Substituted pyrazoles as hepatoselective HMG-CoA reductase inhibitors: Discovery of (3R,5R)-7-[2-(4-fluoro-phenyl)-4-isopropyl-5-(4-methyl- benzylcarbamoyl)-2H-pyrazol-3-yl]-3,5-dihydroxyheptanoic acid (PF-3052334) as a candidate for the treatment of hypercholesterolemia

Article abstract of DOI:10.1021/jm070849r

In light of accumulating evidence that aggressive LDL-lowering therapy may offer increased protection against coronary heart disease, we undertook the design and synthesis of a novel series of HMG-CoA reductase inhibitors based upon a substituted pyrazole template. Optimizing this series using both structure-based design and molecular property considerations afforded a class of highly efficacious and hepatoselective inhibitors resulting in the identification of (3R,5R)-7-[2-(4-fluoro-phenyl)-4-isopropyl-5-(4-methyl-benzyl- carbamoyl)-2H-pyrazol-3-yl]-3,5-dihydroxy-heptanoic (PF-3052334) as a candidate for the treatment of hypercholesterolemia.

Full text of DOI:10.1021/jm070849r

J. Med. Chem. 2008, 51, 31–45
31
Substituted Pyrazoles as Hepatoselective HMG-CoA Reductase Inhibitors: Discovery of
(3R,5R)-7-[2-(4-Fluoro-phenyl)-4-isopropyl-5-(4-methyl-benzylcarbamoyl)-2H-pyrazol-
3-yl]-3,5-dihydroxyheptanoic Acid (PF-3052334) as a Candidate for the Treatment of
Hypercholesterolemia
Jeffrey A. Pfefferkorn,* Chulho Choi, Scott D. Larsen, Bruce Auerbach, Richard Hutchings, William Park, Valerie Askew,
Lisa Dillon, Jeffrey C. Hanselman, Zhiwu Lin, Gina H. Lu, Andrew Robertson, Catherine Sekerke, Melissa S. Harris,
Alexander Pavlovsky, Graeme Bainbridge, Nicole Caspers, Mark Kowala, and Bradley D. Tait
Pfizer Global Research and DeVelopment, 2800 Plymouth Road, Ann Arbor, Michigan 48105
ReceiVed July 12, 2007
In light of accumulating evidence that aggressive LDL-lowering therapy may offer increased protection
against coronary heart disease, we undertook the design and synthesis of a novel series of HMG-CoA reductase
inhibitors based upon a substituted pyrazole template. Optimizing this series using both structure-based
design and molecular property considerations afforded a class of highly efficacious and hepatoselective
inhibitors resulting in the identification of (3R,5R)-7-[2-(4-fluoro-phenyl)-4-isopropyl-5-(4-methyl-benzyl-
carbamoyl)-2H-pyrazol-3-yl]-3,5-dihydroxy-heptanoic (PF-3052334) as a candidate for the treatment of
hypercholesterolemia.
Introduction
Achieving such aggressive LDL-C reductions in patients
Coronary heart disease (CHD)a is a leading cause of death,
and it represents an increasing burden on healthcare resources
worldwide.1 Epidemiological evidence indicates that hyperc-
holesterolemia is an important risk factor for the CHD;2
consequently, significant efforts have been undertaken to
mitigate this condition. Currently, the most common method
of treating hypercholesterolemia is the use of HMG-CoA
reductase inhibitors, also known as statins. These drugs block
the rate-limiting step of cholesterol biosynthesis, ultimately
resulting in up-regulation of the LDL-receptor and the clearing
of low density lipoprotein (LDL) particles from the blood-
stream.3 As a class, statins have proven remarkably safe and
effective for both primary prevention of coronary heart disease
and secondary prevention of coronary events.4
Over the past five years, results from several large clinical
trials including TNT, MIRACAL, IDEAL, and PROVE-IT have
suggested that increasingly aggressive LDL-C lowering therapy
may offer further primary and secondary prevention benefits
relative to previous, less-aggressive treatment standards.5 Ac-
knowledging these findings, the National Cholesterol Education
Program (NCEP-ATP-III) recently modified its treatment guide-
lines for patients at risk for CHD. It is now recommended that
the highest risk patients achieve a treatment goal of LDL-C <
70 mg/dl, down from the previous treatment goal of 100 mg/dl
for this patient group.6 Moreover, other researchers have
suggested that optimal LDL-C levels to prevent atherosclerosis
and CHD might be even lower, in the range of 50–70 mg/dl.7
typically requires the use of high-dose statins alone or in
combination with complementary agents, such as the cholesterol
absorption inhibitor ezetimibe.5,8 A potential limitation of high-
dose statin therapy is statin-induced myalgia, the muscle pain
or weakness that sometimes accompanies statin therapy. While
the overall incidence of myalgia is low (2–7% of patients in
clinical trials), the likelihood of occurrence increases with drug
dose, and it can be a key factor in preventing patient compliance
with a treatment regimen.9a Physiologically, the mechanism of
statin-induced myalgia is complex, but thought to involve, in
part, inhibition of HMG-CoA reductase in nonhepatic tissues
(particularly muscle), thereby disrupting the biosynthesis of
isoprenoid-derived biomolecules important in post-translational
protein modification (i.e., prenylation) and electron transport
(i.e., ubiquinone).9
Fortunately, there is evidence that the likelihood of statin-
induced myalgia can be reduced by targeting HMG-CoA
reductase inhibitors to hepatic tissues and limiting peripheral
exposure.10 Moreover, it has been demonstrated that the
hepatoselectivity of a given HMG-CoA reductase inhibitor is
related to its degree of lipophilicity.10,11 In general, lipophilic
statins tend to achieve higher levels of exposure in nonhepatic
peripheral tissues, whereas more hydrophilic statins tend to be
more hepatoselective. This trend is illustrated experimentally
in Table 1, where three benchmark statins spanning a range of
lipophilicity were evaluated for their ability to inhibit cholesterol
synthesis in both primary rat hepatocyte and rat myocyte
(muscle) cells, with the ratio of these two values taken as a
measure of hepatoselectivity. As shown and consistent with the
earlier findings of others,12 lipophilic statins such as cerivastatin
(which was withdrawn from the market due to high incidences
of myalgia and rhabdomyloysis) tend to be nonselective,
whereas more hydrophilic statins such as pravastatin and
rosuvastatin are hepatoselective. These differences in hepato-
selectivity can be rationalized by the fact that lipophilic statins
passively and nonselectively diffuse into both hepatocyte and
nonhepatocyte cells, while hydrophilic statins (which are
* To whom correspondence should be addressed. Pfizer Global Research
and Development, MS 8220-3020, Eastern Point Road, Groton, CT 06340.
Phone: 1-860-686-3421. Fax: 1-860-715-4608. E-mail: jeffrey.a.pfefferkorn@
pfizer.com.
aAbbreviations: CHD, coronary heart disease; HMG-CoA, 3-hydroxy-
3-methylglutaryl-CoA; IDEAL, incremental decrease in end points through
aggressive lipid-lowering trial; LDL, low density lipoprotein; MIRACAL,
myocardial ischemia reduction with acute cholesterol lowering trial; NECP,
National Cholesterol Education Program; OATP, organic anion transporting
polypeptide; PROVE-IT, pravastatin or atorvastatin evaluation and infection
therapy-thrombolysis in myocardial infarction trial; TNT, treating to new
targets trial.
10.1021/jm070849r CCC: $40.75
2008 American Chemical Society
Published on Web 12/12/2007

32 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1
Pfefferkorn et al.
Table 1. Effect of Inhibitor Lipophilicity on Hepatoselectivity for Selected Known Statins
inhibition of cellular cholesterol synthesisb
LogPa (pH 7.4) cLogD (pH 7.4) HMG-CoAb IC50 (nM) hepatocycte IC50 (nM) myocycte IC50 (nM) myocycte/hepatocycte ratio
cmpd
cerivastatin
pravastatin
rosuvastatin
1.66
1.76
0.70
-2.63
2.8 ( 0.5
13 ( 1
3.1 ( 0.2
1.7 ( 0.2
17 ( 2
0.3 ( 0.1
3.0 ( 0.3
7600 ( 3200
250 ( 20
1.8
450
830
-0.67
NAc
a See ref 11d. b Assay values reported as the mean ( SEM of n g 2 independent determinations. c NA ) not available.
Figure 1. Structures of atorvastatin (1) and pyrazole-based HMG-CoA reductase inhibitors 2 and 3.
inherently less membrane-permeable) rely to a greater extent
on active transport into hepatocyte cells to exert their effects.10,13
Pravastatin and rosuvastatin, among other statins, have been
shown to be substrates for active transport via members of the
organic anion transporting polypeptide (OATP) family of
membrane transporters, which are principally expressed in
hepatocytes.10c,13–15
tion of the amide motif, to optimize its hepatoselectivity and in
vivo efficacy.
Chemistry
Substituted pyrazole analogs for the current studies were
synthesized as outlined in Schemes 1–4. Initial efforts focused
on construction of a heterocyclic core, which could be utilized
as a versatile intermediate for subsequent analog synthesis. As
shown in Scheme 1, 4-fluoroaniline (4) was first converted to its
corresponding diazonium salt by treatment with NaNO2/HCl, and
this intermediate was reacted with methyl 2-chloroacetoacetate to
give hydrazonoyl chloride 5.17 Treatment of 5 with Ag2CO3
resulted in in situ generation of nitrilimine 6, which was engaged
in a stereoselective 1,3-dipolar cycloaddition with E-enone 8 as
the dipolarophile to provide a 5:1 mixture of regioisomeric
dihydropyrazoles, with 9 as the major regioisomer.17,18 The
regiochemistry of 9 was confirmed upon ceric ammonium nitrate
oxidation to pyrazole 10, a crystalline solid that was subjected to
small molecule X-ray analysis, as shown in Scheme 1.
With pyrazole 10 in hand, inhibitor 2 was completed, as
outlined in Scheme 2. The benzyl ester of 10 was first converted
to the carboxaldehyde of 11 using a three-step sequence that
included (a) hydrogenolysis to a carboxylic acid; (b) reduction
to the corresponding alcohol with BH3 ·THF;19 and (c) oxidation
to an aldehyde with Dess-Martin periodinate. The methyl ester
of 11 was saponified with aqueous NaOH to provide carboxylic
acid 12, which was engaged in an EDCI-mediated coupling with
benzyl amine to provide amide 13. Attachment of the 3,5-
dihydroxyhexanoic acid side chain onto the pyrazole core was
anticipated to be accomplished via a Wittig olefination reaction
that first required conversion of 13 into an appropriate ylide
precursor.20 Hence, the carboxaldehyde of 13 was reduced to
One potential limitation of achieving high hepatoselectivity
via reduced inhibitor lipophilicity, however, is that as a
compound becomes more hydrophilic and, therefore, less
permeable, its oral absorption is typically impeded (provided
that no compensating transport mechanism is present in the
gastrointestinal system); consequently, achieving the appropriate
level of inhibitor lipophilicity is pivotal to balancing selectivity
and efficacy.
To meet the challenge of finding novel HMG-CoA reductase
inhibitors with the efficacy and tolerability profiles needed to
help patients achieve aggressive LDL reduction goals, we
undertook a discovery effort to identify a novel class of
inhibitors with best-in-class preclinical efficacy and hepatose-
lectivity. We strategically envisioned meeting this objective
through modification of the atorvastatin (1) template. Specifi-
cally, we sought to replace the pyrrole core of atorvastatin with
alternative five-membered heterocycles of lower inherent lipo-
philicity and/or substitution.16 Of particular interest was a
pyrazole core system, as exemplified by isomeric prototypes 2
and 3 (Figure 1). Synthesis of 2 and 3 (vida infra), followed by
evaluation against HMG-CoA reductase, revealed that pyrazole
2 (IC50 ) 4.7 nM) had activity comparable to the parent
atorvastatin (IC50 ) 12 nM) whereas regioisomer 3 (IC50
)
520 nM) was substantially less active. We subsequently
undertook structure–activity studies on 2, focusing on modifica-

