Introduction of a Methyl Group Curbs Metabolism of Pyrido[3,4- d]pyrimidine Monopolar Spindle 1 (MPS1) Inhibitors and Enables the Discovery of the Phase 1 Clinical Candidate N2-(2-Ethoxy-4-(4-methyl-4 H-1,2,4-triazol-3-yl)phenyl)-6-methyl- N<su

Article abstract of DOI:10.1021/acs.jmedchem.8b00690

Monopolar spindle 1 (MPS1) occupies a central role in mitosis and is one of the main components of the spindle assembly checkpoint. The MPS1 kinase is an attractive cancer target, and herein, we report the discovery of the clinical candidate BOS172722. Th

Full text of DOI:10.1021/acs.jmedchem.8b00690

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Cite This: J. Med. Chem. XXXX, XXX, XXX−XXX
Introduction of a Methyl Group Curbs Metabolism of
Pyrido[3,4‑d]pyrimidine Monopolar Spindle 1 (MPS1) Inhibitors and
Enables the Discovery of the Phase 1 Clinical Candidate N2‑(2-
Ethoxy-4-(4-methyl‑4H‑1,2,4-triazol-3-yl)phenyl)-6-
methyl‑N8‑neopentylpyrido[3,4‑d]pyrimidine-2,8-diamine
(BOS172722)
Hannah L. Woodward,† Paolo Innocenti,† Kwai-Ming J. Cheung,† Angela Hayes,† Jennie Roberts,†
Alan T. Henley,† Amir Faisal,†,⊥ Grace Wing-Yan Mak,† Gary Box,† Isaac M. Westwood,†,‡
Nora Cronin,‡ Michael Carter,† Melanie Valenti,† Alexis De Haven Brandon,† Lisa O’Fee,†
Harry Saville,† Jessica Schmitt,† Rosemary Burke,† Fabio Broccatelli,† Rob L. M. van Montfort,†,‡
Florence I. Raynaud,† Suzanne A. Eccles,† Spiros Linardopoulos,†,§ Julian Blagg,†
and Swen Hoelder*,†
†Cancer Research UK Cancer Therapeutics Unit at The Institute of Cancer Research, London SM2 5NG, United Kingdom
‡Division of Structural Biology, The Institute of Cancer Research, London SW3 6JB, United Kingdom
§The Breakthrough Breast Cancer Research Centre, Division of Breast Cancer Research, The Institute of Cancer Research, London
SW3 6JB, United Kingdom
S
* Supporting Information
ABSTRACT: Monopolar spindle 1 (MPS1) occupies a central
role in mitosis and is one of the main components of the spindle
assembly checkpoint. The MPS1 kinase is an attractive cancer
target, and herein, we report the discovery of the clinical
candidate BOS172722. The starting point for our work was a
series of pyrido[3,4-d]pyrimidine inhibitors that demonstrated
excellent potency and kinase selectivity but suffered from rapid
turnover in human liver microsomes (HLM). Optimizing HLM
stability proved challenging since it was not possible to identify a
consistent site of metabolism and lowering lipophilicity proved
unsuccessful. Key to overcoming this problem was the finding that introduction of a methyl group at the 6-position of the
pyrido[3,4-d]pyrimidine core significantly improved HLM stability. Met ID studies suggested that the methyl group suppressed
metabolism at the distant aniline portion of the molecule, likely by blocking the preferred pharmacophore through which P450
recognized the compound. This work ultimately led to the discovery of BOS172722 as a Phase 1 clinical candidate.
BAY1161909 (1),14 BAY1217389 (2),14 CFI-402257 (3),15
INTRODUCTION
■
and S 81694 (structure undisclosed). We recently reported
MPS1 (monopolar spindle 1, also known as TTK) is a dual-
advanced inhibitors including CCT251455 (4)16 and a series
specificity kinase that occupies a central role in mitosis. MPS1
of pyrido[3,4-d]pyrimidines (34h (5))17 (Figure 1).
is one of the main components of the spindle assembly
checkpoint (SAC)1−4 and ensures cells do not progress from
While the single agent efficacy of MPS1 inhibitors has been
described to be limited,18−21 a number of recent reports have
metaphase to anaphase until the kinetochores are properly
documented that inhibition of MPS1 is particularly effective
attached to the microtubules and under the appropriate
tension at the metaphase plate.2,3 Cancer cells heavily rely on
when used in combination with other drugs, for example,
tubulin-targeting agents or CDK4/6 inhibitors.22−26 We were
MPS1 to cope with aneuploidy resulting from aberrant
numbers of chromosomes.5−8 The kinase has been found to
particularly intrigued by the use of MPS1 inhibitors in
be upregulated in a large number of tumor types6,7,9−13
combination with paclitaxel for triple negative breast cancer.
Paclitaxel is often used for this aggressive and highly
strongly suggesting MPS1 inhibition as a therapeutic approach
for the treatment of cancer. As a result, MPS1 inhibitors have
been pursued by a number of organizations, and accordingly, at
least four compounds have reached Phase 1 clinical trials:
Received: May 1, 2018
© XXXX American Chemical Society
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Figure 1. Published MPS1 inhibitors.
proliferative cancer but, on its own, does frequently not lead to
durable responses, particularly in the metastatic setting.27
Triple negative breast cancer thus remains a high medical need,
and new effective therapeutic regimens are needed.
The work presented here builds upon a series of pyrido[3,4-
d]pyrimidines that we recently disclosed.17 Advanced com-
pounds in this series showed excellent potency in biochemical
and cellular assays, exemplified by 5 (Figure 2); which was
approaches could be used to gain access to the des methyl
compounds. First, the amine was introduced into 7 by
displacement of the chloride, followed by m-CPBA oxidation
to give sulfone 9. Displacement of the sulfone with the
appropriate formamide under NaH/THF conditions gave rise
to the desired des methyl pyrido[3,4-d]pyrimidine compounds
(Scheme 1). Alternatively, the steps could be reversed, carrying
out the m-CPBA oxidation as the first step to afford sulfone 18.
Displacement with the appropriate formamide could then be
carried out as previously, before introducing the amine at the
final step, through reaction of the chloro-intermediate 20 with
neopentylamine in NMP at elevated temperatures.
Compounds in the 6-methyl pyrido[3,4-d]pyrimidine series
were prepared from the key intermediate 8-chloro-2-
(methylthio)pyrido[3,4-d]pyrimidine 22, the synthesis of
which we have previously reported.28 Treatment of this
intermediate with an amine at elevated temperature in NMP
gave rise to sulfides 23−25. Oxidation of these compounds
with m-CPBA afforded sulfones 26−28, which were ideally set
up to undergo selective displacement. The aniline moiety was
introduced using the corresponding formamide with either
NaH in THF or cesium carbonate in DMSO, affording the
desired 6-methyl pyrido[3,4-d]pyrimidines 34−42 (Scheme
2).
Again the order of steps could be reversed with the oxidation
with m-CPBA being carried out first to give sulfone 43, which
could then undergo the same coupling with the appropriate
formamide as previously described using NaH in THF.
Displacement of the chloro intermediate 44 was then carried
out with amines at elevated temperatures in NMP (Scheme 3).
All amines used were commercially available, and the
formamides were synthesized from the corresponding anilines
by refluxing in formic acid. The anilines were prepared by
standard transformations (see Supporting Information for
procedures).
Figure 2. MPS1 inhibitor 5. “P-MPS1” indicates an electro-
chemiluminescence mesoscale discovery (MSD)-based cellular assay
that measured autophosphorylation of ectopically expressed MPS1 in
HCT116 cells. Solubility conditions: HPLC, 1% DMSO, 10 mM PBS,
pH 7.4.
effective in inhibiting MPS1 in vivo.17 However, this series in
general, and 5 in particular, suffered from key liabilities that
prevented further development, specifically high turnover in
human microsomes as well as excessive lipophilicity.
Herein, we describe our optimization of the pyrido[3,4-
d]pyrimidine series17 culminating in the discovery of a Phase 1
clinical candidate compound.
RESULTS AND DISCUSSION
■
CHEMISTRY
We routinely tested our compounds in a caliper-based MPS1
kinase assay at 1 mM ATP. As described in our preceding
publication,17 this relatively high ATP concentration was
■
Des methyl pyrido[3,4-d]pyrimidine compounds were made
using the route shown in Scheme 1. Two complementary
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a
Scheme 1
a
Reagents and conditions: (i) amine, NMP, 100−130 °C; (ii) m-CPBA, CH2Cl2, 0 °C−r.t.; (iii) ArNHCHO 10−13 or 19, NaH, THF, 0 °C−r.t.
necessary due to the high potency of advanced compounds
(Kis < 1 nM) that was beyond the dynamic range of the assay
at lower ATP concentrations. Furthermore, we progressed
compounds of sufficient potency to an MSD-based cellular
assay that measured autophosphorylation of ectopically
expressed MPS1 in HCT116 cells.16 In addition, we routinely
determined selectivity against CDK2, a cell cycle kinase with a
high homology to MPS1 in terms of the ATP binding domain.
Since the CDK2 assay was run at much lower ATP
concentrations, we converted the IC50 values from the MPS1
assay (at 1 mM ATP) and those from the CDK2 assay (at 10
μM ATP) into Ki values and used these calculated Kis to assess
the selectivity ratio.
