Synthesis of bioreductive esters from fungal compounds

Article abstract of DOI:10.1248/cpb.55.930

Four new bioreductive esters (7-10) have been synthesized. Their structures composed of trimethyl lock containing quinone propionic acid with an ester linkage to the fungal cytotoxic compounds; preussomerin G (1), preussomerin I (2), phaseolinone (3) and phomenone (4). The synthesized esters are aimed to act via reductive activation specifically at the cancer cells, resulting from hypoxia and overexpression of reductases. Hence, the toxicity will be lessened during distribution across the normal cells. The anticancer activity was determined in cancer cell lines with reported reductase i.e., BC-1 cells and NCI-H187 as well as in non-reductase containing cancer cells; KB cells. When considering each cell lines, result showed that structure modification giving to 7-10 led to less cytotoxicity than their parent compounds (1-4). Both 7 and 8 were strongly cytotoxic (IC₅₀≤5 μg/ml) to NCI-H187, whereas 9 and 10 were moderately cytotoxic (IC₅₀=6-10 μg/ml) to BC-1 cells. Additional study of stability of represented phenolic ester (8) and an alcoholic ester (9) were performed. Result illustrated that both 8 and 9 were stable in the presence of esterase. Therefore, the cytotoxicity of the synthesized compounds (8-10) might be due to partial bioreductive activation in the cancer cells.

Full text of DOI:10.1248/cpb.55.930

930
Notes
Chem. Pharm. Bull. 55(6) 930—935 (2007)
Vol. 55, No. 6
Synthesis of Bioreductive Esters from Fungal Compounds
Natthida WEERAPREEYAKUL, ,a Rutchayaporn ANORACH,a Thidarut KHUANSAWAD,a Chavi YENJAI,b and
Masahiko ISAKA
*
c
a Faculty of Pharmaceutical Sciences, Khon Kaen University; b Faculty of Sciences, Khon Kaen University; Khon Kaen,
40002 Thailand: and c National Center for Genetic Engineering and Biotechnology (BIOTEC); 113 Paholyothin Road,
Klong 1, Klong Luang, Pathumthani 12120, Thailand. Received January 29, 2007; accepted March 16, 2007
Four new bioreductive esters (7—10) have been synthesized. Their structures composed of trimethyl lock
containing quinone propionic acid with an ester linkage to the fungal cytotoxic compounds; preussomerin G (1),
preussomerin I (2), phaseolinone (3) and phomenone (4). The synthesized esters are aimed to act via reductive
activation specifically at the cancer cells, resulting from hypoxia and overexpression of reductases. Hence, the
toxicity will be lessened during distribution across the normal cells. The anticancer activity was determined in
cancer cell lines with reported reductase i.e., BC-1 cells and NCI-H187 as well as in non-reductase containing
cancer cells; KB cells. When considering each cell lines, result showed that structure modification giving to
7—10 led to less cytotoxicity than their parent compounds (1—4). Both 7 and 8 were strongly cytotoxic
(IC50ꢁ5 mg/ml) to NCI-H187, whereas 9 and 10 were moderately cytotoxic (IC50ꢀ6—10 mg/ml) to BC-1 cells. Ad-
ditional study of stability of represented phenolic ester (8) and an alcoholic ester (9) were performed. Result il-
lustrated that both 8 and 9 were stable in the presence of esterase. Therefore, the cytotoxicity of the synthesized
compounds (8—10) might be due to partial bioreductive activation in the cancer cells.
Key words bioreductive ester; fungal compound; cytotoxic; preussomerin I; preussomerin G; phaseolinone; phomenone
Thailand is known for its biodiversity of natural herbs and city.
fungi. As a consequence, vast varieties of chemical com-
Solid tumors have been reported to be hypoxia and over-
pounds have been discovered. Previously, many cytotoxic expression of reductase enzymes when compared to those in
compounds such as those found in the fungal extracts have the normal cells.3) Irregular shape of the blood vessels and
been screened for anticancer activity.1,2) Preussomerin G, 1 condensed mass in the tumor leads to low levels of oxygen
and preussomerin I, 2 were purified from Xylaria sp. 1067, and nutrient supply, while hypoxia leads to resistance to radi-
while (ꢀ)-phaseolinone, 3 and (ꢀ)-phomenone, 4 were puri- ation therapy.4) The rational design of the bioreductive anti-
fied from Lichenicolous Fungus Microsphaeropsis sp. BCC cancer agents uses these properties as advantages for control-
30501,2) (Fig. 1). Their cytotoxicities to cancer cell lines were ling the bioreductive activation of the bioreductive anticancer
reported as well as in the normal cells. 1, 2, 3 and 4 were agents aiming to specifically release a potent drug only at the
found to exhibit strong cytotoxicity to the KB and BC-1 can- tumor sites. Hence this reduces toxicity during systemic dis-
cer cells, and also showed toxicity to the Vero cells’, which tribution.
