Synthesis and biological evaluation of phenstatin metabolites

Article abstract of DOI:10.1016/j.bmc.2011.08.047

Previous investigations on the incubation of phenstatin with rat and human microsomal fractions revealed the formation of nine main metabolites. The structures of eight of these metabolites have been now confirmed by synthesis and their biological properties have been reported. Eaton's reagent was utilized as a convenient condensing agent, allowing, among others, a simple multigram scale preparation of phenstatin. Synthesized metabolites and related compounds were evaluated for their antiproliferative activity in the NCI-60 cancer cell line panel, and for their effect on microtubule assembly. Metabolite 23 (2′-methoxyphenstatin) exhibited the most potent in vitro cytotoxic activity: inhibition of the growth of K-562, NCI-H322M, NCI-H522, KM12, M14, MDA-MB-435, NCI/ADR-RES, and HS 578T cell lines with GI₅₀ values <10 nM. It also showed more significant tubulin polymerization inhibitory activity than parent phenstatin (3) (IC₅₀ = 3.2 μM vs 15.0 μM) and induced G2/M arrest in murine leukemia DA1-3b cells. The identification of this active metabolite led to the design and synthesis of analogs with potent in vitro cytotoxicity and inhibition of microtubule assembly.

Full text of DOI:10.1016/j.bmc.2011.08.047

Bioorganic & Medicinal Chemistry 19 (2011) 6042–6054
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of phenstatin metabolites
Alina Ghinet a,b, Benoît Rigo a,b, , Jean-Pierre Hénichart a, Delphine Le Broc-Ryckewaert a,c
,
⇑
Jean Pommery a,c, Nicole Pommery a,c, Xavier Thuru d, Bruno Quesnel d,e,f, Philippe Gautret a,b
a Univ Lille Nord de France, F-59000 Lille, France
b UCLille, EA 4481 (GRIIOT), Laboratoire de Pharmacochimie, HEI, 13 rue de Toul, F-59046 Lille, France
c UDSL, EA 4483, Laboratoire de Toxicologie, UFR Pharmacie, 3 rue du Professeur Laguesse, F-59006 Lille, France
d Unité INSERM 837, Institut de Recherche sur le Cancer de Lille, F-59045 Lille, France
e Institut de Médecine Prédictive et de Recherche Thérapeutique, F-59045 Lille, France
f Service des Maladies du Sang, Centre Hospitalier et Universitaire de Lille, F-59037 Lille, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Previous investigations on the incubation of phenstatin with rat and human microsomal fractions
revealed the formation of nine main metabolites. The structures of eight of these metabolites have been
now confirmed by synthesis and their biological properties have been reported. Eaton’s reagent was uti-
lized as a convenient condensing agent, allowing, among others, a simple multigram scale preparation of
phenstatin. Synthesized metabolites and related compounds were evaluated for their antiproliferative
activity in the NCI-60 cancer cell line panel, and for their effect on microtubule assembly. Metabolite
23 (20-methoxyphenstatin) exhibited the most potent in vitro cytotoxic activity: inhibition of the growth
of K-562, NCI-H322M, NCI-H522, KM12, M14, MDA-MB-435, NCI/ADR-RES, and HS 578T cell lines with
GI50 values <10 nM. It also showed more significant tubulin polymerization inhibitory activity than par-
Received 11 May 2011
Revised 16 August 2011
Accepted 19 August 2011
Available online 27 August 2011
Keywords:
Anticancer agent
Tubulin
Phenstatin
Metabolite
ent phenstatin (3) (IC50 = 3.2 lM vs 15.0 lM) and induced G2/M arrest in murine leukemia DA1-3b cells.
The identification of this active metabolite led to the design and synthesis of analogs with potent in vitro
cytotoxicity and inhibition of microtubule assembly.
Murine leukemia
Friedel–Crafts acylation
Eaton’s reagent
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
O
OR
MeO
MeO
Tubulin is a heterodimer of closely related and tightly linked
OR
OMe
MeO
MeO MeO
A
B
A
B
globular
a- and b-tubulin proteins, capable to polymerize in hol-
OMe
low tubes called microtubules. These cytoskeletal structures are in-
volved in many cellular functions.1 One of their key roles is in the
mitotic spindle, intimately involved in cell division, which bridges
between the chromosomes in the center and the centrosomes at
opposite poles of the cell, and leading to, at the metaphase/ana-
phase transition, the separation of the sister chromatids and then
their segregation into the daughter cells. Their importance in cell
division makes microtubules an important target for anticancer
drugs.2 Tubulin-binding agents interfere with the dynamic insta-
bility of microtubules and thereby arrest mitotic cells in the G2/
M phases of the cell division cycle, causing mitotic catastrophy
and finally apoptotic cell death.3 Colchicine and its analogs pre-
dominantly bind to a high affinity site, called colchicine binding
MeO
1 R = H
Combretastatin A-4 (CA-4)
2 R = PO3Na2 Combretastatin A-4P (CA-4P)
3 R = H
Phenstatin
4 R = PO3Na2 Phenstatin-P
Figure 1. Structure of combretastatin and phenstatin derivatives.
for the colchicine binding site, and is one of the most potent antimi-
totic agents,5 also active towards multi-drug-resistant (MDR) cell
lines.6 Research on combretastatin A-4 in order to improve its water
solubility and in vivo activity7 led to its water-soluble prodrug com-
bretastatin A-4 phosphate(CA-4P, Zybrestat) (2) (Fig. 1), presentlyin
phase II human cancer clinical trials8 with phase III trials under way.
CA-4P acts as a vascular disrupting agent (VDA)9: its mechanism of
action is believed to be related to tubulin-binding properties that re-
sult in rapid tumor endothelial cell damage, neovascular shutdown
and subsequent tumor cell death.10 In contrast to colchicine, the
anti-vascular effects of CA-4 in vivo are apparent well below the
maximum tolerated dose, offering a wide therapeutic window.
CA-4P does not induce the common side effects of existing chemo-
therapies such as alopecia and bone marrow toxicity. However,
site, located at the interface between
lumen of the microtubule.
a- and b-tubulin, facing the
Combretastatin A-4 (CA-4) (1) (Fig. 1), extracted from a South
African tree Combretum caffrum by Pettit et al.,4 depicts high affinity
⇑
Corresponding author.
E-mail address: benoit.rigo@hei.fr (B. Rigo).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.08.047

A. Ghinet et al. / Bioorg. Med. Chem. 19 (2011) 6042–6054
6043
cardiovascular toxicity and neurotoxicity are dose limiting for this
compound.11 These significant side effects currently represent the
main obstacles to broad clinical application of CA-4P. For this reason,
it is necessary to develop other CA-4 structurally related compounds
with more specificity for tumor endothelial cells than normal endo-
thelial cells to avoid cardiac toxicity from endothelial damage.12
A closely related compound is phenstatin (3) (Fig. 1), which re-
sulted from a SAR-study on combretastatin A-4 and exhibitedpotent
inhibition of cancer cell growth.13,14 The water-soluble phenstatin
phosphate prodrug (4) and the parent phenstatin (3) (Fig. 1) were
essentially indistinguishable and very similar to the combretastatin
A-4 phosphate prodrug (2) in terms of both potency and differential
cytotoxicity.14 The biotransformation pathways of CA-4 (Fig. 2),15
and the ones of an indole analog of phenstatin (BPR0L075, 13)
(Fig. 3)16 have been published but, curiously, nothing was known
about phenstatin bioconversion before our study, and this
compound has not been advancing in human clinical trials. These
considerations, linked to our interest in the medicinal chemistry of
CA-4 and phenstatin derivatives, led us to study the metabolic fate
of 3 in rat and in human microsomal preparations.17
It was observedthat combretastatinA-4 (CA-4, 1), in thepresence
of rat or human microsomes, was metabolized into compounds 5–
12: one of the main transformations concerned the isomerization
of the double bound leading to the trans-stilbenes 5, 7, 9 and 11.
However, O-demethylases yield phenols 5, 6, 7 and 8, while aromatic
hydroxylases give para diphenols 9 and 10, probably transformed by
oxidases in quinones 11 and 12 (Fig. 2).15 In the case of ketone 13,
O-demethylases yield phenols 15, 17 and 19 and aromatic hydroxy-
lases generate compounds 14, 16 and 18 (Fig. 3).16
OH
OMe
MeO
MeO
OMe
CA-4 1
MeO
MeO
OH
OH
OH
OH
MeO
MeO
MeO
OMe
5
6
MeO
OH
OMe
HO
OH
OMe
HO
MeO
MeO
OMe
7
8
MeO
OH
OH
OMe
MeO
MeO
MeO
MeO
OMe
O
OMe OH
HO
9
O
10
OH
MeO
MeO
MeO
O
MeO
MeO
OMe
MeO
MeO
OMe
OMe
O
O
12
OMe
Phenstatin 3
O
11
Figure 2. Structure of combretastatin A-4 (1) and its main metabolites described in
the literature; the metabolite 8 observed by MS–MS was not synthesized.15
O
O
OMe
MeO
MeO
OMe
OMe
MeO
HO
O
OMe
MeO
OMe
OMe
OMe
OMe
OMe
OMe
N
20
21
MeO
H
OMe
OMe
O
O
OMe
OH
MeO
MeO
OH
MeO
HO
BPR0L075 13
OMe
OMe
OMe
OMe
OMe
23
22
O
O
HO
OH
OH
HO
MeO
HO
O
O
OMe
OMe
OMe
OH
MeO
MeO
HO
OMe
OMe
MeO
N
H
N
H
MeO
24
25
OMe
OMe
OMe
OMe
14
15
OH
OH
OH
OH
OMe
OMe
MeO
MeO
MeO
MeO
O
O
OMe
OMe
MeO
HO
MeO
MeO
OMe
N
N
H
26
27
H
OMe
OMe
17
16
O
OH
OH
MeO
MeO
O
O
MeO
MeO
MeO
MeO
OH
OMe
N
H
N
H
28a
19
18
Figure 4. Structure of phenstatin (3) and its main metabolites 20–27. aThe
Figure 3. Structure of 6-methoxy-3-(30,40,50-trimethoxybenzoyl)-1H-indole (13,
predictable metabolite 28 was found not to be a product of phenstatin
BPR0L075) and its main metabolites described in the literature.16
biotransformation.17

