Novel triacsin C analogs as potential antivirals against rotavirus infections

Article abstract of DOI:10.1016/j.ejmech.2012.02.010

Recently our group has demonstrated that cellular triglyceride (TG) levels play an important role in rotavirus replication. In this study, we further examined the roles of the key enzymes for TG synthesis (lipogenesis) in the replication of rotaviruses by

Full text of DOI:10.1016/j.ejmech.2012.02.010

European Journal of Medicinal Chemistry 50 (2012) 311e318
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel triacsin C analogs as potential antivirals against rotavirus infections
Yunjeong Kim a, David George a, Allan M. Prior b, Keshar Prasain b, Shuanghong Hao b, Duy D. Le b,
,
Duy H. Hua b, Kyeong-Ok Chang a
*
a Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, 1800 Denison Avenue, Manhattan, KS 66506, USA
b Department of Chemistry, Kansas State University, Manhattan, KS 66506, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Recently our group has demonstrated that cellular triglyceride (TG) levels play an important role in
rotavirus replication. In this study, we further examined the roles of the key enzymes for TG synthesis
(lipogenesis) in the replication of rotaviruses by using inhibitors of fatty acid synthase, long chain fatty
acid acyl-CoA synthetase (ACSL), and diacylglycerol acyltransferase and acyl-CoA:cholesterol acyl-
transferase in association with lipid droplets of which TG is a major component. Triacsin C, a natural ACSL
inhibitor from Streptomyces aureofaciens, was found to be highly effective against rotavirus replication.
Thus, novel triacsin C analogs were synthesized and evaluated for their efficacies against the replication
of rotaviruses in cells. Many of the analogs significantly reduced rotavirus replication, and one analog
Received 14 December 2011
Received in revised form
31 January 2012
Accepted 3 February 2012
Available online 11 February 2012
Keywords:
Rotavirus
Antivirals
(1e) was highly effective at a nanomolar concentration range (ED50 0.1 mM) with a high therapeutic index
in cell culture. Our results suggest a crucial role of lipid metabolism in rotavirus replication, and triacsin C
and/or its analogs as potential therapeutic options for rotavirus infections.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Triacsin C analogs
Lipogenesis
1. Introduction
morphogenesis [5,6]. Lipid droplets are cellular organelles for
storage of neutral fats such as TG and cholesterol ester, and play
Rotaviruses are non-enveloped, icosahedral viruses with an 11-
segment double-stranded RNA genome [1]. The capsid of rotavirus
is composed of the outer capsid proteins, VP4 and VP7, and the
major inner capsid protein, VP6. Rotaviruses are divided into 7 (A to
G) antigenically distinct serogroups based on VP6. Among them,
group A rotaviruses are the leading cause of severe gastroenteritis
in infants and children worldwide, associated with over 500,000
deaths in children younger than 5 years of age each year, although
attenuated live vaccines are available [1e3]. Majority of rotavirus
infection-associated mortality occurs in the developing countries.
Nonetheless, nearly 1 in 80 children is hospitalized with rotavirus
gastroenteritis by 5 years of age in the US [1e3]. Since there are no
specific antiviral agents for rotavirus infection, the treatment
options for rotavirus infection are limited to providing oral rehy-
dration solution to restore and maintain hydration until the infec-
tion resolves [4]. However, the development of rotavirus antivirals
to reduce the severity of diseases and duration of rotavirus-related
hospitalization has been impeded by limited information on the
therapeutic targets for rotavirus infections.
a crucial role in regulating cellular lipid levels. Lipid rafts are
microdomains in cell membrane enriched in cholesterol, glyco-
sphingolipids, and proteins. These structures are found important
for infectious virus particle formation of human hepatitis C virus
(HCV) [7,8] and dengue virus [9], suggesting the importance of lipid
homeostasis in virus replication. Recently our group has demon-
strated that rotavirus replication induced an increase in the TG
levels in cells, and suppression of increase in TG levels by the far-
nesoid X receptor (FXR) agonists significantly inhibited rotavirus
replication [10].
Lipogenesis is the process of producing fats from acetyl-CoA,
which is then stored as an energy source. During lipogenesis, fatty
acid (FA) is synthesized (de novo synthesis) from acetyl-CoA, and
subsequently esterified with glycerol to form TG. Numerous
enzymes participate in lipogenesis, which include fatty acid synthase
(FASN), long chain fatty acid acyl-CoA synthetase (ACSL) 1e6, and
diacylglycerol acyltransferase (DGAT) 1/2 and Acyl-CoA:cholesterol
acyltransferase (ACAT)1/2 [11e15]. Lipolysis is the reverse pathway
of lipogenesis to generate acetyl-CoA and energy from TG in which
process multiple enzymes including lipases are involved. In this
study, we found that the commercially available inhibitors for FASN,
ACSL, DGAT and ACAT significantly reduced the replication of rota-
viruses in vitro. Since triacsin C, a fungal metabolite from Strepto-
myces aureofaciens, is an ACSL inhibitor and showed the most potent
Previously it was shown that disruption of lipid rafts and/or lipid
droplets decrease infectious rotaviruses by inhibition of rotavirus
* Corresponding author. Tel.: þ1 785 532 3849; fax: þ1 785 532 4039.
E-mail address: kchang@vet.ksu.edu (K.-O. Chang).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.02.010

312
Y. Kim et al. / European Journal of Medicinal Chemistry 50 (2012) 311e318
inhibition on rotavirus replication, we synthesized its analogs and
examined their antiviral effects against rotavirus. The synthetic
sequence is straightforward and efficient (Schemes 1 and 2), which
would provide flexibility for further optimization. Among the tri-
acsin C and its analogs, 1e is the most potent against rotavirus
replication with the effective dose that reduce 50% of the virus
aluminum hydride followed by oxidation with IBX and DMSO
afforded trienal 1 in 82% overall yield (from ester 6).
