Antitumor activity of FL118, a survivin, Mcl-1, XIAP, and cIAP2 selective inhibitor, is highly dependent on its primary structure and steric configuration

Article abstract of DOI:10.1021/mp4004282

We recently reported the identification and characterization of a novel small chemical molecule designated FL118. FL118 selectively inhibits multiple cancer survival and proliferation-associated antiapoptotic proteins (survivin, Mcl-1, XIAP, cIAP2) and eliminates small and large human tumor xenografts in animal models (Ling et al., PLoS One 2012, 7, e45571). Here, we report a follow-up study on the structure-activity relationship (SAR) of the hydroxyl group in the lactone ring of FL118. We found that the superior antitumor efficacy of FL118 heavily depends on its steric configuration through comparing the antitumor activity of FL118 with FL113 (the racemic mixture of FL118). Consistently, FL118 proved much more effective in inhibiting the expression of survivin, Mcl-1, and cIAP2, both in vitro and in vivo, compared to FL113. Additionally, Tet-on controlled induction of survivin or forced expression of Mcl-1 protects cancer cells from FL118-mediated growth inhibition and cell death. To further explore the SAR, we synthesized seven position 20-esterifiable FL118 and FL113 derivatives. Studies on these seven new compounds revealed that keeping a free hydroxyl group of FL118 is also important for high antitumor efficacy. Together, these studies confirm the superior anticancer activity of FL118 and narrow the window for further SAR studies to generate novel analogues based on FL118 core structure on its other potential chemical positions.

Full text of DOI:10.1021/mp4004282

Article
pubs.acs.org/molecularpharmaceutics
Antitumor Activity of FL118, a Survivin, Mcl-1, XIAP, and cIAP2
Selective Inhibitor, Is Highly Dependent on Its Primary Structure and
Steric Configuration
†
‡
§
‡
†
,†
Jiuyang Zhao, Xiang Ling, Shousong Cao, Xiaojun Liu, Shengbiao Wan, Tao Jiang,*
,‡,∥
and Fengzhi Li*
†Key Laboratory of Marine Drugs, Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, 5
Yushan Road, Qingdao, Shandong 266003 China
‡
§
∥
Departments of Pharmacology & Therapeutics and Medicine and NCI-Supported Experimental Therapeutics Program, Roswell
Park Cancer Institute, Elm and Carlton Streets, Buffalo, New York 14263, United States
*
S Supporting Information
ABSTRACT: We recently reported the identification and
characterization of a novel small chemical molecule designated
FL118. FL118 selectively inhibits multiple cancer survival and
proliferation-associated antiapoptotic proteins (survivin, Mcl-1,
XIAP, cIAP2) and eliminates small and large human tumor
xenografts in animal models (Ling et al., PLoS One 2012, 7,
e45571). Here, we report a follow-up study on the structure−
activity relationship (SAR) of the hydroxyl group in the lactone
ring of FL118. We found that the superior antitumor efficacy of
FL118 heavily depends on its steric configuration through
comparing the antitumor activity of FL118 with FL113 (the
racemic mixture of FL118). Consistently, FL118 proved much
more effective in inhibiting the expression of survivin, Mcl-1, and
cIAP2, both in vitro and in vivo, compared to FL113.
Additionally, Tet-on controlled induction of survivin or forced expression of Mcl-1 protects cancer cells from FL118-mediated
growth inhibition and cell death. To further explore the SAR, we synthesized seven position 20-esterifiable FL118 and FL113
derivatives. Studies on these seven new compounds revealed that keeping a free hydroxyl group of FL118 is also important for
high antitumor efficacy. Together, these studies confirm the superior anticancer activity of FL118 and narrow the window for
further SAR studies to generate novel analogues based on FL118 core structure on its other potential chemical positions.
KEYWORDS: IAP inhibitor, FL118, camptothecin analogue, SAR analysis, human tumor animal models, cancer cells
1
4
1
. INTRODUCTION
survivin, Mcl-1, XIAP, and cIAP2. In this regard, not only
1
−12
16−21
22,23
24−31
survivin,
but also XIAP,
cIAP2,
and Mcl-1,
are
Studies of the inhibitor of apoptosis (IAP) protein survivin and
its gene in the literature have revealed its important role in
anticancer drug resistance and cancer progression.
established and validated genetically modified cancer cell models
in which the luciferase reporter gene was driven under the human
survivin gene regulatory sequences including the untranslated
regulatory sequences of survivin mRNA. Using this assay
system via high throughput screening of chemical compound
libraries, followed by in vitro and in vivo testing, we finally
identified one small chemical molecule designated FL118
involved in anticancer drug resistance and/or prognostic/
diagnostic values in various types of cancer.
The exceptional in vivo antitumor activity of FL118 has not
been recognized until our recent report which demonstrates that
FL118 shows superior and broad antitumor efficacy. This
suggests that FL118 would be a great core structure for
generation of new FL118 analogs. As an additional piece of
evidence, our recent studies revealed that FL118, in an
intravenous (i.v.) compatible formulation, further decreases its
toxicity to animals and increases its therapeutic index (TI). We
hope that, through studies assessing the structure−activity
relationship (SAR) between FL118 and its close structurally
1
−12
We have
14
13
32
(
Figure 1) which possesses superior antitumor activity in animal
14
models of human tumors. Interestingly, while FL118
(
NSC634724) structurally is similar to camptothecin (Figure
15
1) and was considered as a camptothecin analogue, FL118 is
actually an IAP inhibitor and shows poor ability to inhibit
Received: July 25, 2013
topoisomerase 1 (Top1) activity in comparison with SN-38, the
active form/metabolite of irinotecan (Figure 1). We previously
demonstrated that FL118 selectively inhibits the expression of
Revised: October 31, 2013
Accepted: December 15, 2013
Published: December 15, 2013
14
©
2013 American Chemical Society
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Figure 1. Structure of camptothecin, FL118, FL113, irinotecan, SN-38 (the active form of irinotecan), and topotecan.
a
related analogues, we will be able to obtain overall better
anticancer molecules that are highly relevant to the FL118
structure but show an improved TI without increasing adverse
toxicity to normal tissues. Previous studies indicated that the
Scheme 1. Synthesis of Compound FL118 and FL113
1
0,11-methylenedioxycamptothecin (10,11-MDC or FL113)
33
(
Figure 1) exhibits high antitumor activity. In the present
study, we first compared the antitumor activity between FL113
NSC606174) and its S-configuration, FL118 (NSC634724).
