Synthesis and bio-inspired optimization of drimenal: Discovery of chiral drimane fused oxazinones as promising antifungal and antibacterial candidates

Article abstract of DOI:10.1016/j.ejmech.2017.11.051

The synthesis of antifungal natural product drimenal was accomplished. Bio-inspired optimization protruded chiral 8-(R)-drimane fused oxazinone D as a lead, considering favorable physicochemical profiles for novel pesticides. The improved scalable synthesis of scaffold D was implemented by Hofmann rearrangment under mild conditions. Detailed structural optimization was discussed for both antifungal and antibacterial exploration. Substituted groups (SGs) with C₃～C₅ hydrocarbon chain are recommended for exploration of antifungal agents, while substituents with C₄～C₆ carbon length are preferred for antibacterial ingredients. The chiral drimane fused oxazinone D8 was selected as a promising antifungal candidate against Botrytis cirerea, with an EC₅₀ value of 1.18 mg/L, with the enhancement of up to >25 folds and >80 folds than the mother compound D, and acyclic counterpart AB5, respectively. The in vivo bioassay confirmed much better preservative effect of D8 than that of Carbendazim. The chiral oxazinone variant D10 possessed prominent antibacterial activity, with MIC values of 8 mg/L against both Bacillus subtilis and Ralstonia solanacearum, showing advantages over the positive control streptomycin sulfate.

Full text of DOI:10.1016/j.ejmech.2017.11.051

Accepted Manuscript
Synthesis and bio-inspired optimization of drimenal: Discovery of chiral drimane fused
oxazinones as promising antifungal and antibacterial candidates
Dangdang Li, Shasha Zhang, Zehua Song, Wei Li, Feng Zhu, Jiwen Zhang,
Shengkun Li
PII:
S0223-5234(17)30954-6
10.1016/j.ejmech.2017.11.051
EJMECH 9926
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 September 2017
Revised Date: 28 October 2017
Accepted Date: 19 November 2017
Please cite this article as: D. Li, S. Zhang, Z. Song, W. Li, F. Zhu, J. Zhang, S. Li, Synthesis and
bio-inspired optimization of drimenal: Discovery of chiral drimane fused oxazinones as promising
antifungal and antibacterial candidates, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.11.051.
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Graphical Abstract

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Synthesis and Bio-inspired Optimization of Drimenal: Discovery of
Chiral Drimane Fused Oxazinones as Promising Antifungal and
Antibacterial Candidates
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Dangdang Li a, Shasha Zhang a, Zehua Song a, Wei Li a, Feng Zhu c, Jiwen Zhang c,
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Shengkun Li a,b
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a, Department of Pesticide Science, College of Plant Protection, Nanjing Agricultural
University, Weigang 1, Xuanwu District, Nanjing 210095, People’s Republic of
China.
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b, Department of Chemistry, The Chinese University of Hong Kong, Shatin, New
Territories, Hong Kong, People’s Republic of China.
c, College of Chemistry and Pharmacy, Northwest A&F University, No. 3 Taicheng
Road, Yangling, Shaanxi 712100, People’s Republic of China.
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Corresponding Author: Shengkun Li, Email SKL505@outlook.com
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Abstract: The synthesis of antifungal natural product drimenal was accomplished.
Bio-inspired optimization protruded chiral 8-(R)-drimane fused oxazinone D as a lead,
considering favorable physicochemical profiles for novel pesticides. The improved
scalable synthesis of scaffold D was implemented by Hofmann rearrangment under
mild conditions. Detailed structural optimization was discussed for both antifungal
and antibacterial exploration. Substituted groups (SGs) with C3~C5 hydrocarbon chain
are recommended for exploration of antifungal agents, while substituents with C4~C6
carbon length are preferred for antibacterial ingredients. The chiral drimane fused
oxazinone D8 was selected as a promising antifungal candidate against Botrytis
cirerea, with an EC50 value of 1.18 mg/L, with the enhancement of up to >25 folds
and >80 folds than the mother compound D, and acyclic counterpart AB5,
respectively. The in vivo bioassay confirmed much better preservative effect of D8
than that of Carbendazim. The chiral oxazinone variant D10 possessed prominent
antibacterial activity, with MIC values of 8 mg/L against both Bacillus subtilis and
Ralstonia solanacearum, showing advantages over the positive control streptomycin
sulfate.
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Keywords: Chiral Pesticide, Lead Optimization, Drimane, Oxazinone,
Structure-activity Relationship
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1. Introduction
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The ever increasing population coupled with changing dietary habits throughout the
world, required continued improvements in food supplement from limited available
farmland [1, 2]. With an evolving array of agrochemical tools, the agrochemical
industry is a driving force in the green revolution to control a myriad of agricultural
pests. It is desirable for the discovery of new agrochemicals with favorable
environmental and toxicological profiles, which has been playing an undoubtedly
important role in the management of resistance and shifting pest spectra and other
agricultural practices. One of the most important issues and the biggest challenges is
the discovery of novel structures with high potency for a specific target.
The combinatorial chemistry and high-throughput screening (HTS) generated
hope for revolutionizing drug discovery. Structure-based drug design or biotational
design emerged as powerful tools and have been attracting remarkable attention in
both medicines and agrochemicals[3]. Beside the target-oriented tactics, the
ligand-optimization is anastrophic but also powerful and easily operable wepeaon to
address biological problems. Alongside the fruitful achievements in the discovery of a
new drug[4] or pesticides[5] with the old ones as models, natural products are
attracting starting points, not only in the novel scaffold but also can act as a probe for
novel target [6, 7]. Unfortunately, many “hit” compounds were unsuccessfully
converted to “lead” compounds, or unsuitable for further optimization due to the
inherently unfavorable properties, i.e., size, permeability, and conformation. So we
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envisage that simulation of the bioactively important natural products is a good choice
for lead discovery, in which physicochemical profiles should be attached and
compared as important metrics before structure optimization.
Drimane and related meroterpenoids attracted wide attention from synthetic
chemists because of various bioactivities, ranging from medicinal importance to
agrochemical interests[8, 9]. Our interest in drimane related natural products was
triggered by the special structural properties with the enrichments in sp3 fraction,
which is important for improving druggablity[10, 11]. Consistent with that of other
bioactive terpenoids (e.g. artemisinin), the biologically important drimane also
featured specific mono or multi-oxidation, or rigid conformation (fused ring or
chirality) of its core structure[8], such as the antifungal natural products drimenal[12],
polygodial, isotadeonal and marasmal B[13] (Fig.1). Our continuous efforts[14, 15] in
the discovery of novel chiral fungicides candidates based on natural products
encourage us to explore the drimane related natural agents, and the antifungal
drimenal gave us an impetus. Herein, we would like to document the synthesis,
bioactivity and bioinspired optimization of drimenal and drimenol, in which the chiral
drimane fused oxazinones were discovered as promising antifungal and antibacterial
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candidates.
