Curcumin analog cytotoxicity against breast cancer cells: exploitation of a redox-dependent mechanism

Article abstract of DOI:10.1016/j.bmcl.2009.10.023

A series of novel curcumin analogs, symmetrical dienones, were previously shown to possess cytotoxic, anti-angiogenic and anti-tumor activities. Analogs 1 (EF24) and 2 (EF31) share the dienone scaffold and serve as Michael acceptors. We propose that the anti-cancer effects of 1 and 2 are mediated in part by redox-mediated induction of apoptosis. In order to support this concept, 1 and 2 were treated with l-glutathione (GSH) and cysteine-containing dipeptides under mild conditions to form colorless water-soluble adducts, which were identified by LC/MS. Comparison of the cytotoxic action of 1, 2 and the corresponding conjugates, 1-(GSH)₂ and 2-(GSH)₂, illustrated that the two classes of compounds exhibit essentially identical cell killing capabilities. Compared with the yellow, somewhat light sensitive and nearly water insoluble compounds 1 and 2, the glutathione conjugates represent a promising new series of stable and soluble anti-tumor pro-drugs.

Full text of DOI:10.1016/j.bmcl.2009.10.023

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6627–6631
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Curcumin analog cytotoxicity against breast cancer cells: exploitation
of a redox-dependent mechanism
b
a
b
a
b,
Aiming Sun a, , Yang J. Lu , Haipeng Hu , Mamoru Shoji , Dennis C. Liotta , James P. Snyder
*
*
a Department of Chemistry, Emory University, 1510 Clifton Road, Atlanta, GA 30322, United States
b Winship Cancer Institute, Emory University, Atlanta, GA 30322, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel curcumin analogs, symmetrical dienones, were previously shown to possess cytotoxic,
anti-angiogenic and anti-tumor activities. Analogs 1 (EF24) and 2 (EF31) share the dienone scaffold and
serve as Michael acceptors. We propose that the anti-cancer effects of 1 and 2 are mediated in part by
Received 11 August 2009
Revised 5 October 2009
Accepted 5 October 2009
Available online 8 October 2009
redox-mediated induction of apoptosis. In order to support this concept, 1 and 2 were treated with L-glu-
tathione (GSH) and cysteine-containing dipeptides under mild conditions to form colorless water-soluble
adducts, which were identified by LC/MS. Comparison of the cytotoxic action of 1, 2 and the correspond-
ing conjugates, 1-(GSH)2 and 2-(GSH)2, illustrated that the two classes of compounds exhibit essentially
identical cell killing capabilities. Compared with the yellow, somewhat light sensitive and nearly water
insoluble compounds 1 and 2, the glutathione conjugates represent a promising new series of stable
and soluble anti-tumor pro-drugs.
Keywords:
Curcumin analogs
Glutathione
Anti-cancer
Pro-drug
Ó 2009 Elsevier Ltd. All rights reserved.
Curcumin (diferuloylmethane) is a
a
,b-diketone constituent of
apoptosis13 and block the growth of human breast tumors in a
mouse xenograft model with relatively low toxicity.14 Our most re-
turmeric with antioxidant properties. It has been used in tradi-
tional medicine for liver disease, indigestion, urinary tract infec-
tions, rheumatoid arthritis, and insect bites.1 This phytochemical
has also demonstrated both anti-cancer and anti-angiogenic prop-
erties.2,3 Among other things, curcumin blocks several important
cent study identified I-
both compound 1 and curcumin, although the latter is less potent.
Compound 1 rapidly blocks the nuclear translocation of NF-
with an IC50 of 1.3 M compared with curcumin with an IC50 of
13
M.15 In spite of the higher activity of 1 (EF24) and 2 (EF31)
j B kinase (IKKb as an effective target for
j
B
l
cellular targets such as nuclear factor
j
-B (NF-
j
B).4–6 This interac-
l
tion, in turn, induces apoptosis and retards the function of protein
kinase C, epidermal growth factor receptor tyrosine kinase and hu-
man epidermal growth factor receptor-2 (HER-2).7,8 Recent thera-
peutic efficacy against pancreatic cancer in a phase II clinical trial
further supports the use of curcumin as a lead for the development
of a new class of anti-cancer agents.9 Unfortunately, due to the low
potency and poor absorption characteristics of curcumin, its clini-
cal potential may prove to be limited.10 Nonetheless, the com-
pound remains an ideal lead compound for design of derivatives
with improved water solubility.11
About 100 curcumin analogs have been synthesized in our lab-
oratory and tested for anti-cancer and anti-angiogenesis proper-
ties.12 A subset of 10 was further evaluated in the 60 panel NCI
cancer cell lines and in several in vitro anti-angiogenesis screens.
