Fusarielin A as an anti-angiogenic and anti-proliferative agent: Basic biological characterization

Article abstract of DOI:10.1248/cpb.56.298

Fusarielin A shows anti-angiogenic activity in the human umbilical vein endothelial cell (HUVEC) tube formation assay. Structural development studies indicated the importance of the hydroxyl groups in this molecule. A ³H-labeled derivative and a fluorescent affinity-labeling agent were prepared and used to examine the cellular distribution and biological behavior of fusarielin A.

Full text of DOI:10.1248/cpb.56.298

298
Chem. Pharm. Bull. 56(3) 298—304 (2008)
Vol. 56, No. 3
Fusarielin A as an Anti-angiogenic and Anti-proliferative Agent: Basic
Biological Characterization
Haruka FUJIMOTO, Hiroshi AOYAMA, Tomomi NOGUCHI-YACHIDE, Yuichi HASHIMOTO, and
Hisayoshi KOBAYASHI*
Institute of Molecular & Cellular Biosciences, The University of Tokyo; 1–1–1 Yayoi, Bunkyo-ku, Tokyo 113–0032, Japan.
Received October 22, 2007; accepted December 17, 2007; published online December 18, 2007
Fusarielin A shows anti-angiogenic activity in the human umbilical vein endothelial cell (HUVEC) tube for-
mation assay. Structural development studies indicated the importance of the hydroxyl groups in this molecule.
A 3H-labeled derivative and a fluorescent affinity-labeling agent were prepared and used to examine the cellular
distribution and biological behavior of fusarielin A.
Key words fusarielin A; anti-angiogenic activity; tritium-labeling; human umbilical vein endothelial cell; HL-60; binding pro-
tein
Natural products that influence cell proliferation/differen- including rhizoxin,1) griseofulvin,2) nocodazole3) and pho-
tiation and signal transduction systems are of great interest as mopsidin,4) generally show morphological effects, such as
candidate anti-tumor agents. Generally, isolation of bioactive curling, on mycelia germinated from conidia of P. oryzae P-
natural products based on some specific biological activity 2b [Fig. 1, panels (c)—(f)].
requires a specific bioassay system that reflects the corre-
Using the P. oryzae P-2b assay method, we have isolated
sponding biological activity.1—6) For example, tubulin func- an antifungal antibiotic, fusarielin A (1: FSA) (Fig. 2), from
tion disruptors (inhibitors of tubulin polymerization or de- a culture of Fusarium sp. K432.7) The effect of FSA (1) in
polymerization) have generally been isolated with the aid of this bioassay [Fig. 1, panel (b)] was similar to those elicited
microtubule assembly assay,1—4) while signal transduction in- by compounds that interfere with microtubule function, in-
hibitors have generally been isolated with the guidance of in- cluding rhizoxin [panel (c)],1) griseofulvin [panel (d)],2)
hibition assays of specific enzymes, such as protein kinases nocodazole [panel (e)],3) and phomopsidin [panel (f)]4) (Fig.
and phosphatases.5,6) On the other hand, a bioassay system 1). However, FSA (1) did not inhibit the assembly or disas-
that could simultaneously detect various kinds of cell func- sembly of microtubule protein prepared from porcine brain.7)
tion modulators, including cell proliferation inhibitors, cell Further investigation revealed that FSA (1) shows cell type-
differentiation modulators, signal transduction modulators, selective anti-proliferative activity, i.e., it inhibited prolifera-
and so on, would be a superior tool to discover unique bioac- tion of a leukemia cell line HL-60 with an IC50 value of
tive compounds, even if it provided no information as to their 23.6 mM, while it inhibited proliferation of the epidermal cell
specific molecular targets/mechanisms.
One such broad-spectrum bioassay method utilizes the
line HeLa with an IC50 value of 54.6 mM.1)
On the other hand, ICM0301A (3), which is a natural
conidia of Pyricularia oryzae (P. oryzae) P-2b (Fig. 1).7,8) product isolated from the culture broth of Aspergillus sp. F-
Germination of conidia of P. oryzae P-2b is sensitive to vari- 1491 and structurally similar to FSA (1), has recently been
ous kinds of bioactive compounds (details will be published reported to possess anti-angiogenic activity [the anti-angio-
elsewhere). For example, tubulin polymerization inhibitors, genic activity of ICM0301A (3) was assessed by using the in
Fig. 1. Curling on Mycelia Germinated from Conidia of Pyricularia oryzae P-2b
∗ To whom correspondence should be addressed. e-mail: hashimot@iam.u-tokyo.ac.jp
© 2008 Pharmaceutical Society of Japan

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299
vitro angiogenesis model of culturing fragment of rat aorta in 21 d. Based on the results, a total of 2.4 l of culture was kept
three-dimensional fibrin gels, and it is reported to show sig- at 20 °C for 21 d, then the medium was extracted with a mix-
nificant inhibitory activity of 52% inhibition at 1 mg/ml].9,10) ture of acetone and benzene. Successive column chro-
This reported result together with the above-mentioned find- matographies using three types of resins gave 550 mg of FSA
ings prompted us to examine the effect of FSA (1) on the (1) as a white powder (Fig. 3, see Experimental). The struc-
growth and tube formation of human umbilical vein endothe- ture and purity of the isolated FSA (1) were confirmed by
lial cells (HUVEC).
HPLC, NMR spectroscopy and mass spectrometry.
