Synthesis and biological activities of reveromycin A and spirofungin A derivatives

Article abstract of DOI:10.1016/j.bmcl.2008.05.054

Various derivatives of reveromycin A, an inhibitor of eukaryotic cell growth, and spirofungin A, focusing on the 5S hydroxyl group and C18 hemisuccinyl group, were synthesized and their inhibitory effects on both the isoleucyl-tRNA synthetase activity and

Full text of DOI:10.1016/j.bmcl.2008.05.054

Bioorganic & Medicinal Chemistry Letters 18 (2008) 3756–3760
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological activities of reveromycin A and spirofungin
A derivatives
b,
Takeshi Shimizu a, , Takeo Usui , Makoto Fujikura a, Makoto Kawatani b, Tomoharu Satoh a,
*
Kiyotaka Machida b,à, Naoki Kanoh b,§, Je-Tae Woo c, Hiroyuki Osada b, Mikiko Sodeoka a
a Synthetic Organic Chemistry Laboratory, RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama 351-0198, Japan
b Antibiotics Laboratory, RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama 351-0198, Japan
c Department of Biological Chemistry, Chubu University, Matsumoto-cho, Kasugai, Aichi 487-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Various derivatives of reveromycin A, an inhibitor of eukaryotic cell growth, and spirofungin A, focusing
on the 5S hydroxyl group and C18 hemisuccinyl group, were synthesized and their inhibitory effects on
both the isoleucyl-tRNA synthetase activity and the survival of osteoclasts, and activities on the morpho-
logical reversion of srcts-NRK cells were examined. It was found that 2,3-dihydroreveromycin A is the
promising derivative of reveromycin A based on the activity and stability.
Received 8 April 2008
Revised 8 May 2008
Accepted 9 May 2008
Available online 17 May 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Reveromycin A
Spirofungin A
Isoleucyl-tRNA synthetase
Osteoclasts
srcts-NRK cells
Reveromycin A (1, Fig. 1) is a polyketide-type antibiotic isolated
from the soil actinomycete Streptomyces as an inhibitor of mito-
genic activity induced by the epidermal growth factor (EGF) in a
mouse epidermal keratinocyte.1 Reveromycin A (1) showed several
biological activities such as the morphological reversion of srcts-
NRK cells without any noticeable cytotoxicity (EC50 = 1.58 lg/mL),
the anti-proliferative activity against human tumor cell lines
(IC50 = 1.3–2.0 lg/mL) as well as antifungal activity (MIC = 2.0 lg/
mL, pH 3).2 In vitro studies revealed that 1 is a selective inhibitor
of protein synthesis in eukaryotic cells (IC50 = 40 nM).3 In addition,
the molecular target of 1 was identified as the isoleucyl-tRNA syn-
thetase (IleRS) based on yeast genetics and biochemical studies.4
The IC50 of this enzyme was 2.95 ng/mL, while the other amino-
acyl-tRNA synthetases were not inhibited. Recently, it was found
that 1 inhibits bone resorption by specifically inducing apoptosis
in osteoclasts.5
group and two alkyl groups in the structure.6,7 Based on its strong
biological activity as a potential drug and its synthetically chal-
lenging molecular architecture, the first asymmetric total synthesis
of 1 was accomplished by our group.8 We have also reported the
chemical modification of natural reveromycin A (1) and its biolog-
ical activities.9 It was shown that the C5 hydroxyl group and C24
carboxyl group in 1 are particularly important for these activities
from the comparison of the activities of the monoesters 3–5 and
C5 protected derivatives 6–8 (Fig. 1 and Table 1). However, the
C18 methoxy compound 31 among the modified derivatives was
the most active and the inhibitory effect on the IleRS activity was
1/20 of that of 1. We now report the synthesis of the circumstantial
derivatives related to the C5 hydroxyl group and the C18 hemi-
succinyl group in order to create more active and stable com-
pounds, and their biological activities such as cell death
inducibility of the osteoclasts in an addition to inhibition of the
IleRS activity, and morphological reversion activity on srcts-NRK
cells. We have found that 2,3-dihydroreveromycin A is the promis-
ing derivative of 1.
