The discovery of polo-like kinase 4 inhibitors: Identification of (1 R,2 S)-2-(3-((E)-4-(((cis)-2,6-Dimethylmorpholino)methyl)styryl)-1 H -indazol-6-yl)-5′-methoxyspiro[cyclopropane-1,3′-indolin]-2′-one (CFI-400945) as a potent, orally active antitumor agent

Article abstract of DOI:10.1021/jm5005336

Previous publications from our laboratory have introduced novel inhibitors of Polo-like kinase 4 (PLK4), a mitotic kinase identified as a potential target for cancer therapy. The search for potent and selective PLK4 inhibitors yielded (E)-3-((1H-indazol-6-yl)methylene)indolin-2-ones, which were superseded by the bioisosteric 2-(1H-indazol-6-yl)spiro[cyclopropane-1,3′-indolin]-2′-ones, e.g., 3. The later scaffold confers improved drug-like properties and incorporates two stereogenic centers. This work reports the discovery of a novel one-pot double S_N2 displacement reaction for the stereoselective installation of the desired asymmetric centers and confirms the stereochemistry of the most potent stereoisomer, e.g., 44. Subsequent work keys on the optimization of the oral exposure of nanomolar PLK4 inhibitors with potent cancer cell growth inhibitory activity. A short list of compounds with superior potency and pharmacokinetic properties in rodents and dogs was studied in mouse models of tumor growth. We conclude with the identification of compound 48 (designated CFI-400945) as a novel clinical candidate for cancer therapy.

Full text of DOI:10.1021/jm5005336

Article
pubs.acs.org/jmc
The Discovery of Polo-Like Kinase 4 Inhibitors: Identification
of (1R,2S)‑2-(3-((E)‑4-(((cis)‑2,6-Dimethylmorpholino)methyl)styryl)‑
1H‑indazol-6-yl)-5′-methoxyspiro[cyclopropane-1,3′-indolin]-2′-one
(CFI-400945) as a Potent, Orally Active Antitumor Agent
Peter B. Sampson,† Yong Liu,† Bryan Forrest,† Graham Cumming,‡ Sze-Wan Li,† Narendra Kumar Patel,†
Louise Edwards,† Radoslaw Laufer,† Miklos Feher,† Fuqiang Ban,† Donald E. Awrey,† Guodong Mao,†
Olga Plotnikova,† Richard Hodgson,† Irina Beletskaya,† Jacqueline M. Mason,† Xunyi Luo,†
Vincent Nadeem,† Xin Wei,† Reza Kiarash,† Brian Madeira,† Ping Huang,† Tak W. Mak,† Guohua Pan,†
and Henry W. Pauls*,†
†Campbell Family Institute for Breast Cancer Research, University Health Network, TMDT East Tower, MaRS Centre,
101 College Street, Toronto, Ontario MG5 1L7, Canada
‡Celtic Catalysts, 1-03 Nova Centre, Belfield, Dublin 4, Ireland
S
* Supporting Information
ABSTRACT: Previous publications from our laboratory have introduced novel inhibitors of Polo-like kinase 4 (PLK4), a mitotic
kinase identified as a potential target for cancer therapy. The search for potent and selective PLK4 inhibitors yielded (E)-3-((1H-
indazol-6-yl)methylene)indolin-2-ones, which were superseded by the bioisosteric 2-(1H-indazol-6-yl)spiro[cyclopropane-1,3′-
indolin]-2′-ones, e.g., 3. The later scaffold confers improved drug-like properties and incorporates two stereogenic centers. This
work reports the discovery of a novel one-pot double SN2 displacement reaction for the stereoselective installation of the desired
asymmetric centers and confirms the stereochemistry of the most potent stereoisomer, e.g., 44. Subsequent work keys on the
optimization of the oral exposure of nanomolar PLK4 inhibitors with potent cancer cell growth inhibitory activity. A short list of
compounds with superior potency and pharmacokinetic properties in rodents and dogs was studied in mouse models of tumor growth.
We conclude with the identification of compound 48 (designated CFI-400945) as a novel clinical candidate for cancer therapy.
with high homology to the Aurora kinases. PLK4 has a re-
stricted tissue distribution, being present only in proliferating
tissues.
PLK4 localizes to the centrosomes and is a critical regulator
of centriole duplication.5−7 Deregulation of PLK4 results in loss
of centrosome numerical integrity and leads to chromosome
instability (CIN), a characteristic observed in many types of
cancers.8−10 It has been shown that PLK4 is up-regulated in
breast cancer, specifically in the basal-like subtype, and that
INTRODUCTION
■
The Polo-like kinase (PLK) family of serine/threonine kinases
plays a critical role in the regulation of mitosis.1 The family
derives its name from the Polo-box domains (PBD) incorpo-
rated in the proteins, which are distinct and separate from the
kinase (ATP binding) pocket.2 The PBDs bind phosphorylated
peptide sequences, and this interaction plays a role in the
localization of Polo-like kinases to subcellular structures. Of the
five members described to date, PLK1, 2, 3, 4, and 5, PLK1 is
the most studied member. Multiple PLK1 inhibitors have been
described, and several have been taken into the clinic.3 By con-
trast, PLK4 has, as yet, received little attention. This enzyme is
structurally the most distinct member of the family.4 Unlike
PLK1, 2, and 3, PLK4 has one Polo box only and an active site
Special Issue: New Frontiers in Kinases
Received: April 3, 2014
© XXXX American Chemical Society
A
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Scheme 1. Systematic Optimization of PLK4 Inhibitors
Scheme 2. Preparation of Chiral Indazolyl-spiro[cyclopropane-indolinones] via a Double SN2 Displacement
high PLK4 levels are associated with poor patient out-
comes.11−13 We have shown that treatment of breast cancer
cells with PLK4 RNAi prevents centriole duplication and
results in cell death while normal breast cells are unaffected.14,15
Consistent with these observations, RNAi-mediated depletion
of PLK4 in breast cancer xenografts causes growth suppression.
These findings identified PLK4 as a potential target for anti-
cancer therapy and caused us to initiate a drug discovery
effort.16,17
The most common strategy for the inhibition of kinases is
binding to the ATP site; potent inhibitors result but the poten-
tial for cross reactivity with other kinases is well docu-
mented.18−21 An alternative approach to inhibition is possible
for the Polo-like kinase family. A priori, binding to the polo-box
domain could yield specific inhibitors and overcome drug
resistance often associated with ATP competitive kinase
inhibitors.22 The PBD is unique for the family of PLKs and
is essential for PLK functions and so is a potentially useful site
for the development of potent inhibitors for clinical uses.
Unfortunately, the known ligands for PBD sites are generally
rather large phosphorylated peptide sequences.23 These are
unsuitable starting points for lead generation, and thus our
approach was dictated by the availability of drug-like hits for the
inhibition of PLK4, which turned out to be ATP competitive
hinge binders.16,17
Our previous publication on PLK4 inhibition described the
discovery of a novel series of ATP competitive kinase inhibitors,
namely (E)-3-((1H-indazol-6-yl)methylene)indolin-2-ones 1
(Scheme 1). Subsequent optimization of PLK4 activity by sev-
eral orders of magnitude was accomplished by substituting the
indolinone ring and appending an aryl vinyl moiety on the
indazole, giving compound 2.17 The preceding work24 describes
the bioisosteric replacement of the alkene linker with a cyclo-
propane ring to yield (2-(1H-indazol-6-yl)spiro[cyclopropane-
1,3′-indolin]-2′-ones, for example, compound 3. This change
resulted in a substantial improvement in drug-like properties
B
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a
Scheme 3. Synthesis and Chiral Separation of Racemic (1R,2S)-2-(1H-Indazol-6-yl)spiro[cyclopropane-1,3′-indolin]-2′-ones
a
Reagents and conditions: (i) BnCl, DMSO, t-BuOK, 4 h; (ii) BnCl, 150 °C, 8 h; (iii) 4,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxaborolane, Pd(PPh3)2Cl2
(2 mol %), K2CO3, DME/H2O, 80 °C, 7 h; (iv) K2OsO4·2H2O (0.5 mol %), NMO (1.1 equiv), citric acid (2 equiv), t-BuOH/H2O, rt 16 h then
50 °C; (v) SO2Cl2, TEA, CH2Cl2, rt; (vi) MsCl, TEA, CH2Cl2, 0 °C; (vii) NaH (60% in oil), THF; (viii) DMSO, t-BuOK, THF, O2; (ix) chiral
preparative SFC; (x) I2, K2CO3; (xi) Me3S(O)I, NaH, DMF, rt; (xii) ArCHCHBpin, 2:1 PhMe/EtOH, 1 M aq Na2CO3, Pd(PPh3)4 (5 mol %),
microwave 120 °C, 2 h or 1 M aq Na2CO3, LiCl, Pd(PPh3)4 (2.5 mol %), dioxane, reflux, 18 h. Relative and absolute stereochemistry indicated by
unwedged (bold and hashed) and wedged (bold and hashed) lines, respectively.
such as oral bioavailability, which were further refined by morph-
ing the dimethyl amino moiety to a morpholine-containing
compound 4.
was anticipated that only one of these enantiomers was
responsible for the potent PLK4 activity observed. The X-ray
structure presented in the preceding paper showed that
the active enantiomer was likely to have the 1R,2S stereo-
chemistry.24 In fact, our modeling studies indicated that this
same enantiomer would be favored by up to 2 log units of
binding energy. In addition, because development of racemic
mixtures is undesirable, a chiral route to our final compounds
was required.
Besides the documented beneficial effects,24 the change
from an alkene to cyclopropane linker had stereochemical
consequences: the new series has two stereogenic centers.
Fortuitously, the chemistry employed to install the cyclo-
propane ring yielded the more active diastereomer as the
major product, albeit as a racemic mixture of enantiomers. It
C
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Table 1. Effect of Spirocyclopropane Stereochemistry on PLK4 and Cell Growth Inhibition
17
17a
17b
3
3a
3b
4
4a
4b
stereochemistry
PLK4 IC50 (μM)
racemic
0.65
1R,2S
0.29
ND
1S,2R
∼30
ND
racemic
0.002
1R,2S
1S,2R
0.24
0.7
racemic
0.0022
<0.01
1R,2S
0.0009
<0.01
1S,2R
0.12
0.00062
<0.01
MDA-MB-468 GI50 (μM)
ND
<0.01
0.14
A number of strategies for the preparation of asymmetric
spirocyclopropanes were contemplated. The most direct
approach is to apply some chiral version of the ylide chemistry
(Corey−Chaykovsky reaction) used for the racemic synthesis.24
However, cyclopropanations using chiral sulfoxonium ylides do
not provide high levels of asymmetric induction and generally
fail when applied to complex systems.25 Chiral sulfonium ylides
have shown greater success,26 but even simple reagents, such as
dimethylsulfonium methylide, failed to react with the (E)-3-
((1H-indazol-6-yl)methylene)indolin-2-one substrates. Our
approach to the preparation of asymmetric cyclopropanes
was based on the double SN2 displacement of a chiral bis-
electrophile with bis-nucleophile (Scheme 2). A simple racemic
version of this reaction has been described by Marti and
Carreira27 using a cyclic sulfate as the bis-electrophile.28 It was
envisaged that the desired reaction would proceed via an inver-
sion of stereochemistry at the benzylic carbon, i.e., reaction of
the anion of indolinone 6 with the asymmetric cyclic sulfate 7a.
Hydrophobic collapse and/or a favorable π-stacking interaction
during the subsequent ring closure induce formation of the
desired isomer at the quaternary center of the cyclopropane.
This sets the stereochemistry as 1R,2S; removal of the benzyl
protecting group gives compound 5a, the desired isomer.
Conversely, rotation of the indolinone, ring closure and benzyl
deprotection would yield the undesired diastereomer 8a.
In this work, we verify the configuration of the most active
stereoisomer and describe the synthetic chemistry developed
to obtain the same, stereoselectively, with high enantiomeric
purity. This breakthrough, preliminarily disclosed,29 set the
stage for further optimization of the series, resulting in the iden-
tification of compound 48, a potent, orally active PLK4 inhibi-
tor as an antitumor agent for clinical development.
indazole 12, in good yield, which was smoothly converted to
racemic diol 13. Unfortunately, reaction of 13 with thionyl
chloride failed to yield the cyclic sulfate 7. In contrast, treat-
ment of diol 13 with methanesulfonyl chloride readily gener-
ated racemic 14, an alternate version of the required bis-
electrophile. To test the feasibility of the key reaction, 14 and
oxindole 6 were treated with sodium hydride in THF. We
observed rapid conversion to one major product, consistent
with the racemate 15. Similar to the Marti and Carreira result,27
the reaction had proceeded with high relative stereoselectivity
to yield the desired diastereomer (94% de). Debenzylation,
using a mixture of potassium tert-butoxide and an O2-saturated
solution in DMSO/THF,30,31 gave indazole 5, which was resolved
by chiral preparative supercritical fluid chromatography32 into the
presumptive 1R,2S (5a) and 1S,2R (5b) enantiomers. Racemic
5 was smoothly iodinated to give 17; this material was identical
in all respects to a sample of racemic 17 generated by treatment
of 1 with sulfoxonium ylide, as previously described.24 The
racemic (1R,2S)-2-(3-iodo-1H-indazol-6-yl)-5′-methoxyspiro-
[cyclopropane-1,3′-indolin]-2′-one 18 was similarly available
via the cyclopropanation of alkene 1b.
The racemic iodoindazole 17 was resolved as above into the
presumptive 1R,2S (17a) and 1S,2R (17b) enantiomers
(Scheme 3); enantiomers 18a and 18b were similarly obtained.
The tentative assignment was based on correlation of optimal
activity (Table 1) with the co-complex X-ray structure given in
our previous paper.24 Subsequent conversion of 17a and 17b to
compounds 3a and 3b and compounds 18a and 18b to
compounds 4a and 4b, respectively, was effected by Suzuki−
Miyaura reaction.
With these results in hand, preparation of chiral diol 13a
(S-enantiomer), predicted to yield the desired isomer 5a, was
initiated (Scheme 4). Sharpless asymmetric dihydroxylation of
12 using AD-mix-α generated diol 13a as the S-enantiomer in
high enantiomeric excess (>98%).33 The diol was subsequently
converted to the chiral dimesylate 14a in high yield. The key
reaction, i.e., double SN2 displacement of the dimesylate 14a
with the anionic oxindole 6, proceeded cleanly to give the
protected spirocyclopropane 15a with high enantioselectivity
(≥98% ee by chiral HPLC) as the presumptive 1R,2S enan-
tiomer. Similarly, compounds 23−26 were prepared by treat-
ment of 14a with oxindoles 19−22, respectively.
RESULTS AND DISCUSSION
■
Chemical Synthesis. Our preliminary studies were
performed with racemic substrate in order to test the feasibility
of the double displacement chemistry. Scheme 3 outlines the
preparation of the requisite starting material. Treatment of
commercially available bromoindazole 9 with benzyl chloride
and potassium tert-butoxide generated a 1:3 mixture of N2 and
N1-benzylated indazoles, 10 and 11, respectively. The mixture
was isomerized exclusively to the N1 benzylated indazole 11 by
heating to 150 °C in excess benzyl chloride. Suzuki−Miyaura
coupling with vinyl boronic acid pinacol ester gave vinyl
Attempted removal of the benzyl protecting groups of com-
pound 15a, under catalytic hydrogenation conditions, resulted
D
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a
Scheme 4. Stereoselective Synthesis of (1R,2S)-2-(1H-Indazol-6-yl)spiro[cyclopropane-1,3′-indolin]-2′-ones
a
Reagents and conditions: (i) (DHQ)2PHAL, K2OsO4·2H2O (0.5 mol %), K3Fe(CN)6, K2CO3, t-BuOH/H2O, 0 °C, 5 h; (ii) MsCl, TEA, CH2Cl2,
0 °C; (iii) NaH (60% in oil), THF; (iv) DMSO, t-BuOK, THF, O2; (v) I2, K2CO3, DMF; (vi) BnBr, DCM, 50% aq KOH, TBAB;
(vii) ArCH=CHBpin, 2:1 PhMe/EtOH, 1 M Na2CO3, Pd(PPh3)4 (5 mol %), microwave 120 °C, 2 h or 1 M Na2CO3, LiCl, Pd(PPh3)4 (2.5 mol %),
dioxane, reflux, 18 h.
to the materials prepared in Scheme 3 via chiral prep-HPLC. At
this stage, compound 30 was benzylated and the product of the
reaction 33 was recrystallized; single crystal X-ray structure de-
termination was performed on this material (vide infra). PLK4
inhibitors 34−58 were prepared via Suzuki−Miyaura coupling
of iodoindazoles 17a, 18a, and 30−32 with the vinyl boronic
acid pinacol esters in moderate yields.
Cyclopropane Stereochemistry. Our initial assignments
of stereochemistry at positions 1 and 2 of the cyclopropane ring
were based on molecular modeling predictions, wherein the
1R,2S enantiomer scored up to 2 orders of magnitude higher
than the 1S,2R. This tentative assignment was corroborated by
the PLK4 co-complex X-ray structure given in the previous paper
and the chemistry outlined in Scheme 4 which is predicted
to yield the 1R,2S isomer preferentially. This assignment was
confirmed by an X-ray crystallographic study of 33 (see Supporting
Information), which indicates that the 1R,2S stereochemistry of the
Figure 1. ORTEP diagram for compound 33 X-ray structure showing
cyclopropyl enantiomer is derived from the vicinal (S)-diol 13a via
the 1R,2S stereochemistry.
the double SN2 displacement reaction of 14a (Scheme 4). The
corresponding ORTEP diagram is shown in Figure 1.
