Study on the synthesis and bioactivity of novel Mahkoside A derivatives

Article abstract of DOI:10.1080/00397910701489495

A series of novel Mahkoside A derivatives was synthesized, and their in vitro cytotoxic activities were evaluated against the human cancer cell line Ec-9706. A Preliminary structure-activity relationship study showed compounds 7 and 8 have obvious cytotox

Full text of DOI:10.1080/00397910701489495

Synthetic Communicationsw, 37: 3319–3328, 2007
Copyright # Taylor & Francis Group, LLC
ISSN 0039-7911 print/1532-2432 online
DOI: 10.1080/00397910701489495
Study on the Synthesis and Bioactivity
of Novel Mahkoside A Derivatives
Yan-Bing Zhang, Pi-Yong Zhang, Gui-Fu Dai,
and Hong-Min Liu
Department of Chemistry, New Drug Research and Development Center,
Zhengzhou University, Zhengzhou, China
Abstract: A series of novel Mahkoside A derivatives was synthesized, and their in
vitro cytotoxic activities were evaluated against the human cancer cell line Ec-9706.
A Preliminary structure–activity relationship study showed compounds 7 and 8 have
obvious cytotoxic activities (IC50: 30.0 and 12.5 mg mL21, respectively).
.
Keywords: benzophenone, Koenigs–Knorr reaction, Mahkoside A, 2,3,6-tri-O-acetyl-
4-chloro-4-deoxy-b-D-galactoside
1
INTRODUCTION
Mahkota Dewa (Phaleria macrocarpa [Scheff.] Boerl) is widely distributed
in Indonesia. Empirically, the stems, leaves, and fruits of it are used to
control many diseases such as cancer, impotency, hemorrhoids, diabetes
mellitus, allergies, liver and heart diseases, kidney disorders, blood
diseases, rheumatism, high blood pressure, stroke, migraines, various skin
diseases, and acne.[1] There had not been any report about the chemical
constituents of the plant before we first separated a new derivative of
benzophenone, named Mahkoside A,[2] from Mahkota Dewa. It was
reported that this class of compounds is usually used as an ultraviolet
absorber, photo sensitizer, and medical intermediate;[3] exhibits notable
cytotoxicity against the ovarian cancer cell line,[4] the human tumor cell
Received February 2, 2007
Address correspondence to Hong-Min Liu, Department of Chemistry, New Drug
Research and Development Center, Zhengzhou University, Zhengzhou 450052,
China. E-mail: liuhm@zzu.edu.cn
3319

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Y.-B. Zhang et al.
line,[5] the colon cancer cell line,[6] the human submandibular gland adeno-
carcinoma cell line, and primary human gingival fibroblast;[7] combats the
human parasite Trypanosoma cruzi (Chagas’s disease);[8] and displays sig-
nificant antioxidant activity.[9] Hence, we attempted to synthesize various
Mahkoside A derivatives and evaluate the in vitro cytotoxicity of the
product obtained.
2
RESULTS AND DISCUSSION
2.1 Chemistry
To find new Mahkoside A derivatives possessing potent bioactivity, we
needed a variety of structurally modificated compounds to study the
structure–activity relationships of Mahkoside A. Selective protection of
Mahkoside A (1) with benzyl chloride in the presence of anhydrous
K2CO3 afforded 4,40-dibenzyloxy Mahkoside A (2) in 80% yield. Acid
hydrolysis of 2 with aqueous HCl furnished 6-hydroxy-4,40-dibenzyloxy-2-
methoxy benzophenone (3) in 92% yield. In the previous syntheses of
flavonol O-glycosides, all the glycosidations employed glycosyl bromides
or chlorides under either Koenigs–Knorr conditions (promoted by silver
salts) or, later, under phase-transfer-catalyzed (PTC) alkaline conditions.[10]
Although the latter glycosidation yield was relatively low (normally lower
than 50%), it greatly improved and simplified the workup procedures.[10]
We attempted to use the basic PTC conditions (Bu4NBr, K2CO3, CHCl3/
H2O) in the glycosylation of benzophenone 3 with 2,3,6-tri-O-acetyl-4-
chloro-4-deoxy-a-D-galactopyranosidyl bromide and successfully gave 4 in
63% yield. This result might be attributed to the decrease of the side
reactions of the glycosyl bromide under basic conditions, such as elimination
to give the glycal and cleavage of the acyl protecting groups.[10] The 4,40-
dibenzyloxy group on 4 were then completely cleaved by hydrogenolysis
in the catalysis of Pd-C under normal pressure, and the acetate groups on
the sugar moiety were removed with NaOH in methanol to afford new
glycoside 5.
