A Novel Tumor-Activated Prodrug Strategy Targeting Ferrous Iron Is Effective in Multiple Preclinical Cancer Models

Article abstract of DOI:10.1021/acs.jmedchem.6b01470

Here we describe a new approach for tumor targeting in which augmented concentrations of Fe(II) in cancer cells and/or the tumor microenvironment triggers drug release from an Fe(II)-reactive prodrug conjugate. The 1,2,4-trioxolane scaffold developed to enable this approach can in principle be applied to a broad range of cancer therapeutics and is illustrated here with Fe(II)-targeted forms of a microtubule toxin and a duocarmycin-class DNA-alkylating agent. We show that the intrinsic reactivity/toxicity of the duocarmycin analog is masked in the conjugated form and this greatly reduced toxicity in mice. This in turn permitted elevated dosing levels, leading to higher systemic exposure and a significantly improved response in tumor xenograft models. Overall our results suggest that Fe(II)-dependent drug delivery via trioxolane conjugates could have significant utility in expanding the therapeutic index of a range of clinical and preclinical stage cancer chemotherapeutics.

Full text of DOI:10.1021/acs.jmedchem.6b01470

Subscriber access provided by UB + Fachbibliothek Chemie | (FU-Bibliothekssystem)
Article
A Novel Tumor-Activated Prodrug Strategy Targeting Ferrous
Iron is Effective in Multiple Preclinical Cancer Models
Benjamin Spangler, Shaun D. Fontaine, Yihui Shi, Lidia C. Sambucetti,
Aras N. Mattis, Byron Hann, James A. Wells, and Adam R. Renslo
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01470 • Publication Date (Web): 21 Nov 2016
Downloaded from http://pubs.acs.org on November 22, 2016
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 free 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 accessible to all
readers and 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.
Journal of Medicinal Chemistry 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 36
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
A Novel Tumor-Activated Prodrug Strategy
Targeting Ferrous Iron is Effective in Multiple
Preclinical Cancer Models
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
†
,‡
‡,∫
§
§
°
∏
Benjamin Spangler , Shaun D. Fontaine , Yihui Shi , Lidia Sambucetti , Aras N. Mattis , Byron Hann ,
‡,∂
‡, *
James A. Wells , and Adam R. Renslo
†
Graduate Program in Chemistry & Chemical Biology, University of California, San Francisco.
‡
°
§
Department of Pharmaceutical Chemistry, University of California, San Francisco. SRI International.
∏
Department of Pathology, University of California, San Francisco. Preclinical Therapeutic Core,
∂
University of California, San Francisco. Department of Cellular and Molecular Pharmacology, University
of California, San Francisco.
Keywords: Tumor Activated Prodrugs, Drug Delivery, Iron Metabolism, Cancer Chemotherapy
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 36
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
ABSTRACT
Here we describe a new approach for tumor targeting in which augmented concentrations of Fe(II) in
cancer cells and/or the tumor microenvironment trigger drug release from an Fe(II)-reactive prodrug
conjugate. The 1,2,4-trioxolane scaffold developed to enable this approach can in principle be applied
to a broad range of cancer therapeutics and is illustrated here with Fe(II)-targeted forms of a
microtubule toxin and a duocarmycin-class DNA-alkylating agent. We show that the intrinsic
reactivity/toxicity of the duocarmycin analog is masked in the conjugated form and this greatly reduced
toxicity in mice. This in turn permitted elevated dosing levels, leading to higher systemic exposure and a
significantly improved response in tumor xenograft models. Overall our results suggest that Fe(II)-
dependent drug delivery via trioxolane conjugates could have significant utility in expanding the
therapeutic index of a range of clinical and pre-clinical stage cancer chemotherapeutics.
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
ACS Paragon Plus Environment

Page 3 of 36
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
INTRODUCTION
The recent development of molecularly targeted cancer therapeutics has been accompanied by
renewed interest in technologies for the tumor/cell-selective delivery of potent but intrinsically non-
selective cytotoxic agents. These technologies include antibody-drug conjugates (ADCs) that recognize
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
–3
cell-surface antigens and tumor-activated prodrugs (TAPs) that exploit nutrient transporters or
4,5
differences in hypoxia associated with the tumor microenvironment. Accumulating evidence suggests
that an increase in reactive, “labile” intracellular iron is another metabolic signature of cancer, as
6
recently reviewed. Tumor targeting strategies designed to exploit changes in iron homeostasis remain
largely unexplored however, despite clinical precedent for iron-dependent pharmacology in antimalarial
7,8
therapy with artemisinins.
Redox cycling of iron in enzyme cofactors is essential for cellular processes ranging from de novo
nucleotide synthesis to the maintenance of genomic stability, cell cycle regulation, and mitochondrial
6
respiration. However, when unbound and unregulated, redox active iron promotes the
disproportionation of hydrogen peroxide (Fenton reaction) to produce hydroxyl and hydroperoxyl
radicals, reactive oxygen species (ROS) that confer cellular damage and can lead to apoptosis or
9–11
ferroptosis. Iron homeostasis is therefore highly regulated to ensure sufficient labile iron is available
1
2–14
to support essential enzyme function, while limiting exposure to unbound, redox active iron species.
Rapidly proliferating cells have increased requirements for DNA synthesis, repair, and mitochondrial
respiration, and therefore have increased demands for iron cofactor biosynthesis. Accordingly, iron
6
,15–18
acquisition and export pathways are altered in many cancers so as to increase the labile iron pool.
15,19
Furthermore, iron has been shown to contribute to tumor initiation and growth
and epidemiological
evidence has established links between tumor iron metabolism and clinical outcomes in breast cancer
17,20
patients.
Given that labile Fe(II) promotes Fenton chemistry, we sought to develop a tumor-
targeting strategy in which Fenton reaction of a peroxidic prodrug was coupled to release of drug
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 36
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
21–24
payloads. Recognizing that antimalarial agents such as arterolane
exhibit finely tuned iron(II)-
25–28
29, 30
reactivity,
we subsequently developed
an arterolane-inspired small molecule platform (denoted
TRX herein) for Fe(II)-dependent drug delivery. These TRX-drug conjugates function via initial Fe(II)-
promoted fragmentation of a 1,2,4-trioxolane ring to afford a cyclohexanone intermediate, followed by
spontaneous β-elminiation and decarboxylation to release the drug payload (Figure 1 and Supporting
Information, Figure S1). In previous studies, we demonstrated the utility of the TRX scaffold by
efficiently and selectively delivering antimalarial payloads to ferrous iron/heme rich compartments of
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
30,31
32,33
and in vivo.
the malaria parasite, both in vitro
[Suggested location for Figure 1]
Figure 1: Mechanism of iron(II)-dependent payload release from 1,2,4-trioxolane conjugates. Iron(II)-
promoted Fenton-type reduction of the trioxolane ring affords the cyclohexanone species shown which
then undergoes spontaneous β-elimination and decarboxylation to release free payload. This process
occurs with high efficiency and ferrous-iron selectivity in mammalian cancer cell lines, as described
34
recently.
While the iron-dependent pharmacology of 1,2,4-trioxanes and 1,2,4-trioxolanes in malaria is widely
25-28
accepted,
extending this concept to TAPs for oncology applications required a means to assess
whether sufficient labile iron is present in cancer cells to efficiently and selectively activate TRX-based
conjugates. To address this question, we synthesized a TRX conjugate of the aminonucleoside antibiotic
puromycin (i.e., TRX-PURO) as a probe of intracellular labile iron (Figure 1 where R–NH = puromycin).
2
We found that puromycin release from TRX-PURO in cells was dependent on ferrous iron as expected,
34
and was not affected by the addition of other biologically relevant metal ions and reducing agents.
ACS Paragon Plus Environment