represented normal cells.1,2) This led to a limitation for fur-
Quinone delivery systems containing a trimethyl lock have
ther clinically study or for biopharmaceutical uses. More ef- been designed to release toxic moieties selectively and pref-
fort to decrease this bottle neck problem of drug discovery erentially under reductive/hypoxic conditions5—9) (Fig. 2). A
by adopting the bioreductive concept was thus attempted. conformationally constrained bioreductive promoiety was at-
Therefore, in this study, structure modification of 1—4 based tached to a model anticancer agent, melphalan, to create a
on bioreductive activation has been adopted to lessen toxi- tumor targeted drug delivery system (TDDS).5,6) The TDDS
was a substituted benzoquinone, which can also attach to any
drug of interest via an ester or amide linker.5—9) The confor-
mational constraint in the quinone-containing promoiety was
created by the presence of the gem-dimethyl group on the
propionic acid spacer linking the drug and the promoiety;
and the adjacent methyl group on the quinone ring
(“trimethyl lock”). Upon reductive activation, the quinone
was converted to its hydroquinone. The lone pair electrons
form the ortho-OH was used in an intramolecular cyclization
Fig. 2. Structure of Quinone Bioreductive Agent Containing a Trimethyl
Lock as Specified by Dotted Line5,6)
Fig. 1. Fungal Extract Compounds
∗ To whom correspondence should be addressed. e-mail: natthida@kku.ac.th
© 2007 Pharmaceutical Society of Japan

June 2007
931
Fig. 3. Schematic Representing Anticipated Bioreductive Activation of
Bioreductive Agent (Where X is O or N) in the Cancer Cells Containing Re-
ductase Enzyme
Adapted from refs. 5, 6.
Fig. 5. Structures of Synthesized Bioreductive Esters from Fungal Com-
pounds
Fig. 4. Synthesis Steps for Bioreductive Esters when R is the Fungal Cy-
totoxic Compounds 1—4
to release the potent drug resulting in the formation of non- Discussion and Conclusion
toxic lactone species (Fig. 3).5,6) The drug will only be re-
The structures of the bioreductive ester of fungal cytotoxic
leased efficiently in the presence of the tri-methyl lock sys- compounds are shown in Fig. 5. It should be noted that 2.5
tem. The trimethyl lock-containing structure has been re- and 4 eq of quinone propionic acid (6) was required for the
ported to undergo bioreductive activation with rate of reac- reaction with 3 and 4 to get the ester products 9 and 10, res-
tion giving potential specific delivery of many types of pectively. When an attempt to use a lower equivalent of 6
model compounds such as amines, amino acids, peptides, al- (1 eq) was conducted as in the synthesis of bioreductive es-
cohol, fluorophores and other anticancer agents.7,9,10—19)
ters 7 and 8, no bioreductive ester product was formed. After
Various substituents on the benzoquinone ring with differ- structure elucidation, it was found that 9 and 10 were cou-
ent electronic properties such as a methyl-substituted benzo- pled with two molecules of quinone propionic acid. That
quinone were reported to enhance reductase enzyme selectiv- might be due to the structures of 3 and 4 possessing free
ity and stability of the bioreductive promoiety, while redox –OH on both sides of their structures.
properties govern the rate of release of the potent drug.5,6)
The standard anticancer agents, ellipticin and doxorubicin
Therefore, a methyl-substituted benzoquinone containing a were used as positive controls. It was found that ellipticin
trimethyl lock was selected as a template for the design of a and doxorubicin were strongly cytotoxic in all cancer cell
new bioreductive agent from fungal cytotoxic compounds. types as well as in the Vero cell line. Upon bioreductive acti-
Based on the hydroxyl-containing structure of selected fun- vation of the synthesized compounds, it is supposed that the
gal compounds (1—4), structure modification as bioreduc- ester compound is cleaved at ester bond and releases the cy-
tive agent has been designed by using an ester linkage. In this totoxic compound as well as its lactone by-product (Fig. 3).
study, four bioreductive agents were synthesized and struc- Results showed that lactone, 5 exhibited no cytotoxicity as
tures were elucidated by using spectrometry. Then, anti- the IC50 was higher than 50 mg/ml. The cytotoxicity of 6 was
cancer activities in cancer cells as well as Vero cells were also determined in case it might be presented in the cell be-
evaluated in comparison to their parent compounds.
cause of an unpredicted reaction. It was found that 6 did not
show any cytotoxicity as it was inactive (IC50ꢂ50 mg/ml) in
all cell lines studied.

932
Vol. 55, No. 6
Fig. 6. Stability Profile of Compounds 8 and 9 in the Presence of Esterase under 0.05 M Phosphate Buffer Solution, pH 7.4 at 37 °C Which Was Deter-
mined by Gas Chromatography Spectrometer
All data are average of three determinationsꢃS.E.M. The % remaining on Y axis was calculated from the percent of peak area ratio between bioreductive ester and the internal
standard caffeine compared to the peak area of the bioreductive ester in an absence of esterase enzyme.
