Photoactivated enediynes as targeted antitumoral agents: Efficient routes to antibody and gold nanoparticle conjugates

Article abstract of DOI:10.1016/j.bmcl.2007.12.045

Efficient syntheses of a series of functionalized aryl enediynes have been developed. The building blocks were used to effect conjugation to carrier PEG templates which allowed subsequent coupling to a cardiac targeted monoclonal antibody. Immunocompetenc

Full text of DOI:10.1016/j.bmcl.2007.12.045

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 934–937
Photoactivated enediynes as targeted antitumoral agents:
Efficient routes to antibody and gold nanoparticle conjugates
Danielle Falcone, Jane Li, Amit Kale and Graham B. Jones*
Bioorganic and Medicinal Chemistry Laboratories, Department of Chemistry and Chemical Biology,
Northeastern University, Boston, MA 02115, USA
Received 29 October 2007; revised 18 December 2007; accepted 19 December 2007
Available online 7 January 2008
Dedicated to Professor Elias J. Corey on the occasion of his 80th birthday.
Abstract—Efficient syntheses of a series of functionalized aryl enediynes have been developed. The building blocks were used to
effect conjugation to carrier PEG templates which allowed subsequent coupling to a cardiac targeted monoclonal antibody. Immu-
nocompetence of the enediyne-Mab conjugates was demonstrated by ELISA, and both parent enediynes and bioconjugates under-
went successful photo-Bergman cyclization. Finally, surface modified (Au) nanoparticle conjugates were prepared and size
confirmed by TEM analysis. Application as long-circulating photoactivated prodrugs is anticipated.
Ó 2007 Elsevier Ltd. All rights reserved.
The enediynes are a class of potent antitumoral agents
isolated from soil bacteria.1 Over 20 of this class are
now known, two of which have entered clinical evalua-
tion. One of these (MylotargÒ) represents the first ever
monoclonal antibody–cytotoxin conjugate to be ap-
proved by the FDA and is currently used for treatment
of acute myeloid leukemia (AML).2 The natural enediy-
nes use a variety of elaborate triggering mechanisms
which activate the pharmacophore 1 via a cascade pro-
cess ultimately involving Bergman cycloaromatization,
to produce cytotoxic diyl radicals 2, which abstract H
atoms from cellular and nuclear macromolecules en
route to arenes 3 (Scheme 1).3 There has been consider-
able interest in the preparation of designed enediynes,
including the potential for photoactivated prodrugs.
The latter process, commonly referred to as the photo-
Bergman cyclization, has been studied in some depth,4
and parameters affecting photoconversion delineated.5
In order to extend the versatility of the enediynes as con-
trolled cytotoxins, we became interested in the possibil-
ity of assembling photo-Bergman precursors that could
be routinely derivatized, specifically for bioconjugation
chemistry.
Accordingly, a differentially substituted aryl enediyne 4
was firstly prepared from 1,2-di-iodobenzene via sequen-
tial coupling–deprotection (Scheme 2).6 Palladium med-
iated coupling with 4-iodobenzyl alcohol then gave
enediyne 5, reactions readily scaleable through multi-
gram level. The viability of 5 as a photo-Bergman sub-
strate was confirmed (vide infra) as was its shelf-life,
fidelity preserved at room temperature over a period
of weeks. With this building block in hand we elected
to demonstrate versatility via formation of bioconju-
gates under established coupling methods. We firstly
investigated preparation of a C-16 PEG conjugate 6.
PEGylation has become a commonly used strategy for
the delivery of various lipophilic drug candidates.7 En-
hanced circulatory capacity coupled with reported affin-
ity for specific tumor neovasculature suggest such
conjugates hold promise in a number of clinical applica-
tions.7 Conjugate 6 was formed in good yield via carbo-
diimide coupling of the PEG diacid, and displayed
marked solubility in buffered organic and aqueous solu-
tions. One of the expected properties of the conjugate (6)
was facile derivatization to form three component drug-
linker-antibody conjugates, and this was exemplified by
coupling to an available cardio-myosin targeting anti-
body (2-G4) viz. 7 via NHS coupling.8 The immuno-
competence and fidelity of the enediyne bioconjugate 7
was confirmed by ELISA (Fig. 1). To demonstrate ver-
satility of the enediyne building blocks we also devel-
oped a three component approach involving reductive
Keywords: Enediyne; Photo-Bergman; Bioconjugation; Antibody con-
jugate; Nanoparticles.
*
Corresponding author. Tel.: +1 617 373 8619; fax: +1 617 373
8795; e-mail: gr.jones@neu.edu
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.12.045

