Isatin Derived Spirocyclic Analogues with α-Methylene-γ-butyrolactone as Anticancer Agents: A Structure-Activity Relationship Study

Article abstract of DOI:10.1021/acs.jmedchem.6b00400

Design, synthesis, and evaluation of α-methylene-γ-butyrolactone analogues and their evaluation as anticancer agents is described. SAR identified a spirocyclic analogue 19 that inhibited TNFα-induced NF-?°B activity, cancer cell growth and tumor growth in an ovarian cancer model. A second iteration of synthesis and screening identified 29 which inhibited cancer cell growth with low-?M potency. Our data suggest that an isatin-derived spirocyclic α-methylene-γ-butyrolactone is a suitable core for optimization to identify novel anticancer agents.

Full text of DOI:10.1021/acs.jmedchem.6b00400

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Brief Article
Isatin derived spirocyclic analogs with #-methylene-#-butyrolactone
as anticancer agents: A structure activity rela-tionship study
Sandeep Rana, Elizabeth C. Blowers, Calvin Tebbe, Jacob I. Contreras,
Prakash Radhakrishnan, Smitha Kizhake, Tian Zhou, Rajkumar N. Rajule,
Jamie Arnst, Adnan R Munkarah, Ramandeep Rattan, and Amarnath Natarajan
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00400 • Publication Date (Web): 14 Apr 2016
Downloaded from http://pubs.acs.org on April 15, 2016
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Isatin derived spirocyclic analogs with α-methylene-γ-
butyrolactone as anticancer agents: A structure activity rela-
tionship study
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‡,ꢀ
‡,ꢀ
§
ꢀ
Sandeep Rana , Elizabeth C. Blowers , Calvin Tebbe , Jacob I. Contreras , Prakash Radha-
ꢀ
ꢀ
¶
,ꢀ
ꢀ
†
krishnan , Smitha Kizhake , Tian Zhou , Rajkumar N. Rajule , Jamie L. Arnst , Adnan R.
§
§
,ꢀ,¶,⌘,∞
Munkarah , Ramandeep Rattan and Amarnath Natarajan*
ꢀ
¶
Eppley Institute for Research in Cancer and Allied Diseases, Departments of Pharmaceutical Sciences and
⌘
∞
Genetics Cell Biology and Anatomy, Fred and Pamela Buffett Cancer Center, University of Nebraska Medical
Center, Omaha, Nebraska 68022, United States
§
Division of Gynecology Oncology, Department of Women’s Health and Josephine Ford Cancer Center, Henry
Ford Hospital, Detroit, Michigan 48202, United States
ABSTRACT: Design, synthesis and evaluation of α-methylene-γ-butyrolactone analogs and their evaluation as anti-
cancer agents is described. SAR identified a spirocyclic analog 19 that inhibited, TNFα-induced NF-κB activity, cancer
cell growth and tumor growth in an ovarian cancer model. A second iteration of synthesis and screening identified 29
that inhibited cancer cell growth with low-ꢀM potency. Our data suggests that an isatin-derived spirocyclic α-
methylene-γ-butyrolactone is a suitable core for optimization to identify novel anticancer agents.
