04
(2 H, m), 7.42 (1 H, dd, J = 9.0, 3.2 Hz), 4.79 (1 H, m), 3.60 (2 H,
ddd, J = 12.8, 4.0, 3.9 Hz), 3.20−3.44 (2 H, m), 2.65−2.89 (3 H, m),
2.51−2.65 (2 H, m), 1.80−1.89 (6 H, m), 1.68 (1 H, m), 1.31−1.44 (2
H, m), 1.12−1.31 (2 H, m), 1.00 (3 H, d, J = 6.6 Hz). HRMS (ESI)
m/z: calculated for [M + H]+ 457.2822, found 457.2811.
1-(6-Chloropyridin-3-yl)-N,N-dimethylpiperidin-4-amine (58a).
Compound 58a was prepared according to the methods described
in the synthesis of 59a with a 93% yield. 1H NMR (500 MHz, CDCl3)
δ ppm 8.02 (1 H, d, J = 2.9 Hz), 7.19 (1 H, dd, J = 9.8, 2.9 Hz), 7.15
(1 H, d, J = 9.8 Hz), 3.64−3.74 (2 H, m), 2.75−2.81 (2 H, m), 2.31 (6
H, s), 2.16−2.30 (1 H, m), 1.91−1.95 (2 H, m), 1.55−1.78 (2 H, m).
MS (ESI) m/z: 240.1 [M + H]+.
N-(5-(4-(Dimethylamino)piperidin-1-yl)pyridin-2-yl)-9-((1r,4r)-4-
methylcyclohexyl)-9H-pyrido[4′,3′:4,5]pyrrolo[2,3-d]pyrimidin-2-
amine (18). To a solution of 33d (110 mg, 0.39 mmol) in 1,4-dioxane
(4 mL) were added 58a (112 mg, 0.47 mmol), Pd2dba3 (18 mg),
XantPhos (35 mg), and sodium t-butoxide (43 mg, 0.59 mmol). The
reaction mixture was purged with argon then heated at 150 °C under
microwave irradiation for 1 h. The reaction mixture was purified by
flash chromatography on silica gel, eluting with DCM/MeOH/
NH4OH (100:10:1) to give 18 as a yellow solid (140 mg, 74% yield).
1H NMR (400 MHz, DMSO-d6) δ ppm 9.81 (1 H, s), 9.28 (1 H, s),
9.13 (1 H, s), 8.44 (1 H, d, J = 5.3 Hz), 8.21 (1 H, d, J = 9.2 Hz), 8.08
(1 H, d, J = 3.1 Hz), 8.06 (1 H, d, J = 5.3 Hz), 7.51 (1 H, dd, J = 9.2,
3.1 Hz), 4.78 (1 H, m), 3.82−3.85 (2 H, m), 2.77 (6 H, s), 2.72−2.77
(3 H, m), 2.51−2.65 (2 H, m), 2.10−2.14 (2 H, m), 1.72−1.91 (6 H,
m), 1.64 (1 H, m), 1.20−1.29 (2 H, m), 1,01 (3 H, d, J = 6.5 Hz).
HRMS (ESI) m/z: calculated for [M + H]+ 485.3134, found 485.3128.
N-(6-(4-(Dimethylamino)piperidin-1-yl)pyridazin-3-yl)-9-((1r,4r)-
4-methylcyclohexyl)-9H-pyrido[4′,3′:4,5]pyrrolo[2,3-d]pyrimidin-2-
amine (19). Compound 19 was prepared according to the methods
described in the synthesis of 18. 1H NMR (500 MHz, CDCl3) δ ppm
9-((1r,4r)-4-Methylcyclohexyl)-N-(6-(piperazin-1-yl)pyridin-3-yl)-
9H-pyrido[4′,3′:4,5]pyrrolo[2,3-d]pyrimidin-2-amine (15). Com-
pound 15 was prepared according to the methods described in the
1
synthesis of 1. H NMR (400 MHz, DMSO-d6) δ ppm 9.61 (1 H, s),
9.19 (1 H, s), 9.05 (1 H, s), 8.53 (1 H, s), 8.39 (1 H, d, J = 5.1 Hz),
7.87−8.01 (2 H, m), 6.81 (1 H, d, J = 9.0 Hz), 4.75 (1 H, m), 3.28−
3.41 (4 H, m), 2.73−2.88 (4 H, m), 2.53−2.62 (2 H, m), 1.80−1.87 (4
H, m), 1.67 (1 H, m), 1.15−1.25 (2 H, m), 0.99 (3 H, d, J = 6.5 Hz).
HRMS (ESI) m/z: calculated for [M + H]+ 443.2666, found 443.2680.
9-((1r,4r)-4-Methylcyclohexyl)-N-(6-(piperazin-1-yl)pyridazin-3-
yl)-9H-pyrido[4′,3′:4,5]pyrrolo[2,3-d]pyrimidin-2-amine (16). Com-
pound 16 was prepared from 33d and 58e using chemistry similar to
that described in the synthesis of 1. 1H NMR (500 MHz, DMSO-d6) δ
ppm 10.86 (1 H, br s), 9.51 (1 H, s), 9.40 (1 H, s), 8.98 (1 H, br s),
8.62 (1 H, d, J = 5.9 Hz), 8.47 (1 H, d, J = 5.9 Hz), 8.25 (1 H, d, J =
9.8 Hz), 7.58 (1 H, d, J = 9.8 Hz), 4.82 (1 H, t, J = 3.8 Hz), 3.75−3.84
(4 H, m), 3.27−3.33 (4 H, m), 2.49−2.55 (2 H, m), 1.87 (4 H, ddd, J
= 10.1, 1.4, 1.2 Hz), 1.61 (1 H, m), 1.17−1.25 (2 H, m), 0.99 (3 H, d,
J = 6.4 Hz). HRMS (ESI) m/z: calculated for [M + H]+ 444.2619,
found 444.2617.
8-(6-Chloropyridin-3-yl)-1,4-dioxa-8-azaspiro[4.5]decane (59a).
