Facile synthesis of autophagonizer and evaluation of its activity to induce autophagic cell death in apoptosis-defective cell line

Article abstract of DOI:10.1016/j.bmcl.2016.08.035

Some cancer cells are resistant to apoptosis, rendering them irresponsive towards apoptosis-inducing chemotherapy drugs. Another mode of action to kill these apoptosis-defective cells is essential and autophagy, a dynamic process that degrades cytoplasmic contents for cellular maintenance, has been considered as one of the alternate routes. A small molecule inducer of autophagy, autophagonizer was reported to induce cell death through a novel process that is independent of extrinsic apoptosis and the normal signaling pathways of autophagy. Here, we describe an efficient synthetic procedure for the autophagonizer. The newly synthesized autophagonizer (DK-1-49) resulted in an accumulation of autophagy-associated LC3-II and enhanced levels of autophagosomes and acidic vacuoles. Furthermore, cell viability was inhibited by autophagic cell death in not only human cancer cells but also Bax/Bak double-knockout cells. These findings highlight that intrinsic apoptosis is not also involved in the induction of cellular death by the autophagonizer suggesting the autophagonizer is a promising candidate for anticancer therapeutics for cancer cells that are resistant to apoptosis-inducing chemotherapy.

Full text of DOI:10.1016/j.bmcl.2016.08.035

Bioorganic & Medicinal Chemistry Letters 26 (2016) 4753–4756
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Facile synthesis of autophagonizer and evaluation of its activity
to induce autophagic cell death in apoptosis-defective cell line
⇑
Jennifer Nguyen, Luxi Chen, Dhiraj Kumar, Jiyong Lee
Department of Chemistry and Biochemistry, University of Texas at Dallas, 800 W. Campbell Rd., Richardson, TX 75080, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Some cancer cells are resistant to apoptosis, rendering them irresponsive towards apoptosis-inducing
chemotherapy drugs. Another mode of action to kill these apoptosis-defective cells is essential and
autophagy, a dynamic process that degrades cytoplasmic contents for cellular maintenance, has been
considered as one of the alternate routes. A small molecule inducer of autophagy, autophagonizer was
reported to induce cell death through a novel process that is independent of extrinsic apoptosis and
the normal signaling pathways of autophagy. Here, we describe an efficient synthetic procedure for
the autophagonizer. The newly synthesized autophagonizer (DK-1-49) resulted in an accumulation of
autophagy-associated LC3-II and enhanced levels of autophagosomes and acidic vacuoles. Furthermore,
cell viability was inhibited by autophagic cell death in not only human cancer cells but also Bax/Bak dou-
ble-knockout cells. These findings highlight that intrinsic apoptosis is not also involved in the induction
of cellular death by the autophagonizer suggesting the autophagonizer is a promising candidate for anti-
cancer therapeutics for cancer cells that are resistant to apoptosis-inducing chemotherapy.
Ó 2016 Elsevier Ltd. All rights reserved.
Received 20 June 2016
Revised 10 August 2016
Accepted 12 August 2016
Available online 13 August 2016
Keywords:
Autophagonizer
Autophagic cell death
Apoptosis-defective cells
Anticancer therapeutics
Autophagy is a homeostatic process that involves the rearrange-
ment of membrane to sequester intracellular contents into double-
membrane vesicles called autophagosomes. The cargo is then
transferred into lysosomes for degradation by acidic hydrolases
and recycled to sustain cellular metabolism.1,2 Increasing evidence
suggests for the involvement of autophagy in tumorigenesis. In
40–75% of human breast, prostate, and ovarian cancer, the
autophagy-essential gene beclin 1 is monoallelically deleted.3 Par-
tial disruption of beclin 1 decreases autophagy and increases the
cell’s susceptibility to developing into spontaneous tumors such
as lymphomas, lung carcinomas, hepatocellular carcinomas, and
mammary precancerous lesions.4 While reduced levels of beclin
1 increase the likelihood of cancer development, elevated levels
of beclin 1 result in autophagic cell death.5–8 Since autophagy is
commonly associated as a survival strategy, its main role in
tumorigenesis remains controversial and the signaling pathways
from the onset of autophagy to cellular death remain unclear.9,10
Nevertheless, the prospect of actuating cellular death through
the induction of autophagy has attracted considerable attention
resistance may be due to DNA mutation in essential apoptosis
genes, overexpression of anti-apoptosis genes, and/or silencing of
pro-apoptotic genes.14–16 The capability of these cancer cells to
evolve and adopt intrinsic survival strategies is an obstacle that
current researchers face when developing cancer-killing drugs that
rely on apoptotic pathways. Hence, another mode of action to kill
these apoptosis-defective cells is highly desirable.
