Synthesis and antitumor activity of some new xanthotoxin derivatives

Article abstract of DOI:10.1016/j.ejmech.2009.01.006

The condensation of 4-amino-9-methoxy psoralene (4-aminoxanthotoxin) with some aromatic aldehydes led to the formation of 4-arylimine xanthotoxin derivatives 2a-h, which were cyclized with mercaptoacetic acid to afford the thiazolidinone derivatives 3a-h.

Full text of DOI:10.1016/j.ejmech.2009.01.006

European Journal of Medicinal Chemistry 44 (2009) 2967–2974
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antitumor activity of some new xanthotoxin derivatives
,
b
a
Omaima M. Abdel Hafez a , Kamellia M. Amin , Nehad A. Abdel-Latif ,
*
Tahia K. Mohamed a, Eman Y. Ahmed a, Timothy Maher c
a Chemistry of Natural Product Department, National Research Center, El Tahrir Street, Dokki, Cairo, Egypt
b Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Egypt
c Faculty of Pharmacy and Health Science, Boston, MA, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The condensation of 4-amino-9-methoxy psoralene (4-aminoxanthotoxin) with some aromatic alde-
hydes led to the formation of 4-arylimine xanthotoxin derivatives 2a–h, which were cyclized with
mercaptoacetic acid to afford the thiazolidinone derivatives 3a–h. On the other hand, the reaction of
aminoxanthotoxin 1 with some anhydrides afforded 4-imidione derivatives 3a–d. When 1 reacted with
some isothiocyanates, the thiourea derivatives 5a–c were obtained but the thiourea derivative 6 was
obtained when 1 reacted with ammonium thiocyanate. The thiourea derivative 6 was cyclized by the
reaction with monochloroacetic acid in the presence of sodium acetate to give aminothiazolidinone
derivative 7, but when the same reaction is carried out in the presence of pyridine, the thio-
xoimidazolidinone 8 was formed. The condensation of xanthotoxin sulphonamide with aromatic alde-
hydes gave the aryliminosulphonyl derivatives 9a–e. Xanthotoxin sulphonyl hydrazine condensed with
some anhydride afforded sulphonic acid imide derivatives 10a–c. The antitumor and cytotoxic activities
of 9 synthesized derivatives were tested, five compounds were found to be active, they inhibited the
growth of HeLa cells.
Received 16 October 2008
Received in revised form
30 December 2008
Accepted 9 January 2009
Available online 19 January 2009
Keywords:
Xanthotoxin
Schiff’s bases
Thiazolidinones
Anhydrides
Antitumor
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
T-Lymphoblastic Leukemia cell line (CEM) [19] and selective cyto-
toxic agent for leukemia HL-60 (TB) cell line has been reported [20].
Xanthotoxin is a natural compound with antileukodermal
activity and antitumor properties [1–6]. On the other hand, many
publications reported that many Schiff bases with azomethine
linkage show interesting inhibitory activity against experimental
tumor systems [7–10], they could be hydrolyzed selectively by the
tumor cells to serve as alkylating agent at the same time as the
active amine is freed to act as antimetabolite [11]. Indeed the ability
of many candidates containing the 4-thiazolidinone being such
exhibit antiproliferative activity has been documented in numerous
publications and reviews [12–14]; some of thiazolidinones exhibit
All the previous findings generated our interest for preparing new
derivatives by incorporating the xanthotoxin with the above-
mentioned groups with antitumor activity and study the activity of
the new obtained derivatives, with the hope of optimizing the
antitumor and cytotoxic activity of the xanthotoxin.
2. Results and discussion
2.1. Chemistry
the increase of the interaction between Hif-1
a and P300 and
Condensation of 4-aminoxanthotoxin 1 with some aromatic
aldehydes namely, benzaldehyde, p-chlorobenzaldehyde, p-methox-
ybenzaldehyde, p-bromobenzaldehyde, p-nitrobenzaldehyde, furfural,
2-thiophene aldehyde and p-N,N-dimethyl aminobenzaldehyde in
chloroform in the presence of acetic acid yielded the corresponding
4-arylimine-9-methoxy psoralenes 2a–h (Scheme 1).
The evidence for the formation of the 4-arylimine derivatives
2a–h can be achieved by the microanalysis, 1H NMR and IR spectra
(Experimental part).
prevent VEGF gene production in tumor cells under hypoxia
conditions, the compounds are therefore useful for the control of
angiogenesis and tumor growth. In the literature, thiourea deriva-
tives have been described as new drugs with strong cancercidal
activity [15], they have been used for brain cancer treatment [16–
18]and a potent inhibitors of human DNA-topoisomerase II. The
significant effect of the anhydride moiety as cytotoxic agent for
A facile synthesis of thiazolidin-4-ones which involves cyclo-
condensation of the appropriate imine with thioglycolic acid in
refluxing benzene was reported [21,22]. Accordingly, reaction of the
* Corresponding author. Tel.: þ20 237608284.
