Halogenated benzoate derivatives of altholactone with improved anti-fungal activity

Article abstract of DOI:10.1080/10286020.2015.1133611

Abstract: Altholactone exhibited the anti-fungal activity with a high MIC value of 128?μg?ml^?1against Cryptococcus neoformans and Saccharomyces cerevisiae. Fifteen ester derivatives of altholactone 1–15 were modified by esterification and their

Full text of DOI:10.1080/10286020.2015.1133611

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
Halogenated benzoate derivatives of altholactone
with improved anti-fungal activity
Jirayut Euanorasetr, Mayura Junhom, Srisurang Tantimavanich, Onanong
Vorasin, Bamroong Munyoo, Patoomratana Tuchinda & Watanalai
Panbangred
To cite this article: Jirayut Euanorasetr, Mayura Junhom, Srisurang Tantimavanich, Onanong
Vorasin, Bamroong Munyoo, Patoomratana Tuchinda & Watanalai Panbangred (2016):
Halogenated benzoate derivatives of altholactone with improved anti-fungal activity, Journal of
Asian Natural Products Research, DOI: 10.1080/10286020.2015.1133611
To link to this article: http://dx.doi.org/10.1080/10286020.2015.1133611
Published online: 14 Jan 2016.
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Date: 09 April 2016, At: 19:25

Journal of asian natural Products research, 2016
http://dx.doi.org/10.1080/10286020.2015.1133611
Halogenated benzoate derivatives of altholactone with
improved anti-fungal activity
Jirayut Euanorasetra,b , Mayura Junhomb, Srisurang Tantimavanichc,
Onanong Vorasind, Bamroong Munyood, Patoomratana Tuchindad and
Watanalai Panbangreda,b
adepartment of Biotechnology, faculty of science, Mahidol university, Bangkok 10400, thailand;
bMahidol university-osaka university collaborative research center for Bioscience and Biotechnology, faculty
c
of science, Mahidol university, Bangkok 10400, thailand; department of clinical Microbiology, faculty of
d
Medical technology Mahidol university, Bangkok 10400, thailand; department of chemistry, faculty of
science, Mahidol university, Bangkok 10400, thailand
ABSTRACT
ARTICLE HISTORY
received 29 august 2015
accepted 14 december 2015
Altholactone exhibited the anti-fungal activity with a high MIC value
of 128 μg ml−1 against Cryptococcus neoformans and Saccharomyces
cerevisiae. Fifteen ester derivatives of altholactone 1–15 were
modified by esterification and their structures were confirmed by
spectroscopic methods. Most of the ester derivatives exhibited
stronger anti-fungal activities than that of the precursor altholactone.
3-Bromo- and 2,4-dichlorobenzoates (7 and 15) exhibited the lowest
minimal inhibitory concentration (MIC) values against C. neoformans
KEYWORDS
altholactone; anti-fungal
activity; ester derivative;
halogenated benzoate;
Polyalthia crassa
at 16 μg ml−1 while the 4-bromo-, 4-iodo-, and 1-bromo-3-
,
chlorobenzoates (11–13) displayed potent activity against S. cerevisiae
with MIC values of 1 μg ml−1. In conclusion, this analysis indicates that
the anti-fungal activity of altholactone is enhanced by addition of
halogenated benzoyl group to the 3-OH group.
1. Introduction
Currently, the impact of fungal infections on human health is under-appreciated, even
though these infections have a high mortality rate (>50%) resulting in the death of over
1.5 million people annually [1]. Fungi in the genera of Aspergillus, Candida, Cryptococcus,
and Pneumocystis are responsible for nearly 90% of all deaths due to fungal infections [1].
Candida albicans and Cryptococcus neoformans are opportunistic pathogenic yeasts, causing
candidiasis and cryptococcal meningitis in patients that are immunocompromised as a
result of cancer or AIDS [2,3]. Approximately, 46,000 cases of health care-associated invasive
candidiasis are recorded each year in the US [4]. In addition, more than 1 million cases of
cryptococcal meningitis among people with AIDS were reported worldwide in 2006, result-
ing in 625,000 deaths, predominantly in sub-Saharan Africa [5]. Saccharomyces cerevisiae is
CONTACT Watanalai Panbangred
mahidol.ac.th
watanalai.pan@mahidol.ac.th; Patoomratana tuchinda
patoomratana.tuc@
supplemental data for this article can be accessed http://dx.doi.org/10.1080/10286020.2015.1133611.
© 2016 taylor & francis

2
J. EuAnOrASETr ET Al.
Table 1. Mic values of altholactone and ester derivatives (1–15) against three indicator yeasts.
MIC (μg ml−1
)
Compound
C. albicans MT 2014/1
C. neoformans MT 2014/2
S. cerevisiae IFO 10217
Altholactone
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
0.25
128
>128
>128
128
>128
32
32
16
64
32
32
32
>128
32
32
16
0.25
128
32
16
8
16
8
8
8
4
2
4
1
1
1
2
2
0.25
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
amphotericin B
a well-known yeast used in baking and brewing industry and not typically associated with
fungal infections. However, S. cerevisiae-induced vaginitis has been recently identified in
bone marrow transplant patients and other immunocompromised patients [6].
A possible anti-fungal agent is altholactone (goniothalenol), a well-known styryl-lactone
bioactive natural product found in Goniothalamus and Polyalthia (Annonaceae) species.
is furano-pyrone was initially isolated from the bark of an unknown species of Polyalthia
[7] and later from the bark of G. giganteus (Panan chaang) [8]. It was also identified as a
cytotoxic compound present in leaves and twigs of Polyalthia crassa Parker by our research
group [9]. Several studies have characterized the anti-proliferative activity of altholactone
against various human tumor cell lines [10,11]. e cytotoxic mechanism of this styryl-
lactone against mammalian cancer cells has been postulated to be mediated through apop-
tosis, following inhibition of the mitochondrial respiratory chain [12,13]; however, the
precise mechanism remains unclear. In addition to its anti-proliferative effects, altholactone
displays anti-inflammatory [14] and anti-plasmodial [15] activities.
Structure modification of altholactone has the potential to enhance its bioactive prop-
erties as was observed with 3-acetylaltholactone. Inhibition of NADH: oxidase activity by
3-acetylaltholactone, from beef-heart submitochondrial particles, was greater compared to
that of altholactone [12]. Preparation of new altholactone derivatives is possible utilizing
Steglich esterification [16]. erefore, a procedure for the incorporation of various alkyl and
benzoyl groups at the 3-hydroxyl group of altholactone through esterification was designed.
e resulting altholactone ester derivatives were analyzed and many of these compounds
displayed enhanced anti-fungal activity toward C. neoformans and S. cerevisiae.
2. Results and discussion
Altholactone exhibits a high MIC value of 128 μg ml−1 or more toward C. albicans, C. neofor-
mans, and S. cerevisiae as shown in Table 1. e anti-fungal activity of altholactone and its
modified derivatives against these indicator yeasts has not been reported before. erefore,
modification of the hydroxyl group of altholactone to the 15 derivatives by Steglich esterifi-
cation was designed. Under mild conditions, altholactone was reacted with carboxylic acid

