Synthesis of Natural Acylphloroglucinol-Based Antifungal Compounds against Cryptococcus Species

Article abstract of DOI:10.1021/acs.jnatprod.6b00224

Thirty-three natural-product-based acylphloroglucinol derivatives were synthesized to identify antifungal compounds against Cryptococcus spp. that cause the life-threatening disseminated cryptococcosis. In vitro antifungal testing showed that 17 compounds

Full text of DOI:10.1021/acs.jnatprod.6b00224

Article
pubs.acs.org/jnp
Synthesis of Natural Acylphloroglucinol-Based Antifungal
Compounds against Cryptococcus Species
Qian Yu,†,§ Ranga Rao Ravu,† Melissa R. Jacob,† Shabana I. Khan,†,‡ Ameeta K. Agarwal,†,‡ Bo-Yang Yu,§
and Xing-Cong Li*,†,‡
†National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, and
‡Department of Biomolecular Sciences, School of Pharmacy, The University of Mississippi, University, Mississippi 38677, United
States
§Jiangsu Key Laboratory of TCM Evaluation and Translational Research, Department of Complex Prescription of TCM, China
Pharmaceutical University, Nanjing, 211198, People’s Republic of China
S
* Supporting Information
ABSTRACT: Thirty-three natural-product-based acylphloroglucinol derivatives were synthesized to identify antifungal
compounds against Cryptococcus spp. that cause the life-threatening disseminated cryptococcosis. In vitro antifungal testing
showed that 17 compounds were active against C. neoformans ATCC 90113, C. neoformans H99, and C. gattii ATCC 32609, with
minimum inhibitory concentrations (MICs) in the range 1.0−16.7 μg/mL. Analysis of the structure and antifungal activity of
these compounds indicated that the 2,4-diacyl- and 2-acyl-4-alkylphloroglucinols were more active than O-alkyl-
acylphloroglucinols. The most promising compound found was 2-methyl-1-(2,4,6-trihydroxy-3-(4-isopropylbenzyl)phenyl)-
propan-1-one (11j), which exhibited potent antifungal activity (MICs, 1.5−2.1 μg/mL) and low cytotoxicity against the
mammalian Vero and LLC-PK1 cell lines (IC50 values >50 μg/mL). This compound may serve as a template for further synthesis
of new analogues with improved antifungal activity. The findings of the present work may contribute to future antifungal
discovery toward pharmaceutical development of new treatments for cryptococcosis.
ryptococcosis is a fungal infection caused by Cryptococcus
neoformans in immunocompromised patients with AIDS,
methylacryloyl)benzaldehyde, has shown good activity against
C. neoformans with an IC50 value of 2.5 μg/mL. However, this
compound may not be chemically stable due to the presence of
a formyl group on the aromatic ring and the enol functionality
on the side chain.17 During our recent work on the synthesis of
the plant-derived antibacterial acylphloroglucinol psorothatin
C,18 the intermediate 2-isobutyryl-4-isoprenylphloroglucinol
(1), which is also a natural product isolated from the plant
Helichrysum kraussii,19 was found to be active against C.
neoformans with an MIC value of 5.0 μg/mL. The potent
activity of this prenylated acylphlorogluconol prompted the
design and synthesis of additional analogues, with the aim of
identifying structurally simple and chemically stable antifungal
compounds against Cryptococcus spp.
C
cancer, and organ transplants or by Cryptococcus gattii in
immunocompetent hosts. A significant feature of this infection
is that the fungal cells can pass through the blood−brain
barrier, invading the central nervous system, leading to life-
threatening meningitis and meningoencephalitis.1,2 Cryptococ-
cosis is the most prevalent opportunistic invasive mycosis,
affecting greater than 1 million people per year, with a mortality
rate of 20−70%.1 There are few treatment options for this
disease due to a limited number of effective antifungal drugs.2
Therefore, there is a need to discover new lead compounds for
drug development in this therapeutic area.
Naturally occurring acylphloroglucinols constitute a prom-
inent class of structurally diverse compounds that have been
found in plants, marine organisms, and microbes.3 While a large
number of compounds within this structural class have shown
antimicrobial,3−5 anti-HIV,6 antidepressant,7 antiproliferative,8
antiprotozoal,9 antiangiogenic,10 anti-inflammatory,11 antiox-
idant,12 and catalytic activities,13 only a few compounds were
reported to be active against Cryptococcus spp.14−16 A synthetic
formylated acylphloroglucinol, 2,4,6-trihydroxy-3-(3-hydroxy-2-
RESULTS AND DISCUSSION
■
The general design for the synthesis of target compounds was
based on the analysis of structure and activity of compound 1
Received: March 10, 2016
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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and other naturally occurring acylphloroglucinols shown in
Figure 1. Since limited information was available on antifungal
The rationale for the design and synthesis of these particular
compounds is further explained below. First, 2,4-diacylphlor-
oglucinols 5, 9, 10a, and 10b and 2-acyl-4-alkylphloroglucinols
1, 11a, and 11b with different acyl groups were synthesized and
evaluated for in vitro antifungal activity against C. neoformans
ATCC 90113. It was observed that compounds with an
isobutyryl group (1, 5, and 10a) and those with a
corresponding butyryl substituent (9, 10b, and 11a) showed
similar antifungal activity, while compound 11b, with a bulky
pivaloyl group, was inactive. Thus, the isobutyryl group that is
commonly present in natural acylpholoroglucinols3 was
considered as an appropriate acyl group substituted on the
phloroglucinol ring of new analogues to be synthesized. Then,
using isobutyrylated phloroglucinol 8a as the key intermediate,
the 2,4-diacylphloroglucinols 10c−10e were prepared to
examine the effects of aromatic acyl substitutions on antifungal
activity, while the 2-acyl-4-alkylphloroglucinols 11c−11l and
the O-alkylacylphloroglucinols 12a−12i with varying alkyl
chains were synthesized to identify meaningful structure−
activity relationships (SAR).