E-mail address: ommryan@yahoo.com (O.M. Abdel Hafez).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.01.006

2968
O.M. Abdel Hafez et al. / European Journal of Medicinal Chemistry 44 (2009) 2967–2974
NH2
N
Ar
O
ArCHO
O
O
O
O
O
OCH3
(1)
OCH3
(2)
a, Ar=C6H5; b, Ar= C6H4-Cl-p
c, Ar= C6H4-OCH3-p; d, Ar= C6H4-Br-p
e, Ar= C6H4-NO2-p; f, Ar= 2-furyl
g, Ar= 2-thienyl; h, Ar= C6H4-N(CH3)2-p
S
O
Ar
HSCH2COOH
N
O
O
O
OCH3
(3)
a, Ar=C6H5; b, Ar= C6H4-OCH3-p
c, Ar= C6H4-Br-p; d, Ar= 2-furyl
Scheme 1.
Schiff bases 2a, c, d, f with mercaptoacetic acid in dry benzene
afforded the target thiazolidin-4-one derivatives 3a–d in 69–81%
yields (Scheme 1).
The imides were characterized by elemental analysis, in addi-
tion to the IR spectrum (KBr, cmꢀ1) of compound (10a) (Experi-
mental part).
The structures of these compounds were confirmed with
concordant microanalytical and spectral data (Experimental part).
Amr et al. [23] reported the preparation of the imide and
bis-diimide by the reaction of the aminoxanthotoxin derivative
with the appropriate anhydride and dianhydride in acetic acid
(Scheme 2). The structures assigned to the products 4a–d were
substantiated by their microanalysis, 1H NMR, IR and mass spectra
(Experimental part).
In this work, the target thioureas 5a–c were prepared by reac-
tion of the key 4-aminoxanthotoxin with phenyl, ethyl and benzyl
isothiocyanate in dry toluene under reflux (Scheme 3).
The proposed structures of the products 5a–c were confirmed
by elemental analysis, IR and mass spectra (Experimental part).
In this investigation, the target thiourea derivative 6 was
synthesized by refluxing of 4-aminoxanthotoxin and ammonium
thiocyanate in ethanol and diluted hydrochloric acid [24]. Its
structure was confirmed by elemental analysis and mass spectrum
(Experimental part).
2.2. In vitro antitumor activity
Five compounds (Xanthotoxin, 2g, 3c, 5b and 5c) were active,
inhibiting the growth of HeLa cells. In contrast, none of the
compounds showed activity inhibiting the growth of (MCF-7) cells.
IC50 values were calculated using an inhibitory model, with the sum
of squares of the residuals minimized by Excel solver software. The
values obtained for the five compounds against the HeLa cell line
are shown in Table 1.
The viability data used to calculate the IC50 values is presented
in Table 2 and the relationship between percent viability and the
compound concentration is illustrated in Figs. 1–5.
Assay was run on two plates. The ratio of viability in wells
containing 2.5% DMSO compared to wells containing media and
cells but no DMSO in plate 1, in which compounds Xanthotoxin, 2g
and 3c were tested, was 0.77 (control þ DMSO/control ꢀ DMSO,
%RSD 47, 32 respectively) and in plate 2 (compounds 5b and 5c) the
ratio was 0.61 (34, 9).
The results showed that the Schiff base (2g) was the most
effective derivative of all the newly tested compounds, showing
IC50 ¼ 7.2 and percent viability 70 that are closely related to that of
xanthotoxin, 7.6 and 62 respectively.
In this work, the thiazolidinone derivatives 7 were prepared by
the fusion of xanthotoxin thiourea 6 with chloroacetic acid in the
presence of anhydrous sodium acetate [24]. The product was
characterized by its physical, analytical and spectral data. When the
foregoing reaction was applied using chloroacetic acid and the
thiourea derivative 6 in the presence of pyridine, the target
compound 8 was obtained in 60% yield.
The structural assignment for compound 8 was deduced by
elemental and IR spectral analyses (Experimental part).
As previously discussed the Schiff bases 9a–e were prepared by
refluxing 9-methoxy-chromen-7-one-4-sulphonamide with the
appropriate aldehyde in acetic acid to give the target compounds in
good yield after the evaporation of the solvent (Scheme 4).
The structures of the new Schiff bases 9a–e were assigned on
the basis of analytical and spectral data (Experimental part).
As previously described the 4-cyclic imide dione derivatives
10a–c were prepared by reaction of the xanthotoxin sulphonyl
hydrazide with the appropriate anhydride in acetic acid to give the
target compounds in good yield (Scheme 5).
S
N
O
O
O
OMe
Xanthotoxin
O
O
O
OMe
2g

O.M. Abdel Hafez et al. / European Journal of Medicinal Chemistry 44 (2009) 2967–2974
2969
O
H
N
R
S
N
O
H
O
O
O
O
N
RNCS
O
O
O
O
O
OCH3
(4a)
OCH3
(5)
O
N
a) R = C6H5; b, R = C2H5
c) R = C6H5-CH2
O
NH2
NH2
N
O
O
O
O
N
O
NH4SCN
O
O
O
OCH3
(1)
O
O
O
O
O
O
O
O
OCH3
(4b)
OCH3
(1)
NH2
O
O
H
N
S
O
O
O
O
O
O
N
O
O
O
O
H3CO
N
OCH3
O
O
O
OCH3
(6)
O
O
O
(4d)
Chloroacetic acid
Pyridine
Chloroacetic acid
anh. Sod. acetate
H
N
H
N
O
O
O
N
O
O
N
S
N
O
S
O
OCH3
(4c)
O
O
O
O
O
O
Scheme 2.