Analog 1 with ortho-fluoro groups and its 2-pyridine analog 2 ex-
hibit superior cytotoxic activities compared with other members
of the series (Fig. 1). Analog 1 has been shown to inhibit the growth
of cancer cells at a ca. 10-fold lower dose than curcumin,12 induce
by comparison with curcumin, the low bioavailability and fast
metabolism of these analogs still remains a critical problem for fur-
ther development.
Our earlier study showed that 1 induces cell cycle arrest and
apoptosis by means of a redox-dependent process in MDA-MB-
231 human breast cancer and DU-145 human prostate cancer
cells.13 Compound 1, containing a dienone moiety, serves as a Mi-
chael acceptor to deplete L-glutathione (GSH) and GSSG concentra-
tions in both wild type and Bcl-xL overexpressing HT29 human
colon cancer cells. Comparable chemistry is utilized by a series of
novel tyrosine kinase inhibitors developed by Smaill and co-work-
ers, in which a mild Michael acceptor is appended to a quinazoline
H
O
O
O
X
X
MeO
HO
OMe
OH
N
H
1 (EF24): X = C-F
Curcumin
2 (EF31): X = N
* Corresponding authors.
E-mail addresses: asun2@emory.edu (A. Sun), jsnyder@emory.edu (J.P. Snyder).
Figure 1. Structures of curcumin, analogs 1 and 2.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.023

6628
A. Sun et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6627–6631
ring. The compounds engage in a very specific alkylation of Cys-
773 in the ATP pocket of the EGFR. The ,b-unsaturated tyrosine ki-
SH
O
O
O
O
a
H
N
X
X
COOH
nase inhibitors are currently in Phase I clinical trial.16,17 In addi-
tion, Dimmock et al. have long recognized the affinity of
dienones for biological thiols,18 and isolated ethanethiol and 1,2-
ethanedithiol adducts of selected enones.19 Unexplored, however,
are the reversible or partially reversible nature of the compounds,
which may offer potential advantages in terms of target suppres-
sion and in vivo pharmacokinetics.
+
O
N
H
NH3
N
H
1, 2
Glutathione (GSH)
H2O/MeCN
The observation of glutathione depletion suggested a means to
solubilize the curcumin analogs for evaluation in various cell-
based assays. Thus, solubility comparisons indicated that while
aqueous dissolution of curcumin is unaffected by GSH, high con-
centrations of the peptide are capable of drawing 1 into solution.
(Fig. 2) The adduction with glutathione is illustrated in Scheme
1. A number of studies have demonstrated that depletion of thiol
concentrations prior to treatment with various anti-cancer drugs
has increased cell killing compared to the use of drug alone.20,21
Synthesis of GSH conjugates. Compound 1 or 2 dissolved in aceto-
nitrile (CH3CN) was added slowly to 2.1 equiv of GSH in water at
room temperature. The yellow color of the dienone disappears as a
result of eliminating the extended chromophore and the simulta-
neous formation of bis-adducts 1- or 2-(GSH)2. Conjugation of GSH
with 2 takes place instantaneously as reflected by the immediate
disappearance of the yellow color of the unsaturated ketone, while
conjugation with 1 takes several hours to complete. Nevertheless,
both reactions provide bis-adducts as the only products along with
free GSH based on LC/MS (Fig. 3a and b). No mono-adducts were de-
tected under these conditions, although mono-adduct 3 is surely an
intermediate to bis-adduct 4. Accordingly, treatment of 2 with
SG
SG
SG
O
O
X
X
X
X
+
Y
Y
3
4
Scheme 1. Combination of 1 and 2 with GSH to give mono-adduct 3 and bis-adduct
4.