D. Falcone et al. / Bioorg. Med. Chem. Lett. 18 (2008) 934–937
935
H
H
2X-H 2X.
R
R
R
R
R
R
3
2
1
Scheme 1. The Bergman cycloaromatization of 3-hexene-1,5-diynes.
Ph
Ph
1. Pd(PPh3)2Cl2 CuI
n-BuNH2 (59%)
Pd(PPh3)2Cl2
CuI, Et3N (81%)
I
I
Ph
4-iodo benzyl
alcohol
2. Pd(PPh3)2Cl2
CuI, Et3N (57%)
TMS
3. TBAF, THF (100%)
4
H
5
OH
Ph
1. Pd(PPh3)2Cl2
CuI, Et3N
EDCI, DMAP, CH2Cl2
p-I-C6H4-CH2CO2Me (99%)
PEG diacid
(32%)
Ph
O
2. LiOH, THF/H2O
(90%)
O
O
O
OH
12
6
O
Ph
8
EDC, S-NHS
CO2H
2G4 mAb
(70%)
O
1. DCC, CH2Cl2 (96%)
O
2G4
O
O
12
N
H
O
H2N
O
7
n
O
Ph
MW=4000
MeO
OMe
2. Amberlyst-15
CH3COCH3, H2O (90%)
3. NaCNBH3, HEPES
pH 7.4 2G4 mAb
O
O
2G4
N
H
O
N
n
H
9
Scheme 2. Preparation, PEGylation, and bioconjugation of aryl enediyne photo-prodrugs.
ELISA results
differentially substituted (a-amino-x-acetal) PEG with
MW of approx 4000.9 The carboxaldehyde was liberated
using Amberlyst resin, then reductive amination of the
product in the presence of the 2G4 mAb produced bio-
conjugate 9 in >50% yield (based on recovered alde-
hyde). Identical strategy was successfully used to
prepare the bioconjugate of the related mAb 2G5 and
immunocompetency confirmed by ELISA.8
2
1.5
1
2G4
Conjugate
0.5
0
Concentration of Antibody
(ug/mL)
Figure 1. Relative immunoaffinity (7).
With the parent enediynes and two bioconjugates in
hand we were able to assess the impact of derivatization
on photo-Bergman cyclization. Gratifyingly, simple
irradiation of either 5, 6, 7, 8 or 9 resulted in smooth
amination. Specifically 4 was coupled to a phenylacetic
ester, and the (revealed) carboxylate 8 amidated with a

