The protein kinase MPS1 is a crucial component of the spindle assembly checkpoint signal and is aberrantly overexpressed in many human cancers. MPS1 is one of the top 25 genes overexpressed in tumors with chromosomal instability and aneuploidy. PTEN-deficient breast tumor cells are particularly dependent upon MPS1 for their survival, making it a target of significant interest in oncology. We report the discovery and optimization of potent and selective MPS1 inhibitors based on the 1H-pyrrolo[3,2-c]pyridine scaffold, guided by structure-based design and cellular characterization of MPS1 inhibition, leading to 65 (CCT251455). This potent and selective chemical tool stabilizes an inactive conformation of MPS1 with the activation loop ordered in a manner incompatible with ATP and substrate-peptide binding; it displays a favorable oral pharmacokinetic profile, shows dose-dependent inhibition of MPS1 in an HCT116 human tumor xenograft model, and is an attractive tool compound to elucidate further the therapeutic potential of MPS1 inhibition.
The protein kinase MPS1 is a crucial component of the spindle assembly checkpoint signal and is aberrantly overexpressed in many human cancers. MPS1 is one of the top 25 genes overexpressed in tumors with chromosomal instability and aneuploidy. PTEN-deficient breast tumor cells are particularly dependent upon MPS1 for their survival, making it a target of significant interest in oncology. We report the discovery and optimization of potent and selective MPS1 inhibitors based on the 1H-pyrrolo[3,2-c]pyridine scaffold, guided by structure-based design and cellular characterization of MPS1 inhibition, leading to 65 (CCT251455). This potent and selective chemical tool stabilizes an inactive conformation of MPS1 with the activation loop ordered in a manner incompatible with ATP and substrate-peptide binding; it displays a favorable oral pharmacokinetic profile, shows dose-dependent inhibition of MPS1 in an HCT116 human tumor xenograft model, and is an attractive tool compound to elucidate further the therapeutic potential of MPS1 inhibition.
The main role of the
cell cycle is to enable error-free DNA replication,
chromosome segregation, and cytokinesis. Surveillance mechanisms,
the checkpoint pathways, monitor passage through the cell cycle at
several stages. During mitosis, the spindle assembly checkpoint (SAC)
prevents anaphase onset until the appropriate tension and attachment
across kinetochores is achieved.[1,2] One of the first components
of the SAC signal, identified by a genetic screen in budding yeast,
was dubbed MPS1 (monopolar spindle 1, also known as TTK) because of
the monopolar spindles produced by MPS1 mutant cells.[3] Subsequently, the MPS1 gene was shown to encode an essential
dual-specificity kinase conserved from yeast to humans.[4,5] MPS1 activity peaks at the G2/M transition, is enhanced upon activation
of the SAC with nocodazole,[6] and is dependent
upon autophosphorylation of a threonine at position 676 in the activation
loop.[7] MPS1 is required for normal function
of the mitotic spindle checkpoint and subsequent cell division; it
is aberrantly overexpressed in a wide range of human tumors including
bladder, anaplastic thyroid, breast, lung, esophagus, and prostate
cancers.[8−12] In addition, MPS1 has been identified in the signature of the top
25 genes overexpressed in tumors with chromosomal instability[13] and aneuploidy,[14,15] with PTEN-deficient
breast tumor cells particularly dependent upon MPS1 for their survival
such that RNAi-mediated knockdown or chemical inhibition of MPS1
leads to cell death.[14] This body of work
has engendered significant interest in the discovery of selective
small-molecule chemical tools to elucidate further the therapeutic
potential of MPS1 inhibition for the treatment of cancer.First-generation
nonselective inhibitors of MPS1 have been described,
for example, 1 (SP600125), a JNK (c-Jun amino-terminal
kinase) inhibitor that disrupts SAC function in a JNK-independent
manner via the inhibition of MPS1,[16−18] and 2 (reversine),
an MPS1, Aurora A, and Aurora B inhibitor.[19] More recently, several selective small-molecule MPS1 inhibitors
have been utilized to explore the cellular function of MPS1.[20,21] These include 3 (MPI-0479605),[22]4 (AZ3146),[23]5 (NMS-P715),[24,25] a series of selective diaminopyridine-based
inhibitors exemplified by 6(26) that demonstrates inhibition of
growth of A549 human tumor xenografts, and a set of indazole-based
inhibitors represented by 7(27) (Figure 1).
Figure 1
Small-molecule MPS1 inhibitors 1,[16−18]2,[19]3,[22]4,[23]5,[24,25]6,[26] and 7.[27]
Small-molecule MPS1 inhibitors 1,[16−18]2,[19]3,[22]4,[23]5,[24,25]6,[26] and 7.[27]Here, we describe the discovery
of orally bioavailable small-molecule
inhibitors of MPS1 based on the 1H-pyrrolo[3,2-c]pyridine scaffold in a medicinal-chemistry program enabled
by structure-based design and cellular characterization of MPS1 inhibition.
We show that optimized compounds in this series display potent and
selective inhibition of MPS1 in vitro and translate well to cellular
assays of MPS1 autophosphorylation and antiproliferative activity
when compared to other recently reported MPS1 inhibitors. We also
show, by X-ray crystallographic studies, that exemplars of this series
stabilize an inactive conformation of MPS1 in which the activation
loop is ordered in a manner incompatible with ATP and substrate-peptide
binding.Our starting point was the potent but nonselective
and metabolically
unstable compound 8 identified in a high-throughput screen
of an in-house kinase-focused compound library (Figure 2).
Figure 2
MPS1 inhibitor 8 identified by HTS (GSK3β, glycogen
synthase kinase 3β; MLM, mouse liver microsome preparation;
and HLM, human liver microsome preparation).
MPS1 inhibitor 8 identified by HTS (GSK3β, glycogen
synthase kinase 3β; MLM, mouse liver microsome preparation;
and HLM, human liver microsome preparation).
Chemistry
Our synthetic strategy to all desired 1H-pyrrolo[3,2-c]pyridines involved palladium-mediated
Sonagashira coupling
of an appropriately substituted 4-amino-2-bromo-5-iodopyridine, 9, and an alkyne, 10, to generate key pyrrolopyridine
intermediate 11, a domino approach recently exemplified
by Schmidt and colleagues.[28] Subsequent
palladium-mediated displacement of the 6-bromo substituent of intermediates 11 with the appropriate aniline gave the desired products 12 (Scheme 1). This general route was
adapted depending on the identity of the N-1 and C-2 substituents
as described below. We consistently observed that when R1 = H the transformation of 11 to 12 was
low-yielding; therefore, we employed a protecting-group strategy whereby
a Boc substituent was installed at the N-1 position prior to introduction
of the C-6 amino substituent into the 1H-pyrrolo[3,2-c]pyridine scaffold.
Scheme 1
General Synthetic Strategy
Compounds 8 and 21–28 shown in Table 1 were prepared according
to the general strategy depicted in Scheme 1 using palladium-mediated substitution of key 6-bromo-pyrrolopyridine
intermediate 17, which was itself prepared by sequential
Sonagashira cross coupling and base-catalyzed ring closure of sulfonamide 16; introduction of the sulfonamide was necessary to optimize
the efficiency of the domino cyclization reaction, presumably by increasing
the acidity of the remaining anilinic proton (Scheme 2). Cyclization precursor 16 was prepared from
corresponding 4-amino-2-bromopyridine 13; iodination
of 13 was unselective, and desired regioisomer 15 was purified from its partner, 14, by chromatographic
separation in 38 and 37% yields, respectively; subsequent dimesylation
with methanesulfonylchloride and base-mediated removal of one of the
two mesyl groups provided intermediate 16 in 54% yield.
The required tert-butyl 4-ethynyl-1H-pyrazole-1-carboxylate 20 was prepared in 56% overall
yield from 4-iodopyrazole 18 by Boc protection followed
by Sonogashira-mediated coupling with trimethylsilylacetylene and subsequent
TBAF-mediated deprotection of alkyne 19 (Scheme 2).
Table 1
Effect of Aniline
Substituents on
MPS1 Selectivity
n = 1.
Scheme 2
Preparation of 2-(1H-Pyrazol-4-yl)-1H-pyrrolo[3,2-c]pyridines
Preparation of 2-(1H-Pyrazol-4-yl)-1H-pyrrolo[3,2-c]pyridines
Reagents and conditions: (a)
ICl, NaOAc, AcOH, 75 °C; (b) MeSO2Cl, Et3N, CH2Cl2, rt; (c) NaOH, THF/H2O,
rt; (d) (Boc)2O, Et3N, THF, rt; (e) trimethylsilylacetylene, Pd(OAc)2, PPh3, CuI, i-Pr2NH, DMF, 60 °C; (f) TBAF, THF, 0–5 °C;
(g) PdCl2(PPh3)2, CuI, Et3N, DMF, 60 °C; (h) DBU, THF/MeOH, 40 °C; (i) (Boc)2O, Et3N, DMAP, EtOAc, rt; (j) aniline, Pd2(dba)3, Xantphos, Cs2CO3, dioxane,
80 °C; (k) TFA, CH2Cl2, rt.Compounds 30–33, 35, and 36 in Table 2 were
also
prepared according the general procedure (Scheme 1), which necessitated multiple protection/deprotection steps
in addition to the need for bespoke synthesis of the appropriate ethynylheterocycle
(see the Supporting Information). For compounds 29 and 37–44 (Table 2), 48–54 (Table 5), and 61–68 (Table 6) bearing a preferred 1-methylpyrazole substituent
at C-2 of the pyrrolopyridine scaffold, we developed a more efficient
route. Thus, Sonagashira cross coupling of 4-ethynyl-1-methyl-1H-pyrazole 45 with unprotected 4-amino-2-bromo-5-iodopyridine 15 gave cyclization precursor 46 in 88% yield,
which was subjected to smooth base-mediated conversion to the pyrrolopyridine
core followed by substitution of the N-1 position with a tert-butylcarbonate group to give key intermediate 47 in
75% yield (Scheme 3). This two-step, base-mediated
approach to the formation of the pyrrolopyridine scaffold obviated
the need for sulfonamide-mediated activation of the anilinic cyclization
precursor. Subsequent palladium-mediated substitution at the C-6 position,
as described above, gave N-1-Boc derivatives 48–54 (Table 5), 61, and 63–68 (Table 6).
TFA-mediated removal of the N-Boc substituent furnished N-1-unsubstituted
compounds 29, 37–44 (Table 2), and 62 (Table 6), as depicted in Scheme 3.
Table 2
Effect of Pyrrolopyridine C-2 and
Aniline C-4 Substituents on Metabolism
MLM: percentage of parent compound
metabolized after a 30 min incubation in mouse liver microsomes.
n = 1.
Table 5
Effect of the N-1-Boc Substituent
on Cell-Based Potency
MLM/HLM:
percentage of parent compound
metabolized after a 30 min incubation.
ND = not determined.
n = 1.
Table 6
Effect of the Aniline Substituent
on Cell-Based Potency
MLM/HLM:
percentage of parent compound
metabolized after a 30 min incubation in mouse and human liver microsomes.
n = 1.
Scheme 3
Preparation of Preferred tert-Butyl 2-(1-Methyl-1H-pyrazol-4-yl)-1H-pyrrolo[3,2-c]pyridine-1-carboxylates
and 2-(1-Methyl-1H-pyrazol-4-yl)-1H-pyrrolo[3,2-c]pyridines
Preparation of Preferred tert-Butyl 2-(1-Methyl-1H-pyrazol-4-yl)-1H-pyrrolo[3,2-c]pyridine-1-carboxylates
and 2-(1-Methyl-1H-pyrazol-4-yl)-1H-pyrrolo[3,2-c]pyridines
Reagents
and conditions: (a)
PdCl2(PPh3)2, CuI, Et3N, DMF, rt; (b) t-BuOK, NMP, 50 °C; (c) (Boc)2O, Et3N, DMAP, EtOAc, rt; (d) aniline, Pd2(dba)3, Xantphos, Cs2CO3, DMA, 80
°C; (e) TFA, rt or TFA, CH2Cl2, rt.The required alkynes and substituted anilines were
prepared according
to the methods summarized in Scheme 4 (Supporting Information), whereas the appropriate
iodoheterocycles were prepared from commercially available starting
materials by iodination (Supporting Information). However, the 5-iodooxazole required for the synthesis of 1H-pyrrolo[3,2-c]pyridin-2-yl)oxazoles 34 and 55 proved elusive, and an alternative
synthetic route was developed in which desired oxazole intermediate 60 was constructed from acetal 58 in 70% overall
yield by liberation of the aldehyde and reaction with p-toluenesulfonylmethyl isocyanide (TOSMIC) followed by subsequent
N-1 Boc protection. Acetal 58 was obtained from intermediate 16 in 45% yield by Sonogashira cross coupling with 3,3-diethoxyprop-1-yne 56 and concomitant ring closure in a procedure similar to
that described by Le Brazidec et al. for the preparation of 2-substituted
pyrrolopyrimidines followed by base-mediated removal of the N-1 sulfonamide
(Scheme 5).[29]
Compound 8 (Figure 2) exemplifies
a series of 1H-pyrrolo[3,2-c]pyridines
discovered by HTS of an in-house kinase-focused compound library versus
MPS1. Although compound 8 demonstrated potent, ligand-efficient[30] binding to MPS1 (IC50 = 0.025 μM,
LE = 0.43; Table 1) with evidence for cell-based antiproliferative activity in the
HCT116 human colon cancer cell line (MTT assay, GI50 =
0.55 μM), its overall profile suffered from poor selectivity,
particularly against CDK2 (IC50 = 0.043 μM), poor
in vitro metabolic stability in mouse and human liver microsomes,
and significant efflux in Caco-2 permeability assays (Figure 2). An important aim of our initial hit-improvement
strategy was to eradicate activity versus cell cycle kinases (e.g.,
CDK2) and other kinases known to affect mitotic function (e.g., Aurora
kinases A and B) to study the profile of a highly selective MPS1 inhibitor
on mitotic function in cellular mechanistic assays and in vivo. In
addition, we set out to improve the metabolic stability and membrane
permeability of compound 8 to discover a chemical tool
suitable for in vivo PK/PD studies.n = 1.A crystal
structure of the kinase domain of MPS1 with compound 8 (Figure 3) indicated binding of 8, albeit with relatively weak electron density. Nevertheless,
the ligand could be modeled with partial occupancy along with a molecule
of poly(ethylene glycol) wrapped around the active-site Lys553 side
chain, a consequence of the presence of a high concentration of PEG300
in the crystallization conditions. The structure revealed the 6-amino-pyrrolopyridine
motif interacting with the hinge region of the ATP-binding site by
virtue of an H-bond-donor interaction between the backbone amide group
of Gly605 and the pyridine nitrogen hydrogen-bond acceptor of the
pyrrolopyridine scaffold. In addition, the anilinic NH of compound 8 formed a hydrogen bond with the carbonyl group of hinge
residue Gly605, thereby positioning the anilinic moiety at the entrance
of the MPS1 ATP-binding site, stacked above the post-hinge region (residues
606–611) and pointing toward the solvent. Furthermore, it revealed
an H-bond between the C-2 pyrazole and Lys553 as well as a van der
Waals interaction between lipophilic C-3 to C-4 atoms and the gatekeeper
residue, Met602 (Figure 3).