2. Results and discussion
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2.1. Molecular Design and Chemical Synthesis
2.1.1 Synthesis of Drimenal and Drimenol
Degradation of abundant diterpenoids will be a good choice for the
semi-synthesis of drimane related sesquiterpenoids. As shown in Fig. 2, the
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commercially inexpensive (-)-sclareol was chosen as a starting material for the
scalable preparation of (+)-sclareolide in good yield [16]. The 8-OH drimanol was
synthesized in three steps using only one SiO2 column chromatography according to
the previous procedure in a moderate yield [17],[18], with the Baeyer-Villiger
rearrangement as the key translation. The p-TsOH mediated one-step regioselective
dehydration of the tertiary hydroxyl group provided a more facile access to drimenol
than the previous Mitsunobu conditions (DEAD/PPh3)[19], in view of both high yield
and operational simplicity. It can also be achieved effectively through a 3-step
approach reported by Hayakawa and Kigoshi [20]. Oxidation of drimenol with P2O5
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afforded the drimenal in good yield, whose HNMR and 13CNMR spectra were
identical to those previously reported [12].
2.1.2 Divergent synthesis of drimane mimics
As shown in Fig. 3, divergent synthesis was conducted for Drimenol and
8-hydroxyl Drimanol, including different ethers, esters, and carbamates. The
previously founded bioactivity-guided mixture synthesis[14] was applied for the
discovery of the promsing orientation of structural optimization. A number of
halogenated hydrocarbons, acids, and amines were selected to get more precise insight
into the effect of different substituents on bioactivity, and typical derivatives were
listed in Fig. 3. Exposure of these alcohols to halogenated hydrocarbons in refluxing
anhydrous THF with KOH as base furnished different ethers (A, AB). The alcohol
esters of drimenol and 8-hydroxyl drimanol were prepared efficiently with different
organic acids through Steglich-type reaction (B, BB). The carbamates (C, CB) were
prepared easily from the condensation of alcohols and isocyanates with toluene as
media. The 8-hydroxyl drimanol was further oxidized by Jone’s reagent to produce
the drimanic acid, from which the esters (AC) were achieved after the nucleophilic
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attack of carboxylic anion to halogenated hydrocarbons.
2.1.3. Design and synthesis of rigid drimane fused dioxanone
The conformation restricted counterpart 8-(R)-dioxanone (E), was synthesized by
trapping of 8-hydroxyl drimanol with triphosgene under -78
in 87% yield. The
aza-bioisostere D was prepared from sclareolide, through hydrazinolysis followed by
curtius rearrangement, and the 8-epi stereoisomer 8-(S)-Oxazinone (8-epi D) was
constructed based on 8-epi-(+)-sclareolide, which can be prepared efficiently via the
exposure of (+)-sclareolide to a mixture of sulfuric and formic acids [21]. Both
8-(R)-oxazinone and 8-(S)-oxazinone were confirmed by single crystal diffraction,
with the CCDC numbers 1569963 and 1569964 respectively (Fig. 4).
The poor yield of chiral 8-(R)-oxazinone D from curtius rearrangement was due
to the reverse reaction to the corresponding sclareolide and the formation of the
8-formyl drimanyl amine as a byproduct. Fortunately, the improvement in the
synthesis of D was realized by switching the Curtius reaction to Hofmann
rearrangement [22], in which the basic system of PIDA can translate the
homodrimanyl amide to intra-carbamate in up to 92% yield under relatively mild
condition (Fig. 5). The optimization of 8-(R)-oxazinone was initiated with the
decoration of the “NH” in amide subunit, through nucleophilic substitution under
basic conditions. Gratifyingly, the structure and stereochemistry of the oxazinone
derivatives were further determined by acquiring single crystal X-ray structure of the
D12 (CCDC 1569965), other than the MASS, HNMR, and CNMR spectra.
2.2. Antifungal Activity and SAR Discussion
2.2.1. SAR of Drimenal, Drimenol, and Related Derivatives
As shown in Fig. 2, the antifungal activity of drimenal is more promising than
that of its reduced counterpart drimenol, and we envisaged that the antifungal
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bioactivity may be improved with the increment in the oxidative states of drimane
scaffold, examplified by the aforementioned drimanes related natural products. The
drimenal usually served as a starting material to establish many biologically important
drimane quinones, while the instablitiy of drimenal and drivatives thereof made us
explore the more stable and easily accessible precursors, i.e. Drimenol and
8-hydroxyl-drimanol, for structural optimization, biological evaluation, and SAR to
get more promising agrochemical candidates.
The homodirmanyl amides were protruded as promising antifungal agents[14],
showing similar antifungal spectrum to that of Drimenol and Drimenal. The
frequently used substituted groups (SGs) from the commercially available source
were decorated to the core structure of drimane, including saturated and unsaturated
halogenated hydrocarbons, aicds, and isocyanate. After intitial bioactivity- guided
mixed synthesis and antifungal assessment, all the compounds from 8-OH-Drimanol
listed in Fig.3 were synthesized specifically and evaluated against Rhizoctonia solani,
Sclerotinia sclerotiorum, Fusarium graminearum, and Botrytis cirerea (supporting
information). The derivative mixture of 8-OH-drimanol was more active than that of
drimenol, which confirm the crucial role of the hydroxyl group on the antifungal
effect (AB vs A, BB vs B, CB vs C). It is noteworthy that the etherification of
drimenol did not show advantages either in stability and bioactivities. The
hydrophobic and sterically bulky sbustituents were baleful for the inhibtition of all the
tested plant pathogens. As shown in Table 1, all the compounds were prepared and
EC50 values were tested specifically for derivatives of 8-hydroxyl drimanol, and
substituted groups (SGs) were recommended to be shorter than 5-carbon-length.
The oxidation at 9-position (AC) is more beneficial for the improvement in the
antifungal activity against both Sclerotinia sclerotiorum and Botrytis cirerea. The
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formal NH insertion of BB3 or amide insertion of AB5 to get CB2 led to blossom in
antifungal bioactivity, which can be synthesized easily from the condensation of
isocyanate and drimanol. Though both the esters of drimanic acid and carbamates of
8-hydroxyl drimanol showed a comparable antifungal effect against Sclerotinia
sclerotiorum and Botrytis cirerea, utilization of 8-hydroxyl-drimanol as scaffold has
advantages over both drimenol and drimanic acid in synthetic manipulation or/and
antifungal potential.
2.2.2. Discovery of chiral drimane fused oxazinones as potential lead compounds
Flexible molecules may require more energy to adopt a needed binding
conformation, reducing the biological potency. The rigidification or conformational
restriction of ligands has attracted remarkable attention and been widely applied in
both drug [23]and pesticide [24] design, which can minimize the entropic loss for the
ligands in binding with pharmacological target through adopting a preferred
conformation, which leads to enhanced potency and improved selectivity, and reduces
the possibility of undesired metabolism.
