Synthesis and antifungal activity of novel 14-membered benzomacrolides, as galbonolide analogues

Article abstract of DOI:10.1248/cpb.52.163

Asymmetric total synthesis of benzene analogues of galbonolide, a 14-membered antifungal macrolide, possessing a benzene ring instead of a conjugated diene structure, was achieved starting from chiral 1-aryl-1-propanol obtained by enzyme-catalyzed kinetic

Full text of DOI:10.1248/cpb.52.163

January 2004
Communications to the Editor
Chem. Pharm. Bull. 52(1) 163—165 (2004)
163
fungal activity of novel galbonolide analogues linked by a
benzene ring.
Synthesis and Antifungal Activity of
Novel 14-Membered Benzomacrolides, as
Galbonolide Analogues
Our retrosynthetic analysis was shown in Chart 1. Follow-
ing the report by Tse, we planned to achieve the formation of
a 14-membered macrocyclic ring by C2–C3 bond formation
via macro-Dieckmann condensation. The peculiar asymmet-
ric quaternary carbon at C4 on B would be constructed by
making good use of “contra-steric” enolate attack of 5 to
allyl iodide C. The introduction of a trisubstituted double
bond at C6–C7 would be achieved by employing an aldol or
Horner–Emmons reaction of the aldehyde prepared from al-
cohol 6. That is, the chiral alcohol 6 would be the key inter-
mediate in the modification of the substituent on C6. The al-
cohol 6 was expected to be assembled from a benzaldehyde 7
through an optical resolution of a racemic secondary alcohol
at C13 and coupling with a chiral side chain.
In the first place, we tried asymmetric synthesis of a chiral
key intermediate 6 (Chart 2). Treatment of aldehyde 7 with
EtMgBr in Et2O gave racemic alcohol 8 in good yield (92%).
Fortunately, enantioselective acetylation of (rac)-8 using en-
zyme (Lipase LIP®)8) provided (S)-8 as a remaining alcohol,
with a desirable configuration (42%, 95.7% ee).9) Chiral pro-
pionyl amide 11 was prepared from commercially available
optically active hydroxy ester 9 through protection by tert-
butyldimethylsilyl (TBS) group (98%) and installation of a
Weinreb amide in the presence of i-PrMgCl (98%).10) The
coupling reaction of a dianion prepared from (S)-8 with
Weinreb amide 11 was achieved successfully (quantitative
yield), and the corresponding ketone was converted into al-
cohol 12 by protection of the secondary alcohol on C13 by
TBS group (61%) followed by NaBH4 reduction (92%). De-
oxygenation on C9 by way of radical reduction via xanthate
afforded 13 (89%), and subsequent selective deprotection
using p-TsOH led to key intermediate 6 in reasonable yield
*
Hiroki SAKOH, Shunji SAKURABA, Yuichi SUGIMOTO,
Hideaki IMAMURA, Hideki JONA, Koji YAMADA,
Rie BAMBA-NAGANO, Terutaka HASHIZUME, and
Hajime MORISHIMA
Banyu Tsukuba Research Institute, Banyu Pharmaceutical Co.,
Ltd.; Okubo-3, Tsukuba, Ibaraki 300–2611, Japan.
Received September 12, 2003; accepted November 7, 2003
Asymmetric total synthesis of benzene analogues of galbono-
lide, a 14-membered antifungal macrolide, possessing a benzene
ring instead of a conjugated diene structure, was achieved start-
ing from chiral 1-aryl-1-propanol obtained by enzyme-catalyzed
kinetic resolution with high enantioselectivity. Representatively,
a method for the introduction of a methylthio and chloride
function at the vinyl position was also established. The resulting
analogues unfortunately exhibited very little antifungal potency
in comparison with galbonolide A.
Key words galbonolide; inositol phosphoceramide (IPC) synthase; ben-
zene analogue; 14-membered macrolide antibiotic
The novel 14-membered macrolide antibiotics galbonolide
A (rustmicin; 1) and galbonolide B (neorustmicin; 2) were
discovered in a fermentation broth of Micromonospara chal-
cea by Otake et al.1,2) Independently, Achenbach et al. also
isolated galbonolide A and B from Streptomyces galbas.3,4)
In addition to the original potent activity against microorgan-
isms that cause botanical disease, galbonolide A was shown
to have good potency against clinically important fungi in-
cluding Cryptococcus and Candida spp., and then a research-
ing group at Merck found out that the fungicidal activity of
galbonolide A was caused by an inhibitory activity against
inositol phosphoceramide (IPC) synthase.5,6) Galbonolide A
itself is too difficult to develop for clinical application be-
cause of its poor stability under physiological conditions.
