Synthesis of four chiral isomers of β-lactone DU-6622 and inhibition of HMG-CoA synthase by the specific (2R,3R)-isomer

Article abstract of DOI:10.1021/jo9621433

Four chiral forms of the β-lactone DU-6622 (3-hydroxy-2-(hydroxymethyl)-5-[7-(methoxycarbonyl)naphthalen-1-yl]pen tanoic acid 1,3-lactone) were prepared to investigate their inhibitory activity against 3-hydroxy-3-methylglutarly-CoA (HMC-CoA) synthase. The (2R,3R)-β-lactone isomer (+)8a, having the same stereochemistry as that of the fungal β-lactone 1233A, showed the most potent HMG-CoA synthase inhibitory activity (IC₅₀: 0.098 μM). The other three β-lactone isomers, (2S,3R)((-)-8b), (2S,3S)-((-)-8a), and (2R,3S)-isomers ((+)-8b), were weaker inhibitors with larger IC₅₀ values of 9.4, 31, and 360 μM, respectively. Thus, it was concluded that the (2R,3R) stereochemistry of the β-lactone ring is responsible for HMG-CoA synthase inhibition.

Full text of DOI:10.1021/jo9621433

J . Org. Chem. 1997, 62, 2161-2165
2161
Syn th esis of F ou r Ch ir a l Isom er s of â-La cton e DU-6622 a n d
In h ibition of HMG-CoA Syn th a se by th e Sp ecific (2R,3R)-Isom er
Hiroshi Tomoda, Hidetoshi Kumagai, Yuji Ogawa,† Toshiaki Sunazuka,
Hirokazu Hashizume,‡ Hajime Nagashima,§ and Satoshi Oh mura*
Research Center for Biological Function, The Kitasato Institute, and School of Pharmaceutical Sciences,
Kitasato University, Minato-ku 108, J apan, Fuji Chemical Industries Co., Takaoka, Toyama 933, J apan,
and Daiichi Pharmaceutical Co. Ltd., Edogawa-ku, Tokyo 134, J apan
Received November 15, 1996X
Four chiral forms of the â-lactone DU-6622 (3-hydroxy-2-(hydroxymethyl)-5-[7-(methoxycarbonyl)-
naphthalen-1-yl]pentanoic acid 1,3-lactone) were prepared to investigate their inhibitory activity
against 3-hydroxy-3-methylglutarly-CoA (HMG-CoA) synthase. The (2R,3R)-â-lactone isomer (+)-
8a , having the same stereochemistry as that of the fungal â-lactone 1233A, showed the most potent
HMG-CoA synthase inhibitory activity (IC50: 0.098 µM). The other three â-lactone isomers, (2S,3R)-
((-)-8b), (2S,3S)- ((-)-8a ), and (2R,3S)-isomers ((+)-8b), were weaker inhibitors with larger IC50
values of 9.4, 31, and 360 µM, respectively. Thus, it was concluded that the (2R,3R) stereochemistry
of the â-lactone ring is responsible for HMG-CoA synthase inhibition.
In tr od u ction
A fungal â-lactone 1233A (also known as F-244 or
L-659,699) (Figure 1) originally isolated as an antibiotic
by Aldridge et al.1 was rediscovered as a potent and
specific inhibitor of 3-hydroxy-3-methylglutaryl-CoA
(HMG-CoA) synthase.2 The compound inhibited the
enzyme in vitro using a rat liver cytosolic fraction and
in intact cell systems using Vero and HepG2 cells.2
Importantly, it proved orally active in mice by inhibiting
hepatic sterol biosynthesis in a dose-dependent fashion.3
Our early study on 1233A modification revealed that the
2-(hydroxymethyl)-â-lactone moiety of 1233A is essential
for the inhibition.2b The stereochemistry of the â-lactone
was determined to be 2R,3R by Chiang et al.4 Interest-
ingly, we found that ebelactones A and B,5 reported as
esterase inhibitors having a very similar planar structure
to 1233A except for the opposite (2S,3S)-â-lactone ring
(Figure 1), showed no inhibitory activity against HMG-
CoA synthase even at 500 µM.2b Therefore, the impor-
tance of the â-lactone stereochemistry was suggested for
inhibition of HMG-CoA synthase, but the exact correla-
tion between the stereochemistry and the inhibition still
remains to be defined.
† Present Address: Kawai J uku, Sendagaya, Shibuya-ku, Tokyo 151,
J apan.
‡ Fuji Chemical Industries Co. Ltd.
F igu r e 1. Structure of â-lactone stereochemistry of 1233A,
§ Daiichi Pharmaceutical Co. Ltd.
ebelactones, and four chiral DU-6622.
X Abstract published in Advance ACS Abstracts, March 1, 1997.
(1) Aldridge, D. C.; Gile, D.; Turner, W. B. J . Chem. Soc. C 1971,
During our synthetic study of 1233A analogs,6
a
3888.
