Hydroxyurea derivatives of irofulven with improved antitumor efficacy

Article abstract of DOI:10.1016/j.bmcl.2016.02.028

Irofulven is a semi-synthetic derivative of Illudin S, a toxic sesquiterpene isolated from the mushroom Omphalotus illudens. Irofulven has displayed significant antitumor activity in various clinical trials but displayed a limited therapeutic index. A new derivative of irofulven was prepared by reacting hydroxyurea with irofulven under acidic conditions. Acetylation of this new compound with acetic anhydride produced a second derivative. Both of these new derivatives displayed significant antitumor activity in vitro and in vivo comparable to or exceeding that of irofulven.

Full text of DOI:10.1016/j.bmcl.2016.02.028

Accepted Manuscript
Hydroxyurea derivatives of irofulven with improved antitumor efficacy
Michael D. Staake, Alisala Kashinatham, Trevor C. McMorris, Leita A. Estes,
Michael J. Kelner
PII:
S0960-894X(16)30140-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.02.028
BMCL 23579
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
9 December 2015
9 February 2016
10 February 2016
Please cite this article as: Staake, M.D., Kashinatham, A., McMorris, T.C., Estes, L.A., Kelner, M.J., Hydroxyurea
derivatives of irofulven with improved antitumor efficacy, Bioorganic & Medicinal Chemistry Letters (2016), doi:
http://dx.doi.org/10.1016/j.bmcl.2016.02.028
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Hydroxyurea derivatives of irofulven with improved antitumor efficacy
Michael D. Staake, Alisala Kashinatham, Trevor C. McMorris, Leita A. Estes, and Michael J. Kelner

Bioorganic & Medicinal Chemistry Letters
Hydroxyurea derivatives of irofulven with improved antitumor efficacy
a
a
a
b
b,
Michael D. Staake , Alisala Kashinatham , Trevor C. McMorris , Leita A. Estes , and Michael J. Kelner
a
Department of Chemistry & Biochemistry, University of California, San Diego, La Jolla, CA 92093-0358, USA
b
Department of Pathology, University of California, San Diego, MC 7721, La Jolla CA 92093-7721 USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Irofulven is a semi-synthetic derivative of Illudin S, a toxic sesquiterpene isolated from the
mushroom Omphalotus illudens. Irofulven has displayed significant antitumor activity in
various clinical trials but displayed a limited therapeutic index. A new derivative of Irofulven
was prepared by reacting hydroxyurea with irofulven under acidic conditions. Acetylation of
this new compound with acetic anhydride produced a second derivative. Both of these new
derivatives displayed significant antitumor activity in vitro and in vivo comparable to or
exceeding that of Irofulven.
Keywords:
Illudin S
Irofulven
Hydroxyurea
Xenograft
2009 Elsevier Ltd. All rights reserved.
Analog LP-184
The toxic mushroom Omphalotus illudins is the source of the
maximum dose that can be administered to patients. Modifying
the drug to increase its selectivity towards tumor cells versus
normal cells should in theory reduce adverse side effects and
allow administration of higher doses. We describe here the
preparation of two new hydroxyurea derivatives of irofulven with
improved in vitro antitumor activity.
highly toxic sesquiterpenes illudin S 1 and illudin M 2 (Figure
1
1
). These compounds were tested by the National Cancer
Institute and displayed significant antitumor activity but
2
displayed a poor therapeutic index in xenograft studies.
Semisynthetic derivatives of these compounds were developed
which displayed improved efficacy in xenograft studies including
3
4
The drug hydroxyurea 4 (Figure 2) is an inhibitor of DNA
multidrug resistant tumor models. One derivative, irofulven 3
7
synthesis and has been used clinically to treat cancer.
has been extensively investigated in numerous clinical trials and
displayed significant activity against ovarian, prostate, and
Hydroxyurea, however, has limited efficacy as an anticancer drug
as it displays a short half-life in humans and cancer cells readily
develop resistance to the drug. The hydroxyurea derivative
BWB70C 5 was developed and demonstrated superior inhibition
5,6
gastrointestinal cancers including hepatocellular tumors.
8
of tumor growth in vivo.
Figure 1. Structures of Illudin S, Illudin M, and Irofulven
Figure 2: Structures of hydroxyurea and BWB70C
9
Another hydroxyurea derivative, Zileuton 7 has been prepared
Irofulven has proven to be a potent antitumor agent, but its
efficacy is limited by the hematologic side effects that reduce the
N
from benzo[b]thiophen-2-yl-ethanol 6 via an S 1 displacement of
the secondary hydroxyl by hydroxyurea under acidic conditions
—
——

Corresponding author. Tel.: +1-858-657-5731; fax: +1-858-657-5807; e-mail: mkelner@ucsd.edu

