Discovery of Novel Spiro[3H-indole-3,2′-pyrrolidin]-2(1H)-one Compounds as Chemically Stable and Orally Active Inhibitors of the MDM2-p53 Interaction

Article abstract of DOI:10.1021/acs.jmedchem.6b00900

Scaffold modification based on Wang's pioneering MDM2-p53 inhibitors led to novel, chemically stable spiro-oxindole compounds bearing a spiro[3H-indole-3,2′-pyrrolidin]-2(1H)-one scaffold that are not prone to epimerization as observed for the initial spiro[3H-indole-3,3′-pyrrolidin]-2(1H)-one scaffold. Further structure-based optimization inspired by natural product architectures led to a complex fused ring system ideally suited to bind to the MDM2 protein and to interrupt its protein-protein interaction (PPI) with TP53. The compounds are highly selective and show in vivo efficacy in a SJSA-1 xenograft model even when given as a single dose as demonstrated for 4-[(3S,3′S,3′aS,5′R,6′aS)-6-chloro-3′-(3-chloro-2-fluorophenyl)-1′-(cyclopropylmethyl)-2-oxo-1,2,3′,3′a,4′,5′,6′,6′a-octahydro-1′H-spiro[indole-3,2′-pyrrolo[3,2-b]pyrrole]-5′-yl]benzoic acid (BI-0252).

Full text of DOI:10.1021/acs.jmedchem.6b00900

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Article
Discovery of Novel Spiro[3H-indole-3,2´-pyrrolidin]-2(1H)-one Compounds as
Chemically Stable, and Orally Active Inhibitors of the MDM2–p53 Interaction
Andreas Gollner, Dorothea Rudolph, Heribert Arnhof, Markus Bauer, Sophia Maria Blake, Guido Boehmelt,
Xiao-Ling Cockcroft, Georg Dahmann, Peter Ettmayer, Thomas Gerstberger, Jale Karolyi-Oezguer,
Dirk Kessler, Christiane Kofink, Juergen Ramharter, Jörg Rinnenthal, Alexander Savchenko, Renate
Schnitzer, Harald Weinstabl, Ulrike Weyer-Czernilofsky, Tobias Wunberg, and Darryl B. McConnell
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00900 • Publication Date (Web): 24 Oct 2016
Downloaded from http://pubs.acs.org on October 24, 2016
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Discovery of Novel Spiro[3Hꢀindoleꢀ3,2´ꢀ
pyrrolidin]ꢀ2(1H)ꢀone Compounds as Chemically
Stable, and Orally Active Inhibitors of the MDM2–
p53 Interaction
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Andreas Gollner,*, † Dorothea Rudolph†, Heribert Arnhof†, Markus Bauer†, Sophia M. Blake†,
Guido Boehmelt†, XiaoꢀLing Cockroft†, Georg Dahmann‡, Peter Ettmayer†, Thomas
Gerstberger†, Jale KarolyiꢀOezguer†, Dirk Kessler†, Christiane Kofink†, Juergen Ramharter†,
Jörg Rinnenthal†, Alexander Savchenko†, Renate Schnitzer†, Harald Weinstabl†, Ulrike
WeyerꢀCzernilofsky†, Tobias Wunberg†, and Darryl B. McConnell†
† Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria
‡ Boehringer Ingelheim Pharma GmbH & Co KG, Biberach, Germany
ABSTRACT
Scaffold modification based on Wang´s pioneering MDM2ꢀp53 inhibitors led to novel,
chemically stable spiroꢀoxindole compounds bearing a spiro[3Hꢀindoleꢀ3,2´ꢀpyrrolidin]ꢀ
2(1H)ꢀone scaffold that are not prone to epimerization as observed for the initial spiro[3Hꢀ
indoleꢀ3,3´ꢀpyrrolidin]ꢀ2(1H)ꢀone scaffold. Further structure based optimization inspired by
natural product architectures led to a complex fused ring system ideally suited to bind to the
MDM2 protein and to interrupt its proteinꢀprotein interaction (PPI) with TP53. The
compounds are highly selective and show in vivo efficacy in a SJSAꢀ1 xenograft model even
when given as a single dose as demonstrated for 4ꢀ[(3S,3'S,3'aS,5'R,6'aS)ꢀ6ꢀchloroꢀ3'ꢀ(3ꢀ
chloroꢀ2ꢀfluorophenyl)ꢀ1'ꢀ(cyclopropylmethyl)ꢀ2ꢀoxoꢀ1,2,3',3'a,4',5',6',6'aꢀoctahydroꢀ1'Hꢀ
spiro[indoleꢀ3,2'ꢀpyrrolo[3,2ꢀb]pyrrole]ꢀ5'ꢀyl]benzoic acid (BIꢀ0252).
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INTRODUCTION
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The tumor protein p53 (TP53), the so called “guardian of the genome”, is a pivotal tumor
suppressor protein and a mainstay of the body´s cellular antiꢀcancer defense system.1 As a
potent transcription factor, TP53 is activated following cellular stress and regulates multiple
downstream target genes implicated in cellꢀcycle control, apoptosis, senescence and DNA
repair.2 TP53 is one of the most frequently mutated genes in cancer with about 50% of all
human cancers having mutations or deletions in this gene, although the incidence of TP53
mutations differs significantly between different cancer types. 3, 4 Mice lacking TP53 develop
normally but are prone to the spontaneous development of multiple types of cancer,
particularly lymphomas and sarcomas, at an early age.5 Although the remaining 50% of
human cancers have tumors with TP53 wildꢀtype status, the function of TP53 is frequently
attenuated in these cancers by other mechanisms including overexpression of its key negative
regulator HDM2, the human homolog of mouse double minute 2 (MDM2). Mice deficient for
MDM2 die early in embryonic development. Absence of TP53 rescues the embryonic lethality
seen in MDM2ꢀdeficient mice, suggesting a critical role of MDM2 in the regulation of TP53
function.6 MDM2, an E3 ligase, regulates TP53 function and protein stability by three main
mechanisms. First, MDM2 binds to the transactivation domain of TP53 and represses
transcriptional activity of TP53. Secondly, MDM2 transports the transcription factor TP53
from the nucleus to the cytoplasm and third, the E3 ligase function of MDM2 facilitates
proteasomeꢀmediated TP53 protein degradation.7 Recent preclinical data show that genetic
changes in TP53 not only contribute to tumor development but also to continued tumor
growth as restoration of TP53 function in established tumors by genetic means causes tumor
regressions in preclinical models of cancer.8ꢀ10 Based on these data, stabilization and
activation of wildꢀtype TP53 by the inhibition of TP53 binding to its negative regulator
MDM2 has recently been explored as a novel approach to cancer therapy.
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Several MDM2ꢀp53 PPI inhibitors are currently being evaluated in early clinical
development11 with daily dosing regimens, and recent clinical data suggest thrombocytopenia
as an onꢀtarget, doseꢀlimiting toxicity for this class of inhibitors.12ꢀ15 Thrombocytopenia could
limit the clinical utility of MDM2ꢀp53 inhibitors, hence there is a need for a less frequently
dosed MDM2 inhibitor to allow clinical management of side effects. Therefore, we started
investigations to identify a highly potent MDM2 inhibitor capable of achieving efficacy with
less frequent dosing in mice.
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Intense research over the past years has yielded several MDM2ꢀp53 PPI inhibitors both from
academia and industry.16ꢀ20 It is interesting to note that many such inhibitors contain structural
architectures akin to natural products displaying dense, spherical shapes with a high number
of stereo centers rather than compounds prepared in a medicinal chemistry laboratory21ꢀ24
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Notably many scaffolds have been generated by multicomponent reactions in the course of
initial hitꢀfinding initiatives.25ꢀ41 Herein we report the discovery of a new class of spiroꢀ
oxindoles as MDM2ꢀp53 PPI inhibitors inspired by Wang´s spiro[3Hꢀindoleꢀ3,3´ꢀpyrrolidin]ꢀ
2(1H)ꢀone inhibitors31, 42 and natural products43, 44 for which the potency and pharmacokinetic
properties could be optimized to show in vivo efficacy when given as a single dose in a two
week mouse SJSAꢀ1 xenograft study.
