Asymmetric Synthesis of 3,3′-Piperidinoyl Spirooxindoles and Discovery of Stereospecific Cycloadducts as Novel Hedgehog Pathway Modulators

Article abstract of DOI:10.1055/s-0040-1707222

An enantioselective hetero-Diels-Alder reaction of alkylidene oxindoles and 2-aza-3-silyloxy-1,3-butadienes, catalyzed by divalent transition metal complexes with N, N ′-dioxide ligands offered an efficient access to natural-product-based 3,3′-piperidinoyl spirooxindole class of small molecules. exo -Cycloadducts formed via stereospecific cycloaddition with Z -olefin displayed potent activity in modulation of hedgehog pathway.

Full text of DOI:10.1055/s-0040-1707222

SYNTHESIS
0
0
3
9
-
7
8
8
1
1
4
3
7
-
2
1
0
X
© Georg Thieme Verlag Stuttgart · New York
2020, 52, A–M
feature
A
en
M. Rehan et al.
Feature
Synthesis
Asymmetric Synthesis of 3,3′-Piperidinoyl Spirooxindoles and
Discovery of Stereospecific Cycloadducts as Novel Hedgehog
Pathway Modulators
Mohammad Rehana
Jana Flegela,b
Ar2
O
O
R
33 examples
10 mol% Mg(NTf2)2
R
R
TBSO
O
Ar2
Ar2
NH
Ar1
NH
Ar1
11 mol% of 3a
Franziska Heitkampa,b
Jorgelina L. Pergometa
Felix Otteb
Carsten Strohmannb
Kamal Kumar*a,1
N
DCM, 0 °C to rt, 6 h
N
O
O
Ar1
R1
N
N
R1
R1
N
from E-isomer
N
from Z-isomer
O
O
H
yield up to 94%
ee up to 99%
O
iPr
potent modulators of
Hedgehog pathway
O
H
N
N
iPr
iPr
iPr
a Max-Planck-Institute für Molekulare Physiologie, Abteilung Che-
mische Biologie, Otto-Hahn-Straße 11, 44227 Dortmund, Germany
b Fakultät Chemie und Chemische Biologie, Technische Universität
Dortmund, Otto-Hahn-Straße 6, 44227 Dortmund, Germany
Kamal.Kumar@mpi-dortmund.mpg.de
In memory of Prof. Dr. Rolf Huisgen
Received: 12.05.2020
Accepted after revision: 18.06.2020
Published online: 10.08.2020
One of our research programs aimed to develop natural-
product-inspired small molecule collections7 was based on
the core-frameworks of cytotoxic natural products. The
goal was to explore the chemical fingerprint of these bioac-
tive natural products and to develop small molecules that
may offer intriguing modulation of biological functions,
which can be used in drug and probe discovery research. An
interesting subgroup of this class is the piperidinoyl spiroo-
xindole, which consists of up to four consecutive chiral cen-
ters and up to three all-carbon-quaternary centers (Figure
1).8 Piperidinoyl spirooxindole scaffolds exist in biologically
active molecules of natural and synthetic origin, for exam-
ple, in the highly cytotoxic surugatoxin 1 (i) which blocks
the nicotinic acetylcholine receptor or spiroquinazoline
natural product lapatin B (ii).9 Synthetic derivatives of 3,3′-
piperidinoyl spirooxindole appear to be promising antican-
cer agents iii (Figure 1). They inhibit the protein-protein in-
teraction between the tumor suppressor p53 and MDM2.10
However, their limited synthetic exploration did not permit
further exploration of their potential in modulating other
biological functions. Here we report on the asymmetric
synthesis of 3,3′-piperidinoyl spirooxindoles vi via hetero-
Diels–Alder reaction of azadienes iv with alkylidene oxin-
doles v and discovery of these spirocyclic small molecules
as a novel class of modulators of hedgehog signaling path-
way.
DOI: 10.1055/s-0040-1707222; Art ID: ss-2020-z0257-fa
Abstract An enantioselective hetero-Diels–Alder reaction of
alkylidene oxindoles and 2-aza-3-silyloxy-1,3-butadienes, catalyzed by
divalent transition metal complexes with N,N′-dioxide ligands offered
an efficient access to natural-product-based 3,3′-piperidinoyl spiroox-
indole class of small molecules. exo-Cycloadducts formed via stereospe-
cific cycloaddition with Z-olefin displayed potent activity in modulation
of hedgehog pathway.
Key words asymmetric synthesis, hetero-Diels–Alder reaction, spiro-
oxindoles, natural products, hedgehog inhibitors
Small molecules remain the first choice for develop-
ment as drug candidates for various indications despite the
huge strides made by biologics in recent years.2 For in-
stance, in 2019, more than 60% of new molecular entities
approved by the United States Food and Drug Administra-
tion were small molecules.3 Natural products had always
been an important driving force and source of new drug
molecules, especially as anticancer and antimicrobial small
molecules.4 Until 2014, out of 175 anticancer drugs ap-
proved, 85 were either natural products or derived from
them.4a In recent years, small molecules have also been in-
creasingly used as probe molecules to shed light into vari-
ous biological processes to understand the cause, develop-
ment as well as therapeutic solution to various disease
states.5 Thus, discovery of novel biological functions of nat-
ural-product-based small molecules remains one of the
core-interests in medicinal chemistry.6
Hetero-Diels–Alder (HDA) reaction is one of the exten-
sively used chemical transformations in building heterocy-
clic frameworks.11 In the early 1990s, Ghosez and co-work-
ers reported the synthesis and the HDA reaction of 2-aza-3-
silyloxy-1,3-butadienes (2-ASBs iv, Figure 1) with carbonyl
compounds as well as with electron-poor olefins.12 In 1999,
© 2020. Thieme. All rights reserved. Synthesis 2020, 52, A–M
Georg Thieme Verlag KG, Rüdigerstraße 14, 70469 Stuttgart, Germany

B
M. Rehan et al.
Feature
Synthesis
Biographical Sketches
(from left to right) Mohammad Rehan was
born in India. He received his B.Sc. (2008)
and M.Sc. in Organic chemistry (2010) from
Jamia Millia Islamia University, New Delhi, In-
dia. He joined the group of Prof. Prasanta
Ghorai at the Indian Institute of Science Edu-
cation and Research (IISER) Bhopal, for his
doctorate and worked on transition-metal
catalyzed and transition metal-free synthesis
of heterocycles & carbocyclic molecules. In
2017, he obtained his Ph.D. in chemistry
from IISER, Bhopal, India. In 2018, he joined
as a postdoctoral research fellow in the
group of Prof. H. Waldmann at the Max
Planck Institute of Molecular Physiology
(MPI), Dortmund, and worked under the su-
pervision of Dr. Kumar on asymmetric hete-
Jorgelina L. Pergomet was born in Arias
(Córdoba, Argentina). She received her
B.Sc.H. in chemistry from the National Uni-
versity of Rosario (UNR). In 2012, she began
her doctoral studies in Prof. Kaufman’s
group, at Rosario Chemistry Institute
(IQUIR), where she worked on the total syn-
thesis of heterocycles natural products. In
2017, she got a Fellowship from Bunge &
Born Foundation–Max Planck’s Doctoral Fel-
lowship for a short research stay at MPI of
Molecular Physiology, Dortmund, within
Prof. Waldmann’s group and worked under
the supervision of Dr. Kumar. In the same
year, she participated in the Summer School
on Green Chemistry & Sustainable Energy,
Colorado (USA), with a fellowship from the
American Chemical Society. In 2018, she ob-
tained her Ph.D. in Chemistry from UNR and
joined the laboratory of Profs. Mischne and
Riveira at IQUIR, as post-doctoral researcher.
1995 in the group of Prof. Michael Veith. He
was awarded a DFG Heisenberg fellowship,
with which he moved to the Universität
Würzburg (Germany) in 1997. He held tem-
porary professorships at the universities of
Vienna (Austria), Rostock and Würzburg
(Germany) and moved to the Technische
Universität Dortmund (Germany) in 2008
becoming professor of inorganic chemistry.
His work on organolithium chemistry was
honored by the GDCh Arfvedson Schlenk
Prize for 2009. His research interests lie in
the areas of main group chemistry with em-
phasis on lithium and silicon chemistry and
the development and application of highly
polar reagents in synthesis, catalysis and ma-
terials science.
ro-Diels–Alder
reactions
leading
to
Kamal Kumar obtained his Ph.D. degree
from G.N.D. University, Amritsar, India in
2000. He was selected for an Alexander von
Humboldt fellowship to join Prof. Matthias
Beller’s group at the institute of Organic Ca-
talysis, Rostock (Germany) in 2002. He
joined Prof. Herbert Waldmann at the Chem-
ical Biology Department at MPI, Dortmund
(Germany) in 2004. From 2006–2018, he led
his own research group at the same depart-
ment working extensively on developing syn-
thetic methods towards natural-product-
inspired small molecules and exploring their
applications in chemcial biology and medici-
nal chemistry. Currently he leads chemistry
in bacteriology at Aicuris Antiinfective Cures,
Wuppertal Germany and also continues at
MPI Dortmund as a guest researcher. His re-
search interests involve asymmetric synthe-
sis of natural-product-based ring systems,
catalytic annulation reactions, cascade trans-
formations, chemical biology as well as dis-
covery of antibacterial and antiviral small
molecules.
biologically intriguing small molecules.
Jana Flegel was born in 1992 in Germany.
She received her B.Sc. (2015) and M.Sc. de-
gree (2017) in Chemical Biology at the Tech-
nical University Dortmund. By the end of
2017 Jana started her Ph.D. in Chemical Biol-
ogy at MPI in Dortmund. As part of her PhD,
she is currently working on the identification
and characterization of osteoblast differenti-
ation modulators.
Felix Otte is currently a Ph.D. student in the
research group of Professor Carsten
Strohmann in the Inorganic Chemistry De-
partment at the TU Dortmund University,
Germany. He obtained his B.Sc. in 2015 from
the faculty of chemistry and chemical biolo-
gy and M.Sc. in 2017. His research interests
include the characterization of highly reac-
tive and sensitive small molecules via X-ray
crystallography and evaluation of quantum
chemical calculations based on experimental
data.
Franziska Heitkamp was born in Datteln,
Germany and did her bachelor studies in
chemical biology (from October 2013 to
September 2016) at Technical University
Dortmund. During this time, she worked on
the synthesis of tumor-associated MUC4 gly-
copeptides at ISAS Dortmund under the su-
pervision of Dr. Westerlind. During her
master studies at the same university (from
October 2016 to September 2018), she
worked on asymmetric hetero-Diels–Alder
reactions leading to biologically intriguing
small molecules in the research group of Dr.
Kumar at MPI Dortmund.
Carsten Strohmann studied chemistry at
the Technische Universität Braunschweig
(Germany) and Technische Universität Karl-
sruhe (Germany) and received his Ph.D. in
1990 under the supervision of Prof. Reinhold
Tacke. After working as a postdoctoral fellow
with Prof. Dietmar Seyferth at the Massachu-
setts Institute of Technology in Cambridge
(USA), he moved to the Universität des Saar-
landes (Germany) finishing his habilitation in
© 2020. Thieme. All rights reserved. Synthesis 2020, 52, A–M

