Synthesis of bioactive speciosins G and P from hexagonia speciosa

Article abstract of DOI:10.1021/np500341q

The first total synthesis of speciosins P and G, previously isolated from Hexagonia speciosa, is reported. These compounds have been synthesized by Sonogashira coupling from readily available starting materials. Siccayne was also synthesized from the same

Full text of DOI:10.1021/np500341q

Article
pubs.acs.org/jnp
Synthesis of Bioactive Speciosins G and P from Hexagonia speciosa
Guillermo A. Guerrero-Vasquez, Nuria Chinchilla, Jose M. G. Molinillo, and Francisco A. Macías*
́
́
Allelopathy Group, Department of Organic Chemistry, School of Sciences, INBIO Institute of Biomolecules, University of Cadiz,
C/ Republica Saharaui, s/n, 11510-Puerto Real, Cadiz, Spain
́
́
S
* Supporting Information
ABSTRACT: The first total synthesis of speciosins P and G,
previously isolated from Hexagonia speciosa, is reported. These
compounds have been synthesized by Sonogashira coupling
from readily available starting materials. Siccayne was also
synthesized from the same starting material in two steps along
with a number of other derivatives. The compounds were
tested in the wheat coleoptile bioassay. The most active
compound was the intermediate 18, followed by 29 and 17.
The structural requirements for activity in these compounds
are the presence of methoxy groups in the aromatic ring and a formyl or hydroxy group in the side chain.
n 20091 and 2011,2 Jiang and co-workers isolated a series of
Scheme 1. RetrosyntheticAnalysisforSpeciosins G(1) andP (2)
I
substituted hydroquinones, named speciosins A−T, from the
Chinese fungus basidiomycete Hexagonia speciosa. Speciosins G
(1) and P (2) are structurally similar to the biologically active
siccayne (3) (Figure 1), which was isolated from the fungus
Figure 1. Structures of speciosin G (1), ( )-speciosin P (2), and
siccayne (3).
Helminthosporium siccans in 1968 and the marine basidiomycete
Halocyphina villosa in 1981.3,4 Related compounds such as
siccayne, which are present in bacteria, fungi, higher plants, and
mollusks, have a wide range of bioactivities.
Compound 3 showed moderate antibiotic activity, inhibited
mitochondrial respiration in Saccharomyces cerevisiae,5 and
showed cytotoxic activity against the human cancer cell lines
HeLa and HT29,5,6 making speciosins G and P potential targets
in the development of drugs or pesticides.
A retrosynthetic analysis for compounds 1 and 2 is shown in
Scheme 1. The key point in this strategy is the attachment of the
carbon chain to the aromatic nucleus. The starting materials
required to generate the corresponding aryl bromide 6 are
readily available and cheap [p-benzoquinone (4), p-dimethox-
ybenzene (5)].
A second aim of this work was the preparation of analogues of
these compounds, along with siccayne (3), in order to study their
structure−activity relationships.
RESULTS AND DISCUSSION
■
Synthesis. Compound 6a7 was obtained from 4.8 The key
reaction is the Sonogashira cross-coupling with propargyl
Compound 6 could be linked with propargyl alcohol 7 by Pd-
catalyzed Sonogashira coupling to give 8, which in turn would
provide key aldehyde 9 by oxidation. Compound 9 would allow
access to 1 and 2 by functionalization reactions.
Received: April 16, 2014
© XXXX American Chemical Society and
American Society of Pharmacognosy
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alcohol.9−11 This reaction was initially carried out under the
conditions reported for the preparation of siccayne (3). In this
process n-butylamine was used as the solvent and Pd(PPh3)4 as
the catalyst at 78 °C. The yields obtained using these conditions
were about 80%.8 The introduction of Cu(I) as a cocatalyst led to
a decrease in the yield. However, when the reactor was sealed and
heated to 100 °C, the yields increased to 96%, i.e., better than
previously reported yields (Scheme 2). The reaction conditions
Scheme 4. Synthesis of ( )-Speciosin P (2) through Corey−
Chaykovsky Epoxidation
Scheme 2. Conditions for the Pd−Cu-Catalyzed Cross-
Coupling of sp2-C Halides with Terminal Acetylenes
involved the use of equimolecular amounts of aryl bromide 6a
and propargyl alcohol (7) in the presence of the commercially
available catalyst tetrakis(triphenylphosphine)palladium(0)
(3−5 mol %) and n-butylamine as solvent.
The feasibility of the oxidation of 812 to aldehyde 9 was
assessed using the Corey−Suggs reagent, pyridinium chlor-
ochromate (PCC),13−15 Dess−Martin periodinane (DMP),16
and Swern oxidation (Scheme 3).17 The latter conditions were
selected and permitted scale-up to 3.1 mmol with 91% yield.
Scheme 5. Synthesis of Speciosin G (1) through Olefination of
the Key Intermediate 9 and Burgess Dehydration of 15
Scheme 3. Synthesis of the Key Aldehyde 9
Homologation of the side chain was achieved by the Corey−
Chaykovsky reaction (Scheme 4).18−20 Thus, epoxide 10 was
obtained by in situ generation of the dimethylsulfonium
methylide12 and subsequent addition of a dichloromethane
solution of the aldehyde 9. Quantitative acid-mediated hydrolysis
of epoxide 10 was performed in acetone/H2SO4 to give
compound 11. Deprotection of the OMOM groups was carried
out in a solution of HCl in MeOH to give ( )-speciosin P (2) in
90% yield (72% overall yield).2 Simultaneous hydrolysis and
deprotection was not possible. When this reaction was carried
out in MeOH/H2SO4, the methoxy derivative 12 of ( )-spe-
ciosin P (2) was obtained.
aldehydes.22 The modification reported by Takai, in which zinc
dust, trimethylaluminum, and CH2I2 are used, was also employed
to synthesize 14, albeit in a yield less than 15%. Alternatively,
Grignard coupling23 gave a satisfactory yield of 15, and this was
subsequently dehydrated. The commonly used method with
The synthesis of speciosin G requires olefination of aldehyde 9
(Scheme 5). Wittig reaction of 9 with methyltriphenylphospho-
nium iodide under standard conditions21 provided compound 13
after decarbonylation, as observed previously for other alkynyl
B
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POCl3 led to poor yields (17−20%). The use of Burgess’ reagent
(6 equiv added in three portions over 60 h) under reflux and dry
conditions gave 14 in 80% yield.24 Deprotection under acidic
conditions yielded speciosin G (1) in 90% yield (62% overall
yield).
