ATP-citrate lyase as a target for hypolipidemic intervention. 2. Synthesis and evaluation of (3R*,5S*)-ω-substituted-3-carboxy-3,5- dihydroxyalkanoic acids and their γ-lactone prodrugs as inhibitors of the enzyme in vitro and in vivo

Article abstract of DOI:10.1021/jm980091z

A series of (3R*,5S*)-ω-substituted-3-carboxy-3,5-dihydroxyalkanoic acids have been synthesized and evaluated as inhibitors of the recombinant human form of ATP-citrate lyase. The best of these have K(i)'s in the 200- 1000 nM range. As the corresponding thermodynamically favored γ-lactone prodrugs, a number of compounds are able to inhibit cholesterol and fatty acid synthesis in HepG2 cells and reduce plasma triglyceride levels in vivo. The best of these, compound 77, is able to induce clear hypocholesterolemic and hypotriglyceridaemic responses when administered orally to rat and dog. These results provide evidence to support the hypothesis that compounds which inhibit ATP-citrate lyase have the potential to be a novel class of hypolipidemic agent, which possess combined hypocholesterolemic and hypotriglyceridemic activities.

Full text of DOI:10.1021/jm980091z

3582
J . Med. Chem. 1998, 41, 3582-3595
ATP -Citr a te Lya se a s a Ta r get for Hyp olip id em ic In ter ven tion . 2. Syn th esis
a n d Eva lu a tion of (3R*,5S*)-ω-Su bstitu ted -3-ca r boxy-3,5-d ih yd r oxya lk a n oic
Acid s a n d Th eir γ-La cton e P r od r u gs a s In h ibitor s of th e En zym e in Vitr o a n d in
Vivo
Andrew D. Gribble,*,† Robert J . Ife,† Antony Shaw,† David McNair,† Christine E. Novelli,† Susan Bakewell,†
Virendra P. Shah,† Roland E. Dolle,†,‡ Pieter H. Groot,§ Nigel Pearce,§ J ohn Yates,§ David Tew,| Helen Boyd,|
Stephen Ashman,| Drake S. Eggleston,† R. Curtis Haltiwanger, and George Okafo#
Departments of Medicinal Chemistry, Vascular Biology, Mechanistic Enzymology, and Analytical Sciences, SmithKline
Beecham Pharmaceuticals Ltd, New Frontiers Science Park (North), Third Avenue, Harlow, Essex CM19 5AW, U.K.
Received February 11, 1998
A series of (3R*,5S*)-ω-substituted-3-carboxy-3,5-dihydroxyalkanoic acids have been synthesized
and evaluated as inhibitors of the recombinant human form of ATP-citrate lyase. The best of
these have Ki’s in the 200-1000 nM range. As the corresponding thermodynamically favored
γ-lactone prodrugs, a number of compounds are able to inhibit cholesterol and fatty acid
synthesis in HepG2 cells and reduce plasma triglyceride levels in vivo. The best of these,
compound 77, is able to induce clear hypocholesterolemic and hypotriglyceridaemic responses
when administered orally to rat and dog. These results provide evidence to support the
hypothesis that compounds which inhibit ATP-citrate lyase have the potential to be a novel
class of hypolipidemic agent, which possess combined hypocholesterolemic and hypotriglyc-
eridemic activities.
In tr od u ction
based, active-site directed, and tight-binding inhibi-
tors.2b-e In the preceding paper, we described our
design strategy leading to the identification of a series
of 2-substituted butanedioic acids which were reason-
ably potent inhibitors of the enzyme.2a The best of these
were compounds 1a -c, which had Ki values against
ATP-citrate lyase (ACL) catalyzes the cleavage of
citrate in the cytosol of many tissues and is the principal
producer of acetyl CoA units for lipid synthesis.1 Inhi-
bition of hepatic ACL is expected to decrease both
cholesterol and fatty acid synthesis and to precipitate
hypotriglyceridemic and hypocholesterolemic responses
in animals and human by inhibition of very low-density
lipoprotein (VLDL) synthesis and up-regulation of he-
patic low-density lipoprotein (LDL) receptors. A more
detailed discussion of this concept is presented in earlier
communications from these laboratories.2 Such consid-
erations led us to propose and investigate ACL as a
potential target for hypolipidemic intervention. An
inhibitor of ACL would be expected to be a novel and
therapeutically beneficial class of hypolipidemic agent,
which should have advantages over a pure hypocholes-
terolemic agent, particularly in light of the emerging
coimplication of hypertriglyceridemia in coronary heart
disease.
The primary goal of our research effort was to identify
a potent, orally active compound that would allow us
to interrogate the hypothesis that inhibition of ACL in
vivo would result in both hypotriglyceridemic and
hypocholesterolemic responses. In earlier papers, we
have described in detail the mechanism of the enzyme-
mediated reaction and the rationale of our unsuccessful
efforts toward the design and synthesis of mechanism-
both isolated rat and recombinant human forms of the
enzyme in the 1-3 µM range. Unfortunately, in a
HepG2 cell assay used to investigate effects of com-
pounds on cholesterol and fatty acid synthesis, none of
these inhibitors demonstrated any activity. This lack
of activity was attributed to a poor or ineffective
penetration of the inhibitor into the cells, making the
compounds unsuitable for use in vivo. In this paper,
we now describe the results of our continued research
efforts in this area which has led to the identification
of compound 77, a prodrug of the active hydroxy acid
45, as a potent, efficacious, and orally active inhibitor
of ACL. The pharmacology on 77 is consistent with the
hypothesis that inhibition of ACL is a feasible target
for hypolipidemic intervention and that this compound,
or a related analogue, may have potential for develop-
ment as a novel hypolipidemic agent.
† Department of Medicinal Chemistry.
‡ Present address: Pharmacopeia, 2000 Cornwall Rd., Monmouth
J unction, NJ 08852.
Ch em istr y
§ Department of Vascular Biology.
Gen er a l P r ep a r a tion of (3R*,5S*)-ω-Su bstitu ted -
3-ca r boxy-3,5-d ih yd r oxya lk a n oic Acid In h ibitor s.
Compound 45 (Table 1) and its related analogues of
Table 3 were normally prepared by the general route
| Department of Mechanistic Enzymology.
Department of Physical and Structural Chemistry, SmithKline
Beecham Pharmaceuticals, P.O. Box 1539, King of Prussia, PA 19406.
#
Department of Analytical Sciences.
S0022-2623(98)00091-0 CCC: $15.00 © 1998 American Chemical Society
Published on Web 08/19/1998

Hypolipidemic Intervention by ATP-Citrate Lyase
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19 3583
Sch em e 1. General Synthesis of (3R*,5S*)-3,5-Dihydroxybutanedioic Acids and Their Corresponding γ-Lactones
Ta ble 1. Effect of Head Hydroxylation
Ta ble 2. Activities of Resolved Enantiomers of Compounds 45
and 58
a
Microanalyses reported for the disodium salts. Most com-
b
pounds contained varying levels of H2O of crystallization. Mi-
croanalysis not obtained. Purity confirmed by NMR and MECC.
olefin 6. Hydrogenation in the presence of Raney Ni
and boric acid, followed by further hydrogenation of the
olefinic bond with platinum oxide, gave the keto com-
pound 7, without any loss of aryl chlorine substituents.
Reduction of this ketone using sodium triacetoxyboro-
hydride in acetic acid gave a ca. 9:1 mixture of the
3R*,5S*:3R*,5R* diastereomeric diesters 8. An earlier
procedure, producing similar diastereoselectivity, em-
ployed the use of NaBH4 in EtOH in the presence of
CeCl3, but this suffered from competing ester group
reduction. The desired pure 3R*,5S* diastereomer of
the final hydroxy acid product 9 was obtained following
hydrolysis of the diester (NaOH/EtOH) and recrystal-
lization of the resulting disodium salt. The correspond-
ing five-membered ring lactones 10 were prepared as
required for secondary screening by heating the hydroxy
acids in aqueous HCl or in aqueous HCl/THF. An
alternative synthesis of the isoxazole intermediate,
proceeding via 2,4-dichloroheptanaldoxime,2a was used
in earlier preparations of compound 45.
a
Microanalyses reported for the disodium salts. Most com-
b
pounds contained varying levels of H2O of crystallization. See
ref 2a. c Analysis for free diacid. Microanalysis performed on
d
γ-lactone derivative; lactone hydrolyzed to 48 prior to use.
depicted in Scheme 1. ꢀ-Caprolactone was converted to
the oxime 2 by treatment with DIBAL-H followed by
subsequent reaction of the intermediary hydroxy alde-
hyde with hydroxylamine. In situ generation of the
corresponding nitrile oxide (NaOCl, Et3N) and its
subsequent [3 + 2] cycloaddition with dimethyl itacon-
ate3 afforded the isoxazole intermediate 3. Swern
oxidation generated the aldehyde 4, a prerequisite
compound for reaction with the appropriately substi-
tuted phosphonium salt 5 (NaH/DMSO) to give the
Methods for preparations of the di- and trihydroxy
compounds of Table 1 varied depending upon the
position of substitution. The 3R*,5R* diastereomer 46

3584 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19
Gribble et al.
Sch em e 2. Syntheses of the Hydroxylated Analogues of Table 1
iously.2a The 4-hydroxylated derivative 47, and its
higher homologue 48, were prepared by the route shown
in Scheme 2b. The E-olefin 15, prepared from heating
the aldehyde 132a with the phosphorane 14 in toluene,4
was dihydroxylated (OsO4/NMO) to yield the desired
diacids after hydrolytic workup. These were converted
to their respective five-membered ring lactones, as
described above, for purification and hydrolyzed back
to the diacid prior to enzyme screening. The 2-hydroxy
group of compounds 49 and 50 was introduced through
the reaction of the anion of the lactone 17 with 2-(ben-
zenesulfonyl)-3-phenyloxaziridine5 in THF, followed by
separation of the resulting mixture of diastereomers and
hydrolysis (Scheme 2c).
Ta ble 3. Effect of Ortho Substitution of the Phenyl Ring
The ortho O-substituted analogues 60, 61, and 62 of
Table 3 were prepared by a modification of the standard
procedure of Scheme 1. For these, the intermediate
isoxazole 3 was first converted to its corresponding
lactone 19, by analogous procedures to those described
above, and esterified to give 20. This was oxidized
(Swern) to the aldehyde prior to use and reacted with
the phosphonium salt 21 to give 22 (Scheme 3). Simple
hydrolysis of 22 gave 62, while its hydrogenolysis and
hydrolysis afforded compound 60. Methylation of the
hydroxy intermediate 23 (MeI/K2CO3 in DMF) gave the
lactone 25 which was hydrolyzed to compound 61.
The indole derivatives of Table 4 were prepared via
alkylation of the appropriately substituted indole with
either the iodolactone 28 using NaH/DMF or the bro-
moisoxazole 32 using KOH in DMSO (Scheme 4, a and
b). An exception was compound 65 for which the indole
was alkylated with 7-bromoheptanol to give the alcohol
35, the latter oxidized under Swern conditions to the
aldehyde and this converted to the oxime 36 (Scheme
4c). The standard chemistry from these intermediates,
outlined in Scheme 1, was then employed to obtain the
a
Microanalyses reported for the disodium salts. Most com-
b
pounds contained varying levels of H2O of crystallization. Anal.
(C24H26ClFO6Na2‚2.5H2O) C, F, H: calcd 5.62, found 4.97%.
c Microanalysis performed on γ-lactone derivative; lactone hydro-
lyzed to 60 prior to use.
of compound 45 was prepared as a 1:1 mixture with 45
by the route shown in Scheme 2a. Reaction of the
dianion of methyl acetoacetate with the aldehyde 11
gave the keto compound 12 which was converted to the
final product via the cyanohydrin as described prev-