936
D. Falcone et al. / Bioorg. Med. Chem. Lett. 18 (2008) 934–937
Ph
Ph
Ph
hv 450W 3 hr
isopropanol
R
R
11
R
10
R = OH (5)
= O-PEG (6)
= O-PEG-2G4 (7)
= COOH (8)
Scheme 3. Photo-Bergman cyclization of enediyne core and bioconjugates.
and ease of derivatization using standard thiolate
coupling chemistry.12
Photochemical reaction
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
Mitsunobu coupling of 5 with thioacetic acid gave 12
without incident which underwent reduction to 13,
allowing for the Au derivative 14 to be produced via
the Brust two-phase method (Scheme 4).13 Compound
14 was evaluated using transmission electron micros-
copy, which revealed a particle size range of 3–5 nm
(Fig. 3), and fidelity of the conjugate was further sup-
ported by NMR spectroscopy. Preliminary analysis
suggests 14 to be viable in photo-Bergman cycloaro-
matization chemistry,14 and we are currently investigat-
ing the potential for mixed thiol adducts (including thiol
linked PEG-mAb conjugates), where the degree of ened-
iyne loading can be modulated.15
Compound 7
Compound 11
0
0
200
400
600
800
1000
Figure 2. UV/vis data for photolysis [7 (Ç) and 11 (D), R = 2G4] (y
axis = absorbance x axis = wavelength (nm)).
conversion to cyclized adducts 11 in buffered media, pre-
sumably via the intermediate diyl radicals 10 (Scheme 3),
adducts confirmed by LC–MS analysis (unoptimized
yields 18–35%). Conveniently UV–vis spectroscopy
proved an effective means to monitor reaction progress,
which in the case of 7 was complete within 3 h of irradi-
ation as evidenced by consumption of enediyne (Fig. 2).
Preliminary experiments involving incubation of 6 in the
presence of duplex rX174 DNA confirmed photocleav-
ing ability at lM concentrations.10,11 The observation
that both immunocompetence and photo-cyclization
capacity is retained in compound 7 suggests that numer-
ous applications in the design of targeted chemothera-
peutic agents and biochemical reagents may be
attainable.
To further demonstrate the versatility of building block
5 we opted to pursue development of a surface modified
(Au) nanoparticle conjugate. Au nanoparticles have re-
ceived significant attention due to their clinical tolerance
Figure 3. TEM image of gold nanoparticles 14 (y axis = absorbance x
axis = wavelength (nm)).
Ph
Ph
HAuCl4 3H2O
PPh3 (=NCO2i-Pr)2
5
CH3COSH
(58%)
NaBH4
12 R=COCH3
(82%)
LiAlH4
Et2O
S
Au
SR
13 R=H
14
Scheme 4. Preparation of Au modified nanoparticle–enediyne conjugate.

D. Falcone et al. / Bioorg. Med. Chem. Lett. 18 (2008) 934–937
937
6. All new compounds were characterized by appropriate
spectroscopic and analytical methods.
7. For overview see Torchilin, V. P. CHEMTECH 1999, 29,
27.
8. Torchilin, V. P.; Lukyanov, A. N.; Gao, Z.; Papahadjo-
poulos-Sternberg, B. Proc. Nat. Acad. Sci. U.S.A. 2003,
100, 6039.
9. Li, J.; Crasto, C. F.; Weinberg, J.; Amiji, M.; Shenoy, D.;
Sridhar, S.; Bubley, G.; Jones, G. B. Bioorg. Med. Chem.
Lett. 2005, 15, 5558.
In summary, simple readily available linear [aryl]
enediynes can be prepared and converted to versatile
and thermally stable bioconjugates. Subsequent photo-
activation of the systems is equally effective in parent
and (bio)conjugated state, thus allowing the develop-
ment of targeted cytotoxins and biochemical reagents.
The scope and in vitro applications of this technology
will be reported in due course.
10. rX174 RfI DNA (50 lM in base pairs) was irradiated
(Hanovia 450W medium pressure Hg lamp) in Tris–HCl at
pH 8.5 as control or with 6 at 10À6 M for 3 h at 37 °C.
Crude mixtures were subjected to gel electrophoresis (10%
agarose), stained with ethidium bromide, and then sub-
jected to scanning densitometry. Correcting for control
photodegradation, 6 largely induced conversion to type II
DNA (24%), with the onset of minimal conversion to type
III based on random single-stranded cutting events. Full
details, including competition experiments with DNA
binders, will be reported in due course.
Acknowledgments
We thank the NIH (RO1GM57123) and PRF (33920-
AC1) for financial support of this work and Dmitriy
Mongayt for performing ELISAs on 7 and 9.
References and notes
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diradicals, the possibility for damage via a C1–C5
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