Discovery of 4-(4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)benzamides as novel, highly potent and selective, orally bioavailable inhibitors of Tyrosine Threonine Kinase, TTK

Article abstract of DOI:10.1016/j.bmcl.2016.06.021

TTK/Mps1 is a key kinase controlling progression of cell division via participation in the mitotic spindle assembly checkpoint and is overexpressed in a number of human cancers. Herein we report the discovery of 4-(4-aminopyrazolo[1,5-a][1,3,5]triazin-8-y

Full text of DOI:10.1016/j.bmcl.2016.06.021

Accepted Manuscript
Discovery of 4-(4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)benzamides as Nov-
el, highly potent and selective, orally bioavailable inhibitors of Tyrosine Threo-
nine Kinase, TTK
Radoslaw Laufer, Sze-Wan Li, Yong Liu, Grace Ng, Yunhui Lang, Miklos
Feher, Richard Brokx, Irina Beletskaya, Richard Hodgson, Guodong Mao, Olga
Plotnikova, Donald E. Awrey, Jacqueline M. Mason, Xin Wei, Dan Chi-Chia
Lin, Yi Che, Reza Kiarash, Brian Madeira, Graham C. Fletcher, Tak W. Mak,
Mark R. Bray, Henry W. Pauls
PII:
S0960-894X(16)30631-X
http://dx.doi.org/10.1016/j.bmcl.2016.06.021
BMCL 23974
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 April 2016
31 May 2016
8 June 2016
Please cite this article as: Laufer, R., Li, S-W., Liu, Y., Ng, G., Lang, Y., Feher, M., Brokx, R., Beletskaya, I.,
Hodgson, R., Mao, G., Plotnikova, O., Awrey, D.E., Mason, J.M., Wei, X., Lin, D.C-C., Che, Y., Kiarash, R.,
Madeira, B., Fletcher, G.C., Mak, T.W., Bray, M.R., Pauls, H.W., Discovery of 4-(4-aminopyrazolo[1,5-a]
[1,3,5]triazin-8-yl)benzamides as Novel, highly potent and selective, orally bioavailable inhibitors of Tyrosine
Threonine Kinase, TTK, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.
2016.06.021
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Bioorganic & Medicinal Chemistry Letters
Discovery of 4-(4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)benzamides as Novel,
highly potent and selective, orally bioavailable inhibitors of Tyrosine Threonine
Kinase, TTK.
Radoslaw Laufer,a* Sze-Wan Li,a Yong Liu,a Grace Ng,a Yunhui Lang,a Miklos Feher,a Richard Brokx,a
Irina Beletskaya,a Richard Hodgson,a Guodong Mao,a Olga Plotnikova,a Donald E. Awrey,a Jacqueline M.
Mason,a Xin Wei,a Dan Chi-Chia Lin,a Yi Che,a Reza Kiarash,a Brian Madeira,a Graham C. Fletcher,a Tak
W. Mak,a Mark R. Bray,a and Henry W. Paulsa
a
Campbell Family Institute for Breast Cancer Research, University Health Network, TMDT East Tower, MaRS Centre, 101 College Street, Toronto, Ontario,
M5G 1L7, Canada
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
TTK/Mps1 is a key kinase controlling progression of cell division via participation in the mitotic
spindle assembly checkpoint and is overexpressed in a number of human cancers. Herein we
report the discovery of 4-(4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)benzamides as a potent,
novel class of TTK inhibitors. The series was identified by means of bioisosteric replacement of
the related imidazopyrazine and imidazopyridazine scaffolds. Optimization led to the
identification of compounds with excellent potency (Ki = 0.8 nM) and exceptional kinase
selectivity. The SAR indicates a strong dependence of activity on the presence of the N-
cyclopropyl-2-methylbenzamide moiety delineating the geometry for 1½ type kinase inhibitor.
Molecular modeling indicates the extensive and optimal contacts, mediated through H-bonds
and hydrophobic interactions, are responsible for the selectivity and potency of the inhibitors.
The compounds demonstrate a strong anti-proliferative activity in a panel of human cancer cell
lines (HCT116 GI50 < 15 nM) and good rodent pharmacokinetics (oral %F 97 %)
Keywords:
Tyrosine Threonine Kinase (TTK)
Monopolar Spindle 1 Kinase (Mps1)
Antimitotic
Inhibitor
Cancer
2009 Elsevier Ltd. All rights reserved.
———
* Corresponding author. Tel.: +1 647-260-7962; fax: +1-416-977-3000; e-mail: Radek.Laufer@oicr.on.ca

