Discovery of highly potent and selective pan-Aurora kinase inhibitors with enhanced in vivo antitumor therapeutic index

Article abstract of DOI:10.1021/jm201702g

Serine/threonine protein kinases Aurora A, B, and C play essential roles in cell mitosis and cytokinesis. Currently a number of Aurora kinase inhibitors with different isoform selectivities are being evaluated in the clinic. Herein we report the discovery and characterization of 21c (AC014) and 21i (AC081), two structurally novel, potent, kinome-selective pan-Aurora inhibitors. In the human colon cancer cell line HCT-116, both compounds potently inhibit histone H3 phosphorylation and cell proliferation while inducing 8N polyploidy. Both compounds administered intravenously on intermittent schedules displayed potent and durable antitumor activity in a nude rat HCT-116 tumor xenograft model and exhibited good in vivo tolerability. Taken together, these data support further development of both 21c and 21i as potential therapeutic agents for the treatment of solid tumors and hematological malignancies.

Full text of DOI:10.1021/jm201702g

Article
pubs.acs.org/jmc
Discovery of Highly Potent and Selective Pan-Aurora Kinase
Inhibitors with Enhanced in Vivo Antitumor Therapeutic Index
Gang Liu,*,† Sunny Abraham,† Lan Tran,† Troy D. Vickers,† Shimin Xu,† Michael J. Hadd,†
Sheena Quiambao,† Mark W. Holladay,† Helen Hua,‡ Julia M. Ford Pulido,‡ Ruwanthi N. Gunawardane,‡
Mindy I. Davis,§ Shawn R. Eichelberger,⊥ Julius L. Apuy,∥ Dana Gitnick,∥ Michael F. Gardner,∥
Joyce James,∥ Mike A. Breider,∥ Barbara Belli,‡ Robert C. Armstrong,‡ and Daniel K. Treiber§
†
‡
§
∥
Departments of Medicinal Chemistry, Cell Biology and Pharmacology, Technology Development, DMPK and Toxicology, and
⊥CMC, Ambit Biosciences Corporation, 4215 Sorrento Valley Boulevard, San Diego, California 92121, United States
S
* Supporting Information
ABSTRACT: Serine/threonine protein kinases Aurora A, B, and C play essential roles in cell mitosis and cytokinesis. Currently
a number of Aurora kinase inhibitors with different isoform selectivities are being evaluated in the clinic. Herein we report the
discovery and characterization of 21c (AC014) and 21i (AC081), two structurally novel, potent, kinome-selective pan-Aurora
inhibitors. In the human colon cancer cell line HCT-116, both compounds potently inhibit histone H3 phosphorylation and cell
proliferation while inducing 8N polyploidy. Both compounds administered intravenously on intermittent schedules displayed
potent and durable antitumor activity in a nude rat HCT-116 tumor xenograft model and exhibited good in vivo tolerability.
Taken together, these data support further development of both 21c and 21i as potential therapeutic agents for the treatment of
solid tumors and hematological malignancies.
gen phosphate (AZD1152),8 and pan-Aurora inhibitor N-(4-
((4-((5-methyl-1H-pyrazol-3-yl)amino)-6-(4-methylpiperazin-
INTRODUCTION
Aurora kinases are a family of three highly homologous serine/
■
1-yl)pyrimidin-2-yl)thio)phenyl)cyclopropanecarboxamide
threonine kinases that play critical roles during the mitotic stage
(VX-680/MK-0457).9
of the cell cyle.1 Aurora kinases A and B seem to work in
concert during mitosis, whereas the function of Aurora C
during mitosis is much less well-defined. Inhibition of Aurora A
results in mitotic delay and formation of a monopolar spindle
phenotype followed by cell death.2 Inhibition of Aurora B
results in aberrant endoreduplication and abrogation of
cytokinesis, leading to generation of polyploid cells and
apoptosis.3 When both Aurora kinases A and B are inhibited,
the dominant phenotype is the one resulting from the
inhibition of Aurora B.4
Both Aurora A and B gene amplification and protein
overexpression have been frequently detected in a variety of
tumors.5 By targeting components of the mitotic machinery,
Aurora kinases A and B have been suggested as promising
targets for cancer therapy. Intense research over the past decade
or so has resulted in more than 10 structurally diverse small-
molecule Aurora kinase inhibitors entering early human clinical
assessment,6 including Aurora A selective 4-((9-chloro-7-(2,6-
difluorophenyl)-5H-benzo[c]pyrimido[4,5-e]azepin-2-yl)-
amino)benzoic acid (MLN8054),7 Aurora B selective 2-
(ethyl(3-(4-(5-(2-(3-fluorophenylamino)-2-oxoethyl)-1H-pyra-
zol-3-ylamino)quinazolin-7-yloxy)propyl)amino)ethyl dihydro-
We felt that a pan-Aurora kinase inhibitor may provide more
durable efficacy and reduce the chance for the development of
resistance driven by target-modifying mutations.10 Previously, a
series of potent pan-Aurora kinase inhibitors with moderate
intravenous (iv) clearance was described by this laboratory.11
One such compound (1, Figure 1) was found to inhibit tumor
growth in a mouse xenograft model using HCT-116 cells when
dosed intraperitoneally. However, the significant body-weight
loss during the xenograft study complicated the interpretation
of the pharmacological effects. In this paper, we describe the
further optimization of these pyrrolotriazine-based Aurora
kinase inhibitors utilizing a readily accessible, penultimate
intermediate 2 (Figure 1), culminating in the discovery of a
series of highly potent and kinome selective pan-Aurora kinase
inhibitors. Combined with modifications of the in vivo
pharmacology protocol, significant tumor growth inhibition
and enhanced in vivo tolerability were demonstrated with two
lead compounds 21c and 21i in a nude rat model.
Received: December 16, 2011
Published: March 1, 2012
© 2012 American Chemical Society
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Figure 1. Early lead of pyrrolotriazine-based pan-Aurora inhibitor 1 and a penultimate intermediate 2.
a
Scheme 1. General Synthesis of Compounds 5c−h
a
Reagents and conditions: (a) NaH, DMF, alkyl halides, 0 °C to rt; (b) 10% Pd/C, H2, MeOH, rt; (c) aq HCl, THF, rt; (d) 4 N HCl in 1,4-dioxane,
rt; (e) tert-butyl-2,2,2-trichloroacetimidate, THF, rt; (f) cat. Pd(OH)2, H2, MeOH/THF, rt; (g) 2, KI, i-Pr2NEt, DMF, 50 °C.
a
Scheme 2. General Synthesis of Compounds 7a,b
a
Reagents and conditions: (a) (R)-tert-butyl pyrrolidin-3-ylcarbamate, KI, i-Pr2NEt, DMF, 50 °C; (b) 4 N HCl in 1,4-dioxane, rt; (c) i-PrCOCl,
Et3N, THF; (d) CH3SO2Cl, Et3N, THF.
a
Scheme 3. General Synthesis of Compounds 7c−d
CHEMISTRY
■
The analogues described herein could be prepared in a
straightforward fashion via alkylation of the previously
described α-chloroacetamide 212 with appropriate pyrrolidine
derivatives. Schemes 1 and 3 − 5 illustrate the methods for
preparation of a variety of requisite pyrrolidine derivatives prior
to reaction with 2, whereas Scheme 2 illustrates modification of
pyrrolidine functionality following reaction with 2. As shown in
Scheme 1, a suitably protected (R)-3-hydroxypyrrolidine 3 was
subjected to alkylation with various electrophiles to give the
corresponding ethers (3a, 4a−d). Removal of the protecting
groups resulted in the desired pyrrolidine derivatives (3b, 4e−
h). Alkylation of the chloride 2 using these pyrrolidines yielded
the desired analogues 5c−g. Similarly, the formation of the tert-
butyl ester from 4 followed by hydrogenolysis and alkylation
provided compound 5h.
