Synthesis of trichodermin derivatives and their antimicrobial and cytotoxic activities

Article abstract of DOI:10.3390/molecules24203811

Trichothecene mycotoxins are recognized as highly bioactive compounds that can be used in the design of new useful bioactive molecules. In Trichoderma brevicompactum, the first specific step in trichothecene biosynthesis is carried out by a terpene cyclas

Full text of DOI:10.3390/molecules24203811

molecules
Article
Synthesis of Trichodermin Derivatives and Their
Antimicrobial and Cytotoxic Activities
Javier E. Barúa 1,2, Mercedes de la Cruz 3 , Nuria de Pedro 3, Bastien Cautain 3, Rosa Hermosa 4,
Rosa E. Cardoza 5 , Santiago Gutiérrez 5 , Enrique Monte 4, Francisca Vicente 3
and Isidro G. Collado 2,
*
1
Biological Chemistry Department, Chemistry Science Faculty, National University of Asunción, Ruta Mcal.
Estigarribia Km 11.5, San Lorenzo 2160, Paraguay; javierbarua@qui.una.py
Department of Organic Chemistry, Campus of Puerto Real, Science Faculty, University of Cádiz,
11510 Puerto Real, Cádiz, Spain
Fundación Medina, Avda. del Conocimiento 34, Armilla, 18016 Granada, Spain;
mercedes.delacruz@medinaandalucia.es (M.d.l.C.); nuri_dp@hotmail.es (N.d.P.);
bastien.cautain@medinaandalucia.es (B.C.); francisca.vicente@medinaandalucia.es (F.V.)
Department of Microbiology and Genetics, Spanish-Portuguese Institute for Agricultural Research (CIALE),
University of Salamanca, Campus of Villamayor, Rio Duero 12, 37185 Salamanca, Spain; rhp@usal.es (R.H.);
emv@usal.es (E.M.)
2
3
4
5
University of León, Campus of Ponferrada, Superior and Technical Universitary School of Agricultural
Engineers, Area of Microbiology, Avda. Astorga s/n, 24400 Ponferrada, Spain;
re.cardoza@unileon.es (R.E.C.); s.gutierrez@unileon.es (S.G.)
*
Correspondence: isidro.gonzalez@uca.es; Tel.: +34-956-012768
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 1 October 2019; Accepted: 21 October 2019; Published: 22 October 2019
Abstract: Trichothecene mycotoxins are recognized as highly bioactive compounds that can be used
in the design of new useful bioactive molecules. In Trichoderma brevicompactum, the first specific step
in trichothecene biosynthesis is carried out by a terpene cyclase, trichodiene synthase, that catalyzes
the conversion of farnesyl diphosphate to trichodiene and is encoded by the tri5 gene. Overexpression
of tri5 resulted in increased levels of trichodermin, a trichothecene-type toxin, which is a valuable
tool in preparing new molecules with a trichothecene skeleton. In this work, we developed the
hemisynthesis of trichodermin and trichodermol derivatives in order to evaluate their antimicrobial
and cytotoxic activities and to study the chemo-modulation of their bioactivity. Some derivatives with
a short chain at the C-4 position displayed selective antimicrobial activity against Candida albicans and
they showed MIC values similar to those displayed by trichodermin. It is important to highlight the
cytotoxic selectivity observed for compounds
9, 13, and 15, which presented average IC50 values of
2
µg/mL and were cytotoxic against tumorigenic cell line MCF-7 (breast carcinoma) and not against
Fa2N4 (non-tumoral immortalized human hepatocytes).
Keywords: trichothecene; sesquiterpenes; tri genes; synthesis; antimicrobial; cytotoxic bioactivity
1. Introduction
Natural products (NPs, secondary metabolites) are an invaluable source of inspiration in drug
design and development. Having evolved over several millennia to acquire specific ligand–protein
binding motifs, their structures cover a wide range of biologically relevant chemical space that cannot
be efficiently explored by synthetic compounds in commercially available screening libraries [
Of all new drugs approved between 1981 and 2014 (n = 1562), 50% are natural products, are derived
from natural products, or are synthetic molecules inspired by or that mimic natural products [ ].
Terpenoids are the largest class of NPs, with about 60% of NP diversity originating from the terpene
1,2].
3
Molecules 2019, 24, 3811; doi:10.3390/molecules24203811
www.mdpi.com/journal/molecules

Molecules 2019, 24, 3811
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pathway [
on an isopentene skeleton, and most of the chemical intermediates in their biosynthetic pathway are
known [ ]. Typical structures contain carbon skeletons represented by (C5)n and are classified as
4]. All terpenes are derived from the repetitive fusion of branched five-carbon units based
5
hemiterpenes (C5), monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), sesterterpenes (C25),
triterpenes (C30), and tetraterpenes (C40) [6].
Trichothecenes are a particular type of sesquiterpene which are a family of metabolites that have
mainly been isolated from species of Fusarium and certain other fungal genera like Cephalosporium, Isaria,
Microcyclospora, Myrothecium, Spicellum, Stachybotrys, Trichoderma, and Trichothecium. The biosynthesis
of trichothecenes is well documented and has been the focus of several detailed reviews [
trichothecenes contain a common skeleton or tricyclic ring that typically contains an epoxide between
the C-12 and C-13 carbons and a varying number of hydroxyl or acetoxy groups [ ]. This sesquiterpene
7,8]. All
9
family exhibits cytotoxic activity against different eukaryotes cell lines by induction of apoptosis [10],
and also acts as a fungicide [11], an immunosuppressant [12] and a neurotoxin [13].
The biosynthetic pathway of trichothecenes has been widely studied in Fusarium species [14
]
and has recently been described in Trichoderma species [15 16]. In both cases, the genes involved
,
are organized in clusters. The cyclization of farnesyl diphosphate to trichodiene, the first step in
the biosynthetic pathway of approximately 150 different toxic trichothecenes, is catalyzed by the
enzyme trichodiene synthase which is encoded by the tri5 gene. This tri5 gene has been isolated and
characterized in Trichoderma brevicompactum, the only Trichoderma species that is capable of producing
trichodermin (
of 1 and in the antifungal activity exhibited by T. brevicompactum [5].
Trichodermin ( ) is a member of a family of sesquiterpene metabolites that possesses an olefinic
1) [17] and overexpression of the tri5 gene has shown that it is involved in the production
1
group at positions C-9 and C-10 and an epoxide group between C-12 and C-13, on a trichothecene
skeleton, characterizing it as a 12,13-epoxytrichothecene [18]. This compound diffuses rapidly through
the cell membrane and binds to the eukaryotic ribosome to inhibit the translation of proteins by means
of interaction with the peptidyl transferase [19]. It has been shown that trichodermin (
inhibitor of protein synthesis in mammals and its inhibitory activity requires the presence of both the
C9-C10 olefinic group and the characteristic C12-C13 epoxide group [ ]. Trichodermin ( ) has also
exhibited potent antimicrobial activity against filamentous fungi and yeasts and bacteria [ ]. In these
1) is a potent
9
1
5
studies, the overexpression of the tri5 gene resulted in an increase of the level of transcription of three
other trichothecene genes, tri4, tri6, and tri10 [20].
In view of the broad biological activity shown and the pharmacological potential inherent in
the chemical structure of 1, this work deals with the synthesis of trichodermin derivatives to study
the potential chemo-modulation of bioactivity and to evaluate their antimicrobial and cytotoxic
activity against a panel of prokaryotic and eukaryotic organisms and different types of cellular lines,
respectively, through an analysis of the relationship between the chemical structure and activity of
these derivatives.
2. Results and Discussion
2.1. Synthesis of Trichodermin Derivatives
The potency and broad biological activity shown by trichodermin (
amassing adequate amounts of this molecule to complete a study of its biological activity and to perform
chemical transformations on the skeleton with a view to modulating or enhancing this biological
activity. Several efforts have been made to modulate the activity of trichothecenes as antifungal
agents [21 23] with promising results. Total synthesis of trichodermin ( ) [24] and trichodermol
) [25 26] has been proposed. However, this involves several reaction steps, making it expensive
and inefficient.
In recent years, we have worked on obtaining transformants of T. brevicompactum in order to study
the functionality of the genes involved in the biosynthesis of trichothecenes and to obtain mutants to
1) sparked our interest in
1
–
1
(2
,

