Optimization of an imidazopyridazine series of inhibitors of plasmodium falciparum calcium-dependent protein kinase 1 (Pf CDPK1)

Article abstract of DOI:10.1021/jm500342d

A structure-guided design approach using a homology model of Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1) was used to improve the potency of a series of imidazopyridazine inhibitors as potential antimalarial agents. This resulted in high affinity compounds with PfCDPK1 enzyme IC ₅₀ values less than 10 nM and in vitro P. falciparum antiparasite EC₅₀ values down to 12 nM, although these compounds did not have suitable ADME properties to show in vivo efficacy in a mouse model. Structural modifications designed to address the ADME issues, in particular permeability, were initially accompanied by losses in antiparasite potency, but further optimization allowed a good balance in the compound profile to be achieved. Upon testing in vivo in a murine model of efficacy against malaria, high levels of compound exposure relative to their in vitro activities were achieved, and the modest efficacy that resulted raises questions about the level of effect that is achievable through the targeting of PfCDPK1.

Full text of DOI:10.1021/jm500342d

Article
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Optimization of an Imidazopyridazine Series of Inhibitors of
Plasmodium falciparum Calcium-Dependent Protein Kinase 1
(Pf CDPK1)
Timothy M. Chapman,*,† Simon A. Osborne,† Claire Wallace,† Kristian Birchall,† Nathalie Bouloc,†
Hayley M. Jones,† Keith H. Ansell,† Debra L. Taylor,† Barbara Clough,‡ Judith L. Green,‡
and Anthony A. Holder‡
†Centre for Therapeutics Discovery, MRC Technology, 1-3 Burtonhole Lane, Mill Hill, London NW7 1AD, U.K.
‡Division of Parasitology, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, U.K.
ABSTRACT: A structure-guided design approach using a homology model of Plasmodium falciparum calcium-dependent
protein kinase 1 (Pf CDPK1) was used to improve the potency of a series of imidazopyridazine inhibitors as potential antimalarial
agents. This resulted in high affinity compounds with Pf CDPK1 enzyme IC50 values less than 10 nM and in vitro P. falciparum
antiparasite EC50 values down to 12 nM, although these compounds did not have suitable ADME properties to show in vivo
efficacy in a mouse model. Structural modifications designed to address the ADME issues, in particular permeability, were initially
accompanied by losses in antiparasite potency, but further optimization allowed a good balance in the compound profile to be
achieved. Upon testing in vivo in a murine model of efficacy against malaria, high levels of compound exposure relative to their in
vitro activities were achieved, and the modest efficacy that resulted raises questions about the level of effect that is achievable
through the targeting of Pf CDPK1.
host cell invasion, where it is able to phosphorylate components
of the molecular motor that drives parasite invasion of red
blood cells.8,9 The prevention of this invasion process could
break the parasite lifecycle, causing the parasites to die and the
infection to be cleared. Pf CDPK1 therefore represents a novel
target for the potential treatment of malaria and offers promise
for achieving selectivity over the kinases of the human host.
More recently, its role in translational regulation of motor
complex transcripts in gametocytes10 and in schizont develop-
ment11 has also been reported. There has been interest in
CDPKs as drug targets,12 although relatively few inhibitors have
been reported in the literature: Kato et al.7 and Lemercier et
al.13 have reported inhibitors of Pf CDPK1, and inhibitors of
the CDPK1 enzymes from the related Apicomplexan protozoa
Toxoplasma gondii and Cryptosporidium parvum have also been
described.14−16
INTRODUCTION
■
Malaria is one of the most prevalent infectious diseases of the
developing world. In excess of 3 billion people are at risk, and it
currently leads to the deaths of around 655,000 people each
year, with the majority of these occurring in sub-Saharan Africa
among children under five years of age.1 Resistance to existing
antimalarial drugs is widespread,2 and therefore, new
therapeutic approaches are urgently needed. Calcium-depend-
ent protein kinases (CDPKs) are directly regulated by Ca2+ and
are found in plants and organisms in the alveolate lineage,3 but
they are absent in humans. They are present in Apicomplexan
parasites including Plasmodium falciparum, the causative agent
of the most severe form of malaria. CDPKs in Plasmodium are
present as a multigene family containing at least five members,4
and different CDPKs are proposed to be functional at different
stages of the parasite life cycle. P. falciparum calcium-dependent
protein kinase 1 (PfCDPK1), first identified by Zhao et al.,5 is
expressed in the asexual blood stages of the parasite responsible
for disease pathology. It has been shown to be encoded by an
important gene,6,7 and it is implicated in parasite motility and
Received: March 4, 2014
Published: April 1, 2014
© 2014 American Chemical Society
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Figure 1. Summary data for compound 1.
A high throughput screen of our compound collection
against the isolated recombinant Pf CDPK1 enzyme identified a
hit series containing an imidazopyridazine core as the primary
series of interest, and the initial development of the structure−
activity relationship (SAR) in this series has been described
previously.17 Compounds with potent enzyme inhibitory
activity had been generated, which also showed good kinase
selectivity against a human kinase panel and promising in vitro
ADME profiles. In particular, compound 1 (Figure 1)
represented an early lead, with low nanomolar inhibitory
potency against Pf CDPK1, sub-500 nM antiparasite activity,
and modest in vivo efficacy in a P. berghei mouse model of
malaria.
Figure 2. Example of the 2-pyridyl variant with superior predicted
binding affinity from docking studies.
Tyr-148 at the hinge region, a hydrogen bond with the side-
chain carboxylic acid of Asp-212, and an additional hydrogen
bond between the basic amine group and Glu-152 as it points
out toward the solvent.
In order to advance this series, improvements were sought in
the in vitro antiparasite activity and pharmacokinetic profile of
the series while maintaining a good selectivity profile against
human kinases to generate compounds with the potential to
show improved in vivo efficacy.
The top scoring compounds identified from the docking
studies were then synthesized according to the route in Scheme
1. Intermediate 3 was functionalized through nucleophilic
substitution on the chloro- at the 6-position to install the BOC-
protected amine side chains in intermediates 4 and 17. These
were elaborated through Suzuki coupling with the appropriate
boronate reagents to give the 5-aminopyrimidine products 5
and 18, the 5-thiomethylpyrimidine 13 or 5-aminopyridine 20.
The final compounds were prepared by either a Buchwald
coupling with an aryl halide or through oxidation of the
thiomethyl pyrimidine followed by nucleophilic displacement
with the appropriate amine.
The SAR of the synthesized analogues is shown in Table 1.
As predicted by the docking studies, variants containing a 2-
pyridyl or phenyl group at this position both showed increased
binding affinity to the enzyme versus compound 1 (Table 1,
examples 2 and 6). As the potency of the most potent
compounds was now below the limit of detection of our
primary Kinase Glo enzyme assay, a thermal denaturation assay
was used to quantify the differences and define a rank order in
binding affinity between the most potent compounds.20 This
revealed that the 2-pyridyl compound 2 resulted in a larger shift
in the thermal denaturation temperature of the protein (ΔTm)
than its phenyl counterpart 6, and both showed a higher
thermal shift than compound 1 in this assay, which displayed a
ΔTm of 15.7 K. Gratifyingly, this difference in enzyme affinity
was reflected in the potency of the compounds against the P.
falciparum parasite, with compound 2 showing an EC50 of 80
nM compared with 180 nM for compound 6.
RESULTS AND DISCUSSION
■
A structure-guided design approach using a homology model of
PfCDPK1 (based on TgCDPK1, PDB ref: 3I7C)18 was used in
attempting to gain increased binding affinity against the target
and correspondingly increase the cellular potency of the
inhibitors. The homology model had proved effective in
explaining the SAR up to this point, and it was therefore used as
a key component in considering how additional potency could
be gained. In particular, it suggested that the binding pocket
occupied by the isopentyl chain of compound 1 was not
optimally filled and that there was potential to gain additional
beneficial interactions with the enzyme in this region. Virtual
libraries were enumerated with a diverse range of groups at this
position and examined through docking using Glide SP.19 For
enumeration purposes, the basic amine group was set to be
either N-methylpiperidine or 1,4-diaminocyclohexane, which
had been previously demonstrated to be optimal for potency,17
and the heteroaryl linker ring was set as either pyridine or
pyrimidine. Analysis of the docking results suggested that
replacement of the isopentyl group with an aromatic ring
containing a suitably positioned hydrogen-bond acceptor (for
example, a 2-pyridyl group, as in compound 2; Figure 2) could
give increased binding affinity.
In comparison with the isopentyl group in this pocket, the 2-
pyridyl nitrogen atom could potentially form an additional
hydrogen-bond interaction with the backbone N-H of Asp-212
(depicted in Figure 3B), and it also appeared to be an excellent
spatial and electrostatic fit into the rest of this pocket.
Compound 2 was predicted to be able to retain the other
key interactions made by compound 1: a hydrogen bond
between the imidazopyridazine core and the backbone N-H of
Alternative heteroaryl groups were then explored: 2-pyrazine
7 showed good potency, albeit weaker than those of 2 and 6,
but 3-pyridyl 8 and 2-pyrimidyl 9 lost potency against both the
enzyme and parasite. The addition of substituents to the pyridyl
ring was investigated: 3-fluoropyridyl gave a boost in potency
against both the enzyme and the parasite, with compound 10
displaying a high thermal shift of 28.0 K and excellent EC50 of
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Figure 3. Proposed binding modes from the docking of compound 1 (A) and compound 2 (B) illustrating the potential to gain an additional H-
bond interaction with the backbone N-H of Asp-212.
a
Scheme 1. Synthetic Route to Heteroaryl and Aryl Variants
a
Reagents and conditions: (a) trans-cyclohexane-1,4-diamine, NMP, microwave, 180 °C, then di-tert-butyl dicarbonate, DMAP, Et3N, THF, reflux;
(b) 2-aminopyrimidine-5-boronic acid pinacol ester, Pd(dppf)Cl2, Cs2CO3, dioxane/water, 90 °C; (c) aryl/heteroaryl halide, Pd(OAc)2, CyPF-tBu
or Xantphos, NaOtBu or Cs2CO3, DME or dioxane, 80 °C; (d) 4 M HCl/dioxane, MeOH; (e) 2-(thiomethyl)pyrimidine-5-boronic acid,
Pd(dppf)Cl2, Cs2CO3, dioxane/water, 90 °C; (f) m-chloroperoxybenzoic acid, CH2Cl2; (g) 3-(aminomethyl)pyridine or 2-(aminomethyl)pyridine or
(1-methyl-1H-pyrazol-3-yl)methylamine, dioxane, reflux; (h) tert-butyl 4-aminopiperidine-1-carboxylate, DIPEA, NMP, 130 °C; (i) 2-chloro-3-
fluoropyridine, Pd(OAc)2, Xantphos, Cs2CO3, dioxane, microwave, 130 °C or thermal, 90 °C; (j) formaldehyde, AcOH, sodium
triacetoxyborohydride, THF; (k) 2-aminopyridine-5-boronic acid pinacol ester, Pd(dppf)Cl2, Cs2CO3, dioxane/water, 90 °C.
12 nM against the parasite. The introduction of 5-position
substituents to the pyridine ring such as trifluoromethyl (11)
and methyl (12) led to excellent enzyme affinity and increased
thermal shift values relative to 10, although their antiparasite
potency decreased. When a CH2 spacer group was introduced,
the 3-pyridyl variant 14 was relatively weak against the enzyme,
whereas the 2-pyridyl variant 15 and the 3-pyrazole 16 showed
good enzyme inhibitory potency. This was again consistent
with the predictions of the homology model, which suggested
that 15 could form an H-bond with Asp-212, whereas 14 could
not. However, all of these variants were weak against the
parasite. Switching to the N-methylpiperidine basic side chain
(19) gave good potency against the enzyme and the parasite,
while changing the pyrimidine linker ring to a pyridyl linker
ring (21) retained high enzyme potency but led to a loss in
potency against the parasite.
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Table 1. In Vitro Potency Data for Aryl and Heteroaryl Variants
a
The limit of detection of the PfCDPK1 Kinase Glo enzyme assay is 0.010 μM; nt = not tested.
Leading compounds were profiled in in vitro ADME assays,
compounds had low measured log D values and displayed good
and selected data are shown in Table 2. In general, the
stability in both mouse and human microsomes but poor
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the compounds. Piperazine 23 showed weaker enzyme, and
antiparasite potency and no improvement in permeability and
although the N-methylpiperazine 24 (with just one HBD and a
calculated pKa of 7.1) showed improved permeability, it
displayed weak antiparasite activity. The pyrrolidines 25 and
26 showed good enzyme affinity but low permeability. The
more flexible variants 27−29 showed lower enzyme affinity,
and although the weakly basic morpholine 27 showed
improved permeability, it was inactive against the parasite,
whereas piperazine 28 and pyrrolidine 29 both showed low
permeability. As the diaminocyclohexane basic group seemed
optimal for antiparasite activity, cyclization of the free −NH2
into a ring was explored as a way to potentially improve
permeability, through the attenuation of pKa and/or the
reduction in the number of HBDs. The cyclized variants 30
and 31 showed good potency against both the enzyme and
parasite; however, a modest improvement in permeability was
only achieved with morpholine variant 30. Compounds with
oxygen-linked basic amine groups such as piperidine 32 and
pyrrolidine 33 were potent against the enzyme and showed
improved permeability, but they were inactive against the
parasite. Carbon-linked piperidine variant 34 showed good
enzyme inhibitory potency but a weak effect against the
parasite, coupled with low permeability.
In summary, although lowering the pKa and HBD count
resulted in improved permeability in some cases, this tended to
be accompanied by a significant reduction of potency against
the parasite. The best combination was achieved with
compound 30, although it still showed only a weak effect
(22% reduction in parasitemia) when tested for in vivo efficacy
in the P. berghei model under the same dosing regimen as
employed previously. Despite complying with property criteria
that may normally be expected to be sufficient to allow
permeability and oral bioavailability, structure−property
relationships suggested that there were stricter requirements
for this series and that the desired balance in profile could not
be obtained from modifying the basic group alone.
It had been observed that the pyridine linker ring had given
higher permeability than the pyrimidine (comparison of
examples 19 and 21), so further variations at this linker ring
were then explored. Employing a phenyl ring in this position
(35; see Table 4) gave a large increase in permeability versus its
pyrimidine analogue (19), although unfortunately this was
accompanied by a significant loss in antiparasite activity, with
an EC50 of approximately 400 nM. In order to maintain the
phenyl ring but mimic the dipole moments of the pyridyl and
pyrimidyl rings, fluorophenyl and difluorophenyl variants 36
and 37 were investigated, although this approach failed to
restore potent antiparasite activity. Cyclization of the −NH2 of
the diaminocyclohexane side chain into a pyrrolidine ring with
pyridyl or phenyl linker rings gave examples 38 and 39; these
compounds showed good permeability and marginally
Table 2. In Vitro ADME Data for Selected Compounds
PAMPA
a
b
compd HLM (% rem) MLM (% rem) m log D
Papp/nms−1
2
99
89
93
69
92
95
98
77
93
92
85
93
100
95
85
57
1.1
1.6
0.2
1.6
1.1
0.4
1.4
3.1
0
6
2
10
11
12
15
19
21
4
0
nt
nt
1
64
a
b
Percent remaining at 40 min. Percent remaining at 30 min; nt = not
tested.
PAMPA permeability. Kinase selectivity screening against a
human kinase panel revealed that they showed good selectivity,
and the selectivity profile of compound 10 is shown in Figure 4,
in comparison with that of compound 1. Compound 10 also
showed IC50 > 25 μM against CYP-P450 isoforms 1A, 2C9,
2C19, 2D6, and 3A4. However, when 10 was tested for in vivo
efficacy in the 4-day Peters test21 (P. berghei murine model of
malaria) with a 50 mg/kg once daily oral dosing regimen, it
showed no significant reduction in parasitemia levels (4%
reduction). This was thought to be a consequence of low
plasma exposure, consistent with poor absorption in accordance
with its low permeability.
Although the introduction of the 2-pyridyl group gave
improved enzyme and antiparasite potency, poor permeability
was seemingly limiting the bioavailability of the compounds
when dosed in vivo. Efforts were therefore directed at increasing
the permeability to allow sufficient exposure while retaining
high potency. In the first case, the pyrimidine linker ring and
distal 3-fluoro-2-aminopyridyl group present in compound 10
were retained, and the basic amine portion was modified. The
two amino substituents giving the highest potency up to this
point are both highly basic (calculated pKa values for the
conjugate acids of 10 and 19 are >9 in both cases22), and
therefore, attenuating this basicity represents one approach to
potentially gaining improved permeability. Compound 10 also
contains four hydrogen bond donors (HBDs) which may be
leading to poor permeability. Variations at this portion of the
molecule were explored, and selected variants are shown in
Table 3. The requirement for a basic amine side chain in order
to achieve cell-based activity against the parasite was
demonstrated by example 22, in which the basic side chain
was replaced by a methyl group. Although this compound is
still relatively potent against the enzyme with an IC50 of 44 nM,
there is complete loss of potency against the parasite; this effect
is consistent with the observations of Lemercier et al.13 The
high permeability of 22 also confirms that the basic center is
making a significant contribution to the poor permeability of
Figure 4. Kinase selectivity data on compounds 1 (top) and 10 (bottom) screened at 1 μM inhibitor concentration against a 66-member human
kinase panel; green, <50% inhibition; amber, 50−80% inhibition; and red, >80% inhibition.