Cell death by excess autophagy has become one of the main
contenders as an alternate therapeutic strategy against cancer.
Natural alkaloid molecules,17 clozapine,18 arsenic trioxide,19
resveratrol,20 apigenin,21 lapatinib,22 and histone deacetylase inhi-
bitors have demonstrated to induce autophagic cell death and inhi-
bit cell growth in human cancer cell lines.23 A novel small molecule
called autophagonizer was recently reported to induce autophagic
cell death via a unique mechanism that diverges from the tradi-
tional pathways of autophagy.24 Various cancer cell lines were
shown to exhibit sensitivity to autophagonizer. EC50 of the
autophagonizer-induced autophagic cell death was estimated to
be in the range of 3–4 lM. Intriguingly, autophagonizer was
as
a
potential approach towards cancer treatment. Current
described to promote such activity without confiding in apoptotic
machinery, particularly the machinery involved in the extrinsic
pathway.24 Because cancer cells develop insensitivity to apoptosis
in varying degrees, current cancer research aims to develop
anti-cancer drugs that are ideally independent of both extrinsic
and intrinsic apoptosis.11–14 Therefore, we desire to promote
chemotherapy drugs are ineffective to some cancer because they
are resistant to a programmed cell death called apoptosis.11–14 This
⇑
Corresponding author.
E-mail address: jiyong.lee@utdallas.edu (J. Lee).
http://dx.doi.org/10.1016/j.bmcl.2016.08.035
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.

4754
J. Nguyen et al. / Bioorg. Med. Chem. Lett. 26 (2016) 4753–4756
autophagonizer as a potential therapeutic drug by investigating its
unique activity in regards to not only autophagy but also intrinsic
apoptosis. In order to achieve this goal, we have developed the first
synthetic procedure of the autophagonizer because a synthetic
procedure of the compound has not been reported yet. The newly
synthesized autophagonizer was then tested on cancer cells and
apoptosis-defective cells for autophagic activity and cell viability.
Studying the autophagonizer and its peculiar mode of action not
only allows for more discoveries of potential cancer therapeutic
drugs but also brings a new understanding to the complexities of
cellular survival and death.
The synthetic pathways depicted in Scheme 1 outline the chem-
istry of the present study. Ethyl 2-amino-4,5,6,7-tetrahydrobenzo
[b]thiophene-3-carboxylate (1) was easily prepared from the
well-established procedure.25 Compound 1 was then converted
to the corresponding benzylthiourea by reaction with benzylisoth-
iocyanate in ethanol, which were cyclized using potassium hydrox-
ide to give compound 2. Compound 2 underwent nucleophilic
substitution with methyl 2-(chloromethyl)oxazole-4-carboxy-
late26 under basic condition to afford compound 3, which was
hydrolyzed and converted to acid chloride intermediate which
was finally coupled with N-(2-aminoethyl)pyrrolidine giving target
compound autophagonizer (DK-1-49). The overall yield to prepare
the autophagonizer from the starting material (1) was 33%.