JOurnAl OF ASIAn nATurAl PrOduCTS rESEArCH
3
Figure 1. synthesis of ester derivatives of altholactone. reagents and conditions: (a) rcooh, dcc,
dMaP(cat.), ch2cl2, rt; (b) acetic anhydride, dMaP (cat.), rt; (c) rcocl, pyridine, rt.
derivatives in the presence of N,N′-dicyclohexylcarbodiimide (DCC) and 4-dimethylamino-
pyridine (DMAP) as catalyst (cat) in dry dichloromethane to give the desired products (2–9
and 11–15) in 52–94% yield (Figure 1). Compound 1 was prepared through acetylation of
altholactone with acetic anhydride in the presence of DMAP, while esterification of altho-
lactone with 4-chlorobenzoyl chloride in the presence of pyridine gave 10. e structure
of each product was characterized using spectroscopic methods (FTIR, UV, 1H NMR, 13
C
NMR, EI-MS, and HR-TOF-MS) (see Experimental). e 1H NMR and 13C NMR spectra
of 1–15 were provided as shown in Figures S1–S30 of the supporting information.
All modified compounds and the reference drug, amphotericin B, were tested for anti-
fungal activity. e results are shown in Table 1. e majority of the ester derivatives dis-
played enhanced anti-fungal activity compared to the parent compound. When the R was
alkyl group [methyl (Me) or ethyl (Et) in 1 and 2) or dimethoxybenzoyl group (3 and 4),
the MIC values against S. cerevisiae decreased to 8–32 μg ml−1. Most of the halogenated
benzoates 5–15 exhibited MIC values against both C. neoformans (16–32 μg ml−1) and S.

4
J. EuAnOrASETr ET Al.
cerevisiae (1–8 μg ml−1), which were dramatically lower than that of altholactone. An excep-
tion to this was observed with the iodobenzoyl derivative. e 3-iodo benzoate derivative 8
was less active against C. neoformans (≥64 μg ml−1) compared to S. cerevisiae (≥4 μg ml−1),
while the 4-iodobenzoate derivative only displayed activity against S. cerevisiae (≥1 μg ml−1).
In contrast, dihalogenated benzoates (13–15) significantly decreased MIC values to 16–32
and 1–2 μg ml−1 against C. neoformans and S. cerevisiae, respectively. Among these new 15
derivatives, 3-bromo- and 3,5-dichlorobenzoates (7 and 15) were the most potent against C.
neoformans (MIC = 16 μg ml−1) and displayed an eightfold lower MIC value compared to
the parent altholactone. Many of the derivatives were very toxic toward S. cerevisiae, among
these 4-bromo-, 4-iodo-, and 4-bromo-2-chlorobenzoates (11–13, respectively) were 128-fold
more potent (MIC = 1 μg ml−1). ese findings demonstrated that the anti-fungal activities
of altholactone can be significantly improved by modification of the 3-OH group to the halo-
genated benzoate derivatives. To our knowledge, there are no previous reports on the detailed
structure–activity relationship of altholactone derivatives. e current study demonstrated
that several of the new ester derivatives of altholactone possessed enhanced anti-fungal activ-
ity compared to the parent compound. Improved anti-fungal activity against yeasts and molds
has been documented for synthesized halogenated or ester derivatives of other compounds
[17,18]. However, the reason for higher anti-fungal activity of halogenated derivatives is not
well understood but could be caused by better surfactant properties or through other anti-
fungal mechanism(s) [18,19]. Further study on mechanism and cytotoxicity are necessarily
to understand the potential clinical utility of these compounds. In our analysis, altholactone
and the derivatives at a concentration of 128 μg ml−1 could not inhibit growth of C. albicans
MT2014/1. is result is inconsistent with one previous report that demonstrated anti-fungal
activity of altholactone against C. albicans ATCC 10231, with a MIC value of 2.5 μg ml−1 [20].
However, reduced susceptibility of C. albicans clinical isolates, similar to the strain used in this
study, to azole drugs has been reported [21]. C. albicans strain MT2014/1 may also exhibit
enhanced resistance to antibiotic(s) similar to other clinical isolates, although this strain did
not display resistance to amphotericin B (Table 1). Drug resistance in yeast is due to mutations
in genes encoding drug targets and enzymes in metabolic pathways as well as activation of
stress response pathways including drug-efflux pump proteins [22,23]. It is possible that the
failure of altholactone to inhibit growth of the C. albicans clinical isolate used in our analysis
could be due to genetic changes from prior drug exposure.
In conclusion, this study is the first report on the anti-fungal activity of altholactone
against C. neoformans and S. cerevisiae. Ester derivatives of altholactone with modification
at 3-hydroxyl group were prepared and tested against two pathogenic fungi and one strain
of Baker’s yeast. e results demonstrate that most altholactone derivatives, especially the
halogenated benzoates, possess significantly increased anti-fungal activity, with lower MIC
values than that of altholactone.
3. Experimental
3.1. General experimental procedures
Melting points were measured using a digital electrothermal melting point apparatus
(BUCHI, Flawil, Switzerland). Optical rotations were determined on JASCO DIP 370 digital
polarimeter (JASCO, Tokyo, Japan), using a 50-mm microcell (1 ml). UV (in acetonitrile)