Several methodologies for cysteine S-functionalization and protec-
tion have been reported previously.23–26 Dimethyl-thiazolidine
(Dmt) has been successfully employed as a sulfhydryl-amino
masking group for cysteine in the synthesis of glutathione27 and
in the course of natural product syntheses.28,29 The protecting unit
can be removed under mild conditions to yield the corresponding
aminoethanethiol moiety of cysteine. In the present work, Dmt-
cysteine 6 was converted into the Boc acetonide derivative 7. The
latter was coupled with the protected amino acid hydrochloride
salt 8 using EDCI and HOBt in DMF and further treated with diiso-
propyl ethyl amine (DIEA) to give Boc-Dmt-cys-amino acid-ester 9
in medium to good yields. Deprotection of the Dmt group with TFA
delivered the Cys-Phe-methyl ester TFA salt 12. Hydrolysis of 9 un-
der basic conditions (NaOH) in dioxane delivered the Boc-Dmt-cys-
amino acid 10. Removal of the Boc group from 10 under acidic con-
ditions (TFA) and subsequent concentration in the presence of
EtOH and H2O (1:1) led to the complete removal of the acetone
generated and isolation of free amino and thiol-cysteine-dipeptide
TFA salt 11 in excellent yield (95%) (Scheme 2).
1.0 equiv of L-glutathione delivers a mixture of mono- and bis-ad-
ducts, 2-(GSH) and 2-(GSH)2, with the mono-adduct as the major
product by MS. However, due to the similar polarities of 2-(GSH)
and 2-(GSH)2, no clear separation by LC was observed. Thus, a broad
peak presumed to be overlapping bands of the two conjugates was
observed (Fig. 3c).
In order to further investigate the reaction between thiols and
curcumin analogs, several cysteine-containing dipeptides were
prepared and their reactions with curcumin analogs, examined.
Synthesis of cysteine-containing dipeptides. The preparation of
Coupling of Cys-dipeptides with 1 and 2. Compounds 1 and 2 were
treated with dipeptides Cys-Phe (11) and Cys-Gly (12), respec-
tively. Reaction between 2 and Cys-Gly is instantaneous when
the dienone in CH3CN is added dropwise to Cys-Gly in water. The
yellow color of 2 disappears immediately with the formation of
2(Cys-Gly)2 (13d), which was confirmed by LC/MS. By comparison,
reaction between 1 and Cys-Gly as with GSH is much slower,
requiring at least 7 h at room temperature. Both 1 and 2 Cys-Gly
conjugates are white, water-soluble powders upon solvent evapo-
ration. Conjugation with higher mass dipeptides, for instance Cys-
Phe (11), proceeds much more slowly relative to Cys-Gly. Combi-
nation of 2 with Cys-Gly occurs instantly, while conjugation with
Cys-Phe takes several hours to complete. Nonetheless, the prod-
ucts, 13a and 13c are also white, water-soluble powders (Scheme
3).
cysteine-containing dipeptides was initiated with free L-cysteine
5. Due to the sensitivity of the cysteine molecule toward oxidation
and elimination, it was necessary to protect the b-sulfhydryl moi-
ety as well as the amino and/or carboxyl group during synthesis.22
Previous studies of the interaction between curcumin and glu-
tathione have suggested the operation of Michael addition be-
tween the
a,b-unsaturated chromophore of curcumin and GSH.
FAB-MS and MALDI-MS spectra of glutathionated curcumin have
been interpreted in terms of mono- and di-glutathionyl-adducts
as well as cyclic rearrangement products including feruloyl-meth-
ylketone and feruloylaldehyde.30 A recent investigation by Usta
et al. reports that a combination of glutathione and curcumin leads
to the formation of two diastereoisomeric monoglutathionyl cur-
cumin conjugates.31 The structures of both conjugates were identi-
fied by LC/MS and one- and two-dimensional 1H NMR analysis.
Figure 2. Solubility of 1 and curcumin in GSH-doped aqueous solution. Each point
corresponds to 200 mM of compound, and thus a maximum 1:GSH molar ratio of
1:1 and 1:2 at GSH concentrations of 200 and 400 mM, respectively. Powders of the
enones and four GSH concentration were mixed and left at room temperature for
30 min. Insoluble 1 and curcumin were collected by centrifugation, and the pellets
content (% compound undissolved) were determined by bioassay after dissolution
in DMSO. Incomplete solubility of 1-(GSH)2 is due to the short 30 min mixing times
employed in this experiment.

