Discovery of imidazo[1,2- b ]pyridazine derivatives: Selective and orally available Mps1 (TTK) kinase inhibitors exhibiting remarkable antiproliferative activity

Article abstract of DOI:10.1021/jm501599u

Monopolar spindle 1 (Mps1) is an attractive oncology target due to its high expression level in cancer cells as well as the correlation of its expression levels with histological grades of cancers. An imidazo[1,2-a]pyrazine 10a was identified during an HTS campaign. Although 10a exhibited good biochemical activity, its moderate cellular as well as antiproliferative activities needed to be improved. The cocrystal structure of an analogue of 10a guided our lead optimization to introduce substituents at the 6-position of the scaffold, giving the 6-aryl substituted 21b which had improved cellular activity but no oral bioavailability in rat. Property-based optimization at the 6-position and a scaffold change led to the discovery of the imidazo[1,2-b]pyridazine-based 27f, an extremely potent (cellular Mps1 IC₅₀ = 0.70 nM, A549 IC₅₀ = 6.0 nM), selective Mps1 inhibitor over 192 kinases, which could be orally administered and was active in vivo. This 27f demonstrated remarkable antiproliferative activity in the nanomolar range against various tissue cancer cell lines.

Full text of DOI:10.1021/jm501599u

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Article
Discovery of Imidazo[1,2-b]pyridazine Derivatives:
Selective and Orally Available Mps1 (TTK) Kinase
Inhibitors Exhibiting Remarkable Antiproliferative Activity
Ken-ichi Kusakabe, Nobuyuki Ide, Yataro Daigo, Takeshi Itoh, Takahiko Yamamoto, Hiroshi Hashizume,
Kohei Nozu, Hiroshi Yoshida, Genta Tadano, Sachie Tagashira, Kenichi Higashino, Yousuke Okano, Yuji
Sato, Makiko Inoue, Motofumi Iguchi, Takayuki Kanazawa, Yukichi Ishioka, Keiji Dohi, Yasuto Kido, Shingo
Sakamoto, Shigeru Ando, Masahiro Maeda, Masayo Higaki, Yoshiyasu Baba, and Yusuke Nakamura
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 27 Jan 2015
Downloaded from http://pubs.acs.org on January 28, 2015
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Developmental Research Laboratories
Ando, Shigeru; Shionogi Pharmaceutical Research Center, Medicinal
Research Laboratories
Maeda, Masahiro; Shionogi Pharmaceutical Research Center, Innovative
Drug Discovery Research Laboratories
Higaki, Masayo; Shionogi Pharmaceutical Research Center, Innovative
Drug Discovery Research Laboratories
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Baba, Yoshiyasu; Shionogi Pharmaceutical Research Center, Medicinal
Research Laboratories
Nakamura, Yusuke; The University of Tokyo, Institute of Medical Science
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Discovery of Imidazo[1,2ꢀb]pyridazine Derivatives:
Selective and Orally Available Mps1 (TTK) Kinase
Inhibitors Exhibiting Remarkable Antiproliferative
Activity
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Kenꢀichi Kusakabe, Nobuyuki Ide, Yataro Daigo, Takeshi Itoh, Takahiko Yamamoto,
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Hiroshi Hashizume, Kohei Nozu, Hiroshi Yoshida, Genta Tadano, Sachie Tagashira,
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Kenichi Higashino, Yousuke Okano, Yuji Sato, Makiko Inoue, Motofumi Iguchi, Takayuki
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Kanazawa, Yukichi Ishioka, Keiji Dohi, Yasuto Kido, Shingo Sakamoto, Shigeru Ando,
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Masahiro Maeda, Masayo Higaki, Yoshiyasu Baba, and Yusuke Nakamura
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Medicinal Research Laboratories, Drug Developmental Research Laboratories, and Innovative
Drug Discovery Research Laboratories, Shionogi Pharmaceutical Research Center, 1ꢀ1 Futabaꢀ
cho 3ꢀchome, Toyonaka, Osaka 561ꢀ0825, Japan.
‡
Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The
University of Tokyo, 4ꢀ6ꢀ1 Shirokanedai, Minatoꢀku, Tokyo 108ꢀ8639, Japan.
#
Department of Medical Oncology, Shiga University of Medical Science, Seta Tsukinowaꢀcho,
Otsu, Shiga 520ꢀ2192, Japan.
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KEYWORDS: monopolar spindle 1, Mps1, TTK, kinase, inhibitor, imidazo[1,2ꢀb]pyridazine,
Xꢀray structure, cellular activity, antiproliferative activity, cancer
ABSTRACT.
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Monopolar spindle 1 (Mps1) is an attractive oncology target due to its high expression level in
cancer cells as well as the correlation of its expression levels with histological grades of cancers.
An imidazo[1,2ꢀa]pyrazine 10a was identified during an HTS campaign. Although 10a exhibited
good biochemical activity, its moderate cellular as well as antiproliferative activities needed to be
improved. The cocrystal structure of an analogue of 10a guided our lead optimization to
introduce substituents at the 6ꢀposition of the scaffold, giving the 6ꢀaryl substituted 21b which
had improved cellular activity but no oral bioavailability in rat. Propertyꢀbased optimization at
the 6ꢀposition and a scaffold change led to the discovery of the imidazo[1,2ꢀb]pyridazineꢀbased
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7f, an extremely potent (cellular Mps1 IC = 0.70 nM, A549 IC = 6.0 nM) selective Mps1
50 50
inhibitor over 192 kinases, which could be orally administered and was active in vivo. This 27f
demonstrated remarkable antiproliferative activity in the nanomolar range against various tissue
cancer cell lines.
INTRODUCTION
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Genetic instability is a hallmark of cancer cells. This instability is caused by aneuploidy,
with aberrant genomic structures and abnormal numbers of chromosomes. This state is closely
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linked to chromosomal instability (CIN). While aneuploidy and CIN are considered to be
characteristics of cancer cells, normal cells show high intolerance of aneuploidy and CIN. One
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possible explanation for this is that cancer cells acquire the ability to tolerate aneuploidy or CIN.
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Therefore, elucidating the mechanism of how they acquire this ability could offer hints for
developing therapeutic strategies for cancer treatment.
Monopolar spindle 1 (Mps1), also known as TTK, is a dual specificity protein kinase that
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phosphorylates tyrosine, serine, or threonine residues. Mps1 has essential roles in mitosis
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including centrosome duplication, mitotic checkpoint signaling, and the maintenance of CIN.
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Gabrielson et al. reported four important findings related to Mps1 in breast cancer cells. The
first was that Mps1 expression levels were correlated with the histological grades of breast
cancers. Second, the reduction of Mps1 levels by siRNA in cancer cells decreased their survival
and growth resulting in induction of apoptosis, while there was no significant increase in
apoptosis in Mps1ꢀdepleted nonmalignant cells. Reduced growth of xenografts in mice was also
observed after RNAiꢀmediated decrease in Mps1. The third finding was that reduced Mps1 levels
led to aberrant mitoses in breast cancer cells, while a similar level of Mps1 reduction showed
successful mitosis in nonmalignant cells, indicating that high levels of Mps1 would be required
for the cancer cells to normally progress through mitosis. The fourth finding was that reduced
Mps1 levels due to shRNA led to selective survival of cells with less aneuploidy. It was also
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reported that increased expression of Mps1 was observed in a wide range of cancer cells.
Thus, the evidence observed with breast cancer cells could be applied to other types of cancers.
Taken together, high levels of Mps1 seem to support tolerance of aneuploidy in cancer cells. In
other words, inhibition of Mps1 could lead to selective death of cancer cells with aneuploidy,
which would be a promising strategy in the development of cancer therapeutics.
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Potent and selective Mps1 inhibitors have been discovered.
For example, Nerviano
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Medical Sciences and Myrexis (Myriad) have reported on 1 (NMSꢀP715)
and 2 (MPIꢀ
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0479605),
respectively (Chart 1). Recently, Nerviano disclosed its backup compound of 1 as
NMSꢀP153, which showed improved pharmacokinetic profiles and higher in vivo efficacy,
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although its structure is unknown. Both inhibitors 1 and 2 have pyrimidineꢀbased scaffolds and
were shown to inhibit tumor growth in preclinical cancer models. In our efforts to discover Mps1
inhibitors, we identified diaminopyridineꢀbased compound 3 as a selective inhibitor with in vivo
activity. Its high selectivity was explained by its binding to a flippedꢀpeptide conformation at the
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hinge region. Indazoleꢀbased 4, which was designed based on a panꢀkinase inhibitor
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SP600125, was also disclosed. These Mps1 inhibitors were very potent according to
biochemical assay, while their cellular Mps1 as well as antiproliferative activities in tumor cells
showed their moderate in vivo potency. The purpose of our present research is to explore
selective and orally available Mps1 inhibitors that are extremely potent in cancer cells as well as
cancer xenograft models.
