The discovery of orally bioavailable tyrosine threonine kinase (TTK) inhibitors: 3-(4-(heterocyclyl)phenyl)-1H-indazole-5-carboxamides as anticancer agents

Article abstract of DOI:10.1021/jm501740a

The acetamido and carboxamido substituted 3-(1H-indazol-3-yl)benzenesulfonamides are potent TTK inhibitors. However, they display modest ability to attenuate cancer cell growth; their physicochemical properties, and attendant pharmacokinetic parameters, a

Full text of DOI:10.1021/jm501740a

Article
pubs.acs.org/jmc
The Discovery of Orally Bioavailable Tyrosine Threonine Kinase (TTK)
Inhibitors: 3‑(4-(heterocyclyl)phenyl)‑1H‑indazole-5-carboxamides
as Anticancer Agents
Yong Liu,*,† Yunhui Lang,† Narendra Kumar Patel,† Grace Ng,† Radoslaw Laufer,† Sze-Wan Li,†
Louise Edwards,† Bryan Forrest,† Peter B. Sampson,† Miklos Feher,† Fuqiang Ban,† Donald E. Awrey,†
Irina Beletskaya,† Guodong Mao,† Richard Hodgson,† Olga Plotnikova,† Wei Qiu,‡
Nickolay Y. Chirgadze,‡ Jacqueline M. Mason,† Xin Wei,† Dan Chi-Chia Lin,† Yi Che,† Reza Kiarash,†
Brian Madeira,† 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 Centre, 610 University Avenue,
Toronto, Ontario M5G 2C4, Canada
S
* Supporting Information
ABSTRACT: The acetamido and carboxamido substituted 3-(1H-indazol-3-yl)benzenesulfonamides are potent TTK inhibitors.
However, they display modest ability to attenuate cancer cell growth; their physicochemical properties, and attendant pharma-
cokinetic parameters, are not drug-like. By eliminating the polar 3-sulfonamide group and grafting a heterocycle at the 4 position
of the phenyl ring, potent inhibitors with oral exposure were obtained. An X-ray cocrystal structure and a refined binding
model allowed for a structure guided approach. Systematic optimization resulted in novel TTK inhibitors, namely 3-(4-
(heterocyclyl)phenyl)-1H-indazole-5-carboxamides. Compounds incorporating the 3-hydroxy-8-azabicyclo[3.2.1]octan-8-yl
bicyclic system were potent (TTK IC50 < 10 nM, HCT116 GI50 < 0.1 μM), displayed low off-target activity (>500×), and
microsomal stability (T1/2 > 30 min). A subset was tested in rodent PK and mouse xenograft models of human cancer.
Compound 75 (CFI-401870) recapitulated the phenotype of TTK RNAi, demonstrated in vivo tumor growth inhibition upon
oral dosing, and was selected for preclinical evaluation.
histological grade in tumors11 and poor patient outcome.9 High
INTRODUCTION
■
levels of TTK in cancer cells may contribute to their tolerance of
A number of cell cycle and checkpoint kinases have been targeted
the aneuploidy state.11,14,15 Inhibition of TTK impairs the SAC
as potential strategies for the treatment of cancer.1 The spindle
and allows cancer cells to progress through mitosis in the
assembly checkpoint (SAC) is an important and highly
presence of misaligned chromosomes, resulting in an intolerably
orchestrated control mechanism during mitosis; it assures the
high degree of aneuploidy and eventual cell death.11
proper attachment of chromosomes to the mitotic spindle prior
to anaphase.2 Most cancer cells are aneuploid3 and thus take
TTK has been identified as one of the top 25 genes
overexpressed in tumors with chromosomal instability15,16 and
longer than normal diploid cells to align and attach their
aneuploidy.17,18 PTEN-deficient breast tumor cells are partic-
ularly dependent upon TTK for their survival such that RNAi-
mediated knockdown leads to cell death. TTK inhibitors have
been shown to target PTEN-deficient cells more acutely,
presumably exploiting their underlying aneuploidy and genomic
instability.17 Besides its role in mitosis, TTK has been implicated in the
chromosomes to the mitotic spindle.4 It has been demonstrated
that chromosomal instability (CIN) causes aneuploidy, a hall-
mark of cancer,5 therefore the selective suppression of aneuploid
cells should be beneficial.6 The conserved dual-specificity kinase
TTK (threonine tyrosine kinase, also known as Mps1) is essen-
tial for alignment of chromosomes to the metaphase plate7 and
genomic integrity during cell division through establishment and
maintenance of the SAC.8 TTK is overexpressed in a variety of
solid cancers,9−13 and elevated levels of TTK correlate with high
Received: November 10, 2014
© XXXX American Chemical Society
A
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Chart 1. Representative TTK Inhibitors
Hedgehog,19 EGFR,20 and DNA damage signaling pathways.21−23
TTK controls DNA damage responses and genome stability by
phosphorylating the Bloom syndrome helicase (BLM),21 CHK2
in the G2/M checkpoint,22 and c-ABL after oxidative stress.23
TTK first came to our attention as a hit in a genome wide
siRNA screen in which the growth of 7 out of 12 breast cancer
cell lines tested was attenuated.24 Subsequently we were able to
show that siRNA knockdown abrogated cancer cell growth and
caused gross chromosome segregation errors, resulting in
aneuploidy and cell death. In vivo, interfering RNA mediated
depletion suppressed the growth of tumor xenografts in mice.24
Taken together, these observations encouraged us to pursue
TTK as a viable target for cancer therapy.25
TTK has received considerable attention from laboratories
interested in the development of small molecule inhibitors of
this potential cancer target. Representative structures are given
in Chart 1. The structural feature most commonly incorporated
in these inhibitors is the 2-(phenylamino)pyrimidine motif
found in AZ3146,26 reversine,27 Mps1-IN-2, and its analogue 1
(methoxy-Mps1-IN-2, Figure 1),28 2 (NMS-P715),29 and the
Genentech inhibitor.30 The Shionogi-1 compound31 and
CCT25145532 incorporate variations on this theme. These
compounds have been used to further understanding of the
cellular functions of TTK. A patent from Oncotherapy33
describes (alkylamino)imidazo[1,2-b]pyridazines as a novel
series of TTK inhibitors. The related (alkylamino)imidazo[1,2-
a]pyrazines (Mps-BAY2a and Mps-BAY2b) are potent TTK
inhibitors, and the latter has demonstrated complete inhibition
of xenograft tumor growth in combination with paclitaxel.34
Recently, a clinical study with a TTK inhibitor from Bayer, dosed
in combination with paclitaxel, has been initiated.
acetamido and carboxamido substituted 3-(1H-indazol-3-yl)-
benzenesulfonamides.25 Independently, Shionogi has developed
a series of 3-(1H-indazol-3-yl)benzenesulfonamides with very
different substitution patterns on the indazole core (Shionogi-2,
Chart 2).35 We found that the 5-substituted acetamido and
carboxamido 3-(1H-indazol-3-yl)benzenesulfonamides such as 3
and 4, respectively (Chart 2), were potent inhibitors of TTK with
modest ability to attenuate the growth of cancer cells. However,
during the course of our in vivo evaluation of these compounds, it
became apparent that their physicochemical properties, and
attendant pharmacokinetic parameters (Tables 1 and 2), were
not sufficiently drug-like to move these compounds forward. We
postulated that the polar nature of the sulfonamide group (e.g.,
compound 3, PSA = 130) was detrimental to the permeability
of these molecules. We embarked upon a structure guided
modification of phenyl ring substituents while simultaneously
exploring the 5 position of the indazole core. In moving the series
forward, we were cognizant of the desirability of optimizing
PK, while maintaining selectivity and potency, in order to
further explore the unique role of TTK in tumor growth. This
article describes the successful realization of these objectives
and the identification of selective, orally bioavailable inhibitors
of TTK.
RESULTS AND DISCUSSION
■
Inhibitor Design. Figure 1 illustrates the design of our new
inhibitors; the model shows an overlay of 3-sulfonamide 3 with
the Dana-Farber inhibitor 1.28 We sought to reduce the polar
surface area of the molecules by morphing the sulfonamide
(PSA = 130) to a sulfone (PSA = 104) moiety as found in com-
pound 12 (Table 1). Subsequently, we explored the addition of
heterocycles to the 4-position of the phenyl ring. Our modeling
suggested that substitution at position 4 of the phenyl ring is
Our approach built upon hits obtained from a focused
screening exercise which eventually led to the identification of
B
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Figure 1. Overlay of TTK inhibitors. The figure was obtained by aligning compound 1/TTK X-ray structure (PDB: 3H9F)28 with compound 3 docked
into a previously described25 3-(1H-indazol-3-yl)benzenesulfonamide/TTK X-ray structure (PDB: 4JT3).
Chart 2. 3-(1H-Indazol-3-yl)benzenesulfonamide TTK Inhibitors
tolerated and allows for the projection of polar moieties toward
the enzyme/solvent interface. These heterocyclic motifs were
drawn from those found most frequently in known TTK
inhibitors (Chart 1). We also contemplated the removal of the
substituent at the 3-position of the phenyl ring because our
computational analysis supported the hypothesis that 4-mono
substituted compounds would be potent TTK inhibitors.
Chemical Synthesis. The retrosynthesis of the N-(3-(4-
heterocyclyl and/or 3-(methylsulfonyl)phenyl)-1H-indazol-
5-yl)acetamides of Tables 1 and 3 yields the requisite Boc-
protected 3-iodo-1H-indazol-5-amine 9b or N-(3-iodo-1H-
indazol-5-yl)acetamides 10 and boronates 7 (Scheme 1).
When R1 was methyl (5c), direct borylation yielded the desired
boronate 7c, however, when R1 was a proton (5a,b), derivatiza-
tion of the piperidine nitrogen as an amide preceded the
borylation step. Aryl boronates 7 were generally prepared from
aryl halides, such as 6a,b and 8d,e, using either pinacol borane36
or bis(pinocolato)diborane,37 whereas, for the hydroxypiper-
idine R″, piperidinone 8f was borylated and reduced in situ. The
acetamidoindazole iodide coupling partner 10a was assembled
most readily by an amide bond formation from 3-iodo-1-
(tetrahydro-2H-pyran-2-yl)-1H-indol-5-amine 9a. The requisite
carboxylic acids (R‴CO2H) were commercially available as
racemic mixtures or prepared by Petasis reaction25 or
α-alkylation of arylacetate (see Supporting Information).
Compound 10b was prepared similarly, as previously described.25
Subsequent Suzuki−Miyaura coupling of boronates 7 (herein
described) or commercially available boronic acids with 10b
yielded TTK inhibitors 12−18. A THP deprotection step of 10a,
prior to Suzuki−Miyaura coupling, furnished compound 21. For
compounds 19 and 20, the Suzuki−Miyaura coupling with 9b,
followed by Boc deprotection, gave 11; a final amide bond
formation step gave the desired products.
Scheme 2 describes the synthesis of racemic and achiral 3-(4-
(heterocyclyl)phenyl)-1H-indazole-5-carboxamide TTK inhib-
itors of Tables 2−5. Aromatic nucleophilic substitution of
1-fluoro-4-iodobenzene with alcohols 22a−c provided ethers
23a−c.38 When R1 was Boc, 23a, TFA deprotection followed
by amide coupling gave 24, which was converted to its cor-
responding boronate 25a. As for the tropine and pseudotropine
derivatives 23b and 23c, direct borylation36 gave the desired
boronates 25b and 25c, respectively. Compound 28, obtained by
amide coupling of 26 and 27, was the precursor to the achiral
carboxamides of Tables 2, 3, and 5. When racemic amines 31
were not commercially available, they were prepared by re-
ductive amination of ketones 2939 or via a one-pot synthesis
C
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Table 1. Racemic N-(3-(4-(Heterocyclyl and sulfonyl)phenyl)-1H-indazol-5-yl)acetamides
a
b
All entries are racemic mixtures. Compounds 3 and 12 are single determinations with standard errors, the rest are two or more replicates with
standard deviation. <LOD: below limit of detection. ND: not determined.
(Grignard addition and imine reduction)40 from 2-cyanopyr-
idine 30. Suzuki−Miyaura cross-coupling of 28 or 32 with the
requisite aryl boronates furnished the desired achiral compounds
(33, 38, and 39) and racemates (34−37 and 40−48),
respectively.
Achiral compounds 49 and 50 (Table 5) were prepared by
Suzuki−Miyaura cross-coupling of 28 with 3-(4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-8-oxa-3-azabicyclo-
[3.2.1]octane (see Supporting Information) and boronate 60a,
respectively (Scheme 3). Compound 51 (Table 2) was prepared
using methods similar to those described in Scheme 3 from the
enantiomerically enriched 1-(2-chlorophenyl)propan-1-amine
(see Supporting Information).
Scheme 3 outlines the routes used to prepare the chiral
N-(alkyl(aryl)methyl)-3-(4-(heterocyclyl)phenyl)-1H-indazole-
5-carboxamides of Table 5. The asymmetric center in these TTK
inhibitors resides in the 5-substituent of the indazole and is
derived from chiral amine precursors. For the preparation of
chiral amines 55-S and 55-R, we employed both chiral resolution
and enantioselective synthesis. For example, racemic 55 (Ar =
pyridin-2-yl, R = isobutyl) was resolved with (D)- or (L)-DBTA
to provide both R and S enantiomers in good enantiomeric
excess (ee) but poor yield. A more general and efficient
synthesis made use of Ellman’s auxiliary following well
established methods.41,42 Grignard reagent was added to
sulfinamide 53-S in the presence of Me2Zn to give 54-S, which
was subsequently hydrolyzed to afford 55-S in high ee and
moderate yield. Alternatively, for the preparation of (S)-
cyclopentyl(pyridin-2-yl)methanamine, the (R)-2-methylpro-
pane-2-sulfinamide 52-R was converted to the requisite
sufinamide 56-R. Following a similar series of steps, using a
pyridine-2-yl based Grignard reagent, the desired S enantiomer
was obtained. The corresponding (R)-alkyl(aryl)methanamines
(R = cyclopropyl, cyclopentyl, isobutyl) were similarly prepared.
Subsequent amide coupling of the chiral amines so prepared,
with 3-iodo-1H-indazole-5-carboxylic acid 27, afforded the
penultimate intermediates 32-S and 32-R. In some instances,
racemic 32 was subjected to chiral HPLC separation to yield the
desired enantiomeric iodoindazole intermediates 32-S and 32-R.
For the bridged heterocycles, the precursor azabicycloalkanes
58a−d were arylated with 1,4-diiodobenzene in the presence of
CuI catalyst38 to give iodides 59a−d which were converted to
pinacol boronic esters as described above. The homologue 60e
and the tropine−morpholine hybrid 60f were prepared from
ketones 62e and 62f, respectively. Compounds 62e and 62f were
obtained by a one pot Robinson synthesis43 from 61 using
D
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Table 2. 3-(4-(Heterocyclyl)phenyl)-1H-indazole-5-carboxamide TTK Inhibitors
a
b
Compound 51 is enantiomerically enriched; the rest of the entries are achiral or racemic mixtures. Compounds 4, 37, 38, and 51 are single
determinations with standard errors, the rest are two or more replicates with standard deviation. ND: not determined.
glutaraldehyde and 2-(2,2-diethoxyethoxy)-1,1-diethoxyethane,
respectively. Reduction of the ketones with NaBH4 gave
alcohols 63e and 63f, which were subsequently borylated to
provide 60e and 60f. Standard Suzuki−Miyaura coupling
between 32-S or 32-R and boronates 60 completed the
synthesis of the chiral 3-(4-(heterocyclyl)phenyl)-1H-indazole-
5-carboxamides 64−88.
Lead Optimization. Table 1 summarizes our initial findings;
the sulfonamide 3 to sulfone 12 modification was tolerated
in that potent TTK inhibitory activity was maintained. As
anticipated, the reduced polar surface area of sulfone 12 was
reflected in a concomitant increase in exposure upon oral dosing
as compared to the analogous sulfonamide 3. Unfortunately,
this modification resulted in a 4-fold decreased potency in the cell
viability assay, a result also seen in sulfone 14. This tendency
was observed for several additional sulfones (data not shown)
with the lone exception being compound 13 wherein a basic
dimethylaminopropyl group was grafted onto the 4-position
of the phenyl ring. However, this modification served to
abrogate oral exposure, therefore, we turned our attention to
the 4-monosubstituted analogues 15−18.
The TTK IC50 values for compounds 14 and 15 supported the
reasoning, presented in Figure 1 and predicted by molecular
modeling, that substituents would be tolerated at position 4
of the phenyl ring. Furthermore, sulfonamides or sulfones, at
position 3 of the phenyl ring, are not necessary for TTK
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Table 3. Comparison of Acetamide and Carboxamide Linked TTK Inhibitors
kinase IC50 (μM)
GI50 (μM)
microsomal T1/2 (min) mouse/rat HCT116 MDA-MB- 468
a
b
entry
linker
TTK
AURKB/INCENP
AURKA
KDR
c
19
39
20
40
21
41
acetamide
0.012 0.001
0.0077 0.005
0.008 0.0001
0.0027 0.0009
0.010 0.002
0.004 0.0008
ND
2.1 0.4
2.3 0.2
2.3 0.3
2.0 0.3
2.0 0.2
>10
1.2 0.1
3.8 0.3
ND
5.8/10
1.7/1.5
29/15
0.094
0.039
0.059
0.017
0.057
0.024
0.44
0.19
0.19
0.09
0.15
0.13
carboxamide
acetamide
0.75 0.05
ND
carboxamide
acetamide
ND
2.9 0.2
1.5 0.2
21/24
0.071 0.007
0.18 0.01
>60/16
51/38
carboxamide
6.1 0.8
a
b
Compounds 19 and 39 are achiral, the rest of the entries are racemic mixtures. Compounds 19, 21, and 41 are single determinations with standard
c
errors, the rest are two or more replicates with standard deviation. ND: not determined.
a
Scheme 1. Synthesis of (3-(4-Heterocyclyl and/or 3-sulfonyl)phenyl)-1H-indazol-5-yl)acetamides 12−21
a
i
(i) HCO2H or Me2NCH2CO2H, TBTU, Pr2NEt, DMF, 91−95%; (ii) Pd(dppf)Cl2·CH2Cl2, (Bpin)2, KOAc, DMF, 80 °C, or Pd(dba)2,
i
(C6H11)3P·HBF4, (Bpin)2, KOAc, dioxane, 80 °C or Pd(CH3CN)2Cl2, S-Phos, PinBH, dioxane, 100 °C, 29−91%; (iii) R‴CO2H, TBTU, Pr2NEt,
DMF, 23−54%; (iv) TsOH·H2O, MeOH, 125 °C, microwave, 69%; (v) ArB(OH)2 or 7, aq Na2CO3, (Pd(PPh3)4 or Pd(dppf)Cl2·CH2Cl2,
PhMe/EtOH, reflux) or (Pd(PPh3)4, LiCl, dioxane, reflux), 10−58%; (vi) TFA, DCM, 38−55% (2 steps).
inhibition. Subsequently, three additional compounds 16−18,
lacking the 3-substituent, were made to further explore this
hypothesis. All four compounds 15−18 potently inhibited TTK,
had much reduced polar surface area (ranging from 68 to 84),
and were effective in the cell viability assays (HCT116 GI50 ≤
0.057 μM). In addition, these inhibitors achieved markedly
higher exposures than the analogous sulfonamide 3 upon oral
dosing in the mouse. Compounds 16 and 18 were of particular
F
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Scheme 2. Synthesis of Achiral (33, 38, 39) and Racemic (34−37, 40−48) 3-(4-(Heterocyclyl)phenyl)-1H-indazole-5-
a
carboxamides
a
(i) 1-Fluoro-4-iodobenzene, NaH, DMF, 85 °C, 40−55%; (ii) TFA, DCM; (iii) HCO2H, TBTU, DIPEA, DMF, 36% (2 steps); (iv)
Pd(CH3CN)2Cl2, S-Phos, PinBH, dioxane, 110 °C or Pd(dppf)Cl2·CH2Cl2, (Bpin)2, KOAc, DMF, 85−100 °C, 37−75%; (v) TBTU, iPr2NEt, DMF,
81%; (vi) NH4OAc, NaBH3CN, MeOH, 60 °C, 93−100%; (vii) c-PrMgBr, THF, NaBH4, MeOH, 79%; (viii) ArB(OH)2, 7 or 25, aq Na2CO3,
(Pd(PPh3)4 or Pd(dppf)Cl2·CH2Cl2, PhMe/EtOH, reflux) or (Pd(PPh3)4, LiCl, dioxane, reflux), 7−83%.
note, with Cmax of 3.5 and 2.8 μg/mL and AUC of 7.9 and
6.8 μg·h/mL, respectively.
substitute a carbon atom for nitrogen, as in the matched pair,
compounds 17 and 20 (Table 1 and 3). However, the direct
comparison illustrates the beneficial effect of the nitrogen on in
vitro performance; the former is about 2.5-fold more potent
against TTK, which is reflected in its superior activity in the cell
viability assays. In the carboxamides, racemization of the chiral
center, which is alpha to the nitrogen, is less of a concern. In
addition, for the assembly of the asymmetric carboxamides, there
are a number of routes available for the preparation of the
precursor chiral amines.41,42 This is in contrast to the paucity of
suitable methods for the preparation of the chiral α-amino acids
required for asymmetric acetamide synthesis. Finally, Table 3
summarizes the head to head comparison of three matched pairs
of inhibitors: 19/39, 20/40, and 21/41. There is little difference
in overall properties excepting a small but consistent advantage in
potency for the carboxamides. Given these considerations, we
decided to pursue the carboxamide linked inhibitors in our
subsequent lead optimization efforts.
