Structure-activity relationships study of bastadin 6, an anti-angiogenic brominated-tyrosine derived metabolite from marine sponge

Article abstract of DOI:10.1002/ardp.200700231

A structure-activity relationship (SAR) study of bastadin 6 (1), a brominated tyrosine-derived metabolite from Indonesian marine sponge having a potent anti-angiogenic activity, was executed. The syntheses and their biological evaluation of the oxime-modified analogues and bromine-modified analogues revealed that both the oxime moieties and bromine atoms in bastadin 6 (1) play an important role to show the potent and selective anti-proliferative activity against human umbilical vein endothelial cells (HUVECs).

Full text of DOI:10.1002/ardp.200700231

568
Arch. Pharm. Chem. Life Sci. 2008, 341, 568–577
Full Paper
Structure-Activity Relationships Study of Bastadin 6, an Anti-
Angiogenic Brominated-Tyrosine Derived Metabolite from
Marine Sponge
Naoyuki Kotoku, Atsushi Hiramatsu, Hiroaki Tsujita, Yoichi Hirakawa, Mami Sanagawa,
Shunji Aoki, and Motomasa Kobayashi
Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan
A structure-activity relationship (SAR) study of bastadin 6 (1), a brominated tyrosine-derived
metabolite from Indonesian marine sponge having a potent anti-angiogenic activity, was exe-
cuted. The syntheses and their biological evaluation of the oxime-modified analogues and bro-
mine-modified analogues revealed that both the oxime moieties and bromine atoms in bastadin
6 (1) play an important role to show the potent and selective anti-proliferative activity against
human umbilical vein endothelial cells (HUVECs).
Keywords: Angiogenesis / Bastadin / Marine sponge / Structure-activity relationship /
Received: November 12, 2007; accepted: February 1, 2008
DOI 10.1002/ardp.200700231
Introduction
Angiogenesis, a formation of new blood capillaries from
preexisting blood vessels, is critical for tumor growth
and metastasis. A growing tumor needs an extensive net-
work of capillaries to provide nutrients and oxygen, etc.
In addition, the new blood vessels provide a way for
tumor cells to enter in the circulation and to metastasize
to another organ. Therefore, substances that inhibit the
angiogenesis have a considerable potential to be novel
therapeutic agents for the treatment of cancer [1].
In the course of our study on the bioactive substances
from marine organisms, we focused on a search for anti-
angiogenic substances and isolated bastadins [2], a family
Figure 1. Chemical structure of bastadin 6 (1).
of brominated tyrosine-derived metabolites, from the
Indonesian marine sponge of lanthella basta. We found
that bastadin 6 (1, Fig. 1), a major constituent, showed an anti-angiogenic activity in vitro and in vivo, through the
induction of selective apoptosis to endothelial cells [3].
For a further mechanistic study of the anti-angiogenic
effect and a chemical study from the viewpoint of medici-
nal chemistry, we have established a practical synthetic
method of bastadin 6 (1) through the novel Ce(IV)-medi-
ated oxidative coupling of 2,6-dibromophenol derivatives
[4].
Correspondence: Motomasa Kobayashi, Graduate School of Pharma-
ceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-
0871, Japan.
E-mail: kobayasi@phs.osaka-u.ac.jp
Fax: +81 6 6879-8219
Abbreviations: benzyloxycarbonyl (Cbz); diammonium cerium (IV) ni-
trate (CAN); human umbilical vein endothelial cells (HUVECs); structure-
activity relationship (SAR)
More than 20 bastadins have been isolated so far, and
some of them are known to show some interesting bio-
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Arch. Pharm. Chem. Life Sci. 2008, 341, 568–577
Structure-Activity Relationships Study of Bastadin 6
569
logical activities, such as antibacterial [2] and cytotoxic
activities [2], inhibition of inositol-59-phosphate dehydro-
genase [2] and lipoxygenases [5], and interaction with
intracellular ryanodine receptor-1 (RyR-1) calcium chan-
nel complex [2]. In spite of their characteristic chemical
structures and interesting biological activities, there are
few structure-activity relationship (SAR) studies so far.
Thus, we started a SAR study of bastadin 6 (1), in order to
analyze the structural requirements for the anti-angio-
genic activity of 1 and to develop a promising drug lead
for the treatment of cancer. In this paper, we present the
SAR study of bastadin 6 (1) about participation of the
oxime moiety and bromine atom to the anti-angiogenic
activity, through the synthesis of some analogues and
the evaluation of their antiproliferative activity against
human umbilical vein endothelial cells (HUVECs).
Figure 2. Retrosynthesis of oxime-modified analogues.
Results and discussion
Synthesis of oxime-modified analogues
Bastadin 6 (1) has two oxime moieties in the right-half
segment. Firstly, we investigated the synthesis of oxime-
modified analogues of 1 for analyzing the participation
of the oxime moieties in the anti-angiogenic activity of
the compound. Figure 2 shows our synthetic strategy of
the oxime-modified analogues. Following the strategy of
our total synthesis of 1 [4], the macrocyclic structure was
divided into two diaryl ether derivatives, i.e. segments A
2 and B 3. Various oxime-modified analogues could be
efficiently prepared by changing the R group in the seg-
ment B 3. Ce(IV)-mediated oxidative coupling of the corre-
sponding 2,6-dibromophenols would lead to these seg-
ments.
Scheme 1. Synthesis of oxime-modified segment B 3a–e.
The synthesis of the oxime-modified segment B 3a–e is
depicted in Scheme 1. The benzyl ether 3a and methyl
ether 3b derivatives as the right-half segment have
already been in our hand, as the synthetic intermediate
in the total synthesis of 1 [4]. In order to know the partic-
ipation of the oxime-hydroxyl group in 1, the benzyl
ether 3a and methyl ether 3b derivatives were utilized.
An oxime-lacking analogue 3c was synthesized from com-
mercially available 3-(4-hydroxyphenyl)propionic acid in
two steps, i.e. bromination and a subsequent oxidative
coupling reaction of 2,6-dibromophenol 4c [6] with di-
ammonium cerium (IV) nitrate (CAN); though the reac-
tion yield was not so good. Tyrosine-type analogues 4d,
Scheme 2. Synthesis of oxime-modified analogues 8a–g.
ether derivative 3d in moderate yield. In order to clarify
the participation of the steric direction of the amino
4e, having amino groups in place of the oxime moieties, group, D-tyrosine-derived analogue 3e was also prepared
were also prepared. The amino group of 3,5-dibromo-L-
tyrosine was protected by the benzyloxycarbonyl (Cbz)
group to give a coupling precursor 4d [6], and subsequent
in the same way.
Using these segments 3a–3e in our hand, the oxime-
modified analogues of bastadin 6 (1) were synthesized as
treatment with CAN in aqueous CH3CN afforded a diaryl follows (Scheme 2). Condensation with the segment A 2
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Arch. Pharm. Chem. Life Sci. 2008, 341, 568–577
stantial amount of lipophilicity is added, with a clogP
value of 0.863. The overall clogP of 1 is 11.64, which is too
high to be a drug candidate with oral availability, accord-
ing to a Lipinski rule [7]. Our previous SAR study among
naturally occurring bastadins showed that bastadin 5, a
mono-debrominated analogue of 1, exhibited almost sim-
ilar biological activity as 1 [8]. In order to find out how
many bromine atoms are necessary for the anti-angio-
genic activity, in addition, some debrominated ana-
logues were synthesized.
