Synthesis and antifungal activities of trichodermin derivatives as fungicides on rice

Article abstract of DOI:10.1002/cbdv.201200135

Twenty new trichodermin derivatives, 2a-5, containing alkoxy, acyloxy, and Br groups in 4-, 8-, 9-, 10- and 16-positions were synthesized and characterized. The antifungal activities of the new compounds against rice false smut (Ustilaginoidea virens), ri

Full text of DOI:10.1002/cbdv.201200135

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Synthesis and Antifungal Activities of Trichodermin Derivatives as Fungicides
on Rice
by Xiaojun Xua), Jingli Chenga), Yong Zhoua), Chulong Zhanga), Xiaoming Ouc), Weike Sub), Jinhao
Zhao*a)b), and Guonian Zhua)
a) Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310029,
P. R. China (phone: þ86-571-86971220; fax: þ86-571-86430193; e-mail: jinhaozhao@zju.edu.cn)
b) College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou, P. R. China
c) Hunan Research Institute of Chemical Industry, Changsha, China
Twenty new trichodermin derivatives, 2a–5, containing alkoxy, acyloxy, and Br groups in 4-, 8-, 9-,
10- and 16-positions were synthesized and characterized. The antifungal activities of the new compounds
against rice false smut (Ustilaginoidea virens), rice sheath blight (Rhizoctonia solani), and rice blast
(Magnaporthe grisea) were evaluated. The results of bioassays indicated that the antifungal activities
were particularly susceptible to changes at 4-, 8-, and 16-positions, but low to changes at 9- and 10-
positions. Most of these target compounds exhibited good antifungal activities at the concentration of
50 mglꢀ1. Compound 4 (9-formyltrichodermin; EC50 0.80 mglꢀ1) with an CHO group at 9-position
displayed nearly the same level of antifungal activity against Ustilaginoidea virens as the commercial
fungicide prochloraz (EC50 0.82 mglꢀ1), while compound 3f ((8R)-8-{[(E)-3-phenylprop-2-enoyl]oxy}-
trichodermin; EC50 3.58 and 0.74 mglꢀ1) with a cinnamyloxy group at C(8) exhibited much higher
antifungal activities against Rhizoctonia solani and Magnaporthe grisea than the commercial fungicides
prochloraz (EC50 0.96 mglꢀ1) and propiconazole (EC50 5.92 mglꢀ1), respectively. These data reveal that
compounds 3f and 4 possess high antifungal activities and may serve as lead compounds for the
development of fungicides in the future.
Introduction. – First isolated from Trichoderma viride found in New Guinea soil [1],
trichodermin (1; Fig. 1) is a member of a family of fungal metabolites, which possess an
olefinic bond and an epoxy group in a trichothecane ring system and are characterized
as 12,13-epoxytrichothecenes [2]. Trichodermin and other sesquiterpene antibiotics
(trichothecin, trichodermol, verrucarin A, fusarenon X, nivalenol, diacetoxicirpenol,
and T-2 toxin) of the same group are effective inhibitors of the peptidyl transferase
center of eukaryotic ribosomes and, therefore, block peptide-bond formation [3–10],
and they show broad-spectrum antifungal activities with low toxicity to nontarget
organisms such as mammals, vegetables, and fruit [11–14].
Trichodermin-BL, which was applied as pre-sowing seed treatment and on growing
plants to decrease disease incidence, increasing the yield, and raising flax fiber quality,
Fig. 1. Chemical structure of trichodermin (1)
ꢀ 2013 Verlag Helvetica Chimica Acta AG, Zꢁrich

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
601
was widely used in Russia as the first commercial bioproduct [15][16]. Trichodermin
was also used for removing toxic effects of crude oil hydrocarbons on soils and
agricultural plants and increasing productivity of plants [17]. In recent years, it was
reported that trichodermin had good efficacy on disease and pest control against the
gall nematode (Meloidogyne), root rots of cucumber, Rhizoctonia solani, Botrytis
cinerea, and Artemia salina [18][19]. Although some reports had appeared on
trichodermin derivatives and analogs [20–26], their antifungal activities in controlling
rice pathogen have been only rarely reported. With increasing human population,
decreasing amounts of land available for agriculture [27], and growing drug resistance
and new rice pathogens, development of novel and ꢂgreenꢃ chemical fungicides with low
hazard to environment and human being is becoming more and more necessary and
important [28].
In our previous work, a new endophytic fungus Trichoderma taxi sp. nov.
(ZJUF0986) was isolated, which could produce trichodermin in good yield, and
preliminary derivatives had been synthesized [29–32]. To develop novel fungicides
with low toxicity on crops, especially on rice, 29 novel trichodermin derivatives 2a–5
were designed and synthesized, and their antifungal activities against rice false smut
(Ustilaginoidea virens), rice sheath blight (Rhizoctonia solani), and rice blast
(Magnaporthe grisea) were evaluated. Compounds 3f and 4 exhibited high antifungal
activities against rice false smut (Ustilaginoidea virens) and rice blast (Magnaporthe
grisea). In addition, the crystal structure of (8R)-8-{[(E)-3-phenylprop-2-enoyl]oxy}-
trichodermin (3f) was established (cf. Fig. 2 in the Exper. Part).
Results and Discussion. – Design. In our previous work, trichodermin (1) was
modified at C(12) and C(13) by opening the epoxy group, but it was found that the
derivatives obtained had no antifungal activities against the three rice fungi. So we
retained the polycyclic skeleton of trichodermin, including the epoxy group, and
changed the substituents at C(4), C(8), C(9), C(10), and C(16). To investigate the
importance of the ester function at C(4) for activity, we prepared the derivatives 2a–2g
containing saturated, unsaturated, and aromatic acyloxy groups. For comparison, the
ethers 2h and 2j with MeO and EtO groups at C(4) were prepared (Schemes 1 and 2).
Similarly, the effect of the C(9)¼C(10) bond was evaluated by comparing the activity
with that of the dibromo derivative 5 (Scheme 1). The influence of the other functional
groups at C(8) and C(16) of trichodermin on the activity was studied by treatment of
compound 1 with SeO2 in 1,4-dioxane (Scheme 1), and the derivatives with a OH group
at C(8) (3) and an CHO group at C(9) (4) were obtained. Then, the OH group at C(8)
was replaced with saturated, unsaturated, and aromatic acyloxy groups to provide
compounds 3a–3i (Scheme 3).
Chemistry. The synthetic routes to compounds 2–5 are outlined in Scheme 1, and
the target compounds 2a–2i and 3a–3i were obtained in good yields (56.9–91.2%) via
acylation or alkylation of 2 and 3, respectively. To obtain intermediate 3 ((8b)-8-
hydroxytrichodermin) with high purity, the method reported by Kraus and Thomas was
modified by employing AcOEt as the extraction solvent, and petroleum ether and
AcOEt as the eluent for column chromatography [33]. When trichodermin was
oxidized by SeO2 to give compound 3 [33], compound 4, 9-formyltrichodermin, was
obtained as a by-product surprisingly, and it was found to have an excellent antifungal

