Influence of various central moieties on the hypolipidemic properties of long hydrocarbon chain diols and diacids

Article abstract of DOI:10.1021/jm050650j

A series of long (11-15) hydrocarbon chain diols and diacids with various central functional groups and terminal gem-dimethyl or -methyl/aryl substituents was synthesized and evaluated in both in vivo and in vitro assays for its potential to favorably alt

Full text of DOI:10.1021/jm050650j

334
J. Med. Chem. 2006, 49, 334-348
Influence of Various Central Moieties on the Hypolipidemic Properties of Long Hydrocarbon
Chain Diols and Diacids
Daniela C. Oniciu,*,† Jean-Louis H. Dasseux,† Jing Yang,‡ Ralf Mueller,‡ Emil Pop,‡ Anna Denysenko,‡ Caiming Duan,‡
Tian-Bao Huang,‡ Lianhao Zhang,‡ Brian R. Krause,† Sandra L. Drake,† Narendra Lalwani,† Clay T. Cramer,† Brian Goetz,†
Michael E. Pape,† Andrew McKee,† Gregory J. Fici,† Janell M. Lutostanski,† Stephen C. Brown,† and Charles L. Bisgaier†
Esperion Therapeutics, A DiVision of Pfizer Global Research and DeVelopment, 3621 South State Street, 695 KMS Place, Ann Arbor, Michigan
48108, and Alchem Laboratories Corporation, 13305 Rachael BouleVard, Alachua, Florida 32615
ReceiVed July 8, 2005
A series of long (11-15) hydrocarbon chain diols and diacids with various central functional groups and
terminal gem-dimethyl or -methyl/aryl substituents was synthesized and evaluated in both in vivo and in
vitro assays for its potential to favorably alter lipid disorders including metabolic syndrome. Compounds
were assessed for their effects on the de novo incorporation of radiolabeled acetate into lipids in primary
cultures of rat hepatocytes, as well as for their effects on lipid and glycemic variables in obese female
Zucker fatty rats, Crl:(ZUC)-faBR. The most active compounds were hydroxyl-substituted symmetrical diacids
and diols with a 13-atom chain and terminal gem-dimethyl substituents. Furthermore, biological activity
was enhanced by central substitution with O, CdO, S, SdO compared to the methylene analogues and was
diminished for compounds with central functional groups such as carbamate, ester, urea, acetylmethylene,
and hydroxymethylene.
Introduction
of actions have been reported earlier for compounds presenting
HDL-elevating properties in various animal models.5
We have recently identified several series of functionalized
long chain hydrocarbon derivatives as possessing lipid-regulating
activity in an animal model of diabetic dyslipidemia (i.e., the
obese female Zucker fatty rat).1-3 The work was focused on
the design of long hydrocarbon chain ether1 and keto2,3 diols
and diacids with gem-dialkyl,1-3 alkyl/aryl,1,2 and cycloalkyl3
substitution to the terminal acid or hydroxymethylene functions.
The in vivo pharmacologic activity of these compounds is
reflected in serum triglyceride reduction, decreases in non-HDL
cholesterol (non-HDL-C), and increases in HDL cholesterol
(HDL-C). In cell assays (in vitro), activity was also demonstrated
by inhibiting both fatty acid and cholesterol syntheses in cultured
liver cells at micromolar concentrations and by increasing fatty
acid oxidation. Moreover, we reported earlier that this class of
compounds form CoA derivatives in vivo and inhibit fatty acid
synthesis (FAS) at acetyl-CoA-carboxylase (ACC), the rate-
limiting step for FAS, via an allosteric mechanism.4 The
inhibition of fatty acid synthesis produced by such a mechanism
of action (MOA) is consistent with the lowering of serum
triglycerides and enhancement of â-oxidation. We have also
shown that these compounds rapidly block de novo cholesterol
synthesis at a step prior to the formation of mevalonic acid.4
Thus, they are dual inhibitors of lipid synthesis. Statins (HMG-
CoA reductase inhibitors), which only inhibit cholesterol
synthesis, are distinct from these compounds, which also possess
FAS inhibitory activity. When tested in animals, these com-
pounds indeed manifest biological effects related to both
cholesterol and FAS inhibition,4 but in addition they possess
biological activities that may be secondary to the direct targets
(anti-atherosclerosis, anti-diabetic).1c,1d,2b Multiple mechanisms
In our earlier studies,1-3 we have found a chain length of
13-15 atoms to be optimal for biological activity. In addition,
we established that the introduction of an oxygen atom either
as an ether (1) or as a ketone (2) inside the chain increased the
effect of lowering triglycerides and HDL elevation in the Zucker
animal model compared to the methylene analogues. Similarly,
Bisgaier et al. reported that ether-containing diacids were
biologically active lipid regulators in vivo.6,7 Ether diacids of
13-15 atom chain lengths proved to be the most active
compounds. Our findings showed that active compounds
displayed symmetrical or unsymmetrical structures with four
to five methylene groups separating the central ether or ketone
functionalities and the gem-dimethyl, -methyl/aryl, or -cyclo-
propyl substituents.1-3 Bisgaier et al. similarly reported biologi-
cal activity of symmetrical or unsymmetrical ether diacids,6,7
the compound gemcabene having a 13-atom chain length
showing activity in humans.8 Furthermore, biological activity
was found to be greatest in both in vivo and in vitro assays for
gem-tetramethyl-substituted 5,5-keto-diacids and -diols, while
bis(aryl-methyl) derivatives were shown to be the least active.2
Therefore, the purpose of the current study has been to
examine the role of other central moieties for biological
functions. To achieve this goal, we have explored the effects
of various substituents having different bulk, electronic, and
inductive effects and hydrogen bonding as exemplified in Figure
1 on in vivo and in vitro activity.
Results and Discussion
Chemistry. Long hydrocarbon chain compounds with diverse
central moieties (Z, Figure 1) and terminal hydroxyl (X ) H2)
or carboxyl (X ) O) groups were synthesized. The side chains
connected to the central moiety varied both in the number of
methylene spacer units (n ) 3 or 4) and in the attached geminal
modifying groups (R ) Me or Ph). The majority of target
compounds fell in the category of sulfur derivatives (Scheme 1
and Table 1), namely, sulfides (4a-d and 8d),9 sulfoxides (6a-
* To whom correspondence should be addressed. Current address:
Department of Chemistry, University of Florida, Gainesville, FL 32611-
7200. E-mail: oniciu@yahoo.com.
† Esperion Therapeutics, A Division of Pfizer Global Research and
Development.
‡ Alchem Laboratories Corporation.
10.1021/jm050650j CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/08/2005

Influence of Moieties on Hypolipidemic Properties
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 335
Scheme 2. Synthesis of Alcohols 10 and 12a
Figure 1. Overview of compounds.
Scheme 1. Synthesis of Sulfides, Sulfoxides, and Sulfonesa
a Reagents: (a) LiAlH4, MTBE; (b) NaBH4, MeOH; (c) NaOH, water/
EtOH.
Scheme 3. Synthesis of Alcohol 17 and Methyl Ketone 18a
a Reagents: (a) LiBH4, MeOH, CH2Cl2, then 3,4-dihydro-2H-pyran,
pTosOH, CH2Cl2, described in lit. ref 2; (b) Na2S nonahydrate, water/EtOH;
(c) concd HCl, ∆, MeOH; (d) thiourea, KOH, EtOH; (e) Na2S nonahydrate,
water/EtOH; (f) LiAlH4, Et2O; (g) LiBH4, MeOH, CH2Cl2; (h) aq H2O2,
HOAc; (i) MCPBA, CHCl3; (j) KOH, water/EtOH.
a Reagents: (a) diethyl malonate, NaH, TBAI, DMSO; (b) concd HCl,
aq EtOH; (c) KOH, aq EtOH; (d) 200 °C; (e) LiAlH4, THF; (e) MeLi,
THF/Et2O.
Scheme 4. Synthesis of Amine 21 and Hydroxylamine 23a
Table 1. Synthesis of Sulfides, Sulfoxides, and Sulfones
no.
n
R
yield (%)
3c
4a
4b
4c
4d
5a
5b
5d
6a
6b
6c
6d
7c
8d
4
3
3
4
4
3
3
4
3
3
4
4
4
4
Me
Me
Ph
Me
Ph
Me
Ph
Ph
Me
Ph
Me
Ph
Me
Ph
78
98a
54b
77 (53)c
74a
89d
85e
85d
67
69
63
73
51
a Reagents: (a) p-toluenesulfonamide, NaOH, TBAI, 70-80 °C, C6H6/
H2O; (b) Na, naphthalene, DME; (c) concd HCl, MeOH; (d) aq NaOH,
CH2Cl2; (e) benzoyl peroxide, Na2HPO4, 45 °C, MTBE; (f) NaOMe, MeOH.
68
a Prepared according to method f in Scheme 1. b Prepared according to
method g in Scheme 1. c After distillation. d Prepared according to method
d in Scheme 1. e Prepared according to method e in Scheme 1.
for comparison. Urea derivative 42 was also prepared for this
study (Scheme 7).16
Sulfides 4a-d were prepared by two slightly different
pathways starting from bromo esters 1a-d (Scheme 1).1 For
compounds with n ) 4 and R ) Me, bromo ester 1c was
reduced with LiBH4 and MeOH1 and the formed alcohol (not
shown) was subsequently protected as tetrahydropyranyl (THP)
ether 2c (82%). Reaction of 2c with Na2S nonahydrate (0.5
equiv) in aqueous EtOH17 (first at room temperature, then at
reflux) gave sulfide 3c in 78% yield. Removal of the THP
protective groups in 3c by treatment with concd HCl in MeOH
d),10 and a sulfone (7c). Some alcohols (10, 12, and 17, Schemes
2 and 3),11,12 a methyl ketone (18, Scheme 3),13 amine
derivatives (21 and 23, Scheme 4),14,15 an amide (30, Scheme
5),14,15 an ester 33,14 a phosphoric acid ester 35,14 and a
carbamate 3714 (Scheme 6) were prepared and investigated as
well. Previously prepared ether 38,1 ketones 39 and 41,2 and
hydrocarbon analogue 40 (Figure 2)1,2 are included in this work