Our key goal for the optimization of the pyrido[3,4-d]-
pyrimidines series was to significantly improve the low human
liver microsomal stability observed for compound 5 and other
compounds in this series. Extensive attempts to identify major
metabolites showed multiple oxidations and dealkylation
products but failed to identify a consistent site of metabolism.
Furthermore, we had not observed a correlation between
stability in human microsomes and lipophilicity indicating that
lowering LogP was not a promising approach. We thus
suspected that rapid turnover was driven by recognition of a
specific pharmacophore within our series and decided to
systematically derivatize the molecule to discover modifications
that would block this recognition and increase the metabolic
stability.
strated excellent levels of biochemical activity, as well as
showing promising levels of selectivity over CDK2 and cellular
activity. Finally, triazole containing 17, while exhibiting
nanomolar potency for CDK2, stood out in terms of its single
digit nanomolar potency in the cellular assay and represents
one of the most potent MPS1 inhibitors known to us. In fact,
the potency of this compound was beyond the lower range of
our biochemical assay even at 1 mM ATP. The reason why
replacing the pyrazole moiety in 21 with imidazole (15) and
particularly triazole (16) significantly boosted biochemical
activity remained unclear, and crystal structures (vide infra)
did not provide any additional insights.
We tested the human liver microsome (HLM) stability of
compounds 16 and 17, but both still underwent extensive
metabolism showing Clint values of 79 and 92 μL/min/mg
protein, respectively. Nevertheless, the comparably low
molecular weight (418) and lipophilicity (3.6) made 17 an
excellent starting point for further investigation, and we
decided to improve CDK2 selectivity and HLM stability.
In order to develop hypotheses on how to reduce CDK2
activity, we superimposed X-ray structures of our compounds17
with published CDK2 structures (Figure 3).29,30
Fourteen residues are different within the ATP binding
pockets of MPS1 and CDK2, including the gatekeeper residue,
which is Met602 in MPS1 and a bulkier phenylalanine (Phe80)
in CDK2 (Figure 3). We hypothesized that introducing a
methyl group at the 6-position of the pyrido[3,4-d]pyrimidine
core would be less tolerated in CDK2 than in MPS1, due to a
clash with the CDK2 Phe80 gatekeeper residue.
We thus set out to prepare a series of methyl substituted
compounds. The significant investigations required to access
pyrido[3,4-d]pyrimidines with substitution in this position
were disclosed by us recently.28
We started by altering the five-membered ring heterocycle of
5 maintaining a neopentyl substituent in the 8-position of the
pyrido[3,4-d]pyrimidine core. These compounds are summar-
ized in Table 1. Methylated and dimethylated pyrazole
containing compounds (21, 14, and 15) showed only relatively
modest activity in the high ATP assay, as well as in the cellular
assay. The imidazole substituted compound (16) demon-
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a
Scheme 2
a
Reagents and conditions: (i) amine, NMP, 100−130 °C; (ii) m-CPBA, CH2Cl2, 0 °C−r.t.; (iii) ArNHCHO 12, 13, or 29−33, NaH, THF, 0 °C−
r.t.; (iv) ArNHCHO, Cs2CO3, DMSO, 120 °C.
a
and lower but still acceptable levels of inhibition in cells. As
hypothesized, both 6-methylated compounds (34 and 35)
demonstrated a significant improvement in selectivity for
MPS1 over CDK2 (Ki ratio is between 500 and 7600). Even
more importantly and somewhat unexpectedly, we observed a
large improvement in HLM metabolism for the 6-methylated
compounds. Compounds 34 and 35 represented by far the
most stable compounds we had observed and thus a
breakthrough in terms of optimizing the up to this point
persisting HLM liability. We thus decided to focus on the
methylated amino-pyrido[3,4-d]pyrimidine core.
Scheme 3
To understand why these compounds showed greatly
improved stability, we identified the metabolites for the
matched pair 17 and 35. Interestingly, these experiments
showed that the introduction of the methyl group completely
changed the nature of the main metabolites in HLM (Figure
4). Incubation of compound 17 with HLM primarily led to
metabolites in which the triazole and aniline moieties were
oxidized (Figure 4). In sharp contrast, treatment of 35 with
HLM led to oxidation on the neopentyl chain followed by loss
of the entire chain (Figure 4). Importantly, the metabolic
hotspots are not only different for these compounds but, in
both cases, also distant from the position of the newly
introduced methyl group of 35. This observation thus suggests
that the reduction of HLM metabolism is not due to blocking
of a metabolically labile position (a commonly applied strategy,
particularly using fluorine atoms) but instead to blocking of the
a
Reagents and conditions: (i) m-CPBA, CH2Cl2, 0 °C−r.t.; (ii)
ArNHCHO 29, NaH, THF, 0 °C−r.t.; (iii) amine, NMP, 100−130
°C.
Table 2 shows the biochemical and cellular results for two
initial proof-of-concept compounds made as matched pairs to
the corresponding unsubstituted compounds (16 and 17).
Both compounds show potent biochemical inhibition of MPS1
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a
Table 1. Biochemical and Cellular Data for Aniline Modifications on Neopentyl-Substituted Core
a
Results are in nM unless otherwise stated and are mean for n ≥ 3, or mean values of two independent determinations or samples run n = 1. For SD
(for n ≥ 3) and individual determinations (n = 2), see Table S1. “P-MPS1” indicates an electrochemiluminescence mesoscale discovery (MSD)-
based cellular assay that measured autophosphorylation of ectopically expressed MPS1 in HCT116 cells. Kis were calculated from the IC50s using
the Cheng−Prusoff equation.
pharmacophore through which 17 is recognized and bound.
This hypothesis is consistent with the nature of P450 enzymes
where substrate recognition and catalytic sites are spatially
separated.
Interestingly, this matched pair (38 and 39) also demonstrated
a similar increase in selectivity between methoxy and ethoxy
derivatives. Finally, adding a basic dimethylamino group to
improve solubility (40) resulted in loss of cellular potency (P-
MPS1 IC50 230 nM) possibly due to a decrease in cellular
permeability of the more polar dimethylamine tail group,
though the solubility (77.6 μM [HPLC method, 1% DMSO,
10 mM PBS, pH 7.4]) of this compound was greatly improved
in comparison to 35.
From this investigation, 36 emerged as an attractive
compound, and we tested if the overall properties could be
further optimized by modification of the neopentyl amine.
Table 4 shows a representative set of amine substitutions at
the 8-position of the pyrido[3,4-d]pyrimidine core. Compound
45 bears the same branched primary amine used in our
previously reported MPS1 inhibitor 5.17 Compound 45
demonstrated good activity against MPS1 in the biochemical
and cellular assays (Table 4) but exhibits poor solubility (2.2
μM), a possible consequence of the increased ALogP (4.61).
The introduction of secondary amines including pyrrolidine
(46) and substituted azetidines (47, 48, and 42) exhibited
varied activity against MPS1. Pyrrolidine containing 46
displayed a significant decrease in the biochemical and cellular
Compound 35 thus represented a significant step forward,
and we explored whether HLM stability, CDK2 selectivity,
cellular potency, and solubility could be further optimized.
Table 3 summarizes modifications of the methoxy group and
the triazole ring substituents. Based on existing SAR, we
hypothesized that introducing an ethoxy group in place of the
methoxy group of 35 improves selectivity further. The
corresponding ethoxy compound (36) showed similar levels
of biochemical potency (IC50 11 vs 13 nM), albeit with a slight
drop in cellular potency (P-MPS1 IC50 63 vs 30 nM). As
hypothesized, this transformation resulted in an improved
selectivity window over CDK2 (Ki ratio 500 (35) vs 46 (36))
and significantly improved stability in HLM.
Introduction of a methyl group onto the triazole ring (37)
resulted in very similar levels of potency to 35 both in the
biochemical and cellular assays (Table 3), albeit with an
increase in lipophilicity (ALogP = 4.49 vs 3.88). The bicyclic
triazole derivatives 38 and 39 were also potent biochemical
inhibitors but showed significantly weaker inhibition in cells.
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Figure 4. Results of a Met ID study showing oxidation products for
the match pair of compounds 17 and 35 after treatment with HLM.
potency observed. Compound 47 exhibited excellent activity
(P-MPS1 IC50 37 nM) and good levels of selectivity over
CDK2 coupled with an increased solubility of 31.5 μM,
possibly due to the decreased ALogP of 3.85. However,
movement of the gem dimethyl group one carbon round the
ring into the α-position (48) resulted in loss of all MPS1
activity (P-MPS1 IC50 3.8 μM). Cyano-substituted azetidine
compound (42) also showed acceptable levels of potency (P-
MPS1 IC50 110 nM) and selectivity over CDK2 (CDK2 IC50
Figure 3. Top: Superimposed crystal structure of MPS1 (green)
bound to a pyrido[3,4-d]pyrimidine core (carbon atoms colored
green), extracted from PDB code 5EH0, onto the structure of CDK2
(blue), extracted from PDB code 1H08 (ligand hidden for clarity),
showing the different gatekeeper residues present in MPS1 and
CDK2. Bottom: 6-position methyl group on pyrido[3,4-d]pyrimidine
core.
