Antifungal Activity of Griseofulvin Derivatives against Phytopathogenic Fungi in Vitro and in Vivo and Three-Dimensional Quantitative Structure-Activity Relationship Analysis

Article abstract of DOI:10.1021/acs.jafc.9b00606

With environmental pollution, residual hazards accumulate and severe drug resistance and many other problems appear; some highly toxic drugs have been banned, and antifungal agents are far from satisfactory. Natural products play an important role in the discovery and development of new pesticides. The natural product griseofulvin (1) has been known as an antifungal agent in the treatment of dermatomycoses for decades. In this study, a series of new griseofulvin derivatives were synthesized with good yields. Their structures were characterized by ¹H and ¹³C nuclear magnetic resonance and high-resolution mass spectrometry (electrospray ionization). The antifungal activities of griseofulvin analogues were first evaluated against five phytopathogenic fungi (Cytospora sp., Colletotrichum gloeosporioides, Botrytis cinerea, Alternaria solani, and Fusarium solani) in vitro. Of significance is that most of them showed excellent antifungal activities against C. gloeosporioides. The antifungal activities of the four best compounds (6a, 6c, 6e, and 6f) against C. gloeosporioides were further investigated in vivo using infected apples. The results suggested that compounds 6c, 6e, and 6f [half-maximal inhibitory concentration (IC₅₀) = 47.25 ± 1.46, 49.44 ± 1.50, and 53.63 ± 1.74 μg/mL, respectively] were better than thiophanate-methyl (IC₅₀ = 69.66 ± 6.07 μg/mL). Furthermore, comparative molecular field analysis was performed on the basis of the antifungal activity results of all 22 of the compounds against C. gloeosporioides in vitro. The three-dimensional coefficient contour plots revealed that the suitable bulky and electronegative acyl-substituted groups seem to be more favorable for increasing activity at the 4′ position of griseofulvin. The structure-activity relationships were also discussed. Griseofulvin derivatives can be used for the development of highly effective and safe agricultural fungicides.

Full text of DOI:10.1021/acs.jafc.9b00606

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Agricultural and Environmental Chemistry
Antifungal Activity of Griseofulvin Derivatives against
Phytopathogenic Fungi In Vitro, In Vivo, and 3D-QSAR Analysis
Yu-Bin Bai, Yu-Qi Gao, Xiao-Di Nie, Thi Mai Luong Tuong, Ding Li, and Jin-Ming Gao
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00606 • Publication Date (Web): 14 May 2019
Downloaded from http://pubs.acs.org on May 15, 2019
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Antifungal Activity of Griseofulvin Derivatives against Phytopathogenic Fungi In
Vitro, In Vivo, and 3D-QSAR Analysis
Yu-Bin Bai†‡∥, Yu-Qi Gao†∥, Xiao-Di Nie†, Thi-Mai-Luong Tuong†, Ding Li*†,
and Jin-Ming Gao*†
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†Shaanxi Key Laboratory of Natural Products & Chemical Biology, College of Chemistry &
Pharmacy, Northwest A&F University, Yangling 712100, China
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‡Shaanxi Key Laboratory of Chemical Reaction Engineering, College of Chemistry and
Chemical Engineering, Yan’an University, Yan’an 716000, China
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*Corresponding author:
Fax/Tel: +86-29-87092335.
E-mail: jinminggao@nwsuaf.edu.cn (J. M. Gao); jbolid@nwsuaf.edu.cn (D. Li)
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ABSTRACT
With environmental pollution, residual hazards accumulate, and severe drug resistance and
many other problems appear; some highly toxic drugs have been banned, and antifungal agents
are far from satisfactory. Natural products play an important role in the discovery and
development of new pesticides. The natural product griseofulvin (1) is known as an antifungal
agent in the treatment of dermatomycoses for decades. In this study, a series of new griseofulvin
derivatives were synthesized with good yields. Their structures were characterized by 1H and 13C
NMR and HR-MS (ESI). The antifungal activities of griseofulvin analogues were first evaluated
against five phytopathogenic fungi (Cytospora sp., Colletotrichum gloeosporioides, Botrytis
cinerea, Alternaria solani, and Fusarium solani) in vitro. Of significance is that most of them
showed excellent antifungal activities against C. gloeosporioides. The antifungal activities of
four best compounds (6a, 6c, 6e, and 6f) against Colletotrichum gloeosporioides were further
investigated in vivo by using infected apples. The results suggested that compounds 6c, 6e, and
6f (IC50 = 47.25±1.46 μg/mL, IC50 = 49.44±1.50 μg/mL, and IC50 = 53.63±1.74 μg/mL,
respectively) were better than thiophanate-methyl (IC50 = 69.66±6.07 μg/mL). Furthermore,
comparative molecular field analysis (CoMFA) was performed on the basis of the antifungal
activity results of all 22 of the compounds against C. gloeosporioides in vitro. The 3D coefficient
contour plots revealed that the suitable bulky and electronegative acyl substituted groups seem to
be more favorable for increasing activity at the 4 position of griseofulvin. The structure–activity
relationships were also discussed. Griseofulvin derivatives can be used for the development of
highly effective and safe agricultural fungicides.