6044
A. Ghinet et al. / Bioorg. Med. Chem. 19 (2011) 6042–6054
We have examined the metabolic fate of CA-4 (1) (Fig. 2),15 the
benzhydrols 26 (97%) and 2721 (75%). Friedel–Crafts reaction, pro-
moted by Eaton’s reagent, of the protected dimethoxyacids 34 or
35 with the protected phenols 30 or 32 led, respectively, to com-
pounds 36 (41%), 37 (77%) and 38 (80%). The sodium acetate
removing of the chloroacetyl protective group of these ketophenols
yielded metabolite 24 (65%), and compounds 39 (85%) and 40
(84%). Dimethylsulfate methylation of phenstatin (3) gave known
pentamethoxylated metabolite 20 (69%). Synthesis of this ketone
was also described from tin tetrachloride mediated reaction of
trimethoxybenzoyl chloride with veratrole (84%).22 The same
methylation reaction applied to phenol 39 yielded ketone 41
(82%), and then ethanolamine cleavage23 of the remaining carbon-
ate protecting group of intermediates 39, 40 and 41 furnished
metabolites 25 (85%), 2224 (76%) and 21 (78%), respectively. Alter-
natively, cleavage of the central methoxy group of phenstatin (3) in
order to give phenol 25 was realized in 52% by refluxing 3 with
3 equiv of aluminum chloride in dichloromethane.25 Noteworthy,
no such transformation was obtained by using BCl3.
During the syntheses described in Scheme 1, we observed that
chloroacetyl and ethyl carbonate protections of phenol groups
were rather stable when heated in Eaton’s reagent. However, when
the acid 29 was opposed to catechol protected in these ways, many
products were observed by TLC of crude reaction media, and only
monoesterified catechol26 (see Supplementary information sec-
tion) was isolated in 36–63% yield. In a similar procedure, cyclic
carbonate 42 was reacted with trimethoxybenzoic acid 29 in Ea-
ton’s reagent. Thus, the benzophenone 43 was obtained in very
low (3%) yield; deprotection of which gave 51% of potential metab-
olite 28 (Scheme 2).
one of the aromatic ketone BPR0L075 (13) (Fig. 3)16 and the molec-
ular weight of major metabolites observed during the MS-MS
study of phenstatin (3) biotransformation in the same conditions.
This study led us to consider O-methylations (20, 21 and 27), aro-
matic hydroxylations followed by O-methylations (22, 23), ether
demethylations (21, 22, 24, 25, 28), and ketone reductions (26,
27) as possible metabolic processes for phenstatin (3) (Fig. 4).
However, MS–MS study excluded the formation of quinones as
for CA-4. It was later observed that compounds 20–27 were indeed
metabolites of phenstatin, and that catechol 28, which was in-
cluded by comparison with metabolization of CA-4, was not a
product of this biotransformation.
We report here the synthesis of several metabolites of phensta-
tin (3) and their biological evaluation.17 Only three of these com-
pounds had been already described. Using Eaton’s reagent as a
condensing medium, allowed the preparation of this metabolite
in an easier and a more convenient manner. Inter alia, a multigram
synthesis of phenstatin was described. In addition, on the basis of
the insight gained about the major metabolic pathways of phenst-
atin,17 we have determined metabolite 23, which has globally an
improved in vitro efficacy on tubulin polymerization and on cancer
cell lines than that of parent compound 3. Identification of this ac-
tive metabolite of 3 led us to design and synthesize analogs which
exhibit potent in vitro cytotoxicity.
2. Chemistry
The general synthetic procedure for the target compound syn-
thesis is illustrated in Scheme 1. Literature described that Jacobsen
oxidation of CA-4 led to phenstatin (3).14 This compound can also
be obtained by Friedel–Crafts reaction from a protected methoxy-
phenol and trimethoxybenzoic acid in PPA.18 However, this is a
highly viscous medium which is difficult to stir and hydrolyze at
the end of the reaction; then, we decided to use Eaton’s reagent
(MeSO3H/P2O5).19 Thus, phenstatin (3) was obtained by reacting
trimethoxybenzoic acid 29 with protected phenol 30 in Eaton’s re-
agent at 60 °C to give ketone 31 (85%), followed by removal of the
chloroacetyl protection group by using sodium acetate in methanol
(98%) (litt. 91%18). The same sequence applied to the protected
phenol 32 led to ketone 33 (80%), and then to 20-O-methylphenst-
atin 23 (81%). The reduced phenstatin 26 was previously obtained
from Grignard reaction of the corresponding aldehyde,20 however
NaBH4 reduction of ketones 3 and 20 provided a simpler route to
Due to disappointing results obtained in these attempts to pre-
pare ketone 28, another approach was considered: an amino group
was used as a latent phenol function. The Friedel–Crafts acylation
of benzoxazolones with aromatic carboxylic acids in PPA is
known.27 However, by heating at 60 °C a homogeneous mixture
of acid 29 and benzoxazolone 44 in Eaton’s reagent, the sulfonated
benzoxazolone 45 was isolated in low (13%) yield, accompanied by
28% of the pentamethyl rufigallic acid 46. Obtention of the benzo-
quinone 46 with milder condition reactions is of interest since the
formation of such quinones has only been described with sulfuric
acid at very high temperatures.28 In this compound, the position
of the hydroxyl group was deduced from 1H and 13C spectra, and
from HMBC correlations shown in Figure 5. Removing of the meth-
anesulfonyl group in 45 was performed in presence of n-tetrabutyl-
ammonium fluoride in THF at room temperature for 24 h,29 leading
OH
R3
O
R1
R4
OMe
R3
MeO
MeO
OR
R1
R4
(i)
(v)
R2
CO2H
+
OMe
OMe
R2
MeO
MeO
MeO
31 85% R1, R2 = OMe; R3 = H; R4 = OCOCH2Cl
29 R1, R2 = OMe
30 R3 = H; R4 = OCOCH2Cl
32 R3 = OMe; R4 = OCOCH2Cl
26 97% R = H
27 75% R = Me
(ii)
(iii)
34 R1 = OCOCH2Cl; R2 = OMe
35 R1 = OMe; R2 = OCO2Et
3 98% R1, R2 = OMe; R3 = H; R4 = OH
20 69% R1, R2, R4 = OMe; R3 = H
33 80% R1, R2, R3 = OMe; R4 = OCOCH2Cl
(ii)
(ii)
23 81% R1, R2, R3 = OMe; R4 = OH
36 41% R1, R4 = OCOCH2Cl; R2 = OMe; R3 = H
24 65% R1, R4 = OH; R2 = OMe; R3 = H
37 77% R1 = OMe; R2 = OCO2Et; R3 = H; R4 = OCOCH2Cl
39 85% R1 = OMe; R2 = OCO2Et; R3 = H; R4 = OH
41 82% R1 = R4 = OMe; R2 = OCO2Et; R3 = H
(ii)
(iii)
(iv)
(iv)
21 78% R1, R4 = OMe; R2 = OH; R3 = H
25 85% R1 = OMe; R2, R4 = OH; R3 = H
38 80% R1, R3 = OMe; R2 = OCO2Et; R4 = OCOCH2Cl
(ii)
(iv)
40 84% R1, R3 = OMe; R2 = OCO2Et; R4 = OH
22 76% R1, R3 = OMe; R2, R4 = OH
Scheme 1. Reagents and conditions: (i) Eaton’s reagent, 60 °C; (ii) AcONaꢀ3H2O, MeOH, reflux; (iii) Me2SO4, K2CO3, acetone, reflux; (iv) ethanolamine, EtOH 95%, rt; (v) NaBH4
2.2 equiv, EtOH/H2O, rt.

A. Ghinet et al. / Bioorg. Med. Chem. 19 (2011) 6042–6054
6045
O
O
MeO
MeO
MeO
MeO
MeO
MeO
OH
OH
O
O
O
O
(ii)
(i)
(vi)
CO2H
O
O
+
+
MeO
MeO
MeO
MeO
MeO
29
42
43 3%
O
28 51%
O
OH
MeO
MeO
MeO
MeO
OMe
OMe
O
N
O
+
SO2Me
O
MeO
45 13%
46 28%
O
MeO
MeO
CO2H
O
(iii)
28%
N
H
O
O
O
29
44
MeO
MeO
MeO
MeO
OH
MeO
MeO
OH
OH
O
(ii)
(iv)
(v)
O
23%
N
H
NH2
MeO
MeO
MeO
47
48 56%
28 16%
Scheme 2. Reagents and conditions: (i) Eaton’s reagent 4 equiv, 60 °C, 30 h; (ii) Eaton’s reagent 4 equiv (added in small portions), 50 °C, 24 h; (iii) n-(C4H9)4N+Fꢁꢀ3H2O 3 equiv,
THF, rt, 24 h; (iv) NaOH 0.38 N 5 equiv, 80 °C, 10 h; (v) NaIO4, HCl, AcOH, rt, 14 h; (vi) ethanolamine, EtOH 95%, rt, 5 h.
5
H
O
O
15
H3C
H
7
O
O
8
12
13
18
CH3
16
1
9
11 14
19
H3C
17
O
O
4
10
6
H
CH3
O
O
2
3 CH3
46
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
C δ
(ppm)
61.4 56.5 60.9 61.6 56.4 106.1 103.9 157.5 148.5 155.0 121.1 130.7 111.5 130.6 156.0 140.6 158.4 186.7 180.3
4.03 4.00 4.01 4.05 3.99 7.68 7.42 7.68 7.68 7.68 7.68 7.68 12.64 7.42 12.64 12.64 7.42 7.68 7.42
H δ
(ppm)
Figure 5. Main 1H HMBC NMR correlations for the anthraquinone 46 (selected correlations in blue, informative correlations in red).
to 28% of ketone 47 (Scheme 2). It was thought that, because of the
low reactivity of benzoxazolones, self-condensation of trimethoxy-
benzoic acid firstly occurred. Consequently, reaction conditions
were modified, and acid 29 was slowly added by parts to a solution
of benzoxazolone 44 in Eaton’s reagent at 50 °C: benzophenone 47
was then isolated in 23% yield, accompanied by a low amount of an
unknown red by-product.30 Opening of the oxazolone ring was ob-
tained by refluxing compound 47 in sodium hydroxide, and benzo-
phenone 48 was then isolated in 56% yield. Sodium periodate
oxidation31 of 48 was performed, leading to potential metabolite
28 in low 16% yield. A sufficient quantity of this compound 28
was thus obtained allowing the comparison of its structure to those
of known metabolites of phenstatin, confirming that this com-
pound is not such a metabolite. So, no more attempts were realized
in order to improve the yield of these syntheses.
The in vitro anticancer properties obtained for metabolite 23
prompted us to synthesize some analogs to be evaluated in tubulin
polymerization or cytotoxicity. Friedel–Crafts reaction promoted
by Eaton’s reagent of trimethoxybenzoic acid 29 with the protected
aniline 52 led to ketone 53 (41%), and then hydrochloric acid cleav-
age of the acetyl protecting group of intermediate 53 yielded ami-
no analog 54 of phenol 23 (68%). The same sequence of Friedel–
Crafts condensation between anisic acid 55 and aromatic ester 32
led to 53% of protected compound 56. Then, the sodium acetate
removing of the chloroacetyl protective group of 56 yielded ketone
57 (87%), an analog of 23, with only one methoxy group on cycle A.
3. Biological evaluation
The three metabolites 21, 22, and 23, the three intermediate
compounds 45, 46, and 47, and the three 20-methoxybenzophenon-
es 53, 54, and 57 were selected by NCI for screening against 60 hu-
man tumor cell lines. The representative results are summarized in
Tables 1 and 2. Anthraquinone 46 and benzoxazolone 47 generally
showed modest cell growth inhibitory activity at 10ꢁ5 M concen-
tration (only 60% inhibition on OVCAR-4 for compound 46 and
34% inhibition on K-562 for 47) (Table 1). The O-demethylated
metabolites 21 and 22 showed decreased cytotoxicity compared
to parent phenstatin (3). In preliminary screening, 23 showed sig-
nificant cytotoxicity against melanoma cell lines (Fig. 6). In addi-
tion, it also exhibited more potent anti-proliferative activity
against several types of cancer cells, such as DU145, SF-295, and
MCF7 cell lines, than phenstatin (Table 1). Interestingly, the results
obtained from NCI revealed that intermediate 45 had also a mod-
erate cytotoxic effect with IC50 values between 0.29 and 22.3
(Table 1).
lM
In order to examine whether these metabolite and intermediate
compounds interact with tubulin and inhibit in vitro tubulin poly-
merization, we performed a turbidimetric tubulin polymerization
assay. The results are shown in Table 1. Metabolite 23 exhibited
the most potent inhibitory effect and its 50% tubulin polymeriza-
tion inhibition was as low as 3.2
lM, being more active than
phenstatin itself (15.0
l
M) in our testing conditions.32

