Rapid Discovery of Pyrido[3,4-d]pyrimidine Inhibitors of Monopolar Spindle Kinase 1 (MPS1) Using a Structure-Based Hybridization Approach

Article

pubs.acs.org/jmc

Rapid Discovery of Pyrido[3,4‑d]pyrimidine Inhibitors of Monopolar

Spindle Kinase 1 (MPS1) Using a Structure-Based Hybridization

Approach

Paolo Innocenti,^†Hannah L. Woodward,^†Savade Solanki,^†Sebastien Naud,^†Isaac M. Westwood,^†,‡

́

Nora Cronin,^‡Angela Hayes,^†Jennie Roberts,^†Alan T. Henley,^†Ross Baker,^†Amir Faisal,^†,∥

Grace Wing-Yan Mak,^†Gary Box,^†Melanie Valenti,^†Alexis De Haven Brandon,^†Lisa O’Fee,^†

Harry Saville,^†Jessica Schmitt,^†Berry Matijssen,^†Rosemary Burke,^†Rob L. M. van Montfort,^†,‡

Florence I. Raynaud,^†Suzanne A. Eccles,^†Spiros Linardopoulos,^†,§Julian Blagg,^†and Swen Hoelder*^,†

^†Cancer Research UK Cancer Therapeutics Unit, Division of Cancer Therapeutics, The Institute of Cancer Research, 15 Cotswold

Road, Sutton, London, SM2 5NG, United Kingdom

^‡Division of Structural Biology and ^§Breast Cancer Now, Division of Breast Cancer Research, The Institute of Cancer Research, 237

Fulham Road, London, SW3 6JB, United Kingdom

S

* Supporting Information

ABSTRACT: Monopolar spindle 1 (MPS1) plays a central role in the transition of cells from metaphase to anaphase and is one

of the main components of the spindle assembly checkpoint. Chromosomally unstable cancer cells rely heavily on MPS1 to cope

with the stress arising from abnormal numbers of chromosomes and centrosomes and are thus more sensitive to MPS1 inhibition

than normal cells. We report the discovery and optimization of a series of new pyrido[3,4-d]pyrimidine based inhibitors via a

structure-based hybridization approach from our previously reported inhibitor CCT251455 and a modestly potent screening hit.

Compounds in this novel series display excellent potency and selectivity for MPS1, which translates into biomarker modulation

in an in vivo human tumor xenograft model.

surprising that MPS1 is upregulated in a number of tumor types⁴

and that higher levels correlate with higher histological grade,

INTRODUCTION

■

Interfering with mitotic processes has been a successful

therapeutic approach to ﬁght cancer.¹One example of a mitotic

aggressiveness, and poorer patient survival in breast cancer,

glioblastoma, and pancreatic ductal adenocarcinoma.^4a,5Fur-

target is monopolar spindle 1 (MPS1, also known as TTK), a

thermore, phosphatase and tensin homologue (PTEN)-deﬁcient

dual-speciﬁcity kinase that occupies a central role in the

breast cancer cell lines have been reported to be more sensitive to

transition from metaphase to anaphase and is one of the main

MPS1 depletion or kinase inhibition.^3b,6

components of the spindle assembly checkpoint (SAC).²This

kinase prevents cells from progressing through mitosis until the

kinetochores are properly attached to the microtubules and are

under the appropriate tension. While this mechanism is

important to ensure error-free segregation of chromosomes in

normal tissues, aneuploid and chromosomally unstable cancer

cells often overexpress, and are particularly dependent on, MPS1

to cope with the stress arising from abnormal numbers of

chromosomes and centrosomes.³Due to these ﬁndings, it is not

Using advanced inhibitors, including our own 1 (CCT251455,

vide infra),⁷eﬀective dosing schedules have been investigated in

vivo. Importantly, it has recently been shown that MPS1

inhibitors have a relatively narrow therapeutic window^5a,8and

that they are particularly eﬀective when used in combination

Received: November 20, 2015

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Figure 1. Published MPS1 inhibitors.

a

Scheme 1. Synthesis of Isoquinoline Derivatives

a

Reagents and conditions: (a) Furan-2-ylboronic acid or 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole, Na₂CO₃,

t

Pd(dppf)Cl₂·CH₂Cl₂, DME/water, μW, 105 °C; (b) 4-methoxyaniline 12 or 2,4-dimethoxyaniline 13, BuXPhos, Pd₂dba₃, Cs₂CO₃, BuOH/

water, μW, 80−100 °C. Reaction yields are in parentheses.

with, for example, tubulin-targeting agents such as paclitaxel or

CDK4/6 inhibitors.⁹

other cell cycle kinases such as cyclin-dependent kinase 2

(CDK2), Aurora A and B, and more generally against the wider

kinome (K_iratio >100), (d) robustly modulated MPS1 kinase

activity in a human tumor xenograft PK/PD model, and (e)

showed signiﬁcant scope for further modiﬁcation.

Herein we describe the rapid discovery of such a series by

structure-based hybridization of the 1H-pyrrolo[3,2-c]pyridine

series and a modestly potent hit from a focused kinase library

screen. This completely new MPS1 series featured an

unexploited kinase chemotype (the pyrido[3,4-d]pyrimidines)

and advanced examples displayed subnanomolar K_is and

excellent overall selectivity. The favorable in vitro properties

translated into biomarker modulation in an in vivo human tumor

xenograft model.

Several MPS1 inhibitors have been disclosed,¹⁰these include

AstraZeneca’s AZ3146 (2),^10bthe Myrexis compound MPI-

0479605 (3),^8cand the Nerviano compound NMS-P715 (4).¹¹

Also described in the literature are MPS-IN-3 (5),^5aCFI-401870

(6),¹²and the Shionogi compounds (7, 8) (Figure 1).^8a,13

We recently disclosed a series of 1H-pyrrolo[3,2-c]pyridines

exempliﬁed by the potent and selective chemical probe 1.⁷

However, while this compound showed excellent potency in

biochemical and cellular assays, other properties hampered

further development. In particular, 1 has a relatively high

molecular weight (504) and AlogP (5.7) and also featured a tert-

butoxycarbonyl (Boc) moiety that appeared critical for potent

inhibition but unsurprisingly proved to be unstable under

strongly acidic conditions.¹⁴In addition, 1 was a potent

cytochrome P450 (CYP) inhibitor (IC₅₀< 1 μM observed for

CYP 1A2, 3A4, 2C9, 2C19, 2D6) which represented a signiﬁcant

issue not least because MPS1 inhibitors are reported to be

particularly eﬀective in combination with other chemotherapeu-

tic agents, e.g., paclitaxel. To mitigate the risk that these

undesirable features were inherent to the 1H-pyrrolo[3,2-

c]pyridine series, we set out to discover a second structurally

unrelated chemotype featuring a clean CYP proﬁle, no acid labile

groups, and signiﬁcantly lower molecular weight and lip-

ophilicity. We thus set out to discover an additional lead series

that (a) showed potent inhibition of MPS1 in cellular assays

(IC₅₀< 100 nM), (b) showed good ligand eﬃciency (L.E. ∼

0.35),¹⁵(c) displayed excellent selectivity, in particular against

CHEMISTRY

■

The synthesis of isoquinolines 14, 15a and 15b was carried out

from the commercially available 5-bromo-3-chloroisoquinoline 9

through sequential Suzuki couplings and Buchwald reactions

using suitable boronic acids or esters and the required anilines

(Scheme 1).

Compounds 24a−d and 28a−e in the pyrido[3,4-d]-

pyrimidine series were prepared according to a previously

described strategy which makes use of the novel 8-chloro-2-

(methylthio)pyrido[3,4-d]pyrimidine building block 16.¹⁶Thus,

reaction with 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-

lan-2-yl)-1H-pyrazole or cyclopropylboronic acid under Suzuki

conditions yielded intermediates 17 and 18. These thiomethyl

derivatives were in turn oxidized to the corresponding sulfones

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a

Scheme 2. Synthesis of Pyrido[3,4-d]pyrimidine Derivatives

a

Reagents and conditions: (a) 1-Methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole, Na₂CO₃, Pd(dppf)Cl₂·CH₂Cl₂, THF/water, 65

°C (17); (b) cyclopropyl boronic acid, K₃PO₄, Pd(OAc)₂, PCy₃, toluene/water, 95 °C (18); (c) m-CPBA, CH₂Cl₂, rt; (d) N-(4-

methoxyphenyl)formamide 21 or N-(2,4-dimethoxyphenyl)formamide 22, DMSO, Cs₂CO₃, 100 °C (24a,b); (e) 2-methoxy-4-(1-methyl-1H-

pyrazol-4-yl)aniline 23, TFA, 1,2,3-triﬂuoroethanol, μW, 130 °C (24c,d); (f) ArNHCHO (26a−e), NaH, THF, rt; (g) 1-methyl-4-(4,4,5,5-

tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole or phenyl boronic acid, Cs₂CO₃, Pd(PPh₃)₄, 1,4-dioxane/water, 100 °C. Reaction yields are in

parentheses.

using m-CPBA and subsequently coupled with the required

anilines or formamides to aﬀord compounds 24a−d (Scheme 2).

In the majority of cases though, the order of events was reversed.

Oxidation of 8-chloro-2-(methylthio)pyrido[3,4-d]pyrimidine

16 yielded the corresponding sulfone 25,¹⁶which was coupled

with a series of formamides furnishing chloro-intermediates

27a−e (Scheme 2). Final compounds 28a−e were obtained by

means of palladium-catalyzed Suzuki couplings with commer-

cially available boronic acids or esters. Pyrido[3,4-d]pyrimidines

33a and 33b were prepared by direct substitution of amines on

the 8-chloro-2-(methylthio)pyrido[3,4-d]pyrimidine building

block 16 (Scheme 3). The resulting thiomethyl derivatives 29

and 30 were oxidized to the corresponding sulfones 31 and 32

and subsequently coupled with the required formamides to

aﬀord ﬁnal compounds 33a and 33b.

Pyrido[3,4-d]pyrimidines 34a−h were prepared from inter-

mediate 27a¹⁶by displacement using the appropriate nucleo-

philes (Scheme 4). In one instance the synthetic strategy

involved the use of known pyridone 35¹⁶as a starting material:

O-alkylation in the presence of base and bromomethyl

cyclopropane gave intermediate 36. Oxidation using m-CPBA

and subsequent coupling with formamide 26a¹⁶aﬀorded

pyrido[3,4-d]pyrimidine 38 (Scheme 4).

