Development of celecoxib-derived antifungals for crop protection

Article abstract of DOI:10.1016/j.bioorg.2020.103670

Selective COX-2 inhibitor celecoxib was found directly inhibiting the growth of tested phytopathogenic fungi with the inhibitory rate ranging from 30 to 40% at 100 μg/ml. Lead optimization of celecoxib led to the identification of compound 12 among its derivatives as the most active antifungal candidate. The antifungal effect of compound 12 was supposed to be independent of COX-2 inhibition. Transcriptome profiling analysis of Fusarium graminearium (PH-1) treated with compound 12 brought about 406 up-regulated and 572 down-regulated differentially express genes (DEGs) respectively.

Full text of DOI:10.1016/j.bioorg.2020.103670

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Development of celecoxib-derived antifungals for crop protection
Yihui Ma, Lirong Yang, Xiuxiu Liu, Jun Yang, Xianglong Sun
PII:
S0045-2068(19)31461-0
DOI:
https://doi.org/10.1016/j.bioorg.2020.103670
YBIOO 103670
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Bioorganic Chemistry
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7 February 2020
14 February 2020
Please cite this article as: Y. Ma, L. Yang, X. Liu, J. Yang, X. Sun, Development of celecoxib-derived
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Development of celecoxib-derived antifungals for crop protection
Yihui Maa*, Lirong Yanga, Xiuxiu Liua, Jun Yanga, Xianglong Suna,
a Plant Protection Institute, Henan Academy of Agricultural Sciences, Zhengzhou
450002, China
Address for Correspondence:
Yihui Ma, PhD
Plant Protection Institute, Henan Academy of Agricultural Sciences
Zhengzhou 450002, China PR
Tel: 86-371-65731656
Email: ma.150@buckeyemail.osu.edu

Abstract
Selective COX-2 inhibitor celecoxib was found directly inhibiting the growth of
tested phytopathogenic fungi with the inhibitory rate ranging from 30-40% at 100
µg/ml. Lead optimization of celecoxib led to the identification of compound 12
among its derivatives as the most active antifungal candidate. The antifungal
effect of compound 12 was supposed to be independent of COX-2 inhibition.
Transcriptome profiling analysis of Fusarium graminearium (PH-1) treated with
compound 12 brought about 406 up-regulated and 572 down-regulated
differentially express genes (DEGs) respectively.
Keywords
Celecoxib; COX-2 inhibitor; lead optimization; antifungal agrochemical
Graphical abstract

Highlight
 Cyclooxygenase-2 (COX-2) selective inhibitor, celecoxib, was found directly
inhibiting the growth of phytopathogenic fungi at concentrations over 100
µg/ml.
 Derivation of celecoxib leads to the identification of compound 12, a non-
COX-2 inhibitor with increased antifungal activity.
 Transcriptome profiling analysis of compound 12 treated Fusarium
graminearium (PH-1) revealed 406 up-regulated and 572 down-regulated
differentially expressed genes (DEGs) respectively.
 These DEGS were enriched and cofactor binding was the most enriched
gene ontology (GO) category.

Introduction
Phytopathogenic fungi are responsible for a wide range of serious plant disease
and can cause destructive impact to major crops such as wheat, rice and
maize[1, 2]. The majority of plant diseases are caused by fungi pathogens.
Generally, crop disease control relies heavily on chemical fungicides and
fungicides treatment represents an effective approach among the strategies for
the plant disease management. Unlike previously used inorganic fungicides,
recently developed fungicides features lower application rates, less phytotoxicity
and easier to apply by farmers, representing a major improvement in antifungal
pesticide discovery. Since their introduction, significant progress on the research
as well as development of a variety of fungicides with different modes of action
have been seen, due to the rapid growth of the fungicide markets. Recently,
hundred of thousands tons of fungicides in active ingredients have been used
annually [3]. However, long-term and intensive application of these chemical
fungicides have exerted high selective pressure in pathogenic fungi and
increased the risks of resistance development [4-8]. Indeed, many cases of
resistance to almost each class of fungicides with various mode of action have
been documented. Thus develop fungicides with unique structures and/or novel
mode of action is greatly desirable to combat fungicide resistance.