The 2S stereochemistry is set by the SN2 inversion of the
benzylic mesylate as anticipated (Scheme 2). The induction at
in partial ring-opening of the cyclopropane. However, the
benzyl groups were removed cleanly from compounds 15a and
23−26 using the conditions developed for the racemate 15, i.e.,
the spiro position of the cyclopropane to give 1R, virtually
exclusively, was less certain. It would appear that hydrophobic
collapse and/or a π-stacking interaction34 serves to align the
indolinone and indazole in such a way to ensure that ring
closure yields the desired isomer, i.e., the left fork of the
proposed mechanism of Scheme 2. Bis-electrophiles analogous
to 14a react preferentially at the benzylic position.35−37
However, an alternative reaction course, in which the first
t-BuOK in an O2 saturated solution of THF/DMSO. Com-
pound 5a, so prepared, was identical to the material obtained
by preparative chiral chromatography (Scheme 3). Subsequent
iodination of intermediates 5a, 16a, and 27−29 gave 17a, 18a,
and 30−32, respectively, in yields ranging from 45% to 95%.
Enantiomeric excesses were determined at this stage of the
syntheses (chiral HPLC) and were found to be uniformly
≥98%. Compounds 17a and 18a were identical in all respects
E
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Table 2. In vitro Activity of (1R,2S)-2-(1H-indazol-6-yl)spiro[cyclopropane-1,3′-indolin]-2′-ones PLK4 Inhibitors
F
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Table 2. continued
displacement occurs on the primary position followed by
inversion at the benzylic position, cannot be ruled out. The
stereochemical consequences of this sequence would be the
same as long as the latter reaction is controlled by an SN2
process, and the aforementioned π-stacking is operative.
Table 1 summarizes the PLK4 activity for three racemates,
17, 3, and 4, and their constituent enantiomers. It is clear that
the 1R,2S stereoisomer 17a is substantially more active against
PLK4 than the 1S,2R stereoisomer 17b (∼100 fold) and that
the active isomer is 2 times more active than the racemate. This
preference is also seen in the more elaborated and potent
inhibitors 3 and 4 and their constituent enantiomers, 3a,b and
4a,b, respectively. The cell growth inhibitory activity also
reflects this SAR; the PLK4 active isomers are 1−2 orders of
magnitude more effective at attenuating MDA-MB-468 breast
cancer cell growth. Subsequent lead optimization efforts were
focused on the 1R,2S isomer.
to the optically active version as well. This is made clear by the
entries of Table 2, which indicate that excellent biochemical
potency was maintained by the chiral PLK4 inhibitors. Trends
previously established were recapitulated in that substitution
of the indolinone with a methoxy group at the 5′ position was
generally beneficial, as supported by the matched pair 47 and
48. The objective of the work going forward was to optimize
the drug-like characteristics of the compounds in hopes of
identifying an orally bioavailable development candidate for
the treatment of solid tumors. Our efforts were centered on
maintaining the in vitro potency of the compounds while
maximizing PK properties by fine-tuning the indolinone
substituents R and R′ and the aromatic moiety Ar.
Cell growth was determined by measuring total protein con-
tent by SRB assay after 5 days of treatment. In accordance with
other mitotic kinase inhibitors,38,39 PLK4 inhibitors require treat-
ment times of ≥4 days for their effects to manifest.40,41 Figure 2
illustrates the effect of a representative PLK4 inhibitor, namely
compound 47, on the growth inhibition of four breast cancer cell
lines (HCC1954, MCF-7, MDA-MB-231, and MDA-MB-468).
The plot shows % survival (normalized to control) versus log
Lead Optimization. Much of the SAR that had been devel-
oped on the (E)-3-((1H-indazol-6-yl)methylene)indolin-2-
ones17 was transferable to the racemic 2-(1H-indazol-6-yl)-
spiro[cyclo-propane-1,3′-indolin]-2′-ones24 and, as anticipated,
G
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with potent inhibition of PLK4. A series of analogues were
prepared in which this basic motif was retained, i.e., compounds
38−43; biochemical potency ranged an order of magnitude
with the best inhibitors at the subnanomolar level. The 5′-
methoxy compound 38 was the most potent; in contrast, ap-
pending a methyl group at the N-1 position of the indolinone
decreased potency by 2-fold, compound 39. The potential
benefits of N-methylation (described above for 36) were
realized in that compound 39 was the most effective cell
growth inhibitor of the triplet of compounds incorporating the
4-((dialkylamino)methyl)-phenyl group.
Replacement of the dimethyl amine with cyclic variants gave
compounds 40−43; potency against PLK4 was generally main-
tained as was attenuation of the growth of MDA-MB-468 cells.
Curiously, the pyrrolidines (40, 41) were effective against HCC-
1954 cells while the piperidines (42, 43) inhibited the growth of
MCF-7 cell line more strongly; we have no explanation for this
empirical observation. Representatives, 41 and 43, were tested in
mouse PK studies.
The promise of the cyclic inhibitors was explored in addi-
tional variations on this theme, namely the morpholine ana-
logues 4a and 44−53, the oxazepane 54, and the piperazines 55
and 56. The inclusion of the oxygen in this ring system reduces
the pKa of the benzylic nitrogen from 8.9 to 7.1;42 we reasoned
that modulation of the basicity could enhance permeability and
result in improved cell activity and PK parameters. These
inhibitors potently inhibited PLK4 and, excepting MDA-MB-
231, effectively inhibited the growth of the breast cancer cells.
We noted that the meso dimethyl analogues 47−49 were some-
what less effective against PLK4, but good activity against the
cell panel was maintained. However, the 1,3-phenyl substituted
analogues 50 and 51 were less active than their 1,4 substituted
counterparts and were eliminated from further considera-
tion. The methyl, bicylic, and ring expanded analogues 52, 53,
and 54, respectively offered no advantage over the parent
compound 44.
Inclusion of nitrogen into the ring system, i.e., piperazines
55 and 56, shifts the most basic center to the distal nitrogen
and yields compounds with relatively high pKas (8.4−8.8).42
These compounds inhibited PLK4 potently but weakly atten-
uated growth of the HCC-1954 and MCF-7 cell lines. In an effort
to maintain the topology of the morpholinomethyl moiety
while moving the basic center to the distal ring position,
(alkylpiperidin-4-yl)oxy compounds 57 and 58 were prepared.
The latter compound in particular had potent in vitro activities
and was tested more extensively in eADME and in vivo assays.
It should be noted that the growth inhibitory effect of com-
pounds on human mammary epithelial cells (HMEC GI50) was
measured (Table 2). The ability of the PLK4 inhibitors to
inhibit the growth of these cells was poor and generally about 3
orders of magnitude less potent than the effects seen on breast
cancer cell lines such as MDA-MB-468.
eADME and Mouse Plasma Levels. Cytochrome P450,
microsomal stability (T1/2), and mouse plasma levels (PO dosing)
were obtained for selected inhibitors, as given in Table 3. Com-
pounds were routinely tested against the 1A2 and 2D6 isoforms,
however, no inhibition was observed (data not shown). As seen
for the alkene-linked compounds,17 the CYP450 IC50 values were
generally lower for 2C9 and 2C19 as opposed to other isoforms.
Overall, the IC50s against CYP450s were micromolar or higher,
including the all-important 3A4 isoform. In some instances,
values dipped significantly below the single-digit micromolar
range (entries 49, 52, and 56) and these compounds were not
Figure 2. SRB dose−response curves for HCC1954, MCF-7, MDA-
MB-231, and MDA-MB-468 cells upon treatment with compound 47
for 5 days.
concentration of compound 47 in molar (M) and illustrates the
differential cellular responses to compound treatment. In
related work, we have evaluated the effect of our PLK4
inhibitors on growth attenuation in panels of cancer cell lines,
including breast. Distinct outcomes have been observed:
polyploidization followed by cell death (e.g., MDA-MB-468),
polyploidization followed by cell arrest (e.g., MDA-MB-231),
and an intermediate effect.40,41 The sensitivity of the most
responsive cell lines (MDA-MB468 followed by HCC1954 and
MCF-7) to compound 47 is very similar, i.e., the inflection
points for the dose response curves are around 10 nM in each
case. It is difficult to accurately determine the sensitivity of the
MDA-MB-231 cell line in that the survival is high (∼70%) and
the inflection is gradual and indistinct. The GI50s, i.e.,
concentrations of PLK4 inhibitors required to inhibit growth
of these breast cancer cell lines by 50% of the maximal response
has been captured in Table 2.
Our first publication on PLK4 inhibitors17 had demonstrated
the attributes of the 6-(4-methylpiperazin-1-yl)pyridin-3-yl aryl
group vis a vis the pyridinyl aryl group (34), and this was reca-
pitulated in the chiral 2-(1H-indazol-6-yl)spiro[cyclopropane-
1,3′-indolin]-2′-ones. The basic piperazine group in this solvent
accessible region of the molecule is associated with enhanced
cell activity with the side benefit of increased aqueous solubility.
Compounds 35 and 36 are potent against PLK4 and, excepting
MDA-MB-231, are effective in attenuating breast cancer cell
growth across the panel. The latter compound, by methylating
the indolinone nitrogen, has the benefit of removing an
H-bond donor and potentially increasing permeability.
Inspection of our binding models had suggested that this
substitution would be tolerated. Although N-methylation
removes a potential H-bond with Gln-160, this interaction is
solvated and has little thermodynamic value. Because of the
N-Me/carbonyl repulsion, the molecule is pushed away from
Gln-160; deeper insertion of the 5′-methoxy group into the
hydrophobic pocket results, which appears to compensate
somewhat for the loss of a weak H-bond. PLK4 inhibitor 37
was prepared in an effort to remove the potential liability of
the pyridyl nitrogen; the latter maintains excellent cell activity
and solubility. Compounds 34, 35, and 37 were taken forward
into mouse PK studies.
An alternate motif, described in the preceding work,24 is the
4-((dialkylamino)methyl)-phenyl group, found in compound
3a. This moiety also imparts improved aqueous solubility,
especially at acidic pH (pKa = 8.8)42 and is generally associated
H
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a
Table 3. eADME and Mouse Plasma Levels for Selected PLK4 Inhibitors
CYP450 IC50 (μM)
microsomal T1/2 (min)
mouse plasma levels (PO @ 25 mg/kg)
entry
2C9
2C19
3A4 DBF
3A4 BFC
m
r
d
h
Cmax (μg/mL)
AUC (μg·h/mL)
35
36
37
3a
38
39
41
43
44
45
4a
46
47
48
49
52
56
58
2.0
3.8
1.7
1.5
1.2
0.9
5.6
2.9
∼10
0.3
ND
∼5
ND
2.3
>1
6
7
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
11
27
0.2
0.38
1.6
0.34
0.26
0.64
1.1
1.8
1.3
10
0.7
2.8
19
46
0.120
0.11
0.3
>1
>1
∼10
7.5
13
22
12
17
40
15
13
12
24
32
24
10
>60
36
3.5
2.0
1.9
1.5
1.4
0.8
1.2
1.2
1.0
1.7
1.4
0.76
0.9
0.81
1.9
2.6
>1
44
0.51
0.79
0.51
3.5
0.9
∼10
5.4
52
3.4
>1
49
0.7
2.7
6.3
3.0
2.4
20
7.3
4.7
45
1.0
6.2
9
9.5
9.4
56
2.1
5.5
9.5
17
0.8
6.4
8.3
6.4
41
3.5
1.0
6.0
13
37
5.9
0.8
5.0
5.1
3.1
∼10
0.45
∼10
>1
>60
>60
43
>60
>60
>60
53
3.7
10
0.94
0.44
0.44
0.56
2.4
4.6
53
18
2.2
9.8
6.8
16
>1.0
11
>60
3.4
7.2
10
6.8
3.3
1.6
38
30
49
39
>60
>60
1.3
2.2
1.4
1.6
>60
>60
0.51
a
ND: not determined.
taken forward. Mouse microsomal T1/2 was often less than
30 min for the earlier entries, but the half-lives were longer for
the later entries; microsomal T1/2 was also longer in the presence
of human microsomes as compared to mouse. As a predictor of
in vivo performance in the putative toxicology species (rat and
dog), we measured mouse plasma levels obtained upon oral
dosing. Mouse was used for these preliminary exposure studies
being readily available, compound sparing, and the species of
choice for the subsequent xenografts.
The first eight entries in Table 3 attained modest plasma
levels in mouse, with the highest exposure being achieved by
the N-Me indolinone analogues 36 and 39 and the cyclic
inhibitors 41 and 43. It is possible that the increased lipo-
philicity of these entries enhances permeability, however, these
effects are modest. In contrast, the large increase in exposure
for the morpholines 4a and 44−49 is unmistakable. We postu-
late a role for the ring oxygen in attenuating the basicity of the
nitrogen (pKa = 7.1), thus striking a balance between enhanced
solubility at low pH and good permeability at physiological pH.
As a group the morpholino-methyl compounds achieved
exposures an order of magnitude higher than their
dialkylamino-methyl comparators. The more basic piperazinyl
and (alkylpiperidin-4-yl)oxy moieties, as represented by
compounds 56 and 58, respectively, showed similarly lower
exposures.
During the course of the mouse plasma studies, we modeled
the ability of early ADME measures to predict the performance
of our compounds in vivo. Figure 3 gives the correlation of
logAUC (experimental) with logAUC (predicted) for PO
dosing of PLK4 inhibitors in mice. QSAR analysis established
that two independent variables, namely mouse microsomal T1/2
and logD7.4, can describe predicted logAUC with an R2 of 0.43
and RMSE of 0.43. The correlation is low but not unexpected
when modeling in vivo data. The level of variance observed
indicates that additional factors could be important in
describing exposure of our PLK4 inhibitors in mice, such as
the involvement of phase II metabolism, which would not
be captured by microsomal measurements. It appears that the
microsomal metabolism was of some value in guiding compound
Figure 3. Correlation of logAUC (experimental) with logAUC
(predicted) for PO dosing of PLK4 inhibitors in mice. QSAR analysis
established that two independent variables (mouse microsomal T1/2
and logD7.4) describe logAUC (predicted), with an R2 of 0.43 and
RMSE of 0.43 (n = 74).
optimization but of limited applicability in predicting overall in
vivo performance.
Morpholines with a desirable combination of in vitro and
in vivo properties, namely compounds 4a and 44−48, were
advanced to rat and dog pharmacokinetic studies (Table 4).
Compounds 4a, 44, and 45 had relatively high clearance
rates in rat; 4a had a short dog t1/2 and 44 had a low oral
bioavailability in dog. Compound 45 had the additional burden
of being relatively difficult to make, a feature shared by all
compounds with a methyl group at the 5′ position of the indo-
linone. Thus, a short list of three emerged from these studies as
compounds of high interest: 46, 47, and 48. These inhibitors
were tested in an expanded cancer cell line panel including
lung, colon, and ovarian cancers (Table 5). All three com-
pounds had very similar profiles, with responses more depen-
dent on the cancer cell line tested as opposed to the compound
per se; nonetheless, the overall rank order appeared to be in
line with the inherent potency of the compounds against PLK4.
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Table 4. Rat and Dog Pharmacokinetics for Selected PLK4 Inhibitors
4a
4.0
1.1
250
44
3.2
1.4
312
45
3.5
1.6
285
46
1.9
1.3
553
47
0.84
3.9
1234
48
rat IV (1 mg/kg)
Cl (L·h/kg)
t1/2 (h)
1.9
2.0
AUCINF (ng·h/mL)
AUCINF (ng·h/mL)
oral BA (%)
534
rat PO (5 mg/kg)
dog IV (1 mg/kg)
272
22
478
31
410
28
688
25
2807
46
767
21
Cl (L·h/kg)
t1/2 (h)
2.5
0.87
402
2.0
3.2
515
250
9.7
1.4
7.0
795
1.6
3.0
648
0.50
4.5
2050
0.96
2.1
1054
AUCINF (ng·h/mL)
AUCINF (ng·h/mL)
oral BA (%)
dog PO (5 mg/kg)
1423
71
1139
29
1030
32
4987
49
3460
66
Table 5. PLK4 Ki and Growth Inhibition Data for 46, 47, and 48 in an Expanded Panel of Cancer Cell Lines
cancer cell line GI50 (μM)
breast
lung
ovarian
colon
entry
PLK4 Ki (nM)
SKBr-3
Cal-51
BT-20
A549
OVCAR-3
SW620
Colo-205
HCT116+/+
46
47
48
0.10 0.04
0.73 0.28
0.26 0.10
6.5
7.2
5.3
0.11
0.091
0.26
0.055
0.09
0.004
0.009
0.005
0.007
0.015
0.018
0.60
0.35
0.38
0.007
0.057
0.017
0.005
0.008
0.004
0.058
inhibitors of this class. Key interactions, shown in Figure 4A,
include two H-bonds from the indazole to the hinge (Glu90
and Cys92) and two H-bonds from the indolinone carbonyl
and NH to Lys41 and Gln160, respectively. Figure 4B shows a
similar view with a surface representation on the protein; the
morpholine is at the protein−water interface. There is extensive
hydrophobic contact between the enzyme and the ligand,
especially at the aromatic rings. The methoxy group nestles in a
groove bounded by the protein backbone and the side chains of
Leu143 and Glu96.
Kinase Selectivity. The short list of PLK4 inhibitors was
profiled against other members of the Polo-like kinase family;
none of the compounds significantly inhibited PLK1, 2, or 3 at
a concentration of 50 μM. In an effort to distinguish between
compounds, the short list was tested (Millipore radiometric assay)
against a large panel of kinases (see Supporting Information).
Only a subset of the kinases (48 of 274) was inhibited above
50% at 0.1 μM by at least one compound. This data is rep-
resented as a heat map in Figure 5; the PLK4 inhibitor 48
was clearly more selective than compound 46, followed by 47.
For compound 48, biochemical (IC50) and cellular (EC50)40,41
inhibition constants were determined for those human kinases
which were significantly inhibited (Table 6). There is an order
of magnitude separation between the EC50s for PLK4 (12 nM)
and the off-target kinases namely AURKA, AURKB/INCENP,
TIE2/TEK, TRKA, and TRKB. A kinetic analysis of compound
48 (Table 5 and Supporting Information) confirmed the potent
(Ki = 0.26
0.10 nM) and ATP competitive nature of the
binding, not surprising given the X-ray structures of related
compounds.