(2) (a) Oh mura, S.; Tomoda, H.; Kumagai, H.; Greenspan, M. D.;
Yodkovits, J . B.; Chen, J . S.; Albert, A. W.; Martin, I.; Mochales, S.;
Monaghan, R. L.; Chabala, J . C.; Schwartz, R. E.; Patchett, A. A. J .
Antibiot. 1987, 40, 1356. (b) Tomoda, H.; Kumagai, H.; Tanaka, H.;
Oh mura, S. Biochim. Biophys. Acta 1987, 922, 351. (c) Greenspan, M.
D.; Yudkovitz, J . B.; Lo, C. Y. L.; Chen, J . S.; Alberts, A. W.; Hunt, V.
M.; Chang, M. N.; Yang, S. S.; Thompson, K. L.; Chiang, Y.-C. P.;
Chabala, J . C.; Monaghan, R. L.; Schwartz, R. L. Proc. Natl. Acad.
Sci. U.S.A. 1987, 84, 7488.
derivative named trans-DU-6622 (erythro-3-hydroxy-2-
(hydroxymethyl)-5-[7-(methoxycarbonyl)naphthalen-1-yl]-
(6) (a) Sunazuka, T.; Tsuzuki, K.; Kumagai, H.; Tomoda, H.; Tanaka,
H.; Nagashima, H.; Hashizume, H.; Oh mura, S. J . Antibiot. 1992, 45,
1139. (b) Hashizume, H.; Ito, H.; Yamada, K.; Nagashima, H.; Kanao,
M.; Tomoda, H.; Sunazuka, T.; Kumagai, H.; Oh mura, S. Chem. Pharm.
Bull. 1994, 42, 512. (c) Hashizume, H.; Ito, H.; Kanaya, N.; Na-
gashima, H.; Usui, H.; Oshima, R.; Kanao, M.; Tomoda, H.; Sunazuka,
T.; Kumagai, H.; Oh mura, S. Chem. Pharm. Bull. 1994, 42, 1272. (d)
Hashizume, H.; Ito, H.; Kanaya, N.; Nagashima, H.; Usui, H.; Oshima,
R.; Kanao, M.; Tomoda, H.; Sunazuka, T.; Nagamitsu, T.; Kumagai,
H.; Oh mura, S. Heterocycles 1994, 38, 1551. (e) Hashizume, H.; Ito,
H.; Morikawa, T.; Kanaya, N.; Nagashima, H.; Usui, H.; Tomoda, H.;
Sunazuka, T.; Kumagai, H.; Oh mura, S. Chem. Pharm. Bull. 1994, 42,
2097.
(3) Nagashima, H.; Kumagai, H.; Tomoda, H.; Oh mura, S. Life Sci.
1993, 52, 1595.
(4) Chiang, Y.-C. P.; Chang, M. N.; Yang, S. S.; Chabala, J . C.; Heck,
J . V. J . Org. Chem. 1988, 53, 4599.
(5) (a) Umezawa, H.; Aoyagi, T.; Uotani, K.; Hamada, M.; Takeuchi,
T.; Takahashi, T. J . Antibiot. 1980, 33, 1594. (b) Uotani, K.; Naga-
nawa, H.; Kondo, S.; Aoyagi, T.; Umezawa, H. J . Antibiot. 1982, 35,
1495.
S0022-3263(96)02143-3 CCC: $14.00 © 1997 American Chemical Society

2162 J . Org. Chem., Vol. 62, No. 7, 1997
Tomoda et al.
Sch em e 1. Syn th etic Rou te for th e F ou r
Ster eoisom er s of DU-6622a
F igu r e 2. Configurational correlation model for (S)-O-meth-
ylmandalate derivatives (+)-5a and (-)-5a .
Ta ble 1. Non equ iva len t 1H NMR Ch em ica l Sh ifts for
Selected P r oton s of (S)-O-Meth ylm a n d ela tes (+)-5a ,
(-)-5a , (+)-5b, a n d (-)-5b
proton
(+)-5a
2.95
(-)-5a
2.73
(+)-5b
3.17
(-)-5b
3.13
L3 Ha
Hb
3.40, 3.44
3.08, 3.25
3.28, 3.27
3.28, 3.49
L2 Hc
Hd
1.75, 1.90
2.55 2.73
1.92, 2.05
2.88, 3.08
1.94, 2.05
2.88 3.09
1.71, 1.87
2.44, 2.90
lactone isomers) was synthesized as reported previously.6b
Four chiral ((2R,3R)- ((+)-8a ), (2S,3S)- ((-)-8a ), (2R,3S)-
((+)-8b), and (2S,3R)- ((-)-8b)) isomers of DU-6622
(Figure 1) were synthesized by the synthetic sequence
shown in Scheme 1.