Scheme 7. Preparation of the Racemic Syccaine Derivatives
The presence of hydroxy and/or methoxy groups can
markedly change the bioactivity of phenolic compounds.25
Thus, we also prepared all of the intermediates with methoxy
groups in the aromatic ring. Several intermediates of speciosins
were also deprotected to give the corresponding hydroxy
compounds for subsequent assay. The reactions are summarized
in Scheme 6.
Scheme 6. Preparation of the Methoxy and Deprotected
Derivatives
compound 34 by deprotection of 30. The acetylated derivative of
6 was prepared by Sonogashira coupling of 6b.7
The preparation of speciosin G (1) and ( )-speciosin P (2)
was optimized by carrying out a number of Sonogashira reactions
under different conditions, which included changes in temper-
ature, the use of CuI as a cocatalyst, and altering the number of
equivalents of reagents. The results are summarized in Table 1.
The presence of primary hydroxy groups in the alkynyl chain at
100 °C was better tolerated when CuI was not used (entries 3, 6,
16, and 17). The presence of tertiary hydroxy groups did not have
any influence, and better results were obtained on using a
cocatalyst (entries 18 and 24). In general, higher yields were
obtained by increasing the reaction temperature. It was not
necessary to use a temperature above 100 °C, and this led to low
overpressure and allowed the accurate control of the reaction.
Speciosins G (1) and P (2) have been synthesized for the first
time in overall yields of 72% and 62%, respectively, from 6a in five
steps. Siccayne (3) was also synthesized from the same starting
material in two steps with an overall yield of 83%. This procedure
was simpler and more efficient than the reported process.8,27
Bioactivity. A total of 16 compounds were evaluated by the
wheat coleoptile bioassay. This is a rapid test that is sensitive to a
wide range of bioactive substances, including plant growth
regulators, herbicides, antimicrobials, mycotoxins, and assorted
pharmaceuticals.28−30 The results are shown in Figure 2, in which
negative values signify inhibition, positive values denote
activation, and zero represents control.
Siccayne (3) and its derivatives were also target compounds.
Siccayne (3) was synthesized in 94% yield by direct Sonogashira
reaction with the MOM-protected bromohydroquinone 6a.
The initial reaction temperature was 0 °C, and this was increased
gradually to 100 °C due to the volatility of alkyne 25. De-
protection also gave siccayne (3) in good yield (90%) under the
conditions used for speciosins. Modification of the initial chain of
25 to give diol 2626 and acetate 27 permitted the synthesis of
siccayne derivatives with or without methoxy groups in the
aromatic ring (Scheme 7). The acetyl group was removed during
the Sonogashira reaction in all cases. It was not possible to obtain
The most active compound was 18, followed by 29 and 17.
This trend can be seen more clearly by calculating the IC50 values
for the assayed compounds (Table 2), which have values of 323,
670, and 952 μM, respectively.
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Table 1. Optimization of the Conditions for the Sonogashira
Reaction
Table 2. IC50 Values Obtained in the Wheat Coleoptile
Bioassay
a
Pd(PPh3)4
(mol %)
Cu(I)
product (yield,
%)
compound IC50 (μM)
R2
compound IC50 (μM)
R2
entry substrates
(mol %)
T (°C)
1
2568
14 440
1253
0.9867
0.9804
0.9682
0.9857
0.9753
0.974
21
22
23
24
29
31
33
34
2010
62 260
7890
0.9922
0.9958
0.9638
0.9826
0.9174
0.9731
0.977
b
1
6 + 7
10
5
5
5
3
5
3
5
3
5
5
5
5
5
3
5
3
5
3
5
3
3
3
5
3
5
3
3
3
78
8 (82)
2
2
3
100
100
100
100
100
0 to 100
0 to 100
0 to 100
0 to 100
25
8 (59)
3
3
8 (96)
12
17
18
19
20
5799
4354
b
c
4
16 + 7
6 + 25
3
3
17 (62)
17 (52)
17 (95)
28 (87)
28 (92)
29 (72)
29 (94)
29 (75)
30 (94)
30 (94)
31 (94)
31 (12)
31 (99)
31 (81)
30 (80)
30 (31)
30 (65)
30 (62)
30 (44)
30 (41)
31 (91)
31 (34)
31 (72)
31 (45)
31 (33)
31 (30)
952.4
670.2
5
322.6
1055
821
1160
4434
6
0.9703
7
3
3
3
3
5
3
0.9724
8
c
9
16 + 25
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
requirements for activity in these compounds are the presence
of methoxy groups in the aromatic ring and a formyl or hydroxy
group in the chain.
d
6 + 26
100
100
100
100
100
100
100
100
100
100
78
d
16 + 26
3
3
EXPERIMENTAL SECTION
■
General Experimental Procedures. Commercially available
reagents and solvents were analytical grade or were purified by standard
procedures prior to use. Compounds were analyzed by IR, 1H NMR, and
13C NMR spectroscopy and by high-resolution ESI mass spectrometry.
The data obtained are consistent with the proposed structures. Infrared
spectra were recorded on a PerkinElmer FT-IR Spectrum 1000 Mattson
5020 system. HRMS were obtained on a Waters SYNAPT G2 mass
spectrometer (70 eV). 1H NMR, 1H−1H gCOSY, 1H−13C gHSQC, and
1H−13C gHMBC NMR spectra were recorded on Agilent INOVA-400
and INOVA-500 spectrometers using CDCl3 or methanol-d4. Chemical
shifts (δ) are reported in parts per million (ppm) relative to either a
TMS internal standard or solvent signals. HRMS data were obtained
on a Waters SYNAPT G2 mass spectrometer (70 eV). Column
chromatography was performed on silica gel (35−75 mesh), and TLC
analysis was carried out using aluminum precoated silica gel plates.
Synthetic products were purified by preparative HPLC using a
Lichrosorb silica 60 semipreparative column (Lichrospher SiO2,
Merck, 7 and 10 μm, 150 × 10 nm) and Lichrosorb silica 60 analytical
columns (Lichrospher SiO2, Merck, 7 and 10 μm, 250 × 10 mm) in
conjunction with a Hitachi Lachrom D-7000 PLC system with a Hitachi
L-7490 RI detector and Hitachi L-7420 UV detector.
c
6 + 27
3
3
3
78
c
16 + 27
3
3
100
100
100
100
78
3
78
a
The reaction time was 21 h in all cases. The solvent was nBuNH2
except for entries 22, 23, 28, and 29, where TEA, DIPEA, and THF
b
c
d
were used. 1 equiv. 1.2 equiv. 1.1 equiv.