High throughput screening of our kinaseꢀfocused libraries identified imidazo[1,2ꢀa]pyrazine
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10a
as a potent Mps1 inhibitor with a biochemical IC value of 55 nM (Table 1), and this
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was selected as a starting point for our investigation. In parallel with our research, Bayer group
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identified 10a as MpsꢀBAY2b. Further in vitro profiling indicated that 10a showed moderate
activity in cellular Mps1 (cellular Mps1 IC = 276 nM) but lacked antiproliferative activity in
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A549 lung cancer cells (A549 IC = 1112 nM). Therefore, we initiated our medicinal chemistry
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effort to improve the cellular Mps1 as well as the antiproliferative activity of 10a. Herein, we
detail the drug design campaign that led to the discovery of the imidazo[1,2ꢀb]pyridazineꢀbased
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7f, a potent antiproliferative and selective Mps1 inhibitor with in vivo activity.
CHEMISTRY
The syntheses of imidazo[1,2ꢀa]pyrazines 10a−d are illustrated in Scheme 1. Suzuki coupling
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reaction of compound 7 followed by saponification provided carboxylic acid 8, which was then
coupled with amines using HATU followed by oxidation to give 9a−d. Finally, replacement of
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the methylsulfonyl group with isopropyl amine gave compounds 10a−d. Amine derivatives
11a−f were synthesized in a manner similar to 10a (Scheme 2). We introduced various amines to
a reflux condition with 9a to obtain compounds 11a−f.
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To introduce substituents at the 6ꢀposition of the imidazo[1,2ꢀa]pyrazine scaffold, the synthetic
strategy was modified. Starting with compound 12, the 6ꢀsubstituted imidazo[1,2ꢀa]pyrazines
15a and 15b were prepared as shown in Scheme 3. Introduction of the amino group was
accomplished using 4ꢀ(aminomethyl)tetrahydropyran followed by the Suzuki coupling reaction to
obtain compound 14, which was again coupled by the Suzuki reaction with the corresponding
boronic acids to furnish the final compounds 15a and 15b.
The syntheses of imidazo[1,2ꢀb]pyridazines 21a and 21b are illustrated in Scheme 4. Amino
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group addition to 16 followed by protection of tertꢀbutoxycarbonyl (Boc) group afforded 18.
Subsequent iodination of 18 followed by the Suzuki reaction gave the key intermediate 20, which
was then coupled with the corresponding boronic acids and subsequent deprotection to afford
imidazo[1,2ꢀb]pyridazines 21a and 21b.
The syntheses of 6ꢀalkoxy and 6ꢀamino substituted imidazo[1,2ꢀb]pyridazines 24a−e and
27a−f are illustrated in Scheme 5 and 6. Compounds 24a−e were prepared from the Bocꢀ
protected intermediate 18. Sodium alkoxides or phenoxides, prepared using sodium hydride,
were reacted with 18 to afford 22a−e, which was iodinated using iodine followed by the Suzuki
reaction and deprotection of the Boc group to furnish compounds 24a−e. The 6ꢀamino
substituted 27aꢀf were also prepared using intermediate 18 as a starting material (Scheme 6).
Compound 18 was subjected to the BuchwaldꢀHartwig reaction to afford the amines 25a−f.
Conversion of 25a−f to the target compounds 27a−f followed the procedures as described in
Scheme 5.
RESULTS AND DISCUSSION
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SAR of the 3ꢀ and 8ꢀPositions: Identification of Lead 11e. We first examined the effect of
the amide group at the 3ꢀposition of the imidazo[1,2ꢀa]pyrazine scaffold in 10a (Table 1). Even
small changes, such as replacement of the cyclopropyl group with either a cyclobutyl (10b) or an
isopropyl group (10c), resulted in a decrease in activity. The primary amide 10d also led to a 45ꢀ
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fold loss in potency. Along with other data not shown, these results indicated that the
cyclopropyl amide group was optimal for Mps1 activity.
To improve the biochemical Mps1 activity of 10a, a variety of 8ꢀsubstituted imidazo[1,2ꢀ
a]pyrazines were evaluated (Table 2). Branching at the αꢀposition to the nitrogen such as the
isopropyl group (11b) diminished the activity, while a linear chain such as the nꢀpropyl group
was better tolerated (11a). Simple changes to the cyclopropylmethyl group (11d) improved Mps1
potency, indicating that βꢀbranched chains would be important for binding Mps1. Indeed, the
thienylmethyl analogue 11f retained activity relative to 10a, although the benzyl analogue (11c)
resulted in a 2ꢀfold loss in potency. Finally, further exploration of βꢀbranched analogues
identified a promising tetrahydroꢀ2Hꢀpyranylmethyl analogue 11e with an IC value of 35 nM.
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incubation (RLM = 71% remaining) when compared with isobutyl 10a, cyclopropyl 11d, and
thienyl 11f (RLM = 55%, 35%, and 1% remaining, respectively). With good overall properties,
the tetrahydroꢀ2Hꢀpyranylmethyl was chosen for further exploration.
StructureꢀBased Design Using Crystal Structure of Imidazo[1,2ꢀa]pyrazine 11f. To enable
the design of more potent Mps1 inhibitors, we obtained the cocrystal structure of lead analogue
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1f (Figure 1). The binding mode for 11f agrees well with the SAR described above at the 3ꢀ and
8ꢀpositions on the imidazo[1,2ꢀa]pyrazine scaffold. For example, the cyclopropyl amide group
forms suitable van der Waals interactions in the back pocket, also known as the specificity
pocket, explaining why even a slight change such as cyclobutyl 10b and isopropyl 10c could not
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be tolerated at this position (Figure 1A). Additionally, the poor potency of the αꢀbranched
isopropyl 11b can be explained by disruption of the interaction with the hinge region of the
enzyme (Asn606). Finally, the cocrystal structure of 11f with Mps1 suggested that vectors from
the 6ꢀposition of the imidazo[1,2ꢀa]pyrazine would offer the ability to project toward the ribose
binding pocket (Figure 1A). Thus, we hypothesized that introduction of substituents at this
position should provide an opportunity to increase the inhibitory activity of Mps1. As this pocket
is defined by hydrophobic residues such as Ile663 and Pro673, more hydrophobic substituents
would lead to stronger interactions with this pocket.
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Initial SAR at the 6ꢀPosition of 11e. With the structureꢀbased design described above in mind,
we explored substituents at the 6ꢀposition on the imidazo[1,2ꢀa]pyrazine in 11e. In addition to
improving the biochemical activity of Mps1, the initial goal of our SAR was to identify
compounds that were potent in both the cellular Mps1 assay as well as the A549 antiproliferative
assay. To evaluate the cellular inhibition of Mps1, we used an autophosphorylation assay on a
cell line that stably expresses FLAGꢀtagged Mps1 under the control of a tetracyclineꢀsuppressible
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promoter. The antiproliferative activity was measured in the A549 lung carcinoma cell line.
According to the above design, this region was accessed with a focus on hydrophobic
substituents (Table 3). Introduction of the phenyl ring at the 6ꢀposition (15a) led to a 4ꢀfold
increase in biochemical Mps1 IC . Importantly, a significant increase in both cellular Mps1 and
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A549 antiproliferative activity was observed in 15a compared with lead 11e. Further gains in
cellular potency could be found when a cyano group was introduced at the pꢀposition on the
phenyl ring (15b). These results supported the hypothesis that substituents at the 6ꢀposition
occupy the ribose binding pocket and contribute to an increase in activity. Considering the hinge
binding mode of 11f with Mps1 (Figure 1B), the nitrogen at the 1ꢀposition and the amino group
at the 8ꢀposition on imidazo[1,2ꢀa]pyrazine could be expected to be detrimental to the ability to
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bind to the hinge. Therefore, we thought that changing scaffolds retaining these atoms such as
imidazo[1,2ꢀb]pyridazine would maintain the inhibitory activity for Mps1. As expected, changes
from the imidazo[1,2ꢀa]pyrazine to the imidazo[1,2ꢀb]pyridazine (21a) retained both
biochemical and cellular Mps1 activity relative to the corresponding imidazo[1,2ꢀa]pyrazine 15b.
Interestingly, the imidazo[1,2ꢀb]pyridazine 21a showed improved antiproliferative activity (A549
IC = 39 nM) when compared with 15b. Further screening of substituents around 21a revealed
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that incorporation of an amino group at the oꢀposition on the phenyl ring (21b) led to increase in
activity and exhibited a more favorable cellꢀshift.
