Synthesis and cancer cell cytotoxicity of substituted xanthenes

Article abstract of DOI:10.1016/j.bmc.2010.01.018

A series of substituted xanthenes was synthesized and screened for activity using DU-145, MCF-7, and HeLa cancer cell growth inhibition assays. The most potent compound, 9g ([N,N-diethyl]-9-hydroxy-9-(3-methoxyphenyl)-9H-xanthene-3-carboxamide), was found to inhibit cancer cell growth with IC₅₀ values ranging from 36 to 50 μM across all three cancer cell lines. Structure-activity relationship (SAR) data is presented that indicates additional gains in potency may be realized through further derivatization of the compounds (e.g., the incorporation of a 7-fluoro substituent to 9g). Results are also presented that suggest the compounds function through a unique mechanism of action as compared to that of related acridine and xanthone anticancer agents (which have been shown to intercalate into DNA and inhibit topoisomerase II activity). A structural comparison of these compounds suggests the differences in function may be due to the structure of the xanthene heterocycle which adopts a nonplanar conformation about the pyran ring.

Full text of DOI:10.1016/j.bmc.2010.01.018

Bioorganic & Medicinal Chemistry 18 (2010) 1456–1463
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and cancer cell cytotoxicity of substituted xanthenes
Rajan Giri a, John R. Goodell b, Chenguo Xing a, Adam Benoit a, Harneet Kaur a, Hiroshi Hiasa c,
David M. Ferguson a,d,
*
a Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455, United States
b Department of Chemistry and Center for Chemical Methodology and Library Development (CMLD-BU), Boston University, Boston, MA 02215, United States
c Department of Pharmacology, University of Minnesota, Minneapolis, MN 55455, United States
d Center for Drug Design, University of Minnesota, Minneapolis, MN 55455, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of substituted xanthenes was synthesized and screened for activity using DU-145, MCF-7, and
HeLa cancer cell growth inhibition assays. The most potent compound, 9g ([N,N-diethyl]-9-hydroxy-9-
(3-methoxyphenyl)-9H-xanthene-3-carboxamide), was found to inhibit cancer cell growth with IC50 val-
Received 17 August 2009
Revised 6 January 2010
Accepted 7 January 2010
Available online 11 January 2010
ues ranging from 36 to 50 lM across all three cancer cell lines. Structure–activity relationship (SAR) data
is presented that indicates additional gains in potency may be realized through further derivatization of
the compounds (e.g., the incorporation of a 7-fluoro substituent to 9g). Results are also presented that
suggest the compounds function through a unique mechanism of action as compared to that of related
acridine and xanthone anticancer agents (which have been shown to intercalate into DNA and inhibit
topoisomerase II activity). A structural comparison of these compounds suggests the differences in func-
tion may be due to the structure of the xanthene heterocycle which adopts a nonplanar conformation
about the pyran ring.
Keywords:
Xanthene
Xanthone
Cytotoxicity
Cancer cells
MCF-7
DU-145
HeLa
Ó 2010 Elsevier Ltd. All rights reserved.
SAR
Topoisomerase
DNA binding
Intercalation
Drug design
Molecular modeling
1. Introduction
epoxides to the site of alkylation. While a number of other studies
have appeared that show substituted xanthones are cytotoxic to
Xanthones and xanthenes are tricyclic dibenzopyrans with di-
verse physicochemical and pharmacological properties. Although
a large number of xanthone-based natural products have been iso-
lated and characterized,1–3 the development of therapeutic agents
that take advantage of this unique heterocyclic structure has been
limited. Xanthones are active against a variety of pathogens and
show promise for use as antioxidants, antineoplastics, vasodilators,
and anti-inflammatories.4–15 In most cases, the mechanism of ac-
tion is not known, greatly complicating the drug discovery process.
Recently, Na and co-workers reported the synthesis of several xan-
thone-based analogs that were cytotoxic to cancer cells in vitro.4
These compounds contained reactive epoxides which were shown
to block topoisomerase II mediated relaxation of DNA. This mech-
anism of action suggests the planar xanthone structure interacts
with DNA, perhaps through intercalation, directing the reactive
cancer cells,5–9 a common mechanism of action has not yet
emerged.
Xanthones are structurally related to the xanthenes and xan-
then-9-ols as shown in Figure 1. Xanthenes are commonly used
in the production of fluorescent dyes, including fluorescein, rhoda-
mine, and the eosins. Some evidence has surfaced that suggests the
xanthene core structure also may be useful in the design and
development of pharmacological agents.16–18 Starting from 9-
carboxy xanthene, Naya et al. synthesized and evaluated the struc-
ture–activity relationships of a series of 2,9-disubstituted xanth-
enes with potent activity as chemokine receptor (CCR1)
antagonists.16 In an earlier study, Ornstein et al. reported a 9-xan-
thylmethyl amino acid conjugate that was shown to be a selective
and potent antagonist of the metabotropic glutamate receptor.17
Most recently, our lab described the synthesis of a 3-piperazinyl
substituted xanthen-9-ol with low micromolar activity against
* Corresponding author. Tel.: +1 612 626 2601; fax: +1 612 624 0139.
E-mail address: ferguson@umn.edu (D.M. Ferguson).
West Nile Virus.18 Although only
a
small selection of
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.01.018

R. Giri et al. / Bioorg. Med. Chem. 18 (2010) 1456–1463
1457
10
O
4
1
5
8
R1
6
R1
R1
O
O
3
9
2
R2
7
R2
R2
O
OH
Xanthone
Xanthene
Xanthen-9-ol
Figure 1. Xanthone, xanthene, and xanthenol structures.
representative structures were evaluated, the synthetic scheme de-
scribed used a xanthone intermediate, providing an efficient route
to obtaining substituted xanthenes. Compounds of this type have
also been synthesized using aryne insertion reactions of benzalde-
hydes and benzoates.19,20
modeling techniques and screening data derived from topoisomer-
ase II activity.
2. Chemistry
Here, we describe the synthesis of a series of substituted xanth-
enes using a modified Ullmann–Goldberg reaction commonly used
in the synthesis of acridones and acridines.21,22 The approach cou-
ples phenols to aryl halides in the presence of copper to generate
aryl ethers. Cyclization to the xanthone is then accomplished via
Friedel–Crafts-like acylation. One advantage to this approach is
that the intermediate xanthone can be both evaluated for biologi-
cal activity and further converted to the xanthene by any number
of routes. In this report, we use a Grignard reaction to obtain a li-
brary of di- and tri-substituted 9-alkyl xanthenols for evaluation as
potential anticancer agents. The cytotoxicity of the compounds
against three well known cancer cell lines (DU-145, MCF-7, and
HeLa) is reported and compared with previous work on a related
series of substituted acridines. Some insights to the potential
mechanism of action of the compounds are given using molecular
The synthesis of xanthenes proceeds through the generation of
the xanthone intermediate as shown in Schemes 1 and 2. The first
step of the pathway utilizes a modified Ullmann condensation
reaction of 2-iodobenzoic acid (1) or bromoterephthalic acid (5)
with various phenols in the presence of catalytic Cu(0) to form 2-
phenoxybenzoic acid (2) or O-phenylsalicylic acid derivatives 6.