INTRODUCTION
pathway is constitutively activated in a variety of cancers
including pancreatic, breast and ovarian cancers and has
been shown to contribute to anti-apoptosis, prolifera-
tion, tumor progression and chemoresistance.7
Twenty one percent of currently marketed covalent drugs
are used to treat cancer and several new covalent inhibi-
tors are in clinical trials, suggesting the development of
irreversible inhibitors as cancer therapeutics is making a
strong comeback. Covalent inhibitors were not fully
explored as their perceived risk-reward ratio was heavily
biased by concerns regarding their potential idiosyncrat-
ic effects. The resurgence of covalent inhibitors as cancer
therapeutics can be attributed to the successful devel-
opment of currently marketed irreversible inhibitors of
The key proteins in this pathway i.e., kinase IKKβ and
the transcription factor NF-κB, have surface exposed
1-3
179
cysteine residues. Cys found in the activation loop of
IKKβ is primed for targeting as it is between the serine
177
181
residues 177 and 181. Phosphorylation of Ser and Ser
8
results in the activation of IKKβ. Cys38 in NF-κB (p65
subunit) plays an important role in its translocation to
the nucleus to activate gene expression. The sulfhydryl
1
enzyme active sites. The current strategy for the devel-
9
opment of covalent drugs for targeting oncogenic kinases
is to append an electrophilic group to a reversible inhibi-
tor. This electrophilic group on the reversible inhibitor
then forms of a covalent bond with the sulfhydryl group
of a non-catalytic cysteine residue peripheral to the ki-
groups on Cys1 of IKKβ and Cys of NF-κB have been
previously targeted using parthenolide, a sesquiterpene
lactone natural product.1 In a cell-based assay, we re-
cently showed that parthenolide inhibits TNFα-induced-
IKKβ-mediated NF-κB activity with low-ꢀM potency.12
Natural products with the α-methylene-γ-butyrolactone
functionality exhibit a wide-range of biological activities
including anticancer and anti-inflammatory effects.1
The available SAR with parthenolide analogs showed
that the Michael acceptor in the α-methylene-γ-
butyrolactone is critical for activity against the NF-κB
79
38
0,11
4
nase active site. Here we report a biased approach for
the identification of covalent inhibitors and their evalua-
tion as anticancer agents.
Nuclear factor kappa B (NF-κB) is a transcription factor
that plays a key role in innate and adaptive immune re-
sponses, inflammation, cell growth, and apoptosis. In
unstimulated cells, NF-κB is sequestered in the cyto-
plasm by its inhibitor, inhibitor of nuclear factor κB
3-17
5
11
pathway. The Colby lab synthesized fluorinated amino
derivatives of parthenolide and screened them for anti-
(
IκBα). Upon stimulation with pro-inflammatory cyto-
proliferative activities.1
8,19
More recently, the Crooks lab
series of parthenolide and
kines such as tumor necrosis factor alpha (TNFα), IκBα
is phosphorylated by the IκB kinase β (IKKβ), ubiqui-
tinated and rapidly degraded, allowing NF-κB dimers to
translocate to the nucleus and activate transcription.6
Immunohistochemistry (IHC) studies conducted with
surgically resected tumor samples show that TNFα was
found in ~50% of tumors suggesting that the NF-κB
generated
a
melampomagnolide-B analogs and screened them
20-22
against a panel of 60 human cancer cell lines.
The α-
methylene-γ-butyrolactone functionality was appended
to small molecules to covalently link them to their bio-
2
3,24
logical target.
butyrolactone also show anticancer activities.
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Compounds with α-methylene-γ-
25-27
In the

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studies presented here we have expanded on this general inactive compound (15). The methoxy substitution at the
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2-position of 1-napthyl analog (16) was tolerated howev-
er a bigger benzyloxy substitution at the same position
(17) resulted in reduced activity (< 25% inhibition). The
spirocyclic analogs (19 and 20) in which the α-
methylene-γ-butyrolactone is rigid had activities (~75%
inhibition) comparable to the ML-120B indicating that
rigidification of the Michael acceptor is a favorable fea-
ture.
theme via synthesis of α-methylene-γ-butyrolactone con-
taining analogs and screened them to identify pathway
specific inhibitor. Multiple proteins in the NF-κB path-
way have surface exposed cysteine residues; therefore,
we screened our analogs in a TNFα-induced-IKKβ-
mediated NF-κB reporter assay to identify covalent
pathway specific inhibitors.
This exercise led to the identification of an isatin derived
spirocyclic core with an α-methylene-γ-butyrolactone
moiety (19) that inhibits the NF-κB pathway by covalent-
ly binding to IKKβ and NF-κB. This is the first report
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that identified
a
compound with spirocyclic α-
methylene-γ-butyrolactone moiety as a NF-κB inhibitor.
Analog 19 inhibits cancer cell growth in vitro and tumor
growth in an orthotopic ovarian cancer model. Analog 19
is ~4-fold more stable in serum albumin when compared
to parthenolide.
