Synthesis, characterization and in vitro antiproliferative activities of new 13-cis-retinoyl ferrocene derivatives

Article abstract of DOI:10.1016/j.ejmech.2009.01.029

In order to improve biological behavior of the retinoyl derivatives, monoarylferrocenyl alcohols 9a and 9b were synthesized by an improved Suzuki cross-coupling method and their 13-cis-retinoic acid analogues were prepared in moderate to good yields via t

Full text of DOI:10.1016/j.ejmech.2009.01.029

European Journal of Medicinal Chemistry 44 (2009) 2572–2576
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, characterization and in vitro antiproliferative activities of new
3-cis-retinoyl ferrocene derivatives
1
Bohua Long, Shengzong Liang, Dingcheng Xin, Yingbin Yang, Jiannan Xiang*
College of Chemistry and Chemical Engineering and Biomedical Engineering Center, State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University,
Changsha 410082, China
a r t i c l e i n f o
a b s t r a c t
Article history:
In order to improve biological behavior of the retinoyl derivatives, monoarylferrocenyl alcohols 9a and
Received 12 October 2008
Accepted 23 January 2009
Available online 5 February 2009
9
b were synthesized by an improved Suzuki cross-coupling method and their 13-cis-retinoic acid
analogues were prepared in moderate to good yields via the Mitsunobu reaction. Their structures were
1
13
confirmed by IR, H NMR, CNMR, MS spectra and element analysis and their antiproliferative activities
were determined in vitro using human cancer cell lines. The results of bioassay showed that these
organometallic analogues exhibited higher antiproliferative activities than parent 13-cis-retinoic acid
and other retinoyl derivatives.
Keywords:
13-cis-Retinoic acid
Retinoyl ferrocene derivatives
Biological activity
Synthesis
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
organometallic compounds as alternatives of the chemotherapy of
drug-resistance in cancer and tropical diseases [9–11]. The
Retinoids include active metabolites of vitamin A (retinol, 1) as
well as a diverse spectrum of natural or synthetic derivatives. They
play an essential role in vertebrate growth and development,
supporting cell differentiation, embryonic development, the
immune response, and reproduction [1–3]. All-trans-retinoic acid
stability, non-toxicity and readily membrane-permeation of the
ferrocenyl group, the accessibility of a large variety of derivatives,
as well as its favourable electrochemical properties have made
ferrocene and its derivatives very suitable for biological applica-
tions and for conjugation with biomolecules [12,13]. Several
structural modification of established drugs with ferrocenyl
moiety have been reported, such as ferrocene fluconazole [14],
ferrocene aspirin [15], the anti-malarial drugs chloroquine
(termed ferroquine), quinine, mefloquine, and artemisinin [16,17],
and the anti-cancer drug tamixofen (termed ferrocifen) [18].
Accordingly, using ferrocenyl-containing derivatives as medicines
and other chemotherapeutants has long been recognized as an
attractive way.
In order to search novel retinoyl derivatives with potent anti-
proliferative activities, our research group had synthesized some
sugar derivatives with structures of the glycosyl moiety both
directly and indirectly linked with 13-cis-RA and obtained some
biological compounds [19]. The results of biological assays showed
that some compounds possessed some degree of antitumor activ-
ities. Following our continual interest in search of novel analogues
of retinoic acid with potent biological activities, we have sought to
design and synthesize a class of novel retinoates structurally
modified by ferrocenyl group. In the present work, on the basis of
an improved synthetic method of monoarylferrocenyl alcohols 9a
and 9b, we herein reported their chemical and biological screening
results involving their in vitro antitumor activities.
(2, ATRA), 13-cis-retinoic acid (3, 13-cis-RA), and other retinoids are
currently used for treatment of dermatological disorders and as
chemotherapeutic agent against various endothelial cancers, breast
cancer, and endometrial cancer [4,5]. The actions of retinoids are
mediated through binding and activation of the retinoic acid
receptors (RARs) or retinoid X receptors (RXRs), which function as
ligand-dependent transcription factors [6]. However, retinoids,
including retinoic acids, have been found to be too toxic at high
dosage levels to be of practical value for cancer prevention in higher
mammals. Side effects such as teratogenicity, hepatotoxicity, and
headaches have been observed as a result when the most of these
compounds were used [7]. Researchers have been pursuing the
discovery and synthesis of novel retinoic acid analog in order to
increase their therapeutic efficiency and/or reduced toxicity
(Scheme 1) [8].
Recently, increasing interests have been focused on developing
structural variations of established drugs by metallocenic
*
Corresponding author. Fax: þ86 0731 8821740.
E-mail address: jnxiang@hnu.cn (J. Xiang).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.01.029

B. Long et al. / European Journal of Medicinal Chemistry 44 (2009) 2572–2576
2573
1
9
20
1
7
16
preparation of monosubstituted arylferrocenes. For the present
synthesis, we sought to apply the Suzuki Miyaura cross-coupling
method to this more complex side chain. The details of this study
will be disclosed at a later date.
1
R2
5
7
11
, R = H; R = CH OH
retinol
1
1
2
2
1
4
2
3
6
9
13 14
2, R = H; R = COOH all-trans-retinoicacid
3, R1= COOH; R2= H
8
10
12
1 2
5
R
13-cis-retinoicacid
1
18
15
The reduction of the carbonyl group of 8a was facile with
sodium borohydride to give 9a. The physical and spectral charac-
teristics of 9a were identical to the reported literature [25]. The
unreported compound 9b was prepared according to the similar
Scheme 1. Structures of retinol, all-trans-retinoic acid and 13-cis-RA.