Table 1. Cytotoxicity Results for Compounds 1—10
The bioreductive ester of fungal compounds, 7, 8, 9 and 10
demonstrated lesser cytotoxicity in normal cells (e.g., Vero
cell) compared to their parents; 1, 2, 3, and 4, respectively.
IC50 (mg/ml)
Compounds
#
When considering of cytotoxicity in non reductase-contain-
ing cancer cells (e.g., KB cells) between the synthesized
bioreductive esters and their parents (1 vs. 7; 2 vs. 8; 3 vs. 9
and 4 vs. 10), the synthesized bioreductive esters were less
toxic than their parents.
Vero cells KB cells BC-1 cells NCl-H187
Standard ellipticine
Standard doxorubicin
Lactone
0.6
1.9
ꢂ50
ꢂ50
0.1
1.3
0.1
0.6
0.7
4.4
nd
0.27
0.17
ꢂ50
ꢂ50
0.4
0.21
0.14
ꢂ50
ꢂ50
0.7
0.15—0.49
0.02—0.04
ꢂ50
(5)
(6)
(1)
(7)
(2)
(8)
(3)
(9)
(4)
(10)
Quinone propionic acid
Preussomerin G
Preussomerin G ester
Preussomerin I
ꢂ50
It was found that, 1 was strongly toxic to all cell lines with
IC50 ranged from 0.1—0.4 mg/ml. The KB cell (non reduc-
tase-containing cell) and NCI-H187 cancer cells (a reduc-
tase-containing cells) were more sensitive to 1 than other
cancer cell lines. After structural change into bioreductive
ester (7), the NCI-H187 was the highest sensitive cancer cells
to 7 giving IC50 of 0.82 mg/ml, while the KB cells was less
sensitive giving IC50 of 1.15 mg/ml. 2 was strongly toxic to
all cell lines with IC50 ranged from 0.1—0.6 mg/ml. The
NCI-H187 cancer cell was shown to be the most sensitive to
2 than the KB cell and other cancer cell lines. After struc-
tural change into 8, the NCI-H187 was the highest sensitive
cancer cells to 8 yielding IC50 of 1.22 mg/ml, whereas the KB
cells yielding IC50 of 4.8 mg/ml.
0.4
1.15
0.5
2.9
0.82
0.6
0.2
Preussomerin I ester
Phaseolinone
4.8
2.14
0.9
1.22
1.0
1.5
Phaseolinone ester
Phomenone
7.6
5.3
7.04
1.2a)
12.3
0.51a)
7.3
nd
Phomenone ester
9.5
ꢂ20
Values were means of three experiments (ndꢄnot determined). Data interpretation:
IC50 (mg/ml)ꢂ20ꢄinactive, 10—20ꢄweekly active, 6—10ꢄmoderately active and
ꢁ5ꢄstrongly active. Cytotoxicity test in Vero cell, KB cells and BC-1 cells were per-
formed by using sulforhodamine B (SRB) assay, while cytotoxicity test in NCI-H187
cells were using MTT assay. a) Data was obtained from ref. 4.
the cancer cells studied compared to 9 and 10. To rule out
3 was toxic to all cell lines with IC50 ranged from 0.7— that possible hydrolysis reaction, the stability of the biore-
1.5 mg/ml. The BC-1 cancer cells (a reductase-containing ductive esters was thus performed. Due to the low quantity of
cells) was highly sensitive to 3. After structural change into the ester product from the synthesis were obtained, the ester
9, the BC-1 cell was the highest sensitive cells to 9 giving compound 8 and 9 were selected for further stability test.
IC50 of 5.3 mg/ml. 4 was toxic to KB and BC-1 cell lines with The structure of 8 represented a phenolic bioreductive ester,
IC50 ranged from 0.51—1.2 mg/ml. The BC-1 cancer cells (a while 9 represented an alcoholic bioreductive ester. The sta-
reductase-containing cells) was highly sensitive to 4 com- bility of 8 and 9 was conducted in the presence of the es-
pared to the KB cells (non-reductase-containing cancer terase enzyme in a phosphate buffer solution (0.05 M) pH 7.4.
cells). After structural change into 10, the BC-1 cell was the The degradation was determined by using gas chromatogra-
highest sensitive cells to 10 giving IC50 of 7.3 mg/ml.
phy (GC) spectrometer to detect a decrease peak area of 8 or
When compare the chemical structures, the parent pheno- 9 and an increase peak area of lactone that might be occurred
lic compounds (1, 2) were generally more potent than the via the same route of bioreductive activation. Result showed
parent alcoholic compounds (3, 4). And the same pattern of that 8 and 9 were stable under the condition studied (Fig. 6).
cytotoxicity was also observed for the synthesized bioreduc- There was no decreasing of the peak area of 8 or 9 and no
tive esters from the same chemical structures. In other word, lactone peak or any new peak observed based on the GC
the chemosensitivity to the cancer cells of the same set of chromatogram.