A. Sun et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6627–6631
6629
Figure 3. LC spectra following separate addition of 1 equiv of 1 and 2 to 2.1 equiv of GSH; peak mass determined by MS; (a) 1-(GSH)2 and GSH; (b) 2-(GSH)2 and GSH; (c) LC/
MS spectra of 1:1 mixture of 2 and GSH to deliver mono-adduct 2-(GSH) as the major product and bis-product 2-(GSH)2 as the minor product based on the MS.
Ph
O
O
O
O
COOMe
OH
OH
N
H
a
b
HS
OH
c
S
S
S
NH
NBoc
NBoc
NH2
6
7
9
5
Ph
COOH
Ph
COOH
O
O
d
e
N
H
NH
HS
S
NBoc
NH2.CF3COO
11
10
Scheme 2. Preparation of cysteine-containing dipeptides. Reagents and conditions: (a) acetone, reflux; (b) Boc2O, DIEA, MeCN, rt; (c) HOBt, EDCI, DMF, then phenylalanine
methyl esterꢀHCl (8), DIEA; (d) NaOH (1 N), dioxane, then HCl (1 N); (e) TFA–CH2Cl2(3:1), rt, 3 h; then EtOH–H2O (1:1).
Scheme 3. Conjugation of 1 and 2 with cysteine-containing dipeptides.

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A. Sun et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6627–6631
Formation of the two isomers was reported to be reversible,
although the compounds appear to be relatively unstable. Com-
pared with the slow association of curcumin and GSH, the reaction
of either 1 or 2 with GSH is much faster. In addition, the latter are
stable, water-soluble, colorless powders of the bis-adducts 1-
(GSH)2 and 2-(GSH)2 with a potential for releasing drugs 1 and 2
by means of a reversible equilibrium (Scheme 1).
Reversible and competitive equilibria of GSH Michael addition to
curcumin analogs. It is common knowledge that Michael additions
are reversible reactions,32 yet the literature reveals very few inves-
tigations of thiol-mediated equilibria of this type.33 The reversibil-
ity of thiol addition to 1 was demonstrated by treatment of 1-
(GSH)2 with 2 in water at room temperature (Eq (1)):
as efficacious in their cell-kill capacity as the parents 1 and 2. Ana-
logs 1 and 1-(GSH)2 exhibit IC50 values of 1.5 M against MDA-MB-
435 breast cancer cells (Fig. 4a), while 2 and 2-(GSH)2 likewise
show similar IC50 values of 1.0 M (Fig. 4b).
l
l
In summary, thiol conjugates such as 1-(GSH)2 and 2-(GSH)2 are
easily prepared as stable, water-soluble, colorless solids with cyto-
toxic properties comparable to the unconjugated drugs 1 and 2.
The double GSH conjugates can be regarded as a class of pro-drugs
capable of releasing the active agents by means of a readily estab-
lished equilibrium (Scheme 1). While the thiol conjugates of 1 and
2 are in principle capable of existing as a complex mixture of
mono- and bis-GSH diastereoisomers in aqueous solution, we ex-
pect that certain isomers may well dominate in solution, perhaps
as single entities in some cases. Studies underway will explore
the relative bioactivity of other pyridone curcumin analogs, the
bio-effects of replacing GSH with cysteine-containing peptides,
effectiveness against other cell lines, the diastereomeric composi-
tion and the potential of the series as anti-cancer pro-drugs.
ð1Þ
Eq. 1. Reaction of 2 with 1-(GSH)2 in H2O.
The reaction was followed by LC/MS from 5 min to 2 days after
mixing 1-(GSH)2 and 2 in a molar ratio of 1:1. Analysis of the reac-
tion mixture by LC/MS after 5 min revealed only starting materials,
2 and 1-(GSH)2. After 8 h, LC/MS detected 1, 2-GSH and 2-(GSH)2
along with unreacted 2 and 1-(GSH)2. Extending the reaction per-
iod to 48 h gave no evidence of 2, only 1, 2-(GSH)2 and a small
amount of unreacted 1-(GSH)2. The Michael addition of the pyri-
dine analog 2 with glutathione was described above as an instan-
taneous reaction between them, while the corresponding
conjugation of 1 proved to be much slower (Scheme 1). Accord-
ingly, (Eq (1)) implies the retro-Michael addition of 1-(GSH)2
releasing free 1 and glutathione followed by 2’s capture of GSH
to deliver the corresponding mono- and bis-adducts. Thus, the com-
petition confirms that the cross-coupling of 2 with GSH is much
faster than 1.
