Synthesis and Biological Evaluation of Gem-Difluoromethylenated Statin Derivatives as Highly Potent HMG-CoA Reductase Inhibitors

Article abstract of DOI:10.1002/cjoc.201600180

HMG-CoA reductase inhibitors were widely used as lipid-lowing agents through effectively blocking the rate-limiting step of cholesterol biosynthesis. 8 analogs of Rosuvastatin were firstly prepared with different distance and functional group between the

Full text of DOI:10.1002/cjoc.201600180

FULL PAPER
DOI: 10.1002/cjoc.201600180
Synthesis and Biological Evaluation of Gem-Difluoro-
methylenated Statin Derivatives as Highly Potent
HMG-CoA Reductase Inhibitors
a
a
,a,b
a
a
a
Zhao Zhao, Jiaxin Cui, Yan Yin,* Heng Zhang, Yecheng Liu, Rui Zeng,
a
a
,a
,a
Chao Fang, Zhenpeng Kai, Zhonghua Wang,* and Fanhong Wu*
a
School of Chemical and Environmental Engineering, Shanghai Institute of Technology, Shanghai 201418, China
b
Key Laboratory of Organofluorine Chemistry, Shanghai Institute of Organic Chemistry,
Chinese Academy of Sciences, Shanghai 200032, China
HMG-CoA reductase inhibitors were widely used as lipid-lowing agents through effectively blocking the rate-
limiting step of cholesterol biosynthesis. 8 analogs of Rosuvastatin were firstly prepared with different distance and
functional group between the O5-hydroxyl group and terminal COOH group in the hydrophilic side-chain. In pri-
mary and secondary screening of the inhibitory activities against human HMG-CoA reductase, gem-difluorometh-
ylenated derivatives exhibited more than 50% inhibition rate. Then 4 compounds with gem-difluoro group were
further synthesized and evaluated in vitro, three compounds among them exhibited low single digital nmol/L IC50
values against HMG-CoA reductase. Molecular docking also well explained the observed special contribution of the
gem-difluoro group.
Keywords HMG-CoA reductase inhibitors, gem-difluoromethylenated compounds, statin derivatives, hypercho-
lesterolemia, molecular docking
Introduction
tors with novel structure is still needed in medical
community.
Coronary heart disease (CHD) is becoming a leading
cause of death and keeps an increasing pressure on
healthcare worldwide. Numerous and diverse studies
show that hypercholesterolemia is one of the key risk
Since fluorine substituents affect nearly all physical
and adsorption, distribution, metabolism, and excretion
properties of a lead compound, fluorine substituents
have become a widespread and important drug compo-
[
1]
[
2]
factors for the CHD. In recent years, statins as potent
-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA)
[6]
nents. The F substituent in the lipophilic pyrrole core
3
of atorvastatin was found to be superior to ligands with
reductase inhibitors were widely used as CHD drugs
through effectively blocking the rate-limiting step of
[6]
hydroxyl, hydrogen, or methoxy. After long focus on
synthesis, especially on the synthesis of bioactive fluo-
[3]
cholesterol biosynthesis. Except marked HMG-CoA
reductase inhibitors, such as Compactin, Pravastatin,
Lovastatin, Simvastatin, Fluvastatin, Carivastatin, Ato-
vastatin, Rosuvastatin, and Pitavastatin, several research
groups including Mexico group, Zhou group, Nissan
Chemical, BMS, and Pfizer have developed their novel
HMG-CoA reductase inhibitors in recent years (Figure
[7]
rine-containing compounds, we estimated that F sub-
stituents in the hydrophilic side-chain of statin deriva-
tives also cause positive effects on the enzymatic inhib-
itory activity. Herein, we would like to report the full
discovery process including synthesis, in vitro biologi-
cal evaluation, and molecular docking of the gem-
difluoromethylenated statin derivatives as highly potent
HMG-CoA reducatase inhibitors.
[
4]
1
). These HMG-CoA reductase inhibitors consisted of
three parts: hydrophilic β,δ-dihydroxy-pentanoic acid
unit or cyclized β-hydroxy-δ-lactone (red region in Fig-
ure 1), lipophilic aromatic core, and C－C or C＝C
linker (blue region in Figure 1). Although many modi-
fications focused on the lipophilic cores and synthesis
Experimental
Commercially available reagents and anhydrous
solvents were used without further purification unless
otherwise specified. Thin layer chromatography (TLC)
analyses were performed with precoated silica gel.
[
5]
optimization on the hydrophilic side-chain have been
done, discovery of potent HMG-CoA reductase inhibi-
*
E-mail: yinyan@sit.edu.cn; Tel.: 0086-021-60877220; Fax: 0086-021-60877331
Received March 22, 2016; accepted May 14, 2016; published online XXXX, 2016.
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cjoc.201600180 or from the author.
Chin. J. Chem. 2016, XX, 1—8
© 2016 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
1

Zhao et al.
FULL PAPER
F
H C
H C
3
3
H C
3
O
H
H C
3
O
H
H
OH
O
OH
O
OH
O
O
H
H
N
O
O-
O
O
OH
H
H
CH3
CH3
Fluvastatin
Simvastatin
F
Compactin
F
F
H C
3
O
OH
H C
3
O
O-
OH
H
N
N
N
OH
S
O-
O
O
H C
3
N
N
O-
O
N
O
OH
CH3 OH
CH3
H C
3
OH
H C
3
O
Carivastatin
Atorvastatin
Rosuvastatin
Figure 1 HMG-CoA reductase inhibitors.