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Scheme 1. General Synthetic Routes to Compounds 2–5
Scheme 2. General Synthetic Routes to Compounds 2a–2i
Scheme 3. General Synthetic Route to Compounds 3a–3i

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
603
activity. To obtain an anti addition product of compound 5, trichodermin was reacted
with 2 equiv. of Br2 by using pyridine as catalyst (Scheme 1). Compounds 2 and 3 were
reacted with newly prepared acyl chlorides using Et3N and DMAP (¼4-(dimethyl-
amino)pyridine) as base and catalyst, respectively, in dried CH2Cl2 afforded the target
compounds 2a–2g and 3a–3i (Schemes 2 and 3).
1
The main characteristic of the H-NMR spectra of 2a was the presence of broad
multiplets at d(H) 2.66–2.52 and 2.04–1.99 ppm, presumably arising due to the CH2(3)
group linking the two CH(2) and CH(4) groups at. A sharp peak at d(H) 1.88–
1.70 ppm was assigned to the Me group at C(9) directly attached to the C¼C bond of
the six-membered ring.
Biological Activity. Based on the data compiled in Tables 1 and 2, a clearcut, well-
defined relation between chemical structure and biological activity was gradually
developed. The effects of different kinds of electron-withdrawing, electron-donating,
and spatially demanding groups on the fungicidal activity were studied. Table 1
contains the antifungal activities of the target compounds 2a–5, prochloraz, propico-
nazole, and compound 1 against rice false smut (Ustilaginoidea virens), rice sheath
blight (Rhizoctonia solani), and rice blast (Magnaporthe grisea). The results indicated
that most of the designed trichodermin derivatives with modifications at C(4), C(8),
and C(9) had moderate antifungal activities. Table 2 reveals that 3f ((8R)-8-{[(E)-3-
phenylprop-2-enoyl]oxy}trichodermin) and 4 (9-formyltrichodermin) exhibited the
highest antifungal activities against Ustilaginoidea virens, Rhizoctonia solani, and
Magnaporthe grisea than other derivatives, with EC50 values of 1.48, 3.58, and 0.74, and
0.80, 7.40, and 11.82 mglꢀ1, respectively. Fortunately, 3f and 4 had higher antifungal
activities against Ustilaginoidea virens than the parent compound trichodermin (1; EC50
2.30 mglꢀ1), and 3f displayed antifungal activities against Magnaporthe grisea even
about six times better than that of 1 (EC50 4.25 mglꢀ1). While 4 displayed comparable
antifungal activity against Ustilaginoidea virens as prochloraz (EC50 0.82 mglꢀ1), 3f
showed much higher antifungal activities against Rhizoctonia solani and Magnaporthe
grisea than the commercial fungicides propiconazole (EC50 5.92 mglꢀ1) and prochloraz
(EC50 0.96 mglꢀ1), respectively, it could be concluded that the C¼O group at C(9) and
[(E)-3-phenylprop-2-enoyl]oxy group at C(8) increase the antifungal activity. How-
ever, compound 5 brominated at C(9) and C(10) showed no or poor antifungal
activities against the three rice funguses. Interestingly, trichodermin (1), as a starting
material, exhibited the highest antifungal activity against Rhizoctonia solani, with an
EC50 value of 0.72 mglꢀ1
.
StructureꢀActivity Relationship. The results in Tables 1 and 2 showed that the
antifungal activities of derivatives with substituents containing conjugated C¼C bonds
were much better than those with single bonds. For example, the antifungal activities of
compound 3b ((8R)-8-[(3-methylbut-2-enoyl)oxy]trichodermin) against Ustilagino-
idea virens, Rhizoctonia solani, and Magnaporthe grisea at 50 mglꢀ1 concentration were
80.5, 99.7, and 92.6%, respectively, as compared with 52.5, 98.9, and 41.7% for
compound 3c ((8R)-8-[(3-methylbutanoyl)oxy]trichodermin) at the same concentra-
tion. In particular, 3f and 4 with conjugated C¼C bonds had the highest antifungal
activities against the three rice fungi in all the target compounds. At C(4), compounds
2h ((4b)-4-methoxytrichodermin) and 2i ((4b)-4-ethoxytrichodermin) that contained a
MeO or a EtO substituent, respectively, had better antifungal activities than the other

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Table 1. Antifungal Activitiesa) of Compounds 2a–5 against Rice False Smut, Rice Sheath Blight, and
Rice Blast
Compound
Against rice false smut
Against rice sheath blight
Against rice blast
1
2
2a
2b
2c
2d
2e
2h
2g
2h
2i
3
3a
3b
3c
3d
3e
3f
3g
3h
3i
4
5
100
100
87.0
63.0
30.1
57.7
50.5
49.8
32.3
20.0
90.0
87.8
34.0
46.7
99.7
98.9
32.6
76.5
97
100
88.0
77.0
56.0
43.0
37.3
57.5
32.5
68.4
82.3
81.9
56.6
56.7
92.6
41.7
43.1
69.6
100
57.4
31.6
42.7
98
86.3
70.5
69.6
30.8
49.7
32.0
25.4
28.1
73.8
71.5
31.3
29.2
80.5
52.5
36.7
86.3
100
25.3
20.9
40.3
100.0
0.0
60.1
36.2
50.5
95
0.0
0.0
Prochloraz
Propiconazole
100
–
–
100
100
–
a) Antifungal activity [%] indicates the growth rate of rice fungi that the compound, at the concentration
of 50 mglꢀ1, inhibits as compared to the untreated fungus.
Table 2. Antifungal Activities against Rice False Smut, Rice Sheath Blight, and Rice Blast of Compounds
a
1, 3f, 4, Prochloraz, and Propiconazole Expressed as EC50
)
Compound
EC50 [mglꢀ1
]
Toxicities against
rice false smut
Toxicities against
rice sheath blight
Toxicities against
rice blast
1
3f
4
2.30
1.48
0.80
0.82
ꢀ
0.72
3.58
7.40
ꢀ
4.25
0.74
11.82
0.96
ꢀ
Prochloraz
Propiconazole
5.92
a) EC50 [mglꢀ1] indicates the concentration that the compound inhibits the growth rate of rice fungi by
50% as compared to the untreated fungus.