Acknowledgments
This work was financially supported by the National Institutes
of Health (NIH) grants R21 CA82995-01A1 and U.S. Department
of Defense, the Division of U.S. Army DAMD17-00-1-0241 (to M.
Shoji) and 1 U54 HG003918 (to J. P. Snyder).
References and notes
1. Goel, A.; Kunnumakkara, A. B.; Bharat, B. A. Biochem. Pharmacol. 2008, 75, 787.
2. Bemis, D. L.; Katz, A. E.; Buttyan, R. Exp. Opin. Invest. Drugs 2006, 15, 1191.
3. Nonn, L.; Duong, D.; Peehl, D. M. Carcinogenesis 2006, 28, 1188.
4. Tomita, M.; Kawakami, H.; Uchihara, J. N.; Okudaira, T.; Masuda, M.; Takasu, N.;
Matsuda, T.; Ohta, T.; Tanaka, Y.; Ohshiro, K.; Mori, N. Int. J. Cancer 2006, 118,
765.
Comparison of the cytotoxic activity of 1, 2 and the corresponding
GSH conjugates. Treatment of MDA-MB-435 human breast cancer
cells separately with 1, 2, 1-(GSH)2 and 2-(GSH)2 under identical
conditions demonstrates that the GSH bis-conjugated analogs are
5. Jobin, C.; Bradham, C. A.; Russo, M. P.; Juma, B.; Narula, A. S.; Brenner, D. A.;
Sartor, R. B. J. Immunol. 1999, 163, 3474.
6. Shishodia, S.; Potdar, P.; Gariola, C. G.; Aggarwal, B. B. Carcinogenesis 2003, 24,
1269.
7. Aggarwal, B. B.; Shishodia, S. Biochem. Pharmacol. 2006, 71, 1397.
8. Sharma, R. A.; Gescher, A. J.; Steward, W. P. Eur. J. Cancer 2005, 41, 1955.
9. Dhillon, N.; Wolff, R. A.; Abbruzzese, J. L.; Hong, D. S.; Camacho, L. H.; Li, L.;
Braiteh, F. S.; Kurzrock, R. J. Clin. Oncol. 2006, 24, 14151 (Abstract).
10. Shoba, G.; Joy, D.; Joseph, T.; Majeed, M.; Rajendran, R.; Srinivas, P. S. Planta
Med. 1998, 64, 353.
11. Safavy, A.; Raisch, K. P.; Mantena, S.; Sanford, L. L.; Sham, S. W.; Krishna, N. R.;
Bonner, J. A. J. Med. Chem. 2007, 50, 6284.
12. Adams, B. K.; Ferstl, E. M.; Davis, M. C.; Herold, M.; Kurtkaya, S.; Camalier, R. F.;
Hollingshead, M. G.; Kaur, G.; Sausville, E. A.; Rickles, F. R.; Snyder, J. P.; Liotta,
D. C.; Shoji, M. Bioorg. Med. Chem. 2004, 12, 3871.
13. Adams, B. K.; Cai, J.; Armstrong, J.; Herold, M.; Lu, Y.; Sun, A.; Snyder, J. P.;
Liotta, D. C.; Jones, D. P.; Shoji, M. Anticancer Drugs 2005, 16, 263.
14. Shoji, M. et al., unpublished results.
15. Kasinski, A. L.; Du, Y.; Thomas, S. L.; Zhao, J.; Sun, Shi-Y.; Khuri, F. R.; Wang, C.;
Shoji, M.; Sun, A.; Snyder, J. P.; Liotta, D. C.; Fu, H. Mol. Pharmacol. 2008, 74, 654.
16. Fry, D. W.; Bridges, A. J.; Denny, W. A.; Doherty, A.; Greis, K. D.; Hicks, J. L.;
Hook, K. E.; Keller, P. R.; Leopold, W. R.; Loo, J. A.; McNamara, D. J.; Nelson, J. M.;
Sherwood, V.; Smaill, J. B.; Trumpp-Kallmeyer, S.; Dobrusin, E. M. Proc. Natl.
Acad. Sci. 1998, 95, 12022.
17. Klutchko, S. R.; Zhou, H.; Winters, R. T.; Tran, T. P.; Bridges, A. J.; Althaus, I. W.;
Amato, D. M.; Elliott, W. L.; Ellis, P. A.; Meade, M. A.; Roberts, B. J.; Fry, D. W.;
Gonzales, A. J.; Harvey, P. J.; Nelson, J. M.; Sherwood, V.; Han, H.-K.; Pace, G.;
Smaill, J. B.; Denny, W. A.; Hollis, S. H. D. J. Med. Chem. 2006, 49, 1475.
