The discovery of polo-like kinase 4 inhibitors: Design and optimization of spiro[cyclopropane-1,3′[3H]indol]-2′(1′H).ones as orally bioavailable antitumor agents

Article abstract of DOI:10.1021/jm500537u

Polo-like kinase 4 (PLK4), a unique member of the polo-like kinase family of serine-threonine kinases, is a master regulator of centriole duplication that is important for maintaining genome integrity. Overexpression of PLK4 is found in several human cancers and is linked with a predisposition to tumorigenesis. Previous efforts to identify potent and efficacious PLK4 inhibitors resulted in the discovery of (E)-3-((1H-indazol-6-yl)methylene)indolin-2-ones, which are superseded by the bioisosteric 2-(1H-indazol-6-yl)spiro[cyclopropane-1,3′-indolin]-2′-ones reported herein. Optimization of this new cyclopropane-linked series was based on a computational model of a PLK4 X-ray structure and SAR attained from the analogous alkenelinked series. The racemic cyclopropane-linked compounds showed PLK4 affinity and antiproliferative activity comparable to their alkene-linked congeners with improved physicochemical, ADME, and pharmacokinetic properties. Positive xenograft results from the MDA-MB-468 human breast cancer xenograft model for compound 18 support the investigation of PLK4 inhibitors as anticancer therapeutics. A PLK4 X-ray co-structure with racemate 18 revealed preferential binding of the 1R,2S enantiomer to the PLK4 kinase domain.

Full text of DOI:10.1021/jm500537u

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Article
The Discovery of Polo-like Kinase 4 Inhibitors: Design and
Optimization of Spiro[cyclopropane-1,3'[3H]indol]-2'(1'H)-
ones as Orally Bioavailable Antitumor Agents
Peter Brent Sampson, Yong Liu, Narendra Kumar Patel, Miklos Feher, Bryan Forrest, Sze-Wan
Li, Louise Edwards, Radoslaw Laufer, Yunhui Lang, Fuqiang Ban, Donald E. Awrey, Guodong
Mao, Olga Plotnikova, Genie Leung, Richard Hodgson, Jacqueline Mason, Xin Wei, Reza Kiarash,
Erin Green, Wei Qui, Nickolay Y. Chirgadze, Tak W. Mak, Guohua Pan, and Henry W Pauls
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm500537u • Publication Date (Web): 27 May 2014
Downloaded from http://pubs.acs.org on June 21, 2014
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Qui, Wei; University of Toronto, Pharmacology and Toxicology
Chirgadze, Nickolay; University of Toronto, Pharmacology and Toxicology
Mak, Tak; University Health Network, Campbell Family Institute for Breast
Cancer Research
Pan, Guohua; University Health Network, Campbell Family Institute for
Breast Cancer Research
Pauls, Henry; The Campbell Family Institute for Breast Cancer Research,
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The Discovery of Poloꢀlike Kinase 4 Inhibitors: Design
and Optimization of Spiro[cyclopropaneꢀ1,3'[3H]indol]ꢀ
2
'(1'H)ꢀones as Orally Bioavailable Antitumor Agents
,†
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†
Peter B. Sampson,* Yong Liu, Narendra Kumar Patel, Miklos Feher, Bryan Forrest, SzeꢀWan Li,
†
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†
†
†
Louise Edwards, Radoslaw Laufer, Yunhui Lang, Fuqiang Ban, Donald E. Awrey, Guodong Mao,
†
†
†
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†
Olga Plotnikova, Genie Leung, Richard Hodgson, Jacqueline Mason, Xin Wei, Reza Kiarash, Erin
†
‡
‡
†
†
†
Green, Wei Qiu, Nickolay Y. Chirgadze, Tak W. Mak, Guohua Pan, Henry W. Pauls
†
Campbell Family Institute for Breast Cancer Research, TMDT East Tower, MaRS Centre, 101 College
Street, Toronto, Ontario, MG5 1L7, Canada
‡
Department of Pharmacology and Toxicology, University of Toronto, Toronto, Ontario M5S 1A8,
Canada
RECEIVED DATE
RUNNING TITLE: The Discovery of Novel PLK4 Inhibitors as Anticancer Agents.
ABSTRACT: Poloꢀlike kinase 4 (PLK4), a unique member of the poloꢀlike kinase family of serineꢀ
threonine kinases, is a master regulator of centriole duplication that is important for maintaining genome
integrity. Overexpression of PLK4 is found in several human cancers, and is linked with a
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predisposition to tumorigenesis. Previous efforts to identify potent and efficacious PLK4 inhibitors
resulted in the discovery of (E)ꢀ3ꢀ((1Hꢀindazolꢀ6ꢀyl)methylene)indolinꢀ2ꢀones which are superseded by
the bioisosteric 2ꢀ(1Hꢀindazolꢀ6ꢀyl)spiro[cyclopropaneꢀ1,3'ꢀindolin]ꢀ2'ꢀones reported herein.
Optimization of this new cyclopropaneꢀlinked series was based on a computational model of a PLK4 Xꢀ
ray structure and SAR attained from the analogous alkeneꢀlinked series. The racemic (1R,S,2S,R)
cyclopropaneꢀlinked compounds showed PLK4 affinity and antiproliferative activity comparable to their
alkeneꢀlinked congeners with improved physicochemical, ADME and pharmacokinetic properties.
Positive xenograft results from the MDAꢀMBꢀ468 human breast cancer xenograft model for compound
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8 support the investigation of PLK4 inhibitors as anticancer therapeutics. An Xꢀray coꢀstructure
obtained from the racemate 18 revealed preferential binding of the 1R,2S enantiomer to the PLK4 kinase
domain.
KEYWORDS: Poloꢀlike Kinase 4; PLK4 inhibitors; antimitotic agents; Xꢀray structure; breast cancer;
spiro[cyclopropaneꢀ1,3'[3H]indol]ꢀ2'(1'H)ꢀones; MDAꢀMBꢀ468 xenograft.
BRIEFS: Structure guided bioisosteric replacement, resulting in nanomolar PLK4 inhibitors displaying
potent antiproliferative activity, PLK4 coꢀcomplex Xꢀray structure and the results of a xenograft study
are described.
INTRODUCTION:
Blocking cell division through the inhibition of mitosis is one of the most successful clinical strategies
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for the treatment of cancer. Antimitotic drugs, such as the vinca alkaloids and taxanes, have
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demonstrated therapeutic efficacy. The ubiquitous role of tubulin during mitosis in both cancer and
normal cells, however, limits the clinical use of these antimitotics as a result of significant toxicities in
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normal cells. A number of mitotic kinases have been identified as having unique and essential roles in
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cell division, and some of these are the focus of chemotherapeutic drug discovery. The selectivity and
distinct ways in which these new targeted kinase inhibitors interfere with mitosis provide the potential
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to not only overcome certain limitations of current antimitotic therapeutics, but to expand the area of
clinical efficacy established by those drugs. The Aurora kinases, AURKA and AURKB, are established
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druggable targets, with several inhibitors , such as Alisertib (1) currently undergoing clinical
evaluation as anticancer agents. Another class of mitotic kinases that have gained attention as
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therapeutic targets are the Poloꢀlike kinases. The human poloꢀlike kinase (PLK) family consists of five
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serineꢀthreonine kinases, PLK1, PLK2 (Snk), PLK3 (Fnk, Prk), PLK4 (Sak) and PLK5, and are
characterized by the presence of a highly conserved Nꢀterminal catalytic domain and one or two Cꢀ
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,8,9
terminal poloꢀbox domains.
Of the five, PLK1 has been the most extensively studied and there are
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several PLK1 inhibitors currently in Phase 1 and Phase 2 clinical trials.
PLK4 is the most structurally diverse of the PLKs, sharing little homology with the rest of the family,
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possessing only one poloꢀbox domain instead of two.
PLK4 is a low abundance kinase present only
in proliferating tissues, and is a conserved upstream regulator of centriole duplication. PLK4 is
aberrantly expressed in breast cancer, and, in particular, the tripleꢀnegative/basalꢀlike (TNBC) subclass,
as well as other tumor types. Upregulation of PLK4 causes centriole overduplication, and this can result
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in centrosome amplification.
Human cancers frequently contain extra centrosomes, and they are
proposed to play a causative role in genome instability and tumor development. Conversely, depletion
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of PLK4 by RNA interference (RNAi) prevents centriole duplication, resulting in mitotic defects and
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cell death. These observations were corroborated by our internal RNAi results;
PLK4 knockdown
caused breast cancer cell death while normal cells were unaffected. In addition, PLK4 depletion was
shown to cause the suppression of breast cancer tumor growth in mice xenograft experiments. We
rationalized that smallꢀmolecule inhibition of PLK4 would potentially be beneficial therapeutically, and
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recently we have reported our PLK4ꢀdirected drug discovery efforts.
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A number of known kinase inhibitors, such as the Aurora kinase inhibitor VXꢀ680 (2), and the
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antiangiogenic Axitinib (3) are also low nM inhibitors of PLK4. We developed our first generation
PLK4 inhibitors from a screening exercise that yielded simple compounds with micromolar PLK4
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activity. Subsequent iterations of structure based design, synthesis and in vitro screening yielded
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substituted indazolylꢀindolinone compounds such as 4, 5 and 6. As a class, these inhibitors displayed
excellent PLK4 potency, antiproliferative activity and robust tumor growth inhibition upon IP dosing.
Unfortunately, this series displayed poor pharmacokinetic properties, in particular, low oral exposure.
An additional drawback was the configurational lability inherent in these alkeneꢀlinked inhibitors. The
indazolylmethyleneindolinone compounds, while preferentially isolated as the Eꢀisomer, have the
potential to isomerize in vivo, yielding indeterminate mixtures of E/Z isomers. We envisioned that
configurational l stability and physicochemical properties would be improved through bioisosteric
replacement of the double bond with a cyclopropane ring. Cyclopropanes are present in many natural
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products and pharmaceuticals
and have been used in drug discovery programs to prepare
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conformationally restricted analogues of peptidomimetics and SSRIs. Moreover, spirocyclopropylꢀ
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oxindoles have been investigated as reverse transcriptase inhibitors and reported as kinase inhibitors.