Scheme 2 demonstrates the facile synthesis of amine 6
followed by acylation or sulfonylation to afford compounds
7a,b. In Scheme 3, simple transformations of the racemic
pyrrolidine carboxylate 8 generated pyrrolidine derivatives 10
and 12 for the syntheses of analogues 7c and 7d. In Scheme 4,
transformation of 3-pyrrolidinol 13 to tert-butyl thioether 14
and subsequent oxidation and deprotection created the
pyrrolidine derivatives 15−17, which were advanced to
analogues 7e−g.
a
Reagents and conditions: (a) t-BuLi, THF, −78 °C to rt; (b) 10%
Pd/C, H2, MeOH; (c) 2, KI, i-Pr2NEt, DMF, 50 °C; (d) pyrrolidine,
130 °C, 24 h.
Pyrrolidine carboxamides 21b−l can be prepared from the
enantiomerically pure N-Boc β-proline 18, as shown in Scheme
5. Condensation of the acid with appropriate amines yielded
the β-proline carboxamides 19b−l, which were treated with
acid to reveal pyrrolidines 20b−l for the subsequent alkylation.
RESULTS AND DISCUSSION
■
The compounds described herein were tested for their binding
affinity to the catalytic domain of Aurora kinases A and B in a
competition binding assay with an ATP-competitive Aurora
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previously,11 we decided to seek analogues with alternative,
simplified pyrrolidine substituents. 3-Monosubstituted pyrroli-
dines, instead of the 3,4-disubstituted ones, became the focus of
the SAR campaign to allow quicker access to structural
variations. As shown in Table 1, deleting the 4-fluorophenoxy
group of racemic 1 resulted in two enantiomers (5a, 5b) with
biological data showing a mild stereochemistry preference in
favor of the (R)-enantiomer. The decrease in cell activity was
particularly notable with the (S)-enantiomer 5a. Therefore, the
ensuing analogues were pursued with a primary focus on the
(R) absolute stereochemistry.
a
Scheme 4. General Synthesis of Compounds 7e−g
The (R)-3-pyrrolidinol was used as a stepping stone for
accessing ether analogues with neutral or basic hydrophilic
termini, a commonly used SAR maneuver in kinase inhibitor
design. Neutral ethers 5c,d showed marked improvement in
cell potency, with methoxyethyl ether analogue 5d exhibiting
more than 10-fold improvement in both cellular assays
compared to 5b. The corresponding kinome selectivity as
measured by selectivity score deteriorated slightly for these two
compounds. On the other hand, the ether analogues with
terminal basic groups (5e−g) exhibited weaker cell potency
than 5d, regardless the length of the linker or the basicity of the
amino groups. Similar to 5d, the hydrophobic tert-butyl ether
analogue 5h possessed high binding affinity and potent cellular
activity. The kinome selectivity of basic analogues 5e−g was
substantially better than the neutral analogues 5a−d and 5h.
Alternatives to the ether linker at the 3-position of the
pyrrolidine ring were also explored. Initial amide and
sulfonamide analogues (7a, 7b) resulted in moderately active
compounds versus HCT-116 cell proliferation, whereas kinome
selectivity was substantially improved (Table 2). We then
switched to either a carbon or sulfur-based linker. tert-Butyl
ketone analogue 7c possessed encouraging cellular activity,
while racemic pyrrolidine amide compound 7d was even more
potent in cells. A diastereomeric mixture of tert-butyl sulfoxides
(7f) exhibited similar cellular activity to 7d with slightly
improved kinome selectivity. The corresponding sulfide 7e and
sulfone 7g were somewhat less active in cell assays. Encouraged
by the excellent in vitro profile of compound 7d and ease of
diversification, we pursued additional 3-carboxamide-substi-
tuted pyrrolidine derivatives to further define the stereo-
chemistry preference and to fine-tune the suitability for iv
formulation.
a
Reagents and conditions: (a) MsCl, TEA, THF, rt; (b) t-BuSH, NaH,
DMF, 0 °C to rt; (c) 3-chloroperbenzoic acid, DCM, −30 to −20 °C;
(d) 4.5 N HCl in EtOAc/MeOH, 0 °C to rt; (e) 2, KI, i-Pr2NEt,
DMF, 50 °C.
inhibitor bound to a solid surface.13 The cellular activities of the
compounds were assessed in HCT-116 (human colon
carcinoma) cells in two formats, measuring either the inhibition
of histone H3 phosphorylation (pHH3) mediated by Aurora
kinase B14 or the inhibition of cell proliferation. The
compounds described herein were also monitored for their
kinase selectivity in a panel of either 321 or 359 distinct kinase
domains (not counting mutant variants) using the KinomeScan
technology.15,16
As demonstrated by lead compound 1 (Figure 1), the
pyrrolo[1,2-b]triazine core substituted with an aminopyrazole
group at the 4-position and an amidophenylthio group at the 2-
position (solid box) constituted a basic pharmacophore for
achieving high binding affinity and cell activity. The moderately
basic pyrrolidine nitrogen, the pH value of which is attenuated
by the anilide carbonyl group, provided an opportunity for
ionization and improved aqueous solubility as well as expanded
synthetic accessibility.
It is apparent that the structural requirements for the
pyrrolidine portion (dashed box) of 1 are not particularly
stringent with respect to direct interactions with the enzyme. X-
ray structure of the original hit of this series11 in complex with
Aurora A kinase was used as the basis for molecular modeling.
Docking of a compound (similar to 1) described previously11 in
Aurora A revealed that the pyrrolidine ring was located in an
unexplored region flanked by the activation loop and the P
loop.17 It also suggested the possibility of reaching a solvent
exposed space through the gap between these two loops.
Therefore, we focused on optimizing this pyrrolidine motif with
the goal of improving cell potency, pharmacokinetics (PK), and
aqueous solubility for intravenous (iv) administration.
Given the latitude that we had with substituent variations at
both the 3- and 4-positions of pyrrolidine ring as described
Even though two enantiomers of pyrrolidine amides (21a,b)
had very similar binding affinities for Aurora kinases and similar
overall kinome selectivity (Table 3), the (R)-enantiomer 21b
was ∼6-fold more potent in the cellular assays, consistent with
the previously observed stereochemistry preference. Further
exploration of the tertiary carboxamides (21c−e) yielded a
series of highly potent compounds against HCT-116 cell
proliferation with reasonable kinome selectivity. The preference
for lipophilic groups at the terminus of the carboxamide was
evident, since the 4-hydroxypiperidine derivative 21f exhibited a
a
Scheme 5. General Synthesis of Compounds 21b−l
a
Reagents and conditions: (a) HNR1R2, O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU), Et3N, DMF, rt; (b) 4 N
HCl in 1,4-dioxane, rt; (c) 2, KI, NEt-i-Pr2, DMF, 85 °C.