Page 5 of 36
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
These initial studies with TRX-PURO also confirmed that labile iron pools are generally augmented in
34
cancer cells when compared to non-tumorigenic cells. Our findings thus suggested that the TRX
scaffold might indeed be applied to produce a novel class of TAPs for cancer chemotherapy.
Here we describe prototypical TRX-drug conjugates designed to confer tumor-selective delivery of a
microtubule toxin (a combretastatin analog) or DNA-alkylating agent (a duocarmycin analog) in an Fe(II)-
dependent fashion. We show that the intrinsic cytotoxicity of these agents can be ablated in TAP forms
and then restored following activation in cancer cells. We show that cancer cell lines of diverse origins
are generally susceptible to TRX-based TAPs, but that a non-tumorigenic cell line (MCF10A) is highly
resistant. We further show that the resistant MCF10A cell line can be measurably sensitized to the TAP
when transformed with the oncogene cMyc and that this likely derives from oncogenic changes to iron
metabolism. Finally, we show that the TAP of a duocarmycin analog is tolerated in mice at doses up to
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5
0-fold higher than the parent cytotoxin and this, combined with targeted toxin release within tumor,
translates to superior efficacy in PC-3 and MDA-MB-231 xenograft models. These studies provide the
first evidence that reactive iron in tumor cells and/or the tumor microenvironment can be exploited to
afford improved selectivity in the delivery of cancer chemotherapeutics.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 36
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
RESULTS
Conjugate Design, Synthesis and Validation in Cell Culture. To explore ferrous iron-dependent drug
35
delivery in cancer cells we synthesized a known small molecule microtubule toxin (1) and its trioxolane
conjugate 2 (Figure 2a and Supporting Information, Figure S2). A non-peroxidic dioxolane conjugate (3)
was also prepared to confirm that the cytotoxicity of 2 is ablated in conjugated forms and that
intracellular release of active 1 from 2 is peroxide-dependent (Fig. 2a and Supplementary Fig. 2). Finally,
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
29
trioxolane analog 4 lacking the microtubule toxin was prepared to control for intrinsic cytotoxicity of
the TRX moiety (Figure 2a). The cytotoxicity of the trioxolane-conjugate 2 and control compounds 1, 3,
and 4 were then assessed across a small panel of cell lines. The results were unequivocal. In MDA-MB-
231 cells, trioxolane-conjugate 2 displayed activity in the low nM range (EC = 21 nM), three orders of
50
magnitude more potent than either of the negative controls (3 or 4), and nearly as potent as the free
toxin (1) applied directly (EC = 11 nM) (Figure 2b). These results confirm that release of 1 from 2 in
50
cancer cells is both efficient and peroxide-dependent. Moreover, the lack of measurable toxicity
exhibited by 3 demonstrates that the intrinsic cytotoxicity of 1 is effectively blocked in TAP form.
Normalizing the activity of conjugate 2 to that of its cytotoxic payload (1) provided a convenient
metric (“EC ratio”) to compare efficiency of payload release from 2 across different cell lines. In MDA-
50
MB-231 cells, this ratio was found to be 0.54 (EC = 11 nM and 21 nM for 1 and 2, respectively).
50
Amongst the other cell lines examined, U2OS and RKO cells were nearly as susceptible to 2 as MDA-MB-
2
0
31 cells (EC ratio of 0.46) while PC-3 and HeLa cells were somewhat less sensitive (EC ratio of 0.30-
50 50
.32), though 2 was still effective in these cells at therapeutically relevant, low-nM concentrations
(
Figure 2d and Supporting Information, Table S1). Consistent with our previous finding that non-
34
tumorigenic cells possess a smaller reactive iron pool, MCF10A cells were highly resistant to the
trioxolane-conjugate 2 with an EC ratio of just 0.04 (Figure 2c-d).
50
ACS Paragon Plus Environment

Page 7 of 36
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
[
Suggested location for Figure 2]
Figure 2: Exploiting augmented ferrous iron pools for selective drug delivery (a) Chemical structure of
microtubule inhibitor 1 and corresponding conjugates and controls 2-4. (b) Cytotoxicity of compounds 1-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
in MDA-MB-231 cells after 24 h of exposure as determined by cell counting (n=3; error bar, mean ±
s.e.m.). (c) Cytotoxicity of 1 and its trioxolane-conjugate 2 after 24 h of exposure in MDA-MB-231 and
MCF10A cells as determined by cell counting (n=3; error bar, mean ± s.e.m.). The shift in EC value for 1
50
and its trioxolane conjugate 2 is illustrated. Dividing the EC value for 1 by that for 2 in a given cell line
50
produces an “EC ratio” that is used to compare the efficiency of drug release across cell lines. This ratio
50
is 0.54 for sensitive MDA-MB-231 cells (2-fold EC shift), and 0.04 for relatively resistant, non-
50
tumorigenic MCF10A cells (25-fold EC shift). (d) EC ratios calculated as described in (c) for a small
50
50
panel of cell lines. Error bars represent s.e.m. from three individual experiments each conducted in
biological triplicates. (e) EC ratios for an expanded panel of mammalian cell lines. The EC values for 1
50
50
and 2 were determined after a 72 hour incubation using the CellTiter-Glo® cell viability assay. Error bars
represent s.e.m. (n = 3).
Encouraged by these initial findings we further explored the cytotoxicity of 1 and 2 across a larger
panel of cancer cell lines from diverse origins using a CellTiter-Glo® assay to assess cell viability (Figure
2
e). We found EC ratios varied by about 9-fold across the different cell lines, from 0.89 for the most
50
susceptible cells (PC-3) cells to 0.11 for the least susceptible cells (EKVX). Despite the range of
responses, TAP 2 produced EC values in the nM range for even the least responsive cell lines (e.g. EKVX
50
EC = 43 nM, Supporting Information, Table S2). As expected, the EC values for trioxolane control 4
50
50
across this panel were in the ꢀM regime and typically ≥2 log units less potent than 2 (Supporting
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 36
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
Information, Table S2). This confirms that the cytotoxicity of TAP 2 derives from release of 1 and not
significantly from the trioxolane moiety itself.
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
Oncogenic Transformation and Susceptibility to Trioxolane Conjugates. Having found that non-
tumorigenic MCF10A cells were highly resistant to TRX-conjugates, we explored whether oncogenic
transformation of these cells would increase their reactive iron pools and thus sensitize them to
trioxolane conjugates. Bandyopadhyay and co-workers recently reported the generation of a panel of
36
cell lines expressing single oncogenes in MCF10A cells. Using these well-characterized cell lines, we
examined the effects of the oncogenes Ras and Myc, whose effects on iron metabolism have been
6,16,18,37-38
studied previously.
First, we used qRT-PCR to evaluate the transcriptional profile of a panel of
iron regulatory proteins in the MCF10A cells constitutively expressing HRas. In previous work in
different cell types, oncogenic HRas has been variously reported to decrease ferritin mRNA levels and
38
16
increase labile iron or to have no effect on ferritin levels or labile iron. In the MCF10A cells
expressing HRas we observed no significant transcriptional changes to ferritin, transferrin receptor, or
any other of the iron regulatory transcripts probed (Figure 3 and Supporting Information, Figure S3). It
was therefore unsurprising that the HRas-expressing MCF10A cells were no more susceptible to 2 than
MCF10A cells transformed with the empty vector (Figure 3b). In contrast, MCF10A cells transformed
with cMyc showed substantial down regulation of the iron exporter ferroportin and up regulation of the
ferrireductase STEAP3 (Figure 3a). Interestingly, we did not observe an effect of cMyc on ferritin heavy
18
chain mRNA, as has been reported previously. Nevertheless, the changes observed predict for
increased labile iron in the cMyc-transformed MCF10A cells and indeed, these cells were found to be
more sensitive to trioxolane-conjugate 2 than cells transformed with the empty vector, as evidenced by
a significant 3.2-fold shift in the EC ratio (Figure 3b). These results indicate that oncogene induced
50
ACS Paragon Plus Environment

