Eaton's reagent-mediated domino π-cationic arylations of aromatic carboxylic acids to iasi-red polymethoxylated polycyclic aromatic hydrocarbons: Products with unprecedented biological activities as tubulin polymerization inhibitors

Article abstract of DOI:10.1002/chem.201402377

A rapid domino π-cationic arylation of aromatic carboxylic acids, mediated by Eaton's reagent, has been developed for the synthesis of Iasi-red polymethoxylated polycyclic aromatic hydrocarbons (PAHs). This route is currently the easiest method to obtain

Full text of DOI:10.1002/chem.201402377

DOI: 10.1002/chem.201402377
Full Paper
&
Polycycles
Eaton’s Reagent-Mediated Domino p-Cationic Arylations of
Aromatic Carboxylic Acids to Iasi-Red Polymethoxylated Polycyclic
Aromatic Hydrocarbons: Products with Unprecedented Biological
Activities as Tubulin Polymerization Inhibitors
Alina Ghinet,*[a, b, c] Philippe Gautret,[a, b] Nathalie Van Hijfte,[a, b, d] Bertrand Ledꢀ,[e] Jean-
Pierre Hꢀnichart,[a] Elena Bꢁcu,[c] Ulrich Darbost,[f] Benoꢁt Rigo,[a, b] and Adam Da¨ıch*[d]
Abstract: A rapid domino p-cationic arylation of aromatic
carboxylic acids, mediated by Eaton’s reagent, has been de-
veloped for the synthesis of Iasi-red polymethoxylated poly-
cyclic aromatic hydrocarbons (PAHs). This route is currently
the easiest method to obtain such popular PAH compounds,
which bear in addition numerous methoxy groups. The
domino process was generalized, the structure of the ob-
tained red products and the mechanism of their formations
were elucidated, and some of their photophysical properties
were determined. Newly synthesized polymethoxylated-
PAHs were tested for their interaction with tubulin polymeri-
zation as well as for their cytotoxicity on a panel of NCI-60
human cancer cell lines. Interestingly, one of these rubicene
derivatives exhibited remarkable cytotoxicity in vitro, includ-
ing inhibition of leukemia, colon, melanoma, CNS, and ovari-
an cancer cell lines with GI50 values in the low nanomolar
range (GI50 <10 nm).
widely distributed in terrestrial, aquatic, and aerial areas.[1]
Many of them are associated with carcinogenesis, mutagenesis,
or teratogenesis. Consequently, they exert a high impact on
nature and, as a result, on all the vital resources required for
human health and life, including drinks and food.[2] Their me-
tabolism in certain cell tissues, by the action of successive cy-
tochrome P450 monooxygenases and epoxide hydrolase en-
zymes, results in the formation of various polyhydroxylated-
PAHs (OH-PAHs) and vicinal-dihydroxy-PAHs. In many cases,
they will often potentiate the expression of their biological ef-
fects as tumor activators[2a] compared with their parent PAHs.[3]
Importantly, alkoxy groups attached to a PAH platform,
which are obtained by different chemical transformations, are
reported to alter the electronic and photophysical properties
of their parent PAHs essentially because of the extended p-
conjugation that is gained by these p-electron materials. Cer-
tain examples of these products are used as dendritic poly-
mers,[4] liquid-crystalline materials,[5] and electronic organic de-
vices[6] including deep-blue organic light-emitting diodes
(OLEDs),[7] fluorescent tags for labeling biomolecules,[8] and
red-emitting electroluminescent materials.[9] On the other hand
and in contrast to OH-PAHs, no examples of natural bioactive
alkoxy-PAHs bearing one skeleton among PAHs family can be
found in the literature. In the latter case, numerous natural
products based on acephenanthrylene and benzo[j]fluoran-
thene PAH cores (Figure 1) with differing degrees of unsatura-
tion have shown various and interesting biological properties.
This is exemplified by laetevirenol A (1b)[10] and daldinone B
(2a), daldinone A (2b), daldinone G (2c), and hypoxylonol F
(2d).[11] These species, extracted from the roots and stems of
Introduction
Polycyclic aromatic hydrocarbons (PAHs) are characterized by
condensation of numerous (at least three) aromatic rings and
they constitute a large, important family of persistent organic
pollutants. PAHs are mainly formed during the incomplete
combustion of fossil materials, but may also be formed to
a lesser extent by microbes and plants. In addition, they are
[a] Dr. A. Ghinet, Dr. P. Gautret, Dr. N. V. Hijfte, Prof. J.-P. Hꢀnichart,
Prof. B. Rigo
Univ Lille Nord de France, 59000 Lille (France)
E-mail: Alina.GHINET@hei.fr
[b] Dr. A. Ghinet, Dr. P. Gautret, Dr. N. V. Hijfte, Prof. B. Rigo
UCLille, EA 4481 (GRIIOT), Laboratoire de Pharmacochimie, HEI
13 rue de Toul, F-59046 Lille (France)
[c] Dr. A. Ghinet, Prof. E. Bꢁcu
Department of Organic Chemistry, ‘Al. I. Cuza’ University of Iasi
Faculty of Chemistry, Bd. Carol I nr. 11, 700506 Iasi (Romania)
[d] Dr. N. V. Hijfte, Prof. Dr. A. Da¨ıch
URCOM, EA 3221, INC3M CNRS FR-3038
UFR des Sciences & Techniques de l’Universitꢀ du Havre
25 rue Philippe Lebon, BP: 540, 76058 Le Havre Cedex (France)
E-mail: adam.daich@univ-lehavre.fr
[e] Dr. B. Ledꢀ
Holliday-Pigments SAS, BP 50017-203, Route de Wervicq
F-59559, Comines Cedex (France)
[f] Dr. U. Darbost
ICBMS, Equipe CSAp, CNRS UMR 5246
Universitꢀ Lyon 1, 43 Boulevard du 11 Novembre 1918
69622 Villeurbanne (France)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201402377.
Chem. Eur. J. 2014, 20, 10117 – 10130
10117
ꢂ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
arylation with alkyl and aryl systems,[18] and 10) the very popu-
lar and efficient Friedel–Crafts reaction.[11b,19]
Our contribution to this area started from an interesting ser-
endipitous observation we made during the execution of one
of our research projects concerning the elaboration of new in-
hibitors of tubulin polymerization, analogous to the potent
combretastatin A4[20] (CA-4; 3a) and phenstatin (4a; Figure 1
and Scheme 1).[21] Our interest was aroused by the observation
of a strong red-brown dark tint of the reaction media when
some arylcarboxylic acids reacted with aryl compounds under
Friedel–Crafts conditions with acid promoters such as poly-
phosphoric acid (PPA) or Eaton’s reagent, which was generated
from an mixture of P2O5 and MeSO3H (1:10 w/w).[22]
Results and Discussion
In this full account, we document our reinvestigation of the
acid-catalyzed Friedel–Crafts reaction of arylcarboxylic acids
with aromatic nucleophiles. Our attention is especially focused
on the use of Eaton’s reagent to promote cascade Friedel–
Crafts type reactions producing hitherto unpublished struc-
tures belonging to the PAH family. During these investigations,
the domino process was generalized, the structure of the ob-
tained red products and the mechanism of their formations
were elucidated, and some of their photophysical properties
were determined. The results of their biological evaluation for
the inhibition of tubulin and cancer cell growth are also
reported.
Figure 1. Naturally occurring compounds containing the acephenanthrylene
and benzo[j]fluoranthene skeletons belonging to PHA family and combretas-
tatins 3 and phenstatins 4 as the natural potent inhibitors of tubulin.
Parthenocissus laetevirens and the mushroom Hypoxylon trunca-
tum, have displayed strong antioxidant activities and antiproli-
ferative activity against human umbilical vein endothelial cells
(HUVECs) and human umbilical artery endothelial cells
(HUAECs), respectively (Figure 1). In parallel explorations on
the biosynthetic pathways, determination of the immunosup-
pressive polyketides from the broth of mantis-associated Daldi-
nia eschscholzii, identified 2,16-dihydroxybenzo[j]fluoranthene
as a totally aromatic PAH substance, which has recently been
isolated.[12] In addition, certain OH-PAH compounds containing
one or more alkoxy groups (hybrid compounds), named hypo-
xylonols A–E, have been isolated from the same species as hy-
poxylonol F.[11c] Similar hybrid compounds were isolated as sec-
ondary metabolites[11c] from Hypoxylon truncatum (Xylariaceae),
an endophytic fungus residing inside symptomless stems of Ar-
temisia annua, which are the origin of potent cytotoxic sub-
stances.[13] A few examples of alkoxy-PAHs bearing the skele-
tons outlined in Scheme 1 have been used as valuable precur-
sors during the total synthesis of laetevirenol A (1b)[10c] and
benzo[j]fluoranthene derivatives.[11b]
Synthesis and characterization of the red by-product
A detailed view of the synthesis of the key intermediate, pro-
tected phenol-derivative 7, starting from commercially avail-
able 3,4,5-trimethoxybenzoic acid (5) and 2-methoxyphenyl
chloroacetate (6), was based on our previous work
(Scheme 1).[21a] Following the well-known deprotection of
chloroacetic ester in mild alkaline medium [e.g., AcONa·3H2O
(4.5 equiv), MeOH, reflux, 3 h],[23] ketone derivative 7 was sepa-
rated from the dark red-brown mixture to afford the expected
phenstatin (4a) in high yield (91%). It is worth mentioning
that this two-step sequence represents a significant improve-
ment over the first hemisynthesis of phenstatin (4a) from com-
Given the interest in these polycondensed systems, several
synthetic approaches for the elaboration of PAH skeletons as
well as for their peripheral modifications have been devel-
oped.[14] The most generally used methodologies for the con-
struction of the PAH framework include: 1) intramolecular and
intermolecular Diels–Alder cycloaddition,[15] 2) selective radical
cascade cyclization of polyalkynes,[16] 3) flash-vacuum pyrolysis
(FVP) of vinyl compounds,[14] 4) intramolecular photocyclization
of stilbene-type compounds,[14] 5) ring-closing olefin metathe-
sis (RCM),[14] 6) acid-catalyzed benzannulation and electrophilic
cyclization,[14] 7) Lewis-acid promoted oxidative cyclodehydro-
genation,[14] 8) Pd-catalyzed cyclization in tandem or not with
other transformations,[17] 9) decoration of PAHs by direct CꢀH
Scheme 1. Synthetic pathway providing the bioactive phenstatin (4a) and
red by-product 8 in the Friedel–Crafts domino reaction.
Chem. Eur. J. 2014, 20, 10117 – 10130
www.chemeurj.org
10118
ꢂ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
bretastatin A4 (3a) based on Jacobsen’s oxidation.[24] The
method constitutes a competitive approach to phenstatin and
derivatives compared with the PPA-mediated Friedel–Crafts re-
action.[23] Intrigued by the strong tint of the solution in numer-
ous cases, additional purification procedures led to the isola-
tion of a red pigment 8, that we named “Iasi-red”, in 9% aver-
age yield. Interestingly, by repeating this reaction using PPA[23]
at 1008C for 4 h (instead of Eaton’s reagent[25] at 608C;
Scheme 1), the reaction presented the same profile. A dark
red-brown coloration of the reaction media was also observed,
and ketoester 7 was obtained albeit in lower yield (62%),[21a]
together with the red pigment 8, which was produced in com-
parable yield. However, the use of methanesulfonic acid alone,
either with or without solvent, led to the formation of neither
phenstatin (4a) nor the red by-product 8. This result confirmed
that an acidic dehydrating medium, such as PPA or, more con-
Mechanistic aspects of the red-product formation
In our quest to understand the course of the reaction, especial-
ly that providing 8 (Figure 2), various investigations were plan-
ned. Taking into account that the structure of 8, which could
be obtained in an acid-mediated domino reaction, indicates
the presence of three main motifs, presumably derived from
two 3,4,5-trimethoxybenzoic acid (5) and one 2-methoxyphe-
nyl chloroacetate (6). A plausible evolution of the protected
phenstatin 7 in the reaction media was suggested as detailed
below.