Herein we describe the synthesis of 2ꢀ(1Hꢀindazolꢀ6ꢀyl)spiro[cyclopropaneꢀ1,3'ꢀindolin]ꢀ2'ꢀones and
optimization of this series as novel PLKꢀ4 inhibitors.
RESULTS and DISCUSSION:
Chemical
Synthesis.
Treatment
of
indazolꢀ6ꢀylmethyleneindolinꢀ2ꢀone
(7)
with
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trimethylsulfoxonium iodide under modified CoreyꢀChaykovsky
conditions, gave the racemic
cyclopropyl analogues in a 14:1 mixture of trans (8) and cis (9) diastereomers, as shown in Scheme 1,
which were separated by silica gel chromatography. The trans relative stereochemistry was defined
based on the work by Moldvai and coworkers as described for a series of pyridyl
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spirocyclopropylindolones. Irradiation of the 2ꢀH cyclopropane proton resonance only showed a NOE
enhancement to one of the 3ꢀH cyclopropane protons of 8. However, 2ꢀH cyclopropane proton
resonance irradiation of compound 9 gives an NOE enhancement of the signal assigned to the 4′ꢀH of
the indolone ring. Moreover, the 4′ꢀH proton is shifted upfield in the trans conformation compared to
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the cis by 1.2 ppm as a result of anisotropic shielding by the indazole ring. Thus, trans diastereomers
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can readily be distinguished from the cis diastereomers by H NMR.
Compounds carrying the vinylꢀaryl substituent were prepared by various methods, as outlined in
Scheme 2 and Scheme 3. Cyclopropanation of SEMꢀprotected indolinone 10 under CoreyꢀChaykovsky
conditions gave 11. Cyclopropane analogues of indolinones 4 and 5 were prepared via installation of
the vinylꢀaryl substituent under conventional Heck reaction conditions, followed by removal of the SEM
group by stepwise treatment with boron trifluoride etherate and 2 M HCl to give 12 and 13.
Alternatively, indolinones such as 6, 14 and 15 could be treated under CoreyꢀChaykovsky conditions to
give the cyclopropyl analogues 16, 17 and 18 directly.
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In order to prepare analogues via a more convergent route and avoid the use of protecting groups,
three additional synthetic paths were developed in which the cyclopropyl modification was introduced
at an earlier stage, and the vinylꢀaryl group was installed at either the penultimate or final stage.
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Cyclopropyl iodides (20a-g) were prepared under standard conditions from indolinones (19a-g). In
general, Heck coupling to introduce the vinylꢀaryl group to cyclopropyl iodides 20a-g either failed or
generated the desired compound in low yield. However, switching to SuzukiꢀMiyaura conditions with
vinylboronate esters (prepared either via rhodium catalyzed hydroboration of the corresponding terminal
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alkyne or vinyl boronate Heck coupling with the appropriate aryl bromide ) gave the desired
inhibitors 26-37 and 39 directly from iodides 20a-g in better yields.
Secondly, the desired analogues were generated by treatment of 20a under Suzuki Miyaura conditions
with commercially available vinyl boronate ester to give the vinylindazole 21. The vinyl analogue, 21
was then arylated under Heck conditions with the appropriate aryl bromide to give compounds 24, 25
and 38 directly in good yield. The main drawback of this route was the formation of up to 6% of a
branched byproduct, in which the aryl group couples to the Cꢀ1 position of the olefin.
Concurrently to the above two synthetic pathways, a reductive amination approach, was developed.
Compound 23 was prepared in two steps from aldehyde 22 in high selectivity as the Eꢀisomer.
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Inhibitors 40 and 41 were then prepared via reductive amination of 23 with pyrrolidine and piperidine,
respectively.
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Lead Optimization. The rationale for morphing the (indazoꢀ6ꢀlyl)methyleneꢀindolinꢀ2ꢀones to the
structurally analogous (indazolꢀ6ꢀyl)ꢀ3ꢀspirocyclopropylꢀoxindole series was to lock the molecule into a
stable configuration. Our stability studies, empirical evidence and computational models were
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consistent with the E isomer of the (indazoꢀ6ꢀlyl)methyleneꢀindolinꢀ2ꢀones being responsible for the
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observed in vitro activity.
However, it was not possible to unequivocally assign the biological
responses observed to this species alone. Prior to synthesis, the modification was checked
computationally to predict whether compound activity would be retained upon replacing the Eꢀalkene 7
with the transꢀcyclopropane 8 (Figure 3). PLK4 docking, performed using a computational model based
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on the 3COK Xꢀray structure, predicted retention of activity for this bioisosteric replacement. This was
true for molecules with or without the vinylꢀaryl substitution on the indazole. It can be seen that the Eꢀ
form of 7 and the 1R,2S stereoisomer of 8 have nearly identical topology and similar binding poses.
Moreover their interactions with the active site and their docking scores are also similar. However, the
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S,2R enantiomer of 8 exhibits a poorer fit into the site and its docking score is about ½ log unit lower.
In absence of a facile method for enantiomeric resolution, we continued with racemic compounds for
the purposes of compound optimization.
Although there is a possibility of E/Z equilibration of the (indazolꢀ6ꢀyl)methyleneꢀindolinꢀ2ꢀones in
solution, we were pleased to observe that the major product from the CoreyꢀChaykovsky
cyclopropanation was the trans diastereomer (8). The minor cis diastereomer (9) was easily separated
by column chromatography. We were equally pleased to see that the transꢀcyclopropane analogue 8
was active against PLK4 and performed favorably with respect to the Eꢀalkene congener 7 in terms of
selectivity towards Flt3 and KDR. In contrast, the cisꢀcyclopropane diastereomer 9, topologically
similar to the Zꢀalkene, did not inhibit PLK4 in this assay (Table 1).
Furthermore, we found the cyclopropyl modification imparts improved aqueous solubility for 8 vs 7
(8.0 µg/mL vs 0.7 µg/mL at pH 7.4). This improvement is attributed to the nature of the cyclopropane
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ring, being orthogonal to the plane of the indolinone; it serves to counter crystal packing forces. The
cyclopropane disrupts the throughꢀconjugation of the alkene linked system and results in a difference in
color between the alkene linked 7 (yellowꢀorange) to the cyclopropane 8 (off white). Compound 8 also
demonstrated desirable ADME properties, which included an improved profile for five CYP450
isoforms (Table 1), lower plasma protein binding (83% vs >99.9%) and better microsomal stability
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(Table 2). Most significantly, compound 8 achieves up to 100ꢀfold higher level of exposure in mouse
plasma upon IP dosing at 50 mg/kg.
From the preliminary PLK4 IC50 values and our binding hypothesis, we reasoned that the SAR
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observed for the (indazolꢀ6ꢀyl)methyleneꢀindolinꢀ2ꢀones series was likely transferable to the new
indazolꢀ6ꢀyl)ꢀ3ꢀspirocyclopropylꢀoxindole scaffold. Accordingly, syntheses of cyclopropane inhibitors
(
which carry the 3ꢀarylꢀvinyl substitution on the indazole moiety, analogous to some promising leads
from the alkene series (Figure 2), were initiated. With the substituted analogues, there was no
discernible difference in the PLK4 IC50 values (Table 1) or potency in the cellꢀproliferation assays
(MCFꢀ7 and MDAꢀMBꢀ468 cell lines) for cyclopropyl compounds 12, 13 and 16 compared to alkene
compounds 4, 5 and 6, respectively (Table 2). In addition, the trend toward better aqueous solubility,
microsomal stability, lower plasma protein binding and KDR selectivity, held. Moreover, the selectivity
towards other members of the PLK family, observed in the alkene linked compounds, was recapitulated
in the spirocyclopropane modification (Table 3). Unfortunately, the improvement in Flt3 selectivity
observed for 8 (Tables 1 and 5) was not imparted to analogues carrying the vinyl aryl side chain. We
also observed that the 4ꢀpyridinylvinyl analogues 4 and 12 both exhibit very strong inhibition of CYP
2
C9 and 2C19. It appears that the 4ꢀpyridyl function is driving this interaction, independent of the core
structure. Nonetheless, compounds 13 and 16 showed weak CYP450 inhibition for all five isoforms
tested (Table 1).
We were particularly gratified to see that the higher mouse plasma levels seen for the core molecule
carried over for elaborated analogs 12, 13 and 16. For example, although alkene 4 had undetectable
plasma levels at 50 mg/kg IP, a dramatic improvement in exposure was seen for the analogous
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cyclopropane analogue 12 (Table 2). Going forward, efforts were focused on improving kinase
selectivity (e.g. Flt3), maintaining the desirable profiles seen in the first iteration of cyclopropane
inhibitors and to test whether this profile translated into good oral exposure.
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Analogues of 12, in which changing the position of the nitrogen in the ring, or framing the nitrogen
with methyl groups, such as in 17, 24 and 25 had little effect on PLK4 activity. A moderate reduction
in CYP 2C9 inhibition for 24 (IC50 = 400 nM), along with a moderate improvement in cellꢀbased
activity, relative to the marketed anticancer agent 3, was observed. Modeling of 12 in the modified
PLK4 Xꢀray crystal structure (PDB code 3COK, vide infra) revealed that the vinyl group resided in a
hydrophobic groove encompassed by Leu18, and the nitrogen on the pyridine ring was pointing toward
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the solvent interface in the same fashion observed for the alkene series.
It was envisioned that
following the SAR gleaned from the alkeneꢀlinked series, the addition of solubilizing functionalities on
pyridyl ring would not be detrimental to PLK4 activity. Indeed, as can be seen from examples 26 and
2
7, potent PLK4 activity was retained (Table 4). Unfortunately, 26 still exhibited strong CYP 2C9
inhibition (IC = 90 nM).