Some published TTK inhibitors have a propensity to inhib-
it other mitotic kinases such as Aurora kinase B (AURKB/
INCENP), and strategies to mitigate this activity have been
reported.44 Table 3 captures the off-target kinase activity of the
indazole based compounds against Aurora kinase A (AURKA),
AURKB/INCENP, and a receptor tyrosine kinase KDR (kinase
insert domain receptor). Good selectivity against AURKA and
KDR were observed, however, we continued to monitor the
off-target activity against AURKB/INCENP as the in vitro
potency and eADME properties of the series were optimized.
Our initial efforts on improving the properties of the 4-piperidin-
4-yloxy class of TTK inhibitors are captured in Table 4. With the
Previously, we reported25 that the isomeric amide linker, i.e.,
carboxamido, was effective when incorporated into prototypical
sulfonamides such as 4. Accordingly, we prepared representative
carboxamides with the new heterocyclic moieties. The results,
summarized in Table 2, indicate that carboxamides 33, 34, and
36−38, were also potent inhibitors of TTK, with IC50 values
ranging from single- to double-digit nanomolar. As was also seen
for the acetamides, the (1-methylpiperidin-4-yl)oxy group
yielded the most potent inhibitors, compounds 36 and 51.
In contrast, and mirroring the acetamide result, the best
exposure was achieved with compound 34, which incorporates
the morpholino R″ group. Its regioisomer, compound 35, is
20-fold less active, indicating the preference for 4 versus 3
substitution on the phenyl ring. Plasma levels for a representative
sulfonamide 4 were exceedingly low, over 2 orders of magnitude
less than the morpholino analogue 34. A priori, any one of
the R″ heterocyclic moieties could be considered as viable
starting points for subsequent lead optimization studies. Our
initial effort focused on two: the (1-methylpiperidin-4-yl)oxy
group for its potency and the morpholino group for its superior
oral exposure.
An additional consideration was the stereochemistry of the
5-indazole moiety. The acetamido and carboxamido substituted
3-(1H-indazol-3-yl)benzenesulfonamides both incorporate an
asymmetric center which is recapitulated with the new hetero-
cyclic R″ groups. This raises the possibility of racemization, alpha
to the acetamide carbonyl, especially if the substituent is attached
by a nitrogen atom. One way to reduce this liability might be to
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Table 4. 3-(4-(Piperidin-4-yloxy)phenyl)-1H-indazole-5-carboxamide TTK Inhibitors
a
b
All entries are racemic mixtures. Compounds 43 and 45 are two or more replicates with standard deviation, the rest are single determinations with
standard errors. ND: not determined.
starting assumption that the basicity of the piperidine ring
(calculated pKa 8.7) was contributing to low oral exposure, com-
pounds were prepared that modulated this property. For-
mamides 42 and 43 (calculated pKa ≤ 4.3) and amide 44 retained
TTK potency and the latter two were of special interest given
their effectiveness in the HCT116 cell viability assay. Unfortu-
nately, compound 43 proved to be an inhibitor of AURKB/
INCENP whereas the oral exposure of 44 was lower than its
parent 36.
During the course of our work, we obtained a TTK co-complex
X-ray structure with a 3-(4-((1-piperidin-4-yl)oxy)phenyl)-1H-
indazole-5-carboxamide, namely compound 51 (Figure 2).
Inspection of this structure (vide infra) suggested that further
modifications to the piperidine ring were possible. Accordingly,
we prepared a pair of 8-azabicyclo[3.2.1]octane systems bridged
in the endo 45 and exo 46 sense; modeling indicated that either
of these modifications would be tolerated in the enzyme active
site. We anticipated that these larger systems could make better
contact with the enzyme surface, thereby increasing TTK
potency while decreasing off-target activity through steric
clashes. The exo version 46 was less active in vitro and, although
TTK potency was retained for the endo compound 45, selectivity
enhancements were not realized. The good activity for the endo
system was carried over to the formylated versions 47 and 48.
Compound 47 had poor oral exposure, whereas compound 48
achieved respectable plasma levels but suffered from signifi-
cant off-target activity (AURKB/INCENP and CYP 3A4).
In fact, we were not able to achieve the desired potency and
oral exposure while minimizing off-target effects, using this
strategy.
H
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Table 5. 3-(4-(Heterocyclyl)phenyl)-1H-indazole-5-carboxamides Incorporating Bridged Systems
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Table 5. continued
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Table 5. continued
a
b
Compounds 49 and 50 are achiral, the rest of the entries are chiral. Compounds 69, 71, 72, 74, 75, 76, 78, 83, 84, and 86 are two or more
replicates with standard deviation, the rest are single determinations with standard errors.
a
Scheme 3. Synthesis of Chiral 3-(4-(Heterocyclyl)phenyl)-1H-indazole-5-carboxamide TTK Inhibitors 64−88
a
(i) (Arene or cyclopentane)carbaldehyde, CuSO4 or CuSO4/MgSO4, DCM, 61−89%; (ii) RMgBr, Me2Zn, THF, 62−100%; (iii) 2 M HCl in Et2O,
MeOH, 64−87%; (iv) 2-bromopyridine, iPrMgCl·LiCl, THF, 33%; (v) TBTU, iPr2NEt, 3-iodo-1H-indazole-5-carboxylic acid, DMF, 75−98%; (vi) CuI,
1,4-diiodobenzene, K3PO4, BINOL, rt to 50 °C, 8−44%; (vii) Pd(dppf)Cl2·CH2Cl2, (Bpin)2, KOAc, DMF, 80 °C or Pd(CH3CN)2Cl2, S-Phos, PinBH,
dioxane, 100 °C, 44−100% for 60a−d (1 step) and 10−29% for 60e,f (2 steps); (viii) 3-oxopentanedioic acid, HCl, EtOH, 2-(2,2-diethoxyethoxy)-1,1-
diethoxyethane or glutaraldehyde, 15−31%; (ix) NaBH4, MeOH; (x) aq Na2CO3, Pd(PPh3)4 or Pd(dppf)Cl2·CH2Cl2, PhMe/EtOH, reflux; 12−59%.
K
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Figure 2. Active site view of co-complex X-ray structure of compound 51 with TTK (PDB: 4O6L) indicating binding of the R enantiomer to chain A. H-
bonds from indazole to the hinge region (Glu603 and Gly605) and from the amide carbonyl to Lys553 are shown by dotted lines. The ethyl group
nestles in a side pocket, while the phenyl ring makes hydrophobic contact with the protein surface; the protonated piperdine ring extends toward the
solvent interface and makes an interaction with the phosphorylated side chain of Thr675 (Tpo675). The position of the gatekeeper residue (Met602) is
indicated.
Consequently, we turned our attention to the morpholino-
phenyl containing compounds and, based on the reasoning
above, prepared two bridged analogues, compounds 49 and 50
(Table 5). The former (i.e., the 8-oxa-3-azabicyclo[3.2.1]octan-
3-yl system) was less active than its parent 39 (Table 3), however,
the latter (i.e., the 3-oxa-8-azabicyclo[3.2.1]octan-8-yl system)
was not only more potent but about an order of magnitude more
selective against AURKB/INCENP. A number of additional
variations incorporating the latter R″ group were examined,
compounds 64−70. Potency against TTK was generally in the
low nanomolar range; in addition to AURKB/INCENP
selectivity, cell activity, microsomal stability, and cytochrome
P450 activity was routinely monitored and employed as filters to
move compounds forward. In general, the S enantiomer was
marginally more potent against TTK however, the differences
were subtle and AURKB/INCENP selectivity sometimes
favored the R enantiomer. TTK inhibitors with desirable potency
(TTK IC50 < 10 nM), selectivity against AURKB/INCENP and
evaluated. In addition, concerns about their low calculated solu-
bilities (ACD/Stucture Designer)62 prompted the investigation
of compounds incorporating alkyl(pyridinyl)methyl R‴ groups.
These mild bases (67−70) were effective inhibitors of TTK.
Note that the meta and para pyridinyl analogues were not
pursued because previous work with the phenyl sulfonamide
series25 had indicated a loss in activity for these congeners. The
analogue in which the R″ group had been expanded to the 3-oxa-
9-azabicyclo[3.3.1]nonan-9-yl ring system, compound 71, was
less potent and offered no other advantages over its closest
analogue 69. Compounds 68 and 69, deemed to incorporate the
best features from this subclass, were moved into rodent PK
studies.
Concurrently, a similar approach to the hydroxyl-piperidine
R″ yielded a series of bridged bicyclics, compounds 72−88
(Table 5). As a group, these compounds had good in vitro
potency and were generally selective against AURKB/INCENP
(>100-fold). The first two entries in the series, compounds 72
and 73, were moved quickly into mouse PK studies to gauge
the impact of this new R″ group on oral exposure. The results
(Table 6) were sufficiently encouraging to warrant further
investigation of this chemotype. Given their solubility advantage
(vide supra), subsequent analoguing efforts concentrated on R‴
groups with nitrogen containing aromatics. Compounds 74 and
75, isolated as salts, demonstrated good in vitro potency, were
stable to microsomes (Table 5), and otherwise met the criteria
for in vivo study. A related set of compounds, the enantiomeric
CYP 3A4 (IC50 > 500 nM), rodent microsomal stability (T1/2
>
30 min), and cell activity (HCT116 GI50 < 0.1 μM) were selected
for in vivo study.
In common with other compounds incorporating the
(1-morpholinocyclohexyl)methyl R‴ group, TTK inhibitor 50
had a low T1/2 in microsomal stability assays. This liability was
generally mitigated by incorporating alkyl(aryl)methyl R‴
moieties as in compounds 64−66. However, due to their off-
target activities, none of these compounds were further
L
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Table 6. Rodent Pharmacokinetic Data for Selected TTK Inhibitors
PK parameters
68
69
72
73
74
75
76
78
82
86
mouse PO (10 mg/kg)
C
max (μg/mL)
1.5
2.1
2.0
2.8
0.33
0.62
0.48
0.67
1.6
1.6
2.0
3.0
1.5
2.6
0.75
0.97
1.2
1.9
0.8
1.6
AUCINF (h·μg/mL)
rat IV (1 mg/kg)
rat PO (5 mg/kg)
Cl (mL/min/kg)
t1/2 (h)
71
79
ND
ND
ND
ND
ND
ND
ND
ND
ND
66
91
125
0.73
0.13
57
46
0.4
0.24
0.72
0.22
0.78
0.25
0.78
0.19
0.38
0.29
0.90
0.36
AUCINF (h·μg/mL)
AUCINF (h·μg/mL)
oral BA (%)
0.89
75
0.43
39
ND
ND
ND
ND
ND
ND
0.55
44
0.13
13
0.14
21
0.27
19
0.43
24
pair 76 and 77, incorporate the larger cyclopentyl ring, resulting
in enhanced selectivity against AURKB/INCENP. The
S-enantiomer 76 had the most favorable combination of
properties. Further variations on this theme yielded pyrimidines
78 and 79, which showed comparable in vitro activity. They
suffered somewhat from a lower selectivity; again, the more
potent S-isomer was moved into a mouse PK study. Alternatively,
the bulk of the alkyl side chain could be increased by in-
corporating a pendent isobutyl group alpha to the aromatic ring.
Selectivity was maintained, however, enantiomers 80 and 81
demonstrated undesirable CYP 3A4 activity. This was mitigated
by morphing the isobutyl group into a cyclopropylmethyl
moiety, e.g., compound 82, which was selected for in vivo study.
Further variations to the R″ were also examined; ring expan-
sion to a 9-azabicyclo[3.3.1]nonan-3-ol bicyclic system, com-
pound 83, resulted in a loss in potency which could be recovered
by replacing the additional carbon with oxygen as in 84. None-
theless, the potent 3-oxa-9-azabicyclo[3.3.1]nonan-7-ols 84
(poor selectivity) and 85 (off-target CYP450 activity) did not
meet the criteria for in vivo study. The stereochemistry of the
substituent hydroxyl was probed by synthesis of the exo
analogues 86−88. Potency was maintained at least for the
S-isomers, and the compound with the best balance of in vitro
properties, namely 86, was selected for rodent PK studies.
Compound 88 was less stable upon incubation with rodent
microsomes and inhibited CYP 2C9 with an IC50 of 120 nM.
X-ray Structure. The co-complex X-ray structure of
compound 51 with the kinase domain of TTK was obtained at
a resolution of 2.38 Å (PDB: 4O6L, Figure 2). The present
structure is the first reported for TTK in the C2 space group; it
has two molecules in the asymmetric unit related by 2-fold
noncrystallographic symmetry axis. Each protomer active site is
located near the axis without blocking the other’s accessibility,
and in both molecules, the activation loop is positioned away
from any crystallographic contacts. In one of the protomer
molecules (chain A), the activation loop is partially resolved
(similar to our previous structure).25 The electron density is well-
defined up to residue Ser677 and indicates three phosphorylated
residues (Thr675, Thr676, and Ser677). The other protomer
(chain B) is also partially resolved but is unphosphorylated and
away from the ligand in a conformation reminiscent of inactive
kinases. The ligand density is consistent with binding of the
R enantiomer although the crystallization well contained a 68:32
mixture of R and S enantiomers (i.e., 36% ee, see the omit map
in the Supporting Information, Figure S2). Key features of
this structure (Figure 2) include H-bonds from the indazole
nitrogens to the hinge region residues Glu603 and Gly605
and from the amide carbonyl to Lys553. For chain A, the
chlorophenyl and ethyl groups occupy hydrophobic subpockets,
while the phenyl ring makes contact with the protein surface.
The protonated piperdine ring extends toward the solvent
interface and makes an interaction with the phosphorylated side
chain of Thr675.
Computational Chemistry. In the early phases of this work,
the protein structure, cocrystallized with a molecule from the
sulfonamide series,25 was utilized for predictions (PDB code:
4JT3). However, with the availability of the 4O6L structure
(Figure 2), a new predictive model was developed. We found that
the raw GlideXP docking model using literature-standard
docking protocols was unsatisfactory in that 10 of 86 molecules
failed to dock. Achiral compounds such as 19 and 39 were
particularly troublesome; in fact, inhibitors with the
(1-morpholinocyclohexyl)methyl moiety at the 5 position of
the indazole could not be scored. This result is not surprising
when single copy docking is employed,45 however, the model
could be greatly improved by multiple copy docking.46−48 All
inhibitor molecules dock when multiple copies of each input
structure are generated via a conformational search,46 followed
by a post docking minimization step and by selecting the top
scoring solution for each ligand.47 The quality of predictions
(R2, 0.37; rmse, 0.86; n, 86, see Figure S3 in the Supporting
Information) was further improved (Figure 3 left panel: R2, 0.62;
rmse, 0.68; n, 81) by removing the achiral outliers from the
analysis. However, this outlier removal had little effect on ranking
(Spearman’s rho values of 0.79 and 0.71, respectively). Moving
forward, this chemotype was not pursued, thereby increasing our
confidence in the working model which was used as an aid to
prioritize target molecules for synthesis. This approach does not
involve any training, hence it was found to be predictive across
the entire range of TTK ligands in the project. In fact, the quality
of predictions (Figure 3 right panel: R2, 0.57; rmse, 0.69; n, 817)
was sufficient for evaluating virtual libraries and to remove less
promising target molecules from further consideration; the
corresponding Spearman’s rho value for rank correlation was
0.62. We also noted that the experimental pIC50s are better
described by nonlinear models or by omitting points with very
low Glide scores (e.g., > −7.5), however, in absence of an under-
lying explanation, only linear models with all available data were
used.
Rodent PK. The first two entries in Table 6 are from the
3-oxa-8-azabicyclo[3.2.1]octane subclass of TTK inhibitors.
Both demonstrated good oral exposure in the mouse and were
selected for rat PK studies. The next eight entries are from the
3-hydroxy-8-azabicyclo[3.2.1]octane subclass, the first two of
which showed significantly lower exposure than the rest.
Compounds 72 and 73 were more hydrophobic than the latter
entries which incorporated nitrogen in the R‴ aromatic moiety.
Of these, two represent an enantiomeric pair 74 and 75; the latter
isomer, with the best exposure, was subsequently tested in rat.
The remaining compounds showed good mouse plasma levels
M
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Figure 3. Predictive models using the 4O6L X-ray structure: pIC50 (TTK) vs GlideXP docking scores wherein the best GlideXP score (from docking
multiple conformers) is selected asthe docked structure. (left panel) Using compounds from this work (minus achiral outliers) and our previous publication
on indazole based TTK inhibitors25 (R2 = 0.62, rmse = 0.68, n = 81). (right panel) Using all project compounds (R2 = 0.57, rmse = 0.69, n = 817).
Table 7. Mouse Xenograft Results for TTK Inhibitors 69, 75,
and 86
percent tumor growth inhibition
MDA-MB-468
entry
HCT116 cell line
cell line
SW48 cell line
ND
a
b
69 17 @ 25 mg/kg 56 @ 35 mg/kg
75 55 @ 23 mg/kg 77 @ 35 mg/kg
ND
98 @ 30 mg/kg 66 @ 30 mg/kg
b
86 5 @ 100 mg/kg 53 @ 150 mg/kg
ND
ND
a
b
2/8 and 1/8 animals in this arm were lost during the study.
and were also tested in rat. In the rat, compounds 68 and 82 had
short half-lives, whereas compounds 76, 78, and 82 had the
lowest oral exposure. Thus, a short list of three TTK inhibitors
(69, 75, and 86) was identified for subsequent study in mouse
HCT116 xenografts. A pharmacokinetic study of compound 75
was also performed in the dog with key PK parameters as follows:
intravenous t1/2 = 1.2 h; oral BA 43%.
In Vivo Efficacy. The colon HCT116 cell line was chosen for
in vivo screening of the short listed TTK inhibitors because the
corresponding xenograft model was well characterized in our
laboratory. The maximum tolerated dose (MTD) upon oral daily
dosing (QD) was determined for each compound (69, 75, and
86) in athymic CD-1 nude mice. Compounds were dosed QD at
their MTD and 2/3 MTD for 21 days. The results (Table 7)
indicate that the best response, 77% and 55% tumor growth
inhibition (TGI), was observed for compound 75 at 35 and
23 mg/kg, respectively (Figure 4). Mice were lost at the highest
dose for all three compounds; one death was observed for
compound 75 at its putative MTD. On the basis of this result
compound 75 (designated CFI-401870) was tested in two addi-
tional xenograft models (breast cancer cell line MDA-MB-468
and colon cancer cell line SW48), however, the highest dose
was reduced to 30 mg/kg. TGIs of 98% and 66%, respectively,
were observed for these xenografts (Table 7), with minimal
weight loss as compared to vehicle (see Supporting Information,
Table S5) and no signs of overt toxicity. This level of response is
comparable to or better than those previously described for TTK
inhibitors. For example, the Nerviano compound 2, given orally
(90 mg/kg QD for 7 consecutive days), achieved a 53% tumor
growth inhibition at the end of study (day 10) in an A2780 ovary
carcinoma xenograft model.49 The Myriad/Myrexis compound
(MPI-0479605 at 30 mg/kg QD for 21 days) had a TGI of 49% at
day 22 in an HCT-116 xenograft model with one animal lost at
Figure 4. Athymic CD-1 NUDE mice (n = 8) bearing HCT116
xenograft tumors treated daily for 21 days with compound 75 at 23 and
35 mg/kg (PO QD). Plot depicts tumor volume vs day of treatment.
One mouse, receiving the high dose of compound 75, was found dead at
day 14; no adverse effects in the remaining animals were observed (body
weight loss <20%).
day 15.50 It is possible that intermittent dosing strategies50 and/
or combination studies34 will need to be explored to fully exploit
the potential of TKK inhibitors in vivo.
