ATP-citrate lyase as a target for hypolipidemic intervention. Design and synthesis of 2-substituted butanedioic acids as novel, potent inhibitors of the enzyme

Article abstract of DOI:10.1021/jm960167w

ATP-citrate lyase is the primary enzyme responsible for the synthesis of cytosolic acetyl-CoA in many tissues. Inhibitors of the enzyme represent a potentially novel class of hypolipidemic agent, which are anticipated to have combined hypocholesterolemic and hypotriglyceridemic properties. A series of 2-substituted butanedioic acids have been designed and synthesized as inhibitors of the enzyme. The best compounds, 58, 68, 71, 74 have reversible K(i)'s in the 1-3 μM range against the isolated rat enzyme. As representative of this compound class, 58, has been shown to exert its inhibitory action through a mainly competitive mechanism with respect to citrate and a noncompetitive one with respect to CoA. None of the inhibitors were able to inhibit cholesterol and/or fatty acid synthesis in HepG2 cells. This has been attributed to the adverse physicochemical properties of the molecules leading to a lack of cell penetration. Despite this, a lead structural class of compound has been identified with the potential for modification into potent, cell-penetrant, and efficacious inhibitors of ATP- citrate lyase.

Full text of DOI:10.1021/jm960167w

J . Med. Chem. 1996, 39, 3569-3584
3569
ATP -Citr a te Lya se a s a Ta r get for Hyp olip id em ic In ter ven tion . Design a n d
Syn th esis of 2-Su bstitu ted Bu ta n ed ioic Acid s a s Novel, P oten t In h ibitor s of th e
En zym e
Andrew D. Gribble,*,† Roland E. Dolle,†,| Antony Shaw,† David McNair,† Riccardo Novelli,† Christine E. Novelli,†
Brian P. Slingsby,† Virendra P. Shah,† David Tew,‡ Barbara A. Saxty,‡, Mark Allen,‡ Pieter H. Groot,§
Nigel Pearce,§ and J ohn Yates§
Departments of Medicinal Chemistry, Mechanistic Enzymology, and Vascular Biology, SmithKline Beecham Pharmaceuticals
Ltd, The Frythe, Welwyn, Hertfordshire, AL6 9AR, U.K.
Received March 1, 1996X
ATP-citrate lyase is the primary enzyme responsible for the synthesis of cytosolic acetyl-CoA
in many tissues. Inhibitors of the enzyme represent a potentially novel class of hypolipidemic
agent, which are anticipated to have combined hypocholesterolemic and hypotriglyceridemic
properties. A series of 2-substituted butanedioic acids have been designed and synthesized as
inhibitors of the enzyme. The best compounds, 58, 68, 71, 74 have reversible Ki’s in the 1-3
µΜ range against the isolated rat enzyme. As representative of this compound class, 58, has
been shown to exert its inhibitory action through a mainly competitive mechanism with respect
to citrate and a noncompetitive one with respect to CoA. None of the inhibitors were able to
inhibit cholesterol and/or fatty acid synthesis in HepG2 cells. This has been attributed to the
adverse physicochemical properties of the molecules leading to a lack of cell penetration. Despite
this, a lead structural class of compound has been identified with the potential for modification
into potent, cell-penetrant, and efficacious inhibitors of ATP-citrate lyase.
In tr od u ction
There is increased expert opinion, coupled with ever
growing evidence, to support the view that, in man,
elevated plasma cholesterol concentrations in combina-
tion with increased plasma triglyceride levels, repre-
sents a very significant high risk for cardiovascular
disease.
For example, in the PROCAM study, 25% of all
observed myocardial infarct events occurred in subjects
in which the ratio of low density lipoprotein (LDL)/high
density lipoprotein (HDL) cholesterol was >5, and
where triglyceride concentrations exceeded 200 mg/dL.1
The Helsinki heart study has reported that the risk of
pound which could combine the hypocholesterolemic
coronary heart disease is tripled for subjects possessing
properties of an HMG-CoA reductase inhibitor with the
an LDL/HDL ratio >5 and triglycerides >2 mmol/L.2
hypotriglyceridemic effects of a fibrate, would have high
A major advance in therapy for the treatment of
hypercholesterolemia has come with the discovery and
commercial development of clinically effective inhibitors
(e.g. compactin, lovastatin, pravastatin 1a -c) of HMG-
CoA reductase, a key regulatory enzyme in mammalian
cholesterol biosynthesis.3 The fibrates, e.g. lopid (gem-
fibrozil, 2), have been long known as agents that are
effective in lowering plasma triglyceride concentrations.4
However, no marketed drugs or therapeutic strategies
exist which have the ability to decrease effectively both
plasma cholesterol and triglyceride levels. The combi-
nation of an HMG-CoA reductase inhibitor with a
fibrate is not recommended due to increased risk for
myopathies,5 although this combination may be an
option with the more modern fibrates. Thus, a com-
potential as a novel class of hypolipidemic agent for
reducing the mortality and morbidity associated with
coronary heart disease. Such an agent would be an-
ticipated to be the drug of choice for the treatment of
patients with combined type IIb hyperlipidemia.
In this context, we initiated a research program with
the objective of discovering, and evaluating the potential
of, inhibitors of ATP-citrate lyase (ACL; EC 4.1.3.8) as
a novel and therapeutically beneficial class of lipid
lowering agent, capable of combining the desired hypo-
cholesterolemic and hypotriglyceridemic properties in
the same molecule. ACL is a cytosolic enzyme found in
a large variety of animal tissues, and its activity is
particularly high in lipogenic tissues such as the liver.6
It is positioned upstream of HMG-CoA reductase in the
mammalian cholesterol biosynthetic pathway and sup-
plies acetyl units for both cholesterol and fatty acid
synthesis.7 It was this feature which attracted us to
select the enzyme as a target for hypolipidemic inter-
vention.
† Department of Medicinal Chemistry.
‡ Department of Mechanistic Enzymology.
§ Department of Vascular Biology.
| Present address: Pharmacopeia, 2000, Cornwall Rd., Monmouth
J unction, NJ 08852.
Present address: Department of Biochemistry, University of
Edinburgh, Edinburgh, EH8 9XD, Scotland.
X Abstract published in Advance ACS Abstracts, J uly 15, 1996.
Inhibition of ACL would be anticipated to (1) decrease
plasma LDL cholesterol, in a similar manner to HMG-
S0022-2623(96)00167-7 CCC: $12.00 © 1996 American Chemical Society

3570 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18
Gribble et al.
Sch em e 1. Schematic Outlining the Anticipated Effects of Inhibiting ATP-Citrate Lyase
CoA reductase inhibitors, by decreasing cholesterol
synthesis and up-regulating LDL receptor activity,8 and
(2) decrease plasma triglycerides as a consequence of a
reduction in the synthesis of fatty acids, leading to an
ultimate lowering of plasma VLDL. As a precursor for
LDL, a reduction in VLDL may also contribute further
to plasma LDL lowering. Cytosolic levels of malonyl
CoA are also expected to decrease after inhibition of
ACL. As malonyl CoA is known to regulate the entry
of acyl moieties into the mitochondrial â-oxidation spiral
(at the level of carnitine-palmitoyl transferase I), inhibi-
tion of ACL is expected to channel hepatic free fatty
acids into â-oxidation, thereby further decreasing the
pool of fatty acids available for triglyceride and VLDL
synthesis, in a similar manner to that shown for Ro 22-
06549 (Scheme 1).
Some evidence to support the view that inhibition of
ACL should lead to the desired pharmacological profile
can be drawn from studies on (-)-hydroxycitrate (3).10
This citrate analogue is a naturally occurring, potent
(Ki ) 0.15 µΜ, rat ACL) inhibitor of the enzyme.11 We
have demonstrated inhibition of cholesterol synthesis
in HepG2 cells by 3, albeit at relatively high concentra-
tions (IC50 ) 0.1-0.5 mM) compared to its Ki. We have
yield ADP, inorganic phosphate (Pi), oxaloacetate (5),
and acetyl CoA (6), by means of the recently revised
mechanism depicted in Scheme 2.15 This differs from
the originally accepted mechanism for catalysis, which
invoked the necessity for an active site thiol nucleophile
to allow formation of a covalently bound citryl-enzyme
adduct (9) as an intermediary step between 7 and 8.7c
In earlier papers we have described our attempts to
inhibit the enzyme through the rational design of
citrate-based active-site and mechanism-based inhibi-
tors, which were designed to interact with this active-
site thiol nucleophile.16 We have rationalized their lack
of success in light of the revised mechanism for cataly-
sis. We have also described our attempts to design
potential tight-binding inhibitors of the enzyme.17 In
this paper, we now wish to describe our strategies to
design and synthesize a series of 2-substituted butane-
dioic acids e.g. 58, 68, 71, 74 as novel, potent inhibitors
of ATP-citrate lyase.
Design Str a tegy
Following on from our attempts to design tight-
binding, mechanism-based, and active-site directed
inhibitors of ACL, we decided to pursue a third strategy
based upon a multisubstrate analogue approach to
design novel inhibitors. The revised catalytic mecha-
nism of ACL suggests that analogues of either citryl
phosphate (7) or citryl CoA (8) would fit this category.
We considered citryl CoA to be a better target, since it
binds moderately well to the enzyme (Km ) 28 µM; cf.
Km ) 100 µM for citrate)18 and provides ample scope
for modification. Additionally, we were aware of the
MEDICA series of compounds, represented by MEDICA
16 (10), as a potential starting point for the design of
also shown that 3 leads to an increase in HMG-CoA
reductase activity and up-regulation of the LDL receptor
activity in these cells, analogous to mevinolin.12 Al-
though literature reports describe attenuation of lipo-
genesis and cholesterogenesis by 3 in rodents,13 the
compound has many shortcomings as a tool which can
be used to challenge the hypothesis cited above: It is
poorly cell penetrant, has low oral bioavailability, and
stimulates acetyl CoA carboxylase, an enzyme upstream
in the cholesterol biosynthesis pathway.14 Thus, in
order to challenge and fully validate the hypothesis
presented above in appropriate animal models, we
sought an effective, orally active inhibitor of the ATP-
citrate lyase enzyme.
such compounds.19 The MEDICA compounds are simple
R,ω-dicarboxylic acids, possessing hypolipidemic activity
through a combination of several mechanisms of action.
MEDICA 16 is reported to be a citrate competitive
inhibitor of ACL with a Ki of 16 µΜ.19a During the
course of this research effort, we have also established
that it is a competitive inhibitor with respect to CoA
(Ki ) 3 µM). We considered that a possible explanation
ACL catalyses a reversible retro-Claisen reaction
utilizing ATP, citrate (4), and CoASH as substrates to