Here, we describe the anti-angiogenic activity of FSA (1)
Growth-Inhibitory Activity and Anti-angiogenic Activ-
in HUVEC tube formation assay, a preliminary study of the ity of FSA toward HUVEC Next, we investigated the
structure–activity relationship, an analysis of the cellular up- growth-inhibitory activity of FSA (1) on HUVEC. FSA (1)
take and distribution of FSA (1) with biosynthetically pre- inhibited the growth of HUVEC with the IC50 value of
3
pared H-labeled FSA (Fig. 7), and the preparation of an 19.3 mM after 72 h incubation, which is in good accordance
affinity-labeling agent, CAFSA (Coumarin- and Azi- with the IC50 value for HL-60 cells (IC50ꢀ23.6 mM) (Fig. 4a,
dophenyl-substituted FSA derivative: 6, Fig. 9).
Table 1). Anti-angiogenic activity of FSA (1) was assessed
by means of HUVEC tube formation assay.11) FSA (1) exhib-
ited moderate anti-angiogenic activity with the IC50 value of
Results and Discussion
Large-Scale Preparation of FSA (1) To obtain a suffi- 7.1 mM (Figs. 4b, c, Table 1).
cient amount of FSA (1) for our studies, the culture condi-
In our previous studies, fusarielin B (2: FSB) (Fig. 2), in
tions were optimized. Our previous studies indicated that the which one of the two epoxy groups of FSA (1) was opened,
production of FSA (1) by Fusarium sp. K432 is temperature- was co-isolated with FSA (1). FSB (2) did not induce curling
dependent, i.e., FSA (1) was produced at 20 °C, but not at of mycelia of P. oryzae P-2b,3) suggesting that the two epoxy
27 °C.7) Therefore, we investigated the time-course of FSA groups of FSA (1) are essential for the activity. On the other
(1) production by the fungus in standing culture in a potato
dextrose medium at 20 °C. Quantification of FSA (1) in the
culture medium by means of HPLC indicated that the pro-
duction of FSA (1) was time-dependent and saturated after
Fig. 2. Structures of FSA (1), FSB (2) and ICM0301A (3)
Fig. 3. Purification Scheme of FSA (1)
Fig. 4. Effects of FSA (1) on HUVEC Growth and Tube Formation
(a) Growth inhibitory activity of FSA (1) toward HUVEC (incubated for 72 h). (b) Tube formation inhibitory activity of FSA (1) toward HUVEC (treated for 6 h). (c) Micro-
scopic images of HUVEC cells treated with or without 30 mM FSA for 6 h (see Experimental).

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Table 1. HL-60/HUVEC Cell Growth Inhibitory Activity and HUVEC Tube Formation Inhibitory Activity of FSA (1), 3-Bn-FSA (4) and 1,3-Bn2-FSA (5)
FSA
3-Bn-FSA
1,3-Bn2-FSA
HL-60 (IC50: mM)
23.6
19.3
83
15.3
N.D.
98
63.4
ꢁ100
34
Cell growth
inhibitory activity
HUVEC (IC50: mM)
HUVEC: after 72 h incubation at 100 mM (%)
HUVEC: after 6 h incubation at 100 mM (%)
48
46
49
HUVEC tube
formation inhibitory activity
HUVEC: after 6 h (IC50: mM)
7.1
N.D.
N.D.
N.D.ꢀnot determined.
Fig. 5. Synthesis of 3-Bn-FSA (4) and 1,3-Bn2-FSA (5)
Reagents and conditions: (a) tert-butyldimethylchlorosilane, Et3N, DMAP, CH2Cl2, r.t., 23 h (86%); (b) BnBr, NaH, DMF, r.t., 20 h (39%); (c) 0.13 M tetra-n-butylammonium flu-
oride, THF solution, r.t., 25 h (89%); (d) BnBr, NaH, THF, 45 °C, 14 h (74%).
hand, it was not clear whether or not the two hydroxyl groups showed anti-angiogenic activity (Fig. 6A), but lacked
at positions 1 and 3 (numbering is shown in Fig. 7) are nec- growth-inhibitory activity against HUVEC/HL-60 cells. It
essary for the activity. To obtain preliminary information did not show curling-inducing activity on mycelia or growth-
about the structure–activity relationships of FSA (1), deriva- inhibitory activity against P. oryzae P-2b (Fig. 6B). The re-
tives with one or two benzylated hydroxyl group(s) were pre- sults suggest that (i) the free hydroxyl groups are critical for
pared (Fig. 5), i.e., 3-Bn-FSA (4) in which the 3-secondary the curling-inducing activity towards mycelia germinated
alcohol group is protected with a benzyl group and 1,3-Bn2- from conidia of P. oryzae P-2b, (ii) the free hydroxyl groups
FSA (5) in which the 1-primary and 3-secondary alcohol are not necessary for the anti-angiogenic activity, and (iii) the
groups are both protected with benzyl groups (Fig. 5). Be- free hydroxyl groups at the 1-position and the 3-position are
cause azido-functionalized benzyl group was successfully necessary and unnecessary, respectively, for the
applied in identifying the molecular weight of target protein HUVEC/HL-60 cell growth-inhibitory activity. These find-
of dantrolene analog by Hosoya and co-workers,12) we made ings indicate that it may be possible to separate each activity,
a choice for the benzyl group not only to obtain preliminary i.e., cell growth inhibition, anti-angiogenic activity and curl-
information of SAR study of FSA (1), but also to determine ing-inducing activity, by the structural development of FSA
the site of FSA (1) to introduce azido-functionalized benzyl (1). In other words, different structural features, especially
group for a planned photoaffinity labeling (vide infra),12) The the presence of the free hydroxyl groups, appear to be criti-
structures of the synthesized compounds were confirmed by cally recognized by putative target molecules involved in the
1H-NMR spectroscopy and mass spectrometry.
The two derivatives, 3-Bn-FSA (4) and 1,3-Bn2-FSA (5)
three biological activities.