Although inhibitory effects in cellular contexts were demon-
strated for TrkA, TrkB, and Tie2/TEK, these kinases do not
have expression patterns or biological activity that would account
for the effects of the PLK4 inhibitor 48 on the cells tested in
vitro. TrkA and TrkB are highly expressed in neuroblastomas,43
whereas Tie2/TEK is overexpressed in the microvasculature of
solid tumors.44 AURKB is present in all proliferating cells and an
integral component of the chromosome passenger protein
complex (CPPC), thus its inhibition could explain some of the
observed effects of 48 such as the accumulation of polyploid
Figure 4. Binding mode for compound 48 generated by GlideXP
docking in the PLK4 (PDB code 4JXF) active site. (A) Top panel
shows H-bonds from indolinone carbonyl and NH to Lys41 and
Gln160 respectively and H-bonds from indazole to Glu90 and Cys92
of the hinge. (B) Bottom panel shows a similar view with a surface
representation on the protein; morpholine is at protein−water
interface; the OMe group nestles in a groove bounded by the protein
backbone and side chains.
PLK4 Binding Model. A model of compound 48 in the
PLK4 active site is shown in Figure 4 and is representative for
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Figure 5. Heat map showing % inhibition of kinases at a screening concentration of 0.1 μM by PLK4 inhibitors 46, 47, and 48. Only kinases
inhibited >50% by at least one compound (46 of 274) are represented.
Table 6. Biochemical (IC50) and Cellular (EC50) Binding
Constants for Compound 48
schedules.46−48 Accordingly, the maximum tolerated dose
(MTD) for compounds 46, 47, and 48 for a 1-on/3-off schedule
were estimated to be 39, 36, and 39 mg/kg, respectively. Thus,
we were able to increase the total compound dosed over the
21 day experiment from 197.4 mg/kg for 9.4 mg/kg (QD
regimen) to 252 and 273 mg/kg for the 36 and 39 mg/kg (1-on/
3-off regimen) respectively. The short list was dosed at MTD
and 2/3MTD in an HCT116 xenograft model (Figures 7A,B),
with the last dose on day 21. PLK4 inhibitor 48 was the most
effective followed by compounds 46 and 47; the maximal res-
ponse was seen at day 25 (98, 90, and 78% TGI, respectively, at
the MTD). All dosing arms were tolerated. The experiment
continued to day 35 (93% TGI for compound 48 at 39 mg/kg);
at this point, the control tumors had grown to a volume such
that the animals needed to be taken off study and sacrificed.
Compound 48. Compound 48 represents an optimized
structure from the (1R,2S)-2-(3-((E)-4-((dialkylamino)methyl)-
styryl)-1H-indazol-6-yl)spiro[cyclopropane-1,3′-indolin]-2′-one
class of PLK4 inhibitors. Although having comparable in vitro
potency to many other inhibitors listed in Table 2, the mor-
pholino group confers a high level of oral exposure not shared
by the compounds incorporating more basic dialkylamino
moieties. The two methyl substituents on the morpholine con-
fer additional benefit in that clearance in rat and dog is signi-
ficantly reduced. The methoxy substituent on the indolinone
ring is associated with increased potency and selectivity as
evidence by the head to head comparison of compounds 47
and 48. The advantages of compound 48 culminate in its
superior performance in mouse xenografts. On the basis of the
cumulative results (vida supra) and additional preclinical
data,40,41 compound 48 was selected for IND enabling studies.
kinase
PLK4
IC50 (nM) EC50 (nM)
kinase
IC50 (nM) EC50 (nM)
2.8
140
98
12
510
102
TIE2/TEK
TRKA
22
6
117
84
AURKA
AURKB/
INCENP
TRKB
9
88
cells.40,41 Cellular effects of PLK4 inhibition are observed (e.g.,
inhibition of centriole duplication),40,41 and compound 48 is
8.5-fold selective for PLK4 over AURKB in cells. However, we
cannot rule out that inhibition of AURKB, or other off-target
kinases, contribute to the activity of compound 48.
In Vivo Efficacy. Mouse xenograft studies were performed
with the short list, chosen from three pathologies, namely,
breast (MDA-MB-468), colon (SW620), and lung (A549);
these clean cell lines were well established in our laboratories,
demonstrating reproducible tumor growth rates in mice. The
compounds were given orally at a high (9.4 mg/kg) and low
(3.0 mg/kg) dose, once a day, for 21 days. Figure 6 shows the
results for a representative study with compounds 47 and 48
in the breast MDA-MB-468 xenograft model. All doses were
tolerated in that none of the animals were lost during the
course of the experiment (21 days) and none of the mice lost
more than 20% of their body weight. A dose response was
seen for both compounds, and all four treatment arms reached
statistically significant response vis a vis control (p < 0.05).
Table 7 summarizes the results for the shortlisted compounds
in the three xenograft models. Note that all three compounds
were efficacious in the models tested, albeit to varying degrees;
there was a robust response in the SW620 xenograft which
would not have been predicted by the in vitro results (Table 5).
The lone exception was compound 46 in the A549 model; the
high dose was not tolerated and all animals in this arm were
lost. In summary, we deemed these results supported our inter-
est in the shortlisted compounds as potential anticancer agents
but were not discriminatory.
CONCLUSIONS
■
We have verified the configuration of the most active ste-
reoisomer of the (2-(1H-indazol-6-yl)spiro[cyclopropane-1,3′-
indolin]-2′-ones to be 1R,2S and have described a novel synthetic
method to obtain the same, with high enantiomeric and diastereo-
meric purity. This breakthrough set the stage for systematic
optimization of this series to identify the morpholinomethyl-
styryl derivatives with nanomolar activity against PLK4 and con-
comitant inhibitory effects against a panel of breast cancer cell
There are theoretical reasons to suppose that antimitotic
agents can better exert their effects on tumor growth inhibi-
tion by employing an intermittent dosing regimen,45 and
some chemotherapeutic agents perform optimally on such
K
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Figure 6. Tumor volume and average weight vs days of treatment with compounds 47 and 48 in an MDA-MB-468 mouse xenograft model
(PO QD dosing).
been completed and this compound has entered phase I human
trials in Canada and the United States.
Table 7. Mouse Xenograft Results for PLK4 Inhibitors 46,
47, and 48
percent tumor growth inhibition
EXPERIMENTAL SECTION
■
MDA-MB-468
SW620
A549
Computational Methods. The computational processes, model
refinement, and QSAR-type analyses were performed using the MOE
software.49 The PLK4 co-complex X-ray structure, described in the
previous paper,24 was used for the docking experiments described
herein. Ligand preparation, conformer generation, and docking
experiments were as previously described.17
dose 3
dose 9.4
mg/kg
dose 3
mg/kg
dose 9.4
mg/kg
dose 3
mg/kg
dose 9.4
mg/kg
entry
mg/kg
a
a
a
a
a
b
46
47
48
61
96
47
77
46
ND
a
a
a
a
a
45
90
57
80
44
79
a
a
a
a
57
77
7
86
32
70
a
b
Synthetic Chemistry. General Experimental Methods. Commer-
cially available starting materials, reagents, and solvents were used as
received. In general, anhydrous reactions were performed under an
inert atmosphere such as nitrogen or argon. Microwave reactions were
performed using a Biotage Initiator microwave reactor. Reaction
progress was generally monitored by TLC using Merck silica gel plates
with visualization by UV at 254 nm by analytical HPLC or 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. Crude reactions
were partially purified using Waters PoraPak Rxn CX cartridges. Pre-
parative reverse-phase HPLC purification was performed on a Varian
PrepStar model SD-1 HPLC system with a Varian Monochrom 10 μ
C-18 reverse-phase column. Elution was performed using a gradient of
10% MeOH/water to 90% MeOH/water (0.05% TFA) over a 40 min
period at a flow rate of 40 mL/min. Fractions containing the desired
material were concentrated and lyophilized to obtain the final pro-
ducts. All reported yields were not optimized. Proton NMRs were
recorded on a Bruker 400 MHz spectrometer, and mass spectra were
obtained using a Bruker Esquire 4000 spectrometer. Chiral SFC
separations were performed by Lotus Separations, Princeton, NJ.
p < 0.05. All animals in this arm were lost during the study.
lines. These compounds demonstrated high exposure upon oral
dosing in mice, and a subset was taken forward into rat and dog
PK studies from which a short list of three compounds was
selected for detailed study.
Compounds 46, 47, and 48 are single-digit nanomolar PLK4
inhibitors and selective against other members of the PLK
family (>10 μM). The shortlisted compounds attenuate the
growth of a variety of breast, colon, and ovarian cancer cell lines
and are efficacious in mouse xenograft models of tumor growth.
Compound 48 is the most selective shortlisted compound as
determined by a radiometric assay against a panel of 274 human
kinases. Upon intermittent oral dosing, in a mouse model
of colon cancer, compound 48 was an effective inhibitor of
HCT116 tumor growth and was well tolerated. On the strength
of the data presented herein, and additional preclinical
studies,40,41 compound 48 (designated CFI-400945) was
selected for clinical development. IND enabling studies have
L
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Figure 7. HCT-116 mouse xenograft model. Solid arrows indicate treatment days with compounds 46, 47, and 48 (1 on 3 off, PO); 5-fluorouracil
was given at 50 mg/kg IP for 5 days. (A) Top panel shows tumor volume vs day following treatment initiation. (B) Bottom panel shows percent
mean body weight loss vs day following treatment initiation. The solid arrows indicate dosing events.
Optical rotation was measured on an Optical Activity AA-55
polarimeter. Compound purity by analytical HPLC (UV detection at
λ = 270 nm) was performed on a Varian Prostar HPLC or Shimadzu
Prominance HPLC, using a 100 mm × 4.6 mm Phenomenex Luna 3 μ
M
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C-18 column. Elution was performed using a gradient 10% MeOH/
water to 90% MeOH/water (0.05% TFA) between 1 and 10 min at a
flow rate of 1 mL/min. Purity is reported as area percent; the purity of
all final compounds was shown by HPLC to be ≥95%.
A mixture of t-BuOH and water (1:1, 4 L) was added, forming a clear,
biphasic mixture on stirring. The mixture was cooled with an ice
bath before addition of N1-benzyl-6-vinyl-1H-indazole (12) (96 g,
410 mmol). The resulting mixture was vigorously stirred at 0 °C for
4 h, and then warmed to 10 °C for 2 h. The reaction was quenched by
portionwise addition of sodium metabisulfite (1 kg). The mixture was
stirred for 1 h at rt and then filtered through a pad of Celite to remove
precipitated OsO2. The filtrate was extracted with CH2Cl2 (4 × 2 L),
and the combined organic portions dried over MgSO4, filtered, and
concentrated. The crude product was recrystallized from hot toluene
(10 mL/g) to afford the title compound as a white solid (74 g, 68%,
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 spec, using an ESI source. Compounds in DMSO solutions
(5 mg/mL) were diluted to 0.01 mg/mL; 5 uL was injected on to a
Waters BEH C18 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.
Preparation of Indazole Intermediates. N1-Benzyl-6-bromo-1H-
indazole (11). A solution of 6-bromo-1H-indazole (9) (300 g,
1.52 mol) in dry DMSO (1.5 L) was treated with KOtBu (181 mL,
1.8 mol). After 10 min, BnCl (199 g, 1.8 mol) was added, and the
resulting mixture left to stir at rt. After 4 h, the reaction was quenched
with satd NH4Cl (3 L) and extracted with MTBE (2 × 2 L). The
combined organic phases were washed with brine (2 × 1 L) and
concentrated to a red oil. The oil was slurried with heptane to give a
yellow solid as a 3:1 mixture of N1- and N2-benzylated indazoles
(11 and 10). The mixture was isomerized exclusively to the N1 isomer
by dissolving into benzyl chloride (350 mL) and heating to 150 °C for
8 h. Excess benzyl chloride was distilled off at 150 °C to give a red oil
which was treated with heptane (600 mL) to give 11 as a red solid
(335 g, 77%). 1H NMR (400 MHz, CDCl3) δ 8.00 (d, J = 0.6 Hz, 1H),
7.58 (d, J = 8.6 Hz, 1H), 7.52 (s, 1H), 7.36−7.20 (m, 4H), 7.20−7.15
(m, 2H), 5.53 (s, 2H). LCMS (ESI) m/z calcd for [C14H11BrN2 + H]+
287.0 (79Br), 289.0 (81Br); found, 287.0; 289.0.
N1-Benzyl-6-vinyl-1H-indazole (12). A mixture of 11 (300 g,
1.05 mol) and K2CO3 (432 g, 3.1 mol) in DME/water (3:1, 3.3 L) was
purged with nitrogen. 4,4,5,5-Tetramethyl-2-vinyl-1,3,2-dioxaborolane
(300 mL, 1.3 mol) was added, followed by Pd(PPh3)2Cl2 (36 g,
5 mol %), and the mixture was heated to 80 °C for 7 h. The mixture was
diluted with heptane (2 L) and washed with water (3 × 1 L) and brine
(1 L). The organic phase was concentrated to 1 L and cooled to 0 °C.
The resulting precipitate was collected and dried under high vacuum at
40 °C for 12 h to give the title compound as a yellow solid (160 g, 65%).
1H NMR (400 MHz, CDCl3) δ 7.98 (s, 1H), 7.63 (d, J = 8.4 Hz, 1H),
7.29−7.19 (m, 5H), 7.18−7.13 (m, 2H), 6.75 (dd, J = 17.6, 10.9 Hz,
1H), 5.76 (d, J = 17.5 Hz, 1H), 5.53 (s, 2H), 5.26 (d, J = 10.9 Hz, 1H).
LCMS (ESI) m/z calcd for [C16H14N2 + H]+ 235.1; found, 235.0.
1-(N1-Benzyl-1H-indazol-6-yl)-ethane-1,2-diol (13). Compound
12 (5.6 g, 24 mmol) was stirred in a mixture of t-BuOH (25 mL),
water (25 mL), and citric acid (10.1 g, 48.0 mmol). N-Methylmorpho-
line-N-oxide (3.1 g, 26.4 mmol) and K2OsO4·2H2O (44 mg, 0.12 mmol,
0.5 mol %) were added, and the resulting oily, biphasic mixture stirred
overnight at rt. 1H NMR of a sample showed approximately 40%
conversion, and so the mixture was warmed to 50 °C (giving a single
phase) for 7 h. Ethanol (50 mL) was added, and the mixture again
stirred an additional 24 h at rt. The mixture was filtered through Celite,
and the filtrate was concentrated in vacuo. The residue was taken up in
1.0 M aq HCl and EtOAc; the layers were separated and the aqueous
phase extracted with further EtOAc. The combined organic portions
were washed with brine, dried over Na2SO4, filtered, and concentrated.
The crude product was passed through a short silica plug, eluting with
EtOAc, and concentrated to afford racemic 13 as a reddish gum (6.26 g,
97%). 1H NMR (400 MHz, CDCl3) δ 7.96 (s, 1H), 7.65 (d, J = 8.4 Hz,
1 H), 7.38 (s, 1H), 7.27−7.18 (m, 3H), 7.16−7.09 (m, 2H), 7.04
(d, J = 8.4 Hz, 1H), 5.50 (s, 2H), 4.86 (d, J = 7.6 Hz, 1H), 3.78−3.68
(m, 1H), 3.60 (t, J = 9.6 Hz, 1H), 3.46 (s, 1H), 2.84 (s, 1H). LCMS
(ESI) m/z calcd for [C16H16N2O2 + H]+, 269.1; found, 269.0.
1
98% ee), with H NMR and MS results identical to 13. Chiral HPLC
retention time, 16.8 min (Daicel Chiralpak IB (250 mm × 4.6 mm);
isocratic 10% EtOH in n-heptane; 1 mL/min; ambient temperature.
1-(1-Benzyl-1H-indazol-6-yl)ethane-1,2-diyl Dimethanesulfonate
(14). A solution of diol 13 (0.27 g, 1.0 mmol) and TEA (0.35 mL,
2.5 mmol) in dry DCM (5 mL) was cooled in an ice bath followed by
dropwise addition of MsCl (0.15 mL, 2.0 mmol) over 5 min. The
resulting mixture was left to stir for 2 h and warmed to rt. The mixture
was diluted with DCM and washed quickly with 1.0 M aq HCl
(20 mL), satd NaHCO3 (40 mL), and brine (20 mL), dried over
Na2SO4, filtered, and concentrated at 25 °C to afford the crude
product. The crude product was purified by silica gel chromatography
(1:1 Et2O/CH2Cl2) to give 14 as an off-white, gummy foam (0.30 g,
1
71%). H NMR (400 MHz, CDCl3) δ 8.08 (s, 1H), 7.81 (d, J =
8.3 Hz, 1H), 7.42 (s, 1H), 7.34−7.23 (m, 3H), 7.17 (dd, J = 18.1,
7.6 Hz, 3H), 5.89 (dd, J = 8.5, 3.2 Hz, 1H), 5.66 (d, J = 15.8 Hz, 1H),
5.60 (d, J = 15.8 Hz, 1H), 4.52 (dd, J = 11.8, 8.6 Hz, 1H), 4.40 (dd, J =
11.8, 3.2 Hz, 1H), 3.04 (s, 3H), 2.75 (s, 3H). LCMS (ESI) m/z calcd
for [C18H20N2O6S2 + H]+, 425.1; found, 425.0.
(S)-1-(1-Benzyl-1H-indazol-6-yl)ethane-1,2-diyl Dimethanesulfo-
nate (14a). A solution of 13a (134 g, 0.5 mol) and TEA (174 mL,
1.25 mol) in CH2Cl2 (2.5 L) was cooled in an ice bath and treated by
slow addition of MsCl (81 mL, 1.05 mol) over 1 h. The internal
temperature increased to a maximum of 11 °C. The resulting mixture
was left to stir for 30 min. The reaction was quenched with cold 1.0 M
aq HCl (400 mL), the phases separated, and the organic phase washed
with cold 1.0 M aq HCl, aq NaHCO3, and brine, then dried over
MgSO4. The solution was poured onto a short silica gel pad under
suction. The product was eluted using 1:1 Et2O/CH2Cl2 (2 L). Removal
of the solvents under reduced pressure gave a hard white solid, which
was triturated with Et2O (800 mL) overnight. The precipitate was
collected by filtration and washed with further Et2O (2 × 100 mL) to
yield the title compound as a fine white powder (184 g, 87%, 99% ee)
1
with H NMR and MS results identical to 14. Chiral HPLC retention
time, 13.4 min (Daicel Chiralpak IB (250 mm × 4.6 mm); isocratic 30%
EtOH in n-heptane; 1 mL/min; ambient temperature.