5
0
Substituted styryls that carried a solvent exposed solubilizing group, were modeled and were
predicted to retain PLK4 activity compared to the 4ꢀpyridinylvinyl analogues. As can be seen in Table
4
, the nature of the solubilizing group (dimethylamine, piperidine, piperazine or morpholine) was
indiscriminant for PLK4 inhibition in general, although compounds 30 and 31 have measured PLK4
inhibition in the sub nanomolar range. However, these subꢀnanomolar values should be viewed as
apparent values as they approach the nominal concentration of enzyme in the assay (PLK4
concentration = 0.4 nM).
The analogues were tested against a panel of four human breast cancerꢀderived cell lines (Table 4) and
compared to the structurally related compound 3. The pyridyl compounds 12, 16 and 24 show good
potency in the cellꢀbased assay for MCFꢀ7, MDAꢀMBꢀ468 and MDAꢀMBꢀ231 cells, although the
values for SKBrꢀ3 remain comparable to 3. However, potency toward the MCFꢀ7 and MDAꢀMBꢀ468
cell lines was dramatically enhanced when the tertiary amino group is added, as shown with compounds
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2
6 and 27. The enhanced sensitivity, relative to 3, of these two cell lines was also observed for styryl
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analogues 13, 18 and 28-41. Overall, the responsiveness for the SKBrꢀ3 cell line remained comparable
to 3 for all the cyclopropane compounds tested.
A subset of compounds was examined for selectivity against KDR, AURKB and Flt3, compared to
indolinone 4 (Table 5). The pyridyl analogues 12 and 25 showed high selectivity against KDR, but no
selectivity against AURKB and Flt3, compared to 4. Strongly basic amines, such as dimethylamino 13
and piperidine 41, that are unsubstituted on the oxindole ring are again unselective with respect to
AURKB and Flt3. However, substitution of the oxindole ring in the 5'ꢀposition, as shown with
dimethylamino analogues 18 and 28, gives high selectivity for KDR and a moderate 10 fold selectivity
toward AURKB and Flt3. When a less basic solubilizing group such as morpholine is used as in 33, 34
and 37, a 30ꢀ50 fold PLK4/Flt3 selectivity and excellent KDR selectivity was observed. Interestingly,
when a dibasic piperazine group is introduced as in 38, 75ꢀfold PLK4/AURKB selectivity and 50ꢀfold
PLK4/Flt3 selectivity, was attained.
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The pharmacokinetic profile for select PLK4 inhibitors is shown in Table 6. Analogues such as 12,
1
7 and 27, show low oral exposure and strong CYP inhibition for the 2C9 and 2C19 isoforms. CYP
inhibition was improved slightly for styryl analogues 13, 18 and 38, however this was not accompanied
by an increase in oral exposure, likely due to the strongly basic pendent solubilizing groups. A
significant improvement in Cmax and AUC was observed for compounds 33, 34 and 35, which carry the
less basic morpholino substituent. Compound 34 exhibited a high Cmax of 1700 ng/mL and AUC of
4
900 nghr/mL, as well as a respectable CYP inhibition profile, with IC50 values greater than one
micromolar for CYP 1A2, 2C9, 2C19, 2D6 and 3A4.
In vivo Efficacy. To investigate the efficacy of (indazolꢀ6ꢀyl)ꢀ3ꢀspirocyclopropylꢀoxindoles in vivo,
compounds 13 and 18 were administered to mice bearing MDAꢀMBꢀ468 breast cancer tumors in a
xenograft study. Animals were dosed orally (PEG400/H O) at 25 mg/kg QD for 21 days. Antitumor
2
activity was observed in vivo for each case, as shown in Figure 4. Tumor inhibition of 76% for 18 and
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Journal of Medicinal Chemistry
up to 84% for 13 was achieved, with no significant decrease in body weight by the end of the study.
These results compare favorably with 2, a known antiproliferative agent, which showed 73% inhibition
(day 21) in the same xenograft model dosed IP at 50 mg/kg QD.
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PLK4 X-ray Structure. Figure 5 displays the binding site, seen in the PLK4 Xꢀray structure coꢀ
crystallized with 18. A hydrogen bonding network in the hinge region is shown between the indazole
and the backbone carbonyl and NH of Glu90 and Cys92, respectively. Two other hydrogen bonds are
observed between the oxindole and the sidechains of Lys41 and Gln160. As well, there is hydrophobic
contact between the enzyme and the ligand, especially surrounding the indazole (Leu18, Ala39, Val26,
Leu73 and Leu143) and encompassing the vinylꢀphenyl group side chain (e.g. Leu18). The oxindole
group, including the methoxy substituent, is fully embedded in a pocket surrounded by protein backbone
from the top and the sides. One of the main focuses of the lead optimization effort was the placement of
solubilizing functionalities in the inhibitor which could reside in a hydrophilic region when bound to
PLK4. Our modified 3COK PLK4 model predicted that a viable site for inclusion of a solubilizing
group was the aryl ring of the pendent vinylꢀaryl group. As observed in the Xꢀray coꢀstructure, the
solubilizing dimethylamino functionality of 18 resides at the solvent interface.
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Although the PLK4 enzyme was soaked with the racemate of 18, only density for the 1R,2S
enantiomer is observed in the coꢀstructure. The 1S,2R enantiomer does not fit with the experimental
electron density. This result is supported by modeling, which predicted a two log unit binding
preference for the 1R,2S enantiomer compared to the 1S,2R enantiomer.
It is important to note that this Xꢀray structure and the 3COK structure enable significantly different
ligand interactions. The 3COK structure is in a DFGꢀin conformation: the backbone of the activation
loop (Tꢀloop) runs relatively close to the plane of the fused ring system and then branches off, away
from the ligand. In contrast, the coꢀstructure of 18 is in a DFGꢀout conformation and the activation loop
runs close to the indolinone nitrogen. In turn, the Gꢀloop moves away in the coꢀstructure to enable the
activation loop to close down on the ligand. As a result, the 3COK site is substantially open to the
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solvent from the distal side of the indolinone, while in our coꢀstructure this area is essentially shielded
from the solvent, enabling further interactions with the ligand.
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SUMMARY:
In this investigation, (indazolꢀ6ꢀyl)ꢀ3ꢀspirocyclopropylꢀindolinꢀ2ꢀones were prepared as
configurationally stable congeners of the previously described (indazoꢀ6ꢀlyl)methyleneꢀindolinꢀ2ꢀones.
The bioisosteric replacement of the alkene linker with a cyclopropane ring serves to disrupt planarity
and results in potent PLK4 inhibitors with improved physicochemical properties. The (indazolꢀ6ꢀyl)ꢀ3ꢀ
spirocyclopropylꢀindolinꢀ2ꢀone core also displays superior ADME properties, such as reduced CYP
inhibition and plasma protein binding and an enhanced pharmacokinetic profile in mice.
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The first set of molecules, elaborated at the 3 position of the indazole moiety with a pyridinyl vinylic
attachment, showed high affinity for PLK4. However, these compounds exhibited significant CYP450
inhibition, in particular for the 2C9 isoform. Further SAR exploration revealed that there was no
significant difference in PLK4 inhibitory activity between pyridinylvinyl and styryl analogues.
Nonetheless, this change did serve to decrease CYP inhibition for the more sensitive isoforms into the
single digit micromolar range. As a group, the dialkylaminomethyl styryl analogues were potent PLK4
inhibitors, more soluble (especially at low pH) and effective inhibitors of breast cancer cell growth. In
addition, compounds 13 and 18 potently inhibited tumor growth in an MDAꢀMBꢀ468 xenograft model.
Compound 18 exhibited 84 % tumor growth inhibition after 21 days, which compared favourably with
2
, a wellꢀknown antimitotic agent. Pharmacokinetic properties of the inhibitors were generally
improved when morpholine was employed as the solubilizing group, with compounds 33, 34 and 35
showing high exposure in plasma upon oral administration in mice.
Although all compounds tested in this study were cyclopropyl racemates, we now have strong
evidence for the preferential affinity of the 1R,2S enantiomer over the 1S,2R enantiomer. Refinement of
the PLK4/compound 18 complex indicated density consistent with the presence of the 1R,2S enantiomer
in the active site. The subsequent article describes the development of a stereoselective synthesis of the
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Journal of Medicinal Chemistry
(
indazolꢀ6ꢀyl)ꢀ3ꢀspirocyclopropylꢀindolinꢀ2ꢀones and further optimization of the orally bioavailable
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morpholine analogues towards the identification of a development candidate for anticancer therapy.
EXPERIMENTAL SECTION:
Biochemical Assays. Active PLK4, as an amino terminal GST fusion of residues 1ꢀ391 of human
PLK4, was purified from an E. coli expression system. The protein was purified from clarified cell
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o
extracts after induction at 15 C overnight using glutathione sepharose, gel permeation chromatography,
and ion exchange chromatography (Resource Q). The resulting protein was dephosphorylated with
lambda phosphatase (NEB cat. no. P0753) and resolved using glutathione sepharose. The
o
dephosphorylated GSTꢀPLK4 was stored in aliquots at ꢀ80 C until use.
PLK4 activity was measured using an indirect ELISA detection system. Dephosphorylated GSTꢀ
PLK4 (4 nM) was incubated in the presence of 15 ꢁM ATP (Sigma cat. no. A7699), 50 mM HEPESꢀ
2
+
Na pH 7.4, 10 mM MgCl , 0.01% Brij 35 (Sigma cat. no. 03ꢀ3170), in a 96ꢀwell microtitre plate preꢀ
2
coated with MBP (Millipore cat# 30ꢀ011). After 30 min, the plate was washed five times with wash
buffer (50 mM TRISꢀCl pH 7.4 and 0.2% Tween 20), and incubated for 30 min with a 1:3000 dilution
of primary antibody (Cell Signaling cat. no. 9381). The plate was then washed 5 times with wash
buffer, incubated for 30 min in the presence of secondary antibody coupled to horse radish peroxidase
(BioRad cat. no. 1721019, 1:3000 concentration), washed an additional 5 times with Wash Buffer, and
incubated in the presence of TMB substrate (Sigma cat. no. T0440). The colorimetric reaction was
quenched after 5 min, by the addition of stop solution (0.5 N H SO ), and quantified by detection at 450
2
4
nm with either a monochromatic or filter based plate reader (Molecular Devices M5 or Beckman
DTX880, respectively).