to the catalytic Lys553 and the cyclopropyl group engages in
hydrophobic interactions with Met602 and the Lys553 side
Tyrosine Threonine Kinase (TTK, also known as Monopolar
spindle protein 1, Mps1) is a conserved dual-specificity
kinase1 essential for proper distribution of chromosomes to
daughter cells during mitosis via the spindle assembly
checkpoint (SAC).2,3 Elevated levels of TTK contribute to
abnormal number of chromosomes, a trait referred to as
aneuploidy. This characteristics is frequently present in solid
tumors4 and has been identified as predictor of poor prognosis
in a number of cancers.5-8 Furthermore, overexpression of
TTK, common in a variety of tumors,9-13 is correlated with
high histological grade, poor patient prognosis and may
promote initiation, survival of genomically unstable and
aneuploid cancer cells.9 Inhibition of the kinase activity
causes severe chromosome segregation defects that lead to
chain.
Hence, based on the fact that N-cyclopropyl-
benzamide formulates type 1½ binding in a restricted back
pocket,24 selective TTK inhibitors were anticipated.
Chart 2. Scaffold hopping exercise yields pyrazolo[1,5-a]triazines
cancer cell death.
tumorigenesis, we and other groups have been actively
pursuing TTK inhibitors for the treatment of cancer.
number of potent TTK inhibitors have been reported in the
literature (Chart 1) and recently the first two examples have
entered clinical trials.14-18
Recognizing the role of TTK in
A
The 4-(4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)benzamide
scaffold, although not previously described, was deemed to be
accessible from a synthetic standpoint. We demonstrated that
the desired molecules could be synthesized in a highly
convergent sequence starting from 8-bromo-4-chloro-2-
We have previously disclosed our discovery path to CFI-
401870, a small molecule, indazole-based TTK inhibitor that
was selected as a preclinical development candidate (Chart
1).15,19 Having completed our work on the indazole series, we
turned our attention to the development of a backup series.
We considered a diverse set of scaffolds but were particularity
drawn to imidazopyrazines 2 and imidazopyridazine 3
originally disclosed in patent applications by Bayer AG and
Oncotherapy Science, Inc. (Charts 2).20,21 We recognized that
these bicyclic ring systems represented relatively unexplored
templates for the presentation of TTK binding elements.16,22
(methylthio)pyrazolo[1,5-a]-[1,3,5]triazine
5 (Scheme 1).
This highly functionalized core was subjected to three key
steps: two SNAr transformations and one cross-coupling
reaction. More specifically, a SNAr displacement of 4-Cl in 5
with amine 6 was followed by a Suzuki-Miyaura cross
coupling at C8 position of bicycle 8.25
Scheme 1. Synthesis of pyrazolo[1,5-a][1,3,5]triazines
Chart 1. Selected examples of published TTK inhibitors
a. DCM, DIPEA, 0 °C; b. if Z = H then PMBCl, K2CO3, DMF; c. ArBpin
(12), Pd(dppf)Cl2•CH2Cl2, K3PO4, H2O, THF, Δ, μw; d. TFA, DCM; e.
mCPBA, DCM; f. R’R”NH, THF, 35 °C (Hb = R’R”N) or Ar'NH2, NMP,
Δ (Hb = Ar’NH) or Ar’OH, DBU, DME, Δ (Hb = Ar’O) or R’OH, NaH,
DMF/THF 0 °C to rt (Hb = R’O); g. TBTU, DIPEA, RNH2 (for Y =
CO2H) or RCO2H (for Y = NH2); h. Pd(dppf)Cl2•CH2Cl2, B2Pin2, KOAc,
DMF, Δ
Deprotection of the PMB group and an oxidation of 2-
methylthio group in 9 to the corresponding sulfone 10
typically preceded the ultimate SNAr event leading to fully
functionalized inhibitors (4,13-33). Introduction of PMB
group was required to enable the Suzuki-Miyaura cross
coupling of intermediates 8 and 12. Most expeditiously the
protecting group was incorporated in the amine 6. The
required boronate esters 12 were straightforwardly synthesized
from aniline or benzoic acid 11 in an amide coupling followed
by a Miyaura borylation step.
We selected the pyrazolo[1,5-a][1,3,5]triazin-4-amine scaffold
4 (Chart 2) following analysis of molecular docking results
and calculated physicochemical properties of a large set of
bioisosteric rings. Based on molecular docking of 4 into the
active site of TTK23 (Figure 1), multiple hydrogen bond
interactions are predicted between the ligand and the enzyme:
two bonds to the hinge residue Gly605 and two to the solvent
accessible Asp608 and Thr606. The ligand is also expected to
participate in a number of hydrophobic interactions with the
amino acid residue side chains: Val539/Ile663, Pro673 and
Leu654/Ile531 engage in contacts with the phenyl, cyclopentyl
and triazine rings, respectively.
We synthesized 4 as a proof of concept inhibitor and were
gratified to find that it inhibited TTK at IC50 of 5.8 nM with
moderate antiproliferative effects in cell culture (Table 1).
Based on the binding model, the subsequent lead optimization
process focused on three binding vectors: 1. C2-hydrophobic
Most intriguing, is the role of the N-cyclopropyl-2-methyl-
benzamide moiety. This group inserts deep into the back
pocket of the TTK active site; the carbonyl makes an H-bond