The use of a non-nucleophilic base, such as 1,8-diazabicyclo
[5.4.0] undec-7-ene (DBU), was found to be essential for the cou-
pling reaction with phenol to form the diarylethers. Cyclization
of 2-phenoxybenzoic acid (2) and O-phenylsalicylic acid deriva-
tives 6 with concd H2SO4 at 100 °C afforded xanthone (3) and 3-
carboxyxanthone derivatives 7, respectively. Refluxing 7 with
SOCl2 produced the acid chloride intermediate. Subsequent
reaction of the acid chloride with an amine afforded the 3-carbox-
amidexanthone derivatives 8. Xanthone (3) and 3-carboxamidex-
O
O
O
R
HO
OH
c
b
OH
a
O
O
O
I
2
1
3
4
R = -Ph,-(3-OMe)Ph
Scheme 1. Synthesis of the 9-alkylxanthen-9-ols. Reagents and conditions: (a) phenol, Cu, CuI, pyridine, DBU, DMF, reflux; (b) H2SO4, 100 °C; (c) MgBrR, THF, À78 °C 1 h to rt
5 h.
O
O
O
OH
OH
b
a
HO
R1
HO
O
OH
Br
O
O
O
c
O
R1
6
7
5
R1 = -H, -Cl, -F
O
R2 =-OH, N-methylpiperazine,
morpholine, diethylamine
R3 = -CH3, -C2H5, -CH=CH2,
-Ph, -(3-OMe)Ph
HO R3
d
R1
R1
R2
R2
O
O
O
O
8
9
Scheme 2. Synthesis of the di- and tri-substituted xanthenes. Reagents and conditions: (a) phenol, Cu, CuI, pyridine, DBU, DMF, reflux; (b) H2SO4, 100 °C; (c) SOCl2, reflux,
secondary amine, CH2Cl2, 0 °C; (d) MgBrR3, THF, À78 °C 1 h to rt 5 h.

1458
R. Giri et al. / Bioorg. Med. Chem. 18 (2010) 1456–1463
anthone derivatives 8 were reacted with different Grignard re-
As shown in Figure 2, no activity in the relaxation assays was de-
tected. These results are somewhat surprising given the results re-
ported by Na and co-workers that indicated reactive xanthone
derivatives, which include reactive epoxides, inhibit topoisomerase
II activity.4 The differences in their effects on the topoisomerase II
activity might be due to the presence of reactive epoxides. Addi-
tionally, the compounds in the current study were designed, in
part, to possess some of the same structural features found in
our recently published work highlighting acridine-based antican-
cer agents.23 While both the acridines and xanthones have been
shown to intercalate into DNA,4,24 the xanthenes did not interca-
late into DNA (Fig. 3). This observation suggests that the xanthenes
studied here function through an alternative mechanism. A struc-
tural comparison of xanthenol 9e, xanthone 3, and a representative
acridine-based structure is given in Figure 4. The structures were
model built interactively and subsequently energy minimized
using quantum mechanics calculations.25 While the acridine and
xanthone structures adopt a planar conformation that is well sui-
ted to DNA intercalation, the xanthen-9-ol buckles about the pyran
ring and adopts a boat-like conformation thus orienting the phenyl
ring perpendicular to the plane of the heterocycle. This deviation
from planarity may, in part, explain the lack of DNA binding noted
for the xanthenes when compared to related acridines and the par-
ent xanthones.
agents at À75 °C to yield xanthene derivatives
4 and 9,
respectively.
3. Results and discussion
The substituted xanthenes and corresponding activity against
prostate (DU-145), breast (MCF-7), and cervical (HeLa) cancer cell
proliferation are reported in Table 1. While limited, the library was
designed to provide some structure–activity relationship data to
help guide the design of additional xanthene derivatives. The
unsubstituted xanthone (3) and 9-phenyl-xanthenol (4a) were
synthesized for comparison and notably showed no activity. The
simple addition of a 3-methoxy group to the 9-phenyl ring of 4a,
however, was found to have a dramatic impact on activity suggest-
ing this group may contribute to activity. Further analysis of the
activity with respect to the R3 substitution shows that the 3-
methoxyphenyl group is generally favored. One notable exception
is with compound 9b which contains a vinyl group in the R3 posi-
tion. In regards to the R2 substitution, it was found that replace-
ment of the N-methylpiperazine with the diethylamine (9f and
9g, respectively) improved activity against all three cancer cell
lines by several folds. Comparing 4a, 9d, 9f, and 9h (which all con-
tain the 9-phenyl substituent) reveals a clear trend indicating that
the diethylamino group contributes the most favorable to activity.
Additionally, it was found that activity increases in both the
chloro- and fluoro-series when R3 contains a bulky phenyl or 3-
methoxyphenyl substitution. At the R1 position the fluoro
substitutions show the best activity, suggesting that additional
gains in potency may be achieved by adding a fluoro group to
the diethylamine 9g.
4. Conclusion
This study has reported the synthesis and cytotoxicity of a small
library of substituted xanthenes. Although the activity of the com-
pounds is modest, it is important to point out that the xanthenes
described here are novel anticancer agents. Given our previous
work on substituted acridines and the data reported by Woo
et al. for xanthones,4,23,24 the failure of these compounds to inter-
The effect of the compounds on the catalytic activity of human
topoisomerase II has been examined using a DNA relaxation assay.
Table 1
Cancer cell cytotoxicities for the substituted xanthenes and related compoundsa
O
HO
R3
HO
R3
R1
R2
O
O
O
9a-s
3
4a-b
O
Compd
R1
R2
R3
DU-145
MCF-7
HeLa
3
—
—
—
—
—
—
—
Ph
>500
>500
>500
>500
>500
>500
85 15
121 12
74 0.3
4a
4b
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
9p
9q
9r
9s
–Ph(3-OMe)
Methyl
–CH@CH2
–C2H5
–Ph
–Ph(3-OMe)
–Ph
–Ph(3-OMe)
–Ph
–Ph(3-OMe)
Methyl
–CH@CH2
–C2H5
59
4
82
6
–H
–H
–H
–H
–H
–H
–H
–H
–H
–Cl
–Cl
–Cl
–Cl
–Cl
–F
N-Methylpiperazine
N-Methylpiperazine
N-Methylpiperazine
N-Methylpiperazine
N-Methylpiperazine
Diethylamine
Diethylamine
Morpholine
Morpholine
N-Methylpiperazine
N-Methylpiperazine
N-Methylpiperazine
N-Methylpiperazine
N-Methylpiperazine
N-Methylpiperazine
N-Methylpiperazine
N-Methylpiperazine
N-Methylpiperazine
N-Methylpiperazine
119
63
4
5
9
115 13
3
74
2
195
>300
139
218
169
125
62
6
2
7
213
170
111
67
5
4
2
59
3
4
4
2
1
36
3
50
44
125
163
241 0.5
208 12
145 15
4
7
134
151 30
160
>500
180
9
120
6
153 38
264 14
285 25
9
3
153
158
94
3
2
–Ph
117
92
6
95
9
–Ph(3-OMe)
Methyl
–CH@CH2
–C2H5
–Ph
–Ph(3-OMe)
5
72
2
6
>300
>300
139
208
254
138
62
4
8
3
>300
230 16
–F
–F
–F
–F
2
127
109
85
5
6
84
4
9
2
55 0.4
46
4
a
IC50 values (lM).

