Application of a flexible synthesis of (5R)-thiolactomycin to develop new inhibitors of type I fatty acid synthase

Article abstract of DOI:10.1021/jm049389h

Fatty acid synthase (FAS) catalyzes the synthesis of palmitate from the sequential condensation of an acetyl primer with two carbon units added from malonyl-CoA. Inhibition of the β-ketoacyl synthase domain of mammalian FAS leads to selective cytotoxicity

Full text of DOI:10.1021/jm049389h

946
J. Med. Chem. 2005, 48, 946-961
Application of a Flexible Synthesis of (5R)-Thiolactomycin To Develop New
Inhibitors of Type I Fatty Acid Synthase
Jill M. McFadden,† Susan M. Medghalchi,‡ Jagan N. Thupari,§ Michael L. Pinn,§ Aravinda Vadlamudi,‡
Katherine I. Miller,† Francis P. Kuhajda,§ and Craig A. Townsend*,†
Department of Chemistry, Johns Hopkins University, Baltimore, Maryland 21218, FASgen, Inc., Baltimore, Maryland 21224,
and Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
Received July 29, 2004
Fatty acid synthase (FAS) catalyzes the synthesis of palmitate from the sequential condensation
of an acetyl primer with two carbon units added from malonyl-CoA. Inhibition of the â-ketoacyl
synthase domain of mammalian FAS leads to selective cytotoxicity to various cancer cell lines
in vitro and in vivo. Also, inhibitors of FAS can cause reduced food intake and body weight in
mice. Naturally occurring thiolactomycin (TLM) was used as a template to develop a new class
of type I FAS inhibitors. Using a flexible synthesis, families of TLM structural analogues were
obtained that possess selective FAS activity and display anticancer and weight loss effects.
Compounds 13a and 13d inhibit pure FAS (ZR-75-1 breast cancer, IC50 ) e20 µg/mL), are
nontoxic (MCF-7, IC50 ) >50 µg/mL), and display effective weight loss in BalbC mice (>5%).
Another subclass of TLM derivatives (23b-d, 31a) exhibits FAS activity (IC50 ) e15 µg/mL),
causes weight loss (>5%), and is cytotoxic to cancer cells (IC50 < 38 µg/mL). Finally, a third
subclass (16b, 29, 30) is also active against FAS (IC50 ) e20 µg/mL), is cytotoxic to cancer
cells (IC50 < 25 mg/mL), and does not cause weight loss in BalbC mice. These studies identify
thiolactomycin as a promising template for the development of new selective cancer and obesity
treatments.
Introduction
Inhibition of mammalian FAS (multifunctional, type
I; in contrast to the dissociable, bacterial type II FAS)
displays selective cytotoxicity in various cancer cell
lines. For example, administration of cerulenin (1) or
C75 (2) (Figure 1) to MCF7 and SKBR3 breast cancer
cell lines show FAS inhibition, followed by apoptosis.2
Systemic treatment of MCF7 breast cancer xenografts
in nude mice with 2 also shows FAS inhibition, apop-
tosis and reduction in tumor size. Despite its cytotoxicity
to cancer cells, C75 displayed no adverse histological
effects, but caused reversible weight loss in test
animals.2a,b
Recent reports demonstrate that C75 acts as a ma-
lonyl-CoA mimetic and exerts its effects both centrally
to reduce neuropeptide Y (NPY) expression3b and pe-
ripherally as a CPT-1 agonist3a causing reduced food
intake and body weight in mice.3 Furthermore, dimin-
ished adipose tissue and fatty liver in OB/OB mice is
also observed despite elevated levels of malonyl-CoA (a
known inhibitor of CPT-1) produced by FAS inhibition.
Both naturally occurring cerulenin (e.g. Cephalospo-
rium caerulens) and synthetic C75 inactivate bacterial
(type II) and mammalian (type I) FAS systems.2,5
Nature has also produced a unique thiolactone-contain-
ing molecule that is a selective and reversible inhibitor
of the KAS enzymes in type II bacterial FAS systems.6
Thiolactomycin (TLM, e.g. Nocardia sp.) exhibits broad-
spectrum antibiotic activity in vitro against Gram-
positive and Gram-negative bacteria,6 Mycobacterium
tuberculosis,7a,b the malaria parasite, Plasmodium
falciparum,8a-e and African trypanosomes.8a,d Intrigued
by TLM’s reported selectivity for type II FAS systems,
the challenge to develop TLM structural analogues that
show inhibitory activity against type I FAS systems and
Human fatty acid synthase (FAS) is emerging as an
important therapeutic target for carcinomas of the
breast, prostate, endometrium, ovary, and colon.1,2 In
addition, inhibition of FAS, notably in the hypothala-
mus, and the coupled stimulation of fatty acid degrada-
tion in the adipose tissue have been demonstrated to
coordinately mediate profound weight loss in animals.3
FAS is encoded by a single locus in the human genome.4
The design and synthesis of small molecule inhibitors
of this exquisitely evolved polydomainal protein with
the goal to achieve selectivity in their cytotoxic or weight
loss effects is a significant challenge. We address this
question through consideration of reaction mechanism,
X-ray crystal structures, and molecular modeling. Struc-
tural diversification of a natural product template has
been undertaken to achieve both crossover through a
fundamental prokaryotic/eukaryotic activity barrier,
and successful differentiation of cytotoxic and weight
loss effects.
Vastly higher levels of FAS (FAS, EC 2.3.1.85) are
expressed in many human cancers and tumor cells1,2
than in normal tissues in which FAS activity is signifi-
cantly down-regulated and compensated with dietary
fat.4 This difference in expression and activity of FAS
between normal and cancer cells provides an attractive
approach to cancer therapy having the potential for a
large therapeutic index.
* Corresponding author: Craig A. Townsend, Department of Chem-
istry, Johns Hopkins University, 3400 North Charles St., Baltimore,
MD 21218. Phone: 410-516-7444. E-mail: ctownsend@jhu.edu.
† Johns Hopkins University.
‡ FASgen, Inc.
§ Johns Hopkins University School of Medicine.
10.1021/jm049389h CCC: $30.25 © 2005 American Chemical Society
Published on Web 01/20/2005

Inhibitors of Type I Fatty Acid Synthase
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 947
Scheme 1
Figure 1. Inhibitors of fatty acid synthase.
the thiolactone ring was successful in generating TLM
analogues that are inhibitors of type I human FAS.
Moreover, several of these compounds display effective
anticancer activity, while others have been identified
that generally minimize cytotoxicity and cause weight
loss.
Figure 2. Thiolactomycin: a “thiomalonate isostere”.
Chemistry
The asymmetric synthesis of TLM developed earlier
in this laboratory employs Seebach’s self-regeneration
of chirality method using amino acids as the chiral
building blocks (Scheme 1). The key steps involve a thio-
Dieckman reaction of 8 to provide (5R)-thiolactomycin
(3) and a sulfenate-sulfoxide [2,3]-sigmatropic rear-
rangement accompanied by a thermal syn-elimination
to provide diene 7 to achieve almost exclusive trans
stereochemistry at the C-1′ alkene. Finally, optically
pure oxathiolanone 5 can be synthesized from (2S)-
thiolactic acid, which is readily prepared from D-alanine
(Scheme 1).11
First, several derivatives of TLM modified at C-5 were
synthesized to probe the effects of alkyl, alkenyl and
aryl substitutents in this hydrophobic pocket poorly
filled by the short, unsaturated side chain of TLM
(Figure 3). Extended hydrocarbon chains with and
without the 1,3-diene at C-5 were prepared. Formation
of the lithium-enolate of racemic 912 was achieved with
LDA at -78 °C and addition of tiglic aldehyde, 2-methyl-
2-pentenal, trans-2-hexenal, and trans-2-octenal to the
re-face gave 2:1 mixtures of the diastereomeric alcohols
10a (81%), 10b (71%), 10c (88%), and 10d (81%, Scheme
2.
Treatment of these alcohols with 2,4-dinitrobenzene-
sulfenyl chloride and NEt3 in refluxing dichloroethane
provided 11a (75%), 11b (73%), 11c (72%), and 11d
(75%, Scheme 3). This conversion to the 1,3-diene was
advantageous in our synthesis of thiolactomycin since
the [2,3]-sigmatropic rearrangement and elimination
provided the trans stereochemistry at the C-1′ alkene
predominantly (14:1, trans:cis).11,13 This configurational
bias toward trans selectivity was also observed with 11b
(14:1, trans:cis) but less so with 11c and 11d (4:1 trans:
cis). Previously, it was reported that the [2,3]-sigma-
tropic rearrangement of the allyl sulfenate to the allyl
sulfoxide is concerted and reversible with the equilib-
rium lying on the side of the sulfoxide.14
Figure 3. TLM cocrystallized in KAS I of E. coli.
act as anticancer and/or weight loss agents became a
compelling avenue to explore (Figure 2).
Thiolactomycin has been cocrystallized with KAS I
(FabB) from E. coli.9 The structure reveals that TLM
occupies a different region in the active site than
cerulenin and likely binds in the malonyl-ACP pocket
on the basis of kinetic evidence (Figure 2).9 Further-
more, the crystal structure provides insight into the
architecture of the KAS active site and identifies the
presence of hydrophobic and pantetheine binding pock-
ets which are both suboptimally filled by TLM (Figure
3).9
Reports show that TLM analogues with an extended
C-5 hydrocarbon chain are more effective inhibitors of
pea (Pisum sativum) FAS10a and displayed improved
activity against mycobacteria.7a,b Enhanced activity was
also observed against Staphylococcus aureus and Pas-
teurella multocida by appending both the C-3 (acetyl)
and C-5 (aryl or alkyl functionality) of the thiolactone
skeleton.10b
We have recently described an efficient asymmetric
synthesis of (5R)-thiolactomycin which was conceived
to allow the medicinal potential of this template to be
explored.11 This flexible route has been applied to the
preparation of several subclasess of thiolactomycin
structural analogues that are varied at the C-3, C-5, and
C-4 enol loci. Modification of the functionality around

948 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
McFadden et al.
Scheme 2
Scheme 4a
a Reagents and conditions: (a) Cs2CO3, EtOH; (b) AcCl, NEt3,
CH2Cl2; (c) LiHMDS/THF, -78 °C to -5 °C.
Scheme 5a
Scheme 3
a Reagents and conditions: (a) CH3(CH2)nOTf, n ) 5, 7; (b)
NaOEt/EtOH; (c) XCOCl, NEt3, CH2Cl2; (d) LiHMDS, toluene, -78
°C to -5 °C. (e) NaHMDS was used instead LiHMDS in this case.
propionyl, and 4-pentenoyl chloride to give 15a (77%),
15b (79%), 15c (75%), and 15d (86%). Enolate formation
and Dieckman condensation provided thiotetronic de-
rivatives 16a (69%), 16b (73%), 16c (70%), and 16d
(57%).
Next, preferential O-alkylation of 16b was achieved
using NaH in DMF to append the C-4 enol with various
functional groups including alkenes, alkynes, alkyl
halides, and esters (Scheme 6). In the crystal structure
of TLM bound to KAS I, the C-4 enol appears to be
directed into the probable pantethine recognition pocket
of malonyl-ACP (vide infra). Therefore, TLM analogues
with extended polar functionality at the C-4 enol were
thought to further stablize ligand/enzyme binding (Fig-
ure 3).
In a few cases, C-3 and C-2 O-alkylation was ob-
served. Methylation of 16c provided both C-4,C-2 O-
alkylated products as an inseparable mixture 17c/17m
(2.2:1, Scheme 7). Addition of allyl bromide to the
sodium enolate of 16c gave predominately C-alkylated
(17n, 70%, Scheme 7) and C-4,C-2 O-alkylated products
17o/17p (30%, 2.6:1 inseparable mixture) in an 82%
overall yield. Alkylation of 16d provided C-alkylated
In our case, each diastereomeric allylic sulfenate can
give both the E- and Z-sulfoxides. Presumably, the
presence of the 2-methyl group in tiglic aldehyde and
2-methyl-2-pentenal introduces additional steric inter-
actions in the Z-sulfoxides, making them higher in
energy [calcd ∆G ) 1.9 kcal (at 90 °C), 14:1 trans:cis]
than their E counterparts. Thus, dienes 11c,d, which
lack this 2-methyl group, lost their high trans selectivity
as a consequence of a decrease in energy difference
between the E- and Z-sulfoxides [calcd ∆G ) 1.0 kcal
(at 90 °C), 4:1 trans:cis].
The oxathiolanone ring of 11a-d was opened with
Cs2CO3 in EtOH and immediately acylated with acetyl
chloride to yield thioesters 12a-d (63-77%, Scheme 4).
Finally, thio-Dieckman condensation of 12a-d gave 13a
(53%), 13b (49%), 13c (60%), and 13d (41%, Scheme 4).
Thiolactomycin analogues containing a C-5 saturated
hydrocarbon chain were obtained by a slight modifica-
tion of the above procedure. Addition of octyl or hexyl
triflate to 9 provided 14a (83%) and 14b (81%) (Scheme
5). The oxathiolanone ring was cleaved with NaOEt/
EtOH, and the released thiol was acylated with acetyl,