To a solution of 5-bromo-2-chloropyridine (7.70 g, 40 mmol) in
toluene (200 mL) were added 1,4-dioxa-8-azaspiro-[4,5]decane (6.30
g, 44 mmol), sodium t-butoxide (5.76 g, 60 mmol), Pd2dba3 (548 mg,
0.60 mmol), and XantPhos (1388 mg, 2.40 mmol). The mixture was
purged with argon then heated at 100 °C for 5 h. The reaction mixture
was then cooled to room temperature, diluted with ethyl acetate, and
washed with water. The organic layer was concentrated, and the
residue was purified by flash chromatography on silica gel, eluting with
15−50% ethyl acetate in hexanes to give 59a as a light-yellow solid
(7.77 g, 76% yield). 1H NMR (400 MHz, CDCl3) δ ppm 8.03 (1 H, d,
J = 2.2 Hz), 7.19 (1 H, dd, J = 8.0, 2.2 Hz), 7.15 (1 H, d, J = 8.0 Hz),
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9.07 (1 H, s), 8.98 (1 H, s), 8.55 (1 H, d, J = 9.8 Hz), 8.51 (1 H, d, J =
5.4 Hz), 8,25 (1 H, s), 7.84 (1 H, d, J = 5.4 Hz), 7.10 (1 H, d, J = 9.8
Hz), 4.77 (1 H, m), 4.34−4.38 (2 H, m), 2.92−3.04 (2 H, m), 2.46−
2.62 (2 H, m), 2.41 (1 H, m), 2.33 (6 H, s), 1.82−2.03 (6 H, m),
1.55−1.69 (3 H, m), 1.24−1.38 (2 H, m), 1.07 (3 H, d, J = 6.4 Hz).
HRMS (ESI) m/z: calculated for [M + H]+ 486.3087, found 486.3094.
N-(5-(4-(Dimethylamino)piperidin-1-yl)-6-fluoropyridin-2-yl)-9-
((1r,4r)-4-methylcyclohexyl)-9H-pyrido[4′,3′:4,5]pyrrolo[2,3-d]-
pyrimidin-2-amine (20). Compound 20 was prepared according to
temperature then purified by flash chromatography on silica gel to give
(1-(6-chloropyridazin-3-yl)piperidin-4-yl)methyl methanesulfonate
(2.05 g, 98% yield, MS (ESI) m/z: 306.0 [M + H]+). The mesylate
(500 mg, 1.64 mmol) was dissolved in DMF (2 mL), and sodium
methanethiolate (115 mg, 1.64 mmol) was added. The mixture thus
obtained was stirred overnight at room temperature. The reaction
mixture was diluted with ethyl acetate and washed with water and
brine, then dried over MgSO4. The solvent was evaporated under
reduced pressure to give crude 63 (500 mg, 85% purity) which was
used in next step without further purification. MS (ESI) m/z: 258.0
[M + H]+.
3-Chloro-6-(4-((methylsulfonyl)methyl)piperidin-1-yl)pyridazine
(64). To a solution of 63 (500 mg, 85% purity, 1.64 mmol) in 90%
methanol in water was added oxone (2.22 g, 3.61 mmol). The reaction
mixture was stirred overnight at room temperature, then diluted with
ethyl acetate, washed with water and brine, and then dried. The
solvent was evaporated and the residue was purified by flash
chromatography on silica gel eluting with DCM/MeOH/NH4OH-
(200:10:1) to give compound 64 (436 mg, 92% yield). MS (ESI) m/z:
290.0 [M + H]+.
1
the methods described in the synthesis of 18. H NMR (400 MHz,
CD3OD) δ ppm 9.44 (1 H, s), 9.27 (1 H, s), 8.39−8.58 (2 H, m), 8.26
(1 H, d, J = 8.4 Hz), 7.63 (1 H, m), 4.98 (1 H, m), 3.59−3.66 (2 H,
m), 3.39 (1 H, m), 2.96 (6 H, s), 2.85−3.00 (2 H, m), 2.62−2.80 (2 H,
m), 2.20−2.27 (2 H, m), 1.90−2.10 (6 H, m), 1.73 (1 H, m), 1.28−
1.47 (2 H, m), 1.10 (3 H, d, J = 5.6 Hz). HRMS (ESI) m/z: calculated
for [M + H]+ 503.3040, found 503.3044.
N-(5-(4-(Dimethylamino)piperidin-1-yl)-6-methoxypyridin-2-yl)-
9-((1r,4r)-4-methylcyclohexyl)-9H-pyrido[4′,3′:4,5]pyrrolo[2,3-d]-
pyrimidin-2-amine (21). Compound 21 was prepared according to
1
the methods described in the synthesis of 18. H NMR (500 MHz,
CDCl3) δ ppm 9.06 (1 H, s), 8.97 (1 H, s), 8,49 (1 H, d, J = 5.1 Hz),
7.98 (1 H, d, J = 8.3 Hz), 7.90 (1 H, s), 7.82 (1 H, d, J = 5.1 Hz), 7.26
(1 H, d, J = 8.3 Hz), 4.78 (1 H, m), 3.99 (3 H, s), 3.40−3.62 (2 H, m),
2.58−2.77 (4 H, m), 2.35 (6 H, s), 2.34 (1 H, m), 1.91−2.10 (6 H, m),
1.76−1.82 (2 H, m), 1.66 (1 H, m), 1.18−1.39 (2 H, m), 1.07 (3 H, d,
J = 6.6 Hz). HRMS (ESI) m/z: calculated for [M + H]+ 515.3239,
found 515.3239.
9-((1r,4r)-4-Methylcyclohexyl)-N-(6-(4-((methylsulfonyl)methyl)-
piperidin-1-yl)pyridazin-3-yl)-9H-pyrido[4′,3′:4,5]pyrrolo[2,3-d]-
pyrimidin-2-amine (24). Compound 24 was prepared from 33d and
1
64 using similar chemistry described in the synthesis of 1. H NMR
(500 MHz, CDCl3) δ ppm 9.07 (1 H, s), 8.99 (1 H, s), 8.58 (1 H, d, J
= 10.0 Hz), 8.51 (1 H, d, J = 5.3 Hz), 7.85 (1 H, d, J = 5.3 Hz), 7.10 (1
H, d, J = 10.0 Hz), 4.78 (1 H, m), 4.29−4.37 (2 H, m), 3.00−3.09 (5
H, m), 2.98 (3 H, s), 2.40−2.60 (3 H. M), 2.09−2.16 (2 H, m), 1.94−
2.06 (4 H, m), 1.58−1.65 (2 H, m), 1.24−1.36 (2 H, m), 1.07 (3 H, d,
J = 6.5 Hz). HRMS (ESI) m/z: calculated for [M + H]+ 535.2597,
found 535.2601.