In all cases, formamides and anilines were commercially

available (12, 13, 21) or could be synthesized by means of

standard transformations (22, 23,¹⁷and 26a−e, see Exper-

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a

Scheme 3. Functionalization of Pyrido[3,4-d]pyrimidine Core

a

Reagents and conditions: (a) Amine, NMP, 80−135 °C; (b) m-CPBA, CH₂Cl₂, rt; (c) 2-methoxy-4-(1-methyl-1H-pyrazol-4-yl)aniline 23, TFA,

1,2,3-triﬂuoroethanol, μW, 130 °C. Reaction yields are in parentheses.

a

Scheme 4. Synthesis of Derivatives Containing 8-Position N, O, and S Substituents

a

Reagents and conditions: (a) Amine, NMP, 80−135 °C (34a−e, g, h); (b) cyclohexyl thiol, K₂CO₃, DMF, rt (34f); (c) Ag₂CO₃, bromomethyl

cyclopropane, CHCl₃, rt to 60 °C; (d) m-CPBA, CH₂Cl₂, rt; (e) N-(2-methoxy-4-(1-methyl-1H-pyrazol-4-yl)phenyl)formamide 26a, NaH, THF, rt.

Reaction yields are in parentheses.

imental section and Supporting Information for details). In every

case, formylation was carried out by reﬂuxing in formic acid.

we anticipated that 39 would represent a viable starting point for

a second series but that extensive optimization of potency and

selectivity would be required. In order to rapidly discover a new

series, while utilizing previously gained SAR data, we merged the

1H-pyrrolo[3,2-c]pyridine scaﬀold with the screening hit 39

(Figure 3). Docking suggested that both the isoquinoline and

pyrido[3,4-d]pyrimidine scaﬀolds could serve as hinge-binder

elements for such a hybrid series (Figure 3). The isoquinoline

scaﬀold had the advantage that proof-of-concept molecules could

be rapidly prepared using precedented chemistry and commer-

cially available building blocks. The pyrido[3,4-d]pyrimidine

scaﬀold, on the other hand, was attractive from the point of view

of novelty, the signiﬁcantly lower lipophilicity, and the fact that it

incorporated an additional pyridine nitrogen from screening hit

39. This scaﬀold, however, required signiﬁcant investigation into

its synthesis.¹⁶Our plan was thus to prepare a select number of

RESULTS AND DISCUSSION

■

At the start of this work, relatively few inhibitors of MPS1 had

been disclosed. In addition to our 1H-pyrrolo[3,2-c]pyridine

series,⁷we had identiﬁed and crystallized 1H-pyrazolo[4,3-

c]pyridine 39, a modestly potent hit, during an initial screening

campaign. This compound showed low molecular weight (MW =

261) and acceptable ligand eﬃciency (L.E. = 0.43). The crystal

structure of MPS1 in complex with 39 revealed that it binds in a

complementary way to 4, whereby the pyrazole moiety binds to

the hinge region of the protein. The furan moiety occupies the

same region as the carbamate in the 1H-pyrrolo[3,2-c]pyridine

series. The phenyl ring and aniline moiety also occupy similar

parts of the binding pocket (Figure 2).¹⁸Based on these results,

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The data for the initial set of compounds are summarized in

Table 1. The MPS1 inhibition for 14 was modest (IC₅₀= 3.66

μM, Table 1), but replacing the furan with a pyrazole (15a) not

only lowered lipophilicity but also gave a 5-fold increase in

potency (Table 1). Moreover, incorporating an additional

methoxy group led to a signiﬁcant gain in potency, and the

IC₅₀of 15b reached our goal for an initial proof-of-concept

compound. As seen with the 1H-pyrrolo[3,2-c]pyridines,⁷the

latter greatly improved selectivity toward CDK2. The signiﬁcant

gain in potency can be ascribed to the interaction of the aniline’s

2-methoxy substituent with a small hydrophobic pocket formed

by Lys529, Ile531, Gln541, and Cys604.⁷This pocket is not

present in most other kinases including CDK2, explaining the

beneﬁcial eﬀect on kinase selectivity.

Having achieved satisfactory potency and ligand eﬃciency

(L.E. for 15b = 0.36) by merging the 1H-pyrrolo[3,2-c]pyridine

series and screening hit 39, we investigated the less lipophilic

pyrido[3,4-d]pyrimidine scaﬀold. Gratifyingly, 8-substituted 2-

anilinopyrido[3,4-d]pyrimidines proved to be more potent than

their isoquinoline counterparts, reaching the double digit

nanomolar range despite signiﬁcantly lower calculated logP

(Table 1). Isoquinoline 15b along with pyrido[3,4-d]-

pyrimidines 24a and 24b were next tested in an MSD-based

cellular assay. Both 15b and 24b showed only modest inhibition

of the autophosphorylation of ectopically expressed MPS1 in

HCT116 cells, most likely due to suboptimal biochemical

potency. In the 1H-pyrrolo[3,2-c]pyridine series, introduction of

a pyrazole substituent at the 4-position of the aniline caused a

signiﬁcant increase in biochemical and cellular potency.⁷

Gratifyingly, this structural modiﬁcation also resulted in a further

improvement in biochemical potency for the pyrido[3,4-

d]pyrimidine series, with compound 24c achieving a MPS1

IC₅₀= 0.008 μM and a cellular P-MPS1 IC₅₀= 0.604 μM (Table

1).

We solved the crystal structure of MPS1 in complex with

isoquinoline 15b and pyrido[3,4-d]pyrimidine 24b (Figure 4).

The two structures overlaid very well with the crystal structure of

1 (Figure 4A and B), binding to the hinge region through the

same motif as seen with the 1H-pyrrolo[3,2-c]pyridines. This

reinforced our initial hypothesis that merging the 1H-pyrrolo-

[3,2-c]pyridine series and 39 would give a suitable starting point

for an additional series of inhibitors. The aniline portion of all

compounds overlaps as well as the 5-position and 8-position

substituents of the isoquinolines and pyrido[3,4-d]pyrimidines,

Figure 2. Superimposed crystal structure of MPS1 (pale green) bound

to 1 (carbon atoms colored pink), extracted from PDB code 4C4J, onto

the structure of MPS1 (not shown) bound to 39 (carbon atoms colored

yellow), PDB code 5EHY, showing the complementary binding of the

modestly potent hit 39 and MPS1 inhibitor 1 to the hinge region of

MPS1.

proof-of-concept molecules based on the isoquinoline core and

to expand into the less explored and more polar pyrido[3,4-

d]pyrimidine series if the initial isoquinolines showed promising

activity and ligand eﬃciency.

We used our biochemical MPS1 assay at 10 μM ATP

concentration and an in-house electrochemiluminescence Meso

Scale Discovery (MSD)-based cellular assay that measured

autophosphorylation of ectopically expressed MPS1 in HCT116

cells.⁷In addition, we routinely determined selectivity against

CDK2 and Aurora A and B. We did not detect signiﬁcant activity

against either Aurora A or B throughout the series (see

Supporting Information). Furthermore, as the IC₅₀values of

our inhibitors approached the assay enzyme concentrations, and

thus the limit of the dynamic range of our biochemical assay, we

also determined the biochemical potency at a higher (1 mM)

ATP concentration (vide infra). As stated above, our overall goal

for this lead-ﬁnding eﬀort was to show that potent (cellular IC₅₀

< 100 nM) and selective compounds (CDK2/MPS1 K_iratio

>100) could be obtained which exhibit lower molecular weight

(around 450 Da) and improved ligand eﬃciency (L.E. in region

of 0.35) compared with the 1H-pyrrolo[3,2-c]pyridine series.

Moreover, our goal was to show robust activity in our PK/PD

human tumor xenograft model.

Figure 3. Hybridization strategy for the development of a novel chemical series of inhibitors of MPS1, from previously reported inhibitor 1 and

screening hit 39.

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Table 1. Biochemical and Cellular Data for Initial Proof-of-Concept Isoquinolines (14, 15a, 15b) and Pyrido[3,4-d]pyrimidines

a

(24a−c)

a

Results are mean ( SD) for n ≥ 3 or mean values of two independent determinations with individual determinations in parentheses or samples run

n = 1. “P-MPS1″ indicates an electrochemiluminescence MSD-based cellular assay that measured autophosphorylation of ectopically expressed

MPS1 in HCT116 cells. Potency at the lower limit of the dynamic range of the Caliper assay run at 10 μM ATP concentration. IC₅₀value is likely

b

to be lower than that quoted.

respectively, occupying the same region as the carbamate group

of 1. Of note is the diﬀerence in conformation for 15b and 24b

(Figure 4C and D). The introduction of two extra nitrogen atoms

into the aromatic ring, and thus removal of two hydrogen atoms,

results in a much more planar structure for the pyrido[3,4-

d]pyrimidine 24b. We measured the dihedral angles for both of

these compounds: isoquinoline 15b exhibits a dihedral angle of

98° and pyrido[3,4-d]pyrimidine 24b exhibits a dihedral angle of

−20° (Figure 4D). The more coplanar conformation of

pyrido[3,4-d]pyrimidines mirrors the coplanar conformation of

the carbamate function of 1 and is likely to be at least partially

responsible for the improved activity seen with the much less

lipophilic pyrido[3,4-d]pyrimidines. Based on these crystal

structures, we had conﬁdence that with further optimization

we would achieve potent and selective pyrido[3,4-d]pyrimidine

based inhibitors of MPS1.

Through our hybridization strategy and exploration of the

pyrido[3,4-d]pyrimidine scaﬀold, we had achieved a potent

inhibitor (24c, MPS1 IC₅₀= 0.008 μM) and for the ﬁrst time

reached signiﬁcant levels of cellular inhibition with IC₅₀values

below 1 μM. Compound 24c represented a promising starting

point for further optimization of this compound into a novel

series of MPS1 inhibitors.

Since our initial goal was to show target modulation in a

human tumor xenograft model in mice, we investigated the

stability of 24c in mouse liver microsomes. Unfortunately, 24c

showed high turnover (MLM = 73% following 30 min

incubation) and was not suitable for in vivo experiments.