To explore this core structure we generated seven ana-
logs with substitutions at different positions on the isa-
tin-derived spirocyclic core and evaluated their ability to
inhibit cancer cell growth. This led to identification of
analog 29 with low-ꢀM potency and ~2-20 fold more
potency than parthenolide in a panel of cancer cell lines.
RESULTS AND DISCUSSION
We generated a biased library that features the α-
methylene-γ-butyrolactone core using a reported two-
2
8
step synthesis (Scheme 1) and screened analogs at 10
ꢀM (Figure 1B) in a cell-based luciferase assay (A549)
that specifically reports on the ability of the compound to
inhibit TNFα-induced IKKβ-mediated NF-κB activity.12
ML-120B, a well-characterized IKKβ inhibitor, was used
2
9
as a positive control.
SCHEME 1. Synthesis of α-methylene-γ-butyrolactone
containing spiroisatin analog 20
FIGURE 1. (A) Focused library of α-methylene-γ-
butyrolactone analogs and the IKKβ inhibitor ML-120B (B)
A cell-based screen (A549) that reports on the ability of the
inhibitors to specifically block TNFα-induced IKKβ-
mediated NF-κB activity.
(i) Methyl- 2-(bromomethyl)acrylate, In powder, NH Cl,
4
MeOH, 50 °C, 1h (ii) PTSA, CH Cl , 12h. (iii) 4-bromo-1-
2
2
butyne, Cs CO , CH CN, 70 °C, 12h
3
2
3
The unsubstituted, mono- and di-substituted phenyl
compounds (2-10) had modest activity (25-50% inhibi-
tion) with no clear SAR. Pyridine substitution (11) re-
sulted in decreased activity (< 25% inhibition) while a
bulky bromo group ortho to the nitrogen in 12 resulted
in the recovery of activity (~50% inhibition). Interesting-
ly introducing a second α-methylene-γ-butyrolactone in
1
3 did not increase the activity. In the bicyclic fused aryl
ring systems unsubstituted 1- and 2-napthyl substituted
analogs (14 and 18) had modest activity (~50% inhibi-
tion) while the 4-quinolinyl substitution resulted in an
FIGURE 2. (A) Structure of Parthenolide, reduced Par-
thenolide and isatin analogs (B) Evaluation of inhibitors
2
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Journal of Medicinal Chemistry
19 reacts with only Cys and not Lys suggesting a Cys ad-
in TNFα-induced NF-κB activity assay (C) Dose-
response study with analog 19.
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duct.
To determine if the Michael acceptor is critical for activi-
ty, we tested the reduced spirocyclic compound that
lacks the Michael acceptor (21) and compared it with
Parthenolide and reduced Parthenolide analog (Red-P)
(
7
Figure 2A). At 10 ꢀM, analog 19 showed good activity (>
0% inhibition) while reduced analog 21 was inactive (~
5
% inhibition), demonstrating that the Michael acceptor
in 19 is critical for NF-κB inhibitory activity. Par-
thenolide showed remarkable activity (> 95% inhibition)
while Red-P was ~4 fold less active (~ 20% inhibition)
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(
Figure 2B). To confirm whether rigidification indeed
resulted in increased NF-κB inhibition, the isatin derived
acyclic analog (22) was screened under identical condi-
tions. Acyclic analog 22 was ~2 fold less active than the
cyclized version (19) suggesting that rigidification indeed
increases activity against the NF-κB pathway proteins. In
FIGURE 3. (A) Schematic of click chemistry used to
demonstrate covalent binding (left) and covalent binding
of 20 to IKKβ and p65 via click chemistry (right) (B)
Inhibition of IKKβ kinase activity determined by West-
ern blot analyses in MDA-MB-231 cells and (C) Mi-
aPaCa2 cells.
a dose-response study, analog 19 had low-ꢀM (IC50
~
4
ꢀM) inhibitory activity in the TNFα-induced IKKβ-
mediated NF-κB activity assay (Figure 2C), which is
12
comparable to Parthenolide (IC50 = 4.7 ± 1.5 ꢀM). To
To determine if covalent binding to IKKβ results in the
inhibition of the kinase activity of IKKβ, cancer cells
were incubated with analogs 19, 21 and 22 for 2h. The
cells were lysed and the proteins were separated on SDS
PAGE, transferred to a membrane and probed with total
and phosphospecific IκBα antibodies. Since IκBα is a
substrate of IKKβ, inhibition of the kinase activity of
IKKβ should result in reduced IκBα phosphorylation.
