Synthesis and characterization of the 7-(4-aminomethyl-1H-1,2,3-triazol-1- yl) analogue of kabiramide C

Article abstract of DOI:10.1021/np049670z

The 7-(4-aminomethyl-1H-1,2,3-triazol-1-yl) analogue of kabiramide C (5) was synthesized by using the Mitsunobu reaction and 1,3-dipolar cycloaddition. This compound and the intermediate compounds 2 and 4 were shown to bind tightly to G-actin in a 1:1 complex and exhibited the same degree of cytotoxicity as 1. Compound 5 serves as a key intermediate for the synthesis of actin-directed optical probes and drugs.

Full text of DOI:10.1021/np049670z

©
Copyright 2005 by the American Chemical Society and the American Society of Pharmacognosy
Volume 68, Number 2
February 2005
Full Papers
Synthesis and Characterization of the 7-(4-Aminomethyl-1H-1,2,3-triazol-1-yl)
Analogue of Kabiramide C
†
,†
‡
§
,§
Chutima Petchprayoon, Khanit Suwanborirux,* Reagan Miller, Tomoyo Sakata, and Gerard Marriott*
Bioactive Marine Natural Products Chemistry Research Unit (BMNCU), Department of Pharmacognosy, Faculty of
Pharmaceutical Sciences, Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand, Department of Chemistry,
University of Wisconsin, 1101 University Avenue, Madison, Wisconsin 53706, and Department of Physiology, University of
Wisconsin, 1300 University Avenue, Madison, Wisconsin 53706
Received October 7, 2004
The 7-(4-aminomethyl-1H-1,2,3-triazol-1-yl) analogue of kabiramide C (5) was synthesized by using the
Mitsunobu reaction and 1,3-dipolar cycloaddition. This compound and the intermediate compounds 2
and 4 were shown to bind tightly to G-actin in a 1:1 complex and exhibited the same degree of cytotoxicity
as 1. Compound 5 serves as a key intermediate for the synthesis of actin-directed optical probes and
drugs.
Actin is a highly conserved and abundant protein that
plays essential roles in cytokinesis, cell motility, and vesicle
transport. Actin exists in a dynamic equilibrium between
a monomeric state (G-actin) and a polymeric state (F-actin).
In physiological salt solutions, actin exists predominantly
as F-actin, with G-actin being maintained only near the
critical concentration of 0.2 µM. This equilibrium is con-
siderably shifted in living cells, where almost half of the
recently we have shown that some actin-targeted macrolide
drugs, the family of compounds typified by kabiramide C
(KabC), function as unregulated biomimetics of actin
filament (+)-end-capping proteins.3 These studies sug-
gested that optical and chemical probes based on KabC
could provide new information on the regulation of actin
filament dynamics in living cells. An analysis of the high-
resolution structure of the KabC-G-actin complex shows
that optical probes linked to the tail region in the KabC
molecule (Figure 1) would prevent the drug from binding
to actin, and indeed, a previous study showed mixed results
for the binding of mycalolide B and kabiramide D deriva-
,4
200 µM actin exists as G-actin. The shift in equilibrium is
brought about by the binding of different actin-binding
proteins found within cells. These binding proteins exert
a range of activities on actin including sequestering of
G-actin, capping of the (+)-end of the actin filament, and
5
tives of biotin. We argue that the 7-hydroxyl group of KabC
1
accelerating filament disassembly. A major challenge in
would be a far more suitable site because it does not engage
in direct contacts with actin and should therefore not
cell biology is to understand how these actin-binding
protein activities regulate actin filament dynamics and cell
motility.
3
interfere with actin binding. However we are unaware of
any studies on the chemistry of the 7-hydroxyl group of
KabC, although the 5-amino and 5-hydroxy groups are
found naturally in other trisoxazole macrolides, such as
Pharmacological drugs that target sites on actin such
as cytochalasin and latrunculin have played an important
2
role in our understanding of actin function in cells. More
4
halishigamide A and jaspisamide A, respectively. As part
of our approach to the design and synthesis of functional
optical probes of KabC, we elected to introduce an amino
group to the 7-position since this would allow a facile route
for the preparation of a myriad of KabC probes using
commercially available activated carboxylic esters.
*
To whom correspondence should be addressed. (K.S.) Tel: (662)
2
188363. Fax: (662) 2545195. E-mail: skhanit@chula.ac.th. (G.M.) Tel:
(
608) 262-6309. Fax: (608) 265-5512. E-mail: GM@physiology.wisc.edu.
†
Chulalongkorn University.
‡
Department of Chemistry, University of Wisconsin-Madison.
§
Department of Physiology, University of Wisconsin-Madison.
