Discovery and SAR study of hydroxyacetophenone derivatives as potent, non-steroidal farnesoid X receptor (FXR) antagonists

Article abstract of DOI:10.1016/j.bmc.2014.01.032

Compound 1 (IC₅₀ = 35.2 ± 7.2 μM), a moderate FXR antagonist was discovered via high-throughput screening. Structure-activity relationship studies indicated that the shape and the lipophilicity of the substituents of the aromatic ring affect the activity dramatically, increasing the shape and the lipophilicity of the substituents of the aromatic ring enhances the potency of FXR antagonists. Especially, when the OH at C2 position of the aromatic ring was replaced by the OBn substituent (analog 2b), its activity could be improved to IC₅₀ = 1.1 ± 0.1 μM. Besides, the length of the linker and the tetrazole structure are essential for retaining the activity.

Full text of DOI:10.1016/j.bmc.2014.01.032

Bioorganic & Medicinal Chemistry 22 (2014) 1596–1607
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery and SAR study of hydroxyacetophenone derivatives
as potent, non-steroidal farnesoid X receptor (FXR) antagonists
a,b,
Peng Liu a, , Xing Xu b, , Lili Chen b, Lei Ma a, Xu Shen b, , Lihong Hu
⇑
⇑
a Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, PR China
b Shanghai Research Center for Modernization of Traditional Chinese Medicine, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Compound 1 (IC50 = 35.2 7.2 lM), a moderate FXR antagonist was discovered via high-throughput
Received 28 December 2013
Revised 17 January 2014
Accepted 20 January 2014
Available online 30 January 2014
screening. Structure–activity relationship studies indicated that the shape and the lipophilicity of the
substituents of the aromatic ring affect the activity dramatically, increasing the shape and the lipophil-
icity of the substituents of the aromatic ring enhances the potency of FXR antagonists. Especially, when
the OH at C2 position of the aromatic ring was replaced by the OBn substituent (analog 2b), its activity
could be improved to IC50 = 1.1 0.1
essential for retaining the activity.
l
M. Besides, the length of the linker and the tetrazole structure are
Keywords:
FXR antagonist
SAR study
Ó 2014 Elsevier Ltd. All rights reserved.
Hydroxyacetophenone derivatives
Non-steroidal
1. Introduction
which limit their further development.19,20 At the same time, a
variety of FXR antagonists were developed and found promising
results in treatment of cholestasis and hypercholesterolemia.21,22
Although FXR antagonists were believed to be promising drug
candidates, up to now, only a few FXR antagonists were reported
in the literature. Guggulsterone was the first FXR antagonist re-
ported in the literature, which was further proved to be partial
FXR agonist and promiscuous ligand for many nuclear recep-
tors.23–25 Several natural FXR antagonists, lithocholic acid,26 scala-
rane sesterterpene,27 stigmasterol acetate,28 tuberatolide,29
As a member of nuclear hormone receptor superfamily, FXR
(farnesoid X receptor; NR1H4) is a bile acid sensor mainly ex-
pressed in liver, intestine, kidney and adrenals.1 Since the deorph-
anization of FXR in 1999,2 a variety of researches have been
demonstrated on its role in bile acid regulation, lipid and glucose
homeostasis.3–6 FXR is activated by bile acids (BAs) at physiological
concentrations. Bile acid-activated FXR is essential in protecting
hepatic cell from excess BAs at two aspects. BA-activated FXR in-
duces the expression of SHP (small heterodimer partner), leading
33
gorgosterol,30 sulfated sterol,31,32 and 15d-PGJ2 and synthesized
to the down-regulation of CYP7A1 (cholesterol 7
a
-hydroxylase),
FXR antagonists, substituted isoxazole derivatives34 and 1,3,4-tri-
substituted pyrazolone derivatives22 were also described so far.
Limitations of these antagonists, however, include weak antagonist
activity and difficulty in obtaining large amount of materials.
Therefore, more potent, structurally novel and easily obtainable
FXR antagonists were still needed in this field. Herein, we wish
to report our research of discovery and optimization of hydroxy-
acetophenone derivatives as potent FXR antagonists.
Compound 1 was discovered via high–throughput screening
with the moderate FXR antagonist activity (IC50 = 35.2 7.2 lM).
In an attempt to improve the antagonist activity, structure–activity
relationship (SAR) studies were carried out (Fig. 1) and 27 analogs
of compound 1 were synthesized and tested with biology activity.
which is a rate-limiting enzyme in bile acid biosynthesis.7,8 At
the same time, activation of FXR secretes the excess bile acid into
the bile via the inducing of the expression of BSEP (bile salt export
pump) and represses the bile acid uptake via the negative feedback
regulation of NTAP (sodium taurocholate cotransporting polypep-
tide).9,10 Besides its role in the bile acid regulation, FXR has been
demonstrated to connect with the cholesterol disposal, lipid and
triglyceride metabolism, glucose homeostasis, and heart disease.
Further researches imply that FXR ligands show great potential
in treatment of these diseases.11,12 Varieties of potent FXR agonists
have been developed and tested in preclinical and clinical trials.13–18
Reduction of HDL (high density lipoprotein) and increased levels of
LDL (low density lipoprotein) as well as inhibition of basolateral
transporters in the liver are the main drawbacks of these ligands,
2. Chemistry
⇑
The synthesis of the derivatives 2a–2f, 3a–3c, 4a–4f and 5a–5d
was outlined in Scheme 1. Resorcin 6 was assembled by acylation
Corresponding authors. Tel./fax: +86 20231965.
E-mail addresses: lhhu@simm.ac.cn (X. Shen), xshen@mail.shcnc.ac.cn (L. Hu).
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmc.2014.01.032
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

P. Liu et al. / Bioorg. Med. Chem. 22 (2014) 1596–1607
1597
R1
O
O
O
OH
N
HN
R4
R3
N
O
N
H
O
3
n
O
O
R2
2
1
N
3a-3h
2a-2f
HN
4
O
N
N
5
3
OH
O
OH
1
R5
N
HN
N
HN
N
N
N
O
O
N
3
3
4a-4f
R6
Figure 1. The structure and the SAR strategy of HTS hit (1).
5a 5d
-
with various aliphatic acids to afford compounds 7a–7d. Different
acylated resorcins 7a–7d were reduced to 4-alkyl resorcins 8a–8d
with zinc dust in the presence of concentrated hydrochloric acid,
which were subsequently acylated with different aliphatic or aro-
matic acids to give various 1-acyl-5-alkyl resorcins 9a–9j. Nucleo-
philic substitution between isobutyronitrile 11 and different
length of di-halogenated alkanes 10a–10d in the presence of LDA
(lithium diisopropylamide) at À78 °C generated 12a–12d, which
coupled with resorcine analogs 9a–9j to afford compounds 13a–
13m. Compounds 13a–13m were further assembled by cycloaddi-
tion reaction with TMSN3 to generate target products 3a–3c, 4b–4f
and 5a–5d.35 The 1-carbonyl reduction product 4a was achieved
via the reduction of the intermediate 13a (R6, R7 = Me, Me; n = 3)
with zinc dust before assembled with the same cycloaddition pro-
cedure with TMSN3. Etherification of intermediate 13a with differ-
ent halogenated alkanes afforded compounds 14a–14f, which
reacted with TMSN3 to give analogs 2a–2f.
compound 17. The intermediate 17 was reduced with DIBAL-H
(diisobutylaluminum hydride) to give 6-(benzyloxy)hexan-1-ol,
which reacted with TsCl smoothly produced corresponding
tosylate 18. Substitution of 18 with TMSCN in the presence of
CsF generated 7-(benzyloxy)heptanenitrile 19.
a-Me nitiles 20a
and -Et nitiles 20b, accomplished via nucleophilic substitution
a
reaction of 19 with MeI and EtBr, together with compound 19,
were deprotected with hydrogenation before chlorination with
thionyl chloride to give intermediates 21a–21c. Compounds
21a–21c then treated with 1-(5-ethyl-2,4-dihydroxyphenyl)etha-
none 9a to generate intermediates 22a–22c, which reacted with
TMSN3 to afford the derivatives 3d–3f. Isostere derivative 3g was
obtained via hydrolysis of 13a in NaOH/MeOH system, which re-
acted with MeOH catalyzed by H2SO4 to give methyl ester analog
3h.
3. Result and discussion
The analogs 3d–3h were synthesized as outlined in Scheme 2.
Hydrolysis of 6-hexanolactone 16 with MeONa afforded methyl
6-hydroxyhexanoate, which protected with BnBr to give
The binding of compound 1 derivatives were evaluated by their
antagonistic activity against CDCA (chenodeoxycholic acid, FXR
O
OH
OH
O
OH
OH
R7
a
b
a
R6
R6
OH
Cl
d
OH
OH
OH
R6
9a-9j
6
7a-7d
8a-8d
11
c
CN
Br
Cl
+
n
NC
O
n
10a-10d
12a-12d
OR1
O
OH
O
OR1
R7
e
d
H
N
O
O
O
CN
CN
n
3
N
3
R6
N
N
13a-13m
2a-2f
14a-14f
b
e
O
OH
OH
OH
e
R7
H
H
N
O
O
N
O
CN
n
3
N
3
N
R6
3a-3c, 4b-4f, 5a-5d
N
N
N
N
4a
15
Scheme 1. Reagents and conditions: (a) R6COOH or R7COOH, ZnCl2, 90 °C; (b) Zn, concd HCl, EtOH, reflux; (c) LDA, THF, À78 °C; (d) K2CO3, KI, 2-butone, 80 °C; (e) TMSN3,
TBAF, DMF, 100 °C.