336 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Scheme 5. Synthesis of Amide 30a
Oniciu et al.
Scheme 7. Synthesis of Urea 42a
a Reagents: (a) CO2, pyridine, (PhO)3P, 40-55 °C; (b) aq HCl, MeOH,
∆; (c) flash chromatography.
yield,19 respectively, whereas reaction of 1b with Na2S afforded
5b in 85% yield. Reduction of esters 5a, 5b, and 5d with either
LiAlH4 or LiBH4/MeOH gave the corresponding alcohols 4a,
4b, and 4d in varying yields (54-98%). Sulfides 4a-d were
oxidized with H2O2 in acetic acid20 to furnish sulfoxides 6a-d
(63-73%). With 3-chloroperoxybenzoic acid (MCPBA) used
as an oxidizer,21 sulfoxide 7c was obtained from 4c in 51%
yield. The only diacid in the sulfur series, compound 8d, was
produced by saponification of 5d with KOH in aqueous EtOH
(68%).
Alcohols 10 and 12 were synthesized from keto diester 9
(Scheme 2).11 Complete reduction of both ketone and ester
functionalities in 9 was achieved by treatment with LiAlH4 to
give triol 10 in 46% yield. Use of NaBH4 in MeOH as reducing
agent led to selective reduction of the ketone group in 9 to
alcohol diester 11 (92%), which was then saponified under
standard conditions (NaOH, aqueous EtOH) to afford alcohol
diacid 12 in 80% yield.
a Reagents: (a) potassium phthalimide, 80-95 °C, DMF; (b) aq
hydrazine, 78 °C, EtOH; (c) NaCN, 90 °C, DMSO; (d) NaOH, ∆, aq EtOH;
(e) NHS, DCC, CH2Cl2; (f) CH2Cl2; (g) concd HCl, ∆, MeOH.
Scheme 6. Synthesis of Ester 33, Phosphoric Acid Ester 35,
and Carbamate 37a
The synthetic sequence leading to alcohol 17 and methyl
ketone 18 (Scheme 3) started with the dialkylation of diethyl
malonate with alkyl bromide 2c under action of NaH and
tetrabutylammonium iodide (TBAI) in DMSO to afford com-
pound 13 (99%). Removal of the THP protective groups in 13
with concentrated HCl in aqueous EtOH gave 14 (84%), which
was then hydrolyzed (KOH, aqueous EtOH, 60%) to furnish
malonic acid derivative 15. Heating of 15 to 200 °C resulted in
decarboxylation22 to 16 (98%) and subsequent reduction of this
compound with LiAlH4 gave triol 17 in 89% yield. When
carboxylic acid 16 was treated with an excess of methyllithium
at 0 °C,23 a mixture of methyl ketone 18 (41%) and tertiary
alcohol 19 (38%) was formed, which was separated by column
chromatography.
Bromide 2c was also used as a building block for the synthesis
of amine 21 and hydroxylamine 23 (Scheme 4). Amine
hydrochloride 20 was prepared in 37% yield by 2-fold alkylation
of p-toluenesulfonamide with 2c under phase-transfer conditions
(TBAI, aqueous NaOH/benzene, 70-80 °C)24 and subsequent
removal of the N-tosyl protecting group with sodium naphtha-
lenide in DME25 and of the THP groups by acid treatment
(concentrated HCl/MeOH). The free amine 21, obtained by
extraction of 20 with aqueous NaOH solution and CH2Cl2
(82%), was further reacted with benzoyl peroxide and Na2HPO4
to give O-benzoylhydroxylamine 22 in 59% yield, as described
for similar compounds.26 Debenzoylation of 22 was achieved
by treatment with sodium methoxide in MeOH to furnish
hydroxylamine 23 in 42% yield after purification by chroma-
tography and crystallization.
a Reagents: (a) K2CO3, ∆, DMSO/water, described in lit. ref 1; (b) 27,
DCC, DMAP, CH2Cl2; (c) ∆, HOAc/THF/water; (d) POCl3, NEt3, Et2O,
then KHCO3, aq CH3CN; (e) concd HCl, MeOH; (f) CDI, DMAP, CH2Cl2,
then 25, CH3CN; (g) ∆, HOAc/THF/water.
Figure 2. Ether 38, ketone 39, hydrocarbon analogue 40, and keto-
diacid 41.
Amine 25, the synthetic precursor of amide 30 (Scheme 5),
was prepared by Gabriel synthesis27 from 2c via 24 in 52%
yield. Substitution of bromide in 2c by cyanide ion affected by
treatment with NaCN in DMSO gave nitrile 26 (92%), which
was subsequently hydrolyzed (NaOH in aqueous EtOH)28 to
carboxylic acid 27 in 75% yield. After conversion to NHS-ester
28 by reaction with N-hydroxysuccinimide (NHS) and 1,3-
at reflux18 furnished sulfide 4c (77%, 53% after distillation).
For all other sulfur compounds, a shorter approach was chosen.
Accordingly, bromo esters 1a and 1d were reacted with thiourea
and KOH in EtOH to give sulfides 5a and 5d in 89% and 85%

Influence of Moieties on Hypolipidemic Properties
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 337
Table 2. Effect on Lipid Synthesis in Primary Rat Hepatocytes
dicyclohexylcarbodiimide (DCC) (75%),29 this building block
was condensed with the previously prepared amine 25 to give
the THP-protected amide 29. Removal of the THP-protective
groups in 29 under acidic conditions (concd HCl/MeOH)
resulted in formation of amide 30 in 85% yield.
The mono-THP protected diol 31, prepared by hydrolysis of
bromide 2c with K2CO3 in a solvent mixture of DMSO and
water,1 was a common building block for the synthesis of
compounds 33, 35, and 37 (Scheme 6). Coupling of 31 with 27
by means of DCC and 4-(dimethylamino)pyridine (DMAP) in
30
CH2Cl2 led to intermediate 32, which was subsequently
deprotected under mildly acidic conditions (acetic acid/THF/
water)31 at 45 °C to furnish ester 33 in 26% yield over both
steps. Condensation of alcohol 31 with POCl3 and NEt3 in Et2O
followed by treatment with KHCO3 in aqueous acetonitrile32
gave dialkyl phosphate 34 (30%). The THP-protective groups
in 34 were removed with concentrated HCl in MeOH at reflux
to afford phosphoric acid ester 35 in 19% yield. For the synthesis
of carbamate 37, alcohol 31 was condensed with 1,1′-carbon-
33
yldiimidazole (CDI) and DMAP in CH2Cl2 to form an
intermediate imidazole-1-carboxylic acid ester (not shown) that
was further reacted with amine 25 in acetonitrile to give
protected carbamate 36 in 67% yield. Subsequent deprotection
of the hydroxyl groups in a solvent mixture of acetic acid, THF,
and water at 45 °C31 afforded carbamate 37 in 76% yield.
For synthesis of urea 42, amine 25 was first condensed with
CO2 using triphenyl phosphite as a condensing agent in pyridine
(Scheme 7).9 The obtained THP-protected intermediate (not
shown) was then deprotected with concd HCl in MeOH to give
urea 42, which was purified by flash chromatography, in 22%
yield over both steps.
Biological Activity. To evaluate the effect of the central
moiety on biological activity, we largely limited the SAR to
those compounds containing the terminal hydroxymethylene
moiety. In vivo and in vitro tests were used to establish the
SAR for the central substitution series. The hepatic lipid
synthesis assay (HLS) tests the compound effects on the
incorporation of 14C-acetate into total cellular lipids of primary
rat hepatocytes (Table 2). The obese female Zucker rat, an
animal model of diabetic dyslipidemia, was used to test
compound effects on serum lipid variables. Compounds were
administered by oral gavage over a 2-week period at a single
daily dose of 30 or 100 mg/kg (Table 3).
Effect on Lipid Synthesis In Vitro. Compounds in this study
display terminal gem-dimethyl-hydroxymethylene (e.g., 4a, 6c)
and gem-dimethylcarboxylic groups (e.g., 12), or methylphenyl-
hydroxymethylene groups (e.g., 4b, 6b). In vitro HLS studies
demonstrated that dimethyl-substituted derivatives were the most
active, while most of methylphenyl-substituted derivatives had
reduced activity. Therefore, we restricted further SAR studies
to compounds having terminal gem-dimethyl-hydroxymethylene
groups. Replacement of the central oxygen, sulfur, or carbon
atoms with nitrogen resulted in little (23) or no activity (21,
30). Upon introduction of a phosphate group (35) or an amide
group (30), the activity was lost. A central carbamate moiety
(37) or a urea (42) showed similar activity to ketone 39.
Replacing the central carbonyl with hydroxylamine resulted in
activity reduction, for example, hydroxylamine 23 was 40-fold
less active than ketone 39.
a Not active; inhibition of 14C-acetate incorporation into total lipids is
less than 50% at 300 µM. b Test compound was assayed multiple times,
the values represent the average. c Test compound was assayed twice, and
both results are displayed.
Based on the in vitro data, carbamate 37 (IC50 0.8), ketone
39 (IC50 1.5), urea 42 (IC50 3.4), alcohols 10 (IC50 6.4) and 12
(IC50 4.7), and hydrocarbon analogue 40 (IC50 4.2) were likely
to possess in vivo activity as well. In addition, ether 38 (IC50
10.7), sulfide 4a (IC50 15), and sulfone 7c (IC50 8.9) showed
good lipid synthesis inhibition. In contrast, sulfoxide 6a was
not active (although it proved to be active in vivo), while sulfone
7c showed good activity (IC50 8.9).
Effect on Lipid Variables in the Obese Female Zucker
Rat. To test the lipid regulating activity of these compounds,
we used the obese Zucker fatty rat, Crl:(Zuc)-faBR, as a model
of diabetic dyslipidemia, as described in previous articles of
the same group.1-3 The Zucker rat has a mutation in the leptin
receptor that leads to a metabolic disorder similar to human
non-insulin-dependent diabetes mellitus (NIDDM) or type II
Therefore, the activity in the in vitro HLS assay decreased
in the series NH-C(O)-O > CO ≈ CH(OH) ≈ NH-C(O)-
NH > SO2 ≈ S ≈ O > SO (37 > 39 > 12 ≈ 7c ≈ 38 ≈ 4a >
6d).

338 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Table 3. Effect in Female Obese Zucker Ratsa
Oniciu et al.

Influence of Moieties on Hypolipidemic Properties
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 339
Table 3 (Continued)
a / indicates p < 0.05 (paired Student’s t-test); for compounds where n < 3, no statistics were performed. Percent change represents the change of
individual animals averaged. The pretreatment “pre” values are expressed as 100%. Absolute data are shown as mean mg/dL ( standard deviations in
parentheses.
diabetes and causes an age-dependent progression of hypertrig-
lyceridemia, VLDL cholesterol (VLDL-C) elevation, HDL-C
reduction, impaired insulin sensitivity, hyperphagia, and marked
weight gain leading to obesity. Characteristic for this rodent
model is that the non-HDL-C is mainly VLDL-C with es-
sentially no LDL-C with markedly elevated plasma triglycerides.
Thus the lipoproteins in this model are distinct from humans,
where most of the non-HDL-C is carried in the LDL fraction.
The lipid regulating activities of the target compounds in this
model were assessed by administering a single dose of 30 mg/
(kg‚day) or 100 mg/(kg‚day) every day for up to two weeks.
Compounds were evaluated for their ability to reduce non-HDL-
C, elevate HDL-C, and reduce triglycerides (TG), namely, a
less atherogenic serum lipid profile. Because the disease rapidly
progresses in control animals, the non-HDL-C and TG fractions
increase, while the HDL is reduced over the two-week
experimental period. In our animal model, effective test agents
should suppress both non-HDL-C and TG levels, while elevating
HDL. Table 3 summarizes those serum lipid changes induced
by the tested compounds.
Compounds with gem-methylphenyl substitution were not
active, regardless of the central moiety (S for 4b, SO for 6b),
as described earlier for their carbonyl2 and ether1 analogues,
which infers that dimethyl substitution is desirable. It is possible
that the bulk of the methylphenyl moiety does not allow for
inhibition of FAS. The compounds may be unable to form
xenobiotic CoA’s or the ones that are formed are unable to
inhibit ACC and may block compound metabolism required for
the allosteric ACC inhibition.4 The central synthon might also
influence the ability of the compounds to fit into the pocket
and may allow for an easier oxidation of the terminal diol to
the corresponding acid with the eventual formation of the
xenobiotic CoA. We can speculate that the nature of the central
moieties could influence the incorporation of the compounds