The essential carbon skeleton of DU-6622 was formed
by an aldol condensation with 3-hydroxypropionic acid
benzyl ester (1) and the corresponding aldehyde (2) to
afford erythro-(()-3a and threo-(()-3b according to the
method reported previously.6a The selective protection
of the primary alcohol in (()-3a and (()-3b was per-
formed with a triphenylmethyl group. Then, each enan-
tiomer could be isolated by esterification of the secondary
alcohols of (()-4a (or (()-4b) with (S)-O-methylmandelic
acid to form the corresponding diastereomers (+)-5a and
(-)-5a (or (+)-5b and (-)-5b). On the basis of an NMR-
configurational correlation model,4,7 the nonequivalence
of the chemical shifts of the proton resonances from the
groups attached to the carbinyl carbon can be used to
assign the absolute configuration of that stereogenic
center. The assumed low-energy conformation upon
which this model depends is illustrated by the structures
(+)-5a and (-)-5a in Figure 2. The protons of the
substituent that is eclipsed by the phenyl ring always
show an upfield shift possibly due to the shielding effect
of the phenyl ring. In the structures (+)-5a and (-)-5a ,
the substituents are L2 and L3, respectively.
Table 1 summarizes the nonequivalent chemical shifts
for (+)-5a , (-)-5a , (+)-5b, and (-)-5b. The data show
that the protons Ha and Hb in (-)-5a resonate at higher
field than the corresponding protons in (+)-5a . On the
other hand, the Hc and Hd in (+)-5a resonate at higher
field than those in (-)-5a . As illustrated by the correla-
tion model in Figure 2, the shift data are consistent with
the assignment of R- and S-configurations at the second-
ary alcohol in (+)-5a and (-)-5a , respectively. Since
these compounds have erythro-structures, the absolute
a
Abbreviations: LDA, lithium diisopropylamide; TrCl, triphen-
ylmethyl chloride; DMAP, (dimethylamino)pyridine; TEA, triethyl-
amine; DCC, dicyclohexylcarbodiimide; p-TsCl, p-toluenesulfonyl
chloride; TFA, trifluoroacetic acid; BuOH, n-butanol.
pentanoic acid 1,3-lactone) was selected as a potent
HMG-CoA synthase inhibitor. The IC50 value (0.15 µM)
is analogous to that of 1233A (0.20 µM). We chose this
compound as a model inhibitor of HMG-CoA synthase to
answer the above question. In this paper, we describe
the preparation of the four chiral isomers of DU-6622 to
compare their inhibitory activity against HMG-CoA
synthase. Thus, we demonstrate that the (2R,3R)-â-
lactone structure is primarily responsible for HMG-CoA
synthase inhibition.
Resu lts a n d Discu ssion
Syn th esis of F ou r Ch ir a l Isom er s of DU-6622.
trans-DU-6622 (a mixture of (2R,3R)- and (2S,3S)-â-
(7) Dale, J . A.; Mosher, H. S. J . Am. Chem. Soc. 1973, 95, 512.

Synthesis of Four Chiral Isomers of â-Lactone DU-6622
J . Org. Chem., Vol. 62, No. 7, 1997 2163
Ta ble 2. Effect of F ou r Ch ir a l a n d tr a n s-DU-6622 on
Exp er im en ta l Section
HMG-CoA Syn th a se
Ma t er ia ls. Isolation of 1233A from the culture broth of
Scopulariopsis sp. F-244 was performed as reported.9 Lova-
statin was purchased from Merck, Sharp and Dohme (Rahway,
NJ ). Acetyl-CoA, acetoacetyl-CoA, and cholestyramine were
obtained from Sigma (St. Louis, MO). [1-14C]Acetyl-CoA (53
µCi/µmol) was purchased from Dupont/NEN Research Prod-
ucts. All other chemicals used in this work were of the highest
purity available commercially and were used without further
purification.
IC50 (µM)
compd
config (C-2, C-3)
HMG-CoA synthase
(+)-8a
(-)-8a
(+)-8b
(-)-8b
(()-8a
(R,R)
(S,S)
(R,S)
(S,R)
0.098
31
360
9.4
0.15
(R,R) + (S,S)
Syn th eses a n d Ch a r a cter iza tion of An a logs. Four
optically active isomers of DU-6622 were prepared in the
synthetic sequence shown in Scheme 1. All NMR spectra were
measured in CDCl3 at 400 MHz for 1H and at 100 MHz for
13C. Chemical shifts are reported in parts per million relative
to tetramethylsilane used as internal standard.
configurations of (+)-5a and (-)-5a were assigned as
2R,3R and 2S,3S, respectively. Similarly, the absolute
configurations of (+)-5b and (-)-5b were determined to
be 2R,3S and 2S,3R, respectively (Table 1).