HepatoselectiVe HMG-CoA Reductase Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1 33
Scheme 1. Synthesis and X-ray Structure of Pyrazole Intermediate 10a
a Reagents and conditions: (a) (i) NaNO2, HCl, MeOH, 0 °C, 0.25 h; (ii) methyl-2-chloroacetoacetate, MeOH, 25 °C, 12 h, 94%; (b) Ag2CO3, dioxane,
25 °C, 48 h, 73%; (c) BnBr, K2CO3, acetone, 56 °C, 16 h, 98%; (d) ceric ammonium nitrate, THF/H2O, 0 °C, 1 h, 65%.
the corresponding alcohol and then treated with Ph3P·HBr at
elevated temperature to afford phosphonium salt 14 in excellent
yield. In parallel, Swern oxidation of commercially available
alcohol 15 provided aldehyde 16 as a suitable coupling partner.21
In the olefination event, phosphonium salt 14 was deprotonated
with LiHMDS at low temperature, and a solution of aldehyde
16 was added to provide olefin 17 in moderate yield as an
inconsequential mixture of cis/trans isomers. Treatment of 17
with HCl/MeOH effected cleavage of the acetonide protecting
group, and the side chain olefin was then hydrogenated over
10% Pd/C to afford 18. Finally, the terminal ester of 18 was
saponified with aqueous NaOH to provide compound 2 as a
carboxylate sodium salt.
Scheme 3 illustrates the synthesis of inhibitor 3, a regioisomer
of inhibitor 2, utilizing minor modifications to the synthetic route
previously described. Hydrogenolysis of the benzyl ester of 10
provided the corresponding carboxylic acid 19, which was
subjected to an EDCI-mediated coupling with benzyl amine to
give benzyl amide 20. The methyl ester of 20 was then
saponified, and the resulting carboxylic acid reduced with
BH3 ·THF to afford alcohol 22. Treatment of 22 with PBr3
converted this alcohol to an alkyl bromide that was then reacted
with Ph3P at elevated temperature, resulting in the formation
of phosphonium salt 23 in good yield. Notably, this two-step
conversion of 22f23 was found to be more efficient than the
single-step sequence previously described for 13f14 (Scheme
2) owing to the extended reaction time required for this later
transformation. Finally, using the same four-step sequence
(Wittig reaction, acetonide deprotection, hydrogenation, and
saponification) described above, phosphonium salt 23 was
converted to 3, which was isolated as its carboxylate sodium
salt.
Et2BOMe and NaBH4 to produce syn-diol 30.23 Finally, the
synthesis was completed by hydrogenation of the side chain
olefin followed by saponification of the terminal ester to provide
inhibitor 32 as its carboxylate sodium salt.
The synthetic routes outlined in Schemes 14 were subse-
quently utilized for the preparation of all analogs in these studies.
Results and Discussion
Structure–Activity Studies. Our SAR studies in this pyrazole
series focused on optimizing potency, hepatoselectivity, and in
vivo efficacy; importantly and as discussed below, achieving
the necessary balance between these parameters required
manipulation of analog structure and lipophilicity. All new
analogs were first evaluated in an HMG-CoA reductase enzyme
inhibition assay. Additionally, the ability of analogs to block
cholesterol synthesis in both rat hepatocyte and myocyte cell
lines was evaluated; moreover, comparison of these two values
was utilized as a measurement of hepatoselectivity. As a
guideline, inhibitors with potent hepatocyte activity (hepatocyte
IC50 < 10 nM) and good hepatoselectivity (myocyte/hepatocyte
ratio preferably >1000) were selected for further evaluation in
both acute and chronic animal efficacy models. Our acute model,
performed in hamsters, measured a compound’s ability to inhibit
cholesterol synthesis. To get a preliminary assessment of a
compound’s duration of action, two time points were measured
in separate animal groups. In one group (denoted as t ) 2–4 h
group), animals were dosed with drug (10 mpk) at t ) 0 h then
given an intraperitoneal injection of 14C sodium acetate at t )
2 h, and finally, blood samples were obtained at t ) 4 h and
analyzed for 14C cholesterol levels, which were compared to
untreated control animals to determine percent inhibition of acute
cholesterol synthesis. In a second group (denoted as t ) 4–6 h
group), animals were again dosed with drug (10 mpk) at t )
0 h then given an intraperitoneal injection of 14C sodium acetate
at t ) 4 h, and finally, blood samples were obtained at t ) 6 h
and analyzed for 14C cholesterol levels, which were compared
to untreated control animals. Comparison of the results from
these two assays afforded a preliminary measure of a com-
pound’s duration of action, which was utilized for prioritizing
compounds for further evaluation. Based upon these acute
efficacy data, compounds of interest were then selected for
evaluation in a chronic (7 day) LDL lowering study that was
performed in chow-fed guinea pigs. This chronic study was
initially performed at a single dose of drug (10 or 20 mpk),
While the synthetic routes described in Schemes 2 and 3
provided facile assess to many of the analogs described herein,
a second route, employing an alternative method of side chain
attachment, was also utilized for the synthesis of selected
analogs. This alternative route is outlined in Scheme 4 for the
synthesis of inhibitor 32. As shown, amide 26 was prepared as
previously described and engaged in a Wittig olefination reaction
with stabilized ylide 27 (prepared according to the method of
Konoike22) to provide E-olefin 28 in good overall yield. The
TBS-protected side chain hydroxyl of 28 was then liberated by
treatment with aqueous HF to provide ꢀ-hydroxy ketone 29,
which was subjected to a stereoselective syn-reduction with

34 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1
Scheme 2. Synthesis of Pyrazole Prototype Analog 2a
Pfefferkorn et al.
Scheme 3. Synthesis of Pyrazole Prototype Analog 3a
a Reagents and conditions: (a) H2, 10% Pd/C, MeOH, 25 °C, 3 h, 99%;
(b) BnNH2, EDCI, HOBt ·H2O, CH2Cl2, 25 °C, 8 h, 57%; (c) NaOH (aq),
MeOH, 60 °C, 3 h, 98%; (d) BH3 ·THF, THF, 65 °C, 4 h, 35%; (e) PBr3,
CH2Cl2, 25 °C, 12 h, 100%; (f) Ph3P, 80 °C, 14 h, 100%; (g) 16, LiHMDS,
THF, -78 °C, 2.5 h, 63%; (h) HCl (aq), MeOH, 25 °C, 1.5 h, 93%; (i) H2,
10% Pd/C, MeOH, 25 °C, 4 h, 56%; (j) NaOH (aq), MeOH, 25 °C, 16 h,
97%.
a Reagents and conditions: (a) H2, 10% Pd/C, MeOH, 25 °C, 3 h, 99%;
(b) BH3 ·THF, THF, 65 °C, 3.5 h, 90%; (c) Dess-Martin periodinate,
NaHCO3, CH2Cl2, 25 °C, 4 h, 84%; (d) NaOH (aq), MeOH, 60 °C, 4 h,
79%; (e) BnNH2, EDCI, HOBt ·H2O, CH2Cl2, 25 °C, 4 h, 39%; (f) NaBH4,
THF/MeOH, 0 °C, 0.5 h, 87%; (g) Ph3P·HBr, MeCN, 80 °C, 48 h, 98%;
(h) see ref 21; (i) 16, LiHMDS, THF, -78 °C, 5.5 h, 50%; (j) HCl (aq),
MeOH, 25 °C, 3 h, 69%; (k) H2, 10% Pd/C, MeOH, 25 °C, 3 h, 59%; (l)
NaOH (aq), MeOH, 25 °C, 48 h, 94%.
decreased (particularly for 37 and 38), presumably as a result
of reduced oral absorption.
results were compared against the benchmark compound rosu-
vastatin, and analogs with comparable activity were then
evaluated in full dose response studies.
A second type of amide modification explored was N-
alkylation, as highlighted in Table 3. Comparison of secondary
amide 2 to its N-methylated tertiary amide counterpart 39
revealed that the two compounds had similar potency in
hepatocyctes [IC50 ) 0.3 nM (2) vs 0.2 nM (39)], but
interestingly, the N-methyl derivative 39 was 30-fold less active
against myocytes [IC50 ) 100 nM (2) vs 3100 nM (39)]. In
addition to its increased hepatoselectivity, N-methyl amide 39
also exhibited good acute in vivo efficacy, as illustrated in Table
3. Increasing the size of the N-alkyl group to ethyl (40) and
i-propyl (41) resulted in decreased potency for inhibition of
hepatocyte cholesterol synthesis.
As shown in Table 2, preliminary SAR efforts focused on
evaluating substituted secondary benzyl amides. Because the
prototype compound 2, which contained an unsubstituted benzyl
amide, lacked sufficient hepatoselectivity (myocyte IC50/hepa-
tocyte IC50 < 1000) substituents were added to reduce lipo-
philicity and, consequently, improve selectivity. As illustrated,
compounds 35–38 demonstrated sufficient hepatoselectivity to
warrant acute in vivo evaluation. Preliminary acute in vivo
evaluation was done at the t ) 6 h time point with actives
(defined as -10% or greater inhibition) then also evaluated at
the t ) 4 h time point. As shown, analogs 35 and 36 both
exhibited good acute efficacy, whereas the more hydrophilic
compounds 37 and 38 were not acutely efficacious. Interestingly,
inspection of the analogs of Table 2 confirmed that, within this
limited series, hepatoselectivity generally increased as lipophi-
licity decreased (with the exception of 35); additionally, acute
in vivo efficacy tended to also diminish as inhibitor lipophilicity
To further capitalize on the selectivity and efficacy observed
for compound 39, we next sought to investigate the effect of
further substitution on this analog, as shown in Table 4, where
a series of alkyl, alkoxy, and fluoro groups were installed at
the ortho, meta, and para position of the benzyl ring. In nearly
all cases, these compounds had excellent potency and selectivity;
however, optimal acute in vivo efficacy was observed for

HepatoselectiVe HMG-CoA Reductase Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1 35
Scheme 4. Alternative Method for the Synthesis of Pyrazole
Analogs Illustrated for Compound 32a
piperidine, and 3-phenyl piperidine were utilized to prepare 52,
53, and 54, respectively, as 1:1 diastereomeric mixtures. Among
these compounds, 54 provided the best combination of potency,
selectivity, and efficacy. Subsequently, the two individual
stereoisomers of 54 were independently prepared and assayed,
resulting in the identification of 55 as the more active compo-
nent. Encouragingly, this compound had outstanding acute
efficacy; unfortunately, the selectivity of 55 against the myocycte
cell line was somewhat diminished.
Chronic Efficacy Studies To Enable Candidate Selection.
In an effort to better evaluate the therapeutic potential of this
series of HMG-CoA reductase inhibitors, analogs demonstrating
good selectivity and acute efficacy were selected for chronic in
vivo characterization. Specifically, these compounds were
evaluated for their ability to lower LDL cholesterol in a chow-
fed guinea pigs over a 7 day period with daily oral administra-
tion of drug at a single dose (10 or 20 mpk). Efficacy of the
new analogs was benchmarked against rosuvastatin, and com-
pounds with comparable single-dose efficacy were selected for
dose-response studies. As shown in Table 6, analogs 35, 36,
and 49 demonstrated chronic efficacy comparable to rosuvastatin
at the dose evaluated, while the remaining compounds were less
active. As a result of these single dose data 35, 36, and 49 were
then evaluated in dose-response studies.
During the dose-response studies of these three compounds,
compound 36 was found to have inferior efficacy relative to
the benchmark rosuvastatin (data not shown) and was therefore
discarded. By contrast, both 35 and 49 demonstrated efficacy
comparable to if not better than rosuvastatin. Unfortunately,
difficulties obtaining a suitable crystal form of inhibitor 49
prevented further development of this compound. Encourag-
ingly, as shown in Figure 3, comparison of the dose response
of 35 (represented as PF-3052334) versus that of rosuvastatin
and pravastatin revealed that with an ED50 ) 3.6 mpk it was
approximately 3 times more efficacious than rosuvastatin (ED50
) 9.3 mpk) and 27 times more efficacious than pravastatin (ED50
) 100 mpk) in this animal model. Considering both the good
hepatoselectivity (>800-fold) and the excellent LDL-lowering
efficacy of 35, this compound was advanced forward as a
candidate that is currently under evaluation in a phase I trial,
the results of which will be reported separately.
a Reagents and conditions: (a) (2-Me)-PhCH2N(Me)H, EDCI,
HOBt·H2O, CH2Cl2, 25 °C, 6 h, 72%; (b) toluene, 95 °C, 16 h, 98%; (c)
HF (48% aq), MeCN, 25 °C, 16 h, 98%; (d) Et2BOMe, NaBH4, THF/MeOH,
-78 °C, 3 h, 81%; (e) H2, 10% Pd/C, MeOH, 25 °C, 4 h, 66%; (f) NaOH
(aq), MeOH, 25 °C, 12 h, 99%.
Retrospective Analysis of Lipophilicity and Selectivity. As
described at the outset, a key element of our current strategy
was to understand the relationship between inhibitor lipophilicity
and hepatoselectivity and to use the former as a prediction of
the latter to enable efficient analog design and prioritization.
During the course of our studies, we generated a cohort of
pyrazole analogs that spanned a range of lipophilicity (cLogD
) -0.01 to -2.25) but still maintained relatively similar
inherent potencies for inhibition of the HMG-CoA enzyme
reductase enzyme. Furthermore, for each compound, we had a
hepatoselectivity ratio based upon comparison of hepatocyte and
myocyte inhibition values. With this data in hand, we were
positioned to undertake a retrospective analysis on the series to
determine whether or not cLogD was predictive of hepatose-
lectivity for analogs in this series. This analysis is presented
graphically in Figure 4 where a subset of analogs with HMG-
CoA IC50 ) 1–10 nM26 was utilized to generate plots of cLogD
versus log (myocyte IC50) [A], log (hepatocyte IC50) [B], and
log (myocyte/hepatocyte) [C]. Several correlations were noted
and exemplified with trend lines on the plots. First, as exempli-
fied in Plot [A], myocyte potency decreased with increasing
inhibitor hydrophilicity (i.e., lower cLogD). This is consistent
with the fact that membrane permeability typically decreases
compounds 48 and 49 bearing 2,4-F and a 2,3-F substituent
patterns, respectively.
A final type of analog modification focused on the benzylic
position of benzyl amide 37, as illustrated in Table 5. Specif-
ically, we sought to understand the effect of R-methylation of
the benzyl amide, as illustrated by stereosiomers 50 and 51.
While 50 demonstrated superior activity relative to its diaster-
eomeric counterpart 51, its selectivity and efficacy were similar
to the unsubstituted case 39; however, structural biology studies
of 50 revealed an interesting observation.
Figure 2 shows an X-ray structure of compound 50 bound to
the active site of HMG-CoA reductase.24 While the compound
binds in a mode consistent with other known HMG-CoA
reductase inhibitors,25 inspection of the relative positions of the
N-methyl and R-methyl groups reveal that (in the inhibitor’s
bound conformation) these two groups experience a steric
interaction that is intermediate between gauche and eclipsing.
This observation suggested that the bound configuration of 50
was not a low energy conformation. Moreover, it appeared that
the two alkyl groups might be conveniently tethered into a ring
to restrict the amide motif into its active conformation. To
evaluate this hypothesis, racemic 2-phenyl pyrrolidine, 2-phenyl