ATP-Citrate Lyase as a Target for Hypolipidemic Intervention
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18 3571
Sch em e 2. Revised Mechanism of ATP-Citrate Lyase Catalysis
Sch em e 3. Role of HMG-CoA Reductase
for its modest ACL inhibitory activity might be that it
is able to mimic the enzyme bound citryl CoA adduct.
For such a mimickry, one carboxylic acid group could
be associated with the citrate binding site while the
lipophilic alkyl chain and other carboxylic acid group
may associate with the CoA binding domain. On the
basis of this, we rationalized that more potent ACL
inhibitors could be designed by incorporating more
“citrate-like” features into these molecules, coupled with
modifications that would more effectively interact with
the CoA binding site. As a means of identifying
structural modifications that would more effectively
associate with the CoA binding site, we considered the
possibility that there may be structural similarities
between the CoA sites of ACL and HMG-CoA reductase.
The latter enzyme converts HMG-CoA (11) to mevalonic
acid (12), with the concomitant release of CoA in the
process (Scheme 3).20 A study reported in the literature
at the time we initiated this research indicated that the
decalin moiety of HMG-CoA reductase inhibitors, such
as compactin and lovastatin (1a , 1b), plays the role of
a hydrophobic anchor in the binding of these compounds
to the enzyme active site.21 This is shown to occur in a
region normally occupied by CoA. It was further
suggested that HMG-CoA is principally bound by a
3-hydroxy-3-methylglutaryl (HMG) binding region and
a hydrophilic adenine binding domain. The area in the
binding site of the enzyme that links these two domains
is hypothesized to be rather hydrophobic in nature and
to not well accommodate the phosphopantothenic acid
chain of CoA (Figure 1).21 After the enzyme-mediated
reduction has taken place, it is suggested that the CoA
product is bound only in the hydrophilic region and
repelled in the hydrophobic domain. It is postulated
that the lack of complementarity in this region would
F igu r e 1. Hypothetical model of HMG-CoA reductase binding
site, reproduced from reference 21.
assist the expulsion of the product from the active site
and facilitate turnover. We speculated that a similar
scenario may be possible for ACL, where the final stage
of the reaction mechanism relies upon the release of a
similar product, acetyl CoA, from the enzyme bound
citryl CoA adduct. A large number of inhibitors of HMG
CoA reductase are now known in which the hexahy-
dronaphthalene group of 1 has been replaced by a
variety of synthetic lipophilic moieties.22 Compounds
which we were aware of at the time we initiated this
work included 13a -c.23 In these, the decalin ring
system has been replaced by lipophilic (benzyloxy)-
phenyl and biphenyl moieties to give derivatives which
can show potencies ca. three-fold that of compactin.
These compounds can be considered as possessing a
mevalonate-like “head” linked to a hydrophobic anchor.
By analogy, our initial model for the design of ACL
bisubstrate inhibitors, which might make use of a
similar hydrophobic pocket, was based upon a similar
concept of linking a citrate-like “head” to a lipophilic
group. We decided to investigate compounds of this type
in a class in which one carboxylic acid group of citric
acid was replaced by a lipophilic moiety. In the first

3572 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18
Ch a r t 1. Syntheses of the S-Linked Compounds of Tables 1-6
Gribble et al.
targets identified for synthesis (14), we utilized a sulfur
heteroatom to link a citrate-like “head” to a chain
bearing a terminal 2,4-dichlorophenyl ring, to represent
the lipophilic aryl group in 13a and 13b.
intermediate 24 to the hydroxybutanedioic acid in the
usual way, gave the chain S-displaced analogue of
general structure 25, typified by 73. The simple monoac-
ids 23 (e.g. compound 70) were prepared by reaction of
the thiol intermediate with ethyl 4-bromobutanoate,
followed by hydrolysis.
The analogous carbon-linked compounds of general
structure 29 (e.g. 71 and 72) were synthesized by
alkylation of the dianion of methyl acetoacetate (NaH/
nBuLi in THF) with the appropriate aralkyl bromide 27,
followed by subsequent elaboration of the generated
keto ester 28 by the usual method described above
(Chart 2). The keto compounds 33 were derived from
the dihydroisoxazole 31 by treatment with Raney nickel
in aqueous boric acid, followed by base hydrolysis. 31
was prepared from the alcohol intermediate 26 in four
steps via Swern oxidation, oxime (30) formation with
hydroxylamine, in situ generation of the corresponding
nitrile oxide and its subsequent [3 + 2] cycloaddition
with dimethyl itaconate (Chart 2).25
The diethyl ester of 58 (111) and the dimethyl ester
of 68 (113) (Table 7) were obtained either as intermedi-
ates or synthesized by acid-catalyzed esterification of
the diacid with the appropriate alcohol. The diac-
etoxymethyl ester of 58 (112) was prepared from reac-
tion of the diacid with bromomethyl acetate using
potassium carbonate and 18-crown-6 in DMF.
Syn th esis of In ter m ed ia tes. Methods for prepara-
tion of the alcohol, bromide, and thioacetate intermedi-
ates required for the synthetic schemes depicted in
Charts 1 and 2 were dependent upon the functionality
to be introduced in the aryl group and chain. For the
compounds in Tables 1-3, 5, and 6, where the func-
tionality adjacent to the aryl ring is a methylene group,
these intermediates were prepared by the methods
shown in Chart 3. The thioacetates of general structure
Ch em istr y
Syn th esis of F in a l Com p ou n d s. General synthetic
methods for the preparation of the S-linked compounds
of Tables 1-6 are shown in Chart 1. 2-[(Aralkylthio)-
methyl]-2-hydroxybutanedioic acids of general structure
14 were usually prepared from the â-keto ester inter-
mediate 17 via formation of the cyanohydrin and
subsequent acid hydrolysis. Our original method used
cHCl/KCN in ether to generate the cyanohydrin,24 but
we later modified this step to employ KCN in aqueous
potassium dihydrogen phosphate. The â-keto esters
were prepared by the reaction of the thiol 16, generated
in situ from the more stable thioacetates 15, with
methyl 4-chloroacetoacetate. Reduction of the â-keto
ester 17 with NaBH4/CeCl3, followed by base hydrolysis
of the ester groups of 18, gave the descarboxy analogues
19 (e.g. compound 69). The deshydroxy derivatives of
general structure 21 (e.g. compound 68) were obtained
from the Michael addition of in situ-generated thiol to
dimethyl itaconate, followed by acid hydrolysis.
similar procedure using methyl 3-oxo-4-pentenoate as
the Michael acceptor, followed by elaboration of the
A

ATP-Citrate Lyase as a Target for Hypolipidemic Intervention
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18 3573
Ch a r t 2. General Syntheses of the Compounds Not Containing a Thioether Linkage in Table 3
Ch a r t 3. Synthesis of the Intermediates Required for the Preparation of the Compounds of Tables 1, 2, 3, 5, and 6
40 were generated from the reaction of the ω-arylbro-
moalkanes with potassium thioacetate in DMF. The
latter were generally obtained from the appropriate
ω-arylalkanols 39 using NBS/PPh3 in CH2Cl2. For
certain compounds (106, 64, 93, 94, 95), the bromide
intermediates 43 were synthesized directly by Friedel-
Crafts acylation of the substituted benzene 42 with the
appropriate ω-bromoalkanoyl halide, followed by reduc-
tion of the keto group with triethylsilane (Chart 3,
method B).26 The alcohols 39 were prepared by stan-

3574 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18
Gribble et al.
Ch a r t 4. Syntheses of the Intermediates Required To Prepare the Compounds of Table 4
dard methodology which was governed by the substit-
uents and the chain length. Generally, the aryl group
was introduced from the appropriately substituted
benzaldehyde 34 using Wittig chemistry to generate
intermediates of the desired chain length (method A).
These would normally contain a terminal carboxylic acid
or an alcohol group, depending upon the nature of the
phosphonium salts 35 or 37 available. Those bearing
a terminal carboxylic acid group were converted to their
corresponding methyl esters 36 (CH2N2/Et2O or CH3-
OH/cH2SO4) to ease purification and reduced to the
alcohol 38 with DIBAL-H in CH2Cl2. The olefinic bond
was then hydrogenated using either Pd/C or PtO2. The
latter catalyst was found to be necessary for the
halophenyl compounds to prevent dehalogenation. When
the desired ω-hydroxy phosphonium salt 37 was not
available commercially, this was prepared from tri-
phenylphosphine and the R,ω-bromo alcohol in refluxing
toluene.
Compounds containing a functionality other than a
methylene group adjacent to the aryl ring (Table 4) were
also prepared from an appropriate thioacetate interme-
diate by an identical procedure to that described above.
The cis and trans olefins 76 and 77 were prepared as
described above from the separated isomers of 36 (n )
6). The latter were obtained by preparative HPLC on
silica gel using an ether/petroleum ether gradient.
Thioacetate intermediates 47a -d , 48, 50, required for
the synthesis of the other compound classes of Table 4,
were prepared by the general standard methods de-
picted in Chart 4. An exception was the analogue
containing a keto group adjacent to the aryl ring 53.
This compound was prepared via conversion of the olefin

ATP-Citrate Lyase as a Target for Hypolipidemic Intervention
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18 3575
Ta ble 1. Effect of Chain Length in 3-[[[(2,4-Dichlorophenyl)-
alkyl]thio]methyl]-3-hydroxybutan-1,4-dioic Acids on ACL
Inhibitory Activity
the right combination, i.e. 2,4 (58) or 2,3 (65) disubsti-
tuted. The 2-fold increase in potency of the 2,4 analogue
over its 2,3 isomer reflects the difference seen with 3
chloro relative to the other monochloro compounds.
Neither the 3,4 (66) nor the 2,6 (67) dichloro derivatives
show any improvements over the monochloro analogues.
Encouraged by the diversity of activity associated
with the changes so far examined, we embarked upon
a program of synthesis to identify structural features
of the molecule which were important for enzyme
inhibitory activity, with the ultimate goal to improve
potency further. For this analysis, we examined changes
to the molecule in 3 areas: (i) the “citrate head”, (ii)
the linking chain, and (iii) the aryl ring.
no.
n
Ki (µM)
410 ( 9a
480 ( 10
390 ( 28
6.8 ( 0.14
3.3 ( 0.18
7.2 ( 0.36
300 ( 1.1
analyses
54
55
56
57
58
59
60
1
2
3
5
6
7
9
C, H, Cl, S
C, H, S
C,b H, S
C, H, S
C, H
C, H, Cl, S
C, H
Only the results of our inital modifications to the
“citrate head” will be discussed in this manuscript.
These are summarized in Table 3. Some more elaborate
changes that were undertaken are better discussed in
the context of a subsequent paper in this series.
Removal of the tertiary hydroxyl group in 68 gave no
significant change in ACL inhibitory activity. In con-
trast, compounds lacking the tertiary carboxylic acid
group were much less active (69, 70). These results fit
our proposition that compounds of this class are binding
to the citrate site of the enzyme and require the
dicarboxyate functionality to allow binding as the
magnesium chelate, in a similar manner to citrate
itself.7b,27 Further evidence for this postulate arises
from data on another compound (74) discussed below.
a
b
Demonstrates cooperative kinetics. C calcd 45.79%, found
46.58%.
Ta ble 2. Effect of Chlorine Substitution in 3-[[(Phenylhexyl)-
thio]methyl]-3-hydroxybutan-1,4-dioic Acids on ACL Inhibitory
Activity
no.
R1
R2
Ki (µM)
analyses
C, H
61
62
63
64
65
58
66
67
H
H
710 ( 50
69 ( 4
2-Cl
3-Cl
4-Cl
2-Cl
2-Cl
3-Cl
2-Cl
H
H
H
3-Cl
4-Cl
4-Cl
6-Cl
C, H
150 ( 35
71 ( 4
C, H, Cl, S
C, H, Cl, S
C, H, Cl, S
see Table 1
C, H, Cl, S
C, H, Cl, S
Relocation of the sulfur atom in 58 one atom closer
to the aryl ring in 73 gave a compound of equal potency,
as did its replacement by a methylene group in 71. This
suggests no critical role for the heteroatom other than
being part of a spacer group. Other heteroatom replace-
ments for sulfur (e.g. NR, O) were not examined in this
series based on detrimental effects observed in a sim-
pler, related class of compound (data not shown). By a
similar analogy, no advantages from oxidation to the
corresponding sulfoxide or sulfone was anticipated, and
consequently was not examined here.
7.3 ( 1
3.3 ( 0.18
110 ( 15
76 ( 11
51 to the alcohol 52 (BH3, NaBO3), followed by Swern
oxidation and hydrolysis.
Resu lts a n d Discu ssion
Activities of the compounds in Tables 1-6 are pre-
sented as reversible Ki’s for inhibition of purified rat
enzyme unless indicated otherwise. During the course
of our research program, a method for production of
recombinant human ACL was developed. As a conse-
quence, inhibitory data on a few compounds is available
only against the latter form of the enzyme. This is
indicated where appropriate in the tables. Our most
potent inhibitors had almost identical Ki’s against both
rat and human recombinant forms.
The consequences of varying the length of the chain
(n) linking the aromatic group to the “citrate-head” is
demonstrated with the compounds in Table 1. We were
particularly encouraged to discover that 58 (n ) 6, Ki
) 3.3 µΜ) was quite a potent inhibitor of the enzyme.
Its lower and higher homologues 57 and 59 showed
small reductions in potency, while a ca. 100-fold loss in
activity was observed at the other shorter and longer
chain lengths examined. Removal of the two chlorine
atoms in 61 resulted in a 2 orders of magnitude loss of
activity (Table 2). Further examination of the effects
of chlorine substitution on activity are also shown in
Table 2. Mono chlorine substitution is preferred in
either the 2 (62) or 4 (64) positions and both equally
improve activity by 1 order of magnitude over the
unsubstituted parent 61. The 3-chloro isomer 63 is ca.
half as potent as either of these. A second chlorine gives
rise to a further ca. 30-fold increase in activity only in
Replacement of the sulfur by a carbonyl group in 74
gave a modest 3-fold increase in potency (Ki ) 1.2 µΜ).
The critical nature of the chain length is reflected more
so in this analogue, where activity is reduced 25-fold in
the lower homologue 75.
An interesting observation is that the keto compound
74 can act as a substrate for ACL. When used as an
inhibitor of the enzyme, the maximal inhibition of
oxaloacetate 5 formation was approximately 85%. Nor-
mally inhibition approaching 100% is observed as the
inhibitor concentration becomes saturating. In the
absence of citrate, a compound 74-dependent rate of
oxaloacetate formation could be observed. This cor-
related with the apparent incomplete inhibition at
saturating inhibitor concentrations. Omission of all the
usual substrates for the assay (citrate, ATP, CoA) still
resulted in the turnover of 74 to produce oxaloacetate,
but only in the presence of Mg2+. The maximal rate of
turnover was found to be 13% of that for citrate. These
results strongly imply that the “citrate head” of the
inhibitor occupies the citrate binding pocket sufficiently
well to allow an active site base to remove the hydroxyl
proton in the first phase of a retro-Claisen cleavage to
5. The need for Mg2+ is consistent with this deduction
and would be expected based upon its normal require-

