Discovery of Pyrazolo[1,5-a]pyrimidine TTK Inhibitors: CFI-402257 is a Potent, Selective, Bioavailable Anticancer Agent

Article abstract of DOI:10.1021/acsmedchemlett.5b00485

This work describes a scaffold hopping exercise that begins with known imidazo[1,2-a]pyrazines, briefly explores pyrazolo[1,5-a][1,3,5]triazines, and ultimately yields pyrazolo[1,5-a]pyrimidines as a novel class of potent TTK inhibitors. An X-ray structure of a representative compound is consistent with 1¹/₂ type inhibition and provides structural insight to aid subsequent optimization of in vitro activity and physicochemical and pharmacokinetic properties. Incorporation of polar moieties in the hydrophobic and solvent accessible regions modulates physicochemical properties while maintaining potency. Compounds with enhanced oral exposure were identified for xenograft studies. The work culminates in the identification of a potent (TTK K_i = 0.1 nM), highly selective, orally bioavailable anticancer agent (CFI-402257) for IND enabling studies.

Full text of DOI:10.1021/acsmedchemlett.5b00485

Letter
pubs.acs.org/acsmedchemlett
Discovery of Pyrazolo[1,5‑a]pyrimidine TTK Inhibitors: CFI-402257 is
a Potent, Selective, Bioavailable Anticancer Agent
Yong Liu,† Radoslaw Laufer,† Narendra Kumar Patel,† Grace Ng,† Peter B. Sampson,† Sze-Wan Li,†
Yunhui Lang,† Miklos Feher,† Richard Brokx,† Irina Beletskaya,† Richard Hodgson,† Olga Plotnikova,†
Donald E. Awrey,† Wei Qiu,‡ Nickolay Y. Chirgadze,‡ Jacqueline M. Mason,† Xin Wei,†
Dan Chi-Chia Lin,† Yi Che,† Reza Kiarash,† Graham C. Fletcher,† Tak W. Mak,† Mark R. Bray,†
and Henry W. Pauls*,†
†Campbell Family Institute for Breast Cancer Research, University Health Network, TMDT East Tower, MaRS Centre, 101 College
Street, Toronto, Ontario M5G 1L7, Canada
‡Campbell Family Cancer Research Institute, University Health Network, Princess Margaret Cancer Center, 610 University Avenue,
Toronto, Ontario M5G 2C4, Canada
S
* Supporting Information
ABSTRACT: This work describes a scaffold hopping exercise
that begins with known imidazo[1,2-a]pyrazines, briefly
explores pyrazolo[1,5-a][1,3,5]triazines, and ultimately yields
pyrazolo[1,5-a]pyrimidines as a novel class of potent TTK
inhibitors. An X-ray structure of a representative compound is
consistent with 11/2 type inhibition and provides structural
insight to aid subsequent optimization of in vitro activity and
physicochemical and pharmacokinetic properties. Incorpora-
tion of polar moieties in the hydrophobic and solvent accessible regions modulates physicochemical properties while maintaining
potency. Compounds with enhanced oral exposure were identified for xenograft studies. The work culminates in the
identification of a potent (TTK Ki = 0.1 nM), highly selective, orally bioavailable anticancer agent (CFI-402257) for IND
enabling studies.
KEYWORDS: TTK inhibitors, CFI-402257, pyrazolo[1,5-a]pyrimidines, 11/2 type inhibitors
Chart 1. TTK Inhibitor Scaffold Hopping
hreonine tyrosine kinase (TTK) has been a drug discovery
1,2
Tfocus
in our laboratory since it was identified by an
internal RNAi screen.3 The growth of 7 of 12 breast cancer cell
lines was inhibited, and subsequent soft agar experiments showed
that TTK siRNA inhibits colony size and number. These effects
are associated with a disabled mitotic checkpoint, resulting in
chromosome segregation errors, aneuploidy, and cell death.3 In
vivo, interfering RNA meditates growth suppression of breast
cancer xenografts.3 These results are supported by the fact that
TTK is an essential chromosomal regulator4,5 and is overex-
pressed in aneuploid tumors.6−10 High TTK levels correlate with
a high tumor grade11 and poor patient outcomes.6
The potential of this anticancer target is underscored by the
large number of inhibitors disclosed.1,2,12−18 We pursued
indazole based inhibitors which culminated in the identification
of a preclinical candidate.2 The role of TTK is supported by
proof-of-concept studies wherein inhibitors display cell growth
inhibition in vitro and tumor growth inhibition in vivo.1,2,13−19
Moreover, several laboratories have selected TTK inhibitors for
clinical trials.18,19 Our continued interest in TTK inhibitors led to
a scaffold hopping exercise (Chart 1). The imidazo[1,2-
a]pyrazines, originally disclosed in patent applications,20,21
were the starting point for the pyrazolo[1,5-a][1,3,5]triazines.