1
0.1021/np049670z CCC: $30.25
© 2005 American Chemical Society and American Society of Pharmacognosy
Published on Web 01/14/2005

1
58 Journal of Natural Products, 2005, Vol. 68, No. 2
Petchprayoon et al.
Figure 1. Synthesis of 7-aminokabiramide C (3): (i) HN
THF, NH OH/H O, rt or NaBH , THF/MeOH, rt.
3 3 2 2 3 2 3
, PPh , DIAD, dry THF, rt; (ii) SnCl ‚2H O, MeOH, rt or PPh , THF/H O, rt or PPh ,
4
2
4
Figure 2. Synthesis of 7-(4-aminomethyl-1H-1,2,3-triazol-1-yl)kabiramide C (5): (i) THF, 0 °C f rt; (ii) MeOH/H
piperidine in dry CH Cl , rt.
2 3
O, Et N, CuI, rt; (iii) 20% v/v
2
2
Results and Discussion
KabC were identical with KabC data previously reported
in the literature.6
KabC (1) was isolated as a white amorphous solid from
the sponge Pachastrissa nux collected from Sichang Island,
KabC was converted to 7-azido KabC (2) via the Mit-
1
7
Thailand. The H NMR spectrum showed some character-
sunobu reaction by using hydrazoic acid as nucleophile
istic peaks of this class of compound in the downfield region
such as three proton signals of oxazole rings at δ 8.09 (H-
3
in the presence of triphenylphosphine (PPh ) and diisopro-
pyl azadicarboxylate (DIAD; Figure 1). The reaction pro-
ceeded smoothly using the small molecule hydrazoic acid
but failed using the more conventional reagent diphen-
14), 8.03 (H-17), and 7.57 (H-11) ppm and N-formamide
proton signals at δ 8.27 and 8.06 ppm in the ratio 2:1 due
1
8
to the two geometrical forms. The H NMR data of our
ylphosphoryl azide (DPPA), presumably because of steric

Analogue of Kabiramide C
Journal of Natural Products, 2005, Vol. 68, No. 2 159
varying amounts of 5 (0, 10, 100, and 1000 nM) for 16 h.
Cells treated with 5 at a concentration of 1 µM died within
1
6 h, whereas lower concentrations of drug (10 and 100
nM) caused defects in cytokinesis and loss of cell-cell
contacts. Control cells treated with the same volume of
methanol were mainly mononucleate and healthy (Sup-
porting Information).
Experimental Section
General Experimental Procedures. All chemicals used
in this work were purchased from Sigma-Aldrich Corporation.
Dulbecco’s modified Eagle medium (DMEM), fetal bovine
serum (FBS), and penicillin-streptomycin were purchased from
Invitrogen. Acrylodan was purchased from Molecular Probes.
THF was dried over benzophenone and sodium, and CH
2
Cl
2
was passed through Al (activity I) and dried over CaH
2
O
3
2
before use. Optical rotations were measured on a Perkin-Elmer
41 polarimeter. UV spectra were determined with a Shi-
2
madzu UV-1601PC UV-visible spectrophotometer. IR spectra
were obtained from a Perkin-Elmer 2000 FT-IR spectrometer
1
13
and a Mattson Polaris FT-IR spectrometer. H and C NMR
spectra were recorded at 500 and 125 MHz, respectively, on a
Varian INOVA-500 spectrometer and at 300 and 75 MHz on
a Bruker AC-300 spectrometer using TMS as an internal
standard. Mass spectra were recorded on a Micromass LCT
or Micromass AutoSpec. Fluorescence emission spectra were
recorded on an SLM-Aminco AB2 fluorometer.
Figure 3. (A) Stoichiometric binding of 1 µM Prodan-G-actin in
G-buffer with 5 at final concentrations of 0 µM (a), 0.17 µM (b), 0.33
µM (c), 0.50 µM (d), 0.66 µM (e), 0.83 µM (f), 0.99 µM (g), 1.15 µM (h),
Animal Material. The marine sponge was collected at the
depth of 20-25 feet from Sichang Island, Chon Buri Province,
Thailand, in February 2003. The sponge was identified as
Pachastrissa nux by Dr. John N. A. Hooper of Queensland
Museum, Brisbane, Australia. A voucher specimen (QM
G320223) has been deposited at the museum.