Inhibitors of Type I Fatty Acid Synthase
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 949
Scheme 6
Scheme 7a
a Reagents and conditions: (a) NaH, DMF, R ) (CH2)7CH3.
C-3 dialkylated 17n (214.6 ppm, C-4; 204.5 ppm, C-2),
17q (213.5 ppm, C-4; 203.9 ppm, C-2), and C-4,C-2
O-alkylated 17c (195.9 ppm, C-2; 180.2 ppm, C-4)/17q
(202.7 ppm, C-4; 184.9 ppm, C-2).
Acid hydrolysis of 17k-l provided acids 18a (98%)
and 18b (89%, Scheme 8). Coupling reactions of 18 with
EDC, or tris(2,3-dihydro-2-oxobenzoxazol-3-yl)phosphine
oxide16 and the corresponding amines, provided 19
(80%), 21 (68%), and 22 (66%). Significantly, the methyl
ester 19 was hydrolyzed instead of the thiolactone ring
in high yield to give 20 (80%, Scheme 8). This result
suggests that the thiolactone moiety in all of these
analogues should be stable at physiological pH.
Enolate formation of 16b with LiHMDS/THF (-78
°C-rt) and addition of methyl, ethyl, and allyl chloro-
formate provided O-acylated (confirmed by HMQC
NMR) 23a (47%), 23b (70%), 23c (91%), and 23d (67%)
(Scheme 9).
Additionally, C-5 aryl derivatives were also prepared
to ascertain their affinity for the hydrophobic pocket.
Alkaline cleavage (NaOEt/EtOH) and acylation of ben-
zylated 24 yielded 25 (76%). Thio-Dieckman condensa-
tion of 25 gave 26 (45%, Scheme 10). O-Methylation to
give 27 (74%) was achieved with dimethyl sulfate in
DMF (Scheme 10).
Finally, examination of the KAS I/TLM crystal struc-
ture revealed that the C-3 methyl group of thiolacto-
mycin likely occupies the region where the carboxyl
group of malonyl-ACP binds. Also, close inspection of
the crystal structure reveals the presence of two threo-
nines that lie 5.2 Å (Thr302) and 3.9 Å (Thr300) from
the C-3 methyl group of TLM.9 We anticipated that
additional hydrogen bond acceptors/donors will favor-
ably interact with these two threonines or the catalytic
His333 (Figure 4).
Attempts to prepare molecules containing these fea-
tures were made using a Mukaiyama aldol approach.17
Indeed, silyl enol ether 28 was formed quantitatively
from 16b with TMSCl/benzene. Addition of TiCl4 at -78
°C and acetaldehyde provided a mixture of diastereo-
mers 29 and 30, which was separated by column
chromatography (50% overall yield, Scheme 11).
a Mixture of C-4 O-methyl (17c): C-2 O-methyl (17m) 2:2:1.
bMixture of C-4 O-allyl (17h): C-3-dialkylated (17t) (3:1). cMixture
of C-4 O-alkyl (17i): C-3 dialkylated (17q) (3:5:1).
17q as the major product and C-4 O/C-2 O-alkylated
17r/s (17q:17r/s, 5.3:1, 68% overall yield, Scheme 7).
Perhaps, C-2 O- and C-alkylation is favored in 16c due
to the steric congestion of both the adjacent C-5 quar-
ternary center and the C-3 methyl group. Recently, a
study showed that both C-2 and C-4 O-alkylation (C-2
O:C-4 O, 1:2.3-1:9.7) of 3,5-dimethylthiotetronic acid
was observed using various alkyl electrophiles.15 Con-
trary to the reactivity of C-3 methylated 16c, C-4
O-alkylation was the only or predominant route ob-
served in most cases for 16a and 16b bearing a C-3
hydrogen. C-3 Dialkylated 17q and 17t were formed in
minor amounts using allyl bromide as the electrophile
in the alkylation of 16a and 16b (Scheme 6). C-4
O-Alkylation was determined through direct comparison
of 13C NMR carbonyl carbons with those of natural
product thiolactomycin (196.7 ppm, C-2; 179.2 ppm, C-4)

950 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
McFadden et al.
Scheme 8
Scheme 10a
a Reagents and conditions: (a) NaOEt/EtOH; (b) AcCl, NEt3,
CH2Cl2; (c) LiHMDS, -78 °C to -5 °C; (d) DMS, DMF.
Figure 4. (5R)-TLM and nearby residues in E. coli KAS I.
Scheme 11
Scheme 9a
Lewis acids (e.g. ZnCl2, BiCl3/NaI, MgCl2, AlCl3) was
unsuccessful.
Nomura et al. reported an efficient protocol for the
C-3 acylation of tetronic acids.18 This method involves
initial formation of the kinetic O-alkylated product in
the presence of base and a anhydride. Then, catalytic
DMAP-mediated transfer of the acyl group to the C-3
of the tetronic acid occurs over time to provide the
C-alkylated thermodynamic product. Using these condi-
tions 31a (78%), 31b (86%), and 31c (59%, 79% based
on recovered starting material, 16b) were obtained from
16b and the corresponding anhydrides or chlorofor-
mates in good yields (Scheme 12).
a Reagents and conditions: (a) ClCOR, R ) CH2CH3, OCH3,
OCH2CH3, OCH2CHCH2.
Biological Results and Discussion
The biological activity of these thiolactomycin ana-
logues was screened for (i) inhibition of fatty acid
synthesis activity in whole cells, (ii) inhibition of purified
human FAS (ZR-75-1 human breast cancer cells), (iii)
Unfortunately, addition of acid chlorides, chlorofor-
mates, and anhydrides in the presence of TiCl4 or other

Inhibitors of Type I Fatty Acid Synthase
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 951
Scheme 12a
weight loss (2-4%). These data demonstrate a second
class of compounds derived from the TLM scaffold that
are FAS inhibitors with potential antiobesity applica-
tions.
In the crystal structure of TLM/KAS I, the 1,3-diene
of TLM resides between two peptide bonds (Gly391-
Phe392 and Ala271-Pro272) and is possibly stabilized
by π-stacking interactions (Figure 3).9 Also, an edge-to-
face interaction is apparent between the C-1′ alkene and
Phe392.9 To test the importance of π-stacking interac-
tions in TLM binding, benzyl and C-5 alkyl derivatives
(hexyl vs octyl) were prepared and their biological
activity was profiled (Table 2).
a Reagents and conditions: (a)CH3CO2COCH3; (b) CF3CO2COCF3;
(c) ClCO2Me.
Table 1. TLM Analogues with the C-5 1,3-Dienyl Unit
TLM analogue 16b was also an effective inhibitor of
purified FAS (IC50 ) 4.0 µg/mL), suggesting that the
1,3-dienyl group is not essential for effective TLM
binding. Surprisingly, in contrast to the compound
series above, 16b is relatively cytotoxic against MCF-7
cancer cells (IC50 ) 17.6 µg/mL) and does not cause
significant weight loss (2%, 60 mg/kg, ip). Replacement
of the C-3 hydrogen in 16b with a methyl group was
quite detrimental to FAS inhibition 16c (IC50 ) 49.2
µg/mL), yet incorporation of an allyl branch at C-3
displayed FAS inhibition (16d, IC50 ) 2.8 µg/mL).
Perhaps the allyl substituent in 16d is stabilized by
π-stacking with the conserved aromatic residue (Phe229,
E. coli; Tyr229, human/rat) present in the â-ketoacyl
synthase. It has been suggested that Phe229 (E. coli)
helps to promote decarboxylation of malonyl-ACP.9
MCF-7,
MCF-7, wt loss
compd
TLM
R1 R2
R3
14Ca ZR-75-1b,c XTTd
(%)e
2.6
CH3 CH3
{5R}-TLM CH3 CH3
H
H
H
41.8
38.4
40.3
17.3
16.9
30
>80
>80
>80
>80
>80
60
21.1
8.4
neg
neg
8.7
13a
13b
13c
13d
H
H
H
H
CH3
7.8
4.1
CH3 CH3
H
H
CH2CH3
3.2
(CH2)3CH3 22.6
11.0
a Measure 14C acetate incorporation into total lipids in MCF-7
breast cancer cells; average of duplicate runs. b Measure of FAS
activity (overall reaction) by following oxidation of NADPH at 340
nm from purified ZR-5-1 breast cancer cells; neg ) IC50 > 50 µg/
mL. c Error ) <(1. d Measure cytotoxicity using XTT assay
(nonradioactive assay); average of duplicate runs. e Maximum
amount of weight loss with one 60 mg/kg, ip dose (measured over
7 days).
Indeed, the hydrophobic pocket appears to be flexible
and accommodates extended C-5 hydrocarbon units. C-5
Benzylated analogues (26, 27), however, were not active
against FAS, suggesting that aryl groups are not
tolerated in this pocket.
Attempts to mimic the pantetheine arm and optimize
interactions in its proposed binding pocket consisted of
appending to the C-4 OH a range of structural elements
(Figure 3). Several trends were recognized from the data
in Table 2. First, substitution of the C-4 OH with
various acetyl [i.e. acetal (18b); ethyl acetyl- (17d,e);
N-acetyl derivatives of glycinate (19,20), 3-bromopro-
pane (21), and N-allyl (22)] and alkyl groups (17a,b,f-
i)] were inactive against purified FAS under the experi-
mental conditions (The measurement of enzymatic
activity was performed in 10 min. intervals; therefore,
it is possible that these analogues may be slow-binding
inhibitors as was found for C75). Yet, several of these
derivatives conferred inhibition against whole-cell FAS.
Unsaturated analogues 17h,i and 22 were cytotoxic
[17h (IC50 ) 9.0 µg/mL); 17i (IC50 ) 14.5 µg/mL); and
22 (IC50 ) 12.1 µg/mL)], but inactive against whole-cell
and purified FAS. Propargyl analogue 17j was slightly
active against whole-cell FAS (IC50 ) 21.9 µg/mL) and
also very cytotoxic (IC50 ) 8.9 µg/mL). The C-4 OH of
TLM in the crystal structure is stabilized by hydrogen
bonding with the carbonyl of Val270 and the amide of
Gly305 through a network of water molecules. The
inability for these acetyl derivatives to inhibit purified
FAS [ZR-75-1 cells (Table 2)] suggests that this hydro-
gen bonding network is important for TLM binding and
stabilization.
cytotoxicity against cultured MCF-7 human breast
cancer cells (possess high levels of FAS and FAS
activity), and (iv) selected compounds were tested for
weight loss in Balb/C mice. Table 1 displays the inhibi-
tion data of TLM analogues containing the isoprenyl
unit at C-5. Despite previous reports that (+)-thiolac-
tomycin is not active against type I rat FAS,19 both (()
TLM (IC50 ) 30 µg/mL) and (5R)-TLM (IC50 ) 21 µg/
mL) showed considerable activity against purified hu-
man FAS from breast cancer cells (Table 1). Signifi-
cantly, 13a (IC50 ) 8.4 µg/mL) in which the C-3 methyl
group of TLM is replaced with hydrogen was 3.4 times
more potent than TLM against purified human FAS and
relatively noncytotoxic (IC50 > 80 µg/mL). A similar
potency/cytotoxicity profile was observed with the ex-
tended C-5 diene 13d [IC50 ) 8.7 µg/mL (ZR-75-1); IC50
) 60 µg/mL (MCF-7, XTT)]. Shorter-chain dienes (13b,
13c) displayed lower FAS inhibition. These unsaturated
analogues were also screened for weight loss in Balb/C
mice. The compounds were diluted in DMSO at 10 mg/
mL and the mice were injected intraperitoneally (ip)
with 60 mg/kg in 100 µL of DMSO or with vehicle alone.
The mice were observed and weighed daily, and the
experiment was continued until treated animals reached
their pretreatment weights. The most effective FAS
inhibitors in this unsaturated class, 13a and 13d (both
comparatively noncytotoxic) displayed considerable
weight loss with one 60 mg/kg dose (13a, 7.8%; 13b,
11.0%), in contrast to the less active analogues (TLM,
13b, 13c; IC50 > 20 µg/kg ZR-75-1), which displayed less
High activity against both whole-cell and purified
FAS was achieved by directly attaching the carbonyl
moiety to the C-4 OH to provide carbonates 23b-d (IC50

952 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
Table 2. Substitution at C3, C4-OR, and C5 of TLM
McFadden et al.
IC50, µg/mL
compd
n
R1
R2
MCF-7 14Ca
ZR-75-1b
MCF-7, XTTc
wt loss (%)d
11e
C75
16a
16b
16c
16d
17a
17b
17c/m
17d
17e
17f
17 g
17h
17i
-
5
7
7
7
5
7
7
5
7
5
7
5
7
5
7
7
7
7
5
5
5
7
7
7
7
7
7
7
7
7
7
-
-
10.8
16.5
12.6
16.5
34.8
14.0
neg
neg
14.2
10.8
8.6
neg
neg
neg
21.9
neg
neg
neg
13.8
9.8
neg
neg
4.0
49.2
2.8
neg
10.8
>80
17.6
48
44.4
9.4
16.4
17.3
39.6
35.3
20.8
35.3
9.0
H
H
H
CH3
H
2
0
H
CH2CHCH2
H
H
H
CH3
CH3
CH3
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
CH3
H
CH2CO2Et
CH2CO2Et
H
2.7
8.0
H
(CH2)4Cl
(CH2)4Cl
H
H
CH2CHCH2
CH2CHCH2
H
14.5
8.9
17j
H
CH2CCH
-
17n
17o/p
17q
18b
19
CH3, CH2CHCH2
40.5
34.7
40.5
50.3
40.5
>80
21
12.1
26.8
21.6
15.1
37.3
23.9
21.0
12.5
18.4
47.2
>80
61.3
CH3
CH2CHCH2
(CH2CHCH2)2
-
H
CH2CO2H
H
CH2CONHCH2CO2Me
20
H
CH2CONHCH2CO2H
6.6
6.7
3.5
2.4
21
H
CH2CONH(CH2)3Br
22
H
CH2CONHCH2CHCH2
neg
22.6
10.7
14.5
14.2
17.6
21.7
12.0
14.7
18.7
neg
neg
23a
23b
23c
23d
29
H
COCH2CH3
H
CO2CH3
2.1
6.1
7.1
3.4
3.5
0.2
8.2
H
CO2CH2CH3
CO2CH2CHCH2
4.6
6.8
5.7
3.3
H
C(OH)CH3
C(OH)CH3
COCH3
COCF3
CO2CH3
H
H
30
H
31a
31b
31c
26
H
neg
H
41
neg
neg
neg
H
benzylf
benzylf
H
1.6
27
H
CH3
a Measure 14C acetate incorporation into total lipids in MCF-7 breast cancer cells: neg ) IC50 > 50 µg/mL; average of duplicate runs.
b Measure of FAS activity (overall reaction) by following oxidation of NADPH at 340 nm from purified ZR-75-1 breast cancer cells; neg
) IC50 > 50 µg/mL, error ) <(1. c Measure of cytotoxicity using XTT assay (nonradioactive assay); average of duplicate runs. d Maximum
amount of weight loss with one 60 mg/kg ip dose (measured over 7 days). e 20 mg/kg ip dose. f C-5 ) benzylated.
) 2.1, 4.6, and 6.8 µg/mL). Furthermore, linking 16b
with a carbonyl moiety at the C-4 OH provided deriva-
tives that also caused significant weight loss (23b
(6.1%); 23c (7.1%); 23d (3.4%). We anticipated that this
slight modification at the C-4 OH would place the
carbonate moiety closely aligned with first N-acetyl-
pantetheinyl amide of malonyl-CoA in our structural
model (Figure 2), and that these analogues would be
more structurally similar to C75. Indeed the FAS
inhibition, cytotoxicity and weight loss observed by the
carbonates (23b-d) supports this rationale. These data
suggest that appending the TLM skeleton to become
more of a malonyl-CoA mimetic influences the propor-
tion of the observed weight loss. Finally, the loss of FAS
inhibition by propyl analogue 23a suggests that the
presence of the carbonate carbonyl alone does not confer
activity.
previously reported.20 Smith and co-workers present
evidence for an activated water molecule in the active
site that either protonates the malonyl carboxylate,
prior to its release as bicarbonate, or attacks the
malonyl carboxylate to provide a negatively charged
tetrahedral transition state which is stabilized by His
293 (His 298 in E. coli).20 Then, bicarbonate rather than
CO2 is released directly upon breakdown of the tetra-
hedral transition state to provide the acetyl enolate.20
Thus, the active site of â-ketoacyl synthase has to
stablize the formation of three tetrahedral transition
states: (i) the transfer of the acyl-primer to the active
site cysteine reside; (ii) the release of bicarbonate by
attack of water; (iii) the condensation of the acetyl-ACP
enolate and the extended acyl chain. Since thiolacto-
mycin acts as a thiomalonate mimetic, analogues con-
taining C-3 acetyl or alcohol units could be stabilized
by the active site histidines or the residues that reside
in the oxyanion holes. Alcohol derivatives 29 and 30
demonstrated good activity against whole-cell (29, IC50
) 17.6 µg/mL; 30, IC50 ) 21.7 µg/mL) and purified FAS
(29, IC50 ) 5.7 µg/mL; 30, IC50 ) 3.3 µg/mL). Trifluo-
roacetyl analogue 31b displayed modest purified FAS
We anticipated that incorporation of hydrogen bond-
ing donors/acceptors at C-3 could interact with the
active site histidine and/or either of the two threonines
(Figure 4). Recently, a detailed kinetic analysis of the
â-ketoacyl synthase domain of multifunctional fatty acid
synthase provides an alternative mechanism to those

