Design, synthesis and structure-activity relationship studies of novel survivin inhibitors with potent anti-proliferative properties

Article abstract of DOI:10.1371/journal.pone.0129807

The anti-apoptotic protein survivin is highly expressed in most human cancer cells, but has very low expression in normal differentiated cells. Thus survivin is considered as an attractive cancer drug target. Herein we report the design and synthesis of a series of novel survivin inhibitors based on the oxyquinoline scaffold from our recently identified hit compound UC-112. These new analogs were tested against a panel of cancer cell lines including one with multidrug-resistant phenotype. Eight of these new UC-112 analogs showed IC₅₀ values in the nanomole range in anti-proliferative assays. The best three compounds among them along with UC-112 were submitted for NCI-60 cancer cell line screening. The results indicated that structural modification from UC-112 to our best compound 4g has improved activity by four folds (2.2 μM for UC-112 vs. 0.5 μM for 4g, average GI₅₀ values over all cancer cell lines in the NCI-60 panel).Western blot analyses demonstrated the new compounds maintained high selectivity for survivin inhibition over other members in the inhibition of apoptosis protein family. When tested in an A375 human melanoma xenograft model, the most active compound 4g effectively suppressed tumor growth and strongly induced cancer cell apoptosis in tumor tissues. This novel scaffold is promising for the development of selective survivin inhibitors as potential anticancer agents.

Full text of DOI:10.1371/journal.pone.0129807

RESEARCH ARTICLE
Design, Synthesis and Structure-Activity
Relationship Studies of Novel Survivin
Inhibitors with Potent Anti-Proliferative
Properties
Min Xiao1, Jin Wang1, Zongtao Lin1, Yan Lu1, Zhenmei Li2, Stephen W. White2, Duane
D. Miller1, Wei Li1*
1 Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, Memphis,
Tennessee, United States of America, 2 Department of Structure Biology, St. Jude Children’s Research
Hospital, Memphis, Tennessee, United States of America
* wli@uthsc.edu
Abstract
OPEN ACCESS
Citation: Xiao M, Wang J, Lin Z, Lu Y, Li Z, White
SW, et al. (2015) Design, Synthesis and Structure-
Activity Relationship Studies of Novel Survivin
Inhibitors with Potent Anti-Proliferative Properties.
PLoS ONE 10(6): e0129807. doi:10.1371/journal.
pone.0129807
The anti-apoptotic protein survivin is highly expressed in most human cancer cells, but has
very low expression in normal differentiated cells. Thus survivin is considered as an attrac-
tive cancer drug target. Herein we report the design and synthesis of a series of novel survi-
vin inhibitors based on the oxyquinoline scaffold from our recently identified hit compound
UC-112. These new analogs were tested against a panel of cancer cell lines including one
with multidrug-resistant phenotype. Eight of these new UC-112 analogs showed IC50 values
in the nanomole range in anti-proliferative assays. The best three compounds among them
along with UC-112 were submitted for NCI-60 cancer cell line screening. The results indicat-
ed that structural modification from UC-112 to our best compound 4g has improved activity
by four folds (2.2 μM for UC-112 vs. 0.5 μM for 4g, average GI50 values over all cancer cell
lines in the NCI-60 panel).Western blot analyses demonstrated the new compounds main-
tained high selectivity for survivin inhibition over other members in the inhibition of apoptosis
protein family. When tested in an A375 human melanoma xenograft model, the most active
compound 4g effectively suppressed tumor growth and strongly induced cancer cell apo-
ptosis in tumor tissues. This novel scaffold is promising for the development of selective sur-
vivin inhibitors as potential anticancer agents.
Academic Editor: Michal Zmijewski, Medical
University of Gdańsk, POLAND
Received: January 6, 2015
Accepted: May 13, 2015
Published: June 12, 2015
Copyright: © 2015 Xiao et al. This is an open access
article distributed under the terms of the Creative
Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: This work is partially supported by NIH
grants R01CA148706, 1S10OD010678-01,
1S10RR026377-01, and a pilot grant from the Center
for Integrative and Translational Genomics and the
Molecular Resource Center at the University of
Tennessee. The content is solely the responsibility of
the authors and does not necessarily represent the
official views of the National Institutes of Health. The
authors thank the University of Tennessee Research
Foundation for providing funds to send compound 4g
Introduction
Survivin is a unique member of inhibitor of apoptosis protein (IAP) family.[1] It is overex-
pressed in most human cancer cells, but is rarely expressed in adult differentiated tissues.[2–5]
This attribute distinguishes survivin from other IAPs which are usually expressed in both can-
cer and normal cells. Survivin promotes cell proliferation and inhibits apoptosis,[3, 6–9] facili-
tates angiogenesis in tumors,[10–12] and its expression has been shown to strongly correlate
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Novel Small Molecule Survivin Inhibitors for Cancer
to Eurofins Panlabs for profiling its drug-like
properties. Jin Wang acknowledges the support of the
Alma and Hal Reagan Fellowship from University of
Tennessee Health Science Center. The authors thank
Mr. Zongtao Lin for performing the HRMS
characterization for all final compounds. The funders
had no role in study design, data collection and
analysis, decision to publish, or preparation of the
manuscript.
with multiple mechanisms of drug resistance.[13–15] Therefore, survivin is widely considered
to be an ideal cancer drug target. Several molecules in different categories including antisense
oligonucleotides, dominant-negative mutants, ribozymes, small interfering RNAs, cancer vac-
cine and small molecules have been identified as survivin inhibitors.[16] However, due to the
challenging requirement to efficiently disrupt protein-protein interactions, the pool of existing
small molecule survivin inhibitors is quite small.[17] The efficacy of those survivin inhibitors is
also limited.[16] For example, the reported clinical candidate, small molecule survivin inhibitor
YM155 has been shown to be a substrate of P-glycoprotein (Pgp) drug efflux pump, which sug-
gests that YM155 has limited efficacy in multiple drug resistant phenotypes.[18] Therefore, it is
highly significant to develop new survivin inhibitors that can overcome multidrug resistance,
which is an important goal of our research.
Competing Interests: The authors have declared
that no competing interests exist.
We recently discovered that UC-112, [5-((benzyloxy)methyl)-7-(pyrrolidin-1-ylmethyl)
quinolin-8-ol], is a potent, selective survivin inhibitor (Fig 1).[19] UC-112 inhibits tumor cell
growth in several cancer cell lines in vitro and suppresses melanoma tumor growth in vivo.
[19] Mechanistic studies indicated that UC-112 selectively inhibits survivin expression and in-
duces strong cancer cell apoptosis. In order to further optimize this unique oxyquinoline scaf-
fold and explore its structure-activity relationships (SAR), we designed several focused sets of
new UC-112 analogs (Fig 1). We introduced four major modifications to the structure of UC-
112. First, we modified the C ring moiety by introducing substitutions to the phenyl ring or re-
placing it with other groups. Secondly, we modified the D ring moiety with other cyclic amines.
We also explored different chain lengths between the oxygen linker and the C ring moiety. Fi-
nally, the linker between the B ring and the C ring was modified. By performing these targeted
modifications, a series of novel UC-112 analogs were obtained. Biological evaluation of these
new analogs revealed their excellent anti-proliferative activity against several cancer cell lines
including multidrug-resistant phenotypes. Mechanism of action studies confirmed that these
new UC-112 analogs maintained their mechanisms of actions by selectively downregulating
the level of survivin among other IAP family proteins. Preliminary in vivo evaluation for the
most active compound 4g demonstrated its efficacy against human melanoma tumor growth.
Results and Discussion
Chemistry
The general synthesis of C ring substituted UC-112 analogs (4a-4l) is outlined in Fig 2. First
8-hydroxyquinoline reacted with formaldehyde and catalytic zinc chloride in concentrated hy-
drochloric acid to generate salt 2.[20] This step introduced a chloromethyl group to the 5-posi-
tion of quinoline ring. Then salt 2 was allowed to react with different substituted benzyl
alcohols to form ethers 3a-3l, by two different methods. Ethers 3b-3e and ethers 3h-3l were
synthesized using step b in which substituted benzyl alcohols were allowed to react with salt 2
in the presence of sodium hydride in anhydrous THF. Ethers 3a, 3f and 3g were synthesized
through step c and step d. In step c, substituted benzyl alcohols reacted directly with salt 2 with
heating to form different salts which were converted to free base by adjusting pH with NH4OH
solution in step c.[21] The synthesized ethers were than submitted to Mannich reaction condi-
tions with paraformaldehyde and pyrrolidine in ethanol to form the final compounds 4a to 4l.
[22] Compounds 6a-6g with the C ring moiety in UC-112 replaced by different function
groups were prepared as Fig 3 shown. The ethers 5a-5g were first synthesized using similar ap-
proach as shown in Fig 2. Then these ethers were converted to 6a-6g via the Mannich reaction.
The synthesis of D ring modified UC-112 analogs 8a and 8b is shown in Fig 4. Salt 2 first re-
acted with benzyl alcohol to form intermediate 7, which was allowed to react with piperidine
or morpholine via the Mannich reaction to form compounds 8a and 8b. Compounds 10a and
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Novel Small Molecule Survivin Inhibitors for Cancer
Fig 1. Targeted modification to design new UC-112 analogs.
doi:10.1371/journal.pone.0129807.g001
10b which have different chain lengths between oxygen and the phenyl ring from parent com-
pound UC-112 were made using the method shown in Fig 5. First salt 2 reacted with 2-pheny-
lethanol and 3-phenyl-1-propanol respectively to form 9a and 9b, which then underwent the
Mannich reaction with paraformaldehyde and pyrrolidine to form compounds 10a and 10b.
Finally, compounds 12a and 12b, which have different linkers between the B ring and C ring as
compared to the parent compound were made (Fig 6). First salt 2 reacted with benzyl mercap-
tan or N-benzymethylamine to form intermediate 11a and 11b. Then 11a and 11b underwent
the Mannich reaction to form compounds 12a and 12b.
Fig 2. Synthesis of compounds 4a-4l. Reagents and conditions: (a) conc. HCl, ZnCl2, HCHO(37% in H2O);
(b) substituted benzyl alcohol, NaH, THF, reflux; (c) substituted benzyl alcohol, 110 oC; (d) NH4OH, H2O,
Et2O, pH 8–10; (e) paraformaldehyde, pyrrolidine,EtOH, reflux.
doi:10.1371/journal.pone.0129807.g002
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Novel Small Molecule Survivin Inhibitors for Cancer
Fig 3. Synthesis of compounds 6a-6g. Reagents and conditions: (a) R2CH2OH, NaH, THF, reflux; (b)
R2CH2OH, 110 oC; (c) NH4OH, H2O, Et2O, pH 8–10; (d) paraformaldehyde, pyrrolidine,EtOH, reflux.
doi:10.1371/journal.pone.0129807.g003
Biological results and discussion
All UC-112 analogs were evaluated for their cytotoxicity in a panel of human cancer cell lines
including melanoma (A375, M14 and its multidrug resistant daughter line M14/LCC6MDR1)
and prostate cancer (PC-3). UC-112 was also included in the assays, serving as positive control
and basis for comparison. These in vitro biological results are summarized in Tables 1–4 and
discussed in detail in the following sections.