The stability of pure phenstatin derivative 7 was first exam-
ined in Eaton’s reagent (4 equiv) medium at 508C under the
optimized reaction conditions (Scheme 1). The results, shown
in Scheme 2, indicate that the red by-product 8 was not
formed. However, under these conditions extensive decompo-
veniently,
freshly
prepared
Eaton’s reagent, is pivotal for the
formation of red product 8, due
probably to the formation of
mixed anhydride in situ. These
results also confirmed that
Eaton’s reagent is more appro-
priate than PPA for this type
of reaction, as mentioned
elsewhere
compounds.[22,26]
for
related
Scheme 2. Results of the evolution of phenstatin precursor 7 in Eaton’s reagent under the optimized reaction
conditions.
The structure of red by-prod-
uct 8, which was very puzzling,
was established by aggregating
numerous data and spectral characteristics from mono- and bi-
dimensional NMR spectroscopy (Figure 2). However, the struc-
tural assignment was achieved without the aid of X-ray data
sition of the reaction medium occurred. The first events are
cleavage and hydrolysis of the methoxy and ester functions,
respectively, in the para position of ring A and the meta posi-
tion of ring B of the starting phenstatin derivative 7. Both new
phenolic OH groups were then acylated by generating the
chloroacetyl cation in the media and/or sulfonation with
Eaton’s reagent acid to provide chloroacetate diester 9 (23%),
methanesulfonate ester 10 (10%) and diester 11 (12%).[27]
These compounds, which were separable by flash chromatog-
raphy on silica gel column, were accompanied by low amounts
of chloroacetophenone derivative 12 (5%). The latter was
probably formed by a tandem retro-Friedel–Crafts/Friedel–
Crafts reaction sequence. More importantly, no traces of ben-
zoquinone derivatives 13a (R=H) or 13b (R=Me) were de-
tected in the reaction medium. It is worth noting that pentam-
ethyl rufigallic acid (13a, R=H), lacking a methyl group, which
could be considered as an intermediate for the production of
benzo[a]aceanthrylene product 8, was obtained as by-product
in a related condensation of trimethoxybenzoic acid (5) with
benzoxazolone.[21a] To date, such quinones have only been de-
scribed from reactions using sulfuric acid at very high tempera-
tures.[28] These results strongly suggested that a pathway start-
ing from benzophenone 13 as intermediate in the mechanistic
reaction for the formation of 8 should not be eliminated
(Scheme 2).
Figure 2. Established structure of Iasi-red by-product 8, namely 8-hydroxy-
3,5,6,7,10,11,12-heptamethoxybenzo[a]aceanthrylen-2-yl chloroacetate.
because it was not possible to prepare single crystals of 8. Its
solubility was low in DMSO but very high in dichloromethane.
The molecular weight, as obtained by LC-MS (Si-60/MeCN)
analysis, was 612 gmolꢀ1, however, elemental analysis fur-
nished a molecular composition formula of C29H27ClO10, corre-
sponding to MW=571 gmolꢀ1. It was deduced that acetoni-
trile (MW=41 gmolꢀ1), used as solvent, binds sufficiently
strongly to 8 to lead to a combined MS peak.
We then considered the direct reaction of protected phen-
statin 7 with 3,4,5-trimethoxybenzoic acid (5) catalyzed by
Chem. Eur. J. 2014, 20, 10117 – 10130
www.chemeurj.org
10119
ꢂ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
mediary species IV, an intramolecular p-cationic cyclization re-
lated to the Clarkson and Gomberg approach.[29]
The highly activated arylcarboxylic acid intermediate VII,
generated after deprotonation of cation V followed by tauto-
merism equilibration of VI, cyclizes to the pentacyclic ben-
zo[a]aceanthrylene PAH core VIII, again, through intramolecu-
lar Friedel–Crafts aroylation mediated by Eaton’s reagent. Ulti-
mately, the keto-enolic equilibrium in VIII provided the expect-
ed red PAH product 8. Interestingly, during this proposed
domino process, four carbon–carbon bonds were formed, two
intermolecular and two intramolecular, and the integrity of the
chloroacetate group was maintained.
Scheme 3. Reaction of phenstatin precursor 7 with trimethoxybenzoic acid
(5) leading solely to red product 8.
Eaton’s reagent (Scheme 3). To our delight, heating carboxylic
acid 5 with ketone 7 in Eaton’s reagent for 18 h at 608C result-
ed in the formation of Iasi-red 8 as the sole reaction product
in 63% isolated yield. Considering that only one equivalent of
carboxylic acid 5 was used, and that plausible decomposition
of 7 to provide numerous derivatives as mentioned above is
not a favored process (Scheme 2), this reaction confirms that
benzoquinone 13 evoked in Scheme 2 was not an effective in-
termediate in the reaction. Moreover, this finding provides an
elegant way to prepare benzo[a]aceanthrylene PAH 8 in signifi-
cant yield.
The domino reaction seems to proceed predominantly
through the intermediacy of triaryl carbinol II (path A) rather
than the mesomeric form IX of species I. Indeed, it could be
considered that, upon protonation of 7, benzophenone IX cy-
clized to give the corresponding substituted 9H-fluoren-9-ol X
(Scheme 4).[30] This intermediate, with carboxylic acid 5 would
give access to the intermolecular arylation product VII, which
is also evoked in path A. However, we have already described
that, in acid media (CF3SO3Hcat), dismutation of benzhydrols
and 9H-fluoren-9-ol occurs rapidly, leading to diphenylme-
thanes and benzophenones derivatives.[31] A detailed investiga-
tion of the products formed in the synthesis of PAH compound
8 did not reveal any traces of these tricyclic compounds, ren-
dering path B less probable. In addition, the high acidity of
methanesulfonic acid used here (pKa =ꢀ0.6) resulted in the
significant stabilization of the tertiary carbocation III compared
with that generated from 9H-fluoren-9-ol derivative X. This fact
also supports the conclusion that the reaction follows path A
(Scheme 4).[10c,32]
A possible mechanism leading to the formation of ben-
zo[a]aceanthrylene PAH 8 would proceed through domino p-
cationic reactions including successive intermolecular and in-
tramolecular Friedel–Crafts aroylation and arylation, respective-
ly (Scheme 4). Thus, reaction of 3,4,5-trimethoxybenzoic acid
(5) with 2-methoxyphenyl chloroacetate (6) under these condi-
tions resulted in the formation of the standard Friedel–Crafts
aroylation product 7. Protonation of the ketone group fur-
nished cation I, which is in equilibrium with IX (path B). Reac-
tion of cationic species I, with a second equivalent of acid 5,
provided alcohol II through electrophilic addition. In the acidic
medium this triaryl carbinol was never isolated and evolved
into the more stable cation III, which undergoes, via the inter-
Another result that also supports mechanistic path A was
obtained by the reaction of 1,2,3-trimethoxybenzene (14) and
ketone 7 in Eaton’s reagent. In this case, after 6 h heating at
608C, 9H-fluorene product 15 was isolated in 70% yield as the
sole reaction product (Scheme 5). Here, the pathway seems to
Scheme 4. Mechanistic scheme leading to protected phenstatin 7 and benzo[a]aceanthrylene PAH compound 8 from 3,4,5-trimethoxybenzoic acid (5).
Chem. Eur. J. 2014, 20, 10117 – 10130 www.chemeurj.org ꢂ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
10120

Full Paper
agent is effective to provide the benzo[a]aceanthrylene skele-
ton, we next examined this reaction using additional arylcar-
boxylic acids and arenes with the objective of generalizing the
process. To measure the impact of benzoic acid substitution
(mono-, di-, and tri-substituents) and the nature of the aromat-
ic ring used as nucleophile (benzene, benzo[d]oxazol-2-ones)
on the domino reaction, eight additional combinations were
chosen (Table 1).
Generalization of the domino reaction was first realized by
using the optimized reaction conditions [2-methoxyphenyl
chloroacetate (6; 1 equiv), Eaton’s reagent (P2O5/MeSO3H,
4 equiv), 508C, 4–12 h], and changing the arylcarboxylic acid
used (Table 1, entries 1–5). Conducting the same reaction with
carboxylic acid 16 resulted in the formation of ketone 17 ac-
companied by trace amounts of 18 (entry 2). In this case, the
yield of ketone 17 was superior to that obtained for ketone 7
(91 vs. 85%; Table 1). 2-Methoxybenzoic acid (19) also proved
to be less active towards the domino process, with ketone 20
Scheme 5. Reaction of phenstatin precursor 7 with trimethoxybenzene (14)
leading to 9H-fluorene product 15.
proceed through ketone 7 protonation, arene addition, fol-
lowed by intramolecular p-cationic cyclization of the unstable
triaryl carbinol in acid media.[29] Consequently, the benzhydrol
intermediates of type II (Scheme 4) emerged as the probable
intermediate in the formation of PAH compound 8.[32]
Having demonstrated that domino reaction based on Frie-
del–Crafts aroylation and arylation mediated by Eaton’s re-
Table 1. Protected phenstatin derivatives and PAH type compounds produced through the domino reaction based on Friedel–Crafts aroylation and aryla-
tion of arylcarboxylic acids.
Entry
Aryl carboxylic acid
Arene
Phenstatin analogue
Yield [%][a]
PAH compound
Yield [%][a]
1
5
6
7[21a] 85
8
9
2
3
4
16
19
22
6
6
6
17
91
18 trace
20
76
21 trace
23[21a] 77
24
0
5
25
6
26
38
27
10
Chem. Eur. J. 2014, 20, 10117 – 10130
www.chemeurj.org
10121
ꢂ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
Table 1. (Continued)
Entry Aryl carboxylic acid
Arene
Phenstatin analogue
Yield [%][a]
PAH compound
Yield [%][a]
6
5
5
5
28
31
34
29[21a] 80
30
33
36
0
11
7
7
32[21a] 60
8
35
60
[a] Isolated yield after flash chromatography purification on silica gel column (EtOAc/n-heptane).
being isolated in 76% yield accompanied by only trace
amounts of product 21, which was not isolated (entry 3). In
the same line, conducting the domino process with 4-(ethoxy-
carbonyloxy)-3,5-dimethoxybenzoic acid (22) was inoperative,
and only product 23 was isolated (77%).[21a] Furthermore, re-
placement of one methoxy group with a methyl group at the
C4-position on the benzene nuclei (entry 5) did not unfavor-
ably affect the domino reaction, demonstrating that the pKa
and/or the nucleophilicity of the arylcarboxylic acids are impor-
tant parameters for the domino process. Under these condi-
tions (entry 5), acid 25 provided ketone 26 in only 38% and
the PAH derivative 27 in 10% yield.