C
M. Rehan et al.
Feature
Synthesis
a)
O
OH
HN
Cl
HO
HO
OH
HN
NH
O
O
O
N
O
OH
N
O
NH
O
1'NH
2'
N
4
O
3'
NH
OH
5
O
3
3a
OH
O
H
2
O
1
6
N
H
Cl
7a N
O
O
Cl
H
7
N
Br
N
H
H
3,3'-piperidinoyl-
spirooxindole
synthetic
lapatin b
(ii)
surugatoxin 1
anticancer agent
(i)
(iii)
b)
R3
O
R1
R1
R3
R3SiO
NH
four consecutive chiral
centers
O
Lewis acid
R2
N
N
R4
chiral ligand
O
R2
iv
N
R4
v
vi
2-aza-3-silyloxy-1,3-
butadiene (2-ASB)
3,3´-piperidinoyl-
spirooxindole
Figure 1 a) Natural and synthetic bioactive molecules with core piperidinoyl spirooxindole scaffold; b) A hetero-Diels–Alder strategy to piperidinoyl
spirooxindoles.
the same group reported the first asymmetric HDA reaction
of 2-ASBs with conjugated imides.13 The only other asym-
metric reaction of 2-ASBs was reported in 2012 with alde-
hydes.14 Recently, we discovered that chiral metal complex-
es such as a Lewis acid divalent or trivalent salt complexes
with C2-symmetric N,N-dioxide ligand 3 could catalyze the
HDA reaction of 2-ASBs 2 with isatin-derived olefins 1 in an
asymmetric fashion to afford enantiomerically enriched
piperidinoyl spirooxindoles (Scheme 1a).15 The asymmetric
cycloaddition reaction was efficient and could place up to
four contiguous stereogenic centers on the resulting exo-
adducts bearing piperidinoyl spirooxindole core. Interest-
ingly, we observed that the bulkier silyl protecting group at
C3 of the 2-ASB played a key role in successful enantioselec-
tive reactions. Plausibly, nucleophilic 2a may interact and
poison the Lewis acid metal complex that facilitates the
non-catalytic racemic synthesis of 4a. A bulkier silyl group
sterically prevents such an interaction and favors a dieno-
phile–catalyst engagement and thus facilitates the stereo-
selective cycloaddition reaction.
The reported asymmetric HDA reaction between al-
kylidene oxindoles and 2-ASBs from our lab mainly focused
on the use of dienophiles 1 with electron-withdrawing sub-
stituents (CN, CO2Me). This approach strengthens the elec-
trophilic character of the dienophile and therefore facili-
tates the normal electron-demand HDA reaction. However,
adducts with such substitution pattern did not display bio-
logical activity in our screening assays targeting different
signaling pathways. 3,3′-Piperidinoyl spirooxindoles sup-
porting two aromatic residues at the 2′ and 4′ positions
were reported to exhibit promising antitumor activity by
modulating p53-MDM2 interaction.10a We were intrigued
to explore the scope of the above HDA reaction to generate
the 2′,4′-diarylpiperidinoyl spirooxindole class of small
molecules and to unravel its novel biological applications.
The racemic cycloaddition reaction between 2-ASBs 2 and
O
a)
Me
NC
Ph
10 mol% Mg(NTf2)2 or
10 mol% Dy(OTf)3
NH
Ph
NC
N
O
H
N
N
O
O
O
iPr
O
iPr
O
OR
11 mol% of ligand 3
N
H
N
N
N
DCM, 0 °C to rt, 6 h
Me
Boc
iPr
iPr
Boc
1
2
4
3
R = TMS, ee = 0%
R = TBS, ee = >90%
b)
blocks
chelation
competitive
interactions
R1
OtBu
OtBu
R1
O
O
LA
N
N
LA
N
R
N
R1
O
OTBS
O
OTMS
R2
R2
EWG
EWG
Scheme 1 a) Asymmetric HDA reaction between alkylidene oxindole 1 and 2-ASB 2; b) Role of bulky silyl group in directing enantioselective HDA
reaction.
© 2020. Thieme. All rights reserved. Synthesis 2020, 52, A–M

D
M. Rehan et al.
Feature
Synthesis
alkylidene oxindoles 1 was performed without any catalyst
and proceeded at higher temperature and afforded cycload-
duct (±)-5a as the major exo-cycloadduct (Table 1). The
asymmetric HDA reaction of the 2-ASBs 2 with alkylidene
oxindoles was performed using trivalent metal salt
Dy(OTf)3 in combination with the N,N-dioxide ligand 3a.16
However, only low enantioselectivity was observed (Table
1, entry 2) albeit with much improved diastereoselectivity
as compared to racemic reaction (entries 1 vs 2). The struc-
ture of the adduct 5a was assigned with the help of COSY
and HMBC NMR analysis (for details see Supporting Infor-
mation). Further reaction optimization was performed with
other Lewis acid transition metal catalysts.
Upon turning to chiral metal complexes as Lewis acid
catalysts for the HDA reaction, magnesium perchlorate with
3a afforded the desired exo-adduct in moderate enantio-
meric excess (Table 1, entry 3). We observed an increase in
ee using a combination of Mg(OTf)2 or Ni(OTf)2 with ligand
3a (entries 4 and 6). Under the same reaction conditions,
Sc(OTf)3 and La(OTf)3 failed to provide the desired adduct
(entries 5 and 7). To our delight, Mg(NTf2)2 in combination
with 3a steered the HDA reaction efficiently and provided
Table 1 Reaction Condition Optimization for HDA Reaction Between Oxindole 1a and 2-ASB 2aa
O
Me
Ph
Ph
Ph
10 mol% catalyst
NH
Ph
11 mol% ligand 3
N
O
+
DCM, 0 °C to rt, 12 h
O
OTBS
N
N
Boc
Me
Boc
5a
1a
2a
N
O
N
O
H
O
O
N
O
iPr
N
N
iPr
N
O
H
O
O
O
H
O
H
H
N
N
N
O
iPr
N
O
H
N
N
iPr
iPr
iPr
iPr
3c
iPr
3b
3a
O
O
N
N
N
N
O
O
O
H
O
H
iPr
N
N
iPr
O
O
N
N
O
O
O
H
O
H
NH
HN
N
N
3e
3d
3f
iPr
iPr
Entry
Lewis acid
Ligand
Yield (%)b
drc
16:1
ee (%)d
1
2
–
–
63
0
5
Dy(OTf)3
3a
3a
3a
3a
3a
3a
3a
3b
3c
3d
3e
3f
51
20:1
20:1
20:1
–
3
Mg(ClO4)2
Mg(OTf)2
Sc(OTf)3
61
90
94
–
4
69
5
trace
20
6
Ni(OTf)2
4.9:1
–
96
–
7
La(OTf)3
trace
91
8
Mg(NTf2)2
Mg(NTf2)2
Mg(NTf2)2
Mg(NTf2)2
Mg(NTf2)2
Mg(NTf2)2
20:1
5:1
98
54
66
58
0
9
94
10
11
12
13
88
20:1
2.2:1
20:1
20:1
85
68
88
10
a Reaction conditions for racemic synthesis (entry 1): 1a (0.05 mmol) and 2a (0.10 mmol) in toluene (0.5 mL) at 110 °C for 4 h under argon atmosphere. Reaction
conditions for asymmetric synthesis (entries 2–13): 1a (0.05 mmol) and 2a (0.10 mmol) with 10 mol% of catalyst, 11 mol% of ligand in DCM (0.5 mL) and at 0 °C.
b Yield of isolated products.
c Determined by 1H NMR spectroscopy.
d Determined by chiral-phase HPLC for major diastereomer.
© 2020. Thieme. All rights reserved. Synthesis 2020, 52, A–M