Effect of substitutions on the C ring of UC-112
The in vitro activity for compounds 4a-4l which have substitutions on the C ring of UC-112 is
shown in Table 1. Most compounds in this table are more active than the parent compound
UC-112, suggesting that introduction of substitutions to the C ring moiety in UC-112 is favor-
able. Introducing a fluoro group (4a) to the phenyl ring caused the increase of activity in three
of the four cancer cell lines tested(1.5 μM vs 1.9 μM in A375, 1.8 μM vs 2.1 μM in M14 and
0.7 μM vs 1.6 μM in PC-3). Introducing a chloro substitution (4b) resulted in even better activi-
ty in all four cell lines tested. A bromo substitution (4c) further increased activity. Introduction
of an electron withdrawing group, trifluoromethyl (4d), also resulted in an increase in activity
against all the cell lines. As the size of substitution increases from fluoro to bromo substitu-
tions, a general trend of increased activity was observed (i.e. IC50 in M14 cell line. 4a: 1.8 μM;
4b: 1.1 μM; 4c: 1.0 μM). This trend is also true for electron donating group substituted analogs
4e-4g (i.e. IC50 in M14/LCC6MDR1 cell line. 4e: 4.1 μM; 4f: 2.7 μM; 4g: 1.8 μM). But the tert-
Fig 4. Synthesis of compounds 8a-8b. Reagents and conditions: (a) benzyl alcohol, 110 oC; (b) NH4OH,
H2O, Et2O, pH 8–10; (c) paraformaldehyde, piperidine for 8a, morpholine for 8b,EtOH, reflux.
doi:10.1371/journal.pone.0129807.g004
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Novel Small Molecule Survivin Inhibitors for Cancer
Fig 5. Synthesis of compounds 10a-10b. Reagents and conditions: (a) 2-phenylethanol for 9a, 3-phenyl-
1-propanol for 9b, 110 oC; (b) NH4OH, H2O, Et2O, pH 8–10; (c) paraformaldehyde, pyrrolidine, EtOH, reflux.
doi:10.1371/journal.pone.0129807.g005
butyl group substituted compound 4h does not further increase activity compared to iso-propyl
group substituted compound 4g, indicating there is probably a size limit for effective binding
to survivin. In addition, several polar groups which are hydrogen bond acceptors were also test-
ed. However, as Table 1 shows, compounds 4i- 4l do not increase activity significantly com-
pared to parent compound UC-112. Compounds 4j and 4k even had decreased activity
compared to UC-112 (i.e. IC50 in M14/LCC6MDR1 cell line. 4j: 12.1 μM; 4k: 5.5 μM; UC-112:
3.2 μM), suggesting that a bulky substitution is unfavorable. Among all the compounds in this
table, compounds 4c, 4d and 4g are the most potent ones, indicating that the introduction of a
hydrophobic group to the para-position of C ring is favorable for increased activity.
Effect of replacing the phenyl ring of UC-112 with other groups
After we tried different substitutions on the phenyl ring of UC-112, we replaced the phenyl
ring with other groups to further optimize UC-112. Here, we tried seven different groups
which included furan ring, thiophene ring, pyridine ring, cyclohexyl ring, methyl group, ethyl
group, and n-propyl group. The in vitro activity for the seven C ring modified UC-112 analogs
is shown in Table 2. In this table, compounds 6a, 6b and 6c were less active than UC-112 (i.e.
IC50 in A375 cell line. 6a: 2.7 μM; 6b: 2.5 μM; 6c: 8.4 μM; UC-112: 1.9 μM), indicating that re-
placing the phenyl ring with other heterocyclic rings decreases activity. Replacing the phenyl
ring with a cyclohexyl ring resulted in an increase in activity in A375 and M14 cell lines. In
order to see whether a ring is required at the C ring position of UC-112 for activity, compounds
6e, 6f and 6g with only aliphatic chains were synthesized. All these compounds decreased ac-
tivity compared to parent compound UC-112 (i.e. IC50 in A375 cell line. 6e: 6.1 μM; 6f: 5.0 μM;
6g: 3.0 μM; UC-112: 1.9 μM), suggesting a ring system is required at the C ring position of UC-
112. For compounds 6e, 6f and 6g, there is a general trend for activity. As the length of the ali-
phatic chain increases, the activity increases as well. This trend is probably due to the existence
Fig 6. Synthesis of compounds 12a-12b. Reagents and conditions: (a) benzyl mercaptan for 11a,
N-Benzylmethylamine for 11b, NaH, THF, reflux; (b) paraformaldehyde, pyrrolidine,EtOH, reflux.
doi:10.1371/journal.pone.0129807.g006
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Novel Small Molecule Survivin Inhibitors for Cancer
Table 1. In vitro growth inhibitory effects of UC-112 analogs with C ring substitutions.
ID
IC50 values SEM (μM)
M14/LCC6MDR1
A375
M14
PC-3
Resistance Index
4a
1.5 0.2
1.5 0.3
0.9 0.2
0.9 0.3
1.7 0.3
0.9 0.1
0.9 0.1
1.1 0.4
1.8 0.2
2.8 0.1
4.7 0.8
2.8 0.4
1.9 0.6
ND
1.8 0.3
1.1 0.1
1.0 0.2
1.0 0.4
2.3 0.5
0.9 0.1
0.8 0.2
NDa
4.4 0.7
2.4 0.5
1.8 0.6
1.9 0.5
4.1 2.0
2.7 1.0
1.8 0.4
1.5 0.7
3.8 0.6
12.1 0.5
5.5 1.3
>30
0.7 0.2
0.9 0.1
0.7 0.3
1.0 0.1
1.1 0.2
0.9 0.1
0.7 0.1
1.5 0.3
1.0 0.4
ND
2.4
4b
2.2
4c
1.8
4d
1.9
4e
1.8
4f
3.0
4g
2.3
4h
ND
2.5
4i
1.5 0.4
ND
4j
ND
ND
> 8.3
1.5
4k
ND
ND
4l
3.6 1.0
2.1 0.3
0.003
1.6 0.3
1.6 1.0
ND
UC-112
YM-155
3.2 0.5
>10
>2941
a ND: Not Determined. Resistance index is calculated by dividing IC50 values on multidrug-resistant cell line M14/LCC6MDR1 by IC50 values on the
matching sensitive parental cell line M14.
doi:10.1371/journal.pone.0129807.t001
of a hydrophobic pocket in the binding site, which is consistent with previous observation that
introduction of a hydrophobic substitution with bigger space occupation on the phenyl ring is
favorable for activity.
Effect of D ring modification on UC-112
Following C ring modification in the above, we went on with D ring modification of UC-112.
We tried to replace the pyrrolidine ring with two different cyclic amines: piperidine and mor-
pholine at current stage. The in vitro activity for the two D ring modified compounds 8a and
8b is show in Table 3.Compound 8a showed slightly better activity than the parent compound,
while compound 8b was less active than UC-112 (i.e. IC50 in A375 cell line. 8a: 1.1 μM; 8b:
5.0 μM; UC-112: 1.9 μM). This observation means the replacement of pyrrolidine ring in
Table 2. In vitro growth inhibitory effects of C ring modified UC-112 analogs.
ID
IC50 values SEM (μM)
M14/LCC6MDR1
A375
M14
PC-3
Resistance Index
6a
2.7 0.3
2.5 0.5
8.4 1.5
1.0 0.1
6.1 0.7
5.0 0.3
3.0 0.5
1.9 0.6
3.1 0.3
1.9 0.9
8.0 2.2
1.3 0.6
5.1 2.1
5.9 1.2
4.9 1.3
2.1 0.3
15.3 3.1
4.2 0.2
> 30
27.5 2.0
3.4 0.7
> 30
4.9
2.2
>3.8
ND
ND
ND
ND
1.5
6b
6c
6d
NDa
ND
6e
ND
ND
6f
ND
ND
6g
ND
ND
UC-112
3.2 0.5
1.6 1.0
a ND: Not Determined.
doi:10.1371/journal.pone.0129807.t002
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Novel Small Molecule Survivin Inhibitors for Cancer
Table 3. In vitro growth inhibitory effects of D ring modified UC-112 analogs.
ID
IC50 values SEM (μM)
M14/LCC6MDR1
A375
M14
PC-3
Resistance Index
8a
1.1 0.1
5.0 1.6
1.9 0.6
1.3 0.2
4.8 0.5
2.1 0.3
2.1 0.6
3.5 1.0
3.2 0.5
1.3 0.3
NDa
1.6
0.7
1.5
8b
UC-112
1.6 1.0
a ND: Not Determined.
doi:10.1371/journal.pone.0129807.t003
UC-112 with a piperidine ring maintains activity, while replacing pyrrolidine ring with mor-
pholine ring decreases activity. Both compounds 8a and 8b can effectively inhibit the growth
of resistant cell line M14/LCC6MDR1, suggesting that they can overcome the Pgp-mediated
multiple drug resistance. The difference in activity between 8a and 8b indicates that the D ring
position of UC-112 is an important site that further optimization can be carried out.
Effect of chain length between oxygen and C ring of UC-112 analogs
The chain length between oxygen linker and C ring in the parent compound UC-112 is one
carbon. We tried to test the importance of chain length by increasing the carbon numbers.
Two compounds 10a and 10b were prepared and their in vitro activity is shown in Table 4.
Both compounds 10a and 10b were less active than the parental compound, especially the ac-
tivity against resistant cell line M14/LCC6MDR1 reduced from 3.2 to 15~20 μM, suggesting
that increasing the chain length was not a favorable modification for increasing activity.
Effect of linkers between C ring and D ring of UC-112 analogs
After the D ring modification, we went on to optimize the linker. We made two compounds
12a and 12b with two different linkers from the parent compound. The in vitro activity of
those tow compounds is shown in Table 5. Compound 12a which bears a sulfur linker shows
increased activity compared with UC-112 (i.e. 1.4 μM vs 1.9 μM in A375 cell line), meaning
sulfur linker here is favorable. Compound 12b which contains a methylamine linker was less
active (i.e. 7.1 μM vs 1.9 μM in A375 cell line), indicating that a methylamine linker is unfavor-
able. Both compounds 12a and 12b have small resistance indexes. Their activity in the resistant
cell line M14/LCC6MDR1 is comparable with that in the parental cell line M14. While two
compounds can’t determine the optimized linker, at least they give us some information about
the activity requirement of the linker which is very useful for further optimization. Further op-
timization of the linker is now currently being examined.
Table 4. In vitro growth inhibitory effects of UC-112 analogs with different chain lengths.