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
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derivatives against the three rice fungi, but a little lower than the intermediate
trichodermol (2). On the whole, the antifungal activities of trichodermin derivatives
modified at C(4) were not higher than that of those with acryl, 3-methoxylacryl, and
crotonyl substituents [32]. In addition, the C(9)¼C(10) bond exerts great influence on
the activity and should not be modified.
Conclusions and Outlook. – As described above, 20 new trichodermin derivatives
2a–5, synthesized by introducing various substituted groups at C(4), C(8), C(9),
C(10), and C(16), were active fungicides against rice false smut (Ustilaginoidea virens),
rice sheath blight (Rhizoctonia solani), and rice blast (Magnaporthe grisea). The
antifungal activity was acceptable with changes at C(4), C(8), and C(16), but lowered
with changes at C(9) and C(10). Some of them exhibited good antifungal activities at
the concentration of 50 mglꢀ1; particularly the compounds 3f and 4 showed the highest
antifungal activities. Therefore, the present work not only demonstrates that the
antifungal activity of trichodermin derivatives can be significantly improved via
structural modification at some positions, but also paves the way toward the synthesis,
and antifungal-activity studies of new trichodermin derivatives modified at C(8) and
C(16).
This work was supported by grants from the National Key Technology R&D Program of China
(2011BAE06B04–10), the National Natural Science Foundation of China (No. 30700532), and the
Educational Committee Foundation of Zhejiang (No. Y201122463). The authors also thank senior
engineer Jianming Gu (Center of Analysis and Measurement of Zhejiang University) for the X-ray
crystallographic diffraction analysis of compound 3f.
Experimental Part
General. All starting materials and reagents were commercially available and used without further
purification except as indicated. All fungal materials were obtained from the National Engineering
Research Center. M.p.: X-4 binocular microscope melting point apparatus (Beijing Tech Instruments Co.,
Beijing, China); uncorrected. Optical rotation: SEPA-300 spectropolarimeter (Horiba, Ltd., Kyoto,
Japan) equipped with a cell with a 10-cm optical path length at 589 nm and at 288; concentrations of of
samples in MeOH, 5 mgmlꢀ1. 1H- and 13C-NMR (500 and 100 MHz, resp.) spectra: Bruker AVANCE III
in CDCl3; d in ppm rel. to Me4Si as internal standard, J in Hz. HR-MS: FTICR-MS instrument (Ion-
spec7.0T); in m/z. Elemental analyses: Yanaca CHN Corder MT-3 elemental analyzer.
Synthesis of Trichodermol (¼(4b)-12,13-Epoxytrichothec-9-en-4-ol; 2) [32]. A mixture of tricho-
dermin (¼(4b)-12,13-epoxytrichothec-9-en-4-yl acetate; 1; 2.00 g, 6.85 mmol), NaOH (24 ml, 2 mollꢀ1),
and MeOH (30 ml) was stirred at r.t. for 5 h. After the reaction was completed, the solvent was
evaporated under reduced pressure, and the product was purified by flash column chromatography (FC;
silica gel petroleum ether (PE; 60–908)/AcOEt 3 :1) to give 1.52 g of 2 (89%) as colorless crystals. M.p.
117–1198. 1H-NMR (500 MHz, CDCl3): 5.39 (d, J¼5.0, HꢀC(10)); 4.34 (s, HꢀC(4)); 3.84 (d, J¼7.0,
HꢀC(2)); 3.52 (d, J¼6.5, HꢀC(11)); 3.12 (d, J¼5.0, 1 H, CH2(13)); 2.82 (d, J¼5.0, 1 H, CH2(13)); 2.61
(dd, J¼9.0, 19.5, 1 H, CH2(3)); 2.00–1.96 (m, CH2(8)); 1.94–1.91 (m, 1 H, CH2(7)); 1.89–1.87 (m, 1 H,
CH2(3)); 1.71 (s, Me(16)); 1.46–1.43 (m, 1 H, CH2(7)); 0.93 (s, Me(14)); 0.72 (s, Me(15)). 13C-NMR
(100 MHz, CDCl3): 140.04 (C(9)); 118.69 (C(10)); 78.70, 73.91 (C(2,11)); 70.23 (C(4)); 65.71 (C(12));
49.06, 48.34, 47.50 (C(5,13,6)); 40.04 (C(3)); 27.95 (C(8)); 24.36, 23.16 (C(7,16)); 15.75 (C(15)); 6.15
(C(14)). HR-ESI-MS: 273.1471 ([MþNa]þ , C15H22NaO3þ ; calc. 273.1467).
Synthesis of (4b,8b)-8-Hydroxy-12,13-epoxytrichothec-9-en-4-yl Acetate (3) and (4b)-16-Oxo-12,13-
epoxytrichothec-9-en-4-yl Acetate (4). A soln. of 1 (4.00 g, 13.70 mmol) in 1,4-dioxane (30 ml) was added
dropwise during 90 min to a refluxing soln. of SeO2 (2.00 g, 18.02 mmol) in 1,4-dioxane (20 ml) [33].