E
M. Rehan et al.
Feature
Synthesis
the exo-cycloadduct 5a with an excellent ee of 98% (entry
8). Some other ligands17 were also tested for the HDA reac-
tion of 1a with 2a, however, none of them proved better
than Mg(NTf2)2 (entries 9–13). Further reaction screening
using 10 mol% Mg(NTf2)2 and a series of chiral N,N′-dioxide
ligands (entries 9–13) in DCM clearly revealed that 3a was
the best ligand for this cycloaddition reaction.
Based on these results, further enantioselective synthe-
sis of 2′,4′-diaryl-3,3′-piperidinoyl spirooxindoles was tak-
en up using Mg(NTf2)2 complex with ligand 3a as the cata-
lytic system. Both racemic and enantioselective cycloaddi-
tion reactions were performed. The racemic reactions are
required not only to determine the enantiomeric excess in-
duced by the catalysts in the reactions catalyzed by chiral
metal complexes but also for the fact that enantiomers may
exhibit different biological activities and therefore if possi-
ble, both the enantiomers should be screened. However, if
the reactions can deliver only one enantiomer, racemic
compounds must also be screened in order to find the indi-
vidual bioactivity of the enantiomeric forms.
With the optimized reaction conditions and by employ-
ing differently substituted alkylidene oxindoles as well as 2-
ASBs, synthesis of a small collection of substituted 3,3′-
piperidinoyl spirooxindoles 5 with two different aryl
groups at 2′ and 4′ position was taken up (Scheme 2). The
Boc group in the cycloadducts was removed in order to gain
hydrogen bond donor moiety in 5. All of the Lewis acid cat-
alyzed reactions displayed highly diastereoselective forma-
tion of cycloadducts with excellent ee (>90%) (Scheme 2).
The reaction could easily tolerate mono- to penta-substi-
tuted aryl groups on olefinic moiety of oxindoles (Scheme
2) yielding adducts in high yield and with excellent ee. Pyri-
dine substitution (5b) on 4′ position was also well tolerated
under the optimized reaction conditions. Substitution at 4′
position and aryl part of the oxindole ring (5o, 5v, and 5z–
z3) also afforded the spirooxindoles in high yield and with
O
R1
Ar
R2
i) Mg(NTf2)2 (10 mol%)
3a (11 mol%)
DCM, 0 °C to rt, 6 h
NH
Ar
4´
N
2´
R2
R
O
N
ii) DCM/TFA (3:1), 0 °C
OTBS
O
R
R1
N
Boc
H
1
5
2
F
O
O
O
Br
Me
Me
O
O
O
Br
Me
Me
O
Me
N
F
Cl
NH
Ph
NH
Ph
NH
Ph
NH
NH
Ph
NH
Ph
F
O
O
O
O
O
O
N
H
N
N
N
N
H
N
H
H
H
H
(+)-5a
(+)-5b
(+)-5c
92% yield; 99% ee
(+)-5e
(+)-5f
(+)-5g
(+)-5c
93% yield; 97% ee
87% yield; 98% ee
O
86% yield; 98% ee
84% yield; 98% ee
89% yield; 98% ee
F
Cl
Br
O
O
O
O
O
Me
O
Me
F
F
Me
Me
Me
Me
Me
F
R
F
NH
Ph
NH
Ph
NH
Ph
NH
NH
Ph
NH
Ph
NH
Ph
R
Ph
O
Br
F3C
F
Cl
F
O
O
F
O
O
O
O
N
N
H
(–)-5l
N
N
N
H
N
N
R
H
H
H
H
H
(+)-5h; R = Br; 87% yield, 97% ee
(+)-5i; R = F; 92% yield, 95% ee
(+)-5j; R = CF3; 91% yield, 98% ee
(+)-5m
(–)-5n
(+)-5o
(+)-5p; R = Br; 88% yield, 98% ee (+)-5s; R = Br; 93% yield, 98% ee
(+)-5q; R = F; 94% yield, 97% ee
(+)-5r; R = Cl; 90% yield, 99% ee
(+)-5t; R = Cl; 86% yield, 98% ee
91% yield; 97% ee
80% yield; 96% ee
86% yield; 99% ee
89% yield; 98% ee
(+)-5k; R = NMe2; 87% yield, 96% ee
Cl
OMe
Cl
Br
O
F
O
O
O
Br
O
O
Me
O
Me
Me
Me
Me
Me
Me
F
Cl
NH
NH
Ph
NH
NH
Ph
Cl
NH
O
NH
O
NH
Cl
Br
Ph
O
Ph
O
MeO
F
F
F
O
O
O
N
O
N
N
N
H
N
H
N
H
Cl
N
H
H
Cl
H
H
(+)-5z
Cl
(+)-5u
88% yield, 97% ee
(+)-5v
82% yield, 98% ee
(+)-5w
86% yield; 97% ee
(–)-5x
80% yield; 99% ee
(–)-5y
(+)-5z1
84% yield; 99% ee
89% yield; 98% ee
81% yield, 97% ee
Cl
Cl
O
O
F
O
O
O
Br
O
F
Me
Br
O
Me
Me
Me
Me
Me
O
F
N
NH
NH
Ph
NH
Ph
NH
Ph
NH
O
NH
Ph
NH
Cl
Br
F3C
F
Ph
O
F
F
Br
O
F
O
O
O
O
N
H
N
N
N
H
N
N
N
H
H
H
H
H
Cl
(+)-5z2
77% yield, 95% ee
(–)-5z3
82% yield, 99% ee
(+)-5z4
92% yield; 99% ee
(+)-5z5
(+)-5z6
824% yield, 997% ee
(+)-5z7
(+)-5z8
74% yield, 99% ee
89% yield, ee: 98%
Scheme 2 Enantioselective HDA reaction of alkylidene oxindoles 1 and 2-ASBs 2. The reaction was performed in 3 mL rubber fitted screw vial using
alkylidene oxindoles 1 (0.05 mmol) and 2-ASBs 2 (0.10 mmol), Mg(NTf2)2 (10 mol%), 3a (11 mol%), in anhyd DCM under argon atmosphere, affording
2′,4′-diaryl 3,3′-piperidinoyl spirooxindoles 5. Yields mentioned are of isolated products and the enantiomeric excess determined by chiral-phase HPLC
for the major diastereomers.
© 2020. Thieme. All rights reserved. Synthesis 2020, 52, A–M