Table 1
Results of the in vitro human cancer cell growth inhibitiona,b and tubulin polymerization inhibitionc for metabolites 21, 22 and 23 and intermediates 45, 46 and 47
Cell type
Compound
IC50
M) tubulinc
Phenstatin (3)
23
22
21
45
46
47
(
l
15.0 0.2d
3.2 0.2d
14.9 0.9d
>100d
>100d
>100d
>100d
Cell line
GI50 (10ꢁa M)a,b
Cell growth inhibition, % (10ꢁ5 M)
Leukemia
CCRF-CEM
HL-60(TB)
K-562
–
–
–
2.88Eꢁ8
2.35Eꢁ8
<1.00Eꢁ8
8.96Eꢁ7
3.64Eꢁ7
1.53Eꢁ7
4.05Eꢁ6
1.25Eꢁ6
6.66Eꢁ7
3.35Eꢁ6
2.52Eꢁ6
5.88Eꢁ7
0
6.14
0
0
0
33.5
Non-small cell lung cancer
A549/ATCC
EKVX
NCI-H322M
NCI-H460
NCI-H522
–
–
–
4.13Eꢁ8
6.11Eꢁ7
<1.00Eꢁ8
5.07Eꢁ8
<1.00Eꢁ8
6.20Eꢁ6
2.11Eꢁ7
3.20Eꢁ8
1.06Eꢁ8
<1.00Eꢁ8
<1.00Eꢁ8
<1.00Eꢁ8
2.66Eꢁ8
9.46Eꢁ6
2.73Eꢁ8
2.22Eꢁ8
7.30Eꢁ8
6.51Eꢁ7
<1.00Eꢁ8
8.96Eꢁ8
3.47Eꢁ6
4.76Eꢁ6
4.99Eꢁ6
1.97Eꢁ6
2.90Eꢁ7
4.83Eꢁ6
2.89Eꢁ6
1.41Eꢁ6
6.11Eꢁ7
4.09Eꢁ7
4.55Eꢁ7
2.82Eꢁ7
>1.00Eꢁ4
>1.00Eꢁ4
5.01Eꢁ6
3.63Eꢁ7
>1.00Eꢁ4
4.92Eꢁ6
5.15Eꢁ7
9.12Eꢁ7
5.91Eꢁ5
>1.00Eꢁ4
3.44Eꢁ6
3.44Eꢁ6
3.64Eꢁ6
4.68Eꢁ6
4.58Eꢁ6
5.38Eꢁ6
2.64Eꢁ6
2.34Eꢁ6
2.50Eꢁ6
6.75Eꢁ6
2.31Eꢁ6
2.00Eꢁ6
1.15Eꢁ6
1.36Eꢁ6
2.89Eꢁ7
5.11Eꢁ6
1.13Eꢁ5
5.30Eꢁ6
1.56Eꢁ6
2.23Eꢁ5
5.77Eꢁ6
2.03Eꢁ6
2.84Eꢁ6
26.12
13.48
0
9.33
29.5
0
13.74
0
0
0
5.70Eꢁ9e
–
Colon cancer
Melanoma
COLO 205
HCC-2998
HCT-116
HCT-15
4.86Eꢁ6f
1.42Eꢁ5
0
0
0
0
0
0
0
–
–
–
–
N.D.g
2.50Eꢁ6
2.07Eꢁ6
1.09Eꢁ6
9.02Eꢁ7
2.73Eꢁ7
5.29Eꢁ6
9.46Eꢁ6
8.41Eꢁ6
2.33Eꢁ6
>1.00Eꢁ4
1.29Eꢁ5
8.54Eꢁ7
2.20Eꢁ6
3.36Eꢁ6
3.01Eꢁ6
1.88Eꢁ6
6.68Eꢁ6
2.16Eꢁ6
11.78
0
0
KM12
M14
–
–
–
–
–
0
0
MDA-MB-435
SK-MEL-2
SK-MEL-28
UACC-62
16.85
0
14.98
6.13
11.24
0
0
0
Ovarian cancer
Renal cancer
OVCAR-3
OVCAR-4
OVCAR-5
NCI/ADR-RES
SK-OV-3
2.30Eꢁ9e
0
0
0
0
0
0
–
–
–
–
60.25
7.52
20.26
9.78
A498
3.80Eꢁ7e
2.72Eꢁ6
1.20Eꢁ7
1.59Eꢁ8
4.69Eꢁ8
4.89Eꢁ8
1.70Eꢁ6
1.90Eꢁ6
7.55Eꢁ7
1.37Eꢁ5
1.66Eꢁ5
3.11Eꢁ6
2.64Eꢁ6
4.63Eꢁ6
7.79Eꢁ6
6.81Eꢁ6
N.D.g
7.07
2.66
8.73
0
17.01
CAKI-1
RXF 393
SN12C
UO-31
–
–
–
–
0
0
0
0
Prostate cancer
DU145
3.40Eꢁ8f
2.09Eꢁ8
8.18Eꢁ7
4.49Eꢁ6
3.04Eꢁ6
7.83
0
Central nervous system cancer
SF-268
SF-295
SF-539
–
2.11Eꢁ8
1.99Eꢁ8
1.51Eꢁ8
2.64Eꢁ8
2.46Eꢁ8
<1.00Eꢁ8
2.49Eꢁ7
4.25Eꢁ6
1.40Eꢁ6
6.11Eꢁ7
1.01Eꢁ6
2.16Eꢁ6
3.58Eꢁ7
8.64Eꢁ7
7.95Eꢁ6
1.61Eꢁ6
2.36Eꢁ6
4.43Eꢁ6
4.85Eꢁ6
3.16Eꢁ6
2.82Eꢁ6
22.19
37.85
16.23
0
0
0
5.20Eꢁ8e
2.08Eꢁ6
2.96Eꢁ6
4.33Eꢁ7
4.79Eꢁ6
4.92Eꢁ6
1.97Eꢁ6
–
Breast cancer
MCF7
MDA-MB-231/ATCC
HS 578T
4.37Eꢁ7f/3.40Eꢁ8h
26.69
21.33
16.89
22.4
0
0
0
0
–
–
–
MDA-MB-468
a
b
c
Data obtained from NCI’s in vitro 60 cell 5-dose screen.
GI50 is the molar concentration of synthetic compound causing 50% growth inhibition of tumor cells.
See Ref.32 and Supplementary information.
Data obtained from Ref.16
Data obtained from Ref.14
Data obtained from Ref.13
Not determined.
d
e
f
g
h
Data obtained from Ref.33

Table 2
Tubulin polymerization inhibitory activity and cytotoxicity for compounds 53, 54, 57 references 3 (phenstatin) and 1 (CA-4), metabolite 23, and known antitubulin agents 58–61
R3
R4
O
R
X
1
In vitro cell growth inhibition, GI50 (nM)
Compound
TPI IC50 (lM)
O
R2
R1
R2
X
R3
R4
HL-60
A-549
HT-29
MDA-MB-435
MCF7
SF-295
OVCAR-3
A498
DU-145
23
53
54
57
OMe
OMe
OMe
H
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
OMe
OMe
OMe
OMe
OMe
OMe
C@O
C@O
C@O
C@O
C@O
CH@CH
CH@CH
C@O
OMe
OMe
OMe
OMe
H
OH
3.2a
>50a
10a
23.5
41.3
3460
4330
32.3
<10
26.4
9600
10.6
19.9
293
22.2
3300
20.2
N.D.b
7.2e
2720
85.5
20.9
292
NHCOCH3
NH2
OH
OH
OH
OH
H
NH2
OH
3530
33.1
4620
39.0
344
<10
19.9
22.1
27.4
N.D.b
34d
>50a
15.0a
12.7a
1.9h
0.4i
N.D.b
31.0c
31.6f
N.D.b
290c
1200f
N.D.b
1800c
631f
N.D.b
N.D.b
437d
39.8f
N.D.b
163.3e
63.1f
N.D.b
1194e
50.1f
Phenstatin (3)
CA-4 (1)
58
59
60
H
31.6f
39.8f
2.5g
OH
NH2
H
511.5g
36d
42j
33i
23d
C@O
C@O
1.5f
30f
57f
61
OH
0.8g
119.6g
143.6g
Compound 57 showed very modest cell growth inhibition at 10ꢁ5 M concentration (only 26.5% inhibition of renal cancer cell line UO-31 and 23% of HOP-92 (non-small cell lung cancer)).
a
See Ref.40 and Supplementary information.
b
Not determined.
c
Data obtained from Ref.35
d
Data obtained from Ref.36
Data obtained from Ref.14
e
Data obtained from Ref.37
f
Data obtained from Ref.13
g
h
Data obtained from Ref.38
Data obtained from Ref.39
i
Data obtained from Ref.40
j