1
procedure for 9a, whose structure had been confirmed by IR, H
2
. Chemistry
NMR and MS spectra.
2.1. Synthesis of ferrocenyl alcohols 4a–4e and ferrocenyl phenol 4f
2.3. Synthesis of ferrocenyl retinoates 5a–5h
Ferrocenemethanol 4a [20], Ferroceneethanol 4b [20], Ferrocene
The esterification of 13-cis-retinoic acid had been reported in
propanol 4c [20], Ferrocenebutanol 4d [20], p-Ferrocenebenzyl
alcohol 4e [21] were prepared via well established routes. p-ferro-
cenyl phenol 4f was prepared via reaction of diazotized 4-amino
phenol with ferrocene according to a literature procedure [22].
several ways. The reported reaction of 13-cis-RA to react with thi-
onyl chloride (PCl /PCl or other carbonyl chlorides) to form reti-
5
3
noyl chloride, which in turn is reacted with alcohols to produce
retinoates, failed in our laboratory because hydrogen chloride in
solution of retinoyl chloride isomerizes the polyene chain [26].
Initially we envisioned a carbodiimide-mediated coupling using
DCC in combination with a catalytic amount of DMAP. Surprisingly,
the reaction proved to be problematic, providing the desired reti-
noates in less than 60% yield after a prolonged reaction time (48 h)
and use of a large excess (5 equiv) of DCC. Unfortunately, our
attempts to optimize the conditions did not result in a significant
enhancement of the reaction rate or yield. Finally we were
intrigued by the possible application of the Mitsunobu reaction to
construct our desired C–O bond and furnish retinoates. The Mit-
sunobu reaction, discovered in 1967 [27], is a robust and invaluable
synthetic transformation that allows for the stereo-selective
incorporation of azides [28], esters [29], nitriles [30], phthalimides
[31], and sulfonamides [32] with inversion of configuration.
Moreover, various protocols based on the Mitsunobu reaction have
been developed for the coupling of alkyl alcohols with phenols or
carboxylic acids as substrates. Many of these procedures have been
modified for use on solid-phase supports [33]. As such, we envi-
sioned the Mitsunobu reaction as an attractive route to pursue for
the synthesis of retinoates. Gratifyingly, the experiment results
proved that Mitsunobu reaction was a convenient and effective
method for the esterification of 13-cis-RA with various ferrocenyl
alcohols producing the corresponding retinoates in good to excel-
lent yields (as shown in Table 1). It presents the advantages of mild
conditions, high yield, and ease of operation.
2
.2. Synthesis of monoarylferrocenyl alcohols 9a and 9b
The synthetic process of monoarylferrocenyl alcohols 9a and 9b
were performed as shown in Scheme 2. The most frequently used
method is an arylation process involving the reaction of ferrocene
with aryldiazonium salts. However, the yields of mono-
arylferrocenes obtained in these reactions were lower than 40% in
many cases [23]. Furthermore, these reactions are not specific and
0
produce 1, 1 -, 1, 2- and 1, 3-diarylferrocenes which are normally
difficult to separate. Transition metal-catalysed cross-coupling
reactions have proved their value in synthetic and materials
chemistry as tools for C–C bond formation and may be applied to
the synthesis of diaryls containing virtually any functional group
[24]. Introduction of the side chain has efficiently been accom-
plished by an improved Suzuki cross-coupling reaction between
ferroceneboronic acid and aromatic triflates. In an initial experi-
ment, the coupling between 7a and ferroceneboronic acid gave
only 20% of the coupling product under conventional Suzuki
conditions [Pd(PPh
study of various solvents, bases, and catalysts revealed that the
combination of Pd(PPh (0.05 equiv) and K PO (2 equiv) in
refluxing dioxane gave reproducibly high yields of the coupling
reported product 8a [25] and previously unreported compound 8b
under mild conditions.
ꢀ
3
)
4
, K
2
HPO
4
, DMF, THF, 80 C]. A systematic
3
)
4
3
4
The variables in the Suzuki reaction as applied to the synthesis
of arylferrocenes were numerous. With this method, the byproduct
boric acid was easily removed by aqueous extraction and posed no
toxicological concern. This modification of the Suzuki cross-
coupling reaction proved to be a clean and useful method for the
3. Biological studies
All 13-cis-Retinoyl ferrocene derivatives 5a–5h were tested for
antiproliferative activities toward three different cancer cell lines:
human lung cancer cell line (A549), human liver cancer cell line
(
BEL7404), and human tongue cancer cell line (Tca). The results,
R1
R1
expressed as the concentration of the compound required to kill the
tumor cell by 50% (IC50), are listed in Table 2. 13-cis-RA was used as
a reference substance.
Tf O
FcB(OH)2
2
HO
R2
TfO
R2
pyridine
Pd(PPh ) , aq Na CO , DME
3
4
2
3
reflux
The screening data revealed that all selected compounds
showed some antitumor activities, and were stronger than parent
13-cis-retinoic acid. It should be noted that the activity of the fer-
rocenyl retinoates 5a–5d decreased with increasing of the ferro-
cenyl carbon chain. It is interesting to note that the other ferrocenyl
retinoates 5e–5h which include aromatic group showed some
degree of antiproliferative activities, but exhibited lower activities
than those with saturated straight-chain. Compared with our
reported retinoyl sugar derivatives [19], the biological activities of
this type of compounds are increased. It is interesting to note that
the activities in vitro of the title compounds appeared to be weakly
associated with the substituent group on the benzene cycle. The
6
a: R = CHO, R = H
7a: R = CHO, R = H
1 2
1
2
6b: R = OCH , R = CHO
7b: R = OCH , R =CHO
1 3 2
1
3
2
R1
R1
NaBH4
Fc
R2
Fc
R2
8
a: R = CHO, R = H
9a: R = CH OH, R = H
1 2 2
1
2
8
b: R = OCH , R =CHO
9b: R1 = OCH3, R2 =CH2OH
1
3
2
Scheme 2. The synthetic route to monoarylferrocenyl alcohols 9a and 9b.