er yth r o-3-Hydr oxy-5-[7-(m eth oxycar bon yl)n aph th alen -
1-yl]-2-[(tr ityloxy)m eth yl]p en ta n oic Acid Ben zyl Ester
((()-4a ) a n d th r eo-3-Hyd r oxy-5-[7-(m eth oxyca r bon yl)-
n aph th alen -1-yl]-2-[(tr ityloxy)m eth yl]pen tan oic Acid Ben -
zyl Ester ((()-4b). To a cold solution (-40 °C) of diisopro-
pylamine (15.8 mL) in tetrahydrofuran (88.3 mL) was added
a solution of n-butyllithium (15% in hexane) (86.1 mL) drop-
wise over 25 min under an argon atmosphere. After the
addition was complete, the solution was warmed to 0 °C for
15 min and then cooled to -78 °C. A solution of 3-hydroxy-
propionic acid benzyl ester (1) (10.6 g) in tetrahydrofuran (32
mL) was added dropwise over 20 min to the lithium diisopro-
pylamide (LDA) solution at -78 °C. After 10 min, the reaction
mixture was kept at -25 °C for 5 min and then cooled to -78
°C. After 10 min, a solution of 2 (15.67 g) in tetrahydrofuran
(65 mL) was added dropwise over 30 min to the reaction
mixture at -78 °C, and the resulting mixture was stirred at
-78 °C for 50 min. The reaction was quenched by addition of
saturated NH4Cl solution (450 mL), and the mixture was
extracted with ether (3 × 450 mL). The combined extracts
were dried over MgSO4, and the solvent was removed in vacuo
to obtain a yellow oil (24.24 g). This oil was purified by column
chromatography on silica gel (CHCl3-acetone ) 20:1) to afford
erythro-3-hydroxy-2-(hydroxymethyl)-5-[7-(methoxycarbonyl)-
naphthalen-1-yl]pentanoic acid benzyl ester ((()-3a ) (2.85 g,
10%) and threo-3-hydroxy-2-(hydroxymethyl)-5-[7-(methoxy-
carbonyl)naphthalen-1-yl]pentanoic acid benzyl ester ((()-3b)
(3.11 g, 11%).
A solution of (()-3a (2.84 g) in CH2Cl2 was cooled to 0 °C
under argon, 4-(dimethylamino)pyridine (163 mg), triethyl-
amine (1.09 g), and triphenylmethyl chloride (2.81 g) were
added, and then the mixture was stirred at room temperature
overnight. The reaction mixture was diluted with CH2Cl2 (150
mL), washed with saturated NaHCO3 solution (150 mL), and
dried over MgSO4, and the solvent was removed in vacuo. The
residue was purified by column chromatography on silica gel
(CHCl3-diisopropyl ether ) 100:1) to afford (()-4a (4.0 g,
90%): 1H NMR (CDCl3) δ 1.62-1.86 (m, 2H), 2.68-3.35 (m,
4H), 3.47 (d, 2H), 3.85-4.10 (m, 1H), 3.92 (s, 3H), 5.18 (s, 2H),
7.00-8.11 (m, 25H), 8.81 (t, 1H). Anal. Calcd for C44H40O6:
C, 79.49; H, 6.06. Found: C, 79.75; H, 6.32.
Further synthesis was performed according to our
established procedures.6 After removal of the methyl-
mandelic acid ester from each optically pure isomer ((+)-
5a , (-)-5a , (+)-5b, and (-)-5b), they were cyclized by
treatment with p-toluenesulfonyl chloride to form the
â-lactone ((+)-7a , (-)-7a , (+)-7b, and (-)-7b). Finally,
the triphenylmethyl protecting group was removed by
TFA treatment to yield the four DU-6622 isomers ((+)-
8a , (-)-8a , (+)-8b, and (-)-8b, Figure 1).
In h ibition of HMG-CoA Syn th a se by th e Sp ecific
Isom er (2R,3R)-DU-6622. The importance of the ster-
eochemistry of a â-lactone ring was suggested due to the
following evidence using natural â-lactone compounds:
(1) the fungal 1233A with a (2R,3R)-â-lactone ring
showed potent HMG-CoA synthase inhibitory activity,
but (2) microbial ebelactones with an opposite (2R,3R)-
â-lactone ring did not express the inhibitory activity.2b
To make this point clear, the effect of the four optically
active isomers of DU-6622 on HMG-CoA synthase was
tested, and the results are summarized in Table 2.
Among the four stereoisomers, the (2R,3R)-isomer (+)-
8a exhibited the most potent inhibition of HMG-CoA
synthase. The IC50 value was calculated to be 0.098 µM,
indicating that this isomer is more potent than trans-
DU-6622 and 1233A (IC50: 0.15 and 0.20 µM, respec-
tively). However, the other three isomers were substan-
tially weaker inhibitors. Especially, the (2S,3S)-isomer
(-)-8a showed very weak inhibitory activity with an over
300-fold higher IC50 value than (+)-8a . Therefore, we
concluded that the 2R,3R stereochemistry of the â-lactone
ring is responsible for elicitng HMG-CoA synthase inhi-
bition. Furthermore, the (2S,3R)-isomer (-)-8b is more
potent than the (2S,3S)-isomer (-)-8a , suggesting that
the C-3 geometry of the â-lactone is more important for
the inhibitory activity than the C-2 geometry. Miziorko
et al.8 reported that the final product HMG-CoA pos-
sesses inhibitory activity against HMG-CoA synthase by
competing with the substrate acetyl-CoA at the acetyl-
CoA binding site of the enzyme. Therefore, it might be
plausible that DU-6622 is structurally similar to HMG-
CoA as an inhibitor. In fact, if the respective â-lactone
carbons (C-1 to -3, see (+)-8a in Scheme 1) of DU-6622
are fixed to the HOO1C2CH23C(OH)(CH3)- carbons of
HMG-CoA, the C-3 carbon corresponds to the stereogenic
3C carbon and the bulky ethyl naphthalene part of the
inhibitor orients to the same direction as the -CH2-CoA
moiety of HMG-CoA. This might be the reason for the
importance of the C-3 geometry of the â-lactone for HMG-
CoA synthase inhibition.