8-(6-Chloropyridazin-3-yl)-2-thia-8-azaspiro[4.5]decane 2,2-Di-
oxide (66). To a solution of 3,6-dichloropyridazine (5.57 g, 37.4
mmol) and 2-thia-8-azaspiro[4.5]decane 2,2-dioxide hydrochloride
(65)(2.11 g, 9.35 mmol) in 1.4-dioxane (5 mL) was added DIPEA
(5.41 mL, 32.7 mmol). The reaction mixture was heated at 80 °C
overnight and then concentrated. The residue was purified by flash
chromatography on silica gel, eluting with 60−100% ethyl acetate in
hexanes to give 66 as a white solid (2.58 g, 91% yield). NMR (400
MHz, CDCl3) δ ppm 7.24 (1 H, d, J = 9.6 Hz), 6.94 (1 H, d, J = 9.6
Hz), 3.95−4.04 (2 H, m), 3.35 (2 H, ddd, J = 13.7, 10.0, 3.2 Hz), 3.23
(2 H, s), 2.18 (2 H, t, J = 7.6 Hz), 1.88−1.96 (2 H, m), 1.74−1.83 (2
H, m). MS (ESI) m/z: 302.0 [M + H]+.
(R)-1-(Methyl(1-(6-((9-((1r,4r)-4-methylcyclohexyl)-9H-pyrido-
[4′,3′:4,5]pyrrolo[2,3-d]pyrimidin-2-yl)amino)pyridazin-3-yl)-
piperidin-4-yl)amino)propan-2-ol (22). Compound 22 was prepared
1
according to the methods described in the synthesis of 17. H NMR
(500 MHz, CDCl3) δ ppm 9.09 (1 H, s), 8.98 (1 H, s), 8.56 (1 H, d, J
= 9.8 Hz), 8.51 (1 H, d, J = 5.1 Hz), 8.41 (1 H, s), 7.84 (1 H, d, J = 5.1
Hz), 7.10 (1 H, d, J = 9.8 Hz), 5.30 (1 H, s), 4.77 (1 H, m), 4.32−4.56
(2 H, m), 3.78 (1 H, ddd, J = 10.4, 6.1, 3.1 Hz), 2.80−3.03 (2 H, m),
2.69 (1 H, m), 2.46−2.62 (2 H, m), 2.41 (1 H, m), 2.31 (3 H, s), 2.30
(1 H, m), 1.95−2.00 (4 H, m), 1.81 (1 H, m), 1.56−1.77 (3 H, m),
1.20 −1.38 (2 H, m), 1.14 (3 H, d, J = 6.1 Hz), 1.06 (3 H, d, J = 6.4
Hz). HRMS (ESI) m/z: calculated for [M + H]+ 530.3348, found
530.3349.
2-Hydroxy-1-(4-(6-((9-((1r,4r)-4-methylcyclohexyl)-9H-pyrido-
[4′,3′:4,5]pyrrolo[2,3-d]pyrimidin-2-yl)amino)pyridazin-3-yl)-
piperazin-1-yl)ethanone (23). To a solution of 16 (108 mg, 0.243
mmol) in DMF (10 mL) were added glycolic acid (0.023 mL, 0.365
mmol), PyBroP (170 mg, 0.365 mmol), and DIPEA (0.127 mL, 0.730
mmol), and the resulting mixture was stirred at room temperature
overnight. The reaction mixture was concentrated under reduced
pressure, and the residue was purified by flash chromatography on
silica gel, eluting with DCM/MeOH/NH4OH (100:10:1) to give
8-(6-((9-((1r,4r)-4-Methylcyclohexyl)-9H-pyrido[4′,3′:4,5]pyrrolo-
[2,3-d]pyrimidin-2-yl)amino)pyridazin-3-yl)-2-thia-8-azaspiro[4.5]-
decane 2,2-Dioxide (25). Compound 25 was prepared from 33d and
1
66 using similar chemistry described in the synthesis of 1. H NMR
(500 MHz, DMSO-d6) δ ppm 10.18 (1 H, s), 9.26 (1 H, s), 9.10 (1 H,
s), 8.42 (1 H, d, J = 5.3 Hz), 8.19 (1 H, d, J = 9.6 Hz), 8.40 (1 H, d, J =
5.3 Hz), 7.44 (1 H, d, J = 9.6 Hz), 4.77 (1 H, m), 3.81−3.90 (2 H, m),
3.26−3.37 (2 H, m), 3.24 (2 H, t, J = 7.6 Hz), 3.17 (2 H, s), 2.50−2.55
(2 H, m), 2.09 (2 H, t, J = 7.6 Hz), 1.79−1.88 (6 H, m), 1.63−1.69 (3
H, m), 1.19−1.26 (2 H, m), 0.99 (3 H, d, J = 6.6 Hz). HRMS (ESI)
m/z: calculated for [M + H]+ 547.2597, found 547.2607.
1
compound 23 as a yellow solid (85 mg, 70% yield). H NMR (400
MHz, DMSO-d6) δ ppm 10.25 (1 H, s), 9.27 (1 H, s), 9.11 (1 H, s),
8.43 (1 H, d, J = 5.1 Hz), 8.27 (1 H, d, J = 9.8 Hz), 8.04 (1 H, d, J =
5.1 Hz), 7.46 (1 H, d, J = 9.8 Hz), 4.80 (1 H, m), 4.64 (1 H, t, J = 8.2
Hz), 4.16 (2 H, d, J = 8.2 Hz), 3.50−3.65 (8 H, m), 2.50−2.55 (2 H,
m), 1.80−1.90 (4 H, m), 1.66 (1 H, m), 1.20−1.27 (2 H, m), 0.99 (3
H, d, J = 6.5 Hz). HRMS (ESI) m/z: calculated for [M + H]+
502.2673, found 502.2676.
(1-(6-Chloropyridazin-3-yl)piperidin-4-yl)methanol (62). A mix-
ture of 3,6-dichloropyridazine (13.58 g, 91 mmol), DIPEA (11.78 g,
15.92 mL, 91 mmol), and 4-piperidinemethanol (10.50 g, 91 mmol)
was heated at 90 °C for 2 h. The mixture was purified by flash
chromatography on silica gel, eluting with 40−100% ethyl acetate in
hexanes to give 62 as a white solid (20.28 g, 98% yield). 1H NMR (400
MHz, CDCl3) δ ppm 7.19 (1 H, d, J = 9.6 Hz), 6.93 (1 H, d, J = 9.6
Hz), 4.27−4.45 (2 H, m), 3.56 (2 H, d, J = 6.1 Hz), 2.84−3.12 (2 H,
m), 1.65−1.98 (4 H, m), 1.10−1.45 (2 H, m). MS (ESI) m/z: 228.1
[M + H]+.
