Antibacterial Prenylated Acylphloroglucinols from Psorothamnus fremontii

Article abstract of DOI:10.1021/acs.jnatprod.5b00721

Psorothatins A-C (1-3), three antibacterial prenylated acylphloroglucinol derivatives, were isolated from the native American plant Psorothamnus fremontii. They feature an unusual α,β-epoxyketone functionality and a β-hydroxy-α,β-unsaturated ketone struct

Full text of DOI:10.1021/acs.jnatprod.5b00721

Article
pubs.acs.org/jnp
Antibacterial Prenylated Acylphloroglucinols from Psorothamnus
fremontii
†
,§
†
†
†
†
†,‡
Qian Yu, Ranga Rao Ravu, Qiong-Ming Xu, Suresh Ganji, Melissa R. Jacob, Shabana I. Khan,
§
,†,‡
Bo-Yang Yu, and Xing-Cong Li*
†
‡
National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, 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: Psorothatins A−C (1−3), three antibacterial
prenylated acylphloroglucinol derivatives, were isolated from
the native American plant Psorothamnus fremontii. They feature
an unusual α,β-epoxyketone functionality and a β-hydroxy-α,β-
unsaturated ketone structural moiety. The latter forms a pseudo-
six-membered heterocyclic ring due to strong intramolecular
hydrogen bonding, as indicated by the long-range proton−
carbon correlations in the NMR experiments. Psorothatin C (3)
was the most active compound against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium,
with IC50 values in the range 1.4−8.8 μg/mL. The first total synthesis of 3 described herein permits future access to structural
analogues with potentially improved antibacterial activities.
ntibiotic resistance has increasingly posed a profound
threat to human health. The Centers for Disease Control
and Prevention, Atlanta, GA, estimates that drug-resistant
A
bacteria cause two million illnesses leading to approximately
1
2
3 000 people dying each year in the United States alone. The
discovery of new lead compounds from natural products for
antibacterial drug development remains an important approach
2
to combat antibiotic resistance.
In our search for antibacterial compounds from plants, the
MeOH extract of the aerial parts of Psorothamnus fremontii
Torr.) Barneby (Fabaceae) was shown to have IC50 values of
6.7 and 69.7 μg/mL against methicillin-resistant Staphylococcus
aureus (MRSA) ATCC 33591 and methicillin-sensitive S. aureus
RESULTS AND DISCUSSION
The dried twigs, leaves, and flowers of P. fremontii collected in
Utah, USA, were extracted with MeOH. The MeOH extract
was partitioned between hexanes and 90% MeOH in H O. The
hexanes portion that was active against MRSA 33591 (IC ,
0.4 μg/mL) was chromatographed on Sephadex LH-20 using
■
(
5
2
5
0
ATCC 29213, respectively. This plant is a native American
4
3
desert shrub distributed in California and arid areas of Nevada
CHCl −MeOH as an eluting solvent system. The most active
3
and has been used to treat internal hemorrhage and stomach
column fraction (IC < 8.0 μg/mL against MRSA 33591) was
50
4
problems by native Americans. Two isoflavonoids were
chromatographed further on normal-phase and reversed-phase
silica gel and finally purified by HPLC to afford psorothatins
A−C (1−3). All three compounds were isolated as yellow oils
and were not optically active.
5
isolated from its roots and showed antimutagenic activity. In
addition, previous phytochemical investigations of several
Psorothamnus species revealed the isolation of chalcones,
flavonoids, benzofurans, isoflavonoids, pterocarpans, and
triterpenoids, of which some showed anticancer, antiparasitic,
The UV spectrum of psorothatin A (1) showed absorption−
maxima at 255 and 285 nm. Its HRESIMS gave an [M − H]
ion peak at m/z 291.1232, suggesting a molecular formula of
6
−9
−
13
antiprotozoal, and cytotoxic activities.