Triacsin C analogs including hydrazone derivatives 1ae1c and 1ba,
cyano analog 1d, and aminobenzoic acid 1e were synthesized
for bio-evaluation. Hydrazone formation of
1
with chlor-
omethanesulfonohydrazide, 3-chloropropane-1-sulfonohydrazide,
replication (ED50) of 0.1
m
M. Our results suggest that lipogenic
and propane-2-sulfonohydrazide separately in ethanol at room
temperature gave hydrazones 1a, 1b, and 1c, respectively, in good to
excellent yields (Scheme 2). Intramolecular cyclization of 1b with
sodium hydride in N,N-dimethylformamide (DMF) at 25 ꢀCgave 1ba. It
should be noted the aforementioned hydrazones are stable under
silica gel column chromatographic conditions. Cyano compound 1d
was obtained from a HornereWadswortheEmmons olefination of
aldehyde 1 with diethyl cyanomethylphosphonate and sodium
hydride [18]. Reductive amination of aldehyde 1 with 4-aminobenzoic
acid in methanol followed by sodium cyanoborohydride
produced 1e. Chloromethanesulfonohydrazide, 3-chloropropane-1-
sulfonohydrazide, and propane-2-sulfonohydrazide were prepared
by the treatment of chloromethanesulfonyl chloride, 3-
chloropropane-1-sulfonyl chloride, and propane-2-sulfonyl chloride,
respectively, with 40% hydrazine (in aqueous solution) in THF at 25 ꢀC
for 2 h. After aqueous workup, extraction with diethyl ether, and
concentration, the crude hydrazine intermediates were used in
subsequent steps without purification. It should be noted that the
synthesis of triacsin C from aldehyde 1 has been reported by Tanaka
et al. [17] in less than 0.5% yield from a three-step sequence of reac-
tions. 1e not only has a greater bioactivity than triacsin C, its chemical
yield is 44% prepared from aldehyde 1 in one step.
enzymes may represent potential therapeutic targets for rotavirus
infection, and triacsin C analogs may be useful as therapeutic options
for rotavirus infection.
2. Biochemical studies
The effects of commercially available inhibitors for lipogenesis
as well as newly synthesized triacsin C analogs (described below)
were examined against in rotavirus replication using SA11 rotavirus
strain in MA104 cells. Various chemical inhibitors including FASN
inhibitors (cerulenin and C75), ACSL inhibitors (triacsin C and tro-
glitazone), DGAT inhibitors (A922500 and betulinic acid), and ACAT
inhibitors (CI-976, hexadecylamino-p-amino benzoic acid [PHB])
(Fig. 1) were obtained from SigmaeAldrich and Santa Cruz
Biotechnology Inc. (Santa Cruz, CA).
3. Chemistry
Various triascin C analogs were synthesized from (E,E,E)-2,4,7-
undecatrienal (1), which was prepared by a modification of the
reported procedure [16,17] (Scheme 1). Hence, bromination of (E)-
2-hexen-1-ol (2) with phosphorus tribromide in dichloromethane
followed by a displacement reaction with 3-(tetrahydropyranyloxy)
propynyl magnesium bromide and a catalytic amount of copper
cyanide afforded enyne 3. Removal of the THP protecting group of 3
with oxalic acid in refluxing methanol followed by selective
reduction of the propargylic alcohol function with lithium
aluminum hydride in diethyl ether gave (E,E)-2,5-nonadien-1-ol
(4). Oxidation of the hydroxyl function of 4 with o-iodoxybenzoic
acid (IBX) in DMSO provided aldehyde 5. A two-carbon extension
utilizing the HornereWadswortheEmmons protocol was carried
out by the treatment of aldehyde 5 with triethyl phosphonacetate
and sodium hydride in benzene furnished all-trans ester 6 in 57%
yield. When THF was used as solvent, under similar reaction
conditions only (E,E,E)-2,4,6-undecatrienoate (31% yield) was iso-
lated, and 6 was not found. Reduction of ester 6 with lithium
4. Results and discussion
All inhibitors, cerulenin, C75, triacsin C, troglitazone, A922500,
betulinic acid, CI-976 and PHB, significantly reduced the replication
of SA11 rotavirus in cells with ED50 values of 0.2e28.5
The ED50 values of cerulenin, C75, A922500, betulinic acid and PHB
were similar at 11.3e28.5 M, while troglitazone and CI-976 had
lower ED50 values of 5.8 M and 4.3 M, respectively (Table 1). The
TD50 of the inhibitors in MA104 cells varied at 8.5e85.4
(Table 1). Among the tested commercial inhibitors, triacsin C was
the most effective against rotavirus replication with ED50 of 0.2 M.
mM (Table 1).
m
m
m
m
M
m
The in vitro therapeutic index of triacsin C was 248. Similar ED50
values of the inhibitors were observed against Wa rotavirus strain,
and SA11 rotavirus strain was used for further studies. These results
1) Oxalic acid, MeOH
(88% yield)
1) PBr3, CH2Cl2
(89% yield)
OH
OTHP
2) LiAlH4, diethyl ether
2)
THPO CH2
(75% yield)
C
CMgBr
(90% yield)
2
3
(EtO)2POCH2CO2Et
IBX, DMSO
(70% yield)
NaH, benzene
O
OH
(57% yield)
4
5
1) LiAlH4
(85% yield)
OEt
H
2) IBX, DMSO
(96% yield)
O
O
6
1
Scheme 1. Preparation of 2,4,7-undecatrienal (1).

Y. Kim et al. / European Journal of Medicinal Chemistry 50 (2012) 311e318
313
H2NNHSO2CH2Cl
O2
S
EtOH
O
Cl
N
N
H
(100% yield)
1
1a
H2NNHSO2(CH2)3Cl
EtOH
O2
S
Cl
N
N
1
N
H
(98% yield)
1b
H2NNHSO2CH(CH3)2
EtOH
O2
S
1
N
H
(74% yield)
1c
(EtO)2POCH2CN
NaH, benzene
1
CN
(58% yield)
1d
NaH, DMF
25oC
O2
S
N
1b
N
(46% yield)
1ba
1)
H2N
CO2H
H
MeOH
N
1
2) NaCNBH3
(44% yield)
CO2H
1e
Scheme 2. Syntheses of triacsin C analogs.
indicate that disruption of lipogenesis induced by rotavirus repli-
cation at any step significantly inhibited the virus replication.