R. Giri et al. / Bioorg. Med. Chem. 18 (2010) 1456–1463
1459
proliferation in vitro. In addition, no significant preference for can-
cer cell type was noted. These findings support the general hypoth-
esis that these compounds function through specific as opposed to
non-specific binding in mediating cytotoxicity. While the chemis-
try presented here is limited, the SAR suggests that some gains in
activity should be obtainable through further derivatization of
the xanthene scaffold. In particular, fluoro derivatives coupled with
9-(3-methoxyphenyl) substitutions may prove promising in reduc-
ing the IC50 values to the sub-micromolar range. One issue not ad-
dressed here, but central to exploring both the potential
mechanism of action of the compounds and future SAR studies is
chirality. The xanthenes tested in this study are all racemates, sug-
gesting that further gains in potency may be realized by resolving
the enantiomers. Enantioselectivity is a key measure of specificity
in evaluating mechanisms of action and would provide much
needed insight to understanding the function of these compounds
in inhibiting cancer cell growth.
Figure 2. Representative results of topoisomerase II relaxation assay for the
substituted xanthenes.
5. Experimentals
5.1. General
Unless mentioned otherwise all the starting materials and sol-
vents were purchased from commercially available sources. Water
was purified via a Millipore filtration system. Column chromatog-
raphy was conducted using Silica Gel 60 (40–63 l
m). 1H nuclear
magnetic resonance spectra were collected on Varian Mercury
600 MHz instrument using CDCl3 and DMSO-d6 and acetone-d6 as
solvents. High-resolution mass spectra (HRMS) were collected
from a TOF-ESI Agilant LC–MS and analyzed using the Analyst QS
software. All the reactions were monitored using thin layer chro-
matography (TLC) using EMD Chemical Silica Gel 60 F254 on alu-
minum sheets.
Figure 3. Representative results of DNA intercalation assay for the substituted
xanthenes. A, amsacrine.
calate into DNA or block topoisomerase II activity is somewhat sur-
prising. A comparison of the acridine, xanthone, and xanthene
structures, however, indicates this failure is directly related to dif-
ferences in the structures of these compounds. Whereas the acri-
dine and xanthone structures adopt flat, highly conjugated ring
systems, the xanthenes buckle about the pyran ring, placing the
9-phenyl substituent in a perpendicular orientation relative to
the tricyclic ring system. The position of this phenyl ring would
certainly disrupt intercalation and may explain the lack of DNA
binding noted for the xanthenes. This hypothesis could be evalu-
ated by preparing a 9-benzylidene derivative of 4b to further
determine the effect of planarity on function and activity.
5.1.1. 2-Phenoxybenzoic acid (2)
2-Iodo benzoic acid (1) (3.10 g, 12.4 mmol) was added to
100 mL of dimethylformamide (DMF), followed by phenol (2.37 g,
25.2 mmol), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (5.2 mL,
36.9 mmol), pyridine (0.2 mL), copper(0) (0.1 g), and copper(I) io-
dide (0.1 g). The reaction was heated to reflux and monitored via
TLC. After 2 h, TLC showed that all of the 2-iodo benzoic acid was
consumed. The reaction was cooled and diluted with 1 M HCl
(500 mL) until no more precipitate had formed. The resulting pre-
cipitates were filtered and washed with water (100 mL) and dried
under vacuum to give desired product (2.41 g, 93.0%) as brown so-
An analysis of the IC50 values also has shown a correlation exists
between xanthene substitution and the inhibition of cancer cell
Figure 4. Stereoview of xanthenol 9e, xanthone (3) (green), and a representative acridine structure (blue) taken from Refs. 23.26 The structures are overlaid based on an RMS
fit of the tricyclic ring system.

1460
R. Giri et al. / Bioorg. Med. Chem. 18 (2010) 1456–1463
lid: Rf = 0.5 (90:10 CH2Cl2/MeOH); 1H NMR (600 MHz, DMSO-d6) d
6.87–7.81 (m, 9H), 12.1 (s (br s, 1H);APCI-MS: m/z 213.16 [MÀH]À.
C13H9O3 (214.06).
described for 2 to afford the title compound (9.10 g, 81.0%) as
greenish solid: Rf = 0.36 (80:20 CH2Cl2/MeOH); 1H NMR
(600 MHz, DMSO-d6) d 7.01 (dd, 2H, J = 8.8, 2.6 Hz), 7.35–7.39 (m,
2H), 7.79 (d, 1H, J = 8.2 Hz), 7.92 (d, 1H, J = 7.8 Hz), 8.14 (br s,
1H), 12.8 (br s, 2H); APCI-MS: m/z 275.05 [MÀH]À. C14H9O5Cl
(276.04).
5.1.2. Xanthen-9-one (3)
2-Phenoxy-terephthalic acid (2) (3.0 g, 14.0 mmol) was added
to 100 mL of H2SO4 and heated to 100 °C while being stirred for
3 h. The starting material dissolved upon heating. The reaction
was cooled and poured over ice (300 mL), producing a gray solid
that was then filtered and washed with water and dried. The crude
was purified by column chromatography (SiO2, 95:5 CH2Cl2/
MeOH) to give desired product (1.80 g, 67.0%) as white solid: mp
5.1.8. Xanthen-9-one-3-carboxylic acid (7a)
2-Phenoxy-terephthalic acid (6a) (2.70 g, 10.6 mmol) was
added to H2SO4 (100 mL) and heated to 100 °C while being stirred
for 3 h on a water bath. After the completion of reaction, the reac-
tion was cooled and poured over ice (300 mL) producing a gray so-
lid that was then filtered, washed with water and dried to give
desired product (1.98 g, 68.0%) as black solid: Rf = 0.48 (90:10
CH2Cl2/MeOH); 1H NMR (600 MHz, DMSO-d6) d 7.47 (t, 1H,
J = 7.2 Hz), 7.66 (d, 1H, J = 8.4 Hz), 7.86–7.91 (m, 2H), 8.04 (s, 1H),
8.16 (d, 1H, J = 7.8 Hz), 8.23 (d, 1H, J = 13.8 Hz); APCI-MS: m/z
239.04 [MÀH]À. C14H8O4 (240.04).
179–180 °C; 1H NMR (600 MHz, DMSO-d6)
d 7.46 (t, 2H,
J = 7.2 Hz), 7.64 (d, 2H, J = 9.0 Hz), 7.85 (t, 2H, J = 7.2 Hz), 8.17 (d,
2H, J = 7.8 Hz); APCI-MS: m/z 197.06 [M+H]+. C13H8O2 (196.05).