36 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1
Pfefferkorn et al.
Table 2. Structure and Biological Activity of Tertiary Amide Analogs 33–38
inhibition of cellular cholesterol synthesisa inhibition of in vivo cholesterol synthesisb
HMG-CoAa IC50
(nM)
hepatocycte IC50
(nM)
myocycte IC50
(nM)
t ) 2–4 h
(10 mpk)
t ) 4–6 h
(10 mpk)
cLogD
(pH 7.4)
cmpd
R1
R2
rosuvastatin
2
3.1 ( 0.2
4.7 ( 1.0
2.8c
3.8 ( 0.5
1.9 ( 1.1
7.0 ( 1.4
2.2 ( 1.0
25c
0.3 ( 0.1
0.3 ( 0.1
0.4c
250 ( 20
100 ( 10
460c
-76%
-46%
-2.63
-0.47
-0.56
-0.42
-0.01
-0.73
-1.04
-2.25
H
H
H
H
H
OMe
F
33
34
35
36
37
38
0.7c
490 ( 40
730 ( 20
1800 ( 800
2400c
Me
0.9 ( 0.1
0.7 ( 0.1
1.4 ( 0.3
23c
-74%
-71%
-15%
-19%
NAd
CH2OMe H
CN
H
H
C (O) NMe2
21000c
NA
a Assay values reported as the mean ( SEM of n g 2 independent determinations unless otherwise noted. b Data represent the mean of the percent change
from the control group (n ) 8/group). Interanimal variability in 14C cholesterol levels was typically <20%. c Data from n ) 1 determination. d NA ) not
active.
Table 3. Structure and Biological Activity of Tertiary Amide Analogs 39–41
inhibition of cellular cholesterol synthesisa
inhibition of in vivo cholesterol synthesisb
cmpd
R1 HMG-CoAa IC50 (nM) hepatocycte IC50 (nM) myocycte IC50 (nM) t ) 2–4 h (10 mpk) t ) 4–6 h (10 mpk) cLogD (pH 7.4)
rosuvastatin
2
39
40
41
3.1 ( 0.2
4.7 ( 1.0
5.9 ( 1.2
3.8 ( 0.9
9.8c
0.3 ( 0.1
0.3 ( 0.1
0.2 ( 0.1
5.5 ( 0.8
10 ( 2
250 ( 20
100 ( 10
3100 ( 500
4400 ( 500
9800c
-76%
-61%
-43%
-2.63
-0.47
-1.20
-0.67
-0.32
H
Me
Et
-16%
NAd
iPr
a Assay values reported as the mean ( SEM of n g 2 independent determinations, unless otherwise noted. b Data represent the mean of the percent
change from the control group (n ) 8/group). Interanimal variability in 14C cholesterol levels was typically <20%. c Data from n ) 1 determination. d NA
) not active.
as inhibitor hydrophilicity increases; therefore, if these inhibitors
penetrate the myocytes via passive diffusion (as anticipated),
one would expect the correlation that was observed.27 By
contrast, examination of Plot [B] reveals that hepatocyte potency
was somewhat independent of inhibitor lipophilicity. In fact,
there was a slight trend suggesting that cellular potency in
hepatocyctes actually increased as inhibitor hydrophilicity
increased. This is counter to a passive diffusion model and
suggests that these inhibitors are likely experiencing active
transport into the hepatocyte cells. Lastly, the utility of cLogD
as a tool for predicting hepatoselectivity in the current series is
evaluated in Plot [C]. As shown, a modest correlation exits (R2
) 0.6) between cLogD and the hepatoselectivity (defined as
myocyte/hepatocyte activity) such that predicted lipophilicity
might serve as a tool for prioritizing future analogs.
licity as a design tool for predicting the hepatoselectivity of
analogs in this series.
Experimental Section
General. All reagents and solvents were used as received from
commercial sources unless otherwise noted. Alcohol 15 was
purchased from Kanaka Corporation and converted to aldehyde 16,
as previously described.21 [3-14C]-HMG-CoA (57.0 mCi/mmol) was
purchased from Amersham Biosciences, U.K. HMG-CoA, meva-
lonolactone, beta-NADPH (beta-nicotinamide adenine dinucleotide
phosphate, reduced form) were purchased from Sigma Chemical
Co. AG 1–8× resin was purchased from Bio-Rad Laboratory.
All experiments were conducted under an inert nitrogen atmo-
sphere unless otherwise noted. 1H NMR spectra were recorded on
a Varian 400 MHz Nuclear Magnetic Resonance Spectrometer. 1H
NMR spectra were recorded in CDCl3, CD3OD, or d6-DMSO, and
chemical shifts are reported relative to the residual solvent peak.
The following abbreviations were used to assign spectra: s ) singlet,
d ) doublet, t ) triplet, q ) quartet, s ) septet, m ) multiplet.
Mass spectral analysis was conducted on a Waters Micromass ZQ
instrument. HPLC analysis was conducted according to method A,
B, or C, with the retention time (tR) expressed in min at UV
detection of 254 nM. HPLC method A: Agilent 1100 HPLC with
chromatography performed on a Zorbax Eclipse XBD-C8 column
(4.6 × 150 mm, 5 µm) at 25 °C. The mobile phase was a 15 min
binary gradient of acetonitrile (containing 0.1% TFA) and water
(20–90%). HPLC method B: Agilent HPLC with chromatography
Conclusion
In response to the need for increasingly effective yet well-
tolerated HMG-CoA reductase inhibitors, we have described
herein the synthesis and evaluation of a series of hepatoselective
and highly efficacious pyrazole-based inhibitors. These efforts
resulted in the identification of 35 as a candidate for the
treatment of hypercholesterolemia. Finally, through a retrospec-
tive analysis, we demonstrated the utility of calculated lipophi-

HepatoselectiVe HMG-CoA Reductase Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1 37
Table 4. Structure and Biological Activity of Substituted Tertiary Amide Analogs 32, 39, and 42–49
inhibition of cellular cholesterol synthesisa inhibition of in vivo cholesterol synthesisb
HMG-CoAa IC50
(nM)
hepatocycte IC50
(nM)
myocycte IC50
(nM)
t ) 2–4 h
(10 mpk)
t ) 4–6 h
(10 mpk)
cmpd
R1 R2 R3
cLogD (pH 7.4)
rosuvastatin
3.1 ( 0.2
5.9 ( 0.1
16c
0.3 ( 0.1
0.2 ( 0.1
2.1c
250 ( 20
3100 ( 500
2600c
-76%
-61%
-46%
-16%
-18%
-2.63
-1.20
-0.74
-0.74
-0.74
-1.29
-1.15
-1.15
-1.15
-1.06
-1.06
39
32
42
43
44
45
46
47
48
49
H
Me
H
H
H
F
H
H
F
H
H
Me
H
H
H
F
H
H
F
H
H
H
Me
OMe
H
H
F
6.1c
0.6 ( 0.1
4800 ( 2400
-72%
9.9c
12 ( 2
4.8 ( 2.3
3.9 ( 0.9
6.6 ( 1.0
7.0 ( 0.7
9.0 ( 2.7
1.8c
4000c
0.3 ( 0.1
0.5 ( 0.2
0.2 ( 0.1
0.5 ( 0.1
2.1 ( 0.7
0.6 ( 0.1
5800c
-57%
-69%
-65%
-71%
-82%
-81%
17000 ( 8000
6000 ( 300
6000c
4400 ( 2400
5700 ( 1300
-30%
NAd
NA
-17%
-22%
F
H
F
a Assay values reported as the mean ( SEM of n g 2 independent determinations, unless otherwise noted. b Data represent the mean of the percent
change from the control group (n ) 8/group). Interanimal variability in 14C cholesterol levels was typically <20%. c Data from n ) 1 determination. d NA
) not active.
Table 5. Structure and Biological Activity of Analogs 39 and 50–55
a Assay values reported as the mean ( SEM of n g 2 independent determinations, unless otherwise noted. b Data represent the mean of the percent
change from the control group (n ) 8/group). Interanimal variability in 14C cholesterol levels was typically <20%. c Data from n ) 1 determination.
performed on a Phenomenex Develosil Combi-RP C30 140A
column (4.6 × 50 mm, 3 µm) at 45 °C. The mobile phase was a
4.5 min binary gradient of acetonitrile (containing 0.1% HCO2H)
and water (2–50%). HPLC method C: Agilent HPLC with chro-
matography performed on a Phenomenex Develosil Combi-RP C30
140A column (4.6 × 50 mm, 3 µm) at 45 °C. The mobile phase