Inspection of 8-OH-drimanol showed that it has potential in conformational
restriction via the tether of the free teminal hydroxyl groups to reduce the number of
free rotatable bonds (Fig. 4). Considering the Clog P is an important metric to
distinguish fungicides from herbicides and insecticides, the calculated analysis of the
aforementioned scaffolds was conducted before lead optimization, and the results
rationalize our design, with the Clog P values of 2.974 and 2.834 for 8-(R) -oxazinone
(scaffold D) and cyclic carbonate ester (scaffold E), respectively. The synthesized
compounds D, 8-epi-D and E were evaluated against up to 11 kinds of plant
pathogens (see supporting information), to find the valuable one and explore the
effects of chirality, conformational restriction and substitution of “O” by “NH”. The
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conformation constrained variations showed good activity against R. solani, S.
sclerotiorum, F. graminearum and B. Cirerea. The 8-(R)-Oxazinone (D) possessed
much better acitivtity than its diastereoisomer 8-(S)-Oxazinone against most of the
tested fungi, which is a little different from the case our previously reported about
homodrimanyl amides[14]. The importance of the introduction of “NH” was
confirmed by the dramatic decline in the activity when “NH” was replaced by
“O”(scaffold D vs E). With easy preparation, good activity, novel scaffold, favorable
physicochemical profiles and optimizable potential, the chiral drimane fused
oxazinone D can be chosen as a lead compound. The improved synthesis of D via
Hofmann rearrangement laid a solid foundation for scalability and optimization.
The antifungal activity was enhanced with the expansion of hydrocarbon chain
(D1 ~ D8), while the reverse phenomenon was detected when the carbon chain longer
than the n-butyl group (D9 and D10), the antifungal activity was almost lost when
n-dodecyl group was imported (D11), this may be due to the excessive increment in
the Clog P values. Substituted groups (SGs) with C3 ~ C5 hydrocarbon chain are
recommended for detailed exploration, and C3 substituents were chosen as a model
(D3 ~ D6). Sterically hindered isomer is unfavorable in view of reduced yield and
activities (D3 vs D4), desaturation of D3 didn’t produce fruitful improvement either
(D5 and D6). Interestingly, decoration of prenyl (D7), formally dimethylation of D5,
reinforced the activity by more than 2 and 3 folds against Fusarium graminearum and
Botrytis cirerea, respectively. Patulous allyl systems, including benzyl and substituted
benzyl groups, were also brought to the drimane fused oxazinone (D12 ~ D19), and
the activities were strengthened with the simplest benzyl group. It is noteworthy that
the biological activities are very sensitive to the position and electronic properties of
substituted groups on the phenyl ring. Though the para-methyl candidate (D14) can
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provide comparable results, the antifungal activities dropped sharply when it was
switched to meta-position (D13) or replaced by the electron-withdrawing groups,
including fluorine (D15) and chlorine (D18). Gratifyingly, elaborate tuning protrudes
the n-butyl substituted variant D8 as a promising antifungal candidate, possessing
pronounced activities against Sclerotinia sclerotiorum and Botrytis cirerea, with EC50
values of 7.70 mg/L and 1.18 mg/L, respectively (Fig. 5, Table 2). It is worth noting
that the activity against Botrytis cirerea was enhanced >25 folds, >10 folds, and >80
folds than mother compound D, acyclic counterparts CB2 and AB5, respectively.
With the widely used commercial fungicide Carbendazim as a positive control, the in
vivo antifungal effect of the promising oxazinone D8 was conducted against Botrytis
cinerea on cucumber leaf. As shown in Fig. 6, compound D8 showed much better
preventative effect (＞ 4.5 fold) than that of Carbendazim, with preventative rate of
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up to 58% at 100 mg/L, verifying the original molecular design and the potential of
drimane fused oxazinone as a lead for the discovery of novel agrichemicals.
Considering the importance of oxazolidinone in the antibiotic market [25], the
synthesized drimane fused oxazinones, formally with an expanded oxazolidinone ring,
were further explored for the antibacterial possibility against both Gram negative
bacteria and Gram positive bacteria, including Xanthomonas oryzae (-), Erwinia
carotovora (-), Xanthomonas oryzae pv.oryzicola (-), Ralstonia solanacearum (-),
Pseudomonas syringae pv. Lachrymans (-), Bacillus subtilis (+), and Clavibacter
michiganensis subsp. Sepedonicus (+). To our delight, most of the oxazinones
demonstrated moderate to good antibacterial activities against agriculturally important
Bacillus subtilis and Ralstonia solanacearum. There are 19 entries much more active
than the positive control streptomycin sulfate. Similar SAR to that of the antifungal
section was detected (Table 3 vs Table 2), substitution on the NH subunit is necessary
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for the enhancement of antibacterial activity, while the preferred substituents were
extended from C3-C5 to C4-C6 hydrocarbon chain. As can be seen in Table 3, the three
tested bacteria were inhibited by D7 and D10 much easier than the positive control
streptomycin sulfate. Noteworthily, the benzyl variant (D16) also showed much better
activity against Bacillus subtilis and Ralstonia solanacearum than the positive control.
The candidate D10 was most potent and can inhibit efficiently Bacillus subtilis,
Xanthomonas oryzae pv.oryzicola and Ralstonia solanacearum, with the MIC values
of 8, 12.5 and 8 mg/L respectively (Fig.5, Table 3).
3. Conclusions
In summary, the synthesis and antifungal exploration of drimenol and drimenal
were accomplished and followed by bioactivity-guided divergent optimization.
Inspired by the bioactive natural drimane sesquiterpenoids and related quinones or
hydroquinones, the 8-hydroxyl-drimanol was selected as a potent scaffold and further
sublimated to chiral drimane fused oxazinones, considering the favorable
physicochemical profiles before structural optimization. The chiral 8-(R)-drimane
fused oxazinone D was protruded as an optimized lead, with good activity, novel
chiral scaffold, favorable physicochemical profiles as well as optimizable potential.
The improved scalable synthesis of this chiral scaffold was implemented by Hofmann
rearrangment under mild conditions, which facilitated the bioactivity-guided
optimization. Substituted groups (SGs) with C3~C5 hydrocarbon chain are
recommended for the exploration of antifungal agents, while substituents with C4~C6
carbon length are preferred for antibacterial ingredients. Gratifyingly, the chiral
8-(R)-drimane fused oxazinone D8 possessed prominent antifungal activity against
Botrytis cirerea, with EC50 value of 1.18 mg/L, demonstrating the enhancement of up
to > 25 folds, > 10 folds, and > 80 folds than mother compound D, acyclic
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counterparts CB2 and AB5, respectively. The in vivo bioassay demonstrated much
better preventative effect of D8 than that of Carbendazim. The chiral oxazinone
variant D10 was most potent in antibacterial screening, with MIC values of 8 mg/L
against both Bacillus subtilis and Ralstonia solanacearum, showing advantages over
the positive control streptomycin sulfate. Further exploration of those chiral alkaloids
in solving biological problems and related mechanism is in progress and will be
reported in due course.