Compound 1 includes the 6,6-spiroketal core bearing a hemi-
succinate, two unsaturated side chains with a terminal carboxyl
We synthesized derivatives of 1, especially focusing on the ste-
reochemistry of the C5 hydroxyl group and the polarity around the
C5 hydroxyl group. First, the C5-epimer of 1, 5-epireveromycin A
(9), was synthesized by the Dess-Martin oxidation of 1 followed by
the reduction with NaBH4-CeCl3 in 34% overall yield (Fig. 2 and
Scheme1). Some of the derivativesof 1 are unstable and afford a dark
tarunderacidic orbasicconditions. Itwas presumedthatthedecom-
* Corresponding author. Tel.: +81 48 467 9375; fax: +81 48 462 4666.
E-mail address: tshimizu@riken.jp (T. Shimizu).
Present address: Graduate School of Life and Environmental Sciences, University
of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan.
à
Present address: Otsuka Pharmaceutical Co., Ltd, Tokushima, Tokushima 771-
0182, Japan.
§
Present address: Graduate School of Pharmaceutical Sciences, Tohoku University,
Sendai, Miyagi 980-8578, Japan.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.05.054

T. Shimizu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3756–3760
3757
position is a result of the b-elimination of the C5 hydroxyl group
since 1 includes the c-hydroxyl a,b-unsaturated carboxyl group on
the right-side chain. 2,3-Dihydroreveromycin A (10) and 2,3-dihy-
dro-5-epireveromycin A (11) were synthesized to prevent the b-
elimination. The sulfone 20,8 a key intermediate for the total synthe-
sis of 1, was subjected to the one-pot Julia olefination10 with an alde-
hyde 24 to give the allyl esters, which were then deallylated with
Pd(Ph3P)4–Ph3P–pyrrolidine11 followed by desilylation with TBAF
in DMF to afford the 2,3-dihydro derivative 10. The aldehyde 24,
the C1–C6 segment for 10, was prepared from the known ester
2712 in four steps.13 The C5-epimer of 10, 2,3-dihydro-5-epirevero-
mycin A (11) was also synthesized with an aldehyde 25 from the
one-pot Julia olefination in a similar manner for the synthesis of
10. The aldehyde 25 was prepared from the allyl alcohol 2813 using
the Sharpless asymmetric epoxidation with (ꢀ)-DET,14 the Z-selec-
tive Horner–Emmons reaction,15 and palladium-catalyzed selective
hydrogenolysis of the alkenyloxirane with formic acid16 as the key
steps. The C4-hydroxyl derivative 12 might be synthesized from
the sulfone 20 in a manner similar to the syntheses of 10 and 11,
and we were also interested in the polarity and structure activity
relationships. Compound 12 was synthesized using a dihydroxyl
O
O
H
CO2R3
H
Me
Me
Me
18
5
O
CO2R1
R2O2C
O
1
OH
24
Me
Me
1: R1 = R2 =R3 = H: Reveromycin A
3: R1 = Me, R2 = R3 =H
4: R1 = R3 = H, R2 = Me
5: R1 = R2 = H, R3 = Me
Me
Me
1
5
Me
O
CO2H
Me
O
OH
O
CO2H
24
HO2C
O
18
n-Bu
Possible Conformation of 1
O
HO2C
HO2C
O
Me
H
Me
O
18
CO2H
11
O
24
Me
OH
aldehyde 26 prepared from 2,3-isopropylidene-L-threitol in seven
Me
steps. Reveromycin A (1) includes three carboxyl groups, and it
was suggested that the nondissociative form of the carboxylic acids
is necessary for the antifungal activity.2 Reveromycin A (1) also spe-
cifically induced apoptosis in osteoclast progenitors in an acidic
microenvironment, a prominent characteristic of osteoclasts.5 We
were interested in the contribution by each activity of the three car-
boxyl groups. Among the monoesters 3–5, the C1 ester 3 showed
strongest inhibitory effects on the IleRS activity and survival of the
osteoclasts. Base on these results, it was suggested that the effects
oftheC1carboxylgroupamongthesethreecarboxylgroupsare min-
imum. Therefore, simple compounds without the C1 carboxyl group
were synthesized to verify the suggestion. The 3,5-dihydroxyl deriv-
Me
Reveromycin B (2)
O
O
CO2H
H
Me
Me
Me
HO2C
O
CO2H
O
H
OR
Me
Me
6: R = Me
7: R = Ac
8: R = TBS
Figure 1. Structures of reveromycin A (1) and the derivatives.