However, due to their biological activities, galbonolide A and
B remain interesting compounds in medicinal chemistry. The
total synthesis of galbonolide B, a more stable analogue of
galbonolide A, has already been achieved by Tse.7)
In order to improve the stability and to simplify the re-
ported total synthetic method, we attempted to prepare a ben-
zene analogue corresponding to a conjugated diene system as
shown in Fig. 1, focusing on the conversion of a methyl enol
ether part of galbonolide A, which is the most important with
respect to antifungal activity but also the most unstable part,
to other vinyl type substitutes such as methyl thioenol ether
or vinyl chloride. This letter reports the synthesis and anti-
Chart 1
Fig. 1. Structures of Galbonolide A, B and Benzene Analogues
Chart 2
To whom correspondence should be addressed. e-mail: sakouhk@banyu.co.jp
© 2004 Pharmaceutical Society of Japan

164
Vol. 52, No. 1
(70%).
corresponding iodide 17 by the action of a combination of I2,
Another fragment 5 was prepared from the natural a- Ph3P and imidazole (quantitative yield). The coupling reac-
amino acid L-serine by changing the amino group to a hy- tion of allyl iodide 17 with ester 5 using lithium hexamethyl-
droxyl group under the same configuration via Hirth’s proce- disilazide (LHMDS) in a THF-HMPA solvent system12) gave
dure11) (Chart 3). Subsequent esterification gave dihydroxyl adduct 18 in moderate yield (53%), although an excess
ester 14 in good yield (93%). Upon reaction with mesitalde- amount of 5 was needed due to the very low stability of the
hyde and camphorsulfonic acid (CSA) in the presence of lithium enolate of 5. This reaction resulted in the formation
MgSO4 and 4A molecular sieves, 14 was converted to trans- of the peculiar asymmetric quaternary carbon at C4 with ex-
dioxolane 5 and an undesired cis-isomer in a ratio of ca. 3 : 1 cellent selectivity. Deprotection of the TBS group at C13 was
(62%). This ratio was increased to 12 : 1 by recrystallization carried out by treatment of methyl ester 18 with tetrabuty-
from n-hexane (63%).
lammonium fluoride (TBAF) in THF. In this reaction, simul-
Synthesis of a benzene analogue having methylthio enol taneous cleavage of methyl ester to carboxylic acid occurred,
ether structure at C6–7 was actually attempted starting from and it was necessary to convert the resultant carboxylic acid
key intermediate 6 (Chart 4). Alcohol 6 led to aldehyde 15 to methyl ester by means of TMSCHN2 (84% over 2 steps).
by conventional Swern oxidation (96%). The aldol reaction The resulting secondary alcohol was then acetylated to yield
of 15 with lithium enolate of methylthioacetate afforded the a precursor of cyclization, 19 (85%). Because Tse reported in
corresponding hydroxyl esters as a mixture of 4 diastere- case of total synthesis of galobonolide B that a cyclization
omers. Fortunately, following dehydration provided a,b-un- reaction was failed when a precursor having a propionyloxy
saturated ester 16 with exclusively (Z) geometry (69% over 2 group instead of a acetoxy group on C13 was used,10) we de-
steps), probably because the dehydration reaction progressed cided to prepare the acetate 19 as a precursor of cyclization.
to give thermodynamically favored product. The ethyl ester And since the original procedures reported for the synthesis
was then reduced by diisobutylaluminum hydride (DIBAL- of galbonolide B gave poor reproducibility, the solvent was
H) (82%), and the resulting allyl alcohol was changed to the changed from THF to DME and HMPA. The macro-Dieck-
mann condensation of 19 took place very quickly, and com-
pound 20 having a novel galbonolide skeleton was obtained
in moderate yield (59%). Introduction of a methyl group at
C2 using MeI and NaH gave a-methyl form 21 (42%) and
the dimethyl form (14%). Inversion of the stereochemistry at
the C2 position of 21 was carried out by treating 21 with t-
BuOK in DMF followed by protonation to yield b-methyl
form 22 (18%). Finally, the 2,4,6-trimethylbenzylidene acetal
of 22 was removed by using a AcOH–H2O system. This pro-
Chart 3
Chart 5
Chart 4
Table 1. Antifungal Activity of Galbonolide Derivatives
MIC (mg/ml)
Organism
Galbonolide A
Galbonolide B
3
4
Amphotericin B
Candida albicans ATCC90028
Cryptococcus neoformans ATCC90112
Aspergillus funigatus TIMM1776
4
Ͼ64
16
Ͼ64
Ͼ64
Ͼ64
Ͼ64
Ͼ64
Ͼ64
Ͼ64
0.5
0.5
1
Ͻ0.0031
Ͼ64

January 2004
165
2) Otake N., Abe Y., Nakayama H., Shimazu A., Furihata K., Ikeda K.,
Furihata K., Seto H., J. Antibiot., 38, 1810—1812 (1985).