4. Experimental section
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4.1. Chemistry
4.1.1. Instruments, Chemicals and Related Materials.
All solvents and reagents were purchased from commercial sources (Energy or
Meryer Chemicals etc.), they were analytically pure and used as received. Anhydrous
solvents were dried and distilled by standard techniques before use. Silica gel GF254
and column chromatography silica gel for isolation (200 ~ 300 mesh) were both
purchased from Qingdao Broadchem Industrial Co., Ltd. Reaction progress was
monitored by thin-layer chromatography (TLC) on silica gel GF254 with
phosphomolybdic acid and ultraviolet (UV254nm) detection. Yields of all the title
compounds were not optimized. Melting points (m.p.) were recorded on Shenguang
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WRS-1B melting point apparatus and are uncorrected. HNMR and CNMR spectra
were carried out utilizing a Bruker AV 400 or 500 spectrometers with CDCl3 as
solvent and tetramethylsilane as the internal standard. Electrospray ionization mass
spectrometry (ESI-MS) data were obtained with Waters Xevo TQ-S
Micro-Spectrometer. Elemental analyses were performed on a CHN-O-Rapid
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instrument. FTIR data was collected on FTIR Research Spectrometers. The single
crystal diffraction was carried out on Bruker SMART APEX CCD diffractometer.
The fungi and bacteria were provided by the College of Plant Protection, Nanjing
Agricultural University (Nanjing, China).
4.1.2. Synthesis of 8-hydroxyl drimanol
Synthesis of (+)-sclareolide was accomplished through the oxidative
degradation of (-)-sclareol [16]. The 8-hydroxyl drimanol was synthesized in three
steps according to the previous procedures reported by Kane [17] and George [18] in
54% yield, see supporting information for synthetic details.
4.1.3. Synthesis of Drimenol
To a solution of 8-hydroxyl drimanol (1.20 g, 5.0 mmol, 1.0 equiv) in CH2Cl2
(20 mL), p-TsOH·H2O (0.95 g, 5.0 mmol, 1.0 equiv) was added in portions at 0 °C.
The reaction mixture was stirred and allowed to warm to ambient temperature (15 ~
17 °C), the reaction progress was monitored by TLC until the reaction was complete.
The mixture was diluted with CH2Cl2 (30 mL), and quenched by the addition of the
saturated NaHCO3 aqueous solution (30 mL). The organic phase was washed
sequentially with NaHCO3 (30 mL × 2), H2O (30 mL × 3) and brine (30 mL), then
dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by
flash chromatography on silica gel by gradient elution with petroleum ether/EtOAc
(v/v) = 10:0 to 10:2 to yield Drimenol as a white solid (0.64 g, 58 %). M.p.:70.7 °C.
[α]2D8.9 = −25.27 (c 0.1, DCM). 1H NMR (500 MHz, CDCl3) δ 5.54 (d, J = 5.6 Hz, 1H,
C=CH-CH2), 3.85 (dd, J1 = 11.30 Hz, J2 = 3.25 Hz, 1H, CH2OH), 3.73 (dd, J1 =
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11.30 Hz, J2 = 4.90 Hz, 1H, CH2OH), 1.94~2.02 (m, 2H, H in naphthane ring),
1.82~1.91 (m, 2H, H in naphthane ring), 1.78 (s, 3H, CH3C=CH-CH2), 1.57(m, 1H,
H in naphthane ring), 1.41~1.47 (m, 2H, H in naphthane ring), 1.32 (s, br, 1H, OH),
1.17 (td, J1 = 13.65 Hz, J2 = 4.20 Hz, 2H, H in naphthane ring), 1.07 (td, J1 = 13.65
Hz, J2 = 3.90 Hz, 1H, H in naphthane ring), 0.89 (s, 3H, CH3), 0.87 (s, 3H, CH3),
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0.86 (s, 3H, CH3); C NMR (125 MHz, CDCl3) δ 132.88 (C), 124.12 (CH), 60.93
(CH2), 57.28 (CH), 49.89 (CH), 42.13 (CH2), 39.89 (CH2), 36.07 (C), 33.37 (CH3),
32.92 (C), 23.58 (CH2), 22.06 (CH3), 21.95 (CH3), 18.76 (CH2), 14.94 (CH3).
Elemental anal. calcd for C15H26O: C, 81.02; H, 11.79; Found: C, 81.11; H, 11.65;
LC–MS (ESI+) m/z: Calcd. for C15H27O [M+H]+: 223.21, Found: 223.21;
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C15H26ONa [M +Na]+ : 245.19, Found: 245.34.
4.1.4. Synthesis of Drimenal
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To a solution of Drimenol (0.444 g, 2.0 mmol, 1.0 equiv) in CH2Cl2 (20 mL),
was added DMSO (1.0 mL, 14.08 mmol, 7.0 equiv) and P2O5 (3.0 g, 21.13 mmol,
10.5 equiv) successively at 0 °C. The reaction mixture was stirred at 15 ~ 17 °C for
about 0.5 h, and cooled to 0 °C again followed by the addition of the second batch of
NEt3 (1.2 mL, 8.5 mmol, 4.25 equiv). It was stirred at 15~17 °C until the complete
consumption of Drimenol, monitored by TLC. The mixture was quenched by the
addition of cold saturated NH4Cl aqueous solution (10 mL). The organic phase was
separated and the aqueous phase was extracted by CH2Cl2 (10 mL × 2), the combined
organic phase was washed with brine (15 mL), dried over Na2SO4, filtered and
concentrated in vacuo. The residue was purified by flash chromatography on silica gel
1
(200-300m) with PE/EtOAc = 5:1 as eluent to yield drimenal (0.405 g, 92%). H
NMR (400 MHz, CDCl3) δ 0.87 (s, 3H, CH3), 0.92 (s, 3H, CH3), 1.07 (s, 3H, CH3),
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1.15 (dd, J1 = 11.92 Hz, J2 = 5.04 Hz, 1H), 1.22 (dd, J1 = 13.48 Hz, J2 = 4.04 Hz, 1H),
1.28 (td, J1 = 12.96 Hz, J2 = 3.64 Hz, 1H), 1.41~1.48 (m, 2H), 1.54 (m,1H), 1.62 (brs,
3H, CH3), 1.66 (dq, J1 = 12.80 Hz, J2 = 3.20 Hz, 1H), 1.97 (m, 1H), 2.08 (m, 1H),
2.59 (s, 1H), 5.69 (m, 1H), 9.69 (d, J = 5.12 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ
15.73 (CH3), 18.28 (CH2), 21.63 (CH3), 22.08 (CH3), 23.66 (CH2), 33.03 (C), 33.31
(CH3), 37.01 (C), 40.36 (CH2), 41.99 (CH2), 49.05 (CH), 67.58 (CH), 125.47 (CH),
127.78 (C), 206.72 (C). Elemental anal. calcd for C15H24O: C, 81.76; H, 10.98; Found:
C, 81.80; H, 10.91; LC-MS (ESI) m/z: Calcd. for C15H25O [M+H]+: 221.19, Found:
221.21; C15H24ONa [M +Na]+: 243.17, Found: 243.24.