Triacsin C is an analog of polyunsaturated FA, having an 11-
carbon alkenyl chain with a N-hydroxytriazene moiety at the
terminus, and competitively inhibits ACSL 1, 3, and 4 [21]. ACSL is
a family of enzymes that converts long chain FA to acyl-CoA, which
serves as a substrate at each cascading step in the synthesis of
various lipid molecules including TG and cholesteryl ester. In
Fig. 1. Structures of inhibitors specific for FASN, ACSL, DGAT and ACAT.

314
Y. Kim et al. / European Journal of Medicinal Chemistry 50 (2012) 311e318
Table 1
enzymes significantly decreased rotavirus replication. We have also
synthesized novel triacsin analogs by modification of the
The effects of the inhibitors of lipogenic enzymes and triacsin C analogs in the
replication of rotavirus.
C
alkylidene-terminal end for evaluation of anti-rotavirus activity, and
found one of the novel analogs, 1e, was highly effective against
rotavirus replication. The facile and high-yielding synthesis of the
analogs would provide flexibility for further optimization. Based on
these findings, the lipogenic enzymes may serve as potential ther-
apeutic targets for rotavirus infection and triacsin C analogs as
potential therapeutic options for rotavirus infections.
Class
Inhibitors
Rotavirus (SA11)
ED50
(
m
M)a
TD50 (mM)
FASN
Cerulenin
C75
28.5
21.2
0.2
56.7
28.5
49.6
18.5
85.4
27.8
8.5
75.5
74.0
86.3
90.2
78.8
86.4
28.5
ACSL
Triacsin C
Troglitazone
A922500
Betulinic acid
CI-976
PHB
1a
1b
1c
1d
5.8
DGAT
23.2
22.5
4.3
11.3
8.7
11.5
9.8
21.3
2.2
5. Experimental
ACAT
Triacsin C analogs
5.1. General
NMR spectra were obtained from a 400-MHz spectrometer
(Varian Inc.), in CDCl3, unless otherwise indicated, and reported in
ppm. Mass spectra were taken from an API 2000-triple quadrupole
ESI-MS/MS mass spectrometer (from Applied Biosystems). Chem-
icals and solvents including chloromethanesulfonyl chloride, 3-
chloropropane-1-sulfonyl chloride, and propane-2-sulfonyl chlo-
ride were purchased from Fisher Scientific and Aldrich Chemical Co.
A modification of the reported procedure [16,17] was used to
prepare (E,E,E)-2,4,7-undecatrienal (1), and spectral data of all
synthetic intermediates including 13C NMR data (which were not
reported in previous publications [16,17]) are included herein.
1ba
1e
0.1
a
Each ED50 and TD50 value was the average of at least 3 independent tests.
mammals, five isoforms of ACSLs are identified (ACSL 1, 3e6) [12].
In lipid droplet-enriched fractions from huh-7 cells, ACSL 3 is the
most abundant isoform and ACSL 4 is a minor constituent [19]. The
role of ACSL3 seems to be important in the synthesis of neutral
lipids in lipid droplets, suggested by the finding that the addition of
oleic acid as a source of FA in the cells led to the accumulation of
lipid droplets in association with increased levels of ACSL 3 in lipid
droplets [20].
5.2. Representative synthesis
By inhibiting ACSL, triacsin C consequently blocks the synthesis
of TG, diglycerides, and cholesterol esters [22]. Troglitazone,
a member of the thiazolidinediones family, is an anti-diabetic and
anti-inflammatory drug. It is also known to inhibit ACSL4 (but not
other ACSL isoforms), and decreased the size of lipid droplets in
cells [23]. In our study, both triacsin C and troglitazone significantly
reduced the replication of rotavirus. Furthermore, addition of tri-
acsin C directly to the medium of cell culture showed potent anti-
virus effects against rotavirus replication, suggesting that triacsin C
readily penetrates the cells.
Since triacsin C strongly inhibited rotavirus replication, we
synthesized various analogs of triacsin C, and examined their effects
in rotavirus replication and toxicity in MA104 cells. The synthetic
analogs effective against rotaviruses are shown in Table 1 with ED50
and TD50 values, and therapeutic indexes. These analogs have
a conserved 11-carbon chain with various functionalities at the
alkylidene-terminal replacing 1,2,3-triazene of triacsin C. The pres-
ence of various sulfonylhydrazones and cyano functions (1ae1ba) at
the alkylidene-terminal end decreased the antiviral effects
compared to triazene moiety. However, 1e that contains amino-
benzoic acid at the alkylidene-terminal end is highly effective
5.2.1. (E)-1-(2-Tetrahydropyranyl)oxy-nona-5-en-2-yne (3)
To a solution of 10.0 g (0.1 mol) of E-2-hexen-1-ol (2) in 100 mL
of dichloromethane at ꢁ10 ꢀC under argon, was added 13.5 g
(0.05 mol) of PBr3. After stirring at 25 ꢀC for 3 h, the reaction
solution was washed with a saturated aqueous solution of NaHCO3
(50 mL), water, and brine, dried (MgSO4), concentrated to give
14.5 g (89% yield) of E-1-bromo-2-hexene, which was used in the
following step without purification. 1H NMR
d 5.8e5.65 (m, 2H, ]
CH), 3.96 (d, J ¼ 7 Hz, 2H), 2.05 (q, J ¼ 7 Hz, 2H), 1.41 (sextet,
J ¼ 7 Hz, 2H), 0.91 (t, J ¼ 7 Hz, 3H); 13C NMR
d 136.7,126.7, 34.3, 33.9,
22.2, 13.8; MS (electrospray) m/z 165.1 and 163.1 (M þ Hþ; 100%;
bromine isotopes).
