Synthesis and phospholipase A2 inhibitory activity of thielocin B3 derivatives.

Article abstract of DOI:10.1021/jm960437a

We prepared several types of derivatives of thielocin B3, a very potent naturally occurring inhibitor for human nonpancreatic secretory PLA2 (sPLA2-II), and conducted a structure-activity relationship study to identify potent sPLA2-II inhibitors with the aim of developing antiinflammatory drugs. The total number of aromatic rings is critical for sPLA2-II inhibition, and the best result was obtained in the case of six rings. The structure of the central part of the inhibitors was not specific, and potent inhibitors were found among the sulfide, sulfone, ether, methylene, and amino derivatives. Although a diester of the terminal carboxylic acid lost its inhibitory activity, having both of the carboxylic acids was not necessary for expression of activity, as illustrated by a glycine derivative with the benzyl ester group 36. Among the newly synthesized derivatives, 18, 20, 29, and 36 showed very potent human sPLA2-II inhibitory activity comparable to that of natural thielocin B3. Their IC50 values are in the range 0.069-0.14 microM, and they are a class of compounds showing the most potent sPLA2-II inhibition to date.

Full text of DOI:10.1021/jm960437a

J . Med. Chem. 1996, 39, 5183-5191
5183
Syn th esis a n d P h osp h olip a se A2 In h ibitor y Activity of Th ielocin B3 Der iva tives
Isao Teshirogi, Shigeru Matsutani, Kazuhiro Shirahase, Yasuhiko Fujii, Tadashi Yoshida,
Kazushige Tanaka, and Mitsuaki Ohtani*
Shionogi Research Laboratories, Shionogi & Co., Ltd., Fukushima-ku, Osaka 553, J apan
Received J une 13, 1996X
We prepared several types of derivatives of thielocin B3, a very potent naturally occurring
inhibitor for human nonpancreatic secretory PLA2 (sPLA2-II), and conducted a structure-
activity relationship study to identify potent sPLA2-II inhibitors with the aim of developing
antiinflammatory drugs. The total number of aromatic rings is critical for sPLA2-II inhibition,
and the best result was obtained in the case of six rings. The structure of the central part of
the inhibitors was not specific, and potent inhibitors were found among the sulfide, sulfone,
ether, methylene, and amino derivatives. Although a diester of the terminal carboxylic acid
lost its inhibitory activity, having both of the carboxylic acids was not necessary for expression
of activity, as illustrated by a glycine derivative with the benzyl ester group 36. Among the
newly synthesized derivatives, 18, 20, 29, and 36 showed very potent human sPLA2-II inhibitory
activity comparable to that of natural thielocin B3. Their IC50 values are in the range 0.069-
0.14 µM, and they are a class of compounds showing the most potent sPLA2-II inhibition to
date.
In tr od u ction
synthesis was accomplished by the Merck group;20
however, it showed rather weak activity for human
sPLA2-II with an IC50 value of 12 µM.18 Thielocin B3,
on the other hand, is one of the most potent inhibitors
(IC50: 0.076 µM) for human sPLA2-II among inhibitors
known to date21 (Figure 1). Unfortunately, production
of thielocin B3 from fungi is extremely low, and the
pharmacological evaluation is not feasible at this time.19
Phospholipase A2 (PLA2s) is a ubiquitous group of
enzymes which specifically catalyze the hydrolysis of the
sn-2 fatty acyl ester bond of aggregated glycerophos-
pholipids, the major constituents of the cell membrane,
to produce fatty acids and lysophospholipids.1-6 Among
the fatty acids, arachidonic acid is particularly impor-
tant and further metabolized to form prostagladins,
leukotriens, and active molecules which are widely
implicated in the pathophysiology of inflammation. A
second product of PLA2 action is a lysophospholipid
which is believed to be an inducer of some kinds of
inflammation and the precursor of platelet-activating
factor (PAF) inducing a variety of inflammatory disease
states.
These findings prompted us to pursue thielocin B3
derivatives by chemical synthesis, aiming at the pos-
sibility of developing an antiinflammatory drug. De-
scribed here are the synthesis and structure-activity
relationship (SAR) study of thielocin B3 derivatives.
Ch em istr y
There have been many reports regarding low molec-
ular weight (14 kDa) extracellularly secreted enzymes
(sPLA2s) isolated from such sources as mammalian
pancreas (sPLA2-I)7 and human synovial fluid (sPLA2-
II).8 As the latter have been detected not only in the
synovial fluid of patients with rheumatoid arthritis but
also in the serum of patients with sepsis and pancre-
atitis, this enzyme is now considered to be implicated
in many kinds of inflammatory diseases.9-13
Although a number of research groups have been
reporting the isolation, cloning, and mechanism of action
of the intracellular high molecular weight (85 kDa)
PLA2,14 no potent inhibitors of this cytosolic PLA2
(cPLA2) have yet been reported to be undergoing clinical
evaluation. Recently, researchers of Lilly reported very
potent human nonpancreatic sPLA2 (sPLA2-II) inhibi-
tors having an indole structure15 which were prepared
using computer-aided drug design.16 We have isolated
thielocins, potent sPLA2-II inhibitors, from a natural
source17-19 and since then have directed our chemistry
efforts to the synthesis of their derivatives to improve
the sPLA2-II inhibitory activity. Among the thielocins,
thielocin A1 showed very potent sPLA2-II inhibitory
activity in rat with an IC50 value of 0.0033 µM. Its total
On the basis of the structure of thielocin B3, chem-
istry efforts were focused on the synthesis of compounds
depicted by the representative formula A in which X is
shown in Figure 2.
Multisubstituted phenyl ring units were prepared
from ethyl crotonate by procedures reported in the
literature22-24 (Scheme 1). Methylation of 1 with dim-
ethyl sulfate and potassium carbonate, followed by
reduction of the formyl group and usual acetylation,
gave 3 in 69% yield. Coupling reaction of 3 and 4, a
known compound,23 using borontrifluoride etherate
complex afforded 5 in quantitative yield. Then usual
methylation and hydrolysis gave symmetrical dicar-
boxylic acid 6 in 95% yield. Two important building
blocks, 8 and 10, were obtained by deprotection of the
benzyl group using catalytic hydrogenolysis followed by
esterification of the resulting phenolic carboxylic acids
with diphenyldiazomethane in 93 and 84% yield, re-
spectively. Symmetrical esters having phenyl ring,
pyrone ring, or the ethene group in the center of their
molecules were prepared by the diacid chloride forma-
tion of each dicarboxylic acid with oxalyl chloride
followed by esterification with 2 equiv of phenoxide of
the phenolic ester 10. Deprotection of the benzhydryl
esters with trifluoroacetic acid and anisole gave 11, 12,
X Abstract published in Advance ACS Abstracts, December 1, 1996.
S0022-2623(96)00437-2 CCC: $12.00 © 1996 American Chemical Society

5184 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26
Teshirogi et al.
F igu r e 1.
F igu r e 2. Representative formulas of synthesized compounds.
Sch em e 1a
a
Reagents: (a) Me2SO4, K2CO3; (b) NaBH4; (c) Ac2O, pyridine; (d) BF3‚OEt2; (e) KOH; (f) H2, Pd-C; (g) benzyl chloride, K2CO3; (h)
Ph2CN2; (i) oxalyl chloride; (j) n-BuLi.
and 13 in 60, 27, and 80% yield, respectively. Usual
catalytic reduction of 13 gave the saturated ethylenic
compound 14 in 92% yield (Scheme 2). Diphenyl
sulfides and their derivatives were obtained from com-
mercially available 5,5′-thiodisalicylic acid in a manner
similar to that of symmetrical esters described above.