3576 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18
Ta ble 3. Some “Citrate-Head” Modifications to 58
Gribble et al.
a
Microanalysis not obtained, structure consistent by NMR.
Ta ble 4. Phenyl Link Modifications
Ta ble 5. 3-[[(pyridylhexyl)thio]methyl]-3-hydroxybutan-
1,4-dioic Acids
no.
Y
n
Ki (µM)
analyses
no.
Ar
Ki (µM)
analyses
58
76
77
78
79
80
81a
82
83
84
85
CH2
Z-CHdCH
E-CHdCH
O
S
NH
SO
CO
5
4
4
5
5
5
5
5
5
4
4
3.3 ( 0.18
7.4 ( 0.3
33 ( 1.4
67 ( 7
10 ( 2.5
12 ( 0.5
131 ( 21
130 ( 25
64 ( 7
175 ( 25
178 ( 16
see Table 1
C, H
C, H
C, H
C, H
61
Ph
710 ( 50
see Table 2
(320 ( 60a)
86
87
88
89
2-pyridyl
3-pyridyl
4-pyridyl
3,5-diCl-4-pyridyl
30% Inhibn @1 mMa
35% Inhibn @1 mMa
20% Inhibn @1 mMa
121 ( 8a
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, Hb
a
Assayed using hrACL.
C, H
C, H
C, H, N
C, H, N
SO2
-SO2NH-
-SO2N(CH3)-
this position. Most noticable was that polar links, e.g.
sulfonamido 84, were particularly detrimental to activ-
ity.
a
Compound lacks the tertiary OH group in the “head”. This
change is assumed to have little effect on activity based upon the
Our largest synthetic effort in attempts to signifi-
cantly improve the potency of 58 was concentrated on
aryl ring substitution. Simple replacement by a less
lipophilic pyridyl ring reduced activity considerably,
even when chlorines were introduced (Table 5).28 This
result was consistent with our inital hypothesis of the
aryl ring binding to a lipophilic region of the enzyme.
The substituents introduced into the phenyl ring to
replace the two chlorine atoms of 58 are summarized
in Table 6. More polar/less lipophilic substituents than
Cl in the para position reduced activity (e.g. compounds
95, 96, 97, 99). Comparison of 2,4-dichloro (58), 2,4-
dimethyl (106), and 2,4-difluoro (107) compounds sug-
gested that substituents that were both lipophilic and
b
equivalent activities of compounds 58 and 68 (Table 3). Analysis
for 0.57 M acetone.
ment in the citrate cleavage reaction. In the latter case,
citrate binds to the enzyme as its Mg-chelate.27
The majority of other linkage modifications that were
examined in compounds related to 58 concentrated on
changes to the group (Y) linking the alkyl chain directly
to the aryl ring (Table 4). A cis olefinic group in 76 was
preferred to a trans in 77, but neither was preferred
over methylene. None of the modifications in Table 4,
taking into account matching atom number chain
length, were advantageous over a simple methylene in

ATP-Citrate Lyase as a Target for Hypolipidemic Intervention
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18 3577
Ta ble 6. Effect of Aryl Ring Substitution in 3-[[(phenylhexyl)thio]methyl]-3-hydroxybutane-1,4-dioic Acids on ACL Inhibitory Activity
a
b
C: calcd 48.67%; found 49.79%. Analysis for 0.28 M hexane. c Assayed using hr ACL.
electron withdrawing conferred good ACL inhibitory
activity. In the ortho position, a phenyl group in 102
improved activity 40-fold over the unsubstituted parent
and was better than chlorine in this respect. However,
further introduction of chlorines in the 4 and 6 positions
of 103, the substitution pattern present in the lipophilic
portion of the potent synthetic HMG-CoA reductase
inhibitor 13a , did not give rise to the anticipated
increase in potency.29 The presence of a second ortho
substituent appears to be detrimental to activity and
is consistent with the observation of a lack of any
increase in potency for the 2,6-dichloro compound 67
over its 2-chloro analogue 62.
The 2 substituted nitro compound 100 is more potent
than its 4 isomer 98, despite both compounds having
the same lipophilicity. This may suggest a stronger
preference for an electron-withdrawing component to
this substituent in the 2 position over the 4 position.
The 2-nitro-4-chloro compound 105 showed a further
modest increase in activity over 2-nitro alone.
remained that of 2,4-dichloro. The best inhibitors of
ATP-citrate lyase from this series are the S-linked
butanedioic acids 58 and 68, Ki’s 3.3 µΜ and 2.9 µΜ
repectively, the all methylene butanedioic acid 71, Ki
) 3 µΜ, and the keto butanedioic acid 74, Ki ) 1 µΜ.
En zym e Kin etic An a lysis of 58. A more detailed
analysis of the inhibitory profile of 58 was carried out
as representative of the butanedioic acid class of ACL
inhibitor. Competition experiments between 58 and
both citrate and CoA show that 58 is a mixed type
inhibitor with respect to citrate and a noncompetitive
inhibitor with respect to CoA. This is illustrated by the
Lineweaver-Burke plots in Figures 2a (vs citrate) and
2b (vs CoA). Although 58 is definitely a mixed type
inhibitor with respect to citrate (the probability of this
not being the case is 1 × 10-5 as judged by the F-test)
it is clear that 58 exerts its inhibitory action through a
mainly competitive mechanism. The competitive com-
ponent of Ki is 1 order of magnitude smaller than that
for the uncompetitive component. Inhibitors of this
structural type are thus kinetically different to the
However, despite investigating a wide range of changes
(Table 6), the best phenyl ring sustitution pattern

3578 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18
Gribble et al.
should be physiologically advantageous over ones which
are competitive with respect to CoA. However, it could
also be argued that a more effective inhibitor would be
one which was also non- or uncompetitive with repect
to citrate.
HepG2 Cell Assay. Selected compounds that showed
inhibitory activity against isolated ACL enzyme were
assayed further for their ability to inhibit both choles-
terol and fatty acid synthesis in a cell based assay. For
this, we chose to use the well established human
hepatoma cell line, HepG2. For this assay, we found it
necessary to determine inhibition by measuring the
incorporation of 3H2O into cholesterol and fatty acid
synthesis.12 Cellular ATP levels were also measured
in order to determine whether or not any reduction in
cholesterol or fatty acid synthesis was due to a toxic
effect of the test compound. An initial assay, which
measured the incorporation of 14C label from [1,5-14C-]
citrate, gave false positives due to the ability of com-
pounds such as 58 to inhibit uptake of 14C-citrate into
the cell. The results of compound screening are shown
in Table 7. Hydroxycitrate, despite its low Ki (0.15 µΜ)
on the isolated enzyme, is a relatively poor inhibitor of
cholesterol synthesis in the HepG2 cell, IC50 ) 0.25 mM.
Unfortunately, none of our inhibitors were able to
reduce cholesterol or fatty acid synthesis after a 2.5 h
exposure. The reduction in cholesterol (86%) and fatty
acid (84%) synthesis for 57, at 1 mM, was associated
with a significant reduction (16%) of cellular ATP levels
and is likely to be due to a toxic effect.
We considered that the poor efficacy of the inhibitors
in HepG2 cells was likely to be a consequence of
ineffective or limited cell penetration. The compounds
are dicarboxylic acids which will be ionized at neutral
pH and consequently are likely to have difficulty in
passing through cellular membranes. In an attempt to
overcome this, we prepared and evaluated the diethyl
(111) and diacetoxymethyl (112) esters of 58 and the
dimethyl ester (113) of 68 as potential prodrugs of the
diacid inhibitors (Table 7). However, none of these
derivatives showed any significant inhibition of choles-
terol and/or fatty acid synthesis at the concentrations
tested without also having a toxic effect on the cells.
F igu r e 2. (a) Lineweaver-Burke plot demonstrating largely
competitive kinetics of compound 58 versus citrate. (b) Lin-
eweaver-Burke plot demonstrating noncompetitive kinetics
of compound 58 versus CoA.
profile demonstrated by the MEDICA type of compound,
discussed earlier, which we used as our starting point
in this work.
The enzyme kinetic analysis of compounds such as
58 and 74 is consistent with them binding to the citrate
site through the butanedioic acid moiety. Additional
affinity appears to be imparted by their ability to
interact with an adjacently located hydrophobic domain,
rather that through an interaction with the CoA binding
site. Although our initial design strategy made use of
the hypothesis that there may be some similarity
between the CoA binding domains of HMG-CoA reduc-
tase and ACL, the SAR requirements of the lipophilic
group in the HMG-CoA reductase inhibitors and our
ACL inhibitors show marked differences. For example,
the 2,4-dimethylphenyl compound 13c has similar
HMG-CoA reductase inhibitory activity to its 2,4-
dichloro analogue 13a , whereas there is ca. 2 orders of
magnitude difference in ACL inhibitory activity between
compounds 106 and 58. An optimal group for HMG-
CoA reductase inhibitors is 2,4-dichloro-6-phenyl. The
same substituent is not particularly well accomodated
in ACL inhibitors. In fact two ortho substituents in the
phenyl ring are detrimental to activity.
The divergance of lipophilic group SAR between the
two enzymes is not surprising. The kinetic analysis of
the ACL inhibitors strongly suggests that the aryl group
is not binding to the CoA domain, but to some alterna-
tive hydrophobic site on the enzyme. Thus, although
we started with the concept of designing inhibitors to
mimic the bisubstrate intermediate citryl CoA, we have
identified inhibitors which do not now fall into that
descriptive class. The kinetic profile of our inhibitors
Su m m a r y a n d Con clu sion s
The strategy used for the design and identification of
a number of reasonably potent inhibitors of ATP-citrate
lyase has been described. Inhibition of this enzyme in
vivo is anticipated to result in an agent which will have
combined hypocholesterolemic and hypotriglyceridemic
properties. Inhibitor design was based upon the concept
of linking a lipophilic group to a “citrate-like” head. The
best compounds are butanedioic acid derivatives sub-
stituted in the 2 position with an appropriate length (8
atom) spacer group to a 2,4-dichlorophenyl group.
These have reversible Ki
’s in the 1-3 µΜ range.
However, in a cell based assay (HepG2) none of these
inhibitors showed efficacy in reducing cholesterol or
fatty acid synthesis. This has been attributed to the
adverse physicochemical properties of the molecules
leading to a lack of cell penetration. A number of
potential ester prodrugs similarly proved either nonef-
ficacious or toxic to the cells. Despite this, a baseline
for the design of more potent, cell penetrant ACL
inhibitors has been established. Once achieved this
should allow the hypothesis to be challenged that