F
M. Rehan et al.
Feature
Synthesis
excellent ee values. The HDA reaction also delivered ad-
ducts supporting substitution at 2′ and 4′ positions as well
as on the aryl ring of the oxindole framework (5w, 5x, 5z1,
and 5z7) in high yields and with excellent enantioselectivi-
ty. Another interesting adduct bearing aryl ring at 4′ posi-
tion with p-morpholine (5z4) substitution was also formed
in excellent yield and with excellent ee. A representative al-
kyl substitution (cyclopropyl) at 4′ position is demonstrat-
ed in cycloadduct 5z5 that was also formed in high yield and
with excellent enantioselectivity (Scheme 2). The absolute
configuration of the spirooxindoles 5a–5z8 was established
on the basis of X-ray diffraction analysis of 5c (see the Sup-
porting Information).
Formation of exo-adduct is in contrary to the famous
endo-rule for normal demand Diels–Alder reaction and
probably is determined by the nature of the dienophile.18 In
1996, Ghosez et al. observed a dichotomy in the stereo-
chemical courses of cyclic and acyclic electron-poor dieno-
philes in the [4+2] cycloaddition with 2-ASBs. While the cy-
clic dienophiles reacted with 2-ASBs to deliver the expected
endo-product, the acyclic dienophiles afforded the exo-
products. It was claimed that cyclic dienophiles with an s-
trans conformation would be stabilized in the endo transi-
tion state because of secondary molecular orbital interac-
tions. However, the same transition state for acyclic dieno-
philes suffered from repulsive electrostatic interaction be-
tween the lone pairs of the oxygen and the nitrogen and
therefore is not favored (Figure 2a).19
We assume that there is another cause for the exo-selec-
tivity observed in the asymmetric HDA-reactions of al-
kylidene oxindoles and 2-ASBs as depicted in the transition
state in Figure 2b. When the azadiene 2 is 1,4-disubstituted,
either the substituent at C1 (here Ph group, i) in Figure 2b)
or the substituent at C4 (here Me, iii) in Figure 2b) faces ste-
ric hindrance with the aryl groups of the amide function in
the endo-transition state. The exo-transition state depicted
in the middle (ii) is lacking this steric repulsion as well as
the electrostatic repulsion between nitrogen and oxygen
lone pairs and therefore is highly favored. Furthermore, the
proposed transition state (ii) explains the enantioselectivity
of the catalyzed reaction. The Mg2+ is coordinated by the li-
gand with its four oxygen atoms and the dienophile with its
two carbonyl groups forming a distorted octahedral com-
plex. The re-face is blocked and the diene can only approach
from the si-face and leading to the formation of cycloadduct
with excellent enantiomeric excess.20
To explore possible biological activities of the synthe-
sized compounds, adducts 5 were tested in cell-based as-
says with regard to different cellular pathways and cellular
processes such as autophagy.6b,21 Interestingly, some deriv-
atives were identified as inhibitors of the Hedgehog (Hh)-
dependent osteoblast differentiation of murine mesenchy-
a) exo/endo transition state for the reaction between
dienes and dienophiles containing a carbonyl function
Me
TBSO
Me
TBSO
N
N
Ar'
Ar'
repulsive
electrostatic
interaction
O
Ar
Ar
s-cis
BocN
O
N
Boc
83
endo (disfavored)
exo (favored)
b) proposed transition states
HN
iii)
ii)
HN
HN
i)
O
O
O
N
N
N
N
O
O
O
Mg
N
O
O
O
O
O
O
Mg
Mg
O
1
O
O
O
N
Ar
N
O
N
O
O
O
N
N
Ar
Ar
O
Me
TBSO
N
OTBS
Me
TBSO
N
2a
Me
Ph
NH
NH
NH
endo (disfavored)
endo (disfavored)
exo (favored)
O
Me
NH
Ph
Ar
O
N
Boc
(+)- or (–)-5
Figure 2 Proposed interactions and transition state favoring exo-adducts in the asymmetric HDA reaction of alkylidene oxindoles with 2-ASBs
© 2020. Thieme. All rights reserved. Synthesis 2020, 52, A–M

G
M. Rehan et al.
Feature
Synthesis
mal stem cells. Hh signaling is essential during embryonic
development of vertebrates and is required for tissue ho-
meostasis in adult cells.22 However, aberrant activation of
the Hh pathway is involved in different types of cancer such
as medulloblastoma and basal cell carcinoma. Consequent-
ly, the identification of novel Hh pathway inhibitors is an
important aim in medicinal chemistry and chemical biolo-
gy research.23 Here we employed a Hh-dependent osteo-
blast differentiation assay (ODA) using C3H10T1/2 cells for
primary screening and identification of Hh inhibitors.24 In
this assay, C3H10T1/2 cells were treated with the
smoothened agonist purmorphamine to induce Hh signal-
ing, which subsequently initiates the differentiation of
these cells into osteoblasts and thus the expression of alka-
line phosphatase (ALP). The enzymatic activity of this os-
teoblast-specific marker was used as a measure of the dif-
ferentiation status of the cells and an indirect readout of the
Hh pathway activity. The diastereomeric mixture of (±)-5c
displayed intriguing moderate inhibition (around 30%) of
Hh dependent osteoblast differentiation at 10 M. However,
to our surprise, neither (+)-5c enantiomer or (–)-5c (puri-
fied from racemic via HPLC purification) displayed any in-
hibitory activity. A re-analysis of the racemic mixture of di-
astereomers of 5c (obtained via uncatalyzed cycloaddition
reaction) revealed that unlike other adducts, in this particu-
lar case, there were more than two diastereomeric com-
pounds present in the mixture. Therefore, it was of interest
to identify the right compound beholding the bioactivity.
Thus, the racemic HDA reaction of oxindole 1c with 2-ASB
under reflux condition was repeated that led to form three
diastereomers. Purification of three diastereomers by silica
gel column chromatography was possible only after the re-
moval of N-Boc group. The stereochemistry of all three
products could be determined by means of NMR spectral
analysis, in particular, based on 1H-by COSY, and HMBC and
NOESY NMR spectral analysis. The second diastereomer (±)-
5c-2 could cause an inhibition of the Hh induced osteoblast
differentiation down to 60% at a concentration of 10 M
(Scheme 3).
lar to the one explained previously for (+)-5v (see Support-
ing information, Supporting Figures 1 & 2). In the case of dia-
stereomer (±)-5c-2, the NOE signal between the proton at 4′
with the protons at 2′ and 5′ is rather weak. Instead, the
NOE between H4′ and the methyl group is very strong indi-
cating that the aryl group and H4′ at C4′ have changed the
position. Structure of endo-adduct, namely diastereomer
(±)-5c-3 (Supporting Figure 3 in the Supporting Informa-
tion) was assigned accordingly.
Interestingly, the exo-adduct (±)-5c-1 (diastereomer 1
formed from (E)-1c) was formed as the major product in
Mg2+-catalyzed asymmetric reaction but was a minor prod-
uct in the racemic reaction of 1c with 2-ASB. Diastereomer
2, namely (±)-5c-2, that displayed hedgehog inhibition was
the major product in the above racemic reaction (Scheme
3). The stereochemistry of (±)-5c-2 was further corroborat-
ed by X-ray crystallographic analysis.25 It is worth mention-
ing here that one of the major challenges that only rarely is
addressed by synthetic chemists is to build stereochemical
diversity in a screening collection.26 The reaction products
are often the consequence of thermodynamic-driven stabil-
ity of substrates, products or transition states. The efforts to
induce all possible perturbations around stereogenic cen-
ters to access different and sometimes energetically unfa-
vored diastereomers of a desired scaffold often go missing.
Understandably, in the scarcity of such a three-dimensional
chemical diversity in our compound collections, we are
ought to miss numerous biologically interesting diastereo-
meric analogues of biologically inactive compounds. Miss-
ing a diastereomer or an enantiomer may block a small
molecule with immense biological potential forever. Adduct
(±)-5c-2 highlights the significance of stereochemical diver-
sity in screening collection.
Compound (±)-5c-2 has an epimerized chiral center 4′,
and we reasoned that during the reaction of E-alkylidene
oxindole, there might be a heat or light-induced isomeriza-
tion of the olefin forming some amount of Z-isomer that
eventually took part in the HDA reaction to deliver the exo-
adduct (±)-5c-2 (Scheme 4a). A remote possibility that the
adduct 5c-2 is a consequence of single-epimerization under
In Figure 3, a section of the NOESY spectra of diastereo-
mer 1 [(±)-5c-1] and diastereomer 2 [(±)-5c-2] is shown. In
the case of diastereomer (±)-5c-1, the assignment was simi-
O
Me
O
Me
Br
O
Br
Br Me
OTBS
Me
Br
N
NH
Ph
NH
Ph
NH
Ph
Ph
2
F
+
+
F
F
F
i) toluene, reflux, overnight
ii) DCM/TFA (3:1), 0 °C
O
O
O
O
N
N
H
N
H
H
N
Boc
diastereomer 1
diastereomer 3
diastereomer 2
(
)-5c-1 (18%)
(+)-5c-3 (7%)
(+)-5c-2 (37%)
1c
exo-adduct
(from E-1c)
endo-adduct
(from E-1c)
exo-adduct
(from Z-1c)
% age inhibition of ODA
(at 10 mM)
0
>40
15
Scheme 3 Formation of different diastereomers in the racemic cycloaddition reaction of 1c with 2-ASB
© 2020. Thieme. All rights reserved. Synthesis 2020, 52, A–M

