Synthesis of a high-purity chemical library reveals a potent inducer of oxidative stress

Article abstract of DOI:10.1002/cbic.201000193

(Chemical Equation Presented) Needle in a haystack: Synthesis of a high-purity, biogenic, heterocyclic, 936-member library enabled the identification of a small molecule, triazatricyclamide (1), that potently inhibited the proliferation of several cancer cell lines and induced rapid oxidative stress. Unusually, this agent caused cell death by activating both caspase-dependent and -independent pathways.

Full text of DOI:10.1002/cbic.201000193

DOI: 10.1002/cbic.201000193
Synthesis of a High-Purity Chemical Library Reveals a Potent Inducer of
Oxidative Stress
Jiayue Cui,[a] Kenji Matsumoto,[a] Cindy Y. Wang,[b] Marcus E. Peter,[b] and Sergey A. Kozmin*[a]
The high-throughput screening of chemical libraries plays an
important role in biomedical research. The development of
small-molecule libraries that would occupy the regions of vast
chemical space that can be effectively recognized by biological
targets remains challenging. One strategy is to assemble libra-
ries that incorporate validated pharmacophores, also known as
privileged structures,[1] in order to increase the probability of
identifying potent bioactive chemotypes. We describe here the
assembly of a 936-member small-molecule library that was
designed to contain a fusion of benzodiazepine and tetramic
acid subunits. The selection of this structural platform was
guided by the prevalent biological activities of the two hetero-
cyclic fragments,[1,2] as well as their favorable physicochemical
properties. A cell-viability screen of this chemical library identi-
fied a small molecule that rapidly induced oxidative stress,
produced substantial DNA fragmentation, and displayed an
unusual mechanism of cell death initiation that entailed activa-
tion of both caspase-dependent and caspase-independent cel-
lular pathways. The potent activity of this agent (IC50 =0.16–
1.0 mm) is remarkable given the relatively small number of
compounds that were subjected to the primary screen, as well
as the low toxicity of the library.
Supporting Information). Subsequent construction of a focused
library (Figure 2) revealed that the cyclopropyl amide moiety
(R1) was crucial to the potency of 1. para-substitution in the
phenyl group (R2) was well tolerated and resulted in increased
activity of fluorine-substituted analogue. A disubstituted al-
kene to link the phenyl group to the tricyclic core, and an N-
prenyl substituent (R3) were both required for activity.
Cell-cycle analysis on HL-60 cells revealed that 1 induced a
concentration-dependent increase in G0/G1 population and a
The parallel assembly process was based on a protocol origi-
nally described by Matsuo and Tanaka,[3] and entailed the con-
densation of five vinylogous ureas I with 16 aldehydes II to
give 80 tricyclic amines III, followed by chemoselective N-acyla-
tion with 12 acid chlorides IV (Scheme 1). The resulting library
V was prepared in solution on a 2.5 mmol scale (1.0–1.5 mg of
final products) and was rapidly purified by parallel preparative
thin-layer chromatography.[4] Subsequent analysis established
that 936 out of 960 reactions proceeded successfully to deliver
the final compounds in high chemical purity.[5] 1H NMR analysis
was employed to quantify the amount and purity of 120 ran-
domly selected compounds.[5]
Evaluation of the proliferation of the A549 cell line in the
presence of the library revealed that a single library member,
which was termed triazatricyclamide (1) due to its polyhetero-
cyclic architecture, suppressed cellular growth with an IC50 of
160 nm (Figure 1). Similarly potent activity of 1 was observed
in several other cancer cell lines (Figure 1 and Figure S1 in the
[a] J. Cui, K. Matsumoto, Prof. S. A. Kozmin
Department of Chemistry, University of Chicago
5735 South Ellis Avenue, Chicago, IL 60637 (USA)
Fax: (+1)773-702-0805
E-mail: skozmin@uchicago.edu
[b] C. Y. Wang, Prof. M. E. Peter
Ben May Department for Cancer Research, University of Chicago
Chicago, IL 60637 (USA)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201000193.
Scheme 1. Synthesis of 936-member small-molecule library.
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being distinctly different from that of a known apoptosis in-
ducer, staurosporine (STS).[8]
Analysis of the dynamics of cell death induced by 1 revealed
a concentration-dependent build-up of reactive oxygen species
(ROS),[9] which was observed as early as three hours after initial
incubation with 1 (Figures 5 and S6). This effect appeared to
be dependent on the caspase activation, since treatment of
cells with an established caspase inhibitor benzyloxycarbonyl-
valine-alanine-aspartate fluoromethyl ketone (z-VAD-fmk)[10]
substantially suppressed the ROS generation induced by 1
(Figure S6). The absence of changes in mitochondrial mem-
brane potential during the same incubation period (Figure S5)
suggested that mitochondria were not directly involved in the
early release of ROS; this pointed to an unusual mechanism of
cell death initiation.
Figure 1. Growth inhibitory constants (IC50) of 1 in five cancer cell lines.
decrease in S population, while the G2 population was un-
affected (Figure S2). Arrested growth was associated with sub-
stantial chromatin condensation (Figure 3) and DNA fragmen-
tation detected by the standard Nicoletti method (Fig-
ure S3).[6,7] Since fragmented DNA and condensed chromatin
are typical hallmarks of the apoptosis,[6] we next examined the
activity of caspases that are involved in the execution of pro-
grammed cell death. Indeed, treatment of cells with 1 resulted
in both dose- and time-dependent activation of caspases 3, 7
and 8 (Figures 4 and S4), with the dynamics of this process
In addition to caspase activation, the presence of phospha-
tidyl serine (PS) on the outer leaflet of the membrane is highly
indicative of early apoptotic cells.[11] We next analyzed the cel-
lular exposure of PS, as well as the membrane integrity of HL-
60 cells treated with 1. Incubation of cells with 1 resulted in
early (6–12 h after treatment) and caspase-dependent expo-
sure of PS, as shown by the appearance of the annexin V-posi-
tive/propidium iodide-negative
(AV+/PIÀ) population of cells
and the ability of z-VAD-fmk to
block its formation (Figures 6
and S7). The membrane integrity
of cell, however, was progres-
sively lost in caspase-independ-
ent manner during the first 24 h,
since formation of cell popula-
tion, which was positive in both
annexin V (AV+) and propidium
iodide (PI+), could not be inhib-
ited by z-VAD-fmk (Figures 6 and
S7). Additional results indicated
that the cell death induced by 1
was independent of extrinsic
and intrinsic apoptotic pathways.
Similar levels of DNA fragmenta-
tion in response to treatment
with 1 were observed Jurkat A3
(JA3) cells that lacked a Fas-asso-
ciated protein with Death
Domain (JA3 FADDÀ/À) or the
apoptosis initiator caspase
8
(JA3 caspase8À/À) compared to
JA3 parental cells (Figure 7), as
well as MCF7-Fas cells transfect-
ed with Bcl-xL (MCF7-FB) com-
pared to the same cells trans-
fected only with vector control
(MCF7-FV; Figure 8).[12]
Our initial analysis revealed
that 1 activated both caspase-
dependent and -independent
cell-death pathways. The early
Figure 2. Structure–activity relationship of triazatricyclamides measured by their ability to inhibit the growth of
A549 cells; compound 1 was assigned a relative activity of 100%, which corresponded to IC50 =160 nm.
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Figure 3. Chromatin condensation visualized by staining HL-60 cells with
Hoechst 33342.
Figure 6. Effect of z-VAD-fmk on PS exposure and membrane integrity in HL-
60 cells treated with 1.
Figure 4. Time dependence of caspase-3/7 activation in HL-60 cells treated
with 1 and staurosporine (STS). Error bars, standard deviation (SD); n=3.
Figure 7. DNA fragmentation in Jurkat A3 cells lacking key components of
the extrinsic apoptotic pathway upon treatment with 1, leucine zipper-
tagged Fas ligand (LzFasL) and DMSO for 24 h. Error bars, SD; n=3.
Figure 5. Release of ROS in HL-60 cells in response to 1 (20 mm) for 3 h
(shown in black) compared to DMSO control (shown in gray) measured by
monitoring dihydroethidium (DHE) fluorescence.
Figure 8. DNA fragmentation in MCF7-Fas cells overexpressing Bcl-xL upon
treatment with 1, LzFasL and DMSO for 24 h. Error bars, SD; n=3.
events, such as ROS generation and PS exposure, were found
to be highly dependent on caspase activation. It appears,
however, that at some point DNA degradation and cell death
induced by 1 become independent of caspase activity. A
number of caspase-independent forms of cell death have been
described.[13] Most of them involve the release of mitochodrial
proteins such as apoptosis-inducing factor (AIF), high-tempera-
ture requirement protein (HtrA2/OMI), or endonuclease G
(ENDO G), which can cause nuclear changes independently of
caspase activation.[13] However, the release of such factors
would be inhibited by Bcl-xL. The fact that Bcl-xL did not pre-
vent cell death, at least not in MCF7 cells, seems to suggest
that 1 activated a pathway that was independent of mitochon-
dria.[14] To our knowledge, this activity profile does not corre-
late to any of the existing agents that are capable of inducing
either caspase-dependent or -independent cell death.[15]
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ChemBioChem 2010, 11, 1224 – 1227