substitution extending into kinase ribose biding subpocket, 2.
C8 phenylcarboxamide, reaching the kinase catalytic site, and
3. C4-group extending from the hinge region to the protein-
solvent interface (Chart 2).
significantly less active than their corresponding benzamide
isomers 19 (eightfold) and 4 (350-fold). A loss of an order of
magnitude in activity is observed by replacing the cyclopropyl
ring in 21 with an isopropyl group, 19. All together, these
findings are consistent with our binding model i.e. two
favorable hydrogen bonds to the amide and the existence of a
narrow subpocket accommodating the small c-propyl ring.
Gly605
Thr606
An increase in activity was observed with aromatic rings at
C2, as demonstrated by compounds 23-25. This trio also
illustrates the role of the ortho-substituents (R2) of the C8-
benzene ring. A threefold increase in potency for chloro 24
and methyl 25 over the proto congeners 21 and 23 is seen.
Notwithstanding similar IC50 values for 24 and 25, the ortho
methyl substituted 25 demonstrated greater antiproliferative
effects in the panel of human cancer cell lines (Table 1).
Asp608
Met602
The benefit of introducing an aromatic ring at C2 is limited
only to ethers as illustrated by a matched pair of C2-aniline 26
and phenoxy 25. In addition, an attempt to reduce the
hydrophobic character of the C2 arylether resulted in a
decrease in inhibition; for example, 3-acetamidophenyloxy
(27) was about an order of magnitude less active than the
corresponding phenoxy 25. Cell activity was more profoundly
affected. In contrast, introduction of fluorine substituents on
the phenyl ethers afforded compounds with subnanomolar
Lys553
Figure 1. Glide XP-predicted docking pose of compound 4 in a
crystal structure of TTK (PDB code: 4O6L)
For the C2-substituent, nitrogen could be replaced with
oxygen without a loss of activity as demonstrated by 13.
Expansion to six-membered rings was also tolerated, as in
examples 14-15, but without a further improvement over 4. A
conservative increase in polarity was acceptable as for
example, in tetrahydro-2H-pyran-4-amine 14. Further
substitution of the linking C2-nitrogen was detrimental (e.g.
morpholine 17 and N-methyl 16).
activity (28-30).
However, their measured IC50s must be
viewed as apparent values because they approach the nominal
concentration of enzyme in the assay.
The role of the terminal carboxamide (R3) is illustrated by
comparing compounds 4 and 18-21 (Table 1). Of the two
possible amide connectivities, carboxamides 18 and 20 are