Hypolipidemic Intervention by ATP-Citrate Lyase
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19 3585
Sch em e 3. Syntheses of the Ortho-O-Substituted Compounds of Table 3
Sch em e 4. Syntheses of Indole Analogues
final compounds. The halides, 28 and 32, were derived
from the alcohols 27 and 3, respectively, by standard
procedures. The bromoisoxazole intermediate required
for the synthesis of compound 63 was obtained in an
identical manner to its n ) 5 analogue by substituting
δ-valerolactone 26 (n ) 4) for ꢀ-caprolactone in the
reaction sequence of Scheme 1. An equivalent strategy
was used for the synthesis of the carbazole derivatives
in Table 5. The fluorene 76 (Table 5) was prepared as
a mixture of diastereomers by the route depicted in
Scheme 5. For this, 3-chlorofluoren-9-one 37 was
reacted with the Grignard reagent derived from 6-ben-
zyloxyhexyl bromide to give 38, which was hydrogenated
(Pd/C in MeOH/aq HCl) to give alcohol 39. The latter

3586 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19
Ta ble 4. Indole Derivatives
Gribble et al.
enantiomerically pure (+)-enantiomer from a single
crystallization. The (-)-enantiomer was readily ob-
tained in a similar manner by use of the L-(+)-enantio-
meric base 41a . This was readily synthesized by
method of
no.
n
R
R1
5-Cl
5-Cl
5-Cl
5,7-diCl
6-Cl
Ki (µM)
synthesisa analysesb
63
64
65
66
67
68
69
70
71
72
73
4
5
6
5
5
5
5
5
5
5
5
H
H
H
H
H
160 ( 23
3.5 ( 0.37
3.2 ( 0.32
1.8 ( 0.2
41 ( 3.6
2.9 ( 0.2
9.5 ( 0.8
11 ( 1.0
64 ( 10
A
B
C
B
B
B
B
B
A
A
A
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N, Cl
C, H, Nc
C, H, Nd
C, H, Ne
C, H, N
nitration of commercially available (1S,2S)-(+)-1-phen-
yl-2-aminopropane-1,3-diol 41b after first protecting as
its triacetyl derivative. A typical resolution gave excel-
lent yields, with ca. 80% isolation of each pure enanti-
omer from the racemate. Chiral purity of the resultant
hydroxy acids after hydrolysis was established by means
of a micellar electrokinetic capillary chromatography
(MECC) assay developed within our laboratories.6 X-
ray crystallographic analysis on the chloramphenicol
base salt of (+)-77 permitted the assignment of its
absolute stereochemistry as 3S,5R.
3-CH2Ph 5-Cl
3-Me
3-Ph
3-NO2
2-Ph
2-Me
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
0.37 ( 0.03
13 ( 1.2
C, H, N
C, H, N, Cl
a
See Scheme 4. b Microanalyses reported for the disodium salts.
Most compounds contained varying levels of H2O of crystallization.
c Analysis for 1H2O‚2.4SiO2. Analysis for 1H2O‚0.6NaOAc. e Anal-
d
ysis for 1H2O‚0.8SiO2.
Ta ble 5. Tricyclic Compounds
Resu lts a n d Discu ssion
The compounds synthesized in this investigation are
listed in Tables 1-5. Enzyme inhibitory activities are
presented as reversible Ki’s for inhibition of human
recombinant ACL. The latter superceeded our original
screening protocol which employed purified rat enzyme.
Where comparisons were made, potent compounds had
almost identical Ki’s against both forms of the enzyme.
In the preceding paper2a we described the design of
inhibitors of ACL that was based upon the concept of
linking a lipophilic group to a citrate-like “head”. The
best compounds 1a -1c were 2-hydroxybutanedioic acids
substituted in the 2-position with an eight-atom spacer
chain linked to a 2,4-dichlorophenyl group. Enzyme
kinetic analysis was consistent with the hypothesis that
the compounds were interacting with the enzyme by
binding of the butanedioic acid moiety to the citrate site,
while the aryl ring located an adjacent hydrophobic
domain.
a
Microanalyses reported for the disodium salts. Most com-
(-)-Hydroxycitrate7,2a 42a is a known, naturally oc-
b
pounds contained varying levels of H2O of crystallization. 1:1
mixture of 3R*,5S*,9′R* and 3R*,5S*,9′S* diastereomers.
was then converted to the corresponding dihydroisox-
azole intermediate by procedures similar to those shown
for the indole in Scheme 4c and elaborated to compound
76 in the normal way.
curring, competitive inhibitor of ACL.8 Its Ki (0.15 µM,
rat ACL), compared to the Km for citrate (100 µM),2e
suggests that the hydroxyl group is imparting a specific,
favorable interaction to the molecule when binding to
the enzyme. Both the corresponding amino (42b),9 (Ki
) 11 µM10) and thio (42c) (Ki ) 58 µM)2b citrate
analogues are much less potent rat ACL inhibitors,
corroborating this view. As our compounds were citrate
competitive inhibitors,2a we anticipated that their po-
tency might be improved further by introducing ap-
propriately positioned hydroxyl group(s) in the vicinity
of the “citrate-head”. Table 1 shows enzyme inhibitory
activities for a number of hydroxylated analogues that
were prepared for this purpose.
Resolu tion of Com p ou n d s 45 a n d 58. Initial
batches (ca. 10 mg) of the resolved enantiomers of
compounds 45 and 58 were obtained by chiral HPLC
separation of the methyl esters of the corresponding
γ-lactones 77 and 80 (Table 6), followed by hydrolysis
to the hydroxy acid in the normal way. We later
discovered a highly efficient resolution through forma-
tion of the diastereomeric salt of the lactone, 77 or 80,
with chloramphenicol base (D-(-)-threo-2-amino-3-(4-
nitrophenyl)propane-1,3-diol, 40). This produced the
Introduction of a hydroxyl group in the 5-position, as
in compound 45, a single racemic diastereomer whose

Hypolipidemic Intervention by ATP-Citrate Lyase
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19 3587
Sch em e 5. Synthesis of the Fluorene 76
Ta ble 6. Compounds Assayed in HepG2 Cells and/or in Vivo in the Rat
HepG2 cells
% reduction of plasma
triglycerides in the rat
cholesterol fatty acid
synthesis synthesis
ATP
levels
γ-lactone
concn
(µM)
37.5
75
no.
R
n
R1 of compd
% control % control % control
mg/kg/d
mg/kg/d
analyses
42a ((-)-hydroxycitrate)
100
110 ( 19 87 ( 8
100 ( 3
79 ( 11
57 ( 4
99 ( 4
nd
nd
C, H
300
1000
100
3
10
30
5
15
15
30
5
15
15
30
1
40 ( 6
15 ( 9
84 ( 13
98 ( 5
53 ( 7c
9 ( 2c
95 ( 6
52 ( 7c
41 ( 8c
11 ( 6c
105 ( 13
37 ( 7c
33 ( 7c
9 ( 3c
103 ( 3
104 ( 5
104 ( 4
45
nd
31
nd
C, H
C, H, Cl
77
2,4-Cl2C6H3
6
H
45
114 ( 14 97 ( 4
56a
74 ( 19a 97 ( 3
18 ( 3c
113 ( 7
81 ( 9b
69 ( 7c
25 ( 7c
123 ( 8
90 ( 9
94 ( 2a
101 ( 3
100 ( 4
98 ( 2
99 ( 2
101 ( 6
99 ( 5
98 ( 3
98 ( 2
103 ( 10
100 ( 6
106 ( 7
98 ( 3
96 ( 2
100 ( 4
95 ( 6
98 ( 4
96 ( 5
101 ( 4
96 ( 4
nd
(+)-77
(+)-45
nd
nd
nd
nd
C, H
C, H
(-)-77
(-)-45
82 ( 8b
39 ( 3c
78
79
2,4-Cl2C6H3
6
6
OH
H
50
51
nd
nd
nd
nd
C, H
C, H
3
10
3
69 ( 13a
86 ( 69
101 ( 28
65 ( 9b
60 ( 6c
nd
104 ( 13
82 ( 9a
82 ( 5a
nd
74 ( 11
76 ( 13
108 ( 4
75 ( 9b
88 ( 6a
nd
102 ( 8
75 ( 8b
89 ( 2
nd
2-Ph,4-ClC6H3
10
10
30
3
10
10
28.2
nd
nd
nd
80
2-(3,4-dimethylpyrrol-
1-yl)-4-ClC6H3
6
H
58
10
20
C, H, N
81
82
83
2-(C6H5OCH2)-4-ClC6H3
3-Cl-carbazol-9-yl
3-Cl-fluoren-9-yl
6
5
5
H
H
H
62
75
76
nd
nd
nd
nd
nd
nd
22
16
5
34a
33a
16
C, H
C, H, N
C, H
nd
nd
a
b
p < 0.05. p < 0.01. c p < 0.001. nd, not determined.
relative stereochemistry has been determined as 3R*,5S*
by X-ray crystallographic analysis, gives a marginal
increase in potency relative to 1c. Although the 3R*,5R*
diastereomer 46 could not be independently synthesized,
the Ki of 2 µM for the 1:1 mixture of diastereomers,
relative to a Ki of 1 µM for 45, indicates that the 3R*,5R*
diastereomer is at least a 5-10-fold less active inhibitor.
Introduction of the hydroxyl group R to the tertiary
center gives compounds which can be considered to have
a hydroxycitrate-like, rather than a citrate-like, head
linked to the lipophilic group. When the OH group was
located on the lipophilic group side (4-position) in
compound 47, activity was reduced 5-6-fold compared
to compound 45. Relocation of the hydroxyl group to
the 2-position was easiest examined in derivatives of
45. However, both of the possible diastereomers, 49 and
50, showed no distinct advantages in potency over 45,
and certainly not the desired 1-2 orders of magnitude
difference in affinity demonstrated by compound 42a
over citrate.
Even so, compound 45 was one of our most potent
inhibitors of the enzyme. Like 1a , the compound
demonstrates an essentially competitive type of inhibi-
tion with respect to citrate.2a Not surprisingly, like our
earlier compounds,2a it too was ineffective in the HepG2
cell at inhibiting cholesterol or fatty acid synthesis at
concentrations up to 100 µM (Table 6). However, this
compound has the potential for lactonization to either
a five- or six-membered ring lactone which could serve
as a prodrug for the active hydroxy acid form of the
inhibitor. Lactonization of 45 in 25% aqueous HCl or
aqueous HCl/THF, at ca. 60 °C, gave exclusively the
thermodynamically favored five-membered ring lactone
77.11 The latter, as expected, was inactive as an
inhibitor of the isolated enzyme. Encouragingly, com-
pound 77 was found to be efficacious as an inhibitor of
de novo lipid synthesis in HepG2 cells. The IC50’s for
inhibition of de novo cholesterol and fatty acid synthesis
were in the 10-30 µM range (Table 6).12 At higher
concentrations (>30 µM), in the absence of albumin, the