Metabolite ID studies suggested loss of a methyl group, and we

suspected that the methoxy aniline substituent was the most

likely site of demethylation. This methoxy group was important

for both potency and selectivity. After consideration of the X-ray

crystal structures we had in hand, suggesting that larger

substituents at this position would be tolerated, we prepared

and tested a series of compounds in which this putative metabolic

soft spot was replaced with similar moieties (Table 2). The

majority of these compounds showed higher IC₅₀values than 24c

with the exception of the ethoxy derivative 28c, which achieved

comparable potency, while exhibiting signiﬁcantly improved

CDK2 selectivity (>28 fold improvement over 24c) and MLM

stability (MLM = 45%). This increase in selectivity is likely due to

the fact that the aniline 2-position substituent occupies a small

lipophilic pocket present in MPS1, mentioned previously,

consisting of Lys529, Ile531, Gln541 and the gatekeeper+2

residue Cys604. This pocket is not available in CDK2 due to the

presence of the bulkier gatekeeper residue Phe82. The larger 2-

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Figure 4. (A) Superimposed crystal structure of MPS1 (pale green) bound to 1 (carbon atoms colored pink), extracted from PDB code 4C4J, onto the

structure of MPS1 (not shown) bound to 15b (carbon atoms colored cyan), PDB code 5EI6. (B) Superimposed crystal structure of MPS1 (pale green)

bound to 1 (carbon atoms colored pink), extracted from PDB code 4C4J, onto the structure of MPS1 (not shown) bound to 24b (carbons atoms

colored green), PDB code 5EI2. (C) Superimposed crystal structure of MPS1 (pale green) bound to 15b (carbon atoms colored cyan), extracted from

PDB code 5EI6, onto the structure of MPS1 (not shown) bound to 24b (carbon atoms colored green), PDB code 5EI2, showing the extent of the

conformational diﬀerence between the isoquinoline and pyrido[3,4-d]pyrimidine scaﬀolds. (D) Side-by-side view of compound 15b (carbon atoms

colored cyan) and 24b (carbon atoms colored green) showing the diﬀerence in angle of the N-methylpyrazole group. 2Fo − Fc density contoured at 1σ

is overlaid and displayed in the same color as the respective compound carbon atom color. A water molecule interacting with compound 24b is also

displayed as a red sphere. 15b exhibits a dihedral angle of 98°, and 24b exhibits a dihedral angle of −20°.

position ethoxy substituent (28c) clashes with the CDK2

gatekeeper+2 residue, resulting in a better selectivity window

than is seen for the methoxy derivative 24c.

derivative 24d lost considerable activity, possibly due to the fact

that this group is too small to engage in signiﬁcant hydrophobic

interactions. Interestingly, replacement of the pyrazole with a

saturated pyrrolidine (33a) led to an equipotent compound in

the biochemical assay. All compounds showed selectivity against

CDK2 (CDK2/MPS1 ratio >80).

Of the compounds presented in Table 3, we considered the

pyrrolidine derivative (33a) as the most promising for further

optimization. A saturated moiety oﬀered more possibilities to

optimize the three-dimensional hydrophobic interactions in this

subpocket compared with an aromatic ring where substituents

can only be placed in the plane of the ring. Furthermore,

increasing the number of sp³centers has been suggested as a

general approach to improve solubility and drug-like proper-

ties.¹⁹We thus prepared a series of compounds with a saturated

substituent in this position.

Unfortunately, 28c was 5-fold less active in the cellular assay,

which given the similar biochemical potency and physicochem-

ical properties was diﬃcult to rationalize. While we regarded the

ethoxy as a valuable alternative for the methoxy group, with

signiﬁcantly improved selectivity and reduced risk of reactive

intermediate formation through metabolic dealkylation, we

decided to maintain the methoxy in place due to the improved

cellular activity. Instead, we looked to improve the metabolic

stability through modiﬁcation at the 8-position of the pyrido[3,4-

d]pyrimidine scaﬀold. We hypothesized that by introducing

diverse substituents at this position of the scaﬀold, the molecular

recognition of the compounds by metabolic enzymes would be

aﬀected leading to increased stability.

In order to address these aspects we used the structural

information gathered on pyrido[3,4-d]pyrimidine 24c. The

crystal structure of this inhibitor showed that the 8-position

pyrazole group binds to a hydrophobic pocket formed by Ile531,

Val539, Met671, and Pro673 (Figure 5A). This is the same

pocket that is occupied by the carbamate group of 1 and is

suﬃciently large to accommodate a variety of hydrophobic

groups (Figure 5B). It is also of note that the crystal structure of

MPS1 in complex with 24c shows ordering of the activation loop

of MPS1, as is seen with 1.⁷We thus prepared and tested a small

set of compounds with diﬀerent pyrazole replacements including

a saturated pyrrolidine ring.

Importantly, since the IC₅₀values of many compounds were

now approaching the enzyme concentration and thus the limit of

the dynamic range of the biochemical assay, we complemented

the MPS1 kinase assay at 10 μM ATP with testing at 1 mM ATP.

It has been demonstrated that increasing the ATP concentration

shifts the IC₅₀values of ATP competitive inhibitors to higher

values, therefore increasing the dynamic range of the assay.²⁰Due

to the diﬀering ATP concentrations in the high ATP Caliper

assay and the CDK2 assay (1 mM MPS1 vs 10 μM CDK2), the

assays were no longer directly comparable. For this reason, we

used the Cheng−Prusoﬀ equation to calculate the K_ivalues²¹

and, in turn, used these values to determine the selectivity

window.

The data for these compounds are summarized in Table 3. The

phenyl-substituted derivative (28a) showed comparable potency

in the biochemical and cellular assay and, despite higher

lipophilicity, improved microsomal stability. The cyclopropyl

Compound 33b, incorporating a diethylamine substituent,

was signiﬁcantly (6-fold) less potent in the 1 mM ATP Caliper

assay than the pyrrolidine 33a (Table 4). This is likely due to a

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a

Table 2. Biochemical, Cellular, And Mouse Microsomal Turnover Data for Compounds Bearing Alternative Aniline Substituents

a

Results are mean ( SD) for n ≥ 3, or mean values of two independent determinations with individual determinations in parentheses or samples run

n = 1. “P-MPS1″ indicates an electrochemiluminescence MSD-based cellular assay that measured autophosphorylation of ectopically expressed

b

MPS1 in HCT116 cells. Potency at the lower limit of the dynamic range of the Caliper assay run at 10 μM ATP concentration. IC₅₀value is likely

to be lower than that quoted.

potency but also improved microsomal stability, now in an

acceptable range of 30% turnover.

Since saturated amines were well tolerated in this position, we

next prepared and tested derivatives in which the alkyl groups

were linked through a sulfur or oxygen atom (34f and 38).

Remarkably, both lost signiﬁcant activity (at least 10-fold)

compared to the corresponding amine derivatives 34b and 34d.

It is diﬃcult to reconcile the pronounced loss of activity of

compounds 34f and 38 with the wide range of both primary and

secondary amines that are tolerated. Computational conforma-

tional analysis did not suggest a signiﬁcant diﬀerence in the

conformational preference of the oxygen and sulfur-linked

substituents compared to the amine substituents. Furthermore,

analysis of the available crystal structure did not support the

hypothesis that this diﬀerence may be driven by diﬀerent

hydrogen-bond pattern, e.g., to water molecules.

Several amino-substituted compounds did however show

potent IC₅₀values suggesting signiﬁcant scope for further

modiﬁcations. The neopentyl derivative 34e was particularly

promising due to its cellular potency combined with selectivity

and improved mouse microsomal stability. As is apparent from

the MPS1 IC₅₀at 1 mM ATP, it was also the most potent

derivative in this series.

Figure 5. (A) Crystal structure of MPS1 (pale green) bound to 24c

(carbon atoms colored orange), extracted from PDB code 5EI8,

showing residues (Ile531, Val539, Met671, and Pro673) present in the

hydrophobic pocket occupied by 8-position pyrazole. (B) Super-

imposed crystal structure of MPS1 (pale green) bound to 24c (carbon

atoms colored orange), extracted from PDB code 5EI8, onto the

structure of MPS1 (not shown) bound to 1 (carbon atoms colored

pink), PDB code 4C4J, showing that this is the same pocket occupied by

the carbamate group of 1 (carbon atoms colored pink).

higher free energy penalty when binding to the target for this less

constrained compound. Gratifyingly, several amine substituents

(34b, d, and e) not only showed comparable biochemical

H

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Table 3. Biochemical, Cellular, And Mouse Microsomal Turnover Data for Compounds Bearing Alternative 8-Position

a

Substituents

a

Results are mean ( SD) for n ≥ 3, or mean values of two independent determinations with individual determinations in parentheses or samples run

n = 1. “P-MPS1″ indicates an electrochemiluminescence MSD-based cellular assay that measured autophosphorylation of ectopically expressed

b

MPS1 in HCT116 cells. Potency at the lower limit of the dynamic range of the Caliper assay run at 10 μM ATP concentration. IC₅₀value is likely

to be lower than that quoted.

We solved the structure of neopentyl 34e bound into MPS1

(Figure 6). Figure 6 shows that the bulkier, more hydrophobic

neopentyl substituent of 34e addresses the hydrophobic pocket

ﬁrst mentioned in Figure 5A to a greater extent than the pyrazole

substituent of 24c. We hypothesized that an additional increase

in potency could be achieved by further increasing the bulk of the

hydrophobic substituent.

We thus followed up by synthesizing the two enantiomers 34g

and 34h which diﬀer from the neopentyl 34e by an additional

methyl group (Table 5). The (S)-enantiomer (34h) particularly,

translated into an additional gain in biochemical and cellular

potency compared with neopentyl derivative 34e, achieving

cellular modulation of MPS1 in the sub-100 nM range (Table 5).

34h showed suﬃcient selectivity over CDK2 and very good

microsomal stability (MLM = 27%).

With 34h we had achieved our initial potency goal in the

biochemical and cellular assay. Especially considering its

moderate molecular weight of 432 Da, 34h represented an

extremely potent MPS1 inhibitor with an IC₅₀= 0.020 μM at 1

mM ATP (corresponding to a K_iof 0.0002 μM)²¹and a GI₅₀

value in HCT116 cells of 0.16 μM. Furthermore, the combined

SAR suggested signiﬁcant scope for additional modiﬁcations to

further optimize the series. This pyrido[3,4-d]pyrimidine (34h)

was selective over CDK2 (CDK2/MPS1 K_iratio > 1000),²¹

Aurora A and B (Table S1 ), and PLK1 (IC₅₀> 100 μM) as well

as against a wide panel of kinases (Tables S2 −S4 ). Pyrido[3,4-

d]pyrimidine 34h inhibited a small number of kinases, namely

TNK2, JNK1, JNK2, and LRRK at >80% at 1 μM. JNK1 and

JNK2 are considered to be structurally related to MPS1, so follow

up IC₅₀values were obtained (JNK1 IC₅₀= 0.11 μM, JNK2 IC₅₀

= 0.22 μM), showing that 34h was selective for MPS1 over JNK1

and JNK2 by 100- and 200-fold, respectively.