Spiroꢀoxindoles as MDM2 Inhibitors. Spiroꢀoxindoles are promising scaffolds for
anticancer agents45 and have been identified as inhibitors of the MDM2ꢀp53 interaction by
Wang et al.31, 46 employing a structureꢀbased de novo design. The indole ring of Trp23(p53)
,
which fills a hydrophobic cleft in the binding site (Trp23 pocket) and forms a key hydrogen
bond with the backbone carbonyl of Leu54(MDM2), was replaced by an oxindole. Inspired by
complex natural product architectures like Spirotryprostatin B (Scheme 1) the Wang group
utilized additional valency at the C3 (Scheme 2) of the oxindole to install a spiroꢀring and
consequentially spiro[3Hꢀindoleꢀ3,3´ꢀpyrrolidin]ꢀ2(1H)ꢀones were designed. This scaffold
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offered excellent vectors to address the remaining two lipophilic MDM2 cavities (Leu26(p53)
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and Phe19(p53)ꢀpockets) of the TP53 binding site and led to several MDM2ꢀp53 inhibitors16
such as the SanofiꢀAventis clinical spiroindolinone derivative (2'S,3R,4'S,5'R)6ꢀchloroꢀ4'ꢀ(3ꢀ
chloroꢀ2ꢀfluorophenyl)ꢀ2'ꢀ(2,2ꢀdimethylpropyl)ꢀNꢀ(4ꢀhydroxycyclohexyl)ꢀ2ꢀoxoꢀ1,2ꢀ
dihydrospiro[indoleꢀ3,3'ꢀpyrrolidine]ꢀ5'ꢀcarboxamide (SAR405838)47 (Scheme 2, A).
Additional spiro[3Hꢀindoleꢀ3,3´ꢀpyrrolidin]ꢀ2(1H)ꢀone MDM2ꢀp53 inhibitors have been
reported by academia and industry including azaꢀoxindole derivative (2S,3R,4S,5R)ꢀNꢀ(4ꢀ
carbamoylphenyl)ꢀ6'ꢀchloroꢀ4ꢀ(3ꢀchloroꢀ2ꢀfluorophenyl)ꢀ2ꢀ(2,2ꢀdimethylpropyl)ꢀ2'ꢀoxoꢀ1',2'ꢀ
dihydrospiro[pyrrolidineꢀ3,3'ꢀpyrrolo[3,2ꢀc]pyridine]ꢀ5ꢀcarboxamide (RO2468)48 (Scheme 2,
A) along with other potent preclinical compounds from HoffmannꢀLa Roche48, 49 and the
recently disclosed clinical spiroꢀoxindole derivative (3'R,4'S,5'R)ꢀNꢀ[(6S)ꢀ6ꢀcarbamoyloxanꢀ
3ꢀyl]ꢀ6'ꢀchloroꢀ4'ꢀ(2ꢀchloroꢀ3ꢀfluoropyridinꢀ4ꢀyl)ꢀ4,4ꢀdimethylꢀ2''ꢀoxoꢀ1'',2''ꢀ
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dihydrodispiro[cyclohexaneꢀ1,2'ꢀpyrrolidineꢀ3',3''ꢀindole]ꢀ5'ꢀcarboxamide (DSꢀ3032b)50 with
symmetrical pyrrolidine C2 substitution by Daiichi Sankyo (Scheme 2, A). Additional spiroꢀ
oxindoles lead compounds have been identified by hitꢀfinding initiatives25, 51, by the synthesis
of bioisosters of spiro[3Hꢀindoleꢀ3,3´ꢀpyrrolidin]ꢀ2(1H)ꢀones like spiro(oxindoleꢀ
thiazolidine)ꢀbased derivatives52, 53 or recently published spiro[3Hꢀindoleꢀ3,2´ꢀpyrrolidin]ꢀ
2(1H)ꢀone compounds that interestingly show a very different binding mode compared to the
initial compounds34, 54, and others33, 35, 36, 55
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Spiro[3Hꢀindoleꢀ3,3´ꢀpyrrolidin]ꢀ2(1H)ꢀones with a chiral enter at C2 are associated with
chemical instability since they can undergo equilibration to four diastereomers at C2 and C3
in solution by a reversible ringꢀopening retroꢀMannich reaction under various conditions
(Scheme 2, B) which was first recognized and shown by a Xꢀray coꢀcrystal structure by
Popowicz et al (3LBL)56. The Wang group recently approached this liability by installing
symmetrical pyrrolidine substituents which undergo rapid conversion to one stable
diastereomer.42, 57 Our goal was to solve this issue by identifying a novel and stable spiroꢀ
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oxindole scaffold that is suited for further optimization to finally combine potency, chemical
stability and good PK properties (Scheme 1).
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RESULTS
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Design of the New Scaffold. Based on the binding mode of known spiroꢀoxindole MDM2ꢀ
p53 inhibitors we hypothesized that spiro[3Hꢀindoleꢀ3,2´ꢀpyrrolidin]ꢀ2(1H)ꢀones might bind
similarly but would be chemically stable since the ringꢀopening retroꢀMannich reaction
cannot occur. In addition the new scaffold potentially offers vectors better suited to address
additional interactions to improve binding affinity. We therefore prepared spiroꢀoxindole
compounds 5a and 5b which were assumed to occupy the Trp23 pocket with their 6ꢀchloroꢀ
oxindole (ring “A”) and the Leu26 pocket with a 3ꢀchloroꢀphenyl (ring “B”). The racemic
mixture with the aromatic rings “A” and “B” in “trans” configuration indeed showed the
expected biochemical activities with IC50s (MDM2ꢀp53) of 37 µM for 5a and 40 µM for 5b
which were very comparable to the IC50 (MDM2ꢀp53) of 36 µM measured for the racemic
spiro[3Hꢀindoleꢀ3,3´ꢀpyrrolidin]ꢀ2(1H)ꢀone benchmark fragment 4 (Figure 1). The
contribution of the nitroꢀgroup to the binding of 5a and 5b , which was solely incorporated for
synthetic accessibility reasons through dipolar cycloaddition chemistry (see chemistry section)
was not fully examined, however docking results suggested a polar interaction with His96
(Figure 2A). The reduction of 5b to the primary amine gave racemic 1 which showed slightly
weaker potency [IC50 (MDM2ꢀp53) = 74 µM]. The new spiro[3Hꢀindoleꢀ3,2´ꢀpyrrolidin]ꢀ
2(1H)ꢀone (1) was indeed stable under all synthetic conditions tested including acidic
conditions (see chemistry section) and no epimerization was observed.
Addressing the Phe19 pocket. With a stable scaffold now in hand we then focused on
increasing the binding affinity of our compounds to MDM2. Since no Xꢀray coꢀcrystal
structures of the new weakly potent undecorated compounds could be obtained we started our
optimization based on docking results (e.g. Figure 2A) and biochemical measurements. We
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first evaluated the influence of substituents in R1 position of the pyrrolidine ring (Figure 1) to
evaluate if additional polar interactions (e.g. His73, Tyr67) can be addressed or if potentially
the Phe19 pocket can be occupied by using this vector. Lipophilic substituents like the benzyl
group of rac 5d and others (shown in Table 1) only showed moderate improvement in
potency and therefore the attempt of addressing the Phe19 pocket from this position was not
further pursued. Installing side chains to address polar interactions as well resulted in only a
moderate improvement of the IC50 values by a factor of 2ꢀ3 compared to the unsubstituted
compound rac 5a as shown for the racemic compounds 5gꢀ5k in Table 1. The only exception
was compound rac 5f with a (1Hꢀ1,2,4ꢀtriazolꢀ1ꢀyl)methyl sideꢀchain that led to an
improvement by a factor of seven [IC50 (MDM2ꢀp53 )= 5,502 nM] compared to rac 5a. No
Xꢀray structure of 5f in MDM2 could be obtained to help to understand the improved potency
and we subsequently combined this beneficial R1 side chain with substituents on the
pyrrolidine nitrogen (vide infra).