2574
B. Long et al. / European Journal of Medicinal Chemistry 44 (2009) 2572–2576
Table 1
The Mitsunobu reaction of 13-cis-retinoic acid with ferrocenyl alcohols.
R
OH
COOH
COO
R
Ph P, DIAD, THF
3
+
Fe
Fe
Entry
1
Fc–R–OH
Time (h)
2
Product
Yield (%)
83
COO-CH -Fc
(5a)
2
FcCH
FcCH
FcCH
FcCH
Fc
2
2
2
2
OH (4a)
2 2
COO-CH CH
-Fc(5b)
2
3
4
5
6
7
CH
CH
CH
2
2
2
OH (4b)
2.5
2
82.5
82
2 2 2
COO-CH CH CH
-Fc(5c)
CH
CH
2
2
OH (4c)
COO-CH CH CH CH -Fc
2
2
2
2
CH
2
OH (4d)
2
(5d)
81
COO CH2
Fc(5e)
CH OH (4e)
2
80
2
COO
Fc(5f)
OH (4f)
3
75
Fc
Fc
CH OH
2
(9a)
3
COO CH2
COO CH2
(5g)
76
Fc
Fc
OCH3
Fc (5h)
OCH3
8
(9b)
2
78
CH OH
2
derivative 5h with electron-donating groups on phenyl fragment
shows weaker antitumor activities, but the effect is not obvious.
monitored by TLC. Melting points were measured with a XRC-1
melting point apparatus and are uncorrected. FT infrared (IR) spectra
1
were recorded in KBr disks using FD-5DX spectrometer. H NMR and
13
4
. Conclusion
C NMR spectrawere obtained ona VarianINOVA-400 Spectrometer,
using CDCl as a solvent; TMS ( 0.00 ppm) was used as an internal
3
d
Eight 13-cis-RA analogues were synthesized by structural
standard. All NMR chemical shifts are reported as d values inparts per
million (ppm) and coupling constants (J) are given in hertz (Hz). The
splitting pattern abbreviations are as follows: s, singlet; d, doublet; br,
broad peak;and m, multiplet. Massspectrawere carried outonaZAB-
HS mass spectrometer.
modifications with ferrocenyl groups in order to decrease toxicities
of 13-cis-RA and enhance its antiproliferative activities. Secondly,
a new retinoylation method using the Mitsunobu reaction was
developed after evaluating various reaction conditions. All selected
compounds (5a–5h) were tested by MTT assay on growth of three
different tumor cell lines. The results revealed that these
compounds showed strong potential antitumor activities
5.2. General procedure for the cross-couplings of ferroceneboronic
acid with aryl triflates (8a and 8b)
5
. Experimental protocols
A mixture of ferroceneboronic acid (5.5 mmol), aryltriflate
(
5 mmol), Pd(PPh
3
)
4
(0.25 mmol), and K
3
PO
4
(10 mmol) in freshly
until no
5.1. General
distilled dioxane (30 mL) was heated to reflux under N
2
starting material could be detected by TLC. The product was
extracted with ethyl acetate, washed with brine, and dried over
3
13-cis-RA was purchased from Sigma Chemical Co. Ph P and dii-
sopropyl azodicarboxylate (DIAD) were purchased from Alfa Aesar.
Theferroceneand allotherreagentswere ofthe highestcommercially
MgSO
following compounds 8a and 8b.
8a: yield 88%; yellow solid; m.p. 59–60 C IR (KBr): 1684, 1589,
4
. Isolation by column chromatography over silica gel gave
ꢀ
ꢀ
available quality. All retinoids were stored at ꢁ20 C under dry
ꢁ
1 1
nitrogen atmosphere. All operations involved in the preparation,
isolation, purification, and transfer of retinoids were carried out
under an atmosphere of dry nitrogen. All operations were also per-
formed in dim light or photographic darkroom light with containers
wrapped with aluminum foil or black cloth. All reactions were
1495, 1259, 1194, 1105, 1010, 803, 689 cm . H NMR (CDCl
(s, 5 H, Cp); 3.76 (t, 2 H, J ¼ 1.8, C -m); 3.89 (t, 2 H, J ¼ 1.8, C
6.6–7.3 (m, Ar); 9.80 (s, 1 H, CHO).
8b: yield 85%; yellow solid; m.p. 82–83 C; IR (KBr) 1689, 1593,
3
): 3.50
5
H
4
H -o);
5 4
ꢀ
ꢁ
1 1
3
1513, 1266, 813, 704 cm . H NMR (400 MHz, CDCl ) d: 3.97 (s, 3H,

B. Long et al. / European Journal of Medicinal Chemistry 44 (2009) 2572–2576
2575
ꢀ
5b: yield: 82.5 %; orange yellow solid; m.p. 102–103 C. Anal.
Table 2
Cytotoxicity of ferrocenyl retinoates 5a–5h against A549, BEL7404 and Tca cancer
cell.
1
calc. for C32
NMR (400 MHz, d
on C ); 1.58 (m, 2H, H on C
on C ); 2.00–2.05 (m, 2H, H on C
H, J ¼ 6.8 Hz, CH CH ); 4.08 (s, 5H, Cp); 4.14 (t, 2H, J ¼ 14.4 Hz,
OCH CH ); 4.19 (s, 2H, C -m); 4.21 (s, 2H, C -o); 5.70 (s, 1H, H
on C14); 6.22 (d, 1H, H
, J ¼ 13.6 Hz); 6.25 (d, 1H, H10, J ¼ 11.2 Hz);
.28 (d, 1H, J ¼ 11.6 Hz,
J ¼ 10.4 Hz); 7.08 (dd, 1H,
J ¼ 15.2 Hz); 7.68 (d, 1H, H12, J ¼ 15.2 Hz). C NMR (400 MHz, d
DMSO) 12.9, 19.3, 21.0, 21.8, 29.0, 33.1, 34.3, 39.7, 62.0, 68.6, 68.7,
69.5, 76.7, 77.0, 77.3, 116.4, 128.5, 129.4, 130.0, 130.4, 132.2, 137.5,
H
40FeO
2
: C, 74.99; H, 7.87, found C, 75.11; H, 7.79. H
: 1.02 (s, 6H, 2CH ); 1.44–1.45 (m, 2H, H
); 1.70 (s, 3H, CH on C ); 1.97 (s, 3H, CH
); 2.08 (s, 3H, CH on C13); 2.62 (t,
6
-DMSO)
d
3
Compound
Cell line/IC50
A549
(
m
M)
2
3
3
5
3
BEL7404
Tca
9
4
3
2
2
2
5
5
5
5
5
5
5
5
a
b
c
d
e
f
20.4
21.5
24.1
26.0
28.8
29.3
30.7
31.2
35.8
22.3
24.9
25.2
27.6
30.5
31.1
33.0
37.2
42.6
18.6
19.7
20.3
21.7
28.0
30.1
31.8
32.5
39.7
2
2
5
H
4
5 4
H
8
6
H ,
7
11
H ,
13
6
-
g
h
d
13-cis-RA
1
4
37.7, 139.8, 151.4, 166.1. IR (KBr) 2930, 1715, 1232, 1140, 974,
ꢁ
1
þ
92 cm . MS: m/z ¼ 513 ([M þ H] )
5
c: yield: 82 %; orange yellow oil. Anal. calc. for C33
H
42FeO
5.28; H, 8.04; found C, 75.36; H, 8.00; H NMR (400 MHz, d
: 1.02 (s, 6H, 2CH ); 1.43–1.46 (m, 2H, H on C ); 1.56–1.61
m, 2H, H on C ); 1.69 (s, 3H, CH on C ); 1.81 (m, 2H, CH ); 2.00 (s,
); 2.01–2.03 (m, 2H, H on C ); 2.08 (s, 3H, CH on C13);
.35 (t, 2H, J ¼ 7.6 Hz, CH ); 4.05 (s, 5H, Cp); 4.08 (t, 2H, J ¼ 6.8 Hz,
CH ); 4.12 (s, 2H, C -m); 4.20 (s, 2H, C -o); 5.72 (s, 1H, H
on C14); 6.22 (d, 1H, H
, J ¼ 16 Hz); 6.28 (d, 1H, H10, J ¼ 11.2 Hz); 6.31
, J ¼ 6.8 Hz); 7.07 (dd, 1H, H11, J ¼ 11.6 Hz, J ¼ 11.6 Hz); 7.68
2
: C,
6
-
1
7
OCH
3
); 4.10 (s, 5H, Cp); 4.44 (s, 2H, C
5
H
4
-m); 4.93 (s, 2H, C
5
H
4
-o);
DMSO)
d
3
2
7
1
.39 (s, 1H, Ar); 7.40 (s, 1H, Ar); 7.65 (d, 1H, Ar, J ¼ 17.6 Hz); 9.95 (s,
(
3
2
3
3
5
2
H, CHO).
H, CH
3
on C
9
4
3
2
5.3. A general synthetic method for compounds 9a and 9b
OCH
2
2
H
5 4
5 4
H
8
Compound 8 (4.0 mmol) was suspended methanol (100 mL) at
room temperature with stirring. NaBH
(
(
2
1
d, 1H, H
d, 1H, H12, J ¼ 15.6 Hz). C NMR (400 MHz, d
1.0, 21.8, 29.0, 33.1, 34.3, 39.7, 62.0, 68.6, 68.7, 69.5, 76.7, 77.0, 77.3,
7
4
(0.15 g, 4 mmol) was added
13
6
-DMSO) d 12.9, 19.3,
with magnetic stirring. The reaction mixture was stirred for an
additional hour at rt and then poured into ice-water (100 mL). The
pH was adjusted to about 6.0–7.0. The mixture was extracted with
ethyl acetate (150 mL ꢂ 3). The combined organic layers were
16.4, 128.5, 129.4, 130.0, 130.4, 132.2, 137.5, 137.7, 139.8, 151.4, 166.1.
ꢁ1
IR (KBr) 2929, 1730, 1242, 1136, 974, 496 cm . MS: m/z ¼ 527
þ
(
[M þ H] )
washed with water (100 mL ꢂ 2), dried with MgSO
4
, and evapo-
ꢀ
5
d: yield: 81 %; orange yellow solid; m.p. 61–62 C. Anal. calc.
rated by vacuum. The residue was isolated by column chromatog-
1
for C34
H
44FeO
400 MHz, d -DMSO)
); 1.56–1.61 (m, 2H, H on C
on C ); 1.81 (m, 2H, CH
.01–2.03 (m, 2H, H on C ); 2.08 (s, 3H, CH
); 4.03 (s, 5H, Cp); 4.05 (t, 2H, J ¼ 6.8 Hz, OCH
-m); 4.14 (s, 2H, C -o); 5.69 (s, 1H, H on C14);
, J ¼ 16.4 Hz); 6.25 (d, 1H, H10, J ¼ 4.8 Hz); 6.29 (d,
, J ¼ 15.6 Hz); 7.06 (dd, 1H, H11, J ¼ 12 Hz, J ¼ 11.2 Hz); 7.67
2
: C, 75.55; H, 8.20; found C, 75.62; H, 8.14; H NMR
: 1.09 (s, 6H, 2CH ); 1.44–1.45 (m, 2H, H on
); 1.45–1.60 (m, 4H, CH CH ); 1.62
); 2.00 (s, 3H, CH on C );
on C13); 2.40 (t, 2H,
CH );
raphy and gave following compounds 9a and 9b.
(
C
(
2
6
d
3
9
a: yield: 91%; yellow oil. IR (KBr): 3094, 1634, 1385, 1107, 1032,
ꢁ
1 1
2
3
2
2
1
005, 820, 764, 490 cm . H NMR (CDCl
Cp); 4.20(t, 2H, J ¼ 1.8); 4.44 (t, 2 H, J ¼ 1.8); 4.60 (s, 2 H); 7.16–7.28
m, 4 H, Ar). 13C NMR (CDCl
): 64.2, 68.8, 69.0, 70.2, 70.5, 127.2,
28.7, 129.3, 129.7, 131.2, 131.6.
b: yield: 90%; yellow solid. m.p. 125 C. 1H NMR (400 MHz,
CDCl : 3.76 (s, 3H, OCH ); 4.44 (s, 5H, C ); 4.52 (s, 2H, C -m);
.90 (s, 2H, C -o); 7.24–7.31(m, 3 H, Ar). IR (KBr) 3096,1641, 1390,
121, 1029, 500 cm . MS: m/z ¼ 322 ([M þ H] ).
3
): 3.0 (bs, OH); 4.05(s, 5 H,
s, 3H, CH
3
5
2
3
9
4
3
(
1
3
J ¼ 7.6 Hz, CH
2
2
2
4
6
1
(
2
4
.10 (s, 2H, C
.21 (d, 1H, H
H, H
5
H
4
5 4
H
ꢀ
9
8
3
)
d
3
5
H
5
5 4
H
7
4
1
5
H
4
13
d, 1H, H12, J ¼ 15.2 Hz). C NMR (400 MHz, d -DMSO)
0.8, 21.8, 27.3, 28.4, 28.8, 29.1, 32.9, 34.1, 39.2, 39.4, 39.6, 39.8,
0.0, 40.2, 40.4, 63.4, 67.1, 68.0, 68.6, 88.8, 116.5, 128.4, 129.3,
6
d 12.9, 19.0,
ꢁ
1
þ
5
.4. General procedure for the synthesis of 5a–5h
129.9, 130.6, 132.9, 137.3, 137.5, 140.3, 151.5, 165.9. IR (KBr) 2929,
ꢁ1
þ
1
730, 1242, 1136, 974, 496 cm . MS: m/z ¼ 541 ([M þ H] )
ꢀ
To a stirred solution of the ferrocenyl alcohol or phenol
5e: yield: 80 %; orange yellow solid; m.p. 128–129 C. Anal.
1
(
3 mmol), PPh
3
(1.18 g, 4.5 mmol), and 13-cis-RA (3 mmol) in dry
calc. for C37
NMR (400 MHz, d
H on C ); 1.56–1.61 (m, 2H, H on C
(s, 3H, CH
H
42FeO
2
: C, 77.34; H, 7.37; found C, 77.41; H, 7.25;
: 1.03 (s, 6H, 2CH
); 1.62 (s, 3H, CH
); 2.01–2.03 (m, 2H, H on C ); 2.08 (s, 3H, CH
); 4.32 (s, 2H, C -m);
-o); 5.70 (s, 1H, H on C14); 6.21 (d, 1H, H
J ¼ 4.8 Hz); 6.29 (d, 1H,
H
THF (20 mL) was added to DIAD (0.8 g, 4.5 mmol) under N
2
atmo-
6
-DMSO)
d
3
); 1.44–1.45 (m, 2H,
on C ); 2.00
on
ꢀ
sphere at 0 C. The reaction mixture was allowed to warm to rt and
was stirred for additional 2 h. The reaction was monitored by TLC.
The yellow reaction mixture was concentrated on a rotary evapo-
rator (30 C) to give a viscous oil. The residue was purified by
column chromatography (ethyl acetate/petroleum ether, v/v) and
2
3
3
5
3
on C
9
4
3
C
13); 4.05 (s, 5H, Cp); 4.06 (s, 2H, OCH
2
5 4
H
ꢀ
4.66 (s, 2H, C
5
H
4
8
,
H ,
7
J ¼ 16.4 Hz); 6.25 (d, 1H,
H
10
,
gave retinoates 5a–5h in various yields.
J ¼ 15.6 Hz); 7.06 (dd, 1H, H11, J ¼ 12 Hz, J ¼ 11.2 Hz); 7.46 (s, 2H,
ꢀ
13
5
a: yield: 83 %; orange yellow solid; m.p. 105–106 C. Anal. calc.
C
6
H
4
); 7.29(s, 2H, C
6
H
d
4
); 7.82 (d, 1H, H12, J ¼ 15.6 Hz). C NMR
1
for C31
H
38FeO
400 MHz, d -DMSO)
); 1.60 (m, 2H, H on C
); 2.00–2.05 (m, 2H, H on C
); 4.30 (s, 2H, C
.62 (s, 1H, H on C14); 6.15 (d, 1H, H
, J ¼ 16.0 Hz); 6.24 (d, 1H, H10
J ¼ 11.2 Hz); 6.27 (d, 1H, H , J ¼ 10.4 Hz); 6.99 (dd, 1H, H11
J ¼ 11.6 Hz, J ¼ 11.6 Hz); 7.79 (d, 1H, H12, J ¼ 15.2 Hz). C NMR
400 MHz, d -DMSO) 12.9, 19.3, 21.0, 21.8, 29.0, 33.1, 34.3, 39.7,
2.0, 68.6, 68.7, 69.5, 76.7, 77.0, 77.3, 116.4, 128.5, 129.4, 130.0, 130.4,
32.2, 137.5, 137.7, 139.8, 151.4, 166.1. IR (KBr) 2924, 1707, 1232, 1140,
2
: C, 74.69; H, 7.68; found C, 74.76; H, 7.58; H NMR
: 1.03 (s, 6H, 2CH ); 1.30–1.50 (m, 2H, H on
); 1.71 (s, 3H, CH on C ); 1.99 (s, 3H, CH on
); 2.06 (s, 3H, CH on C13); 4.17 (s, 5H,
-m); 4.93 (s, 2H, C -o);
(400 MHz, d -DMSO)
6
12.9, 19.2, 21.0, 21.8, 28.9, 29.0, 33.1, 34.3,
(
C
C
6
d
3
39.6, 65.5, 66.8, 69.3, 69.9, 76.71, 77.0, 77.3, 116.2, 126.3, 128.3,
128.6, 129.3, 130.1, 130.3, 132.4, 137.5, 137.7, 139.3, 151.7. IR (KBr)
2
9
3
3
5
3
ꢁ
1
þ
4
3
2929, 1730, 1242, 1136, 974, 496 cm . MS: m/z ¼ 574 ([M þ H] )
ꢀ
Cp); 4.21 (s, 2H, OCH
2
5
H
4
5
H
4
5f: yield: 75 %; orange yellow solid; m.p. 137–138 C. Anal. calc.
1
5
8
,
,
for C36
(400 MHz, d
); 1.60–1.61 (m, 2H, H on C
CH on C ); 2.02–2.03 (m, 2H, H on C
(s, 5H, Cp); 4.31 (s, 2H, C -m); 4.61 (s, 2H, C
on C14); 6.13 (d, 1H, H
, J ¼ 16 Hz); 6.23 (d, 1H, H10, J ¼ 4.8 Hz); 6.28
(d, 1H, H
, J ¼ 15.6 Hz); 7.03 (dd, 1H, H11, J ¼ 12 Hz, J ¼ 11.2 Hz); 7.06
H40FeO
2
: C, 77.14; H, 7.19; found C, 77.26; H, 7.10; H NMR
7
6
-DMSO)
d
: 1.02 (s, 6H, 2CH
); 1.71 (s, 3H, CH
); 2.17 (s, 3H, CH
-o); 5.87 (s, 1H, H
3
); 1.45–1.47 (m, 2H, H on
on C ); 2.01 (s, 3H,
on C13); 4.05
1
3
C
2
3
3
5
(
6
1
9
6
d
3
9
4
3
H
5 4
5 4
H
8
ꢁ
1
þ
74, 833, 488 cm . MS: m/z ¼ 499 ([M þ H] )
7

2576
B. Long et al. / European Journal of Medicinal Chemistry 44 (2009) 2572–2576
(
s, 2H, C
6
H
4
); 7.48 (s, 2H, C
-DMSO)
6
H
4
); 7.84 (d, 1H, H12, J ¼ 15.2 Hz). 13C
formazan crystals were solubilized with 100 mL of DMSO and the
NMR (400 MHz, d
3
1
6
d
12.9, 19.2, 21.2, 21.7, 29.0, 33.1, 34.3,
solution was vigorously mixed to dissolve the reacted dye. The
absorbance of each well was read on a microplate reader (DYNA-
TECH MR7000 instruments) at 570 nm. The spectrophotometer
was calibrated to zero using culture medium without cells. We
selected inhibitory effect to evaluate side effects of the silica-coated
fluorescent 5a to cells proliferation. The inhibitory effect of 5a was
calculated as percentage inhibition in comparison to the value
obtained in untreated well to which no 5a was added. All the other
compounds were determined by the same method.
9.6, 66.9, 69.5, 70.2, 76.7, 77.0, 77.2, 77.3, 115.2, 121.3, 121.5, 127.0,
29.0, 129.1, 130.2, 133.2, 136.5, 137.4, 137.7, 140.5, 149.1, 153.8, 164.7.
ꢁ
1
IR (KBr) 2925, 1724, 1207, 1120, 960, 818, 503 cm . MS: m/z ¼ 560
þ
(
[M þ H] )
5
g: yield: 76 %; orange yellow oil. Anal. calc. for C37
H
42FeO
7.34; H, 7.37; found C, 77.39; H, 7.31; H NMR (400 MHz, d
: 1.02 (s, 6H, 2CH ); 1.44–1.45 (m, 2H, H on C ); 1.56–1.61
m, 2H, H on C ); 1.62 (s, 3H, CH on C ); 2.00 (s, 3H, CH on C );
.01–2.03 (m, 2H, H on C ); 2.08 (s, 3H, CH on C13); 4.15 (s, 5H,
); 4.31 (s, 2H, C -m); 4.51 (s, 2H, C -o);
.72 (s, 1H, H on C14); 6.21 (d, 1H, H
, J ¼ 16.4 Hz); 6.25 (d, 1H, H10
J ¼ 4.8 Hz); 6.28 (d, 1H, H , J ¼ 15.6 Hz); 7.06 (dd, 1H, H11, J ¼ 12 Hz,
J ¼ 11.2 Hz); 7.25 (s, 2H, C ); 7.41 (s, 2H, C ); 7.82 (d, 1H, H12
J ¼ 15.6 Hz). C NMR (400 MHz, d -DMSO) 12.8, 19.1, 21.2, 21.9,
9.1, 29.3, 33.1, 34.6, 39.8, 65.7, 66.9, 69.6, 67.0, 76.9, 77.1, 77.3,
16.9, 126.8, 128.3, 128.1, 129.9, 130.7, 130.5, 132.6, 137.9, 137.1,
2
: C,
6
-
1
7
DMSO)
(
2
d
3
2
3
3
5
3
9
4
3
Cp); 4.21 (s, 2H, OCH
2
5
H
4
5 4
H
Acknowledgments
5
8
,
7
The authors are grateful for the financial support of 985 Foun-
dation of Ministry of Education. This work was supported by the
National Natural Science Foundation of China (grant no. 20472018),
by the Natural Science Foundation of Hunan (key project no.
6
H
4
6
H
4
,
13
6
d
2
1
1
07JJ3019) and Doctoral Fund of Ministry of Education of China
ꢁ
1
39.9, 152.3. IR (KBr) 2929, 1730, 1242, 1136, 974, 496 cm . MS: m/
(grant no. 20060532022).
þ
z ¼ 574 ([M þ H] )
5
3
h: yield: 78 %; orange yellow oil. Anal. calc. for C38H44FeO : C,
1
7
5.49; H, 7.34; found C, 75.42; H, 7.33; H NMR (400 MHz, d
DMSO) : 1.01 (s, 6H, 2CH ); 1.42–1.45 (m, 2H, H on C ); 1.56–1.58
m, 2H, H on C ); 1.69 (s, 3H, CH on C ); 2.00 (s, 3H, CH on C );
.01–2.03 (m, 2H, H on C ); 2.08 (s, 3H, CH on C13); 3.86 (s, 3H,
); 4.00 (s, 5H, Cp); 4.28 (s, 2H, C -m); 4.76 (s, 2H, C -o);
.10 (s, 2H, OCH ); 5.77 (s, 1H, H on C14); 6.20 (d, 1H, H
J ¼ 16.4 Hz); 6.23 (d, 1H, J ¼ 8.8 Hz); 6.29 (d, 1H,
J ¼ 16.0 Hz); 6.91 (dd, 1H, H11, J ¼ 12 Hz, J ¼ 11.2 Hz); 7.03 (s, 1H,
); 7.25 (s, 1H, C ); 7.52 (d, 1H, C
, J ¼ 7.6 Hz); 7.70 (d, 1H,
12, J ¼ 15.2 Hz). C NMR (400 MHz, d -DMSO) 13.0, 19.0, 20.9,
1.8, 29.1, 32.9, 34.2, 39.2, 39.4, 39.5, 39.6, 39.8, 40.0, 40.2, 40.4,
5.7, 65.3, 68.5, 68.9, 69.5, 82.2, 11.6, 116.2, 120.5, 126.8, 128.6, 129.1,
29.3, 130.0, 130.5, 133.2, 135.6, 137.3, 137.5, 140.5, 152.1, 156.5,
6
-
References
d
3
2
[
[
1] J. Paik, S. Vogel, R. Piantedosi, A. Sykes, W.S. Blaner, K. Swisshelm, Biochem-
istry 39 (2000) 8073–8084.
(
2
OCH
5
3
3
5
3
9
4
3
2] A. Wada, C. Tode, S. Hiraishi, Y. Tanaka, T. Ohfusa, Synthesis
1107–1110.
9 (1995)
3
H
5 4
5 4
H
[
[
[
3] A.B. Barua, J.A. Olson, J. Lipid Res. 26 (1985) 258–262.
4] A.B. Barua, C.A. Huselton, J.A. Olson, Synth. Commun. 26 (1996) 1355–1361.
5] A. Nudelman, A. Rephaeli, J. Med. Chem. 43 (2000) 2962–2966.
2
8
,
H
10
,
7
H ,
[6] R.M. Niles, Nutrition 16 (2000) 1084–1090.
[
7] C.D. Margaret, J. Curley, W. Robert, L. Kevin, L.A. Tephly, V. Sikri, C-linked
analogs of N-(4-hydroxyphenyl) retinamide, US/6117845, September 12, 2000.
8] Y.E. Shealy, J.L. Frye, J. Pharm. Sci. 73 (1984) 745–751.
C
H
2
5
1
1
(
H
6 3
6
H
3
6 3
H
13
6
d
[
[9] D. Dive, C. Biot, ChemMedChem 3 (2008) 383–391.