1
Column chromatography was carried out on silica gel
300－400 mesh). All the products were purified by
1137, 1125, 965, 607, 566 cm ; HRMS calcd for
＋
(
C
18
H23FN
3
O
3
S [M＋H] : 380.1444, found 380.1433.
column chromatography on silica gel with ethyl acetate-
petroleum ether in an appropriate ratio as the eluent. All
compounds were determined to be ＞95% pure by pre-
pared HPLC. NMR spectra were recorded with a
Ethyl 2-bromo-2,2-difluoroacetate (1.89 mmol) was
added slowly at room temperature to a stirred mixture of
THF (2.5 mL) and freshly activated zinc powder (1.89
mmol). Then, a solution of acrylaldehyde 1 (0.63 mmol)
in THF (3 mL) was added dropwisely. After completion
of the addition, the reaction mixture was refluxed for 5
h, and then quenched with saturated ammonium chlo-
ride. The excess zinc powder was removed by suction
filtration and the filter cake was washed with ethyl ace-
tate. The combined filtrate was extracted with ethyl ac-
etate, washed with brine, dried over anhydrous sodium
sulfate, and concentrated in vacuum. The remaining
residue was purified by column chromatography on silica
gel [V(petroleum ether)∶V(ethyl acetate)＝5∶1] to
produce 3 as a yellow solid in 60% yield.
®
®
Bruker 400 MHz spectrometer or Bruker 500 MHz
spectrometer at ambient temperature with the residual
solvent peaks as internal standards. IR spectra (KBr)
were recorded on a Nicolet In10 FT-IR spectrometer in
1
the range of 400－4000 cm . The melting points were
measured on an SGW X-4 melting point instrument.
The HRMS spectra were acquired on a Solaril X70
FT-MS apparatus or Advion expression CMS apparatus.
[7a]
1
0d was obtained as a gift from Dr. Wang.
Synthetic procedures of inhibitors 2－4, 6－9, and
1
0a－10c
NaBH
of N-[4-(4-fluorophenyl)-6-isopropyl-5-(3-oxopropenyl)-
pyrimidin-2-yl]-N-methyl-methanesulfonamide (1
(E)-2,2-Difluoro-5-(4-(4-fluorophenyl)-6-isopropyl-
4
(0.5 mmol) was added dropwise to a solution
2-(N-methylmethylsulfonamido)pyrimidin-5-yl)-3-
1
hydroxypent-4-enoate (3) m.p. 170－171 ℃;
H
1
NMR (400 MHz, CDCl ) δ: 7.68－7.63 (m, 2H), 7.13－
3
mmol, 120.78 mg) in THF (5 mL) and MeOH (3 mL)
under 45 ℃. The reaction mixture was stirred for an-
other 2 h at 45 ℃, and then poured into a saturated
7.08 (m, 2H), 6.80 (d, J＝16.0 Hz, 1H), 5.64 (d, J＝
16.0 Hz, 1H), 4.71－4.64 (m, 1H), 4.34 (q, J＝6.4 Hz,
2H), 3.58 (s, 3H), 3.52 (s, 3H), 3.40－3.34 (m, 1H),
19
NaHCO
ethyl acetate and the combined organic phase was
washed with brine and dried over anhydrous Na SO
3
solution. The aqueous layer was extracted with
1.36 (t, J＝6.4 Hz, 3H), 1.29－1.26 (m, 6H); F NMR
(471 MHz, CDCl ) δ: 106.53 (s, 1F), 107.10 (s, 1F),
3
1
3
2
4
.
3
111.63 (s, 1F); C NMR (125 MHz, CDCl ) δ: 179.7,
After concentrated in vacuum, the residue was purified
by column chromatography on silica gel (petroleum
V(ether)∶V(ethyl acetate)＝4∶1) to provide com-
pound 2 as a yellow liquid in 41% yield.
168.0, 164.2, 158.0, 134.4, 130.7, 116.1, 115.7, 115.5,
77.3, 77.0, 76.8, 69.7, 63.5, 42.6, 33.5, 33.1, 23.7, 21.7,
13.8; IR (KBr) ν: 3475, 2966, 2926, 2850, 1759, 1605,
1548, 1510, 1439, 1338, 1231, 1154, 1079, 962, 844,
1
(E)-N-(4-(4-Fluorophenyl)-5-(3-hydroxyprop-1-en-
27 3 3 5
773, 575, 518 cm ; HRMS calcd for C22H F N O S
＋
1
-yl)-6-isopropylpyrimidin-2-yl)-N-methylmethane-
[M＋H] : 502.1624, found 502.1650.
1
sulfonamide (2) H NMR (400 MHz, CDCl
－
1
3
3
) δ: 7.67
NaOH (1.125 mmol) was added slowly to a solution
of ester 3 (0.225 mmol) in methanol (2 mL) at room
temperature. The resulting mixture was stirred for 2 h.
Then the reaction mixture was concentrated to remove
methanol, extracted with ethyl acetate. The aqueous
7.64 (m, 2H), 7.12－7.08 (m, 2H), 6.61－6.57 (m,
H), 5.69－5.63 (m, 1H), 4.22－4.21 (m, 2H), 3.57 (s,
H), 3.52 (s, 3H), 3.43－3.37 (m, 1H), 1.27 (s, 6H); IR
(KBr) ν: 3443, 2925, 2852, 2026, 1906, 1633, 1401,
2
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© 2016 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2016, XX, 1—8