H
M. Rehan et al.
Feature
Synthesis
Figure 3 NOESY spectra of diastereomer (±)-5c-1 and diastereomer (±)-5c-2 obtained in the racemic synthesis of 5c
the reaction condition and not the cycloaddition product of
Z-olefin 1c was ruled out when no change of adduct 4a into
another diastereomer under reflux condition was observed.
Although our attempts to purify Z-olefin 1c failed and
led only to different ratios of the mixture of (E)- and (Z)-1
compounds, we did observe by NMR experiment that light-
induced isomerization did happen and can favor the forma-
tion of Z-olefin. The bromofluoro-substituted aryl olefin 1c
exists as E/Z in the ratio of 1:0.1 after synthesis. Exposing 1c
to normal light for 4 days hardly changed the E/Z ratio. To
a)
Br
Br
O
O
Br
Me
Me
Ph
(i) Mg(NTf2)2 (10 mol%)
3a (11 mol%)
DCM, 0 °C, 8 h
NH
Ph
NH
Ph
F
N
F
F
O
(ii) DCM/TFA (3:1)
0 °C, 30 min
O
O
OTBS
N
H
N
H
N
Me
Boc
1c
2a
(+)-5c
81% yield
99% ee
(+)-5c-2
4% yield
98% ee
E-isomer
UV
24 h
E/Z = 1:0.62
b)
OTBS
Me
Br
N
O
Br Me
Ph
2
(
)-5c-1
NH
Ph
(18%)
F
i) toluene, reflux, overnight
ii) DCM/TFA (3:1), 0 °C
O
+
F
O
N
N
(
)-5c-3
H
Boc
1c
(7%)
(
)-5c-2
(37%)
E/Z = 1:0.1
Scheme 4 Role of olefin isomerization in the formation of adduct exo-5c-2. a) Attempted asymmetric synthesis of (+)-5c-2. b) Uncatalyzed HDA reac-
tion of 1c and 2-ASB offered desired adduct (±)-5c-2 in moderate yield.
© 2020. Thieme. All rights reserved. Synthesis 2020, 52, A–M

I
M. Rehan et al.
Feature
Synthesis
Figure 4 Light- and heat-induced E/Z iosmerization of different alkylidene oxindole 1c
our pleasure, when E-olefin 1c was exposed to a stronger
ultra violet irradiation for a day, the E/Z ratio changed to
1:0.6 (Figure 4).27 This mixture was used for the asymmet-
ric HDA reaction at 0 °C using Mg salt and 3a as ligand.
However, the desired 5c-2 diastereomer was isolated only in
lower yield and the major product remained the one de-
rived from E-olefin 5c-1. Nevertheless, this was the first in-
stance when under asymmetric catalysis condition we ob-
served formation of 5c-2 with excellent enantiomeric ex-
cess (Scheme 4a).
We suspected that thermodynamic stability of the 1c
with an ortho-disubstituted aryl ring may be lower for Z-
olefin than E-isomer and therefore, the mixture reverted to
(E)-1c at lower temperature. We assumed that heating itself
Br
a)
O
R1
R2
(i) toulene, argon atm.
110 °C, 4 h
F
NH
O
Ar
N
R2
O
(ii) DCM/TFA (3:1)
0 °C, 30 min.
N
OSiR3
Boc
N
H
R1
2
1c
5
Me
O
Br
O
Br
Br
O
Me
Me
O
Br
NH
Ph
NH
Ph
NH
NH
Ph
Br
F
F
F
(
F
F
O
O
O
N
N
H
O
N
H
N
H
H
)-5z11 (24%)
(
)-5z9 (18%)
(
)-5z10 (23%)
(
)-5z12 (11%)
IC50 = 4.73+1.2 mM
IC50 > 10 mM
IC50 = > 10.0 mM
Inactive
Me
Br
O
b)
Me
O
Br
NH
Ph
NH
Ph
F
F
O
N
H
O
N
H
(+)-5c-2
IC50 = 600 100 nM
(–)-5c-2
IC50 = 600 200 nM
Scheme 5 Uncatalyzed HDA reaction between alkylidene oxindole 1c and 2-ASBs and identification of hedgehog modulators. Influence on the Hh
dependent osteoblast differentiation of C3H10T1/2 cells.
© 2020. Thieme. All rights reserved. Synthesis 2020, 52, A–M

J
M. Rehan et al.
Feature
Synthesis
might support such olefinic isomerization. We indeed ob-
served that heating (E)-1c at 110 °C in toluene led to higher
amount of Z-olefin formed (E/Z = 1:0.4; Figure 4 and Sup-
porting Figures 4 and 5 in the SI). Therefore, we decided to
directly use 1c (E/Z = 1:0.1) for a racemic HDA reaction with
2-ASB under reflux in toluene and without any catalyst. To
our pleasure, in this case, we could isolate the desired ad-
duct (±)-5c-2 in 37% isolated yield, along with other two ex-
pected adducts (5c-1 and 5c-3) as minor products (Scheme
4b).
Under the above reaction condition, some more ana-
logues of diastereomer 5c-2 were synthesized using race-
mic reactions of alkylidene oxindole 1c (E/Z = 1:0.1) with
differently substituted 2-ASBs 2 (Scheme 5). Different aryl
as well as alkyl substitutions on azadienes were well toler-
ated and the desired adducts 5z9–5z12 (Scheme 5) were pu-
rified by silica gel column chromatography. Some of the de-
rivatives in the series of 2′,4′-diaryl-3,3′-piperidinoyl spiro-
say. Both (+)-5c-2 and (–)-5c-2 displayed potent ability to
inhibit this process with IC50 of 600 ± 200 nM and 600 ± 100
nM, respectively (Scheme 5b).
To validate the compounds as Hh inhibitors, they were
evaluated in an orthogonal GLI-dependent reporter gene
assay, using Shh-LIGHT2 cells. These cells are stably trans-
fected with a GLI-responsive firefly luciferase and a consti-
tutively expressed Renilla luciferase as a control. In this as-
say (+)-5c-2 and (–)-5c-2 inhibited the Hh induced expres-
sion of the GLI-dependent firefly luciferase with IC50 values
of 12 ± 1.6 M and 11 ± 2.5 M, respectively (Figure 5C).
In conclusion, asymmetric hetero-Diels–Alder reaction
between alkylidene oxindoles and 2-aza-3-silyloxy-1,3-bu-
tadienes was developed as an efficient tool to access a com-
pound collection based on piperidinoyl spirooxindole scaf-
fold, which decorate cytotoxic natural products. In particu-
lar, the target was to develop 2′,4′-diaryl-substituted
spirooxindoles and to unravel their novel biological poten-
tial. Under the transition metal catalysis, specifically, the
divalent magnesium complex with N,N′-dioxide ligand 3a
offered an efficient protocol to generate the desired spiro-
oxindoles 5 in very high yields and with excellent enantio-
meric excess. A cell-based biological screening of the collec-
oxindoles
5
inhibited Hh dependent osteoblast
differentiation with varying potency (Scheme 5 and Sup-
porting Figure 7 in the SI). The two enantiomers of (±)-5c-2
were separated by preparative chiral HPLC and were inves-
tigated in the Hh dependent osteoblast differentiation as-
Figure 5 Influence of selected 3,3′-piperidinoyl spirooxindoles on Hh signaling. A: Chemical structures of (±)-5c-2 and its enantiomers. B: Influence of
adducts on the Hh dependent osteoblast differentiation of C3H10T1/2 cells. Cells were treated with 1.5 M purmorphamine and different concentra-
tions of the compounds or DMSO as a control. After 96 h, the activity of alkaline phosphatase was measured using a chemiluminescent substrate. C: Hh
dependent reporter gene assay. Shh-LIGHT2 cells were treated with 2 M purmorphamine and different concentrations of the compounds or DMSO as
a control for 48 h. B/C: The DMSO-purmorphamine control was set to 100%. Data are mean values of ≥2 biological replicates ± SD.
© 2020. Thieme. All rights reserved. Synthesis 2020, 52, A–M