18. (a) Dimmock, J. R.; Padmanilayam, M. P.; Puthucode, R. N.; Nazarali, A. J.;
Motaganahalli, N. L.; Zello, G. A.; Quail, J. W.; Oloo, E. O.; Kraatz, H.-B.; Prisciak,
J. S.; Allen, T. M.; Santos, C. L.; Balzarini, J.; De Clercq, E.; Manavathu, E. K. J.
Med. Chem. 2001, 44, 586; (b) Pati, H. N.; Das, U.; Quail, J. W.; Kawase, M.;
Sakagami, H.; Dimmock, J. R. Eur. J. Med. Chem. 2008, 43, 1.
19. Dimmock, J. R.; Smith, L. M.; Smith, P. J. Can. J. Chem. 1980, 58, 984.
20. Balendiran, G. K.; Dabur, R.; Fraser, D. Cell Biochem. Funct. 2004, 22, 343.
21. Garcia-Ruiz, C.; Mari, M.; Morals, A.; Colell, A.; Ardite, E.; Fernandez-Checa, J. C.
Hepatology 2000, 32, 56.
22. Alves, D.; Esser, D.; Broadbridge, R.; Beevers, A.; Chapman, C.; Winsor, C.;
Betley, J. J. Pept. Sci. 2003, 9, 221.
23. (a) Brois, J.; Pilot, J.; Barnum, H. J. Am. Chem. Soc. 1970, 92, 7629; (b) Hiskey, R.;
Li, C.; Vunnam, R. J. Org. Chem. 1975, 40, 3697; (c) Kamber, B. Helv. Chim. Acta
1973, 56, 1370; (d) Fotouhi, N.; Galakatos, N.; Kemp, D. J. Org. Chem. 1989, 54,
2803.
Figure 4. Dose–response curves for the treatment of MDA-MB-435 human breast
cancer cells with curcumin analogs and their GSH conjugates: (a) 1 and 1-(GSH)2;
(b) 2 and 2-(GSH)2.

A. Sun et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6627–6631
6631
24. (a) Ruiz-Gayo, M.; Albericio, F.; Pedroso, E.; Giralt, E. J. Chem. Soc., Chem. Commun.
1986, 1501; (b) Corey, E. J.; Gin, D. Y.; Kania, R. S. J. Am. Chem. Soc. 1996, 118, 9202;
(c) Ponsati, B.; Giralt, E.; Andreu, D. Tetrahedron 1990, 46, 8255.
25. West, C.; Estiarte, M.; Rich, R. Org. Lett. 2001, 3, 1205.
26. Katritzky, A.; Angrish, P.; Hür, D.; Suzuki, K. Synthesis 2005, 3, 397.
27. (a) King, F.; Clark-Lewis, J.; Wade, R. J. Chem. Soc. 1957, 880; (b) Sheehan, J.;
Yang, D.-D. J. Am. Chem. Soc. 1958, 80, 1154.
29. West, C.; Estiarte, M.; Rich, R. Org. Lett. 2001, 3, 1205.
30. Awasthi, S.; Pandya, U.; Singhal, S. S.; Lin, J. T.; Thiviyanathan, V.; Seifert, W. E.,
Jr.; Yogesh, Y. C.; Awasthi, C.; Ansari, G. A. Chem. Biol. Interact. 2000, 128, 19.
31. Usta, M.; Wortelboer, H. M.; Vervoort, J.; Boersma, M. G.; Rietjens, I. M. C. M.;
van Bladeren, P. J.; Cnubben, N. H. P. Chem. Res. Toxicol. 2007, 20, 1895.
32. (a) March, J. Advanced Organic Chemistry, 4th ed.; Wiley: New York, 1992. p
796; (b) Bergmann, E. D.; Ginsburg, D.; Pappo, R. Org. React. 1959, 10, 179.
33. Brickley, J. Rubber Age (New York) 1943, 53, 331.
28. Nicolaou, K. C.; Nevalainen, M.; Safina, B.; Zak, M.; Bulat, S. Angew. Chem., Int.
Ed. 2002, 41, 1941.