Discovery of pyrrole-based hepatoselective ligands as potent inhibitors of HMG-CoA reductase

Article abstract of DOI:10.1016/j.bmc.2007.05.031

In an effort to identify hepatoselective inhibitors of HMG-CoA reductase, two series of pyrroles were synthesized and evaluated. Efforts were made to modify (3R,5R)-7-[3-(4-fluorophenyl)-1-isopropyl-4-phenyl-5-phenylcarbamoyl-1H-pyrrol-2-yl]-3,5-dihydroxy

Full text of DOI:10.1016/j.bmc.2007.05.031

Bioorganic & Medicinal Chemistry 15 (2007) 5576–5589
Discovery of pyrrole-based hepatoselective ligands
as potent inhibitors of HMG-CoA reductase
Larry D. Bratton,* Bruce Auerbach, Chulho Choi, Lisa Dillon, Jeffrey C. Hanselman,
Scott D. Larsen, Gina Lu, Karl Olsen, Jeffrey A. Pfefferkorn, Andrew Robertson,
Catherine Sekerke, Bharat K. Trivedi and Paul C. Unangst
Pfizer Global Research and Development, Michigan Laboratories, Ann Arbor, MI 48105, USA
Received 13 March 2007; revised 6 May 2007; accepted 10 May 2007
Available online 17 May 2007
Abstract—In an effort to identify hepatoselective inhibitors of HMG-CoA reductase, two series of pyrroles were synthesized and
evaluated. Efforts were made to modify (3R,5R)-7-[3-(4-fluorophenyl)-1-isopropyl-4-phenyl-5-phenylcarbamoyl-1H-pyrrol-2-yl]-
3,5-dihydroxy-heptanoic acid sodium salt 30 in order to reduce its lipophilicity and therefore increase hepatoselectivity. Two strat-
egies that were explored were replacement of the lipophilic 3-phenyl substituent with either a polar function (pyridyl series) or with
lower alkyl substituents (lower alkyl series) and attachment of additional polar moieties at the 2-position of the pyrrole ring. One
compound was identified to be both highly hepatoselective and active in vivo. We report the discovery, synthesis, and optimization
of substituted pyrrole-based hepatoselective ligands as potent inhibitors of HMG-CoA reductase for reducing low density lipopro-
tein cholesterol (LDL-c) in the treatment of hypercholesterolemia.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
be beneficial for restoring endothelial function, stimulat-
ing bone formation, decreasing vascular inflammation,
and enhancing the stability of plaques associated with
atherosclerosis.4,5
Coronary heart disease (CHD) is the leading cause of
death in most industrialized countries and affects 12–
15 million people in the USA alone.1 The major cause
of CHD is hypercholesterolemia or elevated serum cho-
lesterol levels, particularly non-high density lipoprotein
cholesterol (non-HDL-c), which is most effectively
lowered by the use of statin drugs such as; simvastatin,
atorvastatin, and rosuvastatin.2 Statins work by inhibit-
ing 3-hydroxy-3-methylglutaryl CoA (HMG-CoA)
reductase, the rate limiting enzyme involved in the bio-
synthesis of cholesterol. When cholesterol biosynthesis
is inhibited, the low density lipoprotein cholesterol
(LDL-c) receptor is upregulated and LDL-c is rapidly
cleared from the bloodstream.3 In addition to lowering
LDL-c, statins have been shown to lower very low den-
sity lipoprotein cholesterol (VLDL-c) and triglycerides,
and sometimes raise HDL-c. Furthermore, results from
multiple clinical studies have indicated that statins may
An adverse side effect occasionally associated with all
statins is myalgia, mild muscle pain or weakness which
generally increases with higher doses of the drug.6,7 In
rare cases, statins can cause rhabdomyolysis, a severe
form of myopathy or muscle toxicity, which prompted
cerivastatin’s removal from the market.8 In an effort to
reduce the potential for myalgia, there has been in-
creased emphasis on the design of more hepatoselective
HMG-CoA reductase inhibitors. It is anticipated that
such hepatoselectivity might reduce systemic exposure
and therefore limit a compound’s effect on muscle cells.
One strategy for obtaining hepatoselectivity is to lower
the lipophilicity of the drug. As an affirmation of this
strategy, rosuvastatin (logD = À0.33), the most potent
and hydrophilic statin on the market, was shown in
the clinic to be much more liver-selective than the more
lipophilic statins, simvastatin and cerivastatin
(logD > 1.5).9,10 The basis for greater hepatoselectivity
of hydrophilic statins is presumably due to decreased
passive permeability into non-hepatic cells, while active
uptake into hepatocytes via the organic anion transport-
Keywords: Hepatoselective HMGCoA reductase inhibitors; Substi-
tuted pyrroles; Synthesis; Pyrroles with pyridyl groups; Pyrroles with
lower alkyl substituents.
*
Corresponding author. E-mail: woffendogtoo@chartermi.net
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.05.031

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5577
To obtain a more hepatoselective statin, the lipophilicity
of 30 was reduced by replacing the 4-phenyl substituent
with either a polar pyridyl function or a lower alkyl moi-
ety. Lipophilicity was further reduced by functionalizing
the phenyl group in the amide position with polar sub-
stituents. Herein these changes in 30 will be the focus
of our discussion. The synthesis and hepatoselectivity
profile will also be described.
Figure 1. Compound 30.
ing polypeptides is maintained. To obtain a more potent
and hepatoselective statin, chemical modifications were
implemented on atorvastatin because of its overall effi-
cacy and safety profile compared to other statins cur-
rently on the market. Other coworkers in our group
began making modifications on the core pyrole template
by shifting the nitrogen atom over by one carbon unit
while retaining the same functionality as in atorvastatin.
This modification led to the preparation of 30, which
was found to be quite potent as an inhibitor of choles-
terol in rat hepatocytes (IC50 = 0.43 nM), but did not ex-
hibit the hepatoselectivity profile that we were searching
2. Chemistry
As outlined in Scheme 1, the preparation of the various
pyridyl substituted pyrroles commenced with a Knoeve-
nagel condensation of 2-pyridylacetonitrile 1 and 4-flu-
orobenzaldehyde to generate cyano-styrene 2. Pyrrole
formation was accomplished by treatment of 2 with
ethyl isocyanoacetate in the presence of potassium
tert-butoxide to afford pyrrole 3.12 Subsequently, N-
alkylation of 3 using isopropyl iodide and potassium
hydroxide provided N-isopropyl pyrrole 4. Reduction
11
for (see Fig. 1).
Scheme 1. Reagents and conditions: (a) 4-fluorobenzaldehyde, NaOEt, EtOH, 25 ꢁC, 92%; (b) ethylisocyanoacetate, potassium tert-butoxide, THF,
0 ꢁC, 88%; (c) KOH, i-propyl iodide, DMSO, 25 ꢁC, 74%; (d) LAH, THF, À10 ꢁC, 97%; (e) Ph3PHBr, HCl, DCM, 25 ꢁC, 99%; (f) NaHMDS,
THF:DMSO, 78 ꢁC, 66%; (g) Pd/C, MeOH, 25 ꢁC, 53%; (h) NIS, DMF, 25 ꢁC, 92%.

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L. D. Bratton et al. / Bioorg. Med. Chem. 15 (2007) 5576–5589
of the ethyl ester of pyrrole 4 to the corresponding alco-
hol 5 was accomplished using lithium aluminum hy-
dride. Exposure of alcohol 5 to Ph3PHBr in the
presence of hydrochloric acid resulted in facile conver-
sion to triphenyl phosphonium bromide 6, which was
then engaged in a Wittig olefination reaction with alde-
hyde 7 to generate olefin 8 as an inconsequential mixture
of cis/trans stereoisomers.13 The olefin of intermediate 8
was reduced by catalytic hydrogenation to afford 9,
which was then iodinated with N-iodosuccinimide to
provide iodopyrrole 10 as a key intermediate for the
preparation of analogs 11–15 as highlighted in
Scheme 2. Treatment of iodopyrrole 10 with copper
cyanide and potassium cyanide at elevated temperature
in N,N-dimethylformamide followed by acetonide
removal and subsequent base hydrolysis provided
compound 11. Separately, palladium-mediated carbony-
lative aminations of 10 with various amines (NH3,
EtNH2, PhNH2, and PhCH2NH2)14 afforded, after
deprotection and hydrolysis, amide analogs 12–15.
22, which was introduced into the next reaction without
purification. Synthesis of the various substituted amide
aldehydes 23a–e was investigated using two different
methods starting from their common acid chloride
intermediate.
For example, the formamide aldehyde 23a was prepared
under modified Shotten–Baumann conditions from 22
and ammonia in aqueous ethyl acetate with sodium car-
bonate as base. Initial attempts to prepare amide alde-
hydes 23b–e under these conditions failed to give the
desired products, but instead, resulted in the formation
of a significant amount of either an imine or decarbox-
ylated side product. The remaining amides were eventu-
ally prepared in 54–73% yields when the amidations
were conducted in dichloromethane using N,N-diisopro-
pylethyl amine. Amide aldehydes 23a–e were treated
with the chiral ylide 2418 in refluxing toluene for 3 days
to provide the corresponding Wittig products 25a–e as
inseparable mixtures of cis and trans isomers.18 Reac-
tion times were long and yields were relatively low pre-
sumably due to the steric nature surrounding the
aldehyde function. Cleavage of the silyl ether function
in 25a–e to the alcohol amides 26a–e was accomplished
using either 48% hydrofluoric acid in acetonitrile or HF-
pyridine in tetrahydrofuran.19,20 HF-pyridine proved to
be the reagent of choice when longer reaction times were
required because it appeared to retard the formation of
uncharacterized side products as indicated by thin layer
chromatography. In either case, yields were greater
when the starting materials were consumed in less than
2 h. Stereoselective reduction of the unsaturated ketone
in 26a–e with sodium borohydride, via chelation-control
with diethylmethoxyborane, in a mixture of methanol
and THF gave unsaturated diols 27a–e in 82–88%
yields.20 Reduction of 27a–e to the saturated diols
28a–e was achieved under catalytic hydrogenation con-
ditions over a period of 45 min to 2 h at low hydrogen
pressure. High pressure conditions (50 psi) produced
Preparation of the lower alkyl pyrroles began with com-
mercially available pyrrole ester 16 (Scheme 3), which
was brominated with bromine under basic conditions
to give the brominated pyrrole 17.15 Suzuki cross cou-
pling of 17 with 4-fluoroboronic acid using tetrakis(tri-
phenylphosphine)palladium(0) as catalyst provided the
aryl pyrrole 18 in 74% yield.16 Oxidation of the methyl
substituent to the aldehyde was achieved under acidic
conditions with ceric ammoniumnitrate as the oxidizing
agent to provide the aldehyde pyrrole 19.16,17 The ethyl
group was introduced by treatment of 19 with ethyl io-
dide and cesium carbonate to afford the N-alkylated
pyrrole 20. Saponification of the ester function with lith-
ium hydroxide monohydrate at reflux, followed by acid-
ification with hydrochloric acid, provided the acid
aldehyde 21.17 Acid aldehyde 21 was treated with oxalyl
chloride and a catalytic amount of N,N-dimethylform-
amide in dichloromethane to afford the acid chloride
Scheme 2. Reagents and conditions: (a) CuCN, KCN, DMF, 120 ꢁC, 64%; (b) HCl, MeOH, 25 ꢁC, 49%; (c) NaOH, MeOH, 25 ꢁC, 91%;
(d) i—RNH2, CO (400 psi), Pd(Ph3P)2Cl2, toluene, 100 ꢁC; ii—HCl, MeOH, 25 ꢁC; iii—NaOH, MeOH, 25 ꢁC, 5–34% overall yield over three steps.