Stoichiometric oxidation of the sulfide 18 with m-
chloroperbenzoic acid gave the sulfoxide 19, while use
of excess oxidant afforded the sulfone 20 in high yield.
To ascertain the necessity of the carboxylic acid func-
tion, the (pivaloyloxy)methyl ester of 18 was prepared
with (pivaloyloxy)methyl iodide and potassium carbon-

Synthesis and Activity of Thielocin B3 Derivatives
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26 5185
Sch em e 2a
a
Reagents: (a) oxalyl chloride; (b) TFA, anisole; (c) H2, Pd-C.
Sch em e 3a
a
Reagents: (a) Me2SO4, K2CO3; (b) KOH, aqueous MeOH; (c) oxalyl chloride; (d) TFA, anisole; (e) m-CPBA; (f) (pivaloyloxy)methyl
iodide, K2CO3.
Sch em e 4a
a
Reagents: (a) NaH; (b) CuBr‚SMe2; (c) KOH, aqueous MeOH; (d) oxalyl chloride; (e) TFA, anisole.
ate (Scheme 3). Diphenyl ether derivatives 28, 29, and
30 were synthesized by the Ullmann reaction in the
presence of copper catalyst. After deprotection of benz-
hydryl esters, the desired diphenyl ethers were prepared
in a similar manner as described above (Scheme 4).
Diphenylmethylene derivatives 31, 32, and 33 having
structural similarity to thielocin B3 were prepared from
6 by using 8 or 10 as an aromatic ring unit in 20, 70,
and 61% yield, respectively. In this case, demethylated
derivative 34 was synthesized to examine the effect of
hydroxy and/or methoxy group for sPLA2 inhibitory
activity (Scheme 5). Lastly, structurally diverse com-
pounds having amino groups were prepared by conven-
tional aminomethylene synthesis from thielavin B25
(Scheme 6). Formylation of thielavin B with hexameth-
ylenetetramine and trifluoroacetic acid, followed by
esterification with diphenyldiazomethane to give 35,
which was then subjected to reductive amination with
benzylglycine or optically active phenethylamine and
NaBH3CN, gave 36, 37, or 38. These amino derivatives
were added to examine the effect of an amino group in
the thielocin series on sPLA2 inhibition.
Biologica l Resu lts a n d Discu ssion
Many nonspecific PLA2 inhibitors have been reported
and considered to affect the “quality of the interface”
by modifying phospholipid bilayer properties that render
phospholipid inaccessible to the enzyme. Thielocin A1
and B3 were found to be independent of the concentra-
tion of the substrate.17,18
In addition, the inhibitory activity of thielocin A1 was
independent of the physical state of the substrate such
as Escherichia coli membranes, phospholipids presented
as surfactant mixed micelles, or sonicated liposomes and
of the type of phospholipids (choline, ethanolamine, or
inositol).17
Moreover, 100% of the fluorescence of Naja mocam-
bique PLA2 was quenched with the molar ratio of
thielocin B3/enzyme at 1.0,18 suggesting that inhibition
of sPLA2 by thielocins occurred by direct interaction
with the enzyme.
The characteristic mode of inhibition was also con-
served in the newly synthesized compounds, showing
that the sPLA2 inhibitory activity of thielocin deriva-

5186 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26
Teshirogi et al.
Ta ble 1. PLA2 Inhibitory Activitya
sPLA2 Inhibition: IC50(mM)
Sch em e 5a
group II
human
group I
human
ring
type-
(X or Y)
compound
rat
recombinant recombinant unit
c
thielocin A1 0.0033
thielocin B3 0.012
12
0.076
0.23
5.8
2.2
0.94
4.1
135
18
>100
100
>100
60
100
>100
55
11
9.0
>100
>100
6.5
6.6
>100
90
>100
>100
10
5b
6
5
5
4
4
4
4
6
6
6
6
4
6
6
4
4
6
6
4
4
4
CH2
phenylene
pyrone
CHdCH-(E)
CH2CH2
S
S
S
SO
SO2
Se
O
O
11
12
13
14
0.12
0.88
0.33
0.040
2.1
16
17
2.5
4.8
18
19
20
21
0.049
0.12
0.05
nid
0.13
0.26
0.069
nid
28
3.4
75
29
30
31
0.042
0.035
3.0
0.10
0.24
3.0
2.8
0.19
2.4
0.14
1.8
0.84
76
34
1.5
O
CH2
CH2
CH2
32
4.6
33
34
36
37
0.13
0.95
0.026
0.30
0.36
320
f
CH2
NHg
NHh
NHi
98
60
38
mepacrine
p-BPB
manoalide
6.7
2.0
a
For detailed assay protocol, see the Experimental Section.
According to the criteria used in this study, it contains five rings,
b
a
Reagents: (a) oxalyl chloride; (e) TFA, anisole; (c) BBr3; (d)
but it may be considered to form six rings because of its fused-
Ph2CN2; (e) H2, Pd-C.
d
ring structure. c See Figure 1. No inhibition. e di-POM ester of
g
h
18. f Eight hydroxy groups. Glycine benzyl ester. Phenethyl-
tives is not due to direct interaction with the phosphati-
dylethanolamine substrate.
i
amine derivative (R-isomer). Phenethylamine derivative (S-
isomer).
The newly synthesized thielocin B3 derivatives were
found to be generally very specific sPLA2-II inhibitors
and inactive for human pancreatic secretory PLA2
(sPLA2-I) except for diphenyl ether derivatives 29 and
30 and a sulfone derivative 20. Interestingly, most of
the active compounds showed comparable or several
times less potent activity for human enzyme than that
for rat enzyme (Table 1). Phenylene derivative 11
surprisingly showed potent inhibitory activity with an
IC50 value 3 times higher than that of thielocin B3, while
the pyrone nucleus significantly lowered the activity.
Although introduction of double bond 13 did not improve
the activity, that of ethylenic single bond 14 maintained
it, particularly for rat sPLA2-II. With regard to the total
number of ring units, it was presumed from the limited
number of natural thielocin derivatives that at least five
aromatic rings were necessary for the manifestation of
sPLA2 inhibitory activity: thielocin A1 and B1 which
have five aromatic rings are potent inhibitors, while
thielavins, naturally occurring weak inhibitors, have
only three rings.19 Also among the newly synthesized
compounds, the critical number of rings for sPLA2
inhibition was clearly four, and the best results were
obtained in the case of six aromatic rings. Unnatural
sulfides are potent inhibitors, and the sulfone derivative
20 shows activity comparable to thielocin B3 and
exhibits an IC50 value of 0.069 µM for human sPLA2-II.
The inhibition by 20 was very similar to that of
thielocin B3. It is also a potent inhibitor for rat sPLA2-
II with an IC50 value of 0.05 µM and a weak inhibitor
for human sPLA2-I (IC50 9.0 µM). However, diesterifi-
cation of terminal carboxylic acids of the sulfide 18 (21)
led to complete loss of the activity. Also, diphenyl ethers
29 and 30 show very potent activity, but seem to have
weak cross inhibitory activity for human sPLA2-I.