Figure 3
Crystal structure of
MPS1 with compound 8 bound. Compound 8 is
shown with orange carbon atoms and is modeled with partial
occupancy along with a PEG molecule, shown with orange and cyan carbon
atoms for the two alternate conformers. Selected amino acids that
contact the ligand are shown with green carbon atoms. The electron
density shown in green is from an Fo – Fc omit map and is contoured at 3σ. Key
H-bond interactions are shown as black dotted lines. The interaction
between the C-2 pyrazole and Lys553 has been omitted for clarity.
All structural figures were produced with CCP4MG.[31]
Crystal structure of
MPS1 with compound 8 bound. Compound 8 is
shown with orange carbon atoms and is modeled with partial
occupancy along with a PEG molecule, shown with orange and cyan carbon
atoms for the two alternate conformers. Selected amino acids that
contact the ligand are shown with green carbon atoms. The electron
density shown in green is from an Fo – Fc omit map and is contoured at 3σ. Key
H-bond interactions are shown as black dotted lines. The interaction
between the C-2 pyrazole and Lys553 has been omitted for clarity.
All structural figures were produced with CCP4MG.[31]A striking difference between
the binding of compound 8 and published compound 6 is their respective hydrogen-bond
interactions with the hinge. Whereas the backbone functionalities
of hinge residue Cys604 were not involved in interactions with compound 8, a peptide flip of Cys604 in the structure of MPS1 complexed
with compound 6 (PDB code 3VQU) allowed an H-bond interaction between
the backbone carbonyl of Cys604 and the anilinic NH of compound 6.[26]We initially focused
our attention on modification of the pyrrolopyridine
6-anilino substituent to replace the electron-rich 3,4-dimethoxy aniline,
which we regarded as a metabolic liability. 4-Methoxy analogue 21 proved equipotent and replacement with a range of 4-substituents
maintained activity (compounds 22–24), consistent with the crystal structure of 8 bound
to MPS1, which showed that this vector projects out of the entrance
of the ATP-binding site into the solvent (Figure 3). However, selectivity versus CDK2 remained poor in all of
these compounds. Importantly, the 2-methoxy-, 2-ethoxy-, and 2-chloro-substituted
aniline derivatives, 25–28, maintained
potency while also significantly improving selectivity versus CDK2
and enhancing selectivity versus Aurora A, Aurora B, and GSK3β.
This SAR is consistent with previous reports on the use of 2-substituted
anilines in other chemical series to enhance selectivity versus MPS1
through the exploitation of a small lipophilic pocket adjacent to
Cys604 in the hinge region (see below).[25]Compounds 25–28, although
selective
for MPS1, remained metabolically unstable, and our attention turned
to exploration of the pyrrolopyridine C-2 substituent. This was prompted
by our observation that solutions of compound 8 underwent
slow air oxidation across the double bond between C-2 and C-3 (Supporting InformationFigure
S1). We hypothesized that the unsubstituted C-2 pyrazole rendered
the pyrrole moiety of the pyrrolopyridine scaffold susceptible to
electrophilic attack. Gratifyingly, N-methylation of the C-2 pyrazole
was tolerated with only a 5-fold reduction in potency (compound 29, MPS1 IC50 = 0.12 μM, versus compound 25, MPS1 IC50 = 0.025 μM; Table 2), resulting in an air-stable
compound and a slight improvement in metabolic stability (MLM = 72%
for 29 versus 99% for 25) (Table 2). Similarly, electron-withdrawing trifluoroethyl-substituted
pyrazole analogue 30 maintained potency (MPS1 IC50 = 0.079 μM) and also improved metabolic stability
(53% turnover in MLM); however, corresponding difluoromethyl analogue 31 proved surprisingly weak, with a 6-fold loss of activity
with respect to the trifluoroethyl analogue (IC50 = 0.46
μM for 31 versus 0.079 μM for 30). 1,3- and 1,5-Disubstituted pyrazole analogues (32 and 33) and the 3,5-disubstituted isoxazole 35 also lost potency despite an improvement in metabolic stability;
we rationalized this loss of potency in terms of the sterically encumbered
pocket into which the C-2-pyrazole substituent projects. Imidazole
analogue 36 also lost potency in comparison with pyrazole 25 despite the presence of a potentially isosteric H-bond-donor
or -acceptor interaction with Lys553, depending on the protonation
state of the imidazole. Taken together, these results suggested tight
SAR along the C-2 vector from the pyrrolopyridine scaffold and were
consistent with the crystal structure of MPS1 complexed with compound 8 that showed that the interaction with Lys553 is important
(Figure 3). Unsubstituted oxazole 34 proved to be the only potential C-2 pyrazole replacement that maintained
potency with enhanced metabolic stability. The crystal structure of
MPS1 in complex with oxazole 34 showed unambiguous electron
density for the ligand, supporting the initial binding mode for compound 8 and consistent with the SAR observed for the aniline and
C-2 heterocycle modifications made to the pyrrolopyridine scaffold
(Figure 4). The hinge-binding motif was the
same as that observed with compound 8, and the aniline
C-2-methoxy substituent was positioned, as expected, in a small hydrophobic
pocket lined by Lys529, Ile531, Gln541, and the gatekeeper +2 residue,
Cys604. This pocket is not accessible in many other kinases, including
CDK2, GSK3β, Aurora A, and Aurora B, which have bulkier residues
at the corresponding gatekeeper +2 position (Phe in CDK2 and Tyr in
GSK3β and Aurora A and B). This is consistent with improved
selectivity for MPS1 versus related kinases observed for compounds
with an aniline 2-substituent (Tables 1 and 2).[25] Like the pyrazole
of compound 8, the oxazole of 34 was oriented
toward the catalytic Lys553 residue, and an H-bond was observed between
the pyrrolopyridine N-1 atom and a water molecule. However, despite
the presence of a preferred aniline C-2-substituent, the weak CDK2
activity of 34 coupled with the relative complexity of
the synthetic route to C-2 oxazoles (Scheme 5) disfavored this approach. We selected 1-methylpyrazole in preference
to the 1-trifluoroethyl pyrazole as the optimal pyrrolopyridine C-2
substituent because of its lower lipophilicity and improved ligand
efficiency.
Figure 4
Crystal structure of MPS1 with compound 34 bound.
Compound 34 is shown with orange carbon atoms. Selected
amino acids that contact the ligand are shown with dark green carbon
atoms. H-bond interactions are shown as black dotted lines. The electron
density shown in green is from an Fo – Fc omit map and is contoured at 3σ.
MLM: percentage of parent compound
metabolized after a 30 min incubation in mouse liver microsomes.n = 1.Crystal structure of MPS1 with compound 34 bound.
Compound 34 is shown with orange carbon atoms. Selected
amino acids that contact the ligand are shown with dark green carbon
atoms. H-bond interactions are shown as black dotted lines. The electron
density shown in green is from an Fo – Fc omit map and is contoured at 3σ.We then investigated a range of
aniline substitutions with the
aim of further improving metabolic stability by reduction of both
lipophilicity and electron density in the aniline moiety. 2-Methoxy-5-trifluoromethyl
analogue 37 (IC50 = 4.4 μM; Table 2) illustrates poor tolerance of a 2,5-disubstitution
pattern on the aniline ring. Analysis of the compound 34-bound MPS1 structure suggested that the addition of a CF3 substituent to the 5-position of the aniline ring would induce a
steric clash with Asp608 (Figure 4). This observation
is consistent with the SAR described for a series of Leucine Rich
Repeat Kinase 2 (LRRK2) inhibitors in which a 2,5-disubstituted aniline
was employed to drive selectivity for LRRK2 over MPS1.[32] Exploitation of the aniline C-4 vector, which
extends into the solvent channel (Figure 3),
was more successful and led to the synthesis of compounds 39–44, all of which displayed good potency compared
to their unsubstituted parent 38, improved selectivity,
and in vitro metabolic stability (Table 2).
However, the measured aqueous thermodynamic solubility was low (e.g.,
0.01 mg/mL for compound 42).2-Chloro-4-dimethylcarboxamido-substituted
aniline 39 was selected for pharmacokinetic evaluation
on the basis of its
excellent potency, in vitro selectivity, and improved metabolic stability
in mouse and human liver microsomes (25 and 20% turnover after a 30
min incubation, respectively). This compound displayed an improved
efflux ratio in Caco-2 (10) compared to original hit compound 8 and demonstrated good in vivo pharmacokinetics in mouse
with a low unbound clearance and moderate oral bioavailability, consistent
with our strategy of targeting improved in vitro metabolic stability
versus compound 8 (Table 3).
Table 3
In Vivo Mouse Plasma
Pharmacokinetic
Profile of 39 after Oral and iv Dosing (10 mg/kg)
t1/2 (h)
Cl (mL/min/kg)
PPB (%)
Clu (mL/min/kg)
Vd (L/kg)
F (%)
1.05
4.74
94.1
80
0.32
48
A
crystal structure of compound 39 bound to MPS1 was
obtained by soaking MPS1 crystals for 24 h in a solution containing
1.25 mM of the inhibitor. The crystal structure showed that the overall
binding mode of 39 was very similar to those of HTS hit
compound 8 and oxazole 34. However, the
crystallographic data revealed significant electron density along
a vector aligned from the pyrrolopyridine N-1 position (Figure 5A), which could not be explained by the interacting
water molecule observed in the compound 8-bound and compound 34-bound structures. Analysis of the compound sample used
for the soaking experiment revealed a 2% impurity of the N-Boc synthetic
precursor (48; Table 5), which
fitted well with the additional electron density observed in the crystal
structure. We reasoned that to preferentially occupy the ATP-binding
site in a soaking experiment involving a 50-fold excess of compound 39, compound 48 must be significantly more potent
than compound 39. Subsequent elucidation of the crystal
structure of MPS1 bound to the N-Boc-containing precursor, compound 48, confirmed this hypothesis because the overall binding
mode of 48 was entirely consistent with the other crystal
structures and the Boc group was clearly present in the same location
as the additional electron density in the compound 39-bound structure (Figure 5B). The aniline
C-2-chloro substituent of compound 48 was located in
the same lipophilic pocket as the aniline C-2-methoxy group in compound 34, consistent with the improved selectivity afforded by small
lipophilic substituents in the aniline C-2 position (Tables 1 and 2).
Figure 5
Crystal structure of
MPS1 with compounds 39 (panel
A) and 48 (panel B) bound. Compounds 39 and 48 are shown with orange carbon atoms. Selected amino acids
that contact the ligands are shown with dark green carbon atoms. H-bond
interactions are shown as black dotted lines. The electron density
shown in green in each panel is from an Fo – Fc omit map and is contoured
at 3σ.