Inhibitors of Type I Fatty Acid Synthase
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 953
Figure 5. CLUSTAL W multiple sequence alignment of the â-keto acyl synthase of human/rat FAS with KAS I.
inhibition (IC50 ) 41 µg/mL). All five C-3 analogues (29-
31c, IC50 ) 14-21.7 µg/mL) were effective against
whole-cell FAS. Also, consistent with the correlation of
malonyl-CoA mimetics displaying weight loss, the most
active derivative, 31a, caused significant weight loss
(8.2%). Indeed, 31a would most likely be the best
hydrogen bond acceptor among the C-3 carbonyl-bearing
substituents and therefore be more stabilized by the
active site histidines or threonines present in the
vicinity.
These structure-activity studies identify a fine line
between properties that molecules must possess to
display antiobesity vs anticancer activity. As shown with
C75, inhibition of fatty acid synthase in cancer cells can
be a route to develop new anticancer agents.1,2 Research-
ers have proposed that cancer cells overexpress FAS to
maintain redox balance to compensate for the hypoxic
conditions that malignant cellular phenotypes exhibit.21
High oxidative stress is counterbalanced by the continu-
ous supply of NAD+/NADP+ furnished by dramatic
upregulation of FAS.21 One can imagine that disrupting
this pathway using FAS inhibitors will destablize the
redox balance of cancer cells causing apoptosis and cell
death. We have successfully developed a TLM subclass
of type I FAS inhibitors that displays anticancer activity
with little or no weight loss. Furthermore, we have
identified a second subclass that also inhibits FAS, but
is minimally cytotoxic and causes substantial weight
loss (>5.0%, 60 mg/kg). It appears that fine-tuning the
TLM skeleton to more closely resemble malonyl-CoA is
the determinant of the observed weight loss in several
derivatives including 13a, 13d, 23b-d, and 31a. Ques-
tions still remain whether and to what extent these
analogues cause weight loss through both peripheral
and central mechanisms as C75 does. These studies and
their effects against CPT-1 and NPY are currently
underway.
Structural Modeling of TLM Analogues. Several
compounds described in Tables 1 and 2 were effective
inhibitors of type I human FAS. TLM analogue 13a,
which is virtually identical to TLM with the exception
that the C-3 methyl group is replaced with a hydrogen,
is a more potent (IC50 ) 8.8 mg/mL) inhibitor of human
cancer FAS than (() TLM (IC50 ) 30 µg/mL). Also, the
C-5 saturated version 16b is 11 times more potent than
16c containing a C-3 methyl group. These data suggest
that the presence of a C-3 methyl group in TLM
analogues hinders activity against type I systems and
perhaps is the principal determinant making naturally
occurring thiolactomycin selective for bacterial type II
FAS systems. In an attempt to understand this differ-
ence in observed activity between the methyl-containing
natural product (TLM) and unmethylated 13a and 16b,
we have carried out modeling experiments using the
crystal structure of thiolactomycin bound in KAS I of
E. coli. The crystal structure of KAS I contains four
identical, independent subunits in one asymmetric unit,
which pair around two, 2-fold axes to form a pair of
dimers.22 Each subunit contains 406 amino acids which
fold into an R-â-R domain which, upon dimer forma-
tion, gives an R-â-R-â-R structure.22
A BLASTP pair alignment23 was carried out with
KAS I and the â-ketoacyl synthase regions of human
and rat FAS. Both rat and human FAS are 26%
identical and 42% and 43% similar to KAS I, respec-
tively. The InsightII molecular modeling program was

954 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
McFadden et al.
Figure 6. TLM-human/rat chimera.
used to identify residues within an 8 Å diameter of the
bound ligand in KAS I. Forty residues were identified
in this region. A multiple CLUSTALW alignment24 was
carried out to compare the identity/similarity of these
40 residues in KASI with the â-ketoacyl synthase
regions of rat and human FAS. Twenty-five of the 40
are identical between human/rat FAS and KAS I (Figure
5). Using InsightII, the other 15 nonidentical residues
were mutated in one subunit of the KAS I structure to
correspond to those residues in human/rat FAS. This
human/rat chimera containing TLM was minimized
using Discover. Careful inspection of the minimized
chimeric structure showed that TLM maintains the
important hydrogen bonding interactions that are present
in the KAS I crystal structure. The Phe229Tyr and
Leu335Glu mutations in human/rat incorporate a hy-
drogen-bonding donor/acceptor pair. Rock and co-work-
ers propose that the presence of the aromatic moiety
(Phe229, KAS I) promotes decarboxylation of the ma-
lonyl-ACP through electronic repulsion between the
π-electrons of the aromatic ring and the incoming
carboxylate of malonyl-ACP.9
Several of the mutations are clustered in the hydro-
phobic binding pocket (Figure 6). It has been shown that
slight modifications in this pocket can confer TLM
resistance.25 Recently, six independent TLM-resistant
clones of strain ANS1 were isolated and all expressed
a mutant protein, FabB(F390V). Structural modeling of
this mutant predicted that the valine side chain inter-
fered with positioning of the C-2′ methyl group on the
diene of TLM. Our biological data suggest that the
changes in the hydrophobic pocket between the human
and bacterial â-ketoacyl synthase do not effect binding
of the isoprenoid-like side chain since 13a is still active.
Yet, saturated alkyl analogue, 16b (without the C-2′-
methyl) is slightly more active than 13a. A more
probable explanation for the enhanced activity of 13a
and 16b compared to TLM and 16c is a steric effect
involving the C-3 methyl group. The minimized struc-
ture of the TLM-chimera reveals two mutations that are
in close proximity to the C3 methyl and can possibly
impede TLM binding (Figure 6).
analogues and conferred them with inhibitory activity
against type I FAS systems as was observed in Tables
1 and 2.
Conclusions
In summary, we have demonstrated the flexibility of
our previously described total synthesis to prepare TLM
analogues. The γ-thiolactone skeleton was modified at
C-5, C-3, and C-4 OH to provide structural analogues
that both encompass and successfully partition weight
loss and cytotoxic activities. Several of these derivatives
including 13a, 13d, carbonates (23b-d), and C-4 enol
analogues (16b, 29, 30) are potent inhibitors of human
FAS (IC50 e 20 µg/mL). 13a and 13d are nontoxic to
cancer cells (IC50 > 50 mg/mL) and cause considerable
weight loss with one 60 mg/kg ip dose in BalbC mice
(>5%). 23b-d and 31a display biological activity similar
to C75. Both weight loss (>5%) and cytotoxicity (IC50
<
40 mg/kg) toward cancer cells are observed. Other TLM
compounds including 16b, 29, 30 show effective anti-
cancer activity (IC50 < 25 mg/kg) but do not cause
weight loss. Indeed, a direct correlation between FAS
inhibition and cell death in cancer cells overexpressing
FAS has been shown by us and others.1,2 Evidence
suggests that FAS inhibition and partition into the
hypothalamus and brain in conjunction with a second
or possibly several metabolic pathways (i.e. NPY or
CPT1 or an unknown route) lead to the weight loss.3
Current investigations into distinguishing between
these outcomes and developing molecules that exert
selective activity for each are underway. As an impor-
tant first step we have shown that the γ-thiolactone
skeleton can be used as a template to develop agents to
treat obesity and diabetes-related diseases, or new
therapeutics to treat cancer. Furthermore, we have also
demonstrated how one can synthetically evolve a type
II FAS inhibitor (TLM) into an effective inhibitor of the
higher order type I multifunctional FAS systems.
Experimental Section
Biological Methods. The inhibition of fatty acid synthesis
activity in whole cells;26a-c inhibition of purified FAS (ZR-75-
1, human breast cancer cells);26d,e cytotoxicity against cultured
MCF-7 human breast cancer cells;26f and weight loss in Balb/C
mice3b were assayed as described previously.
Inhibition of FAS from ZR-75-1 Breast Cancer Cells.
Using a 96-well plate and adapting a procedure from Dils et
al.26d and Arslanian et al.,26e FAS activity (the overall reaction)
was determined with a Molecular Devices Spectramax Plus
spectrophotometer at 37 °C by following the malonyl CoA
dependent oxidation of NADPH at 340 nm (at 10 s intervals
for 5 min). The reaction mixture contained 100 mM potassium
Ala206Met is located between the C-3 methyl of TLM
and Met204Leu. Rotation of the Cγ-S-methyl group of
Met206 is restricted due to unfavorable interactions
with the C-3 methyl of TLM and the isobutyl group of
Leu204. Therefore, it is possible that this added steric
congestion in the human protein can interfere with TLM
binding. Thus, substitution of the C-3 methyl group in
TLM for a smaller and less sterically demanding
hydrogen atom enhanced the binding affinity of these