JOurnAl OF ASIAn nATurAl PrOduCTS rESEArCH
5
and IR (KBr) spectra were recorded on a JASCO 530 (JASCO, Tokyo, Japan) and a Perkin-
Elmer 2000 FT-IR spectrometers (Perkin-Elmer, Massachusetts, USA), respectively. e
1H (400 MHz) and 13C (100 MHz) NMR spectra were recorded on Bruker Ascend 400
spectrometer (Bruker, Fallenden, Germany) in CDCl3, using TMS as internal standard.
EIMS were recorded at 70 eV (probe) on a ermo Finnigan Polaris Q mass spectrometer
(ermo scientific, Texas, USA). e HR-TOF-MS were recorded on a Micromass model
VQ-ToF-2 spectrometer (Bruker, Bremen, Germany). Silica gel 60H (Merck, 70–230 mesh
ASTM, Darmstadtt, Germany) was used for column chromatography, while preparative TLC
was carried out with silica gel 60 PF254 (Merck, Darmstadtt, Germany) plates. Altholactone
was isolated from leaves and twigs of P. crassa Parker using the previously described proce-
dure (Tuchinda et al. [9]). All solvents used for extraction and isolation were distilled prior
to use at their boiling point ranges.
3.2. General method for preparation of compound 1
A mixture of altholactone (106 mg, 0.457 mmol), acetic anhydride (0.99 ml, 10.578 mmol),
and 4-dimethylaminopyridine (cat.) was stirred at room temperature for 4 h. e reaction
was quenched with a saturated sodium hydrogen carbonate solution (25 ml). e aqueous
layer was extracted with CH2Cl2 (3×25 ml). e combined organic layer was washed with
H2O, brine and dried over anhydrous magnesium sulfate. Afer filtration and removal of
solvent under reduced pressure, a crude yellow liquid (1.81 g) was obtained. is material
was purified by preparative TLC (50% ethyl acetate-hexanes), followed by recrystallization
from EtOH to give the desired product 1 (75.2 mg, 60% yield) as colorless needles.
3.2.1. (2R,3R,3aR,7aS)-5-Oxo-2-phenyl-3,3a,5,7a-tetrahydro-2H-furo[3,2-b]pyran-3-
yl ethanoate (1)
mp 140.8–141.5 C.[ꢀ]2D8 +126.4 (c 0.088, CHCl ); UV (CH CN) λmax nm (log ε): 202 (5.11).
IR (KBr) υmax cm−1: 1745, 1722, 1632, 1495, 31457, 13783, 1363, 1253, 1223, 1147, 1100,
1056, 1022, 918, 884, 764, 702. 1H NMR (400 MHz, CDCl3) δ: 7.30–7.36 (5H, m, Ar–H),
7.04 (1H, dd, J = 9.8, 5.1 Hz), 6.28 (1H, d, J = 9.8 Hz), 5.39 (1H, brd, J = 3.3 Hz), 4.98 (1H,
d, J = 3.3 Hz), 4.96 (1H, brd, J = 3.6 Hz), 4.63 (1H, dd, J = 5.1, 4.2 Hz), 2.16 (CH3, s). 13
C
NMR (100 MHz, CDCl3): 169.4 (C), 160.4 (C), 139.2 (CH), 137.5 (C), 128.7 (2 × CH), 128.5
(CH), 126.2 (2 × CH), 124.7 (CH), 86.1 (CH), 83.6 (CH), 83.6 (CH), 69.1 (CH), 20.9 (CH3).
EIMS m/z (relative intensity): 275 [M + H]+ (19), 214 (3), 197 (4), 170 (17), 146 (10), 145
(100), 115 (9), 106 (14), 97 (5), 78 (5). HR-TOF-MS (ESI): m/z 297.0748 [M + Na]+ (calcd
for C15H14O5Na, 297.0739)
3.3. General method to prepare compounds 2–15 (except 10)
A mixture of altholactone (1.0 eq), acid derivatives (1.2 eq), N,N′-dicyclohexylcarbodiimide
(1.2 eq), and 4-dimethylaminopyridine (cat.) in dry dichloromethane (2 ml) was stirred at
room temperature for 2 h to overnight. e reaction mixture was monitored by TLC (ethyl
acetate-hexane). Removal of the solvent was performed under reduced pressure and the
residue was further purified by column chromatography on preparative TLC (silica gel) to
give the ester derivatives.

6
J. EuAnOrASETr ET Al.
3.3.1. (2R,3R,3aR,7aS)-5-Oxo-2-phenyl-3,3a,5,7a-tetrahydro-2H-furo[3,2-b]pyran-3-
yl propionate (2)
Compound 2 was prepared by reacting altholactone (50.0 mg, 0.216 mmol) with propanoic
acid (19.2 mg, 0.259 mmol) in the presence of N,N′-dicyclohexylcarbodiimide (53.4 mg,
0.259 mmol) and 4-dimethylaminopyridine (cat.) at room temperature overnight. e reac-
tion mixture was purified by column chromatography (20% ethyl acetate-hexane), followed
by recrystallization from MeOH to give 2 (52.1 mg, 84% yield) as colorless needles; mp
182.4–183.2 °C.[ꢀ]2D8 +70.6 (c 0.088, CHCl ). UV ꢀACN nm (log ε): 206 (4.28). IR ꢀ cm−1:
KBr
max
1744, 1724, 1632, 1495, 1459, 1382, 1368, 31355, 12m4a9x, 1177, 1146, 1100, 1085, 1070, 1054,
1025, 884, 821, 764, 702, 666. 1H NMR (400 MHz, CDCl3) δ: 7.28–7.37 (5H, m, Ar–H), 7.04
(1H, dd, J = 9.8, 5.3 Hz), 6.28 (1H, d, J = 9.8 Hz), 5.40 (1H, brd, J = 3.1 Hz), 4.98 (1H, d,
J = 3.1 Hz), 4.95 (1H, brd, J = 3.3 Hz), 4.64 (1H, dd, J = 5.3, 4.1 Hz), 2.43 (CH2, q, J = 7.5 Hz),
1.18 (CH3, t, J = 7.5 Hz). 13C NMR (100 MHz, CDCl3): δ 172.9 (C), 160.4 (C), 139.2 (CH),
137.5 (C), 128.7 (2 × CH), 128.4 (CH), 126.2 (2 × CH), 124.7 (CH), 86.2 (CH), 83.6 (CH),
83.5 (CH), 69.1 (CH), 27.4 (CH2), 8.9 (CH3). EIMS m/z (relative intensity): 289 [M + H]+
(3), 214 (5), 197 (<1), 170 (20), 146 (5), 145 (100), 116 (7), 106 (6), 78 (10). HR-TOF-MS
(ESI): m/z 311.0893 [M + Na]+ (calcd for C16H16O5Na, 311.0895)
3.3.2. (2R,3R,3aR,7aS)-5-Oxo-2-phenyl-3,3a,5,7a-tetrahydro-2H-furo[3,2-b]pyran-3-
yl 3,5-dimethoxybenzoate (3)
Compound 3 was prepared by reacting altholactone (30.6 mg, 0.132 mmol) with 3,5-dimeth-
oxybenzoic acid (28.8 mg, 0.158 mmol) in the presence of N,N′-dicyclohexylcarbodiimide
(32.6 mg, 0.158 mmol) and 4-dimethylaminopyridine (cat.) at room temperature for 3 h.
e reaction mixture was purified by column chromatography (20% ethyl acetate-hexane)
to yield 3 (44.2 mg, 85% yield) as a colorless oil;[ꢀ]2D8 +20.3 (c 0.127, CHCl ). UV ꢀ
nm
ACN
max
3
KBr
(log ε): 308 (3.47), 253 (3.86), 207 (4.67). IR ꢀ cm−1: 1736, 1596, 1496, 1459, 1429, 1353,
max
1325, 1306, 1224, 1206, 1158, 1105, 1064, 1049, 924, 850, 820, 763, 699. 1H NMR (400 MHz,
CDCl3) δ: 7.31–7.42 (5H, m, Ar–H), 7.19 (2H, d, J = 2.2 Hz), 7.08 (1H, dd, J = 9.8, 5.3 Hz),
6.69 (1H, t, J = 2.2 Hz), 6.31 (1H, d, J = 9.8 Hz), 5.61 (1H, brd, J = 3.2 Hz), 5.14 (1H, d,
J = 3.2 Hz), 5.10 (1H, brd, J = 3.3 Hz), 4.73 (1H, dd, J = 5.3, 4.1 Hz), 3.84 (2xOCH3, s). 13
C
NMR (100 MHz, CDCl3): δ 164.9 (C), 160.8 (2 × C), 160.4 (C), 139.1 (CH), 137.5 (C),
130.7 (C), 128.7 (2 × CH), 128.5 (CH), 126.2 (2 × CH), 124.8 (CH), 107.6 (2 × CH), 106.0
(CH), 86.2 (CH), 84.3 (CH), 83.7 (CH), 69.3 (CH), 55.7 (2 × OCH3). EIMS m/z (relative
intensity): 397 [M + H]+ (1), 214 (2), 184 (11), 182 (100), 166 (28), 138 (14), 122 (8), 108
(5), 78 (9). HR-TOF-MS (ESI): m/z 419.1116 [M + Na]+ (calcd for C22H20O7Na, 419.1107)
3.3.3. (2R, 3R, 3aR, 7aS)-5-Oxo-2-phenyl-3,3a,5,7a-tetrahydro-2H-furo[3,2-b]pyran-
3-yl 3,4-dimethoxybenzoate (4)
Compound 4 was prepared by reacting altholactone (30.6 mg, 0.132 mmol) with 3,4-dimeth-
oxybenzoic acid (28.8 mg, 0.158 mmol) in the presence of N,N′-dicyclohexylcarbodiimide
(32.6 mg, 0.158 mmol) and 4-dimethylaminopyridine (cat.) at room temperature for 6 h.
e reaction mixture was purified by column chromatography (35% ethyl acetate-hexane)
to yield 4 (49.3 mg, 94% yield) as a yellow oil; [ꢀ]2D8 +75.1 (c 0.006, CHCl ). UV ꢀ
nm
ACN
max
3
KBr
(log ε): 293 (3.62), 264 (3.88), 219 (4.19). IR ꢀ cm−1: 1735, 1719, 1600, 1516, 1465, 1459,
max
1453, 1420, 1349, 1295, 1273, 1246, 1220, 1176, 1104, 1022, 876, 821, 760, 700. 1H NMR

