Mechanism of Action of the Cytotoxic Asmarine Alkaloids

Article abstract of DOI:10.1021/acschembio.8b00096

The asmarines are a family of cytotoxic natural products whose mechanism of action is unknown. Here, we used chemical synthesis to reverse engineer the asmarines and understand the functions of their individual components. We found that the potent asmarine analog delmarine arrested the mammalian cell cycle in the G1 phase and that both cell cycle arrest and cytotoxicity were rescued by cotreatment with ferric and ferrous salts. Cellular iron deprivation was clearly indicated by changes in iron-responsive protein markers, and cytotoxicity occurred independently of radical oxygen species (ROS) production. Chemical synthesis allowed for annotation of the distinct structural motifs required for these effects, especially the unusual diazepine, which we found enforced an iron-binding tautomer without distortion of the NCNO dihedral angle out of plane. With this information and a correlation of cytotoxicity with logP, we could replace the diazepine by lipophilic group appendage to N9, which avoided steric clash with the N6-alkyl required to access the aminopyridine. This study transformed the asmarines, scarce marine metabolites, into easily synthesized, modular chemotypes that may complement or succeed iron-selective binders in clinical trials and use.

Full text of DOI:10.1021/acschembio.8b00096

Subscriber access provided by UNIV OF SCIENCES PHILADELPHIA
Mechanism of Action of the Cytotoxic Asmarine Alkaloids
Michael J. Lambrecht, Jeffery W. Kelly, and Ryan A. Shenvi
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00096 • Publication Date (Web): 13 Apr 2018
Downloaded from http://pubs.acs.org on April 14, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 9
ACS Chemical Biology
1
2
3
4
5
6
7
8
9
Mechanism of Action of the Cytotoxic Asmarine Alkaloids
Michael J. Lambrecht, Jeffery W. Kelly, Ryan A. Shenvi*
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Department of Chemistry and Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California
9
2037, United States
ABSTRACT: The asmarines are a family of cytotoxic natural products whose mechanism of action is unknown. Here we used
chemical synthesis to reverse engineer the asmarines and understand the functions of their individual components. We found that
the potent asmarine analog “delmarine” arrested the mammalian cell cycle in G1 phase, and that both cell cycle arrest and cytotoxiꢀ
city was rescued by coꢀtreatment with ferric and ferrous salts. Cellular iron deprivation was clearly indicated by changes in ironꢀ
responsive protein markers, and cytotoxicity occurred independent of radical oxygen species (ROS) production. Chemical synthesis
allowed for annotation of the distinct structural motifs required for these effects, especially the unusual diazepine, which we found
enforced an ironꢀbinding tautomer without distortion of the NCNO dihedral angle out of plane. With this information and a correlaꢀ
tion of cytotoxicity with logP, we could replace the diazepine by lipophilic group appendage to N9, which avoided steric clash with
the N6ꢀalkyl required to access the aminopyridine. This study transformed the asmarines, scarce marine metabolites, into easily
synthesized, modular chemotypes that may complement or succeed ironꢀselective binders in clinical trials and use.
INTRODUCTION
The resources and pressures present in nature differ from
The asmarines (e.g. 1a and 1b, Figure 1) are a small family
1
, 2
those present in rational drug design. As a result, secondary
metabolites occupy areas of chemical space distinct from
of cytotoxic natural products that were first isolated off the
4-7
coast of Eritrea in the late 1990’s. These compounds contain
a unique Nꢀhydroxydiazepine motif connected to a clerodane
core. It is likely that the asmarines originate biosynthetically
from the related agelasine family of natural products (e.g. 2,
Figure 1), which in turn arise from adenylation of a clerodane
2
drugs, even when their functions overlap. The structural comꢀ
plexity of secondary metabolites, however, often impedes
reverse engineering to annotate function for every atom or
3
group. Here we assign a mechanism of action to the asmarine
8, 9
metabolite family and dissect individual structural motifs to
show why they are necessary and how they can be altered.
pyrophosphate (3).
Interestingly, despite high structural
similarity between the asmarines and agelasines, these two
classes of natural products appear to be unrelated with respect
to their cytotoxic mechanisms (vide infra).
The structural complexity of the asmarines (two chiral lobes
connected via an ethyl bridge) makes them some of the most
challenging structures in the purineꢀditerpene class to prepare
and has resulted in interest from chemists seeking to syntheꢀ
10-16
size and understand their cytotoxicities.
Unfortunately, the
lack of any remaining material from the initial isolation has
been a major impediment towards annotation of the biological
17
mechanism of these compounds. Additionally, to this point
their structural complexity has precluded their chemical synꢀ
thesis. However, through the isolation literature and synthesis
of asmarineꢀlike analogs, some structureꢀactivityꢀrelationship
(SAR) trends have been established. For instance, the Nꢀ
hydroxy (NꢀOH) functionality is critical for cytotoxic activity
6
, 7, 13
as similar NꢀH and NꢀOCH diazepines are inactive.
Synꢀ
3
thesis of asmarineꢀlike acyclic compounds (e.g. 4, Figure 1)
has shown the necessity for the diazepine as the cyclic variants
retain potency whereas acyclic compounds are inactive.
This trend suggests that the asmarines and agelasines are unreꢀ
lated functionally as the agelasines inhibit various ATPases
and maintain cytotoxic activity despite the absence of the diꢀ
1
5, 18
9, 19
azepine. Additionally, the parent Nꢀhydroxypurine (HAP, Figꢀ
Figure 1. Structures of asmarine A and related compounds.
ure
1)
heterocycle
is
a
known
ACS Paragon Plus Environment

ACS Chemical Biology
Page 2 of 9
A
B
C
100
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
100
*
*
80
60
40
20
0
ꢀFe3+
DMSO + Fe3+
80
60
+Fe3+
Delmarine + Fe3+
IC50 = 452 ± 28 nM
** **
40
Delmarine
DMSO
20
0
G1
DMSO
S
G2/M
ꢀ1
0
1
2
2
N
4N
log [delmarine], (ꢁM)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
PI Fluorescence
Delmarine
Delmarine + Fe
Figure 2. A) Delmarine induces G1 arrest as assessed by propidium iodide staining and flow cytometry in HeLa cells. Arrest can be resꢀ
cued by addition of 100 µM ferric ammonium citrate. B) Quantification of data in A), N=3. C) 48 h cytotoxicity of delmarine in HT1080
cells as assessed by alamar blue. Cytotoxicity can be rescued by addition of 100 µM ferric ammonium citrate (red curve), N=3. * = p <
ꢀ
4
0
.005, ** = p < 1x10 .