Moreover, the activity of 30, more substituted at the core
of the molecule than 29, was found to be almost
comparable to 29, suggesting that the degree of substi-
tution at this position does not affect the activity.
Compared to symmetrical diphenylmethylene compound
32, the unsymmetrical 31 showed similar activity, which
Sch em e 6a
a
Reagents: (a) hexamethylenetetramine, TFA; (b) Ph2CN2; (c) H2NR; (d) NaBH3CN; (e) TFA, anisole.

Synthesis and Activity of Thielocin B3 Derivatives
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26 5187
Ta ble 2. Effect of Thielocin B3 Derivatives on Arachidonic
Acid and Thromboxane B2 Production by Rat Platelet
Stimulated with A23187
and their IC50 values are the lowest of those reported
to date in the literature.21
% of control releasea
Exp er im en ta l Section
compound
concn (µM) arachidonic acid thromboxane B2
Ma ter ia ls. Thielocin A1 and B3 were prepared as previ-
ously reported.25 1-Palmitoyl-2-[1-14C]linoleoylphosphatidyl-
ethanolamine (2.18 GBq/mmol) was purchased from Amer-
sham Corp. L-R-Phosphatidylethanolamine (from egg yolk)
was purchased from Sigma. Rat PLA2-II was purified from
rat platelets.27 Recombinant human PLA2-I and II were
obtained as previously reported.19 Each of the purified PLA2s
showed a single band of approximately 14 kDa on SDS-
polyacrylamide gel electrophoresis (Coomassie brilliant blue
staining). All other reagents were of analytical grade or better.
[3H]AA (238 Ci/mmol) was purchased from New England
Nuclear. A23187, mepacrine, and p-BPB were purchased from
Sigma. Manoalide was purchased from Wako Chemicals.
Assa y of P h osp h olip a se A2. PLA2 activity was measured
by a method described previously.17 The substrate was
prepared by diluting 1-palmitoyl-2-[1-14C]linoleoylphosphati-
dylethanolamine with L-R-phosphatidylethanolamine to the
specific activity of 2000 dpm/nmol. The reaction was started
by addition of the enzyme. The amount of PLA2s was adjusted
to optimize linear kinetics for quantitation, i.e., hydrolysis of
the substrate was less than 20% in all experiments. Thielocin
B3 and other synthesized compounds were added to the assay
tubes as a DMSO solution (2% of the final volume), using a
DMSO enzyme control. Control experiments showed that
DMSO at this concentration had no effect on enzymatic
activities. IC50 values were determined graphically from plots
of percent inhibition that were obtained from three indepen-
dent experiments, each performed in duplicate, versus log
concentration of test compounds.
thielocin B3
10
10
10
10
10
10
10
10
60.3 ( 3.9**
49.2 ( 8.4**
55.2 ( 5.6*
70.7 ( 7.2**
2.8 ( 3.9***
9.0 ( 7.0**
19.7 ( 5.6***
32.7 ( 6.1**
43.7 ( 8.2**
0.1 ( 12.5*
46.3 ( 14.3*
65.8 ( 12.7
0 ( 14.6**
0 ( 15.4**
0 ( 17.1*
11
12
13
18
29
30
36
0 ( 18.8*
a
See the Experimental Section. [(A23187 + inhibitors)cpm -
(DMSO control)cpm]/[(A23187)cpm - (DMSO control)cpm] × 100.
Each values represents the mean ( SEM for three experiments.
*p < 0.05, **p < 0.01, ***p < 0.001 vs control (Student’s t-test).
was also observed in the case of the diphenyl sulfide
series (16 and 17). Although 33 showed the most potent
activity in the diphenylmethylene series, it could not
exceed that of thielocin B3. Changing all methoxy
groups to hydroxy groups 34 led to significant loss of
activity, suggesting this as the cause of disturbance of
the hydrophobic interaction between the enzyme and
the inhibitor16 and the undesired result. Very interest-
ingly, a glycine derivative 36 having three aromatic
rings and a benzyl ester side chain showed activity
comparable to that of thielocin B3. The contribution of
the amino group to sPLA2 inhibitory activity is consid-
ered to occur at the hydrophilic calcium binding site.
Assa y for Ar a ch id on ic a cid Mobiliza tion . Production
of free arachidonic acid (AA) and thromboxane B2 (TXB2) by
rat platelets was measured using the assay described by
Nakano et al.28 Briefly, platelets were prepared from fresh
rat blood anticoagulated with 0.11 volume of acid citrate
dextrose (85 mM trisodium citrate, 70 mM citric acid, and 110
mM glucose) and mixed with PGE1 (1.5 µg/mL). Platelet-rich
plasma was obtained by centrifugation at 160g for 10 min and
layered on 40% bovine serum albumin. Platelets were sedi-
mented at 1200g for 20 min, resuspended at 2 × 109 cells/mL
in the elution buffer (137 mM NaCl, 2.7 mM KCl, 1.0 mM
MgCl2, 3.8 mM NaH2PO4, 3.8 mM Hepes, 5.6 mM glucose, and
0.035% bovine serum albumin, pH 7.35) containing 1.0 µg/mL
of PGE1, and incubated with [3H]AA (20 µCi/mL) for 2.0 h at
room temperature. Platelets were isolated by gel filtration
through a column of Sepharose 2B and suspended in the
elution buffer to a final concentration of 5 × 108 cells/mL. [3H]-
AA-labeled platelets (250 µL) were incubated with A23187 and
various inhibitors, and stimulation was stopped by adding 0.4
volume of 0.1 N HCl. A23187 (2 µM) was added as a DMSO
solution (0.4% of the final volume). Thielocin B3 derivatives
were also added as a DMSO solution (0.2% of the final volume),
using a DMSO control. Control experiments showed that
DMSO at this concentration had no effect on [3H]AA and [3H]-
TXB2 production. [3H]-labeled compounds were extracted and
separated by thin layer chromatography. The area corre-
sponding to AA and TXB2 was scraped off and the radioactivity
was measured.
Newly synthesized thielocin B3 derivatives showed
no significant human cPLA2 inhibition at the concentra-
tion of 20 µM.26 Thus, they were found to be specific
sPLA2-II inhibitors.
The effect of thielocin B3 derivatives on the release
of arachidonic acid and thromboxane B2 from rat
platelets was also examined. The results of the cellular
assay with respect to release of PLA2 products are
summarized in Table 2. Also in this cellular assay,
compounds 18, 29, and 36 suppressed significantly the
production of arachidonic acid and thromboxane B2,
indicating thielocin derivatives are real and potent PLA2
inhibitors.
Con clu sion
We prepared several types of thielocin B3 derivatives
and conducted their SAR study to identify the potent
sPLA2-II inhibitors. The results showed that the num-
ber of aromatic rings is critical for sPLA2-II inhibition
and more than four rings are necessary, with the best
result occurring with six rings. The structures of the
core moiety of inhibitors are not specific: potent inhibi-
tors were found among the phenylene, sulfide, sulfone,
ether, methylene, and amino derivatives and showed
sPLA2-II inhibitory activity comparable to that of thie-
locin B3. Diesterification of two carboxylic acid func-
tions of thielocin B3 are known to cause almost complete
loss of activity,18 and we could obtain the similar result
again. However, monocarboxylic acid derivative 36
showed very potent inhibitory activity, suggesting that
two carboxylic acid groups of both terminals are not
always necessary for expression of the activity. It is
noteworthy that the newly synthesized thielocin deriva-
tives 18, 20, 29, and 36 show very potent human sPLA2-
II inhibitory activity equivalent to that of thielocin B3,
DMSO control (0.4% of the final volume) released [3H]AA
(3490 ( 347 cpm) and [3H]TXB2 (1268 ( 208 cpm). A23187
(2 µM) released [3H]AA (7797 ( 406 cpm) and [3H]TXB2 (3065
( 376 cpm) from [3H]-labeled rat platelet (means ( SEM from
four independent determinations, each performed in triplicate).