ATP-Citrate Lyase as a Target for Hypolipidemic Intervention
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18 3579
Ta ble 7. Compounds Assayed in HepG2 Cells
cholesterol
synth % control
fatty acid
synth % control
ATP levels
% control
no.
3
concn (µM)
analyses
C, Ha
100
300
1000
1000
20
1000
10
1000
10
110 ( 19
40 ( 6
15 ( 9
116 ( 10
104 ( 8
14 ( 2
108 ( 4
102 ( 7
n/d
n/d
3 ( 1
98 ( 4
13 ( 6
78 ( 6
n/d
87 ( 8
79 ( 11
57 ( 4
107 ( 15
96 ( 6
16 ( 3
98 ( 18
102 ( 5
n/d
n/d
6 ( 0
100 ( 8
16 ( 7
105 ( 12
n/d
100 ( 3
103 ( 3
104 ( 5
100 ( 2
102 ( 3
84 ( 3
102(3
107(3
91 ( 3
86 ( 2
81 ( 1
111 ( 6
60 ( 7
96 ( 3
91 ( 2
58
57
see Table 1
see Table 1
74
see Table 3
C, H, Cl, S
111
30
100
100
1000
10
112
113
C, H
C, H
30
a
Microanalysis performed on the γ-lactone form; lactone hydrolyzed to 3 prior to use.
was fractionated on silica gel (20-30% ether/petroleum ether
40-60 °C) to give the title compound (6.37 g, 80%) as an oil,
comprising a mixture of E and Z isomers.
inhibition of ACL in an appropriate animal model in
vivo will result in a decrease in both plasma cholesterol
and plasma triglyceride levels.
Sep a r a tion of th e (E) a n d (Z) Isom er s of Meth yl 6-(2,4-
Dich lor op h en yl)-5-h exen oa te. The product from a repeat
preparation of 36 (X ) CH, n ) 6, subs ) 2,4-diCl) using 10.0g
(57.2 mmol) of 2,4-dichlorobenzaldehyde and 25.4g (57.2 mmol)
of (4-carboxybutyl)triphenylphosphonium bromide gave a solid
which was fractionated on silica gel (20-30% ether/petroleum
ether 40-60 °C), to give the mixture of E and Z isomers.
Further chromatography on a J obin Yvon Chromatospac Prep
HPLC system, using an ether/petroleum ether 40-60 °C
gradient, gave the Z isomer of the product (3.72 g, 24%) and
the E isomer (7.33 g, 47%), contaminated with ca. 10% of the
Z isomer.
Our subsequent approaches to the modification of
these inhibitors leading to the successful identification
of compounds which demonstrate hypolipidemic activity
in vivo and which validate the hypothesis cited in this
communication will be reported in due course.
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 measured in the Analytical Science
Department of SmithKline Beecham, The Frythe, and are
within 0.4% of theoretical values unless otherwise indicated.
1H NMR spectra were determined on Brucker AM250 or AM
360 spectrometers using tetramethylsilane (TMS) as the
internal standard. IR spectra were recorded on a Perkin-
Elmer Model 298 instrument. Mass spectra were recorded on
a VG-70-250-SEQ instrument. All structural assignments
were consistent with NMR, IR, and mass spectra.
(ii) 6-(2,4-Dich lor op h en yl)-5-h exen -1-ol (38, X ) CH, n
) 6, su bs ) 2,4-d iCl). Di-isobutylaluminium hydride (1.0 M
in dichloromethane, 51.3 mL, 51.3 mmol) was injected into a
stirred solution of methyl 6-(2,4-dichlorophenyl)-5-hexenoate
(6.37 g, 23.3 mmol) in dichloromethane (40 mL) at -78 °C
under argon. After 5 min, the solution was warmed to 0 °C,
stirred for 0.5 h, then cooled again to -78 °C. Water (17 mL)
was injected slowly, while allowing the mixture to warm to
room temperature. When solid had precipitated, ethyl acetate
(50 mL) was added and then excess NaHCO3, and the mixture
was stirred vigorously for 15 min. The solids were filtered off
through hyflo, the solvent was removed under reduced pres-
sure, and the residue was purified by chromatography on silica
gel (30-70% ether/petroleum ether 40-60 °C) to give the title
compound (4.85 g, 85%) as an oil, comprising a mixture of E
and Z isomers.
(-)-Hyd r oxycitr a te (3). Garcinia lactone,10a isolated from
the fruit rind of Garcinia cambogia,30 was dissolved in water
and 3.3 equiv of aqueous sodium hydroxide added. After 10
min, aqueous HCl solution was carefully added to pH 7 and
the solution made up to the desired standard concentration.
Typ ica l P r oced u r e for th e Syn th esis of 2-[(ω-a r yla -
lkylth io)m eth yl]-2-h ydr oxybu tan e-1,4-dioic Acids (Tables
1, 2, 5, 6): (()-2-[[[6-(2,4-Dich lor op h en yl)h exyl]t h io]-
m eth yl]-2-h ydr oxybu tan edioic Acid (58). (i) Meth yl 6-(2,4-
Dich lor op h en yl)-5-h exen oa te (36, X ) CH, n ) 6, su bs )
2,4-d iCl). Sodium hydride (60% dispersion in oil, 2.36 g, 58.9
mmol) was washed with petroleum ether 40-60 °C and then
heated to 80 °C in DMSO (30 mL) under argon until gas
evolution ceased. The solution was cooled in an ice bath, and
a solution of (4-carboxybutyl)triphenylphosphonium bromide
(12.7 g, 28.6 mmol) in DMSO (60 mL) was added. The solution
was stirred for 30 min at room temperature and then cooled
in an ice bath. A solution of 2,4-dichlorobenzaldehyde (5.00
g, 28.6 mmol) in DMSO (10 mL) was added, and the mixture
was stirred at room temperature for 1 h and then poured into
aqueous HCl and extracted with ether. The extracts were
washed with water, saturated aqueous NaCl, and dried
(MgSO4), and then the solvent was removed under reduced
pressure.
Diazomethane was generated from Diazald (12.3 g, 57.2
mmol), carbitol (10 mL), and 60% aqueous KOH (25 mL) in
ether (70 mL) and passed in a stream of ether-saturated
nitrogen through a solution of the crude acid in 10% methanol/
ether (40 mL). When all the acid had reacted (TLC), excess
diazomethane was destroyed with acetic acid, and then the
solution was poured into aqueous NaHCO3 and extracted with
ether. The extracts were washed with water and saturated
aqueous NaCl and dried (MgSO4), and then the solvent was
removed under reduced pressure. The residual yellow solid
(iii) 6-(2,4-Dich lor op h en yl)-1-h exa n ol (39, X ) CH, n
) 6, su bs ) 2,4-d iCl). A solution of the mixture of E and Z
isomers of 6-(2,4-dichlorophenyl)-5-hexen-1-ol (4.85 g, 19.8
mmol) in methanol (40 mL) was shaken under hydrogen (50
psi) with platinum oxide (253 mg, 1.11 mmol, added in
portions) until no starting material could be detected by NMR
spectroscopy. The catalyst was filtered off through hyflo, and
the solvent was removed under reduced pressure. The residue
was dissolved in ether, and the solution was filtered through
a pad of silica gel. The solvent was again removed under
reduced pressure to give the title compound (4.70 g, 96%) as
an oil.
(iv) 1-Br om o-6-(2,4-d ich lor op h en yl)h exa n e (43, n ) 6,
su bs ) 2,4-d iCl). N-Bromosuccinimide (4.27 g, 24.0 mmol)
was added in portions to a stirred solution of 6-(2,4-dichlo-
rophenyl)-1-hexanol (5.38 g, 21.8 mmol) and triphenylphos-
phine (6.29 g, 24.0 mmol) in dichloromethane (60 mL) at 0
°C, and the resulting solution was stirred for 5min at 0 °C
and 20 min at room temperature. The solvent was removed
under reduced pressure, and the residue was triturated with
10% ether/petroleum ether 40-60 °C. The extracts were
filtered through a pad of silica gel. The solvent was removed
from the filtrate under reduced pressure to give the title
compound (6.61 g, 98%) as an oil.