Page 9 of 36
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
changes to the reactive iron pool can sensitize cells to the delivery of potent cytotoxins from TRX-based
TAPs in an otherwise isogenic background.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[
Suggested location for Figure 3]
Figure 3: Profiling the effects of oncogenic transformation on the ferrous iron pool of MCF10A cells.
a) Relative mRNA levels for ferroportin, ferritin, and the ferrireductase STEAP3 in MCF10A cells stably
(
transfected with empty vector (control) or with vectors expressing the oncogenes Myc or Hras (n=3;
error bar, mean ± s.e.m.; **P≤0.01, ****P≤0.0001, Two-Way ANOVA with Dunnett’s multiple
comparisons test). (b) EC ratio (1/2) for MCF10A cells stably transfected with HRas or cMyc oncogenes
50
or the empty vector (control) as assessed by cell counting after 24 h of compound exposure. Error bars
represent s.e.m from three independent experiments each conducted in biological triplicates (**P≤0.01,
One-Way ANOVA with Dunnett’s multiple comparisons test).
In vivo PK/PD Studies of a Duocarmycin Conjugate. The highly cytotoxic cyclopropylbenzindoline
(
CBI) class of natural products like CC-1065 and duocarmycin SA function by alkylating adenine bases in
39-42
duplex DNA.
Synthetic “seco”-CBI analogs are latent cytotoxins that can undergo spontaneous
Winstein-type spirocylization to form the active cyclopropylbenzindoline (CBI) species (i.e., seco-5 ꢀ 5,
Figure 4a and Supporting Information, Figure S4). This activation step can be prevented by acylation or
carbamoylation of the aniline or phenolic function and the ability to “cage” seco-CBI derivatives in this
2
way has made these compounds popular as cytotoxic effectors in antibody-drug conjugates and tumor-
3,41
40
activated prodrugs.
into the desired TAP 6 and the 1,3-dioxolane-CBI conjugate 7 (DXL-CBI), a non-peroxidic control (Figure
a).
We synthesized a known seco-CBI analog and further converted the material
4
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 36
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
We evaluated the cytotoxicity of seco-5, TAP 6 and control 7 following 72 h of exposure in MDA-MB-
31 and PC-3 cells (Figure 4b-c). As expected, seco-5 and its TAP 6 exhibited potent cytotoxic effects in
2
cells while the dioxolane conjugate 7 was non-cytotoxic at all concentrations examined. The inactivity of
confirms that conjugation of seco-5 via its aniline function effectively blocks formation of the activated
and toxic) CBI electrophile. The potent effects of 6 in cells are thus due to peroxide-dependent release
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
7
(
of free seco-5 as desired. We further found that conversion of seco-5 to the TAP 6 dramatically
improves its chemical stability in the extracellular environment. Thus, while free seco-5 degraded over
several hours upon thawing from a DMSO stock solution, TRX conjugate 6 (and DXL conjugate 7) were
chemically stable for more than a year at room temperature in DMSO (Supporting Information, Figure
S5). Stability towards premature hydrolysis likely explains why TAP 6 was measurably more potent than
seco-5 itself in both cell lines examined (Figure 4b-c). Control experiments confirmed that 6 is stable for
more than a week in cell culture media at 37 °C, indicating that its cellular toxicity in the 72 h assays
(Figure 4b-c) results from intracellular release of seco-5 as desired.
[Suggested location of Figure 4]
Figure 4: Structures and in vitro activity of trioxolane-duocarmycin conjugates. (a) Structure of
duocarmycin type DNA-alkylator seco-5 (latent form), the corresponding activated form 5 that reacts
with nucleophilic bases of DNA (:Nu), trioxolane-duocarmycin conjugate 6, and negative control
dioxolane-conjugate 7. (b) Cytotoxicity of compounds 5 and 6 in PC-3 cells after 72 h of exposure as
determined by cell counting (n=3; error bar, mean ± s.e.m.). (c) Cytotoxicity of compounds 5 and 6 in
MDA-MB-231 cells after 72 h of exposure as determined by cell counting (n=3; error bar, mean ± s.e.m.).
ACS Paragon Plus Environment

Page 11 of 36
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
The cell culture studies of 5-7 above revealed the effective ablation of 5-mediated toxicity by chemical
conjugation at an aniline function (Figure 4a-c). We next asked whether this caging effect would
translate to reduced in vivo toxicity for TAP 6 as compared to 5. To determine a maximally tolerated
dose (MTD), non-tumor bearing female NSG mice were administered three i.p. doses of either seco-5 or
6 at four day intervals (Q4d). These studies revealed that seco-5 is highly toxic to mice (MTD ~ 0.3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
41
mg/kg), consistent with previous observations for related duocarmycin analogs. By contrast, TAP 6
could be administered at significantly higher doses, with an MTD of 7.5 mg/kg using the same three
dose Q4d regimen. A subsequent MTD study of 6 with Q7d dosing returned a somewhat higher MTD of
~10 mg/kg.
Previous work with duocarmycin analogs related to 5 have shown dose-limiting hepatotoxicity in
43
44
mice and insufficient therapeutic index leading to failures in human clinical trials. We were therefore
interested in exploring the toxic effects of 5 in mice and whether administration in the TRX-conjugated
form 6 could protect from these toxicities. In fact, mice treated with 0.3 mg/kg seco-5 showed
substantially (~10-fold) higher concentrations of liver transaminase enzymes (ALT and AST) than did
mice treated with 7.5 mg/kg of 6 (Figure 5c). Since hepatotoxicity of duocarmycin analogs has
previously been observed to occur with a delayed onset, mice receiving escalating doses of 5 or 6 were
observed for 50-days post-dosing, then sacrificed, and their livers collected and assessed for altered
morphology and signs of toxicity. Even at well-tolerated doses of 5, mice grossly showed darkened and
roughened capsular and parenchymal changes indicative of hepatotoxicity in addition to enlarged
intestines and serosanguinous ascites near the site of administration. The liver pathology showed
increased hepatic lobular lymphohistiocytic infiltrates, reactive cellular changes, and minimal periportal
fibrosis. In contrast, the livers of mice treated with 7.5 mg/kg of 6 appeared normal and the dose
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 36
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
limiting toxicity appeared instead to be localized toxicity at the site of administration as evidenced by
enlarged intestines. The pathology in these was free of significant inflammation or fibrosis (Figure 5d).
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
[Suggested location for Figure 5]
Figure 5: Pharmacokinetic profile and in vivo tolerability of 5 and its trioxolane conjugate 6. (a) Plasma
concentrations of 5 in female NSG mice following a single 0.3 mg/kg i.p. dose. Three mice were sampled
at each time point. (b) Plasma concentrations of 6, released 5, and the retro-Michael intermediate 16 in
female NSG mice following a single 7.5 mg/kg i.p. dose of 6. An authentic sample of 16 was synthesized
as described in the Supplementary Information file. The study design and numbers of mice per group
was the same as in (a). (c) Measured levels of Alkaline Phosphatase (Alk. Phosp.), serum alanine
aminotransferases (ALT), aspartate aminotransferase (AST), and creatine phosphokinase (CPK) in blood
samples from mice treated with either 5 (0.3 mg/kg) or trioxolane-conjugate 6 (7.5 mg/kg). (d)
Microscopic histology stains of representative mouse liver samples show only mildly increased reactive
changes in the group receiving 0.3 mg/kg 5, including minimally increased lobular lympho-histiocytic
infiltrates. In mice receiving the higher 7.5 mg/kg dose of 6 (right column), only mild simple steatosis
was detected. Top two rows of images show representative H&E sections of centrizonal or periportal
areas. Bottom row of images show representative trichrome stains for evaluation of relative fibrosis,
which shows no significant fibrosis; scale bars in µm as indicated.
To compare the pharmacokinetic properties of seco-5 and its TRX conjugate 6, healthy NSG mice were
administered a single i.p. dose of seco-5 (0.3 mg/kg) or conjugate 6 (7.5 mg/kg), representing the
respective MTD values. Analysis of blood samples showed seco-5 to have a reasonably long half-life (t1/2
=
3.8 h), moderate clearance (CL/F = 20 mL/min/kg) and a high volume of distribution (Vz/F = 6.7 L/Kg)
ACS Paragon Plus Environment