(
We found that the superior antitumor efficacy of FL118 is highly
dependent on the steric configuration of its hydroxyl group in the
lactone ring (Figure 1), since antitumor activity of FL113 is much
weaker than FL118. We then synthesized seven position 20-
esterifiable FL113 and FL118 derivatives for comparison and
found that keeping a free hydroxyl group is also important for
high antitumor efficacy.
2. CHEMISTRY
According to published procedures, 2-amino-4,5-methylenediox-
ybenzaldehyde 3 was prepared from piperonal 1 by nitration and
3
4,35
reduction.
The synthesis of FL118 and FL113 was
accomplished using a Friedlander condensation between
a
Reagents and conditions: (a) HNO , 2−5 °C, 4 h, 68%; (b) FeSO ·
2 4 2
2 h, 65%; (d) p-TsOH, toluene, reflux, 12 h, 67%.
3
4
compound 3 and the known tricyclic keto lactone 4 and 5,
3
6,37
7H O, NH OH, EtOH/H O, 0.5 h, 71%; (c) p-TsOH, toluene, reflux,
1
respectively
(Scheme 1).
Coupling of compound FL113 and FL118 with substituted
benzoic acids 6, catalyzed by EDCI and DMAP, resulted in
position 20-esterifiable FL113 derivatives 7a, 7f, 7g, and FL118
derivatives 7b−7e (Scheme 2).
analog structurally relevant to FL118, Figure 1) at its MTD level
(
100 mg/kg, weekly × 4) in two human colon tumor xenografts
directly established from patients’ colon cancer tissues. However,
FL118 showed strikingly better antitumor activity than FL113
3. RESULTS AND DISCUSSION
(
Figure 2). This indicates that the compound with an R
3
.1. Superior Antitumor Activity of FL118 Heavily
configuration likely has a much weaker antitumor activity than
the compound with an S configuration for the “−OH” group at
the 20-position. This finding is consistent with an earlier finding
that the activity of camptothecin analogs is highly chemical
Depends on Its Steric Configuration. We previously showed
that FL118 is highly effective in inhibiting human head-and-neck
14
and colon tumors in animal xenograft models. Through
comparison of antitumor potential among 16 chemical
structurally relevant FL118 analogues (Supplemental Table
S1), we found that FL113 and FL118 are the top two compounds
showing the highest anticancer potential. To further study the
SAR for compounds that are structurally highly relevant to
FL118, we first compared the antitumor activity between FL118
and FL113 in human tumor animal models. Our studies
indicated that FL118 and its racemic mixture FL113 showed
better antitumor activity at their sub-MTD (maximum tolerated
dose) level [1 mg/kg, of note FL113/118 MTD is ∼1.5 mg/kg in
38
structurally dependent.
FL118, FL113, and irinotecan are structurally related
camptothecin analogues. Early studies demonstrated that
39
40
camptothecin inhibits the synthesis of DNA, RNA, and
41
protein, which was later found to be due to inhibition of DNA
4
2,43
topoisomerase 1 (Top1) activity.
As a proof, certain Top1
44
mutants in cancer cells produced resistance to camptothecin.
For example, the functional activity for irinotecan or topotecan is
unexceptionally linked to inhibiting DNA Top1 activity as a
45−49
major mechanism of action (MOA).
Our previous studies
14
weekly (wk) × 4 schedules ] than irinotecan (a camptothecin
showed that FL118 greatly exhibits superior antitumor efficacy in
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Scheme 2. Synthesis of Compound 7a−7g
Figure 2. Comparison of the in vivo antitumor efficacy among FL113, FL118, and irinotecan (CPT-11): The SCID mouse models of human colon
cancer xenografts were used to evaluate the relative antitumor activity for FL113 and FL118 versus irinotecan (control). Two human primary colon
cancer xenografts 11124 (a) and 14528 (b), which were originally isolated from anonymous colon cancer patients, were first established in SCID mice
and then used in the experiment. Drug treatment was conducted with the clinically relevant schedule of irinotecan (weekly × 4) indicated by the arrows.
The initial drug treatment was designated Day 0, and the treatment was initiated 7 days post subcutaneous tumor implantation, on which tumor size is
3
about 200−250 mm . The tumor curve in each treatment condition is the mean ± SE derived from five individual tumors on five mice. Doses: irinotecan,
100 mg/kg per dose; FL113, 1.0 mg/kg per dose; and FL118, 1.0 mg/kg per dose.
1
4
comparison with irinotecan and topotecan. However, FL118
only showed about half of the Top1 inhibition ability of SN-38,
the active metabolite of irinotecan, at the highest concentration
FaDu-established tumor xenograft animal model to further
explore its potential. Our data revealed that, while the tumors in
the control group (n = 10) treated with the vehicle reached their
maximal sizes allowed by Institutional Animal and Care Use
14
(
1 μM) that can be reached by irinotecan in vivo. Moreover, in
3
contrast to the weak inhibitory effects of FL118 on Top1 activity
at a dose as high as 1 μM, FL118 effectively initiated cancer cell
apoptosis and the inhibition of survivin promoter activity,
surviving expression, and cancer cell growth at 0.1 nM to 10 nM
levels, indicating that the inhibition of Top1 activity by FL118
may not play a major role in its antitumor activity, apoptosis
induction, and cancer cell growth inhibition.
Committee (IACUC) regulations (≤2000 mm ) within two
weeks (Figure 3a), the tumors in the experimental groups (n = 10
per group) treated with FL118 at its sub-MTD (1 mg/kg and
1.25 mg/kg) with the weekly × 4 schedule resulted in tumor
eradication without relapse in a high percentage of mice (Figure
3b, c). Specifically, FL118 eliminated 6 out of 10 tumors in mice
at 1 mg/kg with weekly × 4 schedules (Figure 3b); and 7 out of
10 tumors in mice at 1.25 mg/kg with weekly × 4 schedules
(Figure 3c). Additionally, favorable results were also obtained
using the human colon SW620 cancer cell line-derived tumor
xenograft (Figure 3d, e, f).