Crystal structure of
MPS1 with compounds 39 (panel
A) and 48 (panel B) bound. Compounds 39 and 48 are shown with orange carbon atoms. Selected amino acids
that contact the ligands are shown with dark green carbon atoms. H-bond
interactions are shown as black dotted lines. The electron density
shown in green in each panel is from an Fo – Fc omit map and is contoured
at 3σ.Compound 48 displayed potency at the low end of the
dynamic range of our in vitro MPS1 assay (IC50 = 0.006
μM; Table 5; MPS1 enzyme concentration
= 3–12.5 nM, see the Supporting Information). We therefore set up a high-throughput cell-based assay that measures
the inhibition of ectopic MPS1 autophosphorylation at Thr33 and Ser37
using an MSD electrochemiluminescent readout (see the Supporting Information) to discriminate more
potent compounds. Comparison of compound 39 and its N-Boc
precursor 48 in this assay revealed a 7-fold increase
in cellular potency because of the N-Boc substituent (P-MPS1 IC50 = 0.60 μM for compound 48 versus 4.10
μM for compound 39 and HCT116 GI50 =
2.20 μM for compound 48 versus 9.80 μM for
compound 39; Table 5). We hypothesized
that a combination of increased in vitro potency and lipophilicity-driven
cell penetration was responsible for the increase in cellular potency.
Gratifyingly, compound 48 not only retained metabolic
stability in mouse and human liver microsomes (48 and 34% turnover
after a 30 min incubation, respectively; Table 5) despite increased lipophilicity (48 AlogP = 5.1, 39 AlogP = 3.0) but also displayed reasonable chemical stability
in aqueous acid and base (38% cleavage of the Boc group was observed
after a 75 min incubation of 48 in a simulated gastric
acid fluid at 37 °C, and no cleavage was observed after a 5 h
incubation of 48 in a simulated duodenum solution at
37 °C), indicating that the N-1-Boc substituent of compound 48 may survive gut media on oral administration. Moreover,
the increased lipophilicity of 48 improved passive permeability
(PAMPA >100 × 10–6 cm/s at pH 7.4) and abrogated
Caco-2 efflux (A to B = 17 × 10–6 cm/s, efflux
ratio = 1). In vivo pharmacokinetic profiling in mouse revealed increased
clearance, consistent with higher lipophilicity and MLM metabolic
turnover (48% for 48 at 30 min versus 25% for 39), increased volume of distribution, and higher bioavailability compared
to compound 39, consistent with increased lipophilicity
and passive permeability (Table 4). Finally, we were concerned that increased lipophilicity
imparted by our serendipitous discovery of the influential N-Boc substituent
might erode the in vitro selectivity profile; however, selectivity
versus CDK2 and Aurora A was maintained (Table 5), and we also observed complete selectivity over other mitotic kinases,
for example, NIMA-related kinase 2 (NEK2) and Polo-Like Kinase 1 (PLK1)
(IC50 > 100 μM). Thus, compound 48 resolved
many of our issues with original hit compound 8, and
we elected to maintain the N-1-Boc substituent in further analogues.
We next turned our attention to improvement of the cell-based GI50 in HCT116 cells, which remained relatively weak for compound 48 (GI50 = 2.20 μM; Table 5).
Table 4
In Vivo Mouse Plasma Pharmacokinetic
Profile of 48 after Oral and iv Dosing (5 mg/kg)
t1/2 (h)
Cl (mL/min/kg)
PPB (%)
Clu (mL/min/kg)
Vd (L/kg)
F (%)
3.26
12.44
99.5
2589
1.99
78
MLM/HLM:
percentage of parent compound
metabolized after a 30 min incubation.ND = not determined.n = 1.As expected, further exploration of the aniline C-4 vector in the
N-Boc-substituted pyrrolopyridine series revealed broad tolerance
for a variety of substituents, with optimal translation to cell-based
potency observed for azetidine amide 51, piperidine amides
(52 and 53), and thiomorpholine 1,1-dioxide
amide 54. Consistent with previous SAR, we were pleased
to note that C-2-oxazole 55 was also tolerated in this
series (Table 5), and the crystal structure
of 55 bound to MPS1 confirmed that the oxazole maintains
an interaction with Lys553 (Figure 6), consistent
with the structure of MPS1 with compound 34. However,
neither the C-2-oxazole nor the C-2-pyrazole compounds with variations
at the aniline C-4 vector provided a significant improvement in cell-based
antiproliferative activity (Table 5).
Figure 6
Crystal structure
of MPS1 with compound 55 bound.
Selected amino acids are shown with dark green carbon atoms. Compound 55 is shown with orange carbon atoms. H-bond interactions
are shown as black dotted lines. The electron density shown in green
is from an Fo – Fc omit map and is contoured at 3σ.
Crystal structure
of MPS1 with compound 55 bound.
Selected amino acids are shown with dark green carbon atoms. Compound 55 is shown with orange carbon atoms. H-bond interactions
are shown as black dotted lines. The electron density shown in green
is from an Fo – Fc omit map and is contoured at 3σ.Analysis of the crystal structures of compounds 48 (Figure 5B) and 55 (Figure 6) showed that the aniline C-4-dimethylamido substituent
projected toward the solvent above the Asp608–Ser611 helix-capping
motif in the post-hinge region of the kinase. This suggested that replacement
of the aniline 4-amido substituent with an appropriate heterocycle
may be tolerated, and we were keen to explore the effect of this modification
on cellular potency (Table 6). Gratifyingly, C-4-pyrazolo analogues 61, 62, and 63 proved to be potent inhibitors of
MPS1 in the in vitro biochemical assay, with acceptable metabolic
stability in mouse and human liver microsomes. Analogous to our observations
with compounds 39 and 48, we observed a
significant (43-fold) increase in inhibition of MPS1 autophosphorylation
in cells for N-1-Boc-substituted compound 61 versus its
N-1-H analogue 62 (P-MPS1 IC50 = 0.16 μM
versus 6.90 μM), and this improvement was also observed in an
assay of cell proliferation (61, HCT116 GI50 = 0.50 μM; 62, HCT116 GI50 = 4.60
μM). Although oxazole-substituted analogue 64 and
those substituted with six-membered heterocycles, 66–68, all maintained potent inhibition of MPS1 in vitro, translation
to cell-based activity was not improved compared to pyrazole 61 (Table 6). However, the 1-methyl-imidazol-5-yl
moiety at the 4-position of the aniline (compound 65)
conferred improved translation to cell-based potency (P-MPS1 IC50 = 0.04 μM and HCT116 GI50 = 0.16 μM),
which is comparable to or better than the cell-based potency of reported
MPS1-selective inhibitors tested in our assays (Table 7).
Table 7
Comparison of Compound 65 (CCT251455) with Reported MPS1 Inhibitors
biochemical IC50 (μM)
cellular activity (μM)
compd
MPS1
CDK2
Aurora A
P-MPS1 IC50
HCT116 GI50
65
0.003 ± 0.002
>100
>40
0.043 ± 0.026
0.16 ± 0.09
4
0.007 ± 0.009
36.00a
21.00 ± 11.00
0.72a
1.20a
5
0.007 ± 0.003
>100
>30
0.16 ± 0.12
0.18 ± 0.09
6
0.011 ± 0.004
8.80a
>100
0.56 ± 0.24
1.60 ± 0.75
n = 1.
MLM/HLM:
percentage of parent compound
metabolized after a 30 min incubation in mouse and human liver microsomes.n = 1.n = 1.Compound 65 displayed in vitro potency versus MPS1
at the low end of the dynamic range of our in vitro assay, which together
with an excellent translation to cell-based assays prompted further
analysis of the binding mode of 65 by X-ray crystallography
(Figure 7A). The structure was determined by
cocrystallization of the kinase domain of MPS1 with 65 using PEG3350 as the precipitant instead of PEG300 in an attempt
to remove the artifactual PEG molecule bound in the ATP-binding site.
This resulted in a more physiologically relevant structure without
a PEG molecule wrapped around the active-site Lys553 residue, which
allowed for the formation of the conserved Lys553–Glu571 ion-pair.
Although the binding mode of compound 65 was entirely
consistent with our previous compound-bound crystal structures, MPS1
activation-loop residues Ala668–Thr675 adopt an ordered conformation,
and the ordered loop forms an antiparallel β-sheet interaction
with the P-loop (Figure 7B). In addition, activation-loop
residues Met671–Pro673 form a complementary hydrophobic pocket
wrapped around the N-1-Boc substituent of 65, completely
enclosing the inhibitor in the ATP-binding site. Intriguingly, this
activation-loop conformation has previously been observed in the crystal
structure of MPS1 with a pyrimidodiazepine ligand (PDB code 3H9F)[20] and is incompatible with binding of a PEG molecule around
Lys553 because of steric hindrance. In this structure, a triply phosphorylated
Thr–Thr–Ser motif at residues 675–677 formed
magnesium-mediated crystal contacts, which may have influenced the
activation-loop conformation. A careful analysis of two recently reported
crystal structures of MPS1, one in complex with a diamino-pyridine
inhibitor (PDB code 3VQU)[26] and one with an early indazole-based
inhibitor (PDB code 3W1F),[27] also showed a similarly ordered activation
loop in both structures but with the Thr–Thr–Ser motif
disordered. In our compound 65-bound MPS1 structure,
Thr676 and Ser677 are also disordered, and we did not observe electron
density for a phosphate group on Thr675 or for the mediating magnesium
atoms. This suggests that in our 65-bound MPS1 structure
these residues are not involved in meaningful crystal contacts that
could have an effect on the conformation of the activation loop. Further
analysis of the crystal packing showed that the only residue of a
symmetry-related molecule that is in the vicinity of the activation
loop is Ile738. However, a comparison of several compound-bound structures
(Supporting InformationFigure S2A) showed that Ile738 is located in a region of the
protein with only minor conformational flexibility, whereas the activation
loop in the respective structures shows a wide range of conformations
and a varying degree of order. The absence of any concerted conformational
changes between the activation loop and the symmetry-related Ile738
region in these structures led us to conclude that the ordering of
the activation loop is not influenced by crystal contacts. Nevertheless,
the ordering of the activation loop is clearly prevented by the binding
of a PEG molecule in MPS1 structures resulting from the PEG300-containing
crystallization conditions. This is supported by soaking MPS1 crystals
grown in PEG300 with compound 65, which resulted in a 65-bound MPS1 structure with a disordered activation loop
(data not shown).
Figure 7
Crystal structure of MPS1 with compound 65 bound.
(A) Selected amino acids are shown with dark green carbon atoms. Compound 65 is shown with orange carbon atoms. H-bond interactions
are shown as black dotted lines. The electron density shown in green
is from an Fo – Fc omit map and is contoured at 3σ. (B) Compound 65 is shown as a surface, and MPS1 is shown in dark green
as ribbons. Activation-loop residues Ala668–Thr675 are highlighted
with blue carbon atoms and blue ribbon representation. (C) Superposition
of the compound 65-bound MPS1 structure in green and
the structure of MPS1 complexed with an indazole-based inhibitor (PDB
code 3W1F)[27] in cyan, with an ethoxy-group in similar position
as the N-1 Boc in compound 65. The ordered activation
loops interacting with the respective inhibitors are indicated. (D)
Comparison of the ATP-bound structure of MPS1 (PDB code 3HMN)[34] shown in lilac with the compound 65-bound
structure shown in green. The activation loop in the MPS1-65 complex structure is highlighted in blue, and clashes with all three
phosphates of ATP in the 3HMN structure.
The structures of MPS1 in complex with the
precursors of the diamino-pyridine
and indazole-based inhibitors 6(26) and 7(27) show an ordering
of the activation loop through interactions with the P-loop and an
ethoxy-group, which in both inhibitors is located in a similar position
as the N-1-Boc substituent in 65. Moreover, in the pyrimidodiazepine-bound
MPS1 structure, the inhibitor interacts with the ordered activation
loop via a cyclopentyl moiety in a similar position as the N-1 Boc
in compound 65. Taken together, these findings support
the hypothesis that the ordering of the activation loop might have
a compound-dependent component for inhibitors with substituents similar
to the N-1 Boc in compound 65.It is important
to note that the overall structure of the MPS1
kinase domain in the compound 65-bound structure has
many features of an active kinase conformation. These include the
positioning of the αC-helix and the conserved DFG motif in an
“in” conformation as well as the presence of the canonical
active Lys553–Glu571 ion pair. Further analysis of the conformation
in light of the “spine concept”[33] clearly shows the presence of the catalytic C-spine and shows only
minor distortions in the regulatory R-spine (Supporting
Figure 2B), also indicating that the conformation of MPS1 in
this structure is close to an active kinase conformation. However,
the compound-induced conformation of the activation loop is incompatible
with ATP binding and substrate-peptide binding to the kinase because
it blocks the phosphate-binding region and the peptide binding site
is not formed, a situation that would certainly render MPS1 inactive
(Figure 7D).Crystal structure of MPS1 with compound 65 bound.