Preparation of [3H]FSA As mentioned above, the anti-
(Fig. 5), were investigated for anti-angiogenic activity, angiogenic activity of FSA (1) seems to be specific, at least
growth-inhibitory activity against HUVEC/HL-60 cells in part, and the cell growth-inhibitory activity elicited by
(Table 1, Fig. 6A), and malformation-inducing activity to- FSA (1) seems to be cell type-selective. These results sug-
wards mycelia of P. oryzae P-2b (Fig. 6B). 3-Bn-FSA (4) gest that FSA (1) may elicit its biological activity by binding
showed anti-angiogenic activity and growth-inhibitory activ- to a specific target molecule(s). To examine this working hy-
3
ity against HUVEC/HL-60 with almost the same potency as pothesis, we planned to prepare H-labeled FSA ([3H]FSA).
FSA (1) (Table 1, Fig. 6A), but did not induce curling of FSA (1) is a decaketide, and previous experiments indicated
mycelia [although growth-inhibitory activity against P. the incorporation of both carbons of acetate and the methyl
oryzae P-2b was observed (Fig. 6B)]. 1,3-Bn2-FSA (5) carbon of methionine (Fig. 7).7) To prepare [3H]FSA, we first

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Fig. 7. Schematic Representation of Incorporation of Biosynthetic Precur-
sors into FSA (1)
Fig. 8. Uptake and Distribution of [3H]FSA
[A] Distribution of [3H]FSA in fractionated cell pellet. [B] Distribution of [3H]FSA
in ammonium sulfate precipitate of soluble cytosolic fraction.
Fig. 6. Effects of FSA (1) and Its Benzylated Derivatives [3-Bn-FSA (4)
and 1,3-Bn2-FSA (5)] at 100 mM on HUVEC Tube Formation (A) and Ger-
mination of Conidia of Pyricularia oryzae P-2b (B)
4 °C to separate the supernatant and cell pellet. The cell pel-
let contained about ten times higher concentration of ra-
dioactivity than the supernatant, suggesting efficient uptake
examined the incorporation of methionine-d3, using cultures of [3H]FSA into the cells. Separation of subcellular fractions
of the K432 strain. After standing culture for 21 d, FSA (1) of HL-60 cells and the measurement of [3H]FSA uptake were
1
was extracted and its structure was confirmed by H-NMR performed as described previously.13—15) Briefly, HL-60 cells,
and mass spectrometry. The results showed that methionine- which had been incubated with [3H]FSA (10 mM), were lysed
d3 was incorporated into FSA (1). Therefore, to prepare with hypotonic solution (20 mM Tris–HCl), and the nuclear
[3H]FSA, we added L-methionine, [methyl-3H] (185 MBq) to fraction, soluble cytosolic fraction and membrane fraction
a culture medium which had been pre-cultured for 6 d. The were separated by successive centrifugation. Approximately
culture was continued for a further 8 d, then FSA (1) was ex- 92% of [3H]FSA was distributed to the soluble cytosolic
tracted from the medium and purified by HPLC to give fraction (Fig. 8A). Next, 90% ammonium sulfate was added
[3H]FSA with the specific activity of 75 MBq/mmol.
to the soluble cytosolic fraction, and the mixture was cen-
Distribution of [3H]FSA in HL-60 Cells To examine trifuged to separate the supernatant and pellet. More than
the cellular uptake and distribution of FSA (1), we selected 90% of [3H]FSA was found in the pellet, i.e., the salted-out
HL-60 cells because the HL-60 cell growth inhibition assay protein fraction (Fig. 8B). Therefore, FSA (1) might have
could discriminate FSA (1) and 3-Bn-FSA (4) [active] from been bound to specific protein(s) existing in the soluble cy-
1,3-Bn2-FSA (5) [inactive]. This raises the possibility of de- tosolic fraction.
veloping affinity-labeling agents using the 3-hydroxyl group
as a functional group on which a photolabile group can be in- signed a photoaffinity probe to detect the putative target mol-
troduced (vide infra). ecule(s) of FSA (1) in HL-60 cells. As the free hydroxyl
Photoaffinity Label Based on the above results, we de-
HL-60 cells were incubated for 20 h under 5% CO2 at group at the 3-position of FSA (1) was considered to be un-
37 °C in the presence of [3H]FSA (10 mM). After the incuba- necessary for cell growth-inhibitory activity toward HL-60
tion, the mixture was centrifuged at 2000 rpm for 5 min at cells, we designed and synthesized an FSA derivative with a

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Vol. 56, No. 3
Fig. 9. Synthesis of Photoaffinity Labeling Agent, CAFSA (6)
Reagents and conditions: (a) tert-butyldimethylchlorosilane, Et3N, DMAP, CH2Cl2, r.t., 18 h (98%); (b) 2 N NaOH aq., THF–MeOH, r.t., 3.5 h; (c) piperazine, 1,1ꢂ-carbonyl
diimidazole, DMF, r.t., 15 h (82%, 2 steps); (d) 7-diethylaminocoumarin-2-carboxylic acid, 1,1ꢂ-carbonyldiimidazole, DMF, r.t., 19 h (84%); (e) SnCl2·2H2O, EtOAc, 90 °C, 7 h
(98%); (f) NaNO2, NaN3, 4 N HCl, 0 °C, 20 min (93%); (g) CBr4, P(n-octyl)3, CH2Cl2, r.t., 3 h (59%); (h) sodium hexamethyldisilazide (NaHMDS), DMF, r.t., 24 h (12%).
fluorescent and photoreactive group(s) at the 3-secondary al-
cohol group, i.e., an FSA derivative bearing a coumarin and
an azidophenyl group (6: CAFSA, Fig. 9). The coumarin
skeleton was chosen as a fluorescent chromophore, and an
azidophenyl moiety was introduced as the photoreactive
part.16—18) The probe CAFSA (6) was synthesized as shown
in Fig. 9.