Table 1. In vitro inhibitory activity of pyrazolo[1,5-a][1,3,5]triazin-8-ylbenzamides 10.
Cancer Cell GI50 (µM)
cpd
R1
R2
R3
Hb
TTK IC50 (µM)
MDA-MB 231
HCT116
0.13
OVCAR-3
0.48
4-THP-CH2−*
4-THP-CH2−*
4-THP-CH2−*
4-THP-CH2−*
4-THP-CH2−*
4-THP-CH2−*
4-THP-CH2−*
4-THP-CH2−*
4-THP-CH2−*
4-THP-CH2−*
4-THP-CH2−*
Me
Me
Me
Me
F
(C=O)NHc-Pr
(C=O)NHc-Pr
(C=O)NHc-Pr
(C=O)NHc-Pr
(C=O)NHc-Pr
(C=O)NHc-Pr
NH(C=O)i-Pr
(C=O)NHi-Pr
NH(C=O)c-Pr
(C=O)NHc-Pr
(C=O)NHc-Pr
0.0058
0.0023
0.009
0.0026
0.015
0.032
1.7
0.17
0.23
0.54
0.27
ND
4
0.16
0.28
0.17
ND
0.59
0.78
0.47
ND
0.07
ND
ND
ND
1.7
13
14
15
16
17
18
19
20
21
22
Cl
H
0.45
ND
0.44
ND
H
0.23
ND
ND
Me
H
2.0
ND
ND
0.010
0.007
0.69
1.4
0.39
0.85
F
1.7
4-THP-CH2−*
4-THP-CH2−*
4-THP-CH2−*
4-THP-CH2−*
H
(C=O)NHc-Pr
(C=O)NHc-Pr
(C=O)NHc-Pr
(C=O)NHc-Pr
PhO
PhO
PhO
0.0049
0.0017
0.0014
0.011
0.35
0.23
0.032
1.2
0.20
0.16
0.91
0.40
0.19
1.7
23
24
25
26
Cl
Me
Me
0.013
0.69
PhNH
4-THP-CH2−*
4-THP-CH2−*
Me
Me
(C=O)NHc-Pr
(C=O)NHc-Pr
0.0084
0.0004
1.6
1.3
1.2
27
28
0.012
0.009
0.037
4-THP-CH2−*
4-THP-CH2−*
*
Me
Me
(C=O)NHc-Pr
(C=O)NHc-Pr
0.0007
0.016
0.047
0.009
0.021
0.058
0.19
29
30
0.00094
H
H
(C=O)NHc-Pr
(C=O)NHc-Pr
PhO
PhO
0.014
0.024
ND
ND
ND
31
32
0.96
0.88
0.41
*
*
Me
(C=O)NHc-Pr
0.0022
0.040
0.014
0.17
33
phenyl 28 and meta monofluorinated compound 29 were
equipotent against TTK and cancer cells. However of the two
inhibitors, 29 was significantly more active against CYP 3A4
We noted that several properties were sensitive to subtle
changes to the position of fluorine on the ring. The difluoro