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In parallel we attempted to address the Phe19 pocket by introducing various substituents to
the pyrrolidine nitrogen (R2 in Table 2). Small and branched alkyl chains like the methylꢀ
cyclopropyl group in 6d [IC50 (MDM2ꢀp53) = 563 nM)] led to a dramatic improvement in
potency and to IC50 values below 1 µM as shown for compounds 6aꢀd. For this series
predominantly compounds with a methyl group in R1 have been prepared due to the better
synthetic accessibility (see chemistry section). The compounds with methyl groups in R1
showed very comparable potency to the corresponding compounds with hydrogen in R1 as
shown for the compound pair 5a and 5b (Table 1). Preparation of additional analogs led to
further improvement of potency by a factor of five especially when a 3ꢀethoxyꢀbenzyl
substituent was introduced as shown for compound 6g. Additionally the introduction of a
fluorine atom in R3 to occupy the Leu26 pocket with the 3ꢀchloroꢀ2ꢀfluorophenyl substituent
gave an additional improvement in potency of a factor of up to four as shown for compound
pair 6d and 6e (Table 2). Chiral separation of the racemic mixtures by chiral SFC showed a
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clear eutomer/distomer behavior and the potency generally improved by a factor of two when
comparing the racemate of a compound to its eutomer. This factor was demonstrated for
racemate rac 6g (IC50 = 57 nM) and the eutomer 6g (IC50 = 34 nM) while the distomer was
150 fold less potent.
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The presumed binding mode from docking was confirmed by Xꢀray crystal structures of 6b
and 6g bound to MDM2 (Figure 2) with the 3ꢀchloroꢀphenyl for 6b (respectively 2ꢀfluoroꢀ3ꢀ
chloro phenyl for 6g) deeply buried in the Leu26 pocket forming a key πꢀπ interaction with
His96, and with the 6ꢀchloroꢀindolinone substituent binding to the Trp23 pocket forming a
crucial hydrogen bond with its nitrogen proton to the backbone carbonyl of Leu54. For
compound 6b the neopentyl group binds to the Phe19 pocket as expected, but strikingly the 3ꢀ
ethoxy benzyl substituent of compound 6g induces a change in the protein conformation
which leads to a larger Phe19 pocket compared to the crystal structure of 6b mainly caused by
the new orientation of the Try67 side chain. This additional hydrophobic cleft was to the best
of our knowledge observed the first time in an Xꢀray coꢀcrystal structure with a spiroꢀ
oxindole compound and is filled ideally by the 3ꢀethoxyꢀphenyl substituent explaining the
boost in potency (Figure 2 C and D).
The installation of a Phe19ꢀpocket substituent to the most potent analog that came out of the
R1 screen (rac 5f, Table 1), carrying a (1Hꢀ1,2,4ꢀtriazolꢀ1ꢀyl)methyl substituent unfortunately
did not show a further improvement in potency as shown in Table 2 (rac 6f). This fact is
interesting since it is indicating that the (1Hꢀ1,2,4ꢀtriazolꢀ1ꢀyl)methyl substituent might also
bind to the Phe19 pocket and could compete with the isopentyl group of rac 6f.
Addressing His96 and Lys94. To further improve potency we aimed to address the side
chain of His96 with a hydrogen bond and therefore installed various groups offering hydrogen
bond acceptors in R4. Neither the introduction of an amide (Table 3), nor other functional
groups led to a significant improvement in potency suggesting that the compounds have the
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wrong conformation to address His96 from R4. Compound 9 with a ꢀCH2CO2H group at R4
led to a moderate improvement in potency with an IC50 (MDM2ꢀp53) of 62 nM compared to
the 157 nM measured for compound 6e with no substituent in R4. Attempts to address Lys94
from this scaffold with benzoic acid analog 2 also did not show significantly improved
potency. We concluded this avenue of optimization assuming that the vectors to address
directed polar interactions were not suitable.
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Precise spatial positioning of atoms and functional groups is crucial for pharmacophore
optimization of small molecule inhibitors and in particular for their directed polar interactions
with proteins. Natural products utilize multiple fused rings not only to precisely position
atoms and functional groups but also to reduce the degrees of freedom and possible
conformations of biologically active molecules. Such complex natural product architectures as
Spirotryprostatin B (Scheme 3) served as an inspiration to our design of a rigidified scaffold.
By linking the free ꢀNH2 with the sidechain at the 2ꢀposition of the pyrrolidine we generated
an octahydroꢀpyrrolo[3,2ꢀb]pyrrole ring system (Scheme 3) which offered vectors better
suited to address His96 and Leu94 with additional substituents.58
The unsubstituted octahydroꢀpyrrolo[3,2ꢀb]pyrrole compound 10 (R4 = H, Table 4) showed
no improvement in potency compared to analogs 6e and 8 respectively. However, attaching a
–CH2CO2H group at R4 already led to a clearly improved IC50 (MDM2ꢀp53) of 39 nM. When
we combined this beneficial substituent with the 3ꢀethoxy phenyl substituent in R2 as a Pheꢀ
19ꢀpocket substituent we observed an additional increase in potency to an IC50 (MDM2ꢀp53)
of 2 nM for the enantiomerically pure compound 13. An Xꢀray crystal structure of desꢀfluoro
analog 14 in MDM2 (Figure 3) showed that the carboxylic acid substituent indeed forms a
hydrogen bond interaction with the sidechain of His96 and Lys94 as intended and structurally
explains the increased potency. Interestingly, the two amino acids His96 and Lys94 both
adopted a new conformation compared to the structures obtained thus far (Figure 2). This new
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protein conformation displayed a suboptimal πꢀπ interaction of the His96 side chain with the
Leuꢀpocket substituent (Figure 3B) and also displayed an energetically unfavorable bent
conformation of the Lys94 side chain. We therefore searched for substituents in position R5
(Table 5) which would address Lys94 in its lowꢀenergy extended conformation (as observed
in most Xꢀray coꢀcrystal structures) and would allow ideal πꢀπ interaction with the His96.
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Compounds with 4ꢀphenylꢀCO2H as R5 displayed a dramatic improvement in potency with an
IC50 (MDM2ꢀp53) of 4 nM for compound 3 (BIꢀ0252) with a cyclopropyl substituent in R2.
Compared to compound 2 we gained a factor of 27 in potency by formally only adding one
bond and thereby locking the molecule in an ideal conformation for the interaction with the
MDM2 protein (Scheme 4). Using the 3ꢀethoxyꢀphenyl substituent to address the Phe19
pocket gave compound 17 with an IC50 (MDM2ꢀp53) of 2 nM which is a factor of two more
potent in this assay suggesting that at this point the assayꢀwall of the biochemical assay was
reached. In the Xꢀray coꢀcrystal structure of 3 in MDM2 (Figure 4) we did not observe any
direct interaction of the benzoic acid substituent and the amino group of Lys94 although the
groups are in close proximity. However, the pyrrolidine NH of 3 indeed forms an Hꢀbond
with the sidechain of His96 in this case with the N1ꢀH tautomeric form of the imidazole as
opposed to the N3ꢀH tautomer observed in all interactions thus far. In addition the important
πꢀπ interaction with His96 now shows an ideal orientation in contrast to compound 14 (Figure
4B).
Cellular Potency and Selectivity. Selected compounds were tested for their cellular potency
in the p53 wild type osteosarcoma SJSAꢀ1 cell line proliferation assay (Table 6). Fully
decorated compounds with biochemical IC50 values below 10 nM showed strong cellular
potency with 17 being the most potent compound of this series with 67 nM in the SJSAꢀ1
assay. To assess the selectivity we tested the same set of compounds against the p53 mutant
cell line SKꢀOVꢀ3. Indicative of truly selective MDM2ꢀp53 PPI inhibitors all compounds
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tested showed no effect on the growth of the p53 mutant SKꢀOVꢀ3 cell line up to a
concentration of 25 µM.
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Pharmacokinetic Properties in Mice of 3, 16 and 17. Selected compounds were tested for
their in vivo pharmacokinetic properties in nonꢀtumorꢀbearing female NMRI nude mice.
Single administration iv and po PK experiments were performed at an iv dose of 5 mg/kg and
a po dose of 50 mg/kg for compounds 3, 16 and 17. Respective iv and po PK profiles are
shown in Figure 5 and the derived mean PK parameters are listed in Table 7.
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While compounds 3 and 16 show low clearance in vivo after iv administration, compound 17
shows a comparably high clearance and a rapid decay in plasma concentration. After po
administration compound 3 shows the highest and compound 17 the lowest AUC value (Table
7). Compound 3 was selected for investigation in xenograft models due to its combined high
po in vivo exposure and good cellular potency.