OCH3
(8)
OCH3
(7)
In order to assess the activity of xanthotoxin bearing a hetero-
cyclic ring at position 4, 4-thiazolidonyl xanthotoxin (3c) was
tested and the result revealed that it was moderately active with an
IC50 40 and percent viability 82 at a concentration of 5 mcg/mL.
Scheme 3.
recorded on Nexus 670 FT-IR FT-Raman spectrometer as potassium
bromide discs, at the National Research Centre. The proton nuclear
resonance (1H NMR) spectra were determined on Varian mercury
300 MHz spectrometer, using tetramethylsilane (TMS) as the
internal standard, at Cairo University. The mass spectra were per-
formed on JEOL JMS-AX500 mass spectrometer at the National
Research Centre. The reactions were followed by TLC (Silica gel,
aluminum sheets 60 F254, Merck) using benzene:ethyl acetate (8:2
v/v) as eluent and sprayed with iodine–potassium iodide reagent.
S
C6H4Br
N
O
O
O
O
OMe
3c
3.1.1. Preparation of 4-arylimine-9-methoxy[2,3-g]chromen-7-ones
(Schiff’s bases) (2a–h)
3.1.1.1. General procedure. A mixture of 4-aminoxanthotoxin
(2.31 g, 0.01 mol) and the appropriate aldehydes (0.012 mol),
namely, benzaldehyde, 4-chlorobenzaldehyde, anisaldehyde,
4-bromobenzaldehyde, 4-nitrobenzaldehyde, furfural, 2-thiophene
In case of xanthotoxin thiourea derivatives, the benzyl deriva-
tive (5c), of IC50 ¼ 7.5, elicited higher cytotoxicity than the ethyl
derivative (5b), of IC50 ¼ 23, the two compounds showed % viability
56 and 70 at a concentration of 10 mcg/mL.
The survival curves obtained after treating the selected tumor
cell line with the tested compounds are illustrated in Figs. 1–5.
SO2NH2
SO2N=CHAr
3. Experimental
ArCHO
3.1. Synthesis
O
O
O
O
O
O
OCH3
OCH3
Melting points were determined on Electrothermal IA 9000
apparatus and were uncorrected. Elemental microanalysis were
performed on Elementar, Vario EL, at the Micro Analytical Center,
Faculty of Science, Cairo University. The infrared (IR) spectra were
(9a-e)
Scheme 4.

2970
O.M. Abdel Hafez et al. / European Journal of Medicinal Chemistry 44 (2009) 2967–2974
Table 2
O
N
O
Percent viability against dose concentrations.
H
N
H
N
O
O
O
N
Dose (mcg/mL)
Compound
S
S
N
O
Xanthotoxin
2g
3c
5b
5c
O
O
5
62 (17)a
51 (33)
21 (18)
16 (5)
70 (24)
53 (10)
32 (21)
28 (21)
82 (27)
91 (18)
26 (2)
101 (36)
70 (31)
35 (9)
74 (11)
56 (11)
34 (11)
35 (14)
10
50
100
O
O
O
O
N
O
O
OCH3
(10a)
OCH3
O
(10b)
26 (12)
41 (27)
a
Values given are percent viability with %RSD of three wells/concentration in
O
O
O
parentheses.
O
ppm) 3.9 (3H, s, OCH3 xanthotoxin), 4.12 (3H, s, OCH3 aromatic), 6.5
(1H, d, H-6), 6.7 (1H, d, H-3), 7.2–7.8 (6H, m, 4 Ar–H, H-2, H-5), 8.4
(1H, s, N]CH).
O
O
O
O
O
SO2NHNH2
O
O
3.1.1.2.4. 4-(4-Bromobenzylideamino)-9-methoxy-7H-furo[3,2-
g]chromen-7-one (2d) [26] (C19H12BrNO4, 398.214, 60.54, 280–
285). Anal. Calcd.: C, 57.30; H, 3.04; N, 3.52. Found: C, 57.27; H,
O
O
O
O
O
2.99; N, 3.48 IR (cmꢀ1, KBr): 1739 (C]O
a
-pyrone), 1685 (C]N),
OCH3
1230 (C–O), 1290 (C–N) 616 (C–Br). 1H NMR spectrum (DMSO-d6,
d
,
O
O
ppm) 3.7 (3H, s, OCH3), 6.5 (1H, d, H-6), 6.7 (1H, d, H-3), 7.5–7.9 (6H,
m, 4 Ar–H, H-2, H-5), 8.4 (1H, s, N]CH).
O
N
O
1
2
3
5
O
S
O
3.1.1.2.5. 4-(4-Nitrobenzylidenamino)-9-methoxy-7H-furo[3,2-
g]chromen-7-one (2e) [26] (C19H12N2O6, 364.22, 77, 312–315). Anal.
Calcd.: C, 62.65; H, 3.32; N, 7.69. Found: C, 62.61; H, 3.28; N, 7.66. IR
4
OCH3
H3CO
HN
N
NH
S
O
O
6
O
O
O
O
(cmꢀ1, KBr): 1742 (C]O
a
-pyrone), 1685 (C]N), 1240 (C–O), 1290
(C–N),1522 (N]O). 1H NMR spectrum (DMSO-d6,
d
, ppm) 3.7 (3H, s,
(10c)
OCH3), 6.5 (1H, d, H-6), 6.7 (1H, d, H-3), 7.4 (1H, d, H-5), 7.5–8.2 (5H,
m, 4 Ar–H, H-2), 8.4 (1H, s, N]CH).