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Table 3. In Vitro Potency, Properties, and Permeability Data for Selected Variations on the Basic Amine Side Chain
a
pKa of conjugate acid calculated according to ref 22; nt = not tested.
improved antiparasite activity relative to the N-methylpiper-
idines. Reverting to the diaminocyclohexane basic group with a
free −NH2 (40) gave improved antiparasite activity but
reduced permeability, and variants with a pyrazole linker ring
were also investigated, and these have been described
previously.23
In summary, although modifications of this linker ring led to
significant and steep improvements in PAMPA permeability,
the variations that gave improved permeability versus
compound 10 while maintaining good enzyme inhibitory
potency were also accompanied by a significant loss in
antiparasite activity. Although the desired balance in compound
profile had not yet been achieved through modification of this
group, the higher permeability of these compounds warranted
pharmacokinetic studies to confirm that they could achieve
good in vivo exposure.
ADME and in vivo PK, with compound 35 showing the best PK
profile of those tested (data shown in Table 5).
Compounds 35, 38, and 39 were then advanced to testing in
the P. berghei in vivo mouse model, and despite their lower
antiparasite potency compared to that of earlier examples such
as compound 10, they showed superior efficacy. The observed
efficacy was still only modest, with best results of 44% and 46%
reduction in parasitemia achieved with 35 and 38, respectively,
at a 50 mg/kg oral dose (Table 6).
In order to evaluate the exposure in the efficacy model, blood
samples were taken for compound 35, which revealed that it
had achieved a good exposure with total plasma levels of 1660
ng/mL at 1 h (10-fold over the antiparasite EC50) and 1460
ng/mL at 4 h (8.5-fold over the antiparasite EC50).
Addressing Species Differences between P. falcipa-
rum and P. berghei. As the in vivo efficacy model is performed
with the rodent parasite P. berghei rather than P. falciparum,
there remained a possibility that the low in vivo efficacy
Pharmacokinetics. Pharmacokinetic profiling in rats
revealed that there was a good correlation between in vitro
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a
Table 4. In Vitro Potency and Permeability Data for R2 Linker Ring Variations
a
nt = not tested.
a
Furthermore, based on the residues within 10 Å of any atom of
ATP in the PbCDPK1 crystal structure (PDB ID: 3Q5I), there
is 100% identity. Docking of the inhibitors into the ATP-
binding site of PbCDPK1 predicted that they would bind with
an affinity similar to that for Pf CDPK1. This prediction was
confirmed through the expression of the recombinant
PbCDPK1 enzyme and measurement of the IC50 values,
which showed that key compounds bound with similar affinity
between the two enzymes (representative compounds 1 and 35
showed PbCDPK1 IC50 values of 22 and 15 nM, respectively).
Finally, in order to probe in vivo differences, selected
compounds were tested in a P. falciparum murine model, in
which a severe combined immunodeficient (SCID) mouse can
be injected with human erythrocytes infected with a suitable
strain of P. falciparum parasites.24 Compounds 1 and 35 were
tested in this efficacy model with the same dosing regimen as in
the P. berghei model (50 mg/kg, oral, once daily), but
disappointingly, no efficacy was observed despite good
compound exposure.
In parallel with in vivo testing, efforts had also been focused
on achieving an improved compound profile with respect to the
combination of antiparasite potency and pharmacokinetics.
Although modification of the linker ring had led to improved
pharmacokinetics, this had been achieved at the expense of
antiparasite potency, so attention then turned to the distal
pyridyl ring as a point of modification to potentially restore
potency. The introduction of substituents around this ring had
previously produced compounds with the highest enzyme
affinities as determined by thermal denaturation experiments
(Table 1, compounds 11 and 12), so this approach offered the
potential to yield increased antiparasite potency. Molecular
Table 5. Pharmacokinetics of Compound 35 in Rats
iv t1/2 (h)
4
plasma Cl (mL/min/kg)
blood Cl (mL/min/kg)
Vd (L/kg)
28
14
8
oral BA (%)
70
a
Compound dosed as hydrochloride salt, iv in aqueous vehicle
containing 0.9% (w/v) NaCl at 4.5 mg/kg; po in aqueous vehicle
containing 0.5% (w/v) hydroxypropylmethylcellulose, 0.5% (v/v)
benzyl alcohol, and 0.4% (v/v) Tween-80 at 21 mg/kg.
Table 6. In Vitro ADME and in Vivo Efficacy Data for
Selected Compounds
HLM
MLM
PAMPA
in vivo reduction
in parasitemia
a
b
c
compd (% rem) (% rem) m log D Papp/nms−1
35
38
39
80
71
60
47
88
82
3.2
2.8
3.3
171
92
44
46
34
81
a
b
Percent remaining at 40 min. Percent remaining at 30 min.
c
Measured in the P. berghei mouse model of malaria, 4-day Peters test
with oral dosing once daily at 50 mg/kg; compounds were dosed as
monohydrochloride salts, dissolved in 30:70 EtOH/Tween-80, and
diluted 10-fold with water prior to dosing.
achieved might be due to species differences. Significant efficacy
differences across the parasite species have been observed in the
development of other antimalarial compounds, and therefore, it
was desirable to address this possibility. First, an in silico
assessment of Pf CDPK1 and PbCDPK1 revealed that across
their entire protein sequences PbCDPK1 and Pf CDPK1 have
high homology, with 88% sequence identity and 93% similarity.
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Table 7. In Vitro Potency and ADME Data for Distal Pyridyl Ring Substitution Variants
a
b
Percent remaining at 40 min. Percent remaining at 30 min.
modeling suggested that there was sufficient space in the
binding pocket for small substituents to be accommodated, so a
number of variants were synthesized, and the resulting SAR is
shown in Table 7. With N-methylpiperidine as the basic group
and a phenyl linker ring in place, the addition of 5-chloro (41),
6-methyl (42), or 5-fluoro (43) substituents was well tolerated,
with sub-100 nM antiparasite EC50 values for 41 and 42 and
acceptable in vitro ADME. The 5- and 6-trifluoromethyl
substituted pyridines 44 and 45 also showed excellent
antiparasite EC50 values of 50 and 30 nM, respectively, with
good in vitro ADME characteristics. Compounds 46−48, with
the diaminocyclohexane basic group, also showed a good
balance between antiparasite activity and ADME properties; in
particular, compound 48 demonstrated the best profile with an
antiparasite EC50 of 80 nM combined with high stability in
mouse and human microsomes and acceptable permeability.
Compounds 41 and 48 were advanced to the P. berghei in
vivo mouse model; however, they did not show significantly
improved efficacy compared to that of previous examples
(Table 8), and a maximum of 51% reduction in parasitemia was
achieved with compound 41. Blood samples revealed that
Table 8. In Vivo Efficacy Data and Total Plasma Levels of
Compounds 41 and 48 in the P. berghei Mouse Model
a
compd
41
48
a
in vivo reduction in parasitemia
51
18
621
(18×)
6.54
(0.2×)
plasma concentration at 4 h/ng mL−1 (fold exposure 1610
over in vitro EC50)
(45×)
plasma concentration at 24 h/ng mL−1 (fold exposure 1270
over in vitro EC50)
(35×)
a
Four-day Peters test with once daily oral dosing at 50 mg/kg;
compounds were dosed as monohydrochloride salts, dissolved in
30:70 EtOH/Tween-80, and diluted 10-fold with water prior to
dosing.
compound 41 had achieved a good exposure (total plasma
concentration 45-fold over in vitro antiparasite EC50 at 4 h and
35-fold at 24 h), whereas 48 showed lower exposure, consistent
with its lower observed in vivo efficacy.
CONCLUSIONS
An imidazopyridazine series of Pf CDPK1 inhibitors was
optimized employing a structure-guided design approach with
■
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the water). A flow rate of 3 mL/min was used with UV detection at
254 and 210 nm. The structure of the intermediates and final products
a homology model of PfCDPK1. Initial 2-pyridyl variants
showed excellent potency in assays against the PfCDPK1
enzyme and P. falciparum parasite but did not possess sufficient
permeability to be effective in vivo. Modifications of the basic
side chain and aryl linker rings allowed permeability to be
improved, but this led to a reduction in antiparasite potency.
Finally, further optimization of the linker ring and distal pyridyl
ring led to molecules possessing well-balanced profiles with
respect to potency and in vitro ADME and suitable for in vivo
dosing, with high resulting compound exposures following oral
administration.
1
1
was confirmed by H NMR spectroscopy and mass spectrometry. H
nuclear magnetic resonance (NMR) spectroscopy was carried out
using a JEOL ECX400 spectrometer in the stated solvent at around
room temperature unless otherwise stated. Characteristic chemical
shifts (δ) are given in parts-per-million using conventional
abbreviations for the designation of major peaks: e.g., s, singlet; d,
doublet; t, triplet; q, quartet; dd, doublet of doublets; and br, broad.
Analytical mass spectra were recorded using a MM-ES + APCI or ES
mass spectrometer (G-1956A or G-6120B, manufactured by Agilent).
tert-Butyl {trans-4-[(3-Bromoimidazo[1,2-b]pyridazin-6-yl)-
amino]cyclohexyl}carbamate 4. (a) A solution of 3-bromo-6-
chloroimidazo[1,2-b]pyridazine 3 (1.40 g, 6.02 mmol) in NMP (8
mL) was treated with trans-cyclohexane-1,4-diamine (2.05 g, 18.0
mmol, 3.0 equiv) and stirred with microwave heating at 180 °C for 30
min. It was then diluted with EtOAc (100 mL) and washed with water
(2 × 100 mL). The organic layer was dried and concentrated under
reduced pressure. Chromatography on silica gel (2 M NH3 in MeOH/
EtOAc gradient) gave trans-N-(3-bromo-imidazo[1,2-b]pyridazin-6-
However, despite the improvements made to the compound
profiles compared with those of the early lead compound 1, the
in vivo reduction in parasitemia in a murine P. berghei model of
malaria remained modest, with a maximum reduction of 51%
with an oral dose of 50 mg/kg for compound 41. The reason
for this limited effect remains unclear: although the PfCDPK1
enzyme has been shown to be encoded by an important gene in
the malaria parasite and high levels of in vivo compound
exposure were achieved relative to in vitro antiparasite EC50, it
may be that a higher multiple of this EC50 is required for a
longer period of time to produce higher efficacy or that low
levels of residual CDPK1 enzyme activity may be sufficient for
parasite survival. There may be a level of redundancy allowing a
CDPK1-based inhibitory effect to be circumvented, and indeed,
a recent report has suggested that CDPK1 may not in fact be
required for host cell invasion in the erythrocytic stage in
Plasmodium berghei but rather that it plays an essential role in
the mosquito sexual stages.25 Differences in parasite biology
may also explain the limited effect of the inhibitors in vivo: for
example, P. berghei has only a 24-h multiplication cycle and
develops in reticulocytes in the mouse compared with a 48-h
cycle in mature red cells for P. falciparum, and differences in
synchrony of parasite development may also have contributed
to the reduced efficacy of the compounds. Further studies on
these compounds including understanding their mechanism of
action, the identification of additional CDPK1 substrates, and
the development of better read-outs of activity are ongoing, and
these will be reported in future publications.
1
yl)-cyclohexane-1,4-diamine as a pale yellow solid (981 mg, 52%). H
NMR (400 MHz, DMSO-d6) δ ppm 7.66 (d, J = 10.1 Hz, 1H), 7.45 (s,
1H), 6.95 (br. d, J = 7.3 Hz, 1H), 6.65 (d, J = 9.6 Hz, 1H), 3.63−3.53
(m, 1H), 2.60−2.52 (m, 1H), 2.08−2.04 (m, 2H), 1.82−1.78 (m, 2H),
1.28−1.09 (m, 4H). m/z (ES + APCI)+, 310/312 [M + H]+. (b) A
solution of trans-N-(3-bromo-imidazo[1,2-b]pyridazin-6-yl)-cyclohex-
ane-1,4-diamine (1.80 g, 5.79 mmol) in THF (20 mL) was treated
with Et3N (1.21 mL, 8.70 mmol, 1.5 equiv), di-tert-butyl dicarbonate
(1.90 g, 8.70 mmol, 1.5 equiv), and DMAP (71 mg, 0.58 mmol, 0.1
equiv) and stirred at reflux for 18 h. Concentration under reduced
pressure and silica gel chromatography (20% MeOH/EtOAc) gave 4
as a pale yellow solid (1.60 g, 67%). 1H NMR (400 MHz, DMSO-d6)
δ ppm 7.67 (d, J = 9.6 Hz, 1H), 7.46 (s, 1H), 6.99 (br. d, J = 7.3 Hz,
1H), 6.76 (br. d, J = 8.2 Hz, 1H), 6.65 (d, J = 9.6 Hz, 1H), 3.59−3.52
(m, 1H), 3.28−3.22 (m, 1H), 2.11−2.08 (m, 2H), 1.84−1.81 (m, 2H),
1.38 (s, 9H), 1.30−1.23 (m, 4H). m/z (ES + APCI)+: 410/412 [M +
H]+.
tert-Butyl (trans-4-{[3-(2-Aminopyrimidin-5-yl)imidazo[1,2-b]-
pyridazin-6-yl]amino}cyclohexyl)carbamate 5. A mixture of 4
(1.20 g, 2.92 mmol, 1.0 equiv), 2-aminopyrimidine-5-boronic acid
pinacol ester (970 mg, 4.39 mmol, 1.5 equiv), Cs2CO3 (3.81 g, 11.7
mmol, 4.0 equiv), water (6 mL), and dioxane (12 mL) was degassed
with N2, then Pd(dppf)Cl2 (238 mg, 0.29 mmol, 0.1 equiv) was added,
and the mixture heated at 90 °C for 5 h. The mixture was allowed to
cool, then concentrated to dryness and purified by chromatography on
silica gel (2−20% MeOH/EtOAc) to give 5 as a beige solid (1.07 g,
EXPERIMENTAL SECTION
■
1
86%). H NMR (400 MHz, DMSO-d6) δ ppm 8.96 (s, 2H), 7.78 (s,
Chemistry. All commercial reagents and solvents were used
without further purification. Silica gel chromatography was carried out
using a Biotage SP4 or Isolera MPLC system with prepacked silica gel
cartridges. Preparative HPLC was carried out using an apparatus made
by Agilent. The apparatus is constructed such that the chromatography
(column: either a 19 × 100 mm (5 μm) C-18 Waters Xbridge or a 19
× 100 mm (5 μm) C-6Ph Waters Xbridge column, both at a flow rate
of 40 mL/min) is monitored by a multiwavelength UV detector
(G1365B manufactured by Agilent) and an MM-ES + APCI mass
spectrometer (G-1956A, manufactured by Agilent) connected in
series, and if the appropriate criteria are met, the sample is collected by
an automated fraction collector (G1364B manufactured by Agilent).
Collection was triggered by a combination of UV or mass
spectrometry or based on time. Typical conditions for the separation
process are as follows: the gradient was run over a 7 min period
(gradient at the start, 10% methanol and 90% water; and gradient at
the finish, 100% methanol and 0% water; as buffer, 0.1% formic acid,
0.1% ammonium hydroxide, or 0.1% trifluoroacetic acid was added to
the water). Purity of all final derivatives for biological testing was
confirmed to be >95% as determined using the following conditions:
an Agilent HPLC instrument with a C-18 Xbridge column (3.5 μm,
4.6 × 30 mm, gradient at the start, 10% acetonitrile and 90% water;
gradient at the finish, 100% acetonitrile and 0% water; as buffer, either
0.1% ammonium hydroxide or 0.1% trifluoroacetic acid was added to
1H), 7.70 (d, J = 9.6 Hz, 1H), 6.96 (d, J = 6.9 Hz, 1H), 6.87−6.78 (m,
3H), 6.62 (d, J = 9.6 Hz, 1H), 3.52−3.42 (m, 1H), 3.30−3.21 (m,
1H), 2.17−2.08 (m, 2H), 1.90−1.79 (m, 2H), 1.38 (s, 9H), 1.32−1.21
(m, 4H). m/z (ES + APCI)+: 425 [M + H]+.
trans-N-{3-[2-(Pyridin-2-ylamino)pyrimidin-5-yl]imidazo[1,2-b]-
pyridazin-6-yl}cyclohexane-1,4-diamine 2. Compound 5 (90 mg,
0.21 mmol, 1.1 equiv), 2-chloropyridine (18 μL, 22 mg, 0.19 mmol,
1.0 equiv), and sodium tert-butoxide (73 mg, 0.76 mmol, 4.0 equiv)
were added to a prestirred solution of Pd(OAc)2 (4.3 mg, 0.1 equiv)
and CyPF-tBu (10.5 mg, 10 mol %) in DME (1.5 mL), and the
mixture was heated at 80 °C for 3 h. The mixture was allowed to cool,
diluted with CH2Cl2, and passed through an Isolute silica cartridge (1
g) eluting with CH2Cl2/MeOH (7:3). The eluent was concentrated
under reduced pressure, then stirred in MeOH (3 mL) and 4 M HCl/
dioxane (2 mL) for 40 min, concentrated under reduced pressure, and
purified by prep-HPLC to give 2 as a white solid (5 mg, 6%). 1H NMR
(400 MHz, CD3OD) δ ppm 9.24 (s, 2H), 8.39−8.34 (m, 1H), 8.29−
8.24 (m, 1H), 7.81 (s, 1H), 7.80−7.76 (m, 1H), 7.62 (d, J = 10.1 Hz,
1H), 7.05−7.00 (m, 1H), 6.69 (d, J = 9.6 Hz, 1H), 3.73−3.63 (m,
1H), 2.90−2.80 (m, 1H), 2.31−2.22 (m, 2H), 2.07−1.98 (m, 2H),
1.50−1.30 (m, 4H). m/z (ES + APCI)+: 402 [M + H]+.
trans-N-{3-[2-(Phenylamino)pyrimidin-5-yl]imidazo[1,2-b]-
pyridazin-6-yl}cyclohexane-1,4-diamine 6. Compound 5 (90 mg,
3578
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Journal of Medicinal Chemistry
Article
0.21 mmol, 1.1 equiv), iodobenzene (21 μL, 39 mg, 0.19 mmol, 1.0
equiv), and sodium tert-butoxide (73 mg, 0.76 mmol, 4.0 equiv) were
added to a prestirred solution of Pd(OAc)2 (4.3 mg, 0.1 equiv) and
CyPF-tBu (10.5 mg, 0.1 equiv) in dioxane (1.5 mL), and the mixture
was heated at 80 °C for 40 h. The mixture was allowed to cool, and 4
M HCl/dioxane (1 mL) was added and the mixture stirred for 2 h,
then concentrated to dryness, and purified by prep-HPLC to give 6 as
(m, 1H), 2.65−2.56 (m, 1H), 2.25 (s, 3H), 2.14−2.07 (m, 2H), 1.86−
1.79 (m, 2H), 1.32−1.13 (m, 4H). m/z (ES + APCI)+: 416 [M + H]+.
tert-Butyl [trans-4-({3-[2-(methylsulfanyl)pyrimidin-5-yl]imidazo-
[1,2-b]pyridazin-6-yl}amino)cyclohexyl]carbamate 13. Following
the method for 5 using 2-(thiomethyl)pyrimidine-5-boronic acid, we
1
obtained 13 as a pale yellow solid (90% yield). H NMR (400 MHz,
DMSO-d6) δ ppm 9.38 (s, 2H), 8.03 (s, 1H), 7.77 (d, J = 9.6 Hz, 1H),
7.09 (br. d, J = 6.9 Hz, 1H), 6.83 (br. d, J = 8.2 Hz, 1H), 6.71 (d, J =
9.6 Hz, 1H), 3.53−3.47 (m, 1H), 3.31−3.26 (m, 1H), 2.58 (s, 3H),
2.15−2.11 (m, 2H), 1.87−1.82 (m, 2H), 1.39 (s, 9H), 1.35−1.23 (m,
4H). m/z (ES + APCI)+: 456 [M + H]+.