A preliminary photoaffinity labeling experiment using
CAFSA (6) was performed (Fig. 10) with the soluble cytoso-
lic fraction of cultured HL-60 cells, using a method similar
to that described previously.19—21) The HL-60 cell extract was
incubated with CAFSA (6) [bars (a—c) in Fig. 10] in the
presence [bar (b) in Fig. 10] or absence [bars (a) and (c) in
Fig. 10] of an excess amount of FSA (1), and then the mix-
ture was irradiated at 254 nm with a compact UV lamp (UVP,
4 W). The irradiated mixture was subjected to gel filtration
column chromatography to obtain the macromolecular (pro-
tein) fraction, and the fluorescence intensity of this fraction
was measured (relative fluorescence intensity is indicated by
the length of the bars in Fig. 10). As shown in Fig. 10, the
macromolecular fraction and non-reacted CAFSA (6) were
efficiently separated by the gel filtration column chromatog-
raphy [bars (c) and (d) in Fig. 10]. The high and low levels of
relative fluorescence intensity shown by the bars (a) and (c)
in Fig. 10, respectively, indicate that the covalent binding of
CAFSA (6) to the macromolecular fraction occurred irradia-
tion-dependently. The covalent binding of CAFSA (6) was
inhibited in the presence of an excess of FSA (1) (compare
bars (a) and (b) in Fig. 10), suggesting that CAFSA (6) binds
specifically at the FSA (1) binding site(s). These results sug-
gest that CAFSA (6) is a suitable photoaffinity labeling agent
to identify the target molecule(s) of FSA (1).
Fig. 10. Results of Photoaffinity Labeling Experiments
Experimental
Large-Scale Purification of FSA (1) Fusarium sp. K432 strain was
cultured in dishes each containing 30 ml of potato dextrose medium (potato
200 g, dextrose 20 g per liter of water) at 20 °C for 21 d. The acetone–ben-
zene extract of a total of 2.4 l of culture was separated by silica gel column
chromatography. The column was eluted with ethyl acetate–hexane (3 : 2).
The solution was concentrated, and the residue (2.8 g) was subjected to
Sephadex LH-20 column chromatography. The column was eluted with
MeOH. The solution was concentrated, and the residue was applied to a col-
umn of activated charcoal. The column was eluted with MeOH, and the elu-
ate was concentrated to give FSA (1) as a white powder (550 mg).
Cell Culture HL-60 cells were cultured in RPMI1640 medium supple-
mented with 10% heat-inactivated fetal bovine serum (FBS; Gibco BRL) at
37 °C under a 5% CO2 atmosphere. HUVECs were cultured in EBM-2
medium supplemented with growth factors (hEGF, VEGF, hFGF-B, and
R3IGF-1, as well as FBS) at 37 °C under a 5% CO2 atmosphere.
Growth Inhibitory Activity against HUVEC and HL-60 Each cell
was placed in 12-well plates and treated with various concentrations of the
test compounds at 37 °C for 6 or 72 h. After the incubation, the viability of
the treated cells was measured by direct counting under a microscope.
HUVEC Tube Formation Assay HUVECs were plated on Matrigel
and cultured in the presence of test compounds for 6 h, then tube formation
Further work to identify the macromolecule(s) photoaffin-
ity-labeled with CAFSA (6) is under way.

March 2008
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was measured as previously reported.11) Briefly, six-well plates were coated
with 1.5 ml of the Matrigel basement membrane matrix (Becton Dickinson)
and allowed to gel at 37 °C under a 5% CO2 atmosphere for 30 min. Then,
HUVECs were plated at 5.0ꢃ105 cells/well in Dulbecco’s modified Eagle
medium (DMEM) containing the vehicle (0.5% DMSO) in the presence or
absence of various concentrations of the test compound, and incubation was
continued at 37 °C under a 5% CO2 atmosphere for 6 h. Each well was pho-
tographed using a ꢃ100 objective to analyze tube formation. The correspon-
ding area was evaluated as the number of pixels using MetaMorph software
(Universal Imaging, Downingtown, PA, U.S.A.).
1.49 (1H, ddd, Jꢀ3.0, 10.0, 14.0 Hz), 1.56—1.68 (4H, m), 1.59 (3H, s), 1.62
(3H, s), 1.72—1.82 (2H, m), 2.06—2.18 (1H, m), 2.07 (1H, dd, Jꢀ3.0,
14.0 Hz), 2.44—2.49 (1H, m), 2.72 (1H, s), 2.93 (1H, d, Jꢀ6.0 Hz), 3.50
(1H, d, Jꢀ9.0 Hz), 3.56 (1H, td, Jꢀ6.0, 9.0 Hz), 3.68 (1H, dd, Jꢀ3.0,
10.5 Hz), 4.18 (1H, d, Jꢀ11.5 Hz), 4.32 (1H, d, Jꢀ11.5 Hz), 5.13—5.20
(1H, m), 5.21 (1H, dd, Jꢀ10.5, 15.0 Hz), 5.88 (1H, d, Jꢀ11.0 Hz), 6.18—
6.34 (1H, m), 7.23—7.34 (5H, m). MS (FAB): m/z 606 (M)ꢄ.