Scheme 5.
3.1.1.2.6. 4-(Furan-2-yl-methyleneamino)-9-methoxy-7H-
furo[3,2-g]chromen-7-one (2f) (C17H11NO5, 309.268, 76.7, 134–
135). Anal. Calcd.: C, 66.02; H, 3.58; N, 4.53. Found: C, 66.01; H,
aldehyde or 4-dimethyl aminobenzaldehyde in 20 mL of chloro-
form and 2 mL acetic acid was refluxed for 10–15 h. The solid
obtained by evaporation of the solvent was crystallized from
chloroform/methanol to afford the Schiff bases.
3.51; N, 4.50. IR (cmꢀ1, KBr): 1748 (C]O
a
-pyrone), 1690 (C]N),
1240 (C–O), 1293 (C–N). 1H NMR spectrum (DMSO-d6,
d
, ppm) 4.12
(3H, s, OCH3), 6.4 (1H, d, H-6), 6.6 (1H, d, H-3), 7.5–7.8 (6H, m, 4 Ar–
H, H-2, H-5), 8.6 (1H, s, N]CH).
3.1.1.2. Microanalytical and spectral data of compounds
10a–c (Mol. For., M. wt., yield % and m.p. ꢁC)
3.1.1.2.7. 4-(Thiophen-2-yl-methyleneamino)-9-methoxy-7H-
furo[3,2-g]chromen-7-one (2g) (C17H11NO4S, 325.334, 61.5, 222–
223). Anal. Calcd.: C, 62.76; H, 3.41; N, 4.31. Found: C, 62.71; H,
3.1.1.2.1. 4-(Benzylideneamino)-9-methoxy-7H-furo[3,2-g]chromen-
7-one (2a) [25] (C19H13NO4, 319.298, 79, 198–200). Anal. Calcd.: C,
71.47; H, 4.02; N, 4.39. Found: C, 71.41; H, 4.05; N, 4.30. IR (cmꢀ1, KBr):
3.37; N, 4.29. IR (cmꢀ1, KBr): 1749 (C]O
a
-pyrone), 1709 (C]N),
1749 (C]O
spectrum (DMSO-d6,
(1H, d, H-3), 7.2–7.7 (7H, m, 5 Ar–H, H-2, H-5), 8.5 (1H, s, N]CH).
3.1.1.2.2. 4-(4-Clorobenzylideamino)- 9-methoxy-7H-furo[3,2-
a
-pyrone), 1680 (C]N), 1230 (C–O), 1290 (C–N), 1H NMR
1245 (C–O), 1284 (C–N). 1H NMR spectrum (DMSO-d6,
d, ppm) 3.7
d, ppm) 3.7 (3H, s, OCH3), 6.5 (1H, d, H-6), 6.7
(3H, s, OCH3), 6.2 (1H, d, H-6), 6.7 (1H, d, H-3), 7–7.2 (3H, m,
thiophene protons), 7.4 (1H, d, H-5), 7.5 (1H, d, H-2), 7.6 (1H, s,
N]CH).
g]chromen-7-one (2b) [26] (C19H12ClNO4, 353.755, 62.1, 210–
212). Anal. Calcd.: C, 64.51; H, 3.42; N, 3.96. Found: C, 64.45; H,
3.1.1.2.8. 4-(4-(dimethylamino)benzylideneamino)-9-methoxy-
7H-furo[3,2-g]chromen-7-one (2h) (C21H18N2O4, 362.37, 72,184–
185). Anal. Calcd.: C, 69.60; H, 5.01; N, 7.73. Found: C, 69.57; H,
3.36; N, 3.91. IR (cmꢀ1, KBr): 1749 (C]O
a
-pyrone), 1699 (C]N),
1235 (C–O), 1295 (C–N), 760 (C–Cl). 1H NMR spectrum (DMSO-d6,
d
,
4.94; N, 7.71. IR (cmꢀ1, KBr): 1740 (C]O
a-pyrone), 1695 (C]N),
ppm) 4.12 (3H, s, OCH3), 6.3 (1H, d, H-6), 7.12 (1H, d, H-3), 7.2–7.8
(6H, m, 4 Ar–H, H-2, H-5), 8.7 (1H, s, N]CH).
3.1.1.2.3. 4-(4-Methoxybenzylideneamino)-9-methoxy-7H-
furo[3,2-g]chromen-7-one (2c) [27] (C20H15NO5, 349.328, 65.3, 125–
127). Anal. Calcd.: C, 68.76; H, 4.33; N, 4.00. Found: C, 68.71; H,
70
Xanthotoxin
mcg/mL
100
50
% viability
60
50
40
30
20
10
0
16
21
51
62
4.29; N, 3.96 IR (cmꢀ1, KBr): 1743 (C]O
a-pyrone), 1695 (C]N),
1229 (C–O), 1295 (C–N), 760 (C–Cl). 1H NMR spectrum (DMSO-d6,
d,
10
5
Table 1
IC50 of some newly synthesized compounds.