Inhibition of AurB, FLTꢀ3, KDR PLK1, PLK2 and PLK3 were determined using FRET based
homogenous assays from Invitrogen. The assays were performed according to the manufacturer’s
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specifications with ATP concentrations of 25, 60, 80, 128, 940, and 156 ꢁM, for PLK1, PLK2, PLK3,
AurB, FLT3, and KDR respectively.
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Compound inhibition was determined at either a fixed concentration (10 ꢁM) or at a variable inhibitor
concentration (typically 50 ꢁM to 0.1 ꢁM and 0.1 to 0.0002 ꢁM in a 10 point dose response titration).
Compounds were preincubated in the presence of enzyme for 15 min prior to addition of ATP. The
remaining activity was quantified using the above described activity assay. The percent inhibition of a
compound was determined using the following formula: % inhibition = 100 x (1 – (experimental value –
0
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background value)/(high activity control – background value)). The IC value was determined using a
50
nonꢀlinear 4 point logistic curve fit (XLfit4, IDBS) with the formula; (A+(B/(1+((x/C)^D)))), where A =
background value, B = range, C = inflection point, D = curve fit parameter.
Fluorogenic CYP inhibition studies were conducted at 37 °C in 96ꢀwell, roundꢀbottom, white
polystyrene plates. Supersome mixtures containing CYP protein, insect control supersomes , substrate,
and potassium phosphate buffer (pH 7.4) were prepared with the following final concentrations: CYP
3
A4, 4 nM 3A4+OR+b5 supersomes + 40 ꢀM BFC in 160 mM buffer; CYP 3A4, 0.8 nM 3A4+OR+b5
supersomes + 0.8 ꢀM DBF in 160 mM buffer; CYP 2D6, 6 nM 2D6+OR supersomes + 1.2 ꢀM AMMC
in 40 mM buffer; CYP 2C9, 4 nM CYP 2C9+OR+ b5 + 8 ꢀM MFC in 40 mM buffer; CYP 2C19, 4 nM
CYP 2C19+OR+ b5 + 20 ꢀM CEC in 40 mM buffer; and CYP 1A2, 2 nM CYP 1A2+OR + 4 ꢀM CEC
in 40 mM buffer. Reaction times were verified to be within the limits of kinetics linearity (not shown).
All probe substrate concentrations selected for these determinations were approximately equal to the
Km. The final cofactor concentrations were 1.3 mM NADP, 3.3 mM glucoseꢀ6ꢀphosphate and 0.4 U/mL
o
G6PDH for all enzymes. The final incubation volume was 0.2 mL. The plates were prewarmed at 37 C
for 10 min. The solvent concentration was constant (≤ 0.2% DMSO for test articles, ≤ 2% CH CN for
3
positive control inhibitors) for all conditions within an experiment. Incubations were initiated by the
addition of prewarmed enzyme/substrate mix. After the specified incubation times (10 min for CYP
3
A4/DBF; 15 min for CYP 1A2/CEC; 30 min for CYP 2D6/AMMC, CYP 2C19/MFC and CYP
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3
A4/BFC; 45 min for CYP 2C9/MFC) reactions were quenched by the addition of 75 µL of 80%
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CH CN/20% 0.5 M Tris, with the exception of 3A4/DBF, which was stopped by addition of 75 µL of 2
3
N NaOH. Fluorescence was then measured at the following excitation/emission wavelengths: 3A4
(485/535 nm), 2D6 (390/465 nm), and 2C9 (409/535 nm).
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For data analysis, the fluorescence signals within a dilution series were normalized based on the range
defined by the positive and negative control inhibitors for each isoform/substrate to 0% and 100%
inhibition. The percent inhibition of a compound was determined using the same formula described
above for AurB, FLTꢀ3, KDR PLK1, PLK2 and PLK3.
Microsomal Stability Assay (Microsomal T ): Test compounds were incubated at 1 ꢁM
1
/2
concentration with human, dog, rat, and mouse liver microsomes at 37 °C. The 200 ꢁL of reaction
mixture in 100 mM Tris/HCl, pH 7.5, contained 35 ꢁg of microsomal protein, 9.5 ꢁL of NADPH
Regenerating System Solution A (BD Biosciences cat. no. 451220) and 2 ꢁL of NADPH Regenerating
System Solution B (BD Biosciences cat. no. 451200). After 0, 5, 10, 20, 40, and 60 min incubation, 20
ꢀL aliquots were removed from each reaction and mixed with 80 ꢀL of iceꢀcold CH CN. Stopped
3
reactions were applied to a filter plate, and centrifuged at 500g for 5 min. The filtrate (60 ꢀL) was
transferred to a fresh 96 well plate containing 100 ꢀL water, mixed, and the plate was sealed with a
preslit silicone sealing mat. LCꢀMS (Bruker HCT ion trap coupled to an Agilent 1100 HPLC was used
to quantify the remaining parent. Microsoft Excel was used for the calculations. Curve fitting was used
to determine k (firstꢀorder rate constant) using an exponential fit. The halfꢀlife was calculated as:
Microsomal T1/2 = In(2)/k.
Cell Viability Assay (GI ). MDAꢀMBꢀ231, MDAꢀMBꢀ468, MCFꢀ7 and SKBrꢀ3 breast cancer cells
5
0
were seeded into 96ꢀwell plates at 2500, 3000, 4000, 4000 and 4000 cells per 80 ꢁL, respectively, 24 h
before compound overlay and cultured at 37 °C and 5% CO . Compounds were prepared as 10 mM
2
DMSO stocks. Each 10 mM stock was diluted with DMEM (Dulbecco's Modified Eagle's Medium) cell
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growth medium (Invitrogen, Burlington, ON, Canada), containing 10% FBS (fetal bovine serum) such
that the final concentrations ranged from 50 nM to 250 ꢁM. Aliquots (20 ꢁL) from each concentration
were overlaid to 80 ꢁL of preseeded cells to achieve final concentrations of 10 nM to 50 ꢁM. After 5 d,
the cells were fixed in situ by gently removing the culture media and adding 50 ꢁL of ice cold 10%
TCA per well and incubated at 4 °C for 30 min. The plates were washed with water five times and
allowed to air dry for 5 min. SRB (Sigma, Oakville, ON, Canada) [50 ꢁL of 0.4% (w/v)] solution in 1%
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(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 then air dried for 5 min. The SRB
was solubilized with 100 ꢁL of 10 mM Tris pH 10.5 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 comparison to cells treated with DMSO only. GI s were calculated
5
0
using GraphPad PRISM software (GraphPad Software, Inc., San Diego, CA, USA). Three cell lines
MDAꢀMBꢀ468, MCFꢀ7 and MDAꢀMBꢀ231) generally exhibit comparable sensitivity, with the
(
inflection point for the dose response beginning at about 10 nM. Cell line SKBrꢀ3 is less sensitive,
resulting in typical dose response curves where the inflection point is gradual and indistinct. For this
cell line, the GI50 is calculated as the concentration required to inhibit cell growth at 50% of the
maximal response.
Mouse Plasma Levels. Adult female SCID mice (Princess Margaret Hospital Animal Facility,
Toronto, ON) were used in the experiments. All animal experiments were approved by UHN IACUC.
Nine mice were dosed at either 25 mg/kg (free base equivalents) via oral gavage (examples 13 and 18)
or intraperitoneal injection (water with final pH adjusted to pH 4 with 1 M NaOH) using a volume of
200 ꢁL (example 2). Saphenous vein blood was collected from three mice per time point over an 8 h
period. The plasma samples were analyzed by LCMS/MS for presence of drug and internal standard.
The pharmacokinetic parameters were calculated using Microsoft Excel with pharmacokinetic addꢀin
3
4
functions. Parameters were calculated using plasma concentration time data for composite averages.
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Structural Biology. The kinase domain fragment of human PLK4 (amino acids 6ꢀ269) was subꢀ
cloned into the pET_SUMO vector (Invitrogen) for bacterial expression. The protein was expressed in
E.coli BL21 (DE3) codon plus RIL (Stratagene) cells in TB broth media in the presence of 10% Glucose
overnight at 15 °C with 0.5 mM IPTG.
1
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For the purification, the bacterial pellet was lysed in the lysis buffer (LB) supplemented with 1 mM
imidazole and 1 mM PMSF (25 mM HEPES pH 7.5, 500 mM NaCl, 10% Glycerol, and 1 mM TECEP).
After lysis using an EmulsiFlex C5 homogenizer (Avestin), the lysate was clarified by centrifugation
(50,000g for 1h at 4 °C) and loaded onto a 5 mL HiTrap Chelating column (GE Health Care), charged
0
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2
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7
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9
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2
+
with Ni . The column was washed with 10 column volumes of LB supplemented with 1 mM imidazole,
followed by 10 column volumes of LB supplemented with 50 mM imidazole, and the protein was eluted
with LB supplemented with 300 mM imidazole. SUMO protease (LifeSensors) was added to fractions
containing the PLK4 KD to remove the His6ꢀtag and the cleavage reaction was allowed to proceed
overnight at 4 °C. The cleaved PLK4 KD protein was separated from uncleaved protein, His6_SUMO
protein, and His6_SUMO protease by loading onto a 5 mL HiTrap Chelating column (GE Health Care),
2+
charged with Ni . The cleaved PLK4 KD was eluted from the column with LB supplemented with 50
mM imidazole while uncleaved protein, His6_SUMO protein and His6_SUMO protease were retained
on the column. The protein was loaded onto a Superdex 200 column (GE Health Care) equilibrated with
LB. The protein was further purified to homogeneity by ionꢀexchange chromatography.