The pharmacokinetic (PK) properties of 15b, 21a, and 21b were characterized in male
SpragueꢀDawley (SD) rats after intravenous (iv) and oral dosing (Table 6). The imidazo[1,2ꢀ
b]pyridazine 21a showed lower iv clearance than 15a due to its improved microsomal stability
but exhibited no oral bioavailability in rat. Although the compounds with phenyl groups at the 6ꢀ
position such as 15b, 21a, and 21b satisfied the initial goal of identifying compounds in cellular
assay, these compounds displayed poor pharmacokinetic profiles in rat. Further characterization
revealed that these compounds had poor solubility in buffer containing sodium taurocholate at
pH 6.8. Indeed, compounds 15b, 21a, and 21b exhibited thermodynamic solubilities of 1.0
µg/mL, 0.49 µg/mL, and 2.6 µg/mL, respectively, while the rat liver microsomal (RLM) stability
of 6ꢀsubstituted 21a and 21b showed acceptable values (42% and 59% remaining after 30 min
incubation, respectively) resulting in low to moderate clearance in rat (38 and 23 ml/min/kg,
respectively). In contrast, nonꢀsubstituted 15b exhibited high clearance (145 ml/min/kg)
associated with low metabolic stability (0% remaining), suggesting that increasing the solubility
of 6ꢀsubstituted 21a and 21b might improve the bioavailability.
Optimization at the 6ꢀPosition to Obtain 27f. To improve the oral bioavailability of the 6ꢀ
substituted imidazo[1,2ꢀb]pyridazines such as 21a and 21b by increasing solubility, we focused
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on inserting hetroatoms at the 6ꢀposition. Given that this position occupies the lipophilic ribose
binding pocket, designing substituents with a favorable lipophilicity would be important for
striking a balance between cellular activity and oral bioavailability. As shown in Table 4, we first
examined alkoxy analogues with the imidazo[1,2ꢀb]pyridazine scaffold. Phenoxy 24b was
equipotent with pꢀcyanophenyl 21a, while cyclohexyloxy 24a resulted in a decrease in cellular
activity. Addition of the pꢀfluoro (24c) imparted a 2ꢀfold decrease in both cellular Mps1 and
A549 antiproliferative activities; oꢀfluoro 24d retained cellular potency relative to nonꢀ
substituted 24b. Interestingly, incorporation of the oꢀhydroxyl group (24e) gave excellent cellular
Mps1 potency along with the highest antiproliferative activity in A549 cells with an IC value of
0
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2.0 nM.
The selected alkoxy analogues 24a, 24d, and 24e were assessed for the rat PK profile
following the thermodynamic solubility and RLM stability (Table 6). As expected, 24a and 24d
displayed improved solubility and also retained metabolic stability, which translated into
improved rat oral bioavailability (23% and 16%, respectively). However, 24e with the highest
potency had poor solubility and low metabolic stability regardless of its lower lipophilicity
3
6
relative to 24a and 24d (cLogP : 24a: 4.8; 24d: 4.6; 24e: 3.7), resulting in no oral bioavailability
and high clearance in rat. Taken together, incorporation of an oxygen atom at the 6ꢀposition led
to improved cellular potency and oral bioavailability, which provided a good opportunity to
further optimize potency and PK profiles by using other heteroatoms.
Next, we turned our attention to inserting a nitrogen atom at the 6ꢀposition (Table 5). Unlike
the SAR observed with alkoxy analogues 24a and 24b, cyclohexylamino 27b was more potent
than phenylamino 27a. Cyclopentyl 27c was equipotent to cyclohexyl 27b. Truncating the
cyclohexyl group by an isopropyl (27d) resulted in retained biochemical activity but reduced
cellular activity. Interestingly, bulky tertꢀbutylamino 27e showed improvement of both cellular
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Mps1 and A549 antiproliferative activities when compared with isopropyl 27d. Screening a
series of bulky alkylamino groups led to the identification of compound 27f bearing a CF group,
3
which exhibited excellent cellular potency (cellular Mps1 IC = 0.70 nM, A549 IC = 6.0 nM).
5
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The aqueous solubility of the amino analogue 27f was better than that of aryl and alkoxy
analogues, while the in vitro metabolic stability was comparable to these compounds (Table 6).
Consistent with its improved solubility, compound 27f showed favorable oral exposure at a dose
of 1 mg/kg (Cmax = 38 ng/ml, F = 24%). A similar tendency was confirmed with tertꢀbutylamino
27e that had acceptable solubility of 63 µg/ml, which was translated into oral bioavailability (F =
36%).
Crystal Structure of Imidazo[1,2ꢀb]pyridazine 27b Bound to Mps1. To determine the
binding mode of imidazo[1,2ꢀb]pyridazines with substituents at the 6ꢀposition and to test our
design hypothesis, the crystal structure of 27b, an analogue of 27f, bound to Mps1 was obtained
(Figure 2). The binding mode of 27b is nearly consistent with that in 11f (overlay of 11f and 27b
bound to Mps1 is shown in Supporting Information, Figure S1) As expected, the
cyclohexylamino group at the 6ꢀposition occupies the sugar pocket (Figure 2A and B). Like 11f,
the cyclopropyl amide group forms suitable van der Waals interactions in the back pocket (Figure
+
2
A) and also has hydrogen bond interactions with the amine NH3 of Lsy553 and the amide
carbonyl of Ile663 (Figure 2C and D). The imidazo[1,2ꢀb]pyridazine scaffold in 27b maintains
the key hydrogen bond donorꢀacceptor interaction with the hinge region of Mps1 like the
imidazo[1,2ꢀa]pyrazine in 11f (Figure 2C). Consistent with other Mps1 crystal structures, this
was found to adopt an active kinase conformation because we observed the presence of the
Lys553ꢀGlu571 salt bridge and the DFGꢀin conformation. An important feature of the crystal
structure of Mps1 is an antiparallel βꢀsheet between the activation loop and the phosphateꢀ
binding loop (Pꢀloop): Ser553 in the activation loop forms a hydrogen bond donorꢀacceptor
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interaction with Gln672 in the Pꢀloop, which provides the more defined sugar pocket (Figure 2B).
1
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Similar findings were observed for the crystal structures of Mps1 with Mps1ꢀINꢀ2,
2
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28
29
CCT251455, 3, and 4.
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Correlation between Cellular Mps1 and A549 Antiproliferative Activity. As described in
our previous reports, Mps1 inhibitors with diaminopyridine and indazole scaffolds including 3
and 4 showed excellent correlation between cellular Mps1 and A549 antiproliferative
2
8,29
activity.
To confirm this, we analyzed the relationship using the compounds described in
Tables 1−5 (Figure 3). The potent compounds in the cellular assays (21b, 24e, and 27f) are
highlighted in green. Figure 3A shows a correlative plot of biochemical and cellular Mps1 IC50
2
values (R = 0.917), which implies that these compounds had acceptable permeability to possess
cellular activity. Consistent with our previous Mps1 inhibitors, we observed an excellent
2
correlation between cellular Mps1 and A549 IC values (Figure 3B; R = 0.969), indicating that
5
0
improved antiproliferative activity particularly observed with 21b, 24e, and 27f could be
considered responsible for the increased activity of Mps1.
Kinase Selectivity Profiles of 11e, 24e, and 27f. Understanding the offꢀtarget kinase
inhibition is crucial to help interpret the cellular signaling. The cellular active compounds 24e
and 27f were profiled against a panel of 192 kinases at concentrations of 0.2 µM and 1 µM
(Supporting Information, Table S1). For comparison, 11e, which has no substituent at the 6ꢀ
position, was also profiled against a smaller panel of 122 kinases at a concentration of 1 µM.
Compound 27f displayed excellent selectivity over 192 kinases. Although only two kinases
(
CAMK1 and CLK2) were inhibited by more than 50% by 27f at 1 µM, no significant inhibition
over 192 kinases (>50%) was observed at 0.2 µM. Compound 24e also inhibited two kinases
ABL_G250E and CLK2) out of 192. Unlike 27f, CLK2 was inhibited by more than 50% even at
.2 µM, while ABL_G250E was not.
(
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To gain insight into the effects of the substituents at the 6ꢀposition that occupy the ribose
binding pocket, nonꢀsubstituted 11e was profiled over a panel of 122 kinases. Although this
panel did not include CAMK1 and CLK2 (but did include ABL_G250E), 11e showed no
significant inhibition (>50%) over the kinases, indicating that the substituents at the 6ꢀposition in
the ribose binding pocket would not affect the kinase selectivity. This result was consistent with
our previous results observed with indazole derivatives such as 4; the size of substituents in the
ribose binding pocket did not affect the selectivity, while those in the back pocket were important
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for gaining selectivity. According to the Xꢀray structures of 11b and 27b, the cyclopropyl
amide in compounds 11e, 24e, and 27f have excellent van der Waals contacts with the back
pocket. Indeed, slight structural changes such as cyclobutyl 10b and isopropyl 10c were not
tolerated in this region. Therefore, we hypothesized that the high selectivity of these compounds
may arise from the interaction of the cyclopropyl amide with the back pocket.
Antiproliferative Activity of 27f against Various Cancer Cell Lines. To ascertain the effect
of Mps1 inhibition on tumor growth derived from various tissues, the antiproliferative effects of
compound 27f against a panel of 13 tumor cell lines and a nonꢀtransformed cell line MRCꢀ5 were
3
7
examined as shown in Table 7. For comparison, the antiproliferative activities of paclitaxel (28)
3
8
and MLNꢀ8237 (29), an Aurora A kinase inhibitor, are given in the same Table. After 72 h of
treatment, compound 27f suppressed tumor cell proliferation with IC values ranging from 3.3
5
0
to 320 nM regardless of tumor cell types, which was comparable to 28 and more active than 29.