Table 2. Biochemical and Cellular Data for Two Matched Pairs of Compounds Containing H or Me at the 6-Position of the
a
Pyrido[3,4-d]pyrimidine Core
a
Results are in nM unless otherwise stated and are mean for n ≥ 3, or mean values of two independent determinations or samples run n = 1. For SD
(for n ≥ 3) and individual determinations (n = 2), see Table S2. “P-MPS1” indicates an electrochemiluminescence mesoscale discovery (MSD)-
based cellular assay that measured autophosphorylation of ectopically expressed MPS1 in HCT116 cells. Kis were calculated from the IC50s using
the Cheng−Prusoff equation.
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a
Table 3. Biochemical and Cellular Data for Triazole Modifications Based on 35
a
Results are in nM unless otherwise stated and are mean for n ≥ 3, or mean values of two independent determinations or samples run n = 1. For SD
(for n ≥ 3) and individual determinations (n = 2), see Table S3. “P-MPS1” indicates an electrochemiluminescence mesoscale discovery (MSD)-
based cellular assay that measured autophosphorylation of ectopically expressed MPS1 in HCT116 cells.
2.70 μM), combined with a much lower AlogP of 3.25.
However, this reduced ALogP did not translate into increased
solubility, with 42 only showing a solubility of 6.8 μM.
Introduction of a polar spirocyclic amine (41) resulted in a
dramatic drop in lipophilicity (AlogP = 3.08), which, as
expected, translated into an increase in solubility (56.2 μM).
To understand the observed MPS1 SAR (Table 4), we
solved the crystal structure of compound 36 bound to MPS1
(Figure 5A). As expected, the binding mode of 36 was nearly
identical to that of the previously described pyrido[3,4-
d]pyrimidine inhibitors.17 The pyrido[3,4-d]pyrimidine scaf-
fold of 36 binds to the hinge region, the 6-methyl group
important for CDK2 selectivity and for the reduction in HLM
metabolism, is located close to the side chain of the gatekeeper
residue Met602, and the ethoxy moiety also important for
selectivity binds in the selectivity pocket above the hinge.
Somewhat surprisingly, the triazole moiety was not engaged in
a hydrogen bond, and thus the X-ray structure did not explain
why replacing the pyrazole of 21 with triazole (17, Table 1) led
to a significant increase in activity (vide supra). Together with
previously reported structures of CCT251455 (4) and (5), the
X-ray structure of 36 allowed us to rationalize the SAR
summarized in Table 4. As previously described for compound
5, the neopentyl chain binds to a hydrophobic pocket that is
created by a reordering of the MPS1 activation loop into an
inactive conformation (Figure 5B).16,17 The shape of the
neopentyl chain represents an excellent match to this pocket
resulting in several hydrophobic contacts explaining why this
moiety is critical for activity. The two azetidine derivatives 47
and 48 exemplify the importance of the correct shape of the
amine substituent for potent inhibition. The 3,3-dimethylaze-
tidine substituent of derivative 47 can be regarded as a
constrained mimetic of the neopentyl chain that can likely
engage in similar hydrophobic contacts, and 47 maintains
potent inhibition. The 2,2-dimethylazetidine moiety of 48,
however, differs significantly in its overall shape from the
neopentyl chain leading to less favorable interactions and a 40-
fold higher IC50.
To investigate which of the potent and selective compounds
in Table 4 can be progressed further, we tested the stability in
liver microsomes. Gratifyingly, all compounds tested showed
satisfactory stability (Clint < 45 μL/min/mg protein in mouse
and Clint < 26 μL/min/mg protein in human) (Table 4). This
represented a vast improvement over the human intrinsic
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a
Table 4. Biochemical and Cellular Data for Amine Modifications Based on 36
a
Results are in nM unless otherwise stated and are mean for n ≥ 3, or mean values of two independent determinations or samples run n = 1. For SD
(for n ≥ 3) and individual determinations (n = 2), see Table S4. “P-MPS1” indicates an electrochemiluminescence mesoscale discovery (MSD)-
based cellular assay that measured autophosphorylation of ectopically expressed MPS1 in HCT116 cells.
Figure 5. (A) Crystal structure of 36 bound to MPS1 (pdb code 6H3K). Compound 36 is shown with carbon atoms in yellow. Selected amino
acids are shown in sea green and are labeled. Key hydrogen bonds are indicated as black dotted lines. (B) Close up of the neopentyl binding pocket.
The neopentyl substituent is enveloped by residues Met671 and Pro673 from the activation loop. Protein surface is displayed as a transparent blue
surface. The compound surface is shown in transparent yellow.
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a,b,c
Table 5. Mouse and Rat Blood Pharmacokinetics of 36, 41, 45, and 47 at 5 mg/kg iv and po, unless Otherwise Stated
a
b
c
2.5 mg/kg (iv). 1 mg/kg (iv). Compounds were administered iv and po (Mouse, 0.1 mL/10 g in 10% DMSO, 5% Tween 20 in saline; Rat, 0.05
d
e
mL/10 g in 10% DMSO, 5% Tween 20 in saline). AUClast 6 h for mouse, 24 h for rat, unless otherwise stated. AUClast 6 h.
Figure 6. Bar charts show the ratio of phosphylated-MPS1/total-MPS1 levels (gray) in the Dox-DLD1 model after treatment with 36 (left) and 41
(right) at specified doses and time points. The plasma levels at the respective time points are given in the table below the bar chart.
clearance values seen for compound 5 (Clint 151.2 μL/min/mg
protein), the starting point of our investigation.
(41). This corresponded to a higher blood clearance and
shorter half-life (0.66 h) for 41 compared to 36 (2.68 h).
We thus decided to progress both 36 and 41 to a single dose
pharmacokinetic/pharmacodynamic (PK/PD) experiment in
DLD1 xenografts to investigate if the in vitro and PK profiles
translated into sustained inhibition of MPS1 in vivo (Figure 6).
We recently disclosed32 a xenograft model to assess
modulation of MPS1 activity in vivo. Briefly, this model
(Dox-DLD1) measures the level of MPS1 autophosphorylation
in DLD1 cancer cells and, importantly, overcomes the issue of
low MPS1 levels through doxycycline inducible expression of
the kinase. We tested both compounds at 25 mg/kg and in
addition compound 36 at 50 mg/kg. The data are summarized
in Figure 6. At 25 mg/kg, both compounds led to a
pronounced reduction of MPS1 autophosphorylation after 6
With a number of active, selective, and soluble compounds
in hand, we decided to investigate the mouse and rat
pharmacokinetics (PK) of a selection of compounds (36, 45,
47, and 41) at 5 mg/kg administered both intravenous (iv)
and orally (po) (Table 5).31 The resulting data showed
moderate clearance for all compounds in both mouse (8.4−
22.6 mL/min/kg) and rat (6.00−10.1 mL/min/kg). All
compounds with the exception of 41 showed high oral
bioavailability in both species (63−92%) with moderate
volumes of distribution (Table 5). The plasma protein binding
for all four of these compounds was high (>98%), the lowest
unsurprisingly shown by that presenting the lowest AlogP
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h. Consistent with its longer half-life, only 36 showed
significant inhibition at 24 h. As expected, the 50 mg/kg
dose of 36 resulted in an increased suppression of MPS1
autophosphorylation at 6 and 24 h. The comparison of the
plasma levels determined in the PK/PD study (Figure 6) after
blood to plasma correction with the compound levels
determined in the PK study described above and performed
at lower doses (Table 5) was consistent with linear PK.
The robust modulation of the PD biomarkers observed for
36 prompted us to focus on this compound since a long
duration of action is desirable for cell cycle targets. At 1 mg/kg
iv and 5 mg/kg po, 36 showed complete bioavailability
(100%), low clearance (1.2 mL/min/kg), a moderate volume
of distribution (1.1 L/kg), and a 12 h half-life (Table 6) in a
dog PK study.
CONCLUSIONS
■
We describe herein the discovery of our MPS1 inhibitor 36
(BOS172722). The starting point for the work described here
was a series of previously reported pyrido[3,4-d]pyrimidine
inhibitors. These earlier compounds already showed promising
in vitro potency and selectivity but suffered from a number of
liabilities, particularly high lipophilicity and rapid metabolism
in HLM. Optimizing HLM metabolism proved challenging
since commonly used approaches, such as identification of
metabolites and lowering lipophilicity, did not help. Key to
overcoming this problem was the serendipitous finding that
introduction of a methyl group at the 6-position of the
pyrido[3,4-d]pyrimidine core significantly improved HLM
stability. Met ID studies suggested that the methyl group
suppressed metabolism at the distant aniline portion of the
molecule, likely by blocking the preferred pharmacophore
through which P450 recognized the compound. Compound 17
is thus an interesting example where metabolism is not
primarily driven by hydrophobicity or the presence of a
particular metabolic hotspot but by recognition of a specific
pharmacophore distant from the site of metabolism. These
results underscore the importance of systematic chemical
modification to solve high metabolic turnover.