Extraction and Isolation. The sponge (15 kg, wet wt) was
homogenized and exhaustively extracted with MeOH (11 L ×
1
.32 µM (i), 1.48 µM (j), 1.64 µM (k), 1.80 µM (l), and 1.96 µM (m). (B)
Plot of the integrated intensity in (A) against concentration of 5.
hindrance. Attempts to prepare 7-amino KabC (3) from 2
9
by using various methods such as SnCl
2
‚2H
2
O, PPh
3
/THF/
2 3 4
H O, and PPh /THF/NH OH were unsuccessful. We specu-
late that these reactions also failed because of steric
4
). After filtration and concentration, the residue was parti-
hindrance at this position. In addition, reduction of 2 with
tioned with EtOAc. The EtOAc layer was evaporated to obtain
the residue, which was dissolved in MeOH and partitioned
with hexane. The MeOH extract was concentrated to a give
residue (11.47 g), which was further chromatographed on a
9
d
NaBH
4
caused the complete decomposition of 2. These
findings prompted the synthesis of a 7-modified KabC
derivative harboring an amino group at the end of a longer
SiO
a Sephadex LH-20 column (hexane/CHCl
gel flash column (CHCl /MeOH, 25:1), and a preparative HPLC
O, 78:22) to give kabiramide C (1.61
2
gel vacuum column (step-gradient of CHCl
3
and MeOH),
linker group. For this approach, we coupled 2 with 3-ami-
nopropyne using the 1,3-dipolar cycloaddition reaction10 to
3
/MeOH, 2:1:1), a SiO
2
3
create the 1,2,3-triazole derivative. The commercially
available 3-aminopropyne was protected as 3-(fluoren-9-
ylmethoxycarbonyl)aminopropyne (6) by using N-(9H-fluo-
ren-9-ylmethoxycarbonyloxy)succinimide (FmocNHS). Com-
pound 2 was reacted with 6 in the presence of a catalytic
column (ODS, MeOH/H
g, 0.01% w/w, wet wt).
2
Kabiramide C (1): white amorphous solid; [R]23
+8.33°
D
(
c 0.047, CHCl
film) νmax 3455, 2929, 1716, 1653 cm ; H NMR (CDCl
3
); UV (MeOH) λmax (log ꢀ) 247 (4.37) nm; IR
-1
1
(
3
, 300
amount of copper iodide (CuI) and triethylamine (Et
afford 7-[4-N-(9H-fluoren-9-ylmethoxycarbonyl)aminomethyl-
,2,3-triazol-1-yl]-KabC (4). The presence of the catalytic
3
N) to
MHz) δ 8.27 (0.7H, s, NCOH-35), 8.09 (1H, s, H-14), 8.06 (0.3H,
s, NCOH-35), 8.03 (1H, s, H-17), 7.57 (1H, d, J ) 0.53 Hz,
H-11), 7.45 (1H, ddd, J ) 5.78, 9.69, 15.78, H-20), 7.11 (0.3H,
d, J ) 14.60 Hz, H-35), 6.45 (0.7H, d, J ) 14.60 Hz, H-35),
1
amount of Cu(I) salt in 1,3-dipolar cycloaddition of terminal
alkyne to azide not only catalyzed the reaction but also
improved the regioselectivity to give the 1,4-substituted
6
.28 (1H, d, J ) 15.78 Hz, H-19), 5.30 (1H, m, H-3), 5.14 (1H,
m, H-24), 5.09 (1H, m, H-34), 4.79 (1H, br s, H-9), 3.82 (1H,
m, H-7), 3.65 (1H, m, H-22), 3.44 (3H, s, OCH -9), 3.42 (3H, s,
OCH -22), 3.33 (3H, s, OCH -32), 3.31 (3H, s, OCH -26), 3.24
1H, H-32), 3.02 (1H, s, NCH -35), 3.06 (2H, s, NCH -35), 2.98
1H, H-26), 2.90-2.30 (8H), 2.13 (1H, m, H-8), 2.00-1.20
11H), 1.14 (3H, d, J ) 7.21 Hz, CH -33), 0.98 (3H, d, J ) 6.70
1
1
3
1,2,3-triazole. The structure of this compound was con-
3
3
3
firmed by the presence of aromatic proton signals of Fmoc
(
(
(
3
3
1
in the H NMR spectrum. Finally, deprotection of Fmoc
with 20% piperidine in dry CH
2
Cl
2
gave 7-(4-aminomethyl-
3
1
1
H-1,2,3-triazol-1-yl)kabiramide C (5) (Figure 2). The H
Hz, CH -8), 0.92 (3H, d, J ) 6.47 Hz, CH -5), 0.90 (3H, d, J )
3
3
NMR spectrum confirmed the presence of the triazole
6.89 Hz, CH
3
3
-31), 0.86 (3H, d, J ) 6.92 Hz, CH -23), 0.81 (3H,
1
3
proton signal at δ 7.49 and methylene proton signal at δ
3 3
d, J ) 6.59 Hz, CH -27); C NMR (CDCl , 75 MHz) δ 213.79
(
(
(
(
C), 213.70 (C), 171.21 (C), 162.78 (C), 161.86 (CH), 160.59
4
.01 ppm.