In the current study,
C H O (calcd for C H O , 291.1238). The C NMR
16
20
5
16 19
5
we report the isolation, structure elucidation, and antibacterial
activity of three new prenylated acylphloroglucinol derivatives,
psorothatins A−C (1−3), from this plant, as well as the total
synthesis of 3.
spectrum displayed 16 distinct resonances. The DEPT NMR
Received: August 13, 2015
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.5b00721
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Journal of Natural Products
Article
spectrum permitted identification of five methyls, one
60.0 and C-9 at δ 51.5 suggested that C-1 is oxygenated. An
isobutyryl group in the molecule was evident from the COSY
C
methylene, two methines, and eight quaternary carbons
1
(
Table 2). The H NMR spectrum also showed corresponding
correlations from H-12 at δ 3.97 (1H, m, J = 6.5 Hz) to Me-
H
1
3/Me-14 at δ 1.13/1.14 (3H each, d, J = 7.0 Hz), which was
H
1
a
Table 1. H NMR Data for Compounds 1−3 in CDCl
supported by the HMBC correlations from the two methyl
protons and H-12 to the carbonyl C-11 at δ 207.9. Further
analysis of HMBC correlations indicated that the methyl
protons (Me-17) at δH 1.89 (s) correlated with three
3
C
H
1
2
3
1
4.19, br s
4.21, br s
2.45, d (16)
2.13, d (16)
3.85, m (6.5)
1.77, 1.48, m
1.14, d (7)
1.58, s
4.19, br s
2α
2β
12
13
14
15
16
17
18
20
21
2.42, d (16)
2.10, d (16)
3.97, m (6.5)
2.42, d (16)
2.12, d (16)
3.95, m (6.5)
quaternary carbons at δ 114.3 (C-5), 191.1 (C-6), and 163.0
C
(
C-10), while the hydroxy group proton at δ 19.37 (s) showed
H
cross-peaks with four quaternary carbons at δ 114.3 (C-5),
C
b
b
1.13, d (7)
1.13, d (7)
1
91.1 (C-6), 106.7 (C-7), and 207.9 (C-11) and one methine
b
b
1.14, d (7)
1.14, d (7)
carbon at δ 35.9 (C-12). The remaining quaternary carbon at
C
1.56, s
1.38, s
1.89, s
1.56, s
δ 185.1 (C-8) was correlated with H-1 through a three-bond
C
1.40, s
1.39, s
n
H−C correlation obtained from an HMBC experiment ( J
=
CH
1
0
1.91, s
3.13, d (7)
5.12, t (8)
1.73, s
5
Hz) that emphasized small long-range couplings. This
0.92, t (7.6)
4
experiment also generated a weak J correlation between Me-
CH
1
7 and C-9. These correlations allowed construction of the
1.67, s
carbon skeleton of 1, and its molecular composition requires
the formation of an ether bond between C-10 and C-3 of the
isopentyl group and the presence of an epoxy group between
C-1 and C-9. The proposed planar structure reasonably
explains the chemical shifts for each proton and carbon. For
OH
19.37, s
19.48, s
19.40, s
a
b
Recorded at 400 MHz. δ , ppm, mult (J in Hz). Interchangeable
H
assignment.
Table 2. 13C NMR Data for Compounds 1−3 in CDCl3
example, C-6 was shifted downfield to δ 191.1 due to the
C
a
a
b
strong electron-withdrawing effects of the two carbonyl groups.
1
2
3
In turn, C-9 resonated in the upfield region at δ 51.5 due to
C
C
δC
DEPT
CH
δC
DEPT
CH
δC
DEPT
CH
the strained oxirane ring and the shielding effects of the planar
diene-dione system. The C-6 hydroxy group forms a strong
hydrogen bonding with the C-11 carbonyl group, leading to a
significant shift of the hydroxy group proton to the downfield
region. The long-range correlations observed from this hydroxy
1
60.0
34.8
60.0
34.8
59.9
34.8
2
3
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
CH2
CH2
CH2
80.7
C
80.7
C
80.7
C
114.3
191.1
106.7
185.1
51.5
C
114.4
191.2
105.2
185.3
51.6
C
118.1
190.8
106.9
185.1
51.5
C
C
C
C
n
group proton to C-11 and C-12 in both HMBC experiments ( J
C
C
C
optimized for 5 and 10 Hz) were thus considered to result
CH
C
C
C
from two-bond and three-bond couplings, respectively, via the
C
C
C
hydrogen bond between the C-11 carbonyl group and the C-6
0
1
2
3
4
5
6
7
8
9
0
1
163.0
207.9
35.9
C
162.9
207.4
42.3
C
162.8
207.9
35.8
C
5
hydroxy group (Figure 1), as a J correlation from this
CH
C
C
C
CH
CH3
CH3
CH3
CH3
CH3
CH
CH2
CH3
CH3
CH3
CH3
CH3
CH
CH3
CH3
CH3
CH3
CH2
CH
C
c
c
d
d
18.9
26.7
18.9
19.1
16.6
19.1
30.1
30.1
30.0
31.6
31.6
31.6
8.0
8.0
21.9
12.1
121.2
132.6
18.1
CH3
CH3
26.0
Figure 1. 2D NMR key correlations of 1 and 3.