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
After refluxing for 12 h, the mixture was cooled and concentrated. Then, 50 ml of AcOEt was added, and
the mixture was washed with 5% aq. NaHCO3, dried, and concentrated in vacuo to give 3.50 g of a yellow
liquid. The crude product was purified by FC (SiO2; PE/AcOEt 5 :1) to give 3 (2.10 g, 49.8%). Colorless
crystals. M.p. 138–1418. [a]2D8 ¼ þ3.49 (c¼5, acetone). 1H-NMR (500 MHz, CDCl3): 5.52 (dd, J¼3.5, 7.5,
HꢀC(4)); 5.48–5.46 (m, HꢀC(10)); 4.06 (d, J¼7.5, HꢀC(8)); 3.84 (d, J¼5.0, HꢀC(2)); 3.63 (d, J¼5.5,
HꢀC(11)); 3.13 (d, J¼4.0, 1 H, CH2(13)); 2.85 (d, J¼4.0, 1 H, CH2(13)); 2.53–2.49 (m, 1 H, CH2(3));
2.08 (s, Me); 2.02–1.98 (m, 1 H, CH2(3)); 1.86–1.84 (m, 1 H, CH2(7)); 1.70 (s, Me(16)); 1.53–1.52 (m,
1 H, CH2(7)); 0.98 (s, Me(14)); 0.73 (s, Me(15)). 13C-NMR (100 MHz, CDCl3): 170.94 (Me); 142.24
(C(9)); 121.02 (C(10)); 79.29, 74.11 (C(2,11)); 70.30, 68.92 (C(4,8)); 65.36 (C(12)); 49.03, 48.50, 47.74
(C(5,13,6)); 42.36, 36.61 (C(3,7)); 21.09 (Me); 18.75, 16.87 (C(15,16)); 5.78 (C(14)). HR-ESI-MS:
331.1518 ([MþNa]þ , C17H24NaOþ5 ; calc. 331.1521).
Compound 4 (0.80 g, 19.1%) was obtained as the second product. Pale yellow oil. [a]2D8 ¼ ꢀ1.18 (c¼
5, acetone). 1H-NMR (500 MHz, CDCl3): 9.54 (s, CHO); 6.60–6.65 (m, HꢀC(10)); 5.61 (dd, J¼3.5, 7.5,
HꢀC(4)); 3.89 (d, J¼5.5, HꢀC(2,11)); 3.63 (d, J¼5.5, HꢀC(11)); 3.12 (d, J¼4.0, 1 H, CH2(13)); 2.85 (d,
J¼4.0, 1 H, CH2(13)); 2.58–2.53 (m, 1 H, CH2(3)); 2.48–2.44 (m, 1 H, CH2(3)); 2.08 (s, Me); 2.09–2.03
(m, CH2(8)); 1.87–1.84 (m, 1 H, CH2(7)); 0.97–0.95 (m, 1 H, CH2(7)); 0.92 (s, Me(14)); 0.75 (s, Me(15)).
13C-NMR (100 MHz, CDCl3): 199.22 (CHO); 170.54 (Me); 137.80 (C(9)); 136.74 (C(10)); 79.20 (C(2));
77.04, 74.59 (C(11,4)); 69.75 (C(12)); 49.23, 48.56, 47.74 (C(5,13,6)); 42.36, 40.10 (C(3,7)); 21.01 (Me);
18.75, 18.14 (C(15,8)); 5.28 (C(14)). HR-ESI-MS: 329.1360 ([MþNa]þ , C17H22NaO5þ ; calc. 329.1365).
Synthesis of (4b,9a,10a)-9,10-Dibromo-12,13-epoxytrichothecan-4-yl Acetate (5). In a flask, Br2
(0.22 g, 1.39 mmol) in CH2Cl2 (5 ml) was added dropwise to a soln. of 1 (0.20 g, 0.68 mmol) and
pyridine (0.11 g, 1.39 mmol) in 15 ml of CH2Cl2, and stirred for 3 h at r.t. [34]. The solvent was washed
with dil. HCl and sat. Na2CO3 soln., dried, and concentrated in vacuo to 0.40 g of colorless liquid. The
crude product was purified by FC (silica gel; PE/AcOEt 5 :1) to give 5 0.27 g, 88.0%). White solid. The
solid was filtered and recrystallized from 95.0% EtOH as colorless blocks. M.p. 170–1728. 1H-NMR
(500 MHz, CDCl3): 5.57 (dd, J¼3.5, 8.0, HꢀC(4)); 4.62 (s, HꢀC(10)); 4.02 (s, HꢀC(11)); 3.87 (d, J¼5.0,
HꢀC(2)); 3.21 (d, J¼4.0, 1 H, CH2(13)); 2.94 (d, J¼4.0, 1 H, CH2(13)); 2.49–2.41 (m, 1 H, CH2(13));
2.41–2.35 (m, 1 H, CH2(8)); 2.07 (s, Me); 2.05 (s, Me(16)); 2.01–1.97 (m, HꢀC(3)); 1.96–1.95 (m, 1 H,
CH2(7)); 1.95–1.87 (m, 1 H of CH2(8)); 1.46–1.42 (m, 1 H, CH2(7)); 1.38 (s, Me(14)); 0.71 (s, Me(15)).
13C-NMR (100 MHz, CDCl3): 170.20 (Me); 80.56 (C(2)); 77.35, 74.42 (C(11,4)); 68.78, 64.92 (C(12,9));
52.11 (C(10)); 48.60, 48.23, 47.74 (C(5,13,6)); 42.36, 40.10 (C(3,8)); 35.10, 32.33 (C(16,7)); 21.20 (Me);
18.50, 18.23 (C(15,8)); 6.10 (C14). Anal. calc. for C17H24Br2O4: C 45.16, H 5.35; found: C 45.14, H 5.34.
Synthesis of (4b,8b)-4-(Acetyloxy)-12,13-epoxytrichothec-9-en-8-yl Methylbenzoate (3d). Represen-
tative Procedure. A soln. of 4-methylbenzoyl chloride (0.65 mmol) in dry CH2Cl2 (5 ml) was added
dropwise to a soln. of 3 (0.20 g, 0.65 mmol), Et3N (0.20 g, 1.95 mmol), and DMAP (0.007 g, 0.06 mmol) in
CH2Cl2 (10 ml), and the mixture was stirred at r.t. for 8 h and then was washed successively with H2O, sat.
NaHCO3 soln., and brine. The org. layer was dried (Na2SO4), and the solvent was removed to give a
crude product, which was purified by FC (SiO2; PE/AcOEt 5 :1) to afford 3d (0.24 g, 85.9%). Colorless
crystals. M.p. 132–1348. [a]2D8 ¼ þ11.41 (c¼5, acetone). 1H-NMR (500 MHz, CDCl3): 7.95–7.93 (m, Ar);
7.26–7.24 (m, Ar); 5.63–5.61 (m, HꢀC(10)); 5.60–5.58 (m, HꢀC(8)); 5.56 (dd, J¼4.0, 8.0, HꢀC(4)); 3.88
(d, J¼5.0, HꢀC(2)); 3.69 (d, J¼5.5, HꢀC(11)); 3.13 (d, J¼4.0, 1 H, CH2(13)); 2.81 (d, J¼4.0, 1 H,
CH2(13)); 2.57–2.52 (m, 1 H, CH2(3)); 2.08 (s, Me); 2.05–2.02 (m, 1 H, CH2(3)); 2.01–1.95 (m,
CH2(7)); 1.77 (s, Me(16)); 1.11 (s, Me(14)); 0.72 (s, Me(15)). 13C-NMR (100 MHz, CDCl3): 170.89
(C¼O); 166.29 (C¼O); 143.92 (Ar); 139.17 (C(9)); 129.73, 129.16 (2 Ar); 127.31 (Ar); 122.72 (C(10));
79.41, 74.34, 71.46, 69.96 (C(2,11,8,4)); 65.30 (C(12)); 49.15, 48.42, 47.72 (C(5,13,6)); 42.24 (C(3)); 30.89
(C(7)); 21.64, 21.08, 18.87 (2 Me, C(16)); 16.99 (C(15)); 5.81 (C(14)). Anal. calc. for C25H30O6: C 70.40, H
7.09; found: C 70.38, H 7.11.
Compounds 2a–2g, 3a–3c, and 3e–3i were prepared as described for 3d.
(4b)-12,13-Epoxytrichothec-9-en-4-yl Hexanoate (2a). Yield: 69.8%. Colorless oil. 1H-NMR
(500 MHz, CDCl3): 5.57 (dd, J¼4.0, 8.0, HꢀC(4)); 5.41–5.39 (m, HꢀC(10)); 3.82 (d, J¼5.0, HꢀC(2));
3.61 (d, J¼5.5, HꢀC(11)); 3.12 (d, J¼4.0, 1 H, CH2(13); 2.82 (d, J¼4.0, 1 H, CH2(13)); 2.55–2.51 (m,
1 H, CH2(13)); 2.34–2.31 (m, COCH2); 1.99–1.98 (m, 1 H, CH2(3)); 1.96–1.95 (m, CH2(8)); 1.94–1.92
(m, 1 H, CH2(7)); 1.71 (s, Me(16)); 1.64–1.61 (m, CH2); 1.42–1.40 (m, 1 H, CH2(7)); 1.39–1.32 (m, 2