6048
A. Ghinet et al. / Bioorg. Med. Chem. 19 (2011) 6042–6054
and the effect was observed in a dose-independent manner after
treatment for 24 h with increased amounts of this compound.
About 25.3% of the cells were arrested in the G2/M phase while
47.2% of the cells were found to be in the G2/M phase in presence
of 100 and 1 lM of metabolite 23, respectively. These results con-
firmed that metabolite 23 is a potent antitubulin agent.
Because of the interesting biological activities of 20-methoxy-
phenstatin (23), and the fact that 20-hydroxyphenstatin (61; Table
2) is more active than phenstatin (3),13 it was interesting to reexam-
ine structure-activity relationships concerning the C20 position of
the phenstatin scaffold. Hence, we investigated the effect of intro-
ducing a different substituent at the C30 position of benzophenone
ring B on tubulin polymerization. Methylation of the 20-hydroxyl
group of 61 led to 23, with an increase of six times of its cytotoxicity
against SF-295 and DU-145; substitution of the 30-hydroxyl group of
23 by an amino moiety (54) resulted in a slight decrease of the inhib-
itory activity on tubulin, and in a two-digit increasing of the cellular
growth inhibition for HT-29 and for A498 cell lines. Except for MDA-
MB-435 and DU-145, compound 54 presents an equal or better cyto-
toxicity as phenstatin or CA-4. As it could be predicted, acetylation of
the 3-amino group of 54 lead to amide 53 resulting in lower anti-
proliferative potential, and abolishes the efficacy on microtubule
assembly. The removal of methoxy groups at the C3 and C5 positions
of metabolite 23 ring A (compound 57) resulted in a complete loss of
Figure 6. Dose–response curve of 23 against different melanoma cell lines.
Furthermore, cell cycle analysis of metabolite 23 was performed
using flow cytometry (Fig. 7). We observed that compound 23
strongly induced G2/M arrest in murine leukemia DA1-3b34 cells,
Figure 7. Effects of metabolite 23 on cell cycle progression of DA1-3b cells were determined by flow cytometry analysis. DA1-3b cells were treated with different
concentrations of metabolite 23. The percentage of cells in each cycle phase was indicated.

A. Ghinet et al. / Bioorg. Med. Chem. 19 (2011) 6042–6054
6049
biological activity (Table 2). Thus, this study shows that introduction
of a methoxy group in position 2 of the B-ring leads to compounds 23
and 54 with improved or similar growth inhibitory properties com-
pared to known antitubulin agents as CA-4 (1), phenstatin (3), CA-1
(58), aminobenzophenones 59–60, and hydroxyphenstatin 61
(Table 2).
column chromatography on silica gel to afford pure benzophenon-
es (31, 33, 37, 38, 43, 45, 47, 53, and 56).
5.1.2.1. 2-Methoxy-5-(3,4,5-trimethoxybenzoyl)phenyl chloro-
acetate (31) (Scheme 1).
The general procedure A was followed using 3,4,5-trimethoxy-
benzoic acid 29 (95.19 g, 448.6 mmol), 2-methoxyphenyl chloro-
acetate 30 (60.00 g, 299.1 mmol) and Eaton’s reagent (43.27 g P2O5
in 292.2 mL CH3SO3H). The mixture was heated at 60 °C for 4 h.
Thefinal brown oilwas purified by column chromatography on silica
gel with EtOAc/n-heptane 3/7 to give pure chloroacetate 31 (100.4 g,
85%) (lit. (with PPA)18 80%) as an off-white solid; mp (EtOH/Et2O)
150–152 °C; TLC Rf (EtOAc/n-heptane 7/3) = 0.62; 1H (CDCl3,
200 MHz) d (ppm) 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 (dd, J = 8.6, 2.2 Hz, 1H, ArH). Calcd for
4. Conclusions
Through Friedel–Crafts reaction using Eaton’s reagent, we have
developed a new synthesis of phenstatin which is currently the
easiest method cited in the literature. Syntheses of eight metabo-
lites of phenstatin, and some related compounds were described,
and we showed that phenstatin exhibits a metabolic profile with
significant differences compared to CA-4. It is noteworthy that
metabolite 23 leads tumoral cells to arrest in G2/M, and has potent
cytotoxicity against K-562, NCI-H322M, NCI-H522, HCT-15, KM-
12, M14, MDA-MB-435, NCI/ADR-RES, HS 578T, and DU-145 cells
in the nanomolar concentration range. The same compound
showed a better inhibition of tubulin polymerization than the par-
ent phenstatin (3). Further structure-activity relationship studies
at the 20 position on B ring of phenstatin provided analogs 53,
54, and 57. When acetamido moiety was introduced in place of
the hydroxy group onto the 30 position of metabolite 23, the result-
ing compound showed decreased potency relative to parent com-
pound, while the amino derivative 54 proved to be the most
potent derivative synthesized. Overall, the 20-methoxybenzophe-
nones exhibited interesting biological properties and have poten-
tial for further investigation as anticancer agents.
C19H19O7Cl: C, 57.80; H, 4.85. Found: C, 57.74; H, 4.78.
5.1.2.2. 2,6-Dimethoxy-3-(3,4,5-trimethoxybenzoyl)phenyl chlo-
roacetate (33) (Scheme 1).
The general procedure A was followed using 3,4,5-trimethoxy-
benzoic acid 29 (2.50 g, 11.8 mmol), 2,6-dimethoxyphenyl chloro-
acetate 32 (1.81 g, 7.8 mmol) and Eaton’s reagent (1.14 g P2O5 in
7.67 mL CH3SO3H). The mixture was heated at 60 °C for 5 h. The fi-
nal brown oil was purified on silica gel with EtOAc/n-heptane 4/6
to give pure chloroacetate 33 (2.70 g, 80%) as an off-white solid;
mp (EtOH) 118–120 °C; TLC Rf (EtOAc/n-heptane 4/6) = 0.15; 1H
NMR (CDCl3, 400 MHz) d (ppm) 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 (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
5. Experimental
5.1. Chemistry
(C), 152.9 (2C), 154.3 (C), 165.0 (C), 193.5 (C). IR
m
cmꢁ1: 522,
758, 814, 1093, 1120, 1293, 1290, 1414, 1489, 1667, 1754, 1781.
Calcd for C20H21O8Cl: C, 56.54; H, 4.98. Found: C, 56.37; H, 4.81.
5.1.1. Materials and methods
Starting materials were commercially available. Melting points
were measured on a ElectrothermalÒ apparatus and are uncor-
rected. NMR spectra were acquired at 200 MHz for 1H NMR and
50 MHz for 13C NMR on a Varian Gemini 2000Ò spectrometer, or
at 400 MHz for 1H NMR and 100 MHz for 13C NMR on a Varian
400 MHz Premium ShieldedÒ spectrometer. Chemical shifts (d)
are expressed in ppm relative to TMS as internal standard. Thin
layer chromatographies were performed on Macherey Nagel silica
gel plates with a fluorescent indicator and were visualized with
UV-lamp at 254 and 366 nm. Column chromatographies were per-
5.1.2.3. 5-{4-[(Ethoxycarbonyl)oxy]-3,5-dimethoxybenzoyl}-2-
methoxyphenyl chloroacetate (37) (Scheme 1).
The general procedure A was followed using 4-[(ethoxycar-
bonyl)oxy]-3,5-dimethoxybenzoic acid (see Supplementary infor-
mation) (10.10 g, 37.38 mmol), 2-methoxyphenyl chloroacetate
30 (5.00 g, 24.92 mmol) and Eaton’s reagent (3.67 g P2O5 in
24.80 mL CH3SO3H). The mixture was heated at 50 °C for 3 h. The
resulting brown oil was crystallized in 95% EtOH to give pure prod-
uct 37 (8.70 g, 77%) as a white solid; mp (EtOH) 171–172 °C; 1H
formed on silica gel (40–60 lm; Macherey-Nagel). Elemental anal-
NMR (CDCl3, 200 MHz)
d
(ppm) 1.41 (t, J = 7.1 Hz, 3H,
yses (C, H, N, S) of new compounds were determined by ‘Service de
Microanalyses’, Faculté de Sciences Mirande, Université de Bourgo-
gne, Dijon, France.
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 (dd, J = 8.5, 2.2 Hz, 1H, ArH). 13C NMR (CDCl3, 50 MHz) d
14.0 (CH3), 40.4 (CH2), 56.1 (CH3), 56.3 (CH3), 65.2 (CH2), 106.3
(CH), 106.8 (CH), 111.4 (CH), 111.9 (CH), 124.6 (C), 125.1 (C),
129.8 (CH), 130.3 (C), 135.6 (C), 138.6 (C), 152.0 (2C), 152.5 (C),
154.6 (C), 165.3 (C), 193.3 (C). IR
1253, 1336, 1409, 1597, 1651, 1770, 1792. Calcd for C21H21O9Cl:
C, 55.70; H, 4.67. Found: C, 55.64; H, 4.79.
5.1.2. General procedure A for Friedel–Crafts reactions in the
presence of Eaton’s reagent
Eaton’s reagent was prepared from phosphorus pentoxide
(P2O5) and methanesulfonic acid (CH3SO3H) (weight ratio
P2O5:CH3SO3H 1:10). The mixture was heated at 40 °C under nitro-
gen atmosphere until complete homogeneity. Carboxylic acid
(1.15–1.5 equiv) and aromatic derivative (1.0 equiv) were then
added to Eaton’s reagent. The mixture was heated at 40–80 °C un-
der inert atmosphere for 3–30 h. After cooling to room tempera-
ture, the reaction medium was diluted with dichloromethane and
carefully poured into a separatory funnel containing sodium bicar-
bonate 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 produce a brownish oil. The crude product was purified by
m
cmꢁ1: 760, 1105, 1127, 1209,
5.1.2.4. 3-{4-[(Ethoxycarbonyl)oxy]-3,5-dimethoxybenzoyl}-2,6-
dimethoxyphenyl chloroacetate (38) (Scheme 1).
The general procedure A was followed using 4-[(ethoxycar-
bonyl)oxy]-3,5-dimethoxybenzoic acid (see Supplementary infor-
mation) (1.32 g, 4.88 mmol), 2,6-dimethoxyphenyl chloroacetate
32 (0.75 g, 3.25 mmol) and Eaton’s reagent (0.48 g P2O5 in
3.24 mL CH3SO3H). The resulting mixture was heated at 50 °C for
3 h. The final brown oil was purified by column chromatography