340 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Oniciu et al.
Table 4. Metabolites Observed in Hepatocytes of Multiple Species
in other lipids, such as diglycerides, TG, phospholipids, or
cholesteryl esters, or their regulation of the synthesis of fatty
acids.
metabolite
namea
possible
biotransformation
rat
X
dog
X
monkey
human
X
From analysis of the results presented in Table 3, it is obvious
that the number of atoms between the quaternary substitutions
should be nine or higher. The increasing order of activity for
compounds in terms of HDL-C elevation and TG lowering after
two weeks of treatment was 38 (O) > 6c (SdO) > 10 (CH-
(OH)) > 4c (S) > 39 (CdO) > 40 (CH2). The sulfur substitution
in both sulfide 4c and sulfoxide 6c favorably altered non-HDL-
C, HDL-C, and triglycerides in the Zucker rat model, despite
the in vitro results. Sulfur derivative 4a showed reasonable
HDL-C elevation and TG lowering properties, although the
chain consisted only of seven atoms. Perhaps since the sulfur
atom has a larger atomic radius than either carbon or oxygen,
the compound topology could sterically better mimic a nine-
atom chain, resulting in biological activity.
43
monocarboxylic acid
of 39
X
M2
10
44
glucuronide of 39
reduction of 39
monocarboxylic acid
of 10
X
X
X
X
X
X
X
X
X
X
X
M5
12
glucuronide of 10
dicarboxylic acid
of 10
double glucuronide
of 10
X
X
X
X
X
X
X
M7
M8
M9
M10
M11
45
glucuronide of 44
glucuronide of 44
glucuronide of 43
glucuronide of 43
dicarboxylic acid
of 39
X
X
X
X
X
X
39
parent
A central hydroxyl group as in compound 10 altered
positively the lipid profile compared to the hydrocarbon
analogue 40 and even to the ketone 39. Hydroxyl derivative 10
showed a better profile than the corresponding diacid 12, which
may be due to absorption, metabolism, distribution, and excre-
tion (ADME) factors.
In the ketone series, compound 39 showed good activity,
while compound 18 displaying the double bond in a side chain
showed no activity. Other ketone derivatives have been exten-
sively discussed in earlier publications.2,3
a Measurement of parent and metabolites consisted of organic solvent,
protein precipitation of suspension hepatocyte cultures, C18 reversed-phase
HPLC analyses using standard methods, followed by positive ion ESI-mass
spectral analysis using an LCQ ion trap or TSQ triple quad mass
spectrometer. Sample aliquots were injected and desalted using standard
column switching methods, then eluted under conditions where the
metabolites of interest demonstrated retention times from 7 to 9 min. The
only significant fragmentation observed for all molecules was dehydration,
so ESI source temperatures were kept to ∼220 °C. Molecules were identified
by retention time, molecular ion, and observation of dehydration.
When the central atom in the hydrocarbon chain was replaced
with nitrogen or hydroxylamine, the lipid lowering activity was
weakened or lost compared to the keto, sulfur, and hydroxyl
analogues. Among other central moieties tested, urea 42 and
carbamate 37 positively altered the lipid profile, although less
than sulfur, ketone, and hydroxyl derivatives. The internal
conjugation in the urea functionality and the difference in length
for the C-C-C and C-N-C(dO) bonds definitely induce
steric and topologic modifications compared to the above-
mentioned derivatives, with implications in binding to active
sites and additional hydrogen bonding or hydrogen donation
for the urea derivatives. Since compound 41 with the same
number of carbons in the chain as 42 is extremely active, one
can infer that the steric and electronic changes introduced by
the nitrogen atoms in the chain are detrimental to the activity.
These studies identified compound 39 and putative metabo-
lites in the hepatocyte cultures, including a monocarboxylic acid
(43), the central secondary alcohol of compound 39 (10), and
a mono- and dicarboxylic acid of the secondary alcohol (44,
12). Compound 39 was extensively metabolized by all species,
leaving only 6% or less of the parent compound remaining at
the end of the incubation period for both starting concentrations
(Table 4). The non-glucuronide metabolites were detectable in
all species with the exception of compounds 12 in dog and
monkey and 44 in monkey. The only glucuronide species
detected in human cells were adducts of the test compound 39
and triol metabolite 10. Since oxidation of compound 39 is a
major pathway in hepatocyte metabolism, the prediction is that
metabolites 12, 45, or both will achieve high levels in plasma
or serum after oral dosing of compound 39 in preclinical and
clinical studies.
Pharmacokinetics and Drug Metabolism of 1,13-Dihy-
droxy-2,2,12,12-tetramethyl-tridecan-7-one (39). In a species
comparison study, primary hepatocytes from rats, dogs, mon-
keys, and humans were incubated with compound 39 (parent)
to determine the metabolite profile of the test article. Suspen-
sions of cryo-preserved (dog, monkey, and human) or freshly
isolated (rat) hepatocytes were incubated with 1 or 10 µM
compound 39 in 4-h time-course studies. Cell viability controls
and cell-free incubation controls were also evaluated; the drug
had no effect on viability at these concentrations. Cells and
media were periodically assayed for the parent compound and
metabolites by mass spectrometry (LC/MS). Analytical ranges
of 5 to 50 000 ng/mL for all species were readily attained.
Deuterated standards, in which eight of the internal hydrogens
were substituted to >95% by deuterium, were prepared for use
as internal standards for further quantitation. Deutero (d8)
derivatives were prepared by the same reaction sequence as their
nondeuterated analogues2,11,13 using commercially available 1,4-
dibromo-2,2,3,3-tetradeuteriobutane. The deuterated standards
were used to identify and confirm the metabolites of compound
39 by mass spectroscopy based on retention time and fragmen-
tation pattern.
The major metabolic processes observed were oxidation of
the primary alcohols to carboxylic acids and reduction of the
ketone to a secondary alcohol. Several glucuronide species have
been observed as secondary metabolites but were not extensively
characterized. Other potential secondary metabolites, such as
sulfates or amino acid amides, were not observed. The metabo-
lism of compound 39 is depicted in Figure 3, based on the in
vitro hepatocyte studies detailed above and from rat plasma
analyses after oral and intravenous drug administration.
A study was conducted to identify compound 39 and its
metabolites in male Wistar rats at multiple time points after a
single 100 mg/kg oral (Figure 4) or 10 mg/kg intravenous dose
(Figure 5). Analytical techniques used to identify parent and
metabolites in serum were similar to those described above for
hepatocytes.
Parent compound levels are rapidly removed from circulation,
achieving extremely low or nondetectable levels when compared
to the administered drug (Table 4). Of the predominant
metabolites, the dicarboxylic acid 45 and reduced metabolite
12 exhibited the highest T1/2, Cmax, and AUC values. Cmax values
for 12 achieved 40.6 µg/mL in this study (Table 5). These

Influence of Moieties on Hypolipidemic Properties
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 341
Figure 3. Diagram of compound 39 metabolism. “M#” indicates the metabolite ID number of corresponding glucuronide adducts, indicated by
closed brackets, for example, “[M2]”.
Figure 4. Pharmacokinetics of compound 39 and its major metabolites in serum after oral administration at 100 mg/kg. Each data point represents
the average value from three animals ( standard error of the mean, with the exception of 12 at 6 h where n ) 2. Note that one of the three data
points for 12 at 6 h was three times the average of the other two values and was eliminated in the graph shown. If this data point was included,
the mean value for 12 at 6 h would be 69.1 ( 28.5 µg/mL.
exploratory data suggest high oral bioavailability of compound
39 and rapid conversion to metabolites.
reduced body weights were observed in males at 1000 mg/kg.
Dose-related increases in liver weights at all doses and upon
histological evaluation hepatocyte hypertrophy were observed
in animals treated with g200 mg/kg. Compound 39 was well
tolerated for 2 weeks at doses of 1000 mg/kg, and 200 mg/kg
was considered minimal effect dose. Compound 38 was well
tolerated at 2000 mg/kg with mild liver effects. Following a
2-week treatment, body weight gain suppression was observed.
Dose-dependent increases in liver weights and slightly decreased
red blood cell parameters (red blood cell count, hemoglobin,
and hematocrit) were observed following 2-week treatment.
Compound 4c was well tolerated following a single dose
administration of 5000 mg/kg. Following a 2-week administra-
tion with doses of 50, 100, and 500 mg/kg, decreased body
weight gains and increased liver weights were observed. All
clinical chemistry parameters measured were within normal
Preliminary Toxicology Data. a. Genetic Toxicology.
Mutagenicity was evaluated by AMES assay for compounds
38 and 39. Salmonella typhimurium/Escherichia coli plate
incorporation/preincubation mutation assay was used in the
presence and absence of induced rat liver S-9. Salmonella
typhimurium tester strains, TA98, TA100, TA1535, and TA1537,
and E coli strain WP2 uvrA were used and results demonstrated
that both 38 and 39 were nonmutagenic with and without
metabolic activation.
b. Safety and Tolerability in Rats. Safety and tolerability
of compounds 4c, 38, and 39 were evaluated in rats through
adminstration of compounds by oral gavage as a single dose or
daily doses for 2 weeks. Compound 39 was well tolerated as a
single dose at 2000 mg/kg in both sexes. Upon 2-week dosing,

342 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Oniciu et al.
Figure 5. Pharmacokinetics of compound 39 and its major metabolites in serum after intravenous administration at 10 mg/kg. Each data point
represents the average value from three to five animals ( standard error of the mean, with the exception of 0 h (pretreatment) where n ) 2.
Table 5. Pharmacokinetics of Compound 39 Following Single Oral
Dose Administration (100 mg/kg) in Male Wistar Rats
hydrogen bonds or by having a different molecular geometry
that does not allow interaction with the required active sites
variable
1/2 (h)
39 (parent)
43
2.5
2.0
0.5
44
3.1
21.4
6.0
45
12
(e.g., 21, 23, 30, and 35).
•Position of the central moietyshydroxyl and ketone func-
tionalities should be directly connected to the main chain. Their
presence in the lateral chain diminished the activity (e.g., 17,
18).
•Electronic effectssinductive and electron-donor properties
that lead to conjugation in the center of the molecule may
influence the accessibility of the active sites or lead to
deactivation of the molecule by weak interactions with other
substrates. This may explain why compounds 30, 33, 37, and
42 showed a lower activity as compared to compound 41, having
similar chain length and a central ketone moiety.
Compound 39 was selected for pharmacokinetics and drug
metabolism studies because it was considered the most illustra-
tive of this series. Results showed that compound 39 is
metabolized rapidly to more polar species and their conjugates
with glucuronic acid such that major exposures of compounds
12 and 45 result from oral and intravenous administration of
39. Another example previously published by our group4 showed
the metabolism of the central hydroxyl form of dicarboxylic
acid 41 to its biologically formed CoA ester. This biologically
formed CoA ester, but not its precursor, was shown to be the
active agent causing allosteric inhibition of acyl-CoA-carboxy-
lase (ACC), the rate-limiting enzyme in fatty acid synthesis.
Since CoA esters are only formed within living cells, it was
necessary to invoke a cell- or animal-based assay to screen for
biological activity.
T
a
a
a
a
13.5
14.9
6.0
16.9
40.6
6.0
C
max (µg/mL)
T
max (h)
AUC(0-t)
(µg‚h/mL)
AUC∞
(µg‚h/mL)
volume of
distribution
26.7
289.9
230.3
703.3
a
a
26.8
291.6
344.9
1176.1
2072.1
13670.9
1533.2
5631.9
(mL/kg)
clearance
(mL/h)
a
747.9
69.0
86.8
28.4
a Variables for parent compound 39 could not be determined because
most data points were below quantifiable limits. T1/2 denotes half-life; Cmax
denotes maximal plasma concentrations; Tmax denotes time of maximum
plasma concentration; AUC denotes area under the curve. Concentrations
were determined using co-injected authentic (d8) internal standards.
range including liver enzymes aspartate aminotransferase (AST)
and alanine aminotransferase (ALT). In summary, all com-
pounds tested were well tolerated in the preliminary drug safety
evaluations performed.
Conclusions
The compounds tested showed a large variation of central
moieties and a narrow variation of the length of the chain (11-
15 atoms). The SAR showed that introducing oxygen, sulfur,
carbonyl, and hydroxyl in the center of the molecules favored
a good lipid profile. Compounds with nitrogen or phosphorus
atoms included in the central moieties showed either reduced
or no activity. Compounds containing more than one heteroatom
also had reduced activity, with the exception of urea 42 and
carbamate 37.
Therefore, the SAR was developed in hepatocyte and whole
animal systems wherein test agents can be subject to absorption,
distribution, metabolism, and elimination considerations. Thus,
the biological activity and hence the observed SAR likely result
from combined effects of the test agent and its metabolites.
These biological observations imply that the chemical features
influencing the activity are the following:
Experimental Section
•Steric factorsstetramethyl substitution is favored over
arylmethyl substitution (e.g., 4b, 6b).
Chemistry. Chemical reagents from Sigma-Aldrich, Acros, or
Lancaster were used without further purification. Silica gel for
column chromatography (0.035-0.070 mm, pore diameter ca. 6
nm) was obtained from Acros Organics. ACS grade solvents from
Fisher Scientific or Mallinckrodt were routinely used for chro-
•Chain length and geometrysthe desirable chain length
should be 11-15 atoms.
•Nature of heteroatomssheteroatoms may inflict interactions
with other sites than the desired ones by their ability to form