chemical structures (e.g., a set of 1, 2, 7, 8; and a set of 3, 4,
Structural modification at the hydroxyl group of fungal
9, 10) were similar in each sets. This may be due to the com- compounds via the ester linkage led to less cytotoxicity in all
pounds in each sets are structural related and thus demon- cancer cell lines studied either possessing or not possessing
strating intuitive chemosensitivity to the same cancer cell reductase activity. It is speculated that the ester linkage at the
line.
hydroxyl group of the cytotoxic compound somehow influ-
In general, phenolic esters are more easily hydrolyzed than enced the toxic activity of fungal compounds. The difference
alcohol ester. Our result demonstrated that the synthesized in reductase enzymatic activity between cell lines has been
bioreductive esters 7 and 8 illustrated more cytotoxicity to suggested to be responsible for their variable sensitivity to

June 2007
933
the cytotoxicity of antitumor quinones.20) Different cell lines
might possess different reductase levels and each compound
might have different susceptibility to the bioreductive activa-
tion. When consideration of the reductase-containing cancer
sis steps of the bioreductive agent is shown in Fig. 4.
Lactone (5)5,7—9) Trimethylhydroquinone 0.5 g (3.3 mmol) was added
into methanesulfonic acid 5 ml followed by the addition of 3,3-dimethyl-
acrylic acid 0.4 g (4 mmol). The mixture was allowed to react at 70 °C for
30 min, and then cooled to room temperature. The reaction mixture was
cells (e.g., BC-1 and NCI-H187 cells), almost all of the syn- diluted with water (65 ml) and extracted three times with 25 ml
dichloromethane followed by water, saturated sodium chloride and dried
over anhydrous sodium sulphate. Solvent removal of dichloromethane by ro-
tary evaporator and recrystallization from hexane afforded the pure lactone 5
thesized bioreductive esters showed less toxicity than their
parents. The synthesized esters may undergo partial biore-
ductive activation in reductase-containing cancer cells (e.g.,
as white crystals in 72% yield (1.1 g). Structure elucidation was confirmed
1
BC-1 and NCI-H187 cells) and hence less toxic moieties
were released in the cancer cells compared to what happened
to their parent compounds. However, another possibility that
the new esters themselves expressed the cytotoxicity regard-
less the bioreductive activation could not be ruled out. Toxi-
city of the synthesized bioreductive esters is inconclusive and
more experimental studies need to be performed. Therefore,
further experiments regarding reductase activated bioreduc-
tion of the esters has been now performed in our laboratory.
by using IR, HR-MS and NMR. H-NMR (CDCl3) d: 1.48 (6H, s, H-10ꢆ-
11ꢆ), 2.22 (3H, s, H-12ꢆ), 2.25 (3H, s, H-13ꢆ), 2.39 (3H, s, H-14ꢆ), 2.58 (2H,
s, H-2ꢆ), 4.8 (1H, s, 8ꢆ-OH). 13C-NMR (CDCl3) d: 12.3, 12.5, 14.4, 27.7,
35.5, 46.1, 119.0, 121.9, 123.4, 128.2, 143.5, 148.8, 168.9. IR (KBr) cmꢇ1
:
3441, 2962, 1743, 1614, 1418, 1295, 1261, 1192. HR-MS (ESI-TOF, nega-
tive) m/z: 257.1164 [MꢀNa]ꢀ, Calcd for C14H18O3Na 257.1104. MS m/z:
234 (Mꢀ).
Quinone Propionic Acid (6)5,7—9) N-Bromosuccinimide (NBS) 0.3 g
(1.7 mmol) was dissolved in 30 ml of water. Lactone solution was prepared
by dissolving 0.3 g (1.3 mmol) of lactone in 45 ml of acetonitrile. NBS solu-
tion was added into the lactone solution. The reaction mixture was allowed
to react at room temperature for 45 min, then 75 ml of diethyl ether was
added. The ether layer was extracted with 5% sodium bicarbonate 75 ml, 2
times. The pH of sodium bicarbonate was adjusted to acidic pH, then ex-
tracted with 30 ml of diethyl ether, three times. The ether layer was dried
over anhydrous sodium bicarbonate. Solvent was removed by rotary evapo-
ration. Recrystallization in acetonitrile afforded yellow crystalline quinone
propionic acid 6 in 63% yield (0.2 g). Structure elucidation was confirmed
Experimental
General Experimental Procedure The melting points were measured
with an Electrothermal digital 900 melting point apparatus (U.K.). The IR
spectra were determined with a FT/IR spectrophotometer Spectrum One
(PerkinElmer, U.K.) by a KBr disk method. 1H- and 13C-NMR were recorded
using a NMR Varian AS 400 NMR spectrometer operating at 400 mHz with
residual chloroform (CDCl3) as an internal reference, and chemical shifts
are expressed in d (ppm). TOF mass spectra were measured with a Micro-
mass LCT mass spectrometer (U.S.A.). Optical rotations were measured
using a JASCO DIP-1000 polarimeter. Preparative HPLC was performed on
a WATER 600 instrument (WATERS 996 photodiode array detector). TLC
was conducted with Kieselgel 60 F254 plates (Merck, Germany). Silica gel
GF 254 (Merck, Germany) and Sephadex LH 20 (Amersham Bioscience,
Sweden) were used for column chromatography. Gas chromatography spec-
trometer was performed on a GC (Hewlett® Packard, HP 6890 series), HP-5
Column (Hewlett® Packard, 5% diphenyl and 95% methylpolysiloxane, film
thickness 0.25 mm, length 30 m, column ID 0.32). Nitrogen, helium, hydro-
gen and oxygen gases were from Harris Calorific.