Inhibitors of Type I Fatty Acid Synthase
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 955
phosphate buffer, pH 6.5, acetyl-CoA (61.8 uM), malonyl-CoA
(67.4 uM), NADPH (187.5 uM), DTT (1 mM), and 2 µg of FAS
(purified from ZR-75-1 cells26c) in a total volume 100 uL.
dure A, 10d was obtained (1.1 g, 81%) after flash chromatog-
raphy (10% EtOAc/hexanes) as a 1.2:1 mixture of diastereo-
1
mers at C1′. H NMR (300 MHz, CDCl3) major diastereomer
δ 0.85 (t, J ) 7.2 Hz, 3 H), 0.97 (bs, 9 H), 1.18-1.35 (m, 6 H),
1.56 (s, 3 H), 2.00-2.08 (m, 2 H), 2.38 (d, J ) 5.0 Hz, 1 H),
4.15-4.19 (m, 1 H), 5.13 (s, 1 H), 5.45-5.59 (dd, J ) 7.7, 15.3
Hz, 1 H), 5.72-5.77 (m, 1 H); 13C NMR (75 MHz, CDCl3) δ
13.7, 22.3, 24.7, 28.5, 31.3, 32.1, 35.2, 60.6, 78.8, 87.4, 127.2,
Structural Modeling. All molecular modeling was per-
formed on an SGI Octane 2XR10000 work station. The
â-ketoacyl synthase (KAS I) from E. coli was downloaded from
the PDB data bank (http://www.rcsb.org/pdb/; PDB ID: 1FJ4).
An 8 Å diameter subset around the TLM ligand containing 40
amino acid residues was identitified using InsightII 1997. The
15 nonidentical residues were mutated using InsightII 1997
Builder to correspond to those residues in the â-ketoacyl
synthase regions of human and rat FAS. This human/rat
chimera was energy minimized with Insight 1997 Discover.
Initially the CVFF force field, Steepest Descent algorithm for
1000 iterations was used to minimize large steric interactions.
Then the chimera was energy minimized using the CVFF force
field, conjugate gradients until the maximum RMS derivative
was less than 0.001 kcal/Å. This final structure was compared
with the energy minimized wildtype â-ketoacyl synthase
following the same procedure as above.
General Methods. Air- and moisture-sensitive reactions
were run under inert atmosphere (Ar or N2) using flame-dried
glassware. Diisopropylamine, CH2Cl2, and triethylamine were
distilled from CaH2. THF was distilled from sodium benzophe-
none ketyl. n-Butyllithium in hexanes (nominally 1.6 M) was
purchased from Aldrich and titrated27 before use. All other
solvents were used as received or dried by standard proce-
dures.28 Elemental analyses were carried out by Atlantic
Microlab, Inc. (Norcross, GA). Low- and high-resolution data
were obtained on a VG Instruments 70-S GC/MS at 70 eV and
are tabulated as m/z (intensity expressed as percent of base
peak) or obtained from the Lab for Mass Spectrometry, Ohio
State University. 1H and 13C NMR were acquired on a Bruker
AMX 300 MHz or a Varian Unityplus 400 MHz spectrometer.
Chemical shifts are reported relative to residual CHCl3 (7.25
ppm, 1H; 77.0 ppm 13C). IR spectra were obtained on a Bruker
Vector 22 spectrophotometer. Melting points were determined
in a Thomas-Hoover melting point apparatus and are uncor-
rected. Purity for new target compounds was established by
1H NMR and/or elemental analyses.
General Procedure A. (()-2-(tert-Butyl)-5-(1-hydroxy-
2-methyl-2-pentenyl)-5-methyl-1,3-oxathiolan-4-one (10b).
To a mixture of diisopropylamine (0.6 mL, 4.6 mmol) in THF
(8.0 mL) at -78 °C was added n-BuLi (3.69 mL, 1.2 M in
n-hexane), and the resulting solution was stirred for 30 min
at 0 °C and then cooled to -78 °C. Then (()-912 (800 mg, 4.6
mmol) in THF at -78 °C was added dropwise by cannula and
the resulting solution stirred for 30 min at -78 °C. trans-2-
Methyl-2 pentenal (0.58 mL, 5.1 mmol) in THF (1.4 mL), at
-78 °C was then added via cannula. After stirring at -78 °C
for 1.5 h, 1 N HCl (25 mL) was added and the solution was
extracted with Et2O (3 × 30 mL). The combined organics were
dried (MgSO4), filtered, and evaporated. Flash chromatography
(10% EtOAc/hexanes) gave 10b (884 mg, 71%) as a 1.8:1
mixture of diastereomers at the C1′. 1H NMR (300 MHz,
CDCl3) δ 0.93-0.99 (m, 12 H), 1.40 (s, 3 H), 1.68 (s, 3 H), 2.01-
2.06 (m 2 H), 4.33 (d, J ) 6.9 Hz, 1 H), 5.24 (s, 1 H), 5.48-
5.54 (m, 1 H); 13C NMR (75 MHz, CDCl3) δ 12.6, 13.8, 20.9,
21.1, 24.8, 35.4, 60.6, 81.8, 87.9, 132.6, 133.9, 178.3. IR (NaCl)
2961, 1767 cm-1. Anal. (C14H24O3S) C, H.
1
136.5, 175.7. H NMR (300 MHz, CDCl3) minor diastereomer
1H NMR (300 MHz, CDCl3) δ 0.85 (t, J ) 7.2 Hz, 3 H), 0.97 (s,
9 H), 1.18-1.35 (m, 6 H), 1.40 (s, 3 H), 2.00-2.07 (m, 2 H),
2.31 (d, J ) 5 Hz, 1 H), 4.25-4.30 (m, 1 H), 5.27 (s, 1 H), 5.45-
5.59 (dd, J ) 7.7, 15.3 Hz, 1 H), 5.79-5.83 (m, 1 H); 13C NMR
(75 MHz, CDCl3) δ 13.7, 22.3, 23.9, 24.8, 28.5, 31.2, 32.1, 35.3,
61.1, 78.3, 87.8, 127.2, 137.2, 177.0. IR (NaCl) 2959, 1765 cm-1
Anal. (C16H28O3S) C, H.
.
General Procedure B. (()-2-(tert-Butyl)-5-(2-methyl-
penta-1,3-dienyl)-5-methyl-1,3-oxathiolan-4-one (11b). To
a solution of 10b (500 mg, 1.84 mmol) in Cl(CH2)2Cl (17 mL)
cooled to 0 °C were added NEt3 (0.6 mL, 4.4 mmol) and 2,4-
dinitrobenzenesulfenyl chloride (969 mg, 4.1 mmol). The
solution was warmed to room temperature for 30 min or until
TLC indicated complete formation of the diastereomeric
sulfenate esters. The mixture was then refluxed at 90 °C for
4 h or until complete conversion of the sulfenate ester was
indicated by TLC. After cooling to 0 °C, pentane (50 mL) was
added and this mixture was filtered through Celite and
evaporated. Flash chromatography (2% EtOAc/hexanes) gave
1
pure 11b (342 mg, 73%, 14:1 trans:cis at C1′). H NMR (300
MHz, CDCl3) δ 1.00 (s, 9 H), 1.70 (s, 3 H), 1.75 (d, J ) 6.6 Hz,
3 H), 1.85 (s, 3 H), 5.18 (s, 1 H), 5.57 (s, 1 H), 5.75 (dq, J )
6.6, 15.5 Hz, 1 H), 5.97 (d, J ) 15.5 Hz, 1 H); 13C NMR (75
MHz, CDCl3) d 13.0, 18.0, 25.2, 27.4, 34.8, 53.8, 87.4, 125.4,
129.3, 135.5, 137.8, 176.3. IR (NaCl) 2961, 1770 cm-1. HRMS
(EI) m/z calculated for C14H22O2S (M+) 254.1341, obsd 254.1309.
(()-2-(tert-Butyl)-5-(hexa-1,3-dienyl)-5-methyl-1,3-
oxathiolan-4-one (11c). From (()-10c (690 mg, 2.53 mmol)
following general procedure B, 11c (461 mg, 72%, 4:1 trans:
cis at C-1′) was obtained after flash chromatography (2%
EtOAc/hexanes). 1H NMR (300 MHz, CDCl3) δ 0.95-1.01 (m,
12 H), 1.61 (s, 3 H), 2.07-2.12 (m, 2 H), 5.05 (s, 1 H), 5.58 (d,
J ) 15.2 Hz, 1 H), 5.81 (dt, J ) 6.0, 15.2 Hz, 1 H), 6.00-6.05
(m, 1 H), 6.15-6.24 (dd, J ) 10.0, 15.2 Hz, 1 H); 13C (75 MHz,
CDCl3) δ 13.3, 24.8, 25.3, 25.7, 34.5, 56.1, 87.2, 127.4, 129.4,
130.0, 138.9, 175.1. IR (NaCl) 2966, 1771 cm-1. HRMS (ES)
m/z calculated for C14H22O2SNa+ (M + Na+) 277.1232, obsd
277.1237.
(()-2-(tert-Butyl)-5-(octa-1,3-dienyl)-5-methyl-1,3-
oxathiolan-4-one (11d). From (()-10d (306 mg, 1.00 mmol)
following general procedure B, 11d (212 mg, 75%, 4:1 trans:
cis at C-1′) was obtained after flash chromatography (2%
1
EtOAc/hexanes). H NMR (300 MHz, CDCl3) trans isomer δ
0.84-0.89 (m, 3 H), 1.01 (s, 9 H), 1.22-1.38 (m, 4 H), 1.61 (s,
3 H), 2.04-2.11 (m, 2 H), 5.03 (s, 1 H), 5.58 (d, J ) 15.1 Hz,
1 H), 5.64-5.78 (m, 1 H), 0.96-6.05 (m, 1 H), 6.19 (dd, J )
10.1, 15.1 Hz, 1 H). 13C NMR (75 MHz, CDCl3) trans isomer δ
13.6, 22.0, 22.5, 25.2, 31.2, 32.1, 34.6, 55.9, 87.0, 128.5, 129.6,
130.2, 137.2, 174.7. IR (NaCl) 2959, 1772 cm-1; HRMS (EI)
m/z calculated for C16H26O2S (M+) 282.1653, obsd 282.1681.
General Procedure C. (()-2-Thioacetyl-2,4-dimethyl-
hexa-3,5-dienoic Acid Ethyl Ester (12a). Cesium carbonate
(609 mg, 1.6 mmol) was added directly to a solution of 11a
(380 mg, 1.6 mmol) in EtOH (6.0 mL). After 20 min this
mixture was poured into NH4Cl(sat.)/1 N HCl (15 mL, 3:1) and
extracted with Et2O (3 × 20 mL) and then water (3 × 20 mL).
The combined organics were dried (MgSO4), filtered, evapo-
rated, and redissolved in CH2Cl2 (12 mL). To this precooled
solution at 0 °C were added NEt3 (0.22 mL, 1.0 mmol) and
acetyl chloride (0.11 mL, 1.6 mmol). After 40 min NH4Cl (sat)
(20 mL) was added, and this mixture was extracted with CH2-
Cl2 (3 × 15 mL). The combined organics were dried (MgSO4),
filtered, and evaporated. Flash chromatograpy (5% EtOAc/hex)
(()-2-(tert-Butyl)-5-(1-hydroxy-2-hexenyl)-5-methyl-
1,3-oxathiolan-4-one (10c). From (()-9 (800 mg, 4.59 mmol)
and 2-trans-hexenal (0.56 mL, 4.8 mmol) following general
procedure, 10c (1.3 g, 88%) was obtained after flash chroma-
tography (10% EtOAc/hexanes) as a 2.4:1 mixture of diaster-
1
eomers at C1′. H NMR (300 MHz, CDCl3) δ 0.87 (t, J ) 7.3
Hz, 3 H), 0.99 (s, 9 H), 1.38-1.45 (m, 2 H), 1.41 (s, 3 H), 2.02
(q, J ) 6.5 Hz, 2 H), 4.26-4.31 (m, 1 H), 5.27 (s, 1 H), 5.45-
5.63 (m, 1 H), 5.74-5.83 (m, 1 H); 13C NMR (75 MHz, CDCl3)
δ 13.6, 21.6, 24.1, 24.9, 35.2, 37.2, 61.2, 78.5, 87.9,127.3, 137.3,
179.1. IR (NaCl) 2960 1765 cm-1. Anal. (C14H24O3S), C, H.
(()-2-(tert-Butyl)-5-(1-hydroxy-2-octenyl)-5-methyl-1,3-
oxathiolan-4-one (10d). From (()-9 (800 mg, 4.59) and
2-trans-octenal (0.75 mL, 5.0 mmol) following general proce-
1
gave pure 12a (230 mg, 61%). H NMR (300 MHz, CDCl3) δ
1.25 (t, J ) 7.1 Hz, 3 H), 1.84 (s, 3 H), 1.87 (s, 3 H), 2.24 (s, 3

956 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
McFadden et al.
H), 4.21 (q, J ) 7.1 Hz, 2 H), 5.03 (d, J ) 10.6 Hz, 1 H), 5.21
(d, J ) 17.3 Hz, 1 H), 5.74 (s, 1 H), 6.26-6.35 (dd, J ) 10.6,
17.3 Hz, 1 H); 13C NMR (75 MHz, CDCl3) δ 12.9, 13.9, 25.9,
30.1, 55.8, 62.0, 113.3, 131.3, 138.3, 141.3, 182.3, 194.6. IR
(enol tautomer) δ 13.4, 18.4, 30.7, 59.2, 101.2 (m) 126.2, 128.4,
136.9, 140.6, 190.2, 197.6. IR (NaCl) 2929, 1593 cm-1; Found:
C, 61.8; H, 6.83. HRMS (ES) m/z calculated for C11H14O2SNa+
(M + Na+) 233.0607 obsd 233.0597; Anal. Calcd for C11H14-
O2S: C, 62.8; H, 6.71.
(NaCl) 2982, 1735, 1692 cm-1
.
(()-2-Thioacetyl-2,4-dimethyl-hepta-3,5-dienoic Acid
Ethyl Ester (12b). From 11b (369 mg, 1.5 mmol) and acetyl
chloride (0.10 mL, 1.5 mmol) following general procedure C
gave 12b (271 mg, 77%) after flash chromatography (5%
EtOAc/hexanes). 1H NMR (300 MHz, CDCl3) δ 1.26 (t, J ) 7.1
Hz, 3 H), 1.74 (d, J ) 6.6 Hz, 3 H), 1.81 (s, 3 H), 1.85 (s, 3 H),
2.25 (s, 3 H), 4.17 (q, J ) 7.1 Hz, 2 H), 5.56 (s, 1 H), 5.65-5.73
(dq, J ) 6.6, 15.5 Hz, 1 H), 5.99 (d, J ) 15.5 Hz, 1 H); 13C
NMR (75 MHz, CDCl3) δ 13.8, 14.1, 18.2, 26.2, 30.5, 55.6, 62.0,
125.2, 128.3, 135.7, 138.5, 172.2, 194.8. IR (NaCl) 2926, 1737,
1694 cm-1; HRMS (EI) m/z calculated for C13H20O3S (M+)
256.1133 obsd 256.1118.
(()-2-Thioacetyl-2-methyl-octa-3,5-dienoic Acid Ethyl
Ester (12c). From 11c (567 mg, 2.2 mmol) and acetyl chloride
(174 µL, 2.5 mmol) following general procedure C gave 12c
(374 g, 63%) after flash chromatography (5% EtOAc/hexanes).
1H NMR (300 MHz, CDCl3) δ 0.83 (t, J ) 7.3 Hz, 3 H), 1.24 (t,
J ) 7.1 Hz, 3 H), 1.72 (s, 3 H), 2.03-2.17 (m, 2 H), 2.25 (s, 3
H), 4.17 (q, J ) 7.1 Hz, 2 H), 5.72-5.81 (m, 2 H), 5.95-6.04
(dd, J ) 10.2, 15.3 Hz, 1 H), 6.18-6.27 (dd, J ) 10.2, 15.3 Hz,
1 H); 13C NMR (75 MHz, CDCl3) δ 13.2, 13.9, 22.8, 25.6, 30.2,
56.1, 61.9, 128.2, 128.4, 132.1, 138.5, 171.6, 194.8. IR (NaCl)
2929, 1736, 1693 cm-1; HRMS (ES) m/z calculated for C13H20O3-
SNa+ (M + Na+) 279.1025 obsd 279.1032.
(()-4-Hydroxy-5-methyl-5-hexa-1,3-dienyl-5H-thiophen-
2-one (13c). From 12c (364 mg, 0.46 mmol) following general
1
procedure D, 13c was obtained (180 mg, 60%). H (300 MHz,
CDCl3, exists as a mixture 2.3:1 of the keto:enol tautomer) keto
tautomer: δ 1.00 (t, J ) 7.4 Hz, 3 H); 1.76 (s, 3 H); 2.09-2.16
(m, 2 H); 3.21 (d, J ) 21.1 Hz, 1 H); 3.52 (d, J ) 21.1 Hz, 1 H);
5.70 (d, J ) 15.1 Hz, 1 H); 5.86 (dt, J ) 15.2, 6.4 Hz, 1 H),
6.02 (dd, J ) 10.2, 15.1 Hz, 1 H), 6.38 (dd, J ) 15.1, 10.1 Hz,
1 H); 1H NMR (300 MHz, MeOD) enol tautomer δ 1.09 (t, J )
7.4 Hz, 3 H), 1.87 (s, 3 H), 2.14-2.29 (m, 2 H), 5.78 (d, J )
15.1 Hz, 1 H), 5.87 (dt, J ) 15.2, 6.5 Hz, 1 H), 6.09-6.18 (dd,
J ) 10.2, 15.1, 1 H), 6.38 (dd, J ) 10.2, 15.2 Hz, 1 H); 13C
NMR (75 MHz, MeOD) enol tautomer δ 14.1, 25.2, 26.9, 61.0,
101 (m), 129.7, 131.7, 132.7, 138.9, 188.9, 197.1. IR (NaCl)
2965, 1592 cm-1; Found: C, 62.0; H, 6.94. HRMS (ES) m/z
calculated for C11H14O2SNa+ (M + Na+) 233.0607, obsd
233.0626. Anal. Calcd for C11H14O2S: C, 62.8; H, 6.71; Found:
C, 62.0; H, 6.94.
(()-4-Hydroxy-5-methyl-5-octa-1,3-dienyl-5H-thiophen-
2-one (13d). From 12d (62 mg, 0.22 mmol) following general
1
procedure D, 13d was obtained (21 mg, 41%). H NMR (300
MHz, CDCl3) (keto tautomer) δ 0.88 (t, J ) 6.9 Hz, 3 H), 1.19-
1.41 (m, 4 H), 1.75 (s, 3 H), 2.03-2.19 (m, 2 H), 3.22 (d, J )
21.1 Hz, 1 H), 3.51 (d, J ) 21.1 Hz, 1 H), 5.67 (d, J ) 15.1 Hz,
1 H), 5.80 (dt, J ) 6.9, 14.5 Hz, 1 H), 6.02 (dd, J ) 10.2, 15.1
Hz, 1 H), 6.37 (dd, J ) 10.2, 15.1 Hz, 1 H). 1H NMR (300 MHz,
MeOD) enol tautomer δ 0.97-1.03 (m, 3 H), 1.36-1.53 (m, 4
H), 1.87 (s, 3 H), 2.15-2.22 (m, 2 H), 5.78 (d, J ) 15.4 Hz, 1
H), 5.82-5.90 (m, 1 H), 6.10-6.19 (m, 1 H), 6.38 (dd, J ) 10.3,
15.4 Hz, 1 H); 13C (75 MHz, MeOD) enol tautomer δ 14.4, 23.3,
25.2, 32.6, 33.4, 60.9, 102.1 (m), 130.7, 131.7, 132,7, 137.5,
188.9, 196.9. IR (NaCl) 2927, 1588 cm-1; HRMS (ES) calculated
for C13H18O2SNa+ (M + Na+) 261.0911; obsd 261.0912; Anal.
Calcd for C13H18O2S: C, 65.5; H, 7.61; Found: C, 64.7; H, 7.68.
(()-2-Thioacetyl-2-methyl-deca-3,5-dienoic Acid Ethyl
Ester (12d). From 11d (200 mg, 0.71 mmol) and acetyl
chloride (55µL, 0.78 mmol) following general procedure C gave
12d (119 g 59%) after flash chromatography (5% EtOAc/
1
hexanes). H NMR (300 MHz, CDCl3) δ 0.84-0.89 (m, 3 H),
1.23 (t, J ) 7.1 Hz, 3 H), 1.28-1.38 (m, 4 H), 1.71 (s, 3 H),
2.01-2.08 (m, 2 H), 2.23 (s, 3 H), 4.18 (q, J ) 7.1 Hz, 2 H),
5.66-5.76 (m, 2 H), 5.89-6.03 (m, 1 H), 6.20 (dd, J ) 10.3,
15.3 Hz, 1 H).; 13C NMR (75 MHz, CDCl3) δ 13.8, 13.9, 22.2,
22.8, 29.9, 31.2, 32.3, 56.1, 61.9, 128.4, 129.2, 132.2, 137.1,
171.6, 194.6. IR (NaCl) 2930, 1737, 1694 cm-1. HRMS (ES)
m/z calculated for C15H24O3SNa+ (M + Na+) 307.1338 obsd
307.1339.
(()-5-Benzyl-4-hydroxy-5-methyl-5H-thiophen-2-one (26).
From 25 (1.4 gm, 5.0 mmol) following general procedure D,
26 (500 mg, 45%) was obtained. 1H NMR (300 MHz, CDCl3) δ
1.71 (s, 3 H), 2.89 (ab q, J ) 21.7 Hz, 2 H), 3.17 (ab q, J ) 13.6
Hz, 2 H), 7.26 (m, 5 H); 13C NMR (75 MHz, CDCl3) δ 26.2,
46.6, 48.5, 67.9, 127.7, 128.6, 130.6, 134.9, 195.3, 207.3. HRMS
(ES) calculated for C12H12O2SNa+ (M + Na+) 243.0450; obsd
243.0433.
General Procedure E. (()-2-tert-Butyl-5-methyl-5-
hexyl-[1,3]-oxathiolan-4-one (14a). To a mixture of LiH-
MDS (6.2 mL, 6.20 mmol, 1 M in THF) in THF (9.7 mL) at
-78 °C was added (()-9 (1.00 g, 5.75 mmol) in THF (9.60 mL)
by cannula dropwise, and the resulting solution was stirred
for 30 min at -78 °C. Then, hexyl triflate (1.1 g, 4.7 mmol) in
THF (4 mL) at -78 °C was added via cannula. After stirring
at -78 °C for 2 h, 1 N HCl (10 mL) was added and the solution
was extracted with Et2O (3 × 15 mL). The combined organics
were dried (MgSO4), filtered, and evaporated. Flash chroma-
tography (2% EtOAc/hexanes) gave pure 14a as a 2:1-6:1
mixture of separable diastereomers (1.00 g, 83%). 1H NMR (300
MHz, CDCl3) δ 0.87 (t, J ) 6.5 Hz, 3 H), 0.99 (s, 9 H), 1.24-
1.29 (m, 8 H), 1.54 (s, 3 H), 1.72-1.80 (m, 2 H), 5.13 (s, 1 H);
13C NMR (75 MHz, CDCl3) δ 13.9, 22.5, 24.9, 24.9, 25.1, 25.9,
29.1, 31.6, 41.2, 55.3, 86.7, 177.8. HRMS (EI) m/z calculated
for C14H26O2S+ (M+) 258.1654 obsd 258.1654; Anal. (C14H26O2S)
C, H.
General Procedure D. (()-4-Hydroxy-5-methyl-5-(2-
methyl-buta-1,3-dienyl)-5H-thiophen-2-one (13a). To 12a
(263 mg, 1.09 mmol) in THF (16.4 mL) at -78 °C was added
LiHMDS (1.8 mL, 1.8 mmol, 1.0 M in THF), and the solution
was allowed to slowly warm to -5 °C. The solution was then
poured into 1 N HCl (25 mL) and extracted with EtOAc (3 ×
15 mL). The combined organics were dried (MgSO4), filtered,
and evaporated. This crude mixture was taken up in NaHCO3
(sat, 15 mL) and extracted with Et2O (3 × 10 mL). The aqueous
layer was then acidified to pH 3 (pH paper) with 1 N HCl and
extracted with Et2O (3 × 10 mL) and EtOAc (2 × 10 mL). The
combined organics were dried (MgSO4), filtered, and evapo-
rated to provide pure 13a (114 mg, 53%). 1H NMR (300 MHz,
CDCl3) (keto tautomer) δ 1.78 (s, 3 H), 1.86 (s, 3 H), 3.43 (d, J
) 5.6 Hz, 2 H), 5.12 (d, J ) 10.6 Hz, 1 H), 5.27 (d, J ) 17.3
Hz, 1 H), 5.83 (s, 1 H), 6.27-6.37 (dd, J ) 10.6, 17.3 Hz, 1 H).
1H NMR (300 MHz, MeOD) (enol tautomer) δ 1.79 (s, 3 H),
1.84 (s, 3 H), 5.04 (d, J ) 10.7 Hz, 1 H), 5.25 (d, J ) 17.3 Hz,
1 H), 5.66 (s, 1 H), 6.36 (dd, J ) 10.7, 17.3 Hz, 1 H); 13C NMR
(75 MHz, MeOD) δ 12.6, 30.4, 59.0, 102 (m), 116.9, 131.4,
140.6, 142.3, 189.9, 197.3. HRMS (EI) m/z calculated for
C10H12O2S+ (M+) 196.0552 obsd 196.0552. Anal. Calcd for
C10H12O2S: C, 61.2; H, 6.16; Found: C, 59.5; H, 6.26.
(()-4-Hydroxy-5-methyl-5 (2-methyl-penta-1,3-dienyl)-
5H-thiophen-2-one (13b). From 12b (226 mg, 0.9 mmol)
following general procedure D, 13b (95 mg, 49%) was obtained.
1H NMR (300 MHz, CDCl3) (keto-tautomer) δ 1.75 (s, 3 H),
1.77 (d, J ) 3.2 Hz, 3 H), 1.84 (s, 3 H), 3.42 (d, J ) 1.5 Hz, 2
H), 5.66 (bs, 1 H), 5.78 (m, 1 H), 6.04 (d, J ) 15.4 Hz, 1 H); 1H
NMR (300 MHz, MeOD) (enol tautomer) δ 1.80-1.85 (m, 6
H), 1.90 (s, 3 H), 5.59 (s, 1 H), 5.80-5.95 (dq, J ) 6.6, 15.5 Hz,
1 H), 6.17 (d, J ) 14.9 Hz, 1 H); 13C NMR (75 MHz, MeOD)
(()-2-tert-Butyl-5-methyl-5-octyl-[1,3]-oxathiolan-4-
one (14b). From (()-9 (1.00 g, 5.8 mmol) and octyl triflate
(1.6 g, 6.20 mmol) following general procedure E, 14b (1.33
mg, 81%) was obtained (2% EtOAc/hexanes). 1H NMR (300
MHz, CDCl3) δ 0.86 (t, J ) 6.5 Hz, 3 H), 0.99 (s, 9 H), 1.24-
1.26 (m, 12 H), 1.54 (s, 3 H), 1.72-1.84 (m, 2 H), 5.13 (s, 1 H);
13C NMR (75 MHz,CDCl3) δ 13.9, 22.6, 24.9, 25.1, 25.9, 29.2,
29.3, 29.5, 31.8, 35.2, 41.2, 55.3, 86.5, 177.7. IR (NaCl) 3443,