In Vitro Characterization. A kinetic analysis of TTK
inhibitor 75 (Supporting Information) confirmed the potency
(Ki = 0.7 0.5 nM) and ATP competitive nature of the binding
(i.e., KmApp increases with dose). However, the variation in Vmax
over the concentration range indicates mixed kinetics for this
inhibitor. Compound 75 was tested against an extensive panel
of human kinases, and the results are shown as a heat map in
Figure 5. The compound was substantially more selective than
sunitinib and comparable to imatinib, as measured by the
number of off-target kinases hit. Half-maximal inhibitory
concentrations were determined for those kinases most potently
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Figure 5. Heat map showing % inhibition of 278 kinases at 1.0 μM of compound 75 and two clinically used kinase inhibitors imatinib and sunitinib.
Millipore radiometric assay data for each compound was individually sorted.
Table 8. Biochemical (IC50) and Cellular (EC50) Inhibition Constants for Compound 75
kinase
IC50 (nM)
kinase
IC50 (nM)
kinase
IC50 (nM)
EC50 (nM)
cKit (V560G)
cKit
490
>30000
250
JNK3
68
320
57
TTK
3.1
130
14
MAPK1
MAPK2
MELK
MuSK
PLK4
ND
Ret (V804L)
Ret (V804M)
Ret
AURKA
1300
1300
>6000
>6000
280
160
690
AURKB/INCENP
2800
Figure 6. Cell cycle analysis of HCT116 colon cancer cells treated with compound 75 at given concentrations. Percentage of 8N and 16N cells, at 48 h,
normalized to DMSO control.
Table 9. Growth Inhibition Data for TTK Inhibitor 75 against HMECs and a Panel of Cancer Cell Lines
breast GI50 (μM)
ovarian GI50 (μM)
HMEC GI50 (μM)
MDA-MB-231
BT-474
0.015
SKBr-3
0.008
MCF-7
0.015
T-47D
0.020
A2780
0.020
OV-90
0.030
SKOV-3
0.017
TOV-21G
0.016
>30
0.070
colon GI50 (μM)
prostate GI50 (μM)
melanoma GI50 (μM)
SK-MEL-5 SK-MEL-28
0.020 0.21
lung GI50 (μM)
COLO-205
0.060
HT29
0.018
SW48
0.019
SW620
0.030
PC-3
0.060
DU145
0.017
A549
0.024
H23
0.036
inhibited (Table 8). Although mutant forms of cKit and Ret were
inhibited, the compound was ineffective against the wild-type
enzymes. Of the remaining, JNK3, MAPK2, and PLK4 were most
significantly inhibited, but there was a ≥1.3 log unit separation in
activity and these kinases have been touted as anticancer targets
in their own right. We were particularly interested in the activity
of TTK inhibitor 75 against the mitotic kinases Aurora A and B.
The fold separation was high, and compound 75 did not inhibit
the intracellular activity of these mitotic kinases up to 6 μM
concentration.
These results were reflected in the cell cycle progression of
HCT116 colon cancer cells incubated with 75 (Figure 6); the
profile recapitulated the phenotype of TTK silencing by RNAi.
Compound 75 caused massive aneuploidy and cell death, as
indicated by sub-G1 cells, at about 100−200 nM. Consistent with
potent TTK inhibition and lack of intracellular Aurora B activity,
75 causes aneuploidy at low concentrations with no significant
polyploidy at the highest concentration tested. Inhibition of cell
growth by compound 75 was evaluated in an expanded panel of
breast, colon, ovarian, prostate, melanoma, and lung human
cancer derived cell lines (Table 9). Excepting human mammary
epithelial cells (HMEC GI50 > 30 μM), the compound inhibited
cell growth across the cell line panel; GI50 values ranged between
0.008 and 0.21 μM, with typical GI50 values in the double-digit
nanomolar range.
The effect of compound 75 on chromosome segregation in
colon cancer cells was examined by imaging HCT116 cells
during mitosis (Figure 7). Cells treated with TTK inhibitor 75
for 2 h prior to entry into mitosis exhibited severe defects in their
ability to segregate chromosomes (>60% and >90% of cells at
100 and 500 nM of compound 75, respectively). Despite defects
in chromosome segregation, the majority of TTK inhibitor 75
treated cells complete cytokinesis with a gain or loss in
chromosomes (Figure 8). A decrease in cell viability was observed
as early as 24 h after treatment with compound 75, and at the same
time, increases are induced in cleaved PARP (apoptosis marker),
phospho-H2AX (DNA damage marker), and p53 (data not
shown).
O
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Figure 7. (upper panels) Mitotic figures of HCT116 cells treated with compound 75 or DMSO 6 h post double thymidine block. Centromeres are
stained in green, α-tubulin in red, and DAPI in blue. (lower panel) Graph of the percentage of anaphase and telophase cells with and without lagging
chromosomes. Mean % SD lagging chromosomes for DMSO, 19 2%; 100 nM 75, 61 2%; 500 nM 75, 94 1%. Data are representative of three
independent experiments with a variation <10%.
Figure 8. (left panel) Plot of the chromosome counts on metaphase spreads from HCT116 cells treated with 500 nM 75 or DMSO for 28 h. Data are
representative of three independent experiments. (right panels) Representative images of metaphase spreads from 75 (500 nM) and DMSO treated
HCT116 cells. Chromosomes were stained with DAPI.
Compound 75 was tested in several early ADME assays
including CYP450 and hERG inhibition; separation in activity
between these off-target effects and TTK inhibition approxi-
mated 3 orders of magnitude (Supporting Information, Table S4).
In addition, compound 75 demonstrated good stability (T1/2
>60 min) in mouse, rat, dog, and human microsomes. Plasma
protein binding was observed to be 95.8, 93.1, 88.6, and 97.8% in
mouse, rat, dog, and human plasma, respectively.
accessible and potent in vitro. A molecular model based on the
TTK/compound 51 co-complex X-ray structure (pdb: 4O6L)
was used to prioritize target compounds for synthesis. The SAR
indicated that R″ moieties based on morpholino and hydroxy-
piperidinyl groups were of particular interest. Ten compounds
with desirable potency (TTK IC50 < 10 nM), selectivity against
AURBK/INCENP and CYP 3A4 (IC50 >500 nM), microsomal
stability (T1/2 > 30 min), and cell activity (HCT116 GI50 < 0.1 μM)
were identified for in vivo study. Compounds incorporating
the 8-oxa-3-azabicyclo[3.2.1]octan-3-yl and 3-hydroxy-8-azabicyclo-
[3.2.1]octan-8-yl bicyclic systems demonstrated high exposure upon
oral dosing in mice and were further evaluated in rat PK studies.
Compounds 69, 75, and 86 are single-digit nanomolar TTK
inhibitors, with acceptable off-target activity and good oral
exposure in rodents. The short listed compounds attenuate the
growth of MDA-MB-468 and HCT-116 cancer cells and are
somewhat less effective against OVCAR-3. Compound 75 was
CONCLUSIONS
■
The transformation of the 3-substituted sulfonamido phenyls to
4-substituted heterocyclic phenyl systems on the 1H-indazole
5-(carboxamide and acetamide) cores maintained TTK potency
and greatly improved the PK properties of the molecules. This
breakthrough set the stage for systematic lead optimization of
this series; subsequent efforts focused on enantiomerically
enriched carboxamides because they were more synthetically
P
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in each mobile phases). The purity of all final compounds was shown by
HPLC to be ≥95%.
the most efficacious of the shortlisted compounds in an HCT116
tumor xenograft model (77% TGI at 35 mg/kg PO). This
compound inhibited off-target kinases (panel of 278 human
kinases) with a frequency comparable to clinically used inhibitors
deemed to be selective (e.g., imatinib). Compound 75 inhibited
the growth of a broad panel of human cancer cell lines including
breast, ovarian, colon, prostate, lung, and melanoma and re-
capitulated the phenotype of TTK interfering RNA in HCT116
cells. It demonstrated a reasonable separation of effects between
TTK potency and human CYP and hERG activity (≥1000-fold).
On the strength of the data presented herein, compound 75 was
selected for preclinical development studies.
Suzuki−Miyaura Cross-Coupling of Iodoindazoles: General
Procedure A. A mixture of 3-iodo-1H-indazole (1.0 equiv), aryl
boronic acid, or boronate ester (1−2 equiv), base (2−3 equiv, 1 M aq
Na2CO3), and palladium catalyst (0.05−0.2 equiv PdCl2dppf·CH2Cl2
or Pd(PPh3)4) in appropriate solvents (1:1 to 2:1 EtOH/PhMe or
dioxane) was degassed with argon, sealed, and heated in a Biotage
microwave reactor or oil bath at 100−130 °C for 2−48 h. The reaction
was monitored by LCMS. After aqueous workup, the product was
purified by Biotage silica gel column or by RP prep-HPLC to provide the
desired compound. Ion-exchange PoraPak Rxn columns were used to
assist the purification as necessary. Salts (HCl) were prepared by
dissolution of the free base in MeOH and treatment with 1−2 equiv of
1 M HCl in Et2O. After evaporation of solvents, the process was
repeated several times to obtain the desired mono- or di-HCl salt.
In some instances, trifluoroacetic acid salts were prepared.
EXPERIMENTAL SECTION
■
Computational Methods. Ligands were prepared, and docking
results were analyzed in the MOE software.51 Appropriate protonation
states were generated using the Wash process, and all reasonable
stereoisomers were produced using Corina.52 Low energy conforma-
tions were generated using the Conformer Import option (max
30 conformers within a 3 kcal/mol window) and minimized using the
MMFF94x force field within MOE. The protein structure was prepared
using standard settings with the help of the Protein Preparation Wizard
in the Schrodinger suite53 with all water molecules deleted. For each
molecule, the pregenerated conformations were docked using the
GlideXP program54 and the solutions with the highest docking score
(gscore) were selected. In these docking runs, hydrogen bonding to
one of the two key hinge residues (Glu603 and Gly605) was used as
a constraint and no state penalties for protomers/tautomers were
added to the docking score, otherwise default settings were employed
including postdocking minimization.
Synthesis of Indazole Acetamides: General Procedure B. A
solution of 3-iodo-1H-indazol-5-amine 2,2,2-trifluoroacetate (1.0 equiv),
DIPEA (3 equiv), and RCO2H (1.05 equiv) at 0 °C in DMF was treated
with TBTU (1.05 equiv), added in one portion. The reaction was stirred
and slowly warmed to rt. After several hours or overnight, stirring
the crude reaction was diluted with H2O. Generally, filtration of the
precipitate followed by washing with water provided the desired material
in high purity. Alternatively, the material was purified directly by prep-
HPLC and/or flash chromatography.
N-(3-(3-(Methylsulfonyl)phenyl)-1H-indazol-5-yl)-2-(pyrrolidin-
1-yl)-2-(thiophen-3-yl)acetamide (12). Prepared according to general
procedure A using N-(3-iodo-1H-indazol-5-yl)-2-(pyrrolidin-1-yl)-2-
(thiophen-3-yl)acetamide trifluoroacetate25 (290 mg, 0.51 mmol),
(3-(methylsulfonyl)phenyl)boronic acid (120 mg, 0.6 mmol), PhCH3/
EtOH (3 mL/9 mL), 1 M aq Na2CO3 (1.5 mL, 1.5 mmol), and
Pd(PPh3)4 (11.6 mg, 0.01 mmol, 2 mol %), 1 h at 125 °C, 12 was
obtained as a light-brown solid (TFA salt, 176 mg, 58%). 1H NMR (400
MHz, CD3OD) δ 8.44 (s, 1H), 8.38 (s, 1H), 8.17 (d, J = 7.6 Hz, 1H),
7.90 (d, J = 8.0 Hz, 1H), 7.87 (s, 1H), 7.68 (t, J = 7.8 Hz, 1H), 7.62−7.57
(m, 1H), 7.51 (s, 2H), 7.39 (d, J = 5.2 Hz, 1H), 5.34 (s, 1H), 3.92−3.88
(m, 1H), 3.36−3.00 (m, 6H), 2.25−1.90 (m, 4H). HRMS (ESI) m/z
calcd for [C24H24N4O3S2 + H]+, 481.1368; found, 481.1360.
N-(3-(4-(3-(Dimethylamino)propyl)-3-(methylsulfonyl)-
phenyl)-1H-indazol-5-yl)-2-(pyrrolidin-1-yl)-2-(thiophen-3-yl)-
acetamide (13). Prepared according to general procedure A using
N-(3-iodo-1H-indazol-5-yl)-2-(pyrrolidin-1-yl)-2-(thiophen-3-yl)-
acetamide (60 mg, 0.13 mmol), N,N-dimethyl-3-(2-(methylsulfonyl)-4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)propan-1-amine
(54 mg, 0.15 mmol), PdCl2dppf·CH2Cl2 (5.4 mg, 0.007 mmol), Cs2CO3
(130 mg, 0.4 mmol) in DMF (2.5 mL), and H2O (0.6 mL), 2 h at
130 °C, 13 was obtained as an off-white powder (11 mg, 15%). 1H NMR
(400 MHz, CD3OD) δ 8.56 (d, J = 2.0 Hz, 1H), 8.36 (s, 1H), 8.20 (dd,
J = 8.0, 2.0 Hz, 1H), 7.65 (d, J = 8.0 Hz, 1H), 7.57−7.55 (m, 2H), 7.52
(dd, J = 3.0, 1.2 Hz, 1H), 7.43 (dd, J = 5.0, 3.0 Hz, 1H), 7.35 (dd, J = 5.1,
1.1 Hz, 1H), 4.14 (s, 1H), 3.25 (s, 3H), 3.14−3.07 (m, 2H), 2.74−2.65
(m, 2H), 2.62−2.56 (m, 2H), 2.56−2.48 (m, 2H), 2.37 (s, 6H), 2.03−
1.93 (m, 2H), 1.86 (m, 4H). LCMS (ESI) m/z calcd for [C29H35N5O3S2 +
H]+, 566.2; found, 566.1.
N-(3-(4-(4-Methylpiperazin-1-yl)-3-(methylsulfonyl)phenyl)-
1H-indazol-5-yl)-2-(pyrrolidin-1-yl)-2-(thiophen-3-yl)-
acetamide (14). Prepared according to general procedure A, using
N-(3-iodo-1H-indazol-5-yl)-2-(pyrrolidin-1-yl)-2-(thiophene-3-yl)-
acetamide (75 mg, 0.165 mmol), 1-methyl-4-(2-(methylsulfonyl)-4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) phenyl) piperazine (76 mg,
0.199 mmol), 1 M aq Na2CO3 (0.33 mL, 0.33 mmol), PhMe (2.2 mL),
EtOH (1.1 mL), and Pd(PPh3)4 (10 mg, 0.008 mmol) and purified using
a silica flash column (0−35% MeOH in DCM) followed by reverse
phase C-18 flash chromatography (10−20% MeOH in 0.1% aqueous
TFA) to give 14 as a white solid (TFA salt, 52 mg, 49%). 1H NMR (400
MHz, CD3OD) δ 8.63 (d, J = 2.0 Hz, 1H), 8.43 (d, J = 0.8 Hz, 1H), 8.32
(dd, J = 8.3, 2.3 Hz, 1H), 7.89−7.88 (m, 1H), 7.73 (d, J = 8.4 Hz, 1H),
7.66−7.64 (m, 1H), 7.60−7.53 (m, 2H), 7.39 (d, J = 1.2, 1H), 5.27 (s,
1H), 3.91−3.88 (m, 1H), 3.68 (d, J = 12.0 Hz, 2H), 3.63 (d, J = 11.6 Hz,
General Synthetic Methods. Commercially available starting
materials, reagents, and solvents were used as received. In general,
anhydrous reactions were performed under nitrogen or argon. Micro-
wave reactions were performed using a Biotage Initiator microwave
reactor. Reaction progress was generally monitored by LCMS (Bruker
Esquire 4000). Flash column chromatographic purification of
intermediates or final products was performed on a Biotage Isolera
One purification system using SNAP KP-sil cartridges or SNAP C-18
reverse-phase (RP) columns. Crude reactions were partially purified
using Waters PoraPak Rxn CX cartridges. Preparative reverse-phase
HPLC purification was performed on a Varian PrepStar model SD-1
HPLC system with a Varian Monochrom 10 μm C-18 reverse-phase
column. Elution was performed using a gradient of 10% MeOH/water
to 90% MeOH/water (0.05% TFA) over a 46 min period at a flow rate of
40 mL/min. Fractions containing the desired material were concen-
trated and/or lyophilized to obtain the final products. All reported yields
are unoptimized.
Proton nuclear magnetic resonance (1H NMR) spectra were
recorded on a Bruker AV 400 MHz spectrometer. Chemical shifts are
reported in parts per million (ppm) from tetramethylsilane. Proton
coupling patterns are described as singlet (s), doublet (d), triplet (t),
quartet (q), multiplet (m), and broad (br).
Low resolution mass spectra (MS ESI) were obtained using a Bruker
Esquire 4000 spectrometer under electron spray ionization conditions.
High resolution mass spectra (HRMS) were obtained by a Waters
Acquity UPLC coupled with a Waters Synapt G2 time-of-flight mass
spectrometer, using an ESI source. Compounds in DMSO solutions
(5 mg/mL) were diluted to 0.01 mg/mL; 5 μL was injected on to a
Waters BEH C-18 1.7 μm 2.1 mm × 100 mm column equilibrated at
10% acetonitrile in water, spiked with 0.1% formic acid at a flow rate of
0.5 mL/min. Bound analytes were eluted by a linear gradient of 10−95%
acetonitrile in water between 1 and 8 min. The mass spectrum
was calibrated using formate clusters; leucine enkephalin was used as a
“lock mass” for additional, real-time, accurate-mass correction.
The purity of compounds described within was determined by HPLC
(UV detection at λ = 214 and 254 nm) was performed on a Varian
Prostar HPLC, using a 100 mm × 4.6 mm Phenomenex Luna 3 μm C-18
column with a gradient of 10−90% acetonitrile in water (with 0.1% TFA
Q
DOI: 10.1021/jm501740a
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
2H), 3.55−3.41 (m, 5H), 3.35−3.31 (3H coincident with solvent peak),
3.28−3.13 (m, 2H), 3.01 (s, 3H), 2.18−2.13 (m, 3H), 2.01 (br s, 1H).
HRMS (ESI) m/z calcd for [C29H34N6O3S2 + H]+, 579.2212; found,
579.2214.
2-Cyclopentyl-N-(3-(4-((1-methylpiperidin-4-yl)oxy)phenyl)-
1H-indazol-5-yl)-2-(thiophen-3-yl)acetamide (20). Prepared
according to general procedure B, using 2-cyclopentyl-2-(thiophen-
3-yl)acetic acid (19.0 mg, 0.09 mmol), 3-(4-((1-methylpiperidin-
4-yl)oxy)phenyl)-1H-indazol-5-amine trifluoroacetate (50 mg, 0.09
mmol), TBTU (29.1 mg, 0.09 mmol), DIPEA (0.08 mL, 0.45 mmol),
and DMF (1.5 mL), 20 was obtained as an off-white solid (TFA salt, 31
mg, 54%). 1H NMR (400 MHz, CD3OD) δ 10.11−10.02 (m, 1H), 8.43
(d, J = 1.0 Hz, 1H), 7.91−7.82 (m, 2H), 7.51 (d, J = 9.0 Hz, 1H), 7.42−
7.35 (m, 2H), 7.31 (dd, J = 2.9, 1.1 Hz, 1H), 7.24−7.11 (m, 3H), 4.88−
4.83 (m, 2H), 4.71−4.61 (m, 1H), 3.65−3.54 (m, 2H), 3.44−3.24 (m,
2H), 2.94 (s, 3H), 2.45−2.28 (m, 2H), 2.15−2.07 (m, 3H), 1.75−1.61
(m, 5H), 1.40−1.35 (m, 1H), 1.18−1.13 (m, 1H). LCMS (ESI) m/z
calcd for [C30H34N4O2S + H]+ 515.2; found, 515.5.
2-Cyclopentyl-N-(3-(4-((1-methylpiperidin-4-yl)oxy)phenyl)-
1H-indazol-5-yl)-2-(pyridin-2-yl)acetamide (21). Prepared accord-
ing to general procedure A, using 2-cyclopentyl-N-(3-iodo-1H-indazol-
5-yl)-2-(pyridin-2-yl)acetamide and 1-methyl-4-(4-(4,4,5,5-tetrameth-
yl-1,3,2-dioxaborolan-2-yl)phenoxy)-piperidine, 21 was obtained as a
white solid (18 mg, 32%). 1H NMR (400 MHz, CD3OD) δ 8.53−8.47
(m, 1H), 8.37 (d, J = 1.0 Hz, 1H), 7.85−7.75 (m, 3H), 7.64 (d, J = 8.0
Hz, 1H), 7.52−7.43 (m, 2H), 7.31 (ddd, J = 7.2, 5.0, 1.0 Hz, 1H), 7.05
(d, J = 8.8 Hz, 2H), 4.46 (br s, 1H), 3.61 (d, J = 11.0 Hz, 1H), 2.87−2.64
(m, 3H), 2.39 (br s, 2H), 2.33−2.27 (m, 3H), 2.09−1.90 (m, 3H), 1.89−
1.40 (m, 8H), 1.16−1.05 (m, 1H). LCMS (ESI) m/z calcd for
[C31H35N5O2 + H]+, 510.3; found, 510.2.