Cyclopropane Formation: General Method A. A solution of
indolin-2-one (1 equiv) in dry THF (30 mL/g) was cooled in an ice
bath. Sodium hydride (60 wt % in mineral oil, 2.1 equiv) was added in
four portions, and the mixture was stirred at 0 °C for 15 min. A
solution of 1 equiv of dimesylate (14 or 14a) in THF (30 mL/g) was
added dropwise over 1 h and stirred for 2 h. The reaction was
quenched with satd NH4Cl solution, diluted with water, and extracted
with EtOAc (3 volumes). The organic layer was dried with MgSO4 and
concentrated to a crude product, which was further purified by
trituration or silica gel chromatography.
rac-(1R,2S)-1′-Benzyl-2-(1-benzyl-1H-indazol-6-yl)spiro-
[cyclopropane-1,3′-indolin]-2′-one (15). Prepared according to
general method A using oxindole 6 (45 mg, 0.2 mmol) and dimesylate
14 (85 mg, 0.2 mmol). The crude product was triturated with petro-
leum ether/EtOAc (3:1) and filtered to give the title compound 15 as
a light-brown solid (75 mg, 82%). 1H NMR (400 MHz, CDCl3) δ 7.99
(s, 1H), 7.60 (d, J = 8.3 Hz, 1H), 7.36−7.20 (m, 8H), 7.19 (s, 1H),
7.15−7.10 (m, J = 6.4 Hz, 2H), 6.99 (td, J = 7.8, 0.9 Hz, 1H), 6.92 (d,
J = 8.3 Hz, 1H), 6.74 (d, J = 7.8 Hz, 1H), 6.50 (t, J = 7.4 Hz, 1H), 5.76
(d, J = 7.3 Hz, 1H), 5.61 (d, J = 15.8 Hz, 1H), 5.53 (d, J = 15.8 Hz,
1H), 5.08 (d, J = 15.6 Hz, 1H), 4.97 (d, J = 15.7 Hz, 1H), 3.48 (t, J =
8.5 Hz, 1H), 2.28 (dd, J = 9.0, 4.5 Hz, 1H), 2.02 (dd, J = 8.0, 4.6 Hz,
(S)-1-(N1-Benzyl-1H-indazol-6-yl)-ethane-1,2-diol (13a). A 5 L
clamp-top reaction vessel equipped with an overhead stirrer was
treated with K3Fe(CN)6 (405 g, 1.23 mol), K2CO3 (170 g, 1.23 mol),
(DHQ)2PHAL (3.2 g, 4.1 mmol), and K2OsO4.2H2O (1.54 g, 4.1 mmol).
N
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1H). LCMS (ESI) m/z calcd for [C31H25N3O + H]+, 456.2; found,
456.0.
quenched with 20% aq NH4Cl, extracted with EtOAc, dried, and
evaporated. The product was purified by silica gel chromatography or
recrystallization.
(1R,2S)-1′-Benzyl-2-(1-benzyl-1H-indazol-6-yl)spiro-
[cyclopropane-1,3′-indolin]-2′-one (15a). Prepared according to
general method A using oxindole 6 (57 g, 224 mmol) and dimesylate
14a (95 g, 224 mmol). The crude product was triturated with petro-
leum ether/EtOAc (3:1) and filtered to give the title compound 15a
rac-(1R,2S)-2-(1H-Indazol-6-yl)spiro-[cyclopropane-1,3-indolin]-
2′-one (5). Prepared according to general method B using a solution
of 15 (6.5 g, 14 mmol) in a mixture of DMSO (20 mL) and THF
(200 mL). In this instance, the combined organic portions (4 × 50 mL
EtOAc) were washed with satd aq sodium thiosulfate (50 mL)
and brine (50 mL), dried over Na2SO4, filtered, and concentrated.
The residue was slurried in CH2Cl2 (100 mL) and poured onto a short
silica pad (2 cm depth × 5 cm diameter) under suction. The major
byproduct (Rf 0.6 in 1:1 EtOAc/cyclohexane) was eluted using
CH2Cl2 (ca. 1 L). The product (Rf 0.25 in 1:1 EtOAc/cyclohexane)
was eluted (2% then 5% MeOH/EtOAc) and concentrated to afford
the title compound (2.5 g, 64%) as a pale-brown solid with a diastereo-
meric excess of 94%.
(1R,2S)-2-(1H-Indazol-6-yl)spiro-[cyclopropane-1,3-indolin]-2′-
one (5a). Prepared according to general method B using a solution of
15a (10 g, 22 mmol). The material was purified by dry-flash silica pad
(4 cm depth × 20 cm diameter). Byproducts were eluted using solvent
of increasing eluent strength (0:1, 1:9, 1:4 EtOAc/cyclohexane), and
then the product was eluted using EtOAc to give the title compound
as a beige solid (4.2 g, 70%, >98.5% ee). 1H NMR (400 MHz, DMSO-
d6) δ 13.01 (s, 1H), 10.61 (d, J = 8.3 Hz, 1H), 8.01 (s, 1H), 7.63 (d,
J = 8.3 Hz, 1H), 7.44 (s, 1H), 6.99 (t, J = 7.5 Hz, 1H), 6.92 (d, J =
8.0 Hz, 1H), 6.84 (d, J = 8.0 Hz, 1H), 6.51 (t, J = 7.0 Hz, 1H), 5.98 (d,
J = 8.0 Hz, 1H), 3.20−3.17 (m, 1H), 2.30−2.26 (m, 1H), 2.00−1.95
(m, 1H). LCMS (ESI) m/z calcd for [C17H13N3O + H]+, 276.3;
found, 276.1; chiral HPLC retention time, 14.3 min; Daicel Chiralpak
AS-H (250 mm × 4.6 mm); isocratic 40% EtOH in n-heptane;
1 mL/min; 35 °C.
1
as a light-brown solid (50 g, 46%, 98% ee) with H NMR and MS
results identical to 15. Chiral HPLC retention time, 13.3 min (Daicel
Chiralpak IA, 250 mm × 4.6 mm); isocratic 10% EtOH in n-heptane;
1 mL/min; ambient temperature.
(1R,2S)-1′-Benzyl-2-(1-benzyl-1H-indazol-6-yl)-5′-methoxyspiro-
[cyclopropane-1,3′-indolin]-2′-one (23). Prepared according to
general method A using dimethanesulfonate 14a (95 g, 224 mmol)
and 1-benzyl-5-methoxyindolin-2-one 19 (56 g, 224 mmol). The crude
product was triturated with 3:1 petroleum ether/EtOAc to give 23 as a
white solid (50 g, 46%, >99.9% ee, Chiralpak IA (250 mm × 4.6 mm);
1
1.0 mL/min; hexane/EtOH: 80/20). H NMR (400 MHz, CDCl3)
δ 8.01 (s, 1H), 7.64 (d, J = 8.2 Hz, 1H), 7.36−7.23 (m, 9H), 7.14 (d, J =
7.2 Hz, 2H), 6.94 (d, J = 8.3 Hz, 1H), 6.63 (d, J = 8.3 Hz, 1H), 6.53
(d, J = 8.4 Hz, 1H), 5.61 (d, J = 15.1 Hz, 1H), 5.54 (d, J = 15.4 Hz,
1H), 5.37 (s, 1H), 5.07 (d, J = 15.4 Hz, 1H), 4.95 (d, J = 15.7 Hz, 1H),
3.51 (t, J = 8.1 Hz, 1H), 3.18 (s, 3H), 2.32−2.29 (m, 1H), 2.09−2.00
(m, 1H). LCMS (ESI) m/z calcd for [C32H27N3O2 + H]+, 486.2;
found, 486.3.
(1R,2S)-2-(1-benzyl-1H-indazol-6-yl)-1′-methylspiro-
[cyclopropane-1,3′-indolin]-2′-one (24). Prepared according to the
general method A using 1-methylindolin-2-one 20 (2.33 g, 15.8 mmol)
and a solution of dimesylate 14a (6.70 g, 15.8 mmol) in dry THF
(45 mL). Purification via column chromatography (silica gel, 25−50%
EtOAc in hexane) yielded the title compound as a pale-orange
1
crystalline solid (5.0 g, 84%); H NMR (400 MHz, CDCl3) δ 8.00 (s,
The title compound was also obtained from racemic 5 (25 mg) by
separation using chiral SFC, Chiralpak IA (3 cm × 15 cm), isocratic
30% MeOH (0.1% DEA)/CO2, 70 mL/min) to give a white solid 5a
(11.5 mg, 97% ee, retention time, 5.2 min; Chiralpak IA (15 cm ×
0.46 cm), 3.0 mL/min with isocratic 40% MeOH (0.1% DEA)/CO2,
3.0 mL/min). From this resolution process, the opposite enantiomer,
(1S,2R)-2-(1H-indazol-6-yl)spiro-[cyclopropane-1,3-indolin]-2′-one
(5b) was isolated as a white solid (11.8 mg, >98% ee, retention time:
1H), 7.60 (d, J = 8.3 Hz, 1H), 7.30−7.25 (m, 3H), 7.18 (s, 1H), 7.13−
7.10 (m, 3H), 6.92 (d, J = 8.6 Hz, 1H), 6.85 (d, J = 7.6 Hz, 1H),
6.55 (t, J = 7.0 Hz, 1H), 5.76 (d, J = 7.2 Hz, 1H), 5.63−5.49 (m, 2H),
3.41 (t, J = 8.8 Hz, 1H), 3.33 (s, 3H), 2.22−2.18 (m, 1H), 2.00−1.96
(m, 1H); LCMS (ESI) m/z calcd for [C25H21N3O + H]+ 380.18;
found, 380.2.
(1R,2S)-2-(1-Benzyl-1H-indazol-6-yl)-5′-methoxy-1′-methylspiro-
[cyclopropane-1,3′-indolin]-2′-one (25). Prepared according to the
general method A using dimethanesulfonate 14a (1.44 g, 3.4 mmol)
and 5-methoxy-1-methylindolin-2-one 21 (0.60 g, 3.4 mmol). Purifi-
cation using Biotage Isolera (1−50% EtOAc in hexane, SNAP 25g
column) yielded the title compound as a light-brown solid (1.05 g,
76%). 1H NMR (400 MHz, CDCl3) δ 8.02 (d, J = 0.8 Hz, 1 H), 7.66
(d, J = 8.3 Hz, 1 H), 7.48 (s, 1 H), 7.17−7.31 (m, 3 H), 7.06−7.15 (m,
2 H), 6.92−6.99 (m, 1 H), 6.88 (d, J = 8.3 Hz, 1 H), 6.67 (dd, J = 8.5,
2.5 Hz, 1 H), 5.51−5.68 (m, 2 H), 5.41 (d, J = 2.5 Hz, 1 H),
3.33−3.40 (m, 1 H), 3.28 (s, 3 H), 3.15 (s, 3 H), 2.11−2.24
(m, 2 H). LCMS (ESI) m/z calcd for [C26H23N3O2 + H]+, 410.2;
found, 410.2.
(1R,2S)-1′-Benzyl-2-(1-benzyl-1H-indazol-6-yl)-5′-methylspiro-
[cyclopropane-1,3′-indolin]-2′-one (26). The title compound was
prepared according to the general method A using dimethanesulfonate
14a (4.24 g, 10 mmol) and 1-benzyl-5-methylindolin-2-one 22 (2.37 g,
10 mmol). Purification by Biotage Isolera (20−30% EtOAc in hexane)
gave 26 as a beige solid (3.2 g, 68%). 1H NMR (400 MHz, CDCl3) δ
8.02 (s, 1H), 7.63 (d, J = 8.0 Hz, 1H), 7.36−7.20 (m, 9H), 7.14 (d, J =
6.0 Hz, 2H), 6.94 (d, J = 8.4 Hz, 1H), 6.80 (d, J = 8.0 Hz, 1H), 6.64
(d, J = 7.2 Hz, 1H), 5.62 (d, J = 16.8 Hz, 1H), 5.59 (s, 1H), 5.55 (d,
J = 16.8 Hz, 1H), 5.08 (d, J = 16.0 Hz, 1H), 4.97 (d, J = 15.6 Hz, 1H),
3.48 (t, J = 8.4 Hz, 1H), 2.30−2.25 (m, 1H), 2.02−1.96 (m, 1H), 1.85
(s, 3H). LCMS (ESI) m/z calcd for [C32H27N3O + H]+, 470.2; found,
470.3.
Debenzylation: General Method B. A solution of benzylated
indazol-6-ylspiro[cyclopropane-1,3′-indolin]-2′-ones (1 equiv) in THF
(6 mL/g) was cooled in ice before addition of t-BuOK (1.7 M solution
in THF, 20 equiv). DMSO (19 equiv) was added, and the mixture was
purged gently with oxygen in an ice bath for 3 h and then warmed to rt
overnight under an atmosphere of oxygen. Mixtures were generally
1
2.7 min) with H NMR and LCMS as described for 5a.
(1R,2S)-2-(1H-Indazol-6-yl)-5′-methoxyspiro[cyclopropane-1,3′-
indolin]-2′-one (16a). The title compound was prepared according to
the general method B using 23 (50 g, 103 mmol). Recrystallization
from ethanol gave 16a as a beige solid (22.3 g, 71%, 99.4% ee,
Chiralpak IA (250 mm × 4.6 mm), 1.0 mL/min with isocratic 30%
1
MeOH (0.1% DEA)/70% CO2). H NMR (400 MHz, DMSO-d6) δ
13.02 (br s, 1H), 10.42 (br s, 1H), 8.02 (s, 1H), 7.64 (d, J = 8.3 Hz,
1H), 7.45 (s, 1H), 6.94 (d, J = 7.8 Hz, 1H), 6.73 (d, J = 8.2 Hz, 1H),
6.55 (d, J = 8.6 Hz, 1H), 5.62 (s, 1H), 3.20 (s, 3H), 3.18 (t, J = 8.7 Hz,
1H), 2.34−2.28 (m, 1H), 1.98−1.95 (m, 1H). LCMS (ESI) m/z calcd
for [C18H15N3O2 + H]+, 306.1; found, 306.1. Optical rotation: [α]23
−225° (c 0.44, MeOH).
=
D
(1R,2S)-2-(1H-Indazol-6-yl)-1′-methylspiro[cyclopropane-1,3′-in-
dolin]-2′-one (27). The title compound was prepared according to the
method B using 24 (1.16 g, 3.06 mmol). Purification via column
chromatography (silica gel, 3−6% MeOH in CH2Cl2) yielded the title
compound as a pale-yellow solid (656 mg, 74%). 1H NMR (400 MHz,
CDCl3) δ 10.06 (br. s, 1H), 8.05 (s, 1H), 7.64 (d, J = 7.6 Hz, 1H),
7.36 (s, 1H), 7.14 (t, J = 8.7 Hz, 1H), 6.97 (d, J = 8.7 Hz, 1H), 6.87 (d,
J = 8.2 Hz, 1H), 6.62 (t, J = 7.6 Hz, 1H), 5.91 (d, J = 7.9 Hz, 1H), 3.46
(t, J = 7.8 Hz, 1H), 3.34 (s, 3H), 2.26−2.23 (m, 1H), 2.08−2.04 (m,
1H). LCMS (ESI) m/z calcd for [C18H15N3O + H]+, 290.13; found,
290.1.
(1R,2S)-2-(1H-Indazol-6-yl)-5′-methoxy-1′-methylspiro-
[cyclopropane-1,3′-indolin]-2′-one (28). Prepared according to the
general method B using 25 (0.875 g, 2.1 mmol). The resultant pale-
yellow residue was purified by silica gel column chromatography
using 5−10% EtOAc in hexane to give the title compound as an
1
off-white solid (445 mg, 65%). H NMR (400 MHz, CD3OD) δ 8.02
(s, 1H), 7.67 (d, J = 8.4 Hz, 1H), 7.47 (s, 1H), 6.95−6.90 (m, 2H),
O
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1
6.68 (d, J = 8.8 Hz, 1H), 5.58 (s, 1H), 3.38 (t, J = 8.4 Hz, 1H), 3.32
(s, 3H), 3.20 (s, 3H), 2.28 (dd, J = 9.2, 4.4 Hz, 1H), 2.06 (dd, J = 8.4,
4.8 Hz, 1H). LCMS (ESI) m/z calcd for [C19H17N3O2 + H]+, 320.2;
found, 320.1.
chiral HPLC retention time, 3.4 min) with H NMR and LCMS as
described for 18a.
Alternatively, a solution of 16a (250 g, 0.82 mol) in DMF (500 mL)
and MeOH (500 mL) was treated with potassium carbonate (226 g,
1.64 mol). Iodine (340 g, 1.06 mol) was dissolved into DMF (475
mL) and added to the mixture. The mixture was stirred at rt for 6 h
and quenched with sodium thiosulfate pentahydrate (360 g, 1.46 mol)
in water (5.7 L). The mixture was stirred at rt for 4 h and filtered. The
solid was washed with water (2 L) and dried under vacuum at 50 °C to
give the title compound as a white solid (334 g, 95%, 98% ee) with 1H
NMR and LCMS as described above. Optical rotation: [α]22D = −143°
(c 0.40, MeOH).