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CH2); 0.93 (s, Me(14)); 0.92–0.89 (m, Me); 0.71 (s, Me(15)). 13C-NMR (100 MHz, CDCl3): 173.80
(C¼O); 140.21 (C(9)); 118.67 (C(10)); 79.18, 74.86, 71.53, 70.56 (C(2,11,8,4)); 65.55 (C(12)); 48.90, 47.86
(C(5,6)); 40.46 (C(3)); 36.75 (C(13)); 34.42 (CH2); 31.31, 30.91 (CH2, C(7)); 28.03 (CH2); 24.50 (CH2);
22.31 (C(16)); 16.01 (C(15)); 5.88 (C(14)). HR-ESI-MS: 371.2199 ([MþNa]þ , C21H32NaO4þ ; calc.
371.2198).
(4b)-12,13-Epoxytrichothec-9-en-4-yl 2-(Trifluoromethyl)prop-2-enoate (2b). Yield: 56.9%. Pale-
yellow oil. 1H-NMR (500 MHz, CDCl3): 6.75–6.74 (m, CH2); 6.45 (d, J¼1.0, CH2); 5.67 (dd, J¼3.0, 7.5,
HꢀC(4)); 5.42–5.40 (m, HꢀC(10)); 3.86 (d, J¼5.5, HꢀC(2)); 3.61 (d, J¼5.5, HꢀC(11)); 3.13 (d, J¼4.0,
1 H, CH2(13)); 2.84 (d, J¼4.0, 1 H, CH2(13)); 2.64–2.61 (m, 1 H, CH2(3)); 2.08–2.07 (m, 1 H, CH2(3));
2.06–2.04 (m, CH2(8)); 1.97–1.93 (m, 1 H, CH2(7)); 1.72 (s, Me(16)); 1.45–1.43 (m, 1 H, CH2(7)); 0.95
(s, Me(14)); 0.75 (s, Me(15)). HR-ESI-MS: 395.1446 ([MþNa]þ , C19H23F3NaO4þ ; calc. 395.1450).
(4b)-12,13-Epoxytrichothec-9-en-4-yl 2-Phenylacetate (2c). Yield: 86.1%. Colorless crystals. M.p.
150–1538. [a]D28 ¼ ꢀ8.77 (c¼5, acetone). 1H-NMR (500 MHz, CDCl3): 7.33–7.26 (m, Ph); 5.56 (dd, J¼
3.5, 7.5, HꢀC(4)); 5.39–5.38 (m, HꢀC(10)); 3.82 (d, J¼5.0, HꢀC(2)); 3.65 (d, J¼1.5, 2 CH2); 3.57 (d, J¼
5.5, HꢀC(11)); 3.11 (d, J¼4.0, 1 H, CH2(13)); 2.80 (d, J¼4.0, 1 H, CH2(13)); 2.52–2.50 (m, 1 H,
CH2(3)); 2.00–1.97 (m, 1 H, CH2(3)); 1.97–1.95 (m, CH2(8)); 1.94–1.91 (m, 1 H, CH2(7)); 1.70 (s,
Me(16)); 1.40–1.39 (m, 1 H, CH2(7)); 0.90 (s, Me(14)); 0.64 (s, Me(15)). Anal. calc. for C23H28O4: C
74.97, H 7.66; found: C 75.01, H 7.62.
(4b)-12,13-Epoxytrichothec-9-en-4-yl (2,4,6-Trimethylphenyl)acetate (2d). Yield: 91.2%. Colorless
oil. 1H-NMR (500 MHz, CDCl3): 6.85 (s, Ar); 5.57 (dd, J¼4.0, 8.0, HꢀC(4)); 5.39–5.38 (m, HꢀC(10));
3.82 (d, J¼5.0, HꢀC(2)); 3.67 (s, CH2); 3.57 (d, J¼5.5, HꢀC(11)); 3.11 (d, J¼2.0, 1 H, CH2(13)); 2.80 (d,
J¼4 .0, 1 H, CH2(13)); 2.50 (m, 1 H, CH2(3)); 2.30 (s, Me); 2.01–1.98 (m, 1 H, CH2(3)); 1.97–1.96 (m,
CH2(8)); 1.93–1.90 (m, 1 H, CH2(7)); 1.69 (s, Me(16)); 1.46–1.43 (m, 1 H, CH2(7)); 0.90 (s, Me(14));
0.64 (s, Me(15)). HR-ESI-MS: 433.2355 ([MþNa]þ , C26H34NaO4þ ; calc. 433.2359).
(4b)-12,13-Epoxytrichothec-9-en-4-yl (2E)-3-Phenylprop-2-enoate (2e). Yield: 75.6%. Colorless oil.
1H-NMR (500 MHz, CDCl3): 7.72 (d, J¼6.0, Ph); 7.54–7.52 (m, Ph); 7.38–7.37 (m, Ph); 7.38–7.37 (m,