3588 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19
Gribble et al.
compound was found to be toxic to the cells, as judged
by the effect on cellular ATP levels. The concentration
of (-)-hydroxycitrate 42a required to produce an equiva-
lent reduction in lipid synthesis was at least 10-fold
greater (Table 6). The results are thus consistent with
the lactone facilitating entry into the cell where hy-
drolysis to the active hydroxy acid form of the inhibitor
occurs.
In a complementary approach, we investigated some
N-linked indole analogues as potential inhibitors of the
enzyme. These were regarded as conformationally
restricted derivatives of a moderately potent aniline 43
Encouraged by the results presented above, we con-
tinued our SAR investigation in the hydroxy acid/lactone
class of inhibitor, represented by 45/77, to identify the
best candidate to evaluate in vivo.
(rat ACL, Ki ) 12 µM) which was prepared in the earlier
S-linked series of compounds.2a The compounds syn-
thesized are listed in Table 4. The conformational
restriction imposed by the indole nucleus in 64 did
indeed confer an improvement in activity relative to the
corresponding aniline 43. The critical nature of the
chain length linking the head to the aryl group was
further emphasized with the dramatic loss of activity
of the lower homologue 63. Introduction of a second
chlorine into the indole 66 made only a marginal
difference to activity, unlike the case for compound 45,
suggesting that the indole 3-position is likely to cor-
respond to the normal ortho position in the latter
compound. On the basis of this analysis, substitution
in the 3-position was investigated as a means of
improving activity. However, the compounds in Table
4 indicate that this strategy is not favored, as can be
seen from the lower activity of the 3-methyl (69), phenyl
(70), and nitro (71) analogues. Nevertheless, the 3-
benzyl substituent in compound 68 overcomes this and
restores activity to the level of the parent compound.
This suggests that there may indeed be an additional
binding area for the aryl ring, as discussed earlier for
the benzyloxy compound 62. Thus, identifying ways to
correctly position the phenyl ring was pursued as a
strategy which had high expectations for significantly
improving activity.
En a n tiom er s of 45. Both the (+)- [3S,5R] and (-)-
[3R,5S] enantiomers of compound 45 demonstrated
equivalent potency against both the rat and human
recombinant enzyme (Table 2). Their corresponding
lactones, (+)-77 and (-)-77, repectively, demonstrated
no clear differences in efficacy on de novo lipid synthesis
in HepG2 cells (Table 6). In a similar fashion, the
separate enantiomers of compound 58 (see later) were
both potent inhibitors of hrACL (Table 2). Considering
the stereospecific requirement for exclusive modification
of the citrate pro-S carboxylate in the ACL-catalyzed
cleavage reaction,2b,13 and the deductions that this class
of inhibitor is binding to the citrate site of the enzyme,2a
it is surprising that both enantiomers are equally
effective inhibitors. However, this outcome allowed us
to evaluate the single diastereomeric, but racemic,
analogues in our subsequent SAR studies.
F u r th er SAR. In a preceding paper, in analogues
of compound 1a , we established a requirement for a 2,4-
disubstituted aryl ring, with a preference for a lipophilic
halogen substituent in the para position.2a Less clear
was the scope for modifying the ortho substituent,
although the limited number investigated suggested
that more diverse substituents could be incorporated in
this position. For example, both o-phenyl and o-nitro
gave improvements in activity over the unsubstituted
parent. Continuation of this study in the hydroxy acid
class of inhibitor established that the 2-Ph, 4-Cl sub-
stitution pattern in 51 gave a compound equipotent with
45. However, further substitution in the second phenyl
ring led to decreases in activity (Table 3), which may
be related to the increased bulk of the substituent. The
scope for greater diversity in this substituent was also
demonstrated with the benzyloxy substituent in com-
pound 62. A role for the benzyl group in increasing the
affinity of the inhibitor is indicated by the order of
magnitude improvement in activity relative to a meth-
oxy group in this position in compound 61.
On comparing compounds 68 and 62, most low energy
conformations of the flexible benzyloxy group in 62 can
be mimicked very well by 68. An exception is an
extended all planar conformation (44a ) of the benzyloxy
group of 62 which would be a relatively high energy one
for 68, due to steric interaction between the phenyl and
indole rings (44b). An alternative, but probably higher
energy conformation of the benzyloxy group is that
represented by 44c. This is best mimicked in indole
compounds by substitution in the 2-position (e.g., 44d ).
This proved to be the case when the 2-phenyl group in
compound 72 increased activity to give the most potent
compound in the series (Ki ) 0.37 µM). This is ca. 30
times more potent than the corresponding 3-phenyl
analogue and 1 order of magnitude better than the
unsubstituted parent. By comparison with the 2-me-
thylindole, 73 (Ki ) 13 µM), it is most likely that the
phenyl group is improving activity by making an ad-
We considered that a pyrrol-1-yl group might ap-
proximate as a hybrid of a phenyl ring and nitro group.
This group is reasonably lipophilic (π ) 0.95) and
electron withdrawing (σp ) 0.37, cf. NO2 ) 0.78) on a
phenyl ring.14 Encouragingly, the prototype 57 was a
reasonably potent inhibitor (Ki ) 3.9 µM). An order of
magnitude improvement in potency was achieved on
increasing lipophilicity, by the introduction of methyl
groups in the 3- and 4-positions of the pyrrole ring in
compound 58. This activity was abolished by relocation
of the methyl groups to the R-position in compound 59,
suggesting that the two aryl rings need to lie coplanar
in ortho biaryl systems of this type.

Hypolipidemic Intervention by ATP-Citrate Lyase
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19 3589
ditional binding interaction, rather than by its ability
to control the linking side chain conformation.
By regarding 2,3-ring fused indoles as conformation-
ally restricted analogues of the o-biaryl inhibitors, we
decided to prepare the tricyclic carbazole and fluorene
compounds 74-76 (Table 5). From this study, the
fluorene 76 (Ki ) 0.22 µM) was identified as the most
potent of all the inhibitors of hrACl that we had
synthesized.
effects. Inhibitors of ATP-citrate lyase, like compound
77, thus have the potential to be a novel class of
hypolipidemic agent.
Exp er im en ta l Section
Gen er a l P r oced u r es. Melting points were determined on
a Buchi capillary melting point apparatus and are uncorrected.
Elemental analyses were measures in the Analytical Science
Department of SmithKline Beecham and are within 0.4% of
theoretical values unless otherwise indicated. 1H NMR spectra
were determined on Brucker AM250 or AM360 spectrometers
using tetramethylsilane (TMS) as the internal standard. IR
spectra were recorded on a Perkin-Elmer model 298 instru-
ment. Mass spectra were recorded on a VG-70-250-SEQ
instrument. Optical rotations were measured on a Perkin-
Elmer 241 polarimeter. All structural assignments were
consistent with NMR, IR, and mass spectra.
Or igin a l P r oced u r e for t h e P r ep a r a t ion of (()-
(3R*,5S*)-3-Ca r b oxy-11-(2,4-d ich lor op h en yl)-3,5-d ih y-
d r oxyu n d eca n oic Acid , Disod iu m Sa lt (45). Sodium boro-
hydride (1.47 g, 38.8 mmol) was added in portions to a stirred
solution of (()-methyl 11-(2,4-dichlorophenyl)-3-hydroxy-3-
(methoxycarbonyl)-5-oxoundecanoate2a (16.0 g, 36.9 mmol) and
cerium(III) chloride heptahydrate (14.4 g, 38.8 mmol) in
methanol (200 mL) at 0 °C. The solution was stirred for 0.5 h
and then quenched with aqueous HCl. The mixture was
diluted with water, and extracted with ether. The extracts
were washed with water and saturated aqueous NaCl and
dried (MgSO4), and the solvent was removed under vacuum.
Aqueous NaOH (1.5 M, 100 mL, 150 mmol) was added to a
stirred solution of the crude hydroxy ester in ethanol (100 mL)
at 0 °C. The mixture was stirred for 4 h, diluted with ethanol
(200 mL), and filtered. The solid was recrystallized (aqueous
ethanol) to give the title compound (11.9 g, a monohydrate,
69%) as a white solid, mp indeterminate: 1H NMR (D2O, 250
MHz) δ 7.51 (s, 1H) and 7.30 (s, 2H) (Ar H’s), 3.90 (m, 1H,
CHOH), 2.69 and 2.49 (doublets, 1H each, J ) 15.6 Hz, CH2-
CO2Na), 2.72 (t, 2H, J ) 7.5 Hz, Ar CH2), 1.81 (d, 2H, J ) 5.7
Hz, CH2C(OH)(CO2Na)), 1.6 (m, 2H, CH2CH) and 1.7-1.5 (m,
8H, (CH2)4). Anal. (C18H22Cl2O6Na2‚H2O) C, H.
Typ ica l P r oced u r e for th e P r ep a r a tion of (3R*,5S*)-
ω-Su b st it u t ed -3-ca r b oxy-3,5-d ih yd r oxya lk a n oic Acid s:
(()-(3R*,5S*)-3-Car boxy-3,5-dih ydr oxy-11-[4-ch lor o-2-(3,4-
d im eth ylp yr r ol-1-yl)p h en yl]u n d eca n oic Acid , Disod iu m
Sa lt (58). (i) [[4-Ch lor o-2-(3,4-d im et h ylp yr r ol-1-yl)-
p h en yl]m eth yl]tr ip h en ylp h osp h on iu m Br om id e (5, R )
3,4-d im eth ylp yr r ol-1-yl). 4-Chloro-2-(3,4-dimethylpyrrol-1-
yl)benzoic acid17a was converted to its methyl ester (CH2N2 in
Et2O) and reduced to the benzyl alcohol using DIBAL, and this
was converted to 4-chloro-2-(3,4-dimethylpyrrol-1-yl)benzyl
bromide (NBS/PPh3 in CH2Cl2) by standard procedures (e.g.,
ref 2a). A solution of this bromide (1.76 g, 5.9 mmol) and
triphenylphosphine (1.54 g, 5.9 mmol) in acetonitrile (30 mL)
was heated at reflux for 7 h. The mixture was concentrated
and the residual oil triturated with ether and then filtered to
give the title compound as a white solid (3.00 g, 95%).
(ii) (()-5-(Ca r bom eth oxym eth yl)-3-(5-h yd r oxyp en tyl)-
5-(m eth oxyca r bon yl)-4,5-d ih yd r oisoxa zole (3). Diisobut-
ylaluminum hydride (1.0 M in CH2Cl2, 125 mL) was added by
cannula to a vigorously stirred solution of ꢀ-caprolactone (13.0
g, 0.114 mol) in CH2Cl2 (100 mL) under argon at -78 °C. After
20 min water (41 mL) was added and the mixture allowed to
warm to room temperature. Ethyl acetate was added to make
the viscous mixture stirrable followed by solid sodium bicar-
bonate and this mixture was stirred for 10 min, filtered
through Celite, and concentrated to give an oil. This crude
lactol was dissolved in ether (200 mL), and solid hydroxyl-
amine hydrochloride (25.30 g, 0.364 mol) was added followed
by slow addition of aqueous sodium carbonate (21.2 g, 0.200
mol in 100 mL) and the mixture stirred vigorously for 3 h.
Sodium chloride was added to saturate the aqueous layer and
the ethereal layer separated. The aqueous layer was further
extracted with 1-butanol (3×), and all nonaqueous fractions
were then combined, washed with brine (1×), dried (MgSO4),
Biology
Compounds with Ki’s e 1 µM (50, 51, 58, 62, 75, 76)
were converted to their respective five-membered ring
lactones 78-83 and compared with 77 in their efficacy
in HepG2 cells at inhibiting de novo lipid synthesis and/
or in their ability to reduce plasma triglyceride concen-
trations in the rat after oral administration.15 The
latter in vivo screen was established to determine the
ability of compounds to reduce plasma triglyceride
concentrations 4 h after the second of two doses of
compound given 24 h apart. No compounds were more
active than compound 77 in the HepG2 cell assay (Table
6). Although a number of compounds did demonstrate
hypolipidemic activity in the rat in vivo assay, none
were more potent than 77 (Table 6).
P h a r m a cology of 77. Compound 77 was chosen for
more detailed pharmacological evaluation in the rat and
the dog. The results of these studies will be reported
in detail elsewhere, so are only briefly outlined in this
paper.16 After oral administration to rats, we estab-
lished that the compound is efficiently absorbed by the
liver and converted to the corresponding hydroxy acid
45. Feeding of 77 to rats for a period of 7 days, in
amounts of 0.05 to 0.25% w/w in the diet, resulted in a
dose-dependent decrease in plasma cholesterol (up to
46%) and triglycerides (up to 80%). The rate of VLDL
triglyceride synthesis also decreased, by up to 48%,
suggesting that a decrease in the rate of VLDL produc-
tion contributed to the observed hypolipidemic response.
Administration of 77 to dogs, once a day at doses of 25
mg/kg/day for 15 days, further confirmed the hypolipi-
demic properties of the compound. At this dose, plasma
cholesterol and triglyceride concentrations were de-
creased by up to 23% and 38% respectively.
Su m m a r y a n d Con clu sion s
A series of (3R*,5S*)-ω-substituted-3-carboxy-3,5-
dihydroxyalkanoic acids have been synthesized as in-
hibitors of ATP-citrate lyase and their SAR investigated.
The best compounds have Ki’s for inhibition of human
recombinant enzyme in the 200-1000 nM range. Due
to limited cellular penetration, compounds of this
structural class are ineffective in reducing cholesterol
or triglyceride synthesis in cell-based systems. How-
ever, as their internal, thermodynamically favored,
γ-lactone prodrugs, a number of compounds demon-
strate activity in HepG2 cells and/or are able to reduce
plasma triglyceride levels in rats after oral administra-
tion. The best of these, compound 77, is able to induce
clear hypocholesterolemic and hypotriglyceridemic re-
sponses when administered orally to rat and dog. The
biological data on compound 77 is consistent with our
hypothesis that inhibition of ACL in vivo will result in
combined hypocholesterolemic and hypotriglyceridemic