1598
P. Liu et al. / Bioorg. Med. Chem. 22 (2014) 1596–1607
O
a
b
c
O
BnO
BnO
OBn
COOMe
OTs
18
16
NC
17
d
e
NC
NC
OBn
Cl
R
R
19
20a-20b
21a-21c
O
OH
O
OH
N
f
g
HN
N
CN
N
O
O
R
R
h
22a-22c
3d-3f
O
OH
O
OH
3g, R = H
3h, R = Me
i
O
O
CN
COOR
13a
Scheme 2. Reagents and conditions: (a) (i) MeONa, MeOH, (ii) BnBr, Ag2O, neat; (b) (i) DIBAL-H, THF, 0 °C, (ii) TsCl, Et3N, DCM; (c) TMSCN, CsF, DMF; (d) LDA, RBr or RI, THF,
À78 °C; (e) (i) Pd/C, H2, MeOH; (ii), SOCl2, DCM, 50 °C; (f) K2CO3, KI, 2-butone, 80 °C; (g) TMSN3, TBAF, DMF, 100 °C; (h) 2 N NaOH, MeOH; (i) H2SO4 (cat), MeOH, reflux.
endogeneous agonist) induced FXR
a
LBD coactivator recruitment
mono-Me substituted analog 3e and mono-Et substituted analog
3f all showed reduced potency. The above results shown the struc-
tures of the linker and tetrazole were essential for the derivatives
to maintain the FXR antagonist activity.
The antagonist activities of the analogs 4a–4f and 5a–5d are
shown in Table 3. Increasing the steric bulkiness by extending
the 1-acetyl group with one or more carbon (4b–4e) or introducing
the aromatic ring (4f) improved the potency. Reduction of the
1-acetyl to ethyl (4a) resulted in increasing in antagonist activity
based on HTRF (homogeneous time-resolved florescence).22,33
As shown in Table 1, etherification of 2-OH with different
halogenated alkanes (2a–2f) all resulted in increasing of potency.
2-OMe derivative 2a shown 6-fold improvement in antagonist
potency, 2-benzyl ether derivative 2b was proved to be the most
potent FXR antagonist in this series with the IC50 = 1.1 0.1 lM,
the other two benzyl ether derivatives 2c and 2d shown compara-
ble activity. It deserve to be mentioned that the antagonist activity
of 2b is almost 100-fold more potent than the reference FXR antag-
onist GS. At the same time, the bulky alkyl ether derivative 2e and
2f showed slightly weaker antagonist activities than benzyl ether
derivatives.
As shown in Table 2, shortening or increasing the length of the
linker resulted in loss of the activity. Changing the tetrazole struc-
ture of 1 to acid (3g) or methyl ester (3h) resulted in loss of po-
tency. The dimethyl substituents were essential in maintaining
the FXR antagonist activity while the di-H substituted analog 3d,
to IC50 = 3.1 0.2 lM. Changing the 5-ethyl with various alkyl sub-
stituents affected the activity interestingly. Replacing the 5-ethyl
with n-propyl (5a), iso-butyl (5b) and n-butyl (5c) increased the
potency with the length and bulkiness of substituent increased,
however, further increasing the length to n-hexyl (5d) reduced
the activity, which illustrated the space volume of 5-substituent
was essential for the activity of the derivatives.
The above SAR studies demonstrated that the bulky, lipophilic
substituents of the aromatic ring shown better FXR antagonist
Table 1
FXR antagonist activities of 2a–2f
O
OR1
H
N
O
N
N
N
OR1
IC50
(lM)
Compd
OR1
IC50
(lM)
a
a
Compd
O
O
1
OH
35.2 7.2
2d
1.7 0.3
O
2a
2b
OMe
5.5 0.8
1. 1 0.1
2e
2f
3.3 0.3
2.9 0.4
O
O
2c
1.2 1.3
GSb
/
89.36 12.67
a
The IC50 value is the concentration of the test compound required to decrease the activity of 50
The mixture of Z-GS and E-GS with the ratio about 1:1.
lM CDCA by 50%.
b

P. Liu et al. / Bioorg. Med. Chem. 22 (2014) 1596–1607
1599
Table 2
FXR antagonist activities of 3a–3h
O
OH
R2
R1
R3
O
n
R1, R2
R3
IC50
(
l
M)
Compd
n
R1, R2
H, Me
R3
IC50 (lM)
a
a
Compd
n
N
N
HN
HN
HN
HN
HN
HN
HN
1
3
Me, Me
N
35.2 7.2
3e
3
N
/
N
N
N
N
3a
3b
3c
3d
1
2
4
3
Me, Me
Me, Me
Me, Me
H, H
N
/
/
/
/
3f
3
3
3
H, Et
N
/
N
N
N
N
3g
3h
Me, Me
Me, Me
COOH
>100
/
N
N
N
COOMe
N
N
N
N
a
The IC50 value is the concentration of the test compound required to decrease the activity of 50
lM CDCA by 50%.
Table 3
FXR antagonist activities of 4a–4f and 5a–5d
OH
R1
H
O
N
R2
N
N
N
R1
Et
R2
Et
IC50
(
lM)
Compd
R1
R2
Et
IC50
(l
M)
a
a
Compd
O
4a
3.1 0.2
8.5 7.2
4f
3.1 0.2
Ph
O
O
4b
4c
Et
Et
5a
Ac
25.2 7.8
8.6 0.3
5b
Ac
5.8 0.5
O
O
2
4
4d
4e
Et
Et
5.4 0.4
8.4 1.0
5c
Ac
Ac
1.5 0.3
3.2 0.4
3
5d
5
a
The IC50 value is the concentration of the test compound required to decrease the activity of 50 lM CDCA by 50%.
activity. The best substituents of C1, C2 and C5 of the aromatic ring
and 2-OBn derivative 2b was found to be the most potent one.
The potent, easy obtained antagonist 2b may assist in the research
of the relationship between FXR antagonist and the metabolism
diseases. The further research of the 2b is ongoing and will be re-
ported in the future.
were proved to be ethyl (4a, IC50 = 3.1 0.2
l
M), OBn (3b,
M), respec-
IC50 = 1.1 0.1 M) and n-butyl (5c, IC50 = 1.5 0.3
l
l
tively. To further improve the antagonist activity, the combination
of the three best substituents analogs 1a, 1b and 1c (Fig. 2) was
synthesized with the similar procedure as Scheme 1. However,
the results shown changing the two positions of the aromatic ring
at the same time cannot further improve the antagonist activity
(Fig. 2).
5. Experimental
5.1. Chemistry
4. Conclusion
1H spectra were recorded on Varian-400 MHz spectrometer at
400 MHz with TMS as an internal standard. Chemical shifts (d)
are given in ppm relative to TMS, coupling constants (J) in Hz. Mass
spectra were measured with Thermo Finnigan LCQ-Advantage.
High-resolution mass spectra were recorded with a Finnigan
MAT-8430 instrument. All starting materials and reagents were
commercial products that were used without further purification.
In summary, the SAR of the lead compound 1 was investigated
in detail. Increasing the lipophilicity at C1, C2 and C5 of the aro-
matic ring enhances the potency while changes in the linker and
the tetrazole structure diminish the antagonist activity. The spatial
volume of the aromatic ring is crucial in maintaining the potency

1600
P. Liu et al. / Bioorg. Med. Chem. 22 (2014) 1596–1607
OBn
OH
O
OBn
N
HN
N
N
HN
HN
N
N
N
O
N
O
N
O
N
3
3
3
n-Bu
n-Bu
1a,
1b,
1c,
I C50 = 7.8 1.9 μ M
I C50 = 3.5 0.4 μ M
IC50 = 8.7 1.7 μ M
Figure 2. The combination of the 2-OBn analog 2b, 2-ethyl analog 4a and 5-n-Butyl analog 5c.
5.1.1. 1-(2,4-Dihydroxy-5-propylphenyl)ethanone (9a)
z = 237 [M+H]+. HRMS (ESI): calcd for C14H21O3 [M+H]+ 237.1485,
Resorcinol (1.1 g, 10 mmol) was combined with anhydrous
ZnCl2 (3.1 g, 23.41 mmol) in propionic acid (5 mL) and heated at
95–100 °C for 6 h. After completion of the reaction (monitored by
TLC), The mixture was cooled to 45–50 °C and added to ice-cold
6 N HCl (10 mL). After stirring 30 min, the dark solid was filtered
off, washed with water (20 mL), and dried to yield the crude prod-
uct. The crude product was purified with column chromatography
(petroleum ether (60–90 °C)/acetone) on silica gel to afford the de-
sired products 7a (1.2 g, 73%).
The purified 7a (1.2 g, 7.3 mmol) was mixed with zinc dust
(6.4 g, 100 mmol) in EtOH (10 mL) and concd HCl (10 ml) was
added dropwisely over 10 min. After the addition, the system re-
acted for 2–4 h and filtered, the filtrate was concentrated with re-
duced pressure to afford the crude product 8a (1 g, 90%).
The mixture of the above crude product 8a (1 g, 6.5 mmol) and
anhydrous ZnCl2 (1.9 g, 14 mmol) in acetic acid (5 mL) were
heated at 95–100 °C for 6 h. The mixture was cooled and added
to ice-cold 6 N HCl (10 mL). After stirring 30 min, the dark solid
was filtered off, washed with water (20 mL), and dried to yield
the crude product. The crude product was further purified with
column chromatography (petroleum ether (60–90 °C)/acetone)
on silica gel to afford the desired products 9a (0.9 g, 70%) as a
white solid. 1H NMR (400 MHz, CDCl3) d 12.83 (s, 1H), 7.46 (s,
1H), 6.33 (s, 1H), 5.49 (s, 1H), 2.58 (s, 3H), 2.55 (t, J = 7.4 Hz,
2H), 1.62–1.47 (m, 2H), 0.98 (t, J = 7.3 Hz, 3H). MS (ESI): m/
z = 195 [M+H]+. HRMS (ESI): calcd for C11H15O3 [M+H]+
195.1015, found 195.1017.
found 237.1488.
5.1.5. 1-(5-Ethyl-2,4-dihydroxyphenyl)ethanone (9e)
Compound 9e was prepared with the direct acylation of com-
mercial available 4-ethylresorcinol with glacial acetic acid as de-
scribed in the synthesis of compound 9a with a 72% yield as a
white solid. 1H NMR (400 MHz, CDCl3) d 12.62 (s, 1H), 7.48 (s,
1H), 6.36 (s, 1H), 2.69–2.55 (m, 5H), 1.24 (t, J = 7.5 Hz, 3H). MS
(ESI): m/z = 181 [M+H]+. HRMS (ESI): calcd for C10H13O3 [M+H]+
181.0859, found 181.0861.
5.1.6. 1-(5-Ethyl-2,4-dihydroxyphenyl)propan-1-one (9f)
Compound 9f was prepared with the direct acylation of com-
mercial available 4-ethylresorcinol with propionic acid as de-
scribed in the synthesis of compound 9a with a 79% yield as a
white solid. 1H NMR (400 MHz, CDCl3) d 12.68 (s, 1H), 7.52 (s,
1H), 6.35 (s, 1H), 5.64 (s, 1H), 2.99 (q, J = 7.3 Hz, 2H), 2.60 (q,
J = 7.5 Hz, 2H), 1.25 (t, J = 7.3 Hz, 3H), 1.23 (t, J = 7.5 Hz, 3H). MS
(ESI): m/z = 195 [M+H]+. HRMS (ESI): calcd for C11H15O3 [M+H]+
195.1015, found 195.1015.
5.1.7. 1-(5-Ethyl-2,4-dihydroxyphenyl)-2-methylpropan-1-one
(9g)
Compound 9g was prepared with the direct acylation of com-
mercial available 4-ethylresorcinol with isobutyric acid as de-
scribed in the synthesis of compound 9a with a 71% yield as a
white solid. 1H NMR (400 MHz, CDCl3) d 12.86 (s, 1H), 7.55 (s,
1H), 6.36 (s, 1H), 5.57 (s, 1H), 3.77–3.22 (m, 1H), 2.61 (q,
J = 7.5 Hz, 2H), 1.29–1.21 (m, 9H). MS (ESI): m/z = 209 [M+H]+.
HRMS (ESI): calcd for C12H17O3 [M+H]+ 209.1172, found 209.1173.
5.1.2. 1-(2,4-Dihydroxy-5-isobutylphenyl)ethanone (9b)
Compound 9b was prepared in a similar manner to that de-
scribed in the synthesis of compound 9a with the isobutyric acid
instead of propionic acid in the first acylation reaction. 42% yield
over three steps, white solid. 1H NMR (400 MHz, CDCl3) d 12.53
(s, 1H), 7.43 (s, 1H), 6.34 (s, 1H), 5.49 (s, 1H), 2.58 (s, 3H), 2.45
(d, J = 7.2 Hz, 2H), 1.94–1.87 (m, 1H), 0.95 (d, J = 6.6 Hz, 6H). MS
(ESI): m/z = 209 [M+H]+. HRMS (ESI): calcd for C12H17O3 [M+H]+
209.1172, found 209.1175.
5.1.8. 1-(5-Ethyl-2,4-dihydroxyphenyl)hexan-1-one (9h)
Compound 9h was prepared with the direct acylation of com-
mercial available 4-ethylresorcinol with hexanoic acid as described
in the synthesis of compound 9a with a 65% yield as a white solid.
1H NMR (400 MHz, CDCl3) d 12.73 (s, 1H), 7.51 (s, 1H), 6.35 (s, 1H),
2.92 (t, J = 7.5 Hz, 2H), 2.61 (q, J = 7.6 Hz, 2H), 1.86–1.72 (m, 2H),
1.47–1.35 (m, 4H), 1.25 (t, J = 7.5 Hz, 3H), 0.94 (t, J = 7.2 Hz, 3H).
MS (ESI): m/z = 237 [M+H]+. HRMS (ESI): calcd for C14H21O3
[M+H]+ 237.1485, found 237.1482.
5.1.3. 1-(5-Butyl-2,4-dihydroxyphenyl)ethanone (9c)
Compound 9c was prepared in a similar manner to that de-
scribed in the synthesis of compound 9a with the butyric acid in-
stead of propionic acid in the first acylation reaction. 34% yield
over three steps, white solid. 1H NMR (400 MHz, CDCl3) d 12.56
(s, 1H), 7.47 (s, 1H), 6.34 (s, 1H), 2.67–2.50 (m, 5H), 1.69–1.52
(m, 2H), 1.48–1.32 (m, 2H), 0.98 (t, J = 7.2 Hz, 3H). MS (ESI): m/
z = 209 [M+H]+. HRMS (ESI): calcd for C12H17O3 [M+H]+ 209.1172,
found 209.1170.
5.1.9. 1-(5-Ethyl-2,4-dihydroxyphenyl)octan-1-one (9i)
Compound 9i was prepared with the direct acylation of com-
mercial available 4-ethylresorcinol with octanoic acid as described
in the synthesis of compound 9a with a 60% yield as a white solid.
1H NMR (400 MHz, CDCl3) d 12.74 (s, 1H), 7.51 (s, 1H), 6.35 (s, 1H),
2.92 (t, J = 7.5 Hz, 2H), 2.61 (q, J = 7.7 Hz, 2H), 1.78–1.72 (m, 2H),
1.42–1.29 (m, 8H), 1.25 (t, J = 7.5 Hz, 3H), 0.91 (t, J = 7.3 Hz, 3H).
MS (ESI): m/z = 265 [M+H]+. HRMS (ESI): calcd for C16H25O3
[M+H]+ 265.1798, found 265.1797.
5.1.4. 1-(5-Hexyl-2,4-dihydroxyphenyl)ethanone (9d)
Compound 9d was prepared in a similar manner to that de-
scribed in the synthesis of compound 9a with the hexanoic acid in-
stead of propionic acid in the first acylation reaction. 27% yield
over three steps, white solid. 1H NMR (400 MHz, CDCl3) d 12.56
(s, 1H), 7.47 (s, 1H), 6.34 (s, 1H), 2.67–2.50 (m, 5H), 1.69–1.52
(m, 2H), 1.48–1.32 (m, 2H), 0.98 (t, J = 7.2 Hz, 3H). MS (ESI): m/
5.1.10. (5-Ethyl-2,4-dihydroxyphenyl)(phenyl)methanone (9j)
The solution of 4-ethylresorcinol (180 mg, 1 mmol) and anhy-
drous AlCl3 (269 mg, 2 mmol) in DCM (10 mL) was cooled with
ice-water bath, benzoic chloride (160 mg, 1.1 mmol) was added,