6050
A. Ghinet et al. / Bioorg. Med. Chem. 19 (2011) 6042–6054
(EtOAc/n-heptane 3/7) to give pure product 38 (1.18 g, 80%) as
white solid; TLC Rf (EtOAc/n-heptane 5/5) = 0.39; 1H NMR (CDCl3,
200 MHz) d (ppm) 1.40 (t, J = 7.1 Hz, 3H, OCO2CH2CH3), 3.68 (s,
3H, OCH3), 3.85 (s, 6H, 2OCH3), 3.91 (s, 3H, OCH3), 4.33 (q,
J = 7.1 Hz, 2H, OCOCH2CH3), 4.38 (s, 2H, OCOCH2Cl), 6.81 (d,
J = 8.8 Hz, 1H, ArH), 7.10 (s, 2H, ArH), 7.36 (d, J = 8.8 Hz, 1H, ArH).
d (ppm) 3.99 (s, 3H, OCH3), 4.00 (s, 3H, OCH3), 4.01 (s, 3H, OCH3),
4.03 (s, 3H, OCH3), 4.05 (s, 3H, OCH3), 7.42 (s, 1H, ArH), 7.68 (s,
1H, ArH), 12.64 (s, 1H, ArOH). 13C NMR (CDCl3, 100 MHz) d 56.4
(CH3), 56.5 (CH3), 60.9 (CH3), 61.4 (CH3), 61.6 (CH3), 103.9 (CH),
106.1 (CH), 111.5 (C), 121.1 (C), 130.6 (C), 130.7 (C), 140.6 (C),
148.5 (C), 155.0 (C), 156.0 (C), 157.5 (C), 158.4 (C), 180.3 (C),
186.7 (C); mp (EtOAc/n-heptane) >250 °C. Calcd for C19H18O8: C,
60.96; H, 4.85. Found: C, 60.83; H, 4.78.
IR
m
cmꢁ1: 760, 1105, 1127, 1209, 1253, 1336, 1409, 1597, 1651,
1770, 1792. Calcd for C22H23O10Cl: C, 54.72; H, 4.80. Found: C,
55.08; H, 4.74.
5.1.2.7. 6-(3,4,5-Trimethoxybenzoyl)-1,3-benzoxazol-2(3H)-one
(47) (Scheme 2).
5.1.2.5. 5-(3,4,5-Trimethoxybenzoyl)-1,3-benzodioxol-2-one (43)
(Scheme 2).
The general procedure A was followed using 1,3-benzoxazol-
2(3H)-one 44 (4.00 g, 29.6 mmol) and Eaton’s reagent (4.00 g
P2O5 in 27.00 mL CH3SO3H). 3,4,5-Trimethoxybenzoic acid 29
(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 60 °C for 30 h. The final brown oil was purified by
column chromatography on silica gel with EtOAc/n-heptane 1/9
to give pure compound 47 (2.24 g, 23%); mp (EtOAc/n-heptane)
220–221 °C; 1H (CDCl3, 400 MHz) d (ppm) 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 = 1.7 Hz, 1H, ArH),
8.32 (large s, 1H, NH). 13C NMR (CDCl3 + DMSO-d6, 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),
The general procedure A was followed using 3,4,5-trimethoxy-
benzoic acid 29 (0.30 g, 2.20 mmol), carbonate 42 (0.70 g,
3.31 mmol) and Eaton’s reagent (0.25 g P2O5 in 1.72 mL CH3SO3H).
The mixture was heated at 50 °C for 26 h and turned purple very
quickly. The final brown oil was purified by column chromatogra-
phy on silica gel with EtOAc/n-heptane 2/8 to afford pure 5-(3,4,5-
trimethoxybenzoyl)-1,3-benzodioxol-2-one 43 (21 mg, 3%). This
compound was not characterized and utilized directly in the next
step.
5.1.2.6. 3-(Methylsulfonyl)-6-(3,4,5-trimethoxybenzoyl)-1,3-ben-
zoxazol-2(3H)-one (45) and 1-hydroxy-2,3,5,6,7-pentamethoxy-
anthra-9,10-quinone (46) (Scheme 2).
The general procedure A was followed using 3,4,5-trimethoxy-
benzoic acid 29 (47.11 g, 222.0 mmol), 1,3-benzoxazol-2(3H)-one
44 (20.00 g, 148.0 mmol) and Eaton’s reagent (17.13 g P2O5 in
115.7 mL CH3SO3H). The mixture was heated at 60 °C for 30 h.
The final brown oil was purified by column chromatography on sil-
ica gel with EtOAc/n-heptane 1/9 to give pure compounds 45
(7.84 g, 13%) and 46 (15.51 g, 28%) as yellow solids; Physico-chem-
ical caracteristics of compound 45: 1H (CDCl3, 400 MHz) d (ppm)
3.48 (s, 3H, SO2CH3), 3.88 (s, 6H, 2OCH3), 3.95 (s, 3H, OCH3), 7.02
(s, 2H, ArH), 7.06 (d, J = 8.2 Hz, 1H, ArH), 7.71 (d, J = 1.6 Hz, 1H,
ArH), 7.75 (dd, J = 8.2, 1.6 Hz, 1H, ArH). 13C NMR (CDCl3,
100 MHz) d 28.4 (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), 153.0 (2C), 154.6 (C), 194.1 (C); mp (EtOAc/
n-heptane) 165–168 °C. Calcd for C18H17O8NS: C, 53.07; H, 4.21;
N, 3.44; S, 7.87. Found: C, 53.29; H, 4.60; N, 3.68; S, 7.53. Phys-
ico-chemical caracteristics of compound 46: 1H (CDCl3, 400 MHz)
153.5 (2C), 155.7 (C), 194.7 (C). IR
1104, 1119, 1290, 1413, 1493, 1574, 1643, 1773, 3021. Calcd for
17H15O6N: C, 62.00; H, 4.59; N, 4.25. Found: C, 61.89; H, 4.40; N,
m
cmꢁ1: 583, 704, 815, 922,
C
4.19.
5.1.2.8. N-[2,6-Dimethoxy-3-(3,4,5-trimethoxybenzoyl)phenyl]
acetamide (53) (Scheme 3).
The general procedure A was followed using 3,4,5-trimethoxy-
benzoic acid 29 (1.30 g, 6.1 mmol), N-(2,6-dimethoxyphenyl)acet-
amide 52 (1.00 g, 5.1 mmol) and Eaton’s reagent (0.47 g P2O5 in
3.2 mL CH3SO3H). The mixture was heated at 60 °C for 24 h. The fi-
nal beige oil was purified on silica gel with EtOAc/n-heptane 4/6 to
give pure compound 53 (0.82 g, 41%) as a white solid; mp (EtOAc/
n-heptane) 176–177 °C; TLC Rf (EtOAc) = 0.31; 1H NMR (CDCl3,
400 MHz) d (ppm) 2.23 (large s, 3H, NHCOCH3), 3.66 (s, 3H,
OCH3), 3.88 (s, 6H, 2OCH3), 3.92 (s, 3H, OCH3), 3.93 (s, 3H, OCH3),
6.70 (large s, 1H, ArNH), 6.77 (d, J = 8.6 Hz, 1H, ArH), 7.13 (s, 2H,
O
OMe
MeO
MeO
MeO
OMe
NHCOMe
MeO
MeO
NHCOMe
OMe
(iv)
CO2H
+
OMe
52
MeO
29
53 41%
(v)
(iii) 60%
O
OMe
OH
OH
OMe
NO2
OMe
NH2
MeO
MeO
NH2
(i)
(ii)
NO2
OMe
OMe
OMe
MeO
49
50 99%
51 60%
54 68%
O
OMe
O
OMe
OMe
OH
OCOCH2Cl
OMe
(vi)
(iv)
MeO
CO2H
+
OCOCH2Cl
MeO
OMe
MeO
OMe
32
57 87%
55
56 53%
Scheme 3. Reagents and conditions: (i) Me2SO4 3 equiv, K2CO3 5 equiv, acetone, reflux, 1 h; (ii) SnCl2ꢀ2H2O 5 equiv, conc HCl, EtOH, rt, 30 h; (iii) Ac2O 2 equiv, CH2Cl2, rt, 24 h;
(iv) Eaton’s reagent 4 equiv, 60 °C; (v) HCl 10%, MeOH, reflux, 24 h; (vi) CH3COONaꢀ3H2O 4.5 equiv, MeOH, reflux, 3 h.