1:1
2:1 3:1
A0.6
B
C
0.20
0.15
0.10
0.05
3
:1 2:1
1:1
0.20
0.15
ꢀFe3+
Fe3+
0
0
0
.4
.2
.0
+
0
.10
.05
0
0.00
2
50
350
450
(nm)
550
650
0
5
10
15
20
25
0.2
0.4
0.6
0.8
1.0
λ
[Fe3+], ꢁM
Mole Fraction Delmarine
Figure 3. A) Delmarine binds to iron in phosphate buffered saline as assessed by UVꢀVis spectroscopy. B) delmarine binds to iron with 3:1
ligand:metal stoichiometry as assessed by titration of 20 µM delmarine with increasing concentration of ferric ammonium citrate and abꢀ
sorbance at 330 nm. C) delmarine shows maximal absorbance at 330 nm at a ligand:metal ratio of 3:1 by the method of continuous variaꢀ
tions.
2
0
mutagen as a base analog : an unlikely source of cytotoxicity
for the asmarines. Hydrophobic substitution of the clerodane
core has been shown to be well tolerated and the stereochemꢀ
istry of C13 has been shown to be unimportant for cytotoxiciꢀ
(cervical cancer, Figure 2A,B) and HT1080 (fibrosarcoma,
Supplementary Figure 1A,B) cell lines. At low concentrations
of delmarine, cells passed the G1/S checkpoint and were
found to arrest in S phase (Supplementary Figure 1C).
1
3, 15, 18
ty.
Considering this information, our lab designed a
A number of iron chelators, such as the clinically approved
2
2
23
structurally simplified compound termed “delmarine” (5, Figꢀ
ure 1) and showed that it is a similarly potent toxin as asmaꢀ
rines A and B.
agents hydroxyurea and deferoxamine, are known to arrest
cells in the G1/S phase of the cell cycle. Although iron chelaꢀ
tion affects many cellular proteins and processes, G1/S arrest
induced by these agents is believed to be principally caused by
sequestration of iron needed for the 8 fold upregulation of
15, 18
Here we show that delmarine inhibits DNA synthesis by seꢀ
questration of iron in mammalian cells. The SAR trends deꢀ
scribed above are fully explained by this newly identified
mechanism of action. Additionally, this mechanism clarifies
the unique structural features of these compounds and shows
why evolution arrived at the unusual asmarine motif. With this
information, we redesigned the asmarines using building
blocks that are more easily assembled in a nonꢀcellular enviꢀ
ronment in as few as two synthetic steps.
ribonucleotide reductase necessary for DNA synthesis during
24, 25
S phase.
Due to structural homology between the Nꢀ
hydroxy purine of delmarine and the functionality present in
these other molecules, we hypothesized that iron binding may
be involved in the mechanism of action of delmarine. Screenꢀ
ing of the NCIꢀ60 with delmarine and use of the COMPARE
algorithm returned several iron binding small molecules as
top hits (Supplementary Tables 1 and 2), consistent with
26
2
7
RESULTS AND DISCUSSION
delmarineꢀinduced cell death being related to iron binding.
Delmarine induces iron-dependent G1 arrest. Cell cycle
analysis by flow cytometry can be used to ‘bin’ small moleꢀ
cules according to their effect on the cell state. For example,
antiꢀmitotics such as taxol, colchicine and vinblastine all arrest
the cell cycle in G2/M phase, whereas inhibitors of DNA synꢀ
Therefore, the influence of iron on delmarineꢀinduced cell
cycle arrest was examined next. The addition of excess ferric
ammonium citrate to the cellular growth medium rescued the
G1 arrest of delmarine, but had no effect on untreated cells
(Figure 2A,B and Supplementary Figure 1A,B). Similarly,
addition of excess ferric ammonium citrate to the medium
rescued delmarineꢀinduced cytotoxicity in a nearly quantitaꢀ
tive manner (Figure 1C, Supplementary Figure 1D). Taken
together, this data suggested that iron binding may be involved
in the mechanism of delmarine.
21
thesis such as thymidine and 5ꢀfluorouracil arrest in S phase.
Therefore, in our first interrogation of mechanism, cells treatꢀ
ed with delmarine were subjected to cell cycle analysis by
propidium iodide staining and flow cytometry. Delmarine was
found to arrest cells primarily in G1 phase in both the HeLa
ACS Paragon Plus Environment

Page 3 of 9
ACS Chemical Biology
found to bind to magnesium, manganese or calcium (Figure
A
1
2
3
4
5
6
7
8
9
0.6
4A). Except for iron, the addition of metals involved in celluꢀ
lar processes was found to have little effect on cytotoxicity
induced by delmarine (Figure 4B, Supplementary Figure 3).
These results can likely be explained by the substantially
No Cation
+
+
+
Fe3+
Fe2+
Zn2+
0
0
0
.4
.2
.0
ꢀ
5
ꢀ6
higher concentration of labile cellular iron (10 to 10 M)
+Cu2+
+Ca2+
ꢀ
11
ꢀ15
relative to zinc (10 M) and copper (10 M): while the bindꢀ
ing affinity of delmarine for copper and zinc is significant,
free iron is more available for chelation in cells and is thus
Mn2+
Mg2+
+
+
29, 30
more relevant for cytotoxic activity.