Compound no.
IC50 (mcg/mL)
O
O
O
Xanthotoxin
7.6
7.2
40
23
7.5
OMe
2g
3c
5b
5c
0
50
100
Concentration (mcg/mL)
150
Xanthotoxin
Fig. 1. Survival curve of Xanthotoxin.

O.M. Abdel Hafez et al. / European Journal of Medicinal Chemistry 44 (2009) 2967–2974
2971
80
70
60
50
40
30
20
10
0
120
Compound 2g
mcg/mL % viability
Compound 5b
mcg/mL
100
50
% viability
100
50
10
5
28
32
53
70
100
80
60
40
20
0
41
35
70
10
5
101
S
N
S
HN
NHCH2CH3
O
O
O
O
O
O
0
50
100
Concentration (mcg/mL)
150
OCH3
5b
OCH3
2g
0
20
40
Concentration (mcg/mL)
60
80
100
120
Fig. 4. Survival curve of 5b.
Fig. 2. Survival curve of 2g.
1235 (C–O), 1298 (C–N). 1H NMR spectrum (DMSO-d6,
d
, ppm) 2.48
NMR spectrum (CDCl3, d, ppm) 3.3, 3.4 (2H, dd, CH2 thiazolidinone),
3.73 (3H, s, OCH3 xanthotoxin), 4.1 (3H, s, OCH3 aromatic), 5.9 (1H,
s, CH thiazolidinone), 6.12 (1H, d, H-6), 6.7 (1H, d, H-3), 7.1–7.5 (6H,
m, 4 Ar–H, H-2, H-5). MS: 421 (8%), 231 (100%), 218 (95%), 188
(75%), 120 (70%).
(6H, s, 2CH3), 4.1 (3H, s, OCH3), 6.1 (1H, d, H-6), 6.7 (1H, d, H-3), 7.3
(1H, d, H-5), 7.5 (1H, d, H-2), 8.2–8.4 (4H, 2dd, Ar–H), 8.5 (1H, s,
N]CH).
3.1.2.2.3. 2-(4-Bromophenyl)-3-(9-methoxy-7-oxo-7H-furo[3,2-
g]chromen-4-yl)-thiazolidin-4-one (3c) (C21H14BrNO5, 472.31, 71.7,
217–220). Anal. Calcd.: C, 53.40; H, 2.99; N, 2.96; S, 6.79. Found: C,
53.38; H, 2.96; N, 2.92; S, 6.75. IR (cmꢀ1, KBr): 1749 (C]O
3.1.2. Preparation of (7-oxo-furo[3,2-g]chromen-4-yl)-2-aryl-
thiazolidin-4-ones (3a–d)
3.1.2.1. General procedure. To a well-stirred suspension of the
appropriate arylidene (2a, c, d, f) (0.001 mol) in dry benzene
(15 mL), mercaptoacetic acid (0.42 mL, 0.006 mol) in dry benzene
(5 mL) was added. The reaction mixture was refluxed for 3 h,
cooled; the deposited solid was filtered off and crystallized from
ethanol to give the title compounds (Table 2).
a
-pyrone), 1775 (C]O thiazolidinone), 1225 (C–O), 1295 (C–N), 616
(C–Br). 1H NMR spectrum (CDCl3,
d, ppm) 3.3, 3.4 (2H, dd, CH2
thiazolidinone), 3.7 (3H, s, OCH3), 5.9 (1H, s, CH thiazolidinone),
6.12 (1H, d, H-6), 6.6 (1H, d, H-3), 6.9–7.5 (6H, m, 4 Ar–H, H-2, H-5).
Mþ/Mþ2 472/474 (0.56%, 0.50%), 396/398 (8.53%, 7.29%), 230
(54.76%), 215 (64.03%), 63 (100%).
3.1.2.2. Microanalytical and spectral data of compounds 10a–c (Mol.
For., M. wt., yield % and m.p. ꢁC)
3.1.2.2.4. 2-Furan-2-yl-3-(9-methoxy-7-oxo-7H-furo[3,2-g]chro-
men-4-yl)-thiazolidin-4-one (3d) (C19H13NO6S, 383.364, 80.7, 192–
195). Anal. Calcd.: C, 59.52; H, 3.42; N, 3.65; S, 8.36. Found: C,
59.51; H, 3.39; N, 3.61; S, 8.35. IR (cmꢀ1, KBr): 1742 (C]O
3.1.2.2.1. 3-(9-Methoxy-7-oxo-7H-furo[3,2-g]chromen-4-yl)-2-
phenyl-thiazolidin-4-one (3a) (C21H15NO5S, 393.404, 69, 223–
225). Anal. Calcd.: C, 64.11; H, 3.84; N, 3.56; S, 8.15. Found: C, 64.04;
-pyrone), 1772 (C]O thiazolidinone), 1225 (C–O), 1290 (C–N). 1H
H, 3.79; N, 3.51; S, 8.09. IR (cmꢀ1, KBr): 1740 (C]O
a
-pyrone), 1775
(C]O thiazolidinone), 1223 (C–O), 1294 (C–N). 1H NMR spectrum
(CDCl3, , ppm) 3.3, 3.4 (2H, dd, CH2 thiazolidinone), 3.73 (3H, s,
a
NMR spectrum (CDCl3, d, ppm) 3.3, 3.4 (2H, dd, CH2 thiazolidinone),
3.7 (3H, s, OCH3), 5.9 (1H, s, CH thiazolidinone), 6.12 (1H, d, H-6),
6.2–6.5 (3H, m, furan protons), 6.6 (1H, d, H-3), 7.3–7.5 (2H, d, H-2,
H-5). Mþ 383 (10%), 349 (30%), 215 (95%), 158 (100%), 143 (80%).