Compound (()-3b (3.10 g) was treated in a manner similar
to that described for the preparation of (()-4a to give (()-4b
(3.83 g, 79%): 1H NMR δ 1.62-1.86 (m, 2H), 2.45-3.45 (m,
4H), 3.56 (d, J ) 5.5 Hz, 2H), 3.80-4.20 (m, 1H), 3.91 (s, 3H),
5.14 (s, 2H), 7.00-8.10 (m, 25H), 8.77 (t, J ) 0.7 Hz, 1H). Anal.
Calcd for C44H40O6: C, 79.49; H, 6.06. Found: C, 79.50; H,
6.22.
(+)-er yth r o-3-H yd r oxy-5-[7-(m et h oxyca r b on yl)n a p h -
th a len -1-yl]-2-[(tr ityloxy)m eth yl]p en ta n oic Acid (S)-(+)-
r-Meth oxyp h en yla cetic Acid Ester ((+)-5a ) a n d (-)-
er yth r o-3-Hyd r oxy-5-[7-(m eth oxyca r bon yl)n a p h th a len -
1-yl]-2-[(tr ityloxy)m eth yl]p en ta n oic Acid (S)-(+)-r-Meth -
oxyp h en yla cetic Acid Ester ((-)-5a ). A mixture of (()-4a
(2.0 g), dicyclohexylcarbodiimide (1.24 g), (S)-(+)-R-methoxy-
(9) Tomoda, H.; Kumagai, H.; Takahashi, Y.; Tanaka, Y.; Iwai, Y.;
Oh mura, S. J . Antibiot. 1988, 41, 247. Kumagai, H.; Tomoda, H.;
Oh mura, S. J . Antibiot. 1992, 45, 563.
(8) Miziorko, H. M.; Lane, M. D.; Weidman, S. W. Biochemistry 1979,
18, 399.

2164 J . Org. Chem., Vol. 62, No. 7, 1997
Tomoda et al.
phenylacetic acid (750 mg), and 4-(dimethylamino)pyridine
(375 mg) in CH2Cl2 (3 mL) was stirred at room temperature
for 30 min. The reaction mixture was diluted with ether and
filtered, and then the solvent was removed in vacuo. The
residue was purified by column chromatography on silica gel
(hexane-ethyl acetate ) 10:1-2:1) to afford an oily material.
The oily material was dissolved in EtOH (30 mL), and 5% Pd/C
(2.0 g) was added to the solution. The mixture was stirred
for 1 h at room temperature under a hydrogen atmosphere.
The reaction mixture was filtered through a Celite pad,
concentrated under reduced pressure, and purified by column
chromatography on silica gel (CH2Cl2-MeOH ) 95:5) to afford
(+)-5a (1.05 g, 49%) and (-)-5a (1.05 g, 49%) as colorless solids.
(+)-er yth r o-3-Hyd r oxy-2-(h yd r oxym eth yl)-5-[7-(m eth -
oxyca r bon yl)n a p h th a len -1-yl]p en ta n oic Acid 1,3-La c-
ton e ((+)-8a ). To a solution of (+)-7a (445 mg) in n-butanol
(10 mL) was added trifluoroacetic acid (4.4 mL) dropwise over
10 min at 0 °C, and the mixture was stirred at room
temperature. After 2 h, the mixture was diluted with EtOAc
(150 mL), washed with saturated NaHCO3, and dried over
MgSO4, and the solvent was removed in vacuo. The residue
was purified by column chromatography on silica gel (CHCl3-
acetone ) 50:1) to afford (+)-8a (195 mg, 78%) as a colorless
solid: [R]20 +69.0 (c 1.0, EtOH); MS m/z 314 (M+); HRMS
D
calcd for C18H18O5 314.1154, found 314.1177; 1H NMR δ 2.16-
2.46 (m, 2H), 3.16-3.51 (m, 3H), 3.81-4.18 (m, 2H), 3.96 (s,
3H), 4.67 (dt, J ) 3.6, 6.8 Hz, 1H), 7.30-8.24 (m, 20H), 8.75
(s, 1H). Anal. Calcd for C18H18O5: C, 68.78; H, 5.77. Found:
C, 68.73; H, 5.90.