(1R,2S)-2-(1H-Indazol-6-yl)-5′-methylspiro[cyclopropane-1,3′-in-
dolin]-2′-one (29). Prepared according to the general method B using
26 (1.4 g, 3 mmol). Purification by Biotage Isolera (10−95% EtOAc in
1
hexane) gave the title compound as a light solid (680 mg, 53%). H
NMR (400 MHz, CD3OD) δ 8.02 (s, 1H), 7.67 (d, J = 8.4 Hz, 1H),
7.46 (s, 1H), 6.94 (d, J = 8.4 Hz, 1H), 6.85 (d, J = 8.0 Hz, 1H), 6.81
(d, J = 7.6 Hz, 1H), 5.78 (s, 1H), 3.34−3.03 (m, 1H), 2.20−2.12 (m,
2H), 1.87 (s, 3H). LCMS (ESI) m/z calcd for [C18H15N3O + H]+,
290.1; found, 290.1.
(1R,2S)-2-(3-Iodo-1H-indazol-6-yl)-1′-methylspiro[cyclopropane-
1,3′-indolin]-2′-one (30). Prepared according to the general method
C using 27 (930 mg, 3.21 mmol). Precipitation with EtOAc followed
by filtration and rinsing with EtOAc gave the title compound (970 mg,
Iodination: General Method C. To a solution of cyclopropylindo-
lin-2-one (1 equiv) and K2CO3 (2 equiv) in DMF was added iodine
(1.5 equiv). The resulting mixture was stirred for 18 h at rt and then
poured with stirring into 1% aqueous Na2S2O3·5H2O. The resulting
solid was filtered and washed with water (2×) to give a crude product,
which was further purified by trituration or silica gel chromatography.
rac-(1R,2S)-2-(3-Iodo-1H-indazol-6-yl)spiro[cyclopropane-1,3′-in-
dolin]-2′-one (17). Prepared according to general method C by
treating a solution of 5 (200 mg, 0.72 mmol) and K2CO3 (300 mg,
2.15 mmol) in dry DMF (2 mL) with a solution of I2 (500 mg,
1.97 mmol) in dry DMF (2 mL). The crude product was triturated
with diethyl ether to give a beige solid (225 mg, 78%) identical to the
material previously described.24
1
73%, >98% ee). H NMR (400 MHz, CDCl3) δ 10.96 (br. s, 1H),
7.43−7.39 (m, 2H), 7.16 (t, J = 7.6 Hz, 1H), 7.05 (d, J = 8.0 Hz, 1H),
6.89 (d, J = 7.8 Hz, 1H), 6.66 (t, J = 7.2 Hz, 1H), 5.91 (d, J = 8.0 Hz,
1H), 3.47 (t, J = 8.4 Hz, 1H), 3.35 (s, 3H), 2.30−2.26 (m, 1H), 2.08−
2.04 (m, 1H). LCMS (ESI) m/z calcd for [C18H14IN3O + H]+,
416.03; found, 416.0. Optical rotation: [α]23 = −210° (c 0.4,
D
MeOH). Chiral HPLC retention time, 2.4 min; Phenomenex Lux 5 μ
Amylose-2 150 mm × 4.6 mm (2.5 mL/min with isocratic at 20%
EtOH in hexane for 0.5 min, then gradient 20−50% EtOH in hexane
over 2.5 min, then isocratic at 50% for 1 min).
(1R,2S)-2-(3-Iodo-1H-indazol-6-yl)spiro[cyclopropane-1,3′-indo-
lin]-2′-one (17a) and (1S,2R)-2-(3-Iodo-1H-indazol-6-yl)spiro-
[cyclopropane-1,3′-indolin]-2′-one (17b). The title compounds
were obtained from racemic (1R*,2S*)-2-(3-iodo-1H-indazol-6-yl)-
spiro[cyclopropane-1,3′-indolin]-2′-one (1 g) by separation using
chiral SFC: Chiralcel OJ-H (2 cm × 25 cm), isocratic 40% methanol
(0.1% DEA)/CO2, 100 bar at 50 mL/min), to give a white solid 17a
(478 mg) with >99% ee and a retention time of 1.7 min (Chiralcel OJ-
H (10 cm × 0.46 cm), isocratic 40% methanol (0.1% DEA)/CO2;
3.0 mL/min). From this resolution process, the opposite enantiomer,
17b was isolated as a white solid (426 mg, >99% ee, retention time:
2.5 min).
(1R,2S)-2-(3-Iodo-1H-indazol-6-yl)-5′-methoxy-1′-methylspiro-
[cyclopropane-1,3′-indolin]-2′-one (31). Prepared according to the
general method C using 28 (1.34 g, 4.19 mmol). The title compound
1
was isolated as a cream-colored solid (1.71 g, 91%, 97.9% ee). H
NMR (400 MHz, CDCl3) δ 10.38 (s, 1H), 7.42 (d, J = 8.4 Hz, 1H),
7.35 (s, 1H), 7.04 (d, J = 8.8 Hz, 1H), 6.77 (d, J = 8.4 Hz, 1H), 6.66
(d, J = 8.4 Hz, 1H), 5.53 (s, 1H), 3.46 (t, J = 8.0 Hz, 1H), 3.38 (s,
3H), 3.32 (s, 3H), 2.24 (dd, J = 8.4, 4.8 Hz, 1H), 2.04 (dd, J = 12.4,
4.8 Hz, 1H). LCMS (ESI) m/z calcd for [C19H16IN3O2 + H]+, 446.0;
found, 446.1 [M + 1]+. Optical rotation: [α]22 = −134° (c 0.24,
D
MeOH). Chiral HPLC retention time, 2.6 min; Phenomenex Lux 5μ
Amylose-2 150 mm × 4.6 mm (2.5 mL/min with isocratic at 20%
EtOH in hexane for 0.5 min, then gradient 20−50% EtOH in hexane
over 2.5 min, then isocratic at 50% for 1 min).
Compound 17a was also obtained by direct preparation: 5a (10.6 g,
38 mmol) was dissolved into dioxane (150 mL) and 2 M aq NaOH
(150 mL). Iodine (13.4 g, 53.3 mmol) was added portionwise, and the
reaction was stirred for 12 h. The reaction was quenched with 1%
aqueous Na2S2O3·5H2O and extracted with EtOAc (3 × 150 mL).
Purification by column chromatography on silica (20 cm depth × 4 cm
diameter) using 1:1 EtOAc/cyclohexane gave the title compound 17a
(1R,2S)-2-(3-Iodo-1H-indazol-6-yl)-5′-methylspiro[cyclopropane-
1,3′-indolin]-2′-one (32). Prepared according to the general method
C using 29 (680 mg, 2.35 mmol). Purification by Biotage Isolera
(EtOAc/hexane gradient: 10−90%) gave the title compound as a light-
1
yellow solid (794 mg, 81%, >98% ee). H NMR (400 MHz, DMSO-
1
(7.0 g, 46%, 98.5% ee) as an off-white powder. H NMR (400 MHz,
d6) δ 13.43 (s, 1H), 10.51 (s, 1H), 7.47 (s, 1H), 7.32 (d, J = 8.4 Hz,
1H), 7.02 (d, J = 8.8 Hz, 1H), 6.81 (d, J = 8.4 Hz, 1H), 6.73 (d, J = 7.6
Hz, 1H), 5.86 (s, 1H), 3.18 (t, J = 8.2 Hz, 1H), 2.30−2.20 (m 1H),
2.00−1.90 (m, 1H), 1.85 (s, 3H). LCMS (ESI) m/z calcd for
DMSO-d6) δ 13.47 (s, 1H), 10.62 (s, 1H), 7.47 (s, 1H), 7.30 (d, J =
8.0 Hz, 1H), 7.02−6.98 (m, 2H), 6.84 (d, J = 7.6 Hz, 1H), 6.53 (t, J =
7.6 Hz, 1H), 5.97 (d, J = 7.6 Hz, 1H), 3.18 (q, J = 8.4 Hz, 1H), 2.31
(dd, J = 7.2, 4.8 Hz, 1H), 1.98 (dd, J = 8.8, 4.8 Hz, 1H). LCMS (ESI)
m/z calcd for [C17H12IN3O + H]+, 402.0; found, 402.0. Optical
[C18H14IN3O + H]+, 416.0; found, 416.0. Optical rotation: [α]23
=
D
−145° (c 0.49, MeOH). Chiral HPLC retention time, 9.6 min;
Phenomenex Lux 5μ Cellulose-2 (150 mm × 4.6 mm); isocratic 10%
EtOH in n-hexane (1.75 mL/min at ambient temperature).
rotation: [α]22 = −118° (c 0.57, MeOH). Chiral HPLC retention
D
time, 14.1 min; Daicel Chiralpak AS-H (250 mm × 4.6 mm); isocratic
40% EtOH in n-heptane; 1 mL/min; 35 °C.
(1R,2S)-2-(1-Benzyl-3-iodo-1H-indazol-6-yl)-1′-methylspiro-
[cyclopropane-1,3′-indolin]-2′-one (33). To compound 30 (200 mg,
0.48 mmol) in DCM (3.5 mL) and 50% aq KOH (1 mL) was added
benzyl bromide (69 μL, 0.58 mmol) and TBAB (2 mg, 0.005 mmol).
The mixture was stirred overnight at 0 °C. The reaction was warmed
to rt. DCM (10 mL) was added, and the mixture was washed with
brine (2 × 5 mL), dried over MgSO4, and concentrated. The residue
was purified by Biotage silica gel chromatography (99:1 to 95:5 DCM/
MeOH). The title compound was recrystallized from hot methanol to
(1R,2S)-2-(3-Iodo-1H-indazol-6-yl)-5′-methoxyspiro-
[cyclopropane-1,3′-indolin]-2′-one (18a). The title compound
was obtained from racemic (1R,2S)-2-(3-iodo-1H-indazol-6-yl)-5′-
methoxyspiro[cyclopropane-1,3′-indolin]-2′-one24 (15 g) by separa-
tion using chiral SFC: Chiralcel OJ-H (3 cm × 15 cm), (30% methanol
(0.1% DEA)/CO2, 75 mL/min), to give a white solid 18a (6.75 g, 99%
ee; chiral HPLC retention time, 2.1 min; Chiralpak IA (150 mm ×
4.6 mm), 3.0 mL/min isocratic 40% 2-propanol (0.1% DEA)/CO2).
1H NMR (400 MHz, DMSO-d6) δ 13.48 (br s, 1H), 10.43 (br s, 1H),
1
give a white crystalline solid (150 mg, 63%). H NMR (400 MHz,
7.49 (s, 1H), 7.31 (d, J = 8.6 Hz, 1H), 7.02 (d, J = 8.1 Hz, 1H), 6.73
(d, J = 8.4 Hz, 1H), 6.57 (d, J = 8.6 Hz, 1H), 5.62 (s, 1H), 3.29 (s,
3H), 3.19 (t, J = 8.4 Hz, 1H), 2.36−2.32 (m, 1H), 1.99−1.96 (m 1H).
LCMS (ESI) m/z calcd for [C18H14IN3O2 + H]+, 432.0; found, 432.1
[M + 1]+. From this resolution process, the opposite enantiomer
(1S,2R)-2-(3-iodo-1H-indazol-6-yl)-5′-methoxyspiro[cyclopropane-
1,3′-indolin]-2′-one 18b was isolated as a white solid (6.6 g, 99% ee,
CD3OD) δ ppm 7.35 (d, J = 8.3 Hz, 1H), 7.20−7.32 (m, 4H), 7.07−
7.18 (m, 4H), 6.98 (d, J = 8.3 Hz, 1H), 6.86 (d, J = 8.0 Hz, 1H), 6.56
(t, J = 7.6 Hz, 1H), 5.72 (d, J = 7.55 Hz, 1H), 5.63- 5.49 (m, 2H),
3.39 (t, J = 8.5 Hz, 1H), 3.32 (s, 3H), 2.20 (dd, J = 4.6, 8.4 Hz, 1H),
1.87−1.99 (m, 1H). LCMS (ESI) m/z calcd for [C25H20IN3O + H]+,
506.1; found, 506.1. Single crystal X-ray crystallography structure
determination was performed at the Service Crystallography Laboratory,
P
dx.doi.org/10.1021/jm5005336 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
University of Toronto, Toronto, ON. The crystal and structure refine-
ment data for compound 33 is given in the Supporting Information, and
the corresponding ORTEP diagram is shown in Figure 1.
6.61 (dd, J = 8.5 Hz, 2.2 Hz, 1H), 5.58 (d, J = 2.2 Hz, 1H), 4.39 (s,
2H), 4.09−4.04 (m, 2H), 3.78−3.72 (m, 2H), 3.43−3.33 (m, 3H),
3.27−3.20 (m, 5H), 2.27−2.23 (m, 1H), 2.21−2.16 (m, 1H). HRMS
(ESI) m/z calcd for [C31H30N4O3 + H]+, 507.2396 [M + 1]+; found,
Suzuki−Miyaura Cross Coupling of 3-Iodospirocyclopropa-
neindolinones. General Method D. The appropriate 3-iodospir-
ocyclopropaneindolinone (1 equiv) was dissolved into dioxane and
1 M aq Na2CO3 (5 equiv). The appropriate vinyl boronic ester
(1.2 equiv), LiCl (3 equiv), and catalyst, Pd(PPh3)4 (2.5 mol %), were
added and resulting mixture purged with argon. The mixture was
heated on an oil bath for 18 h, cooled to rt, and diluted with satd
NaHCO3 and extracted with 3 volumes of EtOAc. The combined
organic fractions were dried over MgSO4 and concentrated. After
removal of solvents, the residue was either purified by prep-HPLC
to give the title compound as a TFA salt or by flash chromato-
graphy, followed by treatment with 1 M HCl in ether to generate the
HCl salt.
General Method E. The appropriate 3-iodospirocyclopropaneindo-
linone (1 equiv) was dissolved into 2:1 toluene/ethanol and 1 M aq
Na2CO3 (2 equiv). The appropriate vinyl boronic ester (1.2 equiv)
and catalyst, Pd(PPh3)4 (5 mol %), were added, and the resulting
mixture was heated in a microwave for 2 h at 125 °C. After cooling to
rt, the mixture was diluted with H2O, extracted with EtOAc (×2), and
dried (MgSO4). After removal of solvents, the residue was redissolved
in MeOH and filtered through a Waters CX column and either
purified by prep-HPLC to give the title compound as a TFA salt or by
flash chromatography followed by treatment with 1 M HCl in ether to
generate the HCl salt.
507.2395. Optical rotation: [α]23 = −97° (c 0.5, MeOH). HPLC
D
purity: 97.1% at 270 nM.
(1S,2R)-5′-Methoxy-2-(3-((E)-4-(morpholinomethyl)styryl)-1H-in-
dazol-6-yl)spiro[cyclopropane-1,3′-indolin]-2′-one Hydrochloride
(4b). Prepared according to the general method D using 18b (509 mg,
1.18 mmol) and (E)-4-(4-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)vinyl)benzyl)morpholine (468 mg, 1.42 mmol). Silica gel purifi-
cation (93:7 to 92:8, CH2Cl2/MeOH) gave 374 mg of the free base,
which was dissolved into THF (15 mL) and treated with HCl (0.9 mL
of a 1 M solution in Et2O), and the resulting precipitate was filtered
and washed with Et2O to give the title compound as an off-white
powder (329 mg, 51%) with 1H NMR and MS results identical to 4a.
Optical rotation: [α]24 = +102° (c 0.53, MeOH). HPLC purity:
D
99.2% at 270 nM.
(1R,2S)-2-(3-((E)-2-(Pyridin-3-yl)vinyl)-1H-indazol-6-yl)spiro-
[cyclopropane-1,3′-indolin]-2′-one (34). Prepared according to gen-
eral method D, using 17a (61 mg, 0.147 mmol) and (E)-3-(2-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)vinyl)pyridine (50 mg, 0.2 mmol)
(58 mg, 0.176 mmol). EtOAc (50 mL) was added, and the solu-
tion was washed with water (2 × 5 mL), brine (5 mL) and dried over
MgSO4. Purification by reverse phase prep-HPLC gave the title
1
compound as a yellow solid (20 mg, 44%). H NMR (400 MHz,
DMSO-d6) δ 13.21 (s, 1H), 10.64 (s, 1H), 8.86 (s, 1H), 8.45 (d, J =
3.2 Hz, 1H), 8.15 (d, J = 8.0 Hz, 1H), 8.09 (d, J = 8.4 Hz, 1H), 7.65
(d, J = 16.8 Hz, 1H), 7.49 (d, J = 16.8 Hz, 1H), 7.47 (s, 1H), 7.40 (dd,
J = 8.0 Hz, 4.8 Hz, 1H), 7.03 (d, J = 8.4 Hz, 1H), 6.99 (d, J = 7.2 Hz,
1H), 6.86 (d, J = 7.6 Hz, 1H), 6.53 (t, J = 7.4 Hz, 1H), 6.01 (d, J =
7.6 Hz, 1H), 3.21 (t, J = 8.4 Hz, 1H), 2.32 (dd, J = 7.6 Hz, 4.8 Hz,
1H), 2.00 (dd, J = 9.0 Hz, 4.8 Hz, 1H). MS (ESI) m/z calcd for
[C24H18N4O + H]+, 379.1; found, 379.1. HPLC purity; 95% at 270 nM.
(1R,2S)-5′-Methoxy-2-(3-((E)-2-(6-(4-methylpiperazin-1-yl)-
pyridin-3-yl)vinyl)-1H-indazol-6-yl)-spiro[cyclopropane-1,3′-indo-
lin]-2′-one 2,2,2-trifluoroacetate (35). Prepared according to the
general method D using 18a (130 mg, 0.30 mmol) with (E)-1-methyl-
4-(5-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)vinyl)pyridin-2-
yl)piperazine (119 mg, 0.36 mmol). The reaction mixture was diluted
with MeOH (12.5 mL) and filtered through a plug of silica (5 g),
eluting with 2 M NH3 in MeOH (100 mL). After removal of the
solvents in vacuo, the title compound was purified by prep-HPLC to
(1R,2S)-(E)-2-(3-(4-((Dimethylamino)methyl)styryl)-1H-indazol-6-
yl)spiro[cyclopropane-1,3′-indolin]-2′-one 2,2,2-trifluoroacetate
(3a). To a solution of 17a (125 mg, 0.31 mmol) in DMF (1.6 mL)
and water (0.4 mL) was added (E)-N,N-dimethyl-1-(4-(2-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)vinyl)phenyl) methanamine
(143 mg, 0.5 mmol), potassium fluoride (36 mg, 0.62 mmol), and
Pd(PPh3)4 (18 mg, 0.015 mmol). The mixture was heated to 120 °C
for 2 h under microwave irradiation. EtOAc (50 mL) was added, and
the solution was washed with water (2 × 5 mL) and brine (5 mL) and
dried over MgSO4. Purification by reverse phase prep-HPLC gave the
1
title compound as a yellow solid (73 mg, 44%). H NMR (400 MHz,
CD3OD) δ ppm 8.02 (d, J = 8.3 Hz, 1H), 7.78 (d, J = 8.3 Hz, 2H),
7.56−7.48 (m, 5H), 7.08−7.04 (m, 2H), 6.94 (d, J = 8.3 Hz, 1H), 6.59
(t, J = 7.8 Hz, 1H), 6.00 (d, J = 8.0 Hz, 1H), 4.38 (s, 2H), 3.39−3.33
(m, 1H), 2.90 (s, 6H), 2.28−2.22 (m, 1H), 2.22−2.17 (m, 1H).