38 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1
Pfefferkorn et al.
5.14 (d, J ) 12.4 Hz, 1 H), 5.04 (d, J ) 12.4 Hz, 1 H), 4.58 (d, J
) 4.0 Hz, 1 H), 3.83 (s, 3 H), 3.51–3.49 (m, 1 H), 2.40–2.36 (m,
1 H), 1.00 (d, J ) 6.8 Hz, 3 H), 0.69 (d, J ) 7.2 Hz, 3 H); MS
(APCI+) m/z 399.0 (M + H).
1-(4-Fluoro-phenyl)-4-isopropyl-1H-pyrazole-3,5-dicarboxy-
lic Acid 5-Benzyl Ester 3-Methyl Ester (10). To a solution of 9
(29.3 g, 73.5 mmol) in THF/water (1:1, 500 mL) at 0 °C was slowly
added ceric ammonium nitrate (80.5 g, 147 mmol). The reaction
was stirred at 0 °C for 1 h after which time the THF was removed
under reduced pressure and DCM (500 mL) was added. The organic
layer was separated and washed with water and brine. The organic
layer was dried over Na2SO4 and concentrated, and the product
was purified by silica gel chromatography (5–15% EtOAc/hexanes)
1
to provide 10 (18.95 g, 65%): H NMR (CDCl3) δ 7.29–7.21 (m,
5 H), 7.07 (d, J ) 6.4 Hz, 2 H), 6.94 (t, J ) 3.2 Hz, 2 H), 5.12 (s,
2 H), 3.89 (s, 3 H), 3.84 (sept, J ) 7.2 Hz,1 H), 1.31 (d, J ) 7.2
Hz, 6 H); MS (APCI+) m/z 397.1 (M + H).
Figure 2. X-ray structure of inhibitor 50 bound to the active site of
HMG-CoA reductase (2.1 Å resolution), illustrating the opportunity
for installation of a conformationally restricted amide.
1-(4-Fluoro-phenyl)-5-formyl-4-isopropyl-1H-pyrazole-3-car-
boxylic Acid Methyl Ester (11). To a solution of 10 (18.95 g,
47.8 mmol) in MeOH (300 mL) at 25 °C under N2 was added 10%
Pd/C (700 mg). The reaction vessel was evacuated and filled with
H2 and then stirred at 25 °C for 3 h. Subsequently, the reaction
vessel was flushed with N2 and filtered through a pad of celite.
The filtrate was concentrated to afford 1-(4-fluoro-phenyl)-4-
isopropyl-1H-pyrazole-3,5-dicarboxylic acid 3-methyl ester (14.6g,
was a 4.5 min binary gradient of acetonitrile (containing 0.1%
HCO2H) and water (2–70%). Melting points were recorded on an
Electrothermal Melting Point Apparatus and are reported uncor-
rected. Elemental analysis was performed at Quantitative Technolo-
gies, Inc., Whitehouse, NJ.
For experiments involving the use of animals, all procedures were
carried out in compliance with the NIH Guide for the Care and
Use of Laboratory Animals under a protocol approved by the
Institutional (Pfizer Global Research and Development) Animal
Care and Use Committee.
1
99%): H NMR (CDCl3) δ 7.36–7.31 (m, 2 H), 7.11–7.06 (m, 2
H), 3.90 (s, 3 H), 3.84 (sept, J ) 7.2 Hz, 1 H), 1.43 (d, J ) 7.2
Hz, 6 H); MS (APCI+) m/z 307.1 (M + H).
To a solution of 1-(4-fluoro-phenyl)-4-isopropyl-1H-pyrazole-
3,5-dicarboxylic acid 3-methyl ester (15.9 g, 51.8 mmol) in THF
(300 mL) at 0 °C was slowly added BH3 ·THF (1.0 M solution in
THF, 104 mL, 104 mmol). The reaction was allowed to warm to
25 °C for 30 min and then heated to 65 °C for 3 h. After cooling
to 25 °C, MeOH (50 mL) was slowly added. Subsequently, the
solvent was removed under reduced pressure, a second portion of
MeOH (100 mL) was slowly added, and the solution was stirred at
25 °C for an additional 20 min. The MeOH was then evaporated
and EtOAc was added, and the organic layer was washed with 1 N
NaOH and brine prior to drying over Na2SO4. The organic layer
was concentrated and purified by silica gel chromatography
(15–35% EtOAc/hexanes) to provide 1-(4-fluoro-phenyl)-5-hy-
droxymethyl-4-isopropyl-1H-pyrazole-3-carboxylic acid methyl
ester (13.6 g, 90%): 1H NMR (CDCl3) δ 7.59–7.56 (m, 2 H),
7.15–7.11 (m, 2 H), 4.57 (s, 2 H), 3.89 (s, 3 H), 3.64 (sept, J ) 7.2
Hz, 1 H), 1.36 (d, J ) 7.2 Hz, 6 H); MS (APCI+) m/z 293.1
(M + H).
To a solution of 1-(4-fluoro-phenyl)-5-hydroxymethyl-4-isopro-
pyl-1H-pyrazole-3-carboxylic acid methyl ester (13.6g, 46.7 mmol)
in CH2Cl2 (300 mL) at 25 °C was added solid NaHCO3 (19.6 g,
233 mmol) followed by Dess-Martin periodinane (20.8 g, 49.0
mmol; commercially available from Lancaster). The reaction was
stirred at 25 °C for 4 h after which time saturated sodium bisulfite
(50 mL) was added and the organic layer was separated and washed
with water and brine. The organic layer was dried over Na2SO4and
concentrated to an oil that was purified by silica gel chromatography
(15% EtOAc/hexanes) to afford 11 (11.3 g, 84%): 1H NMR (CDCl3)
δ 9.95 (s, 1 H), 7.42–7.38 (m, 2 H), 7.17–7.13 (m, 2 H), 3.98 (sept,
J ) 7.2 Hz, 1 H), 3.92 (s, 3 H), 1.38 (d, J ) 7.2 Hz, 6 H); MS
(APCI+) m/z 291.2 (M + H).
(4-Fluoro-phenyl)-hydrazono]-chloroacetic Acid Methyl
Ester (5). To a solution of 4-fluoroaniline (10.0 g, 90.0 mmol) in
MeOH (80 mL) was added 6 N HCl (80 mL), and the solution was
cooled to 0 °C. NaNO2 (12.4 g, 180 mmol) was then slowly added
as a solid. The reaction was stirred for 15 min at 0 °C after which
time NaOAc was added as a solid to adjust the reaction to pH 5.
Subsequently, a solution of methyl 2-chloroacetoacetate (10.96 mL,
90.0 mmol) in MeOH (40 mL) was slowly added at 0 °C. The
reaction was then allowed to warm to 25 °C and stirred for 12 h,
after which time the MeOH was removed under reduced pressure
and diethyl ether (300 mL) was added. The organic layer was
separated and washed with saturated NaHCO3 and water prior to
drying over Na2SO4. The organic layer was concentrated to afford
5 (19.42 g, 94%), which was utilized without further purification:
1H NMR (CDCl3) δ 8.37 (bs, 1 H), 7.22–7.12 (m, 2 H), 7.00–6.96
(m, 2 H), 3.87 (s, 3 H); MS (APCI+) m/z 252.9 (M + Na).
(E)-4-Methyl-pent-2-enoic Acid Benzyl Ester (8). To a solution
of 4-methyl-2-pentenoic acid (24.0 g, 210 mmol) in acetone (300
mL) was added K2CO3 (55.8 g, 404 mmol), and the reaction was
stirred at 25 °C for 30 min. A solution of benzyl bromide (25.2
mL, 212 mmol) in acetone (100 mL) was then added dropwise.
The reaction mixture was subsequently heated to reflux for 16 h.
After cooling to 25 °C, the acetone was removed under reduced
pressure, ether (300 mL) and water (300 mL) were added, and the
organic layer was separated, washed with brine, and dried over
Na2SO4. After concentration, the crude product was subjected to
silica gel chromatography (1–5% diethyl ether/hexanes) to afford
1
8 (40.5 g, 98%): H NMR (CDCl3) δ 7.36–7.28 (m, 5 H), 6.96
(dd, J ) 15.6, 6.8 Hz, 1 H), 5.79 (d, J ) 15.6 Hz, 1 H), 5.14 (s,
2 H), 2.42 (sept, J ) 6.8 Hz, 1 H), 1.01 (d, J ) 6.8 Hz, 6 H); MS
(APCI+) m/z 227.1 (M + Na).
1-(4-Fluoro-phenyl)-5-formyl-4-isopropyl-1H-pyrazole-3-car-
boxylic Acid (12). To a solution of 11 (11.34 g, 39.1 mmol) in
MeOH (150 mL) was added NaOH (156 mL of 1 N solution, 156
mmol). The reaction was heated to 60 °C for 4 h. The solvent was
then removed under reduced pressure and water (150 mL) and Et2O
(100 mL) were added. The organic layer was discarded and the
aqueous layer was acidified with 10% HCl to pH 1 and then
extracted with EtOAc (200 mL × 2). The combined organic extracts
were dried over Na2SO4 and concentrated to give 12 (8.55 g, 79%)
as a white solid that required no purification: 1H NMR (CDCl3) δ
10.0 (s, 1 H), 7.48–7.44 (m, 2 H), 7.26–7.15 (m, 2 H), 4.02 (sept,
1-(4-Fluoro-phenyl)-4-isopropyl-4,5-dihydro-1H-pyrazole-3,5-
dicarboxylic Acid 5-Benzyl Ester 3-Methyl Ester (9). To a
solution of 5 (20.93 g, 90.8 mmol) and 8 (18.54 g, 90.8 mmol) in
dioxane (400 mL) at 25 °C was added Ag2CO3 (63.0 g, 227 mmol).
The reaction was protected from light and stirred at 25 °C for 48 h.
Subsequently, the reaction mixture was filtered through a pad of
celite and the filtrate was concentrated. The crude product mixture
was subjected to silica gel chromatography (5–20% EtOAc/hexanes)
1
to afford 9 (29.3 g, 73%): H NMR (CDCl3) δ 7.30–7.24 (m, 3
H), 7.15–7.12 (m, 2 H), 7.01–6.96 (m, 2 H), 6.93–6.88 (m, 2 H),

HepatoselectiVe HMG-CoA Reductase Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1 39
Table 6. LDL-Lowering Efficacy of Selected Compounds upon Chronic 7-Day Dosing
acute inhibition of in vivo cholesterol synthesisa
chronic (7-day) in vivo LDL-C loweringa
cmpd
structure
t ) 2–4 h (10 mpk)
t ) 4–6 h (10 mpk)
reduction
dose (mpk)
hepatoselectivity ratio
rosuvastatin
-76%
-74%
-71%
-61%
-72%
-82%
-81%
-62%
-46%
-15%
-19%
-16%
NAb
-17%
-22%
-10%
-42%
-41%
-42%
-20%
-15%
-32%
-45%
-11%
20
20
20
20
20
10
10
20
830
810
35
36
39
42
48
49
50
Table 2
Table 2
Table 3
Table 4
Table 4
Table 4
Table 5
2600
16000
8000
2100
9500
6300
a Data represent the mean of the percent change from the control group (n ) 8/group). b NA ) not active.
hydrobromide (0.49 g, 1.43 mmol). The reaction was heated to 80
°C for 48 h after which time all starting material was consumed as
determined by TLC. The reaction solvent was removed under
reduced pressure and the resulting white solid was dried under high
vacuum for 12 h to provide 14 (0.977 g, 98%) in sufficient purity
for use in the next step.
(6-{2-[5-Benzylcarbamoyl-2-(4-fluoro-phenyl)-4-isopropyl-
2H-pyrazol-3-yl]-vinyl}-2,2-dimethyl-[1R,3R]dioxan-4-yl)-ace-
tic Acid tert-Butyl Ester (17). To a solution of 14 (0.562 g, 0.811
mmol) in THF/DMSO (50:1, 50 mL) at -78 °C was added 1.0 M
LiHMDS (1.055 mL, 1.055 mmol). An orange color was noted as
the base was added. The reaction mixture was stirred at -78 °C
for 5 min after which time a solution of 16 in THF (10 mL) was
slowly added. After the addition, the reaction mixture was stirred
at -78 °C for 30 min, then allowed to warm to 25 °C, and stirred
at that temperature for 5 h. The reaction was quenched by dropwise
addition of saturated NH4Cl. Ethyl acetate (50 mL) was then added
and the organic layer was separated, washed with water, dried
(Na2SO4), and concentrated. The crude product was purified by
silica gel chromatography (15–20% EtOAc/hexanes) to afford 17
as an inseparable 1:4 mixture of cis/trans-olefin isomers.
Figure 3. Comparison of PF-3052334 (35) versus rosuvatstatin and
pravastatin in chronic plasma LDL reduction study performed in guinea
pigs. Animals were dosed daily with 35, pravastatin, or rosuvastatin
for 7 days. All groups were sacrificed 2 h postdose on the final day of
dosing. ED50 values are reported as 50% of the maximal response in
LDL lowering observed in the study. Data represent the mean ( SEM
of the percent change from the control group (n ) 8/group).
To a solution of 17 (0.360 g, 0.61 mmol) in MeOH (20 mL)
was added 1 N HCl (2 mL), and the solution was stirred for 3 h at
25 °C. Subsequently, the reaction solvent was removed under
reduced pressure and ethyl acetate (50 mL) and saturated NaHCO3
(10 mL) were added. The organic layer was separated, washed with
brine, dried (Na2SO4), and concentrated to afford, after silica gel
chromatography (35% EtOAc/hexanes), 7-[5-benzylcarbamoyl-2-
(4-fluoro-phenyl)-4-isopropyl-2H-pyrazol-3-yl]-3,5-dihydroxy-hept-
6-enoic acid tert-butyl ester (0.231 g, 69%) as a mixture of cis/
trans-isomers.
Subsequently, to a solution of 7-[5-benzylcarbamoyl-2-(4-fluoro-
phenyl)-4-isopropyl-2H-pyrazol-3-yl]-3,5-dihydroxy-hept-6-enoic
acid tert-butyl ester (0.241 g, 0.437 mmol) in MeOH (20 mL) was
added 10% Pd/C (50 mg), and the reaction vessel was evacuated
and filled with hydrogen gas (via balloon) for 3 h. The reaction
mixture was then filtered through a pad of celite. The crude product
was purified by silica gel chromatography (30–50% EtOAc/hexanes)
to afford 18 (0.143 g, 59%): 1H NMR (CDCl3) δ 7.38–7.23 (m, 7
H), 7.16–7.12 (m, 2 H), 4.57 (d, J ) 6.4 Hz, 2 H), 4.13–4.08 (m,
1 H), 3.73–3.72 (m, 1 H), 3.41–3.37 (m, 2 H), 2.83–2.80 (m, 1 H),
2.69–2.67 (m, 1 H), 1.44 (s, 9 H), 1.39 (d, J ) 7.2 Hz, 6 H),
1.51–1.22 (m, 6 H); MS (APCI+) m/z 554.3 (M + H).
J ) 7.2 Hz, 1 H), 1.43 (d, J ) 7.2 Hz, 6 H); MS (APCI+) m/z
277.2 (M + H); Anal. (C14H13F1N2O3) C, H, N; mp 172–174 °C.
1-(4-Fluoro-phenyl)-5-formyl-4-isopropyl-1H-pyrazole-3-car-
boxylic Acid Benzylamide (13). To a solution of 12 (1.25 g, 4.52
mmol) in CH2Cl2 (50 mL) at 25 °C was added 1-(3-dimethylami-
nopropyl)-3-ethylcarbodiimide hydrochloride (1.30 g, 6.79 mmol)
followed by 1-hydroxybenzotriazole hydrate (1.04 g, 6.79 mmol),
and the reaction was stirred for 5 min at 25 °C. Subsequently, benzyl
amine (0.533 g, 4.98 mmol) was added, and the reaction was stirred
for an additional 4 h as a fine white precipitate developed. The
organic layer was washed with 1 N HCl, saturated NaHCO3, and
brine. After drying and concentration, the product was purified by
silica gel chromatography (20% EtOAc/hexanes) to provide 13 (0.65
1
g, 39%): H NMR (CDCl3) δ 9.97 (s, 1 H), 7.37–7.22 (m, 7 H),
7.17–7.12 (m, 2 H), 4.57 (d, J ) 6.0 Hz, 2 H), 4.15 (sept, J ) 7.2
Hz, 1 H), 1.42 (d, J ) 7.2 Hz, 6 H); MS (APCI+) m/z 366.1
(M + H).
[5-Benzylcarbamoyl-2-(4-fluoro-phenyl)-4-isopropyl-2H-pyra-
zol-3-ylmethyl]-triphenyl-phosphonium Bromide (14). To a
solution of 13 (0.620 g, 1.70 mmol) in THF/MeOH (40 mL) at 0
°C was added sodium borohydride (96.3 mg, 2.55 mmol). The
reaction was stirred for 30 min at 0 °C at which point TLC analysis
indicated the reaction was complete and the solvent was removed
under reduced pressure. To the reaction residue was added ethyl
acetate (50 mL) and saturated NaHCO3 (15 mL), and the organic
layer was separated, dried (Na2SO4), and concentrated. The resulting
oil was purified by silica gel chromatography (40% EtOAc/hexanes)
to afford 1-(4-fluoro-phenyl)-5-hydroxymethyl-4-isopropyl-1H-
pyrazole-3-carboxylic acid benzylamide (0.540 g, 87%): 1H NMR
(CDCl3) δ 7.59–7.54 (m, 2 H), 7.32–7.20 (m, 5 H), 7.15–7.10 (m,
2 H), 4.57–4.55 (m, 4 H), 3.72 (sept, J ) 7.2 Hz, 1 H), 1.38 (d, J
) 7.2 Hz, 6 H); MS (APCI+) m/z 368.1 (M + H).
(3R,5R)-7-[5-Benzylcarbamoyl-2-(4-fluoro-phenyl)-4-isopro-
pyl-2H-pyrazol-3-yl]-3,5-dihydroxy-heptanoic Acid Sodium Salt
(2). To a solution of 18 (0.103 g, 0.186 mmol) in MeOH (5 mL)
was added 1.0 N NaOH (0.190 mL, 0.195 mmol), and the reaction
was stirred at 25 °C for 48 h after which time the reaction solvent
was removed under reduced pressure. The resulting solid was then
azeotroped with toluene (3 × 100 mL) and triturated with diethyl
ether to provide a light yellow solid that was dried under vacuum
1
at 60 °C to afford 2 (0.091 g, 94%): H NMR (DMSO-d6) δ 8.55
(t, J ) 2.4 Hz, 1 H), 7.53–7.49 (m, 2 H), 7.33–7.13 (m, 7 H), 4.70
(bs, 1 H), 4.34 (d, J ) 6.4 Hz, 2 H), 3.63–3.57 (m, 1 H), 3.45–3.42
(m, 1 H), 2.74–2.66 (m, 1 H), 2.58–2.50 (m, 1 H), 1.92 (dd, J )
14.8, 4.0 Hz, 1 H), 1.72 (dd, J ) 15.2, 8.0 Hz, 1 H), 1.40–1.10 (m,
To a solution of 1-(4-fluoro-phenyl)-5-hydroxymethyl-4-isopro-
pyl-1H-pyrazole-3-carboxylic acid benzylamide (0.525 g, 1.43
mmol) in acetonitrile (50 mL) was added triphenylphosphine