Single Dose Efficacy of 3 in a SJSAꢀ1 Osteosarcoma Xenograft Model. In vivo efficacy of
compound 3 was tested in the SJSAꢀ1 osteosarcoma xenograft model (Figure 6). Compound 3
was administered orally at a daily dose of 25 mg/kg or at a single dose of 100 mg/kg.
Regressions in all tumors were observed with compound 3 for both dose schedules showing
that efficacy can be achieved with this compound using very different dose schedules. MDM2
inhibitors are expected to lead to TP53 accumulation and eventually induction of TP53 target
genes. To confirm onꢀtarget activity of compound 3 in vivo, a PD biomarker study was
performed in SJSAꢀ1 tumor bearing mice. Tumors were harvested 6 and 24 hours after
administering a single oral dose of 100 mg/kg. Target gene induction was measured using qꢀ
RTꢀPCR or MesoꢀScale assays (Figure 6). Briefly, compound 3 leads to timeꢀdependent
mRNA induction of TP53 target genes including CDKN1a, MDM2 and BBC3 (Figure 6C).
MesoꢀScale assays confirm accumulation of TP53 and MDM2 protein as well as induction of
cleaved CASPASE3, which is a PD biomarker of apoptosis (Figure 6D). In addition,
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histological and immunohistochemical analysis was performed (Figure 7), which confirmed
the findings by qꢀRTꢀPCR and MesoꢀScale analysis. In summary, these results are in line with
the mode of action of a MDM2 inhibitor and demonstrate that compound 3 acts as a MDM2
inhibitor.
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To the best of our knowledge a single dose administration experiment in a mouse SJSAꢀ1
xenograft model with a comparable result has only been reported by Wang et al at a dose of
200 mg/kg and was explained by the induction of apoptosis persisting for 72 hous.47
Thrombocytopenia as an adverse event has been observed in clinical studies with several
MDM2ꢀp53 inhibitors. High grade thrombocytopenia occurred mostly when testing more
continuous dose schedules with these inhibitors in the clinic (e.g. daily dosing for 5ꢀ10, 21 or
28 consecutive days per cycle)13, 59, 60. More recent studies are testing less frequent dose
schedules (e.g. once every 21 days)61 with the intention to manage thrombocytopenia as an
onꢀtarget toxicity and it remains to be shown if this approach is successful. For future studies
we plan to test this working hypothesis.
CHEMISTRY
The spiro[3Hꢀindoleꢀ3,2´ꢀpyrrolidin]ꢀ2(1H)ꢀone core is constructed using a three component
decarboxylative 1,3ꢀdipolar cycloaddition of an isatin, an amino acid, and a nitrostyrene
which generated the core structure with up to four stereocenters in racemic form in only a
single step. The reaction gave moderate to good yields for the desired compounds in many
cases but is strongly dependent on the nature of the amino acid component and produces
regioisomers and diastereomers as byproducts in different ratios which was the subject of
several theoretical62, 63 and experimental studies64ꢀ66. The selectivity favoring the desired
product was completely reversed when glycine (lacking an amino acid sidechain) is used in
the cycloaddition leading to the unwanted regioisomer regioꢀ5a as the major product (48%
isolated yield) while the desired compound 5a was obtained in only 2%. Similar results were
reported for the use of sarcosine as the amino acid component.67 The reaction gave
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significantly better yields for most analogs with larger substituents R1 (R1 ≠ H) as
summarized in Table 8.66 For most examples minor amounts of additional isomers to 5 and
regioꢀ5a have been observed but were not further analyzed.
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Compounds 6aꢀg were prepared by reductive aminations of the cycloaddition products with
the corresponding aldehydes followed by reduction of the nitro groups with In/HCl or Raney
Nickel and H2 in good yields (Scheme 5). The racemates of compounds 6 have been separated
by chiral SFC for compounds 6a, b, d, e, g.
Compound 8 was prepared from amine 6e by amidation with HATU and acetic acid, while
compounds 9 and 2 were prepared by a reductive amination with glyoxylic acid ethylester and
methyl 4ꢀformylbenzoate respectively followed by saponification with NaOH (Scheme 6).
Compound 10 could be prepared from intermediate rac 5m by reductive amination with
cyclopropylcarboxaldehyde and sodium triacetoxyborohydride followed by reduction of the
nitro group with indium powder and HCl and an intramolecular Mitsunobu reaction to
generate the octahydroꢀpyrrolo[3,2ꢀb]pyrrole ring system and chiral separation by SFC.
Analogs 11ꢀ13 were prepared by reduction of compound rac 5 m with Raney Nickel and H2
followed by a Mitsunobu reaction to give the common intermediate rac 18. Selective
acetylation of the sterically less hindered pyrrolidine nitrogen followed by reductive
amination gave compound rac 11. A reaction sequence starting with a selective reductive
amination of rac 18 and glyoxylic acid ethyl ester and subsequent reductive amination of the
remaining secondary amine with cyclopropylcarboxaldehyde or 3ꢀethoxybenzaldehyde and
final saponification delivered analogs rac 12 and 13 respectively after chiral separation
(Scheme 7).
The synthesis of compounds 3 and 16 (Scheme 8) commenced with reductive aminations of
compound rac 5m with cyclopropylcarboxaldehyde or 3ꢀethoxybenzaldehyde respectively
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followed by oxidations with IBX to the corresponding aldehydes 19 and 20. Installation of the
benzoic acid motive was accomplished by iodineꢀmagnesium exchange of 4ꢀiodoꢀbenzoic
acid esters 21 or 22 with iꢀPrMgCl.LiCl and subsequent Grignard addition to aldehydes 19
and 20 to give the corresponding benzylic alcohols as a mixtures of two diasetereomers
favoring the unwanted stereoisomers 23a and 24a (3.5 : 1 for 23a : 23b and 4.5 : 1 for 24a :
24b).68, 69 The diastereomers were separated and oxidation of 23a and 24a to the ketones 25
and 26 followed by reduction with NaBH4 gave mixtures of diastereomers favoring the
desired compounds 23b and 24b (1 : 1.3 for 23a : 23b and 1 : 2.4 for 24a : 24b). Reducing the
nitro groups of 23b with Raney Nickel and H2 and of 24b with indium and HCl, respectively
gave the corresponding primary amines which were used in intramolecular Mitsunobu
reactions to give compounds 14 and 27. Saponification of these analogs delivered compounds
3 and 16. Amidation of 3 with ammonia and HATU gave the primary amide 15.
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DISCUSSION AND CONCLUSIONS
Inspired by Wang´s spiroindolinone inhibitors31, 42 and natural product architectures we were
able to generate highly potent, selective and chemically stable MDM2ꢀp53 PPI inhibitors. The
installation of a multiply fused ring system allowed the precise positioning of functional
groups for optimal hydrogen bond and πꢀπ interactions leading to compounds with the aboveꢀ
mentioned desired properties. As shown for compound 3, tumor regressions in all animals of a
mouse SJSAꢀ1 xenograft study have been achieved ꢀ even with a single dose of compound 3,
with concomitant induction of TP53 target genes and markers of apoptosis.
High grade thrombocytopenia was reported for MDM2ꢀp53 PPI inhibitors as an adverse event
in the clinic in particular when testing continuous dose schedules. For future studies we plan
to test less frequent dose schedules with the intention to manage thrombocytopenia as an onꢀ
target toxicity.
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EXPERIMENTAL SECTION
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Unless otherwise indicated all reactions were carried out in standard commercially available
glassware using standard synthetic chemistry methods. Airꢀsensitive and moistureꢀsensitive
reactions were performed under an atmosphere of dry nitrogen or argon with dried glassware.
Commercial starting materials were used without further purification. Solvents used for
reactions were of commercial “dry”ꢀ or “extraꢀdry” or “analytical” grade. All other solvents
used were reagent grade. Preparative RPꢀHPLC was carried out on Agilent or Gilson systems
using columns from Waters (Sunfire C18 OBD, 5 or 10 µm, 20x50 mm, 30x50 mm or 50x150
mm; XꢀBridge C18 OBD, 5 or 10 µm, 20x50, 30x50, or 50x150 mm) or YMC (Triart C18, 5
or 10 µm, 20x50 mm, or 30x50 mm). Unless otherwise indicated compounds were eluted with
MeCN/water gradients using either acidic (0.2 % HCOOH or TFA) or basic water (5 mL 2 M
NH4HCO3 + 2 mL NH3 (32 %) made up to 1 L with water).