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Table 1. SAR of Monosubstituted Pyrrolidine Derivatives
a
b
Each experiment was run in duplicate, and the values shown are the average of the two. IC50 values are for inhibiting the phosphorylation of
c
d
histone H3 in the HCT-116 cells. IC50 values are for inhibiting the proliferation of HCT-116 cells. The fraction of 321 kinases having <10%
residual activity in the presence of 10 μM inhibitor.
large drop in cell potency despite good binding affinity, perhaps
due to compromised cell permeability. Among the large
number of secondary amides prepared, many were extremely
potent in inhibiting HCT-116 cell proliferation, as exemplified
by analogues 21h−l. Those possessing more hydrophilic amide
moieties, such as 21g, represented the main exceptions.
Some selected analogues were also evaluated in a cellular
Aurora A autophosphorylation assay in HEK-293 cells using
phosphor antibody readout.18 Consistent with the high binding
affinity for Aurora kinase A, compounds 21c and 21i potently
inhibited the formation of pAURKA with IC50 values of 1 and 2
nM, respectively.
(SD) rats as a screening assay to further prioritize compounds.
Many compounds selected for PK assessment showed high
clearance following 1 mg/kg iv bolus dosing. For example, the
clearance values (CL) in SD rats for compounds 5d, 21k, and
21l are 62, 48, and 35 mL min−1 kg−1, respectively. Compounds
21c and 21i exhibited moderate clearance following iv dosing
(Table 4) and, taken together with their favorable biochemical
and cellular profiles, were selected for further evaluation. The
mesylate salts of 21c and 21i could be formulated at
concentrations greater than 5 mg/mL at pH ≈ 5, using a
mixed vehicle TPPW (2% Tween-80, 5% propylene glycol, 10%
PG-400, 83% water) suitable for iv dosing in the xenograft
models. In athymic nude rats, both compounds exhibited
moderate clearance and dose-dependent increase in exposure
Pharmacokinetics. Since quite a few analogues described
herein had very similar in vitro profiles, we used exploratory PK
assessments following iv administration to Sprague−Dawley
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Table 2. SAR of Alternative Linkers of the Pyrrolidine Ring
a
b
Each experiment was run in duplicate, and the values shown are the average of the two. IC50 values are for inhibiting the phosphorylation of
c
d
e
histone H3 in the HCT-116 cells. IC50 values are for inhibiting the proliferation of HCT-116 cells. A mixture of diastereomers. The fraction of
321 kinases having <10% residual activity in the presence of 10 μM inhibitor. The fraction of 359 kinases having <10% residual activity in the
f
presence of 10 μM inhibitor.
following iv injection formulated in TPPW, as shown in Table
4.
Compound 21c was dosed as iv bolus every Monday,
Wednesday, and Friday over the course of three 7-day cycles.
After the dosing was stopped, the animals were observed for 23
additional days. Dose-dependent tumor growth inhibition was
observed for all three dosing groups (5, 10, and 20 mpk)
(Figure 2). Tumor stasis was observed for the 20 mg/kg dosing
group, and it persisted for more than 20 days after dosing. At
the end of the study (day 42), 2 out of 10 animals from the 20
mg/kg group were observed with complete regression (CR).
Tumor growth inhibition (TGI)21 was calculated at 45%, 60%,
and 85% for the 5, 10, and 20 mg/kg groups, respectively.
In contrast, the positive control (irinotecan, 100 mg/kg, ip,
once per week for 3 weeks) group showed efficacy somewhat
less than that of 21c at 10 mg/kg.
Compound 21i was evaluated using the same protocol at
three doses (10, 20, and 40 mpk). Tumor stasis was observed
for the dosing groups at 10 and 20 mg/kg and persisted for
more than 40 days after dosing (Figure 3). At the end of the
study (day 60), 2 animals (out of 10) with CR for the 10 mg/
kg group and 1 CR (out of 10) for the 20 mg/kg group were
observed. TGI was calculated at 77% and 82% for the 10 and 20
mg/kg groups, respectively. Doses at 40 mg/kg were not well
tolerated, with greater than 10% body weight loss observed
(data not shown).
Cellular Mechanistic Study. Aurora kinase B is essential
for chromatin remodeling and cytokinesis. Studies using both
pharmacological and genetic disruption of Aurora kinase B in
cells led to polyploid cells and loss of viability due to
cytokinesis failure.19 In HCT-116 cells treated with either 21c
and 21i, 8N polyploidy was potently induced in a dose-
dependent manner, with EC50 of 3 nM. The observed
phenotype was consistent with the IC50 values derived from
the pHH3 assay and supported the potent Aurora kinase B
inhibition in HCT-116 cells by 21c and 21i.
Efficacy in Tumor Xenograft Model. On the basis of our
experience with compound 1 in a mouse xenograft tumor
model11 and literature reports related to Aurora inhibitor in
vivo evaluations,20 we were keenly aware of the needs for
flexible dosing schedules and drug holidays associated with
many Aurora inhibitors. Earlier maximum tolerated dose
(MTD) studies in nude rats using 21i had established that an
intermittent dosing schedule was necessary for achieving
reasonable tolerability (data not shown). Therefore, two
different dosing schedules were selected for evaluating the
efficacy and tolerability of 21c and 21i as single agents in a
subcutaneous flank-tumor xenograft model in nude rats using
the HCT-116 cell line: QOD (every other day) for 3 weeks and
QD×4/week (4 days on and 3 days off per week) for 3 weeks.
The data from the QOD schedule are summarized below.
Neither body weight loss nor lethality was noted for either
compound at efficacious doses up to 20 mg/kg QOD for 3
weeks. QD×4/week schedule also produced favorable signal of
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Table 3. SAR of 3-Carboxamide Pyrrolidine Derivatives
a
b
Each experiment was run in duplicate, and the values shown are the average of the two values. IC50 values are for inhibiting the phosphorylation of
c
d
histone H3 in the HCT-116 cells. IC50 values are for inhibiting the proliferation of HCT-116 cells. The fraction of 321 kinases having <10%
residual activity in the presence of 10 μM inhibitor. The fraction of 359 kinases having <10% residual activity in the presence of 10 μM inhibitor.
e
tumor growth inhibition and tolerability for both 21c and 21i.22
These results indicate that 21c and 21i are efficacious
compounds in inhibiting HCT-116 tumor growth in nude
rats with enhanced therapeutic index, in comparison to the
earlier Aurora kinase inhibitor 1.
Kinome Selectivity Analysis. Moderate kinome selectivity
expressed as S(10) scores (29% and 33%, respectively, of the
kinases tested showed <10% of control activity) was observed
for both 21c and 21i when they were screened at 10 μM (Table
3). At the efficacious doses in the xenograft studies, the total
plasma concentrations for both compounds were only
transiently above 10 μM. Taking into account the highly
plasma protein-bound nature (∼99% bound) of 21c and 21i,
the unbound plasma efficacious concentrations for both
compounds were estimated to be largely below 100 nM.