1
0
(
Scheme 1). Alkylation occurred specifically at the nitrogen
Table 1: Solid tumor IC50 values (nM) for irofulven and
hydroxyurea derivative 8 from the NCI DTP 60 cell line analysis.
bearing the hydroxyl group.
*
designates equal or increased cytotoxicity by derivative 8.
Log10 IC50
Panel/Cell Line
Non-small cell lung
A549
Irofulven
Compound 8
-6.9
-6.7
-6.8
-6.5
-6.7
-7.1
-6.6
-7.3
-6.8
< -8.0 *
-6.5
EKVX
HOP-62
HOP-92
-7.6 *
-7.4 *
-6.7 *
-7.8 *
-7.0 *
< -8.0 *
-6.0
Scheme 1
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon
We followed an analogous approach to obtain the hydroxyurea
derivative of irofulven. The primary hydroxyl group of irofulven
was previously demonstrated to undergo S
variety of nucleophiles under acidic conditions.
mixture of 2M H SO and acetone the primary hydroxyl group of
irofulven was rapidly replaced by hydroxyurea to yield 8 in 83%
yield (Scheme 2). Alkylation was also observed to occur
exclusively at the nitrogen bearing the hydroxy group. An
acetylated derivative 9 was obtained in quantitative yield by
treating 8 with acetic anhydride and catalytic DMAP.
N
1 displacement by a
11,12
In a 1:1
2
4
COLO 205
HCC-2998
HCT-116
HCT-15
-6.8
-6.8
-6.7
-6.5
-6.6
-6.2
-6.2
-7.6 *
-7.9 *
-6.8 *
-6.9 *
-6.9 *
-6.4 *
-4.8
HT29
KM12
SW-620
Scheme 2
CNS
The antitumor activity of compound 8 was compared to previous
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
-6.6
-6.5
-6.8
-6.5
-6.7
-6.5
-7.7 *
-7.3*
-7.7 *
-6.9 *
-7.5 *
-6.9 *
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
Ovarian
-6.2
-6.7
-6.6
-5.8
-6.7
-6.2
-6.8
-6.6
-6.5 *
-6.9 *
-6.7*
-6.4 *
-6.4
-6.9 *
-7.2 *
-6.6 *
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal
-6.8
-6.6
-6.6
-6.8
-6.7
-6.6
-6.6
-7.6 *
-7.3 *
-7.0 *
-7.6 *
-6.5
irofulven results in the NCI DTP 60 cell line screening assay.
Results indicated that anticancer activity of compound 8 equaled
or exceeded that of irofulven (Table 1) in 43 of 52 comparable
cell lines (cell lines used in the screening assay have varied with
time so that only 52 of the solid tumor lines were tested with both
irofulven and compound 8). Compound 8 retained activity
against multidrug resistant cell lines, such as Ovarian NCI/ADR-
RES, consistent with previous reports on the activity of
-6.8 *
-6.5
786-O
-6.7
-6.7
-6.6
-6.6
-7.3
-6.4
-6.6
-6.7
-6.9 *
-7.5 *
-7.5 *
-7.3 *
-7.8 *
-6.5 *
-7.5 *
-7.6 *
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
3
acylfulvenes against multidrug resistant phenotypes.
The antitumor activities of compounds 8 and 9 were then
compared against that of irofulven using the MV522
adenocarcinoma cell line as the target cell line because these cells
have previously been demonstrated to be resistant to a wide
UO-31
Prostate
Breast
13
PC-3
DU-145
-6.6
-7.0
-6.4
< -8.0 *
variety of conventional chemotherapeutic agents.
The
megakaryocytic cell line CHRF 288-11 was chosen as the non-
target cell line as the primary toxicity of irofulven is
14
MCF-7
-6.6
-6.5
-6.5
-6.5
-6.7
-6.38
-5.9
-6.6 *
-6.3
-6.8 *
-7.2 *
-7.02 *
thrombocytopenia. The in vitro cytotoxicity assay has been
15
MDA-MB-231
HS 578T
BT-549
previously described.
While the hydroxyurea derivatives
retained cytotoxicity towards the target MV522 adenocarcinoma
cell line, the analogs were markedly less toxic to the
megakaryocytic CHRF 288-11 cell line (Table 2).
T-47D
Mean

It is not entirely clear why the hydroxyurea derivatives 8 and 9 of
irofulven display an improved therapeutic index towards solid
tumor cells and not cells of a megakaryocytic lineage. One could
argue that these derivatives are simply functioning as an
equimolar mixture of irofulven and hydroxyurea, however,
hydroxyurea is nontoxic to MV522 cells (IC50 > 650 μM).
Further biological studies will be required to clarify the
mechanism of increased therapeutic ratio for the hydroxyurea
derivatives of irofulven.
Table 2: IC50 values (nM) for irofulven and hydroxyurea
derivatives when tested against MV522 adenocarcinoma cells
a
and CHRF 288-11 megakaryocytic cells.
b
Compound
MV522
70 ± 10
CHRF 288-11
290 ± 20
Ratio
4.1
Irofulven 3
8
9
210 ± 20
130 ± 10
8,800 ± 1,700
1,900 ± 100
41.9
14.6
a
IC50 is the concentration of the irofulven derivative at which
0% inhibition of growth occurs as measured by the trypan blue
Acknowledgements
5
15
This investigation was supported in part by funds provided by
MGI PHARMA, Inc., Minneapolis, Minnesota.
exclusion assay for a 48 hour exposure.
b
The ratio is defined as the CHRF IC50 value/MV522 IC50 value
Supplementary data
Based on the screening results with MV522 and CHRF 288-11
cells, compound 8 was then tested in the MV522 xenograft
model. As a comparator irofulven was tested at 10 mg/kg, the
maximum tolerated dose when administered intraperitoneally at 3
doses per week for 3 weeks. Compound 8 was less toxic than
irofulven, in agreement with the screening results (Table 2) and
could be administered at higher doses (Figure 3). The in vivo
antitumor activity of compound 8 was superior to irofulven when
administered at an equivalent dose of 10 mg/kg (the maximum
tolerated dose for irofulven), and compound 8 could be
administered at a twofold higher dose than irofulven.
1
13
Experimental details for the synthesis and H-NMR and C-NMR
spectra of compounds 8 and 9 can be found in the online version.
Compound 8 has been selected for phase I clinical trials and now
carries the drug designation LP-184 for these trials.
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1
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