A. Ghinet et al. / Bioorg. Med. Chem. 19 (2011) 6042–6054
6051
ArH), 7.35 (d, J = 8.6 Hz, 1H, ArH). 13C NMR (CDCl3, 100 MHz) d 23.5
(CH3), 56.1 (CH3), 56.3 (2CH3), 60.9 (CH3), 62.1 (CH3), 106.1 (CH),
107.4 (2CH), 123.6 (CH), 125.1 (CH), 132.8 (C), 142.5 (C), 152.9
1577, 1607, 1654, 3325. Calcd for C18H20O7: C, 62.06; H, 5.79.
Found: C, 62.00; H, 5.88.
(2C), 155.8 (C), 157.5 (C), 169.2 (C), 194.0 (C), 217.4 (C). IR
cmꢁ1: 1098, 1126, 1464, 1502, 1537, 1576, 1596, 1658. Calcd for
20H23O7N: C, 61.69; H, 5.95; N, 3.60. Found: C, 61.42; H, 5.76;
m
5.1.3.3. (3-Hydroxy-4,5-dimethoxyphenyl)(3-hydroxy-4-methoxy-
phenyl) methanone (24) (Scheme 1).
C
The general procedure B was followed using 36 (1.52 g,
N, 3.45.
3.32 mmol) and sodium acetate (AcONaꢀ3H2O) (2.04 g,
14.95 mmol) in MeOH (25 mL). The resulting reaction mixture
was stirred at reflux for 3 h. The crude solid was poured into water
and the precipitate was filtered, dried and recrystallized from EtOH
to give pure metabolite 24 (0.66 g, 65%) as an off-white solid; mp
(EtOH) 166–167 °C; 1H NMR (CDCl3, 400 MHz) d (ppm) 3.91 (s,
3H, OCH3), 3.98 (s, 3H, OCH3), 3.99 (s, 3H, OCH3), 5.64 (s, 1H, ArOH),
5.82 (s, 1H, ArOH), 6.91 (d, J = 8.3 Hz, 1H, ArH), 6.99 (d, J = 2.0 Hz,
1H, ArH), 7.00 (d, J = 2.0 Hz, 1H, ArH), 7.39 (dd, J = 8.3, 2.0 Hz, 1H,
ArH), 7.43 (d, J = 2.0 Hz, 1H, ArH). 13C NMR (CDCl3, 100 MHz) d
56.1 (CH3), 56.2 (CH3), 61.0 (CH3), 105.7 (CH), 109.7 (CH), 110.6
(CH), 116.2 (CH), 123.7 (CH), 130.9 (C), 133.5 (C), 138.8 (C), 145.2
5.1.2.9. 2,6-Dimethoxy-3-(4-methoxybenzoyl)phenyl chloroace-
tate (56) (Scheme 3).
The general procedure A was followed using 4-methoxybenzoic
acid 55 (7.05 g, 46.3 mmol), 2,6-dimethoxyphenyl chloroacetate 32
(5.34 g, 23.2 mmol) and Eaton’s reagent (2.43 g P2O5 in 16.4 mL
CH3SO3H). The mixture was heated at 60 °C for 3 h. The final beige
oil was purified on silica gel with EtOAc/n-heptane 3/7 to give
chloroacetate 56 (4.48 g, 53%) as a white solid; mp (EtOAc) 48–
50 °C; TLC Rf (EtOAc/n-heptane 3/7) = 0.12; 1H NMR (CDCl3,
200 MHz) d (ppm) 3.65 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 3.89 (s,
3H, OCH3), 4.39 (s, 2H, OCOCH2Cl), 6.88 (d, J = 8.8 Hz, 1H, ArH),
6.93 (d, J = 9.3 Hz, 2H, ArH), 7.32 (d, J = 8.8 Hz, 1H, ArH), 7.82 (d,
J = 9.3 Hz, 2H, ArH). 13C NMR (CDCl3, 100 MHz) d 40.4 (CH2), 55.5
(CH3), 56.3 (CH3), 62.5 (CH3), 106.9 (CH), 113.6 (2CH), 126.2 (C),
128.2 (CH), 130.4 (C), 132.2 (C), 132.4 (2CH), 151.5 (C), 154.1 (C),
163.7 (C), 165.1 (C), 193.2 (C). Calcd for C18H17O6Cl: C, 59.27; H,
4.70. Found: C, 59.48; H, 4.77.
(C), 148.5 (C), 150.2 (C), 152.1 (C), 194.6 (C). IR
m
cmꢁ1: 759,
1113, 1128, 1238, 1253, 1267, 1348, 1580, 1629, 1717, 1755,
3421. Calcd for C16H16O6: C, 63.15; H, 5.30. Found: C, 63.32; H,
5.28.
5.1.3.4. Ethyl 4-(3-hydroxy-4-methoxybenzoyl)-2,6-dimethoxy-
phenyl carbonate (39) (Scheme 1).
The general procedure
B was followed using 37 (3.00 g,
5.1.3. General procedure B for the synthesis of phenols from
chloroacetic esters
6.62 mmol) and sodium acetate (AcONaꢀ3H2O) (4.06 g,
29.81 mmol) in MeOH (35 mL). The resulting reaction mixture
was stirred at reflux for 1 h. The crude solid was poured into water
and the precipitate was filtered, dried and recrystallized from EtOH
to give pure product 39 (2.12 g, 85%) as an off-white solid; mp
(EtOH) 114–116 °C; 1H NMR (CDCl3, 200 MHz) d (ppm) 1.41 (t,
J = 7.1 Hz, 3H, OCO2CH2CH3), 3.87 (s, 6H, 2OCH3), 3.99 (s, 3H,
OCH3), 4.35 (q, J = 7.1 Hz, 2H, OCO2CH2CH3), 5.71 (s, 1H, ArOH),
6.92 (d, J = 8.3 Hz, 1H, ArH), 7.03 (s, 2H, ArH), 7.41 (dd, J = 8.3,
(Mono or di)chloroacetic ester (1 equiv) and sodium acetate
(4.5 equiv or 6 equiv) were dissolved in methanol. The solution
was refluxed for 1–3 h. After cooling at rt, the mixture was concen-
trated under reduced pressure. The residue was taken into distilled
water. The resulting precipitate was filtered, washed with water
several times to remove remaining sodium acetate. The solid was
recrystallized from ethanol.
1.8 Hz, 1H, ArH), 7.47 (d, J = 1.8 Hz, 1H, ArH). IR
m
cmꢁ1: 758,
5.1.3.1. (3-Hydroxy-4-methoxyphenyl)-(3,4,5-trimethoxyphenyl)
methanone (Phenstatin, 3) (Scheme 1).
1052, 1126, 1175, 1202, 1254, 1335, 1414, 1456, 1596, 1649,
1762, 3410. Calcd for C19H20O8: C, 60.64; H, 5.36. Found: C,
61.01; H, 5.42.
The general procedure B was followed using chloroacetate 31
(150.0 g, 0.38 mol) and sodium acetate (AcONaꢀ3H2O) (232.65 g,
1.71 mol) in MeOH (1 L). The reaction mixture was refluxed for
2 h. The formed solid was collected by filtration and recrystallized
from EtOH to obtain phenstatin (3) (118.52 g, 98%) as a white solid
with the same physico-chemical properties as described in the lit-
erature18; TLC Rf (EtOAc/n-heptane 5/5) = 0.32; mp (EtOH) 150–
152 °C; 1H NMR (CDCl3, 200 MHz) d (ppm) 3.89 (s, 6H, 2OCH3),
3.93 (s, 3H, OCH3), 3.99 (s, 3H, OCH3), 5.70 (s, 1H, ArOH), 6.92 (d,
J = 8.2 Hz, 1H, ArH), 7.03 (s, 2H, ArH), 7.39 (dd, J = 8.2, 2.1 Hz, 1H,
ArH), 7.44 (d, J = 2.1 Hz, 1H, ArH). Calcd for C17H18O6: C, 64.14; H,
5.70. Found: C, 64.07; H, 5.76.
5.1.3.5. Ethyl 4-(3-hydroxy-2,4-dimethoxybenzoyl)-2,6-dime-
thoxyphenyl carbonate (40) (Scheme 1).
The general procedure
B was followed using 38 (1.07 g,
2.36 mmol) and sodium acetate (AcONaꢀ3H2O) (1.93 g,
14.17 mmol) in MeOH (20 mL). The resulting reaction mixture
was stirred at reflux for 1 h. The crude solid was poured into water
and the precipitate was filtered, dried and recrystallized from EtOH
to give pure product 40 (0.81 g, 84%) as an off-white solid; mp
(EtOH) 140–143 °C; 1H NMR (CDCl3, 200 MHz) d (ppm) 1.40 (t,
J = 7.1 Hz, 3H, OCO2CH2CH3), 3.81 (s, 3H, OCH3), 3.84 (s, 6H,
2OCH3), 3.97 (s, 3H, OCH3), 4.33 (q, J = 7.1 Hz, 2H, OCO2CH2CH3),
6.70 (d, J = 8.6 Hz, 1H, ArH), 6.96 (d, J = 1.9 Hz, 1H, ArH), 7.10 (s,
5.1.3.2. (3-Hydroxy-2,4-dimethoxyphenyl)-(3,4,5-trimethoxy-
phenyl)methanone (23) (Scheme 1).
2H, ArH). IR
m
cmꢁ1: 776, 1090, 1129, 1268, 1342, 1416, 1468,
The general procedure B was followed using chloroacetate 33
(1.2 g, 2.8 mmol) and sodium acetate (AcONaꢀ3H2O) (1.73 g,
12.7 mmol) in MeOH (10 mL). The reaction mixture was refluxed
for 2 h. The formed solid was collected by filtration and recrystal-
lized from EtOH to obtain 23 (0.80 g, 81%) as white needles; TLC Rf
(EtOAc/n-heptane 5/5) = 0.30; mp (EtOH) 156–157 °C; 1H NMR
(CDCl3, 200 MHz) d (ppm) 3.81 (s, 3H, OCH3), 3.86 (s, 6H, 2OCH3),
3.93 (s, 3H, OCH3), 3.97 (s, 3H, OCH3), 5.74 (s, 1H, ArOH), 6.71 (d,
J = 8.5 Hz, 1H, ArH), 6.93 (d, J = 8.5 Hz, 1H, ArH), 7.10 (s, 2H, ArH).
13C NMR (CDCl3, 100 MHz) d 56.2 (2CH3), 56.3 (CH3), 60.9 (CH3),
61.9 (CH3), 105.7 (CH), 107.6 (2CH), 120.9 (C), 125.7 (C), 133.1
(C), 138.4 (C), 142.5 (C), 145.9 (C), 149.8 (C), 152.8 (2C), 194.2
1600, 1660, 1746, 3447. Calcd for C20H22O9: C, 59.11; H, 5.46.
Found: C, 59.57; H, 5.38.
5.1.3.6. (3-Hydroxy-2,4-dimethoxyphenyl)(4-methoxyphenyl)
methanone (57) (Scheme 3).
The general procedure
B was followed using 56 (2.51 g,
6.88 mmol) and sodium acetate (AcONaꢀ3H2O) (4.68 g, 34.4 mmol)
in MeOH (35 mL). The resulting reaction mixture was stirred at re-
flux for 3 h. The crude solid was poured into water and the precip-
itate was filtered, dried over magnesium sulfate and recrystallized
from EtOH to give pure phenol 57 (1.72 g, 87%) as a white solid; mp
(EtOH) 115–117 °C; TLC Rf (EtOAc/n-heptane 3/7) = 0.66; 1H NMR
(CDCl3, 400 MHz) d (ppm) 3.75 (s, 3H, OCH3), 3.88 (s, 3H, OCH3),
(C). IR
m
cmꢁ1: 611, 1004, 1091, 1131, 1283, 1332, 1409, 1456,