Hydrogenolysis of 1 using Pd-C in the presence of
NaOAc for 30 min gave a mixture of partially debromi-
nated derivatives, and four compounds 9a–d were
Scheme 3. Synthesis of debrominated analogues 9a–d.
obtained in pure form after reversed-phase HPLC separa-
1
[4] using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide tion. From the mass spectrometry and H-NMR analysis,
hydrochloride (EDCI N HCl) and 1-hydroxybenzotriazole the number of the bromine atoms in 9a–d was clarified
(HOBt) gave the amides 5a–e. Reduction of the spirodie- to be 4, 2, 1, and 0, respectively (Scheme 3). However, the
position of the bromine atom(s) in 9a–c could not be
acids 6a–e. Removal of the tert-butoxycarbonyl (Boc) assigned due to the complexity of the NMR spectra.
groups in 6a–e using trifluoroacetic acid (TFA) and subse-
Next, we designed some analogues replacing bromine
none part in 5a–e with Na2S2O4 yielded the carboxylic
quent treatment with HCl/Et2O afforded the HCl salts of atom(s) with polar substituents, aiming to increase the
amines 7a–e, and then, intramolecular macrolactam for- aqueous solubility. As the scaffold, we found that the two
mation was accomplished by the EDCI N HCl and HOBt bromine atoms on the ortho-position of the phenolic
treatment to give the objective oxime-modified ana- hydroxyl group in bastadin 6 (1) were selectively replaced
with nitro groups, by treatment with NaNO2 [9], to give a
removed by the treatment with HBr-AcOH to give tyro- nitro analogue 10. Subsequent reduction of the nitro
logues 8a–e. The Cbz groups in 8d and 8e were further
sine-type analogues 8f and 8g, respectively.
groups in 10 with Na2S2O4 proceeded smoothly to give a
NH2-analogue 11 in good yield. The resulting amino
groups in 11 were further modified to more polar substi-
Synthesis of bromine-modified analogues
Bastadin 6 (1) has six bromine atoms in its molecule. A tution. Thus, coupling of 11 and 2-(4-morpholino)ethyl
halogen residue is known to enhance lipophilicity. When isothiocyanate 14 afforded a thiourea analogue 12, and
a hydrogen atom is replaced by a bromine atom, a sub- treatment of 11 with isothiourea 15 in the presence of
Scheme 4. Synthesis of bromine-
modified analogues 11–13.
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Structure-Activity Relationships Study of Bastadin 6
571
Table 1. Growth inhibitory activity of bastadin analogues.
Hg(II) salt and subsequent deprotection of the Boc groups
with TFA gave a guanidine analogue 13 (Scheme 4).
Analogue
IC50 (lM)
S. I.a)
Biological evaluation of synthetic analogues and
discussion
HUVEC
0.052
KB3-1
1
8a
8b
8c
2.1
A100
A100
6.2
3.0
16
A100
4.3
17
5.0
41
–
–
1.6
3.0
26.2
–
27
35
3
The anti-proliferative effect of the synthetic analogues
against endothelial cells (HUVECs) and KB3-1 cells were
evaluated, as summarized in Table 1. Judging from the
anti-proliferative activity against HUVECs and the selec-
tiveindexbetweenHUVECsandKBcells, theoximemoiety
was found to be an essential function for the anti-angio-
genic activity of bastadin 6 (1) and might form hydrogen
bond(s) between its target protein. Thus, the protection of
the oxime moieties (in the case of analogues 8a and 8b)
resultedinthecompletelossofitsactivity, regardlessofits
bulkiness. In the case of the oxime-lacking analogue 8c,
the anti-proliferative activity against HUVECs dropped
dramatically and no selectivity was observed. The tyro-
sine-type analogues of both L- and D-form (8f and 8g)
showed a less than 10-fold reduced activity against
HUVECs compared to that of 1, although the D-tyrosine-
derivedanalogue8gshowedagoodselectiveindex.
Modification on the bromine atom(s) also affected the
activity of 1. The analogues having more than two bro-
mine atoms on their molecule, 9a or 9b, retained their
selective anti-proliferative activity and selective index,
although less potency than for 1 was observed. On the
contrary, the analogues 9c and 9d, having less than one
bromine atom, showed dramatically reduced activity
and selectivity.
A100
A100
4.0
1.0
0.61
8f
8g
8d, e
9a
9b
9c
9d
11
12
13
A100
0.16
0.48
1.5
2.6
0.96
8.7
3.3
A100
26
3
3.4
–
A100
5.8
4.5
a)
S. I. = Selective index: IC50 against KB3-1 cells / IC50 against
HUVECs
In order to elucidate the influence of the chemical
modification on the conformation of bastadin 6 (1), con-
formational analysis of some representative analogues
synthesized here was executed using the Monte Carlo
Multiple Minimum (MCMM) algorithm and employing
MacroModel ver. 9.0. The conformational search was car-
ried out with 1000 MC steps, and the energy minimiza-
tion was performed using the MMFF force field with
water as the solvent.
In the case of analogue 11, substitution of the two bro-
mine atoms on the neighbor of the phenolic hydroxyl
groups by amino groups resulted in the reduction of anti-
proliferative activity against HUVECs. The introduction
of the thiourea moiety (analogue 12) caused the complete
loss of its anti-proliferative activity.
Firstly, the relationship between the number of bro-
mine atom(s) on the molecule and its conformation was
analyzed. The imaginary analogue 9b9 having two bro-
mine atoms and the non-bromo derivative 9d were
selected as representatives, and those conformations
were compared with that of bastadin 6 (1). The conforma-
tional search for those compounds, 9b9, 9d, 1, resulted in
33, 21, and 18 unique conformations, respectively, out of
the 1000 conformers processed, with a global minimum
energy from 742.8 to 757.4 kJ/mol, 680.4 to 695.4 kJ/mol,
and 858.9 to 872.8 kJ/mol, respectively. The calculation
showed that the number of bromine atoms was closely
related to the conformation of the molecules. Thus, the
average values of pairwise RMSD (root mean sqare devia-
tion) for all the heavy atoms among the unique confor-
mations for these three compounds were 1.81 l 0.81 ꢀ,
2.11 l 0.93 ꢀ, 1.20 l 0.49 ꢀ, respectively. The superim-
posed views of the ten lowest energy conformations for
these compounds showed that the reduced number of
bromine atoms resulted in dispersed conformation
(Fig. 3). These results imply that bromine atom(s) have an
important role in maintaining the rigid conformation of
Recently, we reported the comparative analysis of the
anti-proliferative activity against HUVECs between basta-
din 6 (1) and the isolated related compounds [8]. These
bastadins have been divided into two groups, so-called
bastarane and isobastarane types, based on the difference
in the diphenyl-ether linkage of the left-half segment. We
found that bastarane-type bastadin (e.g. bastadin 6 (1))
showed potent anti-proliferative activity against
HUVECs, while isobastarane-type bastadins like bastadin
13 or 19 showed weaker activity. Conformational search
of those bastadins revealed that isobastarane-type com-
pounds had some flexibility in their conformation,
whereas bastarane skeleton had a relatively rigid confor-
mation [8]. Thus, the macrocyclic structure of bastadins
and their conformational rigidity would be the impor-
tant factor for exhibiting potent anti-angiogenic activity.
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N. Kotoku et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 568–577
Figure 3. The superimposed views of the ten lowest energy conformations for bastadin 6 (1), 2-Br analogue 9b9, 0-Br analogue 9d,
and the NH2 analogue 11.
bastadin 6 (1) suitable for exhibiting potent and selective clic structure to some extent, but it cannot be fully
anti-angiogenic activity.
applied to all the case.