3580 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18
Gribble et al.
(v) S-[6-(2,4-Dich lor op h en yl)h exyl]th ioa ceta te (40, X
) CH, n ) 6, su bs ) 2,4-d iCl). Potassium thioacetate (1.47
g, 12.9 mmol), dried at 60 °C under high vacuum for 1 h, was
added to a stirred solution of 1-bromo-6-(2,4-dichlorophenyl)-
hexane (2.00 g, 6.45 mmol) in dry DMF (20 mL) containing
powdered 4 Å molecular sieves. The mixture was stirred for
2 h under argon and then poured into cold 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 was
chromatographed on silica gel (5-15% ether/petroleum ether
40-60 °C) to give the title compound (1.95 g, 99%) as an oil.
(vi) Meth yl 4-[[6-(2,4-Dich lor op h en yl)h exyl]th io]-3-ox-
obu ta n oa te (17, R ) 2,4-d iClC6H3(CH2)6). Sodium meth-
oxide in methanol (7.40 mL of a 0.470 M solution, 3.48 mmol)
was injected into a stirred solution of S-[6-(2,4-dichlorophenyl)-
hexyl]thioacetate (1.01 g, 3.31 mmol) in methanol (10 mL)
under argon. After 10 min, methyl 4-chloroacetoacetate (344
µL, 2.98 mmol) was injected. The mixture was stirred at room
temperature for 18 h and 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 was
fractionated on silica gel (10-40% ether/petroleum ether 40-
60 °C) to give the title compound (1.14 g, 91%) as an oil.
(vii) (()-2-[[[6-(2,4-Dich lor op h en yl)h exyl]th io]m eth yl]-
2-h yd r oxybu ta n ed ioic Acid (58). A solution of KH2PO4
(9.99 g, 73.4 mmol) in water (60 mL) was injected over 10 min
into a stirred mixture of KCN (4.78 g, 73.4 mmol), methyl 4-[[6-
(2,4-dichlorophenyl)hexyl]thio]-3-oxobutanoate (2.77 g, 7.34
mmol), and ether (25 mL) at 0 °C initially. The flask was
connected to a chloros/NaOH trap system to remove any HCN
evolved. The mixture was stirred vigorously for 18 h and then
the pH adjusted to 4 with concentrated aqueous HCl. The
ether layer was removed by pipette and the aqueous layer
extracted with ether twice more. The solvent was removed
from the extracts under reduced pressure and the residual oil
heated at reflux in 25% aqueous HCl for 3.5 h. After cooling,
the mixture was extracted with ether. The extracts were
washed with 1 M aqueous NaOH twice. The aqueous phase
was washed with ether and then reacidified with 5 M aqueous
HCl. The mixture was extracted with ether once again, and
the extracts were washed with water and saturated aqueous
NaCl and dried (MgSO4). The solvent was removed under
reduced pressure and the crude product recrystallized (ether/
petroleum ether 40-60 °C) to give the title compound (2.28 g,
76%) as a white solid, mp 88-89 °C. 1H NMR (DMSO, 250
MHz) δ 7.55 (m, 1H, Ar), 7.36 (m, 2H, Ar), 5.10 (br s, 1H, OH),
2.81-2.50 (m, 8H, ArCH2, SCH2CH2, SCH2C(OH), CH2CO2H),
1.60-1.20 (m, 8H, SCH2(CH2)4). Anal. C17H22Cl2O5S (C, H).
Ald eh yd es. Most of the aldehydes required for the syn-
theses of the compounds in Tables 1-3, 5, and 6 were
commercially available. Otherwise they were readily prepared
by Swern oxidation of the corresponding benzyl or pyridylm-
ethyl alcohols. 2-Phenyl-4,6-dichlorobenzaldehyde was syn-
thesized by the published procedure.23c
mmol) and triphenylphosphine (11.97 g, 45.6 mmol) in toluene
(15 mL) was heated under reflux for 48 h. On cooling, a gum
separated and the toluene was decanted off. This was washed
several times with toluene and the solvent decanted off. The
residue was triturated under ether to give 37 (n ) 9) (9.03 g,
19.1 mmol, 84%).
9-(2,4-Dich lor op h en yl)-8-n on en -1-ol (38, X ) CH, n )
9, su bs ) 2,4-d iCl). To a mixture of (37, n ) 9) (9 g, 19.1
mmol) in dry THF (135 mL), cooled to -78 °C, under argon,
was added 2.5 M nBuLi in hexanes (15.25 mL, 38.2 mmol),
and the mixture allowed to warm to 0 °C. The resulting red
solution was stirred at 0 °C for 30 min and recooled to -78
°C. 2,4-Dichlorobenzaldehyde (3.84 g, 22 mmol) in THF (90
mL) was then added dropwise and the mixture then allowed
to warm to room temperature. After a further 15 min, the
solution was poured into 1 M aqueous HCl and extracted with
ether (3 × 150 mL). The combined extracts were washed with
water (2x) and brine (2x), dried over MgSO4
, and concentrated
in vacuo. The residue was chromatographed (silica gel, 50%
ether/petroleum ether 40-60 °C) to give 38 (X ) CH, n ) 9,
subs ) 2,4-diCl) (2.21 g, 7.7 mmol, 40%) as a colorless oil.
Syn th eses of 76 a n d 77. The separated E and Z isomers
of 36 (X ) CH, n ) 6, subs ) 2,4-diCl) were reduced to their
respective alcohols with DIBAL-H and converted to 76 and
77 by the general procedures described above. 76: 1H NMR
(CDCl3, 250 MHz) δ 7.39 (m, 1H, Ar), 7.19 (m, 2H, Ar), 6.43
(d, 1H, J ) 11.5 Hz, ArCH), 5.77 (dt, 1H, J ) 11.5, 7.5 Hz,
ArCHdCH), 3.07-2.75 (m, 4H, SCH2C(OH), CH2CO2H), 2.60
(m, 2H, SCH2CH2), 2.17 (m, 2H, CHdCHCH2), 1.65-1.40 (m,
4H, SCH2(CH2)2). Anal. C17H20Cl2O5S (C, H). 77: 1H NMR
(CDCl3, 200 MHz) δ 7.41 (d, 1H, J ) 10.6 Hz, Ar), 7.34 (d, 1H,
J ) 2.7 Hz, Ar), 7.17 (dd, 1H, J ) 10.6, 2.7 Hz, Ar), 6.67 (d,
1H, J ) 15.4 Hz, ArCH), 6.16 (dt, 1H, J ) 15.4, 7.3 Hz,
ArCHdCH), 3.10-2.80 (m, 4H, SCH2C(OH), CH2CO2H), 2.67
(m, 2H, SCH2CH2), 2.25 (m, 2H, CHdCHCH2), 1.70-1.45 (m,
4H, SCH2(CH2)2). Anal. C17H20Cl2O5S (C, H).
(()-Dim et h yl 2-[[[6-(2,4-Dich lor op h en yl)h exyl]t h io]-
m eth yl]bu ta n ed ioa te (113). To a stirred solution of S-[6-
(2,4-dichlorophenyl)hexyl]thioacetate (40, X ) CH, n ) 6, subs
) 2,4-diCl) (3 g, 9.83 mmol) in MeOH (5 mL), under an argon
atmosphere, was added a 0.2 M solution of sodium methoxide
in MeOH (51.6 mL, 10.32 mmol). This was stirred at room
temperature for 1 h and then a solution of dimethyl itaconate
(1.56 g, 9.83 mmol) in MeOH (3 mL) added. This was stirred
at room temperature for 18 h, diluted with 0.5 M HCl solution,
and extracted with ether (×3). The extracts were washed with
water and brine and dried over MgSO4. The solvent was
removed under reduced pressure and the residue chromato-
graphed on silica gel (30% ether/petroleum ether 40-60 °C)
to give 113 (3.14 g,76%) as an oil. 1H NMR (CDCl3, 250 MHz)
δ 7.34 (m, 1H, Ar), 7.14 (m, 2H, Ar), 3.72 (s, 3H, OMe), 3.69
(s, 3H, OMe), 3.06 (m, 1H, CHCO2Me), 2.88 (dd, 1H, J ) 14.0,
5.9 Hz, SCH2CH(CO2Me) or CH2CO2Me), 2.80-2.60 (m, 5H,
ArCH2, SCH2CH(CO2Me), CH2CO2Me), 2.51 (m, 2H, SCH2-
CH2), 1.65-1.30 (m, 8H, ArCH2(CH2)4). Anal. C19H26Cl2O4S
(C, H).
Alter n a tive Syn th eses of In ter m ed ia te Br om id es (43)
(Ch a r t 3, m eth od B). Bromides 43, where subs ) 2,4-diMe,
4-F, 4-Cl, 4-Br, 4-OMe; n ) 6, were prepared from the
substituted benzene and 6-bromohexanoyl halide, by the
acylation/reduction method of J axa-Chamiec et al.26 This
procedure was modified for the 4-halo derivatives whereby the
intermediate ketone was first isolated and then subsequently
reduced with Et3SiH in TFA.
6-(2-Nitr op h en yl)h exyl Br om id e a n d 6-(4-Nitr op h e-
n yl)h exyl Br om id e (43, n ) 6, subs ) 2 and 4 NO2,
respectively). Nitronium tetrafluoroborate (1.58 g, 12 mmol)
was added to a solution of 6-phenylhexyl bromide26 (4.82 g,
20 mmol) in dry CH2Cl2 (20 mL) at 0 °C and stirred for 2 h.
Water was added, the mixture was extracted with ether, and
the combined extracts were dried over MgSO4. The residue
on evaporation in vacuo was chromatographed (silica, 1:1
pentane/CH2Cl2) to separate unreacted starting material from
the 2-nitro (0.7 g) and 4-nitro (1.03 g) isomers.
(()-2-[[[6-(2,4-Dich lor op h e n yl)h e xyl]t h io]m e t h yl]-
bu ta n ed ioic Acid (68). 113 (0.78 g, 1.84 mmol) was stirred
at reflux in 25% HCl solution for 18 h. This was extracted
with ether (×3), and the extracts were washed with water and
brine and then dried over MgSO4. Concentration in vacuo gave
68 (0.71 g, 98%) as an oil. 1H NMR (CDCl3, 250 MHz) δ 7.34
(d, 1H, J ) 1.8 Hz, Ar), 7.14 (m, 2H, Ar), 3.05-2.50 (m, 9H,
ArCH2, SCH2, CH2CO2H, CHCO2H), 1.65-1.30 (m, 8H,
ArCH2(CH2)4). Anal. C17H22Cl2O4S (C, H).
3-Hyd r oxy-4-[[6-(2,4-d ich lor op h en yl)h exyl]th io]bu tyr -
ic Acid (69). (i) Meth yl 3-Hyd r oxy-4-[[6-(2,4-d ich lor op h e-
n yl)h exyl]th io]bu tyr ate (18, R ) 2,4-diClC6H3(CH2)6). NaBH4
(64 mg, 1.7 mmol) was added to a solution of (17, R ) 2,4-
diClC6H3(CH2)6) (0.64 g, 1.7 mmol) and CeCl3‚7H2O (632 mg,
1.7 mmol) in MeOH (10 mL) at 0 °C and then stirred at this
temperature for 1 h. 1 M HCl solution (10 mL) was added
and this extracted with ether (3×). The combined extracts
were washed with water and then brine and dried over MgSO4.
Concentration followed by flash column chromatography (silica,
30-50% ether/petroleum ether 40-60 °C) gave the title
compound (0.54 g, 1.42 mmol, 84%).
Syn th eses of (ω-Hyd r oxya lk yl)tr ip h en ylp h osp h on iu m
Br om id es (37): (8-Hydroxyoctyl)triphenylphosphonium Bro-
mide (37, n ) 9). A solution of 8-bromooctanol (4.77 g, 22.8