_____________________________________________________________________________
KEY WORDS: Griseofulvin derivatives; antifungal activity; phytopathogenic fungi;
structure-activity relationships
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INTRODUCTION
It is well known that plant pathogenic fungi can cause a tremendous loss of global
agricultural production. Despite that synthetic fungicides are effective and play an indispensable
role against pathogenic fungi, the available antifungal agents are far from satisfactory due to
several drawbacks, such as severe drug resistance, drug-related toxicity, and environmental
hazards[1-3] Therefore, novel antifungal agents are needed to effectively control the fungal
diseases of agricultural crops.
Griseofulvin (1, Scheme 1), a spirocyclic benzofuran-3-one natural antifungal product, was
initially isolated from Penicillium griseofulvum in 1939 by Oxford et al.[4]. It was the first oral
antifungal drug in the treatment of dermatomycoses, such as tinea capitis (ringworm of the scalp)
and tinea pedis (athlete’s foot), in animals and humans for decades.[5-8] Griseofulvin has gained a
lot of attention in research and academic circles[9-10]; more than 400 griseofulvin analogues have
been synthesized and used for drug screening.[10-12] In addition to the antifungal effect,
griseofulvin exhibits various other biological activities, including anticancer and antiviral
properties. Moreover, it is also useful against plant pathogenic fungi.[11-13] Zhao et al.[14] reported
that griseofulvin displayed clear inhibition of the growth of 8 different plant pathogenic fungi. In
our previously reported study, griseofulvin, as an isolated natural product, showed a significant
inhibitory activity against the pathogenic fungus Alternaria solani. [15]
In this work, a series of novel griseofulvin derivatives were designed and synthesized from
commercially available griseofulvin via efficient methods (Scheme 2). The antifungal activities
of newly synthesized compounds were investigated for some important agricultural fungi
diseases, including Cytospora sp., Colletotrichum gloeosporioides, Botrytis cinerea, Alternaria
solani, and Fusarium solani in vitro. Active compounds with a high inhibitory effect against
Colletotrichum gloeosporioides were tested in vivo. The structure-antifungal activity
relationships of all of the griseofulvin analogues were also discussed based on 3D-QSAR
CoMFA analysis.
MATERIALS AND METHODS
General Experimental Procedures. The melting points of the products were determined on an
M420 apparatus (Jinan hanon Instruments Co., Ltd., China) and are uncorrected. Nuclear
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magnetic resonance (NMR) spectra were recorded on a Bruker Vance III spectrometer (Unity
plus 500 MHz, Bruker Bios pin, Rheinstetten, Germany) and DD2 400-MR (Agilent NMR
Systems 400 MHz Spectrometer, America) with TMS as the internal standard. Thin-layer
chromatography (TLC) was performed on silica gel 60 F254 (Qingdao Marine Chemical Ltd.,
China), and column chromatography (CC) was performed on silica gel (200-300 mesh, Qingdao
Marine Chemical Ltd.). The high resolution electrospray ionization mass spectrometry
(HRESIMS) data were recorded on a Bruker maXis ESI-Q-TOF (Bruker Daltonics Inc.,
America).
Reagents. Chemicals and reagents are commercially available. Griseofulvin was purchased from
a commercial source (Shanghai Aladdin Bio-Chem Technology Co., Ltd.).
Synthetic Procedures.
General Synthetic Procedure for the Key Intermediates.