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NMR experiments were recorded on Bruker Avance 400 MHz and 500 MHz spectrometers at
298K. Samples were dissolved in 600µL DMSOꢀd6 and TMS was added as an internal
standard. 1D 1H spectra were acquired with 30° excitation pulses and an interpulse delay of
4.2sec with 64k datapoints and 20ppm sweep width. 1D 13C spectra were acquired with
broadband composite pulse decoupling (WALTZ16) and an interpulse delay of 3.3sec with
64k datapoints and a sweep width of 240ppm. Processing and analysis of 1D spectra was
performed with Bruker Topspin 3.0 software. No zero filling was performed and spectra were
manually integrated after automatic baseline correction.
HPLCꢀMS: All samples were analyzed on an Agilent 1100 series LC system coupled with an
Agilent 6140 mass spectrometer. Purity was determined via UV detection with a bandwidth of
170nm in the range from 230ꢀ400nm. LC parameters were as follows: Waters Xbridge C18
column, 2.5µm particle size, 2.1 x 20mm. Run time 2.1 minutes, flow 1ml/min, column
temperature 60°C and 5µl injections. Solvent A (20mM NH4HCO3/ NH3 pH 9), solvent B
(MS grade acetonitrile). Start 10% B, gradient 10% ꢀ 95% B from 0.0 ꢀ 1.5min, 95% B from
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1.5 ꢀ 2.0min, gradient 95% ꢀ 10% B from 2.0 – 2.1min. HRMS data were recorded using a
Thermo Scientific Orbitrap Elite hybrid ion trap/orbitrap spectrometer system with an
Ultimate 3000 series LPGꢀ3400XRS pump system. Mass calibration was performed using the
Pierce LTQ Velos ESI positive ion calibration solution from Thermo Scientific (lot PF200011,
product no. 88323). All biologically evaluated compounds exist in >95% purity as shown by
LC/MS, additionally for 3, 16 and 17 purity > 95% was shown by QꢀNMR.
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Chiral separation by SFC: The separations were carried out on a JASCO SFCꢀsystem under
isocratic conditions. The system is equipped with a CO2ꢀpump PU2088 and a cosolvent pump
PU2086. The sample is injected by an autosampler AS2059SF, the signal is detected by an
UVꢀdetector UV2075 and the backpressure (150bar) was regulated by the BP2080. The
fractions were collected by a fractionꢀcollector FC2088ꢀ08. Mobile phase was CO2 (Air
Liquide, Schwechat) and methanol (Merck, Darmstadt). Chromatographic separation was
obtained by isocratic conditions using several chiral columns (250x20mm; 5µm) from Daicel
(Strassbourg, Europe) at 40°C a flow of 50 mL/min. Inside the chiral columns are chiral
resolving agents based on cellulose or amylose. Docking Studies: GOLD Suite v 5.4 from
CCDC Software Limited was used for the docking of compound 5B (Figure 2). Hydrogen
bonding to the backbone carbonyl oxygen of Leucine 54 was defined as the constraints during
the docking process. Ligands were prepared using CORINA prior to the docking.
Selected Experimental Procedures
Synthesis of Compound 3:
4-[(3S,3'S,3'aS,5'R,6'aS)-6-chloro-3'-(3-chloro-2-fluorophenyl)-1'-(cyclopropylmethyl)-2-oxo-
1,2,3',3'a,4',5',6',6'a-octahydro-1'H-spiro[indole-3,2'-pyrrolo[3,2-b]pyrrole]-5'-yl]benzoic
acid (3)
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To a solution of compound 14 (150 mg, 0.258 mmol) in THF (5 mL) was added NaOH (97
ꢁL, 8 M in H2O) and the reaction mixture was stirred at 60 °C for 18 h. The reaction mixture
was concentrated in vacuo and the crude product was purified by preparative RP HPLC
(Gilson, gradient 10ꢀ55% MeCN in H2O) to give compound 3. 4ꢀ[(3S,3'S,3'aS,5'R,6'aS)ꢀ6ꢀ
chloroꢀ3'ꢀ(3ꢀchloroꢀ2ꢀfluorophenyl)ꢀ1'ꢀ(cyclopropylmethyl)ꢀ2ꢀoxoꢀ1,2,3',3'a,4',5',6',6'aꢀ
octahydroꢀ1'Hꢀspiro[indoleꢀ3,2'ꢀpyrrolo[3,2ꢀb]pyrrole]ꢀ5'ꢀyl]benzoic acid (3) (131 mg, 0.231
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1
mmol, 90%). H NMR (500 MHz, DMSOꢀd6) δ ppm ꢀ0.16 (dq, J=9.46, 4.73 Hz, 1 H) 0.00
(dq, J=9.46, 4.73 Hz, 1 H) 0.23 (tt, J=8.75, 4.65 Hz, 1 H) 0.36 (tt, J=8.59, 4.65 Hz, 1 H) 0.61
ꢀ 0.71 (m, 1 H) 1.55 ꢀ 1.64 (m, 1 H) 2.03 (dd, J=12.61, 7.88 Hz, 1 H) 2.21 (dd, J=12.45, 5.83
Hz, 1 H) 2.37 (dd, J=12.29, 5.36 Hz, 1 H) 3.89 (d, J=9.14 Hz, 1 H) 4.02 (t, J=7.41 Hz, 1 H)
4.52 (dd, J=10.88, 5.20 Hz, 1 H) 4.66 (t, J=8.35 Hz, 1 H) 6.55 (d, J=1.89 Hz, 1 H) 7.04 (dd,
J=7.88, 1.89 Hz, 1 H) 7.13 (t, J=8.04 Hz, 1 H) 7.31 ꢀ 7.36 (m, 1 H) 7.43 ꢀ 7.47 (m, 2 H) 7.49
(d, J=8.20 Hz, 2 H) 7.84 (d, J=8.20 Hz, 2 H) 10.21 (br s, 1 H); 13C NMR (126 MHz, DMSOꢀ
d6) δ ppm 3.92, 5.99, 11.41, 45.03, 49.73, 55.59, 59.62, 66.61, 67.85, 78.10, 109.73, 119.73 (d,
JC-F=19.98 Hz), 122.19, 125.41 (d, JC-F=4.54 Hz), 126.02 (d, JC-F=13.62 Hz), 126.13, 127.37,
127.39, 128.77, 129.65, 129.70, 133.58, 144.00, 156.69 (d, JC-F=247.04 Hz) 178.33; HRMS
(ESI+): calculated for C30H26Cl2FN3O3 [M+H]+ 566.14080, found 566.14246, ꢂ 2.93 ppm;
LC/MS: [M+H]+ = 566; tR = 1.06 min.
Rac-(3S,3'S,4'S,5'S)-6-chloro-3'-(3-chloro-2-fluorophenyl)-5'-(2-hydroxyethyl)-4'-nitro-1,2-
dihydrospiro[indole-3,2'-pyrrolidine]-2-one (rac 5m)
6ꢀChloroisatin (8.0 g, 44.05 mmol), 1ꢀ(2ꢀfluroꢀ3ꢀchlorophenyl)ꢀ2ꢀnitroethene (8.9 g,
44.05 mmol) and Lꢀhomoserin (5.24 g, 44.05 mmol) were stirred under reflux in MeOH (10
mL) for 18 h. The reaction mixture was concentrated in vacuo and the residue titurated in
acetonitrile. The solid was filtered and dried to give rac 5m which was used in the next step
without further purification. Racꢀ(3S,3'S,4'S,5'S)ꢀ6ꢀchloroꢀ3'ꢀ(3ꢀchloroꢀ2ꢀfluorophenyl)ꢀ5'ꢀ(2ꢀ
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hydroxyethyl)ꢀ4'ꢀnitroꢀ1,2ꢀdihydrospiro[indoleꢀ3,2'ꢀpyrrolidine]ꢀ2ꢀone (rac 5m) (8.9 g, 20.2
mmol, 46%). 1H NMR (500 MHz, DMSOꢀd6) δ ppm 1.49 ꢀ 1.63 (m, 2 H) 3.44 ꢀ 3.52 (m, 1 H)
3.52 ꢀ 3.61 (m, 1 H) 4.09 (d, J=7.25 Hz, 1 H) 4.52 ꢀ 4.63 (m, 2 H) 4.74 (d, J=8.51 Hz, 1 H)
6.06 (t, J=8.51 Hz, 1 H) 6.66 (s, 1 H) 7.11 (dd, J=7.88, 0.95 Hz, 1 H) 7.17 (t, J=7.88 Hz, 1 H)
7.44 (br t, J=6.94 Hz, 2 H) 7.56 (d, J=7.88 Hz, 1 H) 10.32 (s, 1 H); LC/MS: [M+H]+ = 440; tR
= 1.21 min.