Compounds 21c and 21i were then screened across a panel of
359 kinases at 100 nM inhibitor concentration. This resulted in
low selectivity scores S(10) at 100 nM of 0.07 and 0.08 for 21c
or 21i, respectively, indicating only a small number of kinases
these two compounds were interacting with at an efficacious
free concentration of 100 nM. By use of 35% of control activity
or below at 100 nM inhibitor concentration as the cutoff, 24
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Table 4. Intravenous PK of Compounds 21c and 21i in Rats
compd
rat species
dose (mg/kg)
clearance (mL min−1 kg−1
)
Vd (L/kg)
AUC(0−inf) (μM·h)
t1/2 (h)
a
21c
SD
1
21.05
19.2
12.5
5.23
16.92
27.9
20.7
14.7
7
1.4
17.5
1.1
3.7
2.9
9.4
1.5
3.8
3.4
b
21c
Nu/Nu
Nu/Nu
Nu/Nu
SD
5
0.7
0.8
0.62
3.49
1.0
1.1
0.84
7.7
b
21c
15
50
1
34.4
181.1
1.8
b
21c
a
21i
b
21i
Nu/Nu
Nu/Nu
3
3.2
b
21i
10
30
14.6
62.5
b
21i
Nu/Nu
a
b
Vehicle: PEG400/water. Vehicle: TPPW.
EXPERIMENTAL SECTION
■
Chemistry. General Methods. Reactions involving air or
moisture sensitive reagents were carried out under an argon
atmosphere. Proton NMR spectra were obtained in the deuterated
solvents indicated on a Bruker Avance 300 or 400, with reference to
tetramethylsilane. Routine LC−MS analyses were carried out on a
Shimadzu LC−MS 2010 EV system. Reverse phase preparative HPLC
purifications were carried out on a Varain preparative HPLC system
using a Varian Pursuit XRs diphenyl column as the stationary phase
and a mixture of water (5% CH3CN, 0.05% AcOH) and CH3CN
(0.05% AcOH) as the mobile phase. Chemical purity for all final
compounds was determined by analytical HPLC with a C18 column
(Phenomenex Luna 5 μm C18(2) 100 Å, 250 mm × 4.6 mm),
detected by UV at 254 nm, ELSD, and MS, confirming ≥95% purity.
Syntheses of Representative Compounds (5h, 7a, 7c, 7f,
21c, and 21i) Listed in Tables 1−3. (R)-3-tert-Butoxypyrrolidine
(4i). To a stirring solution of (R)-(−)-Cbz-3-pyrrolidinol (3) (250 mg,
1.13 mmol) in THF (5 mL) was added tert-butyl 2,2,2-
trichloroacetimidate (0.20 mL, 1.13 mmol). The solution was stirred
at room temperature for 3 h. Then additional tert-butyl 2,2,2-
trichloroacetimidate (0.20 mL, 1.13 mmol) was added and the mixture
was stirred for 0.5 h. Another portion of tert-butyl 2,2,2-
trichloroacetimidate (0.20 mL, 1.13 mmol) was then added, and the
mixture was stirred for an additional 1 h. DCM (15 mL) was then
added, and the mixture was filtered through a Celite plug. The filtrate
was purified by silica gel chromotography, eluting with 30% EtOAc in
hexanes to afford impure (R)-benzyl 3-tert-butoxypyrrolidine-1-
carboxylate (500 mg). LC−MS (ESI) m/z 300 (M + Na)+. To the
impure (R)-benzyl 3-tert-butoxypyrrolidine-1-carboxylate in a mixture
of MeOH/THF (1:1, v/v, 8 mL) was added palladium hydroxide (80
mg) and the mixture was stirred under a hydrogen atmosphere
overnight, and then filtered through a Celite plug. The filtrate was
concentrated under reduced pressure to afford crude (R)-3-tert-
butoxypyrrolidine (4i) (110 mg, 67%). LC−MS (ESI) m/z 144 (M +
Figure 2. Efficacy of compound 21c in a Nu/Nu rat tumor xenograft
model.
Figure 3. Efficacy of compound 21i in a Nu/Nu rat tumor xenograft
model.
and 26 kinases, respectively, for 21c or 21i were picked for the
actual binding affinity determination. The complete list and the
corresponding Kd values are reported in the Supporting
Information.23 In this analysis, both 21c and 21i were also
confirmed to possess high binding affinities for Aurora C, with
estimated Kd values of less than 10 nM.
+
H) .
(R)-2-(3-tert-Butoxypyrrolidin-1-yl)-N-(4-(4-(5-methyl-1H-
pyrazol-3-ylamino)pyrrolo[2,1-f ][1,2,4]triazin-2-yl)thio)-
phenyl)acetamide Diacetate (5h). General Procedure A. To a
stirred mixture of (R)-3-tert-butoxypyrrolidine (4i) (69 mg, 0.48
mmol) in DMF (2 mL) were added potassium iodide (40 mg, 0.24
mmol) and 2-chloro-N-{4-[4-(5-methyl-1H-pyrazol-3-ylamino)-
pyrrolo[2,1-f ][1,2,4]triazin-2-ylsulfanyl]phenyl}acetamide (2) (100
mg, 0.24 mmol), followed by dropwise addition of N,N-diisopropy-
lethylamine (0.041 mL, 0.24 mmol). The resulting solution was stirred
at 50 °C overnight. Then additional N,N-diisopropylethylamine (0.2
mL) and potassium iodide (50 mg) were added and the reaction
mixture was stirred at 50 °C for 24 h. The resulting mixture was
purified by preparative HPLC to afford (R)-2-(3-tert-butoxypyrrolidin-
1-yl)-N-(4-(4-(5-methyl-1H-pyrazol-3-ylamino)pyrrolo[1,2-f ][1,2,4]-
triazin-2-ylthio)phenyl)acetamide as its diacetate salt (5h) (31 mg,
CONCLUSION
■
Optimization of the lead compound 1 was carried out by
installation of carboxamide moieties at the 3-position of the
pyrrolidine ring and by removal of the hydroxyl group at the 4-
position of the pyrrolidine ring. Compounds 21c (AC014) and
21i (AC081) were identified as highly potent and selective pan-
Aurora kinase inhibitors with attractive pharmacokinetic
profiles and pharmaceutical properties. Efficacy and enhanced
tolerability in nude rat xenograft models were demonstrated
with both compounds. Preclinical pharmacology data support
clinical development of either or both of these two compounds
for the potential treatment of hematological malignancies and
solid tumors.
1
22%). H NMR (300 MHz, methanol-d4) δ 8.03 (d, J = 8.5 Hz, 2H),
7.88 (d, J = 8.3 Hz, 2H), 7.73 (br s, 1H), 7.20 (d, J = 3.2 Hz, 1H),
6.76−6.95 (m, 1H), 6.05 (s, 1H), 4.70 (br s, 1H), 3.95 (br s, 2H),
3.31−3.50 (m, 2H), 3.08−3.22 (m, 1H), 2.52 (td, J = 6.9, 13.5 Hz,
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1H), 2.40 (s, 3H), 2.24 (s, 6H), 1.54 (br s, 2H), 1.49 (s, 9H). LC−MS
(ESI) m/z 521 (M + H)+.
(92 mg, 25%) using general procedure A, substituting 2,2-dimethyl-1-
(pyrrolidin-3-yl)propan-1-one (10) for pyrrolidine 4i used in general
1
(R)-2-(3-Aminopyrrolidin-1-yl)-N-(4-((4-((5-methyl-1H-pyra-
zol-3-yl)amino)pyrrolo[2,1-f ][1,2,4]triazin-2-yl)thio)phenyl)-
acetamide Dihydrochloride (6). (R)-tert-Butyl (1-(2-((4-((4-((5-
methyl-1H-pyrazol-3-yl)amino)pyrrolo[2,1-f ][1,2,4]triazin-2-yl)thio)-
phenyl)amino)-2-oxoethyl)pyrrolidin-3-yl)carbamate (prepared using
general procedure A from compound 2 and (R)-tert-butyl pyrrolidin-3-
ylcarbamate, 80 mg, 0.14 mmol) was stirred in EtOAc (1.0 mL) at
room temperature, and 4 N HCl in 1,4-dioxane (1.0 mL) was added.