On the other hand, another method for structural modification of benzo-
phenone 3, alkylamine of 6-OH, was conducted. Intermediate 3 was reacted
with dibromoethane in the refluxing absolute ethanol in the presence of
K2CO3 to give 6-(2-bromoethoxy)-4,40-dibenzyloxy-2-methoxybenzophe-
none 6. Subsequent substitution of the bromo group with different amino
groups led to 6-aminoalkyloxy derivatives 7 and 8 in 74% and 94%, respect-
ively. The final removal of benzyl groups of 8 with catalytic hydrogenolysis
afforded 9 in high yield. When a solution of Mahkoside A (1) in ethanol
was treated with 30% aqueous HCl at reflux temperature, 4,6,40-trihydroxy-
2-methoxybenzophenone (10) was obtained in 95% yield. All procedures
are shown in Scheme 1.

Novel Mahkoside A Derivatives
3321
Scheme 1. BnCl, K2CO3, CH3CH2OH, reflux; (b) HCl, reflux; (c) K2CO3, CHCl3,
(C4H9)4NBr, 408C, 2,3,6-tri-O-acetyl-4-chloro-4-deoxy-b-D-galactosidyl bromide;
(d) i: H2, 10% Pd-C, EtOH-EtOAc, 408C; ii: NaOH, MeOH, rt; (e) BrCH2CH2Br,
K2CO3, C2H5OH, reflux; (f) RH K2CO3, DMF, rt; (g) H2, 10% Pd-C, EtOH-EtOAc,
408C; (h) HCl, reflux.
2.2 Biological Activity
The in vitro cytotoxicity of novel Mahkoside A derivatives 2–4 and 6–10
against human cancer cell line Ec-9706 were evaluated in an assay system.
The results are listed in Table 1. The results showed that compounds 7 and 8
Table 1. In vitro activity of Mahkoside A derivatives
21
)
21
)
.
IC50 (mg mL
.
Inhibition (100 mg mL
Compound
1
2
.100
.100
.100
.100
.100
30.0
18.7
19.2
6.4
3
4
17.9
7.0
6
7
—
—
8
12.5
.100
.100
9
10
33
28.9

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Y.-B. Zhang et al.
21
exhibit higher antitumor activities (IC50 30 mg mL and 12.5 mg mL21),
compound 6 does not show any activity, and others are less bioactive. On the
basis of this result, we can conclude that the 6-aminoalkyloxy group greatly
enhances cytotoxicity and the substitution of two hydroxyl groups at 4,40-
position with two alkyloxy groups increases cytotoxicity also, which are two
effective ways to improve the cytotoxicity. The study on the structure–
activity relationship indicated that the suitable substitutions at benzene rings
greatly improve the bioactivity, but the sugar moiety gives less contribution.
The further structural modification of these compounds to improve the bioac-
tivity is in progress.
.
.
3
EXPERIMENTAL
3.1 General Methods
1H and 13C NMR spectra were acquired on a Bruker Avance DPX-400
spectrometer with chemical shifts (d) given in parts per million relative to
Me4Si as an internal standard. Melting points were determined on a WC-1
melting-point apparatus and are uncorrected. IR spectra were obtained on a
Thermo Nicolet IR200 unit. High-resolution mass spectra (HRMS) were
obtained on a Waters Micromass Q-Tof MicroTM instrument using the ESI
technique. Thin-layer chromatography (TLC) was performed on glass plates
precoated with silica gel GF254 to monitor the reactions and certify the
purity of the reaction products. Visualization was accomplished by spraying
chromatograms with 10% ethanolic sulfuric acid and charring them on a hot
plate. Column chromatography was carried out on silica gel (200–300 mesh).