3590 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19
Gribble et al.
and concentrated. The residue was azeotroped with toluene
to remove traces of 1-butanol to give the crude oxime inter-
mediate. Sodium hypochlorite (15% aqueous solution, 175 mL)
was added dropwise to a stirred mixture of crude oxime,
dimethyl itaconate (22.1 g, 0.140 mL), triethylamine (1 mL,
0.0136 mol), CH2Cl2 (100 mL), and water (20 mL), controlling
the temperature at ambient during addition with a water bath.
After 1 h, the reaction mixture was filtered through Celite and
the organic layer separated. The aqueous layer was extracted
with CH2Cl2 (2×), and the combined organic extracts were then
washed with water (1×) and brine (1×), dried (MgSO4), and
concentrated. The residue was dissolved in ether and then
poured into 3 volumes of petroleum ether (40-60 °C). After
standing, the supernatant was decanted and discarded and
the precipitation procedure applied for a second time to the
residual oil. The residue was dissolved in toluene, filtered
through silica, washing well with 50% ether/petroleum ether
(40-60 °C), followed by ether and then ethyl acetate, to yield
the title compound on concentration as a colorless oil (22-58
g, 69%).
After addition the mixture was left stirring at room temper-
ature for 20 min. To this mixture (recooled to 0 °C) was added
a solution of (()-methyl 11-[4-chloro-2-(3,4-dimethylpyrrol-1-
yl)phenyl]-3-hydroxy-3-(methoxycarbonyl-5-oxoundecanoate
(1.05 g, 2.13 mmol) in acetic acid (6 mL), and this mixture
was allowed to stir for 1 h at room temperature. The mixture
was poured into ice water and extracted with ether (3 × 10
mL). The combined ether extracts were washed with water
(1×) and brine (1×), dried (MgSO4), and concentrated to give
a colorless oil. Aqueous sodium hydroxide (0.26 g, 6.4 mmol
in 3 mL) was added to the crude hydroxy ester in ethanol (25
mL) at 0 °C. The resulting mixture was left to stir overnight
at room temperature, diluted with ethanol (25 mL), and
filtered. The solid was recrystallized from aqueous ethanol
to give the title compound as a white solid (0.74 g, 68%): NMR
(D2O, 250 MHz) δ 7.30 (m, 2H) and 7.16 (s, 1H) (Ar H’s), 6.58
(s, 2H, pyrrole-H’s), 3.87 (m, 1H, CHOH), 2.66 (doublet, 1H, J
) 15.6 Hz) and 2.49 (m 3H) (CH2CO2Na + ArCH2), 2.03 (s,
6H, 2 × CH3), 1.81 (br s, 2H, CH2C(OH)(CO2Na)), 1.38-1.17
(m, 10H, (CH2)5). Anal. (C24H30ClNO6Na2‚1.6H2O) C, H, N.
(iii) (()-5-(Car bom eth oxym eth yl)-5-(m eth oxycar bon yl)-
3-(5-oxop en tyl)-4,5-d ih yd r oisoxa zole (4). Dry DMSO (1.1
mL, 0.0156 mol) was added dropwise to a stirring solution of
oxalyl chloride (0.99 g, 0.0078 mol) in CH2Cl2 (15 mL) at -78
°C under argon. After 10 min, 3 (1.6 g, 0.056 mol) in CH2Cl2
(15 mL) was added, and stirring was continued for 1 h at -78
°C. Triethylamine (3.4 mL, 0.024 mol) was added, and after
10 min the reaction was allowed to warm to room temperature,
poured into 1 M aqueous HCl, and extracted with CH2Cl2 (2
× 25 mL). The combined organic extracts were washed with
water (2×) and brine (1×), dried (MgSO4), and concentrated
to give the title compound as a yellow oil, which was used
directly for next stage.
Typ ica l P r oced u r e for P r ep a r a tion of γ-la cton es: (()-
(3R*,5S*)-3-(Ca r b oxym et h yl)-5-[6-(2,4-d ich lor op h en yl)-
h exyl]-3-h yd r oxytetr a h yd r ofu r a n -2-on e (77). A mixture
of 45 (disodium salt) (11.8 g, 25.1 mmol), aqueous HCl (3 M,
200 mL), and tetrahydrofuran (200 mL) was heated at 60 °C
for 6 h and then cooled. Most of the tetrahydrofuran was
removed under vacuum. The residual mixture was diluted
with water and extracted with ether. The extracts were
washed with water and saturated aqueous NaCl and dried
(MgSO4). The solvent was removed under vacuum, and the
residue was recrystallized (ether/petroleum ether (40-60 °C))
to give the title compound (8.80 g, 90%) as a white solid: mp
1
87-89 °C; H NMR (DMSO-d6, 360 MHz) δ 7.55 (s, 1H) and
7.36-7.39 (m, 2H) (Ar H’s), 4.53 (m, 1H, CH), 2.65-2.75 (m,
4H, ArCH2 + CH2COOH), 2.26 (dd, 1H, J ) 13.4, 5.6 Hz) and
2.06 (dd, 1H, J ) 13.4, 9.8 Hz) (ring CH2), 1.62-1.54 (m, 4H)
and 1.34 (br m, 6H) (CH2)5); MS (FAB positive ion) m/z 389
(iv) (()-5-(Ca r bom eth oxym eth yl)-3-[6-[4-ch lor o-2-(3,4-
d im eth ylp yr r ol-1-yl)p h en yl]h ex-5-en yl]-5-(m eth oxyca r -
bon yl)-4,5-d ih yd r oisoxa zole (6, R ) 3,4-d im eth ylp yr r ol-
1-yl). Sodium hydride (60% dispersion in oil, 0.23 g, 0.058
mol) was stirred with dry DMSO (15 mL) at 80 °C under argon
until hydrogen evolution had ceased. The solution was placed
in an ice bath, a solution of [[4-chloro-2-(3,4-dimethylpyrrol-
1-yl)phenyl]methyl]triphenylphosphonium bromide (3.04 g,
0.056 mol) in dry DMSO (25 mL) was added, and the mixture
was then allowed to warm to room temperature and stir for
30 min. After the mixture was recooled to 0 °C, a solution of
4 (0.056 mol) in dry DMSO (10 mL) was added and the mixture
stirred overnight at room temperature. The reaction was
poured into 1 M HCl and extracted with ether (3 × 25 mL).
The combined ether extracts were washed with H2O (3×) and
brine (1×), dried (MgSO4), and concentrated to give a yellow
oil. This oil was purified by silica gel chromatography (40%
ether/petroleum ether (40-60 °C)) to give title compound as a
yellow oil (mixture of E and Z isomers) (1.58 g, 58%).
(v) (()-Meth yl 11-[4-Ch lor o-2-(3,4-d im eth ylp yr r ol-1-yl)-
ph en yl]-3-h ydr oxy-3-m eth oxycar bon yl-5-oxou n decan oate
(7, R ) 3,4-Dim eth ylp yr r ol-1-yl). A solution of (()-5-
(carbomethoxymethyl)-3-[6-[4-chloro-2-(3,4-dimethylpyrrol-1-
yl)phenyl]hex-5-enyl]-5-(methoxycarbonyl)-4,5-dihydroisox-
azole (1.64 g, 0.0034 mol) and boric acid (0.83 g, 0.014 mol) in
methanol/water (25 mL:3 mL) over Raney nickel (0.02 g) was
shaken under hydrogen for 3 h. The reaction mixture was
filtered through Celite, poured into water, and extracted with
ethyl acetate (3 × 25 mL). The organic solution was washed
with brine (1×), dried (MgSO4), and concentrated to give an
oil. This crude product was dissolved in methanol (30 mL),
Pt2O (ca. 0.05 g) was added, and this mixture was shaken
under hydrogen at 50 psi for 1.5 h. The mixture was filtered
through Celite and concentrated to give a colorless oil. The
title compound was obtained by silica gel chromatography
(40% ether/petroleum ether (40-60 °C)) as a colorless oil (1.08
g, 65%).
((M + H)+); IR (KBr) 3297, 1759, 1707 cm-1. Anal. (C18H22
-
Cl2O5) C, H.
(P h en ylm eth yl)tr iph en ylph osph on iu m Br om ides. The
intermediate phosphonium salts (5), whose syntheses are not
described herein, which were required for the syntheses of the
compounds of Table 3, were prepared in three steps from the
appropriately substituted alkyl benzoates, by analogous pro-
cedures to those described above. Those containing an ortho
(substituted) phenyl group were readily prepared by Suzuki
coupling of the corresponding triflate with the appropriate
boronic acid using standard procedures. The 2-(pyrrol-1-yl)-
4-chlorobenzoates were prepared from the corresponding
2-amino-4-chlorobenzoates by standard literature procedures.17
Syn th esis of (()-(3R*,5R*)-3-Ca r boxy-11-(2,4-d ich lo-
r op h en yl)-3,5-d ih yd r oxyu n d eca n oic Acid , Disod iu m Sa lt
(46) (a s a 1:1 Mixtu r e w ith Its 3R*,5S* Dia ster eom er , 45).
(i) 7-(2,4-Dich lor op h en yl)-1-h ep ta n a l (11). Swern oxida-
tion of 7-(2,4-dichlorophenyl)-1-heptanol2a as described for 4
gave the title compound which was used without further
purification.
(ii) (()-Meth yl 11-(2,4-Dich lor op h en yl)-5-h yd r oxy-3-
oxou n d eca n oa te (12). Methyl acetoacetate (0.247 mL, 2.29
mmol) was added dropwise to a stirred suspension of sodium
hydride (61 mg, mass after removal of mineral oil, 2.52 mmol)
in THF (2 mL) at 0 °C under argon. The solution was stirred
for 0.5 h at room temperature and cooled to 0 °C, and
n-butyllithium (2.5 M in hexanes, 1.01 mL, 2.52 mmol) was
added. This solution was stirred for 0.25 h at room temper-
ature and cooled to -78 °C, and a solution of the crude
aldehyde in tetrahydrofuran (3 mL) was added. The mixture
was stirred for 0.3 h at -78 °C, allowed to warm to room
temperature, poured into aqueous NaHSO4, and extracted with
ether. The extracts were washed with water and saturated
aqueous NaCl and dried (MgSO4). The solvent was removed
under vacuum, and the residue was purified by chromatog-
raphy on silica gel (60-100% ether/petroleum ether (40-60
°C)) to give the title compound (563 mg, 79%) as an oil.
(vi) (()-(3R*,5S*)-3-car boxy-3,5-dih ydr oxy-11-[4-ch lor o-
2-(3,4-d im eth ylp yr r ol-1-yl)p h en yl]u n d eca n oic Acid , Di-
sod iu m Sa lt (58). Solid sodium borohydride (0.32 g, 8.5
mmol) was added in portions to acetic acid (6 mL) at 0 °C.