Another series, using 3,4,5-dimethoxybenzoic acid (5) with
2,6-dimethoxyphenyl 2-chloroacetate (28), benzo[d]oxazol-
2(3H)-one (31), and 3-methylbenzo[d]oxazol-2(3H)-one (34)
was investigated. Surprisingly, next to the expected ketones
29,[21a] 32,[21a] and 35, respectively, no domino product 30
(Table 1, entry 6) was obtained with 28, and in the other cases,
PAH derivatives 33 and 36 were isolated in 11 and 7% yield,
respectively (entries 7 and 8). From these results, it seems that
the nucleophilicity of the aryl carboxylic acids is also important
for the domino process.
Scheme 6. Reaction of the protected phenstatin 7 with benzoic acid (37).
nished only one isolated product with the characteristics of
Iasi-red dye in 37% yield after chromatography purification on
silica gel column.
The structure of this product was established on the basis of
an array of spectroscopic analyses including 1D and 2D NMR
spectroscopy, MS as well as microanalysis, and seems to be, to
our surprise, symmetrical polysubstituted PAH rubicene 39
(Scheme 6). This structure was secured without ambiguity on
the basis of X-ray crystallographic analysis of crystals of Iasi-red
product 39, as shown by the ORTEP drawing in Figure 3.[33]
These crystals, which were difficult to obtain, were grown from
anhydrous chloroform and cyclohexane (5:2) by slow evapora-
tion during two weeks after a large screening of gradients of
all the available solvents. Interestingly, the application of simi-
lar conditions to the other Iasi-red products reported in this
Reaction with benzoic acid
To further validate the proposed domino mechanistic scheme
depicted in Scheme 4, the reaction of benzoic acid (37) with
protected phenstatin derivative 7 was then investigated as
a complement to the reaction reported in Scheme 5. Under
the established conditions, using 5 equivalents of Eaton’s re-
agent (Scheme 6), the reaction after 18 h heating at 508C fur-
Chem. Eur. J. 2014, 20, 10117 – 10130
www.chemeurj.org
10122
ꢂ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
posed in Scheme 4 is plausible. However, the role of benzoic
acid in the course of this reaction is unclear. As a consequence,
this protocol merits serious and full examination with respect
to the development of approaches that can be used to access
large PAH compounds, which are often labor-intensive and as-
sociated with low yields.[34]
Chloroester hydrolysis of PAHs compounds
To increase the number of PAH products available for biologi-
cal testing considerations and establish structure-activity rela-
tionships, benzo[a]aceanthrylene derivative 8 and rubicene 39,
were submitted separately to cleavage of the chloroacetyl pro-
tecting group. The reaction with AcONa·3H2O (4.5 equiv) in
MeOH heated to reflux for 3 h, according to our reports,[21a] led
to two different reaction profiles. Whereas hydrolysis of rubi-
cene 39 provided the symmetrical dihydroxy-rubicene 40 in
near quantitative isolated yield (98%) as expected, benzo[a]-
Figure 3. X-Ray crystal structure of rubicene derivative 39. a),b) Top view
with thermal ellipsoids at 30% probability, c),d) packing structure in the
crystal, together with the crystal unit cell, and e) side view.
aceanthrylene
8 led to 2,3,6,8,9,10-hexamethoxyacenaph-
tho[1,2,3,4-klmn]xanthene-5,11-diol (42) in 31% yield after re-
crystallization from ethyl acetate/n-heptane (Scheme 7). The
mechanism of the formation of this compound was tentatively
proposed to start from sodium acetate mediated removal of
the chloroacetyl protective group to give the expected prod-
uct 41, which was in equilibrium in the media with ketone XI.
Oxidation of the anion formed by sodium acetate with oxygen
dissolved in MeOH and protonation then provided alcohol
XII.[35] Dehydration under the influence of acetic acid produced
in the medium led to the stable cationic species XIII, which un-
derwent an intramolecular oxa-cyclization to XIV. After deme-
thylation of this oxonium species, the resulting derivative XV
ultimately furnished acenaphthoxanthene product 42 through
keto-enol equilibrium. Interestingly, no other products with
definite structure rising from this hydrolysis reaction were de-
tected in the reaction medium; however, large amounts of
starting material were recovered. Ultimately, this behavior to-
wards the hydrolysis reaction seems to be due to the existence
paper did not result in crystallization, and no X-ray crystal
structure could be obtained.
In addition to the information collected from the X-ray anal-
ysis of crystals of 39 (Figure 3), another important point is that
the formation of rubicene 39 does not involve benzoic acid
(37) as a reactant partner. Indeed, red compound 39 seems to
be produced by dimerization through intramolecular domino
Friedel–Crafts reaction initiated by the formation of triaryl car-
binol of type II, as shown in Scheme 4. This is the result of nu-
cleophilic attack by the more nucleophilic trimethoxyphenyl
part of ketone 7, on the carbonyl group of a second molecule
of the same substrate 7. It seems that benzoic acid (37), in
contrast to trimethoxybenzoic acid (5; Table 1, entry 1), is not
sufficiently nucleophilic to attack the carbonyl group of ketone
7, and thus PAH compound 38 is not formed. This also lends
supports to the conclusion that the mechanistic pathway pro-
Scheme 7. Cleavage of the chloroacetyl protecting group of benzo[a]aceanthrylene 8 and rubicene 39 PAH derivatives and a corresponding plausible mecha-
nistic scheme.
Chem. Eur. J. 2014, 20, 10117 – 10130
www.chemeurj.org
10123
ꢂ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
of the hydroxyl phenolic group in the benzo[a]aceanthrylene
structure of 8, which is capable of equilibrating with the
ketone function. This phenomenon does not exist in the struc-
ture of PAH-rubicene derivative 39 and, consequently, only
a classical hydrolysis reaction occurred.
Table 2. In vitro growth inhibition ratio [%] caused by the selected com-
pounds against tumor cell lines in the single-dose assay.
Cell type
leukemia
Cell line
Inhibition ratio at 10 mm [%][a]
8
27 33
36
39
40
CCRF-CEM
HL-60 (TB)
MOLT-4
K-562
RPMI-8226
SNB-75
HCT-15
HT29
20 57 n.d.[b] 41
n.d.
n.d.
n.d.
n.d.
n.d.
74
n.d.
n.d.
n.d.
n.d.
n.d.
32
11 34
35 48 11
34 10
9
33
24
26
58
42
10
–
UV/Vis measurements
9
[c]
18 32
–
The optical absorption of benzo[a]aceanthrylene 27, rubicenes
39 and 40, and xanthene 42 are shown in Figure 4. All com-
pounds absorb in the blue (380–401 nm) and green (504–
CNS cancer
colon cancer
–
–
14
16 n.d.
29
–
8
–
87
62
100[d] 100
100[e] 56
KM12
13 19
–
17
non-small cell
lung cancer
melanoma
HOP-92
15
5
23
n.d. n.d.
n.d.
LOX IMVI
SK-MEL-5
OVCAR-5
OVCAR-8
ACHN
–
9
–
–
–
32
31 13
–
52 13
11
5
7
83
40
31
35
21
12
52
77
37
100[f] 27
ovarian cancer
renal cancer
–
57
87
72
72
80
96
3
71
43
6
22
56
6
UO-31
14 29 11
26 34 42
prostate cancer PC-3
breast cancer
MDA-MB-231/ 22 27 16
ATCC
T-47D
Mean [%][g]
38 37 33
18
40
18
42
81
79
22
0
1
[a] Data obtained from NCI’s in vitro disease-oriented human tumor cell
screen at 10 mm concentration.[37] [b] Not determined. [c] Inactive. [d] Posi-
tive cytotoxic effect: cell growth percent of ꢀ10%. [e] Positive cytotoxic
effect: cell growth percent of ꢀ4%. [f] Positive cytotoxic effect: cell
growth percent of ꢀ17%. [g] Average percentage of inhibition of all cell
lines for the tested compounds.
Figure 4. UV/Vis absorption spectra of compounds 27, 39, 40, and 42
(10 mm) in CH2Cl2 as solvent.
570 nm) regions, which is in accordance with their red color.
However, xanthene 42 absorbs much less in the green region
compared with other studied compounds. This may be ex-
plained by the length of conjugation, which, in this case, is
shorter because of the newly formed oxygen-containing ring.
Moreover, its red color is less intense relative to other
compounds.
pounds: 57% inhibition on leukemia CCRF-CEM and 52% in-
hibition on ovarian OVCAR-8 cell lines for pentamethoxy deriv-
ative 27 and 58% inhibition on leukemia RPMI-8226 and 77%
inhibition on prostate PC-3 cell lines for N-methyl-benzoxazo-
lone 36. Their analogues, heptamethoxy derivative 8 and ben-
zoxazolone 33, lost their cytostatic potential. In the rubicene
series, chloroacetic ester 39 and phenol 40 showed significant
cytostatic activity, especially on colon cancer cell lines (total in-
hibition of the growth of HT29 cell lines).
The effect of the pH on the UV/Vis spectra of the studied
compounds was explored, but no influence was observed
(spectra available in the Support-
ing Information).
Biological activity
Six PAH type compounds, 8, 27,
33, 36, 39 and 40, were selected
by NCI for screening against 60
human tumor cell lines.[36] The
compounds were tested initially
at a single high dose (10 mm) in
the full 60-cell panel. The repre-
sentative results are summarized
in Table 2 and Figure 5. In the
pentacyclic PAH series, com-
pounds 27 and 36 exhibited the
most interesting cell growth in-
Figure 5. Representative biological activity of dichloroacetyl rubicene 39 (front row) versus deprotected rubicene
hibition among the tested com- 40 (back row), tested at a single high dose (10ꢀ5 m) in the full NCI 60 cell panel.
Chem. Eur. J. 2014, 20, 10117 – 10130
www.chemeurj.org
10124
ꢂ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
Generally, the deprotected compounds were more active
than their precursors; the free hydroxyl group may be involved
in interactions with different amino acids in the binding site of
the biological target, which we assumed to be, in this case, the
tubulin. Unexpectedly, deprotected rubicene 40 exhibited sig-
nificantly decreased cellular activity on most tested cancer cell
lines compared with protected precursor 39 (Figure 5). There-
fore, only protected rubicene 39 satisfied predetermined
threshold inhibition criteria and progressed to the 5-dose
screen to evaluate its GI50 values (Table 2). This rubicene deriva-
tive exhibited the most important in vitro cytotoxicity among
the studied compounds: inhibition of HL-60(TB), K-562, SR,
HCT-15, KM-12, SW-620, M14, MDA-MB-435, MALME-3M, NCI-
ADR/RES, and SF-539 cell lines with GI50 values in the low
nanomolar range (GI50 <10 nm; Table 3). It is important to
note that the same compound 39 proved to be inactive on mi-
crotubules assembly (Table 4). Together, these results suggest
a different mode of action for rubicene 39 that does not in-
volve the tubulin. However, given its remarkable cytotoxicity
and simple synthesis, this compound deserves further
investigation, both in terms of biological mechanism and
biotransformation.
Table 4. Inhibitory activities on tubulin polymerization.
Entry
Product
TPI [%][a,b]
IC50 [mm][b]
1
2
3
4
5
6
7
8
8
27
33
36
39
40
51
64
42
21
49
13
88
95
n.d.[c]
45.0
n.d.
n.d.
n.d.
n.d.