3.2 Preparation of 2,3,6-tri-O-acetyl-4-chloro-4-deoxy-a-D-
galactopyranosidyl Bromide
2,3,6-Tri-O-acetyl-4-chloro-4-deoxy-a-D-galactopyranosidyl bromide was
synthesized by following the literature methods.[11,12]
3.3 4,40-Dibenzyloxy Mahkoside A (2)
To a mixture of Mahkoside A (1, 422 mg, 1.00 mmol) and dry K2CO3
(276 mg, 2.00 mmol) in dry ethanol (10 mL), benzyl chloride (1 mL) was
added. The mixture was refluxed for 3 h (TLC showed the disappearance of
Mahkoside A). The resulting mixture was diluted with water (10 mL) and
extracted with EtOAc (3 ꢀ 10 mL). The organic layer was washed with
brine and dried over anhydrous Na2SO4. The residue obtained after evapor-
ation of the solvent was purified by flash chromatography on silica gel (6:1

Novel Mahkoside A Derivatives
3323
CHCl3-CH3OH) to afford 2 (480 mg, 80% yield) as a yellow solid. Mp
1
86–888C; IR (KBr): 3402, 1646, 1599, 1454, 1162, 1076, 923 cm21; H
NMR (CDCl3, 400 MHz): d 3.72 (s, 3H, CH3O), 3.43–3.81 (m, 10H), 4.82
(d, J ¼ 8.4 Hz, 1H, H-10-gal), 4.85 (s, 2H, ArCH2), 5.03 (s, 2H, ArCH2),
6.21 (s, 1H, H-3), 6.44 (s, 1H, H-5), 6.87 (d, J ¼ 8.4 Hz, 2H, H-30, H-50),
7.38–6.89 (m, 10H, Ar-H), 7.75 (d, J ¼ 8.6 Hz, 2H, H-20, H-60); 13C NMR
(CDCl3, 400 MHz): d 55.6 (CH3O) 61.9 (C-60-gal), 69.7 (C-40-gal), 70.1
(2ArCH2), 73.5 (C-20-gal), 75.8 (C-30-gal), 76.0 (C-50-gal), 94.8 (C-5), 95.7
(C-3), 103.3 (C-10-gal), 112.8 (C-1), 114.4 (C-30,50), 127.5, 127.6, 128.1,
128.2, 128.3, 128.6 (Ar), 131.8 (C-10), 132.2 (C-20,60), 136.1 (Ar), 136.2
(Ar), 157.3 (C-6), 157.7 (C-2), 162.7 (C-40), 162.9 (C-4), 194.3 (C-7);
HRMS (ESI) calcd. for C34H34O10: m/z 625.2052 [M þ Na]þ. Found:
625.2041.
3.4 6-Hydroxy-4,40-dibenzyloxy-2-methoxybenzophenone (3)
To a solution of 2 (151 mg, 0.25 mmol) in 95% ethanol (10 mL), 30% aq HCl
(1 mL) was added. The mixture was refluxed for 2 h, then cooled to room
temperature and neutralized with satd. aq. NaHCO3. The resulting mixture
was extracted with CH2Cl2. The organic layer was washed with water and
dried over anhydrous Na2SO4. Subsequent concentration gave a residue.
The residue was purified by flash chromatography on silica gel (4:1
petroleum ether–EtOAc) to afford 3 (101 mg, 92% yield) as a yellow solid.
1
Mp 110–1128C; IR (KBr): 3160, 1634, 1605, 1508, 1172 cm21; H NMR
(CDCl3, 400 MHz): d 3.85 (s, 3H, CH3O), 4.83 (s, 2H, Ar-CH2), 5.01
(s, 2H, ArCH2), 6.04 (d, J ¼ 2.3 Hz, 1H, H-3), 6.19 (d, J ¼ 2.2 Hz, 1H,
H-5), 6.76 (d, J ¼ 7.0 Hz, 2H, H-30, H-50), 6.85–7.41 (m, 10H, Ar-H), 7.57
(dd, J ¼ 6.9 Hz, J ¼ 1.8 Hz, 2H, H-20, H-60); 13C NMR (CDCl3, 400 MHz):
d 55.6 (CH3O), 70.1 (ArCH2), 70.0 (ArCH2), 92.4 (C-5), 93.8 (C-3), 106.1
(C-1), 113.8 (C-30,50), 126.0, 127.4, 127.5, 128.0, 128.1, 128.6 (Ar), 130.6
(C-20,60), 134.4 (C-10), 135.6 (Ar), 136.4 (Ar), 160.5 (C-6), 161.5 (C-2),
165.2 (C-40), 165.8 (C-4), 197.9 (C-7). HRMS (ESI) calcd. for C28H24O5:
m/z 463.1522 [M þ Na]þ. Found: 463.1521.