General Procedure for the Sonogashira Coupling.8,10,11
Compounds 6a31 and 168 were synthesized according to literature
procedures. Aryl halide 6a or 16 (9.21 mmol) in n-butylamine (6.4 mL)
was placed in a flame-dried round-bottomed flask under an argon
atmosphere. A mixture of terminal alkynes 7, 25, 26, or 27 (9.21 mmol)
in n-butylamine (10 mL) and Pd(Ph3)4 (5% or 3%) was added, with the
optional addition of CuI (3%) where appropriate. The mixture was
heated for 21 h at 98 °C and poured into H2O (80 mL). The product was
extracted with EtOAc (3 × 80 mL). The combined organic layers were
washed with brine, dried over anhydrous Na2SO4, and evaporated under
reduced pressure. The crude product was purified by silica gel column
chromatography (EtOAc/hexanes, 10−50%).
3-[2,5-Bis(methoxymethoxy)phenyl]prop-2-yn-1-ol12 (8). Yield
96%; colorless oil; IR (KBr) νmax 3310, 2230 cm−1; 1H NMR (CDCl3,
400 MHz) δ 3.46 (3H, s, H-4b), 3.51 (3H, s, H-1b), 4.51 (2H, s, H-1a),
5.09 (2H, s, H-4a), 5.17 (2H, s, H-1a), 6.95 (1H, dd, J = 9 and 3.0 Hz,
H-5), 7.03 (1H, d, J = 9.0 Hz, H-6), 7.10 (1H, d, J = 3.0 Hz, H-3); 13C
NMR (CDCl3, 100 MHz) δ 51.81 (C-9), 56.05 (C-4b), 56.38 (C-1b),
81.74 (C-7), 91.56 (C-8), 95.14 (C-4a), 95.88 (C-4b), 114.19 (C-2),
117.13 (C-5), 118.50 (C-3), 121.20 (C-6), 151.95 (C-4), 153.06 (C-1);
HRESIMS m/z 275.0900 [M + Na]+ (calcd for C13H16O5 275.0896).
3-(2,5-Dimethoxyphenyl)prop-2-yn-1-ol12 (17). Yield 95%; yellow
microcrystalline solid; mp 67−68 °C; IR (KBr) νmax 2230, 3396 cm−1;
1H NMR (CDCl3, 400 MHz) δ 3.73 (3H, s, H-4a), 3.82 (3H, s, H-1a),
4.52 (2H, s, H-9), 6.78 (1H, d, J = 8.3 Hz, H-6), 6.83 (1H, dd, J = 9.0 and
3.0 Hz, H-5), 6.94 (1H, d, J = 3.0 Hz, H-3); 13C NMR (CDCl3, 100
MHz) δ 51.76 (C-9), 55.84 (C-4a), 56.43 (C-1a), 81.80 (C-7), 91.62
(C-8), 111.99 (C-3), 112.24 (C-2), 115.92 (C-5), 118.43 (C-6), 153.23
Figure 2. Effect of the compounds tested on etiolated wheat coleoptiles.
The bioassays were carried out in triplicate.
Regarding the structural requirements for activity, comparison
of pairs (29, 3), (31, 34), and (21, 1) shows that the presence of
the methoxy group led to an increase in activity. On the other
hand, the compounds with only one hydroxy group in the chain
are more active than those that contain two hydroxy groups
[cf. (22, 19) and (2, 24)]. Comparison of the siccayne (3, 29, 31,
and 34) and speciosin (1, 2, 21, and 22) derivatives revealed that
the siccayne derivatives are more active than the speciosins.
In summary, a number of derivatives were prepared and tested
in the wheat coleoptile bioassay. The optimum structural
D
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(C-1), 154.49 (C-4); HRESIMS m/z 215.0694 [M + Na]+ (calcd for
C11H12O3 215.0685).
1,4-Bis(methoxymethoxy)-2-(3-methylbut-3-en-1-yn-1-yl)-
benzene8 (28). Yield 92%; colorless oil; IR (KBr) νmax 2194, 1493 cm−1;
1H NMR (CDCl3, 400 MHz) δ 1.99 (3H, s, 10-H), 3.46 (3H, s, 4b-H)*,
3.77 (3H, s, H-4a), 3.88 (3H, s, H-1a), 6.86 (1H, d, J = 9.0 Hz, H-6), 7.01
(1H, dd, J = 9.0 and 3.1 Hz, H-5), 7.05 (1H, d, J = 3.0 Hz, H-3), 9.45
(1H, s, H-9); 13C NMR (CDCl3, 100 MHz) δ 56.02 (C-4a), 56.48
(C-1a), 92.37 (C-8), 92.56 (C-7), 108.94 (C-2), 112.36 (C-6), 119.05
(C-5), 119.97 (C-3), 153.29 (C-1), 156.49 (C-4), 176.92 (C-9);
HRESIMS m/z 213.0529 [M + Na]+ (calcd. for C11H10O3 213.0528).
General Procedure for Acetylation. Acetylations were carried out
using the standard methodology, which involved dissolving the
compound in dry pyridine (5 mL) and adding an excess of Ac2O at
0 °C. The reaction was complete after 16 h, warmed from 0 °C to room
temperature, and quenched by adding distilled H2O. The product was
extracted with EtOAc (3 × 25 mL), and the combined organic layers
were washed several times with saturated aqueous CuSO4 until the
pyridine had been removed. The organic layer was then dried over
anhydrous NaSO4, filtered, and concentrated under vacuum.
3.51 (3H, s, 1b-H)*, 5.09 (2H, s, 4a-H), 5.17 (2H, s, 1a-H), 6.95 (1H, d,
J = 9.0 and 3.0 Hz, 5-H), 7.03 (d, J = 9.0 Hz, 1H, 6-H), 7.10 (1H, d, J =
3.0 Hz, 3-H); 13C NMR (CDCl3, 100 MHz) δ 23.40 (C-10), 55.92 (C-
4a)*, 56.38 (C-1a)*, 84.44 (C-7), 94.51 (C-8), 95.01 (C-4b)*, 95.81
(C-1b)*, 113.30 (C-2), 116.35 (C-5), 117.48 (C-3), 121.39 (C-6),
121.94 (C-11), 126.85 (C-9), 151.91 (C-4), 152.70 (C-1); HRESIMS
m/z 263.1286 [M + H]+ (calcd. for C15H18O4 263.1284).
1,4-Dimethoxy-2-(3-methylbut-3-en-1-yn-1-yl)benzene32 (29).