Gem-Difluoromethylenated Statin Derivatives as Potent HMG-CoA Reductase Inhibitors
layer was washed with 10% HCl, extracted a second
time with ethyl acetate, washed with brine, dried over
anhydrous sodium sulfate, and concentrated in vacuum
to give a residue. The residue was purified by column
chromatography on silica gel [V(petroleum ether)∶
V(ethyl acetate)＝1∶1] to give 4 as a yellow oil in 59%
yield.
acetate. The combined organic phase was washed with
water, dried over anhydrous sodium sulfate and evapo-
rated under reduced pressure to give an oil residue. The
oil was purified by column chromatography on silica gel
[V(petroleum ether)∶V(ethyl acetate)＝5∶1) to pro-
vide 7 as a yellow oil in 21% yield.
(E)-tert-Butyl-4,4,5-trifluoro-7-(4-(4-fluorophenyl)-6-
(
E)-2,2-Difluoro-5-(4-(4-fluorophenyl)-6-isopropyl-
isopropyl-2-(N-methylmethylsulfonamido)pyrimidin-
1
2
-(N-methylmethylsulfonamido)pyrimidin-5-yl)-3-
hydroxypent-4-enoic acid (4) H NMR (DMSO-d
5-yl)-3-hydroxyhept-6-enoate (7)
MHz, CDCl
H NMR (500
1
6
,
3
) δ: 7.55 (d, J＝8.5 Hz, 2H), 7.20 (d, J＝
4
6
1
1
1
1
00 MHz) δ: 7.77－7.73 (m, 2H), 7.28－7.24 (m, 2H),
8.5 Hz, 2H), 6.49－6.38 (m, 1H), 6.31－6.17 (m, 1H),
5.98－5.86 (m, 1H), 4.19 (s, 1H), 3.60 (s, 3H), 3.54 (s,
3H), 3.48－3.35 (m, 1H), 2.67－2.44 (m, 2H), 1.49 (s,
.67－6.60 (m, 1H), 6.36 (br, 1H, OH), 5.86－5.60 (m,
H), 4.40－4.34 (m, 1H), 3.54 (s, 3H), 3.45 (s, 3H),
.23－1.19 (m, 6H); IR (KBr) ν: 3412, 2967, 2929,
648, 1604, 1545, 1509, 1437, 1382, 1337, 1229, 1154,
19
3
9H), 1.32－1.29 (m, 6H); F NMR (471 MHz, CDCl )
δ: 105.28－106.99 (m, 1F), 112.01－113.75 (m,
1
13
068, 965, 814, 775, 576, 566, 520 cm ; HRMS calcd
2F), 170.69－173.69 (m, 1F); C NMR (125 MHz,
＋
for C20
H F N O
23 3 3 5
S [M＋H] : 474.1311, found 474.1350.
3
CDCl ) δ: 178.6, 166.6, 163.9, 158.6, 137.1, 134.5,
Keeping the inner temperature at 78 ℃, n-butyl-
lithium (2.2 mol/L, 10.9 mL) was added to a solution of
diisopropylethylamine (3.4 mL, 24 mmol) in THF (5
131.2, 127.5, 116.6, 87.9, 86.5, 81.9, 60.4, 42.5, 32.9,
2
2
9
7.9, 22.7, 21.4, 21.0, 14.2, 1.0; IR (KBr) ν: 3359, 3192,
955, 2922, 2851, 1659, 1632, 1548, 1510, 1466, 1156,
mL) over 20 min under N atmosphere. The reaction
1
2
62, 668, 649 cm ; HRMS calcd for C26
H
34
F
4
N
3
O
5
S
mixture was stirred at 30 to 40 ℃ for 30 min.
tert-Butyl acetate (6.5 mL, 24 mmol) was added over 10
min. The reaction mixture was stirred for 30 min. Then
the mixture was cooled to 78 ℃, and a solution of 3 (8
mmol) in THF was added over 30 min. The reaction
mixture was stirred for 2 h, and then the temperature
was raised to 0 ℃ over 30 min. The reaction was
quenched with saturated aqueous ammonium chloride
solution and stirred for 15 min. The aqueous layer was
extracted with ethyl acetate and the combined organic
layer was washed with brine and dried over anhydrous
sodium sulfate. After evaporation of the solvent, the
residue 5 was used for the next step without further pu-
rification.
＋
[
M＋H] : 576.2155, found 576.2150.
TFA (15 mmol) was added to a solution of ester 7
0.5 mmol) in dichloromethane (15 mL), and the mix-
(
ture was stirred at 0 ℃ for 1 h，and at room tempera-
ture for 12 h. The mixture was then poured into a stir-
ring mixture of saturated aqueous NaHCO . The mix-
3
ture was extracted with dichloromethane. The organic
phase was washed with brine, dried over anhydrous so-
dium sulfate and evaporated under reduced pressure to
give an oil residue. The residue was purified by column
chromatography on silica gel [V(petroleum ether) ∶
V(ethyl acetate)＝1∶1) to provide 8 as a yellow oil in
4
6% yield.
E)-N-(5-(2-(3,3-Difluoro-4-hydroxy-6-oxotetra-
Following the synthesis procedure of 2 from 1, 6
was obtained from 5 as a yellow solid in 50% yield after
two steps.
(
hydro-2H-pyran-2-yl)vinyl)-4-(4-fluorophenyl)-6-
isopropylpyrimidin-2-yl)-N-methylmethanesulfon-
amide (8) H NMR (500 MHz, CDCl
8
Hz, 1H), 5.66 (d, J＝16.2 Hz, 1H), 5.38－5.28 (m, 1H),
4.29 (s, 1H), 3.60 (s, 3H), 3.54 (s, 3H), 3.44－3.35 (m,
(E)-tert-Putyl-4,4-difluoro-7-(4-(4-fluorophenyl)-6-
1
3
) δ: 7.67 (d, J＝
isopropyl-2-(N-methylmethylsulfonamido)pyrimidin-
.6 Hz, 2H), 7.12 (d, J＝8.6 Hz, 2H), 6.86 (d, J＝16.2
5
1
2
-yl)-3,5-dihydroxyhept-6-enoate (6) m.p. 111 －
1
12 ℃; H NMR (500 MHz, CDCl
3
) δ: 7.69－7.61 (m,
H), 7.11－7.07 (m, 2H), 6.82 (d, J＝16.0 Hz,1H), 5.69
d, J＝16.0 Hz, 1H), 4.64 (s, 1H), 3.57 (s, 3H), 3.52 (s,
19
(
1H), 3.07－2.89 (m, 2H), 1.29－1.26 (m, 6H); F NMR
471 MHz, CDCl ) δ: 111.11 (s, 1F), 123.91 (s, 1F),
3
9
H), 3.38－3.34 (m, 1H), 2.62－2.58 (m, 2H),1.48 (s,
H), 1.28 (d, J＝3.3 Hz, 3H), 1.27 (d, J＝3.3 Hz, 3H);
(