Extraction, Separation and Purification of Preussomerin G (1) and
Preussomerin I (2) Separation and purification of 1 and 2 followed the
method of Seephonkai et al.1) Briefly, Microsphaeropsis sp. BCC 3050 was
macerated with 1 l methanol at room temperature for 2 d. The methanolic ex-
tract was mixed with water and washed with hexane. The methanol layer
was evaporated to concentrate and was dissolved in ethyl acetate. The crude
extract from the ethyl acetate layer was further separated by using column
chromatography (CC) (3.5ꢅ26 cm) using Sephadex LH 20 as the stationary
phase and 100% methanol as the mobile phase. Fraction numbers 150—325
were collected for further CC separation by using a silica gel column with
stepwise gradient elution: dichloromethene/hexane in order of 20 : 80,
30 : 70, 40 : 60, 50 : 50, 80 : 20, respectively. Four fractions were collected
and fraction number 2 contained 1, while fraction number 3 contained 1, 2
and other compounds. Fraction number 2 was purified by using a silica gel
column with gradient mobile phase: dichloromethene/hexane to get pure
compound, 1. Fraction number 3 was purified by using silica gel column
with gradient mobile phase: CH2Cl2/hexane to get pure compound, 2. The
products 1 and 2 were yellow crystals. The structure elucidation of 1 and 2
were identified and were found to be identical to those previously reported.1)
Extraction, Separation and Purification of Phaseolinone (3) and
Phomenone (4) Separation and purification of 3 and 4 followed the
method of Isaka et al.2) Briefly, Xylaria sp. BCC 1067 was macerated with
ethyl acetate. The ethyl acetate crude extract was separated by using two co-
lumn chromatography systems; Sephadex LH 20 as the stationary phase and
100% methanol as the mobile phase; and silica gel 60H with a gradient
mobile phase (100% dichloromethene-dichloromethene/methanolꢄ95 : 5).
Preparative HPLC with a reverse phase column (Prep Nova-Pak® HR C18
column, 6 mm, 40ꢅ100 mm), 80% water/20% methanol as mobile phase,
flow rate of 10 ml/min, and UV-detector at 220 nm was conducted. Pure (ꢀ)-
phaseolinone, 3 and (ꢀ)-phomenone, 4 were eluted at a retention time of
14 min and 23 min, respectively. The products 3 and 4 were colorless crys-
tals. The structure elucidation of 3 and 4 were identified and were identical
to those previously reported.2)
1
by using IR, MS and NMR. H-NMR (CDCl3) d: 1.43 (6H, s, H-10ꢆ-11ꢆ),
1.93 (3H, s, H-12ꢆ), 1.95 (3H, s, H-13ꢆ), 2.14 (3H, s, H-14ꢆ), 3.02 (2H, s, H-
2ꢆ). 13C-NMR (CDCl3) d: 12.0, 12.4, 14.2, 28.8, 37.9, 47.0, 138.4, 139.0,
142.9, 151.9, 177.1, 187.4, 190.8. IR (KBr) cmꢇ1: 3434, 2971, 1706, 1645,
1436, 1379, 1226. HR-MS (ESI-TOF, negative) m/z: 273.1095 [MꢀNa]ꢀ,
(Calcd for C14H18O4Na 273.1103). MS m/z: 250 (Mꢀ).
Bioreductive Esters The syntheses of bioreductive ester compounds
were accomplished within one step by esterification of purified fungal com-
pounds with quinone propionic acid, 6 based on the previously reported
methods with modification (Fig. 4).5,7—9,21)
Preussomerin G Ester (7) and Preussomerin I Ester (8) Quinone pro-
pionic acid (53 mg, 0.2 mmol) was dissolved in dry dichloromethane and
reacted with preussomerin G or preussomerin I (0.05 mmol) under nitro-
gen gas. 4-(N,N-dimethylamino)pyridine (DMAP) (0.04 mmol) was added
into reaction mixture followed by N,Nꢈ-dicyclohexylcarbodiimide (DCC)
(0.26 mmol) in dry dichloromethane solution. The resulting reaction mixture
was stirred at 40 °C for 2 h with observed formation of a white precipitate.
The filtrate was collected after filtering, cold 1 N HCl was added and it was
then extracted with dichloromethane and adjusted to neutral pH by 5%
sodium bicarbonate. The aqueous layers was washed with dichloromethane
and dried over anhydrous sodium sulphate. Solvent was removed by rotary
evaporation. The residue was purified by column chromatography (Sephadex
LH 20: 100% methanol) giving yellow crystals of 7 and 8. Percent yields of
7 and 8 were 38% (12 mg) and 80% (24 mg), respectively. Structure elucida-
tion was confirmed by using IR, MS and NMR.