In order to validate biological activity of the newly synthesized
autophagonizer (DK-1-49), the compound was then tested on HeLa
cervical cancer cells for autophagic cell death activity. After 24 h
treatment of HeLa cells with DK-1-49 at various dosages,
autophagosomes and DNA were stained with Cyto-ID reagent
and Hoechst nuclear stain, respectively (Fig. 1A). Fluorescence
images reveal that DK-1-49-treated cells exhibited higher fluores-
cent intensity by Cyto-ID-labeled autophagosomes compared with
vehicle control (DMSO)-treated cells, suggesting that autophagy
was induced upon treatment with DK-1-49. A sigmoidal dose-
response curve indicates that DK-1-49 promotes autophagosome
formation in a dose-dependent manner, and the EC50 was approx-
imated to be 3.5 lM (Fig. 1B). Monodansylcadaverine (MDC), a
specific marker for acidic vacuoles, was also used to confirm the
induction of autophagy in DK-1-49-treated cells. Prominent punc-
tate patterns of blue fluorescence representing the presence of
acidic vacuoles were observed (Fig. 1C), further supporting autop-
hagic activity by DK-1-49. The EC50 was calculated to be approxi-
mately 1.3 lM (Fig. 1D). Immunoblot analysis was finally utilized
to assess the conversion of light chain 3-I (LC3-I) for additional
validation of DK-1-49 for its autophagy induction activity. Upon
Scheme 1. Synthesis of autophagonizer (DK-1-49). Reagents and conditions: (a)
BnNCS, KOH, EtOH, Reflux, 16 h, 90%; (b) K2CO3, THF, RT, 16 h, 70%; (c) 1 M LiOH,
THF/MeOH (9:1), RT, 16 h, 94%; (d) Oxalyl chloride, DCM, DMF (cat.), RT, 4 h,
quantitative; (e) TEA, THF, RT, 16 h, 55%.
A
B
9
DMSO
0.25 µM
0.5 µM
1.0 µM
2.0 µM
6.0 µM
EC50 = 3.5 µM
8
7
6
5
4
-7.0
-6.5
-6.0
-5.5
-5.0
-4.5
Log(concentration [M])
C
D
DMSO
0.25 µM
0.5 µM
1.0 µM
2.0 µM
6.0 µM
8.0 µM
E
F
DK-1-49 (µM)
5.0 2.5
2.5 EC50
= 1.3 µM
100
50
0
DMSO Untreated
2.0
1.5
1.0
LC3-I
LC3-II
EC50 = 3.5 µM
Actin
-7.0
-6.5
-6.0
-5.5
-5.0
-4.5
-7.0
-6.5
-6.0
-5.5
-5.0
-4.5
Log (concentration [M])
Log (concentration [M])
Figure 1. Autophagonizer (DK-1-49) induces autophagic cell death of cancer cells. HeLa cells were treated with DK-1-49 for 24 h at various dosages. (A) Treated cells were
stained with the Hoechst 33342 nuclear stain and green Cyto-ID reagent. Images were taken under the DAPI and GFP filter. (B) Fluorescence was read at 340/480 nm (Ex/Em)
for Hoechst nuclear staining and 480/539 nm (Ex/Em) for Cyto-ID autophagosome staining. Fluorescence per cell was taken relative to DMSO-treated cells and used to
construct a sigmoidal dose–response curve. (C) Treated cells were stained with MDC for acidic vacuoles. Images were taken under the DAPI filter. (D) MDC fluorescence was
read at 335/512 nm (Ex/Em). Fluorescence was first normalized against the number of viable cells measured by MTT assay. Fluorescence per cell was then taken relative to
DMSO-treated cells to obtain fold intensity and a sigmoidal dose–response curve was constructed. (E) Immunoblot analysis for conversion of LC3-I to LC3-II. (F) MTT cell
viability assay. The number of viable cells was normalized against DMSO-treated cells to obtain the percentage of viable cells.