40 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1
Pfefferkorn et al.
Figure 4. Relationships between predicted lipophilicity and myocyte activity [Plot A], hepatocyte activity [Plot B], and hepatoselectivity [Plot C].
10 H); HRMS (ESI) calcd for C27H32FN3O5 [(M + H)+], 498.2399;
found, 498.2393; HPLC (method B) tR ) 1.86 min (98.2%).
5-(Benzylcarbamoyl)-1-(4-fluorophenyl)-4-isopropyl-1H-pyra-
zole-3-carboxylic Acid (21). To a solution of 10 (9.40 g, 23.7
mmol) in MeOH (150 mL) at 25 °C under N2 was added 10% Pd/C
(500 mg). The reaction vessel was evacuated and filled with H2
and then stirred at 25 °C for 3 h. Subsequently, the reaction vessel
was flushed with N2 and filtered through a pad of celite. The filtrate
was concentrated to afford 19 (7.20 g, 99%).
To a solution of 19 (3.00 g, 9.79 mmol) in CH2Cl2 (100 mL) at
25 °C was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (2.82 g, 14.7 mmol) followed by 1-hydroxybenzo-
triazole hydrate (2.25 g, 14.7 mmol), and the reaction was stirred
for 5 min at 25 °C. Subsequently, benzyl amine (1.26 g, 11.8 mmol)
was added, and the reaction was stirred for an additional 8 h. The
organic layer was washed with 1 N HCl, saturated NaHCO3, and
brine. After drying and concentration, the product was purified by
silica gel chromatography (15% EtOAc/hexanes) to provide 20 (2.20
g, 57%).
To a solution of 20 (2.20 g, 5.56 mmol) in MeOH (50 mL) was
added NaOH (22.3 mL of 1 N solution, 22.3 mmol). The reaction
was heated to 60 °C for 3 h. The solvent was then removed under
reduced pressure and water (50 mL) and Et2O (100 mL) were added.
The organic layer was discarded and the aqueous layer was acidified
with 10% HCl to pH 1 and then extracted with EtOAc (200 mL ×
2). The combined organic extracts were dried over Na2SO4 and
concentrated to give 21 (2.07 g, 98%) as a white solid that required
no purification: 1H NMR (CDCl3) δ 7.45–7.41 (m, 2 H), 7.25–7.21
(m, 3 H), 7.06–7.01 (m, 4 H), 5.86 (bs, 1 H), 4.42 (d, J ) 5.6 Hz,
2 H), 3.55 (sept, J ) 7.2 Hz, 1 H), 1.31 (d, J ) 7.2 Hz, 6 H).
N-Benzyl-1-(4-fluorophenyl)-3-(hydroxymethyl)-4-isopropyl-
1H-pyrazole-5-carboxamide (22). To a solution of 21 (2.07 g,
5.43 mmol) in THF (100 mL) at 0 °C was slowly added BH3 ·THF
(8.14 mL of 1.0 M solution in THF, 8.14 mmol). The reaction was
allowed to warm to 25 °C for 30 min and then heated to 65 °C for
4 h. After cooling to 25 °C, MeOH (20 mL) was slowly added.
Subsequently, the solvent was removed under reduced pressure, a
second portion of MeOH (50 mL) was slowly added, and the
solution was stirred at 25 °C for an additional 20 min. The MeOH
was then evaporated, EtOAc was added, and the organic layer was
washed with 1 N NaOH and brine prior to drying over Na2SO4.
The organic layer was concentrated and purified by silica gel
chromatography (15–35% EtOAc/hexanes) to provide 22 (0.69 g,
35%): MS(APCI+) m/z 368.1 (M + H).
(3R,5R)-tert-Butyl 7-(5-(benzylcarbamoyl)-1-(4-fluorophenyl)-
4-isopropyl-1H-pyrazol-3-yl)-3,5-dihydroxyheptanoate (25). To
a solution of 22 (0.69 g, 1.88 mmol) in CH2Cl2 (50 mL) at 25 °C
was slowly added PBr3 (0.94 mL of 1.0 M solution in CH2Cl2,
0.94 mmol). The resulting reaction mixture was then stirred at 25
°C for 12 h, after which time water (10 mL) was added, and the
reaction mixture was stirred for an additional 0.5 h at 25 °C. The
organic layer was separated, washed with saturated NaHCO3
solution (2 × 40 mL), washed with brine (1 × 40 mL), dried
(Na2SO4), and concentrated to afford N-benzyl-3-(bromomethyl)-
1-(4-fluorophenyl)-4-isopropyl-1H-pyrazole-5-carboxamide (0.810
g, 100%), which was utilized without additional purification.
To a solution of N-benzyl-3-(bromomethyl)-1-(4-fluorophenyl)-
4-isopropyl-1H-pyrazole-5-carboxamide (0.810 g, 1.88 mmol) in
toluene (50 mL) at 25 °C was added triphenyl phosphine (0.494 g,
1.88 mmol). The resulting reaction mixture was then heated to 80
°C for 14 h. The reaction mixture was then cooled to 25 °C, the
solvent was removed under reduced pressure, CH2Cl2 (100 mL)
was added, and the solution was concentrated under reduced
pressure to afford 23 (1.30 g, 100%), which was utilized without
additional purification.
To a solution of 23 (1.30 g, 1.88 mmol) in THF (50 mL) at -78
°C was added 1.0 M LiHMDS (2.44 mL, 2.44 mmol). The reaction
mixture was stirred at -78 °C for 5 min, after which time a solution
of 16 (0.582 g, 2.25 mmol) in THF (10 mL) was slowly added.
After the addition, the reaction mixture was stirred at -78 °C for
30 min then allowed to warm to 25 °C and stirred at that
temperature for 2 h. The reaction was quenched by dropwise
addition of saturated NH4Cl. Ethyl acetate (75 mL) was then added,
and organic layer was separated, washed with water, dried (Na2SO4),
and concentrated. The crude product was purified by silica gel
chromatography (10% EtOAc/hexanes) to afford 24 (0.70 g, 63%)
as an inseparable about 1:3 mixture of cis/trans-olefin isomers.
To a solution of 24 (0.70 g, 1.18 mmol) in MeOH (15 mL) was
added 1 N HCl (2 mL), and the solution was stirred for 1.5 h at 25
°C. Subsequently, the reaction solvent was removed under reduced