Hypolipidemic Intervention by ATP-Citrate Lyase
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19 3591
(iii) 1:1 Mixtu r e of (()-(3R*,5R*)- a n d (()-(3R*,5S*)-3-
Car boxy-11-(2,4-dich lor oph en yl)-3,5-dih ydr oxyu n decan o-
ic Acid , Disod iu m Sa lt (46). 12 was converted to a 1:1
mixture of the title compound diastereomers via cyanohydrin
formation (KCN/NaH2PO4) and acid hydrolysis, as described
previously.2a Anal. (C18H22Cl2O6‚NNa2‚1.3H2O) C, H.
P r oced u r e for th e Syn th eses of 47 a n d 48: Syn th esis
of 48. (i) (E)-Meth yl 12-(2,4-Dich lor op h en yl)-3-(m eth -
oxyca r bon yl)-3-d od ecen oa te (15, n ) 8). Swern oxidation,
analogous to 11, of 9-(2,4-dichlorophenyl)-1-nonanol (2.00 g,
6.91 mmol) (see ref 2a for a typical synthesis) gave the
corresponding crude aldehyde, which was heated in toluene
(25 mL) with 1,2-bis(methoxycarbonyl)ethylenetriphenylphos-
phorane (14) (4.21 g, 10.4 mmol) at 100 °C for 24 h and then
cooled. The solvent was removed under vacuum, and the
residue was purified by chromatography on silica gel (10-40%
ether/petroleum ether (40-60 °C)) to give the title compound
(2.46 g, 86%) as an oil.
on e (18). n-Butyllithium (3.27 mL, 8.18 mmol, 2.5 M in
hexanes) was added into a stirred solution of hexamethyld-
isilazane (1.73 mL, 8.18 mmol) in tetrahydrofuran (15 mL) at
0 °C under argon. After 5 min, the solution was cooled to -78
°C and a solution of 17 (1.15 g, 2.85 mmol) in tetrahydrofuran
(10 mL) was added via cannula slowly. After 1 h at -78 °C,
a solution of 2-(benzenesulfonyl)-3-phenyloxaziridine5a (1.07
g, 4.09 mmol) in tetrahydrofuran (7 mL) was added by cannula.
The solution was stirred at -78 °C for 2 h and at -50 °C for
2.5 h, and then allowed to warm to 0 °C. Aqueous HCl was
added, and the mixture was extracted with ether. The extracts
were washed with water and saturated aqueous NaCl and
dried (MgSO4). The solvent was removed under vacuum, and
the residue was purified by chromatography on silica gel (60-
100% ether/petroleum ether (40-60 °C)) to afford the sepa-
rated (1′R*,3R*,5S*) (236 mg, 20%) and (1′R*,3S*,5R*) (145
mg, 12%) diastereoisomers, both as oils.
(iii)
(()-(2R*,3R*,5S*)-3-Ca r b oxy-11-(2,4-d ich lor o-
p h en yl)-2,3,5-tr ih yd r oxyu n d eca n oic Acid , Disod iu m Sa lt
(49). Aqueous NaOH (1 M, 2.25 mL, 2.25 mmol) was added
dropwise to a stirred solution of (()-(1′R*,3R*,5S*)-3-[(car-
bomethoxy)hydroxymethyl]-5-[6-(2,4-dichlorophenyl)hexyl]-3-
hydroxytetrahydrofuran-2-one (235 mg, 0.56 mmol) in metha-
nol (6 mL) at 0 °C. The solution was stirred for 10 min at 0
°C and then for 3 h at room temperature. Methanol was
removed under vacuum, and the residue was diluted with
water and acidified with aqueous HCl. The mixture was
extracted with ether, and the extracts were washed with water.
The solvent was removed under vacuum, and the wet residue
was dissolved in ethanol (20 mL). Aqueous NaOH (1 M, 1.4
mL, 1.4 mmol) was added. The mixture was allowed to stand
for 45 min, then boiled, and cooled to 0 °C. The solid was
filtered off, washed with 5% water/ethanol, and dried to give
the title compound (181 mg, 69%), mp indeterminate: 1H NMR
(D2O, 250 MHz) δ 7.42 (s, 1H) and 7.23(s, 2H) (Ar H’s), 4.09
(s, 1H, CH(OH)CO2Na), 3.81 (m, 1H, CH2CH(OH)CH2), 2.65
(t, 2H, J ) 7.5 Hz, ArCH2), 2.00 (m, 2H, CH2C(OH)(CO2Na)),
1.6-1.3 (m, 10H, (CH2)5); MS (FAB positive ion) m/z 467 ((M
(ii) (()-(3R*,4S*)-Meth yl 12-(2,4-Dich lor op h en yl)-3,4-
d ih yd r oxy-3-(m eth oxyca r bon yl)d od eca n oa te (16, n ) 8).
A mixture of (E)-methyl 12-(2,4-dichlorophenyl)-3-(methoxy-
carbonyl)-3-dodecenoate (1.50 g, 3.61 mmol), osmium tetroxide
(0.229 mL of a 2% solution in tert-butyl alcohol, 0.018 mmol),
N-methylmorpholine N-oxide (634 mg, 5.42 mmol), water (2
mL), and acetone (2 mL) was stirred at room temperature for
64 h. Aqueous NaHSO3 (1.1 M, 6 mL) was added, and the
mixture filtered, after 20 min, through a plug of silica gel. The
silica gel plug was washed with ether, and then the filtrate
was washed with aqueous HCl, water, and saturated aqueous
NaCl. After drying (MgSO4), the solvent was removed under
vacuum, and the residue was purified by chromatography on
silica gel (50-100% ether/petroleum ether (40-60 °C)) to give
the title compound (1.33 g).
(iii) (()-(4R*,5S*)-4-Ca r boxy-5-[8-(2,4-d ich lor op h en yl)-
octyl]-4-h ydr oxytetr ah ydr ofu r an -2-on e (γ-Lacton e of 48).
(()-(3R*,4S*)-Methyl 12-(2,4-dichlorophenyl)-3,4-dihydroxy-3-
(methoxycarbonyl)-3-dodecanoate (0.95 g, 2.11 mmol) was
heated under reflux in aqueous HCl (7.7 M) for 3.5 h. The
mixture was cooled, diluted with water, and extracted with
ether. The extracts were washed with aqueous NaOH, and
then the aqueous extracts were washed with ether, acidified
(aqueous HCl), and extracted again with ether. The combined
extracts were washed with water and saturated aqueous NaCl
and dried (MgSO4). The solvent was removed under vacuum.
The crude diacid was stirred in ether with silica gel (5 g)
impregnated with 0.5 mL of 2 M aqueous H2SO4 at room
temperature for 18 h, and then the mixture was filtered and
the solvent removed under vacuum from the filtrate. The
residue was recrystallized (dichloromethane/petroleum ether
(40-60 °C)) to give the title compound (485 mg, 57%): 1H NMR
(DMSO-d6, 250 MHz) δ 7.55 (s, 1H) and 7.36 (s, 2H) (Ar H’s),
6.20 br s, 1H, OH), 4.26 (m, 1H, CH), 2.98 and 2.65 (doublets,
1H each, J ) 17 Hz, ring CH2), 2.63 (t, 2H, J ) 8 Hz, ArCH2),
1.55-1.20 (m, 14H, (CH2)7); MS (FAB positive ion) m/z 403
((M + H)+); IR (KBr) 3489, 1731 cm-1. Anal. (C19H24Cl2O5)
C, H.
+ H)+); IR (KBr) 3399, 1599 cm-1
. Anal. (C18H22Cl2Na2O7‚
0.84H2O) C, H.
(iv)
(()-(2R*,3S*,5R*)-3-Ca r b oxy-11-(2,4-d ich lor o-
p h en yl)-2,3,5-tr ih yd r oxyu n d eca n oic Acid , Disod iu m Sa lt
(50). (()-(1′R*,3S*,5R*)-3-[(Carbomethoxy)hydroxymethyl]-
5-[6-(2,4-dichlorophenyl)hexyl]-3-hydroxytetrahydrofuran-2-
one (145 mg, 0.346 mmol) was hydrolyzed by a procedure
analogous to that described above for 49 to give 50 (111 mg,
69%), contaminated with 20% of the (2R*,3R*,5S*) diastere-
oisomer, mp indeterminate: 1H NMR (D2O, 250 MHz) δ 7.43
(s, 1H) and 7.25 (s, 2H) (Ar H’s), 4.14 (s, 1H, CH(OH)CO2Na),
3.81 (m, 1H, CH2CH(OH)CH2), 2.69 (t, 2H, J ) 7.5 Hz, ArCH2),
1.96 (m, 2H, CH2C(OH)(CO2Na)), 1.6-1.3 (m, 10H, (CH2)5);
MS (FAB positive ion) m/z 467 ((M + H)+); IR (KBr) 3403, 1597
cm-1. Anal. (C18H22Cl2O7Na2‚0.9H2O) C, H.
(()-(3R*,5S*)-11-[2-(Ben zyloxy)-4-ch lor op h en yl]-3-ca r -
boxy-3,5-d ih yd r oxyu n d eca n oic Acid , Disod iu m Sa lt (62).
(i) [2-(Ben zyloxy)-4-ch lor oben zyl]tr iph en ylph osph on iu m
Br om id e (21). This was prepared from ethyl 2-(benzyloxy)-
4-chlorobenzoate by the general procedure exemplified for
4-chloro-2-(3,4-dimethylpyrrol-1-yl)benzyl bromide above.
This lactone was hydrolyzed with excess aqueous NaOH to
generate the corresponding hydroxy acid (48) prior to adding
to the buffer mix for enzyme screening.
(ii) (()-(3R*,5S*)-3-(Ca r bom eth oxym eth yl)-3-h yd r oxy-
5-(5-h yd r oxyp en tyl)tetr a h yd r ofu r a n -2-on e (20). The di-
hydroisoxazole 3 was converted to the lactone 19 by the general
procedures described above. This was converted to the title
methyl ester using MeOH/H2SO4, followed by chromatography
on silica gel (0-80% EtOAc/Et2O).
(iii) (()-(3R*,5S*)-5-[6-[2-(Ben zyloxy)-4-ch lor op h en yl]-
h exyl]-3-(ca r bom eth oxym eth yl)-3-h yd r oxytetr a h yd r ofu -
r a n -2-on e (22). Swern oxidation of 20 (0.5 g, 1.92 mmol), as
described above for 4, gave the corresponding aldehyde.
Sodium hydride (60% oil suspension, 63 mg, 1.58 mmol) was
washed with petroleum ether (40-60 °C) under argon, and
then heated with DMSO (2 mL) at 70-90 °C until all solid
had dissolved. After the mixture was cooled in a water bath,
a solution of 21 (0.86 g, 1.5 mmol) in DMSO (10 mL) was added
Syn t h eses of 49 a n d 50: (i) (()-(3R*,5S*)-3-(Ca r b o-
m et h oxym et h yl)-5-[6-(2,4-d ich lor op h en yl)h exyl]-3-h y-
d r oxytetr a h yd r ofu r a n -2-on e (17). Diazomethane, gener-
ated from diazald (1.37 g, 6.38 mmol) and 60% aqueous KOH
(6 mL) in carbitol (6 mL) and ether (6 mL), was bubbled in a
stream of ether saturated nitrogen through a solution of 77
(1.24 g, 3.19 mmol) in 10% methanol/ether (12 mL). When a
yellow color appeared in the solution, the excess diazomethane
was quenched with acetic acid, and then the solvent was
removed under vacuum. The residue was purified by chro-
matography on silica gel (50-100% ether/petroleum ether (40-
60 °C)) to give the title compound (1.15 g, 90%) as an oil.
(ii) (()-(1′R*,3R*,5S*) a n d (()-(1′R*,3S*,5R*) Dia ster -
eom er s of 3-[(Ca r bom eth oxy)h yd r oxym eth yl]-5-[6-(2,4-
d ich lor op h e n yl)h e xyl]-3-h yd r oxyt e t r a h yd r ofu r a n -2-