All 33 synthetic compounds were tested for in vitro
antifungal activity against C. neoformans ATCC 90113.
Compounds exhibiting greater than 50% growth inhibition
(IC50) at a concentration of 20 μg/mL were considered to be
active, which were further tested against this strain as well as C.
neoformans H99 and C. gattii ATCC 32609 to obtain MIC
values. The results showed that 17 active compounds had
overall MIC values in the range 1.0−16.7 μg/mL (Table 1).
For comparison, the antifungal drugs fluconazole and
amphotericin B gave MIC values in the range 6.3−25.0 and
0.3−0.4 μg/mL, respectively, against the three fungal strains.
Cytotoxicity testing against the mammalian Vero and LLC-PK1
cell lines showed that all these antifungal compounds had IC50
values of greater than 10 μg/mL (Table 1), indicating they
were moderately or marginally cytotoxic.
Analysis of the potency of individual antifungal compounds
revealed SAR information. Within the chemotype of 2,4-
diacylphloroglucinols, the aliphatically acylated compounds 5
and 9 showed potent activity with MIC values in the range
1.5−3.3 μg/mL. Replacement of one aliphatic acyl group with
an aromatic acyl group in the molecule decreased antifungal
activity by 3−4-fold, e.g., 5 versus 10a. Additional substitutions
on the aromatic ring with either an electron-withdrawing or an
electron-donating group such as in 10c and 10d altered
antifungal activity slightly when compared with 10a. However,
compound 10e, with a bulky tri-OMe-substituted aromatic acyl
group, was inactive at 20 μg/mL. This suggested that the
antifungal activity of this chemotype of compounds is
associated with acyl groups, and aliphatic acyl substitutions
appear to improve potency.
For 2-acyl-4-alkylphloroglucinols, a short alkyl chain that
contains a double bond, a triple bond, a cyano group, or an
ester functionality disabled antifungal activity as in the cases of
compounds 11c−11f, which were inactive at 20 μg/mL.
Compound 2, with a C10 geranyl group, was more potent than
1, with a C5 isoprenyl group (Table 1). Compound 11l, with an
extended C15 farnesyl group, showed decreased activity by 3−7-
fold when compared with 2. Although 2 and 1 are potent
antifungal compounds against Cryptococcus spp., their chemical
instability is a disadvantage, as they can undergo readily
intramolecular cyclization to form corresponding chromane
derivatives.29 Such cyclized products have been isolated and
demonstrated to be inactive at 20 μg/mL in this study (data
Figure 1. Structures of antimicrobial acylphloroglucinols.
activity of this class of compounds against Cryptococcus spp., the
structures of those active against other fungal pathogens or
bacteria were considered as synthetic templates, with
assumptions that compounds with similar core structures
would have antifungal activity or exhibit both antifungal and
antibacterial activities. In fact, compound 1 and its synthetic
analogues with different acyl substituents have demonstrated
antifungal and antibacterial activities.20 The structurally similar
geranylated acylphloroglucinols 2 and 3 isolated from Hyper-
icum punctatum21 and Hypericum olympicum,22 respectively,
showed potent antibacterial activity, but their antifungal activity
has not been explored. 2,4-Diacetylphloroglucinol (4), isolated
from the red marine alga Corpus spongiosum, and its synthetic
analogue 2,4-diisobutyrylphloroglucinol (5), which showed
antibacterial activity,23,24 may possess antifungal activity.
Finally, the microbial metabolite bis(2,4-diacetylphloroglucyl)-
methane (6) isolated from Pseudomonas aurantiaca25 was also
considered as a template because it represents a large number
of structurally similar dimers or trimers formed through a
methylene linker within this class.3,7 Mimicking the afore-
mentioned templates, three chemotypes of compounds, 2,4-
diacylphloroglucinols (5, 9, and 10a−10e), 2-acyl-4-alkylphlor-
oglucinols (1, 2, 11a−11l), and O-alkylacylphloroglucinols
(12a−12i), were synthesized.
The synthetic approach for preparation of these compounds
is shown in Scheme 1, which is an adaptation from synthetic
methodologies available in the literature for preparation of
alkylated acylphloroglucinols.26−28 Thus, Friedel−Crafts acyla-
tion of phloroglucinol (7) with acyl chloride (RCOCl) and
AlCl3 in nitrobenzene afforded the monoacylated products 8a−
8c and the diacylated products 5 and 9. Further acylation of
8a−8b with a different acylating reagent gave the diacylated
products 10a−10e, while treatment of 8a−8c with alkyl halide
yielded the C-alkylated products 1, 2 and 11a−11l and the O-
alkylated products 12a−12i.
B
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a
Scheme 1. Synthesis of Acylphloroglucinols
a
Reagents and conditions: (a) RCOCl, AlCl3, PhNO2, 65 °C, 21 h. (b) RX, DBU, THF, rt, 40 h. (c) RBr, K2CO3, acetone, 65 °C, 6 h.
not shown). Compounds 11g−11k with varying aromatic-
containing alkyl chains showed strong antifungal activity (Table
1), which is of interest, as these compounds are chemically
stable compared to compounds 1 and 2 and structurally simple
compared to template 6. It appears that different substituents
and substitution patterns on the aromatic ring in 11g−11k
influence antifungal activity. Compound 11j demonstrated the
best activity profiles due to its strong antifungal activity against
the three fungal pathogens, with MIC values in the range 1.5−
2.1 μg/mL, as well as low cytotoxicity against the two
mammalian cell lines, Vero and LLC-PK1, with IC50 values
greater than 50 μg/mL. It was also interesting to note that there
was a 2−4-fold difference in antifungal activity between 11j and
11k, with a para-isopropyl group and a para-nitro group on the
aromatic ring, respectively, indicating that further modifications
of the aromatic-containing alkyl chain may afford compounds
with improved antifungal activity.