ATP-Citrate Lyase as a Target for Hypolipidemic Intervention
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18 3581
(ii) 3-Hyd r oxy-4-[[(2,4-d ich lor op h en yl)h exyl]th io]bu -
tyr ic Acid (69). 18 (R ) 2,4-diClC6H3(CH2)6) (0.4 g, 1.05
mmol) was stirred in a solution of NaOH (84 mg, 2.1 mmol) in
absolute ethanol (1 mL) and water (1 mL) at room temperature
for 30 min. The pH was adjusted to ca. 1 with 1 M HCl and
extracted with CH2Cl2 (3×). The combined extracts were
washed with water and brine and then dried over MgSO4.
Concentration gave the title compound as a clear oil (0.363 g,
0.99 mmol, 94%). 1H NMR (CDCl3, 250 MHz) δ 7.34 (m, 1H,
Ar), 7.14 (m, 2H, Ar), 4.12 (m, 1H, CH(OH)), 2.80-2.50 (m,
8H, CH2CO2H, CH2CH(OH), SCH2CH2, ArCH2), 1.70-1.30 (m,
8H, ArCH2(CH2)4). Anal. C16H22Cl2O3S (C, H, Cl).
Syn th esis of 4-[[6-(2,4-Dich lor op h en yl)h exyl]th io]bu -
tyr ic a cid (70). (i) Meth yl 4-[6-(2,4-Dich lor op h en yl)-
h exyl]th io]bu tyr a te (22, R ) 2,4-d iClC6H3(CH2)6). To a
solution of 40 (X ) CH, n ) 6, subs ) 2,4-diCl) (800 mg, 2.62
mmol) in MeOH (10 mL), under argon, was added a freshly
prepared (from Na and MeOH) 0.2 M solution of NaOMe in
MeOH (14.4 mL, 2.88 mmol). This was stirred at room
temperature for 30 min and then ethyl 4-bromobutyrate (0.413
mL, 2.88 mmol) added and the mixture stirred for a further
18 h. 0.5 M HCl was added and this was then extracted with
ether (3×), and the combined extracts were washed with water
and brine and dried over MgSO4. Flash chromatography (30%
ether/petroleum ether 40-60 °C) gave the title compound (0.83
g, 2.28 mmol, 87%).
solution of oxalyl chloride (9.35 mL, 107 mmol) in dichlo-
romethane (150 mL) at -78 °C under argon. After 5 min, a
solution of 7-(2,4-dichlorophenyl)-1-heptanol (20.0 g, 76.6
mmol) in dichloromethane (100 mL) was added by cannula.
After stirring 0.5h at -78 °C, triethylamine (47 mL, 337 mmol)
was injected. The mixture was stirred 5 min, allowed to warm
to room temperature, and then poured into 1 M aqueous
NaHSO4. The product was extracted with ether. The extracts
were washed with water and saturated aqueous NaCl, and
then the solvent was removed under vacuum. A solution of
the crude aldehyde product in ether (120 mL) was added to a
stirred suspension of hydroxylamine hydrochloride (17.0 g, 245
mmol) in water (10 mL) at 0 °C, followed by aqueous Na2CO3
(2.7 M, 50 mL, 135 mmol). The mixture was stirred at room
temperature for 2.5 h, poured into 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 purified by chromatography
on silica gel (30-50% ether/petroleum ether 40-60 °C) to give
the title compound (17.3 g, 82%) as a mixture of E and Z
isomers.
(iii) (()-5-(Car bom eth oxym eth yl)-3-[6-(2,4-dich lor oph e-
n yl)h exyl]-5-(m eth oxycar bon yl)-4,5-dih ydr oisoxazole (31,
n ) 6). Aqueous NaOCl (2.0 M, 340 mL, 680 mmol) and
triethylamine (2.5 mL, 18.0 mmol) were added in 4 portions
separately over 40 h to a stirred solution of 7-(2,4-dichlorophe-
nyl)heptanaldoxime (17.3 g, 63.1 mmol) and dimethyl itaconate
(23.0 g, 145 mmol) in dichloromethane (200 mL). The mixture
was stirred vigorously at room temperature over this period
and then poured into water and extracted with ether. The
extracts were washed with water and saturated aqueous NaCl
(ii) 4-[[6-(2,4-Dich lor op h en yl)h exyl]th io]bu tyr ic Acid
(70). Hydrolysis of 22 (R ) 2,4-diClC6H3(CH2)6) with NaOH
in EtOH/H2O, as described for 69, gave 70 as a white solid
(0.72 g, 2.06 mmol, 95%), mp 48-49 °C. 1H NMR (CDCl3, 250
MHz) δ 7.34 (d, 1H, J ) 1.7 Hz, Ar), 7.14 (m, 2H, Ar), 2.71-
2.47 (m, 8H, ArCH2, SCH2, CH2CO2H), 1.91 (m, 2H, CH2CH2-
CO2H), 1.65-1.30 (m, 8H, ArCH2(CH2)4). Anal. C16H22Cl2O2S
(C, H, Cl).
and dried (MgSO4
). The solvent was removed under vacuum
and the residue purified by chromatography on silica gel (30-
60% ether/petroleum ether 40-60 °C) to give the title com-
pound (19.5 g, 72%) as an oil.
Syn th eses of Com p ou n d s 71 a n d 72 (Ch a r t 2): (()-3-
Car boxy-10-(2,4-dich lor oph en yl)-3-h ydr oxydecan oic Acid
(72). (i) Meth yl 10-(2,4-Dich lor op h en yl)-3-oxod eca n oa te
(28, n ) 6). Methyl acetoacetate (696 µL, 6.45 mmol) was
injected into a stirred slurry of NaH (284 mg of a 60% oil
dispersion, 7.10 mmol), washed free of oil with petroleum ether
40-60 °C, in THF (8 mL) at 0 °C under argon. The mixture
was stirred at room temperature for 30 min and cooled back
to 0 °C, and n-butyllithium (2.84 mL, 2.5 M in hexanes, 7.10
mmol) was injected dropwise. The resulting solution was
stirred at room temperature for 15 min and again cooled to 0
°C. A solution of 1-bromo-6-(2,4-dichlorophenyl)hexane (43,
n ) 6, subs ) 2,4-diCl) (2.00 g, 6.45 mmol) in THF (5 mL) was
added by cannula, and the mixture was stirred for 2 h at room
temperature and then poured into cold aqueous HCl and
extracted with ether. The extracts were washed with water,
saturated aqueous NaCl, and dried (MgSO4). The solvent was
removed under reduced pressure and the residue chromato-
graphed on silica gel (10-40% ether/petroleum ether 40-60
°C) to give the title compound (1.20 g, 54%) as an oil.
(ii) (()-3-Ca r boxy-10-(2,4-d ich lor op h en yl)-3-h yd r oxy-
d eca n oic Acid (72). The procedure described for 58 was
carried out, using methyl 10-(2,4-dichlorophenyl)-3-oxode-
canoate (28, n ) 6) in place of methyl 4-[[6-(2,4-dichlorophe-
nyl)hexyl]thio]-3-oxobutanoate to give the title compound
(84%) as a white solid, mp 92-94 °C (ether/petroleum ether
40-60 °C). 1H NMR (DMSO, 250 MHz) δ 12.4 (br s, 2H,
CO2H), 7.54 (m, 1H, Ar), 7.35 (m, 2H, Ar), 4.77 (br s, 1H, OH),
2.71 (d, 1H, J ) 15.4 Hz, CH2CO2H), 2.65 (m, 2H, ArCH2),
2.44 (d, 1H, J ) 15.4 Hz, CH2CO2H), 1.66-1.05 (m, 12H,
ArCH2(CH2)6). Anal. C17H22Cl2O5 (C, H).
(iv) (()-Met h yl 11-(2,4-Dich lor op h en yl)-3-h yd r oxy-3-
(m eth oxyca r bon yl)-5-oxo-u n d eca n oa te (32, n ) 6).
A
solution of (()-5-(carbomethoxymethyl)-3-[6-(2,4-dichlorophe-
nyl)hexyl]-5-(methoxycarbonyl)-4,5-dihydroisoxazole (19.5 g,
45.3 mmol) and boric acid (8.39 g, 136 mmol) in methanol was
shaken with Raney nickel (50% slurry in water, 8 g) under
hydrogen (50 psi) at room temperature for 2 h. The catalyst
was removed by filtration, and most of the solvent was
removed under vacuum. The mixture was diluted with water
and extracted with ethyl acetate. 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 (50-80% ether/
petroleum ether 40-60 °C) to give the title compound (16.7 g,
85%) as an oil.
(v) (()-11-(2,4-Dich lor op h en yl)-3-h yd r oxy-3-ca r boxy-
5-oxou n d eca n oic a cid (74). A solution of NaOH (602 mg,
15.1 mmol) in water (22 mL) was added slowly to a stirred
solution of diester (32, n ) 6) (1.62 g, 3.74 mmol) in ethanol
(23 mL) at 0 °C. The solution was stirred 5 min at 0 °C and
then 1 h at room temperature. After recooling to 0 °C, the
pH was adjusted to 2 with 1 M aqueous HCl, 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
recrystallized (ether/petroleum ether 40-60 °C) to give the title
compound (1.05 g, 69%) as a white solid, mp 91-93 °C. 1H
NMR (CDCl3, 250 MHz) δ 7.33 (m, 1H, Ar), 7.13 (m, 2H, Ar),
3.08-2.78 (m, 4H, COCH2C(OH), CH2CO2H), 2.65 (m, 2H,
ArCH2), 2.44 (m, 2H, COCH2CH2), 1.60-1.20 (m, 8H,
ArCH2(CH2)4). Anal. C18H22Cl2O6 (C, H).
Compound 71 was synthesized by an analogous procedure
from the higher homologue of 43 (n ) 7). 1H NMR (CDCl3,
250 MHz) δ 7.33 (m, 1H, Ar), 7.13 (m, 2H, Ar), 3.02 (d, 1H, J
) 17.1 Hz, CH2CO2H), 2.77 (d, 1H, J ) 17.1 Hz, CH2CO2H),
2.66 (m, 2H, ArCH2), 1.80-1.20 (m, 14H, ArCH2(CH2)7). Anal.
C18H24Cl2O5 (C, H).
Syn th esis of 74 a n d 75 (Ch a r t 2). (i) 7-(2,4-Dich lo-
r op h en yl)-1-h ep ta n ol (26, n ) 6). This was prepared by an
analogous procedure to that described above for the lower
homologue (39, X ) CH, n ) 6, subs ) 2,4-diCl).
Syn th esis of (()-3-Ca r boxy-5-[5-(2,4-d ich lor op h en yl)-
p en t yl]t h io]-3-h yd r oxyp en t a n oic Acid (73). S-[5-(2,4-
Dichlorophenyl)pentyl] thioacetate (40, X ) CH, n ) 5, subs
) 2,4-diCl) was prepared by the method described for (40, X
) CH, n ) 6, subs ) 2,4-diCl) above. To this in MeOH (5 mL)
was injected a solution of NaOMe in MeOH (0.47M, 7.65 mL,
3.60 mmol) at room temperature. After 5 min, a solution of
methyl 3-oxo-4-pentenoate31 (440 mg, 3.43 mmol) in methanol
(5 mL) was added by cannula and stirring continued for 2 h.
The mixture was partitioned between aqueous HCl and ether.
The ether extracts were washed with water and saturated
(ii) 7-(2,4-Dich lor op h en yl)h ep ta n a ld oxim e (30, n ) 6).
DMSO (15.2 mL, 214 mmol) was added slowly to a stirred