Conformational rigidity is, however, not the only one
In summary, the SAR study of bastadin 6 (1) was exe-
requirement for the anti-angiogenic activity of bastadins. cuted through the synthesis of some analogues and the
For example, the NH2-analogue 11, which exhibited the evaluation of their anti-proliferative activities against
reduced anti-proliferative activity against HUVECs with endothelial cells. It revealed that both the oxime moiety
no selectivity, also showed good convergence among the and bromine atoms are needed for the potent anti-prolif-
ten lowest energy conformations (Fig. 3). Thus, the con- erative activity of 1. Recently, Molinski et al. reported a
formational search for the analogue 11 resulted in 16 SAR study of bastadin 5 for modulation of the RyR1/
unique conformers with a total energy of 821.4 to FKBP12 Ca2+ channel complex [10]. They revealed that a
835.3 kJ/mol, and the average RMSD value for all unique simplified 18-membered cyclic analogue acted as a
conformations was 1.17 l 0.59 ꢀ. These results indicate potent antagonist of the RyR1/FKBP12 receptor, and con-
that the potent anti-angiogenic activity of bastadin 6 (1) cluded that the essential structural element for its bind-
is related to its conformational property of the macrocy- ing activity is not the oxime moiety but the bromocate-
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Arch. Pharm. Chem. Life Sci. 2008, 341, 568–577
Structure-Activity Relationships Study of Bastadin 6
573
2.0 Hz), 6.24 (1H, d, J = 2.0 Hz), 6.18 (1H, d, J = 2.5 Hz), 6.11 (1H, t,
J = 7.0 Hz), 4.02 (3H, s), 3.96 (3H, s), 3.83 (2H, s), 3.43 (1H, d, J =
20.0 Hz), 3.38 (2H, q, J = 7.0 Hz), 3.37 (2H, q, J = 7.0 Hz), 3.19 (1H,
d, J = 20.0 Hz), 2.86 (2H, t, J = 7.0 Hz), 2.65 (2H, t, J = 7.0 Hz), 1.37
(9H, s). 13C-NMR (125 MHz, acetone-d6) d: 172.5, 163.6, 162.8,
156.6, 151.5, 148.3 (2C), 147.6, 146.8, 146.1, 145.7, 143.2, 141.9,
138.9, 134.8 (2C), 134.6 (2C), 132.4, 127.5, 124.2, 120.1, 118.3
(2C), 117.5 (2C), 113.9, 110.8, 79.1, 78.7, 64.3, 63.6, 42.1, 41.5,
35.6, 35.2, 34.8, 32.3, 28.7 (3C).
chol ether unit. On the contrary, our results strongly sug-
gested that the oxime moiety plays a crucial role for the
potent anti-angiogenic activity of bastadins, indicating
that the target molecule of bastadin 6 (1) should not be
the RyR1/FKBP12 receptor. The mechanism of action of
compound 1 is currently under investigation.
This study was financially supported by Grant-in-Aid for scien-
tific research from the Ministry of Education, Science, Sport,
and Culture of Japan. The authors are also grateful to the
Takeda Science Foundation and the Tokyo Biochemical
Research Foundation for financial support.
3-[3-Bromo-5-(2,6-dibromo-4-{2-[2-(3-bromo-5-{2,6-
dibromo-4-[2-(tert-butoxycarbonylamino)ethyl]phenoxy}-
4-hydroxyphenyl)ethylcarbamoyl]-2-
methoxyiminoethyl}phenoxy)-4-hydroxyphenyl]-2-
methoxyiminopropionic acid 6b
The authors have declared no conflict of interest.
Na2S2O4 (44 mg, 0.25 mmol) in H2O (3 mL) was added to a solu-
tion of 5b (104 mg, 0.084 mmol) in CH3CN (6.0 mL), and the
whole mixture was stirred for 10 min at 08C. Brine was added to
the mixture and the whole mixture was extracted with AcOEt.
The AcOEt extract was dried over Na2SO4, and the solvent was
removed from the AcOEt extract under reduced pressure to give
6b (104 mg, 99%) as white powder. ESI-Q-TOF MS: m/z 1261, 1263,
1265, 1267, 1269, 1271, 1273 [M + Na]+. HR-ESI-Q-TOF MS: m/z
1266.7630, calcd for C41H4079Br381Br3N4O11Na, found: 1266.7632.
IR mmax (KBr) cm– 1: 3277, 2978, 1696, 1669, 1589, 1283, 1252. 1H-
NMR (500 MHz, DMSO-d6) d: 10.06 (1H, s), 9.92 (1H, s), 8.17 (1H, t, J
= 6.5 Hz), 7.61 (2H, s), 7.55 (2H, s), 7.04 (1H, d, J = 1.5 Hz), 6.96 (1H,
d, J = 1.5 Hz), 6.91 (1H, t, J = 6.5 Hz), 6.13 (1H, d, J = 1.5 Hz), 6.06
(1H, d, J = 1.5 Hz), 3.97 (3H, s), 3.81 (2H, s), 3.70 (3H, s), 3.49 (2H, s),
3.18 (4H, t-like, J = 6.5 Hz), 2.72 (2H, t, J = 6.5 Hz), 2.53 (2H, t, J =
6.5 Hz), 1.33 (9H, s). 13C-NMR (125 MHz, acetone-d6) d: 164.2,
162.8, 156.6, 151.6, 150.6, 147.8, 147.5, 145.6, 145.4, 143.4,
143.2, 141.8, 138.5, 134.6 (2C), 134.5 (2C), 132.3, 129.1, 127.7,
127.4, 118.4 (2C), 118.3 (2C), 114.5, 113.8, 110.7, 110.4, 78.7, 63.5,
63.4, 41.9, 41.4, 35.6, 35.1, 30.5, 30.2, 28.6 (3C).
Experimental
The following instruments were used to obtain physical data: a
JASCO DIP-370 digital polarimeter for specific rotations; a JASCO
FT/IR-5300 infrared spectrometer for IR spectra (Jasco, Tokyo,
Japan); a JEOL JMS SX-102 mass spectrometer for FAB MS (Jeol,
Japan); a Waters Q-Tof Ultima API mass spectrometer for ESI-Q-
TOF MS (Waters Corporation, Milford, MA, USA); a JEOL JNM AL-
500 or JNM AL-300 NMR spectrometer for 1H- and 13C-NMR using
tetramethylsilane as an internal standard. HPLC was performed
using a Hitachi L-6000 pump equipped with Hitachi L-4000H UV
detector (Hitachi Instruments, Tokyo, Japan). Silica gel (Merck,
Germany, 60–230 mesh) and pre-coated thin layer chromatogra-
phy (TLC) plates (Merck, 60F254) were used for column chromatog-
raphy and TLC. Spots on TLC plates were detected by spraying
ninhydrin solution (2 g ninhydrin in 100 mL of sat. n-BuOH aq.)
and acidic p-anisaldehyde solution (p-anisaldehyde: 25 mL, c-
H2SO4: 25 mL, AcOH: 5 mL, EtOH: 425 mL) with subsequent heat-
ing.
Methyl ether analogue 8b
TFA (2.0 mL) was added to a stirred solution of 6b (400 mg,
0.32 mmol) in CH2Cl2 (15 mL), and the whole mixture was stirred
for 2 h at 08C. The solvent was removed under reduced pressure
to give TFA salt of 7b. Then, the TFA salt was treated with 2.0 M
HCl / Et2O (10 mL64) to furnish a HCl salt of 7b. Et3N (0.1 mL,
0.64 mmol), HOBt (100 mg, 0.64 mmol), EDCI (80 mg,
0.39 mmol) was added to the HCl salt in anhydrous THF (30 mL)
at 08C, and the reaction mixture was gradually warmed to rt
with stirring for 21 h. HCl (5%) was added to the mixture, and
the whole mixture was extracted with AcOEt. The AcOEt extract
was washed with brine and dried over Na2SO4. Removal of the
solvent from the EtOAc extract under reduced pressure gave a
crude product, which was purified by SiO2 column (n-hexane/
AcOEt = 1 : 1) to give 8b (91 mg, 25%) as a white powder. ESI-Q-
TOF MS: m/z 1143, 1145, 1147, 1149, 1151, 1153, 1155 [M + Na]+.