To a solution of 15 g (0.18 mol) of dihydropyrane and 2 mg of p-
toluenesulfonic acid was added 10 g (0.18 mol) of 2-propyn-1-ol.
After stirring at 25 ꢀC for 3 h, the reaction solution was diluted with
100 mL of diethyl ether and washed with 10% aqueous NaHCO3,
water, and brine, dried (MgSO4), concentrated to give 20.5 g (82%
yield) of 2-(2-propynyloxy)-tetrahydropyrane. 1H NMR
d 4.83 (t,
J ¼ 3.6 Hz, 1H, CHO), 4.29 (dd, J ¼ 16, 3 Hz, 1H, CH2O), 4.26 (dd,
J ¼ 16, 3 Hz, 1H, CH2O), 3.88e3.82 (m, 1H, CHO), 3.58e3.52 (m, 1H,
CHO), 2.42 (d, J ¼ 3 Hz, 1H, ^CH), 1.90e1.50 (m, 6H); 13C NMR
against rotavirus replication with lower ED50 (0.1
mM) and higher
d 97.0, 79.9, 74.2, 62.1, 54.1, 30.4, 25.5, 19.1; MS (electrospray;
in vitro therapeutic index (285), compared to those of triacsin C. Anti-
rotaviral effects of triacsin C, 1ba, and 1e were also confirmed with
Western blot analysis and IFA (Fig. 2). The pre-treatment of SA11
negative mode) m/z 139.0 (M ꢁ Hꢁ, 100%). MS (electrospray; posi-
tive mode) m/z 163.1 (M þ Naþ; 100%).
To a solution of 18 mL (18 mmol) of 1 M ethylmagnesium
bromide in THF under argon at 0 ꢀC, were added 1.7 g (12 mmol) of
2-(2-propynyloxy)-tetrahydropyrane and 4 mg of CuCN. After stir-
ring for 0.5 h, 2.0 g (12 mmol) of E-1-bromo-2-hexene was added,
and the resulting solution was stirred at 25 ꢀC for 0.5 h, neutralized
with 0.1 N HCl, and extracted with diethyl ether three times. The
combined ether extract was washed with water and brine, dried
(MgSO4), concentrated, and column chromatographed on silica gel
using a mixture of hexane and diethyl ether (20:1) to give 2.0 g
rotavirus with triacsin C (100 mM) or 1e (100 mM) did not affect the
viral titer, indicating that the antiviral effects of triacsin C and 1e are
not the result of direct viral neutralization or virucidal effect on
rotavirus. The suppression of an increase in lipid droplets by triacsin
C, 1ba and 1e was dose-dependent in Huh-7 cells and positively
correlated (r2 ¼ 0.89 and 0.87 for 1ba and 1e, respectively) with
reduction of rotavirus replication in MA104 cells (Fig. 3). The results
suggest that the inhibition of virus replication by these compounds
is associated with the suppression of lipid accumulation in the cells
triggered by viral infection.
(75% yield) of compound 3: 1H NMR
d
5.66 (dt, J ¼ 16, 7 Hz, 1H, ]
CH), 5.40 (dt, J ¼ 16, 5.6 Hz, 1H, ]CH), 4.83 (t, J ¼ 3 Hz, 1H, CHO),
4.33 (dt, J ¼ 16, 2.4 Hz, 1H, CH2O), 4.24 (dd, J ¼ 16, 2.4 Hz, 1H, CH2O),
3.89e3.82 (m, 1H, CHO), 3.57e3.51 (m,, CHO), 2.95 (d, J ¼ 3.6 Hz,
In this study, we demonstrated that rotavirus replication is
closely associated with lipid metabolism, and inhibition of lipogenic

Y. Kim et al. / European Journal of Medicinal Chemistry 50 (2012) 311e318
315
Fig. 2. The effects of triacsin C, and its analogs in rotavirus (SA11) replication determined by Western blot analysis (A) and IFA (B). SA11 rotavirus was inoculated to the cells at a MOI
of 2, and cells were incubated with or without each inhibitor (triacsin C, 1e or 1ba). Cell lysates were collected at 12 h post infection (A) and virus infected cells were fixed at 12 h
post infection (B) at the indicated concentrations.
2H), 2.00 (q, J ¼ 7 Hz, 2H), 1.90e1.50 (m, 6H), 1.39 (sextet, J ¼ 7 Hz,
5.2.2. (E,E)-2,5-Nanodien-1-ol (4)
2H), 0.90 (t, J ¼ 7 Hz, 3H); 13C NMR
d
132.5, 124.1, 96.9, 84.4, 77.6,
A solution of 7.2 g (32 mmol) of compound 3 in 60 mL of 2%
oxalic acid in methanol was heated under reflux for 2 h, and most of
the methanol was removed under a rotary evaporator. The residue
62.2, 54.9, 34.6, 30.5, 25.6, 22.6, 22.3, 19.3, 13.9; MS (electrospray)
m/z 245.2 (M þ Naþ; 100%).
