TX-2152: A conformationally rigid and electron-rich diyne analogue of FTY720 with in vivo antiangiogenic activity

Article abstract of DOI:10.1016/j.bmc.2008.07.003

We designed FTY720 analogues with conformationally rigid and electron-rich acetylenic chains as antiangiogenic agents (the monoyne 1: TX-2148, the diyne 2: TX-2152, the triyne 3: TX-2256). Molecular orbital (MO) calculations of our designed acetylenic ana

Full text of DOI:10.1016/j.bmc.2008.07.003

Bioorganic & Medicinal Chemistry 16 (2008) 7705–7714
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
TX-2152: A conformationally rigid and electron-rich diyne analogue
of FTY720 with in vivo antiangiogenic activity
Shinichi Nakayama a, Yoshihiro Uto a, Kanako Tanimoto a, Yasuhiro Okuno a, Yuki Sasaki a,
Hideko Nagasawa b, Eiji Nakata a, Ken Arai a, Kaori Momose a, Tetsuro Fujita c, Toshihiro Hashimoto d,
Yasuko Okamoto d, Yoshinori Asakawa d, Satoru Goto e, Hitoshi Hori a,
*
a Department of Life System, Institute of Technology and Science, Graduate School, The University of Tokushima, Minamijosanjimacho 2, Tokushima 770-8506, Japan
b Laboratory of Pharmaceutical Chemistry, Gifu Pharmaceutical University, Mitahorahigashi-5, Gifu 502-8585, Japan
c Research Institute for Production Development, 15 Shimogamo Morimoto-cho, Sakyo-ku, Kyoto 606-0805, Japan
d Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan
e Department of Pharmaceutical Sciences, International University of Health and Welfare, 2600-1 Kitakanemaru, Ohtawara, Tochigi 324-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We designed FTY720 analogues with conformationally rigid and electron-rich acetylenic chains as anti-
angiogenic agents (the monoyne 1: TX-2148, the diyne 2: TX-2152, the triyne 3: TX-2256). Molecular
orbital (MO) calculations of our designed acetylenic analogues and FTY720 showed that the localization
of the lowest unoccupied MO and the highest occupied MO increased from phenyl ring to acetylenic
chain compared with that of FTY720. These acetylenic analogues were synthesized from p-hydroxyphe-
nylethanol as a starting material. The construction of the acetylenic chain was carried out by an iterative
strategy using a Sonogashira cross-coupling reaction and desilylative bromination in two steps. The cor-
responding overall yields of the monoyne 1, the diyne 2, and the triyne 3 were 27% (11 steps), 13% (13
steps), and 10% (15 steps). The in vivo antiangiogenic activities of these acetylenic analogues and
FTY720 were evaluated by the chick embryo chorioallantoic membrane (CAM) assay and compared to
the activities of the known antiangiogenic agent TNP-470. The diyne 2 showed more potent antiangio-
genic activity (90% inhibition) than FTY720 (77% inhibition) and other acetylenic analogues (the monoyne
Received 18 June 2008
Revised 1 July 2008
Accepted 2 July 2008
Available online 8 July 2008
Keywords:
TX-2152
FTY720
Acetylenic analogue
Antiangiogenic agent
Chick embryo CAM assay
1: 42% inhibition, the triyne 3: 60% inhibition), and TNP-470 (82% inhibition) at a dose of 10
without showing toxicity. The diyne 2 also had potent inhibitory activity at a dose of 5 and 2.5
l
g/CAM,
g/
l
CAM. These results indicate that the flexibility of C8 alkyl chain of FTY720 is not required for its antian-
giogenic activity. We suggest that the diyne 2 (TX-2152) may be a promising candidate as an antiangio-
genic agent for antineoplastic drug discovery.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
FTY720 (Fig. 1),1–3 an immunosuppressant, is currently in clin-
ical phase III trials for the treatment of relapsing-remitting multi-
ple sclerosis in oral administration.4 FTY720 exerts its
immunosuppressive activity as the phosphate ester formed by
phosphorylation in vivo.3,5 The phosphate interacts with sphingo-
sine-1-phosphate (S1P) receptors.6,7 Recently, it was reported that
FTY720 was able to abrogate the stimulating effect of vascular
endothelial growth factor (VEGF: 20 ng/mL) even at subnanomolar
concentrations in a human umbilical vein endothelial cell spheroid
model.8 It was also reported that FTY720 potently inhibited VEGF
and S1P induced angiogenesis in vivo in growth factor implant
Figure 1. The structure of FTY720.
and corneal models in mice at well-tolerated and clinically rele-
vant doses.9
Angiogenesis, the formation of a new capillary network from
pre-existing blood vessels, plays a crucial role in the growth of a
neoplasm.10,11 Since the neoplasm actively induces angiogenesis
for its own growth,12 an antiangiogenic agent is considered to be
an antineoplastic agent. In the past, many antiangiogenic agents
have been developed and some of these are currently undergoing
* Corresponding author. Tel.: +81 88 656 7514; fax: +81 88 656 9164.
E-mail address: hori@bio.tokushima-u.ac.jp (H. Hori).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.07.003

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S. Nakayama et al. / Bioorg. Med. Chem. 16 (2008) 7705–7714
clinical trials.10,13 Bevacizumab, which is a monoclonal antibody
against VEGF, was the first antiangiogenic agent approved for colon
cancer by the FDA.14,15 About 60% of human neoplasms express
VEGF but many neoplasms produce other angiogenic proteins as
well.16 Furthermore, some neoplasms normally produce only
VEGF, but mutation allows them to produce new angiogenic pro-
teins. If more patients respond well to bevacizumab therapy and
bevacizumab receives FDA approval for other tumors, we think
that co-administration of other antiangiogenic inhibitors should
be explored.13,17 There is a continuing need for the development
of new antiangiogenic agents.
Previously, we have developed a series of antiangiogenic agents
and have evaluated their in vitro and in vivo antiangiogenic activ-
ities.18–25 We have used the chick embryo chorioallantoic mem-
brane (CAM) assay which is widely employed in this area for
evaluation of in vivo antiangiogenic activity.26 It is well known that
the angiogenic endothelium of the CAM expresses VEGF and its
recepter-2.27–29 From this and our previous studies, we were con-
fident that FTY720 would also show antiangiogenic activity in the
CAM assay. For these reasons, we decided to design FTY720 ana-
logues as antiangiogenic agents.
From previous structure–activity relationship (SAR) studies of
FTY720 analogues, the 2-amino-1,3-propane diol moiety is known
to be critical for its immunosuppressive activity.2 We assume that
this moiety has also important role for its antiangiogenic activity.