P. Liu et al. / Bioorg. Med. Chem. 22 (2014) 1596–1607
1601
then warmed to rt and stirred for 20 h. The mixture was quenched
5.1.16. 7-(2-Ethyl-5-hydroxy-4-propionylphenoxy)-2,2-
dimethylheptanenitrile (13b)
with water, washed with 6 N HCl, water, brine, dried with Na2SO4
and concentrated in vacuo to afford the crude product. Purification
by column chromatography (petroleum ether (60–90 °C)/EtOAc)
Compound 13b was prepared with the etherification of 9f and
12a in a similar manner as described in the synthesis of 13a with
the yield of 65% as a white solid. 1H NMR (400 MHz, CDCl3) d
12.80 (s, 1H), 7.48 (s, 1H), 6.39 (s, 1H), 4.02 (t, J = 6.3 Hz, 2H),
2.98 (q, J = 7.3 Hz, 2H), 2.58 (q, J = 7.5 Hz, 2H), 1.94–1.83 (m, 2H),
1.57–1.51 (m, 6H), 1.38 (s, 6H), 1.25 (t, J = 7.3 Hz, 3H), 1.20 (t,
J = 7.5 Hz, 3H). MS (ESI): m/z = 332 [M+H]+. HRMS (ESI): calcd for
gave desired product 9j (58%) as
a
white solid. 1H NMR
(400 MHz, CDCl3) d 12.50 (s, 1H), 7.77–7.46 (m, 5H), 7.37 (s, 1H),
6.45 (s, 1H), 5.79 (s, 1H), 2.55 (q, J = 7.5 Hz, 2H), 1.16 (t,
J = 7.5 Hz, 3H). MS (ESI): m/z = 243 [M+H]+. HRMS (ESI): calcd for
C
15H15O3 [M+H]+ 243.1015, found 243.1018.
C
20H30NO3 [M+H]+ 332.2220, found 332.2221.
5.1.11. 7-Chloro-2,2-dimethylheptanenitrile (12a)
The solution of isobutyronitrile (0.7 g, 10 mmol) in THF (20 mL)
was cooled to À80 °C and LDA (Lithium diisopropylamide) solution
(2.0 M in hexane, 6 mL, 12 mmol) was added over 10 min. The
solution was stirred 30 min, and then a solution of 1-bromo-5-
chloropentane (1.8 g, 10 mmol) in THF (10 mL) was added over
5 min. After stirring for 90 min, 6 N HCl (6 mL) was added slowly
and the temperature allowed to rise to rt. The residue was concen-
trated under reduced pressure and EtOAc (20 mL) was added. The
separated organic layer was washed with water (10 mL). The or-
ganic solvent was dried with Na2SO4 and concentrated in vacuo
to give the crude product. Purification by column chromatography
(petroleum ether (60–90 °C)/EtOAc) gave 1.5 g of 2,2-dimethyl-7-
chloroheptanenitrile (88%) as a colorless oil. 1H NMR (400 MHz,
CDCl3) d 3.57 (t, J = 6.6 Hz, 2H), 1.92–1.72 (m, 2H), 1.58–1.44 (m,
6H), 1.36 (s, 6H).
5.1.17. 7-(2-Ethyl-5-hydroxy-4-isobutyrylphenoxy)-2,2-
dimethylheptanenitrile (13c)
Compound 13c was prepared with the etherification of 9g and
12a in a similar manner as described in the synthesis of 13a with
the yield of 79% as a colorless oil. 1H NMR (400 MHz, CDCl3) d
12.76 (s, 1H), 7.57 (s, 1H), 6.35 (s, 1H), 4.00 (t, J = 6.2 Hz, 2H),
3.79–3.66 (m, 1H), 2.44 (q, J = 7.5 Hz, 2H), 1.75–1.55 (m, 4H),
1.38 (s, 6H), 1.35–1.27 (m, 4H), 1.18–1.09 (m, 9H). MS (ESI): m/
z = 346 [M+H]+. HRMS (ESI): calcd for C21H32NO3 [M+H]+
346.2376, found 346.2372.
5.1.18. 7-(2-Ethyl-4-hexanoyl-5-hydroxyphenoxy)-2,2-
dimethylheptanenitrile (13d)
Compound 13b was prepared with the etherification of 9h and
12a in a similar manner as described in the synthesis of 13a with
the yield of 88% as a colorless oil. 1H NMR (400 MHz, CDCl3) d
12.88 (s, 1H), 7.47 (s, 1H), 6.38 (s, 1H), 4.02 (t, J = 6.2 Hz, 2H),
3.02 (t, J = 7.2 Hz, 2H), 2.58 (q, J = 7.5 Hz, 2H), 1.80–1.62 (m, 4H),
1.59–1.50 (m, 4H), 1.47–1.27 (m, 12H), 1.20 (t, J = 7.5 Hz, 3H),
0.93 (t, J = 7.2 Hz, 3H). MS (ESI): m/z = 374 [M+H]+. HRMS (ESI):
calcd for C23H36NO3 [M+H]+ 374.2689, found 374.2691.
5.1.12. 5-Chloro-2,2-dimethylpentanenitrile (12b)
Compound 12b was prepared using 1-bromo-3-chloropropane
instead of 1-bromo-5-chloropentane in a similar manner as de-
scribed in the synthesis of 12a with a 78% yield as a colorless oil.
1H NMR (400 MHz, CDCl3) d 3.48 (t, J = 6.6 Hz, 2H), 1.87–1.70 (m,
2H), 1.52–1.41 (m, 2H), 1.35 (s, 6H).
5.1.19. 7-(2-Ethyl-5-hydroxy-4-octanoylphenoxy)-2,2-
dimethylheptanenitrile (13e)
5.1.13. 6-Chloro-2,2-dimethylhexanenitrile (12c)
Compound 12c was prepared using 1-bromo-4-chlorobutane
instead of 1-bromo-5-chloropentane in a similar manner as de-
scribed in the synthesis of 12a with a 71% yield as a colorless oil.
1H NMR (400 MHz, CDCl3) d 3.52 (t, J = 6.6 Hz, 2H), 1.85–1.70 (m,
2H), 1.62–1.43 (m, 4H), 1.35 (s, 6H).
Compound 13e was prepared with the etherification of 9i and
12a in a similar manner as described in the synthesis of 13a with
the yield of 74% as a colorless oil. 1H NMR (400 MHz, CDCl3) d
12.87 (s, 1H), 7.47 (s, 1H), 6.38 (s, 1H), 4.02 (t, J = 6.3 Hz, 2H),
2.94–2.89 (m, 2H), 2.59 (q, J = 7.5 Hz, 2H), 1.99–1.82 (m, 2H),
1.81–1.69 (m, 2H), 1.59–1.52 (m, 6H), 1.37 (s, 6H), 1.37–1.29 (m,
8H), 1.20 (t, J = 7.5 Hz, 3H), 0.91 (t, J = 6.8 Hz, 3H). MS (ESI): m/
z = 402 [M+H]+. HRMS (ESI): calcd for C25H40NO3 [M+H]+
402.3002, found 402.3005.
5.1.14. 8-Chloro-2,2-dimethyloctanenitrile (12d)
Compound 12d was prepared using 1-bromo-6-chlorohexane
instead of 1-bromo-5-chloropentane in a similar manner as de-
scribed in the synthesis of 12a with a 76% yield as a colorless oil.
1H NMR (400 MHz, CDCl3) d 3.55 (t, J = 6.6 Hz, 2H), 1.83–1.65 (m,
2H), 1.78–1.41 (m, 8H), 1.38 (s, 6H).
5.1.20. 7-(4-Benzoyl-2-ethyl-5-hydroxyphenoxy)-2,2-
dimethylheptanenitrile (13f)
Compound 13f was prepared with the etherification of 9j and
12a in a similar manner as described in the synthesis of 13a with
the yield of 89% as a colorless oil. 1H NMR (400 MHz, CDCl3) d
12.68 (s, 1H), 7.72–7.51 (m, 5H), 7.34 (s, 1H), 6.49 (s, 1H), 4.07 (t,
J = 6.2 Hz, 2H), 2.53 (q, J = 7.4 Hz, 2H), 1.94–1.86 (m, 2H), 1.62–
1.57 (m, 6H), 1.38 (s, 6H), 1.12 (t, J = 7.5 Hz, 3H). MS (ESI): m/
z = 380 [M+H]+. HRMS (ESI): calcd for C24H30NO3 [M+H]+
380.2220, found 380.2223.
5.1.15. 7-(4-Acetyl-2-ethyl-5-hydroxyphenoxy)-2,2-
dimethylheptanenitrile (13a)
To a solution of ethanone 9e (180 mg, 1 mmol) and nitrile 12a
(190 mg, 1.1 mmol) in MEK (5 mL) were added K2CO3 (280 mg,
2 mmol), KI (26 mg, 0.2 mol), and the mixture was heated at reflux
with stirring for 30 h. The mixture was then cooled and diluted
with water (10 mL). The product was extracted with EtOAc
(2 Â 15 mL). The organic solution was washed with 0.5 N NaOH
(5 mL), 1 N HCl (5 mL), water (3 mL), and brine (10 mL), and then
dried with Na2SO4. The residue was concentrated by vacuo to yield
crude product which was further purified with column chromatog-
raphy (petroleum ether (60–90 °C)/acetone) on silica gel to afford
the desired products 13a (260 mg, 82%) as a white solid. 1H NMR
(400 MHz, CDCl3) d 12.72 (s, 1H), 7.44 (s, 1H), 6.38 (s, 1H), 4.03
(t, J = 6.3 Hz, 2H), 2.65–2.50 (m, 5H), 1.93–1.82 (m, 2H), 1.57–
1.50 (m, 6H), 1.38 (s, 6H), 1.20 (t, J = 7.5 Hz, 3H). MS (ESI): m/
z = 318 [M+H]+. HRMS (ESI): calcd for C19H28NO3 [M+H]+
318.2063, found 318.2061.
5.1.21. 7-(4-Acetyl-5-hydroxy-2-propylphenoxy)-2,2-
dimethylheptanenitrile (13g)
Compound 13g was prepared with the etherification of 9a and
12a in a similar manner as described in the synthesis of 13a with
the yield of 69% as a colorless oil. 1H NMR (400 MHz, CDCl3) d
12.64 (s, 1H), 7.66 (s, 1H), 6.38 (s, 1H), 4.08 (t, J = 6.3 Hz, 2H),
2.60 (s, 3H), 2.53 (t, J = 7.6 Hz, 2H), 1.99–1.83 (m, 2H), 1.66–1.51
(m, 8H), 1.35 (s, 6H), 0.93 (t, J = 7.3 Hz, 3H). MS (ESI): m/z = 332
[M+H]+. HRMS (ESI): calcd for C20H30NO3 [M+H]+ 332.2220, found
332.2223.