(+)-5a : [R]23D + 7.2 (c 1.0, EtOH); 1H NMR δ 1.60-1.95 (m,
2H), 2.50-3.50 (m, 5H), 3.33 (s, 3H), 3.90 (s, 3H), 4.69 (s, 1H),
5.24-5.55 (m, 1H), 7.10-8.10 (m, 25H), 8.55 (s, 1H). Anal.
Calcd for C46H42O8: C, 76.43; H, 5.85. Found: C, 76.58; H,
(-)-er yth r o-3-Hyd r oxy-2-(h yd r oxym eth yl)-5-[7-(m eth -
oxyca r bon yl)n a p h th a len -1-yl]p en ta n oic Acid 1,3-La c-
ton e ((-)-8a ). Compound (-)-7a (480 mg) was treated in a
manner similar to that described for the preparation of (+)-
5.95. (-)-5a : [R]23 +3.6 (c 1.0, EtOH); 1H NMR (CDCl3, δ
D
ppm) 1.70-2.20 (m, 2H), 2.50-3.60 (m, 5H), 3.33 (s, 3H), 3.95
(s, 3H), 4.73 (s, 1H), 5.24-5.54 (m, 1H), 7.10-8.12 (m, 25H),
8.75 (s, 1H). Anal. Calcd for C46H42O8: C, 76.43; H, 5.85.
Found: C, 76.60; H, 5.92.
8a to give (-)-8a (210 mg, 78%) as a colorless solid: [R]20
D
-68.9 (c 1.0, EtOH); MS m/z 314 (M+); HRMS calcd for
C18H18O5 314.1154, found 314.1172; 1H NMR δ 2.16-2.46 (m,
2H), 3.16-3.51 (m, 3H), 3.81-4.18 (m, 2H), 3.96 (s, 3H), 4.67
(dt, J ) 3.6, 6.8 Hz, 1H), 7.30-8.24 (m, 20H), 8.75 (s, 1H).
Anal. Calcd for C18H18O5: C, 68.78; H, 5.77. Found: C, 68.50;
H, 5.90.
(+)-er yth r o-3-H yd r oxy-5-[7-(m et h oxyca r b on yl)n a p h -
th a la n -1-yl]-2-[(tr ityloxy)m eth yl]p en ta n oic Acid ((+)-6a ).
To a solution of (+)-5a (700 mg) in MeOH (7 mL) was added
4 M CH3ONa (1 mL), and the mixture was stirred at room
temperature for 3 h. The pH of the reaction mixture was
adjusted to 2.0 by addition of 1 M HCl, and the mixture was
extracted with CH2Cl2. The CH2Cl2 solution was dried over
MgSO4, and the solvent was removed in vacuo. The residue
was purified by column chromatography on silica gel (CH2-
Cl2-MeOH ) 9.1) to afford (+)-6a (500 mg, 90%) as a colorless
(+)-th r eo-3-Hyd r oxy-5-[7-(m eth oxyca r bon yl)n a p h th a -
len -1-yl]-2-[(tr ityloxy)m eth yl]p en ta n oic Acid (S)-(+)-r-
Meth oxyp h en yla cetic Acid Ester ((+)-5b) a n d (-)-th r eo-
3-H yd r oxy-5-[7-(m et h oxyca r b on yl)n a p h t h a len -1-yl]-2-
[(t r it yloxy)m et h yl]p en t a n oic Acid (S)-(+)-rMet h oxy-
p h en yla cetic Acid Ester ((-)-5b). Compound (()-4b (1.5
g) was treated in a manner similar to that described for the
preparation of (+)-5a and (-)-5b to give (+)-5b (680 mg, 42%)
and (-)-5b (680 mg, 42%).
solid: [R]23 +13.0 (c 1.0, EtOH); 1H NMR δ 1.68-2.00 (m,
D
2H), 2.60-2.87 (m, 1H), 3.05-3.33 (m, 2H), 3.50 (d, J ) 5.9
Hz, 2H), 3.70-4.20 (m, 1H), 3.88 (s, 3H), 6.95-8.10 (m, 20H),
8.80 (s, 1H). Anal. Calcd for C37H34O6: C, 77.33; H, 5.96.
Found: C, 77.51; H, 5.65.
(+)-5b: [R]20D +31.0 (c 1.0, EtOH); 1H NMR (CDCl3) δ 1.70-
2.10 (m, 2H), 2.20-3.70 (m, 5H), 3.42 (s, 3H), 3.98 (s, 3H), 4.58
(s, 1H), 5.12-5.42 (m, 1H), 7.05-8.10 (m, 25H), 8.63 (s, 1H).
(-)-er yth r o-3-H yd r oxy-5-[7-(m et h oxyca r b on yl)n a p h -
th a len -1yl]-2-[(tr ityloxy)m eth yl]p en ta n oic Acid ((-)-6a ).