Within the chemotype of O-alkylacylphloroglucinols, only
12f and 12h showed antifungal activity against the three
Cryptococcus strains, with MIC values in the range 5.0−8.3 μg/
mL. Structural comparison of the active compound 12h and the
inactive compound 12g with the same (2-chloro-6-
fluorophenyl)methyl group substituted at the ortho and para
positions of the phloroglucinol ring, respectively, indicated that
different substitution patterns are associated with antifungal
activity of this chemotype of compounds. Considering the low
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a
Table 1. In Vitro Antifungal Activity and Cytotoxicity of Synthetic Acylphloroglucinols
cytotoxicity
(IC50, μg/mL)
b
c
d
antifungal activity (MIC, μg/mL)
SI
C. neoformans
C. neoformans
C. gattii
LLC-
Vero IC50/ fungal
LLC-PK1 IC50/fungal
e
f
g
g
ATCC 90113
H99
ATCC 32609
Vero
PK1
MIC
MIC
1
5.0
1.7
2.5
2.1
10.0
6.7
8.3
13.3
3.3
6.7
1.0
1.7
1.5
6.7
7.5
8.3
5.0
0.4
6.3
−
8.3
1.7
5.0
1.0
2.1
1.5
8.3
5.0
6.7
8.3
5.0
10.0
2.1
4.2
2.1
5.0
7.5
5.0
5.0
0.3
25.0
−
18.0
22.5
11.5
14.5
23
31.0
24.5
17.5
24.5
42.5
27
3.6
13.2
4.6
6.2
14.4
7.0
2
5
3.3
9
2.5
6.9
11.7
4.3
10a
10.0
6.7
2.3
10b
14.3
13.5
10.5
16.3
28
2.1
4.0
10c
11.7
10.0
8.3
27.5
24.8
28.0
28.5
29.5
26.3
>50
28.0
27.0
34.5
>50
−
1.6
3.3
10d
0.8
1.9
11a
4.9
8.5
11g
16.7
4.2
4.2
4.3
11h
27.5
28
27.5
16.5
>33.3
2.7
29.5
15.5
>33.3
4.2
11i
6.7
11j
2.1
>50
11k
10.0
5.0
18.0
25.0
32.5
11l
3.3
3.6
12f
5.0
3.9
4.2
12h
8.3
>50
>10
>10
h
amphotericin B
fluconazole
doxorubicin
0.3
−
−
−
−
−
−
−
12.5
−
−
>10
−
1.3
a
b
Both antifungal activity and cytotoxicity data are expressed as mean values of three experimental data. MIC: minimum inhibitory concentration
(the lowest concentration that allows no detectable growth). The highest test concentration for synthetic acylphloroglucinols, amphotericin B, and
c
fluconazole were 20, 5, and 50 μg/mL, respectively. IC50: concentration responsible for 50% growth inhibition of mammalian cells. The highest test
d
e
concentration for synthetic acylphloroglucinols and the positive control doxorubicin was 50 and 10 μg/mL, respectively. Selectivity index. African
f
g
h
green monkey kidney fibroblasts. Pig kidney epithelial cells. Calculated by using the C. neoformans ATCC 90113 strain. Not tested.
cytotoxicity of 12h (IC50 values of >50 μg/mL) against the two
mammalian cell lines tested, this antifungal compound may
warrant further studies.
EXPERIMENTAL SECTION
■
General Experimental Procedures. The NMR spectra using
standard pulse programs were recorded at room temperature on a
Bruker Avance DPX-400 spectrometer operating at 400 (1H) and 100
(13C) MHz. The chemical shift (δ, ppm) values were calibrated using
the residual NMR solvent. High-resolution TOFMS were measured on
an Agilent series 1100 SL spectrometer equipped with an ESI source.
TLC was performed on silica gel aluminum sheets (silica gel 60 F254,
Merck, Darmstadt, Germany) and reversed-phase silica gel glass plates
(RP-18 F254S, Merck, Darmstadt, Germany) and visualized by UV
254 nm and spraying 10% H2SO4, followed by heating. Flash column
chromatography was done on normal-phase silica gel (230 × 400
mesh, J. T. Baker, Center Valley, PA, USA) and reversed-phase silica
gel (RP-18, 40 μm, J. T. Baker). Reactants and reagents including
anhydrous phloroglucinol, isobutyryl chloride, trimethylacetyl chloride,
butanoyl chloride, 3,3-dimethylallyl bromide, 3-methyl-2-butenal,
aluminum chloride (AlCl3), 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU), benzoyl chloride, 4-nitrobenzyl bromide, 3-bromo-1-propene,
bromoacetonitrile, 3-bromo-1-propyne, ethyl bromoacetate, butyryl
chloride, 4-fluorobenzoyl chloride, 4-methoxybenzoyl chloride, 3,4,5-
trimethoxybenzoyl chloride, geranyl bromide, farnesyl bromide, n-tert-
butyl-2-chloroacetamide, nitrobenzene, tetrahydrofuran, and sodium
sulfate were purchased from Sigma-Aldrich (St. Louis, MO, USA) in
appropriate grades and were used without further purification. 2,6-
Dichlorobenzyl chloride, 2-chloro-6-fluorobenzyl chloride, and 4-
isopropylbenzyl chloride were purchased from Otava, Ltd. (Vaughan,
Ontario, Canada). The yield of each synthetic product after column
chromatography is reported. The 1H and 13C NMR assignments for all
synthetic products and key intermediates with appropriate numbering
systems are shown in the Supporting Information.