Influence of Moieties on Hypolipidemic Properties
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 343
matographic purifications and extractions. Melting points (uncor-
rected) were determined on either a Thomas-Hoover capillary or
Haake-Buchler melting point apparatus. 1H NMR spectra were
recorded at 300 MHz and 13C NMR spectra at 75 MHz and ambient
temperature on Varian NMR spectrometers. Chemical shifts for
proton NMR are given in parts per million downfield from an
internal tetramethylsilane standard, and 13C chemical shifts are
calibrated on the CDCl3 resonance at 77.23 ppm, unless otherwise
specified. Coupling constants (J) are given in hertz. The purity of
target compounds was analyzed using Shimadzu HPLC systems
equipped with UV or RI detection or both.
Bis-(5,5-dimethyl-6-tetrahydropyranyloxyhexyl)-sulfide (3c).
A solution of 2c (14.9 g, 50.8 mmol) in EtOH (100 mL) was added
dropwise over 30 min to a solution of Na2S nonahydrate (6.1 g,
25.4 mmol) in water (10 mL) at room temperature under N2
atmosphere. The reaction mixture was stirred for 18 h and then
heated to reflux for 3.5 h. The solution was concentrated in vacuo,
5% aqueous NaOH solution (100 mL) was added, and the reaction
mixture was extracted with CH2Cl2 (200 mL). The organic layer
was dried over MgSO4, concentrated in vacuo, and dried in high
vacuum to give 3c (9.2 g, 78%) as a slightly yellowish oil. 1H NMR
(CDCl3): δ 4.54 (t, 2H, J ) 2.9), 3.83 (m, 2H), 3.48 (m, 2H), 3.45
(d, 2H, J ) 9.2), 2.98 (d, 2H, J ) 9.2), 2.50 (t, 4H, J ) 7.3), 1.82
(m, 2H), 1.75-1.44 (m, 16H), 1.42-1.18 (m, 10H), 0.89 (s, 6H),
0.89 (s, 6H). 13C NMR (CDCl3): δ 98.90, 76.26, 61.68, 38.77,
34.04, 32.00, 30.51, 25.44, 24.42, 24.36, 23.20, 19.28. HRMS:
calcd for C26H51SO4 (MH+) 459.3508; found 459.3504.
1-1.18 (m, 8H), 0.86 (s, 12H). 13C NMR (CDCl3): δ 71.43, 37.88,
34.86, 31.90, 30.20, 23.78, 22.93. HRMS: calcd for C16H35SO2
(MH+) 291.2358; found 291.2353. Anal. (C16H34O2S): C, H, S.
On a larger scale, deprotection of 3c (265.0 g, 0.58 mol) as above
gave crude 4c (152.0 g), which was distilled in high vacuum,
affording 4c (90.0 g, 53%). Bp 175-180 °C, 0.1-0.2 mm. HPLC:
99.2% pure.
6-(6-Hydroxy-5-methyl-5-phenylhexylsulfanyl)-2-methyl-2-
phenylhexan-1-ol (4d). Under N2-atmosphere, MeOH (1.41 g,
43.98 mmol) was added dropwise to a stirred suspension of LiBH4
(0.97 g, 43.98 mmol) in anhydrous CH2Cl2 (80 mL) at room
temperature. A solution of 5d (7.3 g, 14.66 mmol) in anhydrous
CH2Cl2 (60 mL) was added dropwise. The reaction mixture was
heated to reflux overnight. The mixture was cooled to 5 °C, and
ice (30 g) and saturated NH4Cl solution (100 mL) were added. The
aqueous layer was extracted with CH2Cl2 (3 × 50 mL). The
combined organic layers were washed with water (2 × 100 mL),
dried over MgSO4, and concentrated in vacuo to yield the crude
product (6.7 g). Purification by column chromatography (silica gel,
EtOAc/heptane )1/10 to 1/2) furnished 4d (4.5 g, 74% yield) as a
1
colorless viscous oil. H NMR (CDCl3): δ 7.35-7.18 (m, 10H),
3.68 (d, J ) 11.1, 2H), 3.52 (d, J ) 11.1, 2H), 2.37 (t, J ) 7.5,
4H), 1.82-1.69 (m, 2H), 1.55-1.42 (m, 8H), 1.33-1.22 (m, 2H),
1.33 (s, 6H), 1.07-1.03 (m, 2H). 13C NMR (CDCl3): δ 144.7,
128.5, 126.7, 126.2, 72.4, 43.5, 38.2, 32.1, 30.5, 23.4, 21.8.
HRMS: calcd for C26H39O2S (MH+) 415.2671; found 415.2670.
Anal. (C26H38O2S): C, H, S.
5-(5-Hydroxy-4,4-dimethylpentylsulfanyl)-2,2-dimethylpen-
tan-1-ol (4a). Under N2-atmosphere, LiAlH4 (1.0 g, 26 mmol) was
added to anhydrous Et2O (100 mL) and stirred for 10 min. To this
solution was added a solution of 5a (3.10 g, 8.90 mmol) in Et2O
(50 mL). The reaction mixture was heated to reflux for 3 h and
stirred at room temperature overnight. The excess of LiAlH4 was
decomposed by addition of EtOAc (20 mL). The sludge was
dissolved in aqueous HCl (5 N, 5 mL) and water (50 mL). The
mixture was extracted with Et2O (3 × 20 mL). The combined
organic layers were washed with saturated NH4Cl solution (20 mL),
dried over MgSO4, concentrated in vacuo, and dried in high vacuum
5-(4-Ethoxycarbonyl-4-methylpentylsufanyl)-2,2-dimethyl-
pentanoic Acid Ethyl Ester (5a). A solution of 1a (9.10 g, 38.4
mmol), KOH (85%, 2.75 g, 41.7 mmol), and thiourea (3.65 g, 48.0
mmol) in EtOH (100 mL) was stirred at room temperature for 20
min and then heated to 40-45 °C for 1 h. The solution was cooled,
and ice (100 g), concd aqueous HCl (100 mL), and water (100
mL) were added. The solution was extracted with CH2Cl2 (3 ×
200 mL). The combined organic phases were washed with 5%
NaHCO3 solution (2 × 200 mL) and saturated NH4Cl solution (300
mL), then dried over Na2SO4 and concentrated in vacuo to 5a (6.52
1
1
to give 4a (2.3 g, 98%) as an oil. H NMR (CDCl3): δ 3.31 (s,
g, 89%) as a clear, pale-yellow oil. H NMR (CDCl3): δ 4.11 (q,
4H), 2.72 (br, 2H), 2.50 (t, 4H, J ) 7.3), 1.62-1.50 (m, 4H), 1.37-
1.25 (m, 4H), 0.87 (s, 12H). 13C NMR (CDCl3): δ 71.61, 37.86,
35.16, 33.21, 24.36, 24.02. HRMS: calcd for C14H31O2S (MH+)
263.2045; found 263.2044. HPLC: 92.7% pure.
4H, J ) 7.1), 2.47 (t, 4H, J ) 6.9), 1.70-1.46 (m, 8H), 1.25 (t,
6H, J ) 7.1), 1.17 (s, 12H). 13C NMR (CDCl3): δ 177.5, 60.1,
41.9, 39.7, 32.3, 25.00, 24.95, 14.1. HRMS: calcd for C18H35O4S
(MH+) 347.2256; found 347.2261.
5-(5-Hydroxy-4-methyl-4-phenylpentylsulfanyl)-2-methyl-2-
phenylpentan-1-ol (4b). Under N2-atmosphere, MeOH (2.96 g,
92.6 mmol) was added dropwise to a stirred suspension of LiBH4
(2.04 g, 92.6 mmol) in anhydrous CH2Cl2 (160 mL) at room
temperature. A solution of 5b (14.5 g, 30.9 mmol) in anhydrous
CH2Cl2 (140 mL) was added dropwise. The reaction mixture was
heated to reflux overnight. The reaction mixture was cooled to 5
°C, and saturated NH4Cl solution (200 mL) was added. The aqueous
layer was extracted with CH2Cl2 (3 × 300 mL). The combined
organic layers were washed with water (2 × 200 mL), dried over
MgSO4, and concentrated in vacuo to yield the crude product (12.5
g). Purification by column chromatography (silica gel, EtOAc/
heptane ) 1/9 to 1/3) furnished 4b (6.39 g, 54%) as a colorless,
5-(4-Ethoxycarbonyl-4-phenylpentylsulfanyl)-2-methyl-2-phe-
nylpentanoic Acid Ethyl Ester (5b). A solution of Na2S nonahy-
drate (2.65 g, 11 mmol) and 1b (6.49 g, 22 mol) in water (50 mL)
and EtOH (5 mL) was stirred at 40 °C for 20 h and heated to reflux
for 1 h. The solution was concentrated in vacuo and 5% aqueous
NaOH solution (50 mL) was added. The mixture was extracted
with CH2Cl2 (3 × 50 mL); the combined organic layers were
washed with water (2 × 100 mL) and dried over anhydrous MgSO4.
The solution was filtered through basic alumina (100 g), eluting
with CH2Cl2 (250 mL). The filtrate was concentrated and dried
(100 °C, 1 mmHg) to produce 5b (4.18 g, 85%) as a colorless,
viscous oil, which was used without further purification in the next
step. 1H NMR (CDCl3): δ 7.32-7.19 (m, 10H), 4.11 (q, 4H, J )
6.9), 2.46-2.40 (m, 4H), 2.09-1.98 (m, 4H), 1.54 (s, 6H), 1.48
(m, 4H), 1.17 (t, 6H, J ) 6.9). 13C NMR (CDCl3): δ 176.08,
143.78, 128.47, 126.79, 126.07, 60.92, 50.14, 38.64, 32.54, 24.95,
22.89, 14.25.
6-(5-Ethoxycarbonyl-5-phenylhexylsulfanyl)-2-methyl-2-phe-
nylhexanoic acid ethyl ester (5d). According to the procedure
given for 5a, 1d (18.5 g, 59.1 mmol) was reacted with thiourea
(7.0 g, 92.0 mmol) and KOH (85%, 6.1 g, 92.4 mmol) in EtOH
(200 mL). After workup, the residue was purified by column
chromatography (silica gel, hexanes/EtOAc ) 10/1) to give 5d (12.5
g, 85%) as a yellow oil. 1H NMR (CDCl3): δ 7.42-7.15 (m, 10H),
4.20-4.05 (q, 4H, J ) 7.1), 2.44 (t, 4H, J ) 7.6), 2.15-1.95 (m,
2H), 1.95-1.75 (m, 2H), 1.62-1.40 (m, 10H), 1.35-1.10 (m, 4H),
1.18 (t, 6H, J ) 7.1). 13C NMR (CDCl3): δ 176.06, 143.89, 128.25,
1
viscous oil. H NMR (CDCl3): δ 7.29-7.19 (m, 10H), 3.64 (d, J
) 9.9, 2H), 3.51 (m, 2H), 2.32 (t, J ) 6.9, 4H), 1.81-1.77 (m,
2H), 1.58-1.37 (m, 6H), 1.31 (s, 6H), 1.27-1.21 (m, 2H). 13C
NMR (CDCl3): δ 144.5, 128.5, 126.7, 126.2, 72.5, 43.4, 37.6, 32.8,
24.0, 21.7. HRMS: calcd for C24H35O2S (MH+) 387.2358; found
387.2350. HPLC: 95.6% pure. Anal. (C24H34O2S) C, H, S.
6-(5,5-Dimethyl-6-hydroxyhexylsulfanyl)-2,2-dimethylhexan-
1-ol (4c). A solution of 3c (9.2 g, 20.0 mmol) in MeOH (100 mL)
and concd HCl (10 mL) was heated to reflux for 1.5 h under N2
atmosphere. The reaction mixture was cooled to room temperature,
concentrated in vacuo, diluted with CH2Cl2 (250 mL), and extracted
with saturated NaHCO3 solution (2 × 100 mL) and brine (100 mL).
The organic layer was dried over MgSO4 and concentrated in vacuo
to give 4c (4.5 g, 77%) as a malodorous oil. 1H NMR (CDCl3): δ
3.30 (s, 4H), 2.52 (t, 4H, J ) 7.1), 2.24 (s, 2H), 1.57 (m, 4H),