250
350
450
(nm)
550
650
λ
A
B
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
B
2.5
100
No Cation
*
+
+
Fe3+
Fe2+
2.0
1.5
8
6
0
0
H2O2
NS
NS
NS
+Zn2+
DFO
1
.0
0.5
.0
+
+
Cu2+
Ca2+
Triapine
40
2
0
0
+Mn2+
Mg2+
Delmarine
DMSO
0
+
e
n
DFO
2
O
i
p
a
O
S
e
H 2
M
in
ar
D
Tr i
ꢀ1
0
1
2
lm
e
C
D
log [delmarine], (ꢁM)
8
00
00
00
6
4
2
+
3+
2+
2+
Figure 4. A) Delmarine binds to Fe , Fe , Zn , and Cu in
phosphate buffered saline but not to Mn , Mg , or Ca as asꢀ
sessed by UVꢀVis spectroscopy. B) Only addition of Fe and
Fe rescues delmarineꢀinduced cell death in HT1080 cells as
assessed by alamar blue (48 h), N = 3.
EDTA
2
+
2+
2+
Delmarine
Triapine
DFO
3+
200
0
2
+
ꢀ200
Delmarine binds iron with a 3:1 stoichiometry. The abilꢀ
ity of delmarine to bind iron in aqueous buffer and its preꢀ
ferred stoichiometry of iron binding was next probed. Delmaꢀ
rine was found to directly bind to iron as assessed by UVꢀ
Visible spectroscopy (Figure 3A), and this interaction was
found to occur with 3 : 1 delmarine : iron stoichiometry by
titration with increasing concentrations of iron (Figure 3B), as
well as with the method of continuous variations (Figure 3C).
The formation constant of delmarine for iron was approximatꢀ
ed by EDTA titration (Supplementary Figure 2) and found to
0.1
1
3
Chelator:Iron Ratio (IBE)
D
100
80
Delmarine
Delmarine + NAC
Triapine
60
Triapine + NAC
DFO
40
20
DFO + NAC
0
ꢀ1
0
1
2
23
log [Compound], (ꢁM)
be 7.9x10 , a value similar in magnitude to that of other bivaꢀ
lent iron chelators.
2
8
Figure 6. A) ROS induction as assessed by the ROSꢀresponsive
dye H2ꢀDCFDA by flow cytometry. B) Quantification of data in
A), N = 3, * = p < 0.001. C) Significant ROS production is not
seen in vitro by the ascorbate oxidation assay, N = 3. D) The antiꢀ
oxidant Nꢀacetyl cysteine (NAC) does not protect from cell death
induced by iron chelators as assessed in HeLa cells at 48 h by
alamar blue, N = 3.
Delmarine (ꢁM)
0.01 0.03 0.1 0.3
0
1
3
10
FTH1
IRP2
Cells treated with delmarine display proteomic changes
consistent with altered iron homeostasis. Having shown that
delmarine binds to iron and that its cell cycle and cytotoxic
effects can be rescued by exogenous addition of iron, we next
asked whether cellular processes consistent with iron deprivaꢀ
tion were observed upon treatment with delmarine. A variety
of protein markers have been demonstrated to correlate with
iron homeostasis in cells, including those of the iron storage
protein ferritin (FTH1), the iron responsive element binding
protein (IRP2) and the transferrin receptor (CD71). Treatment
of HT1080 cells with delmarine and analysis of cell lysates by
western blot showed a doseꢀdependent reduction of protein
levels of FTH1, whereas levels of CD71 and IRP2 were found
to be increased (Figure 5). Similar trends were seen in HeLa
cells (Supplementary Fig. 4), though the effects were more
bimodal in this cell line.
CD71
GAPDH
HT1080
Figure 5. Changes of proteins associated with cellular iron levels
in HT1080 cells as assessed by western blot. Representative reꢀ
sults of one of two replicates shown.
The ability of delmarine to bind to other cellular metal cations
and the ability of these complexes to modulate delmarine cytoꢀ
toxicity was next examined. In bulk solvent, binding was seen
with metals other than ferric iron including ferrous iron (Fe ),
copper (Cu ), and zinc (Zn ) (Figure 4A). Delmarine was not
2+
2+
2+
ACS Paragon Plus Environment

ACS Chemical Biology
Page 4 of 9
9
N
N
9
7
N
N
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
N
N
N
RꢀX, MeMgCl
2ꢀ95%
RꢀNHOH
1ꢀ66%
50
5
50
35
20
5
N
N
Me
N
6
N
7
1
1
6
5
N
N
4
R
N
R
Cl
9
7
R
OH
N
OH
N
N
7,8
9ꢀ12
0.5
0.05
N
H
1
6
R
Cl
R
9
N
N
9
N
Ph
6
ꢀchloropurine (6)
RꢀX, NaH
55ꢀ64%
N
RꢀNHOH
53ꢀ97%
Delmarine
N
clogP = 4.05
N
N
1
N
1
7
7
6
6
N
Cl
R
OH
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13,14
15ꢀ18
5
0
50
50
5
50
50
5
50
N
N
N
N
N
N
N
N
N
N
N
5
35
20
5
35
20
5
N
35
20
5
N
N
Me
Me
OH
0.5
N
0.5
0.05
0.5
0.05
OH
Ph
Me
OH
Me
0
.05
9
10
logP = 1.83
11
logP = 3.24
logP = 0.02
50
5
50
35
20
5
50
5
50
35
20
5
50
50
35
20
5
Me
N
N
N
N
N
N
Me
N
5
N
N
N
N
N
N
N
Ph
OH
N
0.5
0.05
0.5
OH
0
.5
Me
15
OH
0
.05
0.05
12
16
logP = 2.09
logP = 5.05
logP = 0.28
N
N
N
Ph
NH
50
5
50
35
20
5
50
5
50
35
20
5
50
50
Ph
N
N
N
N
N
OH
N
N
5
0.5
35
20
5
N
N
Me
N
N
0.5
0.5
0.05
OH
Me
OH
0.05
0.05
Ph
17
18
19
logP = 3.64
logP = 5.45
logP = 4.80
Figure 7. Synthesis and evaluation of delmarine analogs. IC50 values represent 48 h values assessed by alamar blue and are reported in µM.
Calcein EC50 values represent the concentration of compound required to increase fluorescence of 1 µM calcein in the presence of 3 µM
ferric ammonium citrate to 50% of ironꢀfree 1 µM calcein.
Delmarine-induced cytotoxicity is not driven by ROS.