Importantly, a nonꢀtransformed cell line MRCꢀ5 was less sensitive to 27f with an IC value of
5
0
>
10 µM, while treatment of 28 in MRCꢀ5 elicited an antiproliferative effect with an IC value of
50
80 nM. Like 27f, no significant decrease in cell viability was observed when treated with Aurora
kinase inhibitor 29 in MRCꢀ5.
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Tumor Xenograft Study of 27f. On the basis of this promising profile, 27f was further
characterized though a PK study conducted with a preclinical species of nu/nu mice. Consistent
with the rat PK profile, good oral exposure in the mice was observed when dosed at 10 and 100
mg/kg (Table 8). To determine the unbound drug level, the plasma protein binding of 27f was
determined. As a result, the free plasma concentration of 27f at 10 mg/kg was found to be 24 nM
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(fu = 0.51%), which was sufficient to induce an in vivo effect considering the antiproliferative
IC values. Multiple doses of 27f were administered daily for 2 weeks to nude mice bearing
5
0
NCIꢀH460 lung cancer xenografts (n = 6). Based on the above data, the doses chosen were 2.5, 5,
and 10 mg/kg. When dosed at 5 and 10 mg/kg, significant body weight loss and death were
observed (Supporting Information, Figure S2; body weight loss of 25 and 32% relative to
vehicleꢀtreated mice, one and three deaths, respectively). The lowest dose of 2.5 mg/kg induced
tumor growth inhibition (tumor growth inhibition factor T/C = 57%) without significant body
weight loss, and doses of 5 and 10 mg/kg also exhibited T/C values of 40% and 19%,
respectively, associated with toxicity as shown in Figure 4.
23
28
Recently, Mps1 inhibitors 1 and 3 were shown to have significant antitumor activity with
no significant body weight loss. In contrast, administration of compound 2 to mice led to tumor
2
5
growth inhibition associated with significant toxicity (body weight loss and death). As for in
vitro studies, when Mp1ꢀINIꢀ1 was treated with HCT116 and normal colorectal cells, both cell
1
8
lines showed loss of viability, while there was no significant effect in nonmalignant MCF10A
5
cells after reduction of Mps1 levels by siRNA. Thus, contradictory results were reported when
inhibiting the Mps1 function in both in vitro and in vivo studies. Although the cause of the
toxicity observed with 27f is unclear, combination therapy, such as with tubulinꢀtargeting agents,
may provide an opportunity to not only reduce toxicity but also enhance efficacy as discussed in
25,32(c),41
other reports.
The imidazo[1,2ꢀb]pyridazine derivatives disclosed in this report such as
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7f showed one of the highest cellular Mps1 and antiproliferative activities of known Mps1
inhibitors. More importantly, a clear correlation between cellular Mps1 and antiproliferative IC50
was seen in our series, and 27f was selective over 192 kinases, indicating that the
antiproliferative activity may derive from Mps1 inhibition as shown in above. Therefore, these
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selective Mps1 inhibitors, particularly 27f, can serve as important probes for investigating the
biological role and the cancer therapeutic potential of Mps1.
CONCLUSIONS
We identified the selective Mps1 inhibitor 27f with remarkable antiproliferative activity,
starting from an HTS hit 10a with an imidazo[1,2ꢀa]pyrazine scaffold. Introduction of
substituents at the 6ꢀpositon and the change to an imidazo[1,2ꢀb]pyridazine scaffold led to
significant improvement of biochemical and cellular activities, as found with compound 21b.
Further propertyꢀbased design around 21b led to the discovery of imidazo[1,2ꢀb]pyridazine
derivatives with good oral bioavailability and excellent cellular activity. We observed a strong
correlation between cellular Mps1 and A549 IC values, and representative compounds such as
5
0
11e, 24f, and 27f demonstrated excellent kinase selectivity, indicating that the improved
antiproliferative activity observed in our series would be responsible for the increased activity of
Mps1. The in vivo result of 27f showed significant tumor growth inhibition associated with
toxicity. Further investigation on dose regimens and combination drugs such as tubulinꢀtargeting
agents may improve the therapeutic potentials of 27f. Finally, we believe that compound 27f can
contribute to further clarifying the biological roles of Mps1 due to its excellent cellular activity
and kinase selectivity.
EXPERIMENTAL SECTION
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General Chemistry. All commercial reagents and solvents were used as received unless
otherwise noted. Thin layer chromatography (TLC) analysis was performed using Merck silica
gel 60 F254 thin layer plates (250 µm in thickness). Flash column chromatography was carried out
0
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on an automated purification system using Yamazen prepacked silica gel columns. H NMR
spectra were recorded on a Varian Gemini 300 MHz. Spectral data are reported as follows:
chemical shift (as ppm referenced to tetramethylsilane), multiplicity (s = singlet, d = doublet, dd
=
double doublets, dt = double triplet, t = triplet, q = quartet, m = multiplet, br = broad peak),
coupling constant, and integration value. Analytical LC/MS was performed on a Waters Xꢀ
Bridge (C18, 5 µm, 4.6 mm × 50 mm, a linear gradient from 10% to 100% B over 3 min and then
1
00% B for 1 min (A = H O + 0.1% formic acid, B = MeCN + 0.1% formic acid), flow rate 3.0
2
mL/min) using a Waters system equipped with a ZQ2000 mass spectrometer, 2525 binary
gradient module, 2996 photodiode array detector (detection at 254 nm), and 2777 sample
manager. Preparative LC/MS was performed on a Waters XꢀBridge (C18, 5 µm, 19 mm × 50 mm,
a linear gradient from 10% to 100% B over 5 min and then 100% B for 2 min (A = H O + 0.1%
2
formic acid, B = MeCN + 0.1% formic acid), flow rate 25 mL/min) using a Waters system
equipped with a ZQ2000 mass spectrometer, 2525 binary gradient module, 2996 photodiode
array detector (detection at 254 nm), and 2777 sample manager. The purity of all compounds
used in the bioassays was determined by this method to be >95%. High resolution mass spectra
were recorded on a Thermo Fisher Scientific LTQ Orbitrap using electrospray positive ionization.
4ꢀ(8ꢀ(Methylthio)imidazo[1,2ꢀa]pyrazinꢀ3ꢀyl)benzoic acid (8). A mixture of 7 (3.00 g, 12.3
mmol), 4ꢀmethoxycarbonylphenylboronic acid pinacol ester (4.19 g, 16.0 mmol),
PdCl (dppf)·DCM (1.00 g. 1.23 mmol), and Na CO (2 M solution in water, 12.3 mL, 24.6
2
2
3
mmol) in dioxane (60 mL) was heated to reflux for 3 h under a nitrogen atmosphere. The
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reaction mixture was allowed to cool to room temperature and diluted with water and EtOAc.
The aqueous layer was separated and extracted with EtOAc. The combined organic extracts were
washed with water and brine, dried over MgSO , filtered, and concentrated. The residue was
4
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purified by flash chromatography (silica gel; EtOAc/hexane, gradient: 20−60% EtOAc) to afford
1
methyl 4ꢀ(8ꢀ(methylthio)imidazo[1,2ꢀa]pyrazinꢀ3ꢀyl)benzoate (3.03 g, 82%) as a white solid. H
NMR (400 MHz, DMSOꢀd ) δ 2.62 (s, 3H), 3.90 (s, 3H), 7.85 (d, J = 4.5 Hz, 1H), 7.88 (d, J =
6
8.0 Hz, 2H), 8.03 (s, 1H), 8.11 (d, J = 8.0 Hz, 2H), 8.41 (d, J = 4.5 Hz, 1H). MSꢀESI (m/z) = 300
+
[
M+H] . To a solution of this compound (309 mg, 1.03 mmol) in THF/methanol (1:1, 18 mL)
was added aqueous LiOH solution (4 M; 516 µL, 2.06 mmol) at room temperature. The reaction
mixture was stirred overnight and then acidified with aqueous HCl solution (2 M; 1.1 mL). The
organic solvents were evaporated, and the resulting mixture was diluted with EtOAc. The
aqueous layer was separated and extracted with EtOAc. The combined organic extracts were
washed with water and brine, dried over MgSO , filtered, and concentrated to afford 8 (301 mg,
4
1
quant.) as an offꢀwhite solid. H NMR (400 MHz, DMSOꢀd ) δ 2.62 (s, 3H), 7.82−7.89 (m, 4H),
6
8.02 (s, 1H), 8.10 (d, J = 8.0 Hz, 2H), 8.41 (d, J = 4.8 Hz, 1H), 13.16 (br. s., 1H). MSꢀESI (m/z)
+
=
286 [M+H] .
NꢀCyclopropylꢀ4ꢀ(8ꢀ(methylsulfonyl)imidazo[1,2ꢀa]pyrazinꢀ3ꢀyl)benzamide (9a). To a
solution of 8 (100 mg, 0.350 mmol), DIEA (122 µL, 0.701 mmol), and HATU (160 mg, 0.421
mmol) in DCM (2 mL) was added cyclopropylamine (30 µL, 0.421 mmol) at room temperature.