CH), 156.96 (C), 156.06 (C), 155.07 (C), 141.62 (CH), 141.03
C), 137.02 (CH), 136.71 (CH), 135.44 (CH), 130.80 (C), 129.59
C), 128.49 (CH), 124.48 (CH), 115.28 (CH), 112.93 (CH),
The binding of compounds 2, 4, and 5 to Prodan-G-actin
was confirmed by measuring the change in the fluorescence
emission spectrum of Prodan-G-actin as a function of drug
concentration. The data shown in Figure 3A,B show that
each KabC derivative was capable of tightly binding
G-actin with the same 1:1 stoichiometry as KabC.3 The
toxicity of 5 on human cervix carcinoma (HeLa) cells was
evaluated by treating the medium bathing HeLa cells with
1
7
5
4
11.16 (CH), 87.22 (CH), 87.14 (CH), 81.86 (CH), 79.03 (CH),
8.24 (CH), 73.84 (CH), 73.08 (CH), 69.13 (CH), 61.24 (CH ),
7.76 (CH ), 57.52 (CH ), 57.37 (CH ), 49.07 (CH), 48.98 (CH),
4.76 (CH ), 43.55 (CH ), 42.83 (CH ), 42.33 (CH ), 42.26 (CH ),
3
3
3
3
-5
2
2
2
2
2
40.41 (CH), 37.58 (CH), 37.52 (CH), 37.38 (CH), 34.49 (CH),
33.88 (CH ), 33.10 (CH ), 32.86 (CH ), 27.59 (CH ), 24.99 (CH),
2
3
2
3

1
60 Journal of Natural Products, 2005, Vol. 68, No. 2
Petchprayoon et al.
2
4.89 (CH
2
), 19.36 (CH
), 8.52 (CH ); ESIMS m/z [M + Na] 964.4866 (calcd
14Na, 964.4895).
-Azidokabiramide C (2). A paste of sodium azide (260
mg, 4 mmol) in H O (260 µL) was stirred on an ice bath, and
3
), 18.40 (CH
3
), 15.56 (CH
3
), 13.58 (CH
3
),
6.52 Hz, CH
3
-33), 1.03 (3H, d, J ) 5.92 Hz, CH
3 3
3
-8), 0.95-0.83
+
13
1
0.88 (CH
3
3
(12H, 4 × CH
); C NMR (CDCl , 75 MHz) δ 214.20 (C), 214.11
for C48
H
71
N
5
O
(C), 171.68 (C), 163.19 (C), 162.13 (CH), 160.85 (CH), 157.09
(C), 156.36 (C), 156.36 (C), 155.48 (C), 144.53 (C), 143.81 (C),
143.81 (C), 141.93 (CH), 141.93 (C), 141.19 (C), 141.19 (C),
137.37 (CH), 136.94 (CH), 135.48 (CH), 130.94 (C), 129.73 (C),
128.70 (CH), 127.63 (CH), 127.63 (CH), 126.97 (CH), 126.97
(CH), 125.03 (CH), 125.03 (CH), 124.68 (CH), 121.27 (CH),
119.91 (CH), 119.91 (CH), 115.63 (CH), 113.06 (CH), 111.28
(CH), 87.33 CH), 87.24 (CH), 81.89 (CH), 80.71 (CH), 79.40
7
2
benzene (1.6 mL) was added. Concentrated sulfuric acid (106
µL, 2 mmol) was carefully added dropwise to the reaction
mixture. After stirring for 10 min, the organic layer containing
hydrazoic acid was separated and dried over anhydrous
MgSO
4 3
. To the mixture of KabC (286 mg, 304 µmol) and PPh
(
158 mg, 602 µmol) in dry THF (3 mL) stirred on an ice bath
(CH), 73.90 (CH), 69.48 (CH), 66.89 (CH
(CH ), 58.09 (CH ), 57.93 (CH ), 57.38 (CH
(CH), 47.11 (CH), 42.59 (CH ), 42.37 (CH ), 41.84 (CH
(CH), 39.89 (CH ), 39.20 (CH), 37.59 (CH), 37.37 (CH), 36.66
2
), 62.23 (CH), 61.32
), 49.12 (CH), 49.03
), 40.24
under a nitrogen atmosphere was added the solution of
hydrazoic acid in benzene (300 µL). After stirring for 15 min,
DIAD (117 µL, 604 µmol) was added dropwise over 1 min to
the reaction mixture. The reaction mixture was allowed to
warm to room temperature and stirred for 4 h. The reaction
mixture was concentrated and purified by a Sephadex LH-20
3
3
3
3
2
2
2
2
(CH
(CH
2
), 34.35 (CH), 33.67 (CH
), 26.15 (CH), 24.72 (CH
2
), 33.08 (CH
3
), 32.28 (CH
), 19.31 (CH
2
), 27.56
), 15.53
3
2
), 20.96 (CH
3
3
(CH
3
), 13.54 (CH
3
), 8.64 (CH ), 6.81 (CH
3
3
); ESIMS m/z [M +
+
column (hexane/CH
TLC (CH
(
λ
2
Cl
2
/MeOH, 2:1:1) and preparative SiO
2
gel
Na] 1266.6033 (calcd for C66H N O15Na, 1266.6063).