The resulting mixture was stirred at room temperature for 8 h. Diethyl
ether (10 mL) was then added to the mixture, and the precipitate was
collected via filtration, washed with diethyl ether, and dried in a
vacuum oven to give (R)-2-(3-aminopyrrolidin-1-yl)-N-(4-((4-((5-
methyl-1H-pyrazol-3-yl)amino)pyrrolo[2,1-f ][1,2,4]triazin-2-yl)thio)-
phenyl)acetamide dihydrochloride (6) as a white solid (55 mg, 72%).
1H NMR (300 MHz, DMSO-d6) δ 11.20 (br s, 1H), 10.79−11.05 (m,
1H), 10.70 (br s, 1H), 8.67−8.86 (m, 2H), 8.60 (br s, 1H), 7.77 (d, J =
8.3 Hz, 2H), 7.65 (br s, 1H), 7.61 (d, J = 7.3 Hz, 2H), 7.23 (br s, 1H),
6.60 (d, J = 2.3 Hz, 1H), 5.69 (br s, 1H), 4.47 (d, J = 16.4 Hz, 2H),
3.82−4.23 (m, 2H), 3.50−3.81 (m, 2H), 3.38 (d, J = 6.2 Hz, 1H), 2.28
(br s, 1H), 2.08 (br s, 3H). LC−MS (ESI) m/z 462 (M − H)−.
(R)-N-(1-(2-((4-((4-((5-Methyl-1H-pyrazol-3-yl)amino)pyrrolo-
[2,1-f ][1,2,4]triazin-2-yl)thio)phenyl)amino)-2-oxoethyl)-
pyrrolidin-3-yl)isobutyramide (7a). General Procedure B. To a
suspension of compound 6 (92 mg, 0.17 mmol) in anhydrous THF (2
mL) at 0 °C was added dropwise Et3N (95 μL, 0.68 mmol), followed
by a solution of isobutyryl chloride (21 μL, 0.20 mmol) in THF. The
reaction mixture was then stirred at room temperature for 2 h. The
resulting mixture was quenched with water and extracted by EtOAc
(15 mL). The organic layer was separated, dried, and concentrated
under reduced pressure. The residue was purified by silica gel
chromatography, eluting with DCM in MeOH (20:1 to 10:1, v:v) to
give (R)-N-(1-(2-((4-((4-((5-methyl-1H-pyrazol-3-yl)amino)pyrrolo-
[2,1-f ][1,2,4]triazin-2-yl)thio)phenyl)amino)-2-oxoethyl)pyrrolidin-3-
yl)isobutyramide (7a) (35 mg, 30% yield). 1H NMR (400 MHz,
DMSO-d6) δ 12.13 (s, 1H), 10.82 (s, 1H), 10.64 (d, J = 1.2 Hz, 1H),
10.35 (t, J = 1.6 Hz, 1H), 8.15 (d, J = 1.2 Hz, 1H), 7.71 (d, J = 8.4 Hz,
2H), 7.63 (d, J = 8.4 Hz, 2H), 7.57 (s, 1H), 7.21 (q, J = 1.2 Hz, 1H),
6.58 (d, J = 2.8 Hz, 1H), 5.68 (s, 1H), 4.31 (d, J = 2.0 Hz, 2H), 3.30−
3.94 (overlapping with solvent, m, 2H), 2.38 (m, 1H), 2.05 (s, 3H),
1.88−1.94 (m, 1H), 1.01 (d, J = 6.8 Hz, 6H). LC−MS (ESI) m/z 534
(M + H)+.
procedure A. H NMR (300 MHz, DMSO-d6) δ 12.08 (br s, 1H),
10.63 (s, 1H), 10.08 (br s, 1H), 7.81 (d, J = 8.3 Hz, 2H), 7.52−7.64
(m, 3H), 7.22 (br s, 1H), 6.58 (br s, 1H), 5.63 (s, 1H), 3.56−3.75 (m,
1H), 3.34−3.43 (m, 1H), 3.18−3.30 (m, 1H), 2.91 (t, J = 8.6 Hz, 1H),
2.77−2.87 (m, 1H), 2.58−2.67 (m, 1H), 2.56 (br s, 1H), 2.02 (s, 3H),
1.90−2.01 (m, 1H), 1.67−1.86 (m, 1H), 1.10 (s, 9H). LC−MS (ESI)
m/z 533 (M + H)+.
(R)-tert-Butyl 3-(tert-Butylthio)pyrrolidine-1-carboxylate
(14). Step 1. To a stirred solution of (S)-tert-butyl 3-hydroxypyrro-
lidine-1-carboxylate (0.90 g, 4.8 mmol) in THF (5 mL) at 0 °C was
added Et3N (1.39 mL, 10.0 mmol), followed by dropwise addition of
methanesulfonyl chloride (0.68 g, 5.8 mmol). The resulting mixture
was then stirred at room temperature for 8 h before it was partitioned
between EtOAc (50 mL) and water (25 mL). The organic layer was
washed with brine (25 mL), dried over MgSO4, filtered, and
concentrated under reduced pressure to give (S)-tert-butyl 3-
((methylsulfonyl)oxy)pyrrolidine-1-carboxylate (1.07 g, 81%). LC−
MS (ESI) m/z 266 (M + H)+.
Step 2. To a suspension of NaH (60%, 211 mg, 5.27 mmol) in DMF
(3 mL) was added dropwise 2-methylpropane-2-thiol (237 mg, 2.64
mmol) at 0 °C. The reaction mixture was allowed to warm to room
temperature and stirred for 20 min. The resulting mixture was added
to a solution of (S)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl methanesul-
fonate (350 mg, 1.32 mmol) in DMF (2 mL) at 0 °C. The reaction
mixture was stirred at room temperature for 8 h and then quenched
with 10% aqueous citric acid solution. The reaction mixture was
concentrated under reduced pressure and the residue was purified by
flash column chromatography, eluting with 5% EtOAc in petroleum
ether to afford (R)-tert-butyl 3-(tert-butylthio)pyrrolidine-1-carbox-
ylate (14) (300 mg, 88% yield). LC−MS m/z 260 (M + H)+.
(R)-3-(tert-Butylsulfinyl)pyrrolidine Hydrochloride (16). Step
1. To a solution of 14 (400 mg, 1.54 mmol) in DCM (5 mL) at −20
°C was added portionwise 70% 3-chloroperbenzoic acid (417 mg, 1.69
mmol). The reaction mixture was stirred at −30 °C for 20 min, then
quenched with aqueous Na2SO3 (5 mL). The resulting mixture was
partitioned between DCM (25 mL) and 10% NaOH (10 mL). The
organic layer was washed with brine, dried over Na2SO4, filtered, and
concentrated under reduced pressure. The residue was purified by
flash column chromatography, eluting with 10% EtOAc in dichloro-
methane to give (R)-tert-butyl 3-(tert-butylsulfinyl)pyrrolidine-1-
carboxylate (220 mg, 52% yield). LC−MS m/z 276 (M + H)+.