1
3
1
3
124.10 (s, 1F); C NMR (125 MHz, DMSO-d
74.8, 172.5, 167.4, 163.3, 157.5, 132.6, 126.6, 121.7,
15.6, 79.7, 79.2, 42.0, 33.6, 32.1, 31.7, 29.5, 21.9, 21.7,
6
) δ:
1
9
F NMR (471 MHz, CDCl
3
) δ: 110.86 (s, 1F),
13

117.91 (d, 1F), 127.55 (d, 1F); C NMR (125 MHz,
1
CDCl ) δ: 175.0, 171.9, 163.7, 157.7, 134.0, 132.2,
3
1
1
5
4.5; IR (KBr) ν: 3467, 2964, 2928, 1712, 1605, 1546,
1
3
2
1
28.0, 120.9, 115.3, 82.5, 77.3, 42.4, 34.4, 34.1, 33.1,
2.1, 29.5, 28.0, 21.7, 14.3, 1.0; IR (KBr) ν: 3442, 2981,
934, 1716, 1604, 1556, 1509, 1445, 1401, 1383, 1197,
509, 1439, 1381, 1335, 1230, 1155, 1066, 963, 845,

1
25 3 3 5
76, 520 cm ; HRMS calcd for C22H F N O S [M＋
＋
1
H] : 500.1467, found 500.1461.
Following the synthesis procedure of 7 from 6, 9
was obtained from 8 as a yellow oil in 22%.
(R,E)-N-(5-(2-(4,4-Difluoro-7-oxo-2,3,4,7-tetra-
hydro-oxepin-3-yl)vinyl)-4-(4-fluorophenyl)-6-
isopropylpyrimidin-2-yl)-N-methylmethane sulfon-
amide (9) H NMR (500 MHz, CDCl
(m, 2H), 7.17－7.07 (m, 2H), 6.94－6.90 (m, 1H),
6.87－6.83 (m, 1H), 6.36－6.34 (m, 1H), 5.72－5.68
156, 1096, 1066, 905, 840, 756, 567, 519 cm ; HRMS
＋
calcd for C H F N O S [M＋H] : 574.2199, found
2
6
35
3
3
6
5
74.2181.
To a mixture of DAST (3.78 mmol) in THF (10 mL)
at 78 ℃, a solution of 6（1.26 mmol）in THF (10 mL)
was added slowly. The mixture was stirred at 78 ℃
for 5 h, at 0 ℃ for 1 h, and at room temperature for 1 h.
The reaction mixture was then poured into saturated
1
3
) δ: 7.70－7.60
3
aqueous NaHCO . The mixture was extracted with ethyl
Chin. J. Chem. 2016, XX, 1—8
© 2016 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
3

Zhao et al.
FULL PAPER
(
(
6
1
m, 1H), 5.14－5.06 (m, 1H), 4.22－4.01 (m, 2H), 3.60
s, 3H), 3.54 (s, 3H), 3.42－3.36 (m, 1H), 1.30 (d, J＝
1H), 5.82－5.74 (m, 1H), 4.43－4.27 (m, 2H), 4.11－
4.03 (m, 1H), 2.53－2.33 (m, 1H), 2.33－2.23 (m, 1H),
19
.3 Hz, 6H); F NMR (471 MHz, CDCl
3
) δ: -106.51 (s,
2.15－2.07 (m, 1H), 1.25－1.23 (m, 2H), 1.03－1.01
1
3
19
F), -108.65 (s, 1F), 110.78 (s, 1F); C NMR (125
) δ: 175.0, 171.1, 163.9, 157.8, 137.9,
32.1, 125.1, 119.8, 115.5, 79.7, 60.4, 42.4, 33.1, 32.2,
(m, 2H); F NMR (471 MHz, DMSO-d
6
) δ: 116.59 (s,
1
3
MHz, CDCl
3
1F), 116.70 (s, 1F), 123.07 (s, 1F); C NMR (125
MHz, DMSO-d ) δ: 175.4, 163.9, 161.9, 147.1, 131.6,
1
2
2
6
9.7, 23.4, 21.7, 21.0, 14.1; IR (KBr) ν: 2961, 2925,
131.3, 130.9, 130.8, 130.3, 128.3, 126.7, 126.1, 125.8,
116.1, 90.1, 88.8, 60.6, 29.7, 21.0, 16.1, 14.2, 10.7; IR
(KBr) ν: 3362, 2977, 2851, 1887, 1659, 1500, 1456,
1402, 1437, 1347, 1107, 1095, 999, 971, 941, 839, 815,
854, 1750, 1605, 1547, 1509, 1439, 1382, 1338, 1229,
1
1
156, 1106, 1073, 962, 845, 815, 772, 575, 520 cm .
＋
HRMS calcd for C22
found 482.1354.
H
23
F N
3 3
O
4
S [M＋H] : 482.1356,
1
23 3 4
739, 564, 523 cm . HRMS calcd for C25H F NO [M
＋
Following the synthesis procedure of 4 from 3, 10a
was obtained from 6 as a yellow oil in 49% yield.
＋H] : 458.1579, found 458.1571.
[
10]
Biological evaluation in vitro
(
E)-4,4-Difluoro-7-(4-(4-fluorophenyl)-6-isopropyl-
2
-(N-methylmethylsulfonamido)pyrimidin-5-yl)-3,5-
All inhibitors were dissolved with NaOH/THF (1/1)
at 0.1 mol/L concentration and stored in a 20 ℃
fridge. Then statin inhibitors were diluted with the buff-
1
dihydroxyhept-6-enoic acid (10a) H NMR (500
MHz, DMSO-d ) δ: 7.76－7.71 (m, 2H), 7.29－7.24 (m,
H), 6.74 (d, J＝16.0 Hz, 1H), 5.65 (d, J＝16.0 Hz, 1H),
6
4
5
6
2
4
3
1
er to the concentration of 1×10 , 1×10 , 1×10 ,
7
8
.55－4.43 (m, 1H), 4.16－4.01 (m, 1H), 3.55 (s, 3H),
.45 (s, 3H), 2.58－2.54 (m, 1H), 2.36－2.30 (m, 1H),
1×10 , or 1×10 mol/L. The spectrophotometer was
seated at 37 ℃ and 340 nm, with a kinetic program.
Then 181 μL assay buffer, 1 μL inhibitor or pravastatin,
4 μL NADPH, 12 μL HMG-CoA, and HMG-CoA re-
ductase were added to the well, respectively. Then the
samples mixture was mixed thoroughly and the kinetics
program was started immediately. The enzymatic reac-
tion was initiated by the addition of HMG-CoA. The
UV absorbance was measured twice for each sample.
The inhibitory rate values were worked out by the equa-
tion in Figure 2. The IC50 values were worked out
through graphpad prism 5 nonlinear regression calcula-
tion.
19
.23－1.21 (m, 6H); F NMR (471 MHz, DMSO-d
6
) δ:

111.53－111.92 (m, 1F), 118.87 (d, J＝250.6 Hz,
13
1
F), 122.38 (d, J＝250.6 Hz, 1F); C NMR (125 MHz,
DMSO-d ) δ: 174.9, 163.3, 162.1, 157.5, 134.6, 132.6,
26.9, 121.7, 115.5, 70.4, 67.3, 60.2, 55.1, 42.0, 36.5,
6
1
3
2
1
5
5
3.6, 31.8, 21.9, 14.1; IR (KBr) ν: 3466, 2969, 2932,
872, 2389, 2251, 1711, 1605, 1545, 1510, 1438, 1381,
333, 1231, 1155, 1067, 964, 901, 845, 777, 576, 566,
1
＋
20 cm ; HRMS calcd for C22
18.1573, found 518.1565.
H
27
F
3
N
3
O
6
S [M＋H] :
Following the synthesis procedure of 10a from 1,
0b was obtained from 3-[3-(4-fluorophenyl)-1-iso-
1
propyl-1H-indol-2-yl]-propenal as a yellow oil in 11%
yield after 4 steps.
7
-[2-Cyclopropyl-4-(4-fluorophenyl)-quinolin-3-
yl]-4,4-difluoro-3,5-dihydroxyhept-6-enoic acid (10b)
1
H NMR (500 MHz, DMSO-d
.45－7.42 (m, 3H), 7.25－7.21 (m, 2H), 7.18－7.14
m, 1H), 7.06－7.02 (m, 1H), 6.86－6.82 (m, 1H,),
.97－5.90 (m, 1H), 5.83－5.77 (m, 2H), 4.95－4.88
m, 1H), 4.56－4.45 (m, 1H), 4.18－4.10 (m, 1H), 2.60
2.58 (m, 1H), 2.38－2.24 (m, 1H), 1.68－1.65 (m,
6
) δ: 7.68－7.66 (m, 1H),
7
(
5
(
－
19
6

H); F NMR (471 MHz, DMSO-d ) δ: -113.86 (s, 1F),
6
13
123.43 － -123.54 (m, 2F); C NMR (125 MHz,
DMSO) δ: 172.6, 162.1, 160.2, 135.4, 133.6, 132.1,
1
6
3
1
5
4
27.9, 122.3, 120.1, 119.3, 115.9, 114.0, 112.5, 69.3,
6.0, 60.2, 56.3, 47.6, 35.9, 21.5, 14.5; IR (KBr) ν: 3392,
068, 2927, 2855, 1709, 1636, 1574, 1513, 1489, 1411,
365, 1261, 1158, 1093, 1057, 1023, 932, 841, 765, 619,
Figure 2 Formula of inhibition rate.
Molecular docking

1
＋
58, 530 cm ; HRMS calcd for C H F NO [M＋H] :
24
25
3
4
The ligand preparation was performed using SYBYL
.0. Energy minimization was optimized using Tripos
48.1736, found 448.1731.
Following the synthesis procedure of 10a from 1,
0c was obtained from 3-[2-cyclopropyl-4-(4-fluoro-
2
molecular mechanics force field with energy gradient
criterion of 0.005 kcal (Å•mol), and the atom charges of
Tripos force field were calculated by the Gasteiger-
Huckel method. The crystal structures of HMGR for
molecular docking were downloaded from RCSB Pro-
tein Data Bank (http:/www.rcsb.org/pdb/home/home.do,
PDB: 2Q1L). All water molecules were removed and
hydrogen atoms were added. In the docking process, the
protein was fixed while the ligands were flexible, the
1
phenyl)-quinolin-3-yl]-propenal as a yellow oil in 15%
yield after 4 steps.
(
E)-4,4-Difluoro-7-[3-(4-fluorophenyl)-1-isopro-
pyl-1H-indol-2-yl]-3,5-dihydroxyhept-6-enoic acid
) δ: 7.85－7.86
m, 1H), 7.68－7.67 (m, 1H), 7.45－7.44 (m, 1H), 7.31
1
(
10c) H NMR (500 MHz, DMSO-d
6
(
－
7.39 (m, 4H), 7.25－7.27 (m, 1H), 6.73－6.67 (m,
4
www.cjc.wiley-vch.de
© 2016 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2016, XX, 1—8