Page 13 of 36
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(
Figure 5a-b, Supporting Information, Table S3). Conjugate 6 exhibited a significantly longer half-life (t1/2
=
20.4 h), moderate-high clearance (CL/F = 31.3 ml/min/kg) and a very high volume of distribution (Vz/F
= 55 L/kg). In animals receiving 6, total exposure to 6 (AUC = 5050 h*ng/mL) exceeded that of released
seco-5 by approximately 8-fold, indicating a small degree of drug release in normal tissues of healthy
mice. Maximum plasma concentrations of free seco-5 in mice receiving 7.5 mg/kg of 6 were still lower
than those in mice treated with 0.3 mg/kg of seco-5 directly (Cmax = 48 ng/mL vs 108 ng/mL). Most
importantly, the total systemic exposure of 6 was ~20-fold greater than for seco-5 when both agents
were administered at their respective MTD (AUC0-24 h = 5050 h*ng/mL and 246 h*ng/mL respectively).
The higher in vivo exposure achievable with TAP 6 was thus expected to result in superior efficacy in
tumor bearing mice, where 6 would be converted to the active agent (5) selectively in the tumor.
To see whether the higher in vivo exposure levels achievable with conjugate 6 translated to improved
in vivo efficacy, we treated PC-3 and MDA-MB-231 xenograft-bearing mice with 6 or seco-5 and
compared body weights and rates of tumor growth under different dosing regimens (Figure 6a-d). In
MDA-MB-231 xenograft mice, three 0.3 mg/kg doses of seco-5 given Q4d produced only a minor
reduction in the rate of tumor growth (Figure 6a). Significantly, the mice in this group showed
substantial weight loss over the course of the study, presumably reflecting compound-related toxicity
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(
Figure 6b). Comparatively, mice treated under the same regimen with 2.5 mg/kg of 6 exhibited a similar
reduction in tumor growth rate but unlike 5-treated mice had negligible weight loss, suggesting reduced
toxicity at a under a comparably efficacious regimen (Figure 7a). Using the same regimen but increasing
the dose of 6 to its MTD of 7.5 mg/kg produced a substantially improved effect on tumor growth and
with much less severe weight loss than was observed with a 25-fold lower dose of seco-5 (Figure 6a-b).
These findings were replicated in a follow-up study that also included the evaluation of dioxolane
conjugate 7 (Supporting Information, Figure S6). As expected, compound 7 at 10 mg/kg had no effect
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 36
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
on tumor growth or mouse weight, indicating that the beneficial in vivo effects of 6 result from
peroxide-dependent activation and release of 5 in tumor.
The results described above for MDA-MB-231 xenograft mice were qualitatively replicated in PC-3
xenograft bearing mice. Thus, administration of 6 at 7.5 mg/kg (3x, Q4d, i.p.) produced comparable if
not superior effects on tumor growth rate without the weight loss observed in mice receiving seco-5 at
0.3 mg/kg (Figure 6c-d). Since the MTD and pathology studies had suggested that the dose-limiting
toxicity of TAP 6 was localized G.I. toxicity near the site of administration, we considered that an altered
dosing regimen might mitigate this toxicity and enable a higher dose. Indeed, by increasing the dose of
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
6
to 15 mg/kg but extending the dosing interval from Q4d to Q7d (once weekly), we observed robust
tumor regression (Figure 6c, orange line), albeit with weight loss that was similar to the animals
receiving the 0.3 mg/kg Q4d regimen of 5. The tumor regression produced with 15 mg/kg Q7d dosing of
6
was found to be quite durable, with no evidence of further tumor growth observed for the remainder
of the study, several weeks after the third and final dose.
[Suggested location for Figure 6]
Figure 6: Efficacy of 5 and 6 in mouse xenograft models. (a) Changes in tumor volume over time as
determined by calipers in MDA-MB-231 xenograft bearing female SCID-beige mice treated with the
indicated doses of 5 or 6 via i.p. administration on a Q4d dosing schedule (3 total doses) as compared to
mice treated with vehicle. Error bars represent s.e.m from dosing cohorts of 8 mice per condition. (b)
Changes in mouse weight over time for the mice in (a). (c) Changes in tumor volume over time as
determined by calipers in PC-3 xenograft bearing female nude mice (NCR nu/nu, Taconic) treated with
the indicated doses of 5 or 6 via i.p. administration on a Q4d dosing schedule (3 total doses) as
compared to mice treated with vehicle. Mice treated with the highest dose of 6 (15 mg/kg, orange line)
ACS Paragon Plus Environment