1,8-Diazaspiro[4.5]decan-2-one hydrochloride (68). To a solution
of 67 (268 mg, 1.06 mmol) in 1,4-dioxane (6 mL) was added 4 M HCl
in dioxane (6 mL), and the solution thus obtained was stirred 2 h at 50
°C. The reaction mixture was concentrated, and the residue was dried
under high vacuum to give 68 as a white solid (201 mg, 100% yield).
1H NMR (500 MHz, DMSO-d6) δ ppm 8.85 (1 H, br s), 3.05−3.15 (4
H, m), 2.22 (2 H, t, J = 8.1 Hz), 1.88 (2 H, t, J = 8.1 Hz), 1.69−1.82 (4
H, m). MS (ESI) m/z: 155.1 [M + H]+.
8-(6-Chloropyridin-3-yl)-1,8-diazaspiro[4.5]decan-2-one (69a).
Compound 69a was prepared from 56a and 68 using similar chemistry
described in the synthesis of 34a. 1H NMR (500 MHz, CDCl3) δ ppm
8.04 (1 H, d, J = 2.7 Hz), 2.7 (1 H, s), 7.16−7.23 (2 H, m), 6.23 (1 H,
br s), 3.19−3.31 (4 H, m), 2.45 (2 H, t, J = 8.1 Hz), 2.04 (2 H, t, J =
8.1 Hz), 1.83−1.89 (4 H, m). MS (ESI) m/z: 266.0 [M + H]+.
8-(6-Chloro-2-methoxypyridin-3-yl)-1,8-diazaspiro[4.5]decan-2-
one (69b). Compound 69b was prepared from 56d and 68 using
3-Chloro-6-(4-((methylthio)methyl)piperidin-1-yl)pyridazine (63).
To a solution of 62 (1.55 g, 6.81 mmol) in DCM (10 mL) were added
triethylamine (1.14 mL, 8.17 mmol) and methane sulfonyl chloride
(0.579 mL, 7.49 mmol). The mixture was stirred for 3 h at room
3445
dx.doi.org/10.1021/jm500118j | J. Med. Chem. 2014, 57, 3430−3449

Journal of Medicinal Chemistry
Article
1
similar chemistry described in the synthesis of 34a. H NMR (500
MHz, DMSO-d6) δ ppm 7.95 (1 H, br s), 7.23 (1 H, d, J = 8.1 Hz),
6.98 (1 H, d, J = 8.1 Hz), 3.87 (3 H, s), 3.08−3.15 (2 H, m), 2.80−
2.90 (2 H, m), 2.21 (2 H, t, J = 8.0 Hz), 1.86 (2 H, t, J = 8.0 Hz),
1.60−1.75 (4 H, m). MS (ESI) m/z: 296.1 [M + H]+.
H, m), 1.48 (9H, s), 1.22−1.34 (2 H, m), 1.06 (3H, d, J = 6.4 Hz).
HRMS (ESI) m/z: calculated for [M + H]+ 514.2925, found 514.2928.
9-((1r,4r)-4-Methylcyclohexyl)-N-(5,6,7,8-tetrahydro-1,6-naph-
thyridin-2-yl)-9H-pyrido[4′,3′:4,5]pyrrolo[2,3-d]pyrimidin-2-amine
(73). To a suspension of 72 (4.65 g, 9.05 mmol) in MeOH (30 mL)
were added concentrated HCl (6.74 mL) and water (14 mL). The
mixture thus obtained was stirred at room temperature overnight.
Analysis by HPLC-MS indicated that the reaction was complete. Then
50% NaOH in water (4.8 mL) was added at 0 °C to the reaction
mixture to adjust the pH value to 9. The precipitated yellow solid was
collected by filtration, rinsed with water (25 mL), and air-dried for 3
days to give compound 73 (3.75 g, 100% yield). 1H NMR (400 MHz,
CDCl3) δ ppm 9.08 (1 H, s), 8.96 (1 H, s), 8.50 (1 H, d, J = 5.3 Hz),
8.34 (1 H, d, J = 8.4 Hz), 8.03 (1 H, br s), 7.84 (1 H, d, J = 5.3 Hz),
7.41 (1 H, d, J = 8.4 Hz), 4.71 (1 H, m), 4.04 (2 H, s), 3.26 (2 H, t, J =
6.0 Hz), 2,89 (2 H, t, J = 6.0 Hz), 2.53−2.69 (2 H, m), 2.02 (2 H, s),
1.95−2.00 (3 H, m), 1.25−1.29 (3 H, m), 1.07 (3 H, d, J = 6.5 Hz).
HRMS (ESI) m/z: calculated for [M + H]+ 414.2401, found 414.2404.
2,5-Dioxopyrrolidin-1-yl 2-acetoxyacetate (74). A 3-neck round-
bottom flask equipped with a mechanical stirrer, thermocouple, and
addition funnel with nitrogen inlet was charged with N-hydroxysucci-
nimide (211 g, 1.83 mol) and DCM (2.25 L) at room temperature,
resulting in a suspension. Pyridine (178 mL, 2.2 mol) was added in
one portion with no change in the internal temperature. A solution of
acetoxyacetyl chloride (197 mL, 1.83 mol) in DCM (225 mL) was
added dropwise over 60 min, and the temperature rose to 35 °C.
Stirring was continued at room temperature for 2.5 h. The stirring was
stopped, and the reaction mixture was washed with water (1 L), 1 N
HCl (2 L), and brine (1 L). The organic layer was concentrated under
vacuum and azeotroped with toluene (1 L) to obtain compound 74 as
a white solid (367 g, 93% yield). 1H NMR (400 MHz, CDCl3) δ ppm
4.96 (2 H, s), 2.86 (4 H, s), 2.19 (3 H, s) ppm. LCMS m/z: 238.0 [M
+ Na]+.
8-(6-((9-((1r,4r)-4-Methylcyclohexyl)-9H-pyrido[4′,3′:4,5]pyrrolo-
[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)-1,8-diazaspiro[4.5]decan-
2-one (26). Compound 26 was prepared from 33d and 69a using
similar chemistry described in the synthesis of 1. 1H NMR (500 MHz,
DMSO-d6) δ ppm 9.73 (1 H, s), 9.26 (1 H, s), 9.10 (1 H, s), 8.42 (1
H, d, J = 8.1 Hz), 8.19 (1 H, d, J = 9.8 Hz), 8.04 (1 H, d, J = 5.0 Hz),
8.03 (1 H, d, J = 8.1 Hz), 8.00 (1 H, s), 7.48 (1 H, dd, J = 9.8, 5.0 Hz),
4.80 (1 H, m), 3.30−3.40 (2 H, m), 3.10−3.14 (2 H, m), 2.54−2.64 (2
H, m), 2.23 (2 H, t, J = 8.1 Hz), 1.90 (2 H, t, J = 8.1 Hz), 1.82−1.91 (4
H, m), 1.62−1.77 (5 H, m), 1.22−1.29 (2 H, m), 1.01 (3 H, d, J = 6.5
Hz). HRMS (ESI) m/z: calculated for [M + H]+ 511.2927, found
511.2931.