Pyrazole motif is a very useful structural unit for molecule design in medicinal
chemistry and frequently present in both agrochemicals and pharmaceuticals [9,
10]. Pyrazole derivatives are well-known for their broad-spectrum of biological
activities such as anti-inflammatory[11], anti-analgesic[12], antifungal[13] and
anticancer effect [14]. Celecoxib, a pyrazole derivative, is a COX-2 selective
nonsteroidal anti-inflammatory drug (NSAID) used to treat pain or inflammation
caused by many conditions such as arthritis and menstrual pain [12]. Besides,
celecoxib also exhibited off-target activity such as antibacterial [15-18] and
anticancer effect [19]. And many active antibiotics and anticancer agents with
higher level of activity than celecoxib have been under development by structural
optimization of celecoxib’s scaffold. In addition, celecoxib was also reported to
reduce the fungal burden in the lungs and spleen of mice infected with
Histoplasma capsulatum as well as increased the survival of mice after infection
with a lethal inoculum of H. capsulatum [20]. Interestingly, this apparent
antifungal activity was still mediated through its effect against COX-2 and the
subsequent inhibition of prostaglandins. Thus, direct effect of celecoxib against
fungal pathogens has not been well explored. Inspired by the broad-spectrum
biological activities of celecoxib which may arise from its versatile 1,5-diaryl-3-
trifluoromethyl skeleton, and in the continuation of our current effort to seek
antifungal chemicals for crop protection, it is very interesting to profile the
antifungal activity of celecoxib against some representative plant pathogenic

fungi and further explore the potential of this unique scaffold to develop
practically effective fungicides through structural optimization. In this study,
celecoxib was found moderately active against four selected species of fungi
phytopathogens. Lead optimization of celecoxib for more potent fungicidal
pesticides gave rise to 19 analogs, which were screened for their antifungal
activity. Among them, compounds 12, which has a -COOH in the para position of
the phenyl ring attached to the N1 atom of the pyrazole, demonstrates the
enhanced activity over celecoxib and was more active than any other members
in the library.
Chemistry:
The synthesis of compounds 1-19 was following the scheme 1. Generally,
acetonphenones were condensed with ethyl trifluoroacetate in anhydrous
tetrahydrofuran with sodium hydride as base to give 1,3-dione adducts, which
was cyclized with various substituted phenylhydrazine hydrochloride in ethanol to
give corresponding final compounds. Compounds 12, 18 and 19 which bear a
free –COOH were prepared in N,N-dimethylformide to avoid the formation of
1
corresponding ethyl ester. All the compounds were characterized with H 13C
NMR and HRMS. The spectroscopic data (supplementary materials) was in full
accordance with their depicted structures and consistent with those reported [12].

(a) ethyl trifluoroacetate, NaH/THF; (b) substituted phenylhydrazine-HCl, EtOH, reflux.
Scheme 1 Synthetic route to compound 1-19.
Result and Discussion
Celecoxib exhibited broad-spectrum antifungal effect against tested
phytophathogenic fungi.
To examine whether celecoxib has any antifungal activity, potato agrose dishes
(PDA) assay was used to test its inhibitory effect on the growth of four selected
species of pathogenic fungi available in our lab at a broad concentration range
across 0.1, 1, 10, 100 µg/ml and the results is depicted in figure 1. Interestingly,
our results revealed that celecoxib, a selective COX-2 inhibitor, exhibited
moderate but clear inhibition against each tested pathogens. Starting 1 µg/ml,
celecoxib showed slight activity against Fusarium graminearium Sehw and
Myrothecium roridum with the inhibition of 14% and 9% respectively. The
inhibitory rate rose to 20%-30% at concentration of 10 µg/ml against all the

tested fungi except for Helminthosporium maydis Nisik & Miy which did not
demonstrated any susceptibility to celcoxib at this concentration. In contrast, 100
µg/ml celecoxib obviously inhibited the growth of Helminthosporium maydis Nisik
& Miy with an inhibition of 50%, indicating the sensitivity of this fungi to the tested
concentrations. There was also slight improvement in potency for celecoxib at
100 µg/ml against the other three fungi with the enhanced inhibition rate of 30-
40% compared to that at 10 µg/ml. Further increasing celecoxib’s concentration
up to 400 µg/ml did not bring about significant improvement in inhibition (data not
shown). Even though, this observed direct inhibition effect associated with
celecoxib is different from its host-directed antifungal effect against Histoplasma
capsulatum infection, which also resulted from its original COX-2 inhibition
property. Thus, this finding underscores the potential of celecoxib acting as a
lead compound to develop novel antifungals for agricultural purpose.