To assess whether these antifungal compounds possess
antibacterial activity, 5, 11j, and 12h, representing the
aforementioned three chemotypes of acylphloroglucinols,
were tested using a published protocol18 for in vitro activity
against two Gram-positive bacteria (Staphylococcus aureus and
Mycobacterium intracellulare) and two Gram-negative bacteria
(Escherichia coli and Pseudomonas aeruginosa). The three
compounds were active only against S. aureus (data shown in
the Supporting Information). While compound 5 showed the
most potent activity, with an MIC value of 1.3 μg/mL, which is
consistent with the data reported previously in the literature,24
11j was the least antibacterial compound, with an MIC value of
10.0 μg/mL. Taking into account its antifungal potency,
selectivity over mammalian cell lines (selectivity indices >33.3,
Table 1) and bacteria, and chemical stability, 11j is considered
to be the most promising compound and may serve as a
template for further synthesis of new analogues with improved
antifungal properties.
In conclusion, the present work with a focus on natural-
products-based chemical synthesis has led to the discovery of
several potent antifungal acylphloroglucinols against Crypto-
coccus spp. and a promising antifungal template. These findings
may contribute to future antifungal discovery toward
pharmaceutical development of new treatments for cryptococ-
cosis.
General Procedure for the Preparation of the 2-Acylphlor-
oglucinols 8a−8c and 2,4-Diacylphloroglucinols 5 and 9. To a
solution of anhydrous phloroglucinol (7, 79.4 mmol) in nitrobenzene
(80 mL) was added AlCl3 (317 mmol). The reaction mixture was
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stirred at room temperature under nitrogen for 30 min. Isobutyryl
chloride or n-butyryl chloride (87.2 mmol) was added dropwise, and
the reaction mixture was stirred at 65 °C for 21 h for production of 8a
and 5 or 8b and 9, respectively.27 Acylation using trimethylacetyl
chloride occurred rapidly, and the reaction mixture was stirred at room
temperature for 20 min to afford the monoacylated product 8c. After
addition of ice−water (40 mL), the reaction mixture was extracted
with EtOAc (100 mL × 3). The combined EtOAc layers were treated
with 2 M NaOH (160 mL × 2). The aqueous layers were neutralized
with 2 M HCl and extracted with EtOAc (100 mL × 3). The
combined EtOAc layers were washed with water and brine, dried over
Na2SO4, filtered, and concentrated under reduced pressure to afford a
residue, which was chromatographed on silica gel using CHCl3−
MeOH (19:1 for 5 and 8b, 9:1 for 8a, and 10:0 for 9) or CHCl3−
acetone (9:1 for 8c) to give the respective purified compounds.
Identification of compounds 8a,27 8b,30 8c,31 5,24 and 924 was made by
comparison of their NMR spectroscopic and high-resolution ESIMS
data (see Supporting Information) with those reported in the
literature.
Na2SO4. Removal of the solvent afforded a residue, which was
chromatographed on silica gel using hexanes−EtOAc (19:1) or
CHCl3−acetone (10:0−9:1) to afford purified compounds. Note that
some reactions afforded only 2-acyl-4-alkylphloroglucinols (1, 11a−
11e, 11g, and 11k) or O-alkyl-acyl-phloroglucinols (12d/12f), while
others produced both chemotypes of compounds, including 11f/12a/
12b/12c, 11h/12f, 11i/12g/12h, and 11j/12i. The known com-
pounds 116 and 11a32 were identified by comparison of their NMR
and HRESIMS spectroscopic data (see Supporting Information) with
those reported in the literature. The new compounds were
1
characterized by spectroscopic methods as follows (the detailed H
and 13C NMR assignments are shown in the Supporting Information).
2,2-Dimethyl-1-(2,4,6-trihydroxy-3-(3-methylbut-2-en-1-yl)-
phenyl)propan-1-one (11b): 14% yield, yellow powder; 1H NMR
(MeOH-d4) δ 5.91 (1H, s), 5.11 (1H, t, J = 7.0 Hz), 3.17 (2H, d, J =
6.5 Hz), 1.68 (3H, s), 1.60 (3H, s), 1.21 (9H, s); 13C NMR (MeOH-
d4) δ 217.9, 159.2, 156.5, 155.1, 132.0, 124.4, 110.8, 108.6, 96.1, 46.2,
27.8 (3C), 26.0, 22.7, 18.0; HRESIMS m/z 277.1480 [M − H]− (calcd
−
for C16H21O4 , 277.1445).
1-(3-Allyl-2,4,6-trihydroxyphenyl)-2-methylpropan-1-one (11c):
General Procedure for the Preparation of the 2,4-
Diacylphloroglucinols 10a−10e. Compound 8a or 8b was acylated
with an aromatic acyl chloride using the same reaction conditions
described above for preparation of the starting material. Similar
workup and purification procedures of the reaction mixture afforded
the respective purified compounds.
1
10% yield, red powder; H NMR (MeOH-d4) δ 5.87 (1H, s), 5.81
(1H, m), 4.89 (1H, br d, J = 17.2 Hz), 4.80 (1H, br d, J = 9.8 Hz), 3.96
(1H, m), 3.19 (2H, d, J = 6.5 Hz), 1.08 (6H, d, J = 7.0 Hz); 13C NMR
(MeOH-d4) δ 211.9, 165.4, 163.7, 161.5, 138.0, 114.1, 106.3, 104.4,
95.0, 40.0, 27.4, 19.9 (2C); HRESIMS m/z 235.0976 [M − H]− (calcd
−
for C13H15O4 , 235.0976).