344 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Oniciu et al.
126.52, 125.84, 60.65, 50.04, 38.77, 31.80, 30.11, 24.06, 22.61,
14.01. HRMS: calcd for C30H43O4S1 (MH+) 499.2882; found
499.2868.
(m, 2H), 2.52 (m, 4H), 1.80-1.53 (m, 10 H), 1.33 (s, 6H), 1.33-
1.23 (m, 4H). 13C NMR (CDCl3): δ 144.60, 128.61, 126.69, 126.34,
72.47, 72.26, 52.33, 43.55, 43.50, 38.15, 23.51, 22.06, 21.79.
HRMS: calcd for C26H39O3S1 (MH+) 431.2620; found 431.2611.
HPLC: 93.0% pure. Anal. (C26H38O3S1) C, H, S.
5-(5-Hydroxy-4,4-dimethylpentyl-1-sulfinyl)-2,2-dimethylpen-
tan-1-ol (6a). To a solution of 4a (10.0 g, 38.0 mmol) in glacial
acetic acid (50 mL) was added H2O2 (50 wt. % in water, 2.64 g,
38.0 mmol) under cooling in an ice-water bath. The reaction
mixture was stirred for 4 h and stored in a refrigerator overnight.
The reaction mixture was diluted with water (500 mL) and extracted
with CHCl3 (2 × 250 mL). The combined organic phases were
washed with saturated NaHCO3 solution (2 × 300 mL) and
saturated NaCl solution (200 mL), dried over MgSO4, and
concentrated in vacuo. The residue (12.8 g) was dissolved in hot
EtOAc (ca. 20 mL) and filtered. Hexanes was slowly added to this
solution until a slight turbidity was produced, which was cleared
by addition of a small portion of EtOAc. The solution was allowed
to cool to room temperature and stored at -5 °C overnight to give
6-(5,5-Dimethyl-6-hydroxyhexane-1-sulfonyl)-2,2-dimethyl-
hexan-1-ol (7c). To a solution of 4c (7.6 g, 26.2 mmol) in CHCl3
(300 mL) was added a solution of MCPBA (17.6 g, 78.6 mmol) in
CHCl3 (150 mL) over 1 h at room temperature. The reaction mixture
was stirred for 17 h at room temperature and then washed with
saturated NaHCO3 solution (3 × 200 mL) and saturated NaCl
solution (200 mL). The organic layer was dried over MgSO4 and
concentrated in vacuo. The residue was purified by flash chroma-
tography (silica; CHCl3/MeOH ) 95:5) to yield 7c (4.3 g, 51%)
1
as an oil, which solidified upon standing. H NMR (CDCl3): δ
3.30 (s, 4H), 2.97 (m, 4H), 2.32 (s br, 2H), 1.82 (m, 4H), 1.40 (m,
4H), 1.26 (m, 4H), 0.86 (s, 12H). 13C NMR (CDCl3): δ 71.49,
52.66, 37.95, 35.16, 24.11, 23.03, 22.87. HRMS: calcd for C16H35-
SO4 (MH+) 323.2256; found 323.2261. HPLC: 97.8% pure.
6-(5-Carboxy-5-phenyl-hexylsulfanyl)-2-methyl-2-phenylhex-
anoic Acid (8d). A solution of 5d (6.0 g, 12.0 mmol) and NaOH
(3.1 g, 74.0 mmol) in EtOH (40 mL) and deionized water (4 mL)
was heated to reflux for 2 h. The EtOH was evaporated in vacuo,
and the residue was dissolved in water (20 mL) and extracted with
Et2O (20 mL). The aqueous layer was acidified with 2 N aqueous
HCl (15 mL) to pH 2-3 and extracted with Et2O (4 × 50 mL).
The combined organic layers were washed with brine (15 mL),
dried over MgSO4, and concentrated in vacuo. The residue (4.5 g)
was purified by column chromatography (silica gel, hexanes/EtOAc
) 10/1) to give 8d (3.6 g, 68%) as a yellowish oil. 1H NMR
(CDCl3): δ: 7.42-7.20 (m, 10H), 2.45 (t, 4H, J ) 7.3), 2.15-
1.80 (m, 4H), 1.65-1.45 (m, 4H), 1.56 (s, 6H), 1.40-1.15 (m,
4H). 13C NMR (CDCl3): δ 182.43, 142.89, 128.41, 126.91, 126.05,
50.00, 38.53, 31.80, 30.00, 24.03, 22.40. HRMS: calcd for
C26H35O4S1 (MH+) 443.2256; found 443.2231. HPLC: 91.9% pure.
2,2,12,12-Tetramethyltridecane-1,7,13-triol (10). Under Ar
atmosphere, methyl tert-butyl ether (MTBE, 50 mL) was added to
LiAlH4 (2.56 g, 67.5 mmol), and the gray suspension was stirred
under cooling with an ice bath. A solution of 9 (87% pure by GC,
10.0 g, 27.0 mmol) in MTBE (50 mL) was added dropwise,
followed by additional MTBE (100 mL). After 2 h at 0 °C, the
reaction mixture was quenched by addition of EtOAc (30 mL) and
allowed to warm to room temperature overnight. The mixture was
cooled with an ice-bath and carefully hydrolyzed by addition of
crushed ice (47 g) and water (50 mL). The pH was adjusted to 1
by addition of 2 N H2SO4 (140 mL), and the solution was stirred
at room temperature for 15 min. The layers were separated, and
the aqueous layer was extracted with MTBE (100 mL). The
combined organic layers were washed with deionized water (100
mL), saturated NaHCO3 solution (100 mL), and saturated NaCl
solution (100 mL), dried over MgSO4, concentrated in vacuo, and
dried in high vacuum. The residue (6.6 g) was crystallized from
hot CH2Cl2 (65 mL). The crystals were filtered, washed with ice-
cold CH2Cl2 (3 × 10 mL), and dried in high vacuum to give 10
1
6a (7.1 g, 67%) as a white, crystalline solid. Mp 53-54 °C. H
NMR (CDCl3): δ 3.31 (m, 4H), 2.78-2.50 (m, 4H), 2.45 (br, 2H),
1.67-1.80 (m, 4H), 1.50-1.25 (m, 4H), 0.87 (s, 6H), 0.86 (s, 6H).
13C NMR (CDCl3): δ 70.82, 52.82, 37.35, 35.16, 24.16, 23.86,
17.43. HRMS: calcd for C14H31O3S1 (MH+) 279.1994; found
279.2015. HPLC: 96.7% pure. Anal. (C14H30O3S) C, H, S.
5-(5-Hydroxy-4-methyl-4-phenylpentylsulfinyl)-2-methyl-2-
phenylpentan-1-ol (6b). To a solution of 4b (4.3 g, 11.14 mmol)
in glacial acetic acid (120 mL) was added H2O2 (50 wt % in water,
0.79 mL, 11.14 mmol). The reaction mixture was stirred at room
temperature for 4 h, then diluted with water (500 mL) and extracted
with CH2Cl2 (3 × 150 mL). The combined organic phases were
washed with saturated NaHCO3 solution (3 × 150 mL) and brine
(150 mL), dried over MgSO4, and concentrated in vacuo to yield
the crude product (4.2 g). Purification by column chromatography
(silica gel, EtOAc/CH2Cl2 ) 1/1) furnished 6b (3.1 g, 69%) as a
1
colorless oil. H NMR (CDCl3): δ 7.33-7.21 (m, 10H), 3.70-
3.50 (m, 4H), 2.60-2.32 (m, 4H), 2.00-1.80 (m, 4H), 1.80-1.52
(m, 4H), 1.52-1.31 (m, 2H), 1.35 (s, 6H). 13C NMR (CDCl3): δ
144.3, 128.7, 126.6, 126.5, 72.0, 71.7, 53.0, 52.9, 43.5, 37.7, 37.5,
22.2, 21.8, 18.0, 17.8. HRMS: calcd for C24H35O3S1 (MH+)
403.2307; found 403.2295. HPLC: 99.4% pure.
6-(5,5-Dimethyl-6-hydroxyhexane-1-sulfinyl)-2,2-dimethylhexan-
1-ol (6c). To a solution of 4c (12.0 g, 41.3 mmol) in glacial acetic
acid (100 mL) was added H2O2 (50 wt % in water, 2.7 g, 39.7
mmol) under cooling with an ice bath. The reaction mixture was
kept in the refrigerator overnight, then diluted with water (400 mL)
and extracted with CHCl3 (2 × 150 mL). The combined organic
phases were subsequently washed with saturated NaHCO3 solution
(3 × 200 mL) and saturated NaCl solution (200 mL), dried over
MgSO4, and concentrated in vacuo. The residue (12.0 g) was
dissolved in warm Et2O (ca. 20 mL), and some pentane was added
slowly until the solution became turbid. The turbidity was cleared
by addition of a small portion of Et2O, and the solution was cooled
to room temperature, then to -5 °C to give a cream-white,
crystalline solid. Additional crystallization from warm EtOAc (ca.
10 mL) and some pentane gave 6c (8.0 g, 63%) as a white,
crystalline solid. Mp 43-44 °C. 1H NMR (CDCl3): δ 3.29 (d, 2H,
J ) 12.2), 3.27 (d, 2H, J ) 12.2), 2.70 (m, 4H), 2.48 (s br, 2H),
1.76 (m, 4H), 1.44 (m, 4H), 1.30 (4H), 0.87 (s, 6H), 0.86 (s, 6H).
13C NMR (CDCl3): δ 71.39, 52.20, 38.08, 35.15, 24.21, 24.03,
23.66, 23.26. HRMS: calcd for C16H35SO3 (MH+) 307.2307; found
307.2309. HPLC: 98.9% pure.
1
(3.57 g, 46%) as an off-white solid. Mp 70 °C. H NMR (CD3-
OD): δ 3.50 (m, 1H), 3.22 (s, 4H), 1.55-1.15 (m, 16H), 0.84 (s,
12H). 13C NMR (CD3OD): δ 72.38, 72.01, 39.97, 38.47, 35.98,
27.85, 25.09, 24.64, 24.60. HRMS: calcd for C17H37O3 (MH+)
289.2743; found 289.2756. HPLC: 95.0% pure. Anal. (C17H36O3)
C, H.
6-(6-Hydroxy-5-methyl-5-phenylhexylsulfinyl)-2-methyl-2-
phenylhexan-1-ol (6d). To a solution of 4d (3.8 g, 9.2 mmol) in
glacial acetic acid (100 mL) was added hydrogen peroxide (50 wt
% in water, 0.64 mL, 9.2 mmol). The reaction mixture was stirred
at room temperature for 4 h, then diluted with water (500 mL) and
extracted with CH2Cl2 (3 × 150 mL). The combined organic phases
were washed with saturated NaHCO3 solution (3 × 150 mL) and
brine (150 mL), dried over MgSO4, and concentrated in vacuo.
Purification by column chromatography (silica gel, EtOAc/CH2-
7-Hydroxy-2,2,12,12-tetramethyltridecanedioic acid diethyl
ester (11). To a solution of 10 (9.2 g, 24.8 mmol) in MeOH (200
mL) was added NaBH4 (0.95 g, 25.1 mmol) under cooling with an
ice-water bath. After 2 h, another portion of NaBH4 (0.95 g, 25.1
mmol) was added and stirring was continued for 2 h. The reaction
mixture was hydrolyzed with water (200 mL) and extracted with
CH2Cl2 (3 × 150 mL). The combined organic layers were dried
over MgSO4 and concentrated in vacuo to give 11 (8.5 g, 92%) as
an oil. 1H NMR (CDCl3): δ 4.11 (q, 4H, J ) 7.0), 3.60-3.50 (m,
1H), 1.66-1.32 (m, 11H), 1.24 (t, 12H, J ) 7.0), 1.15 (s, 12H).
13C NMR (CDCl3): δ 178.0, 71.7, 60.1, 42.1, 40.6, 37.3, 26.0,
1
Cl2 ) 1/1) furnished 6d (2.9 g, 73%) as a colorless oil. H NMR
(CDCl3): δ 7.31-7.21 (m, 10 H), 3.70 (d, J ) 10.5 Hz, 2H), 3.57