HepatoselectiVe HMG-CoA Reductase Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1 41
pressure and ethyl acetate (50 mL) and saturated NaHCO3 solution
(15 mL) were added. The organic layer was separated, washed with
brine, dried (Na2SO4), and concentrated to afford, after silica gel
chromatography (40% EtOAc/hexanes), (3R,5R)-tert-butyl 7-(5-
(benzylcarbamoyl)-1-(4-fluorophenyl)-4-isopropyl-1H-pyrazol-3-
yl)-3,5-dihydroxyhept-6-enoate (0.65 g, 93%) as a mixture of cis/
trans-isomers.
EtOAc/hexanes) to afford 28 (2.48 g, 98.1%). To a solution of 28
(2.48 g, 3.82 mmol) in MeCN (40 mL) at 25 °C was added HF
(2.5 mL of 48% in water). The reaction was stirred at 25 °C for
16 h. Subsequently, EtOAc (100 mL) and water (100 mL) were
added, and the organic layer was separated, dried, and concentrated.
Product was purified by column chromatography (30–50%
EtOAc-hexanes) to afford 29 (2.03 g, 98.0%): 1H NMR (CDCl3)
[mixture of rotamers at 25 °C] δ 7.41–7.43 (m), 7.33–7.09 (m),
6.26 (d), 6.20 (d), 4.74 (s), 4.66 (s), 4.49 (bs), 3.69 (s), 3.38–3.37
(m), 3.17–3.13 (m), 3.03 (s), 2.91 (s), 2.76–2.74 (m), 2.54–2.53
(m), 2.35 (s), 2.31 (s), 1.39–1.36 (m), 1.24–1.22 (m); MS (APCI+)
536.4 m/z (M + H).
Subsequently, to a solution of (3R,5R)-tert-butyl 7-(5-(benzyl-
carbamoyl)-1-(4-fluorophenyl)-4-isopropyl-1H-pyrazol-3-yl)-3,5-di-
hydroxyhept-6-enoate (0.605 g, 1.10 mmol) in MeOH (15 mL) was
added 10% Pd/C (70 mg), and the reaction vessel was evacuated
and filled with hydrogen gas (via balloon) for 4 h. The reaction
mixture was then filtered through a pad of celite. The crude product
was purified by silica gel chromatography (45% EtOAc/hexanes)
to 25 (0.34 g, 56%): 1H NMR (CDCl3) δ 7.34–7.30 (m, 2 H),
7.23–7.19 (m, 3 H), 7.01–6.93 (m, 4 H), 5.86 (t, J ) 5.6 Hz, 1 H),
4.42 (d, J ) 6.0 Hz, 2 H), 4.18–4.16 (m, 1 H), 3.92–3.88 (m, 1 H),
3.01 (sept, J ) 6.8 Hz, 1 H), 2.75 (t, J ) 7.6 Hz, 2 H), 2.34–2.32
(m, 1 H), 1.84–1.79 (m, 2 H), 1.58–1.54 (m, 2 H), 1.40 (s, 9 H),
1.24 (d, J ) 6.8 Hz, 6 H); MS (APCI+) m/z 554.2 (M + H).
(3R,5R)-7-(5-(Benzylcarbamoyl)-1-(4-fluorophenyl)-4-isopro-
pyl-1H-pyrazol-3-yl)-3,5-dihydroxyheptanoic Acid Sodium Salt
(3). To a solution of 25 (0.428 g, 0.773 mmol) in MeOH (5 mL)
was added 1.0 N NaOH (0.788 mL, 0.788 mmol), and the reaction
was stirred at 25 °C for 16 h after which time the reaction was
solvent was removed under reduced pressure. The resulting solid
was then azeotroped with toluene (2 × 50 mL) to provide a white
solid that was dried under vacuum at 60 °C to afford 3 (0.39 g,
7-{2-(4-Fluoro-phenyl)-4-isopropyl-5-[methyl-(2-methyl-ben-
zyl)-carbamoyl]-2H-pyrazol-3-yl}-3,5-dihydroxy-hept-6-enoic Acid
Methyl Ester (30). To a solution of 29 (2.04 g, 3.81 mmol) in
THF (100 mL) and MeOH (30 mL) at -78 °C was added
diethylmethoxyborane (4.95 mL of 1.0 M solution, 4.95 mmol).
The reaction was stirred at -78 °C for 1 h, after which time NaBH4
(166 mg, 4.38 mmol) was added. The reaction mixture was stirred
at -78 °C for 2 h and then warmed to 0 °C, after which time glacial
acetic acid (0.5 mL) was added. The reaction was diluted with
EtOAc and water, and the organic layer was separated and washed
with satd NaHCO3 and brine prior to concentration. The resulting
crude oil was then dissolved in MeOH (50 mL) and evaporated
again. Subsequently, a second amount of MeOH (50 mL) was added
and the solution was stirred at 25 °C for 12 h, after which time it
was concentrated and purified by column chromatography (40–60%
1
EtOAc/hexanes) to afford 30 (1.66 g, 81.1%): H NMR (CDCl3)
1
[mixture of rotamers at 25 °C] δ 7.40–7.36 (m), 7.30–7.03 (m),
6.44 (d), 5.79 (dd), 5.70 (dd), 4.78 (s), 4.70 (s), 4.51–4.44 (m),
4.27–4.24 (m), 3.71 (s), 3.68–3.54 (m), 3.05 (sept), 3.01 (s), 2.92
(s), 2.52–2.49 (m), 2.35 (s), 2.25 (s), 1.71–1.52 (m), 1.37–1.31 (m);
MS (APCI+) 538.2 m/z (M + H).
97%): H NMR (DMSO-d6) δ 7.55 (s, 1 H), 7.36–7.33 (m, 2 H),
7.21–7.05 (m, 7 H), 4.69 (bs, 1 H), 3.70–3.63 (m, 2 H), 3.15 (sept,
J ) 7.2 Hz, 1 H), 3.71–3.59 (m, 1 H), 3.51–3.41 (m, 1 H), 1.93
(dd, J ) 14.8, 3.6 Hz, 1 H), 1.79–1.25 (m, 5 H), 1.14 (d, J ) 7.2
Hz, 6 H); HRMS (ESI) calcd for C27H32FN3O5 [(M + H)+],
498.2399; found, 498.2397; HPLC (method A) tR ) 6.57 min
(98.2%).
7-{2-(4-Fluoro-phenyl)-4-isopropyl-5-[methyl-(2-methyl-ben-
zyl)-carbamoyl]-2H-pyrazol-3-yl}-3,5-dihydroxy-heptanoic Acid
Methyl Ester (31). To a solution of 30 (1.19 g, 2.21 mmol) in
MeOH (50 mL) was added Pd/C (100 mg). The reaction vessel
was evacuated, flushed with nitrogen, and then filled with hydrogen
(via balloon). The reaction was stirred at 25 °C for 4 h, then flushed
with nitrogen, and filtered through a pad of celite. The filtrate was
concentrated and purified by column chromatography (30–40%
1-(4-Fluoro-phenyl)-5-formyl-4-isopropyl-1H-pyrazole-3-car-
boxylic Acid Methyl-(2-methyl-benzyl)-amide (26). To a solution
of 2-methylbenzaldehyde (10.0 g, 83.23 mmol) in MeOH at 25 °C
was added methylamine (40% in H2O, 25.85 g, 332.9 mmol). The
reaction mixture was stirred at 25 °C for 30 min, after which time
it was cooled to 0 °C and NaBH4 (6.30 g, 166.5 mmol) was added
portionwise. The reaction mixture was then warmed to 25 °C and
stirred for an additional 1 h. The solvent was removed under reduced
pressure, and water and CH2Cl2 were added. The organic layer was
separated and washed with saturated NaHCO3 and brine. After
drying over Na2SO4, the organic phase was concentrated to afford
the desired methyl-(2-methyl-benzyl)-amine (10.49 g, 93.2%) as
1
EtOAc/hexanes) to afford 31 (0.792 g, 66%): H NMR (CDCl3)
[mixture of rotamers at 25 °C] δ 7.39–7.36 (m), 7.29–7.07 (m),
5.28 (s), 4.77 (s), 4.71 (s), 4.18–4.11 (m), 3.79–3.71 (m), 3.69 (s),
3.15–3.05 (m), 3.04–2.78 (m), 2.76–2.61 (m), 2.43 (s), 2.40 (s),
2.35 (s), 2.20 (s), 1.53–1.16 (m); MS (APCI+) 540.2 m/z
(M + H).
1
an oil of sufficient purity for use in the next reaction: H NMR
(3R,5R)-7-{2-(4-Fluoro-phenyl)-4-isopropyl-5-[methyl-(2-methyl-
benzyl)-carbamoyl]-2H-pyrazol-3-yl}-3,5-dihydroxy-heptanoic
Acid Sodium Salt (32). To a solution of 31 (0.792 g, 1.47 mmol)
in MeOH (5 mL), NaOH solution (1.028 N, 1.50 mL, 1.54 mmol)
was added. The reaction mixture was stirred at 25 °C for 12 h.
After the solvent was evaporated, the crude product was dried
azeotopically with toluene (3 × 10 mL). It was dissolved in a
MeOH/CH2Cl2 mixture (1:9, 10 mL) and diluted with CH2Cl2 (15
mL). It was filtered through cotton, and the filtrate was concentrated
and triturated with Et2O for 12 h. The product was isolated by
filtration and dried under vacuum at 60 °C for 12 h to afford 32
(CDCl3) δ 7.35–7.08 (m, 3 H), 3.73 (s, 2 H), 2.49 (s, 3 H), 2.34 (s,
3 H); MS (APCI+) 136.3 m/z (M + H).
To a solution of 12 (1.50 g, 5.43 mmol) in CH2Cl2 (100 mL) at
25 °C was added EDCI (1.56 g, 8.14 mmol), followed by
HOBt·H2O (1.25 g, 8.14 mmol), and the reaction was stirred for
10 min at 25 °C. Subsequently, methyl-(2-methyl-benzyl)-amine
(1.10 g, 8.14 mmol) was added, and the reaction was stirred for an
additional 6 h at 25 °C. The organic layer was then washed with
HCl, saturated NaHCO3, and brine. After drying over Na2SO4 and
concentrated, the product was purified by column chromatography
(10–25% EtOAc/hexanes) to afford 26 (1.53 g, 71.6%) as an oil:
1H NMR (CDCl3) [mixture of rotamers at 25 °C] δ 9.36 (s), 9.14
(s), 7.44–7.40 (m), 7.31–7.11 (m) 4.80 (s), 4.65 (s), 3.56 (sept),
3.04 (s), 2.89 (s) 2.35 (s), 2.19 (s), 1.40–1.38 (m); MS (APCI+)
394.2 m/z (M + H).
1
(798 mg, 99%): H NMR (DMSO-d6) [mixture of rotamers at 25
°C] δ 7.79–7.65 (m), 7.58–7.46 (m), 7.41–7.24 (m), 7.22–7.07 (m),
4.85–4.60 (m), 3.62–3.58 (m), 3.46–3.44 (m), 2.89–2.85 (m), 2.84
(s), 2.74–2.66 (m), 2.54–2.45 (m), 2.29 (s), 1.93–1.88 (m),
1.73–1.67 (m), 1.53–1.10 (m); HRMS (ESI) calcd for C29H36FN3O5
[(M + H)+], 526.2712; found, 526.2703; HPLC (method A) tR )
7.39 min (95.4%).
(3R,5R)-7-[2-(4-Fluoro-phenyl)-4-isopropyl-5-(4-methoxy-ben-
zylcarbamoyl)-2H-pyrazol-3-yl]-3,5-dihydroxy-heptanoic Acid
Sodium Salt (33). 1H NMR (DMSO-d6) δ 7.51–7.48 (m, 2 H),
7.32–7.28 (m, 2 H), 7.21–7.16 (m, 3 H), 6.80 (d, J ) 8.8 Hz, H),
4.27 (s, 2 H), 3.65 (s, 3 H), 3.64–3.49 (m, 3 H), 2.73–2.69 (m, 1
H), 2.57–2.49 (m, 1 H), 1.94 (dd, J ) 14.8, 4.0 Hz, 1 H), 1.74 (dd,
7-{2-(4-Fluoro-phenyl)-4-isopropyl-5-[methyl-(2-methyl-ben-
zyl)-carbamoyl]-2H-pyrazol-3-yl}-5-hydroxy-3-oxo-hept-6-eno-
ic Acid Methyl Ester (29). To a solution of 26 (1.53 g, 3.89 mmol)
in toluene (80 mL) was added 3-(tert-butyl-dimethyl-silanyloxy)-
5-oxo-6-(triphenyl-l5-phosphanylidene)-hexanoic acid methyl es-
ter22 (2.70 g, 5.06 mmol), and the reaction was heated to 95 °C for
16 h. After cooling to 25 °C, the solvent was removed under reduced
pressure and the product was run through pad of silica gel (20–30%