Inhibition of Celecoxib against Phytopathogenic Fungi
Fusarium oxysporum.f.sp.niveum
Fusarium graminearium Sehw
Myrothecium roridum.
Helminthosporium maydis Nisik & Miy.
100
10
1
0.1
Concentrations (µg/ml)
Fig 1. The effects of lead compound celecoxib on four species of crop pathogens were measured using
PDA assay at concentrations of 0.1,1,10,100 µg/ml.
Exploration the Antifungal Activity of celecoxib Derivatives
Encouraged by the above-described findings of the direct antifungal activity of
celecoxib against phytopathogenic fungi, we performed lead optimization by
exploiting its scaffold as a molecular platform to develop potent antifungal
agrochemicals for crop protection. Accordingly, we designed and synthesized a
focused compound library consisting of 19 derivatives of celecoxib. At first, to
examine the steric and electronic effect of the substituents on the phenyl group
attached to the N1 of pyrazole ring on fungicidal activity, substituents with various
bulkiness and electron properties were introduced to generate compound 1-17.
SAR analysis reveled an exclusive preference to –COOH among various

substituents in R1 as compound 12 exhibited higher level of activity against each
fungus than any other members within the compound series and noticeable
potency improvement relative to lead compound celecoxib. For example, the
inhibition of compound 12 against Fusarium oxysporum.f.sp.niveum, Fusarium
graminearium Sehw and Myrothecium roridum reached 83%, 84% and 73%,
comparing to the respective inhibition rates of 37%, 30%, and 35% associated
with celecoxib. It is also encouraging that the growth of Helminthosporium
maydis Nisik & Miy was completed inhibited by compound 12. However, the
results for the rest analogues were disappointing as they generally displayed
much lower level of potency regardless of substitution patterns. Interestingly,
further optimization of toluene moiety revealed there is certain flexibility in
selecting R2, as either electron-donating or electron-withdrawing functional group
apparently both elevated the antifungal activity of the corresponding compound
18 and 19 compared to celecoxib. And between them, compound 18
demonstrated slightly more promising antifungal effect than that of compound 19.
On the other hand, Myrothecium roridum was most susceptible to these
chemicals and several derivatives including compound 8, 11, 12, all revealed
similar inhibition rate over 70%. While for the most tolerable strain
Helminthosporium maydis Nisik & Miy, none of these analogues displayed any
inhibition rate greater than 17% with the exception of compound 12, 18 and 19,
which by contrast greatly inhibited its growth. Compared to two controls,

compound 12 demonstrated comparable antifungal potency with that of
triadimenfon, but was still less active than pyraclostrobin, which completed
inhibited the growth of all the tested fungi. Even through, the increased antifungal
activity of compound 12 over the lead compound celecoxib indicated that
celecoxib could be structurally optimized to develop potent antifungals and its
1,5-diaryl scaffold may represent a novel potential pharmacophore for antifungals
development by appropriately selecting the substituents on the two aromatic
rings.
Table 1. Inhibition rate of celecoxib and its derivatives against fungi pathogens at 100
mg/ml.
% Growth Inhibition
Fusarium
Fusarium
Compounds
R1
R2
Myrothecium
roridum
Helminthosporium maydis
Nisik & Miy
oxysporum.f.s graminearium
p.niveum
37%
Sehw
30%
29%
21%
36%
21%
21%
celecoxib
35%
55%
36%
45%
36%
55%
50%
0%
1
2
3
4
5
Me
Me
Me
Me
Me
2-Cl
3-Cl
24%
14%
0%
4-Cl
24%
15%
17%
0%
2,4-dichloro
2,6-dichloro
19%
19%