L. D. Bratton et al. / Bioorg. Med. Chem. 15 (2007) 5576–5589
5579
Scheme 3. Reagents and conditions: (a) Br2, pyridine, DCM, 5 ꢁC, 81%; (b) 4-fluorophenyl boronic acid, aq DMF, Na2CO3, Pd(PPh3)4, reflux, 71%;
(c) (NH4)2Ce(NO3)6, acetic acid, aq THF, 25 ꢁC, 65%; (d) EtI, Cs2CO3, acetonitrile, 25 ꢁC, 87%; (e) i—LiOH–H2O, aq THF, reflux, ii—aq HCl, 99%;
(f) oxalyl chloride, DMF (cat), DCM, 25 ꢁC, 100%; (g) R2NH2; (i-Pr)2NEt, DCM, 0–25 ꢁC, 54–73%; (h) NH3, Na2CO3, MeOH, aq ethyl acetate,
À5 ꢁC, 56%.
some side product identified as the monoalcohol of 28a–
e due to reductive elimination of the hydroxyl group at
the 5-position of the side chain. However, shorter reac-
tion times and lower hydrogen pressure (5 psi) mini-
mized the formation of this unwanted side product.
Finally, saponification of 28a–e with sodium hydroxide
in methanol at room temperature provided the target
carboxylates 29a–e in 62–95% yields. Similarly, 28d
was converted to the target dicarboxylate 29f using ex-
cess base in a mixture of methanol and water at reflux
(Scheme 4).
showed good hepatocyte activity suggesting that they
may be actively transported. The lower alkyl analogs
29c–f displayed the same lipophilic-hepatoselective
trend as above. The only exception was found for com-
pounds 29a and 29b. For 29a loss in selectivity was due
to decreased potency in liver hepatocytes. This loss in
potency is not surprising since optimal binding to the
receptor was not achieved due to the absence of the large
phenyl group in the amide position. Similarly, dimin-
ished potency was also demonstrated for analogs 11–
13, which also lack the large lipophilic functionality in
the 2-position of the pyrole ring.
3. Biology
Most of the compounds shown in Table 1 were tested
in vivo using the mouse acute inhibition of cholesterol
synthesis (MAICS) model. Two hepatoselective analogs
11 and 12 in the pyridyl series were found to maintain
excellent in vivo activity. In the lower alkyl series, com-
pound 29d was the only analog to show modest in vivo
activity (>60% inhibition) at the dose tested.
All new analogs were evaluated in a microsomal HMG-
CoA reductase assay as well as in both hepatocyte and
myocyte cellular assays.21 The ratio of inhibition of cho-
lesterol synthesis in heptaocytes versus myocytes was
used to determine the hepatoselectivity of individual
analogs. Compounds with sufficient in vitro potency
and selectivity were then evaluated in an in vivo efficacy
model for measuring inhibition of cholesterol synthesis.
For the analogs 11–13 and 15 containing the pyridyl
function, there was a correlation between lipophilicity
and hepatoselectivity (Table 1). More polar compounds
were less likely to inhibit cholesterol biosynthesis in
muscle myocytes. This is likely a result of poor mem-
brane permeability. In contrast, such compounds
4. Conclusion
In summary, we have demonstrated that the hepatose-
lectivity of pyrrole-based HMG-CoA reductase inhibi-
tors can in general be increased by decreasing their
lipophilicity, consistent with literature precedent for
other statins. Although most of the new compounds

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L. D. Bratton et al. / Bioorg. Med. Chem. 15 (2007) 5576–5589
Scheme 4. Reagents and conditions: (a) toluene, reflux, 53–67%; (b) HF–pyridine, THF, 25 ꢁC, 35–68%; (c) 48% HF, acetonitrile, 5–25 ꢁC, 70–74%;
(d) diethylmethoxyborane, NaBH4, 4:1 THF/MeOH, À60 ꢁC, 82–88%; (e) 10% Pd/C, H2 (5 psi), 1:1 THF/MeOH, 48–61%; (f) NaOH, aq MeOH,
25 ꢁC, 62–95%; (g) excess NaOH, aq MeOH, reflux, 57%.
Table 1. Inhibition of cholesterol synthesis in rat liver microsomes, rat hepatocytes, and L6 myocytes in order of decreasing hepato selectivity and
mouse acute inhibition of cholesterol synthesis
a
RM IC50 (nM)
a
HEP IC50 (nM)
a
L6 IC50 (nM)
a
L6/HEP IC50 (nM)
Compound
ClogD (pH 7.4)
MAICS (%)
Ros
30
À0.338
3.6
12
0.23
0.43
1.3
720
157
229
7
3130
374
175
4
À82
À75
À44
À92
0.31
Sim
Cer
>1.58
>1.58
18
2.8
1.7
Pyridyl
12
13
Analogs
À1.83
À1.13
À0.75
0.42
5.3
21
4.9
5.1
18,609
10,821
1730
28
3798
2122
824
À73
nt
11
15
4.8
3.7
(2.4)
(2.1)
0.15
0.17
À72
nt
187
165
14
À1.04
28
nt
Lower
29f
29e
Alkyl
À1.15
À1.16
0.80
Analogs
0.85
4.7
(0.11)
0.35
0.24
1760
5225
184
16,000
14,929
770
À27
À52
nt
29c
1.6
30
29a
29b
29d
À1.36
À0.57
0.86
32
(0.29)
(0.28)
22,701
102
26
709
349
nt
2.8
1.6
À53
À66
93
a Values in parentheses are given as an average of 2–3 experiments, Sim, simvastatin; Ros, rosuvastatin; Cer, cerivastatin; nt, not tested; ns, not
selective; RM, HEP, and L6 are inhibition of cholesterol synthesis: of rat microsomal HMG CoA Reductase, in rat liver hepatocytes, and in rat
muscle myocytes, respectively; L6/HEP, liver selective cholesterol inhibition; MAICS, mouse acute inhibition of cholesterol synthesis. All com-
pounds were tested at a dose of 30 mg/kg in the MAICS assay with the exception of the following compounds: Ros, 10 mg/kg and 29e, 100 mg/kg.