5.1.3. 9-Phenyl-xanthen-9-ol (4a)
Xanthen-9-one (3) (0.52 g, 2.70 mmol) was taken into dry THF
and cooled to À78 °C. Phenyl magnesium bromide (1.10 g,
6.0 mmol) was added into the reaction with continued stirring
while maintaining the temperature of reaction at À78 °C in
30 min. The reaction was warmed to rt with continued stirring
overnight. The reaction was quenched with the addition of satd
NH4Cl followed by extraction with EtOAc (2 Â 50 mL). The organic
layers were combined and washed with water (20 mL). The organic
layer was then dried over MgSO4 evaporated in vacuo. Crude prod-
uct was purified by column chromatography (Sio2, 95:5 CH2Cl2/
MeOH) to afford the desired product (0.40 g, 56.0%) as white solid:
mp 143–145 °C; 1H NMR (600 MHz, DMSO-d6) d 6.73 (br s, 1H),
7.07 (t, 2H, J = 15 Hz), 7.11 (t, 1H, J = 13.2 Hz), 7.19–7.30 (m, 8H),
7.35 (d, 2H, J = 7.8 Hz); APCI-MS: m/z 275.10 [M+H]+. C19H14O2
(274.09).
5.1.9. 2-Chloro-xanthen-9-one-6-carboxylic acid (7b)
2-(4-Chlorophenoxy) terephthalic acid (6b) (5.0 g, 17.1 mmol)
was treated with H2SO4 using the same procedure described for
7a to afford the title compound (3.67 g, 78.0%) as dark green solid:
Rf = 0.43 (90:10 CH2Cl2/MeOH); 1H NMR (600 MHz, DMSO-d6) d
7.73 (d, 1H, J = 9.0 Hz), 7.89–7.93 (m, 2H), 8.01 (s, 1H), 8.05 (s,
1H), 8.07 (d, 1H, J = 2.4 Hz), 8.23 (d, 1H, J = 3.6 Hz); APCI-MS: m/z
273.01 [MÀH]À. C14H7O4Cl (274.00).
5.1.10. 2-Fluoro-xanthen-9-one-6-carboxylic acid (7c)
2-(4-Fluorophenoxy) terephthalic acid (6c) (5.0 g, 18.1 mmol)-
was treated with H2SO4 using the same procedure described for
7a to afford the title compound (2.90 g, 62.0%) as dark green solid:
Rf = 0.42 (90:10 CH2Cl2/MeOH); 1H NMR (600 MHz, DMSO-d6) d
7.71–7.80 (m, 2H), 7.83 (d, 1H, J = 7.2 Hz), 7.93 (d, 1H, J = 7.8 Hz),
8.01 (s, 1H), 8.05 (s, 1H), 8.23 (d, 1H, J = 8.4 Hz); APCI-MS: m/z
257.01 [MÀH]À. C14H7O4F (258.03).
5.1.4. 9-(3-Methoxyphenyl)-xanthen-9-ol (4b)
Xanthen-9-one (3) (0.52 g, 2.7 mmol) was reacted with 3-
methoxyphenyl magnesium bromide (1.10 g, 6.0 mmol) using the
same procedure described for 4a to afford title compound
(0.73 g, 89.0%) as white solid: mp 127–128 °C; Rf = 0.64 (95:5
CH2Cl2/MeOH), 1H NMR (600 MHz, DMSO-d6) d 3.23 (s, 3H), 6.19
(d, 1H, J = 8.4 Hz), 6.25 (dd, 1H, J = 10.2, 2.4 Hz), 6.28 (s, 1H), 6.49
(br s, 1H), 6.62–6.68 (m, 3H), 6.75 (d, 2H, J = 8.4 Hz), 6.85 (dd,
2H, J = 15.6, 7.2 Hz), 6.91 (d, 2H, J = 7.8 Hz); APCI-MS: m/z 303.10
[MÀH]-. C20H16O3 (304.11).
5.1.11. 3-(4-Methylpiperazine-1-carbonyl)-xanthen-9-one (8a)
Thionyl chloride (25 mL) was added to a flask containing xan-
then-9-one-3-carboxylic acid (7a) (0.26 g, 1.10 mmol) and was
set to reflux for 1 h. Excess thionyl chloride was removed under re-
duced pressure. The resulting residue was dissolved in CH2Cl2
(15 mL) and cooled to 0 °C on an ice bath. Next, N-methylpipera-
zine (0.22 mL, 1.60 mmol) was added dropwise while stirring.
The reaction was allowed to reach rt with continued stirring for
1 h. An additional 30.0 mL of CH2Cl2 was added, and the reaction
was extracted with 1 M HCl. The aqueous layer was made basic
with 5% NaOH and extracted into EtOAc, dried with MgSO4, and
concentrated in vacuo to afford the desired product (0.25 g,
71.0%) as slightly yellow solid: Rf = 0.7 (95:5 CH2Cl2/MeOH); 1H
NMR (600 MHz, CDCl3) d 2.34 (s, 3H), 2.38 (br s, 2H), 2.53 (br s,
2H), 3.45 (br s, 2H), 3.85 (br s, 2H), 7.38–7.43 (m, 2H), 7.51–7.55
(m, 2H), 7.75 (m, 1H), 8.35 (d, 1H, J = 7.2 Hz), 8.37 (d, 1H,
J = 8.4 Hz); APCI-MS: m/z 323.21 [M+H]+. C19H18O2 (322.13).
5.1.5. 2-Phenoxyterephthalic acid (6a)
Bromotherethphalic acid (5) (10.0 g, 41.0 mmol) was reacted
with phenol (7.7 g, 82.0 mmol) using the same procedure de-
scribed for 2 to afford the title compound (9.70 g, 93.0%) as green-
ish solid: Rf = 0.5 (80:20 CH2Cl2/MeOH), 1H NMR (600 MHz, DMSO-
d6) d 6.91 (d, 2H, J = 7.8 Hz), 7.15 (t, 1H, J = 7.2 Hz), 7.42 (dd, 2H,
J = 12, 7.3 Hz), 7.71 (d, 1H, J = 7.8 Hz), 7.87 (d, 1H, J = 9.6 Hz), 8.12
(s, 1H), 13.28 (br s, 2H); ESI-MS: m/z 257.06 [MÀH]À. C14H10O5
(258.05).
5.1.6. 2-(4-Chlorophenoxy) terephthalic acid (6b)
Bromotherethphalic acid (5) (10.0 g, 40.8 mmol) was reacted
with 4-chloro phenol (10.5 g, 81.6 mmol) using the same proce-
dure described for 2 to afford the title compound (11.0 g, 91.0%)
as greenish solid; Rf = 0.4 (80:20 CH2Cl2/MeOH), 1H NMR
(600 MHz, DMSO-d6) d 6.98 (dd, 2H, J = 9.0, 2.4 Hz), 7.40 -7.41
(m, 2H), 7.77–7.78 (m, 1H), 7.89 (d, 1H, J = 7.8 Hz), 8.01 (s, 1H),
13.2 (br s, 2H); APCI-MS: m/z 293.02 [M+H]+. C14H9O5Cl (292.04).
5.1.12. [N,N-Diethyl]-xanthen-9-one-3-carboxamide (8b)
Thionyl chloride (25 mL) was added to a flask containing xan-
then-9-one-3-carboxylic acid (7a) (0.26 g, 1.10 mmol) and was
set to reflux for 1 h. Excess thionyl chloride was removed under re-
duced pressure. The resulting residue was dissolved in CH2Cl2
(15 mL) and cooled to 0 °C on an ice bath. Next, Diethylamine
(1.7 mL, 1.60 mmol) was added dropwise while stirring. The reac-
tion was allowed to reach rt under continued stirring for about 1 h.