JOurnAl OF ASIAn nATurAl PrOduCTS rESEArCH
7
(400 MHz, CDCl3) δ: 7.71 (1H, dd, J = 8.5, 1.9 Hz), 7.55 (1H, d, J = 1.9 Hz), 7.31–7.43 (5H,
m, Ar–H), 7.08 (1H, dd, J = 9.8, 5.4 Hz), 6.92 (1H, d, J = 8.5 Hz), 6.31 (1H, d, J = 9.8 Hz),
5.61 (1H, brd, J = 3.4 Hz), 5.16 (1H, d, J = 3.4 Hz), 5.12 (1H, dd, J = 4.1, 0.8 Hz), 4.75 (1H,
dd, J = 5.4, 4.1 Hz), 3.96 (OCH3, s), 3.94 (OCH3, s). 13C NMR (100 MHz, CDCl3): δ 164.9
(C), 160.5 (C), 153.7 (C), 148.9 (C), 139.2 (CH), 137.6 (C), 128.7 (2 × CH), 128.5 (CH), 126.2
(2 × CH), 124.8 (CH), 123.9 (CH), 121.3 (C), 112.2 (CH), 110.3 (CH), 86.2 (CH), 84.0 (CH),
83.7 (CH), 69.3 (CH), 56.1 (2 × OCH3). EIMS m/z (relative intensity): m/z 397 [M + H]+
(<1), 214 (1), 184 (10), 183 (100), 165 (22), 145 (6), 122 (4), 96 (4), 78 (13). HR-TOF-MS
(ESI): m/z 419.1106 [M + Na]+ (calcd for C22H20O7Na, 419.1107)
3.3.4. (2R,3R,3aR,7aS)-5-Oxo-2-phenyl-3,3a,5,7a-tetrahydro-2H-furo[3,2-b]pyran-3-
yl 3-fluorobenzoate (5)
Compound 5 was prepared by reacting altholactone (32.6 mg, 0.141 mmol) with 3-fluoroben-
zoic acid (26.3 mg, 0.169 mmol) in the presence of N,N′-dicyclohexylcarbodiimide (34.8 mg,
0.169 mmol) and 4-dimethylaminopyridine (cat.) at room temperature overnight. e reac-
tion mixture was purified by column chromatography (25% ethyl acetate-hexane) to yield
5 (29.4 mg, 59% yield) as a yellow oil; [ꢀ]2D8 + 109.2 (c 0.012, CHCl ). UV ꢀ
nm (log ε):
ACN
max
3
KBr
279 (2.94), 227 (3.82). IR ꢀ cm−1: 1736, 1638, 1593, 1450, 1291, 1271, 1245, 1200, 1105,
1068, 821, 754, 699. H NMR (400 MHz, CDCl3) δ: 7.89 (1H, brd, J = 7.8 Hz), 7.76 (1H,
max
1
brd, J = 8.5 Hz), 7.49 (1H, m), 7.32–7.44 (6H, m), 7.10 (1H, dd, J = 9.8, 5.3 Hz), 6.25 (1H,
d, J = 9.8 Hz), 5.65 (1H, brd, J = 3.3 Hz), 5.16 (1H, d, J = 3.3 Hz), 5.14 (1H, brd, J = 4.1 Hz),
4.67 (1H, dd, J = 5.3, 4.1 Hz). 13C NMR (100 MHz, CDCl3): δ 160.3 (C), 139.1 (CH), 137.3
(C), 130.4 (C), 130.3 (C), 128.8 (2 × CH), 128.6 (CH), 126.2 (2 × CH), 125.6 (C), 124.8
(CH), 121.0 (CH), 120.8 (CH), 116.8 (CH), 116.6 (CH), 86.1 (CH), 84.4 (CH), 83.6 (CH),
69.3 (CH). EIMS m/z (relative intensity): 355 [M]+ (22), 214 (6), 197 (11), 170 (24), 145 (43),
144 (100), 123 (33), 105 (28), 95 (16), 77 (11). HR-TOF-MS (ESI): m/z 377.0804 [M + Na]+
(calcd for C20H15FO5Na, 377.0801)
3.3.5. (2R,3R,3aR,7aS)-5-Oxo-2-phenyl-3,3a,5,7a-tetrahydro-2H-furo[3,2-b]pyran-3-
yl 3-chlorobenzoate (6)
Compound 6was prepared by reacting altholactone (29.7 mg, 0.128 mmol) with 3-chloroben-
zoic acid (24.0 mg, 0.154 mmol) in the presence of N,N′-dicyclohexylcarbodiimide (31.7 mg,
0.154 mmol) and 4-dimethylaminopyridine (cat.) at room temperature for 3 h. e reaction
mixture was purified by column chromatography (15% ethyl acetate-hexane) to yield 6
(35.9 mg, 76% yield) as a yellow oil; [ꢀ]2D8 +95.5 (c 0.031, CHCl ). UV ꢀ
nm (log ε): 283
ACN
max
3
KBr
(2.95), 231 (3.84). IR ꢀ cm−1: 1735, 1638, 1575, 1496, 1452, 1428, 1384, 1293, 1243, 1129,
max
1104, 1070, 1025, 909, 881, 820, 747, 699. 1H NMR (400 MHz, CDCl3), δ: 7.94 (1H, brs), 7.87
(1H, brd, J = 7.8 Hz), 7.51 (1H, dd, J = 8.0, 1.0 Hz), 7.21–7.37 (6H, m, Ar–H), 6.99 (1H, dd,
J = 9.8, 5.3 Hz), 6.23 (1H, d, J = 9.8 Hz), 5.54 (1H, brd, J = 3.3 Hz), 5.06 (1H, d, J = 3.3 Hz),
5.02 (1H, dd, J = 4.2, 0.8 Hz), 4.65 (1H, dd, J = 5.3, 4.2 Hz). 13C NMR (100 MHz, CDCl3):
δ 163.9 (C), 160.4 (C), 139.1 (CH), 137.4 (C), 134.8 (C) 133.8 (CH), 130.7 (C), 130.0 (CH),
129.8 (CH), 128.7 (2 × CH), 128.6 (CH), 128.0 (CH), 126.2 (2 × CH), 124.8 (CH), 86.0
(CH), 84.4 (CH), 83.6 (CH), 69.3 (CH). EIMS m/z (relative intensity): 371 [M + H]+ (<1),
214 (5), 170 (19), 145 (100), 139 (15), 116 (10), 105 (10), 76 (16). HR-TOF-MS (ESI): m/z
393.0502 [M + Na]+ (calcd for C20H15ClO5Na, 393.0506)