12.7
15.0
combretastatin A4[d] 3a
phenstatin[d] 4a
[a] Inhibition of tubulin polymerization at 50 mm concentration. [b] Values
represent the mean of two experiments. [c] Not determined. [d] Concern-
ing the tubulin polymerization inhibition measure, see ref. [21a] and the
Supporting Information.
the results of their biological evaluation on tubulin and cancer
cells lines have been reported. These new derivatives showed
significant activities against cellular proliferation and tubulin
polymerization. Rubicene 39 proved to be the most potent
compound synthesized, with cytotoxicity activity against HL-
60(TB), K-562, SR, HCT-15, KM-12, SW-620, M14, MDA-MB-435,
MALME-3M, NCI-ADR/RES, and SF-539 cells in the low nanomo-
lar concentration range. Unexpectedly, the deprotected rubi-
cene 40 showed decreased potency relative to chloroacetic
precursor 39. These results indicate that rubicene derivative 39
is an interesting starting point for further investigation as
a new cytotoxic agent. This compound will be evaluated in the
future against other biological targets to elucidate the mecha-
nism of action and develop structural analogues with im-
proved characteristics.
From the results highlighted in Table 3, compounds with sig-
nificant cytostatic and cytotoxic potency in the single-dose cel-
lular assays were further tested for their ability to interact with
tubulin[37] (Table 4). Interestingly and to our total surprise, the
newly synthesized PAHs such as benzo[a]aceanthrylenes 8 and
27 showed antitubulin properties. Their biological potential
was lower than that of parent phenstatin or CA-4, which are
known for their remarkable bioactivity to this end and are
used as references. However, these derivatives could serve as
lead compounds for designing novel anticancer agents that
target microtubules.
Experimental Section
General remarks
Conclusion
Starting materials are commercially available and were used with-
out further purification. Melting points were measured with a MPA
100 OptiMelt apparatus and are uncorrected. NMR spectra were ac-
quired at 400 MHz for 1H NMR and 100 MHz for 13C NMR with
a Varian 400 MHz Premium Shielded spectrometer. Chemical shifts
(d) are given in parts per million relative to CDCl3 (7.26 ppm;
77.1 ppm). Splitting patterns are designed: s, singlet; d, doublet;
dd, doublet of doublets; t, triplet; m, multiplet, and sym m, sym-
metric multiplet. Coupling constants J are reported in hertz (Hz).
We have developed a rapid domino p-cationic arylation of aro-
matic carboxylic acids, mediated by Eaton’s reagent, for the
synthesis of Iasi-red polymethoxylated-PAHs. This synthetic
route is currently the most straightforward method for obtain-
ing such methoxylated-PAHs. The domino process was general-
ized, the structures of the obtained red products were eluci-
dated and the mechanism for their formation was investigated.
Some of their photophysical properties were established, and
Table 3. GI50 values [nm] for rubicene 39 on different tumor cell lines (see the Supporting Information for reports of biological evaluation and all details of
the measurements).
Cell type
Leukemia
CNS cancer
Colon cancer
Cell line
39: GI50 (nm)
Cell type
Cell line
39: GI50 (nm)
Cell type
HL-60(TB)
<10
Non-Small Cell Lung cancer
HOP-62
40
Renal cancer
786-0
70
K-562
<10
SR
<10
SNB-75
20
Melanoma
M14
<10
Prostate cancer
SF-539
<10
U251
20
SW-620
<10
HCT-15
<10
KM-12
<10
Ovarian cancer
OVCAR-3
20
Breast cancer
MCF-7
NCI-H460
30
NCI-H23
40
MDA-MB-435
<10
MALME-3M
<10
NCI-ADR/RES
<10
SK-OV-3
10
Cell line
39: GI50 (nm)
CAKI-1
600
UO-31
50
PC-3
40
DU-145
30
BT-549
20
HS 578T
20
30
Chem. Eur. J. 2014, 20, 10117 – 10130
www.chemeurj.org
10125
ꢂ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
1
COSY and NOESY techniques were used in the assignment of H–
oxybenzoic acid 16 (5.0 g, 27.4 mmol), 2-methoxyphenyl chloroace-
tate 6 (3.67 g, 18.3 mmol), and Eaton’s reagent (1.82 g P2O5 in
12.3 mL CH3SO3H). The mixture was heated at 508C for 8 h and the
final product was recrystallized from absolute ethanol to give pure
chloroacetate 17 (6.07 g, 91%) as a white solid; m.p. 112–1148C
(EtOH); 1H NMR (CDCl3, 200 MHz): d=3.93 (s, 3H; OCH3), 3.94 (s,
3H; OCH3), 3.97 (s, 3H; OCH3), 4.36 (s, 2H; OCOCH2Cl), 6.92 (d, J=
8.2 Hz, 1H; ArH), 7.06 (d, J=8.6 Hz, 1H; ArH), 7.38 (dd, J=8.2,
1.9 Hz, 1H; ArH), 7.42 (d, J=2.0 Hz, 1H; ArH), 7.58 (d, J=1.9 Hz,
1H; ArH), 7.76 ppm (dd, J=8.6, 2.0 Hz, 1H; ArH); 13C NMR (CDCl3,
100 MHz): d=40.5 (CH2), 56.0 (CH3), 56.1 (CH3), 56.2 (CH3), 109.9
(CH), 111.6 (CH), 112.2 (CH), 124.6 (CH), 124.8 (CH), 130.1 (CH),
130.2 (C), 130.8 (C), 138.7 (C), 149.0 (C), 152.8 (C), 154.2 (C), 165.3
(C), 193.3 ppm (C); IR: n˜ =770, 820, 865, 1021, 1104, 1130, 1226,
1262, 1424, 1509, 1597, 1635, 1789cm–1; elemental analysis calcd
(%) for C18H15O6Cl: C 59.27, H 4.70; found: C 59.32, H 4.49.
1H relationships. HSQC and HMBC techniques were used through-
out for the assignment of the H–13C relationships. Thin-layer chro-
1
matography was conducted on Macherey–Nagel silica gel plates
with a fluorescent indicator and visualized with a UV-lamp at 254
and 366 nm. Column chromatography was performed with a Com-
biFlash Rf Companion (Teledyne-Isco System) using RediSep
packed columns. IR spectra were recorded with a Varian 640-IR
FTIR Spectrometer. UV/Vis spectra were recorded with a Cary UV
100 (Varian) spectrophotometer with cells of 1 cm path length, at
258C. A flow cell was used to allow pH adjustments. Elemental
analyses (C, H, N) of new compounds were determined by the
‘Pꢃle Chimie Molꢀculaire’, Facultꢀ de Sciences Mirande, Universitꢀ
de Bourgogne, Dijon, France.
General procedure A: Friedel–Crafts reaction in the presence
of Eaton’s reagent
2-Methoxy-5-(2-methoxybenzoyl)phenyl
chloroacetate
(20;
Table 1): General procedure A was followed by using 2-methoxy-
benzoic acid (19; 1.31 g, 8.59 mmol), 2-methoxyphenyl chloroace-
tate (6; 1.50 g, 7.48 mmol) and Eaton’s reagent (0.48 g P2O5 in
3.21 mL CH3SO3H). The mixture was heated at 508C for 4 h, and
the final product was recrystallized from absolute ethanol to give
pure chloroacetate 20 (1.9 g, 76%) as an off-white solid; 1H NMR
(CDCl3, 200 MHz): d=3.74 (s, 3H; OCH3), 3.90 (s, 3H; OCH3), 4.33 (s,
2H; OCOCH2Cl), 6.99 (d, J=8.6 Hz, 2H; ArH), 7.04 (td, J=7.6,
1.2 Hz, 1H; ArH), 7.33 (dd, J=7.4, 1.6 Hz, 1H; ArH), 7.46 (td, J=8.6,
1.6 Hz, 1H; ArH), 7.56 (d, J=2.0 Hz, 1H; ArH), 7.75 ppm (dd, J=8.6,
2.0 Hz, 1H; ArH); 13C NMR (CDCl3, 100 MHz): d=40.5 (CH2), 55.6
(CH3), 56.2 (CH3), 111.4 (CH), 111.5 (CH), 111.6 (CH), 120.5 (CH), 124.3
(CH), 129.4 (C), 130.2 (CH), 131.0 (C), 131.8 (CH), 138.9 (C), 154.8 (C),
157.1 (C), 165.2 (C), 194.1 ppm (C); elemental analysis calcd (%) for
C17H15O5Cl: C 61.00, H 4.52; found: C 60.74, H 4.48.
Eaton’s reagent was prepared from phosphorus pentoxide (P2O5)
and methanesulfonic acid (CH3SO3H; weight ratio P2O5/CH3SO3H,
1:10). The mixture was heated at 408C under a nitrogen atmos-
phere until complete homogeneity. Carboxylic acid (1.15–1.5 equiv)
and aromatic derivative (1.0 equiv) were then added and the mix-
ture was heated at 50–608C under an inert atmosphere for 3–30 h.
After cooling to RT, the reaction medium was diluted with di-
chloromethane and carefully poured into a separatory funnel con-
taining aqueous sodium bicarbonate solution (50% NaHCO3). The
aqueous solution was extracted with dichloromethane and the
combined organic layers were dried (MgSO4). Solvent was removed
under reduced pressure to produce a brownish oil. The crude
product was purified by column chromatography on silica gel to
afford pure benzophenones and red by-products.
2-Methoxy-5-(3,4,5-trimethoxybenzoyl)phenyl chloroacetate (7;
Scheme 1):[21a] General procedure A was followed by using 5
(11.42 g, 53.8 mmol), 6 (9.00 g, 44.9 mmol) and Eaton’s reagent
(4.09 g P2O5 in 27.6 mL CH3SO3H). The mixture was heated at 508C
for 3 h. The final brown oil was purified by column chromatogra-
phy on silica gel (EtOAc/n-heptane, 3:7) to give pure chloroacetate
7 (15.1 g, 85%; lit. (with PPA)[23a] 80%) as an off-white solid; m.p.
150–1528C (EtOH/Et2O); TLC: Rf =0.62 (EtOAc/n-heptane, 7:3);
1H NMR (CDCl3, 200 MHz): d=3.89 (s, 6H; 2OCH3), 3.94 (s, 6H;
2OCH3), 4.36 (s, 2H; OCOCH2Cl), 7.03 (s, 2H; ArH), 7.07 (d, J=
8.6 Hz, 1H; ArH), 7.62 (d, J=2.2 Hz, 1H; ArH), 7.79 ppm (dd, J=8.6,
2.2 Hz, 1H; ArH); elemental analysis calcd (%) for C19H19O7Cl: C
57.80, H 4.85; found: C 57.74, H 4.78.