See supporting information for synthetic procedure for ethers (Series A and AB),
esters (Series B and BB) and carbamates (Series C and CB) of Drimenol and
8-OH-Drimanol; and the synthesis of esters of 8-OH-Drimanic Acid (Series AC).
4.1.5. Synthesis of drimane fused oxazinone (scaffolds D and 8-epi-D).
Curtius Rearrangement Approach. To a solution of (+)-sclareolide (2.50 g, 10.0
mmol, 1.0 equiv) in ethanol (20 mL) was added hydrazine hydrate (5 mL, 0.54 mmol,
1.0 equiv) dropwise with an ice-bath. The reaction mixture was then stirred at room
temperature for about 60 min until the reaction was complete monitored by TLC. The
solvent was removed under reduced pressure to give crude products. The residue was
purified by flash chromatography on silica gel (200 ~ 300 m) (EtOAc to
MeOH/EtOAc = 1:20, v/v) to give the 8-(R)-acylhydrazine intermediate as a white
solid (2.62 g, yield 93%), Rf = 0.13 (MeOH/Ethyl Acetate = 1:10), M.p.
150.5-153.5 . 1HNMR (400 MHz, CDCl3) δ 0.79 (s, 6H, 2×CH3), 0.87 (s, 3H, CH3),
0.95~0.99 (m, 2H, H in naphthane ring), 1.14 (s, 3H, CH3), 1.15 (td, J1 = 13.48 Hz, J2
= 4.08 Hz, 1H, H in naphthane ring), 1.21~1.62 (m, 8H, H in naphthane ring and NH2),
1.68 (m, 1H, H in naphthane ring), 1.77 (dd, J1 = 5.32 Hz, J2 = 4.40 Hz, 1H, H in
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naphthane ring), 1.93 (dt, J1 = 12.48 Hz, J2 = 3.28 Hz, 1H, H in naphthane ring), 2.14
(dd, J1 = 15.36 Hz, J2 = 4.40 Hz, 1H, CH2-C=O), 2.37 (dd, J1 = 15.36 Hz, J2 = 5.32
Hz, 1H, CH2-C=O), 7.47 (br, s, 1H, NH-C=O). LC–MS (ESI+) m/z: Calcd. for
C16H29N2O [M-H2O+H]+ 265.23, Found:265.34; C16H30N2O2Na [M+Na]+305.22,
Found:305.32.
The 8-epi-acylhydrazine counterpart was prepared starting from 8-epi-sclareolide
through similar manipulation in 92% yield.
To a solution of the synthesized 8-(R)-acylhydrazine (564 mg, 2.0 mmol, 1.0
equiv) in THF/H2O (10 mL/ 10 mL) were added NaNO2 (207 mg, 3.0 mmol, 1.5
equiv). The reaction was cooled to 0 oC with ice-bath, then 5.76 mL of 0.5 M aqueous
solution of HCl was added, and the reaction mixture was stirred until the full
conversion of acylhydrazine monitored by TLC. The reaction mixture was then
extracted with DCM (20 mL × 3). The combined organic phase was washed with 5%
NaHCO3 (20 mL × 2), water (20 mL × 2) and brine (20 mL), dried over anhydrous
Na2SO4, filtered and concentrated under reduced pressure to give crude products. The
resultant residues were purified by chromatography on silica gel (200 ~ 300m) with
carefully gradient elution to give the 8-(R)-oxazinone D in 36 % yield. Rf = 0.58
(MeOH/Ethyl Acetate = 1:10), M.p. 188.2-189.3 . 1H NMR (400 MHz, CDCl3) δ
0.82 (s, 3H, CH3), 0.87 (s, 3H, CH3), 0.90 (s, 3H, CH3), 1.01~1.07 (m, 2H, H in
naphthane ring), 1.18 (td, J1 = 14.80 Hz, J2 = 3.84 Hz, 1H, H in naphthane ring), 1.31
(m, 1H, H in naphthane ring), 1.39 (s, 3H, CH3), 1.42-1.55 (m, 3H, H in naphthane
ring), 1.60 (m, 1H, H in naphthane ring), 1.64-1.71 (m, 2H, H in naphthane ring), 1.76
(m, 1H), 2.02 (dt, J1 = 12.64 Hz, J2 = 3.16 Hz, 1H), 3.19 (m, 1H), 3.27 (m,1H), 6.28
(br, s, 1H, NH). Elemental anal. calcd for C16H27 NO2: C, 72.41; H, 10.25; N, 5.28;
Found: C, 72.36; H, 10.19; N, 5.33; LC–MS (ESI+) m/z: Calcd. for C16H27NO2Na
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[M+Na]+ 288.19, Found:288.35. The white solid was re-dissolved in CH2Cl2 to afford
colorless square crystal easily, which was confirmed by X-ray single crystal
diffraction (CCDC: 1569963).
The 8-(S)-oxazinone 8-epi-D was prepared accordingly in 23% yield. Rf = 0.58
(MeOH / Ethyl Acetate = 1:10), M.p. 248.6-249.3 . 1H NMR (400 MHz, CDCl3) δ
0.84 (s, 3H, CH3), 0.89 (s, 3H, CH3), 1.02 (s, 3H, CH3), 1.14 (td, J1 = 14.32 Hz, J2 =
4.68 Hz, 1H, H in naphthane ring), 1.27 (m, 1H, H in naphthane ring), 1.38 (s, 3H,
CH3), 1.39~1.70 (m, 8H, H in naphthane ring), 1.74 (m, 1H), 2.11 (dt, J1 = 13.80 Hz,
J2 = 2.64 Hz, 1H), 3.38 (dd, J1 = 12.68 Hz, J2 = 3.80 Hz, 1H), 3.48 (dd, J1 = 12.68 Hz,
J2 = 7.08 Hz, 1H), 5.78 (br, s, 1H, NH). Elemental anal. calcd for C16H27 NO2: C,
72.41; H, 10.25; N, 5.28; Found: C, 72.44; H, 10.15; N, 5.25; LC–MS (ESI+) m/z:
Calcd. for C16H27NO2Na [M+Na]+ 288.19, Found:288.32. This compound was also
confirmed by X-ray single crystal diffraction (CCDC: 1569964).