HR-ESI-Q-TOF MS: m/z 1148.7000, calcd for C36H3079Br381Br3N4O8-
Na, found: 1148.7020. IR mmax (KBr) cm– 1: 3405, 2938, 1671, 1589,
1279, 1248. 1H-NMR (500 MHz, CDCl3) d: 7.52 (2H, s), 7.44 (2H, s),
7.17 (1H, d, J = 2.0 Hz), 7.06 (1H, d, J = 2.0 Hz), 6.72 (1H, t, J =
6.5 Hz), 6.69 (1H, t, J = 6.5 Hz), 6.32 (1H, d, J = 2.0 Hz), 6.30 (1H, d, J
= 2.0 Hz), 6.04 (1H, s), 5.93 (1H, s), 4.04 (3H, s), 3.83 (2H, s), 3.70
(2H, s), 3.58 (3H, s), 3.53 (2H, q, J = 6.5 Hz), 3.45 (2H, q, J = 6.5 Hz),
2.79 (2H, t, J = 6.5 Hz), 2.69 (2H, t, J = 6.5 Hz). 13C-NMR (125 MHz,
CDCl3) d: 162.1, 161.9, 151.4, 150.2, 147.1 (2C), 143.9, 143.8,
Synthesis
(2-{3,5-Dibromo-4-[3-bromo-5-(2-{3-[3,5-dibromo-4-(9-
bromo-3-methoxyimino-2,8-dioxo-1-oxaspiro[4.5]deca-
6,9-dien-7-yloxy)phenyl]-2-methoxyiminopropionyl-
amino}ethyl)-2-hydroxyphenoxy]phenyl}ethyl)carbamic
acid tert-butyl ester 5b
HOBt (55 mg, 0.36 mmol), EDCI (100 mg, 0.52 mmol) was added
to a solution of segment A [4] (2, 115 mg, 0.19 mmol) and 3b
(110 mg, 0.17 mmol) in anhydrous THF (4.0 mL), then the whole
mixture was stirred for 2 h at rt under nitrogen atmosphere.
HCl (5%)was added to the mixture, and the whole mixture was
extracted with AcOEt. The AcOEt extract was washed with brine
and dried over Na2SO4. Removal of the solvent from the EtOAc
extract under reduced pressure gave a crude product, which was
purified by SiO2 column (CHCl3) to give 5b (105 mg, 50%) as a
white powder. ESI-Q-TOF MS: m/z 1259, 1261, 1263, 1265, 1267,
1269, 1271 [M + Na]+. HR-ESI-Q-TOF MS: m/z 1264.7474, calcd for
C41H3879Br381Br3N4O11Na, found: 1264.7467. IR mmax (KBr) cm– 1
3420, 2940, 1784, 1696, 1669, 1615, 1586, 1281, 1250. H-NMR
(500 MHz, acetone-d6) d: 8.68 (1H, br s), 7.80 (1H, d, J = 2.5 Hz),
7.60 (2H, s), 7.59 (1H, t, J = 7.0 Hz), 7.57 (2H, s), 7.11 (1H, d, J =
:
1
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141.7, 141.5, 139.7, 136.7, 133.6 (4C), 130.7, 128.7, 127.7, 126.6,
118.1 (2C), 117.9 (2C), 113.6, 112.7, 109.5, 109.0, 63.3, 62.5, 40.0,
39.6, 34.6 (2C), 28.9, 28.5.
(125 MHz, acetone-d6) d: 173.6, 171.8, 156.7, 147.6, 147.5, 145.7,
145.6, 143.4, 143.1, 143.0, 141.8, 134.5 (2C), 134.3, 134.1 (2C),
132.6, 127.3, 127.1, 118.3 (4C), 113.9, 113.5, 110.7, 110.5, 78.7,
42.0, 41.1, 37.8, 35.9, 35.6, 35.3, 31.1, 30.4, 28.7 (3C).
3-[3,5-Dibromo-4-(9-bromo-2,8-dioxo-1-oxaspiro[4.5]-
deca-6,9-dien-7-yloxy)phenyl]propionic acid 3c
Oxime-lacking analogue 8c
In the same procedure as 8b, 6c (310 mg, 0.28 mmol) was con-
verted to 8c (125 mg, 42%), which was obtained as a white pow-
der. ESI-Q-TOF MS: m/z 1035, 1037, 1039, 1041, 1043, 1045, 1047
[M + H]+. HR-ESI-Q-TOF MS: m/z 1040.7064, calcd for
C34H2979Br381Br3N2O6, found: 1040.7076. IR mmax (KBr) cm– 1: 3297,
A solution of CAN (680 mg, 1.24 mmol) in H2O (4.0 mL) was
added dropwise to a cooled (08C) solution of 4c (200 mg,
0.62 mmol) in CH3CN (8.0 mL). After stirring for 5 min, brine was
added to the mixture and the whole mixture was extracted with
AcOEt. The AcOEt extract was dried over Na2SO4, and the
removal of the solvent from the AcOEt extract under reduced
pressure gave a crude product, which was purified by SiO2 col-
umn (CHCl3 / MeOH = 20 : 1) to give 3c (75 mg, 43%) as a white
powder. ESI-Q-TOF MS: m/z 585, 587, 589, 591 [M + Na]+. HR-ESI-Q-
TOF MS: m/z 586.8139, calcd for C18H1379Br281BrO6Na, found:
586.8145. IR mmax (KBr) cm– 1: 3025, 1784, 1694, 1615, 1254. 1H-NMR
(300 MHz, acetone-d6) d: 7.78 (1H, d, J = 3.0 Hz), 7.66 (2H, s), 5.99 (1H, d,
J = 3.0 Hz), 2.94 (2H, t, J = 7.5 Hz), 2.88–2.79 (2H, m), 2.67 (2H, t, J =
7.5 Hz), 2.72–2.44 (2H, m). 13C-NMR (125 MHz, acetone-d6) d: 175.7,
173.5, 172.6, 149.0, 146.8, 146.6, 143.6, 134.2 (2C), 123.7, 120.7, 117.5
(2C), 81.6, 35.1, 32.6, 30.2, 28.2.
1
2928, 1647, 1551, 1281, 1250. H-NMR (500 MHz, DMSO-d6) d:
9.99 (1H, s), 9.94 (1H, s), 7.85 (1H, t, J = 6.0 Hz), 7.80 (1H, t, J =
6.0 Hz), 7.59 (2H, s), 7.55 (2H, s), 6.99 (1H, d, J = 2.0 Hz), 6.96 (1H,
d, J = 1.5 Hz), 5.95 (1H, d, J = 2.0 Hz), 5.91 (1H, d, J = 1.5 Hz), 3.26
(2H, q, J = 6.0 Hz), 3.01 (2H, q, J = 6.0 Hz), 2.80 (2H, t, J = 6.5 Hz),
2.64 (2H, t, J = 6.0 Hz), 2.37 (2H, dd-like), 2.26 (2H, t, J = 7.5 Hz),
2.23 (2H, t, J = 7.5 Hz), 2.10 (2H, dd-like).
(2S)-2-Benzyloxycarbonylamino-3-(3,5-dibromo-4-
hydroxyphenyl)propionic acid 4d
According to the literature [10], L-tyrosine (170 mg, 0.938 mmol)
was converted to 4d (445 mg, quant.), which was obtained as a
white powder. ESI-Q-TOF MS: m/z 494, 496, 498 [M + Na]+. HR-ESI-
Q-TOF MS: m/z 495.9194, calcd for C17H15NO579Br81BrNa, found:
495.9184. IR mmax (KBr) cm– 1: 3322, 2920, 1667, 1593, 1507. 1H-
NMR (500 MHz, acetone-d6) d: 7.49 (2H, s), 7.26–7.35 (5H, m), 6.66
(1H, d, J = 8.0 Hz), 5.03 (2H, ABq, J = 13.0 Hz), 4.48 (1H, ddd, J = 5.0,
8.0, 9.0 Hz), 3.18 (1H, dd, J = 5.0, 14.0 Hz), 2.95 (1H, dd, J = 9.0,
14.0 Hz). 13C-NMR (125 MHz, acetone-d6) d: 182.6, 172.8, 156.9,
150.2, 134.0 (2C), 133.0 (2C), 129.2 (2C), 128.6, 128.4, 111.2 (2C),
66.7, 55.9, 36.5. [a]D25 +3.87 (c = 1.11, MeOH).