1
an off-white solid (15 mg, 18%). H NMR (400 MHz, DMSO-d6) δ
ppm 9.91 (s, 1H), 9.24 (s, 2H), 7.91−7.88 (m, 1H), 7.83−7.77 (m,
2H), 7.76−7.71 (m, 1H), 7.33−7.26 (m, 2H), 7.03−6.92 (m, 2H),
6.67 (d, J = 9.6 Hz, 1H), 3.58−3.46 (m, 1H), 2.66−2.56 (m, 1H),
2.15−2.07 (m, 2H), 1.87−1.78 (m, 2H), 1.33−1.10 (m, 4H). m/z (ES
+ APCI)+: 401 [M + H]+.
trans-N-(3-{2-[(Pyridin-3-ylmethyl)amino]pyrimidin-5-yl}imidazo-
[1,2-b]pyridazin-6-yl)cyclohexane-1,4-diamine 14. Compound 13
(2.00 g, 4.39 mmol, 1.0 equiv) in CH2Cl2 (60 mL) was treated with
mCPBA (2.38 g, 9.66 mmol, 2.2 equiv) and stirred at rt for 2 h. The
mixture was diluted with saturated aqueous Na2SO3 (20 mL) and
CH2Cl2 (50 mL) and washed with saturated aqueous NaHCO3 (80
mL). The aqueous layer was extracted with CH2Cl2 (50 mL), then the
combined organic layers were washed with brine (60 mL), dried, and
trans-N-{3-[2-(Pyrazin-2-ylamino)pyrimidin-5-yl]imidazo[1,2-b]-
pyridazin-6-yl}cyclohexane-1,4-diamine 7. Compound 5 (80 mg,
0.19 mmol, 1.0 equiv), 2-chloropyrazine (22 mg, 0.19 mmol, 1.0
equiv), Pd(OAc)2 (4.3 mg, 0.1 equiv), Xantphos (11 mg, 0.1 equiv),
and Cs2CO3 (248 mg, 0.76 mmol, 4.0 equiv) in dioxane (1 mL) under
N2 were heated at 90 °C for 10 h. The mixture was allowed to cool,
and 4 M HCl/dioxane (1 mL) was added and the mixture stirred for 2
h then concentrated to dryness. Purification by prep-HPLC gave 7 as a
1
concentrated to give an orange solid (2.1 g, 98%). H NMR (400
MHz, DMSO-d6) δ ppm 9.80 (s, 2H), 8.30 (s, 1H), 7.85 (d, J = 9.6
Hz, 1H), 7.24 (br. d, J = 6.9 Hz, 1H), 6.85 (br. d, J = 8.2 Hz, 1H), 6.82
(d, J = 10.3 Hz, 1H), 3.60−3.52 (m, 1H), 3.44 (s, 3H), 3.33−3.25 (m,
1H), 2.17−2.13 (m, 2H), 1.88−1.83 (m, 2H), 1.39 (s, 9H), 1.37−1.23
(m, 4H). A solution of the intermediate (60 mg, 0.12 mmol, 1.0 equiv)
in dioxane (2 mL) was treated with 1-(pyridin-3-yl)methanamine (54
mg, 0.49 mmol, 4.0 equiv) and stirred at reflux for 4 h. Concentration
under reduced pressure, purification by prep-HPLC, and treatment
with 4 M HCl/dioxane (1 mL) followed by elution through an Isolute
aminopropyl cartridge gave 14 as an off-white solid (12 mg, 24%). 1H
NMR (400 MHz, DMSO-d6) δ ppm 9.02 (br. s, 2H), 8.58 (d, J = 1.8
Hz, 1H), 8.48−8.38 (m, 1H), 8.08 (t, J = 6.4 Hz, 1H), 7.78−7.68 (m,
3H), 7.43−7.27 (m, 1H), 6.94 (d, J = 6.9 Hz, 1H), 6.62 (d, J = 9.6 Hz,
1H), 4.55 (d, J = 6.4 Hz, 2H), 3.53−3.41 (m, 1H), 2.64−2.55 (m,
1H), 2.13−2.01 (m, 2H), 1.88−1.75 (m, 2H), 1.33−1.08 (m, 4H). m/
z (ES + APCI)+: 416 [M + H]+.
1
white solid (8 mg, 10%). H NMR (400 MHz, DMSO-d6) δ ppm
10.52 (br. s, 1H), 9.52 (d, J = 1.4 Hz, 1H), 9.34 (s, 2H), 8.36−8.34
(m, 1H), 8.24 (d, J = 2.3 Hz, 1H), 7.97 (s, 1H), 7.77 (d, J = 9.6 Hz,
1H), 7.04 (d, J = 6.9 Hz, 1H), 6.70 (d, J = 9.6 Hz, 1H), 3.58−3.49 (m,
1H), 2.76−2.65 (m, 1H), 2.17−2.09 (m, 2H), 1.90−1.82 (m, 2H),
1.34−1.16 (m, 4H). m/z (ES + APCI)+: 403 [M + H]+.
trans-N-{3-[2-(Pyridin-3-ylamino)pyrimidin-5-yl]imidazo[1,2-b]-
pyridazin-6-yl}cyclohexane-1,4-diamine 8. Following the method for
6 using 5 and 3-bromopyridine, we obtained 8 as an orange/brown
1
solid (28% yield). H NMR (400 MHz, DMSO-d6) δ ppm 10.14−
10.08 (m, 1H), 9.29 (s, 2H), 8.94 (d, J = 1.8 Hz, 1H), 8.28−8.24 (m,
1H), 8.19−8.16 (m, 1H), 7.93 (s, 1H), 7.75 (d, J = 9.6 Hz, 1H), 7.36−
7.32 (m, 1H), 7.03 (d, J = 6.9 Hz, 1H), 6.67 (d, J = 9.6 Hz, 1H), 3.57−
3.47 (m, 1H), 2.66−2.58 (m, 1H), 2.16−2.08 (m, 2H), 1.88−1.80 (m,
2H), 1.32−1.11 (m, 4H). m/z (ES + APCI)+: 402 [M + H]+.
trans-N-{3-[2-(Pyrimidin-2-ylamino)pyrimidin-5-yl]imidazo[1,2-
b]pyridazin-6-yl}cyclohexane-1,4-diamine 9. Following the method
for 7 using 5 and 2-chloropyrimidine, we obtained 9 as a white solid
trans-N-(3-{2-[(Pyridin-2-ylmethyl)amino]pyrimidin-5-yl}imidazo-
[1,2-b]pyridazin-6-yl)cyclohexane-1,4-diamine 15. Following the
method for 14 using 1-(pyridin-2-yl)methanamine, we obtained 15
1
as an off-white solid (27% yield). H NMR (400 MHz, DMSO-d6) δ
1
(10% yield). H NMR (400 MHz, DMSO-d6) δ ppm 10.45 (br. s,
ppm 9.00 (br. s, 2H), 8.50 (dt, J = 0.9, 2.5 Hz, 1H), 8.04−7.96 (m,
1H), 7.78−7.67 (m, 3H), 7.32 (d, J = 7.8 Hz, 1H), 7.24 (ddd, J = 0.9,
4.8, 7.6 Hz, 1H), 6.93 (d, J = 6.9 Hz, 1H), 6.62 (d, J = 9.6 Hz, 1H),
4.64 (d, J = 6.4 Hz, 2H), 3.55−3.40 (m, 1H), 2.66−2.56 (m, 1H),
2.18−2.02 (m, 2H), 1.89−1.76 (m, 2H), 1.31−1.06 (m, 4H). m/z (ES
+ APCI)+: 416 [M + H]+.
trans-N-[3-(2-{[(1-Methyl-1H-pyrazol-3-yl)methyl]amino}-
pyrimidin-5-yl)imidazo[1,2-b]pyridazin-6-yl]cyclohexane-1,4-dia-
mine 16. Following the method for 14 using 1-(1-methyl-1H-pyrazol-
3-yl)methanamine, we obtained 16 as an off-white solid (11% yield).
1H NMR (400 MHz, DMSO-d6) δ ppm 9.00 (br. s, 2H), 7.81−7.65
(m, 3H), 7.55 (d, J = 2.3 Hz, 1H), 6.93 (d, J = 6.9 Hz, 1H), 6.62 (d, J
= 9.6 Hz, 1H), 6.13 (d, J = 2.3 Hz, 1H), 4.47 (d, J = 6.0 Hz, 2H), 3.77
(s, 3H), 3.55−3.40 (m, 1H), 2.64−2.54 (m, 1H), 2.15−2.03 (m, 2H),
1.89−1.74 (m, 2H), 1.37−1.08 (m, 4H). m/z (ES + APCI)+: 419 [M
+ H]+.
tert-Butyl 4-[(3-bromoimidazo[1,2-b]pyridazin-6-yl)amino]-
piperidine-1-carboxylate 17. A solution of 3 (3.00 g, 12.9 mmol
1.0 equiv) in NMP (15 mL) was treated with tert-butyl 4-
aminopiperidine-1-carboxylate (5.10 g, 25.8 mmol, 2.0 equiv),
DIPEA (5.60 mL, 32.3 mmol, 2.5 equiv), and heated at 130 °C for
5 h. The reaction mixture was diluted with CH2Cl2 (100 mL) and
washed with deionized water (3 × 150 mL). The separated organic
layer was concentrated under reduced pressure and column
chromatography (10−80% EtOAc/pet ether) to give 17 as a brown
solid (3.01 g, 59%). 1H NMR (400 MHz, DMSO-d6) δ ppm 7.71 (d, J
= 9.6 Hz, 1H), 7.48 (s, 1H), 7.10 (br. d, J = 7.3 Hz, 1H), 6.68 (d, J =
10.1 Hz, 1H), 3.87−3.82 (m, 3H), 3.02−2.93 (m, 2H), 2.02−1.98 (m,
2H), 1.41 (s, 9H), 1.40−1.31 (m, 2H). m/z (ES + APCI)+: 396/398
[M + H]+.
1H), 9.30 (s, 2H), 8.60 (d, J = 5.0 Hz, 2H), 7.97 (s, 1H), 7.78 (d, J =
9.6 Hz, 1H), 7.08−7.03 (m, 2H), 6.70 (d, J = 9.6 Hz, 1H), 3.60−3.50
(m, 1H), 2.82−2.73 (m, 1H), 2.18−2.09 (m, 2H), 1.92−1.84 (m, 2H),
1.34−1.20 (m, 4H). m/z (ES + APCI)+: 403 [M + H]+.
trans-N-(3-{2-[(3-Fluoropyridin-2-yl)amino]pyrimidin-5-yl}-
imidazo[1,2-b]pyridazin-6-yl)cyclohexane-1,4-diamine 10. Follow-
ing the method for 7 using 5 and 2-chloro-3-fluoropyridine, we
obtained 10 as a yellow solid (3% yield). 1H NMR (400 MHz, DMSO-
d6) δ ppm 10.04 (br. s, 1H), 9.16 (s, 2H), 8.25−8.19 (m, 1H), 7.87 (s,
1H), 7.77−7.69 (m, 2H), 7.32−7.23 (m, 1H), 6.99 (d, J = 6.9 Hz,
1H), 6.67 (d, J = 10.5 Hz, 1H), 3.57−3.44 (m, 1H), 2.65−2.56 (m,
1H), 2.14−2.03 (m, 2H), 1.86−1.76 (m, 2H), 1.31−1.08 (m, 4H). m/
z (ES + APCI)+: 420 [M + H]+.
trans-N-[3-(2-{[3-Fluoro-5-(trifluoromethyl)pyridin-2-yl]amino}-
pyrimidin-5-yl)imidazo[1,2-b]pyridazin-6-yl]cyclohexane-1,4-dia-
mine 11. Following the method for 7 using 2-bromo-3-fluoro-5-
(trifluoromethyl)pyridine, we obtained 11 as a yellow solid (14%
1
yield). H NMR (400 MHz, DMSO-d6) δ ppm 9.26 (s, 2H), 8.61 (s,
1H), 8.25 (dd, J = 10.5, 1.8 Hz, 1H), 7.94 (s, 1H), 7.75 (d, J = 9.6 Hz,
1H), 7.03 (d, J = 6.9 Hz, 1H), 6.68 (d, J = 9.6 Hz, 1H), 3.56−3.46 (m,
1H), 2.65−2.57 (m, 1H), 2.13−2.05 (m, 2H), 1.86−1.78 (m, 2H),
1.31−1.09 (m, 4H). m/z (ES + APCI)+: 488 [M + H]+.
trans-N-(3-{2-[(5-Methylpyridin-2-yl)amino]pyrimidin-5-yl}-
imidazo[1,2-b]pyridazin-6-yl)cyclohexane-1,4-diamine 12. Follow-
ing the method for 7 using 2-chloro-5-methylpyridine, we obtained 12
1
as an off-white solid (5% yield). H NMR (400 MHz, DMSO-d6) δ
ppm 9.92 (s, 1H), 9.26 (s, 2H), 8.17 (d, J = 8.7 Hz, 1H), 8.14−8.11
(m, 1H), 7.92 (s, 1H), 7.75 (d, J = 9.6 Hz, 1H), 7.60 (dd, J = 2.3, 8.7
Hz, 1H), 7.00 (d, J = 6.9 Hz, 1H), 6.67 (d, J = 9.6 Hz, 1H), 3.57−3.47
3579
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tert-Butyl 4-{[3-(2-aminopyrimidin-5-yl)imidazo[1,2-b]pyridazin-
(d, J = 2.3 Hz, 1H), 8.41 (d, J = 9.2 Hz, 1H), 8.32 (d, J = 2.3 Hz, 1H),
7.75 (d, J = 9.2 Hz, 1H), 2.65 (s, 3H). b) To a solution of 6-
methylimidazo[1,2-b]pyridazine (4.0 g, 30.0 mmol, 1.0 equiv) in
MeCN (40 mL) was added N-bromosuccinimide (5.9 g, 33.0 mmol,
1.1 equiv) portionwise. The reaction mixture was stirred overnight at
rt, then concentrated under reduced pressure, dissolved in CH2Cl2
(200 mL), and washed with deionized water (3 × 200 mL). The
organic phase was separated and dried (MgSO4), and purification by
column chromatography (0−10% MeOH in CH2Cl2) gave 3-bromo-6-
6-yl]amino}piperidine-1-carboxylate 18. Following the procedure for
1
5 using 17, we obtained 18 as a beige solid (100% yield). H NMR
(400 MHz, DMSO-d6) δ ppm 8.95 (s, 2H), 7.79 (s, 1H), 7.74 (d, J =
9.6 Hz, 1H), 7.06 (d, J = 6.4 Hz, 1H), 6.85 (s, 2H), 6.65 (d, J = 9.6 Hz,
1H), 3.93−3.83 (m, 2H), 3.80−3.71 (m, 1H), 3.01−2.88 (m, 2H),
2.06−2.00 (m, 2H), 1.42 (s, 9H), 1.39−1.31 (m, 2H). m/z (ES +
APCI)+: 411 [M + H]+.
3-{2-[(3-Fluoropyridin-2-yl)amino]pyrimidin-5-yl}-N-(1-methylpi-
peridin-4-yl)imidazo[1,2-b]pyridazin-6-amine 19. (a) To a mixture
of 18 (100 mg, 0.24 mmol, 1.0 equiv), 2-chloro-3-fluoropyridine (28
mg, 0.30 mmol, 1.2 equiv) and Cs2CO3 (316 mg, 0.97 mmol, 4.0
equiv) in dioxane (3.0 mL) was added Pd(OAc)2 (6 mg, 0.1 equiv)
and Xantphos (16 mg, 0.1 equiv). The reaction mixture was heated in
the microwave at 130 °C for 50 min, diluted with CH2Cl2 (20 mL),
and washed with deionized water (20 mL). The organic was separated
and treated with 4 M HCl/dioxane (2 mL) and methanol (5 mL). The
product was purified by prep-HPLC give a white solid (24 mg, 24%).