Preussomerin G Ester (7) (Chart 1): 1H-NMR (CDCl3) d: 1.51 (3H, s, H-
10ꢆ), 1.59 (3H, s, H-11ꢆ), 1.89 (6H, s, H-12ꢆ, 13ꢆ), 2.18 (3H, s, H-14ꢆ), 3.22
(H, d, Jꢄ17 Hz, H-2ꢆ), 3.40 (1H, d, Jꢄ17 Hz, H-2ꢆ), 3.81 (1H, d, Jꢄ4.3 Hz,
H-2), 4.19 (1H, d, Jꢄ4.3 Hz, H-3), 6.60 (1H, d, Jꢄ9.75 Hz, H-2ꢈ), 6.98 (1H,
Synthesis of Bioreductive Agents A schematic diagram of the synthe-
Chart 1

934
Vol. 55, No. 6
(6H, s, H-13ꢆ), 2.06 (1H, m, H-5b), 2.12 (3H, s, H-14ꢆ), 2.14 (3H, s, H-14ꢆ),
2.30 (1H, m, H-4a), 2.51 (1H, m, H-4b), 2.82 (1H, d, Jꢄ16.7 Hz, H-2aꢆ),
3.03 (1H, d, Jꢄ16.7 Hz, H-2bꢆ), 3.30 (1H, s, H-9), 4.63 (1H, d, Jꢄ13.2 Hz,
H-12a), 4.70 (1H, d, Jꢄ13.2 Hz, H-12b), 4.78 (1H, dt, Jꢄ11, 4.3 Hz, H-6),
5.32 (1H, s, H-15a), 5.36 (1H, s, H-15b), 5.73 (1H, s, H-2). 13C-NMR
(CDCl3) d: 11.2, 12.1, 12.6, 12.7, 14.3, 18.4, 28.6, 28.7, 28.8, 28.9, 29.7,
30.5, 31.4, 37.8, 38.3, 41.2, 41.4, 47.5, 47.9, 61.3, 64.7, 68.4, 72.7, 117.3,
121.1, 138.4, 138.5, 138.7, 138.8, 142.8, 142.9, 152.2, 152.4, 162.2, 172.1,
172.4, 187.4, 190.6, 190.9, 191.9. IR (KBr) cmꢇ1: 2927, 1642, 1533, 1450,
1374, 1223. HR-MS (ESI-TOF, negative) m/z: 751.3465 [MꢀNa]ꢀ, Calcd
for C43H52O10Na 751.3458. MS m/z: 728 (Mꢀ). [a]D28: ꢀ72.5°(cꢄ0.0038,
acetonitrile).
Cytotoxicity Study Cytotoxicity was evaluated in cancer cell lines that
has been reported to contain reductase enzymes (human breast cancer BC-1
and human lung cancer NCI-H187), or without reductase (human epider-
moid carcinoma, KB) as well as in normal cells (African green monkey kid-
ney fibroblast, Vero cells).1) The cytotoxicities of the bioreductive esters
Chart 2
d, Jꢄ8.58 Hz, H-8), 7.01 (1H, d, Jꢄ8.17 Hz, H-7ꢈ), 7.04 (1H, d, Jꢄ8.58 Hz, were compared to their unchanged form (parent compounds) by using col-
H-7), 7.21 (1H, d, Jꢄ9.75 Hz, H-3ꢈ), 7.40 (1H, dd, Jꢄ8.17, 6.6 Hz, H-8ꢈ), orimetric methods; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
7.62 (1H, d, Jꢄ6.63 Hz, H-9ꢈ). 13C-NMR (CDCl3) d: 12.0, 12.5, 14.3, 28.9, bromide (MTT) assay22) and Sulforhodamine B (SRB).23) MTT was used for
29.1, 38.1, 47.3, 52.1, 52.6, 53.4, 89.9, 93.9, 119.0, 120.0, 120.9, 121.0,
study in NCI-H187 cells, while SRB was used for study in Vero, KB and
122.8, 127.1, 130.2, 131.2, 133.6, 138.6, 139.2, 140.4, 142.7, 142.8, 148.1, BC-1 cells.
148.7, 151.8, 171.4, 183.6, 187.5, 189.5, 190.7. IR (KBr) cmꢇ1: 2963, 1725,
In this assay, NCI-H187 cells were seeded on 96-well plates (105
1645, 1439, 1187, 1136. HR-MS (ESI-TOF, negative) m/z: 617.1424 cells/well) containing 200 ml RPMI 1640 media supplemented with 10%
[MꢀNa]ꢀ, Calcd for C34H26O10Na 617.1424. MS m/z: 594 (Mꢀ). [a]D28
:
FBS, 1% NEAA, 0.5% penicillin–streptomycin solution (ꢅ100) at 37 °C
with 5% carbon dioxide. Vero, KB and BC-1 cells were seeded on 96-well
plates (105 cells/well) containing 200-ml DMEM media supplemented with
10% FBS, 1% non-essential amino acid, 0.5% penicillin–streptomycin at
ꢀ65° (cꢄ0.0045, acetonitrile).