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P. Liu et al. / Bioorg. Med. Chem. 22 (2014) 1596–1607
5.1.22. 7-(4-Acetyl-5-hydroxy-2-isobutylphenoxy)-2,2-
dimethylheptanenitrile (13h)
5.1.28. 1-(5-Ethyl-2-hydroxy-4-((6-methyl-6-(1H-tetrazol-5-
yl)heptyl)oxy)phenyl)ethanone (1)
Compound 13h was prepared with the etherification of 9b and
12a in a similar manner as described in the synthesis of 13a with
the yield of 77% as a colorless oil. 1H NMR (400 MHz, CDCl3) d
12.69 (s, 1H), 7.39 (s, 1H), 6.38 (s, 1H), 4.01 (t, J = 6.2 Hz, 2H),
2.57 (s, 3H), 2.43 (d, J = 7.1 Hz, 2H), 1.91–1.81 (m, 3H), 1.58–1.50
(m, 6H), 1.38 (s, 6H), 0.92 (d, J = 6.6 Hz, 6H). MS (ESI): m/z = 346
[M+H]+. HRMS (ESI): calcd for C21H32NO3 [M+H]+ 346.2376, found
346.2379.
Compound 13a (63 mg, 0.2 mmol) was combined with TMSN3
(500 mg, 4 mmol) in a flame-dried round-bottom flask. The tube
was sealed and then evacuated and backfilled with nitrogen (3
times). DMF (1 mL) and TBAFÁ3H2O (1.2 g, 4 mmol) was added
via syringe into the reaction system, then the temperature was
raised to 100 °C and kept at this temperature for 20 h. After the
completing of the reaction (monitored by TLC), the reaction was
cooled to rt and Water (10 mL) was added to dilute the reaction.
The product was extracted with EtOAc (2 Â 15 mL), the organic
layer was washed with brine, dried with Na2SO4 and concentrated
in vacuo to yield the crude product which was further purified
with column chromatography (petroleum ether (60–90 °C)/ace-
tone/acetic acid) to give the compound 1 with the yield of 72% as
a white solid. 1H NMR (400 MHz, CDCl3) d 7.61 (s, 1H), 6.44 (s,
1H), 3.99 (t, J = 6.2 Hz, 2H), 2.55 (s, 3H), 2.50–2.45 (m, 2H), 1.72–
1.63 (m, 4H), 1.40–1.30 (m, 8H), 1.10 (t, J = 7.5 Hz, 3H). MS (ESI):
m/z = 361 [M+H]+. HRMS (ESI): calcd for C19H29N4O3 [M+H]+
361.2234, found 361.2231.
5.1.23. 7-(4-Acetyl-2-butyl-5-hydroxyphenoxy)-2,2-
dimethylheptanenitrile (13i)
Compound 13i was prepared with the etherification of 9c and
12a in a similar manner as described in the synthesis of 13a with
the yield of 83% as a colorless oil. 1H NMR (400 MHz, CDCl3) d
12.70 (s, 1H), 7.43 (s, 1H), 6.38 (s, 1H), 4.02 (t, J = 6.3 Hz, 2H),
2.59–2.52 (m, 5H), 1.93–1.74 (m, 2H), 1.57–1.49 (m, 8H), 1.44–
1.33 (m, 8H), 0.96 (t, J = 7.3 Hz, 3H). MS (ESI): m/z = 346 [M+H]+.
HRMS (ESI): calcd for
346.2377.
C
21H32NO3 [M+H]+ 346.2376, found
5.1.29. 1-(5-Ethyl-2-hydroxy-4-((4-methyl-4-(1H-tetrazol-5-
yl)pentyl)oxy)phenyl)ethanone (3a)
5.1.24. 7-(4-Acetyl-2-hexyl-5-hydroxyphenoxy)-2,2-
dimethylheptanenitrile (13j)
Compound 3a was prepared with the [3+2] cyclization reaction
of 13k with TMSN3 in a similar manner as described in the synthe-
sis of 3a with the yield of 82% as a white solid. 1H NMR (400 MHz,
CDCl3) d 12.82 (s, 1H), 7.43 (s, 1H), 6.35 (s, 1H), 3.97 (t, J = 6.0 Hz,
2H), 2.63–2.48 (m, 5H), 2.04–1.95 (m, 2H), 1.83–1.68 (m, 2H),
1.56 (s, 6H), 1.18 (t, J = 7.4 Hz, 3H). MS (ESI): m/z = 333 [M+H]+.
Compound 13j was prepared with the etherification of 9d and
12a in a similar manner as described in the synthesis of 13a with
the yield of 83% as a colorless oil. 1H NMR (400 MHz, CDCl3) d
12.65 (s, 1H), 7.62 (s, 1H), 6.44 (s, 1H), 3.98 (t, J = 5.3 Hz, 2H),
2.61–2.37 (m, 5H), 1.72–1.64 (m, 4H), 1.52–1.43 (m, 2H), 1.42–
0.78 (m, 19H). MS (ESI): m/z = 374 [M+H]+. HRMS (ESI): calcd for
HRMS (ESI): calcd for
333.1923.
C
17H25N4O3 [M+H]+ 333.1921, found
C
23H36NO3 [M+H]+ 374.2689, found 374.2687.
5.1.25. 5-(4-Acetyl-2-ethyl-5-hydroxyphenoxy)-2,2-
dimethylpentanenitrile (13k)
5.1.30. 1-(5-Ethyl-2-hydroxy-4-((5-methyl-5-(1H-tetrazol-5-
yl)hexyl)oxy)phenyl)ethanone (3b)
Compound 13k was prepared with the etherification of 9e
and 12b in a similar manner as described in the synthesis of
13a with the yield of 78% as
(400 MHz, CDCl3) d 12.72 (s, 1H), 7.45 (s, 1H), 6.37 (s, 1H),
4.06 (t, J = 6.1 Hz, 2H), 2.65–2.53 (m, 5H), 2.11–2.00 (m, 2H),
1.82–1.70 (m, 2H), 1.43 (s, 6H), 1.21 (t, J = 7.5 Hz, 3H). MS
(ESI): m/z = 290 [M+H]+. HRMS (ESI): calcd for C17H24NO3
[M+H]+ 290.1750, found 290.1747.
Compound 3b was prepared with the [3+2] cyclization reaction
of 13l with TMSN3 in a similar manner as described in the synthe-
sis of 3a with the yield of 65% as a white solid. 1H NMR (400 MHz,
CDCl3) d 12.79 (s, 1H), 7.43 (s, 1H), 6.36 (s, 1H), 3.98 (t, J = 6.2 Hz,
2H), 2.58 (s, 3H), 2.52 (q, J = 7.5 Hz, 2H), 1.93–1.75 (m, 4H), 1.51
(s, 6H), 1.44–1.30 (m, 2H), 1.15 (t, J = 7.5 Hz, 3H). MS (ESI): m/
z = 347 [M+H]+. HRMS (ESI): calcd for C18H27N4O3 [M+H]+
347.2407, found 347.2409.
a
colorless oil. 1H NMR
5.1.26. 6-(4-Acetyl-2-ethyl-5-hydroxyphenoxy)-2,2-
dimethylhexanenitrile (13l)
5.1.31. 1-(5-Ethyl-2-hydroxy-4-((7-methyl-7-(1H-tetrazol-5-
yl)octyl)oxy)phenyl)ethanone (3c)
Compound 13l was prepared with the etherification of 9e and
12c in a similar manner as described in the synthesis of 13a with
the yield of 92% as a colorless oil. 1H NMR (400 MHz, CDCl3) d
12.73 (s, 1H), 7.45 (s, 1H), 6.38 (s, 1H), 4.05 (t, J = 6.1 Hz, 2H),
2.59 (q, J = 7.6 Hz, 2H), 2.58 (s, 3H), 1.94–1.85 (m, 2H), 1.79–1.66
(m, 2H), 1.66–1.60 (m, 2H), 1.38 (s, 6H), 1.20 (t, J = 7.5 Hz, 3H).
MS (ESI): m/z = 304 [M+H]+. HRMS (ESI): calcd for C18H26NO3
[M+H]+ 304.1907, found 304.1909.
Compound 3c was prepared with the [3+2] cyclization reaction
of 13m with TMSN3 in a similar manner as described in the synthe-
sis of 3a with the yield of 68% as a white solid. 1H NMR (400 MHz,
CDCl3) d 12.88 (s, 1H), 7.44 (s, 1H), 6.38 (s, 1H), 3.97 (t, J = 6.4 Hz,
2H), 2.58 (s, 3H), 2.54 (q, J = 7.5 Hz, 1H), 1.90–1.71 (m, 4H), 1.51
(s, 6H), 1.49–1.20 (m, 6H), 1.17 (t, J = 7.5 Hz, 3H). MS (ESI): m/
z = 375 [M+H]+. HRMS (ESI): calcd for C20H31N4O3 [M+H]+
375.2390, found 375.2395.
5.1.27. 8-(4-Acetyl-2-ethyl-5-hydroxyphenoxy)-2,2-
dimethyloctanenitrile (13m)
5.1.32. 1-(5-Ethyl-2-hydroxy-4-((6-methyl-6-(1H-tetrazol-5-
yl)heptyl)oxy)phenyl)propan-1-one (4b)
Compound 13m was prepared with the etherification of 9e and
12d in a similar manner as described in the synthesis of 13a with
the yield of 92% as a colorless oil. 1H NMR (400 MHz, CDCl3) d 12.73
(s, 1H), 7.44 (s, 1H), 6.38 (s, 1H), 4.02 (t, J = 6.3 Hz, 2H), 2.65–2.51
(m, 5H), 1.93–1.75 (m, 2H), 1.58–1.48 (m, 6H), 1.50–1.39 (m,
2H), 1.37 (s, 6H), 1.20 (t, J = 7.5 Hz, 3H). MS (ESI): m/z = 332
[M+H]+. HRMS (ESI): calcd for C19H28NO3 [M+H]+ 332.2220, found
332.2224.
Compound 4b was prepared with the [3+2] cyclization reaction
of 13b with TMSN3 in a similar manner as described in the synthe-
sis of 3a with the yield of 54% as a white solid. 1H NMR (400 MHz,
DMSO-d6) d 7.63 (s, 1H), 6.44 (s, 1H), 3.98 (t, J = 6.2 Hz, 2H), 3.02 (q,
J = 7.2 Hz, 2H), 2.46 (q, J = 7.5 Hz, 2H), 1.73–1.62 (m, 4H), 1.35 (s,
6H), 1.38–1.29 (m, 4H),1.12–1.06 (m, 6H). MS (ESI): m/z = 375
[M+H]+. HRMS (ESI): calcd for C20H31N4O3 [M+H]+ 375.2390, found
375.2393.