Molecules 2019, 24, 3811
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efficiently produce large quantities of trichodermin (1) or trichodermol (2), the final precursor of 1 in
its biosynthetic pathway. To that end, our group has analyzed the production of trichothecenes by
transformants overexpressing the tri5 gene. We found that trichodermin ( ) production in these strains
increased dramatically compared to that of the parent strain, achieving yields up to 32.3% of dry crude
extract compared to the wild strain that reached levels of 11.3 mg/mL after 14 days of fermentation [ ].
With this knowledge, and to obtain adequate amounts of trichodermin ( ) needed for the synthesis
of its derivatives, we fermented T. brevicompactum IBT40841-derived tri5-overexpressing transformant
Tb41tri5 [ ] in PDB medium for 14 days, which produced, by extraction of the fermentation broth with
ethyl acetate, 1.4 g of crude extract. This was preliminarily analyzed by TLC and 1H-NMR. From the
chromatographic separation of the crude extract, a compound –enriched fraction was obtained, which
1
5
1
5
1
was then purified by HPLC and characterized by extensive NMR experiments to yield 815 mg of the
pure compound.
Scheme 1 shows the chemical transformations performed on 1. Modifications were made on the
olefin located on the C9–C10 carbons and on the C12–C13 epoxide. Furthermore, a group of nine
compounds with different substituents at the C4 carbon were prepared by esterifying the hydroxyl
group at this position. All the products obtained were purified by HPLC, and their structures were
elucidated by spectroscopic techniques.
Scheme 1. Chemical transformations carried out on trichodermin (
preparation of compounds 3–16.
1) and trichodermol (2) for the
Product
3 was prepared at a yield of 49% by treating 1 with a stirred aqueous solution of HCl
(10%) for 15 h. Its molecular formula was confirmed by High Resolution Mass Spectrometry (HRMS)
where an m/z ion [C17H26O5 + H]+ 311.1837 was observed. Furthermore, the opening of the epoxide at
C12-C13 of
2H corresponding to the two H-13 protons.
Products and were prepared by treating
acid (MCPBA). The reaction yielded two epoxidation products corresponding to C9-C-10-
) and the epoxide with orientation ( ), with yields of 7.2% and 82.3%, respectively. The loss of the
1 δ 3.88 ppm, which integrated for
was revealed by the signals in its 1H-NMR spectrum at
4
5
1, dissolved in chloroform, with m-chloroperbenzoic
α
-epoxide
(
4
β
5
signals corresponding to the C9-C-10 olefin of trichodermin (1
) was confirmed by means of 1H-NMR
and 13C-NMR spectroscopy. HRMS data confirmed the expected molecular formula C17H24O5 for
both products. Lastly, stereochemistry was assigned based on observed nuclear Overhauser effect

Molecules 2019, 24, 3811
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(NOE) interactions. Thus, in NOESY-1D experiments performed on
5, irradiation of H-10 produced
enhancements at H-11, H-15, and H-16, determining the
Trichodermol ( ) was obtained from the alkaline treatment of
of NaOH (2M). The reaction product was confirmed by comparing its NMR signals with those
β
orientation of the epoxide for this derivative.
2
1
by stirring in a methanolic solution
2
described in the literature [27]. The loss of the acetyl group at position C-4 left 4-OH free, an easily
accessible function for the introduction of various substituents. This product was used to prepare
derivatives 6–16.
Product
one described for 4 and 5. All its physical constants were concordant with the proposed structure.
Products 15 were prepared from the esterification of the hydroxyl group at C-4 of compound
, with the corresponding acyl chlorides in the presence of pyridine. Good yields of all the products
6 was prepared from trichodermol (2) by means of an epoxidation reaction similar to the
7–
2
were obtained, and their spectroscopic data coincided with the proposed structures. Product 16 was
obtained from a reaction of 2 with an excess of propanoyl chloride in the presence of pyridine.
The acid medium, probably due to a large excess of the reagent, led to the opening of the
12,13-epoxide of 2, simultaneously with the introduction of a propanoyl group at position C-4.
The mass spectrum showed an ion m/z 347.1830 (calculated for C18H28O5Na [M + Na]+ 347.1834),
verifying the expected molecular formula.
2.2. Evaluation of the Cytotoxic and Antimicrobial Activity of Trichodermin (1) Derivatives
Antimicrobial and cytotoxic activity tests were performed against a panel of microorganisms
and human cell lines to evaluate the biological activity of trichodermin (
1), trichodermol (2),
and derivatives 16, respectively. Table 1 summarizes the results of the antimicrobial activity
3
–
expressed as Minimum Inhibitory Concentrations (MIC). None of the compounds showed significant
activity against Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, or Bacillus subtilis.
These results are presented as Supplementary Materials (Table S2). Compounds 9–11 mildly inhibited
the growth of Escherichia coli 5746.
Table 1. Antimicrobial activity of compounds 1, 2
in µg/mL.
, 7–9, and 10–15. The results are expressed as MIC
Entry
E. coli MB5746
MRSA MB5393
C. albicans MY1055
1
2
7
9
10
11
12
13
14
15
>64
>64
>64
64
32
32
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
<4
8–16
16–32
4–8
8
>64
4
4
8–16
8
MRSA = Methicillin-resistant S. aureus. MIC = minimal inhibitory concentration.
However, as was expected [
albicans (<4 g/mL). Compounds
activity of compounds 10, and 15 can be considered interesting with values between 4–8
Special mention should be made of the activity of compounds 12 and 13 with values of 4 g/mL, the most
active of the series except for trichodermin ( ). Compounds and 12 were previously evaluated in
5
], trichodermin (
1
) exhibited high inhibition against the yeast Candida
g/mL), while the
g/mL.
µ
2,
7, and 14 exhibited moderate activity (8–32
µ
9
,
µ
µ
1
9
antifungal assays against Botrytis cinerea, Peronophythora litchii, Rhizoctonia solani, Sclerotonia sclerotiorum,
and Magnaporthe grisea [21].