The intermediate 2 ((2S,6'R)-7-chloro-4-hydroxy-2',6-dimethoxy-6'-methyl-2-spiro[benzofuran-
2,1'-cyclohex[2]ene]-3,4'-dione) was synthesized from griseofulvin by a reported procedure.[8, 16]
Iodine pills (1.83 g, 7.2 mmol) were added to a solution of magnesium turnings (0.52 g, 21.6
mmol) in anhydrous Et2O (4 mL) and toluene (8 mL). This solution was refluxed at 80°C for
about 30 min. until the reaction mixture turned to be a colorless solution under argon. The
resulting solution was added to a solution of griseofulvin (1.41 g, 4 mmol) in dry toluene (10
mL) and heated to 80°C for 4 h. Then, H2O (20 mL) and Na2S2O3 were added and the reaction
mixture was poured into 5% hydrochloric acid (10 mL). The mixture was extracted with EtOAc
(3 × 20 mL), dried under MgSO4 and concentrated.[8, 16] The residue was purified by column
chromatography (CH2Cl2: MeOH: AcOH 97:2:1) to yield the title compound as a white solid
(1.12 g, 81%). m.p. 130.7 – 133.2°C; [α]19.6 D = + 397.2 (c = 0.11 in CH3OH); 1H NMR (500
MHz, CDCl3) δ 6.18 (s, 1H, H-5), 5.56 (s, 1H, H-3), 3.97 (s, 3H, H-10), 3.65 (s, 3H, H-11), 2.95
(dd, J = 16.2, 13.2 Hz, 1H, H-5), 2.87 (dtd, J = 10.8, 6.4, 4.1 Hz, 1H, H-6), 2.48 (dd, J = 16.2,
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4.1 Hz, 1H, H-5), 0.97 (d, J = 6.5 Hz, 3H, H-8); C NMR (125 MHz, CDCl3) δ 196.8 (C-4),
196.1 (C-3), 170.3 (C-2), 167.4 (C-7a), 165.7 (C-6), 156.1 (C-4), 105.1 (C-3), 104.1 (C-3a),
96.7 (C-7), 93.9 (C-5), 91.5 (C-2), 57.4 (C-10), 57.0 (C-11), 40.2 (C-5), 36.4 (C-6), 14.4 (C-8).
MS (ESI): m/z for C16H14ClO6: 337.05; found: 337.45 [M-H]-.
General synthetic procedure for intermediate 4 by the conventional method[8].
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Griseofulvin (3.52 g, 10 mmol) was dissolved in a mixture of ethanol (200 mL) and water (60
mL). Then, hydroxyl ammonium hydrochloride (2.26 g, 35 mmol) and sodium acetate trihydrate
(5.88 g, 43 mmol) were added to this solution. The mixture was refluxed for 6 h and then cooled
to room temperature, followed by the addition of cold water. The mixture was extracted with
Et2O (3 × 100 mL), dried (MgSO4), and concentrated[18-20]. The residue was purified by column
chromatography on silica gel, eluting with PE-actone (2:1) to yield the final compound as a light
yellow solid 3.42 g, yield 93%. m.p. 228.1 – 230.4°C; [α]19.4 D= +379.7 (c = 0.19 in CH3OH);
MS (ESI): m/z calcd. for C17H18ClNO6H: 368.09; found: 368.10 [M+H]+.
General synthetic procedure for compound 5 by the same method with intermediate 4.
(2S,6'R)-7-chloro-2',4,6-trimethoxy-4'-(methoxyimino)-6'-methyl-2-spiro[benzofuran-2,1'-cyclo
hex[2]ene]-3-one (5). Yield, 92%; white solid; m.p. 188.6 – 189.5°C; [α]19.6 D= +388.8 (c 0.15
in CH3OH). The product was a mixture of cis and trans isomers with a molar ratio about 5a: 5b =
1
0.8:1. 5a H NMR (500 MHz, CDCl3) δ 6.15 (s, 0.8H, H-5), 6.09 (s, 0.8H, H-3′), 4.00 (s, 3H,
H-10), 3.95 (s, 2.4H, H-9), 3.87 (s, 2.4H, -NOCH3), 3.57 (s, 2.4H, H-11), 2.99 – 2.93 (m, 0.8H,
H-5′), 2.54 (m, 0.8H, H-6′), 2.37 (dd, J = 15.1, 4.2 Hz, 0.8H, H-5′), 0.93 (d, J = 6.8, 2.4H, H-8).
13C NMR (125 MHz, CDCl3) δ 194.2 (C-3), 169.6 (C-2′), 164.4 (C-7a), 161.1 (C-4′), 157.6 (C-6),
151.1 (C-4), 105.8 (C-3a), 97.3 (C-7), 93.3 (C-3′), 91.6 (C-2), 89.3 (C-5), 61.5 (-NOCH3), 57.0
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(C-10), 56.4 (C-9), 56.2 (C-11), 36.6 (C-5′), 26.2 (C-6′), 14.4 (C-8). 5b H NMR (500 MHz,
CDCl3) δ 6.09 (s, 1H, H-5), 5.59 (s, 1H, H-3′), 4.00 (s, 3H, H-10), 3.95 (s, 3H, H-9), 3.88 (s, 3H,
-NOCH3), 3.53 (s, 3H, H-11), 3.01 (dd, J = 16.8, 4.9 Hz, 1H, H-5′), 2.68 – 2.60 (m, 1H, H-6′),
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2.54 (m, 1H, H-5′), 0.91 (d, J = 6.7 Hz, 3H, H-8). C NMR (125 MHz, CDCl3) δ 194.3 (C-3),
169.6 (C-2′), 164.4 (C-7a), 158.5 (C-4′), 157.6 (C-6), 154.4 (C-4), 105.8 (C-3a), 99.3 (C-3′), 97.3
(C-7), 91.5 (C-2), 89.3 (C-5), 61.8 (-NOCH3), 57.0 (C-10), 56.4 (C-9), 56.0 (C-11), 35.5 (C-5′),
31.2 (C-6′), 14.5 (C-8). HR-MS (ESI): m/z calcd. for C18H20ClNO6Na: 404.0877; found:
404.0873 [M+Na]+.