Table 6. Dog Blood Pharmacokinetics not Dogblood 36 at 1
a
mg/kg iv and 5 mg/kg po
Further optimization led to a set of compounds with
promising in vitro profile, and we progressed a number of
selected compounds to PK and subsequently PK/PD experi-
ments. Compound 36 emerged as our candidate showing
excellent PK in mouse, rat, and dog. Data showing robust
efficacy of 36 in combination with paclitaxel in in vivo models
will be published shortly.
Interestingly, 36 showed very good bioavailability in all three
species despite very modest solubility at physiological pH. We
attribute this observation to the weakly basic character of 36
(pKa = 6.22) and other compounds in this series, which is
likely accelerating their dissolution in the acidic environment
of the stomach.
a
The compound was administered as the dichloride salt in saline
b
containing 10% DMSO. AUClast 48 h.
We were intrigued by the high bioavailability of 36,
particularly given its poor solubility. Interestingly, pKa values
for 36 were determined as 6.22 and 2.63. This suggests that,
while the compound primarily exists as the free base at
physiological pH, it is protonated in the acidic environment of
the stomach, likely accelerating the dissolution and enabling
high bioavailability despite modest solubility at physiological
pH. This is an attractive feature of the pyrido [3,4-
d]pyrimidine scaffold since it avoids the well-recognized risk
associated with drugs carrying a positive charge at physio-
logical pH, namely, hERG inhibition, efflux, and detrimental
effects on passive permeation while still allowing high
bioavailability and salt formation. To shed light on the
question of which atom represents the weakly basic center, we
performed a calculation using MoKa.33 This calculation
predicted that the nitrogen atom of the pyridine ring is by
far the most basic atom.
To complete the in vitro profiling, 36 was tested in a wide
panel of more than 400 kinases (Supporting Information,
Tables S5−S8). As we had seen with our previous MPS1
inhibitor 5,17 only a small number of other kinases were
inhibited by 36, in particular JNK1, JNK2, JNK3, and LRRK2
at >80% at 1 μM. Follow up IC50 values were obtained (JNK1
IC50 = 92 nM, JNK2 IC50 = 76 nM, JNK3 IC50 = 242 nM, and
LRRK2 IC50 = 48 nM) showing that 36 is selective for MPS1
over these other kinases. Furthermore, 36 also showed a clean
CYP and hERG profile (Supporting Information, Tables S9
and S10).
The synthesis of 36 has been scaled to the kilogram range,
and the compound is currently in Phase 1 clinical trials.
EXPERIMENTAL SECTION
■
General Chemistry Information. Starting materials, reagents,
and solvents for reactions were reagent grade and used as purchased.
Chromatography solvents were HPLC grade and were used without
further purification. Thin layer chromatography (TLC) analysis was
performed using silica gel 60 F-254 thin layer plates. Flash column
chromatography was carried out using columns prepacked with 40−
63 μm silica. Microwave-assisted reactions were carried out using a
Biotage Initiator microwave system. LCMS and HRMS analyses were
performed on a HPLC system with diode array detector operating at
254 nm, fitted with a reverse-phase 50 × 4.6 mm column at a
temperature of 22 °C, connected to a time of flight (ToF) mass
spectrometer (ESI). The following solvent system, at a flow rate of 2
mL/min, was used: solvent A, methanol; solvent B, 0.1% formic acid
in water. 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 then reversion back to 1:9 (A:B) over
0.3 min, 1:9 (A:B) for 0.2 min. 1H NMR spectra were recorded on a
Bruker Avance 500 MHz spectrometer using an internal deuterium
lock. NMR data is given as follows: chemical shift (δ) in ppm,
multiplicity, coupling constants (J) given in Hz, and integration. The
purity of final compounds was determined by HPLC as described
above and is ≥95% unless specified otherwise.
Following extensive in vivo testing, particularly in combina-
tion with paclitaxel, the results of which will be published in
due course (manuscript in preparation), we nominated 36 as
our candidate. The synthesis of 36 has been scaled up into the
kilogram range, and the drug is currently undergoing Phase 1
clinical trials.
Compounds 8-chloro-2-(methylthio)pyrido[3,4-d]pyrimidine28 7,
2-(methylthio)-N-neopentylpyrido[3,4-d]pyrimidin-8-amine28 8, 2-
(methylsulfonyl)-N-neopentylpyrido[3,4-d]pyrimidin-8-amine28 9, 8-
chloro-2-(methylsulfonyl)pyrido[3,4-d]pyrimidine28 18, N-(2-me-
J
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Article
thoxy-4-(1-methyl-1H-pyrazol-4-yl)phenyl)formamide28 19, 8-chloro-
N-(2-methoxy-4-(1-methyl-1H-pyrazol-4-yl)phenyl)pyrido[3,4-d]-
pyrimidin-2-amine28 20, N2-(2-methoxy-4-(1-methyl-1H-pyrazol-4-
yl)phenyl)-N8-neopentylpyrido[3,4-d]pyrimidine-2,8-diamine17,28 21,
8-chloro-6-methyl-2-(methythio)pyrido[3,4-d]pyrimidine28 22, 6-
methyl-2-(methylthio)-N-neopentylpyrido[3,4-d]pyrimidin-8-amine28
23, and 6-methyl-2-(methylsulfonyl)-N-neopentylpyrido[3,4-d]-
pyrimidin-8-amine28 26 were synthesized by previously reported
methods.
chromatography (0−50% EtOAc in cyclohexane) to afford the title
compounds.
2-(6-Methyl-2-(methylthio)pyrido[3,4-d]pyrimidin-8-yl)-7-oxa-2-
azaspiro[3.5]nonane 24. Using 7-oxa-2-azaspiro[3.5]nonane. Yield =
40%. HRMS (ESI) m/z calcd for C16H21N4OS (M + H) 317.1431,
found 317.1422; 1H NMR (500 MHz, CD3OD) δ 9.00 (s, 1H), 6.76
(app s, 1H), 4.33 (br s, 4H), 3.71 (app t, J = 5.0 Hz, 4H), 2.65 (s,
3H), 2.44 (d, J = 0.5 Hz, 3H), 1.90 (app t, J = 5.0 Hz, 4H).
3-Methyl-1-(6-methyl-2-(methylthio)pyrido[3,4-d]pyrimidin-8-
yl)azetidine-3-carbonitrile 25. Using 3-methylazetidine-3-carbon-
itrile hydrochloride. Yield = 66%. HRMS (ESI) m/z calcd for
C14H16N5S (M + H) 268.1121, found 268.1119; 1H NMR (500 MHz,
CD3OD) δ 9.05 (s, 1H), 6.89 (app s, 1H), 4.83 (br d, J = 7.5 Hz,
2H), 4.48 (br d, J = 7.5 Hz, 2H), 2.64 (s, 3H), 2.47 (d, J = 0.5 Hz,
3H), 1.78 (s, 3H).
General Procedure for m-CPBA Oxidation on 23−25. To a
cooled (0 °C) solution of appropriate sulfide (1 equiv) in CH2Cl2 (10
mL) was added m-CPBA (3 equiv). The reaction mixture was stirred
for 18 h, while slowly warming to r.t. In some instances, further m-
CPBA was needed to achieve full conversion. The reaction mixture
was quenched with water and extracted with CH2Cl2. The combined
organic layers were washed with aq. sat. NaHCO3, dried (MgSO4),
and concentrated in vacuo. The residue was purified by flash column
chromatography (0−10% MeOH in CH2Cl2).
Preparation of Compounds in Scheme 1. General Procedure
for NaH Mediated Displacement on 9 (Compounds 14−17). To a
cooled (0 °C) solution of appropriate formamide (1 equiv) in THF
(3 mL) was added sodium hydride (60% dispersion in oil, 1.6 equiv).
The reaction mixture was stirred at r.t. for 10 min. The reaction
mixture was cooled to 0 °C, and appropriate sulfone (1.2 equiv) was
added. The reaction mixture was stirred for 18 h, while slowly
warming to r.t. aq NaOH (1 M, 1 mL) and MeOH (1 mL) were
added, and the reaction mixture was stirred at r.t. for 1 h. The reaction
mixture was concentrated in vacuo, and the residue was diluted with
EtOAc and water. The aqueous layer was re-extracted with EtOAc
and CH2Cl2. The combined organic layers were washed with water
and brine, dried (MgSO4), and concentrated in vacuo. The residue
was purified by flash column chromatography (0−10% MeOH in
EtOAc or CH2Cl2) and, if necessary, followed by SCX-2 cartridge
(MeOH, 1 M NH3 in MeOH) to afford the title compounds.
N2-(4-(1,3-Dimethyl-1H-pyrazol-4-yl)-2-methoxyphenyl)-N8-
neopentylpyrido[3,4-d]pyrimidine-2,8-diamine 14. Using N-(4-
(1,3-dimethyl-1H-pyrazol-4-yl)-2-methoxyphenyl)formamide 10 and
2-(methylsulfonyl)-N-neopentylpyrido[3,4-d]pyrimidin-8-amine 9.28
Yield = 21%. HRMS (ESI) m/z calcd for C24H30N7O (M + H)
2-(6-Methyl-2-(methylsulfonyl)pyrido[3,4-d]pyrimidin-8-yl)-7-
oxa-2-azaspiro[3.5]nonane 27. Using 2-(6-methyl-2-(methylthio)-
pyrido[3,4-d]pyrimidin-8-yl)-7-oxa-2-azaspiro[3.5]nonane 24. Yield =
28%. HRMS (ESI) m/z calcd for C16H21N4O3S (M + H) 349.1329,
found 349.1318; 1H NMR (500 MHz, CD3OD) δ 9.38 (s, 1H), 6.94
(s, 1H), 4.29 (br s, 4H), 3.73 (app t, J = 5.5 Hz, 4H), 3.43 (s, 3H),
2.53 (s, 3H), 1.92 (app t, J = 5.5 Hz, 4H).