An additional 30 mL of CH2Cl2 was added, and the reaction was ex-
tracted with 1 M HCl. The organic layer was dried with MgSO4, and
concentrated in vacuo to afford desired product (0.22 g, 68.0%) as
5.1.7. 2-(4-Fluorophenoxy) terephthalic acid (6c)
Bromotherethphalic acid (5) (10.0 g, 40.8 mmol) was reacted
with 4-fluoro phenol (9.20 g, 81.6 mmol) using the same procedure

R. Giri et al. / Bioorg. Med. Chem. 18 (2010) 1456–1463
1461
slightly yellow solid: Rf = 0.64 (95:5 CH2Cl2/MeOH); 1H NMR
(600 MHz, CDCl3) d 1.12 (br s, 3H), 1.24 (br s, 3H), 3.17 (br s, 2H),
3.43 (br s, 2H), 7.22–7.45 (m, 6H), 7.71 (d, 1H, J = 7.4 Hz); APCI-
MS: m/z 294.12 [MÀH]À. C18H17O3 (295.12).
7.71 (s, 1H), 7.75 (d, 1H, J = 7.8 Hz); APCI-MS: m/z 351.17 [M +
H]+. C21H22N2O3 (350.16).
5.1.18. 3-(4-Methylpiperazine-1-carbonyl)-9-ethyl-9-hydroxy-
9H-xanthene (9c)
5.1.13. 3-(Morpholine-4-carbonyl)xanthen-9-one (8c)
Xanthen-9-one-3-carboxylic acid (7a) (0.26 g, 1.08 mmol) was
reacted with SOCl2 (25 mL) followed by the addition of morpholine
(1.3 mL, 1.6 mmol) using the same procedure described for 8a to
afford the title compound (0.78 g, 62.0%) as slightly yellow solid:
Rf = 0.72 (95:5 CH2Cl2/MeOH); 1H NMR (600 MHz, CDCl3) d 2.47
(br s, 4H), 3.48 (br s, 4H), 3.42 (br s, 2H), 7.18–7.23 (m, 3H), 7.36
(dd, 1H, J = 12.6, 3.0 Hz), 7.68 (d, 1H, J = 7.8 Hz); APCI-MS: m/z
310.08 [M+H]+. C18H17O3 (309.10).
3-(4-Methylpiperazine-1-carbonyl)-xanthen-9-one (8a) (0.30 g,
0.90 mmol) was reacted with ethyl magnesium bromide (0.27 g,
2.0 mmol) using the same procedure described for 4a to afford title
compound (0.28 g, 46.0%) as an off white solid: mp 148–152 °C;
Rf = 0.52 (95:5 CH2Cl2/MeOH), 1H NMR (600 MHz, DMSO-d6) d
0.36 (t, 3H, J = 7.2 Hz), 1.83 (q, 2H, J = 7.6 Hz), 2.03 (s, 3H), 2.13
(br s, 4H), 2.24 (br s, 4H), 4.89 (s, 1H), 6.98–7.21 (m, 5H), 7.60
(dd, 1H, J = 9.0, 1.8 Hz), 7.64 (d, 1H, 7.8 Hz); ESI-MS: m/z 353.20
[M+H]+. C21H24N2O3 (352.18).
5.1.14. 2-Chloro-6-(4-methylpiperazine-1-carbonyl)xanthen-9-
one (8d)
5.1.19. 3-(4-Methylpiperazine-1-carbonyl)-9-hydroxy-9-
phenyl-9H-xanthene (9d)
2-Chloro-xanthen-9-one-6-carboxylic
acid
(7b)
(2.0 g,
3-(4-Methylpiperazine-1-carbonyl)-xanthen-9-one (8a) (0.50 g,
1.50 mmol) was reacted with phenyl magnesium bromide (0.60 g,
3.3 mmol) using the same procedure described for 4a to afford title
compound (0.30 g, 57.0%) as an off white solid: mp 187–189 °C;
Rf = 0.71 (95:5 CH2Cl2/MeOH), 1H NMR (600 MHz, DMSO-d6) d
2.17 (s, 3H), 2.25 (br s, 2H), 2.32 (br s, 2H), 3.30 (br s, 2H), 3.59
(br s, 2H), 6.81 (s, 1H), 7.07–7.14 (m, 3H), 7.15–7.26 (m, 5H),
7.30–7.34 (m, 2H), 7.39 (d, 1H, 8.4 Hz); 13C NMR (DMSO-d6, d
ppm): 168.581, 149.995, 149.713, 149.504, 137.019, 130.275,
129.847, 129.557, 129.423, 128.946, 128.690, 128.578, 127.205,
127.150, 126.316, 124.326, 122.525, 116.569, 115.107, 69.308,
55.290, 54.888, 47.719, 46.272, 42.147; APCI-MS: m/z 401.20
[M+H]+. C25H24N2O3 (400.17).
7.3 mmol) was reacted with SOCl2 (80 mL) followed by N-methyl
piperazine (1.22 mL, 11.0 mmol) using the same procedure de-
scribed for 8a to afford the title compound (1.56 g, 64.0%) as
slightly yellow solid: Rf = 0.6 (95:5 (95:5 CH2Cl2/MeOH)); 1H
NMR (600 MHz, CDCl3) d 2.34 (s, 3H), 2.37 (br s, 2H), 2.51 (br s,
2H), 3.42 (br s, 2H), 3.73 (br s, 2H), 7.38–7.43 (m, 4H), 7.65 (d,
1H, J = 7.6 Hz), 7.81 (d, 1H, J = 7.8 Hz); APCI-MS: m/z 357.19 [M +
H]+. C19H17ClN2O3 (356.09).
5.1.15. 2-Fluoro-6-(4-methylpiperazine-1-carbonyl)xanthen-9-
one (8e)
2-Fluoro-xanthen-9-one-6-carboxylic
acid
(7c)
(0.32 g,
1.20 mmol) was reacted with SOCl2 (10 mL) followed by N-methyl
piperazine (0.18 mL, 1.60 mmol) using the same procedure de-
scribed for 8a to afford the title compound (0.28 g, 66.0%) as a
slightly yellow solid: Rf = 0.65 (95:5 CH2Cl2/MeOH); 1H NMR
(600 MHz, CDCl3) d 2.34 (s, 3H), 2.37 (br s, 2H), 2.51 (br s, 2H),
3.42 (br s, 2H), 3.73 (br s, 2H), 7.18–7.23 (m, 4H), 7.36 (dd, 1H,
J = 12.6, 3.0 Hz), 7.68 (d, 1H, J = 7.8 Hz); APCI-MS: m/z 341.17 [M
+ H]+. C19H17FN2O3 (340.12).