These inhibitors possess desirable attributes including
biochemical potency, cell activity, and oral bioavailability (vide
infra). However, rodent pharmacokinetic (PK) data indicates
that certain members suffer from dissolution limiting exposure.
To address this problem a further variation on the theme was
Received: December 14, 2015
Accepted: April 26, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.5b00485
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
examined. Inspired by the imidazo[1,2-b]pyridazines20−22 we
reasoned that a carbon for nitrogen switch at position 3 of the
pyrazolo[1,5-a][1,3,5]-triazines23 would result in potent TTK
inhibitors (Chart 1), i.e., the pyrazolo[1,5-a]pyrimidines
described herein. A secondary benefit could accrue in that the
basicity and solubility (at low pH) of the latter could be increased
relative to the former.
work24 had demonstrated the strict requirement for the N-
cyclopropyl-2-methylbenzamide at position 3 of the imidazo-
[1,2-b]pyridazine inhibitors, ruling out further modifications
here. Therefore, we modulated physicochemical properties by
incorporating polar, basic, solubilizing elements into the
hydrophobic and solvent exposed areas of the compounds.
To this end we applied the strategy of appending a moderately
basic morpholino function at the solvent accessible region.
Compounds 9 and 10 were potent TTK inhibitors, but only the
latter matched the inhibition of cancer cell growth achieved by 7.
Compound 10 was orally bioavailable in mice (Table 1) and
displayed a dose-dependent increase in plasma concentrations;
an MTD of 6.5 mg/kg QD was established.
The synthesis of pyrazolo[1,5-a]pyrimidines (Scheme 1)
begins with 3-bromo-5,7-dichloro-pyrazolo[1,5-a]pyrimidine 3
a
Scheme 1. Synthesis of Pyrazolopyrimidines
In an effort to better understand the TTK binding interactions
of our pyrazolo[1,5-a]pyrimidine inhibitors, a cocrystallized X-
ray structure was obtained with compound 9 (Figure 1). A
unique feature of this structure is a bound water not previously
observed.1,2 The molecule is positioned to interact with NHs of
the activation loop and the side chain of Glu571, but its role is
uncertain in that the angles for the formation of hydrogen bonds
are not ideal. The phenyloxy ring makes extensive hydrophobic
interactions with Pro673 and the side chain of Val539.
The cyclopropylbenzamide carbonyl and NH coordinate with
the side chain of Lys553 and Ile663 carbonyl, respectively. This
indicates a highly structured activation loop and is consistent
with the strict requirement for the cyclopropane amido group in
this region. Two hydrogen bonds from the anilino and pyrazolo
nitrogens of 9 to the hinge (Gly605) are observed. The
morpholino group is in a solvent exposed region suggesting that
polar substituents will be tolerated here.
a
Reagents and conditions: (a) DCM, DIPEA, 0 °C, 84−100%; (b)
Boc2O, TEA, DCM, 40−100%; (c) R1R2NH, DIPEA or ArOH,
K2CO3, 46−100%; (d) N-cyclopropyl-2-methyl-4-(4,4,5,5-tetrameth-
yl-1,3,2-dioxaborolan-2-yl)benzamide, Pd(dppf)Cl2·CH2Cl2, K3PO4,
H2O, THF; (e) TFA, DCE or TFA, DCM, 10−68% over 2 steps.
Further inspection of the X-ray structure indicated that the
“hydrophobic region”, occupied by Z, was bounded by polar
residues. This pocket corresponds to the ribose binding region of
the enzyme, suggesting that polar moieties would be tolerated.