1
4
3
.2. FL118 Exhibits a High Ability To Eradicate Human
Tumors in Animal Models at a Sub-MTD Dose. To confirm
the antitumor efficacy of FL118 at its sub-MTD condition (<1.5
mg/kg), we used the human head-and-neck cancer cell line
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Figure 3. Antitumor activity of FL118 at its sub-MTD: FL118 treatment (designated Day 0, weekly × 4, indicated with arrows) initiated 7 days after
3
subcutaneous tumor implantation, when individual tumors grew to ∼200−250 mm . (a, b, and c) Antitumor activity of FL118 at its sub-MTD (its MTD:
1
.5 mg/kg weekly × 4). Antitumor activity of FL118 in individual mice (N = 10 per group) at the dose of 1 mg/kg (b) and 1.25 mg/kg (c) versus the
vehicle control (a) in mice bearing human FaDu head and neck tumor xenografts is shown. (d, e, and f) Antitumor activity of FL118 in individual mice at
the dose of 1 mg/kg (e) and 1.25 mg/kg (f) versus the vehicle control (d) in mice bearing human SW620 tumor xenografts is shown.
Our results showed that FL118 treatment at its MTD (1.5 mg/
kg, weekly × 4) eradicates up to 100% human tumors in animal
models using a human primary head-and-neck cancer tissue-
14
established xenograft tumor. Here, our new data revealed that a
significant percentage of tumors could be eliminated by FL118
even at a dose under its MTD (Figure 3).
3.3. FL118 Appears To Be a Much Better Compound to
Inhibit Its Downstream Targets than FL113 in Vitro and in
Vivo. Our previous studies revealed that, consistent with its
superior antitumor activity, FL118 selectively inhibits multiple
cancer cell survival and proliferation-associated antiapoptotic
14
proteins including survivin, Mcl-1, XIAP, and cIAP2. To
determine the possibility that the lower efficacy of FL113 in
antitumor activity in comparison with FL118 (Figure 2) is due to
the lower effectiveness of FL113 in the downregulation of FL118
targets (survivin, Mcl-1, XIAP, and cIAP2), human head-and-
neck FaDu cancer cells were treated with and without FL113 (10
nM) or FL118 (10 nM) for 48, 72, and 96 h, followed by
determining FL118 target protein expression using Western
blots. The results showed that FL118 appears to be much more
effective in inhibiting these protein targets (Figure 4). To further
investigate whether FL113 and FL118 treatment could display
similar results as observed in vitro using cultured cancer cells
Figure 4. Effects of FL113 and FL118 on the expression of survivin, Mcl-
1, XIAP, and cIAP2: (a) Subconfluent human head-and-neck FaDu
cancer cells were treated with or without FL113 (10 nM) or FL118 (10
nM) for 48, 72, and 96 h. Cells were then lysed and analyzed by Western
blots using antibodies for survivin, Mcl-1, XIAP, or cIAP2. Actin is the
internal control for total protein loading. (b) Human head-and-neck
FaDu cancer cell-derived xenograft tumors were isolated from SCID
mice with or without FL113 or FL118 treatment for 72 h (one time i.p.
injection, 1.5 mg/kg). The isolated tumors were then lysed using a
motor-driven homogenizer and analyzed by Western blots with
antibodies for survivin, Mcl-1, XIAP, cIAP2, or actin (internal control
for total protein loading). Results from two independent tumors from
two mice are shown in parallel.
(
Figure 4a), we collected human FaDu tumor tissues from
xenograft mice 72 h post FL113 and FL118 treatment and
analyzed relevant gene expression by Western blots. We found
that consistent with the different in vivo antitumor activity
between FL118 and FL113 (Figure 2), FL118 was much more
effective in inhibiting relevant gene expression in vivo as well
(Figure 4b). Interestingly, while FL118 was shown to inhibit
XIAP in cultured cancer cells, FL118 exhibited no inhibitory
effect on XIAP from the in vivo tumor tissues (Figure 4b). This is
an intriguing observation and would be worthy of further
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investigation to determine whether this is a cancer cell type-
specific phenomenon or evidence that the results derived from in
vitro studies may not be fully consistent with the results derived
from the in vivo studies. Alternatively, downregulation of XIAP in
vivo may need a much longer time. Nevertheless, based on
previous studies in the literature, we know that, while multiple
antiapoptotic proteins from the IAP and Bcl-2 families (e.g.,
survivin, Mcl-1, XIAP, or cIAP2) could be simultaneously
expressed in cancer, the essential role of these individual
molecules in cancer initiation and development can be cancer
type- and/or individual cancer patient-dependent. To induce
cancer cell death, in many cases, it is unnecessary to inhibit the
expression of all these antiapoptotic proteins for cancer cell
apoptosis.
We would like to point out a differential level of inhibition in
survivin and Mcl-1 expression between the two tumor tissue
samples at 72 h following FL118 treatment (Figure 4b). This
variation is consistent with the variation observed during the in
vivo tumor regression shown in Figure 3. While most tumors
regressed, some tumors were only inhibited (Figure 3).
Therefore, the variation in FL118 target inhibition and tumor
regression could be due to the distinct interaction between tumor
and individual mice. This may result in such as vascular density
differences between tumors and individual mice. Further studies
are required to explore this phenomenon.
Figure 5. Role of survivin and Mcl-1 in FL118-mediated cancer cell
growth inhibition and cell death: (a) Semiquantitative RT-PCR
(semiQPCR) shows representative Tet-on induced survivin expression
cell clones. The process of generation of individual cell clones with Tet-
on controlled/Dox-inducible expression of survivin in tumorigenic L929
fibrosarcoma cells was described in the Experimental Section. Cell
clones were induced with 4 μg/mL Dox for 48 h; survivin mRNA
expression was determined by semiQPCR and displayed on 1% agarose
gels. GAPDH mRNA expression was used as an internal control during
semiQPCR. (b) Induction of survivin expression by Dox protects L929
cells from FL118-mediated growth inhibition. The Western blot insert
shows survivin protein induction by Dox (the insert in b). L929#54 cells
were induced with and without Dox at 4 μg/mL for 48 h and then
treated with FL118 in a series of concentrations as indicated in the
presence and absence of Dox for 72 h. Cell viability was determined by
MTT assay. (c) Induction of survivin expression by Dox protects L929
cells from FL118-mediated cell death. L929#54 cells were induced with
and without Dox at 4 μg/mL for 48 h and then treated with 100 nM
FL118 in the presence and absence of Dox for 72 h. The DNA
fragmentation-induced cell death was determined by the DNA
fragmentation cell death ELISA (Roche). (d) Forced expression of
Mcl-1 in H2122 lung cancer cells protects cells from FL118-mediated
growth inhibition in a limited range of FL118 concentrations. H2122
cells were transiently transfected with pcDNA3.1 control vectors or with
pcDNA3.1-Mcl-1 expression vectors for 16 h. The transfected cells were
then treated with different concentrations of FL118 as indicated for 72 h.