6
Me
Me
4-Br
4-F
14%
24%
19%
14%
24%
14%
83%
10%
0%
36%
43%
29%
43%
43%
29%
84%
29%
21%
29%
29%
36%
57%
54%
100%
100%
18%
64%
71%
55%
73%
72%
73%
27%
18%
55%
55%
73%
66%
56%
21%
100%
0%
19%
10%
0%
7
8
Me
2,4-difluoro
2-NO2
9
Me
10
Me
4-NO2
33%
0%
11
Me
4-OMe
4-COOH
4-CN
12
Me
100%
14%
0%
13
Me
14
Me
4-OCF3
2-CH3
15
Me
29%
5%
17%
12%
0%
16
Me
4-CF3
17
18
Me
4-SO2Me
4-COOH
4-COOH
29%
68%
57%
66%
100%
4'-F
4'-OMe
60%
64%
33%
100%
19
Triadimefon
Pyraclostrobin
Differentially expressed genes (DEGs) in PH-1 was identified upon treatment of
compound 12
As our initial attempt to explore the mode of action underlying the antifungal
effect of compound 12, transcriptomic profiling analysis of Fusarium
graminearum (PH-1) was employed to identified the differentially expressed
genes after it was treated with compound 12. PH-1 was selected as a model

fungus for its genome has been extensively studied. RNA-seq expression
profiling of PH-1 was performed 3h after treatment with 30 µg/ml of compound 12
to allow for the extraction of high quality RNA as any longer period of treatment
and higher concentration of compound would further dissolve or inhibit the
growth of hyphae. One biological replicate with paired-end was sequenced for
compound 12-treated PH-1 as well as the untreated. A total of 46413308 and
52319584 raw reads leading to 45510240 and 50755126 clean reads were
collected from compound 12-treated PH-1 as well as the untreated respectively
(table 2).
Table 2 Transcripome statistics of cDNA libraries form compound 12 treated PH-1 and
untreated PH-1.
sample
name
GC
raw_reads1 clean_reads2 clean_bases3 error_rate4
Q205
97.72
97.85
Q306
93.69
93.97
content7
Comp. 12
treated
PH-1
Untreated
PH-1
46413308
52319584
45510240
50755126
6.83G
7.61G
0.03
0.03
54.13
54.01
1 The numbers of original data sequence
2 The filtered data sequence
3 Qphred = -10log10(e)
4 The sequence length multiplied by the number of sequencing
5 The percentage of bases with a Phred value>20
6 The percentage of bases with a Phred value>30
7 The percentage of bases number of G and C
The sequence reads were mapped to the PH-1 genome (Table 3). Among the
transcripts from two conditions, up to 97.01% and 97.2% of the total reads were

uniquely mapped to the PH-1 genome respectively. Small proportions (less than
0.5%) for both conditions were mapped to multiple locations in the PH-1 genome.
All uniquely mapped reads were used to calculate FPKM (Fragments Per
Kilobase Million), which can be used to normalize expression levels. Accordingly,
Log2-fold DEGs between the two conditions were identified with corrected P-
value of 0.05. A total of 978 DEGs were detected between compound 12-treated
and control, with 406 DEGs up-regulated and 572 down-regulated.
Table 3. Summary of mapping the sequenced reads to the PH-1 genome form compound 12 treated
PH-1 and the untreated PH-1
Sample
Event of mapping
Comp. 12 treated
PH-1
Untreated treated PH-1
Total reads
45510240
50755126
49529691(97.59%)
196812(0.39%)
49332879(97.2%)
24694718(48.65%)
24638161(48.54%)
24644099(48.55%)
24688780(48.64%)
Total map1
44366473(97.49%)
215899(0.47%)
44150574(97.01%)
22058738(48.47%)
22091836(48.54%)
22044279(48.44%)
22106295(48.57%)
Multi map2
Unique map3
Read1 map4
Read2 map5
Positive map6
Negative map7
1Total number of reads mapped on the PH-1
2Total number of reads mapped to multiple locations in the PH-1
3Total number of reads mapped to uniquely locations in the PH-1
4The two directions of paired-end sequencing
5The two directions of paired-end sequencing
6Total number of reads mapped to positive strand of PH-1
7Total number of reads mapped to negative strand of PH-1