Hofmann Rearrangement Approach. The (+)-sclareolide (1.0 g, 4.0 mmol, 1.0
equiv) was dissolved in 7 M ammonia in MeOH (4.0 mL, 28 mmol, 7.0 equiv),
followed by the addition of NaOCH3 (0.216 g, 4.0 mmol, 1.0 equiv) at ambient
temperature, then the flask was immersed into the pre-heated oil bath at 60
and
stirred for 24 hours until the full conversion of sclareolide tracked by TLC. The
solvent was removed under reduced pressure to give crude products, which was
purified by flash chromatography on silica gel (200 ~ 300 m) (EtOAc to MeOH /
EtOAc = 1:20, as eluent, v/v) to give the 8-(R)-homodrimanyl amide intermediate as
white solid (0.94 g, yield 88%), Rf = 0.39 (MeOH/Ethyl Acetate = 1:10), M.p.
1
119.0-120.6 . H-NMR (400 MHz, CDCl3) δ 0.79 (s, 6H, 2 × CH3), 0.87 (s, 3H,
CH3), 0.97~1.03 (m, 2H, H in naphthane ring), 1.14 (td, J1 = 14.24 Hz, J2 = 4.12 Hz,
1H, H in naphthane ring), 1.15 (s, 3H, CH3), 1.26 (m, 1H, H in naphthane ring),
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1.35~1.70 (m, 6H, H in naphthane ring), 1.76~1.82 (m, 2H, H in naphthane ring), 1.93
(dt, J1 = 12.48 Hz, J2 = 3.24 Hz, 1H), 2.16 (dd, J1 = 15.56 Hz, J2 = 4.28 Hz, 1H), 2.43
(dd, J1 = 15.56 Hz, J2 = 5.08 Hz, 1H), 2.44 (br, s, 1H, OH), 5.49 (br, s, 1H, NH),
5.99(br, s, 1H, NH). LC–MS (ESI+) m/z: Calcd. for C16H28NO [M-H2O+H]+ 250.22,
Found 250.31; Calcd. for C16H29NNaO2 [M+Na]+ 290.21, found 290.31; Calcd. for
C32H58N2NaO4 [2M+Na]+ 557.43, found 557.51.
To the synthesized 8-(R)-homodrimanyl amide (267 mg, 1.0 mmol, 1.0 equiv) in
MeOH (10 mL) was added KOH (140 mg, 2.5 mmol, 2.5 equiv) at room temperature.
The reaction was cooled to 0oC, PIDA (354mg,1.1mol, 1.1equiv) was added in
portions, and the reaction mixture was then stirred until the reaction was complete
tracked by TLC. The volatile solvent was removed under reduced pressure and then
extracted with DCM (20 mL × 3). The combined organic phase was washed with
saturated aqueous solution of NH4Cl (15 mL × 3), water (15 mL × 3) and brine (15
mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure
to give crude products, which were purified by flash chromatography on silica gel
(200~300 m) to give the 8-(R)-oxazinone D as a white solid in 96% yield.
The improved synthesis of 8-epi-oxazinone (8-epi-D) was furnished accordingly
in 91% yield.
4.1.6. Synthesis of 8-(R)-dioxanone (Compound E) from 8-hydroxyl-Drimanol
To a solution of 8-OH-Drimanol (240 mg, 1.0 mmol, 1.0 equiv) in DCM (10 mL)
was added pyridine slowly (0.50 mL, 6.21 mmol, 6.21 equiv) at room temperature.
o
The reaction was cooled to -78 C, a solution of triphosgene (296 mg, 1.0 mmol, 1.0
equiv) in DCM (10 mL) was added dropwise, then the mixture was allowed to warm
gradually to room temperature and stirred for about 4 hours. The reaction was
quenched by the addition of 0.5 M HCl (12 mL), the inorganic phase was extracted
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with DCM (10 mL ×3). The combined organic phase was washed with water (15 mL
× 3) and brine (15 mL), dried over anhydrous Na2SO4, filtered and concentrated under
reduced pressure to give crude products which were purified by flash chromatography
on silica gel (200 ~ 300 m) with petroleum ether/EtOAc = 1:2 (V/V) as eluent to give
8-(R)- dioxanone E as white solid (231 mg, 87% yield). M.p.196.6-196.9 . 1H NMR
(400 MHz, CDCl3) δ 0.83 (s, 3H, CH3), 0.90 (s, 3H, CH3), 0.91 (s, 3H, CH3),
1.04-1.39 (m, 5H, H in naphthane ring), 1.42-1.58 (m, 3H, H in naphthane ring), 1.49
(s, 3H, CH3), 1.67 (td, J1 = 13.20 Hz, J2 = 4.40 Hz, 1H, H in naphthane ring), 1.81 (m,
1H, H in naphthane ring), 1.89 (m, 1H, H in naphthane ring), 2.05 (dt, J1 = 12.68 Hz,
J2 = 3.48 Hz, 1H, H in naphthane ring), 4.33-4.43 (m, 2H, OCH2). 13C NMR (100
MHz, CDCl3) δ 15.52, 18.14, 19.52, 21.38, 21.72, 33.08, 33.30, 36.38, 39.01, 39.85,
41.53, 51.24, 55.85, 66.88, 82.15, 149.12. Elemental anal. calcd for C16H26O3: C,
72.14; H, 9.84; Found: C, 72.24; H, 9.91; LC–MS (ESI+) m/z: Calcd. for C16H26NaO3
[M+Na]+ 289.18, Found:289.24.
4.1.7. General Procedure for the N-alkylation of Oxazinone.
To a solution of 8-(R)-oxazinone D (265 mg, 1.0 mmol, 1.0equiv) in anhydrous
THF (10 mL) was added NaH (60% dispersion in mineral oil, 0.080 g, 2.0 mmol, 2.0
o
equiv) at 0 C. The reaction mixture was then stirred for about 30 min, then benzyl
chloride (0.14 mL, 1.2 mmol, 1.2 equiv) was added and the mixture was refluxed until
the full consumption of 8-(R)-oxazinone monitored by TLC. The reaction was then
cooled to room temperature and quenched by careful addition of saturated NH4Cl
aqueous solution (adjusted pH ~7). The volatile solvent was removed under reduced
pressure and then extracted with EtOAc (20 mL ×3). The combined organic phase was
washed with water (20 mL ×3) and brine (20 mL), dried over anhydrous Na2SO4,
filtered and concentrated under reduced pressure to give crude products which were
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purified by flash chromatography on silica gel (200 ~ 300m) (petroleum ether/EtOAc
= 2:1, v/v) to give compound D12 as a colorless square crystal (298 mg, 84% yield).