3-(4-(4-Methylpiperazin-1-yl)phenyl)-N-((1-morpholinocyclohexyl)-
methyl)-1H-indazole-5-carboxamide (33). Prepared according to
general procedure A, using 3-iodo-N-((1-morpholinocyclohexyl)-
methyl)-1H-indazole-5-carboxamide trifluoroacetate (95 mg, 0.16 mmol)
and 1-methyl-4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-
piperazine (65 mg, 0.21 mmol), 33 was obtained as a white solid (TFA
salt, 84 mg, 83%). 1H NMR (400 MHz, CD3OD) δ 8.70 (s, 1H), 8.01
(dd, J = 8.9, 1.6 Hz, 1H), 7.96 (d, J = 8.8 Hz, 2H), 7.66 (d, J = 8.8 Hz,
1H), 7.23 (d, J = 8.8 Hz, 2H), 4.21−4.12 (m, 2H), 4.04−3.96 (m, 2H),
3.94 (s, 2H), 3.94−3.83 (m, 2H), 3.71−3.64 (m, 4H), 3.28−3.38 (m,
4H), 3.22−3.08 (m, 2H), 3.01 (s, 3H), 2.05−1.55 (m, 9H), 1.40−1.22
(m, 1H). HRMS (ESI) m/z [C30H40N6O2 + H]+, 517.3291; found,
517.3287.
N-(Cyclopropyl(phenyl)methyl)-3-(4-morpholinophenyl)-
1H-indazole-5-carboxamide (34). Prepared according to general
procedure A, using N-(cyclopropyl(phenyl)methyl)-3-iodo-1H-inda-
zole-5-carboxamide (50 mg, 0.12 mmol) and 4-morpholinophenylbor-
onic acid pinacol ester (40 mg, 0.14 mmol), 34 was obtained as a beige
solid (TFA salt, 11 mg, 16%). 1H NMR (400 MHz, CD3OD) δ 8.60 (s,
1H), 7.98−7.87 (m, 3H), 7.58 (d, J = 8.8 Hz, 1H), 7.47 (d, J = 7.0 Hz,
2H), 7.31 (t, J = 7.2 Hz, 2H), 7.22 (t, J = 6.8 Hz, 1H), 7.15 (d, J = 7.8 Hz,
2H), 4.48 (d, J = 9.5 Hz, 1H), 3.86 (br s, 4H), 3.25 (br s, 4H), 1.44−1.34
(m, 1H), 0.71−0.59 (m, 2H), 0.55−0.40 (m, 2H). HRMS (ESI) m/z
calcd for [C28H28N4O2 + H]+, 453.2291; found, 453.2282.
N-(3-(4-(4-Methylpiperazin-1-yl)phenyl)-1H-indazol-5-yl)-2-
(pyrrolidin-1-yl)-2-(thiophen-3-yl)acetamide (15). Prepared
according to general procedure A, using N-(3-iodo-1H-indazol-5-yl)-
2-(pyrrolidin-1-yl)-2-(thiophen-3-yl)acetamide (50 mg, 0.11 mmol),
1-methyl-4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-
piperazine (40 mg, 0.13 mmol), Pd(PPh3)4 (6.35 mg, 0.005 mmol), and
1 M aq Na2CO3 (0.22 mL) in PhMe/EtOH (2.25 mL, 2:1 mixture), 15
1
was obtained as a pale-yellow solid (TFA salt, 21 mg, 26%). H NMR
(400 MHz, CD3OD) δ 8.35 (s, 1H), 7.85−7.83 (m, 3H), 7.64 (br s, 1H),
7.54−7.49 (m, 2H), 7.38 (dd, J = 4.4 Hz, 1H), 7.19 (d, J = 8.4 Hz, 2H),
5.26 (s, 1H), 3.98−3.86 (m, 3H), 3.67−3.60 (m, 2H), 3.41−3.25 (3H
coincident with solvent peak), 3.17−3.11 (m, 4H), 3.00 (s, 3H), 2.26−
2.14 (m, 3H), 2.02−2.01 (br s, 1H). HRMS (ESI) m/z calcd for
[C28H32N6OS + H]+, 501.2437; found, 501.2426.
N-(3-(4-Morpholinophenyl)-1H-indazol-5-yl)-2-(pyrrolidin-1-yl)-
2-(thiophen-3-yl)acetamide (16). Prepared according to general
procedure A, using N-(3-iodo-1H-indazol-5-yl)-2-(pyrrolidin-1-yl)-
2-(thiophen-3-yl)acetamide (300 mg, 0.66 mmol) and (4-
morpholinophenyl)boronic acid (191 mg, 0.93 mmol), 16 was obtained
1
as a yellow powder (TFA salt, 265 mg, 56%). H NMR (400 MHz,
CD3OD) δ 8.38 (d, J = 1.2 Hz, 1H), 7.87 (dd, J = 3.0, 1.2 Hz, 1H), 7.80
(d, J = 8.8 Hz, 2H), 7.67 (dd, J = 5.0, 3.0 Hz, 1H), 7.53 (d, J = 9.0 Hz,
1H), 7.48 (dd, J = 9.0, 1.7 Hz, 1H), 7.38 (dd, J = 5.0, 1.2 Hz, 1H), 7.20
(d, J = 8.8 Hz, 2H), 5.20 (s, 1H), 3.90−3.84 (m, 5H), 3.30−3.25 (m,
4H), 3.24−3.13 (m, 1H), 3.10 (br s, 1H), 2.27−2.13 (m, 3H), 1.99 (br s,
1H). HRMS (ESI) m/z calcd for [C27H29N5O2S + H]+, 488.2120;
found, 488.2116.
N-(3-(4-((1-Methylpiperidin-4-yl)oxy)phenyl)-1H-indazol-5-yl)-
2-(pyrrolidin-1-yl)-2-(thiophen-3-yl)acetamide (17). Prepared ac-
cording to general procedure A, using N-(3-iodo-1H-indazol-5-yl)-2-
(pyrrolidin-1-yl)-2-(thiophen-3-yl)acetamide (400 mg, 0.88 mmol) and
1-methyl-4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)-
piperidine (368 mg, 1.05 mmol), 17 was obtained as an off-white solid
(TFA salt, 545 mg, 28%). 1H NMR (400 MHz, CD3OD) δ 8.36 (s, 1H),
7.91−7.82 (m, 3H), 7.64 (dd, J = 5.0, 3.0 Hz, 1H), 7.57−7.49 (m, 2H),
7.38 (dd, J = 5.1, 1.1 Hz, 1H), 7.22−7.11 (m, 2H), 5.29 (s, 1H), 4.73−
4.62 (m, 0.33H), 3.87 (br s, 1H), 3.60−3.69 (m, 0.67H), 3.49−3.34 (m,
3.33H), 3.27−3.02 (m, 3H), 2.93 (s, 3H), 2.43 (d, J = 14.3 Hz, 0.67H),
2.34−1.86 (m, 8H). HRMS (ESI) m/z calcd for [C29H33N5O2S + H]+,
516.2433; found, 516.2437.
N-(3-(4-(4-Hydroxypiperidin-1-yl)phenyl)-1H-indazol-5-yl)-
2-(pyrrolidin-1-yl)-2-(thiophen-3-yl)acetamide (18). Prepared
according to general procedure A, using N-(3-iodo-1H-indazol-5-yl)-
2-(pyrrolidin-1-yl)-2-thiophen-3-yl) acetamide (250 mg, 0.55 mmol),
1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)piperidin-4-
ol (168 mg, 0.55 mmol), Pd(PPh3)4 (48 mg, 0.041 mmol), and 1 M aq
Na2CO3 (1.11 mL) in toluene/EtOH (7.5 mL, 1:0.5 mixture), 3.5 h at
130 °C, 18 was obtained as a light-brown solid (TFA salt, 40 mg, 10%).
1H NMR (400 MHz, CD3OD) δ 8.42 (m, 1H), 8.13 (dd, J = 6.8, 2.0 Hz,
2H), 7.89 (dd, J = 2.8, 1.2 Hz, 1H), 7.79 (s, 1H), 7.77 (s, 1H), 7.64−7.62
(m, 1H), 7.59−7.53 (m, 2H), 7.39 (dd, J = 4.8, 1.2 Hz, 1H), 5.36 (s, 1H),
4.12−4.10 (m, 1H), 3.90−3.85 (m, 3H), 3.66−3.61(m, 2H), 3.30−3.05
(m, 2H), 2.29−2.01(m, 8H). HRMS (ESI) m/z calcd for [C28H31N5O2
S + H]+, 502.2277; found, 502.2270.
N-(Cyclopropyl(phenyl)methyl)-3-(3-morpholinophenyl)-
1H-indazole-5-carboxamide (35). Prepared according to general
procedure A, using N-(cyclopropyl(phenyl)methyl)-3-iodo-1H-indazole-
5-carboxamide (100 mg, 0.24 mmol) and 4-(3-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)phenyl)morpholine (69 mg, 0.24 mmol), 35 was
obtained as a beige solid (39 mg, 36%). 1H NMR (400 MHz, CD3OD) δ
8.58 (s, 1H), 7.94 (dd, J = 8.8, 1.4 Hz, 1H), 7.62 (d, J = 8.8 Hz, 1H), 7.55−
7.44 (m, 5H), 7.34 (t, J = 7.6 Hz, 2H), 7.24 (t, J = 7.3 Hz, 1H), 7.10−7.05
(m, 1H), 4.48 (d, J = 8.5 Hz, 1H), 3.89−3.84 (m, 4H), 3.27−3.22 (m,
4H), 1.44−1.33 (m, 1H), 0.69−0.62 (m, 2H), 0.54−0.41 (m, 2H). LCMS
(ESI) m/z calcd for [C28H28N4O2 + H]+, 453.2; found, 453.2.
N-(Cyclopropyl(phenyl)methyl)-3-(4-((1-methylpiperidin-4-
yl)oxy)phenyl)-1H-indazole-5-carboxamide (36). Prepared ac-
cording to general procedure A, using N-(cyclopropyl(phenyl)-
methyl)-3-iodo-1H-indazole-5-carboxamide (200 mg, 0.48 mmol) and
(4-((1-methylpiperidin-4-yl)oxy)phenyl)boronic acid pinacol ester
(152 mg, 0.48 mmol), 36 was obtained as a white solid (80 mg, 35%).
1H NMR (400 MHz, CD3OD) δ 8.60 (s, 1H), 7.95 (d, J = 9.0 Hz, 1H),
2-(1-Morpholinocyclohexyl)-N-(3-(4-morpholinophenyl)-1H-
indazol-5-yl)acetamide (19). Prepared according to general
procedure B, using 2-(1-morpholinocyclohexyl)acetic acid hydro-
chloride (100 mg, 0.34 mmol), 3-(4-morpholinophenyl)-1H-indazol-
5-amine (106 mg, 0.34 mmol), TBTU (109 mg, 0.34 mmol), DIPEA
(0.3 mL, 1.70 mmol), and DMF (3 mL), 19 was obtained as a yellow
solid (di-TFA salt, 87 mg, 35%). 1H NMR (400 MHz, CD3OD) δ 10.62
(s, 1 H), 8.41 (s, 1H), 7.91 (d, J = 8.8 Hz, 2H), 7.58−7.52 (m, 2H), 7.30
(d, J = 8.8 Hz, 2H), 4.18 (d, J = 11.6 Hz, 2H), 3.93−3.81 (m, 6H), 3.64
(d, J = 12.0 Hz, 2H), 3.37−3.24 (m, 5H), 3.20 (s, 2H), 1.94−1.80 (m,
7H), 1.68−1.62 (m, 2H), 1.36−1.25 (m, 1H). LCMS (ESI) m/z calcd
for [C29H37N5O3 + H]+, 504.3; found, 504.3.
7.90 (d, J = 8.8 Hz, 2H), 7.59 (d, J = 8.8 Hz, 1H), 7.47 (d, J = 7.5 Hz,
2H), 7.31 (t, J = 7.5 Hz, 2H), 7.22 (t, J = 7.5 Hz, 1H), 7.08 (d, J = 8.8 Hz,
R
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Journal of Medicinal Chemistry
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2H), 4.55 (br s, 1H), 4.47 (d, J = 9.3 Hz, 1H), 3.03−2.91 (m, 2H), 2.76−
2.63 (m, 2H), 2.50 (s, 3H), 2.14−2.02 (m, 2H), 1.98−1.85 (m, 2H),
1.44−1.34 (m, 1H), 0.64 (d, J = 8.3 Hz, 2H), 0.45 (dd, J = 9.5, 4.5 Hz,
2H). LCMS (ESI) m/z calcd for [C30H32N4O2 + H]+, 481.3; found,
481.4.
(R)-N-(1-(2-Chlorophenyl)propyl)-3-(4-((1-methylpiperidin-
4-yl)oxy)phenyl)-1H-indazole-5-carboxamide (51). Prepared ac-
cording to general procedure A, using (R)-N-(1-(2-chlorophenyl)-
propyl)-3-iodo-1H-indazole-5-carboxamide (58 mg, 0.13 mmol, 36% ee)
and 1-methyl-4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
phenoxy)piperidine (55 mg, 0.17 mmol), 51 was obtained as an orange
solid (TFA salt, 39 mg, 59%). 1H NMR (400 MHz, CD3OD) δ 8.57 (s,
1H), 7.95−7.91 (m, 3H), 7.61 (d, J = 8.8 Hz, 1H), 7.55 (d, J = 7.0 Hz,
1H), 7.39 (d, J = 8.0 Hz, 1H), 7.30−7.14 (m, 4H), 5.47−5.43 (m, 1H),
4.87 (br s, 1H), 3.65−3.17 (m, 4H), 2.93−2.92 (m, 3H), 2.44−2.27 (m,
2H), 2.15−1.86 (m, 4H), 1.07 (t, J = 7.2 Hz, 3H). HRMS (ESI) m/z
calcd for [C29H31ClN4O2 + H]+, 503.2214; found, 503.2214.
N-(Cyclopropyl(thiophen-3-yl)methyl)-3-(4-(4-hydroxypi-
peridin-1-yl)phenyl)-1H-indazole-5-carboxamide (37). Prepared
according to general procedure A, using N-(cyclopropyl(thiophen-
3-yl)methyl)-3-iodo-1H-indazole-5-carboxamide (50 mg, 0.12 mmol)
and 1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-
piperidin-4-ol (54 mg, 80% pure, 0.14 mmol), 37 was obtained as a
pale-pink solid (TFA salt, 27 mg, 39%). 1H NMR (400 MHz, CD3OD) δ
8.63 (s, 1H), 8.24 (d, J = 8.8 Hz, 2H), 7.99 (dd, J = 8.9, 1.4 Hz, 1H), 7.78
(d, J = 8.8 Hz, 2H), 7.67 (d, J = 8.8 Hz, 1H), 7.40−7.36 (m, 2H), 7.21 (t,
J = 3.3 Hz, 1H), 5.49 (s, 1H), 4.64 (d, J = 9.3 Hz, 1H), 4.11 (tt, J = 7.0, 3.3
Hz, 1H), 3.91 (ddd, J = 12.0, 8.4, 3.4 Hz, 2H), 3.65 (ddd, J = 11.9, 7.7, 3.5
Hz, 2H), 2.31−2.21 (m, 2H), 2.04 (dtd, J = 14.2, 7.2, 3.5 Hz, 2H), 1.51−
1.41 (m, 1H), 0.74 (td, J = 8.3, 3.8 Hz, 1H), 0.65 (td, J = 8.3, 4.8 Hz, 1H),
0.56−0.45 (m, 2H). HRMS (ESI) m/z calcd for [C27H28N4O2S + H]+,
473.2011; found, 473.2017.
3-(4-(4-Hydroxypiperidin-1-yl)phenyl)-N-((1-morpholino-
cyclohexyl)methyl)-1H-indazole-5-carboxamide (38). Prepared
according to general procedure A, using 3-iodo-N-((1-
morpholinocyclohexyl)methyl)-1H-indazole-5-carboxamide trifluoroa-
cetate (80 mg, 0.136 mmol), 1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxabor-
olan-2-yl)phenyl)piperidin-4-ol (70 mg, 0.18 mmol), and Pd(PPh3)4
(19 mg, 0.016 mmol), 38 was obtained as a pale-yellow solid (TFA salt,
31 mg, 30%). 1H NMR (400 MHz, CD3OD) δ 8.73 (s, 1H), 8.25 (d, J =
8.8 Hz, 2H), 8.04 (dd, J = 8.8, 1.6 Hz, 1H), 8.83 (d, J = 8.8 Hz, 2H), 7.71
(d, J = 8.8 Hz, 1H), 4.11−4.95 (m, 3H), 3.98−3.86 (m, 6H), 3.70−3.64
(m, 4H), 3.36−3.21 (1H coincident with solvent peak), 2.66 (s, 2H),
2.31−2.25 (m, 2H), 2.09−1.98 (m, 4H), 1.91−1.81(m, 2H), 1.81−1.75
(m,5H),1.42−1.21 (m, 1H). LCMS (ESI) m/z calcd for [C30H39N5O3 +H]+,
518.3; found, 518.2.
N-(Cyclopentyl(pyridin-2-yl)methyl)-3-(4-((1-methylpiperidin-
4-yl)oxy)phenyl)-1H-indazole-5-carboxamide (41). Prepared ac-
cording to general procedure A, using N-(cyclopentyl(pyridin-2-
yl)methyl)-3-iodo-1H-indazole-5-carboxamide (100 mg, 0.22 mmol),
and (4-((1-methylpiperidin-4-yl)oxy)phenyl)boronic acid pinacol ester
(70 mg, 0.22 mmol), and Pd(PPh3)4 (13 mg, 0.011 mmol), 41 was
obtained as a white solid (43 mg, 38%). 1H NMR (400 MHz, CD3OD) δ
8.57 (s, 1H), 8.51 (d, J = 4.3 Hz, 1H), 7.94−7.86 (m, 3H), 7.79 (t, J = 7.6
Hz, 1H), 7.59 (d, J = 8.8 Hz, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.29 (dd, J =
7.0, 5.0 Hz, 1H), 7.10 (d, J = 8.5 Hz, 2H), 5.01 (d, J = 10.3 Hz, 1H), 4.52
(br s, 1H), 2.82 (br s, 2H), 2.58−2.46 (m, 3H), 2.39 (s, 3H), 2.12−1.81
(m, 5H), 1.76−1.45 (m, 5H), 1.41−1.20 (m, 2H). LCMS (ESI) m/z
calcd for [C31H35N5O2 + H]+, 510.3; found, 510.2.
N-(Cyclopropyl(phenyl)methyl)-3-(4-((1-formylpiperidin-4-
yl)oxy)phenyl)-1H-indazole-5-carboxamide (42). Prepared ac-
cording to general procedure A, using N-(cyclopropyl(phenyl)-
methyl)-3-iodo-1H-indazole-5-carboxamide (43 mg, 0.10 mmol) and
4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)piperidine-
1-carbaldehyde (41 mg, 0.12 mmol), 42 was obtained as a yellow solid
(14 mg, 27%). 1H NMR (400 MHz, CD3OD) δ 8.61 (s, 1H), 8.03 (s,
1H), 7.98−7.89 (m, 3H), 7.59 (d, J = 8.8 Hz, 1H), 7.48 (d, J = 7.3 Hz,
2H), 7.32 (t, J = 7.5 Hz, 2H), 7.23 (m, J = 7.5 Hz, 1H), 7.12 (d, J = 8.8
Hz, 2H), 4.77−4.69 (m, 1H), 4.51−4.45 (m, 1H), 3.79−3.62 (m, 2H),
3.58−3.49 (m, 1H), 3.46−3.37 (m, 1H), 2.08−1.91 (m, 2H), 1.91−
1.72 (m, 2H), 1.46−1.34 (m, 1H), 0.65 (d, J = 8.3 Hz, 2H), 0.53−0.41
(m, 2H). LCMS (ESI) m/z calcd for [C30H30N4O3 + H]+, 495.2; found,
495.3.