8-(2-Methoxy-6-((9-((1r,4r)-4-methylcyclohexyl)-9H-pyrido-
[4′,3′:4,5]pyrrolo[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)-1,8-
diazaspiro[4.5]decan-2-one (27). Compound 27 was prepared from
33d and 69b using similar chemistry described in the synthesis of 1.
1H NMR (500 MHz, DMSO-d6) δ ppm 9.55 (1 H, s), 9.29 (1 H, s),
9.12 (1 H, s), 8.43 (1 H, d, J = 5.3 Hz), 8.05 (1 H, d, J = 5.3 Hz), 7.97
(1 H, br s), 7.84 (1 H, d, J = 9.8 Hz), 7.28 (1 H, d, J = 9.8 Hz), 4.83 (1
H, m), 3.92 (3 H, s), 3.30−3.38 (2 H, m), 3.10−3.14 (2 H, m), 2.56−
2.64 (2 H, m), 2.23 (2 H, d, J = 8.1 Hz), 1.90 (2 H, t, J = 8.1 Hz),
1.82−1.90 (4 H, m), 1.66−1.80 (5 H, m), 1.23−1.30 (2 H, m), 1.02 (3
H, d, J = 6.5 Hz). HRMS (ESI) m/z: calculated for [M + H]+
541.3039, found 541.3040.
t-Butyl 2-Chloro-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxy-
late (71). To a slurry of 2-chloro-5,6,7,8-tetrahydro-1,6-naphthyridine
hydrochloride (106.1 g, 517 mmol) and N,N-diisopropylethylamine
(80 g, 108 mL, 621 mmol, 1.2 equiv) in DCM (1 L) was added a
solution of di-tert-butyl dicarbonate (119 g, 543 mmol, 1.05 equiv) in
DCM (100 mL) via an addition funnel within 1 h. The reaction
mixture became a clear solution, and the solution thus obtained was
stirred at room temperature for an additional hour and monitored
using LCMS. Upon completion, the reaction mixture was concen-
trated. The residue was dissolved in ethyl acetate (1 L) and washed
with water (3 × 300 mL), brine (300 mL), and dried over MgSO4.
The solvent was evaporated under vacuum to give compound 71 as an
2-(2-((9-((1r,4r)-4-Methylcyclohexyl)-9H-pyrido[4′,3′:4,5]pyrrolo-
[2,3-d]pyrimidin-2-yl)amino)-7,8-dihydro-1,6-naphthyridin-6(5H)-
yl)-2-oxoethyl acetate (75). To a suspension of 73 (827 mg, 2.0
mmol) in chloroform (10 mL) were added diisopropylethylamine
(258 mg, 348 uL, 2.0 mmol) and 74 (560 mg, 2.6 mmol). The
reaction mixture thus obtained was stirred at room temperature for 30
min, whereupon the mixture became a yellow solution. HPLC-MS
analysis indicated that the reaction was complete. The reaction mixture
was concentrated. MeOH (5 mL) and water (6 mL) were added to
form a slurry, which was stirred at room temperature for 1 h. The solid
was collected by filtration to give compound 75 as a light-yellow solid
1
off-white solid (139 g, 100% yield). H NMR (400 MHz, CDCl3) δ
ppm 7.38 (1 H, d, J = 8.0 Hz), 7.17 (1 H, d, J = 8.0 Hz), 4.57 (2 H, s),
3.73 (2 H, t, J = 5.9 Hz), 2.97 (2 H, t, J = 5.9 Hz), 1.49 (9H, s). LCMS
(ESI) m/z: 269 [M + H]+.
1
(1.04 g, 98% yield). H NMR (400 MHz, CDCl3, rotamers) δ ppm
9.11 (1 H, s), 8.98 (1 H, s), 8.52 (1 H, d, J = 5.3 Hz), 8.40−8.49 (1 H,
m), 8.10−8.21 (1 H, m), 7.86 (1 h, d, J = 5.3 Hz), 7.45−7.57 (1 H,
m), 4.82−4.90 (2 H, m), 4.65−4.80 (2 H, m), 4.59 (1 H, m), 3.97 (1
H, t, J = 5.9 Hz), 3.75 (1 H, t, J = 5.9 Hz), 2.93−3.08 (2 H, m), 2.52−
2.69 (2 H, m), 2.22 (3 H, s), 1.97−2.03 (4 H, m), 1.20−1.37 (2 H, m),
1.08 (3 H, d, J = 6.5 Hz). HRMS (ESI) m/z: calculated for [M + H]+
514.2561, found 514.2563.
t-Butyl 2-((9-((1r,4r)-4-Methylcyclohexyl)-9H-pyrido[4′,3′:4,5]-
pyrrolo[2,3-d]pyrimidin-2-yl)amino)-7,8-dihydro-1,6-naphthyri-
dine-6(5H)-carboxylate (72). To a solution of 33d (2.81 g, 10 mmol)
in 1,4-dioxane (45 mL) were added 71 (2.57 g, 9.55 mmol), XantPhos
(231 mg, 0.40 mmol), and sodium t-butoxide (1.44 g, 15 mmol).