5-{4-[(Ethoxycarbonyl)oxy]-3,5-dimethoxybenzoyl}-2-methoxy-
phenyl chloroacetate (23; Table 1):[21a] General procedure A was
followed by using 4-[(ethoxycarbonyl)oxy]-3,5-dimethoxybenzoic
acid (22; 10.10 g, 37.38 mmol), 2-methoxyphenyl chloroacetate (6;
5.00 g, 24.92 mmol), and Eaton’s reagent (3.67 g P2O5 in 24.80 mL
CH3SO3H). The mixture was heated at 508C for 3 h and the result-
ing brown oil was crystallized in 95% EtOH to give pure product
1
23 (8.70 g, 77%) as a white solid; m.p. 171–1728C (EtOH); H NMR
(CDCl3, 200 MHz): d=1.41 (t, J=7.1 Hz, 3H; OCO2CH2CH3), 3.87 (s,
6H; 2OCH3), 3.94 (s, 3H; OCH3), 4.35 (q, J=7.1 Hz, 2H;
OCOCH2CH3), 4.37 (s, 2H; OCOCH2Cl), 7.02 (s, 2H; ArH), 7.07 (d, J=
8.5 Hz, 1H; ArH), 7.63 (d, J=2.2 Hz, 1H; ArH), 7.81 ppm (dd, J=8.5,
2.2 Hz, 1H; ArH); 13C NMR (CDCl3, 50 MHz): d=13.9 (CH3), 40.2
(CH2), 56.0 (2CH3), 56.2 (CH3), 65.0 (CH2), 106.5 (2CH), 111.5 (CH),
124.6 (CH), 129.7 (CH), 129.9 (C), 130.3 (C), 135.4 (C), 138.5 (C),
151.9 (2C), 152.3 (C), 154.4 (C), 165.2 (C), 193.2 ppm (C); IR: n˜ =
760, 1105, 1127, 1209, 1253, 1336, 1409, 1597, 1651, 1770,
1792 cm–1; elemental analysis calcd (%) for C21H21O9Cl: C 55.70, H
4.67; found: C 55.64, H 4.79.
8-Hydroxy-3,5,6,7,10,11,12-heptamethoxybenzo[a]aceanthrylen-
2-yl chloroacetate (8; Table 1): By-product from the synthesis of 7.
Red solid (2.3 g, 9%); m.p. 182–1858C (EtOAc/n-heptane); Rf =0.74
1
(EtOAc); H NMR (CDCl3, 400 MHz): d=3.84 (s, 3H; OCH3), 4.00 (s,
3H; OCH3), 4.06 (s, 6H; 2OCH3), 4.15 (s, 3H; OCH3), 4.22 (s, 3H;
OCH3), 4.34 (s, 3H; OCH3), 4.45 (s, 2H; OCOCH2Cl), 7.58 (s, 1H; ArH),
7.88 (s, 1H; ArH), 8.48 (s, 1H; ArH), 9.80 ppm (s, 1H; ArOH);
13C NMR (CDCl3, 100 MHz): d=41.0 (CH2), 55.9 (CH3), 56.2 (CH3),
61.4 (CH3), 61.4 (CH3), 61.6 (CH3), 61.6 (CH3), 62.3 (CH3), 77.2 (CH),
98.0 (CH), 106.8 (C), 107.4 (CH), 119.1 (C), 119.2 (C), 120.4 (CH),
121.2 (C), 123.5 (C), 127.6 (C), 132.9 (C), 135.0 (C), 138.4 (C), 142.6
(C), 144.8 (C), 148.2 (C), 148.4 (C), 149.0 (C), 150.2 (C), 150.9 (C),
166.1 ppm (C); IR: n˜ =847, 1069, 1112, 1254, 1291, 1312, 1453,
1606, 1674, 3341 cm–1; LC/MS (APCI+): m/z: 571.8 [M+H]+; elemen-
tal analysis calcd (%) for C29H27O10Cl: C 60.71, H 5.09; found: C
60.76, H 5.00.
5-(3,5-Dimethoxy-4-methylbenzoyl)-2-methoxyphenyl chloroace-
tate (26; Table 1): General procedure A was followed by using 3,5-
dimethoxy-4-methyllbenzoic acid (25; 3.80 g, 19.5 mmol), 2-me-
thoxyphenyl chloroacetate (6; 3.60 g, 17.7 mmol), and Eaton’s re-
agent (1.82 g P2O5 in 12.3 mL CH3SO3H). The mixture was heated at
608C for 4 h and the final brown oil was purified by column chro-
matography on silica gel (EtOAc/n-heptane, 15:85!3:7) and the
resulting solid was recrystallized from diethyl ether to give pure
chloroacetate 26 (2.58 g, 38%) as white crystals; m.p. 125–1308C
(Et2O); 1H NMR (CDCl3, 400 MHz): d=2.17 (s, 3H; ArCH3), 2.85 (s,
6H; 2OCH3), 3.94 (s, 3H; OCH3), 4.36 (s, 2H; OCOCH2Cl), 6.95 (s,
2H; 2ArH), 7.06 (d, J=8.6 Hz, 1H; ArH), 7.63 (d, J=1.9 Hz, 1H;
5-(3,4-Dimethoxybenzoyl)-2-methoxyphenyl chloroacetate (17;
Table 1): General procedure A was followed by using 3,4-dimeth-
Chem. Eur. J. 2014, 20, 10117 – 10130
www.chemeurj.org
10126
ꢂ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
ArH), 7.81 ppm (dd, J=8.6, 1.9 Hz, 1H; ArH); 13C NMR (CDCl3,
100 MHz): d=8.7 (CH3), 40.5 (CH2), 55.9 (2CH3), 56.2 (CH3), 105.3
(2CH), 111.7 (CH), 119.8 (C), 124.9 (CH), 130.3 (CH), 130.6 (C), 135.8
(C), 138.7 (C), 154.4 (C), 158.0 (2C), 165.4 (C), 194.3 ppm (C); IR: n˜ =
760, 821, 1015, 1135, 1234, 1268, 1406, 1606, 1639, 1788 cm–1; ele-
mental analysis calcd (%) for C19H19O6Cl: C 60.24, H 5.06; found: C
60.55, H 4.98.
OCH3), 3.99 (s, 3H; OCH3), 4.02 (s, 3H; OCH3), 4.04 (s, 3H; OCH3),
4.18 (s, 3H; OCH3), 4.25 (s, 3H; OCH3), 7.62 (s, 1H; ArH), 7.81 (s, 1H;
ArH), 8.49 (s, 1H; ArH), 10.06 (s, 1H; ArOH), 11.62 ppm (s, 1H;
ArNH); 13C NMR (CDCl3 + [D6]DMSO, 100 MHz): d=55.7 (CH3), 61.1
(CH3), 61.2 (CH3), 61.4 (2CH3), 62.2 (CH3), 98.2 (CH), 104.7 (C), 106.5
(C), 110.4 (C), 119.0 (C), 119.2 (C), 120.6 (C), 123.2 (C), 127.0 (C),
127.5 (CH), 131.8 (C), 134.2 (CH), 142.5 (C), 143.6 (C), 144.7 (C),
147.9 (C), 148.5 (C), 150.3 (C), 150.4 (C), 150.6 (C), 155.9 ppm (C);
IR: n˜ =850, 938, 1112, 1129, 1298, 1425, 1503, 1599, 1638, 1793,
3019, 3339 cm–1; LC/MS (APCI+): m/z: 506.1 [M+H]+; elemental
analysis calcd (%) for C27H23O9N: C 64.16, H 4.59, N 2.77; found: C
64.52, H 4.63, N 2.89.
8-Hydroxy-3,5,7,10,12-pentamethoxy-6,11-dimethylbenzo[a]-
aceanthrylen-2-yl chloroacetate (27; Table 1): By-product from
the synthesis of compound 26. Red solid (0.95 g, 10% yield); m.p.
175–1808C (EtOAc/n-heptane); 1H NMR (CDCl3, 400 MHz): d=2.45
(s, 3H; ArCH3), 2.53 (s, 3H; ArCH3), 3.68 (s, 3H; OCH3), 4.00 (s, 3H;
OCH3), 4.03 (s, 3H; OCH3), 4.09 (s, 3H; OCH3), 4.10 (s, 3H; OCH3),
4.46 (s, 2H; OCOCH2Cl), 7.54 (s, 1H; ArH), 7.85 (s, 1H; ArH), 8.50 (s,
1H; ArH), 9.77 ppm (s, 1H; ArOH); 13C NMR (CDCl3, 100 MHz): d=
9.7 (CH3), 9.8 (CH3), 41.0 (CH2), 55.6 (CH3), 56.2 (CH3), 60.9 (CH3),
61.1 (CH3), 62.2 (CH3), 96.2 (CH), 107.3 (CH), 107.5 (CH), 119.3 (C),
120.5 (CH), 121.4 (C), 121.7 (C), 121.9 (C), 122.2 (C), 124.2 (C), 130.2
(C), 133.3 (C), 134.9 (C), 138.5 (C), 148.3 (C), 149.7 (C), 154.2 (C),
154.8 (C), 155.2 (C), 155.3 (C), 166.1 ppm (C); IR: n˜ =837, 979, 1090,
1120, 1141, 1210, 1292, 1462, 1612, 1780, 3345 cm–1; elemental
analysis calcd (%) for C29H27O8Cl: C 64.63, H 5.05; found: C 65.01, H
5.17.
3-Methyl-6-(3,4,5-trimethoxybenzoyl)-1,3-benzoxazol-2(3H)-one
(35; Table 1): General procedure A was followed by using 3,4,5-tri-
methoxybenzoic acid (5; 1.64 g, 7.73 mmol), 3-methyl-1,3-benzoxa-
zol-2(3H)-one (34; 1.0 g, 6.7 mmol), and Eaton’s reagent (0.60 g
P2O5 in 4.0 mL CH3SO3H). The mixture was heated at 608C for 20 h
and the final brown oil was purified by column chromatography
on silica gel (EtOAc/n-heptane, 4:6) to give pure 35 (1.38 g, 60%)
as a white solid; m.p. 155–1568C (EtOAc/n-heptane); 1H NMR
(CDCl3, 400 MHz): d=3.49 (s, 3H; NCH3), 3.89 (s, 6H; 2OCH3), 3.95
(s, 3H; OCH3), 7.02 (s, 2H; ArH), 7.06 (d, J=8.1 Hz, 1H; ArH), 7.72
(dd, J=3.6, 1.6 Hz, 1H; ArH), 7.74 ppm (dd, J=10.9, 1.5 Hz, 1H;
ArH); 13C NMR (CDCl3, 100 MHz): d=24.4 (CH3), 56.3 (2CH3), 61.0
(CH3), 107.3 (CH), 107.6 (2CH), 111.6 (CH), 127.2 (CH), 132.5 (C),
132.6 (C), 135.4 (C), 142.1 (C), 142.3 (C), 152.9 (2C), 154.6 (C),
194.1 ppm (C); elemental analysis calcd (%) for C18H17O6N: C 62.97,
H 4.99, N 4.08; found: C 62.87, H 4.91, N 4.29.