(A) Selected amino acids are shown with dark green carbon atoms. Compound 65 is shown with orange carbon atoms. H-bond interactions
are shown as black dotted lines. The electron density shown in green
is from an Fo – Fc omit map and is contoured at 3σ. (B) Compound 65 is shown as a surface, and MPS1 is shown in dark green
as ribbons. Activation-loop residues Ala668–Thr675 are highlighted
with blue carbon atoms and blue ribbon representation. (C) Superposition
of the compound 65-bound MPS1 structure in green and
the structure of MPS1 complexed with an indazole-based inhibitor (PDB
code 3W1F)[27] in cyan, with an ethoxy-group in similar position
as the N-1 Boc in compound 65. The ordered activation
loops interacting with the respective inhibitors are indicated. (D)
Comparison of the ATP-bound structure of MPS1 (PDB code 3HMN)[34] shown in lilac with the compound 65-bound
structure shown in green. The activation loop in the MPS1-65 complex structure is highlighted in blue, and clashes with all three
phosphates of ATP in the 3HMN structure.Introduction of the methylimidazole into compound 65 resulted in compromised passive permeability (PAMPA = 18 ×
10–6 cm/s at pH 7.4) and increased efflux (Caco-2
A to B = 5 × 10–6 cm/s, B to A = 12 ×
10–6 cm/s, ER = 2.5) compared to compound 48. Although the thermodynamic aqueous solubility of compound 65 was very low (<0.001 mg/mL), solubility in fasted- and
fed-state-simulated intestinal fluid (FaSSIF and FeSSIF) was 0.01
and ∼0.55 mg/mL, respectively, consistent with the presence
of a weakly basic center (the methylimidazole ring). All other in
vitro properties were maintained, and in view of its favorable in
vitro profile, compound 65 was selected for more extensive
in vitro profiling versus a panel of 121 kinases (Supporting InformationTable S1) and in vivo pharmacokinetic evaluation. Of the 121 kinases in the
panel, MPS1 showed the greatest inhibition by 65, and
only three other kinases showed inhibition greater than 80%. Mouse
and rat blood pharmacokinetics revealed a favorable profile with moderate
clearance and good to moderate oral bioavailability (Table 8). This compound was
progressed to a human tumor xenograft model to test whether pharmacodynamic
biomarker modulation could be achieved in vivo. Oral administration
of two doses of compound 65 at 50, 75, and 100 mg/kg
b.i.d. to mice bearing HCT116 human colon carcinoma xenografts demonstrated
dose-dependent modulation of MPS1-driven phospho-histone H3 levels
versus control animals at 2 and 10 h but not at 72 h after the last
dose, consistent with engagement of MPS1 in vivo (Figure 8A). The compound was well-tolerated at all doses,
and the observed decrease in phospho-histone H3 inhibition over time
by compound 65 tracks with a decrease in total plasma
and tumor tissue exposure measured in the same experiment (Figure 8B,C).
Table 8
Mouse and Rat in
Vivo PK Profile of
Compound 65 Dosed at 5 mg/kg iv/po
species
t1/2 (h)
Cl (mL/min/kg)
PPB (%)
Vd (L/kg)
F (%)
mouse
4.2
23.8
99.93
2.3
83
rat
3.1
7.04
ND
1.75
32
ND = not
determined.
Figure 8
(A) Representative immunoblots
of phospho-histone H3 showing dose-dependent
PD modulation in HCT116 human tumor xenografts following two oral
doses of 65 (50, 75, and 100 mg/kg) or vehicle. Total
histone H3, cleaved poly ADP ribose polymerase (PARP, a measure of
apoptosis), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, for
protein loading) are also shown. (B, C) Plasma and tumor exposure
levels of 65 from the same experiment measured 2 (light
gray bars) or 10 h (dark gray bars) after the last dose.
ND = not
determined.(A) Representative immunoblots
of phospho-histone H3 showing dose-dependent
PD modulation in HCT116 human tumor xenografts following two oral
doses of 65 (50, 75, and 100 mg/kg) or vehicle. Total
histone H3, cleaved poly ADP ribose polymerase (PARP, a measure of
apoptosis), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, for
protein loading) are also shown. (B, C) Plasma and tumor exposure
levels of 65 from the same experiment measured 2 (light
gray bars) or 10 h (dark gray bars) after the last dose.
Conclusions
We describe the structure-based
optimization of a potent but nonselective
and metabolically unstable 1H-pyrrolo[3,2-c]pyridine HTS hit 8 to compound 65, a highly potent inhibitor of MPS1 that demonstrates high selectivity
versus kinases tested in a broad kinome profiling panel. We observed
excellent translation of in vitro biochemical potency versus isolated
MPS1 enzyme to cell-based potency (P-MPS1 IC50 = 0.04 μM
and HCT116 GI50 = 0.16 μM), which is comparable to
or better than the cell-based potency of other literature-reported
MPS1-selective inhibitors tested side-by-side in our assays. Medicinal-chemistry
optimization to 65 was educated by structure-based design;
in particular, incorporation of an N-1-carbamate substituent was inspired
by our observation that the activation loop of MPS1 became ordered
in the presence of this substituent. The crystal structure of 65 in MPS1 confirmed this activation-loop stabilization, which
results in an occluded ATP-binding site and is incompatible with ATP
and substrate binding. Despite the increased lipophilicity imparted
by the carbamate moiety, 65 demonstrates a good oral
pharmacokinetic profile in mouse and rat as well as inhibition of
MPS1 activity in vivo following oral administration; 65 is a suitable chemical probe[35] for cell-based
assays and in vivo evaluation of the effect of MPS1 inhibition in
human tumor xenograft models.
Experimental Section
Chemistry
Commercially available starting materials,
reagents and dry solvents were used as supplied. Flash column chromatography
was performed using Merck silica gel 60 (0.025–0.04 mm). Column
chromatography was also performed on a FlashMaster personal unit using
isolute Flash silica columns or a Biotage SP1 purification system
using Merck or Biotage Flash silica cartridges. Preparative TLC was
performed on Analtech or Merck plates. Ion-exchange chromatography
was performed using acidic Isolute Flash SCX-II columns, Isolute Si-carbonate
columns, or basic Isolute Flash NH2 columns. Preparative
HPLC was conducted using a Phenomenex Luna column (5 μm, 250
× 21.2 mm, C18, Phenomenex, Torrance, USA) using a Gilson GX-281
liquid handler system combined with a Gilson 322 HPLC pump (Gilson,
Middleton, USA) over a 15 min gradient elution from 10:90 to 100:0
MeOH/water (both modified with 0.1% formic acid) at a flow rate of
20 mL/min or over a 15 min gradient elution from 40:60 to 100:0 MeOH/water
(both modified with 0.1% formic acid) at a flow rate of 20 mL/min.
UV–vis spectra were acquired at 254 nm on a Gilson 156 UV–vis
detector (Gilson, Middleton, USA). Collection was triggered by UV
signal and collected using a Gilson GX-281 liquid handler system (Gilson,
Middleton, USA). Raw data was processed using Gilson Trilution Software. 1H NMR spectra were recorded on a Bruker Avance-500. Samples
were prepared as solutions in a deuterated solvent and referenced
to the appropriate internal deuterated solvent peak or tetramethylsilane.
Chemical shifts were recorded in ppm (δ) downfield of tetramethylsilane.
LC/MS analysis was performed on a Waters Alliance 2795 Separations
Module and Waters 2487 dual-wavelength absorbance detector coupled
to a Waters/Micromass LCt time-of-flight mass spectrometer with ESI
source. Analytical separation was carried out at 30 °C either
on a Merck Chromolith SpeedROD column (RP-18e, 50 × 4.6 mm) using
a flow rate of 2 mL/min in a 3.5 min gradient elution with detection
at 254 nm or on a Merck Purospher STAR column (RP-18e, 30 × 4
mm) using a flow rate of 1.5 mL/min in a 3.5 min gradient elution
with detection at 254 nm. The mobile phase was a mixture of MeOH (solvent
A) and water (solvent B), both containing formic acid at 0.1%. Gradient
elution was as follows: 1:9 (A/B) to 9:1 (A/B) over 2.25 min, 9:1
(A/B) for 0.75 min, and then reversion back to 1:9 (A/B) over 0.3
min, and finally 1:9 (A/B) for 0.2 min (also referred to as ESI-HRMS
method A). LC/MS and HRMS analyses were performed on an Agilent 1200
series HPLC and diode array detector coupled to a 6210 time-of-flight
mass spectrometer with dual multimode atmospheric pressure CI/ESI
source. Analytical separation was carried out at 30 °C either
on a Merck Chromolith SpeedROD column (RP-18e, 50 × 4.6 mm) using
a flow rate of 2 mL/min in a 4 min gradient elution with detection
at 254 nm or on a Merck Purospher STAR column (RP-18e, 30 × 4
mm) using a flow rate of 1.5 mL/min in a 4 min gradient elution with
detection at 254 nm. The mobile phase was a mixture of MeOH (solvent
A) and water (solvent B), both containing formic acid at 0.1%. Gradient
elution was as follows: 1:9 (A/B) to 9:1 (A/B) over 2.5 min, 9:1 (A/B)
for 1 min, and then reversion back to 1:9 (A/B) over 0.3 min, and
finally 1:9 (A/B) for 0.2 min (default method, also referred to as
ESI-HRMS method B). The following references masses were used for
HRMS analysis: caffeine [M + H]+ 195.087652, (hexakis(1H,1H,3H-tetrafluoropentoxy)phosphazene
[M + H]+ 922.009798), and hexakis(2,2-difluoroethoxy)phosphazene
[M + H]+ 622.02896 or reserpine [M + H]+ 609.280657.
All tested compounds gave >95% purity as determined by either method
A or method B.
Preparation of Compounds in Table 1 (Exemplified
by the Preparation of Compound 3)
2-Bromo-5-iodopyridin-4-amine
(15)
4-Amino-2-bromopyridine 13 (22.8 g, 131.8 mmol) and sodium acetate (20.8 g, 254 mmol)
were stirred in AcOH (82 mL), and a solution of iodine monochloride
(1 M in AcOH, 134 mL, 134 mmol) was added. The mixture was stirred
and heated at 75 °C for 3 h. Most of the AcOH was evaporated,
and the residue was partitioned between water and EtOAc. The aqueous
fraction was again extracted with EtOAc. The combined extracts were
washed twice with 10% sodium carbonate solution, 10% sodium thiosulfate
solution, water, and brine, dried, and evaporated. This gave 40.3
g of a crude product that was combined with the product from a reaction
on 7.5 g of 4-amino-2-bromopyridine. Purification by chromatography
on a silica column (9 cm internal diameter with 28 cm bed of silica)
eluting with 5% EtOAc in CH2Cl2, then 10% EtOAc
in CH2Cl2, and then 20% EtOAc in CH2Cl2 gave desired isomer 15 (20.2 g, 38%). 1H NMR (500 MHz, CDCl3): δ 4.74 (br s, 2H,
NH2), 6.80 (s, 1H), 8.34 (s, 1H). LC (method A)-MS (ESI, m/z) tR 1.82
min, 299 [(M + H+), 100%] and subsequently with EtOAc/CH2Cl2 (1:1) to give undesired isomer 4-amino-2-bromo-3-iodopyridine 14 (19.3 g, 37%). LC (method A)-MS (ESI, m/z) tR 1.62 min, 299
[(M + H+), 100%].
4-Amino-2-bromo-5-iodopyridine 15 (3.055 g, 10.2 mmol) was stirred in CH2Cl2 (34 mL), and Et3N (6.9 mL, 49.1 mmol) was added. The
mixture was cooled in ice. To the cold solution was added dropwise
a solution of methanesulfonyl chloride (3.2 mL, 40.6 mmol) in CH2Cl2 (11.5 mL) over a period of 14 min. The cold
bath was removed, and the reaction was stirred at rt for 1.5 h. The
reaction was diluted with CH2Cl2 and washed
twice with water. The solution was dried and evaporated. Trituration
with ether gave a solid (5.01 g). The crude product was passed in
5% EtOAc in CH2Cl2 through a 2.5 cm pad of silica
in a 10 cm diameter sinter to give N-(2-bromo-5-iodopyridin-4-yl)-N-(methylsulfonyl)methanesulfonamide (3.01 g, 64%). 1H NMR (500 MHz, CDCl3): δ 3.60 (s, 6H), 7.53
(s, 1H), 8.89 (s, 1H). LC (method A)-MS (ESI, m/z) tR 1.91 min, 455 [(M + H+), 100%]. N-(2-Bromo-5-iodopyridin-4-yl)-N-(methylsulfonyl)methanesulfonamide (228 mg, 0.50 mmol)
was stirred with THF (1.3 mL) and 10% sodium hydroxide in water (1.3
mL) at rt for 3 h. The THF was evaporated, and the aqueous phase was
neutralized using a 10% citric acid solution. The deposited white
solid was filtered off, washed with water, and dried in a vacuum desiccator
over sodium hydroxide to give 16 (159 mg, 84%). 1H NMR (500 MHz, DMSO-d6): δ
3.29 (s, 3H), 7.54 (s, 1H), 8.64 (s, 1H). LC (method A)-MS (ESI, m/z) tR 1.89
min, 377 [(M + H+), 100%].
4-Iodopyrazole 18 (7.85 g,
40.4 mmol) was dissolved in THF (120 mL), and
Et3N (8.5 mL, 60.5 mmol) and di-tert-butyl
dicarbonate (9.7 g, 44.5 mmol) were added. The reaction was stirred
at rt for 3 h. The THF was evaporated, and EtOAc was added. The solution
was washed with water and brine, dried, and evaporated to leave an
oil (14.2 g). The crude product was purified by chromatography on
a pad of silica in a sinter (10 cm diameter, 6 cm thick) eluted with
10% EtOAc in cyclohexane and then 20% EtOAc in cyclohexane to give tert-butyl 4-iodo-1H-pyrazole-1-carboxylate
(11.66 g, 98%). 1H NMR (500 MHz, CDCl3): δ
1.68 (s, 9H), 7.73 (s, 1H), 8.17 (s, 1H). tert-Butyl
4-iodo-1H-pyrazole-1-carboxylate (4.67 g, 15.9 mmol)
and trimethylsilylacetylene (2.18 g, 22.2 mmol) were dissolved in
DMF (22 mL) and placed under argon. Diisopropylamine (2.9 mL, 20.7
mmol), copper(I) iodide (197 mg, 1.03 mmol), triphenylphosphine (832
mg, 3.18 mmol), and palladium acetate (239 mg, 1.06 mmol) were added,
and the flask was flushed again with argon. The reaction was heated
at 60 °C for 1.25 h. The reaction was cooled and added to water.