Argon was bubbled through the mixture for 10 min. Pd2dba3 (183 mg,
0.20 mmol) was added, and argon was again bubbled through the
mixture for 5 min. The reaction mixture thus obtained was stirred at
100 °C for 3 h, whereupon HPLC-MS analysis indicated that the
reaction was complete. The reaction mixture was cooled to 40 °C,
diluted with DCM (90 mL), and treated with Si-triamine (function-
alized silica gel) (2.8 g) overnight at room temperature. Celite brand
filter aid 545 (6 g) was added, the mixture was filtered with a sintered
glass funnel, and the solid phase was rinsed with DCM (100 mL). The
filtrate was concentrated to 25 mL on a rotary evaporator and diluted
with a mixture of ethyl acetate and hexane (20 mL, 4:1). The resulting
slurry was stirred at room temperature for 5 h. The solid was collected
by filtration, washed with a mixture of ethyl acetate and hexane (20
mL, 1:1), and air-dried for a few hours to provide compound 72 as an
2-Hydroxy-1-(2-((9-((1r,4r)-4-methylcyclohexyl)-9H-pyrido-
[4′,3′:4,5]pyrrolo[2,3-d]pyrimidin-2-yl)amino)-7,8-dihydro-1,6-
naphthyridin-6(5H)-yl)ethanone (28). To a solution of 75 (514 mg,
1.0 mmol) in DCM (7.5 mL) and MeOH (2.5 mL) was added 0.5 M
sodium methoxide solution in MeOH (0.30 mL, 0.15 mmol), and the
reaction mixture was stirred at room temperature for 1 h, monitored
using LCMS. Upon completion, the reaction mixture was concen-
trated. The residue was treated with EtOH (5 mL) and water (10 mL)
to provide a solid which was collected by filtration, washed with water,
and dried in a vacuum oven at 55 °C overnight to give compound 28
as a white solid (468 mg, 99% yield). 1H NMR (500 MHz, acetic acid-
d4, 373 K) δ ppm 9.43 (1 H, s), 9.35 (1 H, s), 8.63 (1H, d, J = 6.0 Hz),
8.40 (1 H, d, J = 6.0 Hz), 8.33 (1 H, d, J = 8.5 Hz), 7.76 (1 H, d, J =
8.5 Hz), 4.90 (1 H, m), 4.77 (2 H, br s), 4.46 (2 H, br s), 3.88 (2 H, br
s), 3.10 (2 H, t, J = 5.4 Hz), 2.68 (2 H, dq, J = 12.7, 3.3 Hz), 2.06−
2.13 (2 H, m), 1.99−2.03 (2 H, m), 1.70−1.80 (1 H, m), 1.31−1.43 (2
H, m), 1.09 (3H, d, J = 6.5 Hz). HRMS (ESI) m/z: calculated for [M
+ H]+ 472.2455, found 472.2461.
1
off-white solid (4.90 g, 100% yield). H NMR (500 MHz, CD2Cl2) δ
ppm 9.10 (1 H, s), 8.97 (1 H, s), 8.46 (1 H, d, J = 5.1 Hz), 8.42 (1 H,
d, J = 8.6 Hz), 8.10 (1 H, br s), 7.85 (1 H, d, J = 5.1 Hz), 7.51 (1 H, d,
J = 8.6 Hz), 4.83 (1 H, m), 4.57 (2 H, s), 3.74 (2 H, t, J = 6.0 Hz), 2,88
(2 H, t, J = 6.0 Hz), 2.60−2.66 (2 H, m), 1.93−2.02 (4 H, m), 1.67 (1
3446
dx.doi.org/10.1021/jm500118j | J. Med. Chem. 2014, 57, 3430−3449

Journal of Medicinal Chemistry
Article
FLT3 Kinase Assay. The inhibitory activity of FLT3 was
determined using a time-resolved fluorescence resonance energy
transfer (TR-FRET) assay. The FLT3 enzyme (GST-fused human
FLT3 cytoplasmic domain, amino acids 564−993) was purchased from
Carna Biosciences (Natick, MA). An ULight-labeled synthetic peptide
(ULight-JAK1, PerkinElmer, Waltham, MA) derived from human
Janus kinase 1 (amino acids 1015−1027) was used as the
phosphoacceptor substrate. The FLT3 assay was conducted in a
384-well white OptiPlate (PerkinElmer) in a total volume of 20 μL.
The reaction mixture contained 50 nM ULight-JAK1, 116 μM ATP
(equal to Km), 0.5 nM FLT3, and serially diluted test compounds in a
reaction buffer of 50 mM Hepes, pH 7.6, 1 mM EGTA, 10 mM
MgCl2, 2 mM DTT, and 0.005% Tween 20. The reaction was allowed
to proceed for 1 h at room temperature and stopped by adding 20 μL
of 20 mM EDTA and 4 nM LANCE Eu-W1024 antiphospho-tyrosine
antibody (PerkinElmer) in LANCE detection buffer (PerkinElmer).
The plates were incubated at room temperature for 2 h after addition
of detection reagents and were then read on an Envision multimode
reader (PerkinElmer). Fluorescence signals measured at 615 nm (8.5
nm bandwidth) and 665 nm (7.5 nm bandwidth) with a 60 μs delay
after excitation at 320 nm (75 nm bandwidth). The signal ratio at 665/
615 nm (APC/Eu) was used in all data analyses. IC50 values were
obtained by analyzing competition curves using a four-parameter
sigmoidal model in GraphPad Prism v5.01 (GraphPad).
CDK Kinases Assay. CDK4/Cyclin D1, CDK6/Cyclin D1, CDK1/
Cyclin B, and CDK2/Cyclin A were purchased from Cell Signaling
Technology (Danvers, MA). For CDK4 and CDK6 kinase assays, Rb
(amino acids 773−928), and histone H1 were from Millipore
(Bedford, MA) and used as substrate for CDK4/6 and CDK1/2,
respectively. [r-33P]-ATP was purchased from PerkinElmer (Shelton,
CT). Assays were performed in 96-well filter plates (MSDVN6B50,
Millipore, Bedford, MA) with a final volume of 100 μL, containing 1
μg Rb, 25 ng CDK4/cyclin D1, 25 μM ATP, 1 μCi [r-33P]-ATP, and
compound in kinase reaction buffer (20 mM Tris-HCl, pH 7.4, 10 mM
magnesium chloride, 5 mM β-glycerophosphate, 1 mM dithiothreitol,
and 0.1% bovine serum albumin). Plates with reaction mix were
incubated at room temperature for 60 min, and reactions were
terminated by addition of 200 μL of 20% trichloroacetic acid. Wells
were washed with 200 μL of 10% trichloroacetic acid and left to dry at
room temperature. The bottom of the plates were sealed with tape,
and 100 μL of MicroScint-20 scintillation cocktail (PerkinElmer,
Shelton, CT) were added to each well. Plates were read on a
TopCount (PerkinElmer, Shelton, CT) to determine radioactive
incorporation. IC50 values were calculated by nonlinear regression
curve fitting using GraphPad Prism v5.01 (GraphPad).
cells per well and treated with compounds at indicated concentrations
for 24 h. Cells were then stained with the reagents in the kit and
analyzed by flow cytometry. The Sytox Green fluorescence versus APC
fluorescence dot plot shows resolution of live, apoptotic and dead cells,
which were quantified using Flowjo software.