Molecules 2019, 24, 3811
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In other studies, compound 10 exhibited good antifungal activity against Ustilaginoidea virens, R.
solani, and M. grisea [22].
The specificity of the activity of the compounds herein described against C. albicans should be
highlighted. They exhibited good inhibition activity against this yeast and low or no activity against
the bacterial groups. A comparison of activities and chemical structures showed that the hydroxyl
at C-4 must be esterified with a short-chain ester. However, the presence of a double bond at C-20 in
12 and 13 produces an increase in biological activity close to
1. Compounds 3–6 and 16 completely
lost the inhibitory capacity of their precursor trichodermin ( ), confirming that both the 12,13-epoxide
1
group and the C9–C10 double bond play a fundamental role in the toxicity of these compounds [28].
Table 2 summarizes the results of the cytotoxic effect observed from trichothecene derivatives on
different human cell lines. The results are presented as IC50 in
None of the compounds showed significant cytotoxic activity against the A549 (lung carcinoma) or
HT29 (colon carcinoma) lines after 24 h of incubation (Table S3). Trichodermin ( ) showed intense
µg/mL, measured after 24 h of incubation.
1
cytotoxic activity against the MCF-7 line (breast carcinoma), and moderately inhibited RCC4-VA (renal
carcinoma) and Fa2N4 (immortalized hepatocytes). Unfortunately, this compound exhibited little
specificity in terms of its cytotoxic activity, acting indistinctly against both the carcinoma lines and
the non-tumoral Fa2N4 line. It is well known that trichodermin is a very potent inhibitor of protein
synthesis [29]. However, it is important to highlight the cytotoxicity of derivatives
with approximate IC50 values of between 2–4 g/mL. This can be considered promising as they were
selective against the tumorigenic cell line MCF-7, without affecting the non-tumor line Fa2N4.
9, 10, 12, 13, and 15
µ
Table 2. Cytotoxicity of compounds
incubation. IC50 (µg/mL).
1, 2, 7, 9–15 in different human cell lines, measured at 24 h of
Entry
HepG2
MCF-7
RCC4-VA
RCC4-VHL
Fa2N4
1
2
7
9
10
11
12
13
14
15
>20
>20
>20
>20
14.20
>20
>20
17.48
>20
>20
<1.25
4.42
13.52
1.93
3.59
11.26
3.78
2.15
5.22
2.03
14.19
>20
>20
>20
>20
>20
6.02
>20
>20
17.14
>20
>20
>20
>20
>20
>20
8.00
>20
>20
>20
12.09
>20
>20
>20
>20
>20
>20
>20
>20
>20
HepG2 = liver cancer (CCL-8065); MCF-7 = breast carcinoma (HTB-22); RCC4-VA and –VHL = renal carcinoma;
Fa2N4 = immortalized human hepatocytes.
A natural product called 8-deoxytrichothecin (17) was isolated from Trichothecium roseum [30] and
Spicellum roseum [31,32] and induced the accumulation of glucosylceramide (GlcCer) and simultaneous
reduction in the formation of lactosylceramide (LacCer) in complex gangliosides in primary cultured
neurons [33]. It also significantly reduced the cell viabilities of HL-60, SMMC-7721, A549, MCF-7 and
SW480 cell lines [34]. Compound 13 is an isomer of 17 with an E configuration in the double bond
C20–C30.
These results lead to the proposal of new chemical transformations that enhance the activities of
these derivatives. Compared to antimicrobial activity results, the high toxicity of
1 against eukaryotic
cells can be confirmed, with no noticeable effect on bacteria.

Molecules 2019, 24, 3811
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Compounds
3
and 16 lost all their activity in all the cell lines tested. These derivatives lost the
12,13-epoxide group of trichodermin (
activity of these trichothecenes. Likewise,
the importance of the C9-C10 double bond in the activity of these trichothecenes. Cell line MCF-7
proved to be especially sensitive to several of the compounds tested. Trichodermol ( ) was less active
compared to trichodermin ( ) due to the loss of the acetyl group, although it still exhibited good
15, obtained by the esterification of at C4–OH, all have good IC50 values,
(4-pentanoyltrichodermol) with activity of 1.93 g/mL, the most active of this
1), confirming that the presence of this group is key to the
5
is inactive under the conditions tested, demonstrating
2
1
activity. Derivatives
especially compound
7
9
–
2
µ
series, followed by compounds 15 (2.03 µg/mL) and 13 (2.15 µg/mL).
3. Experimental Section
3.1. General
Unless otherwise noted, materials and reagents were obtained from commercial suppliers and
were used without further purification. Air- and moisture-sensitive reactions were performed under an
argon atmosphere. Optical rotations were determined on a Perkin-Elmer 341 polarimeter (Perkin-Elmer,
Seville, Spain). IR spectra were recorded on a Perkin-Elmer Spectrum BX FT-IR spectrophotometer
1
(Perkin-Elmer, Seville, Spain). H and 13C-NMR spectra were obtained on a Varian INOVA 400 MHz
NMR spectrometer (Varian, Palo Alto, CA, USA) using tetramethylsilane as an internal reference.
NMR assignments were made using a combination of 1D and 2D techniques and, where appropriate,
by comparison with assignments available in the literature for previously described compounds.
High-Resolution Mass Spectroscopy (HRMS) was performed with a QTOF mass spectrometer in
positive ion ESI or APCI (APGC+ for sample analyses using GC chromatography (Waters, Manchester,
UK). HPLC was performed with a Hitachi/Merck L-6270 device equipped with a UV-VIS detector (L
4250) and a differential refractometer detector (RI-71) (VWR, Barcelona, Spain). TLC was performed on
a Merck Kieselgel 60 F254, 0.2 mm thick, Silica gel (Merck KGaA, Darmstadt, Germany) was used for
column chromatography. Purification by high-pressure liquid chromatography (HPLC) was performed
using a Si gel column (LiChrospher Si 60, 10 µm, 1 cm wide, 25 cm long, (VWR, Barcelona, Spain).
3.2. Fungal Strain and Culture Conditions
Trichoderma brevicompactum IBT 40841 (=IBT40841) (IBT Culture Collection, Department of
Biotechnology, Technical University of Denmarck, Kongens Lyngby, Denmark) and tri5-overexpressing
transformant Tb41tri5 [5] were used throughout this study. This strain was maintained on potato
dextrose agar medium (PDA) (Difco Becton Dickinson, Sparks, MD, USA).
Six micelial plugs (6 mm each) of Tb41tri5 transformant strain were used to inoculate 0.5-L flasks
containing 250 mL of potato dextrose broth (PDB) (Difco Becton Dickinson). Each flask was cultured at
25 ◦C and 250 rpm in the dark for 14 days. Sixteen 0.5-L flasks were used for trichodermin (
1) extraction.
The yeast Candida albicans MY1055 used as the target in antimicrobial assays was maintained on
Sabouraud dextrose agar (SDA) (Difco Becton Dickinson).
3.3. Microorganism Used in the Bioassays
The following microorganisms were used in the antimicrobial bioassays: Methicillin-resistant
Staphylococcus aureus MRSA 5393, Staphylococcus aureus EPI167, Acinetobacter baumannii MB5973,
Escherichia coli MB2884 and MB5746, Pseudomonas aeruginosa MB5919, Candida albicans MY1055,
and Bacillus subtilis MB964.