Fig. 3. Lipid droplets monitored with NBD-cholesterol in rotavirus (SA11) infected MA104 cells (A) or uninfected Huh-7 cells (B). A. Lipid droplets in MA104 cells infected with SA11
rotavirus with a MOI of 10. Panels, a and b: cells incubated with mock-medium (a) or SA11 rotavirus (b) for 10 h. Panels, c and d: SA11 rotavirus infected cells incubated in the
presence of triacsin C (1
mM) (c) or 1e (1
mM) (d) for 10 h. B. The development of lipid droplets in Huh-7 cells incubated with mock-medium, triacsin C, or 1e. Panel a: 1 day old cells;
b: 4 day old cells; c and d: 4 day old cells incubated in the presence of triacsin C (1
m
M) (c) or 1e (1 M) (d).
m

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Y. Kim et al. / European Journal of Medicinal Chemistry 50 (2012) 311e318
was diluted with diethyl ether (100 mL), washed with water and
brine, dried (MgSO4), concentrated, and column chromatographed
on silica gel using a gradient mixture of hexane and diethyl ether as
eluant to give 3.94 g (88% yield) of E-5-nonen-2-yn-1-ol. 1H NMR
carefully, and extracted with diethyl ether twice. The combined
extracts were washed with water and brine, dried (MgSO4), and
concentrated to give 0.43 g (85% yield) of (E,E,E)-2,4,7-undecan-1-
ol. 1H NMR
d
6.23 (dd, J ¼ 15, 11 Hz, 1H, ]CH), 6.05 (dd, J ¼ 15,
d
5.67 (dt, J ¼ 16, 7 Hz, 1H, ]CH), 5.39 (dt, J ¼ 16, 5.6 Hz, 1H, ]CH),
11 Hz,1H, ]CH), 5.78e5.67 (m, 2H, ]CH), 5.47e5.37 (m, 2H, ]CH),
4.16 (q, J ¼ 6 Hz, 2H, CHO), 2.78 (t, J ¼ 6 Hz, 2H, CH2), 1.98 (q,
J ¼ 7 Hz, 2H), 1.37 (sextet, J ¼ 7 Hz, 2H), 0.89 (t, J ¼ 7 Hz, 3H); 13C
4.29 (dt, J ¼ 6, 2 Hz, 2H, CH2O), 2.96 (d, J ¼ 2 Hz, 2H), 2.00 (q,
J ¼ 7 Hz, 2H), 1.39 (sextet, J ¼ 7 Hz, 2H), 0.90 (t, J ¼ 7 Hz, 3H); 13C
NMR
d
132.6, 123.9, 84.4, 80.0, 51.7, 34.6, 22.6, 22.2, 13.9; MS
NMR d 134.1 (2 C), 132.1, 130.1 (2 C), 129.9, 127.7, 63.7, 35.8, 34.9,
(electrospray) m/z 161.1 (M þ Naþ; 100%).
22.8, 13.9; MS (electrospray) m/z 205.1 (M þ Kþ; 100%), 189.1
To a solution of 5.5 g (40 mmol) of E-5-nonen-2-yn-1-ol in
50 mL of diethyl ether under argon was added 1.5 g (39 mmol) of
LiAlH4 in portions. After stirring at 25 ꢀC for 24 h, the reaction
mixture was quenched with 50 mL of 1 N NaOH, and extracted with
diethyl ether three times (50 mL each). The combined ether
extracts were washed with water and brine, dried (MgSO4),
concentrated to give 5.0 g (90% yield) of compound 4, which was
used in the next step without further purification. 1H NMR
(M þ Naþ).
To a solution of 0.43 g (2.6 mmol) of (E,E,E)-2,4,7-undecan-1-ol
in 3 mL of DMSO under argon was added 0.91 g (3.3 mmol) of
IBX, and the solution was stirred at 25 ꢀC for 4 h, diluted with
dichloromethane (50 mL), and washed with water (50 mL). The
aqueous layer was extracted with dichloromethane, and the
combined organic layers were washed with brine (20 mL), dried
(MgSO4), concentrated, and column chromatographed on silica gel
using hexane and diethyl ether (20:1) as eluant to give 0.41 g (96%
d
5.75e5.6 (m, 2H, ]CH), 5.50e5.35 (m, 2H, ]CH), 4.12 (t, J ¼ 4 Hz,
2H, CH2O), 2.75 (t, J ¼ 5 Hz, 2H), 1.99 (q, J ¼ 7 Hz, 2H), 1.38 (sextet,
yield) of aldehyde 1. 1H NMR
d
9.55 (d, J ¼ 8 Hz, 1H, CHO), 7.10 (dd,
J ¼ 7 Hz, 2H), 0.89 (t, J ¼ 7 Hz, 3H); 13C NMR
d
131.9 (2 C), 129.6,
J ¼ 15, 9 Hz, 1H, ]CH), 6.37e6.24 (m, 2H, ]CH), 6.10 (dd, J ¼ 15,
7.6 Hz, 1H, ]CH), 5.55e5.38 (m, 2H, ]CH), 2.91 (t, J ¼ 9.6 Hz, 2H,
CH2), 2.01 (q, J ¼ 7 Hz, 2H), 1.40 (sextet, J ¼ 7 Hz, 2H), 0.91 (t,
127.8, 63.9, 35.4, 34.9, 22.8, 22.2, 13.9; MS (electrospray) m/z 163.1
(M þ Naþ; 100%).
J ¼ 7 Hz, 3H); 13C NMR
d 194.1, 152.8, 145.4, 133.4, 130.6, 129.0,
5.2.3. (E,E)-2,5-Nonadienal (5)
126.0, 36.3, 34.9, 22.7, 13.9; MS (electrospray) m/z 187.1 (M þ Naþ,
To a solution of 5.0 g (36 mmol) of compound 4 in 13 mL of
DMSO under argon at 25 ꢀC was added 12.5 g (45 mmol) of o-
iodoxybenzoic acid (IBX), and the solution was stirred for 4 h,
diluted with diethyl ether (100 mL), and washed with water twice
(50 mL each). The aqueous layers were extracted with diethyl
ether, and the combined ether layers were washed with brine,
dried (MgSO4), concentrated, and column chromatographed on
silica gel using hexane and diethyl ether (20:1) as eluant to give
100%).