ID
IC50 values SEM (μM)
A375
M14
M14/LCC6MDR1
Resistance Index
10a
2.8 0.9
2.4 0.7
1.9 0.6
2.9 0.2
2.3 0.8
2.1 0.3
15.0 2.5
20.0 1.6
3.2 0.5
5.2
8.7
1.5
10b
UC-112
doi:10.1371/journal.pone.0129807.t004
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Novel Small Molecule Survivin Inhibitors for Cancer
Table 5. In vitro growth inhibitory effects of UC-112 analogs with different linkers.
ID
IC50 values SEM (μM)
M14/LCC6MDR1
A375
M14
PC-3
Resistance Index
12a
1.4 0.3
7.1 0.7
1.9 0.6
1.4 0.2
6.2 1.4
2.1 0.3
2.9 0.6
8.0 1.0
3.2 0.5
1.1 0.1
2.5 0.6
1.6 1.0
2.1
1.3
1.5
12b
UC-112
doi:10.1371/journal.pone.0129807.t005
UC-112 analogs can overcome multidrug resistance
In order to determine whether the new analogs we made can overcome multidrug resistance
(MDR), we compared the activity of those analogs against multidrug-resistant melanoma cells
(M14/LCCMDR1) and their parental sensitive cancer cells (M14). This pair of cell lines have
been well validated and widely used to assess abilities of drugs overcoming Pgp-mediated
MDR.[23–25] Our three best compounds 4c, 4d and 4g together with the most successful
small molecule survivin inhibitor YM155 were tested on both the MDR melanoma cells and
their parental cells (Table 1). The resistance index is calculated by dividing the IC50 value of the
resistant cell line by IC50 value of the sensitive parental cell line. So the smaller this value, the
better resistance overcoming effect obtained. As this table shows, compounds 4c, 4d and 4g all
have very small resistance indexes (1.8, 1.9 and 2.3 respectively). Their activity in the resistant
cell line is comparable with that in the parental cell line. For existing survivin inhibitor,
YM155, although it is very potent in parental cell line (IC50 at 3 nM), its activity in resistant
cell line is considerably lower (IC50 is higher than 10 μM, resistance index is higher than 2941).
This data indicate that the new UC-112 analogs can circumvent Pgp-mediated multi-drug re-
sistance and are distinct from that of YM-155.
UC-112 analogs show good anti-proliferation effects with selectivity in
NCI-60 cell line screening
UC-112 and three potent analogs 4c, 4g and 12a were submitted to NCI for its one-concentra-
tion (10 μM) screening against the NCI-60 cell lines. All four compounds showed good activity
and were selected for the subsequent five doses testing to determine their growth inhibition po-
tency (GI50) in the NCI-60 cell lines. As shown in Fig 7, structure modification from UC-112
to our best compound 4g has improved the average GI50 by nearly four times (2.2 μM for UC-
112 vs. 0.5 μM for 4g).
The heat map in Fig 8 summarizes the compound growth inhibition pattern which is char-
acterized by the GI50 mean values from NCI-60 screening (S1 Fig, S2 Fig, S3 Fig, and S4 Fig).
UC-112 and its new analogs showed interesting selective growth inhibition behavior within the
NCI 60 cell lines. The GI50value of compound 4g in renal cancer cell line UO-31 was as low as
52.5 nM. Interestingly, two other cell lines, HCT-15 (colon cancer) and NCI/ADR-RES (ovari-
an cancer), were also particularly sensitive to the treatment of UC-112 and its analogs, with
lowest GI50 value (highest activity) for compound 4g at 46.8 nM and 50.1 nM, respectively.
Since colorectal adenocarcinoma HCT-15 cells intrinsically expresses moderate levels of Pgp,
multidrug-resistance-associated protein (MRP) and lung-resistance-associated protein (LRP)
[26], and ovarian cancer NCI/ADR-RES cells are naturally over-expressing MDR1 and resis-
tant to various chemotherapies including doxorubicin [27], these data have supported our find-
ings that UC-112 and its analogs could effectively overcome the multidrug resistance in vitro.
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Novel Small Molecule Survivin Inhibitors for Cancer
Fig 7. Average GI50 data for UC-112, compound 12c, 4c and 4g tested in NCI-60 anti-proliferative screening. Data is shown with mean SD as
bar graph.
doi:10.1371/journal.pone.0129807.g007
Compound 4g possesses good drug-like properties
Adsorption, distribution, metabolism and elimination (ADME) properties are key elements in
small molecule drug development. Poor ADME property is one of the major contributors of
failure in advancing new compounds towards drug approval. To preliminarily evaluate the
overall drug-like properties for the UC-112 scaffold, we sent our best compound 4g to an exter-
nal contract research company (Eurofins Panlabs Inc., Redmond, WA) to determine its aque-
ous solubility and in vitro ADME properties. The aqueous solubility for compound 4g was
tested in PBS solution at pH 7.4 (Table 6) and the result showed that compound 4g had very
good aqueous solubility of 148.7 μM. The in vitro microsomal stability for compound 4g was
determined in human liver microsomes. The result showed that it had a reasonable half life
time at 51 minutes with a medium clearance rate at 136.6 μL/min/mg. Drug metabolism studies
using cytochrome P450 inhibition by compound 4g (Table 7) showed weak inhibition for most
cytochrome P450 enzymes (less than 30%) except CYP2D6. This study suggests that the UC-
112 scaffold demonstrated by compound 4g has good drug-like properties, which clearly war-
rants its further development.
Mechanism of action studies
In our recently report, [19] UC-112 induces apoptosis by selectively inhibiting the expression
of survivin in cancer cells. In order to determine whether the new UC-112 analogs maintained
the same mechanism of action, we performed the caspases activation and western blotting
assay for two potent new analogs 4f and 4g in two cancer cell lines (Fig 9). Both compounds 4f
and 4g dose-dependently suppressed survivin level in these two cancer cell lines, while the lev-
els of other IAPs were minimally affected. The presence of compound 4g at 300 nM reduced
survivin levels over 50% in both A375 and PC-3 cells as shown in the lane density data quanti-
fied by ImageJ software (S5 Fig). The IC50 values for survivin inhibition were estimated to be in
the low nanomolar range. Furthermore, incubation of compound 12c, 4c or 4g at concentra-
tion of 1 μM for 24 h significantly activated the executioner caspase 3/7 up to 4 folds higher
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Novel Small Molecule Survivin Inhibitors for Cancer
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Novel Small Molecule Survivin Inhibitors for Cancer
Fig 8. Heat map showing the GI50 values (nM) for UC-112 and three analogs in the NCI-60 screening.
High intensity (blue) cells indicate high activity and low intensity (red) cells indicate low activity. Average GI50
values were calculated for each compound, separately.
doi:10.1371/journal.pone.0129807.g008
than the DMSO control group (Fig 10). This is consistent with the expected enhanced apopto-
sis induction capability due to survivin inhibition.
Molecular modeling study
To understand the observed improvement in potency of compound 4g over its parental com-
pound UC-112, we developed a molecular model and performed the molecular docking study
using the complex of human survivin-SMAC AVPI (PDB entry: 3UIH). As shown in Fig 11,
UC-112 displayed several interesting interactions with the survivin protein BIR domain: (1)
two hydrogen bonding interactions between the A/B-ring of UC-112 and residue Asp71; (2)
one hydrogen bonding interaction between the D-ring nitrogen and residue Glu68; and (3) an
π- π stacking interaction between the A/B-ring of UC-112 and residue Typ67 (Fig 11A). Exam-
ination of this proposed binding pose clearly suggests that the un-substituted phenyl ring of
UC-112 failed to occupy a hydrophobic groove (cycled with green dash line in Fig 11A), and a
properly sized, non-polar para-substitution (e.g., an isopropyl moiety as in compound 4g) to
this ring would fill this groove and also provide excellent overlap with the bioactive AVPI pep-
tide (Fig 11B). This is consistent with our experimental observations. Further refinement to
this model, and ultimately an X-ray crystal structure will greatly facilitate the future optimiza-
tion of this scaffold.
Drug affinity responsive target stability (DARTS) assay
To investigate that whether the survivin down-regulation effect is caused by direct interaction
between survivin protein and UC-112 analogs, we performed DARTS assay which is a well-es-
tablished target identification method [28–31]. DARTS assay relies on the increasing of prote-
olysis resistance of the target protein generated by the interaction with small molecular ligand.
We utilized immunoblotting to detect the abundances of several proteins including survivin in
M14 or A375 cell lysates either undigested or digested by different concentrations of pronase.
Representative data shown in Fig 12 clearly indicated that the protease susceptibility of survivin
is significantly reduced in cell lysates pre-treated with 20 μM 4g, comparing with the GAPDH
control group. Consistent with its selective survivin inhibition, 4g has negligible effects to pro-
tease susceptibility for other IAPs such as XIAP, cIAP1 or cIAP2. This observation suggested a
possible direct interaction between 4g and survivin protein in cell lysates that will be confirmed
by X-ray crystallography in the future.
Table 6. Aqueous solubility and in vitro metabolism properties of compound 4g.
ID
Solubility a (μM)
Half-Life b (min)
Clint (μL/min/mg)
4g
148.7
51.0
136.6
a test concentration is 200 μM
b test concentration is 100 nM.
doi:10.1371/journal.pone.0129807.t006
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Novel Small Molecule Survivin Inhibitors for Cancer
Table 7. Cytochrome P450 inhibition effects of compound 4g.
CYP enzyme
Substrate
% inhibition of control values
CYP1A
phenacetin
bupropion
18
21
21
17
45
87
3
CYP2B6
CYP2C8
CYP2C9
CYP2C19
CYP2D6
CYP3A
paclitaxel
diclofenac
omeprazole
dextromethorphan
midazolam
testosterone
CYP3A
29
doi:10.1371/journal.pone.0129807.t007
In vivo anti-tumor efficacy assessment
Since our in vitro study showed that compound 4g has the highest anti-proliferative potency in
this series of UC-112 analogs, we selected 4g to test its in vivo efficacy against tumor growth in
a human melanoma A375 xenograft model through i.p injection. As shown in Fig 13A, com-
pound 4g inhibited the growth of A375 xenograft tumor in a dose-dependent manner during
the three weeks of continuous treatment. The tumor growth in compound 4g 20 mg/kg and
40 mg/kg treatment group is 53% and 79% slower than the vehicle control group, respectively.
Furthermore, Western blotting analysis revealed that the expression levels of survivin and
XIAP protein decreased in tumor tissues which were freshly collected from compound 4g treat-
ed groups (Fig 13B). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)
assay, which measured the nuclear DNA fragmentation, showed that a high dose (40 mg/kg)
of compound 4g treatment caused the widely-spread cell apoptosis inside the tumor tissues
(Fig 13C).
Fig 9. Western blotting assay of A375 and PC-3 cells treated with gradient increasing dose of 4f or 4g for 24 h. Left panel is in A375 cancer cell line.