Some iron chelators are thought to possess a cytotoxic mechaꢀ
nism strongly associated with radical oxygen species (ROS)
It has been suggested that triꢀ and bidentate
chelators can cause increases in cellular ROS through redox
cycling whereas hexadentate chelators exhibit no such phenoꢀ
type. The ability of delmarine (bidentate) as well as the small
molecule chelators triapine (tridentate) and deferoxamine
DFO, hexadentate) to affect the generation of cellular ROS
was examined. Preloading of HT1080 cells with the ROSꢀ
responsive probe H2ꢀDCFDA followed by incubation with
delmarine and the small molecule chelators showed a slight
but not statistically significant increase in ROS by flow cyꢀ
tometry, with no significant difference between the different
iron chelators (Figure 6A,B). Further, while EDTA showed
activity in an in vitro ascorbate oxidation ROS generation
assay, no such activity was seen for delmarine, triapine or
deferoxamine (Figure 6C). Finally, delmarineꢀinduced cell
death was not protected by coꢀtreatment with the ROS scavenꢀ
of ROS induction by triapine has been debated in the literaꢀ
33, 36
ture;
and ROS generation often occurs more generally in
37, 38
concert with cell death.
3
1-33
production.
Dissection of the asmarine scaffold. The determination
that the asmarines induce cytotoxicity primarily through iron
chelation allowed for the further dissection of the functionality
present in these natural products. We reasoned that in all likeꢀ
lihood the nitrogen atom of Nꢀ1 and the Nꢀ6 hydroxylamine
comprise the motif that is principally responsible for iron
binding and that large changes could be made to the rest of the
asmarine structure without sacrificing activity so long as the
iron binding motif was retained. An effort to chemically synꢀ
thesize simplified analogs with asmarineꢀlike function was
therefore undertaken (Figure 7).
34
(
To retain similarity to the asmarines (Nꢀ6 and Nꢀ7 substitutꢀ
ed), a series of analogs substituted at the Nꢀ6 and Nꢀ7 posiꢀ
tions were first made. A regioselective Nꢀ7 alkylation of 6ꢀ
chloropurine (6) followed by displacement of the chlorides of
compounds 7 and 8 with various hydroxylamines provided
compounds 9ꢀ12. Compounds 9ꢀ12 were designed to have
varying values of logP as we had previously found this to be
3
1
39
35
ger Nꢀacetylcysteine (NAC) (Figure 6D). Taken together,
this data suggests that delmarine does not induce substantial
ROS as part of its cytotoxic mechanism. The presence or lack
ACS Paragon Plus Environment

Page 5 of 9
ACS Chemical Biology
Also, increased potency of iron
1
5, 18
an important parameter.
iron and are not cytotoxic. This result suggests a second role
for the asmarine diazepine: to provide N6ꢀsubstitution and
force the ironꢀbinding Nꢀhydroxy aminopyridine tautomer in
solution (Figure 8).
1
2
3
4
5
6
7
8
9
binders with increased logP is a known phenomenon linked to
the ability of more hydrophobic chelators being better able to
4
0, 41
transport iron across cellular membranes.
Interestingly, all
these Nꢀ7 substituted analogs were found to have little activity
in HeLa or HT1080 cells (Figure 7), even when their logP
matched or exceeded that of delmarine. The reason for this
inactivity soon became clear, as these compounds were shown
to be unable to bind iron in aqueous buffer with anywhere near
the affinity of delmarine as determined by a calcein competiꢀ
tion assay (Figure 7). It was reasoned that steric interactions
between substituents at the Nꢀ6 and Nꢀ7 positions that do not
exist in the diazepine structure force the NO bond out of plane
in the simplified analogs (Figure 8). We reasoned that analogs
substituted at Nꢀ7 lost the ability to bind iron efficiently as a
result of this loss of planarity.
Finally, the logP trends associated with the synthetic comꢀ
pounds (Figure 7) provide a rationale for the existence of the
clerodane core in the asmarine structure. The clerodane core
imparts substantial hydrophobicity on the asmarine system
(logP of asmarine A = 5.79, logP of delmarine = 4.05). As we
and others have already shown that this motif can be substitutꢀ
13, 15, 18
ed without loss of cytotoxic activity,
it is likely that the
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
clerodane is present for its hydrophobic nature to enable iron
movement across cellular membranes. In this context, the purꢀ
ported tenꢀfold potency increase from asmarine A to B is curiꢀ
ous. In this context, the purported tenꢀfold potency increase
from asmarine A to B is curious. If real, the difference may
result from altered transport across membranes, or may point
to a minor, secondary pharmacology. Secondary effects might
result from the unusual reactivity of the asmarine phramacoꢀ
Compounds with Nꢀ9 substituents were synthesized next
4
2, 43
(Figure 7) using standard conditions
to provide regioselecꢀ
tive Nꢀ9 substitution. Analogous to the Nꢀ7 series, displaceꢀ
ment of the chlorides in compounds 13 and 14 with hydroxylꢀ
amines provided analogs 15ꢀ18. The iron binding ability of the
Nꢀ9 series was improved (Figure 7) and the potency of these
compounds in HeLa and HT1080 cells was proportional to
their logP. Compounds with higher logP values such as 17 and
5,15
hophore or metalloprotein binding.
CONCLUSION
Here we have shown that the asmarine alkaloids are optiꢀ
mized for cytotoxic iron binding through three main structural
features. First, appendage of a terpenyl substituent is necesꢀ
sary to increase hydrophobicity and is likely present to enꢀ
hance membrane permeability. Second, N6 alkylation enforces
an ironꢀbinding ‘hydroxyꢀaniline’ tautomer, whereas nonꢀ
alkylated analogs populate an ‘oxime’ tautomer that binds
poorly. Third, the diazepine ring of the asmarines allows the
hydroxylꢀaniline group to rotate into the plane of the purine
without steric repulsion by the N7 substituent. It appears that
evolution of cytotoxicity requires these three related structural
features to be present to accommodate the restrictions of bioꢀ
synthetic starting materials. Now that the ironꢀbinding funcꢀ
tion of the asmarines has been discovered, a functionꢀoriented
remodeling of the asmarine structure is possible using a greatꢀ
er breadth of building blocks than is available within cellular
environments. We have shown that the hydrophobic chain can
be rearranged to N9 which allows the N6ꢀalkylated hydroxylꢀ
amine tautomer to remain in plane, retaining iron binding caꢀ
pacity, as demonstrated by competition with calcein and
mammalian cytotoxicity. Not only can chemical synthesis in
the modern era deliver large quantities of complex molecules,
but it also can fully annotate every substructure in a complex
metabolite4 and simplify such molecules to smaller
1
8 were found to have cytotoxic activity on par with or exꢀ
ceeding that of delmarine (Figure 7).