Purified PLK4 KD was crystallized in the presence of 18 using the hanging drop vapour diffusion
method at 22 °C by mixing equal volume of the protein solution and the reservoir solution. The PLK4 ꢀ
1
8 binary complex (protein:compound at a molar ratio of 1:5) was crystallized in 20% PEG 4000, 0.2 M
magnesium chloride hexahydrate, and 0.1 M Tris pH 8.5. All the crystals were soaked in the mother
liquor supplemented with 15% Glycerol as cryoprotectant before freezing in liquid nitrogen.
X-ray Data Collection and Processing. The Xꢀray data set of PLK4/compound 18 complex was
collected at an inꢀhouse facility using HFꢀ007 Rigaku rotating anode (CuK ) equipped with Osmic
a
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Journal of Medicinal Chemistry
Page 18 of 54
VariMax focusing system and MARꢀ345 image detector at temperature of 100K. The diffraction data
1
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7
8
9
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1
1
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1
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5
were reduced and scaled with the program XDS. The summary of crystallographic data and refinement
statistics are reported in the supplemental data.
Structure Determination and Crystallographic Refinement. The crystals of PLK4 complex
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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8
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8
9
0
belonged to I23 space group. The initial phase of PLK4 complex was determined by molecular
3
6
replacement using Phaser from Phenix package. Following the initial rigid body refinement,
37
36
interactive cycles of model building and refinement were carried out using COOT and phenix.refine
3
8
or busterꢀTNT. The coordinates and topologies of the ligands from this study were generated using
36
phenix.elbow. Ligands were introduced at the later stages of refinement after most of the protein
models were built. Water molecules, as well as other solvent ligands, were added based on 2mF ꢀDFc
o
map in COOT and refined with phenix.refine or busterꢀTNT.
Computational Methods. In much of this work, the 3COK Xꢀray structure was used for modeling
19
purposes. As described previously, this structure was modified to enable hydrogen bonding between
the side chain of Lys41 and the indolinone carbonyl. The protein structure was prepared using standard
3
9
settings with the help of the Protein Preparation Wizard in the Schrodinger suite. All water molecules
were deleted because none of them were judged as structural water (i.e. part of the binding pattern).
4
0
Ligands were prepared and docking results were analyzed in the MOE software, appropriate
protonation states were generated using the Wash process, all reasonable stereoisomers were obtained
4
1
using Corina, low energy conformations were generated using Conformer Import (max. 30 conformers
within a 1 kcal/mol window) and minimized using the MMFF94x forcefield within MOE. These
conformers were all docked using GlideXP and the highest scoring solution was selected, as had been
4
2,43
recommended in order to minimize sensitivity to initial conditions.
For cyclopropane compounds,
note that docking conditions typically favoured the 1R,2S form, which had ½ to 2 orders of magnitude
better docking scores than the 1S,2R enantiomer, hence only these forms appeared for comparison in the
4
4
docking scores. Mostly default conditions were applied in GlideXP docking, but with the ring
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Journal of Medicinal Chemistry
conformational search switched off. Docking scores were calculated without the Epik state penalties
and using protein hydrogen bond constraints to the hinge (at least 1 of the hydrogen bonds to Glu90 or
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
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6
Cys92 had to be satisfied). This model was sufficiently predictive on PLK4 ligands to enable ranking
2
(
the correlation coefficient (R ) between docking score and PLK4 pIC was 0.53 with an RMSE of 0.91
5
0
and n = 553).
During the course of the study, an Xꢀray coꢀstructure with 18 was obtained and a
0
1
2
3
4
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6
7
8
9
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0
docking model was generated based on this new structure. This coꢀstructure already indicated a
hydrogen bond between Lys41 and the ligand, so it was applied for generating a docking model without
2
further modifications. This model provided superior performance on the same ligand set (R of 0.60
with RMSE of 0.84 with n = 563) and was used in the latter stages of this work.
Synthesis. General Experimental Methods. Commercially 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 using 230ꢀ400
mesh silica gel 60 from EMD chemicals or on a Biotage Isolera One purification system using SNAP
KPꢀsil cartridges. Crude reactions were partially purified using Waters PoraPak Rxn CX cartridges.
Preparative reverseꢀphase HPLC purification was performed on a Varian PrepStar model SDꢀ1 HPLC
system with a Varian Monochrom 10µ Cꢀ18 reverseꢀphase column. Elution was performed using a
gradient of 10% MeOH/water to 90% MeOH/water (0.05% TFA) over a 40ꢀmin period at a flow rate of
4
0 mL/min. Fractions containing the desired material were concentrated and lyophilized to obtain the
final products. Compound purity by analytical HPLC (UV detection at λ = 214 and 254 nm) was
performed on a Varian Prostar HPLC, using a 100 x 4.6 mm Phenomenex Luna 3µ Cꢀ18 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
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Journal of Medicinal Chemistry
Page 20 of 54
compounds was shown by HPLC to be ≥95%. Proton NMRs were recorded on a Bruker 400 MHz
spectrometer, and mass spectra were obtained using a Bruker Esquire 4000 spectrometer. All reported
yields are not optimized.
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Preparation of 3-spirocyclopropyl-oxindoles.
trimethylsulfoxonium iodide (2 eq.) in anhydrous DMF (40 mL) was added NaH (60% dispersion in oil)
6 eq.) at 0 °C. The mixture was stirred for 15 min after which time the appropriate methyleneꢀindolinꢀ
ꢀone (1 eq.) was added. The solution was stirred overnight at rt. The reaction was quenched with sat.
aq. NH Cl solution, extracted with 4 volumes EtOAc, dried over MgSO and concentrated to dryness.
General Procedure A. To a solution of
0
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7
8
9
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8
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8
9
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4
5
6
7
8
9
0
(
2
4
4
The major trans diastereomer was isolated by either by silica gel chromatography or preparative HPLC.
(1R*,2S*)-2-(1H-indazol-6-yl)spiro[cyclopropane-1,3'-indolin]-2'-one (8) The title compound was
19
prepared in a manner similar to general procedure A by utilizing 7 (151 mg, 0.58 mmol). The major
diastereomer 8 was isolated by silica gel chromatography (50% EtOAc in hexanes) as a beige solid (44
1
mg, 28%). H NMR (400 MHz, DMSOꢀd ) δ 13.01 (s, 1H), 10.61 (d, J = 8.3 Hz, 1H), 8.01 (s, 1H),
6
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 [C H N O+ H] 276.1; found, 276.1.
1
7
13
3
(1R*,2R*)-2-(1H-indazol-6-yl)spiro[cyclopropane-1,3'-indolin]-2'-one (9) The minor diastereomer
1
from the reaction of Example 8 was isolated as beige solid (3.5 mg, 2%). H NMR (400 MHz, DMSOꢀ
d6) δ 12.97 (s, 1H), 10.33 (d, J = 8.3 Hz, 1H), 7.99 (s, 1H), 7.59 (d, J = 8.2 Hz, 1H), 7.41 (s, 1H), 7.18–
7
.12 (m, 2H), 6.99–6.94 (m, 2H), 6.86 (d, J = 7.8 Hz, 1H), 3.32 (t, J = 8.3 Hz, 1H), 2.27–2.23 (m, 1H),
+
2
.18–2.15 (m, 1H); LCMS (ESI) m/z calcd for [C H N O+ H] 276.1; found, 276.1.
17
13
3
(1R*,2S*)-2-(3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indazol-6-yl)spiro[cyclo-propane-
1,3'-indolin]-2'-one (11) To a solution of trimethylsulfoxonium iodide (1.89 g, 8.6 mmol) in anhydrous
DMF (40 mL) was added NaH (60% dispersion in oil) (1.03 g, 25.8 mmol) at 0°C. The mixture was
stirred for 15 min after which time 10 (2.2 g, 4.3 mmol) was added. The solution was stirred overnight
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Journal of Medicinal Chemistry
at rt. The reaction was quenched with sat. aq. NH Cl solution (50 mL), extracted with EtOAc (4 x 100
4
1
2
3
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
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2
2
2
2
2
2
2
2
3
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3
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4
4
4
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4
4
4
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5
5
5
5
5
5
5
5
5
5
6
mL), dried over MgSO and concentrated to dryness. The title compound was isolated by silica gel
4
1
chromatography (2% MeOH in CH Cl ) as a yellow solid (1.5 g, 66%). H NMR (400 MHz, CDCl ) δ
2
2
3
7
.46 (s, 1H), 7.39 (d, J = 8.3 Hz, 1H), 7.09 (t, J = 7.5 Hz, 1H), 7.04 (d, J = 8.0 Hz, 1H), 6.92 (d, J = 7.8
Hz, 1H), 6.61 (t, J = 8.0 Hz, 1H), 5.90 (d, J = 8.0 Hz, 1H), 5.70 (s, 2H), 3.57–3.53 (m, 2H), 3.49–3.44
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(m, 1H), 2.31–2.28 (m, 1H), 2.12–2.09 (m, 1H), 0.89–0.84 (m, 2H), ꢀ0.05 (s, 9H); LCMS (ESI) m/z
+
calcd for [C H IN O Si+ H] 532.1; found, 532.1.