To develop FTY720 analogues, we planned to perform conforma-
tional rigidification of the flexible C8 alkyl chain of FTY720. As
shown in Figure 2A, the flexible sp3 carbon chain of FTY720 fills a
large space. The structural flexibility generates a vast number of
conformations.30,31 Conformational flexibility can be a complicating
factor in drug development, and conformational rigidification can
be a very important strategy in drug design. To achieve effective
rigidification, we selected acetylenic structures, a structural motif
widely distributed in nature.32,33 Acetylenic compounds including
polyynes have unique structural properties that include rigidity
and high electron-densities,34 and show diverse biological activities
such as antiangiogenesis,35 antitumor,36 antiinflammatory,37 anti-
biotic,38 and anti-HIV activities.39 As can be seen in Figure 2B, such
acetylenic units contribute to substantial rigidification of conforma-
tions. There are no examples of the use of polyynes for drug rigidifi-
cation beyond the use of diynes in medicinal chemistry.
acetylenic chains. We also show the in vivo antiangiogenic activi-
ties of our designed acetylenic FTY720 analogues evaluated by
the CAM assay.
2. Results
2.1. Design
As shown in Figure 3, we designed acetylenic FTY720 analogues
such as the monoyne 1 (TX-2148), the diyne 2 (TX-2152), and the
triyne 3 (TX-2256) wherein the flexible C8 alkyl chain of FTY720
has been replaced with a conformationally rigid and electron-rich
acetylenic chain. We expected that this molecular design could
change the molecular properties and electronic states of the mole-
cules in a manner that would improve the pharmacodynamic prop-
erties. We felt that such changes could also affect and possibly
increase antiangiogenic activity.
We carried out molecular orbital (MO) calculations to aid in the
design of the acetylenic FTY720 analogues. In particular, the MO
calculations enabled us to examine the extent to which the acety-
lenic units influenced the overall electronic states of the molecules.
The MO calculations were done using both MOPAC 2000 with PM3
method40 and Gaussian 03 with B3LYP/6-31G(d).41 As shown in
Figure 4, the lowest unoccupied MO (LUMO) and the highest occu-
pied MO (HOMO) of FTY720 were localized at the phenyl moiety. In
contrast, localizations of LUMO and HOMO of the acetylenic
FTY720 analogues extended from the phenyl moiety to the acety-
lenic chain. The energy levels of the LUMOs (ELUMO) of the acety-
lenic FTY720 analogues were at lower energy than those of
FTY720, while the energy levels of their HOMOs (EHOMO) were
higher than those of FTY720. The calculated logP (clogP) values
of these acetylenic FTY720 analogues were obtained using OSIRIS
Property Explorer.42 As shown in Table 1, the corresponding clogP
values of the monoyne 1 (TX-2148), the diyne 2 (TX-2152), and the
triyne 3 (TX-2256) were 0.58, 1.06, and 1.54.
2.2. Synthesis
The synthesis of a key intermediate 11 began with commer-
cially available p-hydroxyphenylethanol as shown in Scheme 1.
Selective protection of the primary alcohol gave the phenol 4.43
Silylation of the phenol 4 followed by selective deprotection of
the acetyl group produced the alcohol 5. Iodination of the alcohol
5 gave the ethyl iodide 6, which was condensed with diethyl ace-
tamidomalonate to give the diester 7. The reduction of the diester
7 with LiAlH4 followed by acetylation gave the triacetate 8. Desily-
In this paper, we describe the design and synthesis of FTY720
analogues that have conformationally rigid and electron-rich
Figure 3. The structures of our designed acetylenic FTY720 analogues, the
monoyne 1 (TX-2148), the diyne 2 (TX-2152), and the triyne 3 (TX-2256).
Figure 2. Filled space images of (A) sp3 carbon chain and (B) sp carbon chain.

S. Nakayama et al. / Bioorg. Med. Chem. 16 (2008) 7705–7714
7707
Figure 4. Molecular orbitals and their energy levels of FTY720 and acetylenic FTY720 analogues. aMOPAC 2000 with PM3. bGaussian 03 with B3LYP/6-31G(d).
lation of the triacetate 8 afforded the phenol 9. The phenol 9 was
converted to the triflate 10, which was subjected to a modified pal-
ladium-catalyzed Sonogashira cross-coupling44 with trimethylsi-
lylacetylene to provide the TMS-monoyne 11.
stituent hydrophobic constant (
logP and , we calculated the clogP values of 11, 13, and 15. As
shown in Table 1, the corresponding clogP values of 11, 13, and
15 were 4.32, 4.80, and 5.27. The estimated of the ethynyl group
p) per acetylenic bond. To estimate
p
p
The homologation of the acetylenic chain was carried out using
the two-step acetylene homologation sequence as shown in
Scheme 2.45 An in situ desilylation–bromination reaction of the
TMS-monoyne 11 gave the bromo-monoyne 12.46 The bromo-
monoyne 12 was treated with trimethylsilylacetylene under Sono-
gashira cross-coupling conditions to give the TMS-diyne 13. The
TMS-triyne 15 was similarly obtained from the TMS-diyne 13. As
shown in Scheme 3, the TMS-triyne 15 was also synthesized via
one-step reaction from the bromo-monoyne 12 using bis-
trimethylsilylbutadiyne but the yield of this transformation was
relatively low.47 All the acetylenic FTY720 analogues, including
the monoyne 1 (TX-2148), the diyne 2 (TX-2152), and the triyne
3 (TX-2256) were obtained by hydrolysis of the corresponding tri-
methylsilyl alkynes. The corresponding overall yields of the mono-
yne 1 (TX-2148), the diyne 2 (TX-2152), and the triyne 3 (TX-2256)
were 27% (11 steps), 13% (13 steps), and 10% (15 steps) from
p-hydroxyphenylethanol.
obtained from these clogP values of the acetylenic FTY720 ana-
logues was approximately 0.48. The corresponding logPow values
of 11, 13, and 15 were 4.07, 4.97, and 5.90. The
p of ethynyl group
obtained from these logPow values was approximately 0.92.
2.4. In vivo antiangiogenic activity
In vivo antiangiogenic activity of FTY720 and the acetylenic
FTY720 analogues were evaluated by the CAM assay. The known
antiangiogenic inhibitor TNP-47048,49 was also evaluated as a posi-
tive control. In this assay, we determined that the more potent
antiangiogenic agent showed its inhibitory activity of more than
60% with a dose lower than 100 lg/CAM. The antiangiogenic activ-
ities of FTY720, the acetylenic FTY720 analogues, and TNP-470 are
listed in Table 2. FTY720 showed 100% inhibition at a dose of
100
10
poor activity at all doses of 10, 5, and 1
2 (TX-2152) had potent inhibitory activities from 10 to 2.5
CAM. The triyne 3 (TX-2256) also showed potent inhibitory activity
at 2.5 g/CAM (67% inhibition) but its activity did not increase at
its higher dose. At 10 g/CAM, the diyne 2 (TX-2152) showed more
potent inhibitory activity than FTY720 and TNP-470. But the
l
l
g/CAM. FTY720 also showed potent inhibitory activity at
g/CAM (77% inhibition). The monoyne 1 (TX-2148) showed
g/CAM tested. The diyne
g/
l
2.3. Determination of calculated and observed hydrophobicity
of ethynyl group
l
l
We measured the observed logP (logPow) values of 11, 13, and
15 by an HPLC method to determine the gain in an observed sub-
l

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S. Nakayama et al. / Bioorg. Med. Chem. 16 (2008) 7705–7714
Table 1
monoyne 1 (TX-2148) and the triyne 3 (TX-2256) were less potent
than FTY720 and TNP-470. As shown in Figure 5, the diyne 2 (TX-
2152) clearly inhibited antiangiogenesis compared with control. As
noted above, the diyne 2 (TX-2152) was the most potent antiangio-
genic agent among all the acetylenic FTY720 analogues tested.
