Synthesis and biological activity of a novel squalene epoxidase inhibitor, FR194738

Article abstract of DOI:10.1016/j.bmcl.2003.11.072

The synthesis and biological properties of a novel squalene epoxidase inhibitor, FR194738, are described. This compound displayed potent in vitro inhibitory activities against squalene epoxidase and cholesterol synthesis, and lowered plasma cholesterol and triglyceride levels in dogs.

Full text of DOI:10.1016/j.bmcl.2003.11.072

Bioorganic & Medicinal Chemistry Letters 14 (2004) 633–637
Synthesis and biological activity of a novel squalene epoxidase
inhibitor, FR194738
Masae Sawada,a,* Ken-ichi Washizukab and Hiroyuki Okumurab
aMedicinal Biology Research Laboratories, Fujisawa Pharmaceutical Co. Ltd, 2-1-6Kashima, Yodogawa-ku,
Osaka 532-8514, Japan
bMedicinal Chemistry Research Laboratories, Fujisawa Pharmaceutical Co. Ltd, 2-1-6Kashima, Yodogawa-ku,
Osaka 532-8514, Japan
Received 29 September 2003; accepted 18 November 2003
Abstract—The synthesis and biological properties of a novel squalene epoxidase inhibitor, FR194738, are described. This com-
pound displayed potent in vitro inhibitory activities against squalene epoxidase and cholesterol synthesis, and lowered plasma
cholesterol and triglyceride levels in dogs.
# 2003 Elsevier Ltd. All rights reserved.
1. Introduction
antifungal compounds have been shown to be specific
inhibitors of SE.9 Depending on their precise chemical
structure, allylamine derivatives can be highly selective
for either fungal or mammalian SE. A number of efforts
have been directed at the discovery of potent SE inhibi-
tors for treatment of hypercholesterolemia. NB-598 was
the first example of a potent and specific inhibitor of
mammalian SE with an allylamine structure.10 Other
scaffolds have also been described.11,12
Hypercholesterolemia is a major risk factor for the
developmentof coronary heartdisease (CHD). 1 Clinical
trials have demonstrated that lowering plasma choles-
terol levels by diet or drug intervention leads to benefits
for hypercholesterolemic patients.2 Although several
inhibitors of HMG-CoA reductase, a rate-limiting
enzyme in cholesterol biosynthesis, have been developed
and used for this purpose,3,4 they reduce non-sterol
products such as farnesol, ubiquinone, dolichol and
isoprenylated proteins,5 some of which play important
roles in the multivalent feedback inhibition of HMG-
CoA reductase.6 The resultant lack of feedback inhibi-
tion may lead to the induction of HMG-CoA reductase
itself, and thereby attenuate the efficacy or even abolish
the cholesterol lowering effects of HMG-CoA reductase
inhibitors.7,8
In the course of our research aimed at discovery of new
SE inhibitors, we aimed to improve the lipophilic prop-
erties of NB-598 by modifying the thienyl–thienyl
structure and to introduce polar substituents to increase
polarity. As a result, we have discovered the novel SE
inhibitor FR194738. In this paper, we describe the
synthesis and biological properties of FR194738 (Fig. 1).
Squalene epoxidase (SE) is a microsomal enzyme that
catalyzes the conversion of squalene to 2,3-oxidosqua-
lene. SE is located downstream in the cholesterol
synthesis pathway, past the branch points for the bio-
synthesis of the non-sterol products mentioned above.
Inhibition of SE should therefore have no effect on their
production, but at the same time lead to cholesterol
lowering with great efficacy. The allylamine class of
* Corresponding author. Tel.: +81-6-6390-5482; fax: +81-6-6304-
5367; e-mail: masae_sawada@po.fujisawa.co.jp
Figure 1.
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.11.072