23
26
3
2
(1R*,2S*)-(E)-2-(3-(2-(pyridin-4-yl)vinyl)-1H-indazol-6-yl)spiro-[cyclo-propane-1,3'-indolin]-2'-
one (12) To a solution of 11 (375 mg, 0.7 mmol), 4ꢀvinylpyridine (110 mg, 1.05 mmol), DIPEA (0.25
mL, 1.4 mmol) and DMF (2.5 mL) was added Pd(OAc) (8 mg, 0.035 mmol) and P(oꢀtol) (22 mg, 0.07
2
3
mmol). The mixture was heated under microwave irradiation (130° C) for 2 h. Ethyl acetate (150 mL)
was added and the solution was washed with water (2 x 20 mL) and brine (20 mL), dried over MgSO4
and concentrated. The residue was purified by silica gel chromatography (1:1 hexanes/EtOAc to 100%
EtOAc) to give SEMꢀprotected intermediate as a beige solid (320 mg). The material was dissolved in
CH Cl (15 mL) under an atmosphere of N . Boron trifluoride etherate (1 mL) was added dropwise and
2
2
2
the reaction was stirred for 2 h. Methylene chloride was removed in vacuo, and a 2:1 mixture of EtOH /
o
2
M HCl (5 mL) was added and the reaction heated to 50 C for 2 h. The reaction was cooled with an
iceꢀbath and neutralized with 1 M NH OH to pH~8. Ethanol was removed in vacuo and the resulting
4
precipitate was collected which was further purified by silica gel chromatography (15% MeOH in
1
CH Cl ) to give the title compound as a yellow solid (220 mg, 81%). H NMR (400 MHz, DMSOꢀd ) δ
2
2
6
1
3.31 (s, 1H), 10.63 (s, 1H), 8.57–8.50 (m, 2H), 8.10 (d, J = 8.1 Hz, 1H), 7.80 (d, J = 16.6 Hz, 1H),
7.75–7.70 (m, 2H), 7.50–7.42 (m, 2H), 7.09–6.99 (m, 2H), 6.85 (d, J = 7.6 Hz, 1H), 6.53 (t, J = 7.5 Hz,
1
H), 6.01 (d, J = 7.8 Hz, 1H) 3.23–3.19 (m, 1H), 2.35–2.31 (m, 1H), 2.02–1.98 (m, 1H); HRMS (ESI)
+
m/z calcd for [C H N O + H] 379.1559; found, 379.1559; HPLC purity: 98.8% at 214 nM.
24 18
4
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(
1R*,2S*)-(E)-2-(3-(4-((dimethylamino)methyl)styryl)-1H-indazol-6-yl)spiro[cyclopropane-1,3'-
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
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5
5
6
indolin]-2'-one (13) To solution of 11 (250 mg, 0.5 mmol), N,Nꢀdimethylꢀ1ꢀ(4ꢀ
a
vinylphenyl)methanamine (125 mg, 0.78 mmol), DIPEA (0.25 mL, 1.5 mmol) and DMF (1 mL) was
added Pd(OAc) (5 mg, 0.025 mmol) and P(oꢀtol) (15 mg, 0.05 mmol). The mixture was heated under
2
3
microwave irradiation (130 °C) for 2 h. Ethyl acetate (20 mL) was added and the solution was washed
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
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7
8
9
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1
2
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4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
with water (2 x 5 mL) and brine (5 mL), dried over MgSO and concentrated. The residue was purified
4
by silica gel chromatography (50% to 100% EtOAc in hexanes) to give SEMꢀprotected intermediate as
a beige solid (95 mg). The material was dissolved in CH Cl (3 mL) under an atmosphere of N . Boron
2
2
2
trifluoride etherate (0.2 mL) was added dropwise and the reaction was stirred for 2 h. Methylene
chloride was removed in vacuo, and a 2:1 mixture of EtOH / 2M HCl (0.5 mL) was added and the
o
reaction heated to 50 C for 2 h. The reaction was cooled with an iceꢀbath and neutralized with 1 M
NH OH to pH~8. Ethanol was removed in vacuo and the resulting precipitate was collected which was
4
1
further purified by reverse phase HPLC to give the title compound as a yellow solid (63 mg, 23%). H
NMR (400 MHz, CD OD)
δ
8.02 (d, J = 8.3 Hz, 1H), 7.78 (d, J = 8.3 Hz, 2H), 7.56–7.48 (m, 5H),
3
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
+
[
C H N O + H] 435.2185; found, 435.2183; HPLC purity: 98.8% at 214 nM.
2
8
26
4
(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'-indolin]-2'-one (16) The title compound was prepared according to the
19
general procedure A using compound 6 (155 mg, 0.28 mmol) The crude product was purified by silica
gel chromatography (5–7.5% 2 M NH ꢀMeOH in DCM) to yield an 85:15 mixture of the title compound
3
and the cisꢀdiastereomer. The major isomer was isolated by silica gel chromatography (3–5% MeOH
and 2% Et N in CHCl ) to yield a sticky yellow solid which was triturated with Et O to give the 16 as a
3
3
2
1
yellow powder (57 mg, 41%). H NMR (400 MHz, CD OD)
δ
8.26 (s, 1 H), 8.00 (d, J = 8.4 Hz, 1 H),
3
7
.92 (d, J = 9.2 Hz, 1 H), 7.45 (s, 1 H), 7.40 (d, J = 16.8 Hz, 1 H), 7.28 (d, J = 16.8 Hz, 1 H), 7.03 (d, J
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Page 23 of 54
Journal of Medicinal Chemistry
=
7.6 Hz, 1 H), 6.87 (d, J = 9.2 Hz, 1 H), 6.83 (d, J = 8.4 Hz, 1 H), 6.61 (dd, J = 8.4, 2.4 Hz, 1 H), 5.58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
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3
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3
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3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(d, J = 2.0 Hz, 1 H), 3.61 (s, 4 H), 3.39–3.37 (m, 1 H), 3.26 (s, 3 H), 2.59–2.57 (m, 4 H), 2.36 (s, 3 H),
+
2.25–2.17 (m, 2 H); HRMS (ESI) m/z calcd for [C H N O + H] 507.2508; found, 507.2802; HPLC
3
0
30
6
2
purity: 95.6% at 214 nM.
1R*,2S*)-2-(3-((E)-2-(pyridin-3-yl)vinyl)-1H-indazol-6-yl)spiro-[cyclopropane-1,3'-indolin]-2'-
0
1
2
3
4
5
6
7
8
9
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1
2
3
4
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7
8
9
0
(
one (17) To a mixture of trimethylsulfoxonium iodide (176 mg, 0.8 mmol) and 60% NaH (96 mg, 2.4
mmol) in a RBF was added DMF (5 mL). The resulting mixture was stirred for 2 min at rt then cooled
to 0 °C. A solution of (E)ꢀ3ꢀ((3ꢀ((E)ꢀ2ꢀ(pyridinꢀ3ꢀyl)vinyl)ꢀ1Hꢀindazolꢀ6ꢀyl)methylene)indolinꢀ2ꢀone
1
9
(
14) (122 mg, 0.33 mmol)) in DMF (20 mL) was added via pipette. After addition, the resulting
mixture was heated at 55 °C for 2 h and cooled to rt. Additional trimethylsulfoxonium iodide (176 mg,
.8 mmol) and 60% NaH (96 mg, 2.4 mmol) were added and the resulting mixture was stirred O/N at rt
0
and poured onto ice (80 mL). The reaction mixture was acidified with sat. aq. NH Cl and extracted with
4
EtOAc (3 x 40 mL). The combined extracts were dried (Na SO ) and concentrated to give a light brown
2
4
liquid. This residue was purified by flash chromatography (eluent: CH Cl to CH Cl /MeOH/TEA =
2
2
2
2
200:10:1) to give the crude title compound as a light yellow solid which was triturated with MeOH (5
1
mL) and suction filtered to give the title compound as a yellow solid (43 mg, 34%). H NMR (400 MHz,
DMSOꢀd ) 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,
6
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
1
3
.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,
H), 2.00 (dd, J = 9.0 Hz, 4.8 Hz, 1H); HRMS (ESI) m/z calcd for [C H N O + H]+ 379.1559; found,
2
4
18
4
79.1559; HPLC purity: 99% at 254 nM.
(1R*,2S*)-(E)-2-(3-(4-((dimethylamino)methyl)styryl)-1H-indazol-6-yl)-5'-methoxyspiro
[cyclopropane-1,3'-indolin]-2'-one (18) DMF (3 mL) was added to a mixture of NaH (60%, 85 mg,
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2
.1 mmol) and trimethylsulfoxonium iodide (132 mg, 0.6 mmol). The resulting mixture was stirred at rt
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
for 10 min followed by the addition of (E)ꢀ3ꢀ((3ꢀ(4ꢀ((dimethylamino)methyl)ꢀstyryl)ꢀ1Hꢀindazolꢀ6ꢀ
1
9
yl)methylene)ꢀ5ꢀmethoxyindolinꢀ2ꢀone (15) (163 mg, 0.29 mmol) as a solution in DMF (6 mL,
divided for transfer and vial rinse). The reaction was not complete after stirring at rt for 24 h. The
o
mixture was heated at 55 C for 1 h but was still not complete. After cooling to rt, NaH (60%, 44 mg,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
.1 mmol) and trimethylsulfoxonium iodide (69.5 mg, 0.31 mmol) was added and the mixture was
o
heated at 55 C for 1 h prior to quenching by addition of water (25 mL) and brine (25 mL). The mixture
was extracted with EtOAc (300mL) and the organic layer was washed with brine (2 x 25 mL), dried
over Na SO and concentrated in vacuo. The crude product was purified by silica gel chromatography
2
4
(5–7.5% 2 M NH ꢀMeOH in DCM), followed by prepꢀHPLC to yield the title compound as a yellow
3
1
TFA salt (43 mg, 32%). H NMR (400 MHz, CD OD) δ 8.02 (d, J = 8.5 Hz, 1H), 7.76 (d, J = 8.3 Hz,
3
2
H), 7.57–7.46 (m, 5H), 7.05 (d, J = 8.0 Hz, 1H), 6.84 (d, J = 8.3 Hz, 1H), 6.61 (dd, J = 2.26, 8.5 Hz,
1H), 5.58 (d, J = 2.5 Hz, 1H), 4.33 (s, 2H), 3.37 (t, J = 8.4 Hz, 1H), 3.26 (s, 3H), 2.88 (s, 6H), 2.26–2.23
+
(m, 1H), 2.22–2.15 (m, 1H); HRMS (ESI) m/z calcd for [C H N O + H] 465.2290; found, 465.2289;
2
9
28
4
2
HPLC purity: 95.8 % at 254 nM.