These compounds were not observed to be toxic to developing
chick embryo in the CAM assay.
The clogP and the logPow values of acetylenic FTY720 analogues
Compound clogP
logPow
0.58
1.06
1.54
4.32
4.80
5.27
—
3. Discussion
In this study, we have demonstrated that the acetylenic FTY720
analogues have potent antiangiogenic activity comparable to TNP-
470 in vivo in the CAM assay. We also showed that the C8 alkyl
chain of FTY720 could be replaced by the diyne or the triyne with
respect to antiangiogenic activity, and introducing conformational
rigidity did not diminish antiangiogenic activity. Since S1P did not
show any antiangiogenic activities in the CAM assay from our pre-
vious study (data not shown), FTY720 and the acetylenic FTY720
analogues are thought to exert these antiangiogenic activities
without involving S1P receptors.
In the past, various acetylenic coupling reactions have been
developed and applied50 to natural product synthesis.32,34 To install
the acetylenic chains in our new FTY720 analogues, we used an iter-
ative homologation strategy based on the Sonogashira cross-cou-
pling reaction together with desilylative bromination.45,51 An
advantage of this strategy is the ability to add both even- and
odd-numbers of acetylenic units by controlling the number of iter-
ations of the chain growth cycles. This allows the establishment of a
SAR study of the effect of the length of the acetylenic chain.
Parish et al. isolated the ene-triyne, an amide of the C-9 ene-tri-
yne carboxylic acid with p-aminobenzoic acid, from the Basidomyc-
ete fungus Baeospora myosura.38 The ene-triyne showed extremely
potent antibiotic activity against the Gram-positive bacteria Staph-
ylococcus aureus, while the reduced analogue of the ene-triyne and
4-acetimidobenzoic acid were not active. It is not clear that the al-
tered activity results from loss of conformational rigidity, the elec-
tronic character of acetylenic structure, and/or other factors.
Despite this, introduction of the acetylenic structure greatly influ-
—
—
4.07
4.97
5.90
Scheme 1. Reagents and conditions: (a) NaHSO4ꢀSiO2/n-hexane/EtOAc, reflux, 24 h; (b) TBSCl/imidazole/CH2Cl2, rt, 15 min; (c) LiAlH4/THF, rt, 15 min; (d) imidazole/PPh3/I2/
CH2Cl2, rt, 1.5 h; (e) AcNH(COOEt)2/NaH/DMF, rt, 4 h; (f) LiAlH4/THF, rt, 6 h; (g) Ac2O/Et3N/DMAP/THF, rt, 15 min; (h) (n-Bu)4NF/THF, rt, 20 min; (i) Tf2O/pyridine/CH2Cl2,
0 °C–rt, 15 min; (j) TMSCCH/Et3N/PdCl2(PPh3)2/CH3CN, reflux, 1.5 h.

S. Nakayama et al. / Bioorg. Med. Chem. 16 (2008) 7705–7714
7709
Scheme 2. Reagents and conditions: (a) NBS/AgNO3/acetone, rt, 2 h; (b) TMSCCH/Et3N/PdCl2(PPh3)2/CuI/THF, rt, 2 h; (c) LiOH/MeOH, rt, 40 min–23 h.
enced the biological activity. This and our present results have
shown the utility of our approach. A number of acetylenic moieties
have been used as architectures of several compounds.34 However,
there were no examples of drug design in which acetylenic units
were extended beyond diyne structures as pharmacophoric
descriptors. Our present study may become a new application strat-
egy for primary SAR exploration for drug development.52
our initial screening of antiangiogenic agents using the CAM assay,
this will reduce the risk of failure at a later phase of our research.
Yamaguchi et al. carried out an SAR study of caryonencin ana-
logues including compounds possessing acetylenic moieties.53
Their study showed that the tetrayne analogue exhibited potent
antibacterial and antifungal activities in vitro, while the triyne
and diyne analogues were less active. These results indicate that
SAR studies of acetylenic compounds related to the number of
acetylenic units present can reveal important information on the
biological activity of acetylenic compounds that should be useful
in drug development. The results of the CAM assay of our acety-
lenic FTY720 analogues also showed that the length of the acety-
lenic structure (monoyne, diyne, triyne) influenced the in vivo
antiangiogenic activity. Although many unsaturated fatty acids
found in nature contain one or more acetylenic structures,36,54–57
the pharmacological, especially pharmacokinetic and pharmacody-
namic, properties related to this functionality are still largely un-
known. The SAR study of these acetylenic compounds should add
to the knowledge of the effects and potential of acetylenic groups
in drug development.
Good in vitro activity for a target is one important criterion upon
which medicinal chemists base drug development. This criterion
alone can be misleading. Our previous study indicated that potent
antiangiogenic activities shown in vitro did not always correlate
with similar tendencies in vivo.18–25 However, compounds which
had potent activity in the CAM assay advanced to the next stage of
development with no difficulty. We think that it is mainly because
of support from surrounding tissues in the in vivo enviroment.10,12
In general, it is well documented that many promising candidate
compounds with good in vitro activity have been dropped based
on poor in vivo results. Given this perspective, we suggest the use
of the in vivo CAM assay as an early or even initial screening proce-
dure for antiangiogenic agents. In vitro assays evaluate individual
antiangiogenic properties such as effects on cell proliferation,
migration, tube formation, as well as toxicity.26 Their environments
differ greatly from that of in vivo. Furthermore, these tests are time
consuming, expensive, and technically difficult. In vivo assays using
animals such as mice and rats are also expensive, time consuming,
and require special facilities. On the other hand, the CAM assay
can easily evaluate the total antiangiogenic activity with a small
amount of drug at a very low cost in an incubator and is suitable
for large-scale screening.26 In addition, this assay will concurrently
provide information on toxicity based on the life or death of CAM.
For these reasons, we have taken the position that if we carry out
The hydrophobic constant
substituent has been shown to be 0.4, but the
p
of the ethynyl group as an aromatic
of the extended
p
acetylenic substituents greater than the diyne has not been deter-
mined.58 The differences between the clogP values of 11, 13, and
15 and the logPow values show the importance of the experimental
measurement of logPow values. There are few reported values of
logPow of acetylenic compounds in spite of the importance of their
substituent hydrophobic constants in their quantitative SAR. Our
determination of the substituent hydrophobic constants for these
acetylenic compounds based on the experimental measurement
of logPow will be very useful for further rational drug design.