6052
A. Ghinet et al. / Bioorg. Med. Chem. 19 (2011) 6042–6054
3.96 (s, 3H, OCH3), 5.72 (s, 1H, ArOH), 6.71 (d, J = 8.6 Hz, 1H, ArH),
6.92 (d, J = 8.6 Hz, 1H, ArH), 6.93 (d, J = 8.9 Hz, 2H, ArH), 7.83 (d,
J = 8.9 Hz, 2H, ArH). 13C NMR (CDCl3, 100 MHz) d 55.5 (CH3), 56.3
(CH3), 61.8 (CH3), 106.0 (CH), 113.5 (2CH), 120.7 (CH), 126.2 (C),
130.8 (C), 132.4 (2CH), 138.3 (C), 145.6 (C), 149.6 (C), 163.5 (C),
194.0 (C). Calcd for C16H16O5: C, 66.66; H, 5.59. Found: C, 66.56;
H, 5.62.
on silica gel eluting EtOAc/n-heptane 6/4 to isolate phenol 22
(0.25 g, 76%) as a white cotton; mp (EtOAc/n-heptane) 162–
164 °C; TLC Rf (EtOAc/n-heptane 6/4) = 0.27; 1H NMR (CDCl3,
200 MHz) d (ppm) 3.80 (s, 3H, OCH3), 3.90 (s, 6H, 2OCH3), 3.97 (s,
3H, OCH3), 5.74 (s, 1H, ArOH), 5.98 (s, 1H, ArOH), 6.71 (d,
J = 8.6 Hz, 1H, ArH), 6.92 (d, J = 8.6 Hz, 1H, ArH), 7.15 (s, 2H, ArH).
13C NMR (CDCl3, 50 MHz) d 56.0 (CH3), 56.1 (CH3), 56.3 (2CH3),
107.0 (CH), 107.5 (2CH), 120.1 (C), 120.7 (C), 125.6 (C), 128.9
5.1.4. General procedure C for the synthesis of benzhydrols by
reduction of the corresponding ketones
(CH), 138.1 (C), 139.4 (C), 146.3 (2C), 149.4 (C), 193.7 (C). IR m
cmꢁ1: 602, 751, 1089, 1121, 1203, 1288, 1336, 1450, 1609, 1651,
3468. Calcd for C17H18O7: C, 61.07; H, 5.43. Found: C, 60.66; H,
5.39.
An aqueous solution of sodium borohydride (1.1–2.2 equiv) was
added dropwise to a solution of benzophenone (1 equiv) in abso-
lute ethanol. After stirring for 5–8 h at room temperature, the reac-
tion media is neutralized with a hydrochloric acid solution (1.5 M)
and extracted with ethyl acetate (3 ꢂ 25 mL). Combined organic
layers were dried over magnesium sulfate and concentrated under
reduced pressure. The residue was purified by column chromatog-
raphy on silica gel or recrystallized from H2O to afford the required
benzhydrols 26 or 27.
5.1.5.2. (4-Hydroxy-3,5-dimethoxyphenyl)(3-hydroxy-4-methoxy-
phenyl)methanone (25) (Scheme 1).
The general procedure D was followed using ethyl 4-(3-hydro-
xy-4-methoxybenzoyl)-2,6-dimethoxyphenyl carbonate 39 (2.0 g,
5.3 mmol) and ethanolamine (1.6 mL, 26.6 mmol) in EtOH 95%
(30 mL). The residue was recrystallized from acetone to give ben-
zophenone 25 (1.37 g, 85%) as an off-white compact solid; mp
(acetone) 162–164 °C; 1H NMR (CDCl3, 400 MHz) d (ppm) 3.92 (s,
6H, 2OCH3), 3.99 (s, 3H, OCH3), 5.68 (s, 1H, ArOH), 5.91 (s, 1H,
ArOH), 6.93 (d, J = 8.5 Hz, 1H, ArH), 7.10 (s, 2H, ArH), 7.37 (dd,
5.1.4.1. 5-[Hydroxy(3,4,5-trimethoxyphenyl)methyl]-2-methoxy-
phenol (26) (Scheme 1).
The general procedure C was followed using NaBH4 (0.26 g,
6.9 mmol) in water (10 mL) and phenstatin (3) (1.0 g, 3.1 mmol)
in EtOH (30 mL). The mixture was stirred at rt for 5 h. The residue
was recrystallized from H2O to give benzhydrol 26 (0.98 g, 97%) as
a brown solid with the same physical properties as described in the
literature20; mp (H2O) 145–147 °C; 1H NMR (CDCl3, 200 MHz) d
(ppm) 3.84 (s, 9H, 3OCH3), 3.89 (s, 3H, OCH3), 5.20 (s, 1H, ArOH),
5.69 (s, 1H, ArOH), 6.60 (s, 1H, ArCHAr), 6.61 (s, 2H, ArH), 6.80–
J = 8.5, 2.0 Hz, 1H, ArH), 7.42 (d, J = 2.0 Hz, 1H, ArH). IR
m :
cmꢁ1
752, 1026, 1112, 1274, 1291, 1319, 1415, 1462, 1488, 1552,
1619, 3101. Calcd for C16H16O6, 5/3 H2O: C, 57.48; H, 5.83. Found:
C, 57.10; H, 6.22.
5.1.5.3. (3,4-Dimethoxyphenyl)(4-hydroxy-3,5-dimethoxyphenyl)
methanone (21) (Scheme 1).
6.95 (m, 3H, ArH). IR
m
cmꢁ1: 1121, 1422, 1465, 1507, 1591,
The general procedure D was followed using ethyl 4-(3,4-dim-
3249, 3480. Calcd for C17H20O6: C, 63.74; H, 6.29. Found: C,
ethoxybenzoyl)-2,6-dimethoxyphenyl
carbonate
41
(0.4 g,
63.50; H, 6.11.
1.0 mmol) and ethanolamine (0.31 mL, 5.1 mmol) in EtOH 95%
(15 mL). The residue was purified by chromatography on silica
gel, eluting with EtOAc/n-heptane 6/4 to give pure benzophenone
21 (0.25 g, 78%) as an off-white solid; mp (EtOAc/n-heptane)
150–153 °C; 1H NMR (CDCl3, 400 MHz) d (ppm) 3.93 (s, 6H,
2OCH3), 3.95 (s, 3H, OCH3), 3.97 (s, 3H, OCH3), 5.91 (s, 1H, ArOH),
6.92 (d, J = 8.6 Hz, 1H, ArH), 7.10 (s, 2H, ArH), 7.39 (dd, J = 8.5,
2.0 Hz, 1H, ArH), 7.43 (d, J = 2.0 Hz, 1H, ArH). 13C NMR (CDCl3,
100 MHz) d 56.0 (CH3), 56.1 (CH3), 56.5 (2CH3), 107.6 (2CH),
109.8 (CH), 112.4 (CH), 124.7 (CH), 129.2 (C), 130.7 (C), 138.9 (C),
5.1.4.2. (3,4-Dimethoxyphenyl)(3,4,5-trimethoxyphenyl)metha-
nol (27) (Scheme 1).
The general procedure C was followed using NaBH4 (0.05 g,
0.6 mmol) in water (3 mL) and benzophenone 20 (0.20 g,
1.30 mmol) in EtOH (10 mL). The mixture was stirred at rt for
8 h. The residue was purified by chromatography on silica gel, elut-
ing EtOAc/n-heptane 4/6 to deliver pure benzhydrol 27 (0.15 g,
75%) as a viscous beige oil; TLC Rf (EtOAc/n-heptane 4/6) = 0.32;
1H NMR (CDCl3, 200 MHz) d (ppm) 3.82 (s, 3H, OCH3), 3.83 (s,
6H, 2OCH3), 3.87 (s, 6H, 2OCH3), 5.24 (s, 1H, ArOH), 6.57 (s, 2H,
ArH), 6.60 (s, 1H, ArCHAr), 6.81 (dd, J = 7.8, 1.6 Hz, 1H, ArH), 6.86
(d, J = 7.8 Hz, 1H, ArH), 6.91 (d, J = 1.6 Hz, 1H, ArH). Calcd for
146.5 (2C), 148.9 (C), 152.6 (C), 194.5 (C). IR
m
cmꢁ1: 739, 754,
1023, 1117, 1216, 1264, 1330, 1414, 1465, 1511, 1578, 1641,
3432. Calcd for C17H18O6: C, 64.14; H, 5.70. Found: C, 64.01; H,
5.38.
C18H22O6: C, 64.66; H, 6.63. Found: C, 64.58; H, 7.19.
5.1.5.4. (3,4-Dihydroxyphenyl)(3,4,5-trimethoxyphenyl)metha-
none (28) (Scheme 2).
5.1.5. General procedure D for the synthesis of phenols by
cleavage of ethyl carbonates
From benzophenone 43: A mixture of benzophenone 43 (0.021 g,
0.063 mmol) and ethanolamine (0.1 mL, 1.656 mmol) in EtOH 95%
was stirred at room temperature for 5 h. The final suspension was
concentrated in vacuo, treated with distilled water, and then neu-
tralized to pH 7 by adding slowly concentrated HCl. The aqueous
solution was extracted with dichloromethane. The organic phase
was dried (MgSO4), filtered and concentrated in vacuo. The residue
was purified by column chromatography on silica gel (EtOAc/n-
heptane 5/5) affording pure compound 28 (9.9 mg, 51%) as a red
solid.
Ethylcarbonate (1 equiv) and ethanolamine (5 equiv) were dis-
solved in 95% ethanol. The resulting yellow solution was stirred
at rt for 5 h. After reaction completion, the final suspension was
concentrated in vacuo, treated with distilled water, and then neu-
tralized to pH 7 by adding slowly concentrated HCl. The aqueous
solution was extracted with dichloromethane. The organic phase
was dried (MgSO4), filtered and concentrated in vacuo. The residue
was purified by chromatography on silica gel or recrystallized from
acetone to afford phenol.
From aminophenol 48: A solution of 48 (0.25 g, 0.82 mmol) in
AcOH (100 mL) was added to a vigorously stirred solution of so-
dium metaperiodate (3.84 g, 17.95 mmol) in 0.1 N HCl (269 mL)
at rt for 4 h. The reaction mixture was extracted with CHCl3. The
organic layer was washed with water and, after addition of AcOH
(10 mL) and KI (1.25 g, 7.53 mmol), was stirred at rt for 10 h. The
iodine formed was reduced with aqueous NaHSO3. The organic
5.1.5.1. (3-Hydroxy-2,4-dimethoxyphenyl)(4-hydroxy-3,5-dime-
thoxyphenyl)methanone (22) (Scheme 1).
The general procedure D was followed using ethyl 4-(3-hydro-
xy-2,4-dimethoxybenzoyl)-2,6-dimethoxyphenyl carbonate 40
(0.4 g, 1.0 mmol) and ethanolamine (0.3 mL, 4.9 mmol) in EtOH
95% (25 mL). The final residue was purified by chromatography