COCH); 6.50 (d, J¼16.0, CH); 5.72 (dd, J¼3.5, 7.5, HꢀC(4)); 5.44–5.42 (m, HꢀC(10)); 3.86 (d, J¼5.0,
HꢀC(2)); 3.65 (d, J¼5.5, HꢀC(11)); 3.15 (d, J¼4.0, 1 H, CH2(13)); 2.86 (d, J¼4.0, 1 H, CH2(13)); 2.61–
2.60 (m, 1 H, CH2(3)); 2.11–2.10 (m, 1 H, CH2(3)); 2.02–1.99 (m, CH2(8)); 1.96 (s, 1 H of CH2(7)); 1.73
(s, Me(16)); 1.46–1.43 (m, 1 H of CH2(7)); 0.98 (s, Me(14)); 0.77 (s, Me(15)). 13C-NMR (100 MHz,
CDCl3): 166.85 (C¼O); 145.17 (CH); 140.24 (C(9)); 134.40 (Ph); 130.33 (Ph); 128.88 (Ph); 128.17 (Ph);
118.67 (C(10)); 118.11 (COCH); 79.24 (C(2)); 75.24 (C(13)); 70.60 (C(4)); 65.63 (C(12)); 49.28 (C(5));
47.92 (C(11)); 40.52 (C(6)); 36.76 (C(3)); 28.04 (C(8)); 24.53 (C(7)); 23.27 (C(16)); 16.05 (C(15)); 6.67
(C(14)). HR-ESI-MS: 403.1886 ([MþNa]þ , C24H28NaO4þ ; calc. 403.1882).
(4b)-12,13-Epoxytrichothec-9-en-4-yl 2-(Naphthalen-2-yl)acetate (2f). Yield: 84.9%. Colorless oil.
1H-NMR (500 MHz, CDCl3): 8.03–8.00 (m, Naph); 7.86–7.84 (m, Naph); 7.53–7.26 (m, Naph); 5.58 (dd,
J¼3.5, 8.0, HꢀC(4)); 5.37–5.36 (m, HꢀC(10)); 4.10 (s, CH2); 3.81 (d, J¼5.0, HꢀC(2)); 3.55 (d, J¼6.0,
HꢀC(11)); 3.11 (d, J¼4.0, 1 H, CH2(13)); 2.80 (d, J¼4.0, 1 H, CH2(13)); 2.52–2.51 (m, 1 H, CH2(3));
2.01–1.99 (m, 1 H, CH2(3)); 2.00–1.96 (m, J¼4.0, CH2(8)); 1.99–1.95 (m, 1 H, CH2(7)); 1.68 (s,
Me(16)); 1.37–1.25 (m, 1 H, CH2(7)); 0.85 (s, Me(14)); 0.60 (s, Me(15)). HR-ESI-MS: 441.2042 ([Mþ
Na]þ , C20H30NaOþ4 ; calc. 441.2045).
(4b)-12,13-Epoxytrichothec-9-en-4-yl 2,6-Dichloro-5-fluoropyridine-3-carboxylate (2g). Yield:
1
71.1%. Colorless crystals. M.p. 141–1448. [a]2D8 ¼ ꢀ7.17 (c¼5, acetone). H-NMR (500 MHz, CDCl3):
8.05 (d, J¼7.0, Py); 5.79 (dd, J¼3.0, 7.5, HꢀC(4)); 5.43 (d, J¼4.5, HꢀC(10)); 3.90 (d, J¼5.0, HꢀC(2));
3.64 (d, J¼5.5, HꢀC(11)); 3.17 (d, J¼4.0, 1 H, CH2(13)); 2.87 (d, J¼4.0, 1 H, CH2(13)); 2.86–2.65 (m,
1 H, CH2(3)); 2.16–2.11 (m, 1 H, CH2(3)); 2.01–1.96 (m, CH2(8)); 2.00–1.95 (m, 1 H, CH2(7)); 1.73 (s,
Me(16)); 1.47–1.44 (m, 1 H, CH2(7)); 0.99 (s, Me(14)); 0.79 (s, Me(15)). Anal. calc. for C21H22Cl2FNO4:
C 57.02, H 5.01, N 3.17; found: C 57.26, H 4.97, N 3.16.
(4b,8b)-4-(Acetyloxy)-12,13-epoxytrichothec-9-en-8-yl Butanoate (3a). Yield: 80.0%. Colorless oil.
1H-NMR (500 MHz, CDCl3): 5.60–5.58 (m, HꢀC(10)); 5.53 (dd, J¼3.5, 8.0, HꢀC(4)); 5.40–5.37 (m,
HꢀC(8)); 4.26–4.21 (m, HꢀC(2)); 3.86 (d, J¼5.0, 1 H, CH2(13)); 3.66 (d, J¼5.5, HꢀC(11)); 3.13 (d, J¼
4.0, 1 H, CH2(13)); 2.80–2.78 (m, 1 H, CH2(7)); 2.54–2.50 (m, 1 H, CH2(3)); 2.08 (s, Me); 2.02–2.00 (m,