Inhibitors of Type I Fatty Acid Synthase
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 957
1
2929, 1829, 1769 cm-1; HRMS (EI) m/z calculated for
C16H30O2S+ (M+) 286.1967 obsd 286.1969; Anal. (C16H30O2S)
C, H.
2.26 (m, 2 H), 3.35 (s, 2 H). H NMR (300 MHz, MeOD) (enol
tautomer) δ 0.89 (t, J ) 6.5 Hz, 3 H), 1.21-1.36 (m, 7 H), 1.46-
1.54 (m, 1 H), 1.64 (s, 3 H), 1.80-1.90 (m, 2 H); 13C NMR (75
MHz, MeOD) δ 14.6, 23.8, 26.3, 27.1, 30.5, 32.9, 39.8, 61.3,
103.5 (m), 189.8, 197.8. Anal. (C11H18O2S) C, H.
General Procedure F. (()-2-Acetylsulfanyl-2-methyl-
octanoic Acid Ethyl Ester (15a). To 14a (940 mg, 3.6 mmol)
in EtOH (14.6 mL) was added NaOEt (2.1 M, 2.3 mL, 4.72
mmol) [freshly prepared from Na metal (200 mg, 8.3 mmol)
in EtOH (4.0 mL)], and the solution was allowed to stir at room
temperature. After 2 h, the solution was poured into NH4-
Cl(sat)/1 N HCl (25 mL, 3:1) and extracted with Et2O (3 × 20
mL). The combined organics were then washed with H2O (3
× 25 mL), dried (MgSO4), filtered, evaporated, and redissolved
in CH2Cl2 (26 mL). To this precooled solution (0 °C) were added
NEt3 (0.5 mL, 3.6 mmol) and acetyl chloride (0.26 mL, 3.6
mmol). After 40 min at 0 °C, NH4Cl(sat) (30 mL) was added
and the solution was extracted with CH2Cl2. The combined
organics were dried (MgSO4), filtered, and evaporated. Flash
chromatography (5% EtOAc/hexanes) gave pure 15a (943 mg,
(()-4-Hydroxy-5-methyl-5-octyl-5H-thiophen-2-one(16b).
From 15b (500 mg, 1.7 mmol) following general procedure G,
16b (308 mg, 73%) was obtained after flash chromatography
(20% EtOAc/2% CH3CO2H/hexanes). 1H NMR (300 MHz,
CDCl3) (keto-tautomer) δ 0.86 (t, J ) 6.7 Hz, 3 H), 1.19-1.24
(m, 10 H), 1.48-1.53 (m, 2 H), 1.65 (s, 3 H), 1.77-1.85 (m, 1
H), 1.94-2.01 (m, 1 H), 3.36 (s, 2 H); 1H NMR (300 MHz,
MeOD) (enol tautomer) 0.87-0.89 (m, 3 H), 1.29 (m, 10 H),
3.29 (s, 3 H), 1.81-1.87 (m, 2 H); 13C NMR (75 MHz, MeOD)
(enol tautomer) δ 14.7, 23.8, 26.4, 27.1, 30.5, 30.6, 30.8, 33.2,
39.8, 61.3, 103.1 (m), 189.8, 197.8. IR (NaCl) 3422, 1593 cm-1
Anal. (C13H22O2S), C, H.
;
(()-4-Hydroxy-3,5-dimethyl-5-octyl-5H-thiophen-2-
one (16c). From 15c (469 mg, 1.55 mmol) and NaHMDS (3.87
mL, 3.87 mmol, 1.0 M in THF) following general procedure G,
1
77%). H NMR (300 MHz, CDCl3) d 0.86 (t, J ) 6.9 Hz, 3 H),
1.22-1.27 (m, 11 H), 1.61 (s, 3 H), 1.75-1.79 (m, 2 H), 2.25 (s,
3 H), 4.17 (q, J ) 7.1 Hz, 2 H); 13C NMR (75 MHz, CDCl3) δ
13.9, 14.1, 22.4, 23.4, 24.4, 29.3, 30.3, 31.5, 38.4, 55.7, 61.5
173.0, 194.7. IR (NaCl) 3449, 1736, 1694 cm-1; Anal. (C13H24O3S)
C, H.
1
16c (397 mg, 70%) was obtained. H NMR (300 MHz, CDCl3)
(enol tautomer) δ 0.86 (t, J ) 6.8 Hz, 3 H), 1.23 (s, 11 H), 1.30-
1.45 (m, 1 H), 1.59 (s, 3 H), 1.74 (s, 3 H), 1.84-1.88 (m, 2 H);
13C NMR (75 MHz, CDCl3) δ 7.48, 14.0, 22.6, 25.2, 25.9, 29.2,
29.4, 29.6, 31.8, 38.5, 58.2, 110.5, 180.9, 198.0. IR (NaCl) 2927,
1601 cm-1. HRMS (ES) calculated for C14H24O2SNa+ (M + Na+)
279.1389; obsd 279.1380; Anal. (C14H24O2S), C, H.
(()-2-Acetylsulfanyl-2-methyl-decanoic Acid Ethyl Es-
ter (15b). From 14b (904 mg, 3.2 mmol) and acetyl chloride
(0.2 mL, 3.3 mmol) following general procedure F, 15b (725
mg, 80%) was obtained after flash chromatography (5% EtOAc/
(()-4-Hydroxy-3-(2-propenyl)-5-methyl-5-octyl-5H-
thiophen-2-one (16d). From 15d (1.29 g, 6.04 mmol) follow-
ing general procedure G, 16d (629 mg, 57%) was obtained. 1H
NMR (300 MHz, CDCl3) δ 0.85 (t, J ) 6.9 Hz, 3 H), 1.24 (m,
12 H), 1.65 (s, 3 H), 1.81-1.86 (m, 2 H), 3.02 (d, J ) 6.4 Hz,
2 H), 5.12 (dq, J ) 10.6, 1.5 Hz, 1 H), 5.20 (dq, J ) 17.3, 1.5
Hz, 1 H), 5.84 (ddt, J ) 10.6, 17.3, 6.4 Hz, 1H). 13C NMR (100
MHz, CDCl3) δ 14.1, 22.6, 25.2, 26.1, 26.9, 29.1, 29.3, 29.5,
31.8, 38.5, 57.5, 111.5, 117.4, 134.4, 180.8, 195.4.
1
hexanes). H NMR (300 MHz, CDCl3) δ 0.87 (t, J ) 6.9 Hz, 3
H),; 1.22-1.27 (m, 15 H), 1.61 (s, 3 H), 1.75-1.84 (m, 2 H),
2.26 (s, 3 H), 4.18 (q, J ) 7.1 Hz, 2 H); 13C NMR (75 MHz,
CDCl3) δ 13.9, 14.1, 22.6, 23.4, 24.4, 29.1, 29.2, 29.6, 30.3, 31.8,
38.3, 55.8, 61.5, 173.1, 195.8. IR (NaCl) 3430, 1868, 1693, 1644
cm-1; Anal. (C15H28O3S) C, H.
(()-2-Propionylsulfanyl-2-methyldecanoic Acid Ethyl
Ester (15c). From 14b (613 mg, 2.14 mmol) and propionyl
chloride (0.19 mL, 2.14 mmol) following general procedure F,
15c (484 mg, 75%) was obtained after flash chromatography
General Procedure H. (()-4- Methoxy-5-methyl-5-hexyl-
5H-thiophen-2-one (17a). To 16a (40 mg, 0.19 mmol) in DMF
(0.8 mL) cooled to -40 °C was added NaH (11 mg, 0.26 mmol,
60% in mineral oil), and the solution was allowed to warm and
stir at 0 °C for 30 min. Dimethyl sulfate (35 µL, 0.38 mmol)
was then added directly and the mixture was allowed to warm
and stir for 2.5 h at room temperature. NH4Cl(sat)/1 N HCl (3:
1, 10 mL) was added and the solution was extracted with Et2O
(3 × 10 mL). The combined organics were washed with H2O
(3 × 15 mL), dried (MgSO4), filtered, and evaporated. Flash
chromatography (15% EtOAc/hexanes) gave pure 17a (24.7 mg,
1
(5% EtOAc/hexanes). H NMR (300 MHz, CDCl3) δ 0.84 (t, J
) 6.9 Hz, 3 H), 1.10 (t, J ) 7.5 Hz, 3 H), 1.19-1.24 (m, 15 H),
1.58 (s, 3 H), 1.72-1.77 (m, 2 H), 2.48 (q, J ) 7.5 Hz, 2 H),
4.17 (q, J ) 7.1 Hz, 2 H); 13C NMR (75 MHz, CDCl3) δ 9.45,
14.1, 14.1, 22.6, 23.5, 24.5, 29.1, 29.3, 29.7, 31.8, 36.9, 38.5,
55.5, 61.4, 173.2, 199.2. Anal. (C16H30O3S) C, H.
(()-2-(4-Pentenoyl)sulfanyl-2-methyldecanoic Acid Eth-
yl Ester (15d). From 14b (1.3 g, 4.54 mmol) and 4-pentenoyl
chloride (0.65 mL, 5.90 mmol) following general procedure F,
15d (1.29 g, 86%) was obtained after flash chromatography
1
58%). H NMR (300 MHz, CDCl3) δ 0.86 (t, J ) 6.7 Hz, 3 H),
1
(5% EtOAc/hexanes). H NMR (300 MHz, CDCl3) d 0.86 (t, J
1.08-1.13 (m, 1 H), 1.24 (s, 6 H), 1.35-1.39 (m, 1 H), 1.61 (s,
3 H), 1.75-1.82 (m, 2 H), 3.81 (s, 3 H), 5.30 (s, 1 H); 13C NMR
(75 Mz, CDCl3) δ 14.0, 22.5, 25.1, 26.4, 29.2, 31.5, 38.9, 59.4,
59.4, 101.3, 187.3, 193.8. HRMS (ES) calculated for C12H20O2-
SNa+ (M + Na+) 251.1076; obsd 251.1076; Anal. (C12H20O2S),
C, H
) 6.9 Hz, 3 H), 1.23 (m, 15 H), 1.60 (s, 3 H), 1.76-1.78 (m, 2
H), 2.34-2.36 (m, 2 H), 2.53-2.59 (m, 2 H), 4.16 (q, J ) 7.2
Hz, 2 H), 4.98 (d, J ) 10.3 Hz, 1 H), 5.01 (d, J ) 17.6 Hz, 1 H),
5.77 (ddt, J ) 10.3, 17.6, 6.3 Hz, 1 H).
(()-2-Acetylsulfanyl-2-methyl-3-phenyldecanoic Acid
Ethyl Ester (25). From 5-benzyl-2-tert-butyl-5-methyl-[1,3]-
oxathiolan-4-one11 (1.2 g, 4.7 mmol) following general proce-
dure F, 25 (954 mg, 76%) was obtained after flash chroma-
(()-4-Methoxy-5-methyl-5-octyl-5H-thiophen-2-one(17b).
From 16b (70 mg, 0.27 mmol) and dimethyl sulfate (50 µL,
0.55 mmol) following general procedure H, 17b (59 mg, 80%)
was obtained was obtained after flash chromatography (15%
EtOAc/hexanes). 1H NMR (300 MHz, CDCl3) δ 0.85 (t, J ) 7.1
Hz, 3 H); 1.07-1.18 (m, 1 H), 1.23 (s, 10 H), 1.43-1.49 (m, 1
H), 1.61 (s, 3 H), 1.74-1.81 (m, 2 H), 3.81 (s, 3 H), 5.29 (s, 1
H); 13C NMR (75 MHz, CDCl3) δ 14.0, 22.6, 25.1, 26.4, 29.1,
29.3, 29.5, 31.8. 38.8, 59.3, 59.4, 101.3, 187.3, 193.8. IR (NaCl)
2927, 1682, 1608 cm-1. Anal. (C14H24O2S) C, H.
1
tography (5% EtOAc/hexanes). H NMR (300 MHz, CDCl3) δ
1.19 (t, J ) 7.2 Hz, 3 H), 1.55 (s, 3 H), 2.26 (s, 3 H), 3.13 (q, J
) 11.9 Hz, 2 H), 4.13 (q, J ) 7.2 Hz, 2 H), 7.1 (m, 2 H), 7.2 (m,
3 H); 13C NMR (75 MHz, CDCl3) δ 14.0, 23.1, 30.3, 43.6, 56.3,
61.7, 127.2, 128.1, 130.7, 135.4, 172.8, 194.8. HRMS (ES)
calculated for C14H18O3SNa+ (M + Na+) 289.0869; obsd
289.0892.
General Procedure G. (()-4-Hydroxy-5-methyl-5-hexyl-
5H-thiophen-2-one (16a). To 15a (715 mg, 2.8 mmol) in
toluene (43 mL) at -78 °C was added LiHMDS (6.3 mL, 6.3
mmol, 1.0 M in THF), and the solution was allowed to slowly
warm to -5 °C. The solution was then poured into 1 N HCl
(40 mL) and extracted with EtOAc (3 × 25 mL). The combined
organics were dried (MgSO4), filtered, and evaporated. Flash
chromatography (20% EtOAc/2% CH3CO2H/hexanes) gave 16a
(()-4-Methoxy-3,5-dimethyl-5-octyl-5H-thiophen-2-
one (17c); (()-5-Methoxy-2,4-dimethyl-2-octyl-2,3-dihy-
dro-3-thiophenone (17m). From 16c (50 mg, 0.17 mmol), and
dimethyl sulfate (28 µL, 0.30 mmol) following general proce-
1
dure H, 17c/17m (2.2:1, 39 mg, 72%) was obtained H NMR
(300 MHz, CDCl3) (17c) δ 0.86 (t, J ) 6.3 Hz, 3 H), 1.06-1.09
(m, 1 H), 1.24 (bs, 10 H), 1.41-1.48 (m, 1 H), 1.55 (s, 3 H),
1.71-1.79 (m, 2 H), 1.98 (s, 3 H), 4.09 (s, 3 H); (17m) d 0.86 (t,
J ) 6.3 Hz, 3 H), 1.06-1.09 (m, 1 H), 1.24 (bs, 10 H), 1.41-
1.48 (m, 1H), 1.48 (s, 3H), 1.71-1.79 (m, 2 H), 1.66 (s, 3 H),
1
(402 mg, 69%). H NMR (300 MHz, CDCl3) δ (keto tautomer)
0.86 (t, J ) 6.8 Hz, 3 H), 1.27 (bs, 8 H), 1.68 (s, 3 H), 1.94-