(2-{3,5-Dibromo-4-[3-bromo-5-(2-{3-[3,5-dibromo-4-(9-
bromo-2,8-dioxo-1-oxaspiro[4.5]deca-6,9-dien-7-
yloxy)phenyl]propionylamino}ethyl)-2-hydroxyphenoxy]-
phenyl}ethyl)carbamic acid tert-butyl ester 5c
In the same procedure as 5b, 3c (460 mg, 0.81 mmol) was con-
verted to 5c (516 mg, 55%), which was obtained as a white pow-
der. ESI-Q-TOF MS: m/z 1173, 1175, 1177, 1179, 1181, 1183, 1185
[M + Na]+. HR-ESI-Q-TOF MS: m/z 1178.7357, calcd for
C39H3679Br381Br3N2O9Na, found: 1178.7363. IR mmax (KBr) cm– 1
:
3368, 2976, 1786, 1694, 1659, 1252. 1H-NMR (500 MHz, acetone-
d6) d: 8.64 (1H, br s), 7.77 (1H, d, J = 2.5 Hz), 7.59 (2H, s), 7.58 (2H,
s), 7.06 (1H, d, J = 2.0 Hz), 7.02 (1H, br s, J = 7.0 Hz), 6.23 (1H, d, J =
2.0 Hz), 6.14 (1H, br s, J = 7.0 Hz), 5.95 (1H, d, J = 2.5 Hz), 3.36 (2H,
q-like, J = 7.0 Hz), 3.27 (2H, q-like, J = 7.0 Hz), 2.90–2.75 (5H, m),
2.68–2.58 (2H, m), 2.55 (2H, t, J = 7.0 Hz), 2.48-2.44 (1H, m), 2.42
(2H, t, J = 7.0 Hz), 1.38 (9H, s). 13C-NMR (125 MHz, acetone-d6) d:
175.7, 172.7, 171.6, 156.7, 149.0 (2C), 147.7, 147.0, 146.6, 145.8,
144.2, 143.2, 142.0, 134.7 (2C), 134.3 (2C), 132.7, 127.4, 123.8,
120.8, 118.4 (2C), 117.5, 114.0, 110.8, 81.7, 78.7, 42.1, 41.2, 37.8,
35.7, 35.4, 32.7, 31.1, 28.8 (3C), 28.3.
(2S)-2-Benzyloxycarbonylamino-3-{4-[(3S,5RS)-3-
benzyloxycarbonylamino-9-bromo-2,8-dioxo-1-
oxaspiro[4.5]deca-6,9-dien-7-yloxy]-3,5-
dibromophenyl}propionic acid 3d
In the same procedure as 3c, 4d (2.0 g, 4.23 mmol) was converted
to 3d (1 : 1 mixture of two diastereomers, 770 mg, 42%), which
was obtained as a white powder. ESI-Q-TOF MS: m/z 883, 885, 887,
889 [M + Na]+. HR-ESI-Q-TOF MS: m/z 886.9073, calcd for
C34H2779Br81Br2N2O10Na, found: 886.9080. IR mmax (KBr) cm– 1: 3345,
1698, 1613, 1258. 1H-NMR (500 MHz, acetone-d6) d: 7.98 (1H, d, J =
2.0 Hz), 7.71 (2H, s), 7.70 (2H, s), 7.66 (1H, d, J = 2.0 Hz), 7.36–7.29
(20H, m), 7.15 (1H, d, J = 8.0 Hz), 7.00 (1H, d, J = 8.0 Hz), 6.78 (1H,
d, J = 8.0 Hz), 6.73 (1H, d, J = 8.0 Hz), 6.20 (1H, d, J = 2.0 Hz), 5.78
(1H, d, J = 2.0 Hz), 5.12–4.98 (8H, m), 4.96–4.90 (1H, m), 4.66 (1H,
q-like, J = 9.5 Hz), 4.56–4.48 (2H, m), 3.32-3.26 (2H, m), 3.07 (1H,
dd, J = 9.5, 14.0 Hz), 3.01 (1H, dd, J = 9.5, 14.0 Hz), 2.94 (1H, dd, J =
9.5, 13.0 Hz), 2.82–2.74 (2H, m), 2.57 (1H, dd, J = 11.0, 13.0 Hz).
3-[3-Bromo-5-(2,6-dibromo-4-{2-[2-(3-bromo-5-{2,6-
dibromo-4-[2-(tert-butoxycarbonylamino)ethyl]phenoxy}-
4-hydroxyphenyl)ethylcarbamoyl]ethyl}phenoxy)-4-
hydroxyphenyl]propionic acid 6c
In the same procedure as 6b, 5c (400 mg, 0.35 mmol) was con-
verted to 6c (400 mg, quant.), which was obtained as a white
powder. ESI-Q-TOF MS: m/z 1175, 1177, 1179, 1181, 1183, 1185,
1187 [M + Na]+. HR-ESI-Q-TOF MS: m/z 1180.7514, calcd for
[(3S,5RS)-7-(4-{(2S)-2-Benzyloxycarbonylamino-2-[2-(3-
bromo-5-{2,6-dibromo-4-[2-(tert-
butoxycarbonylamino)ethyl]phenoxy}-4-
hydroxyphenyl)ethylcarbamoyl]ethyl}-2,6-
dibromophenoxy)-9-bromo-2,8-dioxo-1-oxaspiro[4.5]-
deca-6,9-dien-3-yl]carbamic acid benzyl ester 5d
C39H3879Br381Br3N2O9Na, found: 1180.7498. IR mmax (KBr) cm– 1
:
3320, 2930, 1688, 1644, 1591, 1281, 1248. 1H-NMR (500 MHz, ace-
tone-d6) d: 8.68 (1H, br s), 7.57 (4H, s), 7.14 (1H, t, J = 6.5 Hz), 7.11
(1H, d, J = 2.0 Hz), 7.06 (1H, d, J = 2.0 Hz), 6.26 (1H, d, J = 2.0 Hz),
6.23 (1H, d, J = 2.0 Hz), 6.20 (1H, t, J = 6.5 Hz), 3.36 (2H, q, J =
6.5 Hz), 3.28 (2H, q, J = 6.5 Hz), 2.90 (2H, t, J = 7.5 Hz), 2.83 (2H, t,
J = 6.5 Hz), 2.66 (2H, t, J = 7.5 Hz), 2.54 (2H, t, J = 6.5 Hz), 2.45 (2H,
t, J = 7.5 Hz), 2.43 (2H, t, J = 7.5 Hz), 1.38 (9H, s). 13C-NMR
In the same procedure as 5b, 3d (40 mg, 0.046 mmol) was con-
verted to 5d (1 : 1 mixture of two diastereomers, 41 mg, 61%),
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Arch. Pharm. Chem. Life Sci. 2008, 341, 568–577
Structure-Activity Relationships Study of Bastadin 6
575
which was obtained as a white powder. ESI-Q-TOF MS: m/z 1471,
1473, 1475, 1477, 1479, 1481, 1483 [M + Na]+. HR-ESI-Q-TOF MS:
m/z 1476.8311, calcd for C55H5079Br381Br3N4O13Na, found:
NMR (500 MHz, CD3OD) d: 7.57 (4H, br s), 7.11 (1H, d, J = 2.0 Hz),
7.08 (1H, d, J = 2.0 Hz), 6.03 (1H, d, J = 2.0 Hz), 5.98 (1H, d, J =
2.0 Hz), 3.64–3.33 (2H, m), 3.23–3.18 (1H, m), 3.13–3.08 (1H, m),
3.00 (1H, dd, J = 5.0, 14.0 Hz), 2.86–2.79 (3H, m), 2.73–2.68 (1H,
m), 2.62 (1H, dd, J = 4.0, 13.5 Hz), 2.34–2.24 (4H, m).
D-Tyrosine-type analogue 8g was also prepared by the same
manner as 8f.