1H NMR (400 MHz, DMSO-d6) δ ppm 9.99 (s, 1H), 9.13 (s, 2H),
8.23 (td, J = 4.6, 1.4 Hz, 1H), 7.87 (s, 1H), 7.77 (d, J = 9.6 Hz, 1H),
7.76−7.70 (m, 1H), 7.29 (ddd, J = 8.4, 4.9, 3.7 Hz, 1H), 7.10 (d, J =
6.9 Hz, 1H), 6.69 (d, J = 9.6 Hz, 1H), 3.83−3.61 (m, 1H), 3.13−3.03
(m, 2H), 2.78−2.63 (m, 2H), 2.14−1.94 (m, 2H), 1.59−1.31 (m, 2H).
m/z (ES + APCI)+: 406 [M + H]+. (b) To a solution of 3-{2-[(3-
fluoropyridin-2-yl)amino]pyrimidin-5-yl}-N-(piperidin-4-yl)imidazo-
[1,2-b]pyridazin-6-amine (30 mg, 0.07 mmol, 1.0 equiv) in THF (0.5
mL) was added formaldehyde (37% aq., 6 μL, 0.07 mmol, 1.0 equiv),
AcOH (8 μL, 0.148 mmol, 2.0 equiv), and sodium triacetoxyborohy-
dride (31 mg, 0.015 mmol, 2.0 equiv). The reaction mixture was
stirred for 2 h at rt and concentrated under reduced pressure.
Purification by prep-HPLC gave 19 as an off-white solid (12 mg, 39%).
1H NMR (400 MHz, DMSO-d6) δ ppm 10.01 (d, J = 4.1 Hz, 1H),
1
methylimidazo[1,2-b]pyridazine as a red solid (2.0 g, 31%). H NMR
(400 MHz, DMSO-d6) δ ppm 8.05 (d, J = 9.2 Hz, 1H), 7.83 (s, 1H),
7.21 (d, J = 9.2 Hz, 1H), 2.58 (s, 3H). (c) To a solution of 3-bromo-6-
methylimidazo[1,2-b]pyridazine (2.0 g, 9.43 mmol, 1.0 equiv) in
dioxane (10 mL) was added 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)pyrimidin-2-amine (2.5 g, 11.3 mmol, 1.2 equiv), bis(di-tert-
butyl(4- dimethylaminophenyl) phosphine) dichloropalladium(II)
(332 mg, 0.47 mmol, 0.05 equiv), and 2 M sodium carbonate (18.8
mL, 37.7 mmol, 4.0 equiv). The reaction mixture was heated at 80 °C
for 6 h, concentrated under reduced pressure, and the residue taken up
in CH2Cl2 (100 mL). The organic phase was washed with deionized
water (3 × 100 mL) and dried (MgSO4), and purification by column
chromatography (0−10% MeOH in CH2Cl2) gave 5-(6-
methylimidazo[1,2-b]pyridazin-3-yl)pyrimidin-2-amine as an off-
1
white solid (205 mg, 10%). H NMR (400 MHz, DMSO-d6) δ ppm
8.92 (s, 2H), 8.12−8.00 (m, 2H), 7.16 (d, J = 9.2 Hz, 1H), 6.93 (s,
2H), 2.57 (s, 3H). m/z (ES + APCI)+: 227 [M + H]+. (d) A mixture
of 5-(6-methylimidazo[1,2-b]pyridazin-3-yl)pyrimidin-2-amine (95
mg, 0.42 mmol, 1.0 equiv), 2-chloro-3-fluoropyridine (82 mg, 0.63
mmol, 1.5 equiv), Pd(OAc)2 (19 mg, 0.08 mmol, 0.2 equiv), Xantphos
(49 mg, 0.08 mmol, 0.2 equiv), and Cs2CO3 (547 mg, 1.68 mmol, 4.0
equiv) in dioxane (3 mL) was heated at 120 °C for 2 h. The reaction
mixture was diluted with CH2Cl2 (100 mL) and washed with
deionized water (3 × 100 mL). Purification by column chromatog-
raphy (2−20% MeOH in CH2Cl2) gave 22 as a yellow solid (36 mg,
28%). 1H NMR (400 MHz, DMSO-d6) δ ppm 10.06 (s, 1H), 9.16 (s,
2H), 8.25 (td, J = 1.3, 4.7 Hz, 1H), 8.19 (s, 1H), 8.10 (d, J = 9.2 Hz,
1H), 7.76 (ddd, J = 10.5, 8.2, 1.4 Hz, 1H), 7.31 (ddd, J = 3.9, 4.8, 8.2
Hz, 1H), 7.20 (d, J = 9.6 Hz, 1H), 2.58 (s, 3H). m/z (ES + APCI)+:
322 [M + H]+.
9.15−9.12 (m, 2H), 8.24−8.21 (m, 1H), 7.87 (s, 1H), 7.77−7.69 (m,
2H), 7.29 (ddd, J = 8.1, 4.7, 3.7 Hz, 1H), 7.09−7.03 (m, 1H), 6.69 (d,
J = 9.6 Hz, 1H), 3.64−3.50 (m, 1H), 2.82−2.70 (m, 2H), 2.18 (s, 3H),
2.09−1.90 (m, 4H), 1.54−1.31 (m, 2H). m/z (ES + APCI)+: 420 [M
+ H]+.
tert-Butyl 4-{[3-(6-aminopyridin-3-yl)imidazo[1,2-b]pyridazin-6-
yl]amino}piperidine-1-carboxylate 20. Following the method for 5
using 17 and 2-aminopyridine-5-boronic acid pinacol ester, we
N-(3-Fluoropyridin-2-yl)-5-[6-(piperazin-1-yl)imidazo[1,2-b]-
pyridazin-3-yl]pyrimidin-2-amine 23. (a) Following the method for
17 using tert-butyl piperazine-1-carboxylate, we obtained tert-butyl 4-
(3-bromoimidazo[1,2-b]pyridazin-6-yl)piperazine-1-carboxylate as a
1
obtained 20 as an off-white solid (91% yield). H NMR (400 MHz,
DMSO-d6) δ ppm 8.69 (d, J = 1.8 Hz, 1H), 8.07 (dd, J = 2.3, 8.7 Hz,
1H), 7.74−7.65 (m, 2H), 7.00 (d, J = 6.4 Hz, 1H), 6.61 (d, J = 10.1
Hz, 1H), 6.54 (dd, J = 0.9, 8.7 Hz, 1H), 6.12 (s, 2H), 3.96−3.83 (m,
2H), 3.82−3.68 (m, 1H), 3.04−2.84 (m, 2H), 2.11−1.96 (m, 2H),
1.47−1.27 (m, 11H). m/z (ES + APCI)+: 410 [M + H]+.
1
yellow solid (33% yield). H NMR (400 MHz, DMSO-d6) δ ppm
7.91 (d, J = 10.1 Hz, 1H), 7.62 (s, 1H), 7.24 (d, J = 10.1 Hz, 1H),
3.62−3.42 (m, 8H), 1.43 (s, 9H). m/z (ES + APCI)+: 382/384 [M +
H]+. (b) Following the method for 5 using tert-butyl 4-(3-
bromoimidazo[1,2-b]pyridazin-6-yl)piperazine-1-carboxylate, we ob-
tained tert-butyl 4-[3-(2-aminopyrimidin-5-yl)imidazo[1,2-b]-
pyridazin-6-yl]piperazine-1-carboxylate as a brown solid (902 mg,
77%). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.91 (s, 2H), 7.93 (d, J
= 9.6 Hz, 1H), 7.90 (s, 1H), 7.20 (d, J = 10.1 Hz, 1H), 6.86 (s, 2H),
3.49 (br. s, 8H), 1.42 (s, 9H). m/z (ES + APCI)+: 397 [M + H]+. (c)
To a solution of tert-butyl 4-[3-(2-aminopyrimidin-5-yl)imidazo[1,2-
b]pyridazin-6-yl]piperazine-1-carboxylate (300 mg, 0.76 mmol, 1.0
equiv), 2-chloro-3-fluoropyridine (15 mg, 1.14 mmol, 1.5 equiv), and
Cs2CO3 (990 mg, 3.03 mmol, 4.0 equiv) in dioxane (10 mL) was
added Pd(OAc)2 (50 mg, 0.23 mmol, 0.3 equiv) and Xantphos (130
mg, 0.23 mmol, 0.3 equiv). The reaction mixture was heated at reflux
for 2 h, diluted with CH2Cl2 (100 mL), and washed with deionized
water (3 × 100 mL). The combined organics were dried and
concentrated under reduced pressure. Purification by column
chromatography (2−20% MeOH/DCM) gave 112 mg of Boc-
protected intermediate, which was treated with 4 M HCl in dioxane
(2 mL). The reaction mixture was basified with triethylamine and
concentrated under reduced pressure. Purification by column
chromatography (5−30% MeOH/DCM) gave 23 as an off-white
solid (45 mg, 15%). 1H NMR (400 MHz, DMSO-d6) δ ppm 10.00 (s,
1H), 9.14 (s, 2H), 8.24 (td, J = 4.6, 1.4 Hz, 1H), 8.00 (s, 1H), 7.91 (d,
J = 9.6 Hz, 1H), 7.75 (ddd, J = 10.5, 8.2, 1.4 Hz, 1H), 7.30 (ddd, J =
3-{6-[(3-Fluoropyridin-2-yl)amino]pyridin-3-yl}-N-(piperidin-4-yl)-
imidazo[1,2-b]pyridazin-6-amine 21. Following the method for 7
1
using 20, we obtained an off-white solid (33% yield). H NMR (400
MHz, DMSO-d6) δ ppm 9.30 (br. s, 1H), 9.13−9.01 (m, 1H), 8.42
(dd, J = 2.5, 8.9 Hz, 1H), 8.19−8.09 (m, 1H), 8.09−8.04 (m, 1H),
7.87 (s, 1H), 7.74 (d, J = 9.6 Hz, 1H), 7.65 (ddd, J = 1.4, 8.0, 11.2 Hz,
1H), 7.09−6.94 (m, 2H), 6.68 (d, J = 10.1 Hz, 1H), 3.77−3.59 (m,
1H), 3.06−2.89 (m, 2H), 2.63−2.52 (m, 2H), 2.07−1.87 (m, 2H),
1.43−1.20 (m, 2H). m/z (ES + APCI)+: 405 [M + H]+. This was then
treated following the method for 19b, which gave 21 as a yellow solid
1
(22 mg, 76%). H NMR (400 MHz, DMSO-d6) δ ppm 9.31 (s, 1H),
9.07 (d, J = 2.3 Hz, 1H), 8.45−8.36 (m, 1H), 8.15−8.09 (m, 1H), 8.06
(d, J = 8.2 Hz, 1H), 7.87 (s, 1H), 7.75 (d, J = 9.6 Hz, 1H), 7.65 (ddd, J
= 1.4, 8.0, 11.2 Hz, 1H), 7.07−6.94 (m, 2H), 6.69 (d, J = 9.6 Hz, 1H),
3.60 (d, J = 6.4 Hz, 1H), 2.81−2.70 (m, 2H), 2.19 (s, 3H), 2.12−1.97
(m, 4H), 1.60−1.38 (m, 2H). m/z (ES + APCI)+: 419 [M + H]+.
N-(3-Fluoropyridin-2-yl)-5-(6-methylimidazo[1,2-b]pyridazin-3-
yl)pyrimidin-2-amine 22. (a) To a solution of 3-amino-6-methylpyr-
idazine (5.0 g, 45.8 mmol, 1.0 equiv) in butan-1-ol (10 mL) was added
50% chloroacetaldehyde in water (6.4 mL, 50.4 mmol, 1.1 equiv). The
reaction mixture was heated at reflux for 4 h, then concentrated under
reduced pressure, and purification by column chromatography (0−
10% MeOH in CH2Cl2) gave 6-methylimidazo[1,2-b]pyridazine as a
beige solid (4.2 g, 69%). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.62
3580
dx.doi.org/10.1021/jm500342d | J. Med. Chem. 2014, 57, 3570−3587

Journal of Medicinal Chemistry
Article
8.2, 4.6, 3.7 Hz, 1H), 7.22 (d, J = 10.1 Hz, 1H), 3.47−3.37 (m, 4H),
2.90−2.78 (m, 4H). m/z (ES + APCI)+: 392 [M + H]+.
3-{2-[(3-Fluoropyridin-2-yl)amino]pyrimidin-5-yl}-N-[2-(morpho-
lin-4-yl)ethyl]imidazo[1,2-b]pyridazin-6-amine 27. (a) Following the
method for 17 using 2-(morpholin-4-yl)ethanamine, we obtained 3-
bromo-N-[2-(morpholin-4-yl)ethyl]imidazo[1,2-b]pyridazin-6-amine
N-(3-Fluoropyridin-2-yl)-5-[6-(4-methylpiperazin-1-yl)imidazo-
[1,2-b]pyridazin-3-yl]pyrimidin-2-amine 24. Following the method
for 19b using 23, we obtained 24 as an off-white solid (41% yield). 1H
NMR (400 MHz, DMSO-d6) δ ppm 10.00 (s, 1H), 9.13 (s, 2H), 8.24
(td, J = 4.9, 1.2 Hz, 1H), 8.00 (s, 1H), 7.93 (d, J = 10.1 Hz, 1H), 7.75
(ddd, J = 10.5, 8.0, 1.6 Hz, 1H), 7.30 (ddd, J = 8.1, 4.7, 3.7 Hz, 1H),
7.25 (d, J = 10.1 Hz, 1H), 3.59−3.44 (m, 4H), 2.48−2.39 (m, 4H),
2.22 (s, 3H). m/z (ES + APCI)+: 406 [M + H]+.
1
as a yellow oil (41% yield). H NMR (400 MHz, DMSO-d6) δ ppm
7.69 (d, J = 9.6 Hz, 1H), 7.47 (s, 1H), 7.09 (t, J = 5.5 Hz, 1H), 6.74 (d,
J = 9.6 Hz, 1H), 3.64−3.49 (m, 4H), 3.45−3.36 (m, 2H), 2.54 (t, J =
6.6 Hz, 2H), 2.48−2.39 (m, 4H). m/z (ES + APCI)+: 326/328 [M +
H]+. (b) Following the method for 5 using 3-bromo-N-[2-(morpholin-
4-yl)ethyl]imidazo[1,2-b]pyridazin-6-amine, we obtained 3-(2-amino-
pyrimidin-5-yl)-N-[2-(morpholin-4-yl)ethyl]imidazo[1,2-b]pyridazin-
6-amine as an off-white solid (91% yield). 1H NMR (400 MHz,
DMSO-d6) δ ppm 8.93 (s, 2H), 7.76 (s, 1H), 7.71 (d, J = 9.6 Hz, 1H),
7.03 (t, J = 5.3 Hz, 1H), 6.83 (s, 2H), 6.70 (d, J = 9.6 Hz, 1H), 3.58 (t,
J = 4.4 Hz, 4H), 3.43−3.31 (m, 2H), 2.55 (t, J = 6.6 Hz, 2H), 2.47−
2.34 (m, 4H). m/z (ES + APCI)+: 341 [M + H]+. c) Following the
method for 25c using 3-(2-aminopyrimidin-5-yl)-N-[2-(morpholin-4-
yl)ethyl]imidazo[1,2-b]pyridazin-6-amine with purification by prep-
(R,S)-3-{2-[(3-Fluoropyridin-2-yl)amino]pyrimidin-5-yl}-N-(pyrroli-
din-3-yl)imidazo[1,2-b]pyridazin-6-amine 25. (a) Compound 3
(1.00 g, 4.30 mmol, 1.0 equiv), (R,S)-3-amino-1-N-Boc-pyrrolidine
(2.00 g, 2.5 equiv), and NMP (4 mL) were heated at 140 °C for 16 h.
The mixture was diluted with EtOAc (80 mL) and washed with water
(60 mL). The aqueous layer was extracted with EtOAc (30 mL), and
the combined organic layers were dried and concentrated under
reduced pressure. Purification by silica gel chromatography (0.2−3.5%
MeOH/EtOAc) gave (R,S)-tert-butyl 3-[(3-bromoimidazo[1,2-b]-
pyridazin-6-yl)amino]pyrrolidine-1-carboxylate as a brown-yellow
1
HPLC, we obtained 27 as an off-white solid (32% yield). H NMR
(400 MHz, DMSO-d6) δ ppm 9.97 (s, 1H), 9.16 (s, 2H), 8.23 (td, J =
4.6, 1.4 Hz, 1H), 7.88 (s, 1H), 7.79−7.69 (m, 2H), 7.30 (ddd, J = 8.2,
4.6, 3.7 Hz, 1H), 7.09 (t, J = 5.5 Hz, 1H), 6.74 (d, J = 9.6 Hz, 1H),
3.59−3.49 (m, 4H), 3.44−3.34 (m, 2H), 2.54 (t, J = 6.6 Hz, 2H),
2.47−2.35 (m, 4H). m/z (ES + APCI)+: 436 [M + H]+.