1-(3-Benzoyl-2,4,6-trihydroxyphenyl)-2-methylpropan-1-one
(10a): 20% yield, yellow powder; 1H NMR (CDCl3) δ 7.92 (2H, d, J =
8.0 Hz), 7.43 (1H, t, J = 8.0 Hz), 7.24 (2H, t, J = 8.0 Hz), 5.76 (1H, s),
3.76 (1H, m), 1.01 (6H, d, J = 7.0 Hz); 13C NMR (MeOH-d4) δ
211.5, 199.5, 172.3 (2C), 170.4, 140.6, 134.0, 130.4, 128.7, 103.9,
103.8, 96.3, 39.6, 19.3 (2C); HRESIMS m/z 299.0930 [M − H]−
2-Methyl-1-(2,4,6-trihydroxy-3-(prop-2-yn-1-yl)phenyl)propan-1-
one (11d): 28% yield, red powder; 1H NMR (MeOH-d4) δ 5.87 (1H,
s), 3.95 (1H, m), 3.30 (2H, s), 1.96 (1H, t, J = 2.6 Hz, H-13), 1.08
(6H, d, J = 7.0 Hz); 13C NMR (MeOH-d4) δ 211.9, 165.3, 163.2,
161.9, 104.4, 103.7, 95.0, 84.2, 67.0, 40.0, 19.8 (2C), 12.3; HRESIMS
−
m/z 233.0806 [M − H]− (calcd for C13H13O4 , 233.0819).
−
(calcd for C17H15O5 , 299.0925).
2-(2,4,6-Trihydroxy-3-isobutyrylphenyl)acetonitrile (11e): 21%
1-(3-Benzoyl-2,4,6-trihydroxyphenyl)butan-1-one (10b): 29%
1
1
yield, red powder; H NMR (MeOH-d4) δ 5.93 (1H, s), 3.90 (1H,
yield, yellow powder; H NMR (MeOH-d4) δ 7.52 (2H, d, J = 8.0
m), 3.51 (2H, s), 1.08 (6H, d, J = 7.0 Hz); 13C NMR (MeOH-d4) δ
211.9, 165.7, 163.4, 163.0, 120.0, 104.3, 97.4, 94.9, 40.1, 19.8 (2C),
11.2; HRESIMS m/z 234.0765 [M − H]− (calcd for C12H12O4N−,
234.0772).
Hz), 7.39 (1H, t, J = 8.0 Hz), 7.29 (2H, t, J = 8.0 Hz), 5.83 (1H, s),
2.96 (1H, t, J = 7.8 Hz), 1.59 (2H, m), 0.90 (3H, t, J = 7.5 Hz); 13C
NMR (MeOH-d4) δ 207.6, 200.4, 169.9, 169.5, 167.5, 142.1, 132.5,
129.3 (2C), 128.8 (2C), 105.5, 104.9, 95.9, 48.7, 18.9, 14.4; HRESIMS
−
m/z 299.0939 [M − H]− (calcd for C17H15O5 , 299.0925).
Ethyl 2-(2,4,6-trihydroxy-3-isobutyrylphenyl)acetate (11f). 9%
yield, yellow powder; 1H NMR (MeOH-d4) δ 5.78 (1H, s), 4.42
(1H, s), 4.09 (1H, q, J = 7.8 Hz), 3.66 (2H, m), 1.12 (3H, t, J = 7.5
Hz), 0.95 (6H, d, J = 7.0 Hz); 13C NMR (MeOH-d4) δ 211.9, 175.6,
165.4, 163.5, 162.0, 104.6, 101.1, 95.9, 62.0, 39.8, 28.8, 19.6 (2C),
1-(3-(4-Fluorobenzoyl)-2,4,6-trihydroxyphenyl)-2-methylpropan-
1
1-one (10c): 38% yield, yellow powder; H NMR (CDCl3) δ 7.61
(2H, t, J = 8.0 Hz), 7.05 (2H, t, J = 8.0 Hz), 5.86 (1H, s), 3.91 (1H,
m), 1.18 (6H, d, J = 7.0 Hz); 13C NMR (CDCl3) δ 212.2/211.5,
198.8/198.1, 172.1/170.3, 166.4/163.9, 166.2/163.7,136.9/136.5,
133.0/132.9, 131.00/131.96/130.91/130.87 (2C), 115.9/115.7/
115.4/115.3/115.2/115.1 (2C), 103.8/103.7, 102.6, 96.0, 39.6, 19.3
(2C); HRESIMS m/z 317.0812 [M − H]− (calcd for C17H14O5F−,
317.0831).
−
14.4; HRESIMS m/z 281.1066 [M − H]− (calcd for C14H17O6 ,
281.1031).
1-(3-Benzyl-2,4,6-trihydroxyphenyl)-2-methylpropan-1-one
(11g): 57% yield, yellow powder; 1H NMR (MeOH-d4) δ 7.19 (2H, d,
J = 7.4 Hz), 7.11 (2H, t, J = 7.4 Hz), 7.00 (1H, t, J = 7.2 Hz), 5.90
(1H, s), 3.96 (1H, m), 3.80 (2H, s), 1.09 (6H, d, J = 7.0 Hz); 13C
NMR (MeOH-d4) δ 211.9, 165.7, 163.8, 161.6, 143.5, 129.6 (2C),
128.9 (2C), 126.3, 108.1, 104.6, 95.1, 40.1, 28.9, 19.9 (2C); HRESIMS
2-Methyl-1-(2,4,6-trihydroxy-3-(4-methoxybenzoyl)phenyl)-
1
propan-1-one (10d): 11% yield, yellow powder; H NMR (MeOH-
d4) δ 7.65 (2H, d, J = 8.0 Hz), 6.87 (2H, d, J = 8.0 Hz), 5.92 (1H, s),
3.93 (1H, m), 3.78 (3H, s), 1.10 (6H, d, J = 7.0 Hz); 13C NMR
(MeOH-d4) δ 212.0, 197.8, 167.9, 167.1, 165.6, 164.8, 133.3, 132.5
(2C), 114.2 (2C), 107.0, 104.1, 95.7, 55.9, 40.2, 19.5 (2C); HRESIMS
−
m/z 285.1149 [M − H]− (calcd for C17H17O4 , 285.1132).