a,b
Recorded at 100 and 125 MHz, respectively. δc, ppm.
c,d
Interchangeable assignment.
hydroxy group proton to C-12 is unlikely to occur through a
five-bond coupling. This is of particular significance, since, to
the best of our knowledge, this kind of HMBC correlation has
not been described previously in the literature. The chemical
resonances, in addition to a characteristic resonance signifi-
cantly shifted downfield at δH 19.37 (1H, s) arising from a
1
13
hydroxy group proton (Table 1). Analysis of these H and C
NMR chemical shifts enabled the conclusion to be made that
this C molecule is unprecedented, and 2D NMR experiments
shift of C-6 (δ 191.1), which may be considered to result from
C
a carbonyl carbon resonance, also supports a pseudo-six-
membered heterocyclic ring involving an intramolecular
hydrogen bond.
16
were thus employed to establish structural moieties and C−C
connectivity of the molecule. The presence of an oxygenated
isopentyl unit in 1 was determined from a COSY correlation
from H-1 at δ 4.19 (br s) to H-2 at δ 2.10 and 2.42 (1H
−
The HRESIMS of psorothatin B (2) gave an [M − H] ion
peak at m/z 305.1397, which was consistent with a molecular
13
formula of C H O . The C NMR spectrum displayed 17
H
H
17 22
5
each, d, J = 16.0 Hz) as well as HMBC correlations from both
Me-15 at δH 1.56 (s) and Me-16 at δH 1.38 (s) to the
resonances, most of which were closely comparable to those of
psorothatin A (Table 2). The difference between the two
compounds is that psorothatin B bears a 2-methylbutyryl group
oxygenated quaternary C-3 at δ 80.7 and the methylene C-2 at
C
1
δC 34.8. Additional HMBC correlations from H-2 to C-1 at δC
replacing the isobutyryl group at C-7 in psorothatin A. The H
B
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Journal of Natural Products
Article
and 13C NMR chemical shifts of the 2-methylbutyryl group
observed were consistent with the reported data in the
Scheme 1. Hypothetical Biosynthesis of 3
1
1
literature.
Psorothatin C (3) gave a molecular formula of C H O as
20
26
5
−
determined by HRESIMS at m/z 345.1710 [M − H] (calcd
−
13
for C H O , 345.1707). The C NMR spectrum showed 20
20
25
5
carbon resonances, which were classified into six methyl, two
methylene, three methine, and nine quaternary carbons by
1
13
DEPT NMR. Comparison of its H and C NMR spectra with
those of psorothatin A (1) indicated that they are analogues,
and a C isoprenyl unit was substituted at C-5 in 3 (Table 2).
5
This was supported by detailed 2D NMR analysis as shown in
Figure 1. In particular, a reasonable NOESY spectrum for this
1
compound was obtained, which facilitated H NMR assign-
ments and understanding of the relative configuration and
conformation of the molecule. The C-2 methylene proton at δH
2
1
.12 (H-2β) showed NOE correlations with both H-1 and Me-
6 (δ 1.39). Another methylene proton at δ 2.42 (H-2α)
H
H
showed NOE correlations with H-1 as well as Me-15 (strong)
and Me-16 (δH 1.39) (weak). This indicated that the
tetrahydropyran ring with an epoxy functionality between C-1
and C-9 should adopt a half-chair conformation, as shown in
Figure 2. In this geometry optimized by ChemBio 3D Ultra
ruled out for these compounds to possess alternative structures
formed via possible keto−enol tautomerization. The optical
inactivity of all three compounds indicates that they are
racemates, suggesting that they were generated via a non-
stereoselective oxidation mechanism. Similar phenomena were
observed in naturally occurring racemized phenolic compounds
13,14
with an oxygenated prenyl moiety.
Although hundreds of
structurally diverse acylphloroglucinols and their derivatives
11,15−17
have been isolated from natural sources,
this is the first
report for this class of compounds with such an unprecedented
oxidation pattern.