P. Liu et al. / Bioorg. Med. Chem. 22 (2014) 1596–1607
1603
5.1.33. 1-(5-Ethyl-2-hydroxy-4-((6-methyl-6-(1H-tetrazol-5-
5.1.39. 1-(5-Butyl-2-hydroxy-4-((6-methyl-6-(1H-tetrazol-5-
yl)heptyl)oxy)phenyl)-2-methylpropan-1-one (4c)
yl)heptyl)oxy)phenyl)ethanone (5c)
Compound 4c was prepared with the [3+2] cyclization reaction
of 13c with TMSN3 in a similar manner as described in the synthe-
sis of 3a with the yield of 66% as a white solid. 1H NMR (400 MHz,
DMSO-d6) d 12.79 (s, 1H), 7.68 (s, 1H), 6.46 (s, 1H), 4.00 (t,
J = 6.2 Hz, 2H), 3.73–3.62 (m, 1H), 2.48 (q, J = 7.5 Hz, 2H), 1.75–
1.55 (m, 4H), 1.36 (s, 6H), 1.35–1.27 (m, 4H), 1.15–1.07 (m, 9H).
MS (ESI): m/z = 389 [M+H]+. HRMS (ESI): calcd for C21H33N4O3
[M+H]+ 389.2547, found 389.2545.
Compound 5c was prepared with the [3+2] cyclization reaction
of 13i with TMSN3 in a similar manner as described in the synthe-
sis of 3a with the yield of 67% as a white solid. 1H NMR (400 MHz,
DMSO-d6) d 12.61 (s, 1H), 7.62 (s, 1H), 6.44 (s, 1H), 3.98 (t,
J = 6.1 Hz, 2H), 2.55 (s, 3H), 2.52–2.43 (m, 2H), 1.74–1.63 (m, 4H),
1.51–1.42 (m, 2H), 1.36 (s, 6H), 1.33–1.25 (m, 4H), 1.17–1.06 (m,
2H), 0.89 (t, J = 7.3 Hz, 3H). MS (ESI): m/z = 389 [M+H]+. HRMS
(ESI): calcd for C21H33N4O3 [M+H]+ 389.2547, found 389.2546.
5.1.34. 1-(5-Ethyl-2-hydroxy-4-((6-methyl-6-(1H-tetrazol-5-
yl)heptyl)oxy)phenyl)pentan-1-one (4d)
5.1.40. 1-(5-Hexyl-2-hydroxy-4-((6-methyl-6-(1H-tetrazol-5-
yl)heptyl)oxy)phenyl)ethanone (5d)
Compound 4d was prepared with the [3+2] cyclization reaction
of 13d with TMSN3 in a similar manner as described in the synthe-
sis of 3a with the yield of 78% as a white solid. 1H NMR (400 MHz,
CDCl3) d 13.05 (s, 1H), 7.47 (s, 1H), 6.38 (s, 1H), 3.97 (t, J = 6.4 Hz,
2H), 2.97–2.87 (m, 2H), 2.56 (q, J = 7.5 Hz, 2H), 1.89–1.57 (m,
6H), 1.51 (s, 6H), 1.51–1.23 (m, 8H), 1.18 (t, J = 7.5 Hz, 3H), 0.94
(t, J = 7.0 Hz, 3H). MS (ESI): m/z = 403 [M+H]+. HRMS (ESI): calcd
for C22H35N4O3 [M+H]+ 403.2703, found 403.2701.
Compound 5d was prepared with the [3+2] cyclization reaction
of 13i with TMSN3 in a similar manner as described in the synthe-
sis of 3a with the yield of 67% as a white solid. 1H NMR (400 MHz,
DMSO-d6) d 12.61 (s, 1H), 7.62 (s, 1H), 6.44 (s, 1H), 3.98 (t,
J = 5.3 Hz, 2H), 2.61–2.37 (m, 5H), 1.72–1.64 (m, 4H), 1.52–1.43
(m, 2H), 1.42–0.78 (m, 19H). MS (ESI): m/z = 417 [M+H]+. HRMS
(ESI): calcd for C23H37N4O3 [M+H]+ 417.2860, found 417.2865.
5.1.41. 7-(4-Acetyl-2-ethyl-5-methoxyphenoxy)-2,2-
5.1.35. 1-(5-Ethyl-2-hydroxy-4-((6-methyl-6-(1H-tetrazol-5-
yl)heptyl)oxy)phenyl)heptan-1-one (4e)
dimethylheptanenitrile (14a)
To a solution of 13a (62 mg, 0.2 mmol) and iodomethane
(70 mg, 0.5 mmol) in MEK (5 mL) and DMSO (1 mL) were added
K2CO3 (140 mg, 1 mmol), and the mixture was heated at reflux
with stirring for 30 h. The mixture was then cooled and diluted
with water (10 mL). The product was extracted with EtOAc
(2 Â 15 mL). The organic solution was washed with water (3 mL),
and brine (10 mL), and then dried with Na2SO4. The residue was
concentrated by vacuo to yield crude product which was further
purified with column chromatography (petroleum ether (60–
90 °C)/acetone) on silica gel to afford the desired products 14a
(60 mg, 88%) as a white solid. 1H NMR (400 MHz, CDCl3) d 7.69
(s, 1H), 6.41 (s, 1H), 4.06 (t, J = 6.2 Hz, 2H), 3.93 (s, 3H), 2.64–2.51
(m, 5H), 1.95–1.86 (m, 2H), 1.67–1.52 (m, 6H), 1.38 (s, 6H), 1.18
(t, J = 7.5 Hz, 3H). MS (ESI): m/z = 332 [M+H]+. HRMS (ESI): calcd
for C20H30NO3 [M+H]+ 332.2220, found 332.2221.
Compound 4e was prepared with the [3+2] cyclization reaction
of 13e with TMSN3 in a similar manner as described in the synthe-
sis of 3a with the yield of 56% as a white solid. 1H NMR (400 MHz,
CDCl3) d 7.64 (s, 1H), 6.43 (s, 1H), 3.98 (t, J = 6.2 Hz, 2H), 2.96 (t,
J = 7.4 Hz, 2H), 2.47 (q, J = 7.4 Hz, 2H), 1.72–1.45 (m, 8H), 1.35 (s,
6H), 1.32–1.20 (m, 10H), 1.10 (t, J = 7.4 Hz, 3H), 0.85 (t, J = 6.8 Hz,
3H). MS (ESI): m/z = 431 [M+H]+. HRMS (ESI): calcd for
C
24H39N4O3 [M+H]+ 431.3016, found 431.3013.
5.1.36. (5-Ethyl-2-hydroxy-4-((6-methyl-6-(1H-tetrazol-5-
yl)heptyl)oxy)phenyl)(phenyl)methanone (4f)
Compound 4f was prepared with the [3+2] cyclization reaction
of 13f with TMSN3 in a similar manner as described in the synthe-
sis of 3a with the yield of 51% as a white solid. 1H NMR (400 MHz,
DMSO-d6) d 12.14 (s, 1H), 7.67–7.53 (m, 3H), 7.24 (s, 1H), 6.57 (s,
1H), 4.03 (t, J = 6.4 Hz, 1H), 2.43 (q, J = 7.5 Hz, 2H), 1.76–1.62 (m,
2H), 1.36 (s, 4H), 1.18–1.08 (m, 2H), 1.03 (t, J = 7.5 Hz, 3H). MS
(ESI): m/z = 423 [M+H]+. HRMS (ESI): calcd for C24H31N4O3
[M+H]+ 423.2390, found 423.2387.
5.1.42. 7-(4-Acetyl-5-(benzyloxy)-2-ethylphenoxy)-2,2-
dimethylheptanenitrile (14b)
Compound 14b was prepared with etherification reaction of
13a with BnBr in a similar manner as described in the synthesis
of 14a with the yield of 82% as a white solid. 1H NMR (400 MHz,
CDCl3) d 7.71 (s, 1H), 7.49–7.36 (m, 5H), 6.46 (s, 1H), 5.19 (s, 2H),
3.99 (t, J = 6.2 Hz, 2H), 2.63–2.56 (m, 5H), 2.00–1.75 (m, 2H),
1.57–1.51 (m, 6H), 1.38 (s, 6H), 1.18 (t, J = 7.5 Hz, 3H). MS (ESI):
m/z = 408 [M+H]+. HRMS (ESI): calcd for C26H34NO3 [M+H]+
408.2533, found 408.2536.
5.1.37. 1-(2-Hydroxy-4-((6-methyl-6-(1H-tetrazol-5-
yl)heptyl)oxy)-5-propylphenyl)ethanone (5a)
Compound 5a was prepared with the [3+2] cyclization reaction
of 13g with TMSN3 in a similar manner as described in the syn-
thesis of 3a with the yield of 63% as a white solid. 1H NMR
(400 MHz, CDCl3) d 12.90 (s, 1H), 7.42 (s, 1H), 6.37 (s, 1H), 3.95
(t, J = 6.4 Hz, 2H), 2.58 (s, 3H), 2.52–2.46 (m, 2H), 1.91–1.72 (m,
4H), 1.62–1.54 (m, 2H), 1.53 (s, 6H), 1.49–1.40 (m, 2H), 1.31–
1.22 (m, 2H), 0.93 (t, J = 7.4 Hz, 3H). MS (ESI): m/z = 375 [M+H]+.
5.1.43. 7-(4-Acetyl-2-ethyl-5-((3-methylbenzyl)oxy)phenoxy)-
2,2-dimethylheptanenitrile (14c)
Compound 14c was prepared with etherification reaction of 13a
with m-MeBnBr in a similar manner as described in the synthesis
of 14a with the yield of 75% as a white solid. 1H NMR (400 MHz,
CDCl3) d 7.70 (s, 1H), 7.40–7.09 (m, 4H), 6.46 (s, 1H), 5.14 (s, 2H),
3.99 (t, J = 6.2 Hz, 2H), 2.59 (s, 3H), 2.59 (q, J = 7.5 Hz, 2H), 2.40
(s, 3H), 1.91–1.78 (m, 2H), 1.59–1.50 (m, 6H), 1.38 (s, 6H), 1.18
(t, J = 7.5 Hz, 3H). MS (ESI): m/z = 422 [M+H]+. HRMS (ESI): calcd
for C27H36NO3 [M+H]+ 422.2689, found 422.2692.
HRMS (ESI): calcd for
375.2393.
C
20H31N4O3 [M+H]+ 375.2390, found
5.1.38. 1-(2-Hydroxy-5-isobutyl-4-((6-methyl-6-(1H-tetrazol-5-
yl)heptyl)oxy)phenyl)ethanone (5b)
Compound 5b was prepared with the [3+2] cyclization reaction
of 13h with TMSN3 in a similar manner as described in the synthe-
sis of 3a with the yield of 73% as a white solid. 1H NMR (400 MHz,
DMSO-d6) d 12.60 (s, 1H), 7.58 (s, 1H), 6.44 (s, 1H), 3.97 (t,
J = 6.3 Hz, 2H), 2.55 (s, 3H), 2.38–2.30 (m, 2H), 1.84–1.75 (m, 1H),
1.70–1.64 (m, 4H), 1.42 - 1.31 (s, 8H), 1.15–1.08 (m, 2H), 0.84 (d,
J = 6.6 Hz, 6H). MS (ESI): m/z = 389 [M+H]+. HRMS (ESI): calcd for
5.1.44. 7-(4-Acetyl-2-ethyl-5-((3-methylbenzyl)oxy)phenoxy)-
2,2-dimethylheptanenitrile (14d)
Compound 14d was prepared with etherification reaction of
13a with (1-bromoethyl)benzene in a similar manner as described
in the synthesis of 14a with the yield of 79% as a white solid. 1H
C
21H33N4O3 [M+H]+ 389.2547, found 389.2544.