Right panel is in PC-3 cancer cell line.
doi:10.1371/journal.pone.0129807.g009
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Novel Small Molecule Survivin Inhibitors for Cancer
Fig 10. Relative in vitro caspases 3/7 activity of human melanoma A375 or human prostate cancer PC-
3 cells were evaluated after treatment of UC-112 analogs (1 μM) for 24 h (N = 3). The luminescence unit
data was adjusted according to the cell viability results read from the same well in 96-well plate.
***P < 0.001, **P < 0.01 compared with corresponded results from control group.
doi:10.1371/journal.pone.0129807.g010
Materials and Methods
General
All animal studies described in this manuscript have been approved by the IACUC at the Uni-
versity of Tennessee Health Science Center. All reagents were purchased from Sigma-Aldrich
Chemical Co., Alfa Aesar (Ward Hill, MA), and AK Scientific (Mountain View, CA) and were
used without further purification. Routine thin layer chromatography (TLC) was performed
on aluminum-backed Uniplates (Analtech, Newark, DE). NMR spectra were obtained on a
Varian Inova-500 spectrometer (Agilent Technologies, Santa Clara, CA) or a Bruker Ascend
400 (Billerica, MA) spectrometer. Chemical shifts are reported as parts per million (ppm) rela-
tive to TMS in CDCl3. High Resolution Mass spectra were collected in positive detection mode
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Novel Small Molecule Survivin Inhibitors for Cancer
Fig 11. Potential binding pose of UC-112 and compound 4g in the SMAC N-terminus tetra-peptide
AVPI binding site of survivin crystal structure (PDB entry: 3UIH). The surface of AVPI binding site in
survivin was colored according to residue charge (blue for positive while red for negative). (a) UC-112 (green
tube) formed hydrogen bonds with residue Asp71 and Glu68 on the survivin protein BIR domain. (b)
Compound 4g (orange tube) displayed similar binding pose with UC-112 but had better occupied the grove
toward the N-terminus of survivin protein. And this pose was overlapping well with the binding mode of native
ligand, SMAC AVPI (green stick).
doi:10.1371/journal.pone.0129807.g011
on a Waters Xevo G2-S Tof instrument equipped with an electron-spray ionization (ESI)
source (Milford, MA).
Synthesis
Preparation of 5-chloromethyl-8-quinolinol hydrochloride (2). A mixture of 5.84 g
(40.0 mmol) of 8-quinolinol, 50 ml of concentrated hydrochloric acid, and 6.4 ml of 37% form-
aldehyde was treated with 0.6 g of zinc chloride and stirred for 12 h. The mixture was filtered,
washed with copious acetone and dried to give compound 2 as a yellow solid (7.2g, 78%). 1H
NMR (400 MHz, Deuterium Oxide) δ 9.12 (dd, J = 8.7, 1.4 Hz, 1H), 8.88 (dd, J = 5.5, 1.4 Hz,
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Novel Small Molecule Survivin Inhibitors for Cancer
Fig 12. Representative drug affinity responsive target stability (DARTS) results for pronase-digested
A375 or M14 cell lysates. Immunoblotting showed protection of the target protein, survivin, by incubation
with compound 4g at the concentration of 20 μM, whereas digestion of the non-target proteins like GAPDH
was unchanged.
doi:10.1371/journal.pone.0129807.g012
1H), 7.97 (dd, J = 8.7, 5.4 Hz, 1H), 7.59 (d, J = 8.0 Hz, 1H), 7.24 (d, J = 7.9 Hz, 1H), 4.93
(s, 2H).
General procedure for the synthesis of compounds (3a-3l). Method A(only for 3a,3f and
3g): 5-chloromethyl-8-quinolinol hydrochloride 2 (1.15 g, 5 mmol) was suspended in substi-
tuted benzyl alcohol 3 (25 mmol) and the mixture was heated at 90°C for 2 h, during which
time the solution became completely homogeneous. The solution was then poured to 200 ml
diethyl ether. The corresponding hydrochloride salt was recovered by filtration as a bright yel-
low solid. The yellow solid was then suspended in diethyl ether and aqueous NH4OH (2.5%
v/v, 7.5 mL, ~1.2 equiv.) was added drop by drop under vigorous stirring until the aqueous
phase was basic (pH 8–10). The organic phase was washed with water, dried with anhydrous
sodium sulfate and evaporated to dryness to afford the desired ethers.
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Novel Small Molecule Survivin Inhibitors for Cancer
Fig 13. In vivo anti-tumor efficacy of compound 4g (N = 6). (a) 4g effectively inhibited the growth of A375
xenograft tumor after three weeks continuous treatment (i.p. injection) in a dose-dependent manner (left
panel) without causing obvious decrease of mice body weight (right panel). (b) Western blotting results on the
A375 xenograft tumor tissues (each lane represents one single mice). (c) Representative images of TUNEL
assay using the formalin-fixed tumor sections.
doi:10.1371/journal.pone.0129807.g013
Method B (for 3b-e and 3h-l): To a solution of substituted benzyl alcohol 3(6 mmol) in
anhydrous THF(30ml) was added sodium hydride (60% dispersion in mineral oil, 0.72g,
18 mmol) at 0 oC. The suspension was stirred at 0 oC for 30 minutes. Salt 2 (1.15g, 5mmol)
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Novel Small Molecule Survivin Inhibitors for Cancer
was added to the suspension. The mixture was stirred at r.t for 3 hours. Water was added to the
suspension, the mixture became homogeneous. The mixture was extracted by ethyl acetate and
washed with brine, dried over anhydrous sodium sulfate and concentrated to get the crude.
The crude compound was purified by flash chromatography (ethyl acetate: hexane 3:1).
5-(((4-fluorobenzyl)oxy)methyl)quinolin-8-ol(3a). 1H NMR (400 MHz, Chloroform-d)
δ 8.81 (dd, J = 4.2, 1.6 Hz, 1H), 8.47 (dd, J = 8.5, 1.5 Hz, 1H), 7.49 (dd, J = 8.5, 4.2 Hz, 1H), 7.41
(d, J = 7.8 Hz, 1H), 7.32 – 7.27 (m, 2H), 7.11 (d, J = 7.7 Hz, 1H), 7.06 – 6.98 (m, 2H), 4.87 (s,
2H), 4.50 (s, 2H).
5-(((4-chlorobenzyl)oxy)methyl)quinolin-8-ol(3b). 1H NMR (400 MHz, Chloroform-d)
δ 8.83 (dd, J = 4.2, 1.6 Hz, 1H), 8.51 (dd, J = 8.5, 1.6 Hz, 1H), 7.52 (dd, J = 8.5, 4.2 Hz, 1H), 7.44
(d, J = 7.7 Hz, 1H), 7.36 – 7.31 (m, 5H), 7.14 (d, J = 7.7 Hz, 1H), 4.90 (s, 2H), 4.53 (s, 2H).
5-(((4-bromobenzyl)oxy)methyl)quinolin-8-ol(3c). 1H NMR (400 MHz, Chloroform-d)
δ 8.81 (dd, J = 4.2, 1.6 Hz, 1H), 8.47 (dd, J = 8.5, 1.6 Hz, 1H), 7.51 – 7.45 (m, 3H), 7.41 (d,
J = 7.7 Hz, 1H), 7.22 – 7.17 (m, 2H), 7.11 (d, J = 7.7 Hz, 1H), 4.87 (s, 2H), 4.48 (s, 2H).
5-(((4-(trifluoromethyl)benzyl)oxy)methyl)quinolin-8-ol(3d). 1H NMR (400 MHz,
Chloroform-d) δ 8.75 (dd, J = 4.2, 1.6 Hz, 1H), 8.45 (dd, J = 8.5, 1.6 Hz, 1H), 7.56 – 7.49 (m,
2H), 7.45 (dd, J = 8.5, 4.3 Hz, 1H), 7.36 (ddd, J = 7.8, 2.0, 1.2 Hz, 3H), 7.08 (d, J = 7.7 Hz, 1H),
4.85 (s, 2H), 4.51 (s, 2H).
5-(((4-methylbenzyl)oxy)methyl)quinolin-8-ol(3e). 1H NMR (500 MHz, Chloroform-d)
δ 8.72 (dd, J = 4.2, 1.6 Hz, 1H), 8.40 (dd, J = 8.5, 1.6 Hz, 1H), 7.40 (dd, J = 8.5, 4.1 Hz, 1H), 7.34
(d, J = 7.7 Hz, 1H), 7.15 (d, J = 7.9 Hz, 2H), 7.09 (d, J = 7.8 Hz, 2H), 7.03 (d, J = 7.7 Hz, 1H),
4.78 (s, 2H), 4.44 (s, 2H), 2.28 (s, 3H).
5-(((4-ethylbenzyl)oxy)methyl)quinolin-8-ol(3f). 1H NMR (400 MHz, Chloroform-d) δ
8.72 (dd, J = 4.2, 1.6 Hz, 1H), 8.41 (dd, J = 8.5, 1.5 Hz, 1H), 7.40 (dd, J = 8.5, 4.2 Hz, 1H), 7.35
(d, J = 7.7 Hz, 1H), 7.17 (d, J = 2.1 Hz, 2H), 7.11 (d, J = 8.0 Hz, 2H), 7.03 (d, J = 7.7 Hz, 1H),
4.79 (s, 2H), 4.44 (s, 2H), 2.58 (q, J = 7.6 Hz, 2H), 1.16 (t, J = 7.6 Hz, 3H).
5-(((4-isopropylbenzyl)oxy)methyl)quinolin-8-ol(3g). 1H NMR (400 MHz, Chloroform-
d) δ 8.72 (dd, J = 4.2, 1.6 Hz, 1H), 8.41 (dd, J = 8.5, 1.6 Hz, 1H), 7.40 (dd, J = 8.5, 4.2 Hz, 1H),
7.35 (d, J = 7.7 Hz, 1H), 7.18 (d, J = 4.2 Hz, 2H), 7.14 (d, J = 8.2 Hz, 2H), 7.03 (d, J = 7.7 Hz,
1H), 4.80 (s, 2H), 4.44 (s, 2H), 2.83 (p, J = 6.9 Hz, 1H), 1.17 (d, J = 7.0 Hz, 6H).
5-(((4-(tert-butyl)benzyl)oxy)methyl)quinolin-8-ol(3h). 1H NMR (400 MHz, Chloro-
form-d) δ 8.67 (dd, J = 4.2, 1.6 Hz, 1H), 8.37 (dd, J = 8.5, 1.6 Hz, 1H), 7.35 (dd, J = 8.5, 4.2 Hz,
1H), 7.31 (d, J = 7.7 Hz, 1H), 7.25 (d, J = 8.5 Hz, 2H), 7.17–7.13 (m, 2H), 6.98 (d, J = 7.7 Hz,
1H), 4.76 (s, 2H), 4.40 (s, 2H), 1.20 (s, 9H).