Influence of Moieties on Hypolipidemic Properties
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 345
25.1, 24.9, 14.2. HRMS: calcd for C21H41O5 (MH+) 373.2954;
found 373.2936.
The reaction mixture was stirred for 2 h at room temperature to
give a dark-green solution of sodium naphthalenide. A portion of
this solution (ca. 40 mL) was added dropwise to a solution of N,N-
bis-(5,5-dimethyl-6-tetrahydropyranyloxyhexyl)-p-toluenesulfona-
mide (7.0 g, 11.7 mmol) in anhydrous DME (200 mL) at -78 °C
until the greenish color persisted. After 15 min, the reaction mixture
was hydrolyzed with saturated NaHCO3 solution (20 mL) and
warmed to room temperature. K2CO3 (100 g) was added, and the
reaction mixture was stirred for 1.5 h. The solids were removed
by filtration and washed with Et2O (2 × 200 mL). The filtrate was
dried over Na2SO4 and concentrated to give bis-(5,5-dimethyl-6-
tetrahydropyranyloxyhexyl)-amine (13.5 g) as an oil. This oil (13.5
g) was dissolved in MeOH (100 mL), concd HCl (10 mL) was
added, and the reaction mixture was heated to reflux under N2
atmosphere for 2 h. After the mixture cooled to room temperature,
water (200 mL) was added, and the non-salts were removed by
extraction with CH2Cl2 (3 × 100 mL). The pH of the aqueous layer
was adjusted to 11 with solid Na2CO3. The aqueous layer was
extracted with CH2Cl2 (3 × 100 mL). The combined organic layers
were dried over Na2SO4 and concentrated in vacuo to give 21 as a
red oil. This oil was dissolved in EtOH (20 mL) and acidified with
concd HCl (2 mL) to pH 1. The solvents were removed in high
vacuum, affording 20 (1.45 g, 37% over three steps) as a reddish
7-Hydroxy-2,2,12,12-tetramethyltridecanedioic Acid (12). A
mixture of 11 (91.6 g, 0.246 mol) and NaOH (24.6 g, 0.62 mol) in
EtOH (60 mL) and water (19 mL) was refluxed for 2.5 h. The
mixture was diluted with water (150 mL) and extracted with MTBE
(2 × 100 mL). The aqueous solution was acidified with 6 M H2-
SO4 (ca. 100 mL) to pH 2 and extracted with MTBE (2 × 200
mL). The combined organic layers were washed with water (200
mL), dried over MgSO4, and concentrated in vacuo. The residue
(77.0 g) was purified by column chromatography (silica gel,
heptane/EtOAc ) 9:1, 4:1, 2:1, 3:2) to yield two fractions of 12
(10.0 g, 96.3% pure by HPLC; 51.8 g, 97.9% pure by HPLC.
Combined yield 80%), both as oils. 1H NMR (CDCl3): δ 8.10 (br,
3H), 3.58 (br, 1H), 1.62-1.22 (m, 16H), 1.18 (s, 12H). 13C NMR
(CDCl3): δ 184.3, 71.8, 42.1, 40.5, 36.9, 25.9, 25.0, 24.9. HRMS:
calcd for C17H33O5 (MH+) 317.2328; found 317.2330. Anal.
(C17H32O5) C, H.
7-Hydroxymethyl-2,2,12,12-tetramethyltridecane-1,13-diol (17).
Under N2 atmosphere, to a solution of LiAlH4 (1.09 g, 28.8 mmol)
in anhydrous THF (100 mL) was added dropwise a solution of 16
(3.64 g, 11.5 mmol) in THF (40 mL) at room temperature. The
reaction mixture was heated to reflux for 5 h and kept at room
temperature overnight. Water (100 mL) was added carefully to the
reaction mixture under cooling with a water bath. The pH of the
mixture was adjusted to 1 with 2 M aqueous HCl. The product
was extracted with Et2O (3 × 60 mL). The combined organic layers
were washed with water (3 × 50 mL) and brine (50 mL), dried
over Na2SO4, and concentrated in vacuo. The residue (3.2 g) was
purified by chromatography (silica, EtOH) to give 17 (3.10 g, 89%)
1
glass. H NMR (CD3OD): δ 3.24 (s, 4H), 3.00 (m, 4H), 1.70 (m,
4H), 1.48-1.22 (m, 8H), 0.88 (s, 12H). 13C NMR (CD3OD): δ
71.66, 49.06, 39.34, 36.02, 28.25, 24.65, 22.25. HRMS: calcd for
C16H36NO2 (MH+) 274.2746; found 274.2746. GC: 95.5% pure.
6-(6-Hydroxy-5,5-dimethylhexylamino)-2,2-dimethylhexan-1-
ol (21). Compound 20 (7.68 g, 24.78 mmol) was extracted with
10% aqueous NaOH solution (100 mL) and CH2Cl2 (80 mL). The
layers were separated, and the aqueous layer was extracted with
CH2Cl2 (2 × 80 mL). The combined organic layers were washed
with saturated NaCl solution (50 mL), dried over Na2SO4,
concentrated in vacuo, and dried in high vacuum to afford 21 (5.55
1
as a yellow oil. H NMR (CD3OD): δ 4.88 (s, 3H), 3.44 (d, 2H,
J ) 4.8), 1.50-1.10 (m, 17H), 0.85 (s, 12H). 13C NMR (CD3OD):
δ 72.0, 65.7, 41.7, 40.0, 36.0, 32.2, 29.0, 25.4, 24.7, 24.6. HRMS:
calcd for C18H39O3 (MH+) 303.2899; found 303.2901. HPLC:
94.6% pure. Anal. (C18H38O3): C, H.
1
g, 82%) as an orange, viscous oil. H NMR (CDCl3): δ 3.27 (s,
4H), 3.1-2.4 (br, OH, NH), 2.60 (t, 4H, J ) 7.1), 1.48 (m, 4H),
1.25 (m, 8H), 0.85 (s, 12H). 13C NMR (CDCl3): δ 71.15, 49.62,
38.05, 35.13, 30.35, 24.29, 21.36. HRMS: calcd for C16H36NO2
(MH+) 274.2746; found 274.2746. Anal. (C16H35NO2) C, H, N.
6-[Benzoyloxy-(6-hydroxy-5,5-dimethylhexyl)-amino]-2,2-di-
methylhexan-1-ol (22). Under Ar atmosphere, to a stirred suspen-
sion of 21 (2.62 g, 9.58 mmol) and Na2HPO4 (6.95 g, 48.93 mmol)
in MTBE (50 mL) was added dropwise over 45 min a solution of
benzoyl peroxide (2.55 g, 10.54 mmol) in MTBE (90 mL) at room
temperature. The mixture was heated to 45 °C for 17 h, cooled to
room temperature, diluted with MTBE (100 mL), and extracted
with 10% Na2CO3 solution (2 × 100 mL) and brine (50 mL). The
organic layer was dried over MgSO4 and concentrated in vacuo.
The residue was purified by flash chromatography (silica, hexanes/
EtOAc ) 50/50) to afford 22 (2.24 g, 59%) as a viscous, slightly
9-Hydroxy-3-(6-hydroxy-5,5-dimethylhexyl)-8,8-dimethylnonan-
2-one (18) and 7-(1-Hydroxy-1-methylethyl)-2,2,12,12-tetra-
methyltridecan-1,13-diol (19). A solution of 16 (1.0 g, 3.16 mmol)
in THF (40 mL) was cooled in an ice-water bath and methyllithium
(1.4 M in Et2O, 27 mL, 37.8 mmol) was added. The mixture was
kept at 0 °C for 2 h, and then was poured into diluted HCl (5 mL
concd HCl/60 mL water). The organic layer was separated, and
the aqueous layer was extracted with Et2O (2 × 50 mL). The
combined organic layers were dried over Na2SO4 and concentrated.
The residue (1.0 g) was purified by column chromatography
(hexanes/EtOAc ) 80/20, then 50/50) to give 18 (0.41 g, 41%) as
1
an oil and 19 (0.40 g, 38%) as a white solid. Compound 18. H
NMR (CDCl3): δ 3.26 (s, 4H), 2.45-2.30 (m, 1H), 2.08 (s, 3H),
1.86 (br, 2H), 1.62-1.30 (m, 4H), 1.30-1.05 (m, 12H), 0.82 (s,
12H). 13C NMR (CDCl3): δ 213.4, 71.7, 53.2, 38.3, 34.9, 31.6,
28.7, 28.3, 23.8. HRMS: calcd for C19H39O3 (MH+) 315.2899;
found 315.2866. HPLC: 90.5% pure. Anal. (C19H38O3) C, H.
Compound 19. Mp 72-74 °C. 1H NMR (CDCl3): δ 3.24 (s, 4H),
2.59 (br, 3H), 1.55-0.95 (m, 17H), 1.11 (s, 6H), 0.81 (s, 12H).
13C NMR (CDCl3): δ 74.0, 71.5, 49.6, 38.4, 34.9, 31.2, 30.3, 27.1,
24.3, 23.9, 23.8. HRMS: calcd for C20H43O3 (MH+) 331.3212;
found 331.3205. HPLC: 96.4% pure.
6-(6-Hydroxy-5,5-dimethylhexylamino)-2,2-dimethylhexan-1-
ol Hydrochloride (20). A mixture of 2c (15.2 g, 51.8 mmol),
p-toluenesulfonamide (4.43 g, 25.9 mmol), NaOH (2.60 g, 64.75
mmol), TBAI (480 mg, 1.30 mmol), benzene (175 mL), and water
(50 mL) was stirred vigorously and heated to 70 °C under N2
atmosphere. Additional TBAI (400 mg, 1.08 mol) was added after
20 h and stirring was continued at 80 °C. After 24 h, the mixture
was cooled to room temperature, the layers were separated, and
the organic layer was washed with water (100 mL). The organic
layer was dried over MgSO4, concentrated, and dried in vacuo to
furnish N,N-bis-(5,5-dimethyl-6-tetrahydropyranyloxyhexyl)-p-tolu-
enesulfonamide (14.5 g) as a colorless, viscous oil. Under N2
atmosphere, anhydrous DME (75 mL) was added to a mixture of
Na (2.15 g, 93.6 mmol) and naphthalene (14.8 g, 115.5 mmol).
1
yellowish oil. H NMR (CDCl3): δ 8.01 (m, 2H), 7.97 (m, 1H),
7.44 (m, 2H), 3.29 (s, 4H), 2.98 (t, 4H, J ) 7.3), 2.62 (s, 2H), 1.56
(m, 4H), 1.42-1.16 (m, 8H), 0.83 (s, 12H). 13C NMR (CDCl3): δ
165.92, 133.17, 129.56, 129.17, 128.51, 71.20, 59.33, 37.97, 35.04,
27.46, 24.20, 21.28. HRMS: calcd for C23H40NO4 (MH+) 394.2957;
found 394.2954.
6-[Hydroxy-(6-hydroxy-5,5-dimethylhexyl)-amino]-2,2-di-
methylhexan-1-ol (23). Under Ar atmosphere, to a solution of 22
(2.0 g, 5.08 mmol) in anhydrous MeOH (20 mL) was added a
solution of NaOMe in anhydrous MeOH (0.5 M, 20.4 mL, 10.16
mmol) at room temperature. The mixture was stirred for 4 h, diluted
with saturated NH4Cl solution (200 mL), and extracted with CH2-
Cl2 (2 × 50 mL). The combined organic layers were washed with
saturated NaCl solution (100 mL), dried over MgSO4, concentrated
in vacuo, and dried in high vacuum. The residue (1.6 g) was purified
by flash chromatography (silica, hexanes/EtOAc ) 25/75) to afford
23 (710 mg, 48%) as a white solid. Crystallization from MTBE/
hexanes (10 mL, 50/50) at - 5 °C furnished the product (620 mg,
42%) in the form of white crystals. Mp 73 °C. 1H NMR (CDCl3):
δ 3.30 (s, 4H), 2.67 (t, 4H, J ) 7.1), 1.58 (m, 4H), 1.27 (m, 4H),
0.85 (s, 12H). 13C NMR (CDCl3): δ 71.59, 60.57, 38.31, 35.21,