8
J. EuAnOrASETr ET Al.
3.3.6. (2R,3R,3aR,7aS)-5-Oxo-2-phenyl-3,3a,5,7a-tetrahydro-2H-furo[3,2-b]pyran-3-
yl 3-bromobenzoate (7)
Compound 7 was prepared by reacting altholactone (31.3 mg, 0.135 mmol) with 3-bro-
mobenzoic acid (32.5 mg, 0.162 mmol) in the presence of N,N′-dicyclohexylcarbodiimide
(33.4 mg, 0.162 mmol) and 4-dimethylaminopyridine (cat.) at room temperature overnight.
e reaction mixture was purified by column chromatography (10% ethyl acetate-hexane)
to yield 7 (37.2 mg, 66% yield) as a yellow oil;[ꢀ]2D8 +108.4 (c 0.0096, CHCl ). UV ꢀ
nm
ACN
max
3
KBr
(log ε): 280 (3.70), 228 (4.59), 206 (5.13). IR ꢀ cm−1: 1735, 1719, 1638, 1571, 1560, 1459,
max
1291, 1243, 1123, 1103, 1067, 820, 746, 700. 1H NMR (400 MHz, CDCl3), δ: 8.18 (1H, dd,
J = 1.9, 1.5 Hz), 8.00 (1H, ddd, J = 8.1, 1.5, 1.2 Hz), 7.75 (1H, ddd, J = 8.0, 1.9, 1.2 Hz),
7.29–7.42 (6H, m), 7.08 (1H, dd, J = 9.8, 5.4 Hz), 6.32 (1H, d, J = 9.8 Hz), 5.63 (1H, dd,
J = 3.6, 1.1 Hz), 5.14 (1H, d, J = 3.6 Hz), 5.11 (1H, dd, J = 4.1, 1.1 Hz), 4.74 (1H, dd, J = 5.4,
4.1 Hz). 13C NMR (100 MHz, CDCl3): δ 163.8 (C), 160.3 (C), 139.1 (CH), 137.3 (C), 136.7
(CH), 132.7 (CH), 130.8 (C), 130.3 (CH), 128.8 (2 × CH), 128.6 (CH), 128.5 (CH), 126.2
(2 × CH), 124.8 (CH), 122.7 (C), 86.1 (CH), 84.4 (CH), 83.6 (CH), 69.3 (CH). EIMS m/z
(relative intensity): 414 [M]+ (1), 214 (2), 186 (3), 185 (43), 183 (44), 170 (7), 146 (13), 144
(100), 143 (16), 115 (15), 76 (10). HR-TOF-MS (ESI): m/z 437.0000/438.9978 [M + Na]+
(79Br/81Br) (calcd for C20H15BrO5Na, 437.0001/438.9980)
3.3.7. (2R,3R,3aR,7aS)-5-Oxo-2-phenyl-3,3a,5,7a-tetrahydro-2H-furo[3,2-b]pyran-3-
yl 3-iodobenzoate (8)
Compound 8 was prepared by reacting altholactone (31.6 mg, 0.136 mmol) with 3-iodo-
benzoic acid (40.43 mg, 0.163 mmol) in the presence of N,N′-dicyclohexylcarbodiimide
(33.6 mg, 0.163 mmol) and 4-dimethylaminopyridine (cat.) at room temperature for 2.5 h.
e reaction mixture was purified by column chromatography (20% ethyl acetate-hexane)
to yield 8 (33.6 mg, 53% yield) as a yellow oil;[ꢀ]2D8 +90.4 (c 0.023, CHCl ). UV
ꢀ
nm (log
ACN
max
3
ε): 287 (2.66), 218 (4.08). IR ꢀKBr cm−1: 1734, 1565, 1561, 1419, 1289, 1242, 1102, 819, 743,
699. 1H NMR (400 MHz, CDmCalx3), δ: 8.37 (1H, brt, J = 1.6 Hz), 8.03 (1H, brd, J = 7.8 Hz),
7.95 (1H, brd, J = 8.1 Hz), 7.29–7.42 (5H, m, Ar–H), 7.24 (1H, t, J = 7.9 Hz), 7.08 (1H, dd,
J = 9.8, 5.4 Hz), 6.32 (1H, d, J = 9.8 Hz), 5.62 (1H, dd, J = 3.5, 1.0 Hz), 5.14 (1H, d, J = 3.5 Hz),
5.11 (1H, dd, J = 4.1, 1.0 Hz), 4.74 (1H, dd, J = 5.4, 4.1 Hz). 13C NMR (100 MHz, CDCl3): δ
163.6 (C), 160.4 (C), 142.6 (CH), 139.1 (CH), 138.5 (CH), 137.3 (C), 130.8 (C), 130.3 (CH),
129.1 (CH), 128.7 (2 × CH), 128.6 (CH), 126.2 (2 × CH), 124.8 (CH), 94.0 (C), 86.1 (CH),
84.4 (CH), 83.6 (CH), 69.3 (CH). EIMS m/z (relative intensity): 463 [M]+ (<1), 232 (7),
231 (82), 214 (10), 203 (16), 171 (46), 145 (40), 144 (100), 105 (22), 77 (16). HR-TOF-MS
(ESI): m/z 484.9862 [M + Na]+ (calcd for C20H15IO5Na, 484.9862)
3.3.8. (2R,3R,3aR,7aS)-5-Oxo-2-phenyl-3,3a,5,7a-tetrahydro-2H-furo[3,2-b]pyran-3-
yl 4-fluorobenzoate (9)
Compound 9 was prepared by reacting altholactone (29.8 mg, 0.128 mmol) with 4-fluoroben-
zoic acid (21.6 mg, 0.154 mmol) in the presence of N,N′-dicyclohexylcarbodiimide (31.8 mg,
0.154 mmol) and 4-dimethylaminopyridine (cat.) at room temperature for 3 h. e reaction
mixture was purified by column chromatography (20% ethyl acetate-hexane), followed by
recrystallization from MeOH to yield 9 (31.5 mg, 70% yield) as white needles; mp 112.9–
113.8 °C.[ꢀ]2D8 +189.9 (c 0.015, CHCl ). UV ꢀ
nm (log ε): 230 (4.66). IR ꢀ cm−1: 1729,
ACN
KBr
max
max
3
1602, 1508, 1453, 1412, 1375, 1366, 1351, 1267, 1248, 1153, 1112, 1092, 1067, 858, 819, 764,