The cell viability was then determined by MTT assay. The Western blot
insert shows survivin protein expression in Mcl-1 transfected and
untransfected H2122 lung cancer cells at the 48 h time point. *,
represents the p-value < 0.05.
Finally, computational molecular modeling of the physical
interaction of FL118 with the survivin X-ray crystal structure
(PDB ID: 1F3H, 2.58 Å resolution) using SYBYL-X software
package (Tripos, Inc., St. Louis, MO) indicated that FL118 forms
several hydrogen bonds with survivin. However, using tritium-
3
labeled FL118 (H −FL118, Moravek Biochemicals) as a probe
to hybridize the ProtoArray that displays over 9000 proteins
(
Invitrogen), we failed to find a physical interaction of FL118
with the human survivin protein. Of course, for many reasons, the
FL118−survivin interaction could be missed in this protein array
experiment. This includes but may not be limited to the fact that
the affinity between FL118 and survivin is too low (i.e., K >
d
μM); FL118 may only recognize the survivin protein when
survivin is within a protein complex associated with other
proteins, and/or the survivin protein may be in an unrecogniz-
able confirmation on the ProtoArray surface. Therefore, studies
are required in the future to explore this interesting area.
3.4. Alternative Demonstration of a Role of Survivin
and Mcl-1 in FL118-Mediated Cancer Cell Growth
Inhibition and Cell Death. Previously, we demonstrated that
silencing of survivin by survivin-specific shRNA significantly
increases FL118-mediated cancer cell growth inhibition and
14
apoptosis induction (increased Annexin V staining). Similarly,
FL118-induced cell growth inhibition in comparison with
pcDNA3.1 control vector transfected cells (Figure 5d). These
data alternatively confirmed that survivin and Mcl-1 play a role in
FL118 function and are downstream targets of FL118. Of note,
we found that the L929 cell line is good for obtaining a Tet-on
controlled survivin induction system and that the H2122 cell line
is good for transient transfection of the pcDNA3.1-Mcl-1
expression vector.
3.5. Presence of a Free Hydroxyl Group (−OH) in the
Lactone Ring of FL118 and FL113 Is Critical for FL118 or
FL113 To Show Superior Antitumor Efficacy. Based on the
fact that both FL113 and FL118 showed stronger antitumor
activity than irinotecan (Figure 2), it is appropriate to do a
comparison among FL113- and FL118-derived analogs. We
synthesized three FL113 derivatives and four FL118 derivatives
by esterification of the hydroxyl group at the 20-position with
shRNA knockdown of Mcl-1 expression significantly increased
14
FL118-mediated PARP cleavage, a hallmark of apoptosis, while
overexpression of XIAP or cIAP2 decreases PARP cleavage,
14
caspase 3 activation, and Annexin V staining, suggesting these
gene products play a role in FL118 function. Here, we generated
a Tet-on controlled, doxycycline (Dox)-inducible survivin
expression system (Figure 5a). Using this system we
demonstrated that induction of survivin expression by Dox in
the tumorigenic L929 fibrosarcoma cells significantly increases
L929 cell resistance to FL118-induced growth inhibition in
comparison with L929 cells without Dox induction (Figure 5b).
Induction of survivin expression by Dox also significantly
blocked FL118-mediated DNA fragmentation-induced cell
death (Figure 5c). Similarly, forced expression of pcDNA3-
Mcl-1 in H2122 lung cancer cells increased cell resistance to
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various substituted benzoic acids (Schemes 1 and 2). Then, we
determined their ability to inhibit survivin promoter activity and
cancer cell growth. Our results showed that most of these
compounds exhibit the inhibition of survivin promoter-driven
luciferase activity at a concentration of 1000 nM in 2008 ovarian
cancer cells (Figure 6). Consistently, the inhibitory activity of the
Figure 6. Effects of the seven derivatives of FL113 and FL118 on
survivin promoter-driven luciferase activity: Ovarian cancer cells (2008)
that stably express the survivin promoter-luciferase cassette at 50−70%
subconfluence were treated with vehicle (control) or with one of the
seven FL113 or FL118 derivatives at three concentrations as shown for
Figure 7. Effects of the seven derivatives derived from FL113 or FL118
on cancer cell growth: Three cancer cell lines (FaDu, SW620, HCT-8)
as shown were used in this study. Cells at 50−70% subconfluence were
treated with vehicle or with one of the seven FL113 or FL118 derivatives
at three concentrations as shown for 72 h. Cell growth and viability were
then determined by MTT assay. Data obtained from each cancer cell line
were plotted as a histogram. Individual bars are the mean ± SD
24 h. Cells were then lysed, followed by the luciferase activity assay as
described in the Methods section. Data are presented in histograms, and
each bar is the mean ± SD (standard deviation) derived from three
independent assays. The asterisk (*) denotes typical comparisons with
p-value <0.05.