L. D. Bratton et al. / Bioorg. Med. Chem. 15 (2007) 5576–5589
5581
had in vivo activity inferior to the less hepatoselective
lead 30, we were successful in identifying one compound
12 with hepatoselectivity 10-fold higher than 30 that
maintained robust in vivo activity.
ether (300 mL) and water (100 mL) were then added and
the organic layer was separated, dried (Na2SO4), and
concentrated to give a crude oil, which was purified by
silica gel chromatography (10–40% EtOAc/hexane) to
give the title compound (23.2 g, 74%): MS(APCI+): m/z
353 (M+H);
H
NMR (DMSO-d6)
d
8.42 (d,
5. Experimental
J = 4.0 Hz, 1H), 7.79 (s, 1H), 7.47–7.43 (m, 1H), 7.20–
7.04 (m, 5H), 6.65 (d, J = 12 Hz, 1H), 5.27 (sept,
J = 7.9 Hz, 1H), 3.91 (q, J = 8.0 Hz, 2H), 1.46 (d,
J = 8.0 Hz, 6H), 0.81 (t, J = 8.0 Hz, 3H).
Melting points were determined on an Electro thermal
Melting Point Apparatus and are uncorrected. Samples
were characterized on a 400 MHz Nuclear Magnetic
Resonance Spectrometer using either deuterated
dimethylsulfoxide or deuterated chloroform. Mass spec-
tra were determined on an LC Platform Mass Spectrom-
eter using Chemical Ionization. A Rotoray Evaporator
was used to remove solvents under reduced pressure.
The precursor to intermediate 7 was purchased from
Kaneka. Compound 24 was purchased from Bridge
Organics. Standard sodium hydroxide solutions were
purchased from Aldrich. All other reagents and starting
materials were obtained from commercially available
sources.
5.3. [3-(4-Fluorophenyl)-1-isopropyl-4-pyridin-2-yl-1H-
pyrrol-2-yl]-methanol (5)
To a solution of 4 (6.50 g, 18.4 mmol) in THF (120 mL)
at À10 ꢁC was slowly added lithium aluminum hydride
(46.1 mL of 1.0 M in Et2O, 46.1 mmol). The reaction
mixture was stirred at À10 ꢁC for 1 h after which time
it was carefully quenched by slow addition of saturated
NH4Cl. Once the quench was complete, water was
slowly added and the reaction mixture was extracted
with ethyl acetate. The organic layer was dried over
Na2SO4 and concentrated. The product was purified
by silica gel chromatography (50–75% EtOAc/hexane)
to afford the title compound (5.54 g, 97%) as a white so-
lid: MS(APCI+): m/z 311 (M+H); H NMR (CDCl3) d
8.47 (d, J = 7.6 Hz, 1H), 7.37 (br s, 1H), 7.30 (t,
J = 14.0 Hz, 1H), 7.22–7.17 (m, 3H), 7.04–6.92 (m,
3H), 6.69 (d, J = 8.0 Hz, 1H), 4.59 (sept, J = 6.8 Hz,
1H), 4.49–4.48 (m, 2H), 1.51 (d, J = 6.8 Hz, 6H).
5.1. (Z)-3-(4-Fluorophenyl)-2-pyridin-2-yl-acrylonitrile
(2)
To
a
solution of 4-fluorobenzaldehyde (52.5 g,
423 mmol) in EtOH (200 mL) at 25 ꢁC were added pyri-
din-2-yl-acetonitrile 1 (50.0 g, 423 mmol) and NaOEt
(151 g of 21% solution, 466 mmol). The reaction mixture
was stirred at 25 ꢁC for 0.5 h during which time a light
brown precipitate developed. The solid was isolated by
filtration and washed with EtOH (75 mL). The product
was then dried under vacuum to afford the title com-
pound (87 g, 92%), which was used without further puri-
fication: MS(APCI+): m/z 225 (M+H); H NMR (CDCl3)
d 8.62 (d, J = 4.0 Hz, 1H), 8.40 (s, 1H), 8.06–8.01 (m,
2H), 7.92–7.88 (m, 1H), 7.79 (d, J = 12.0 Hz, 1H),
7.41–7.33 (m, 3H).
5.4. ((4R,6S-6-{(E,Z)-2-[3-(4-Fluorophenyl)-1-isopropyl-
4-pyridin-2-yl-1H-pyrrol-2-yl]-vinyl}-2,2-dimethyl-[1,3]-
dioxin-4-yl)-acetic acid tert-butyl ester (8)
To a solution of 5 (3.15 g, 10.1 mol) in CH2Cl2 (150 mL)
were added triphenylphosphine hydrobromide (3.48 g,
10.2 mmol) and HCl (5.1 mL of 2.0 M solution in
Et2O, 10.1 mmol). The reaction mixture was stirred at
25 ꢁC for 1 h after which time all starting material was
consumed as determined by TLC. The organic layer
was then washed with saturated NaHCO3 and dried
over Na2SO4. The organic layer was then concentrated
under reduced pressure and dried under high vacuum
for 12 h to afford [3-(4-fluorophenyl)-1-isopropyl-4-pyri-
din-2-yl-1H-pyrrol-2-ylmethyl]-triphenylphosphonium
bromide 6 (6.36 g, 99%) as a yellow solid. To a solution
of 6 (6.00 g, 8.93 mmol) in THF/DMSO (500 mL, 25:1)
at À78 ꢁC was added NaHMDS (9.12 mL of a 1.0 M
solution in THF, 9.12 mmol). An orange color was
noted as the base was added to the reaction mixture.
The reaction mixture was stirred at À78 ꢁC for 5 min
after which time a solution of 7 (2.16 g, 8.35 mmol) in
THF (20 mL) was added. The reaction mixture was stir-
red at À78 ꢁC for 0.5 h and then allowed to warm to
25 ꢁC over 1.5 h. The reaction was quenched by addition
of saturated NH4Cl. Ethyl acetate was then added and
the organic layer was washed with water, dried
(Na2SO4), and concentrated. The resulting oil was puri-
fied by silica gel chromatography (20–25% EtOAc/hex-
ane) to provide the title compound (2.69 g, 66%) as a
mixture of cis/trans isomers: MS(APCI+): m/z 535
(M+H); H NMR [mixture cis/trans isomers] (CDCl3) d
5.2. 3-(4-Fluorophenyl)-1-isopropyl-4-pyridin-2-yl-1H-
pyrrole-2-carboxylic acid ethyl ester (4)
A solution of 2 (25.0 g, 112 mmol) and ethyl isocyanoac-
etate (12.3 mL, 112 mmol) in THF (300 mL) was slowly
added to a solution of KOt-Bu (223 mL of 1.0 M solu-
tion, 223 mmol) in THF (100 mL) at 0 ꢁC. The resulting
reaction mixture was stirred at 0 ꢁC for 1.5 h after which
time TLC indicated that the reaction was complete. The
reaction mixture was transferred to a separatory funnel
and ethyl acetate (500 mL) and water (200 mL) were
added. The organic layer was separated, washed with
brine, and dried over Na2SO4. Upon concentration of
the organic layer, the crude product solidified to give
3-(4-fluorophenyl)-4-pyridin-2-yl-1H-pyrrole-2-carbox-
ylic acid ethyl ester 3 (30.5 g, 88%) as a brown solid,
which was utilized without further purification. To a
solution of 3 (30.5 g, 98.3 mmol) in DMSO (100 mL)
at 25 ꢁC was added powdered KOH (24.8 g, 442 mmol)
and the reaction mixture was stirred at 25 ꢁC for 0.5 h.
Subsequently, 2-iodopropane (26.5 mL, 265 mmol) was
added dropwise to the suspension and the reaction mix-
ture was stirred for an additional 0.5 h at 25 ꢁC. Diethyl

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L. D. Bratton et al. / Bioorg. Med. Chem. 15 (2007) 5576–5589
8.51–8.49, 7.36–7.13, 7.01–6.94, 6.71–6.61, 6.36–6.26,
5.58–5.53, 5.37–5.35, 4.29–4.28, 4.10–4.07, 2.37–2.35,
2.26–2.29, 2.12–2.03, 1.49–1.34, 1.21–1.11.
by silica gel chromatography (50% EtOAc/hexane) to
give (3R,5R)-7-[5-cyano-3-(4-fluorophenyl)-1-isopropyl-
4-pyridin-2-yl-1H-pyrrol-2-yl]-3,5-dihydroxyheptanoic
acid tert-butyl ester (0.082 g, 49%). To a solution of this
compound in MeOH (10 mL) was added 1.03 N NaOH
(0.159 mL, 0.163 mmol) and the reaction was stirred at
25 ꢁC for 48 h. The reaction mixture was then concen-
trated and azeotroped with toluene (3· 25 mL). The
product was dried under vacuum at 60 ꢁC to give the ti-
tle compound (0.069 g, 91%): MS(APCI+): m/z 466
(M+H); H NMR (DMSO-d6) d 8.49 (d, J = 7.6 Hz,
1H), 7.58–7.49 (m, 2H), 7.19–7.15 (m, 1H), 7.12–7.07
(m, 3H), 6.84 (d, J = 9.2 Hz, 1H), 4.79 (br s, 1H), 4.67
(sept, J = 6.8 Hz, 1H), 4.24 (m, 1H), 3.67–3.65 (m,
1H), 3.27–3.21 (m, 1H), 2.65–2.61 (m, 1H), 2.47–2.42
(m, 1H), 1.93 (dd, J = 15.2, 4.0 Hz, 1H), 1.72 (dd,
J = 15.2, 8.4 Hz, 1H), 1.58–1.16 (m, 10H).
5.5. ((4R,6R)-6-{2-[3-(4-Fluorophenyl)-1-isopropyl-4-
pyridin-2-yl-1H-pyrrol-2-yl]-ethyl}-2,2-dimethyl-
[1,3]dioxin-4-yl)-acetic acid tert-butyl ester (9)
To a solution of 8 (3.11 g, 5.82 mmol) in MeOH
(100 mL) was added 10% Pd/C (300 mg). The reaction
vessel was then evacuated and treated with hydrogen
(50 psi) for 12 h at 25 ꢁC. The reaction mixture was then
filtered through a pad of Celite filter aid and the filtrate
was concentrated. The resulting oil was purified by silica
gel chromatography (30–50% EtOAc/hexane) to provide
the title compound (1.65 g, 53%): MS(APCI+): m/z 537.7
(M+H); H NMR (CDCl3) d 8.45 (d, J = 6.8 Hz, 1H),
7.33–7.14 (m, 5H), 7.01–6.88 (m, 3H), 6.65 (d,
J = 8.0 Hz, 1H), 4.33 (sept, J = 6.8 Hz, 1H), 4.12–4.09
(m, 1H), 3.65–3.61 (m, 1H), 2.65–2.61 (m, 1H), 2.51
(dd, J = 15.2, 7.8 Hz, 1H), 2.49–2.46 (m, 1H), 2.32 (dd,
J = 15.2, 4.0 Hz, 1H), 1.61–1.21 (m, 24H).
5.8. (3R,5R)-7-[5-carbamoyl-3-(4-fluoro-phenyl)-1-iso-
propyl-4-pyridin-2-yl-1H-pyrrol-2-yl]-3,5-dihydroxy-
heptanoic acid sodium salt (12)
A high pressure reactor was charged with 10 (0.465 g,
0.702 mmol), Pd(PPh3)2Cl2 (0.064 g), and toluene
(25 ml). The reactor was pressurized with ammonia
(85 psi) and CO (400 psi) and then heated to 100 ꢁC for
15 h. After cooling to 25 ꢁC, the reaction solvent was re-
moved under reduced pressure and the resulting residue
was purified by silica gel chromatography to give (6-{2-
[5-carbamoyl-3-(4-fluorophenyl)-1-isopropyl-4-pyridin-
2-yl-1H-pyrrol-2-yl]-ethyl}-2,2-dimethyl-[1,3]dioxan-4-yl)-
acetic acid tert-butyl ester (0.103 g, 25%). To a solution of
this compound in MeOH (5 mL) was added 1 N HCl
(0.533 mL, 0.533 mmol). The reaction mixture was stirred
at 25 ꢁC for 2 h after which time the solvent was removed
by evaporation and ethyl acetate (20 mL) was added. The
organic layer was washed with saturated NaHCO3, water,
and brine prior to drying over Na2SO4. After concentra-
tion in vacuo, the product was purified by silica gel chro-
matography (50–100% EtOAc/hexane) to give (6-{2-[5-
carbamoyl-3-(4-fluorophenyl)-1-isopropyl-4-pyridin-2-yl-
1H-pyrrol-2-yl]-ethyl}-2,2-dimethyl-[1,3]dioxan-4-yl)-
acetic acid tert-butyl ester (0.057 g, 59%). To a solution of
this compound in MeOH (5 mL) was added 1.02 N
NaOH (0.108 mL, 0.111 mmol) and the reaction mixture
was stirred at 25 ꢁC for 72 h. The reaction mixture was
concentrated in vacuo and azeotroped with toluene
(3· 25 mL). The product was dried under vacuum at
60 ꢁC to give the title compound as a white solid
(0.051 g, 96%): MS(APCI+): m/z 484 (M+H); H NMR
(DMSO-d6) d 8.40 (d, J = 7.6 Hz, 1H), 7.59–7.44 (m,
3H), 7.11–6.83 (m, 6 H), 4.74 (br s, 1H), 4.64 (sept,
J = 6.8 Hz, 1H), 4.22 (m, 1H), 3.66–3.62 (m, 1H), 3.51–
3.47 (m, 1 H), 2.66–2.62 (m, 1H), 2.49–2.43 (m, 1H),
1.93 (dd, J = 15.2, 4.0 Hz, 1H), 1.72 (dd, J = 14.8,
8.4 Hz, 1H), 1.53–1.14 (m, 10H).
5.6. ((4R,6R)-6-{2-[3-(4-Fluorophenyl)-5-iodo-1-isoprop-
yl-4-pyridin-2-yl-1H-pyrrol-2-yl]-ethyl}-2,2-dimethyl-
[1,3]dioxin-4-yl)-acetic acid tert-butyl ester (10)
To a solution of 9 (0.80 g, 1.49 mmol) in DMF (8 mL)
at 25 ꢁC was added N-iodosuccinimide (0.309 g,
1.79 mmol). The reaction mixture was stirred at 25 ꢁC
for 1.5 h after which time DCM (50 mL) and saturated
NaHCO3 (50 mL) were then added and the organic layer
was separated, washed with brine, and dried over
Na2SO4. The organic layer was concentrated and the
product was purified by silica gel chromatography
(10% EtOAc/hexane) to give the title compound
(0.912 g, 92%): MS(APCI+): m/z 663 (M+H); H NMR
(CDCl3) d 8.44 (d, J = 6.8 Hz, 1H), 7.55–7.52 (m, 1H),
7.07–7.05 (m, 1H), 7.01–6.96 (m, 5H), 4.59–4.55 (m,
1H), 4.17–4.14 (m, 1H), 3.83–3.79 (m, 1H), 3.21–3.18
(m, 1H), 2.35–2.11 (m, 2H), 1.69–1.24 (m, 24H).
5.7. (3R,5R)-7-[5-cyano-3-(4-fluoro-phenyl)-1-isopropyl-
4-pyridin-2-yl-1H-pyrrol-2-yl]-3,5-dihydroxy-heptanoic
acid sodium salt (11)
To a solution of 10 (0.153 g, 1.71 mmol) and KCN
(0.111 g, 1.71 mmol) in 10 mL of DMF was added
CuCN (0.183 g, 2.05 mmol). The reaction mixture was
heated to 120 ꢁC for 2 h. After cooling to 25 ꢁC, the
reaction solvent was removed under reduced pressure
and the resulting residue was purified by silica gel chro-
matography (20–50% EtOAc/hexane) to give (6-{2-[5-
cyano-3-(4-fluorophenyl)-1-isopropyl-4-pyridin-2-yl-
1H-pyrrol-2-yl]-ethyl}-2,2-dimethyl-[1,3]dioxan-4-yl)-
acetic acid tert-butyl ester (0.512 g, 64%). To a solution
of this compound in MeOH (10 mL) at 25 ꢁC was added
1 N HCl (1.62 mL, 1.62 mmol). The reaction mixture
was stirred at 25 ꢁC for 2 h after which time the solvent
was removed by evaporation and ethyl acetate (20 mL)
was added. The organic layer was washed with saturated
NaHCO3, water, and brine prior to drying over Na2SO4.
After concentration in vacuo, the product was purified
5.9. 7-[5-Ethylcarbamoyl-3-(4-fluoro-phenyl)-1-isopropyl-
4-pyridin-2-yl-1H-pyrrol-2-yl]-3,5-dihydroxy-heptanoic
acid sodium salt (13)
Compound 13 was prepared according to the method
described for 12 using ethyl amine in place of ammonia