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
1 H, CH2(3)); 1.99–1.83 (m, 2 CH2); 1.86–1.83 (m, 1 H, CH2(7)); 1.72 (s, Me(16)); 1.06–1.03 (m, Me);
1.04 (s, Me(14)); 0.71 (s, Me(15)). HR-ESI-MS: 401.1940 ([MþNa]þ , C21H30NaO6þ ; calc. 401.1943).
(4b,8b)-4-(Acetyloxy)-12,13-epoxytrichothec-9-en-8-yl 3-Methylbut-2-enoate (3b). Yield: 67.2%.
Colorless oil. 1H-NMR (500 MHz, CDCl3): 6.09 (s, CH); 5.53–5.58 (m, CH2(8)); 5.41–5.39 (m,
HꢀC(10)); 3.83 (d, J¼5.5, HꢀC(2)); 3.62 (d, J¼5.5, HꢀC(11)); 3.09 (d, J¼4.0, 1 H, CH2(13)); 2.78 (d,
J¼4.0, 1 H, CH2(13)); 2.53 (dd, J¼7.5, 15.5, 1 H, CH2(3)); 2.18 (s, Me); 2.08 (s, Me); 2.01–1.97 (m,
HꢀC(3,7)); 1.91 (s, Me); 1.88 (s, Me(16)); 1.05 (s, Me(14)); 0.70 (s, Me(15)). HR-ESI-MS: 413.1940
([MþNa]þ , C22H30NaOþ6 ; calc. 413.1944).
(4b,8b)-4-(Acetyloxy)-12,13-epoxytrichothec-9-en-8-yl 3-Methylbutanoate (3c). Yield: 74.3%. Pale
1
yellow oil. H-NMR (500 MHz, CDCl3): 5.57–5.56 (m, HꢀC(4)), 5.53 (dd, J¼4.0, 8.0, HꢀC(8)); 5.36–
5.33 (m, HꢀC(10)); 3.85 (d, J¼5.0, HꢀC(2)); 3.64 (d, J¼5.0, HꢀC(11)); 3.13 (d, J¼4.0, 1 H, CH2(13));
2.80 (d, J¼4.0, 1 H, CH2(13)); 2.54 (dd, J¼8.0, 15.5, 1 H, CH2(3)); 2.22 (d, J¼5.0, CH2); 2.13–2.10 (m,
CH); 2.08 (s, Me); 2.02–2.10 (m, 1 H, CH2(3)); 2.00–1.98 (m, CH2(7)); 1.70 (s, Me(16)); 1.15 (s,
Me(14)); 0.97 (d, J¼7.0, 2 Me); 0.71 (s, Me(15)). HR-ESI-MS: 415.2097 ([MþNa]þ , C22H32NaO6þ ; calc.
415.2102).
(4b,8b)-4-(Acetyloxy)-12,13-epoxytrichothec-9-en-8-yl 4-Fluorobenzoate (3e). Yield: 70.0%. Color-
less crystals. M.p. 142–1448. [a]2D8 ¼ þ3.61 (c¼5, acetone). 1H-NMR (500 MHz, CDCl3): 8.08–8.05 (m,
Ar); 7.15–7.11 (m, Ar); 5.64–5.63 (m, HꢀC(10)); 5.60–5.58 (m, HꢀC(8)); 5.56 (dd, J¼3.5, 8.0, HꢀC(4));
3.88 (d, J¼5.0, HꢀC(2)); 3.69 (d, J¼5.5, HꢀC(11)); 3.13 (d, J¼4.0, 1 H, CH2(13)); 2.81(d, J¼4.0, 1 H,
CH2(13)); 2.55 (dd, J¼8.0, 15.5, 1 H, CH2(3)); 2.08 (s, Me); 2.04–1.99 (m, 1 H, CH2(3)); 1.99–1.97 (m,
CH2(7)); 1.76 (s, Me(16)); 1.11 (s, Me(14)); 0.73 (s, Me(15)). Anal. calc. for C24H27FO6: C 66.96, H 6.32;
found: C 66.89, H 6.38.
(4b,8b)-4-(Acetyloxy)-12,13-epoxytrichothec-9-en-8-yl (2E)-3-Phenylprop-2-enoate (3f). Yield:
85.3%. Colorless crystals. M.p. 100–1028. 1H-NMR (500 MHz, CDCl3): 7.72 (d, J¼16.0, COCH);
7.54–7.52 (m, Ph); 7.40–7.39 (m, Ph); 6.45 (d, J¼16, CH); 5.61 (d, J¼5.5, HꢀC(10)); 5.55 (dd, J¼3.5, 8.0,
HꢀC(4)); 5.51–5.48 (m, HꢀC(8)); 3.87 (d, J¼5.0, HꢀC(2)); 3.68 (d, J¼5.5, HꢀC(11)); 3.14 (d, J¼4.0,
1 H, CH2(13)); 2.83 (d, J¼4.0, 1 H, CH2(13)); 2.56–2.51 (m, 1 H of CH2(3)); 2.08 (s, Me); 2.04–1.98 (m,
CH2(7)); 1.93–1.91 (m, 1 H, CH2(3)); 1.75 (s, Me(16)); 1.08 (s, Me(14)); 0.73 (s, Me(15)). 13C-NMR
(100 MHz, CDCl3): 170.90 (C¼O); 166.60 (COCH); 145.31 (CH); 139.04 (C(9)); 134.31 (Ph); 130.49
(Ph); 128.96, 128.16 (Ph); 122.81 (C(10)); 117.93 (CHCO); 79.42, 74.37, 71.07, 69.96 (C(2,11,8,4)); 65.33
(C(12)); 49.17, 47.75 (C(5,6)); 42.22 (C(3)); 36.69 (C(13)); 31.01 (C(7)); 21.10, 18.83 (Me, C(16)); 16.99
(C(15)); 5.84 (C(14)). Anal. calc. for C26H30O6: C 71.21, H 6.90; found: C 71.18, H 6.83.
(4b,8b)-4-(Acetyloxy)-12,13-epoxytrichothec-9-en-8-yl 2,2-Dimethyl-3-(2-methylprop-1-en-1-yl)cy-
clopropanecarboxylate (3g). Yield: 76.8%. Colorless oil. [a]2D8 ¼ þ3.24 (c¼5, acetone). 1H-NMR
(500 MHz, CDCl3): 5.56–5.55 (m, HꢀC(10)); 5.53 (dd, J¼4.0, 8.0, HꢀC(4)); 5.41–5.38 (m, HꢀC(8));
4.91 (d, J¼8.0, CH); 3.85 (d, J¼5.0, HꢀC(2)); 3.64 (d, J¼5.0, HꢀC(11)); 3.12 (d, J¼4.0, 1 H, CH2(13));
2.81 (d, J¼4.0, 1 H, CH2(13)); 2.54–2.49 (m, 1 H, CH2(3)); 2.07 (s, Me); 2.02–1.97 (m, 1 H of CH2(3));
1.96–1.91 (m, 1 H, CH2(7)); 1.83–1.80 (m, 1 H, CH2(7)); 1.71 (s, Me(16)); 1.70–1.76 (m, 2 Me); 1.80–
1.79 (m, CH); 1.42–1.41 (m, COCH); 1.25 (s, Me); 1.15 (s, Me); 1.03 (s, Me(14)); 0.70 (s, Me(15)). HR-
ESI-MS: 481.2562 ([MþNa]þ , C27H38NaO6þ ; calc. 481.2566).
(4b,8b)-4-(Acetyloxy)-12,13-epoxytrichothec-9-en-8-yl 3-(2,2-Dibromoethenyl)-2,2-dimethylcyclo-
propanecarboxylate (3h). Yield: 60.8%. Colorless oil. 1H-NMR (500 MHz, CDCl3): 6.79 (d, J¼8.5,
Br2CCH); 5.58–5.56 (m, HꢀC(10)); 5.53 (dd, J¼4.0, 8.0, HꢀC(4)); 5.37–5.35 (m, HꢀC(8)); 3.85 (d, J¼
5.5, HꢀC(2)); 3.64 (d, J¼5.5, HꢀC(11)); 3.14 (d, J¼4.0, 1 H, CH2(13)); 2.80 (d, J¼4.0, 1 H, CH2(13));
2.56–2.51 (m, 1 H, CH2(3)); 2.18–2.16 (m, CH); 2.08 (s, Me); 2.03–1.92 (m, 1 H, CH2(3), CH2(7));
1.87–1.80 (m, COCH); 1.72 (s, Me(16)); 1.30–1.21 (m, 2 Me); 1.03 (s, Me(14)); 0.71 (s, Me(15)). HR-
ESI-MS: 609.0464 ([MþNa]þ , C25H32Br2NaO6þ ; calc. 609.0461).
(4b,8b)-4-(Acetyloxy)-12,13-epoxytrichothec-9-en-8-yl 2,6-Dichloro-5-fluoropyridine-3-carboxylate
(3i). Yield: 78.3%. Colorless crystals. M.p. 86–888. [a]2D8 ¼ þ6.13 (c¼5, acetone). 1H-NMR
(500 MHz, CDCl3): 5.02 (d, J¼7.0, Py); 5.67–5.65 (m, HꢀC(10)); 5.64–5.62 (m, HꢀC(8)); 5.56 (dd,
J¼3.5, 8.0, HꢀC(4)); 3.87 (d, J¼5.0, HꢀC(2)); 3.70 (d, J¼5.5, HꢀC(11)); 3.13 (d, J¼4.0, 1 H, CH2(13));
2.80 (d, J¼4.0, 1 H, CH2(13)); 2.57–2.52 (m, 1 H, CH2(3)); 2.08 (s, Me); 2.05–2.00 (m, 1 H, CH2(3));