5.1.20. 3-(4-Methylpiperazine-1-carbonyl)-9-hydroxy-9-(3-
methoxyphenyl)-9H-xanthene (9e)
3-(4-Methylpiperazine-1-carbonyl)-xanthen-9-one (8a) (0.50 g,
1.5 mmol) was reacted with 3-methoxy phenyl magnesium bro-
mide (0.52 g, 2.5 mmol) using the same procedure described for
4a to afford title compound (0.54 g, 81.0%) as off an white solid:
mp 165–167 °C; Rf = 0.56 (95:5 CH2Cl2/MeOH), 1H NMR
(600 MHz, DMSO-d6) d 2.05 (s, 3H), 2.21 (s, 3H), 2.34 (br s, 4H),
2.85 (br s, 1H), 3.41 (br s, 2H), 3.63 (br s, 2H), 7.11–7.15 (m, 3H),
7.19–7.21 (m, 2H), 7.26–7.32 (m, 3H), 7.38 (d, 2H, J = 7.8 Hz),
5.1.16. 3-(4-Methylpiperazine-1-carbonyl)-9hydroxy-9-methyl-
9H-xanthene (9a)
7.46 (d, 1H, J = 8.4 Hz); APCI-MS: m/z 431.18 [M
+
H]+.
3-(4-Methylpiperazine-1-carbonyl)-xanthen-9-one (8a) (0.50 g,
1.50 mmol) was reacted with methyl magnesium bromide (0.55 g,
3.30 mmol) using the same procedure described for 4a to afford ti-
tle compound (0.35 g, 57.0%) as an off white solid: mp 94–98 °C;
Rf = 0.6 (95:5 CH2Cl2/MeOH), 1H NMR (600 MHz, DMSO-d6) d 1.48
(s, 3H), 2.02 (s, 3H), 2.17 (br s, 4H), 3.24 (br s, 2H), 3.45 (br s,
2H), 5.48 (s, 1H), 6.89 (s, 1H), 6.98 (d, 1H, J = 8.4 Hz), 7.02–7.07
(m, 2H), 7.17–7.18 (m, 1H), 7.64–7.68 (m, 2H); 13C NMR (ace-
tone-d6, d ppm): 168.589, 149.601, 149.490, 137.079, 130.848,
129.482, 128.679, 127.221, 126.986, 123.791, 122.142, 115.989,
114.762, 65.547, 56.518, 56.507, 54.997, 45.540, 34.919, 34.420;
APCI-MS: m/z 339.17 [M+H]+. C20H22N2O3 (338.16).
C26H26N2O4 (430.18).
5.1.21. [N,N-Diethyl]-9-hydroxy-phenyl-9H-xanthene-
carboxamide (9f)
[N,N-Diethyl]-xanthen-9-one-3-carboxamide
(8b)
(0.41 g,
1.4 mmol) was reacted with phenyl magnesium bromide (0.50 g,
2.80 mmol) using the same procedure described for 4a to afford ti-
tle compound (0.30 g, 58.0%) as an off white solid: mp 139–142 °C;
Rf = 0.76 (95:5 CH2Cl2/MeOH), 1H NMR (600 MHz, DMSO-d6) d 1.12
(br s, 3H), 1.24 (br s, 3H), 2.91 (s, 1H), 3.27 (br s, 2H), 3.53 (br s, 2H),
7.01 (d, 1H, J = 7.8 Hz), 7.08 (t, 1H, J = 7.2 Hz), 7.16 (s, 1H), 7.18–
7.20 (m, 2H), 7.26–7.32 (m, 4H), 7.36–7.39 (m, 3H); APCI-MS: m/
z 374.19 [M+H]+. C24H24NO3 (373.44).
5.1.17. 3-(4-Methylpiperazine-1-carbonyl)-9-hydroxy-9-vinyl-
9H-xanthene (9b)
5.1.22. [N,N-Diethyl]-9-hydroxy-(3-methoxyphenyl)-9H-
xanthene-carboxamide (9g)
3-(4-Methylpiperazine-1-carbonyl)-xanthen-9-one (8a) (0.50 g,
1.5 mmol) was reacted with vinyl magnesium bromide (0.44 g,
3.3 mmol) using the same procedure described for 4a to afford title
compound (0.20 g, 37.0%) as an off white solid: mp 145–147 °C;
Rf = 0.52 (95:5 CH2Cl2/MeOH), 1H NMR (600 MHz, DMSO-d6) d
2.11 (s, 3H), 2.27 (br s, 2H), 2.37 (d, 2H, J = 16.8 Hz), 2.48 (br s,
4H), 4.01 (br s, 1H), 4.90 (d, 1H, J = 16.8 Hz), 5.1 (d, 1H,
J = 10.4 Hz), 5.99–6.05 (m, 1H), 7.19 (q, 2H, J = 8.0 Hz), 7.35 (t,
1H, J = 7.2 Hz), 7.56 (d, 1H, J = 7.2 Hz), 7.67 (d, 1H, J = 7.8 Hz),
[N,N-Diethyl]-xanthen-9-one-3-carboxamide
(8b)
(0.41 g,
1.4 mmol) was reacted with 3-methoxy phenyl magnesium bro-
mide (0.59 g, 2.8 mmol) using the same procedure described for
4a to afford title compound (0.36 g, 65.0%) as an off white solid:
mp >250 °C; Rf = 0.64 (95:5 CH2Cl2/MeOH), 1H NMR (600 MHz,
DMSO-d6) d 1.12 (br s, 3H), 1.23 (br s, 3H), 2.96 (s, 1H), 3.26 (br
s, 2H), 3.53 (br s, 2H), 3.76 (s, 3H), 6.73 (dd, 1H, J = 9.6, 1.8 Hz),
6.86 (d, 1H, J = 7.8 Hz), 7.01 (d, 1H, J = 7.8 Hz), 7.06–7.09 (m, 1H),

1462
R. Giri et al. / Bioorg. Med. Chem. 18 (2010) 1456–1463
7.14–7.20 (m, 2H), 7.26 (s, 1H), 7.29–7.40 (m, 4H); APCI-MS: m/z
5.1.28. 2-Chloro-6-(4-methylpiperazine-1-carbonyl)-9-
hydropxy-9-phenyl-9H-xanthene (9m)
404.19 [M+H]+. C25H25NO4 (403.18).
2-Chloro-6-(4-methylpiperazine-1-carbonyl)xanthen-9-one
(8d) (0.16 g, 0.50 mmol) was reacted with phenyl magnesium bro-
mide (0.16 g, 0.9 mmol) using the same procedure described for 4a
to afford title compound (0.15 g, 77.0%) as an off white solid: mp
124 °C; Rf = 0.52 (95:5 CH2Cl2/MeOH), 1H NMR (600 MHz, ace-
tone-d6) d 2.21 (s, 3H), 2.34 (br s, 4H), 3.40 (br s, 2H), 3.63 (br s,
2H), 5.94 (s, 1H), 7.10–7.15 (m, 3H), 7.19–7.21 (m, 2H), 7.26–
7.32 (m, 3H), 7.40 (d, 2H, J = 7.8 Hz), 7.48 (d, 1H, J = 7.8 Hz);
APCI-MS: m/z 435.14 [M+H]+. C25H23N2O3Cl (434.14).