346 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Oniciu et al.
27.85, 24.29, 21.68. HRMS: calcd for C16H36NO3 (MH+) 290.2695;
found 290.2676. Anal. (C16H35NO3) C, H, N.
the reaction mixture was stirred at room temperature for 3.5 h. The
reaction mixture was diluted with water (250 mL) and extracted
with Et2O (250 mL). The aqueous layer was acidified with concd
HCl (7 mL) to pH 1 and then extracted with Et2O (2 × 250 mL).
The combined organic phases were washed with saturated NaCl
solution (100 mL), dried over MgSO4, concentrated in vacuo, and
dried in high vacuum to give 34 (4.1 g, 30%) as a viscous oil,
6,6-Dimethyl-7-(tetrahydropyran-2-yloxy)-heptanoic Acid [5,5-
Dimethyl-6-(tetrahydropyran-2-yloxy)-hexyl]-amide (29). To a
solution of 25 (1.0 g, 4.36 mmol) in CH2Cl2 (600 mL) was added
dropwise a solution of 28 (1.55 g, 4.36 mmol) in CH2Cl2 (200 mL).
The mixture was stirred for 48 h at room temperature. The solvent
was removed in vacuo. The residue was dissolved in Et2O, and the
solution was filtered to remove leftover DCU from the previous
step. The filtrate was concentrated in vacuo and purified by flash
chromatography (silica gel, hexanes/EtOAc ) 2/1) to furnish 29
1
which was used for the next step without further purification. H
NMR (CDCl3): δ 4.54 (t, 2H, J ) 3.3), 4.02 (m, 4H), 3.82 (m,
2H), 3.50 (m, 2H), 3.45 (d, 2H, J ) 9.2), 2.98 (d, 2H, J ) 9.2),
1.92-1.20 (m, 24H), 0.89 (s, 6H), 0.88 (s, 6H). 13C NMR
(CDCl3): δ 99.33, 76.64, 67.76 (J ) 6), 62.14, 39.00, 34.40, 31.28
(J ) 7), 30.82, 25.72, 24.67, 20.10, 19.61.
1
(1.56 g, 76%) as a colorless oil. H NMR (CDCl3): δ 5.50 (br,
1H), 4.50 (m, 2H), 3.75 (m, 2H), 3.40 (m, 4H), 3.15 (m, 2H), 2.95
(d, 2H, J ) 9.2), 2.12 (t, 2H, J ) 7.5), 1.90-1.10 (m, 24H), 0.85
(s, 12H). 13C NMR (CDCl3): δ 173.17, 99.27, 99.20, 76.50, 76.43,
62.12, 62.01, 39.44, 38.97, 38.85, 36.85, 34.20, 30.72, 30.52, 26.83,
25.60, 24.54, 23.70, 21.29, 19.60, 19.53. HRMS: calcd for
C27H52O5N (MH+) 470.3845; found 470.3839.
Phosphoric Acid Bis-(5,5-dimethyl-6-hydroxyhexyl)-ester (35).
A solution of 34 (4.0 g, 7.65 mmol) in MeOH (100 mL) and concd
HCl (10 mL) was refluxed for 2 h. The solution was diluted with
water (200 mL) and concentrated under reduced pressure to a
volume of ca. 100 mL. The solution was extracted with CH2Cl2 (3
× 100 mL). The combined organic layers were extracted with
saturated NaHCO3 solution (2 × 50 mL), then acidified with concd
HCl (10 mL) to pH 1 and extracted with CH2Cl2 (3 × 75 mL).
The combined organic layers were dried over MgSO4, concentrated
in vacuo, and dried in high vacuum to give 35 (1.73 g, 64%) as a
viscous oil. 1H NMR (CDCl3): δ 6.18 (br, 3H), 4.05 (m, 4H), 3.33
(s, 4H), 1.67 (m, 4H), 1.48-1.22 (m, 8H), 0.87 (s, 12H). 13C NMR
(CDCl3): δ 71.15, 67.29 (J ) 6), 37.70, 35.14, 30.94 (J ) 7),
24.38, 19.76. HRMS: calcd for C16H36PO6 (MH+) 355.2250; found
355.2245.
7-Hydroxy-6,6-dimethylheptanoic Acid (6-Hydroxy-5,5-di-
methylhexyl)-amide (30). To a solution of 29 (1.41 g, 3.0 mmol)
in MeOH (50 mL) was added concd aqueous HCl (9 mL). The
mixture was refluxed for 2 h, cooled to room temperature, diluted
with water (50 mL), and concentrated in vacuo to a volume of ca.
60 mL. The solution was extracted with CH2Cl2 (3 × 100 mL).
The combined organic layers were washed with saturated NaHCO3
solution (3 × 100 mL) and brine (50 mL), dried over MgSO4, and
concentrated in vacuo. The residual oil was purified by flash
chromatography (silica, EtOAc/hexanes ) 1/2, then CH2Cl2/MeOH
1
) 10/1) to furnish 30 (0.77 g, 85%) as a yellowish oil. H NMR
[5,5-Dimethyl-6-(tetrahydropyran-2-yloxy)-hexyl]-carbamic
Acid 5,5-Dimethyl-6-(tetrahydropyran-2-yloxy)-hexyl Ester (36).
To a solution of 31 (4.00 g, 17.4 mmol) and CDI (3.52 g, 21.7
mmol) in anhydrous CH2Cl2 (200 mL) was added DMAP (0. g,
3.4 mmol) at room temperature under N2 atmosphere. The reaction
mixture was stirred for 2 h and concentrated in vacuo. A portion
of the residue (2.7 g of 5.0 g) was dissolved in anhydrous CH3CN
(160 mL), and 25 (8.25 g, 36.00 mmol) in anhydrous CH3CN (20
mL) was added dropwise. The reaction mixture was stirred for 24
h at room temperature, then washed with 15% aqueous citric acid
solution (2 × 75 mL) and 1% aqueous HCl (100 mL). The organic
layer was dried over MgSO4 and concentrated in vacuo to afford
36 (3.05 g, 67%) as an oil, which was used without further
(CDCl3): δ 6.05 (m, 1 H), 3.28 (s, 4 H), 3.25 (m, 2 H), 2.58 (br,
2 H), 2.19 (t, 2 H, J ) 7.0), 1.61 (m, 2 H), 1.48 (m, 2 H), 1.25 (m,
8 H), 0.85 (s, 12 H). 13C NMR (CDCl3): δ 173.80, 71.35, 71.15,
39.10, 37.99, 37.82, 36.60, 35.17, 30.55, 26.69, 24.40, 24.30, 23.41,
20.80. HRMS: calcd for C17H36NO3 (MH+) 302.2695; found
302.2723. HPLC: 97.0% pure.
6,6-Dimethyl-7-(tetrahydropyran-2-yloxy)-heptanoic Acid 5,5-
Dimethyl-6-(tetrahydropyran-2-yloxy)-hexyl Ester (32). A mix-
ture of 31 (8.1 g, 35.2 mmol), 27 (10.0 g, 38.7 mmol), DCC (8.8
g, 0.7 mmol), and DMAP (1.1 g, 9.0 mmol) in CH2Cl2 (600 mL)
under N2 atmosphere was stirred at room temperature for 18 h.
The precipitated DCU was removed by filtration. The filtrate was
concentrated in vacuo, and the residue was purified by column
chromatography (silica, hexanes/EtOAc ) 5/1), affording 32 (11.0
g, 66%) as an oil, which was used without further purification for
the next step. 1H NMR (CDCl3): δ 4.50 (m, 2H), 4.08 (t, 2H, J )
7.5), 3.75 (m, 2H), 3.40 (m, 4H), 2.95 (m, 2H), 2.25 (t, 2H, J )
7.5), 1.90-1.10 (m, 24H), 0.85 (s, 12H). 13C NMR (CDCl3): δ
174.46, 99.64, 77.00, 64.90, 62.43, 39.57, 35.46, 34.96, 31.21,
30.10, 26.93, 25.99, 24.26, 24.12, 20.94, 19.99. HPLC: 56.7% pure.
7-Hydroxy-6,6-dimethylheptanoic Acid 6-Hydroxy-5,5-di-
methylhexyl Ester (33). A solution of 32 (10.0 g, 21.2 mmol) in
a solvent mixture of acetic acid, THF, and water (4/2/1, 440 mL)
was heated to 45 °C for 4 h. The solution was concentrated in vacuo.
Et2O (300 mL) was added, and the solid (DCU) was removed by
filtration. The filtrate was concentrated in vacuo and purified by
flash chromatography (silica, EtOAc/hexanes ) 1/2) to furnish 33
(2.5 g, 39%) as an oil. 1H NMR (CDCl3): δ 4.08 (t, 2H, J ) 6.5),
3.29 (s, 4H), 2.29 (m, 2H), 1.61 (m, 4H), 1.26 (m, 8H), 0.87 (s,
6H), 0.86 (s, 6H). 13C NMR (CDCl3): δ 174.22, 71.71, 64.39,
38.26, 35.10, 34.40, 29.62, 25.92, 23.98, 23.50, 20.38. HRMS
(LSIMS, gly): calcd for C17H35O4 (MH+) 303.2535; found
303.2528. HPLC: 97.6% pure. Anal. (C17H34O) C, H.
1
purification for the next step. H NMR (CDCl3): δ 4.75 (br, 1H),
4.48 (m, 2H), 3.99 (t, 2H, J ) 6.5,), 3.77 (m, 2H), 3. (m, 4H), 3.11
(m, 2H), 2.94 (d, 2H, J ) 9.0), 1.78-1.46 (m, 16H), 1.22 (m, 8H),
0.83 (s, 12H). 13C NMR (CDCl3): δ 156.94, 99.38, 99.16, 76.63,
76.45, 64.98, 62.28, 62.05, 40.98, 39.17, 38.90, 34.39, 30.85, 30.14,
25.72, 24.79, 24.68, 21.18, 20.51, 19.71, 19.60. HRMS: calcd for
C27H52NO6 (MH+) 486.3795; found 486.3775.
(6-Hydroxy-5,5-dimethylhexyl)-carbamic Acid 6-Hydroxy-
5,5-dimethylhexyl Ester (37). A solution of 36 (7.35 g, 15.13
mmol) in acetic acid/THF/water (236 mL/118 mL/59 mL) was
heated to 45 °C for 24 h. The reaction mixture was poured into
ice-water (200 g) and extracted with CH2Cl2 (3 × 100 mL). The
organic layers were washed with saturated NaHCO3 solution (2 ×
100 mL) and brine (150 mL), dried over MgSO4, and concentrated
in vacuo. The residue was purified by flash chromatography (silica
gel, hexanes, then EtOAc/hexanes ) 1/10, 1/2, 1/1), affording 37
1
(3.65 g, 76%) as an oil. H NMR (CDCl3): δ 4.95 (br, 1H), 4.08
(t, 2H, J ) 6.5), 3.25 (s, 4H), 3.15 (m, 2H), 2.17 (br, 2H), 1.53 (m,
2H), 1.41 (m, 2H), 1.21 (m, 8H), 0.81 (s, 12H). 13C NMR
(CDCl3): δ 157.29, 71.61, 71.40, 64.83, 40.71, 38.22, 37.99, 35.19,
30.97, 30.07, 24.21, 24.12, 20.92, 20.31. HRMS: calcd for C17H36-
NO4 (MH+) 318.2644; found 318.2663. HPLC: 99.0% pure. Anal.
(C17H35NO4) C, H, N.
Phosphoric Acid Bis-[5,5-dimethyl-6-(tetrahydropyran-2-
yloxy)-hexyl]-ester (34). POCl3 (4.06 g, 2.5 mL, 26.48 mmol) was
added dropwise to a solution of 31 (12.2 g, 52.97 mmol) and NEt3
(5.36 g, 7.4 mL, 52.97 mmol) in anhydrous Et2O (200 mL) at room
temperature under N2 atmosphere. The reaction mixture was stirred
for 17 h. The ammonium salts were removed by filtration and
washed with Et2O (100 mL). The filtrate was concentrated in vacuo.
To a solution of the residue (15.0 g) in water (100 mL) and
acetonitrile (100 mL) was added KHCO3 (13.3 g, 133 mmol), and
1,3-Bis(6-hydroxy-5,5-dimethylhexyl)urea (42). To a solution
of 25 (14.9 g, 64.96 mmol) in pyridine (200 mL) was added
triphenyl phosphite (10.1 g, 32.48 mmol). The solution was heated
to 40-50 °C, and CO2 gas was introduced for ca. 8 h through a
gas immersion tube. The reaction mixture was cooled to room
temperature after 18 h, and a sample was taken for monitoring of
the reaction progress by NMR spectroscopy (ratio P/SM . 1/2).