K
M. Rehan et al.
Feature
Synthesis
tion to identify hedgehog modulators led to the identifica-
tion of a (±)-5c-2 – a stereospecific cycloadduct formed via
hetero-Diels–Alder reaction of Z-isomer of alkylidene oxin-
dole 1c with azadiene 2; as the most potent inhibitor of
hedgehog pathway in the compound collection. Some ana-
logues of the latter adduct also displayed potential hedge-
hog modulating activity. Thus, development of a synthetic
methodology enabled us to identify a new biological anno-
tation to piperidinoyl spirooxindoles, which previously are
known to exhibit inhibition of p53-MDM2 interaction. This
work also demonstrates the significance to develop meth-
ods to various possible stereoisomers of a desired product
with stereogenic centers. As there is yet no efficient meth-
od available to selectively get access to Z-alkylidene oxin-
doles, which may offer access to analogues of various re-
ported cycloaddition products or new products, synthesis
and potential of corresponding cycloadducts remain under-
explored. Work in this direction is interesting as well as
challenging and calls for further developments in synthetic
methodologies. We believe the biological potential of piper-
idinoyl spirooxindoles reported in this work will also gain
attention from medicinal chemists to explore their further
applications.
3,3′-piperidinoyl spirooxindoles prepared, see the Supporting Infor-
mation.
(2′R,3R,4′S,5′S)-5'-Methyl-2′,4′-diphenylspiro[indoline-3,3′-piperi-
dine]-2,6′-dione [(+)-5a]
White solid; yield: 17.78 mg (93%); dr >20:1; ee of 5a was determined
by chiral HPLC column: 97% ee. Chiralpak IA: isohex-
ane/2%EtOH/DCM, 15/85, flow rate 0.5 mL/min,  = 254 nm, tR (major)
= 18.82 min, tR (minor) = 28.28 min; []D20 +73 (c 1.0 in CHCl3).
1H NMR (500 MHz, CDCl3):  = 7.57 (s, 1 H), 7.51 (d, J = 7.6 Hz, 1 H),
7.28 (s, 1 H), 7.17–7.14 (2 H), 7.12–7.05 (5 H), 7.07–6.97 (4 H), 6.79 (d,
J = 5.4 Hz, 1 H), 6.49 (d, J = 7.7 Hz, 1 H), 5.20 (s, 1 H), 4.29 (d, J = 9.3 Hz,
1 H), 3.45–3.23 (m, 1 H), 1.24 (d, J = 7.5 Hz, 3 H).
13C NMR (125 MHz, CDCl3):  = 177.73, 177.57, 140.76, 136.73,
134.31, 129.47, 128.84, 128.70, 128.11, 128.05, 127.48, 127.21,
127.16, 126.62, 121.94, 109.84, 62.89, 59.47, 50.86, 38.36, 14.51.
HRMS (ESI): m/z calcd for C25H23N2O2 [M + H]+: 383.17595; found:
383.17549.
(2′R,3R,4′S,5′S)-5′-Methyl-2′-phenyl-4′-(pyridin-4-yl)spiro[indo-
line-3,3′-piperidine]-2,6′-dione [(+)-5b]
White solid; yield: 16.68 mg (87%); dr >20:1; ee of 5b was deter-
mined by chiral HPLC column: 98% ee. Chiralpak IA: isohex-
ane/2%EtOH/DCM, 20/80, flow rate 0.5 mL/min,  = 254 nm, tR (major)
= 21.33 min, tR (minor) = 46.00 min; []D20 +41 (c 1.0 in CHCl3).
1H NMR (400 MHz, CDCl3):  = 8.28 (d, J = 2.6 Hz, 2 H), 7.50–48 (2 H),
7.19–7.12 (2 H), 7.10–7.04 (m, 2 H), 7.00 (td, J = 7.7, 1.1 Hz, 1 H), 6.95–
6.92 (2 H), 6.73 (d, J = 4.8 Hz, 2 H), 6.48 (d, J = 1.8 Hz, 1 H), 6.28 (s, 1
H), 5.17 (s, 14 H), 4.26 (d, J = 9.4 Hz, 1 H), 3.43–3.27 (m, 1 H), 1.21 (d,
J = 7.5 Hz, 3 H).
13C NMR (100 MHz, CD2Cl2):  = 179.28, 176.51, 176.49, 162.46,
159.99, 141.10, 134.55, 129.84, 129.74, 129.23, 129.21, 128.48,
128.46, 128.26, 127.99, 127.42, 126.67, 126.62, 126.57, 126.54,
126.39, 126.20, 121.56, 115.37, 115.13, 109.29, 61.62, 61.04, 49.70,
38.19, 11.98.
Solvents and reagents used for reactions, column chromatography,
and HPLC were obtained from commercial suppliers. TLC was per-
formed on precoated Merck silica gel aluminum plates with 60F254
indicator, visualized by irradiation with UV light. Ceric ammonium
molybdate (CAM) and alkaline KMnO4 were used as TLC staining solu-
tion. Unless otherwise noted, 1H NMR and 13C NMR spectra were re-
corded on Bruker DRX400 (400 MHz), DRX500 (500 MHz), DRX600
(600 MHz), and DRX700 (700 MHz) spectrometers. High-resolution
mass spectra were recorded on LTQ Orbitrap mass spectrometer cou-
pled to an Acceka HPLC-System (HPLC column: Hypersil GOLD, 50 mm
× 1 mm, 1.9 m). The enantiomeric excesses were obtained by HPLC
analysis on a chiral stationary phase column (CHIRALPAK IA column
using Agilent 1100 series instrument with PDA detector).
HRMS (ESI): m/z calcd for C24H22N3O2 [M + H]+: 384.17120; found:
384.17078.
(2′R,3R,4′R,5′S)-4′-(2-Bromo-6-fluorophenyl)-5′-methyl-2′-phe-
nylspiro[indoline-3,3′-piperidine]-2,6′-dione [(+)-5c]
3,3′-Piperidinoyl Spirooxindoles; General Procedure
White solid; yield: 22.05 mg (92%); dr >20:1; ee of 5c was determined
by chiral HPLC column: 99% ee. Chiralpak IA: isohex-
ane/2%EtOH/DCM, 20/80, flow rate 0.5 mL/min,  = 254 nm), tR (ma-
jor) = 21.33 min, tR (minor) = 46.00 min; []D20 +15 (c 1.0 in CHCl3).
1H NMR (400 MHz, CD2Cl2):  = 8.07 (s, 1 H), 7.51 (dd, J = 7.5, 2.7 Hz, 2
H), 7.09–7.03 (m, 2 H), 7.02–6.91 (m, 4 H), 6.88–6.81 (m, 2 H), 6.85–
6.75 (m, 2 H), 6.71 (td, J = 7.6, 1.1 Hz, 1 H), 6.50 (s, 1 H), 6.33 (d, J = 7.7
Hz, 1 H), 5.10 (d, J = 1.7 Hz, 1 H), 4.91 (d, J = 9.7 Hz, 1 H), 3.33 (dq, J =
9.6, 6.9 Hz, 2 H), 1.01 (d, J = 7.0 Hz, 3 H).
13C NMR (100 MHz, CD2Cl2):  = 179.28, 176.51, 176.49, 162.46,
159.99, 141.10, 134.55, 129.84, 129.74, 129.23, 129.21, 128.48,
128.46, 128.26, 127.99, 127.42, 126.67, 126.62, 126.57, 126.54,
126.39, 126.20, 121.56, 115.37, 115.13, 109.29, 61.62, 61.04, 49.70,
38.19, 11.98.
A solution of Mg(NTf2)2 (2.923 mg, 0.005 mmol, 0.1 equiv) and ligand
(3.85 mg, 0.0055 mmol, 0.11 equiv) in DCM (0.5 mL) was stirred for
60 min at r.t. To this was added a solution of Boc-protected alkylidine
oxindole 1 (0.05 mmol, 1 equiv) in DCM (0.5 mL), followed by a solu-
tion of azadiene 215 (0.10 mmol, 2 equiv) in DCM (0.5 mL) at 0 °C and
the reaction mixture was slowly warmed to r.t. and the reaction mix-
ture was stirred continously at r.t. till the complete conversion of
starting materials (monitored by TLC). The Boc-protecting group was
subsequently removed by adding a 3:1 mixture of DCM/TFA at 0 °C to
the mixture. After 30 min, ice-cold H2O was added to the mixture,
which was extracted with EtOAc (3 ×). The products 5 were obtained
as a white solid after silica gel column chromatographic purification
(DCM/MeOH 99.5:0.5 → 97:3). Chiral HPLC with chiral pack IA col-
umn was used to determine the enantiomeric ratio of cycloadducts.
HRMS (ESI): m/z calcd for C25H21BrFN2O2 [M + H]+: 479.07704; found:
479.07637.
The preparation and properties of Boc-protected alkylidene oxindoles
1 are described in the Supporting Information. Some selected 3,3′-
piperidinoyl spirooxindoles are presented below; for the rest of the
© 2020. Thieme. All rights reserved. Synthesis 2020, 52, A–M