3.5 4,40-Dibenzyloxy-2-methoxybenzophenon-6-yl 2,3,6-tri-O-
acetyl-4-chloro-4-deoxy-b-D-galactopyranoside (4)
A mixture of 3 (73 mg, 0.165 mmol), a catalytic amount of Bu4NBr, and
K2CO3 (46 mg, 0.33 mmol) in 2:1 CHCl3–H2O (10 mL) was stirred at 408C
for 1 h. Then, 2,3,6-tri-O-acetyl-4-chloro-4-deoxy-b-D-galactosidyl bromide
(128 mg, 0.33 mmol) was added, and stirring continued at 408C for 1 day.
The reaction mixture was diluted with CH2Cl2 (10 mL) and washed with
water (10 mL) and brine (10 mL). The organic layer was then dried over

3324
Y.-B. Zhang et al.
Na2SO4 and concentrated in vacuo. The residue was purified by flash chrom-
atography on silica gel (10:1 CHCl3–EtOAc) to afford 4 (78 mg, 63% yield)
as a yellow solid. Mp 61–638C; IR (KBr): 1751, 1660, 1601, 1507, 1231,
1
1094, 910 cm21; H NMR (CDCl3, 400 MHz): d 1.96 (s, 3H, CH3CO) 2.08
(s, 3H, CH3CO), 2.10 (s, 3H, CH3CO), 3.79 (s, 3H, CH3O); 4.06 (t, 1H,
J ¼ 5.9 Hz, H-50-gal), 4.24 (dd, 1H, J ¼ 5.2 Hz, J ¼ 11.5 Hz, H-60-gal),
4.37 (dd, 1H, J ¼ 7.0 Hz, J ¼ 11.5 Hz, H-60a-gal), 4.49 (d, 1H, J ¼ 3.6 Hz,
H-40-gal), 4.91 (d, 1H, J ¼ 8.0 Hz, H-10-gal), 4.95 (d, 1H, J ¼ 4.0 Hz, H-30-
gal), 4.97 (s, ArCH2, 2H), 5.12 (s, ArCH2, 2H), 5.33 (dd, 1H, J ¼ 8.0 Hz,
J ¼ 10.0 Hz, H-20-gal), 6.29 (d, 1H, J ¼ 2.0 Hz, H-3), 6.49 (d, 1H,
J ¼ 2.0 Hz, H-5), 6.96 (d, 2H, J ¼ 8.8 Hz, 2H, H-30,50), 7.77–7.05
(m, 10H, 2Ar-H), 7.78 (d, 2H, J ¼ 8.8 Hz, H-2060); 13C NMR (CDCl3,
400 MHz): d 20.5 (CH3CO), 55.5 (CH3O), 57.2 (C-40-gal), 63.2 (C-60-gal),
67.2 (C-20-gal), 70.1 (ArCH2), 70.2 (ArCH2), 71.5 (C-30-gal), 71.8 (C-50-
gal), 94.9 (C-5), 96.8 (C-3), 101.0 (C-10-gal), 114.1 (C-1), 114.4 (C-30,50),
126.7, 127.5, 127.7, 128.2, 128.4, 128.7 (Ar), 131.3 (C-10), 131.9 (C-20,60),
135.6 (Ar), 136.4 (Ar), 155.5 (C-6), 157.1 (C-2), 161.7 (C-40), 162.8 (C-4),
169.0 (CH3CO), 170.3 (CH3CO), 170.4 (CH3CO), 192.5 (C-7). HRMS
(ESI) calcd. for C40H39ClO12: m/z 769.2028 [M þ Na]þ. Found: 769.2014.
3.6 4,40-Dihydroxy-2-methoxybenzophenon-6-yl-4-chloro-4-
deoxy-b-D-galactopyranoside (5)
A solution of 4 (60 mg, 0.08 mmol) in 1:1 EtOH–EtOAc (80 mL) was treated
with a catalytic amount of 10% Pd-C. After stirring under 1 atm of H2 at 408C
for 7 h, the reaction mixture was filtered and concentrated in vacuo. The
residue was dissolved in dry CH3OH (10 mL), followed by addition of a
catalytic amount of NaOH. After stirring for 1 h at room temperature, the
reaction mixture was filtered and solid was washed with CH3OH (10 mL).