Step 2. To a solution of (R)-tert-butyl 3-(tert-butylsulfinyl)-
pyrrolidine-1-carboxylate (275 mg, 1.00 mmol) in MeOH (2 mL)
was added dropwise 4.5 N HCl in EtOAc at 0 °C. The mixture was
allowed to warm to room temperature and was stirred for 1 h. The
organic solvents were removed under reduced pressure and the residue
was dried to afford (R)-3-(tert-butylsulfinyl)pyrrolidine hydrochloride
(16) (190 mg, 90% yield). LC−MS m/z 176 (M + H)+. The crude
product was used directly in the next step without further purification.
2-((R)-3-(tert-Butylsulfinyl)pyrrolidin-1-yl)-N-(4-((4-((5-meth-
yl-1H-pyrazol-3-yl)amino)pyrrolo[2,1-f ][1,2,4]triazin-2-yl)thio)-
phenyl)acetamide (7f). Compound 7f was prepared as an off-white
solid (22 mg, 21% yield) using general procedure A, substituting (R)-
3-(tert-butylsulfinyl)pyrrolidine hydrochloride (16) for pyrrolidine 4i
used in general procedure A. 1H NMR (400 MHz, DMSO-d6) δ 12.08
(s, 1H), 10.63 (s, 1H), 10.09 (s, 1H), 7.85 (dd, J = 8.0 Hz, 2H), 7.78
(m, 3H), 7.22 (s, 1H), 6.57 (s, 1H), 5.62 (s, 1H), 4.03(d, J = 7.2 Hz,
1H), 3.47 (s, 2H), 3.17−2.64 (m, 4H), 2.32−1.92 (m, 5H), 1.14 (s,
9H). LC−MS m/z 553 (M + H)+.
(1-(1-Benzylpyrrolidin-3-yl)-2,2-dimethylpropan-1-one (9).
To a stirred solution of ethyl 1-benzylpyrrolidine-3-carboxylate (400
mg, 1.71 mmol) in THF (5 mL) at −78 °C was added tert-
butyllithium in THF (1.7 M, 1.0 mL, 1.7 mmol) dropwise. The
resulting mixture was left at −78 °C for 30 min before it was allowed
to warm to room temperature. After another 90 min, the reaction
mixture was quenched with saturated aqueous NH4Cl (15 mL) and
extracted with EtOAc (50 mL). The organic layer was washed with
brine (15 mL), dried over Na2SO4, and evaporated under reduced
pressure. The residue was purified by silica gel column chromatog-
raphy, eluting with 30−60% EtOAc in hexanes to give (1-(1-
benzylpyrrolidin-3-yl)-2,2-dimethylpropan-1-one (9) as a colorless
oil (200 mg, 48% yield). 1H NMR (300 MHz, DMSO-d6) δ 7.32 (d, J
= 4.0 Hz, 5H), 3.64 (s, 2H), 3.49−3.61 (m, 1H), 2.80−3.02 (m, 2H),
2.29−2.55 (m, 2H), 1.96−2.12 (m, 1H), 1.79−1.95 (m, 1H), 1.12 (s,
9H). LC−MS (ESI) m/z 246 (M + H)+.
2,2-Dimethyl-1-(pyrrolidin-3-yl)propan-1-one (10). General
Procedure C. Compound 9 (210 mg, 0.86 mmol) was dissolved in 1
mL of MeOH. Then 10% Pd/C (35 mg) was added under argon
atmosphere. The mixture was stirred under a hydrogen atmosphere at
60 °C for 2 h. The reaction mixture was then filtered through a Celite
plug and the filtrate was evaporated under reduced pressure to give
2,2-dimethyl-1-(pyrrolidin-3-yl)propan-1-one (10) as a colorless oil
(120 mg, 90% yield). LC−MS (ESI) m/z 156 (M + H)+.
(R)-tert-Butyl 3-(3,3-Difluoropyrrolidine-1-carbonyl)-
pyrrolidine-1-carboxylate (19c). To a stirred solution of (R)-1-
(tert-butoxycarbonyl)pyrrolidine-3-carboxylic acid (3.8 g, 17.7 mmol)
in DMF (20 mL) were added 3,3-difluoropyrrolidine hydrochloride
(2.8 g, 19.5 mmol) and O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-
tetramethyluronium hexafluorophosphate (8.1 g, 21.3 mmol) sequen-
tially. Triethylamine (7.6 mL, 53.1 mmol) was then slowly added to
the reaction mixture. The resulting mixture was then stirred at room
temperature overnight. The reaction mixture was quenched with water
N-(4-((4-((5-Methyl-1H-pyrazol-3-yl)amino)pyrrolo[2,1-f ]-
[1,2,4]triazin-2-yl)thio)phenyl)-2-(3-pivaloylpyrrolidin-1-yl)-
acetamide (7c). Compound 7c was prepared as an off-white solid
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(50 mL) and extracted with EtOAc (2 × 50 mL). The combined
organic layers were washed with water (25 mL) and brine (25 mL),
dried over Na2SO4, and evaporated under reduced pressure. The
residue was purified by silica gel flash column chromatography, eluting
with 0−15% EtOAc in hexanes to give (R)-tert-butyl 3-(3,3-
difluoropyrrolidine-1-carbonyl)pyrrolidine-1-carboxylate (19c) (4.8 g,
analogous to the ones described for the preparation of 21c,
substituting cyclopentylamine for 3,3-difluoropyrrolidine hydrochlor-
ide used in the preparation of 19c. 1H NMR (300 MHz, DMSO-d6) δ
12.04 (s, 1H), 10.60 (s, 1H), 10.12 (s, 1H), 7.71−7.92 (m, 3H), 7.46−
7.65 (m, 3H), 7.21 (br s, 1H), 6.58 (dd, J = 2.5, 4.2 Hz, 1H), 5.65 (s,
1H), 4.00 (qd, J = 6.9, 13.7 Hz, 1H), 3.37 (s, 1H), 3.21 (d, J = 16.2 Hz,
1H), 2.57−2.95 (m, 5H), 2.03 (s, 3H), 1.86−2.00 (m, 2H), 1.78 (d, J
= 4.3 Hz, 2H), 1.61 (d, J = 6.6 Hz, 2H), 1.42−1.55 (m, 2H), 1.36 (dd,
J = 5.6, 12.0 Hz, 2H). LC−MS (ESI) m/z 560 (M + H)+.
1
88% yield) as a white solid. H NMR (300 MHz, chloroform-d) δ
3.65−3.92 (m, 5H), 3.40−3.64 (m, 3H), 3.34 (br s, 1H), 2.89−3.15
(m, 1H), 2.46 (d, J = 5.9 Hz, 1H), 2.29−2.42 (m, 1H), 1.96−2.28 (m,
2H), 1.46 (s, 9H). LC−MS (ESI) m/z 305 (M + H)+.
(R)-N-Cyclopentyl-1-(2-((4-((4-((5-methyl-1H-pyrazol-3-yl)-
amino)pyrrolo[2,1-f ][1,2,4]triazin-2-yl)thio)phenyl)amino)-2-
oxoethyl)pyrrolidine-3-carboxamide Methanesulfonate (21i
Mesylate Salt). Compound 21i methanesulfonate (7.4 g, 67%) was
prepared as a light yellow powder according to general procedure E,
substituting 21i free base for 21c free base used in general procedure
(R)-(3,3-Difluoropyrrolidin-1-yl)(pyrrolidin-3-yl)methanone
Hydrochloride (20c). To a stirred mixture of 19c (4.75 g, 15.6
mmol) in EtOAc (10 mL) was added 30 mL of 4 N HCl in 1,4-
dioxane. The resulting mixture was stirred at room temperature
overnight. The volatile solvents were evaporated under reduced
pressure, and the residue was evaporated with CH3CN (2 × 25 mL).