L
M. Rehan et al.
Feature
Synthesis
2′R,3R,4′S,5′S)-4′-[4-(Allyloxy)phenyl]-5′-methyl-2′-phenyl-
spiro[indoline-3,3′-piperidine]-2,6′-dione [(+)-5e]
Acknowledgment
JLP thanks Bunge & Born Foundation–Max Planck’s Doctoral Fellow-
ship for a short research stay at MPI. Authors thank Dr. S. Jayakumar
and Dr. S. Ziegler (MPI Dortmund) for their help in initial synthesis ex-
periments and biological data analysis, respectively. The Compound
Management and Screening Center (COMAS, Dortmund) is acknowl-
edged for carrying out the screening and data analysis. Authors are
grateful to Prof. H. Waldmann (MPI Dortmund) for his support and
encouragement.
White solid; yield: 18.85 mg (86%); dr >20:1; ee of 5e was determined
by chiral HPLC column: 98% ee. Chiralpak IA: isohex-
ane/2%EtOH/DCM, 15/85, flow rate 0.5 mL/min,  = 254 nm, tR (major)
= 24.35 min, tR (minor) = 43.78 min; []D20 +426 (c 1.0 in CHCl3).
1H NMR (500 MHz, CDCl3):  = 7.51 (d, J = 7.6 Hz, 1 H), 7.25 (s, 1 H),
7.17–7.02 (m, 4 H), 7.03–6.92 (m, 3 H), 6.66–6.63 (3 H), 6.59–6.58 (2
H), 6.45 (d, J = 7.7 Hz, 1 H), 5.97 (ddt, J = 15.9, 10.6, 5.3 Hz, 1 H), 5.43–
5.26 (m, 1 H), 5.23–5.21 (m, 1 H), 5.17 (s, 1 H), 4.38 (d, J = 5.3 Hz, 2 H),
4.22 (d, J = 9.4 Hz, 1 H), 3.49–3.14 (m, 1 H), 1.21 (d, J = 7.6 Hz, 3 H).
13C NMR (125 MHz, CDCl3):  = 177.50, 177.15, 157.55, 140.77,
134.66, 133.13, 128.98, 128.81, 128.63, 128.11, 127.46, 127.17,
126.67, 121.91, 117.69, 114.08, 109.72, 68.60, 62.86, 59.52, 50.17,
38.61, 14.72.
Supporting Information
Supporting information for this article is available online at
https://doi.org/10.1055/s-0040-1707222.
S
u
p
p
orit
n
gInformati
o
n
S
u
p
p
orit
n
gInformati
o
n
HRMS (ESI): m/z calcd for C28H27N2O3 [M + H]+: 439.20217; found:
439.20135.
References
(2′R,3R,4′R,5′S)-4′-(2-Bromo-4-fluorophenyl)-5′-methyl-2′-phe-
nylspiro[indoline-3,3′-piperidine]-2,6′-dione [(+)-5f]
(1) Present Address: Aicuris Anti-infective Cures GmbH, Friedrich-
Ebert-Str. 475, 42117 Wuppertal, Germany.
(2) Eder, J.; Sedrani, R.; Wiesmann, C. Nat. Rev. Drug Discov. 2014,
13, 577.
(3) (a) U.S. Food and Drug Administration (FDA); Available online:
http://www.accessdata.fda.gov/scripts/cder/daf/ (b) de la Torre,
B. G.; Albericio, F. Molecules 2020, 25, 745.
(4) (a) Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2016, 79, 629.
(b) Cragg, G. M.; Grothaus, P. G.; Newman, D. J. Chem. Rev. 2009,
109, 3012. (c) Ojima, I. J. Med. Chem. 2008, 51, 2587.
(d) Newman, D. J. J. Med. Chem. 2008, 51, 2589. (e) Wilson, R.
M.; Danishefsky, S. J. J. Org. Chem. 2006, 71, 8329. (f) Gullo, V.;
McAlpine, J.; Lam, K.; Baker, D.; Petersen, F. J. Ind. Microbiol. Bio-
technol. 2006, 33, 523.
White solid; yield: 20.13 mg (84%); dr >20:1; ee of 5f was determined
by chiral HPLC column: 98% ee. Chiralpak IA: isohex-
ane/2%EtOH/DCM, 15/85, flow rate 0.5 mL/min,  = 254 nm, tR (major)
= 18.78 min, tR (minor) = 22.66 min; []D20 +103 (c 1.0 in CHCl3).
1H NMR (500 MHz, CDCl3):  = 7.68 (s, 1 H), 7.26 (s, 1 H), 7.23 (d, J =
7.6 Hz, 1 H), 7.15–7.12 (2 H), 7.10–7.03 (4 H), 6.93 (d, J = 7.6 Hz, 3 H),
6.81–6.73 (2 H), 6.51 (d, J = 7.8 Hz, 1 H), 5.21 (s, 1 H), 4.77 (d, J = 8.6
Hz, 1 H), 3.53 (pent, J = 7.6 Hz, 1 H), 1.16 (d, J = 7.4 Hz, 3 H).
13C NMR (125 MHz, CDCl3):  = 178.03, 177.64, 161.90, 159.90,
140.78, 133.58, 132.97, 130.38, 130.32, 128.98, 128.78, 128.07,
127.48, 127.00, 126.28, 126.16, 126.09, 122.04, 120.45, 120.26,
114.21, 114.04, 110.00, 77.30, 77.05, 76.79, 63.46, 58.62, 48.05, 37.01,
14.06.
(5) (a) Kumar, K.; Waldmann, H. Isr. J. Chem. 2019, 59, 41. (b) Rizzo,
S.; Waldmann, H. Chem. Rev. 2014, 114, 4621.
HRMS (ESI): m/z calcd for C25H21BrFN2O2 [M + H]+: 479.07704; found:
479.07650.
(6) (a) Karageorgis, G.; Reckzeh, E. S.; Ceballos, J.; Schwalfenberg,
M.; Sievers, S.; Ostermann, C.; Pahl, A.; Ziegler, S.; Waldmann, H.
Nat. Chem. 2018, 10, 1103. (b) Sheremet, M.; Kapoor, S.;
Schroder, P.; Kumar, K.; Ziegler, S.; Waldmann, H. ChemBioChem
2017, 18, 1797. (c) Laraia, L.; Ohsawa, K.; Konstantinidis, G.;
Robke, L.; Wu, Y. W.; Kumar, K.; Waldmann, H. Angew. Chem. Int.
Ed. 2017, 56, 2145. (d) Newman, D. J. Pharmacol. Therap. 2016,
162, 1. (e) Zhang, J.; Wu, J.; Hong, B.; Ai, W.; Wang, X.; Li, H.; Lei,
X. Nat. Commun. 2014, 5, 4614. (f) Over, B.; Wetzel, S.; Grutter,
C.; Nakai, Y.; Renner, S.; Rauh, D.; Waldmann, H. Nat. Chem.
2013, 5, 21.
(7) (a) Schroder, P.; Bauer, J. O.; Strohmann, C.; Kumar, K.;
Waldmann, H. J. Org. Chem. 2016, 81, 10242. (b) Schroder, P.;
Forster, T.; Kleine, S.; Becker, C.; Richters, A.; Ziegler, S.; Rauh,
D.; Kumar, K.; Waldmann, H. Angew. Chem. Int. Ed. 2015, 54,
12398. (c) Kolundzic, F.; Murali, A.; Perez-Galan, P.; Bauer, J. O.;
Strohmann, C.; Kumar, K.; Waldmann, H. Angew. Chem. Int. Ed.
2014, 53, 8122. (d) Eschenbrenner-Lux, V.; Kuchler, P.; Ziegler,
S.; Kumar, K.; Waldmann, H. Angew. Chem. Int. Ed. 2014, 53,
2134. (e) Dakas, P. Y.; Parga, J. A.; Hoing, S.; Scholer, H. R.;
Sterneckert, J.; Kumar, K.; Waldmann, H. Angew. Chem. Int. Ed.
2013, 52, 9576. (f) Duckert, H.; Pries, V.; Khedkar, V.;
Menninger, S.; Bruss, H.; Bird, A. W.; Maliga, Z.; Brockmeyer, A.;
Janning, P.; Hyman, A.; Grimme, S.; Schurmann, M.; Preut, H.;
Hubel, K.; Ziegler, S.; Kumar, K.; Waldmann, H. Nat. Chem. Biol.
2012, 8, 179.
(2′R,3R,4′S,5′S)-4′-(3-Chloro-4-fluorophenyl)-5′-methyl-2′-phenyl-
spiro[indoline-3,3′-piperidine]-2,6′-dione [(+)-5g]
White solid; yield: 19.3 mg (89%); dr >20:1; ee of 5g was determined
by chiral HPLC column: 98% ee. Chiralpak IA: isohex-
ane/2%EtOH/DCM, 15/85, flow rate 0.5 mL/min,  = 254 nm, tR (major)
= 23.02 min, tR (minor) = 43.74 min; []D20 +87 (c 1.0 in CHCl3).
1H NMR (500 MHz, CDCl3):  = 7.45 (s, 1H), 7.42 (d, J = 7.6 Hz, 1H),
7.10–7.07 (2H), 7.02–6.96 (4H), 6.88 (d, J = 7.6 Hz, 2H), 6.78–6.74 (t,
J = 8.7 Hz, 2H), 6.58 (s, 1H), 6.45 (d, J = 7.7 Hz, 1H), 5.09 (s, 1H), 4.16
(d, J = 9.5 Hz, 1H), 3.43–3.14 (m, 1H), 1.14 (d, J = 7.5 Hz, 3H).
13C NMR (125 MHz, CDCl3):  = 177.25, 158.17, 156.19, 140.72,
133.99, 133.76, 129.32, 128.88, 128.21, 127.46, 127.00, 125.90,
122.26, 120.57, 120.43, 116.19, 116.02, 110.12, 62.71, 59.36, 49.84,
37.97 14.52.
HRMS (ESI): m/z calcd for C25H21ClFN2O2 [M + H]+: 435.12756; found:
435.12691.
Funding Information
This research work was supported by the Max Planck Gesellschaft.
M
a
x
P
l
a
n
c
k
G
ese
l
l
sc
h
af
t
()
© 2020. Thieme. All rights reserved. Synthesis 2020, 52, A–M