5.2.6. (E)-Chloro-N0-((2E,4E,7E)-undeca-2,4,7-trienylidene)
methanesulfonohydrazide (1a)
A solution of 20 mg (0.12 mmol) of (E,E,E)-2,4,7-undecatrienal
(1) and 35 mg (0.24 mmol) of chloromethanesulfonohydrazide in
1 mL of ethanol was stirred under argon at 25 ꢀC for 2 h, and
ethanol was removed under a rotary evaporator. The residue was
diluted with 20 mL of diethyl ether, washed with water (15 mL)
three times, and brine (15 mL). The organic layer was dried
(MgSO4), concentrated, and column chromatographed on silica gel
using a 1:1 mixture of hexane and diethyl ether as eluent to give
3.45 g (70% yield) of aldehyde 5. 1H NMR
d
9.53 (d, J ¼ 7 Hz, 1H,
CHO), 6.85 (dt, J ¼ 16, 7 Hz, 1H, ]CH), 6.13 (dd, J ¼ 16, 8 Hz, 1H, ]
CH), 5.55 (dt, J ¼ 15, 5 Hz, 1H, ]CH), 5.43 (dt, J ¼ 15, 5 Hz, 1H, ]
CH), 3.03 (t, J ¼ 7 Hz, 2H, CH2), 2.01 (q, J ¼ 7 Hz, 2H), 1.39 (sextet,
J ¼ 7 Hz, 2H), 0.91 (t, J ¼ 7 Hz, 3H); 13C NMR
d
194.2, 157.3, 134.4,
35 mg (100% yield) of 1a: 1H NMR (CDCl3)
d 7.91 (s, 1H, NH), 7.55 (d,
133.2, 124.6, 35.8, 34.8, 22.6, 13.8; MS (electrospray) m/z 161.2
(M þ Naþ; 100%).
J ¼ 10 Hz,1H, N]CH), 6.56 (dd, J ¼ 15,10 Hz,1H), 6.27e6.15 (m, 2H),
5.97 (dt, J ¼ 12, 7 Hz, 1H), 5.47e5.38 (m, 2H), 4.70 (s, 2H, CH2Cl),
2.85 (t, J ¼ 7 Hz, 2H), 1.99 (q, J ¼ 7 Hz, 2H), 1.39 (sextet, J ¼ 7 Hz, 2H),
5.2.4. Ethyl (E,E,E)-2,4,7-Undecatrienoate (6)
0.90 (t, J ¼ 7 Hz, 3H); 13C NMR (CDCl3)
d 152.0, 142.3, 140.0, 132.8,
129.7, 126.7, 125.3, 53.0, 36.1, 34.9, 22.7, 13.9; MS (electrospray) m/z
To a mixture of 0.35 g (8.7 mmol) of sodium hydride (60% in oil;
pre-washed with diethyl ether twice to remove mineral oil) in
20 mL of benzene under argon was added 1.95 g (8.7 mmol) of
triethyl phosphonacetate, and the solution was stirred at 25 ꢀC for
2 h. To it, a solution of 1.0 g (7.2 mmol) of compound 5 in 2 mL of
benzene was added via cannula under argon. After stirring for 0.5 h,
the reaction solution was neutralized with 0.1 N HCl, and extracted
twice with diethyl ether. The combined extract was washed with
water and brine, dried (MgSO4), concentrated, and column chro-
matographed on silica gel using hexane and diethyl ether (30:1) as
eluant to give 0.64 g (57% yield based on recovered starting mate-
rial) of compound 6 and 0.25 g (25% recovery) of compound alde-
313.0 (M þ Naþ; 100%).
5.2.7. (E)-3-Chloro-N0-((2E,4E,7E)-undeca-2,4,7-trienylidene)
propane-1-sulfonohydrazide (1b)
A similar procedure to that described above was followed. From
20 mg (0.12 mmol) of aldehyde 1 and 42 mg (0.24 mmol) of 40%
hydrazine, 38 mg (98% yield) of 1b was obtained after column
chromatographic purification. 1H NMR (CDCl3)
d 8.08 (s, 1H, NH),
7.49 (d, J ¼ 10 Hz, 1H, N]CH), 6.52 (dd, J ¼ 15, 10 Hz, 1H), 6.27e6.14
(m, 2H), 5.94 (dt, J ¼ 12, 7 Hz, 1H), 5.49e5.38 (m, 2H), 3.68 (t,
J ¼ 7 Hz, 2H, CH2Cl), 3.43 (dd, J ¼ 7, 3 Hz, 2H, CH2S), 2.84 (t, J ¼ 7 Hz,
2H), 2.31 (pentet, J ¼ 7 Hz, 2H), 1.99 (q, J ¼ 7 Hz, 2H), 1.37 (sextet,
hyde 5. 1H NMR
d
7.27 (dd, J ¼ 16, 10 Hz, 1H, ]CH), 6.22e6.09 (m,
2H, ]CH), 5.80 (d, J ¼ 16 Hz, 1H, ]CH), 5.52e5.37 (m, 2H, ]CH),
4.20 (q, J ¼ 7 Hz, 2H), 2.86 (t, J ¼ 6 Hz, 2H, CH2),1.99 (q, J ¼ 7 Hz, 2H),
1.39 (sextet, J ¼ 7 Hz, 2H), 1.30 (t, J ¼ 7 Hz, 3H), 0.90 (t, J ¼ 7 Hz, 3H);
J ¼ 7 Hz, 2H), 0.89 (t, J ¼ 7 Hz, 3H); 13C NMR (CDCl3)
d 150.4, 141.3,
139.2,132.7,129.8,126.9,125.7, 48.8, 42.8, 36.0, 34.9, 26.7, 22.7,13.9;
MS (electrospray) m/z 341.0 (M þ Naþ; 100%).
13C NMR
d 167.5, 145.0, 142.8, 132.9, 128.8, 126.5, 119.8, 60.4, 36.1,
34.9, 22.7, 14.5, 13.9; MS (electrospray) m/z 231.0 and 163.1
5.2.8. (E)-N0-[(2E,4E,7E)-Undeca-2,4,7-trienylidene]propane-2-
sulfonohydrazide (1c)
Following a similar reaction sequence as that described for 1a,
from 25 mg (0.15 mmol) of aldehyde 1 and 42 mg (0.31 mmol) of
propane-2-sulfonohydrazide, gave 32 mg (74% yield) of 1c. 1H NMR
(M þ Naþ; 100%), 209.2 (M þ Hþ).
