Synthesis and SAR of new pyrazolo[4,3-h]quinazoline-3-carboxamide derivatives as potent and selective MPS1 kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.05.122

The synthesis and SAR of a series of novel pyrazolo-quinazolines as potent and selective MPS1 inhibitors are reported. We describe the optimization of the initial hit, identified by screening the internal library collection, into an orally available, potent and selective MPS1 inhibitor.

Full text of DOI:10.1016/j.bmcl.2011.05.122

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4507–4511
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and SAR of new pyrazolo[4,3-h]quinazoline-3-carboxamide
derivatives as potent and selective MPS1 kinase inhibitors
⇑
Marina Caldarelli , Mauro Angiolini, Teresa Disingrini, Daniele Donati, Marco Guanci, Stefano Nuvoloni,
Helena Posteri, Francesca Quartieri, Marco Silvagni, Riccardo Colombo
Nerviano Medical Sciences srl, Business Unit Oncology, Viale Pasteur 10, 20014 Nerviano, MI, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and SAR of a series of novel pyrazolo-quinazolines as potent and selective MPS1 inhibitors
are reported. We describe the optimization of the initial hit, identified by screening the internal library
collection, into an orally available, potent and selective MPS1 inhibitor.
Received 27 April 2011
Revised 30 May 2011
Accepted 31 May 2011
Available online 14 June 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
MPS1
TTK
Anticancer
Kinase selectivity
SAR
MPS1 (Monopolar Spindle-1 kinase), also known as TTK, plays
critical roles in the proper execution of mitosis, is frequently
over-expressed in human tumors, and is required for tumor cell
proliferation.1–3 Specifically, MPS1 kinase activity regulates the
spindle assembly checkpoint (SAC), which in mitosis is responsible
for the correct chromosome segregation, and its inhibition leads to
rapid cell division with massive aneuploidy not compatible with
cell survival.4–7 Selective inhibitors of the target may provide an
innovative therapy for the treatment of tumors and SAC inhibition
which could be a way to selectively target aneuploid tumor prolif-
eration.8,9 We report the synthetic process and the structure–activ-
ity relationship that led from the initial hit (1) to the identification
of a potent, orally available and highly selective MPS1 inhibitor,
which shows tumor growth inhibition in xenograft mouse
models.10
6
5
7 N
8
4
O
3
HN
4'
N
R2
R1
2'
N
N
N2
H
1
R3
Figure 1. 4,5-Dihydro-pyrazolo[4,3-h]quinazoline-3-carboxamide core.
previously prepared and reported as a potent inhibitor of other cell
cycle kinases (Aur-A, CDK2/A and PLK1)11,12 with moderate anti-
proliferative activity against the ovarian cancer cell line A2780
The tricyclic core ring system of 4,5-dihydro-pyrazolo[4,3-h]-
quinazoline (Fig. 1) emerged as one of the most promising
scaffolds from our HTS campaign for the identification of MPS1
inhibitors. This core was previously identified as an inhibitor of
other cell cycle kinases like Aur-A, CDK211 and PLK1.12 Thus, our
initial efforts were directed at gaining selectivity for MPS1 versus
other kinases that had shown high affinity for this chemical class.
According to its enzymatic activity against MPS1 (IC50: 0.500 lM),
compound 1 (Table 1) was considered a reasonable starting point
to undertake a medicinal chemistry program. Compound 1 was
(IC50: 0.500 lM) indicating its cell permeability.
The envisioned optimization strategy was first to modify the dif-
ferent parts of the template independently and then to combine the
best substituents into a single compound that would possess an im-
proved overall profile. The template explorations were mainly fo-
cused on the ortho-residue (R1), on the solubilizing portion (R3)
on the phenyl ring and on the amide (R2) of the core (Fig. 1).
The synthesis of all the compounds was adapted from previ-
ously described methods11,12 and is outlined in Schemes 1 and 2.
Enaminone 26, iodoquinazoline 27 and aminoquinazoline 28 were
prepared according to already reported methods11,12 starting from
commercially available 1,2-cyclohexanedione. Compound 26 was
condensed with phenylguanidine in DMF to form the desired
⇑
Corresponding author. Tel.: +39 331 581551; fax: +39 331 581347.
E-mail address: marina.caldarelli@nervianoms.com (M. Caldarelli).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.122