The general procedure of synthesizing compounds 3a-3g was a reported method[17], and
general synthetic procedures of compounds 6a-6k, 7 were based on a conventional method;
please see these details in supporting information.
Antifungal Activity Assay in Vitro
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The antifungal activities of griseofulvin and its derivatives were evaluated against five plant
phytopathogenic fungi: Cytospora sp, Colletotrichum gloeosporioides, Botrytis cinerea,
Alternaria solani, and Fusarium solani by the mycelial growth inhibitory rate method according
to previously reported approaches.[2] PDA medium was prepared and sterilized. The compounds
were dissolved in acetone before mixing with molten agar at 40°C; the concentration of the
compounds in the medium was fixed at 100 μg/mL. The mixture was then poured into sterilized
Petri dishes. After cooling, a mycelial disk of approximately 4 mm diameter from the culture
medium of test fungi was picked up with a sterilized inoculation needle and inoculated in the
center of the fresh PDA Petri dishes. The Petri dishes were incubated at 28°C for 3–4 d. Acetone
was used as the negative control, while the hymexazol (Hy) and thiophanate-methyl (Tpm), both
commercially available agricultural fungicides for direct application, were used as the positive
controls. Each sample was measured in triplicate, and each colony diameter of all triplicates was
measured 4 times by the cross bracketing method. After the mycelia grew completely, the
diameters of the mycelia were measured and the inhibition rate was calculated according to the
formula:
Inhibition rate (%) = (C－T)/ (C－4 mm) × 100%,
where C represents the diameter of fungal growth on untreated PDA, and T represents the
diameter of fungal growth on treated PDA.
The compounds with high activity against the target fungi were prepared in 7 fresh PDA Petri
dishes with different concentrations (100, 50, 25, 12.5, 6.25, 3.125, and 1.5625 μg/mL), and their
precise antifungal effects (IC50 value) were further investigated.
Antifungal Activity Assay in Vivo
According to the results of in vitro antifungal activity, the 4 best activity compounds 6a, 6c,
6e, and 6f against Colletotrichum gloeosporioides were further tested in vivo. The same kind of
ripened apples with similar shapes were picked in the same orchard for testing. The apples were
soaked in 1% sodium hypochlorite for 2 minutes to disinfect them before they were well rinsed
under running water for 5 minutes. Then, a hole (3 mm × 3 mm) was punched on the apple
surface. After the drying of holes, a dose of 20 μL solution (each compound was dissolved in
acetone and five concentration gradients 100, 50, 25, 12.5, and 6.25 ug/mL were set) was added
to separate samples. As the acetone solution of the compound dried, a 20-μL spore suspension
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solution (CFU = 5 × 104 /mL) was added to each specimen. Upon drying, all of the apples
were placed in green room at 28°C, and moisture was retained for 7–8 d. A spore suspension
solution served as the blank while sterile water was a negative control. The commercially
available agricultural fungicide thiophanate-methyl (Tpm) was used as a positive control. Each
sample was measured in triplicate. The same method was followed for the preparation of
solutions of different concentrations, and the results were analyzed in vitro.
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3D-QSAR Analysis
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Data Sets for 3D-QSAR Analysis. All of the molecular modeling and calculations were
performed using SYBYL-X1.3 software (Tripos, Inc.) in a CCNUGrid-based computational
environment[21]. The results of biological activities of all 22 griseofulvin analogues were used to
derive the CoMFA analyses model listed in Table 4. The structure of griseofulvin was used as a
template to construct the other molecular structures by using the “SKETCH” option function in
SYBYL. The Gasteiger-Hückel charge was used to calculate the partial atomic charges. Each
structure was fully geometrically-optimized using a conjugate gradient procedure based on the
TRIPOS force field with the Powell conjugate gradient minimization algorithm and a
convergence criterion of 0.05 kcal/mol·Å. These compounds share a common skeleton; the
cyclization atoms marked with an asterisk (Scheme 1) were used as the reference atoms, and GF
was chosen as template molecule to fit the remaining compounds by using the Database Align
function in SYBYL.
CoMFA Descriptors. The CoMFA modeling was done by following a previously described
procedure[22, 23]. The CoMFA steric and electrostatic interaction fields were calculated on a 3D
cubic lattice with a regularly spaced grid of 2.0 Å. The grid pattern was generated automatically
by the SYBYL/CoMFA routine, and an sp3 carbon atom probe with a van der Waals radius of
1.52 Å and a charge of +1.0 was used to calculate the steric (Lennard-Jones 6–12 potential) field
energies and electrostatic (Coulombic potential) fields with a distance-dependent dielectric at
each lattice point. Values of the steric and electrostatic fields were truncated at 30.0 kcal/mol.