The effect of various inhibitors on the increase of [3H]AA and
[3H]TXB2 production by A23187-stimulated rat platelets is
expressed as “% of increase”: [(A23187 + inhibitors)cpm -
(DMSO control)cpm]/[(A23187)cpm - (DMSO control)cpm] ×
100. Each values represents the mean ( SEM for three
experiments; *p < 0.05, **p < 0.01, ***p < 0.001 vs control
(Student’s t-test).
Ch em istr y. Melting points are uncorrected. 1H NMR and
13C NMR spectra were determined at 200 and 50.3 MHz,

5188 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26
Teshirogi et al.
respectively. Liquid secondary ion mass spectra (LSIMS) and
high-resolution (HR)-LSIMS were determined using m-ni-
trobenzyl alcohol as a matrix. Unless otherwise stated, all
reactions were carried out under a nitrogen atmosphere with
anhydrous solvents that had been dried over type 4A molecular
sieves. Drying of an organic phase over anhydrous sodium
sulfate is simply indicated by the word “dried”. Column
chromatography using Merck silica gel 60 or a Merck Lobar
column is referred to as “chromatography on silica gel”.
Meth yl 5-(Acetoxym eth yl)-2,4-d im eth oxy-3,6-d im eth -
ylben zoa te (3). A mixture of 1 (3.70 g, 16.5 mmol), Me2SO4
(4.74 mL, 16.5 mmol × 3), K2CO3 (11.58 g, 16.5 mmol × 5),
and 200 mL of acetone was heated under reflux for 2 h, and
after the mixture was cooled, the resultant solid was filtered
off. The filtrate was partitioned between ethyl acetate and 2
N hydrochloric acid, and the organic phase was washed with
water, dried over Na2SO4, and then concentrated in vacuo to
yield the crude product, which was recrystallized from ether-
n-hexane to provide 6.8 g of 2 as a red oil. The oil was
dissolved in 30 mL of isopropyl alcohol, to the solution was
added NaBH4 (0.94 g, 16.5 mmol × 1.5), and the reaction
mixture was stirred and then partitioned between ethyl
acetate and water. The organic phase was washed with 2 N
hydrochloric acid, followed by with water, dried, and then
concentrated in vacuo. The resultant residue was subjected
to column chromatography (70 g of SiO2, eluent: n-hexane-
ethyl acetate (4:1 to 1:1)) to yield 3.18 g of the alcohol. Then,
the oil was dissolved in 20 mL of CH2Cl2, and to the solution
was added 6 mL of acetic anhydride, 10 mL of pyridine, and
50 mg of (dimethylamino)pyridine, and then the mixture was
stirred at room temperature for 2 h. The reaction mixture was
partitioned between ethyl acetate and 2 N hydrochloric acid,
and the organic phase was washed with water, dried, and then
concentrated in vacuo to yield the crude product, which was
recrystallized from ether-n-hexane to yield 3.35 g (69%) of
the acetate 3: colorless pillars; mp 69-70 °C; 1H NMR (CDCl3)
δ 2.07 (s, 3H), 2.22 (s, 3H), 2.24 (s, 3H), 3.74 (s, 3H), 3.78 (s,
3H), 3.93 (s, 3H), 5.18 (s, 2H). Anal. (C15H20O6) C, H.
column chromatography (80 g of SiO2; eluent, n-hexane-ethyl
acetate (19:1 to 1:1)) to yield 5.76 g (93%) of the ester 8:
colorless pillars; mp 125-127 °C; H NMR (CDCl3) δ 2.06 (s,
3H), 2.11 (s, 3H), 2.15 (s, 3H), 3.52 (s, 3H), 4.82 (s, 1H), 7.17
(s, 1H), 7.22-7.50 (m, 10H); IR (Nujol) 3360, 1695, 1585, 1290,
1205 cm-1. Anal. (C24H24O4) C, H.
1
4-((4-(Be n zh yd r yloxyca r b on yl)-3-m e t h oxy-2,5,6-t r i-
m eth ylph en oxy)car bon yl)-3-m eth oxy-2,5,6-tr im eth ylph e-
n ol (10). Compound 7 (4.19 g, 13.28 mmol × 1.05) was
dissolved in 20 mL of CH2Cl2, oxalyl chloride (4.25 mL, 13.28
mmol × 1.05 × 3.5) was added at room temperature, the
resultant mixture was stirred at room temperature for 30 min,
and then the mixture was gently warmed under reflux for 30
min. After the mixture was concentrated in vacuo, the residue
was dissolved in THF, and the solution was again concentrated
in vacuo. Alternatively, compound 8 (5.00 g, 13.28 mmol) was
dissolved in 20 mL of THF, n-BuLi (1.6 M solution in hexane,
8.30 mL, 13.28 mmol) was added gradually to the solution at
-78 °C, and then the solution was stirred at the same
temperature for 30 min. To the reaction mixture was added
a solution of the acid chloride of 7 obtained above in THF (30
mL) at -78 °C, and the mixture was stirred at the same
temperature for 10 min. The mixture was allowed to warm
slowly to room temperature and left to stand overnight. The
reaction mixture was partitioned between ethyl acetate and 1
N hydrochloric acid, and the organic phase was washed with
water and then dried. After the mixture was concentrated in
vacuo, the residue was subjected to column chromatography
(200 g of SiO2; eluent, toluene-ethyl acetate (0-10%)) to yield
1
7.32 g of 9 (84%) as a colorless crystal: mp 183-185 °C; H
NMR (CDCl3) δ 2.09 (s, 3H), 2.22 (s, 3H), 2.25 (s, 3H), 2.26 (s,
3H), 2.28 (s, 3H), 2.37 (s, 3H), 3.58 (s, 3H), 3.81 (s, 3H), 4.80
(s, 2H), 7.20 (s, 1H), 7.28-7.55 (m, 15H); IR (Nujol) 1760, 1723,
1573, 1455, 1156, 740, 697 cm-1. Anal. (C42H42O7) C, H.