L. D. Bratton et al. / Bioorg. Med. Chem. 15 (2007) 5576–5589
5583
to afford the title compound in 5% overall yield over
three steps. MS(APCI+): m/z 512 (M+H); H NMR
(DMSO-d6) d 8.39–8.37 (m, 1H), 8.07–8.05 (m, 1H),
7.75–7.71 (m, 1H), 7.45–7.42 (m, 1H), 7.05–6.96 (m,
4H), 6.81–6.79 (m, 1H), 4.79–4.76 (m, 1H), 4.55–4.51
(m, 1H), 4.11 (m, 1H), 3.64–3.61 (m, 1H), 3.53–3.44
(m, 1H), 2.97 (q, J = 7.1 Hz, 2H), 2.63–2.59 (m, 1H),
1.96–1.91 (m, 1H), 1.73–1.69 (m, 1H), 1.49 (d,
J = 6.8 Hz, 6H), 1.54–1.21 (m, 4H), 0.88 (t, J = 7.0 Hz,
5H).
(q, J = 7.07, 2H), 2.13 (s, 3H), 2.11 (s, 3H), 1.22 (t,
J = 7.08 Hz, 3H).
5.13. 4-(4-Fluorophenyl)-3,5-dimethyl-1H-pyrrole-2-car-
boxylic acid ethyl ester (18)
To a solution of 17 (27 g, 108 mmol) in 300 mL of N,N-
dimethylformamide was added 4-fluoroboronic acid
(22 g, 157 mmol), a solution of sodium carbonate
(29 g, 278 mmol) in 75–80 mL of water, followed by
tetrakis(triphenylphosphine)palladium(0) (4.2 g, 3.6 mmol).
The reaction mixture was stirred at reflux for 19 h.
The reaction mixture was diluted with 1 L of ethyl
acetate and filtered through a bed of Celite filter aid.
The filtrate was washed with 5% aqueous sodium car-
bonate (3· 1 L) and brine (3· 1 L). The organic layer
was separated, dried (sodium sulfate), filtered, and then
the filtrate was evaporated to give a gray solid. Recrys-
tallization from 2.4 L of 66% aqueous acetonitrile trea-
ted with activated charcoal gave 20.2 g (71%) as light
tan crystals; mp 174–175 ꢁC; MS(APCIÀ) m/z 260
(MÀ1); 1H NMR (d6-DMSO) d 11.37 (s, 1H), 7.12–
7.23 (m, 4H), 4.18 (q, J = 7.07, 2H), 2.13 (s 3H), 2.10
(s, 3H), 1.23 (t, J = 7.14, 3H).
5.10. (3R,5R)-7-[3-(4-fluoro-phenyl)-1-isopropyl-5-phe-
nylcarbamoyl-4-pyridin-2-yl-1H-pyrrol-2-yl]-3,5-dihy-
droxy-heptanoic acid sodium salt (14)
Compound 14 was prepared according to the method
described for 12 using aniline in place of ammonia to af-
ford the title compound in 6% overall yield over three
steps. MS(APCI+): m/z 560 (M+H); H NMR (DMSO-
d6) d 10.3 (s, 1H), 8.35 (d, J = 4.0 Hz, 1H), 7.68 (s,
1H), 7.44–7.39 (m, 3H), 7.24–7.14 (m, 2H), 7.06–6.93
(m, 5H), 6.79 (d, J = 8.0 Hz, 1H), 4.77 (br s, 1H), 4.64
(sept, J = 8.0 Hz, 1H), 4.22 (m, 1H), 3.66–3.60 (m,
1H), 3.58–3.53 (m, 1H), 2.70–2.61 (m, 1H), 2.41–2.47
(m, 1H), 1.94 (dd, J = 15.2, 4.0 Hz, 1H), 1.72 (dd,
J = 15.2, 8.0 Hz 1H), 1.55–1.18 (m, 10H).
5.14. 4-(4-Fluorophenyl)-5-formyl-3-methyl-1H-pyrrole-
2-carboxylic acid ethyl ester (19)
5.11. (3R,5R)-7-[5-Benzylcarbamoyl-3-(4-fluoro-phenyl)-
1-isopropyl-4-pyridin-2-yl-1H-pyrrol-2-yl]-3,5-dihydroxy-
heptanoic acid (15)
To a mixture of 18 (9.6 g, 37 mmol) in a mixture of
300 mL of tetrahydrofuran, 45 mL of acetic acid, and
100 mL of water was added ceric ammonium nitrate
(80 g, 24 mmol) in portions for a period of a few min-
utes. The reaction mixture was stirred at 24 ꢁC for 2 h
and then poured into 1 kg of ice and water. The mixture
was extracted with dichloromethane (4· 300 mL). The
combined organic layers were washed with 5% aqueous
sodium bicarbonate (4· 500 mL) and brine (500 mL).
The organic layer was dried (sodium sulfate) and evap-
orated to provide an orange solid. Recrystallization
from 40% aqueous acetonitrile afforded 6.6 g (65%) as
crystals (yellow plates); mp 144–146 ꢁC; MS(APCI+)
Compound 15 was prepared according to the method
described for 12 using benzyl amine in place of ammonia
to afford the title compound in 34% overall yield over
three steps. MS(APCI+): m/z 574 (M+H); H NMR
(DMSO-d6)
d 8.71 (t, J = 5.6 Hz, 1H), 8.24 (d,
J = 4.0 Hz, 1H), 7.55–6.77 (m, 13H), 4.74 (s, 1H),
4.61–4.55 (m, 1H), 4.18 (d, J = 5.2 Hz, 2H), 3.75–3.59
(m, 1H), 3.57–3.43 (m, 1H), 2.77–2.58 (m, 1H), 2.55–
2.38 (m, 1H), 1.95 (dd, J = 15.2, 4.0 Hz, 1H), 1.73 (dd,
J = 14.8, 8.0 Hz, 1H) 1.58–1.03 (m, 4), 1.48 (d,
J = 6.4 Hz, 6H).
1
m/z 276 (M+1); H NMR (d6-DMSO) d 12.60 (s, 1H),
9.49 (s, 1H), 7.33–7.41 (m, 2H), 7.17–7.26 (m, 2H),
4.26 (q, J = 7.09 Hz, 2H), 2.14 (s, 3H), 1.27 (t,
J = 7.15 Hz, 3H).
5.12. 4-Bromo-3,5-dimethyl-1H-pyrrole-2-carboxylic acid
ethyl ester (17)
To a cold (5 ꢁC) solution of 3,5-dimethyl-2-pyrrole-car-
boxylate 16 (23 g, 135 mmol) in 350 mL of dichloro-
methane was added pyridine (22 g, 284 mmol),
followed by a solution of bromine (24 g, 148 mmol) in
100 mL of dichloromethane. The reaction mixture was
stirred for 15 min and then poured into 1 L of ice cold
2.0 N aqueous sodium thiosulfate. The layers were sep-
arated and then the aqueous layer was extracted with
dichloromethane (3· 250 mL). The combined organic
layers were washed with ice cold 2.0 N hydrochloric acid
(3· 500 mL), followed by 5% aqueous sodium bicarbon-
ate (2· 500 mL) and brine (500 mL). The organic layer
was dried (sodium sulfate), filtered, and then the filtrate
was evaporated to afford an orange solid. Recrystalliza-
tion from 750 mL of hexane provided 26.8 g (81%) as
crystals; mp 138 ꢁC (dec); MS(APCI+) m/z 248 (M+2);
5.15. 1-Ethyl-4-(4-fluorophenyl)-5-formyl-3-methyl-1H-
pyrrole-2 carboxylic acid ethyl ester (20)
To a solution of 19 (6.6 g, 24 mmol) in 250 mL of aceto-
nitrile was added cesium carbonate (12 g, 36 mmol),
followed by 7.5 mL of ethyl iodide (15 g, 94 mmol).
The reaction mixture was stirred at 24 ꢁC for 21 h and
then the mixture was filtered to remove insoluble mate-
rial, which was washed several times with acetonitrile.
The filtrate was evaporated to give a residue, which
was dissolved in 250 mL of ethyl acetate. The organic
layer was washed with brine (2· 250 mL), dried (sodium
sulfate), filtered, and then the filtrate was evaporated to
afford a residue. Purification by flash chromatography
(silica gel, 10% ethyl acetate in hexane) gave 6.3 g
(87%) of a viscous oil, which rapidly solidified into a
solid; mp 69–71 ꢁC; MS(APCI+) m/z 304 (M+1); 1H NMR
1H NMR (d6-DMSO)
d
11.70 (s, 1H), 4.16