1
432.2506, found 432.2502; H NMR (500 MHz, CD3OD) δ 9.07 (s,
3-Methyl-1-(6-methyl-2-(methylsulfonyl)pyrido[3,4-d]pyrimidin-
8-yl)azetidine-3-carbonitrile 28. Using 3-methyl-1-(6-methyl-2-
(methylthio)pyrido[3,4-d]pyrimidin-8-yl)azetidine-3-carbonitrile 25.
Yield = 77%. HRMS (ESI) m/z calcd for C14H16N5O2S (M + H)
318.1019, found 318.1009; H NMR (500 MHz, CD3OD) δ 9.46 (s,
1H), 7.08 (s, 1H), 4.54 (br s, 4H), 3.44 (s, 3H), 2.56 (s, 3H), 1.80 (s,
1H), 8.42 (d, J = 8.0 Hz, 1H), 7.73 (s, 1H), 7.71 (d, J = 6.0 Hz, 1H),
7.10 (d, J = 1.5 Hz, 1H), 7.03 (dd, J = 8.0, 1.5 Hz, 1H), 6.85 (d, J =
6.0 Hz, 1H), 4.00 (s, 3H), 3.88 (s, 3H), 3.40 (s, 2H), 2.40 (s, 3H),
1.09 (s, 9H).
1
N2-(4-(1,5-Dimethyl-1H-pyrazol-4-yl)-2-methoxyphenyl)-N8-
neopentylpyrido[3,4-d]pyrimidine-2,8-diamine 15. Using N-(4-
(1,5-dimethyl-1H-pyrazol-4-yl)-2-methoxyphenyl)formamide 11 and
2-(methylsulfonyl)-N-neopentylpyrido[3,4-d]pyrimidin-8-amine 9.28
Yield = 8%. HRMS (ESI) m/z calcd for C24H30N7O (M + H)
3H).
N2-(4-(1,2-Dimethyl-1H-imidazol-5-yl)-2-methoxyphenyl)-6-
methyl-N8-neopentylpyrido[3,4-d]pyrimidine-2,8-diamine 34. To a
cooled (0 °C) solution of N-(4-(1,2-dimethyl-1H-imidazol-5-yl)-2-
methoxyphenyl)formamide 12 (17 mg, 0.069 mmol) in THF (3 mL)
was added sodium hydride (2.7 mg, 0.11 mmol, 60% dispersion in
oil). The reaction mixture was stirred at r.t. for 10 min. The reaction
mixture was cooled to 0 °C, and 6-methyl-2-(methylsulfonyl)-N-
neopentylpyrido[3,4-d]pyrimidin-8-amine 2628 (25.6 mg, 0.083
mmol) was added. The reaction mixture was stirred for 18 h, while
slowly warming to r.t. aq NaOH (1 M, 1 mL) and MeOH (1 mL)
were added, and the reaction mixture was stirred at r.t. for 1 h. The
reaction mixture was concentrated in vacuo, and the residue diluted
with EtOAc and water. The aqueous layer was re-extracted with
EtOAc and CH2Cl2. The combined organic layers were washed with
water and brine, dried (MgSO4), and concentrated in vacuo. The
residue was purified by flash column chromatography (0−10%
MeOH in EtOAc) and followed by SCX-2 cartridge (MeOH, 1 M
NH3 in MeOH) to afford the title compound (3.3 mg, 11%). HRMS
(ESI) m/z calcd for C25H32N7O (M + H) 446.2663, found 446.2648;
1H NMR (500 MHz, CD3OD) δ 9.02 (s, 1H), 8.57 (d, J = 8.5 Hz,
1
432.2506, found 432.2504; H NMR (500 MHz, CDCl3) δ 9.02 (s,
1H), 8.51 (br s, 1H), 8.05 (m, 1H), 7.84 (m, 1H), 7.58 (s, 1H), 6.99
(dd, J = 8.2, 1.8 Hz, 1H), 6.96 (d, J = 1.8 Hz, 1H), 6.80 (br s, 1H),
3.99 (s, 3H), 3.88 (s, 3H), 3.64 (br s, 2H), 2.42 (s, 3H), 1.14 (s, 9H).
N2-(4-(1,2-Dimethyl-1H-imidazol-5-yl)-2-methoxyphenyl)-N8-
neopentylpyrido[3,4-d]pyrimidine-2,8-diamine 16. Using N-(4-
(1,2-dimethyl-1H-imidazol-5-yl)-2-methoxyphenyl)formamide 12
and 2-(methylsulfonyl)-N-neopentylpyrido[3,4-d]pyrimidin-8-amine
9.28 Yield = 21%. HRMS (ESI) m/z calcd for C24H30N7O (M +
1
H) 432.2506, found 432.2504; H NMR (500 MHz, CDCl3) δ 9.04
(s, 1H), 8.76 (d, J = 8.0 Hz, 1H), 8.12 (s, 1H), 7.94 (d, J = 6.0 Hz,
1H), 7.21 (s, 1H), 6.99 (dd, J = 8.0, 2.0 Hz, 1H), 6.91 (d, J = 2.0 Hz,
1H), 6.79 (d, J = 6.0 Hz, 1H), 6.58 (br s, 1H), 4.02 (s, 3H), 3.67 (s,
3H), 3.50 (d, J = 6.0 Hz, 2H), 2.82 (s, 3H), 1.12 (s, 9H).
N2-(2-Methoxy-4-(4-methyl-4H-1,2,4-triazol-3-yl)phenyl)-N8-
neopentylpyrido[3,4-d]pyrimidine-2,8-diamine 17. Using N-(2-
methoxy-4-(4-methyl-4H-1,2,4-triazol-3-yl)phenyl)formamide 13
and 2-(methylsulfonyl)-N-neopentylpyrido[3,4-d]pyrimidin-8-amine
9.28 Yield = 12%. HRMS (ESI) m/z calcd for C22H27N8O (M +
H) 419.2302, found 419.2288; 1H NMR (500 MHz, CD3OD) δ 9.15
(s, 1H), 8.74 (d, J = 8.5 Hz, 1H), 8.57 (s, 1H), 7.77 (d, J = 6.0 Hz,
1H), 7.43 (d, J = 1.5 Hz, 1H), 7.35 (dd, J = 8.5, 1.5 Hz, 1H), 6.90 (d,
J = 6.0 Hz, 1H), 4.07 (s, 3H), 3.87 (s, 3H), 3.44 (s, 2H), 1.10 (s, 9H).
Preparation of Compounds in Scheme 2. General Procedure
for Amine Displacement on 22. To a solution of 8-chloro-6-methyl-
2-(methythio)pyrido[3,4-d]pyrimidine 22 (1 equiv) in NMP (20
mL) was added appropriate amine (2 equiv) and triethylamine (5
equiv). The reaction mixture was heated to 100 °C for 36 h. The
reaction mixture was diluted with EtOAc and water, dried (MgSO4),
and concentrated in vacuo. The residue was purified by flash column
1H), 7.09 (d, J = 2.0 Hz, 1H), 7.02 (dd, J = 8.5, 2.0 Hz, 1H), 6.88 (s,
1H), 6.70 (d, J = 1.0 Hz, 1H), 4.02 (s, 3H), 3.61 (s, 3H), 3.45 (s,
2H), 2.45 (s, 3H), 2.44 (d, J = 1.0 Hz, 3H), 1.09 (s, 9H).
General Procedure for Cesium Carbonate Mediated Substitution
on 26, 27, or 28. To a solution of the appropriate sulfone (1 equiv)
in DMSO (20 mg sulfone/mL) was added appropriate formamide
(1.2 equiv) and cesium carbonate (2 equiv). The reaction mixture was
heated to 120 °C in a closed cap vial for 18 h. The reaction mixture
was diluted with EtOAc and water. The aqueous layer was re-
extracted with EtOAc, and the combined organic layers were dried
(MgSO4) and concentrated in vacuo. The residue was purified by flash
column chromatography (0−10% MeOH in CH2Cl2) and if necessary
K
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Article
followed by SCX-2 cartridge (MeOH, 1 M NH3 in MeOH) to afford
the title compounds.
N-(2-Ethoxy-4-(4-methyl-4H-1,2,4-triazol-3-yl)phenyl)-6-methyl-
8-(7-oxa-2-azaspiro[3.5]nonan-2-yl)pyrido[3,4-d]pyrimidin-2-
amine 41. Using 2-(6-methyl-2-(methylsulfonyl)pyrido[3,4-d]-
pyrimidin-8-yl)-7-oxa-2-azaspiro[3.5]nonane 27 and N-(2-ethoxy-4-
(4-methyl-4H-1,2,4-triazol-3-yl)phenyl)formamide 29. Yield = 46%.