1
Yield 94%; yellow oil; IR (KBr) νmax 1670, 2192 cm−1; H NMR
(CDCl3, 400 MHz) δ 2.0 (3H, s, H-11), 3.75 (3H, s, H-4a), 3.82 (3H, s,
H-1a), 5.29 (1H, d, J = 1.6 Hz, H-10a), 5.41 (1H, d, J = 1.6 Hz, H-10b),
6.78 (1H, d, J = 9.0 Hz, H-6), 6.81 (1H, dd, J = 9.0 and 3.0 Hz, H-5), 7.11
(1H, d, J = 3.0 Hz, H-3); 13C NMR (CDCl3, 100 MHz) δ 23.47 (C-11),
55.71 (C-4a), 56.42 (C-1a), 84.56 (C-7), 94.54 (C-8), 112.07 (C-3),
112.87 (C-2), 115.60 (C-5), 118.00 (C-3), 121.93 (C-10), 126.87 (C-
9), 153.15 (C-1), 154.32 (C-4); HRESIMS m/z 203.1073 [M + H]+
(calcd for C13H14O2 203.1073).
2-Hydroxy-2-methylbut-3-yn-1-yl acetate33 (27). Yield 89%; color-
1
less oil; IR (KBr) νmax 3278, 2116, 1737 cm−1; H NMR (CDCl3,
400 MHz) δ 1.48 (3H, s, H-4), 2.11 (3H, s, H-7), 2.45 (1H, s, H-1), 4.10
(2H, dd, J = 7.4 and 11.1 Hz, H-5); 13C NMR (CDCl3, 100 MHz) δ
20.91 (C-7), 25.98 (C-4), 66.64 (C-1), 70.82 (C-3), 72.53 (C-5), 84.83
(C-2), 171.04 (C-6); HRESIMS m/z 157.0856 [M + H]+ (calcd for
C8H12O3 157.0865).
4-[2,5-Bis(methoxymethoxy)phenyl]-2-methylbut-3-yne-1,2-diol
(30). Yield 94%; colorless oil; IR (KBr) νmax 3412, 2193 cm−1; 1H NMR
(CDCl3, 400 MHz) δ 1.53 (3H, s, H-11), 3.45 (3H, s, H-4b), 3.49 (3H,
s, H-1b), 3.56, 3.73 (each 1H, d, J = 11.0 Hz, H-10), 5.08 (2H, s, H-4a),
5.15 (2H, s, H-1a), 6.93 (1H, d, J = 9.0 and 2.9 Hz, H-5), 6.98 (1H, d,
J = 9.0 Hz, H-6), 7.07 (1H, d, J = 2.9 Hz, H-3); 13C NMR (CDCl3,
100 MHz) δ 24.93 (C-11), 55.88 (C-1a), 56.32 (C-4a), 69.26 (C-9),
70.98 (C-10), 80.62 (C-7), 94.88 (C-8), 94.95 (C-1b), 95.85 (C-4b),
113.76 (C-2), 117.03 (C-6), 118.45 (C-5), 120.50 (C-3), 151.82 (C-1),
152.88 (C-4); HRESIMS m/z 279.1235 [M − H2O + H]+ (calcd for
C15H20O6 279.1233).
4-(2,5-Dimethoxyphenyl)-2-methylbut-3-yne-1,2-diol (31). Yield
12%; yellow oil; IR (KBr) νmax 2226, 3394 cm−1; 1H NMR (CDCl3, 400
MHz) δ 1.54 (3H, s, H-11), 3.73 (3H, s, H-4a), 3.80 (3H, s, H-1a), 3.55,
3.73 (each 1H, d, J = 11.0 Hz, H-10), 6.76 (1H, d, J = 8.0 Hz, H-6), 6.83
(1H, dd, J = 9.0 and 3.0 Hz, H-5), 6.89 (1H, d, J = 3.0 Hz, H-3); 13C
NMR (CDCl3, 100 MHz) δ 24.87 (C-11), 55.70 (C-1a), 56.34 (C-4a),
69.33 (C-9), 71.03 (C-10), 80.87 (C-7), 94.94 (C-8), 111.83 (C-2),
111.89 (C-6), 115.90 (C-5), 117.75 (C-3), 153.12 (C-1), 154.39 (C-4);
HRESIMS m/z 219.0973 [M − H2O + H]+ (calcd for C13H16O4
235.0971).
4-[2,5-Bis(methoxymethoxy)phenyl]-2-hydroxy-2-methylbut-3-
yn-1-yl acetate (32). Yield 85%; colorless oil; IR (KBr) νmax 3278, 2116,
1
1737 cm−1; H NMR (CDCl3, 400 MHz) δ 1.57 (3H, s, H-11), 2.11
(3H, s, H-13), 3.43 (3H, s, H-4b), 3.48 (3H, s, H-1b), 4.10, 4.31 (each
1H, d, J = 11.1 Hz, H-10), 5.07 (3H, s, H-4a), 5.13 (3H, s, H-1a), 6.92
(1H, dd, J = 9.0 and 2.9 Hz, H-5), 6.98 (1H, d, J = 9.0 Hz, H-6), 7.05
(1H, d, J = 2.9 Hz, H-3); 13C NMR (CDCl3, 100 MHz) δ 20.78 (C-13),
25.94 (C-11), 55.85 (C-1a), 56.15 (C-4a), 67.21 (C-9), 70.86 (C-10),
80.34 (C-7), 93.74 (C-8), 94.89 (C-4b), 95.74 (C-4b), 113.81 (C-2),
117.32 (C-5), 118.45 (C-3), 120.84 (C-6), 151.79 (C-4), 152.88 (C-1),
170.77 (C-12); HRESIMS m/z 321.1342 [M − H2O + H]+ (calcd for
C17H22O7 321.1366).
4-(2,5-Dimethoxyphenyl)-2-hydroxy-2-methylbut-3-yn-1-yl ace-
tate (33). Yield 90%; yellow oil; IR (KBr) νmax 3445, 2228, 1747
cm−1; 1H NMR (CDCl3, 400 MHz) δ 1.60 (3H, s, H-11), 2.14 (3H, s, H-
13), 3.74 (3H, s, H-4a), 3.80 (3H, s, H-1a), 4.11, 4.37 (each 1H, d, J =
11.1 Hz, H-10), 6.78 (1H, d, J = 9.0 Hz, H-6), 6.83 (1H, dd, J = 9.0 and
3.1 Hz, H-3), 6.94 (1H, d, J = 3.6 Hz, H-5); 13C NMR (CDCl3, 100
MHz) δ 20.85 (C-13), 25.94 (C-11), 55.77 (C-4a), 56.38 (C-1a), 67.38
(C-9), 70.95 (C-10), 80.63 (C-7), 93.62 (C-8), 111.9 (C-3), 112.18
(C-2), 116.04 (C-5), 118.24 (C-6), 153.11 (C-1), 154.59 (C-4), 170.83
(C-12); HRESIMS m/z 261.1138 [M − H2O + H]+ (calcd for C15H18O5
261.1154).