1-(3-(2,6-Dichlorobenzyl)-2,4,6-trihydroxyphenyl)-2-methylpro-
1
pan-1-one (11h): 16% yield, yellow powder; H NMR (MeOH-d4) δ
−
m/z 329.1043 [M − H]− (calcd for C18H17O6 , 329.1031).
7.16 (2H, d, J = 8.0 Hz), 6.98 (1H, t, J = 8.0 Hz), 5.82 (1H, s), 4.09
(2H, s), 3.95 (1H, m), 1.07 (6H, d, J = 7.0 Hz); 13C NMR (MeOH-
d4) δ 211.9, 166.2, 164.1, 161.6, 139.1, 137.5 (2C), 129.0 (2C), 128.1,
105.3, 104.5, 95.1, 40.0, 26.3, 19.9 (2C); HRESIMS m/z 353.0380 [M
2-Methyl-1-(2,4,6-trihydroxy-3-(3,4,5-trimethoxybenzoyl)phenyl)-
1
propan-1-one (10e): 7% yield, yellow powder; H NMR (MeOH-d4)
δ 7.29 (2H, s), 5.81 (1H, s), 3.93 (1H, m, J = 7.8 Hz), 3.87 (6H, s),
3.86 (3H, s), 1.14 (6H, d, J = 7.0 Hz); 13C NMR (MeOH-d4) δ 212.4,
205.4, 172.8, 170.5, 170.1, 149.0 (2C), 142.0, 108.2, 105.3, 104.1 (2C),
96.0, 58.3, 56.9 (2C), 40.5, 19.7 (2C); HRESIMS m/z 389.1272 [M −
−
− H]− (calcd for C17H15O4Cl2 , 353.0353).
1-(3-(2-Chloro-6-fluorobenzyl)-2,4,6-trihydroxyphenyl)-2-methyl-
propan-1-one (11i): 14% yield, yellow powder; 1H NMR (MeOH-d4)
δ 7.02 (2H, m), 6.82 (1H, t, J = 8.0 Hz), 5.84 (1H, s), 3.97 (1H, m),
3.94 (2H, s, H-11), 1.07 (6H, d, J = 7.0 Hz); 13C NMR (MeOH-d4) δ
212.0, 166.2, 164.1, 163.1, 162.4, 161.7, 136.9/136.8, 129.1/129.0,
128.3/128.2, 126.1/126.0, 114.7/114.5, 105.6, 104.5, 95.1, 40.0, 21.3,
20.0 (2C); HRESIMS m/z 337.0674 [M − H]− (calcd for
C17H15O4ClF−, 337.0648).
−
H]− (calcd for C20H21O8 , 389.1242).
General Procedure for the Preparation of the 2-Acyl-4-
alkylphloroglucinols 1 and 11a−11k and the O-Alkyl-
acylphloroglucinols 12a−12i. A solution of 8a or 8b or 8c (2.8
mmol), alkyl halide (3 mmol), and DBU (3.1 mmol) in dry
tetrahydrofuran (THF) (12.5 mL) was stirred at room temperature
under nitrogen for 48 h. The reaction was quenched with 2 M HCl
(12.5 mL), and the solution was extracted with EtOAc (12.5 mL × 3).
The combined EtOAc layers were washed with brine and dried over
2-Methyl-1-(2,4,6-trihydroxy-3-(4-isopropylbenzyl)phenyl)-
propan-1-one (11j): 15% yield, yellow powder; 1H NMR (MeOH-d4)
δ 7.14 (2H, d, J = 8.0 Hz), 7.02 (1H, d, J = 8.0 Hz), 5.93 (1H, s), 4.00
E
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Article
(1H, m), 3.79 (2H, s), 2.80 (1H, m), 1.20 (6H, d, J = 7.0 Hz), 1.12
(6H, d, J = 7.0 Hz); 13C NMR (MeOH-d4) δ 211.8, 165.6, 163.7,
161.4, 146.8, 140.7, 129.4 (2C), 126.7 (2C), 108.2, 104.5, 95.0, 39.9,
35.0, 28.3, 24.6 (2C), 19.8 (2C); HRESIMS m/z 327.1642 [M − H]−
(6H, d, J = 7.0 Hz); 13C NMR (MeOH-d4) δ 212.1, 166.4, 165.6 (2C),
150.2, 135.4, 128.9 (2C), 127.7 (2C), 105.6, 95.4 (2C), 71.1, 40.2,
35.3, 24.5 (2C), 19.7 (2C); HRESIMS m/z 327.1603 [M − H]−
(calcd for C20H23O4, 327.1602).
−
(calcd for C20H23O4 , 327.1602).