Table 1
IC50 on IleRS activity, cell death inducibility of osteoclasts, and morphological reversion of srcts-NRK cells by reveromycin derivatives
Compound
Molecular
weight
IC50 on IleRS
activity
Morphological reversion of srcts-NRK cells concentration
(lg/mL)
Cell death inducibility
of osteoclasts (lg/mL)
(ng/mL)
100
30
10
++
3
+
1
0.3
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
30
31
32
33
34
35
660.79
660.79
702.83
702.83
702.83
674.82
702.83
775.05
660.79
662.81
662.81
662.76
620.77
562.69
536.65
496.59
636.73
576.68
560.72
574.75
620.84
502.64
502.64
602.71
2.95
+++a
ꢀ
+++
ꢀ
0.06
>15
2.00
3.10
2.50
>15
0.46
5.90
21.5
0.22
0.82
1.01
6.31
ntb
nt
nt
>15
nt
11.5
nt
>1000
211.0
>1000
292.8
374.0
>1000
>1000
378.3
11.6
58.3
14.4
22.9
560.3
>1000
>1000
>1000
>1000
94.6
+++
+++
+++
ꢀ
+++
+++
+++
+++
+++
++
+
ꢀ
ꢀ
ꢀ
+++
+++
ꢀ
+++
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
++
ꢀ
+
ꢀ
+
ꢀ
+++
++
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+++
+++
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
57.9
+++
+++
+++
ꢀ
+++
+
+
+
ꢀ
497.4
564.5
>1000
>1000
9.60
>30
>30
12.4
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
tox
ꢀ
a
Rate of reversed cells was presented as follows: +++, >80%; ++, 50–80%; +, 20–50%; , 10–20%; ꢀ, <10%.
b
Not tested.

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T. Shimizu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3756–3760
O
O
O
O
CO2H
H
O
H
CO2H
H
Me
Me
Me
Me
2
Me
HO2C
Me
HO2C
5
O
O
CO2H
CO2H
3
O
H
OH
OH
Me
Me
Me
Me
2,3-Dihydroreveromycin A (10)
5-Epireveromycin A (9)
O
O
O
H
CO2H
H
O
H
CO2H
H
Me
OH
Me
Me
Me
HO2C
Me
HO2C
O
O
CO2H
CO2H
4
O
O
OH
OH
Me
Me
Me
Me
12
2,3-Dihydro-5-epireveromycin A (11)
O
O
O
H
CO2H
H
O
H
CO2H
Me
Me
Me
Me
HO2C
Me
HO2C
H
5
O
OH
O
OH
5
O
O
3
OH
Me
Me
Me
Me
13
14
O
O
O
O
CO2H
H
O
H
CO2H
H
Me
Me
HO2C
Me
HO2C
O
OH
O
OH
O
9
7
H
Me
Me
Me
Me
16
15
O
O
O
CO2H
H
O
CO2H
H
Me
Me
Me
HO2C
Me
HO2C
O
O
O
CO2H
CO2H
5
O
O
7
H
H
O
Me
Me
Me
Me
18
17
Figure 2. Reveromycin A derivatives having the modified right-side chain.