85 9
2
Cl
2
/MeOH, 20:1) to give 2 as a white amorphous solid
7-(4-Aminomethyl-1H-1,2,3-triazol-1-yl)kabiramide C
(5). To remove the Fmoc protecting group, compound 4 (97
2
3
119 mg, 40.46%): [R]
D
-6.27° (c 0.047, CHCl
3
); UV (MeOH)
max (log ꢀ) 247 (4.12) nm; IR (CHCl
3
) νmax 3464, 3347, 3166,
mg, 78 µmol) was treated with 20% v/v piperidine in dry CH
Cl
reaction mixture was extracted with CH
2
-
-1 1
3
020-2935, 2104, 1720, 1657 cm ; H NMR (CDCl
3
, 300 MHz)
2
(2 mL) for 20 min. Water (3 mL) was added, and the
Cl
δ 8.27 (0.7H, s, NCOH-35), 8.10 (1H, s, H-14), 8.08 (0.3H, s,
NCOH-35), 8.04 (1H, s, H-17), 7.61 (1H, d, J ) 1.18 Hz, H-11),
2
2
(3 mL × 4). The
organic layers were combined, washed with brine, dried, and
concentrated to give a residue, which was purified by prepara-
7
.51 (1H, ddd, J ) 5.89, 9.99, 16.00, H-20), 7.13 (0.3H, d, J )
4.30 Hz, H-35), 6.46 (0.7H, d, J ) 14.30 Hz, H-35), 6.29 (1H,
1
tive SiO
amorphous solid (25 mg, 30.76%): [R]
CHCl ); UV (MeOH) λmax (log ꢀ) 246 (4.12); IR (CHCl
3
3464, 3352, 3072-2884, 1722, 1655 cm ; H NMR (CDCl , 500
2 2 2
gel TLC (CH Cl /MeOH, 20:3) to give 5 as a white
23
d, J ) 16.00 Hz, H-19), 5.31 (1H, m, H-3), 5.14 (1H, m, H-24),
D
-3.87° (c 0.012,
5
3
2
.10 (1H, m, H-34), 4.42 (1H, br s, H-9), 3.72 (1H, m, H-22),
.52 (3H, s, OCH -9), 3.52-3.44 (1H, H-7), 3.44 (3H, s, OCH
2), 3.34 (3H, s, OCH
3
3
) νmax
-1 1
3
3
-
3
-32), 3.33 (3H, s, OCH
3
-26), 3.34-3.32
MHz) δ 8.29 (0.7H, s, NCOH-35), 8.10 (1H, s, H-14), 8.07 (0.3H,
s, NCOH-35), 8.06 (1H, s, H-17), 7.64 (1H, s, H-11), 7.51 (1H,
m, H-20), 7.49 (s, 1H, triazole H-5), 7.13 (0.3H, d, J ) 14.20
Hz, H-35), 6.46 (0.7H, d, J ) 14.20 Hz, H-35), 6.31 (1H, d, J )
15.41 Hz, H-19), 5.30 (1H, m, H-3), 5.13 (1H, m, H-24), 5.10
(
1H, H-32), 3.08 (1H, s, NCH
3
3
-35), 3.04 (2H, s, NCH -35), 2.99
(
1H, H-26), 2.90-2.20 (8H), 2.00-1.20 (12H), 1.16 (3H, d, J )
7
.07 Hz, CH
3
-33), 1.03 (3H, d, J ) 6.10 Hz, CH
3 3
3
-8), 0.95-0.81
13
(
(
(
(
(
12H, 4 × CH
); C NMR (CDCl , 75 MHz) δ 214.16 (C), 214.07
C), 171.57 (C), 163.13 (C), 162.08(CH), 160.80 (CH), 157.19
C), 156.28 (C), 155.50 (C), 142.35 (C), 142.04 (CH), 137.11
CH), 136.70 (CH), 135.52 (CH), 131.00 (C), 129.80 (C), 128.66