3592 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19
Gribble et al.
by cannula, and the mixture was stirred at room temperature
for 15 min. A solution of the aldehyde in DMSO (4 mL) was
added, and the mixture was stirred for 1 h, then poured into
aqueous HCl, and extracted with ether. The extracts were
washed with water and saturated aqueous NaCl, and dried
(MgSO4). The solvent was removed under reduced pressure,
and the residue columned on silica gel (50-90% ether/
petroleum ether (40-60 °C)) to give (()-(3R*,5S*)-5-[6-[2-
(benzyloxy)-4-chlorophenyl]-5-hexenyl]-3-(carbomethoxymethyl)-
3-hydroxytetrahydrofuran-2-one (0.42 g, 65%) as a mixture of
E and Z isomers. This compound (0.4 g, 0.83 mmol) in
methanol (5 mL) was shaken with PtO2 under hydrogen at 50
psi for 3 h, and then the hydrogen replaced with nitrogen. The
catalyst was filtered off through Hyflo, and the solvent was
removed under reduced pressure. The residue was columned
on silica gel (ether) to give the title compound (0.39 g, 99%)
as an oil.
3,5-d ih yd r oxyu n d eca n oic Acid , Disod iu m Sa lt (61). (i)
(()-(3R*,5S*)-3-(Ca r bom eth oxym eth yl)-5-[6-(4-ch lor o-2-
m e t h oxyp h e n yl)h e xyl]-3-h yd r oxyt e t r a h yd r ofu r a n -2-
on e (25). Iodomethane (0.045 mL, 0.728 mmol) was injected
into a stirred mixture of 23 (200 mg, 0.52 mmol), potassium
carbonate (108 mg, 0.780 mmol), and dimethylformamide (3
mL) under argon, and the mixture was stirred for 5 h.
Aqueous HCl was added and the mixture extracted with ether.
The extracts were washed with water and saturated aqueous
NaCl and dried (MgSO4). The solvent was removed under
reduced pressure, and the residue was columned on silica gel
(50-70% ether/petroleum ether (40-60 °C)) to give the title
compound (181 mg, 87%) as an oil.
(ii) (()-(3R*,5S*)-3-Ca r b oxy-11-(4-ch lor o-2-m et h oxy-
p h en yl)-3,5-d ih yd r oxyu n d eca n oic Acid , Disod iu m Sa lt
(61). Hydrolysis of 25 as described for other lactones above
gave the title compound as a solid, mp >250 °C. Anal. (C19H25
ClO7Na2) C, H.
-
(iv) (()-(3R*,5S*)-11-[2-(Ben zyloxy)-4-ch lor op h en yl]-3-
ca r boxy-3,5-d ih yd r oxyu n d eca n oic Acid , Disod iu m Sa lt
(62). A solution of NaOH (95 mg, 2.37 mmol) in aqueous
ethanol (1:1, 2 mL) was added dropwise to a stirred solution
of 22 (282 mg, 0.594 mmol) in ethanol (6 mL) at 0 °C. The
mixture was stirred for 20 min at 0 °C and then for 5 h at
room temperature. Ethanol (6 mL) was added and the solid
filtered off and then recrystallized (water) to give the title
compound (217 mg, 70%) as a solid: mp >250 °C; 1H NMR
(D2O, 360 MHz) δ 7.47 (m, 5H, OBz), 7.20 (d, 1H, J ) 8 Hz),
7.13 (s, 1H) and 7.01 (d, 1H, J ) 8 Hz) (Ar H’s), 5.25 (s, 2H,
ArCH2O), 3.88 (m, 1H, CHOH), 2.66 and 2.48 (doublets, 1H
each, J ) 15.3 Hz, CH2CO2Na), 2.58 (t, 2H, J ) 7.5 Hz, ArCH2),
1.82 (d, 2H, J ) 5.7 Hz, CH2C(OH)(CO2Na)), 1.53 (m, 2H,
ArCH2CH2), 1.37-1.27 (m, 8H, (CH2)4); MS (FAB negative ion)
Typ ica l P r oced u r es for th e Syn th eses of th e In d oles
of Ta ble 4. In d ole In ter m ed ia tes (29). 5,7-Dichloroin-
dole,18 3-methyl-5-chloroindole,19 and 3-phenyl-5-chloroindole20
were prepared by published procedures.
3-Nitr o-5-ch lor oin d ole. A solution of benzoyl chloride
(0.64 g, 4.55 mmol) in CH3CN (1 mL) was added to a stirred
mixture of silver nitrate (0.85 g, 5 mmol) and CH3CN (5 mL)
at 0 °C, and the mixture was stirred for 20 min. Stirring was
stopped and the precipitate allowed to settle. The supernatant
liquor was transferred to a stirred solution of 5-chloroindole
(647 mg, 4.27 mmol) in acetonitrile (8 mL) cooled in an ice/
salt bath at -5 °C. The resulting solution was stirred under
argon for 30 min at -5 °C and at room temperature for 18 h
and then poured into water. The mixture was extracted with
ether, and the extracts were washed with aqueous Na2CO3,
water and saturated aqueous NaCl and dried (MgSO4). The
solvent was removed under reduced pressure, and the residue
was columned on silica gel (60-100% ether/petroleum ether
(40-60 °C)) to give the title compound (328 mg, 39%).
2-P h en yl-5-ch lor oin d ole. A mixture of 4-chloroaniline
(10.0 g, 78.4 mmol), 1-phenylethanediol (4.33 g, 31.4 mmol),
bis(triphenylphosphine)ruthenium dichloride (300 mg, 0.314
mmol), and 2-methoxyethyl ether (20 mL) was heated at reflux
with stirring under argon for 18 h, cooled, and poured into
aqueous HCl. The mixture was extracted with ether, and the
extracts were washed with water and saturated aqueous NaCl
and dried (MgSO4). The solvent was removed under reduced
pressure and the residue columned on silica gel twice (10-
100% ether/petroleum ether (40-60 °C), then 10-50% dichlo-
romethane/petroleum ether) to give the title compound (0.47
g, 7%) as a solid, mp 186-191 °C.
(a ) Meth od A (Sch em e 4a ): (()-(3R*,5S*)-3-Ca r boxy-
10-(5-ch lor o-3-n it r oin d ol-1-yl)-3,5-d ih yd r oxyd eca n oic
Acid , Disod iu m Sa lt (71). (i) (()-(3R*,5S*)-3-(Ca r -
b om et h oxym et h yl)-3-h yd r oxy-5-(5-iod op en t yl)t et r a h y-
d r ofu r a n -2-on e (28, n ) 5). N-Bromosuccinimide (752 mg,
4.22 mmol) was added to a stirred solution of 20 (1.00 g, 3.84
mmol) and triphenylphosphine (1.11 g, 4.22 mmol) in CH2Cl2
(25 mL) at 0 °C under argon. After 1 h, toluene (5 mL) was
added to the solution, and the CH2Cl2 was removed under
reduced pressure. The residual solution was loaded onto a
column of silica gel and the bromide eluted with 60-100%
ether/petroleum ether (40-60 °C). Sodium iodide (4.55 g, 30.3
mmol) was added to a stirred solution of the bromide in acetone
(20 mL) under argon. The mixture was stirred for 18 h, and
then the solvent was removed under reduced pressure. The
residue was partitioned between water and ether, and the
ethereal extracts were washed with water and saturated
aqueous NaCl and then dried (MgSO4). The solvent was
removed under reduced pressure to give the title compound
(1.11 g, 78%) as an oil.
m/z 477 (free acid, (M - H)-); IR (Nujol mull) 1572, 1457 cm-1
Anal. (C25H29ClO7Na2‚0.85H2O) C, H.
.
(()-(3R*,5S*)-11-(2-Hydr oxy-4-ch lor oph en yl)-3-car boxy-
3,5-d ih yd r oxyu n d eca n oic Acid (60). (i) (()-(3R*,5S*)-3-
(Ca r bom eth oxym eth yl)-5-[6-(4-ch lor o-2-h yd r oxyp h en yl)-
h exyl]-3-h yd r oxytetr a h yd r ofu r a n -2-on e (23). A solution
of 22 (300 mg, 0.634 mmol) in methanol (20 mL) containing
concentrated HCl (0.02 mL) was shaken with palladium on
charcoal (5%, 125 mg, 0.059 mmol) under hydrogen at 50 psi
for 7 h. The atmosphere was then replaced with nitrogen and
the catalyst filtered off through Hyflo. The solvent was
removed from the filtrate under reduced pressure and the
residue columned on silica gel (70-90% ether/petroleum ether
(40-60 °C)) to give the title compound (190 mg, 78%) as an
oil.
(ii) (()-(3R*,5S*)-3-(Ca r b oxym et h yl)-5-[6-(4-ch lor o-2-
h yd r oxyp h e n yl)h e xyl]-3-h yd r oxyt e t r a h yd r ofu r a n -2-
on e (24). A solution of NaOH (73 mg, 1.83 mmol) in aqueous
ethanol (1:1, 2 mL) was added dropwise to a stirred solution
of 23 (176 mg, 0.457 mmol) in ethanol (6 mL) at 0 °C, and the
mixture was stirred at room temperature for 20 h. Ethanol
(6 mL) was added, and the solid was filtered off. The crude
disodium salt was heated in aqueous HCl (10 mL) at 60 °C
for 18 h, then cooled, and extracted with ether. The extracts
were washed with water and saturated aqueous NaCl and
dried (MgSO4). The solvent was removed under reduced
pressure, and the residue was columned on silica gel (0-40%
ethyl acetate/ether + 0.5% acetic acid). The material was
further purified by recrystallization (chloroform/hexane) to give
the title compound (120 mg, 71%) as a solid: mp 156-158 °C;
1H NMR (DMSO-d6, 360 MHz) δ 7.05 (d, 1H, J ) 8 Hz), 6.79
(s, 1H) and 6.74 (d, 1H, J ) 8 Hz) (Ar H’s), 4.54 (m, 1H, CH),
2.69 (s, 2H, CH2CO2H), 2.48 (t, 2H, ArCH2), 2.25 (dd, 1H, J )
13.4, 5.6 Hz) and 2.05 (dd, 1H, J ) 13.4, 9.8 Hz) (ring CH2),
1.23-1.62 (m, 10H, (CH2)5); MS (EI) m/z 370 (M+), 352, 334,
306, 141; IR (Nujol mull) 1746, 1693 cm-1. Anal. (C18H23ClO6)
C, H.
(iii) (()-(3R*,5S*)-11-(2-H yd r oxy-4-ch lor op h en yl)-3-
ca r boxy-3,5-d ih yd r oxyu n d eca n oic Acid (60). The lactone
24 was hydrolyzed to the hydroxy acid form for enzyme
screening by analogous procedures to those described above.
(ii) (()-(3R*,5S*)-3-(Ca r bom eth oxym eth yl)-5-[5-(5-ch lo-
r o-3-n itr oin d ol-1-yl)p en tyl]-3-h yd r oxytetr a h yd r ofu r a n -
2-on e (30, n ) 5, R ) 3-NO2, R1 ) 5-Cl). A solution of 3-nitro-
5-chloroindole (100 mg, 0.509 mmol) in dimethylformamide (2
mL) was added to a stirred suspension of sodium hydride (13
(()-(3R*,5S*)-3-Car boxy-11-(4-ch lor o-2-m eth oxyph en yl)-