Compound 9 (7.32 g, 11.1 mmol) was subject to hydrogena-
tion by a procedure similar to that of 8, the resultant crude
product (4.31 g, 10.7 mmol) was benzhydrylated by a similar
procedure to that of 8, and the resultant product was recrys-
tallized from ether-n-hexane to yield 5.31 g (84%) of 10:
(3-Ca r bom eth oxy-4,6-d ih yd r oxy-2,5-d im eth ylp h en yl)-
(3-ca r b om et h oxy-4,6-d im et h oxy-2,5-d im et h ylp h en yl)-
m eth a n e (5). Compound 4 (1.45 g, 6.74 mmol × 1.1), which
is known in the literature and the acetate 3 (2.00 g, 6.74 mmol)
were dissolved in 25 mL of toluene, to the solution was added
BF3‚OEt2 (0.83 mL, 6.74 mmol), and the mixture was stirred
at room temperature for 15 min. To the reaction mixture was
added ethyl acetate, and then the mixture was washed with
water, dried, and concentrated in vacuo. The residue was
subjected to column chromatography (SiO2; Merck, Lobar B,
eluent: n-hexane-ethyl acetate) to yield 2.91 g of 5 (100%):
1
colorless pillars; mp 195-197 °C; H NMR (CDCl3) δ 2.08 (s,
3H), 2.19-2.24 (m, 12H), 2.37 (s, 3H), 3.56 (s, 3H), 3.80 (s,
3H), 4.95 (s, 1H), 7.19 (s, 1H), 7.28-7.52 (m, 10H); IR (Nujol)
3390, 1737, 1706, 1285, 1156, 759, 700 cm-1. Anal. (C35H36O7)
C, H.
Bis[4-[(4-ca r boxy-3-m eth oxy-2,5,6-tr im eth ylp h en oxy)-
ca r bon yl]-3-m eth oxy-2,5,6-tr im eth ylp h en oxy]isop h th a -
la te (11). Compounds 11, 12, and 13 were prepared by a
procedure similar to that of 10 described above. Compound
11 (yield 60% from isophthalic acid and 2 equiv of 10):
colorless prisms; mp 267-269 °C; 1H NMR (DMSO-d6) δ 2.08-
2.26 (m, 30H), 2.37 (s, 6H), 3.73 (s, 6H), 3.79 (s, 6H), 7.98 (t,
J ) 9 Hz, 1H), 8.66 (dd, J ) 9, 1 Hz, 2H), 8.93 (m, 1H); IR
1
colorless prisms; mp 169-170 °C; H NMR (CDCl3) δ 2.07 (s,
6H), 2.25 (s, 3H), 2.55 (s, 3H), 3.75 (s, 6H), 3.88 (s, 3H), 3.92
(s, 3H), 3.95 (s, 2H), 7.20 (s, 1H), 11.38 (s, 1H); IR (Nujol) 3285,
1743, 1644 cm-1. Anal. (C23H28O8) C, H.
(KBr) 3430, 2940, 1745, 1700, 1222, 1160 cm-1
.
Anal.
Bis(3-ca r b oxy-4,6-d im e t h oxy-2,5-d im e t h ylp h e n yl)-
m eth a n e (6). Compound 5 (2.91 g, 7.4 mmol) was methox-
ylated in a procedure similar to that of compound 3, and the
crude residue was subjected to column chromatography (60 g
of SiO2, eluent: toluene-ethyl acetate (9:1 to 4:1)) to yield 2.97
g of methylated product. The product (600 mg, 130 mmol) was
hydrolyzed in a usual procedure using KOH in aqueous DMSO
to yield 559 mg (95%) of 6: colorless crystal; mp 265-267 °C;
1H NMR (DMSO-d6) δ 1.96 (s, 6H), 2.12 (s, 6H), 3.48 (s, 6H),
3.66 (s, 6H), 3.98 (s, 2H); IR (KBr) 3670-2400, 3430, 2940,
1710, 1217, 1105 cm-1. Anal. (C23H28O8‚0.4H2O) C, H.
4-Hyd r oxy-2-m eth oxy-3,5,6-tr im eth ylben zoic Acid (8).
Compound 7 (5.0 g, 16.6 mmol), which is known in the
literature, was subject to hydrogenation by a usual procedure
using Pd-C in a mixture of AcOEt and MeOH to yield 3.8 g
of the hydroxy compound. The product (3.36 g, 16.0 mmol)
was dissolved in 30 mL of ethyl acetate, diphenyldiazomethane
(5.9 g, 16 mmol) was added at room temperature, and the
mixture was left to stand overnight. An excess of diphenyl-
diazomethane was decomposed by adding 2 N hydrochloric
acid, and the resultant mixture was extracted with ethyl
acetate. The organic phase was washed with water, dried, and
then concentrated in vacuo. The residue was subjected to
(C52H54O16‚H2O) C, H.
Bis[4-[(4-ca r boxy-3-m eth oxy-2,5,6-tr im eth ylp h en oxy)-
ca r b on yl]-3-m et h oxy-2,5,6-t r im et h ylp h en oxy]ch elid o-
n a te (12): colorless plates; mp 294-296 °C; 1H NMR (CDCl3)
δ 2.16 (s, 6H), 2.21 (s, 6H), 2.26 (s, 6H), 2.29 (s, 6H), 2.36 (s,
6H), 2.42 (s, 6H), 3.84 (s, 6H), 3.87 (s, 6H), 7.52 (s, 2H). Anal.
(C51H52O18) C, H.
(E)-1,2-Bis[4-[(4-car boxy-3-m eth oxy-2,5,6-tr im eth ylph e-
n oxy)ca r b on yl]-3-m et h oxy-2,5,6-t r im et h ylp h en oxyca r -
bon yl]eth en e (13). Compound 13 was recrystallized from
N,N-dimethylformamide: colorless plates; mp > 300 °C; 1H
NMR (DMSO-d6) δ 2.45 (s, 6H), 2.48 (s, 6H), 2.52 (s, 6H), 2.53
(s, 12H), 2.70 (s, 6H), 4.07 (s, 6H), 4.11 (s, 6H), 7.78 (s, 2H).
Anal. (C48H52O16‚1/2H2O‚1/2DMF) C, H.
1,2-Bis[[4-[(4-ca r b oxy-3-m et h oxy-2,5,6-t r im et h ylp h e-
n oxy)ca r bon yl]-3-m eth oxy-2,5,6-tr im eth ylp h en oxy]ca r -
bon yl]eth a n e (14). Compound 14 was obtain by catalytic
reduction using Pd-C as a catalyst in a mixture of MeOH and
acetic acid in 92% yield: colorless plates; mp > 270 °C dec; 1H
NMR (DMSO-d6) δ 2.04 (s, 6H), 2.07 (s, 6H), 2.17 (s, 6H), 2.18
(s, 6H), 2.19 (s, 6H), 2.32 (s, 6H), 3.18 (s, 4H), 3.72 (s, 6H),
3.73 (s, 6H). Anal. (C48H54O6) C, H.

Synthesis and Activity of Thielocin B3 Derivatives
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26 5189
3-Ca r boxy-4-m eth oxyp h en yl 3-[[4-[(4-Ca r boxy-3-m eth -
oxy-2,5,6-tr im eth ylp h en oxy)ca r bon yl]-3-m eth oxy-2,5,6-
t r im et h ylp h en oxy]ca r bon yl]-4-m et h oxyp h en yl Su lfid e
(16). 5,5′-Thiodisalicylic acid was methylated with dimethyl
sulfate and K2CO3, and the resulting diester was hydrolyzed
with KOH in aqueous MeOH as described in the case of 6 to
give the dimethoxy dicarboxylic acid 15 in 76% yield: colorless
prisms; mp 158-159 °C. Anal. (C16H14O6S) C, H, S.
temperature for 16 h. To the mixture was added 200 mL of
ice-water, and the resultant mixture was extracted with ethyl
acetate. The organic phase was washed with water and dried
(Na2SO4), and the solvent was evaporated in vacuo to yield
0.65 g of the crude product. The product was recrystallized
from ethyl acetate-n-hexane to provide 0.516 g (85%) of 21:
mp 120 °C dec; 1H NMR (CDCl3) δ 1.26 (s, 18H), 2.13 (s, 6H),
2.17 (s, 6H), 2.23 (s, 12H), 2.26 (s, 6H), 2.39 (s, 6H), 3.79 (s,
6H), 3.81 (s, 6H), 3.96 (s, 6H), 6.00 (s, 4H), 7.05 (d, J ) 9.0
Hz, 2H), 7.59 (dd, J ) 2.5, 8.7 Hz, 2H), 8.08 (d, J ) 2.4 Hz,
2H). Anal. (C72H82O22S‚1/2hexane) C, H, S.