N-(Cyclopropyl(pyridin-2-yl)methyl)-3-(4-((1-formylpiperi-
din-4-yl)oxy)phenyl)-1H-indazole-5-carboxamide (43). Prepared
according to general procedure A, using N-(cyclopropyl(pyridin-2-
yl)methyl)-3-iodo-1H-indazole-5-carboxamide (84 mg, 0.2 mmol) and
4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)piperidine-
1-carbaldehyde (74 mg, 0.22 mmol), 43 was obtained as a white solid
(36 mg, 29%). 1H NMR (400 MHz, CD3OD) δ 8.77 (dd, J = 5.7, 0.7 Hz,
1H), 8.70 (s, 1H), 8.56 (t, J = 7.1 Hz, 1H), 8.20 (d, J = 8.0 Hz, 1H), 8.05
(s, 1H), 8.01−7.90 (m, 4H), 7.63 (d, J = 8.8 Hz, 1H), 7.15 (d, J = 8.8 Hz,
2H), 4.80−4.72 (m, 1H), 4.49 (d, J = 10.0 Hz, 1H), 3.82−3.64 (m, 2H),
3.58−3.39 (m, 2H), 2.10−1.92 (m, 2H), 1.90−1.71 (m, 2H), 1.58−1.45
(m, 1H), 0.95−0.84 (m, 1H), 0.81−0.58 (m, 3H). HRMS (ESI) m/z
calcd for [C29H29N5O3 + H]+, 496.2349; found, 496.2355.
N-(Cyclopropyl(phenyl)methyl)-3-(4-((1-(2-(dimethylamino)-
acetyl)piperidin-4-yl)oxy)phenyl)-1H-indazole-5-carboxamide
(44). Prepared according to general procedure A, using N-cyclopropyl-
(phenyl)methyl)-3-iodo-1H-indazole-5-carboxamide (89 mg, 0.21
mmol) and 2-(dimethylamino)-1-(4-(4-(4,4,5,5-tetramethyl-1,3,2-diox-
aborolan-2-yl)phenoxy)piperidin-1-yl)ethanone (91 mg, 0.24 mmol),
1
44 was obtained as a pale-yellow solid (66 mg, 56%). H NMR (400
MHz, CD3OD) δ 8.60 (s, 1H), 7.95 (m, J = 8.5 Hz, 3H), 7.61 (d, J = 8.8
Hz, 1H), 7.50 (d, J = 7.0 Hz, 2H), 7.35 (t, J = 7.6 Hz, 2H), 7.25 (m, J =
7.3 Hz, 1H), 7.16 (d, J = 8.8 Hz, 2H), 4.80−4.73 (m, 1H), 4.49 (d, J = 9.5
Hz, 1H), 3.92−3.74 (m, 2H), 3.67 (s, 3H), 3.55−3.47 (m, 1H), 2.58 (s,
6H), 2.14−1.96 (m, 2H), 1.93−1.76 (m, 2H), 1.47−1.37 (m, 1H),
0.70−0.64 (m, 2H), 0.53−0.44 (m, 2H). LCMS (ESI) m/z calcd for
[C33H37N5O3 + H]+, 552.3; found, 552.5.
N-(Cyclopropyl(phenyl)methyl)-3-(4-((endo)-8-methyl-8-
azabicyclo[3.2.1]octan-3-yl)oxy)phenyl)-1H-indazole-5-car-
boxamide (45). Prepared according to general procedure A, using
2-cyclopropyl-N-(3-iodo-1H-indazol-5-yl)-2-phenylacetamide (90 mg,
0.22 mmol) and (endo)-8-methyl-3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxa-
borolan-2-yl)phenoxy)-8-azabicyclo[3.2.1]octane (0.14 g, 0.31 mmol),
45 was obtained as a white powder (7.5 mg, 7%). 1H NMR (400 MHz,
CD3OD) δ 8.61 (s, 1 H), 8.00−7.89 (m, 3H), 7.61 (d, J = 8.8 Hz, 1H),
7.49 (d, J = 7.2 Hz, 2H), 7.33 (t, J = 7.5 Hz, 2H), 7.24 (t, J = 7.3 Hz, 1H),
7.04 (d, J = 8.8 Hz, 2H), 4.69 (t, J = 4.7 Hz, 1H), 4.48 (d, J = 9.2 Hz, 1H),
3.44 (br s, 2H), 2.50 (s, 3H), 2.37−2.02 (m, 8H), 1.48−1.37 (m, 1H),
0.70−0.62 (m, 2H), 0.52−0.42 (m, 2H). HRMS (ESI) m/z calcd for
[C32H34N4O2 + H]+, 507.2760; found, 507.2758.
N-((1-Morpholinocyclohexyl)methyl)-3-(4-morpholinophenyl)-
1H-indazole-5-carboxamide (39). Prepared according to general
procedure A, using 3-iodo-N-((1-morpholinocyclohexyl)methyl)-1H-
indazole-5-carboxamide trifluoroacetate (70 mg, 0.12 mmol) and
(4-morpholinophenyl)boronic acid (35 mg, 0.17 mmol), 39 was
1
obtained as pale-yellow solid (TFA salt, 46 mg, 74%). H NMR (400
MHz, CD3OD) δ 8.54 (s, 1H), 7.89 (d, J = 9.0 Hz, 3H), 7.63 (d, J = 8.8
Hz, 1H), 7.13 (d, J = 9.0 Hz, 2H), 3.90−3.79 (m, 4H), 3.71−3.45 (m,
2H), 3.28−3.17 (m, 4H), 2.97−2.56 (m, 2H), 1.84−1.25 (m, 16H).
LCMS (ESI) m/z calcd for [C29H37N5O3 + H]+, 504.3; found, 504.3.
N-(Cyclopentyl(thiophen-3-yl)methyl)-3-(4-((1-methylpiperidin-
4-yl)oxy)phenyl)-1H-indazole-5-carboxamide (40). Prepared accord-
ing to general procedure A, using N-(cyclopentyl(thiophen-3-yl)-
methyl)-3-iodo-1H-indazole-5-carboxamide (150 mg, 0.332 mmol) and
1-methyl-4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)-
piperidine (106 mg, 0.334 mmol), 40 was obtained as an off-white solid
(TFA salt, 60 mg, 28%). 1H NMR (400 MHz, CD3OD) δ 8.52 (s, 1H),
7.94−7.88 (m, 3H), 7.60 (d, J = 8.8 Hz, 1H), 7.36−7.32 (m, 2H), 7.20−
7.13 (m, 3H), 5.08 (d, J = 6.0 Hz, 1H),4.85 (s, 1H), 3.66 (t, J = 13.6 Hz,
1H), 3.44−3.32 (m, 5H), 3.23 (br s, 1H), 2.59−2.53 (m, 1H),
2.45−2.27 (m, 2H), 2.16−1.87 (m, 3H), 1.74−1.44 (m, 6H), 1.28−1.25
(m, 1H). LCMS (ESI) m/z calcd for [C30H34N4O2S + H]+, 515.2;
found, 515.4.
N-(Cyclopropyl(phenyl)methyl)-3-(4-((exo)-8-methyl-8-
azabicyclo[3.2.1]octan-3-yl)oxy)phenyl)-1H-indazole-5-car-
boxamide (46). Prepared according to general procedure A, using
N-(cyclopropyl(phenyl)methyl)-3-iodo-1H-indazole-5-carboxamide
S
DOI: 10.1021/jm501740a
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(208 mg, 0.5 mmol) and (exo)-8-methyl-3-(4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)phenoxy)-8-azabicyclo[3.2.1]octane (137 mg, 0.4
mmol), 46 was obtained as a white solid (29 mg, 14%). 1H NMR (400
MHz, CD3OD) δ 8.62 (s, 1H), 7.96 (dd, J = 8.8, 1.6 Hz, 1H), 7.89 (d, J =
8.4 Hz, 2H), 7.59 (d, J = 8.8 Hz, 1H), 7.47 (d, J = 7.2 Hz, 2H), 7.32 (t, J =
7.6 Hz, 2H), 7.22 (t, J = 7.2 Hz, 1H), 7.05 (d, J = 8.8 Hz, 2H), 4.70−4.60
(m, 1H), 4.48 (d, J = 9.6 Hz, 1H), 3.30−3.23 (m, 2H), 2.33 (s, 3H), 2.17−
2.04 (m, 4H), 1.85−1.74 (m, 4H), 1.45−1.35 (m, 1H), 0.68−0.60 (m,
2H), 0.52−0.40 (m, 2H). HRMS (ESI) m/z calcd for [C32H34N4O2 +
H]+, 507.2760; found, 507.2752.
N-(Cyclobutyl(thiophen-3-yl)methyl)-3-(4-(((endo)-8-formyl-
8-azabicyclo[3.2.1]octan-3-yl)oxy)phenyl)-1H-indazole-5-car-
boxamide (47). Prepared according to general procedure A, using
N-(cyclobutyl(thiophen-3-yl)methyl)-3-iodo-1H-indazole-5-carboxamide
(56 mg, 0.13 mmol) and (endo)-3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)phenoxy)-8-azabicyclo[3.2.1]octane-8-carbaldehyde (50 mg,
0.14 mmol), 47 was obtained as a white solid (17 mg, 25%). 1H
NMR (400 MHz, CD3OD) δ 8.56 (s, 1H), 8.10 (s, 1H), 7.95−7.85 (m,
3H), 7.58 (d, J = 8.8 Hz, 1H), 7.32 (dd, J = 4.8, 3.0 Hz, 1H), 7.26 (s, 1H),
7.12 (d, J = 5.0 Hz, 1H), 6.98 (d, J = 8.8 Hz, 2H), 5.27 (d, J = 10.5 Hz,
1H), 4.74 (br s, 1H), 4.57−4.45 (m, 1H), 4.15 (br s, 1H), 2.93 (d, J = 9.8
Hz, 1H), 2.34−1.74 (m, 14H). HRMS (ESI) m/z calcd for [C31H32N4O3S +
H]+, 541.2273; found, 541.2275.
0.17 mmol), and Pd(PPh3)4 (15 mg, 0.012 mmol), 64 was obtained
as a yellow solid (HCl salt, 54 mg, 59%). 1H NMR (400 MHz, CD3OD)
δ 8.62 (s, 1H), 8.27 (d, J = 8.4 Hz, 2H), 7.99 (d, J = 8.8 Hz, 1H), 7.81 (d,
J = 8.4 Hz, 2H), 7.68 (d, J = 8.4 Hz, 1H), 7.58 (t, J = 6.4 Hz, 1H), 7.31−
7.26 (m, 1H), 7.17 (t, J = 7.6 Hz, 1H), 7.11−7.06 (m, 1H), 4.77 (d, J =
9.6 Hz, 1H), 4.71 (br s, 2H), 4.25 (d, J = 12.4 Hz, 2H), 3.99 (d, J = 12.8
Hz, 2H), 2.37−2.35 (m, 2H), 2.22−2.19 (m, 2H), 1.48−1.46 (m, 1H),
0.73−0.70 (m, 1H), 0.69−0.59 (m, 1H), 0.54−0.45 (m, 2H). LCMS
(ESI) m/z calcd for [C30H29FN4O2 + H]+, 497.2; found, 497.4.
3-(4-(3-Oxa-8-azabicyclo[3.2.1]octan-8-yl)phenyl)-N-((R)-
cyclopropyl(2-fluorophenyl)methyl)-1H-indazole-5-carboxa-
mide (65). Prepared according to general procedure A, using
(R)-N-(cyclopropyl(2-fluorophenyl)methyl)-3-iodo-1H-indazole-5-
carboxamide (80 mg, 0.18 mmol), 8-(4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)phenyl)-3-oxa-8-azabicyclo[3.2.1]octane (65 mg,
0.2 mmol), and Pd(PPh3)4 (10 mg, 0.009 mmol), 65 was obtained as
a yellow solid (TFA salt, 13 mg, 12%). 1H NMR (400 MHz, CD3OD) δ
8.61 (s, 1H), 7.97−7.84 (m, 3H), 7.63−7.50 (m, 2H), 7.31−7.22 (m,
1H), 7.16 (t, J = 7.2 Hz, 1H), 7.12−7.03 (m, 2H), 4.75 (d, J = 9.5 Hz,
1H), 4.24 (br s, 2H), 3.93 (d, J = 11.0 Hz, 2H), 3.57 (br s, 2H), 2.15−
1.97 (m, 4H), 1.48−1.40 (m, 1H), 0.72−0.62 (m, 1H), 0.62−0.53 (m,
1H), 0.53−0.38 (m, 3H). LCMS (ESI) m/z calcd for [C30H29FN4O2 + H]+,
497.2; found, 497.4.
3-((4-((endo)-8-Formyl-8-azabicyclo[3.2.1]octan-3-yl)oxy)-
phenyl)-N-(3-methyl-1-(pyridin-2-yl)butyl)-1H-indazole-5-car-
boxamide (48). Prepared according to general procedure A, using
3-iodo-N-(3-methyl-1-(pyridin-2-yl)butyl)-1H-indazole-5-carboxamide
(55 mg, 0.13 mmol) and (endo)-3-(4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)phenoxy)-8-azabicyclo[3.2.1]octane-8-carbaldehyde
(50 mg, 0.14 mmol), 48 was obtained as a white solid (32 mg, 47%). 1H
NMR (400 MHz, CD3OD) δ 8.65 (s, 1H), 8.49 (d, J = 5.0 Hz, 1H), 8.08
(s, 1H), 7.97 (dd, J = 8.8, 1.5 Hz, 1H), 7.90−7.86 (m, 2H), 7.78−7.71
(m, 1H), 7.59 (d, J = 8.8 Hz, 1H), 7.47 (d, J = 7.8 Hz, 1H), 7.29−7.20
(m, 1H), 6.98−6.68 (m, 2H), 5.36 (dd, J = 9.8, 5.3 Hz, 1H), 4.68 (br s,
1H), 4.50 (br s, 1H), 4.11 (br s, 1H), 2.29−2.15 (m, 2H), 2.15−1.97 (m,
4H), 1.97−1.81 (m, 3H), 1.81−1.62 (m, 2H), 1.06−0.91 (m, 6H).
LCMS (ESI) m/z calcd for [C32H35N5O3 + H]+, 538.3; found, 538.4.
3-(4-(8-Oxa-3-azabicyclo[3.2.1]octan-3-yl)phenyl)-N-((1-
morpholinocyclohexyl)methyl)-1H-indazole-5-carboxamide
(49). Prepared according to general procedure A, using 3-iodo-N-((1-
morpholinocyclohexyl)methyl)-1H-indazole-5-carboxamide (80 mg,
0.17 mmol), 3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-8-
oxa-3-azabicyclo[3.2.1]octane (54 mg, 0.17 mmol), and PdCl2dppf·CH2Cl2
(14 mg, 0.017 mmol), 49 was obtained as a yellow solid (di-TFA salt,
36 mg, 31%). 1H NMR (400 MHz, CD3OD) δ 8.68 (s, 1H), 8.00 (d, J =
9.3 Hz, 1H), 7.87 (d, J = 8.3 Hz, 2H), 7.64 (d, J = 8.8 Hz, 1H), 7.03 (d,
J = 8.0 Hz, 2H), 4.51 (br s, 2H), 4.16 (d, J = 12.8 Hz, 2H), 3.98−3.82 (m,
4H), 3.66 (d, J = 12.3 Hz, 2H), 3.51 (d, J = 11.5 Hz, 2H), 2.98 (d, J = 11.5
Hz, 2H), 2.06−1.85 (m, 9H), 1.84−1.55 (m, 6H), 1.39−1.24 (m, 1H).
HRMS (ESI) m/z calcd for [C31H39N5O3 + H]+, 530.3131; found,
530.3125.
3-(4-(3-Oxa-8-azabicyclo[3.2.1]octan-8-yl)phenyl)-N-((S)-1-
(2-chlorophenyl)-2-methylpropyl)-1H-indazole-5-carboxamide
(66). Prepared according to general procedure A, using (S)-N-(1-(2-
chlorophenyl)-2-methylpropyl)-3-iodo-1H-indazole-5-carboxamide
(100 mg, 0.22 mmol) and 8-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)phenyl)-3-oxa-8-azabicyclo[3.2.1]octane (69 mg, 0.22 mmol), 66
was obtained as a yellow solid (TFA salt, 51 mg, 37%). 1H NMR (400
MHz, CD3OD) δ 8.54 (s, 1H), 7.93 (br s, 2H), 7.90−7.86 (m, 1H), 7.58
(d, J = 8.5 Hz, 1H), 7.54 (dd, J = 7.7, 1.6 Hz, 1H), 7.38 (dd, J = 7.9, 1.4
Hz, 1H), 7.32−7.27 (m, 1H), 7.25−7.13 (m, 3H), 5.37 (d, J = 9.8 Hz,
1H), 4.32 (br s, 2H), 4.00−3.95 (m, 2H), 3.64 (br s, 2H), 2.33−2.22 (m,
1H), 2.18−2.02 (m, 4H), 1.16 (d, J = 6.8 Hz, 3H), 0.86 (d, J = 6.5 Hz,
3H). LCMS (ESI) m/z calcd for [C30H31ClN4O2 + H]+, 515.2; found,
515.6.
3-(4-(3-Oxa-8-azabicyclo[3.2.1]octan-8-yl)phenyl)-N-((S)-
cyclopropyl(pyridin-2-yl)methyl)-1H-indazole-5-carboxamide
(67). Prepared according to general procedure A, using (S)-N-
(cyclopropyl(pyridin-2-yl)methyl)-3-iodo-1H-indazole-5-carboxamide
(100 mg, 0.24 mmol) and 8-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)phenyl)-3-oxa-8-azabicyclo[3.2.1]octane (76 mg, 0.24 mmol), 67
was obtained as a yellow solid (di-TFA salt, 64 mg, 38%). 1H NMR (400
MHz, CD3OD) δ 8.73−8.64 (m, 2H), 8.40 (td, J = 7.9, 1.8 Hz, 1H), 8.05
(d, J = 8.0 Hz, 1H), 7.96 (dd, J = 8.8, 1.5 Hz, 1H), 7.87 (d, J = 8.0 Hz,
2H), 7.83−7.77 (m, 1H), 7.60 (d, J = 8.8 Hz, 1H), 7.02 (br s, 2H), 4.48
(d, J = 10.0 Hz, 1H), 4.19 (br s, 2H), 3.90 (d, J = 10.8 Hz, 2H), 3.53 (br s,
2H), 2.12−1.96 (m, 4H), 1.54−1.42 (m, 1H), 0.89−0.80 (m, 1H),
0.75−0.56 (m, 3H). LCMS (ESI) m/z calcd for [C29H29N5O2 + H]+,
480.2; found, 480.2.
3-(4-(3-Oxa-8-azabicyclo[3.2.1]octan-8-yl)phenyl)-N-((R)-
cyclopropyl(pyridin-2-yl)methyl)-1H-indazole-5-carboxamide
(68). Prepared according to general procedure A, using (R)-N-
(cyclopropyl(pyridin-2-yl)methyl)-3-iodo-1H-indazole-5-carboxamide
(100 mg, 0.24 mmol) and 8-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)phenyl)-3-oxa-8-azabicyclo[3.2.1]octane (76 mg, 0.24 mmol), 68
was obtained as a yellow solid (di-HCl salt, 68 mg, 51%). 1H NMR (400
MHz, CD3OD) δ 8.83−8.78 (m, 2H), 8.66 (t, J = 8.4 Hz, 1H), 8.32 (d,
J = 8.0 Hz, 1 H), 8.28 (d, J = 8.5 Hz, 2H), 8.05−8.00 (m, 2H), 7.75 (d, J =
8.5 Hz, 2H), 7.69 (d, J = 8.8 Hz, 1H), 4.68 (br s, 2H), 4.51 (d, J = 10.3
Hz, 1H), 4.21 (d, J = 12.5 Hz, 2H), 3.93 (d, J = 12.0 Hz, 2H), 2.38−2.30
(m, 2H), 2.24−2.16 (m, 2H), 1.65−1.56 (m, 1H), 0.98−0.89 (m, 1H),
0.82−0.62 (m, 3H). HRMS (ESI) m/z calcd for [C29H29N5O2 + H]+,
480.2400; found, 480.2399.
3-(4-(3-Oxa-8-azabicyclo[3.2.1]octan-8-yl)phenyl)-N-((1-
morpholinocyclohexyl)methyl)-1H-indazole-5-carboxamide
(50). Prepared according to general procedure A, using 3-iodo-N-((1-
morpholinocyclohexyl)methyl)-1H-indazole-5-carboxamide (100 mg,
0.21 mmol) and 8-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
phenyl)-3-oxa-8-azabicyclo[3.2.1]octane (67 mg, 0.21 mmol), 50 was
1
obtained as a yellow solid (di-TFA salt, 70 mg, 44%). H NMR (400
MHz, CD3OD) δ 8.67 (s, 1H), 7.99 (dd, J = 8.9, 1.6 Hz, 1H), 7.86 (d, J =
8.8 Hz, 2 H), 7.63 (dd, J = 8.9, 0.6 Hz, 1H), 7.03 (d, J = 8.5 Hz, 2H), 4.21
(br s, 2H), 4.15 (d, J = 12.5 Hz, 2H), 3.95−3.82 (m, 6H), 3.65 (d, J =
11.8 Hz, 2H), 3.54 (d, J = 10.8 Hz, 2H), 3.33−3.30 (m, 2H), 2.13−2.00
(m, 4H), 1.99−1.93 (m, 2H), 1.92−1.84 (m, 2H), 1.83−1.70 (m, 3H),
1.69−1.55 (m, 2H), 1.36−1.23 (m, 1H). HRMS (ESI) m/z calcd for
[C31H39N5O3 + H]+, 530.3131; found, 530.3124.