HRMS (ESI) m/z calcd for C26H31N8O2 (M + H) 487.2564, found
487.2511; 1H NMR (500 MHz, CD3OD) δ 9.06 (s, 1H), 8.57 (d, J =
8.5 Hz, 1H), 8.56 (s, 1H), 7.39 (d, J = 2.0 Hz, 1H), 7.36 (dd, J = 8.5,
2.0 Hz, 1H), 6.78 (s, 1H), 4.31−4.27 (m, 6H), 3.87 (s, 3H), 3.69
(app t, J = 5.5 Hz, 4H), 2.44 (s, 3H), 1.89 (app t, J = 5.5 Hz, 4H),
1.54 (t, J = 7.0 Hz, 3H).
N2-(2-Methoxy-4-(4-methyl-4H-1,2,4-triazol-3-yl)phenyl)-6-
methyl-N8-neopentylpyrido[3,4-d]pyrimidine-2,8-diamine 35.
Using 6-methyl-2-(methylsulfonyl)-N-neopentylpyrido[3,4-d]-
pyrimidin-8-amine 26 and N-(2-methoxy-4-(4-methyl-4H-1,2,4-tria-
zol-3-yl)phenyl)formamide 13. Yield = 48%. HRMS (ESI) m/z calcd
1
for C23H29N8O (M + H) 433.2459, found 433.2447; H NMR (500
MHz, CD3OD) δ 9.05 (s, 1H), 8.75 (d, J = 8.5 Hz, 1H), 8.56 (s, 1H),
7.42 (d, J = 2.0 Hz, 1H), 7.34 (dd, J = 8.5, 2.0 Hz, 1H), 6.71 (d, J =
1.0 Hz, 1H), 4.07 (s, 3H), 3.87 (s, 3H), 3.48 (s, 2H), 2.44 (app s,
3H), 1.10 (s, 9H).
1-(2-((2-Ethoxy-4-(4-methyl-4H-1,2,4-triazol-3-yl)phenyl)-
amino)-6-methylpyrido[3,4-d]pyrimidin-8-yl)-3-methylazetidine-3-
carbonitrile 42. Using 3-methyl-1-(6-methyl-2-(methylsulfonyl)-
pyrido[3,4-d]pyrimidin-8-yl)azetidine-3-carbonitrile 28 and N-(2-
ethoxy-4-(4-methyl-4H-1,2,4-triazol-3-yl)phenyl)formamide 29.
Yield = 38%. HRMS (ESI) m/z calcd for C24H26N9O (M + H)
N2-(2-Ethoxy-4-(4-methyl-4H-1,2,4-triazol-3-yl)phenyl)-6-meth-
yl-N8-neopentylpyrido[3,4-d]pyrimidine-2,8-diamine 36. Using 6-
methyl-2-(methylsulfonyl)-N-neopentylpyrido[3,4-d]pyrimidin-8-
amine 26 and N-(2-ethoxy-4-(4-methyl-4H-1,2,4-triazol-3-yl)phenyl)-
formamide 29. Yield = 70%. HRMS (ESI) m/z calcd for C24H31N8O
1
456.2255, found 456.2180; H NMR (500 MHz, CD3OD) δ 9.10 (s,
1
(M + H) 447.2615, found 447.2629; H NMR (500 MHz, acetone-
1H), 8.56 (s, 1H), 8.46 (d, J = 8.5 Hz, 1H), 7.40 (dd, J = 8.5, 2.0 Hz,
1H), 7.39 (d, J = 2.0 Hz, 1H), 6.91 (s, 1H), 4.75 (d, J = 9.0 Hz, 2H),
4.39 (d, J = 9.0 Hz, 2H), 4.28 (q, J = 7.0 Hz, 2H), 3.88 (s, 3H), 2.47
(s, 3H), 1.75 (s, 3H), 1.53 (t, J = 7.0 Hz, 3H).
d6) δ 9.11 (s, 1H), 8.80 (d, J = 8.5 Hz, 1H), 8.37 (s, 1H), 8.21 (br s,
1H), 7.46 (d, J = 2.0 Hz, 1H), 7.40 (dd, J = 8.5, 2.0 Hz, 1H), 6.74 (d,
J = 0.5 Hz, 1H), 4.35 (q, J = 7.0 Hz, 2H), 3.90 (s, 3H), 3.51 (s, 2H),
2.41 (d, J = 0.5 Hz, 3H), 1.55 (t, J = 7.0 Hz, 3H), 1.08 (s, 9H).
N2-(4-(4,5-Dimethyl-4H-1,2,4-triazol-3-yl)-2-ethoxyphenyl)-6-
methyl-N8-neopentylpyrido[3,4-d]pyrimidine-2,8-diamine 37.
Using 6-methyl-2-(methylsulfonyl)-N-neopentylpyrido[3,4-d]-
pyrimidin-8-amine 2628 and N-(2-ethoxy-4-(4,5-dimethyl-4H-1,2,4-
triazol-3-yl)phenyl)formamide 30. Yield = 56%. HRMS (ESI) m/z
Preparation of Compounds in Scheme 3. 8-Chloro-6-methyl-
2-(methylsulfonyl)pyrido[3,4-d]pyrimidine 43. A suspension of 8-
chloro-6-methyl-2-(methylthio)pyrido[3,4-d]pyrimidine 22 (1.13 g,
5.01 mmol) in CH2Cl2 (50 mL) was treated with m-CPBA (77% w/
w, 2.60 g, 11.57 mmol) at 0 °C. The reaction mixture was stirred for
18 h, while slowly warming to r.t. The reaction was quenched with
water and extracted with CH2Cl2. The combined organic layers were
washed with aq. sat. NaHCO3, dried (MgSO4), and concentrated in
vacuo. The residue was purified by flash column chromatography (0−
70% EtOAc in cyclohexane) to afford the title compound (972 mg,
75%). HRMS (ESI) m/z calcd for C9H9ClN3O2S (M + H) 258.0099,
found 258.0092; 1H NMR (500 MHz, CD3OD) δ 9.82 (s, 1H), 7.96
(d, J = 0.5 Hz, 1H), 3.54 (s, 3H), 2.78 (d, J = 0.5 Hz, 3H).
8-Chloro-N-(2-ethoxy-4-(4-methyl-4H-1,2,4-triazol-3-yl)phenyl)-
6-methylpyrido[3,4-d]pyrimidin-2-amine 44. To a solution of N-(2-
ethoxy-4-(4-methyl-4H-1,2,4-triazol-3-yl)phenyl)formamide 29 (1.88
g, 7.63 mmol) in THF (70 mL) was added NaH (60% w/w, 500 mg,
12.50 mmol) at 0 °C. The reaction mixture was stirred at r.t. for 30
min. The mixture was cooled to 0 °C, and 8-chloro-6-methyl-2-
(methylsulfonyl)pyrido[3,4-d]pyrimidine 43 (2.50 g, 9.70 mmol) was
added. The reaction mixture was stirred for 18 h, while slowly
warming to r.t. aq. NaOH (2 M, 25 mL) and MeOH (25 mL) were
added, and the resulting mixture stirred at r.t. for 1 h. The reaction
mixture was concentrated in vacuo, and the residue was diluted with
CH2Cl2 and water. The aqueous layer was extracted with CH2Cl2, and
the combined organic layers were dried (MgSO4) and concentrated in
vacuo. The residue was purified by flash column chromatography (0−
6% MeOH in CH2Cl2) to afford the title compound (3.24 g, quant).
HRMS (ESI) m/z calcd for C19H19ClN7O (M + H) 396.1339, found
396.1335; 1H NMR (500 MHz, (CD3)2SO) δ 9.46 (s, 1H), 8.85 (d, J
= 8.3 Hz, 1H), 8.79 (s, 1H), 8.56 (s, 1H), 7.74 (d, J = 1.0 Hz, 1H),
7.49−7.36 (m, 2H), 4.25 (q, J = 7.0 Hz, 2H), 3.80 (s, 3H), 2.58 (s,
3H), 1.43 (t, J = 7.0 Hz, 3H).
1
calcd for C25H33N8O (M + H) 461.2772, found 461.2756; H NMR
(500 MHz, CD3OD) δ 9.06 (s, 1H), 8.75 (d, J = 8.5 Hz, 1H), 7.34 (d,
J = 1.9 Hz, 1H), 7.28 (dd, J = 8.5, 1.9 Hz, 1H), 6.72 (s, 1H), 4.30 (q, J
= 7.2 Hz, 2H), 3.71 (s, 3H), 3.48 (s, 2H), 2.53 (s, 3H), 2.45 (s, 3H),
1.56 (t, J = 7.2 Hz, 3H), 1.10 (s, 9H).