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S. Nakayama et al. / Bioorg. Med. Chem. 16 (2008) 7705–7714
Table 2
Electron transfers in molecules are important contributing fac-
tors to their chemical properties and reactivity. Kovacic and Pozos
coined the term ‘bioelectronome’ to characterize the multiple reac-
tions undergone by electrons in biological systems, including elec-
tron transfer and redox processes.59 Because of the abundance and
In vivo antiangiogenic activities of FTY720, acetylenic FTY720 analogues, and TNP-
470 in the CAM assay
Compound
Dosea
(l
g/CAM)
Inhibition (%)
reactivity of
p electrons in acetylenic structures that would be
100
10
1
100
77
40
available for diverse biological effects, we considered this moiety
to be a bioelectronome. This was a factor in our decision to intro-
duce conformationally rigid and electron-rich acetylenic chains
into known drugs or biomolecules as a means to change their elec-
tronic structures in order to create new bioelectronomes or new
‘electronic drugs’.
Because pharmacodynamic properties are greatly influenced
by electrostatic and steric alterations, the alteration of electronic
state and the rigidification of conformation in our new analogues
should have an effect on biological or pharmacological activities.
Our molecular design strategy, the structural transfer from sp3
hybrid to sp hybrid, should be applicable to other known drugs.
Although this strategy represents simple modifications, it should
lead to substantial changes in biological or pharmacological
activities.
10
5
1
42
17
17
10
5
2.5
1
90
79
71
35
In conclusion, we have presented the design and synthesis of
conformationally rigid and electron-rich acetylenic analogues of
FTY720, based on our new molecular drug design strategy. The
length of acetylenic chain affected in vivo antiangiogenic activities
in the CAM assay. The diyne 2 (TX-2152) showed more potent
antiangiogenic activity than FTY720, the monoyne 1 (TX-2148),
the triyne 3 (TX-2256), and TNP-470, without showing toxicity.
We suggest that diyne 2 (TX-2152), as an acetylenic FTY720
analogue, shows promise to be an antiangiogenic agent candidate
for antineoplastic drug discovery.
10
5
2.5
1
60
67
67
40
10
5
1
82c
78c
50
4. Experimental
4.1. General procedures
Controlb
0
0
1H NMR spectra were recorded on a JEOL JNM-EX400 spectrom-
eter (400 MHz) with tetramethylsilane as the internal standard.
Chemical shifts are reported in ppm. Coupling constants are re-
ported in Hz. Mass spectra were measured on a Shimazu GC-MS
QP-1000 mass spectrometer using an electron ionization (EI) meth-
od. HRMS were measured on a JOEL JMS-700 mass spectrometer
using a CI method. IR spectra were measured on a Perkin-Elmer
spectrum RX 1. Elemental analyses were performed with a Yanako
CHN recorder MT-5. Reactions were monitored by analytical TLC
with use of Merck silica gel 60 F254 glass plates. Column chromatog-
raphy was performed on Kanto Chemical silica gel 60 N (230–
a
The test compounds were dissolved in DMSO, and then mixed in 1% methyl
cellulose/0.9% NaCl (0.05% DMSO).
b
In a control experiment, 0.05% DMSO mixed in 1% methyl cellulose/0.9% NaCl
without the test compound was used.
c
The inhibition values were from Ref. 23.
Scheme 3. Reagents and conditions: (a) MeLiꢀLiBr, THF, 0 °C, 1 h, then CuBr, 0 °C–rt,
30 min; (b) 12, pyridine, rt, 4 h.
400 mesh). HPLC was performed on C18 column (InertsilÒ, 5
lm,
4.6 mm I.D. ꢁ 250 mm, GL Sciences Inc.) with a JASCO PU-1586
pump equipped with a UV–vis detector JASCO UV-1570. The clogP
values were determined by OSIRIS Property Exploler42 which avail-
able free on the Web.60,61 All chemicals were purchased from Wako
Pure Chemical Industries, Ltd (Osaka, Japan), Kanto Chemical Co.,
Inc. (Tokyo, Japan), Tokyo Chemical Industry Co., Ltd (Tokyo, Japan),
or Sigma–Aldrich Japan (Tokyo, Japan). The fertilized chicken eggs
were purchased from Goto hatchery, Inc. (Gifu, Japan).
4.2. Molecular modeling methods
The semi-empirical MO calculation was performed with the
PM3 methods of Stewart using MOPAC 2000 in a WinMOPAC ver.
3.0 package.40 The ab initio MO calculation was performed with
the B3LYP hybrid density functional in conjugation with the 6-
31G(d) basis set using the GAUSSIAN 03 suite of programs.41 The
visualization of the molecular geometries calculated by GAUSSIAN
03 were carried out with MOLKEL 4.3.62
Figure 5. The pictures of CAM treated with the diyne 2 (TX-2152) or a control.

S. Nakayama et al. / Bioorg. Med. Chem. 16 (2008) 7705–7714
7711
4.3. Synthesis
4.3.5. Diethyl 2-(acetylamino)-2-(4-{[1-(tert-butyl)-1,1-
dimethylsilyl]oxy}phenethyl)malonate (7)
4.3.1. Preparation of the NaHSO4ꢀSiO2 Catalyst43
To a suspension of NaH (60% dispersion in mineral oil, 1.4 g,
33 mmol) in DMF (35 mL) was added a solution of diethyl ace-
tamidomalonate (7.2 g, 33 mmol) in DMF (24 mL). After 1 h,
the ethyl iodide 6 (3.0 g, 8.3 mmol) in DMF (4 mL) was added.
The suspension was stirred for 4 h, poured into water at 0 °C
and extracted with Et2O. The organic layer was washed with
brine, dried with MgSO4, and evaporated. The residue was puri-
fied by silica gel column chromatography (n-hexane/EtOAc = 4:1)
to give the diester 7 (3.3 g, 89%) as a colorless oil. 1H NMR
(CDCl3): d 6.99 (d, 2H, J = 8.5 Hz), 6.77 (s, 1H), 6.73 (d, 2H,
J = 8.5 Hz), 4.21 (m, 4H), 2.65 (m, 2H), 2.41 (m, 2H), 2.00 (s,
3H), 1.25 (t, 6H, J = 7.1 Hz), 0.97 (s, 9H), 0.17 (s, 6H); FT-IR
(KBr): 3372, 2960, 2933, 2896, 2863, 1758, 1741, 1666, 1612,
1513, 1474, 1463, 1454, 1391, 1371, 1308, 1260, 1194, 1171,
1097, 1082, 1031, 1016, 923, 841, 811, 781, 692, 590 cmꢂ1; EI-
MS m/z: 451 (M+).
To a solution of NaHSO4ꢀSiO2 (12 g, 89 mmol) in water (60 mL)
was added SiO2 (30 g). The mixture was stirred for 15 min and then
gently heated on a hot plate, with intermittent swirling, until a
free-flowing white solid was obtained. The catalyst was further
dried at 120 °C for at least 48 h prior to use.