A. Ghinet et al. / Bioorg. Med. Chem. 19 (2011) 6042–6054
6053
layer was washed with water, acidified with AcOH (5 mL) and
dried (MgSO4). After evaporation of the solvent, the residue was
purified by column chromatography on silica gel with EtOAc/n-
heptane 3/7 to give pure compound 28 (40.1 mg, 16%); 1H (CDCl3,
400 MHz) d (ppm) 3.84 (s, 6H, 2OCH3), 3.96 (s, 3H, OCH3), 5.64
(large s, 2H, ArOH), 6.53 (s, 1H, ArH), 7.07 (s, 2H, ArH), 7.40 (d,
J = 8.2 Hz, 1H, ArH), 7.91 (d, J = 8.2 Hz, 1H, ArH). This compound
was not further analyzed, but directly utilized in the studies on
metabolization of phenstatin.
desired O-methylated product 48 (0.26 g, 56%) as a beige solid;
1H (CDCl3, 400 MHz) d (ppm) 3.88 (s, 6H, 2OCH3), 3.93 (s, 3H,
OCH3), 6.44 (s large, 1H, ArOH), 6.69 (d, J = 8.2 Hz, 1H, ArH), 7.00
(s, 2H, ArH), 7.31 (dd, J = 8.2, 1.8 Hz, 1H, ArH), 7.45 (d, J = 1.8 Hz,
1H, ArH). Calcd for C16H17O5N: C, 63.36; H, 5.65; N, 4.62. Found:
C, 63.61; H, 5.77; N, 4.80. This compound was not further analyzed,
but utilized directly in the next step.
5.1.6.4.
(3-Amino-2,4-dimethoxyphenyl)(3,4,5-trimethoxy-
phenyl)methanone (54) (Scheme 3).
5.1.6. General procedure E for methylation of phenolic
functions
A mixture of acetamide 53 (0.50 g, 1.28 mmol) and a 10% hydro-
chloric acid solution (15 mL) in methanol (20 mL) was stirred at
80 °C for 24 h. The final suspension was neutralized to pH 7 by
slow addition of an ammoniacal solution. The aqueous solution
was extracted with dichloromethane. The organic phase was dried
(MgSO4), filtered and concentrated in vacuo. The residue was
recrystallized from Et2O to afford pure aniline 54 (0.30 g, 68%) as
a beige solid; mp (Et2O) 145–147 °C; TLC Rf (EtOAc/n-heptane 4/
6) = 0.26; 1H (CDCl3, 400 MHz) d (ppm) 3.72 (s, 3H, OCH3), 3.86
(s, 6H, 2OCH3), 3.93 (s, 6H, 2OCH3), 4.05 (large s, 2H, ArNH2),
6.63 (d, J = 8.6 Hz, 1H, ArH), 6.78 (d, J = 8.6 Hz, 1H, ArH), 7.11 (s,
2H, ArH). 13C NMR (CDCl3, 100 MHz) d 55.8 (CH3), 56.2 (2CH3),
60.9 (CH3), 61.3 (CH3), 105.1 (CH), 107.7 (2CH), 119.1 (CH), 125.1
(C), 129.8 (C), 133.2 (C), 142.3 (C), 145.8 (C), 150.1 (C), 152.7
Benzophenones (1 equiv) were dissolved in anhydrous acetone,
under inert atmosphere. Potassium carbonate (3–5 equiv) was
then added to the mixture. Dimethyl sulfate (1–2.5 equiv) was
added dropwise to the resulting suspension. The reaction mixture
was refluxed for 2 h, then cooled at rt, and finally concentrated un-
der reduced pressure. The finally residue was recrystallized from
water to afford O-methylated compounds.
5.1.6.1. (3,4-Dimethoxyphenyl)(3,4,5-trimethoxyphenyl)metha-
none (20) (Scheme 1).
The general procedure E was followed using phenstatin (3)
(0.25 g, 0.8 mmol), K2CO3 (0.33 g, 2.4 mmol), dimethyl sulfate
(0.11 mL, 1.2 mmol) and acetone (20 mL). The mixture was re-
fluxed for 2 h. The final precipitate was recrystallized from water
to give the corresponding O-methylated ketone 20 (0.18 g, 69%)
as an off-white powder; mp (H2O) 121–122 °C (lit. 122–123 °C,22a
119–120 °C22b); 1H NMR (CDCl3, 200 MHz) d (ppm) 3.89 (s, 6H,
2OCH3), 3.94 (s, 3H, OCH3), 3.96 (s, 3H, OCH3), 3.98 (s, 3H, OCH3),
6.92 (d, J = 8.3 Hz, 1H, ArH), 7.04 (s, 2H, ArH), 7.41 (dd, J = 8.3,
1.8 Hz, 1H, ArH), 7.47 (d, J = 1.8 Hz, 1H, ArH). 13C NMR (CDCl3,
100 MHz) d 56.1 (2CH3), 56.3 (2CH3), 61.0 (CH3), 107.5 (2CH),
109.8 (CH), 112.3 (CH), 125.1 (CH), 130.3 (C), 133.3 (C), 141.6 (C),
(2C), 197.1 (C). IR
Calcd for C18H21O6N: C, 62.24; H, 6.09; N, 4.03. Found: C, 62.14;
H, 6.11; N, 4.34.
m
cmꢁ1: 1123, 1459, 1500, 1580, 1596, 1657.
5.2. Tubulin studies
All our compounds and the two reference compounds (phenst-
atin and CA-4) have been tested in identical operating conditions.32
Turbidimetric assays of microtubules were performed as de-
scribed,41 except when 50–150
lL quartz microcuvettes were uti-
149.0 (C), 152.8 (2C), 152.9 (C), 194.6 (C). IR
1118, 1242, 1265, 1329, 1412, 1578, 1632, 1732. Calcd for
18H20O6: C, 65.05; H, 6.07. Found: C, 65.17; H, 6.38.
m
cmꢁ1: 763, 998,
lized instead of 96-well plates. Lyophilized bovine brain tubulin
(97%) was purchased from Cytoskeleton Inc. and was reconstituted
to 5 mg/mL with the following buffer: 80 mM PIPES, pH 6.9, 2 mM
C
MgCl2, 0.5 mM EGTA, 1.0 mM GTP, 5% glycerol. To 63
lL of this
5.1.6.2. 4-(3,4-Dimethoxybenzoyl)-2,6-dimethoxyphenyl ethyl
carbonate (41) (Scheme 1).
solution in ultra micro quartz cuvette at 0 °C was added 7
lL of test
compound in dimethyl sulfoxide. The increase in absorbance was
monitored at 340 nm and 37 °C on a HekIOS Gamma&Delta. The
IC50 was defined as the compound molar concentration that inhib-
ited the extent of assembly by 50% after 30 min incubation.
The general procedure E was followed using ethyl 4-(3-hydro-
xy-4-methoxybenzoyl)-2,6-dimethoxyphenyl
carbonate
39
(0.50 g, 1.3 mmol), K2CO3 (0.55 g, 4.0 mmol), dimethyl sulfate
(0.19 mL, 2.0 mmol) and acetone (20 mL). The reaction mixture
was stirred and refluxed for 2 h. The final precipitate was recrystal-
lized from water to give the corresponding O-methylated ketone
41 (0.43 g, 82%) as an off-white solid; mp (H2O) 142–144 °C; 1H
5.3. Cell proliferation assay
Six metabolites and intermediates compounds were tested
against a panel of 60 human cancer cell lines at the National Cancer
Institute, Bethesda, MD.42a The cytotoxicity studies were con-
ducted using a 48 h exposure protocol using the sulforhodamine
B assay.42b
NMR (CDCl3, 400 MHz)
d
(ppm) 1.41 (t, J = 7.1 Hz, 3H,
OCO2CH2CH3), 3.87 (s, 6H, 2OCH3), 3.96 (s, 3H, OCH3), 3.98 (s, 3H,
OCH3), 4.35 (q, J = 7.1 Hz, 2H, OCO2CH2CH3), 6.91 (d, J = 8.4 Hz,
1H, ArH), 7.03 (s, 2H, ArH), 7.42 (dd, J = 8.3, 1.9 Hz, 1H, ArH), 7.49
(d, J = 1.9 Hz, 1H, ArH). 13C NMR (CDCl3, 100 MHz) d 14.1 (CH3),
56.1 (2CH3), 56.4 (2CH3), 65.2 (CH2), 106.7 (2CH), 109.8 (CH),
112.1 (CH), 125.3 (CH), 129.9 (CH), 132.1 (C), 136.3 (C), 149.1 (C),
152.0 (2C), 152.6 (C), 153.1 (C), 194.4 (C). Calcd for C20H22O8: C,
61.53; H, 5.68. Found: C, 61.14; H, 5.73.
5.4. Cell cycle analysis for metabolite 23
The frequency of G2/M cells was assessed by studying the cell
cycle after fixation overnight at 4 °C with 70% ice-cold ethanol/
phosphate-buffered saline (PBS) followed by staining with a solu-
5.1.6.3.
(4-Amino-3-hydroxyphenyl)(3,4,5-trimethoxyphenyl)
tion containing PI (50 lg/mL) and RNase A (0.5 mg/mL), and ana-
methanone (48) (Scheme 2).
lyzed with a Becton Dickinson FACScan cytofluorometer. DA1-3b
cells were incubated for 24 h in presence of metabolite 23 and then
analyzed.
A mixture of benzoxazolone 47 (0.50 g, 1.52 mmol) and sodium
hydroxide (0.30 g, 7.50 mmol) in water (20 mL) was stirred at 80 °C
for 10 h. The final suspension was neutralized to pH 7 by adding
slowly concentrated HCl. The aqueous solution was extracted with
dichloromethane. The organic phase was dried (MgSO4), filtered
and concentrated in vacuo. The residue was purified by column
chromatography on silica gel (EtOAc/n-heptane 5/5) affording
Acknowledgements
The authors gratefully acknowledge the ‘Conseil Régional Nord-
Pas-de-Calais’ for the A.G.’s PhD scholarship, the ‘Ministère de

6054
A. Ghinet et al. / Bioorg. Med. Chem. 19 (2011) 6042–6054
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Consulting and Solution GmbH; however, no bibliographic reference is
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l’Enseignement Supérieur et de la Recherche’ for the D.L.B.-R.’s PhD
scholarship, the ARC (Association pour la Recherche sur le Cancer)
for the additional financial support of this project (No. 4941) and
the NCI (National Cancer Institute) for the biological evaluation
of some compounds on their 60-cell panel.
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Supplementary data
Supplementary data (Syntheses and physico-chemical charac-
terization of all intermediates and by-products are related in this
section) associated with this article can be found, in the online ver-
sion, at doi:10.1016/j.bmc.2011.08.047.
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