634
M. Sawada et al. / Bioorg. Med. Chem. Lett. 14 (2004) 633–637
Scheme 1. (a) BrCH2CO2Et, KI, K2CO3, acetone, reflux, quant.; (b) NaBH4, EtOH, 0 ꢀC, 63%; (c) TBDMSCl, imidazole, DMF, 0 ꢀC, quant.; (d)
MeMgBr, THF, 0 ꢀC, quant.; (e) (1) NaH, DMF, 0 ꢀC to rt; (2) 3-bromomethylthiophene, 0 ꢀC to rt, 67%; (f) TBAF, THF, 0 ꢀC, 82%; (g) MsCl,
Et3N, THF, quant.; (h) (E)-N-ethyl-6,6-dimethyl-2-hepten-4-yn-1-amine, K2CO3, DMF, rt, 54%; (i) (1) HCl/AcOEt, rt; (2) recryst. from Hex/
AcOEt, 63%.
[14C]acetate was added. After 2 h of incubation, the cells
were lysed and saponified with ethanolic KOH. Digito-
2. Synthesis
The synthesis of our new SE inhibitor, FR194738, is
outlined as shown in Scheme 1. Alkylation of m-hydro-
xybenzaldehyde with ethyl bromoacetate, followed by
aldehyde reduction with sodium borohydride afforded
the primary alcohol 3 in 63% yield over two steps. Pro-
tection of the alcohol as TBDMS, followed by reaction
with excess methylmagnesium bromide afforded the
tertiary alcohol 5 in quantitative yield. Alkylation of
the alcohol moiety of 5 with 3-bromomethylthiophene
in the presence of NaH–DMF, followed by deprotection
of the silyl protecting group with TBAF in tetra-
hydrofuran afforded the alcohol 7 in good yield.
Finally, activation of the alcohol as the mesylate (MsCl–
Et3N), followed by coupling with (E)-N-ethyl-6,6-dime-
thyl-2-hepten-4-yn-1-amine in the presence of potassium
carbonate and conversion of the resulting free amine
into the hydrochloride salt, afforded FR194738 as a
white crystalline solid. The derivatives shown in Table 1
and 2 were prepared in a similar manner.
nin was added to the non-saponifiable lipids. Radio-
activity of the digitonin coprecipitable sterols was
counted.
3.3. Induction of HMG-CoAreductase activity in HepG2
cells
HepG2 cells were incubated for 18 h in a medium con-
taining 10% human lipoprotein-deficient serum with or
without drug. Thereafter, the cells were washed, scalped
and suspended in buffer containing potassium phos-
phate, EDTA, KCl and Brij 97. After incubation for 15
min at37 ꢀC, the suspension was centrifuged at 12,000g
for 15 min and the supernatant was used for measuring
HMG-CoA reductase activity. HMG-CoA reductase
Table 1. Squalene epoxidase inhibitory activity of compounds with
heteroatom-based linkers
3. Biological methods
3.1. In vitro SE assay
Compd
A
R
Squalene epoxidase
inhibitory activity
IC50 (nM)
ClogP
Dog or rat liver microsomes and the homogenate of
HepG2 cells, which are considered to be a suitable
model for investigating lipid metabolism in human
liver,13 were solubilized with 2% Triton X-100. The
assay mixture containing microsomes or cell homo-
genate, AMO-1680, FAD, NADPH, EDTA, [3H]squa-
lene and drug was incubated for 90 min at 37 ꢀC. The
reaction was terminated by the addition of ethanolic
KOH. Non-saponifiable materials were extracted and
separated on a silica gel TLC plate, and the radio-
activity of 2,3-oxidosqualene was counted.
Human
Dog
9
Et23
Et5.4
Me
370
34
7.48
10a
11
12
7.30
11
31
7.2
77
51
43
6.78
7.83
8.19
Pr
3.2. Cholesterol synthesis in HepG2 cells
HepG2 cells were incubated overnight in a medium
containing 10% human lipoprotein-deficient serum. The
cells were preincubated for 1 h with drug and then
NB-598
a Free form of FR194738.