5584
L. D. Bratton et al. / Bioorg. Med. Chem. 15 (2007) 5576–5589
(d6-DMSO) d 9.37 (s, 1H), 7.32–7.39 (m, 2H), 7.21–7.28
(m, 2H), 4.62 (q, J = 6.90 Hz, 2H), 4.29 (q, J = 7.08 Hz,
2H), 2.06 (s, 3H), 1.22–1.32 (m, 6H).
5% aqueous sodium bicarbonate (300 mL) and brine
(300 mL). The organic layer was dried (sodium sulfate),
filtered, and then the filtrate was evaporated to afford a
residue. Purification by flash chromatography (silica gel,
20–50% ethyl acetate in hexane) afforded 5.2 g (73%). A
sample recrystallized from ethyl acetate/hexane gave
crystals; mp 216–218 ꢁC; MS(APCI+) m/z 351 (M+1);
1H NMR (d6-DMSO) d 10.63 (s, 1H); 9.35 (s, 1H),
7.72–7.64 (m, 2H), 7.44–7.22 (m, 6H), 7.12–7.05 (m,
1H), 4.40 (q, J = 7.11 Hz, 2H), 1.99 (s, 3H), 1.26 (t,
J = 7.08 Hz, 3H).
5.16. 1-Ethyl-4-(4-fluorophenyl)-5-formyl-3-methyl-1H-
pyrrole-2-carboxylic acid (21)
To a solution of 20 (7.0 g, 23 mmol) in 182 mL of 70%
aqueous tetrahydrofuran was added lithium hydroxide
monohydrate (5.3 g, 127 mmol). The reaction mixture
was heated at reflux for 20 h and then added to
500 mL of water. The mixture was acidified with 1 N
HCl to pH 2 to form a precipitate, which was triturated
at 24 ꢁC for 30 min. The mixture was filtered to collect a
solid and then rinsed with 500 mL of water. The mate-
rial was dried to provide 6.3 g (99%) of a yellow solid;
5.19. 1-Ethyl-4-(4-fluorophenyl)-5-formyl-3-methyl-1H-
pyrrole-2-carboxylic acid 4-methoxy-benzylamide (23c)
Compound 23c was prepared by the procedure
described for the preparation of compound 23b using
4-methoxybenzylamine. Purification by flash chroma-
tography (silica gel, 35% ethyl acetate in hexane) provided
3.7 g (60%) as an orange foamy solid; MS(APCI+) m/z
1
mp 116–118 ꢁC; MS(APCI+) m/z 276 (M+1); H NMR
(d6-DMSO) d 12.38 (s, 1H), 9.36 (s, 1H), 7.32–7.38 (m,
2H), 7.20–7.28 (m, 2H), 4.66 (q, J = 6.94 Hz, 2H), 2.06
(s, 3H), 1.24 (t, J = 6.87 Hz, 3H).
1
395 (M+1); H NMR (d6-DMSO) d 9.29 (s, 1H), 9.04
5.17. 1-Ethyl-4-(4-fluorophenyl)-5-formyl-3-methyl-1H-
pyrrole-2-carboxylic acid amide (23a)
(t, J = 6.09 Hz, 1H), 7.37–7.29 (m, 2H), 7.28–7.18 (m,
4H), 6.89–6.80 (m, 2H), 4.39–4.29 (m, 4H), 3.68 (s,
3H), 1.91 (s, 3H), 1.19 (t, J = 7.09 Hz, 3H).
To a mixture of 21 (3.2 g, 12 mmol) in 125 mL of dichlo-
romethane were added 4 drops of N,N-dimethylform-
amide (cat), followed by 1.5 mL of oxalyl chloride
(2.2 g, 17 mmol). The reaction mixture was stirred at
24 ꢁC for 18 h and then evaporated to afford 3.4 g
(100%) of a crude green oil as 22 1-ethyl-4-(4-fluorophen-
yl)-5-formyl-3-methyl-1H-pyrrol-2-carbonyl chloride.
To 55 mL of 80% aqueous ethyl acetate was added
6.4 mL of a 2 M solution of ammonia (0.22 g, 13 mmol)
in methanol, followed by sodium carbonate (1.8 g,
17 mmol). The two-phase reaction mixture was cooled
to À5 ꢁC and then a solution of 22 (3.4 g, 12 mmol) in
11 mL of ethyl acetate was added. The reaction mixture
was vigorously stirred at À5 ꢁC for 3 h and then at room
temperature for 3 days. The reaction mixture was di-
luted with 300 mL of ethyl acetate and then washed with
1 N hydrochloric acid (3· 50 mL), followed by saturated
sodium bicarbonate (2· 50 mL) and brine (50 mL). The
organic layer was dried (sodium sulfate), filtered, and
then the filtrate was evaporated to provide a crude yel-
low-brown solid. Purification by flash chromatography
(silica gel, 60% ethyl acetate in hexane) gave 1.78 g
(56%) as a yellow solid; mp 181–182 ꢁC; MS(APCI+)
5.20. 4-({[1-Ethyl-4-(4-fluorophenyl)-5-formyl-3-methyl-
1H-pyrrole-2-carbonyl]-amino}-methyl)-benzoic acid
methyl ester (23d)
Compound 23d was prepared by the procedure de-
scribed for the preparation of compound 23b using 4-
(aminomethyl)benzoate hydrochloride. Purification by
flash chromatography (silica gel, 35% ethyl acetate in
hexane) gave 4.1 g (54%) as a tan solid; mp 143–
145 ꢁC; MS(APCI+) m/z 423 (M+1); 1H NMR (d6-
DMSO) d 9.30 (s, 1H), 9.18 (t, J = 5.93 Hz, 1H), 7.93–
7.87 (m, 2H), 7.47–7.41 (m, 2H), 7.37–7.31 (m, 2H),
7.28–7.21 (m, 2H), 4.51 (d, J = 6.00 Hz, 2H), 4.34 (q,
J = 7.05 Hz, 2H), 3.79 (s, 3H), 1.94 (s, 3H), 1.19 (t,
J = 7.04 Hz, 3H).
5.21. 1-Ethyl-4-(4-fluorophenyl)-5-formyl-3-methyl-1H-
pyrrole-2-carboxylic acid (5-methyl-pyrazin-2-ylmethyl)
amide (23e)
Compound 23e was prepared by the procedure de-
scribed for the preparation of compound 23b using 2-
(aminomethyl)-5-methylpyrazine. Purification by flash
chromatography (silica gel, 60% ethyl acetate in hexane)
gave 2.4 g (57%) as a tan solid; mp 91–93 ꢁC; MS(AP-
1
m/z 275 (M+1); H NMR (d6-DMSO) d 9.32 (s, 1H),
7.92 (d, J = 39.96 Hz, 2H), 7.40–7.32 (m, 2H), 7.31–
7.22 (m, 2H), 4.42 (q, J = 7.16 Hz, 2H), 1.98 (s, 3H),
1.25 (t, J = 7.08 Hz, 3H).
1
CI+) m/z 381 (M+1); H NMR (d6-DMSO) d 9.30 (s,
1H), 9.16 (t, J = 5.85 Hz, 1H), 8.48 (s, 1H), 8.44 (s,
1H), 7.39–7.20 (m, 4H), 4.55 (d, J = 5.94 Hz, 2H), 4.36
(q, J = 7.05 Hz, 2H), 2.42 (s, 3H), 1.96 (s, 3H), 1.20 (t,
J = 7.04 Hz, 3H).
5.18. 1-Ethyl-4-(4-fluorophenyl)-5-formyl-3-methyl-1H-
pyrrole-2-carboxylic acid phenylamide (23b)
To a cold (0 ꢁC) solution of aniline (2.2 g, 24 mmol) and
N,N-diisopropylethylamine (‘Hunig’s base’) in 125 mL
of dichloromethane was added a solution of 22 (6.0 g,
20 mmol) in 125 mL of dichloromethane. The reaction
mixture was stirred as the ice bath slowly melted for
22 h. The reaction mixture was diluted with 125 mL of
dichloromethane and then the organic layer was washed
with 2.0 N hydrochloric acid (3· 300 mL), followed by
5.22. (E/Z)-(R)-3-(Tert-butyldimethylsilanyloxy)-7-[5-
carbamoyl-1-ethyl-3-(4-fluorophenyl)-4-methyl-1H-pyr-
rol-2-yl]-5-oxo-hept-6-enoic acid methyl ester (25a)
To a solution of 23a (1.6 g, 5.8 mmol) in 100 mL of tol-
uene was added the wittig reagent 24 (4.6 g, 8.7 mmol).
The reaction mixture was heated at reflux for 3 days