The product was extracted with ether. The combined extracts were washed
with water and brine, dried, and evaporated. The crude product was
purified by flash chromatography (silica, eluting with 10% EtOAc in
cyclohexane) to give 19 (3.88 g, 92%). 1H
NMR (500 MHz, CDCl3): δ 0.25 (s, 6H), 1.67 (s, 9H),
7.77 (d, J = 0.6 Hz, 1H), 8.20 (d, J = 0.6 Hz, 1H).
tert-Butyl 4-((trimethylsilyl)ethynyl)-1H-pyrazole-1-carboxylate 19 (3.88 g, 14.69
mmol) was dissolved in THF (40 mL) and cooled to 0–5 °C.
A 1 M solution of tetrabutylammonium fluoride in THF (16 mL, 16 mmol)
was added, and the reaction was stirred for 20 min. The THF was evaporated,
and the residue was taken up in EtOAc and washed with water and brine,
dried, and evaporated. The residue was purified on a flash column
(silica, eluting with 15% EtOAc in cyclohexane) to give 20 (1.765 g, 62%). 1H NMR (500 MHz, CDCl3): δ
1.68 (s, 9H), 3.11 (s, 1H), 7.79 (s, 1H), 8.24 (s, 1H). LC (method
A)-MS (ESI, m/z) tR 2.27 min, no ion recorded.
To a mixture of N-(2-bromo-5-iodopyridin-4-yl)methanesulfonamide 16 (419 mg, 1.11 mmol) and tert-butyl-4-ethynyl-1H-pyrazole-1-carboxylate 20 (277 mg, 1.44 mmol)
were added copper(I) iodide (7.4 mg, 0.039 mmol) and DMF (4 mL) followed
by Et3N (0.69 mL, 4.92 mmol). The reaction was flushed
with nitrogen. Bis(triphenylphosphine)palladium dichloride (27 mg,
0.038 mmol) was added, the reaction flushed with nitrogen, and it
was heated at 60 °C for 70 min. The reaction was added to water
and extracted with EtOAc. The combined extracts were washed with water
and brine, dried, and evaporated. The residue was purified on four
2 mm, 20 × 20 cm silica prep TLC plates and eluted with EtOAc/cyclohexane
(3:1). The product band was recovered with acetone to give tert-butyl 4-(6-bromo-1-(methylsulfonyl)-1H-pyrrolo[3,2-c]pyridin-2-yl)-1H-pyrazole-1-carboxylate (251 mg, 51%). 1H NMR (500 MHz,
CDCl3): δ 1.73 (s, 9H), 3.02 (s, 3H), 6.81 (s, 1H),
7.97 (s, 1H), 8.26 (s, 1H), 8.43 (s, 1H), 8.69 (s, 1H). LC (method
A)-MS (ESI, m/z) tR 2.55 min, 441 [(M + H+), 100%]. tert-Butyl-4-(6-bromo-1-(methylsulfonyl)-1H-pyrrolo[3,2-c]pyridin-2-yl)-1H-pyrazole-1-carboxylate
(1.48 g, 3.35 mmol) was stirred in THF (20 mL), and DBU (0.51 mL,
3.4 mmol) was added. The reaction was warmed at 40 °C for 1 h.
The reaction was cooled, and THF was evaporated. The residue was dissolved
in EtOAc (50 mL), washed with water and brine, dried, and evaporated. 1H NMR of the residue revealed incomplete conversion. The material
was redissolved in THF (20 mL), and DBU (0.3 mL) was added. The reaction
was heated at 40 °C for 1.5 h. MeOH (1 mL) was added, and heating
was continued for 0.5 h. The solution was evaporated. and EtOAc was
added. The solution was washed with water. The organic solution was
washed again with water and brine, dried, and evaporated. 1H NMR of the residue revealed both demesylated and completely deprotected
products. To this material were added EtOAc and di-tert-butyl dicarbonate (1.11 g, 5.1 mmol) followed by Et3N
(0.72 mL, 5.1 mmol) and a crystal of DMAP. The reaction was stirred
at rt for 1 h, more di-tert-butyl dicarbonate (414
mg, 1.9 mmol) was added, and stirring was continued for a further
2 h. The solution was evaporated, and the residue was kept at ambient
temperature overnight. It was adsorbed from CH2Cl2 onto flash silica, packed onto a flash column made in 20% EtOAc
in cyclohexane, and eluted with this solvent and then with 40% EtOAc
in cyclohexane to give 17 (1.2 g, 77%). 1H
NMR (500 MHz, CDCl3): δ 1.58 (s, 9H), 1.69 (s, 9H),
6.67 (d, J = 1.0 Hz, 1H), 7.84 (d, J = 1.0 Hz, 1H), 8.24 (t, J = 0.6 Hz, 1H), 8.27 (d, J = 0.6 Hz, 1H), 8.61 (d, J = 0.6 Hz, 1H).
LC (method B)-MS (ESI, m/z) tR 3.31 min, 407 [(M + H+-C4H8), 100%].
To tert-butyl 6-bromo-2-(1-(tert-butoxycarbonyl)-1H-pyrazol-4-yl)-1H-pyrrolo[3,2-c]pyridine-1-carboxylate 17 (101 mg, 0.218
mmol) was added 3,4-dimethoxyaniline (42 mg, 0.275 mmol) followed
by cesium carbonate (140 mg, 0.432 mmol) and Xantphos (12.4 mg, 0.0216
mmol). Dioxane (2.4 mL) was added, and the flask flushed with nitrogen.
Tris(dibenzylideneacetone)dipalladium(0) (10 mg, 0.0108 mmol) was
added, and the flask was flushed again with nitrogen and heated at
80 °C for 5.25 h. The reaction was cooled and diluted with EtOAc.
The solution was washed with water and brine, dried, and evaporated.
The residue was applied in chloroform to three 1 mm, 20 × 20
cm silica prep plates that were eluted with CH2Cl2/EtOAc (9:1). The product band was recovered with acetone to give tert-butyl-2-(1-(tert-butoxycarbonyl)-1H-pyrazol-4-yl)-6-(3,4-dimethoxyphenylamino)-1H-pyrrolo[3,2-c]pyridine-1-carboxylate (91 mg, 77%). 1H NMR (500 MHz, CDCl3): δ 1.46 (s, 9H), 1.68
(s, 9H), 3.90 (s, 3H), 3.91 (s, 3H), 6.45 (br s, 1H), 6.56 (d, J = 1.0 Hz, 1H), 6.89 (m, 2H), 6.96 (m, 1H), 7.45 (m, 1H),
7.81 (d, J = 0.6 Hz, 1H), 8.20 (d, J = 1.0 Hz, 1H), 8.42 (d, J = 1.0 Hz, 1H). ESI-HRMS
calcd for C28H34N5O6 [M
+ H]+, 536.2504; found, 536.2528. tert-Butyl-2-(1-(tert-butoxycarbonyl)-1H-pyrazol-4-yl)-6-(3,4-dimethoxyphenylamino)-1H-pyrrolo[3,2-c]pyridine-1-carboxylate (88 mg, 0.164 mmol) was dissolved
in CH2Cl2 (0.8 mL), TFA (0.8 mL) was added to
the solution, and the mixture was stirred at rt for 3.5 h. The solution
was evaporated, and the residue dried in vacuum over NaOH for 1 h.
The residue was dissolved in MeOH, the solution was evaporated, and
the residue again was dried in a vacuum desiccator. The glassy material
was taken up in MeOH, and 1 M sodium hydroxide solution (180 μL,
0.18 mmol) was added. The solution was applied to a 2 g SCX-2 column,
and the column washed with more MeOH. The product was recovered using
2 M ammonia in MeOH to give a gum after evaporation (56 mg). This
was triturated with ether to give 8 as a solid (49 mg,
89%). 1H NMR (500 MHz, DMSO-d6): δ 3.74 (s, 3H), 3.74 (s, 3H), 6.50 (s, 1H), 6.72 (s, 1H),
6.84 (d, J = 8.5 Hz, 1H), 7.06 (dd, J = 8.5, 2.2 Hz, 1H), 7.21 (d, J = 2.2 Hz, 1H), 8.01
(s, 1H), 8.25 (s, 1H), 8.33 (s, 1H), 11.11 (br s, 1H), 12.96 (br s,
1H). ESI-HRMS calcd for C18H18N5O2 [M + H]+, 336.1455; found, 336.1468.
Preparation
of Compounds in Tables 2 and 5 (Exemplified by the Preparation of Compounds 39, 40, and 48)
4-Ethynyl-1-methyl-1H-pyrazole (45)
4-Iodopyrazole 18 (5.0 g, 25.7 mmol) was
dissolved in DMF (50 mL), and potassium carbonate (4.26 g, 30.9 mmol)
was added and stirred (2 min) before iodomethane (1.76 mL, 28.3 mmol)
was added. The reaction was stirred rapidly at rt for 17 h. It was
filtered through a Celite pad, and the filtrate was evaporated to
10 mL using a rotary evaporator. Water was added to the residue, and
the resultant mixture was extracted with EtOAc. The combined organics
were washed with water and brine, dried, and evaporated to give 1-methyl-4-iodopyrazole
as a solid (4.56 g, 85%). 1H NMR (500 MHz, CDCl3): δ 3.93 (s, 3H), 7.42 (s, 1H), 7.50 (s, 1H). LC (method A)-MS
(ESI, m/z) tR 1.74 min, 209 [(M + H+), 100%]. 1-Methyl-4-iodo-pyrazole
(5.0 g, 24.04 mmol) was dissolved in DMF (32 mL), and trimethylsilylacetylene
(4.76 mL, 33.7 mmol) was added followed by diisopropylamine (4.46
mL, 31.78 mmol), copper(I) iodide (304 mg, 1.59 mmol), and triphenylphosphine
(1.26 g, 4.81 mmol). The reaction was flushed with argon. Palladium
acetate (351 mg, 1.56 mmol) was added, and the reaction was again
flushed with argon. It was heated at 60 °C for 1 h. The reaction
was cooled, added to water, and extracted with ether. The organic
solution was filtered from a brown solid, which was washed with a
little more ether. The organic solution was washed with water and
brine, dried, and evaporated. The crude product was chromatographed
(silica gel) using EtOAc/cyclohexane (1:4) and then EtOAc/cyclohexane
(1:3) to give 1-methyl-4-((trimethylsilyl)ethynyl)-1H-pyrazole as a solid (2.85 g, 67%). 1H NMR (500 MHz, CDCl3): δ 0.24 (s, 9H), 3.87 (s, 3H), 7.50 (s, 1H), 7.58
(s, 1H). LC (method A)-MS (ESI, m/z) tR 2.44 min, 179 [(M + H+), 100%]. 1-Methyl-4-((trimethylsilyl)ethynyl)-1H-pyrazole (6.86 g, 38.5 mmol) was dissolved in MeOH (77 mL), and
potassium carbonate (385 mg, 2.79 mmol) was added. The reaction was
stirred at rt for 2 h. MeOH was evaporated to a small volume. EtOAc
was added, and the solution washed with water and brine. Each aqueous
phase was backwashed with a single 40 mL portion of EtOAc. The EtOAc
solution was dried and evaporated, and the residue was chromatographed
(silica) and eluted with EtOAc/cyclohexane (1:3) and EtOAc/cyclohexane
(1:1) to give 45 (3.18 g, 77%). 1H NMR (500
MHz, CDCl3): δ 3.00 (s, 1H), 3.88 (s, 3H), 7.52 (s,
1H), 7.59 (s, 1H). LC (method A)-MS (ESI, m/z) tR 1.34 min, no ion recorded.
4-Amino-2-bromo-5-iodopyridine 15 (2.58 g, 8.63 mmol), copper(I) iodide (164 mg, 0.86 mmol), and bis(triphenyphosphine)palladium
dichloride (216 mg, 0.432 mmol) were weighed into a 100 mL flask,
and DMF (25 mL) with Et3N (22 mL) was added. The mixture
was stirred at rt for 15 min under nitrogen. 4-Ethynyl-1-methyl-1H-pyrazole 45 (945 mg, 8.91 mmol) in DMF (10
mL) and Et3N (5 mL) were added to the flask. The reaction
was stirred at rt for 1.75 h. The reaction was diluted with EtOAc,
and the solution was washed with water and brine, dried, and evaporated.
The residue was purified by flash chromatography (silica/EtOAc) to
give 46 (2.11 g, 88%). 1H NMR (500 MHz, CDCl3): δ 3.94 (s, 3H), 4.80 (br s, 2H), 6.79 (s, 1H), 7.59
(s, 1H), 7.66 (s, 1H), 8.15 (s, 1H). LC (method A)-MS (ESI, m/z) tR 1.83
min, 277 [(M + H+), 100%].