Molecules 2019, 24, 3811
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3.4. Chemical Procedures
3.4.1. Trichodermin (1) Extraction
Four liters of fermentation broth were filtered through 200
µm Nylon filters and saturated with
NaCl, and the aqueous phase was extracted with ethyl acetate (EtOAc). The EtOAc extract was washed
three times with H2O and then dried over anhydrous Na2SO4. Evaporation of the solvent under
reduced pressure afforded a dense oil that was separated by means of flash chromatography on silica
gel, with a mixture of ethyl acetate/hexane (20% in EtOAc). The fractions were collected and purified
by HPLC rendering 815 mg of pure trichodermin (1
). Spectroscopic methods, specifically 1H NMR and
13C NMR, were then employed to identify the molecule. Spectroscopic data for compound
identical to those described in the literature [18].
1 were
3.4.2. Preparation of Compound 3
An aqueous solution of hydrochloric acid (HCl) (5 mL, 10% v/v) was added to trichodermin (1)
(20.0 mg, 0.068 mmol). The mixture was stirred overnight at room temperature, and when TLC analysis
indicated completion of the reaction (14 h), a diluted solution of sodium hydroxide (NaOH) (1 M,)
was added until pH = 7. The aqueous phase was extracted with ethyl acetate (3 × 20 mL), dried over
anhydrous sodium sulfate and filtered. Evaporation of the solvent gave a crude product that was
purified by silica gel column chromatography (petroleum ether/EtOAc, 80:20), to yield compound 3.
(4R,12S)-12-Hydroxy-12-(hydroxymethyl)trichothec-9-en-4-yl acetate (
3
) [21] (10.3 mg, 49%). White solid;
IR (film) υmax 3461, 2968, 1734, 1238 cm−1; 1H-NMR (CDCl3, 400 MHz)
δ
5.50 (1H, m, H-10), 5.12 (1H,
dd, J = 5.6 Hz, 10.0, H-4), 4.15 (1H, dd, J = 6.1, 11.3 Hz, H-2), 3.88 (1H, dd, J = 26.6, 7.3 Hz, H-130), 3.88
(1H, dd, J = 7.3, 1.8 Hz, H-130’), 3.75 (1H, d, J = 5.2 Hz, H-11), 2.57 (1H, dt, J = 5.8, 11.8, H-3), 2.14 (1H, t,
OH-13), 2.05 (3H, s, H-20), 1.96 (3H, m, H-3, H-8), 1.71 (1H, dd, H-70), 1.71 (3H, s, H-16), 1.27 (1H, m,
H-70’), 1.16 (3H, s, H-14), 0.76 (3H, s, H-15); 13C NMR (CDCl3, 100 MHz)
δ
170.2 (C-10), 141.0 (C-9),
117.3 (C-10), 90.4 (C-12), 77.7 (C-11), 73.0 (C-4), 64.6 (C-13), 63.1 (C-2), 58.0 (C-5), 43.6 (C-6), 38.7 (C-3),
27.8 (C-8), 25.3 (C-7), 23.3 (C-16), 21.0 (C-20), 13.6 (C-15), 10.8 (C-14); HRMS(ESI+): m/z 311.1837 [M +
H]+ (calcd for C17H27O5, 311.1858).
3.4.3. General Procedure for the Preparation of Compounds 4–5 [21]
On a stirring solution of trichodermin (1) (74 mg, 0.25 mmol) in dry chloroform (CHCl3, 6 mL),
m-chloroperbenzoic acid (mCPBA) (123 mg, 70%, 0.5 mmol) was added at room temperature, and the
reaction was monitored by TLC. After 6 h, 2 mL of a saturated solution of sodium hydrogen carbonate
(NaHCO3) was added to the reaction mixture and was then followed by the addition of sodium
sulphite (NaSO3). Distilled water (5 mL) was added, and the aqueous phase was extracted with CHCl3.
The organic layer was dried over anhydrous Na2SO4, filtered, and the solvent was evaporated under
reduced pressure. Column chromatography of the crude reaction mixture on silica gel, eluted with
a gradient mixture of petroleum ether–EtOAc, produced a mixture of compounds
4 and 5. Further
purification with normal phase HPLC with petroleum ether/EtOAc (80:20) as mobile phase yielded
pure compounds 4 and 5.
20
(4R,9S,10R)-9,10-Epoxy-12,13-epoxytrichothec-9-en-4-yl acetate (4). (5.6 mg, 7.2%) Colourless oil; [α]
D
−
12.9◦ (c 0.09, CHCl3); IR (film) υmax 2959, 1736, 1244, 1219, 1089 cm−1; 1H-NMR (CDCl3, 400 MHz)
5.55 (1H, dd, J = 3.6, 7.8 Hz, H-4), 3.84 (1H, d, J = 5.1 Hz, H-2), 3.78 (1H, br. s, H-11), 3.11 (1H, d,
δ
J = 4.0 Hz, H-13), 2.93 (1H, d, J = 1.8 Hz, H-10), 2.79 (1H, d, J = 4.0 Hz, H-13), 2.50 (1H, dd, J = 7.8,
15.5 Hz, H-3), 2.05 (3H, s, H-20), 2.00 (2H, m, H-3, H-7), 1.76 (2H, m, H-7,H-8), 1.33 (3H, s, H-16), 1.20
(1H, m, H-8), 1.05 (3H, s, H-15), 0.64 (3H, s, H-14); 13C-NMR (CDCl3, 100 MHz)
δ
170.8 (C-10), 79.2
(C-2), 74.7 (C-4), 70.2 (C-11), 65.0 (C-12), 61.5 (C-10), 57.3 (C-9), 49.5 (C-5), 47.8 (C-13), 40.3 (C-6), 36.3
(C-3), 25.8 (C-7), 24.2 (C-16), 24.2 (C-8), 21.1 (C-20), 16.2 (C-15), 5.5 (C-14); HRMS (APGC+): m/z 309.1706
[M + H]+ (calcd for C17H25O5, 309.1702);