L. D. Bratton et al. / Bioorg. Med. Chem. 15 (2007) 5576–5589
5585
and then evaporated to give a crude red oil. Purification
by flash chromatography (silica gel, 60–70% ethyl ace-
tate in hexane) provided 1.62 g (53%) of a yellow foamy
solid as a mixture of cis and trans isomers; mp 114–
115 ꢁC; MS(APCI+) m/z 531 (M+1); 1H NMR (d6-
DMSO) d 7.75, 7.47, 7.42–7.27, 6.10, 4.58–4.59, 4.41,
3.66, 2.76, 2.54, 2.04, 1.35, 0.83, 0.06, 0.00.
combined organic layers were washed with ice cold 5%
sodium bicarbonate (3· 300 mL) and brine (300 mL).
The organic layer was dried (sodium sulfate), filtered,
and evaporated to afford a residue. Purification by flash
chromatography (silica gel, 40–75% ethyl acetate in hex-
ane) provided 1.75 g (74%) of an orange solid as the
trans isomer; mp 139–141; MS(APCI+) m/z 493
(M+1); 1H NMR (d6-DMSO) d 10.33 (s, 1H), 7.71–
7.62 (m, 2H), 7.41–7.18 (m, 7H), 7.09–6.99 (m, 1H),
6.02 (d, J = 16.18 Hz, 1H), 4.86 (d, J = 5.82 Hz, 1H),
4.27 (q, J = 7.23 Hz, 2H), 4.23–4.14 (m, 1H), 3.52 (s,
3H), 2.67–2.51 (m, 2H), 2.43–2.24 (m, 2H), 1.94 (s,
3H), 1.26 (t, J = 7.02 Hz, 3H).
5.23. (E/Z)-(R)-3-(Tert-butyldimethylsilanyloxy)-7-[1-
ethyl-3-(4-fluorophenyl)-5-(4-methoxybenzyl-carbamoyl)-
4-methyl-1H-pyrrol-2-yl]-5-oxo-hept-5-enoic acid methyl
ester (25c)
Compound 25c was prepared from 23c and 24 (2 equiv)
by the procedure described for the preparation of 25a to
afford 3.95 g (67%) of a red tacky solid as a mixture of
5.26. (E/Z)-(R)-7-[1-Ethyl-3-(4-fluorophenyl)-5-(4-meth-
oxybenzylcarbamoyl)-4-methyl-1H-pyrrol-2-yl]-3-hydro-
xy-5-oxo-hept-6-enoic acid methyl ester (26c)
1
cis and trans isomers; MS(APCI+) m/z 651 (M+1); H
NMR (d6-DMSO) d 8.89, 7.46, 7.42–7.27, 7.03–6.95,
6.10, 4.58–4.45, 4.35, 3.82, 3.66, 2.76, 2.54, 2.00, 1.33,
0.83, 0.06, 0.00.
Compound 26c was prepared from 25c by the procedure
described for the preparation of 26a. Purification by flash
chromatography (silica gel, 60% ethyl acetate in hexane)
afforded 3.07 g (68%) of a red tacky solid as a mixture
of cis and trans isomers; MS(APCI+) m/z 537 (M+1);
1H NMR (d6-DMSO) d 8.73, 7.38–6.99, 6.92–6.79, 5.97,
4.84, 4.34, 4.27–4.12, 3.68, 3.52, 2.64–2.23, 1.86, 1.20.
5.24. (E/Z)-(R)-7-[5-Carbamoyl-1-ethyl-3-(4-fluoro-phe-
nyl)-4-methyl-1H-pyrrol-2-yl]-3-hydroxy-5-oxo-hept-6-
enoic acid methyl ester (26a)
To a solution of 25a (2.1 g, 4.0 mmol) in 38 mL of anhy-
drous tetrahydrofuran was added 5.2 mL of a 70% solu-
tion of hydrofluoric acid in pyridine (5.6 g, 195 mmol).
The reaction mixture was stirred in a plastic vessel at room
temperature for 4 h and then the mixture was quenched
with solid sodium bicarbonate to pH 8. The mixture
was extracted with dichloromethane (100 mL) and then
washed with brine (100 mL). The organic layer was dried
(sodium sulfate), filtered, and then the filtrate was evapo-
rated to afford a residue. Purification by flash chromatog-
raphy (silica gel, 80–100% ethyl acetate in hexane,
followed by 95% ethyl acetate in methanol) gave 1.08 g
(65%) of a yellow solid as a mixture of cis and trans iso-
5.27. 4-({[1-Ethyl-4-(4-fluorophenyl)-5-((E/Z)-(R)-
5-hydroxy-6-methoxycarbonyl-3-oxo-hex-1-enyl)-
3-methyl-1H-pyrrole-2-carbonyl]-amino}-methyl)-
benzoic acid methyl ester (26d)
Compound 26d was prepared from 23d and 24 by the
procedure described for the preparation of 26b. Purifica-
tion by flash chromatography (silica gel, 50–65% ethyl
acetate in hexane) afforded 3.4 g (70%) of a red-orange
foam as a mixture of cis and trans isomers; MS(APCI+)
m/z 565 (M+1); 1H NMR (d6-DMSO) d 8.85, 7.94–7.85,
7.47–6.98, 5.97, 4.84, 4.49, 4.27–4.12, 3.79, 3.27, 2.64–
2.22, 1.90, 1.20.
1
mers; mp 150–152 ꢁC; MS(APCI+) m/z 417 (M+1); H
NMR (d6-DMSO) d 7.60, 7.32, 7.28–6.99, 5.97, 4.84,
4.26, 4.21–4.12, 3.52, 2.64–2.48, 2.42–2.22, 1.90, 1.22.
5.28. (E/Z)-(R)-7-{1-ethyl-3-(4-fluorophenyl)-4-methyl-5-
[(5-methylpyrazin-2-ylmethyl)-carbamoyl]-1H-pyrrol-2-
yl}-3-hydroxy-5-oxo-hept-6-enoic acid methylester (26e)
5.25. (E)-(R)-7-[1-Ethyl-3-(4-fluorophenyl)-4-methyl-5-
phenylcarbamoyl-1H-pyrrol-2-yl]-3-hydroxy-5-oxo-
hept-6-enoic acid methyl ester (26b)
Compound 26e was prepared from 23e and 24 by the pro-
cedure described for the preparation of 26b using a solu-
tion of hydrofluoric acid in pyridine. Purification by
flash chromatography (silica gel, 80% ethyl acetate in hex-
ane) afforded 1.06 g (35%) of a yellow tacky solid as a mix-
ture of cis and trans isomers; MS(APCI+) m/z 5.23 (M+1);
1H NMR (d6-DMSO) d 8.83, 8.49–8.41, 7.36–7.15, 5.98,
4.84, 4.53, 4.27–4.12, 3.52, 2.63–2.23, 1.92, 1.21.
To a solution of 23b (2.5 g, 7.1 mmol) in 100 mL of tolu-
ene was added the Wittig reagent 24 (7.6 g, 14 mmol).
The reaction mixture was heated at reflux for 2 days
and then evaporated to give a crude orange oil. Purifica-
tion by flash chromatography (silica gel, 20% ethyl ace-
tate in hexane) provided 2.9 g (67%) of a mixture of cis
and trans isomers as intermediate 25b (E/Z)-(R)-3-(tert-
butyldimethylsilanyloxy)-7-[1-ethyl-3-(4-fluorophenyl)-
4-methyl-5-phenylcarbamoyl-1H-pyrrol-2-yl]-5-oxo-
hept-6-enoic acid methyl ester. To a cold (5 ꢁC) solution
of 25b (2.9 g, 4.8 mmol) in 50 mL of acetonitrile was
added a solution of 1.1 mL of 48% aqueous hydrofluoric
acid (1.3 g, 30 mmol) in 12 mL of acetonitrile. The reac-
tion mixture was stirred as it warmed to room tempera-
ture for 1.5 h. The reaction mixture was added to
300 mL of ice cold 5% aqueous sodium bicarbonate
and then extracted with ethyl acetate (4· 100 mL). The
5.29. (E/Z)-(3R,5S)-7-[5-Carbamoyl-1-ethyl-3-(4-fluoro-
phenyl)-4-methyl-1H-pyrrol-2-yl]-3,5-dihydroxy-hept-6-
enoic acid methyl ester (27a)
To a cold (À60 ꢁC) solution of 26a (0.97 g, 2.3 mmol) in
37.5 mL of 80% tetrahydrofuran in methanol was added
2.3 mL of a 1 M solution of diethylmethoxyborane
(0.23 g, 1 mmol) in tetrahydrofuran. The reaction mix-
ture was stirred for 30 min and then powdered sodium
borohydride (0.09 g, 2.3 mmol) was added. The reaction