The CoMFA steric and electrostatic fields generated were scaled by the CoMFA-STD method in
SYBYL.
A partial least-squares (PLS) approach was used to derive the 3D-QSAR, in which the
CoMFA descriptors were used as independent variables, and the IC50 values were used as
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dependent variables. The cross-validation was carried out with the leave-one-out (LOO) option
and the SAMPLS program, rather than column filtering, to obtain the optimal number of
components to be used in the final analysis. After the determination of the optimal number of
components, a non-cross-validated analysis was performed without column filtering. The
modeling capability (goodness of fit) was judged by the correlation coefficient square r2, and the
prediction capability (goodness of prediction) was implied by the cross-validated r2 (q2).
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RESULTS AND DISCUSSION
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Chemistry
The synthesis procedures of the compounds are outlined in Scheme 2. Intermediate 2 was
obtained by MgI2, which can selectively remove the methyl of griseofulvin at position 4 with a
high yield (81%). The corresponding compounds 3a~3g were synthesized with various acyl
chlorides and sodium hydride in dry acetone with yields of 71% ~ 81%. The intermediate 4 and
derivative 5 were synthesized by oximation with the addition of hydroxylamine hydrochloride
and methoxyamine hydrochloride with high yields of 93% and 92%, respectively. Furthermore,
the analogues 6a~6k were obtained by esterification through the use of triethylamine and
different acetyl chlorides in dry acetone with good yields (74%~95%). Compound 7 was
obtained (with a good yield, 90%) when intermediate 4 and metallic sodium were refluxed in
ethyl alcohol. It should be noticed that the methods to synthesize these series of compounds are
simple and effective.
All of these compounds, 2 intermediates, and the newly prepared 19 derivatives were
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confirmed by H NMR, C NMR, and MS, as well as HRESIMS spectral data. Out of the 22
analogues, 17 compounds (3b-3g, 5, 6a-6c, 6e-6k, 7) are new based on searching in SciFinder
web. The antifungal activities of all of the synthesized analogues, including known compounds,
are also reported here for the first time.
Evaluation of Inhibitory Efficacy In Vitro
The preliminary inhibition rates of all of the title compounds,[24] including griseofulvin and
intermediates 2 and 4, and two commercial agricultural fungicides, against five plant-pathogenic
fungi (Cytospora sp., Colletotrichum gloeosporioides, Botrytis cinerea, Alternaria solani, and
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Fusarium solani) are shown in supporting information. The results suggested that some
synthesized compounds have significant activities with a 65% higher inhibition rate against five
plant pathogenic fungi at a concentration of 100 μg/mL. Furthermore, in order to evaluate their
antifungal effects precisely, their IC50 (the half-maximal inhibitory concentration) values were
determined by the method presented in the antifungal activity assay part, and the data are listed
in Table 1. According to the data, griseofulvin showed more remarkable inhibitory activities
against Colletotrichum gloeosporioides (IC50 = 11.06±0.42 μg/mL) and Alternaria solani (IC50 =
2.68±0.33 μg/mL) than the positive control hymexazol (IC50  100 μg/mL for Colletotrichum
gloeosporioides, IC50 = 44.62±0.59 μg/mL for Alternaria solani), but slightly less activity against
B. cinerea than hymexazol. While compared to thiophanate-methyl, griseofulvin was similar or a
bit more potent against Colletotrichum gloeosporioides, Botrytis cinereal, and Alternaria solani,
but less against Cytospora sp. and Fusarium solani. The intermediate 2 reduced the antifungal
activity of griseofulvin and only had moderate activity with respect to Colletotrichum
gloeosporioides (IC50 = 33.07±0.83 μg/mL). The intermediate 4 was barely able to maintain the
antifungal activity against Colletotrichum gloeosporioides (IC50 = 24.38±0.59 μg/mL) and
Alternaria solani (IC50 = 19.50±0.99 μg/mL). The overall results revealed that the modifications
at position 4 of the griseofulvin molecule decreased the antifungal activity against all five plant
pathogenic fungi species analyzed, while the modifications at the 4 position would maintain or
improve the antifungal potency of the parent compound up to 10-fold.
For instance, some compounds in series 3 (compounds 3a~3g) including intermediate 2, were
exceptionally less active than griseofulvin and most of them were completely inactive.
Fortunately, some maintained specific activity against Colletotrichum gloeosporioides, and
compound 3b had the highest activities (IC50 = 35.56±1.3 μg/mL). Moreover, among these
compounds, it was observed that the activities of aliphatic acyl analogues (3a, IC50 = 46.74±0.49
μg/mL) were superior to those of the aromatic acyl analogues (3f, IC50 = 68.52±0.11 μg/mL), and
acyl analogues were more significant in inhibitory efficacy than sulfonyl analogues.