Molecules 2019, 24, 3811
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20
(4R,9R,10S)-9,10-Epoxy-12,13-epoxytrichothec-9-en-4-yl acetate (
5
). (63.4 mg, 82.3%). Colourless oil; [
α
]
D
−
1.5◦ (c 0.65, CHCl3); IR (film) υmax 2971, 1732, 1245, 1220, 1093 cm−1; 1H-NMR (CDCl3, 400 MHz)
δ
5.55 (1H, dd, J = 3.7, 7.9 Hz, H-4), 3.89 (1H, d, J = 5.2 Hz, H-2), 3.60 (1H, dd, J = 2.2, 5.6 Hz, H-11), 3.15
(1H, d, J = 4.0 Hz, H-13), 3.05 (1H, d, J = 5.6 Hz, H-10), 2.75 (1H, d, J = 4.0 Hz, H-13), 2.48 (1H, dd,
J = 7.9, 15.4 Hz, H-3), 2.06 (3H, s, H-20), 1.98 (1H, m, H-3), 1.88 (1H, m, H-7), 1.70 (2H, m, H-7,H-8),
1.33 (3H, s, H-16), 1.09 (1H, m, H-8), 0.88 (3H, s, H-15), 0.64 (3H, s, H-14); 13C NMR (CDCl3, 100 MHz)
δ
170.9 (C-10), 78.8 (C-2), 74.8 (C-4), 70.3 (C-11), 64.9 (C-12), 57.2 (C-10), 57.2 (C-9), 48.6 (C-5), 47.7
(C-13), 40.1 (C-6), 36.4 (C-3), 26.5 (C-7), 22.4 (C-16), 21.4 (C-8), 21.1 (C-20), 16.8 (C-15), 5.7 (C-14); HRMS
(APGC+): m/z 309.1706 [M+H]+ (calcd for C17H25O5, 309.1702).
3.4.4. Preparation of Trichodermol (2)
A methanolic solution of sodium hydroxide (NaOH) (2 M, 6 mL) was added to trichodermin (1)
(50 mg, 0.171 mmol), and the mixture was stirred at room temperature. After 45 min, an aqueous
solution of hydrochloric acid (HCl) 1 N was added dropwise until reaching pH = 7. The methanol was
evaporated under reduced pressure, and the aqueous suspension was extracted with EtOAc. The organic
layer was dried over anhydrous Na2SO4, filtered, and the solvent was evaporated under reduced
pressure. Column chromatography of the crude reaction mixture on silica gel, eluted with a mixture
of petroleum ether–EtOAc (50:50), yielded white crystals (42.5 mg, 99%). Spectroscopic methods,
specifically 1H-NMR and 13C-NMR, were then employed to identify the molecule. Spectroscopic data
for the compound were identical to those described in the literature for trichodermol 2 [27].
3.4.5. Preparation of Compound 6
On a stirring solution of trichodermol (2) (20 mg, 0.08 mmol) in dry dichloromethane (CH2Cl2,
1 mL), m-chloroperbenzoic acid (mCPBA) (39.4 mg, 70%, 0.16 mmol) was added at room temperature,
and the reaction was monitored by TLC. After 6 h, 1 mL of a saturated solution of sodium hydrogen
carbonate (NaHCO3) was added to the reaction mixture, followed by sodium sulfite (NaSO3). Distilled
water (5 mL) was added, and the aqueous phase was extracted with CH2Cl2. The organic layer was
dried over anhydrous Na2SO4, filtered, and the solvent was evaporated under reduced pressure.
Column chromatography of the crude reaction mixture on silica gel, eluted with a gradient mixture of
petroleum ether–EtOAc, yielded pure compound 6.
20
(4R,9R,10S)-9,10-Epoxy-12,13-epoxytrichothec-9-en-4-ol (
6
). (63.4 mg, 82.3%). White solid; [
α
]
−
17.8◦
D
(c 0.17, CHCl3); IR (film) υmax 3508, 2964, 2935, 1086, 746 cm−1; 1H-NMR (CDCl3, 400 MHz)
δ
4.29 (1H,
dd, J = 3.2, 7.6 Hz, H-4), 3.90 (1H, d, J = 5.2 Hz, H-2), 3.51 (1H, dd, J = 2.3, 5.6 Hz, H-11), 3.13 (1H, d,
J = 3.8 Hz, H-13), 3.03 (1H, d, J = 5.6 Hz, H-10), 2.74 (1H, d, J = 3.8 Hz, H-13), 2.56 (1H, dd, J = 7.6,
15.8 Hz, H-3), 1.90 (2H, m, H-3, H-7), 1.71 (2H, m, H-7,H-8), 1.32 (3H, s, H-16), 1.12 (1H, m, H-8), 0.81
(3H, s, H-15), 0.73 (3H, s, H-14).; 13C-NMR (CDCl3, 100 MHz)
δ 78.38 (C-2), 73.98 (C-4), 70.05 (C-11),
65.17 (C-12), 57.25 (C-10), 57.18 (C-9), 48.82 (C-5), 47.42 (C-13), 39.96 (C-3), 39.49 (C-6), 26.60 (C-7), 22.43
(C-16), 21.35 (C-8), 16.62 (C-15), 6.23 (C-14); HRMS (ESI+): m/z 267.1591 [M + H]+ (calcd for C15H23O4,
267.1596).
3.4.6. General Procedure for the Preparation of Trichodermol (2) C-4 derivatives (7–15)
A solution of trichodermol (2) in dry CH2Cl2 was prepared in a Schlenk flask under an inert argon
atmosphere. Dry pyridine (d = 0.982 g/mL) was added, followed by the dropwise addition of acyl
halide. The suspension was stirred at room temperature and monitored by TLC. When the reaction
was completed, the crude mixture was supported on silica gel, and the products were separated by
column chromatography and eluted with a gradient mixture of petroleum ether–EtOAc. Each product
(7–15) was purified by HPLC. See supporting information, S2, for more details and yields.
20
(4R)-12,13-Epoxytrichothec-9-en-4-yl propionate (
7
). Colourless oil; [
α
]
D
−
7.6◦ (c 0.25, CHCl3); IR (film)
υmax 3392, 2979, 1731, 1192 cm−1; 1H-NMR (CDCl3, 400 MHz)
δ
5.56 (1H, dd, J = 3.8, 7.9 Hz, H-4), 5.39