3-(4-(3-Oxa-8-azabicyclo[3.2.1]octan-8-yl)phenyl)-N-((S)-
cyclopropyl(2-fluorophenyl)methyl)-1H-indazole-5-carboxa-
mide (64). Prepared according to general procedure A, using
(S)-N-(cyclopropyl(2-fluorophenyl)methyl)-3-iodo-1H-indazole-5-car-
boxamide (75 mg, 0.17 mmol), 8-(4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)phenyl)-3-oxa-8-azabicyclo[3.2.1]octane (55 mg,
3-(4-(3-Oxa-8-azabicyclo[3.2.1]octan-8-yl)phenyl)-N-((S)-
cyclopentyl(pyridin-2-yl)methyl)-1H-indazole-5-carboxamide
(69). Prepared according to general procedure A, using (S)-N-
(cyclopentyl(pyridin-2-yl)methyl)-3-iodo-1H-indazole-5-carboxamide
(100 mg, 0.22 mmol) and 8-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)phenyl)-3-oxa-8-azabicyclo[3.2.1]octane (69 mg, 0.22 mmol),
T
DOI: 10.1021/jm501740a
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Journal of Medicinal Chemistry
Article
69 was obtained as a yellow solid (di-TFA salt, 32 mg, 20%, >99.9% ee).
1H NMR (400 MHz, CD3OD) δ 8.78 (d, J = 6.0 Hz, 1H), 8.61 (s, 1H),
8.55 (t, J = 7.9 Hz, 1H), 8.09 (d, J = 8.0 Hz, 1H), 7.98−7.81 (m, 4H),
7.60 (d, J = 8.3 Hz, 1H), 7.08 (br s, 2H), 5.01 (d, J = 10.8 Hz, 1H), 4.25
(br s, 2H), 3.94 (d, J = 11.0 Hz, 2H), 3.58 (br s, 2H), 2.68−2.55 (m, 1H),
2.26−2.15 (m, 1H), 2.14−2.00 (m, 4H), 1.84−1.67 (m, 3H), 1.67−1.53
(m, 2H), 1.49−1.39 (m, 1H), 1.29−1.18 (m, 1H). HRMS (ESI) m/z
calcd for [C31H33N5O2 + H]+, 508.2713; found, 508.2709. Optical
rotation (69-2HCl): [α]23D = +90.9° (c 0.473, MeOH). Chiral HPLC
retention time (69-2HCl): 16.6 min; Daicel Chiralpak IA (150 mm ×
N-((S)-Cyclopropyl(pyridin-2-yl)methyl)-3-(4-((endo)-3-hy-
droxy-8-azabicyclo[3.2.1]octan-8-yl)phenyl)-1H-indazole-5-
carboxamide (74). Prepared according to general procedure A, using
(S)-N-(cyclopropyl(pyridin-2-yl)methyl)-3-iodo-1H-indazole-5-car-
boxamide (106 mg, 0.25 mmol) and (endo)-8-(4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)phenyl)-8-azabicyclo[3.2.1]octan-3-ol (100 mg,
0.3 mmol), 74 was obtained as a yellow solid (13 mg, 10%, >99.9% ee). 1H
NMR (400 MHz, CD3OD) δ 8.67 (d, J = 0.8 Hz, 1H), 8.51 (d, J = 4.0 Hz,
1H), 7.95 (dd, J = 8.8, 1.5 Hz, 1H), 7.88−7.76 (m, 3H), 7.57 (d, J = 8.5
Hz, 1H), 7.53 (d, J = 8.0 Hz, 1H), 7.33−7.25 (m, 1H), 6.94 (d, J = 8.8 Hz,
2H), 4.51 (d, J = 9.5 Hz, 1H), 4.25 (br s, 2H), 3.93 (s, 1H), 2.36 (d, J = 7.3
Hz, 2H), 2.22 (d, J = 14.3 Hz, 2H), 2.09−2.00 (m, 2H), 1.63 (d, J = 14.6
Hz, 2H), 1.49−1.34 (m, 1H), 0.72−0.63 (m, 1H), 0.61−0.45 (m, 3H).
HRMS (ESI) m/z calcd for [C30H31N5O2 + H]+, 494.2556; found,
494.2565. [α]23D = +80.4° (c 0.746, MeOH). Chiral HPLC retention time
(74-2HCl): 10.6 min; Daicel Chiralpak IA (150 mm × 4.6 mm); isocratic
40% iPrOH (0.5% Et2NH) in hexane; 1 mL/min, λ = 254 nm.
N-((R)-Cyclopropyl(pyridin-2-yl)methyl)-3-(4-((endo)-3-hy-
droxy-8-azabicyclo[3.2.1]octan-8-yl)phenyl)-1H-indazole-5-
carboxamide (75). A mixture of (R)-N-(cyclopropyl(pyridin-2-
yl)methyl)-3-iodo-1H-indazole-5-carboxamide (4.95 g, 11.8 mmol)
and (endo)-8-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-
8-azabicyclo[3.2.1]octan-3-ol (4.29 g, 13.04 mmol) was treated with
EtOH/PhMe (100 mL/50 mL), 1 M aq Na2CO3 (23.7 mL, 2.7 mmol,
2 equiv), followed by Pd(PPh3)4 (685 mg, 0.593 mmol, 5 mol %). The
resulting mixture was refluxed (oil bath temperature at 110 °C) under
argon for 1.5 days; LCMS showed the reaction was incomplete.
Additional portions of 1 M aq Na2CO3 (14.2 mL, 1.2 equiv) and
Pd(PPh3)4 (685 mg, 0.59 mmol) were added, and the resulting mixture
was refluxed (oil bath temperature at 110 °C) for 1 day. Water (100 mL)
and brine (50 mL) were added, followed by EtOAc (300 mL). Upon
separation, the aqueous layer was extracted with EtOAc (2 × 60 mL)
and the combined organic layers were dried over Na2SO4 and
concentrated. The resulting dark-brown oil was purified by flash
chromatography using two Biotage 100g SiO2 columns (10−100%
EtOAc in hexanes, followed by 0−10% MeOH in DCM). The impure
fractions were combined and purified by flash chromatography for a
second time. All clean fractions were combined and dried to give 75
(2.64 g, 45%, >99.9% ee) as a light-brown foam. 1H NMR (400 MHz,
CD3OD) 8.67 (s, 1H), 8.51 (d, J = 4.8 Hz, 1H), 7.95 (d, J = 8.4 Hz, 1H),
7.85 (d, J = 8.8 Hz, 2H), 7.80 (dt, J = 8.0, 1.6 Hz, 1H), 7.57 (d, J = 8.8 Hz,
1H), 7.53 (d, J = 8.0 Hz, 1H), 7.30 (dd, J = 5.2, 2.0 Hz, 1H), 6.94 (d, J =
8.8 Hz, 2H), 4.50 (d, J = 9.2 Hz, 1H), 4.25 (br s, 2H), 3.93 (br t, J = 4.8
Hz, 1H), 2.39−2.36 (m, 2H), 2.26−2.18 (m, 2H), 2.07−2.02 (m, 2H),
1.63 (d, J = 14.8 Hz, 2H), 1.46−1.36 (m, 1H), 0.71−0.66 (m, 1H),
0.61−0.50 (m, 3H). HRMS (ESI) m/z calcd for [C30H31N5O2 + H]+,
494.2556; found, 494.2552. Optical rotation (75-2HCl): [α]23D = −81.2°
(c 1.13, MeOH). Chiral HPLC retention time (75-2HCl): 7.9 min; Daicel
Chiralpak IA (150 mm × 4.6 mm); isocratic 40% iPrOH (0.5% Et2NH) in
hexane; 1 mL/min, λ = 254 nm.
i
4.6 mm); isocratic 40% PrOH (0.5% Et2NH) in hexane; 1 mL/min,
λ = 254 nm.
3-(4-(3-Oxa-8-azabicyclo[3.2.1]octan-8-yl)phenyl)-N-((R)-
cyclopentyl(pyridin-2-yl)methyl)-1H-indazole-5-carboxamide
(70). Prepared according to general procedure A, using (R)-N-
(cyclopentyl(pyridin-2-yl)methyl)-3-iodo-1H-indazole-5-carboxamide
(100 mg, 0.22 mmol) and 8-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)phenyl)-3-oxa-8-azabicyclo[3.2.1]octane (69 mg, 0.22 mmol), 70
was obtained as a yellow solid (di-TFA salt, 93 mg, 58%). 1H NMR (400
MHz, CD3OD) δ 8.76 (d, J = 6.0 Hz, 1H), 8.60 (s, 1H), 8.53−8.46 (m,
1H), 8.05 (d, J = 8.3 Hz, 1H), 7.93−7.83 (m, 4H), 7.60 (d, J = 8.8 Hz,
1H), 7.10−7.01 (m, 2H), 5.01 (d, J = 10.8 Hz, 1 H), 4.24 (br s, 2H), 3.93
(d, J = 11.3 Hz, 2H), 3.57 (d, J = 11.5 Hz, 2H), 2.66−2.55 (m, 1H),
2.23−2.13 (m, 1H), 2.10 (br s, 4H), 1.84−1.67 (m, 3H), 1.67−1.54 (m,
2H), 1.48−1.38 (m, 1H), 1.30−1.19 (m, 1H). LCMS (ESI) m/z calcd
for [C31H33N5O2 + H]+, 508.3; found, 508.3.
3-(4-(3-Oxa-9-azabicyclo[3.3.1]nonan-9-yl)phenyl)-N-((S)-
cyclopentyl(pyridin-2-yl)methyl)-1H-indazole-5-carboxamide
(71). Prepared according to general procedure A, using (S)-N-
(cyclopentyl(pyridin-2-yl)methyl)-3-iodo-1H-indazole-5-carboxamide
(100 mg, 0.22 mmol) and 9-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)phenyl)-3-oxa-9-azabicyclo[3.3.1]nonane (74 mg, 0.22 mmol), 71
was obtained as a yellow solid (di-TFA salt, 49 mg, 30%). 1H NMR (400
MHz, CD3OD) δ 8.78 (d, J = 5.8 Hz, 1H), 8.61 (s, 1H), 8.56 (t, J = 7.3
Hz, 1H), 8.09 (d, J = 8.0 Hz, 1H), 7.95 (t, J = 6.8 Hz, 1H), 7.92−7.82 (m,
3H), 7.59 (d, J = 8.8 Hz, 1H), 7.04 (d, J = 8.5 Hz, 2H), 5.01 (d, J = 10.8
Hz, 1H), 4.05 (d, J = 11.0 Hz, 2H), 3.98 (d, J = 11.3 Hz, 2H), 3.92 (br s,
2H), 2.70−2.55 (m, 2H), 2.25−2.15 (m, 1H), 2.12−1.99 (m, 2H),
1.84−1.69 (m, 5H), 1.67−1.54 (m, 3H), 1.49−1.39 (m, 1H), 1.30−1.16
(m, 1H). LCMS (ESI) m/z calcd for [C32H35N5O2 + H]+, 522.3; found,
522.4.
N-((S)-1-(2-Chlorophenyl)-2-methylpropyl)-3-(4-((endo)-3-
hydroxy-8-azabicyclo[3.2.1]octan-8-yl)phenyl)-1H-indazole-5-
carboxamide (72). Prepared according to general procedure A, using
(S)-N-(1-(2-chlorophenyl)-2-methylpropyl)-3-iodo-1H-indazole-5-car-
boxamide (69 mg, 0.15 mmol) and (endo)-8-(4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)phenyl)-8-azabicyclo[3.2.1]octan-3-ol (50 mg,
0.15 mmol), 72 was obtained as a yellow solid (15 mg, 19%). 1H NMR
(400 MHz, CD3OD) δ 8.55 (dd, J = 1.6, 0.9 Hz, 1H), 7.87 (dd, J = 8.8,
1.5 Hz, 1H), 7.78 (d, J = 8.8 Hz, 2H), 7.58−7.50 (m, 2H), 7.36 (dd, J =
8.0, 1.3 Hz, 1H), 7.30−7.23 (m, 1H), 7.20 (dd, J = 8.0, 1.8 Hz, 1H), 6.89
(d, J = 8.8 Hz, 2H), 5.38 (d, J = 9.8 Hz, 1H), 4.21 (br s, 2H), 3.91 (s, 1H),
2.35 (d, J = 7.0 Hz, 2H), 2.31−2.23 (m, 1H), 2.19 (d, J = 14.8 Hz, 2H),
2.06−1.98 (m, 2H), 1.61 (d, J = 14.6 Hz, 2H), 1.14 (d, J = 6.5 Hz, 3H),
0.85 (d, J = 6.8 Hz, 3H). HRMS (ESI) m/z calcd for [C31H33ClN4O2 +
H]+, 529.2370; found, 529.2366.
N-((S)-Cyclopropyl(phenyl)methyl)-3-(4-((endo)-3-hydroxy-
8-azabicyclo[3.2.1]octan-8-yl)phenyl)-1H-indazole-5-carboxa-
mide (73). Prepared according to general procedure A, using (S)-N-
(cyclopropyl(phenyl)methyl)-3-iodo-1H-indazole-5-carboxamide (115 mg,
0.28 mmol) and (endo)-8-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)phenyl)-8-azabicyclo[3.2.1]octan-3-ol (100 mg, 0.3 mmol), 73 was
obtained as a yellow solid (40 mg, 29%). 1H NMR (400 MHz, CD3OD)
δ 8.64 (d, J = 0.8 Hz, 1H), 7.95 (dd, J = 8.9, 1.4 Hz, 1H), 7.82 (d, J = 8.5
Hz, 2H), 7.56 (d, J = 8.8 Hz, 1H), 7.47 (d, J = 7.5 Hz, 2H), 7.30 (t, J = 7.5
Hz, 2H), 7.21 (m, J = 7.5 Hz, 1H), 6.90 (d, J = 8.8 Hz, 2H), 4.47 (d, J =
9.5 Hz, 1H), 4.20 (br s, 2 H), 3.92−3.87 (m, 1 H), 2.34 (m, J = 7.0 Hz,
2H), 2.23−2.13 (m, 2H), 2.06−1.97 (m, 2H), 1.60 (d, J = 14.3 Hz, 2H),
1.45−1.31 (m, 1H), 0.67−0.56 (m, 2H), 0.51−0.36 (m, 2H). HRMS
(ESI) m/z calcd for [C31H32N4O2 + H]+, 493.2604; found, 493.2604.
N-((S)-Cyclopentyl(pyridin-2-yl)methyl)-3-(4-((endo)-3-hy-
droxy-8-azabicyclo[3.2.1]octan-8-yl)phenyl)-1H-indazole-5-
carboxamide (76). Prepared according to general procedure A, using
(S)-N-(cyclopentyl(pyridin-2-yl)methyl)-3-iodo-1H-indazole-5-car-
boxamide (86 mg, 0.19 mmol) and (endo)-8-(4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)phenyl)-8-azabicyclo[3.2.1]octan-3-ol (70 mg,
0.21 mmol), 76 was obtained as a white solid (33 mg, 33%). 1H NMR
(400 MHz, CD3OD) δ 8.61 (dd, J = 1.5, 0.8 Hz, 1H), 8.54−8.48 (m,
1H), 7.91 (dd, J = 8.8, 1.5 Hz, 1H), 7.84−7.73 (m, 3H), 7.56 (dd, J = 8.9,
0.6 Hz, 1H), 7.50−7.45 (m, 1H), 7.28 (ddd, J = 7.5, 5.0, 1.3 Hz, 1H),
6.90 (d, J = 8.8 Hz, 2H), 5.01 (d, J = 10.3 Hz, 1H), 4.22 (br s, 2H), 3.91
(t, J = 4.5 Hz, 1H), 2.58−2.46 (m, 1H), 2.40−2.29 (m, 2H), 2.19 (d, J =
11.0 Hz, 2H), 2.07−1.88 (m, 3H), 1.74−1.43 (m, 7H), 1.40−1.19 (m,
2H). HRMS (ESI) m/z calcd for [C32H35N5O2 + H]+, 522.2869; found,
522.2844
N-((R)-Cyclopentyl(pyridin-2-yl)methyl)-3-(4-((endo)-3-hy-
droxy-8-azabicyclo[3.2.1]octan-8-yl)phenyl)-1H-indazole-5-
carboxamide (77). Prepared according to general procedure A, using
(R)-N-(cyclopentyl(pyridin-2-yl)methyl)-3-iodo-1H-indazole-5-car-
boxamide (86 mg, 0.19 mmol) and (endo)-8-(4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)phenyl)-8-azabicyclo[3.2.1]octan-3-ol (70 mg,
U
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Journal of Medicinal Chemistry
Article
0.21 mmol), 77 was obtained as a yellow solid (34 mg, 24%). 1H NMR
(400 MHz, CD3OD) δ 8.61 (dd, J = 1.5, 0.8 Hz, 1H), 8.54−8.48 (m,
1H), 7.91 (dd, J = 8.8, 1.5 Hz, 1H), 7.84−7.73 (m, 3H), 7.56 (dd, J = 8.9,
0.6 Hz, 1H), 7.50−7.45 (m, 1H), 7.28 (ddd, J = 7.5, 5.0, 1.3 Hz, 1H),
6.90 (d, J = 8.8 Hz, 2H), 5.01 (d, J = 10.3 Hz, 1H), 4.22 (br s, 2H), 3.91 (t, J =
4.5 Hz, 1H), 2.58−2.46 (m, 1H), 2.40−2.29 (m, 2H), 2.19 (d, J = 11.0 Hz,
2H), 2.07−1.88 (m, 3H), 1.74−1.43 (m, 7H), 1.40−1.19 (m, 2H). LCMS
(ESI) m/z calcd for [C32H35N5O2 + H]+, 522.2869; found, 522.2844.
N-((S)-Cyclopentyl(pyrimidin-2-yl)methyl)-3-(4-((endo)-3-hy-
droxy-8-azabicyclo[3.2.1]octan-8-yl)phenyl)-1H-indazole-5-
carboxamide (78). Prepared according to general procedure A, using
(S)-N-(cyclopentyl(pyrimidin-2-yl)methyl)-3-iodo-1H-indazole-5-car-
boxamide (70 mg, 0.16 mmol) and (endo)-8-(4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)phenyl)-8-azabicyclo[3.2.1]octan-3-ol (52 mg,
0.16 mmol), 78 was obtained as a yellow solid (19 mg, 23%). 1H NMR
(400 MHz, CD3OD) δ 8.76 (d, J = 5.0 Hz, 2H), 8.63 (d, J = 0.5 Hz, 1H),
7.92 (dd, J = 8.9, 1.6 Hz, 1H), 7.83 (d, J = 8.8 Hz, 2H), 7.57 (d, J = 8.8
Hz, 1H), 7.35 (t, J = 4.9 Hz, 1H), 6.93 (d, J = 8.8 Hz, 2H), 5.18 (d, J = 9.5
Hz, 1H), 4.24 (br s, 2H), 3.92 (t, J = 4.4 Hz, 1H), 2.57 (m, J = 9.0 Hz,
1H), 2.40−2.31 (m, 2H), 2.21 (d, J = 15.1 Hz, 2H), 2.08−2.00 (m, 2H),
1.92 (d, J = 7.3 Hz, 1H), 1.75−1.47 (m, 7H), 1.45−1.36 (m, 2H). HRMS
(ESI) m/z calcd for [C31H34N6O2 + H]+, 523.2821; found, 523.2801.
N-((R)-Cyclopentyl(pyrimidin-2-yl)methyl)-3-(4-((endo)-3-hy-
droxy-8-azabicyclo[3.2.1]octan-8-yl)phenyl)-1H-indazole-5-
carboxamide (79). Prepared according to general procedure A, using
(R)-N-(cyclopentyl(pyrimidin-2-yl)methyl)-3-iodo-1H-indazole-5-car-
boxamide (70 mg, 0.16 mmol) and (endo)-8-(4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)phenyl)-8-azabicyclo[3.2.1]octan-3-ol (52 mg,
0.16 mmol), 79 was obtained as a yellow solid (30 mg, 37%). 1H NMR
(400 MHz, CD3OD) δ 8.80−8.72 (m, 2H), 8.65−8.60 (m, 1H), 7.92
(dd, J = 8.8, 1.5 Hz, 1H), 7.84 (d, J = 8.8 Hz, 2H), 7.57 (d, J = 8.8 Hz,
1H), 7.36 (t, J = 4.9 Hz, 1H), 6.94 (d, J = 8.8 Hz, 2H), 5.18 (d, J = 9.8 Hz,
1H), 4.25 (br s, 2H), 3.93 (s, 1H), 2.66−2.48 (m, 1H), 2.36 (d, J = 7.3
Hz, 2H), 2.21 (d, J = 14.6 Hz, 2H), 2.08−2.00 (m, 2H), 1.98−1.88 (m,
1H), 1.75−1.47 (m, 7H), 1.46−1.33 (m, 2H). LCMS (ESI) m/z calcd
for [C31H34N6O2 + H]+, 523.3; found, 523.4.