Next we proﬁled 34h against a panel of cell lines (Table 6). As

expected for an MPS1 inhibitor, 34h showed potent growth

inhibition for all cancer cell lines but importantly a signiﬁcantly

higher GI₅₀for the nontransformed line PNT2.

34h did not inhibit CYP or hERG (Tables S5 and S6).

Importantly, pyrido[3,4-d]pyrimidine 34h, showed low turnover

in mouse and rat liver microsomes (27 and 24%, after 30 min

incubation, respectively), and we progressed the compound to

mouse and rat PK experiments, despite a 70% turnover in human

liver microsomes, in order to evaluate its suitability for proof of

mechanism in vivo experiments. 34h showed moderate clearance

(28 and 24 mL/min/kg in mouse and rat, respectively) and high

oral bioavailability (68 and 100% in mouse and rat, respectively)

with moderate to high volumes of distribution (Table 7).

We thus performed a 3 day pharmacokinetic/pharmacody-

namic (PK/PD) study to determine whether biomarker

modulation could be achieved in vivo. MPS1 inhibition results

in premature exit of cells from mitosis,^10eand we therefore chose

the mitotic marker phospho-histone H3 as a readout. Histone H3

is speciﬁcally phosphorylated at Ser 10 during mitosis.²²

Gratifyingly, oral administration of 100 mg/kg of 34h b.i.d. for

3 days to mice bearing HCT116 human colon carcinoma

xenografts caused a reduction of the phospho-histone H3 levels

compared with vehicle control treated animals at 2 and 6 h,

consistent with MPS1 inhibition (Figure 7).

Compound 34h fulﬁlled all of the criteria we had initially set

and compared favorably with our best-in-class 1H-pyrrolo[3,2-

c]pyridine (1). Particularly, 34h was devoid of CYP inhibition,

did not require an acid labile Boc group for biochemical and

I

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Table 4. Biochemical, Cellular, And Mouse Microsomal Turnover Data for Compounds Bearing a Saturated Substituent at the 8-

a

Position

a

Results are mean ( SD) for n ≥ 3, or mean values of two independent determinations with individual determinations in parentheses or samples run

n = 1. “P-MPS1″ indicates an electrochemiluminescence MSD-based cellular assay that measured autophosphorylation of ectopically expressed

MPS1 in HCT116 cells. “HCT116 GI₅₀” indicates a cell proliferation assay carried out by colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT) assay in HCT116 cells. Potency at the lower limit of the dynamic range of the Caliper assay run at 10 μM

b

ATP concentration. IC₅₀value is likely to be lower than that quoted.

cellular potency, and showed excellent overall selectivity. Its

physicochemical properties (ALogP = 4.65 and MW = 432)

made 34h a better starting point for further optimization

especially given that we had already achieved the targeted

potency. Surprisingly, given that it represented a pyrimidine-

based kinase scaﬀold, the 2-amino pyrido[3,4-d]pyrimidine

chemotype was relatively unexplored when we initiated this

work, with only one published kinase patent application in the

public domain.²³Our previously published chemical route¹⁶

facilitated the synthesis of a large range of derivatives as well as

upscaling of advanced derivatives. We thus nominated 34h as an

advanced lead compound.

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Table 6. GI₅₀Values for 34h in 3 Day MTT Assay Carried out

a

on Panel of Cell Lines

origin

colon

cell line

GI₅₀(μM)

SW620

CAL27

CAL51

Miapaca-2

RMG1

PNT2

0.065

0.23

head and neck

breast

0.068

0.25

pancreatic

ovarian

0.110

3.95

prostate

a

“GI₅₀” indicates a cell proliferation assay carried out by colorimetric

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)

assay in a panel of cell lines.

Figure 6. Superimposed crystal structure of MPS1 (pale green) bound

to 24c (carbon atoms colored orange), extracted from PDB code 5EI8,

onto the structure of MPS1 (not shown) bound to 34e (carbon atoms

colored blue), PDB code 5EH0, showing the hydrophobic pocket

formed by Ile531, Val539, Met671, and Pro673. The bulkier neopentyl

substituent of 34e addresses the pocket to a greater extent than the

pyrazole substituent of 24c.

pyridines. Moreover, our optimized compound 34h was devoid

of CYP inhibition and proved to be extremely potent in the

MPS1 biochemical assay with the ability to target this kinase in

cells and to induce signiﬁcant growth inhibition at nanomolar

concentrations. A screen against a large sample of the human

kinome revealed a high level of selectivity, especially with regard

to mitotic kinases. Most importantly, pyrido[3,4-d]pyrimidine

34h showed a satisfactory pharmacokinetic proﬁle in rodents and

eﬀectively inhibited MPS1 activity in vivo.

Optimization of the remaining issues associated with 34h, in

particular HLM instability and plasma protein binding, as well as

the investigation of advanced compounds in combination

eﬃcacy models, is ongoing and the results will be reported in

due course.

CONCLUSIONS

■

By merging two distinct chemical series, namely 1H-pyrrolo[3,2-

c]pyridines and 1H-pyrazolo[4,3-c]pyridines, we successfully

fulﬁlled our aim to discover a new class of MPS1 inhibitors that

did not require an acid labile Boc group for potent inhibition.

The pyrido[3,4-d]pyrimidine core was unprecedented for

kinases,¹⁶and our structure guided optimization resulted in

potent MPS1 inhibitors of substantially reduced size and

lipophilicity compared with the parent 1H-pyrrolo[3,2-c]-

a

Table 5. Biochemical, Cellular, And Mouse Microsomal Turnover Data for Enantiomers 34g and 34h

a

Results are mean ( SD) for n ≥ 3, or mean values of two independent determinations with individual determinations in parentheses or samples run

n = 1. “P-MPS1″ indicates an electrochemiluminescence MSD-based cellular assay that measured autophosphorylation of ectopically expressed

MPS1 in HCT116 cells. “HCT116 GI₅₀” indicates a cell proliferation assay carried out by colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT) assay in HCT116 cells. Potency at the lower limit of the dynamic range of the Caliper assay run at 10 μM

b

ATP concentration. IC₅₀value is likely to be lower than that quoted.

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Table 7. Mouse and Rat Blood Pharmacokinetics of 34h at 5 mg/kg iv and po

species

t_1/2(h)

Cl (mL/min/kg)

Cmax_po(nmol/L)

AUC_po(nmol·h/L)

PPB (%)

V_ss(L/kg)

F (%)

mouse

rat

8.2

28

24

770

560

4800

9300

99.98

99.97

14.7

4.64

68

a

2.5

100

a

Only detected to 4 h.

Figure 7. (A,B) Representative immunoblots of phospho-histone H3 showing dose-dependent PD modulation in HCT116 human tumor xenografts,

following 100 mg/kg b.i.d. dosing of 34h for 3 consecutive days. Total histone H3, cleaved poly ADP ribose polymerase (PARP, a measure of apoptosis)

and glyceraldehyde 3-phosphate dehydrogenase (GADPH, for protein loading) are also shown. (C) Phospho-histone H3 versus total histone H3 ratio

for control and treated samples at 2 and 6 h after last dose. Asterisks show statistically signiﬁcant diﬀerences from control groups as determined by one-

way ANOVA.

7.99−7.90 (m, 2H), 7.71−7.55 (m, 2H), 6.79 (dd, J = 3.4, 0.6 Hz, 1H),

6.62 (dd, J = 3.4, 1.8 Hz, 1H).

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 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 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

3-Chloro-5-(1-methyl-1H-pyrazol-4-yl)isoquinoline 11. A suspen-

sion of 5-bromo-3-chloroisoquinoline 9 (300 mg, 1.24 mmol), 1-

methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole

(309 mg, 1.49 mmol), and Pd(dppf)Cl₂·CH₂Cl₂(105 mg, 0.124 mmol)

in DME (2.5 mL) and sodium carbonate (2 M, 1.24 mL) was heated to

105 °C under microwave irradiation for 1.5 h. The mixture was

concentrated onto silica gel and puriﬁed by ﬂash column chromatog-

raphy (0−100% EtOAc in cyclohexane) to give the title compound (324

mg, quant.). HRMS (ESI) m/z calcd for C₁₃H₁₁ClN₃(M + H)

244.0636, found 244.0639. ¹H NMR (500 MHz, CDCl₃) δ 9.08 (s, 1H),

7.96 (s, 1H), 7.90 (d, J = 8.1 Hz, 1H), 7.73 (s, 1H), 7.67−7.55 (m, 3H),

4.05 (s, 3H).

5-(Furan-2-yl)-N-(4-methoxyphenyl)isoquinolin-3-amine 14. A

suspension of 3-chloro-5-(furan-2-yl)isoquinoline 10 (41 mg, 0.18

mmol), 4-methoxyaniline 12 (29 mg, 0.23 mmol), cesium carbonate

(204 mg, 0.626 mmol), ^tBuXPhos (30 mg, 0.071 mmol), Pd₂(dba)₃(16

mg, 0.018 mmol), and ^tBuOH (3% H₂O) (1 mL) was heated to 80 °C

under microwave irradiation for 3 h. The mixture was concentrated onto

silica gel and puriﬁed by ﬂash column chromatography (0−100% EtOAc

in cyclohexane) to give the title compound (10 mg, 18%). HRMS (ESI)

1

reversion back to 1:9 (A:B) over 0.3 min, 1:9 (A:B) for 0.2 min. H

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.

Compounds 2-methoxy-4-(1-methyl-1H-pyrazol-4-yl)aniline (23),¹⁷

8-chloro-2-(methylsulfonyl)pyrido[3,4-d]pyrimidine (25),¹⁶N-(2-me-

thoxy-4-(1-methyl-1H-pyrazol-4-yl)phenyl)formamide (26a),¹⁶8-

chloro-N-(2-methoxy-4-(1-methyl-1H-pyrazol-4-yl)phenyl)pyrido[3,4-

d]pyrimidin-2-amine (27a),¹⁶and 2-(methylthio)pyrido[3,4-d]-

pyrimidin-8(7H)-one (35)¹⁶were synthesized using previously

published procedures.