Molecules 2019, 24, 3811
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(1H, m, H-10), 3.80 (1H, d, J = 5.3 Hz, H-2), 3.59 (1H, d, J = 5.6 Hz, H-11), 3.10 (1H, d, J = 4.1 Hz, H-13),
2.81 (1H, d, J = 4.1 Hz, H-13), 2.52 (1H, dd, J = 7.9, 15.5 Hz, H-3), 2.35 (2H, q, J = 7.6 Hz, H-20), 1.94 (4H,
m, H-3, H-7, H-8), 1.70 (3H, s, H-16), 1.39 (1H, m, H-7), 1.14 (3H, t, J = 7.6 Hz, H-30), 0.92 (3H, s, H-15),
0.69 (3H, s, H-14).; 13C-NMR (CDCl3, 100 MHz)
δ
174.3 (C-10), 140.2 (C-9), 118.6 (C-10), 79.1 (C-2), 74.8
(C-4), 70.5 (C-11), 65.5 (C-12), 48.9 (C-5), 47.8 (C-13), 40.4 (C-6), 36.7 (C-3), 27.9 (C-8), 27.7 (C-2), 24.4
(C-7), 23.2 (C-16), 15.9 (C-15), 9.1 (C-30), 5.8 (C-14); HRMS (APGC+): m/z 307.1917 [M + H]+ (calcd for
C18H27O4, 307.1909).
20
(4R)-12,13-Epoxytrichothec-9-en-4-yl butyrate (
8
). Colourless oil; [
α
]
D
−
4.6◦ (c 0.06, CHCl3); IR (film)
υmax 2961, 1729, 1176, 1078 cm−1; 1H-NMR (CDCl3, 400 MHz)
δ
5.56 (1H, dd, J = 4.0, 8.0 Hz, H-4), 5.39
(1H, m, H-10), 3.81 (1H, d, J = 5.2 Hz, H-2), 3.60 (1H, d, J = 5.6 Hz, H-11), 3.11 (1H, d, J = 4.0 Hz, H-13),
2.81 (1H, d, J = 4.0 Hz, H-13), 2.52 (1H, dd, J = 7.6, 15.2 Hz, H-3), 2.31 (2H, t, J = 7.2 Hz, H-20), 1.92
(4H, m, H-3, H-7, H-8), 1.70 (3H, s, H-16), 1.65 (2H, td, J = 7.6 Hz, H-30), 1.40 (1H, m, H-7), 0.94 (3H,
t, J = 7.2 Hz, H-40), 0.92 (3H, s, H-15), 0.69 (3H, s, H-14).; 13C-NMR (CDCl3, 100 MHz) 173.5 (C-10),
δ
140.2 (C-9), 118.6 (C-10), 79.1 (C-2), 74.8 (C-4), 70.5 (C-11), 65.5 (C-12), 48.9 (C-5), 47.8 (C-13), 40.4 (C-6),
36.7 (C-3), 36.3 (C-20), 28.0 (C-8), 24.4 (C-7), 23.2 (C-16), 18.4 (C-30), 16.0 (C-15), 13.6 (C-40), 5.8 (C-14);
HRMS (APGC+): m/z 321.2077 [M + H]+ (calcd for C19H29O4, 321.2066).
20
(4R)-12,13-Epoxytrichothec-9-en-4-yl pentanoate (
9
) [21]. Colourless oil; [
α
]
−
12.5◦ (c 0.22, CHCl3); IR
D
(film) υmax 2959, 1731, 1172, 1079 cm−1; 1H-NMR (CDCl3, 400 MHz)
δ
5.55 (1H, dd, J = 3.6, 7.8 Hz, H-4),
5.38 (1H, m, H-10), 3.80 (1H, d, J = 5.2 Hz, H-2), 3.59 (1H, d, J = 5.2 Hz, H-11), 3.10 (1H, d, J = 4.0 Hz,
H-13), 2.80 (1H, d, J = 4.0 Hz, H-13), 2.51 (1H, dd, J = 7.8, 15.4 Hz, H-3), 2.32 (2H, td, J = 7.4 Hz, H-20),
1.90 (4H, m, H-3, H-7, H-8), 1.69 (3H, s, H-16), 1.59 (2H, m, J = 7.6 Hz, H-30), 1.31 (3H, m, H-7, H-40),
0.92 (3H, s, H-15), 0.89 (3H, t, J = 7.4 Hz, H-50), 0.69 (3H, s, H-14); 13C-NMR (CDCl3, 100 MHz)
δ 173.7
(C-10), 140.1 (C-9), 118.6 (C-10), 79.1 (C-2), 74.8 (C-4), 70.4 (C-11), 65.5 (C-12), 48.9 (C-5), 47.8 (C-13), 40.4
(C-6), 36.7 (C-3), 34.1 (C-20), 27.9 (C-8), 26.9 (C-30), 24.4 (C-7), 23.2 (C-16), 22.2 (C-40), 15.9 (C-15), 13.6
(C-50), 5.8 (C-14); HRMS (APGC+): m/z 335.2237 [M + H]+ (calcd for C20H31O4, 335.2222).
20
(4R)-12,13-Epoxytrichothec-9-en-4-yl hexanoate (10). Colourless oil; [
(film) υmax 2957, 1732, 1171, 1080 cm−1; 1H-NMR (CDCl3, 400 MHz)
α
]
D
−
14.5◦ (c 0.25, CHCl3); IR
δ
5.55 (1H, dd, J = 3.6, 7.8 Hz, H-4),
5.38 (1H, m, H-10), 3.80 (1H, d, J = 5.2 Hz, H-2), 3.58 (1H, d, J = 5.5 Hz, H-11), 3.10 (1H, d, J = 4.0 Hz,
H-13), 2.80 (1H, d, J = 4.0 Hz, H-13), 2.51 (1H, dd, J = 7.8, 15.4 Hz, H-3), 2.31 (2H, td, J = 7.4 Hz, H-20),
1.92 (4H, m, H-3, H-7, H-8), 1.69 (3H, s, H-16), 1.61 (2H, m, H-30), 1.38 (1H, m, H-7), 1.28 (4H, m, H-40,
H-50), 0.92 (3H, s, H-15), 0.87 (3H, t, J = 7.2 Hz, H-60), 0.69 (3H, s, H-14); 13C-NMR (CDCl3, 100 MHz)
δ
173.7 (C-10), 140.1 (C-9), 118.5 (C-10), 79.1 (C-2), 74.8 (C-4), 70.5 (C-11), 65.5 (C-12), 48.9 (C-5), 47.8
(C-13), 40.4 (C-6), 36.6 (C-3), 34.3 (C-20), 31.2 (C-40),27.9 (C-8), 24.5 (C-30), 24.4 (C-7), 23.2 (C-16), 22.2
(C-50), 15.9 (C-15), 13.8 (C-60), 5.8 (C-14); HRMS (APGC+): m/z 349.2390 [M + H]+ (calcd for C21H33O4,
349.2379).
20
(4R)-12,13-Epoxytrichothec-9-en-4-yl heptanoate (11). Colourless oil; [
α
]
−
11.7◦ (c 0.25, CHCl3); IR
D
(film) υmax 2931, 1731, 1168, 1081 cm−1; 1H-NMR (CDCl3, 400 MHz)
δ
5.55 (1H, dd, J = 3.8, 7.9 Hz, H-4),
5.38 (1H, m, H-10), 3.80 (1H, d, J = 5.3 Hz, H-2), 3.59 (1H, d, J = 5.3 Hz, H-11), 3.10 (1H, d, J = 4.1 Hz,
H-13), 2.80 (1H, d, J = 4.1 Hz, H-13), 2.51 (1H, dd, J = 7.9, 15.5 Hz, H-3), 2.31 (2H, t, J = 7.3 Hz, H-20),
1.95 (4H, m, H-3, H-7, H-8), 1.69 (3H, s, H-16), 1.60 (2H, m, H-30), 1.39 (1H, m, H-7), 1.27 (6H, m, H-40,
H-50, H-60), 0.92 (3H, s, H-15), 0.86 (3H, m, H-70), 0.69 (3H, s, H-14).; 13C-NMR (CDCl3, 100 MHz)
δ
173.8 (C-10), 140.2 (C-9), 118.5 (C-10), 79.1 (C-2), 74.8 (C-4), 70.5 (C-11), 65.5 (C-12), 48.9 (C-5), 47.8
(C-13), 40.4 (C-6), 36.7 (C-3), 34.4 (C-20), 31.4 (C-50), 28.7 (40), 27.9 (C-8), 24.8 (C-30), 24.4 (C-7), 23.2
(C-16), 22.4 (C-60), 15.9 (C-15), 13.9 (C-70), 5.8 (C-14); HRMS (APGC+): m/z 363.2537 [M + H]+ (calcd for
C22H35O4, 363.2535).
20
(4R)-12,13-Epoxytrichothec-9-en-4-yl acrylate (12) [21]. Colourless oil; [
α
]
−
14.9◦ (c 0.13, CHCl3); IR
D
(film) υmax 2972, 1717, 1192, 1079 cm−1; 1H-NMR (CDCl3, 400 MHz)
δ
6.42 (1H, dd, J = 1.5, 17.3 Hz,
H-30), 6.14 (1H, dd, J = 10.4, 17.3 Hz, H-20), 5.83 (1H, dd, J = 1.5, 10.4 Hz, H-30), 5.63 (1H, dd, J = 3.5,