CH), 124.66 (CH), 115.31 (CH), 113.02 (CH), 111.22 (CH),
(1H, m, H-34), 4.48 (1H, br s, H-9), 4.01 (2H, s, NH
(1H, m, H-22), 3.49-3.44 (1H, H-7), 3.43 (3H, s, OCH
3.35 (3H, s, OCH -32), 3.33 (3H, s, OCH -26), 3.35-3.30 (1H,
H-32), 3.15 (3H, s, OCH -9), 3.08 (1H, s, NCH -35), 3.04 (2H,
s, NCH -35), 2.99 (1H, H-26), 2.83-2.30 (8H), 2.10-1.20 (12H),
1.16 (3H, d, J ) 6.49 Hz, CH -33), 1.01 (3H, CH -8), 0.95-
); C NMR (CDCl , 125 MHz) δ 214.25
2
CH
2
), 3.65
3
-22),
3
3
3
3
8
7
5
4
3
3
2
8
7.28 (CH), 87.21 (CH), 82.39 (CH), 81.95 (CH), 78.98 (CH),
3
3.87 (CH), 69.25 (CH), 64.42 (CH), 61.28 (CH
7.77 (CH ), 57.32 (CH ), 49.05 (CH), 48.97 (CH), 42.89 (CH
2.46 (CH ), 42.34 (CH ), 42.26 (CH ), 40.36 (CH), 39.51 (CH
8.56 (CH), 37.54 (CH), 37.35 (CH), 34.47 (CH), 34.42 (CH),
3.65 (CH ), 33.03 (CH ), 32.81 (CH ), 27.51 (CH ), 25.56 (CH),
4.81 (CH ), 20.92 (CH ), 19.27 (CH ), 15.45 (CH ), 13.48 (CH ),
.43 (CH ), 6.23 (CH ); ESIMS m/z [M + Na] 989.4691 (calcd
13Na, 989.4690).
3
), 58.50 (CH
3
),
2
3
3
1
3
3
3
),
0.83 (12H, 4 × CH
3
3
2
2
2
2
),
(C), 214.15 (C), 171.67 (C), 163.26 (C), 162.16 (CH), 160.87
(CH), 157.14 (C), 156.44 (C), 155.52 (C), 142.28 (C), 142.28 (C),
141.82 (CH), 137.36 (CH), 136.98 (CH), 135.44 (CH), 131.11
(C), 129.86 (C), 128.75 (CH), 124.74 (CH), 120.33 (CH), 115.70
(CH), 113.11 (CH), 111.32 (CH), 87.38 (CH), 87.28 (CH), 81.96
(CH), 80.81 (CH), 79.48 (CH), 73.94 (CH), 69.52 (CH), 62.19
2
3
2
3
2
3
3
3
3
+
3
3
for C48
70 8
H N O
7
-[4-N-(9H-Fluoren-9-ylmethoxycarbonyl)aminomethyl-
(CH), 61.36 (CH
(CH), 49.07 (CH), 42.64 (CH
(CH ), 40.38 (CH), 40.12 (CH
(CH), 34.41 (CH), 33.77 (CH
(CH ), 26.46 (CH), 24.77 (CH
(CH ), 13.57 (CH ), 8.44 (CH
H] 1022.5574 (calcd for C51
3
), 58.29 (CH
3
), 57.97 (CH
3 3
), 57.43 (CH ), 49.16
1
,2,3-triazol-1-yl]kabiramide C (4). Compound 2 (119 mg,
23 µmol) was dissolved in a small volume of MeOH followed
2
), 42.43 (CH
2
), 42.38 (CH
2
), 41.97
1
2
2
), 39.58 (CH), 37.43 (CH), 37.32
by water (6 mL). To the suspension of 2 were added compound
2
), 33.12 (CH
3
), 32.40 (CH
2
), 27.60
6
(51 mg, 184 µmol), Et
3
N (35 µL, 251 µmol), and Cu(I)I (3
3
2
), 21.72 (CH
3
), 19.35 (CH
); ESIMS m/z [M +
76 9
H N O13, 1022.5562).