1
solid (588 mg, 36%). H NMR (400 MHz, DMSO-d6) δ ppm 7.73
(d, J = 9.6 Hz, 1H), 7.50 (s, 1H), 7.45−7.38 (m, 1H), 6.71 (d, J = 9.6
Hz, 1H), 4.31−4.20 (m, 1H), 3.73−3.58 (m, 1H), 3.42−3.13 (m, 3H),
2.21−2.12 (m, 1H), 1.95−1.83 (m, 1H), 1.43−1.36 (m, 9H). (b)
Following the method for 5 using (R,S)-tert-butyl 3-[(3-
bromoimidazo[1,2-b]pyridazin-6-yl)amino]pyrrolidine-1-carboxylate,
we obtained (R,S)-tert-butyl 3-{[3-(2-aminopyrimidin-5-yl)imidazo-
[1,2-b]pyridazin-6-yl]amino}pyrrolidine-1-carboxylate as a pale yellow
solid (272 mg, 45%). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.94 (d,
J = 7.8 Hz, 2H), 7.81−7.73 (m, 2H), 7.35−7.28 (m, 1H), 6.84 (br. s,
2H), 6.66 (d, J = 9.6 Hz, 1H), 4.26−4.19 (m, 1H), 3.68−3.46 (m,
1H), 3.41−3.34 (m, 2H), 3.30−3.18 (m, 1H), 2.21−2.06 (m, 1H),
2.04−1.87 (m, 1H), 1.39 (d, J = 12.8 Hz, 9H). m/z (ES)+: 397 [M +
H]+. (c) (R,S)-tert-Butyl 3-{[3-(2-aminopyrimidin-5-yl)imidazo[1,2-
b]pyridazin-6-yl]amino}pyrrolidine-1-carboxylate (100 mg, 0.25
mmol, 1.0 equiv), 2-chloro-3-fluoropyridine (35 μL, 46 mg, 0.35
mmol, 1.4 equiv), Pd(OAc)2 (11 mg, 0.2 equiv), Xantphos (0.2 equiv),
and Cs2CO3 (326 mg, 1.00 mmol, 4.0 equiv) in dioxane (1.5 mL) were
heated at 100 °C for 8 h. The mixture was allowed to cool and
concentrated under reduced pressure, and purification by silica
chromatography (1−10% MeOH/EtOAc) gave (R,S)-tert-butyl 3-
[(3-{2-[(3-fluoropyridin-2-yl)amino]pyrimidin-5-yl}imidazo[1,2-b]-
pyridazin-6-yl)amino]pyrrolidine-1-carboxylate as a pale yellow solid
3-{2-[(3-Fluoropyridin-2-yl)amino]pyrimidin-5-yl}-N-[2-(4-methyl-
piperazin-1-yl)ethyl]imidazo[1,2-b]pyridazin-6-amine 28. (a) Fol-
lowing the method for 17 using 1-(2-aminoethyl)-4-methylpiperazine,
we obtained 3-bromo-N-[2-(4-methylpiperazin-1-yl)ethyl]imidazo-
[1,2-b]pyridazin-6-amine as a brown solid (40% yield). 1H NMR
(400 MHz, DMSO-d6) δ ppm 7.68 (d, J = 9.6 Hz, 1H), 7.47 (s, 1H),
7.05 (t, J = 5.3 Hz, 1H), 6.74 (d, J = 9.6 Hz, 1H), 3.42−3.34 (m, 2H),
2.57−2.51 (m, 2H), 2.50−2.21 (m, 8H), 2.14 (s, 3H). m/z (ES +
APCI)+: 339/341 [M + H]+. (b) Following the method for 5 using 3-
bromo-N-[2-(4-methylpiperazin-1-yl)ethyl]imidazo[1,2-b]pyridazin-6-
1
amine, we obtained an off-white solid (68% yield). H NMR (400
MHz, DMSO-d6) δ ppm 8.93 (s, 2H), 7.76 (s, 1H), 7.72 (d, J = 10.1
Hz, 1H), 7.29−7.05 (m, 1H), 6.83 (s, 2H), 6.71 (d, J = 9.6 Hz, 1H),
3.45−3.35 (m, 2H), 3.02−2.52 (m, 13H). m/z (ES + APCI)+: 354 [M
+ H]+. (c) Following the method for 25c using 3-(2-aminopyrimidin-
5-yl)-N-[2-(4-methylpiperazin-1-yl)ethyl]imidazo[1,2-b]pyridazin-6-
amine, we obtained 28 after purification by prep-HPLC as an off-white
solid (11% yield). 1H NMR (400 MHz, CD3OD) δ ppm 9.16 (s, 2H),
8.19 (d, J = 4.6 Hz, 1H), 7.79 (s, 1H), 7.68−7.59 (m, 2H), 7.23 (ddd, J
= 8.1, 4.7, 3.7 Hz, 1H), 6.73 (d, J = 9.6 Hz, 1H), 3.51 (t, J = 6.9 Hz,
2H), 2.69−2.63 (m, 2H), 2.72−2.36 (m, 8H), 2.29 (s, 3H). m/z (ES +
APCI)+: 449 [M + H]+.
1
(68 mg, 55%). H NMR (400 MHz, DMSO-d6) δ ppm 9.99 (br. s,
1H), 9.17−9.08 (m, 2H), 8.25−8.20 (m, 1H), 7.90 (s, 1H), 7.81 (d, J
= 10.1 Hz, 1H), 7.77−7.69 (m, 1H), 7.41−7.34 (m, 1H), 7.33−7.24
(m, 1H), 6.72 (d, J = 10.1 Hz, 1H), 4.30−4.20 (m, 1H), 3.71−3.46
(m, 1H), 3.40−3.35 (m, 2H), 3.30−3.15 (m, 1H), 2.20−2.08 (m, 1H),
2.02−1.92 (m, 1H), 1.43−1.31 (m, 9H). m/z (ES)+: 397 [M + H]+.
(d) (R,S)-tert-Butyl 3-[(3-{2-[(3-fluoropyridin-2-yl)amino]pyrimidin-
5-yl}imidazo[1,2-b]pyridazin-6-yl)amino]pyrrolidine-1-carboxylate
(67 mg, 0.14 mmol, 1.0 equiv) was stirred with 4 M HCl/dioxane (1.5
mL) and MeOH (1.5 mL) for 5 h. The mixture was concentrated
under reduced pressure, then dissolved in MeOH/CH2Cl2 and eluted
through an Isolute aminopropyl cartridge (1 g), which gave 25 as a
3-{2-[(3-Fluoropyridin-2-yl)amino]pyrimidin-5-yl}-N-[2-(pyrroli-
din-1-yl)ethyl]imidazo[1,2-b]pyridazin-6-amine 29. (a) 3-Bromo-6-
chloro-imidazo[1,2-b]pyridazine (500 mg, 2.15 mmol, 1.0 equiv), 1,2-
aminoethylpyrrolidine (682 μL, 614 mg, 5.38 mmol, 2.5 equiv), and
NMP (3 mL) were heated at 170 °C under microwave irradiation for
60 min. The mixture was diluted with EtOAc (60 mL) and washed
with water (50 mL). The aqueous layer was basified to pH 10 with 2
M NaOH (aq.), extracted with EtOAc (40 mL), and the combined
organic layers were dried (MgSO4) and concentrated under reduced
pressure. Purification by silica gel chromatography (2−20% MeOH/
EtOAc with 1% conc. aqueous ammonia) gave 3-bromo-N-[2-
(pyrrolidin-1-yl)ethyl]imidazo[1,2-b]pyridazin-6-amine as a yellow
1
beige solid (52 mg, 98%). H NMR (400 MHz, DMSO-d6) δ ppm
9.99 (br. s, 1H), 9.20−9.12 (m, 2H), 8.26−8.20 (m, 1H), 7.89 (s, 1H),
7.78−7.71 (m, 2H), 7.32−7.26 (m, 1H), 7.23 (d, J = 6.0 Hz, 1H), 6.69
(d, J = 9.6 Hz, 1H), 4.16−4.08 (m, 1H), 3.10 (dd, J = 11.4, 6.4 Hz,
1H), 2.95−2.72 (m, 3H), 2.10−2.00 (m, 1H), 1.73−1.63 (m, 1H). m/
z (ES)+: 392 [M + H]+.
(R,S)-3-{2-[(3-Fluoropyridin-2-yl)amino]pyrimidin-5-yl}-N-(1-
methylpyrrolidin-3-yl)imidazo[1,2-b]pyridazin-6-amine 26. Follow-
ing the method for 19b using 25, we obtained 26 as a white solid (37%
yield). 1H NMR (400 MHz, CD3OD) δ ppm 9.15 (s, 2H), 8.19 (d, J =
5.0 Hz, 1H), 7.82 (s, 1H), 7.71−7.59 (m, 2H), 7.26−7.19 (m, 1H),
6.76 (d, J = 9.6 Hz, 1H), 4.46−4.37 (m, 1H), 3.27−3.20 (m, 1H),
3.14−3.03 (m, 1H), 3.01−2.93 (m, 1H), 2.92−2.83 (m, 1H), 2.59 (s,
3H), 2.55−2.42 (m, 1H), 2.03−1.91 (m, 1H). m/z (ES)+: 406 [M +
H]+.
1
solid (450 mg, 68%). H NMR (400 MHz, DMSO-d6) δ ppm 7.67
(d, J = 9.6 Hz, 1H), 7.46 (s, 1H), 7.11 (t, J = 5.3 Hz, 1H), 6.75 (d, J =
9.6 Hz, 1H), 3.42−3.35 (m, 2H), 2.66 (t, J = 6.6 Hz, 2H), 2.54−2.49
(m, 4H), 1.72−1.65 (m, 4H). m/z (ES + APCI)+: 310/312 [M + H]+.
(b) Following the procedure for 5 using 3-bromo-N-[2-(pyrrolidin-1-
yl)ethyl]imidazo[1,2-b]pyridazin-6-amine, we obtained 3-(2-amino-
pyrimidin-5-yl)-N-[2-(pyrrolidin-1-yl)ethyl]imidazo[1,2-b]pyridazin-
6-amine as an off-white solid (91% yield). 1H NMR (400 MHz,
DMSO-d6) δ ppm 8.93 (s, 2H), 7.78−7.72 (m, 2H), 7.15 (br. s, 1H),
6.83 (s, 2H), 6.70 (d, J = 9.6 Hz, 1H), 3.45 (br. s, 2H), 3.09−2.61 (m,
6H), 1.85−1.70 (m, 4H). m/z (ES + APCI)+: 325 [M + H]+. (c)
Following the procedure for 25c using 3-(2-aminopyrimidin-5-yl)-N-
3581
dx.doi.org/10.1021/jm500342d | J. Med. Chem. 2014, 57, 3570−3587

Journal of Medicinal Chemistry
Article
[2-(pyrrolidin-1-yl)ethyl]imidazo[1,2-b]pyridazin-6-amine, we ob-
tained 29 as a pale yellow solid (60% yield). H NMR (400 MHz,
and the reaction mixture was heated at 100 °C for 4 h. The mixture
was cooled to rt, concentrated, and purified by neutral alumina
chromatography (10−40% MeOH/CHCl3) to give 3-(2-amino-
pyrimidin-5-yl)-N-(4-(pyrrolidin-1-yl)-trans-cyclohexyl)imidazo[1,2-
1
DMSO-d6) δ ppm 9.97 (s, 1 H), 9.17 (s, 2H), 8.24 (td, J = 1.4, 5.0 Hz,
1H), 7.89 (s, 1H), 7.77−7.70 (m, 2H), 7.32−7.27 (m, 1H), 7.10 (t, J =
5.5 Hz, 1H), 6.76 (d, J = 9.6 Hz, 1H), 3.41−3.33 (m, 2H), 2.67 (t, J =
6.9 Hz, 2H), 2.48−2.45 (m, 4H), 1.70−1.63 (m, 4H). m/z (ES +
APCI)+: 420 [M + H]+.
1
b]pyridazin-6-amine as an off-white solid (100 mg, 64%). H NMR
(400 MHz, DMSO-d6) δ ppm 8.99 (s, 2H), 7.80 (s, 1H), 6.99 (d, J =
3.2 Hz, 1H), 6.86 (s, 2H), 6.71 (s, 1 H), 6.62 (d, J = 9.6 Hz, 1 H), 3.54
(s, 1H), 2.11−2.01 (m, 5H), 1.67 (s, 4H), 1.33−1.23 (m, 4H), 1.06 (s,
4H). m/z (APCI)+: 379 [M + H]+. (c) A mixture of 3-(2-
aminopyrimidin-5-yl)-N-(4-(pyrrolidin-1-yl)-trans-cyclohexyl)imidazo-
[1,2-b]pyridazin-6-amine (100 mg, 0.26 mmol, 1.0 equiv), 2-chloro-3-
fluoropyridine (52 μL, 69 mg, 0.52 mmol, 2.0 equiv), Xantphos (15
mg, 0.02 mmol, 0.1 equiv), and Cs2CO3 (338 mg, 1.04 mmol, 4.0
equiv) in 1,4-dioxane (5 mL) was degassed using argon for 30 min.
Pd(PPh3)4 (30 mg, 0.02 mmol, 0.1 equiv) was then added and the
mixture further degassed for 15 min. The reaction mixture was heated
at 130 °C in a sealed tube for 6 h, then cooled to rt, concentrated, and
purified by prep-HPLC to give 31 as an off-white solid (50 mg, 40%).
1H NMR (400 MHz, CDCl3) δ ppm 12.45 (s, 1H), 9.14 (s, 2H), 8.29
3-(2-(3-Fluoropyridin-2-ylamino)pyrimidin-5-yl)-N-(4-morpholi-
no-trans-cyclohexyl)imidazo[1,2-b]pyridazin-6-amine 30. (a) To a
stirred solution of trans-N-(3-bromo-imidazo[1,2-b]pyridazin-6-yl)-
cyclohexane-1,4-diamine (1.00 g, 3.24 mmol, 1.0 equiv) in n-BuOH
(15 mL) was added K2CO3 (2.20 g, 15.94 mmol, 5.0 equiv) and
potassium iodide (1.18 g, 7.11 mmol, 2.2 equiv) at 0 °C. After 15 min,
1-chloro-2-(2-chloroethoxy)ethane (925 mg, 6.47 mmol, 2.0 equiv)
was added, and the reaction mixture was heated at 100 °C for 16 h.
The reaction mixture was cooled to rt and filtered. The filtrate was
diluted with dichloromethane (50 mL) and washed with H2O (2 × 20
mL) and a brine solution (20 mL). The organic layer was dried
(Na2SO4) and concentrated, and purification by silica gel chromatog-
raphy (5% MeOH/CHCl3) gave 3-bromo-N-(4-morpholino-trans-
cyclohexyl)imidazo[1,2-b]pyridazin-6-amine as a yellow solid (600 mg,
49%). 1H NMR (400 MHz, CDCl3) δ ppm 7.58 (d, J = 10.0 Hz, 1H),
7.47 (s, 1H), 6.40 (d, J = 9.6 Hz, 1H), 4.27 (d, J = 6.8 Hz, 1H), 3.75−
3.69 (m, 5H), 2.60−2.55 (m, 4H), 2.40−2.20 (m, 3H), 2.05−2.01 (m,
2H), 1.45−1.40 (m, 2H), 1.15−1.10 (m, 2H). m/z (ES)+: 381 [M +
H]+. (b) Following the method for 5 using 3-bromo-N-(4-
morpholino-trans-cyclohexyl)imidazo[1,2-b]pyridazin-6-amine (1.30
g, 3.42 mmol, 1.0 equiv), we obtained 3-(2-aminopyridin-5-yl)-N-(4-
morpholino-trans-cyclohexyl)imidazo[1,2-b]pyridazin-6-amine as an
(d, J = 5.2 Hz, 1H), 7.77−7.70 (m, 2H), 7.49−7.46 (m, 1H), 7.08−
7.03 (m, 1H), 6.48 (d, J = 10.4 Hz, 1H), 4.40 (d, J=6.8 Hz, 1H), 3.72
(m, 2H), 3.08−2.91 (m, 3H), 2.42−2.26 (m, 6H), 2.01−1.85 (m, 4H),
1.43−1.31 (m, 3H). m/z (ES)+: 474 [M + H]+.
N-(3-Fluoropyridin-2-yl)-5-{6-[(1-methylpiperidin-4-yl)oxy]-
imidazo[1,2-b]pyridazin-3-yl}pyrimidin-2-amine 32. (a) To a
solution of 1-methylpiperidin-4-ol (375 mg, 3.2 mmol, 1.5 equiv) in
anhydrous THF (15 mL) was added NaH (60% in mineral oil, 130
mg, 3.2 mmol, 1.5 equiv) at 0 °C. The mixture was allowed to stir at rt
for 30 min, then 3-bromo-6-chloro-imidazo[1,2-b]pyridazine (500 mg,
2.14 mmol, 1.0 equiv) was added and heated to 65 °C for 4 h. The
mixture was allowed to cool, diluted with EtOAc (100 mL), and
washed with water (50 mL). The organic layer was dried (Na2SO4)
and concentrated under reduced pressure, and purification by silica gel
chromatography (5−10% MeOH/CHCl3) gave 3-bromo-6-(1-meth-
ylpiperidin-4-yloxy)imidazo[1,2-b]pyridazine as a pale yellow solid
1
off-white solid (950 mg, 70%). H NMR (400 MHz, DMSO-d6) δ
ppm 8.98 (s, 2H), 7.79 (s, 1H), 7.70 (d, J = 10.0 Hz, 1H), 6.97 (d, J =
6.8 Hz, 1H), 6.86 (s, 2H), 6.62 (d, J = 10.0 Hz, 1H), 3.57−3.49 (m,
4H), 2.50−2.40 (m, 4H), 2.30−2.10 (m, 4H), 2.00−1.90 (m, 2H),
1.40−1.20 (m, 4H). m/z (APCI)+: 395 [M + H]+. (c) A mixture of 3-
(2-aminopyridin-5-yl)-N-(4-morpholino-trans-cyclohexyl)imidazo[1,2-
b]pyridazin-6-amine (150 mg, 0.38 mmol, 1.0 equiv), 2-chloro-3-
fluoropyridine (100 mg, 0.76 mmol, 2.0 equiv), Xantphos (22 mg, 0.04
mmol, 0.1 equiv), and Cs2CO3 (496 mg, 1.52 mmol, 4.0 equiv) in 1,4-
dioxane (8 mL) was degassed using argon for 45 min. Pd(PPh3)4 (44
mg, 0.04 mmol, 0.1 equiv) was added and the mixture further degassed
for 45 min. The reaction mixture was heated at 130 °C in a sealed tube
for 6 h then cooled to rt, H2O was added (30 mL) and extracted with
20% MeOH/CHCl3 (2 × 20 mL). The combined organic extracts
were washed with brine solution (20 mL), dried (Na2SO4), and
concentrated. The crude compound was purified by neutral alumina
chromatography (2% MeOH/CHCl3) to give 30 as a pale yellow solid
1
(100 mg, 15%). H NMR (400 MHz, DMSO-d6) δ ppm 8.04 (d, J =
10.0 Hz, 1H), 7.74 (s, 1H), 6.93 (d, J = 9.6 Hz, 1H), 5.04−5.00 (m,
1H), 2.75−2.65 (m, 2H), 2.32−2.24 (m, 5H), 2.15−2.05 (m, 2H),
1.85−1.75 (m, 2H). m/z (APCI)+: 311/313 [M + H]+. (b) Following
the method for 5 using 3-bromo-6-(1-methylpiperidin-4-yloxy)-
imidazo[1,2-b]pyridazine, we obtained 5-(6-(1-methylpiperidin-4-
yloxy)imidazo[1,2-b]pyridazin-3-yl)pyrimidin-2-amine as a pale yellow
1
solid (44% yield). H NMR (400 MHz, DMSO-d6) δ ppm 8.93 (s,
2H), 8.06−8.01 (m, 2H), 6.99 (s, 2H), 6.86 (d, J = 9.6 Hz, 1H), 4.95−
4.85 (m, 1H), 2.75−2.65 (m, 2H), 2.19−2.08 (m, 7H), 1.79−1.74 (m,
2H). m/z (APCI)+: 326 [M + H]+. (c) Following the method for 30c
using 5-(6-(1-methylpiperidin-4-yloxy)imidazo[1,2-b]pyridazin-3-yl)-
pyrimidin-2-amine, we obtained 32 as a pale yellow solid (42%
yield). 1H NMR (400 MHz, CDCl3) δ ppm 9.16 (s, 2H), 8.30 (d, J =
4.4 Hz, 1H), 7.87 (d, J = 8.2 Hz, 2H), 7.72 (s, 1H), 7.49−7.44 (m,
1H), 7.08−7.04 (m, 1H), 6.75 (d, J = 10.0 Hz, 1H), 5.10−5.00 (m,
1H), 2.70−2.65 (m, 2H), 2.45−2.35 (m, 2H), 2.33 (s, 3H), 2.13−2.10
(m, 2H), 1.94−1.92 (m, 2H). m/z (APCI)+: 421 [M + H]+.