HRMS (ESI) m/z calcd for [C28H26N4O + H]+, 435.2185; found,
435.2192. HPLC: 99.1% at 270 nM.
1
yield a yellow solid (104 mg, 34%). H NMR (400 MHz, CD3OD) δ
8.26 (s, 1H), 8.00 (d, J = 8.4 Hz, 1H), 7.92 (d, J = 9.2 Hz, 1H), 7.45
(s, 1H), 7.40 (d, J = 16.8 Hz, 1H), 7.28 (d, J = 16.8 Hz, 1H), 7.03 (d,
J = 7.6 Hz, 1H), 6.87 (d, J = 9.2 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H),
6.61 (dd, J = 8.4, 2.4 Hz, 1H), 5.58 (d, J = 2.0 Hz, 1H), 3.61 (m, 4H),
3.38 (m, 1H), 3.26 (s, 3H), 2.57 (m, 4H), 2.36 (s, 3H), 2.21 (m, 2H).
HRMS (ESI) m/z calcd for [C30H30N6O2 + H]+, 507.2508; found,
507.2497. HPLC: 99.9% at 270 nM.
(1R,2S)-1′-Methyl-2-(3-((E)-2-(6-(4-methylpiperazin-1-yl)pyridin-
3-yl)vinyl)-1H-indazol-6-yl)spiro[cyclopropane-1,3′-indolin]-2′-one
bis(2,2,2-trifluoroacetate) (36). Prepared according to general
method D, using 30 (61 mg, 0.147 mmol) and (E)-1-methyl-4-(5-
(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)vinyl)pyridin-2-yl)-
piperazine (58 mg, 0.176 mmol). Purification by prep-HPLC resulted
in a pale-yellow solid, which was sonicated with Et2O and filtered to
give the title compound (21 mg, 20%). 1H NMR (400 MHz, CD3OD)
δ 8.35 (s, 1H), 8.05 (d, J = 8.4 Hz, 1H), 7.98 (d, J = 8.2 Hz, 1H),
7.44−7.33 (m, 3H), 7.14 (t, J = 7.5 Hz, 1H), 7.06−6.99 (m, 3H), 6.63
(t, J = 6.7 Hz, 1H), 6.02 (d, J = 7.7 Hz, 1H), 4.60−4.30 (m, 4H),
3.60−3.08 (m, 4H), 3.47−3.33 (m, 4H), 2.97 (s, 3H), 2.28−2.25 (m,
1H), 2.21−2.18 (m, 1H). HRMS (ESI) m/z calcd for [C30H30N6O +
H]+, 491.2559; found, 491.2560. HPLC: 98.5% at 270 nM.
(1S,2R)-(E)-2-(3-(4-((Dimethylamino)methyl)styryl)-1H-indazol-6-
yl)spiro[cyclopropane-1,3′-indolin]-2′-one 2,2,2-trifluoroacetate
(3b). To a solution of 17b (20 mg, 0.05 mmol) in DMF (0.4 mL)
and water (0.1 mL) was added (E)-N,N-dimethyl-1-(4-(2-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)vinyl)phenyl) methanamine (25 mg,
0.08 mmol), potassium fluoride (6 mg, 0.1 mmol), and Pd(PPh3)4
(3 mg, 0.002 mmol). The mixture was heated to 120 °C for 2 h under
microwave irradiation. EtOAc (50 mL) was added, and the solution
was washed with water (2 × 5 mL) and brine (5 mL) and dried over
MgSO4. Purification by reverse phase prep-HPLC gave the title com-
1
pound as a yellow solid (4 mg, 15%) with H NMR and MS results
identical to 3a. HPLC purity: 99.8% at 270 nM.
(1R,2S)-5′-Methoxy-2-(3-(4-(morpholinomethyl)styryl)-1H-inda-
zol-6-yl)spiro[cyclopropane-1,3′-indolin]-2′-one Hydrochloride
(4a). Prepared according to general method D using 18a (5.3 g,
12.3 mmol) and (E)-4-(4-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)vinyl)benzyl)morpholine (4.86 g, 14.8 mmol). The residue was
purified by column chromatography (silica gel, CH2Cl2/MeOH, 9:1)
to give a solid, which was sonicated with Et2O and filtered to give a
white solid. The solid was dissolved into THF (50 mL), and then HCl
(9 mL of a 1 M solution in Et2O) was added. A precipitate formed
immediately, and further precipitate was obtained on addition of more
Et2O (150 mL). The solid was quickly filtered and washed with Et2O
(1R,2S)-(E)-2-(3-(4-(Piperazin-1-yl)styryl)-1H-indazol-6-yl)spiro-
[cyclopropane-1,3′-indolin]-2′-one 2,2,2-trifluoroacetate (37). The
title compound was synthesized according to the general method E
using 17a (40 mg, 0.1 mmol) and (E)-tert-butyl 4-(4-(2-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)vinyl)phenyl)piperazine-1-carbox-
ylate (50 mg, 0.12 mmol). The crude Boc-protected amine from the
1
to give an off-white solid (3.2 g, 48%, 99% ee). H NMR (400 MHz,
CD3OD) δ 8.04 (d, J = 8.4 Hz, 1H), 7.78 (d, J = 8.2 Hz, 2H), 7.75−
7.50 (m, 5H), 7.07 (d, J = 8.6 Hz, 1H), 6.83 (d, J = 8.5 Hz, 1H),
Q
dx.doi.org/10.1021/jm5005336 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
reaction was dissolved into CH2Cl2 (10 mL), and TFA (1 mL) was
added. The reaction was stirred for 3 h, the solvent was removed and
the residue purified by prep-HPLC to yield the title compound
7.73 (d, J = 7.8 Hz, 2H), 7.55−7.45 (m, 5H), 7.06−7.00 (m, 2H), 6.93
(d, J = 7.7 Hz, 1H), 6.56 (t, J = 7.5 Hz, 1H), 5.97 (d, J = 7.5 Hz, 1H),
5.46 (d, J = 52.4 Hz, 1H), 4.46 (s, 2H), 3.73−3.31 (m, 5H), 2.65−2.44
(m, 2H), 2.26−2.15 (m, 2H). HRMS (ESI) m/z calcd for
[C30H27FN4O + H]+, 479.2247; found, 479.2242. Optical rotation:
1
(23 mg, 40%). H NMR (400 MHz, CD3OD) δ 7.97 (d, J = 8.1 Hz,
1H), 7.57 (d, J = 8.6 Hz, 2H), 7.47−7.42 (m, 2H), 7.30 (d, J = 17 Hz,
1H), 7.06−7.01 (m, 4H), 6.93 (d, J = 7.6 Hz, 1H), 6.58 (t, J = 7.6 Hz,
1H), 5.98 (d, J = 8.0 Hz, 1H), 3.49−3.44 (m, 4H), 3.40−3.31 (m,
5H), 2.26−2.22 (m, 1H), 2.19−2.16 (m, 1H). HRMS (ESI) m/z calcd
for [C29H27N5O + H]+, 462.2294; found, 462.2290. HPLC: 97.8% at
270 nM.
[α]22 = −125° (c 0.44, MeOH). HPLC: 99.2% at 270 nM.
D
(1R,2S)-(E)-2-(3-(4-(Piperidin-1-ylmethyl)styryl)-1H-indazol-6-yl)-
spiro[cyclopropane-1,3′-indolin]-2′-one 2,2,2-trifluoroacetate (42).
The title compound was synthesized according to the general method
E using 17a (175 mg, 0.4 mmol) and (E)-4-(4-(2-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)vinyl)benzyl)piperidine (225 mg, 0.67 mmol).
Purification by reverse phase prep-HPLC gave the title compound as a
yellow TFA salt (140 mg, 60%). 1H NMR (400 MHz, CD3OD) δ 8.02
(d, J = 8.8 Hz, 1H), 7.77 (d, J = 8.6 Hz, 2H), 7.75−7.48 (m, 5H),
7.08−7.05 (m, 2H), 6.94 (d, J = 7.5 Hz, 1H), 6.59 (t, J = 7.7 Hz, 1H),
5.99 (d, J = 7.3 Hz, 1H) 4.31 (s, 2H), 3.53−3.45 (m, 2H), 3.39−3.34
(m, 1H), 3.04−2.93 (m, 2H) 2.27−2.17 (m, 2H), 2.02−1.95 (m, 2H),
1.88−1.71 (m, 3H), 1.57−1.45 (m, 1H). HRMS (ESI) m/z calcd for
[C31H30N4O + H]+, 475.2498; found, 475.2501. Optical rotation:
(1R,2S)-(E)-2-(3-(4-((Dimethylamino)methyl)styryl)-1H-indazol-6-
yl)-5′-methoxyspiro[cyclopropane-1,3′-indolin]-2′-one Hydrochlor-
ide (38). The title compound was synthesized according to the
general method E using 18a (251 mg, 0.58 mmol) and (E)-N,N-
dimethyl-1-(4-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)vinyl)-
phenyl)methanamine (191 mg, 0.67 mmol). The product was ex-
tracted using EtOAc (40 mL) with a Varian 3 mL ChemElut cartridge.
After removal of the solvents in vacuo, the title compound was
purified by chromatography on Biotage (silica, SNAP-25g, 5−20%
MeOH in DCM). Trituration with 1:1 Et2O/DCM yielded the title
compound (92 mg, 34%). HCl (1 M in Et2O, 0.25 mL, 0.25 mmol)
was added in a dropwise manner to an ice cooled solution of the
free base (92 mg, 0.20 mmol) in THF (10 mL), and the resulting
mixture was allowed to stir in ice for 40 min, then Et2O (10 mL) was
added to the mixture. Filtration under vacuum yielded the title
compound as the hydrochloride salt (orange−red solid, 79 mg,
[α]23 = −109° (c 0.35, MeOH). HPLC: 98.2% at 270 nM.
D
(1R,2S)-(E)-5′-Methoxy-2-(3-(4-(piperidin-1-ylmethyl)styryl)-1H-
indazol-6-yl)spiro[cyclopropane-1,3′-indolin]-2′-one 2,2,2-trifluor-
oacetate (43). The title compound was synthesized according to
the general method E using 18a (175 mg, 0.4 mmol) and (E)-4-(4-(2-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)vinyl)benzyl)piperidine
(225 mg, 0.67 mmol). Purification by reverse phase prep-HPLC gave a
1
yellow powder (90 mg, 36%). H NMR (400 MHz, CD3OD) δ 8.02
1
79%). H NMR (400 MHz, CD3OD) δ 8.05 (d, J = 8.5 Hz, 1H),
(d, J = 8.3 Hz, 1H), 7.75 (d, J = 8.6 Hz, 2H), 7.53−7.49 (m, 5H), 7.05
(d, J = 8.5 Hz, 1H), 6.84 (d, J = 8.5 Hz, 1H), 6.61 (dd, J = 8.4, 2.3 Hz,
1H), 5.58 (d, J = 2.3 Hz, 1H), 4.30 (s, 2H), 3.52−3.44 (m, 2H), 3.38−
3.34 (m, 1H), 3.26 (s, 3H), 3.01−2.93 (m, 2H), 2.26−2.17 (m, 4H),
2.00−1.91 (m, 2H), 1.89−1.67 (m, 3H), 1.58−1.46 (m, 1H). HRMS
(ESI) m/z calcd for [C32H32N4O2 + H]+, 505.2604; found, 505.2602.
7.77 (d, J = 8.0 Hz, 2H), 7.48−7.63 (m, 5H), 7.09 (d, J = 8.3 Hz, 1H),
6.84 (d, J = 8.5 Hz, 1H), 6.61 (dd, J = 8.3, 2.3 Hz, 1H), 5.61 (d, J =
2.3 Hz, 1H), 4.36 (s, 2 H), 3.35 (m, 1H), 3.28 (s, 3H), 2.89 (s, 6H),
2.26 (dd, J = 7.7, 5.1 Hz, 1H), 2.18 (dd, J = 8.8, 4.8 Hz, 1H). HRMS
(ESI) m/z calcd for [C29H28N4O2 + H]+, 465.2291; found, 465.2288.
Optical rotation: [α]24 = −70° (c 0.44, MeOH). HPLC: 99.8%
Optical rotation: [α]23 = −69° (c 0.29, MeOH). HPLC: 99.9% at
D
D
at 270 nM.
270 nM.
(1R,2S)-(E)-2-(3-(4-((Dimethylamino)methyl)styryl)-1H-indazol-6-
yl)-1′-methylspiro[cyclopropane-1,3′-indolin]-2′-one 2,2,2-trifluor-
oacetate (39). Prepared according to the general method D using
30 (134 mg, 0.32 mmol) and (E)-N,N-dimethyl-1-(4-(2-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)vinyl)phenyl)methanamine (111 mg,
0.39 mmol). Purification by prep-HPLC resulted in a pale-yellow
solid, which was triturated with Et2O and filtered to give the title
(1R,2S)-(E)-2-(3-(4-(Morpholinomethyl)styryl)-1H-indazol-6-yl)-
spiro[cyclopropane-1,3′-indolin]-2′-one Hydrochloride (44). The
title compound prepared according to the general method E, using
iodide 17a (2.5 g, 6.3 mmol) and (E)-4-(4-(2-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)vinyl)benzyl)morpholine (2.5 g, 7.5 mmol).
Purification by Biotage silica gel chromatography (gradient 0−10%
MeOH in CH2Cl2) gave the free base, which was dissolved into
THF (50 mL) and treated with 1 M HCl in ether (6 mL) and diluted
with diethyl ether (200 mL). The resulting precipitate was collected
by filtration to give the title compound as a yellow solid (2.6 g,
1
compound (42 mg, 23%). H NMR (400 MHz, CD3OD) δ 8.00 (d,
J = 8.0 Hz, 1H), 7.76 (d, J = 7.6 Hz, 2H), 7.54−7.47 (m, 5H), 7.15 (t,
J = 8.2 Hz, 1H), 7.03 (d, J = 7.8 Hz, 2H), 6.64 (t, J = 7.5 Hz, 1H), 6.02
(d, J = 7.0 Hz, 1H), 4.33 (s, 2H), 3.41−3.35 (m, 4H), 2.88 (s, 6H),
2.29−2.26 (m, 1H), 2.22−2.19 (m, 1H). HRMS (ESI) m/z calcd for
[C29H28N4O + H]+, 449.2341; found, 449.2336. Optical rotation:
1
79%). H NMR (400 MHz, CD3OD) δ 8.03 (d, J = 8.3 Hz, 1H),
7.78 (d, J = 7.5 Hz, 2H), 7.49−7.61 (m, 5H), 7.05 (t, J = 8.3 Hz,
2H), 6.94 (d, J = 8.3 Hz, 1H), 6.58 (t, J = 7.4 Hz, 1H), 5.99 (d, J = 7.0
Hz, 1H), 4.40 (s, 2H), 4.08−4.05 (m, 2H), 3.80−3.74 (m, 2H),
3.43−3.36 (m, 3H), 3.27−3.21 (m, 2H), 2.26−2.18 (m, 2H). HRMS
(ESI) m/z calcd for [C30H28N4O2 + H]+, 477.2291; found, 477.2284.
[α]22 = −152° (c 0.42, MeOH). HPLC: 96.8% at 270 nM.
D
(1R,2S)-(E)-2-(3-(4-(Pyrolidin-1-ylmethyl)styryl)-1H-indazol-6-yl)-
spiro[cyclopropane-1,3′-indolin]-2′-one 2,2,2-trifluoroacetate (40).
The title compound was synthesized according to the general method
E, using 17a (600 mg, 1.5 mmol) and (E)-4-(4-(2-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)vinyl)benzyl)pyrrolidine (460 mg, 1.5 mmol).
Purification by reverse phase prep-HPLC gave the title compound as a
yellow solid (337 mg, 45%). 1H NMR (400 MHz, CD3OD) δ 8.02 (d,
J = 8.6 Hz, 1H), 7.76 (d, J = 8.6 Hz, 2H), 7.55−7.48 (m, 5H), 7.08−
7.05 (m, 2H), 6.94 (d, J = 7.8 Hz, 1H), 6.59 (t, J = 7.5 Hz, 1H), 5.99 (d,
J = 7.5 Hz, 1H) 4.40 (s, 2H), 3.55−3.46 (m, 2H), 3.38−3.34 (m, 1H),
3.27−3.16 (m, 2H), 2.27−2.17 (m, 4H), 2.06−1.98 (m, 2H). HRMS
(ESI) m/z calcd for [C30H28N4O + H]+, 461.2341; found, 461.2336.
Optical rotation: [α]24D = −117° (c 0.52, MeOH). HPLC purity: 98.8%
at 270 nM.
Optical rotation [α]24 = −79° (c 0.33, MeOH). HPLC: 95.1%
D
at 270 nM.