Hypolipidemic Intervention by ATP-Citrate Lyase
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19 3593
mg of a 60% oil dispersion, 0.56 mmol) in dimethylformamide
(1 mL) at room temperature. The mixture was stirred for 5
min, and then a solution of (()-(3R*,5S*)-3-(carbomethoxym-
ethyl)-3-hydroxy-5-(5-iodopentyl)tetrahydrofuran-2-one (207
mg, 0.56 mmol) in dimethylformamide (2 mL) was added. The
resulting solution was stirred under argon for 55 h and then
poured into dilute aqueous HCl. The aqueous mixture was
extracted with ether, and the extracts were washed with water
and saturated aqueous NaCl and dried (MgSO4). The solvent
was removed under reduced pressure, and the residue was
columned on silica gel (40-80% ethyl acetate/hexane) to give
the title compound (146 mg, 65%) as an oil.
(iii) (()-(3R*,5S*)-3-Ca r boxy-10-(5-ch lor o-3-n itr oin d ol-
1-yl)-3,5-d ih yd r oxyd eca n oic Acid , Disod iu m Sa lt (71).
Aqueous NaOH (7.10 mL, 1 M, 7.10 mmol) was added dropwise
to a stirred solution of (()-(3R*,5S*)-3-(carbomethoxymethyl)-
5-[5-(5-chloro-3-nitroindol-1-yl)pentyl]-3-hydroxytetrahydro-
furan-2-one (779 mg, 1.78 mmol) in ethanol (50 mL) at 0 °C.
The mixture was stirred at 0 °C for 30 min and at room
temperature for 24 h. Ethanol (50 mL) was added, and then
the solid was filtered off and recrystallized (H2O/EtOH) to give
the title compound (742 mg, 86%) as a solid: mp > 250 °C; 1H
NMR (D2O, 400 MHz) δ 8.45 (s, 1H, indole-2H), 8.03 (s, 1H,
indole-4H), 7.55 (d, 1H, J ) 9 Hz, indole-7H), 7.37 (d, 1H, J )
9 Hz, indole-6H), 4.25 (t, 2H, J ) 7 Hz, NCH2), 3.86 (m, 1H,
CHOH), 2.68 and 2.47 (doublets, 1H each, J ) 15.6 Hz, CH2-
CO2Na), 1.90-1.83 (m, 4H, NCH2CH2 and CH2C(OH)(CO2Na)),
1.46-1.30 (m, 6H, (CH2)3); MS (FAB positive ion) m/z 465 ((M
+ H)+ of monosodium salt); IR (Nujol mull) 1573, 1526, 1456
cm-1. Anal. (C19H21ClN2O8Na2‚0.5H2O) C, H, N.
(b) Meth od B (Sch em e 4b): (()-(3R*,5S*)-3-Ca r boxy-
10-(5,7-d ich lor oin d ol-1-yl)-3,5-d ih yd r oxyd eca n oic Acid ,
Disod iu m Sa lt (66). (i) (()-3-(5-Br om op en tyl)-5-(ca r -
bom eth oxym eth yl)-5-(m eth oxycar bon yl)-4,5-dih ydr oisox-
a zole (32). N-Bromosuccinimide (4.50 g, 25.3 mmol) was
added in portions to a stirred solution of 3 (6.61 g, 23.0 mmol)
and triphenylphosphine (6.64 g, 25.3 mmol) in CH2Cl2 (70 mL)
at 0 °C. The solution was stirred at room temperature under
argon for 1.5 h, then toluene (20 mL) was added, and the CH2-
Cl2 was removed under reduced pressure. The residual
solution was loaded onto a column of silica gel, and the
products were eluted with 50-80% ether/petroleum ether (40-
60 °C). This gave the title compound (6.93 g, 86%) as an oil.
(ii) (()-5-(Ca r bom eth oxym eth yl)-3-[5-(5,7-d ich lor oin -
d ol-1-yl)p en t yl]-5-(m et h oxyca r b on yl)-4,5-d ih yd r oisox-
a zole (33, R ) H, R1 ) 5,7-d iCl). Freshly ground potassium
hydroxide (206 mg, 3.67 mmol) was stirred in DMSO (20 mL)
under argon for 5 min, 5,7-dichloroindole18 (580 mg, 3.12 mmol)
was added, and the mixture was stirred for 45 min. A solution
of 32 (1.31 g, 3.74 mmol) in DMSO (5 mL) was added by
cannula, and the mixture was stirred for 1 h. Water was
added, and the mixture was extracted with ether. The extracts
were washed with water and saturated aqueous NaCl and
dried (MgSO4). The solvent was removed under reduced
pressure, and the residue columned on silica gel (50% ether/
petroleum ether (40-60 °C)) to give the title compound (1.07
g, 75%) as an oil.
above for (()-5-(carbomethoxymethyl)-3-[5-(5,7-dichloroindol-
1-yl)pentyl]-5-(methoxycarbonyl)-4,5-dihydroisoxazole. This
was converted to the title compound, via the dihydroisoxazole
intermediate, by the general procedures described above.
Anal. (C20H24ClNO6Na2‚H2O) C, H, N.
Syn th eses of 74 a n d 75. The procedure described for 66
was employed, using either 3-chlorocarbazole21 or 6-chlorotet-
rahydrocarbazole22 in place of the indole.
(()-(3R*,5S*,9′R*)- a n d (()-(3R*,5S*,9′S*)-3-Ca r boxy-
10-(3′-ch lor oflu or en -9′-yl)-3,5-d ih yd r oxyd eca n oic Acid ,
Disod iu m Sa lt (a s a 1:1 Mixtu r e of Dia ster eom er s) (76).
(i) 3-Ch lor o-9-[6′-(b en zyloxy)h ex-1′-yl]-9-h yd r oxyflu o-
r en e (38). 6-(Benzyloxy)hexyl bromide (3.00 g, 0.011mol) was
added to magnesium turnings (2.98 g, 0.122 mol) in anhydrous
ether (10 mL), under an argon atmosphere. Reaction was
initiated by adding a crystal of iodine and heating. Further
6-(benzyloxy)hexyl bromide (28.68 g, 0.106 mol) in anhydrous
ether (50 mL) was added at such a rate to maintain reflux.
After addition, the reaction was chilled (-78 °C), 3-chlorof-
luorenone (12.07 g, 0.056 mol) in THF (50 mL) was added by
cannula, and the mixture was left to warm to room temper-
ature and stir overnight. The reaction mixture was poured
into 1 M aqueous HCl and extracted with ether (3×). The
combined ether extracts were washed with water (3×) and
brine (1×), dried (MgSO4), and concentrated to give an oil. This
was purified by silica gel chromatography (20% ether/
petroleum ether (40-60 °C)) to give the title compound as a
colorless gum (15.86 g, 33%).
(ii) (()-6-(3′-Ch lor oflu or en -9′-yl)h exa n -1-ol (39). A so-
lution of (()-3-chloro-9-[6′-(benzyloxy)hex-1′-yl]-9-hydroxyfluo-
rene (15.76 g, 0.0387 mol) in methanol (100 mL) and concen-
trated aqueous HCl (3 mL), mixed with 10% palladium on
carbon (2.0 g), was shaken at room temperature under
hydrogen at 50 psi for a total of 24 h. The reaction mixture
was filtered through Celite, poured into water, and extracted
with ether (2×). The combined ether extracts were washed
with water (2×) and brine (1×), dried (MgSO4), and concen-
trated to give an oil. This was purified by silica gel chroma-
tography (20% ether and then 50% ether/petroleum ether (40-
60 °C)) to give the title compound as an oil (5.16 g, 44%).
(iii) (()-(3R*,5S*,9′R*)- a n d (()-(3R*,5S*,9′S*)-3-Ca r -
b oxy-10-(3′-ch lor oflu or en -9′-yl)-3,5-d ih yd r oxyd eca n oic
Acid , Disod iu m Sa lt (a s 1:1 Mixtu r e of Dia ster eom er s)
(76). 39 was converted to the title compound, via the corre-
sponding dihydroisoxazole intermediate, using the general
procedures described above.
1H NMR (D2O, 400 MHz at 340 K) δ 8.02 (m, 2H), 7.82 (m,
1H), 7.74 (d, 1H, J ) 8 Hz), 7.63-7.56 (m, 2H) and 7.55 (d,
1H, J ) 8 Hz) (all fluorene ring H’s), 4.22 (m, 1H, CH), 3.97
(m, CHOH), 2.80 and 2.62 (doublets, 1H each, J ) 15.3 Hz,
CH2CO2Na), 2.20 (m, 2H, CHCH2), 1.93 (m, CH2C(OH)(CO2-
Na), 1.49-1.17 (m, 8H, (CH2)4); MS (FAB negative ion) m/z
427 ((M - H)- - H2O), 393 (Cl/H ex); IR (Nujol mull) 1572,
1457, 1445 cm-1. Anal. (C24H25ClO6Na2‚0.84H2O) C, H, Cl.
R esolu t ion of 77 a n d 45. (S)-(3R*,5S*)-3-(Ca r b oxy-
m eth yl)-5-[6-(2,4-d ich lor op h en yl)h exyl]-3-h yd r oxytetr a -
h yd r ofu r a n -2-on e ((+)-77). The racemic lactone 77 (15 g,
38.5 mmol) was dissolved in ethanol-water (96:4) (50 mL).
To this was added a solution of d-(-)-threo-2-amino-1-(4-
nitrophenyl)propane-1,3-diol (8.17 g, 38.5 mmol) in hot etha-
nol-water (96:4) (210 mL). This was allowed to cool to room
temperature, depositing a precipitate, and left to stand
overnight in the body of a refridgerator. The crystallized solid
(10.31 g) was collected, washed with ethanol-water (96:4), and
dried in vacuo. This salt was suspended in water and 2 M
HCl was added to pH 1-2. This was extracted with ether (3×),
and the combined extracts were washed with water (1×) and
dried over MgSO4. Concentration gave a white solid (6.40 g)
which was recrystallized from CHCl3/hexane to give the pure
(+)-77 (6.23 g, 16 mmol, 83% of theoretical); mp 87-88.5 °C;
(iii) (()-(3R*,5S*)-3-Ca r b oxy-10-(5,7-d ich lor oin d ol-1-
yl)-3,5-d ih yd r oxyd eca n oic Acid , Disod iu m Sa lt (66). The
dihydroisoxazole intermediate 33 (R ) H, R1 ) 5,7-diCl) was
converted to the title compound, mp >250 °C, by the general
procedures described above: 1H NMR (D2O, 360 MHz) δ 7.58
(s, 1H, indole 6-H), 7.34 (d, 1H, J ) 3 Hz, indole 2-H), 7.25 (s,
1H, indole 4-H), 6.52 (d, 1H, J ) 3 Hz, indole 3-H), 4.48 (t,
2H, J ) 7 Hz, NCH2), 3.85 (m, 1H, CHOH), 2.68 and 2.47
(doublets, 1H each, J ) 15.6 Hz, CH2CO2Na), 1.80 (m, 4H,
NCH2CH2 and CH2C(OH)(CO2Na)), 1.41-1.28 (m, 6H, (CH2)3);
MS (FAB positive ion) m/z 432 ((M + H)+ of free acid); (FAB
negative ion) m/z 430 (M - H)- of free acid); IR (Nujol mull)
1574, 1463 cm-1. Anal. (C19H21Cl2NO6Na2‚1.1H2O) C, H, N.
[R]25 ) +19.4 (c ) 0.5% w/v; EtOH). Anal. (C18H22Cl2O5) C,
D
(c) Meth od C (Sch em e 4c): (()-(3R*,5S*)-3-Ca r boxy-
11-(5-ch lor oin d ol-1-yl)-3,5-d ih yd r oxyu n d eca n oic Acid ,
Disod iu m Sa lt (65). 5-Chloroindole was alkylated with
7-bromoheptanol using KOH in DMSO by the procedure used
H.
(R )-(3R *,5S *)-3-(Ca r b oxym e t h yl)-5-[6-(2,4-d ich lor o-
p h en yl)h exyl]-3-h yd r oxytetr a h yd r ofu r a n -2-on e ((-)-77).