Antibacterial testing showed that the isolated compounds 1−
had IC50 values of 11.1, 6.2, and 1.4 μg/mL, respectively,
3
against MRSA 33591 and 10.0, 5.6, and 2.4 μg/mL,
respectively, against S. aureus ATCC 29213. Since compound
3
was the most active one, we embarked on a chemical
Figure 2. Optimized geometry of 3.
synthesis of this compound. Taking advantage of available
synthetic routes for similar prenylated acylphlorogluco-
18−20
1
4.0, the calculated distances from H-2β to H-1 and equatorial
nols,
Scheme 2. Friedel−Crafts reaction of anhydrous phloroglucinol
9) with isobutyryl chloride and AlCl in nitrobenzene gave
a synthetic strategy for 3 was designed as shown in
Me-16 are 2.646 and 2.389 Å, respectively, while the distances
from H-2α to H-1, Me-16, and axial Me-15 are 2.601, 2.614,
and 2.471 Å, respectively, explaining the observed NOE
correlations. The ring fusion of this molecule requires the
expoxy group (α-oriented) and H-1 (β-oriented) to take
opposite orientations, thereby defining the relative config-
uration of C-1 and C-9. Since psorothatins A (1) and B (2)
have the same NMR behaviors as psorothatin C (3) for their
bicyclic ring system (Tables 1 and 2), they should also possess
the same relative configuration and conformation.
(
3
Scheme 2. Total Synthesis of 3
Psorothatins A−C (1−3) are novel natural products
featuring a unique α,β-epoxyketone functionality and a β-
hydroxy-α,β-unsaturated ketone structural moiety. They are
12
likely biosynthesized from acylphloroglucinol. A possible
biosynthetic pathway for 3 is proposed as shown in Scheme 1.
Prenylation of isobutyrylphloroglucinol (4) would yield
prenylated metabolite 5, which could undergo an intra-
molecular cyclization followed by dehydrogenation to afford
the prenylated chromene 6. Tautomerization of 6 into the key
intermediate 7 would make epoxidation of the exo-double bond
possible to form 8, which may be tautomerized further to the
final product 3 as a more stable tautomer. Compounds 1 and 2
1
would also be biosynthesized in a similar fashion. Since the H
and 13C NMR spectra of compounds 1−3 indicated that only
one compound was predominantly present in the NMR solvent
at room temperature (see Supporting Information), it can be
C
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Journal of Natural Products
Article
a
Table 3. In Vitro Antibacterial Activities and Cytotoxicity of Synthetic 3
b
c
antibacterial activity (IC , μg/mL)
cytotoxicity (IC , μg/mL)
50
50
SA
9213
MRSA
e
MRSA
f
MRSA
f
MRSA
f
EF
29212
VRE
51299
VRE
700221
LLC-
d
g
h
i
j
k
l
2
33591
1696
1708
1717
HEPG2
PK1
Vero
3
2.4
0.5
−
1.4
>50
−
8.8
3.0
−
8.6
3.2
−
1.6
26.9
−
5.3
2.8
−
3.3
1.6
−
>20
31
−
−
30
−
−
21
−
−
m
methicillin
vancomycin
doxorubicin
−
1.4
−
−
−
−
−
−
−
−
0.4
0.5
5.4
a
b
Both antibacterial activity and cytotoxicity are expressed as mean values of three experimental data. IC : concentration responsible for 50%
50
growth inhibition of bacterial cells. The highest test concentrations for compound 3, methicillin, and vancomycin were 20, 50, and 20 μg/mL,
c
respectively. IC : concentration responsible for 50% growth inhibition of mammalian cells. The highest test concentrations for compound 3 and
50
d
e
the positive control doxorubicin were 50 and 10 μg/mL, respectively. Methicillin-sensitive Staphylococcus aureus ATCC 29213. Standard
f
methicillin-resistant S. aureus ATCC 33591. Methicillin-resistant S. aureus ATCC strains with various degrees of resistance: 1696 is the community-
acquired USA400 strain, 1708 is a mupirocin-resistant strain, and 1717 is the community-acquired USA300 strain. Vancomycin-sensitive
Enterococcus faecalis ATCC 29212. Low-level vancomycin-resistant E. faecalis ATCC 51299. Vancomycin-resistant E. faecium ATCC 700221.
Human hepatic carcinoma. Pig kidney epithelial. African green monkey kidney fibroblasts. Not tested.
g
h
i
j
k
l
m
acylphloroglucinol 4. Alkylation of 4 with prenyl bromide in the
presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and dry
tetrahydrofuran (THF) afforded prenylated acylphloroglucinol
aluminum sheets (silica gel 60 F254, Merck, Darmstadt, Germany)
and reversed-phase glass plates (RP-18 F254S, Merck) and visualized
by UV 254 nm and spraying with 10% H SO , followed by heating.