1
Preussomerin I Ester (8) (Chart 2): H-NMR (CDCl3) d: 1.51 (3H, s, H-
10ꢆ), 1.58 (3H, s, H-11ꢆ), 1.89 (6H, s, H-12ꢆ, 13ꢆ), 3.10 (1H, dd, Jꢄ17.9,
2.7 Hz, H-2ꢈa), 3.22 (1H, d, Jꢄ16.7 Hz, H-2ꢆa), 3.37 (1H, dd, Jꢄ17.9, 37 °C with 5% carbon dioxide. The test compounds were added into cultured
2.7 Hz, H-2ꢈb), 3.38 (1H, d, Jꢄ17.1 Hz, H-2ꢆb), 3.53 (3H, s, 3ꢈ-OCH3), 3.80 cells, 24 h after seeding.
(1H, d, Jꢄ4.3 Hz, H-2), 4.25 (1H, d, Jꢄ4.3 Hz, H-3), 4.33 (1H, t, Jꢄ2.7 Hz,
H-3ꢈ), 7.00 (1H, d, Jꢄ8.9 Hz, H-8), 7.04 (1H, d, Jꢄ8.9 Hz, H-7), 7.05 (1H,
d, Jꢄ8.5 Hz, H-7ꢈ), 7.39 (1H, t, Jꢄ8 Hz, H-8ꢈ), 7.65 (1H, d, Jꢄ7.8 Hz, H-
9ꢈ). 13C-NMR (CDCl3) d: 12.1, 12.5, 14.3, 28.9, 29.1, 38.1, 40.3, 47.3, 52.6,
MTT Method: After treatment for 21 h, the MTT solution (5 mg/ml of
MTT) was added to each well. After 3 more hours (treatment of total 24 h),
100 ml of solution mixture (10% SDS and 0.01 N HCl) was added to each
well. Cell viability of the treated cells was determined by measuring ab-
53.4, 59.3, 79.1, 93.5, 94.7, 119.3, 120.5, 120.8, 121.9, 122.5, 127.1, 130.7, sorbance of the blue formazan at 570 nm using an ELISA plate reader with a
131.3, 138.6, 139.2, 142.4, 142.7, 142.9, 147.6, 149.7, 151.8, 171.4, 187.4, reference wavelength of 620 nm for background subtraction. The experi-
189.7, 190.7, 193.2. IR (KBr) cmꢇ1: 2927, 1761, 1696, 1642, 1476, 1284, ments were performed as triplicates. The MTT transformation of MTT by
1107. HR-MS (ESI-TOF, negative) m/z: 649.1699 [MꢀNa]ꢀ, Calcd for
C35H30O11Na 649.1686. MS m/z: 626 (Mꢀ). [a]D28: ꢇ258° (cꢄ0.0044, ace-
tonitrile).
the media-treated cells was set as zero percent-cytotoxicity.
Sulforhodamine B (SRB) Method: After exposure of the cells with test
compound for 24 h, the medium was removed by suction. The cells were
fixed with 40% trichloroacetic acid (TCA) (Sigma, St. Louis, MO, U.S.A.)
Phaseolinone Ester (9) or Phomenone Ester (10) Synthesis of phase-
olinone ester (9) or phomenone ester (10) was similar to the method used for and the plates were kept at 4 °C for 1 h. The TCA was removed by suction,
synthesis of 7 and 8. For synthesis of phaseolinone ester (9), quinone propi-
onic acid (0.08 mmol) in dry dichloromethane, phaseolinone (11.4 mg,
0.03 mmol), DMAP (0.011 mmol) and DCC (0.11 mmol) were used for the
and the plates were rinsed with water and stained with SRB (Sigma) 0.4%
w/v in 1% acetic acid for 30 min. Excess dye was washed out by 1% acetic
acid (Bio Lab Ltd., Jerusalem, Israel), repeatedly 4 times. The stained cells
reaction. Preparative thin layer chromatography was used to purify the were then extracted and solubilized with 10 nM Tris base (tris(hydroxy-
residue of 9 by using silica gel as the stationary phase and ethyl
acetate : dichloromethane (5 : 95) as the mobile phase giving yellow crystals.
The Rf value of 9 was 0.59. For the synthesis of phomenone (10), quinone
propionic acid (0.14 mol) in dry dichloromethane, phomenone (9 mg,
0.34 mmol), DMAP (0.017 mmol) and DCC (0.17 mmol) were used for the
methyl)aminomethane) (Sigma) (0.5—2.0 ml) and then was measured by
using a microtiter plate reader at 564 nm. The numbers of viable cells were
expressed as a percentage of the untreated control cell cultures.