5586
L. D. Bratton et al. / Bioorg. Med. Chem. 15 (2007) 5576–5589
mixture was stirred for 3 h at À60 ꢁC and then warmed
to 10 ꢁC. Glacial acetic acid (3 mL) was added and then
the mixture was stirred for 18 h as it warmed to room
temperature. The mixture was diluted with 300 mL of
ethyl acetate and then the organic layer was washed with
saturated sodium bicarbonate (3· 50 mL) and brine
(100 mL). The organic layer was dried (sodium sulfate),
filtered, and then the filtrate was evaporated to give an
oily residue. Purification by flash chromatography (silica
gel, 100–95% ethyl acetate in methanol) provided
833 mg (86%) of a yellow solid as a mixture of cis and
trans isomers; mp 82–84 ꢁC; MS(APCI+) m/z 401
chromatography (silica gel, 95% ethyl acetate in metha-
nol) gave 755 mg (87%) of a yellow solid as a mixture of
cis and trans isomers; mp 47–50 ꢁC; MS(APCI+) m/z 525
(M+1); 1H NMR (d6-DMSO) d 8.48–8.40, 7.20–7.08,
6.31, 5.40, 4.76, 4.66, 4.50, 4.15–4.00, 3.81–3.67, 3.52,
2.42, 2.39–2.16, 1.96, 1.53–1.19, 1.14.
5.34. (3R,5R)-7-[5-Carbamoyl-1-ethyl-3-(4-fluorophenyl)-
4-methyl-1H-pyrrol-2-yl}-3,5-dihydroxy-heptanoic acid
methyl ester (28a)
To a solution of 27a (0.74 g, 1.8 mmol) in 35 mL of 50%
tetrahydrofuran in methanol was added 10% Pd/C
(0.10 g, 0.09 mmol). The reaction mixture was stirred
at room temperature under hydrogen (5 psi) atmosphere
between 45 min and 2 h. Purification by flash chroma-
tography (silica gel, 95% ethyl acetate in methanol) pro-
vided 423 mg (57%) as an off-white solid; mp 98–100 ꢁC;
1
(M+1); H NMR (d6-DMSO) d 7.23, 7.16–7.11, 6.30,
5.38, 4.75, 4.66, 4.14, 4.08–4.00, 3.82–3.66, 3.52, 2.39–
2.15, 1.94, 1.52–1.05.
5.30. (E/Z)-(3R,5S)-7-[1-Ethyl-3-(4-fluorophenyl)-4-
methyl-5-phenylcarbamoyl-1H-pyrrol-2-yl]-3,5-dihy-
droxy-hept-6-enoic acid methyl ester (27b)
1
MS(APCI+) m/z 421 (M+1); H NMR (d6-DMSO) d
7.18–7.12(m, 4H), 7.08 (s, 2H), 4.70 (d, J = 5.7 Hz,
1H), 4.52 (d, J = 5.11 Hz, 1H), 4.11 (q, J = 7.17 Hz,
2H), 3.93–3.82 (m, 1H), 3.54–3.43 (m, 4H), 2.66–2.13
(m, 4H), 1.96 (s, 3H), 1.53–1.26 (m, 4H), 1.14 (t,
J = 7.03 Hz, 3H).
Compound 27b was prepared by the procedure de-
scribed for the preparation of 27a. Purification by flash
chromatography (silica gel, 60–75% ethyl acetate in hex-
ane) provided 1.50 g (88%) of an off-white foam as a
mixture of cis and trans isomers; MS(APCI+) m/z 495
(M+1); H NMR (d6-DMSO) d 10.00, 7.70–7.61, 7.31–
7.10, 7.05–6.96, 6.35, 5.45, 4.79, 4.67, 4.18–4.03, 3.81–
3.70, 3.52, 2.39–2.18, 1.98, 1.53–1.13.
1
5.35. (3R,5R)-7-[1-Ethyl-3-(4-fluorophenyl)-4-methyl-5-
phenylcarbamoyl-1H-pyrrol-2-yl]-3,5-dihydroxy-hepta-
noic acid methyl ester (28b)
5.31. (E/Z)-(3R,5S)-7-[1-Ethyl-3-(4-fluorophenyl)-5-(4-
methoxybenzylcarbamoyl)-4-methyl-1H-pyrrol-2-yl]-
3,5-dihydroxy-hept-6-enoic acid methyl ester (27c)
Compound 28b was prepared by the procedure de-
scribed for the preparation of 28a. Purification by flash
chromatography (silica gel, 50–75% ethyl acetate in hex-
ane, gradient elution) provided 580 mg (58%) as a white
foam; MS(APCI+) m/z 497 (M+1); 1H NMR (d6-
DMSO) d 9.88 (s, 1H), 7.66–7.62 (m, 2H). 7.31–7.12
(m, 6H), 7.04–6.95 (m, 1H), 4.70 (d, J = 5.21, 1H),
4.54 (d, J = 5.03 Hz, 1H), 4.10 (q, J = 7.12 Hz, 2H),
3.94–3.83 (m, 1H), 3.55–3.44 (m, 4H), 2.68–2.54 (m,
1H), 2.49–2.15 (m, 3H), 2.00 (s, 3H), 1.57–1.28 (m,
4H), 1.19 (t, J = 6.94 Hz, 3H).
Compound 27c was prepared by the procedure de-
scribed for the preparation of 27a. Purification by flash
chromatography (silica gel, 70% ethyl acetate in hexane)
afforded 2.43 g (86%) of a yellow foam as a mixture of
cis and trans isomers; mp 49–51 ꢁC; MS(APCI+) m/z
539 (M+1); 1H NMR (d6-DMSO) d 8.35, 7.25–7.09,
6.89–6.79, 6.30, 5.39, 4.75, 4.66, 4.32, 4.15–3.99, 3.80–
3.69, 3.67, 3.26, 2.39–2.15, 1.90, 1.51–1.18, 1.13.
5.36. (3R,5R)-7-(1-Ethyl-3-(4-fluorophenyl)-5-(4-meth-
oxybenzylcarbamoyl)-4-methyl-1H-pyrrol-2-yl]-3,5-
dihydroxy-heptanoic acid methyl ester (28c)
5.32. 4-({[5-((E/Z)-(3S,5R)-3,5-Dihydroxy-6-methoxy-
carbonyl-hex-1-enyl)-1-ethyl-4-(4-fluorophenyl)-3-
methyl-1H-pyrrole-2-carbonyl]-amino}-methyl)-
benzoic acid methyl ester (27d)
Compound 28c was prepared by the procedure de-
scribed for the preparation of 28a. Purification by flash
chromatography (silica gel, 80% ethyl acetate in hexane)
afforded 673 mg (67%) as a white foam; MS(APCI+) m/z
541 (M+1); 1H NMR (d6-DMSO) d 8.19 (t, J = 6.02 Hz,
1H), 7.24–7.09 (m, 6H), 6.87-6.79 (m, 2H), 4.69 (d,
J = 5.14 Hz, 1H), 4.52 (d, J = 5.13 Hz, 1H), 4.31 (d,
J = 6.03 Hz, 2H), 4.06 (q, J = 7.16 Hz, 2H), 3.93–3.82
(m, 1H), 3.67 (s, 3H), 3.54–3.43 (m, 4H), 2.64–2.14 (m,
4H), 1.93 (s, 3H), 1.54–1.27 (m, 4H), 1.11 (t,
J = 7.01 Hz, 3H).
Compound 27d was prepared by the procedure described
for the preparation of 27a. Purification by flash chroma-
tography (silica gel, 60–75% ethyl acetate in hexane, gra-
dient elution) gave 2.7 g (82%) of a light yellow foam as a
mixture of cis and trans isomers; MS(APCI+) m/z 549
1
(M+1)+; H NMR (d6-DMSO) d 8.47, 7.91–7.85, 7.46–
7.38, 7.19–7.10, 6.31, 5.39, 4.76, 4.66, 4.47, 4.17–3.98,
3.79, 3.77–3.68, 3.52, 2.39–2.16, 1.94, 1.52–1.17, 1.13.
5.33. (E/Z)-(3R,5S)-7-{1-Ethyl-3-(4-fluorophenyl)-4-
methyl-5-[(5-methyl-pyrazin-2-ylmethyl)-carbamoyl]-
1H-pyrrol-2-yl}-3,5-dihydroxy-hept-6-enoic
acid methyl ester (27e)
5.37. 4-({[5-((3R,5R)-3,5-Dihydroxy-6-methoxycarbonyl-
hexyl)-1-ethyl-4-(4-fluorophenyl)-3-methyl-1H-pyrrole-2-
carbonyl]-amino}-methyl)-benzoic acid methyl ester (28d)
Compound 27e was prepared by the procedure de-
scribed for the preparation of 27a. Purification by flash
Compound 28d was prepared by the procedure de-
scribed for the preparation of 28a. Purification by flash

L. D. Bratton et al. / Bioorg. Med. Chem. 15 (2007) 5576–5589
5587
chromatography (silica gel, 75–80% ethyl acetate in hex-
ane, gradient elution) gave 930 mg (48%) as a white
foam; MS(APCI+) m/z 569 (M+1); 1H NMR (d6-
DMSO) d 8.32 (t, J = 6.01 Hz, 1H), 7.93–7.84 (m, 2H),
7.47–7.38 (m, 2H), 7.20–7.10 (m, 4H), 4.69 (d,
J = 5.20 Hz, 1H), 4.52 (d, J = 5.11 Hz, 1H), 4.46 (d,
J = 6.11 Hz, 2H), 4.07 (q, J = 7.21 Hz, 2H), 3.93–3.83
(m, 1H), 3.79 (s, 3H), 3.53–3.44 (m, 4H), 2.66–2.13 (m,
4H), 1.97 (s, 3H), 1.55–1.26 (m, 4H), 1.10 (t,
J = 6.94 Hz, 3H).
5.41. (3R,5R)-7-[1-Ethyl-3-(4-fluorophenyl)-5-(4-meth-
oxybenzylcarbamoyl)-4-methyl-1H-pyrrol-2-yl]-3,5-dihy-
droxy-heptanoic acid sodium salt (29c)
Compound 29c was prepared by the procedure de-
scribed for the preparation of 29a to provide 544 mg
(95%) as a white solid; MS(APCIÀ) m/z 527 (MÀNa);
1H NMR (d6-DMSO) d 8.22 (t, J = 6.02 Hz, 1H), 7.57
(s, 1H), 7.24–7.10 (m, 6H), 6.86–6.78 (m, 2H), 4.70 (s,
1H), 4.31 (d, J = 6.10 Hz, 2H), 4.06 (q, J = 7.08 Hz,
2H), 3.70–3.58 (m, 4H), 3.53–3.43 (m, 1H), 2.64–2.31
(m, 2H), 1.96–1.65 (m, 5H), 1.50–1.06 (m, 7H).
5.38. (3R,5R)-7-{1-Ethyl-3-(4-fluorophenyl)-4-methyl-5-
[(5-methyl-pyrazin-2-ylmethyl)-carbamoyl]-1H-pyrrol-2-
yl}-3,5-dihydroxy-heptanoic acid methyl ester (28e)
5.42. (3R,5R)-7-[1-Ethyl-3-(4-fluorophenyl)-5-(4-meth-
oxycarbonyl-benzylcarbamoyl)-4-methyl-1H-pyrrol-2-yl]-
3,5-dihydroxy-heptanoic acid sodium salt (29d)
Compound 28e was prepared by the procedure de-
scribed for the preparation of 28a. Purification by flash
chromatography (silica gel, 95% ethyl acetate in metha-
nol) afforded 261 mg (61%) of an off-white tacky solid;
Compound 29d was prepared by the procedure de-
scribed for the preparation of 29b to provide 190 mg
(62%) as a white solid; MS(APCIÀ) m/z 553 (MÀNa);
1H NMR (d6-DMSO) d 8.35 (t, J = 5.95 Hz, 1H),
7.91–7.83 (m, 2H), 7.59 (br s, 1H), 7.45–7.38 (m, 2H),
7.20–7.10 (m, 4H), 4.70 (br s, 1H), 4.45 (d,
J = 5.99 Hz, 2H), 4.06 (q, J = 7.00 Hz, 2H), 3.80 (s,
3H), 3.69–3.58 (m, 1H), 3.53–3.42 (m, 1H), 2.64–2.32
(m, 2H), 2.00–1.65 (m, 5H), 1.51–1.06 (m, 7H).
1
MS(APCI+) m/z 527 (M+1); H NMR (d6-DMSO) d
8.44 (s, 1H), 8.41 (s, 1H), 8.30 (t, J = 5.90 Hz, 1H),
7.19–7.11 (m, 4H), 4.70 (d, J = 5.20 Hz, 1H), 4.52 (d,
J = 5.08 Hz, 1H), 4.49 (d, J = 5.83 Hz, 2H), 4.07 (q,
J = 7.25 Hz, 2H), 3.93–3.82 (m, 1H), 3.54–3.43 (m,
4H), 2.64–3.13 (m, 7H), 1.99 (s, 3H), 1.55–1.27 (m,
4H), 1.11 (t, J = 6.97 Hz, 3H).
5.39. (3R,5R)-7-[5-Carbamoyl-1-ethyl-3-(4-fluorophenyl)-
4-methyl-1H-pyrrol-2-yl]-3,5-dihydroxy-heptanoic acid
sodium salt (29a)
5.43. (3R,5R)-7-{1-Ethyl-3-(4-fluorophenyl)-4-methyl-5-
[(5-methyl-pyrazin-2-ylmethyl)-carbamoyl]-1H-pyrrol-2-
yl}-3,5-dihydroxy-heptanoic acid sodium salt (29e)
To a solution of 28a (0.36 g, 0.85 mmol) in 10 mL of
methanol was added 0.91 mL of a 1.028 N aqueous solu-
tion of sodium hydroxide (0.04 g, 0.94 mmol). The reac-
tion mixture was stirred at room temperature for 3 h
and then evaporated in vacuo to give a semi-solid resi-
due. Purification by triturating in 50 mL of diethyl ether
gave 326 mg (89%) as a white solid; mp 150 ꢁC (dec);
Compound 29e was prepared by the procedure de-
scribed for the preparation of 29a to provide 189 mg
(90%) as an off-white solid; MS(APCIÀ) m/z 511
1
(MÀNa); H NMR (d6-DMSO) d 8.44 (s, 1H), 8.41 (s,
1H), 8.34 (t, J = 5.87 Hz, 1H), 7.60 (s, 1H), 7.19–7.11
(m, 4H), 4.71 (s, 1H), 4.48 (d, J = 5.58 Hz, 2H), 4.06
(q, J = 7.05 Hz, 2H), 3.68–3.57 (m, 1H), 3.53–3.43 (m,
1H), 2.66–2.30 (m, 5H), 1.98 (s, 3H), 1.94–1.65 (m,
2H), 1.52–1.06 (m, 7H).
1
MS(APCIÀ) m/z 405 (MÀNa); H NMR (d6-DMSO) d
7.53 (s, 1H), 7.19–7.11 (m, 4H), 7.08 (s, 2H), 4.70 (s,
1H), 4.10 (q, J = 6.96 Hz, 2H), 3.69–3.59 (m, 1H),
3.54–3.43 (m, 1H), 2.63–2.30 (m, 2H), 2.01–1.65 (m,
5H), 1.51-0.99 (m, 7H).
5.44. (3R,5R)-7-[5-(4-carboxy-benzylcarbamoyl)-ethyl-3-
(4-fluorophenyl)-4-methyl-1H-pyrrol-2-yl]-3,5-dihydroxy-
heptanoic acid disodium salt (29f)
5.40. (3R,5R)-7-[1-Ethyl-3-(4-fluorophenyl)-4-methyl-5-
phenylcarbamoyl-1H-pyrrol-2-yl]-3,5-dihydroxy-hepta-
noic acid sodium salt (29b)
To a solution of 28d (0.60 g, 1.1 mmol) in a mixture of
10 mL of methanol and 2 mL of water was added
5.2 mL of a 1.028 M aqueous solution of sodium
hydroxide (0.21 g, 5.3 mmol). The reaction mixture
was stirred at reflux for 2 h and then evaporated to
give a semi-solid residue, which was suspended in
100 mL of 20% methanol in dichloromethane. The
mixture was filtered, the filtrate evaporated, and the
final residue was triturated in 75 mL of diethyl ether
for 18 h. The solid was filtered and washed several
times with fresh diethyl ether to provide, after drying,
350 mg (57%); mp >280 ꢁC; MS(APCIÀ) m/z 538
(MÀNa+1); 1H NMR (d6-DMSO) d 8.26 (t, J =
6.04 Hz, 1H), 7.79–7.71 (m, 2H), 7.44 (s, 1H), 7.22–
7.10 (m, 6H), 4.69 (s, 1H), 4.38 (d, J = 5.71 Hz, 2H),
4.07 (q, J = 6.94 Hz, 2H), 3.70–3.59 (m, 1H), 3.54–3.43
(m, 1H), 2.65–2.32 (m, 2H), 2.02–1.66 (m, 5H), 1.52–
1.07 (m, 7H).
To a solution of 28b (0.53 g, 1.1 mmol) in 18 mL of
66% aqueous absolute ethanol was added 1.04 mL of
a 1.028 N aqueous solution of sodium hydroxide
(0.04 g, 1.1 mmol). The reaction mixture was stirred
at room temperature for 2 h and then evaporated in
vacuo to give a semi-solid residue. The residue was
suspended in acetone and evaporated again three
times. Purification by triturating from 75 mL of
diethyl ether gave 470 mg (87%) as a white solid; mp
1
198–200 ꢁC; MS(APCIÀ) m/z 481 (MÀNa); H NMR
(d6-DMSO) d 9.92 (s, 1H), 7.72–7.33 (m, 3H), 7.31–
7.08 (m, 6H), 7.05–6.92 (m, 1H), 4.72 (s, 1H), 4.18–
3.99 (m, 2H), 3.71–3.57 (m, 1H), 3.56–3.43 (m, 1H),
2.71–2.23 (m, 2H), 2.09–1.64 (m, 5H), 1.54–0.95 (m,
7H).