(Table 2). By contrast the ortho monofluorinated compound
30 was not a potent inhibitor of CYP3A4. This advantage also
held in comparison to the des fluoro analog 25.
A kinetic analysis of TTK inhibitor 25 confirmed the potency
(Ki = 0.77 ± 0.5 nM) and ATP competitive nature of the
binding.25 Compound 25 was also tested against a panel of
protein kinases and, as illustrated by the heat map in Figure 2,
did not inhibit any of the fifty five targets at concentration of 1
µM.25 In the dose response assays, the compound exhibited
IC50 greater than 50 µM against PLK4, AURKA,
AURKB/INCENP. Based on a cell cycle analysis, we
concluded that compound 25 caused massive aneuploidy and
cell death, at concentrations between 100 and 200 nM (Figure
3). The compound exhibited low to moderate levels of
inhibition of Cyp450 isoforms (Table 2) with over three orders
of magnitude separation over TTK activity.
Table 2. ADME and pharmacokinetic properties of
pyrazolo[1,5-a][1,3,5]triazin-8-ylbenzamides.
mouse PK (10 mg/kg)
CYP 3A4
IC50 (μM)
microsomal T1/2
(min) m/r/h
entry
AUC
Cmax (μg/mL)
(μg•h/mL)
19
0.8
14/11/>60
46/>60/>60
28/40/>60
30/39/>60
17/43/>60
0.43
0.89
1.60
ND
0.53
1.53
4.0
4
25
28
29
30
>20
0.47
9.3
ND
1.9
0.61
For groups at C4 (R1), the vector projects towards the protein-
solvent interface, this region was not extensively explored.
Nonetheless the cyclic N-((tetrahydro-2H-pyran-4-yl)methyl)
was preferred over its acyclic variant (e.g. 31 vs 23, Table 1).
For basic solubilizing groups (e.g. morpholinoethyl) over an
order of magnitude loss in TTK inhibition was observed
(compound 32). Activity could be recouped in this case by
using difluorophenoxy ring as the C-2 hydrophobe, as in entry
33.
Figure 3. Cell cycle analysis of HCT116 colon cancer cells treated
with compound 25 at given concentrations. Percentage of 8 N and 16 N
cells, at 48 h, normalized to DMSO control.
In summary, a scaffold hopping exercise led to the
discovery of a new class of TTK inhibitor, namely 4-(4-
aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)benzamides. The lead
optimization of this novel scaffold resulted in preparation of
very potent and strikingly selective molecules with good oral
exposure in rodents. The strength of these results set stage to
our work on related pyrazolo[1,5-a]pyrimidines, culminating
in the identification of a development candidate, CFI-
402257.27
A subset of TTK inhibitors (4, 25, 29, 30 and 33) were tested
against additional human cancer cell lines from colon, ovary
and breast (Table 3). The results show that these molecules
were particularly effective against HT-29 and PA-1 and less
active against MDA-MB-468. Nonetheless these data indicate
a general applicability of this class of inhibitors against a
broader spectrum of cancers.
Supplementary Material
Table 3. Cancer cell growth inhibition (GI50) for selected
compounds
Synthetic, analytical methods and data are provided.
Inhibition data of 55-member kinase panel for compounds 25.
Steady State kinetics results for compound 25. This material is
available free of charge via the Internet at http://....
GI50 (µM)
4
25
29
30
33
colon HT-29
0.084 0.015
0.053 0.030
0.007
0.020
0.021
ovarian PA-1
0.006
0.23
0.017
0.12
0.017
0.15
Funding Sources
breast MDA-MB-468
0.32
0.16
The Canadian Institutes of Health Research and the Princess
Margaret Cancer Foundation are acknowledged for financial
support.
The pharmacokinetic properties of selected compounds were
assessed in rodents. In mouse, compound 25 as well as other
phenyl ethers (25, 28, 30, Table 2) displayed high oral
exposure. In the rat, high bioavailability (97 %), low
clearance (19 mL/(min.kg)) and moderate half-life (1.5 h)
were observed for compound 25. Compound 25 displayed
relatively high plasma protein binding (98.4 % in mouse, 97.2
% in human). It should be noted that the linear dose-AUC
dependence for 25 only extended to 70 mg/kg effectively
thwarting our efforts to establish the maximum tolerated dose,
a prerequisite to the in vivo efficacy studies.26
Acknowledgments
We thank Dr. Homer Pearce for helpful discussion during the
course of the work.
Abbreviations
Ar, aryl; ATP, Adenosine-5'-triphosphate; AUC, area under the
curve; BA, bioavailability; CYP, Cytochrome P450; DBU,
1,8-diazabicyclo[5.4.0]undec-7-ene; DCM, dichloromethane;
DIPEA, diisopropylethylamine;
Δ
heat; DME, 1,2-
dimethoxyethane; DMF, N,N-dimethylformamide; DMSO,
dimethyl sulfoxide; dppf, 1,1'-bis(diphenylphosphino)
ferrocene; h, hour or human (in MS); GI50, half maximal cell
growth inhibitory concentration; Hb, hydrophobe; IC50, half
maximal inhibitory concentration; m, mouse; mCPBA, meta-
chloroperoxybenzoic acid; µw, microwave irradiation; Mps1,
Monopolar spindle protein 1; ND, not determined; PDB,
Figure 2. Heat map showing % inhibition of 55 human kinases at a
screening concentration of 1 µM for compound 25 (bottom row) in
comparison to 2 (top). For each kinase [ATP] = Km. Compounds were
individually sorted.

inhibitors with potent anti-tumor activity. AACR Annual
Meeting 2015, 3090.
protein data bank; PK, pharmacokinetics; pin, pinacol; PMB,
para–methoxybenzyl; r, rat; rt, room temperature; SNAr,
Nucleophilic Aromatic Substitution; TBTU, O-(benzotriazol-
1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate; TFA,
trifluoroacetic acid; THF, tetrahydrofuran; THP, tetrahydro-
2H-pyran; TTK, Threonine Tyrosine Kinase.
19. Laufer, R.; Ng, G.; Liu, Y.; Patel, N. K.; Edwards, L.; Lang,
Y.; Li, S.-W.; Feher, M.; Awrey, D.; Leung, G.; Beletskaya, I.;
Plotnikova, O.; Mason, J.; Hodgson, R.; Wei, X.; Mao, G; Luo,
X.; Huang, P.; Green, E.; Kiarash, R.; Lin, D. C. -C.; Harris-
Brandts, M.; Nadeem, V.; Mak, T. W.; Pan, J. G.; Qui, W.;
Chirgadze, N. Y.; Pauls, H. W. Bioorg. Med. Chem. 2014, 22,
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R.; Lienau, P.; Siemeister, G.; Wengner, A. M. PCT. Int. Appl.
WO2012032031, 2012.
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