958 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
McFadden et al.
4.03 (s, 3 H); 13C NMR (100 MHz, CDCl3) (17c) δ 9.59, 14.1,
22.6, 25.1, 26.5, 29.2, 29.3, 29.5, 31.8, 38.9, 57.3, 59.8, 111.3,
180.1, 195.8. (17m) d 7.72, 9.57, 14.1, 22.6, 25.1, 25.3, 29.2,
29.5, 29.6, 38.9, 58.8, 64.8, 107.4, 184.9, 202.7. (17c/17m
mixture): IR (NaCl) 2927, 1676, 1631, 1582 cm-1. HRMS (ES)
calculated for C15H26O2SNa+ (M + Na+) 293.1545; obsd
293.1552. Anal. (C15H26O2S) C, H.
193.9. HRMS (ES) calcd for C14H22O2SNa+ (M + Na+) 277.1232;
obsd 277.1241; Anal. (C14H22O2S) C, H. (17t): 1H NMR (300
MHz, CDCl3) d 0.86 (t, J ) 6.8 Hz, 3 H), 1.24 (bs, 8 H), 1.54
(s, 3 H), 1.81-1.84 (m, 2 H), 2.42-2.48 (m, 2 H), 5.05-5.10
(m, 4 H), 5.56-5.67(m, 2 H).
(()-4-Allyloxy-5-methyl-5-octyl-5H-thiophen-2-one (17i).
From 16b (197 mg, 0.91 mmol) and allyl bromide (140 µL, 1.62
mmol) following general procedure H, 17i and C-3 dialkylated
17q (173 mg, 76%) were obtained as a 3.5:1 mixture which
could be separated and purified using flash chromatography
(15% EtOAc/hexanes). (17i) (O-alkylation) 1H NMR (300 MHz,
CDCl3) δ 0.86 (t, J ) 6.8 Hz, 3 H), 1.12-1.17 (m, 1 H), 1.24 (s,
10 H), 1.45-1.49 (m, 1 H), 1.64 (s, 3 H), 1.77-1.84 (m, 2 H),
4.47 (d, J ) 5.6 Hz, 2 H), 5.29 (s, 1 H), 5.31 (d, J ) 10.4 Hz,
1 H), 5.39 (d, J ) 16.9 Hz, 1 H), 5.90-5.99 (ddd, J ) 5.6, 10.4,
16.9 Hz, 1 H); 13C NMR (75 MHz, CDCl3) δ 14.1, 22.6, 25.1,
26.5, 29.2, 29.3, 29.5, 31.8, 38.9, 59.7, 72.8, 102.0, 119.5, 130.8,
185.8, 193.8. IR (NaCl) 3441, 1682, 1608 cm-1. Anal. (C16H26O2S)
C, H.
(()-5-Benzyl-4-methoxy-5-methyl-5H-thiophen-2-one
(27). From 26 (50 mg, 0.23 mmol), and dimethyl sulfate (44
µL, 0.45 mmol) following general procedure H, 27 (38 mg, 74%)
1
was obtained. H NMR (300 MHz, CDCl3) δ 1.65 (s, 3 H), 3.1
(q, J ) 7.1 Hz, 2 H), 3.84 (s, 3 H), 5.19 (s, 1 H), 7.21 (m, 5 H);
13C NMR (75 MHz, CDCl3) δ 26.0, 45.0, 59.3, 59.9, 101.9, 127.2,
128.0, 130.4, 135.9, 186.5, 192.9. HRMS (ES) calcd for C13H14O2-
SNa+ (M + Na+) 257.0606; obsd 257.0615; Anal. (C13H14O2S)
C, H
(()- 5-Methyl-5-hexyl-2-oxo-thiophen-4-yloxy)acetic
Acid Ethyl Ester (17d). From 16a (20 mg, 0.09 mmol) and
ethyl bromoacetate (20 µL, 0.2 mmol) following general
procedure H, 17d (18 mg, 67%) was obtained after flash
chromatography (15% EtOAc/hexanes). 1H NMR (300 MHz,
CDCl3) d 0.86 (t, J ) 6.8 Hz, 3 H), 1.24-1.27 (m, 7 H), 1.32 (t,
J ) 7.1 Hz, 3 H), 1.47-1.48 (m, 1 H), 1.68 (s, 3 H), 1.84-1.88
(m, 2 H); 4.25 (q, J ) 7.1 Hz, 2 H), 4.54 (s, 2 H), 5.21 (s, 1 H);
13C NMR (75 MHz, CDCl3) δ 14.1, 14.1, 22.5, 25.1, 26.4, 29.2,
31.6, 38.9, 59.7, 61.9, 68.0, 102.3, 166.2, 185.3, 193.3. IR (NaCl)
2932, 1762, 1682, 1612 cm-1. HRMS (ES) calcd for C15H24O4-
SNa+ (M + Na+) 323.1287; obsd 323.1286; Anal. (C15H24O4S)
C, H.
(()-3-(2-Propenyl)-3,5-Dimethyl-5-octyl-thiophene-2,4-
dione (17n); (()-4-Allyloxy-3,5-dimethyl-5-octyl-5H-thio-
phen-2-one (17o)/5-Allyloxy-2,4-dimethyl-2-octyl-2,3-di-
hydro-3-thiopenone (17p). From 16c (70.1 mg, 0.27 mmol)
and allyl bromide (47 µL, 0.55 mmol) following general
procedure H, 17n and 17o,p (17o:17p, 2.6:1) (82% overall)
were obtained as a 2.3:1 mixture. 17n was easily separated
from the C-4 and C-2 O-alkylated mixture (17o, 17p) using
flash chromatography (20% EtOAc/hexanes).
1
(17n). H NMR (300 MHz, CDCl3) δ 0.86 (t, J ) 6.6 Hz, 3
H), 1.16-1.47 (m, 15 H), 1.57 (s, 3 H), 1.74-1.96 (m, 2 H),
2.42-2.46 (m, 2 H),. 5.04-5.10 (m, 2 H), 5.53-5.67 (m, 1 H).
13C NMR (100 MHz, CDCl3) δ 14.0, 22.2, 22.6, 25.5, 26.2, 29.1,
29.2, 29.5, 31.7, 41.3, 42.0, 58.4, 65.1, 120.2, 131.4, 204.5, 214.6.
IR (NaCl) 2928, 1742, 1698 cm-1. Anal. Calcd for C17H28O2S:
C, 68.9; H, 9.52; Found: C, 69.8; H, 9.85
(()-5-Methyl-5-octyl-2-oxo-thiophen-4-yloxy)acetic Acid
Ethyl Ester (17e). From 16b (39 mg, 0.16 mmol) and ethyl
bromoacetate (36 µL, 0.32 mmol) following general procedure
H, 17e (39 mg, 73%) was obtained after flash chromatography
(15% EtOAc/hexanes). 1H NMR (300 MHz, CDCl3) δ 0.86 (t, J
) 6.6 Hz, 3 H), 1.24 (s, 11 H), 1.29 (t, J ) 7.2 Hz, 3 H), 1.47-
1.48 (m, 1 H), 1.68 (s, 3 H), 1.85-1.88 (m, 2 H), 4.25 (q, J )
7.1 Hz, 2 H), 4.54 (s, 2 H), 5.20 (s, 1 H); 13C NMR (75 MHz,
CDCl3) δ 14.1, 14.1, 22.6, 25.1, 26.4, 29.2, 29.3, 29.5, 31.8, 38.8,
59.7, 61.9, 67.9, 102.3 166.2, 185.3, 193.4. IR (NaCl) 2928,
1762, 1682, 1612 cm-1. Anal. (C17H28O4S), C, H.
(()-4-(4-Chlorobutoxy)-5-methyl-5-hexyl-5H-thiophen-
2-one (17f). From 16a (36 mg, 0.17 mmol) and 3-iodo-1-
chlorobutane (40 µL, 0.34 mmol) following general procedure
H, 17f (32 mg, 75%) was obtained after flash chromatography
(20% EtOAc/hexanes). 1H NMR (400 MHz, CDCl3) δ 0.86 (t, J
) 5.1 Hz, 3 H), 1.09-1.14 (m, 1 H), 1.25 (s, 6 H), 1.44-1.53
(m, 1 H), 1.63 (s, 3 H), 1.77-1.85 (m, 2 H), 1.90-2.00 (m, 4
H), 3.59 (t, J ) 4.5 Hz, 2 H), 3.95-3.99 (m, 2 H), 5.28 (s, 1 H).
13C NMR (75 MHz, CDCl3) δ 13.7, 22.3, 25.1, 26.1, 26.4, 29.1,
29.1, 31.5, 39.0, 43.9, 59.5, 71.6, 101.5, 185.9, 192.9. IR (NaCl)
2927, 1683, 1607 cm-1. Anal. (C15H25ClO2S), C, H.
1
(17o/17p mixture) H NMR (300 MHz, CDCl3) δ 0.86 (t, J
) 6.3 Hz, 3 H), 1.06-1.48 (m, 12 H), 1.58 (s, 3 H), 1.71-1.82
(m, 2 H), 1.94 (s, 3 H), 4.80-4.82 (m, 2 H), 5.28-5.40 (m, 2
H), 5.89-6.03 (m, 1 H); (17p) d 0.86 (t, J ) 6.3 Hz, 3 H), 1.06-
1.48 (m, 12 H), 1.49 (s, 3 H), 1.71-1.82 (m, 2 H), 1.69 (s, 3 H),
4.73-4.75 (m, 2 H), 5.32-5.46 (m, 2 H), 5.89-6.03 (m, 1 H).
(17o) 13C NMR (75 MHz, CDCl3) δ 9.65, 14.0, 22.6, 25.2, 26.6,
29.2, 29.3, 29.6, 31.8, 39.0, 57.5, 72.5, 111.8, 118.2, 132.6, 179.4,
195.7. (17p) 9.65, 14.0, 22.6, 25.2, 26.6, 29.2, 29.3, 29.6, 31.8,
39.0, 64.9, 75.5, 107.9, 119.5, 131.3, 185.1, 202.8. (17o/17p
mixture): IR (NaCl) 2855, 1676, 1628, 1580 cm-1; Anal.
(C17H28O2S) C, H.
(()-5-Methyl-5-4-prop-2-ynyloxy-5H-thiophen-2-one (17j).
From 16a (45 mg, 0.21 mmol) and propargyl bromide (37 µL,
0.21 mmol) following general procedure H, 17j (21 mg, 40%)
1
was obtained. H NMR (300 MHz, CDCl3) d 0.86 (t, J ) 6.9
Hz, 3 H), 1.11-1.20 (m, 1 H), 1.24 (s, 6 H), 1.41-1.49 (m, 1
H), 1.63 (s, 3 H), 1.76-1.86 (m, 2 H), 2.59 (t, J ) 2.5 Hz, 1 H),
4.62 (d, J ) 3.7 Hz, 1 H), 4.63 (d, J ) 3.7 Hz, 1 H), 5.43 (s, 1
H); 13C NMR (100 MHz, CDCl3) δ 13.9, 22.5, 24.8, 26.4, 29.0,
31.5, 38.7, 59.3, 59.5, 75.6, 77.4, 102.9, 184.4,193.6. IR (NaCl)
(()-4-(4-Chlorobutoxy)-5-methyl-5-octyl-5H-thiophen-
2-one (17g). From 16b (47 mg, 0.18 mmol) and 3-iodo-1-
chlorobutane (40 µL, 0.36 mmol) following general procedure
H, 17g (46 mg, 85%) was obtained after flash chromatography
(20% EtOAc/hexanes). 1H NMR (300 MHz, CDCl3) δ 0.86 (t, J
) 7.3 Hz, 3 H), 1.07-1.27 (m, 1 H), 1.24 (s, 10 H) 1.48-1.51
(m, 1 H), 1.62 (s, 3 H), 1.75-1.82 (m, 2 H), 1.89-1.98 (m, 4
H), 3.59 (t, J ) 5.9 Hz, 2 H), 3.95-3.98 (m, 2 H), 5.28 (s, 1 H);
13C NMR (75 MHz, CDCl3) δ 14.1, 22.6, 25.1, 26.0, 26.5, 29.0,
29.2, 29.3, 29.5, 29.7, 31.8, 44.1, 59.6, 71.7, 101.6, 186.1, 193.8.
IR (NaCl) 2926, 1682, 1608 cm-1. Anal. (C17H29ClO2S) C, H.
(()-4-Allyloxy-5-methyl-5-hexyl-5H-thiophen-2-one(17h).
From 16a (270 mg, 1.30 mmol) and allyl bromide (0.2 mL, 2.52
mmol) following general procedure H, 17h and C-3 dialkylated
17t were obtained (205 mg, 58%) as a 3:1 (17h:17t) mixture
which could be separated and purified using flash chromatog-
raphy (15% EtOAc/hexanes). 1H NMR (300 MHz, CDCl3) (17h)
(O-alkylation) δ 0.84 (t, J ) 6.9 Hz, 3 H), 1.09-1.17 (m, 1 H),
1.23 (s, 6 H), 1.40-1.51 (m, 1 H), 1.62 (s, 3 H), 1.73-1.83 (m,
2 H), 4.46 (d, J ) 5.6 Hz, 2 H), 5.33 (d, J ) 10.0 Hz, 1 H), 5.38
(d, J ) 17.3 Hz, 1 H), 5.28 (s, 1 H), 5.87-5.98 (ddd, J ) 5.6,
10.2, 17.3 Hz, 1 H); 13C NMR (75 MHz, CDCl3) δ 14.0, 22.5,
25.1, 26.5, 29.2, 31.6, 38.9, 59.7, 72.8, 101.9, 119.6, 130.7, 185.8,
2130, 1676, 1607 cm-1
.
(()-5-Methyl-5-hexyl-2-oxo-thiophen-4-yloxy)acetic Acid
tert-Butyl Ester (17k). From 16a (169 mg, 0.79 mmol) and
tert-butyl bromoacetate (0.23 mL, 1.58 mmol) following general
procedure H, 17k (206 mg, 80%) was obtained after flash
chromatography (15% EtOAc/hexanes). 1H NMR (300 MHz,
CDCl3) δ 0.82 (t, J ) 6.8 Hz, 3 H), 1.21 (s, 8 H), 1.47 (s, 9 H),
1.64 (s, 3 H), 1.78-1.83 (m, 2 H), 4.41 (s, 2 H), 5.15 (s, 1 H);
13C NMR (75 MHz, CDCl3) δ 14.0, 22.5, 25.1, 26.3, 28.0, 29.1,
31.5, 38.9, 59.6, 68.4, 83.4, 102.1, 165.2, 185.5, 193.4. HRMS
(ES) calcd for C17H28O4SNa+ (M + Na+) 351.1600; obsd
351.1612.
(()-5-Methyl-5-octyl-2-oxo-thiophen-4-yloxy)acetic Acid
tert-Butyl Ester (17l). From 16b (60 mg, 0.25 mmol) and tert-
butyl bromoacetate (73 µL, 0.49 mmol) following general
procedure H, 17l (62 mg, 70%) was obtained after flash
chromatography (15% EtOAc/hexanes). 1H NMR (300 MHz,
CDCl3) δ 0.86 (t, J ) 6.9 Hz, 3 H), 1.24 (s, 12 H), 1.49 (s, 9 H),