JOurnAl OF ASIAn nATurAl PrOduCTS rESEArCH
9
701, 607. 1H NMR (400 MHz, CDCl3), δ: 8.10 (1H, d, J = 8.7 Hz), 8.08 (1H, d, J = 8.6 Hz),
7.29–7.42 (5H, m, Ar–H), 7.17 (1H, d, J = 8.6 Hz), 7.15 (1H, d, J = 8.7 Hz), 7.08 (1H, dd,
J = 9.8, 5.3 Hz), 6.32 (1H, d, J = 9.8 Hz), 5.62 (1H, brd, J = 3.3 Hz), 5.15 (1H, d, J = 3.3 Hz),
5.11 (1H, brd, J = 4.0 Hz), 4.74 (1H, dd, J = 5.3, 4.0 Hz). 13C NMR (100 MHz, CDCl3): δ
167.5 (C), 164.9 (C), 164.1 (C), 160.4 (C), 139.1 (CH), 137.4 (C), 132.5 (CH), 132.4 (CH),
128.7 (2 × CH), 128.5 (CH), 126.2 (2 × CH), 124.8 (CH), 116.0 (CH), 115.8 (CH), 86.1
(CH), 84.2 (CH), 83.6 (CH), 69.3 (CH). EIMS m/z (relative intensity): 355 [M]+ (<1), 214
(5), 170 (22), 145 (15), 144 (100), 123 (28), 105 (10), 96 (24), 75 (14). HR-TOF-MS (ESI):
m/z 377.0807 [M + Na]+ (calcd for C20H15FO5Na, 377.0801)
3.3.9. (2R,3R,3aR,7aS)-5-Oxo-2-phenyl-3,3a,5,7a-tetrahydro-2H-furo[3,2-b]pyran-3-
yl 4-bromobenzoate (11)
Compound 11 was prepared by reacting altholactone (22.3 mg, 0.096 mmol) with 4-bro-
mobenzoic acid (23.2 mg, 0.115 mmol) in the presence of N,N′-dicyclohexylcarbodiimide
(23.8 mg, 0.115 mmol) and 4-dimethylaminopyridine (cat.) at room temperature for 3 h.
e reaction mixture was purified by column chromatography (20% ethyl acetate-hexane)
to yield 11 (31.6 mg, 80% yield) as a colorless oil;[ꢀ]2D8 +123.1 (c 0.011, CHCl ). UV
ꢀ
nm
ACN
max
3
(log ε): 246 (4.23), 205 (4.32). IR ꢀKBr cm−1: 1726, 1591, 1483, 1457, 1398, 1376, 1319, 1272,
1252, 1115, 1101, 1062, 1029, 845,m8ax17, 768, 752, 704, 681. 1H NMR (400 MHz, CDCl3), δ:
7.92 (2H, d, J = 8.6 Hz), 7.63 (2H, d, J = 8.6 Hz), 7.30–7.42 (5H, m, Ar–H), 7.07 (1H, dd,
J = 9.8, 5.4 Hz), 6.31 (1H, d, J = 9.8 Hz), 5.62 (1H, dd, J = 3.6, 1.0 Hz), 5.14 (1H, d, J = 3.6 Hz),
5.11 (1H, dd, J = 4.2, 1.0 Hz), 4.73 (1H, dd, J = 5.4, 4.2 Hz). 13C NMR (100 MHz, CDCl3):
δ 164.4 (C), 160.3 (C), 139.1 (CH), 137.3 (C), 132.0 (2 × CH), 131.3 (2 × CH), 129.1 (C),
128.7 (2 × CH), 128.6 (CH), 127.8 (C), 126.2 (2 × CH), 124.8 (CH), 86.0 (CH), 84.3 (CH),
83.6 (CH), 69.3 (CH). EIMS m/z (relative intensity): 415 [M]+ (<1), 214 (6), 186 (6), 185
(54), 184 (52), 171 (33), 145 (100), 144 (51), 105 (16), 97 (2), 78 (13). HR-TOF-MS (ESI):
m/z 437.0007/448.9983 [M + Na]+ (79Br/81Br) (calcd for C20H15BrO5Na, 437.0001/438.9980)
3.3.10. (2R,3R,3aR,7aS)-5-Oxo-2-phenyl-3,3a,5,7a-tetrahydro-2H-furo[3,2-b]pyran-
3-yl 4-iodobenzoate (12)
Compound 12 was prepared from altholactone (32.6 mg, 0.141 mmol) reacted with 4-iodo-
benzoic acid (41.8 mg, 0.169 mmol) in the presence of N,N′-dicyclohexylcarbodiimide
(34.7 mg, 0.169 mmol) and 4-dimethylaminopyridine (cat.) at room temperature for 3 h.
e reaction mixture was purified by column chromatography (20% ethyl acetate-hexane),
followed by recrystallization from MeOH to yield 12 (38.4 mg, 59% yield) as colorless
needles; mp 186.4–187.2 °C. [ꢀ]2D8 +36.6 (c 0.093, CHCl ). UV ꢀ
nm (log ε): 257 (4.56),
ACN
max
3
KBr
209 (4.58). IR ꢀ cm−1: 1724, 1587, 1481, 1457, 1395, 1376, 1272, 1253, 1115, 1101, 1063,
1010, 817, 767, 749, 703. H NMR (400 MHz, CDCl3), δ: 7.85 (2H, d, J = 8.5 Hz), 7.76
max
1
(2H, d, J = 8.5 Hz), 7.29–7.41 (5H, m, Ar–H), 7.07 (1H, dd, J = 9.8, 5.3 Hz), 6.31 (1H, d,
J = 9.8 Hz), 5.61 (1H, brd, J = 3.3 Hz), 5.13 (1H, d, J = 3.3 Hz), 5.11 (1H, brd, J = 4.1 Hz),
4.73 (1H, dd, J = 5.3, 4.1 Hz). 13C NMR (100 MHz, CDCl3): δ 164.7 (C), 160.3 (C), 139.0
(CH), 138.0 (2 × CH), 137.3 (C) 131.1 (2 × CH), 128.7 (2 × CH), 128.6 (CH), 128.4 (C),
126.2 (2 × CH), 124.8 (CH), 101.8 (C), 86.0 (CH), 84.3 (CH), 83.6 (CH), 69.3 (CH). EIMS
m/z (relative intensity): 464 [M + H]+ (<1), 233 (4), 231 (74), 215 (6), 203 (2), 171 (28),
146 (34), 145 (100), 105 (14), 77 (14). HR-TOF-MS (ESI): m/z 484.9863 [M + Na]+ (calcd
for C20H15IO5Na, 484.9862)