3-Bn-FSA (4) A mixture of 8 (13.0 mg, 0.021 mmol) and tetra-n-butyl-
ammonium fluoride (0.13 M THF solution, 0.46 ml, 0.06 mmol) was stirred
at ambient temperature for overnight. The reaction mixture was applied di-
rectly to silica-gel and purified by silica-gel column chromatography
(hexane/ethyl acetateꢀ3/1) to give 3-Bn-FSA (4) (9.4 mg, 0.019 mmol,
89%) as a colorless oil; 1H-NMR (DMSO-d6, 50 °C) d: 0.70 (3H, d,
Jꢀ7.5 Hz), 1.13 (3H, s), 1.18—1.29 (1H, m), 1.27 (3H, s), 1.51 (1H, ddd,
Jꢀ3.0, 10.0, 14.0 Hz), 1.56—1.67 (5H, m), 1.60 (3H, s), 1.63 (3H, s),
1.68—1.83 (2H, m), 2.08 (1H, dd, Jꢀ3.0, 14.0 Hz), 2.14 (1H, ddd, Jꢀ5.0,
10.0, 10.0 Hz), 2.44—2.51 (1H, m), 2.71 (1H, s), 2.93 (1H, d, Jꢀ5.0 Hz),
3.33 (1H, td, Jꢀ6.5, 10.0 Hz), 3.49 (1H, d, Jꢀ9.0 Hz), 3.61 (1H, td, Jꢀ4.5,
10.5 Hz), 4.165 (1H, t, Jꢀ5.0 Hz), 4.166 (1H, d, Jꢀ12.0 Hz), 4.31 (1H, d,
Jꢀ12.0 Hz), 5.14—5.24 (1H, m), 5.21 (1H, dd, Jꢀ10.0, 15.0 Hz), 5.89 (1H,
d, Jꢀ11.0 Hz), 6.26 (1H, dd, Jꢀ11.0, 15.0 Hz), 7.22—7.35 (5H, m). HR-MS
(FAB, [MꢅH]ꢄ) Calcd for C32H43O4: 491.3161, Found: 491.3206.
Curling of Mycelia of P. oryzae P-2b Conidia of P. oryzae P-2b germi-
nated at 27 °C in water suspension containing 0.02% yeast extract and were
easily observed under an optical microscope. A time point of 16 h was the
most appropriate for observation of hyphal growth.8)
Preparation of [3H]FSA L-Methionine, [methyl-3H] (185 MBq) in
EtOH was left on a dish for several days. After the EtOH had completely
evaporated, 6 d culture of the K432 strain was added to the dish. After a fur-
ther 8 d, the culture was extracted with acetone–benzene. The organic solu-
tion was evaporated, and the residue was purified and subjected to HPLC.
The yield of [3H]FSA was 33.6 mg, and the specific activity was
75 MBq/mmol.
Distribution of [3H]FSA in HL-60 Cells HL-60 cells were incubated in
RPMI1640 medium in the presence or absence of [3H]FSA (10 mM) for 20 h.
Treated HL-60 cells were centrifuged at 2000 rpm for 5 min at 4 °C. The ra-
dioactivity of the supernatant was measured. The pellet was resuspended in
buffer (20 mM Tris–HCl, 0.6 M KCl), and centrifuged at 2000 rpm for 5 min
at 4 °C. The radioactivity of the resulting pellet was measured.
HL-60 cells were incubated in RPMI1640 medium in the presence or ab-
sence of [3H]FSA (10 mM) for 20 h. Treated HL-60 cells were centrifuged at
2000 rpm for 5 min at 4 °C. The pellet was resuspended in buffer, and cen-
trifuged at 2000 rpm for 5 min at 4 °C. The pellet was resuspended in hypo-
tonic solution (20 mM Tris–HCl), vortexed if necessary, and kept on ice for
45 min. After microscopic confirmation of cell lysis, the suspension was
centrifuged at 2500 rpm for 15 min at 4 °C. The pellet was resuspended in
buffer and centrifuged at 2500 rpm for 15 min at 4 °C. The resulting pellet
was designated as the nuclear fraction. The supernatant was centrifuged at
50000 rpm for 1 h at 4 °C. The resulting supernatant was designated as the
soluble cytosolic fraction, and the pellet was designated as the membrane
fraction. The radioactivity of each fraction was measured with a liquid scin-
tillation counter.
1,3-Bn2-FSA (5) To a solution of 1-pivaloyloxy-FSA (9)7) (27.9 mg,
0.057 mmol) in DMF (0.4 ml) was added NaH (55—72% contained, 2.3 mg).
After 30 min, to this was added benzyl bromide (15.0 ml, 0.126 mmol) and
the mixture was stirred at 40 °C for 2 h. Further NaH (55—72% contained,
4.6 mg) was then added and the mixture was stirred at the same temperature
for 15 h. The reaction was quenched by adding H2O and the mixture was ex-
tracted with hexane. The product was purified by silica-gel column chro-
matography (hexane/ethyl acetateꢀ10/1, then 6/1) to give 1,3-Bn2-FSA (5)
(24.6 mg, 0.042 mmol, 74%) as a colorless oil; 1H-NMR (DMSO-d6) d: 0.75
(3H, d, Jꢀ7.0 Hz), 1.01 (1H, dd, Jꢀ7.0, 9.5 Hz), 1.12 (3H, s), 1.26 (3H, s),
1.49 (1H, ddd, Jꢀ3.5, 10.5, 15.0 Hz), 1.59 (3H, s), 1.59—1.67 (5H, m), 1.64
(3H, s), 1.76 (1H, td, Jꢀ5.0, 15.0 Hz), 1.86—1.95 (1H, m), 2.07 (1H, dd,
Jꢀ3.0, 13.5 Hz), 2.07—2.17 (1H, m), 2.39—2.55 (1H, m), 2.72 (1H, s),
2.93 (1H, d, Jꢀ6.5 Hz), 3.41 (1H, dd, Jꢀ6.5, 9.0 Hz), 3.53 (1H, d,
Jꢀ9.5 Hz), 3.55 (1H, dd, Jꢀ3.0, 9.0 Hz), 4.12 (1H, d, Jꢀ12.0 Hz), 4.31 (1H,
d, Jꢀ12.0 Hz), 4.39 (1H, d, Jꢀ12.0 Hz), 4.44 (1H, d, Jꢀ12.0 Hz), 5.12—
5.23 (1H, m), 5.21 (1H, dd, Jꢀ9.5, 15.0 Hz), 5.90 (1H, d, Jꢀ10.5 Hz),
6.18—6.33 (1H, m), 7.16—7.36 (10H, m). HR-MS [FAB, (M)ꢄ] Calcd for
C39H50O4: 582.3709, Found: 582.3679.