(1R*,2S*)-2-(3-iodo-1H-indazol-6-yl)spiro[cyclopropane-1,3'-indolin]-2'-one (20a) The title
1
9
compound was prepared according to the general procedure A using 19a (500 mg, 1.3 mmol) to yield
1
2
0a as a white solid (330 mg, 64%). H NMR (400 MHz, DMSOꢀd ) δ 13.47 (s, 1H), 10.62 (s, 1H),
6
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,
1
H), 5.97 (d, J = 7.6 Hz, 1H), 3.18 (t, J = 8.4 Hz, 1H), 2.31 (dd, J = 7.2 Hz, 4.8 Hz, 1H), 1.98 (dd, J =
+
+
8
.8 Hz, 4.8 Hz, 1H); LCMS (ESI) m/z calcd for [C H IN O + H] 402.0; found, 402.0 [M + 1] .
17
12
3
(1R*,2S*)-2-(3-iodo-1H-indazol-6-yl)-5'-methoxyspiro-[cyclopropane-1,3'-indolin]-2'-one (20b)
19
The title compound was prepared according to the general procedure A using 19b (658 mg, 1.6 mmol)
1
to yield 20b as a white solid (470 mg, 69%). H NMR (400 MHz, DMSOꢀd ) δ 13.48 (s, 1H), 10.43 (s,
6
1
H), 7.49 (s, 1H), 7.33 (d, J = 8.4 Hz, 1H), 7.02 (d, J = 8.4 Hz, 1H), 6.74 (d, J = 8.4 Hz, 1H), 6.57 (dd, J
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Journal of Medicinal Chemistry
=
8.4 Hz, 2.4 Hz, 1H), 5.62 (d, J = 2.4 Hz, 1H), 3.29 (s, 3H), 3.18 (t, J = 8.2 Hz, 1H), 2.34 (dd, J = 7.8
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
Hz, 4.6 Hz, 1H), 1.98 (dd, J = 9.2 Hz, 4.8 Hz, 1H); LCMS (ESI) m/z calcd for [C H IN O + H]
18
14
3
2
432.1; found, 432.0.
(1R*,2S*)-5'-fluoro-2-(3-iodo-1H-indazol-6-yl)spiro[cyclopropane-1,3'-indolin]-2'-one (20c) The
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
9
title compound was prepared according to the general procedure A using 19c (160 mg, 0.39 mmol) to
1
yield 20c as a cream solid (89 mg, 54%). H NMR (400 MHz, DMSOꢀd ) δ 13.50 (s, 1H), 10.65 (s,
6
1
H), 7.50 (s, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.00 (d, J = 8.4 Hz, 1H), 6.85–6.81 (m, 2H), 5.81 (d, J = 8.4
Hz, 1H), 3.23 (t, J = 8.4 Hz, 1H), 2.45–2.41 (m, 1H), 2.03–2.00 (m, 1H); LCMS (ESI) m/z calcd for
+
[
C H FIN O + H] 420.0; found, 420.0.
1
7
11
3
(1R*,2S*)-5'-ethyl-2-(3-iodo-1H-indazol-6-yl)spiro[cyclopropane-1,3'-indolin]-2'-one (20d) To a
mixture of 3ꢀiodoꢀ1Hꢀindazoleꢀ6ꢀcarbaldehyde (1.36 g, 5 mmol) and 5ꢀethylindolinꢀ2ꢀone (885 mg, 5.5
mmol) in MeOH (25 mL) was added piperidine (0.1 mL, 1 mmol). The resulting mixture was refluxed
for 90 min, then cooled to rt. The resulting red precipitate was collected by suction filtration and dried to
give the indolinone 19d, which was used without further purification to prepare 20d, according to the
1
general procedure A, as a light orange solid (710 mg, 33%). H NMR (400 MHz, DMSOꢀd )
δ
13.44 (s,
6
1
H), 10.51 (s, 1H), 7.44 (s, 1H), 7.30 (d, J = 8.4 Hz, 1H), 6.99 (d, J = 8.8 Hz, 1H), 6.80 (d, J = 7.2 Hz
1H), 6.72 (d, J = 7.6 Hz, 1H), 5.76 (s, 1H), 3.17 (t, J = 7.8 Hz, 1H), 2.29 (dd, J = 8.0 Hz, 4.8 Hz, 1H),
2
.18–2.04 (m, 2H), 1.98 (dd, J = 8.6 Hz, 4.8 Hz, 1H), 0.60 (t, J = 7.4 Hz, 3H); LCMS (ESI) m/z calcd
+
for for [C H IN O + H] 430.0; found, 430.0.
19 16
3
(1R*,2S*)-5'-(difluoromethoxy)-2-(3-iodo-1H-indazol-6-yl)spiro[cyclopropane-1,3'-indolin]-2'-
one (20e) The title compound was prepared in a manner similar to general procedure A by utilizing 19e
(150 mg, 0.33 mmol) to yield 20e as a white solid (70 mg, 45%) after purification by Biotage silica
1
column chromatography (0 to 30% acetone gradient in hexanes). H NMR (400 MHz, CDCl )
δ 9.31 (s,
3
1
H), 7.40–7.43 (m, 2 H), 7.00 (d, J = 8.4 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 6.84–6.86 (m, 1 H), 5.99 (t,
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J = 74 Hz, 1H), 5.69 (s, 1H), 3.50 (t, J = 8.8 Hz, 1 H), 2.31–2.35 (m, 1 H), 2.10–2.15 (m, 1 H); LCMS
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
(
ESI) m/z calcd for [C H F IN O + H] 468.0; found, 468.0.
18 12
2
3
2
(1R*,2S*)-5'-chloro-2-(3-iodo-1H-indazol-6-yl)spiro[cyclopropane-1,3'-indolin]-2'-one (20f) To
a mixture of 3ꢀiodoꢀ1Hꢀindazoleꢀ6ꢀcarbaldehyde (1.36 g, 5 mmol) and 5ꢀchloroindolinꢀ2ꢀone (880 mg,
.5 mmol) in MeOH (25 mL) was added piperidine (0.1 mL, 1 mmol). The resulting mixture was
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
refluxed for 90 min, then cooled to rt. The resulting yellow precipitate was collected by suction filtration
and dried to give the indolinone 19f, which was used without further purification to prepare 20f,
1
according to the general procedure A, as a light beige solid (2.29 g, quantitative). H NMR (400 MHz,
DMSOꢀd6)
δ 13.51 (s, 1H), 10.76 (s, 1H), 7.52 (s, 1H), 7.33 (d, J = 8.0 Hz, 1H), 7.04 (t, J = 8.8 Hz, 2H),
6
.84 (d, J = 8.4 Hz, 1H), 6.03 (s, 1H), 3.23 (t, J = 8.0 Hz, 1H), 2.06–1.97 (m, 2H); LCMS (ESI) m/z
+
calcd for [C H ClIN O + H] 436.0; found, 436.2.
17
11
3
(1R*,2S*)-2-(3-iodo-1H-indazol-6-yl)-5'-methylspiro[cyclopropane-1,3'-indolin]-2'-one (20g) To
a mixture of 3ꢀiodoꢀ1Hꢀindazoleꢀ6ꢀcarbaldehyde (1.36 g, 5 mmol) and 5ꢀmethylindolinꢀ2ꢀone (772 mg,
5.25 mmol) in MeOH (25 mL) was added piperidine (0.1 mL, 1 mmol). The resulting mixture was
refluxed for 90 min, then cooled to rt. The resulting yellow precipitate was collected by suction filtration
and dried to give the indolinone 19g, which was used without further purification to prepare 20f,
according to the general procedure A, as a yellow solid and a 6:1 mixture of diastereomers (2.06 g,
1
9
9%) which was used without further purification. H NMR (400 MHz, DMSOꢀd )
δ
13.43 (s, 1H),
6
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
1
.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),
+
.85 (s, 3H); LCMS (ESI) m/z calcd for [C H IN O + H] 416.0; found, 416.1.
18
14
3
(1R*,2S*)-2-(3-vinyl-1H-indazol-6-yl)spiro[cyclopropane-1,3'-indolin]-2'-one (21) To a mixture
of 20a (802 mg, 2 mmol) and 4,4,5,5ꢀtetramethylꢀ2ꢀvinylꢀ1,3,2ꢀdioxaborolane (462 mg, 3 mmol) in a 20
mL microwave vial was added PhCH /EtOH (8 mL/4 mL), followed by 1 M aq. Na CO (3 mL, 3
3
2
3
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Journal of Medicinal Chemistry
mmol) and Pd(PPh ) (46 mg, 0.04 mmol, 2 mol%) and the resulting mixture was purged with argon
3
4
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
o
and microwaved 3 h at 120 C. After aqueous workup, the solution was extracted with EtOAc and was
purified by flash chromatography (50% hexanes in EtOAc) to give the crude title compound as a light
1
yellow foam (512 mg) which was used without further purification. H NMR (400 MHz, CDCl )
δ
7.74
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(d, J = 8.4 Hz, 1H), 7.35 (s, 1H), 7.10–6.88 (m, 5H), 6.54 (t, J = 7.4 Hz, 1H), 6.06 (d, J = 18.0 Hz, 1H),
5
.92 (d, J = 7.6 Hz, 1H), 5.49 (d, J = 7.6 Hz, 1H), 3.46 (d, J = 8.2 Hz, 1H), 2.30–2.18 (m, 2H); LCMS
+
(
ESI) m/z calcd for [C H N O + H] 302.1; found, 302.0.
1
9
15
3
4-((E)-2-(6-((1R*,2S*)-2'-oxospiro[cyclopropane-1,3'-indoline]-2-yl)-1H-indazol-3-
yl)vinyl)benzaldehyde (23) Diethyl 4ꢀcyanobenzylphosphonate (600 mg, 2.4 mmol) was dissolved in
DMF (5 mL) at 0 °C. Potassium tertꢀbutoxide (540 mg, 4.8 mmol) was added and the mixture was
stirred for
5
min.