N2-(4-(6,7-Dihydro-5H-pyrrolo[2,1-c][1,2,4]triazol-3-yl)-2-me-
thoxyphenyl)-6-methyl-N8-neopentylpyrido[3,4-d]pyrimidine-2,8-
diamine 38. Using 6-methyl-2-(methylsulfonyl)-N-neopentylpyrido-
[3,4-d]pyrimidin-8-amine 2628 and N-(4-(6,7-dihydro-5H-pyrrolo-
[2,1-c][1,2,4]triazol-3-yl)-2-methoxyphenyl)formamide 31. Purified
by SCX-2 cartridge (MeOH, 1 M NH3 in MeOH) followed by
purification by HPLC. Yield = 31%. HRMS (ESI) m/z calcd for
C25H31N8O (M + H) 459.2615, found 459.2571; 1H NMR (500
MHz, CDCl3) δ 8.92 (s, 1H), 8.71 (d, J = 8.3 Hz, 1H), 8.07 (s, 1H),
7.66 (d, J = 1.9 Hz, 1H), 7.26 (br d, J = 1.9 Hz, 1H), 6.59 (d, J = 0.9
Hz, 1H), 6.53 (br t, J = 5.9 Hz, 1H), 4.24 (t, J = 6.9 Hz, 2H), 4.04 (s,
3H), 3.49 (d, J = 5.9 Hz, 2H), 3.06 (dd, J = 8.3, 6.9 Hz, 2H), 2.95−
2.80 (m, 2H), 2.48 (d, J = 0.9 Hz, 3H), 1.11 (s, 9H).
N2-(4-(6,7-Dihydro-5H-pyrrolo[2,1-c][1,2,4]triazol-3-yl)-2-ethoxy-
phenyl)-6-methyl-N8-neopentylpyrido[3,4-d]pyrimidine-2,8-dia-
mine 39. Using 6-methyl-2-(methylsulfonyl)-N-neopentylpyrido[3,4-
d]pyrimidin-8-amine 2628 and N-(4-(6,7-dihydro-5H-pyrrolo[2,1-
c][1,2,4]triazol-3-yl)-2-ethoxyphenyl)formamide 32. Yield = 35%.
HRMS (ESI) m/z calcd for C26H33N8O (M+H) 473.2777, found
473.2769; 1H NMR (500 MHz, CD3OD) δ 9.00 (s, 1H), 8.67 (d, J =
8.5 Hz, 1H), 7.51 (d, J = 1.8 Hz, 1H), 7.39 (dd, J = 8.5, 1.8 Hz, 1H),
6.67 (s, 1H), 4.35−4.28 (m, 4H), 3.47 (s, 2H), 3.04−3.01 (m, 2H),
2.91−2.87 (m, 2H), 2.43 (s, 3H), 1.57 (t, J = 6.9 Hz, 3H), 1.10 (s,
9H).
General Procedure for Amine Displacement on 44 (Compounds
45−48). To a solution of the appropriate chloro compound (1 equiv)
in NMP (3 mL) was added the appropriate amine or salt thereof (2−
5 equiv) and triethylamine (5 equiv). The reaction mixture was
heated to 100 °C in a closed cap vial for 18 h. The reaction mixture
was diluted with EtOAc and water. The organic layer was washed with
brine, dried (MgSO4), and concentrated in vacuo. The residue was
purified by flash column chromatography (eluting with the
appropriate solvent system) and, if necessary, followed by SCX-2
cartridge (MeOH, 1 M NH3 in MeOH).
N2-(4-(5-(2-(Dimethylamino)ethyl)-4-methyl-4H-1,2,4-triazol-3-
yl)-2-ethoxyphenyl)-6-methyl-N8-neopentylpyrido[3,4-d]-
pyrimidine-2,8-diamine 40. Using 6-methyl-2-(methylsulfonyl)-N-
neopentylpyrido[3,4-d]pyrimidin-8-amine 2628 and N-(4-(5-(2-
(dimethylamino)ethyl)-4-methyl-4H-1,2,4-triazol-3-yl)-2-
ethoxyphenyl)formamide 33. Purified by reverse phase flash column
chromatography (0−100% water in MeOH, followed by 0−30% 2 M
methanolic ammonia in CH2Cl2). Yield = 18%. HRMS (ESI) m/z
1
(S)-N8-(3,3-Dimethylbutan-2-yl)-N2-(2-ethoxy-4-(4-methyl-4H-
1,2,4-triazol-3-yl)phenyl)-6-methylpyrido[3,4-d]pyrimidine-2,8-dia-
mine 45. Using 8-chloro-N-(2-ethoxy-4-(4-methyl-4H-1,2,4-triazol-3-
yl)phenyl)-6-methylpyrido[3,4-d]pyrimidin-2-amine 44 and (S)-3,3-
dimethylbutan-2-amine at 130 °C for 2 days. Yield = 52%. HRMS
(ESI) m/z calcd for C25H33N8O (M + H) 461.2777, found 461.2777;
calcd for C28H40N9O (M + H) 518.3356, found 518.3370; H NMR
(500 MHz, CD3OD) δ 9.05 (s, 1H), 8.75 (d, J = 8.5 Hz, 1H), 7.34 (d,
J = 1.8 Hz, 1H), 7.28 (dd, J = 8.5, 1.8 Hz, 1H), 6.71 (s, 1H), 4.30 (q, J
= 7.0 Hz, 2H), 3.73 (s, 3H), 3.47 (s, 2H), 3.09 (app t, J = 7.5 Hz,
2H), 2.91 (app t, J = 7.5 Hz, 2H), 2.44 (s, 3H), 2.42 (s, 6H), 1.56 (t, J
= 7.0 Hz, 3H), 1.09 (s, 9H).
L
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Article
1H NMR (500 MHz, (CD3)2SO) δ 9.15 (s, 1H), 8.57 (s, 1H), 8.43
points over the 24 h post dose and spotted on Whatman B cards
(VWR) together with a standard curve and quality controls spiked in
control blood. Cards were allowed to dry at r.t. for at least 6 h. Cards
were punched and 6 mm discs were extracted with 200 μL of
methanol containing 500 nM olomoucine as an internal standard.
Following centrifugation, extracts were analyzed by multiple reaction
monitoring of precursor and product ions by ESI-LCMS/MS on a
QTRAP 4000 (ABSciex) following gradient separation with 0.1%
formic acid in methanol on a Phenomenex Kinetex C18 UPLC
column (50 × 2.1 mm, 2.6 μM). Quantitation was carried out with an
external calibration (typically 8 points ranging from 1 nM to 25 μM).
Quality controls were included (three concentrations) at the
beginning and the end of the analytical run and were within 20% of
nominal concentrations.
(d, J = 8.3 Hz, 1H), 7.41 (d, J = 1.9 Hz, 1H), 7.31 (dd, J = 8.2, 1.9 Hz,
1H), 6.72 (d, J = 1.0 Hz, 1H), 6.43 (d, J = 9.5 Hz, 1H), 4.23 (q, J =
6.9 Hz, 2H), 4.13 (dq, J = 9.4, 6.6 Hz, 1H), 3.78 (s, 3H), 2.38 (s,
3H), 1.41 (t, J = 6.9 Hz, 3H), 1.16 (d, J = 6.6 Hz, 3H), 1.00 (s, 9H).
N-(2-Ethoxy-4-(4-methyl-4H-1,2,4-triazol-3-yl)phenyl)-6-methyl-
8-(pyrrolidin-1-yl)pyrido[3,4-d]pyrimidin-2-amine 46. Using 8-
chloro-N-(2-ethoxy-4-(4-methyl-4H-1,2,4-triazol-3-yl)phenyl)-6-
methylpyrido[3,4-d]pyrimidin-2-amine 44 and pyrrolidine at 130 °C.
Yield = 70%. HRMS (ESI) m/z calcd for C23H27N8O (M + H)
1
431.2308, found 431.2315; H NMR (500 MHz, (CD3)2SO) δ 9.13
(s, 1H), 8.56 (s, 1H), 8.34 (s, 1H), 8.23 (d, J = 8.2 Hz, 1H), 7.39−
7.29 (m, 2H), 6.76 (d, J = 0.8 Hz, 1H), 4.21 (q, J = 6.9 Hz, 2H), 3.88
(s, 4H), 3.78 (s, 3H), 2.37 (s, 3H), 1.94−1.83 (m, 4H), 1.41 (t, J =
6.9 Hz, 3H).
Pharmacokinetic parameters were derived from noncompartmental
analysis using Phoenix Pharsight WinNonlin (model 200 and 201)
version 6.1/6.3.
8-(3,3-Dimethylazetidin-1-yl)-N-(2-ethoxy-4-(4-methyl-4H-1,2,4-
triazol-3-yl)phenyl)-6-methylpyrido[3,4-d]pyrimidin-2-amine 47.
Using 8-chloro-N-(2-ethoxy-4-(4-methyl-4H-1,2,4-triazol-3-yl)-
phenyl)-6-methylpyrido[3,4-d]pyrimidin-2-amine 44 and 3,3-dime-
thylazetidine hydrochloride. Yield = 70%. HRMS (ESI) m/z calcd for
All experiments using animals were performed in accordance with
the local Animal Welfare and Ethical Review Board, the UK Home
Office Animals Scientific Procedures Act 1986, and with the United
Kingdom National Cancer Research Institute Guidelines for the
Welfare of Animals in Cancer Research.31 The ICR does not
undertake research in nonrodent species and requires internal ethical
review when such studies are sponsored by organizations with whom
we collaborate. Collaborator-sponsored nonrodent pharmacology
studies of compound 36 necessary for the prediction of therapeutic
window and application to the clinic were approved by the ICR
Animal Welfare and Ethics Review Board and were conducted in full
compliance with national regulations at AAALAC accredited R&D
sites.