Compound (-)-5a (722 mg) was treated in a manner similar
to that described for the preparation of (+)-6a to give (-)-6a
Anal. Calcd for C46H42O8: C, 76.43; H, 5.85. Found: C, 76.45;
1
H, 5.80. (-)-5b: [R]20 +8.2 (c 1.0, EtOH); H NMR δ 1.86-
D
2.16 (m, 2H), 2.60-3.40 (m, 5H), 3.32 (s, 3H), 4.00 (s, 3H), 4.72
(s, 1H), 5.10-5.40 (m, 1H), 7.10-8.15 (m, 25H), 8.75 (s, 1H).
Anal. Calcd for C46H42O8: C, 76.43; H, 5.85. Found: C, 76.40;
H, 5.85.
(517 mg, 90%) as a colorless solid: [R]20 -13.9 (c 1.0, EtOH);
D
1H NMR δ 1.68-2.00 (m, 2H), 2.60-2.87 (m, 1H), 3.05-3.33
(m, 2H), 3.50 (d, J ) 5.9 Hz, 2H), 3.70-4.20 (m, 1H), 3.88 (s,
3H), 6.95-8.10 (m, 20H), 8.80 (s, 1H). Anal. Calcd for
(+)-th r eo-3-Hyd r oxy-5-[7-(m eth oxyca r bon yl)n a p h th a -
len -1-yl]-2-[(tr ityloxy)m eth yl]p en ta n oic Acid 1,3-La cton e
((+)-7b). Compound (+)-5b (55 mg) was treated in a manner
similar to that described for the preparation of (+)-6a to give
(+)-6b (36 mg, 82%) and then treated in a manner similar to
that described for the preparation of (+)-7a to give (+)-7b (30
C
37H34O6: C, 77.33; H, 5.96. Found: C, 77.52; H, 5.73.
(+)-er yth r o-3-H yd r oxy-5-[7-(m et h oxyca r b on yl)n a p h -
th a len -1-yl]-2-[(tr ityloxy)m eth yl]p en ta n oic Acid 1,3-La c-
ton e ((+)-7a ). To a solution of (+)-6a (500 mg) in pyridine
(1 mL) was added p-toluenesulfonyl chloride (700 mg) at 0 °C,
and the mixture was stirred at 3 °C overnight. MeOH (5 mL)
was added to the mixture, and the mixture was stirred at room
temperature for an additional 30 min and diluted with CH2-
Cl2 (50 mL). The solution was washed with saturated NaCl
solution (50 mL) and dried over MgSO4, and the solvent was
removed in vacuo. The residue was purified by column
chromatography on silica gel (CHCl3-MeOH ) 10:1) to afford
mg, 86%): [R]20 +13.3 (c 1.0 EtOH); 1H NMR δ 1.92-2.40
D
(m, 2H), 2.87-3.76 (m, 4H), 3.82-4.07 (m, 1H), 3.95 (s, 3H),
4.61 (dt, J ) 7.9, 6.9 Hz, 1H), 7.00-8.15 (m, 20H), 8.64 (s,
1H). Anal. Calcd for C37H32O5: C, 79.83; H, 5.79. Found: C,
79.88; H, 5.58.
(-)-th r eo-3-Hyd r oxy-5-[7-(m eth oxyca r bon yl)n a p h th a -
len -1-yl]-2-[(tr ityloxy)m eth yl]p en ta n oic Acid 1,3-La cton e
((-)-7b). Compound (-)-5b (68 mg) was treated in a manner
similar to that described for the preparation of (+)-6a to give
(-)-6b (45 mg, 86%) and then treated in a manner similar to
that described for the preparation of (+)-7a to give (-)-7b (30
mg, 67%): [R]20D -5.0 (c 1.0, EtOH); 1H NMR δ 1.92-2.40 (m,
2H), 2.87-3.76 (m, 4H), 3.82-4.07 (m, 1H), 3.95 (s, 3H), 4.61
(dt, J ) 7.9, 6.9 Hz, 1H), 7.00-8.15 (m, 20H), 8.64 (s, 1H).
Anal. Calcd for C37H32O5: C, 79.83; H, 5.79. Found: C, 79.70;
H, 5.71.
(+)-7a (445 mg, 92%) as colorless solid: [R]20 +33.2 (c 1.0,
D
EtOH); MS m/z 556 (M+); HRMS calcd for C37H32O5 556.2250,
found 556.2237; 1H NMR δ 2.10-2.41 (m, 2H), 3.10-3.38 (m,
4H), 3.42-3.67 (m, 1H), 3.95 (s, 3H), 4.58 (dt, J ) 4.1, 6.6 Hz,
1H), 7.00-8.21 (m, 20H), 8.75 (s, 1H). Anal. Calcd for
C
37H32O5: C, 79.83; H, 5.79. Found: C, 79.77; H, 5.70.
