The synthesis of a c(RGDyK) targeted SN38 prodrug with an indolequinone structure for bioreductive drug release

Article abstract of DOI:10.1021/ol1002626

(Figure Presented) Preparation of a novel c(RGDyK) targeted SN38 prodrug incorporating an indolequinone structure for bloreductively triggered drug release is described. This design yields a prodrug that targets surface molecules on tumor cells (a_v

Full text of DOI:10.1021/ol1002626

ORGANIC
LETTERS
2010
Vol. 12, No. 7
1384-1387
The Synthesis of a c(RGDyK) Targeted
SN38 Prodrug with an Indolequinone
Structure for Bioreductive Drug Release
Baohua Huang, Ankur Desai, Shengzhuang Tang, Thommey P. Thomas, and
James R. Baker, Jr.*
Michigan Nanotechnology Institute for Medicine and Biological Sciences,
UniVersity of Michigan, Ann Arbor, Michigan 48109
jbakerjr@med.umich.edu
Received November 10, 2009
ABSTRACT
Preparation of a novel c(RGDyK) targeted SN38 prodrug incorporating an indolequinone structure for bioreductively triggered drug release is
described. This design yields a prodrug that targets surface molecules on tumor cells (rvꢀ3 integrins) and releases drug under bioreductive
conditions. There are three moieties in the prodrug design, namely a therapeutic drug SN38, an indolequinone structure serving as a drug
releasing trigger, and an rvꢀ3 integrin targeting peptide c(RGDyK). Preliminary studies showed that SN38 is released in the presence of a
bioreductive enzyme (DT-diaphorase).
There is a tremendous interest in using targeted drug
delivery for cancer therapies.1 Targeted delivery can
minimize the undesirable drug side effects while retaining
desirable therapeutic activities. Advances in molecular
biology have facilitated the identification of tumor markers
that can function as potential therapeutic targets.2,3 An
example of this is the identification of molecular markers
that can differentiate newly formed capillaries from their
mature counterparts, providing a strategy for targeted delivery
of cytotoxic agents to the tumor vasculature.4,5 The Rvꢀ3
integrin is one of the most specific markers of tumor-
associated vasculature. This marker can be recognized by
targeting agents that are restricted to the vascular space
during angiogenesis. Cyclic pentapeptide containing RGD
is highly selective for Rvꢀ3 and the D amino acid following
Asp in the pentapeptide is essential for the 1000 times higher
activity in comparison to the linear RGD.6-8 Because of this,
there has been growing interest in cyclic RGD targeted gene
delivery,9 imaging applications,10 and tumor therapy.11
(4) Baillie, C. T.; Winslet, M. C.; Bradley, N. J. Br. J. Cancer 1995, 72,
257
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Kessler, H. Febs Lett. 1991, 291, 50
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A.; Kessler, H. J. Am. Chem. Soc. 1996, 118, 7461
(8) Pfaff, M.; Tangemann, K.; Mueller, B.; Gurrath, M.; Mueller, G.;
Kessler, H.; Timpl, R.; Engel, J. J. Biol. Chem. 1994, 269, 20233
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(1) Han, H. K.; Amidon, G. L. AAPS Pharmsci. 2000, 2 (1), 48.
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10.1021/ol1002626  2010 American Chemical Society
Published on Web 03/01/2010