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Methyl 4-[(3S,3'S,3'aS,5'R,6'aS)-6-chloro-3'-(3-chloro-2-fluorophenyl)-1'-(cyclopropylmethyl)
-2-oxo-1,2,3',3'a,4',5',6',6'a-octahydro-1'H-spiro[indole-3,2'-pyrrolo[3,2-b]pyrrole]-5'-
yl]benzoate (14)
A solution of intermediate 23b (830 mg, 1,321 mmol) in MeOH (10 mL) and DCM (8 mL)
was stirred in Büchi laboratory autoclave with Raney Ni (78 mg) under a H2 pressure of 8 bar
for 18 h at room temperature. The Raney Ni was filtered of carefully and the solvent removed
in vacuo. The crude product was purified by preparative RP HPLC (Gilson, 40ꢀ85 % MeCN
in H2O) to give methyl 4ꢀ[(1R)ꢀ2ꢀ[(3S,3'S,4'S,5'S)ꢀ4'ꢀaminoꢀ6ꢀchloroꢀ3'ꢀ(3ꢀchloroꢀ2ꢀ
fluorophenyl)ꢀ1'ꢀ(cyclopropylmethyl)ꢀ2ꢀoxoꢀ1,2ꢀdihydrospiro[indoleꢀ3,2'ꢀpyrrolidine]ꢀ5'ꢀyl]ꢀ
1ꢀhydroxyethyl]benzoate (593 mg, 0.990 mmol, 75%). 1H NMR (500 MHz, DMSOꢀd6) δ ppm
ꢀ0.28 ꢀ ꢀ0.21 (m, 1 H) ꢀ0.04 ꢀ 0.05 (m, 1 H) 0.08 ꢀ 0.17 (m, 1 H) 0.25 ꢀ 0.32 (m, 1 H) 0.45 ꢀ
0.53 (m, 1 H) 1.99 (br d, J=14.50 Hz, 1 H) 2.14 ꢀ 2.19 (m, 1 H) 2.20 ꢀ 2.24 (m, 1 H) 2.25 ꢀ
2.29 (m, 1 H) 2.25 ꢀ 2.29 (m, 1 H) 2.35 (br d, J=8.51 Hz, 1 H) 2.46 ꢀ 2.48 (m, 1 H) 3.58 (br t,
J=8.35 Hz, 1 H) 3.75 (d, J=11.35 Hz, 1 H) 3.79 (s, 2 H) 4.52 ꢀ 4.60 (m, 1 H) 4.80 (dd,
J=10.72, 2.52 Hz, 1 H) 6.49 (d, J=1.89 Hz, 1 H) 6.98 (dd, J=7.88, 1.89 Hz, 1 H) 7.11 (t,
J=7.88 Hz, 1 H) 7.21 (d, J=7.88 Hz, 1 H) 7.30 ꢀ 7.37 (m, 2 H) 7.49 (d, J=8.51 Hz, 1 H) 7.47 ꢀ
7.53 (m, 1 H) 7.88 (d, J=8.20 Hz, 1 H) 7.86 ꢀ 7.89 (m, 1 H) 7.87 ꢀ 7.90 (m, 1 H) 10.09 (s, 1 H).
LC/MS: [M+H]+ = 598; tR = 1.48 min. To a solution of methyl 4ꢀ[(1R)ꢀ2ꢀ[(3S,3'S,4'S,5'S)ꢀ4'ꢀ
aminoꢀ6ꢀchloroꢀ3'ꢀ(3ꢀchloroꢀ2ꢀfluorophenyl)ꢀ1'ꢀ(cyclopropylmethyl)ꢀ2ꢀoxoꢀ1,2ꢀ
dihydrospiro[indoleꢀ3,2'ꢀpyrrolidine]ꢀ5'ꢀyl]ꢀ1ꢀhydroxyethyl]benzoate (213 mg, 0.356 mmol)
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in DCM (4 mL) was added triphenylphosphine (102 mg, 0.39 mmol) and DEAD (195 µL,
0.43 mmol, 40 % in toluene) and the reaction mixture was stirred for 18 h at rt. Saturated
aqueous NaHCO3 solution was added to the reaction mixture and the mixture was extracted
with DCM. The combined organic layer was dried (MgSO4), filtered, concentrated in vacuo
and the crude product was purified by preparative RP HPLC (Gilson, gradient 50ꢀ90% MeCN
in H2O) to give methyl 4ꢀ[(3S,3'S,3'aS,5'R,6'aS)ꢀ6ꢀchloroꢀ3'ꢀ(3ꢀchloroꢀ2ꢀfluorophenyl)ꢀ1'ꢀ
(cyclopropylmethyl)ꢀ2ꢀoxoꢀ1,2,3',3'a,4',5',6',6'aꢀoctahydroꢀ1'Hꢀspiro[indoleꢀ3,2'ꢀpyrrolo[3,2ꢀ
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b]pyrrole]ꢀ5'ꢀyl]benzoate (14) (55 mg, 0.267 mmol, 75%). H NMR (500 MHz, DMSOꢀd6) δ
ppm ꢀ0.16 (dq, J=9.46, 4.73 Hz, 1 H) 0.01 (dq, J=9.46, 4.73 Hz, 1 H) 0.23 (tt, J=8.75, 4.65 Hz,
1 H) 0.32 ꢀ 0.40 (m, 1 H) 0.61 ꢀ 0.70 (m, 1 H) 1.59 (td, J=11.74, 6.78 Hz, 1 H) 2.03 (dd,
J=12.45, 7.72 Hz, 1 H) 2.21 (dd, J=12.45, 5.83 Hz, 1 H) 2.37 (dd, J=12.29, 5.36 Hz, 1 H)
2.92 (br dd, J=8.98, 3.94 Hz, 1 H) 3.80 (s, 3 H) 3.89 (d, J=9.14 Hz, 1 H) 4.02 (t, J=7.41 Hz, 1
H) 4.50 ꢀ 4.58 (m, 1 H) 4.66 (td, J=8.28, 3.63 Hz, 1 H) 6.54 (d, J=1.89 Hz, 1 H) 7.04 (dd,
J=7.88, 1.89 Hz, 1 H) 7.13 (s, 1 H) 7.31 ꢀ 7.37 (m, 1 H) 7.41 ꢀ 7.48 (m, 2 H) 7.54 (d, J=8.51
Hz, 2 H) 7.87 (d, J=8.51 Hz, 2 H) 10.20 (s, 1 H); LC/MS: [M+H]+ = 580; tR = 1.59 min.