2,6-Dimethoxy-3-(3,4,5-trimethoxybenzoyl)phenyl chloroacetate
(29; Table 1):[21a] General procedure A was followed by using 3,4,5-
trimethoxybenzoic acid (5; 1.25 g, 5.9 mmol), 2,6-dimethoxyphenyl
chloroacetate (28; 0.90 g, 3.9 mmol), and Eaton’s reagent (0.57 g
P2O5 in 3.83 mL CH3SO3H). The mixture was heated at 608C for 5 h
and the final brown oil was purified on silica gel (EtOAc/n-heptane,
4:6) to give pure chloroacetate 29 (1.35 g, 80%) as an off-white
5-Hydroxy-1,2,3,6,7,8-hexamethoxy-10-methylbenzo-
[1,2]fluorantheno[8,9-d]-[1,3]oxazol-11(10H)-one (36; Table 1):
By-product from the synthesis of 35. Red solid (0.24 g, 7% yield);
m.p.>2508C (EtOAc/n-heptane); 1H NMR (CDCl3, 400 MHz): d=
3.53 (s, 3H; NCH3), 3.85 (s, 3H; OCH3), 4.07 (s, 6H; 2OCH3), 4.18 (s,
3H; OCH3), 4.26 (s, 3H; OCH3), 4.34 (s, 3H; OCH3), 7.61 (s, 1H; ArH),
7.80 (s, 1H; ArH), 8.72 (s, 1H; ArH), 9.86 ppm (s, 1H; ArOH);
13C NMR (CDCl3, 100 MHz): d=28.3 (CH3), 55.9 (CH3), 61.3 (CH3),
61.4 (CH3), 61.6 (CH3), 61.6 (CH3), 62.3 (CH3), 98.1 (CH), 102.9 (CH),
106.6 (C), 108.6 (CH), 119.2 (C), 119.3 (C), 120.8 (C), 123.7 (C), 127.2
(C), 128.9 (C), 132.1 (C), 135.1 (C), 142.4 (C), 142.6 (C), 145.1 (C),
148.1 (C), 148.8 (C), 150.5 (C), 150.6 (C), 150.9 (C), 155.6 ppm (C);
LC/MS (APCI+): m/z: 520.2 [M+H]+; elemental analysis calcd (%) for
C28H25O9N: C 64.74, H 4.85, N 2.70; found: C 64.36, H 5.03, N 3.06.
1
solid; m.p. 118–1208C (EtOH); H NMR (CDCl3, 400 MHz): d=3.68 (s,
3H; OCH3), 3.86 (s, 6H; 2OCH3), 3.91 (s, 3H; OCH3), 3.93 (s, 3H;
OCH3), 4.38 (s, 2H; OCOCH2Cl), 6.81 (d, J=8.6 Hz, 1H; ArH), 7.10 (s,
2H; ArH), 7.33 ppm (d, J=8.6 Hz, 1H; ArH); 13C NMR (CDCl3,
100 MHz): d=40.4 (CH2), 56.2 (2CH3), 56.3 (CH3), 60.9 (CH3), 62.6
(CH3), 106.9 (CH), 107.4 (2CH), 125.7 (C), 128.3 (CH), 132.3 (C), 132.5
(C), 142.6 (C), 151.8 (C), 152.9 (2C), 154.3 (C), 165.0 (C), 193.5 ppm
(C). IR: n˜ =522, 758, 814, 1093, 1120, 1293, 1290, 1414, 1489, 1667,
1754, 1781 cm–1; elemental analysis calcd (%) for C20H21O8Cl: C
56.54, H 4.98; found: C 56.37, H 4.81.
6-(3,4,5-Trimethoxybenzoyl)-1,3-benzoxazol-2(3H)-one
(32;
Table 1):[21a] General procedure A was followed by using 1,3-ben-
zoxazol-2(3H)-one (31; 4.00 g, 29.6 mmol) and Eaton’s reagent
(4.00 g P2O5 in 27.00 mL CH3SO3H). 3,4,5-Trimethoxybenzoic acid
(5; 10.99 g, 51.8 mmol) was added to the reaction mixture in small
portions (1.0 g every 30 min) and the resulting viscous solution
was heated at 608C for 30 h. The final brown oil was purified by
column chromatography on silica gel (EtOAc/n-heptane, 1:9) to
give pure compound 32 (2.24 g, 23%) as a beige solid; m.p. 220–
2218C (EtOAc/n-heptane). 1H NMR (CDCl3, 400 MHz): d=3.89 (s,
6H; 2OCH3), 3.95 (s, 3H; OCH3), 7.02 (s, 2H; ArH), 7.16 (d, J=
8.6 Hz, 1H; ArH), 7.71 (dd, J=8.6, 1.7 Hz, 1H; ArH), 7.73 (d, J=
5-{4-[(Chloroacetyl)oxy]-3,5-dimethoxybenzoyl}-2-methoxyphe-
nyl chloro-acetate (9), 5-{3,5-dimethoxy-4-[(methylsulfonyl)oxy]-
benzoyl}-2-methoxy-phenyl chloroacetate (10), 2,6-dimethoxy-4-
{4-methoxy-3-[(methylsulfonyl)-oxy]benzoyl}phenyl methanesul-
fonate (11), and 5-(chloroacetyl)-2-methoxy-phenyl chloroace-
tate (12; Scheme 2): Eaton’s reagent was prepared from phospho-
rus pentoxide (P2O5) and methanesulfonic acid (CH3SO3H; weight
ratio P2O5/CH3SO3H, 1:10). The mixture was heated at 408C under
a nitrogen atmosphere until complete homogeneity. Chloroacetate
(7; 5 g, 12.7 mmol) was then added to Eaton’s reagent and the
mixture was heated at 608C under an inert atmosphere for 18 h.
After cooling to RT, the reaction medium was diluted with di-
chloromethane and carefully poured into a separatory funnel con-
taining sodium bicarbonate aqueous solution (50% NaHCO3). The
aqueous solution was extracted with dichloromethane and the
combined organic layers were dried (MgSO4). Solvent was removed
under reduced pressure to give a brownish oil. No red coloration
of the media was observed, but a multitude of products were
formed, from which four compounds were isolated and character-
ized. The crude product was purified by column chromatography
on silica gel (EtOAc/n-heptane, 1:9) to give pure compounds 9
1.7 Hz, 1H; ArH), 8.32 ppm (large s, 1H; NH); 13C NMR (CDCl3
+
[D6]DMSO, 100 MHz): d=56.9 (2CH3), 61.4 (CH3), 108.1 (2CH), 109.8
(CH), 111.5 (CH), 127.8 (CH), 132.3 (C), 133.5 (C), 135.4 (C), 142.3 (C),
144.3 (C), 153.5 (2C), 155.7 (C), 194.7 ppm (C); IR: n˜ =583, 704, 815,
922, 1104, 1119, 1290, 1413, 1493, 1574, 1643, 1773, 3021 cm–1; ele-
mental analysis calcd (%) for C17H15O6N: C 62.00, H 4.59, N 4.25;
found: C 61.89, H 4.40, N 4.19.
5-Hydroxy-1,2,3,6,7,8-hexamethoxybenzo[1,2]fluorantheno[8,9-
d][1,3]oxazol-11(10H)-one (33; Table 1): By-product from the syn-
thesis of compound 32. Red solid (1.65 g, 11% yield); m.p.>2508C
(EtOAc/n-heptane); 1H NMR ([D6]DMSO, 400 MHz): d=3.77 (s, 3H;
Chem. Eur. J. 2014, 20, 10117 – 10130
www.chemeurj.org
10127
ꢂ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
(1.33 g, 23%), 10 (0.58 g, 10%), 11 (0.70 g, 12%), and 12 (0.17 g,
5%).
General procedure B: synthesis of phenols from chloroacetic
esters
Compound 9: White solid; 1H NMR (CDCl3, 400 MHz): d=3.85 (s,
6H; 2OCH3), 3.94 (s, 3H; OCH3), 4.36 (s, 2H; OCOCH2Cl), 4.41 (s,
2H; OCOCH2Cl), 7.02 (s, 2H; ArH), 7.07 (d, J=8.8 Hz, 1H; ArH), 7.63
(d, J=2.4 Hz, 1H; ArH), 7.81 ppm (dd, J=8.8, 2.4 Hz, 1H; ArH);
13C NMR (CDCl3, 100 MHz): d=40.4 (CH2), 40.5 (CH2), 56.3 (CH3),
56.4 (2CH3), 106.6 (2CH), 111.8 (CH), 125.0 (CH), 129.9 (C), 130.4
(CH), 131.4 (C), 136.1 (C), 138.8 (C), 151.9 (2C), 154.7 (C), 164.8 (C),
165.4 (C), 193.3 ppm (C); elemental analysis calcd (%) for
C20H18O8Cl2: C 52.53, H 3.97; found: C 52.56, H 3.97.
(Mono or di)chloroacetic ester (1.0 equiv) and sodium acetate (AcO-
Na·3H2O; 4.5 equiv for monochloroacetic ester and 9.0 equiv for di-
chloroacetic ester) were dissolved in MeOH. The solution was
heated to refluxed for 5 h. After cooling to RT, the mixture was
concentrated under reduced pressure and the residue was taken
into distilled water. The resulting precipitate was filtered, washed
with water several times to remove remaining sodium acetate, and
recrystallized from absolute EtOH or purified by flash chromatogra-
phy to obtain pure phenols.
1
Compound 10: Colorless solid; H NMR (CDCl3, 400 MHz): d=3.36
(s, 3H; OSO2CH3), 3.92 (s, 6H; 2OCH3), 3.94 (s, 3H; OCH3), 4.37 (s,
2H; OCOCH2Cl), 7.02 (s, 2H; ArH), 7.07 (d, J=8.8 Hz, 1H; ArH), 7.62
(d, J=2.0 Hz, 1H; ArH), 7.80 ppm (dd, J=8.8, 2.0 Hz, 1H; ArH);
13C NMR (CDCl3, 100 MHz): d=40.2 (CH3), 40.5 (CH2), 56.3 (CH3),
56.6 (2CH3), 106.7 (2CH), 111.9 (CH), 124.9 (CH), 129.7 (C), 130.4
(CH), 131.0 (C), 136.6 (C), 138.7 (C), 153.1 (2C), 154.9 (C), 165.4 (C),
193.1 ppm (C); elemental analysis calcd (%) for C19H19ClO9S: C
49.73, H 4.17, S 6.99; found: C 49.68, H 4.02, S 7.41.
1,2,3,5,8,9,10,12-Octamethoxyrubicene-6,13-diol (40; Scheme 7):
General procedure B was followed by using dichloroacetate 39
(0.08 g, 0.106 mmol) and sodium acetate (AcONa·3H2O; 0.13 g,
0.958 mmol) in MeOH (5 mL). The resulting solid was collected by
filtration and recrystallized from absolute EtOH to obtain diphenol
1
40 (52 mg, 82%) as a dark-red solid. H NMR (CDCl3, 400 MHz): d=
4.05 (s, 6H; 2OCH3), 4.08 (s, 6H; 2OCH3), 4.16 (s, 6H; 2OCH3), 4.23
(s, 6H; 2OCH3), 5.70 (s, 2H; 2ArOH), 7.76 (s, 2H; 2ArH), 8.50 ppm (s,
2H; 2ArH); 13C NMR (CDCl3, 100 MHz): d=56.2 (2CH3), 61.2 (2CH3),
61.5 (2CH3), 61.7 (2CH3), 106.7 (2CH), 113.4 (2CH), 119.7 (2C), 123.1
(2C), 130.2 (2C), 130.8 (2C), 131.6 (2C), 132.4 (2C), 145.0 (2C),
146.0 (2C), 147.6 (2C), 149.5 (2C), 149.8 ppm (C); LC/MS (APCI+):
m/z: 599.2 [M+H]+.