Molecules 2019, 24, 3811
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7.9 Hz, H-4), 5.40 (1H, m, H-10), 3.83 (1H, d, J = 5.3 Hz, H-2), 3.62 (1H, d, J = 5.6 Hz, H-11), 3.12 (1H, d,
J = 4.1 Hz, H-13), 2.82 (1H, d, J = 4.1 Hz, H-13), 2.56 (1H, dd, J = 7.9, 15.5 Hz, H-3), 1.98 (4H, m, H-3,
H-7, H-8), 1.71 (3H, s, H-16), 1.41 (1H, m, H-7), 0.94 (3H, s, H-15), 0.72 (3H, s, H-14).; 13C-NMR (CDCl3,
100 MHz) δ
166.0 (C-10), 140.2 (C-9), 131.0 (C-30), 128.4 (C-20), 118.6 (C-10), 79.1 (C-2), 75.3 (C-4), 70.5
(C-11), 65.5 (C-12), 49.2 (C-5), 47.8 (C-13), 40.4 (C-6), 36.6 (C-3), 28.0 (C-8), 24.4 (C-7), 23.2 (C-16), 16.0
(C-15), 5.8 (C-14); HRMS (APGC+): m/z 305.1748 [M + H]+ (calcd for C18H25O4, 305.1753).
20
(4R)-12,13-Epoxytrichothec-9-en-4-yl (E)-but-2-enoate (13) [21]. Colourless oil; [
α
δ
]
−
17.8◦ (c 0.18,
D
CHCl3); IR (film) υmax 2968, 1716, 1180, 1079 cm−1; 1H-NMR (CDCl3, 400 MHz)
6.98 (1H, dq, J = 7.0,
15.4 Hz, H-30), 5.86 (1H, dq, J = 1.8, 15.4 Hz, H-20), 5.60 (1H, dd, J = 3.6, 8 Hz, H-4), 5.39 (1H, m, H-10),
3.81 (1H, d, J = 5.2 Hz, H-2), 3.61 (1H, d, J = 5.2 Hz, H-11), 3.11 (1H, d, J = 4.0 Hz, H-13), 2.81 (1H, d,
J = 4.0 Hz, H-13), 2.54 (1H, dd, J = 7.6, 15.2 Hz, H-3), 1.97 (4H, m, H-3, H-7, H-8), 1.86 (3H, dd, J = 1.8,
7 Hz, H-40), 1.70 (3H, s, H-16), 1.40 (1H, m, H-7), 0.93 (3H, s, H-15), 0.70 (3H, s, H-14); 13C-NMR (CDCl3,
100 MHz) δ
166.2 (C-10), 145.1 (C-30), 140.2 (C-9), 122.7 (C-20), 118.6 (C-10), 79.1 (C-2), 74.7 (C-4), 70.5
(C-11), 65.5 (C-12), 49.1 (C-5), 47.8 (C-13), 40.4 (C-6), 36.7 (C-3), 28.0 (C-8), 24.4 (C-7), 23.2 (C-16), 17.9
(C-40), 16.0 (C-15), 5.8 (C-14); HRMS (APGC+): m/z 319.1912 [M + H]+ (calcd for C19H27O4, 319.1909).
20
(4R)-12,13-Epoxytrichothec-9-en-4-yl 3-methylbutanoate (14). Colourless oil; [
IR (film) υmax 2960, 1731, 1189, 1080 cm−1; 1H-NMR (CDCl3, 400 MHz)
α
]
D
−
16.8◦ (c 0.21, CHCl3);
δ
5.55 (1H, dd, J = 3.6, 7.8 Hz,
H-4), 5.39 (1H, m, H-10), 3.80 (1H, d, J = 5.2 Hz, H-2), 3.59 (1H, d, J = 5.5 Hz, H-11), 3.10 (1H, d,
J = 4.0 Hz, H-13), 2.81 (1H, d, J = 4.0 Hz, H-13), 2.53 (1H, dd, J = 7.9, 15.5 Hz, H-3), 2.21 (2H, d, J = 6.9 Hz,
H-20), 2.10 (1H, m, H-30), 1.94 (4H, m, H-3, H-7, H-8), 1.70 (3H, s, H-16), 1.40 (1H, m, H-7), 0.95 (3H, s,
H-40*), 0.94 (3H, s, H-50*), 0.92 (3H, s, H-15), 0.70 (3H, s, H-14); 13C-NMR (CDCl3, 100 MHz)
δ 173.0
(C-10), 140.1 (C-9), 118.6 (C-10), 79.1 (C-2), 74.8 (C-4), 70.5 (C-11), 65.5 (C-12), 48.8 (C-5), 47.8 (C-13), 43.4
(C-20), 40.4 (C-6), 36.8 (C-3), 27.9 (C-8), 25.5 (C-30), 24.4 (C-7), 23.2 (C-16), 22.4 (C-40*), 22.3 (C-50*), 15.9
(C-15), 5.9 (C-14); HRMS (APGC+): m/z 335.2222 [M + H]+ (calcd for C20H28O4, 335.2222).
20
(4R)-12,13-Epoxytrichothec-9-en-4-yl 2-(benzyloxy)acetate (15). Colourless oil; [
α
]
−
11.8◦ (c 0.27,
D
CHCl3); IR (film) υmax 3332, 2970, 1749, 1731, 1200, 1125, 1079 cm−1; 1H-NMR (CDCl3, 400 MHz)
δ
7.36 (5H, m, H-50, H-60, H-70, H-80, H-90), 5.67 (1H, dd, J = 3.6, 7.8 Hz, H-4), 5.40 (1H, m, H-10), 4.64
(2H, s, H-30), 4.12 (2H, s, H-20), 3.82 (1H, d, J = 5.2 Hz, H-2), 3.59 (1H, d, J = 5.6 Hz, H-11), 3.11 (1H, d,
J = 4.0 Hz, H-13), 2.80 (1H, d, J = 4.0 Hz, H-13), 2.55 (1H, dd, J = 7.8, 15.4 Hz, H-3), 1.96 (4H, m, H-3,
H-7, H-8), 1.70 (3H, s, H-16), 1.40 (1H, m, H-7), 0.94 (3H, s, H-15), 0.70 (3H, s, H-14); 13C-NMR (CDCl3,
100 MHz)
δ
170.3 (C-10), 140.2 (C-9), 137.0 (C-40), 128.4 (C-50, C-90), 128.0 (C-60, C80), 127.9 (C-70), 118.5
(C-10), 79.0 (C-2), 75.6 (C-4), 73.3 (C-30), 70.5 (C-11), 67.0 (C-20), 65.4 (C-12), 49.1 (C-5), 47.8 (C-13), 40.4
(C-6), 36.5 (C-3), 27.9 (C-8), 24.4 (C-7), 23.2 (C-16), 16.0 (C-15), 5.8 (C-14); HRMS (APGC+): m/z 399.2174
[M + H]+ (calcd for C24H31O5, 399.2171).
3.4.7. Preparation of Compound 16
A solution of trichodermol (
2) (10 mg, 0.04 mmol) in dry CH2Cl2 (3 mL) was prepared in a Schlenk
flask. Dry pyridine (100 L, d = 0.982 g/mL) was added, followed by propanoyl chloride (100
µ
µL, 98%,
d = 1.059, 1.1 mmol). The suspension was stirred at room temperature overnight. The crude mixture
was supported on silica gel, and the product was separated by column chromatography and eluted
with a gradient mixture of petroleum ether–EtOAc. Further purification with normal phase HPLC
with petroleum ether/EtOAc (80:20) as the mobile phase yielded pure compound 16.
(4R,12S)-12-Hydroxy-12-hydroxymethyltrichothec-9-en-4-yl propionate (16) Colourless oil; [
0.33, CHCl3); IR (film) υmax 3479, 2969, 1732, 1183 cm−1; 1H-NMR (CDCl3, 400 MHz)
α]
20 + 5.9◦ (c
D
δ
5.50 (1H, m,
H-10), 5.13 (1H, dd, J = 4.0, 8.0 Hz, H-4), 4.15 (1H, dd, J = 6.0, 11.2 Hz, H-2), 3.87 (2H, m, H-13), 3.75
(1H, d, J = 5.2 Hz, H-11), 2.57 (1H, dt, J = 5.9, 11.9 Hz, H-3), 2.33 (2H, q, J = 10.4 Hz, H-20), 2.14 (1H, t,
OH-13), 1.93 (3H, m, H-3, H-8), 1.71 (1H, m, H-7), 1.71 (3H, s, H-16), 1.27 (1H, m, H-7), 1.16 (3H, s,
H-14), 1.14 (3H, t, J = 7.6 Hz, H-30), 0.75 (3H, s, H-15); 13C-NMR (CDCl3, 100 MHz)
δ
173.5 (C-10), 141.0