1
Rf = 0.84 (Petroleum/Ethyl Acetate = 1:1), M.p. 138.6-139.4 . H NMR (400 MHz,
CDCl3) δ 0.79 (s, 3H, CH3), 0.80 (s, 3H, CH3), 0.93~1.02 (m, 2H, H in naphthane
ring), 1.14 (td, J1 = 14.16 Hz, J2 = 4.56 Hz, 1H, H in naphthane ring), 1.28 (m, 1H, H
in naphthane ring), 1.33 (s, 3H, CH3), 1.35-1.44 ( m, 3H, H in naphthane ring), 1.53
(m, 1H, H in naphthane ring), 1.65 (td, J1 = 13.68 Hz, J2 = 4.44 Hz, 1H, H in
naphthane ring), 1.71-1.78 (m, 2H, H in naphthane ring), 2.02 (dt, J1 = 12.72 Hz, J2 =
3.20 Hz, 1H, H in naphthane ring), 2.97-3.09 (m, 2H, NCH2CH), 4.48 (dd, J = 14.88
Hz, 1H, CH2Ph), 4.66 (dd, J = 14.88 Hz, 1H, CH2Ph), 7.22-7.36(m, 5H, aromatic H).
13C NMR (100 MHz, CDCl3) δ 15.20 (CH3), 18.22 (CH2), 19.45 (CH2), 21.41 (CH3),
21.45 (CH3), 33.06 (C), 33.33 (CH3), 36.27 (C), 39.06 (CH2), 40.08 (CH2), 41.59
(CH2), 42.45 (CH2), 51.73 (CH), 52.73 (CH2), 55.74 (CH), 80.02 (C), 127.56 (CH),
127.87 (2 × CH), 128.69 (2 × CH), 136.89 (C), 153.82 (C). Elemental anal. calcd for
C23H33NO2: C, 77.70; H, 9.36; N, 3.94; Found: C, 77.79; H, 9.51; N, 3.86; LC–MS
(ESI+) m/z: Calcd. for C23H34NO2 [M+H]+ 356.26, Found 356.33; C23H33NO2Na
[M+Na]+ 378.24, Found: 378.31. The compound D12 was confirmed by X-ray single
crystal diffraction (CCDC: 1569965).
All the derivatives in Table 2 were synthesized through similar manipulation.
4.2. Bioassay
4.2.1. General Procedure for Antifungal Bioassay
The antifungal activity of the target compounds was tested in vitro against the
eleven plant pathogenic fungi using the mycelium growth rate test, which we used
previously[14]. The in vivo biological assay against Botrytis cinerea of the D8 and the
positive control (carbendazim) was carried out on the cucumber leaf accordingly. See
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supporting information for details.
4.2.2. General Procedure for Antibacterial Bioassay
Antibacterial activities of the target compounds were tested in vitro against the
seven phytopathogenic bacteria (Xanthomonas oryzae pv. oryzae, Bacillus subtilis,
Erwinia carotovora, Xanthomonas oryzae pv. Oryzicola, Ralstonia solanacearum,
Pseudomonas syringae pv. lachrymans and Clavibacter michiganensis subsp.
sepedonicus) using the filter paper dispersion method (K-B method) for initial
screening. All the tested compounds were dissolved in DMSO at a concentration of 10
mg/L. Round filter papers with the diameter of 5mm were put on the plating medium
containing 108 CFU/mL of the indicator strains, then 2 µL of the stock solution
(10mg/L) was added to each filter paper, with sterile water as the blank control. The
plating medium was cultured at 28 for 16-24 h. Then diameter (mm) of inhibition
zone was detected. The average value was calculated to evaluate the antibacterial
effects with three repetitions.
The agar diffusion dilution method (ADM)[26] was further applied to confirm
the minimum inhibitory concentration (MIC) of the target compounds. Stock solution
was diluted to 100, 50, 25, 12.5, 6.25, 3.125, 1.5625 µg/mL or 128, 64, 32, 16, 8,
4µg/mL according to the same ADM adopted to assess MIC. Subsequently, 2 µL of
the nutrient broth containing 108 CFU/mL of the indicator strains were inoculated to
all plates. Plates were then cultured at 28
for 16~24 h. MIC was defined as the
lowest concentration that did not result in any visible growth of the microorganism in
comparison with the growth in the control plate; the presence of one or two colonies
was not taken into account for the final assessment of MIC. Three bacterial strains
were chosen for the determination of MICs (µg/mL), which were listed in Table 3.
The above experiments were repeated for three times and streptomycin sulfate was
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chosen as positive control simultaneously.
The statistical analyses of Antifungal Bioassay and Antibacterial Bioassay were
performed by SPSS software version 20.0.
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Author Contributions
S. Li conceived and designed this work, D. Li performed the chemical synthesis
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and biological experiments, S. Zhang helped to perform the derivative synthesis and
biological test. D. Li, S. Zhang, Z. Song and W. Li carried out NMR and MASS
detection and elemental analysis. F. Zhu and J. Zhang conducted the FTIR test. D. Li
and S. Li analyzed the data. S. Li wrote the paper.
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Acknowledgement
This work was financially supported by National Natural Science Foundation of
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China (No. 31401777), the Fundamental Research Funds for the Central Universities,
Natural Science Foundation of Jiangsu Province (No. BK20140684), and Hong
Kong Scholars Program for S. Li. (XJ2016011).
Appendix A. Supplementary data
List of synthesized compounds, antifungal, and antibacterial data, selected
antifungal pictures, NMR spectra and X-ray spectra are available.
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599
600
601
602
603
604
605
606
607
608
609
610
611
Figures
612
613
614
Fig.1. Drimane and typical bioactive natural products with specific oxidation and chirality
615
616
Fig.2. Synthesis and antifungal activity of Drimenol and Drimenal
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617
618
619
620
Fig.3. Diverse Synthesis and SAR of Drimenol and 8-OH-Drimanol
621
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622
Fig.4. Design, synthesis and antifungal activity of rigid drimanol analogs
623
624
Fig.5. Improved synthesis and optimization of chiral drimane fused oxazinones
625
626
627
628
Fig.6. In vivo antifungal activity of D8 against Botrytis cinerea
629
630
631
632
633
Tables
Table 1. EC50 values of the 8-OH-drimenol derivitives in vitro (unit: µg/mL)
R. solani
S. sclerotiorum
F. graminearum
B. cirerea
Compd.