3
), 15.56
mg, 16 µmol). The reaction mixture was stirred at room
temperature for 1 h and then extracted with EtOAc (6 mL ×
3
3
3 3
), 6.97 (CH
+
4
). The combined extracts were washed with brine, dried, and
3-(Fluoren-9-ylmethoxycarbonyl)aminopropyne (6). A
suspension of N-(9H-fluoren-9-ylmethoxycarbonyloxy)succin-
imide (168 mg, 0.5 mmol) in dry THF was cooled with an ice
bath, and 3-aminopropyne (propargylamine, 35 µL, 0.55 mmol)
was added dropwise. The reaction mixture was stirred and
allowed to warm to room temperature over 2 h. The solvent
was removed under reduced pressure to give a residue. The
residue was dissolved in EtOAc and washed with water. The
organic layer was dried and concentrated. Recrystallization
from EtOAc gave 6 as white needles (102 mg, 73.65%): UV
(MeOH) λmax (log ꢀ) 266 (4.24), 290 (3.65), 300 (3.74) nm; IR
concentrated to give a crude product, which was purified by
preparative SiO gel TLC (CH Cl /MeOH, 20:1) to give 4 as a
white amorphous solid (97 mg, 63.41%): [R]
CHCl ); UV (MeOH) λmax (log ꢀ) 255 (4.49), 299 (3.82) nm; IR
CHCl ) νmax 3453, 3348, 3072, 3006-2883, 1721, 1655, 1513
2
2
2
23
D
+3.06° (c 0.032,
3
(
3
1 1
-
3
cm ; H NMR (CDCl , 300 MHz) δ 8.29 (0.7H, s, NCOH-35),
8
7
7
.07 (1H, s, H-14), 8.07 (0.3H, s, NCOH-35), 8.03 (1H, s, H-17),
.76 (2H, d, J ) 7.16 Hz, Fmoc H-4 and H-5), 7.58 (2H, d, J )
.16 Hz, Fmoc H-1 and H-8), 7.57 (1H, s, H-11), 7.53 (1H, m,
H-20), 7.41 (1H, s, triazole H-5), 7.40 (2H, t, J ) 7.16 Hz, Fmoc
H-3 and H-6), 7.30 (2H, t, J ) 7.16 Hz, Fmoc H-2 and H-7),
-1 1
(CHCl ) νmax 3453, 3307, 3066-2954, 1725, 1510 cm ; H NMR
3
(CDCl , 300 MHz) δ 7.77 (2H, d, J ) 7.42 Hz, Fmoc H-4 and
3
7
.13 (0.3H, d, J ) 14.58 Hz, H-35), 6.46 (0.7H, d, J ) 14.58
Hz, H-35), 6.29 (1H, d, J ) 16.27 Hz, H-19), 5.47 (1H, m, NH),
H-5), 7.59 (2H, d, J ) 7.42 Hz, Fmoc H-1 and H-8), 7.41 (2H,
dt, J ) 0.83, 7.42 Hz, Fmoc H-3 and H-6), 7.32 (2H, dt, J )
1.31, 7.42 Hz, Fmoc H-2 and H-7), 4.95 (1H, NH), 4.44 (2H, d,
5
.31 (1H, m, H-3), 5.14 (1H, m, H-24), 5.12 (1H, m, H-34), 4.53
3H, H-9 and NHCH ), 4.40 (2H, d, J ) 7.15 Hz, Fmoc CH ),
.22 (1H, t, J ) 7.15 Hz, Fmoc H-9), 3.71 (1H, m, H-22), 3.49-
.43 (1H, H-7), 3.43 (3H, s, OCH -22), 3.34 (3H, s, OCH -32),
.32 (3H, s, OCH -26), 3.34-3.32 (1H, H-32), 3.09 (3H, s,
-9), 3.08 (1H, s, NCH -35), 3.04 (2H, s, NCH -35), 3.00
1H, H-26), 2.83-2.30 (8H), 2.15-1.20 (12H), 1.16 (3H, d, J )
(
2
2
4
3
3
J ) 6.69 Hz, Fmoc CH
2
), 4.23 (1H, t, J ) 6.69 Hz, Fmoc H-9),
3
3
4.01 (2H, dd, J ) 2.37, 5.39 Hz, H-3), 2.26 (1H, t, J ) 2.37 Hz,
1
3
3
H-1); C NMR (CDCl , 75 MHz) δ 155.86 (C), 143.70 (C),
3
OCH
(
3
3
3
143.70 (C), 141.22 (C), 141.22 (C), 127.66 (CH), 127.66 (CH),
126.99 (CH), 126.99 (CH), 124.95 (CH), 124.95 (CH), 119.93

Analogue of Kabiramide C
Journal of Natural Products, 2005, Vol. 68, No. 2 161
References and Notes
(
CH), 119.93 (CH), 79.59 (C), 71.62 (CH), 66.97 (CH
2
), 47.03
+
(
CH), 30.75 (CH
, 277.1103).
G-Actin Binding Assay. G-Actin was prepared from rabbit
2
); EIMS m/z [M] 277.1114 (calcd for C18H -
15
(
1) Pollard, T. D. Nature 2003, 422, 741-745.
NO
2
(2) Forscher, P.; Smith, S. J. J. Cell Biol. 1988, 107, 1505-1516.
(3) Klenchin, V. A.; Allingham, J. S.; King, R.; Tanaka, J.; Marriott, G.;
Rayment, I. Nat. Struct. Biol. 2003, 10, 1058-1063.