d
OCH3), 5.7 (1H, s, CH thiazolidinone), 6.2 (1H, d, H-6), 6.7 (1H, d, H-
3), 7–7.5 (7H, m, 4 Ar–H, H-2, H-5). MS: Mþ 393 (2%), 295 (10%), 230
(19%), 215 (28%), 393 (2%), 105 (100%), 77 (50%).
3.1.3. Preparation of 4-cyclic imide dione chromene-7-one
derivatives (4a–d)
To a solution of aminoxanthotoxin (1) (2.31 g, 0.01 mol) in acetic
acid, the appropriate anhydrides (0.01 mol) namely phthalic
anhydride, pyridine anhydride, 1,8-napthaleneanhydride or
benzene dianhydride were added. The reaction mixture was
3.1.2.2.2. 3-(9-Methoxy-7-oxo-7H-furo[3,2-g]chromen-4-yl)-2-
(4-methoxyphenyl) thiazolidin-4-one (3b) (C22H17NO6S, 423.434,
76.1, 238–240). Anal Calcd.: C, 62.40; H, 4.05; N, 3.31; S, 7.57. Found:
C, 62.37; H, 4.01; N, 3.30; S, 7.53. IR (cmꢀ1, KBr): 1740 (C]O
a-
pyrone), 1770 (C]O thiazolidinone), 1220 (C–O), 1295 (C–N). 1H
100
Compound 3c
90
80
70
60
50
40
30
20
10
0
mcg/mL
100
50
% viability
Compound 5c
26
26
91
82
80
mcg/mL
100
50
% viability
70
60
50
40
30
20
10
0
35
34
56
74
10
5
10
5
S
S
O
C6H5Br
N
NHCH2Ph
HN
O
O
O
20
40
60
Concentration (mcg/mL)
100 120
0
80
O
O
OCH3
O
0
50
Concentration (mcg/mL)
100
150
OCH3
Fig. 3. Survival curve of 3c.
Fig. 5. Survival curve of 5c.

2972
O.M. Abdel Hafez et al. / European Journal of Medicinal Chemistry 44 (2009) 2967–2974
refluxed for 5–10 h; the formed precipitate by cooling was filtered
off and crystallized from glacial acetic acid to give the corre-
sponding cyclic imide dione derivatives.
Calcd.: C, 63.14; H, 4.24; N, 7.36; S, 8.43. Found: C, 63.11; H, 4.22; N,
7.33; S, 8.41. IR (cmꢀ1, KBr): 1747 (C]O
-pyrone), 3470 (NH, br s),
1260 (C]S), 1285 (C–N). MS: 381 (5%), 252 (25%), 230 (72%), 216
a
(100%), 188 (27%).
3.1.3.1. Microanalytical and spectral data of compounds 10a–c (Mol.
For., M. wt., yield % and m.p. ꢁC)
3.1.5. Preparation of 9-methoxy-7-oxo-7H-furo[3,2-g]chromen-4-
yl thiourea (6)
3.1.3.1.1. 6-(9-Methoxy-7-oxo-7H-furo[3,2-g]chromen-4-yl)-iso-
indole-1,3-dione (4a) (C20H11NO6, 361.301, 88.4, 227–230). Anal.
Calcd.: C, 66.48; H, 3.07; N, 3.88. Found: C, 66.42; H, 3.11; N, 3.86.
To a solution of 4-aminoxanthotoxin (1) (1 g, 0.0043 mol) in
ethanol (20 mL) and dilute hydrochloric acid (20 mL), ammonium
thiocyanate (0.5 g, 0.005 mol) was added. The mixture was refluxed
for 10 h. The solution was cooled. The precipitated solid was filtered
off, washed with water and crystallized from DMF/water, yield 78%,
m.p. above 300 ꢁC.
Analysis for C13H10N2O4S (M. wt. 290.31): Calculated %: C, 53.78;
H, 3.47; N, 9.65; S, 11.05. Found %: C, 53.74; H, 3.5; N, 9.67; S, 11.10.
Mass spectrum of 6: Mþ 290 (3.99%), 259 (2.8%), 231 (64.0%), 215
(100%), 187 (22.8%), 158 (23%), 130 (3%), 102 (6%) and 77 (13%).
IR (cmꢀ1, KBr): 1740 (C]O
a
-pyrone), 1750 (C]O imide), 1225
(C–O), 1290 (C–N). 1H NMR spectrum (DMSO-d6,
d
, ppm) 4.0 (3H, s,
OCH3), 6.12 (1H, d, H-6), 6.6 (1H, d, H-3), 7.3 (1H, d, H-5), 7.5 (1H, d,
H-2), 8–8.2 (4H, m, 4 Ar–H). MS: Mþ 361 (5%), 330 (71%), 215
(100%).