The reaction mixture was stirred for 4 h and then diluted with water and DCM. The aqueous
layer was separated and extracted with DCM. The combined organic extracts were dried over
MgSO , filtered, and concentrated to afford Nꢀcyclopropylꢀ4ꢀ(8ꢀ(methylthio)imidazo[1,2ꢀ
4
1
a]pyrazinꢀ3ꢀyl)benzamide (127 mg) as a pale yellow solid. H NMR (400 MHz, DMSOꢀd ) δ
6
0.57−0.63 (m, 2H), 0.72 (s, 2 H), 2.59−2.65 (m, 3H), 2.84−2.94 (m, 1H), 7.80 (d, J = 8.0 Hz, 2H),
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.84 (d, J = 4.5 Hz, 1H), 7.96−8.04 (m, 3H), 8.37 (d, J = 4.5 Hz, 1H), 8.57 (d, J = 3.8 Hz, 1H).
+
MSꢀESI (m/z) = 325 [M+H] . A mixture of this compound (121 mg, 0.373 mmol) and mꢀCPBA
(69% wt, 280 mg, 1.12 mmol) in DCM (3 mL) was stirred at room temperature for 3 h. The
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reaction mixture was diluted with water and DCM. The aqueous layer was separated and
extracted with DCM. The combined organic extracts were washed with saturated aqueous
NaHCO solution and brine, dried over MgSO , and concentrated. The residue was purified by
3
4
flash chromatography (silica gel; EtOAc/hexane, gradient: 50−100% EtOAc) to afford 9a (110
1
mg, 88%) as a yellow amorphous substance. H NMR (400 MHz, CDCl ) δ 0.65−0.71 (br m, 2H),
3
0.89−0.96 (m, 2H), 2.92−3.00 (br m, 1H), 3.58 (s, 3H), 7.64 (d, J = 7.5 Hz, 2H), 7.96 (d, J = 7.5
Hz, 2H), 8.06 (d, J = 4.4 Hz, 1H), 8.10 (s, 1H), 8.43 (d, J = 4.4 Hz, 1H). MSꢀESI (m/z) = 357
+
[M+H] .
NꢀCyclopropylꢀ4ꢀ(8ꢀ(isobutylamino)imidazo[1,2ꢀa]pyrazinꢀ3ꢀyl)benzamide
(10a).
A
mixture of 9a (62.7 mg, 0.176 mmol) and isobutylamine (87 µL, 0.88 mmol) in dioxane (1.0 mL)
was heated to reflux for 12 h. The reaction mixture was allowed to cool to room temperature and
diluted with water and EtOAc. The aqueous layer was separated and extracted with EtOAc The
combined organic extracts were washed with water and brine, dried over MgSO , and
4
concentrated. The residue was purified by flash chromatography (silica gel; EtOAc/hexane,
1
gradient, 50−90% EtOAc) to afford 10a (58.3 mg, 95%) as a yellow solid. H NMR (400 MHz,
DMSOꢀd ) δ 0.58−0.62 (m, 2H), 0.70−0.74 (m, 2H), 0.91 (d, J = 6.8 Hz, 6H), 2.00−2.07 (m, 1H),
6
2.85−2.92 (m, 1H), 3.29−3.35 (m, 2H), 7.35 (d, J = 4.8 Hz, 1H), 7.57 (t, J = 5.8 Hz, 1H),
7
.74−7.76 (m, 3H), 7.80 (s, 1H), 7.98 (d, J = 8.0 Hz, 2H), 8.54 (d, J = 3.8 Hz, 1H). HRMSꢀESI
+
(m/z): [M+H] calcd for C H N O, 350.1975; found, 350.1975.
20 24
5
6ꢀBromoꢀ3ꢀiodoꢀNꢀ((tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)methyl)imidazo[1,2ꢀa]pyrazinꢀ8ꢀamine
(13). A solution of 12 (1.58 g, 3.92 mmol) and 4ꢀ(aminomethyl)tetrahydropyran (1.34 g, 11.8
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mmol) in DMA (7.9 mL) was heated in microwave reactor at 130 °C for 20 min. The reaction
mixture was diluted with water, and the resulting solid was collected on a glass filter to afford 13
1
(
1.66 g, 97%) as a white solid. H NMR (300 MHz, DMSOꢀd ) δ 1.21 (ddd, J = 24.2, 11.9, 3.8
6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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29
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42
43
44
45
46
47
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53
54
55
56
57
58
59
60
Hz, 2H), 1.59 (d, J = 12.5 Hz, 2H), 1.89−2.00 (br m, 1H), 3.25 (t, J = 11.4 Hz, 2H), 3.83 (d, J =
+
1
0.5 Hz, 2H), 7.60 (s, 1H), 7.64 (s, 1H), 8.15 (t, J = 5.6 Hz, 1H). MSꢀESI (m/z) = 436.9 [M+H] .
4ꢀ(6ꢀBromoꢀ8ꢀ((tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)methylamino)imidazo[1,2ꢀa]pyrazinꢀ3ꢀyl)ꢀNꢀ
cyclopropylbenzamide (14). A mixture of 13 (1.66 g, 3.80 mmol), Pd(PPh ) (220 mg, 0.190
3
4
mmol), Na CO (2 M in water, 3.8 mL), and 4ꢀ(cyclopropylaminocarbonyl)phenylboronic acid
2
3
pinacol ester (1.09 g, 3.80 mmol) was heated in microwave reactor at 130 °C for 20 min. The
reaction mixture was diluted with CHCl and water, and then filtered through celite. The aqueous
3
layer was separated and extracted with CHCl . The combined extracts were washed with water
3
and brine, dried over MgSO , filtered, and concentrated. The resulting solid was collected on a
4
1
glass filter to afford 14 (1.47 g, 82%) as a tan solid. H NMR (400 MHz, DMSOꢀd ) δ 0.58−0.62
6
(m, 2H), 0.72 (q, J = 6.1 Hz, 2H), 1.24 (ddd, J = 24.4, 12.0, 3.8 Hz, 2H), 1.61 (d, J = 12.3 Hz,
2H), 1.91−2.02 (br m, 1H), 2.85−2.91 (m, 1H), 3.27 (t, J = 11.4 Hz, 2H), 3.85 (d, J = 9.8 Hz, 2H),
7.74 (d, J = 8.0 Hz, 2H), 7.80 (br s, 2H), 7.98 (d, J = 8.0 Hz, 2H), 8.16 (t, J = 5.6 Hz, 1H), 8.54
+
(d, J = 3.8 Hz, 1H). MSꢀESI (m/z) = 470 [M+H] .
4
ꢀ(6ꢀ(4ꢀCyanophenyl)ꢀ8ꢀ((tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)methylamino)imidazo[1,2ꢀa]pyrazinꢀ
ꢀyl)ꢀNꢀcyclopropylbenzamide (15b). A mixture of 14 (188 mg, 0.400 mmol), 4ꢀ
cyanophenylboronic acid (58.7 mg, 0.400 mmol), Na CO (2 M in water, 0.40 mL), and
3
2
3
PdCl (PPh ) (14.0 mg, 0.0200 mmol) was heated in microwave reactor at 130 °C for 10 min.
2
3 4
The reaction mixture was diluted with CHCl and water, and then filtered through celite. The
3
aqueous layer was separated and extracted with CHCl . The combined extracts were washed with
3
water and brine, dried over MgSO , filtered, and concentrated. The mixture was purified by
4
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preparative LC/MS to afford 15b (107 mg, 54%) as a tan solid. H NMR (400 MHz, DMSOꢀd )
6
δ 0.58−0.62 (m, 2H), 0.72 (q, J = 6.1 Hz, 2H), 1.30 (ddd, J = 24.3, 12.0, 3.7 Hz, 2H), 1.66 (d, J =
1
2.8 Hz, 2H), 1.98−2.09 (br m, 1H), 2.86−2.93 (m, 1H), 3.27 (t, J = 11.5 Hz, 2H), 3.53 (t, J = 6.3
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
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7
8
9
0
1
2
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Hz, 2H), 3.86 (d, J = 9.8 Hz, 2H), 7.82−7.84 (m, 3H), 7.88−7.91 (m, 3H), 8.01 (d, J = 7.8 Hz,
2
H), 8.22 (d, J = 8.0 Hz, 2H), 8.30 (s, 1H), 8.56 (d, J = 4.0 Hz, 1H). MSꢀESI (m/z) = 493
+
[
M+H] . HRMSꢀESI (m/z): [M+H]+ calcd for C H N O , 493.2347; found, 493.2349.