From the results, it is obvious that compounds in series 6 (compounds 6a~6j) had meaningful
activities against Colletotrichum gloeosporioides, Botrytis cinereal, and Alternaria solani. The
most aliphatic acyl derivatives, obtained by modifications at the 4 position, improved antifungal
activities, as compared to griseofulvin, against Colletotrichum gloeosporioides and Botrytis
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cinerea. In particular, compound 6c displayed the most promising inhibitory activities against
four out of five targeted fungi. Furthermore, the antifungal activities of aliphatic acyl analogues
were better than those of aromatic acyl analogues. Compared with the positive control, six
analogues (6a~6e and 6i) in this series had more influential activities against Colletotrichum
gloeosporioides and Alternaria solani, specifically Colletotrichum gloeosporioides. Although
compounds 6a~6c, 6e and 6f were less active than hymexazol against Botrytis cinerea, they still
showed similar or even higher inhibition potency than thiophanate-methyl. As presented in Table
1, compound 6c had the most extensive and remarkable activity against almost all of the target
fungal species. In general, the results revealed that the griseofulvin analogues obtained by
modifications at position 4′ are more significant for antifungal activity and might be worthy of
further development as potential agricultural fungicides.
Evaluation of Inhibitory Efficacy In Vivo.
The 4 compounds (6a, 6c, 6e, and 6f) with the highest activities against Colletotrichum
gloeosporioides in vitro were further investigated in vivo. The results of in vivo inhibition
activities are summarized in Table 2 and showed that the antifungal activities in vivo were less
pronounced than those in vitro. All of the above 4 compounds not only presented significant
inhibition against C. gloeosporioides but they also protected the fruit from the infection of the
fungal pathogen. The results for 7 days inhibitory efficacy of compounds are presented in Table
3. It was found that compounds 6c (IC50 = 47.25±1.46 μg/mL), 6e (IC50 = 49.44±1.50 μg/mL)
and 6f (IC50 = 53.63±1.74 μg/mL) possessed better restraint of fungal strains than the positive
control Thiophanate-methyl (IC50 = 69.66±6.07 μg/mL). The present results also demonstrated
that compound 6e has the highest in vitro and in vivo antifungal activities against Colletotrichum
gloeosporioides, while compound 6c showed very good in vitro and in vivo antifungal activities
against Colletotrichum gloeosporioides and the highest in vitro antifungal activities against other
fungal species. These findings strongly suggest that griseofulvin derivatives can be used for
development as potential fungicides.
3D-QSAR CoMFA Analysis
Comparative Molecular Field Analysis (CoMFA) mapped the interaction fields that
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surrounded the structures according to their impact on given activities.[25] To derive a reliable
CoMFA analysis model, all 22 of the griseofulvin analogues with distinct in vivo activity against
Colletotrichum gloeosporioides were studied by cross validation with the LOO option, and the
SAMPLS program was used to determine the optimal number of components in CoMFA
3D-QSAR analyses. Then, a non-cross-validated analysis was performed without column
filtering. The model had three components q2 (cross-validated r2) = 0.648, r2 (non-crossvalidated
r2) = 0.929, and SEE (standard error of estimate) = 0.151, defined in SYBYL. The observed and
calculated activity values are listed in Table 4. The models exhibited a good predictability for
these compounds. The steric and electrostatic contribution contour maps of CoMFA are plotted
in Figure 1 and 2, respectively. The 3D coefficient contour plots showed the field effect on the
target property; they were helpful to identify important regions that changed in the steric and
electrostatic fields. These can help to calculate the possible interaction sites. Compound 6c was
illustrated to explain the field contributions of different properties obtained from the CoMFA
analyses.
Figure 1 shows that there are two steric unfavorable yellow contours: one near the 4 position
and the other located beside the propionyl group of 6c. The first steric yellow contours showed
that an increase in steric bulk would decrease the activity, which revealed that the more bulky
group modifications at 4 position of griseofulvin would result in a decreased antifungal activity.
The second steric yellow contours together with the green polyhedral were the most important
characteristic of Figure 1, which indicates that too small or bulky substituents were not
inappropriate in the 4′ position of griseofulvin. The potency difference between 4 and 6c (6c and
6g) could be explained by changes from the -H to -COCH2CH3 (-COCH2CH3 to -COC6H4-CH3)
group.
The blue contour near the 4 position of 6c insinuated that increasing electron density was
unfavorable in this region, as depicted in Figure 2. Besides the blue contours, a predominant
feature of the electrostatic plot was the presence of the red contour proximal to the acyl carbonyl
oxygen of 6c. It could be reasonably presumed that there was a significant electrostatic
interaction between the acyl carbonyl oxygen and the possible receptor.