1
C24H30N8O (M + 2H)/2 223.1266, found 223.1261; H NMR (500
MHz, CD3OD) δ 9.05 (s, 1H), 8.56 (d, J = 8.5 Hz, 1H), 8.54 (s, 1H),
7.37 (d, J = 1.5 Hz, 1H), 7.34 (dd, J = 8.5, 1.5 Hz, 1H), 6.77 (s, 1H),
4.29 (q, J = 7.0 Hz, 2H), 4.21 (br s, 4H), 3.87 (s, 3H), 2.44 (s, 3H),
1.54 (t, J = 7.0 Hz, 3H), 1.38 (s, 6H).
8-(2,2-Dimethylazetidin-1-yl)-N-(2-ethoxy-4-(4-methyl-4H-1,2,4-
triazol-3-yl)phenyl)-6-methylpyrido[3,4-d]pyrimidin-2-amine 48.
Using 8-chloro-N-(2-ethoxy-4-(4-methyl-4H-1,2,4-triazol-3-yl)-
phenyl)-6-methylpyrido[3,4-d]pyrimidin-2-amine 44 and 2,2-dime-
thylazetidine. Yield = 89%. HRMS (ESI) m/z calcd for C24H29N8O
(M + H) 445.2459, found 445.2461; 1H NMR (500 MHz, CD3OD) δ
8.98 (s, 1H), 8.54 (s, 1H), 8.52 (d, J = 8.0 Hz, 1H), 7.35 (d, J = 2.0
Hz, 1H), 7.33 (dd, J = 8.0, 2.0 Hz, 1H), 6.65 (s, 1H), 4.70−4.66 (m,
2H), 4.27 (d, J = 7.0 Hz, 2H), 3.85 (s, 3H), 2.39 (s, 3H), 2.22 (dd, J =
9.0, 7.5 Hz, 2H), 1.71 (s, 6H), 1.54 (t, J = 7.0 Hz, 3H).
PK/PD Experiments. These experiments were conducted as
previously described.32
ASSOCIATED CONTENT
* Supporting Information
■
S
Biochemical Assays. MPS1 and CDK2 counterscreen assay were
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.8b00690.
performed as reported previously.16
MSD Assay. An electrochemiluminescence assay (Meso Scale
Discovery, MSD) for detection of autophosphorylation of MPS1 at
pTpS33/37 sites in cell lysates has been described previously.16
Autophosphorylation of MPS1 at pTpS33/37 and total MPS1-GFP
levels in MPS1-doxycycline inducible DLD-1 xenografts were
determined by MSD assays as described previously.32 Results were
presented as the ratio of Phospho-MPS1/Total MPS1.
Molecular formula strings (CSV)
Experimental procedures and analytical data for all
formamides (10−13 and 29−33) and corresponding
intermediates, CYP, hERG activity, and P-gp efflux data
as well as kinase selectivity profiling of 36. Tables with
standard deviations for all compounds as well as
antiproliferative (GI50) data for several compounds
(PDF)
Microsomal Metabolism. Microsomal clearance was determined
in female CD1 mice, female Sprague−Dawley rats, and mixed gender
human liver microsomes obtained from Tebu-Bio (Peterborough,
U.K.) following incubation of 1 μM compound at 37 °C in 1 mg/mL
microsomal protein, 3 mmol/L MgCl2, 1 mmol/L NADPH, 2.5
mmol/L, UDP-glucuronic acid, and 10 mmol/L phosphate buffer
(pH 7.4) (all purchased from Sigma-Aldrich, Gillingham, U.K).
Reactions were started by addition of the cofactors following 10 min
preincubation of microsomes with test compound and were
terminated at −1, 0, 5, 10, 15, and 30 min with three volumes of
ice-cold methanol containing internal standard. Samples were
centrifuged at 2800g for 30 min at 4 °C and the supernatants
analyzed. Control incubations were prepared as above with omission
of cofactors. Compound measurements were performed by LCMS on
an Agilent quadrupole time-of-flight instrument (Agilent 6510)
following separation with a 6 min gradient of 0.1% formic acid in
methanol on a 50 × 2.1 mm 2.6 μm C18 column (Kinetex
Phenomenex). For metabolite identification, the gradient was
extended to 20 min and MS/MS carried out with fragment
elucidation for ions of interest.
Accession Codes
The PDB ID code for 36 bound to MPS1 is 6H3K.
AUTHOR INFORMATION
Corresponding Author
*Tel: +44 (0)20 8722 4353. E-mail: swen.hoelder@icr.ac.uk.
■
ORCID
Julian Blagg: 0000-0002-7409-0323
Swen Hoelder: 0000-0001-8636-1488
Present Address
⊥(A.F.) SBA School of Science and Engineering, Lahore
University of Management Sciences, D.H.A., Lahore 54792,
Pakistan.
Pharmacokinetic Studies. Animals were adapted to laboratory
conditions for at least 1 week prior to dosing and were allowed food
and water ad libitum. Compounds were administered iv or po (mouse,
0.1 mL/10 g in 10% DMSO, 5% tween 20 in saline; rat, 0.05 mL/10 g
in 10% DMSO, 5% tween 20 in saline; dog, 10% in DMSO in saline).
Blood samples were collected from the tail vein (20 μL) at 8 time
Notes
The authors declare the following competing financial
interest(s): The authors are current or former employees of
The Institute of Cancer Research, which has a commercial
interest in the development of kinase inhibitors.
M
DOI: 10.1021/acs.jmedchem.8b00690
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Journal of Medicinal Chemistry
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proliferation and chromosomal instability signature in anaplastic
ACKNOWLEDGMENTS
■
thyroid carcinoma. Cancer Res. 2007, 67, 10148−10158.
This work was supported by Cancer Research UK [grant
number C309/A11566]. We also acknowledge the Cancer
Research Technology Pioneer Fund and Sixth Element Capital
for funding (to P.I.) and NHS funding to the NIHR
Biomedical Research Centre. S.L. is also supported by
Breakthrough Breast Cancer. We thank Dr. Amin Mirza, Dr.
Maggie Liu, and Mr. Meirion Richards for their help with LC,
NMR, and mass spectrometry. We are grateful to the staff of
DIAMOND Light Source for their support during data
collection.
̈
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ABBREVIATIONS USED
■
CDK2, cyclin-dependent kinase 2; Cl, clearance; hERG,
̀
human Ether-a-go-go-Related Gene; HLM, human liver
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V.; Atrash, B.; Cheung, K.-M. J.; Liu, M.; Hayes, A.; Schmitt, J.;
Wood, A.; Choi, V.; Boxall, K.; Mak, G.; Gurden, M.; Valenti, M.; de
Haven Brandon, A.; Henley, A.; Baker, R.; McAndrew, C.; Matijssen,
B.; Burke, R.; Hoelder, S.; Eccles, S. A.; Raynaud, F. I.; Linardopoulos,
S.; van Montfort, R. L. M.; Blagg, J. Structure-based design of orally
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Westwood, I. M.; Cronin, N.; Hayes, A.; Roberts, J.; Henley, A. T.;
Baker, R.; Faisal, A.; Mak, G. W.-Y.; Box, G.; Valenti, M.; De Haven
Brandon, A.; O’Fee, L.; Saville, H.; Schmitt, J.; Matijssen, B.; Burke,
R.; van Montfort, R. L. M.; Raynaud, F. I.; Eccles, S. A.;
Linardopoulos, S.; Blagg, J.; Hoelder, S. Rapid discovery of
pyrido[3,4-d]pyrimidine inhibitors of monopolar spindle kinase 1
(MPS1) using a structure-based hybridization approach. J. Med. Chem.
2016, 59, 3671−3688.
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M.; McKinnon, R.; Peterson, A.; Douce, T. B.; Robinson, R.;
Dorweiler, I.; Davis, T.; Hess, M. A.; Ostanin, K.; Papac, D. I.;
Baichwal, V.; McAlexander, I.; Willardsen, J. A.; Saunders, M.;
Christophe, H.; Kumar, D. V.; Wettstein, D. A.; Carlson, R. O.;
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Hagemann, C.; Vandertop, W. P.; Van Tellingen, O.; Noske, D. P.;
Gray, N. S.; Wurdinger, T. Effects of the selective MPS1 inhibitor
̈
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(20) Kusakabe, K.-I.; Ide, N.; Daigo, Y.; Itoh, T.; Yamamoto, T.;
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Dohi, K.; Kido, Y.; Sakamoto, S.; Ando, S.; Maeda, M.; Higaki, M.;
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microsomes; L.E., ligand efficiency; MLM, mouse liver
microsomes; MPS, monopolar spindle kinase; MSD, Meso-
Scale Discovery; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide; PARP, poly ADP ribose polymerase;
PLK1, polo-like kinase; PTEN, phosphatase and tensin
homologue; SAC, spindle assembly checkpoint; RLM, rat
liver microsomes; Vss, volume of distribution
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DOI: 10.1021/acs.jmedchem.8b00690
J. Med. Chem. XXXX, XXX, XXX−XXX