1

m/z calcd for C₂₀H₁₇N₂O₂(M + H) 317.1285, found 317.1282. H

NMR (500 MHz, CDCl₃) δ 8.95 (s, 1H), 7.84−7.75 (m, 2H), 7.61 (s,

1H), 7.57 (s, 1H), 7.58−7.25 (m, 3H), 7.17−6.90 (m, 2H), 6.77−6.60

(m, 2H), 6.54 (dd, J = 3.3, 1.8 Hz, 1H), 3.85 (s, 3H).

N-(4-Methoxyphenyl)-5-(1-methyl-1H-pyrazol-4-yl)isoquinolin-3-

amine 15a. A suspension of 3-chloro-5-(1-methyl-1H-pyrazol-4-

yl)isoquinoline 11 (55 mg, 0.23 mmol), 4-methoxyaniline 12 (36 mg,

3-Chloro-5-(furan-2-yl)isoquinoline 10. A suspension of 5-bromo-

3-chloroisoquinoline 9 (58 mg, 0.24 mmol), furan-2-ylboronic acid (32

mg, 0.29 mmol), and Pd(dppf)Cl₂·CH₂Cl₂(20 mg, 0.024 mmol) in

DME (0.5 mL) and sodium carbonate (2 M, 0.24 mL) was heated to 105

°C under microwave irradiation for 1 h. The mixture was concentrated

onto silica gel and puriﬁed by ﬂash column chromatography (0−50%

EtOAc in cyclohexane) to give the title compound (41 mg, 75%).

HRMS (ESI) m/z calcd for C₁₃H₉ClNO (M + H) 230.0367, found

t

0.29 mmol), cesium carbonate (257 mg, 0.789 mmol), BuXPhos (38

t

mg, 0.090 mmol), Pd₂(dba)₃(21 mg, 0.023 mmol), and BuOH (3%

H₂O) (1 mL) was heated to 80 °C under microwave irradiation for 1.5 h.

The mixture was concentrated onto silica gel and puriﬁed by ﬂash

column chromatography (0−100% EtOAc in cyclohexane) to give the

title compound (6 mg, 8%). HRMS (ESI) m/z calcd for C₂₀H₁₉N₄O (M

+ H) 331.1553, found 331.1546. ¹H NMR (500 MHz, CDCl₃) δ 8.95 (s,

1H), 7.75 (d, J = 8.2 Hz, 1H), 7.68 (s, 1H), 7.52 (s, 1H), 7.50−7.46 (m,

1

230.0343. H NMR (500 MHz, CDCl₃) δ 9.09 (s, 1H), 8.33 (s, 1H),

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2H), 7.31−7.21 (m, 3H), 7.03−6.89 (m, 2H), 6.48 (s, 1H), 3.98 (s, 3H),

3.83 (s, 3H).

N-(4-Methoxyphenyl)-8-(1-methyl-1H-pyrazol-4-yl)pyrido[3,4-d]-

pyrimidin-2-amine 24a. To a solution of 8-(1-methyl-1H-pyrazol-4-

yl)-2-(methylsulfonyl)pyrido[3,4-d]pyrimidine 19 (29 mg, 0.10 mmol)

in DMSO (4 mL) was added cesium carbonate (59 mg, 0.18 mmol) and

N-(4-methoxyphenyl)formamide (15 mg, 0.10 mmol). The mixture was

heated to 100 °C for 18 h. The mixture was diluted with EtOAc and

water. The aqueous layer was re-extracted with EtOAc. The combined

organics were washed with brine, dried, and concentrated in vacuo. The

residue was puriﬁed by ﬂash column chromatography (0−10% MeOH

in CH₂Cl₂), followed by SCX-2 cartridge (MeOH - 1 M NH₃in MeOH)

to give the title compound (8 mg, 24%). HRMS (ESI) m/z calcd

C₁₈H₁₆N₆O (M + H) 333.1464, found 333.1450. ¹H NMR (500 MHz,

CD₃OD) δ 9.24 (s, 1H), 8.38 (s, 1H), 8.29 (d, J = 5.4 Hz, 1H), 7.63−

7.59 (m, 3H), 7.54 (d, J = 5.4 Hz, 1H), 7.05 (d, J = 8.9 Hz, 2H), 3.91 (s,

3H), 3.87 (s, 3H).

8-Cyclopropyl-N-(2-methoxy-4-(1-methyl-1H-pyrazol-4-yl)-

phenyl)pyrido[3,4-d]pyrimidin-2-amine 24d. A solution of 2-

methoxy-4-(1-methyl-1H-pyrazol-4-yl)aniline 23 (50 mg, 0.25 mmol),

TFA (45 μL, 0.60 mmol), and 8-cyclopropyl-2-(methylsulfonyl)pyrido-

[3,4-d]pyrimidine 20 (31 mg, 0.12 mmol) in 2,2,2-triﬂuoroethanol (0.7

mL) was heated to 130 °C under microwave irradiation for 1.5 h. The

reaction was diluted with EtOAc and quenched with aqueous sat.

sodium bicarbonate. The aqueous layer was extracted with EtOAc, and

the combined organics were washed with water and brine, dried, and

concentrated in vacuo. The residue was puriﬁed by ﬂash column

chromatography (0−60% EtOAc in cyclohexane) to give the title

compound (20 mg, 43%). HRMS (ESI) m/z calcd for C₂₁H₂₁N₆O (M +

H) 373.1771, found 373.1773. ¹H NMR (500 MHz, (CD₃)₂SO) δ 9.40

(s, 1H), 8.54 (s, 1H), 8.41 (d, J = 8.3 Hz, 1H), 8.28 (d, J = 5.3 Hz, 1H),

8.17 (d, J = 0.9 Hz, 1H), 7.90 (d, J = 0.8 Hz, 1H), 7.56 (d, J = 5.3 Hz,

1H), 7.30 (d, J = 1.8 Hz, 1H), 7.25 (dd, J = 8.2, 1.8 Hz, 1H), 3.96 (s, 3H),

3.88 (s, 3H), 3.24 (m, 1H), 1.16−1.08 (m, 4H).

Preparation of Formamides 26b−e (Exempliﬁed by Prepara-

tion of Compound 26b). N-(2-Methyl-4-(1-methyl-1H-pyrazol-4-

yl)phenyl)formamide 26b. A solution of 2-methyl-4-(1-methyl-1H-

pyrazol-4-yl)aniline S1 (100 mg, 0.534 mmol) in formic acid (3 mL) was

heated to 100 °C for 3 h. The mixture was concentrated in vacuo. The

residue was partitioned between aqueous sat. sodium bicarbonate and

EtOAc. The aqueous layer was re-extracted with EtOAc. The combined

organics were washed with water and brine, dried, and concentrated in

vacuo. The residue was puriﬁed by ﬂash column chromatography (0−

10% MeOH in EtOAc) to give the title compound (160 mg, 34%).

HRMS (ESI) m/z calcd for C₁₂H₁₄N₃O (M + H) 216.1131, found

216.1141. ¹H NMR (500 MHz, CD₃OD) δ 8.31 (s, 1H), 7.93 (s, 1H),

7.80 (s, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.44 (d, J = 2.0 Hz, 1H), 7.38 (dd, J

= 8.0, 2.0, Hz, 1H), 3.92 (s, 3H), 2.31 (s, 3H).

8-Chloro-N-(2-methyl-4-(1-methyl-1H-pyrazol-4-yl)phenyl)-

pyrido[3,4-d]pyrimidin-2-amine 27b. To a cooled (0 °C) suspension

of N-(2-methyl-4-(1-methyl-1H-pyrazol-4-yl)phenyl)formamide 26b

(40 mg, 0.19 mmol) in THF (4 mL) was added sodium hydride (60%

w/w dispersion in oil, 12 mg, 0.30 mmol). The reaction mixture was

stirred at rt for 10 min. The mixture was cooled to 0 °C, and 8-chloro-2-

(methylthio)pyrido[3,4-d]pyrimidine 25¹⁶(60 mg, 0.24 mmol) was

added. The mixture was stirred for 18 h while slowly warming to rt and

then concentrated in vacuo, and the residue was partitioned between

EtOAc and water. The aqueous layer was extracted with EtOAc and

CH₂Cl₂. The combined organics were washed with water and brine,

dried, and concentrated in vacuo. The residue was puriﬁed by ﬂash

column chromatography (0−5% MeOH in EtOAc) to give the title

compound (79 mg, 97%). HRMS (ESI) m/z calcd for C₁₉H₁₆ClN₆(M +

H) 351.1119, found 351.1111. ¹H NMR (500 MHz, CDCl₃) δ 9.17 (s,

1H), 8.26 (d, J = 5.0 Hz, 1H), 7.79 (s, 1H), 7.63 (s, 1H), 7.52 (d, J = 5.0

Hz, 1H), 7.47 (dd, J = 8.0, 2.0 Hz, 1H), 7.42 (m, 1H), 7.39 (d, J = 2.0 Hz,

1H), 3.98 (s, 3H), 2.44 (s, 3H).

N-(2,4-Dimethoxyphenyl)-5-(1-methyl-1H-pyrazol-4-yl)-

isoquinolin-3-amine 15b. A suspension of 3-chloro-5-(1-methyl-1H-

pyrazol-4-yl)isoquinoline 11 (55 mg, 0.23 mmol), 2,4-dimethoxyaniline

13 (45 mg, 0.29 mmol), cesium carbonate (257 mg, 0.789 mmol),

^tBuXPhos (38 mg, 0.090 mmol), Pd₂(dba)₃(21 mg, 0.023 mmol), and

^tBuOH (3% H₂O) (1 mL) was heated to 80 °C under microwave

irradiation for 1.5 h and then to 100 °C for 1.5 h. The mixture was

concentrated onto silica gel and puriﬁed by ﬂash column chromatog-

raphy (0−100% EtOAc in cyclohexane) to give the title compound (22

mg, 27%). HRMS (ESI) m/z calcd for C₂₁H₂₁N₄O₂(M + H) 361.1659,

found 361.1661. ¹H NMR (500 MHz, CDCl₃) δ 8.96 (s, 1H), 7.78−7.69

(m, 2H), 7.61 (d, J = 8.6 Hz, 1H), 7.54 (s, 1H), 7.47 (dd, J = 7.0, 1.0 Hz,

1H), 7.33−7.23 (m, 2H), 6.73 (s, 1H), 6.58−6.49 (m, 2H), 3.99 (s, 3H),

3.85 (s, 3H), 3.83 (s, 3H).