The residue was dried under high vacuum overnight to give (R)-(3,3-
difluoropyrrolidin-1-yl)(pyrrolidin-3-yl)methanone hydrochloride 20c
(3.8 g, 100%) as a light yellow thick oil. 1H NMR (499 MHz,
chloroform-d) δ 9.96 (br s, 1H), 9.47 (br s, 1H), 3.03−4.70 (m, 6H),
2.13−2.97 (m, 4H), 1.69−2.13 (m, 3H). LC−MS (ESI) m/z 205 (M
+ H)+.
1
E. H NMR (300 MHz, DMSO-d6) δ 12.09 (br s, 1H), 10.69−10.87
(m, 1H), 10.65 (s, 1H), 10.27 (br s, 1H), 8.03−8.25 (m, 1H), 7.73 (d,
J = 8.85 Hz, 2H), 7.64 (d, J = 8.85 Hz, 2H), 7.58 (s, 1H), 7.22 (br s,
1H), 6.59 (dd, J = 2.64, 4.33 Hz, 1H), 5.68 (br s, 1H), 4.14−4.46 (m,
2H), 3.53−4.09 (m, 3H), 2.98−3.33 (m, 4H), 2.30 (s, 3H), 2.06 (s,
3H), 1.72−1.89 (m, 2H), 1.63 (d, J = 6.97 Hz, 2H), 1.46−1.58 (m,
2H), 1.27−1.45 (m, 2H). LC−MS (ESI) m/z 560 (M + H)+.
Kinase Competition Binding. KinomeScan competition binding
assays (www.kinomescan.com) were performed as described pre-
viously.13
(R)-2-(3-(3,3-Difluoropyrrolidine-1-carbonyl)pyrrolidin-1-yl)-
N-(4-((4-((5-methyl-1H-pyrazol-3-yl)amino)pyrrolo[2,1-f ]-
[1,2,4]triazin-2-yl)thio)phenyl)acetamide (21c). General Proce-
dure D. To a stirred solution of compound 2 (6.1 g, 14.8 mmol) in
DMF (25 mL) was added compound 20c (3.8 g, 15.8 mmol) in DMF
(10 mL), followed by N,N-diisopropylethylamine (6.4 mL, 36.8
mmol) and potassium iodide (0.24 g, 1.4 mmol). The reaction mixture
was heated at 85 °C for 4 h. LC−MS indicated the completion of the
reaction. The reaction mixture was then cooled to room temperature,
quenched with water (50 mL), and extracted with EtOAc (3 × 50
mL). The combined organic layers were washed with water (50 mL)
and brine (50 mL), dried over Na2SO4, and evaporated under reduced
pressure. The residue was purified by silica gel flash column
chromatography, eluting with 0−5% MeOH (containing 2 N NH3)/
EtOAc to give (R)-2-(3-(3,3-difluoropyrrolidine-1-carbonyl)-
pyrrolidin-1-yl)-N-(4-((4-((5-methyl-1H-pyrazol-3-yl)amino)pyrrolo-
[2,1-f ][1,2,4]triazin-2-yl)thio)phenyl)acetamide (21c) as a white solid
HTC-116 pHH3 Cell Assay. HCT-116 cells derived from a human
colorectal carcinoma cell line (ACT no. CLL-247) were grown to 80−
90% confluency in McCoy’s 5A complete medium (Gibco catalog no.
16600-108) supplemented with 10% FBS and penicillin (100 U/mL)/
streptomycin (100 μg/mL) and seeded at 4 × 104 cells/well into
prewarmed 96-well poly-D-lysine-coated plates and then incubated at
37 °C, 5% CO2 for 4−6 h. At the end of the incubation period,
nocodazole was added to the wells and maintained at a final
concentration of 66 ng/mL to arrest the cells in mitosis. The cells were
incubated with nocodazole overnight at 37 °C, 5% CO2 for 16−18 h.
At the end of the incubation period, the cells were added to a 96-well
propylene plate containing compounds in DMSO that were serially
diluted 3-fold down the row nine times for the generation of a nine-
point curve. The cells were treated with compounds for 2 h at 37 °C,
5% CO2. To harvest the HH3 protein, the medium was first aspirated
from cells and the plate washed with cold PBS. The cells were lysed
with cell extraction buffer (Biosource, catalog no. FNN0011)
supplemented with phosphatase inhibitor (Roche catalog no. 11 83
580 001), and the plate was shaken at 5 °C for 30 min followed by
centrifugation at 3000g for 20 min. The samples were then transferred
to a Nunc PS 96-well plate and diluted in standard dilution buffer
provided in the Path Scan phospho histone H3 (Ser 10) sandwich
ELISA kit (Cell Signaling catalog no. 7155). The diluted samples were
directly used following the protocol for the Path Scan phospho histone
H3 (Ser 10) sandwich ELISA kit. The phosphorylation state of HH3
was detected using an anti-phospho-histone H3 (Ser10) antibody in a
colorimetric sandwich ELISA. The reaction product was quantified by
measuring the absorbance of the samples at 450 nm using a
SpectraMax Plus 384 plate reader. The concentration of a compound
that inhibited the phosphorylation of HH3 by 50% was reported as the
pHH3 IC50 for that compound.
HTC-116 Cell Proliferation Assay. HCT-116 cells derived from a
human colorectal carcinoma cell line (ATCC no. CCL-247) were
grown to 50−70% confluency in McCoy’s 5A complete medium
(Gibco catalog no. 16600-108) with 10% FBS and penicillin (100 U/
mL)/streptomycin (100 μg/mL). Cells were seeded at 800 cells/well
in a 96-well plate and were allowed to incubate at 37 °C, 5% CO2
overnight. On the following day, compounds in DMSO were serially
diluted 3-fold down the row nine times for the generation of a nine-
point curve. The diluted compounds were added to the plate
containing cells and allowed to incubate for 72 h at 37 °C, 5% CO2. At
the end of the incubation period, the cells were directly used following
the bromodeoxyuridine (BrdU) ELISA protocol described in the cell
proliferation ELISA, BrdU (colorimetric) kit (catalog no.
11647229001, Roche Applied Science). The peroxidase reaction
1
(7.1 g, 83% yield). H NMR (300 MHz, DMSO-d6) δ 12.07 (br s,
1H), 10.63 (br s, 1H), 10.13 (br s, 1H), 7.84 (d, J = 8.1 Hz, 2H),
7.50−7.76 (m, 3H), 7.23 (br s, 1H), 6.59 (d, J = 2.4 Hz, 1H), 5.64 (br
s, 1H), 3.97 (t, J = 13.0 Hz, 1H), 3.67−3.87 (m, 2H), 3.51−3.66 (m,
2H), 3.06−3.28 (m, 2H), 2.62−3.01 (m, 5H), 2.29−2.47 (m, 1H),
2.12−2.23 (m, 1H), 2.03 (br s, 3H), 1.74−1.96 (m, 1H). LC−MS
(ESI) m/z 582 (M + H)+.