4508
M. Caldarelli et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4507–4511
Table 1
Results for compounds 1–11
N
O
N
HN
N
O
N
H
N
F
F
O
N
HN
N
NH2
N
N
F
O
NH
N
15
1
Initial hit
NMS-P715
R1
R2
IC50
(lM) MPS1
IC50
(l
M) Aur-A
IC50
(l
M) CDK2/A
IC50 (lM) PLK1
a
a
a
a
Compd
1
2
3
4
5
6
7
8
9
–H
–H
–H
–H
–H
0.500
0.241
0.066
1.218
0.530
2.170
>10
>10
>10
0.525
0.823
0.050
0.605
0.607
>10
0.152
0.312
>10
0.374
0.677
3.168
>10
0.002
0.003
0.090
>10
0.068
0.015
0.042
0.117
>10
4.215
>10
>10
–CH3
–OCH3
–OCF3
–H
–H
–H
–H
–H
OCH3
OCF3
2,6-Diethylphenyl
-CH3
Phenyl
>10
0.019
0.574
1.053
0.697
>10
Benzyl
2-Phenylethyl
2,6-Diethylphenyl
2,6-Diethylphenyl
>10
>10
>10
10
11
>10
a
Values are means of two or more experiments, standard deviation is <20%.
O
N
O
a
O
OH
O
N
N
O
N
O
N
N
R1
N
N
26
N
N
c
N
N
R1
N
H
O
29 R1=H
32 R1=H
NH
30 R1=OCH3
31 R1=OCF3
33 R1=OCH3
O
34 R1=OCF3
N
I
N
N
b
d
27
O
R2
1
3
4
R1=H
R2=H
R2=H
N
H
R1=OCH3
R1=OCF3
R1
R2=H
N
N
N
5
R1=H
R1=H
R2=2,6-Diethylphenyl
R2=CH3
N
6
N
H
7
8
9
R1=H
R1=H
R1=H
R2=Phenyl
R2=Benzyl
R2=2-Phenylethyl
R2=2,6-Diethylphenyl
10 R1=OCH3
11 R1=OCF3
R2=2,6-Diethylphenyl
Scheme 1. Synthesis of final compounds 1 and 3–11. Reagents and conditions: (a) phenylguanidine carbonate, DMF, 110 °C, 72%; (b) anilines, Pd(OAc)2, ( )-BINAP, K2CO3,
DMF 80 °C, 48–54%, (c) KOH/EtOH, reflux, quant.; (d) amines, EDC, HOBt, DMF, DIPEA, rt, 60–93%.
pyrimidine 29 (Scheme 1). Alternatively, different aromatic rings at
position 8 of the scaffold were inserted by Buchwald–Hartwig cou-
pling between iodo-intermediate 27 and substituted anilines using
Pd(OAc)2 and ( )-BINAP. Final amides 1 and 3–11 were obtained by
hydrolysis of ethyl esters with KOH in EtOH to give acids 32–34,
followed by condensation with amines in the presence of HOBt
and EDC in DMF. Compound 2 was prepared from iodoamide-qui-
nazoline as previously described.12 Para-substituted amides (R3,
Fig. 1) were prepared starting from intermediate 28 (Scheme 2)
which was hydrolyzed to the corresponding acid 35 followed by
amidation using 2,6-diethylaniline, HOBt and EDC in DMF to give
36. Alternatively, compound 36 was obtained by ester aminolysis
using 2,6-diethylaniline and NaN(TMS)2 in THF.13 4-t-Butoxycar-
bonylaryl moieties were inserted by Buchwald–Hartwig coupling
on quinazoline 36 with the suitable iodo-derivatives 45–46 in
the presence of Pd2(dba)3 and X-Phos in dioxane. Finally, t-butyl
esters 37–38 were hydrolyzed to acids 39–40, which were subse-
quently converted, in the presence of amine, TBTU and DIPEA in
DMF, to the corresponding final compounds 12–25 (Scheme 2).
The iodo-intermediates 45–46 were in turn prepared from com-
mercially available anilines 41–42 using KI and NaNO2, and then
protecting the carboxylic group of 43–44 under standard condi-
tions (Scheme 3).
We observed the effect of the ortho-substitution (R1, Fig. 1) on
the phenyl ring (Table 1) and observed that, while the methyl-
group (2) decreased the activity on Aur-A only, the methoxy-group
(3) provided an increased inhibitory efficacy towards MPS1 if com-
pared to CDK2/A and Aur-A but still maintained activity against