Compound 11: Colorless solid; 1H NMR (CDCl3, 400 MHz): d=3.25
(s, 3H; OSO2CH3), 3.35 (s, 3H; OSO2CH3), 3.93 (s, 6H; 2OCH3), 4.01
(s, 3H; OCH3), 7.06 (s, 2H; ArH), 7.13 (d, J=8.8 Hz, 1H; ArH), 7.81
(d, J=2.0 Hz, 1H; ArH), 7.88 ppm (dd, J=8.8, 2.0 Hz, 1H; ArH);
13C NMR (CDCl3, 100 MHz): d=38.8 (CH3), 40.2 (CH3), 56.4 (CH3),
56.6 (2CH3), 106.9 (2CH), 112.6 (CH), 127.0 (CH), 130.0 (C), 130.9
(CH), 131.2 (C), 136.1 (C), 137.4 (C), 153.2 (2C), 155.4 (C), 192.6 ppm
(C); elemental analysis calcd (%) for C18H20O10S2: C 46.95, H 4.38, S
13.93; found: C 46.81, H 4.41, S 13.79.
5-Hydroxy-2,3,6,8,9,10-hexamethoxyacenaphtho[1,2,3,4-
klmn]xanthen-11(11cH)-one (42; Scheme 7): General procedure B
was followed by using chloroacetate 8 (0.2 g, 0.35 mmol) and
sodium acetate (AcONa·3H2O; 0.21 g, 1.54 mmol) in MeOH (5 mL).
After evaporation of the methanol in vacuo, the residue was
poured in distilled water. The residue was purified by column chro-
matography (EtOAc/n-heptane, 15:85) and recrystallized from EtOH
to obtain heterocycle 42 (52 mg, 31%) as a red solid. 1H NMR
(CDCl3, 400 MHz): d=3.48 (s, 3H; OCH3), 3.61 (s, 3H; OCH3), 3.80 (s,
3H; OCH3), 3.91 (s, 3H; OCH3), 3.95 (s, 3H; OCH3), 4.16 (s, 3H;
OCH3), 4.76 (s, 1H; ArH), 6.86 (s, 1H; ArH), 7.35 ppm (s, 1H; ArH);
13C NMR (CDCl3, 100 MHz): d=54.9 (CH3), 56.1 (CH3), 60.6 (CH3),
60.7 (CH3), 61.0 (CH3), 61.5 (CH3), 62.0 (CH3), 97.6 (CH), 107.4 (CH),
115.5 (CH), 117.2 (C), 118.7 (C), 121.6 (C), 129.1 (C), 132.3 (C), 132.5
(C), 143.4 (C), 145.3 (C), 145.5 (C), 149.9 (C), 154.0 (C), 154.2 (C),
155.1 (C), 156.8 (C), 178.3 (C), 195.9 ppm (C); LC/MS (APCI+): m/z:
479.1 [M+H]+.
Compound 12: Beige solid; 1H NMR (CDCl3, 400 MHz): d=3.93 (s,
3H; OCH3), 4.36 (s, 2H; OCOCH2Cl), 4.63 (s, 2H; OCOCH2Cl), 7.05 (d,
J=8.6 Hz, 1H; ArH), 7.73 (d, J=2.1 Hz, 1H; ArH), 7.91 ppm (dd, J=
8.6, 2.1 Hz, 1H; ArH); 13C NMR (CDCl3, 100 MHz): d=40.4 (CH2), 45.4
(CH2), 56.3 (CH3), 112.0 (CH), 123.2 (CH), 127.4 (C), 129.0 (CH), 139.4
(C), 155.6 (C), 165.1 (C), 188.9 ppm (C); elemental analysis calcd (%)
for C11H10O4Cl2: C 47.68, H 3.64; found: C 48.00, H 3.99.
13-[(Chloroacetyl)oxy]-1,2,3,5,8,9,10,12-octamethoxyrubicen-6-yl
chloroacetate (39; Scheme 6): Eaton’s reagent was prepared from
phosphorus pentoxide (P2O5) and methanesulfonic acid (CH3SO3H;
weight ratio P2O5/CH3SO3H, 1:10). The mixture was heated at 408C
under a nitrogen atmosphere until complete homogeneity. Chlor-
oacetate 7 (0.3 g, 0.76 mmol) and benzoic acid 37 (0.093 g,
0.76 mmol) were then added and the mixture was heated at 508C
under an inert atmosphere for 18 h. The mixture became red very
quickly. After cooling to RT, the reaction medium was diluted with
dichloromethane and carefully poured into a separatory funnel
containing aqueous sodium bicarbonate solution (50% NaHCO3).
The aqueous solution was extracted with dichloromethane, and
the combined organic layers were dried (MgSO4). Solvent was re-
moved under reduced pressure to give a red dark oil. The crude
product was purified by column chromatography on silica gel
(EtOAc/n-heptane, 2:8!6:4) to obtain rubicene derivative 39
Cell proliferation assay:[36] The compounds were tested against
a panel of 60 human cancer cell lines at the National Cancer Insti-
tute, Rockville, MD. The cytotoxicity studies were conducted using
a 48 h exposure protocol using the sulforhodamine B assay.
Tubulin studies: All the studied compounds and the reference
compounds (phenstatin and CA-4) were tested under identical op-
erating conditions. Turbidimetric assays of microtubules were per-
formed as described,[37] except when 50–150 mL quartz microcuv-
ettes were utilized instead of 96-well plates. Lyophilized bovine
brain tubulin (97%) was purchased from Cytoskeleton Inc. and was
reconstituted to 5 mgmLꢀ1 with buffer (80 mm PIPES, pH 6.9, 2 mm
MgCl2, 0.5 mm EGTA, 1.0 mm GTP, 5% glycerol). To this solution
(63 mL) in an ultramicro quartz cuvette at 08C was added test com-
pound in dimethyl sulfoxide (7 mL). The increase in absorbance was
monitored at 340 nm and 378C with a HekIOS Gamma&Delta. The
samples were prepared in duplicate. The IC50 value was defined as
the compound molar concentration that inhibited the extent of as-
sembly by 50% after 30 min incubation.
1
(106 mg, 37%) as a red solid; H NMR (CDCl3, 400 MHz): d=4.02 (s,
6H; 2OCH3), 4.04 (s, 6H; 2OCH3), 4.18 (s, 6H; 2OCH3), 4.26 (s, 6H;
2OCH3), 4.45 (s, 4H; 2OCOCH2Cl), 7.89 (s, 2H; 2ArH), 8.58 ppm (s,
2H; 2ArH); 13C NMR (CDCl3, 100 MHz): d=40.9 (2CH2), 56.2 (2CH3),
61.3 (2CH3), 61.6 (2CH3), 61.9 (2CH3), 107.5 (2CH), 119.5 (2C), 121.0
(2CH), 123.8 (2C), 130.3 (2C), 130.9 (2C), 131.2 (2C), 137.2 (2C),
138.2 (2C), 147.5 (2C), 150.2 (2C), 150.9 (2C), 151.1 (2C),
166.0 ppm (2C); elemental analysis calcd (%) for C38H32Cl2O12: C
60.73, H 4.29; found: C 61.01, H 4.40.
Chem. Eur. J. 2014, 20, 10117 – 10130
www.chemeurj.org
10128
ꢂ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
[15] C. Kitamura, Y. Abe, T. Ohara, A. Yoneda, T. Kawase, A. Kobayashi, H.
Naito, Chem. Eur. J. 2010, 16, 890–898.
Acknowledgements
[16] P. M. Byers, I. V. Alabugin, J. Am. Chem. Soc. 2012, 134, 9609–9614, and
references cited therein.
The authors gratefully acknowledge the Conseil Rꢀgional
Nord-Pas-de-Calais and Janssen Research & Development (Divi-
sion of Janssen Pharmaceutica NV) for financial support. This
work was also supported by a grant of the Romanian Ministry
of Education, CNCS-UEFISCDI, project number PN-II-RU-PD-
2012-3-0426. The authors acknowledge the National Cancer In-
stitute (NCI) for biological evaluation of compounds on their
60-cell panel: the testing was performed by the Developmen-
tal Therapeutics Program, Division of Cancer Treatment and Di-
agnosis (the URL to the Program’s website: http://dtp.cancer.-
gov). The authors would like to thank warmly Dr. J. Pommery
for the biological evaluation of compounds on tubulin
polymerization.
[17] a) H.-H. Hseuh, M.-Y. Hsu, T.-L. Wu, R.-S. Liu, J. Org. Chem. 2009, 74,
8448–8451; b) Y.-H. Kung, Y.-S. Cheng, C.-C. Tai, W.-S. Liu, C.-C. Shin, C.-
C. Ma, Y.-C. Tsai, T.-C. Wu, M.-Y. Kuo, Y.-T. Wu, Chem. Eur. J. 2010, 16,
5909–5919; c) I. Ullah, M. Nawaz, A. Villinger, P. Langer, Tetrahedron Lett.
2011, 52, 1888–1890; d) A. R. Mohebbi, F. Wudl, Chem. Eur. J. 2011, 17,
2642–2646; e) B. Kumar, C. E. Strasser, B. T. King, J. Org. Chem. 2012, 77,
311–316.
[18] a) M. Hashimoto, T. Horiuchi, S. Igawa, J. Kamatani, M. Nakasu, S. Okada,
N. Yamada, Jpn Patent WO2008/140132, 2008; b) A. H. Sato, M. Maeda,
S. Mihara, T. Iwasawa, Tetrahedron Lett. 2011, 52, 6284–6287; c) K. Kawa-
sumi, K. Mochida, T. Kajino, Y. Segawa, K. Itami, Org. Lett. 2012, 14, 418–
421.
[19] a) A. V. Zabula, C. Dubceac, A. S. Filatov, M. A. Petrukhina, J. Org. Chem.
2011, 76, 9572–9576; b) J. A. Clement, R. Sivasakthikumaran, A. K. Mo-
hanakrishnan, S. Sundaramoorthy, D. Velmurugan, Eur. J. Org. Chem.
2011, 569–577; c) Y. Li, W.-K. Heng, B. S. Lee, N. Aratani, J. L. Zafra, N.
Bao, R. Lee, Y. M. Sung, Z. Sun, K.-W. Huang, R. D. Webster, J. T. L. Navar-
rete, D. Kim, A. Osuka, J. Casado, J. Ding, J. Wu, J. Am. Chem. Soc. 2012,
134, 14913–14922; d) P. Sarkar, P. Dechambenoit, F. Durola, H. Bock,
Asian J. Org. Chem. 2012, 1, 366–376.
Keywords: antitumor agents
·
conjugation
·
domino
reactions · dyes/pigments · polycycles
[20] G. R. Pettit, S. B. Singh, E. Hamel, C. M. Lin, D. S. Alberts, D. Garcia-Ken-
dall, Experientia 1989, 45, 209–211.
[1] a) D. P. Arfsten, D. J. Schaeffer, D. C. Mulveny, Ecotoxicol. Environ. Saf.
1996, 33, 1–24; b) C. F. Moffat, K. J. Whittle, in Environmental Contami-
nants in Food (Eds.: C. F. Moffat, K. J. Whittle), Sheffield Academic Press,
Sheffield, 1999, p.364; c) R. Eisler, Handbook of Chemical Risk Assess-
ment: Health Hazards to Humans, Plants and Animals, Vol. 2, Lewis, Boca
Raton, 2000, p.1500.
[2] a) R. G. Harvey, Cambridge Monographs on Cancer Research, Polycyclic Ar-
omatic Hydrocarbons: Chemistry and Carcinogenicity, Cambridge Univer-
sity Press, Cambridge, 1991; b) R. G. Harvey, in Chemical Carcinogenesis
(Ed.: T. M. Penning), Humana, New York, 2011; Chapter 1, pp. 1–26.