growth when compared with vehicle control cells were calculated
from sigmoidal plots of cell viability versus the logarithm of drug
concentration.
In closing, we have demonstrated that the high-throughput
synthesis of a chemical library based on the fusion of two privi-
leged biogenic subunits resulted in the identification of a
potent chemical agent that impaired cellular viability by an un-
usual mechanism involving both caspase-dependent and -in-
dependent cell-death pathways. Detailed investigation of the
mechanism of action and elucidation of the cellular target of
triazatricyclamide are under investigation.
Chromatin condensation: HL-60 cells were harvested by centrifu-
gation (250g, 5 min) and resuspended in complete growth
medium. Cells were counted by using a hemacytometer. For each
sample, approximately 1ꢁ106 cells were transferred into an un-
treated polystyrene culture dish. Complete growth media contain-
ing various concentrations of drug as well as DMSO vector only
were added (6 mL total volume, 0.1% DMSO). After incubation for
24 h, the cells were harvested by centrifugation (250g, 5 min) and
washed with phosphate-buffered saline (PBS, 1 mL). The cells were
then resuspended in PBS (1 mL) and treated with a solution of
Hoechst 33342 (1 mL, 5 mgmLÀ1). The samples were protected
from light and incubated on ice for 30 min before analysis on an
Olympus IX81 fluorescence microscope.
Experimental Section
Library synthesis: The following procedure represents the synthe-
sis of the first set of 96 compounds. One of the five vinylogous
ureas I (256 mmol) was dissolved in acetonitrile (1.92 mL) and CDCl3
(3.2 mL). The resulting solution was divided into eight equal batch-
es (640 mL each) that were treated individually with eight alde-
hydes II (40–60 mmol each). Following subsequent treatment of
each reaction mixture with 12n aqueous HCl (1 mL), the solutions
were left at 208C until the starting material had been completely
consumed. Each reaction mixture was treated with Et3N (15 mL)
and divided into twelve equal batches (each 50 mL batch contain-
ing 2.5 mmol of one benzodiazepine III), which were transferred
into a polypropylene 96-well PCR plate. Each well was treated with
12 acid chlorides IV (0.60–1.2 mL) according to the plate map
shown in the Supporting Information. Upon complete consump-
tion of diamines III, each well was treated with a solution of 2n
aq. NaOH/MeOH (1:4, 15 mL). The reaction mixtures were trans-
ferred onto preparative TLC plates by using a multichannel pipet-
tor with adjustable gaps. The plates were developed in acetone/
dichloromethane (1:6), containing 1% of NEt3. The products were
detected under UV light and removed from TLC plates as circular
silica gel pellets. Each compound was eluted from the silica gel
with MeOH (0.6 mL). The solvent was removed in vacuo, and 12
randomly selected compounds were dissolved in CD3OD (0.5 mL)
ROS production: HL-60 cells (0.25ꢁ106 cells in 1 mL of medium)
were seeded into 12-well plates and treated with desired stimuli
(0.2% DMSO in all samples). After the indicated incubation time,
cells were harvested by centrifugation, washed with PBS (1 mL)
and resuspended in a solution of dihydroethidium (DHE) in PBS
(0.5 mL, 10 mm). The cells were protected from light and incubated
at 378C for 15 min, followed by analysis on a BD FACSCanto flow
cytometer.
Acknowledgements
This work was partially supported by NIH grant P50 GM086145.
S.A.K. thanks the Sloan Foundation, the Dreyfus Foundation,
Amgen, and GSK for additional financial support.
Keywords: apoptosis
·
caspases
·
chemical libraries
·
cytotoxicity · reactive oxygen species
1
and analyzed by H NMR. The amount of material in each sample
was determined by integration using residual MeOH as a precali-
brated internal standard.
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1
Triazatricyclamide (1): H NMR (400 MHz, CD3OD): d=7.54 (s, 1H),
7.43 (s, 1H), 7.16–7.24 (m, 5H), 6.47 (d, J=3.6 Hz, 1H), 6.13–6.15
(m, 2H), 5.20 (t, J=6.6 Hz, 1H), 4.01 (d, J=6.4 Hz, 2H), 1.76 (s, 3H),
1.71 (s, 3H), 1.52 (s, 3H), 1.42 (s, 3H), 1.30–1.32 (m, 1H), 0.94–0.96
(m, 1H), 0.77–0.87 (m, 2H), 0.62–0.64 (m, 1H); 13C NMR (125 MHz,
[D6]DMSO): d=171.40, 167.61, 157.14, 140.02, 135.91, 133.26,
132.56, 132.17, 130.47, 129.03, 128.98, 127.74, 126.36, 123.27,
122.51, 121.24,102.75, 61.20, 52.52, 35.89, 25.41, 24.09, 23.51, 17.62,
12.61, 8.39, 8.18.; MS (APCI) calcd for C30H31Cl2N3O2: 535.18 [M]+,
found 536.1 [M+H]+.
Cellular growth inhibition: All assays were performed in at least
three replicate wells for each compound concentration tested.
Threefold serial dilutions in DMSO were performed for each active
compound by using stock DMSO solutions with NMR-calibrated
concentrations. We seeded cells in 96-well white plates at a density
of 1000 cells per well (A549, PC3, HCT116, MCF7 cell lines) or 3000
cells per well (HL-60 cell line) in the appropriate cell culture
medium (100 mL). Adherent A549, PC3, HCT116 and MCF7 cells
were allowed to attach and grow for 24 h before treatment with
serially diluted compound solutions, and incubation for a further
48 h. HL-60 cells were treated with the compound solutions at the
time of seeding and incubated for 48 h. After incubation, the
number of cells was determined by using CellTiter-Glo (Promega).
Compound concentrations that gave a 50% reduction in cell
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Received: March 26, 2010
Published online on May 11, 2010
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