ative 13 was easilysynthesized via theJulia olefinationof thesulfone
20 with an aldehyde 238 followed by deprotection of the silyl groups
and hydrogenolysis of the allyl esters. The primary C5 hydroxyl
derivative 14 was in turn prepared from the triallyl esters of 1 via
the retrograde aldol reaction with NaH. The resulting unsaturated
aldehyde was reduced with NaBH4-CeCl3 and the allyl groups were
removed by the palladium (0)-catalyzed hydrogenolysis to afford
14. The triallyl esters of 1 were prepared using the allyl alcohol
and EDCI under high pressure.9 Although the trimethyl esters of 1
were easily prepared by the esterification with TMSCHN2 at room
temperature, the deprotection of the dimethyl esters to obtain 14
was difficult after the retrograde aldol reaction. Since the formation
of the hydrogen bond between the C7 hydroxyl group and C24 car-
boxyl group might be low, there is a slight contribution of the C7 hy-
droxyl group to the stability. The C7 hydroxyl derivative 15 without
the C5 hydroxyl group was prepared from the alcohol 198 in four
steps including the Wittig reaction of the corresponding aldehyde
with an ylide 21 followed by reduction with Zn(BH4)2 and deallyla-
tion. The hemisuccinate of the C7 hydroxyl group of 15, a tricarbox-
ylic acid 17, was directly synthesized from 15 using succinic
anhydride. The C5 carboxylic acid 18 was prepared from 19 via
two Wittig reactions with ylides 21 and 22. For the hemisuccinate
of the C18 tertiary hydroxyl group, we have already reported the
preparations and inhibitions on the IleRS activity of the hydroxyl
(30), methoxy (31), and methylthiomethyl (32) derivatives.9 Inhibi-
tory effects of 30 and 31 on the IleRS activity still existed. However,
the effects were 1/20–30 of that of 1. The C18 hydrogen derivative
33, spirofunginA, whichwasisolatedasasecondarymetabolitefrom
Streptomyces violaceusniger Tü 4113, showed various antifungal
activities, particularly against yeast.17 Since spirofungin A was ini-
tially isolated as a equilibrium mixture with spirofungin B (34), a
C15 epimer of 33, compounds 33 and 34 were synthesized in the
pure form for the study of the structure–activity relationships.12
A
C5-hemisuccinate 35 of 33, the tricarboxylic acid as well as revero-
mycin A (1), was synthesized from 33 in three steps. The carboxyl
groups of 33 were esterified with 2-(trimethysilyl)ethanol and EDCI
and the C5 hydroxyl group was esterified with the mono-trimethysi-
lylethyl succinate and EDCI. Finally, the trimethysilylethyl groups
were cleaved with TBAF to afford the hemisuccinate 35 (see Fig. 3).
We have already reported that reveromycin A (1) induces the
morphological reversion activity of srcts-NRK cells from spherical
transformed cells to flat cells at 32 °C and the molecular target of 1

T. Shimizu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3756–3760
OR
3759
c, d, b, e
a, b
Me
Me
9
1
14
Me
H
18
O
CO2H
HO2C
O
H
OH
Me
Me
h
16
15
a, f, g, e
17
30 : R = H
31 : R = Me
e
32 : R = MTM
O
O
Me
Me
Me
H
18
O
15
O
H
CO2allyl
H
CO2H
HO2C
O
1
Me
allylO2C
a, f, i, e
H
OH
24
O
OH
Me
18
Me
Spirofungin A (33)
Me
Me
19
ref. 8
Me
Me
Me
H
O
O
O
CO2H
15
HO2C
O
O
CO2allyl
H
H
OH
Me
O2
S
Me
Me
allylO2C
Me
S
O
j, k, e
13
Spirofungin B (34)
N
H
Me
Me
20
Me
Me
Me
O
H
O
5
o, e, p
n, e, m
CO2H
CO2H
l, e, m
HO2C
O
H
O
Me
Me
10
11
12
35
Me
Figure 3. The C18 hemisuccinate derivatives.