10
J. EuAnOrASETr ET Al.
3.3.11. (2R,3R,3aR,7aS)-5-Oxo-2-phenyl-3,3a,5,7a-tetrahydro-2H-furo[3,2-b]pyran-
3-yl 4-bromo-2-chlorobenzoate (13)
Compound 13 was prepared by reacting altholactone (29.1 mg, 0.125 mmol) with
4-bromo-2-chlorobenzoic acid (35.2 mg, 0.150 mmol) in the presence of N,N′-
dicyclohexylcarbodiimide (30.9 mg, 0.150 mmol) and 4-dimethylaminopyridine (cat.) at
room temperature for 3 h. e reaction mixture was purified by column chromatography
(15% ethyl acetate-hexane) to yield 13 (43.7 mg, 78% yield) as a yellow oil; [ꢀ]2D8 +72.9 (c
ACN
KBr
0.017, CHCl ). UV ꢀ
nm (log ε): 282 (2.97), 246 (4.02), 209 (4.46). IR ꢀ cm−1: 1736,
max
max
3
1580, 1496, 1469, 1451, 1371, 1289, 1240, 1124, 1103, 1048, 877, 819, 766, 699. 1H NMR
(400 MHz, CDCl3), δ: 7.68 (1H, d, J = 8.4 Hz), 7.6 (1H, d, J = 1.7 Hz), 7.43 (1H, dd, J = 8.4,
1.7 Hz), 7.22–7.34 (5H, m, Ar–H), 6.99 (1H, dd, J = 9.8, 5.3 Hz), 6.23 (1H, d, J = 9.8 Hz),
5.54 (1H, brd, J = 3.2 Hz), 5.08 (1H, d, J = 3.2 Hz), 5.02 (1H, brd, J = 4.0 Hz), 4.63 (1H, dd,
J = 5.3, 4.0 Hz). 13C NMR (100 MHz, CDCl3): δ 163.7 (C), 160.3 (C), 139.1 (CH), 137.3
(C), 135.0 (C), 134.1 (CH), 132.9 (CH), 130.3 (CH), 128.7 (2 × CH), 128.6 (CH), 127.6 (C),
127.5 (C), 126.2 (2 × CH), 124.8 (CH), 86.1 (CH), 84.8 (CH), 83.4 (CH), 69.3 (CH). EIMS
m/z (relative intensity): 451 [M + H]+ (<1), 221 (12), 220 (49), 218 (36), 171 (11), 146 (22),
145 (100), 143 (6), 106 (10), 78 (7). HR-TOF-MS (ESI): m/z 470.9617 [M + Na]+ (calcd for
C20H14BrClO5Na, 470.9611)
3.3.12. (2R,3R,3aR,7aS)-5-Oxo-2-phenyl-3,3a,5,7a-tetrahydro-2H-furo[3,2-b]pyran-
3-yl 2-fluoro-5-iodobenzoate (14)
Compound 14 was prepared by reacting altholactone (29.8 mg, 0.128 mmol)
with 2-fluoro-5-iodobenzoic acid (40.9 mg, 0.154 mmol) in the presence of N,N′-
dicyclohexylcarbodiimide (31.7 mg, 0.154 mmol) and 4-dimethylaminopyridine (cat.)
at room temperature for 2 h. e reaction mixture was purified by column chromatog-
raphy (15% ethyl acetate-hexane), followed by recrystallization from MeOH to yield 14
(32.1 mg, 52% yield) as colorless needles; mp 105.3–106.0 °C.[ꢀ]2D8 +98.1 (c 0.018, CHCl ).
3
ACN
KBr
UV ꢀ
nm (log ε): 216 (4.04). IR ꢀ cm−1: 1741, 1732, 1600, 1484, 1449, 1386, 1365,
max
max
1
1293, 1275, 1235, 1140, 1108, 1083, 1068, 1059, 915, 880, 819, 736, 700, 610. H NMR
(400 MHz, CDCl3), δ: 8.25 (1H, dd, J = 5.2, 2.4 Hz), 7.86 (1H, ddd, J = 8.8, 5.2, 2.4 Hz),
7.30–7.42 (5H, m, Ar–H), 7.07 (1H, dd, J = 9.8, 5.4 Hz), 6.96 (1H, dd, J = 10.3, 8.8 Hz), 6.31
(1H, d, J = 9.8 Hz), 5.62 (1H, brd, J = 3.3 Hz), 5.13 (1H, d, J = 3.3 Hz), 5.10 (1H, dd, J = 4.1,
0.9 Hz), 4.72 (1H, dd, J = 5.4, 4.1 Hz). 13C NMR (100 MHz, CDCl3): δ 160.3 (2 × C), 144.1
(CH), 144.0 (CH), 140.8 (CH), 139.1 (CH), 137.3 (C), 128.7 (2 × CH), 128.6 (CH), 126.2
(2 × CH), 124.8 (CH), 119.6 (C), 119.5 (CH), 119.3 (CH), 86.1 (CH), 84.8 (CH), 83.4 (CH),
69.3 (CH). EIMS m/z (relative intensity): 481 [M + H]+ (<1), 250 (46), 249 (25), 215 (6), 171
(12), 170 (21), 146 (27), 145 (100), 123 (11), 106 (13), 95 (11), 94 (7). HR-TOF-MS (ESI):
m/z 502.9767 [M + Na]+ (calcd for C20H14FIO5Na, 502.9768)
3.3.13. (2R,3R,3aR,7aS)-5-Oxo-2-phenyl-3,3a,5,7a-tetrahydro-2H-furo[3,2-b]pyran-
3-yl 3,5-dichlorobenzoate (15)
Compound 15 was prepared by reacting altholactone (31.2 mg, 0.134 mmol)
with 3,5-dichlorobenzoic acid (30.8 mg, 0.161 mmol) in the presence of N,N′-
dicyclohexylcarbodiimide (33.13 mg, 0.161 mmol) and 4-dimethylaminopyridine (cat.) at
room temperature for 3 h. e reaction mixture was purified by column chromatography
(15% ethyl acetate-hexane), followed by recrystallization from EtOH to yield 15 (47.9 mg,