Potassium tert-butoxide (315 mg, 2.81 mmol) was dissolved in NMP (3
mL), and 2-bromo-5-((1-methyl-1H-pyrazol-4-yl)ethynyl)pyridin-4-amine 46 (375 mg, 1.35 mmol) was added to the stirred solution.
The reaction was placed under nitrogen and warmed at 50 °C for
3 h. The reaction was cooled, and 10% ammonium chloride (3 mL) added.
Water (21 mL) was heated to about 60 °C, and the product solution
in NMP/water was added to the water; a solid immediately crashes out.
The suspension was allowed to cool to rt and filtered, and the solid
was washed with water. Drying in a vacuum desiccator over KOH for
3 days gave product (347 mg) that was azeotroped with EtOH and toluene
to give 6-bromo-2-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[3,2-c]pyridine (315 mg, 84%). 1H NMR (500 MHz, DMSO-d6): δ
3.90 (s, 3H), 6.69 (d, J = 1.0 Hz, 1H), 7.47 (t, J = 1.0 Hz, 1H), 7.94 (d, J = 0.6 Hz, 1H),
8.18 (s, 1H), 8.50 (d, J = 1.0 Hz, 1H), 11.92 (br
s, 1H). LC (method A)-MS (ESI, m/z) tR 1.67 min, 277 [(M + H+), 100%]. 6-Bromo-2-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[3,2-c]pyridine (7.22 g, 26.1
mmol) was stirred in EtOAc (93 mL) and Et3N (5.3 mL, 37.8
mmol). To the suspension were added DMAP (622 mg, 5.1 mmol) and di-tert-butyl dicarbonate (8.30 g, 38.1 mmol). After 25 min,
solid was deposited from solution, and the suspension was evaporated
to dryness. The residue was chromatographed (silica, EtOAc/cyclohexane
(1:1) then EtOAc/cyclohexane (3:1) then pure EtOAc) to give 47 (8.8 g, 89%). 1H NMR (500 MHz, CDCl3): δ 1.57 (s, 9H), 3.98 (s, 3H), 6.57 (d, J = 0.6 Hz, 1H), 7.59 (s, 1H), 7.63 (d, J = 0.6 Hz,
1H), 8.19 (t, J = 0.6 Hz), 8.58 (d, J = 0.6 Hz, 1H). LC (method A)-MS (ESI, m/z) tR 2.75 min, 321 (M + H+-C4H8), 100%].
Tris(dibenzylideneacetone)dipalladium(0)
(6.2 mg, 6.76 μmol) was added to a mixture of tert-butyl 6-bromo-2-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[3,2-c]pyridine-1-carboxylate 47 (51 mg, 0.135 mmol), cesium carbonate (88 mg, 0.270 mmol),
4-amino-3-chloro-N,N-dimethylbenzamide
(32 mg, 0.162 mmol), and Xantphos (7.8 mg, 0.014 mmol) in dioxane
(1.5 mL). The vial was flushed with dry argon, and the reaction mixture
was heated at 80 °C 3 h. Another 0.2 equiv of Pd and 0.4 equiv
of ligand were added, and the reaction mixture was heated at 80 °C
for 3 h. The reaction mixture was then filtered on SCX-2 column and
concentrated under vacuum. The residue was purified via Biotage silica
gel column chromatography, eluting with CH2Cl2/EtOH (99:1 to 90:10) to afford 48 as a white solid
(19 mg, 28%). 1H NMR (500 MHz, CDCl3): δ
1.52 (s, 9H), 3.10 (s, 6H), 3.98 (s, 3H), 6.53 (s, 1H), 7.08 (s, 1H),
7.34 (dd, J = 8.5, 1.9 Hz, 1H), 7.55 (d, J = 1.9 Hz, 1H), 7.56 (s, 1H), 7.61 (s, 1H), 7.72 (s, 1H),
8.15 (d, J = 8.5 Hz, 1H), 8.53 (s, 1H). ESI-HRMS
calcd for C25H28ClN6O3 [M+H]+, 495.1906; found, 495.1900.
Tris(dibenzylideneacetone)dipalladium(0)
(24.8 mg, 0.027 mmol) was
added to a mixture of tert-butyl 6-bromo-2-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[3,2-c]pyridine-1-carboxylate 47 (51 mg, 0.135 mmol),
cesium carbonate (88 mg, 0.270 mmol), 4-amino-3-methoxy-N,N-dimethylbenzamide (32 mg, 0.162 mmol), and Xantphos
(31 mg, 0.054 mmol) in dioxane (1.5 mL). The vial was flushed with
dry argon, and the reaction mixture was heated at 80 °C for 6
h. It was then filtered on a SCX-2 column and concentrated under vacuum.
The residue was purified via Biotage silica gel column chromatography,
eluting with CH2Cl2/EtOH (99:1 to 90:10) to
afford tert-butyl-6-(4-(dimethylcarbamoyl)-2-methoxyphenylamino)-2-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[3,2-c]pyridine-1-carboxylate as a white solid (13 mg, 20%). 1H NMR (500 MHz, CDCl3): δ 1.52 (s, 9H), 3.11
(s, 6H), 3.95 (s, 3H), 3.97 (s, 3H), 6.50 (s, 1H), 7.05 (dd, J = 8.2, 1.7 Hz, 1H), 7.08 (d, J = 1.7
Hz, 1H), 7.22 (s, 1H), 7.54 (s, 1H), 7.60 (s, 1H), 7.69 (s, 1H), 8.10
(d, J = 8.2 Hz, 1H), 8.49 (s, 1H). LC (method B)-MS
(ESI, m/z) tR 2.40 min, 491 [(M + H+), 100%]. tert-Butyl-6-(4-(dimethylcarbamoyl)-2-methoxyphenylamino)-2-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[3,2-c]pyridine-1-carboxylate (13 mg, 0.027 mmol) in TFA (265
μL) was stirred for 30 min at rt. It was then concentrated and
filtered on an Isolute Flash NH2 column. The residue was
purified via Biotage silica gel column chromatography, eluting with
10% MeOH/aq NH3 (10:1) in CH2Cl2 to
afford 40 as a white solid (7 mg, 68%). 1H
NMR (500 MHz, CD3OD): δ 3.11 (s, 6H), 3.95 (s, 3H),
3.96 (s, 3H), 6.59 (d, J = 0.8 Hz, 1H), 7.01 (dd, J = 8.2, 1.8 Hz, 1H), 7.06–7.09 (m, 2H), 7.65 (d, J = 8.2 Hz, 1H), 7.85 (s, 1H), 7.95 (s, 1H), 8.40 (d, J = 0.8 Hz, 1H). ESI-HRMS calcd for C21H23N6O2 [M+H]+, 391.1877; found,
391.1873.
4-Amino-3-methoxy-N,N-dimethylbenzamide
HATU (0.296 g, 0.778 mmol)
was added to a solution of 4-amino-3-methoxybenzoic
acid (0.1 g, 0.598 mmol), DIPEA (0.15 mL, 0.897 mmol), and dimethylamine
(2 M in THF, 0.60 mL, 1.196 mmol) in THF (1.6 mL) under argon. The
reaction mixture was stirred overnight. It was then partitioned between
EtOAc and water. The separated organic phase was washed with water,
dried over Na2SO4, and evaporated in vacuum.
The crude was purified via Biotage silica gel column chromatography,
eluting with CH2Cl2/EtOAc (60:40 to 40:60),
and was then filtered on a SCX-2 column to afford the title compound
as a colorless oil (69 mg, 59%). 1H NMR (500 MHz, CDCl3): δ 3.06 (s, 6H), 3.87 (s, 1H), 3.99 (br s, 2H), 6.65
(d, J = 7.9 Hz, 1H), 6.89 (dd, J = 7.9, 1.7 Hz, 1H), 6.96 (d, J = 1.7 Hz). LC (method
B)-MS (ESI, m/z) tR 1.23 min, 195 [(M + H+), 100%].
Preparation
of Compounds in Scheme 5 (Exemplified
by Compounds 34 and 55)
To DMF (3.4 mL) containing Et3N (0.64 mL, 4.5
mmol) were
added propargylaldehyde diethyl acetal 56 (183 μL,
1.27 mmol) and N-(2-bromo-5-iodopyridin-4-yl)methanesulfonamide 16 (400 mg, 1.06 mmol) followed by copper(I) iodide (7.1 mg,
0.037 mmol). The reaction was placed under nitrogen. Bis(triphenylphosphine)palladium
dichloride (26.1 mg, 0.037 mmol) was added, and the reaction was flushed
again with nitrogen and then heated at 60 °C for 2 h. The reaction
was cooled and added to water containing a NaHCO3 solution.
The reaction was extracted with EtOAc. The combined organic layers
were washed with water containing NaHCO3 solution and brine,
and concentrated in vacuo. The residue was purified using silica gel
column chromatography, eluting with 100% CH2Cl2 to 5% EtOAc in CH2Cl2 to 10% EtOAc in CH2Cl2 to give 57 (207 mg, 51%). 1H NMR (500 MHz, CDCl3): δ 1.32 (t, J = 6.9 Hz, 6H), 3.38 (s, 3H), 3.75 (m, 2H), 3.84 (m, 2H),
5.86 (d, J = 1.0 Hz, 1H), 6.94 (t, J = 1.0 Hz, 1H), 8.14 (t, J = 1.0 Hz, 1H), 8.67 (d, J = 1.0 Hz, 1H). LC (method A)-MS (ESI, m/z) tR 2.44 min, 377
[(M + H+), 100%].
6-Bromo-2-(diethoxymethyl)-1-(methylsulfonyl)-1H-pyrrolo[3,2-c]pyridine 57 (202 mg, 0.54 mmol) was stirred in MeOH (2.4 mL), and 1 M sodium
hydroxide in water (0.62 mL, 0.62 mmol) was added. The reaction was
stirred at 25 °C for 6 h. The MeOH was evaporated, and the residue
taken up in EtOAc. The solution was washed with water and brine, and
the organic layer was concentrated in vacuo to afford 58 (142 mg, 89%). 1H NMR (500 MHz, CDCl3): δ
1.27 (t, J = 6.9 Hz, 6H), 3.58–3.72 (m, 4H),
5.73 (m, 1H), 6.59 (m, 1H), 7.48 (m, 1H), 8.65 (s, 1H), 8.84 (br s,
1H). LC (method A)-MS (ESI, m/z) tR 2.08 min, 253 [(M + H+-EtOH), 100%].
To a solution of
6-bromo-2-(diethoxymethyl)-1H-pyrrolo[3,2-c]pyridine 58 (142 mg, 0.47 mmol) in THF (1.4
mL) and water (0.28 mL) was added tosic acid hydrate (134 mg, 0.705
mmol), and the reaction was stirred at 25 °C for 55 min. The
reaction was partitioned between EtOAc and NaHCO3. The
layers were separated, and the aqueous layer again was extracted with
EtOAc. The combined organic layers were washed with NaHCO3 and brine and concentrated in vacuo to afford 6-bromo-1H-pyrrolo[3,2-c]pyridine-2-carbaldehyde (112 mg,
over theory, residual solvent). 1H NMR (500 MHz, CDCl3): δ 7.37 (s, 1H), 7.62 (t, J = 1.0
Hz, 1H), 8.88 (d, J = 1.0 Hz, 1H), 9.32 (br s, 1H),
9.93 (s, 1H). LC (method A)-MS (ESI, m/z) tR 1.59 min, 225 [(M + H+), 100%]. 6-Bromo-1H-pyrrolo[3,2-c]pyridine-2-carbaldehyde (604 mg, 2.68 mmol), TOSMIC (1.05 g, 5.37
mmol), and potassium carbonate (759 mg, 5.5 mmol) in MeOH (30 mL)
was stirred and heated at 65 °C for 110 min. The MeOH was evaporated,
and the residue was partitioned between EtOAc and water. The layers
were separated, and the organic solution was washed with water and
brine and concentrated in vacuo. The residue was purified using silica
gel column chromatography, eluting with EtOAc to afford 59 (561 mg, 79%). 1H NMR (500 MHz, acetone-d6): δ 7.02 (d, J = 1.0 Hz, 1H),
7.60 (t, J = 1.0 Hz, 1H), 7.62 (s, 1H), 8.30 (s,
1H), 8.67 (d, J = 1.0 Hz, 1H). LC (method A)-MS (ESI, m/z) tR 1.83
min, 264 [(M + H+), 100%].
5-(6-Bromo-1H-pyrrolo[3,2-c]pyridin-2-yl)oxazole 59 (152 mg, 0.58 mmol)
was stirred in EtOAc (2 mL). Et3N (140 μL, 1.0 mmol)
was added followed by a crystal of DMAP and di-tert-butyl dicarbonate (190 mg, 0.87 mmol). The reaction was stirred
at 25 °C for 60 min. Further di-tert-butyl dicarbonate
(54 mg, 0.25 mmol) was added, and the reaction allowed to stir at
rt overnight. The reaction was concentrated in vacuo, and the residue
applied in chloroform to a preparative TLC plate. The product was
eluted with EtOAc/cyclohexane (1:1, three times) to afford 60 (150 mg, 71%). 1H NMR (500 MHz, CDCl3): δ
1.54 (s, 9H), 6.90 (d, J = 1.0 Hz, 1H), 7.40 (s,
1H), 8.02 (s, 1H), 8.31 (s, 1H), 8.68 (s, 1H)/; LC (method A)-MS (ESI, m/z) tR 2.58
min, 308 [(M + H+-C4H8), 100%].