Entries 11−16 in Table 1 explore the effect of introducing
hydroxy alkyl amino hydrophobes. Compound 11 was
significantly less potent than 8, but the SAR predicted that we
could increase potency by incorporating additional hydrophobic
bulk. This expectation was realized (compounds 12−14) and
optimized by judicious selection of stereochemistry (cf. 13 and
14). Further variations on this theme yielded inhibitors 15 and
16 with equivalent enzymatic potency. However, the former
suffered from reduced cell activity and oral exposure.
and proceeds by sequential displacement of halides. Intermediate
5 is obtained by nucleophilic aromatic substitution by amine 4 at
the 7-position followed by protection of the nitrogen, as
required. Subsequent displacement at the 5-position is affected
by amines or aryloxy anions to give secondary anilines or ethers,
respectively. Intermediate 6 is subjected to Suzuki−Miyaura
coupling and deprotection to yield the desired products 7−24.
A third approach to modulating physicochemical properties
while maintaining in vitro potency and bioavailability was
pursued. Hydroxyl functions (e.g., cyclohexanol) are added to
the solvent accessible region of the molecule. Simultaneously, a
weak base (pyridyl) is incorporated into the aromatic hydro-
phobe. Both stereoisomers, 17 and 18, are potent TTK inhibitors
but only the cis isomer 17 demonstrated desirable cell activity
and oral exposure. Unfortunately 17 was rapidly converted to the
trans isomer in vivo.
Isomerization was not seen in the cyclobutane analogues 19
and 20 but at the cost of significantly lower oral exposure. We
postulated that oral bioavailability would be increased by
morphing the hydroxyl cyclobutanes to tertiary alcohols as is
found in compounds 21−24. In fact, these compounds are
potent inhibitors of TTK; the cis congeners 23 and 24 proved to
be the most orally bioavailable in mice. The latter also had the
best activity against our sentinel panel of cancer cell lines.
Head to head, the solubilities of the hydroxyl containing
compounds, at physiologic pH, were not higher than their
tetrahydropyranyl counterparts. However, with the incorpo-
Figure 1. TTK/compound 9 cocrystallized X-ray structure (PDB:
4ZEG) with intermolecular H-bonds indicated.
Pyrazolopyrimidines 7 and 8 were compared to their
respective pyrazolotriazine congeners 1 and 2 (Table 1). The
former pair was comparable to the latter in potency (IC50) with
much improved cell activity (GI50) and higher oral exposure in
mice. Nonetheless, it appears that the bioavailability of
compound 7 is dissolution limited as we were unable to achieve
plasma levels high enough to reach a maximum tolerated dose
(MTD). Accordingly, a lead optimization program designed to
modify the physicochemical properties was initiated. Related
B
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ACS Medicinal Chemistry Letters
Letter
Table 1. In Vitro and in Vivo Data for Pyrazolo[1,5-a][1,3,5]triazine and Pyrazolo[1,5-a]pyrimidine TTK Inhibitors
ration of weak bases, increases in solubility were manifest at the
acidic pH of the gastric milieu (Table 1) and allowed for the
preparation of salts.
We chose four potent compounds with good oral exposure for
further evaluation (Table 2). They were tested against a panel of
human kinases at 1 μM (Table S3). Compound 10 was the least
C
DOI: 10.1021/acsmedchemlett.5b00485
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ACS Medicinal Chemistry Letters
Letter
single agent activity upon daily oral dosing is comparable to
published TTK inhibitors.17,18 On the strength of the cumulative
data, CFI-402257 was selected for IND enabling studies.
Table 2. In Vitro Data, Rat and Dog PK, and Mouse Xenograft
Results for Selected TTK Inhibitors
study\entry
10
12
16
24
a
kinases selectivity
6/262
0.78
34
1/262
3.0
4/262
2.0
0/262
0.51
14
ASSOCIATED CONTENT
* Supporting Information
■
CYP 3A4 IC50 (μM)
S
b
rat PK
Cl (mL/min/kg)
66
22
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.5b00485.
PO AUCINF
1.9
0.49
3.7
3.4
(h·μg/mL)
BA (%)
77
29
38
13
94
7.9
14
53
4.9
25
b
dog PK
Cl (mL/min/kg)
Experimental methods, extended GI50 panel (Table S1),
X-ray data (Table S2), kinase screen (Table S3), and
HCT116 xenograft (Figure S1) (PDF)
PO AUCINF
(h·μg/mL)
BA (%)
2.2
6.9
77
98
95
114
HCT116/HT29 xenografts % 72/41 @ 70/38 @ 72/53 @ 68/60 @
c
TGI @ MTD (mg/kg)
6.5
15
15
6.5
AUTHOR INFORMATION
Corresponding Author
*E-mail: henry.pauls@cogeco.ca. Phone: 905-337-3446.