(-)-er yth r o-3-H yd r oxy-5-[7-(m et h oxyca r b on yl)n a p h -
th a len -1-yl]-2-[(tr ityloxy)m eth yl]p en ta n oic Acid 1,3-La c-
ton e ((-)-7a ). Compound (-)-6a (500 mg) was treated in a
manner similar to that described for the preparation of (+)-
(+)-th r eo-3-Hyd r oxy-2-(h yd r oxym eth yl)5-[7-(m eth oxy-
ca r b on yl)n a p h t h a len -1-yl]p en t a n oic Acid 1,3-La ct on e
((+)-8b). Compound (+)-7b (30 mg) was treated in a manner
similar to that described for the preparation of (+)-8a to give
7a to give (-)-7a (480 mg, 96%) as a colorless solid: [R]20
D
-17.4 (c 1.0, EtOH); MS m/z 556 (M+); HRMS calcd for
C
37H32O5 556.2250, found 556.2234; 1H NMR δ 2.10-2.41 (m,
(+)-8b (10 mg, 59%): [R]20 +62.9 (c 1.0, EtOH); MS m/z 314
2H), 3.10-3.38 (m, 4H), 3.42-3.67 (m, 1H), 3.95 (s, 3H), 4.58
(dt, J ) 4.1, 6.6 Hz, 1H), 7.00-8.21 (m, 20H), 8.75 (s, 1H).
Anal. Calcd for C37H32O5: C, 79.83; H, 5.79. Found: C, 79.85;
H, 5.66.
D
(M+); HRMS calcd for C18H18O5 314.1154, found 314.1179; 1H
NMR δ 2.24-2.53 (m, 2H), 2.65 (brs, 1H), 3.20-3.42 (m, 2H),
3.72-4.35 (m, 3H), 3.98 (s, 3H), 4.71 (dt, J ) 5.9, 6.8 Hz, 1H),

Synthesis of Four Chiral Isomers of â-Lactone DU-6622
J . Org. Chem., Vol. 62, No. 7, 1997 2165
7.31-8.14 (m, 5H), 8.88 (s, 1H). Anal. Calcd for C18H18O5:
C, 68.78; H, 5.77. Found: C, 68.72; H, 5.82.
potassium phosphate elutes contained â-ketothiolase and
HMG-CoA synthase, respectively. The enzyme was concen-
trated by ammonium sulfate precipitation, dissolved in the
buffer used for elution, and stored at -70 °C.
(-)-th r eo-3-Hydr oxy-2-(h yd r oxym eth yl)-5-[7-(m eth oxy-
ca r b on yl)n a p h t h a len -1-yl]p en t a n oic Acid 1,3-La ct on e
((-)-8b). Compound (-)-7b (30 mg) was treated in a manner
similar to that described for the preparation of (+)-8a to give
HMG-CoA synthase was assayed as described previously.2b
The reaction mixture contained 100 mM Tris-HCl (pH 8.2),
100 µM EDTA, 100 µM acetoacetyl-CoA, 200 µM [1-14C]acetyl-
CoA (0.06 µCi), â-lactone compounds (dissolved in 5 µL of
EtOH), and enzyme protein in a total volume of 200 µL. The
reaction was initiated by addition of the enzyme. The reaction
mixture was incubated at 37 °C for 30 min and then stopped
by the removal of 40 µL of the reaction mixture to 100 µL of 6
M HCl in a glass counting vial. The vials were heated to
dryness at 90 °C for 90 min. The residues were determined
(-)-8b (8 mg, 47%): [R]20 -49.3 (c 1.0, EtOH); MS m/z 314
D
(M+); HRMS calcd for C18H18O5 314.1154, found 314.1162; 1H
NMR δ 2.24-2.53 (m, 2H), 2.65 (brs, 1H), 3.20-3.42 (m, 2H),
3.72-4.35 (m, 3H), 3.98 (s, 3H), 4.71 (dt, J ) 5.9, 6.8 Hz, 1H),
7.31-8.14 (m, 5H), 8.88 (s, 1H). Anal. Calcd for C18H18O5:
C, 68.78; H, 5.77. Found: C, 68.81; H, 5.85.
Assa y for HMG-CoA Syn th a se. HMG-CoA synthase was
prepared from livers of rats fed lovastatin (0.1%) and
cholestyramine (5%) for 7 days. The enzyme was purified
through the DEAE-cellulose step according to the method of
Mehrabian et al.10 with some modification. The 30-55%
ammonium sulfate precipitation and a dialysis were carried
out prior to the DEAE-cellulose step. The 60 and 100 mM
for radioactivity with
(Aloka).
a liquid scintillation spectrometer
Ack n ow led gm en t. This work was supported in part
by grants from the J apan Society for the Promotion of
Science, from the Ministry of Education, Science and
Culture of J apan, and from J apan Keirin Association.
(10) Mehrabian, M.; Callaway, K. A.; Clarke, C. E.; Tanaka, R. D.;
Greenspan, M.; Lusis, A.; Sparkes, R. S.; Mohandas, T.; Edmond, J .;
Edwards, P. A. J . Biol. Chem. 1986, 261, 16249.
J O9621433