To tert-butyl 6-bromo-2-(oxazol-5-yl)-1H-pyrrolo[3,2-c]pyridine-1-carboxylate 60 (49 mg, 0.135 mmol) were added cesium carbonate (88 mg,
0.27 mmol) and Xantphos (7.8 mg, 0.0135 mmol) and then 2-methoxyaniline
(20 mg, 0.163 mmol) in dioxane (1.5 mL). The flask was flushed with
nitrogen. Tris(dibenzylideneacetone)dipalladium(0) (6.3 mg, 0.0068
mmol) was added, and the flask was flushed again with nitrogen and
heated at 80 °C for 3 h. The reaction was cooled and diluted
with EtOAc. The organic solution was washed with water and brine,
dried, and evaporated. The residue was applied to two 1 mm, 20 ×
20 cm silica prep TLC plates, which were eluted with EtOAc. The product
band was recovered with acetone to afford tert-butyl-6-(2-methoxyphenylamino)-2-(oxazol-5-yl)-1H-pyrrolo[3,2-c]pyridine-1-carboxylate
(44 mg, 80%). 1H NMR (500 MHz, CDCl3): δ
1.50 (s, 9H), 3.92 (s, 3H), 6.80 (d, J = 0.6 Hz,
1H), 6.92–7.02 (m, 3H), 7.14 (br s, 1H), 7.30 (s, 1H), 7.70
(t, J = 1.0 Hz, 1H), 7.99 (m, 2H), 8.55 (d, J = 1.0 Hz, 1H). ESI-HRMS calcd for C22H23N4O4 [M+H]+, 407.1714; found,
407.1707. tert-Butyl-6-(2-methoxyphenylamino)-2-(oxazol-5-yl)-1H-pyrrolo[3,2-c]pyridine-1-carboxylate
(44 mg) was dissolved in CH2Cl2 (1 mL) and TFA
(1 mL) and stirred at rt for 3.25 h. The solvents were evaporated,
and the residue was kept for 1.5 h in a vacuum desiccator over NaOH.
The residue was partitioned between EtOAc and a saturated NaHCO3 solution. The layers were shaken and separated, and the aqueous
layer was washed with more EtOAc. The combined organics were dried
and evaporated, leaving a solid residue (33 mg). This was triturated
with ether to give, after removal of the mother liquors and drying,
compound 34 (27 mg, 81%). 1H NMR (500 MHz,
DMSO-d6): δ 3.86 (s, 3H), 6.78 (d, J = 1.0 Hz, 1H), 6.84–6.91 (m, 2H), 6.96 (t, J = 1.0 Hz, 1H), 6.98–7.01 (m, 1H), 7.53 (s, 1H),
7.70 (br s, 1H), 8.04–8.07 (m, 1H), 8.47 (s, 1H), 8.49 (s,
1H), 11.65 (br s, 1H). ESI-HRMS calcd for C17H15N4O2 [M+H]+, 307.1190; found, 307.1186.
To tert-Butyl 6-bromo-2-(oxazol-5-yl)-1H-pyrrolo[3,2-c]pyridine-1-carboxylate 60 (49 mg, 0.135 mmol) were added cesium carbonate (88 mg,
0.27 mmol), Xantphos (7.8 mg, 0.0135 mmol), and 4-amino-3-methoxy-N,N-dimethylbenzamide (32.1 mg solids,
0.162 mmol) in DMA (1.5 mL). The flask was flushed with argon. Tris(dibenzylideneacetone)dipalladium(0)
(6.3 mg, 0.0068 mmol) was added, and the flask was flushed again with
argon and heated at 80 °C for 3 h. The reaction was cooled and
added to water. The product was extracted with EtOAc. The combined
organics were washed with water and with brine, dried, and evaporated.
The residue was applied to two 1 mm, 20 × 20 cm silica prep TLC
plates, which were eluted twice with EtOAc. The product band was recovered
with acetone to give impure product (45 mg). This still contains some
of the aniline. This material was applied to one 1 mm, 20 × 20
cm silica prep TLC plate, which was eluted twice with EtOAc/CH2Cl2 (1:1). The product was recovered with acetone,
giving 55 (39 mg, 60%). 1H NMR (500 MHz, CDCl3): δ 1.50 (s, 9H), 3.10 (s, 6H), 6.84 (d, J = 0.6 Hz, 1H), 7.17 (br s, 1H), 7.37 (dd, J = 8.5,
1.9 Hz, 1H), 7.56 (d, J = 1.9 Hz, 1H), 7.77 (s, 1H),
8.01 (s, 1H), 8.21 (d, J = 8.5 Hz, 1H), 8.61 (d, J = 1.0 Hz, 1H). ESI-HRMS calcd for C24H25ClN5O4 [M+H]+, 482.1590;
found, 482.1586.
Tris(dibenzylideneacetone)dipalladium(0)
(6.1 mg, 6.63 μmol) was added to a mixture of tert-butyl 6-bromo-2-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[3,2-c]pyridine-1-carboxylate 47 (50 mg, 0.133 mmol), cesium carbonate (86 mg, 0.265 mmol),
2-chloro-4-(1-methyl-1H-imidazol-5-yl)aniline (33
mg, 0.159 mmol), and Xantphos (7.7 mg, 0.013 mmol) in DMA (1.4 mL).
The vial was flushed with dry argon, and the reaction mixture was
heated at 80 °C for 3 h. It was then filtered on a SCX-2 column
and concentrated under vacuum. The residue was purified via Biotage
silica gel column chromatography, eluting with CH2Cl2/EtOH (99:1 to 95:5) to afford 65 as a white
solid (40 mg, 60%). 1H NMR (500 MHz, CDCl3):
δ 1.52 (s, 9H), 3.69 (s, 3H), 3.98 (s, 3H), 6.53 (d, J = 0.9 Hz, 1H), 7.04 (s, 1H), 7.09 (br s, 1H), 7.27 (dd, J = 8.5, 2.0 Hz, 1H), 7.45 (d, J = 2.0
Hz, 1H), 7.53 (br s, 1H), 7.56 (s, 1H), 7.61 (s, 1H), 7.72 (t, J = 0.9 Hz, 1H), 8.18 (d, J = 8.5 Hz, 1H),
8.52 (d, J = 0.9 Hz, 1H). ESI-HRMS calcd for C26H27ClN7O2 [M+H]+, 504.1909; found, 504.1885.
2-Chloro-4-(1-methyl-1H-imidazol-5-yl)aniline
Tetrakis(triphenylphosphine)palladium
(0.046 g, 0.039 mmol) was
added to a solution of 2-chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline
(0.1 g, 0.394 mmol), 5-iodo-1-methyl-1H-imidazole
(0.123 g, 0.592 mmol), and sodium carbonate (0.125 g, 1.183 mmol)
in DME/H2O (3:1, 2.0 mL). The vial was flushed with dry
argon, and the reaction mixture was heated at 135 °C for 1 h
under microwave irradiation. It was then diluted with EtOAc and quenched
with water. The layers were separated, and the aqueous layer was extracted
with EtOAc. The combined organic layers were dried (Na2SO4), filtered, and concentrated under reduced pressure.
The crude mixture was purified via Biotage silica gel column chromatography,
eluting with CH2Cl2/EtOH (99:1 to 95:5) to afford
the title product as a white solid (50 mg, 61%). 1H NMR
(500 MHz, CDCl3): δ 3.62 (s, 3H), 4.23 (br s, 2H),
6.81 (d, J = 8.3 Hz, 1H), 7.01 (d, J = 1.2 Hz, 1H), 7.08 (dd, J = 8.2, 2.0 Hz, 1H),
7.27 (d, J = 2.0 Hz, 1H), 7.47 (s, 1H). LC (method
B)-MS (ESI, m/z) tR 0.93 min, 208 [(M + H+), 100%].
Protein
Production for Full-Length MPS1
The coding
sequence for full-length human MPS1 was amplified by PCR using a plasmid
containing MPS1 as a template (kindly provided by the laboratory of
Prof. Dr. Eric Nigg, University of Basel, Basel, Switzerland). The
PCR product was inserted into a modified version of pFastBac1 that
encodes an N-terminal 6× His-tag followed by
a GST-tag and then an HRV 3C protease site. Recombinant baculovirus
was generated according to Bac-to-Bac protocols (Invitrogen, Paisley,
UK). For protein production, Sf9 insect cells were grown in sf-900
II media to a cell density of around 2 × 106 cells
per milliliter and infected with 30–100 μL of virus per
107 cells. Infected cell cultures were harvested 3 days
postinfection. Cell pellets were resuspended in 3 volumes of lysis
buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 1 mM MgCl2, and
10% (v/v) glycerol) containing 1× complete EDTA-free protease
inhibitors, 20 mM β-glycerophosphate, 10 mM NaF, 2 mM Na3VO4, and 25 U/mL benzonase nuclease, and the resuspended
cells were lysed by sonication. Following centrifugation, the supernatant
was purified over 10 mL of Talon resin using a batch/gravity protocol,
eluting with 5 column volumes (CV) of 50 mM HEPES, pH 7.0, 300 mM
NaCl, 250 mM imidazole, 10% (v/v) glycerol, and 1× complete EDTA-free
protease inhibitors. The Talon eluate was subsequently purified over
a 5 mL GSTrap FF column equilibrated in GSH buffer A (10 mM HEPES,
pH 7.0, 150 mM NaCl, 1 mM DTT, 0.1 mM EDTA, and 0.0001% Tween 20)
and eluted with 4 CV of GSH Buffer B (GSH buffer A + 10 mM glutathione).
N-terminal His and GST tags were removed by overnight incubation at
4 °C with HRV 3C protease. Cleaved protein was concentrated to
0.5 mL and applied to a Superdex 200 HR 10/30 column in series with
a 5 mL GSTrap FF column that was equilibrated with 10 mM HEPES, pH
7.0, 150 mM NaCl, 1 mM DTT, 0.1 mM EDTA, and 0.0001% Tween 20. Selected
fractions were pooled, concentrated to 1 to 2 mg/mL, and frozen.
MPS1 Kinase Assay
The enzyme reaction (10 μL
total volume) was carried out in black 384-well low-volume plates
containing full-length MPS1 (LifeTechnologies or in-house, in a range
from 3 to 12.5 nM to obtain 10% total conversion during the assay),
fluorescent-labeled peptide [H236, sequence: 5FAM-DHTGFLTEYVATR-CONH2, Pepceuticals Ltd., Enderby, UK] (5 μM), ATP (10 μM),
either 1% (v/v) DMSO or the test compound (in the range from 0.25
nM to 100 μM in 1% (v/v) DMSO), and assay buffer (50 mM HEPES,
pH 7.0, 0.02% (w/v) NaN3, 0.01% (w/v) BSA, 0.1 mM orthovanadate,
10 μM MgCl2, 1 μM DTT, and Roche protease inhibitor).
The reaction was carried out for either 60 or 90 min at room temperature
and stopped by the addition of buffer (10 μL) containing 20
mM EDTA and 0.05% (v/v) Brij-35, in 0.1 M HEPES-buffered saline (Free
acid, Sigma, UK). The plate was read on a Caliper EZ reader II (PerkinElmer
Life Sciences, Waltham, MA, USA). The reader provides a Software package
(‘Reviewer’) that converts the peak heights into percent
conversion by measuring both the product and substrate peaks, and
it also allows selection of control wells that represent 0 and 100%
inhibition, respectively. The percent inhibition of the compounds
was calculated relative to the mean values of selected control wells.
IC50 values were determined by testing the compounds at
a range of concentrations from 0.25 nM to 100 μM. The percent
inhibition at each concentration was then fitted to a four-parameter
logistic fit using the Studies package (Dotmatics, Bishops Stortford,
UK): y = (a + ((b – a)/(1 + ((c/x)))), where a = asym
min, b = asym max, c = IC50, and d = Hill coefficient.
Aurora
A and Aurora B Kinase Assays
The enzyme reactions
(10 μL total volume) were carried out in black 384-well low-volume
plates containing full-length AurB/INCENP complex (1 nM, Carna Biosciences,
Japan) or N-terminal HIS-tagged AurA (5 nM, in-house), fluorescent-labeled
peptide [FL-Peptide 21, PerkinElmer, sequence: 5FAM-LRRASLG-CONH2] (1.5 μM), ATP (15 or 20 μM), either 1% (v/v)
DMSO or the test compound (in the range from 0.25 nM to 100 μM
in 1% (v/v) DMSO), and assay buffer (50 mM Tris, pH 7.4, 200 mM NaCl,
5 mM MgCl2, 2 mM DTT, and 0.1% (v/v) Tween 20). The reaction
was carried out for 60 min at room temperature and stopped by the
addition of buffer (10 μL) containing 20 mM EDTA and 0.05% (v/v)
Brij-35 in 0.1 M HEPES-buffered saline (Free acid, Sigma, UK). The
plate was read on a Caliper EZ reader II (PerkinElmer). The reader
provides a Software package (‘Reviewer’) that converts
the peak heights into percent conversion by measuring both the product
and substrate peaks. The percentage inhibition was calculated relative
to blank wells (containing no enzyme and 1% (v/v) DMSO). IC50 values were determined by testing the compounds at a range of concentrations
from 0.25 nM to 100 μM. The percent inhibition at each concentration
was then fitted to a four-parameter logistic fit using the Studies
package (Dotmatics, Bishops Stortford, UK): y = (a + ((b – a)/(1
+ ((c/x)))), where a = asym min, b = asym
max, c = IC50, and d =
Hill coefficient.