M. Sawada et al. / Bioorg. Med. Chem. Lett. 14 (2004) 633–637
635
activity was determined according to the method of
14
Brown etal.
lower activity. Even a methylene group (compound 15)
was five times weaker against human SE. The hydro-
chloride salt(FR194738) of compound 10 was prepared
and selected for further evaluation.
3.4. Cholesterol-lowering activity
Male beagle dogs weighing 9–11 kg (n=3) were treated
orally with FR194738 twice a day for 28 days. Blood
samples were taken on the indicated days. On day 14,
serum lipoprotein fractions were separated using an
ultracentrifugation procedure. Plasma and lipoprotein
lipid levels were determined enzymatically.
5. Results and discussion
The novel derivative FR194738 was found to potently
inhibitSE in HepG2 cell homogenaet and liver micro-
somes in dogs and rats (Table 3), and the inhibitory
activities were comparable to NB-598. The pharmaco-
kinetic parameters in rats and dogs are shown in Tables
4 and 5. In rats, Cmax, AUC0-8 h and bioavailability of
FR194738 were higher than for NB-598. In dogs, NB-
598 was undetectable in the plasma during the experi-
ment. The superior pharmacokinetic properties of
4. Structure–activity relationships
Table 1 outlines some of our results on the determina-
tion of SAR for our new series of compounds. NB-598
is a highly lipophilic compound (ClogP=8.19). Since
the highly lipophilic properties of compounds are rela-
ted to poor solubility, permeation and absorption,15 we
attempted to increase polarity by incorporation of het-
eroatom-based linkers between the terminal thiophene,
shown to be essential,10 and the aryl amine portion of
the molecule. The ester compound 9 was about3 itmes
weaker than NB-598 towards human SE, however, the
ether 10 was slightly more potent (5.4 vs 7.2 nM). Of the
N-alkyl analogues prepared, the N-methyl analogue 11
had the lowest lipophilicity, but the N-ethyl analogue 10
was most potent.
Table 4. Pharmacokinetic parameters in rats
Compd
Cmax
(mg/mL)
Tmax (h)
AUC0À8 h
(mg h/mL)
BA
(%)
.
FR194738
NB-598
0.486
0.329
1.17
1.67
1.873
1.392
32.9
16.5
Rats were given the compound orally, as a suspension in 0.5%
methylcellulose at a dose of 10 mg/kg
Table 5. Plasma concentration in dogs
Compd
Dose (mg/kg)
Plasma concn (mg/mL)
Variation of the geminal dimethyl moiety of 10 is shown
in Table 2. It is clear from this table that only methyl is
suitable at this position as other groups afforded much
1 h
4 h
24 h
FR194738
NB-598
5
16
10
0.199
0.910
<0.05
0.138
0.717
<0.05
<0.05
<0.05
<0.05
Table 2. Compounds with replacements for the geminal dimethyl
moiety
Dogs were given the compound orally, as a suspension in 0.5%
methylcellulose.
Compd
R
Squalene epoxidase inhibitory
activity IC50 (nM)
ClogP
7.30
Human
5.4
Dog
10a
13
14
15
Me
Et120
Allyl
H
34
ND
8.36
160
25
7.2
ND
230
43
8.45
6.60
8.19
NB-598
ND, notdetermined
a Free form of FR194738.
Table 3. Squalene epoxidase inhibitory activity
Compd
Squalene epoxidase inhibitory
activity IC50 (nM)
ClogP
Human
Dog
Rat
FR194738
NB-598
9.8
7.2
14
43
25
36
7.30a
8.19
Figure 2. Inhibition of sterol biosynthesis in HepG2 cells by
FR194738 and HMG-CoA reductase inhibitors.
a Value for free form.