Bis(3-ca r boxy-4-m eth oxyp h en yl) Eth er (26) a n d 5-(3-
Ca r b oxy-4-m et h oxy-5-m et h ylp h en oxy)-2,4-d im et h oxy-
ben zoic Acid (27). Compound 22 (200 mg, 1.1 mmol) was
dissolved in 5 mL of DMF, 46 mg of NaH (60% in oil) (1.15
mmol) was added, and the mixture was stirred for 1.5 h. After
690 mg (3.36 mmol) of copper(I) bromide-dimethyl sulfide
complex was added to the mixture, 270 mg of the bromide 24
(1.1 mg) which had been dissolved in 0.5 mL of DMF was
added, and the resultant mixture was refluxed with stirring
for 22 h. After cooling, the reaction mixture was partitioned
between 1 N hydrochloric acid and ethyl acetate, and the
organic phase was washed with 1 N hydrochloric acid, water,
and the saturated brine, successively, and then dried. The
solvent was evaporated to dryness, and the residue (383 mg)
was applied to silica gel chromatography (40 g of SiO2; eluent,
ethyl acetate-n-hexane (2:3)) to yield 163 mg of methyl ester
of 26. The methyl ester was hydrolyzed by usual procedure
using KOH in aqueous MeOH to give the dicarboxylic acid 26
in 38% yield: colorless plates; mp 178-180 °C; 1H NMR
(DMSO-d6): δ 3.81 (s, 6H), 7.10-7.23 (m, 6H). Anal. (C16H14O7)
C, H.
Compound 15 (1.31 g, 3.9 mmol) was dissolved in 50 mL of
methylene chloride, and to the solution was added 0.8 g (4.1
mmol) of diphenyldiazomethane under cooling with ice-water,
and the mixture was stirred for 1.8 h. The solvent was
evaporated in vacuo, the residue was applied to column
chromatography (SiO2, 150 g), and it was eluted with benzene-
ethyl acetate (5:1) and then chloroform-methanol (9:1) to yield
1.06 g of monobenzhydryl ester. The half-ester was reacted
with 10 in a manner similar to that of 11 to give the
unsymmetrical sulfide 16 in 17% overall yield: colorless plates;
mp 192-194 °C; 1H NMR (CDCl3) δ 2.14 (s, 3H), 2.19 (s, 3H),
2.25 (s, 3H), 2.29 (s, 3H), 2.35 (s, 3H), 2.39 (s, 3H), 3.83 (s,
3H), 3.85 (s, 3H), 3.97 (s, 3H), 4.08 (s, 3H), 7.03 (d, J ) 8.8
Hz, 1H), 7.05 (d, J ) 8.8 Hz, 1H), 7.55 (dd, J ) 2.4, 8.8 Hz,
1H), 7.60 (dd, J ) 2.4, 8.8 Hz, 1H), 8.08 (d, J ) 2.4 Hz, 1H),
8.18 (d, J ) 2.4 Hz, 1H). Anal. (C38H38O12S) C, H, S.
Bis[3-[(4-ca r boxy-3-m eth oxy-2,5,6-tr im eth ylp h en oxy)-
ca r bon yl]-4-m eth oxyp h en yl] Su lfid e (17). Compound 17
was obtained from 15 by the reaction with 2 equiv of 8 and
deprotection in 67% yield: colorless plates; mp 237-239 °C;
1H NMR (CDCl3) δ 2.08 (s, 6H), 2.09 (s, 6H), 2.32 (s, 6H), 3.81
(s, 6H), 3.95 (s, 6H), 7.03 (d, J ) 8.8 Hz, 2H), 7.60 (dd, J )
2.4, 8.8 Hz, 2H), 8.09 (d, J ) 2.4 Hz, 2H). Anal. (C38H38O12S‚1/
2benzene) C, H, S.
Compound 27 was prepared in a manner similar to that
described above from 23 and 25 in 13% yield: pale yellow
plates; mp 171-174 °C; 1H NMR (DMSO-d6) δ 2.22 (s, 3H),
3.69 (s, 3H), 3.86 (s, 3H), 3.90 (s, 3H), 6.82 (d, J ) 3.0 Hz,
1H), 6.87 (s, 1H), 6.95 (d, J ) 3.0 Hz, 1H), 7.39 (s, 1H); IR
Bis[3-[[4-[(4-ca r b oxy-3-m et h oxy-2,5,6-t r im et h ylp h e-
n oxy)ca r bon yl]-3-m eth oxy-2,5,6-tr im eth ylp h en oxy]ca r -
bon yl]-4-m eth oxyp h en yl] Su lfid e (18). Compound 18 was
prepared in a manner similar to that of 17 using 2 equiv of 10
1
in 35% yield: colorless plates; mp 278-280 °C dec; H NMR
(KBr) 3680-2320, 3440, 2960, 1695, 1616, 1215, 1120 cm-1
Anal. (C18H18O8‚0.3H2O) C, H.
.
(CDCl3) δ 2.14 (s, 6H), 2.18 (s, 6H), 2.25 (s, 6H), 2.29 (s, 6H),
2.36 (s, 6H), 2.40 (s, 6H), 3.83 (s, 6H), 3.86 (s, 6H), 3.97 (s,
6H), 7.05 (d, J ) 8.8 Hz, 2H), 7.60 (dd, J ) 2.0, 8.8 Hz, 2H),
8.10 (d, J ) 2.0 Hz, 2H). Anal. (C60H62O18S‚1/2H2O) C, H, S.
Bis[3-[[4-[(4-ca r b oxy-3-m et h oxy-2,5,6-t r im et h ylp h e-
n oxy)ca r bon yl]-3-m eth oxy-2,5,6-tr im eth ylp h en oxy]ca r -
bon yl]-4-m eth oxyp h en yl] Su lfoxid e (19). Dibenzhydryl
ester of 18 (200 mg, 0.14 mmol) was dissolved in 5 mL of
methylene chloride, to the solution was added 30 mg (0.14
mmol) of m-chloroperbenzoic acid (80%) under cooling with
ice-water, and the mixture was stirred at 0 °C for 4.5 h. The
reaction mixture was washed with 5% aqueous sodium thio-
sulfate, an aqueous saturated sodium bicarbonate, and water,
successively, and dried, and then the solvent was evaporated
in vacuo to yield 245 mg of the crude product. The product
was purified by column chromatography (40 g of SiO2; eluent,
ethyl acetate-n-hexane (2:1)) to yield 151 mg of the dibenz-
hydryl ester of 19. Deprotection of the ester gave 95 mg (60%)
Bis[3-[(4-ca r boxy-3-m eth oxy-2,5,6-tr im eth ylp h en oxy)-
ca r bon yl]-4-m eth oxyp h en yl] Eth er (28). Compound 28
was obtained by a manner similar to that of 17, a thia analogue
of 28, in 63% yield: colorless plates; mp 194-195 °C; 1H NMR
(CDCl3) δ 2.11 (s, 6H), 2.12 (s, 6H), 2.33 (s, 6H), 3.82 (s, 6H),
3.95 (s, 6H), 7.06 (d, J ) 9.2 Hz, 2H), 7.27 (dd, J ) 3.0, 9.2
Hz, 2H), 7.73 (d, J ) 3.0 Hz, 2H). Anal. (C38H38O13‚H2O) C,
H.