4.3.2. 4-Hydroxyphenethyl acetate (4)
p-Hydroxyphenylethanol (40.4 g, 0.292 mol) was dissolved in a
33% solution by volume of EtOAc in n-hexane (900 mL). To this
solution was added NaHSO4ꢀSiO2 (35 g). The mixture was heated
at reflux for 24 h, then cooled to room temperature and filtered
through Celite. The filtrate was evaporated, and the residue was
purified by silica gel column chromatography (n-hexane/
EtOAc = 9:1) to give the phenol 4 (50.7 g, 96%) as white solid. 1H
NMR (CDCl3): d 7.05 (d, 2H, J = 8.3 Hz), 6.78 (d, 2H, J = 8.3 Hz),
4.24 (t, 2H, J = 7.1 Hz), 2.85 (t, 2H, J = 7.1 Hz), 2.04 (s, 3H); FT-IR
(KBr): 3382, 3043, 2972, 2951, 2890, 2360, 1713, 1615, 1516,
1445, 1268, 1173, 830, 650 cmꢂ1; EI-MS m/z: 180 (M+).
4.3.6. 2-(Acetylamino)-2-[(acetyloxy)methyl]-4-(4-{[1-(tert-
butyl)-1,1-dimethylsilyl]oxy}phenyl)butyl acetate (8)
To a solution of the diester 7 (5.03 g, 11.1 mmol) in THF
(60 mL) cooled to 0 °C was added dropwise a solution of 1.0 M
LiAlH4 in Et2O (32 mL, 32 mmol). The mixture was warmed to
room temperature. After 6 h, the reaction was quenched with
saturated aqueous Na2SO3. The mixture was filtered through Cel-
ite and evaporated. The residue was purified by silica gel column
chromatography (EtOAc) to give 2-amino-2-(4-{[1-(tert-butyl)-
1,1-dimethylsilyl]oxy}phenethyl)-1,3-propanediol (2.82 g, 69%)
as a white solid. 1H NMR (CDCl3): d 7.04 (d, 2H, J = 8.5 Hz),
6.76 (d, 2H, J = 8.5 Hz), 5.87 (s, 1H), 3.86 (d, 2 H, J = 10.5 Hz),
3.84 (s, 2H), 3.62 (d, 2H, J = 10.5 Hz), 2.59 (m, 2H), 1.96 (s, 3H),
1.93 (m, 2H), 0.97 (s, 9H), 0.17 (s, 6H); FT-IR (KBr): 3499,
3311, 3095, 3034, 2955, 2931, 2887, 2881, 1630, 1554, 1510,
1473, 1461, 1434, 1417, 1378, 1322, 1303, 1263, 1200, 1174,
1106, 1062, 997, 978, 912, 840, 782, 646, 616, 588 cmꢂ1; EI-MS
m/z: 367 (M+).
4.3.3. 2-(4-{[1-(tert-Butyl)-1,1-dimethylsilyl]oxy}phenyl)-1-
ethanol (5)
To a solution of the phenol 4 (39.5 g, 0.220 mol) dissolved in
CH2Cl2 (600 mL) and cooled to 0 °C were added tert-butyldimethyl-
silylchloride (40 g, 0.26 mol) and imidazole (22 g, 0.33 mol). After
15 min, H2O was added, and the mixture was extracted with
CH2Cl2. The organic layer was washed with brine, dried with anhy-
drous MgSO4, and evaporated. The residue was purified by silica
gel column chromatography (n-hexane/EtOAc = 9:1) to give 4-
{[1-(tert-butyl)-1,1-dimethylsilyl]oxy}phenethyl acetate (65.8 g,
quant.) as
a d 7.06 (d, 2H,
white solid. 1H NMR (CDCl3):
J = 8.5 Hz), 6.77 (d, 2 H, J = 8.5 Hz), 4.23 (t, 2H, J = 7.3 Hz), 2.86 (t,
2H, J = 7.3 Hz), 2.03 (s, 3H), 0.98 (s, 9H), 0.18 (s, 6H); FT-IR (KBr):
3470, 3034, 2960, 2934, 2899, 2861, 2361, 1743, 1612, 1513,
1390, 1364, 1258, 1171, 1107, 1035, 919, 841, 810, 782, 689 cmꢂ1
;
EI-MS m/z: 294 (M+).
To a solution of the diol (610 mg, 1.66 mmol) in THF (5.5 mL)
The intermediate (2.00 g, 6.79 mmol) was dissolved in THF
(23 mL). LiAlH4 (1.0 M solution in Et2O, 7.5 mL, 7.5 mmol) was
added dropwise to the solution at 0 °C. The mixture was warmed
to room temperature. After 15 min, the reaction was quenched
with saturated aqueous Na2SO3. The mixture was filtered through
Celite and evaporated. The residue was purified by silica gel col-
umn chromatography (n-hexane/EtOAc = 3:1) to give the alcohol
5 (1.65 g, 96%) as a colorless oil. 1H NMR (CDCl3): d 7.07 (d, 2H,
J = 8.5 Hz), 6.78 (d, 2H, J = 8.5 Hz), 3.82 (t, 2H, J = 6.6 Hz), 2.80 (t,
2H, J = 6.6 Hz), 0.98 (s, 9H), 0.19 (s, 6H); FT-IR (KBr): 3369, 2959,
2934, 2889, 2860, 2364, 1612, 1513, 1473, 1464, 1391, 1363,
1262, 1170, 1104, 1047, 918, 840, 811, 780, 690 cmꢂ1; EI-MS m/
z: 252 (M+).
were added Ac2O (610
6.6 mmol), and DMAP (10 mg, 80
l
L, 6.6 mmol), triethylamine (900
lL,
lmol). After 15 min, the reaction
was quenched with H2O and the mixture was extracted with
EtOAc. The organic layer was washed with brine, dried with
MgSO4, and evaporated. The residue was purified by silica gel col-
umn chromatography (EtOAc) to give the triacetate 8 (751 mg,
quant.) as
a d 7.62 (d, 2H,
white solid. 1H NMR (CDCl3):
J = 8.3 Hz), 6.75 (d, 2H, J = 8.3 Hz), 5.64 (s, 1H), 4.34 (s, 4H), 2.54
(m, 2H), 2.17 (m, 2H), 2.09 (s, 6H), 1.96 (s, 3H), 0.97 (s, 9H), 0.18
(s, 6H); FT-IR (KBr): 3485, 3312, 2955, 2861, 2357, 2343, 1738,
1651, 1558, 1513, 1470, 1382, 1259, 1229, 1054, 922, 842,
780 cmꢂ1; EI-MS m/z: 451 (M+).