Indeed we observed a complete inhibition of IκBα phos-
phorylation in cells treated with 19 but no inhibition of
IκBα phosphorylation in cells treated with the reduced
compound 21 that lacks the Michael acceptor (Figure 3B
and C). Interestingly the acyclic analog 22 showed par-
tial inhibitory activity (Figure 3B), which indicates that
rigidification of the Michael acceptor results in increased
inhibition of IKKβ.
summarize, our synthesis and screening effort identified
an isatin derived spirocyclic compound with the α-
methylene-γ-butyrolactone as a potent NF-κB inhibitor.
The acyclic analog 22 adopts multiple conformations
when compared to the rigidified analog 19 and therefore
could bind to additional targets, which explains the in-
creased the NF-κB inhibitory activity observed with 19.
One of the limitations with the use of parthenolide is the
short half-life in serum. A recent study characterized the
covalent binding of parthenolide through the α-
methylene-γ-butyrolactone to the free cysteine (34) resi-
due of serum albumin by MS analyses. They also deter-
mined the half-life of parthenolide to be ~37 minutes.3
To determine the stability of our analog 19 we conducted
a head-to-head comparison of analog 19 and par-
thenolide for serum albumin binding using HPLC (Fig-
ure S1). Our results show that half-life of 19 is 290 min
as compared to 66 min for parthenolide indicating the
spirocyclic disposition in 19 as opposed to a fused dispo-
sition in parthenolide could contribute to the observed
increased serum stability.
0
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Next, we used click chemistry to determine if com-
pound 19 indeed irreversibly binds to IKKβ and NF-κB.
Recombinant IKKβ and NF-κB (p65) were incubated
with analog 20, which is analog 19 with an alkyne linker,
for 1h at room temperature. Rhodamine azide and click
reagents were added to the reaction mixture and incu-
bated for an additional hour. At the end of the second
hour, the mixture was subjected to SDS PAGE and the
gel was imaged using the Typhoon 9410 Variable Mode
Imager, an imager that produces digital images of fluo-
rescent samples. The data summarized in Figure 3A
shows fluorescent bands at molecular weights that corre-
spond to IKKβ and NF-κB (p65 protein truncated at C-
terminus and has L159V, P180S, F309S, A439V and
V462M mutations runs at ~50 kDa – accession no.
AAA36408) demonstrating that analog 20 is a covalent
inhibitor of IKKβ and NF-κB. To determine if this is a
Cys adduct we conducted a HPLC study wherein Boc
protected Cys or Lys amino acids incubated with analog
FIGURE 4. Dose-dependent (A) and time-dependent
(B) effects on the growth of breast cancer cells by analogs
19 and 21. Cell viability was assessed using PrestoBlue
dye after 3 days treatment.
To determine if the IKKβ-NFκB inhibitory activity trans-
lates to anticancer activity, we subjected a panel of can-
cer cell lines to 19, 21 and three previously reported
IKKβ inhibitors 13-197,3 Bayer VIII and TPCA1.
2
33-36
Analog 19 showed dose and time dependent inhibition of
cancer cell growth while analog 21 did not (Figure 4A
and 4B) further demonstrating that the Michael acceptor
is critical for anticancer activity. Importantly, the growth
inhibitory activity of 19 was comparable to known IKKβ
inhibitors (Table S1).
1
9 (data not shown). Our results showed that analog
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demonstrates synergism in the low-ꢀM ranges with cis-
platin toward the inhibition of ovarian cancer cell
growth.
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FIGURE 6. Dose-dependent effects on ovarian (A2780)
tumor growth by analog 19, cisplatin and the combina-
tion in an orthotopic model of ovarian cancer (left) and
NF-κB(p65) staining of the excised tumors (right top)
and quantification (right bottom) (n=10) **p < 0.01, *p <
0.05.