Scheme 1. Synthesis of Non-Targeted SN38 Prodrug
Tissue hypoxia due to inadequate blood supply is a
common feature of solid tumors. Unfortunately, hypoxic
tumor cells appear to be resistant to both radiotherapy and
chemotherapy.12 However, tumor hypoxia provides a unique
strategy for cancer therapy. Several therapeutics have been
designed to form prodrugs which can be activated by hypoxia
under bioreductive conditions.13,14 In this regard, prodrugs
with indolequinone structures have been studied inten-
sively.15,16 There are two mechanisms through which in-
dolequinone-based prodrugs can induce cytotoxicity. First,
the indolequinone structure itself can be converted to a
reactive, cytotoxic species following reduction to a hydro-
quinone.17 Alternatively, the indolequinone moiety can be
used to form a prodrug that selectively releases other
cytotoxic agents to hypoxic (i.e., bioreductive) tissues.15
SN38 belongs to the class of 20(s)-camptothecin (CPT)
group of compounds that act as potent topoisomerase I
inhibitors. Due to its general toxicity and poor solubility,
SN38 cannot be systemically administered to cancer patients.
Numerous modifications have been made to enhance the
drugs solubility including liposomal formulation,18 antibody
conjugation,19 and PEG functionalization.20 CPT-11 (Irino-
tecan) is a SN38 derivative and clinically approved for the
treatment of colorectal carcinoma.21 However, only 2-5%
of of the injected dose of CPT-11 is converted to active SN38
and the drug has serious side effects including gastrointestinal
toxicity and neutropenia.22
the prodrug will be water-soluble due to the presence of the
targeting peptide and the short polyethylene glycol tether.
Second, the prodrug will be specifically delivered to cells
that overexpress the Rvꢀ3 integrin.11 Finally, the active drug
SN38 will be released under bioreductive conditions pre-
sented in tumor tissues.24 This design will enhance drug
specificity toward tumor cells that overexpress the Rvꢀ3
integrin and thus reduce side effects.
The synthesis of the nontargeted SN38 prodrug 12 is
demonstrated in Scheme 1. The N-1 methyl analogue of the
indolequinone structure has been synthesized by Naylor et
(9) Kunath, K.; Merdan, T.; Hegener, O.; Haberlein, H.; Kissel, T.
J. Gene Med. 2003, 5, 588.
(10) Haubner, R.; Bruchertseifer, F.; Bock, M.; Kessler, H.; Schwaiger,
M.; Wester, H. Nuklearmedizin-Nuclear Med. 2004, 43, 26.
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J. R. Chem. Commun. 2005, 46, 5739.
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Denny, W. A. J. Med. Chem. 1996, 39, 2508
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P. Free Radical Res. 1996, 25, 99
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Patel, K. B.; Everett, S. A.; Adams, G. E.; Stratford, I. J. J. Med. Chem.
1997, 40, 2335
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Hansen, H. J.; Goldenberg, D. M. J. Med. Chem. 2008, 51, 6916.
(20) Zhao, H.; Rubio, B.; Sapra, P.; Wu, D. C.; Reddy, P.; Sai, P.;
Martinez, A.; Gao, Y.; Lozanguiez, Y.; Longley, C.; Greenberger, L. M.;
Horak, I. D. Bioconjugate Chem. 2008, 19, 849.
Here we report the design, synthesis and preliminary drug
release study of a cyclic RGDyK (denoted by c(RGDyK))23
targeted SN38 prodrug possessing an indolequinone structure
for bioreductively triggered drug release (compound 1,
Scheme 3). There are three moieties in the prodrug design,
namely a therapeutic drug SN38, an indolequinone structure
serving as a drug releasing trigger, and an Rvꢀ3 integrin
targeting peptide c(RGDyK). We envision that this design
will impart the following advantages over the free drug. First,
(21) Vanhoefer, U.; Harstrick, A.; Achterrath, W.; Cao, S. S.; Seeber,
S.; Rustum, Y. M. J. Clin. Oncol. 2001, 19, 1501.
(22) Oreilly, S.; Rowinsky, E. K. Crit. ReV. Oncol. Hemat. 1996, 24, 47.
(23) Auernheimer, J.; Haubner, R.; Schottelius, M.; Wester, H. J.;
Kessler, H. HelV. Chim. Acta 2006, 89, 833.
(24) Everett, S. A.; Swann, E.; Naylor, M. A.; Stratford, M. R. L.; Patel,
K. B.; Tian, N.; Newman, R. G.; Vojnovic, B.; Moody, C. J.; Wardman, P.
Biochem. Pharmacol. 2002, 63, 1629.
Org. Lett., Vol. 12, No. 7, 2010
1385