The filtrate and washings were combined and concentrated. The residue was
purified by flash chromatography on silica gel (4:1 CH2Cl2–CH3OH) to
give 5 (21 mg, 61% yield) as a brown solid. Mp 190–1928C; IR (KBr):
1
3423, 1621, 1600, 1433, 1163, 1081 cm21; H NMR (CD3OD, 400 MHz):
d 3.46 (dd, 1H, J ¼ 7.9 Hz, J ¼ 9.2 Hz, H-20-gal), 3.65 (dd, 1H, J ¼ 5.8 Hz,
J ¼ 11.2 Hz, H-60-gal), 3.72 (dd, 1H, J ¼ 6.8 Hz, J ¼ 11.2 Hz, H-60-gal),
3.77 (d, 1H, J ¼ 3.6 Hz, H-30-gal), 3.80 (s, 3H,CH3-O); 3.89 (t, 1H,
J ¼ 6.4 Hz, H-50-gal), 4.31 (d, 1H, J ¼ 3.6 Hz, H-40-gal), 4.88 (d, 1H,
J ¼ 7.6 Hz, H-10-gal), 6.16 (d, 1H, J ¼ 2.0 Hz, H-3), 6.39 (d, 1H,
J ¼ 2.0 Hz, H-5), 6.78 (d, 2H, J ¼ 8.4 Hz, H-30,50), 7.68 (d, 2H, J ¼ 8.8 Hz,
13
H-2060); C NMR (CD3OD, 400 MHz) d: 55.9 (CH3O), 62.6 (C-40-gal),
63.2 (C-60-gal), 71.6 (C-20-gal), 73.6 (C-30-gal), 95.0 (C-5), 96.8 (C-3),
103.1 (C-10-gal), 111.6 (C-1), 115.9 (C-30,50), 131.9 (C-10), 158.3 (C-6),
159.3 (C-2), 163.8 (C-40), 164.3 (C-4), 197.1 (C-7); HRMS (ESI) calcd. for
C20H21ClO9: m/z 463.0772 [M þ Na]þ. Found: 463.0756.

Novel Mahkoside A Derivatives
3325
3.7 6-(2-Bromoethoxy)-4,40-dibenzyloxy-2-
methoxybenzophenone (6)
A solution of 3 (110 mg, 0.25 mmol), K2CO3 (69 mg, 0.5 mmol), and
dibromoethane (1 mL) in dry ethanol (10 mL) was refluxed for 6 h. The
reaction mixture was filtered, and the solid was washed with ethanol
(10 mL). The filtrate and washings were combined and concentrated. The
residue was purified by flash chromatography on silica gel (4:1 petroleum
ether–EtOAc) to give 6 (88 mg, 65% yield) as a yellow solid. Mp 97–
1008C; IR (KBr): 1659, 1602, 1448, 1161 cm21
;
1H NMR (CDCl3,
400 MHz): d 3.38 (t, 2H, J ¼ 6.8 Hz, the protons of carbon connected with
bromine), 3.80 (s, 3H, CH3O), 4.19 (t, 2H, J ¼ 6.8 Hz, OCH2), 5.01 (s, 2H,
ArCH2), 5.12 (s, 2H, ArCH2), 6.14 (d, 1H, J ¼ 1.6 Hz, H-3), 6.21 (d, 1H,
J ¼ 1.6 Hz, H-5), 6.97 (d, 2H, J ¼ 8.8 Hz, H-30,50), 7.44–7.09 (m, 10H,
2Ar-H), 7.81 (d, 2H, J ¼ 8.8 Hz, H-2060); 13C NMR (CDCl3, 400 MHz)
d: 11.1 (2-CH2CH3), 47.5 (2CH2CH3), 50.9 (C-CH2-N), 55.5 (CH3O), 66.7
(O-CH2-C), 70.1 (ArCH2), 70.3 (ArCH2), 91.8 (C-5), 92.8 (C-3), 112.0
(C-1), 114.4 (C-30,50), 126.7, 127.5, 127.6, 128.2, 128.3, 128.7 (Ar), 131.7
(C-20,60), 131.9 (C-10), 136.3 (Ar), 136.6 (Ar), 157.5 (C-6), 157.6 (C-2),
162.1 (C-40), 162.6 (C-4), 193.3 (C-7). HRMS (ESI) calcd. for C30H27BrO5:
m/z 547.1120 [M þ H]þ. Found: 547.1110.