Influence of Moieties on Hypolipidemic Properties
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 347
Additional triphenyl phosphite (10.2 g, 32.80 mmol) was added,
CO2 was introduced for ca. 18 h, and the reaction mixture was
heated to 55 °C for 23 h. The solvent was removed in vacuo, and
the reaction mixture concentrated in high vacuum to give crude
1,3-bis[5,5-dimethyl-6-(tetrahydropyran-2-yloxy)-hexyl]urea (53 g)
as a yellow oil. This crude intermediate was dissolved in MeOH
(200 mL) and concd. HCl (20 mL) and heated to reflux for 3 h.
The solution was cooled to room temperature, diluted with water
(200 mL), and concentrated in vacuo to a volume of ca. 300 mL.
The solution was extracted with CH2Cl2 (3 × 100 mL). The
combined organic layers were washed with 10% NaOH solution
(2 × 100 mL) and saturated NaCl solution (2 × 100 mL), dried
over MgSO4, concentrated in vacuo, and dried in high vacuum.
The residual oil was purified by flash chromatography (silica;
hexanes/EtOAc ) 40/60, then 20/80, followed by CHCl3/MeOH
) 95/5) to furnish 42 (2.24 g, 22% over two steps) as a yellow oil.
1H NMR (CDCl3): δ 3.26 (s, 4H), 3.11 (t, 4H, J ) 6.8), 1.51-
1.38 (m, 4H), 1.36-1.18 (m, 8H), 0.85 (s, 12H). 13C NMR
(CDCl3): δ 161.50, 71.97, 41.08, 39.67, 36.07, 32.49, 24.59, 22.33.
HRMS: calcd for C17H37N2O3 (MH+) 317.2804, found 317.2793.
Biological Methods. In Vitro Measurement of Lipid Synthesis
in Isolated Hepatocytes. Compounds were tested for inhibition
of lipid synthesis in primary cultures of rat hepatocytes. Male
Sprague-Dawley rats were anesthetized with intraperitoneal injec-
tion of sodium pentobarbital (80 mg/kg). Rat hepatocytes were
isolated essentially as described by the method of Seglen,34 which
includes hepatic exposure to dexamethasone. Hepatocytes were
suspended in Dulbecco’s modified Eagle’s medium containing 25
mM D-glucose, 14 mM HEPES, 5 mM l-glutamine, 5 mM leucine,
5 mM alanine, 10 mM lactate, 1 mM pyruvate, 0.2% bovine serum
albumin, 17.4 mM nonessential amino acids, 20% fetal bovine
serum, 100 nM insulin, 100 nM dexamethasone, and 20 µg/mL
gentamycin and plated at a density of 1.5 × 105 cells/cm2 on
collagen-coated 96-well plates. Four hours after plating, media was
replaced with the same media without serum. Cells were grown
overnight to allow formation of monolayer cultures. Lipid synthesis
incubation conditions were initially assessed to ensure the linearity
of [1-14C]-acetate incorporation into hepatocyte lipids for up to 4
h. Hepatocyte lipid synthesis inhibitory activity was assessed during
incubations in the presence of 0.25 µCi of [1-14C]-acetate/well (final
radiospecific activity in assay is 1 Ci/mol) and 0, 1, 3, 10, 30, 100,
or 300 µM compounds for 4 h. At the end of the 4-h incubation
period, medium was discarded, and cells were washed twice with
ice-cold phosphate-buffered saline and stored frozen prior to
analysis. To determine total lipid synthesis, 170 µL of MicroScint-E
and 50 µL of water was added to each well to extract and partition
the lipid-soluble products to the upper organic phase containing
the scintillant. Lipid radioactivity was assessed by scintillation
spectroscopy in a Packard TopCount NXT. Lipid synthesis rates
were used to determine the IC50’s of the compounds.
In Vivo Effects on Lipid Variables in Female Obese Zucker
Fatty Rats. Ten- to twelve-week-old (400-500 g) female Zucker
fatty rats, Crl:(Zuc)-faBR, were obtained from Charles River
Laboratories. Animals were acclimated to the laboratory environ-
ment for 7 days. During the acclimation and study period, animals
were housed by group in shoebox polycarbonate cages on Cellu-
Dri bedding. The temperature and humidity in the animals' quarters
(68-78 °F; 30-75% RH) were monitored, and the airflow in the
room was sufficient to provide several exchanges per hour with
100% fresh filtered air. An automatic timing device provided an
alternating 12-hour cycle of light and dark. Rats received pelleted
Purina Laboratory Rodent Chow (5001) prior to and during the
drug intervention period except for a 6-hour phase prior to blood
sampling. Fresh water was supplied ad libitum via an automatic
watering system. Compounds were suspended by mixing in a dosing
vehicle consisting of 20% ethanol and 80% poly(ethylene glycol)-
200 [v/v]. Dose volume of vehicle or vehicle plus each compound
was set at 0.25% of body weight to deliver the appropriate dose.
Doses were administered daily by oral gavage, approximately
between 8 and 10 AM. Regarding blood sampling, animals were
fasted for 6 h prior to all blood collections. Prior to and after 7
days of dosing, a 1.0-2.0-mL sample of blood was collected by
administering O2/CO2 anesthesia and bleeding from the orbital
venous plexus. Following 14 days of dosing, blood was collected
by cardiac puncture after euthanasia with CO2. All blood samples
were processed for separation of serum and stored at -80 °C until
analysis. Commercially available kits were used to determine serum
triglycerides (Roche Diagnostic Corporation, kit no. 148899, or
Boehringer Mannheim, kit no. 1488872), total cholesterol (Roche
Diagnostic Corporation, kit no. 450061), non-esterified fatty acids
(Wako Chemicals, kit no. 994-75409), and â-hydroxybutyrate
(Wako Chemicals, kit no. 417-73501, or Sigma, kit. no. 310-0) on
a Hitachi 912 automatic analyzer (Roche Diagnostic Corporation).
In some instances, an in-house cholesterol reagent was used to
determine total serum cholesterol levels. Serum lipoprotein cho-
lesterol levels were determined by lipoprotein profile analysis.
Lipoprotein profiles were analyzed using gel-filtration chromatog-
raphy on a Superose 6HR (1 × 30 cm2) column equipped with
on-line detection of total cholesterol as described by Kieft et al.35
The total cholesterol content of each lipoprotein was calculated by
multiplying the independent values determined for serum total
cholesterol by the percent area of each lipoprotein in the profile.
Pharmacokinetics and Drug Metabolism of 1,13-Dihydroxy-
2,2,12,12-tetramethyl-tridecan-7-one (39). Primary hepatocytes
from male donors were isolated and suspended in cold sHMM
medium (Williams’ E media supplemented with 0.1 µM insulin,
0.1 µM dexamethasone, 50 µg/mL gentamycin, and 50 ng/mL
amphotericin B) to a final concentration of ∼0.8 × 106 cells/mL.
Cell viability was assessed by exclusion of trypan blue. Test article
was diluted from a concentrated stock solution into sHMM medium
such that residual organic solvent was <0.5% at final working
solutions of 2 and 20 µM drug. Cell suspensions (0.25 mL,
∼200 000 cells) were incubated in 24-well culture plates in a 37
°C water-jacketed incubator with 95% air/5% CO2 for 30 min before
addition of drug. Equal volumes of cell suspensions and test article
solutions were gently mixed in triplicate at 37 °C. Negative controls
were done in parallel, containing every experimental component
except cells. Positive controls were also done in parallel, using 20
µM bufuralol to demonstrate viability of both primary and second-
ary metabolism characteristics typical of each species. Incubations
were quenched at 0, 0.5, 1, 2, and 4 h by addition of ice-cold
acetonitrile (0.25 mL), then submitted to LCMS analyses. Quenched
cell suspensions were centrifuged (13 000 rpm, 5 °C, 15 min), and
200 µL of supernatant was transferred to an HPLC injection vial.
Thirty microliters was injected onto a loading column and desalted,
then transferred by loop injection onto a Zorbax XDB C18 (4.6 ×
30 mm2, 3.5 µ dP) column and eluted at 1 mL/min using a shallow
gradient (50%-75% B, 12 min; solvent A 0.2% acetic acid in water;
solvent B methanol) into a Finnigan LCQ ion trap mass spectrom-
eter. Column effluent was electrosprayed after a 1:4 split, and
positive ions were detected in full scan mode (m/z ) 200-800).
Total ion current was monitored, as well as XIC of characteristic
[M + H]+1 ions (compound 39, 287 m/z (tR 7.52 min); compound
10, 289 m/z (tR 8.62 min); compound 45, 315 m/z (tR 7.17 min);
compound 12, 317 m/z (tR 7.92 min)). Retention times and mass
spectral fingerprints were verified by co-injection of authentic
standards for these compounds. Intermediate metabolites (com-
pounds 43 and 44) were inferred from retention times and mass
spectra. Multiple dehydrations were the principle in-source frag-
mentations observed for both parent and primary metabolites.
Relative quantities of parent and metabolites were estimated
assuming equal detector sensitivities for all species and reported
as percentages of the integrated ion intensities of the parent (39) at
incubation time 0. For in vivo experiments, structures of all primary
metabolites were verified and quantitated using co-injected deu-
terated (d8) internal standards and reported as mg/mL of serum.
Acknowledgment. We thank Ms. Ping Yang Han and Mr.
Bruce H. McCosar of Alchem Laboratories for their assistance
in the synthesis of some compounds. The authors acknowledge
Michelle Washburn, Rose Ackermann, Leigh-Ann Jankowski,

348 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
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Supporting Information Available: Details on the syntheses
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JM050650J