R
R1=
CH3
41.06±0.56
33.69±0.62
41.13±0.26
20.10±0.20
49.87±0.18
>100
74.86±0.04
33.69±0.62
23.77±0.36
18.91±0.06
65.98±0.68
>100
46.22±1.10
38.14±0.36
66.52±0.22
25.58±0.37
>100
60.80±0.16
22.54±0.14
40.20±0.21
23.94±0.26
>100
AB1
AB2
AB3
AB4
AB5
AB7
AB8
AB9
AB10
AC1
AC2
AC3
AC4
AC5
AC7
AC8
CH2CH3
CH=CH2
CH≡CH
(CH2)2CH3
(CH2)4CH3
Ph
>100
>100
>100
>100
>100
>100
4-CH3Ph
4-FPh
>100
>100
>100
>100
>100
>100
>100
>100
CH3
>100
68.69±0.03
20.07±0.08
16.65±0.18
18.4±0.07
12.74±0.24
>100
46.24±0.14
43.12±0.12
41.43±0.19
31.8±0.22
42.87±0.13
>100
68.69±0.03
20.07±0.08
16.65±0.18
12.76±0.12
13.04±0.13
>100
CH2CH3
CH=CH2
45.35±0.10
32.09±0.09
27.66±0.14
43.10±0.12
>100
CH≡CH
(CH2)2CH3
(CH2)4CH3
Ph
>100
>100
>100
>100
R2=
CH3
BB1
66.82±1.84
>100
64.86±0.12
61.40±0.05
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CH2CH3
(CH2)3CH3
Ph
BB2
BB3
BB4
BB9
BB10
77.15±0.13
61.20±0.56
>100
64.42±0.03
59.63±0.05
>100
>100
>100
>100
>100
>100
46.97±0.08
21.72±0.14
>100
4-CH3Ph
4-FPh
>100
>100
>100
>100
>100
50.83±0.38
R3=
(CH3)2CH
(CH2)3CH3
Ph
43.51±0.25
>100
>100
>100
>100
>100
CB1
CB2
CB3
11.39±0.38
12.62±0.17
59.71±0.96
13.90±0.30
>100
53.09±1.42
634
635
636
637
638
639
640
641
Table 2 EC50 values of the chiral drimane fused oxazinones in vitro (unit: µg/mL)
R
R. solani
S. sclerotiorum
F. graminearum
B. cirerea
Compd.
H
-CH3
19.25±0.58
20.05±0.18
60.86±0.05
13.41±0.11
25.66±0.21
35.44±0.10
27.42±0.22
20.45±0.32
11.40±0.39
22.05±0.13
31.83±0.34
>100
35.32±0.12
39.88±0.04
26.04±0.06
12.49±0.09
42.98±0.03
19.52±0.12
13.91±0.07
35.97±0.04
7.70±0.15
9.08±0.33
26.28±0.38
>100
32.00±0.23
73.62±0.04
56.12±0.04
38.31±0.06
33.31±0.21
40.99±0.20
29.18±0.04
16.12±0.12
11.91±0.12
26.93±0.28
26.99±0.39
>100
33.23±0.03
35.54±0.21
34.56±0.08
23.26±0.16
19.30±0.12
33.11±0.04
20.72±0.16
9.14±0.37
1.18±0.70
9.37±0.26
5.71±0.56
>100
D
D1
-CH2CH3
-CH2CH2CH3
-CH(CH3)2
-CH2C=CH2
-CH2C≡CH
-CH2CH=C(CH3)2
-(CH2)3CH3
-(CH2)4CH3
-(CH2)5CH3
-(CH2)11CH3
Bn-
D2
D3
D4
D5
D6
D7
D8
D9
D10
D11
D12
D13
D14
D15
D16
D17
D18
D19
15.28±0.15
>100
48.16±0.05
>100
13.36±0.36
>100
11.37±0.22
>100
3-CH3Bn-
4- CH3Bn-
4-FBn-
11.71±0.61
>100
43.98±0.51
>100
19.99±0.48
>100
10.17±0.33
>100
2-ClBn-
31.43±0.83
>100
>100
67.55±0.40
>100
>100
3-ClBn-
>100
>100
4-ClBn-
14.20±0.26
>100
>100
>100
>100
3,5-FBn-
>100
>100
>100
642
643
644
645
646
Note: See supporting information for EC90 values.
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649
650
651
652
653
654
655
656
657
658
659
660
661
662
Table 3 Antibacterial activities (MIC, mg/L) of the chiral drimane fused oxazinones
Xanthomonas oryzae
Ralstonia
R
Bacillus subtilis(+)
Compd.
pv.oryzicola (-)
solanacearum (-)
100 (0.377)
100 (0.358)
100 (0.341)
25 (0.081)
H
100 (0.377)
＞100 (＞0.358)
＞100 (＞0.341)
25 (0.081)
100 (0.377)
＞100 (＞0.358)
＞100 (＞0.341)
25 (0.081)
D
-CH3
D1
-CH2CH3
D2
-CH2CH2CH3
-CH(CH3)2
-CH2C=CH2
-CH2C≡CH
-CH2CH=C(CH3)2
-(CH2)3CH3
-(CH2)4CH3
-(CH2)5CH3
-(CH2)11CH3
Bn-
D3
25 (0.081)
25 (0.081)
25 (0.081)
D4
50 (0.164)
50 (0.164)
50 (0.164)
D5
100 (0.330)
8 (0.024)
D6
＞100 (＞0.330)
8 (0.024)
＞100 (＞0.330)
25 (0.075)
D7
D8
12.5 (0.039)
25 (0.074)
12.5 (0.039)
12.5 (0.039)
25 (0.074)
25 (0.074)
D9
D10
D11
D12
D13
D14
D15
D16
D17
D18
D19
8 (0.023)
12.5 (0.036)
8 (0.023)
＞100 (＞0.230)
12.5 (0.035)
＞100 (＞0.271)
100 (0.271)
＞100 (＞0.230)
100 (0.281)
＞100 (＞0.230)
8 (0.022)
3-CH3Bn-
4- CH3Bn-
4-FBn-
100 (0.271)
100 (0.271)
＞100 (＞0.268)
25 (0.064)
＞100 (＞0.271)
＞100 (＞0.271)
＞100 (＞0.268)
25 (0.064)
＞100 (＞0.268)
12.5 (0.032)
＞100 (＞0.256)
100 (0.256)
2-ClBn-
3-ClBn-
＞100 (＞0.256)
100 (0.256)
＞100 (＞0.256)
100 (0.256)
＞100 (＞0.255)
50 (0.034)
4-ClBn-
3,5-2FBn-
streptomycin sulfate
100 (0.255)
＞100 (＞0.255)
100 (0.069)
25 (0.017)
663
664
665
666
667
668
Note: Data in parentheses was presented in molar concentration (mmol/L); The molecular weight (MW) of
streptomycin sulfate is 1457.38, and the MICs of streptomycin monomer are 0.034, 0.138 and 0.068 mmol/L
against the listed three bacteria respectively.
28