General Procedure for the Corey−Chaykovsky Epoxida-
tion.20 Potassium hydride (1.8 mmol) as a 30% mineral oil dispersion
was placed in a 25 mL two-necked, round-bottomed flask and washed
four times with 10 mL portions of hexanes by swirling, allowing the
hydride to settle, and decanting, in order to remove the mineral oil. In a
separate 25 mL two-necked round-bottomed flask, dry CH2Cl2 (1 mL)
was added to trimethylsulfonium methyl sulfate20 (1.8 mmol) under an
argon atmosphere. This solution was added to the first vessel, and the
mixture was stirred at rt for 1 h to obtain dimethylsulfonium methylide.
A solution of aldehyde 9 (114 mg, 0.6 mmol) or 18 (150 mg, 0.6 mmol)
in dry CH2Cl2 (3.5 mL) was added, and the reaction mixture was heated
under reflux at 60 °C for 6 h under argon to generate the epoxides. Water
(10 mL) was added at rt, and the solution was stirred for 30 min. The
organic phase was decanted off, and the aqueous phase was extracted
with CH2Cl2 (3 × 10 mL). The combined organic phases were washed
with brine and dried over anhydrous Na2SO4. Evaporation of the solvent
gave a residue, which was purified on Sephadex LH-20 eluting with 95%
CH2Cl2/hexanes.
Generation of the Key Aldehyde.17 Oxalyl chloride (272.3 μL,
3.12 mmol) in dry CH2Cl2 (9 mL) was added to a stirred solution of
DMSO (332 μL, 4.68 mmol) in dry CH2Cl2 (1.5 mL) under an argon
atmosphere at −78 °C. The mixture was stirred for 15 min, and the
alcohol 8 (393.5 mg, 1.56 mmol) or alcohol 17 (300 mg, 1.56 mmol) in
dry CH2Cl2 (12 mL) was added dropwise (Note: Swern oxidation could
be scaled-up to 1.56 mmol of starting material). After the starting
material had been consumed (nearly 2 h), Et3N (1.88 mL, 7.8 mmol)
was added. The reaction mixture was stirred at −78 °C for a further
30 min and was allowed to warm to rt and quenched with saturated
NH4Cl and H2O, and the mixture was stirred for 30 min. The organic
phase was decanted off, and the aqueous layer was extracted with
CH2Cl2 (3 × 30 mL). The combined organic layers were washed with
brine, dried over anhydrous Na2SO4, and evaporated under reduced
pressure.
3-[2,5-Bis(methoxymethoxy)phenyl]prop-2-ynal (9). Yield 91%;
colorless oil; IR (KBr) νmax 1660, 2194 cm−1; 1H NMR (CDCl3,
400 MHz) δ 3.46 (3H, s, H-4b), 3.51 (3H, s, H-1b), 5.10 (2H, s, H-4a),
5.21 (2H, s, H-1a), 7.09 (1H, dd, J = 9.2 and 1.2 Hz, H-6), 7.12 (1H, dd,
J = 9.1 and 2.2 Hz, H-5), 7.22 (1H, dd, J = 2.2 and 1.3 Hz, H-3), 9.44
(1H, s, H-9); 13C NMR (CDCl3, 100 MHz) δ 56.18 (C-4b), 56.54 (C-
1b), 92.05 (C-8), 92.27 (C-7), 95.22 (C-4a), 95.58 (C-1a), 110.70 (C-
2), 116.72 (C-6), 122.0 (C-5), 122.09 (C-3), 151.85 (C-4), 154.88 (C-
1), 176.92 (C-9); HRESIMS m/z 273.0741 [M + Na]+ (calcd for
C13H14O5 273.0739).
2-{[2,5-Bis(methoxymethoxy)phenyl]ethynyl}oxirane (10). Yield
1
85%; IR (KBr) νmax 2231 cm−1; H NMR (CDCl3, 400 MHz) δ 3.01
(2H, m, H-10), 3.47 (3H, s, H-4b), 3.51 (3H, s, H-1b), 3.57 (1H, t, J =
3.4 Hz, H-9), 5.01 (2H, s, H-4a), 5.13 (2H, s, H-1a), 6.93 (1H, dd, J = 9.0
and 2.9 Hz, H-6), 6.99 (1H, dd, J = 9.0 and 3 Hz, H-5), 7.08 (1H, dd, J =
2.9 and 2.9 Hz, H-3); 13C NMR (CDCl3, 100 MHz) δ 40.19 (C-9),
48.97 (C-10), 55.76 (C-4b), 56.07 (C-1b), 79.41 (C-7), 89.55 (C-8),
3-(2,5-Dimethoxyphenyl)propiolaldehyde (18). Yield 91%; color-
less oil; IR (KBr) νmax 1656, 2186 cm−1; 1H NMR (CDCl3, 400 MHz) δ
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Journal of Natural Products
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94.86 (C-4a), 95.57 (C-1a), 113.30 (C-2), 116.84 (C-6), 118.66 (C-5),
121.00 (C-3), 151.65 (C-1), 153.24 (C-4); HRESIMS m/z 287.0905
[M + Na]+ (calcd for C14H16O5 287.0895).
(1H, q, J = 6.6 Hz, H-9), 5.06 (3H, s, H-4a), 5.14 (3H, s, H-1a), 6.90
(1H, dd, J = 9.0 and 3.0 Hz, H-5), 6.98 (1H, d, J = 9.0 Hz, H-6), 7.06
(1H, d, J = 3.0 Hz, H-3); 13C NMR (CDCl3, 100 MHz) δ 24.28 (C-10),
55.91 (C-4b), 56.25 (C-1b), 58.70 (C-9), 79.86 (C-7), 94.96 (C-4a),
95.41 (C-8), 95.83 (C-1a), 114.33 (C-2), 117.25 (C-6), 118.23 (C-5),
120.94 (C-3), 151.87 (C-4), 152.81 (C-1); HRESIMS m/z 289.1057
[M + Na]+ (calcd for C14H18O5 289.1154).