Preparation of Compounds in Scheme 2 (Exempliﬁed by the

Preparation of Compounds 24a, 24d, 28a, and 28b). 8-(1-Methyl-

1H-pyrazol-4-yl)-2-(methylthio)pyrido[3,4-d]pyrimidine 17. A solu-

tion of 8-chloro-2-(methylthio)pyrido[3,4-d]pyrimidine 16 (480 mg,

2.27 mmol), (1-methyl-1H-pyrazol-4-yl)boronic acid pinacol ester (940

mg, 4.52 mmol), and Pd(dppf)Cl₂·CH₂Cl₂(100 mg, 0.122 mmol) in

THF (15 mL) and sodium carbonate (2 M, 5 mL) was heated to 65 °C

for 18 h. The mixture was diluted with EtOAc and quenched with brine.

The aqueous layer was extracted with EtOAc three times. The combined

organics were washed with water and brine, dried, and concentrated in

vacuo. The residue was puriﬁed by ﬂash column chromatography (0−

4% MeOH in CH₂Cl₂) to give the title compound (658 mg, quant).

HRMS (ESI) m/z calcd for C₁₂H₁₂N₅S (M + H) 258.0813, found

1

258.0817. H NMR (500 MHz, CDCl₃) δ 9.22 (s, 1H), 8.67 (s, 1H),

8.63−8.56 (m, 2H), 7.47 (d, J = 5.3 Hz, 1H), 4.05 (s, 3H), 2.78 (s, 3H).

8-Cyclopropyl-2-(methylthio)pyrido[3,4-d]pyrimidine 18. A solu-

tion of 8-chloro-2-(methylthio)pyrido[3,4-d]pyrimidine 16 (20 mg,

0.094 mmol), cyclopropyl boronic acid (11 mg, 0.13 mmol), PCy₃(3

mg, 11 μmol), K₃PO₄(70 mg, 0.32 mmol), and Pd(OAc)₂(1.0 mg, 4.5

μmol) was dissolved in toluene/water (6:1, 1 mL) and heated to 95 °C

for 18 h. The mixture was diluted with EtOAc and quenched with brine.

The aqueous layer was extracted with EtOAc three times. The combined

organics were washed with water and brine, dried, and concentrated in

vacuo. The residue was puriﬁed by ﬂash column chromatography (0−

20% EtOAc in cyclohexane) to give the title compound (13 mg, 62%).

HRMS (ESI) m/z calcd C₁₁H₁₂N₃S (M + H) 218.0746, found 218.0751.

¹H NMR (500 MHz, CDCl₃) δ 9.18 (s, 1H), 8.46 (d, J = 5.4 Hz, 1H),

7.37 (d, J = 5.5 Hz, 1H), 3.46 (tt, J = 8.2, 4.8 Hz, 1H), 2.74 (s, 3H), 1.34−

1.27 (m, 2H), 1.25−1.17 (m, 2H).

8-(1-Methyl-1H-pyrazol-4-yl)-2-(methylsulfonyl)pyrido[3,4-d]-

pyrimidine 19. A suspension of 8-(1-methyl-1H-pyrazol-4-yl)-2-

(methylthio)pyrido[3,4-d]pyrimidine 17 (584 mg, 2.27 mmol) in

CH₂Cl₂(22 mL) was treated with m-CPBA (77% w/w, 1.12 g, 4.98

mmol) at 0 °C and then allowed to reach rt over 18 h. The mixture was

quenched with water and extracted with CH₂Cl₂. The combined

organics were washed with water, dried, and concentrated in vacuo. The

residue was puriﬁed by ﬂash column chromatography (0−100% EtOAc

in cyclohexane) to give the title compound (408 mg, 62%). HRMS

(ESI) m/z calcd for C₁₂H₁₂N₅O₂S (M + H) 290.0706, found 290.0722.

¹H NMR (500 MHz, (CD₃)₂SO) δ 10.00 (s, 1H), 8.91 (d, J = 5.4 Hz,

1H), 8.81 (s, 1H), 8.53 (s, 1H), 7.99 (d, J = 5.4 Hz, 1H), 3.99 (s, 3H),

3.59 (s, 3H).

8-Cyclopropyl-2-(methylsulfonyl)pyrido[3,4-d]pyrimidine 20. A

suspension of 8-cyclopropyl-2-(methylthio)pyrido[3,4-d]pyrimidine

18 (127 mg, 0.584 mmol) in CH₂Cl₂(5 mL) was treated with m-

CPBA (77% w/w, 290 mg, 1.29 mmol) at 0 °C and then allowed to reach

rt over 18 h. The mixture was quenched with water and extracted with

CH₂Cl₂. The combined organics were washed with water, dried, and

concentrated onto silica. The residue was puriﬁed by ﬂash column

chromatography (0−70% EtOAc in cyclohexane) to give the title

compound (128 mg, 88%). HRMS (ESI) m/z calcd C₁₁H₁₂N₃O₂S (M +

H) 250.0645, found 250.0648. ¹H NMR (500 MHz, (CD₃)₂SO) δ 9.99

(s, 1H), 8.79 (d, J = 5.5 Hz, 1H), 7.94 (d, J = 5.5 Hz, 1H), 3.56 (s, 3H),

3.44 (m, 1H), 1.30−1.25 (m, 2H), 1.24−1.20 (m, 2H).

N-(2-Methoxy-4-(1-methyl-1H-pyrazol-4-yl)phenyl)-8-

phenylpyrido[3,4-d]pyrimidin-2-amine 28a. To a solution of 8-

chloro-N-(2-methoxy-4-(1-methyl-1H-pyrazol-4-yl)phenyl)pyrido[3,4-

d]pyrimidin-2-amine 27a¹⁶(25 mg, 0.068 mmol) in 1,4-dioxane/water

(2:1, 3 mL) was added phenyl boronic acid (17 mg, 0.14 mmol),

Pd(PPh₃)₄(16 mg, 0.014 mmol), and cesium carbonate (33 mg, 0.10

M

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Article

mmol). The reaction was heated to 100 °C under microwave irradiation

for 30 min. The reaction was diluted with EtOAc and water, dried, and

concentrated in vacuo. The residue was puriﬁed by ﬂash column

chromatography (0−100% EtOAc in cyclohexane) followed by SCX-2

cartridge (MeOH - 1 M NH₃in MeOH) to give the title compound (8

mg, 29%). HRMS (ESI) m/z calcd for C₂₄H₂₁N₆O (M + H) 409.1777,

found 409.1771. ¹H NMR (500 MHz, CD₃OD) δ 9.37 (s, 1H), 8.56 (d, J

= 8.5 Hz, 1H), 8.50 (d, J = 5.5 Hz, 1H), 8.11−8.09 (m, 2H), 7.97 (s, 1H),

7.82 (s, 1H), 7.79 (d, J = 5.5 Hz, 1H), 7.61−7.59 (m, 3H), 7.19 (d, J = 2.0

Hz, 1H), 6.99 (dd, J = 8.5, 2.0 Hz, 1H), 4.02 (s, 3H), 3.95 (s, 3H).

8-(1-Methyl-1H-pyrazol-4-yl)-N-(2-methyl-4-(1-methyl-1H-pyra-

zol-4-yl)phenyl)pyrido[3,4-d]pyrimidin-2-amine 28b. To a solution of

8-chloro-N-(2-methyl-4-(1-methyl-1H-pyrazol-4-yl)phenyl)pyrido-

[3,4-d]pyrimidin-2-amine 27b (12 mg, 0.034 mmol) in 1,4-dioxane/

water (2:1, 3 mL) were added 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-

dioxaborolan-2-yl)-1H-pyrazole (14 mg, 0.068 mmol), cesium carbo-

nate (17 mg, 0.051 mmol), and Pd(PPh₃)₄(2 mg, 1.7 μmol). The

reaction mixture was heated to 100 °C under microwave conditions for

30 min. The reaction mixture was diluted with EtOAc and water. The

combined organics were washed with water and brine, dried, and

concentrated in vacuo. The residue was puriﬁed by ﬂash column

chromatography (0−15% MeOH in EtOAc) to give the title compound

(7 mg, 52%). HRMS (ESI) m/z calcd for C₂₂H₂₁N₈(M + H) 397.1884,

found 397.1878. ¹H NMR (500 MHz, CDCl₃) δ 9.16 (s, 1H), 8.43 (d, J

= 5.5 Hz, 1H), 8.40 (d, J = 5.5 Hz, 2H), 7.86 (d, J = 8.0 Hz, 1H), 7.82 (s,

1H), 7.67 (s, 1H), 7.50−7.45 (m, 2H), 7.36 (d, J = 5.5 Hz, 1H), 7.05 (br

s, 1H), 4.00 (s, 3H), 3.77 (s, 3H), 2.39 (s, 3H).

Preparation of Compounds in Scheme 3 (Exempliﬁed by the

Preparation of 33a). 2-(Methylsulfonyl)-8-(pyrrolidin-1-yl)pyrido-

[3,4-d]pyrimidine 31. A mixture of 8-chloro-2-(methylthio)pyrido[3,4-

d]pyrimidine 16 (105 mg, 0.496 mmol) and pyrrolidine (425 μL, 5.10

mmol) in NMP (2.5 mL) was stirred at 135 °C for 3 h. The mixture was

quenched with aqueous sat. sodium bicarbonate and extracted with

EtOAc. The combined organics were washed with water and brine,

dried, and concentrated in vacuo to aﬀord the crude sulﬁde.

A suspension of crude sulﬁde 29 (ca. 0.496 mmol) in CH₂Cl₂(4 mL)

was treated with m-CPBA (77% w/w, 250 mg, 1.11 mmol) at 0 °C and

then allowed to reach rt for 18 h. An additional portion of m-CPBA (77%

w/w, 60 mg, 0.27 mmol) was added at rt, and the mixture stirred for 2 h.

The mixture was quenched with water and extracted with CH₂Cl₂. The

combined organics were washed with aqueous sat. sodium bicarbonate

and brine, dried, and concentrated in vacuo. The residue was puriﬁed by

ﬂash column chromatography (0−70% EtOAc in cyclohexane) to give

the title compound (62 mg, 45% over two steps). LCMS (ESI) m/z 279

(M + H). ¹H NMR (500 MHz, (CD₃)₂SO) δ 9.62 (s, 1H), 8.30 (d, J =

5.4 Hz, 1H), 7.11 (d, J = 5.5 Hz, 1H), 3.97 (br s, 4H), 3.45 (s, 3H), 1.98

(s, 4H).