3) Achenbach H., Muhlenfeld A., Fauth U., Zahner H., Tetrahedron Lett.,
26, 6167—6170 (1985).
4) Achenbach H., Muhlenfeld A., Fauth U., Zahner H., J. Antibiot., 39,
1760—1764 (1986).
5) Mandala S. M., Thornton R. A., Milligan J., Rosenbach M., Garcia-
Calvo M., Bull H. G., Harris G., Abruzzo G. K., Flattery A. M., Gill
C. J., Bartizal K., Dreikorn S., Kurtz M. B., J. Biol. Chem., 273,
14942—14949 (1998).
6) Harris G. H., Shafiee A., Cabello M. A., Curotto J. E., Genilloud O.,
Goklen K. E., Kurtz M. B., Rosenbach M., Salmon P. M., Thornton R.
A., Zink D. L., Mandala S. M., J. Antibiot., 51, 837—844 (1998).
7) Tse B., J. Am. Chem. Soc., 118, 7094—7100 (1996).
8) Sugimoto Y., Imamura H., Shimizu A., Nakano M., Nakajima S., Abe
S., Yamada K., Morishima H., Tetrahedron: Asymmetry, 11, 3609—
3617 (2000). Lipase LIP® was purchased from TOYOBO.
9) The configuration of (S)-8 was determined by conversion to a chiral
compound, (S)-(Ϫ)-1-phenyl-1-propanol by following procedure, then
its specific rotation was compared with that of commercially available
sample which was purchased from Aldrich.
cedure afforded a novel galbonolide analogue 3 possessing a
benzene ring and a methylthio enol ether part instead of the
conjugated diene and methyl enol ether in 80% yield.13)
Next, the synthesis of another analogue having a vinyl
chloride group at C6–C7 was attempted by a similar method
(Chart 5). The Horner–Emmons reaction of aldehyde 15
using triethyl phosphonoacetate and NaH in the presence of
N-chlorosuccinimide (NCS)14) was performed to assemble a
trisubstituted double bond including a chlorine atom, to pro-
vide the desired isomer (Z)-23 as a minor product (29%).
(Z)-23 led to iodide 24 by DIBAL-H reduction (89%) and
subsequent iodination (77%). The allyl iodide was reacted
with excess lithium enolate prepared from 5 to yield ester 25
(57%). TBS ether 25 was converted into acetate 26 by the
same procedure as above (65% over 3 steps), and subsequent
macro-Dieckmann cyclization of 26 using LHMDS in reflux-
ing DME in the presence of HMPA gave compound 27 in
good yield (74%). A methyl group was introduced at C2 by
the action of MeI and t-BuOK in DMF. However, an unde-
sired a-methyl form 28 was obtained exclusively yet again
(83%) and a dimethyl form was not generated. After 28 was
treated with t-BuOK followed by protonation to invert the
stereochemistry at C2, removal of 2,4,6-trimethylbenzylidene
acetal provided a novel galbonolide analogue 4 having a
vinyl chloride function at C6–7 in good yield (85% over 2
steps).15)
The in vitro antifungal activities of the galbonolide deriva-
tives obtained above were evaluated against Candida albi-
cans (ATCC90028), Cryptococcus neoformans (ATCC90112)
and Aspergills fumigatus (TIMM1776). Serial dilution tech-
niques were employed for the minimum inhibitory concen-
tration (MIC) determinations.16) Galbonolide A, B and am-
photericin B were used as reference compounds. Unfortu-
nately, the antifungal activities of galbonolide derivatives 3
and 4 were significantly lower than that of the original gal-
bonolide A (Table 1).
In conclusion, we succeeded in the asymmetric total syn-
thesis of novel galbonolide analogues possessing a benzene
ring instead of a conjugated diene structure via the following
transformations: optical resolution of benzylic alcohol (rac)-
8, construction of a trisubstituted double bond with a
methylthio ether or chlorine atom, assembly of an asymmet-
ric quaternary carbon, and finally macro-Dieckmann cycliza-
tion. Unfortunately, the synthesized novel compounds 3 and
4 exhibited no significant antifungal activity. Although, it is
not clear that which of transformations, conjugated diene to
benzene ring and/or MeO to MeS or Cl, was the cause of
such a loss of potency so far, we could establish a method for
construction of galbonolide skeleton which possess replaced
C6 functional group by this work. So, by applying the
10) Williams J. M., Jobson R. B., Yasuda N., Marchesini G., Dolling Ulf-
H., Grabowski E. J. J., Tetrahedron Lett., 36, 5461—5464 (1995).