Gem-Difluoromethylenated Statin Derivatives as Potent HMG-CoA Reductase Inhibitors
ligands were docked into the ATP-binding site of
HMGR by the Surflex-Dock module and the docked
complex was evaluated by empirical scoring function.
By default, 30 conformations were generated because
each ligand and their scores were forecasted based on
the strength of receptor-ligand interactions.
our F-substituted modifications for it has the best IC
50
[8]
value among those listed drugs in Figure 1. The dis-
tance and functional group between the O5-hydroxyl
group and the terminal COOH group were first studied.
Eight compounds (2－4, 6－9, and 10a) with different
chain lengths and functional groups were firstly synthe-
sized and submitted to the preliminary biological evalu-
ation in vitro. All of them exhibited inhibitory activities
against HMG-CoA reductase at the concentration of
Results and Discussion
Chemistry
6
5
×10 mol/L (Table 1). The inhibition rates were 44%
for 2 with CH OH as the side-chain, 21% for 3 with
γ-hydroxy-β,β-difluoro-ethyl ester as the side-chain,
1% for 4 with γ-hydroxy-β,β-difluoropropionic acid as
The synthesis of target compounds 2－4, 6－9, and
0a－10d was presented in Scheme 1. Commercial
2
1
available 3-substituted propenals 1 was reduced to
-substituted allylic alcohol (2) by sodium borohydride
2
3
the side-chain, 77% for 6 with β,δ-diols-γ,γ-difluoro-
tert-butyl ester as the side-chain, 50% for 7 with
δ,γ,γ-trifluoro-β-hydroxyl-tert-butyl ester as the side-
chain, 59% for 8 with γ,γ-difluro-β-hydroxy-δ-lactone as
the tail, 88% for 9 with γ,γ-difluro-α,β-unsaturated-δ-
lactone as the side-chain, 80% for 10a with β,δ-dihy-
droxy-γ,γ-difluoropentanoic acid as the side-chain, re-
spectively. Shorting the chain length from 3 to 1 meth-
ylene unit obviously decreased the activity (Entries 4, 5
and 8 vs. Entries 2 and 3). Although all of them (Ester 6,
eliminated lactone 8, and acid 10a) exhibited good in-
hibitory activities for the enzymatic hydrolysis to car-
boxylate anion, the inhibition rates between them were
different due to the hydrolysis degree and 10a had the
highest inhibition rate among them (Entry 8 vs. Entries
in methanol. Zinc-mediated Reformatsky reaction of 1
with ethyl bromodifluoroacetate gave the ester 3. Sub-
sequent ester 3 was hydrolyzed to acid 4 with aqueous
sodium hydroxide in ethanol. Treatment of ester 3 with
acetic acid tert-butyl ester and lithium diisopropylamide
(
LDA) gave the δ-hydroxy-β-keto ester (5). Reduction
of carbonyl group afforded the β,δ-diols tert-butyl ester
6). Compound 6 was converted into the δ,γ,γ-trifluoro
(
ester (7) by the treatment with diethylamido sulfur tri-
fluoride (DAST). To our expectation, compound 7 un-
derwent cyclization to give lactone 8 in the presence of
TFA in CH Cl , and no hydrolysis product was obtained.
2
2
Reaction of compound 8 and DAST gave the hydroxyl
elimination product 9. Finally, β,δ-diol-ester (6) was
hydrolyzed by sodium hydroxide to produce compound
1
0.
Bioassay
In this study, all the synthesized compounds were
4
and 6). The replacement of methylene unit to
gem-difluoromethylene slightly increased the inhibition
rate from 77% to 80% (Entry 8 vs. Entry 9). However,
the replacement of O5-hydroxy to fluoro caused lower
inhibition rate (Entry 4 vs. Entry 5). It should be pointed
out that compounds (2－4, 6－9, and 10a) were all
racemates. Five compounds (6－9, and 10a) exhibited
more than 50% inhibition rate at the concentration of 5
evaluated for their ability to inhibit human HMG-CoA
reductase. The assay is based on the spectrophotometric
measurement of the decrease in absorbance at 340 nm,
which represents the oxidation of NADPH by the cata-
lytic subunit of HMG-CoA reductase in the presence of
the substrate of HMG-CoA. The test was performed by
using a spectrophotometric method according to a
commercial HMG-CoA reductase assay kit (Sigma-
Aldrich).
6
×
10 mol/L, therefore they were diluted to lower con-
centration and submitted to second round in vitro bio-
logical evaluation.

7
Rosuvastatin was chosen as the lead compound for
At the concentration of 5×10 mol/L, all com-
4 2
Scheme 1 Reagents and condition: (a) NaBH , MeOH, 0 ℃; (b) BrCF COOEt, Zn, THF, reflux; (c) 1 equiv. NaOH, MeOH; then 2
equiv. HCl; (d) acetic acid tert-butyl ester, LDA, -78 ℃; (e) DAST, -78 ℃; (f) TFA, DCM
OH OH
O
O
a
Ar
d
OH
Ar
OH
Ar
H
F
F
10
1
2
b
c
OH
O
OH
F
O
O
OH OH O
a
Ar
O
Ar
F
O
Ar
O
F
F
F
F F
6
3
5
e
c
OH
F
F
OH O
OH
O
F
e
f
F
Ar
O
Ar
OH
Ar
O
O
F F
F
F
Ar
O
O
4
9
8
7
Chin. J. Chem. 2016, XX, 1—8
© 2016 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
5

Zhao et al.
FULL PAPER
Table1 Inhibition rates
Continued
a
a
Entry
Compound
Inhibition rate /%
Entry
Compound
Inhibition rate /%
F
1
44
21
21
77
50
59
88
8
80
O
N
N
2
OH
S
N
O
F
OH
O
2
9
77
O
N
O
F
F
S
O
N
N
3
a
Inhibition rates were means of three or more experiments with
errors within 30% of them, and the concentration was 5×10
mol/L.