Me
OTES
Ph3P
CO2allyl
O
O
Ph3P
OTBS
OTBS
23
22
21
Me
carboxyl groups in 1 are essential for the activities and the free hy-
droxyl group at C5 and the C24 carboxyl group are particularly
important for the strong activities based on the activities of the tri-
carboxylic acid mono- (3–5), di-, and tri-esters and C5 hydroxyl
derivatives such as the methoxy (6), acetoxy (7), and silyloxy (8)
derivatives.9 It was anticipated that the hydrogen bond between
the C5 hydroxyl group and the C24 carboxyl group might contribute
to the stability and the activity of 1. The activity of 5-epireveromycin
A (9) was surprisingly reduced to 1/128 of that of 1 (IC50 = 378.3 ng/
mL). It was assumed that the formation of the hydrogen bond be-
tween the C5R hydroxyl group and C24 carboxyl group might be dif-
ficult and even the hydrogen bond could be formed, therefore the
a,b-unsaturated C1 carboxyl group was placed at a disadvantage
for the reciprocal action with the target molecule. On the other hand,
2,3-dihydroreveromycin A (10) showed an equivalent activity to 1
(IC50 = 11.6 ng/mL), suggesting that the fixed conformation of C1
carboxyl group by a,b-unsaturated bond is not required for IleRS
inhibition. Since 10 is free from the b-elimination, the 2,3-dihydro
derivatives are promising as potent drugs. Interestingly, 2,3-dihy-
dro-5-epireveromycin A (11), the 2,3-dihydrated 5-epireveromycin
(9), recoveredtheactivity(IC50 = 58.3 ng/mL). Thismightresultfrom
the flexibility of the conformation of the a,b-saturated C1 carboxyl
group. The 4-hydroxyl derivative 12 strongly inhibited the IleRS
activity as expected (IC50 = 14.4 ng/mL). The 3,5-dihydroxyl deriva-
tive 13 showed a strong inhibitory effect on IleRS (IC50 = 22.9 ng/mL)
in spite of the absence of the C1 carboxyl group. Since a simple 5-hy-
droxyl derivative 14 showed a weak activity (IC50 = 560.3 ng/mL),
the C3 hydroxyl group in 13 might play an important role in the
activity. The primary 7-hydroxyl (15) and 9-hydroxyl (16) deriva-
tives only slightly inhibited the IleRS activity (IC50 > 1000 ng/mL).
Thehemisuccinate17of theC7 hydroxylderivative 15, whichis a tri-
carboxylic acid like 1 and excludes the C5 hydroxyl group, did not
show any activity even at the concentration of 1000 ng/mL. The C5
carboxylic acid 18, a tricarboxylic acid without the C5 hydroxyl
group, also did not show any activity. Being related to the C18 hemi-
succinyl group in 1, the C18 hydroxyl derivative 30 and the C18
Me
CO2allyl
O
CO2allyl
O
CO2allyl
O
OTBS
OTBS
26
OTBS
25
24
q-s, a
t-x, q, r, s, a
y, a, z, aa, x, s, a
Me Me
Me
Me
OH
O
O
MPMO
CO2Me
MPMO
28
OTBS
27
HO
OH
29
Scheme 1. Reagents and conditions: (a) Dess-Martin Periodinane, MS4A, CH2Cl2,
(99% for 24, 92% for 25, 99% for 26); (b) NaBH4, CeCl3ꢁ7H2O, rt (34% for 9, 22% for 14,
two steps); (c) allyl alcohol, EDCl, DMAP, CH2Cl2, 1.5 GPa, rt (36%); (d) NaH, THF,
0 °C; (e) Pd(Ph3P)4, Ph3P, pyrrolidine, CH2Cl2, 0 °C–rt (68% for 13, 74% for 14, 76% for
15, 74% for 16, 78% for 18); (f) 21, toluene, 110 °C (96% for 15, 18, two steps); (g)