1604
P. Liu et al. / Bioorg. Med. Chem. 22 (2014) 1596–1607
NMR (400 MHz, CDCl3) d 7.66 (s, 1H), 7.45–7.31 (m, 5H), 6.20 (s,
1H), 5.38 (q, J = 6.1 Hz, 1H), 3.90–3.75 (m, 1H), 3.68–3.47 (m,
0H), 2.74 (s, 3H), 2.51 (q, J = 7.5 Hz, 2H), 1.74 (d, J = 6.1 Hz, 3H),
1.73–1.66 (m, 2H), 1.63–1.58 (m, 6H), 1.36 (s, 6H), 1.13 (t,
J = 7.5 Hz, 3H). MS (ESI): m/z = 422 [M+H]+. HRMS (ESI): calcd for
5.1.50. 1-(5-Ethyl-4-((6-methyl-6-(1H-tetrazol-5-yl)heptyl)oxy)-
2-(1-phenylethoxy)phenyl)ethanone (2d)
Compound 2d was prepared via a [3+2] cyclization reaction of
14d with TMSN3 in a similar manner as described in the synthesis
of 3a with the yield of 45% as a white solid. 1H NMR (400 MHz,
CDCl3) d 7.64 (s, 1H), 7.39–7.31 (m, 5H), 6.17 (s, 1H), 5.37 (q,
J = 6.4 Hz, 1H), 3.80–3.68 (m, 1H), 3.61–3.50 (m, 1H), 2.77 (s,
3H), 2.42 (q, J = 7.5 Hz, 2H), 1.85–1.77 (m, 2H), 1.72 (d,
J = 6.4 Hz, 3H), 1.62–1.53 (m, 2H), 1.51 (s, 6H), 1.37–1.27 (m,
2H), 1.21–1.10 (m, 2H), 1.04 (t, J = 7.5 Hz, 3H). MS (ESI): m/
z = 465 [M+H]+. HRMS (ESI): calcd for C27H37N4O3 [M+H]+
465.2860, found 465.2856.
C
27H36NO3 [M+H]+ 422.2689, found 422.2690.
5.1.45. 7-(4-Acetyl-5-(cyclopentyloxy)-2-ethylphenoxy)-2,2-
dimethylheptanenitrile (14e)
Compound 14e was prepared with etherification reaction of 13a
with iodocyclopentane in a similar manner as described in the syn-
thesis of 14a with the yield of 49% as a white solid. 1H NMR
(400 MHz, CDCl3) d 7.69 (s, 1H), 6.38 (s, 1H), 4.95–4.83 (m, 1H),
4.03 (t, J = 6.2 Hz, 2H), 2.67–2.47 (m, 5H), 2.02–1.94 (m, 4H),
1.93–1.79 (m, 4H), 1.77–1.66 (m, 2H), 1.59–1.52 (m, 6H), 1.38 (s,
6H), 1.18 (t, J = 7.5 Hz, 3H), 0.94–0.83 (m, 2H). MS (ESI): m/
z = 386 [M+H]+. HRMS (ESI): calcd for C24H36NO3 [M+H]+
386.2689, found 386.2686.
5.1.51. 1-(2-(Cyclopentyloxy)-5-ethyl-4-((6-methyl-6-(1H-
tetrazol-5-yl)heptyl)oxy)phenyl)ethanone (2e)
Compound 2e was prepared via a [3+2] cyclization reaction of
14e with TMSN3 in a similar manner as described in the synthesis
of 3a with the yield of 38% as a white solid. 1H NMR (400 MHz,
CDCl3) d 7.67 (s, 1H), 6.33 (s, 1H), 4.95–4.81 (m, 1H), 3.95 (t,
J = 6.1 Hz, 2H), 2.61 (s, 3H), 2.50 (q, J = 7.5 Hz, 2H), 1.98–1.91 (m,
4H), 1.89–1.62 (m, 8H), 1.51 (s, 6H), 1.49–1.39 (m, 2H), 1.30–
1.18 (m, 2H), 1.11 (t, J = 7.5 Hz, 3H). MS (ESI): m/z = 429 [M+H]+.
HRMS (ESI): calcd for C24H37N4O3 [M+H]+ 429.2860, found
429.2864.
5.1.46. 7-(4-Acetyl-5-(cyclohexylmethoxy)-2-ethylphenoxy)-
2,2-dimethylheptanenitrile (14f)
Compound 14f was prepared with etherification reaction of 13a
with (bromomethyl)cyclohexane in a similar manner as described
in the synthesis of 14a with the yield of 55% as a white solid. 1H
NMR (400 MHz, CDCl3) d 7.69 (s, 1H), 6.37 (s, 1H), 4.04 (t,
J = 6.2 Hz, 2H), 3.86 (d, J = 5.8 Hz, 2H), 2.63 (s, 3H), 2.58 (q,
J = 7.5 Hz, 2H), 1.95–1.69 (m, 8H), 1.59–1.52 (m, 4H), 1.38 (s, 6H),
1.36–1.23 (m, 4H), 1.18 (t, J = 7.5 Hz, 3H), 0.93–0.84 (m, 3H). MS
(ESI): m/z = 414 [M+H]+. HRMS (ESI): calcd for C26H40NO3 [M+H]+
414.3002, found 414.3005.
5.1.52. 1-(2-(Cyclohexylmethoxy)-5-ethyl-4-((6-methyl-6-(1H-
tetrazol-5-yl)heptyl)oxy)phenyl)ethanone (2f)
Compound 2f was prepared via a [3+2] cyclization reaction of
14f with TMSN3 in a similar manner as described in the synthesis
of 3a with the yield of 62% as a white solid. 1H NMR (400 MHz,
CDCl3) d 7.67 (s, 1H), 6.32 (s, 1H), 3.95 (t, J = 6.1 Hz, 2H), 3.85 (d,
J = 5.8 Hz, 2H), 2.67 (s, 3H), 2.49 (q, J = 7.4 Hz, 2H), 1.98–1.68 (m,
12H), 1.53 (s, 6H), 1.50–1.12 (m, 7H), 1.10 (t, J = 7.4 Hz, 3H). MS
(ESI): m/z = 457 [M+H]+. HRMS (ESI): calcd for C26H41N4O3
[M+H]+ 457.3173, found 457.3177.
5.1.47. 1-(5-Ethyl-2-methoxy-4-((6-methyl-6-(1H-tetrazol-5-
yl)heptyl)oxy)phenyl)ethanone (2a)
Compound 2a was prepared via a [3+2] cyclization reaction of
14a with TMSN3 in a similar manner as described in the synthesis
of 3a with the yield of 76% as a white solid. 1H NMR (400 MHz,
DMSO-d6) d 7.47 (s, 1H), 6.63 (s, 1H), 4.05 (t, J = 6.1 Hz, 2H), 3.91
(s, 3H), 2.49–2.44 (m, 5H), 1.76–1.63 (m, 4H), 1.44–1.32 (m, 8H),
1.18–1.09 (m, 2H), 1.08 (t, J = 7.5 Hz, 3H). MS (ESI): m/z = 375
[M+H]+. HRMS (ESI): calcd for C20H31N4O3 [M+H]+ 375.2390, found
375.2386.
5.1.53. 7-(2,4-Diethyl-5-hydroxyphenoxy)-2,2-
dimethylheptanenitrile (15)
Compound 15 was prepared via a reduction reaction of 13a
with Zn dust in a similar manner as described in the synthesis of
8a with the yield of 83% as a colorless oil. 1H NMR (400 MHz,
CDCl3) d 6.89 (s, 1H), 6.37 (s, 1H), 4.72 (s, 1H), 3.93 (t, J = 6.3 Hz,
2H), 2.57 (q, J = 7.6 Hz, 4H), 1.88–1.74 (m, 2H), 1.59–1.48 (m,
4H), 1.37 (s, 6H), 1.23 (t, J = 7.6 Hz, 3H), 1.18 (t, J = 7.6 Hz, 3H),
0.94–0.84 (m, 2H). MS (ESI): m/z = 304 [M+H]+. HRMS (ESI): calcd
for C19H30NO3 [M+H]+ 304.2271, found 304.2274.
5.1.48. 1-(2-(Benzyloxy)-5-ethyl-4-((6-methyl-6-(1H-tetrazol-5-
yl)heptyl)oxy)phenyl)ethanone (2b)
Compound 2b was prepared via a [3+2] cyclization reaction of
14b with TMSN3 in a similar manner as described in the synthesis
of 3a with the yield of 59% as a white solid. 1H NMR (400 MHz,
DMSO-d6) d 7.58–7.30 (m, 6H), 6.74 (s, 1H), 5.27 (s, 2H), 4.03 (t,
J = 6.2 Hz, 2H), 2.49–2.41 (m, 5H), 1.77–1.60 (m, 4H), 1.41–1.29
(m, 8H), 1.22–1.06 (m, 2H), 1.08 (t, J = 7.4 Hz, 3H). MS (ESI): m/
z = 451 [M+H]+. HRMS (ESI): calcd for C26H35N4O3 [M+H]+
451.2703, found 451.2705.
5.1.54. 2,4-Diethyl-5-((6-methyl-6-(1H-tetrazol-5-
yl)heptyl)oxy)phenol (4a)
Compound 4a was prepared via a [3+2] cyclization reaction of
15 with TMSN3 in a similar manner as described in the synthesis
of 3a with the yield of 77% as a white solid. 1H NMR (400 MHz,
CDCl3) d 6.85 (s, 1H), 6.39 (s, 1H), 5.14 (s, 1H), 3.75 (t, J = 6.9 Hz,
2H), 2.58 (q, J = 7.5 Hz, 2H), 2.50 (q, J = 7.5 Hz, 2H), 1.69–1.52 (m,
4H), 1.50 (s, 6H), 1.39–1.16 (m, 4H), 1.12 (t, J = 7.5 Hz, 3H), 0.85
(t, J = 7.5 Hz, 3H). MS (ESI): m/z = 347 [M+H]+. HRMS (ESI): calcd
for C19H31N4O3 [M+H]+ 347.2441, found 347.2444.
5.1.49. 1-(5-Ethyl-4-((6-methyl-6-(1H-tetrazol-5-yl)heptyl)oxy)-
2-((3-methylbenzyl)oxy)phenyl)ethanone (2c)
Compound 2c was prepared via a [3+2] cyclization reaction of
14c with TMSN3 in a similar manner as described in the synthesis
of 3a with the yield of 46% as a white solid. 1H NMR (400 MHz,
CDCl3) d 7.68 (s, 1H), 7.38–7.12 (m, 4H), 6.42 (s, 1H), 5.13 (s, 2H),
3.91 (t, J = 6.2 Hz, 2H), 2.61 (s, 3H), 2.52 (q, J = 7.5 Hz, 2H), 2.39
(s, 3H), 1.86–1.65 (m, 4H), 1.51 (s, 6H), 1.47–1.20 (m, 4H), 1.12
(t, J = 7.5 Hz, 3H). MS (ESI): m/z = 465 [M+H]+. HRMS (ESI): calcd
for C27H37N4O3 [M+H]+ 465.2860, found 465.2863.
5.1.55. Methyl 6-(benzyloxy)hexanoate (17)
6-Hexanolactone 16 (11.4 g, 100 mmol) was dissolved in MeOH
(150 mL), then NaOMe (10.8 g, 200 mmol) was added in the system
at rt. After stirred for 0.5 h, the solvent was evaporated in vacuo,
1 N HCl was added to neutralize the base. The product was