5-(((4-methoxybenzyl)oxy)methyl)quinolin-8-ol(3i). 1H NMR (400 MHz, Chloroform-
d) δ 8.80 (dd, J = 4.2, 1.6 Hz, 1H), 8.47 (dd, J = 8.5, 1.6 Hz, 1H), 7.48 (dd, J = 8.5, 4.2 Hz, 1H),
7.41 (d, J = 7.8 Hz, 1H), 7.28–7.23 (m, 2H), 7.10 (d, J = 7.7 Hz, 1H), 6.93–6.82 (m, 2H), 4.84 (s,
2H), 4.48 (s, 2H), 3.81 (s, 3H).
5-(((4-(benzyloxy)benzyl)oxy)methyl)quinolin-8-ol(3j). 1H NMR (500 MHz, Chloro-
form-d) δ 8.84 (d, J = 4.2 Hz, 1H), 8.51 (d, J = 8.5 Hz, 1H), 7.53–7.41 (m, 6H), 7.37 (t, J = 7.3
Hz, 1H), 7.34–7.27 (m, 2H), 7.16 (d, J = 7.6 Hz, 1H), 7.06–6.98 (m, 2H), 5.12 (s, 2H), 4.89 (s,
2H), 4.53 (s, 2H).
N-(4-(((8-hydroxyquinolin-5-yl)methoxy)methyl)phenyl)acetamide(3k). 1H NMR (400
MHz, DMSO-d6) δ 9.93 (s, 1H), 9.86 (s, 1H), 8.87 (dd, J = 4.1, 1.6 Hz, 1H), 8.47 (dd, J = 8.6, 1.6
Hz, 1H), 7.60 (dd, J = 8.6, 4.2 Hz, 1H), 7.57–7.52 (m, 2H), 7.44 (d, J = 7.8 Hz, 1H), 7.28–7.21
(m, 2H), 7.02 (d, J = 7.7 Hz, 1H), 4.83 (s, 2H), 4.47 (s, 2H), 2.03 (s, 3H).
5-(((3,4,5-trimethoxybenzyl)oxy)methyl)quinolin-8-ol (3l). 1H NMR (400 MHz, Chlo-
roform-d) δ 8.85 (dd, J = 4.3, 1.5 Hz, 1H), 8.57 (dd, J = 8.5, 1.5 Hz, 1H), 7.53 (dd, J = 8.5, 4.2
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Novel Small Molecule Survivin Inhibitors for Cancer
Hz, 1H), 7.47 (d, J = 7.8 Hz, 1H), 7.19 (d, J = 7.6 Hz, 1H), 6.56 (s, 2H), 4.92 (s, 2H), 4.50 (s,
2H), 3.86 (s, 3H), 3.84 (s, 6H)
General Procure for the preparation of compounds (4a-4l). An equimolar mixture of
the substrates 3, paraformaldehyde, and the pyrrolidine in anhydrous ethanol (30 mL) was re-
fluxed for 4 hours under argon. After cooling, the solvent was evaporated under reduced pres-
sure. The crude compound was purified by flash chromatography (Dichloromethane:
methanol 20: 1).
5-(((4-fluorobenzyl)oxy)methyl)-7-(pyrrolidin-1-ylmethyl)quinolin-8-ol(4a). 1H NMR
(400 MHz, Chloroform-d) δ 8.91 (dd, J = 4.1, 1.6 Hz, 1H), 8.40 (dd, J = 8.5, 1.7 Hz, 1H), 7.44
(dd, J = 8.5, 4.2 Hz, 1H), 7.35 – 7.30 (m, 2H), 7.26 (s, 1H), 7.10 – 6.97 (m, 2H), 4.87 (s, 2H),
4.55 (s, 2H), 4.02 (s, 2H), 2.84 – 2.61 (m, 4H), 2.01 – 1.79 (m, 4H). HRMS (ESI): m/z calculated
for C22H23FN2O2 + H+ [M + H+]: 367.1822; Found: 367.1828.
5-(((4-chlorobenzyl)oxy)methyl)-7-(pyrrolidin-1-ylmethyl)quinolin-8-ol(4b). 1H NMR
(400 MHz, Chloroform-d) δ 8.82 (dd, J = 4.1, 1.6 Hz, 1H), 8.30 (dd, J = 8.5, 1.7 Hz, 1H), 7.34
(dd, J = 8.5, 4.1 Hz, 1H), 7.27 – 7.22 (m, 2H), 7.21 – 7.17 (m, 2H), 7.15 (s, 1H), 4.77 (s, 2H),
4.45 (s, 2H), 3.92 (s, 2H), 2.70 – 2.56 (m, 4H), 1.88 – 1.74 (m, 4H). HRMS (ESI): m/z calculated
for C22H23ClN2O2 + H+ [M + H+]: 383.1526; Found: 383.1521.
5-(((4-bromobenzyl)oxy)methyl)-7-(pyrrolidin-1-ylmethyl)quinolin-8-ol(4c). 1H NMR
(400 MHz, Chloroform-d) δ 8.89 (dd, J = 4.2, 1.7 Hz, 1H), 8.38 (dd, J = 8.5, 1.7 Hz, 1H),
7.49 – 7.44 (m, 2H), 7.42 (dd, J = 8.5, 4.1 Hz, 1H), 7.25 (s, 1H), 7.23 – 7.17 (m, 2H), 4.85 (s,
2H), 4.51 (s, 2H), 4.01 (s, 2H), 2.84 – 2.64 (m, 4H), 1.97 – 1.81 (m, 4H). HRMS (ESI): m/z cal-
culated for C22H23BrN2O2 + H+ [M + H+]: 427.1021; Found: 427.1015.
7-(pyrrolidin-1-ylmethyl)-5-(((4-(trifluoromethyl)benzyl)oxy)methyl)quinolin-8-ol
(4d). 1H NMR (500 MHz, Chloroform-d) δ 8.91 (dd, J = 4.1, 1.6 Hz, 1H), 8.42 (dd, J = 8.5, 1.7
Hz, 1H), 7.61 (d, J = 8.0 Hz, 2H), 7.49 – 7.40 (m, 3H), 7.27 (d, J = 3.1 Hz, 1H), 4.90 (s, 2H), 4.62
(s, 2H), 4.02 (s, 2H), 2.82 – 2.68 (m, 4H), 1.97 – 1.85 (m, 4H). HRMS (ESI): m/z calculated for
C23H23F3N2O2 + H+ [M + H+]: 417.1790; Found: 417.1792.
5-(((4-methylbenzyl)oxy)methyl)-7-(pyrrolidin-1-ylmethyl)quinolin-8-ol(4e). 1H NMR
(500 MHz, Chloroform-d) δ 8.80 (dd, J = 4.2, 1.7 Hz, 1H), 8.31 (dd, J = 8.5, 1.7 Hz, 1H), 7.34
(dd, J = 8.5, 4.1 Hz, 1H), 7.23 (s, 1H), 7.16 (d, J = 7.9 Hz, 2H), 7.09 (d, J = 7.7 Hz, 2H), 4.75 (s,
2H), 4.46 (s, 2H), 3.96 (s, 2H), 2.80 – 2.65 (m, 4H), 2.28 (s, 3H), 1.88–1.78 (m, 4H). HRMS
(ESI): m/z calculated for C23H26N2O2 + H+ [M + H+]: 363.2073; Found: 363.2078.
5-(((4-ethylbenzyl)oxy)methyl)-7-(pyrrolidin-1-ylmethyl)quinolin-8-ol(4f). 1H NMR
(400 MHz, Chloroform-d) δ 8.81 (dd, J = 4.2, 1.7 Hz, 1H), 8.30 (dd, J = 8.5, 1.7 Hz, 1H), 7.33
(dd, J = 8.5, 4.1 Hz, 1H), 7.20 (s, 1H), 7.18 (d, J = 5.4 Hz, 2H), 7.12 (d, J = 8.1 Hz, 2H), 4.76 (s,
2H), 4.47 (s, 2H), 3.93 (s, 2H), 2.70 – 2.62 (m, 4H), 2.58 (q, J = 7.6 Hz, 2H), 1.88 – 1.74 m, 4H),
1.16 (t, J = 7.6 Hz, 3H). HRMS (ESI): m/z calculated for C24H28N2O2 + H+ [M + H+]:
377.2229; Found: 377.2240.
5-(((4-isopropylbenzyl)oxy)methyl)-7-(pyrrolidin-1-ylmethyl)quinolin-8-ol(4g). 1H
NMR (400 MHz, Chloroform-d) δ 8.89 (dd, J = 4.1, 1.6 Hz, 1H), 8.38 (dd, J = 8.5, 1.6 Hz, 1H),
7.40 (dd, J = 8.5, 4.1 Hz, 1H), 7.29 (s, 1H), 7.26 – 7.20 (m, 4H), 4.85 (s, 2H), 4.55 (s, 2H), 4.00
(s, 2H), 2.92 (p, J = 6.9 Hz, 1H), 2.78 – 2.60 (m, 4H), 1.96 – 1.80 (m, 4H), 1.25 (d, J = 6.9 Hz,
6H). HRMS (ESI): m/z calculated for C25H30N2O2 + H+ [M + H+]: 391.2386; Found: 391.2377.
5-(((4-(tert-butyl)benzyl)oxy)methyl)-7-(pyrrolidin-1-ylmethyl)quinolin-8-ol(4h). 1H
NMR (400 MHz, Chloroform-d) δ 8.81 (dd, J = 4.1, 1.6 Hz, 1H), 8.31 (dd, J = 8.5, 1.7 Hz, 1H),
7.36 – 7.28 (m, 3H), 7.23 – 7.19 (m, 2H), 4.78 (s, 2H), 4.47 (s, 2H), 3.94 (s, 2H), 2.77 – 2.50 (m,
4H), 1.90 – 1.74 (m, 4H), 1.25 (s, 9H). HRMS (ESI): m/z calculated for C26H32N2O2 + H+ [M +
H+]: 405.2542; Found: 405.2535.
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Novel Small Molecule Survivin Inhibitors for Cancer
5-(((4-methoxybenzyl)oxy)methyl)-7-(pyrrolidin-1-ylmethyl)quinolin-8-ol(4i). 1H
NMR (500 MHz, Chloroform-d) δ 8.89 (dd, J = 4.1, 1.7 Hz, 1H), 8.38 (dd, J = 8.5, 1.7 Hz, 1H),
7.41 (dd, J = 8.5, 4.1 Hz, 1H), 7.30 – 7.27 (m, 2H), 7.25 (s, 1H), 6.97 – 6.83 (m, 2H), 4.83 (s,
2H), 4.52 (s, 2H), 4.01 (s, 2H), 3.82 (s, 3H), 2.86 – 2.62 (m, 4H), 1.96 – 1.79 (m, 4H). HRMS
(ESI): m/z calculated for C23H26N2O3 + H+ [M + H+]: 379.2022; Found: 379.2025.