5.2.5. (E,E,E)-2,4,7-Undecatrienal (1)
To a solution of 0.63 g (3.0 mmol) of compound 6 in 20 mL of
diethyl ether under argon at 0 ꢀC was added 0.12 g (3.0 mmol) of
LiAlH4, and the solution was stirred for 1 h, diluted with water
(CDCl3)
d
7.90 (s, 1H, NH), 7.48 (d, J ¼ 10 Hz, 1H, N]CH), 6.50 (dd,
J ¼ 15, 10 Hz, 1H), 6.24 (dd, J ¼ 15, 10 Hz, 1H), 6.16 (dd, J ¼ 14, 10 Hz,

Y. Kim et al. / European Journal of Medicinal Chemistry 50 (2012) 311e318
317
1H), 5.92 (dt, J ¼ 15, 7 Hz, 1H), 5.46e5.37 (m, 2H), 3.53 (heptet,
6. Biochemical studies
J ¼ 7 Hz, 1H, CH), 2.83 (t, J ¼ 7 Hz, 2H), 1.99 (q, J ¼ 7 Hz, 2H), 1.41 (d,
J ¼ 7 Hz, 6H), 1.38 (sextet, J ¼ 7 Hz, 2H), 0.89 (t, J ¼ 7 Hz, 3H); 13C
6.1. Cells, antisera and reagents
NMR (CDCl3)
d 149.4, 140.6, 138.8, 132.6, 129.9, 126.9, 126.0, 52.4,
36.0, 34.9, 22.7, 16.4, 13.9; MS (electrospray) m/z 307.1 (M þ Naþ;
The MA104 cell line was maintained in minimum essential
medium (MEM) containing 5% fetal bovine serum and antibiotics
(penicillin and streptomycin). Antibodies specific to rotavirus VP6 or
100%).
5.2.9. (2E,4E,6E,9E)-Trideca-2,4,6,9-tetraenenitrile (1d)
b-actin were obtained from Santa Cruz Biotechnology Inc. Rotavirus
To a mixture of 15 mg (0.36 mmol) of sodium hydride (washed
twice with distilled anhydrous diethyl ether) in 1 mL of benzene
under argon was added 64 mg (0.36 mmol) of diethyl cyanome-
thylphosphonate, and the solution was stirred at 25 ꢀC for 2 h. To it,
a solution of 40 mg (0.24 mmol) of aldehyde 1 in 1 mL of benzene
was added via syringe. The resulting solution was stirred for 1 h,
diluted with water (10 mL), neutralized with 0.1 N HCl solution, and
extracted with diethyl ether three times (20 mL each). The
combined extract was washed with brine, dried (MgSO4), concen-
trated, and column chromatographed on silica gel using a 30:1
mixture of hexane and diethyl ether as eluant to give 26 mg (58%
strains including Wa (human G1) and SA11 (primate G3) were ob-
tained from American Type Culture Collection (ATCC, Manassas, VA).
6.2. Nonspecific cytotoxic effects
Confluent MA104 grown in 24-well plates were treated with
various concentrations of each compound for 24 h. Cell cytotoxicity
was measured by a CytoTox 96Ò non-radioactive cytotoxicity assay
kit (Promega, Madison, WI), and TD50 value of each compound was
determined using the method of Litchfield and Wilcoxon [24].
yield) of 1d. 1H NMR (CDCl3)
d
7.00 (dd, J ¼ 15, 10 Hz, 1H), 6.52 (dd,
6.3. Rotavirus infection and treatments in cell culture
J ¼ 15, 10 Hz, 1H), 6.22e6.05 (m, 2H), 6.00 (dt, J ¼ 15, 7 Hz, 1H),
5.46e5.38 (m, 2H), 5.28 (d, J ¼ 15 Hz, 1H), 2.86 (t, J ¼ 7 Hz, 2H), 1.99
(q, J ¼ 7 Hz, 2H), 1.39 (sextet, J ¼ 7 Hz, 2H), 0.90 (t, J ¼ 7 Hz, 3H); 13C
Fully confluent monolayered MA104 cells grown in 6- or 12-well
plates were inoculated with Wa or SA11 rotavirus at high (2) or low
(0.01) multiplicity of infection (MOI) for 1 h. Following washing step
with phosphate buffered saline (PBS), MEM containing each
compound or DMSO (0.1%) was added to each well. In addition,
NMR (CDCl3)
d 150.6, 142.0, 140.7, 132.8, 129.7, 127.5, 126.7, 195.0,
97.2, 36.1, 34.9, 22.7, 13.9; MS (electrospray) m/z 210.1 (M þ Naþ;
100%).
trypsin (SigmaeAldrich) was included at 1
rotavirus replication. The concentration of each compound in the
cells were less than 50 M for cerulenin, C75, triacsin C, various
triacsin C analogs, A922500 and PHB, and less than 20 M for
betulinic acid, and CI-976. As a control, nitazoxanide was used at
0.5e5 M. Virus replication was monitored at 12, 24 and 48 h post
infection by immunofluorescent assay (IFA) or Western blot analysis
with antibody against VP6, and the 50% tissue culture infectious dose
(TCID50)/mL was also determined. The ED50 was calculated based on
TCID50 titers at 24 h post infection (with 0.05 MOI) for each
compound using the method of Litchfield and Wilcoxon [24].