2-[(2,5-Dimethoxyphenyl)ethynyl]oxirane (20). Yield 93%; color-
less oil; IR (KBr) νmax 2192 cm−1; 1H NMR (CDCl3, 400 MHz) δ 3.03
(2H, m, H-10), 3.64 (1H, m, H-9), 3.75 (3H, s, H-4a), 3.84 (3H, s, H-
1a), 6.80 (1H, d, J = 9.1 Hz, H-6), 6.86 (1H, dd, J = 9.0 and 3.1 Hz, H-5),
6.95 (1H, d, J = 3.1 Hz, H-3); 13C NMR (CDCl3, 100 MHz) δ 40.60
(C-9), 49.38 (C-10), 55.96 (C-4a), 56.54 (C-1a), 79.88 (C-7), 89.83
(C-8), 111.67 (C-2), 112.15 (C-6), 116.57 (C-5), 118.54 (C-3), 153.27
(C-4), 155.08 (C-1); HRESIMS m/z 205.0865 [M + H]+ (calcd for
C12H12O3 205.0865).
Epoxide Hydrolysis. 4-[2,5-Bis(methoxymethoxy)phenyl]but-3-
yne-1,2-diol (11). Compound 10 (42 mg, 0.16 mmol) in H2O/acetone
(1:1) (5 mL) was placed in a flame-dried round-bottomed flask under an
argon atmosphere. H2SO4 (80 μL) was added dropwise to the solution.
The reaction mixture was stirred at rt for 90 min under argon, and the
mixture quenched with saturated NH4Cl (5 mL) and stirred for a further
30 min. The organic layer was separated. The aqueous layer was
extracted with EtOAc (4 × 10 mL). The combined organic layers were
washed with brine, dried over anhydrous Na2SO4, and evaporated under
reduced pressure. The crude material was purified by flash silica gel
column chromatography, eluting with 40% CHCl3/acetone to give
compound 11 (40.4 mg, 90% yield) as a gray, amorphous solid: IR
(KBr) νmax 3336, 2197 cm−1; 1H NMR (CD3OD, 400 MHz) δ 3.42 (3H,
s, H-4b), 3.48 (3H, s, H-1b), 3.64 (1H, dd, J = 7.2 and 11.2 Hz, H-10a),
3.70 (1H, dd, J = 4.7 and 10.1 Hz, H-10b), 4.55 (1H, d, J = 4.7 and 7.2
Hz, H-9), 5.09 (2H, s, H-4a), 5.15 (2H, s, H-4b), 6.80 (1H, dd, J = 3.0
and 9.1 Hz, H-5), 6.86 (1H, d, J = 9.1 Hz, H-6), 6.94 (1H, d, J = 3.0 Hz,
H-3); 13C NMR (CD3OD, 100 MHz) δ 56.12 (C-4a), 56.55 (C-1a),
64.78 (C-9), 67.38 (C-10), 81.98 (C-7), 93.14 (C-8), 96.09 (C-1a),
96.97 (C-4a), 115.62 (C-2), 118.49 (C-6), 119.30 (C-5), 121.93 (C-3),
153.33 (C-1), 156.24 (C-4); HRESIMS m/z 282.1053 [M + Na + H]+
(calcd for C12H18O6 282.1080).
4-(2,5-Dimethoxyphenyl)but-3-yne-1,2-diol (22). Compound 20
(20 mg, 0.1 mmol) in dry CH2Cl2 (2 mL) was placed in a flame-dried
round-bottomed flask under an argon atmosphere, and H2SO4 (1M,
8 mL) was added to the solution. The reaction mixture was heated under
reflux at 50 °C for 24 h under argon. The mixture was cooled to rt and
stirred for 16 h, the mixture was cooled to 0 °C, NaHCO3 was added to
give pH = 7, and the solution was stirred for 30 min. The organic phase
was decanted off, and the aqueous layer was extracted with EtOAc
(3 × 10 mL). The combined organic layers were washed with brine,
dried over anhydrous Na2SO4, and evaporated under reduced pressure.
The crude material was purified by silica gel column chromatography,
eluting with 60% EtOAc/hexanes, to give compound 22 (18.9 mg, 87%
yield) as a gray, amorphous solid: IR (KBr) νmax 3346, 2187 cm−1; 1H
NMR (CDCl3, 400 MHz) δ 3.75 (3H, s, H-4a), 3.81 (2H, s, H-10), 3.82
(3H, s, H-1a), 4.70 (1H, m, H-9), 6.79 (1H, d, J = 9.0 Hz, H-6), 6.85
(1H, dd, J = 9.0 and 3.0 Hz, H-5), 6.94 (1H, d, J = 3.0 Hz, H-3); 13C
NMR (CDCl3, 100 MHz) δ 55.80 (C-4a), 56.33 (C-1a), 64.05 (C-9),
66.65 (C-10), 82.77 (C-7), 90.83 (C-8), 111.57 (C-2), 11.87 (C-6),
116.23 (C-5), 118.09 (C-3), 153.16 (C-1), 154.62 (C-4); HRESIMS
m/z 205.0871 [M − H2O]+ (calcd for C12H14O4 205.0892).
4-(2,5-Dimethoxyphenyl)but-3-yn-2-ol23 (19). IR (KBr) νmax 3427,
2193 cm−1; 1H NMR (CDCl3, 400 MHz) δ 1.57 (3H, d, J = 7.0 Hz, H-
10), 3.76 (3H, s, H-4a), 3.83 (3H, s, H-1a), 4.80 (1H, q, J = 6.6 Hz, H-9),
6.79 (1H, d, J = 9.0 Hz, H-6), 6.84 (1H, dd, J = 9.0 and 3.0 Hz, H-5), 6.94
(1H, d, J = 3.0 Hz, H-3); 13C NMR (CDCl3, 100 MHz) δ 24.46 (C-10),
55.93 (C-4a), 56.55 (C-1a), 59.14 (C-9), 80.35 (C-7), 95.22 (C-8),
112.16 (C-6), 112.35 (C-2), 116.0 (C-5), 118.46 (C-3), 153.31 (C-1),
154.42 (C-4); HRESIMS m/z 189.0918 [M − H2O + H]+ (calcd for
C12H14O3 189.0943).
Dehydration Methodology.24 (Carboxysulfamoyl)triethyl-
ammonium hydroxide inner salt methyl ester34,35 (Burgess’ reagent)
(70 mg, 0.29 mmol, 3.9 equiv) in dry benzene (2 mL) and 15 (20 mg,
0.19 mmol) in dry benzene (1 mL) were placed in a flame-dried round-
bottomed flask under an argon atmosphere. The mixture was heated
under reflux at 110 °C for 18 h. A further portion of Burgess’ reagent
(54 mg, 0.23 mmol, 3 equiv) was added to the reaction mixture, and this
was heated under reflux for 24 h. Finally, an additional 3 equiv of
Burgess’ reagent was added to the reaction mixture, and this was heated
under reflux for 24 h. The reaction mixture was cooled to rt, quenched
with saturated brine (5 mL), and stirred for a further 30 min, and the
organic layer was decanted off. The aqueous layer was extracted with
EtOAc (3 × 20 mL), and the combined organic layers were washed with
brine, dried over anhydrous MgSO4, and evaporated under reduced
pressure. The crude material was purified by silica gel column
chromatography, eluting with 15% EtOAc/hexanes, to give compound
14 (15.1 mg, 81% yield) as a yellow oil.