Molecules 2019, 24, 3811
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(C-9), 117.3 (C-10), 90.5 (C-12), 77.7 (C-11), 72.8 (C-4), 64.6 (C-13), 63.2 (C-2), 58.1 (C-5), 43.6 (C-6), 38.7
(C-3), 27.8 (C-8), 27.8 (C-20), 25.3 (C-7), 23.3 (C-16), 13.7 (C-15), 10.8 (C-14), 9.1 (C-30); HRMS (ESI+): m/z
347.1830 [M + Na]+ (calcd for C18H28O5Na, 347.1834).
3.5. Evaluation of Antimicrobial Activity
3.5.1. Antifungal Assay
Sabouraud Dextrose Agar (SDA) plates were inoculated with C. albicans MY1055 and incubated
for 24 h at 35 ◦C. The raised colonies were collected from the SDA plates and resuspended in modified
RPMI-1640 medium, which was prepared with 20.8 g of RPMI medium (Sigma-Aldrich, Madrid, Spain),
13.4 g of YNB (Yeast Nitrogen Base), 80 mL of 1M HEPES, and 72 mL of 50% glucose. The volume
was adjusted to 2 L with MilliQ water and filtered. The OD at 660 nm of the colony suspension was
adjusted to 0.25 using RPMI-1640 modified medium as diluent and blank. This inoculum was then
again diluted to 1:10 and kept on ice until it was used to inoculate the 96-well microtiter plates. For
the assay, 90
µL/well of diluted 1:10 inoculum was mixed with 1.6
µL/well of compound solution
in DMSO and 8.4
µL/well of modified RPMI-1640 medium. Amphotericin B and penicillin G were
used as positive and negative internal controls, respectively. After distribution to the test plates of
the inoculum, samples, and controls, the absorbance at T0 (zero time) was measured at a 612 nm DO
with a Tecan Ultraevolution spectrophotometer. The plates were then incubated at 37 ◦C for 20 h.
Following this incubation period, the plates were shaken on a Micromix-5 shaker (Marshall Scientific,
Hampton, NH, USA), and the absorbance at Tf (final time) was again measured at the same OD as
initially. Percent growth inhibition (PGI) was then calculated.
Compounds
10 concentrations starting at 64
or antifungal compound that inhibited
Data were analyzed using the Screener Genedata program (Genedata AG, Basel, Switzerland). In all
the experiments performed in this work, the RZ ‘factor obtained was between 0.85 and 0.95.
1
–
16 were serially diluted in DMSO with a dilution factor of 2 to provide
g/mL. MIC was defined as the lowest concentration of an antimicrobial
95% growth of a microorganism after overnight incubation.
µ
≥
3.5.2. Antibacterial Test
The bacterial strains (Methicillin-resistant Staphylococcus aureus MRSA MB5393, Acinetobacter
baumannii MB5973, Escherichia coli MB2884 and MB5746, and Pseudomonas aeruginosa MB5919) were
re-plated on Luria-Bertani agar plates (LBA, 40 g/L) and incubated at 37 ◦C overnight to obtain isolated
colonies. The individual colonies of each microorganism were inoculated with 25 mL of Luria-Bertani
medium (LB, 25 g/L) in 250 mL Erlenmeyer flasks and incubated overnight at 37 ◦C with shaking
at 220 rpm. The bacterial inocula were then diluted to obtain the corresponding equivalents for
the assay of approximately 1.1
and P. aeruginosa). For the assay, 90
each compound dissolved in DMSO and 8.4
×
106 CFU/mL (MRSA) or 5–6
L/well of diluted inoculum were mixed with 1.6
L/well of LB medium. Kanamycin and amphotericin
×
105 CFU/mL (A. baumannii, E. coli
µ
µL/well of
µ
B (MRSA), rifampicin and amphotericin B (A. baumannii), novobiocin and amphotericin B (E. coli),
and ciprofloxacin and amphotericin B (P. aeruginosa) were included as positive and negative internal
controls respectively on each plate. The absorbance at T0 (zero time) was measured at 612 nm with a
Tecan Ultra Evolution spectrophotometer and immediately thereafter, the plates were incubated at 37
◦C for 20 h. After this period, the assay plates were shaken using a Micromix-5 agitator and once again
the absorbance at Tf (final time) was measured at an OD of 612 nm. Percent growth inhibition was
calculated and the MIC was defined in the same way as described for C. albicans. The data were also
analyzed using the Screener Genedata program (Genedata AG, Switzerland). In all the experiments
performed in this work, the RZ ‘factor obtained was between 0.85 and 0.95. Assays on agar plates were
conducted to determine antimicrobial susceptibility against vancomycin resistant B. subtilis MB964
and
β
-lactam antibiotics. B. subtilis MB964 assay plates were prepared by adding 1 mL (1.5
×
108
CFU/mL) of a suspension of B. subtilis spores directly to 1 L of yeast extract and nutrient agar medium

Molecules 2019, 24, 3811
12 of 14
(NAYE, nutrient agar 23 g/L, yeast extract 2 g/L) and pouring 30 mL of this mixture into each test plate.
The compounds were serially diluted in 20% DMSO with a dilution factor of 2 to provide 10 compound
concentrations starting at 64 µg/mL. Ten microliters of each compound concentration were added to
the agar plate containing the pathogenic microorganism. Activity was determined by measuring the
size of the inhibition halos.
3.5.3. MTT Cytotoxicity Assay
The MTT tetrazole reduction rate is an indicator of the functional integrity of the mitochondria and,
hence, of cellular viability. Day 1: Cells were seeded at their corresponding concentrations in 96-well
plates and allowed to incubate for 24 h. Day 2: The media was stripped from the plates, and then the
1
previously prepared compounds were added in 100% DMSO, serial dilutions. Compounds were
2
added in a 1:200 dilution (2
µL of compound/600
µL of medium) using a BiomekFX
™ multi-well
automatic plate dispenser (Beckman Coulter). Of this mixture, 200
µL were pipetted to three-cell plates,
resulting in each compound tested in triplicate with a constant DMSO percentage of 10.5%. We used
MMS (2 mM) as a positive control and DMSO as negative control at the same concentration at which
the compounds and controls had been dissolved. Day 3: Compounds were incubated with the cells for
24 h. The MTT was then added to a final concentration of 0.5 mg/mL and allowed to incubate with
the cells for 3 h. The chromogenic formazan concentration generated by spectrophotometry at 570
nm was then determined in a Victor2
™ multi-well plate reader (PerkinElmer). Data were analyzed
using the ScreenerTM (Genedata) program for the calculation of IC50 (dose causing 50% cell death),
taking as treatment 100% cell death (positive control) with 1 mM MMS, and as 0% cell death (negative
control) 0.5% DMSO. The percentage of cell death for each concentration was calculated. Then, using
the Levenberg-Marquardt algorithm to adjust the dose-response curve, the IC50 of each compound
was calculated by interpolating the curve thus obtained.
4. Conclusions
The results presented in this paper point to trichodermin (
a model in the development of new drugs. Our results show that, through simple modifications on
the trichodermin skeleton ( ), it is possible to modulate bioactivity and obtain derivatives that are of
1) as a molecule with great potential as
1
great interest in the research and development of new molecules with potential use as antimicrobial
agents and/or cytotoxic cancer treatment drugs. The specificity observed in the biological activity of
these trichothecenes should spark further study of their mechanisms of action through the use of other
study models.
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/24/20/3811/s1:
Part A: General conditions for the preparation of compounds 7–15. Part B: Antimicrobial and cytotoxic activities
of compounds 1–16. 1H and 13C-NMR spectra for compounds 3–16.
Author Contributions: I.G.C. and F.V. conceived and directed this project. E.M. and S.G. designed and directed
the molecular experiments of overexpression of the tri 5 gene. J.E.B. synthetized the trichodermin derivatives and
wrote the paper. M.D.L.C., N.D.P., and B.C. performed the cytotoxic and antimicrobial experiments. R.H. and
R.E.C. performed the preparation of the transformants of T. brevicompactum.
Funding: The MEDINA authors disclosed the receipt of financial support from Fundación MEDINA,
a public-private partnership of Merck Sharp & Dohme de España S.A./Universidad de Granada/Junta de
Andalucía.
Acknowledgments: This research was supported by grants from the Spanish Government MINECO-FEDER
(RTI2018-097356-B-C21) and the Regional Government of Castile and Leon (SA270P18). J.E.B. is grateful to
ITAIPU Binacional (Paraguay) and CONACYT-Paraguay for his research fellowship. Use of NMR and mass
spectrometry (QTOF) facilities at Servicio Centralizado de Ciencia y Tecnología (SCCYT) of the University of
Cádiz is acknowledged.
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2019, 24, 3811
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).