M. Caldarelli et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4507–4511
4509
O
O
N
H
O
O
OEt
N
H
OH
N
N
N
N
N
N
e
f
h
N
HN
N
N
R1
N
37 R1=OCH3
38 R1=OCF3
N
N
N
N
N
N
28
35
36
NH2
NH2
NH2
g
O
O
i
O
O
N
N
H
H
N
N
N
N
N
N
N
N
HN
HN
R1
R1
l
39 R1=OCH3
40 R1=OCF3
12-25
R3
OH
O
Scheme 2. Synthesis of final compounds 12–25. Reagents and conditions: (e) KOH/EtOH, reflux, quant.; (f) 2,6-diethylaniline, EDC, HOBt, DMF, DIPEA, rt, 70%; (g) 2,6-
diethylaniline, NaN(TMS)2, THF, 0 °C, 80%; (h) 45–46%, Pd2(dba)3, X-Phos, Cs2CO3, dioxane, 90 °C, 85–88%; (i) TFA, DMF, rt, quant.; (l) amines, TBTU, DMF, DIPEA, rt, 72–88%.
NH2
I
I
0.541 and 2.858
low solubility of at <1
l
M, respectively), but in both cases displayed
M.
R1
R1
R1
l
m
n
In order to improve solubility, we then started an exploration of
solubilizing groups both on the o-methoxy and on the o-trifluoro-
methoxy series. Although the crystal structure of an inhibitor
bound to MPS1 was not available during these investigations, we
assumed (as was later confirmed),10 a similar binding mode and
orientation in the ATP pocket to the one observed for other
kinases.12,13 For this reason, we explored the solvent accessible re-
gion introducing several solubilizing groups at the 40-position of
the phenyl ring in position 8 of the tricyclic scaffold.
COOH
COOH
COOtBu
41 R1=OCH3
42 R1=OCF3
43 R1=OCH3
44 R1=OCF3
45 R1=OCH3
46 R1=OCF3
Scheme 3. Synthesis of intermediates 45 and 46. Reagents and conditions: (m) KI,
NaNO2, HCl, 0 °C, 46–75%; (n) Boc2O, t-BuOH, DMAP, DCM, reflux, 62–91%.
PLK1. Replacement of the methoxy- with trifluoromethoxy-substi-
tuent (4) instead, abolished the activity on Aur-A and CDK2/A but
also the inhibitory effect towards PLK1 and MPS1 was reduced (Ta-
ble 1).
The introduction of primary amides at position 40 (R3, Fig. 1, 12–
13) increased the activity on the target while maintaining selectiv-
ity versus other kinases. All the other compounds described in
Table 2 (both secondary and tertiary amides) were active in the
range of 20–400 nM. A completely selective profile was observed
for compounds bearing the trifluoromethoxy-group (13, 15, 17,
19, 21, 23 and 25), as opposed to the corresponding methoxy-ana-
logs (12, 14, 16, 18, 20, 22 and 24). The presence of an asymmetric
center on the amide portion had no impact on activity against
MPS1 (18 vs 20 and 19 vs 21). The influence on solubility at pH
7 was not predictable, but methoxy-derivatives were, in general,
more soluble than the corresponding trifluoromethoxy com-
pounds, except for 14 and 15. Moreover, compound 15 proved to
be highly selective against a panel of more than 60 kinases.10
Compounds that showed reasonable potency against MPS1,
selectivity and cell growth inhibition against a human tumor cell
line (A2780), were then selected for further evaluation and tested
in mechanism of action studies (MoA). Nocodazole arrested U2OS
osteosarcoma cells were then treated, confirming the capability
of our MPS1 inhibitors to promote mitotic override10 (Table 2).
Among the best compounds which showed, together with solubil-
ity, complete selectivity, good cellular activity and MoA, 15 and 25
were selected and further analyzed in preliminary pharmacoki-
netic (PK) experiments (Table 3). While compound 25 showed a
We then analyzed another portion of the molecule and ob-
served how the modulation of the amide in position 3 of the core
(R2, Fig. 1) influenced both potency and selectivity. The introduc-
tion of 2,6-diethylphenylamide group on the scaffold resulted in
a significant improvement in selectivity. Specifically compound 5,
was equipotent as 1 against MPS1, but was completely inactive
on CDK2/A and PLK1 with a threefold lower activity against
Aur-A than 1. By contrast, replacement of the primary amide with
other secondary alkyl amides did not show the same effect. For
example, the introduction of a methyl (6) decreased the activity
about 2–4-fold on all kinases except for CDK2/A. Other groups such
as phenyl (7), benzyl (8) and phenylethyl (9) were instead not
active on MPS1.
Having found favorable substitution elements for both the
amide and the phenyl ring system, we next combined the best
modifications and verified their compatibility. Compounds 10
and 11 (Table 1) were both active on the target and highly selective
towards our target kinase MPS1. In spite of compound 10 being
more potent than 11, the trifluoromethoxy moiety gave a cleaner
profile versus the kinases analyzed. In addition, compound 10
and 11 showed moderate cellular activity against A2780 (IC50
:
high iv clearance and a low oral exposure, compound 15