The large differences in iron binding and cytotoxic activity
between Nꢀ7 analogs versus Nꢀ9 analogs assigned an imꢀ
portant role to the asmarine diazepine: the ring allows N6 and
N7 alkyl groups to occupy the same plane by alleviating steric
repulsion through covalent bond formation (Figure 8). Altꢀ
hough the same effects can be achieved by appendage of lipoꢀ
philic groups to N9, available biosynthetic intermediates guide
the evolution of function. No similar purine metabolites have
been isolated that contain an N9ꢀterpenyl linkage, whereas N7
connections are common.
Figure 8. Iron binding requirements for the asmarines and asmaꢀ
rine analogs.
4
fragments. This exploration has uncovered the detailed strucꢀ
While the necessity of the diazepine to enforce a proper
ironꢀbinding conformation on the asmarines provided some
clarity with respect to its cytotoxicity, it did not necessarily
explain the inactivity of acyclic agelasineꢀlike intermediates
(e.g. 4, Figure 1). To better understand such compounds, we
assessed compound 19 (Figure 7), the immediate synthetic
tural requirements for asmarine cytotoxicity; other natural
products undoubtedly hold their own trove of secrets.
METHODS
Cell Lines and Reagents. HeLa and HT1080 cells were
cultured in RPMI 1640 or MEM, respectively. Media was
supplemented with 10% FBS and 1% penicillin/streptomycin
1
5
precursor to delmarine in both the calcein ironꢀbinding comꢀ
petition assay as well as for cytotoxicity. Compound 19 was
found to possess diminished cytotoxicity in cells relative to
delmarine and compounds 17 and 18 and was found to not
bind iron in the calcein assay (Figure 7). The lack of iron bindꢀ
ing of compounds such as 19 which lack Nꢀsubstitution can be
explained by the preference of such compounds for oxime
tautomers over Nꢀhydroxy aminopyridine tautomers in soluꢀ
tion (Figure 8). As a result, if iron binding does not offset the
energy of tautomerization, these linear purines do not bind
at 37°C and 5% CO and MEM was additionally supplementꢀ
2
ed with 2 mM glutamax.
Cell Death Assessment by Alamar Blue. HeLa cells or
HT1080 cells were seeded in 96ꢀwell plates in 50 µL of the
appropriate medium at cell numbers of 2,500 cells/well for
HeLa or 5,000 cells/well for HT1080. Cells were allowed to
adhere for 6ꢀ8 h and compounds were added in 50 µL of meꢀ
dium from DMSO stocks to final concentrations of 50ꢀ0.016
ACS Paragon Plus Environment

ACS Chemical Biology
µM with a final DMSO concentration of 0.5% (v/v). Cells
Page 6 of 9
Binding of Delmarine to Other Metals. Delmarine was
added to a final concentration of 20 µM in PBS with final
DMSO = 2% (v/v). Solutions of the appropriate cation were
added from 10 mM stocks to final concentrations of 20 µM.
Samples were incubated for 30 min and analyzed by UVꢀ
visible spectroscopy.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
were incubated for 48 h and resazurin (10 µL of a 1 mg/mL
solution in water) was added. Staurosporine (10 µM) was used
as the positive control for 100% cell death. For experiments
involving supplementation of media, appropriate metal salts
were added directly to the media to final concentrations of 100
µM. For these experiments, media was removed and replaced
with fresh media containing resauzrin after 48 h. For NAC
protection, Nꢀacetyl cysteine was added to a final concentraꢀ
tion of 5 mM. Data was analyzed in Prism and IC50 values
were estimated in cases where dose response curves failed to
converge.
Calcein Competition Assay. Solutions of analogs were
added to a 96 well plate at concentrations of 50ꢀ0.39 µM from
DMSO stocks (final DMSO = 5% v/v). A solution of calcein
(1 µM) and ferric ammonium citrate (3 µM) in PBS was added
and the solutions were incubated for 1 h in the dark. Calcein
fluorescence was read on a plate reader and the percentage of
ligand bound was determined by normalizing to ligand free
and iron free solutions.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Cell Cycle Analysis by Flow Cytometry. HeLa or HT1080
cells (200,000) were plated in 6ꢀwell plates in a volume of 2
mL and allowed to adhere 4ꢀ8 h. Prior to treatment, the media
was removed and replaced and delmarine was added to the
appropriate concentration (10 µM unless otherwise specified).
The compounds were incubated for 18 h at which point the
media was collected and the cells were trypsinized and harꢀ
vested. The cells were centrifuged at 1000 x g for 5 min,
washed with 3 mL of PBS, resuspended in 500 µL of PBS,
and fixed by dropwise addition to 3 mL of 70% EtOH at –
LogP Calculation. LogP values were calculated using the
property
http://fafdrugs3.mti.univꢀparisꢀdiderot.fr/index.html).
Ascorbate Oxidation Assay. The assay was performed as
calculation
software
FAFDrugs4:
45
(
31
described previously. Briefly, ferric ammonium citrate was
dissolved to a final concentration of 30 µM. Ligands were
added to final concentrations of iron binding equivalents of 0,
0.1, 1, or 3:1. Ascorbate was added to a final concentration of
100 µM and A260 was measured at 0 and 30 min. Change in
A260 between the 0 and 30 min time points was measured and
plotted for each point relative to control (0 iron binding equivꢀ
alents).