3.8 6-(2-Diethylaminoethoxy)-4,40-dibenzyloxy-2-
methoxybenzophenone (8)
A solution of 6 (60 mg, 0.11 mmol), K2CO3 (30 mg, 0.22 mmol), and
diethylamine (1 mL) in dry DMF was stirred for 10 h at room temperature.
The mixture was filtered. The filtrate was diluted with water (10 mL) and
extracted with CH2Cl2 (3 ꢀ 10 mL). The organic layer was dried over
Na2SO4 and concentrated in vacuo. The residue was purified by flash
chromatography on silica gel (4:1 CH2Cl2-CH3OH) to give 8 (56 mg, 94%
yield) as a yellow syrup. IR (KBr): 3032, 1661, 1600, 1455, 1250,
1
1159 cm21; H NMR (CDCl3, 400 MHz): d 0.93 (t, 6H, J ¼ 7.1 Hz, 2CH3);
2.56 (d, 4H, J ¼ 6.0 Hz, 2CH2CH3), 2.81 (s, 2H, the protons of carbon
connected with diethylamino), 3.79 (s, 3H, OCH3), 4.09 (s, 2H, OCH2),
4.99 (s, 2H, ArCH2), 5.13 (s, 2H, ArCH2), 6.16 (d, 1H, J ¼ 1.8 Hz, H-3),
6.18 (d, 1H, J ¼ 1.8 Hz, H-5), 6.96 (d, 2H, J ¼ 8.8 Hz, H-30,50), 7.44–7.09
(m, 10H, 2Ar-H), 7.81 (d, 2H, J ¼ 8.8 Hz, H-20,60); 13C NMR (CDCl3,
400 MHz) d: 11.1 (2CH2CH3), 47.5 (2CH2CH3), 50.9 (C-CH2-N), 55.5
(CH3O), 66.7 (O-CH2-C), 70.1 (ArCH2), 70.3 (ArCH2), 91.8 (C-5), 92.8
(C-3), 112.0 (C-1), 114.4 (C-30,50), 126.7, 127.5, 127.6, 128.2, 128.3, 128.7
(Ar), 131.7 (C-20,60), 131.9 (C-10), 136.3 (Ar), 136.6 (Ar), 157.5 (C-6),
157.6 (C-2), 162.1 (C-40), 162.6 (C-4), 193.3 (C-7). HRMS (ESI) calcd. for
C34H37NO5: m/z 540.2750 [M þ H]þ. Found: 540.2770.

3326
Y.-B. Zhang et al.
3.9 6-[2-(Piperid-1-yl)-ethyloxy]-4,40-dibenzyloxy-2-
methoxybenzophenone (7)
A similar procedure as described previously for the preparation of 8 from
6 was used for the preparation of 7. Yield 74% as a yellow syrup. IR
;
(KBr): 3388, 1659, 1599, 1455, 1251, 1160 cm21 1H NMR (CDCl3,
400 MHz) d: 3.05–2.68 (d, 10H, CH2NCH2), 3.78 (s, 3H, OCH3), 4.02
(s, 2H, OCH2), 4.98 (s, 2H, ArCH2), 5.14 (s, 2H, ArCH2), 6.12 (s, 1H,
H-3), 6.19 (s, 1H, H-5), 6.97 (d, 2H, J ¼ 7.2 Hz, H-30,50), 7.45–7.08
(m, 10H, 2Ar-H), 7.78 (d, 2H, J ¼ 7.3 Hz, H-2060); 13C NMR (CDCl3,
400 MHz) d: 43.2 (NCH2CH2N), 49.9 (NCH2CH2N), 55.5 (CH3O), 56.2
(CH2NR1R2), 67.2 (O-CH2-C), 70.2 (ArCH2), 70.3 (ArCH2), 92.2 (C-5),
92.9 (C-3), 112.2 (C-1), 114.5 (C-30,50), 126.8, 127.5, 127.7, 128.2,
128.4, 128.7, (Ar), 131.8 (C-20,60), 131.9 (C-10), 131.7 (C-10), 136.3
(Ar), 136.7 (Ar), 157.4 (C-6), 157.5 (C-2), 162.1 (C-40), 162.8 (C-4),
193.4 (C-7); HRMS (ESI) calcd. for C34H36N2O5: m/z 553.2702
[M þ H]þ. Found: 553.2706.