5.1.23. 3-(Morpholino-4-carbonyl)-9-hydroxy-9-phenyl-9H-
xanthene (9h)
3-(Morpholine-4-carbonyl)xanthen-9-one
(8c)
(0.44 g,
1.40 mmol) was reacted with phenyl magnesium bromide
(0.57 g, 3.12 mmol) using the same procedure described for 4a to
afford title compound (0.38 g, 68.0%) as
a white solid: mp
>250 °C; Rf = 0.56 (95:5 CH2Cl2/MeOH), 1H NMR (600 MHz, ace-
tone-d6) d 3.65 (br s, 8H), 7.10–7.23 (m, 6H), 7.26–7.33 (m, 4H),
7.39–7.41 (m, 3H), 7.45–7.48 (m, 1H); APCI-MS: m/z 388.15
[M+H]+. C24H21NO4 (387.15).
5.1.29. 2-Chloro-6-(4-methylpiperazine-1-carbonyl)-9-
hydroxy-(3-methoxyphenyl)-9H-xanthene (9n)
5.1.24. 3-(Morpholino-4-carbonyl)-9-hydroxy-(3-
methoxyphenyl)-9H-xanthene (9i)
2-Chloro-6-(4-methylpiperazine-1-carbonyl)xanthen-9-one
(8d) (0.30 g, 0.84 mmol) was reacted with 3-methoxy phenyl mag-
nesium bromide (0.40 g, 1.85 mmol) using the same procedure de-
scribed for 4a to afford title compound (0.20 g, 67.0%) as an off
white solid: mp 174–177 °C; Rf = 0.50 (95:5 CH2Cl2/MeOH), 1H
NMR (600 MHz, acetone-d6) d 2.24 (s, 3H), 2.36 (br s, 4H), 3.44
(br s, 2H), 3.63 (br s, 2H), 3.76 (s, 3H), 5.83 (s, 1H), 6.77 (dd, 1H,
J = 10.2, 2.4 Hz), 6.82 (d, 1H, J = 7.8 Hz), 7.10–7.14 (m, 4H), 7.18–
7.20 (m, 2H), 7.25–7.28 (m, 1H), 7.48 (d, 1H, J = 7.8 Hz); APCI-
MS: m/z 465.17 [M+H]+. C26H25N2O4Cl (464.15).
3-(Morpholine-4-carbonyl)xanthen-9-one
(8c)
(0.25 g,
0.81 mmol) was reacted with 3-methoxy phenyl magnesium bro-
mide (0.38 g, 1.6 mmol) using the same procedure described for
4a to afford title compound (0.20 g, 57.0%) as a white solid: mp
111–114 °C; Rf = 0.62 (95:5 CH2Cl2/MeOH), 1H NMR (600 MHz,
DMSO-d6) d 2.10 (br s, 4H), 2.81 (br s, 5H), 3.75 (s, 3H), 6.06 (s,
1H), 6.80 (d, 1H, J = 7.8 Hz), 6.86 (d, 1H, J = 8.4 Hz), 7.10 (s, 1H),
7.22 (d, 2H, J = 7.2 Hz), 7.31 (s, 1H), 7.47 (d, 1H, J = 8.4 Hz), 7.54
(d, 1H, J = 7.2 Hz), 7.73 (d, 1H, J = 7.2 Hz), 7.82 (s, 1H); ESI-MS: m/
z 418.13 [M+H]+. C25H23NO5 (417.16).
5.1.30. 2-Fluoro-6-(4-methylpiperazine-1-carbonyl)-9-
hydroxy-methyl-9H-xanthene (9o)
5.1.25. 2-Chloro-6-(4-methylpiperazine-1-carbonyl)-9-
hydroxy-9-methyl-9H-xanthene (9j)
2-Fluoro-6-(4-methylpiperazine-1-carbonyl)xanthen-9-one
(8e) (0.30 g, 0.88 mmol) was reacted with methyl magnesium bro-
mide (0.33 g, 1.94 mmol) using the same procedure described for
4a to afford title compound (0.20 g, 64.0%) as an off white solid:
mp 143–144 °C; Rf = 0.58 (95:5 CH2Cl2/MeOH), 1H NMR
(600 MHz, acetone-d6) d 1.51 (s, 3H), 2.11 (s, 3H), 2.23 (br s, 4H),
3.32 (br s, 2H), 3.51 (br s, 2H), 5.11 (br s, 1H), 6.96–7.10 (m, 4H),
7.35 (dd, 1H, J = 12.6, 3.0 Hz), 7.68 (d, 1H, J = 7.8 Hz); APCI-MS:
m/z 357.15 [M+H]+. C20H21FN2O3 (356.15).
2-Chloro-6-(4-methylpiperazine-1-carbonyl)xanthen-9-one
(8d) (0.17 g, 0.5 mmol) was reacted with methyl magnesium bro-
mide using (0.17 g, 1.10 mmol) the same procedure described for
4a to afford title compound (0.10 g, 57.0%) as an off white solid:
Rf = 0.65 (95:5 CH2Cl2/MeOH), 1H NMR (600 MHz, acetone-d6) d:
1.64 (s, 3H), 2.36 (s, 3H), 2.24 (br s, 4H), 2.82 (s, 1H), 3.60 (br
s, 4H), 7.10–7.22 (m, 4H), 7.48 (dd, 1H, J = 12.6, 4.8 Hz), 7.82
(d, 1H, J = 9.0 Hz); APCI-MS: m/z 373.14 [M+H]+. C20H21N2O3Cl
(372.12).
5.1.31. 2-Fluoro-6-(4-methylpiperazine-1-carbonyl)-9-
hydroxy-vinyl-9H-xanthene (9p)
2-Fluoro-6-(4-methylpiperazine-1-carbonyl)xanthen-9-one
(8e) (0.50 g, 1.5 mmol) was reacted with vinyl magnesium bro-
mide (0.42 g, 3.23 mmol) using the same procedure described for
4a to afford title compound (0.35 g, 65.0%) as an off white solid:
mp 147–149 °C; Rf = 0.48 (95:5 CH2Cl2/MeOH), 1H NMR
(600 MHz, acetone-d6) d 2.23 (s, 3H), 2.35 (br s, 4H), 3.43 (br s,
2H), 3.65 (br s, 2H), 5.13–5.16 (m, 2H), 5.51 (s, 1H), 6.11 (q, 1H,
J = 11.0 Hz), 7.14–7.22 (m, 4H), 7.34 (dd, 1H, J = 12.6, 3.0 Hz), 7.67
(d, 1H, J = 8.4 Hz); ESI-MS: m/z 369.13 [M+H]+. C21H21FN2O3
(358.15).
5.1.26. 2-Chloro-6-(4-methylpiperazine-1-carbonyl)-9-
hydroxy-9-vinyl-9H-xanthene (9k)
2-Chloro-6-(4-methylpiperazine-1-carbonyl)xanthen-9-one
(8d) (0.50 g, 1.40 mmol) was reacted with vinyl magnesium bro-
mide (0.41 g, 3.30 mmol) using the same procedure described for
4a to afford title compound (0.31 g, 57.0%) as an off white solid:
mp 143–145 °C; Rf = 0.44 (95:5 CH2Cl2/MeOH), 1H NMR
(600 MHz, acetone-d6) d 2.23 (s, 3H), 2.35 (br s, 4H), 2.82 (s, 1H),
3.43 (br s, 2H), 3.65 (br s, 2H), 5.15 (d, 2H, J = 16.8 Hz), 6.08–6.13
(m, 1H), 7.14–7.17 (m, 4H), 7.34 (dd, 1H, J = 12.6, 3.6 Hz), 7.68 (d,
1H, J = 8.4 Hz); APCI-MS: m/z 385.15 [M + H]+. C21H21N2O3Cl
(384.12).