FIGURE 5. (A) Dose-dependent effects on colony for-
mation of HeLa cells by analogs 19 and 21 (B) Caspase
3/7 activity induced by 19, cisplatin and the combination
in SKOV3 cells (C) The effects of 19, 21 and Etoposide
(
positive control) on PARP cleavage in HeLa cells (D) A
combination index (CI) versus fraction affected plot
demonstrating synergistic growth inhibition with 19 and
cisplatin *p < 0.05 vs. control.
Our in vitro studies clearly demonstrate the anticancer
effects induced by 19 are dependent on the presence of
the Michael acceptor. We next investigated if these ef-
fects translate to an ovarian cancer mouse model.41
The ability of analog 19 and 21 to inhibit colony for-
mation was accessed using a clonogenic assay. Cells were
sparsely plated and allowed to grow in the presence or
absence of 19 or 21 for 7 days. Colonies were then
stained using crystal violet and quantified (Figure 5A). In
the plates treated with 19, we observed a dose-
dependent decrease in the number of colonies while no
such effect was observed with 21. This demonstrates that
the Michael acceptor functionality is critical for 19 to
inhibit colony formation.
Our first goal was to determine if analog 19 has anti-
cancer activity as a single agent and to define an optimal
dose for both cisplatin and 19 in an orthotopic ovarian
42,43
cancer model.
Ovarian cancer cells (A2780) were
injected into the peritoneal cavity of nude mice and the
tumors were allowed to establish. On day 3, mice were
divided into four groups and the groups were treated
with vehicle, 1, 2.5 or 5 mg/kg of 19, the dose and route
of administration were selected based on literature re-
ports with parthenolide.4
4-46
The mice were treated in-
Inhibition of the NF-κB pathway has been explored as a
therapeutic strategy to sensitize cancers to current
traperitoneally 5 days a week for 4 weeks. At the end of
the study the mice were sacrificed and the tumor weights
determined. None of the mice treated with 19 showed
any overt toxicity. A dose dependent effect on tumor
growth (~ 10 – 40%) was observed in mice treated with
19. A ~40% reduction in tumor weights in mice treated
with 19 at the highest dose (5 mg/kg). The ~10% reduc-
tion in tumor growth at 1 mg/kg is consistent with the
effects observed with parthenolide at an equivalent dose
in the prophylactic metastasis study.45 Consistent with
our previous studies, we observed a ~25% reduction of
tumor weights in mice treated with cisplatin (2 mg/kg)
when compared to the controls (Figure 6A).41
To determine if 19 can chemosensitize ovarian tumors to
cisplatin, we performed a follow up study in which mice
were treated with 2.5 mg/kg of 19 (resulted in ~21% re-
duction in tumor growth bar #3 Figure 6A) and 2 mg/kg
of cisplatin (resulted in ~30% reduction in tumor growth
bar #5 Figure 6A). Therefore, a reduction of > 51% in
tumor growth with the combination of 2.5 mg/kg of 19
and 2.0 mg/kg of cisplatin will indicate synergism. In-
deed the combination was synergistic with ~65% (bar #7
Figure 6A) reduction in tumor weights compared to the
controls. The reduction of tumor weights by treatment
with 19 + cisplatin is significant compared to treatment
with either drug alone (One-way ANOVA, P < 0.001).
These studies clearly demonstrate that analog 19 has
anticancer activity as a single agent and also demon-
3
7
chemotherapeutics. The NF-κB inhibitor BAY 11-7085
that targets the NF-κB pathway proteins through cova-
lent inhibition via its Michael acceptor sensitizes ovarian
3
8
cancer cells to cisplatin-induced apoptosis. Two key
events during apoptosis are the activation of caspase 3/7
and poly (ADP-ribose) polymerase (PARP) cleavage. In
cells treated with 19 we observed modest induction of
caspase 3/7 by itself and synergistic induction in the
presence of cisplatin (Figure 5B). This is consistent with
reports that implicate activation of NF-κB in chemo-
3
9
resistance. We also observed decreased/cleaved PARP
in cells treated with 19. Importantly no such effects with
21 treated cells (Figure 5C and Figure S2) were observed.