2-But-3-en-1-yn-1-yl-1,4-bis(methoxymethoxy)benzene (14). IR
1
(KBr) νmax 2330, 1741 cm−1; H NMR (CDCl3, 400 MHz) δ 3.46
(3H, s, H-4b), 3.52 (3H, s, H-1b), 5.10 (3H, s, H-4a), 5.17 (3H, s, H-1a),
5.53 (1H, dd, J = 11.1 and 2.1 Hz, H-10), 5.73 (1H, dd, J = 17.5 and
2.1 Hz, H-10), 6.04 (1H, dd, J = 17.5 and 11.1 Hz, H-9), 6.93 (1H, dd,
J = 9.0 and 3.0 Hz, H-5), 7.02 (1H, d, J = 9.0 Hz, H-6), 7.11 (1H, d, J =
3.0 Hz, H-3); 13C NMR (CDCl3, 100 MHz) δ 56.08 (C-4b), 56.39 (C-
1b), 86.21 (C-8), 92.17 (C-7), 95.17 (C-4a), 96.03 (C-1a), 114.97 (C-
2), 117.40 (C-6), 117.47 (C-9), 118.40 (C-5), 120.96 (C-3), 127.06 (C-
10), 152.05 (C-4), 152.95 (C-1); HRESIMS m/z 287.0905 [M + Na]+
(calcd for C14H16O5 287.0896).
2-But-3-en-1-yn-1-yl-1,4-dimethoxybenzene (21). Yield 80%;
colorless oil; IR (KBr) νmax 2194 cm−1; 1H NMR (CDCl3, 500 MHz)
δ 3.75 (1H, s, H-4a), 3.84 (1H, s, H-1a), 5.53 (1H, dd, J = 11.2 and 2.1
Hz, H-10), 5.75 (1H, dd, J = 17.5 and 2.1 Hz, H-10), 6.06 (1H, dd, J =
17.5 and 11.2 Hz, H-9), 6.79 (1H, d, J = 9.0 Hz, H-6), 6.83 (1H, dd, J =
9.0 and 2.9 Hz, H-5), 6.95 (1H, d, J = 2.9 Hz, H-3); 13C NMR (CDCl3,
125 MHz) δ 56.43 (C-1a), 55.79 (C-4a), 86.17 (C-7), 92.09 (C-8),
111.97 (C-2), 112.75 (C-9), 115.84 (C-6), 117.33 (C-5), 118.1 (C-3),
126.84 (C-10), 153.18 (C-4), 154.38 (C-1); HRESIMS m/z 189.0920
[M + H]+ (calcd for C12H12O2 189.0916).
General Procedure for the Grignard Reaction.23 Compound 9
(150 mg, 0.599 mmol) or 18 (50 mg, 0.26 mmol) in dry THF (7.5 mL)
was placed in a flame-dried round-bottomed flask under an argon
atmosphere at 0 °C. The mixture was stirred for 5 min, and MeMgBr in
THF (1M, 1.5 equiv) was added dropwise to the solution over 5 min.
The reaction mixture was warmed to rt and stirred for 2.5 h. The mixture
was cooled to 0 °C, quenched with saturated NH4Cl, and stirred for a
further 30 min. The aqueous layer was extracted with EtOAc (3 × 15 mL).
The combined organic layers were washed with brine and dried over
anhydrous Na2SO4. Evaporation of the solvent gave a residue, which was
purified on Sephadex LH-20 eluting with 95% CH2Cl2/hexanes to give
compound 15 (159 mg, 99% yield) or compound 19 (54 mg, 99% yield)
as a yellow oil.
Deprotection Study. 4-[2,5-Bis(methoxymethoxy)phenyl]-2-me-
thoxy-2-methylbut-3-yn-1-ol (12). Compound 10 (0.1 mmol) in
MeOH (2.8 mL) was placed in a flame-dried round-bottomed flask
under an argon atmosphere. H2SO4 (95 μL) was added dropwise to the
solution, and this was stirred for 30 min. The reaction mixture was
quenched with saturated NH4Cl and stirred for a further 30 min, and the
organic layer was separated. The aqueous layer was extracted with
EtOAc (3 × 10 mL). The combined organic layers were washed with
brine, dried over anhydrous Na2SO4, and evaporated under reduced
pressure. The crude material was purified by silica gel column
chromatography, eluting with 60% EtOAc/hexanes, to give compound
12 (27.9 mg, 90% yield) as a colorless oil, which was highly air-sensitive
(the compound needs to be frozen and kept in the dark under argon). IR
(KBr) νmax 3336, 2197 cm−1; 1H NMR (CD3OD, 400 MHz) δ 3.43 (3H,
s, H-11), 3.70 (2H, m, H-10), 4.26 (1H, dd, J = 6.3 and 5.1 Hz, H-9),
6.73 (1H, s, H-3), 6.73 (1H, d, J = 1.0 Hz, H-5), 6.78 (1H, dd, J = 2.4 and
4-[2,5-Bis(methoxymethoxy)phenyl]but-3-yn-2-ol23 (15). IR
1
(KBr) νmax 3398, 2226 cm−1; H NMR (CDCl3, 400 MHz) δ 1.51
(3H, d, J = 6.6 Hz, H-10), 3.43 (3H, s, H-4b), 3.48 (3H, s, H-1b), 4.74
F
dx.doi.org/10.1021/np500341q | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
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1.1 Hz, H-6); 13C NMR (CD3OD, 100 MHz) δ 55.75 (C-11), 64.83 (C-
10), 73.28 (C-9), 82.53 (C-7), 90.46 (C-8), 109.74 (C-2), 116.16 (C-6),
117.50 (C-5), 117.97 (C-3), 149.95 (C-4), 151.53 (C-1); HRESIMS
m/z 207.0714 [M − H]+ (calcd for C11H12O4 207.0736).
ASSOCIATED CONTENT
■
S
* Supporting Information
Analytical data including 1H and 13C NMR spectra and selected
mass spectra for new compounds are illustrated. This material is
available free of charge via the Internet at http://pubs.acs.org.