5588
L. D. Bratton et al. / Bioorg. Med. Chem. 15 (2007) 5576–5589
[3-14C]-HMGCoA (57.0 mCi/mmol) was purchased
from Amersham Biosciences, UK. HMGCoA, mevalon-
olactone, Beta-NADPH (Beta-Nicotinamide Adenine
Dinucleotide Phosphate, Reduced form) were purchased
from Sigma Chemical Co. AG 1-8X resin was purchased
from Bio-Rad Laboratory.
(FBS) and 10 mM Hepes (N-2-hydroxyethyl-piperazie-
N1-2-ethane sulfonic acid) (Gibco No. 15630-080) for
24 h. The cells were pre-incubated with compounds for
4 h and then labeled by incubating in medium contain-
ing 1 lCi/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) solu-
tion containing 150 mM NaCl and 1 nM EDTA, and
collected in the lysis buffer containing 10% KOH and
80% ethanol by volume. In order to separate labeled
cholesterol from labeled non-cholesterol lipids, the cells
lysate were subjected 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 30 min of vigor-
ous shaking. After the separation of the 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 Graph-
Pad software (Prism 3.03).
5.45. 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 homogenized in a
sucrose homogenizing solution in an ice bath 10 times.
Homogenates were diluted into a final volume of
200 mL and centrifuged for 15 min. with a Sorvall Cen-
trifuge at 5 ꢁC, 10,000 rpm (12,000g). The upper fat
layer was removed and the supernatant decanted into
fresh tubes. This step was repeated one more time before
transferring the supernatant into ultracentrifuge tubes
and centrifuged at 36,000 rpm (105,000g) for an hour
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 homogenized gently by hand
about 10 times. 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 Chem-
ical Company. Aliquots (1 mL) of microsomes were
kept frozen in liquid nitrogen.
5.48. Procedure for sterol biosynthesis (cell assay) in L6
rat myoblasts, cell culture, compound treatment, cell
labeling, cholesterol extraction, and data analysis
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 60,000 cells
per well. The cells were grown in Dulbecco’s modified
Eagle’s medium (DMEM) containing 10% heat inacti-
vated fetal bovine serum (FBS) for 72 h until reaching
confluence. The cells were pre-incubated in media with
compound and 0.20% DMSO (dimethylsulfoxide) for
3 h and then labeled by incubating in medium contain-
ing compound, 0.20% DMSO, and 1 lCi/mL of 14C ace-
tic acid for an additional 3 h. After labeling, the cells
were washed once with 1· PBS and then lysed overnight
at 4 ꢁC in buffer containing 10% KOH and 78% ethanol
by volume. Lipid ester bonds were hydrolyzed by sapon-
ification 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 pip-
ette 6 times. The upper organic phase solution was col-
lected and combined with an equal volume of 1 N KOH
in 50% methanol and mixed by pipette 6 times. 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 formula: y = Bmax(1 À (Xn/Kn + Xn)) + Y2, where
K is the IC50 for the inhibition curve, X is inhibitor con-
centration, Y is the response being inhibited, and
Bmax + Y2 is the limiting response as X approaches zero.
5.46. HMG-CoA reductase assay (in vitro) procedure
DMSO (1 lL) or 1 lL of DMSO containing a test com-
pound at a concentration of between 0.1 and 1 nM was
placed into each well of a Corning 96-well plate. A volume
of 34 lL buffer [100 mM NaH2PO4, 10 mM imidazole,
and 10 mM EDTA, (ethylenediamine-tetraacetic acid)]
containing 35 lg/mL rat liver microsomes was added into
each well. After incubation for 30 min on ice, 15 lL of
14C-HMGCoA (0.024 lCi) with 15 mM NADPH,
25 mM DTT, (dithiothreitol) was added and incubated
at 37 ꢁC for an additional 45 min. The reaction was termi-
nated by the addition of 10 lL of 2 M HCl, followed by
the addition of 5 lL of 0.10 M mevalonolactone. Plates
were incubated at room temperature for 60 min to allow
lactonization of mevalonate to mevalonolactone. The
incubated samples were applied to columns containing
300 lL of AGI-X8 anion exchange resin in a Corning fil-
ter 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).
5.47. Procedure for sterol biosynthesis (cell assay) in rat
hepatocytes, cell culture, compound treatment, cell label-
ing, cholesterol extraction, and data analysis
Frozen rat hepatocyctes purchased from Xeno Tech
were seeded on a 6-well collagen 1 coated plates at a
density of 105 cells/per well. The cells were grown in
Dulbecco’s modified Eagle’s medium (DMEM) (Gibco,
No. 11054-020) containing 10% fetal bovine serum
5.49. Procedure for cholesterol synthesis inhibition effects
of the test compounds in mice
5.49.1. In vivo study procedure. Male C57/BL6 mice 8
weeks old obtained from Charles River Laboratories

L. D. Bratton et al. / Bioorg. Med. Chem. 15 (2007) 5576–5589
5589
were randomly placed into nine groups with 10 mice per
Acknowledgments
group and acclimated for a minimum of 7 days to a re-
versed 12 h light (6:00 a.m. to 6:00 p.m.), 12 h dark (6:00
p.m. to 6:00 a.m.) since natural peak cholesterol synthe-
sis is mid-diurnal during the dark period in mice. On the
test day, a single dose of the test compounds or vehicle
(control group) was administered by oral gavage 2 h
prior to the middle of the dark period. A single intra-
peritoneal injection of 25 lCi 14C sodium acetate (Amer-
sham Biosciences UK Limited) was administered 30 min
after drug administration. At 4 h post 14C sodium ace-
tate administration, mice were euthanized by CO2
asphyxiation and exsanguinated by cardiac puncture
for plasma collection. Individual animal plasma samples
were acquired by placing 0.8 mL of whole blood into
centrifuge tubes containing 20 lL EDTA and centri-
fuged. Plasma was then transferred into new tubes and
stored at À20 ꢁC until assayed for 14C labeled
cholesterol.
Thanks to the staff from our high pressure lab for con-
ducting the catalytic hydrogenation experiments.
References and notes
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5.49.3. Assay for plasma 14C labeled cholesterol. Frozen
plasma samples (0.4 mL) were allowed to thaw, and an
equal volume of physiological saline was added to each
3
sample along with 0.025 lCi H-cholesterol (Perkin-El-
mer Life Sciences Inc., Boston, Mass). Freshly prepared
10% KOH solution was then added at 2.5 mL per sample.
Samples were then vortexed and saponified at 75 ꢁC for
1 h. After the samples cooled, saponified 14C labeled syn-
thesized cholesterol was extracted by adding 2.5 mL
petroleum ether (Mallinckrodt No. 4976 Petroleum
Ether) per sample, shaken for 10 min, followed by centri-
fuging for 10 min at 0 ꢁC. The organic phase (1.4 mL) was
transferred to new tubes and 5 mL of the scintillation
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3
achieved. The percent recovery of the H-cholesterol in
each sample was used to mathematically correct the 14C
counts for any differences in recovery.
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5.49.4. Data analysis. Effects of the test samples on per-
cent inhibition of cholesterol synthesis measured in plas-
ma were determined by comparing the amount of 14C
sodium acetate incorporated into 14C labeled cholesterol
in the plasma of compound treated mice versus the
amount in vehicle treated mice.