Synthesis of the 2-Acyl-4-alkylphloroglucinols 2 and 11l. A
solution of 8a (2.6 mmol), geranyl bromide or farnesyl bromide (1.2
mmol), and anhydrous K2CO3 (5.4 mmol) in acetone (10 mL) was
stirred at 65 °C under nitrogen for 6 h. After removal of the solvent
and addition of a 2 M HCl solution (20 mL), the reaction mixture was
extracted with EtOAc (20 mL × 3). The combined EtOAc layers were
washed with brine and dried over Na2SO4. Removal of the solvent
afforded a crude product, which was purified by column chromatog-
raphy on silica gel using CHCl3−acetone (19:1). The known
compound 2 was identified by comparison of its NMR spectroscopic
data (see Supporting Information) with those reported in the
literature.33
2-Methyl-1-(2,4,6-trihydroxy-3-(4-nitrobenzyl)phenyl)propan-1-
1
one (11k): 25% yield, yellow powder; H NMR (MeOH-d4) δ 7.92
(2H, d, J = 8.8 Hz), 7.36 (2H, d, J = 8.7 Hz), 5.92 (1H, s), 3.92 (1H,
m, J = 7.8 Hz), 3.85 (2H, s), 1.07 (6H, d, J = 6.7 Hz); 13C NMR
(MeOH-d4) δ 211.8, 165.0, 163.7, 162.0, 151.9, 147.1, 130.5 (2C),
124.1 (2C), 106.5, 104.5, 95.0, 40.1, 29.0, 19.9 (2C); HRESIMS m/z
330.0992 [M − H]− (calcd for C17H16O6N−, 330.0983).
Ethyl 2-(3,5-dihydroxy-4-isobutyrylphenoxy)acetate (12a): 16%
yield, yellow powder; 1H NMR (CDCl3) δ 5.96 (2H, s), 4.59 (2H, s),
4.27 (2H, q, J = 7.5 Hz), 3.85 (1H, m), 1.30 (3H, t, J = 7.5 Hz), 1.12
(6H, d, J = 7.0 Hz); 13C NMR (CDCl3) δ 211.1, 169.5, 163.9, 163.2
(2C), 105.0, 94.9 (2C), 64.9, 62.4, 39.5, 19.3 (2C), 14.3; HRESIMS
2-Methyl-1-(2,4,6-trihydroxy-3-((2E,6E)-3,7,11-trimethyldodeca-
2,6,10-trien-1-yl)phenyl)propan-1-one (11l): 25% yield, yellow solid;
1H NMR (MeOH-d4) δ 5.86 (1H, s), 5.14 (1H, t, J = 6.7 Hz), 4.99
−
m/z 281.1046 [M − H]− (calcd for C14H17O6 , 281.1031).
Ethyl 2-(3,5-dihydroxy-2-isobutyrylphenoxy)acetate (12b): 16%
yield, yellow powder; 1H NMR (CDCl3) δ 5.99 (1H, s), 5.74 (1H, s),
4.60 (2H, s), 4.24 (2H, q, J = 7.5 Hz), 3.94 (1H, m), 1.27 (3H, t, J =
7.5 Hz), 1.12 (6H, d, J = 7.0 Hz); 13C NMR (CDCl3) δ 211.1, 168.5,
167.5, 163.2, 160.9, 104.9, 97.7, 91.9, 65.7, 62.2, 39.6, 19.4 (2C), 14.2;
(2H, br t, J = 6.0 Hz), 3.95 (1H, m), 3.15 (2H, d, J = 7.0 Hz), 2.10−
1.75 (8H, m), 1.69 (3H, s), 1.59 (3H, s), 1.51 (3H, s), 1.49 (3H, s),
1.08 (6H, d, J = 7.0 Hz); 13C NMR (MeOH-d4) δ 211.9, 165.4, 163.4,
161.0, 135.8, 134.7, 131.9,125.5 (2C), 124.8, 108.4, 104.7, 95.2, 40.93,
40.87, 40.0, 27.8, 27.6, 26.1, 22.3, 20.0 (2C), 17.9, 17.9, 16.4, 16.3;
−
HRESIMS m/z 281.1056 [M − H]− (calcd for C14H17O6 , 281.1031).
−
HRESIMS m/z 399.2527 [M − H]− (calcd for C25H35O4 , 399.2541).
Diethyl 2,2′-((5-hydroxy-4-isobutyryl-1,3-phenylene)bis(oxy))-
diacetate (12c): 14% yield, yellow powder; 1H NMR (CDCl3) δ
5.89 (1H, s), 5.80 (1H, s), 4.55 (2H, s), 4.50 (2H, s), 4.16 (4H, q, J =
7.5 Hz), 3.90 (1H, m), 1.20 (6H, t, J = 7.5 Hz), 1.06 (6H, d, J = 7.0
Hz); 13C NMR (CDCl3) δ 210.3, 167.7, 167.6, 167.2, 163.1, 160.0,
105.3, 94.8, 91.7, 65.3, 64.7, 61.3 (2C), 39.3, 19.0 (2C), 13.9 (2C);
In Vitro Antifungal Assay. A modified version of the CLSI
(formerly NCCLS) method was used for susceptibility testing. The
fungal strains C. neoformans ATCC 90113 and C. gattii ATCC 32609
were obtained from the American Type Culture Collection (ATCC)
(Manassas, VA, USA). C. neoformans H99 was obtained from the
Department of Molecular Genetics and Microbiology at Duke
University School of Medicine. Amphotericin B (ICN Biomedicals,
Aurora, OH, USA) and fluconazole (ICN Biomedicals) were used as
positive controls. The detailed procedure has been described
previously.34,35
−
HRESIMS m/z 367.1371 [M − H]− (calcd for C18H23O8 , 367.1398).