5-(((4-(benzyloxy)benzyl)oxy)methyl)-7-(pyrrolidin-1-ylmethyl)quinolin-8-ol(4j). 1H
NMR (400 MHz, Chloroform-d) δ 8.80 (dd, J = 4.1, 1.6 Hz, 1H), 8.30 (dd, J = 8.5, 1.7 Hz, 1H),
7.39–7.29 (m, 5H), 7.28–7.20 (m, 4H), 6.94–6.85 (m, 2H), 5.00 (s, 2H), 4.75 (s, 2H), 4.44 (s,
2H), 3.97 (s, 2H), 2.80–2.60 (m, 4H), 1.90–1.76 (m, 4H). HRMS (ESI): m/z calculated for
C29H30N2O3 + H+ [M + H+]: 455.2335; Found: 455.2335.
N-(4-(((8-hydroxy-7-(pyrrolidin-1-ylmethyl)quinolin-5-yl)methoxy) methyl)phenyl)
acetamide(4k). 1H NMR (400 MHz, Chloroform-d) δ 8.81 (dd, J = 4.1, 1.7 Hz, 1H), 8.30 (dd,
J = 8.5, 1.7 Hz, 1H), 7.42 (d, J = 8.4 Hz, 2H), 7.34 (dd, J = 8.5, 4.1 Hz, 1H), 7.25–7.20 (m, 2H),
7.18 (s, 1H), 4.75 (s, 2H), 4.46 (s, 2H), 3.94 (s, 2H), 2.76–2.66 (m, 4H), 2.11 (s, 3H), 1.88–1.76
(m, 4H). HRMS (ESI): m/z calculated for C24H27N3O3 + H+ [M + H+]: 406.2131; Found:
406.2136.
7-(pyrrolidin-1-ylmethyl)-5-(((3,4,5-trimethoxybenzyl)oxy)methyl)quinolin-8-ol
(4l). 1H NMR (400 MHz, Chloroform-d) δ 8.82 (dd, J = 4.1, 1.6 Hz, 1H), 8.34 (dd, J = 8.5, 1.7
Hz, 1H), 7.34 (dd, J = 8.5, 4.1 Hz, 1H), 7.19 (s, 1H), 6.48 (s, 2H), 4.79 (s, 2H), 4.42 (s, 2H), 3.93
(s, 2H), 3.77 (s, 3H), 3.76 (s, 6H), 2.78 – 2.48 (m, 4H), 1.89 – 1.66 (m, 4H). HRMS (ESI): m/z
calculated for C25H30N2O5 + H+ [M + H+]: 439.2233; Found: 439.2245.
General Procure for the synthesis of compounds (5a-5g). Method A (for 5a-5d): To a so-
lution of alcohols (6 mmol) in anhydrous THF(30ml) was added sodium hydride (60% disper-
sion in mineral oil, 0.72g, 18 mmol) at 0 oC. The suspension was stirred at 0 oC for 30 minutes.
Salt 2 (1.15g, 5mmol) was added to the suspension. The mixture was stirred at r.t for 3 hours.
Water was added to the suspension, the mixture became homogeneous. The mixture was ex-
tracted by ethyl acetate and washed with brine, dried over anhydrous sodium sulfate and con-
centrated to get the crude. The crude compound was purified by flash chromatography (ethyl
acetate: hexane 3:1).
Method B(for 5e-5g): 5-chloromethyl-8-quinolinol hydrochloride 2 (1.15 g, 5 mmol) was
suspended in different alcohols (25 mmol) and the mixture was heated at 90°C for 2 h, during
which time the solution became completely homogeneous. The solution was then poured to
200 ml diethyl ether. The corresponding hydrochloride salt was recovered by filtration as a
bright yellow solid. The yellow solid was then suspended in diethyl ether and aqueous NH4OH
(2.5% v/v, 7.5 mL, ~1.2 equiv.) was added drop by drop under vigorous stirring until the aque-
ous phase was basic (pH 8–10). The organic phase was washed with water, dried with anhy-
drous sodium sulfate and evaporated to dryness to afford the desired products.
5-((furan-2-ylmethoxy)methyl)quinolin-8-ol(5a). 1H NMR (400 MHz, Chloroform-d) δ
8.72 (dd, J = 4.3, 1.6 Hz, 1H), 8.37 (dd, J = 8.5, 1.5 Hz, 1H), 7.41 (dd, J = 8.5, 4.2 Hz, 1H), 7.38–
7.33 (m, 2H), 7.03 (d, J = 7.7 Hz, 1H), 6.32–6.24 (m, 2H), 4.79 (s, 2H), 4.42 (s, 2H).
5-((thiophen-2-ylmethoxy)methyl)quinolin-8-ol(5b). 1H NMR (400 MHz, Chloroform-
d) δ 8.80 (dd, J = 4.2, 1.6 Hz, 1H), 8.48 (dd, J = 8.5, 1.6 Hz, 1H), 7.48 (dd, J = 8.5, 4.2 Hz, 1H),
7.42 (d, J = 7.7 Hz, 1H), 7.31 (dd, J = 4.8, 1.5 Hz, 1H), 7.10 (d, J = 7.7 Hz, 1H), 7.00–6.97 (m,
2H), 4.88 (s, 2H), 4.70 (s, 2H).
5-((pyridin-2-ylmethoxy)methyl)quinolin-8-ol(5c). 1H NMR (400 MHz, Chloroform-d)
δ 8.80 (dd, J = 4.2, 1.6 Hz, 1H), 8.58–8.52 (m, 2H), 7.67 (td, J = 7.7, 1.8 Hz, 1H), 7.50 (dd,
J = 8.5, 4.2 Hz, 1H), 7.46 (d, J = 7.7 Hz, 1H), 7.40 (dt, J = 7.8, 1.0 Hz, 1H), 7.21–7.16 (m, 1H),
7.11 (d, J = 7.7 Hz, 1H), 4.98 (s, 2H), 4.70 (s, 2H).
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Novel Small Molecule Survivin Inhibitors for Cancer
5-((cyclohexylmethoxy)methyl)quinolin-8-ol(5d). 1H NMR (400 MHz, Chloroform-d) δ
8.72 (dd, J = 4.2, 1.6 Hz, 1H), 8.43 (dd, J = 8.5, 1.6 Hz, 1H), 7.42 (dd, J = 8.5, 4.2 Hz, 1H), 7.34
(d, J = 7.7 Hz, 1H), 7.02 (d, J = 7.7 Hz, 1H), 4.73 (s, 2H), 3.21 (d, J = 6.5 Hz, 2H), 1.79 – 1.41
(m, H), 1.23 – 0.97 (m, 3H), 0.91 – 0.73 (m, 2H).
5-(ethoxymethyl)quinolin-8-ol(5e). 1H NMR (400 MHz, Chloroform-d) δ 8.73 (dd,
J = 4.2, 1.6 Hz, 1H), 8.43 (dd, J = 8.5, 1.6 Hz, 1H), 7.42 (dd, J = 8.5, 4.2 Hz, 1H), 7.35 (d, J = 7.6
Hz, 1H), 7.03 (d, J = 7.7 Hz, 1H), 4.76 (s, 2H), 3.50 (q, J = 7.0 Hz, 2H), 1.16 (t, J = 7.0 Hz, 3H).
5-(propoxymethyl)quinolin-8-ol(5f). 1H NMR (400 MHz, Chloroform-d) δ 8.72 (dd,
J = 4.1, 1.6 Hz, 1H), 8.44 (dd, J = 8.5, 1.6 Hz, 1H), 7.42 (dd, J = 8.6, 4.2 Hz, 1H), 7.34 (d, J = 7.7
Hz, 1H), 7.02 (d, J = 7.7 Hz, 1H), 4.75 (s, 2H), 3.38 (t, J = 6.7 Hz, 2H), 1.55 (dtd, J = 14.0, 7.4,
6.7 Hz, 2H), 0.83 (t, J = 7.4 Hz, 3H).
5-(butoxymethyl)quinolin-8-ol(5g). 1H NMR (400 MHz, Chloroform-d) δ 8.72 (dd,
J = 4.2, 1.6 Hz, 1H), 8.43 (dd, J = 8.5, 1.6 Hz, 1H), 7.42 (dd, J = 8.6, 4.2 Hz, 1H), 7.34 (d, J = 7.7
Hz, 1H), 7.02 (d, J = 7.7 Hz, 1H), 4.75 (s, 2H), 3.42 (t, J = 6.6 Hz, 2H), 1.51 (ddt, J = 8.9, 7.9, 6.4
Hz, 2H), 1.34 – 1.22 (m, 2H), 0.81 (t, J = 7.4 Hz, 3H).
General Procure for the preparation of compounds (6a-6g). An equal molar mixture of
the substrates 5, paraformaldehyde, and the pyrrolidine in anhydrous ethanol (30 mL) was re-
fluxed for 4 hours under argon. After cooling, the solvent was evaporated under reduced pres-
sure. The crude compound was purified by flash chromatography (Dichloromethane:
methanol 20: 1) to generate pure products.
5-((furan-2-ylmethoxy)methyl)-7-(pyrrolidin-1-ylmethyl)quinolin-8-ol(6a). 1H NMR
(400 MHz, Chloroform-d) δ 8.87 (dd, J = 4.1, 1.6 Hz, 1H), 8.33 (dd, J = 8.5, 1.6 Hz, 1H), 7.44
(dd, J = 1.9, 0.9 Hz, 1H), 7.40 (dd, J = 8.5, 4.1 Hz, 1H), 7.25 (s, 1H), 4.83 (s, 2H), 4.52 (s, 2H),
4.00 (s, 2H), 2.78–2.68 (m, 4H), 1.95 – 1.79 (m, 4H). HRMS (ESI): m/z calculated for
C20H22N2O3 + H+ [M + H+]: 339.1709; Found: 339.1711.
7-(pyrrolidin-1-ylmethyl)-5-((thiophen-2-ylmethoxy)methyl)quinolin-8-ol(6b). 1H
NMR (400 MHz, Chloroform-d) δ 8.88 (dd, J = 4.1, 1.6 Hz, 1H), 8.37 (dd, J = 8.5, 1.7 Hz, 1H),
7.40 (dd, J = 8.5, 4.1 Hz, 1H), 7.31 (dd, J = 4.9, 1.3 Hz, 1H), 7.22 (s, 1H), 7.04 – 6.96 (m, 2H),
4.85 (s, 2H), 4.73 (s, 2H), 3.99 (s, 2H), 2.81 – 2.56 (m, 4H), 1.99 – 1.74 (m, 4H). HRMS (ESI):
m/z calculated for C20H22N2O2S + H+ [M + H+]: 355.1480; Found: 355.1473.
5-((pyridin-2-ylmethoxy)methyl)-7-(pyrrolidin-1-ylmethyl)quinolin-8-ol(6c). 1H NMR
(400 MHz, Chloroform-d) δ 8.89 (dd, J = 4.2, 1.6 Hz, 1H), 8.56 (ddd, J = 4.9, 1.8, 0.9 Hz, 1H),
8.46 (dd, J = 8.5, 1.6 Hz, 1H), 7.67 (td, J = 7.7, 1.8 Hz, 1H), 7.45 – 7.39 (m, 2H), 7.29 (s, 1H),
7.18 (ddd, J = 7.6, 4.9, 1.2 Hz, 1H), 4.96 (s, 2H), 4.71 (s, 2H), 4.02 (s, 2H), 2.82 – 2.66 (m, 4H),
1.95 – 1.84 (m, 4H). HRMS (ESI): m/z calculated for C21H23N3O2 + H+ [M + H+]: 350.1869;
Found: 350.1859.