(standard deviation) derived from independent assays (N = 4) in at least
triplicates. (a) Results from the FaDu head and neck cancer cell line. (b)
Results from the SW620 colon cancer cell line. (c) Results from the
HCT-8 colon cancer cell line. The asterisk (*) denotes typical
comparisons with p-value <0.05.
pure S-configuration derivatives of 7b and 7c is higher than that
of the mixture of R- and S-configuration derivatives, 7a, 7f, and
7g (Figure 6). We further tested their potential to inhibit cell
growth in three different cancer cell lines and found that similar
to their ability to inhibit survivin promoter activity shown in
Figure 6, most of these compounds at a concentration of 1000
nM could inhibit cancer cell growth (Figure 7). For the head and
neck FaDu cell line, the inhibitory activity of 7a to 7f derivatives
is higher than that of 7g. For the colon HCT-8 cell line, the S-
configuration 7b is the most active compound among these seven
compounds. However, these results (Figures 6 and 7) are about
inhibition of survivin promoter-luciferase activity and/or cell
proliferation in vitro assay at the micromolar concentration level
(Figures 6 and 7). The loss of antitumor activity for FL118
derivatives is likely due to the lack of appropriate hydrolytic
enzymes to release the added chemical groups and restore the
free hydroxyl group at position 20 of the FL118 molecule in
animal models. This result suggests that the presence of a free
hydroxyl group is important for FL118 to exhibit superior
antitumor effects. In short, our studies are not fully consistent
with the previous notion that modification of the free hydroxyl
group in camptothecin or its analogues could improve in vivo
antitumor activity and pharmacokinetic profile and reduce
14
1
000 to 10,000 fold less effective than FL118. Next, we selected
the compounds 7b and 7c to determine their antitumor activity
in both head-and-neck (FaDu) and colon (SW620) tumors. The
in vivo animal model experiments revealed that while these
derivatives significantly decrease toxicity to animals (i.e., an
increased MTD), they nearly lost antitumor activity (Figure 8a,
b).
51−54
gastrointestinal toxicity.
Our studies indicate that the
The initial idea for replacement of the hydrogen atom in the
hydroxyl group with various small chemical groups was to
generate new FL118 derivatives, with the hope of further
decreasing toxicity to animals, without affecting antitumor
activity by temporarily sealing the hydroxyl group during drug
delivery. Although previous studies suggest that modification of
the free hydroxyl group in camptothecin analogs via ester binds
could be a promising way to improve in vivo antitumor activity
and pharmacokinetic profile and reduce gastrointestinal
overall decrease of toxicity (increased MTD) from position 20
FL118 derivatives could not override the loss of antitumor
activity (Figure 8), suggesting FL118 is a unique core structure
platform for generating novel analogues.
4
. CONCLUSION
Based on our previous study of FL118, we conducted a follow-up
study on the SAR of the hydroxyl group in the lactone ring of
FL118. We found that the superior antitumor efficacy of FL118
heavily depends on both its primary structure and its steric
configuration. FL118 exhibits a high ability to eradicate human
tumors in animal models at doses even lower than its MTD. We
synthesized seven position 20 esterifiable FL113 and FL118
derivatives and tested their in vitro and in vivo antitumor
5
0−53
toxicity,
our studies with 20 O-linked benzoic acid ester
derivatives of either FL118 or FL113 resulted in almost complete
loss of antitumor activity in vivo, although toxicity was
significantly lowered (MTD increased, Figure 8), and some of
the FL113 and FL118 derivatives (e.g., 7a, 7b, 7c) showed
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measured on a WRX-1S melting-point apparatus and were
1
13
uncorrected. H NMR and C NMR spectra were taken on Jnm-
Ecp-600 spectrometer (Jeol Ltd., Tokyo, Japan) with tetrame-
thylsilane (Me Si) as the internal standard. Mass spectra were
4
recorded on a Q-TOF Global mass spectrometer (Waters Co.,
Milford, MA, USA).
5
.1.2. General Procedure for the Synthesis of Compounds
7
a−7g. At room temperature, EDCI (423 mg, 2.21 mmol),
DMAP (62 mg, 0.51 mmol), and substituted benzoic acid 6 (1.02
mmol) were added slowly to a stirring solution of FL113 or
FL118 (100 mg, 0.255 mmol) in dry dichloromethane (60 mL),
and then the resulting mixture suspension was heated to reflux
under N atmosphere for 12 h. After being cooled to room
2
temperature, the mixture solution was then washed with 0.1 M
HCl, water, and brine, and the organic layer was dried over
anhydrous MgSO for 12 h. After filtration to remove MgSO ,
4
4
the solvent was removed under reduced pressure. The residue
was purified by flash chromatography on a silica gel (CH Cl /
2
2
CH OH = 97/3, v/v) to yield the desired compound, 7a−7g,
3
respectively.
5.1.3. 10,11-Methylenedioxycamptothecin-20 (RS)-O-(4-
bromobenzoate) (7a). The general synthetic method described
1
above afforded 7a as yellow solid (42%), mp > 250 °C; H NMR
(
DMSO-d , 600 MHz) δ: 8.44 (s, 1 H), 8.05 (d, J = 8.76 Hz, 2
6
H), 7.85 (d, J = 8.82 Hz, 2 H), 7.49 (s, 1 H), 7.43 (s, 1 H), 6.93 (s,
H), 6.25 (d, J = 4.44 Hz, 2 H), 5.55 (s, 2 H), 5.21 (s, 2 H), 2.31
1
13
(
m, 2 H), 1.03 (t, J = 7.8 Hz, 3 H); C NMR (CF COOD, 150
3
MHz) δ: 171.4, 167.6, 158.7, 153.2, 148.3, 141.1, 140.1, 139.5,
Figure 8. Antitumor activity and toxicity (body weight loss) of the
selected two FL118 derivatives (7b, 7c) generated by replacement of the
hydrogen atom of the hydroxyl group in the lactone ring of FL118.
1
1
5
39.2, 132.9, 131.7, 131.5, 129.8, 129.6, 125.6, 122.6, 105.4,
04.2, 102.5, 97.2, 77.4, 67.5, 54.6, 51.8, 31.7, 6.3; MS(ESI): m/z,
+
76.1 [M + H] .
Treatment was initiated 7 days after xenograft tumor implantation
3
5.1.4. 10,11-Methylenedioxycamptothecin-20 (S)-O-(4-io-
(
designated Day 0) at which time the tumor size is about 200−250 mm .
dobenzoate) (7b). The general synthetic method described
Treatment with the schedule of every other day for five times (q2 × 5) is
indicated with arrows. (a and b) The mean tumor curves derived from
five tumors on five mice treated with vehicle (control) or with one of the
two FL118 derivatives (7b, 7c) for FaDu head and neck (a) or SW620
colon (b) tumors in SCID mouse models. The standard error (SE) of
tumor variation is within 15% in the same group. (c) The mean mouse
body weight curves derived from five mice treated with vehicle (control)
or with one of the two FL118 derivatives (7b, 7c). Of note, the standard
error (SE) of body weight loss variation is within 10% among each
group.