According to the CoMFA/PLS analysis, the steric and electrostatic field properties
contributed in a 52/48 ratio to the total variance, which specified that both the steric field and
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electrostatic descriptors have the same importance in explaining the dependent variable of the
CoMFA model. The 3D coefficient contour plots revealed that the suitable bulky and
electronegative acyl substituted groups seemed to be more favorable for increasing activity at the
4 position of griseofulvin. Although the CoMFA model was constructed using only the
antifungal activities against Colletotrichum gloeosporioides, it seemed to be helpful to
understand structure–activity relationships of these griseofulvin analogues against three other
target fungi. Most importantly, these results offered important new insights into designing highly
active compounds prior to future synthesis.
Conclusion
A series of griseofulvin derivatives were synthesized by effective approaches, with good yields,
and 17 of the 22 were novel. Antifungal activities results indicated that griseofulvin and the
majority derivatives have the strongest activities against different phytopathogenic fungi in vitro.
In particular, compounds (6a~6f, 6i) were found to have significant antifungal potential and were
superior to commercial fungicides hymexazol and thiophanate-methyl. Inhibitory efficacy in vivo
further demonstrated that the griseofulvin derivatives can be used as potential fungicides. The
SAR of the 4 position derivatives against all five fungi had the same trend, with the majority
being less active than griseofulvin. The modification of the 4′ position can improve the
antifungal activity against Colletotrichum gloeosporioides and Botrytis cinerea, even up to
10-fold more than the inhibition potency of the parent compound. To further explore the
structure–activity relationship, comparative molecular field analysis (CoMFA) was performed on
the basis of all 22 compounds with their inhibition of C. gloeosporioides. The 3D coefficient
contour plots revealed that the suitable bulky and electronegative acyl substituted groups at the 4
position of griseofulvin seem to be more favorable for increasing activity. In conclusion,
griseofulvin derivatives can be used for the development of potential agricultural fungicides.
■ AUTHOR INFORMATION
To whom Correspondence should be addressed.
Tel: +86-29-87092335.
E-mail: jinminggao@nwsuaf.edu.cn (J.M. Gao); jbolid@nwsuaf.edu.cn (D. Li)
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ORCID
Jin-Ming Gao: 0000-0003-4801-6514
Ding Li: 0000-0003-3130-6099
Author Contributions
∥
Yu-Bin Bai and Yu-Qi Gao contributed equally to this work.
Funding
This work was supported by the Natural Science Foundation of China (21502152), Natural
Science Foundation of Shaanxi Province (2014JQ2075), China Postdoctoral Science Foundation
(2014M562452), the Program of Unified Planning Innovation Engineering of Science &
Technology in Shaanxi Province (No. 2015KTCQ02-14), and Doctoral Scientific Research
Start-Up Fund of NWSUAF (1012013BSJJ037).
Notes
The authors declare that they do not have any competing financial interests.
■ ACKNOWLEDGMENTS
The authors would like to express their thanks to Prof. Jian Wan at Central China Normal
University for providing computing resources.
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9
OMe
OMe
*O
4
3'
2'
3a
5
*
*
*
*
*
3
*
*
*
2
4'
O
*
*
*
*
O
10
MeO
*
*
6
7a
5'
7
6'
Cl
8
1
440
441
442
Scheme 1. Structure of griseofulvin
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Scheme 2. Synthetic Route of Griseofulvin Analoguesa
OMe
O
O
OR1
OH
OMe
O
b
a
O
O
MeO
O
MeO
Cl 3a~3g
Cl
2
OMe
OMe
O
OMe
OMe
O
OMe
OMe
O
O
O
e
O
MeO
NOR2
NOH
Cl
O
MeO
c1
MeO
1
Cl 6a~6k
Cl
4
d
OEt
OMe
OMe
O
OMe
O
c2
NOH
NOMe
O
O
EtO
MeO
Cl
7
5
Cl
444
445
a
Reagents and conditions: (a) MgI2, Et2O/tolune; (b) NaH, THF; (c1) NH2OH·HCl, AcONa·3H2O /EtOH;
446
447
(c2) MeONHOH·HCl, AcONa·3H2O /EtOH; (d) Na, EtOH reflux; (e) triethylamine, acetone.
R1=
3a: -COCH3
3e: -COC6H4-NO2 (p)
3f: -COC6H4-F (p)
448
449
450
451
452
453
454
455
456
457
3b: -COCH2CH3
3c: -COCH2CH2CH3
3d: -COCH2CH2CH2CH3
3g: -COC6H3-F2 (3, 4)
R2=
6a: -COCH3
6g: -COC6H4-CH3 (p)
6b: -COCH2Cl
6c: -COCH2CH3
6d: -COCHCH2
6e: -COCH2CH2CH3
6f: -COCH2CH2CH2CH3
6h: -SO2C6H4-CH3 (p)
6i: -COC6H4-F (p)
6j: -COC6H4-Cl (p)
6k: -COC6H4-Br (p)
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459
460
461
462
Figure 1. Steric contour maps from the CoMFA model. Compound 6c is shown inside the field. Green
contours (70% contribution) encompass regions where the steric interaction is favored, whereas in yellow
contoured areas (30% contribution), the steric interaction is disfavored.