5-((cyclohexylmethoxy)methyl)-7-(pyrrolidin-1-ylmethyl)quinolin-8-ol(6d). 1H NMR
(400 MHz, Chloroform-d) δ 8.80 (dd, J = 4.1, 1.7 Hz, 1H), 8.34 (dd, J = 8.5, 1.7 Hz, 1H), 7.36
(dd, J = 8.5, 4.1 Hz, 1H), 7.24 (s, 1H), 4.71 (s, 2H), 3.98 (s, 2H), 3.24 (d, J = 6.5 Hz, 2H),
2.80 – 2.65 (m, 4H), 1.92 – 1.78 (m, 4H), 1.74 – 1.44 (m, 6H), 1.25 – 1.00 (m, 3H), 0.90 – 0.70
(m, 2H). HRMS (ESI): m/z calculated for C22H30N2O2 + H+ [M + H+]: 355.2386; Found:
355.2385.
5-(ethoxymethyl)-7-(pyrrolidin-1-ylmethyl)quinolin-8-ol(6e). 1H NMR (400 MHz,
Chloroform-d) δ 8.90 (dd, J = 4.2, 1.6 Hz, 1H), 8.43 (dd, J = 8.5, 1.7 Hz, 1H), 7.45 (dd, J = 8.5,
4.1 Hz, 1H), 7.29 (d, J = 1.2 Hz, 1H), 4.82 (s, 2H), 4.03 (s, 2H), 3.62 (q, J = 7.0 Hz, 2H),
2.88 – 2.61 (m, 4H), 2.04 – 1.73 (m, 4H), 1.27 (t, J = 7.0 Hz, 3H). HRMS (ESI): m/z calculated
for C17H22N2O2 + H+ [M + H+]: 287.1760; Found: 287.1753.
5-(propoxymethyl)-7-(pyrrolidin-1-ylmethyl)quinolin-8-ol(6f). 1H NMR (400 MHz,
Chloroform-d) δ 8.81 (dd, J = 4.2, 1.6 Hz, 1H), 8.34 (dd, J = 8.5, 1.7 Hz, 1H), 7.35 (dd, J = 8.5,
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Novel Small Molecule Survivin Inhibitors for Cancer
4.1 Hz, 1H), 7.19 (s, 1H), 4.72 (s, 2H), 3.94 (s, 2H), 3.41 (t, J = 6.7 Hz, 2H), 2.75 – 2.58 (m, 4H),
1.87 – 1.76 (m, 4H), 1.56 (dtd, J = 14.0, 7.4, 6.6 Hz, 2H), 0.85 (t, J = 7.4 Hz, 3H). HRMS (ESI):
m/z calculated for C18H24N2O2 + H+ [M + H+]: 315.2073; Found: 315.2059.
5-(butoxymethyl)-7-(pyrrolidin-1-ylmethyl)quinolin-8-ol(6g). 1H NMR (400 MHz,
Chloroform-d) δ 8.81 (dd, J = 4.1, 1.6 Hz, 1H), 8.34 (dd, J = 8.5, 1.7 Hz, 1H), 7.35 (dd, J = 8.5,
4.1 Hz, 1H), 7.20 (s, 1H), 4.72 (s, 2H), 3.95 (s, 2H), 3.45 (t, J = 6.6 Hz, 2H), 2.74 – 2.58 (m, 4H),
1.90 – 1.73 (m, 4H), 1.52 (ddt, J = 8.9, 7.9, 6.4 Hz, 2H), 1.36 – 1.23 (m, 2H), 0.82 (t, J = 7.4
Hz, 3H). HRMS (ESI): m/z calculated for C19H26N2O2 + H+ [M + H+]: 301.1916; Found:
301.1906.
General Procure for the preparation of compounds (8a-8b). To a solution of compound
7 (1 mmol) was added paraformaldehyde (1 mmol) and piperidine (1 mmol for 8a) or mor-
pholine (1 mmol for 8b). The mixture was stirred under reflux for 4 hours. The solvent was
then evaporated to get the crude product. The crude product was purified by flash chromatog-
raphy (Dichloromethane: methanol 20: 1).
5-((benzyloxy)methyl)-7-(piperidin-1-ylmethyl)quinolin-8-ol(8a). 1H NMR (400 MHz,
Chloroform-d) δ 8.82 (dd, J = 4.1, 1.7 Hz, 1H), 8.29 (dd, J = 8.5, 1.7 Hz, 1H), 7.32 (dd, J = 8.5,
4.2 Hz, 1H), 7.29–7.20 (m, 5H), 7.09 (s, 1H), 4.76 (s, 2H), 4.49 (s, 2H), 3.77 (s, 2H), 2.52 (s,
4H), 1.61 (p, J = 5.6 Hz, 4H), 1.44 (s, 2H). HRMS (ESI): m/z calculated for C22H24N2O3 + H+
[M + H+]: 363.2073; Found: 363.2080.
5-((benzyloxy)methyl)-7-(morpholinomethyl)quinolin-8-ol(8b). 1H NMR (400 MHz,
Chloroform-d) δ 8.88 (dd, J = 4.1, 1.6 Hz, 1H), 8.40 (dd, J = 8.5, 1.6 Hz, 1H), 7.43 (dd, J = 8.5,
4.2 Hz, 1H), 7.38–7.29 (m, 5H), 7.28 (s, 1H), 4.85 (s, 2H), 4.58 (s, 2H), 3.86 (s, 2H), 3.81–3.75
(m, 4H), 2.70–2.55 (m, 4H). HRMS (ESI): m/z calculated for C23H26N2O2 + H+ [M + H+]:
365.1865; Found: 365.1850.
General Procure for the preparation of compounds (9a-9b). The method to make com-
pounds 9a-9b was the same as the way to make compounds 5e- 5g.
5-(phenethoxymethyl)quinolin-8-ol(9a). 1H NMR (400 MHz, Chloroform-d) δ 8.77 (dd,
J = 4.2, 1.6 Hz, 1H), 8.30 (dd, J = 8.5, 1.5 Hz, 1H), 7.40–7.35 (m, 2H), 7.28–7.14 (m, 5H), 7.07
(d, J = 7.7 Hz, 1H), 4.82 (s, 2H), 3.72 (t, J = 7.0 Hz, 2H), 2.90 (t, J = 7.0 Hz, 2H).
5-((3-phenylpropoxy)methyl)quinolin-8-ol(9b). 1H NMR (400 MHz, Chloroform-d) δ
8.80 (dd, J = 4.2, 1.6 Hz, 1H), 8.51 (dd, J = 8.5, 1.6 Hz, 1H), 7.50 (dd, J = 8.5, 4.2 Hz, 1H), 7.41
(d, J = 7.7 Hz, 1H), 7.25 – 7.22 (m, 2H), 7.20 – 7.14 (m, 1H), 7.13 – 7.08 (m, 3H), 4.83 (s, 2H),
3.50 (t, J = 6.3 Hz, 2H), 2.65 (t, J = 8.6 Hz, 2H), 1.97 – 1.86 (m, 2H).
General Procure for the preparation of compounds (10a-10b). An equimolar mixture of
the substrates 9, paraformaldehyde, and the pyrrolidine in anhydrous ethanol (30 mL) was re-
fluxed for 4 hours under argon. After cooling, the solvent was evaporated under reduced pres-
sure. The crude product was purified by flash chromatography (Dichloromethane: methanol
20: 1) to generate pure products.
5-(phenethoxymethyl)-7-(pyrrolidin-1-ylmethyl)quinolin-8-ol(10a). 1H NMR (400
MHz, Chloroform-d) δ 8.86 (dd, J = 4.2, 1.7 Hz, 1H), 8.23 (dd, J = 8.5, 1.7 Hz, 1H), 7.32 (dd,
J = 8.5, 4.2 Hz, 1H), 7.27 (d, J = 2.4 Hz, 1H), 7.26–7.16 (m, 5H), 4.80 (s, 2H), 3.98 (s, 2H), 3.74
(t, J = 7.0 Hz, 2H), 2.91 (t, J = 7.0 Hz, 2H), 2.75–2.65 (m, 4H), 1.95–1.80 (m, 4H). HRMS (ESI):
m/z calculated for C23H26N2O2 + H+ [M + H+]: 363.2073; Found: 363.2060.
5-((3-phenylpropoxy)methyl)-7-(pyrrolidin-1-ylmethyl)quinolin-8-ol(10b). 1H NMR
(400 MHz, Chloroform-d) δ 8.89 (dd, J = 4.1, 1.7 Hz, 1H), 8.42 (dd, J = 8.5, 1.6 Hz, 1H), 7.43
(dd, J = 8.5, 4.1 Hz, 1H), 7.26 – 7.19 (m, 3H), 7.19 – 7.14 (m, 1H), 7.14 – 7.10 (m, 2H), 4.80
(s, 2H), 3.99 (s, 2H), 3.53 (t, J = 6.4 Hz, 2H), 2.75 – 2.64 (m, 6H), 1.99 – 1.83 (m, 6H).
HRMS (ESI): m/z calculated for C24H28N2O2 + H+ [M + H+]: 377.2229; Found: 377.2238.
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Novel Small Molecule Survivin Inhibitors for Cancer
General Procure for the preparation of compounds (11a-11b). To a solution of benzyl
mercaptan (6 mmol for 12a) or N-benzymethylamine (6 mmol for 12b) in anhydrous THF
(30ml) was added sodium hydride (60% dispersion in mineral oil, 0.72g, 18 mmol) at 0 oC.
The suspension was stirred at 0 oC for 30 minutes. Salt 2 (1.15g, 5mmol) was added to the sus-
pension. The mixture was stirred at r.t for 3 hours. Water was added to the suspension, the
mixture became homogeneous. The mixture was extracted by ethyl acetate and washed with
brine, dried over anhydrous sodium sulfate and concentrated to get the crude. The crude prod-
uct was purified by flash chromatography (ethyl acetate: hexane 3:1).
5-((benzylthio)methyl)-7-(pyrrolidin-1-ylmethyl)quinolin-8-ol(11a). 1H NMR (400
MHz, Chloroform-d) δ 8.78 (dd, J = 4.2, 1.6 Hz, 1H), 8.30 (dd, J = 8.6, 1.5 Hz, 1H), 7.44 (dd,
J = 8.5, 4.2 Hz, 1H), 7.37–7.26 (m, 5H), 7.06 (d, J = 7.8 Hz, 1H), 3.96 (s, 2H), 3.65 (s, 2H).