mg/mL in medium for
5.2.10. 2-[(2E,4E,7E)-Undeca-2,4,7-trienylideneamino]-1,1-dioxo-
1-isothiazolidine (1ba)
m
A solution of 15 mg (47
m
mol) of 1b and 2.0 mg (50
m
mol) of
m
sodium hydride in 0.5 mL of DMF (distilled) under argon was
stirred at 25 ꢀC for 3 days. The resulting solution was neutralized
with diluted aqueous HCl solution, and extracted with diethyl
ether twice (25 mL each). The combined extract was washed with
water three times (20 mL each), and brine (20 mL), dried
(MgSO4), concentrated, and column chromatographed on silica
gel using a 1:1 mixture of hexane and diethyl ether as eluant to
m
give 6 mg (46% yield) of 1ba. 1H NMR (CDCl3)
d
7.54 (d, J ¼ 10 Hz,
1H, N]CH), 6.54 (dd, J ¼ 15, 10 Hz, 1H), 6.32 (dd, J ¼ 15, 10 Hz,
1H), 6.18 (dd, J ¼ 15, 10 Hz, 1H), 5.91 (dt, J ¼ 15, 7 Hz, 1H),
5.46e5.40 (m, 2H), 3.59 (t, J ¼ 7 Hz, 2H), 3.22 (t, J ¼ 7 Hz, 2H), 2.84
(t, J ¼ 7 Hz, 2H), 2.49 (pentet, J ¼ 7 Hz, 2H), 1.99 (q, J ¼ 7 Hz, 2H),
1.38 (sextet, J ¼ 7 Hz, 2H), 0.89 (t, J ¼ 7 Hz, 3H); 13C NMR (CDCl3)
6.4. Pre-treatment of viruses with triacsin C or 1e
To investigate if triacsin C or its analog 1e has virucidal effects on
rotavirus, SA11 rotavirus of high titer (>109 TCID50/mL) was pre-
incubated with PBS (or DMSO), triacsin
C (200 mM) or 1e
d
151.5, 140.8, 138.6 (2C), 132.6, 130.1, 126.8, 46.3, 45.3, 36.1, 34.9,
(200
m
M) at 37 ꢀC for 2 h. Then the mixture was diluted up to 100
30.6, 22.8, 18.0, 13.9; MS (electrospray) m/z 305.2 (M þ Naþ;
times for cell inoculation. Virus infected cells were incubated with
medium containing trypsin for up to 24 or 48 h, and the virus
replication was monitored by the titration of progeny viruses using
the TCID50 method.
100%).
5.2.11. 4-((2E,4E,7E)-Undeca-2,4,7-trienylamino)benzoic acid (1e)
A solution of 30 mg (0.18 mmol) of aldehyde 1 and 24 mg
(0.18 mmol) of 4-aminobenzoic acid in 5 mL of methanol was stir-
6.5. Determination of viral replication using IFA, Western blot
analysis and TCID50 method
red under argon at 25 ꢀC for 1 h. To it, 10
mL of acetic acid and 30 mg
(0.48 mmol) of sodium cyanoborohydride were added sequentially.
After stirring at 25 ꢀC for 12 h, the reaction solution was diluted
with aqueous ammonium chloride, and extracted with diethyl
ether twice (25 mL each). The combined extract was washed with
brine, dried (MgSO4), concentrated and column chromatographed
on silica gel using a gradient mixture of hexane and ethyl acetate as
Virus replication in cell culture was assessed by the IFA or
Western blot analysis at 12 or 24 h post infection before the
extensive cytopathic effects (CPE) are induced by viral infection.
Viral replication was also titrated with the TCID50 method at 24 and
48 h post infection after cells were lysed with repeated freezing and
thawing. IFA: cells were fixed with 100% methanol at room
temperature for 30 min. Then, monoclonal antibody specific for
rotavirus VP6 was applied to the cells followed by goat anti-mouse
IgG conjugated fluorescein isothiocyanate (FITC). The stained cells
were observed under a fluorescence microscope. Western blot
analysis: The expression levels of VP6 in the presence of each
compound were also assessed by Western blot analysis. The cells
were lysed at 12 h post infection, and the cell lysates were prepared
eluant to give 23 mg (44% yield) of 1e. 1H NMR (CDCl3)
d 7.93 (d,
J ¼ 8 Hz, 2H, Ar), 6.58 (d, J ¼ 8 Hz, 2H, Ar), 6.25 (dd, J ¼ 15, 10 Hz,
1H), 6.06 (dd, J ¼ 15, 10 Hz, 1H), 5.75e5.64 (m, 2H), 5.45e5.40 (m,
2H), 3.86 (d, J ¼ 7 Hz, 2H, CH2H), 2.78 (t, J ¼ 7 Hz, 2H), 1.98 (q,
J ¼ 7 Hz, 2H), 1.38 (sextet, J ¼ 7 Hz, 2H), 0.90 (t, J ¼ 7 Hz, 3H); 13C
NMR (CDCl3)
d 171.8, 152.6, 134.0, 132.9, 132.5, 132.1, 129.7, 127.6,
127.1, 117.6, 111.9, 45.3, 35.8, 34.9, 22.8, 13.9; MS (electrospray) m/z
308.1 (M þ Naþ; 100%).

318
Y. Kim et al. / European Journal of Medicinal Chemistry 50 (2012) 311e318
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m
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In human liver carcinoma Huh-7 cells, lipid droplets develop
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Acknowledgments
This work was supported by NIH U01AI081891. The authors
thank Samira Najm for technical assistance. SH would like to thank
the International Cooperation Program for Excellent Lectures of
Shandong Provincial Education Department, P. R. China.
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Appendix. Supplementary material
Supplementary data associated with this article can be found, in
[25] L.J. Reed, H. Muench, A simple method of estimating fifty percent endpoints,
Am. J. Hyg. 27 (1938) 493e497.
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High density lipoprotein-mediated cholesterol uptake and targeting to lipid
droplets in intact L-cell fibroblasts. A single- and multiphoton fluorescence
approach, J. Biol. Chem. 275 (2000) 12769e12780.
the online version, at doi:10.1016/j.ejmech.2012.02.010. 1H and 13
C
NMR spectra of compounds 1a e 1e are included in the Supple-
mentary data.
[27] P.M. McDonough, R.M. Agustin, R.S. Ingermanson, P.A. Loy, B.M. Buehrer,
J.B. Nicoll, N.L. Prigozhina, I. Mikic, J.H. Price, Quantification of lipid droplets
and associated proteins in cellular models of obesity via high-content/high-
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