Page 15 of 36
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
received three i.p. doses on a Q7d schedule. Error bars represent s.e.m from dosing cohorts of 8 mice
per condition. (d) Changes in mouse weight over time for the mice in (c).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
DISCUSSION
The search for more effective and better tolerated cancer therapies has yielded the first molecularly
45,46
targeted agents
(e.g., kinase inhibitors, PARP inhibitors) and new technologies to more selectively
deliver potent cytotoxins to tumors (e.g., antibody-drug conjugates). Herein we have described a new
class of tumor activated prodrugs engineered to release a drug payload upon encountering reactive
iron(II) in the tumor microenvironment. The trioxolane-based scaffold employed in these studies is the
same one we used previously to study intracellular labile ferrous iron in cells, revealing larger iron(II)
34
pools in cancer-derived cell lines compared to non-tumorigenic lines. Here we exploited this
knowledge and the tools we developed to deliver cytotoxic payloads to diverse cancer cell lines and to
target tumors in two different mouse xenograft models.
The trioxolane TAP scaffold used here was engineered to confer “traceless” release of drug payloads,
thereby enabling a broad scope of potential applications encompassing molecularly targeted and
generally cytotoxic agents possessing suitable amine or alcohol functionality for conjugation. When
employing this strategy, the site of drug conjugation is selected so that the intrinsic activity/toxicity of
tethered drug is ablated, thereby preventing or minimizing exposure of active drug in non-targeted
tissues. To exemplify this approach, we prepared amine-linked TAP conjugates 2 and 6 from the
microtubule inhibitor 1 and duocarmycin analog seco-5, respectively. We confirmed that the
activity/toxicity of these agents was ablated in the TAP form by preparing dioxolane-drug conjugates 3
and 7, which proved inactive at the highest concentrations tested (Figure 2b and Figure 4b-c). The lack
of measurable activity for 3 and 7 thus confirms that drug release from 2 and 6 is peroxide dependent.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 36
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
Consistent with the expected mechanism of iron(II)-dependent drug release, the relative sensitivity of
cells to 2 (Figure 2d) largely mirrored the response of the TRX-PURO probe to intracellular labile iron in
34
the same cell lines.
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
We found that sensitivity to TRX-CMB conjugate 2 varied by about 9-fold across a panel of cancer cell
lines from diverse origins (Figure 2e). Further interrogation of this data may provide insight into the
specific alterations of iron metabolism that predict for increased tumor susceptibility towards TRX-drug
conjugates. In our preliminary study of oncogenic changes and related effects on iron metabolism, we
observed that Myc-driven changes in MCF10A cells produced increased sensitivity to trioxolane
conjugate 2. Consistent with these observations, several of the most 2-susceptible cell lines examined
36,47
here, such as RKO and MDA-MB-231, are known to overexpress Myc.
Thus, iron(II)-dependent drug
delivery could find utility in targeting Myc-driven tumors indirectly via the alteration of iron metabolism
induced by this prevalent oncogene. These findings are particularly relevant given how intractable Myc
48
driven tumors have been towards other targeted therapies.
To examine the utility of trioxolane-mediated drug delivery in vivo, we prepared trioxolane TAP 6 in
which a potent duocarmycin-class cytotoxin (seco-5) is stabilized chemically and biologically inactivated
(while in prodrug form). Significantly, conjugate 6 was tolerated in mice at doses up to 50-fold higher
than seco-5 and produced superior efficacy in two different xenograft models when administered at or
near its MTD. A pharmacokinetic study in healthy mice revealed that administration of seco-5 in the
form 6 not only limited exposure to free drug (to ~15% of the total dose) but also significantly improved
distribution to tissues and the total duration of drug exposure. These properties of 6 enabled safe
administration at a relatively high dose of 15 mg/kg once weekly and this dosing regimen produced a
particularly robust and durable tumor regression in a PC-3 xenograft model. Overall, our studies indicate
that trioxolane-based TAPs release their drug payload in proportion to the concentration of labile iron(II)
encountered in different cell/tissue types. While intracellular iron(II) pools are likely implicated in TAP
ACS Paragon Plus Environment

Page 17 of 36
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
activation, it is possible that other aspects of tumor biology in vivo (e.g., hypoxia, macrophage
infiltration, tumor necrosis) contribute to the presence of excess reactive iron(II) in the tumor
microenvironment.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In conclusion, trioxolane-mediated iron(II)-dependent drug delivery is a new approach for cell/tissue
selective drug targeting that leverages altered reactive iron(II) concentrations in tumor cells and in the
tumor microenvironment. Here we described two prototypical trioxolane-drug conjugates bearing
cytotoxins with distinct mechanisms of cellular toxicity. We confirmed that the intrinsic toxicity of these
agents could be ablated in conjugated forms and yet fully realized following cell- or tumor-selective
release at their intended site of action. These results should encourage further study of this concept to
identify drugs and tumor types that best leverage this new drug delivery approach.
EXPERIMENTAL SECTION
35
40
The known cytotoxic agents 1 and seco-5 were prepared as previously described. These compounds
29
30
were coupled to known trioxolane and dioxolane intermediates via activated nitrophenyl carbonate
29-31
or isocyanate intermediates as we have described previously
Supporting Information.
and as further detailed in the
All compounds tested in cells or animals were judged to be of >95% purity as determined using a
Waters Micromass ZQTM, equipped with Waters 2795 Separation Module, Waters 2996 Photodiode
Array Detector (254 nm), and Waters 2424 ELS detector. Separations were carried out with an XTerra
MS C18, 5ꢀm, 4.6 x 50 mm column, at ambient temperature (unregulated) using a mobile phase of
water-methanol containing a constant 0.10 % formic acid. Representative LC chromatograms are
provided in the Supporting Information.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 36
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
Mammalian cell lines were maintained in an atmosphere of 5% CO in RPMI 1640 media purchased
2
from HyClone supplemented with 10% FBS (Gibco), Pen/Strep (1x final concentration, Gemini Bio-
Products), and non-essential amino acids (UCSF Cell Culture Facility). Unless otherwise noted, cell lines
were obtained from ATCC and verified by STR profiling. Graphing and analysis of data was done using
GraphPad Prism 6 Software and Microsoft Excel 2010. Figures were prepared with Adobe Design
Standard CS6 software.
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
Statistics: Error bars in all figures represent s.e.m. unless otherwise indicated. When three or more
means were compared, one or two-way ANOVA tests were applied as required with Dunnett’s multiple
comparisons tests used to determine significance. Statistical significance is indicated as follows: * =
P≤0.05, ** = P≤0.01, *** = P≤0.001, **** = P≤0.0001.
Toxicity by Nuclei Counting: Cells were plated in 96-well greiner black µClear tissue culture plates at
3
000-6000 cells per well in RPMI 1640 cell culture media (or the appropriate growth medium as
specified) and incubated at 37 °C in 5% CO incubators for at least 16 h prior to exposure to compounds.
2
Cells were then treated in triplicate with escalating concentrations of compounds performed in media
containing 0.1% DMSO (100 µL of media, per well). 24-72 h post–treatment (as specified) media was
removed and cells were washed with 100 µL of PBS then fixed in 4% paraformaldehyde for 10 min at rt
and stained with Hoechst nuclear stain at a final concentration of 10 µg/mL in PBS for 10 min at rt. After
fixing the cells were and stored in 100 µL of PBS for imaging. Wells were imaged with an inCell 2000
automated cell imager at 10x magnification with 9 images per well (complete coverage) in bright field
and DAPI channel fluorescence and images were analyzed for nuclei count by IN Cell developer
software. EC values were calculated in GraphPad Prism from normalized dose response curves.
50
Toxicity by CellTiter-Glo®: Cells were harvested, re-suspended and plated with a Wellmate liquid
handler (Thermo Scientific) into 384-well plates and cultured for 24 hours before dosing. Master
compound plates were made with a Janus (PerkinElmer), then further diluted to achieve uniform final
ACS Paragon Plus Environment