2-[(3S,3'S,4'S,5'S)-6-chloro-3'-(3-chloro-2-fluorophenyl)-1'-(cyclopropylmethyl)-4'-nitro-2-
oxo-1,2-dihydrospiro[indole-3,2'-pyrrolidine]-5'-yl]acetaldehyde (19)
To a solution of cyclopropylcarboxaldehyde (508 µL, 6.81 mmol) in DMF (2 mL) was added
intermediate rac 5m (1.0 g, 2.27 mmol) and AcOH (2.6 mL, 45.43 mmol) and the reaction
mixture was stirred for 15 min at room temperature. Sodium triacetoxyborohydride (1.52 g,
6.81 mmol) was added and the reaction mixture was stirred for 18 h. Water was added to the
reaction mixture and it was extracted with EtOAc. The combined organic layer was dried
(MgSO4), filtered, concentrated in vacuo to give racꢀ(3S,3'S,4'S,5'S)ꢀ6ꢀchloroꢀ3'ꢀ(3ꢀchloroꢀ2ꢀ
fluorophenyl)ꢀ1'ꢀ(cyclopropylmethyl)ꢀ5'ꢀ(2ꢀhydroxyethyl)ꢀ4'ꢀnitroꢀ1,2ꢀdihydrospiro[indoleꢀ
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3,2'‐pyrrolidine]ꢀ2ꢀone (1.03 g, 2.078 mmol, 92%) which was separated by chiral SFC to
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(3S,3'S,4'S,5'S)ꢀ6ꢀchloroꢀ3'ꢀ(3ꢀchloroꢀ2ꢀfluorophenyl)ꢀ1'ꢀ(cyclopropylmethyl)ꢀ5'ꢀ(2ꢀ
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hydroxyethyl)ꢀ4'ꢀnitroꢀ1,2ꢀdihydrospiro[indoleꢀ3,2'‐pyrrolidine]ꢀ2ꢀone (>95% ee). H NMR
(400 MHz, DMSOꢀd6) δ ppm ꢀ0.20 (dt, J=9.57, 4.72 Hz, 1 H) 0.01 (dq, J=9.44, 4.88 Hz, 1 H)
0.09 ꢀ 0.18 (m, 1 H) 0.23 ꢀ 0.33 (m, 1 H) 0.45 ꢀ 0.57 (m, 1 H) 1.77 (dq, J=13.91, 6.69 Hz, 1 H)
1.92 ꢀ 2.02 (m, 1 H) 2.31 (dd, J=13.69, 7.10 Hz, 1 H) 2.58 ꢀ 2.64 (m, 1 H) 3.54 ꢀ 3.68 (m, 2 H)
4.21 (ddd, J=9.50, 6.84, 4.94 Hz, 1 H) 4.71 (t, J=4.94 Hz, 1 H) 4.78 (d, J=11.41 Hz, 1 H) 6.42
(dd, J=11.28, 9.76 Hz, 1 H) 6.69 (d, J=1.77 Hz, 1 H) 7.19 (dd, J=7.98, 1.90 Hz, 1 H) 7.23 (t,
J=7.98 Hz, 1 H) 7.43 ꢀ 7.49 (m, 1 H) 7.51 ꢀ 7.56 (m, 1 H) 7.56 ꢀ 7.60 (m, 1 H) 7.57 (d, J=8.11
Hz, 1 H) 10.57 (s, 1 H); LC/MS: [M+H]+ = 494; tR = 1.39 min. To a solution of
(3S,3'S,4'S,5'S)ꢀ6ꢀchloroꢀ3'ꢀ(3ꢀchloroꢀ2ꢀfluorophenyl)ꢀ1'ꢀ(cyclopropylmethyl)ꢀ5'ꢀ(2ꢀ
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hydroxyethyl)ꢀ4'ꢀnitroꢀ1,2ꢀdihydrospiro[indoleꢀ3,2'‐pyrrolidine]ꢀ2ꢀone (1.00 g, 2.02 mmol) in
CH3CN (13 mL) was added IBX (680 mg, 2.43 mmol) and the reaction mixture was stirred
for 60 min at 75 °C. The reaction mixture was cooled to 0 °C and kept at this temperature for
1 h without stirring before it was filtered through Celite® and concentrated in vacuo. The
crude product was used without further purification for the next step. 2ꢀ[(3S,3'S,4'S,5'S)ꢀ6ꢀ
chloroꢀ3'ꢀ(3ꢀchloroꢀ2ꢀfluorophenyl)ꢀ1'ꢀ(cyclopropylmethyl)ꢀ4'ꢀnitroꢀ2ꢀoxoꢀ1,2ꢀ
1
dihydrospiro[indoleꢀ3,2'ꢀpyrrolidine]ꢀ5'ꢀyl]acetaldehyde (19) (926 mg, 1.88 mmol, 93%). H
NMR (400 MHz, DMSOꢀd6) δ ppm ꢀ0.01 (dq, J=9.38, 4.73 Hz, 1 H) 0.18 (dq, J=9.38, 4.73
Hz, 1 H) 0.34 ꢀ 0.42 (m, 1 H) 0.43 ꢀ 0.54 (m, 1 H) 0.65 ꢀ 0.77 (m, 1 H) 2.38 ꢀ 2.48 (m, 1 H)
2.52 ꢀ 2.60 (m, 1 H) 3.08 ꢀ 3.17 (m, 1 H) 3.19 ꢀ 3.28 (m, 1 H) 4.83 (td, J=8.93, 3.17 Hz, 1 H)
4.98 (d, J=11.15 Hz, 1 H) 6.58 (t, J=10.27 Hz, 1 H) 6.85 (d, J=1.77 Hz, 1 H) 7.34 (dd, J=8.11,
1.77 Hz, 1 H) 7.38 (t, J=8.11 Hz, 1 H) 7.62 (br t, J=7.22 Hz, 1 H) 7.67 (br t, J=7.48 Hz, 1 H)
7.75 (d, J=7.86 Hz, 1 H) 9.94 (s, 1 H) 10.75 (s, 1 H); LC/MS: [M+H]+ = 492; tR = 1.44 min.
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Methyl 4-[(1R)-2-[(3S,3'S,4'S,5'S)-6-chloro-3'-(3-chloro-2-fluorophenyl)-1'-
(cyclopropylmethyl)-4'-nitro-2-oxo-1,2-dihydrospiro[indole-3,2'-pyrrolidine]-5'-yl]-1-
hydroxyethyl]benzoate (23b)
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To a solution of methyl 4ꢀiodobenzoate 21 (3.85 g, 14.381 mmol) in dry THF (15 mL) under
argon atmosphere at ꢀ 40 °C was added isopropylmagnesium chloride lithium chloride
complex (11.1 mL, 14.38 mmol, 1.3 M in THF) dropwise and the reaction mixture was stirred
for 1 h. A solution of aldehyde 19 (2.36 mg, 4.79 mmol) in THF (40 mL) was added slowly at
ꢀ 40 °C and the reaction mixture was stirred for 1.5 h and was slowly warmed to rt overnight.
