A direct synthesis of atractylodinol, a potent inhibitor of PRRSV, and its biological evaluation

Article abstract of DOI:10.1016/j.tetlet.2016.10.014

A direct synthesis of atractylodinol from 2-furylbutenyne and bromoacetylene 6 is reported. Both compounds 1 and 8 showed greater than 99% virus inhibition.

Full text of DOI:10.1016/j.tetlet.2016.10.014

Tetrahedron Letters 57 (2016) 5185–5187
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
A direct synthesis of atractylodinol, a potent inhibitor of PRRSV, and
its biological evaluation
a
a
b
b
George A. Kraus a, , Pengfei Dong , Yang Qu , Alyssa Evans , Susan Carpenter
⇑
a Department of Chemistry, Iowa State University, Ames IA 50011, United States
b Department of Animal Science, Iowa State University, Ames, IA 50011, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A direct synthesis of atractylodinol from 2-furylbutenyne and bromoacetylene 6 is reported. Both com-
pounds 1 and 8 showed greater than 99% virus inhibition.
Received 16 August 2016
Revised 4 October 2016
Accepted 6 October 2016
Available online 8 October 2016
Ó 2016 Elsevier Ltd. All rights reserved.
Keywords:
Total synthesis
Atractylodinol
PRRSV
Antiviral activity
In vitro
Porcine reproductive and respiratory syndrome (PRRS) is
caused by porcine reproductive and respiratory syndrome virus
(PRRSV). PRRS is a widespread disease affecting domestic pigs,
and is considered one of the most economically significant diseases
in the global swine industry.1 In 2005, the annual costs of PRRS for
the American swine industry were approximately 560 million dol-
lars.2,3 Although vaccines are used to treat PRRSV, genetic diversity
of PRRSV reduces the effectiveness of vaccines and contributes to
PRRSV persistence in the field. There is an urgent need for alternate
strategies to reduce the economic burden of PRRS. An alternative
strategy is the development of potent antiviral drugs. Unlike vacci-
nes, antiviral drugs can provide almost immediate treatment for
animals. The antiviral drugs can also be used in periods of
future biological activity tests in animals. A patent using cobalt-
complexed acetylenes to prepare dienediynes was reported in
2016.6 The related hydrocarbon analog atractylodin was synthe-
sized using a bis-silylated diacetylene.7
In our synthetic plan, compound 1 could be synthesized by cou-
pling reaction of furyl enyne 4 and bromo alcohol 6. Enyne 4 could
be converted by a Corey–Fuchs reaction8 from commercially avail-
able 3-(2-furyl)acrolein (3). Compound 6 is accessible through
unsaturated aldehyde 5.
Commercially available 3-(2-furyl)acrolein 3 was subjected to
the Corey–Fuchs protocol as shown in Scheme 1 to obtain the ter-
minal alkyne 4 in 67% overall yield. Treatment of unsaturated alde-
hyde 5 (generated from cis-butenediol in two steps)9 with carbon
increased susceptibility.
A
few natural products have been
tetrabromide and triphenylphosphine afforded a 1,1-dibro-
reported to significantly inactivate the PRRS virus. Their structures
are depicted in Figure 1.
moalkene. Treatment of the 1,1-dibromoalkene with tetra-n-buty-
lammonium fluoride in THF at 45 °C resulted in both deprotection
of the TBS group and the elimination of bromide to make alcohol 6
in 66% yield.10
With the two coupling components in hand, we focused on
finding the most suitable coupling condition. After several modifi-
cations, utilizing a copper (I) catalyst with ethylamine and hydrox-
ylamine hydrochloride in methanol, the Cadiot–Chodkiewicz
coupling reaction of compounds 4 and 6 furnished the desired nat-
ural product 1 in 25% isolated yield.10 Although the yield was mod-
est for the final step, it was the only effective conditions that we
found for this two-component coupling. With the possible excep-
tion of the recent patent, no total synthesis route of atractylodinol
Atractylodinol (1) and Ethoxysanguinarine (2) were reported as
anti-PRRSV drugs by Li with IC50 values of 7.9 and 39.4 lmol/L,
respectively.4 Atractylodinol was isolated from rhizomes of
Atractylodes lancea. A. lancea and is widely used in traditional Chi-
nese and Japanese medicines against rheumatic diseases, digestive
disorders, night blindness, and influenza.5 The reported isolation
procedure gives 9.6 mg of impure 1 from 570 g of dried and pow-
ered A. lancea rhizomes.5 The low natural abundance inhibits
⇑
Corresponding author. Tel.: +1 515 294 7794; fax: +1 515 294 0105.
E-mail address: gakraus@iastate.edu (G.A. Kraus).
http://dx.doi.org/10.1016/j.tetlet.2016.10.014
0040-4039/Ó 2016 Elsevier Ltd. All rights reserved.