3.1.3.1.2. 2-(9-Methoxy-7-oxo-7H-furo[3,2-g]chromen-4-yl)-pyr-
rolo[3,4-b]pyridin-5,7-dione (4b) (C19H10N2O6, 362.29, 94.34, 240–
243). Anal. Calcd.: C, 62.98; H, 2.78; N, 7.73. Found: C, 62.92; H,
2.72; N, 7.71. IR (cmꢀ1, KBr): 1735 (C]O
a
-pyrone), 1753 (C]O
imide), 1228 (C–O), 1298 (C–N). 1H NMR spectrum (DMSO-d6,
d
,
3.1.6. Preparation of 2-(9-methoxy-7-oxo-7H-furo[3,2-g]-
chromen-4-yl-amino)-thiazolidine-4-one (7)
ppm) 3.9 (3H, s, OCH3), 6.12 (1H, d, H-6), 6.4 (1H, d, H-3), 7.2 (1H, d,
H-5), 7.4 (1H, d, H-2), 8–9 (3H, m, pyridine protons). MS: Mþ 361
(2%), 272 (100%), 229 (90%), 216 (98%), 173 (50%), 120 (62%).
3.1.3.1.3. 2-(9-Methoxy-7-oxo-7H-furo[3,2-g]chromen-4-yl)-ben-
zo[de]isoqunolin-1,3-dione (4c) (C24H13NO6, 411.35, 83.09, 23-
2). Anal. Calcd.: C, 70.07; H, 3.18; N, 3.40. Found: C, 70.05; H, 3.16;
A mixture of thiourea derivative (6) (1 g, 3.4 mol), anhydrous
sodium acetate (0.5 g) and monochloroacetic acid (0.4 g, 4 mol)
was fused for 6 h and filtered off to give a solid product, which was
crystallized from DMF/H2O, yield 65%, m.p. above 300 ꢁC.
Analysis for C15H10N2O5S (M. wt. 330.3): Calculated %: C, 54.87;
H, 3.07; N, 8.53; S, 9.77. Found %: C, 54.89; H, 3.10; N, 8.60; S, 9.71.
Mass spectrum: Mþ 330 (5.46%), 245 (0.9%), 231 (44.6%), 216
(72.7%), 215 (100%), 187 (16%). IR (KBr, cmꢀ1): 3451 (NH), 3123
N, 3.39. IR (cmꢀ1, KBr): 1743 (C]O
a
-pyrone), 1752 (C]O imide),
, ppm) 3.9
1222 (C–O), 1295 (C–N). 1H NMR spectrum (DMSO-d6,
d
(3H, s, OCH3), 6.12 (1H, d, H-6), 6.7 (1H, d, H-3), 7.2 (1H, d, H-5), 7.4–
8 (7H, m, 6 Ar–H, H-2). MS: Mþ 411 (84.6%), 410 (50.9%), 213
(28.7%), 212 (34.7%), 180 (85.8%), 179 (100%), 170 (30.2%), 152
(36.2%).
(C–H, stretching), 1777 (C]O, thiazolidine), 1759 (C]O,
1667 (C]N).
a-pyrone),
3.1.3.1.4. 2,6-Bis-(9-methoxy-7-oxo-7H-furo[3,2-g]chromen-4-
3.1.7. Preparation of 3-(9-methoxy-7-oxo-7H-furo[3,2-g]-
chromen-4-yl)-2-thioxo-imidazolidine-4-one (8)
yl)-pyrrolo[3,4-f]-isoindol-1,3,5,7-tetraone
644.49, 84.3, 300). Anal. Calcd.: C, 63.36; H, 2.50; N, 4.35. Found: C,
63.33; H, 2.49; N, 4.31. IR (cmꢀ1, KBr): 1742 (C]O
-pyrone), 1752
(C]O imide), 1227 (C–O), 1298 (C–N). 1H NMR spectrum (DMSO-d6,
(4d)
(C34H16N2O12,
A mixture of thiourea derivative (6) (1 g, 3.4 mol), mono-
chloroacetic acid (0.4 g, 4 mol) and drops of pyridine was fused for
6 h, poured on crushed ice then filtered off to give a solid product
which was crystallized from DMF/water, yield 60%, m.p. above
300 ꢁC.
a
d
, ppm) 3.8 (3H, s, OCH3), 6.2 (1H, d, H-6), 6.7 (1H, d, H-3), 7.3 (1H, d,
H-5), 7.4 (1H, d, H-2), 8.9 (2H, s, 2 Ar–H). MS: Mþ 644 (10%), 215
(100%), 187 (58%).
Analysis for C15H10N2O5S (M. wt. 330.3): Calculated %: C, 54.87;
H, 3.07; N, 8.53; S, 9.77. Found %: C, 54.70; H, 3.15; N, 8.40; S, 9.72.
3.1.4. Preparation of 7-oxo-furochromen-4-yl thiourea derivatives
(5a–c)
IR (KBr, cmꢀ1): 3448 (NH), 3123 (C–H, stretching),1743 (C]O,
pyrone), 1769 (C]O, imidazolidine).
a-
To a solution of aminoxanthotoxin (1) (2.31 g, 0.01 mol) in
40 mL dry toluene, the appropriate isothiocyanates (0.015 mol)
namely phenyl, ethyl or benzyl isothiocyanate respectively were
added. The mixture was refluxed for 3 h, cooled, filtered and crys-
tallized from methanol to afford the title compounds.