2
9
29
6
2
6
ꢀChloroꢀNꢀ((tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)methyl)imidazo[1,2ꢀb]pyridazinꢀ8ꢀamine (17). A
mixture of 16 (4.85 g, 15.9 mmol), 4ꢀaminomethyltetrahydropyran (3.11 g, 27 mmol), and DIEA
8.32 mL, 47.6 mmol) in ethanol (40 mL) was heated to reflux for 15 h. The reaction mixture
(
was allowed to cool to room temperature and then diluted with water and EtOAc. The aqueous
layer was separated and extracted with EtOAc. The combined extracts were washed with brine,
dried over MgSO , filtered, and evaporated. The residue was purified by flash column
4
chromatography (silica gel, EtOAc/hexane, gradient: 25−70% EtOAc) to afford 17 (2.99 g, 71%)
1
as a brown solid. H NMR (300 MHz, DMSOꢀd ) δ 1.21 (ddd, J = 24.8, 11.9, 4.4 Hz, 2H), 1.60
6
(
dd, J = 12.7, 1.9 Hz, 2H), 1.87−1.95 (m, 1H), 3.20−3.28 (m, 4H), 3.82 (dd, J = 11.4, 2.7 Hz,
2
H), 6.23 (s, 1H), 7.49 (d, J = 1.2 Hz, 1H), 7.95 (t, J = 5.6 Hz, 1H), 7.97 (d, J = 1.2 Hz, 1H).
+
MSꢀESI (m/z) = 267 [M+H] .
tertꢀButyl
6ꢀchloroimidazo[1,2ꢀb]pyridazinꢀ8ꢀyl((tetrahydroꢀ2Hꢀpyranꢀ4ꢀ
yl)methyl)carbamate (18). A solution of 17 (15.0 g, 56.2 mmol), Boc O (26.5 mL, 124 mmol),
2
and DMAP (0.687 g, 5.62 mmol) in THF (150 mL) was heated at 50 °C for 1 h. The reaction
mixture was allowed to cool to room temperature and then diluted with EtOAc and water. The
aqueous layer was separated and extracted with EtOAc. The combined extracts were washed with
brine, dried over MgSO , filtered, and evaporated. The residue was purified by flash column
4
chromatography (silica gel; EtOAc/hexane, gradient: 10−70% EtOAc) to afford 18 (22.7 g,
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10%) as a yellow oil. H NMR (300 MHz, DMSOꢀd ) δ 1.06−1.20 (m, 2H), 1.35 (s, 9H), 1.54
6
(
d, J = 11.7 Hz, 2H), 1.63−1.72 (m, 1H), 3.15 (t, J = 10.8 Hz, 2H), 3.76 (dd, J = 11.3, 2.9 Hz,
2
H), 3.93 (d, J = 7.3 Hz, 2H), 7.46 (s, 1H), 7.81 (t, J = 0.9 Hz, 1H), 8.32 (d, J = 1.1 Hz, 1H).
10
11
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15
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17
18
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20
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26
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28
29
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
MSꢀESI (m/z) = 367 [M+H] .
tertꢀButyl
6ꢀchloroꢀ3ꢀiodoimidazo[1,2ꢀb]pyridazinꢀ8ꢀyl((tetrahydroꢀ2Hꢀpyranꢀ4ꢀ
yl)methyl)carbamate (19). A solution of 18 (70.9 g, 193 mmol) and NIS (51.4 g, 228 mmol) in
DMF (567 mL) was heated at 80 °C for 9 h. The mixture was allowed to cool to room
temperature and diluted with EtOAc and water. The aqueous layer was separated and extracted
with EtOAc. The combined extracts were washed with water, aqueous Na S O solution, and
2
2
3
brine, dried over MgSO , filtered, and evaporated. The residue was purified by flash
4
chromatography (silica gel; EtOAc/hexane, 15% EtOAc) to afford 19 (83.9 g, 89%) as a pale
1
yellow solid. H NMR (300 MHz, CDCl ) δ 1.28−1.33 (m, 2H), 1.50 (s, 9H), 1.58 (dd, J = 12.8,
3
1
.8 Hz, 2H), 1.74−1.79 (m, 1H), 3.30 (td, J = 11.7, 2.1 Hz, 2H), 3.93 (dd, J = 11.4, 2.5 Hz, 2H),
4.18 (d, J = 7.2 Hz, 2H), 7.19 (s, 1H), 7.80 (s, 1H).
tertꢀButyl
6ꢀchloroꢀ3ꢀ(4ꢀ(cyclopropylcarbamoyl)phenyl)imidazo[1,2ꢀb]pyridazinꢀ8ꢀ
yl((tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)methyl)carbamate (20). A mixture of 19 (71.0 g, 144 mmol), 4ꢀ
(cyclopropylaminocarbonyl)phenylboronic acid pinacol ester (49.7 g, 173 mmol), Pd(PPh3)4
(
8.33 g, 7.20 mmol), and Na CO (21.4 g, 202 mmol) in DMF/H O (426 mL/101 mL) was heated
2 3 2
at 100 °C for 7 h. The reaction mixture was allowed to cool to room temperature and diluted with
EtOAc and water. The aqueous layer was separated and extracted with EtOAc. The combined
extracts were washed with water, dried over MgSO , filtered, and concentrated. The residue was
4
purified by flash column chromatography (silica gel; EtOAc/hexane, gradient: 40−60% EtOAc)
1
to afford 20 (41.7 g, 55%) as a yellow amorphous substance. H NMR (300 MHz, CDCl ) δ
3
0
.63−0.68 (m, 2H), 0.87−0.93 (m, 2H), 1.26−1.37 (m, 2H), 1.50 (s, 9H), 1.59 (d, J = 12.4 Hz,
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H), 1.72−1.83 (m, 1H), 2.90−2.98 (m, 1H), 3.25−3.34 (m, 2H), 3.92 (dd, J = 11.7, 2.8 Hz, 2H),
.18 (d, J = 7.2 Hz, 2H), 6.39 (br s, 1H), 7.15 (s, 1H), 7.88 (d, J = 8.4 Hz, 2H), 8.04 (s, 1H), 8.12
4
(d, J = 8.4 Hz, 2H).
0
1
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8
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0
1
2
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4ꢀ(6ꢀ(4ꢀCyanophenyl)ꢀ8ꢀ((tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)methylamino)imidazo[1,2ꢀ
b]pyridazinꢀ3ꢀyl)ꢀNꢀcyclopropylbenzamide (21a). A mixture of 20 (245 mg, 0.467 mmol), 4ꢀ
cyanophenylboronic acid (129 mg, 0.877 mmol), PdCl (dppf)·DCM (35.8 mg, 0.044 mmol), and
2
Na CO (65 mg, 0.614 mmol) in ethanol/H O (2.5 mL/0.31 mL) was heated to reflux for 1 h. The
2
3
2
reaction mixture was allowed to cool to room temperature and then diluted with EtOAc and
water. The aqueous layer was separated and extracted with EtOAc. The combined extracts were
washed with water and brine, dried over MgSO , filtered, and concentrated. The residue was
4
purified by flash column chromatography (silica gel; EtOAc/hexane, gradient: 65−75% EtOAc)
to
afford
tertꢀbutyl
6ꢀ(4ꢀcyanophenyl)ꢀ3ꢀ(4ꢀ(cyclopropylcarbamoyl)phenyl)imidazo[1,2ꢀ
b]pyridazinꢀ8ꢀyl((tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)methyl)carbamate (277 mg, 107%) as a yellow solid.
1
H NMR (300 MHz, CDCl ) δ 0.68 (br s, 2H), 0.84−0.90 (br m, 2H), 1.26−1.39 (m, 2H), 1.54 (s,
3
9
H), 1.63 (d, J = 12.6 Hz, 2H), 1.74−1.90 (br m, 1H), 2.89−2.98 (br m, 1H), 3.29 (t, J = 11.3 Hz,
H), 3.92 (d, J = 10.1 Hz, 2H), 4.23 (d, J = 6.9 Hz, 2H), 6.91 (s, 1H), 7.61 (s, 1H), 7.81 (d, J =
2
8.1 Hz, 2H), 7.94 (d, J = 8.1 Hz, 2H), 8.09−8.17 (m, 5H). To this compound in DCM (2.4 mL)
was added TFA (2.4 mL) at room temperature. The reaction mixture was stirred for 20 min and
then poured into aqueous K CO solution. The mixture was diluted with DCM, THF, and water.
2
3
The aqueous layer was separated and extracted with DCM/THF (2:5). The mixture was washed
with brine, dried over Na SO , filtered, and concentrated. The residue was diluted with EtOAc
2
4
(8.0 mL) and heated to reflux. After cooling, the resulting solid was collected on a glass filter to
1
afford 21a (139 mg, 60%) as an offꢀwhite solid. H NMR (300 MHz, DMSOꢀd ) δ 0.56−0.63 (m,
6
2H), 0.67−0.74 (m, 2H), 1.22−1.36 (m, 2H), 1.66 (d, J = 11.9 Hz, 2H), 1.94−2.05 (m, 1H),
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.83−2.91 (m, 1H), 3.22−3.39 (m, 4H), 3.85 (d, J = 9.9 Hz, 2H), 6.82 (s, 1H), 7.75−7.80 (m, 1H),
.95 (d, J = 8.4 Hz, 2H), 8.01 (d, J = 8.4 Hz, 2H), 8.12 (s, 1H), 8.26−8.31 (m, 4H), 8.48 (d, J =
+
.1 Hz, 1H). MSꢀESI (m/z) = 493 [M+H] .