5186
G. A. Kraus et al. / Tetrahedron Letters 57 (2016) 5185–5187
Figure 1. Natural product inhibitors of PRRSV.
Figure 2. Antiviral activity was tested using 10 ug of each compound against 102
FFU PRRSV. Percent virus inhibition is reported as the mean percent reduction in
compound-treated wells compared to virus-only control wells. Error bars repre-
sent one standard deviation of the mean of replicates.
Scheme 1. Total synthesis of atractylodinol.
synthetic route through ethyl ester 8 was also reported. To the best
of our knowledge, neither of these two efficient synthetic routes
has been reported.
Acknowledgments
We thank the Department of Chemistry for support for PD.
References and notes
Scheme 2. Alternate synthetic route.
1. Islam, Z. U.; Bishop, S. C.; Savill, N. J.; Rowland, R. R. R.; Lunney, J. K.; Trible, B.;
Doeschl-Wilson, A. B. PLoS One 2013, 8 e83567.
2. Neumann, E. J.; Kliebenstein, J. B.; Johnson, C. D.; Mabry, J. W.; Bush, E. J.;
Seitzinger, A. H.; Green, A. L.; Zimmerman, J. J. J. Am. Vet. Med. Assoc. 2005, 227,
385–392.
had been reported in the literature. Despite the modest yield of the
coupling reaction, the overall yield from commercially available
materials was 11%.
To improve the synthetic route for natural product 1, as well as
to make analogs 7 and 8, we designed an alternate route as shown
below in Scheme 2. Coupling of 3-iodopropargyl alcohol11 with 4
provided alcohol 7 in 37% isolated yield. Alcohol oxidation using
the Dess–Martin reagent followed immediately by a Wittig reac-
tion using commercially available phosphorane generated ester 8
in 80% yield over two steps. Reduction of ester 8 using DIBAL12
from À78 °C to room temperature afforded 1 in 88% yield. The
advantages of this route included a significantly improved coupling
reaction with easily prepared 3-iodopropargyl alcohol and the high
yield ester reduction with DIBAL. This route would be the preferred
route for the scaling up of 1.14–17
3. Holtkamp, D.P.; Kliebenstein J.B.; Zimmerman J.J.; Neumann E.; Rotto H.; Yoder
T.K.; Wang C.; Yeske P.; Mowrer C.L.; Haley C.; Animal Industry Report AS 658,
2012 ASL R2671.
4. Li, W.; Dai, F.; Cheng, Y.; Yin, G.; Bi, J.; Li, D. Chem. Res. Chin. Univ. 2013, 29, 290–
293.
5. Nishikawa, Y.; Yasuda, I.; Watanabe, Y.; Seto, T. Yakugaku Zasshi 1976, 96,
1322–1326.
6. Aoki, K.; Yomura, K. Jpn. Kokai Tokkyo Koho (2016), JP 2016044161
20160404.
A
7. Fiandanese, V.; Bottalico, D.; Marchese, G.; Punzi, A. Tetrahedron 2006, 62,
5126–5132.
8. Corey, E. J.; Fuchs, P. L. Tetrahedron Lett. 1972, 13, 3769–3772.
9. Bhunia, N.; Das, B. Synthesis 2015, 47, 1499–1509.
10. Takamura, H.; Wada, H.; Lu, N.; Ohno, O.; Suenaga, K.; Kadota, I. J. Org. Chem.
2013, 78, 2443–2454.
11. Xu, R.; Schweizer, W. B.; Frauenrath, H. Chem. Eur. J. 2009, 15, 9105–9116.
12. Takeuchi, R.; Tanabe, K.; Tanaka, S. J. Org. Chem. 2000, 65, 1558–1561.
13. Experimental procedure for the synthesis of (E)-2-(But-1-en-3-yn-1-yl)furan
(4): To a solution of CBr4 (2.653 g, 8.0 mmol) in CH2Cl2 (50 mL) at 0 °C, PPh3
(4.197 g, 16 mmol) was added in portions over 3 minutes. The color of solution
changed to dark brown. After adding, the mixture was stirred for 10 minutes
under argon gas protection. Acrolein 3 (0.489 g, 4 mmol) was added to the
The antiviral activity of compounds 1, 4, and 8 was evaluated
in vitro using 10 l
g of each compound and 102 focus forming units
(FFU) of PRRSV strain NVSL97-7895. DMSO was used as a control.18
The compound plus virus mixtures were incubated at 37° for one
hour, and then inoculated in triplicate onto MARC-145 cells. At
24 h post infection, cells were fixed and immunocytochemistry
performed to detect foci of PRRSV-infected cells. Virus inhibition
was calculated as the percent reduction in FFU in compound-trea-
ted wells compared to virus-only control wells. The percent virus
inhibition (PVI) for compound 1 was 100.0 0.7%, which demon-
strates that our synthetic compound 1 is effective toward PRRSV
as was reported. The PVI of compounds 4 and 8 were 93.4 4.0%
and 99.3 0.7%, respectively. The DMSO had little to no inhibitory
effect on PRRSV, with PVI of 0.78 14.3%, indicating the inhibitory
activity is due to the compounds, and not DMSO (Fig. 2).
resulting solution over
5 minutes portionwise. After another 30 minutes
stirring at 0 °C, TLC was checked to make sure the reaction was done.