1
1476.8330. IR mmax (KBr) cm– 1: 3331, 2936, 1698, 1252. H-NMR
(500 MHz, acetone-d6) d: 8.70 (1H, br s), 8.67 (1H, br s), 7.95 (1H, d,
J = 2.0 Hz), 7.62 (4H, br s), 7.57 (4H, s), 7.44 (2H, br s), 7.36–7.28
(22H, m), 7.09 (3H, br s), 6.56 (1H, d, J = 8.5 Hz), 6.53 (1H, d, J =
8.5 Hz), 6.29 (1H, s), 6.27 (1H, s), 6.13 (3H, d-like), 5.75 (1H, d, J =
2.0 Hz), 5.09–4.93 (8H, m), 4.90–4.87 (1H, m), 4.65 (1H, q-like, J =
8.0 Hz), 4.30–4.25 (2H, m), 3.37–3.36 (4H, m), 3.32–3.28 (2H, m),
3.25–3.18 (2H, m), 3.11–3.05 (2H, m), 2.96–2.89 (2H, m), 2.86–
2.82 (6H, m), 2.77–2.75 (1H, m), 2.67 (1H, dd, J = 10.0, 13.5 Hz),
2.59–2.54 (4H, m), 1.38 (18H, br s).
D-Cbz-Tyrosine-type analogue 8e
ESI-Q-TOF MS: m/z 1355, 1357, 1359, 1361, 1363, 1365, 1367
[M + Na]+. HR-ESI-Q-TOF MS: m/z 1360.7837, calcd for
C50H4279Br381Br3N4O10Na, found: 1360.7809. NMR and IR spectra
were same as those of 8d.
(2S)-2-Benzyloxycarbonylamino-3-[3-(4-{(2S)-2-
benzyloxycarbonylamino-2-[2-(3-bromo-5-{2,6-dibromo-
4-[2-(tert-butoxycarbonylamino)ethyl]phenoxy}-4-
hydroxyphenyl)ethylcarbamoyl]ethyl}-2,6-dibromo-
D-Tyrosine-type analogue 8g
ESI-Q-TOF MS: m/z 1065, 1067, 1069, 1071, 1073, 1075, 1077
[M + H]+. HR-ESI-Q-TOF MS: m/z 1070.7282, calcd for
C34H3179Br381Br3N4O6, found: 1070.7306. NMR and IR spectra were
same as those of 8f.
phenoxy)-5-bromo-4-hydroxyphenyl]propionic acid 6d
In the same procedure as 5a, 5d (15 mg, 0.01 mmol) was con-
verted to 6d (15 mg, quant.), which was obtained as a white pow-
der. ESI-Q-TOF MS: m/z 1473, 1475, 1477, 1479, 1481, 1483, 1485
Synthesis of debromo analogues
NaOAc (13 mg, 0.161 mmol) and 10% Pd/C (5 mg) was added to a
solution of bastadin 6 (1) (59 mg, 0.054 mmol) in MeOH (5.4 mL),
and the whole mixture was stirred for 30 min under hydrogen
atmosphere; then, the catalyst was filtered off through celite
pad. Brine was added to the filtrate and the whole mixture was
extracted with AcOEt. The AcOEt extract was washed with brine
and dried over Na2SO4. Removal of the solvent from the EtOAc
extract under reduced pressure gave a crude product, which was
purified by reversed-phase HPLC (Cosmosil 5C18-AR column,
250 mm610 mm i. d., MeOH / H2O = 7 : 3; Nacalai, Japan) to give
4-Br analogue (9a, 4.5 mg), 2-Br analogue (9b, 4 mg), 1-Br ana-
logue (9c, 3.5 mg), and 0-Br analogue (9d, 4.5 mg).
[M
+
Na]+. HR-ESI-Q-TOF MS: m/z 1478.8467, calcd for
C55H5279Br381Br3N4O13Na, found: 1478.8455. IR mmax (KBr) cm– 1
:
3310, 2936, 1690, 1645, 1534, 1501, 1279, 1252. 1H-NMR
(500 MHz, acetone-d6) d: 8.74 (1H, br s), 8.69 (1H, br s), 7.56 (4H, s),
7.51 (1H, t-like, J = 5.5 Hz), 7.36–7.28 (10H, m), 7.13 (1H, d, J =
2.0 Hz), 7.10 (1H, s), 6.56 (1H, d-like, J = 8.0 Hz), 6.32-6.30 (2H, m),
6.27 (1H, s), 6.15 (1H, t-like, J = 5.5 Hz), 5.03 (4H, s), 4.39–4.34 (2H,
m), 3.38–3.32 (4H, m), 3.09-3.05 (1H, m), 3.00 (1H, dd, J = 5.0,
14.0 Hz), 2.92 (1H, dd, J = 8.5, 14.0 Hz), 2.82 (3H, t-like), 2.61-2.58
(2H, m), 1.38 (9H, s). [a]D25 +22.2 (c = 2.67, dioxane).
L-Cbz-Tyrosine-type analogue 8d
4-Bromo analogue 9a
In the same procedure as 8b, 6d (18.5 mg, 0.013 mmol) was con-
verted to 8d (6.0 mg, 34%), which was obtained as a white pow-
der. ESI-Q-TOF MS: m/z 1355, 1357, 1359, 1361, 1363, 1365, 1367
[M + Na]+. HR-ESI-Q-TOF MS: m/z 1360.7837, calcd for
White powder. FAB MS: m/z 959, 961, 963, 965, 967 [M + Na]+. 1H-
NMR (500 MHz, acetone-d6) d: 7.65 (1H, br s), 7.61 (2H, s), 7.18 (1H,
br s), 7.17 (2H, d, J = 8.5 Hz), 7.16 (1H, d, J = 1.5 Hz), 6.91 (1H, d, J =
8.5 Hz), 6.89 (1H, d, J = 8.0 Hz), 6.84 (1H, dd, J = 2.0, 8.0 Hz), 6.79
(1H, d, J = 2.0 Hz), 6.41 (1H, d, J = 2.0 Hz), 3.82 (2H, s), 3.65 (2H, s),
3.48 (2H, q-like, J = 6.5 Hz), 3.39 (2H, q-like, J = 6.5 Hz), 2.72–2.79
(4H, m).
C50H4279Br381Br3N4O10Na, found: 1360.7826. IR mmax (KBr) cm– 1
:
3297, 2926, 1692, 1645, 1279, 1256. 1H-NMR (500 MHz, acetone-
d6) d: 8.77 (1H, br s), 8.66 (1H, br s), 7.64 (2H, br s), 7.56 (2H, br s),
7.37–7.28 (10H, m), 7.16 (1H, br s), 7.10 (1H, s), 7.08 (1H, s), 6.56
(1H, d-like, J = 7.5 Hz), 6.24 (1H, d-like, J = 6.0 Hz), 6.11 (1H, s), 6.03
(1H, s), 5.10–4.95 (4H, m), 4.28 (1H, m), 4.14 (1H, m), 3.56–3.55
(1H, m), 3.40 (1H, m), 3.24 (2H, m), 3.10 (1H, m), 2.93 (1H, m), 2.78
(2H, m), 2.70 (1H, dd, J = 4.5, 14.0 Hz), 2.61 (1H, m), 2.45 (2H, m).
2-Bromo analogue 9b
White powder. ESI-Q-TOF MS: m/z 803, 805, 807 [M + Na]+. HR-ESI-
Q-TOF MS: m/z 805.0308, calcd for C34H3079Br81BrN4O8Na, found:
805.0345. IR mmax (KBr) cm– 1: 3210, 2926, 1663, 1507, 1281, 1229.
1H-NMR (500 MHz, DMSO-d6) d: 11.89 (1H, s), 11.80 (1H, s), 9.91
(1H, s), 9.27 (1H, s), 8.01 (1H, t, J = 6.0 Hz), 7.90 (1H, t, J = 6.0 Hz),
7.57 (1H, d, J = 1.5 Hz), 7.25 (1H, d, J = 2.0 Hz), 7.10 (2H, d, J =
8.5 Hz), 7.08 (1H, dd, J = 1.5, 8.0 Hz), 6.90 (1H, d, J = 8.0 Hz), 6.87
(1H, dd, J = 1.5, 8.0 Hz), 6.82 (2H, d, J = 8.5 Hz), 6.76 (1H, d, J =
8.5 Hz), 6.71 (1H, d, J = 1.5 Hz), 6.59 (1H, d, J = 1.5 Hz), 3.70 (2H, s),
3.64 (2H, s), 3.35-3.43 (2H, m), 3.29 (2H, q-like, J = 7.0 Hz), 2.70
(2H, t, J = 6.5 Hz), 2.66 (2H, t, J = 7.0 Hz).