Fig 2. Volcano of DEGs between compound 12 treated PH-1 and untreated PH-1. The y-axis corresponds
to the mean expression value of log10 (p-value), and the x-axis displays the log2 fold change value. The
red dots represent up-regulated DEGs, the green dots represent down-regulated DEGs.
The DEGs were then annotated and assigned to different functional GO (gene
ontology) categories which fall into three groups of biological process (BP),
cellular component (CC) and molecular function (MF). For each group, several
most significant terms were

down
up
down
up
down
up
BP
CC
MF
Fig 3. Functional annotation of DEGs based on gene ontology (GO) categorization.
depicted in figure 3. Specifically, for biological process, the dominant categories
were organic substance catabolic process with 32 DEGs; catalytic complex had
the most DEGs of 11 for the group cellular component. Notably, the largest
category of cofactor binding in molecular function group was the most significant
among all the gene categories in the gene enrichment analysis, indicating the
importance of these pathway in the compound-12 induced antifungal effect.

Although many work still need to be done to explore the exact mode of action of
compound 12, this assay provided global and useful information as to identify the
direct target of this kind of antifungal chemicals.
Conclusion
Resistance development in phytopathogenic fungi has posed a big challenge for
plant disease control and calls for novel types of fungicide for effective plant
disease management. Keeping development of novel chemicals with highly
potent activity or novel mode of action would always be awaited for resistance
management. From drug development perspective, unique structure different
from that of the currently used agrochemicals would have the potential to
facilitate the fight against resistance. On the other hand, repurposing an existing
drug to find new uses represent a promising alternative strategy for novel drug
development in both pharmaceutical and agricultural area as it can accelerate
the development process as well as reduces the research and development
cost[21-24]. Here, we present that a widely used COX-2 inhibitor celecoxib
showed direct antifungal effect on some selected phytopathogenic fungi.
Structural modification led to the identification of compound 12 whose activity
was significantly elevated over the lead compound celecoxib, and this
improvement was closely related to the presence of the substituent of 4-COOH,

as any other substitution pattern did not deliver any apparent antifungal effect.
More importantly, the structure of this kind of celecoxib-derived antifungal is
different from any class of mainstream of fungicidal pesticides currently used in
the market. Thus, this unique structure may be accompanied with novel mode of
action, which would be instrumental to resistance management.
Regarding the mode of action, previous study showed that the COX-2 inhibition
of compound 12 was significantly decreased compared to parent compound
celecoxib [25], indicating the independence of the antifungal activity of these
novel fungicides on their inhibition against COX-2. This promise was further
confirmed by the lack of antifungal effect of compound 17, which is a potent
COX-2 inhibitor with even longer plasma half-life [12]. Thus their putative
antifungal target(s) must be different from COX-2-like enzyme in fungal
pathogens. Our profiling analysis of compound 12’s antifungal effect on PH-1
revealed that 406 differentially expressed gene were up-regulated while 572
down-regulated. All these DEGs were enriched and the result showed than
cofactor-binding related DEGs were the most, followed by organic substance
catabolic process. In contrast, cell component related DEGs were much less.
Furthermore, it was reported that compound 12, with a carboxylic acid in the
place of sulfonamide of celecoxib, showed higher level of metabolic stability that

that of celecoxib [25], suggesting compound 12 might have a longer duration of
activity, which is a desirable property for their application in the field.
Although this work is about antifungal pesticides development, it may also have
far-reaching implications in pharmaceutical areas. As celecoxib itself has been
used in clinical for many years and in light of its outstanding physical properties,
development of celecoxib-based fungicide to treat infectious disease may be a
better start point due to the direct antifungal effect associated with its skeleton. In
this context, it would be also interesting to examine as well the contribution
resulted from the direct antifungal effect of celecoxib to the mice’s defense
improvement against Histoplasma capsulatum infection in addition to those
mediated through host COX-2 inhibition[20].
Experiment
5.1 Chemistry
All chemical reagents were commercially available without further purification and flash
column chromatography was performed over silica gel 200-300 mesh. Analytical thin-
layer chromatography (TLC) was performed with silica gel plates using silica gel 60
GF254 (Qingdao Haiyang Chemical Co., Ltd). Melting points were determined with an X-
4 melting points apparatus (Shanghai) and were uncorrected. High-resolution mass
spectra were obtained on a Q-TOF Mass Spectrometer with ESI ionization. 1H NMR and