(R)-2-(3-(3,3-Difluoropyrrolidine-1-carbonyl)pyrrolidin-1-yl)-
N-(4-((4-((5-methyl-1H-pyrazol-3-yl)amino)pyrrolo[2,1-f ]-
[1,2,4]triazin-2-yl)thio)phenyl)acetamide Methanesulfonate
(21c Mesylate Salt). General Procedure E. To a stirred suspension
of compound 21c free base (7.1 g, 12.2 mmol) in EtOH (40 mL) at
room temperature was added methanesulfonic acid (1.23 g, 12.8
mmol) in EtOH (10 mL) dropwise. The resulting mixture was heated
at 65 °C, and the reaction mixture became clear soon after the heating
was initiated. After heating at 65 °C overnight, the reaction mixture
was cooled to room temperature, and Et2O (20 mL) was added with
stirring. The white precipitates were collected via filtration, washed
with cold EtOH/Et2O (1:1, v/v), and dried in a vacuum oven at 50 °C
for 1 day to give (R)-2-(3-(3,3-difluoropyrrolidine-1-carbonyl)-
pyrrolidin-1-yl)-N-(4-((4-((5-methyl-1H-pyrazol-3-yl)amino)pyrrolo-
[2,1-f ][1,2,4]triazin-2-yl)thio)phenyl)acetamide methanesulfonate
1
(6.4 g, 77%). H NMR (300 MHz, DMSO-d6) δ 12.10 (br s, 1H),
10.82 (br s, 1H), 10.65 (s, 1H), 10.32 (br s, 1H), 7.73 (d, J = 8.67 Hz,
2H), 7.64 (d, J = 8.67 Hz, 2H), 7.54−7.60 (m, 1H), 7.22 (br s, 1H),
6.59 (dd, J = 2.64, 4.33 Hz, 1H), 5.68 (s, 1H), 4.16−4.52 (m, 2H),
3.62−4.10 (m, 4H), 3.08−3.62 (m, 5H), 2.34−2.62 (m, 2H), 2.32 (s,
3H), 1.80−2.21 (m, 4H). LC−MS (ESI) m/z 582 (M + H)+.
(R)-N-Cyclopentyl-1-(2-((4-((4-((5-methyl-1H-pyrazol-3-yl)-
amino)pyrrolo[2,1-f ][1,2,4]triazin-2-yl)thio)phenyl)amino)-2-
oxoethyl)pyrrolidine-3-carboxamide (21i). Compound 21i (9.4 g,
81% yield) was prepared as a light yellow powder using procedures
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product was quantified by measuring the absorbance of the samples at
450 and 690 nm using a SpectraMax Plus 384 plate reader. The
concentration of a compound that inhibited the proliferation of HCT-
116 by 50% was reported as the antiproliferation IC50 for that
compound.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 858-334-2164. Fax: 858-334-2192. E-mail: gliu@
ambitbio.com.
Pharmacokinetics. Precatheterized male SD rats (jugular vein,
230−300 g; Charles River, Hollister, CA) and female athymic Nu/Nu
rat (tail vein, 225−250 g; Charles River, Hollister, CA) were
acclimated in the vivarium for at least 3 days following delivery and
prior to entering a study. Rats were fasted overnight before dosing.
Compounds were administered iv to SD rats at 1 mg/kg in PEG400/
water (3:1, v/v) and to Nu/Nu rats at 5, 15, and 50 mpk (compound
21c) or 3, 10, and 30 mg/kg (compound 21i) in Tween-80/PEG200/
polyethylene glycol/water (2:10:5:83, v/v/v/v). Three rats were used
per study arm. Blood was collected into K3 EDTA tubes at 5 min, 15
min, 30 min, 1 h, 2 h, 4 h, 6 h, and 24 h postdose. Collection tubes
were mixed gently and spun at 4 °C to isolate the plasma. Plasma
samples, calibration, and quality control standards (20 μL) were
extracted with six volumes of acetonitrile (ACN) containing an
internal standard and analyzed by LC−MS/MS (Sciex 3200). Sample
separation was achieved on a 5 μm Zorbax SB-C8 column (4.6 mm ×
50 mm) using a 1.6 mL/min flow rate and a 1 min gradient (1.5 min
cycle time) from 5% to 95% ACN containing 0.05% formic acid. The
parent compound to fragment mass transitions of 582.1 → 217.2
(21c) and 560.2 → 195.3 (21i) were monitored. Peak areas were
quantified using Analyst 1.4.1, and pharmacokinetic parameters were
calculated from the normalized LC−MS/MS peak areas using a
noncompartmental model and the linear trapezoidal estimation
method with WinNonlin (Pharsight, version 5.2).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Drs. Shripad S. Bhagwat and Wendell D. Wierenga
for guidance and support. We thank the Ambit High
Throughput Screening team (now a division of DiscoveRx)
for kinase profiling and Kd measurements. We also thank
Shanghai Syncores Technologies, Inc. for assistance in
synthesizing some of the compounds listed herein. We also
thank Piedmont Research Center, LLC for performing the
nude rat xenograft studies.
ABBREVIATIONS USED
■
CAN, acetonitrile; pAURKA, phosphorylated Aurora A kinase;
BrdU, bromodeoxyuridine; CL, clearance value; CR, complete
regression; pHH3, histone H3 phosphorylation; QOD, every
other day dosing; SD, Sprague−Dawley rat; TBTU, O-
(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoro-
borate; TGI, tumor growth inhibition; TPPW, 2% Tween-80,
5% propylene glycol, 10% PG-400, 83% water
In Vivo Tumor Xenograft Studies. Female athymic nude rats
(Nu/Nu Harlan Laboratories, Indianapolis, IN) between 8 and 9
weeks of age were implanted subcutaneously on day 0 with 5 × 106
HCT-116 cells in 200 μL of PBS. Ten days later (designated as Day 1)
rats were sorted into groups of 10 animals each with a mean tumor
volume per group of 301−306 mm3. Both compounds 21c and 21i
were formulated in TPPW and were administered by lateral tail vein
injection. Each dose of the drug was delivered at a volume of 10 mL/
kg and was adjusted for the body weight of the animal. Compound 21c
was given either at 5, 10, or 20 mg/kg on days 1, 3, 5, 8, 10, 12, 15, 17,
19 or at 2.5, 5, or 10 mg/kg on days 1−4, 8−11, 15−18. Compound
21i was given at 10 or 20 mg/kg on days 1, 3, 5, 8, 10, 12, 15, 17, 19
and at 40 mg/kg on days 1, 3, 5, 8, and 10 and then at 30 mg/kg on
days 15, 17, 19 because of a tolerability issue. An alternative schedule
with compound 21i was given at 5, 10, or 20 mg/kg on days 1−4, 8−
11, 15−18. Camptosar (irinotecan) lot OAPWR was received as a
commercial dosage formulation at 20 mg/mL in 5% dextrose in water
(D5W). Animals were treated with Camptosar at 100 mg/kg ip qw×3.
Animals with tumors in excess of 10 000 mg or with excessive
ulcerated tumors were euthanized, as were those found in obvious
distress or in a moribund condition. Body weights and tumor
measurements were recorded twice weekly. Tumor burden was
estimated from caliper measurements by the formula for the volume of
a prolate ellipsoid, assuming unit density: tumor burden (mm3) =
(LW2)/2, where L and W are the respective orthogonal tumor length
and width measurements in mm.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Molecular modeling of a previously described Aurora inhibitor
in complex with Aurora A; experimental data for compounds 2,
5a−d,f,g, 7b−e,g, 21a,b,d−h,j−l; complete list of additional
kinase binding affinities determined for compounds 21c and
21i; additional nude rat xenograft efficacy figures for
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charge via the Internet at http://pubs.acs.org.
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