25
[a]D –10.0 (c = 0.45, dioxane).
L-Tyrosine-type analogue 8f
An amount of 0.5 mL of 25% HBr in AcOH was added to a solu-
tion of 8d (33 mg, 0.025 mmol) in dioxane (2.5 mL), and the
whole mixture was stirred for 2 h at rt. Saturated aqueous
NaHCO3 was added to the mixture, and the whole mixture was
extracted with AcOEt. The AcOEt extract was washed with brine
and dried over Na2SO4. Removal of the solvent from the EtOAc
extract under reduced pressure. The residue was purified by
washing with Et2O to give 8f (18 mg, 68%) as a white powder. ESI-
Q-TOF MS: m/z 1065, 1067, 1069, 1071, 1073, 1075, 1077 [M + H]+.
HR-ESI-Q-TOF MS: m/z 1070.7282, calcd for C34H3179Br381Br3N4O6,
found: 1070.7277. IR mmax (KBr) cm– 1: 3331, 2928, 1651, 1576. 1H-
1-Bromo analogue 9c
White powder. ESI-Q-TOF MS: m/z 725, 727 [M + Na]+. HR-ESI-Q-
TOF MS: m/z 725.1223, calcd for C34H3179BrN4O8Na, found:
725.1215. IR mmax (KBr) cm– 1: 3227, 1665, 1510, 1294, 1231. H-
1
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576
N. Kotoku et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 568–577
NMR (500 MHz, DMSO-d6) d: 11.89 (1H, s), 11.74 (1H, s), 9.55 (1H,
s), 9.29 (1H, s), 7.97 (1H, t, J = 6.0 Hz), 7.84 (1H, t, J = 6.0 Hz), 7.51
(1H, d, J = 2.0 Hz), 7.06 (2H, d, J = 8.5 Hz), 7.00 (2H, dd, J = 2.0,
8.5 Hz), 6.93 (1H, d, J = 8.5 Hz), 6.90 (1H, d, J = 8.5 Hz), 6.87 (1H,
dd, J = 2.0, 8.5 Hz), 6.80 (2H, d, J = 8.5 Hz), 6.75 (1H, d, J = 1.5 Hz),
6.65 (1H, d, J = 1.5 Hz), 6.52 (1H, d, J = 8.5 Hz), 3.75 (2H, s), 3.65
(2H, s), 3.35-3.47 (2H, m), 3.30 (2H, q, J = 6.0 Hz), 2.67 (2H, t, J =
6.0 Hz), 2.66 (2H, t, J = 6.0 Hz).
166.1, 165.6, 153.6, 152.1, 149.1, 149.0, 146.0, 145.8, 140.9,
138.4, 137.4, 136.9, 134.9, 134.7 (2C), 134.5 (2C), 133.7, 130.9,
128.8, 119.3 (2C), 119.1 (2C), 113.3, 112.0, 105.9, 105.4, 41.7, 41.2,
35.6, 35.5, 29.7, 29.1.
Thiourea analogue 12
Triethylamine (3 lL, 0.022 mmol) and 2-(4-morpholino)ethyl iso-
thiocyanate (14, 3 lL, 0.020 mmol) was added to a solution of 11
(8 mg, 0.0085 mmol) in anhydrous THF (0.8 mL), and the whole
mixture was stirred for 12 h at 508C under nitrogen atmosphere.
The solvent was removed from the reaction mixture under
reduced pressure, and the residue was purified by successive
washing with Et2O and CHCl3 to give 12 (11 mg, 98%) as a white
powder. ESI-Q-TOF MS: m/z 1311, 1313, 1315, 1317, 1319 [M + H]+.
0-Bromo analogue 9d
White powder. ESI-Q-TOF MS: m/z 647 [M + Na]+. HR-ESI-Q-TOF MS:
m/z 647.2118, calcd for C34H32N4O8Na, found: 647.2125. IR mmax
(KBr) cm– 1: 3322, 2920, 1667, 1593, 1507. 1H-NMR (500 MHz, ace-
tone-d6) d: 10.89 (1H, s), 10.75 (1H, s), 8.03 (1H, s), 8.02 (1H, s), 7.28
(1H, t, J = 6.5 Hz), 7.22 (1H, t, J = 6.5 Hz), 7.14 (2H, d, J = 8.5 Hz),
7.07 (2H, d, J = 8.5 Hz), 7.01 (1H, dd, J = 2.0, 8.5 Hz), 6.91-6.84 (4H,
m), 6.80 (2H, d, J = 8.5 Hz), 6.74 (2H, d, J = 8.5 Hz), 6.68 (1H, d, J =
1.5 Hz), 3.81 (4H, s), 3.46 (2H, t, J = 6.5 Hz), 3.43 (2H, t, J = 6.5 Hz),
2.74 (2H, t, J = 6.5 Hz), 2.68 (2H, t, J = 6.5 Hz).
HR-ESI-Q-TOF
MS:
m/z
1315.0237,
calcd
for
:
C48H5579Br281Br2N10O10S2, found: 1315.0206. IR mmax (KBr) cm– 1
3268, 2926, 2855, 1661, 1613, 1524, 1252, 1211. 1H-NMR
(500 MHz, acetone-d6) d: 7.72 (1H, br s), 7.61 (2H, s), 7.57 (2H, s),
7.42 (1H, br s), 7.20 (1H, br s), 7.11 (1H, br s), 6.29 (1H, s), 6.25 (1H,
s), 3.92 (2H, s), 3.69 (6H, br s), 3.58 (8H, br s), 3.48 (2H, t, J = 6.5 Hz),
3.41 (2H, t, J = 6.5 Hz), 2.88 (2H, t, J = 7.0 Hz), 2.75 (2H, t, J =
7.0 Hz), 2.58 (4H, br s), 2.44 (8H, br s).
NO2-analogue 10
A solution of NaNO2 (90 mg, 1.28 mmol) in H2O (1 mL) was added
dropwise to a solution of 1 (140 mg, 0.13 mmol) in AcOH (10 mL)
and H2O (4 mL). After stirring for 1 h at 158C, brine was added to
the mixture and the whole mixture was extracted with AcOEt.
The AcOEt extract was washed with brine and dried over Na2SO4.
Removal of the solvent from the EtOAc extract under reduced
pressure, and the residue was suspended to CHCl3, filtered
through celite pad, and the filtrate was concentrated to give 10
(130 mg, 99%) as a yellow powder. ESI-Q-TOF MS: m/z 1049, 1051,
1053, 1055, 1057 [M + Na]+. HR-ESI-Q-TOF MS: m/z 1052.8199,
calcd for C34H2679Br281Br2N6O12Na, found: 1052.8199. IR mmax (KBr)
cm– 1: 3227, 3063, 2928, 1663, 1632, 1545, 1256. 1H-NMR
(500 MHz, CDCl3) d: 10.78 (1H, s), 10.71 (1H, s), 9.52 (1H, br s), 8.51
(1H, br s), 7.82 (1H, d, J = 2.0 Hz), 7.68 (1H, d, J = 2.0 Hz), 7.44 (2H,
s), 7.38 (2H, s), 7.16 (1H, t, J = 5.5 Hz), 6.70 (1H, d, J = 2.0 Hz), 6.64
(1H, t, J = 6.0 Hz), 6.46 (1H, d, J = 1.0 Hz), 3.93 (2H, s), 3.76 (2H, s),
3.55 (2H, q, J = 6.0 Hz), 3.70 (2H, m), 2.89 (2H, t, J = 6.0 Hz), 2.80
(2H, dd-like). 13C-NMR (125 MHz, CDCl3) d: 164.7, 162.5, 151.7,
151.1, 147.4, 146.7 (3C), 144.8, 144.6, 139.8, 136.9, 134.3, 134.1,
133.7 (2C), 133.0 (2C), 128.6, 128.2, 121.9, 119.5, 118.5, 118.2
(3C), 117.8 (2C), 40.6, 39.6, 34.9, 33.3, 29.3, 28.1.