2
0°C. The fixed cells were centrifuged, washed once with 3
mL PBS, 500 µL of FxCycle PI/RNAse solution (Therꢀ
moFisher) was added, and the cells were incubated at room
temperature for 30 min. The cells were analyzed by flow cyꢀ
tometry using a NovoCyte flow cytometer and 15,000 events
were collected.
Immunoblotting. HeLa or HT1080 cells (200,000 or
3
50,000 cells, respectively) were added to a 6 well plate and
Detection of ROS by Flow Cytometry. HT1080 cells
(100,000) were plated in 2 mL of MEM in wells of a sixꢀwell
plate. The cells were allowed to adhere for 6 h and were treatꢀ
ed with 20 µM H2ꢀDCFDA 1 h. The media was then removed
and replaced with media containing delmarine (10 µM), Triapꢀ
ine (10 µM) or DFO (100 µM). Cells were incubated 16 h, the
media collected, the cells trypsinized, and centrifuged at 1000
x g for 5 min. The cell pellet was washed with 1 mL HBSS,
centrifuged again, resuspended in 450 µL HBSS and analyzed
by flow cytometry.
allowed to adhere for 4ꢀ8 h. Cells were treated with the indiꢀ
cated concentrations of compound (final DMSO = 1% v/v)
and incubated for 18 h. After 18 h the media was removed, the
cells were washed once with cold PBS, trypsinized and harꢀ
vested. The cells were lysed in 50 µL of ice cold RIPA buffer
for 30 min. After 30 min, the cells were centrifuged at 14,000
x g and the supernatant was resolved by SDSꢀPAGE. After
transfer to a membrane, the blots were blocked in 5% milk in
TBST for 1 h and incubated with primary antibody (1:500
dilution in TBST with 5% BSA for FTH1, IRP2, and CD71 or
3
+
UV Titration of Delmarine with Fe . Delmarine was diꢀ
luted to a final concentration of 20 µM in 1 mL of PBS with
final DMSO concentration = 1% (v/v). Ferric ammonium citꢀ
rate was added from a concentrated stock of 1 mM to final
concentrations of 1.5ꢀ24 µM. Additionally, delmarineꢀfree
solutions with corresponding amounts of iron were prepared in
PBS to allow for blanking of the absorbance of iron. Samples
were incubated for 30 min at room temperature and a specꢀ
trum of each solution was collected in a quartz cuvette.
1
:5,000 dilution in 2.5% milk for GAPDH) overnight at 4 °C.
The blots were washed with TBST, incubated with antiꢀrabbit
HRP secondary antibody (1:5,000 for FTH1, IRP2, and CD71,
1
:10,000 for GAPDH) for 1 h, washed with TBST, treated
with luminol/peroxide, and imaged.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
UV Titration of Delmarine Complex with EDTA. EDTA
was added to concentrations of 10ꢀ0.0001 µM from 50x stocks
to solutions of delmarine (20 µM) and ferric ammonium citꢀ
rate (6.67 µM). The solutions were incubated for 30 min and
analyzed by UVꢀVis spectroscopy. Maximal binding was deꢀ
fined as no added EDTA and minimal binding was determined
by addition of no ferric ammonium citrate to the ligand.
Supporting Figures. Chemical and biological procedures. Characꢀ
terization data for new compounds. (PDF)
AUTHOR INFORMATION
3
+
Corresponding Author
Delmarine-Fe Assessment by the Method of Continu-
ous Variations. Delmarine was added from DMSO stocks to
final concentrations of 0ꢀ20 µM with DMSO = 2% (v/v). Ferꢀ
ric ammonium citrate was added so that the sum of the conꢀ
centration of delmarine and ferric ammonium citrate was alꢀ
ways 20 µM. Samples were incubated for 30 min and anaꢀ
lyzed by UVꢀvisible spectroscopy.
*rshenvi@scripps.edu
Author Contributions
All authors have given approval to the final version of the manuꢀ
script.
ACKNOWLEDGMENT
ACS Paragon Plus Environment

Page 7 of 9
ACS Chemical Biology
Financial support for this work was provided by the NIH
(17) Kashman, Y. Personal Communication.
1
2
3
4
5
6
7
8
9
(1R35GM122606ꢀ01 to R.A.S.) and the Arnold and Mabel Beckꢀ
man Foundation (Postdoctoral Fellowship to M.J.L.). We thank C.
Fearns for critical review of the manuscript.
(18) Wan, K. K., and Shenvi, R. A. (2016) Conjuring a
Supernatural Product – DelMarine. Synlett 27, 1145ꢀ1164.
(
19) Pimentel, A. A., Felibertt, P., Sojo, F., Colman, L., Mayora,
A., Silva, M. L., Rojas, H., Dipolo, R., Suarez, A. I., Compagnone, R.
S., Arvelo, F., GalindoꢀCastro, I., De Sanctis, J. B., Chirino, P., and
Benaim, G. (2012) The marine sponge toxin agelasine B increases the
ABBREVIATIONS
SAR, StructureꢀActivity Relationship; ROS, Radical Oxygen
Species; NAC, NꢀAcetyl Cysteine; DFO, Deferoxamine.
2+
intracellular Ca concentration and induces apoptosis in human
breast cancer cells (MCFꢀ7). Cancer Chemother. Pharmacol. 69, 71ꢀ
83.
(
20) Kozmin, S. G., Schaaper, R. M., Shcherbakova, P. V.,
REFERENCES
Kulikov, V. N., Noskov, V. N., Guetsova, M. L., Alenin, V. V.,
Rogozin, I. B., Makarova, K. S., and Pavlov, Y. I. (1998) Multiple
antimutagenesis mechanisms affect mutagenic activity and specificity
of the base analog 6ꢀNꢀhydroxylaminopurine in bacteria and yeast.
Mutat. Res. Fund. Mol. Mech. Mut. 402, 41ꢀ50.
(21) Hung, D. T., Jamison, T. F., and Schreiber, S. L. (1996)
Understanding and controlling the cell cycle with natural products.
Chem. Biol. 3, 623ꢀ639.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
1) Dewick, P. M. (2009) Medicinal Natural Products A
Biosynthetic Approach, 3 ed., John Wiley & Sons, West Sussex,
United Kingdom.