J. Nguyen et al. / Bioorg. Med. Chem. Lett. 26 (2016) 4753–4756
4755
activation of autophagy, the microtubule-associated form of LC3,
LC3-I, is converted to the autophagy-associated form, LC3-II.1 After
24 h of treatment with DMSO or DK-1-49, changes in LC3 levels
were evaluated (Fig. 1E). DK-1-49-treated cells exhibited an accu-
mulation of LC3-II while DMSO-treated cells exhibited no signifi-
cant conversion of LC3-I to LC3-II. Note that the increase of MDC
fluorescence and LC3-II accumulation of cancer cells by DK-1-49
was consistent with the previous Letter.24
cells. As mentioned previously, DK-1-71 stimulates weaker autop-
hagy compared to DK-1-49 in HeLa cells. Similar results were
observed in both wild-type and DKO MEFs. Minimal MDC and
Cyto-ID fluorescence were seen (Fig. 3A) and low conversion of
LC3-I to LC3-II was detected (Fig. 3B). Additionally, cytotoxic
effects of DK-1-71 were weaker than those of DK-1-49 by approx-
imately 10-fold in both wild-type cells and apoptosis-defective
cells. The EC50 was determined to be 28.6 lM and 24.2 lM respec-
The autophagonizer was originally reported to induce autopha-
gic cell death. To validate that our autophagonizer exerts similar
cytotoxic effects, 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetra-
zolium bromide (MTT) assay was employed. MTT results showed
that cell death occurred in cells after 24 h treatment with DK-1-
49 (Fig. 1F). The percentage of viable cells dramatically declined
tively (Fig. 3C). These results on apoptosis-defective MEFs further
accentuate that the modification of the pyrrolidine nitrogen to a
carbon markedly reduced autophagic cell death, suggesting that
this nitrogen may be vital for autophagonizer activity. According
to a previous study, autophagonizer promotes cell death in cancer
cells even under the presence of Z-VAD-FMK, a pan-caspase
inhibitor.24 This finding implicates that the extrinsic pathway of
apoptosis is not involved in the mechanism towards autophago-
nizer-induced cell death. Based on our results regarding Bax/Bak
DKO cells, the intrinsic pathway of apoptosis is also not involved
because cell death still occurred after treatment with the
autophagonizer.
by 5-fold after compound treatment at a dosage of 8
l
M. From
the dose–response curve, EC50 was estimated to be 3.5
lM, which
is similar to the previously reported value.24 Taken together from
these results of autophagic activity and cell viability, the DK-1-49
synthesized using the scheme described above was validated to
induce autophagic cell death in cancer cell line.
Autophagonizer was previously reported to undergo a mecha-
nism that is unique from the traditional pathways towards the
onset of autophagy. In order to gain additional insight, we are cur-
rently performing structure–activity relationship (SAR) analysis.
Derivatives of autophagonizer were generated, each bearing a
modification in various regions of the structure. Additional
detailed studies on SAR are necessary and will be reported in due
course. However, we have identified a less-active derivative of
the autophagonizer, DK-1-71 (Fig. 2A). The nitrogen of pyrrolidine
moiety of DK-1-49 was replaced with a carbon in DK-1-71. This
modification resulted in a marked reduction in autophagic cell
death activity in HeLa cells. No sufficient increase in MDC fluores-
cence was observed in cells treated with DK-1-71 compared to
cells treated with DK-1-49 (Fig. 2B), suggesting that DK-1-71
induces weaker autophagy. This was also reflected by DK-1-71’s
autophagic cell death activity as the EC50 of the compound was cal-
In summary, in order to gain a further understanding of
autophagonizer-induced autophagic cell death, we have success-
fully developed the first synthetic procedure of the autophago-
nizer. The synthesized autophagonizer (DK-1-49) was validated
to induce autophagic cell death in cancer cells as similarly
described in the previous study. We have also shown that DK-1-
49 can induce cell death in Bax/Bak DKO MEFs in which the intrin-
sic apoptosis was disabled. This result, combined with the result in
A
B
culated to be approximately 25
lM, about 7-folds higher than DK-
1-49’s EC50 of 3.5 M (Fig. 2C). Because DK-1-71 proves to be less
l
DMSO
DMSO
1.0 µM
3.0 µM
6.0 µM
active in inducing autophagic cell death than DK-1-49 after the
replacement of the nitrogen of pyrrolidine moiety, DK-1-71 was
assessed alongside DK-1-49 in the following studies.