10
11
12
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14
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18
19
20
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58
59
60
tertꢀButyl
6ꢀ(cyclohexyloxy)imidazo[1,2ꢀb]pyridazinꢀ8ꢀyl((tetrahydroꢀ2Hꢀpyranꢀ4ꢀ
yl)methyl)carbamate (22a). To a solution of cyclohexanol (259 ꢁL, 2.45 mmol) in NMP (1.5
mL) was added NaH (60% in mineral oil; 82 mg, 2.04 mmol) at room temperature. The mixture
was heated to 50 °C for 10 min and then cooled to 0 °C. A solution of 18 (300 mg, 0.818 mmol)
in NMP (1.5 mL) was added to this solution, and the reaction mixture was stirred at room
temperature for 1.5 h. The mixture was diluted with aqueous citric acid solution and EtOAc. The
aqueous layer was separated and extracted with EtOAc. The combined organic layer was washed
with water and brine, dired over Na SO , filtered, and concentrated. The residue was purifeid by
2
4
flash column chromatography (silica gel; EtOAc/hexane = 1/1) to afford 22a (239 mg, 68%) as a
1
white amorphous substance. H NMR (400 MHz, CDCl ) δ 1.24−1.64 (m, 9H), 1.73−1.82 (m,
3
3H), 2.04−2.09 (m, 2H), 3.29 (td, J = 11.7, 2.0 Hz, 2H), 3.91 (dd, J = 11.4, 2.8 Hz, 2H), 4.04 (d,
J = 7.1 Hz, 2H), 4.92−4.98 (m, 1H), 6.61 (s, 1H), 7.53 (d, J = 1.0 Hz, 1H), 7.70 (d, J = 1.0 Hz,
+
1
H). MSꢀESI (m/z) = 431 [M+H] .
tertꢀButyl 6ꢀ(cyclohexyloxy)ꢀ3ꢀiodoimidazo[1,2ꢀb]pyridazinꢀ8ꢀyl((tetrahydroꢀ2Hꢀpyranꢀ4ꢀ
yl)methyl)carbamate (23a). To a solution of 22a (238 mg, 0.553 mmol) in DMF (5 mL) was
added NIS (137 mg, 0.608 mmol). The reaction mixture was stirred at room temperature for 5 h
and then diluted with EtOAc and water. The aqueous layer was separated and extracted with
EtOAc. The combined organic extracts were washed with water and brine, dried over Na SO ,
2
4
filtered, and concentrated. The residue was purified by flash column chromatography (silica gel;
1
EtOAc/hexane = 1/1) to afford 23a (268 mg, 87%) as a white amorphous substance. H NMR
(
400 MHz, CDCl3) δ 1.23−1.67 (m, 9H), 1.70−1.86 (m, 3H), 2.10−2.13 (m, 2H), 3.28 (td, J =
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1.5, 1.7 Hz, 2H), 3.91 (dd, J = 11.4, 2.8 Hz, 2H), 4.00 (d, J = 7.1 Hz, 2H), 5.03−5.09 (m, 1H),
+
6
.67 (s, 1H), 7.61 (s, 1H). MSꢀESI (m/z) = 557 [M+H] .
4
ꢀ(6ꢀ(Cyclohexyloxy)ꢀ8ꢀ((tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)methylamino)imidazo[1,2ꢀ
b]pyridazinꢀ3ꢀyl)ꢀNꢀcyclopropylbenzamide (24a). A mixture of 23a (267 mg, 0.480 mmol), 4ꢀ
cyclopropylaminocarbonyl)phenylboronic acid pinacol ester (207 mg, 0.720 mmol),
PdCl (dtbpf) (15.6 mg, 0.024 mmol), and K CO (199 mg, 1.44 mmol) in DMF/H O (3 mL/0.3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
2
2
3
2
mL) was heated at 50 °C for 2 h. The reaction mixture was allowed to cool to room temperature
and then diluted with EtOAc and water. The aqueous layer was separated and extracted with
EtOAc. The combined organic extracts were washed with water and brine, dried over Na SO ,
2
4
filtered, and concentrated. The residue was purified by flash column chromatography (silica gel;
EtOAc/hexane 4/1) to afford tertꢀbutyl 6ꢀ(cyclohexyloxy)ꢀ3ꢀ(4ꢀ
cyclopropylcarbamoyl)phenyl)imidazo[1,2ꢀb]pyridazinꢀ8ꢀyl((tetrahydroꢀ2Hꢀpyranꢀ4ꢀ
=
(
yl)methyl)carbamate (283 mg, 100%) as a white amorphous substance. MSꢀESI (m/z) = 590
+
[M+H] . To a solution of this compound (283 mg) in DCM (3 mL) was added TFA (3 mL). The
reaction mixture was stirred at room temperature for 30 min and then poured into aqueous
K CO solution. The mixture was extracted with EtOAc, and the combined organic extracts were
2
3
then washed with water and brine, dried over Na SO , filtered, and concentrated. The residue
2
4
was triturated with EtOAc, and the resulting solid was collected on a glass filter to afford 24a
1
(182 mg, 77%) as a white solid. H NMR (400 MHz, CDCl ) δ 0.62−0.68 (m, 2H), 0.90 (q, J =
3
6.3 Hz, 2H), 1.23−2.18 (m, 15H), 2.90−2.98 (m, 1H), 3.20 (t, J = 6.6 Hz, 2H), 3.40 (t, J = 11.2
Hz, 2H), 4.01 (dd, J = 3.5, 11.7 Hz, 2H), 4.90−4.99 (m, 1H), 5.65 (s, 1H), 5.72 (t, J = 6.1 Hz,
1
H), 6.26 (s, 1H), 7.73 (s, 1H), 7.81 (d, J = 8.1 Hz, 2H), 8.16 (d, J = 8.1 Hz, 2H). MSꢀESI (m/z)
+
= 490 [M+H] .
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tertꢀButyl
(tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)methyl(6ꢀ(1,1,1ꢀtrifluoroꢀ2ꢀmethylpropanꢀ2ꢀ
ylamino)imidazo[1,2ꢀb]pyridazinꢀ8ꢀyl)carbamate (25f). A suspension of RuPhos (12.7 g, 27.3
mmol) and Pd (dba) (6.24 g, 6.81 mmol) in dioxane (250 mL) was stirred at room temperature
2
3
10
11
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for 5 min. Sodium tꢀbutoxide (21.0 g, 218 mmol), 18 (25 g, 68.1 mmol), and 1,1,1ꢀtrifluoroꢀ2ꢀ
methylpropanꢀ2ꢀamine hydrochloride (22.3 g, 136 mmol) was added to the suspension, and the
reaction mixture was heated to reflux for 6 h. After cooling, the mixture was diluted with EtOAc
and water. The aqueous layer was separated and extracted with EtOAc. The combined extracts
were washed with water and brine, dried over MgSO , filtered, and concentrated. The residue
4
was purified by flash column chromatography (silica gel, EtOAc/hexane, gradient: 30−100%
1
EtOAc) to afford 25f (22.1 g, 71%) as a tan solid. H NMR (300 MHz, DMSOꢀd ) δ 1.36 (s, 9H),
6
1.64 (s, 6H), 3.17 (t, J = 10.8 Hz, 2H), 3.74−3.83 (m, 4H), 6.74 (s, 1H), 6.90 (s, 1H), 7.39 (d, J =
+
1
.1 Hz, 1H), 7.82 (d, J = 1.2 Hz, 1H). MSꢀESI (m/z) = 458 [M+H] .
tertꢀButyl 3ꢀiodoꢀ6ꢀ(1,1,1ꢀtrifluoroꢀ2ꢀmethylpropanꢀ2ꢀylamino)imidazo[1,2ꢀb]pyridazinꢀ8ꢀ
yl((tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)methyl)carbamate (26f). A mixture of 25f (22.1 g, 48.3 mmol)
and NIS (12.5g, 55.6 mmol) in DMF (200 mL) was stirred at room temperature for 14 h. The
mixture was poured into aqueous Na S O solution and then diluted with EtOAc. The aqueous
2
2
3
layer was separated and extracted with EtOAc. The combined organic layer was washed with
water and brine, dried over MgSO4, filtered, and concentrated to afford 25f (26.9 g, 95%) as a
1
tan solid. H NMR (300 MHz, DMSOꢀd ) δ 1.35 (s, 9H), 1.71 (s, 6H), 3.17 (t, J = 10.8 Hz, 2H),
6
+
3.75−3.78 (m, 4H), 6.80 (s, 1H), 7.10 (s, 1H), 7.51 (s, 1H). MSꢀESI (m/z) = 584 [M+H] .