■
a
b
Number with >50% inhibition @ 1 μM, [ATP] = Km. IV dose: 1
c
mg/kg. PO dose: 5 mg/kg. Results at day 21.
Funding
selective, yet only inhibited six of the 262 kinases tested (>50% @
1 μM), whereas compound 24 inhibited none. The high
selectivity is attributed, in part, to the 11/2 type nature of the
binding; the N-cyclopropyl-2-methylbenzamide group inserts
deeply into the ATP binding site of TTK. As illustrated by our X-
ray structure, the cyclopropyl-2-methylbenzamide moiety and its
binding pocket are highly complementary.
The short listed compounds were tested in a pair of xenograft
models at their estimated maximum tolerated dose (MTD).
Similar results were obtained for all four compounds in the
HCT116 xenograft, i.e., between 68 and 72% tumor growth
inhibition (TGI). A wider range of TGI was observed in the
HT29 model, but all four compounds had modest responses
overall. In contrast the PK parameters determined for dog
spanned an order of magnitude. The very high oral exposure seen
in mouse with compound 12 did not carry over to rat, the low
AUC apparently driven by high clearance. On balance, two
compounds (16, 24) distinguished themselves from their
comparators (10, 12) with high oral exposure and low clearance
in rat and dog.
Given its in vitro potency (TTK IC50 = 1.7 nM), exquisite
selectivity, and PK parameters, pyrazolo[1,5-a]pyrimidine 24
was deemed to have the best balance of properties for subsequent
preclinical evaluation. One potential issue, CYP inhibition, was
examined in more detail. Compound 24, had no activity against
CYPs 1A2 and 2D6, inhibited 2C9 and 2C19 with IC50s of 13 and
8 μM, respectively. The level of CYP 3A4 activity observed was
substrate dependent with IC50s of 0.51 and 14 μM for BFC and
DBF, respectively. In comparison, the TTK Ki for compound 24
equals 0.1 nM; however, further studies may be required to
understand its potential for drug−drug interactions.
Compound 24 was tested against a panel of cancer cell lines
from different pathologies and shown to be a potent inhibitor of
cell growth (Table S1). The molecule is an intracellular inhibitor
of exogenous TTK autophosphorylation with no effect on
Aurora kinase A or B or Histone H3 phosphorylation.26 This
selective inhibitor has the expected mechanism of action in
cancer cells and a phenotype consistent with the inhibition of
TTK;26 experimental details will be disclosed in due course.
The performance of compound 24 (designated CFI-402257)
in the HCT116 colorectal cancer xenograft model is illustrated in
Figure S1. A dose-dependent increase in TGI versus vehicle was
observed at day 21: TGI = 39% @ 5.5 mg/kg (p = 0.08) as
compared to 68% at 6.5 mg/kg (p = 0.01). There was minimal
weight loss (<10%), and no signs of overt toxicity. This level of
The Canadian Institutes of Health Research (DR1-01477),
Princess Margaret Cancer Foundation funded this work.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
ATP, adenosine-5′-triphosphate; AUC, area under the curve;
BA, bioavailability; BFC, 7-benzyloxy-4-trifluoromethylcoumar-
in; Boc, tert-butoxycarbonyl; Cl, clearance; CYP, cytochrome
P450; DBF, dibenzylfluorescein; DCE, dichloroethylene; DCM,
dichloromethane; DIPEA, N,N-diisopropylethylamine; dppf,
1,1′-bis(diphenylphosphino)ferrocene; GI50, half-maximal cell
growth inhibitory concentration; IV, intravenous; MTD,
maximum tolerated dose; Mps1, monopolar spindle 1; PDB,
Protein Data Bank; PG, protecting group; PMB, p-methoxy-
benzyl; PO, per os (by mouth); QD, quaque die (once daily);
RNAi, RNA interference; SAR, structure−activity relationship;
siRNA, small interfering RNA; TGI, tumor growth inhibition;
THF, tetrahydrofuran; TEA, triethylamine; TFA, trifluoroacetic
acid; TTK, threonine tyrosine kinase
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