(
2) Lachance, H., Wetzel, S., Kumar, K., and Waldmann, H.
(
2012) Charting, Navigating, and Populating Natural Product
Chemical Space for Drug Discovery. J. Med. Chem. 55, 5989ꢀ6001.
(3) Gerry, C. J., Hua, B. K., Wawer, M. J., Knowles, J. P., Nelson
Jr, S. D., Verho, O., Dandapani, S., Wagner, B. K., Clemons, P. A.,
BookerꢀMilburn, K. I., Boskovic, Z. V., and Schreiber, S. L. (2016)
RealꢀTime Biological Annotation of Synthetic Compounds. J. Am.
Chem. Soc. 138, 8920ꢀ8927.
(4) Yosief, T., Rudi, A., Stein, Z., Goldberg, I., Gravalos, G. M.
D., Schleyer, M., and Kashman, Y. (1998) Asmarines AꢀC; Three
novel cytotoxic metabolites from the marine sponge Raspailia sp.
Tetrahedron Lett. 39, 3323ꢀ3326.
(
22) Adams, R. L. P., and Lindsay, J. G. (1967) Hydroxyurea:
Reversal of Inhibition and Use as a CellꢀSynchronizing Agent. J.
Biol. Chem. 242, 1314ꢀ1317.
(23) Brodie, C., Siriwardana, G., Lucas, J., Schleicher, R., Terada,
N., Szepesi, A., Gelfand, E., and Seligman, P. (1993) Neuroblastoma
Sensitivity to Growth Inhibition by Deferrioxamine: Evidence for a
Block in G1 Phase of the Cell Cycle. Cancer Res. 53, 3968ꢀ3975.
(
24) Eriksson, S., Gräslund, A., Skog, S., Thelander, L., and
Tribukait, B. (1984) Cell cycleꢀdependent regulation of mammalian
ribonucleotide reductase. The S phaseꢀcorrelated increase in subunit
M2 is regulated by de novo protein synthesis. J. Biol. Chem. 259,
(
5) Yosief, T., Rudi, A., and Kashman, Y. (2000) Asmarines A−F,
Novel Cytotoxic Compounds from the Marine Sponge Raspailia
Species. J. Nat. Prod. 63, 299ꢀ304.
1
1695ꢀ11700.
25) Engström, Y., Eriksson, S., Jildevik, I., Skog, S., Thelander,
(
6) Rudi, A., Shalom, H., Schleyer, M., Benayahu, Y., and
(
Kashman, Y. (2004) Asmarines G and H and Barekol, Three New
Compounds from the Marine Sponge Raspailia sp. J. Nat. Prod. 67,
L., and Tribukait, B. (1985) Cell cycleꢀdependent expression of
mammalian ribonucleotide reductase. Differential regulation of the
two subunits. J. Biol. Chem. 260, 9114ꢀ9116.
1
06ꢀ109.
(7) Rudi, A., Aknin, M., Gaydou, E., and Kashman, Y. (2004)
Asmarines I, J, and K and Nosyberkol:ꢁ Four New Compounds from
the Marine Sponge Raspailia sp. J. Nat. Prod. 67, 1932ꢀ1935.
(
26) Paull, K. D., Shoemaker, R. H., Hodes, L., Monks, A.,
Scudiero, D. A., Rubinstein, L., Plowman, J., and Boyd, M. R. (1989)
Display and Analysis of Patterns of Differential Activity of Drugs
Against Human Tumor Cell Lines: Development of Mean Graph and
COMPARE Algorithm. J. Natl. Cancer Inst. 81, 1088ꢀ1092.
(
8) Hertiani, T., EdradaꢀEbel, R., Ortlepp, S., van Soest, R. W. M.,
de Voogd, N. J., Wray, V., Hentschel, U., Kozytska, S., Müller, W. E.
G., and Proksch, P. (2010) From antiꢀfouling to biofilm inhibition:
New cytotoxic secondary metabolites from two Indonesian Agelas
sponges. Bioorg. Med. Chem. 18, 1297ꢀ1311.
(
27) Sun, D., Melman, G., LeTourneau, N. J., Hays, A. M., and
Melman, A. (2010) Synthesis and antiproliferating activity of iron
chelators of hydroxyaminoꢀ1,3,5ꢀtriazine family. Bioorg. Med. Chem.
Lett. 20, 458ꢀ460.
(28) Grillo, A. S., SantaMaria, A. M., Kafina, M. D., Cioffi, A. G.,
Huston, N. C., Han, M., Seo, Y. A., Yien, Y. Y., Nardone, C., Menon,
A. V., Fan, J., Svoboda, D. C., Anderson, J. B., Hong, J. D., Nicolau,
B. G., Subedi, K., Gewirth, A. A., WesslingꢀResnick, M., Kim, J.,
Paw, B. H., and Burke, M. D. (2017) Restored iron transport by a
small molecule promotes absorption and hemoglobinization in
animals. Science 356, 608ꢀ616.
(
9) Nakamura, H., Wu, H., Ohizumi, Y., and Hirata, Y. (1984)
AgelasineꢀA, ꢀB, ꢀC and ꢀD, novel bicyclic diterpenoids with a 9ꢀ
methyladeninium unit possessing inhibitory effects on Na,KꢀATPase
from the Okinawa sea sponge Agelas sp. Tetrahedron Lett. 25, 2989ꢀ
2
992.
10) Pappo, D., Rudi, A., and Kashman, Y. (2001) A synthetic
(
approach towards the synthesis of asmarine analogues. Tetrahedron
Lett. 42, 5941ꢀ5943.
(11) Ohba, M., and Tashiro, T. (2002) Preparatory Study for the
Synthesis of the Marine Sponge Alkaloids Asmarines AꢀF: Synthesis
of Their Heterocyclic Portions. Heterocycles 57, 1235.
(
29) Ba, L. A., Doering, M., Burkholz, T., and Jacob, C. (2009)
Metal trafficking: from maintaining the metal homeostasis to future
drug design. Metallomics 1, 292ꢀ311.
(30) Finney, L. A., and O'Halloran, T. V. (2003) Transition Metal
Speciation in the Cell: Insights from the Chemistry of Metal Ion
Receptors. Science 300, 931ꢀ936.