1
(120 mg, 64%). H NMR (400 MHz, DMSO-d6) δ ppm 10.02 (s,
1H), 9.16 (s, 2H), 8.22 (d, J = 4.4 Hz, 1H), 7.88 (s, 1 H), 7.73−7.67
(m, 2H), 7.31−7.27 (m, 1H), 7.06−7.04 (m, 1H), 6.66 (d, J = 10.0
Hz, 1H), 3.72−3.40 (m, 5H), 2.50−2.40 (m, 4H), 2.30−2.10 (m, 3H),
2.01−1.80 (m, 2H), 1.40−1.20 (m, 4H). m/z (ES)+: 490 [M + H]+.
3-(2-(3-Fluoropyridin-2-ylamino)pyrimidin-5-yl)-N-(4-(pyrrolidin-
1-yl)-trans-cyclohexyl) imidazo [1,2-b] pyridazin-6-amine 31. (a)
To a solution of 3 (500 mg, 1.61 mmol 1.0 equiv) and 1,4-
dibromobutane (291 μL, 524 mg, 1.5 equiv) in anhydrous DMF (4
mL) was added K2CO3 (670 mg, 4.85 mmol, 3.0 equiv), and the
reaction mixture was heated at 70 °C for 4 h. The reaction mixture was
cooled to rt and concentrated, and purification by neutral alumina
chromatography (10% MeOH/CHCl3) gave 3-bromo-N-4-(pyrroli-
din-1-yl)-trans-cyclohexyl)imidazo[1,2-b]pyridazin-6-amine as an off-
white solid (150 mg, 25%). 1H NMR (400 MHz, CDCl3) δ ppm 7.58
(d, J = 9.6 Hz, 1H), 7.47 (s, 1H), 6.41 (d, J = 10.0 Hz, 1H), 4.29 (d, J
= 6.8 Hz, 1H), 3.77−3.71 (m, 1H), 2.62 (s, 4H), 2.27−2.24 (m, 2H),
2.12−2.08 (m, 3H), 1.8 (s, 4H), 1.52−1.46 (m, 2H), 1.29−1.23 (m,
2H). m/z (APCI)+: 364 [M + H]+. (b) A mixture of 3-bromo-N-(4-
(pyrrolidin-1-yl)-trans-cyclohexyl)imidazo[1,2-b]pyridazin-6-amine
(150 mg, 0.41 mmol, 1.0 equiv), 2-aminopyrimidine-5-boronic acid
pinacol ester (136 mg, 0.61 mmol, 1.5 equiv), Na2CO3 (175 mg, 1.65
mmol, 4.0 equiv) in DMF (5 mL), and water (1 mL) was degassed
using argon for 30 min. (Aphos)2PdCl2 (30 mg, 5 mol %) was added,
(R,S)-N-(3-Fluoropyridin-2-yl)-5-{6-[(1-methylpyrrolidin-3-yl)oxy]-
imidazo[1,2-b]pyridazin-3-yl}pyrimidin-2-amine 33. (a) Following
the method for 32a using (R,S)-1-methylpyrrolidin-3-ol, we obtained
(R,S)-3-bromo-6-[(1-methylpyrrolidin-3-yl)oxy]imidazo[1,2-b]-
1
pyridazine as a pale yellow solid (1.4 g, crude). H NMR (400 MHz,
CDCl3) δ ppm 7.74 (d, J = 9.6 Hz, 1H), 7.59 (s, 1H), 6.73 (d, J = 8.0
Hz, 1H), 5.48−5.44 (m, 1H), 2.97−2.89 (m, 2H), 2.82−2.78 (m, 1H),
2.52−2.45 (m, 1H), 2.42 (s, 3H), 2.40−2.34 (m, 1H), 2.10−2.04 (m,
1H). m/z (APCI)+: 297/299 [M + H]+. (b) Following the method for
5 using (R,S)-3-bromo-6-[(1-methylpyrrolidin-3-yl)oxy]imidazo[1,2-
b]pyridazine, we obtained (R,S)-5-(6-(1-methylpyrrolidin-3-yloxy)-
imidazo[1,2-b]pyridazin-3-yl)pyrimidin-2-amine as a pale yellow
solid (39%). 1H NMR (400 MHz, CDCl3) δ ppm 8.93 (s, 2H),
7.84 (d, J = 9.2 Hz, 1H), 7.81 (s, 1H), 6.75 (d, J = 8.0 Hz, 1H), 5.40−
5.35 (m, 1H), 5.22 (br. s, 2H), 3.05−2.97 (m, 2H), 2.70−2.65 (m,
1H), 2.42 (s, 3H), 2.35−2.30 (m, 1H), 2.11−2.03 (m, 2H). m/z
(APCI)+: 312 [M + H]+. (c) Following the method for 30c using
3582
dx.doi.org/10.1021/jm500342d | J. Med. Chem. 2014, 57, 3570−3587

Journal of Medicinal Chemistry
Article
(R,S)-5-(6-(1-methylpyrrolidin-3-yloxy)imidazo[1,2-b]pyridazin-3-yl)-
pyrimidin-2-amine, we obtained 33 as a pale yellow solid (90 mg,
53%). H NMR (400 MHz, CDCl3) δ ppm 9.18 (s, 2H), 8.30 (d, J =
4.4 Hz, 1H), 7.88 (s, 1H), 7.86 (d, J = 9.6 Hz, 1H), 7.74 (s, 1H), 7.47
(t, J = 8.0 Hz, 1H), 7.07 (m, 1H), 6.78 (d, J = 8.0 Hz, 1H), 5.45−5.40
(m, 1H), 3.04−2.94 (m, 2H), 2.70−2.65 (m, 1H), 2.51−2.43 (m, 1H),
2.41 (s, 3H), 2.36−2.27 (m, 1H), 2.11−2.04 (m, 1H). m/z (APCI)+:
325 [M + H]+.
+ APCI)+: 405 [M + H]+. (f) Following the procedure for 19b, we
obtained 34 as a yellow solid (34 mg, 65%). H NMR (400 MHz,
CD3OD) δ ppm 9.16 (s, 2H), 8.25−8.17 (m, 1H), 8.10 (s, 1H), 8.01
(d, J = 9.1 Hz, 1H), 7.66 (ddd, J = 10.1, 8.2, 1.0 Hz, 1H), 7.30−7.21
(m, 2H), 3.48 (m, 2H) 3.09−2.97 (m, 2H), 2.94 (d, J = 6.8 Hz, 2H),
2.84 (s, 3H), 2.31−2.13 (m, 1H), 2.02 (m, 2H), 1.71−1.53 (m, 2H).
m/z (ES + APCI)+: 419 [M + H]+.
1
1
3-{4-[(3-Fluoropyridin-2-yl)amino]phenyl}-N-(1-methylpiperidin-
4-yl)imidazo[1,2-b]pyridazin-6-amine 35. (a) Following the method
for 5 using 17 and 4-aminophenylboronic acid pinacol ester, we
obtained tert-butyl 4-{[3-(4-aminophenyl)imidazo[1,2-b]pyridazin-6-
N-(3-Fluoropyridin-2-yl)-5-{6-[(1-methylpiperidin-4-yl)methyl]-
imidazo[1,2-b]pyridazin-3-yl}pyrimidin-2-amine 34. (a) To a
solution of 1-Boc-4-methylene-piperidine (1.29 g, 6.5 mmol, 2.0
equiv) in dry THF (10 mL) under nitrogen was added 9-BBN (0.5 M
in THF, 16.3 mL, 8.16 mmol, 2.5 equiv). The reaction mixture was
heated at 75 °C for 3 h. After cooling, the resulting solution was added
to a mixture of 6-chloroimidazo[1,2-b]pyridazine (0.5 g, 3.2 mmol),
Pd(dppf)Cl2 (133 mg, 0.16 mmol, 0.05 equiv), and K2CO3 (1.35 g, 9.8
mmol, 3.0 equiv) in DMF (5 mL) and water (1 mL). The reaction
mixture was heated at 75 °C for 16 h then concentrated under reduced
pressure. The residue was dissolved in EtOAc and washed with water
(50 mL) and brine (20 mL). The organics were dried over MgSO4 and
concentrated under reduced pressure. Purification by chromatography
on silica gel gave tert-butyl 4-(imidazo[1,2-b]pyridazin-6-ylmethyl)-
piperidine-1-carboxylate as a solid (0.406 g, 40%). 1H NMR (400
MHz, DMSO-d6) δ ppm 8.21 (s, 1H), 8.03 (d, J = 9.1 Hz, 1H), 7.71
(s, 1H), 7.16 (d, J = 9.1 Hz, 1H), 3.91 (m, 2H), 2.79−2.55 (m, 4H),
1.92 (m, 1H), 1.65−1.52 (m, 2H), 1.38 (s, 9H), 1.22−0.99 (m, 2H).
m/z (ES + APCI)+: 317 [M + H]+. (b) To a solution of tert-butyl 4-
(imidazo[1,2-b]pyridazin-6-ylmethyl)piperidine-1-carboxylate (0.40 g,
1.26 mmol) in dry CH2Cl2 (6 mL) was added dropwise a solution of
NBS (0.246 g, 1.39 mmol, 1.1 equiv) in dry acetonitrile (6 mL). The
reaction mixture was stirred at room temperature for 1 h. NBS (24 mg,
0.13 mmol) was added, and the reaction mixture was stirred for a
further hour. The reaction mixture was concentrated under reduced
pressure. The residue was dissolved in CH2Cl2 (100 mL) and washed
with water (2 × 30 mL). The organics were dried over MgSO4 and
concentrated under reduced pressure. Purification by chromatography
on silica gel gave tert-butyl 4-[(3-bromoimidazo[1,2-b]pyridazin-6-
1
yl]amino}piperidine-1-carboxylate as a yellow solid (84% yield). H
NMR (400 MHz, DMSO-d6) δ ppm 7.88−7.78 (m, 2H), 7.68 (d, J =
9.6 Hz, 1H), 7.63 (s, 1H), 6.94 (d, J = 6.4 Hz, 1H), 6.67−6.60 (m,
2H), 6.58 (d, J = 9.6 Hz, 1H), 5.27 (s, 2H), 4.01−3.85 (m, 2H), 3.85−
3.70 (m, 1H), 3.05−2.87 (m, 2H), 2.13−2.00 (m, 2H), 1.48−1.27 (m,
11H). m/z (ES + APCI)+: 409 [M + H]+. (b) Following the method
for 7 using tert-butyl 4-{[3-(4-aminophenyl)imidazo[1,2-b]pyridazin-
6-yl]amino}piperidine-1-carboxylate, we obtained 3-{4-[(3-fluoropyr-
idin-2-yl)amino]phenyl}-N-(piperidin-4-yl)imidazo[1,2-b]pyridazin-6-
amine as an off-white solid (25% yield). 1H NMR (400 MHz, DMSO-
d6) δ ppm 8.97 (d, J = 1.8 Hz, 1H), 8.15−8.06 (m, J = 8.7 Hz, 2H),
8.05−7.98 (m, 1H), 7.92−7.84 (m, J = 8.7 Hz, 2H), 7.80 (s, 1H), 7.72
(d, J = 9.6 Hz, 1H), 7.57 (ddd, J = 1.4, 7.9, 11.8 Hz, 1H), 6.96 (d, J =
6.9 Hz, 1H), 6.83 (ddd, J = 3.2, 4.8, 8.0 Hz, 1H), 6.65 (d, J = 9.6 Hz,
1H), 3.79−3.62 (m, 1H), 3.09−2.96 (m, 2H), 2.66−2.56 (m, 2H),
2.12−1.98 (m, 2H), 1.44−1.25 (m, 2H). m/z (ES + APCI)+: 404 [M
+ H]+. (c) Following the method for 19b using 3-{4-[(3-fluoropyridin-
2-yl)amino]phenyl}-N-(piperidin-4-yl)imidazo[1,2-b]pyridazin-6-
1
amine, we obtained 35 as a yellow solid (70% yield). H NMR (400
MHz, DMSO-d6) δ ppm 9.00 (d, J = 1.8 Hz, 1H), 8.14−8.06 (m, 2H),
8.04−7.97 (m, 1H), 7.94−7.85 (m, 2H), 7.80 (s, 1H), 7.72 (d, J = 9.6
Hz, 1H), 7.62−7.51 (m, 1H), 6.97 (d, J = 6.4 Hz, 1H), 6.84 (ddd, J =
3.4, 4.7, 7.9 Hz, 1H), 6.65 (d, J = 9.6 Hz, 1H), 3.68−3.47 (m, 1H),
2.89−2.74 (m, 2H), 2.20 (s, 3H), 2.12−1.99 (m, 4H), 1.60−1.42 (m,
2H). m/z (ES + APCI)+: 418 [M + H]+.