In order to determine if the observed synergistic induc-
tion of caspase 3/7 leads to growth inhibition, we sub-
jected ovarian cancer cells to either 19 or cisplatin alone
and their combination and monitored their effects on the
cancer cell growth over a 3-day period (Figure 5D). The
combination index (CI) values for the various combina-
tions were derived from median effect plot and dose ef-
fect curves (Figure S3) using calcusyn (biosoft.com). CI <
1
indicates synergism, CI = 1 indicates additive effects
40
and CI > 1 indicates antagonism. Concentration com-
binations of 19 and cisplatin in the 1-10 ꢀM range had CI
< 1 indicating synergistic inhibition of ovarian cancer cell
growth (Figure 5D). These studies demonstrate that 19
sensitizes cancer cells to cisplatin induced apoptosis and
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Journal of Medicinal Chemistry
strates the ability to chemosensitize ovarian tumors to
cisplatin (Figure 6A).
0.72
>100
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2.0 ± 0.12
Red-P
To determine if 19 affects NF-κB (p65) levels and NF-κB
regulated proteins (Mcl-1) in the tumors, we conducted
IHC studies with p65 and Mcl-1 antibodies with the ex-
cised tumor tissue. IHC studies showed reduced p65
staining in tumors of animals treated with 19 and cispla-
tin individually and the combination (Figure 6B). Similar
effects were observed in Mcl-1 levels (Figure S4). These
studies suggest that the antitumor effects of 19 in vivo
are mediated by the inhibition of the NF-κB pathway.
Since, in vitro and in vivo studies clearly demonstrate
the anticancer effects of compound 19, we functionalized
the spirocyclic oxindole core with substitutions at vari-
ous positions to generate seven additional analogs (Fig-
ure 7). We screened these for inhibition of cancer cell
growth in A2780 cell lines (Table 1). In this cell line we
observed a ~four-fold higher potency in cell growth inhi-
bition with 19 compared to parthenolide which corre-
lates with ~ 4-fold higher serum stability.
Alkylation (20, 23, 24) of the nitrogen atom on the ox-
indole had modest effects on the growth inhibitory activ-
ity. Analog 25 with a phenyl substitution on the lactone
ring did not have a significant effect on the activity.
Likewise, substitutions at 4-7 positions (26-29) on the
phenyl ring of the oxindole had modest effects when
compared to 19 on the growth of A2780 cells. Only me-
thyl substitutions at the both 4- and 7- positions of the
oxindole (29) showed > 2-fold improvement in the
growth inhibitory effects when compared to analog 19.
As expected, the corresponding reduced spirocyclic ana-
log 30 was inactive (IC50 > 100 ꢀM). Importantly, under
our assay conditions, the growth inhibitory activity of 29
was ~13 fold better (IC50 values 1.0 ꢀM vs. 12.9 ꢀM) than
parthenolide and ~ 20 fold better than ibrutinib. Sur-
prisingly, reducing the Michael acceptor in ibrutinib
We also compared the efficacy of analog 29, its reduced
analog 30, parthenolide, reduced parthenolide (Red-P),
ibrutinib and reduced ibrutinib (Red-I) in a panel of can-
cer cell lines (Table 2, Figure S6). Compound 29 was ~2-
10 fold more potent than parthenolide and ~5-20 fold
more potent than ibrutinib. Reduced compounds 30 and
Red-P resulted in >10-fold loss of activity. However, re-
duced ibrutinib (Red-I) showed modest improvement in
the growth inhibitory activity when compared to ibru-
tinib in all the lines. At the present time, we do not have
an explanation for the results observed with ibrutinib
and its reduced analog. On the other hand the cell-based
activity of both parthenolide and 29 are largely depend-
ent on the presence of the Michael acceptor.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
TABLE 2. Inhibition of HeLaGFP, MiaPaCa-2 and
SW480 cancer cell growth.