M
M. Rehan et al.
Feature
Synthesis
(8) (a) Galliford, C. V.; Scheidt, K. A. Angew. Chem. Int. Ed. 2007, 46,
8748. (b) Hong, L.; Wang, R. Adv. Synth. Catal. 2013, 355, 1023.
(c) Ye, N.; Chen, H. Y.; Wold, E. A.; Shi, P. Y.; Zhou, J. ACS Infect.
Dis. 2016, 2, 382. (d) Yu, B.; Yu, D. Q.; Liu, H. M. Eur. J. Med. Chem.
2015, 97, 673.
(9) (a) Hayashi, E.; Yamada, S. Brit. J. Pharmacol. 1975, 53, 207.
(b) Hayashi, E.; Yamada, S.; Brown, D. A. Jpn. J. Pharmacol. 1976,
26, P147. (c) Inoue, S.; Okada, K.; Tanino, H.; Hashizume, K.;
Kakoi, H.; Mizuno, M. Heterocycles 1984, 21, 729. (d) Larsen, T.
O.; Petersen, B. O.; Duus, J. Ø.; Sørensen, D.; Frisvad, J. C.;
Hansen, M. E. J. Nat. Prod. 2005, 68, 871.
(10) (a) Chen, L.; Ding, Q.; Liu, J.-J.; Yang, S.; Zhang, Z. U.S. Pat. Appl.
Publ US 20080009486, 2008. (b) Chen, L.; Han, X.; Yang, S.;
Zhang, Z. Can. Pat. Appl. Pub CA 20102758640, 2010.
(11) (a) Bodnar, B. S.; Miller, M. J. Angew. Chem. Int. Ed. 2011, 50,
5629. (b) Eschenbrenner-Lux, V.; Kumar, K.; Waldmann, H.
Angew. Chem. Int. Ed. 2014, 53, 11146. (c) Rowland, G. B.;
Rowland, E. B.; Zhang, Q.; Antilla, J. C. Curr. Org. Chem. 2006, 10,
981.
(12) (a) Adam, J. M.; Ghosez, L.; Houk, K. N. Angew. Chem. Int. Ed.
1999, 38, 2728. (b) Beaudegnies, R.; Ghosez, L. Tetrahedron:
Asymmetry 1994, 5, 557. (c) Ghosez, L.; Bayard, P.;
Nshimyumukiza, P.; Gouverneur, V.; Sainte, F.; Beaudegnies, R.;
Rivera, H.; Frisquehesbain, A. M.; Wynants, C. Tetrahedron 1995,
51, 11021. (d) Ntirampebura, D.; Ghosez, L. Tetrahedron Lett.
1999, 40, 7079. (e) Pouilhes, A.; Langlois, Y.; Nshimyumukiza,
P.; Mbiya, K.; Ghosez, L. Bull. Soc. Chim. Fr. 1993, 130, 304.
(13) Jnoff, E.; Ghosez, L. J. Am. Chem. Soc. 1999, 121, 2617.
(14) Watanabe, Y.; Washio, T.; Krishnamurthi, J.; Anada, M.;
Hashimoto, S. Chem. Commun. 2012, 48, 6969.
S.; Feng, X. M. Angew. Chem. Int. Ed. 2008, 47, 1308. (c) Zheng, J.
F.; Lin, L. L.; Fu, K.; Zhang, Y. L.; Liu, X. H.; Feng, X. M. Chem. Eur.
J. 2014, 20, 14493. (d) Zheng, J. F.; Lin, L. L.; Kuang, Y. L.; Zhao, J.
N.; Liu, X. H.; Feng, X. M. Chem. Commun. 2014, 50, 994. (e) Lian,
X.; Lin, L.; Fu, K.; Ma, B.; Liu, X.; Feng, X. Chem. Sci. 2017, 8, 1238.
(f) Li, J.; Lin, L.; Hu, B.; Zhou, P.; Huang, T.; Liu, X.; Feng, X.
Angew. Chem. Int. Ed. 2017, 56, 885. (g) Yao, Q.; Liao, Y.; Lin, L.;
Lin, X.; Ji, J.; Liu, X.; Feng, X. Angew. Chem. Int. Ed. 2016, 55,
1859.
(18) Ho, G.-M.; Huang, C.-J.; Li, E. Y.-T.; Hsu, S.-K.; Wu, T.; Zulueta, M.
M. L.; Wu, K. B.; Hung, S.-C. Sci. Rep. 2016, 6, 35147.
(19) Ghosez, L. Pure Appl. Chem. 1996, 68, 15.
(20) (a) Liu, X. H.; Lin, L. L.; Feng, X. M. Org. Chem. Front. 2014, 1, 298.
(b) Zheng, K.; Yin, C.; Liu, X.; Lin, L.; Fen, X. Angew. Chem. Int. Ed.
2011, 50, 2573. (c) Li, W.; Liu, X.; Hao, X.; Cai, Y.; Lin, L.; Feng, X.
Angew. Chem. Int. Ed. 2012, 51, 8644.
(21) (a) Garcia-Castro, M.; Kremer, L.; Reinkemeier, C. D.; Unkelbach,
C.; Strohmann, C.; Ziegler, S.; Ostermann, C.; Kumar, K. Nat.
Commun. 2015, 6, 6516. (b) Lee, Y. C.; Patil, S.; Golz, C.;
Strohmann, C.; Ziegler, S.; Kumar, K.; Waldmann, H. Nat.
Commun. 2017, 8, 14043.
(22) Briscoe, J.; Therond, P. P. Nat. Rev. Mol. Cell Biol. 2013, 14, 416.
(23) (a) Mahindroo, N.; Punchihewa, C.; Fujii, N. J. Med. Chem. 2009,
52, 3829. (b) Rimkus, T. K.; Carpenter, R. L.; Qasem, S.; Chan, M.;
Lo, H. W. Cancers (Basel) 2016, 8, 22.
(24) Wu, X.; Ding, S.; Ding, Q.; Gray, N. S.; Schultz, P. G. J. Am. Chem.
Soc. 2002, 124, 14520.
(25) CCDC 1992928 [(±)-5c-2] and CCDC 1992929 [(+)-5c] contain
the supplementary crystallographic data for this paper. The
data can be obtained free of charge from The Cambridge Crys-
tallographic Data Centre via www.ccdc.cam.ac.uk/getstruc-
tures.
(15) Jayakumar, S.; Louven, K.; Strohmann, C.; Kumar, K. Angew.
Chem. Int. Ed. 2017, 56, 15945.
(16) (a) Liu, X.; Zheng, H.; Xia, Y.; Lin, L.; Feng, X. Acc. Chem. Res.
2017, 50, 2621. (b) Liu, X.; Lin, L.; Feng, X. Acc. Chem. Res. 2011,
44, 574.
(17) For some selected reports from Feng group using N,N-dioxide
ligands, see: (a) Lin, L. L.; Kuang, Y. L.; Liu, X. H.; Feng, X. M. Org.
Lett. 2011, 13, 3868. (b) Yu, Z. P.; Liu, X. H.; Dong, Z. H.; Xie, M.
(26) Galloway, W. R. J. D.; Isidro-Llobet, A.; Spring, D. R. Nat.
Commun. 2010, 1, 80.
(27) Roke, D.; Sen, M.; Danowski, W.; Wezenberg, S. J.; Feringa, B. L.
J. Am. Chem. Soc. 2019, 141, 7622.
© 2020. Thieme. All rights reserved. Synthesis 2020, 52, A–M