Page 19 of 36
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
concentrations of DMSO of 0.1% in media for all treatment conditions. Compound treatments in media
were added to the cell plates with a Matrix Platemate (Thermo Scientific). Cell viabilities were
determined 72 hours after treatment by Cell-Titer Glo Assay (Promega) on the Envision Multilabel Plate
Reader (Perkin Elmer). Relative Luminescent Units (RLU) were plotted against corresponding drug
concentrations and fitted with a standard four parameter sigmoidal curve with GraphPad Prism 6. Data
was further fit for EC shift parameters in GraphPad Prism 6 to determine EC ratios for 1/2. Data is
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
50
50
reported as the EC ratio, error bars represent s.e.m. (n=3). Data for cell lines in which EC ratio fits
50
50
2
were ambiguous, R values were less than 0.9, or response to the free drug was less than 40% was not
reported.
Quantitative PCR: Cells were seeded 300,000 cells per well in 6-well plates and grown to confluence
then collected with trypsin (0.05%), washed with PBS, and snap frozen in liq. N then stored at -78 °C.
2
Cell pellets were processed for mRNA isolation using Qiagen RNeasy Mini Kit with QIAshredder lysate
homogenizers and on column DNA digest. Isolated mRNA was analyzed for concentration and purity on
a ThermoScientific NanoDrop 2000c spectrophotometer and 1000 ng of mRNA from each sample was
translated to cDNA using Invitrogen SuperScript® III First-Strand Synthesis System. The resulting cDNA
®
was used for qRT-PCR analysis (10 ng/reaction) with SsoAdvanced™ Universal SYBR Green Supermix in a
Roche LightCycler 480. GAPDH was used as an endogenous control and relative mRNA levels were
calculated from a standard curve of pooled samples with LightCycler 480 software used for 2nd
derivative maximum analysis and standard curve fitting. Samples were prepared and run in biological
triplicates and error bars represent s.e.m. A list of the gene specific primers used is provided in the
Supporting Information file.
Maximum Tolerable Dose Studies: To evaluate the tolerability of the experimental agents, groups of
three NSG (NOD scid gamma) mice were treated with three i.p. injections of test article (Q4d or Q7d in
separate studies) in a formulation comprising 50:40:10 PEG 400:20% 2-hydroxypropyl-cylcodextrine in
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 36
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
water: DMSO. Individual groups (n = 3) received doses that increased in 2- or 3-fold steps until a given
dose caused one or more mice in the group to reach protocol limits for tolerance (>20% weight loss) at
any point post dosing.
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
Pharmacokinetics Studies: To evaluate the in vivo pharmacokinetic properties of the experimental
agents, female NSG mice were treated with a single i.p. injection of test compounds formulated in
50:40:10 PEG 400:20% 2-hydroxypropyl-cylcodextrine in water: DMSO. Blood samples were collected 5
min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h post dosing (3 mice were sampled per time point;
each group of mice was sampled ≤ 3 times over 24 h) and analyzed for plasma concentrations of each
compound via MS/MS analysis conducted by Integrated Analytical Solutions, Inc. (Berkeley, CA). The
resulting data was analyzed with WinNonlin software to calculate standard PK parameters.
Efficacy Studies: To evaluate the in vivo properties of the experimental agents, we used the
heterotopic indirect tumor xenograft model in nude mice (NCR nu/nu, Taconic) and SCID-beige mice.
Early passage PC-3 cells were harvested and a cell suspension (1:1 serum free DMEM: Matrigel) was
6
injected subcutaneously (s.c.) into the right flank of anesthetized donor nude mice (10 cells/mouse in
0
.1 ml). For MDA-MB-231 xenografts, cells were injected into the mammary fat pad of anesthetized
6
female SCID-beige mice (10 cells/mouse in 0.1 ml of PBS). When the mean tumor volume was 250-400
3
mm , tumor-bearing mice were treated with the indicated doses of compounds formulated in 50:40:10
PEG 400:20% 2-hydroxypropyl-cylcodextrine in water: DMSO via i.p. administration with the indicated
frequency. Tumor volume (by caliper) and mouse weight was monitored twice weekly.
AUTHOR INFORMATION
Corresponding Author
*
Phone: 415-514-9698; Fax: 415-514-4507; E-mail: adam.renslo@ucsf.edu.
ACS Paragon Plus Environment

Page 21 of 36
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Present Address
∫
present address: Prolynx LLC, San Francisco, CA, 94158
Author Contributions
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
B.S., J.A.W. and A.R.R designed compounds and conceived experiments. B.S. and S.D.F. synthesized
compounds. B.S., S.D.F, Y.S., and B.H. acquired data. All authors analyzed data. A.R.R. supervised the
project. B.S. and A.R.R wrote the manuscript. All authors reviewed, edited and commented on the
manuscript.
ASSOCIATED CONTENT
Supporting Information. Supplemental Tables, Figures and Schemes. Synthetic procedures and
compound characterization for new compounds. Animal Welfare statement and a list of gene-specific
primers used for qRT-PCR analysis. This material is available free of charge via the Internet at
http://pubs.acs.org.
ACKNOWLEDGMENT
A. R. Renslo acknowledges funding from the US National Institutes of Health (R01 Grant AI105106) and
from the NIH National Center for Advancing Translational Science (UCSF-CTSI Grant UL1 TR000004). B.
Spangler acknowledges funding from the NIH Research Training Grant in Chemistry and Chemical
Biology (T32 GM064337). The authors would like to thank Prof. S. Bandyopadhyay (UCSF) for providing
the MCF10A cell lines used in these studies, Mr. Steven Chen for technical support in automated cell
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 36
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
imaging and image analysis with InCell Developer software, Dr. Tetsuya Matsuguchi for his assistance
with qRT-PCR set up and analysis, Dr. Alan Wolfe for his assistance in calculating pharmacokinetic
parameters, and the Preclinical Therapeutic Core at UCSF where in vivo experiments were performed.
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
ABBREVIATIONS USED
TAP, tumor-activated prodrug; ADC, antibody-drug conjugate; TRX, a 1,2,4-trioxolane scaffold for
payload delivery; DXL, 1,3-dioxolane scaffold used as non-peroxidic controls; PURO, puromycin; qRT-
PCR, quantitative real-time polymerase chain reaction; CBI, cyclopropylbenzindoline; MTD, maximally
tolerated dose; Q4d, dosed every 4 days; Q7d, dosed every 7 days; ALT, alanine transaminase; AST,
aspartate transaminase; CPK, creatine phosphokinase; H&E, hematoxylin and eosin stained; CL,
clearance; F, fraction of drug in achieving systemic exposure following an i.p. dose; Vz, volume of
distribution; AUC, area under the curve.
REFERENCES
(
1)
Lorusso, P. M.; Edelman, M. J.; Bever, S. L.; Forman, K. M.; Pilat, M.; Quinn, M. F.; Li, J.; Heath, E.
I.; Malburg, L. M.; Klein, P. J.; Leamon, C. P.; Messmann, R. A.; Sausville, E. A. Phase I study of folate
conjugate EC145 (vintafolide) in patients with refractory solid tumors. J. Clin. Oncol. 2012, 30, 4011–
4
016.
(
(
2)
Beck, A.; Reichert, J. M. Antibody-drug conjugates: present and future. MAbs. 2014, 6, 15–17.
3)
Kratz, F.; Müller, I. A.; Ryppa, C.; Warnecke, A. Prodrug strategies in anticancer chemotherapy.
ChemMedChem 2008, 3, 20–53.
(
4)
Brown, J. M; Giaccia, A. J. The unique physiology of solid tumors: opportunities (and problems)
for cancer therapy. Cancer Res. 1998, 58, 1408–1416.
ACS Paragon Plus Environment