42 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1
Pfefferkorn et al.
J ) 14.8, 8.4 Hz, 1 H), 1.24 (d, J ) 7.2 Hz, 6 H), 1.37–1.10 (m,
4 H); HRMS (ESI) calcd for C28H34FN3O5 [(M + H)+], 528.2504;
found, 528.2454; HPLC (method B) tR ) 1.76 min (>99%).
C29H36FN3O5 [(M + H)+], 526.2712; found, 526.2687; HPLC
(method C) tR ) 3.15 min (98.0%).
(3R,5R)- 7-[5-(Benzyl-isopropyl-carbamoyl)-2-(4-fluoro-phen-
yl)-4-isopropyl-2H-pyrazol-3-yl]-3,5-dihydroxy-heptanoic Acid
Sodium Salt (41). 1H NMR (DMSO-d6) δ 7.66–7.59 (m, 2 H),
7.48–7.19 (m, 7 H), 4.71 (bs, 1 H), 4.56 (s, 1 H), 4.44 (s, 1 H),
3.83–3.81 (m, 1 H), 3.46–3.43 (m, 1 H), 3.27–3.19 (m, 1 h),
2.90–2.88 (m, 1 H), 2.79–2.72 (m, 1 H), 2.65–2.60 (m, 1 H), 1.91
(dd, J ) 14.8, 4.0 Hz, 1 H), 1.70 (dd, J ) 14.8, 8.4 Hz, 1 H),
1.41–1.21 (m, 4 H), 1.18 (d, J ) 6.0 Hz, 6 H), 1.02 (d, J ) 6.8 Hz,
6 H); HRMS (ESI) calcd for C30H38FN3O5 [(M + H)+], 540.2868;
found, 540.2847; HPLC (method C) tR ) 3.34 min (95.2%).
(3R,5R)- 7-{2-(4-Fluoro-phenyl)-4-isopropyl-5-[methyl-(3-
methyl-benzyl)-carbamoyl]-2H-pyrazol-3-yl}-3,5-dihydroxy-hep-
tanoic Acid Sodium Salt (42). 1H NMR (DMSO-d6) δ 7.47–7.41
(m, 2 H), 7.31–7.27 (m, 1 H), 7.21–7.07 (m, 5 H), 4.71 (bs, 1 H),
4.58 (s, 1 H), 4.47 (s, 1 H), 3.61–3.59 (m, 1 H), 3.46–3.43 (m, 1
H), 2.89–2.87 (m, 1 H), 2.80 (s, 3 H), 2.78–2.75 (m 1 H), 2.56–2.52
(m, 1 H), 2.21 (s, 3 H), 1.91 (dd, J ) 14.8, 4.0 Hz, 1 H), 1.69 (dd,
J ) 14.8, 8.2 Hz, 1 H), 1.36–1.10 (m, 4 H), 1.16 (d, J ) 6.8 Hz,
6 H); HRMS (ESI) calcd for C29H36FN3O5 [(M + H)+], 526.2712;
found, 526.2702; HPLC (method C) tR ) 3.67 min (>99%).
(3R,5R)-7-{2-(4-Fluoro-phenyl)-4-isopropyl-5-[methyl-(4-methyl-
benzyl)-carbamoyl]-2H-pyrazol-3-yl}-3,5-dihydroxy-heptanoic
(3R,5R)-7-[2-(4-Fluoro-phenyl)-4-isopropyl-5-(4-fluoro-ben-
zylcarbamoyl)-2H-pyrazol-3-yl]-3,5-dihydroxy-heptanoic Acid
Sodium Salt (34). 1H NMR (DMSO-d6) δ 7.52–7.48 (m, 2 H),
7.32–7.26 (m, 4 H), 7.06 (t, J ) 8.8 Hz, 2 H), 4.31 (s, 2 H),
3.61–3.58 (m, 1 H), 3.45–3.42 (m, 1 H), 3.23–3.11 (m, 1 H),
2.73–2.61 (m, 1 H), 2.57–2.49 (m, 1 H), 1.89 (dd, J ) 14.8, 4.0
Hz, 1 H), 1.67 (dd, J ) 14.8, 8.4 Hz, 1 H), 1.24 (d, J ) 7.2 Hz, 6
H), 1.35–1.05 (m, 4 H); HRMS (ESI) calcd for C27H31F2N3O5 [(M
+ H)+], 516.2305; found, 516.2309; HPLC (method B) tR ) 1.91
min (>99%).
(3R,5R)-7-[2-(4-Fluoro-phenyl)-4-isopropyl-5-(4-methyl-ben-
zylcarbamoyl)-2H-pyrazol-3-yl]-3,5-dihydroxy-heptanoic Acid
Sodium Salt (35). 1H NMR (DMSO-d6) δ 7.52–7.48 (m, 2 H),
7.29 (t, J ) 8.8 Hz, 2 H), 7.14 (J ) 8.0 Hz, 2 H), 7.03 (d, J ) 8.0
Hz, 2 H), 4.29 (s, 2 H), 3.62–3.56 (m, 1 H), 3.45–3.42 (m, 1 H),
3.28 (bs, 1 H), 2.73–2.66 (m, 1 H), 2.57–2.51 (m, 1 H), 2.20 (s, 3
H), 1.90 (dd, J ) 14.8, 4.0 Hz, 1 H), 1.70 (dd, J ) 14.8, 8.4 Hz,
1 H), 1.24 (d, J ) 7.2 Hz, 6 H), 1.53–1.11 (m, 4 H); MS (APCI+)
m/z 512.2 (M + H); Anal. (C28H33F1N3O5Na) C, H, N; HPLC
(method A) tR ) 8.29 min (99.5%); mp 240–241 °C.
(3R,5R)- 7-[2-(4-Fluoro-phenyl)-4-isopropyl-5-(3-methoxym-
ethyl-benzylcarbamoyl)-2H-pyrazol-3-yl]-3,5-dihydroxy-hep-
1
Acid Sodium Salt (43). H NMR (DMSO-d6) δ 7.47–7.41 (m, 2
H), 7.31–7.25 (m, 2 H), 7.18–7.06 (m, 4 H), 4.71 (bs, 1 H), 4.57
(s, 1 H), 4.45 (s, 1 H), 3.65–3.61 (m, 1 H), 3.45–3.42 (m, 1 H),
2.88–2.85 (m, 1 H), 2.78 (s, 3 H), 2.77–2.72 (m, 1 H), 2.55–2.52
(m, 1 H), 2.22–2.20 (m, 3 H), 1.91 (dd, J ) 14.8, 4.0 Hz, 1 H),
1.70 (dd, J ) 14.8, 8.4 Hz, 1 H), 1.35–1.10 (m, 4 H), 1.16 (d, J )
6.4 Hz, 6 H); HRMS (ESI) calcd for C29H36FN3O5 [(M + H)+],
526.2711; found, 526.2699; Anal. (C29H36FN3O5Na ·1.9H2O) C, H,
N; HPLC (method B) tR ) 1.93 min (>99%).
1
tanoic Acid Sodium Salt (36). H NMR (DMSO-d6) δ 8.56 (t, J
) 6.0 Hz, 1 H), 7.55–7.52 (m, 2 H), 7.35–7.11 (m, 6 H), 4.73 (bs,
1 H), 4.37–4.33 (m, 4 H), 3.64–3.62 (m, 1 H), 3.48–3.46 (m, 1 H),
3.23 (s, 3 H), 2.72–2.69 (m, 1 H), 2.59–2.45 (m, 1 H), 1.95 (dd, J
) 14.8, 4.0 Hz, 1 H), 1.73 (dd, J ) 14.8, 8.0 Hz, 1 H), 1.40–1.26
(d, J ) 6.8 Hz, 6 H), 1.41–1.13 (m, 4 H); HRMS (ESI) calcd for
C29H36FN3O6 [(M + H)+], 542.2661; found, 542.2649; Anal.
(C28H33FN3O5Na·0.6 H2O) C, H, N; HPLC (method A) tR ) 7.13
min (98.2%).
(3R,5R)-7-{2-(4-Fluoro-phenyl)-4-isopropyl-5-[(4-methoxy-
benzyl)-methyl-carbamoyl]-2H-pyrazol-3-yl}-3,5-dihydroxy-hep-
tanoic Acid Sodium Salt (44). 1H NMR (DMSO-d6) δ 7.47–7.40
(m, 2 H), 7.13–7.12 (m, 4 H), 6.87–6.81 (m, 2 H), 4.71 (bs, 1 H),
4.54 (s, 1 H), 4.41 (s, 1 H), 3.68–3.66 (m, 3 H), 3.60–3.58 (m, 1
H), 3.45–3.41 (m, 1 H), 2.89–2.86 (m, 1 H), 2.77–2.76 (m, 1 H),
2.75–2.68 (m, 1 H), 2.58–2.52 (m, 1 H), 1.90 (dd, J ) 14.8, 4.0
Hz, 1 H), 1.71 (dd, J ) 14.8, 8.2 Hz, 1 H), 1.39–1.19 (m, 4 H),
1.14 (d, J ) 6.8 Hz, 6 H); HRMS (ESI) calcd for C29H36FN3O5
[(M + H)+], 542.2660; found, 542.2586; HPLC (method C) tR )
3.45 min (98.5%).
(3R,5R)-7-[5-(3-Cyano-benzylcarbamoyl)-2-(4-fluoro-phenyl)-
4-isopropyl-2H-pyrazol-3-yl]-3,5-dihydroxy-heptanoic Acid So-
dium Salt (37). 1H NMR (DMSO-d6) δ 8.78–8.75 (m, 1 H),
7.77–7.60 (m, 2 H), 7.59–7.40 (m, 4 H), 7.38–7.23 (m, 2 H),
4.76–4.72 (m, 1 H), 4.43–4.38 (m, 2 H), 3.68–3.58 (m, 1 H),
3.57–3.42 (m, 1 H), 3.23–3.16 (m, 2 H), 2.80–2.62 (m, 1 H),
2.61–2.50 (m, 1 H), 1.93 (dd, J ) 14.8, 4.0 Hz, 1 H), 1.73 (dd, J
) 14.8, 8.4 Hz, 1 H), 1.26 (d, J ) 6.8 Hz, 6 H), 1.43–1.08 (m, 4
H); HRMS (ESI) calcd for C28H31FN4O5 [(M + H)+], 523.2351;
found, 523.2344; HPLC (method A) tR ) 6.91 min (98.0%).
(3R,5R)-7-[5-(4-Dimethylcarbamoyl-benzylcarbamoyl)-2-(4-
fluoro-phenyl)-4-isopropyl-2H-pyrazol-3-yl]-3,5-dihydroxy-hep-
tanoic Acid Sodium Salt (38). 1H NMR (DMSO-d6) δ 8.64–8.59
(m, 1 H), 7.67–7.35 (m, 4 H), 7.30–7.05 (m, 4 H), 4.72 (s, 1 H),
4.36 (d, J ) 6.0 Hz, 2 H), 3.65–3.35 (m, 2 H), 2.90 (s, 3 H), 2.82
(s, 3 H), 1.88 (dd, J ) 14.8, 4.0 Hz, 1 H), 1.66 (dd, J ) 14.8, 8.2
Hz, 1 H), 1.23 (d, J ) 6.8 Hz, 6 H), 1.40–1.05 (m, 4 H); HRMS
(ESI) calcd for C30H37FN4O5 [(M + H)+], 569.2769; found,
569.2778; HPLC (method C) tR ) 2.14 min (>99%).
(3R,5R)-7-[5-[(2-Fluoro-benzyl)-methyl-carbamoyl]-2-(4-fluoro-
phenyl)-4-isopropyl-2H-pyrazol-3-yl]-3,5-dihydroxy-heptanoic
1
Acid Sodium Salt (45). H NMR (DMSO-d6) δ 7.55–7.10 (m, 8
H), 4.74 (bs, 1 H), 4.70 (s, 1 H), 4.67 (s, 1 H), 3.65–3.61 (m, 1 H),
3.49–3.45 (m, 1 H), 2.92–2.91 (m, 4 H), 2.88–2.82 (m 1 H),
2.59–2.54 (m, 1 H), 1.92 (dd, J ) 14.8, 4.0 Hz, 1 H), 1.71 (dd, J
) 14.8, 8.2 Hz, 1 H), 1.42–1.21 (m, 3 H), 1.17–1.11 (m, 6 H);
HRMS (ESI) calcd for C28H33F2N3O5 [(M + H)+], 530.2461; found,
530.2450; Anal. (C28H33F2N3O5Na·1.8H2O) C, H, N; HPLC
(method A) tR ) 7.01 min (98.0%).
(3R,5R)-7-[5-(Benzyl-methyl-carbamoyl)-2-(4-fluoro-phenyl)-
4-isopropyl-2H-pyrazol-3-yl]-3,5-dihydroxy-heptanoic Acid So-
dium Salt (39). 1H NMR (DMSO-d6) δ 7.42–7.06 (m, 9 H), 4.61
(s, 1 H), 4.49 (s, 1 H), 3.87–3.71 (m, 4 H), 3.47–3.42 (m, 1 H),
2.80–2.69 (m, 3 H), 1.95 (dd, J ) 14.8, 4.0 Hz, 1 H), 1.77 (dd, J
) 14.8, 7.6 Hz, 1 H), 1.17 (d, J ) 7.2 Hz, 6), 1.57–1.21 (m, 4 H);
HRMS (ESI) calcd for C28H34FN3O5 [(M + H)+], 512.2555; found,
512.2545; Anal. (C28H33FN3O6Na) C, H, N; HPLC (method A) tR
) 6.79 min (98.2%).
(3R,5R)- 7-[5-(Benzyl-ethyl-carbamoyl)-2-(4-fluoro-phenyl)-
4-isopropyl-2H-pyrazol-3-yl]-3,5-dihydroxy-heptanoic Acid So-
dium Salt (40). 1H NMR (DMSO-d6) δ 7.47–7.21 (m, 9 H), 4.71
(bs, 1 H), 4.62 (s, 1 H), 4.47 (s, 1 H), 3.76–3.74 (m, 1 H), 3.59–3.56
(m, 1 H), 3.29–3.17 (m, 2 H), 2.87–2.85 (m, 1 H), 2.71–2.67 (m,
1 H), 2.57–2.52 (m, 1 H), 1.90 (dd, J ) 14.8, 4.0 Hz, 1 H), 1.70
(dd, J ) 14.8, 8.0 Hz, 1 H), 1.41–1.19 (m, 4 H), 1.16 (d, J ) 6.8
Hz, 6 H), 1.00 (t, J ) 5.2 Hz, 3 H); HRMS (ESI) calcd for
(3R,5R)-7-[5-[(3-Fluoro-benzyl)-methyl-carbamoyl]-2-(4-fluoro-
phenyl)-4-isopropyl-2H-pyrazol-3-yl]-3,5-dihydroxy-heptanoic
Acid Sodium Salt (46). H NMR (DMSO-d6) δ 7.55–7.10 (m, 8
H), 4.73 (bs, 1 H), 4.66 (s, 1 H), 4.55 (s, 1 H), 3.62–3.59 (m, 1 H),
3.48–3.45 (m, 1 H), 2.94–2.91 (m, 1 H), 2.88–2.82 (m 1 H), 2.86
(s, 3 H), 2.57–2.53 (m, 1 H), 1.91 (dd, J ) 14.8, 4.0 Hz, 1 H),
1.72 (dd, J ) 14.8, 8.0 Hz, 1 H), 1.43–1.15 (m, 4 H), 1.17 (d, J )
6.8 Hz, 6 H); HRMS (ESI) calcd for C28H33F2N3O5 [(M + H)+],
530.2461; found, 530.2448; HPLC (method A) tR ) 7.06 min
(96.2%).
1
(3R,5R)-7-[5-[(4-Fluoro-benzyl)-methyl-carbamoyl]-2-(4-fluoro-
phenyl)-4-isopropyl-2H-pyrazol-3-yl]-3,5-dihydroxy-heptanoic
1
Acid Sodium Salt (47). H NMR (DMSO-d6) δ 7.52–7.08 (m, 8
H), 4.71 (bs, 1 H), 4.60 (s, 1 H), 4.48 (s, 1 H), 3.61–3.58 (m, 1 H),
3.47–3.45 (m, 1 H), 2.89–2.86 (m, 1 H), 2.80 (s, 3 H), 2.70–2.61(m,
1 H), 2.57–2.54 (m, 1 H), 1.89 (dd, J ) 14.8, 4.0 Hz, 1 H), 1.69
(dd, J ) 14.8, 8.2 Hz, 1 H), 1.42–1.10 (m, 4 H), 1.16–1.11 (m, 6