IC50 ± SEM (ꢀM)
Inhibitors
HeLaGFP
16.8 ± 1.7
15.1 ± 0.1
MiaPaCa-2
16.6 ± 2.4
8.2 ± 1.4
9.8 ± .05
84.3 ± 11.1
0.9 ± 0.1
>100
SW480
25.6 ± 0.3
13.4 ± 0.2
15.3 ± 0.1
>100
Ibrutinib
Red-I
Parthenolide 5.7 ± 3.3
Red-P
9
0
66.1± 1.9
3.2 ± 0.03
>100
2
3
1.2 ± 0.2
>100
In conclusion, a cell-based based pathway screen with a
focused library of α-methylene-γ-butyrolactone contain-
ing-analogs led to the identification of the isatin derived
spirocyclic analog 19 as a potent inhibitor of TNFα-
induced IKKβ-mediated NF-κB activation. SAR studies
revealed that rigidification of the α-methylene-γ-
butyrolactone and the Michael acceptor in the spirocyclic
system are critical features required for activity. Chang-
ing the context of the α-methylene-γ-butyrolactone from
a fused to a spirocyclic system could explain the in-
creased stability of 19 in serum when compared to par-
thenolide. Using click chemistry we show that the inhibi-
tion of TNFα-induced IKKβ-mediated NF-κB activation
is due to covalent binding of 19 to IKKβ and NF-κB.
Analog 19 inhibited the phosphorylation of IκBα in can-
cer cells and exhibited anticancer activities that was
comparable to known IKKβ inhibitors. On the other
hand, analog 21, a reduced form of 19, was inactive in all
the assays. Analog 19 inhibits ovarian tumor growth as a
single agent and sensitizes ovarian tumors to cisplatin in
an orthotopic model. Our studies clearly demonstrate
that α-methylene-γ-butyrolactone containing spirocyclic
oxindole analog 19 phenocopies the effects of natural
product parthenolide. A second iteration of synthesis
and screening led to the identification of a dimethyl ana-
log 29, which inhibited cancer cell growth with low-ꢀM
potency. Depending on cell lines, analog 29 was ~2-20
fold better than parthenolide. Studies to expand the SAR
of α-methylene-γ-butyrolactone containing spirocyclic
oxindole analogs for the identification of suitable pre-
therapeutic lead compounds with anticancer effects are
currently underway and the results from these studies
will be reported in due course.
(
Red-I) did not alter the growth inhibitory effects. How-
ever, reduction of the Michael acceptor in parthenolide
resulted in loss of activity (Table 1, Figure S5). Although
limited in number, this preliminary study clearly demon-
strates that the spirocyclic oxindole core with the α-
methylene-γ-butyrolactone core can be functionalized to
improve biological activity and possible drug like proper-
ties.
FIGURE 7. Focused library of substituted α-methylene-
γ-butyrolactone – oxindole analogs.
TABLE 1. Inhibition of A2780 cancer cell growth by
substituted α-methylene-γ-butyrolactone – oxindoles.
EC50 values
EC50 values
Analogs
Analogs
(ꢀM)
(ꢀM)
2
2
2
0
3
4
5.5 ±0.58
3.9 ±0.45
2.7 ±0.23
19
29
30
2.3 ± 1.4
1.0 ± 0.16
>100
2
0.1 ±
.26
2
5
3.8 ± 0.21
ibrutinib
1
2
2
6
7
2.2 ± 0.24
3.1 ± 0.32
Red-I
parthenolide
15.2 ± 1.11
12.9 ±
ASSOCIATED CONTENT
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parthenolide. Tetrahedron 1965, 21, 1509ꢀ1519.
1. Kwok, B. H.; Koh, B.; Ndubuisi, M. I.; Elofsson, M.; Crews,
Govindachari, T. R. J., B. S.; Kamat, V. N. Structure of
SUPPORTING INFORMATION
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The supporting Information is available free of charge via
the Internet at http://pubs.acs.org. Experimental for west-
ern blotting, cell viability assay, kB-luciferase assay, click
chemistry, PARP cleavage, Caspase 3/7 assay, colony for-
mation assay, mouse studies, Immunohistochemistry, syn-
thetic procedures, NMR spectra.
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AUTHOR INFORMATION
Corresponding Author
A.N.: phone, +402 559 3793; fax, +402 559 8270; E-mail,
anatarajan@unmc.edu.