Flash column chromatography was performed 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). HPLC was
performed on a Waters 2795 series HPLC system composed of an LC-
20AT pump with an SPD-20A detector (Shimadzu Corp., Kyoto,
Japan). A Phenomenex C18 semipreparative HPLC column (250 ×
10.0 mm i.d., 5 μm, Palo Alto, CA, USA) was used. The mobile phase
2
4
10. Compound 10 reacted with 3-methyl-2-butenal in ethyl-
enediamine diacetate (EDDA) and CH Cl yielded a mixture of
2
2
prenylated acylchromenes (6 and 11). Treatment of 6 with
oxygen in EDDA and CH Cl furnished the final product 3.
2
2
However, similar treatment of 11 did not produce the
corresponding epoxide. This can be explained by the strong
intramolecular hydrogen bonding in 11 preventing tautomeri-
zation, whereas in 6 there is a free hydroxy group that facilitates
tautomerization for oxidation (Scheme 1).
Synthetic psorothatin C (3) showed equivalent antibacterial
activity to that of the natural product in the initial testing
against the aforementioned Staphylococcus strains. It was further
tested against multiple drug-sensitive and -resistant S. aureus
and Enterococcus strains. As shown in Table 3, compound 3 was
active against methicillin-resistant S. aureus and vancomycin-
resistant E. faecium with IC50 values in the range 1.4−8.8 μg/
mL. In vitro cytotoxicity testing showed that 3 exhibited IC50
values in the range 21−31 μg/mL against three mammalian cell
lines (HEPG2, LLC-PK1, and Vero), indicating favorable
antibacterial selectivity for this compound.
In conclusion, three novel antibacterial prenylated acylphlor-
oglucinols, psorothatins A−C (1−3), have been isolated from
P. fremontii. Compound 3 has been synthesized from
phloroglucinol by a facile four-step reaction sequence. This
study has provided a novel chemically tractable natural product
template for the synthesis of analogues with potentially
improved antibacterial properties against drug-resistant strains.
was composed of solvent A (5% H O) and solvent B (95%
2
acetonitrile). The flow rate, injection volume, column temperature,
and UV wavelength were set at 1.8 mL/min, 25 μL, 25 °C, and 325
nm, respectively. Reactants or reagents for synthesis including
anhydrous phloroglucinol, isobutyryl chloride, 3,3-dimethylallyl bro-
mide, 3-methyl-2-butenal, aluminum chloride, 1,8-diazabicyclo[5.4.0]-
undec-7-ene, ethylenediamine diacetate, nitrobenzene, tetrahydrofur-
an, and sodium sulfate were purchased from Sigma-Aldrich
Corporation (St. Louis, MO, USA) in appropriate grades and were
used without further purification.
Plant Material. The twigs, leaves, and flowers of Psorothamnus
fremontii were collected in Garfield, Utah, USA, with coordinates of
3
8°09′00″ N, 112°00′00″ W by Elray Nixon in June 2011, who also
identified this plant (specimen no. NIX0200611). An authentic sample
of the plant material is deposited at the National Center for Natural
Products Research at The University of Mississippi (NIX20061-1-A).
Extraction and Isolation. The dried twigs, leaves, and flowers
(
1
350 g) were extracted with MeOH (1 L × 3) at room temperature for
2 h. Removal of the solvent by a rotary evaporator under vacuum at
40 °C yielded an MeOH extract (56.5 g), which was dissolved in 90%
aqueous MeOH (500 mL) and partitioned with hexanes (500 mL ×
3). The hexanes extract (12.5 g) was active against MRSA ATCC
3
3591, with an IC50 value of 40.4 μg/mL. It was chromatographed on
a Sephadex LH-20 column using a CHCl −MeOH gradient eluting
3
solvent system to yield pooled fractions A−F. Fraction D (5.09 g, IC
5
0
EXPERIMENTAL SECTION
■
< 8 μg/mL against MRSA ATCC 33591) was chromatographed by
General Experimental Procedures. Optical rotations were
measured on a SEPA-3000 automatic digital polarimeter. UV spectra
were measured on a Hewlett-Packard 8453 spectrometer. IR spectra
were run on an ATI Mattson Genesis Series FTIR spectrometer. The
NMR spectra using standard pulse programs were recorded at room
temperature on a Bruker Avance DPX-400 spectrometer operating at
normal-phase silica gel using hexanes−CHCl (9:1−3:7, 5 L) as the
3
gradient eluting solvent system, followed by MeOH (1 L) to give
pooled fractions D1−D12. The combined active fractions D7−D9
(350 mg, IC50 ≈ 0.8 μg/mL against MRSA ATCC 33591) were
separated by a reversed-phase silica gel column with aqueous CH CN
3
(55−100%) followed by semipreparative reversed-phase HPLC
1
13
4
00 ( H) and 100 ( C) MHz or a Bruker Avance DRX 500 FT
purification using 95% CH CN in H O, to give psorothatins A (1)
3
2
1
13
spectrometer operating at 500 ( H) and 125 ( C) MHz. The
chemical shift (δ, ppm) values were calibrated using the residual NMR
solvent. 2D NMR spectra were measured with standard pulse
programs and acquisition parameters, except for the HMBC
(3.5 mg, t 10.5 min), B (2) (2.3 mg, t 11.7 min), and C (3) (2.2 mg,
R
R
tR 13.5 min).