Methyl 3-(tert-Butyldimethylsilyloxymethyl)-5-nitrobenzoate (11) To
a solution of methyl 3-(hydroxymethyl)-5-nitrobenzoate (10)16,17) (422 mg,
2.00 mmol) in CH2Cl2 (5.0 ml) were added tert-butyldimethylchlorosilane
(330 mg, 2.19 mmol), triethylamine (340 ml, 2.44 mmol) and N,N-dimethy-
laminopyridine (10.0 ml, 137 mmol), and the mixture was stirred at ambient
temperature for 18 h. The reaction was quenched by adding H2O and the
mixture was extracted with ethyl acetate. The product was purified by silica-
gel column chromatography (hexane/ethyl acetateꢀ7/1) to give methyl
Derivatives of FSA. General 1H-NMR (500 MHz) spectra was
recorded on a JEOL JNM-a500 spectrometer. Mass spectra were obtained
on JEOL JMA-HX 110 spectrometer with m-nitrobenzyl alcohol. Melting
points were determined on Yanaco MP-J3 micro melting point apparatus,
and are uncorrected. Flash column chromatography was performed on silica
gel 60 (Kanto Kagaku, 40—100 mm).
1-(tert-Butyldimethylsilyloxy)-FSA (7) To a solution of FSA (1)7)
(58.6 mg, 0.146 mmol) in CH2Cl2 (0.4 ml) were added tert-butyldimethyl-
chlorosilane (24.0 mg, 0.159 mmol), triethylamine (25.0 ml, 0.179 mmol),
and N,N-dimethylaminopyridine (1.0 mg, 0.008 mmol). The mixture was
stirred at ambient temperature for 23 h, then applied directly to silica-gel
and purified by silica-gel column chromatography (hexane/ethyl acetateꢀ
7/1, then 4/1) to give 1-(tert-butyldimethylsilyloxy)-FSA (7) (64.3 mg,
3-(tert-butyldimethylsilyloxymethyl)-5-nitrobenzoate
(11)
(636 mg,
1
1.96 mmol, 98%) as a white foram; H-NMR (CDCl3) d: 0.12 (6H, s), 0.95
(9H, s), 3.96 (3H, s), 4.84 (2H, s), 8.25—8.31 (1H, m), 8.36—8.42 (1H, m),
8.69—8.75 (1H, m). MS (FAB): m/z 326 (MꢄH)ꢄ.
1
0.124 mmol, 86%) as a pale-yellow oil; H-NMR (DMSO-d6) d: 0.00 (3H,
3-(tert-Butyldimethylsilyloxymethyl)-5-nitrobenzoic Acid (12) To a
solution of 11 (632 mg, 1.94 mmol) in THF (5.0 ml) and H2O (2.5 ml) was
added 2 N aqueous NaOH (1.1 ml, 2.20 mmol) and the mixture was stirred at
ambient temperature for 3.5 h. Evaporation under reduced pressure afforded
an aqueous residue which was acidified with 2 N HCl (ca. pH 2) and ex-
tracted with ethyl acetate. The organic solution was washed with brine, dried
(MgSO4), and concentrated under reduced pressure to give 3-(tert-butyl-
dimethylsilyloxymethyl)-5-nitrobenzoic acid (12) (583 mg, 1.87 mmol, 96%)
as a white solid which was used without further purification; 1H-NMR
(CDCl3) d: 0.14 (6H, s), 0.96 (9H, s), 4.87 (2H, s), 8.35 (1H, s), 8.44 (1H,
s), 8.80 (1H, s).
3-(tert-Butyldimethylsilyloxymethyl)-5-nitrobenzoyl Piperazine (13)
To a solution of 12 (581 mg, 1.87 mmol) in DMF (6.0 ml) was added
1,1ꢂ-carbonylbis-1H-imidazole (366 mg, 2.26 mmol) and the mixture was
stirred at ambient temperature for 40 min. To this was added piperazine
(483 mg, 5.61 mmol) and the mixture was stirred at the same temperature
for 15 h. The reaction was quenched by adding H2O and the mixture was
extracted with ether, dried (MgSO4), and concentrated under reduced
pressure. The product was purified by silica-gel column chromatography
(CH2Cl2/methanolꢀ19/1) to give 3-(tert-butyldimethylsilyloxymethyl)-5-ni-
trobenzoyl piperazine (13) (603 mg, 1.59 mmol, 85%) as a pale-yellow solid;
s), 0.01 (3H, s), 0.69 (3H, d, Jꢀ7.0 Hz), 0.85 (9H, s), 0.92—1.00 (1H, m),
1.11 (3H, s), 1.19—1.28 (1H, m), 1.27 (3H, s), 1.47 (1H, ddd, Jꢀ3.0, 10.5,
14.0 Hz), 1.54—1.75 (6H, m), 1.59 (3H, s), 1.61 (3H, s), 2.02—2.14 (1H,
m), 2.06 (1H, dd, Jꢀ3.0, 14.0 Hz), 2.43—2.52 (1H, m), 2.70 (1H, s), 2.90
(1H, d, Jꢀ5.5 Hz), 3.43 (1H, dd, Jꢀ7.0, 9.5 Hz), 3.61 (1H, dd, Jꢀ4.0,
8.5 Hz), 3.70 (1H, dd, Jꢀ4.0, 9.5 Hz), 4.62 (1H, d, Jꢀ4.0 Hz), 5.13 (1H, dd,
Jꢀ10.5, 15.0 Hz), 5.13—5.20 (1H, m), 5.79 (1H, d, Jꢀ10.5 Hz), 6.13—6.24
(1H, m). MS (FAB): m/z 539 (MꢄNa)ꢄ.