4.3.7. 2-(Acetylamino)-2-[(acetyloxy)methyl]-4-(4-
4.3.4. tert-Butyl[4-(2-iodoethyl)phenoxy]dimethylsilane (6)
To a solution of the alcohol 5 (2.06 g, 8.16 mmol) in CH2Cl2
(80 mL) cooled to 0 °C were added PPh3 (4.3 g, 16 mmol), imidazole
(1.1 g, 17 mmol), and I2 (4.2 g, 16 mmol). After 1.5 h, the reaction
was quenched with Na2SO3. The mixture was extracted with
CH2Cl2. The water layer was back-extracted with CH2Cl2 and the
combined organic layer was washed with brine, dried with anhy-
drous MgSO4, and evaporated. The residue was purified by silica
gel column chromatography (n-hexane) to give the ethyl iodide 6
(2.91 g, 98%) as a colorless oil. 1H NMR (CDCl3): d 7.03 (d, 2 H,
J = 7.3 Hz), 6.77 (d, 2H, J = 7.3 Hz), 3.30 (t, 2H, J = 7.1 Hz), 3.10 (t,
2H, J = 7.1 Hz), 0.98 (s, 9H), 0.19 (s, 6H). FT-IR (KBr): 2960, 2932,
2888, 2860, 1610, 1510, 1473, 1464, 1264, 1170, 917, 841, 814,
781, 688, 591 cmꢂ1; EI-MS m/z: 362 (M+).
hydroxyphenyl)butyl acetate (9)
The triacetate 8 (2.73 g, 6.03 mmol) was dissolved in THF
(90 mL). Tetrabutylammonium fluoride (1.0 M solution in Et2O,
6.0 mL, 6.0 mmol) was added dropwise to the solution. After
20 min, H2O was added and the mixture was extracted with
Et2O. The organic layer was washed with brine, dried with MgSO4,
and evaporated. The residue was purified by silica gel column chro-
matography (n-hexane/EtOAc = 1:4) to give the phenol 9 (1.99 g,
98%) as a white solid. 1H NMR (CDCl3): d 7.03 (d, 2H, J = 8.5 Hz),
6.76 (d, 2H, J = 8.5 Hz), 5.77 (s, 1H), 4.34 (s, 4H), 2.53 (m, 2H),
2.20 (m, 2H), 2.10 (s, 6H), 1.99 (s, 3H); FT-IR (KBr): 3352, 3104,
3018, 2962, 2932, 2902, 2859, 1744, 1658, 1616, 1598, 1550,
1518, 1455, 1379, 1260, 1173, 1044, 868, 838, 776, 668, 608 cmꢂ1
;
EI-MS m/z: 337 (M+).

7712
S. Nakayama et al. / Bioorg. Med. Chem. 16 (2008) 7705–7714
4.3.8. 2-(Acetylamino)-2-[(acetyloxy)methyl]-4-(4-
{[(trifluoromethanesulfonyl)oxy]phenyl}butyl acetate (10)
3168, 3082, 3032, 2972, 2203, 1739, 1654, 1558, 1511, 1470,
1430, 1382, 1316, 1259, 1230, 1194, 1058, 1034, 968, 848, 821,
To a chilled (0 °C) solution of the phenol 9 (555 mg, 1.64 mmol)
654, 626, 543 cmꢂ1
.
in CH2Cl2 (3.3 mL) was added pyridine (637
l
L, 7.87 mmol). Tf2O
The bromo-monoyne 12 (165 mg, 0.389 mmol) was dissolved in
THF (4.0 mL), and trimethylsilylacetylene (84 L, 0.59 mmol),
PdCl2(PPh3)2 (14 mg, 20 mol), CuI (3.8 mg, 20 mol), and Et3N
(110 L, 0.78 mmol) were added. After 2 h, the reaction was
(331 L, 1.97 mmol) in CH2Cl2 (720 L) was then added dropwise
l
l
l
l
at 0 °C. The reaction mixture was warmed to room temperature.
After 15 min, the reaction was quenched with saturated aqueous
NaHCO3, extracted with CH2Cl2, washed with 10% HCl, washed
with brine, dried with MgSO4, and evaporated. The residue was
purified by silica gel column chromatography (n-hexane/
EtOAc = 1:2) to give the triflate 10 (769 mg, quant.) as a light yel-
low solid. 1H NMR (CDCl3): d 7.26 (d, 2H, J = 8.5 Hz), 7.18 (d, 2H,
J = 8.5 Hz), 5.71 (s, 1H), 4.33 (s, 4H), 2.64 (m, 2H), 2.23 (m, 2H),
2.10 (s, 6H), 1.98 (s, 3H); FT-IR (KBr): 3339, 3216, 3082, 3012,
2996, 2963, 2944, 2870, 1754, 1657, 1547, 1505, 1427, 1394,
1381, 1328, 1248, 1143, 1045, 1020, 903, 831, 640, 607, 503,
478 cmꢂ1; EI-MS m/z: 469 (M+).
l
l
quenched with saturated aqueous NH4Cl. The mixture was ex-
tracted with Et2O and the organic layer was washed with brine,
dried with MgSO4, and evaporated. The residue was purified by sil-
ica gel column chromatography (n-hexane/EtOAc = 2:1 to 1:1 gra-
dient) to give the TMS-diyne 13 (132 mg, 77%) as a white solid. 1
H
NMR (CDCl3): d 7.39 (d, 2H, J = 7.8 Hz), 7.13 (d, 2H, J = 7.8 Hz), 5.78
(s, 1H), 4.32 (s, 4H), 2.60 (m, 2H), 2.19 (m, 2H), 2.09 (s, 6H), 1.97 (s,
3H), 0.23 (s, 9H); 13C NMR (CD3CN): d 171.9, 171.8, 146.2, 134.2,
130.2, 119.6, 92.0, 88.9, 78.3, 74.5, 65.1, 59.1, 34.3, 30.5, 24.3,
21.4, 0.0; FT-IR (KBr): 3316, 3084, 2963, 2364, 2342, 2207, 2106,
1747, 1654, 1558, 1511, 1468, 1379, 1251, 1228, 1049, 1016,
848, 820, 761, 700, 606 cmꢂ1; EI-MS m/z: 441 (M+); Anal. Calcd
for C24H31NO5Si: C, 65.28; H, 7.08; N, 3.17. Found: C, 65.02; H,
7.14; N, 3.22.
4.3.9. 2-(Acetylamino)-2-[(acetyloxy)methyl]-4-{4-[2-(1,1,1-
trimethylsilyl)-1-ethynyl]phenyl}butyl acetate (11)
The triflate 10 (1.0 g, 2.1 mmol) was dissolved in CH3CN
(28 mL), and trimethylsilylacetylene (400
lL, 3.2 mmol),
The TMS-diyne 13 (33.6 mg, 0.0761 mmol) was dissolved in
PdCl2(PPh3)2 (74 mg, 0.11 mmol), and Et3N (1.3 mL, 9.3 mmol)
were added. The reaction mixture was heated at reflux. After
1.5 h, the mixture was cooled, and then filtered through Celite.
The filtrate was evaporated and the residue was purified by silica
gel column chromatography (n-hexane/EtOAc = 1:2 to 1:4 gradi-
ent) to give the TMS-monoyne 11 (538 mg, 61%) as a white solid.
1H NMR (CDCl3): d 7.37 (d, 2H, J = 8.0 Hz), 7.11 (d, 2H, J = 8.0 Hz),
5.70 (s, 1H), 4.33 (s, 4H), 2.59 (m, 2H), 2.19 (m, 2H), 2.09 (s, 6H),
1.97 (s, 3H), 0.24 (s, 9H); 13C NMR (CD3CN): d 171.4, 171.4,
144.5, 132.7, 129.6, 121.3, 106.1, 94.3, 64.6, 58.6, 33.9, 29.9, 23.9,
21.0, 0.0; FT-IR (KBr): 3450, 3338, 3302, 3081, 2958, 2155, 1747,
1653, 1560, 1376, 1249, 1228, 1041, 866, 844 cmꢂ1; EI-MS m/z:
417 (M+); Anal. Calcd for C22H31NO5Si: C, 63.28; H, 7.48; N, 3.35.
Found: C, 63.21; H, 7.45; N, 3.33.
MeOH (320 lL), and 2 N aqueous LiOH (300 lL) was added drop-
wise at room temperature. After 40 min, the mixture was evapo-
rated. The residue was purified by silica gel column
chromatography (CH2Cl2/MeOH = 3:1 to 2:1 gradient) to give the
diyne 2 (9.8 mg, 53%) as a green solid. 1H NMR (CD3OD): d 7.42
(d, 2H, J = 8.3 Hz), 7.24 (d, 2H, J = 8.3 Hz), 3.49 (dd, 4H, J = 21.5,
10.7 Hz), 2.67 (m, 2H), 1.68 (m, 2H); FT-IR (KBr): 3295, 3282,
2939, 2896, 2868, 2208, 1914, 1646, 1609, 1556, 1509, 1456,
1436, 1413, 1376, 1343, 1329, 1319, 1298, 1263, 1242, 1206,
1174, 1062, 1019, 986, 966, 853, 820, 703, 666, 614, 607 cmꢂ1
;
CI-HRMS m/z calcd for C15H18NO2 [(M+H)+] 244.1338, found
244.1349. 2ꢀHCl: 1H NMR (CD3OD): d 7.45 (d, 2H, J = 8.3 Hz), 7.26
(d, 2H, J = 8.3 Hz), 3.68 (s, 4H), 3.12 (s, 1H), 2.69 (m, 2H), 1.94 (m,
2H).
4.3.10. 2-Amino-2-[4-(1-ethynyl)phenethyl]-1,3-propanediol
(1: TX-2148)
4.3.12. 2-Amino-2-[4-(1,3,5-hexatriynyl)phenethyl]-1,3-
propanediol (3: TX-2256)
The TMS-monoyne 11 (100 mg, 0.240 mmol) was dissolved in
MeOH (1.0 mL) and 2 N aqueous LiOH (810 lL) was added drop-
The TMS-diyne 13 (117 mg, 0.265 mmol) was dissolved in ace-
tone (3.5 mL), and NBS (87 mg, 0.49 mmol) and AgNO3 (42 mg,
0.25 mmol) were added. After 2 h, H2O was added at 0 °C. The mix-
ture was extracted with Et2O, the extracts were washed with brine,
dried with MgSO4, and evaporated. The residue was purified by sil-
ica gel column chromatography (n-hexane/EtOAc = 1:2) to give the
bromo-diyne 14 (123 mg, quant.) as a yellow solid. 1H NMR
(CDCl3): d 7.42 (d, 2H, J = 8.3 Hz), 7.14 (d, 2H, J = 8.3 Hz), 5.68 (s,
1H), 4.32 (s, 4H), 2.61 (m, 2H), 2.20 (m, 2H), 2.09 (s, 6H), 1.97 (s,
3H); FT-IR (KBr): 3380, 3072, 2927, 2857, 2219, 2130, 1745,
1711, 1695, 1662, 1551, 1509, 1457, 1420, 1375, 1247, 1185,
wise at room temperature. After 2 h, the mixture was evaporated.
The residue was purified by silica gel column chromatography
(CH2Cl2/MeOH = 1:4) to give the monoyne 1 (42.1 mg, 80%) as a
white solid. 1H NMR (CD3CN): d 7.39 (d, 2H, J = 8.3 Hz), 7.21 (d,
2H, J = 8.3 Hz), 3.39 (dd, 4H, J = 18.5, 10.7 Hz), 3.32 (s, 1H), 2.64
(m, 2 H), 1.61 (m, 2H); FT-IR (KBr): 3318, 3073, 3060, 3024,
3000, 2947, 2923, 2868, 2856, 2646, 2110, 1928, 1814, 1575,
1503, 1476, 1465, 1454, 1438, 1415, 1401, 1355, 1312, 1297,
1281, 1238, 1190, 1160, 1148, 1108, 1083, 1068, 1047, 1023,
966, 923, 853, 827, 810, 793, 763, 649, 641, 629, 556, 514 cmꢂ1
;
1143, 1047, 891, 850, 822, 638, 605, 546 cmꢂ1
The bromo-diyne 14 (50.6 mg, 0.113 mmol) was dissolved in
THF (1.1 mL), and trimethylsilylacetylene (19 L, 0.13 mmol),
PdCl2(PPh3)2 (0.8 mg, 1 mol), CuI (0.2 mg, 1 mol), and Et3N
(31 L, 0.22 mmol) were added. After 2 h, the reaction was
.
CI-HRMS m/z calcd for C13H18NO2 [(M+H)+] 220.1337, found
220.1330.
l
l
l
4.3.11. 2-Amino-2-[4-(1,3-butadiynyl)phenethyl]-1,3-
l
propanediol (2: TX-2152)
quenched with saturated aqueous NH4Cl. The mixture was ex-
tracted with Et2O and the organic layer was washed with brine,
dried with MgSO4, and evaporated. The residue was purified by sil-
ica gel column chromatography (n-hexane/EtOAc = 1:1) to give the
TMS-triyne 15 (34.5 mg, 67%) as a white solid. 1H NMR (CDCl3): d
7.43 (d, 2H, J = 8.5 Hz), 7.15 (d, 2H, J = 8.5 Hz), 5.68 (s, 1H), 4.32
(s, 4H), 2.61 (m, 2H), 2.20 (m, 2H), 2.09 (s, 6H), 1.97 (s, 3H), 0.22
(s, 9H); 13C NMR (CD3CN): d 172.0, 172.0, 147.1, 134.8, 130.6,
118.9, 91.2, 88.8, 79.1, 74.5, 67.8, 65.2, 62.8, 59.2, 34.4, 30.8,
30.4, 24.5, 21.6, 0.0; FT-IR (KBr): 3315, 3208, 3081, 2962, 2931,
2858, 2175, 2077, 1742, 1654, 1551, 1511, 1469, 1437, 1376,
The TMS-monoyne 11 (446 mg, 1.07 mmol) was dissolved in
acetone (3.6 mL), and NBS (209 mg, 1.18 mmol) and AgNO3
(182 mg, 1.07 mmol) were added. After 2 h, H2O was added at
0 °C. The mixture was extracted with Et2O, the extracts were
washed with brine, dried with Na2SO4, and evaporated. The residue
was purified by silica gel column chromatography (n-hexane/
EtOAc = 1:1) to give the bromo-monoyne 12 (428 mg, 94%) as a
light yellow solid. 1H NMR (CDCl3): d 7.36 (d, 2H, J = 8.3 Hz), 7.13
(d, 2H, J = 8.3 Hz), 5.69 (s, 1H), 4.33 (s, 4H), 2.60 (m, 2H), 2.20 (m,
2H), 2.09 (s, 6H), 1.97 (s, 3H); FT-IR (KBr): 3321, 3300, 3212,

S. Nakayama et al. / Bioorg. Med. Chem. 16 (2008) 7705–7714
7713
1248, 1141, 1049, 845, 761, 642, 603 cmꢂ1; EI-MS m/z: 465 (M+);
Anal. Calcd for C26H31NO5Si: C, 67.07; H, 6.71; N, 3.01. Found: C,
66.82; H, 6.80; N, 2.97.