(
12) Pappo, D., and Kashman, Y. (2003) Synthesis of 9ꢀsubstituted
tetrahydrodiazepinopurines—asmarine A analogues. Tetrahedron 59,
6
9
493ꢀ6501.
(13) Pappo, D., Shimony, S., and Kashman, Y. (2005) Synthesis of
ꢀSubstituted Tetrahydrodiazepinopurines:ꢁ Studies toward the Total
(
31) Dean, R. T., and Nicholson, P. (1994) The Action of Nine
Chelators on IronꢀDependent Radical Damage. Free Rad. Res. 20, 83ꢀ
01.
(32) Chaston, T. B., Lovejoy, D. B., Watts, R. N., and Richardson,
Synthesis of Asmarines. J. Org. Chem. 70, 199ꢀ206.
14) Vik, A., and Gundersen, L.ꢀL. (2007) Synthetic studies
directed towards asmarines; construction of the
1
(
D. R. (2003) Examination of the Antiproliferative Activity of Iron
Chelators. Clin. Cancer Res. 9, 402ꢀ414.
tetrahydrodiazepinopurine moiety by ring closing metathesis.
Tetrahedron Lett. 48, 1931ꢀ1934.
(
33) Shao, J., Zhou, B., Di Bilio, A. J., Zhu, L., Wang, T., Qi, C.,
(
15) Wan, K. K., Iwasaki, K., Umotoy, J. C., Wolan, D. W., and
Shih, J., and Yen, Y. (2006) A Ferrousꢀtriapine complex mediates
formation of reactive oxygen species that inactivate human
ribonucleotide reductase. Mol. Cancer. Ther. 5, 586ꢀ592.
Shenvi, R. A. (2015) Nitrosopurines en route to potently cytotoxic
asmarines. Angew. Chem. Int. Ed. Engl. 54, 2410ꢀ2415.
(16) Rodgen, S. A., and Schaus, S. E. (2006) Efficient
Construction of the Clerodane Decalin Core by an Asymmetric
Morita–Baylis–Hillman Reaction/Lewis Acid Promoted Annulation
Strategy. Angew. Chem. Int. Ed. Engl. 45, 4929ꢀ4932.
(
34) Bogdan, A. R., Miyazawa, M., Hashimoto, K., and Tsuji, Y.
2016) Regulators of Iron Homeostasis: New Players in Metabolism,
Cell Death, and Disease. Trends Biochem. Sci. 41, 274ꢀ286.
(
ACS Paragon Plus Environment

ACS Chemical Biology
Page 8 of 9
(
35) Bair, J. S., Palchaudhuri, R., and Hergenrother, P. J. (2010)
(43) Yang, W., Chen, Y., Zhou, X., Gu, Y., Qian, W., Zhang, F.,
Han, W., Lu, T., and Tang, W. (2015) Design, synthesis and
biological evaluation of bisꢀaryl ureas and amides based on 2ꢀaminoꢀ
3ꢀpurinylpyridine scaffold as DFGꢀout BꢀRaf kinase inhibitors. Eur.
J. Med. Chem. 89, 581ꢀ596.
(44) Crane Erika, A., and Gademann, K. (2016) Capturing
Biological Activity in Natural Product Fragments by Chemical
Synthesis. Angew. Chem. Int. Ed. Engl. 55, 3882ꢀ3902.
(45) Néron, B., Ménager, H., Maufrais, C., Joly, N., Maupetit, J.,
Letort, S., Carrere, S., Tuffery, P., and Letondal, C. (2009) Mobyle: a
new full web bioinformatics framework. Bioinformatics 25, 3005ꢀ
3011.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Chemistry and Biology of Deoxynyboquinone, a Potent Inducer of
Cancer Cell Death. J. Am. Chem. Soc. 132, 5469ꢀ5478.
(
Jungwirth, U., Mair, G. E., Steinborn, R., Enyedy, E. A., Jakupec, M.
A., Berger, W., and Keppler, B. K. (2013) Triapine and a More Potent
Dimethyl Derivative Induce ER Stress in Cancer Cells. Mol.
Pharmacol. 85, 451ꢀ459.
36) Trondl, R., Flocke, L. S., Kowol, C. R., Heffeter, P.,
(
37) Cai, J., and Jones, D. P. (1998) Superoxide in Apoptosis:
Mitochondrial Generation Triggered by cytochrome c loss. J. Biol.
Chem. 273, 11401ꢀ11404.
(38) Simon, H.ꢀU., HajꢀYehia, A., and LeviꢀSchaffer, F. (2000)
Role of reactive oxygen species (ROS) in apoptosis induction.
Apoptosis 5, 415ꢀ418.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
39) Chen, S., Graceffa, R. F., and Boezio, A. A. (2016) Direct,
Regioselective NꢀAlkylation of 1,3ꢀAzoles. Org. Lett. 18, 16ꢀ19.
(40) Baker, E., Vitolo, M. L., and Webb, J. (1985) Iron chelation
by pyridoxal isonicotinoyl hydrazone and analogues in hepatocytes in
culture. Biochem. Pharmacol. 34, 3011ꢀ3017.
(
41) Richardson, D., Tran, E., and Ponka, P. (1995) The potential
of iron chelators of the pyridoxal isonicotinoyl hydrazone class as
effective antiproliferative agents. Blood 86, 4295ꢀ4306.
(42) de Ligt, R. A. F., van der Klein, P. A. M., von Frijtag Drabbe
Künzel, J. K., Lorenzen, A., Ait El Maate, F., Fujikawa, S., van
Westhoven, R., van den Hoven, T., Brussee, J., and Ijzerman, A. P.
(
2004) Synthesis and biological evaluation of disubstituted N6ꢀ
cyclopentyladenine analogues: the search for a neutral antagonist with
high affinity for the adenosine A1 receptor. Bioorg. Med. Chem. 12,
139ꢀ149.
ACS Paragon Plus Environment

Page 9 of 9
ACS Chemical Biology
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
TOC graphic
4
97x194mm (72 x 72 DPI)
ACS Paragon Plus Environment