1-(tert-Butyldimethylsilyl)-3-Bn-FSA (8) To a solution of 7 (28.9 mg,
0.056 mmol) in DMF (0.4 ml) was added NaH (55—72% contained, 2.3 mg).
After 10 min, to this was added benzyl bromide (15.0 ml, 0.126 mmol), and
the mixture was stirred at ambient temperature for 16 h. Further NaH (55—
72% contained, 2.3 mg) and benzyl bromide (15.0 ml, 0.126 mmol) were
added and the mixture was stirred at the same temperature for 4 h. The reac-
tion was quenched by adding H2O and the mixture was extracted with
hexane. The product was purified by silica-gel column chromatography
(hexane/ethyl acetateꢀ19/1, then 9/1) to give 1-(tert-butyldimethylsilyloxy)-
3-Bn-FSA (8) (13.1 mg, 0.022 mmol, 39%) as a colorless oil; 1H-NMR
(DMSO-d6) d: ꢅ0.01 (6H, s), 0.70 (3H, d, Jꢀ7.0 Hz), 0.85 (9H, s), 1.01
(1H, dd, Jꢀ11.0, 15.0 Hz), 1.12 (3H, s), 1.20—1.25 (1H, m), 1.26 (3H, s),

304
Vol. 56, No. 3
mp 91—93 °C. 1H-NMR (CDCl3) d: 0.13 (6H, s), 0.95 (9H, s), 2.83 (2H, br
s), 2.97 (2H, br s), 3.38 (2H, br s), 3.78 (2H, br s), 4.83 (2H, s), 7.69 (1H, s),
8.13 (1H, s), 8.23 (1H, s). MS (FAB): m/z 380 (MꢄH)ꢄ.
(5H, m), 1.57 (1H, ddd, Jꢀ3.0, 10.5, 13.5 Hz), 1.65 (3H, s), 1.71 (3H, s),
1.90—1.99 (1H, m), 2.09—2.22 (5H, m), 2.42—2.52 (1H, m), 2.65—2.70
(1H, br), 2.86 (1H, d, Jꢀ6.0 Hz), 3.40—3.90 (14H, br), 3.53 (4H, q,
Jꢀ7.0 Hz), 4.53 (1H, d, Jꢀ12.5 Hz), 4.57 (1H, d, Jꢀ12.5 Hz), 5.16—5.30
(1H, m), 5.21 (1H, dd, Jꢀ10.5, 15.0 Hz), 5.91 (1H, d, Jꢀ10.5 Hz), 6.20—
6.32 (1H, m), 6.52 (1H, s), 6.76 (1H, dd, Jꢀ2.0, 9.0 Hz), 7.06 (1H, s), 7.15
(1H, s), 7.24 (1H, s), 7.47 (1H, d, Jꢀ9.0 Hz), 7.88 (1H, d, Jꢀ2.0 Hz). HR-
MS [FAB, (MꢅH)ꢄ] Calcd for C51H63N6O8: 887.4707, Found: 887.4675.
PhotoAffinity Labeling Soluble cytosolic fraction of HL-60 cells
(0.2 mg/ml, prepared as described above) was incubated with the photoaffin-
ity probe CAFSA (6) (10 mM) in a 0.6 ml Eppendorf tube in the presence or
absence of fusarielin A (FSA) (1 mM). The mixture (final volumeꢀ200 ml)
was photoirradiated with UV light (254 nm, 4 W, UVP UVGL-25) for 5 min
from a distance of 1.7 cm at 25 °C. A part of the mixture (100 ml) was de-
canted onto gel (Sephadex G-25 Medium) and gel filtration was carried out
using Tris–buffer (20 mM Tris–HCl, 600 mM KCl, 5 mM dithiothreitol
(DTT), pH 7.5) as the eluent. The eluates were collected in fractions of 3
drops (about 100 ml) and the fluorescence intensity of each fraction was
measured.
1-[3-(tert-Butyldimethylsilyloxymethyl)-5-nitrobenzoyl]-4-(7-diethyl-
aminocoumarin-3-carbonyl)piperazine (14) To a solution of 7-diethyl-
aminocoumarin-3-carboxylic acid18) (209 mg, 0.800 mmol) in dimethylfor-
mamide (DMF) (4.0 ml) was added 1,1ꢂ-carbonylbis-1H-imidazole (156 mg,
0.960 mmol) and the mixture was stirred at ambient temperature for 40 min.