[3] a) R. Dabestani, I. N. lvanov, Photochem. Photobiol. 1999, 70, 10–34;
b) A. Wu, D. Xu, D. Lu, T. M. Penning, I. A. Blair, R. G. Harvey, Tetrahedron
2012, 68, 7217–7233, and references cited therein; c) A. I. Barrado, S.
Garcia, Y. Castrillejo, R. M. Perez, Atmos. Pollut. Res. 2012, 3, 81–87, and
references cited therein.
[21] a) A. Ghinet, B. Rigo, J.-P. Hꢀnichart, D. Le Broc-Ryckewaert, J. Pommery,
N. Pommery, X. Thuru, B. Quesnel, P. Gautret, Bioorg. Med. Chem. 2011,
19, 6042–6054; b) A. Ghinet, A. Tourteau, B. Rigo, V. Stocker, M. Leman,
A. Farce, J. Dubois, P. Gautret, Bioorg. Med. Chem. 2013, 21, 2932–2940;
c) V. Stocker, A. Ghinet, M. Leman, B. Rigo, R. Millet, A. Farce, C. Water-
lot, P. Gautret, D. Desravines, J. Dubois, RSC Adv. 2013, 3, 3683–3696.
[22] a) A. Ghinet, N. Van Hijfte, P. Gautret, B. Rigo, H. Oulyadi, J. Rousseau,
Tetrahedron 2012, 68, 1109–1116, and references cited therein; b) A.
Ghinet, P. Gautret, B. Rigo, Rev. Roum. Chim. 2012, 57, 1065–1072.
[23] a) M. Wu, Q. Ji, C. Yang, Y. Xie, Org. Prep. Proced. Int. 2005, 37, 272–275;
b) J.-F. Masson, Energy Fuels 2008, 22, 2637–2640.
[24] G. R. Pettit, B. Toki, D. L. Herald, P. Verdier-Pinard, M. R. Boyd, E. Hamel,
R. K. Pettit, J. Med. Chem. 1998, 41, 1688–1695.
[25] a) P. E. Eaton, C. R. Carlson, J. T. Lee, J. Org. Chem. 1973, 38, 4071–4073;
b) D. L. Boger, J. Org. Chem. 1978, 43, 2296–2297.
[4] J. M. Riley, S. Alkan, A. Chen, M. Shapiro, W. A. Khan, W. R. Murphy, Jr.,
J. E. Hanson, Macromolecules 2001, 34, 1797–1809.
[26] Eaton’s reagent (see ref. [25]) is a 1:10 solution by weight of phospho-
rus pentoxide in methanesulfonic acid. Organic compounds often dis-
solve rapidly in this mobile liquid and the solutions obtained can be
easily stirred. This condensing agent is often more efficient than poly-
phosphoric acid (PPA), and is rapidly hydrolyzed at the end of the reac-
tion. According to Eaton, methanesulfonic anhydride is formed in this
mixture, but other species are also present, such as polyphosphoric
and mixed anhydrides of polyphosphoric and methanesulfonic acid. For
discussions on these aspects, see: a) D. Zhao, D. L. Hughes, D. R.
Bender, A. M. De Marco, P. J. Reider, J. Org. Chem. 1991, 56, 3001–3006;
b) Y.-H. So, J.-P. Heeschen, J. Org. Chem. 1997, 62, 3552–3561.
[27] We have already encountered such reactions in another context, see
refs. [21a] and [22].
[5] a) X. Dou, X. Yang, G. J. Bodwell, M. Wagner, V. Enkelmann, K. Mꢄllen,
Org. Lett. 2007, 9, 2485–2488; b) R. Chaudhuri, M.-Y. Hsu, C.-W. Li, C.-I.
Wang, C.-J. Chen, C. K. Lai, L.-Y. Chen, S.-H. Liu, C.-C. Wu, R.-S. Liu, Org.
Lett. 2008, 10, 3053–3056; c) S. H. Wadumethrige, R. Rathore, Org. Lett.
2008, 10, 5139–5142; d) R. Yamaguchi, S. Hiroto, H. Shinokubo, Org.
Lett. 2012, 14, 2472–2475.
[6] a) Z. Li, L. Zhi, N. T. Lucas, Z. Wang, Tetrahedron 2009, 65, 3417–3424;
b) Z. U. Levi, T. D. Tilley, J. Am. Chem. Soc. 2010, 132, 11012–11014.
[7] H. Wettach, S. S. Jester, A. Colsmann, U. Lemmer, N. Rehmann, K. Meer-
holz, S. Hçger, Synth. Met. 2010, 160, 691–700.
[8] J. F. Fuini, A. B. Surampudi, M. A. Penick, M. P. D. Mahindaratne, G. R. Ne-
grete, L. Brancaleon, Dyes Pigm. 2011, 88, 204–211.
[28] a) J. Grimshaw, R. D. Haworth, J. Chem. Soc. 1956, 4225–4232; b) H. K.
Bisoyi, S. Kumar, Tetrahedron Lett. 2007, 48, 4399–4402.
[9] T.-H. Huang, J. T. Lin, Y.-T. Tao, C.-H. Chuen, Chem. Mater. 2003, 15,
4854–4862.
[29] a) R. G. Clarkson, M. Gomberg, J. Am. Chem. Soc. 1930, 52, 2881–2896;
b) M. E. Walters, W. F. Richey, K. S. Clement, S. L. Brewster, E. L. Tasset,
P. M. Puckett, V. R. Durvasula, H. A. Nguyen, U. S. Patent 789. 232, 1995;
c) we have already encountered the cyclization of a stabilized benzhy-
dryl cation to give a benzofluorene structure, see: A. Legrand, B. Rigo,
J.-P. Hꢀnichart, B. Norberg, F. Camus, F. Durant, D. Couturier, J. Hetero-
cycl. Chem. 2000, 37, 215–227.
[30] For numerous cyclizations of benzophenones substituted by methoxy
groups, see: a) S. A. Snyder, A. L. Zografos, Y. Lin, Angew. Chem. 2007,
119, 8334–8339; Angew. Chem. Int. Ed. 2007, 46, 8186–8191; b) A. S.
Snyder, S. P. Breazzano, A. G. Ross, Y. Lin, A. L. Zografos, J. Am. Chem.
Soc. 2009, 131, 1753–1765.
[10] a) S. A. Adesanya, R. Nia, M.-T. Martin, N. Boukamcha, A. Montagnac, M.
Pais, J. Nat. Prod. 1999, 62, 1694–1695; b) S. He, B. Wu, Y. Pan, L. Jiang,
J. Org. Chem. 2008, 73, 5233–5241; c) Y. L. Choi, B. T. Kim, J.-N. Heo, J.
Org. Chem. 2012, 77, 8762–8767.
[11] a) D. N. Quang, T. Hashimoto, M. Tanaka, M. Baumgartner, M. Stadler, Y.
Asakawa, J. Nat. Prod. 2002, 65, 1869–1874; b) M.-Y. Chang, T.-W. Lee,
M.-H. Wu, Org. Lett. 2012, 14, 2198–2201; c) M. Fukai, M. Tsukada, K.
Miki, T. Suzuki, T. Sugita, K. Kinoshita, K. Takahashi, M. Shiro, K. Koyama,
J. Nat. Prod. 2012, 75, 22–25, and references cited therein.
[12] Y. L. Zhang, J. Zhang, N. Jiang, Y. H. Lu, L. Wang, S. H. Xu, W. Wang, G. F.
Zhang, Q. Xu, H. M. Ge, J. Ma, Y. C. Song, R. X. Tan, J. Am. Chem. Soc.
2011, 133, 5931–5940.
[13] W. Gu, H. M. Ge, Y. C. Song, H. Ding, H. L. Zhu, X. A. Zhao, R. X. Tan, J.
Nat. Prod. 2007, 70, 114–117.
[14] X. Feng, W. Pisula, K. Mꢄllen, Pure Appl. Chem. 2009, 81, 2203–2224.
[31] a) P. Gautret, S. El-Ghammarti, A. Legrand, D. Couturier, B. Rigo, Synth.
Commun. 1996, 26, 707–713; b) C. Waterlot, D. Couturier, M. De Backer,
B. Rigo, Can. J. Chem. 2000, 78, 1242–1246.
Chem. Eur. J. 2014, 20, 10117 – 10130
www.chemeurj.org
10129
ꢂ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
[32] For examples for the synthesis of 9,9’-spirobifluorene compounds by
electrophilic addition using H2SO4, PPA, and particularly methanesulfon-
ic acid and/or Eaton’s reagent, see: a) X. Cheng, G.-H. Hou, J.-H. Xie, Q.-
L. Zhou, Org. Lett. 2004, 6, 2381–2383; b) K.-T. Wong, Y.-M. Chen, Y.-T.
Lin, H.-C. Su, C.-C. Wu, Org. Lett. 2005, 7, 5361–5364; c) C.-L. Chiang, C.-
F. Shu, C.-T. Chen, Org. Lett. 2005, 7, 3717–3720; d) L.-H. Xie, F. Liu, C.
Tang, X.-Y. Hou, Y.-R. Hua, Q.-L. Fan, W. Huang, Org. Lett. 2006, 8, 2787–
2790; e) P.-I. Shih, C.-L. Chiang, A. K. Dixit, C.-K. Chen, M.-C. Yuan, R.-Y.
Lee, C.-T. Chen, E. W.-G. Diau, C.-F. Shu, Org. Lett. 2006, 8, 2799–2802;
f) X. Cheng, S.-F. Zhu, X.-Ch. Qiao, P.-C. Yan, Q.-L. Zhou, Tetrahedron
2006, 62, 8077–8082, and references cited therein; g) Ch. Wang, N. Li,
D. W. Shin, S. Y. Lee, N. R. Kang, Y. M. Lee, M. D. Guiver, Macromolecules
2011, 44, 7296–7306.
[34] S. Pogodin, I. Agranat, Org. Lett. 1999, 1, 1387–1390, and references
cited therein.
[35] We have already encountered many cases of anion oxidation by
oxygen dissolved in MeOH, see: a) A. Bourry, D. Couturier, G. Sanz, L.
Van Hijfte, J.-P. Hꢀnichart, B. Rigo, Tetrahedron 2006, 62, 4400–4407;
b) R. Akuꢀ-Gꢀdu, D. Couturier, J.-P. Hꢀnichart, B. Rigo, G. Sanz, L. Van
Hijfte, A. Bourry, Tetrahedron 2012, 68, 1117–1127, and references cited
therein.
[36] For a recent reference in this field, see: R. H. Shoemaker, Nat. Rev.
Cancer 2006, 6, 813–823, and references cited therein.
[37] a) M. L. Shelanski, F. Gaskin, C. R. Cantor, Proc. Natl. Acad. Sci. USA 1973,
70, 765–768; b) J. C. Lee, S. N. Timasheff, Biochemistry 1977, 16, 1754–
1764.
[33] CCDC-984664 (39) contains the supplementary crystallographic data for
this paper. These data can be obtained free of charge from The Cam-
bridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_re-
quest/cif.
Received: February 26, 2014
Published online on July 7, 2014
Chem. Eur. J. 2014, 20, 10117 – 10130
www.chemeurj.org
10130
ꢂ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim