Synthesis of the new pseudo-symmetrical tamoxifen derivatives and their anti-tumor activity

Article abstract of DOI:10.1016/j.bmcl.2007.02.037

Three new pseudo-symmetrical tamoxifen derivatives, RID-B (15), C (16), and D (17), were synthesized via the novel three-component coupling reaction, and the structure-activity relationships of the pseudo-symmetrical tamoxifen derivatives were examined. I

Full text of DOI:10.1016/j.bmcl.2007.02.037

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2421–2424
Synthesis of the new pseudo-symmetrical tamoxifen derivatives
and their anti-tumor activity
Isamu Shiina,a,* Yoshiyuki Sano,a Kenya Nakata,a Takaaki Kikuchi,a
Akane Sasaki,a Masahiko Ikekitab,* and Yoshimune Hasomeb
aDepartment of Applied Chemistry, Faculty of Science, Tokyo University of Science,
Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan
bDepartment of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science,
Yamazaki, Noda, Chiba 278-8510, Japan
Received 2 November 2006; revised 13 February 2007; accepted 15 February 2007
Available online 17 February 2007
Abstract—Three new pseudo-symmetrical tamoxifen derivatives, RID-B (15), C (16), and D (17), were synthesized via the novel
three-component coupling reaction, and the structure–activity relationships of the pseudo-symmetrical tamoxifen derivatives were
examined. It was discovered that 15 strongly inhibits the viability of HL-60 human acute promyelocytic leukemia, whereas 16 pos-
sesses medium activity against the cell line and 17 has no effect on the cell viability. The agarose gel electrophoresis for DNA cleav-
age showed the cell death might be induced by apoptosis.
Ó 2007 Elsevier Ltd. All rights reserved.
Tamoxifen (1, Fig. 1),1 the early generation of SERMs
(selective estrogen receptor modulators), has been used
as the first-line agent for the treatment of estrogen-
dependent breast cancer since the 1970s. Accumulative
risk-benefit assessment of tamoxifen therapy and com-
parative studies of 1 and other new types of drugs also
established its efficacy and safety. Therefore, the devel-
opment of an expeditious synthetic route for producing
new tamoxifen-type drugs followed by the systematic
studies of their biological activities is significantly
required. In this communication, we report a novel
short-step synthesis of a new class of anti-cancer agents,
which are pseudo-symmetrical tamoxifen derivatives, via
the three-component coupling reaction, and their
cytotoxic activity against HL-60 cancer cells.
Friedel–Crafts type alkylation reaction can be effectively
applied to the preparation of 1 and its halogenated
derivatives.3 The coupling reaction of benzaldehyde,
cinnamyltrimethylsilane (4), and anisole promoted by
1 equivalent of HfCl4 and 50 mol% of TMSOTf at
room temperature afforded 3,4,4-triarylbutene 5, which
is the basic skeleton of the tamoxifen derivatives, in a
satisfactory yield (Scheme 1). The coupling reaction
also proceeded using 3-pivaloyloxybenzaldehyde and
b-chlorophenetole as a second nucleophile, which
implemented a new route to droloxifene (2), one of
3-hydroxytamoxifen derivatives, through only three
steps.4
Recently, we have established a novel three-component
coupling reaction among aromatic aldehydes, cinnamyl-
trimethylsilane (4), and aromatic nucleophiles in the
presence of a Lewis acid catalyst.2 Furthermore, we
reported that the sequential one-pot allylation and
Keywords: Tamoxifen; SERMs; Selective estrogen receptor modula-
tors; pseudo-Symmetrical structure; Anti-tumor activity; Apoptosis;
Synthesis.
*
Corresponding authors. Fax: +81 3 3260 5609; e-mail addresses:
shiina@rs.kagu.tus.ac.jp; masalab@rs.noda.tus.ac.jp
Figure 1. Structures of tamoxifen (1) and droloxifene (2).
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.02.037

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I. Shiina et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2421–2424
migration reaction and successive deprotection of the
O-methyl group by BBr3 in CH2Cl2 at 0 °C. Next, 11
was treated with 60% NaH followed by an excess
amount of 2-pyrroridinoethylchloride hydrochloric acid
salt (12), 2-piperidinoethylchloride hydrochloric acid
salt (13), or 2-morphorinoethylchloride hydrochloric
acid salt (14) in DMF to afford the novel tamoxifen
derivatives, RID-B (15),6 RID-C (16),7 or RID-D
(17),8 respectively.
It is well known that E-isomer of tamoxifen is not anti-
estrogenic but functions as an estrogen agonist,
although (Z)-tamoxifen is effective in treating estrogen-
dependent breast cancer. Therefore, the efficiency of
the synthesis of tamoxifen derivatives usually depends
on the stereoselectivity of the olefination step to gener-
ate the desired Z-isomer; however, it is not required to
use stereogenic reactions to produce the pseudo-symmet-
rical system included in 15, 16, and 17.
Scheme 1. Reagents and conditions (for X = H): (a) 4, HfCl4 (1 equiv),
TMSOTf (50 mol%), anisole, rt, 2 h, 57%; (b) t-BuOK, DMSO, rt,
15 min, 96%; (c) BBr3, CH2Cl2, ꢀ78 °C, 2 h, 98%; (d) 8, NaH, DMF,
50 °C, 30 min, 95% (Z/E = 54:46); (e) TfOH, CH2Cl2, 0 °C, 3 h (51% of
1, 46% of 10).
Next, the potency of the anti-tumor activities of 15, 16,
and 17 was assessed in this study. In order to evaluate
the anti-tumor activities of these newly prepared com-
pounds against HL-60 human acute promyelocytic leu-
kemia, we tried to determine the efficiency of the
pseudo-symmetrical compounds decreasing the cell via-
bility by MTT assay, a method of determining cell death
by measuring the mitochondrial succinic dehydrogenase
activity. Furthermore, it was examined whether this cell
death was due to apoptosis or necrosis by agarose gel
electrophoresis for oligonucleosomal DNA cleavage.9
We first investigated the effects on the viability of the
HL-60 cells treated with 15, 16, and 17 at various concen-
trations for 0–6 h (Fig. 2). RID-B (15) in the final concen-
trations of 5, 7.5, and 10 lM decreased the cell viability in
a time-dependent manner. A 6-h incubation with 5, 7.5,
and 10 lM final concentrations of 15 inhibited the cell
viability in more than 90% asmeasured by the MTT assay.
RID-C (16) in 7.5 and 10 lM concentrations also
inhibited the cell viability in more than 80% after a 6-h
treatment. Contrarily, RID-D (17) showed no effect on
the viability of the HL-60 cells after 6 h. Among them,
15 and 16 clearly induced, dose-dependently, cell death
to a greater extent than 17 as shown in Figure 2. Although
15 and 16 have strong cytotoxic activity inducing
apoptosisof the HL-60 cells, 17 includingmorpholine side
chain shows no activity on the same cells; therefore, it is
indicated that oxygen in morpholine side chain varied
cytotoxic characters of 15 and 16 to non-cytotoxic by
lowering its basicity.10 The specific moieties as well as
the hydrophobicity derived from the long alkyl side chain
might be both important for the strong cytotoxic activity
against the HL-60 cells.
Scheme 2. Reagents and conditions: (a) 4, HfCl4 (1 equiv), anisole, rt,
2 h, 78%; (b) t-BuOK, DMSO, 90 °C, 1 h, 93%; (c) BBr3, CH2Cl2, 0 °C,
2 h, 86%; (d) NaH, 12, DMF, 50 °C, 30 min, 84% (for 15); NaH, 13,
DMF, 50 °C, 30 min, 95% (for 16); NaH, 14, DMF, 50 °C, 30 min,
95% (for 17).
Based on the preliminary results for the synthesis of
tamoxifen (1) and droloxifene (2), the coupling reaction
among 4-pivaloyloxybenzaldehyde (9), cinnamyltri-
methylsilane (4), and anisole was carried out for the
preparation of pseudo-symmetrical tamoxifen analogues
(Scheme 2). The three-component coupling reaction
smoothly proceeded in the presence of a stoichiometric
amount of HfCl4 at room temperature to afford the cou-
pling product 10 in high yield. Bisphenol 115 was pre-
pared from 10 by heating with an excess amount of
t-BuOK in DMSO via the base-catalyzed double-bond
The effect of tamoxifen itself on growth of the HL-60
human cell line was measured by the MTT assay at
5 lg/mL after a 4-h incubation. The final growth rate
of the cells was inhibited to be ca. 50% content
compared with the growth rate at 0 h. On the other
hand, the biological activity of 5 lg/mL 15 is very strong
to inhibit the cell viability in more than 90% after 4 h
under the same conditions.

I. Shiina et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2421–2424
2423
Figure 3. Results of determination of the nature of the cell death assay
of pseudo-symmetrical tamoxifens for 6 h [for only medium (lane 1),
tamoxifen (lane 2), RID-B (15) (lane 3), RID-C (16) (lane 4), ethanol
(lane 5), or actinomycin D (lane 6)].
with a 9 lM final concentration of any of the compounds
(Fig. 3). The HL-60 cells were treated with 15 (lane 3), 16
(lane 4), and with only medium, tamoxifen, ethanol, or
actinomycin D as the controls (lanes 1, 2, 5, or 6, respec-
tively). The cell death assay of all molecules was carried
out for 6 h although the time required for the DNA frag-
mentation clearly varied for each tested sample.
DNA fragmentation was observed after treatment of the
HL-60 cells with the pseudo-symmetrical compounds 15
and 16 using the chromatin DNA analysis, and the
DNA ladder patterns of 15 (lane 3) and 16 (lane 4) are
similar to that of tamoxifen (lane 2). Recently, it has
been reported that tamoxifen and some derivatives such
as nafoxidine induce apoptosis in HL-60 leukemic
cells;11 therefore, these results support the conclusion
that 15 and 16 induced cell death by apoptosis.
In summary, we developed the effective synthesis of the
new pseudo-symmetrical tamoxifen derivatives RID-B
(15), C (16), and D (17) from very simple molecules in four
steps. Although 17 (involving morpholine part) does not
have any biological activity in the MTT assay, 15 (involv-
ing pyrrolidine moiety) and 16 (involving piperidine
moiety) showed significant anti-tumor activities. The
latter two compounds 15 and 16 might be potent candi-
dates as anti-tumor agents with different modes of action
from existing drugs for cancer treatment. It was also dis-
covered that the time-dependent activity of 15 is superior
to that of 16 as shown in Figure 2. The synthesis of other
derivatives and further studies of the anti-tumor activities
of these new entries as pseudo-symmetrical tamoxifen
agents are now in progress.
Figure 2. Effects of RID-B (15), C (16), and D (17) on growth of cells.
Time-dependent effects of 15, 16, and 17 on HL-60 cells were measured
by the MTT assay. Data are expressed as growth rate (growth rate has
shown that absorbance of formazan at 0 h was 1.0), and error bars
show SD (n = 5). HL-60 cells were treated at the following four
different final concentrations: 2.5, 5, 7.5, and 10 lM, respectively.
The median growth-inhibitory concentrations (IC50s
)
against the HL-60 cells after a 6-h incubation with 15
and 16 were as follows: 3 and 5 lM final concentrations,
respectively. Furthermore, the IC50 of 15 after a 3-h
treatment was a 4 lM final concentration. We also
determined the IC50s by neutral red assay. Neutral red
solution is taken through the cell membrane by living
cells and accumulated in the lysosomes, whereas MTT
is a substrate for mitochondrial succinic dehydrogenase.
The IC50s determined by the neutral red assay were low-
er than those determined by the MTT assay for almost
all of the tested compounds, however, the two most
effective products were still 15 and 16. On the basis of
these results, we selected these two pseudo-symmetrical
compounds 15 and 16 for further examinations.
Acknowledgments
This study was partially supported by a Research Grant
from the Center for Green Photo-Science and Technol-
ogy, and Grants-in-Aid for Scientific Research from the
Ministry of Education, Science, Sports and Culture,
Japan.
References and notes
To clarify whether the two products (15 and 16) induced
cell death by apoptosis or necrosis, we found evidence ob-
tained by agarose gel electrophoresis for the oligonucleos-
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I. Shiina et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2421–2424
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836, 820 cmꢀ1; 1H NMR (CDCl3): d 7.18–7.07 (m, 7H, Ar),
6.88 (d, J = 8.7 Hz, 2H, Ar), 6.75 (d, J = 8.7 Hz, 2H, Ar),
6.54 (d, J = 8.7 Hz, 2H, Ar), 4.12 and 3.96 (t, J = 6.0 Hz,
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127.8, 125.8, 114.0, 113.3, 66.9, 66.7, 54.7, 54.7, 55.1, 55.1,
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