P. Liu et al. / Bioorg. Med. Chem. 22 (2014) 1596–1607
1605
extracted with EtOAc (2 Â 50 mL), washed with brine and dried
with Na2SO4 before concentrated to obtain the crude product
methyl 6-hydroxyhexanoate. The combination of methyl 6-
hydroxyhexanoate (from last step) and BnBr (34 g, 200 mmol)
was cooled with ice-water bath, then Ag2O (23 g, 100 mmol) was
added in portions over 20 min. After the addition, the reaction
was warmed to rt, and stirred for another 2 h to finish the reaction.
The mixture was filtrated, the filter cake was washed with EtOAc
(20 mL), and the filtrate was concentrated in vacuo to afford the
crude product. Further purification via column chromatography
(petroleum ether (60–90 °C)/acetone) on silica gel to afford the de-
sired products 17 (18 g, 76%) as a colorless oil. 1H NMR (400 MHz,
CDCl3) d 7.38–7.33 (m, 5H), 4.52 (s, 2H), 3.69 (s, 3H), 3.49 (t,
J = 6.5 Hz, 2H), 2.34 (t, J = 7.5 Hz, 2H), 1.70–1.62 (m, 4H), 1.52–
1.36 (m, 2H). MS (ESI): m/z = 237 [M+H]+.
J = 6.4 Hz, 2H), 2.69–2.50 (m, 1H), 1.70–1.40 (m, 8H), 1.33 (d,
J = 7.1 Hz, 3H). MS (ESI): m/z = 232 [M+H]+. HRMS (ESI): calcd for
C
15H22NO [M+H]+ 231.1695, found 231.1693.
5.1.59. 6-(Benzyloxy)-2-ethylhexanenitrile (20b)
Compound 20b was prepared via a reaction of 19 with EtBr in a
similar manner as described in the synthesis of 20a with the yield
of 69% as a colorless oil. 1H NMR (400 MHz, CDCl3) d 7.43–7.29 (m,
5H), 4.53 (s, 2H), 3.50 (t, J = 6.4 Hz, 2H), 2.46–2.36 (m, 1H), 1.84–
1.35 (m, 10H), 1.07 (t, J = 7.2 Hz, 3H). MS (ESI): m/z = 246 [M+H]+.
HRMS (ESI): calcd for C16H24NO [M+H]+ 245.1852, found 246,1853.
5.1.60. 6-Chlorohexanenitrile (21a)
The solution of 6-(benzyloxy)hexanenitrile 19 (203 mg,
1 mmol) in MeOH (5 mL) was evacuated and backfilled with nitro-
gen (3 times). Pd/C (10%, 20 mg) was added into the system, then
backfilled with hydrogen (1 atm). After 2 h, the mixture was fil-
trated and concentrated in vacuo. The residue was dissolved in tol-
uene (5 mL), then thionyl chloride (0.3 mL, 3 mmol) was added.
The mixture was stirred overnight, then evaporated in vacuo. The
residue was purified by column chromatography (petroleum ether
(60–90 °C)/acetone) on silica gel to give the desired product 21a
(74 mg, 51%) as a colorless oil. 1H NMR (400 MHz, CDCl3) d 3.57
(t, J = 6.6 Hz, 2H), 2.37 (t, J = 7.1 Hz, 2H), 1.77–1.65 (m, 2H), 1.65–
1.56 (m, 2H), 1.56–1.40 (m, 4H). MS (ESI): m/z = 146 [M+H]+.
5.1.56. 5-(Benzyloxy)pentyl 4-methylbenzenesulfonate (18)
The solution of methyl 6-(benzyloxy)hexanoate
7 (2.4 g,
10 mmol) in anhydrous THF (20 mL) was cooled over ice-water
bath, then DIBAL-H (1.0 M, 20 mL, 20 mmol) was added over
20 min. After the addition, the reaction was stirred at 0 °C for 2 h
to complete the reaction. After the reaction, 1 N HCl was added
to quench the reaction. The mixture was diluted with EtOAc
(50 mL), washed with water, brine, dried over Na2SO4, and concen-
trated in vacuo to yield the crude product. The crude product was
combined with pyridine (1.6 g, 20 mmol) in DCM (50 mL), then
TsCl (2.3 g, 12 mmol) was added in portions, and then stirred for
2–3 h. After the reaction, the mixture was washed with 1 N HCl,
water, brine and dried over Na2SO4 to give the crude product.
The crude product was purified via column chromatography
(petroleum ether (60–90 °C)/acetone) on silica gel to afford the de-
sired products 18 (2.8 g, 82%) as a colorless oil. 1H NMR (400 MHz,
CDCl3) d 7.81 (d, J = 8.3 Hz, 2H), 7.41–7.29 (m, 7H), 4.50 (s, 2H),
4.03 (t, J = 6.5 Hz, 2H), 3.45 (t, J = 6.5 Hz, 2H), 2.46 (s, 3H), 1.72–
1.61 (m, 2H), 1.64–1.53 (m, 2H), 1.40–1.28 (m, 2H). MS (ESI): m/
z = 349 [M+H]+.
5.1.61. 7-Chloro-2-methylheptanenitrile (21b)
Compound 21b was prepared via a hydrogenation reaction fol-
lowed by chlorination of 20a in a similar manner as described in
the synthesis of 21a with the yield of 39% as a colorless oil. 1H
NMR (400 MHz, CDCl3) d 3.63 (t, J = 6.6 Hz, 2H), 2.69–2.54 (m,
1H), 1.70–1.39 (m, 8H), 1.34 (d, J = 7.1 Hz, 3H). MS (ESI): m/
z = 160 [M+H]+. HRMS (ESI): calcd for C8H15ClN [M+H]+ 160.0887,
found 160.0889.
5.1.62. 7-Chloro-2-ethylheptanenitrile (21c)
Compound 21c was prepared via a hydrogenation reaction fol-
lowed by chlorination of 20b in a similar manner as described in
the synthesis of 21a with the yield of 45% as a colorless oil. 1H
NMR (400 MHz, CDCl3) d 3.68 (t, J = 6.5 Hz, 2H), 2.55–2.43 (m,
1H), 1.72–1.55 (m, 8H), 1.54–1.34 (m, 2H), 1.10 (t, J = 7.4 Hz, 3H).
MS (ESI): m/z = 174 [M+H]+. HRMS (ESI): calcd for C9H17ClN
[M+H]+ 174.1044, found 174.1046.
5.1.57. 6-(Benzyloxy)hexanenitrile (19)
The solution of 5-(benzyloxy)pentyl 4-methylbenzenesulfonate
18 (350 mg, 1 mmol) and CsF (750 mg, 5 mmol) in DMF (5 mL) was
heated to 50 °C, then TMSCN (500 mg, 5 mmol) was added into the
system via syringe. The reaction was stirred at 50 °C for 10 h before
finishing. Then, the reaction was cooled to rt, water (20 mL) was
added in the system, and the mixture was extracted with EtOAc
(2 Â 20 mL). The organic layer was washed with water, brine, dried
over Na2SO4, and concentrated in vacuo before column chromatog-
raphy (petroleum ether (60–90 °C)/acetone) on silica gel to give
the desired product 19 (170 mg, 85%) as a colorless oil. 1H NMR
(400 MHz, CDCl3) d 7.44–7.29 (m, 5H), 4.52 (s, 2H), 3.49 (t,
J = 6.2 Hz, 2H), 2.35 (t, J = 7.1 Hz, 2H), 1.74–1.61 (m, 4H), 1.56–
1.39 (m, 4H). MS (ESI): m/z = 204 [M+H]+.
5.1.63. 7-(4-Acetyl-2-ethyl-5-hydroxyphenoxy)heptanenitrile
(22a)
Compound 22a was prepared with etherification reaction of 21a
with 9a in a similar manner as described in the synthesis of 14a
with the yield of 75% as a colorless oil. 1H NMR (400 MHz, CDCl3)
d 12.72 (s, 1H), 7.44 (s, 1H), 6.38 (s, 1H), 4.02 (t, J = 6.3 Hz, 2H),
2.82–2.58 (m, 7H), 1.93–1.80 (m, 2H), 1.71–1.51 (m, 4H), 1.32–
1.21 (m, 2H), 1.20 (t, J = 7.3 Hz, 3H). MS (ESI): m/z = 290 [M+H]+.
5.1.58. 7-(Benzyloxy)-2-methylheptanenitrile (20a)
HRMS (ESI): calcd for
290.1753.
C
17H24NO3 [M+H]+ 290.1750, found
The solution of 6-(benzyloxy)hexanenitrile 19 (203 mg,
1 mmol) in THF (5 mL) was cooled to À78 °C, then LDA (2.0 M,
0.6 mL, 1.2 mmol) was added into the system dropwisely. After
the addition, the reaction was stirred for 0.5 h at À78 °C, then
MeI (210 mg, 1.5 mmol) was added into the mixture. After stirred
for 2 h, 1 N HCl was added into the system to quench the reaction,
warmed to rt, and diluted with EtOAc (15 mL). The mixture was
washed with water, brine, dried over Na2SO4, and concentrated
in vacuo. The residue was purified by column chromatography
(petroleum ether (60–90 °C)/acetone) on silica gel to give the de-
sired product 20a (135 mg, 62%) as a colorless oil. 1H NMR
(400 MHz, CDCl3) d 7.42–7.29 (m, 5H), 4.52 (s, 2H), 3.50 (t,
5.1.64. 7-(4-Acetyl-2-ethyl-5-hydroxyphenoxy)-2-
methylheptanenitrile (22b)
Compound 22b was prepared with etherification reaction of
21b with 9a in a similar manner as described in the synthesis of
14a with the yield of 67% as a colorless oil. 1H NMR (400 MHz,
CDCl3) d 12.72 (s, 1H), 7.44 (s, 1H), 6.38 (s, 1H), 4.02 (t, J = 6.3 Hz,
2H), 2.72–2.50 (m, 6H), 1.93–1.80 (m, 2H), 1.71–1.51 (m, 4H),
1.35 (d, J = 7.0 Hz, 3H), 1.32–1.24 (m, 2H), 1.20 (t, J = 7.5 Hz, 3H).
MS (ESI): m/z = 304 [M+H]+. HRMS (ESI): calcd for C18H26NO3
[M+H]+ 304.1907, found 304.1905.