Bis[3-[[4-[(4-ca r b oxy-3-m et h oxy-2,5,6-t r im et h ylp h e-
n oxy)ca r bon yl]-3-m eth oxy-2,5,6-tr im eth ylp h en oxy]ca r -
bon yl]-4-m eth oxyph en yl] Eth er (29) an d 3-[[4-[(4-Car boxy-
3-m eth oxy-2,5,6-tr im eth ylph en oxy)ca r bon yl]-3-m eth oxy-
2,5,6-t r im et h ylp h en oxy]ca r b on yl]-4-m et h oxy-5-m et h -
ylp h en yl 5-[[4-[(4-Ca r b oxy-3-m et h oxy-2,5,6-t r im et h yl-
p h en oxy)ca r bon yl]-3-m eth oxy-2,5,6-tr im eth ylp h en oxy]-
ca r bon yl]-2,4-d im eth oxyp h en yl Eth er (30). Compound 29
was prepared by a manner similar to that of 28 using 2 equiv
of 10. The yield was 83% from 26: colorless plates; mp 258-
1
of 19: mp 193-195 °C; H NMR (CDCl3) δ 2.11 (s, 6H), 2.16
(s, 6H), 2.23 (s, 6H), 2.27 (s, 6H), 2.34 (s, 6H), 2.38 (s, 6H),
3.81 (s, 6H), 3.85 (s, 6H), 4.01 (s, 6H), 7.21 (d, J ) 9.0 Hz,
2H), 7.92 (dd, J ) 2.2, 9.0 Hz, 2H), 8.35 (d, J ) 2.2 Hz, 2H).
Anal. (C60H62O19S‚H2O) C, H, S.
1
260 °C; H NMR (CDCl3) δ 2.16 (s, 6H), 2.20 (s, 6H), 2.26 (s,
6H), 2.29 (s, 6H), 2.36 (s, 6H), 2.40 (s, 6H), 3.83 (s, 6H), 3.86
(s, 6H), 3.97 (s, 6H), 7.08 (d, J ) 9.2 Hz, 2H), 7.29 (dd, J )
3.2, 9.2 Hz, 2H), 7.74 (d, J ) 3.2 Hz, 2H). Anal. (C60H62O19‚1/
2EtOH) C, H.
Bis[3-[[4-[(4-ca r b oxy-3-m et h oxy-2,5,6-t r im et h ylp h e-
n oxy)ca r bon yl]-3-m eth oxy-2,5,6-tr im eth ylp h en oxy]ca r -
bon yl]-4-m eth oxyp h en yl] Su lfon e (20). Compound 18 was
oxidized with 4 equiv of m-chloroperbenzoic acid in a manner
similar to that of 19 to give 20 in 81% yield: colorless plates;
Compound 30 was also prepared from 23 and 25 via the
coupling product 27 in a manner similar to that of 29 in 5.5%
overall yield: colorless powder (hygroscopic); 1H NMR (DMSO-
d6) δ 1.90 (s, 3H), 1.98-2.90 (m, 27H), 2.28-2.37 (m, 9H),
3.68-3.81 (m, 15H), 3.96 (s, 3H), 4.00 (s, 3H), 7.03 (s, 1H),
7.18 (d, J ) 2.8 Hz, 1H), 7.28 (d, J ) 2.8 Hz, 1H), 7.80 (s, 1H);
1
mp 294-295 °C dec; H NMR (CDCl3) δ 2.12 (s, 6H), 2.16 (s,
6H), 2.23 (s, 6H), 2.27 (s, 6H), 2.34 (s, 6H), 2.38 (s, 6H), 3.80
(s, 6H), 3.85 (s, 6H), 4.04 (s, 6H), 7.20 (d, J ) 9.0 Hz, 2H),
8.20 (dd, J ) 2.2, 9.0 Hz, 2H), 8.65 (d, J ) 2.6 Hz, 2H). Anal.
(C60H62O20S‚1/2H2O) C, H, S.
IR (KBr) 3430, 2930, 1745, 1575, 1462, 1150 cm-1
. Anal.
(C62H66O20‚3.5H2O) C, H.
Bis[3-[[4-[[4-[[[(p iva loyloxy)m et h yl]oxy]ca r b on yl]-3-
m eth oxy-2,5,6-tr im eth ylp h en oxy]ca r bon yl]-3-m eth oxy-
2,5,6-tr im eth ylph en oxy]car bon yl]-4-m eth oxyph en yl] Su l-
fid e (21). A mixture containing 500 mg (0.45 mmol) of 18,
265 mg (1.09 mmol) of (pivaloyloxy)methyl iodide, 216 mg (1.56
mmol) of K2CO3, and 25 mL of acetone was stirred at room
(5-Ca r b oxy-2,4-d im et h oxy-3,6-d im et h ylp h en yl)[5-[[4-
[(4-car boxy-3-m eth oxy-2,5,6-tr im eth ylph en oxy)car bon yl]-
3-m e t h oxy-2,5,6-t r im e t h ylp h e n oxy]ca r b on yl]-2,4-d i-
m eth oxy-3,6-d im eth ylp h en yl]m eth a n e (31). The methyl-
enic compound 31 was obtained from 6 by half-ester formation
with diphenyldiazomethane, followed by coupling reaction with

5190 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26
10. This procedure is similar to that of 16, a thia analogue of
Teshirogi et al.
mL of dichloromethane, and then 0.6 mL (5.5 mmol) of anisole
was added. To the mixture was slowly added dropwise a
solution of 0.85 mL (11 mmol) of trifluoroacetic acid in 4 mL
of dichloromethane in an ice cooling bath. The mixture was
directly stirred for 4 h and concentrated in vacuo, 1 N
hydrochloric acid was added to the residue, and the precipi-
tated solid was collected by filtration. The solid was washed
with water repeatedly, dissolved in 50 mL of ethyl acetate,
washed with 0.1% aqueous phosphate solution and with water,
and then dried. The solvent was evaporated to yield 865 mg
of a yellow oil. The oil was subjected to silica gel chromatog-
raphy (SiO2; 50 g), eluting with chloroform-methanol (10:1),
to yield 720 mg of 36 as pale yellow plates (88%): mp 134-
136 °C dec. Anal. (C41H45NO12‚EtOH‚H2O) C, H, N.
1
31: colorless powder; H NMR (CD3OD) δ 2.00 (s, 3H), 2.11
(s, 3H), 2.18-2.34 (m, 21H), 2.41 (s, 3H), 3.57 (s, 3H), 3.63 (s,
3H), 3.77 (s, 3H), 3.82 (s, 3H), 3.83 (s, 3H), 4.18 (s, 2H); IR
(KBr): 3440, 2950, 1745, 1703, 1570, 1460, 1145, 1095, 1075
cm-1; MS (LSI) 817 (M + H)+.