3-[3-Fluoro-4-(pyridin-2-ylamino)phenyl]-N-(1-methylpiperidin-
4-yl)imidazo[1,2-b]pyridazin-6-amine 36. (a) Following the method
for 5 using 17 and 4-amino-3-fluorophenylboronic acid pinacol ester,
we obtained tert-butyl 4-{[3-(4-amino-3-fluorophenyl)imidazo[1,2-
b]pyridazin-6-yl]amino}piperidine-1-carboxylate as an off-white solid
(56% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 7.98 (dd, J = 2.1,
14.0 Hz, 1H), 7.78 (s, 1H), 7.73 (d, J = 9.6 Hz, 1H), 7.64 (dd, J = 1.8,
8.2 Hz, 1H), 7.08 (d, J = 6.4 Hz, 1H), 6.83 (dd, J = 8.2, 9.6 Hz, 1H),
6.64 (d, J = 9.6 Hz, 1H), 5.35 (br. s, 2H), 4.04−3.88 (m, 2H), 3.84−
3.69 (m, 1H), 3.05−2.85 (m, 2H), 2.14−2.04 (m, 2H), 1.45−1.28 (m,
11H). m/z (ES + APCI)+: 427 [M + H]+. (b) Following the method
for 7 using tert-butyl 4-{[3-(4-amino-3-fluorophenyl)imidazo[1,2-
b]pyridazin-6-yl]amino}piperidine-1-carboxylate and 2-chloropyridine,
we obtained 3-[3-fluoro-4-(pyridin-2-ylamino)phenyl]-N-(piperidin-4-
1
yl)methyl]piperidine-1-carboxylate as a solid (0.42 g, 83%). H NMR
(400 MHz, DMSO-d6) δ ppm 8.09 (d, J = 9.1 Hz, 1H), 7.86 (s, 1H),
7.26 (d, J = 9.1 Hz, 1H), 3.96−3.82 (m, 2H), 2.80 (d, J = 6.9 Hz, 2H),
2.77−2.59 (m, 2H), 1.98−1.87 (m, 1H), 1.64−1.55 (m, 2H), 1.38 (s,
9H), 1.24−1.01 (m, 2H). (c) Following the method for 5, we obtained
tert-butyl 4-{[3-(2-aminopyrimidin-5-yl)imidazo[1,2-b]pyridazin-6-yl]-
1
methyl}piperidine-1-carboxylate as a yellow solid (0.170 g, 70%). H
NMR (400 MHz, DMSO-d6) δ ppm 8.93 (s, 2H), 8.10 (s, 1H), 8.09
(d, J = 9.1 Hz, 1H) 7.18 (d, J = 9.1 Hz, 1 H), 6.93 (br. s, 2H), 3.95−
3.85 (m, 2H), 2.79 (d, J = 7.3 Hz, 2H), 2.75−2.55 (m, 2H), 2.03−1.89
(m, 1H), 1.70−1.58 (m, 2H), 1.38 (s, 9H), 1.21−1.03 (m, 2H). m/z
(ES + APCI)+: 410 [M + H]+. (d) Following the method for 25c, we
obtained tert-butyl 4-[(3-{2-[(3-fluoropyridin-2-yl)amino]pyrimidin-5-
yl}imidazo[1,2-b]pyridazin-6-yl)methyl]piperidine-1-carboxylate as a
yellow solid (0.10 g, 48%). 1H NMR (400 MHz, CD3OD) δ ppm 9.21
(s, 2H). 8.24−8.20 (m, 1H), 8.12 (s, 1H), 8.01 (d, J = 9.6 Hz, 1H),
7.67 (ddd, J = 10.2, 8.4, 1.6 Hz, 1H), 7.31−7.18 (m, 2H), 4.13−4.01
(m, 2H), 2.87 (d, J = 7.3 Hz, 2H), 2.85−2.65 (m, 2H), 2.15−2.05 (m,
1H), 1.80−1.65 (m, 2H), 1.44 (s, 9H), 1.32−1.15 (m, 2H). m/z (ES +
APCI)+: 505 [M + H]+. (e) To a solution of tert-butyl 4-[(3-{2-[(3-
fluoropyridin-2-yl)amino]pyrimidin-5-yl}imidazo[1,2-b]pyridazin-6-
yl)methyl]piperidine-1-carboxylate (0.10 g, 0.19 mmol) in MeOH (2
mL) was added 4 M HCl (2 mL), and the reaction mixture was stirred
at room temperature for 2 h. The reaction mixture was concentrated
under reduced pressure. The residue was purified by prep-LCMS to
give the product as a TFA salt, which was eluted through an Isolute
aminopropyl cartridge to give N-(3-fluoropyridin-2-yl)-5-[6-(piper-
idin-4-ylmethyl)imidazo[1,2-b]pyridazin-3-yl]pyrimidin-2-amine as a
1
yl)imidazo[1,2-b]pyridazin-6-amine as a yellow solid (28% yield). H
NMR (400 MHz, DMSO-d6) δ ppm 8.89−8.75 (m, 1H), 8.36−8.25
(m, 2H), 8.16 (dd, J = 1.4, 5.0 Hz, 1H), 7.90 (s, 1H), 7.84 (dd, J = 1.8,
8.7 Hz, 1H), 7.74 (d, J = 10.1 Hz, 1H), 7.59 (ddd, J = 1.8, 7.0, 8.6 Hz,
1H), 7.08−6.98 (m, 2H), 6.79 (ddd, J = 0.9, 5.5, 6.4 Hz, 1H), 6.67 (d,
J = 9.6 Hz, 1H), 3.77−3.62 (m, 1H), 3.08−2.94 (m, 2H), 2.65−2.54
(m, 2H), 2.16−1.97 (m, 2H), 1.42−1.25 (m, 2H). m/z (ES + APCI)+:
404 [M + H]+. (c) Following the method for 19b using 3-[3-fluoro-4-
(pyridin-2-ylamino)phenyl]-N-(piperidin-4-yl)imidazo[1,2-b]-
1
pyridazin-6-amine, we obtained 36 as a yellow solid (62% yield). H
NMR (400 MHz, DMSO-d6) δ ppm 8.84 (s, 1H), 8.39−8.27 (m, 2H),
8.16 (td, J = 1.4, 5.0 Hz, 1H), 7.91 (s, 1H), 7.82 (dd, J = 1.8, 8.7 Hz,
1H), 7.75 (d, J = 9.6 Hz, 1H), 7.63−7.54 (m, 1H), 7.11−6.99 (m,
2H), 6.79 (ddd, J = 0.9, 5.5, 6.4 Hz, 1H), 6.68 (d, J = 9.6 Hz, 1H),
3.70−3.51 (m, 1H), 2.89−2.75 (m, 2H), 2.19 (s, 3H), 2.16−1.99 (m,
4H), 1.60−1.40 (m, 2H). m/z (ES + APCI)+: 418 [M + H]+.
3-[3,5-Difluoro-4-(pyridin-2-ylamino)phenyl]-N-(1-methylpiperi-
din-4-yl)imidazo[1,2-b]pyridazin-6-amine 37. (a) A solution of 4-
bromo-2,6-difluoroaniline (2.50 g, 12.0 mmol, 1.0 equiv), bis-
(pinacolato)diboron (3.36 g, 13.2 mmol, 1.1 equiv), Pd(dppf)Cl2
1
solid (75 mg, 94%). H NMR (400 MHz, CD3OD) δ ppm 9.20 (s,
2H), 8.24−8.19 (m, 1H), 8.12 (s, 1H), 8.01 (d, J = 9.1 Hz, 1H), 7.67
(ddd, J = 10.5, 8.2, 1.3 Hz, 1H), 7.31−7.21 (m, 2H), 3.13−3.04 (m,
2H), 2.88 (d, J = 7.3 Hz, 2H), 2.67 (td, J = 12.5, 2.7 Hz, 2H), 2.30−
2.07 (m, 1H), 1.77 (m, 2H), 1.34 (qd, J = 12.5, 4.1 Hz, 2H). m/z (ES
3583
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Journal of Medicinal Chemistry
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(150 mg, 0.18 mmol, 15 mol %), and potassium acetate (3.54 g, 36.06
mmol, 3.0 equiv) in DMSO was heated at 80 °C under N2 for 90 min.
The reaction mixture was partitioned between EtOAc (100 mL) and
saturated aqueous bicarbonate (100 mL). The organic layer was
washed with brine (3 × 100 mL), dried over MgSO4, and concentrated
under reduced pressure to give 2,6-difluoro-4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)aniline as a brown solid (3.0 g, 97%). 1H
NMR (400 MHz, DMSO-d6) δ ppm 7.07−6.94 (m, 2H), 5.66 (s, 2H),
1.28−1.17 (m, 12H). m/z (ES + APCI)+: 256 [M + H]+. (b) To a
solution of 1-methylpiperidine-4-amine (11.8 g, 103 mmol, 3.0 equiv)
in NMP (32 mL) was added 3-bromo-6-chloroimidazo[1,2-b]-
pyridazine (8.0 g, 34.0 mmol, 1.0 equiv). The reaction mixture was
heated in a microwave at 180 °C for 35 min. The reaction mixture was
diluted with ethyl acetate (200 mL) and washed with deionized water
(3 × 250 mL). The organic layer was separated, dried (MgSO4),
filtered, and concentrated under reduced pressure. Purification by
chromatography on silica gel (2.5%−25% 2 M NH3 in MeOH/
EtOAc) gave 3-bromo-N-(1-methylpiperidin-4-yl)imidazo[1,2-b]-
pyridazin-6-amine as a pale yellow solid (4.8 g, 45%). 1H NMR
(400 MHz, CD3OD) δ ppm 7.52 (d, J = 9.6 Hz, 1H), 7.37 (s, 1H),
6.67 (d, J = 9.6 Hz, 1H), 3.91−3.66 (m, 1H), 2.97−2.78 (m, 2H), 2.30
(s, 3H), 2.26−2.08 (m, 4H), 1.79−1.72 (m, 2H). m/z (ES + APCI)+:
310 [M + H]+. (c) Following the method for 5 using 3-bromo-N-(1-
methylpiperidin-4-yl)imidazo[1,2-b]pyridazin-6-amine and 2,6-di-
fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline, we ob-
tained 3-(4-amino-3,5-difluorophenyl)-N-(1-methylpiperidin-4-yl)-
imidazo[1,2-b]pyridazin-6-amine as a yellow solid (521 mg, 45%).
1H NMR (400 MHz, DMSO-d6) δ ppm 7.96−7.80 (m, 3H), 7.71 (d, J
1.36−1.13 (m, 5H). m/z (ES + APCI)+: 377 [M + H]+. (b) Following
the method for 25c using 3-(4-aminophenyl)-N-[trans-4-(pyrrolidin-1-
yl)cyclohexyl]imidazo[1,2-b]pyridazin-6-amine, we obtained 39 as an
1
off-white solid (27% yield). H NMR (400 MHz, DMSO-d6) δ ppm
8.99 (d, J = 1.8 Hz, 1H), 8.18−8.06 (m, 2H), 8.00 (d, J = 5.0 Hz, 1H),
7.94−7.85 (m, J = 9.2 Hz, 2H), 7.80 (s, 1H), 7.71 (d, J = 9.6 Hz, 1H),
7.57 (ddd, J = 1.6, 8.1, 11.8 Hz, 1H), 6.94 (d, J = 6.9 Hz, 1H), 6.84
(ddd, J = 3.2, 4.8, 8.0 Hz, 1H), 6.63 (d, J = 9.6 Hz, 1H), 3.66−3.51 (m,
1H), 2.58−2.52 (m, 3H), 2.24−2.11 (m, 2H), 2.10−1.95 (m, 3H),
1.74−1.61 (m, 4H), 1.40−1.20 (m, 5H). m/z (ES + APCI)+: 472 [M
+ H]+.
trans-N-(3-{4-[(3-Fluoropyridin-2-yl)amino]phenyl}imidazo[1,2-
b]pyridazin-6-yl)cyclohexane-1,4-diamine 40. (a) Following the
method for 5 using 4 and 4-aminophenylboronic acid pinacol ester,
we obtained tert-butyl (trans-4-{[3-(4-aminophenyl)imidazo[1,2-b]-
pyridazin-6-yl]amino}cyclohexyl)carbamate as a yellow solid (66%
1
yield). H NMR (400 MHz, DMSO-d6) δ ppm 7.95−7.76 (m, 2H),
7.65 (d, J = 9.6 Hz, 1H), 7.63−7.58 (m, 1H), 6.84 (d, J = 6.9 Hz, 1H),
6.79 (s, 1H), 6.70−6.59 (m, 2H), 6.56 (d, J = 10.1 Hz, 1H), 5.26 (s,
2H), 3.64−3.42 (m, 1H), 3.39−3.13 (m, 1H), 2.24−2.10 (m, 2H),
1.93−1.76 (m, 2H), 1.39 (s, 9H), 1.35−1.17 (m, 4H). m/z (ES +
APCI)+: 423 [M + H]+. (b) Following the method for 7 using tert-
butyl (trans-4-{[3-(4-aminophenyl)imidazo[1,2-b]pyridazin-6-yl]-
amino}cyclohexyl)carbamate, we obtained 40 as a yellow solid (17%
1
yield). H NMR (400 MHz, DMSO-d6) δ ppm 9.00 (d, J = 1.8 Hz,
1H), 8.15−8.07 (m, 2H), 8.05−7.97 (m, 1H), 7.93−7.85 (m, 2H),
7.79 (s, 1H), 7.70 (d, J = 9.6 Hz, 1H), 7.57 (ddd, J = 1.6, 8.1, 11.8 Hz,
1H), 6.89 (d, J = 6.9 Hz, 1H), 6.83 (ddd, J = 3.2, 4.8, 8.0 Hz, 1H), 6.63
(d, J = 9.6 Hz, 1H), 3.61−3.46 (m, 1H), 2.69−2.55 (m, 1H), 2.19−
2.09 (m, 2H), 1.90−1.79 (m, 2H), 1.34−1.10 (m, 4H). m/z (ES +
APCI)+: 418 [M + H]+.
= 9.6 Hz, 1H), 7.05 (d, J = 6.9 Hz, 1H), 6.64 (d, J = 9.6 Hz, 1H), 5.40
(s, 2H), 3.67−3.42 (m, 1H), 2.91−2.76 (m, 2H), 2.19 (s, 3H), 2.15−
1.94 (m, 4H), 1.59−1.34 (m, 2H). m/z (ES + APCI)+: 359 [M + H]+.
(d) Following the method for 25c using 3-(4-amino-3,5-difluor-
ophenyl)-N-(1-methylpiperidin-4-yl)imidazo[1,2-b]pyridazin-6-amine
and 2-chloropyridine, we obtained 37 as a yellow solid (9% yield). 1H
NMR (400 MHz, DMSO-d6) δ ppm 8.54 (s, 1H), 8.07−8.00 (m, 3H),
7.98 (dd, J = 1.4, 5.0 Hz, 1H), 7.75 (d, J = 9.6 Hz, 1H), 7.52 (ddd, J =
1.8, 7.0, 8.6 Hz, 1H), 7.13 (d, J = 6.4 Hz, 1H), 6.77−6.60 (m, 3H),
3.67−3.43 (m, 1H), 2.93−2.69 (m, 2H), 2.16 (br. s, 3H), 2.12−1.92
(m, 4H), 1.61−1.32 (m, 2H). m/z (ES + APCI)+: 436 [M + H]+.
3-{6-[(3-Fluoropyridin-2-yl)amino]pyridin-3-yl}-N-[trans-4-(pyrro-
lidin-1-yl)cyclohexyl]imidazo[1,2-b]pyridazin-6-amine 38. (a) Fol-
lowing the procedure for 5 using 3-bromo-N-4-(pyrrolidin-1-yl)-trans-
cyclohexyl)imidazo[1,2-b]pyridazin-6-amine (500 mg, 1.37 mmol, 1.0
equiv) and 2-aminopyridine-5-boronic acid pinacol ester (452 mg, 2.06
mmol, 1.5 equiv), we obtained 3-(6-aminopyridin-3-yl)-N-[trans-4-
(pyrrolidin-1-yl)cyclohexyl]imidazo[1,2-b]pyridazin-6-amine as a
3-{4-[(5-Chloro-3-fluoropyridin-2-yl)amino]phenyl}-N-(1-methyl-
piperidin-4-yl)imidazo[1,2-b]pyridazin-6-amine 41. (a) Following
the method for 5 using 3-bromo-N-(1-methylpiperidin-4-yl)imidazo-
[1,2-b]pyridazin-6-amine and 4-aminophenylboronic acid pinacol
ester, we obtained 3-(4-aminophenyl)-N-(1-methylpiperidin-4-yl)-
imidazo[1,2-b]pyridazin-6-amine as a yellow solid (42% yield). 1H
NMR (400 MHz, DMSO-d6) δ ppm 7.93−7.79 (m, 2H), 7.66 (d, J =
9.6 Hz, 1H), 7.63 (s, 1H), 6.89 (d, J = 6.4 Hz, 1H), 6.66−6.60 (m,
2H), 6.58 (d, J = 9.6 Hz, 1H), 5.28 (s, 2H), 3.70−3.47 (m, 1H), 2.88−
2.77 (m, 2H), 2.21 (s, 3H), 2.11−1.93 (m, 4H), 1.59−1.35 (m, 2H).
m/z (ES + APCI)+: 323 [M + H]+. (b) Following the method for 25c
using 3-(4-aminophenyl)-N-(1-methylpiperidin-4-yl)imidazo[1,2-b]-
1
pyridazin-6-amine, we obtained 41 as a yellow solid (32% yield). H
NMR (400 MHz, DMSO-d6) δ ppm 9.18 (d, J = 1.8 Hz, 1H), 8.22−
8.02 (m, 3H), 7.97−7.77 (m, 4H), 7.72 (d, J = 9.6 Hz, 1H), 6.97 (d, J
= 6.4 Hz, 1H), 6.65 (d, J = 9.6 Hz, 1H), 3.73−3.51 (m, 1H), 2.91−
2.72 (m, 2H), 2.21 (s, 3H), 2.13−1.95 (m, 4H), 1.61−1.35 (m, 2H).
m/z (ES + APCI)+: 452 [M + H]+.
1
beige solid (468 mg, 90%). H NMR (400 MHz, DMSO-d6) δ ppm
8.76−8.73 (m, 1H), 8.08 (dd, J = 2.3, 8.7 Hz, 1H), 7.70−7.66 (m,
2H), 6.93 (d, J = 6.9 Hz, 1H), 6.59 (d, J = 9.6 Hz, 1H), 6.53 (d, J = 8.7
Hz, 1H), 6.11 (s, 2H), 3.64−3.46 (m, 1H), 2.88−2.61 (m, 3H), 2.21−
2.00 (m, 5H), 1.84−1.63 (m, 4H), 1.44−1.19 (m, 5H). m/z (ES +
APCI)+: 378 [M + H]+. (b) Following the method for 25c using 3-(6-
aminopyridin-3-yl)-N-[trans-4-(pyrrolidin-1-yl)cyclohexyl]imidazo-
[1,2-b]pyridazin-6-amine, we obtained 38 as a pale yellow solid (24%
3-{4-[(3-Fluoro-6-methylpyridin-2-yl)amino]phenyl}-N-(1-methyl-
piperidin-4-yl)imidazo[1,2-b]pyridazin-6-amine 42. Following the
method for 41 using 2-bromo-3-fluoro-6-methylpyridine, we obtained
1
42 as a yellow solid (15% yield). H NMR (400 MHz, DMSO-d6) δ
ppm 8.91 (d, J = 2.3 Hz, 1H), 8.21−8.05 (m, 2H), 8.03−7.89 (m, 2H),
7.82 (s, 1H), 7.72 (d, J = 9.6 Hz, 1H), 7.44 (dd, J = 8.0, 11.7 Hz, 1H),
6.98 (d, J = 6.4 Hz, 1H), 6.76−6.54 (m, 2H), 3.70−3.48 (m, 1H),
2.92−2.77 (m, 2H), 2.39 (s, 3H), 2.20 (s, 3H), 2.15−1.94 (m, 4H),
1.58−1.39 (m, 2H). m/z (ES + APCI)+: 432 [M + H]+.