To this was added 13 (334 mg, 0.880 mmol) and the mixture was stirred at
the same temperature for 22 h. The reaction was quenched by adding H2O
and brine, and the resulting precipitate was collected by filtration, and dried
in vacuo. The product was purified by silica-gel column chromatography
(CH2Cl2/ethyl acetateꢀ5/2) to give 1-[3-(tert-butyldimethylsilyloxymethyl)-
5-nitrobenzoyl]-4-(7-diethylaminocoumarin-3-carbonyl)piperazine
(14)
(488 mg, 0.783 mmol, 98%) as a yellow foam; 1H-NMR (CDCl3) d: 0.12
(6H, s), 0.94 (9H, s), 1.21 (6H, t, Jꢀ7.0 Hz), 3.35—4.00 (8H, br), 3.42 (4H,
q, Jꢀ7.0 Hz), 4.82 (2H, s), 6.45 (1H, br s), 6.59 (1H, d, Jꢀ9.0 Hz), 7.29 (1H,
d, Jꢀ9.0 Hz), 7.68 (1H, s), 7.90 (1H, s), 8.13 (1H, br s), 8.25 (1H, s). MS
(FAB): m/z 623 (MꢄH)ꢄ.
1-[3-Amino-5-(hydroxymethyl)benzoyl]-4-(7-diethylaminocoumarin-
3-carbonyl)piperazine (15) To a solution of 14 (203 mg, 0.326 mmol) in
ethyl acetate (3.0 ml) was added SnCl2·2H2O (210 mg, 0.931 mmol) and the
mixture was refluxed for 7 h. The reaction was quenched by adding saturate
aqueous NaHCO3 solution and the resulting precipitate was removed by fil-
tration through a pad of Celite. The filtrate was extracted with CH2Cl2, dried
(MgSO4), and concentrated under reduced pressure. The crude product was
purified by silica-gel column chromatography (CH2Cl2/methanolꢀ19/1 then
9/1) to give 1-[3-amino-5-(hydroxymethyl)-benzoyl]-4-(7-diethylamino-
coumarin-3-carbonyl)piperazine (15) (153 mg, 0.319 mmol, 98%) as a yel-
low foam; 1H-NMR (CDCl3) d: 1.20 (6H, t, Jꢀ7.0 Hz), 1.84 (1H, br s),
3.25—3.95 (10H, br), 3.41 (4H, q, Jꢀ7.0 Hz), 4.59 (2H, br s), 6.45 (1H, br
s), 6.58 (1H, d, Jꢀ9.0 Hz), 6.59 (1H, s), 6.72 (2H, s), 7.28 (1H, d,
Jꢀ9.0 Hz), 7.87 (1H, s). MS (FAB): m/z 479 (MꢄH)ꢄ.
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methyl)benzoyl]-4-(7-diethylaminocoumarin-3-carbonyl)piperazine
(16)
(304 mg, 0.603 mmol, 93%) as a yellow solid; mp 108—111 °C. 1H-NMR
(CDCl3) d: 1.20 (6H, t, Jꢀ7.0 Hz), 2.01 (1H, br s), 3.20—4.00 (8H, br), 3.41
(4H, q, Jꢀ7.0 Hz), 4.70 (2H, br s), 6.45 (1H, br s), 6.59 (1H, d, Jꢀ9.0 Hz),
6.95 (1H, s), 7.10 (1H, s), 7.13 (1H, s), 7.29 (1H, d, Jꢀ9.0 Hz), 7.88 (1H, s).
MS (FAB): m/z 505 (MꢄH)ꢄ.
1-[3-Azido-5-(bromomethyl)benzoyl]-4-(7-diethylaminocoumarin-3-
carbonyl)piperazine (17) To a solution of 16 (20.0 mg, 0.040 mmol) in
CH2Cl2 (0.5 ml) was added CBr4 (26.5 mg, 0.080 mmol) and tri-n-octylphos-
phine (42.0 mg, 0.113 mmol), and the mixture was stirred for 2 h at the am-
bient temperature. The reaction mixture was directly applied to silica-gel
and the product was purified by silica-gel column chromatography
(CH2Cl2/acetoneꢀ9/1) to give 1-[3-azido-5-(bromomethyl)benzoyl]-4-(7-di-
ethylaminocoumarin-3-carbonyl)piperazine (17) (13.2 mg, 0.023 mmol,
59%) as a yellow foam; 1H-NMR (CDCl3) d: 1.21 (6H, t, Jꢀ7.0 Hz), 3.23—
3.95 (8H, br), 3.42 (4H, q, Jꢀ7.0 Hz), 4.42 (2H, s), 6.45 (1H, br s), 6.59
(1H, dd, Jꢀ1.5, 9.0 Hz), 6.99 (1H, d, Jꢀ1.5 Hz), 7.08 (1H, br s), 7.16 (1H,
s), 7.29 (1H, d, Jꢀ9.0 Hz), 7.89 (1H, s). MS (FAB): m/z 567 (MꢄH)ꢄ.
Fluorescent Photoaffinity Labeling Probe: CAFSA (6) To a solution
of 7 (28.8 mg, 0.056 mmol) in DMF (0.3 ml) was added NaHMDS (0.5 M
tetrahydrofran (THF) solution, 100 ml, 0.050 mmol) at 0 °C and the mixture
was stirred for 1 h at the same temperature. To this was added a solution of
17 (13.2 mg, 0.023 mmol) in DMF (0.2 ml) and the mixture was stirred for
5 h at the same temperature. The reaction was quenched by adding H2O and
the mixture was extracted with CH2Cl2, dried (MgSO4), and concentrated
under reduced pressure. The product was purified by silica-gel column chro-
matography (CH2Cl2/methanolꢀ9/1) to give CAFSA (6) (2.5 mg,
0.003 mmol, 12%) as a yellow oil; 1H-NMR (acetone-d6) d: 0.84 (3H, d,
Jꢀ6.5 Hz), 1.15 (2H, br s), 1.21 (6H, t, Jꢀ7.0 Hz), 1.26 (3H, s), 1.52—1.80
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