JOurnAl OF ASIAn nATurAl PrOduCTS rESEArCH
11
88% yield) as colorless needles; mp 144.4–145.4 °C. [ꢀ]2D8 +143.9 (c 0.035, CHCl3). UV
ACN
ꢀ
nm (log ε): 294 (2.69), 237 (3.72), 209 (4.46). IR ꢀKBr cm−1: 1739, 1571, 1497, 1450, 1435,
max
1400, 1372, 1351, 1265, 1244, 1152, 1100, 1032, 1022m, 9ax07, 878, 829, 804, 758, 737, 702. 1H
NMR (400 MHz, CDCl3), ꢀ: 7.84 (2H, d, J = 1.8 Hz), 7.53 (1H, t, J = 1.8 Hz), 7.22–7.33 (5H,
m, Ar–H), 6.99 (1H, dd, J = 9.8, 5.0 Hz), 6.23 (1H, d, J = 9.8 Hz), 5.54 (1H, brd, J = 3.8 Hz),
5.02–5.04 (2H, overlapping signal), 4.66 (1H, dd, J = 5.1, 4.8 Hz). 13C NMR (100 MHz,
CDCl3): δ 162.8 (C), 160.2 (C), 139.0 (CH), 137.2 (C), 135.6 (2 × C), 133.6 (CH), 131.7
(C), 128.8 (2 × CH), 128.6 (CH), 128.2 (2 × CH), 126.2 (2 × CH), 124.8 (CH), 85.9 (CH),
84.7 (CH), 83.5 (CH), 69.2 (CH). EIMS m/z (relative intensity): 404.68 [M]+ (<1), 214 (6),
175 (30), 173 (43), 170 (25), 144 (100), 109 (8), 105 (18), 77 (12). HR-TOF-MS (ESI): m/z
427.0113 [M + Na]+ (calcd for C20H14Cl2O5Na, 427.0116)
3.4. General method for preparation of compound 10
A mixture of altholactone (50 mg, 0.216 mmol), and 4-chlorobenzoyl chloride (0.15 ml,
0.259 mmol) in pyridine (1 ml) was stirred at room temperature overnight. e reaction
was quenched with aqueous ammonium chloride (10 ml). e aqueous layer was extracted
with CH2Cl2 (3×25 ml). e combined organic layer was washed with H2O, brine and dried
over anhydrous magnesium sulfate. Afer filtration and removal of solvent under reduced
pressure, a crude product (148.3 mg) was obtained and purified by preparative TLC (25%
ethyl acetate-hexane), followed by recrystallization from MeOH to afford the desired prod-
uct 10 (61.8 mg, 78% yield) as colorless needles.
3.4.1. (2R,3R,3aR,7aS)-5-Oxo-2-phenyl-3,3a,5,7a-tetrahydro-2H-furo[3,2-b]pyran-3-
yl 4-chlorobenzoate (10)
mp 134.8–135.6 °C. [ꢀ]D28 +158.7 (c 0.049, CHCl ). UV ꢀ
nm (log ε): 241 (4.24). IR
ACN
max
3
ꢀKBr cm−1: 1725, 1591, 1489, 1401, 1291, 1260, 1226, 1173, 1104, 1090, 1063, 1015, 956,
8m78ax, 853, 827, 819, 757, 743, 699. 1H NMR (400 MHz, CDCl3), ꢀ: 8.0 (2H, d, J = 8.5 Hz),
7.47 (2H, d, J = 8.5 Hz), 7.30–7.41 (5H, m, Ar–H), 7.08 (1H, dd, J = 9.8, 5.2 Hz), 6.32 (1H,
d, J = 9.8 Hz), 5.62 (1H, brd, J = 3.4 Hz), 5.14 (1H, d, J = 3.4 Hz), 5.11 (1H, brd, J = 4.4 Hz),
4.74 (1H, dd, J = 5.2, 4.4 Hz). 13C NMR (100 MHz, CDCl3): δ 164.3 (C), 160.4 (C), 140.4
(C), 139.1 (CH), 137.4 (C), 131.2 (2 × CH), 129.0 (2 × CH), 128.7 (2 × CH), 128.6 (CH),
127.4 (C), 126.2 (2 × CH), 124.8 (CH), 86.0 (CH), 84.3 (CH), 83.6 (CH), 69.3 (CH). EIMS
m/z (relative intensity): 371 [M + H]+ (4), 215 (4), 198 (4), 171 (19), 145 (100), 140 (23),
139 (10), 112 (10), 105 (6), 77 (5). HR-TOF-MS (ESI): m/z 393.0509 [M + Na]+ (calcd for
C20H15BrO5Na, 393.0506)
3.5. Anti-fungal susceptibility testing
Altholactone and its derivatives were tested against three yeast species: C. albicans clinical
isolate MT 2013/1, Cryptococccus neoformans clinical isolate MT 2013/2, and Saccharomyces
cerevisiae IFO 10217. C. albicans and C. neoformans were from yeast stock cultures stored at
the Faculty of Medical Technology, Mahidol University, ailand. C. albicans and C. neofor-
mans were cultivated and maintained on potato dextrose agar (PDA) (Merck, Darmstadt,
Germany) at 37 °C, whereas S. cerevisiae was cultivated on the same media at 30 °C.

12
J. EuAnOrASETr ET Al.
Minimal inhibition concentrations (MICs) were determined using the broth microdilu-
tion method, in 96-well microplates (ExtraGENE, California, USA), based on the guideline
from the European Committee on Antimicrobial Susceptibility Testing (EUCAST) definitive
document EDef 7.2 [24]. e procedure was carried out by preparing overnight cultures
of indicator yeast on PDA agar followed by colony suspension in sterile water to obtain
0.5 McFarland standard turbidity (1–5 × 106 CFU ml−1),and samples were further diluted
10-fold to obtain a cell density of 1–5 × 105 CFU ml−1. Compounds dissolved in 100%
DMSO (Merck, Hohenbrunn, Germany) were prepared, using twofold serial dilutions in
RPMI 1640 broth (Sigma-Aldrich, Missouri, USA) with 2% glucose to obtain a concentra-
tion ranging from 256 to 0.5 μg ml−1. e highest concentration of DMSO in the test was
2.5%. Yeast cultures (100 μl) and compounds (100 μl) were added to each well of microtiter
plates and incubated at 37 °C for 1 day for C. albicans and 3 days for C. neoformans. In case
of S. cerevisaie, YPD (yeast peptone dextrose broth contained yeast extract 10 g, peptone
20 g, and dextrose 20 g in 1.0 liter distilled water) was used and incubation was performed
at 30 °C for 1 day. Following incubation, the growth inhibition was monitored using a
Wallac 1420 Victor2 microplate reader (Perkin Elmer, Turku, Finland). Blank medium
was used as the sterility control. DMSO alone (under the same dilution condition) was
also employed as a negative control. Amphotericin B (Biolab, ailand) was included in
the test as a positive control. All experiments were performed in triplicate and results are
from two independent trials.
Acknowledgments
We are grateful to Dr Laran T. Jensen (Department of Biochemistry, Faculty of Science, Mahidol
University) for critically proofreading of the manuscript.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
is study was financially supported by the ailand Research Fund through the Royal Golden Jubilee
PhD Program [Grant number PHD/0103/2552] to JE and WP; the Science Achievement Scholarship
of ailand (SAST), Faculty of Science, Mahidol University to OV.
ORCID
Jirayut Euanorasetr
Watanalai Panbangred
http://orcid.org/0000-0003-2300-797X
http://orcid.org/0000-0003-0141-9279
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