11) Hirth G., Walther W., Helv. Chim. Acta, 68, 1863—1871 (1985).
12) Seebach D., Aebi J. D., Gandner-Coquoz M., Naef R., Helv. Chim.
Acta, 70, 1194—1216 (1987). As regards this reaction, Tse proposed
the chelation model of lithiated 5 in ref. 4. Excellent stereoselectivity
was observed by preferentially nucleophilic attack from the less hin-
dered convex side.
13) Data of compound 3: 1H-NMR (300 MHz, C6D6) d: 0.82 (3H, t,
Jϭ7.3 Hz), 0.92 (3H, d, Jϭ6.7 Hz), 1.41 (3H, d, Jϭ7.0 Hz), 1.78 (3H,
s), 1.88 (2H, m), 2.00 (1H, m), 2.25 (1H, dd, Jϭ12.5, 8.8 Hz), 2.31
(1H, br s), 2.62 (2H, s), 2.73 (1H, dd, Jϭ12.5, 4.6 Hz), 3.15 (1H, m),
3.55 (1H, q, Jϭ7.0 Hz), 3.63 (1H, d, Jϭ11.3 Hz), 3.72 (1H, m), 5.42
(1H, d, Jϭ9.5 Hz), 5.50 (1H, t, Jϭ7.6 Hz), 6.85 (1H, d, Jϭ7.6 Hz),
7.02 (1H, d, Jϭ7.6 Hz), 7.10 (1H, dd, Jϭ7.6, 7.6 Hz), 7.25 (1H, s).
FAB-MS m/z: 429 (MϩNaϩ).
14) Braum N. A., Klein I., Spitzner D., Vogler B., Braum S., Borrmann H.,
Simon A., Liebigs. Ann., 1995, 2165—2169 (1995).
15) Data of compound 4: 1H-NMR (300 MHz, C6D6) d: 0.80 (3H, t,
Jϭ7.7 Hz), 0.82 (3H, d, Jϭ7.2 Hz), 1.40 (3H, d, Jϭ7.5 Hz), 1.68—
1.97 (2H, m), 2.12 (1H, dd, Jϭ13.6, 9.4 Hz), 2.44 (1H, d, Jϭ15.1 Hz),
2.65 (1H, dd, Jϭ13.6, 5.7 Hz), 2.76—2.94 (1H, m), 2.86 (1H, d,
Jϭ15.1 Hz), 3.46 (1H, q, Jϭ7.5 Hz), 3.55 (1H, d, Jϭ9.8 Hz), 3.61
(1H, d, Jϭ9.8 Hz), 5.28 (1H, d, Jϭ9.1 Hz), 5.45 (1H, t, Jϭ7.7 Hz),
6.82 (1H, d, Jϭ7.8 Hz), 7.02—7.15 (3H, m). FAB-MS m/z: 417
(MϩNaϩ).
demonstrated method, further modification of this analogue 16) MICs were determined by microbroth dilution method using YNBP
medium, comprising Yeast Nitrogen Base (Difco Laboratories, Detroit,
Mich.), 1% glucose, and 0.25% KH2PO4. Candida albicans or Crypto-
coccus neoformans cells were suspended in YNBP liquid to give a
to include compounds with a 1,3-diene system as in the nat-
ural product is currently under investigation, and will be re-
ported in the near future.
final concentration of approximately 6—10ϫ103 cells/ml. Aspergillus
fumigatus conidia were suspended in YNBP agar (0.2%) to give a final
concentration of approximately 2.5ϫ103 conidia/ml. Test samples were
dissolved and serially twofold diluted in dimethyl sulfoxide. Aliquots
of 2 ml were distributed to a 96-well, flat-bottomed plate, then plates
were filled with 200 ml of cell or conidia solution. The MIC was de-
fined as the lowest concentration of samples that completely prevented
visible growth was inhibited.
Acknowledgments We indebted to Mr. Shinnosuke Abe for FAB-MS
analyses and to Dr. Shigeru Nakajima for NMR measurements. We are also
grateful to Dr. Bruno Tse, Merck & Co., Inc. for his precious information
about derivations of galbonolides.
References and Notes
1) Otake N., Takatsu T., Nakayama H., Shimazu A., Furihata K., Ikeda
K., Furihata K., Seto H., J. Antibiot., 38, 1806—1809 (1985).