6
3
pounds (6－9, 10a, and pitavastatin) exhibited lower
inhibitory activities compared with the concentration of
×10 mol/L, and the inhibition rates order from high
to low was 72% for inhibitor 10a, 70% for inhibitor 6,
2% for pitavastatin, 46% for inhibitor 7, 40% for in-
hibitor 9, and 29% for inhibitor 8 (Table 2). Again
gem-difluoromethylenated ester 6 exhibited the similar
good inhibition rate as acid 10a for ester was easy to
hydrolyze to carboxylate anion under test conditions
(Entry 1 vs. 5), however gem-difluoromethylenated
lactone 8 and 9 showed ＜50% inhibition rates proba-
ble due to the hydrolysis was too lower compared with
ester (Entries 3 and 4 vs. Entry1). Trifluoro-containing
inhibitor 7 also showed ＜50% inhibition rates (Entry
6
5
F
6
OH OH
O
4
O
N
O
F
F
S
O
N
N
6
5
2
).
Table 2 Inhibition rates
a
Entry
Compound
Inhibition rate /%
1
2
3
4
5
6
6
7
8
9
70
46
29
40
72
62
6
a
10
pitavastatin
a
Inhibition rates were means of three or more experiments with
errors within 30% of them, and the concentration was 5×10
mol/L.

7
7
Since 10a displayed the best inhibition rate in both
primary and second screening, it was treated as the new
lead compound, and the modification on the lipophilic
region of 10a was then investigated (Table 3). For the
6
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© 2016 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2016, XX, 1—8

Gem-Difluoromethylenated Statin Derivatives as Potent HMG-CoA Reductase Inhibitors
convenience of the synthesis, the preparation of gem-di-
fluoro-substituted inhibitors 10a－10d was started from
commercial available 3-substituted (E)-propenals.
Rosuvastatin derivative 10a, fluvastatin derivative 10b,
and pitavastatin derivative 10c had a little bit better in-
hibitory activities against HMG-CoA reductase com-
pared with pitavastatin (IC ＝2.8 nmol/L for 10a, 3.1
nmol/L for 10b, and 4.7 nmol/L for 10c vs. 8.4 nmol/L
for pitavastatin), however the simplification of the lip-
ophilic part to phenyl ring caused the totally lost of the
inhibitory activities in the test window, probably due to
the missing of the interaction between 4-fluorophenyl
group and enzyme, and polar interaction between the
arginine ε-nitrogen atoms and the fluorine atoms (En-
tries 4－5).
[8]
According to the structure-activity relationship (SAR)
investigation of compounds 2－4, 6－9, and 10a－10d,
several informations were concluded that keeping chain
length as 3 methylene units between O5-hydroxyl group
and the terminal COOH group was necessary for the
potency, gem-difluoro modification at γ-position of the
hydrophilic side-chain benefited the inhibitory activity,
the replacement of O5-hydroxyl by F generally de-
creased the activity, compounds with terminal COOH
group had higher inhibition rate than esters or lactones,
and the interaction between 4-fluoro-phenyl group in
lipophilic region and enzyme was one of the key factors
for the high potency of inhibitors.
50
Table 3 IC50 values
a
－1
Entry
Compound
IC50 /(nmol•L
)
F
Molecular docking
Computational approaches have aided our drug dis-
OH OH
O
[
9]
covery process for a long time. Molecular docking,
which is widely used to explore the binding affinities of
ligands, was used to guide and better explain the SAR
for this series of gem-difluoromethylenated statin deriv-
atives as highly potent HMG-CoA reductase inhibitors.
1
2.8
3.1
4.7
O
N
OH
F
F
S
N
N
O
1
0a
1
0a as the most active inhibitor was docked into the
catalytic domain of human HMG-CoA reductase (PDB:
Q1L). As shown in Figure 3, the binding motif of
3R,5S)-10a revealed 9 H-bond interactions were
2
(
2
formed between ligand and protein: Lys691 and gem-
3
4
b
NR
F
OH OH
O
5
8.4
OH
N
pitavastatin
a
IC50 values were means of three or more experiments with errors
within 30% of them, and compounds were tested at five concen-
Figure 3 Docking results of (3R,5S)-10a. Hydrogen interac-
tions were shown as green lines, and hydrophobic interactions
were shown as pink lines.

6



10
trations (5×10 , 5×10 , 5×10 , 5×10 , and 5×10
b
mol/L). Out of the test window.
Chin. J. Chem. 2016, XX, 1—8
© 2016 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
7