N-(tert-Butyl)-2-(3,5-dihydroxy-4-isobutyrylphenoxy)acetamide
1
(12d): 27% yield, yellow powder; H NMR (MeOH-d4) δ 5.98 (2H,
s), 4.63 (2H, s), 3.99 (1H, m), 1.37 (9H, s), 1.14 (6H, d, J = 7.0 Hz);
13C NMR (MeOH-d4) δ 212.3, 171.5, 170.3, 165.6, 164.8, 106.3, 95.2
(2C), 67.7, 51.9, 40.4, 29.2 (3C), 19.7 (2C); HRESIMS m/z 308.1573
[M − H]− (calcd for C16H22O5N−, 308.1503).
In Vitro Cytotoxicity Assay. The two mammalian cell lines Vero
(African green monkey kidney fibroblast) and LLC-PK1 (pig kidney
epithelial) were obtained from ATCC. Cytotoxicity was determined by
the neutral red method.36 The detailed assay procedure has been
described previously.34,37
N-(tert-Butyl)-2-(3,5-dihydroxy-2-isobutyrylphenoxy)acetamide
(12e): 14% yield, yellow powder; 1H NMR (MeOH-d4) δ 5.93 (1H, s),
5.81 (1H, s), 4.69 (2H, s), 3.95 (1H, m), 1.34 (9H, s), 1.11 (6H, d, J =
7.0 Hz); 13C NMR (MeOH-d4) δ 211.4, 171.1, 165.7, 162.2, 106.0,
98.2, 93.3, 68.2, 52.0, 40.8, 29.2 (3C), 19.9 (2C); HRESIMS m/z
308.1508 [M − H]− (calcd for C16H22O5N−, 308.1503).
ASSOCIATED CONTENT
* Supporting Information
■
S
1-(2-((2,6-Dichlorobenzyl)oxy)-4,6-dihydroxyphenyl)-2-methyl-
1
propan-1-one (12f): 7% yield, yellow powder; H NMR (MeOH-d4)
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.6b00224.
δ 7.47 (2H, d, J = 8.0 Hz), 7.39 (1H, t, J = 8.0 Hz), 6.16 (1H, s), 5.96
(1H, s), 5.34 (2H, s), 3.49 (1H, m), 0.85 (6H, d, J = 7.0 Hz); 13C
NMR (MeOH-d4) δ 211.4, 168.4, 166.3, 163.5, 138.2 (2C), 132.8,
132.5, 129.9 (2C), 105.7, 97.6, 92.9, 66.7, 40.4, 19.7 (2C); HRESIMS
In vitro antibacterial data of compounds 5, 11j, and 12h
and original NMR and HRESIMS spectra of synthetic
compounds (PDF)
−
m/z 353.0365 [M − H]− (calcd for C17H15O4Cl2 , 353.0353).
1-(4-((2-Chloro-6-fluorobenzyl)oxy)-2,6-dihydroxyphenyl)-2-
methylpropan-1-one (12g): 6% yield, yellow powder; 1H NMR
(MeOH-d4) δ 7.40 (1H, q, J = 8.0 Hz), 7.31 (1H, d, J = 8.0 Hz), 7.15
(1H, t, J = 8.0 Hz), 6.03 (2H, s), 5.17 (2H, s), 3.99 (1H, m), 1.15 (6H,
d, J = 7.0 Hz); 13C NMR (MeOH-d4) δ 212.2, 166.2, 165.7 (2C),
164.7, 162.3, 137.7, 132.7, 126.9, 123.4/123.3, 115.7/115.5, 105.9,
95.2 (2C), 62.2, 40.3, 19.7 (2C); HRESIMS m/z 337.0655 [M − H]−
(calcd for C17H15O4ClF−, 337.0648).
AUTHOR INFORMATION
Corresponding Author
*Tel: 662-915-6742. Fax: 662-915-7989. E-mail: xcli7@olemiss.
edu (X.-C. Li).
■
Notes
1-(2-((2-Chloro-6-fluorobenzyl)oxy)-4,6-dihydroxyphenyl)-2-
methylpropan-1-one (12h): 12%, yield, yellow powder; 1H NMR
(MeOH-d4) δ 7.41 (1H, q, J = 8.0 Hz), 7.31 (1H, d, J = 8.0 Hz), 7.15
(1H, t, J = 8.0 Hz), 6.15 (1H, s), 5.96 (1H, s), 5.21 (2H, s), 3.50 (1H,
m), 0.87 (6H, d, J = 7.0 Hz); 13C NMR (MeOH-d4) δ 211.4, 168.3,
166.2, 163.4, 164.7/162.2, 137.6/137.5, 132.9/132.8, 126.9/126.8,
123.0/122.9, 115.7/115.5, 105.7, 97.7, 93.0, 62.7, 40.4, 19.7 (2C);
HRESIMS m/z 337.0674 [M − H]− (calcd for C17H15O4ClF−,
337.0648).
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank Dr. J. Heitman at Duke University for
providing the fungal strain C. neoformans H99, Ms. M. Wright
for in vitro antifungal testing, Mr. J. Trott for cytotoxicity
testing, and Dr. B. Avula for high-resolution mass spectrometric
analyses. This work was supported by the NIH, NIAID, Grant
No. AI27094, the USDA Agricultural Research Service Specific
Cooperative Agreement No. 58-6408-1-603, and the China
Scholarship Council.
1-(2,6-Dihydroxy-4-((4-isopropylbenzyl)oxy)phenyl)-2-methyl-
1
propan-1-one (12i): 9% yield, yellow solid; H NMR (MeOH-d4) δ
7.30 (2H, d, J = 8.0 Hz), 7.22 (2H, d, J = 8.0 Hz), 5.98 (2H, s), 5.00
(2H, s), 3.97 (1H, m), 2.90 (1H, m), 1.23 (6H, d, J = 7.0 Hz), 1.12
F
DOI: 10.1021/acs.jnatprod.6b00224
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Journal of Natural Products
Article
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