5.1.32. 2-Fluoro-6-(4-methylpiperazine-1-carbonyl)-9-ethyl-9-
hydroxy-9H-xanthene (9q)
2-Fluoro-6-(4-methylpiperazine-1-carbonyl)-9H-xanthen-9-
one (8e) (0.18 g, 0.53 mmol) was reacted with ethyl magnesium
bromide (0.16 g, 1.2 mmol) using the same procedure described
for 4a to afford title compound (0.12 g, 63.0%) as an off white solid:
mp 139–42 °C; Rf = 0.51 (95:5 CH2Cl2/MeOH), 1H NMR (600 MHz,
acetone-d6) d 0.51 (t, 3H, J = 7.8 Hz), 1.96 (q, 2H, J = 7.4 Hz), 2.25
(s, 3H), 2.38 (br s, 4H), 2.83 (br s, 4H), 5.51 (s, 1H), 7.13 (s, 1H),
7.17 (d, 1H, J = 9.0 Hz), 7.24 (d, 1H, J = 7.8 Hz), 7.36 (dd, 1H,
J = 11.4, 2.4 Hz), 7.70 (d, 1H, J = 1.8 Hz), 7.77 (d, 1H, J = 7.8 Hz);
13C NMR (CDCl3, d ppm): 170.172, 159.324, 149.581, 149.436,
149.287, 137.922, 129.325, 129.209, 129.061, 128.897, 127.997,
126.951, 123.826, 121.393, 119.128, 116.442, 114.529, 112.149,
111.996, 70.181, 55.230, 43.281, 39.304, 14.285, 12.857; ESI-MS:
m/z 371.17 [M+H]+. C21H23N2O3F (371.17).
5.1.27. 2-Chloro-6-(4-methylpiperazine-1-carbonyl)-9-ethyl-9-
hydroxy-xanthene (9l)
2-Chloro-6-(4-methylpiperazine-1-carbonyl)xanthen-9-one
(8d) (0.40 g, 1.1 mmol) was reacted with ethyl magnesium bro-
mide (0.33 g, 2.5 mmol) using the same procedure described for
4a to afford title compound (0.26 g, 61.0%) as an off white solid:
mp 169–172 °C; Rf = 0.46 (95:5 CH2Cl2/MeOH), 1H NMR
(600 MHz, acetone-d6) d 0.51 (t, 3H, J = 7.8 Hz), 1.96 (q, 2H,
J = 7.5 Hz), 2.05 (br s, 1H), 2.25 (s, 3H), 2.40 (br s, 4H), 2.83 (br
s, 4H), 7.13 (s, 1H), 7.17 (d, 1H, J = 9.0 Hz), 7.24 (d, 1H,
J = 7.8 Hz), 7.36 (dd, 1H, J = 11, 2.1 Hz), 7.70 (d, 1H, J = 2.4 Hz),
7.77 (d, 1H, J = 7.8); APCI-MS: m/z 387.16 [M+H]+. C21H23N2O3Cl
(386.14).

R. Giri et al. / Bioorg. Med. Chem. 18 (2010) 1456–1463
1463
5.1.33. 2-Fluoro-6-(4-methylpiperazine-1-carbonyl)-9-
hydroxy-phenyl-9H-xanthene (9r)
drug were incubated at 37 °C for 30 min. Reactions were termi-
nated by adding EDTA to 25 mM and the DNA products were ana-
lyzed by electrophoresis through vertical 1.2% agarose gels at 2 V/
cm for 15 h in TAE buffer. Gels were stained with ethidium bro-
mide and photographed using an Eagle Eye II system (Stratagene).
The DNA intercalation assay was performed as described
previously.27
2-Fluoro-6-(4-methylpiperazine-1-carbonyl)xanthen-9-one
(8e) (0.28 g, 0.82 mmol) was reacted with phenyl magnesium bro-
mide (0.33 g, 1.81 mmol) using the same procedure described for
4a to afford title compound (0.36 g, 55.0%) as an off white solid:
mp 175–178 °C; Rf = 0.63 (95:5 CH2Cl2/MeOH), 1H NMR
(600 MHz, acetone-d6) d 2.21 (s, 3H), 2.32 (br s, 4H), 3.41 (br s,
2H), 3.63 (br s, 2H), 5.94 (s, 1H), 7.10–7.15 (m, 3H), 7.19–7.21
(m, 2H), 7.26–7.32 (m, 3H), 7.40 (d, 2H, J = 7.8 Hz), 7.47(d, 1H,
J = 7.8 Hz); APCI-MS: m/z 419.11 [M+H]+. C25H23N2O3F (418.17).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.01.018.
5.1.34. 2-Fluoro-6-(4-methylpiperazine-1-carbonyl)-9-
hydroxy-(3-methoxyphenyl)-9H-xanthene (9s)
References and notes
2-Fluoro-6-(4-methylpiperazine-1-carbonyl)xanthen-9-one
(8e) (0.3 g, 0.88 mmol) was reacted with 3-methoxy phenyl mag-
nesium bromide (0.41 g, 1.94 mmol) using the same procedure de-
scribed for 4a to afford title compound (0.21 g, 53.0%) as an off
white solid: mp 108–111 °C; Rf = 0.52 (95:5 CH2Cl2/MeOH), 1H
NMR (600 MHz, acetone-d6) d 2.24 (s, 3H), 2.36 (br s, 4H), 3.44
(br s, 2H), 3.64 (br s, 2H), 3.76 (s, 3H), 5.83 (s, 1H), 6.77 (dd, 1H,
J = 10.2, 2.4 Hz), 6.82 (d, 1H, J = 7.8 Hz), 7.10–7.14 (m, 4H), 7.18–
7.20 (m, 2H), 7.25–7.28 (m, 1H), 7.49 (d, 1H, J = 7.8 Hz); APCI-
MS: m/z 450.10 [M+H]+. C26H25N2O4F (448.49).
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5.2. Biology
The in vitro cytotoxicity of compounds was assayed by deter-
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In brief, 104 cells/well were plated in a 96-well plate. Twenty-four
hours after plating, a series of compounds in varied concentrations
with 1% (v/v) DMSO in the final cell medium were used for the cell
treatment (cells treated with medium containing 1% DMSO served
as a control). After a 48 h treatment, the relative viable cell mass in
each well was determined by using CellTiter-Blue Cell Viability As-
say kit (Promega, Madison, WI), which is based on the ability of
viable cells to reduce resazurin to the fluorescent end product res-
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5.3. Topoisomerase activity
Standard relaxation reaction mixtures (20
50 mM Tris–HCl (pH 8.0), 10 mM MgCl2, 200 mM potassium gluta-
mate, 10 mM dithiothreitol, 50 g/mL bovine serum albumin,
1 mM ATP, 0.3 g of pBR322 plasmid DNA, two units of human
topoisomerase II (Topogen), and the indicated concentrations of
ll) containing
l
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