3-{4-[(3,5-Difluoropyridin-2-yl)amino]phenyl}-N-(1-methylpiperi-
din-4-yl)imidazo[1,2-b]pyridazin-6-amine 43. Following the method
for 41 using 2-bromo-3,5-difluoropyridine, we obtained 43 as a yellow
solid (27% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 9.02 (d, J =
1.8 Hz, 1H), 8.15−8.04 (m, 3H), 7.89−7.78 (m, 4H), 7.72 (d, J = 9.6
Hz, 1H), 6.97 (d, J = 6.4 Hz, 1H), 6.65 (d, J = 9.6 Hz, 1H), 3.68−3.51
(m, 1H), 2.85−2.74 (m, 2H), 2.20 (s, 3H), 2.12−1.98 (m, 4H), 1.56−
1.43 (m, 2H). m/z (ES + APCI)+: 436 [M + H]+.
1
yield). H NMR (400 MHz, DMSO-d6) δ ppm 9.37−9.32 (m, 1H),
9.11 (d, J = 1.8 Hz, 1H), 8.42 (dd, J = 2.3, 8.7 Hz, 1H), 8.11−8.06 (m,
2H), 7.88 (s, 1H), 7.73 (d, J = 9.6 Hz, 1H), 7.67−7.61 (m, 1H), 7.04−
6.96 (m, 2H), 6.66 (d, J = 9.6 Hz, 1H), 3.58 (br. s, 1H), 2.74−2.51 (m,
3H), 2.19−2.08 (m, J = 8.7 Hz, 2H), 2.07−1.96 (m, 3H), 1.72−1.63
(m, 4H), 1.37−1.20 (m, 5H). m/z (ES + APCI)+: 473 [M + H]+.
3-{4-[(3-Fluoropyridin-2-yl)amino]phenyl}-N-[trans-4-(pyrrolidin-
1-yl)cyclohexyl]imidazo[1,2-b]pyridazin-6-amine 39. (a) Following
the method for 5 using 3-bromo-N-[trans-4-(pyrrolidin-1-yl)-
cyclohexyl]imidazo[1,2-b]pyridazin-6-amine and 4-aminophenylbor-
onic acid pinacol ester, we obtained 3-(4-aminophenyl)-N-[trans-4-
(pyrrolidin-1-yl)cyclohexyl]imidazo[1,2-b]pyridazin-6-amine as an off-
white solid (37% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm
7.90−7.81 (m, 2H), 7.69−7.60 (m, 2H), 6.84 (d, J = 6.9 Hz, 1H),
6.67−6.59 (m, 2H), 6.59−6.50 (m, 2H), 5.27 (s, 2H), 2.61−2.52 (m,
3H), 2.23−2.09 (m, 2H), 2.09−1.94 (m, 3H), 1.74−1.62 (m, 4H),
N-(1-Methylpiperidin-4-yl)-3-(4-{[5-(trifluoromethyl)pyridin-2-yl]-
amino}phenyl)imidazo[1,2-b]pyridazin-6-amine 44. Following the
method for 41 using 2-chloro-5-trifluoromethylpyridine, we obtained
1
44 as an off-white solid (7% yield). H NMR (400 MHz, CD3OD) δ
3584
dx.doi.org/10.1021/jm500342d | J. Med. Chem. 2014, 57, 3570−3587

Journal of Medicinal Chemistry
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ppm 8.45−8.40 (m, 1H), 8.13−8.06 (m, 2H), 7.79−7.68 (m, 4H),
7.61 (d, J = 9.6 Hz, 1H), 6.92 (d, J = 8.7 Hz, 1H), 6.68 (d, J = 9.6 Hz,
1H), 3.84−3.74 (m, 1H), 2.99−2.90 (m, 2H), 2.36 (s, 3H), 2.33−2.16
(m, 4H), 1.72−1.57 (m, 2H). m/z (ES + APCI)+: 468 [M + H]+.
3-(4-{[3-Fluoro-6-(trifluoromethyl)pyridin-2-yl]amino}phenyl)-N-
(1-methylpiperidin-4-yl)imidazo[1,2-b]pyridazin-6-amine 45. Fol-
lowing the procedure for 41 using 2-chloro-6-trifluoromethylpyridine,
we obtained 45 as an off-white solid (12% yield). 1H NMR (400 MHz,
CD3OD) δ ppm 8.06−8.01 (m, 2H), 7.91−7.85 (m, 2H), 7.76−7.70
(m, 2H), 7.67 (d, J = 9.6 Hz, 1H), 7.14 (d, J = 7.3 Hz, 1H), 7.03 (d, J
= 8.7 Hz, 1H), 6.70 (d, J = 9.6 Hz, 1H), 4.05−3.95 (m, 1H), 3.57−
3.40 (m, 2H), 3.21−3.01 (m, 2H), 2.83 (s, 3H), 2.53−2.38 (m, 2H),
1.99−1.69 (m, 2H) m/z (ES + APCI)+: 468 [M + H]+.
trans-N-(3-{4-[(3,5-Difluoropyridin-2-yl)amino]phenyl}imidazo-
[1,2-b]pyridazin-6-yl)cyclohexane-1,4-diamine 46. Following the
method for 40 using 2-bromo-3,5-difluoropyridine, we obtained 46
as a yellow solid (31% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm
9.03 (d, J = 1.8 Hz, 1H), 8.16−8.04 (m, 3H), 7.89−7.78 (m, 4H), 7.70
(d, J = 9.6 Hz, 1H), 6.90 (d, J = 6.9 Hz, 1H), 6.62 (d, J = 9.6 Hz, 1H),
3.60−3.48 (m, 1H), 2.68−2.57 (m, 1H), 2.19−2.10 (m, 2H), 1.90−
1.80 (m, 2H), 1.32−1.13 (m, 4H). m/z (ES + APCI)+: 436 [M + H]+.
trans-N-(3-{4-[(5-Chloro-3-fluoropyridin-2-yl)amino]phenyl}-
imidazo[1,2-b]pyridazin-6-yl)cyclohexane-1,4-diamine 47. Follow-
ing the method for 40 using 2,5-dichloro-3-fluoropyridine, we
obtained 47 as a yellow solid (13% yield). 1H NMR (400 MHz,
DMSO-d6) δ ppm 9.19 (d, J = 1.8 Hz, 1H), 8.18−8.05 (m, 3H), 7.90−
7.77 (m, 4H), 7.71 (d, J = 10.1 Hz, 1H), 6.91 (d, J = 6.9 Hz, 1H), 6.63
(d, J = 9.6 Hz, 1H), 3.62−3.43 (m, 1H), 2.72−2.57 (m, 1H), 2.21−
2.08 (m, 2H), 1.91−1.76 (m, 3H), 1.33−1.13 (m, 4H). m/z (ES +
APCI)+: 452 [M + H]+.
identify the fluorescence maxima and minima, and the midpoints of
melting curves were determined by fitting to the Boltzmann equation
(XLfit add-in, IDBS software). Results were expressed as ΔTm values
relative to DMSO controls where ΔTm = Tm (inhibitor) − Tm (DMSO
controls).
P. falciparum in Vitro Parasite Assay. P. falciparum EC50 values
were measured using an in vitro model of malaria parasite growth
which measures merozoite invasion of red blood cells. Test cultures
were set up at 0.5% parasitemia and 2% hematocrit from a
synchronized stock culture of 3D7 P. falciparum. Compounds were
diluted into 2% DMSO and added to parasites 24 h postinvasion using
a 95 μL parasite culture in a 96-well plate and incubated under static
conditions. Compounds were tested at least in duplicate. Cells were
recovered 48 h later and processed for FACS analysis: 50 μL of
parasite culture was transferred into a FACS tube and mixed with 500
μL of 500 μg/mL hydroethidine in PBS to stain parasite DNA. The
parasites were incubated for 20 min at 37 °C, then diluted with 1 mL
of PBS, and stored on ice prior to FACS analysis. The data were
acquired using CellQuest Pro software on a FACSCalibur (Becton
Dickinson). Growth inhibition was calculated using the following
formula: % growth inhibition = (1 − [parasitemia of culture/
parasitemia of control culture]) × 100.
PbCDPK1 Enzyme Assay. To establish activity of the compounds
against recombinant P. berghei CDPK1 enzyme, ATPase activity was
measured using a biosensor sensitive to ADP (rhodamine-labeled
ParM, gift of M. Webb, NIMR). The progress of the reactions was
monitored by an increase in fluorescence corresponding to the
accumulation of ADP using a Pherastar plate reader (BMG Labtech).
Kinase Selectivity. Kinase selectivity profiling was carried out at
the National Centre for Protein Kinase Profiling in the MRC Protein
Phosphorylation Unit at the University of Dundee.
trans-N-(3-{4-[(3-Fluoro-6-methylpyridin-2-yl)amino]phenyl}-
imidazo[1,2-b]pyridazin-6-yl)cyclohexane-1,4-diamine 48. Follow-
ing the method for 40 using 2-bromo-3-fluoro-6-methylpyridine, we
obtained 48 as a yellow solid (16% yield). 1H NMR (400 MHz,
DMSO-d6) δ ppm 8.91 (d, J = 1.8 Hz, 1H), 8.19−8.03 (m, 2H), 8.03−
7.86 (m, 2H), 7.81 (s, 1H), 7.70 (d, J = 9.6 Hz, 1H), 7.43 (dd, J = 7.8,
11.5 Hz, 1H), 6.91 (d, J = 6.4 Hz, 1H), 6.72−6.55 (m, 2H), 3.63−3.46
(m, 1H), 2.70−2.57 (m, 1H), 2.38 (s, 3H), 2.25−2.11 (m, 2H), 1.92−
1.81 (m, 2H), 1.39−1.14 (m, 4H). m/z (ES + APCI)+: 432 [M + H]+.
Pf CDPK1 Enzyme Assay. IC50 determinations were performed
using Kinase Glo (Promega) to measure ATP depletion resulting from
the kinase reaction. Compounds were added to 22 μL reaction
mixtures in white 384-well plates containing 10 nM full length
recombinant Pf CDPK1 and 8 μM MyoA-Tail domain Interacting
Protein (MTIP) in assay buffer (Tris-HCl buffer at pH 8.0 containing
0.1 mM EGTA, 0.2 mM CaCl2, 1 mM DTT, and 0.01% Triton X-100)
and incubated for 30 min at rt prior to initiating the reaction with 10
μM ATP (Km) and 20 mM MgCl2 at final concentrations. Reactions
were allowed to proceed for 120 min at ambient temperature and
stopped by the addition of 22 μL of Kinase Glo Plus detection reagent.
Luminescence proportional to the remaining ATP at the end of the
reaction was measured using a Pherastar plate reader (BMG Labtech).
Ten point dose−response curves were obtained from half-log dilutions
of the test compound diluted in assay buffer at a constant final DMSO
concentration of 1%.
Thermal Denaturation Assay. Purified recombinant PfCDPK1
was diluted to 1 μM in 10 mM HEPES buffer at pH 7.5 containing 150
mM NaCl, 1 mM Ca2+, and 1/1000 SYPRO Orange dye (Invitrogen).
Test compounds were prediluted to 400 μM in 40% v/v DMSO in
water, and 1 μL of diluted compound was added to 39 μL of enzyme/
dye mix in white 96-well quantitative PCR plates (Thermo Scientific)
to give a final compound concentration of 10 μM and preincubated for
30 min at rt. Reference melting temperatures were obtained in parallel
by the addition of 1 μL of 40% v/v DMSO to 39 μL of diluted
PfCDPK1. The plates were sealed with transparent adhesive covers
(Biorad) and subjected to a temperature gradient from 25 to 95 K at a
rate of 1 K/min using a quantitative PCR machine (MX3005P,
Stratagene). Fluorescence data were acquired at 1 min intervals using
the FAM/ROX filter set (Ex 492 nm/Em 610 nm) and the raw data
exported to Excel (Microsoft) for analysis. Data were processed to
P. berghei Murine in Vivo Efficacy Protocol. Plasmodium berghei
ANKA strain expressing GFP26 was used. Mice were NMRI females
(20−22 g). Compounds were solubilized or suspended in a solution
consisting of 70% Tween-80 and 30% ethanol, followed by a 10-fold
dilution in H2O. Chloroquine (Sigma C6628) was used as a control
drug. Test procedure: Day 0, from a donor mouse with approximately
30% parasitemia, heparinized blood (containing 50 μL of 200 u/mL
Heparin) is taken and diluted in physiological saline to 108 parasitized
erythrocytes per mL. Of this suspension, 0.2 mL was injected
intravenously (i.v.) into experimental groups of 3 mice and a control
group of 3 mice. Four hours postinfection, the experimental groups
were treated with a single dose of compound by the oral (p.o.) route.
Days 1−3: 24, 48, and 72 h postinfection, the experimental groups
were treated with a single daily dose of compound p.o. at 50 mg/kg.
Day 4: 24 h after the last drug treatment, 1 μL of tail blood was taken
and suspended in 1 mL of PBS buffer. Parasitemia was determined
with a FACScan (Becton Dickinson) by counting 100′000 red blood
cells. The difference of the mean infection rate of the control group (=
100%) to the test group was calculated and expressed as percent
reduction. The results are expressed as the reduction of parasitemia on
day 4 in % as compared to the untreated control group. As an example,
activity determination with a mean of, e.g., 2% parasitemia in treated
mice and a mean of, e.g., 40% parasitemia in the control animals is
calculated as follows: (40% − 2%)/40% *100 = 95% reduction in
parasitemia.
P. falciparum Murine in Vivo Efficacy Protocol. Described in
ref 24.
In Silico Studies. BLAST was used with the Pf CDPK1 sequence
(UniProt: P62344) to search the PDB to identify suitable templates
for homology modeling. Sequence alignments were generated and
structures inspected using the Schrodinger molecular modeling suite,
2010 version.19 The structure of TgCDPK1 (PDB: 3I7C) was selected
as the most suitable template for a variety of reasons including good
crystallographic quality and high sequence identity, particularly around
the ATP site; of the 49 residues within 6 Å of the crystal ligand, 69%
are identical, and 92% are homologous. Homology modeling was
carried out using Prime,19 and further refinements were not required
due to the excellent correspondence between the target and template.
The resultant structure was prepared for docking studies involving
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solvent removal, valence and charge assignment, addition of hydrogen
atoms, orientation of ambiguous groups (e.g., amide groups of Asn and
Gln), and restrained minimization to relieve residual strain. A docking
grid was generated with size and location based on the 3I7C crystal
ligand, including optional constraints to prominent H-bonding groups
including Tyr148 at the hinge, Glu152 at the entrance to the pocket,
and Asp212 of the DFG-loop. Virtual libraries of compounds were
enumerated and prepared for docking using LigPrep19 then docked
flexibly using GlideSP19 with a H-bonding constraint to Tyr148 N-H,
outputting up to three diverse poses per ligand. High scoring candidate
molecules were prioritized for synthesis following manual inspection
and consideration of additional factors such as favorable/unfavorable
contacts and ligand strain.
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AUTHOR INFORMATION
Corresponding Author
■
*Phone: +44 0 20 8906 7100. Fax: +44 0 20 8906 7200. E-mail:
tim.chapman@tech.mrc.ac.uk.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank David Tickle and Sadhia Mahmood at MRCT for in
vitro ADME, David Whalley for in vitro testing against P. berghei
CDPK1, and Munira Grainger at NIMR for the provision of P.
falciparum parasites. We are grateful to the Medicines for
Malaria Venture for providing support for this project,
including Paul Willis, Didier Leroy, and Simon Campbell for
their input, Sergio Wittlin at the Swiss Tropical and Public
Health Institute for conducting P. berghei in vivo efficacy
studies, Sue Charman and Karen White at the Centre for Drug
Candidate Optimisation at Monash University for PK studies,
and GlaxoSmithKline Tres Cantos for running the P. falciparum
SCID mouse model. A.A.H. is funded by the MRC
(U117532067) and the EU FP7 Grant agreement 242095
(EviMalar).
(12) Kugelstadt, D.; Derrer, B.; Kappes, B. Calcium-Dependent
Protein Kinases as Drug Targets. In Drug Discovery in Infectious
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Kugelstadt, D.; Bomke, J.; Domostoj, M.; Schwarz, M. K.; Scheer,
A.; Kappes, B.; Leroy, D. Identification and characterization of novel
small molecules as potent inhibitors of the plasmodial calcium-
dependent protein kinase 1. Biochemistry 2009, 48, 6379−6389.
(14) Lourido, S.; Zhang, C.; Lopez, M. S.; Tang, K.; Barks, J.; Wang,
Q.; Wildman, S. A.; Shokat, K. M.; Sibley, L. D. Optimizing small
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(15) Johnson, S. M.; Murphy, R. C.; Geiger, J. A.; DeRocher, A. E.;
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(16) Zhang, Z.; Ojo, K. K.; Johnson, S. M.; Larson, E. T.; He, P.;
Geiger, J. A.; Castellanos-Gonzalez, A.; White, A. C.; Parsons, M.;
Merritt, E. A.; Maly, D. J.; Verlinde, C. L. M. J.; Van Voorhis, W. C.;
Fan, E. Benzoylbenzimidazole-based selective inhibitors targeting
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ABBREVIATIONS USED
■
Pf, Plasmodium falciparum; Pb, Plasmodium berghei; CDPK,
calcium-dependent protein kinase; MLM, mouse liver micro-
somes; HLM, human liver microsomes; (A-Phos)2PdCl2,
bis(di-tert-butyl(4-dimethylaminophenyl)phosphine)-
dichloropalladium(II); CyPF-tBu, (dicyclohexylphosphino)-
ferrocenyl]ethyldi-tert-butylphosphine; DIPEA, N,N-diisopro-
pylethylamine; Pd2(dba)3, tris(dibenzylideneacetone)-
dipalladium(0); Pd(dppf)Cl2, 1,1′-bis(diphenylphosphino)-
ferrocenedichloropalladium(II); SCX, strong cation exchange;
Xantphos, 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene;
9-BBN, 9-borabicyclo[3.3.1] nonane
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