3-(4-((endo)-3-Hydroxy-8-azabicyclo[3.2.1]octan-8-yl)-
phenyl)-N-((S)-3-methyl-1-(pyridin-2-yl)butyl)-1H-indazole-5-
carboxamide (80). Prepared according to general procedure A, using
(S)-3-iodo-N-(3-methyl-1-(pyridin-2-yl)butyl)-1H-indazole-5-carboxa-
mide (133 mg, 0.3 mmol) and (endo)-8-(4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)phenyl)-8-azabicyclo[3.2.1]octan-3-ol (120 mg, 0.36
mmol), 80 was obtained as a yellow solid (di-HCl salt, 44 mg, 25%). 1H
NMR (free base, 400 MHz, CD3OD) δ 8.65 (d, J = 0.8 Hz, 1H), 8.52−
8.49 (m, 1H), 7.95 (dd, J = 8.8, 1.6 Hz, 1H), 7.81 (d, J = 8.8 Hz, 2H),
7.77 (dt, J = 7.6, 1.7 Hz, 1H), 7.57 (d, J = 8.8 Hz, 1H), 7.48 (d, J = 8.0 Hz,
1H), 7.30−7.26 (m, 1H), 6.92 (d, J = 8.8 Hz, 2H), 5.34 (dd, J = 10.0, 5.2
Hz, 1H), 4.25−4.18 (m, 2H), 3.92 (t, J = 4.6 Hz, 1H), 2.38−2.31 (m,
2H), 2.23−2.16 (m, 2H), 2.06−1.98 (m, 2H), 1.96−1.85 (m, 1H),
1.80−1.66 (m, 2H), 1.62 (d, J = 14.4 Hz, 2H), 1.03−0.97 (m, 6H).
LCMS (ESI) m/z calcd for [C31H35N5O2 + H]+, 510.3; found, 510.4.
3-(4-((endo)-3-Hydroxy-8-azabicyclo[3.2.1]octan-8-yl)-
phenyl)-N-((R)-3-methyl-1-(pyridin-2-yl)butyl)-1H-indazole-5-
carboxamide (81). Prepared according to general procedure A, using
(R)-3-iodo-N-(3-methyl-1-(pyridin-2-yl)butyl)-1H-indazole-5-carboxa-
mide (130 mg, 0.3 mmol) and (endo)-8-(4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)phenyl)-8-azabicyclo[3.2.1]octan-3-ol (109 mg, 0.33
mmol), 81 was obtained as a yellow solid (di-HCl salt, 52 mg, 30%). 1H
NMR (free base, 400 MHz, CD3OD) δ 8.66 (d, J = 0.8 Hz, 1H), 8.52−
8.49 (m, 1H), 7.95 (dd, J = 8.8, 1.6 Hz, 1H), 7.82 (d, J = 8.8 Hz, 2H),
7.77 (dt, J = 7.6, 1.7 Hz, 1H), 7.57 (d, J = 8.8 Hz, 1H), 7.48 (d, J = 8.0 Hz,
1H), 7.29−7.25 (m, 1H), 6.91 (d, J = 8.8 Hz, 2H), 5.35 (dd, J = 10.0, 5.2
Hz, 1H), 4.25−4.18 (m, 2H), 3.91 (t, J = 4.6 Hz, 1H), 2.38−2.31 (m,
2H), 2.23−2.16 (m, 2H), 2.06−1.98 (m, 2H), 1.96−1.85 (m, 1H),
1.80−1.66 (m, 2H), 1.61 (d, J = 14.4 Hz, 2H), 1.02−0.97 (m, 6H).
LCMS (ESI) m/z calcd for [C31H35N5O2 + H]+, 510.3; found, 510.3.
N-((R)-2-Cyclopropyl-1-(pyridin-2-yl)ethyl)-3-(4-((endo)-3-hy-
droxy-8-azabicyclo[3.2.1]octan-8-yl)phenyl)-1H-indazole-5-
carboxamide (82). Prepared according to general procedure A, using
(R)-N-(2-cyclopropyl-1-(pyridin-2-yl)ethyl)-3-iodo-1H-indazole-5-car-
boxamide (166 mg, 0.4 mmol) and (endo)-8-(4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)phenyl)-8-azabicyclo[3.2.1]octan-3-ol (132 mg,
0.4 mmol), 82 was obtained as a yellow solid (di-HCl salt, 67 mg, 29%).
1H NMR (free base, 400 MHz, CD3OD) δ 8.68−8.61 (m, 1H), 8.53 (d,
J = 5.0 Hz, 1H), 7.94 (d, J = 8.5 Hz, 1H), 7.86 (d, J = 8.5 Hz, 2H), 7.82
(td, J = 7.7, 1.88 Hz, 1H), 7.59 (d, J = 9.0 Hz, 1H), 7.52 (d, J = 7.8 Hz,
1H), 7.34−7.28 (m, 1H), 6.99 (d, J = 8.8 Hz, 2H), 5.34 (dd, J = 8.7, 6.40
Hz, 1H), 4.32−4.26 (m, 2H), 3.99−3.93 (m, 1H), 2.42−2.35 (m, 2H),
2.30−2.22 (m, 2H), 2.10−2.03 (m, 2H), 1.99−1.90 (m, 1H), 1.88−1.78
(m, 1H), 1.70−1.63 (m, 2H), 0.85−0.76 (m, 1H), 0.56−0.39 (m, 2H),
0.25−0.17 (m, 1H), 0.11−0.03 (m, 1H). HRMS (ESI) m/z calcd for
[C31H33N5O2 + H]+, 508.2713; found, 508.2712.
3-(4-((endo)-3-Hydroxy-9-azabicyclo[3.3.1]nonan-9-yl)-
phenyl)-N-((S)-3-methyl-1-(pyridin-2-yl)butyl)-1H-indazole-5-
carboxamide (83). Prepared according to general procedure A, using
(S)-3-iodo-N-(3-methyl-1-(pyridin-2-yl)butyl)-1H-indazole-5-carboxa-
mide (130 mg, 0.3 mmol) and (endo)-9-(4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)phenyl)-9-azabicyclo[3.3.1]nonan-3-ol (113 mg, 0.33
mmol), 83 was obtained as a yellow solid (di-HCl salt, 36 mg, 20%). 1H
NMR (free base, 400 MHz, CD3OD) δ 8.66 (d, J = 0.4 Hz, 1H), 8.52−8.48
(m, 1H), 7.95(dd, J =9.0, 1.4 Hz, 1H), 7.80(d, J = 8.4 Hz, 2H), 7.76 (dd, J =
7.8, 1.8 Hz, 1H), 7.58 (d, J = 8.8 Hz, 1H), 7.48 (d, J =7.6Hz, 1H), 7.29−7.25
(m, 1H), 7.98 (d, J = 8.8 Hz, 2H), 5.38−5.32 (m, 1H), 4.35−4.27 (m, 2H),
3.75−3.66 (m, 1H), 2.47−2.37 (m, 2H), 2.30−2.15 (m, 1H), 1.95−1.85 (m,
1H), 1.85−1.77 (m, 4H), 1.68−1.40 (m, 5H), 1.05−0.97 (m, 6H). LCMS
(ESI) m/z calcd for [C32H37N5O2 + H]+, 524.3; found, 524.3.
3-(4-((endo)-7-Hydroxy-3-oxa-9-azabicyclo[3.3.1]nonan-9-
yl)phenyl)-N-((S)-3-methyl-1-(pyridin-2-yl)butyl)-1H-indazole-
5-carboxamide (84). Prepared according to general procedure A,
using (S)-3-iodo-N-(3-methyl-1-(pyridin-2-yl)butyl)-1H-indazole-5-
carboxamide (130 mg, 0.3 mmol) and (endo)-9-(4-(4,4,5,5-tetrameth-
yl-1,3,2-dioxaborolan-2-yl)phenyl)-3-oxa-9-azabicyclo[3.3.1]nonan-7-
ol (99 mg, 0.3 mmol), 84 was obtained as a yellow solid (di-HCl salt, 59
mg, 33%). 1H NMR (400 MHz, CD3OD) δ 8.88 (s, 1H), 8.32 (d, J = 4.8
Hz, 1H), 8.70−8.62 (m, 1H), 8.28 (d, J = 6.4 Hz, 1H), 8.20 (d, J = 8.8
Hz, 1H), 8.08−7.98 (m, 3H), 7.76 (d, J = 8.8 Hz, 1H), 7.22 (d, J = 6.4
Hz, 2H), 5.53−5.45 (m, 1H), 4.19 (br s, 2H), 4.07−3.95 (m, 4H), 3.92−
3.87 (m, 1H), 2.40−2.31 (m, 3H), 1.91−1.78 (m, 4H), 1.08 (d, J = 5.2
Hz, 3H), 1.06 (d, J = 5.2 Hz, 3H). LCMS (ESI) m/z calcd for
[C31H35N5O3 + H]+, 526.3; found, 526.4.
N-((R)-Cyclopentyl(pyridin-2-yl)methyl)-3-(4-((endo)-7-hy-
droxy-3-oxa-9-azabicyclo[3.3.1]nonan-9-yl)phenyl)-1H-inda-
zole-5-carboxamide (85). Prepared according to general procedure
A, using (R)-N-(cyclopentyl(pyridin-2-yl)methyl)-3-iodo-1H-indazole-
5-carboxamide (100 mg, 0.22 mmol) and (endo)-9-(4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-3-oxa-9-azabicyclo[3.3.1]-
nonan-7-ol (76 mg, 0.22 mmol), 85 was obtained as a yellow solid (di-
TFA salt, 68 mg, 40%). 1H NMR (400 MHz, CD3OD) δ 8.76 (d, J = 5.0
Hz, 1H), 8.60 (s, 1H), 8.48 (td, J = 7.9, 1.5 Hz, 1H), 8.04 (d, J = 8.0 Hz, 1H),
7.93−7.84 (m, 4H), 7.59 (d, J = 8.8 Hz, 1H), 7.05 (d, J = 8.5 Hz, 2H), 5.01
(d, J = 10.8 Hz, 1H), 4.09−4.03 (m, 2H), 4.02−3.91 (m, 4H), 3.88 (br s,
1H), 2.66−2.54 (m, 1H), 2.37−2.27 (m, 2H), 2.22−2.13 (m, 1H), 1.82−
1.67 (m, 5H), 1.66−1.53 (m, 2H), 1.48−1.38 (m, 1H), 1.29−1.18 (m, 1H).
LCMS (ESI) m/z calcd for [C32H35N5O3 + H]+, 538.3; found, 538.5.
N-((S)-Cyclopropyl(pyridin-2-yl)methyl)-3-(4-((exo)-3-hy-
droxy-8-azabicyclo[3.2.1]octan-8-yl)phenyl)-1H-indazole-5-
carboxamide (86). Prepared according to general procedure A, using
(S)-N-(cyclopropyl(pyridin-2-yl)methyl)-3-iodo-1H-indazole-5-car-
boxamide (125 mg, 0.3 mmol) and (exo)-8-(4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)phenyl)-8-azabicyclo[3.2.1]octan-3-ol (99 mg,
0.3 mmol), 86 was obtained as a yellow solid (di-HCl salt, 65 mg, 38%,
1
>99.9% ee). H NMR (free base, 400 MHz, CD3OD) δ 8.67 (s, 1H),
8.51 (d, J = 4.8 Hz, 1H), 7.95 (dd, J = 8.8, 1.2 Hz, 1H), 7.86 (d, J = 8.8
Hz, 2H), 7.79 (dt, J = 8.0, 1.7 Hz, 1H), 7.57 (d, J = 7.2 Hz, 1H), 7.52 (d,
J = 8.0 Hz, 1H), 7.29 (dd, J = 7.4, 5.0 Hz, 1H), 6.96 (d, J = 8.8 Hz, 2H),
4.51 (d, J = 8.8 Hz, 1H), 4.33 (s, 2H), 4.20−4.08 (m, 1H), 2.14−1.97
(m, 2H), 1.90−1.68 (m, 6H), 1.45−1.35 (m, 1H), 0.72−0.64 (m, 1H),
0.60−0.48 (m, 3H). HRMS (ESI) m/z calcd for [C30H31N5O2 + H]+,
494.2556; found, 494.2563. Optical rotation (86-2HCl): [α]23D = +82.0°
(c 0.305, MeOH). Chiral HPLC retention time (86-2HCl): 13.7 min;
Daicel Chiralpak IA (150 mm × 4.6 mm); isocratic 40% iPrOH (0.5%
Et2NH) in hexane; 1 mL/min, λ = 254 nm.
V
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Journal of Medicinal Chemistry
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N-((R)-Cyclopropyl(pyridin-2-yl)methyl)-3-(4-((exo)-3-hy-
droxy-8-azabicyclo[3.2.1]octan-8-yl)phenyl)-1H-indazole-5-
carboxamide (87). Prepared according to general procedure A, using
(R)-N-(cyclopropyl(pyridin-2-yl)methyl)-3-iodo-1H-indazole-5-car-
boxamide (92 mg, 0.22 mmol) and (exo)-8-(4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)phenyl)-8-azabicyclo[3.2.1]octan-3-ol (80 mg,
0.24 mmol), 87 was obtained as a yellow solid (58 mg, 53%, >99.9% ee).
1H NMR (400 MHz, CD3OD) δ 8.68 (d, J = 0.8 Hz, 1H), 8.53−8.45 (m,
microtiter plates, coated with 5 μg/well His6-SUMO-TTK-N, as
previously described.25
Compound inhibition was determined at either a fixed concentration
(10 μM) or at a variable inhibitor concentration (typically 50 μM to
0.1 μM in a 10 point dose response titration). Compounds were
preincubated in the presence of enzyme for 15 min prior to addition of
ATP, and the activity remaining quantified using the above-described
activity assay. The % inhibition of a compound was determined using the
following formula: % inhibition = 100(1 − (experimental value−
background value)/(high activity control−background value)). The
IC50 value was determined using a nonlinear 4 point logistic curve fit
(XLfit4, IDBS) with the formula; (A + (B/(1 + ((x/C)D)))), where A =
background value, B = range, C = inflection point, D = curve fit parameter.
The apparent Ki for compound 75 was estimated to be 0.7 0.5 nM
by varying the ATP concentration in a series of TTK inhibition experi-
ments at 2.2, 6.6, and 11 nM of inhibitor and applying nonlinear
regression analysis (see Supporting Information).
1H), 7.95 (dd, J = 8.9, 1.4 Hz, 1H), 7.84 (d, J = 8.5 Hz, 2H), 7.76 (td, J =
7.8, 1.8 Hz, 1H), 7.56 (d, J = 8.8 Hz, 1H), 7.50 (d, J = 7.8 Hz, 1H), 7.26
(ddd, J = 7.5, 5.0, 1.0 Hz, 1H), 6.92 (d, J = 8.8 Hz, 2H), 4.51 (d, J = 9.3
Hz, 1H), 4.29 (br s, 2H), 4.12 (tt, J = 11.0, 5.2 Hz, 1H), 2.07−1.97 (m,
2H), 1.84−1.65 (m, 6H), 1.48−1.28 (m, 1H), 0.70−0.62 (m, 1H),
0.59−0.46 (m, 3H). LCMS (ESI) m/z calcd for [C30H31N5O2 + H]+,
494.3; found, 494.5. [α]23D = −85.8° (c 0.746, MeOH). Chiral HPLC
retention time (87-2HCl): 9.15 min; Daicel Chiralpak IA (150 mm ×
i
4.6 mm); isocratic 40% PrOH (0.5% Et2NH) in hexane; 1 mL/min,
Biochemical Assays. Active PLK4 was purified and used to measure
PLK4 activity, using an indirect ELISA detection system, as previously
described.56 KDR, AURKA, and AURKB/INCENP compound inhibition
were determined using FRET based homogeneous assay kits from
Invitrogen (Life Technologies, Burlington, Ontario). Recombinant
Aurora A and KDR were purchased from Invitrogen; AURKB/INCENP
was purchased from Millipore (Billerica, MA). The assays were performed
according to the manufacturer’s specifications with ATP concentrations
of 156, 20, and 128 μM, respectively for KDR, AURKA, and AURKB/
INCENP.25,56 Compound inhibition was determined at either a fixed
concentration (10 μM) or at a variable inhibitor concentrations (as
described above) to determine an IC50. The KDR, AURKA, and AURKB/
INCENP IC50 values given in the Tables 1−5 are the result of single
determinations wherein standard fitting errors are given.
The selectivity of inhibitor 75 against 278 protein kinases was
assessed by Millipore (Dundee, Scotland) using a radiometric assay.
Inhibition was determined at 0.1 μM inhibitor concentration with ATP
at Km; % inhibition was determined (see Supporting Information). For
compound 75, the inhibition constants (IC50s) were determined by
Millipore, as per the suppliers specifications, against selected kinases:
cKit (V560G), cKit, Ret (V804L), Ret (V804M), Ret, JNK3, MAPK1,
MAPK2, MELK, and MUSK.
λ = 254 nm.
N-((S)-Cyclopentyl(pyridin-2-yl)methyl)-3-(4-((exo)-3-hy-
droxy-8-azabicyclo[3.2.1]octan-8-yl)phenyl)-1H-indazole-5-
carboxamide (88). Prepared according to general procedure A, using
(S)-N-(cyclopentyl(pyridin-2-yl)methyl)-3-iodo-1H-indazole-5-car-
boxamide (99 mg, 0.22 mmol) and (exo)-8-(4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)phenyl)-8-azabicyclo[3.2.1]octan-3-ol (80 mg,
0.24 mmol), 88 was obtained as a yellow solid (66 mg, 57%). 1H NMR
(400 MHz, CD3OD) δ 8.59 (s, 1 H), 8.53 (d, J = 4.0 Hz, 1H), 7.91 (d, J =
8.8 Hz, 1H), 7.86 (d, J = 8.8 Hz, 2H), 7.83−7.79 (m, 1H), 7.58 (d,
J = 8.8 Hz, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.35−7.27 (m, 1H), 7.01 (d, J =
8.8 Hz, 2H), 5.02 (d, J = 10.3 Hz, 1H), 4.38 (br s, 1H), 4.23−4.10 (m,
1H), 2.64−2.47 (m, 1H), 2.09 (d, J = 5.5 Hz, 2H), 2.04−1.93 (m, 1H),
1.89−1.48 (m, 12H), 1.43−1.24 (m, 2H). LCMS (ESI) m/z calcd for
[C32H35N5O2 + H]+, 522.3; found, 522.5.
Purification of TTK-N Protein Substrate. An N-terminal
fragment of human TTK (TTK-N; amino acids 1−275) was subcloned
into the pET-SUMO vector (Life Technologies, Burlington, ON) for
bacterial expression. The TTK- N protein was expressed in Escherichia
coli BL21 (DE3) CodonPlus-RIPL (Life Technologies) cells in TB
Broth media overnight at 15 °C with 1.0 mM IPTG.
The bacterial pellet was lysed in lysis buffer (30 mM Tris pH 7.5,
500 mM NaCl, 10% (v/v) glycerol, 1 mM TCEP), supplemented with 1
mM PMSF, Complete Protease Inhibitor Tablets EDTA-free (Roche
Diagnostics, Mississagua, ON) (1 tablet per 50 mL), and 0.01 μL/mL
benzonase nuclease (Sigma, Oakville, ON). The cell suspensions were
stirred for 30 min on ice and kept at 4 °C for DNA cleavage and after that
lysed by 3× passage using an EmulsiFlex C5 homogenizer (Avestin,
Ottawa, ON). The lysed cells were centrifuged 40000g, 45 min, 4 °C.
Cell lysate supernatants were treated with 1 g/50 mL Whatman DE52
resin (GE Life Sciences, Baie d’Urfe, QC), which had been preswollen
and pre-equilibrated with lysis buffer. Lysate was allowed to contact the
resin for 20 min on ice to bind and remove DNA. Lysate was vacuum-
filtered to remove DE52 resin beads and loaded onto a 5 mL HisTrap
column (GE Life Sciences) for FPLC purification of His-tagged
proteins. The column was pre-equilibrated with lysis buffer after being
charged with Ni2+ using 0.1 M NiSO4 as per manufacturer’s instructions.