Water and Et2O were added to the reaction mixture and the phases were separated. The
aqueous phase was extracted with Et2O and the combined organic layer was dried (MgSO4),
filtered, concentrated in vacuo. The crude product was purified by normal phase MPLC
chromatography (10ꢀ22 % EtOAc in cyclohexane) to give compounds 23a and 23b. Methyl 4ꢀ
[(1S)ꢀ2ꢀ[(3S,3'S,4'S,5'S)ꢀ6ꢀchloroꢀ3'ꢀ(3ꢀchloroꢀ2ꢀfluorophenyl)ꢀ1'ꢀ(cyclopropylmethyl)ꢀ4'ꢀ
nitroꢀ2ꢀoxoꢀ1,2ꢀdihydrospiro[indoleꢀ3,2'ꢀpyrrolidine]ꢀ5'ꢀyl]ꢀ1ꢀhydroxyethyl]benzoate
(23a)
(1.92 g, 3,055 mmol, 64%). 1H NMR (500 MHz, DMSOꢀd6) δ ppm ꢀ0.19 (dq, J=9.42, 4.74 Hz,
1 H) ꢀ0.02 ꢀ 0.11 (m, 2 H) 0.17 ꢀ 0.26 (m, 1 H) 0.40 ꢀ 0.49 (m, 1 H) 1.73 ꢀ 1.82 (m, 1 H) 2.09 ꢀ
2.17 (m, 1 H) 2.51 (dd, J=13.87, 7.25 Hz, 1 H) 2.85 (dd, J=13.56, 5.99 Hz, 1 H) 3.96 (s, 3 H)
4.22 (td, J=8.51, 5.04 Hz, 1 H) 4.85 (d, J=11.66 Hz, 1 H) 4.93 ꢀ 5.01 (m, 1 H) 5.80 (d, J=4.73
Hz, 1 H) 6.53 (dd, J=11.66, 9.46 Hz, 1 H) 6.72 (d, J=1.89 Hz, 1 H) 7.23 ꢀ 7.31 (m, 2 H) 7.42 ꢀ
7.49 (m, 1 H) 7.54 ꢀ 7.60 (m, 1 H) 7.66 (d, J=8.20 Hz, 2 H) 7.75 (d, J=7.88 Hz, 1 H) 8.08 (d,
J=8.20 Hz, 2 H) 10.59 (br s, 1 H); LC/MS: [M+H]+ = 628; tR = 1.56 min. Methyl 4ꢀ[(1R)ꢀ2ꢀ
[(3S,3'S,4'S,5'S)ꢀ6ꢀchloroꢀ3'ꢀ(3ꢀchloroꢀ2ꢀfluorophenyl)ꢀ1'ꢀ(cyclopropylmethyl)ꢀ4'ꢀnitroꢀ2ꢀoxoꢀ
1,2ꢀdihydrospiro[indoleꢀ3,2'ꢀpyrrolidine]ꢀ5'ꢀyl]ꢀ1ꢀhydroxyethyl]benzoate (23b) (532 mg,
1
0.846 mmol, 18%). H NMR (500 MHz, DMSOꢀd6) δ ppm ꢀ0.20 (dq, J=9.69, 4.76 Hz, 1 H)
0.01 (dq, J=9.62, 4.78 Hz, 1 H) 0.14 ꢀ 0.23 (m, 1 H) 0.27 ꢀ 0.36 (m, 1 H) 0.55 ꢀ 0.64 (m, 1 H)
1.91 (br dd, J=14.82, 9.46 Hz, 1 H) 2.09 ꢀ 2.22 (m, 2 H) 2.55 (d, J=1.89 Hz, 1 H) 3.88 (s, 3 H)
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4.45 (t, J=8.98 Hz, 1 H) 4.67 (br dd, J=10.09, 4.10 Hz, 1 H) 4.87 (d, J=11.03 Hz, 1 H) 5.76 (d,
J=4.10 Hz, 1 H) 6.44 ꢀ 6.50 (m, 1 H) 6.67 (d, J=1.89 Hz, 1 H) 7.14 (dd, J=8.20, 1.89 Hz, 1 H)
7.22 (t, J=7.88 Hz, 1 H) 7.42 (d, J=8.20 Hz, 2 H) 7.46 (t, J=6.78 Hz, 1 H) 7.48 ꢀ 7.53 (m, 1 H)
7.55 (d, J=8.20 Hz, 1 H) 7.98 (d, J=8.20 Hz, 2 H) 10.52 (br s, 1 H); LC/MS: [M+H]+ = 628;
tR = 1.61 min. To a solution of 23a (1.92 g, 3.06 mmol) in a MeCN (15 mL) was added IBX
(984 mg, 3.51 mmol) and the reaction mixture was stirred for 2 hours at 75 °C. The reaction
mixture was cooled to 0 °C and kept at this temperature for one hour without stirring before it
was filtered through Celite® and concentrated in vacuo. The crude ketone 25 (1.76 g, 2.80
mmol, 92%) was used without further purification for the next step. To a solution of 25 (1.76
g, 2.80 mmol) in MeOH (10 mL) was slowly added sodium borohydride (106 mg, 2.80 mmol)
at 0 °C and the reaction mixture was stirred at the same temperature for 6 h. The reaction
mixture was quenched with water and saturated aqueous NaHCO3 solution was added to the
mixture. The aqueous phase was extracted with EtOAc and the combined organic layer was
dried (MgSO4), filtered, concentrated in vacuo .The crude product was purified by preparative
RP HPLC (Gilson, gradient 50ꢀ90% MeCN in H2O) to give compounds 23a (630 mg, 1.002
mmol, 36%) and 23b. 831 mg (1.322 mmol, 47%)
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ASSOCIATED CONTENT
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Supporting Information.
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Synthesis procedures; method description for biochemical and cell proliferation assays;
method description for in vivo studies and biomarker studies; Histopathological analyses and
immunohistochemistry; NMR spectra; crystallographic data collection and refinement
statistics (PDF); molecular formula string (CSV). This material is available free of charge on
the ACS Publications website at http://pubs.acs.org.
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Accession Codes
The atomic coordinates and structure factors have been deposited in the RCSB Protein Data
Bank (compound 6b: PDB code 5LAV; compound 6g: PDB code 5LAY; compound 14: PDB
code 5LAW; and compound BIꢀ0252 (3): PDB code 5LAZ). Authors will release the atomic
coordinates and experimental data upon article publication.
AUTHOR INFORMATION
* corresponding author, email: andreas.gollner@boehringerꢀingelheim.com
Phone: +43 (1) 80 105ꢀ2596
Notes
The authors declare the following competing financial interest: all authors were full
Boehringer Ingelheim fullꢀtime employees at the time when this study was performed.
BoehringerꢀIngelheim is grateful for financial support by the Austrian Research Promotion
Agency FFG (grant: 832260, 837815 and 842856).
AUTHOR CONTRIBUTION
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A.G. supervised the medicinal chemistry team, designed the compounds and synthetic
strategies. A.G. and D.R. wrote the manuscript. G.D., P.E., A.G., C.K., J.Ra., H.W. T.W.
contributed to the medicinal chemistry strategy and compound design. D.R. supervised the
biology team and designed and supervised the animal experiments. G.B., D.R., R.S., U.W.ꢀC.,
S.M.B. designed and supervised biological experiments, A.S. performed histopathological
analyses and IHC. D.K. analysed the crystal structures. XꢀL.C. performed the docking studies.
T.G. supervised the biochemical Alpha assays and cell proliferation assays. M.B. and J.K.ꢀOe.
synthesized the compounds and developed the chemistry routes. J.Ri. supervised PK studies
and wrote the PK section. H.A. supervised the HRMS measurements and chemical stability
studies, D.B.McC. was responsible for the medicinal chemistry strategy and reviewed the
manuscript.
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ACKNOWLEDGMENTS
The authors thank Christoph Albrecht, Christian Aumeyer, Gerd Bader, Alexandra Beran,
Helmut Berger, Christoph Burdack, Tuncay Ciftci, Ida Dinold, Nora Dürkop, Norbert Eidkum,
Wolfgang Egermann, Gerlinde Flotzinger, Geraldine Garavel, Gerhard Gmaschitz, Gregor
Grotemeier, Eric Haaksma, Petra Handler, Daniela Härnig, Wolfgang Hela, Cederic Kalinski,
Matthew Kennedy, Matthias Klemencic, Michael Kulhanek, Roland Kousek, Norbert Kraut,
Moriz Mayer, Katharina Mayr, Sabine Olt, Christina Puri, Alexander Wlachos, Christina Puri,
Christoph Peinsipp, Oliver Petermann, Jens Quant, Carlos Roberto RamirezꢀSanta Cruz,
Maria Rieger, Günther Ross, Christian Salamon, Otmar Schaaf, Maike Scheller, Yvonne
Scherbatin, Andreas Schrenk, Norbert Schweifer, Gorana Sijan, David Sill, Steffen Steurer,
Michaela Streicher, Harald Studensky, Steffen Weik, Irene Weiner, Anika Weiss, Andreas
Wernitznig, Alexander Wlachos, Susanne WollnerꢀGaida, Markus Zeeb, and Andreas Zöphel
ABBREVIATIONS USED
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BBC3, BCL2 binding component 3; BCL2, Bꢀcell lymphoma 2, CL, clearance; cꢀPr,
cyclopropyl; CDKN1a, CyclinꢀDependent Kinase Inhibitor 1A; DEAD, diethyl
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azodicarboxylate; DIPEA, N,Nꢀdiisopropylethylamine; HATU, Oꢀ(7ꢀAzabenzotriazolꢀ1ꢀyl)ꢀ
N,N,N′,N′ꢀtetramethyluroniumꢀhexafluorphosphat; HCl, hydrochloric acid; HDM2, human
analogue of MDM2; HPꢀβꢀCD , 2ꢀhydroxypropylꢀβꢀcyclodextin; IBX, 2ꢀiodoxybenzoic acid;
In, indium; MDM2, murine double minute 2, MeCN, acetonitrile; NaOH, sodium hydroxide;
PhMe, toluene; PPI, proteinꢀprotein interaction; rac, racemic; RPꢀHPLC, reversed phase highꢀ
performance liquid chromatography; Vss, volume of distribution steady state.
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6b: PDB code 5LAV; compound 6g: PDB code 5LAY
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compound 14: PDB code 5LAW; and
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compound BIꢀ0252 (3): PDB code 5LAZ). Authors will release the atomic coordinates and
experimental data upon article publication.
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