Speciosin P. Compound 11 (22 mg, 0.078 mmol) in MeOH (1 mL)
was placed in a flame-dried round-bottomed flask under an argon
atmosphere, and HCl[33%] (10 μL) was added to the solution. The
reaction mixture was heated under reflux at 70 °C for 2 h. The mixture
was cooled to rt, and saturated KHCO3 (5 mL) was added to adjust the
pH of the solution to 7. The aqueous phase was extracted with EtOAc
(4 × 10 mL). The combined organic layers were washed with brine,
dried over anhydrous Na2SO4, and evaporated under reduced pressure.
The crude material was purified by silica gel column chromatography,
eluting with 40% CHCl3/acetone, to give the natural product ( )-speciosin
P (2) (15.1 mg, 90% yield) as a colorless oil. The spectroscopic data
corresponded to the literature data.2
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +34-956012770. Fax: +34-956016288. E-mail: famacias@
uca.es.
Notes
The authors declare no competing financial interest.
2-(3-Hydroxyprop-1-yn-1-yl)benzene-1,4-diol (23). Compound 8
(20 mg, 0.079 mmol) in MeOH (1 mL) was placed in a flame-dried
round-bottomed flask under an argon atmosphere, and HCl[33%]
(10 μL) was added to the solution. The reaction mixture was heated
under reflux at 70 °C for 2 h under argon. The mixture was cooled to rt,
and saturated KHCO3 (5 mL) was added to adjust the pH of the
solution to 7. The aqueous phase was extracted with EtOAc (4 × 10
mL), and the combined organic layers were washed with brine, dried
over anhydrous Na2SO4, and evaporated under reduced pressure. The
crude material was purified by silica gel column chromatography, eluting
with 40% CHCl3/acetone, to give 23 (12 mg, 94% yield) as a colorless
oil: IR (KBr) νmax 3270, 2193 cm−1; 1H NMR (CD3OD, 400 MHz) δ
4.41 (2H, s, 9-H), 6.63 (1H, dd, J = 8.0 and 2.6 Hz, 3-H), 6.67 (1H, d, J =
8.0 Hz, 5-H), 6.71 (1H, dd, J = 2.6 Hz, 3-H); 13C NMR (CD3OD, 100
MHz) δ 51.43 (C-9), 81.92 (C-7), 92.58 (C-8), 111.53 (C-2), 117.15
(C-6), 118.22 (C-3), 119.40 (C-5), 151.03 (C-4), 152.47 (C-1);
HRESIMS m/z 163.0387 [M − H]+ (calcd for C9H8O3 163.0395).
2-(3-Hydroxybut-1-yn-1-yl)benzene-1,4-diol (24). Yield 90%;
ACKNOWLEDGMENTS
■
The authors gratefully acknowledge the Spanish Ministry of Science
and Technology, Project No. AGL2009-08864/AGR, and
́
́
Consejeria de Innovacion, Ciencia e Industria (P07-FQM-03031).
We also acknowledge the Spanish Ministry of Science and
Technology for fellowships.
REFERENCES
■
(1) Jiang, M. Y.; Zhang, L.; Liu, R.; Dong, Z. J.; Liu, J. K. J. Nat. Prod.
2009, 72, 1405−1409.
(2) Jiang, M.-Y.; Li, Y.; Wang, F.; Liu, J.-K. Phytochemistry 2011, 72,
923−928.
(3) Ishibashi, K.; Nose, K.; Shindo, T.; Arai, M.; Mishima, H. Sankyo
Kenkyusho Nempo 1968, 20, 76−79.
(4) Kupka, J.; Anke, T.; Steglich, W.; Zechlin, L. J. Antibiot. 1981, 34,
298−304.
1
colorless oil; IR (KBr) νmax 3342, 2197 cm−1; H NMR (C2D6CO,
(5) Kim, J. H.; Mahoney, N.; Chan, K. L.; Molyneux, R. J.; Campbell, B.
C. Curr. Microbiol. 2004, 49, 282−287.
500 MHz) δ 1.44 (3H, d, J = 6.6 Hz, H-10), 4.69 (1H, q, J = 6.6 Hz, H-9),
6.70 (1H, dd, J = 8.7 and 2.7 Hz, H-5), 6.73 (1H, dd, J = 8.7 and 0.8 Hz, H-
5), 6.75 (1H, dd, J = 2.7 and 0.8 Hz, H-3); 13C NMR (C2D6CO, 125 MHz)
δ 24.94 (C-10), 58.64 (C-9), 79.33 (C-7), 97.73 (C-8), 111.10 (C-2),
116.99 (C-6), 118.05 (C-5), 118.78 (C-3), 150.89 (C-4), 152.06 (C-1);
HRESIMS m/z 179.0713 [M + H]+ (calcd for C10H10O3 179.0709).
Speciosin G. Compound 14 (20 mg, 0.081 mmol) in MeOH (2 mL)
was placed in a flame-dried round-bottomed flask under an argon
atmosphere, HCl[33%] (20 μL) was added to the solution, and the
reaction mixture was heated under reflux at 70 °C for 90 min. The
mixture was cooled to rt, and saturated aqueous KHCO3 (5 mL) was
added to adjust the pH of the solution to 7. The aqueous phase was
extracted with EtOAc (4 × 10 mL). The combined organic layers were
washed with brine, dried over anhydrous Na2SO4, and evaporated under
reduced pressure. The crude material was purified by HPLC D7000,
eluting with 30% EtOAc/hexanes, to give the natural product speciosin
G (1) (11.6 mg, 90% yield) (retention time 12.3 min) as a colorless oil.
The spectroscopic data corresponded to the literature data.1
Wheat Coleoptile Bioassays. Wheat seeds (Triticum aestivum L.
cv. Catervo) were sown in 15 cm diameter Petri dishes moistened with
H2O and grown in the dark at 22 1 °C for 3 days.36 The roots and
caryopses were removed from the shoots and were placed in a Van der
Weij guillotine, and the apical 2 mm was cut off and discarded. The next
4 mm of the coleoptiles was removed and used for the bioassay. All
manipulations were performed under a green safelight.37 Test solutions
of each compound were prepared in a phosphate-citrate buffer solution
containing 2% sucrose adjusted to pH 5.6 at concentrations of 1000,
333, 100, 33, and 10 μM. Parallel controls were also run. Five coleoptiles
were placed in each test tube containing test or control solutions (2 mL)
(three replicates per dilution). The test tubes were rotated at 0.25 rpm
on a roller tube apparatus for 24 h at 22 °C in the dark. The coleoptiles were
digitally photographed and measured. Data were statistically analyzed using
Welch’s test,38 and results are presented as percentage differences from
control. Thus, zero represents the control, positive values represent growth
stimulation, and negative values represent growth inhibition.
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