3594 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19
Gribble et al.
The ethanol-water liquors containing the enrched (-)-77 salt
from the above were concentrated in vacuo and to the residue
added water and then 2 M aqueous HCl to acidify. This was
extracted with ether (3×), and the extracts were washed with
2 M HCl and then water and dried over MgSO4. Concentration
gave an oil which was triturated with hexane to give enriched
(-)-77 (8.12 g, 20.8 mmol) as a solid. This was dissolved in
ethanol-water (96:4) (25 mL), and a hot solution of (L-(+)-
threo-2-amino-1-(4-nitrophenyl)propane-1,3-diol, 41a (4.41 g,
20.8 mmol), in the same solvent (120 mL) was added. The
resulting precipitate was collected, after standing overnight
in the body of a refridgerator, and the pure (-)-77 (5.83 g, 15
mmol, 78% of theoretical after recrystallization) was regener-
tables of fractional atomic coordinates, interatomic distances
and angles, and thermal parameters (11 pages). Ordering
information is given on any current masthead page.
Refer en ces
(1) (a) Srere, P. A. The Enzymology of the Formation and Break-
down of Citrate. Adv. Enzymol. 1975, 43, 57-101. (b) Houston,
B.; Nimmo, H. G. Purification and Some Kinetic Properties of
Rat Liver ATP-Citrate Lyase. Biochem. J . 1984, 224, 437-443.
(c) Plowman, K. M.; Cleland, W. W. Purification and Kinetic
Studies of the Citrate Cleavage Enzyme. J . Biol. Chem. 1967,
242, 4239-4247. (d) Walsh, C. Enzyme Reaction Mechanisms;
W. H. Freeman and Co.: San Francisco, 1979; pp 766-768.
(2) (a) Gribble, A. D.; Dolle, R. E.; Shaw, A.; McNair, D.; Novelli,
R.; Novelli, C. E.; Slingsby, B. P.; Shah, V. P.; Tew, D.; Saxty,
B. A.; Allen, M.; Groot, P. H.; Pearce, N.; Yates, J . ATP-Citrate
Lyase as a Target for Hypolipidemic Intervention. Design and
Synthesis of 2-Substituted Butanedioic Acids as Novel, Potent
Inhibitors of the Enzyme. J . Med. Chem. 1996, 39, 3569-3584.
(b) Dolle, R. E.; Gribble, A.; Wilks, T.; Kruse, L. I.; Eggleston,
D.; Saxty, B. A.; Wells, T. N. C.; Groot, P. H. E. Synthesis of
Novel Thiol-Containing Citric Acid Analogues. Kinetic Evalua-
tion of These and Other Potential Active-Site-Directed and
Mechanism-Based Inhibitors of ATP-Citrate Lyase. J . Med.
Chem. 1995, 38, 537-543. (c) Dolle, R. E.; McNair, D.; Hughes,
M. J .; Kruse, L. I.; Eggleston, D.; Saxty, B. A.; Wells, T. N. C.;
Groot, P. H. E. ATP-Citrate Lyase as a Target for Hypolipidemic
Intervention. Sulfoximine and 3-Hydroxy-â-lactam Containing
Analogues of Citric Acid as Potential Tight-Binding Inhibitors.
J . Med. Chem. 1992, 35, 4875-4884. (d) Saxty, B. A.; Novelli,
R.; Dolle, R. E.; Kruse, L. I.; Reid, D. G.; Camilleri, P.; Wells, T.
N. C. Synthesis and Evaluation of (+)- and (-)-2,2-Difluoroci-
trate as Inhibitors of Rat-liver ATP-Citrate Lyase and Porcine-
Heart Aconitase. Eur. J . Biochem. 1992, 202, 889-896. (e) Dolle,
R. E.; Novelli, R.; Saxty, B. A.; Wells, T. N. C.; Kruse, L. I.;
Camilleri, P.; Eggleston, D. Preparation of (+)-(Erythro)- and
(+)-(Threo)-2-Vinyl Citric Acids as Potential Mechanism-Based
Inhibitors of ATP-Citrate Lyase. Tetrahedron Lett. 1991, 32,
4587-4590.
ated from the salt as described above: mp 87-88.5 °C; [R]25
D
) -19.5 (c ) 0.5% w/v; EtOH). Anal. (C18H22Cl2O5) C, H.
(S)-(3R*,5S*)-3-Ca r boxy-11-(2,4-d ich lor op h en yl)-3,5-d i-
h yd r oxyu n d eca n oic Acid , Disod iu m Sa lt ((+)-45). (+)-
77 (160 mg) was dissolved in ethanol (2.5 mL), and 5% aqueous
NaOH solution (0.64 mL) in water (1.86 mL) was added with
stirring. After ca. 10 min, the precipitated solid was collected,
washed with 1:1 EtOH-H2O, and recrystallized from water-
ethanol to give the (+)-45 (100 mg): [R]25D ) + 23.3 (c ) 0.16%
w/v; H2O). Anal. (C18H22Cl2O6Na2‚H2O) C, H.
(R)-(3R*,5S*)-3-Ca r boxy-11-(2,4-d ich lor op h en yl)-3,5-d i-
h yd r oxyu n d eca n oic Acid , Disod iu m Sa lt ((-)-45). Hy-
drolysis of (-)-77 (100 mg) as described for (+)-77 above gave
(-)-45 (65 mg), [R]25 ) -20.6 (c ) 0.1% w/v; H2O). Anal.
D
(C18H22Cl2O6Na2‚H2O) C, H.
Estim a tion of En a n tiom er ic P u r ity of Resolved En a n -
tiom er s by Micella r Electr ok in etic Ca p illa r y Ch r om a -
togr a p h y (MECC). Resolved lactone chloramphenicol base
salts of 77 and 80 were analyzed for enantiomeric purity by
MECC prior to regeneration of the free lactone or subsequent
hydrolysis to the hydroxy acid. Free hydroxy acids were
dissolved in water, and the lactones, or their salts, were
hydrolyzed to the hydroxy acids by addition of excess NaOH
to the compound in water. MECC separation was carried out
on this solution according to the method developed by Okafo
et al.6 on a Beckman P/ACE instument 570 mm × 50 µm id
capillary, using the following conditions: capillary, 570 mm
× 50 µm id (effective length 500 mm); buffer, 30 mM sodium
phosphate, 10 mM boric acid, 50 mM taurodeoxycholic acid,
20 mM â-cyclodextrin, adjusted to pH 7 with 0.1 M NaOH;
applied voltage, 25 kV; detection, UV absorbance at 214 nM;
temperature, 25 °C. The resolved (+) and (-) enantiomers of
both 45 and 58, obtained from hydrolysis of 77 and 80,
repectively, showed one single peak by MECC, with no trace
of the other enantiomer (level of detection >99.5%).
(3) Curran, D. P. In Advances in Cycloaddition; Curran, D. P., Ed.;
J AI Press: Greenwich, CT, 1988; Vol. 1, pp 129-189.
(4) For typical reaction see (a) Kim, T. H.; Sachihiko, I. Total
Synthesis of (()-(Ε)-8â, 17-Epoxylabd-12-ene-15,16,-dial. J .
Chem. Soc., Chem Commun. 1983, 730-1. For synthesis of the
phosphorane, see: (b) Cameron, A. F.; Duncanson, F. D.; Freer,
A. A. J . Chem. Soc., Perkin Trans. 2 1975, 1030-1036.
(5) (a) Davis, F. A.; Chattopadhyay, S.; Towson, J . C.; Lal, S.; Reddy,
T. Chemistry of Oxaziridines. 9. Synthesis of 2-Sulfonyl and
2-Sulfamyloxaziridines Using Potassium Peroxymonosulfate
(Oxone). J . Org. Chem. 1988, 53, 2087-2089. (b) Davis, F. A.;
Vishwakarma, L. C.; Billmers, J . M. Synthesis of R-Hydroxy
Carbonyl Compounds (Acyloins): Direct Oxidation of Enolates
Using 2-Sulfonyloxaziridines. J . Org. Chem. 1984, 49, 3241-
3243.
(6) Okafo, G. N.; Bintz, C.; Clarke, S. E.; Camilleri, P. J . Chem.
Soc., Chem. Commun. 1992, 1189-1192.
En zym e Assa y a n d Hep G2 Cell Assa y. Both assays were
(7) (a) Lewis, Y. S. Isolation and Properties of Hydroxycitric Acid.
Methods Enzymol. 1969, 13, 613-619. (b) Stallings, W. C.;
Blount, J . F.; Srere, P. A.; Glusker, J . P. Structural Studies of
Hydroxycitrates and Their Relevance to Certain Enzymatic
Mechanisms. Arch. Biochem. Biophys. 1969, 193, 431-448.
(8) (a) Sullivan, A. C.; Singh, M.; Srere, P. A..; Glusker, J . P.
Reactivity and Inhibitor Potential of Hydroxycitrate Isomers
with Citrate Synthase, Citrate Lyase and ATP-Citrate Lyase.
J . Biol. Chem. 1977, 252, 7583-7590. (b) Cheema-Dhadli, S.;
Halperin, M. L.; Leznoff, C. C. Inhibition of Enzymes Which
Interact With Citrate by (-)-Hydroxycitrate and 1,2,3-Tricar-
boxybenzene. Eur J . Biochem. 1973, 38, 98-102. (c) Sullivan,
A. C.; Hamilton, J . G.; Miller, O. N.; Wheatley, V. R. Inhibition
of Lipogenesis in Rat Liver by (-)-Hydroxycitrate. Arch. Bio-
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carried out as described previously.2
Mea su r em en t of Effect of Com p ou n d s on In Vivo
Tr iglycer id e Con cen tr a tion s in Ra ts. The procedures
involving animals in this study were subject to both internal
review and UK Home Office regulations. Male Sprague-
Dawley rats were maintained on a reverse light cycle (12 h
light, 12 h dark) and fed a standard RM1 powdered diet
(Special Diet Service, Witham, Essex, UK) with free access to
food and water. Compound was administered as two doses,
given 24 h apart by oral gavage at the dose levels indicated in
the table. Blood samples were obtained mid dark cycle (4 h
post second dose), placed into EDTA plasma tubes and plasma
isolated by centrifugation at 1500g for 15 min. Plasma
triglyceride concentrations were measured enzymatically, us-
ing the Merck GPO-PAP test kit for trigylceride. Results are
expressed as the percentage reduction of plasma triglyceride
concentration from control levels. Significant changes from
control were determined by analysis of variance.
(9) Guthrie, R. W.; Kierstead, R. W. Amino Citric Acid Derivatives.
U.S. Patent 3, 960,933, 1976.
(10) SB laboratories, unpublished results.
(11) Our initial lactonization method utilised stirring 45 in ether at
room temperature with silica gel which had been impregnated
with concentrated sulfuric acid. This produced a ca. 4:1 mixture,
respectively, of the 5 and 6 ring lactones. Conversion to their
repective methyl esters with diazomethane allowed ready sepa-
ration of the two by flash column chromatography. However,
when subjected to acid hydrolysis (25% HCl/∆) to regenerate the
carboxylic acids, both esters gave solely the thermodynamically
favored 5 ring lactone 77. Indeed, we noticed that interconver-
sion of the 6 ring lactone to 77 even occurred in the solid form
of the 4:1 mixture of the two over a period of several months.
Ack n ow led gm en t. We would like to acknowledge
the contributions of our colleagues in the Analytical
Sciences Department for providing the microanalytical
and spectral data.
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails of the X-ray structure determination of (+)-77, including

Hypolipidemic Intervention by ATP-Citrate Lyase
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19 3595
(12) 77 has also been found to be efficacious as an inhibitor of de
novo synthesis of cholesterol and fatty acids in primary rat
hepatocytes. G. Gibbons, University of Oxford, unpublished
results.
(13) Wells, T. N. C. ATP-Citrate Lyase From Rat Liver. Characteri-
sation of the Citryl-Enzyme Complexes. Eur. J . Biochem. 1991,
199, 163-186.
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Zalay, A. W.; Oesterlin, R.; Clemens, S. D.; Dumas, D. J .
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Oesterlin, R.; Bell, M. R.; McGarry, R. H.; Hlavac, A. G.
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dro-1,3,4,6,6-pentamethylcyclopenta[c]pyrrole-4-carboxamides.
J . Med. Chem. 1997, 20, 11068-1071.
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(15) We were unable to evaluate the γ-lactone of 72 due to insufficient
compound being available.
(16) (a) Pearce, N. J .; Yates, J . W.; Berkhout, T. A.; J ackson, B.; Tew,
D.; Boyd, H.; Camilleri, P.; Sweeney, P.; Gribble, A. D.; Shaw,
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Regulation of Plasma Lipids. Hypolipidemic Effects of SB-
204990, a Lactone Prodrug of the Potent ATP Citrate-lyase
Inhibitor SB-201076. Biochem. J ., in press. (b) These data have
been presented in a poster: Groot, P. H. E.; Pearce, N. J .;
Gribble, A. D.; Shaw, A. N.; Tew, D. G.; Wiggins, D.; Gibbons,
G. F. Hepatic ATP-Citrate Lyase as a Target for Hypolipidaemic
Intervention., Xth International Symposium on Atherosclerosis,
Montreal, October 1994.
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