6ꢀ((1R*,2S*)ꢀ2'ꢀoxospiro[cyclopropaneꢀ1,3'ꢀindoline]ꢀ2ꢀyl)ꢀ1Hꢀindazoleꢀ3ꢀ
carbaldehyde 22 (200 mg, 0.66 mmol) was dissolved into DMF (5 mL), added dropwise and the
reaction mixture was stirred for 90 min. The reaction was neutralized with aq. HCl (0.1 N) and the
resulting precipitate collected. The precipitate was dissolved in EtOAc (100 mL) and washed with H O
2
(
2 x 10 mL), brine (10 mL), dried over MgSO and concentrated to dryness. The residue was purified
4
by silica gel chromatography to give 4ꢀ((E)ꢀ2ꢀ(6ꢀ((1R*,2S*)ꢀ2'ꢀoxospiro[cyclopropaneꢀ1,3'ꢀindoline]ꢀ2ꢀ
1
yl)ꢀ1Hꢀindazolꢀ3ꢀyl)vinyl)benzonitrile as a white solid (100 mg, 38%). H NMR (400 MHz, CD OD) δ
3
8
.04 (d, J = 8.5 Hz, 1H), 7.82 (d, J = 8.6 Hz, 2H), 7.74 (d, J = 8.6 Hz, 2H), 7.66–7.53 (m, 2H), 7.48 (s,
1H), 7.08–7.04 (m, 2H), 6.94 (d, J = 8.3 Hz, 1H), 6.59 (t, J = 7.6 Hz, 1H), 5.99 (d, J = 7.5 Hz, 1H),
3
4
.38–3.34 (m, 1H), 2.27–2.18 (m, 2H); LCMS (ESI) m/z calcd for [C H N O + H]+ 403.1; found
26 18 4
03.1. The nitrile (100 mg, 0.25 mmol) was then dissolved in pyridine (3 mL), acetic acid (0.8 mL) and
water (1 mL). Raney Nickel (100 mg), was added, following by dropwise addition of aqueous sodium
hypophosphite (180 mg, 2.1 mmol in 1 mL of water) and the reaction was stirred overnight at 60 °C.
The product was extracted into EtOAc (30 mL), washed with brine (5 mL), dried over MgSO and
4
concentrated to dryness. The residue was purified by silica gel chromatography (99:1 CH Cl /MeOH)
2
2
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1
to give the title compound as an orange solid (95 mg, 94%). H NMR (400 MHz, CDCl ) δ 10.03 (s,
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
H), 8.20 (s, 1H), 7.86–7.97 (m, 2H), 7.74 (d, J = 8.3 Hz, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.41 (s, 1H),
7.07–7.13 (m, 2H), 6.88–7.03 (m, 2H), 6.63 (t, J = 7.5 Hz, 1H), 5.94 (d, J = 7.8 Hz, 1H), 3.40 (t, J = 7.1
+
Hz, 1H), 2.35–2.44 (m, 1H), 2.08–2.02 (m, 1H); LCMS (ESI) m/z calcd for [C H N O + H] 406.2;
26
19
3
2
found, 406.2.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Heck Arylation of 3-vinylspirocyclopropaneindolinones. General Procedure B.
To a mixture of 2ꢀ(3ꢀvinylꢀ1Hꢀindazolꢀ6ꢀyl)spiro[cyclopropaneꢀ1,3'ꢀindolin]ꢀ2'ꢀone (1 eq.) and
arylbromide (1 eq.) in DMF was added DIPEA (2 eq.), followed by Pd(OAc) (5 mol%) and P(oꢀtol)
2
3
o
(10 mol%). The resulting mixture was purged with argon, and then microwaved 2 h at 125 C. The
mixture was filtered through a Waters PoraPak CX column and then purified by preparative HPLC.
(1R,2S)-2-(3-((E)-2-(6-methylpyridin-3-yl)vinyl)-1H-indazol-6-yl)spiro[cyclopropane-1,3'-
indolin]-2'-one (24) The title compound was prepared according to general procedure B using 21 (60
mg, 0.2 mmol) and 5ꢀbromoꢀ2ꢀmethylpyridine (34.4 mg, 0.2 mmol). Purification by preparative HPLC
1
gave 24 as a light yellow solid (25 mg, 25%) contaminated with ~6% of the branched isomer. H NMR
(400 MHz, CD OD) δ 8.90 (s, 1H), 7.74 (dd, J = 8.4 Hz, 1.6 Hz, 1H), 8.02 (d, J = 8.4 Hz, 1H), 7.87 (d,
3
J = 8.4 Hz, 1H), 7.78 (d, J = 16.8 Hz, 1H), 7.55 (d, J = 16.8 Hz, 1H), 7.49 (s, 1H), 7.07–7.00 (m, 2H),
6.94 (d, J = 8.0 Hz, 1H), 6.55 (t, J = 7.6 Hz, 1H), 5.97 (d, J = 7.6 Hz, 1H), 3.34 (t, J = 8.4 Hz, 1H), 2.78
(s, 3H), 2.23 (dd, J = 7.6 Hz, 4.8 Hz, 1H), 2.18 (dd, J = 8.8 Hz, 4.8 Hz, 1H); HRMS (ESI) m/z calcd for
+
[
C H N O + H] 393.1715; found, 393.1711; HPLC purity: 94% at 214 nM.
2
5
20
4
(1R*,2S*)-2-(3-((E)-2-(2,6-dimethylpyridin-3-yl)vinyl)-1H-indazol-6-yl)spiro[cyclopropane-1,3'-
indolin]-2'-one (25) The title compound was prepared according to general procedure B using 21 (60
mg, 0.2 mmol) and 3ꢀbromoꢀ2,6ꢀdimethylpyridine (37 mg, 0.2 mmol). Purification by preparative
1
HPLC gave 25 as a light yellow solid (23 mg, 22%) contaminated with ~3% of the branched isomer. H
NMR (400 MHz, CD OD)
δ
8.73 (d, J = 8.0 Hz, 1H), 7.97 (d, J = 8.4 Hz, 1H), 7.72 (d, J = 8.4 Hz, 1H),
3
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7
.68 (d, J = 16.4 Hz, 1H), 7.59 (d, J = 16.4 Hz, 1H), 7.50 (s, 1H), 7.06 (d, J = 8.8 Hz, 1H), 7.05 (t, J =
.6 Hz, 1H), 6.94 (d, J = 8.0 Hz, 1H), 6.56 (t, J = 7.6 Hz, 1H), 5.98 (d, J = 7.6 Hz, 1H), 3.35 (t, J = 8.4
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
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Hz, 1H), 2.86 (s, 3H), 2.76 (s, 3H), 2.24 (dd, J = 7.6 Hz, 4.8 Hz, 1H), 2.18 (dd, J = 9.0 Hz, 5.0 Hz, 1H);
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HRMS (ESI) m/z calcd for [C H N O + H] 407.1872; found, 407.1877; HPLC purity: 96.9% at 214
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22
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nM.
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Suzuki-Miyaura cross coupling of 3-iodospirocyclopropaneindolinones. General Procedure C.
The appropriate 2ꢀ(3ꢀiodoꢀ1Hꢀindazolꢀ6ꢀyl)spiro[cyclopropaneꢀ1,3'ꢀindolin]ꢀ2'ꢀone 20 (1 eq.) was
dissolved into 2:1 PhCH /EtOH and 1 M Na CO (2 eq.). The appropriate vinyl boronic ester (1.2 eq.)
3
2
3
and catalyst, Pd(PPh ) (5 mol%) were added and resulting mixture was heated in a microwave for 2 h
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4
o
at 125 C. After cooling to rt, the mixture was diluted with H O, extracted with EtOAc (x 2) and dried
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(MgSO ). After removal of solvents, the residue was redissolved in MeOH and filtered through a
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Waters CX column and purified by preparative HPLC to give the title compound as a TFA salt.
(1R*,2S*)-(E)-2-(3-((E)-2-(6-(piperidin-1-ylmethyl)pyridin-3-yl)vinyl)-1H-indazol-6-
yl)spiro[cyclopropane-1,3'-indolin]-2'-one (26) The title compound was synthesized according to the
general procedure C using 20a (50 mg, 0.12 mmol) and (E)ꢀ2ꢀ(piperidinꢀ1ꢀylmethyl)ꢀ5ꢀ(2ꢀ(4,4,5,5ꢀ
tetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)vinyl)pyridine (65 mg, 0.2 mmol). Purification by reverse phase
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preparative HPLC gave 26 as a yellow TFA salt (9 mg, 15%). H NMR (400 MHz, CD OD) δ 8.91 (s,
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1
6
H), 8.20 (dd, J = 8.2, 2.0 Hz, 1H), 8.04 (d, J = 8.0 Hz, 1H), 7.68–7.49 (m, 4H), 7.08–7.04 (m, 2H),
.95 (d, J = 7.8 Hz, 1H), 6.60–6.56 (m, 1H), 5.99 (d, J = 7.5 Hz, 1H), 4.45 (s, 2H) 3.39–3.10 (m, 3H),
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2.27–2.18 (m, 2H), 1.95–1.60 (m, 8H); HRMS (ESI) m/z calcd for [C H N O + H] 476.2450; found,
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29
5
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76.2448; HPLC purity: 97.8% at 214 nM.
(1R*,2S*)-5'-methoxy-2-(3-((E)-2-(6-(piperidin-1-ylmethyl)pyridin-3-yl)vinyl)-1H-indazol-6-
yl)spiro[cyclopropane-1,3'-indolin]-2'-one (27) The title compound was synthesized according to the
general procedure C using 20b (51 mg, 0.12 mmol) and (E)ꢀ2ꢀ(piperidinꢀ1ꢀylmethyl)ꢀ5ꢀ(2ꢀ(4,4,5,5ꢀ
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