Natural products as sources of new fungicides (III): Antifungal activity of 2,4-dihydroxy-5-methylacetophenone derivatives

Article abstract of DOI:10.1016/j.bmcl.2016.03.073

A series of new 2,4-dihydroxy-5-methylacetophenone 2 derivatives were synthesized, and characterized by ¹H, ¹³C NMR and ESI-MS. Their antifungal activities were evaluated in vitro against five important plant fungal pathogens including Cytospora sp., Glomerella cingulate, Pyricularia oryzaecar, Botrytis cinerea and Alternaria solani by the mycelial growth inhibitory rate assay. Compounds 2b-d, 2g and 2h displayed a broad-spectrum activity. The log P value of these active compounds is ranging from 1.71 to 2.54. Especially, isopropyl ketone 2g (log P 2.27) was found to be the most active to the tested organisms with IC₅₀ values of 17.28-32.32 μg/mL. The results suggest that compound 2g might be a promising candidate in the development of new agrochemical antifungal agents. Preliminary structure-activity relationship (SAR) studies of the acetophenone derivatives are also discussed.

Full text of DOI:10.1016/j.bmcl.2016.03.073

Accepted Manuscript
Natural Products as Sources of New Fungicides (III): Antifungal activity of 2,4-
dihydroxy-5-methylacetophenone derivatives
Wei Shi, Wen-Jia Dan, Jiang-Jiang Tang, Yan Zhang, Tseden Nandinsuren, An-
Ling Zhang, Jin-Ming Gao
PII:
S0960-894X(16)30305-5
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.03.073
BMCL 23716
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 January 2016
29 February 2016
18 March 2016
Please cite this article as: Shi, W., Dan, W-J., Tang, J-J., Zhang, Y., Nandinsuren, T., Zhang, A-L., Gao, J-M.,
Natural Products as Sources of New Fungicides (III): Antifungal activity of 2,4-dihydroxy-5-methylacetophenone
derivatives, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.03.073
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Natural Products as Sources of New Fungicides (III): Antifungal
activity of 2,4-dihydroxy-5-methylacetophenone derivatives
Wei Shi, Wen-Jia Dan, Jiang-Jiang Tang, Yan Zhang, Tseden Nandinsuren, An-Ling
Zhang*, and Jin-Ming Gao*
Shaanxi Key Labotory of Natural Products & Chemical Biology, College of Science,
Northwest A&F University, Yangling 712100, Peoples Republic of China
* to whom correspondence should be addressed:
Fax: 86-29-87092515; Email: jinminggao@nwsuaf.edu.cn (A.L. Zhang, J.M. Gao)

Abstract:
A series of new 2,4-dihydroxy-5-methylacetophenone 2 derivatives were synthesized,
1
and characterized by H, 13C NMR and ESI-MS. Their antifungal activities were
evaluated in vitro against five important plant fungal pathogens including Cytospora
sp., Glomerella cingulate, Pyricularia oryzaecar, Botrytis cinerea and Alternaria
solani by the mycelial growth inhibitory rate assay. Compounds 2b-d, 2g and 2h
displayed a broad-spectrum activity. The log P value of these active compounds is
ranging from 1.71 to 2.54. Especially, isopropyl ketone 2g (log P 2.27) was found to
be the most active to the tested organisms with IC50 values of 17.28 to 32.32 μg/mL.
The results suggest that compound 2g might be a promising candidate in the
development of new agrochemical antifungal agents. Preliminary structure-activity
relationship (SAR) studies of the acetophenone derivatives are also discussed.
Keywords: Natural products; agrochemicals; acetophenone derivatives;
phytopathogenic fungi; antifungal agents; structure-activity relationship