Psorothatin A (1) [6-hydroxy-7-isobutyryl-3,3,5-trimethyl-1a,2-
2
0
D
dihydro-3H,8H-oxireno[2,3-d]chromen-8-one]: yellow oil, [α] 0 (c
experiments, which were set for the delay times of 50 and 100 ms,
1.0, CHCl ); UV (MeOH) λ log (ε) 255 (4.04), 285 (3.22) nm; IR
3
max
n
corresponding to J coupling constants of 10 and 5 Hz, respectively.
(CHCl ) 3750, 2975, 2929, 1662, 1526, 1466, 1124, 889, 810, 719
CH
3
−
1
HRESIMS was conducted on an Agilent series 1100 SL spectrometer
equipped with an ESI source. TLC was performed on silica gel
cm ; NMR (CDCl ) data, see Tables 1 and 2; HRESIMS m/z
3
−
−
291.1232 [M − H] (calcd for C H O , 291.1238).
16
19
5
D
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1
Psorothatin B (2) [6-hydroxy-3,3,5-trimethyl-7-(2-methylbutano-
yl)-1a,2-dihydro-3H,8H-oxireno[2,3-d]chromen-8-one]: yellow oil,
Compound 11: pale yellow oil; H NMR (400 MHz, CDCl ) δ
3
14.28 (s, OH), 6.55 (1H, d, J = 9.9 Hz, H-1), 6.35 (1H, br s, OH),
5.44 (1H, d, J = 9.9 Hz, H-2), 5.27 (1H, t, J = 7.0 Hz, H-18), 3.90 (1H,
m, J = 7.8 Hz, H-12), 3.38 (2H, d, J = 7.0 Hz, H-17), 1.82 (3H, s, Me-
20), 1.77 (3H, s, Me-21), 1.48 (6H, s, Me-15,16), 1.18 (6H, d, J = 7.0
Hz, Me-13,14); C NMR (100 MHz, CDCl
(C-10), 157.3 (C-6), 154.8 (C-8), 136.6 (C-19), 124.9 (C-2), 122.0
(C-18), 116.8 (C-1), 105.5 (C-7), 105.0 (C-5), 102.0 (C-9), 77.9 (C-
3), 39.5 (C-12), 28.0 (C-15), 27.9 (C-16), 26.1 (C-20), 21.8 (C-17)+,
19.8 (C-13,14), 18.0 (C-21); HRESIMS m/z 331.1965 [M + H]
(calcd for C H O , 331.1904). The NMR assignments of this new
synthetic compound (with a numbering system similar to psorothatins,
which is shown in the Supporting Information) were made by
20
[
α] 0 (c 1.0 CHCl ); UV (MeOH) λ log (ε) 255 (4.06), 290
D 3 max
(
3.28) nm; IR (CHCl ) 3726, 2971, 2932, 1662, 1622, 1525, 1467,
3
−1
1
2
3
120, 973, 879, 798, 668 cm ; NMR (CDCl ) data, see Tables 1 and
3
−
−
13
; HRESIMS m/z 305.1397 [M − H] (calcd for C H O ,
3
) δ 210.9 (C-11), 163.6
17
21
5
05.1394).