Page 23 of 36
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(
5)
Brown, J. M.; Wilson, W. R. Exploiting tumour hypoxia in cancer treatment. Nat. Rev. Cancer
2
004, 4, 437–447.
(6)
Torti, S. V.; Torti, F. M. Iron and cancer: more ore to be mined. Nat. Rev. Cancer 2013, 13, 342–
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
55.
(
7)
Chaturvedi, D.; Goswami, A.; Saikia, P. P.; Barua, N. C.; Rao, P. G. Artemisinin and its derivatives:
a novel class of anti-malarial and anti-cancer agents. Chem. Soc. Rev. 2010, 39, 435–454.
(
8)
O’Neill, P. M.; Posner, G. H. A medicinal chemistry perspective on artemisinin and related
endoperoxides. J. Med. Chem. 2004, 47, 2945–2964.
(
9)
Mercer, A. E.; Copple, I. M.; Maggs, J. L.; O’Neill, P. M.; Park, B. K. The role of heme and the
mitochondrion in the chemical and molecular mechanisms of mammalian cell death induced by the
artemisinin antimalarials. J. Biol. Chem. 2011, 286, 987–996.
(10)
Mercer, A. E.; Maggs, J. L.; Sun, X-M.; Cohen, G. M.; Chadwick, J.; O’Neill P. M.; Park, B. K.
Evidence for the involvement of carbon-centered radicals in the induction of apoptotic cell death by
artemisinin compounds. J. Biol. Chem. 2007, 282, 9372–9382.
(
11)
Dixon, SJ.; Stockwell, B. R. The role of iron and reactive oxygen species in cell death. Nat. Chem.
Biol. 2014, 10, 9–17.
(
12)
Pantopoulos, K. Iron metabolism and the IRE/IRP regulatory system: an update. Ann NY Acad.
Sci. 2004, 1012, 1–13.
(
13)
Wang, J.; Pantopoulos, K. Regulation of cellular iron metabolism. Biochem. J. 2011, 434, 365–
381.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 36
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
(
14)
Richardson, D. R.; Ponka, P. The molecular mechanisms of the metabolism and transport of iron
in normal and neoplastic cells. Biochim. Biophys. Acta. 1997, 1331, 1–40.
(15)
Boult, J.; Roberts, K.; Brookes, M. J.; Hughes, S.; Bury, J. P.; Cross, S. S.; Anderson, G. J.; Spychal,
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
R.; Iqbal, T.; Tselepis, C. Overexpression of cellular iron import proteins is associated with malignant
progression of esophageal adenocarcinoma. Clin. Cancer. Res. 2008, 14, 379–387.
(
16)
and cell responsiveness to H-ras-induced growth via expansion of the labile iron pool. Biochem. J. 2002,
36, 431–436.
Kakhlon, O.; Gruenbaum, Y.; Cabantchik, Z. Ferritin expression modulates cell cycle dynamics
4
(
17)
Pinnix, Z. K.; Miller, L. D.; Wang, W.; D’Agostino, R.; Kute, T.; Willingham, M. C.; Hatcher, H.;
Tesfay, L.; Sui, G.; Di, X.; Torti, S. V.; Torti, F. M. Ferroportin and iron regulation in breast cancer
progression and prognosis. Sci. Transl. Med. 2010, 2, 43ra56–43ra56.
(
18)
Wu, K.-J.; Polack, A., Dalla-Favera, R. Coordinated Regulation of Iron-controlling genes, H-ferritin
and IRP2, by c-MYC. Science 1999, 283, 676–679.
(
19)
Toyokuni, S. Role of iron in carcinogenesis: cancer as a ferrotoxic disease. Cancer Sci. 2009, 100,
9–16.
(20)
Miller, L. D.; Coffman, L. G.; Chou, J. W.; Black, M. A.; Bergh, J.; D’Agostino, R.; Torti, S. V.; Torti,
F. M. An iron regulatory gene signature predicts outcome in breast cancer. Cancer Res. 2011, 71, 6728–
737.
6
(
21)
Valecha, N.; Looareesuwan, S.; Martensson, A.; Abdulla, S. M.; Krudsood, S.; Tangpukdee, N.;
Mohanty, S.; Mishra, S. K.; Tyagi, P. K.; Sharma, S. K.; Moehrle, J.; Gautam, A.; Roy, A.; Paliwal, J. K.;
Kothari, M.; Saha, N.; Dash, A. P.; Björkman, A. Arterolane, a new synthetic trioxolane for treatment of
ACS Paragon Plus Environment

Page 25 of 36
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
uncomplicated Plasmodium falciparum malaria: a phase II, multicenter, randomized, dose-finding
clinical trial. Clin. Infect. Dis. 2010, 51, 684–691.
(22)
Borstnik, K.; Paik, I.; Shapiro, T. A.; Posner, G. H. Antimalarial chemotherapeutic peroxides:
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
artemisinin, yingzhaosu A and related compounds. Int. J. Parasitol. 2002, 32, 1661–1667.
(
23)
Vennerstrom, J. L.; Arbe-Barnes, S.; Brun, R.; Charman, S. A.; Chiu, F. C.; Chollet, J.; Dong, Y.;
Dorn, A.; Hunziker, D.; Matile, H.; McIntosh, K.; Padmanilayam, M.; Santo Tomas, J; Scheurer, C.;
Scorneaux, B.; Tang, Y.; Urwyler, H.; Wittlin, S.; Charman, W.N. Identification of an antimalarial synthetic
trioxolane drug development candidate. Nature. 2004, 430, 900–904.
(
24)
Valecha, N.; Krudsood, S.; Tangpukdee, N.; Mohanty, S.; Sharma, S. K.; Tyagi, P. K.; Anvikar, A.;
Mohanty, R.; Rao, B. S.; Jha, A. C.; Shahi, B.; Singh, J. P. N.; Roy, A.; Kaur, P.; Kothari, M.; Mehta, S.;
Gautam, A.; Paliwal, J. K.; Arora, S.; Saha, N. Arterolane maleate plus piperaquine phosphate for
treatment of uncomplicated plasmodium falciparum malaria: A comparative, multicenter, randomized
clinical trial. Clin. Infect. Dis. 2012, 55, 663–671.
(
25)
Creek, D. J.; Charman, W. N.; Chiu, F. C. K.; Prankerd, R. J.; Dong, Y.; Vennerstrom, J. L.; Charman,
S. A. Relationship between antimalarial activity and heme alkylation for spiro- and dispiro-1,2,4-
trioxolane antimalarials. Antimicrob. Agents Chemother. 2008, 52, 1291–1296.
(
26)
Creek, D.; Charman, W.; Chiu, F. C. K.; Prankerd, R. J.; Mccullough, K. J.; Dong Y.; Vennerstrom, J.
L.; Charman, S. A. Iron‐mediated degradation kinetics of substituted dispiro‐1, 2, 4‐trioxolane
antimalarials. J. Pharm. Sci. 2007, 96, 2945–56.
(
27)
Tang, Y.; Dong, Y.; Wang, X.; Sriraghavan, K.; Wood, J. K.; Vennerstrom, J. L. Dispiro-1,2,4-
trioxane analogues of a prototype dispiro-1,2,4-trioxolane: mechanistic comparators for artemisinin in
the context of reaction pathways with iron(II). J. Org. Chem. 2005, 70, 5103–5110.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 36
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
(
28)
Wang, X.; Creek, D. J.; Schiaffo, C. E.; Dong, Y.; Chollet J.; Scheurer C.; Wittlin, S.; Charman, S. A.;
Dussault, P. H.; Wood, J. K.; Vennerstrom, J. L. Spiroadamantyl 1,2,4-trioxolane, 1,2,4-trioxane, and
1,2,4-trioxepane pairs: relationship between peroxide bond iron(II) reactivity, heme alkylation efficiency,
and antimalarial activity. Bioorg. Med. Chem. Lett. 2009, 19, 4542–4545.
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
(
29)
Fontaine, S. D.; Dipasquale, A. G.; Renslo, A. R. Efficient and Stereocontrolled Synthesis of 1,2,4-
trioxolanes useful for ferrous iron-dependent drug delivery. Org. Lett. 2014, 16, 5776–5779.
(30)
Fontaine, S. D.; Spangler, B.; Gut, J.; Lauterwasser, E. M. W.; Rosenthal, P. J.; Renslo, A. R. Drug
delivery to the malaria parasite using an arterolane-like scaffold. ChemMedChem 2015, 10, 47–51.
(
31)
Mahajan, S. S.; Deu, E.; Lauterwasser, E. M. W.; Leyva, M. J.; Ellman, J. A.; Bogyo, M.; Renslo, A.
R. A fragmenting hybrid approach for targeted delivery of multiple therapeutic agents to the malaria
parasite. ChemMedChem 2011, 6, 415–419.
(
32)
Deu, E.; Chen, I. T.; Lauterwasser, E. M. W.; Valderramos, J.; Li, H.; Edgington, L. E.; Renslo, A. R.;
Bogyo, M. Ferrous iron-dependent drug delivery enables controlled and selective release of therapeutic
agents in vivo. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 18244–18249.
(33)
Lauterwasser, E. M. W.; Fontaine, S. D.; Li, H.; Gut, J.; Katneni, K.; Charman, S. A.; Rosenthal, P.
J.; Bogyo, M.; Renslo, A. R. Trioxolane-mediated delivery of mefloquine limits brain exposure in a mouse
model of malaria. ACS Med. Chem. Lett. 2015, 6, 1145–1149.
(
34)
A. R. A reactivity-based probe of the intracellular labile ferrous iron pool. Nat. Chem. Biol. 2016, 12, 680–
85.
Spangler, B.; Morgan, C. W.; Fontaine, S. D.; Vander Wal, M. N.; Chang, C. J.; Wells, J. A.; Renslo,
6
(
35)
Odlo, K.; Hentzen, J.; dit Chabert, JF.; Ducki, S.; Gani, O. A. B. S. M.; Sylte, I.; Skrede, M.; Florenes,
V. A.; Hansen, T. V. 1,5-Disubstituted 1,2,3-triazoles as cis-restricted analogues of combretastatin A-4:
ACS Paragon Plus Environment