Sorafenib-Resistant MOLM13 Cells. Sorafenib resistant
MOLM13 (MOLM13SR) was isolated by passaging the cells in growth
medium containing increasing concentrations of sorafenib (1−1000
nM) over 3−4 months. The resistant cells were cloned by limiting
dilution. RNA was isolated from each clone, and cDNA was sequenced
to identify FLT3 kinase domain mutations.
Xenograft Tumor Models. CrTac:NCR-Foxn1nu (NCR) nude
mice were treated with ip injection of 100 μL of antiasialo GM
(WAKO Chemicals) antibody to abolish NK activity and allow for
enhanced growth of subsequently inoculated tumor cells. The
following day, 7.5 million MOLM13 or U937 tumor cells in PBS
were formulated as a 1:1 mixture with matrigel (BD Biosciences) and
injected into the subcutaneous space on the right flank of the mice.
Tumors were measured with PRO-MAX electronic digital caliper
(Japan Micrometer Mfg. Co. LTD), and the mice were weighed every
other day prior to each tumor measurement. Tumor volumes were
calculated as follows: tumor volume (mm3) = [(W2 × L)/2], where
width (W) is defined as the smaller of the two measurements and
length (L) is defined as the larger of the two measurements. Tumor
growth inhibition (TGI) is calculated as 100 − [(measured − initial
volume)/(control − initial volume) × 100].
Statistical Analysis of Tumor Growth in Vivo. Tumor volumes
are expressed as means plus or minus standard errors and plotted as a
function of time. Statistical significance of observed differences
between growth curves was evaluated by repeated measures analysis
of covariance of the log transformed tumor volume data with Dunnett
adjusted multiple comparisons. The analysis was done using SAS proc
mixed with model effects of baseline log tumor volume, day, treatment,
and day-by-treatment interaction; a repeated statement where day was
a repeated value, animal the subject, and a Toeplitz covariance
structure; and an lsmeans statement to do a Dunnett analysis
comparing the control group to the other treatment groups. The data
was log transformed because larger volumes tended to have larger
variances, and baseline log tumor volume was included as a covariate
in the model to account for possible pretreatment tumor volume
differences. All statistical calculations were made through the use of
JMP software v7.0 interfaced with SAS v9.1 (SAS Institute Inc., Cary,
NC).
Analysis of pRb and pSTAT5 in Tumor Samples. Tumors were
harvested by dissection and snap-frozen. Frozen tumors were weighed
and lysed in 150 mM NaCl, 20 mM Tris pH 7.5, 1 mM EDTA, 1 mM
EGTA, 1% Triton-X-100, and 2× protease and phosphatase inhibitors
(Meso Scale Discovery) at twice the volume of tumor mass. Samples
were processed on a Genogrinder (Spec SamplePrep) and shaken
twice for 30 s at 1200 rpm with cooling on ice between shaking.
Samples were then centrifuged at 1200 rpm, and cleared lysates were
analyzed using assay kits for phosphorylated STAT5 (Tyr694), total
STAT5a and b, phosphorylated Rb (Ser780)/total Rb, and total
Cell Growth Inhibition Assay. Cell growth was measured by a
DNA synthesis assay. Cells were seeded in a 96-well Cytostar T plate
(GE Healthcare Biosciences, Piscataway, NJ) at a density of 5 × 103
cells/well in a total volume of 160 μL. The plates were incubated
overnight to allow cells to attach. Test compounds were serially diluted
into the plate (20 μL/well), and 20 μL/0.1 μCi of [14C]-thymidine
(GE Healthcare Biosciences, Piscataway, NJ) were added to each well.
Isotope incorporation was determined using a β plate counter (Wallac,
Gaithersburg, MD) after a further 72 h. IC50 values were determined as
described above.
p38MAPK
.
X-ray Crystallography. The CDK6-Vcyclin complex was ex-
pressed and crystallized, using minor modifications on the methods
described by Lu and Schulze-Gahmen.29 In short, the CDK6 and
Vcyclin proteins were expressed separately in baculovirus infected Hi5
cells. The proteins were mixed, and the complex was purified. The
purified complex was concentrated to 10−15 mg/mL, and unliganded
CDK6-Vcyclin was crystallized from 10 mM Tris pH 7.9, 100 mM
calcium acetate, 10 mM DTT, 8−12% PEG 3350, and 100 mM
NDSB-201. Crystals were soaked with 0.25 mM compound 1 and
cryoprotected with glycerol prior to flash cooling. A 2.9 Å X-ray
diffraction data set was collected at beamline 5.0.2 at the Berkeley
Advanced Light Source and processed with iMOSFLM.30 The
structure was solved by molecular replacement with MOLREP31
using 2EUF as the search model. Final refinement and model building
were carried out using PHENIX32 and Coot,33 respectively. Atomic
Analysis of pSTAT5 and pRb in Vitro. To determine levels of
pSTAT5 and pRb, cells were seeded in a 96-well cell culture plate at a
density of 5000−10000 cells per well in a total volume of 180 μL and
incubated overnight in medium containing 10% fetal bovine serum
(Life Technologies, Rockville, MD). Then 20 μL of serially diluted
compounds were added to each well the next day. Cell lysates were
harvested after 24 h and pSTAT5 or pRb was quantified using assay
kits for pSTAT5 (Tyr694)/total STAT5 and pRb (Ser780)/total Rb
(Meso Scale Discovery) following the manufacturer’s protocols.
Finally, 10 μg of total protein from each sample was analyzed.
Apoptosis Assay. Apoptosis of the AML cells treated with
compounds was assayed by using a Vybrant Apoptosis Assay Kit no. 9
following the manufacturer’s protocol (Invitrogen, catalogue no.
V35113). Briefly, the MOLM13, MOLM13SR, or U937 cells in
exponential growth phase were seeded into a 6-well plate at 5 × 105
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coordinates have been deposited in the RCSB Protein Data Bank
under accession code 4P41.
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1/Flk2) in human leukemia. Blood 1996, 87, 1089−1096.
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A.; Ehninger, G.; Illmer, T. Analysis of FLT3-activating mutations in
979 patients with acute myelogenous leukemia: association with FAB
subtypes and identification of subgroups with poor prognosis. Blood
2002, 99, 4326−4335.
ASSOCIATED CONTENT
* Supporting Information
■
S
POC kinase selectivity data of 28, Kd data for 28 for the set of
kinases with POC less than 20 from the initial POC screen,
terminal exposure levels of 28 in the MOLM13 xenograft
model, and average body weights for the 28 treated mouse
group in the MOLM13 xenograft model. This material is
available free of charge via the Internet at http://pubs.acs.org.