5-((benzyl(methyl)amino)methyl)quinolin-8-ol(11b). 1H NMR (400 MHz, Chloroform-
d) δ 8.82 (dd, J = 4.2, 1.6 Hz, 1H), 8.62 (dd, J = 8.5, 1.6 Hz, 1H), 7.47 (dd, J = 8.6, 4.2 Hz, 1H),
7.40 (d, J = 7.7 Hz, 1H), 7.37–7.32 (m, 4H), 7.32–7.26 (m, 1H), 7.13 (d, J = 7.7 Hz, 1H), 3.85 (s,
2H), 3.58 (s, 2H), 2.20 (s, 3H).
General Procure for the preparation of compounds (12a-12b). To a solution of com-
pound 11 (1 mmol) was added paraformaldehyde (1 mmol) and pyrrolidine (1mmol). The
mixture was stirred under reflux for 4 hours. The solvent was then evaporated to get the crude
product. The crude product was purified by flash chromatography (Dichloromethane: metha-
nol 20: 1) to get a light yellow solid.
5-((benzylthio)methyl)-7-(pyrrolidin-1-ylmethyl)quinolin-8-ol(12a). 1H NMR (400
MHz, Chloroform-d) δ 8.78 (dd, J = 4.2, 1.6 Hz, 1H), 8.17 (dd, J = 8.6, 1.6 Hz, 1H), 7.32 (dd,
J = 8.6, 4.1 Hz, 1H), 7.28 – 7.15 (m, 5H), 7.14 (s, 1H), 3.97 (s, 2H), 3.89 (s, 2H), 3.60 (s, 2H),
2.82 – 2.65 (m, 4H), 1.90– 1.80 (m, 4H). HRMS (ESI): m/z calculated for C22H24N2OS + H+
[M + H+]: 365.1688; Found: 365.1689.
5-((benzyl(methyl)amino)methyl)-7-(pyrrolidin-1-ylmethyl)quinolin-8-ol(12b). 1H
NMR (400 MHz, Chloroform-d) δ 8.87 (dd, J = 4.1, 1.6 Hz, 1H), 8.57 (dd, J = 8.6, 1.7 Hz, 1H),
7.43–7.31 (m, 5H), 7.31–7.27 (m, 1H), 7.24 (s, 1H), 3.93 (s, 2H), 3.91 (s, 2H), 3.68 (s, 2H),
2.60–2.48 (m, 4H), 2.30 (s, 3H), 1.85–1.70 (m, 4H). HRMS (ESI): m/z calculated for
C23H27N3O + H+ [M + H+]: 362.2232; Found: 362.2224.
Cell culture and cell viability assay
The anti-proliferative effect of UC-112 and its analogs were tested in human melanoma (A375,
M14 and M14/LCC6MDR1) and human prostate cancer (PC-3) cell lines. All the cell lines
were purchased from ATCC (American Type Culture Collection, Manassas, VA). The cancer
cells were cultured using the supplemented cell culture medium as described before [32] at
37°C in a humidified atmosphere containing 5% CO2. 5000 cells in logarithm growing phase
were seeded overnight into each well of a 96-well plate. Then the cells were continuously incu-
bated with sequential diluted compound solution (10 nM to 100 μM, 100 μl per well) in cell
culture medium. After 48 h treatment, the cell viability was determined in MTS assay and IC50
was calculated (n = 4) as described before [19, 33].
NCI-60 screening
Four compounds including the parental compound UC-112 were submitted to National Can-
cer Institute for its NCI-60 cell line screening, initially tested at one concentration (10 μM),
and subsequently selected for full five concentration testing following the standard protocols
disclosed by NCI [34]. In brief, cells were plated into 96 well micro-titer plates 24 h prior to the
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Novel Small Molecule Survivin Inhibitors for Cancer
treatment of compound solution for 48 h, then the cell viability was read out through absor-
bance of sulforhodamine B (SRB) staining.
Drug-like property profiling for compound 4g
All these experiments were performed by a contract research service company, Eurofins Pan-
labs Inc. (Study Number 100017964). Aqueous solubility (μM) was determined using the
shake-flask method by comparing the peak area of the principal peak in a calibration standard
(200 μM) containing organic solvent (methanol/water, 60/40, v/v) with the peak area of the
corresponding peak in a buffer sample. Metabolic stability study was carried out by incubating
compound 4g with human liver microsomes. Metabolic stability which is expressed as percent
of the parent compound remaining, was calculated by comparing the peak area of the com-
pound at the time point relative to that at time-0. The half-life (T1/2) was estimated from the
slope of the initial linear range of the logarithmic curve of compound remaining (%) vs. time.
In the cytochrome P450 inhibition study, human liver microsomes (0.1 mg/mL) were incubat-
ed with different substrates and compound 4g for 10 minutes. After the incubation, peak areas
corresponding to the metabolite of each substrate were recorded. The percent of control activi-
ty was then calculated by comparing the peak area obtained in the presence of compound 4g to
that obtained in the absence of compound 4g. Subsequently, the percent inhibition was calcu-
lated by subtracting the percent control activity from 100.
Caspase functional assay
The caspase 3/7 activity of cancer cells treated by DMSO control or compound of interest was
analyzed using Glo Caspase-Glo 3/7 kit from Promega Corporation (Madison, WI) as per
manufacturer’s instructions in similar protocol as described before [19]. The readings of rela-
tive luminescence unit were normalized by the cell viability read from the same well deter-
mined by compatible CytoTox-Fluor Cytotoxicity assay kit (Promega, WI).
Western blotting
Lysates of A375 cells treated by the compound solution for 24 h were used to determine the
change of IAP protein levels through western blotting. Primary rabbit antibodies against survi-
vin, XIAP, cIAP1, cIAP2, livin and GAPDH were purchased from Cell Signaling Technology,
Inc. (Danvers, MA) and used according to manufacture instructions as reported previously
[19]. Protein lane intensities were quantified by ImageJ software (US National Institutes of
Health, Bethesda, MD).
Molecular modeling
The molecular docking studies were performed following similar procedure as described before
[19, 35]in Schrodinger Molecular Modeling Suite 2014 (Schrodinger Inc., Portland, OR). All
the ligands were prepared to generate various conformation before being docked into the
SMAC AVPI binding site of a human survivin crystal structure (Protein Data Bank entry:
3UIH). Molecular dynamic calculation was done after the docking to minimize the energy of
potential ligand binding poses. Results were visualized using the Maestro interface of the
Schrodinger software.
DARTS assay
Drug affinity responsive target stability (DARTS) assay was performed to identify the protein
targets of UC-112 analogs in A375 or M14 cell lysates following the protocols described in the
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Novel Small Molecule Survivin Inhibitors for Cancer
literature [28–31]. Briefly, A375 or M14 lysates were prepared in non-denaturing M-PER lysis
buffer (Thermo Fisher Scientific, Waltham, MA) with protease and phosphatase inhibitors.
Then TNC (Tris, NaCl, CaCl2) buffer was added into cell lysates before the total protein con-
centration of lysate being determined by BCA protein assay kit (Thermo Fisher Scientific, Wal-
tham, MA). The lysates were split into two groups: one group was added with compound of
interest solution, the other group was added with same amount of vehicle (DMSO). The sam-
ples were mixed thoroughly and incubated at room temperature for 1 h. Then 10 mg/mL pro-
nase stock solution was added into both compound treated or vehicle control groups to achieve
the final dilution of 1:100, 1:300, 1:1000 and 1:3000. One aliquot of each group was kept as un-
digested control. The proteolysis was performed at room temperature for 30 min before ice
cold protease inhibitor stock being added into the mixture. Then SDS loading buffer was added
into all the samples and heated to 70°C for 10 min. Finally, the results were analyzed by
SDS-PAGE and immunoblotting.
In vivo anti-tumor efficacy
3 × 106 A375 cells were implanted into the left-side dorsal flank of each nude mouse (n = 5) to
establish the human melanoma A375 tumor xenograft model as described previously [19, 36].
Compound 4g were first dissolved in DMSO then diluted by 2% methylcellulose PBS buffer.
The proportion of DMSO in final solution was kept at lower than 5%. Mice body weight and
the size of tumor were closely monitored during the 3 week continuous treatment (i.p. injec-
tion, one dose per day, five days per week). At the end point of the treatment, mice were sacri-
ficed after anesthesia. A375 tumor tissues were isolated then freshly lysed on ice to check the
targeted protein levels through Western blotting.
TUNEL assay
A375 tumor tissues collected from the in vivo efficacy study in the above were fixed in formalin
phosphate buffer for one week. Then the tissues were processed to get paraffin embedded sec-
tions. TUNEL assay was performed using DeadEnd Fluorometric kit (Promega Corporation,
Madison, WI) following manufacturer’s instructions. By the end of the experiment, VECTA-
SHIELD Hard Set mounting medium with DAPI (Vector Lab, Inc., Burlingame, CA) was used
to mount the tumor slides and stain the nuclei. The final slides were analyzed immediately
under a fluorescence microscope (EVOS FL Cell Imaging System, Thermo Fisher Scientific
Inc., NY).
Conclusion
In summary, a series of novel analogs were designed and synthesized based on targeted struc-
tural modification of the lead compound UC-112. Structure-activity relationships (Fig 14)
were investigated by making modifications to C ring, D ring and the linker. Several analogs
showed excellent anti-proliferative activities and could also effectively overcome Pgp-mediated
multiple drug resistance. This scaffold as demonstrated by our most potent compound 4g has
showed good drug-like properties which is important for further development. Preliminary
studies of action mechanism confirmed that the new analogs maintain their mode of action by
selectively down-regulating the level of survivin as the parent compound UC-112. Compound
4g effectively inhibited the growth of A375 xenograft tumor in vivo. Further optimization of
this scaffold and efforts to produce X-ray crystal structures of survivin protein in complex with
these novel survivin inhibitors are currently being carried out to generate more potent and se-
lective survivin inhibitors based on this unique platform.
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Novel Small Molecule Survivin Inhibitors for Cancer
Fig 14. Structure activity relationships of UC-112 analogs.
doi:10.1371/journal.pone.0129807.g014
Supporting Information
S1 Fig. Mean graph report for UC-112 (NSC D-782181) from NCI-60 cell line five dose
anti-proliferative screening.
(TIF)
S2 Fig. Mean graph report for compound 12a (NSC D-782184) from NCI-60 cell line five
dose anti-proliferative screening.
(TIF)
S3 Fig. Mean graph report for compound 4c (NSC D-782182) from NCI-60 cell line five
dose anti-proliferative screening.
(TIF)
S4 Fig. Mean graph report for compound 4g (NSC D-782180) from NCI-60 cell line five
dose anti-proliferative screening.
(TIF)
S5 Fig. ImageJ quantification data of western blotting analysis results in Fig 2.
(TIF)
Author Contributions
Conceived and designed the experiments: MX JW WL. Performed the experiments: MX JW
ZTL WL. Analyzed the data: MX JW ZTL YL DDM WL. Contributed reagents/materials/anal-
ysis tools: DDM SWW WL ZML. Wrote the paper: MX JW ZTL YL DDM WL.
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Novel Small Molecule Survivin Inhibitors for Cancer
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