HepatoselectiVe HMG-CoA Reductase Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1 43
H); HRMS (ESI) calcd for C28H33F2N3O5 [(M + H)+], 530.2461;
found, 530.2452; HPLC (method B) tR ) 1.57 min (99.0%).
(3R,5R)-7-[5-[(2,4-Difluoro-benzyl)-methyl-carbamoyl]-2-(4-
fluoro-phenyl)-4-isopropyl-2H-pyrazol-3-yl]-3,5-dihydroxy-hep-
tanoic Acid Sodium Salt (48). 1H NMR (DMSO-d6) δ 7.53–7.00
(m, 7 H), 4.71 (bs, 1 H), 4.63 (s, 1 H), 4.60 (s, 1 H), 3.61–3.58 (m,
1 H), 3.47–3.42 (m, 1 H), 2.87–2.84 (m, 4 H), 2.78–2.75 (m, 1 H),
2.69–2.65 (m, 1 H), 1.89 (dd, J ) 14.8, 4.0 Hz, 1 H), 1.71 (dd, J
) 14.8, 8.2 Hz, 1 H), 1.36–1.11 (m, 4 H), 1.13–1.09 (m, 6 H);
HRMS (ESI) calcd for C28H32F3N3O5 [(M + H)+], 548.2366; found,
548.2355; HPLC (method A) tR ) 7.37 min (>99%).
(3R,5R)-7-[5-[(2,3-Difluoro-benzyl)-methyl-carbamoyl]-2-(4-
fluoro-phenyl)-4-isopropyl-2H-pyrazol-3-yl]-3,5-dihydroxy-hep-
tanoic Acid Sodium Salt (49). 1H NMR (DMSO-d6) δ 7.48–7.02
(m, 7 H), 4.71–4.69 (m, 3 H), 3.59–3.57 (m, 1 H), 3.45–3.43 (m,
1 H), 2.91–2.84 (m, 4 H), 2.72–2.68 (m, 1 H), 2.58–2.53 (m, 1 H),
1.90 (dd, J ) 14.8, 4.0 Hz, 1 H), 1.69 (dd, J ) 14.8, 8.0 Hz, 1 H),
1.40–1.08 (m, 4 H), 1.14–1.09 (m, 6 H); HRMS (ESI) calcd for
C28H32F3N3O5 [(M + H)+], 548.2366; found, 548.2352; Anal.
(C27H30F3N3O5Na) C, H, N; HPLC (method A) tR ) 7.33 min
(98.6%).
(3R,5R)-7-{2-(4-Fluoro-phenyl)-4-isopropyl-5-[N-methyl-(R)-
r-methyl-benzylcarbamoyl]-2H-pyrazol-3-yl}-3,5-dihydroxy-
heptanoic Acid Sodium Salt (50). 1H NMR (DMSO-d6) δ
7.46–7.41 (m, 2 H), 7.35–7.10 (m, 7 H), 5.91 (t, J ) 6.8 Hz, 0.6
H), 5.29 (t, J ) 6.8 Hz, 0.4 H), 4.70 (bs, 1 H), 3.62–3.60 (m, 1 H),
3.45–3.42 (m, 1 H), 2.90–2.83 (m, 1 H), 2.73–2.69 (m, 1 H),
2.56–2.45 (m, 4 H), 1.93 (dd, J ) 14.8, 3.6 Hz, 1 H), 1.72 (dd, J
) 14.8, 8.4 Hz, 1 H), 1.47 (t, J ) 6.8 Hz, 3 H), 1.41–1.05 (m, 10
H); HRMS (ESI) calcd for C29H36FN3O5 [(M + H)+], 526.2711;
found, 526.2708; Anal. (C28H33FN3O5Na ·1.6H2O) C, H, N; HPLC
(method A) tR ) 7.33 min (97.4%).
(3R,5R)-7-{2-(4-Fluoro-phenyl)-4-isopropyl-5-[N-methyl-(S)-
r-methyl-benzylcarbamoyl]-2H-pyrazol-3-yl}-3,5-dihydroxy-
heptanoic Acid Sodium Salt (51). 1H NMR (DMSO-d6) δ
7.46–7.42 (m, 2 H), 7.35–7.12 (m, 7 H), 5.94 (t, J ) 6.4 Hz, 0.5
H), 5.24 (t, J ) 6.8 Hz, 0.5 H), 3.62–3.58 (m, 1 H), 3.45–3.34 (m,
1 H), 2.92–2.85 (m, 1 H), 2.71–2.65 (m, 1 H), 2.59–2.45 (m, 4 H),
1.90 (dd, J ) 14.8, 4.0 Hz, 1 H), 1.71 (dd, J ) 14.8, 8.2 Hz, 1 H),
1.47 (t, J ) 7.2 Hz, 3 H), 1.35–1.05 (m, 10 H); HRMS (ESI) calcd
for C29H36FN3O5 [(M + H)+], 526.2711; found 526.2700; HPLC
(method B) tR ) 1.73 min (>99%).
homogenized in a sucrose homogenizing solution in an ice bath.
Homogenates were diluted into a final volume of 200 mL and
centrifuged for 0.25 h with a Sorvall centrifuge at 5 °C, 10000
rpm (12000× G). The upper fat layer was removed and the
supernatant was decanted into fresh tubes. This step was repeated
twice prior to transferring the supernatant into ultracentrifuge tubes,
which were centrifuged at 36000 rpm (105000× G) for 1 h at 5
°C. The resulting supernatant was discarded and the pellet was
added to a total of 15 mL of 0.1 M KH2PO4. Pellets were then
homogenized gently by hand. Samples were pooled and diluted
into a total of 60 mL buffer. The protein concentration of the
homogenate was determined by the Lowry method using a BCA
(bicinchoninic acid) kit from Pierce Chemical Company. Aliquots
(1 mL) of microsomes were subsequently kept frozen in liquid
nitrogen.
HMG-CoA Reductase Assay Procedure. DMSO (1 µL) or
DMSO (1 µL) containing a test compound at a concentration of
between 0.1 to 1 nM was placed into each well of a Corning 96
well plate. A volume of 34 µL buffer [100 mM NaH2PO4, 10 mM
imidazole and 10 mM EDTA (ethylenediamine-tetraacetic acid)]
containing 35 µg/mL rat liver microsomes was added into each
well. After incubation for 0.5 h on ice, 15 µL of 14C-HMGCoA
(0.024 µCi) with 15 mM NADPH, 25 mM DTT, (dithiothreitol)
was added and incubated at 37 °C for an additional 0.75 h. The
reaction was terminated by the addition of 10 µL of 2 M HCl,
followed by the addition of 5 µL of 0.10 M mevalonolactone. Plates
were incubated at room temperature for 1 h to allow lactonization
of mevalonate to mevalonolactone. The incubated samples were
applied to columns containing 300 µL of AGI-X8 anion exchange
resin in a Corning filter plate. The eluates were collected into
Corning 96 well capture plates. Scintillation cocktail (Ultima-Flo-
M) was added into each well and plates counted on a Trilux
Microbeta Counter. The IC50 values were calculated with GraphPad
software (Prism).
Procedure for Sterol Biosynthesis Assay in Rat Hepatocytes.
Frozen rat hepatocyctes purchased from Xeno Tech were seeded
on a six-well collagen 1 coated plates at a density of 105 cells/
well. The cells were grown in DMEM (Dulbecco’s modified Eagle
medium, Gibco, 11054-020) containing 10% FBS (fetal bovine
serum) and 10 mM HEPES (N-2-hydroxyethyl-piperazie-N1-2-
ethane sulfonic acid, Gibco 15630-080) for 24 h. The cells were
preincubated with compounds for 4 h and then labeled by incubating
in medium containing 1 µCi/mL of 14C acetic acid for an additional
4 h. After labeling, the cells were washed twice with 5 nM MOPS
(3-[N-morpholino]propane sulfonic acid) solution containing 150
mM NaCl and 1 nM EDTA and collected in the lysis buffer
containing 10% KOH and 80% ethanol. To separate labeled
cholesterol from labeled noncholesterol lipids, the cells lysates were
subject to saponification at 60 °C for 2 h. The lysates were then
combined with 0.5 volume of H2O and 2 volumes of hexane,
followed by 0.5 h of vigorous shaking. After the separation of two
phases, the upper-phase solution was collected and combined with
5 volumes of scintillation cocktail. The amount of 14C cholesterol
was quantified by liquid scintillation counting. The IC50 values were
calculated with GraphPad software (Prism 3.03).
Procedure for Sterol Biosynthesis L6 Rat Myoblasts. L6 rat
myoblasts purchased from ATCC (CRL-1458) were grown in T-150
vented culture flasks and seeded on 12-well culture plates at a
density of 60000 cells per well. The cells were grown in DMEM
containing 10% heat inactivated FBS for 72 h until reaching
confluence. The cells were preincubated in media with compound
and 0.20% DMSO for 3 h and then labeled by incubating in medium
containing compound, 0.20% DMSO and 1 µCi/mL of 14C acetic
acid for an additional 3 h. After labeling, the cells were washed
once with PBS then lysed overnight at 4 °C in buffer containing
10% KOH and 78% ethanol. Lipid ester bonds were hydrolyzed
by saponification of the lysates at 60 °C for 2 h. Sterols (including
cholesterol) were extracted from saponified lysates by combining
with 3 volumes of hexane and mixing by pipet six times. The upper
organic phase solution was collected and combined with an equal
volume of 1 N KOH in 50% methanol and mixed by pipet six times.
(3R,5R)-7-[2-(4-Fluoro-phenyl)-4-isopropyl-5-(2-phenyl-pyr-
rolidine-1-carbonyl)-2H-pyrazol-3-yl]-3,5-dihydroxy-heptano-
ic Acid Sodium Salt (52). 1H NMR (DMSO-d6) [mixture of
diastereomers] δ 7.51–7.47 (m), 7.33–7.06 (m), 6.93–6.92 (m),
5.37–5.32 (m), 5.14–5.11 (m), 3.79–3.28 (m), 2.96–2.89 (m),
2.78–2.19 (m), 1.92–1.56 (m), 1.24–0.87 (m); HRMS (ESI) calcd
for C30H36FN3O5 [(M + H)+], 538.2711; found, 538.2700; HPLC
(method A) tR ) 7.01 min (>99%).
(3R,5R)-7-[2-(4-Fluoro-phenyl)-4-isopropyl-5-(2-phenyl-pip-
eridine-1-carbonyl)-2H-pyrazol-3-yl]-3,5-dihydroxy-heptanoic
Acid Sodium Salt (54). 1H NMR (DMSO-d6) [mixture of diaster-
eomers] δ 7.59–7.50 (m), 7.32–7.23 (m) 5.91–5.89 (m), 4.74–4.72
(m), 3.65–3.61 (m), 3.35–3.32 (m), 2.91–2.47 (m), 1.94–1.90 (m)
1.85–1.62 (m), 1.59–1.10 (m); HRMS (ESI) calcd for C31H38FN3O5
[(M + H)+], 552.2868; found, 552.2854; HPLC (method A) tR )
7.75 min (95.9%).
(3R,5R)-7-[2-(4-Fluoro-phenyl)-4-isopropyl-5-(3-phenyl-pip-
eridine-1-carbonyl)-2H-pyrazol-3-yl]-3,5-dihydroxy-heptanoic
1
Acid Sodium Salt (55). H NMR (DMSO-d6) δ 7.54–7.48 (m, 2
H), 7.24 (t, J ) 4.8 Hz, 1 H), 7.39–7.11 (m, 6 H), 4.73 (d, J ) 7.6
Hz, 1 H), 3.62 (dd, J ) 18.0, 4.0 Hz, 1 H), 3.79–3.68 (m, 2 H),
3.61–3.58 (m, 1 H), 3.25–3.23 (m, 1 H), 3.05–2.99 (m, 1 H),
2.91–2.79 (m, 1 H), 2.78–2.52 (m, 3 H), 1.98–1.85 (m, 2 H),
1.84–1.61 (m, 3 H), 1.55–1.10 (m, 10 H); HRMS (ESI) calcd for
C31H38FN3O5 [(M + H)+], 552.2868; found, 552.2851; HPLC
(method A) tR ) 7.74 min (98.0%).
Isolation of Rat Liver Microsomes. Male Charles River
Sprague–Dawley rats were fed with 2.5% cholestyramine in rat
chow diets for 7 days before sacrificing. Livers were minced and

44 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1
Pfefferkorn et al.
The upper organic phase was collected in a scintilant-coated plate,
and hexanes were removed by evaporation at room temperature
for 3 h. The amount of 14C cholesterol was quantified by
scintillation counting in a Triflux 1450 plate reader (Wallac). The
IC50 values were calculated from % inhibitions relative to negative
controls versus compound concentration on Microsoft excel 2000
data analysis wizard using a sigmoid inhibition curve model with
the following formula: y ) Bmax(1 - (Xn/Kn + Xn)) + Y2, where
K is the IC50 for the inhibition curve, X is inhibitor concentration,
Y is the response being inhibited, and Bmax + Y2 is the limiting
response as X approaches zero.
CO2 approximately 2 h postdose. Blood was collected in EDTA
anticoagulant for analysis of plasma drug levels, total cholesterol,
triglycerides, and lipid profile.
Acknowledgment. We would like to acknowledge the
technical contributions made by Fang Sun, Loola Al-Kassim,
Susan Holly, Cindy Spessard, and Siradanahalli Guru. We would
also like to thank Barry Finzel for his assistance with the X-ray
structure of compound 50.
Supporting Information Available: Elemental analysis or
HRMS and HPLC purity data for final compounds 2, 3, 32–52,
and 54, and 55. This material is available free of charge via the
Internet at http://pubs.acs.org.
Hamster Acute In Vivo Study Protocol..28 Male Golden Syrian
Hamsters were acquired from Charles River for use in these studies.
Upon delivery, the hamsters were randomly divided into treatment
and control groups of eight animals each and acclimated to a reverse
light cycle for 5–11 days. While acclimating to the reverse light
cycle, the hamsters were allowed to consume ad libitum normal
rodent chow (Research Diets, Purina 5001). Three days prior to
the date of the study, the hamsters were transferred to wire-bottom
flush caging, and the normal rodent chow was replaced by normal
rodent chow with the addition of 2.5% cholestyramine. Hamsters
were allowed access to food and water throughout the study period.
Each group (n ) 8) of hamsters were dosed with either vehicle
or vehicle plus test compound via oral gavage at a volume
equivalent to 0.25% body weight. Either 2 (t ) 2–4 h assay) or 4 h
(t ) 4– 6h assay) postdose, each animal was injected (i.p.) with 80
µCi of 14C sodium acetate (Amersham Biosciences, CFQ30013)
in 1 mL of 0.9% NaCl. At 4 (t ) 2–4 h assay) or 6 h (t ) 4–6 h
assay) postdose, the hamsters were euthanized via CO2, followed
by blood collection via cardiac stick. Each sample of blood collected
was centrifuged for 10 min at 3000 RPM at 5 °C. After separation,
400 µL of plasma were removed from each tube and placed in a
16 × 100 M culture tube for future saponification and extraction
of cholesterol esters.
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To each 400 µL sample of plasma were added 500 µL of saline,
100 µL of 3H cholesterol (internal standard for extraction), and
2.5 mL of 10% KOH. After vortexing, the rack of sample tubes
was placed in a water bath preheated to 75 °C for one hour to
complete the saponification process. The sample rack was removed
from the water bath after one hour and allowed to cool for
approximately 15 min. At this point, 2.5 mL of petroleum ether
was added to extract the cholesterol esters. The rack was placed
on a shaker, and the solutions were mixed for 10 min. The tubes
were then centrifuged for 10 min at 3000 RPM and 5 °C to separate
the organic and aqueous phases. Using a 2500 µL pipettor and
proceeding in groups, 1.2 mL of the organic supernatant was drawn
from each culture tube and expressed into a correspondingly
numbered 7 mL scintillation vial. Each vial had 5.1 mL of
scintillation fluid added to it and was capped for further analysis.
Included in the sample analysis were three 7 mL scintillation vials
containing 6.3 mL of scintillation fluid serving as blanks, three 7
3
mL scintillation vials containing 100 µL of H cholesterol added
to 6.3 mL of scintillation fluid to provide the denominator in the
calculation of the percent extraction of the internal standard, and 2
µL of the 80 µCi of 14C sodium acetate/0.9% NaCl solution in 6.3
mL of scintillation fluid to measure consistency of count between
aliquots.
Seven-Day Guinea Pig LDL-Lowering Assay. Male Hartley
guinea pigs (300–500 g initial body weight) were obtained from
Charles River, pair housed in guinea pig caging under a 12 h light
and dark cycle, and given Guinea Pig Laboratory Diet 5025 and
water ad libitum. Animals were allowed to acclimate in house for
at least 1 week prior to the study. During the experiment, the
animals were fed guinea pig chow (Laboratory diet 5025). Prior to
the start of dosing, animals were identified using implantable chips
(Bio Medic Data Inc., model IMI-1000). Body weights were
obtained at the start of each week to calculate daily doses. Animals
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