3582 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18
Gribble et al.
aqueous NaCl and dried (MgSO4). The solvent was removed
under reduced pressure and the residue fractionated on silica
gel (20-50% ether/petroleum ether 40-60 °C) to give methyl
5-[[5-(2,4-dichlorophenyl)pentyl]thio]-3-oxopentanoate (24, R
) 2,4-diClC6H3(CH2)5) (1.10 g, 85%) as a colorless oil. The
latter compound was converted to 73 by the general procedures
described above. 1H NMR (CDCl3, 250 MHz) δ 7.34 (d, 1H, J
) 1.8 Hz, Ar), 7.14 (m, 2H, Ar), 3.03 (d, 1H, J ) 16.9 Hz, CH2-
CO2H), 2.79 (d, 1H, J ) 16.9 Hz, CH2CO2H), 2.71-2.43 (m,
6H, ArCH2, SCH2), 2.11-1.95 (m, 2H, CH2CH2C(OH)), 1.67-
1.37 (m, 6H, ArCH2(CH2)3). Anal. C17H22Cl2O5S (C, H).
Syn th eses of In ter m ed ia tes for th e P r ep a r a tion of
Com p ou n d s of Ta ble 4 (Ch a r t 4). S-[5-(2,4-Dich lor op h e-
n oxy)p en tyl] th ioa ceta te (47a , n ) 5). Potassium carbonate
(5.00 g, 36.2 mmol) and potassium iodide (2-3 crystals) were
added to a solution of 2,4-dichlorophenol (5 g, 31 mmol) and
1,5-dibromopentane (21.39 g, 93 mmol) in acetone (50 mL),
and the mixture was heated at reflux for 1 h and then cooled.
The solids were filtered off, the solvent was removed under
reduced pressure, and the residue was partitioned between
EtOAc and water. The aqueous layer was washed further with
EtOAc. The combined organic extracts were washed with
NaHCO3 solution, water, and brine and dried over MgSO4.
Concentration in vacuo, followed by flash chromatography
(silica, 0-10% ether/petroleum ether 40-60 °C gradient) gave
5-(2,4-dichlorophenoxy)pentyl bromide (45a , n ) 5) (8.5 g, 27.2
mmol, 88%). The bromide was converted to the corresponding
title thioacetate by the standard procedure described above.
S-[5-[(2,4-Dich lor oph en yl)th io]pen tyl] th ioacetate (47b,
n ) 5). Potassium carbonate (5.00 g, 36.2 mmol) and potas-
sium iodide (46 mg, 0.28 mmol) were added to a solution of
2,4-dichlorobenzenethiol (4.96 g, 27.7 mmol) and 1,5-dibro-
mopentane (19.3 g, 84.3 mmol) in acetone (50 mL), and the
mixture was heated at reflux for 1 h and then cooled. The
solids were filtered off, and solvent was removed from the
filtrate under reduced pressure. The residue was fractionated
on silica gel (0-5% ether/petroleum ether 40-60 °C). The solid
product was triturated with petroleum ether 40-60 °C to
remove traces of 1,5-dibromopentane leaving 5-bromopentyl
(2,4-dichlorophenyl sulfide (45b, n ) 5) (2.58 g, 28%) as a solid,
mp 59-62 °C. The bromide was converted to the correspond-
ing title thioacetate by the standard procedure described
above.
at 0 °C under argon. The solution was heated at reflux for 3
h and cooled, and 1 M aqueous HCl (8.5 mL) was added. The
resulting mixture was heated while allowing the THF to distill
off. The residual aqueous phase was then cooled, diluted with
water, and extracted with ether. The extracts were washed
with water and dried (MgSO4), and the solvent was removed
under reduced pressure to leave crude 1-bromo-5-(2,4-dichlo-
roanilino)pentane (1.11 g), sufficiently pure for the next step.
This bromide was converted to the corresponding title thio-
acetate by the standard procedure described above.
S-[[4-(2,4-Dich lor oben zen esu lfon yl)a m in o]bu tyl] th io-
a ceta te (50, n ) 4). To a solution of 2,4-dichlorobenzene-
sulfonyl chloride (4 g, 16.3 mmol) in pyridine (14 mL) at 0 °C
was slowly added a solution of 4-aminobutan-1-ol (1.82 g, 20.36
mmol) in pyridine (6 mL). The solution turned orange-red in
color and was stirred at room temperature for 36 h. Water
was added, the pH adjusted to 7 with aqueous HCl, and this
extracted with ether (3×). The combined ether extracts were
washed with 1 M HCl (2×), water, and brine and dried over
MgSO4. The residue on evaporation was flash chromato-
graphed (silica, 70-100% ether/petroleum ether 40-60 °C) to
give 4-[(2,4-dichlorobenzenesulfonyl)amino]butan-1-ol (49, n )
4) (1.29 g, 4.33 mmol, 27%). This alcohol was converted to
the corresponding thioacetate by the general procedures
described above.
Syn th esis of (()-2-[[[6-(2,4-d ich lor op h en yl)-6-oxoh exy-
l]th io]m eth yl]-2-h yd r oxybu ta n ed ioic Acid (82). (i) (()-
(E)-Dim eth yl 2-[[[6-(2,4-Dich lor op h en yl)h ex-5-en yl]th io]-
m eth yl]-2-h ydr oxybu tan edioate (51, n ) 5). Diazomethane,
generated from diazald (5.36 g, 25.0 mmol) and 60% KOH (24
mL) in ether/carbitol (1:1, 48 mL), was bubbled in an ether-
saturated nitrogen stream into a solution of 77 (2.55 g, 6.26
mmol) in 10% methanol/ether (30 mL) at room temperature.
When the reaction solution turned yellow, the flow was stopped
and the excess reagent quenched by the addition of acetic acid.
The solvent was removed under reduced pressure and the
residue fractionated on silica gel (40-60% ether/petroleum
ether 40-60 °C) to give the title compound (2.36 g, 87%) as
an oil.
(ii) (()-Dim eth yl 2-[[[6-(2,4-dich lor oph en yl)-6-h ydr oxy-
h exyl]th io]m eth yl]-2-h yd r oxybu ta n ed ioa te (52, n ) 5).
A solution of borane in THF (1 M, 3.37 mL, 3.37 mmol) was
injected in four portions over 4 h into a stirred solution of 51
(n ) 5) (1.00 g, 2.30 mmol) in THF (2 mL) at 0 °C under argon.
Thirty minutes after the final addition, water (3 mL) was
added and the mixture stirred for five minutes. Sodium
perborate (708 mg, 4.60 mmol) was added and stirring
continued for 1.5 h. The 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 reduced pressure and the residue fraction-
ated on silica gel (50-80% ether/petroleum ether 40-60 °C)
to give the title compound (459 mg, 44%, colorless oil) as a
mixture of diastereomers.
S-[5-(2,4-Dich lor oben zen esu lfin yl)p en tyl] th ioa ceta te
(47c, n ) 5). To a solution of 5-bromopentyl 2,4-dichlorophe-
nyl sulfide (45b, n ) 5) (1.38 g, 4.2 mmol) in CH2Cl2 (50 mL),
at -78 °C, was added 55% m-chloroperbenzoic acid (1.32 g,
4.2 mmol), and the solution was stirred for a further 1 h at
this temperature and then left in the body of a freezer for 18
h. An aqueous solution of Na2SO3/NaHCO3 was added, the
organic layer separated, and the aqueous solution extracted
further with CH2Cl2. The combined organic extracts were
washed with water and brine, dried (MgSO4), and flash
chromatographed (silica, 80% ether/petroleum ether 40-60 °C)
to give 5-(2,4-dichlorobenzenesulfinyl)pentyl bromide (46, n )
5, m ) 1) (1.05 g, 3.05 mmol, 72%). The bromide was converted
to the corresponding title thioacetate by the standard proce-
dure described above.
(iii) (()-2-[[[6-(2,4-Dich lor op h en yl)-6-oxoh exyl]t h io]-
m eth yl]-2-h yd r oxybu ta n ed ioic Acid (82). DMSO (0.161
mL, 2.26 mmol) was injected dropwise into a stirred solution
of oxalyl chloride (0.099 mL, 1.13 mmol) in CH2Cl2 (3 mL) at
-78 °C under argon. After 5 min, a solution of 52 (n ) 5)
(427 mg, 0.942 mmol) in CH2Cl2 (2 mL) was added by cannula
over 5 min and the mixture stirred 0.5 h. Triethylamine (0.495
mL, 3.55 mmol) was injected. The mixture was stirred while
warming to room temperature and then poured into 1 M
aqueous NaHSO4 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 fractionated on silica gel (40-70% ether/
petroleum ether 40-60 °C) to give 53 (n ) 5) (326 mg, 77%)
as a colorless oil.. This (311 mg, 0.689 mmol) was hydrolyzed
using NaOH in aqueous MeOH, as described above, to give
82 (291 mg, 100%) as a gummy solid. 1H NMR (CDCl3, 250
MHz) δ 7.43 (m, 2H, Ar), 7.31 (dd, 1H, J ) 8.3 Hz, 1.9 Hz,
Ar), 3.08-2.82 (m, 6H, COCH2, SCH2C(OH), CH2CO2H), 2.64
(m, 2H, SCH2CH2), 1.75-1.35 (m, 6H, SCH2(CH2)3). Anal.
C17H20Cl2O6S (C, H).
S -[[5-(2,4-D i c h lo r o b e n z e n e s u lfo n y l]p e n t y l]t h i o -
a ceta te (47d , n ) 5). This compound was obtained by an
analogous procedure to that used for 47c (n ) 5), but using 2
equiv of MCPBA at 0 °C, followed by stirring at room
temperature for 3 h, for the oxidation step.
S-[5-(2,4-Dich lor oph en yl)am in o]pen tyl] th ioacetate (48,
n ) 5). 5-Bromopentanoyl chloride (6.16 g, 30.9 mmol) was
added slowly to a stirred solution of 2,4-dichloroaniline (5.00
g, 30.9 mmol) and 4-(dimethylamino)pyridine (0.75 g, 6.14
mmol) in pyridine (40 mL) at 0 °C. The mixture was stirred
at room temperature for 18 h and then poured into aqueous
HCl and extracted with ether. The extracts were washed with
aqueous HCl, water, and saturated aqueous NaCl and dried
(MgSO4). The solvent was removed under reduced pressure
and the residue fractionated on silica gel (30-50% ether/
petroleum ether 40-60 °C) to give 5-bromo-N-(2,4-dichlo-
rophenyl)pentanamide (1.28 g, 13%) as an oil. The latter (1.14
g, 3.51 mmol) in THF (45 mL) was added by cannula to a
stirred solution of borane in THF (1 M, 8.51 mL, 8.51 mmol)
(()-Diet h yl
2-[[[6-(2,4-d ich lor op h en yl)h exyl]t h io]-
m eth yl]bu ta n ed ioa te (111). To a solution of 68 (250 mg,