[
[
10] U. Schatzschneider, N. Metzler-Nolte, Angew. Chem., Int. Ed. Engl. 45 (2006)
1504–1507.
11] S. Top, A. Vessieres, G. Leclercq, J. Quivy, J. Tang, J. Vaissermann, M. Huche,
G. Jaouen, Chem. Eur. J. 9 (2003) 5223–5236.
ꢁ
1
65.7. IR (KBr) 2930,1739, 1240, 1145, 970, 500 cm . MS: m/z ¼ 594
þ
[
[
[
12] D.R. van Staveren, N. Metzler-Nolte, Chem. Rev. 104 (2004) 5931–5986.
13] T. Moriuchi, T. Hirao, Chem. Soc. Rev. 33 (2004) 294–301.
14] C. Biot, N. Francois, L. Maciejewski, J. Brocard, D. Poulain, Bioorg. Med. Chem.
Lett. 10 (2000) 839–841.
[M þ H] )
5
.5. In vitro cytotoxicity assay
[
[
15] R. Epton, G. Marr, G.K. Rogers, J. Organomet. Chem. 150 (1978) 93–100.
16] C. Biot, L. Delhaes, L.A. Maciejewski, M. Mortuaire, D. Camus, D. Dive,
J.S. Brocard, Eur. J. Med. Chem. 35 (2000) 707–714.
The antiproliferative activities of 5a–5h were assessed by use of
the MTT assay. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazol-ium bromide) assay is a simple nonradioactive colori-
metric assay to measure cell cytotoxicity, proliferation, or viability.
MTT is a yellow, water-soluble, tetrazolium salt. Metabolically
active cells are able to convert this dye into a water-insoluble dark
blue formazan by reductive cleavage of the tetrazolium ring. For-
mazan crystals then can be dissolved and quantified by measuring
the absorbance of the solution at 570 nm, and the resultant value is
related to the number of living cells.
[
[
[
[
[
17] L. Delhaes, C. Biot, L. Berry, L.A. Maciejewski, D. Camus, J.S. Brocard, D. Dive,
Bioorg. Med. Chem. 8 (2000) 2739–2745.
18] S. Top, A. Vessieres, C. Cabestaing, I. Laios, G. Leclercq, C. Provot, G. Jaouen, J.
Organomet. Chem. 637–639 (2001) 500–506.
19] J.-N. Xiang, L.-H. Jiang, C.-Y. Chen, Z. Fu, J.-F. Duan, X.-X. He, J. Carbohydr. Chem.
25 (2006) 595–614.
20] W.L. Davis, R.F. Shago, H.G. Langner, J.C. Swarts, Polyhedron 24 (2005)
1611–1616.
21] K.M. Roth, A.A. Yasseri, Z. Liu, R.B. Dabke, V. Malinovskii, K.H. Schweikart, L. Yu,
H. Tiznado, F. Zaera, J.S. Lindsey, W.G. Kuhr, D.F. Bocian, J. Am. Chem. Soc. 125
(2003) 505–517.
[
[
22] V. Weinmayr, J. Am. Chem. Soc. 77 (1955) 3012–3014.
23] C. Lmrie, C. Loubser, P. Engelbrecht, J. Chem. Soc., Perkin Trans. 1 (1999)
2513–2523.
The effect of 5a on the cells’ proliferation efficiency was deter-
mined after 24 h incubation with cells. To determine cell prolifer-
ation, the A549 cell lines, BEL7404 cell lines, and Tca cell lines were
[24] E. Poetsch, Kontakte, Darmstadt 2 (1988) 15–18.
[25] M.R. Buchmeiser, N. Schuler, G. Kaltenhauser, K.H. Ongania, I. Lagoja, K. Wurst,
H. Schottenberger, Macromolecules 31 (1998) 3175–3183.
4
individually plated at a density of 1 ꢂ10 cells/well in 96 well plates
ꢀ
at 37 C in 5% CO
2
atmosphere. After 24 h of culture, the medium in
[
26] C.A. Maryanoff, H.P. New, Process for the preparation of N-(4-hydroxyphenyl)-
the wells was replaced with the fresh medium containing 5a of
varying concentrations respectively. The 5a concentration given
upon is the final concentration in the well. Every concentration
retinamide, US/5399757, March 21, 1995.
[
[
[
27] O. Mitsunobu, M. Yamada, Bull. Chem. Soc. Jpn. 40 (1967) 2380–2382.
28] H. Loibner, E. Zbiral, Helv. Chim. Acta 59 (1976) 2100–2113.
29] S.F. Martin, J.A. Dodge, Tetrahedron Lett. 32 (1991) 3017–3020.
added five wells as parallel control. After 24, 10
m
L of MTT dye
[30] B.K. Wilk, Synth. Commun. 23 (1993) 2481–2484.
[
[
31] O.W. Mitsunobu, T. Sano, J. Am. Chem. Soc. 94 (1972) 679–680.
32] T. Fukuyama, M. Cheung, C.K. Jow, Y. Hidai, T. Kan, Tetrahedron Lett. 38 (1997)
solution (5 mg/mL in phosphate buffer pH 7.4) was added to each
ꢀ
well and incubated for 4 h at 37 C and 5% CO
2
for exponentially
5831–5834.
growing cells and 10 min for steady-state confluent cells. The
[33] A.P. Marchand, P.R. Dave, J. Org. Chem. 53 (1988) 1212–1218.