The TMS-triyne 15 (29.7 mg, 0.064 mmol) was dissolved in
MeOH (270 lL), and 2 N aqueous LiOH (144 lL) was added to the
window was about 1 cm. The windows were closed with stain-
less-steel caps. The eggs were incubated in a humidified incubator
at 39.0 °C. After 1 day, the cap was removed to check if the size of
the CAM had become 2–3 mm. A silicon ring (3.0 mm external
diameter, 5.0 mm internal diameter, 1.5 mm thickness) was placed
solution dropwise at room temperature. After 23 h, the mixture
on the CAM, and then 10 lL of test compound solution or control
was evaporated. The residue was purified by silica gel column
solution mixed in 1% methyl cellulose/0.9% NaCl (0.05% DMSO)
was applied into the silicon ring. The windows were closed with
stainless-steel caps. The eggs were incubated in a humidified incu-
bator at 39.0 °C. After 1 day, the position of silicon ring was
checked, and temperature of incubator was raised to 39.5 °C. The
eggs were incubated for 1day and then the shell was cut out with
scissors to make the CAM easy to observe. Inhibition of angiogen-
esis was evaluated after visualization by injection of a fat emulsion
IntraliposÒ (1 mL) into the CAM. The angiogenic inhibition was
indicated by an avascular zone around a ring of 3 mm diameter.
It was evaluated in five stages. The egg whose vessels around a ring
showed no inhibition of angiogenesis was designated minus. The
egg whose vessels were not clear as to whether these were positive
or negative was designated plus-minus. The egg whose capillaries
were partially inhibited was designated plus, while the egg whose
capillaries were completely inhibited was double plus. The egg
whose large vessels showed inhibition was designated triple plus.
The percent inhibition of angiogenesis was calculated by the fol-
lowing equation.
chromatography (CH2Cl2/MeOH = 2:1) to give the triyne
3
(10.8 mg, 63%) as a light brown solid. 1H NMR (CD3CN): d 7.42
(d, 2H, J = 8.5 Hz), 7.22 (d, 2H, J = 8.5 Hz), 3.37 (dd, 4H, J = 21.0,
10.7 Hz), 2.65 (m, 2H), 1.59 (m, 2H); FT-IR (KBr): 3394, 3355,
3336, 3306, 3057, 2932, 2864, 2855, 2362, 2195, 2134, 1624,
1576, 1510, 1466, 1455, 1400, 1146, 1129, 1085, 1022, 1067,
1049, 829, 807, 723, 639, 545 cmꢂ1; CI-HRMS m/z calcd for
C
17H18NO2 [(M+H)+] 268.1338, found 268.1342.
4.3.13. 2-(Acetylamino)-2-[(acetyloxy)methyl]-4-{4-[6-(1,1,1-
trimethylsilyl)-1,3,5-hexatriynyl]phenyl}butyl acetate (15)
Bistrimethylsilylbutadiyne (229 mg, 1.2 mmol) was dissolved in
THF (2.3 mL), and MeLiꢀLiBr (1.5 M solution in THF, 790
ll,
1.2 mmol) was added at 0 °C. After 1 h, CuBr (169 mg, 1.2 mmol)
was added, and then the solution was warmed to room tempera-
ture. After 30 min, the solution was evaporated. The bromo-mono-
yne 12 (500 mg, 1.2 mmol) in pyridine (3.0 mL) was added to the
residue at room temperature. After 4 h, the reaction was quenched
with saturated aqueous NH4Cl. The mixture was extracted with
Et2O, and the organic layer was washed with brine, dried with
Na2SO4, and evaporated. The residue was purified by silica gel col-
umn chromatography (n-hexane/EtOAc = 2:1 to 1:1 gradient) to
give the TMS-triyne 15 (244 mg, 44%) as a brown solid.
Inhibition ð%Þ ¼ Ni=Ns ꢁ 100
where Ni is number of eggs evaluated greater than or equal to plus,
Ns is number of total survived eggs.
Acknowledgments
4.4. Determination of logPow
The authors thank Ms. Maki Nakamura, Ms. Kayoko Yamashita,
Ms. Emiko Okayama and the staff of our Faculty for measurement
of NMR, MS, and elemental analysis. We also thank Dr. Hiroyuki
Konno of our faculty for useful advice. We also thank Dr. Kenneth
L. Kirk (NIH) for his critical and valuable comments. We also thank
Takeda Pharmaceutical Co., Ltd (Osaka, Japan) for providing TNP-
470.
The measurement of logPow values was carried out using an
HPLC method. UV detection was performed at 254 nm. Methanol
and water in a ratio 3:1 (v/v) were employed as the mobile phase
at a flow rate of 1.0 mL/min. The test compounds and the reference
compounds were dissolved in the mobile phase for their injection.
The logPow is deduced from the logarithm of the capacity factor k
(logk), calculated according to the following equation.
References
k ¼ ðtr ꢂ t0Þ=t0
where tr is the retention time and t0 is the dead time. Thiourea dis-
solved in the mobile phase was used to determine the t0. In order to
correlate the logk of a compound with its logPow value, a calibra-
tion graph using 11 reference compounds, ranging in logPow values
from 0.3 to 5.7, was established. Since the calibration graph showed
strong correlation (R2 = 0.9806) between log k and logPow, the
logPow value of any compound exceeding 5.7 was determined by
extrapolation.
1. Adachi, K.; Kohara, T.; Nakao, N.; Arita, M.; Chiba, K.; Mishina, T.; Sasaki, S.;
Fujita, T. Bioorg. Med. Chem. Lett. 1995, 5, 853.
2. Kiuchi, M.; Adachi, K.; Kohara, T.; Minoguchi, M.; Hanano, T.; Aoki, Y.; Mishina,
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Antiangiogenic activity was assayed using the CAM assay
according to the method described by Oikawa.63 All compounds
tested were dissolved in DMSO. In a control experiment, a test
compound solution was replaced with DMSO. Fertilized chicken
eggs were incubated in a humidified incubator at 37.6 °C for 4 days
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