(1R,2S)-(E)-5′-Methyl-2-(3-(4-(morpholinomethyl)styryl)-1H-inda-
zol-6-yl)spiro[cyclopropane-1,3′-indolin]-2′-one 2,2,2-trifluoroace-
tate (45). Prepared according to the general method E using 32
(415 mg, 1 mmol) and (E)-4-(4-(2-(4,4,5,5-tetramethyl-1,3,2-dioxabor-
olan-2-yl)vinyl)benzyl)morpholine (329 mg, 1 mmol). Purification by
1
prep-HPLC gave title compound as a yellow solid (470 mg, 78%). H
NMR (400 MHz, CD3OD) δ 7.73 (d, J = 8.4 Hz, 1H), 7.53 (d, J = 8.0
Hz, 2H), 7.44 (d, J = 8.0 Hz, 2H), 7.38 (s, 1H), 7.32 (d, J = 16.8 Hz,
1H), 7.27 (d, J = 16.8 Hz, 1H), 6.82 (d, J = 8.4 Hz, 1H), 6.77 (d, J =
8.0 Hz, 1H), 6.73 (d, J = 8.0 Hz, 1H), 5.78 (s, 1H), 4.29 (s, 2H), 3.99
(d, J = 11.2 Hz, 2H), 3.75 (t, J = 11.6 Hz, 2H), 3.42−3.32 (m, 2H), 3.21
(t, J = 8.4 Hz, 1H), 3.18−3.08 (m, 2H), 2.09−2.01 (m, 2H), 1.72 (s,
3H). HRMS (ESI) m/z calcd for [C31H30N4O2 + H]+, 491.2447; found,
(1R,2S)-(E)-2-(3-(4-(((R)-3-Fluoropyrrolidin-1-yl)methyl)styryl)-1H-
indazol-6-yl)spiro[cyclopropane-1,3′-indolin]-2′-one 2,2,2-trifluor-
oacetate (41). The title compound was synthesized according to
the general method D, using 17a (630 mg, 1.57 mmol) and (3R)-3-
fluoro-1-(4-((E)-2-(4,4,6-trimethyl-1,3,2-dioxaborinan-2-yl)vinyl)-
benzyl)pyrrolidine (622 mg, 1.88 mmol). The title product was
obtained as a pale-yellow solid after prep-HPLC purification (338 mg,
491.2445. Optical rotation [α]23 = −89° (c 0.28, MeOH). HPLC:
D
99.5% at 270 nM.
(1R,2S)-(E)-5′-Methoxy-1′-methyl-2-(3-(4-(morpholinomethyl)-
styryl)-1H-indazol-6-yl)spiro-[cyclopropane-1,3′-indolin]-2′-one Hy-
drochloride (46). The title compound was synthesized according to
1
48%). H NMR (400 MHz, CD3OD) δ 7.98 (d, J = 8.3 Hz, 1H),
R
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the general method D using 31 (5 g, 11.2 mmol) and (E)-4-(4-(2-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)vinyl)benzyl)morpholine
(4.8 g, 14.6 mmol). Purification by column chromatography (silica
gel, CH2Cl2/MeOH, 95:5 to 92:8) gave the free base as a yellow
solid, which was dissolved into THF (100 mL) and treated with
(11.2 mL of a 1 M solution in Et2O) which gave after drying, the title
0.23 mmol) and (E)-4-(4-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)vinyl)benzyl)morpholine (100 mg, 0.26 mmol). Purification by
reverse phase prep-HPLC gave the title compound as a yellow solid
1
(72 mg, 49%). H NMR (400 MHz, CD3OD) δ 7.93 (d, J = 8.5 Hz,
1H), 7.71 (d, J = 7.8 Hz, 2H), 7.52 (d, J = 7.8 Hz, 2H), 7.49−7.45 (m,
3H), 6.95 (d, J = 8.5 Hz, 1H), 6.89 (d, J = 8.5 Hz, 1H), 6.66 (d, J =
8.5 Hz, 1H), 5.62 (s, 1H), 4.35 (s, 2H), 3.90−3.82 (s, 2H), 3.34−3.43
(m, 2H), 3.30 (s, 3H), 3.26 (s, 3H), 2.76 (t, J = 11.5 Hz, 2H), 2.13−
2.28 (m, 2H), 1.23 (d, J = 6.0 Hz, 6H). HRMS (ESI) m/z calcd for
[C34H36N4O3 + H]+, 549.2866; found, 549.2855. Optical rotation:
1
product as a pale-yellow solid (3.9 g, 67%). H NMR (400 MHz,
CD3OD) δ 8.02 (d, J = 8.6 Hz, 1H), 7.77 (d, J = 8.0 Hz, 2H), 7.56−
7.49 (m, 5H), 7.04 (d, J = 8.4 Hz, 1H), 6.92 (d, J = 7.8 Hz, 1H), 6.69
(d, J = 8.6 Hz, 1H), 5.63 (s, 1H), 4.39 (s, 2H), 4.08−4.05 (m, 2H),
3.77−3.71 (m, 2H), 3.35−3.23 (m, 11H), 2.27−2.25 (m, 1H), 2.22−
2.19 (m, 1H). HRMS (ESI) m/z calcd for [C32H32N4O3 + H]+,
[α]23 = −69° (c 0.41, MeOH). HPLC: 98.2% at 270 nM.
D
(1R,2S)-(E)-2-(3-(3-(Morpholinomethyl)styryl)-1H-indazol-6-yl)-
spiro[cyclopropane-1,3′-indolin]-2′-one 2,2,2-trifluoroacetate (50).
The title compound was synthesized according to the general method
E using 17a (160 mg, 0.4 mmol) and (E)-4-(3-(2-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)vinyl)benzyl)morpholine (184 mg, 0.56 mmol).
Purification by prep-HPLC gave the title compound as a yellow solid
521.2553; found, 521.2551. Optical rotation: [α]22 = −85° (c 0.59,
D
MeOH). HPLC: 97.0% at 270 nM. Anal. (C32H36ClN4O3·1.5H2O)
C, H, N.
(1R,2S)-(E)-2-(3-(4-cis-2,6-Dimethylmorpholino)methyl)styryl)-
1H-indazol-6-yl)spiro[cyclopropane-1,3′-indolin]-2′-one Hydro-
chloride (47). Prepared according to the general method E
using 17a (8 g, 20 mmol) and cis-2,6-dimethyl-4-(4-((E)-2-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)vinyl)benzyl)morpholine (8.4 g,
24 mmol). The residue was purified by Biotage silica gel chro-
matography using a gradient of 0−10% methanol in DCM. The free
base was dissolved in THF (100 mL) and treated with 1 M HCl in
ether (12 mL). Diethyl ether (200 mL) was added, and the precipitate
was collected by filtration to give a yellow solid (5.6 g, 52%). 1H NMR
(400 MHz, CD3OD) δ 8.03 (d, J = 8.3 Hz, 1H), 7.78 (d, J = 8.3 Hz,
2H), 7.49−7.64 (m, 5H), 7.00−7.10 (m, 2H), 6.94 (d, J = 7.8 Hz,
1H), 6.58 (t, J = 7.5 Hz, 1H), 5.99 (d, J = 7.5 Hz, 1H), 4.37 (s, 2H),
3.81−3.95 (m, 2H), 3.34−3.41 (m, 3H), 2.79 (t, J = 11.8 Hz, 2H),
2.14−2.29 (m, 2H), 1.24 (d, J = 6.3 Hz, 6H). HRMS (ESI) m/z calcd
for [C32H32N4O2 + H]+, 505.2604; found, 505.2601. Optical rotation
1
(158 mg, 67%). H NMR (400 MHz, CD3OD) δ 7.88 (d, J = 8.4 Hz,
1H), 7.73 (s, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.50−7.38 (m, 5H), 6.98
(t, J = 7.8 Hz, 1H), 6.91 (d, J = 8.0 Hz, 2H), 6.48 (t, J = 7.6 Hz, 1H),
5.92 (d, J = 7.2 Hz, 1H), 4.36 (s, 2H), 4.03 (d, J = 11.6 Hz, 2H), 3.76
(t, J = 12.0 Hz, 2H), 3.45−3.14 (m, 5H), 2.17−2.08 (m, 2H). HRMS
(ESI) m/z calcd for [C30H28N4O2 + H]+, 477.2291; found, 477.2284.
Optical rotation [α]23 = −144° (c 0.34, MeOH). HPLC: 97.7%
D
at 270 nM.
(1R,2S)-2-(3-((E)-3-((cis-2,6-Dimethylmorpholino)methyl)styryl)-
1H-indazol-6-yl)spiro[cyclopropane-1,3′-indolin]-2′-one 2,2,2-tri-
fluoroacetate (51). The title compound was synthesized according
to the general method E using 17a and (cis)-2,6-dimethyl-4-(3-((E)-2-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)vinyl)benzyl)morpholine
(105 mg, 0.3 mmol). Purification by reverse phase prep-HPLC gave
1
[α]23 = −134° (c 0.27, MeOH). HPLC: 99.9% at 270 nM. Anal.
the title compound as a yellow TFA salt (65 mg, 41%). H NMR
D
(400 MHz, CD3OD) δ 7.95 (d, J = 8.5 Hz, 1H), 7.67−7.81 (m, 2H),
7.36−7.57 (m, 5H), 6.88−7.08 (m, 3H), 6.53 (t, J = 7.6 Hz, 1H), 5.96
(d, J = 7.5 Hz, 1H), 4.37 (s, 2H), 3.87 (s, 2H), 3.34−3.45 (m, 3H),
2.81−2.75 (m, 2H), 2.05−2.26 (m, 2H), 1.22 (d, J = 6.0 Hz, 6H).
HRMS (ESI) m/z calcd for [C32H32N4O2 + H]+, 505.2604; found,
(C32H33ClN4O2·2.5H2O) C, H, N.
(1R,2S)-(E)-2-(3-(4-cis-2,6-Dimethylmorpholino)methyl)styryl)-
1H-indazol-6-yl)-5′-methoxyspiro[cyclopropane-1,3′-indolin]-2′-
one Fumarate (48). To a solution of 18a (20 g, 46 mmol) and
(E)-4-(4-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)vinyl)-
benzyl)morpholine (18.2 g, 51 mmol) in dioxane (400 mL) and water
(80 mL) was added Na2CO3 (9.8 g, 92.8 mmol) and Pd(PPh3)4 (1.61 g,
1.39 mmol). The mixture was heated to 85 °C for 24 h, cooled to rt,
and diluted with water (320 mL). The mixture was concentrated to a
volume of 450 mL and EtOAc (320 mL) was added and the organic
layer was separated and washed with water (80 mL). The solution was
concentrated and redissolved into EtOAc (300 mL) and treated with
charcoal (3 g) at 40 °C for 1 h. The charcoal was filtered off and TMT
(2.8 g) was added and the mixture stirred at 40 °C for 16 h. The TMT
was filtered off, and the organic phase was washed with 0.5 N aq NaOH
(3 × 120 mL) and brine (120 mL). The solvents were removed in
vacuo, and the resulting residue was recrystallized from methyl acetate
(65 mL) to give the free base as a white solid (11.2 g, 48%). The free
base (9.0 g, 16.8 mmol) was dissolved into acetone (36 mL) and
heated to 50 °C. In a separate flask, fumaric acid (1.94 g, 16.8 mmol)
was dissolved into acetone (120 mL), heated to 50 °C, and added to
the free base solution. The solution was concentrated to a volume of
50 mL, and MTBE was added (25 mL). The solution was cooled to rt
and stirred 24 h. The filtrate was collected and dried under high
vacuum at 70 °C to give the title compound as a white crystalline solid
(10.1 g, 92%, 99.9% ee). 1H NMR (400 MHz, CD3OD) δ 8.00 (d, J =
8.4 Hz, 1H), 7.68 (d, J = 8.0 Hz, 2H), 7.50−7.45 (m, 5H), 7.03 (d, J =
8.1 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 6.73 (s, 2H), 6.60 (dd, J = 8.4,
2.5 Hz, 1H), 5.58 (s, 1H), 4.00 (s, 2H), 3.82−3.78 (m, 2H), 3.36 (t,
J = 8.4 Hz, 1H), 3.26 (s, 3H), 3.13−3.10 (m, 2H), 2.34 (t, J = 11.4 Hz,
2H), 2.25−2.16 (m, 2H), 1.01 (d, J = 6.0 Hz, 6H). HRMS (ESI) m/z
calcd for [C33H34N4O3 + H]+, 535.2709; found, 535.2712. Optical
505.2600. Optical rotation: [α]23 = −128° (c 0.5, MeOH). HPLC:
D
99.5% at 270 nM.
(1R,2S)-2-(3-((E)-4-((S)-1-Morpholinoethyl)styryl)-1H-indazol-6-
yl)spiro[cyclopropane-1,3′-indolin]-2′-one Hydrochloride (52). The
title compound was synthesized according to the general method D,
using 17a (244 mg, 0.61 mmol) and 4-((1S)-1-(4-((E)-2-(4,4,6-trimethyl-
1,3,2-dioxaborinan-2-yl)vinyl)phenyl)ethyl)morpholine (230 mg,
0.67 mmol). Silica gel purification (94:6 to 95:5, CH2Cl2/MeOH)
gave 105 mg, 35% of the free base which was dissolved into THF
(10 mL) and treated with HCl (0.5 mL of a 1 M solution in Et2O),
and the resulting precipitate was filtered and washed with Et21O to give
the title compound as an off-white powder (87 mg, 29%). H NMR
(400 MHz, CD3OD) δ 8.01 (d, J = 8.4 Hz, 1H), 7.78 (d, J = 8.3 Hz,
2H), 7.56−7.47 (m, 5H), 7.07−7.03 (m, 2H), 6.93 (d, J = 7.5 Hz,
1H), 6.57 (t, J = 7.6 Hz, 1H), 5.98 (d, J = 7.7 Hz, 1H), 4.50 (q, J =
6.9 Hz, 1H), 4.12−4.09 (m, 1H), 4.00−3.96 (m, 1H), 3.87−3.81 (m,
1H), 3.73−3.67 (m, 2H), 3.35 (t, J = 8.2 Hz, 1H), 3.20−3.03 (m, 3H),
2.26−2.22 (m, 1H), 2.20−2.17 (m, 1H), 1.79 (d, J = 6.9 Hz, 3H).
HRMS (ESI) m/z calcd for [C31H30N4O2 + H]+, 491.2447; found,
491.2446. Optical rotation: [α]23 = −183° (c 0.58, MeOH). HPLC:
D
99.1% at 270 nM.
(1R,2S)-2-(3-((E)-4-(8-Oxa-3-azabicyclo[3.2.1]octan-3-ylmethyl)-
styryl)-1H-indazol-6-yl)spiro[cyclopropane-1,3′-indolin]-2′-one Hy-
drochloride (53). The title compound was synthesized according to
the general method D using 17a (250 mg, 0.62 mmol) and (E)-3-(4-
(2-(4,4,6-trimethyl-1,3,2-dioxaborinan-2-yl)vinyl)benzyl)-8-oxa-3-
azabicyclo[3.2.1]octane (332 mg, 0.93 mmol). Silica gel purification
(96:4 to 95:5, CH2Cl2/MeOH) gave a crude material which was
triturated with 4:1 hexanes/EtOAc to give 110 mg of the free base
which was then dissolved into THF (2 mL) and treated with HCl
(0.3 mL, 1 M in Et2O), and the resulting precipitate was filtered and
washed with Et2O to give the title compound as an off-white powder
rotation [α]20 = −97° (c 0.5, MeOH). HPLC: 99.7% at 270 nm.
D
Anal. (C37H38N4O7) C, H, N.
(1R,2S)-2-(3-((E)-(4-cis-2,6-Dimethylmorpholino)methyl)styryl)-
1H-indazol-6-yl)-5′-methoxy-1′-methylspiro[cyclopropane-1,3′-in-
dolin]-2′-one 2,2,2-trifluoroacetate (49). The title compound was
synthesized according to the general method E, using 31 (100 mg,
1
(97 mg, 31%). H NMR (400 MHz, CD3OD) δ 8.01 (d, J = 8.7 Hz,
S
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1H), 7.76 (d, J = 8.2 Hz, 2H), 7.58−7.48 (m, 5H), 7.07−7.03 (m,
2H), 6.93 (d, J = 7.8 Hz, 1H), 6.58 (t, J = 7.5 H, 1H), 5.98 (d, J =
7.5 Hz, 1H), 4.53 (bs, 2H), 4.35 (s, 2H), 3.36 (t, J = 8.8 Hz, 1H),
3.30−3.25 (m, 4H), 2.26−2.23 (m, 1H), 2.20−2.17 (m, 1H), 2.14−
1.87 (m, 4H). HRMS (ESI) m/z calcd for [C32H30N4O2 + H]+,
prep-HPLC gave the title compound as a yellow powder (73 mg,
49%). H NMR (400 MHz, CD3OD) δ 7.98 (d, J = 8.0 Hz, 1H),
1
7.55−7.64 (m, 2H), 7.42−7.50 (m, 2H), 7.27−7.36 (m, 1H), 6.98−
7.09 (m, 4H), 6.94 (d, J = 7.8 Hz, 1H), 6.58 (t, J = 7.4 Hz, 1H), 5.99
(d, J = 7.3 Hz, 1H), 4.83 (bs, 1H), 3.60−3.54 (m, 2H), 3.41−3.35 (m,
2H), 3.29−3.17 (m, 1H), 2.47−2.43 (m, 1H), 2.37−2.10 (m, 5H),
1.94−1.88 (m, 1H), 1.33−1.44 (m, 6H). HRMS (ESI) m/z calcd for
[C33H34N4O2 + H]+, 519.2760; found, 519.2758. Optical rotation:
503.2447; found, 503.2443. Optical rotation: [α]23 = −174° (c 0.54,
D
MeOH). HPLC: 99.6% at 270 nM.
(1R,2S)-(E)-2-(3-(4-((1,4-Oxazepan-4-yl)methyl)styryl)-1H-inda-
zol-6-yl)spiro[cyclopropane-1,3′-indolin]-2′-one 2,2,2-trifluoroace-
tate Salt (54). The title compound was synthesized according to the
general method E, using 17a (100 mg, 0.25 mmol) and (E)-4-(4-(2-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)vinyl)benzyl)-1,4-oxaze-
pane (103 mg, 0.30 mmol). Purification by reverse phase prep-HPLC
[α]23 = −127° (c 0.48, MeOH). HPLC: 95.6% at 270 nM.