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M. Sawada et al. / Bioorg. Med. Chem. Lett. 14 (2004) 633–637
Table 6. Effects of FR194738 on plasma lipoprotein cholesterol
levels and atherogenic index in dogs
Compd
Dose
(mg/kg/day) cholesterol
LDL
HDL
cholesterol
Atherogenic
index
(% change) (% change) (% change)
Control
FR194738
—
3.2
10
32
+5
À2
+1
0
+3
À2
À47
À61
À14
À38
À47
À27
Dogs were orally given the compound twice a day. On day 14, blood
samples were taken and lipoprotein fractions were separated.
Atherogenic index=[LDL cholesterol]/[HDL cholesterol].
Figures represent% change from pre-value.
FR194738 compared with NB-598 may reflect the dif-
ference in lipophilicity and/or better metabolic stability.
The inhibitory effect of FR194738 in comparison to the
clinically used HMG-CoA reductase inhibitors, simvas-
tatin, fluvastatin and pravastatin, on cholesterol bio-
synthesis in HepG2 cells was next examined (Fig. 2).
Among these compounds, FR194738 was the most
potent, with an IC50 of 2.1 nM. The IC50s of simvasta-
tin, fluvastatin and pravastatin were 40, 28 and 5100
nM, respectively.
Figure 3. Induction of HMG-CoA reductase activity in HepG2 cells
by FR194738 and simvastatin.
HMG-CoA reductase activity in HepG2 cells after long-
term incubation with FR194738 or simvastatin is shown
in Figure 3. Simvastatin caused a concentration-dependent
increase of HMG-CoA reductase activity with a max-
imum increase of 18.8-fold. FR194738 also increased
the enzyme activity but the degree of the change was less
than simvastatin. The maximum increase by FR194738
was 4.6-fold. The difference between the two drugs
could be attributed to the mechanism of action. HMG-
CoA reductase activity is controlled through multi-
valent feedback regulation mediated by cholesterol and
non-sterol metabolites of mevalonate.6
In vivo lipid lowering effects of FR194738 in dogs are
shown in Figure 4. FR194738 dose-dependently lowered
plasma cholesterol and triglyceride levels. The max-
imum decrease for plasma cholesterol levels was 40%,
and that for plasma triglyceride levels was over 80%.
Reduced secretion of VLDL, triglyceride-rich particles,
by FR194738 might be responsible for the reduction in
triglyceride levels in dogs. Khan reported that choles-
terol is required for the secretion of VLDL.16 In addi-
tion, FR194738 decreased LDL cholesterol levels more
than HDL cholesterol levels, resulting in an improved
atherogenic index (LDL/HDL ratio) (Table 6).
In summary, FR194738 possessed comparable in vitro
potency to NB-598, but improved lipophilic and phar-
macokinetic properties. FR194738 inhibited cholesterol
biosynthesis in HepG2 cells more potently than the
HMG-CoA reductase inhibitors, simvastatin, fluvasta-
tin and pravastatin, with a minimal increase of HMG-
CoA reductase activity. FR194738 lowered plasma
cholesterol levels and improved atherogenic index in
dogs, and has potential to be highly effective in the
treatment of hypercholesterolemia.
Figure 4. Lipid-lowering effects of FR194738 in dogs.

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637
Acknowledgements
6. Brown, M. S.; Goldstein, J. L. J. Lipid Res. 1980, 21, 505.
7. Pazzucconi, F.; Dorigotti, F.; Gianfranceschi, G.; Cam-
pagnoli, G.; Sirtori, M.; Franceschini, G.; Sirtori, C. R.
Atherosclerosis 1995, 117, 189.
8. Fujioka, T.; Nara, F.; Tsujita, Y.; Fukushige, J.; Fukami,
M.; Kuroda, M. Biochim. Biophys. Acta 1995, 1254, 7.
9. Abe, I.; Tomesch, J. C.; Wattanasin, S.; Prestwich, G. D.
Natural Product Reports 1994, 11, 279.
The authors are grateful to Dr. David Barrett in the
Medicinal Chemistry Research Laboratories for useful
advice during the preparation of this manuscript.
References and notes
10. Iwasawa, Y.; Horie, M. Drugs of the Future 1993, 18, 911.
11. Gotteland, J. P.; Loubat, C.; Planty, B.; Junquero, D.;
´
1. Steinberg, D.; Witztum, J. L. JAMA 1990, 264, 3047.
2. Blankenhorn, D. H.; Hodis, H. N. West. J. Med. 1993,
159, 172.
3. Scandinavian Simvastatin Survival Study Group Lancet
1994, 344, 1383.
4. Shepherd, J.; Cobbe, S. M.; Ford, I.; Isles, C. G.; Lor-
imer, A. R.; Macfarlane, P. W.; McKillop, J. H.; Packard,
C. J. N. Eng. J. Med. 1995, 333, 1301.
5. Ghirlanda, G.; Oradei, A.; Manto, A.; Lippa, S.; Uccioli,
L.; Caputo, S.; Greco, A. V.; Littarru, G. P. J. Clin.
Pharmacol. 1993, 33, 226.
Delhon, A.; Halazy, S. Bioorg. Med. Chem. Lett. 1998, 8,
1337.
12. Chugh, A.; Ray, A.; Gupta, J. B. Prog. Lipid Res. 2003,
42, 37.
13. Erickson, S. K.; Fielding, P. E. J. Lipid Res. 1986, 27, 875.
14. Brown, M. S.; Dana, S. E.; Goldstein, J. L. J. Biol. Chem.
1974, 249, 789.
15. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney,
P. J. Adv. Drug Deliv. Rev. 1997, 23, 3.
16. Khan, B. V.; Fungwe, T. V.; Wilcon, H. G.; Heimberg,
M. Biochim. Biophys. Acta 1990, 1044, 297.