Agricultural crops are facing tremendous losses due to pests, diseases, and weed
damages which result in direct economic losses including reduction in grain yield and
quality.1,2 Despite the availability of effective synthetic fungicides, new fungicides
with novel chemical structures which have higher potency and broader spectrum of
activity against resistant fungal strains are still needed. Recent studies have
demonstrated that acetophenones and their derivatives can be seen as important
intermediates for the synthesis of some agrochemicals and pharmaceuticals. For
example, two antifungal agents (Figure 1) 2-hydroxy-4,6-dimethoxyacetophenone
(xanthoxylin, 1) and 2,4-dihydroxy-5-methylacetophenone (2) were isolated from a
medicinal plant Melicope borbonica3 and from the higher fungus Polyporus picipes
(Polyporaceae),4 respectively. In our previous studies,4-6 compound 2 exhibited in
vitro significant growth inhibitory activity, and initial structure-activity relationship
(SAR) studies also showed the importance of 2,4-dihydroxy groups for antifungal
activity.
It has been reported that the mode of antimicrobial action of some new antifungal
agent, such as alkyl parabens (p-hydroxybenzoic acid alkyl esters),7 which depends on
their ability to disrupt the native membrane due to their hydrophobicity.8 To explore
the effects of the improved hydrophobicity on antifungal activity of acetophenone
derivatives, and as a continuation of our research on the synthesis of structurally
diverse antifungal compounds, in this Letter, we report the synthesis of lipophilic
acetophenone-like compounds through aryl acetoxylation reaction with two methods,
and evaluated antifungal activities against several fungal phytopathogens in vitro.
Figure 1. Structures of two antifungal natural products 1 (xanthoxylin) and 2
(2,4-dihydroxy-5-methylacetophenone).

The synthesis of 2,4-dihydroxy-5-methylacetophenone derivatives commenced
from starting material 2,4-dihydroxybenzaldehyde. In a conventional procedure,
2,4-dihydroxybenzaldehyde was reduced to 2,4-dihydroxytoluene with NaBH3CN and
HCl in THF in high yield (95%). With 2,4-dihydroxytoluene in hands, two methods
were conducted to obtain these derivatives 2a–s (Scheme 1). Method A for
compounds 2a–h employed Fries-type rearrangement of appropriate phenolic esters to
hydroxyaryl ketones by catalysis of Lewis acids. A mixture of 2,4-dihydroxytoluene,
corresponding acid anhydride and boron trifluoride-diethyl ether was refluxed. Then
reaction mixture was poured into crushed ice and extracted with ethyl acetate. The
organic layer was dried and concentrated under reduced pressure. The resulting oil
was purified by silica gel column chromatography; Method B for compounds 2i–s
involved the reaction of 2,4-dihydroxytoluene with appropriate acyl chlorides to yield
ortho-acyl phenols via Friedel–Crafts acylation in a single step as reported in our
earlier study.5 Commercially unavailable acyl chlorides were prepared according to
the reported methods.9 In the case of compounds 2i–s, besides the desired ortho-acyl
phenols, we observed the formation of the very small amount of the corresponding
esters as byproducts, and the target phenols were difficult to obtain; as a result, a
further purification by crystallization after flash chromatography was needed. The
structures of the derivatives 2a–s were shown in Scheme 1.10 (see the Supporting
Material). The tested five phytopathogenic fungi, namely, Cytospora sp., Glomerella
cingulata Schr, Pyricularia oryzaecar, Botrytis cinerea Pers et Tris and Alternaria
solani were provided by College of Science, Northwest A&F University.

R
log P
R
log P
-CH3
1.05
-(CH3)C=CH2
2.09
2 (2a)
2b
2k
2l
-CH2CH3
1.71
1.78
-CH2CH2CH3
-CH2(CH2)2CH3
-CH2(CH2)3CH3
-CH2(CH2)4CH3
-CH(CH3)2
-C(CH3)3
2.13
2.54
2.96
3.38
2.27
2.98
1.58
2.20
-p-F-Ph
-p-Cl-Ph
-p-Br-Ph
3.11
3.51
3.78
4.31
2.21
2.83
3.44
2c
2d
2e
2f
2g
2h
2i
2m
2n
2o
2p
2q
2r
-p-I-Ph
-p-NO2-Ph
-p-OCH3-Ph
-p-CH3-Ph
-CH2Cl
-CF3
2s
2j
Scheme1. Synthetic route of the title compounds and log P values.
We estimated the role of alkyl and aryl ketone derivatives against a panel of plant
pathogenic fungi.11 As shown in Table 1, aryl ketone derivatives were not effective
against all the fungal strains tested in this study. As for alkyl ketone derivatives,
methyl, ethyl, propyl and butyl ketones showed strong to moderate activities, an
increase in the chain length of linear alkyl ketones led to enhanced antifungal activity.
Among the linear alkyl ketones (2a–f), butyl ketone (2d) had the highest activity; the
decrease of activities of compounds 2e (R = pentyl) and 2f (R = hexyl) possibly due
to either to an unfavorable steric hindrance or to an increased lipophilicity. Consistent
with the antifungal activity of alkyl ketones, branched alkyl ketones showed stronger
activity than linear alkyl ketones. Compounds 2g and 2h showed the increased
antifungal activities in comparison with the compounds 2c and 2d, respectively. This
result suggested that the introduction of methyl group in the linear carbon chain is