ATP-Citrate Lyase as a Target for Hypolipidemic Intervention
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 18 3583
(2) Mannien V.; Tenkanen, L.; Koskinen, P.; Huttunen, J . K.;
Manntari, M.; Heinonen, O. P.; Frick M. H. J oint Effects of
Serum Triglyceride and LDL Cholesterol and HDL Cholesterol
on Coronary Heart Disease Risk in the Helsinki Heart Study.
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(3) (a) Grundy, S. M. HMG-CoA Reductase Inhibitors for Treatment
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Lee, T. J .; Synthesis, SARs and Therapeutic Potential of HMG-
CoA Reductase Inhibitors. Trends Pharm Sci. 1987, 8, 442-446.
(c) Kathawala, F. G. HMG-CoA Reductase Inhibitors: An
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(4) See for example: Illingworth, D. R. Lipid Lowering Drugs. An
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(5) International Task Force for Prevention of Coronary Heart
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vasc. Dis. 1992, 2, 113-156.
(6) Srere, P. A. The Enzymology of the Formation and Breakdown
of Citrate. Adv. Enzymol. 1975, 43, 57-101.
(7) (a) Houston, B.; Nimmo, H. G. Purification and Some Kinetic
properties of Rat Liver ATP-Citrate Lyase. Biochem. J . 1984,
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and Kinetic Studies of the Citrate Cleavage Enzyme. J . Biol.
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Mechanisms; W. H. Freeman and Co.: San Francisco, 1979; pp
766-768.
(8) Kovanen, P. T.; Bilheimer, D. W.; Goldstein, J . L.; J aramillo, J .
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U.S.A. 1981, 78, 1194-1198.
0.64 mmol) in absolute ethanol (4 mL) was added p-toluene-
sulfonic acid (12 mg, 0.064 mmol) and the solution stirred at
reflux for 18 h. The solvent was removed under reduced
pressure and the residue chromatographed on silica gel (50-
100% ether/petroleum ether 40-60 °C) to give the title
compound (223 mg, 78%). Anal. C21H30Cl2O4S (C, H). 1H
NMR (CDCl3, 200 MHz) δ 7.34 (s, 1H, Ar), 7.14 (m, 2H, Ar),
4.28 (q, 2H, J ) 7.1 Hz, CO2CH2), 4.14 (q, 2H, J ) 7.1 Hz,
CO2CH2), 3.93 (s, 1H, OH), 2.97-2.57 (m, 8H, ArCH2, CH2-
SCH2, CH2CO2), 1.57-1.21 (m, 14H, ArCH2(CH2)4, 2 × CH3).
(()-2-[[[6-(2,4-Dich lor op h en yl)h exyl]t h io]m et h yl]-2-
h yd r oxybu ta n ed ioic Acid , Dia cetoxym eth yl Ester (112).
Bromomethyl acetate (0.62 mL, 6.4 mmol) was added by
syringe to a stirring mixture of 58 (0.26 g, 6.4 mmol),
potassium carbonate (0.18 g, 1.27 mmol), 18-crown-6 (0.05 g),
and powdered 4A sieves in dry DMF (7 mL) under argon and
then stirred at room temperature for 5 days. 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
under reduced pressure. The residual oil was chromato-
graphed on silica gel (40% ether/petroleum ether 40-60 °C)
to yield title compound (0.21 g, 60%). Anal. C23H30Cl2O9S (C,
H). 1H NMR (CDCl3, 250 MHz) δ 7.34 (s, 1H, Ar), 7.18-7.19
(m, 2H, Ar), 5.83-5.79 (m, 2H, CO2CH2OCOCH3), 5.75-5.68
(m, 2H, CH2CO2CH2OCOCH3), 3.75 (s, 1H, OH), 3.01-2.77 (m,
4H, CH2C(OH)(CO2CH2)CH2), 2.70-2.58 (m, 4H, SCH2-
(CH2)4CH2), 2.13, 2.12 (2s, 6H, 2 × OCOCH3), 1.64-1.56 (m,
4H, SCH2CH2(CH2)2CH2), 1.38-1.35 (m, 4H, S(CH2)2(CH2)2).
En zym e Assa ys a n d An a lysis of Kin etic Da ta . Rat ACL
and recombinant human ACL were purified as described
previously.15,32 ACL activity was measured by the maleate
dehydrogenase catalyzed reduction of oxaloacetate by NADH.
Briefly, ACL was added to buffer containing Tris (100 mM),
pH ) 8.0, MgCl2 (10 mM), KCl (10 mM), dithiothreitol (10
mM), ATP (250 mM), NADH (35 mM), and maleate dehydro-
genase. For initial Ki determinations CoA was present at 200
(9) Yamamoto, M.; Fukuda, N.; Triscari, J .; Sullivan, A. C.; Ontko,
J . A. Decreased Hepatic Production of Very Low Density
Lipoproteins Following Activation of Fatty Acid Oxidation by
Ro 22-0654. J . Lipid Res. 1985, 26, 1196-1204.
(10) (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. 1979, 193, 431-448.
(11) (a) Sullivan, A. C.; Singh, M.; Srere, P. A.; Glusker, J . P.
Reactivity and Inhibitor Potential of Hydroxycitrate Isomers
with Citrate Synthetase, 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-
chem. Biophys. 1977, 150, 183-190. (d) Rudney, H.; Sexton, R.
C. Regulation of Cholesterol Biosynthesis. Annu. Rev. Nutr.
1986, 6, 245-272.
µM and citrate was added to 100 µM (Km
start the reaction. Rates were measured over 20 min by
monitoring the decrease in absorbance at 340 nm. To routinely
) 100 µM) to
(citrate)
determine Ki values, data was fitted to the equation v ) Vmax
/
(2 + [I]/Ki) using Grafit 3.0.33 This assumes that compounds
were purely citrate competitive. For substrate competition
experiments data was initially fitted to the general equation
v ) Vmax([S]/Km)/(1 + [S]/Km + [I]/Kei + [S][I]/KmKesi). The
significance of either Kei or Kesi was then determined by curve
fitting in the absence of one of these parameters and applying
an F test to the result again using Grafit 3.0.
(12) Berkhout, T. A.; Havekes, L. M.; Pearce, N. J .; Groot, P. H. E.
The Effect of (-)-Hydroxycitrate on the Activity of the Low-
Density-Lipoprotein Receptor and 3-Hydroxy-3-methylglutaryl-
CoA Reductase Levels in the Human Hepatoma Cell Line Hep
G2. Biochem. J . 1990, 272, 181-186.
Mea su r em en t of Effect of Com p ou n d s on Ch olester ol
a n d F a tty Acid Syn th esis in Hep G2 Cells. HepG2 cells
were cultured in 24-well cell culture plates in DMEM (Dul-
becco’s Modified Eagle’s Medium) containing Hepes (20 mM),
bicarbonate (10 mM), glutamine (2 mM), and fetal calf serum
(10% w/v). Once the cells had grown to between 80% and 90%
confluence, the medium was replaced by DMEM without the
addition of fetal calf serum and the cells incubated overnight.
The cells were then incubated for 2.5 h after addition of either
vehicle or test compound to the final desired concentration.
The rates of cholesterol and fatty acid synthesis were then
(13) (a) Pullinger, C. R.; Gibbens, G. F. Regulation of Cholesterol
Synthesis in the Liver and Mammary Gland of the Lactating
Rat. Biochem. J . 1983, 210, 625-632. (b) Mathisa, M. M.;
Sullivan, A. C.; Hamilton, J . G. Fatty Acid and Cholesterol
Synthesis From Specifically Labeled Leucine by Isolated Rat
Hepatocytes. Lipids 1981, 16, 739-743. (c) Sullivan, A. C.;
Triscari, J .; Hamilton, J . G.; Kritchevsky, D. Hypolipidemic
Activity of (-)-Hydroxycitrate. Lipids 1977, 12, 1-9. (d) Lowen-
stein, J . M.; Brunengraber, H. In Methods in Enzymology;
Lowenstein, J . M., Ed.; Academic Press: New York, 1981; pp
486-497. (e) Sullivan, A. C.; Triscari, J .; Hamilton, J . G.; Miller,
0.; Wheatley, V. R. Effects of (-)-Hydroxycitrate Upon the
Accumulation of Lipid in the Rat. I. Lipogenesis. Lipids 1973,
9, 121-128. (f) Sullivan, A. C.; Triscari, J .; Hamilton, J . G.;
Miller, O. Effects of (-)-Hydroxycitrate Upon the Accumulation
of Lipid in the Rat. II. Appetite. Lipids 1973, 9, 129-134.
(14) Triscari, J .; Sullivan, A. Comparitive Effects of (-)-Hydroxyci-
trate and (+)-Allo Hydroxycitrate on Acetyl-CoA Carboxylase
and Fatty Acid and Cholesterol Synthesis in vivo. Lipids 1977,
12, 357-363.
3
measured by the addition of H2O, to a specific radioactivity
of 71 µCi/ mmol, for the final 90 min of the incubation.
Incubations were terminated and the rates of cholesterol and
fatty acid synthesis determined from the amounts of 3H
incorporated into cellular cholesterol and fatty acids essentially
as described previously.12
Ack n ow led gm en t. We would like to acknowledge
the contributions of our colleagues in the Analytical
Sciences Department for providing microanalytical and
spectral data. We would also like to acknowledge the
help and advice provided by Dr. T. N. C. Wells, Ms.
Helen Saul, and Ms. Katherine Clare.
(15) Wells, T. N. C. ATP-Citrate Lyase From Rat Liver. Characteri-
sation of the Citryl-Enzyme Complexes. Eur. J . Biochem. 1991,
199, 163-186.
(16) (a) Dolle, R. E.; Novelli, R.; Saxty, B. A.; Wells, T. N. C. Wells,
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. (b) 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-Difluorocitrate as
Inhibitors of Rat-Liver ATP-Citrate Lyase and Porcine-Heart
Aconitase. Eur. J . Biochem. 1992, 202, 889-896. (c) Dolle, R.
E.; Gribble, A.; Wilks, T.; Kruse, L. I.; Eggleston, D.; Saxty, B.
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A.; Wells, T. N. C.; Groot, P. H. Synthesis of Novel Thiol-
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1989, 32, 2072-2084. (c) Rose-Khan, G.; Bar-Tana, J . Inhibition
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(24) Beach, R. L.; Aogaichi, T.; Plaut, G. W. E. Identification of
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(20) For an overview of the role and mechanism of HMG-CoA
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zyme A Reductase; CRC Series in Enzyme Biology; Sabine, J .
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(26) J axa-Chamiec, A.; Shah, V. P.; Kruse, L. I. Aromatic
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(27) K. M. Plowman and W. W. Cleland have shown the actual citrate
(22) Although by no means a comprehensive list, see for example
references 23a-c and (a) Roth, B. D.; Ortwine, D. F.; Hoefle, M.
L.; Stratton, C. D.; Sliskovic, D. R.; Wilson, M. W.; Newton, R.
S. Inhibitors of Cholesterol Biosynthesis. 1. trans-6-(2-Pyrrol-l-
ylethyl)-4-hydroxypyran-2-ones, a Novel Series of HMG-CoA
Reductase Inhibitors. 1. Effects of Structural Modifications at
the 2- and 5-Positions of the Pyrrole Nucleus. J . Med. Chem.
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38. (c) Roth, B. D.; Blankley, C. J .;Chucholowski, A. W.;
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Sekerke, C. S.; Sliskovic, D. R.; Stratton, C. D.; Wilson, M. W.
Inhibitors of Cholesterol Biosynthesis. 3. Tetrahydro-4-hydroxy-
6-[2-(lH-pyrrol-l-yl)ethyl]-2H-pyran-2-one Inhibitors of HMG-
CoA Reductase. 2. Effects of Introducing Substituents at Posi-
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chelate [ref 7b]. In the
Mg2+ chelate, the central carboxylate together with the tertiary
alcohol and a terminal carboxylate of citric acid act as metal
ligands to form a tridentate complex. The Mg2+-citrate complex
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(28) These data are only available against the recombinant human
enzyme.
(29) In a related series of compounds, to be reported in a subsequent
paper,
a 2-Ph,4-Cl substitution pattern did indeed give a
significant improvement in activity over 2-Ph alone.
(30) Garcinia cambogia was obtained from J oseph Flach & Sons Ltd.,
140 Falkland Rd., London N8 ONP.
(31) Trost, B. M.; Kunz, R. A. New Synthetic Reactions. A Convenient
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(32) (a) Wraight, C.; Day, A.; Hoogenraad, N.; Scopes, R. A Two-Step
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(33) Leatherbarrow R. J . Grafit Version 3.0, Erithacus Software Ltd,
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J M960167W