Stability Study The bioreductive esters 8 and 9 were selected as repre-
sentative of phenolic and alcoholic structures. Stability study of 8 (200 mM)
reaction. The residue of 10 from the reaction was purified by preparative or 9 (1600 mM) was conducted separately under the condition containing the
TLC with silica gel as the stationary phase and ethyl acetate : dichloro-
methane (5 : 95) as the mobile phase giving yellow crystals and Rf value of
0.55. Percent yields of 9 and 10 were 73% (18 mg) and 71% (20 mg), respec-
esterase enzyme (4 and 8 units/ml, respectively) in the phosphate buffer
0.05 M, pH 7.4 at 37 °C. At each time points, the reaction mixture was pipet-
ted out to another tube containing acetonitrile to stop the reaction. The reac-
tively. The organic solvent was removed and structures were elucidated by tion mixture was frozen, thawed, and centrifuged at 12000 rpm for 15 min to
using IR, MS and NMR.
Phaseolinone Ester (9): H-NMR (CDCl3) d: 1.04 (3H, d, Jꢄ6.6 Hz, H-
precipitate the enzyme. Only 100 ml of supernatant was pipetted and deter-
1
mined by using the GC and caffeine was used as an internal standard with a
13), 1.12 (3H, s, H-14), 1.34 (1H, m, H-5a), 1.41 (6H, s, H-10ꢆ), 1.43 (6H, s, final concentration of 100 mg/ml.
H-11ꢆ), 1.93—1.96 (12H, s, H-12ꢆ, 12ꢆ, 13ꢆ, 13ꢆ), 2.01—2.07 (2H, m, H-7,
5b), 2.12 (3H, s, H-14ꢆ), 2.14 (3H, s, H-14ꢆ), 2.31 (1H, m, H-4a), 2.42 (1H,
m, H-4b), 2.57 (1H, d, Jꢄ5.0 Hz, H-15a), 2.94 (H, d, Jꢄ5 Hz, H-15b), 2.99
GC Chromatographic Assay: In order to follow the degradation of biore-
ductive esters, the detection of a decrease peak area of the bioreductive ester
as well as an increase peak area of the lactone must be detected. Briefly, the
(4H, m, H-2ꢆ), 3.02 (4H, s, H-2ꢆ, 2ꢆ), 3.50 (1H, s, H-9), 4.34 (1H, d, injection port set to splitless mode was adopted for the quantitative analysis
Jꢄ12 Hz, H-12a), 4.54 (1H, d, Jꢄ12 Hz, H-12b), 4.74 (1H, m, H-6), 5.70
(1H, m, H-2). 13C-NMR (CDCl3) d: 11.2, 12.1, 12.5, 12.6, 14.2, 14.3, 18.5,
28.7, 28.8, 28.9, 29.6, 30.4, 31.1, 37.9, 38.3, 41.1, 41.3, 47.4, 47.8, 48.6,
of those two peaks. HP-5 (5% diphenyl and 95% methylpolysiloxane) col-
umn and flame ionization detector (FID) were used. Nitrogen gas was used
as a carrier gas with flow rate of 1.0 ml/min. The injected volume of 8 and 9
54.8, 61.7, 62.5, 63.4, 72.6, 120.6, 120.8, 138.4, 138.7, 138.8, 138.9, 142.8, was 2 ml. The syringe temperature was kept at 290 °C. Volatile samples were
142.9, 152.2, 152.3, 162.5, 172.0, 172.4, 187.4, 187.5, 190.6, 190.8, 192.3.
separated on the HP-5 column. For compound 8, the column was kept at
IR (KBr) cmꢇ1: 2934, 1638, 1526, 1450, 1374, 1226. HR-MS (ESI-TOF, 200 °C for 5 min. The temperature was then increased at 280 °C with the rate
negative) m/z : 745.3591 [MꢀH]ꢀ, Calcd for C43H52O11H 745.3588. MS of 30 °C/min and kept for 9 min at this temperature. For compound 9, the
m/z: 744 (Mꢀ). [a]D28: ꢀ82° (cꢄ0.0039, acetonitrile).
column was kept at 150 °C for 5 min. The temperature was then increased at
280 °C with the rate of 30 °C/min and kept for 7 min at this temperature. The
1
Phomenone Ester (10): H-NMR (CDCl3) d: 1.07 (3H, d, Jꢄ6.63 Hz, H-
13), 1.27 (3H, s, H-14), 1.32 (1H, m, H-5a), 1.38 (3H, s, H-10ꢆ), 1.39 (3H, s, column outlet was connected to an FID detector, which was set at 290 °C.
H-10ꢆ), 1.44 (6H, s, H-11ꢆ), 1.89 (3H, s, H-12ꢆ), 1.94 (3H, s, H-12ꢆ), 1.97

June 2007
935
Acknowledgement Financial support from the Thai Research Fund and
the Commission on Higher Education (MRG4680181) is gratefully ac-
knowledged.
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Vyas D. M., Bioorg. Med. Chem. Lett., 3, 1761—1766 (1993).
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