Psorothatin C (3) [6-hydroxy-7-isobutyryl-3,3-dimethyl-5-(3-
methylbut-2-en-1-yl)-1a,2-dihydro-3H,8H-oxireno[2,3-d]chromen-
8
2
1
-one]: yellow oil, [α]20 0 (c 1.0, CHCl ); UV (MeOH) λ log (ε)
D 3 max
+
4
30 (4.68), 290 (3.13) nm; IR (CHCl ) 3736, 2974, 2924, 1662, 1622,
3
20 27
−1
525, 1442, 1376, 1250, 889, 798, 749 cm ; NMR (CDCl ) data, see
3
−
Tables 1 and 2; HRESIMS m/z 345.1710 [M − H] (calcd for
C H O , 345.1707).
−
22
comparison with similar compounds in the literature.
Synthesis of Psorothatin C (3). To a solution of compound 6
20
25
5
Synthesis of Isobutylphloroglucinol (4). To a solution of
anhydrous phloroglucinol (9, 5.0 g, 39.7 mmol) in nitrobenzene (40
(62.7 mg, 0.189 mmol) in CH
.06 mmol). The reaction mixture was stirred at room temperature
under oxygen for 24 h. After addition of H O (5 mL), the reaction
Cl (5 mL) was added EDDA (0.009 g,
2 2
0
mL) was added AlCl (21.2 g, 158.5 mmol). The reaction mixture was
3
stirred at room temperature under nitrogen for 30 min. Isobutyryl
chloride (4.57 mL, 43.6 mmol) was slowly added, and the reaction
mixture was heated at 65 °C for 21 h. The reaction was quenched by
addition of ice−water (20 mL). The reaction mixture was extracted
with EtOAc (50 mL × 3). The combined EtOAc layers were treated
with 2 M NaOH (80 mL × 2). The aqueous layers were neutralized
with 2 M HCl and extracted with EtOAc (50 mL × 3). The combined
EtOAc layers were washed with water and brine, dried over Na SO ,
2
mixture was extracted with CH Cl2 (8 mL × 3). The combined
2
CH Cl layers were dried over Na SO . Evaporation of the solvent
2
2
2
4
gave a crude product, which was purified by column chromatography
on silica gel using hexanes−acetone (95:5) to give 3 (15 mg, 23%
yield): yellow oil; its H and C NMR spectra are identical with those
of the natural product.
In Vitro Antibacterial Assays. Microorganisms were obtained
from the American Type Culture Collection (Manassas, VA, USA)
including Staphylococcus aureus ATCC 29213 (methicillin-sensitive),
Staphylococcus aureus ATCC 33591 (standard methicillin-resistant
strain), Staphylococcus aureus ATCC BAA-1696 (the community-
acquired USA400 strain), Staphylococcus aureus ATCC BAA-1708
mupirocin-resistant strain), Staphylococcus aureus ATCC BAA-1717
the community-acquired USA300 strain), vancomycin-sensitive
Enterococcus faecalis ATCC 29212, low-level vancomycin-resistant
Enterococcus faecalis ATCC 51299, and vancomycin-resistant Enter-
ococcus faecium ATCC 700221. All organisms were tested using
1
13
2
4
filtered, and concentrated under reduced pressure to afford a crude
product, which was purified by column chromatography on silica gel
using CHCl −MeOH (9:1) to give 4 (4.5 g, 58% yield): pale yellow
3
solid. Identification of compound 4 was made by comparison of its
18
NMR data with those reported in the literature.
(
(
Synthesis of 2-Methyl-1-(2,4,6-trihydroxy-3-(3-methylbut-2-
en-1-yl)phenyl)propan-1-one (10). A mixture of 4 (2.21 g, 11.3
mmol), prenyl bromide (1.79 g, 12 mmol), and DBU (2.01 g, 13.2
mmol) in dry THF (50 mL) was stirred at room temperature under
nitrogen for 48 h. After addition of a 2 N HCl solution (50 mL), the
reaction mixture was extracted with EtOAc (50 mL × 3). The
combined EtOAc layers were washed with brine and dried over
Na SO . Removal of the solvent afforded a crude product, which was
23
modified versions of the CLSI (formerly NCCLS) method, which
has been described previously. Briefly, samples (dissolved in DMSO)
24
were serially diluted in 20% DMSO−saline and transferred (10 μL) in
duplicate to 96-well flat-bottom microplates. Inocula were prepared by
adjusting the OD630 of microbe suspensions in the assay to afford the
final desired CFU/mL. The positive control drugs included methicillin
and vancomycin (from ICN Biomedicals, Aurora, OH, USA). The
highest test concentrations of compounds and the control drugs are
indicated in Table 3. IC50 values (concentration that affords 50%
inhibition relative to controls) were calculated using XLfit software
IDBS, Alameda, CA, USA) with Fit Model 201.