Evaporation of CH2Cl2, followed by a quick flash column chromatography
(silica gel, EtOAc:hexanes 1:1) to remove most of salt gave a pale gray crude
solid product. The purification of crude product by flash column
chromatography (silica gel, EtOAc:hexanes 1:4) afforded dibromo compound
as a light yellow solid in 77% yield; 1H NMR (300 MHz, CDCl3) d = 7.47–7.40 (m,
1H), 7.02 (dd, J = 10.4, 0.5 Hz, 1H), 6.68 (dd, J = 15.5, 10.4 Hz, 1H), 6.54–6.36 (m,
3H).
To a solution of prepared dibromo compound (0.560 g, 2.02 mmol) in 15 mL
THF, n-BuLi (2.5 M in hexane, 1.62 mL, 4.03 mmol) was added dropwise over
10 min at À78 °C under argon protection. The resulting mixture stirred at
À78 °C for 1 h, the temperature was increased to rt. After 1 h stirring at rt,
saturated NH4Cl aqueous solution was added slowly to quench the reaction.
The reaction mixture was extracted with diethyl ether (3 Â 30 mL). The organic
layers were collected, dried with anhydrous MgSO4, filtered and concentrated
in vacuo. The residue was purified by flash column chromatography (pentane)
Atractylodinol (1) was successfully synthesized in seven steps
and all the characterization spectra (NMR and MS) of final product
were identical to the literature spectra. One more efficient six-step

G. A. Kraus et al. / Tetrahedron Letters 57 (2016) 5185–5187
5187
to afforded 4 as colorless oil in 86% yield; 1H NMR (300 MHz, CDCl3) d = 7.38 (d,
J = 1.8 Hz, 1H), 6.78 (d, J = 16.1 Hz, 1H), 6.45–6.31 (m, 2H), 6.03 (d, J = 18.4 Hz,
1H), 3.09 (d, J = 2.4 Hz, 1H); 13C NMR (75 MHz, CDCl3) d = 152.0, 143.4, 130.3,
112.1, 110.7, 105.3, 83.0, 80.3;
2.00 (s, 1H); 13C NMR (150 MHz, CDCl3) d = 151.9, 143.9, 131.8, 112.3, 111.7,
104.4, 81.6, 78.5, 76.3, 70.9, 51.9; HRMS (ESI-QTOF) calcd for C11H8O2 [M+H]+
173.0597, found 173.0599.
17. Experimental procedure for the synthesis of Ethyl (2E,8E)-9-(furan-2-yl)
nona-2,8-dien-4,6-diynoate (8): To a solution of 7 (50.0 mg, 0.29 mmol) in
CH2Cl2 (4 mL) was added Dess-Martin Periodinane (135 mg, 0.32 mmol) at
0 °C, the mixture was warm up to room temperature and stirred for an
additional hour. (Carbethoxymethylene)triphenylphosphorane (121.8 mg,
0.35 mmol) in CH2Cl2 (1 mL) was then added to the mixture and stirred for
another hour. Concentration and flash column chromatography (silica gel,
EtOAc:hexanes 1:10) gave compound 8 in 80% yield; 1H NMR (400 MHz, CDCl3)
d = 7.41 (s, 1H), 6.87 (m, 2H), 6.44 (m, 2H), 6.32 (d, J = 15.8 Hz, 1H), 6.14 (d,
J = 15.9 Hz, 1H), 4.23 (q, J = 7.1 Hz, 2H), 1.30 (t, J = 7.1 Hz, 3H); 13C NMR
(100 MHz, CDCl3) d = 165.7, 151.8, 144.2, 132.8, 132.3, 124.1, 112.5, 112.3,
104.2, 85.3, 82.9, 79.4, 76.5, 61.2, 14.4; HRMS (ESI-QTOF) calcd for C15H12O3 [M
+H]+ 241,0859, found 241.0856.
HRMS (ESI-QTOF) calcd for C8H6O [M+H]+ 119.0491, found 119.0493.
14. Experimental procedure for the synthesis of (E)-5-Bromopent-2-en-4-yn-1-ol
(6): Dibromo Compound was prepared using previous described procedure for
compound 4 in 96% yield; 1H NMR (300 MHz, CDCl3) d = 6.96 (d, J = 10.3 Hz,
1H), 6.42–6.26 (m, 1H), 6.14–6.00 (m, 1H), 4.00 (d, J = 7.8 Hz, 2H), 0.91 (s, 9H),
0.08 (d, J = 7.5 Hz, 3H).