Zhao et al.
FULL PAPER
difluoro group at the side-chain, Arg590 and the fluo-
rine atom in the lipophlic region, Arg590 and O3-hy-
droxyl group, His752 and O5-hydroxyl group, Lys735/
Ser684 and carbonyl group, Ala751 and terminal OH,
Asp690 amd H of C3. In addition to the H-bond interac-
tions, an important contributor to the high potency of
Prachavasittikul, V. Eur. Food Res. Technol. 2015, 240, 1.
[
2] (a) Han, K. H.; Rha, S. W.; Kang, H. J.; Bae, J. W.; Choi, B. J.; Choi,
S. Y.; Gwon, H. C.; Bae, J. H.; Hong, B. K.; Choi, D. H.; Han, K. R.
J. Clin. Lipidol. 2012, 6, 340; (b) Bowles, D. M.; Boyles, D. C.;
Choi, C.; Pfefferkom, J. A.; Schuyler, S. Org. Process Res. Dev.
2
011, 15, 148.
[
[
3] (a) Tani, S.; Nagao, K.; Hirayama, A. Am. J. Cardiovasc. Drugs
2012, 12, 349; (b) Besseling, J.; Capelleveen, J. V.; Kastelein, J. J. P.;
Hovingh, G. K. Drugs 2013, 73, 293; (c) Tiwari, R.; Pathak, K. J.
Pharm. Pharmacol. 2011, 63, 983.
1
0a was the hydrophobic interactions. 6 hydrophobic
interactions were formed: the 4-fluorophenyl moiety
and Val683, isopropyl group and His752/Leu562/
Cys561, pyrimidine moiety and Leu853. Similar
H-bond interactions were also observed between protein
and the gem-difluoro group in 10b and 10c. So the
docking results well explained the observed SAR that
the introduction of gem-difluoromethylenated moiety at
the side chain contributed the HMG-CoA reductase in-
hibitory activity. Meanwhile, the presence of O5-hy-
droxyl group was essential.
4] (a) Zhao, S. K.; Zhou, W. C.; Liu, J. Bioorg. Med. Chem. 2009, 17,
7
915; (b) Pfefferkorn, J. A.; Choi, C.; Larsen, S. D.; Auerbach, B.;
Hutchings, R.; Park, W.; Askew, V.; Dillon, L.; Hanselman, J. C.;
Lin, Z. W.; Lu, G. H.; Robertson, A.; Sekerke, C.; Harris, M. S.;
Pavlovsky, A.; Bainbrigde, G.; Caspers, N.; Kowala, M.; Tait, B. D.
J. Med. Chem. 2008, 51, 31; (c) Ahmad, S.; Madsen, C. S.; Stein, P.
D.; Janovitz, E.; Huang, C.; Ngu, K.; Bisaha, S.; Kennedy, K. J.;
Chen, B. C.; Zhao, R. L.; Sitkoff, D.; Monshizadegan, H.; Yin, X. H.;
Ryan, C. S.; Zhang, R. G.; Giancarli, M.; Bird, E.; Chang, M.; Chen,
X.; Setters, R.; Search, D.; Zhuang, S.; Nguyen-Tran, V.; Cuff, C. A.;
Harrity, T.; Darienzo, C. J.; Li, T.; Reeves, R. A.; Blannar, M. A.;
Barrish, J. C.; Zahler, R.; Robl, J. A. J. Med. Chem. 2008, 51, 2722;
Conclusions
(
d) Park, W. K. C.; Kennedy, R. M.; Larsen, S. D.; Miller, S.; Roth,
In this work, 11 compounds were synthesized and
evaluated against human HMG-CoA reductase. The ob-
tained SAR demonstrated that gem-difluoro group on
the hydrophobic side-chain of the statin derivatives in-
creased the inhibition efficiency against the HMG-CoA
reductase. Molecular modelling studies indicated that
gem-difluoromethylenated Rosuvastatin derivative 10a
had H-bond interaction between the gem-difluoro group
and protein, which probable well explained the higher
potency of these series inhibitors. More biological
evaluation about these gem-difluoromethylenated statin
derivatives 10a－10c including cell assay, PK, and
small animal models is underway and will be reported
in due course.
B. D.; Song, Y. T.; Steinbaugh, B. A.; Sun, K.; Tait, B. D.; Kowala,
M. C.; Trivedi, K.; Auerbach, B.; Askew, V.; Dillon, L.; Hanselman,
J. C.; Lin, Z. W.; Lu, G. H.; Robertson, A.; Sekerke, C. Bioorg. Med.
Chem. Lett. 2008, 18, 1151; (e) Suzuki, M.; Iwasaki, H.; Fujikawa,
Y.; Kitahara, M.; Sakashita, M.; Sakoda, R. Bioorg. Med. Chem.
2001, 9, 2727.
[5] (a) Wu, Y.; Xiong, F. J.; Chen, F. E. Tetrahedron 2015, 71, 8487; (b)
Luo, Y. F.; Roy, I. D.; Madec, A. G. E.; Lam, H. W. Angew, Chem.,
Int. Ed. 2014, 53, 4186; (c) Hao, Q.; Pan, J.; Li, Y. J.; Cai, Z. Y.;
Zhou, W. C. Org. Process Res. Dev. 2013, 17, 921.
6] Muller, K.; Faeh, C.; Diederich, F. Science 2007, 317, 1881.
7] (a) Wang, X. G.; Fang, X.; Xiao, H. Y.; Yin, Y.; Xiao, H. M.; Wu, F.
H. J. Fluorine Chem. 2012, 133, 178; (b) Wang, Z.; Cheng, L. P.;
Kai, Z. P.; Wu, F. H.; Liu, Z. Y.; Cai, M. F. Bioorg. Med. Chem. Lett.
2014, 24, 2725; (c) Yin, Y.; Sun, G. F.; Zhang, H.; Zhou, H.; Wu, F.
H. Chin. J. Chem. 2014, 32, 365.
[
[
[
[
8] Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160.
9] (a) Yin, Y.; Zheng, K.; Eid, N.; Howard, S.; Jeong, J. H.; Yi, F.; Guo,
J.; Guo, J.; Park, C. M.; Bibian, M.; Wu, W. L.; Hernandez, P.; Park,
H.; Wu, Y. T.; Luo, J. L.; LoGrasso, P. V.; Feng, Y. B. J. Med. Chem.
Acknowledgement
The work was supported by National Natural Sci-
ence foundation of China (Nos. 21502117, 21172148),
and Shanghai Municipal Education Commission (Plat-
eau Discipline Construction Program).
2
015, 58, 1846; (b) Ding, M.; Yin, Y.; Wu, F. H.; Cui, J. X.; Zhou, H.;
Sun, G. F.; Jiang, Y.; Feng, Y. B. Bioorg. Med. Chem. 2015, 23, 2505;
c) Cui, J. X.; Yin, Y.; Deng, W.; Ding, M.; Wang, Z. H.; Jiang, Y.;
Zhou, H.; Sun, G. F.; Feng, Y. B. Bioorg. Med. Chem. 2015, 23, 7464;
d) Yin, Y.; Lin, L.; Ruiz, C.; Khan, S.; Cameron, S. K.; Grant, W.;
(
(
Pocas, J.; Eid, N.; Park, H.; Schroeter, T.; LoGrasso, P. V.; Feng, Y.
B. J. Med. Chem. 2013, 56, 3568.
References
[
1] (a) Gelsinger, C.; Steinhagen-Thiessen, E.; Kassner, U. Drugs 2012,
2, 1445; (b) Sinthupoom, N.; Prachavasittikul, V.; Ruchirawat, S.;
[10] Check the Sigma-Aldrich’s product information of HMG-CoA re-
ductase assay kit for detail.
7
(Pan, B.; Qin, X.)
8
www.cjc.wiley-vch.de
© 2016 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2016, XX, 1—8