1
2
muscle as described by Spudich and Watt. Actin was labeled
with the thiol probe, Acrylodan, as described by Marriott et
al. For the G-actin binding study, Prodan-G-actin was
(
4) Tanaka, J.; Yan, Y.; Choi, J.; Bai, J.; Klenchin, V. A.; Rayment, I.;
Marriott, G. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 13851-
13856.
1
3
14
(5) Wada, S.; Matsunaga, S.; Saito, S.; Fusetani, N.; Watanabe, S. J.
Biochem. 1998, 123, 946-952.
diluted to 1 µM in G-buffer and then titrated with increasing
amounts of the test compound from 0.17 to 2 µM. Fluorescence
emission spectra were conducted by exciting Prodan at 385
nm and recording the emission between 400 and 650 nm.
Cell Culture. HeLa cells were cultured in DMEM supple-
mented with 10% v/v heat-activated FBS, 1% v/v penicillin
(
6) (a) Matsunaga, S.; Fusetani, N.; Hashimoto, K. J. Am. Chem. Soc.
1
986, 108, 847-849. (b) Matsunaga, S.; Fusetani, N.; Hashimoto, K.;
Koseki, K.; Noma, M.; Noguchi, H.; Sankawa, U. J. Org. Chem. 1989,
4, 1360-1363.
7) (a) Mitsunobu, O. Synthesis 1981, 1, 1-28. (b) Ko, S. Y. J. Org. Chem.
5
(
2
002, 67, 2689-2691. (c) Stachel, S. J.; Chappell, M. D.; Lee, C. B.;
(
10 000 unit/mL), and streptomycin (10 mg/mL) at 37 °C in a
Danishefsky, S. J.; Chou, T.; He, L.; Horwitz, S. B. Org. Lett. 2000,
2, 1637-1639.
humidified atmosphere containing 5% CO . Compound 5 was
2
(
8) Jeong, E. J.; Kang, E. J.; Sung, L. T.; Hong, S. K.; Lee, E. J. Am.
Chem. Soc. 2002, 124, 14655-14662.
dissolved in MeOH and diluted to 10, 100, and 1000 nM in
culture medium immediately prior to use. The final concentra-
tion of MeOH in this experiment was 0.1% v/v and was
nontoxic to the cells. Phase contrast images of control and
drug-treated cells were recorded after 16 h.
(
9) (a) Felpin, F.; Girard, S.; Vo-Thanh, G.; Robins, R. J.; Villi e´ ras, J.;
Lebreton, J. J. Org. Chem. 2001, 66, 6305-6312. (b) Makabe, H.;
Kong, L. K.; Hirota, M. Org. Lett. 2003, 5, 27-29. (c) DeNinno, M.
P.; Masamune, H.; Chenard, L. K.; DiRico, K. J.; Eller, C.; Etienne,
J. B.; Tickner, J. E.; Kennedy, S. P.; Knight, D. R.; Kong, J.; Oleynek,
J. J.; Tracey, W. R.; Hill, R. J. J. Med. Chem. 2003, 46, 353-355.
Acknowledgment. This research was financially sup-
ported by the Thailand Research Fund for the 2001 Golden
Jubilee Ph.D. Program Scholarship (PHD/0115/2544) and set
up funds from the UW-Madison provided to G.M. The NSF
(
d) Fringuelli, F.; Pizzo, F.; Vaccaro, L. Synthesis 2000, 5, 646-
650.
10) (a) Horne, W. S.; Stout, C. D.; Ghadiri, M. R. J. Am. Chem. Soc. 2003,
25, 9372-9376.
(
(
1
11) (a) Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002,
67, 3057-3064. (b) Rostovsev, V. V.; Green, L. G.; Fokin, V. V.;
Sharpless, K. B. Angew. Chem., Int. Ed. 2002, 41, 2596-2599. (c)
Wang, Z.; Qin, H. Chem. Commun. 2003, 19, 2450-2451.
(
0
CHE-9208463 and CHE-9629688) and NIH (1 S10 RR0 8389-
1) are greatly acknowledged for NMR spectrometry instru-
mentation. BMNCU was also supported by a grant for Center
of Excellence from Chulalongkorn University.
(
12) Spudich, J. A.; Watt, S. J. Biol. Chem. 1971, 246, 4866-4871.
(13) Marriott, G.; Zechel, K.; Jovin T. M. Biochemistry 1988, 27, 6214-
6
220.
Supporting Information Available: 1H NMR spectra of com-
pounds 1, 2, 4, and 5. Images of cells treated with varying amounts of
compound 5 are also shown. These materials are available free of
charge via the Internet at http://pubs.acs.org.
(14) G-buffer contains 5 mM Tris, 0.1 mM CaCl
2
, 0.2 mM ATP, and 1 mM
dithiothreitol (DTT).
NP049670Z