D
Biochemical Assays. Active PLK4 was purified and used to mea-
sure PLK4 activity, using an indirect ELISA detection system, as previ-
ously described.17 PLK1, PLK2, PLK3, AURKA, and AUKB/INCENP
compound inhibition were determined using FRET-based homoge-
neous assay kits from Invitrogen (Burlington, Ontario, Canada). Re-
combinant kinase enzymes were purchased from Invitrogen, with the
exception of AURKB/INCENP, which was purchased from Millipore
(Billerica, MA). The assays were performed according to the manu-
facturer’s specifications with ATP concentrations of 25, 60, and 80 μM,
respectively, for PLK1, PLK2, and PLK3 and ATP concentrations of
20 and 128 μM, respectively, for AURKA and AURKB/INCENP.
Compound inhibition was determined at either a fixed concen-
tration (10 μM) or at a variable inhibitor concentration (typically 50−
0.1 μM in a 10-point dose response titration). Compounds were mixed
with enzyme 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 four-parameter logistic curve fit (XLfit4, IDBS, Guildford,
Surrey, UK) with the formula: (A + (B/(1 + ((x/C)D))), where A =
background value, B = range, C = inflection point, D = curve fit
parameter. Unless otherwise designated, the kinase IC50 values given in
Tables 1, 2, and 3 are the results of a single determination. For values
determined by averaging three replicates, the standard deviation is
given in the PLK4 IC50 column.
1
gave the title compound as a yellow powder (38 mg, 26%). H NMR
(400 MHz, CD3OD) δ 8.02 (d, J = 9.0 Hz, 1H), 7.78 (d, J = 8.3 Hz,
2H), 7.61−7.53 (m, 4H), 7.48 (s, 1H), 7.08−7.04 (m, 2H), 6.94 (d,
J = 7.5 Hz, 1H), 6.60−6.56 (m, 1H), 5.99 (d, J = 7.8 Hz, 1H), 4.45 (s,
2H), 4.02−3.80 (m, 4H), 3.65−3.60 (m, 1H), 3.57−3.35 (m, 4H),
2.29−2.12 (m, 4H). HRMS (ESI) m/z calcd for [C31H30N4O2 + H]+,
491.2447; found, 491.2440. Optical rotation [α]23 = −146° (c 0.39,
D
MeOH). HPLC: 95.1% at 270 nM.
(1R,2S)-2-(3-((E)-4-(((3S,5R)-3,4,5-Trimethylpiperazin-1-yl)-
methyl)styryl)-1H-indazol-6-yl)spiro[cyclopropane-1,3′-indolin]-2′-
one 2,2,2-trifluoroacetate (55). The title compound was synthesized
according to the general method E using 17a (600 mg, 1.5 mmol) and
(2S,6R)-1,2,6-trimethyl-4-(4-((E)-2-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-
lan-2-yl)vinyl)benzyl)piperazine (618 mg, 1.8 mmol). Purification by
reverse phase prep-HPLC gave the title compound as a yellow powder
(296 mg, 31%). 1H NMR (400 MHz, CD3OD) δ 7.97 (d, J = 8.5 Hz, 1H),
7.67 (d, J = 7.8 Hz, 2H), 7.51−7.41 (m, 5H), 7.08−6.98 (m, 2H), 6.94 (d,
J = 8.0 Hz, 1H), 6.56 (t, J = 7.5 Hz, 1H), 5.98 (d, J = 7.3 Hz, 1H), 4.00 (s,
2H), 3.62−3.57 (m, 2H), 3.41−3.33 (m, 3H), 2.92 (s, 3H), 2.79 (t, J =
12.3 Hz, 2H), 2.26−2.12 (m, 2H), 1.41 (d, J = 6.3 Hz, 6H). HRMS (ESI)
m/z calcd for [C33H35N5O + H]+, 518.2920; found, 518.2920. Optical
rotation: [α]23 = −113° (c 0.37, MeOH). HPLC: 96.3% at 270 nM.
D
(1R,2S)-5′-Methoxy-2-(3-((E)-4-(((2R,6S)-2,4,6-trimethylpiperazin-
1-yl)methyl)styryl)-1H-indazol-6-yl)spiro[cyclopropane-1,3′-indo-
lin]-2′-one 2,2,2-trifluoroacetate (56). The title compound was synthe-
sized according to the general method E using 18a (60 mg, 0.14 mmol)
and (2R,6S)-2,4,6-trimethyl-1-(4-((E)-2-(4,4,5,5-tetramethyl-1,3,2-dioxa-
borolan-2-yl)vinyl)-benzyl)piperazine (60 mg, 0.16 mmol). Purification
by reverse phase prep-HPLC gave the title compound as a yellow powder
(35 mg, 46%). 1H NMR (400 MHz, CD3OD) δ 8.00 (d, J = 8.5 Hz, 1H),
7.58−7.67 (m, J = 8.0 Hz, 2H), 7.44−7.50 (m, 3H), 7.36−7.44 (m, 2H),
7.03 (d, J = 7.5 Hz, 1H), 6.83 (d, J = 8.5 Hz, 1H), 6.61 (dd, J = 8.4, 2.4
Hz, 1H), 5.59 (d, J = 2.5 Hz, 1H), 3.75 (s, 2H), 3.44 (bs, 2H), 3.36 (t, J =
8.5 Hz, 1H), 3.26 (s, 3H), 3.17−3.14 (m, 2H), 2.90 (s, 3H), 2.46−2.40
(m, 2H), 2.28−2.14 (m, 2H), 1.37 (d, J = 6.5 Hz, 6H). HRMS (ESI) m/z
calcd for [C34H37N5O2 + H]+, 548.3026; found, 548.3021. HPLC: 99.2%
at 270 nM.
The apparent Kis for compounds 46, 47, and 48 against PLK4 were
estimated to be 0.10, 0.73, and 0.26 nM, respectively, by varying the
ATP concentration in a series of PLK4 inhibition experiments at 0, 2.0,
and 10 μM and applying nonlinear regression analysis (see Supporting
Information).
The selectivity of inhibitors 46, 47, and 48 against 274 protein
kinases was assessed by Millipore (Dundee, Scotland) using a radio-
metric assay. Inhibition was determined at 0.1 μM inhibitor concen-
tration with ATP at Km; % inhibition was determined as stated in the
preceding paragraph (see Supporting Information). For compound 48,
the inhibition constants (IC50s) were determined against selected
kinases: TIE2, TRKA, and TRKB.
Fluorogenic cytochrome P450 inhibition studies were conducted at
37 °C in 96-well plates, as previously described,17 for CYP 1A2, CYP
2D6, CYP 2C9, CYP 2C19, and CYP 3A4 (BFC and DBF substrates).
Cell Viability Assay (GI50). MDA-MB-468, MCF-7, HCC1954,
MDA-MB-231, SKBr-3, Cal-51, and BT-20 breast cancer cells were
seeded into 96-well plates at 3000, 4000, 4000, 2500, 4000, 3000, and
6000 cells per 80 μL, respectively, 24 h before compound overlay and
cultured at 37 °C and 5% CO2. Compounds were prepared as 10 mM
stocks in 100% DMSO. Each 10 mM stock was diluted with DMEM
(Dulbecco’s Modified Eagle’s Medium) cell growth medium
(Invitrogen, Burlington, ON, Canada) containing 10% FBS (fetal
bovine serum) such that the final concentrations ranged from 50 nM
to 250 μM. Aliquots (20 μL) from each concentration were overlaid to
80 μL of preseeded cells to achieve final concentrations of 10 nM to
50 μM. After 5 d, the cells were fixed in situ by gently removing the
culture media and adding 50 μL of ice-cold 10% trichloroacetic acid
(TCA) per well and incubation at 4 °C for 30 min. The plates were
washed with water five times and allowed to air-dry for 5 min. Then
50 μL of 0.4% (w/v) sulforhodamine B (SRB) (Sigma, Oakville, ON,
Canada) solution in 1% (v/v) acetic acid was added to each well,
followed by incubation for 30 min at rt. The plates were washed four
times with 1% acetic acid to remove unbound SRB and air-dried for
5 min. The SRB was solubilized with 100 μL of 10 mM Tris pH 10.5
(1R,2S)-(E)-2-(3-(4-(1-Methylpiperidin-4-yloxy)styryl)-1H-indazol-
6-yl)spiro[cyclopropane-1,3′-indolin]-2′-one 2,2,2-trifluoroacetate
Salt (57). The title compound was synthesized according to the
general method E using 17a (100 mg, 0.25 mmol) and (E)-1-methyl-
4-(4-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)vinyl)phenoxy)-
piperidine (105 mg, 0.3 mmol). Purification by reverse phase prep-
HPLC gave the title compound as a yellow powder (35 mg, 25%). 1H
NMR (CD3OD) δ 7.99−7.97 (m, 1H), 7.63−7.56 (m, 2H), 7.50−7.42
(m, 2H), 7.36−7.28 (m, 1H), 7.10−7.01 (m, 4H), 6.97−6.91 (m, 1H),
6.59 (t, J = 7.7 Hz, 1H), 5.99 (d, J = 7.8 Hz, 1H), 4.84−4.79 (m,
0.5H), 4.65−4.60 (m, 0.5H), 3.65−3.62 (m, 1H), 3.47−3.34 (m, 2H),
3.25−3.13 (m, 1H), 2.94 (s, 3H), 2.44−2.40 (m, 1H), 2.32−2.01 (m,
5H), 1.96−1.81 (m, 1H). LCMS (ESI) m/z calcd for [C31H30N4O2 +
H]+, 491.2447; found, 491.2446. Optical rotation [α]22 = −154°
D
(c 0.43, MeOH). HPLC: 98.9% at 270 nM.
(1R,2S)-2-(3-((E)-4-((1-Isopropylpiperidin-4-yl)oxy)styryl)-1H-inda-
zol-6-yl)spiro[cyclopropane-1,3′-indolin]-2′-one 2,2,2-trifluoroace-
tate (58). The title compound was synthesized according to the
general method E using 17a (100 mg, 0.25 mmol) and ((E)-1-
isopropyl-4-(4-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)vinyl)-
phenoxy)piperidine (111 mg, 0.3 mmol). Purification by reverse phase
T
dx.doi.org/10.1021/jm5005336 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
per well, and absorbance was read at 570 nm using a SpectraPlus
microplate reader (Molecular Devices Corporation). The percentage
(%) of relative inhibition of cell viability was calculated by comparing
to DMSO treated only cells with the maximal response pegged at
100%. GI50s (concentrations required to inhibit growth by 50%) were
calculated using GraphPad PRISM software (GraphPad Software, Inc.,
San Diego, CA, USA).
In a similar manner, lung (A549), ovarian (OVCAR-3), and colon
(SW620, Colo-205, and HCT116+/+) cancer cell lines were seeded at
1500, 6000, 1500, 3000, and 800 cells, respectively, overlaid with
compounds 45, 46, and 48, incubated (5 d), fixed, and read using the
SRB assay.
Microsomal Stability Assay. Microsomal half-life (T1/2) for selected
compounds (Table 3) was determined with human (h), dog (d), rat (r),
and mouse (m) liver microsomes as previously described.23 Curve fitting
was used to determine k (first-order rate constant) using an exponential
fit; the half-life was calculated as microsomal T1/2 = ln(2)/k.
samples were collected via cannulated jugular veins and stored frozen
until analysis.
Seven male beagle dogs (6.5−9 kg), were purchased from Suzhou
Xishan Zhongke laboratory animal breeding center and with
Qualification no. SCXK 2007-0005, 0014369. The IV dose (3 dogs)
was conducted via cephalic vein injection, PO (3 dosage), via intra-
gastric administration. Approximately 0.5 mL blood/time point was
collected from cephalic or saphenous veins into precold EDTA tubes.
Blood samples were put on wet ice and centrifuged at 4 °C to obtain
plasma within 30 min of sample collection and stored frozen until
analysis. One animal, not placed on study, was used for collection of
blank blood (5−10 mL). The resulting blank plasma was used for the
development of the bioanalytical method and standard curve.
Diluted time point samples were analyzed by LCMSMS-02 (API
4000, triple quadrupole) using a standard curve. The PK parameters
were determined by a noncompartmental model using WinNonlin
Professional 5.2.
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 30% PEG400/70%
deionized water 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 three 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.50
Parameters were calculated using plasma concentration time data for
composite averages.
ASSOCIATED CONTENT
* Supporting Information
■
S
Double reciprocal plot of PLK4 inhibition by compound 48;
synthesis of compound intermediates; atomic coordinates,
bond lengths and angles, and anisotropic displacement
parameters for an X-ray structure of compound 33; inhibition
data for the shortlisted compounds (46, 47, and 48) at 0.1 μM
against a 274 member kinase panel. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: 416-581-7620. E-mail: hpauls@uhnresearch.ca.
Notes
■
Xenograft Models. MDA-MB-468 breast cancer cells (Basal, HER-2
negative), A549 lung cancer cells, and SW620 colon cancer cells were
purchased from ATCC (American Type Culture Collections) and
cultured in DMEM (Dulbecco’s Modified Eagle Medium, purchased
from GIBCO) supplemented with 10% fetal calf serum. Five million
MDA-MB468, 5 million A549, and 3 million SW620 were injected
subcutaneously into the right flank of 6−8 week old female CB-17
SCID mice supplied by Charles River. When the tumor volume
reached 80−120 mm3, mice were randomized into groups (n = 8) and
received either vehicle or compound at the doses indicated. Com-
pounds 46, 47, and 48 were weighed and dissolved in 30% PEG 400/
70% water. The compound was dispensed in aliquots and stored at
−20 C for duration of the study, and each aliquot was thawed at rt
before each dose. The mice were dosed via oral gavage daily for 21 d.
Tumor volume and body weight were measured at least 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
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Homer Pearce for helpful discussion during the
course of the work and Dr. Alan Lough, X-ray Crystallography,
University of Toronto, for X-ray data collection and structure
refinement. The Princess Margaret Cancer Foundation, the
Canadian Institute of Health Research, and the Canadian
Foundation for Innovation are acknowledged for financial
support.
ABBREVIATIONS USED
■
aq, aqueous; anhyd, anhydrous; Ar, argon; ATP, adenosine-5′-
triphosphate; Boc, tert-butoxycarbonyl; b, broad; calcd,
calculated; CYP, cytochrome P450; d, doublet; DBU, 1,8-
diazabicycloundec-7-ene; DCM, dichloromethane; DIPEA,
diisopropylethylamine; DMAP, 4-dimethyl-aminopyridine;
DME, dimethoxyethane; DMF, N,N-dimethylformamide;
DMSO, dimethyl sulfoxide; dppf, 1,1′-bis(diphenylphosphino)
ferrocene; FLT3, FMS-related tyrosine kinase 3; GI50, half-
maximal cell growth inhibitory concentration; GST, glutathione
S-transferase; h, hour; HEPES, 4-(2-hydroxyethyl)-1-piperazi-
neethanesulfonic acid; HOBt, hydroxybenzotriazole; HMBC,
heteronuclear multiple-bond correlation spectroscopy; HPLC,
high performance liquid chromatography; HRMS, high-
resolution mass spectrometry; IP, intraperitoneal; KDR, kinase
insert domain receptor; LCMS, liquid chromatography coupled
to mass spectrometry; MBP, myelin basic protein; min, minute;
m, multiplet; MS ESI, electrospray ionization mass spectrom-
etry; NMR, nuclear magnetic resonance; NBS, N-bromosucci-
nimide; PAMPA, parallel artificial membrane permeability
assay; PBS, phosphate buffered saline; PCR polymerase chain
compound was calculated by TGI = 100 × 1 − (tumor volumefinal
tumor volumeinitial for compound treated group)/(tumor volumefinal
tumor volumeinitial for compound control group).
−
−
HCT116 colon cancer cells were purchased, cultured, and injected
(1 million HCT116 cancer cells) into the right flank of 6−8 week old
female athymic CD1 nude mice (Charles River) as described above.
Upon reaching a tumor volume of 80−120 mm3, mice were random-
ized into groups (n = 8) and received either vehicle or weighed
compounds 46, 47, and 48 dissolved in 30% PEG 400/70% water as
described above. The mice were dosed via oral gavage on days 1, 5, 9,
13, 17, and 21 and sacrificed on day 35. Tumor volume, body weight,
and percent growth inhibition were determined as described above.
Rat and Dog Pharmacokinetic Studies. The pharmacokinetic
studies were performed by Shanghai Chempartner, 720 Cailun Road,
Pudong New Area, Shanghai, China. Compounds were dissolved
in 10% NMP/40% PEG/50% water. Male SD rats (200−230 g,
purchased from SLAC Laboratory Animal Co. LTD) were
administered IV (foot dorsal vein) and PO (oral gavage); blood
U
dx.doi.org/10.1021/jm5005336 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Sampson, P.; Li, S.-W.; Liu, Y.; Lang, Y.; Pauls, H. W.; Mak, T. W.;
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reaction; PDB, Protein Data Bank; PEG400, poly(ethylene
glycol) 400; pin, pinacol; PMB, p-methoxybenzyl; PPB, plasma
protein binding; prep, preparative; QSAR, quantitative
structure−activity relationship; RBF, round-bottomed flask;
RNAi, RNA interference; rt, room temperature; RP, reverse
phase; s, singlet; satd, saturated; SEM, 2-(trimethylsilyl)ethoxy]-
methyl; siRNA, small interfering RNA; SMs, starting materials; t,
triplet; TBAB, tetra-n-butylammonium bromide; TEA, triethyl-
amine; temp, temperature; TFA, trifluoroacetic acid; TGI, tumor
growth inhibition; THF, tetrahydrofuran; TLC, thin layer
chromatography; TMT, 2,4,6-trimercaptotriazine; q, quartet;
UPLC, ultra performance liquid chromatography; UHN IACUC,
University Health Network Institutional Animal Care and Use
Committee
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