3.10 6-(2-Diethylaminoethoxy)-4,40-dihydroxy-2-
methoxybenzophenone (9)
A suspension of 8 (72 mg, 0.13 mmol) and a catalytic amount of 10%
Pd-C in 1:1 EtOH–EtOAc (80 mL) was stirred under 1 atm of H2 at
408C for 7 h. The reaction mixture was filtered and concentrated in
vacuo. The residue was purified by silica-gel chromatography (2:1
CH2Cl2-CH3OH) to afford 9 (46 mg, 96% yield) as a yellow syrup. IR
(KBr): 3436, 1660, 1604, 1432, 1274, 1157 cm21
;
1H NMR
(CD3COCD3, 400 MHz) d: 0.84 (t, 6H, J ¼ 7.2 Hz, 2CH2CH3), 1.23
(d, 4H, J ¼ 4.6 Hz, 2CH2CH3), 2.70 (s, 2H, the protons of carbon
connected with diethylamino), 3.74 (s, 3H, OCH3), 3.99 (t, 2H,
J ¼ 5.2 Hz, OCH2), 6.12 (s, 1H, H-3), 6.14 (s, 1H, H-5), 6.80 (d, 2H,
J ¼ 8.8 Hz, H-30,50), 7.55 (d, 2H, J ¼ 8.4 Hz, H-2060); 13C NMR
(CD3COCD3, 400 MHz) d: 8.2 (2CH2CH3) 47.1 (2CH2CH3), 49.6 (C-
CH2-N), 55.2 (CH3O), 63.6 (O-CH2-C), 91.7 (C-5), 94.6 (C-3), 107.8
(C-1), 115.0 (C-30,50), 131.5 (C-20,60), 131.7 (C-10), 158.6 (C-6), 160.8
(C-2), 161.9(C-40), 164.1 (C-4), 195.0 (C-7). HRMS (ESI) calcd. for
C20H25NO5: m/z 360.1811[M þ H]þ. Found: 360.1813.
3.11 4,40,6-Trihydroxy-2-methoxybenzophenone (10)
A similar procedure as described previously for the preparation of 3 from 2
was used for the preparation of 10 from 1. Yield 95% as a yellow solid. Mp
1
83–858C; IR (KBr): 3347, 1634, 1608, 1438, 1162; H NMR (CD3COCD3,

Novel Mahkoside A Derivatives
3327
400 MHz) d: 3.80 (s, 3H,OCH3), 6.04 (s, 2H, H-3,5), 6.85 (d, 2H, J ¼ 8.4 Hz,
H-30,50), 7.61 (d, 2H, J ¼ 8.8 Hz, H-2060); 13C NMR (CD3COCD3, 400 MHz)
d: 55.6 (CH3O), 94.3 (C-5), 94.3 (C-3), 115.0 (C-1), 115.1 (C-30,50), 132.3
(C-20,60), 132.9 (C-10), 161.8 (C-6), 162.1 (C-2), 162.4 (C-40), 165.9 (C-4),
197.6 (C-7). HRMS (ESI) calcd. for C14H12 O5: m/z 283.0583 [M þ Na]þ.
Found: 283.0583.
3.12 Biological Activity Assays
In vitro cytotoxicity study: The Ec-9706 cell line was kindly presented by the
Henan Institute of Medical Sciences, China. The culture medium was RPMI-
1640 supplemented with 10% (v/v) fetal calf serum, 100 U/mL penicillin,
and 100 mg/mL streptomycin.
Sensitivity test: Cancer cells, growing in monolayer cultures at 378C
under 5% CO2, were trypsinized, rinsed with PBS (w/o Ca2þ and Mg2þ),
and plated into 96-well plates (7 ꢀ 103 cells/well). The next day, test sub-
stances were freshly dissolved in DMSO, resulting in 10-mg/mL stock
solutions. Stock solutions were diluted in the culture medium and added
(200 mL/well) at various concentrations to the wells, resulting in five final
concentrations between 6.25 and 100 mg/mL. The MTT-test was performed
48 h later.
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