1
above afforded 7b as yellow solid (56%), mp > 250 °C; H NMR
(DMSO-d , 600 MHz) δ: 8.45 (s, 1 H), 8.03 (d, J = 8.82 Hz, 2
6
H), 7.87 (d, J = 8.22 Hz, 2 H), 7.49 (s, 1 H), 7.44 (s, 1 H), 6.91 (s,
1
H), 6.25 (d, J = 4.38 Hz, 2 H), 5.55 (s, 2 H), 5.22 (s, 2 H), 2.30
13
(
m, 2 H), 1.03 (t, J = 7.68 Hz, 3 H); C NMR (DMSO-d , 150
6
MHz) δ: 167.6, 164.6, 157.1, 151.9, 150.0, 149.2, 147.1, 146.9,
1
1
45.8, 138.8, 131.8, 130.8, 128.9, 128.1, 126.3, 118.4, 105.2,
03.9, 103.5, 103.0, 95.7, 77.1, 66.8, 49.1, 30.7, 8.3; MS(ESI): m/
+
z, 623.4 [M + H] .
5
.1.5. 10,11-Methylenedioxycamptothecin-20 (S)-O-(4-ni-
activities. Although these new FL118 derivatives decrease the
drug toxicity (increased MTD) to animals, they are about 1000
to 10,000 fold less effective than FL118. Therefore, overall, the
presence of a free hydroxyl group is important for FL118 to
exhibit superior antitumor effects. These findings narrow the
window for further SAR studies on other potential chemical
positions in the FL118 core structure molecule, which are
currently being pursued by our research teams.
trobenzoate) (7c). The general synthetic method described
above afforded 7c as yellow solid (31%), mp > 250 °C; H NMR
(CF COOD, 600 MHz) δ: 8.97 (s, 1 H), 8.42 (d, J = 8.82 Hz, 2
1
3
H), 8.35 (d, J = 8.76 Hz, 2 H), 7.74 (s, 1 H), 7.52 (s, 1 H), 7.46 (s,
1 H), 6.34 (s, 2 H), 5.98 (d, J = 18.18 Hz, 1 H), 5.69 (d, J = 17.04
Hz, 1 H), 5.68 (s, 2 H), 2.57 (m, 1 H), 2.45 (m, 1 H), 1.19 (t, J =
7.14 Hz, 3 H); 13C NMR (CF COOD, 150 MHz) δ: 170.7,
3
165.1, 158.4, 158.0, 152.9, 151.3, 147.5, 140.7, 139.9, 139.2,
1
1
H] .
38.9, 132.9, 131.3, 129.4, 129.3, 123.9, 122.5, 105.1, 103.8,
5
. EXPERIMENTAL SECTION
02.1, 96.9, 77.5, 67.2, 51.5, 31.4, 5.9; MS(ESI): m/z, 542.3 [M +
+
5.1. Chemistry. 5.1.1. Materials and Methods Used. All
reagents used in the experiments were obtained from commercial
sources and purified in a conventional manner. Published
procedures were used for the preparation of 2-amino-4,5-
methylenedioxybenzaldehyde (3), 20-S-10, 11-methylenediox-
ycamptothecin (FL118), and 10,11-methylenedioxycamptothe-
5.1.6. 10,11-Methylenedioxycamptothecin-20 (S)-O-(3-
bromobenzoate) (7d). The general synthetic method described
above afforded 7d as yellow solid (58%), mp > 250 °C; H NMR
(DMSO-d , 600 MHz) δ: 8.45 (s, 1 H), 8.26 (s, 1 H), 8.12 (d, J =
1
6
8.22 Hz, 1 H), 8.01 (d, J = 7.68 Hz, 1 H), 7.60 (t, J = 7.74 Hz, 1
H), 7.49 (s, 1 H), 7.43 (s, 1 H), 6.99 (s, 1 H), 6.25 (d, J = 4.38 Hz,
2 H), 5.55 (s, 2 H), 5.22 (s, 2 H), 2.33 (m, 2 H), 1.03 (t, J = 7.2
3
4−37
cin (FL113).
Thin-layer chromatography (TLC) was
performed on Merck Silica Gel 60 F254 plates. Flash column
chromatography was performed on a silica gel (200−300 mesh;
Qingdao Makall Group, Qingdao, China). Melting points were
Hz, 3 H); 13C NMR (DMSO-d , 150 MHz) δ: 167.6, 163.8,
6
157.1, 151.9, 150.2, 149.3, 147.2, 147.0, 145.7, 137.8, 132.6,
4
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Article
1
1
5
32.0, 130.9, 130.8, 129.4, 129.0, 126.3, 122.8, 118.5, 105.2,
5.2.2. Animal Models Used for Human Tumor Xenograft
Experiments. Six to 12-week-old female athymic nude mice (nu/
nu, body weight 20−25 g) were purchased from Charles River
Laboratories International, Inc. (Wilmington, MA) or Harlan
Sprague−Dawley Inc. (Indianapolis, IN). Six to 12-week-old
female SCID mice were purchased from the RPCI Laboratory
Animal Facility (LAR). Mice were housed at 5 mice per cage with
water and food ad libitum. All animal experiments were
performed in accordance with our IACUC (Institute Animal
Care and Use Committee)-approved animal protocol.
03.7, 103.2, 94.3, 77.5, 66.9, 49.2, 30.7, 8.3; MS(ESI): m/z,
+
76.4 [M + H] .
5.1.7. 10,11-Methylenedioxycamptothecin-20 (S)-O-(3-io-
dobenzoate) (7e). The general synthetic method described
1
above afforded 7e as yellow solid (62%), mp > 250 °C; H NMR
(
DMSO-d , 600 MHz) δ: 8.45 (s, 1 H), 8.42 (s, 1 H), 8.16 (d, J =
6
8
7
.28 Hz, 1 H), 8.12 (d, J = 7.74 Hz, 1 H), 7.48 (s, 1 H), 7.44 (t, J =
.68 Hz, 2 H), 6.97 (s, 1 H), 6.26 (d, J = 4.44 Hz, 2 H), 5.76 (s, 2
13
H), 5.55 (s, 2 H), 2.33 (m, 2 H), 1.02 (t, J = 7.14 Hz, 3 H); C
NMR (DMSO-d , 150 MHz) δ: 167.6, 163.7, 157.1, 151.9, 150.1,
5.2.3. Determination of the Maximum Tolerated Dose
6
1
1
5
49.3, 147.1, 147.0, 145.8, 143.6, 138.3, 131.8, 130.8, 129.6,
29.0, 126.2, 118.4, 105.2, 103.6, 103.1, 95.8, 94.3, 77.4, 66.9,
5.4, 50.8, 30.8, 8.3; MS(ESI): m/z, 623.0 [M + H] .