An obstacle to chemotherapy is some cancer cells’ resistance
against apoptosis, rendering them unresponsive to anti-cancer
drugs that target the apoptosis pathway. In order to determine
whether DK-1-49 holds suppressive effects against apoptosis-
defective cells through autophagic cell death, Bax/Bak double-
knockout (DKO) mouse embryonic fibroblasts (MEFs) were used.27
Bax and Bak are pro-apoptotic Bcl-2 family members that perme-
abilize mitochondria to induce apoptosis.28 Therefore, Bax/Bak
DKO MEFs are defective in activating mitochondrial intrinsic apop-
tosis. We have expected that, if DK-1-49 induces cell death through
the intrinsic apoptosis pathway, DK-1-49 may induce less cell
death in Bax/Bak DKO MEFs compared to wild type MEFs. After
15 h of treatment, wild-type and DKO MEFs were stained with
MDC and Cyto-ID reagent (Fig. 3A). Upon treatment with DK-1-
49, an accumulation of acidic vacuoles and autophagosomes was
observed in both wild-type and DKO MEFs. Furthermore, LC3-I to
LC3-II conversion was detected by Western blot, confirming that
autophagy was induced in DK-1-49-treated cells (Fig. 3B). The
wild-type and DKO MEFs were subsequently assessed with MTT
assay for effects on cell viability (Fig. 3C). DK-1-49 was shown to
have potent cytotoxicity in both wild-type and DKO MEFs. The
apparent EC50 calculated from the dose-response curve for the
25.0 µM
C
EC50 = ~ 25 µM
Figure 2. Identification of the less active form of autophagonizer, DK-1-71.
(A) Structure of DK-1-71. The arrow indicates the site of modification of DK-1-49
in DK-1-71. (B) After 24 h treatment, cells were stained with MDC for acidic
vacuoles. Images were taken under the DAPI filter. (C) Cytotoxicity of DK-1-71 in
HeLa cells was measured by MTT assay. Viable DK-1-71-treated cells were
normalized against DMSO-treated cells to obtain the percentage of viable cells.
wild-type and DKO MEFs were essentially identical, 2.1
2.2 respectively. These results further suggest DK-1-49’s
potentiality as an anti-cancer drug against apoptosis-defective
lM and
lM

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J. Nguyen et al. / Bioorg. Med. Chem. Lett. 26 (2016) 4753–4756
A
B
DKO
DK-1-71
(µM)
WT
DK-1-71
(µM)
2.5 DMSO
DK-1-49 (µM)
DK-1-71 (µM)
DK-1-49
(µM)
5
DK-1-49
(µM)
DMSO
2
2
4
4
2.5
5
2.5 DMSO
5
2.5
5
LC3-I
LC3-II
C
DK-1-49
DK-1-71
EC50 (WT) = 2.1 µM
EC50 (DKO) = 2.2 µM
EC50 (WT) = 28.6 µM
EC50 (DKO) = 24.2 µM
150
100
50
150
100
50
DKO
WT
DKO
WT
0
0
-8
-7
-6
-5
-4
-3
-2
-7.0
-6.5
-6.0
-5.5
-5.0
-4.5
Log (concentration [M])
Log (concentration [M])
Figure 3. Autophagic cell death of apoptosis-defective cells by DK-1-49. Wild-type (WT) and Bax/Bak double knockout (DKO) MEFs were treated for 15 h with DK-1-49, DK-
1-71, and vehicle control (DMSO) at the indicated concentrations. (A) Treated cells were stained with Cyto-ID (green fluorescence) reagent for autophagosomes and MDC
(blue fluorescence) for acidic vacuoles. (B) Immunoblot analysis for the conversion of LC3-I to LC3-II. (C) Cytotoxicity of DK-1-49 and DK-1-71 in wild-type and Bax/Bak DKO
MEFs. Cell viability was measured by MTT assay and normalized against DMSO-treated cells to obtain the percentage of viable cells.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.08.
035. These data include MOL files and InChiKeys of the most
important compounds described in this article.
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