13C NMR spectra were recorded on a Bruker AM-400 spectrometer at 400 MHz and 100
MHz respectively. Chemical shifts are reported in parts per million relative to internal
tetramethylsilane(TMS). All the tested compounds hold a purity of at least 95%.
Analytical HPLC was performed on the Thermo Ultimate 3000 equipped with Thermo
Scientific Acclaim-C18 column and UV wavelength set at 254 nm. Eluent: 40% MeOH in
H2O; flow rate = 1 ml/min.
5.1.1 General procedure for the preparation of trifluoro-1-(4-substituted phenyl)butane-
1,3-dione
To a tetrahydrofuran solution of acetophenones (100 mmol) was added NaH (200 mmol),
and the mixture was stirred for 5 min. Ethyl trifluoroacetate (250 mmol) was then added.
The reaction mixture was stirred at room temperature for 4 h and neutralized with 2N
HCl. The precipitate formed was collected, washed with methanol and dried, which was
directly used for the next step without further purification.
5.1.2 General procedure for methyl 1,5-disubstituted-3-trifluoromethyl-1H-pyrazole (1-
19)
A mixture of 1,3-dione adducts (0.05 mol) with selected substituted phenylhydrazines
(0.06 mol) in ethanol or N,N-Dimethylformamide (DMF) (200 ml) was refluxed for 8 h.
Water was then added after the reaction mixture was cooled down to room temperature.
The resulting precipitate was collected and recrystallized from methanol to give target
compounds.

5.2 Antifungal susceptibility assays
The antifungal activity of newly synthesized compounds was tested following a
modification of previously described protocol [26]. Briefly, to 900 ml of double-distilled
water was added 26 g of potato dextrose agar (PDA) to give PDA medium. The medium
prepared was autoclaved at 120 ˚ C for 20 min before cooled down to 55 ˚ C. The
compounds stock solutions in DMSO diluted by mixing with the medium to give final
indicated concentrations were poured into petri dishes (9 cm diameter). A treatment with
vehicle (DMSO) was included as reference, and three replicates were performed for
each treatment. Each petri dish was inoculated with a mycelia disc (1 mm diameter) pin-
picked from the margin of freshly prepared source colonies of the respective fungus
growing on PDA medium. After incubation in the dark, all three diameters of the
mycelium for each treatment were measured, averaged and converted to percentage
activity relative to vehicle treatment.
5.3 Transcriptome profiling assay
The transcriptome profiling assay and data process was performed according to the
discribed protocal of Yang [27] with minor modifications. Briefly, liquid cultures of
Fusarium graminearum (PH-1) were grown in YEPD (yeast extract peptone dextrose
medium) on a rotary shaker (175 rpm at 25 ºC) for three days, The cultures was then
added compound 12 with a final concentration of 30 µmol/L and incubated for 3 hours.
Fresh mycelia were collected and washed three times with sterile deionized water. The

smaple were then immediately frozen and stored in liquid nitrogen untl analysis. Total
RNA was extracted from these materials using RNAiso Plus (Total RNA extraction
reagent) (TaKaRa, Otsu, Japan). RNA purity was verified using a NanoPhotometer
spectrophotometer (Implen, Westlake Village, CA, USA). RNA concentration was
measured using a Qubit RNA Assay Kit in a Qubit 2.0 Fluorometer (Life Technologies,
Grand Island, NY, USA). RNA integrity was assessed using the RNA Nano 6000 Assay
Kit of the Bioanalyzer 2100 system (Agilent Technologies, Wilmington, DE, USA).
Library construction, RNA-seq sequencing, Read mapping to the reference genome,
Differential expression analysis and Gene Ontology (GO) enrichment analysis all
followed the protocol of Yang[27].
Acknowledgments
This work was supported by special fund from Henan Academy of Agricultural
Sciences (Grant No. 20188109).
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Highlight
 Cyclooxygenase-2 (COX-2) selective inhibitor, celecoxib, was found directly
inhibiting the growth of phytopathogenic fungi at concentrations over 100
µg/ml.
 Derivation of celecoxib leads to the identification of compound 12, a non-
COX-2 inhibitor with increased antifungal activity.
 Transcriptome profiling analysis of compound 12 treated Fusarium
graminearium (PH-1) revealed 406 up-regulated and 572 down-regulated
differentially expressed genes (DEGs) respectively.
 These DEGS were enriched and cofactor binding was the most enriched
gene ontology (GO) category.