In Vitro Cytotoxicity Assays. A panel of mammalian cell lines
obtained from ATCC (Manassas, VA, USA) included human hepatic
carcinoma (HEPG2), pig kidney epithelial (LLC-PK ), and African
2
4
purified by column chromatography on silica gel using hexanes−
EtOAc (7:3) to give 10 (1.3 g, 43% yield): pale yellow solid.
Identification of compound 10 was made by comparison of its NMR
21
data with those reported in the literature.
Synthesis of 1-(5,7-Dihydroxy-2,2-dimethyl-8-(3-methylbut-
2
(
-en-1-yl)-2H-chromen-6-yl)-2-methylpropan-1-one (6) and 1-
5,7-Dihydroxy-2,2-dimethyl-6-(3-methylbut-2-en-1-yl)-2H-
(
chromen-8-yl)-2-methylpropan-1-one (11). To a solution of
compound 10 (649.8 mg, 2.46 mmol) and 3-methyl-2-butenal (0.3
mL, 5.1 mmol) in CH Cl2 (30 mL) at room temperature under
2
1
nitrogen was slowly added EDDA (0.09 g, 0.6 mmol). The reaction
green monkey kidney fibroblasts (Vero). The detailed assay procedure
has been described in our previous papers.
mixture was stirred at room temperature for 12 h. H O (30 mL) was
25,26
2
added, and the reaction mixture was extracted with CH Cl (30 mL ×
2
2
3
). The combined CH Cl layers were dried over Na SO . Evaporation
2 2 2 4
ASSOCIATED CONTENT
of the solvent under reduced pressure afforded a residue, which was
chromatographed on silica gel using hexanes−EtOAC (8:2) to give 6
■
*
S
Supporting Information
(
147 mg, 18% yield) and 11 (301 mg, 37% yield).
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.5b00721.
1
Compound 6: pale yellow oil; H NMR (400 MHz, CDCl ) δ
3
13.27 (s, OH), 6.65 (1H, d, J = 9.9 Hz, H-1), 5.46 (1H, d, J = 9.9 Hz,
H-2), 5.19 (1H, t, J = 6.5 Hz, H-18), 3.90 (1H, m, J = 7.8 Hz, H-12),
3
2
.35 (2H, d, J = 6 Hz, H-17), 1.85 (3H, s, Me-20), 1.79 (3H, s, Me-
1), 1.42 (6H, s, Me-13,14), 1.16 (6H, d, J = 7.0 Hz, Me-15,16);
NMR and ESIMS spectra of compounds 1−4, 6, 10, and
11 (PDF)
13
C
NMR (100 MHz, CDCl ) δ 211.0 (C-11), 163.6 (C-6), 158.9 (C-10),
3
1
1
57.0 (C-8), 136.2 (C-19), 125.6 (C-2), 121.7 (C-18), 116.5 (C-1),
05.6 (C-7), 104.3 (C-5), 102.8 (C-9), 77.9 (C-3), 39.6 (C-12), 28.4
AUTHOR INFORMATION
■
*
(
(
3
C-15), 27.9 (C-16), 26.1 (C-20), 21.9 (C-17), 19.7 (C-13,14), 18.1
C-21); HRESIMS m/z 331.1982 [M + H] (calcd for C H O ,
31.1904). The NMR assignments of this new synthetic compound
Corresponding Author
Tel: 662-915-6742. Fax: 662-915-7989. E-mail: xcli7@olemiss.
edu (X.-C. Li).
+
+
20
27
4
(
with a numbering system similar to psorothatins, which is shown in
Notes
the Supporting Information) were made by comparison with similar
compounds in the literature.
22
The authors declare no competing financial interest.
E
DOI: 10.1021/acs.jnatprod.5b00721
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
ACKNOWLEDGMENTS
■
The authors thank Ms. M. Wright for in vitro antimicrobial
testing, Mr. J. Trott for cytotoxicity testing, Dr. B. Avula for
mass spectrometry analyses, and Mr. Frank T. Wiggers for 2D
NMR data. This work was supported by the USDA Agricultural
Research Service Specific Cooperative Agreement No. 58-6408-
2-0009 and China Scholarship Council.
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DOI: 10.1021/acs.jnatprod.5b00721
J. Nat. Prod. XXXX, XXX, XXX−XXX