CDK2 Kinase Assay
The enzyme reaction
(10 μL
total volume) was carried out in black 384-well low-volume plates
containing full-length CDK2/CyclinA complex (2 nM, LifeTechnologies),
fluorescent-labeled peptide [FL-Peptide18, PerkinElmer, sequence:
5FAM-QSPKKG-CONH2] (1.5 μM), ATP (25 μM), either
1% (v/v) DMSO or the test compound (in the range from 0.25 nM to 100
μM in 1% (v/v) DMSO), and assay buffer (50 mM HEPES, pH 7.0,
0.02% (w/v) NaN3, 0.01% (w/v) BSA, 0.1 mM orthovandate,
10 μM MgCl2, and 1 μM DTT). The reaction was
carried out for 60 min at room temperature and stopped by the addition
of buffer (10 μL) containing 20 mM EDTA and 0.05% (v/v) Brij-35
in 0.1 M HEPES-buffered saline (Free acid, Sigma, UK). The plate was
read on a Caliper EZ reader II (PerkinElmer). The reader provides
a Software package (‘Reviewer’) that converts the peak
heights into percent conversion by measuring both the product and
substrate peaks. The percentage inhibition was calculated relative
to blank wells (containing no enzyme and 1% (v/v) DMSO). IC50 values were determined by testing the compounds at a range of concentrations
from 0.25 nM to 100 μM. The percent inhibition at each concentration
was then fitted to a four-parameter logistic fit using the Studies
package (Dotmatics, Bishops Stortford, UK): y = (a + ((b – a)/(1
+ ((c/x)))), where a = asym min, b = asym
max, c = IC50, and d =
Hill coefficient.
GSK3β Kinase Assay
All GSK3β
percentage
inhibitions at 1 μM were performed in duplicates by Invitrogen
in a Z’LYTE activity assay using their SelectScreen biochemical
kinase profiling service.
Cell Viability Assay
Cell proliferation assays were
carried out by colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay (Sigma). Briefly, cells were plated in 96-well
plates at 1500 cells per well in 100 μL of culture medium in
triplicate. On the next day, 2-fold dilutions of the compounds to
be tested were made in culture medium so that when diluted 5×
the final concentration in the wells ranged from 0 to 20 μM.
Twenty-five microliters of compounds dilutions in the medium was added
to 100 μL of cells and incubated at 37 °C and 5% CO2 for 3 more days (72 h). Cells were then incubated with 40
μL of 5 mg/mL solution of MTT reagent at 37 °C for 3 h.
Media was carefully removed, and crystals were dissolved in 100 μL
of DMSO. The absorbance was measured at 570 nm with the Wallac VICTOR2
1420 multilabel counter (PerkinElmer), and analysis was performed
to calculate the GI50 using GraphPad PRISM.
MSD Assay
for MPS1 Autophosphorylation
Cellular IC50 values
of MPS1 autophosphorylation inhibition were measured
by an in-house electrochemiluminescence (Meso Scale Discovery, MSD)
assay that measured phosphorylation of ectopic MPS1 at the Thr33 and
Ser37 sites.[36,37] On day 1, 3 × 104 HCT116 cells per well in a 96-well plate were reverse-transfected
with 100 ng of wild-type Myc-MPS1 using Lipofectamine LTX (Invitrogen).
On the next day, cells were treated with 50 ng/mL of nocodazole. On
the following day, cells were treated with 2-fold dilutions of test
compounds ranging from 0 to 10 μM for 2 h in the presence of
10 μM MG132. After treatment, cells were washed with PBS and
lysed with 60 μL per well of complete lysis buffer (50 mM NaCl,
20 mM Tris, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1% (v/v) Triton X100, 10
mM NaF, protease inhibitor tablet, and phosphatase inhibitor cocktails)
on ice for 30 min with shaking. Cell lysates were mixed thoroughly
by pipetting up and down, and 25 μL of lysate was loaded onto
MSD plates that were precoated with total MPS1 antibody (mouse monoclonal,
Invitrogen, cat. no. 35-9100) and blocked with 3% (w/v) BSA. After
a 1 h incubation at room temperature on a shaker, plates were washed
three times with MSD wash buffer, and 25 μL of pThr33/pSer37
antibody (Invitrogen, cat. no. 44-1325G) diluted in 1% (w/v) BSA was
added followed by incubation for a further 1 h at room temperature.
Plates were washed again three times with MSD wash buffer and incubated
with 25 μL of anti-rabbit sulfo-TAG antibody (Meso Scale Discovery,
cat. no. R32AB) diluted in 1% (w/v) BSA) for 1 h. After the final
incubation, plates were washed three times with MSD wash buffer and
read in the presence of 1× MSD read buffer. IC50 values
were determined using GraphPad PRISM.
Solubility Assays
Aqueous, fed-state-simulated intestinal
fluid (FeSSIF) and fasted-state-simulated intestinal fluid (FaSSIF)
solubilities were determined at Pharmorphix.
Crystal
Structure Determination of MPS1 with Ligands
The kinase domain
(residues 519–808) of MPS1 was produced
in E. coli and purified as described
previously.[38] Purified MPS1 was crystallized
in the apo form at 18 °C using the sitting-drop vapor-diffusion
method. The crystallization drops were composed of 2 μL of protein
(8.9 mg/mL) and 2 μL of reservoir solution placed over 200 μL
of reservoir solution of 30–45% (v/v) aqueous PEG300 in 48-well
plates. Apo crystals typically grew in 72 h. For compounds 8, 34, 39, 48, and 55, apo-protein crystals were soaked for 24 h at 18 °C in 4 μL
drops composed of 1–10 mM compound in 35% (v/v) PEG300, 0.1
M HEPES, pH 7.5, and 5% (v/v) DMSO. Soaked protein crystals were briefly
transferred to cryoprotectant solution containing 40% (v/v) PEG300,
0.1 M HEPES, pH 7.5, and 20% (w/v) ethylene glycol prior to cryocooling
in liquid nitrogen.MPS1 was cocrystallized with compound 65 by mixing 2 μL of protein solution, premixed with
1 mM 65 for 30 min on ice, with 2 μL of reservoir
solution placed over 200 μL of reservoir solution consisting
of 14% (w/v) PEG8000, 0.05 M magnesium acetate, and 0.1 M sodium acetate.
Cocrystals of MPS1 with 65 formed within 72 h at 18 °C
and were briefly transferred to a cryoprotectant solution containing
10% (w/v) PEG8000, 0.05 M magnesium acetate, 0.1 M sodium acetate,
and 25% (v/v) glycerol prior to cryocooling in liquid nitrogen.X-ray data were collected at Diamond Light Source, Oxfordshire,
UK, at beamlines I04 and I04-1. Crystals belonged to the space group I222 and diffracted to a resolution between 2.36 and 2.80
Å. Data were integrated with MOSFLM[39,40] or XDS[41] and scaled and merged with AIMLESS.[42] The structures were solved by molecular replacement
using PHASER,[40,43] and a publicly available MPS1
structure (PDB code 4BI1)[38] with ligand and water molecules removed
was used as the molecular replacement model. The protein–ligand
structures were manually rebuilt in COOT[44] and refined with BUSTER[45] in iterative
cycles. Ligand restraints were generated with grade[46] and Mogul.[47] The quality of
the structures was assessed with MOLPROBITY.[48] The data collection and refinement statistics are presented in Supporting InformationTable
S2.
Mouse Liver Microsomal Stability
Compounds (10 μM)
were incubated with male CD1 mouse liver microsomes (1 mg/mL) protein
in the presence of 1 mM NADPH, 2.5 mM UDP-glucuronic acid (UDPGA),
and 3 mM MgCl2 in 10 mM PBS at 37 °C. Incubations
were conducted for 0 and 30 min. Control incubations were generated
by the omission of NADPH and UDPGA from the incubation reaction. The
percentage of compound remaining was determined after analysis by
LC/MS.
Human Liver Microsomal Stability
Compounds (10 μM)
were incubated with mixed-gender pooled human liver microsomes (1
mg/mL) protein in the presence of 1 mM NADPH, 2.5 mM UDPGA, and 3
mM MgCl2 in 10 mM PBS at 37 °C. Incubations were conducted
for 0 and 30 min. Control incubations were generated by the omission
of NADPH and UDPGA from the incubation reaction. The percentage of
compound remaining was determined after analysis by LC/MS.
In
Vivo Mouse PK
All procedures involving animals were
carried out within The ICR’s Animal Ethics Committee and national
guidelines.[49] Mice (female Balb/C) were
dosed po or iv (5 or 10 mg/kg) in 10% (v/v) DMSO and 5% (v/v) Tween
20 in saline. After administration, mice were culled at 5, 15, and
30 min and 1, 2, 4, 6, and 24 h. Blood was removed by cardiac puncture
and centrifuged to obtain plasma samples. Plasma samples (100 μL)
were added to the analytical internal standard (olomoucine; IS) followed
by protein precipitation with 300 μL of methanol. Following
centrifugation (1200g, 30 min, 4 °C), the resulting
supernatants were analyzed for compound levels by LC/MS. For blood
pharmacokinetics, 20 μL was spotted on a Whatman B card and
allowed to dry for at least 12 h at room temperature and 6 mm diameter
disks were punched and extracted with 200 μL of methanol containing
500 nM olomoucine used as internal standard. Sample extracts were
analyzed by LC/MSMS against calibration curves (six levels) and six
quality controls (three levels in duplicate). Separation was carried
out by an Acquity UPLC binary system (Waters) on a reverse-phase Kinetex
C18 (Phenomenex 50 × 2.1 mm, 1.7 μm particles) analytical
column. Elution was achieved with a 4.5 min gradient of 0.1% formic
acid/methanol (95% formic acid to 0%) following a 0.5 min isocratic
period. Detection was performed in positive ion mode ESI multiple-reaction
monitoring (MRM) on a QTRAP 4000 (AB-SCIEX).
In Vivo
Mouse PK/PD Study
Human HCT116 colon carcinoma
cells (3 × 106) were sc injected bilaterally in the
flanks of female CrTac:NCr-Fox1(nu) athymic mice.
Once tumors reached a mean diameter of ∼8 mm (day 15), mice
were dosed twice at a 12 h intervals with 50, 75, or 100 mg/kg of
compound 65 in 10% (v/v) DMSO and 5% (v/v) Tween 20 in
saline. Mice were culled (n = 3 per group) at 2,
10, and 72 h after the second dose. Tumors were snap-frozen and stored
at −80 °C until analysis. Tumor samples were homogenized
in PBS (3 vol/tumor weight).
Authors: Keith D Tardif; Aaron Rogers; Jared Cassiano; Bruce L Roth; Daniel M Cimbora; Rena McKinnon; Ashley Peterson; Thomas B Douce; Rosann Robinson; Irene Dorweiler; Thaylon Davis; Mark A Hess; Kirill Ostanin; Damon I Papac; Vijay Baichwal; Ian McAlexander; J Adam Willardsen; Michael Saunders; Hoarau Christophe; D Vijay Kumar; Daniel A Wettstein; Robert O Carlson; Brandi L Williams Journal: Mol Cancer Ther Date: 2011-10-06 Impact factor: 6.261
Authors: Russell K Dorer; Sheng Zhong; John A Tallarico; Wing Hung Wong; Timothy J Mitchison; Andrew W Murray Journal: Curr Biol Date: 2005-06-07 Impact factor: 10.834
Authors: Laura Hewitt; Anthony Tighe; Stefano Santaguida; Anne M White; Clifford D Jones; Andrea Musacchio; Stephen Green; Stephen S Taylor Journal: J Cell Biol Date: 2010-07-12 Impact factor: 10.539
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Authors: Yong Liu; Radoslaw Laufer; Narendra Kumar Patel; Grace Ng; Peter B Sampson; Sze-Wan Li; Yunhui Lang; Miklos Feher; Richard Brokx; Irina Beletskaya; Richard Hodgson; Olga Plotnikova; Donald E Awrey; Wei Qiu; Nickolay Y Chirgadze; Jacqueline M Mason; Xin Wei; Dan Chi-Chia Lin; Yi Che; Reza Kiarash; Graham C Fletcher; Tak W Mak; Mark R Bray; Henry W Pauls Journal: ACS Med Chem Lett Date: 2016-05-06 Impact factor: 4.345
Authors: Pramodkumar P Gupta; Virupaksha A Bastikar; Dalius Kuciauskas; Shanker Lal Kothari; Jonas Cicenas; Mindaugas Valius Journal: Med Oncol Date: 2017-09-06 Impact factor: 3.064
Authors: Daniel Ian McSkimming; Shima Dastgheib; Timothy R Baffi; Dominic P Byrne; Samantha Ferries; Steven Thomas Scott; Alexandra C Newton; Claire E Eyers; Krzysztof J Kochut; Patrick A Eyers; Natarajan Kannan Journal: Mol Biosyst Date: 2016-11-15
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Figure 1
Figure 2
Scheme 1
Scheme 2
Scheme 3
Scheme 4
Scheme 5
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8