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1.99–1.95 (m, CH2(7)); 1.78 (s, Me(16)); 1.10 (s, Me(14)); 0.73 (s, Me(15)). Anal. calc. for
C23H24Cl2FNO6: C 55.21, H 4.83, N 2.80; found: C 55.27, H 4.73, N 2.82.
Synthesis of (4b)-4-Methoxy-12,13-epoxytrichothec-9-ene (2h). A soln. of MeI (0.23 g, 1.63 mmol) in
5 ml of dry DMF was added dropwise to a soln. of 2 (0.10 g, 0.40 mmol) and NaH (0.029 g, 1.21 mmol) in
DMF (10 ml) at 08, and the mixture was stirred for 6 h. The solvent was removed under reduced pressure,
and AcOEt (20 ml) was added. The AcOEt soln. was washed with H2O and brine, and dried, filtered, and
concentrated in vacuo to give a crude product, which was purified by FC (SiO2; PE/AcOEt 8 :1) to afford
0.09 g (85.2%) of 2h. Colorless oil. 1H-NMR (500 MHz, CDCl3): 5.40–5.38 (m, HꢀC(10)); 3.88 (dd, J¼
3.5, 7.0, HꢀC(4)); 3.82 (d, J¼5.0, HꢀC(2)); 3.48 (d, J¼5.5, HꢀC(11)); 3.35 (s, Me); 3.09 (d, J¼4.0, 1 H,
CH2(13)); 2.80 (d, J¼4.0, 1 H, CH2(13)); 2.39–2.35 (m, 1 H, CH2(3)); 1.99–1.97 (m, 1 H, CH2(3)); 1.96–
1.94 (m, CH2(8)); 1.94–1.93 (m, 1 H, CH2(7)); 1.71 (s, Me(16)); 1.43–1.41 (m, 1 H of CH2(7)); 0.86 (s,
Me(14)); 0.76 (s, Me(15)). 13C-NMR (100 MHz, CDCl3): 140.10 (C(9)); 119.00 (C(10)); 80.13, 78.70,
74.53 (C(2,11,4)); 68.88 (C(12)); 59.46 (Me); 55.08, 48.96, 46.79 (C(5,13,6)); 39.56 (C(3)); 28.53 (C(8));
24.74, 23.10 (C(7,16)); 16.10 (C(15)); 6.78 (C(14)). HR-ESI-MS: 287.1626 ([MþNa]þ , C16H24NaO3þ ;
calc. 287.1623).
Synthesis of (4b)-4-Ethoxy-12,13-epoxytrichothec-9-ene (2i). Compound 2i was prepared as
described for 2h to give a colorless oil (0.08 g, 75.0%). 1H-NMR (500 MHz, CDCl3): 5.39–5.38 (m,
HꢀC(10)); 3.97 (dd, J¼3.5, 7.5, HꢀC(4)); 3.79 (d, J¼5.0, HꢀC(2)); 3.61–3.56 (m, CH2O); 3.43–3.39 (m,
CH2O); 3.49 (d, J¼5.5, HꢀC(11)); 3.08 (d, J¼4.0, 1 H, CH2(13)); 2.80 (d, J¼4.0, 1 H, CH2(13)); 2.40–
2.35 (m, 1 H, CH2(3)); 1.99–1.97 (m, 1 H, CH2(3)); 1.96–1.94 (m, CH2(8)); 1.94–1.93 (m, 1 H, CH2(7));
1.70 (s, Me(16)); 1.43–1.41 (m, 1 H, CH2(7)); 1.21–1.18 (m, Me); 0.86 (s, Me(14)), 0.76 (s, Me(15)).
13C-NMR (100 MHz, CDCl3): 140.02 (C(9)); 118.51 (C(10)); 81.40, 78.70, 72.53 (C(2,11,4)); 68.50, 64.53
(C(12), MeCH2O); 54.89, 48.40, 46.10 (C(5,13,6)); 39.17 (C(3)); 27.89 (C(8)); 24.39, 22.95 (C(7,16));
16.13, 15.32 (C(15), MeCH2O); 6.90 (C(14)). HR-ESI-MS: 301.1784 ([MþNa]þ , C17H26NaO3þ ; calc.
301.1780).
Antifungal Activity. Magnaporthe grisea, Ustilaginoidea virens, and Rhizoctonia solani were routinely
maintained on potato dextrose agar (PDA) slants, and kept for stock at 48. The antifungal activities were
tested according to the method described in [35][36]. Cultures were obtained by transplanting mycelium
disks, 10 mm in diameter, from a single culture in stationary phase. These were incubated at 25ꢁ18 on
PDA (pH 5.6ꢁ0.2) on thin sterile sheets of cellophane, until the logarithmic phase of growth was
reached, and then transferred to Petri dishes containing the medium supplemented with the compound to
be tested. Each compound was dissolved in DMF, and a proper dilution was aseptically added to the
medium at 458 to obtain a final concentration of 50 mgmlꢀ1. The DMF concentration of the final soln. was
adjusted to 0.1%. Controls were set up with equivalent quantities (0.1%) of acetone. The growth rate was
determined by measuring daily colony diameter for 4 d after the transport of the fungus onto dishes
containing the substance to be tested. Four replicates were used for each concentration. Percentage
inhibition was expressed as the mean values obtained in three independent experiments. For comparative
purposes, the commercial fungicides prochloraz and propiconazole were tested under the same
conditions as the title compounds. Compounds 1, 3f, and 4, with high antifungal activities, were tested at
10, 5, 2.5, 1.25, and 0.625 mgmlꢀ1 concentration.
X-Ray Crystal Structure Analysis of 3f (Fig. 2). Crystal data were collected with a Rigaku R-AXIS-
RAPID diffractometer equipped with a graphite-monochromatized MoKa radiation (l¼0.71073 ꢄ).
Compound 3f was recrystallized from acetone to give colorless crystals, and its crystal structure was
established using X-ray crystallographic diffraction analysis as shown in Fig. 2. The crystal was
monoclinic with space group C2, and the cell parameters were a¼23.2430(13) ꢄ, b¼7.8970(7) ꢄ, c¼
14.2170(9) ꢄ, b¼99.233(2)8, V¼2575.3(3) ꢄ3, Z¼2. The final R1 ¼0.0619, wR2 ¼0.1674, S¼1.102, the
difference densities were 0.26 e ꢄꢀ3 (max.) and ꢀ0.42 e ꢄꢀ3 (min.). The CCDC No. is 883766. The H-
atoms of H2O were located from a difference Fourier map with OꢀH distance of 0.88 ꢄ, and the other
H-atoms were placed in calculated positions with CꢀH distances between 0.93 and 0.98 ꢄ according to
their own hybrid way. All the H-atoms were included in the final cycles of refinement in riding model,
with Uiso(H) of 1.2 or 1.5 Ueq of the carrier atoms. Surprisingly, there exists a positive hole that contains
H2O occupying a third of the total volume. It plays an important role in the formation of the single crystal.

610
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Fig. 2. Molecular structure and packing diagram of 3f

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
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Received December 9, 2012