Zn(BH4)2, Et2O, 0 °C (99%); (h) succinic anhydride, DMAP, pyridine, CH2Cl2, rt (95%);
(i) 22, toluene, 100 °C (61%); (j) 23, LHMDS, THF, ꢀ78 °C (90%); (k) TBAF, THF, 0 °C
(79%); (l) 24, KHMDS, THF, ꢀ78 °C (55%); (m) TBAF, DMF, rt (48% for 10, 75% for 11,
each two steps); (n) 25, LHMDS, THF, ꢀ78 °C (72%); (o) 26, LHMDS, THF, ꢀ78 °C,
(41%); (p) TBAF, DMPU, rt (35 % for 23, two steps); (q) H2, Pd/C, EtOAc, rt, (96% for
24, 93% for 25); (r) allyl alcohol, ClBu2SnOSnBu2OH, toluene, 110 °C, (89% for 24,
92% for 25); (s) DDQ, CH2Cl2-H2O, rt, (95% for 24, 95% for 25, 99% for 26); (t) TBHP,
(ꢀ)-DET, Ti(O-i-Pr)4, M-4A, CH2Cl2, ꢀ25 °C, (79%); (u) (COCl)2, DMSO, CH2Cl2 then
Et3N, ꢀ78 °C; (v) (CF3CH2O)2POCH2CO2Me, KN(TMS)2, 18-crown-6, ꢀ78 °C, (87%,
two steps); (w) Pd2(dba)3ꢁCHCl3, n-Bu3P, HCO2H, Et3N, dioxane, rt, (91%); (x) TBSCl,
imidazole, DMF, rt, (94% for 25, 94% for 26); (y) MPMCl, NaH, THF, 0 °C–rt, (88%); (z)
22, CH2Cl2, rt; (aa) TsOH, MeOH, rt–60 °C, (40%, three steps).
is the isoleucyl-tRNA synthetase (IleRS).2,4 Recently, it was also
found that 1 inhibits bone resorption by specifically inducing apop-
tosis in osteoclasts.5 Therefore, we tested these biological activities
of the synthesized compounds for the discovery of compounds more
active and stable than 1, and the results are shown in Table 1.
Reveromycin A (1) strongly inhibits the IleRS activity and the IC50
on this enzyme was 2.95 ng/mL. It was revealed that two of the three

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T. Shimizu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3756–3760
methoxyderivative31stillinhibitedtheIleRSactivityandtheeffects
were 1/20–32 of the activity of 1 (IC50 = 94.6, 57.9 ng/mL, respec-
tively). The C18 methoxy derivative 31 is expected to be a stable
drug. The C18 MTM ether derivative 22 weakly exhibited any activ-
ities. Although a mixture of spirofungin A (33) and B (34) was re-
ported to inhibit the growth of Candida albicans and Rhodotorula
rubra (minimal concentration: 15 lg/mL),17 the synthesized pure
compound 33 very weakly inhibited the IleR activity and the effect
was 1/191 of that of 1 (IC50 = 564.5 ng/mL). It is noteworthy that
the strong inhibition of 33 on the growth of several human cancer
cell lines including HL-60 and PC3 is consistent with the cell-based
behavior of reveromycin A (1).18 Spirofungin B 34 did not inhibit
the IleRS as presumed from the structure, in which the hydrogen
bond between the C5 hydroxyl group and C24 carboxyl group might
be negligible. Moreover, the C5-hemisuccinate 35 of 33, a tricarbox-
ylic acid, did not inhibit the activity. It became clear that the C5
hemisuccinyl group in 35 decreased the inhibition activity toward
IleRS, such as the C5 acetoxy (7) and C5 silyloxy (8) derivatives of
1. Based on the effects of the IleRS activity, 2,3-dihydroreveromycin
A (10), the 4-hydroxyl derivative 12, and the 3,5-dihydroxyl deriva-
tive 13 were expected to be promising derivatives of 1.