1606
P. Liu et al. / Bioorg. Med. Chem. 22 (2014) 1596–1607
5.1.65. 7-(4-Acetyl-2-ethyl-5-hydroxyphenoxy)-2-
ethylheptanenitrile (22c)
5.1.70. Methyl 7-(4-acetyl-2-ethyl-5-hydroxyphenoxy)-2,2-
dimethylheptanoate (3h)
Compound 22c was prepared with etherification reaction of 21c
with 9a in a similar manner as described in the synthesis of 14a
with the yield of 82% as a colorless oil. 1H NMR (400 MHz, CDCl3)
d 12.73 (s, 1H), 7.44 (s, 1H), 6.38 (s, 1H), 4.02 (t, J = 6.2 Hz, 2H),
2.64–2.54 (m, 5H), 2.54–2.44 (m, 1H), 1.93–1.77 (m, 2H), 1.73–
1.60 (m, 6H), 1.60–1.48 (m, 2H), 1.20 (t, J = 7.5 Hz, 3H), 1.11 (t,
J = 7.4 Hz, 3H). MS (ESI): m/z = 318 [M+H]+. HRMS (ESI): calcd for
A solution of compound 3h (160 mg, 0.5 mmol) in absolute
MeOH (5 mL) was stirred at rt. 2 drop of commercial sulfuric acid
was added in the mixture. The system was heated at reflux
temperature and stirred for 5 h. The mixture was concentrated in
vacuo. The residue was purified by column chromatography
(petroleum ether (60–90 °C)/acetone) on silica gel to give the
desired product 3h (154 mg, 88%) as a white solid. 1H NMR
(400 MHz, CDCl3) d 12.73 (s, 1H), 7.44 (s, 1H), 6.38 (s, 1H), 4.00
(t, J = 6.4 Hz, 2H), 3.68 (s, 3H), 2.63–2.51 (m, 5H), 1.89–1.78 (m,
2H), 1.59–1.53 (m, 2H), 1.53–1.42 (m, 2H), 1.37–1.25 (m, 2H),
1.23–1.16 (m, 9H). MS (ESI): m/z = 351 [M+H]+. HRMS (ESI): calcd
for C20H31O5 [M+H]+ 351.2166, found 351.2164.
C
19H28NO3 [M+H]+ 318.2063, found 318.2066.
5.1.66. 1-(4-((6-(1H-Tetrazol-5-yl)hexyl)oxy)-5-ethyl-2-
hydroxyphenyl)ethanone (3d)
Compound 3d was prepared via a [3+2] cyclization reaction of
22a with TMSN3 in a similar manner as described in the synthesis
of 3a with the yield of 79% as a white solid. 1H NMR (400 MHz,
DMSO-d6) d 12.63 (s, 1H), 7.63 (s, 1H), 6.47 (s, 1H), 4.03 (t,
J = 6.1 Hz, 2H), 2.88 (t, J = 7.4 Hz, 2H), 2.56 (s, 3H), 2.54–2.47 (m,
2H), 1.82–1.62 (m, 4H), 1.54–1.29 (m, 4H), 1.12 (t, J = 7.4 Hz, 3H).
MS (ESI): m/z = 333 [M+H]+. HRMS (ESI): calcd for C17H25N4O3
[M+H]+ 333.1921, found 333.1924.
5.1.71. 5-(7-(5-(Benzyloxy)-2,4-diethylphenoxy)-2-
methylheptan-2-yl)-1H-tetrazole (1a)
Compound 1a was prepared via a reaction of 4a with BnBr in a
similar manner as described in the synthesis of 20a with the yield
of 63% as a white solid. 1H NMR (400 MHz, DMSO-d6) d 7.52–7.27
(m, 5H), 6.86 (s, 1H), 6.61 (s, 1H), 5.10 (s, 2H), 3.88 (t, J = 6.3 Hz,
2H), 2.46 (q, J = 7.5 Hz, 2H), 2.44 (q, J = 7.5 Hz, 2H), 1.75–1.54 (m,
4H), 1.36 (s, 6H), 1.36–1.32 (m, 2H), 1.15–1.01 (m, 8H). MS (ESI):
m/z = 437 [M+H]+. HRMS (ESI): calcd for C26H37N4O2 [M+H]+
437.2911, found 437.2913.
5.1.67. 1-(4-((6-(1H-tetrazol-5-yl)heptyl)oxy)-5-ethyl-2-
hydroxyphenyl)ethanone (3e)
Compound 3e was prepared via a [3+2] cyclization reaction of
22b with TMSN3 in a similar manner as described in the synthesis
of 3a with the yield of 65% as a white solid. 1H NMR (400 MHz,
DMSO-d6) d 12.62 (s, 1H), 7.62 (s, 1H), 6.45 (s, 1H), 4.01 (t,
J = 6.3 Hz, 2H), 3.23–3.10 (m, 1H), 2.56 (s, 3H), 2.53–2.46 (m, 2H),
1.79–1.58 (m, 4H), 1.47–1.37 (m, 2H), 1.29 (d, J = 7.0 Hz, 3H),
1.27–1.16 (m, 2H), 1.11 (t, J = 7.5 Hz, 3H). MS (ESI): m/z = 347
[M+H]+. HRMS (ESI): calcd for C18H27N4O3 [M+H]+ 347.2077, found
347.2079.
5.1.72. 4-Butyl-2-ethyl-5-((6-methyl-6-(1H-tetrazol-5-
yl)heptyl)oxy)phenol (1b)
Compound 1b was prepared via a reduction reaction of 5c with
Zn dust in a similar manner as described in the synthesis of 8a with
the yield of 63% as a white solid. 1H NMR (400 MHz, DMSO-d6) d
8.91 (s, 1H), 6.73 (s, 1H), 6.33 (s, 1H), 3.78 (t, J = 6.2 Hz, 2H),
2.46–2.32 (m, 4H), 1.73–1.57 (m, 4H), 1.48–1.35 (m, 4H), 1.36 (s,
6H), 1.31–1.19 (m, 2H), 1.15–1.07 (m, 2H), 1.06 (t, J = 7.5 Hz, 3H),
0.86 (t, J = 7.3 Hz, 3H). MS (ESI): m/z = 375 [M+H]+. HRMS (ESI):
calcd for C21H35N4O2 [M+H]+ 375.2754, found 375.2751.
5.1.68. 1-(4-((6-(1H-Tetrazol-5-yl)octyl)oxy)-5-ethyl-2-
hydroxyphenyl)ethanone (3f)
Compound 3f was prepared via a [3+2] cyclization reaction of
22c with TMSN3 in a similar manner as described in the synthesis
of 3a with the yield of 45% as a white solid. 1H NMR (400 MHz,
DMSO-d6) d 12.62 (s, 1H), 7.62 (s, 1H), 6.45 (s, 1H), 4.00 (t,
J = 6.2 Hz, 2H), 3.03–2.94 (m, 1H), 2.56 (s, 3H), 2.51–2.46 (overlap,
2H), 1.79–1.59 (m, 6H), 1.46–1.33 (m, 2H), 1.30–1.18 (m, 2H), 1.11
(t, J = 7.4 Hz, 3H), 0.73 (t, J = 7.4 Hz, 3H). MS (ESI): m/z = 361
[M+H]+. HRMS (ESI): calcd for C19H29N4O3 [M+H]+ 361.2234, found
361.2236.
5.1.73. 1-(2-(Benzyloxy)-5-butyl-4-((6-methyl-6-(1H-tetrazol-5-
yl)heptyl)oxy)phenyl)ethanone (1c)
Compound 1c was prepared via a reaction of 5c with BnBr in a
similar manner as described in the synthesis of 20a with the yield
of 67% as a white solid. 1H NMR (400 MHz, CDCl3) d 7.69 (s, 1H),
7.55–7.34 (m, 5H), 6.45 (s, 1H), 5.18 (s, 2H), 3.98 (t, J = 6.2 Hz,
2H), 2.68–2.33 (m, 5H), 1.94–1.78 (m, 2H), 1.61–1.49 (m, 8H),
1.42–1.24 (m, 8H), 0.94 (t, J = 7.3 Hz, 3H). MS (ESI): m/z = 479
[M+H]+. HRMS (ESI): calcd for C28H39N4O2 [M+H]+ 479.3016, found
479.3019.
5.1.69. 7-(4-Acetyl-2-ethyl-5-hydroxyphenoxy)-2,2-
dimethylheptanoic acid (3g)
A mixture of Compound 13a (320 mg, 1 mmol) and an aqueous
solution of sodium hydroxide (10%, 5 mL) was heated with mag-
netic stirring in an oil bath at reflux temperature for 10 h. After
the solution was cooled to rt, it was extracted with EtOAc (10 mL)
to remove nonacidic material. 1 N HCl was added into the system
and the product was extracted with EtOAc (2 Â 10 mL). The com-
bined organic layer was washed with water, brine, dried with Na2-
SO4 and concentrated in vacuo. The residue was purified by column
chromatography (petroleum ether (60–90 °C)/acetone/acetic acid)
on silica gel to give the desired product 3f (178 mg, 54%) as a white
solid. 1H NMR (400 MHz, CDCl3) d 12.72 (s, 1H), 7.44 (s, 1H), 6.38 (s,
1H), 4.01 (t, J = 6.4 Hz, 2H), 2.58 (q, J = 7.5 Hz, 2H), 2.58 (s, 3H),
1.91–1.78 (m, 2H), 1.64–1.57 (m, 2H), 1.54–1.33 (m, 4H), 1.28 (s,
6H), 1.20 (t, J = 7.5 Hz, 3H). MS (ESI): m/z = 337 [M+H]+. HRMS
(ESI): calcd for C19H29O5 [M+H]+ 337.2009, found 337.2006.
5.2. In vitro pharmacological studies
5.2.1. Binding assay
The compounds binding to FXRaLBD were determined by coac-
tivator recruitment assay based on homogenous time-resolved flo-
rescence (HTRF) as described before.22,33 The experiments were
conducted with 10 nM GST-FXRa LBD and 100 nM Bio-SRC-1 in
the presence of 50 mM CDCA (FXR endogenous agonist) in a buffer
containing 100 mM HEPES, 125 mM KF, 0.125% (w/v) CHAPS, 0.05%
dry milk, 0.83 nM anti-GST-(Eu)K, 41.75 nM SA/XL665. Different
compounds at 20 lM or different concentrations of indicated com-
pounds were incubated with the mixture for 30 min at room tem-
perature. And then fluorescence was measured on a SpectraMax
M5 instrument (Molecular Devices). All values were reported as
mean SD of triple measurements.

P. Liu et al. / Bioorg. Med. Chem. 22 (2014) 1596–1607
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Acknowledgments
This work was supported by the National Natural Science
Foundation of China (Grants 81273397, 81373462, 81173105),
the Chinese National Science & Technology Major Project ‘Key
New Drug Creation and Manufacturing Program’ (Grants
2013ZX09508104, 2012ZX09103-101-018, 2014ZX09301-306-
03), the Science Foundation of Shanghai (Grant 12XD1405700)
and the Fundamental Research Funds for the Central Universities
(WY1114026).
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