al. Extensive structure-activity studies have been per-
formed.15 We use an ethoxycarbonyl methyl functional group
to replace the methyl group at the N-1 position. This synthetic
design allows the N-1 site to serve as an open reaction
position to facilitate the coupling of a long chain tether with
an azido functional group. The azido group is then reacted
with an alkyne modified targeting moiety via click chemistry.
Commercially available 5-methoxy-2-methylindole was first
treated with Vilsmeier reagents to give 3-formyl compound
2 with excellent yield. Alkylation of position 1 was then
carried out with sodium hydride and ethyl bromo-acetate.
Nitration at the desired position 4 could be achieved at a
low reaction concentration and with a large excess of nitric
acid. However, a 6-nitro isomer was present at a low ratio
(about 15%). The mixture of 6-nitro and 4-nitro had a very
poor solubility in organic solvents, making it impossible to
purify through chromatography. The mixture was reduced
with Sn/HCl to give a mixture of 4- and 6-amine products.
The desired 4-amino compound 5 was then purified using
chromatography.
Scheme 2. Modification of c(RGDyK) Peptide
activated with TSTU and reacted with the c(RGDyK). The
resulting mixture was purified with semipreparative HPLC.
The collected fractions were lyophilized to give 14 (66%
yield) with a retention time of 17.4 min on analytical HPLC.
The synthesis of c(RGDyK) targeted SN38 prodrug is
demonstrated in Scheme 3. After the click reaction, the
Fremy’s salt was used to covert the aromatic ring to a
quinone structure with quantitative yield. The crucial step
in this process is the reduction of the 3-carboxaldehyde to a
hydroxyl group because several other sites in the structure
can also be reduced, particularily the ethyl ester. Selective
reduction of the aldehyde rather than the ester could only
be achieved by using 5 equiv of NaBH4 at 0 °C over 8 min
and the yield of this reduction was 67%. Higher temperatures
and prolonged reaction times decrease the yield drastically
by reducing other structures. Ethyl ester 7 was then hydro-
lyzed with 1 equivalent of LiOH to give carboxylic acid
compound 8. Compound 8 was then coupled with 11-azido-
3,6,9-trioxaundecan-1-amine with HATU to give compound
9. First we activated the 3-methylhydoxyl of compound 9
with 4-nitrophenyl chloroformate and let it reacted with the
phenolic hydroxyl of SN38 directly via a carbonate bond.
However, a preliminary stability study showed that the
carbonate bond is not stable in PBS buffer, releasing the drug
molecule within 2 h (data not shown). To enhance the
stability of the prodrug, a small diamine spacer was
incorporated between the indolequinone structure and the
drug molecule that forms two, more stable carbamate bonds.
Moreover, the spacer can potentially reduce steric interfer-
ence from the drug on the linker, thus enhancing the release
rate when hypoxia is present.25 To achieve this, the activated
4-nitrophenyl carbonate of compound 9 was condensed with
an excess of N,N′-dimethylethylene diamine to give com-
pound 10. Remarkably, only 3 equivalents of diamine was
used in the reaction and there was minimal dimerization.
The secondary amine of compound 10 was then converted
to a carbamoyl chloride with triphosgene, which was
subsequently reacted with SN38 to form the nontargeted
prodrug 12 with good yield.
Scheme 3. Synthesis of c(RGDyK) Targeted SN38 Prodrug
desired product 1 was purified using a semipreparative
HPLC. Compound 1 was lyophilized as a brownish red fluffy
solid (48% yield). The retention time of 1 is 25.1 min on an
analytical HPLC. Electrospray mass spectrum gave peaks
at 1782.1 (M + H) and 902.5 (M + H + Na) (see Supporting
Information).
The release of SN38 from prodrug 1 in the presence of
DT-diaphorase (DTD) was tested. DTD is an obligate two
electron quinine reductase that uses either NADH or NADPH
as electron donors. DTD has received great deal of attention
as a target for anticancer prodrug development and has been
implicated in the bioreductive activation of quinone-contain-
ing compounds.26 Upon reduction, the electron density at
the indole nitrogen increases drastically, triggering expulsion
of the exiting group in a reverse-Michael-like process,
releasing the drug coupled to a small spacer. The ability of
The synthesis of alkyne modified c(RGDyK) is shown in
Scheme 2. A bifunctional small linker 13 was prepared from
propargyl amine and glutaric anhydride. 13 was then
(26) Phillips, R. M.; Naylor, M. A.; Jaffar, M.; Doughty, S. W.; Everett,
S. A.; Breen, A. G.; Choudry, G. A.; Stratford, I. J. J. Med. Chem. 1999,
42, 4071.
(25) Saari, W. S.; Schwering, J. E.; Lyle, P. A.; Smith, S. J.; Engelhardt,
E. L. J. Med. Chem. 1990, 33, 97.
1386
Org. Lett., Vol. 12, No. 7, 2010

DTD to activate certain quinones, in conjunction with the
fact that certain tumor types contain elevated levels of DTD,
suggests that it could mediate hypoxic induced, enzyme-
directed bioreductive drug delivery.27 SN38 release in the
presence of DTD was characterized and monitored using
HPLC. The results showed that in the presence of DTD,
SN38 was efficiently released from the prodrug. (Figure 1)
Figure 2. Cytotoxicity of 12 in the presence of DTD.
whereas there was ∼50-70% decrease in cell growth in the
presence of DTD.
To summarize, we have synthesized a novel c(RGDyK)
targeted SN38 prodrug with an indolequinone structure for
bioreductive drug release. Preliminary studies showed that
free drug can be released from prodrug 1 in the presence of
DTD. This indicates that prodrug 1 can be used to deliver
anticancer drugs specifically under bioreductive conditions.
The in Vitro toxicity studies of prodrug 1 toward cancer cell
lines expressing the Rvꢀ3 integrin receptor under hypoxia
conditions are under investigation.
Figure 1. Release of SN38 from prodrug 1 under DTD.
The prodrug hydrolysis over the same time frame in PBS
without the enzyme was very limited. We further tested if
12 acts as a prodrug (minimally active) and if the released
SN38 is still active. For this, we tested the cytotoxicity of
12 in the human cervical carcinoma KB cells before and
after treatment with DTD. We incubated 12 with recombinant
human DTD (2mU/µL) added into the cell growth medium,
in presence of different concentration of 12 for three days
and cytotoxicity was determined by XTT assay. As shown
in Figure 2, in the absence of DTD, the compound was
essentially noncytotoxic to up to ∼300 nM concentration.,
Acknowledgment. This project has been funded in whole
or in part with Federal funds from the National Cancer
Institute, National Institutes of Health, under award 1 R01
CA119409.
Supporting Information Available: Detailed preparetive
procedures, analytical data. This material is available free
of charge via the Internet at http://pubs.acs.org.
(27) Workman, P. Oncol. Res. 1994, 6, 461.
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