The derivatives which contain the C24 carboxyl group and the
C5S hydroxyl group, the 2,3-dihydroxyreveromycin A (10) and 4-hy-
droxy reveromycin A (12), exhibited morphological reversion activ-
ities on srcts-NRK cells at the concentration of 10 lg/mL as the C1
monoester 3 and the C40 monoester 5. On the contrary, the 5-epire-
veromycin A (9) did not show the morphological reversion at the
concentration of 100 lg/mL, as expected from the loss of IleRS inhi-
bition. Furthermore, the 2,3-dihydro-5-epireveromycin A (11)
weakly exhibited the reversion (++ at 100 lg/mL). It might be due
to the flexibility of the conformation on the right-side chain in 11
for the inhibition on the IleRS. The 3,5-dihydroxyl derivative 13, a
24,40-dicarboxylic acid, moderately showed the reversion. Spirofun-
gin A (33), a 1,24-dicarboxylic acid without the C18-hemisuccinyl
group as the C18 methoxy- (31) and C18 MTM (32) derivatives,
strongly exhibited morphological reversion activities on srcts-NRK
cells, while 34 did not. The C5-hemisuccinate 35 of 33 did not show
the reversion activity at the concentration of 100 lg/mL, while the
C5 acetate 7 of reveromycin A (1) induced strong morphological
reversion activities. It was suggested that the C5 acetate was easily
hydrolyzed by cellular esterase to afford 1 after being taken into
the cells, showed these activities and the C5-hemisuccinate 35 re-
sisted hydrolysis.
line RAW264 under neutral conditions (data not shown). Therefore,
8 is an attractive compound as a prodrug which was cleaved to give 1
under acidic conditions around the osteoclasts. The activity of 5-
epireveromycin A (9) was also reduced as the inhibition on the IleRS
was reduced. The 2,3-dihydroreveromycin A (10) inhibition was as
strong as 1 (IC50 = 0.22 lg/mL) and even 2,3-dihydro-5-epirevero-
mycin
A (11) strongly induced apoptosis in the osteoclasts
(IC50 = 0.82 lg/mL). The 4-hydroxyl derivative 12 also produced a
strong inhibition (IC50 = 1.01 lg/mL). The inhibition effect of the
3,5-dihydroxylderivative13, adicarboxylicacid, wasmoderate. Sur-
prisingly, the dicarboxylic acid, spirofungin A (33) and B (34) did not
inhibit the survival of the osteoclasts. It is important to note that the
hemisuccinate 35, a tricarboxylic acid, moderately inhibited the sur-
vivalof theosteoclasts although 35did notinhibittheIleRS. Basedon
theeffects onthe survival of osteoclasts, it was shownthat the tricar-
boxylic acid is essential to exhibit a strong activity and the C5 TBS
ether 8 is an attractive compound as a prodrug.
Various derivatives of reveromycin A and spirofungin A focus-
ing on the 5S hydroxyl group and C18 hemisuccinyl group were
synthesized and their inhibitory effects on both the isoleucyl-tRNA
synthetase activity and the survival of osteoclasts, and activities on
the morphological reversion of srcts-NRK cells were examined. 2,3-
Dihydroreveromycin A (10) and the 4-hydroxyl derivative 12 are
expected to be the attractive derivatives of 1 based on their activ-
ities and stabilities. The C5 TBS ether 8 is a promising derivative as
a prodrug for osteoporosis.
Acknowledgments
This work was supported in part by a Grant-in-Aid for Scientific
Research (18590027) to T.S. from the Ministry of Education, Cul-
ture, Sports, Science and Technology, Japan, and the RIKEN Chem-
ical Biology program. We thank Dr. H. Koshino for the NMR
measurements and Ms. K. Harata for the mass spectrum
measurements.
References and notes
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