NꢀCyclopropylꢀ4ꢀ(8ꢀ((tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)methylamino)ꢀ6ꢀ(1,1,1ꢀtrifluoroꢀ2ꢀ
methylpropanꢀ2ꢀylamino)imidazo[1,2ꢀb]pyridazinꢀ3ꢀyl)benzamide (27f). A mixture of 26f
(26.9 g, 46.1 mmol), 4ꢀ(cyclopropylaminocarbonyl)phenylboronic acid pinacol ester (15.9 g, 55.3
mmol), PdCl (dtbpf) (3.77 g, 4.61 mmol), and aqueous Na CO (2 M solution; 69.2 mL, 138
2
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mmol) in DMF (250 mL) was heated at 50 °C for 4 h. The reaction mixture was allowed to cool
to room temperature and then diluted with EtOAc and water. The insoluble materials were
filtered off, and the aqueous layer was separated and extracted with EtOAc. The combined
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organic extracts were washed with water and brine, dried over MgSO , filtered, and concentrated.
4
The residue was purified by flash column chromatography (silica gel; EtOAc/hexane, gradient:
20−70% EtOAc) to afford tertꢀbutyl 3ꢀ(4ꢀ(cyclopropylcarbamoyl)phenyl)ꢀ6ꢀ(1,1,1ꢀtrifluoroꢀ2ꢀ
methylpropanꢀ2ꢀylamino)imidazo[1,2ꢀb]pyridazinꢀ8ꢀyl((tetrahydroꢀ2Hꢀpyranꢀ4ꢀ
1
yl)methyl)carbamate (26.8 g, 94%) as a brown solid. H NMR (300 MHz, DMSOꢀd ) δ
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0.56−0.61 (m, 2H), 0.67ꢀ0.73 (m, 2H), 1.37 (s, 9H), 1.55 (d, J = 12.0 Hz, 2H), 1.66 (s, 6H), 1.98
(s, 2H), 2.81ꢀ2.90 (m, 1H), 3.19 (t, J = 10.8 Hz, 2H), 3.75−3.84 (m, 4H), 6.85 (s, 1H), 7.10 (s,
1H), 7.91 (d, J = 8.7 Hz, 2H), 7.96 (s, 1H), 8.12 (d, J = 8.7 Hz, 2H), 8.49 (d, J = 4.3 Hz, 1H).
+
MSꢀESI (m/z) = 617 [M+H] . To a solution of this compound (26.8 g) in DCM (90 mL) was
added TFA (33.5 mL). The reaction mixture was stirred at room temperature for 15 h, diluted
with CHCl (300 mL), and then basified with aqueous NaHCO solution. The aqueous layer was
3
3
separated and extracted with CHCl . The combined organic extracts were washed with water,
3
dried over MgSO , filtered, and concentrated. The residue was crystallized from methanol/ether
4
1
to afford 27f (17.5 g, 78%) as a tan solid. H NMR (300 MHz, DMSOꢀd ) δ 0.54−0.74 (m, 4H),
6
1.16−1.32 (m, 2H), 1.56ꢀ1.70 (m, 2H), 1.64 (s, 6H), 1.95 (m, 1H), 2.85 (m, 1H), 3.08 (t, J = 6.0
Hz, 2H), 3.22−3.34 (m, 2H), 3.81−3.92 (m, 2H), 5.80 (s, 1H), 6.47 (s, 1H), 7.11 (t, J = 6.0 Hz,
1H), 7.79 (s, 1H), 7.88 (d, J = 8.4 Hz, 2H), 8.15 (d, J = 8.4 Hz, 2H), 8.45 (d, J = 4.2 Hz, 1H).
+
HRMSꢀESI (m/z): [M+H] calcd for C H F N O , 517.2533; found, 517.2535.
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2
®
Biochemical Mps1 Assays. Mps1 kinase activity was measured by the DELFIA
(dissociationꢀenhanced lanthanide fluorescence immunoassay) method that monitors
phosphorylation of the p38 MAPK peptide (biotinꢀAGAGLARHTDDEMTGYVA) using a
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phosphorylated site specific antibody as described previously. The protein preparation method
for Mps1 was also reported in ref 28. Reported values are means of n ≥ 2 determinations.
Cellular Mps1 Autophosphorylation Assays. Mps1 cellular activity was measured by
detecting inhibition of autophosphorylation using RERFꢀLCꢀAI cells (RIKEN) that stably
express FLAGꢀtagged Mps1 under the control of a tetracycline (Tet)ꢀsuppressible promoter. The
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detailed method was reported previously. The reported values are means of n = 2
determinations. In all cases, individual measurements were within 2ꢀfold for each compound.
Antiproliferative Assays. Antiproliferative activity was measured by the MTT (3ꢀ(4,5ꢀdiꢀ
methylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide) method using lung carcinoma cells A549
(
ATCC), NCIꢀH460 (ATCC), Lu99 (RIKEN), and a normal cell MRCꢀ5 (ATCC) as described
2
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previously. The MRCꢀ5 cells were seeded in a 96ꢀwell plate at 10000 cells/100 ꢁL /well and
incubated overnight. The reported values are means of n ≥ 2 determinations. In all cases,
individual measurements were within 2ꢀfold for each compound. Antiproliferative assays using
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other cell lines were conducted at the National Cancer Center.
Tumor Xenograft Models. NCIꢀH460 cells were cultured in the medium described in ref 28.
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NCIꢀH460 cells (1 × 10 to 1 × 10 cells) were inoculated subcutaneously into the flank of female
nude mice (BALB/cA Jclꢀnu/nu, 7 weeks old, CLEA Japan). The treatment was started when the
3
tumor volumes reached around 200 mm . The test compound suspended in 0.5% methylcellulose
solution (Wako) was administered po daily for 14 days (n = 6). Details have been described
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previously. All mice had access to water and food ad libitum and all experiments were
performed with the approval of the Shionogi Animal Care and Use Committee.
nd
Solubility Assay. Japanese Pharmacopeia JPꢀ2 (JPꢀ2) solution (pH 6.8) containing 20
mmol/L of sodium taurocholate was used as the aqueous buffer. The buffer was prepared as
follows: JPꢀ2 solution: phosphate buffer 500 mL and water 500 mL; JPꢀ2 solution containing
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sodium taurocholate: sodium taurocholate (1.08 g) and the JPꢀ2 solution to reach 100 mL. For
each compound, 0.2 mL of the JPꢀ2 solution containing sodium taurocholate solution was added
to 0.5 mg of dry compound, and the mixture was shaken for 1 h at 37 °C. The solution was
filtered with a membrane filter (0.45 µm), and 0.1 mL of methanol was added to the filtrate (0.1
mL) so that the filtrate was diluted 2ꢀfold. Quantification was performed by HPLC with an
absolute calibration method.
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Rat Microsomal Stability Studies. Rat microsomes were prepared from male Spragueꢀ
Dawley rats (8 weeks). The metabolic stability of test compounds in rat liver microsomes was
determined at one concentration (0.1 µM). The compounds were incubated with 0.5 mg
protein/mL in suspension in 50 mM TrisꢀHCl buffer (pH 7.4), 150 mM KCl, 10 mM MgCl , 1
2
mM βꢀNADPH at 37 °C. Microsomal incubations were initiated by the addition of 100ꢀfold
concentrated solution of the compounds. Incubations was terminated by addition of 2ꢀfold
volume of organic solvent (MeCN/MeOH = 1/1) after 0 and 30 minutes of incubation at 37 °C.
The preparation protein was removed by centrifugation. The supernatants were analyzed by
LC/MS/MS. All incubations were conducted in duplicate, and the percentage of compound
remaining at the end of the incubation was determined from the LC/MS/MS peak area ratio.
Pharmacokinetic Studies. Male SpragueꢀDawley rat (8 weeks) were purchased from Charles
River Laboratories. Compounds were formulated as suspensions in 0.5% methylcellulose (0.2
mg/mL) and dosed orally at 1 mg/kg (n = 2) under the nonfasted condition. For iv study, the
compounds were formulated as solutions in DMA/propylene glycol (1:1, 0.5 mg/mL) and dosed
intravenously from the tail vein at 0.5 mg/kg (n = 2) under isoflurane anesthesia under the
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nonfasted condition. Details of the method has been described previously. All experiments
were performed with the approval of the Shionogi Animal Care and Use Committee.
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Cocrystallization of Mps1 with 11f and 27b. Protein expression and purification were
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performed as described previously. Cocrystals of Mps1 in complex with 11f and 27b were
prepared using the sittingꢀdrop vapor diffusion method. Equal volumes of protein solution (11.4
mg/mL containing 0.5 mM of 11f, 6.8 mg/mL containing 0.5 mM of 27b) and the mother liquor
were mixed in a single droplet and equilibrated against 0.1 mL of mother liquor at 293 K. The
mother liquor conditions were 0.1 M PIPES pH 6.5, 12.3% w/v PEG 5000, and 0.082 M
magnesium sulfate for 11f and 0.1 M Tris hydrochloride pH 7.5, 8.0% w/v triethylene glycol, and
0.12 M potassium chloride for 27b. Data collection and structure solution were performed as
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described previously.
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