(MTD) for FL118 Analogues. In this study, we took advantage of
14
the known MTD for FL118 reported previously as a start point
by escalating with 0.5 mg/kg intervals until the MTD was
achieved. Each dose was tested on a cohort of 5 mice in individual
independent experiments. The MTD was defined as the highest
drug dose at the defined drug administration schedule and route
causing no drug-related lethality in mice with a body weight loss
≤20% of original body weight with reversible and temporary
toxicities.
+
5.1.8. 10,11-Methylenedioxycamptothecin-20 (RS)-O-(4-
chlorobenzoate) (7f). The general synthetic method described
1
above afforded 7f as yellow solid (55%), mp > 250 °C; H NMR
(
DMSO-d , 600 MHz) δ: 8.46 (s, 1 H), 8.14 (d, J = 8.82 Hz, 2
6
H), 7.72 (d, J = 8.82 Hz, 2 H), 7.49 (s, 1 H), 7.43 (s, 1 H), 6.93 (s,
H), 6.26 (d, J = 4.44 Hz, 2 H), 5.55 (s, 2 H), 5.23 (s, 2 H), 2.31
1
1
3
(m, 2 H), 1.03 (t, J = 7.68 Hz, 3 H); C NMR (DMSO-d , 150
5.2.4. Documentation of Tumor Sizes and Anticancer
Activity. During animal experiments, tumor size and animal body
weight were documented daily during week days until the end of
the experiments, or for the first 3 weeks and then every other day
(Monday, Wednesday, and Friday) until the end of the
experiments. Two dimensions (mm) of a tumor (L, longest
axis; W, shortest axis) were measured with a Vernier caliper.
6
MHz) δ: 167.6, 164.4, 157.1, 151.9, 150.1, 149.3, 147.2, 147.0,
1
1
45.8, 139.2, 132.1, 130.7, 129.9, 129.0, 127.5, 127.0, 118.4,
14.3, 106.4, 105.2, 103.6, 103.1, 77.2, 66.9, 50.8, 30.7, 8.3;
+
MS(ESI): m/z, 531.1 [M + H] .
5.1.9. 10,11-Methylenedioxycamptothecin-20 (RS)-O-(3,4-
dimethoxybenzoate) (7g). The general synthetic method
described above afforded 7g as yellow solid (34%), mp > 250
3
Tumor volume (mm ) was estimated using the formula of
1
3
2
°
9
8
C; H NMR (DMSO-d , 600 MHz) δ: 8.45 (s, 1 H), 7.81 (d, J =
.9 Hz, 1 H), 7.50 (d, J = 4.2 Hz, 2 H), 7.44 (s, 1 H), 7.19 (d, J =
.82 Hz, 1 H), 6.91 (s, 1 H), 6.26 (d, J = 3.3 Hz, 2 H), 5.54 (s, 2
“tumor volume (mm ) = 1/2(LW )”. All figures were made using
Sigma Plot software.
6
5.2.5. Cancer Cell Lines Used and Cell Culture. The human
head and neck squamous cell carcinoma cell line FaDu, the
human ileocecal adenocarcinoma (HCT-8), and colon cancer
(SW620) cell lines were from American Type Culture Collection
(ATCC, Manassas, VA). The human ovarian cancer cell line
2008 (a gift from Dr. Kunle Odunsi, Roswell Park Cancer
Institute) was originally derived from a patient with ovary
H), 5.23 (s, 2 H), 3.88 (s, 3 H), 3.82 (s, 3 H), 2.29 (m, 2 H), 1.03
1
3
(
t, J = 6.24 Hz, 3 H); C NMR (CF COOD, 150 MHz) δ: 171.6,
3
167.9, 158.6, 155.1, 153.2, 148.6, 148.5, 141.1, 140.1, 139.5,
139.1, 129.8, 129.5, 126.3, 122.5, 119.5, 112.8, 111.3, 105.4,
104.1, 102.6, 97.2, 77.2, 67.5, 55.4, 51.8, 31.7, 6.3; MS(ESI): m/z,
5
+
57.2 [M + H] .
50
5.2. Biological Studies. 5.2.1. Reagents, Compounds, and
cystadenocarcinoma. Tumorigenic L-929 fibrosarcoma cells
were purchased from ATCC. H2122 lung cancer cells were from
Dr. Daniel Chan (University of Colorado, Aurora) as a gift. Cells
were grown either in RPMI 1640 medium (HCT-8, SW620,
2008, L929, H2122) or in Eagle’s minimum essential medium
(FaDu), which were supplemented with 10% heat inactivated
FCS, 100 U/mL of penicillin, and 0.1 μg/mL of streptomycin.
Tumor Types Used. Survivin antibody (FL-142) was purchased
from Santa Cruz. Antibodies for Mcl-1, XIAP, and cIAP2 were
purchased from Cell Signaling. Actin antibody (A2066) was
purchased from Sigma. The pcDNA3.1 vector was purchased
from Invitrogen. pcDNA3.1-Mcl-1 was purchased from Addgene
(
(
http://www.addgene.org). The Tet-on system and doxycycline
Dox) were purchased from Clontech.
Cells were cultured in a 5% CO incubator at 37 °C and
2
Compounds used for cell culture studies were first dissolved in
subcultured every 3−4 days. All cell lines were mycoplasma-free,
confirmed by MycoSensor PCR Assay kit (Stratagene).
5.2.6. Immunoblotting Analysis (Western Blot). Western
DMSO at a 1000× stock solution and then directly diluted for
1
concentration. The compounds used for the in vivo testing of
antitumor activity and toxicity were formulated in the same
000× with cell culture medium to the finally needed
5
5
blots were performed as previously described. Proteins of
interest were detected using Western Lightning-ECL (Perkin-
Elmer, Waltham, MA) and visualized by exposure for various
times (5−60 s). Actin was used as an additional internal control
for equal protein loading.