Inhibitors of Type I Fatty Acid Synthase
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 959
1.68 (s, 3 H), 1.83-1.86 (m, 2 H), 4.43 (s, 2 H), 5.19 (s, 1 H);
13C NMR (75 MHz, CDCl3) δ 14.0, 22.6, 25.2, 26.3, 28.1, 29.2,
29.3, 29.5, 31.8, 38.9, 59.7, 68.5, 83.4, 102.1, 165.2, 185.5, 193.4.
Anal. (C19H32O4S) C, H.
(()-N-(4-Bromobutyl)(5-methyl-5-hexyl-2-oxo-thiophen-
4-yloxy)acetamide (21). To 18a (61 mg, 0.22 mmol) and
1-aminopropanol hydrobromide (50 mg, 0.23 mmol) following
general procedure J, 21 (65 mg, 74%) was obtained after flash
chromatography (50% EtOAc/hexanes). 1H NMR (300 MHz,
CDCl3) δ 0.86 (t, J ) 6.9 Hz, 3 H), 1.12-1.15 (m, 1 H), 1.23-
1.28 (s, 6 H), 1.46-1.53 (m, 1 H), 1.69 (s, 3 H), 1.82-1.88 (m,
2 H), 2.14 (quint. J ) 6.3 Hz, 2 H), 3.42 (m, 2 H), 3.54 (q, J )
6.3 Hz, 2 H), 4.43 (s, 2 H), 5.35 (s, 1H) 6.45 (bs, 1 H). 13C NMR
(75 MHz, CDCl3) δ 22.5, 25.3, 25.3, 26.4, 29.2, 30.7, 31.5, 31.7,
38.1, 39.0, 59.4, 70.4, 103.3, 167.9, 184.2, 192.9. IR (NaCl)
1666, 1607, 1543 cm-1. Anal. (C16H26NO3BrS) C, H.
(()-N-Allyl-(5-methyl-5-octyl-2-oxo-thiophen-4-yloxy)-
acetamide (22). To a cooled solution (0 °C) of 18b (64 mg,
0.21 mmol) in CH2Cl2 (1.1 mL) were added 1-[3-(dimethylami-
no)propyl]-3-ethylcarbodiimide hydrochloride (EDC) (49 mg,
0.25 mmol), DMAP (3 mg, 0.02 mmol), and allylamine (18 µL,
0.25 mmol), and the mixture was allowed to warm to room
temperature and stir for 12 h. The solution was poured into a
solution of 1 N HCl/(sat) (1:3) and extracted with Et2O (3 × 10
mL). The combined organics were dried (MgSO4), filtered, and
evaporated to give crude 22. Flash chromatography (50%
EtOAc/hexanes) gave pure 22 (50 mg, 66%). 1H NMR (300
MHz, CDCl3) δ 0.86 (t, J ) 6.9 Hz, 3 H), 1.12-1.22 (m, 1 H),
1.24 (s, 10 H), 1.41-1.51 (m, 1 H), 1.68 (s, 3 H), 1.82-1.87 (m,
2 H), 3.98 (app t, J ) 5.7 Hz, 2 H), 4.50 (s, 2 H), 5.20 (d, J )
10.2 Hz, 1 H), 5.22 (d, J ) 17.3 Hz, 1 H), 5.35 (s, 1 H), 5.80-
5.90 (ddt, J ) 5.4, 10.2, 17.3 Hz, 1 H), 6.19 (bs, 1 H); 13C NMR
(75 MHz, CDCl3) δ 14.0, 22.6, 25.3, 26.5, 29.2, 29.4, 29.5, 31.8,
39.1, 41.6, 59.3, 70.3, 103.4, 117.2, 133.2, 165.3, 183.9, 192.8.
IR (NaCl) 2957, 1692, 1605, 1539 cm-1. Anal. (C18H29NO3S)
C, H.
(()-3,3-Diallyl-5-methyl-5-octyltetrahydro-2,4-thio-
phenedione (17q). 3-Allyl-4-allyoxyl-5-methyl-5-octyl-2,5-
dihydro-2-thiophenone (17r); 4-Allyl-5-allyloxy-2-methyl-
2-octyl-2,3-dihydro-3-thiophenone (17s). From 16d (374
mg, 1.33 mmol) and allyl bromide (184 µL, 2.13 mmol)
following general procedure H, 17q and 17r/17s (2.6:1) (68%,
5.3:1 17q:17r/17s) was obtained. 17q was separated from 17r/
17s mixture after flash chromatography (15% EtOAc/hexanes).
(17q): 1H NMR (300 MHz, CDCl3) δ 0.86 (t, J ) 6.5 Hz, 3 H),
1.25 (m, 11 H), 1.43-1.47 (m, 1 H), 1.54 (s, 3 H), 1.79-1.84
(m, 2 H), 2.43-2.47 (m, 4 H), 5.05-5.11 (m, 4 H), 5.57-5.69
(m, 2 H). 13C NMR (100 MHz, CDCl3) δ 14.1, 22.6, 25.1, 25.8,
29.1, 29.2, 29.5, 31.8, 40.2, 40.7, 41.3, 62.8, 64.8, 120.3, 120.4,
131.2, 131.2, 203.9, 213.5.
General Procedure I. (()-5-Methyl-5-hexyl-2-oxo-thio-
phen-4-yloxy)acetic Acid (18a). To 17k (177 mg, 0.54 mmol)
dissolved in CH2Cl2 (3.9 mL) was added trifluoroacetic acid
(TFA) (2.6 mL), and the solution was stirred at room temper-
ature for 4 h. The solvents were evaporated, and the crude
material was chromatographed (20% EtOAc/2% CH3CO2H/
hexanes) to give pure 18a (144 mg, 98%). 1H NMR (300 MHz,
CDCl3) δ 0.85 (t, J ) 6.8 Hz, 3 H), 1.24 (s, 7 H), 1.44-1.47 (m,
1 H), 1.68 (s, 3 H), 1.84-1.91 (m, 2 H), 4.62 (s, 2 H), 5.33 (s, 1
H); 13C NMR (75 MHz, CDCl3) δ 14.1, 22.6, 25.1, 26.1, 29.2,
31.6, 38.9, 60.3, 67.7, 102.4, 169.8, 185.9, 196.1. HRMS (ES)
calcd for C13H20O4SNa+ (M + Na+) 295.0974; obsd 295.0950.
(()-5-Methyl-5-octyl-2-oxo-thiophen-4-yloxy)acetic Acid
(18b). To 17l (65 mg, 0.18 mmol) and trifluoroacetic acid (TFA)
(0.7 mL) following general procedure I, 18b (48 mg, 89%) was
obtained after flash chromatography (20% EtOAc/2% CH3-
CO2H/hexanes). 1H NMR (300 MHz, CDCl3) δ 0.86 (t, J ) 6.9
Hz, 3 H), 1.24 (s, 11 H), 1.47-1.48 (m, 1 H), 1.68 (s, 3 H), 1.84-
1.88 (m, 2 H), 4.62 (s, 2 H), 5.31 (s, 1 H); 13C NMR (75 MHz,
CDCl3) δ 14.1, 22.6, 25.1, 26.1, 29.2, 29.3, 29.5, 31.8, 38.9, 60.1,
General Procedure K. (()-4-Propionyl-5-methyl-5-oc-
tyl-5H-thiophen-2-one (23a). To a solution of 16b (40 mg,
0.17 mmol) in THF (0.78 mL) cooled to -78 °C was added
LiHMDS (0.24 mL, 0.25 mmol, 1 M in THF), and the solution
was allowed to stir for 30 min at -78 °C. Propionyl chloride
(20 µL, 0.62 mmol) was then added, and the mixture was
transferred to an ice bath and allowed to slowly warm to room
temperature. After 1 h at room temperature, the mixture was
67.7, 102.4, 169.8, 185.8, 195.4. IR (NaCl) 3442, 1645 cm-1
Anal. (C15H24O4S) C, H.
;
poured into a solution of HCl (1 N)/NH4Cl
(10 mL) and
(sat)
General Procedure J. (()-(5-Methyl-5-hexyl-2-oxo-thio-
phen-4-yloxy)acetylmethyl Glycinate (19). To a solution
of 18a (42.4 mg, 0.15 mmol) in CH3CN (0.86 mL) were added
tris(2-oxo-3-oxazolinyl)phosphine oxide16 (91 mg, 0.20 mmol),
methyl glycinate hydrochloride (19.7 mg, 0.16 mmol), and NEt3
(43 µL, 0.31 mmol), and the solution was allowed to stir at
room temperature for 20 min. The mixture was poured into a
solution of NH4Cl(sat)/1 N HCl (10 mL) and extracted with Et2O
(3 × 10 mL). The combined organics were dried (MgSO4),
filtered, evaporated, and chromatographed (40-50% EtOAc/
extracted with Et2O (3 × 10 mL). The combined organics were
dried (MgSO4), filtered, evaporated, and chromatographed
(15% EtOAc/hexanes) to give pure 23a (23.1 mg, 47%). 1H
NMR (300 MHz, CDCl3) δ 0.85 (t, J ) 6.9 Hz, 3 H), 1.12-1.25
(m, 13 H), 1.42-1.49 (m, 2 H), 1.64 (s, 3 H), 1.78-1.84 (m, 2
H), 2.57 (q, J ) 7.5 Hz, 2 H), 6.39 (s, 1 H); 13C NMR (75 MHz,
CDCl3) δ 8.71, 14.0, 22.6, 25.1, 25.9, 27.9, 29.1, 29.3, 29.5, 31.8,
38.6, 60.4, 113.8, 169.1, 177.0, 179.9. IR (NaCl) 2928, 1787,
1688 cm-1; Anal. (C16H26O3S) C, H.
1
(()-4-Carbonic Acid Methyl Ester, 5-Methyl-5-octyl-
5H-thiophen-2-one (23b). From 16b (73 mg, 0.30 mmol) and
methyl chloroformate (37 µL, 0.48 mmol) following general
procedure K, 23b (63 mg, 70%) was obtained after flash
chromatography (20% EtOAc/hexanes).1H NMR (300 MHz,
CDCl3) δ 0.85 (t, J ) 6.4 Hz, 3 H), 1.15-1.21 (m, 1 H), 1.22 (s,
10 H), 1.41-1.51 (m, 1 H), 1.66 (s, 3 H), 1.81 (d, J ) 9.0 Hz,
hexanes) to give pure 19 (43 mg, 80%). H NMR (300 MHz,
CDCl3) δ 0.85 (t, J ) 6.8 Hz, 3 H), 1.23-1.26 (m, 7 H), 1.49-
1.55 (m, 1 H), 1.65 (s, 3 H), 1.84-1.90 (m, 2 H), 3.79 (s, 3 H),
4.11 (d, J ) 5.0 Hz, 1 H), 4.02-4.09 (m, 2 H), 4.47 (s, 2 H),
5.36 (s, 1 H), 6.76 (bs, 1 H). 13C NMR (75 MHz, CDCl3) δ 13.8,
22.4, 25.2, 26.2, 29.0, 31.4, 38.9, 40.8, 52.4, 59.3, 70.1, 103.1,
165.6, 169.6, 184.2, 192.7; IR (NaCl) 1752, 1689, 1612, 1535
cm-1; Anal. (C16H25NO5S) C, H.
1 H), 1.83 (d, J ) 9.0 Hz, 1 H), 3.92 (s, 3 H), 6.39 (s, 1 H); 13
C
NMR (75 MHz, CDCl3) δ 14.1, 22.6, 25.2, 25.9, 29.2, 29.3, 29.4,
31.8, 38.4, 56.2, 60.2, 112.9, 150.9, 175.5, 194.1. IR (NaCl)
3382, 1626, 1560, 1542 cm-1. Anal. (C15H24O4S) C, H.
(()-(5-Methyl-5-hexyl-2-oxo-thiophen-4-yloxy)acetyl
Glycinate (20). To 18a (22 mg, 0.06 mmol) dissolved in THF/
H2O (0.5 mL, 3:1), cooled to 0 °C, was added LiOH (3 mg, 0.07
mmol), and this solution was allowed to stir for 45 min. Then,
the mixture was poured into a solution of HCl (10 mL, 1 N)
and extracted with Et2O (3 × 10 mL). The combined organics
were dried (MgSO4), filtered, and evaporated to give crude 20.
Flash chromatography (50% EtOAc/2%CH3CO2H/hexanes)
gave pure 20 (19 mg, 86%). 1H NMR (300 MHz, CDCl3) δ 0.85
(t, J ) 6.7 Hz, 3 H), 1.25 (s, 7 H), 1.48-1.52 (m, 2 H), 1.68 (s,
3 H), 2.08-2.10 (m, 2 H), 4.05 (s, 2 H), 4.56 (s, 2 H), 5.41 (s, 1
H). 13C NMR (75 MHz, CDCl3) δ 14.0, 22.5, 25.3, 25.9, 29.1,
31.5, 38.8, 40.9, 59.9, 69.9, 103.2, 166.8, 171.9, 184.9, 194.9;
Anal. Calcd for C15H23NO5S: C, 54.6; H, 7.16; Found: C, 53.4;
H, 7.16.
(()-4-Carbonic Acid Ethyl Ester, 5-Methyl-5-octyl-
5H-thiophen-2-one (23c). From 16b (95 mg, 0.39 mmol) and
ethyl chloroformate (60 µL, 0.32 mmol) following general
procedure K, 23c (111 mg, 91%) was obtained after flash
chromatography (15% EtOAc/hexanes). 1H NMR (300 MHz,
CDCl3) δ 0.85 (t, J ) 6.9 Hz, 3 H), 1.12-1.17 (m, 11 H), 1.38
(t, J ) 7.1 Hz, 3 H), 1.42-1.50 (m, 1 H), 1.67 (s, 3 H), 1.82 (d,
J ) 9.0 Hz, 1 H), 1.85 (d, J ) 9.0 Hz, 1 H), 4.33 (q, J ) 7.1 Hz,
2 H), 6.38 (s, 1 H); 13C NMR (75 MHz, CDCl3) δ 14.0, 14.0,
22.6, 25.2, 25.8, 29.1, 29.2, 30.4, 31.8, 38.4, 60.1, 66.0, 112.8,
150.2, 175.6, 193.9. IR (NaCl) 2928, 1782, 1690, 1625 cm-1
Anal. (C16H26O4S) C, H.
.