The column was washed with 10 column volumes of lysis buffer,
followed by 10 column volumes of lysis buffer supplemented with
10 mM imidazole. His6-SUMO-TTK-N protein was eluted with lysis
buffer supplemented with 500 mM imidazole. Fractions were collected
and analyzed by NuPAGE (Life Technologies). Appropriate fractions
were pooled for size-exclusion chromatography on a Superdex
200 26/60 column (GE Life Sciences) equilibrated with 30 mM Tris
pH 7.5, 200 mM NaCl, and 0.5 mM TCEP.
Fluorogenic cytochrome P450 inhibition studies were conducted at
37 °C in 96-well plates, as previously described,25,56 for CYP 1A2, CYP
2D6, CYP 2C9, CYP 2C19, and CYP 3A4 (BFC and DBF substrates).
Purification and Crystallization of TTK Kinase Domain. The
kinase domain fragment of human TTK (TTK KD) (amino acids 513−
795) was subcloned into the pET-SUMO vector (Life Technologies
Inc., Burlington, ON) for bacterial expression. The protein was ex-
pressed in E. coli BL21 AI (Life Technologies) cells in Terrific Broth
(TB) media overnight at 15 °C with 0.2% (w/v) L-arabinose and 1.0 mM
IPTG. His6-SUMO-TTK-KD was purified as for His6-SUMO-TTK-N
(see above), except the lysis buffer was 50 mM HEPES, pH 7.5, 500 mM
NaCl, 5% (v/v) glycerol, and 1 mM TCEP. Clarified lysates were loaded
onto a 5 mL HisTrap equilibrated with lysis buffer containing 50 mM
imidazole, and the His6-SUMO-TTK-KD was eluted with 300 mM
imidazole. Size exclusion chromatography was performed on a Superdex
200 26/60 column equilibrated with 50 mM HEPES, pH 7.5, 250 mM
NaCl, and 1 mM TCEP. Elution was confirmed by SDS PAGE.
SUMO protease (LifeSensors Inc., Malvern, PA) was added to
fractions containing His6-SUMO-TTK KD to remove the His6-SUMO
tag, and the cleavage reaction was allowed to proceed overnight at 4 °C.
The cleaved TTK-KD was separated from uncleaved protein, His6-SUMO
protein, and His6-SUMO protease by loading onto a 5 mL HisTrap column.
Uncleaved protein, His6-SUMO protein, and His6-SUMO protease were
retained on the column, whereas cleaved TTK KD was collected in the
flow-through. The TTK KD was further purified to homogeneity by ion-
exchange chromatography (IEX). Cleaved TTK KD fractions were diluted
20× volume with IEX buffer (50 mM Tris, pH 8.0, 1 mM TCEP) and
loaded onto FPLC IEX Resource Q (GE Life Sciences), 6 mL column.
Elution with a gradient 12.5−500 mM NaCl was monitored by NuPAGE
and TTK KD peak, which eluted at approximately 250 mM NaCl, was
pooled and concentrated.
TTK Inhibition Assay. TTK activity was determined using the
N-terminal portion (residues 1−275) as a polypeptide substrate.55 His6-
SUMO-TTK-N (see above) was as effective as a substrate as the cleaved
version and thus was used without cleavage of the His6-SUMO fusion
tag. Active TTK was purchased as an amino terminal GST fusion of full
length human TTK (Life Technologies, Burlington, ON). TTK activity
was measured using an indirect ELISA detection system using 96-well
W
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Journal of Medicinal Chemistry
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Purified TTK KD was crystallized in the presence of compound 51
using the hanging drop vapor diffusion method at 22 °C by mixing equal
volumes of the protein solution and the reservoir solution. The TTK
KD−compound 51 binary complex (protein:compound at a molar ratio
of 1:5) was crystallized in 17% (v/v) PEG 10000, 0.2 M ammonium
sulfate, and 0.1 M Bis-Tris pH 5.5. The crystals were soaked in the
mother liquor supplemented with 15% (v/v) glycerol as cryoprotectant
before freezing in liquid nitrogen.
X-ray Structure Determination. The X-ray data set of the
TTK−compound 51 complex was collected at a temperature of 100 K
using beamline 17-ID at the Advanced Photon Source, Argonne
National Laboratory. The diffraction data was reduced and scaled with
XDS software;57 the crystal of this TTK complex belongs to the
C2 space group. The initial phases of the TTK−inhibitor complex was
determined by molecular replacement using PDB 3CEK set of
coordinates as a search model and Phaser from the Phenix program
suite.58 Following the initial rigid body refinement, interactive cycles of
model building and refinement were carried out using COOT,59
phenix.refine,58 and buster-TNT.60 The coordinates and topology of the
ligand from this study were generated using phenix.elbow.58 The ligand
(compound 51) was introduced in the later stages of refinement after
most of the protein model had been built. Water molecules as well as
other solvent ligands were added based on the 2mFo − DFc map in
COOT and refined with phenix.refine or buster-TNT. The final
structure was refined to 2.38 Å resolution with an Rcryst of 20.97% and an
Rfree of 24.99% and deposited in the PDB as 4O6L; additional
crystallographic data and refinement statistics are presented in the
Supporting Information.
Cell Lines. All cell lines were obtained from American Type Culture
Collection (ATCC, Manassas, VA), and maintained according to the
supplier’s instructions. Short tandem repeat (STR) profiling was used to
verify authenticity of the cell lines. Sixteen STR loci were simultaneously
amplified in a multiplex PCR reaction (The Hospital for Sick Children,
Toronto), and the ATCC database was used for comparison. Cell
lines were routinely tested for mycoplasma and used at low passage
numbers (<15).
Cell Viability Assay (GI50). Cancer cell lines were seeded into
96-well plates at 800−6000 cells per 80 μL, depending on the cell
growth rate (see Supporting Information, Table S3), 24 h before
compound overlay, cultured, then fixed in situ and processed for
sulforhodamine B (SRB) (Sigma, Oakville, ON) assay as previously
described.25 GI50s were calculated using GraphPad PRISM software
(GraphPad Software, Inc., San Diego, CA).
Antibodies. Antibodies targeting the following were used according
to the manufacturers’ instructions: α-tubulin (mouse monoclonal
T6199; Sigma, Oakville, ON), centromere (human polyclonal 15−
234−0001; Antibodies Inc., Davis, CA), phospho-Aurora A (Thr288)/
Aurora B (Thr232)/Aurora C (Thr198) (rabbit monoclonal 2914; Cell
Signaling Technology Inc., Danvers, MA), TTK (mouse monoclonal
05−682; Millipore, Billerica, MA), and GAPDH (mouse monoclonal
MAB374; Millipore, Billerica, MA). Phospho-TTK (Thr12/Ser15)
rabbit polyclonal antibodies were produced and affinity purified by
Agro-Bio (La Ferte Saint Aubin, France) by using the synthetic peptide
CGRELpTIDpSIMNKVRDIK coupled to keyhole limpet hemocyanin
as antigen.
Cellular Kinase Inhibition Assay (EC50). HCT116 colon cancer
cells transfected with wild-type TTK (Origene; Rockville, MD) were
treated with nocodazole (200 ng/mL; Sigma, Oakville, ON) for 17 h.
Cells were pretreated with MG132 (15 μM; Sigma, Oakville, ON) for
30 min before treatment with 75 or DMSO for 4 h. Protein lysates were
prepared using ice-cold radioimmunoprecipitation assay (RIPA) lysis
buffer. A total of 75 mg of cell lysate was loaded onto Bis-Tris gels
(Invitrogen, Carlsbad, CA) with full-range molecular weight marker
(Invitrogen, Carlsbad, CA) as a size reference and resolved by SDS-
PAGE electrophoresis. Proteins were transferred to nitrocellulose mem-
brane (Millipore, Billerica, MA), blocked, and probed with antibodies
for phospho-TTK (Thr12/Ser15), phospho-Aurora A (Thr288)/
Aurora B (Thr232)/Aurora C (Thr198), TTK, and GAPDH. Secondary
antibodies were diluted 1 in 15000 and incubated for 1 h at room tem-
perature. Protein bands were visualized and quantified using Odyssey
near-infrared imager (LI-COR, Lincoln, NE). EC50s were calculated
using GraphPad PRISM software (GraphPad Software Inc., San Diego, CA).
Cell Cycle Analysis. HCT116 colon cancer cells in 6-well plates
were treated with 75 or DMSO at the indicated concentrations and
cultured at 37 °C and 5% CO2. After 2 days, the cells were harvested in
trypsin/EDTA (0.25%/5 mM; Invitrogen, Carlsbad, CA), washed once
in phosphate buffer saline containing 10 mM phosphate buffer, 2.7 mM
potassium chloride, 137 mM sodium chloride, and 1.76 mM potassium
phosphate and adjusted to pH 7.4 (1× PBS), centrifuged at 1000 rpm
for 5 min, fixed in cold (−20 °C) 70% (v/v) ethanol in 1× PBS, and
stored at 4 °C for at least 1 h. The cells were then centrifuged at
1500 rpm for 5 min, washed twice in 1× PBS, and stained with 50 μg/mL
propidium iodide (Sigma, Oakville, ON) in 1× PBS containing RNaseA
(100 μg/mL; Invitrogen, Carlsbad, CA) overnight at 4 °C. The DNA
content was measured using a FACSCalibur flow cytometer (Becton,
Dickinson and Company−Canada, Mississauga, ON) and data was
analyzed using FloJo software (Tree Star, Inc., Ashland, OR).
Immunofluorescence Microscopy. HCT116 colon cancer cells
treated with 75 or DMSO in 4-well chamber slides (NalgeNunc
International, Rochester, NY) were washed twice in 1× PBS, fixed in
cold methanol (−20 °C) for 10 min and then washed four times in
1× PBS at 5 min intervals. Cells were incubated in blocking buffer (1%
BSA (v/v), 0.5% (v/v/)Triton X-100 in 1× PBS) for 1 h at rt. Antibodies
targeting centromeres and α-tubulin in blocking buffer were added to
cells for 2 h at rt followed by three 1× PBS washes at 5 min intervals.
Goat antirabbit and goat antimouse secondary antibodies conjugated
to Alexa 488 and Alexa 555 were used to detect primary antibodies.
DNA was detected with DAPI and cells were mounted in Permafluor
(Invitrogen, Carlsbad, CA). Slides were analyzed using a Leica DM5000
B microscope equipped with fluorescence optics with a Leica X63 oil-
immersion objective.
Metaphase Chromosome Spread. After 24 h of treatment with
75 or DMSO, HCT116 colon cancer cells were treated for 4 h with
nocodazole (200 ng/mL) and MG132 (15 μM). Cells were then lysed in
hypotonic solution (0.56% (v/v) KCl) for 10 min at 37 °C and fixed in
methanol/glacial acetic acid (3:1) solution for 10 min at 4 °C. One drop
of suspended cells was applied to glass slides. DNA was detected with
DAPI, and cells were mounted in Permafluor (Invitrogen, Carlsbad,
CA). Slides were analyzed using a Leica DM5000 B microscope equipped
with fluorescence optics with a Leica X63 oil-immersion objective, and
chromosomes were counted using ImageJ analysis software.
Mouse Plasma Levels. Adult female athymic CD1 nude mice
(Charles River) were used in the experiments; all animal experiments
were approved by UHN Animal Care Committee. Dosing solutions
were prepared by dissolving the compound in 10% NMP/40% PEG400/
50% DI water (v/v) such that the dose volume was 10 mL/kg of animal
body weight. In some instances, brief sonication or vortexing and/or gentle
warming was required to ensure dissolution and/or dispersion. Nine mice
were dosed with compounds at 10 mg/kg (free base equivalents) via oral
gavage by standard methods. Saphenous vein blood was collected from
3 mice per time point over an 8 h period. The plasma samples were
analyzed for drug and internal standard via LCMS/MS protocol using a
Waters (Millford, MA, USA) UPLC coupled to either a Q-ToF Premier
or a XevoTQ mass spectrometer. The pharmacokinetic parameters were
calculated using Microsoft Excel with pharmacokinetic add-in functions
from Usansky et al.61 Parameters were calculated using plasma concen-
tration time data for composite averages.
Xenograft Model. To determine the maximum tolerated dose
(MTD) in athymic CD-1 nude female mice, for use in the efficacy
studies, compounds were formulated in 10% NMP/40% PEG400/50%
DI water (v/v) and dosed daily by oral gavage. A range of compound
concentrations, plus vehicle control, were dosed at 10 mL/kg body
weight. The MTD was defined as the highest daily dose for which test
subjects (n = 3) do not demonstrate body weight loss in excess 20% and
are not deemed moribund, as evaluated in accordance with UHN
IACUC humane end point guidelines standard. MTDs for compounds
69, 75, and 86 were estimated to be 35, 35, and 150 mg/kg, respectively.
HCT116 colon cancer cells were purchased from ATCC (American
Type Culture Collections) and cultured in DMEM (GIBCO, Life
Technologies Inc., Burlington, ON) supplemented with 10% (v/v) fetal
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Journal of Medicinal Chemistry
Article
calf serum. Five million cells were injected subcutaneously in the right
flank of 6−8 week old female athymic CD-1 NUDE mice purchased
from Charles River laboratories. When the mean tumor volume reached
80−120 mm3 (95−100% take rate), mice were randomized into groups
(n = 8). Compounds 69, 75, and 86 were weighed and dissolved in 10%
NMP/40% PEG400/50% DI water and sonicated at rt for 30 min.
The compound was dispensed in aliquots and stored at −20 °C for the
duration of the study, and each aliquot was thawed at rt before each dose.
The mice received oral daily doses of vehicle or compound for 21 days at
the doses indicated (i.e., the MTD and ∼2/3 MTD).
MDA-MB-468 breast cancer cells were purchased from ATCC and
grown as detailed above; 10 million cells were injected subcutaneously in
the right flank of 6−8 week old female CB-17 SCID mice supplied by
UHN animal facility. When the mean tumor volume reached 80−120
mm3 (85% take rate), mice were randomized into groups (n = 5) and
treated with vehicle or compound 75 as described above.
SW48 colon cancer cells were purchased from ATCC and grown as
detailed above; 5 million cells were injected subcutaneously in the right
flank of 6−8 week old female CD-1 Nude mice purchased from Charles
River laboratories (Wilmington, MA). When the mean tumor volume
reached 80−120 mm3 (95% take rate), mice were randomized into
groups (n = 7) and treated with vehicle or compound 75 as described
above.
Tumor volume and body weight were measured three times weekly.
Efficacy data were graphically represented as the mean tumor volume.
Tumor volume was calculated by the following formula: x2y/2. Percent
tumor growth inhibition after initiation of treatment with compound
was calculated by TGI = 100 × 1 − (tumor volumefinal − tumor
volumeinitial for compound treated group)/(tumor volumefinal − tumor
volumeinitial for compound control group).
Foundation are acknowledged for financial support. The use of
the IMCA-CAT beamline 17-ID at the Advanced Photon Source
was supported by the companies of the Industrial Macro-
molecular Crystallography Association through a contract with
the Center for Advanced Radiation Sources at the University of
Chicago. Use of the Advanced Photon Source was supported by
the U.S. Department of Energy, Office of Science, Office of Basic
Energy Sciences, under contract no. W-31-109-Eng-38.
ABBREVIATIONS USED
■
A%, area percent; ADME, absorption, distribution, metabolism
and excretion; anh, anhydrous; aq, aqueous; Ar, aromatic;
ATCC, American Type Culture Collection; ATP, adenosine-5′-
triphosphate; AURKA, Aurora kinase A; AURKB/INCENP,
Aurora kinase B/inner centromere protein; b, broad; BA,
bioavailability; BFC, 7-benzyloxy-4-trifluoromethylcoumarin;
BLM, Bloom syndrome helicase; Boc, tert-butoxycarbonyl;
BSA, bovine serum albumin; calcd, calculated; CHK2,
checkpoint kinase 2; CIN, chromosomal instability; CYP,
cytochrome P450; d, doublet; DAPI, 4′,6-diamidino-2-phenyl-
indole; (D)- or (L)-DBTA, Dibenzoyl-D or L-tartaric acid; DBF,
dibenzylfluorescein; DBU, 1,8-diazabicycloundec-7-ene; DCM,
dichloromethane; DI, deionized; DIPEA, diisopropylethylamine;
DMAP, 4-dimethylaminopyridine; DME, dimethoxyethane;
DMEM, Dulbecco’s Modified Eagle’s Medium; DMF, N,N-
dimethylformamide; DMSO, dimethyl sulfoxide; dppf, 1,1′-
bis(diphenylphosphino) ferrocene; eADME, early ADME; ee,
enantiomeric excess; EGFR, epidermal growth factor receptor;
EGTA, ethylene glycol tetraacetic acid; ELISA, enzyme-linked
immunosorbent assay; FLT3, fms-related tyrosine kinase 3; GI50,
half-maximal cell growth inhibitory concentration; GST,
glutathione S-transferase; h, hour; hERG, human ether-a-go-go
related gene; HEPES, 4-(2-hydroxyethyl)-1-piperazineethane-
sulfonic acid; HOBt, hydroxybenzotriazole; HMBC, hetero-
nuclear multiple-bond correlation spectroscopy; HPLC, high-
performance liquid chromatography; HRMS, high-resolution
mass spectrometry; IEX, ion exchange; IP, intraperitoneal;
IPTG, Isopropyl β-D-thiogalactopyranoside; JNK3, c-Jun N-
terminal kinase 3; KD, kinase domain; KDR, kinase insert
domain receptor; LB, lysis buffer; LCMS, liquid chromatography
coupled to mass spectrometry; MAPK2, mitogen-activated
protein kinase 2; MBP, myelin basic protein; min, minute; m,
multiplet; Mps1, monopolar spindle 1; MS ESI, electrospray
ionization mass spectrometry; MTD, maximum tolerated dose;
NMP, N-methylpyrrolidone; NMR, nuclear magnetic resonance;
NBS, N-bromosuccinimide; PAMPA, parallel artificial mem-
brane permeability assay; PARP, poly ADP ribose polymerase;
PBS, phosphate buffered saline; PCR, polymerase chain reaction;
PDB, Protein Data Bank; PEG400, polyethylene glycol 400; pin,
pinacol; PK, pharmacokinetics; PLK4, Polo-like kinase 4; PMB,
p-methoxybenzyl; PMSF, phenylmethanesulfonyl-fluoride; PPB,
plasma protein binding; prep, preparative; PTEN, phosphatase
and tensin homologue; PSA, polar surface area; QD, quaque die
(once daily); QSAR, quantitative structure−activity relation-
ship; Ra−Ni, Raney nickel; RBF, round bottomed flask;
RIPA, radio-immunoprecipitation assay; rmse, root-mean-
square error; rt, room temperature; RP, reverse phase; s,
singlet; SAC, spindle assembly checkpoint; satd, saturated;
SEM, 2-(trimethylsilyl)ethoxy]methyl; siRNA, small interfer-
ing RNA; SMs, starting materials; t, triplet; SRB, sulforhod-
amine B; STR, short tandem repeat; SUMO, small ubiquitin-
like modifier; TB, terrific broth; TBAB, tetra-n-butylammo-
nium bromide; TBAF, tetra-n-butylammonium fluoride;
Rat Pharmacokinetic Studies. The pharmacokinetic studies were
performed by Shanghai Chempartner (Pudong New Area, Shanghai,
China). Compounds were dissolved in 10% NMP/40% PEG/50% DI
water. Male SD rats (200−230 g, purchased from SLAC Laboratory
Animal Co. LTD, Shanghai) were administered IV (foot dorsal vein)
and PO (oral gavage); blood samples were collected via cannulated
jugular veins and stored frozen. Diluted time point samples were
analyzed by LCMSMS-02 (API 4000, triple quadrupole) using a
standard curve. The PK parameters were determined by a non-
compartmental model using WinNonlin Professional 5.2.
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthesis of compound intermediates; double reciprocal plot of TTK
inhibition by compound 75; inhibition of 278 kinase panel by 75;
summary of crystallographic data and refinement statistics for TTK-
compound 51 co-complex (PDB accession code: 4O6L); omit map
for TTK−compound 51 co-complex; correlation of pIC50(TTK) vs
GlideXP docking scores (n = 86); cell seeding data for cancer cell
lines; early ADME data for compound 75; mouse weights upon oral
QD dosing of compounds 69, 75, and 86. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
■
*For H.W.P.: phone, 416-581-7620; E-mail, hpauls@uhnresearch.ca.
*For Y.L.: phone, 416-581-8550 (8450); E-mail, yongl@
uhnresearch.ca.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Richard Brokx for editorial comment during
manuscript preparation and Dr. Homer Pearce for helpful advice
and discussion during the course of the work. The Canadian
Institutes of Health Research and the Princess Margaret Cancer
Y
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