27a¹⁶(27 mg, 0.074 mmol) and 2-methylpropan-1-amine (100 μL, 1.0

mmol) in NMP (0.7 mL) was stirred at 130 °C in a closed cap vial for 5

h. The reaction mixture was quenched with aqueous sat. sodium

bicarbonate and extracted with EtOAc. The combined organics were

washed with water and brine, dried, and concentrated in vacuo. The

residue was puriﬁed by ﬂash column chromatography (0−80% EtOAc in

cyclohexane) to give the title compound (19 mg, 63%). HRMS (ESI)

m/z calcd for C₂₂H₂₆N₇O (M + H) 404.2193, found 404.2177. ¹H NMR

(500 MHz, (CD₃)₂SO) δ 9.16 (s, 1H), 8.43 (s, 1H), 8.19 (d, J = 8.3 Hz,

1H), 8.15 (d, J = 0.8 Hz, 1H), 7.88 (d, J = 0.8 Hz, 1H), 7.77 (d, J = 5.7

Hz, 1H), 7.27 (d, J = 1.9 Hz, 1H), 7.18 (dd, J = 8.2, 1.9 Hz, 1H), 6.88−

6.85 (m, 2H), 3.93 (s, 3H), 3.87 (s, 3H), 3.39−3.28 (m, 2H), 2.00 (hept,

J = 6.8 Hz, 1H), 0.95 (d, J = 6.7 Hz, 6H).

8-(Cyclohexylthio)-N-(2-methoxy-4-(1-methyl-1H-pyrazol-4-yl)-

phenyl)pyrido[3,4-d]pyrimidin-2-amine 34f. A mixture of 8-chloro-N-

(2-methoxy-4-(1-methyl-1H-pyrazol-4-yl)phenyl)pyrido[3,4-d]-

pyrimidin-2-amine 27a¹⁶(26 mg, 0.071 mmol) and potassium

carbonate (15 mg, 0.11 mmol) in DMF (0.35 mL) was treated with

cyclohexanethiol (12 μL, 0.098 mmol) and stirred at rt for 4 d. An

additional batch of potassium carbonate (10 mg, 0.070 mmol) and

cyclohexanethiol (12 μL, 0.098 mmol) was added, and the mixture

stirred at 50 °C for 18 h. The reaction was quenched with brine and

extracted with EtOAc. The combined organics were washed with water

and brine, dried, and concentrated in vacuo. The residue was puriﬁed by

ﬂash column chromatography (0−80% EtOAc in cyclohexane) to give

the title compound (30 mg, 94%). HRMS (ESI) m/z calcd for

C₂₄H₂₇N₆OS (M + H) 447.1962, found 447.1948. ¹H NMR (500 MHz,

(CD₃)₂SO) δ 9.35 (s, 1H), 8.55 (br s, 1H), 8.29 (d, J = 5.4 Hz, 1H), 8.20

(d, J = 0.9 Hz, 1H), 7.93 (d, J = 0.8 Hz, 1H), 7.48 (d, J = 5.4 Hz, 1H),

7.29 (d, J = 1.9 Hz, 1H), 7.26 (dd, J = 8.2, 1.9 Hz, 1H), 3.96 (br s, 4H),

3.87 (s, 3H), 2.17−2.05 (m, 2H), 1.82−1.72 (m, 2H), 1.65 (m, 1H),

1.59−1.40 (m, 4H), 1.35 (m, 1H).

8-(Cyclopropylmethoxy)-2-(methylthio)pyrido[3,4-d]pyrimidine

36. A suspension of 2-(methylthio)pyrido[3,4-d]pyrimidin-8(7H)-one

35¹⁶(502 mg, 2.60 mmol) and silver carbonate (988 mg, 3.58 mmol) in

CHCl₃(25 mL) was treated with bromomethyl cyclopropane (310 μL,

3.19 mmol) and stirred at rt for 18 h. The mixture was heated to 60 °C

for 4 h, and an additional batch of bromomethyl cyclopropane (310 μL,

3.19 mmol) was added. The reaction was heated to 60 °C for 18 h. An

additional batch of bromomethyl cyclopropane (310 μL, 3.19 mmol)

was added and heated for a further 2 h. Et₃N was added (6 mL), the

mixture ﬁltered through Celite (CH₂Cl₂), and concentrated in vacuo.

The residue was puriﬁed by ﬂash column chromatography (0−80%

EtOAc in cyclohexane) to give the title compound (112 mg, 17%).

LCMS (ESI) m/z 248 (M + H). ¹H NMR (500 MHz, CDCl₃) δ 9.14 (s,

1H), 8.07 (d, J = 5.6 Hz, 1H), 7.18 (d, J = 5.6 Hz, 1H), 4.43 (d, J = 7.0

Hz, 2H), 2.74 (s, 3H), 1.48 (m, 1H), 0.73−0.61 (m, 2H), 0.54−0.43 (m,

2H).

N-(2-Methoxy-4-(1-methyl-1H-pyrazol-4-yl)phenyl)-8-(pyrrolidin-

1-yl)pyrido[3,4-d]pyrimidin-2-amine 33a. A solution of 2-methoxy-4-

(1-methyl-1H-pyrazol-4-yl)aniline 23 (52 mg, 0.26 mmol), TFA (50 μL,

0.65 mmol) and 2-(methylsulfonyl)-8-(pyrrolidin-1-yl)pyrido[3,4-d]-

pyrimidine 31 (35 mg, 0.13 mmol) in 2,2,2-triﬂuoroethanol (0.6 mL)

was heated to 130 °C under microwave irradiation for 2 h. An additional

portion of TFA (50 μL, 0.65 mmol) was added, and the mixture was

heated to 180 °C under microwave irradiation for 2 h. The reaction

mixture was diluted with EtOAc, quenched with aqueous sat. sodium

bicarbonate, and the aqueous layer extracted with EtOAc. The

combined organics were washed with water and brine, dried,

concentrated in vacuo, and puriﬁed by ﬂash column chromatography

(0−100% EtOAc in cyclohexane) to give the title compound (10 mg,

20%). HRMS (ESI) m/z calcd for C₂₂H₂₄N₇O (M + H) 402.2037, found

402.2040. ¹H NMR (500 MHz, (CD₃)₂SO) δ 9.12 (s, 1H), 8.37 (s, 1H),

8.14 (s, 1H), 7.88 (d, J = 0.9 Hz, 1H), 7.87−7.79 (m, 2H), 7.24 (d, J = 1.9

Hz, 1H), 7.17 (dd, J = 8.2, 1.9 Hz, 1H), 6.86 (d, J = 5.4 Hz, 1H), 3.89 (s,

3H), 3.87 (s, 3H), 3.84−3.76 (m, 4H), 1.91−1.81 (m, 4H).

8-(Cyclopropylmethoxy)-2-(methylsulfonyl)pyrido[3,4-d]-

pyrimidine 37. A suspension of 8-(cyclopropylmethoxy)-2-

(methylthio)pyrido[3,4-d]pyrimidine 31 (110 mg, 0.445 mmol) in

CH₂Cl₂(4 mL) was treated with m-CPBA (77% w/w, 325 mg, 1.35

mmol) at 0 °C and then allowed to reach rt for 18 h. The mixture was

quenched with water and extracted with CH₂Cl₂. The combined

organics were washed with aqueous sat. sodium bicarbonate, dried, and

concentrated in vacuo. The residue was puriﬁed by ﬂash column

chromatography (0−55% EtOAc in cyclohexane) to give the title

compound (95 mg, 77%). HRMS (ESI) m/z calcd C₁₂H₁₄N₃O₃S (M +

H) 280.0750, found 280.0748. ¹H NMR (500 MHz, (CD₃)₂SO) δ 9.92

(s, 1H), 8.44 (d, J = 5.7 Hz, 1H), 7.72 (d, J = 5.7 Hz, 1H), 4.43 (d, J = 7.2

Hz, 2H), 3.50 (s, 3H), 1.41(m, 1H), 0.69−0.57 (m, 2H), 0.49−0.40 (m,

2H).

8-(Cyclopropylmethoxy)-N-(2-methoxy-4-(1-methyl-1H-pyrazol-

4-yl)phenyl)pyrido[3,4-d]pyrimidin-2-amine 38. A solution of N-(2-

methoxy-4-(1-methyl-1H-pyrazol-4-yl)phenyl)formamide 26a (30 mg,

0.13 mmol) in THF (1 mL) was treated with sodium hydride (60% w/w

dispersion in oil, 8 mg, 0.20 mmol) at 0 °C. After stirring for 40 min at rt,

the mixture was cooled to 0 °C, and 8-(cyclopropylmethoxy)-2-

(methylsulfonyl)pyrido[3,4-d]pyrimidine 37 (46 mg, 0.17 mmol) was

added. The reaction was allowed to reach rt and stirred for 18 h. Sodium

Preparation of Compounds in Scheme 4 (Exempliﬁed by the

Preparation of Compounds 34a, 34f, and 38). N⁸-Isobutyl-N²-(2-

methoxy-4-(1-methyl-1H-pyrazol-4-yl)phenyl)pyrido[3,4-d]-

pyrimidine-2,8-diamine 34a. A mixture of 8-chloro-N-(2-methoxy-4-

(1-methyl-1H-pyrazol-4-yl)phenyl)pyrido[3,4-d]pyrimidin-2-amine

N

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Journal of Medicinal Chemistry

Article

hydroxide (2 M, 1 mL) and MeOH (1 mL) were added, and the

resulting mixture stirred at rt for 3 h. The mixture was concentrated in

vacuo. The residue was partitioned between EtOAc and water. The

aqueous layer was extracted with EtOAc, and the combined organics

were washed with water and brine, dried, and concentrated in vacuo.

The residue was puriﬁed by ﬂash column chromatography (0−75%

EtOAc in cyclohexane) to give the title compound (27 mg, 52%).

HRMS (ESI) m/z calcd for C₂₂H₂₃N₆O₂(M + H) 403.1877, found

403.1871. ¹H NMR (500 MHz, (CD₃)₂SO) δ 9.31 (s, 1H), 8.61 (s, 1H),

8.39 (s, 1H), 8.17 (d, J = 0.8 Hz, 1H), 7.94−7.84 (m, 2H), 7.35 (d, J = 5.6

Hz, 1H), 7.28 (d, J = 1.8 Hz, 1H), 7.19 (dd, J = 8.3, 1.8 Hz, 1H), 4.33 (d,

J = 6.9 Hz, 2H), 3.94 (s, 3H), 3.88 (s, 3H), 1.40 (m, 1H), 0.68−0.59 (m,

2H), 0.49−0.39 (m, 2H).