960 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
McFadden et al.
(()-4-Carbonic Acid Allyl Ester, 5-methyl-5-octyl-
5H-thiophen-2-one (23d). From 16b (51.5 mg, 0.21 mmol)
and allyl chloroformate (33 µL, 0.32 mmol) following general
procedure K, 23d (46.3 mg, 67%) was obtained after flash
chromatography (15% EtOAc/hexanes). 1H NMR (300 MHz,
CDCl3) δ 0.85 (t, J ) 6.9 Hz, 3 H), 1.16-1.23 (bs, 10 H), 1.41-
1.51 (m, 2 H), 1.67 (s, 3 H), 1.81-1.87 (m, 2 H), 4.74 (app dt,
J ) 6.1, 1.3 Hz, 2 H), 5.37 (app dq, J ) 10.3, 1.0 Hz, 1 H), 5.44
(app dq, J ) 15.9, 1.0 Hz, 1 H), 5.90-6.0 (m, 1 H), 6.39 (s, 1
H); 13C NMR (75 MHz, CDCl3) 14.0, 22.6, 25.2, 25.8, 29.1, 29.2,
29.4, 31.8, 38.4, 60.1, 70.2, 112.9, 120.6, 130.2, 150.0, 175.5,
193.7. IR (NaCl) 2927, 1782, 1691, 1606 cm-1. Anal. (C17H26O4S)
C, H.
4-Hydroxy-3-(1-hydroxyethyl)-5-methyl-5-octyl-5H-
thiophen-2-one (29, 30). To 16b (247 mg, 1.02 mmol)
dissolved in hexanes were added triethylamine (0.23 mL, 1.68
mmol) and trimethylsilyl chloride (0.21 mL, 1.64 mmol), and
the solution was allowed to stir at room temperature for 4 h.
The mixture was filtered over Celite and evaporated to provide
5-methyl-5-octyl-4-trimethylsilanyloxy-5H-thiophen-2-one. To
a solution of TiCl4 (0.7 mL, 0.7 mmol) in CH2Cl2 (1.95 mL) at
-78 °C was added acetaldehyde (54 µL, 0.97 mmol), and this
solution was allowed to stir for 5 min at -78 °C. Then,
5-methyl-5-octyl-4-trimethylsilanyloxy-5H-thiophen-2-one dis-
solved in CH2Cl2 (0.4 mL) was cannulated into TiCl4/acetal-
dehyde solution giving a bright orange color. This mixture was
allowed to warm and stir for 20 min at 0 °C. The mixture was
poured into NH4Cl(sat) (15 mL) and extracted with CH2Cl2 (3
× 15 mL). The organics were combined, dried (MgSO4), filtered,
and evaporated. Flash chromatography (10% EtOAc/hexanes)
provided pure 29 (34 mg) and 30 (24 mg) (50%). (29) 1H NMR
(300 MHz, CDCl3) δ 0.86 (t, J ) 6.9 Hz, 3 H), 1.05-1.08 (m, 1
H), 1.24 (bs, 11 H), 1.49 (d, J ) 6.5 Hz, 3 H, rotamer) 1.55 (d,
J ) 5.2 Hz, 3 H, rotamer), 1.62 (s, 3 H), 1.78-1.82 (m, 2 H),
4.68 (q, J ) 6.5 Hz, 1 H, rotamer), 5.04 (q, J ) 5.2 Hz, 1 H,
rotamer). HRMS (ES) m/z calculated for C16H28O3SNa+ (M +
CH2 + Na+) 323.1660 obsd 323.1660.
C15H21F3O3S; C, 53.2; H, 6.26; Found: C, 52.0; H, 6.15. HRMS
(EI) m/z calculated for C15H21F3O3S+ (M+) 338.1158 obsd
338.1171.
4-Hydroxy-5-methyl-5-octyl-2-oxo-2,5-dihydro-thio-
phene-3-carboxylic Acid Methyl Ester (31c). From 16b (91
mg, 0.37 mmol), methyl chloroformate (63 µL, 0.81 mmol),
(dimethylamino)pyridine (DMAP) (23 mg, 0.18 mmol), and
NEt3 (108 µL, 0.77 mmol) following General Procedure L, 31c
(66 mg, 59%, 79% based on recovered starting material) was
obtained after flash chromatography (30% EtOAc/2%AcOH/
hexanes-10%THF/2%AcOH/EtOAc). 1H NMR (300 MHz,
MeOD) δ 0.86 (t, J ) 6.9 Hz, 3 H), 1.20 (bs, 12 H), 1.35 (s, 3
H), 1.55 (m, 1 H), 1.71-1.75 (m, 1 H), 3.59 (s, 3 H); 13C NMR
(100 MHz, MeOD) δ 13.9, 22.1, 24.6, 27.3, 28.5, 28.6, 28.8, 29.2,
31.3, 50.9, 58.4, 97.1, 168.4, 187.9, 203.2. HRMS (EI) m/z
calculated for C15H24O4S+ (M+) 300.1389 obsd 300.1375.
Acknowledgment. We thank the National Insti-
tutes of Health (CA 91632 to J.M.M. and in part
1R43DK65423 and 1R44CA99435 to FASgen Inc.) and
FASgen Inc. Under a license agreement between FAS-
gen, Inc. and The Johns Hopkins University, F.P.K. and
C.A.T. are entitled to share royalty received by the
University on sales of products described in this article.
F.P.K. and C.A.T. own FASgen, Inc., stock, which is
subject to certain restrictions under university policy.
F.P.K. and C.A.T. are consultants to FASgen. The Johns
Hopkins University, in accordance with its conflict of
interest policies, is managing the terms of this arrange-
ment.
1
Supporting Information Available: Copies of H NMR
spectra for compounds 13a-d, 16a-d, 17a-c/m, 17d-j, 17n,
17o/p, 17q, 18b, 19, 20, 21, 22, 23a-d, 26, 27, 29, 30, 31a-
c. This material is available free of charge via the Internet at
http://pubs.acs.org.
(30) 1H NMR (300 MHz, CDCl3) δ 0.85 (t, J ) 6.9 Hz, 3 H),
1.24 (bs, 12 H), 1.47 (d, J ) 6.6 Hz, 3 H, rotamer), 1.54 (d, J
) 5.4 Hz, 3 H, rotamer), 1.59 (s, 3 H), 1.76-1.82 (m, 2 H),
4.65 (q, J ) 6.3 Hz, 1 H), 5.06 (q, J ) 5.4 Hz, 1 H). HRMS
(ES) m/z calculated for C16H28O3SNa+ (M + CH2 + Na+)
323.1660 obsd 323.1660.
References
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octyl-5H-thiophen-2-one. (31a). To 16b (94 mg, 0.38 mmol)
in CH2Cl2 (1.9 mL) at 0 °C was added NEt3 (58 µL, 0.42 mmol),
(dimethylamino)pyridine (DMAP) (19 mg, 0.15 mmol) and
acetic anhydride (43 µL, 0.47 mmol). The solution stirred at 0
°C for 15 min then was allowed to warm and stir at room
temperature for 2-14 h or until TLC indicated completion of
the reaction. The mixture was poured into NH4Cl(sat)/HCl (1
N) (3:1, 8 mL) and extracted with CH2Cl2 (3 × 10 mL). The
organics were combined, dried (MgSO4), filtered, and evapo-
rated to give crude 31a. Flash chromatography 30%EtOAc/
2%AcOH/hex (Rf ) 0.44) gave pure 31a (83 mg, 78%). 1H NMR
(300 MHz, CDCl3) δ 0.84 (m, 3 H), 1.22 (bs, 10 H), 1.48 (m, 2
H), 1.65 (s, 3 H), 1.77-1.92 (m, 2 H), 2.55 (s, 3 H). 13C NMR
(75 MHz, CDCl3) δ 13.9, 22.6, 23.8, 25.1, 26.3, 29.1, 29.2, 29.5,
31.7, 39.4, 59.7, 109.7, 190.5, 195.5, 204.9. Anal. Calcd for
C15H24O3S: C, 63.3; H, 8.51; Found: C, 59.5; H, 7.83. HRMS
(EI) m/z calculated for C15H24O3S+ (M+) 284.1441 obsd 284.1414.
4-Hydroxy-5-methyl-5-octyl-3-(2,2,2-trifluoro-acetyl)-
5H-thiophen-2-one (31b). From 16b (90 mg, 0.37 mmol),
trifluoroacetic anhydride (114 µL, 0.81 mmol), (dimethylami-
no)pyridine (DMAP) (18 mg, 0.15 mmol), and NEt3 (108 µL,
0.77 mmol) following General Procedure L, 31b (107 mg, 86%)
was obtained after flash chromatography (40%Hex/10% THF/
1
2%AcOH/EtOAc). H NMR (300 MHz, MeOD) δ 0.85 (t, J )
6.9 Hz, 3 H), 1.09 (m, 1 H), 1.21 (bs, 11 H), 1.38 (s, 3 H), 1.51-
1.60 (m, 1 H), 1.65-1.71 (m, 1 H). 13C NMR (100 MHz, MeOD)
d 14.6, 23.8, 26.3, 27.8, 30.5, 30.7, 30.9, 33.1, 41.2, 61.2, 105.7,
152.9 (q, J ) 57 Hz), 174.3, 196.1, 203.5. Anal. Calcd for

Inhibitors of Type I Fatty Acid Synthase
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