important for these derivatives to show the antifungal effects. Notably, 2g, bearing an
isopropyl substituent, had the best activity against all the five organisms and
displayed significantly more potent potency for Cytospora sp., G. cingulata, P.
oryzaecar and A. solani than a commercial fungicide, thiabendazole.
Table 1. IC50 values (μg/mL) of derivatives against five plant pathogenic fungi.
Compd.
Glomerella
cingulata
Alternaria
solani
Pyricularia
oryzaecar
>50
Botrytis
cinerea
>50
Cytospora sp.
>50
>50
2 (2a)
47.25 ± 1.42
>50
2b
>50
49.53 ± 1.52
23.63 ± 0.87
>100
42.64 ± 0.34
23.18 ± 1.25
21.30 ± 1.07
>50
>50
36.62 ± 0.86
24.17 ± 1.18
>50
48.45 ± 1.13
45.23 ± 1.29
35.95 ± 0.52
>50
46.58 ± 1.32
2c
2d
42.52 ± 1.16
>50
2e
>100
37.45 ± 0.87
>50
2f
>100
>100
>100
>100
2g
24.73 ± 1.56
33.52 ± 1.85
>50
19.84 ± 0.72
25.43 ± 0.96
46.12 ± 1.24
>100
17.28 ± 0.75
33.16 ± 1.26
>50
32.32 ± 1.56
35.46 ± 1.73
>50
2h
>50
2i
>100
>50
2j
>100
>100
>100
2k
45.59± 1.55
44.41± 1.18
>100
47.20 ± 0.52
>50
48.97± 1.28
>50
>50
>50
2l
>50
>100
>100
>100
>100
>200
>100
>100
>50
2m
>100
>100
>50
2n
>100
>100
>100
>100
2o
>100
>100
>100
>100
2p
>100
>100
>100
>100
2q
>200
>200
>200
>200
2r
2s
>100
>100
>100
>100
>100
>100
>100
>100
thiabendazole
>50
30.13 ± 1.12
>50
24.78 ± 0.52
Thiabendazole was used as the positive control.
All data represent the mean ± S.D. of at least three independent experiments performed in
triplicates.
The results listed in the Table 1 also revealed that the introduction of a chlorine
atom (2i) or an unsaturated aliphatic chain (2k) at the position of the C=O group
generally did not affect the activity compared to compound 2a. Thus, we can
hypothesize that the Cl group or a conjugated (-MeC=CH-C=O-) unit in the alkyl
chain is sensitive to the tested pathogens, especially G. cingulata. A similar result was
observed as we introduced a cyclopropyl group (2l) on the side chain (2l vs. 2a). In
addition, compounds 2b–2d, 2h and 2k were significantly more active than the

positive control, thiabendazole, for some fungi. These results suggested the possibility
of the alkyl acetophenones as antifungal agents.
To gain a further insight into the SAR, we calculated the lipophilicity values (log
P) of the compounds 2a–2s (Scheme 1). According to Ghose and Crippen’s
fragmentation method using ChemDrawUltra software, log P was expressed as
logarithm of partition coefficient in octan-1-ol/H2O.12 As shown in Scheme 1,
compounds 2c (log P 2.13), 2d (log P 2.54) and 2g (log P 2.27) exhibited obviously
higher antifungal activities than lipophilic compounds 2e (log P 2.96) and 2f (log P
3.38), while 2b (log P 1.71) and 2k (log P 2.09) displayed high antifungal activities.
The chloromethyl derivative 2i (log P 1.58) and the cyclopropyl derivative 2l (log P
1.78) exhibited better antifungal activities than the parent product 2a (log P 1.05) on a
wide range of organisms. The introduction of benzene rings (i.e., 2m, log P 3.11; 2n,
log P 3.51) led to a decrease in antifungal activity. The results revealed that the most
active compounds have a log P value in the range of 1.71–2.54, the two exceptions
being compounds 2j (log P 2.2) and 2q (log P 2.21) which decreased antifungal
effects, despite their reasonable log P value. This was most likely due to the
electron-attracting substituent in the side chain; the decrease in antifungal activity for
compounds with log P> 2.5 could be due to an unfavorable steric hindrance. This
result suggests that the addition of the aliphatic side chain increases the lipophilicity
of the compounds, leading to an improved antifungal activity, which might be
associated with better penetration through the plasmatic membrane of the fungi.
In conclusion, we synthesized a series of new acetophenone analogs and assayed
their antifungal activities in vitro. Among the synthetic compounds, alkyl ketone
derivatives exerted broad-spectrum antifungal activities to several plant pathogens,
and an increase in the chain length of linear alkyl ketones led to enhanced antifungal
activities; branched alkyl ketones exhibited stronger activity than linear alkyl ketones.
And these active compounds have a log P value ranging from 1.71 to 2.54. In
particular, compound 2g (log P 2.27), with an isobutyryl group, produced a
remarkable in vitro antifungal activity, which might be considered a promising
candidate in the development of new fungicides.