(6) Pratz, K. W.; Levis, M. J. Bench to bedside targeting of FLT3 in
acute leukemia. Curr. Drug Targets 2010, 11, 781−789.
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in AML; still challenging after all these years. Blood 2010, 9, 5089−
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Levis, M. J.; Perl, A. E.; Travers, K. J.; Wang, S.; Hunt, J. P.; Zarrinkar,
P. P.; Schadt, E. E.; Kasarskis, A.; Kuriyan, J.; Shah, N. P. Validation of
ITD mutations in FLT3 as a therapeutic target in human acute
myeloid leukaemia. Nature 2012, 485, 260−263.
(9) Knapper, S. FLT3 inhibition in acute myeloid leukaemia. Br. J.
Haematol. 2007, 138, 687−699.
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D. F.; Liu, T.; Caligiuri, M. A.; Briesewitz, R. Pharmacologic inhibition
of CDK4/6: mechanistic evidence for selective activity or acquired
resistance in acute myeloid leukemia. Blood 2007, 110, 2075−2083.
(11) Drexler, H. G. Review of alterations of the cyclin-dependent
kinase inhibitor INK4 family genes p15, p16, p18 and p19 in human
leukemia-lymphoma cells. Leukemia 1998, 12, 845−859.
(12) Malumbres, M.; Barbacid, M. Cell cycle, CDKs and cancer: a
changing paradigm. Nature Rev. Cancer 2009, 9, 253−266.
(13) Li, Z.; Dai, K.; Keegan, K.; Ma, J.; Ragains, M.; Kaizerman, J.;
McMinn, D.; Fu, J., Fisher, B.; Gribble, M. et al. CDK4/FLT3 dual
inhibitors as potential therapeutics for acute myeloid leukemia. In Drug
Discovery and Lead Optimization; Proceedings of the American
Association for Cancer Research, AACR Annual Meeting 2013,
Washington, DC, April 6−10, 2013; Abstract 2351, p 575.
(14) All software programs used for molecular modeling are from
Accession Codes
The coordinates of 1 with CDK6 have been deposited in the
PDB with accession code 4P41.
AUTHOR INFORMATION
Corresponding Author
*Phone: 650-244-2501. E-mail: zhihongl@amgen.com.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank David Chaw for assistance of NMR spectra, Guifen
Xu for bioanalytical support, Simon Wong for determination of
CYP IC50 values, and Dhanashri Bagal and Iain Campuzano for
determination of high-resolution mass spectra. We also thank
Xiaolin Hao for useful discussion.
ABBREVIATIONS USED
■
AML, acute myeloid leukemia; ATP, adensoine triphosphate;
AUC, area under curve; CDK4, cyclin-dependent kinase 4; CL,
clearance; Cmax, maxium concentration; CYP3A4, cytochrome
p450 3A4; DCM, dichloromethane; DIPEA, N,N-diisopropy-
lethylamine; DMAP, dimethylaminopyridine; DMF, dimethyl-
formamide; dppf, ferrocene,1,1′-bis(diphenylphosphino)−; ED,
effective dose; EDTA, ethylenediamine-N,N,N′,N′- tetraacetic
acid; EGTA, ethylene glycol bis(2-aminoethyl ether)-
N,N,N′,N′-tetraacetic acid; F, bioavailability; Fe(acac)3, tris-
(acetylacetonato) iron(III); FLT3, FMS-like tyrosine kinase 3;
Schrodinger (New York, NY). The homology model for FLT3 with
̈
the activation loop in the DFG-in conformation was built with cKit as
the scaffold (PDB 1PKG) using the Prime module. Compound 1 was
modeled into the ATP binding site of the FLT3 model using Glide.
Protein representations were prepared via PyMOL 1.5.0.5.
(15) Malumbres, M.; Pevarello, P.; Barbacid, M.; Bischoff, J. R. CDK
inhibitors in cancer therapy: What is next? Trends Pharmacol. Sci.
2008, 29, 16−21.
̀
hERG, human ether-a-go-go-related gene; IC50, half-maximal
inhibitory concentration; KHMDS, potassium bis-
(trimethylsilyl)amide; LiHMDS, lithium hexamethyldisilazide;
LiTMP, 1-lithio-2,2,6,6-tetramethylpiperidine; NIS, n-iodosuc-
cinimide; NMP, N-methyl-2-pyrrolidone; PBS, phosphate
buffered saline; PI3K, phosphatidylinositide 3 kinase; PK,
pharmacokinetic; PPTS, pyridinium p-toluenesulfonate; Rb,
retinoblastoma protein; SAR, structure−activity relationship;
TEA, triethylamine; TFA, trifluoroacetic acid; THF, tetrahy-
dofuran; TLC, thin layer chromatography; Vss, volume of
distribution; XantPhos, 4,5-bis(diphenylphosphino)-9,9-dime-
thylxanthene; X-Phos, 2-dicyclohexylphosphophino-2′4′6′-trii-
sopropylbiphenyl
(16) Detailed docking studies of our compound to the CYP3A4
active site will be published elsewhere in due course.
(17) Takacs-Novak, K.; Szasz, G. ion pair partition of quaternary
́ ́
ammonium drugs: the influence of counter ions of different
lipophilicity, size, and flexibility. Pharm. Res. 1999, 16, 1633−1638.
(18) KINOMEscan Assay Platform; DiscoveRx Corporation: Fremont,
CA; http://www.discoverx.com/technologies-platforms/competitive-
binding-technology/kinomescan-technology-platform.
(19) Sorafenib and palbociclib were purchased from AdooQ
BioScience (Irvine, CA).
(20) More detailed in vitro and in vivo studies of 28 and its abilities
to simultaneously inhibit CDK4 and FLT3 and suppress mutations in
FLT3 will be published elsewhere in due course.
(21) Zuccotto, F.; Ardini, E.; Casale, E.; Angiolini, M. Through the
“gatekeeper door”: Exploiting active kinase conformation. J. Med.
Chem. 2010, 53, 2681−2694.
(22) Liu, Y.; Gray, N. S. Rational design of inhibitors that bind to
inactive kinase conformations. Nature Chem. Biol. 2006, 2, 358−364.
(23) FLT3 apo structure (PDB 1RJB) used for DFG-out kinase with
Sorafenib modeled with Glide. FLT3 DFG-in homology model (cKIT,
PDB 1PKG) used to model 28.
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