4510
M. Caldarelli et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4507–4511
Table 2
Results for compounds 12–25
O
N
H
R1
N
N
N
N
R3
N
H
Compd R1
R3
IC50
(l
M)
IC50
(l
M)
IC50
(
l
M)
IC50
PLK1
(lM)
IC50
(
lM)
IC50
MoA
(lM)
Solubility (
pH7
lM)
a
a
a
a
a
a
MPS1
Aur-A
CDK2/A
A2780
12
13
OCH3 –CONH2
OCF3
0.036
0.140
0.768
>10
5.568
>10
3.034
>10
0.135
0.298
0.004
0.16
<1
1
14
15
OCH3
OCF3
O
0.084
0.182
1.450
>10
>10
>10
0.237
>10
0.150
1.000
0.016
0.032
3
45
N
H
N
16
17
OCH3
OCF3
0.033
0.130
0.750
1.376
>10
>10
1.652
>10
0.101
0.771
0.012
0.11
44
27
O
N
N
H
18
19
OCH3
OCF3
0.103
1.376
>10
>10
>10
>10
>10
0.083
0.422
0.011
0.21
110
65
O
0.391b
N
N
20
21
OCH3
OCF3
O
0.072
0.491
>10
>10
>10
>10
>10
0.073
0.407
0.013
0.26
96
69
0.202b
N
N
22
23
OCH3
OCF3
O
O
0.019
0.206
0.484
>10
>10
>10
>10
>10
0.083
0.485
0.002
0.040
116
70
N
N
N
24
25
OCH3
OCF3
0.021
0.146
0.372
>10
3.625
>10
>10
>10
<0.016
0.244
0.002
0.032
59
46
N
a
Values are means of two or more experiments, standard deviation is <20%.
Single data.
b
Table 3
PK parameters for compounds 15 and 25a
Compd
In vivo PK (mouse) 10 mg/Kg iv
CL (mL/min/kg) AUC ( M h)
In vivo PK (mouse) 10 mg/Kg os
Cmax M) AUC ( M h)
t1/2 (h)
l
Vss (L/Kg)
t1/2 (h)
(
l
l
F (%)
15
25
7.65 0.83
1.61 0.14
10.0 0.24
77.5 17.8
22.4 0.81
2.9 0.72
5.45 0.55
8.41 1.42
8.01 1.32
2.92 0.89
0.600 0.150
0.245 0.12
8.66 2.09
0.64 0.25
37.9
22.4
a
n = 3 animals per study.
(NMS-P715) showed a good half life value and oral bioavailability.
On the basis of its favorable PK profile, 15 was therefore selected
for in vivo efficacy studies in mice bearing A2780 tumor xenograft.
When dosed per os, daily at 90 mg/Kg for seven consecutive days,
at the end of the treatment it showed 53% tumor growth inhibition
with no sign of body weight loss.10
In summary, a series of 4,5-dihydro-[4,3-h]quinazoline-3-car-
boxamides was synthesized and evaluated as MPS1 inhibitors.
Compound 11 was found to be a selective inhibitor with an IC50
of 820 nM. Introduction of a solubilizing group on the core scaffold
through an amide linkage not only improved the solubility but also
significantly increased the enzymatic inhibitory potency and the
cellular activity against A2780 cell line. Among the compounds
prepared, 15 (NMS-P715) was found to have favorable pharmaco-
kinetic parameters, good potency and selectivity profile. On the ba-
sis of the above results, compound 15 was selected for efficacy
studies and for the first time, the in vivo antitumor activity of an
MPS1 inhibitor was demonstrated. These results support the use
of selective MPS1 inhibitors for cancer therapy.
Acknowledgments
We thank Jürgen Moll for useful discussion, Daniele Pezzetta
(Accelera) for PK characterization, the group of Assay Development
and Biochemical Screening for the biochemical assay on kinase pa-
nel, the group of Cell Screening for cell proliferation, the Experi-
mental Therapy group for the in vivo experiment, Gianluca Papeo
for critical reading and Susan Watts for revision of the manuscript.
Supplementary data
Supplementary data (experimental methods and characteriza-
tion of compounds) associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.05.122.

M. Caldarelli et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4507–4511
4511
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