Bis[5-[(4-ca r boxy-3-m eth oxy-2,5,6-tr im eth ylp h en oxy)-
ca r b on yl]-2,4-d im et h oxy-3,6-d im et h ylp h en yl]m et h a n e
(32). Compound 32 or 33 was prepared from 6 in a manner
similar to that of 17 or 18, respectively: yield (70%); colorless
plates; mp 267-269 °C; 1H NMR (DMSO-d6) δ 2.04-2.28 (m,
30H), 3.52 (s, 6H), 3.70 (s, 6H), 3.74 (s, 6H), 4.11 (s, 2H); IR
(KBr) 3440, 2940, 1740, 1700, 1460, 1155 cm-1
(C45H52O14) C, H.
.
Anal.
(R)-4-[[[4-[[[5-[(sec-P h en eth yla m in o)m eth yl]-2,4-d ih y-
dr oxy-3,6-dim eth ylph en yl]car bon yl]oxy]-2-m eth oxy-3,5,6-
t r im e t h ylp h e n yl]ca r b on yl]oxy]-2-m e t h oxy-3,5,6-t r i-
m eth ylben zoic Acid (37) a n d (S)-4-[[[4-[[[5-[(sec-P h en -
eth yla m in o)m eth yl]-2,4-d ih yd r oxy-3,6-d im eth ylp h en yl]-
car bon yl]oxy]-2-m eth oxy-3,5,6-tr im eth ylph en yl]car bon yl]-
Bis[5-[[4-[(4-ca r b oxy-3-m et h oxy-2,5,6-t r im et h ylp h e-
n oxy)ca r bon yl]-3-m eth oxy-2,5,6-tr im eth ylp h en oxy]ca r -
bon yl]-2,4-d im eth oxy-3,6-d im eth ylp h en yl]m eth a n e (33):
yield (61%); colorless powder; 1H NMR (DMSO-d6) δ 2.12-
2.40 (m, 48H), 3.54 (s, 6H), 3.73 (s, 6H), 3.77 (s, 12H), 4.14 (s,
2H); IR (KBr) 3680-2400, 3450, 2940, 1743, 1697, 1140 cm-1
.
oxy]-2-m et h oxy-3,5,6-t r im et h ylb en zoic
Acid
(38).
Anal. (C67H76O20) C, H.
Compound 35 was reacted in a procedure similar to that of
compound 36 except that (R)-phenethylamine was used in-
stead of glycine benzyl ester 37: yield 13%; mp 208-210 °C
dec; [R]24D +49.5 ( 0.9° (c 1, dioxane); 1H NMR (CDCl3) δ 1.57
(d, J ) 6.8 Hz, 3H), 2.13 (s, 3H), 2.15 (s, 6H), 2.23 (s, 3H),
2.26 (s, 3H), 2.29 (s, 3H), 2.40 (s, 3H), 2.42 (s, 3H), 3.83 (s,
3H), 3.84 (s, 3H), 3.94 (m, 2H), 7.37 (m, 5H); MS (LSI) 700 (M
+ H) +. Anal. (C40H45NO10‚0.5H2O) C, H, N.
Bis[5-[[4-[(4-ca r b oxy-3-h yd r oxy-2,5,6-t r im et h ylp h en -
oxy)ca r bon yl]-3-h yd r oxy-2,5,6-tr im eth ylp h en oxy]ca r bo-
n yl]-2,4-d ih yd r oxy-3,6-d im eth ylp h en yl]m eth a n e (34). A
mixture of the benzhydryl ester of 33 (500 mg, 326 µM), BBr3
(371 µL, 326 µM × 12), and CH2Cl2 (15 mL) was stirred at
room temperature for 6 h, the reaction was quenched with
water, and then the resultant mixture was partitioned between
ethyl acetate and brine. The organic phase was washed with
brine three times, dried, and concentrated in vacuo. The
residue was dissolved in ethyl acetate (6 mL), and the solution
was subjected to esterification with diphenyldiazomethane.
After purification by SiO2 column chromatography, the product
was subjected to catalytic hydrogenolysis using Pd-C to yield
(S)-Isomer 38 was obtained by a procedure similar to that
described above: mp 192-194 °C dec; [R]25 -51.0 ( 1.8˚ (c
D
0.5, dioxane). Anal. (C40H45NO10‚1.5H2O) C, H, N.
Ack n ow led gm en t. The authors gratefully acknowl-
edge Dr. Hitoshi Arita, General Manager of Discovery
Research Laboratories II, Shionogi Co. & LTD., for
helpful discussions and for providing us with the
recombinant proteins.
1
34 in 15% overall yield: colorless plates; mp 162 °C dec; H
NMR (DMSO-d6) δ 1.96-2.40 (m, 48H), 4.09 (s, 2H), 9.43 (s,
2H), 9.90 (s, 2H); IR (Nujol) 3700-2560, 1670, 1455, 1155 cm-1
;
MS (LSI) 1088 (M•+), 1089 (M + H+). Anal. (C59H60O20‚3.5H2O)
C, H.
4-[[[4-[[[5-[[[(Ben zyloxycar bon yl)m eth yl]am in o]m eth yl]-
2,4-dih ydr oxy-3,6-dim eth ylph en yl]car bon yl]oxy]-2-m eth -
oxy-3,5,6-tr im eth ylph en yl]car bon yl]oxy]-2-m eth oxy-3,5,6-
tr im eth ylben zoic Acid (36). Compound 35 was prepared
from thielavin B by a known procedure in the literature,24 and
2 g (2.6 mmol) of 35 and 8.9 g (26 mmol) of glycine benzyl
ester p-toluenesulfonate were dissolved in 30 mL of a dried
dimethylformamide. To the solution was add 4.3 g (52 mmol)
of sodium acetate, and the mixture was stirred for 3.5 h. The
reaction mixture was poured into 300 mL of cooled water to
precipitate crystals, which were collected by filtration. The
crystals were dissolved in 100 mL of ethyl acetate, and the
solution was washed with 100 mL of water and 100 mL of brine
and then dried. The solvent was evaporated to yield 2.58 g of
the formimino derivative as a yellow foam. The material was
recrystallized from ethyl acetate-n-hexane (1:2) to yield 2.2
g of the desired compound (92%) as yellow pillars: mp 153-
155 °C. Anal. (C54H53NO12) C, H, N.
The formimino derivative (2.15 g, 2.37 mmol) obtained in
the above step was dissolved in 30 mL of dried dimethylform-
amide, and then 80 mL of dried methanol and 2 mL of acetic
acid were added. A solution of 0.3 g of sodium cyanoborohy-
dride in 4 mL of methanol was added to the mixture in a
stream of nitrogen, under cooling in a ice bath over 15 min.
The resulting mixture was directly stirred for 3 h and then
allowed to stand overnight at 4 °C. The methanol was
evaporated in vacuo, and the residue was poured into cooled
water. The mixture was acidified with 1 N hydrochloric acid
and then basified with a saturated sodium bicarbonate solu-
tion. The precipitated crystals were collected by filtration and
dissolved in 200 mL of ethyl acetate, and the solution was
washed with 100 mL of brine and dried. The solvent was
evaporated to yield 2.33 g of the yellow oil. The oil was
subjected to silica gel chromatography (SiO2; 120 g), eluting
with ethyl acetate-n-hexane (1:2) to yield 1.07 g of the amino
derivative as a pale yellow oil (50%).
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