14
recipe as previously reported by us.
Human 11124 and 14528 colon cancer xenograft tumors were
initially isolated from cancer patients at Roswell Park Cancer
Institute (RPCI) and established in SCID (severe combined
immunodeficient) mice. Human cancer cell line-derived tumor
xenografts (human head and neck FaDu and human colon
SW620) were established by subcutaneously injecting 1−3
million cultured cancer cells. The xenografts were passed through
5.2.7. Generation of Tet-On-Controlled Survivin Expression
in Tumorigenic L929 Fibrosarcoma Cells. Survivin cDNA was
subcloned from pcDNA3-survivin expression vector into
pTREhyg vector (Clontech) at BamH I and Sal I sites in the
cis-direction to generate pTREhyg-survivin expression vector
(hygromycin resistant). The pTREhyg-survivin vectors were
then cotransfected with pTet-On vector (G418 resistant,
Clontech) into the tumorigenic L929 fibrosarcoma cells.
Transfected cells were grown in the presence of both G418
and hygromycin to select G418/hygromycin-resistant cell
1−3 generations by subtransplanting 40−50 mg non-necrotic
tumor tissues via a trocar from the passage xenograft tumors. The
derived xenograft tumors were used for in vivo experiments.
Xenograft tumor mice for experiments were prepared using the
same method as xenograft tumor passage.
4
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Molecular Pharmaceutics
Article
clones. Each of the isolated G418/hygromycin-resistant cell
clones was grown in the presence (to turn survivin on) and
absence (to turn survivin off) of doxycycline (Dox) for 48 h,
respectively. Survivin mRNA expression in individual cell clones
was determined by semiquantitative RT-PCR (semiQPCR),
followed by display of the PCR products on agarose gel (results
from representative cell clones are shown in Figure 5a) as
previously described. The semiQPCR-identified positive cell
clone that was used for experiments was further confirmed by
Western blotting analysis (Figure 5b, insert).
AUTHOR INFORMATION
■
Corresponding Authors
E-mail: jiangtao@ouc.edu.cn. Tel.: 0086-532-82032712. Fax:
086-532-82033054.
E-mail: fengzhi.li@roswellpark.org. Tel.: (716)-845-4398. Fax:
716)-845-8857.
*
0
*
(
56
Author Contributions
J.Z., X. Ling, S.C., and X. Liu made equal contributions in this
work.
5
.2.8. MTT Assays. Cancer cell growth/viability was
Notes
56
determined by a MTT assay. MTT, a tetrazolium salt with
the chemical definition of 3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide, was used as a colorimetric
substrate for measuring cell viability. Seventy-two hours post
treatment with vehicle or individual compounds, MTT was
added to a final concentration of 0.5 mg/mL onto treated cells in
The authors declare the following competing financial
interest(s): FL118 will be further developed in Canget
BioTekpharma Company (www.canget-biotek.com). F.L. is the
founder of Canget, and F.L., X.L., X.J.L. and T.J. are initial
investors in Canget.
9
6-well plates. Cells were further incubated in a 5% CO2
ACKNOWLEDGMENTS
■
incubator at 37 °C for an additional 4 h and then lysed
thoroughly with lysis buffer (20% SDS, 50% N,N-dimethylfor-
mamide, pH 4.7, 100 μL/well). The absorbance in the relevant
wells was measured at 570 nm using an Ultra Microplate Reader
We would like to thank the Animal Center (DLAR) staff and
attending veterinarians for daily care of our animals at RPCI and
helping us to discover possible issues during our experiments. We
also would like to thank Ms. Sumei Ren and Xiuli Zhang from the
School of Pharmacy, Ocean University of China for their MS and
NMR analysis. Additionally, we would like to thank other lab
members from the Li and Jiang laboratories for being a part of
our team and for their cooperation and support of these studies.
We also would like to thank Editor David Hadbawnik of the
English Department from the State University of New York
(SUNY) at Buffalo (UB) for reading, correcting, and editing this
manuscript. Finally, we sincerely thank Dr. Suzanne M. Hess
(Research Support Services, Roswell Park Cancer Institute,
Buffalo, NY) for critically reading and revision of this manuscript
before publication. This work was sponsored in part by grants
from Natural Science Foundation of China (NSF) (21171154)
and a Special Fund for Marine Scientific Research in the Public
Interest of China (201005024) to T.J.; by grants from US Army
Department of Defense (PC110408), Mesothelioma Applied
Research Foundation (Alexandria, VA), and the Roswell Park
Alliance Foundation (Buffalo, NY) to F.L.; and by shared
resources supported by NCI Cancer Center Core Support Grant
to Roswell Park Cancer Institute (CA016056). Of note, S.C. was
paid by grants in part from a grant to F.L. during this work.
(
Bio-Tek Instruments). The data derived from luciferase activity
and MTT assays were analyzed using Excel 2010. Each bar is
presented as mean ± standard deviation (SD).
5.2.9. DNA Fragmentation Cell Death ELISA Assays. Tet-on
survivin L929 cells were induced with Dox (4 μg/mL) for 48 h
and then treated with 50 nM FL118 for 72 h. Cells were then
analyzed for DNA fragmentation-induced cell death determi-
Plus
nation using a Cell Death Detection ELISA assay kit (Roche,
Indianapolis, IN) following the recommended protocol as
5
6
previously described. Briefly, ∼4000 Dox-induced and
uninduced cells were seeded per well in a 96-well plate overnight
and then treated with or without 50 nM FL118 for 72 h in the
presence or absence of Dox. Cells were then lysed, and the lysates
were cleared by centrifugation; the resulting supernatant was
used for testing DNA fragmentation-induced cell death as
56
previously described.
.2.10. Luciferase Activity Assays. Luciferase activity assays
were performed following the protocol described in our
5
5
7,58
previously publications.
Briefly, 2008 ovarian cancer cells
that were stably transfected with the pLuc-4080 survivin
promoter−luciferase construct were seeded and grown to a
subconfluent state in 48 well plates. Cells were then treated with
the defined concentration of drugs in triplicate for 24 h and then
followed by processing of the luciferase assays using the
PAE4550 Luciferase Reporter Assay System (Promega). Data
were normalized to equal total protein amounts as arbitrary units
to show relative promoter activity.
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