13. Ramachandran, P. V.; YipꢀSchneider, M.; Schmidt, C. M.
Natural and synthetic alpha,betaꢀunsaturated carbonyls for NFꢀkappaB
inhibition. Future Med. Chem. 2009, 1, 179ꢀ200.
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Wang, D.; Rathe, S. K.; Meece, F. A.; Noble, K. E.; Sachs, Z.;
Largaespada, D. A.; Harki, D. A. Synthesis and antileukemic activities of
C1ꢀC10ꢀmodified parthenolide analogues. Bioorg. Med. Chem. 2015, 23,
4
1
Islam, M. A.; Singh, S. K.; Vishwakarma, R. A.; Pilcher, L. A. Design,
synthesis and anticancer activity of Michaelꢀtype thiol adducts of alphaꢀ
santonin analogue with exocyclic methylene. Eur. J. Med. Chem. 2015,
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Kempema, A. M.; Widen, J. C.; Hexum, J. K.; Andrews, T. E.;
Present Addresses
737ꢀ4745.
5.
†
Ashland Inc., 601-608, Plot No. 17-18, Platinum Techno
Khazir, J.; Riley, D. L.; Chashoo, G.; Mir, B. A.; Liles, D.;
Park Sector 30-A, Vashi, Navi Mumbai, India
Author Contributions
‡
Equal contribution
1
1
01, 769ꢀ779.
6. Qin, X. Y.; Chen, B. Y.; Fu, J. J.; Shan, L.; Lei, X. G.; Zhang,
ACKNOWLEDGMENT
W. D. Synthesis, cytotoxicity and inhibition of NO production of
ivangustin enantiomer analogues. Eur. J. Med. Chem. 2015, 102, 256ꢀ265.
1
Huang, D. J.; Chen, J. J.; Gao, K. Design, synthesis and biological
evaluation of novel sesquiterpene mustards as potential anticancer agents.
Eur. J. Med. Chem. 2015, 94, 284ꢀ297.
This work was supported in part by CA182820, CA127297,
Nebraska Research Initiative to A.N., LB506 to P.R. and
DOD to R.R. We would like to thank Ed Ezell for NMR,
Krishna Kattel for Mass spectrometry and the Natarajan lab
members for helpful discussions. We would like to thank
Fred & Pamela Buffett Cancer Center Support Grant
7.
Xu, Y. Z.; Gu, X. Y.; Peng, S. J.; Fang, J. G.; Zhang, Y. M.;
18.
Woods, J. R.; Mo, H.; Bieberich, A. A.; Alavanja, T.; Colby,
D. A. Fluorinated aminoꢀderivatives of the sesquiterpene lactone,
parthenolide, as (19)f NMR probes in deuteriumꢀfree environments. J.
Med. Chem. 2011, 54, 7934ꢀ7941.
(
P30CA036727) for NMR core facility.
1
9.
James R. Woods; Huaping Mo; Andrew A. Bieberich;
ABBREVIATIONS
Alavanja, T.; Colby, a. D. A. Aminoꢀderivatives of the sesquiterpene
lactone class of natural products as prodrugs. MedChemComm. 2013, 4,
2
20.
Crooks, P. A. Antiꢀcancer activity of carbamate derivatives of
melampomagnolide B. Bioorg. Med. Chem. Lett. 2014, 24, 3499ꢀ3502.
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Penthala, N. R.; Crooks, P. A. Dimers of Melampomagnolide B Exhibit
Potent Anticancer Activity against Hematological and Solid Tumor Cells.
J. Med. Chem. 2015, 58, 8896−8906.
SAR, Structure Activity Relationship; TNFα, tumor ne-
crosis factor alpha; NF-κB, Nuclear factor kappa B;
IκBα, inhibitor of nuclear factor κB; IKKβ, IκB kinase β;
PARP, poly ADP ribose polymerase; IHC, immunohisto-
chemistry.
7ꢀ33.
Janganati, V.; Penthala, N. R.; Madadi, N. R.; Chen, Z.;
Janganati, V.; Ponder, J.; Jordan, C. T.; Borrelli, M. J.;
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