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465
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Figure 2. Electrostatic contour maps from the CoMFA model. Compound 6c is shown inside the field. Blue
contours (70% contribution) encompass regions where negative charge will decrease inhibitory activity,
whereas in red contoured areas (30% contribution), negative charges are favorable.
468
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Table 1. Antifungal activity of compounds in vitro
IC50±SD/ (μg /mL)
Compd
C.s.
>50
C. g.
B.c.
>50
A.s.
2.68±0.33
>50
F.s.
>50
11.06±0.42
33.07±0.83
46.74±0.49
35.56±1.3
51.65±2.49
48.58±0.14
44.07±2.28
68.52±0.11
52.51±0.22
24.38±0.59
1
2
>50
>50
>50
>100
>100
>100
>100
>100
>100
>100
>50
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>50
3a
3b
3c
3d
3e
3f
>100
>100
>100
>100
>100
>100
3g
4
56.62±1.97 19.50±0.99
108.49±5.84 126.33±3.48
14.19±1.65
22.28±0.32
26.57±0.66
19.78±0.27
>50
>100
29.94±0.51
>50
5
>50
>50
3.03±0.11
5.53±0.48
3.38±0.30
5.11±0.32
1.80±0.18
4.18±0.30
11.13±1.64
50.21±2.34
9.67±0.50
16.26±0.22
13.58±0.10
>100
19.43±0.43
9.21±0.16
6.24±0.21
21.90±0.82
>100
6a
6b
6c
6d
6e
6f
>50
>50
24.94±2.50
>50
>50
>50
33.35±2.22
36.61±2.07
>50
>50
>50
26.49±0.92
41.95±2.12
60.22±3.79
16.87±0.35
53.68±7.47
>50
>50
>50
>100
>100
>50
6g
6h
6i
>100
>50
>100
>50
>100
>100
>100
62.22±1.47
>50
>100
>100
6j
>100
6k
Hy
Tpm
7.11±0.16
>100
44.62±0.59 88.63±2.45
>100 48.00±3.46
<6.25
470
471
472
Note: C.s., Cytospora sp.; C.g., Colletotrichum gloeosporioides; B.c., Botrytis cinerea; A.s., Alternaria solani;
F.s.; Fusarium solani; Hy, Hymexazol; Tpm, Thiophanate-methyl, (a commercial available agricultural
fungicide for direct application bought from a pesticide shop, effective activity constituent is 75%).
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Table 2. The half-maximal inhibitory concentration values of compounds in vivo
Compds against C. g.
6a
6c
6e
6f
Tpm
IC50±SD/ (μg /mL)
79.14±7.07
47.25±1.46
49.44±1.50
53.63±1.74
69.66±6.07
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Table 3. Inhibitory efficacy of compounds in vivo
Pictures of 3 days inhibitory
efficacy of compound 6f 1
Pictures of 7 days inhibitory efficacy of compounds
Concentration
(μg/mL)
6f
6a
6c
6e
6f
Tpm
100
50
25
12.5
6.25
Sterile water
(negative
control)
Spore
suspension
(blank control)
476
1: 3 days inhibitory efficacy of compounds 6a, 6c and 6e is identical or similar to that of compound 6f.
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Table 4. Data Sets for 3D-QSAR Analysis.
pIC50
Compd.
Actual Calculated
4.5037 4.2012
4.0104 4.1339
4.1786 4.023
Residual
1
0.3025
-0.1235
0.1556
-0.3058
-0.1619
0.0063
-0.0745
-0.0231
0.1756
-0.086
0.0398
0.0898
-0.0357
-0.078
-0.1217
0.16
2
4
5
3.4803 3.7861
3a
3b
3c
3d
3e
3f
3g
6a
6b
6c
6d
6e
6f
6g
6h
6i
3.911
4.0729
4.0454 4.0391
3.8985 3.9729
3.9397 3.9628
4.0441 3.8685
3.8277 3.9137
3.9599 3.9201
5.1311 5.0414
4.9049 4.9406
5.0983 5.1763
4.9167 5.0384
5.3861 5.2261
5.0339 5.2146
4.6401 4.577
4.0169 3.9314
4.7047 4.6458
4.4933 4.5314
4.6081 4.5163
-0.1807
0.063
0.0855
0.0589
-0.038
0.0918
6j
6k
479
480
481
482
483
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