Acknowledgements
This work was supported by grants from the Program of Unified Planning
Innovation Engineering of Science & Technology in Shaanxi Province
(No.2015KTCQ02-14) and the National Natural Science Foundation of China (No.
21542016, 30971882).
Conflicts of Interest
The authors declare no conflict of interest.
Supplementary data
Supplementary data associated with this article can be found, in the online
version, at http://.

References and notes
1. Villegas, A. M.; Peralta, K. D.; Tapia, C. G.; Marin, K. C.; Catalan, L. E. Nat. Prod. Res.2015, 29,
586.
2. Thind, T. S. Indian Phytopathology 2012,65, 109.
3. Simonsen, H. T.; Adsersen, A.; Bremner, P.; Heinrich, M.; Smitt, U. W.; Jaroszewski, J. W.
Phytother. Res. 2004, 18, 542.
4. Gao J.-M., L. Y., Xie S.-D., Zhang A.-L., Shi X.-W., Zhang W.-L. China Patent; 2009,
200910020963.7.
5. Nandinsuren, T.; Shi, W.; Zhang, A. L.; Bai, Y. B.; Gao, J. M. Nat. Prod.
Res.2015,DOI:10.1080/14786419.2015.1041140.
6. Ma, Y. T.; Fan, H. F.; Gao, Y. Q.; Li, H.; Zhang, A. L.; Gao, J. M. Chem. Biol. Drug Des. 2013, 81,
545.
7. Ito, S.; Yazawa, S.; Nakagawa, Y.; Sasaki, Y.; Yajima, S. Bioorg. Med. Chem. Lett. 2015, 25, 1774.
8. Ma, Y.; Marquis, R. E. Lett. Appl. Microbiol. 1996, 23, 329.
9. Liu, K.; Xu, L.; Berger, J. P.; MacNaul, K. L.; Zhou, G.; Doebber, T. W.; Forrest, M. J.; Moller, D.
E.; Jones, A. B. J. Med. Chem. 2005, 48, 2262.
10. 1H and 13C NMR spectra were recorded with a Bruker Avance 500 MHz. MS spectra was taken on
a Bruker Daltonics esquire 6000 (ESI-ION TRAP). Spectral data of representative compound 2g. Light
yellow solid, yield 70%, m.p. 119.2-120.7 °C. 1H NMR (500 MHz, CD3OD) δ 7.71(s, 1 arom. H), 6.51
(s, 1 arom. H), 3.63-3.52 (m, 1H, CH), 2.07 (s, 3H, CH3), 1.17 (6H, d, J = 12.8 Hz, 2CH3); 13C NMR
(125 MHz，CD3OD) δ210.1, 164.7, 163.0, 130.0, 112.1, 112.1, 107.8, 35.0, 19.9, 7.8; HRESI-MS: m/z
[M-H]- 193.1834 (calcd. for C11H13O3, 193.1825).
11. Antifungal bioassay: The in vitro antifungal activity was determined by measuring half
maximal inhibitory concentration (IC50) according to the reported method.5,13 Briefly, after
incubation with these compounds for 72 h, the mycelial growth of fungi (mm) in both treated (T)
and control (C) Petri dishes was measured diametrically in three different directions (radial
fungal-growth assay) till the fungal growth in the control dishes was almost complete. The
percentage of growth inhibition (I) was calculated using the following formula. I (%) = [(C-T)/C]
× 100%.
12. Ghose, A. K.; Crippen, G. M. J. Chem. Inf. Comput. Sci. 1987,27, 21.
13. Bai, Y. B.; Zhang, A. L.; Tang, J. J.; Gao, J. M. J. Agr. Food Chem.2013, 61, 2789.

Graphical Abstract