Page 27 of 36
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Synthesis, molecular modeling and evaluation as cytotoxic agents and inhibitors of tubulin. Bioorg. Med.
Chem. 2008, 16, 4829–4838.
(36) Martins, M. M.; Zhou, A. Y; Corella, A.; Horiuchi, D.; Yau, C.; Rakshandehroo, T.; Gordan, J. D.;
Levin, R. S.; Johnson, J.; Jascur, J.; Shales, M.; Sorrentino, A.; Cheah, J.; Clemons, P. A.; Shamji, A. F.;
Schreiber, S. L.; Krogan, N. J.; Shokat, K. M.; McCormick, F. Gogo, A.; Bandyopadhyay, S. Linking tumor
mutations to drug responses via a quantitative chemical-genetic interaction map. Cancer Discov. 2015,
5, 154–167.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(
37) Radulescu, S.; Brookes, M. J.; Salgueiro, P.; Ridgway, R. A.; McGhee, E.; Anderson, K.; Ford, S. J.;
Stones, D. H.; Iqbal, T. H.; Tselepis, C.; Sansom, O. J. Luminal iron levels govern intestinal tumorigenesis
after Apc loss in vivo. Cell Rep. 2012, 2, 270–282.
(38)
dependent, nonapoptotic cell death in oncogenic-RAS-harboring cancer cells. Chem. Biol. 2008, 15,
34–245.
Yang, W. S.; Stockwell, B. R. Synthetic lethal screening identifies compounds activating iron-
2
(
39)
Boger, D. L.; Johnson, D. S. CC-1065 and the duocarmycins: unraveling the keys to a new class of
naturally derived DNA alkylating agents. Proc. Natl. Acad. Sci. U. S. A. 1995, 92, 3642–3649.
(
40)
Yang, S.; Denny, W. A. A new short synthesis of 3-substituted 5-amino-1-(chloromethyl)-1,2-
dihydro-3H-benzo[e]indoles (amino-CBIs). J. Org. Chem. 2002, 67, 8958–8961.
(
41)
Atwell, G. J.; Milbank, J. J.; Wilson, W. R.; Hogg, A.; Denny, W. A. 5-Amino-1-(chloromethyl)-1,2-
dihydro-3H-benz[e]indoles: relationships between structure and cytotoxicity for analogues bearing
different DNA minor groove binding subunits. J. Med. Chem. 1999, 42, 3400–3411.
(
42)
Jeffrey, S. C.; Torgov, M. Y.; Andreyka, J. B.; Boddington, L.; Cerveny, C. G.; Denny, W. A.;
Gordon, K. A.; Gustin, D.; Haugen, J.; Kline, T.; Nguyen, M. T.; Senter, P. D. Design, synthesis, and in vitro
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 36
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
evaluation of dipeptide-based antibody minor groove binder conjugates. J. Med. Chem. 2005, 48, 1344–
1
358.
(43)
McGovren, J. P.; Clark, G. L.; Pratt, E. A.; DeKoning, T. F. Preliminary Toxicity Studies with the
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
DNA-binding antibiotic, CC-1065. J. Antibiot. (Tokyo). 1984, 37, 63–70.
(
44)
Markovic, S. N.; Suman, V. J.; Vukov, A. M.; Fitch, T. R.; Hillman, D. W.; Adjei, A. A.; Alberts, S. R.;
Kaur, J. S.; Braich, T. A.; Leitch, J. M.; Creagan, E. T. Phase II trial of KW2189 in patients with advanced
malignant melanoma. Am. J. Clin. Oncol. 2002, 25, 308–312.
(45)
Workman, P. Genomics and the second golden era of cancer drug development. Mol. Biosyst.
2
005, 1, 17–26.
(
46)
Collins, I.; Workman, P. New approaches to molecular cancer therapeutics. Nat. Chem. Biol.
2
006, 2, 689–700.
(47)
Taylor, C. W.; Kim, Y. S.; Childress-Fields, K. E.; Yeoman, L. C. Sensitivity of nuclear c-Myc levels
and induction to differentiation-inducing agents in human colon tumor cell lines. Cancer Lett. 1992, 62,
5–105.
9
(
48)
Horiuchi, D.; Anderton, B.; Goga, A. Taking on Challenging Targets: Making Myc druggable. Am.
Soc. Clin. Oncol. Educ. B. 2014, 34, e497–502.
ACS Paragon Plus Environment

Page 29 of 36
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Table of Contents Graphic
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
traceless
linker
Fe(II)-reactive
endoperoxide
caged CBI
payload
HN CBI
vehicle
O
O
O
O
O
H N-CBI
2
TRX-CBI
TRX-CBI
ACS Paragon Plus Environment