Biochemical Assays. MPS1, CDK2, and Aurora A and B

counterscreen assays were performed as reported previously.⁷

MSD Assay. IC₅₀of MPS1 autophosphorylation inhibition at

pTpS^33/37sites in HCT116 cells was determined by an electro-

chemiluminescence assay (Meso Scale Discovery, MSD) as described

previously.⁷

Cell Viability Assay. Cell proliferation assays were carried out by

colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-

mide (MTT) assay (Sigma) in HCT116 cells as described previously.⁷

Crystallization. The kinase domain (residues 519−808) of MPS1

was produced in E. coli and puriﬁed as described previously.^10cFor

compound 39, apo crystals of MPS1 were grown in PEG300 as

previously described⁷prior to soaking in a fresh solution containing 35%

PEG300, 1 mM inhibitor, and 1% (v/v) DMSO for 24 h. The crystal was

cryo-protected in soak solution supplemented with 22.5% ethylene

glycol prior to ﬂash cooling in liquid nitrogen.

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 10 mM

ammonium acetate in methanol on a 50 × 2.1 mm 2.6μm particles 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.

Pharmacokinetic Studies. All in vivo studies were performed in

accordance with U.K. Home Oﬃce regulations, ICR ethical review

processes, and U.K. National Cancer Research Institute guidelines.³⁴

Female Balb/C mice and Sprague−Dawley rats were obtained from

Charles River (Margate, U.K.). 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/10g in 10% DMSO, 5% tween 20 in saline, rat: 0.05 mL/10g in 10%

DMSO, 5% tween 20 in saline). Blood samples were collected from the

tail vein (20 μL) at 8 time points over the 24 h post dose and spotted on

Whatman B cards together with a standard curve and quality controls

spiked in control blood. Cards were allowed to dry at rt for at least 6 h.

Cards were punched, and 6 mm discs were extracted with 200 μL

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 separation as above. Quantitation

was carried out with an external calibration (8 points ranging from 1 nM

to 25 μM). Quality controls were included (3 concentrations) at the

beginning and the end of the analytical run and were within 20% of

nominal concentrations.

Pharmacokinetic parameters were derived from noncompartmental

analysis WiniNONlin (model 200 and 201) Pharsight version.

In Vivo Proof-of-Concept Studies. Animals (6−8 week old female

NCr athymic mice) were supplied by a commercial breeder and fed

sterilized food and water ad libitum.

PK/PD Study. Three million HCT116 human colorectal carcinoma

cells were injected s.c. bilaterally into the ﬂanks. Dosing commenced

when tumors reached a mean diameter of 8−10 mm (day 14). Animals

(n = 6 per group) were dosed twice daily with compound 34h (100 mg/

kg po) or vehicle (10% DMSO, 5% Tween 20, 85% saline) over 3 days

(6 doses), and groups of three were culled at 2 or 6 h after the ﬁnal dose.

Heparinized plasma and tumor samples were collected and snap frozen

for pharmacokinetic (PK) and pharmacodynamic (PD) biomarker

analysis.

PD Assays. For PD analysis, frozen tumor samples were

homogenized in RIPA lysis buﬀer (150 mM NaCl, 50 mM Tris pH

7.5, 1 mM EDTA pH 8.0, 1% NP40, 1% sodium deoxycholate, 1%

sodium dodecyl sulfate, and supplemented with protease and

phosphatase inhibitors), sonicated, and centrifuged to clear the debris.

Protein concentrations of the supernatants were measured, and 5 μg

protein for each sample was loaded onto LDS-PAGE (Life

technologies). Proteins were separated, transferred to nitrocellulose

membrane, and probed with phospho-histone H3 (Millipore), total-

histone H3 (Abcam), cleaved-PARP (Cell Signaling), and GAPDH

(Millipore) antibodies. Blots were quantiﬁed using ImageJ and analyzed

with Graphpad Prism.

Co-crystals of MPS1 with compound S11 were grown at 18 °C using

the sitting-drop vapor-diﬀusion method. The crystallization drops were

composed of 2 μL of protein/ligand solution (8.9 mg/mL protein and 5

mM S11) and 2 μL of reservoir solution placed over 200 μL of reservoir

solution of 18−26% (w/v) PEG3350, 0.1 M bis-Tris propane pH 7.5,

0.1 M MgCl₂, and 0.1 M sodium formate in 48-well plates. Co-crystals

typically grew in 12−16 h. Crystals of MPS1 with S11 were transferred

to backsoaking solutions containing reservoir solution also containing

200 mM of inhibitor and up to 20% (v/v) DMSO and incubated at 18

°C for 24−48 h. Crystals were cryo-protected with paratone-N oil prior

to ﬂash cooling in liquid nitrogen.

Co-crystals of MPS1 with compound 34e were grown at 18 °C using

the sitting-drop vapor-diﬀusion method. The crystallization drops were

composed of 2 μL of protein/ligand solution (11.4 mg/mL protein and

2 mM 34e) with 20% (w/v) PEG3350, 0.1 M bis-Tris propane pH 7.5,

and 0.2 M sodium formate. Crystals were cryo-protected with paratone-

N oil prior to ﬂash cooling in liquid nitrogen.

Data Collection, Structure Solution, and Reﬁnement. X-ray

diﬀraction data were collected at 100 K at Diamond Light Source

(Oxfordshire, U.K.) or in-house on a Rigaku FRX with Pilatus 300 K

detector. Data were integrated with XDS²⁴or MOSFLM (S11 data set

only).²⁵All data were imported to MTZ format with POINTLESS,²⁶

then scaled and merged with AIMLESS²⁶in the CCP4 suite.²⁵The

structures were solved by molecular replacement with PHASER,²⁷with

the PDB structure 4C4J⁷as the search model after removal of all

nonprotein atoms. Structures were reﬁned in iterative cycles of model

building with COOT²⁸and reﬁnement with BUSTER.²⁹TLS groups

were selected with PHENIX phenix.ﬁnd_tls_groups.³⁰Ligand restraints

were generated with GRADE³¹and MOGUL.³²The ﬁnal structure

quality was checked with MOLPROBITY.³³The data collection and

reﬁnement statistics are presented in Table S7.

Microsomal Metabolism. Microsomal turnover was carried out in

male CD1 mice, female Sprague−Dawley rats, and pooled human liver

microsomes obtained from Tebu-Bio (Peterborough, U.K.) following

30 min incubation of 10 μM compound 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, at 37 °C,

were started by addition of the test compound and were terminated at 0

and 30 min by the addition of 3 volumes of ice-cold methanol containing

internal standard. Samples were centrifuged at 2800g for 30 min at 4 °C

ASSOCIATED CONTENT

■

S

* Supporting Information

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.5b01811.

Additional data (CSV)

Experimental procedures and analytical data for ﬁnal

compounds 24b, 24c, 28c, 28d, 28e, 33b, 34b−e, 34g,

34h, intermediates and formamides. Aurora A and B

inhibition data available for all compounds. CYP and

hERG activity as well as kinase selectivity proﬁling of 34h.

O

DOI: 10.1021/acs.jmedchem.5b01811

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

5389. (b) Brough, R.; Frankum, J. R.; Sims, D.; MacKay, A.; Mendes-

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Cerone, M. A.; Natrajan, R.; Sharpe, R.; Shiu, K.-K.; Wetterskog, D.;

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aneuploidy in cancer. Nat. Rev. Genet. 2012, 13, 189−203.

Crystallographic analysis of compounds 15b, 24b, 24c,

34e, 39 bound to MPS1 (PDF)

Accession Codes

Atomic coordinates and structure factors for compounds 1, 15b,

24b, 24c, 34e and 39 can be accessed using PBD codes 4C4J,

5EI6, 5EI2, 5EI8, 5EH0, 5EHY, respectively.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: swen.hoelder@icr.ac.uk. Phone: +44 (0)2087224353.

■

Present Address

^∥SBA School of Science and Engineering, Lahore University of

Management Sciences, D.H.A., Lahore 54792, Pakistan.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

This work was supported by Cancer Research U.K. [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 (recently merged with Breast Cancer Campaign forming

Breast Cancer Now). We thank the staﬀ of DIAMOND Light

Source for their support during data collection. We thank Dr.

Amin Mirza, Dr. Maggie Liu, and Mr. Meirion Richards for their

help with LC, NMR, and mass spectrometry.

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̈

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ABBREVIATIONS USED

CDK2, cyclin-dependent kinase 2; Cl, clearance; hERG, human

■

ether-a-

̀

go-go-related gene; HLM, human liver microsomes;

JNK1, c-Jun N-terminal kinase 1; JNK2, c-Jun N-terminal kinase

2; L.E., ligand eﬃciency; L.L.E, lipophilic ligand eﬃciency;

LRRK, leucine-rich repeat kinase; MLM, mouse liver micro-

somes; MPS1, monopolar spindle kinase 1; MSD, Meso Scale

Discovery; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-

trazolium bromide; PARP, poly ADP ribose polymerase;

PLK1, polo-like kinase 1; PTEN, phosphatase and tensin

homologue; SAC, spindle assembly checkpoint; RLM, rat liver

microsomes; TGI, tumor growth inhibition; V_ss, volume of

distribution

(7) Naud, S.; Westwood, I. M.; Faisal, A.; Sheldrake, P.; Bavetsias, V.;

Atrash, B.; Cheung, K.-M. J.; Liu, M.; Hayes, A.; Schmitt, J.; Wood, A.;

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Montfort, R. L. M.; Blagg, J. Structure-Based Design of Orally

Bioavailable 1H-Pyrrolo[3,2-c]pyridine Inhibitors of Mitotic Kinase

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Higaki, M.; Baba, Y.; Nakamura, Y. Discovery of Imidazo[1,2-

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N. K. B.; Edwards, L. G.; Lang, Y.; Li, S.-W.; Feher, M.; Awrey, D. E.;

Leung, G.; Beletskaya, I.; Plotnikova, O.; Mason, J. M.; Hodgson, R.;

Wei, X.; Mao, G.; Luo, X.; Huang, P.; Green, E.; Kiarash, R.; Lin, D. C.-

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Article Doi

Rapid Discovery of Pyrido[3,4-d]pyrimidine Inhibitors of Monopolar Spindle Kinase 1 (MPS1) Using a Structure-Based Hybridization Approach

DOI: 10.1021/acs.jmedchem.5b01811

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