Guanidine analogue 13
1,3-Bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea
(15,
8 mg, 0.028 mmol), Et3N (7 lL, 0.049 mmol) and HgCl2 (13.5 mg,
0.049 mmol) was added to a solution of 11 (12 mg, 0.012 mmol)
in anhydrous THF (1.0 mL), and the whole mixture was stirred
for 14 h at rt under argon atmosphere. The Hg salt was filtered
off through celite pad. Brine was added to the filtrate and the
whole mixture was extracted with AcOEt. The AcOEt extract was
washed with brine and dried over Na2SO4. Removal of the solvent
from the EtOAc extract under reduced pressure gave a crude
product, which was purified by SiO2 column (n-hexane / AcOEt =
3 : 2) to give Boc-protected compound (17 mg, 93%) as a white
powder. ESI-Q-TOF MS: m/z 1473, 1475, 1477, 1479, 1481
[M + Na]+. HR-ESI-Q-TOF MS: m/z 1477.1248, calcd for
C56H6679Br281Br2N10O16Na, found: 1477.1239. IR mmax (KBr) cm– 1
:
3586, 3526, 3261, 1726, 1663, 1613, 1252, 1152. 1H-NMR
(500 MHz, acetone-d6) d: 11.73 (1H, br s), 11.71 (1H, br s), 11.27
(1H, br s), 10.69 (1H, s), 10.60 (1H, s), 10.35 (1H, s), 10.21 (1H, br s),
9.78 (1H, br s), 7.70 (1H, t, J = 6.0 Hz), 7.61 (2H, s), 7.57 (2H, s), 7.48
(1H, d-like), 7.40 (1H, t, J = 6.0 Hz), 7.30 (1H, d-like), 6.26 (1H, d, J =
2.0 Hz), 6.22 (1H, d, J = 1.5 Hz), 3.91 (2H, s), 3.70 (2H, s), 3.48 (2H,
q-like), 3.42 (2H, q-like), 2.87 (2H, t, J = 7.0 Hz), 2.75 (2H, t, J =
7.0 Hz), 1.59 (18H, br s), 1.44 (18H, br s). 13C-NMR (125 MHz,
CDCl3) d: 164.4, 162.8, 161.0 (2C), 154.3, 153.7, 153.2, 153.0,
151.9, 151.4, 148.1, 147.9, 147.5, 146.9, 139.4, 139.0, 138.3,
136.4, 133.4 (2C), 132.6 (2C), 129.5, 128.7, 124.4, 124.1, 120.0,
119.3, 118.6 (2C), 118.2 (2C), 113.6, 111.7, 85.0, 84.7, 81.9, 81.2,
40.0, 39.6, 34.3, 33.6, 29.2, 28.2, 28.1 (3C), 28.03 (3C), 28.00 (3C),
27.96 (3C).
NH2-analogue 11
Na2S2O4 (13 mg, 0.076 mmol) in H2O (1 mL) was added to a solu-
tion of 10 (13 mg, 0.013 mmol) in MeOH (2 mL), and the whole
mixture was stirred for 10 min at 08C. Brine was added to the
mixture and the whole mixture was extracted with AcOEt. The
AcOEt extract was washed with brine and dried over Na2SO4.
Removal of the solvent from the EtOAc extract under reduced
pressure gave a crude product, which was purified by prepara-
tive TLC (CHCl3 / MeOH / Et3N = 80 : 10 : 1) to give 11 (10 mg, 80%)
as a white powder. ESI-Q-TOF MS: m/z 989, 991, 993, 995, 997 [M +
TFA (0.5 mL) was added to a solution of the above product
(18 mg, 0.012 mmol) in CH2Cl2 (2.0 mL), and the whole mixture
was stirred for 2.5 h at 08C. The solvent was removed from the
reaction mixture under reduced pressure to give 13 (15 mg,
quant.) as a white powder. ESI-Q-TOF MS: m/z 526, 527, 528, 529,
530 [M + 2H]2+. HR-ESI-Q-TOF MS: m/z 527.9705, calcd for 1/
Na]+.
C34H3079Br281Br2N6O8Na, found: 992.8734. IR mmax (KBr) cm– 1
HR-ESI-Q-TOF
MS:
m/z
992.8715,
calcd
for
:
3225, 2926, 1667, 1620, 1520, 1453. 1H-NMR (500 MHz, acetone-
d6) d: 7.63 (1H, br s), 7.55 (2H, s), 7.53 (2H, s), 7.27 (1H, t, J = 6.0 Hz),
6.40 (1H, d, J = 2.0 Hz), 6.28 (1H, d, J = 2.0 Hz), 5.69 (1H, d, J =
1.5 Hz), 5.62 (1H, d, J = 1.5 Hz), 4.40 (2H, br s), 4.36 (2H, br s), 3.89
(2H, s), 3.56 (2H, s), 3.46 (2H, q-like), 3.33 (2H, q-like), 2.85 (2H, t, J
= 7.0 Hz), 2.59 (2H, t, J = 7.5 Hz). 13C-NMR (125 MHz, CD3OD) d:
2(C36H3679Br281Br2N10O8), found: 527.9694. IR mmax (KBr) cm– 1
:
3351, 2926, 2853, 1665, 1613, 1263, 1213. 1H-NMR (500 MHz, ace-
tone-d6) d: 11.53 (1H, s), 10.60 (1H, s), 10.14 (1H, s), 9.96 (1H, s),
8.91 (2H, br s), 7.85 (3H, br s), 7.69 (1H, t, J = 6.5 Hz), 7.60 (2H, s),
i 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2008, 341, 568–577
Structure-Activity Relationships Study of Bastadin 6
577
7.57 (2H, s), 7.48 (1H, t, J = 6.5 Hz), 6.97 (1H, d, J = 1.5 Hz), 6.90
(1H, d, J = 2.0 Hz), 6.39 (1H, d, J = 2.0 Hz), 6.37 (1H, d, J = 2.0 Hz),
5.46 (1H, br s), 3.89 (2H, s), 3.67 (2H, s), 3.46 (2H, q, J = 7.0 Hz), 3.41
(2H, q, J = 6.5 Hz), 3.06 (2H, br s), 2.85 (2H, t, J = 7.0 Hz), 2.76 (2H, t,
J = 7.0 Hz).
formations with energy difference of 15 kJ/mol from the global
minimum were saved. Extended cut-off distances were defined
at 8 ꢀ for van-der-Waals, 20 ꢀ for electrostatics, and 4 ꢀ for H-
bonds.
Cell culture
HUVECs (56105 cells/vial) were obtained from Kurabo Inc.
(Osaka, Japan) and grown in the HuMedia-EG2 medium with
growth supplements (Kurabo Inc.). Human KB epidermoid carci-
noma cells (KB3-1) were cultured in the RPMI 1640 medium sup-
plemented with heat-inactivated 10% fetal bovine serum (FBS)
and kanamycin (50 lg/mL) in a humidified atmosphere of 5%
CO2 at 378C.
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Growth inhibition assay
A suspension of HUVECs in the proliferation medium (HuMedia-
EG2) with growth supplements was plated into well of 96-well
plate (26103 cells/well/100 lL). After 24 h, the culture medium
was removed and replaced with fresh essential minimal
medium (HuMedia-EB2) with growth factor [bFGF (30 ng/mL) or
VEGF (30 ng/mL)] of endothelial cells and various concentrations
of bastadin analogues. Plates were incubated for an additional
72 h in a humidified atmosphere of 5% CO2 at 378C, and cell pro-
liferation was detected by WST-8 colorimetric reagent. KB 3-1
cells in RPMI 1640 medium (26103 cells/well/100 lL) were also
inoculated into well of 96-well plate and treated as same as in
the case of HUVECs for the evaluation of anti-proliferative effect
of bastadins. The IC50 value was determined by linear interpola-
tion from the growth inhibition curve. We selectively assessed of
anti-proliferative activity [selective index (S. I.)] from the differ-
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Molecular modeling experiments were executed using Macro-
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i 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com