To a solution of the dibromoalkene obtained above (0.383 g, 1.08 mmol) in THF
was added TBAF (1.0 M solution in THF, 0.4 mL, 0.4 mmol) at rt, then the
mixture was stirred at 45 °C for 18 h and diluted with Et2O. The mixture was
washed with saturated NH4Cl, H2O, and brine and then dried over MgSO4.
Concentration gave the mixture of the corresponding TBS- deprotected
bromoacetylene 6; 1H NMR (300 MHz, CDCl3) d = 6.38–6.23 (m, 1H), 5.72 (dt,
J = 15.9, 1.9 Hz, 1H), 4.20 (dd, J = 5.0, 1.9 Hz, 2H).
18. Antiviral Activity Compounds were diluted in DMSO and screened for anti-
PRRSV activity using a focus-reduction assay adapted from Wu et al.13 Briefly,
MARC-145 cells were seeded at 3 Â 105 cells/well in a 12-well plate 24 h prior
to the anti-viral assay, and media changed to 1 ml/well directly before
15. Atractylodinol (1): To a solution of EtNH2 (70% aqueous solution, 2.2 mL) in
MeOH (3 mL) was added CuCl (13.0 mg, 131.4 lmol) at rt that resulted in the
formation of a blue solution. To the resulting mixture was added NH2OHÁHCl
(54.8 mg, 0.788 mmol) at room temperature to discharge the blue color. The
resulting colorless solution indicated the presence of Cu(I) salt. To the resulting
mixture was added 4 (108.6 mg, 0.920 mmol) in MeOH (2 mL) at rt, and the
mixture was stirred at rt for 10 min that resulted in the formation of a yellow
suspension. To the resulting mixture was added bromoacetylene 6 (42.3 mg,
0.263 mmol) in MeOH (2 mL) at À78 °C, and mixture was stirred at the same
temperature for 30 min. The mixture was allowed to warm to rt for 3 h. The
mixture was diluted with Et2O, washed with H2O and brine, and then dried
over MgSO4. Concentration and flash column chromatography (silica gel,
EtOAc:hexanes 1:4) gave the corresponding product 1; 1H NMR (300 MHz,
CDCl3) d = 7.39 (s, 1H), 6.81 (d, J = 16.0 Hz, 1H), 6.41 (dt, J = 11.1, 4.5 Hz, 3H),
6.11 (d, J = 15.9 Hz, 1H), 5.88 (d, J = 15.9 Hz, 1H), 4.26 (d, J = 3.3 Hz, 2H); 13C
NMR (75 MHz, CDCl3) d = 152.0, 145.4, 143.9, 131.3, 112.4, 111.6, 109.3, 104.8,
81.3, 81.1, 77.1, 75.0, 63.0. HRMS (ESI-QTOF) calcd for C13H8O [M+H-H2O]+
181.0848, found 181.0646.
infection. For each compound, 35
units (FFU) of PRRSV in volume of 1.2 ml (incubation volume), for an
incubation concentration of 29.17 g/ml of the compound. In addition, a virus-
lg (35 ll) was added to 700 focus-forming
a
l
only sample with 700 FFU PRRSV in 1.2 ml media was used as a control. The
virus-compound mixtures and virus-only control were incubated at 37° for one
hour. Samples were brought to
inoculated per well in triplicate, resulting in each well containing 10
compound and 200 FFU in a well volume of 2 ml for a final well concentration
of 5 g/ml of compound. The plates were incubated at 37° supplemented with
a total volume of 3.5 ml, and 1 ml was
l
g
l
5% CO2. At 24 h post infection, cells were fixed in ice-cold methanol:acetone
and immunocytochemistry performed using the PRRSV N protein specific
monoclonal antibody SDOW17 (Rural Technology) as the primary antibody
and sheep anti-mouse IgG conjugated to HRP (Jackson ImmunoResearch) as
the secondary antibody. Following addition of the HRP substrate, cells were
rinsed with distilled water, air-dried, and foci of infected cells enumerated by
light microscopy. Percent virus inhibition was calculated compared to virus-
only control wells. Standard deviations were calculated using the means of
repeated experiments.
16. Experimental procedure for the synthesis of (E)-7-(furan-2-yl)hepta-6-en-
2,4-diyn-1-ol (7): Alcohol compound was prepared using similar procedure for
compound 1 in 37% yield; 1H NMR (600 MHz, CDCl3) d = 7.39 (s, 1H), 6.82 (d,
J = 16.0 Hz, 1H), 6.40 (d, J = 18.0 Hz, 2H), 6.07 (d, J = 16.0 Hz, 1H), 4.40 (s, 2H),