3.1.8. Preparation of 4-aryliminosulphonyl-furochromen-7-one
derivatives (Schiff’s bases) (9a–e)
To a solution of xanthotoxin sulfonamide 8 (2.95 g, 0.01 mol)
in acetic acid, the appropriate aldehydes (0.12 mol) namely
benzaldehyde, 4-chlorobenzaldehyde, anisaldehyde, 4-bromo-
benzaldehyde or furfural respectively were added. The mixture
was refluxed for 6 h. The solid obtained by evaporation of the
solvent was crystallized from benzene to afford the Schiff bases
(9a–e).
3.1.4.1. Microanalytical and spectral data of compounds 10a–c (Mol.
For., M. wt., yield % and m.p. ꢁC)
3.1.4.1.1. 1-(9-Methoxy-7-oxo-7H-furo[3,2-g]chromen-4-yl)-3-
phenyl thiourea (5a) (C19H14N2O4S, 366.391, 35.5, 241–244). Anal.
Calcd.: C, 62.28; H, 3.85; N, 7.65; S, 8.75. Found: C, 62.22; H, 3.81;
N, 7.62; S, 8.70. IR (cmꢀ1, KBr): 1743 (C]O
a
-pyrone), 3478, 3384
3.1.8.1. Microanalytical and spectral data of compounds 10a–c (Mol.
For., M. wt., yield % and m.p. ꢁC)
(Two NH groups, br s), 1285 (C]S), 1290 (C–N). MS: Mþ 366
(7.78%), 277 (8.73%), 231 (62.49%), 216 (100%), 188 (43%), 231
(12%), 215 (64%).
3.1.8.1.1. 9-Methoxy-7-oxo-7H-furo[3,2-g]chromen-4-sulfonic
acid benzylidine-amide (9a) (C19H13NO6S, 383.36, 46.2, 227–
229). Anal. Calcd.: C, 59.52; H, 3.42; N, 3.65; S, 8.36. Found: C,
59.49; H, 3.39; N, 3.61; S, 8.32. IR (cmꢀ1, KBr): 1731 (C]O
3.1.4.1.2. 1-Ethyl-3-(9-methoxy-7-oxo-7H-furo[3,2-g]chromen-4-
yl)thiourea (5b) (C15H14N2O4S, 318.351, 72.2, 233–235). Anal. Calcd.:
C, 56.59; H, 4.43; N, 8.80; S, 10.07. Found: C, 56.56; H, 4.41; N, 8.79;
a
-pyrone), 1370, 1170 (SO2–N), 1669 (C]N). MS: Mþ 383 (1%), 312
S, 10.02. IR (cmꢀ1, KBr): 1745 (C]O
a
-pyrone), 3384 (NH, br s), 1260
(50%), 249 (49%), 252 (100%), 161 (75%), 146 (88%).
(C]S), 1295 (C–N). MS: Mþ 318 (15%), 231 (72%), 216 (100%),188
(30%).
3.1.8.1.2. 9-Methoxy-7-oxo-7H-furo[3,2-g]chromen-4-sulfonica-
cid-4-chlorobenzylidine-amide (9b) (C19H12ClNO6S, 417.82, 48, 242–
243). Anal. Calcd.: C, 54.61; H, 2.89; N, 3.35; S, 7.67. Found: C, 54.59;
3.1.4.1.3. 1-Benzyl-3-(9-methoxy-7-oxo-7H-furo[3,2-g]chromen-
4-yl)thiourea (5c) (C20H16N2O4S, 380.421, 57.9, 238–240). Anal.
H, 2.87; N, 3.29; S, 7.65. IR (cmꢀ1, KBr): 1728 (C]O
a-pyrone), 1397,

O.M. Abdel Hafez et al. / European Journal of Medicinal Chemistry 44 (2009) 2967–2974
2973
1169 (SO2–N), 1667 (C]N), 755 (C–Cl). MS: Mþ/Mþ2 417/419
(0.76%), 294 (13.49%), 215 (13.3%), 63 (100%).
1.5 g/L sodium bicarbonate. The medium was supplemented with
10% fetal bovine serum and the medium for MCF-7 contained
additionally 0.01 mg/mL bovine insulin. Cultures were maintained
in T-75 flasks in an atmosphere of 5.0% CO2 at 37 ꢁC. Media were
changed every other day in cultures of MCF-7 cells and cells were
passaged once a week at a subcultivation ratio of 1:3 after release
from the flasks with 0.25% Trypsin–0.53 mM EDTA solution. HeLa
cells were passaged every other day or on the third day after
a weekend at a ratio of 1:8.
3.1.8.1.3. 9-Methoxy-7-oxo-7H-furo[3,2-g]chromen-4-sulfonica-
cid-4-methoxybenzylidine-amide (9c) (C20H15NO7S, 413.39, 58.6,
258–260). Anal. Calcd.: C, 58.10; H, 3.66; N, 3.39; S, 7.76. Found: C,
58.08; H, 3.62; N, 3.35; S, 7.75. IR (cmꢀ1, KBr): 1730 (C]O
a-
pyrone), 1390, 1170 (SO2–N), 1673 (C]N). MS: Mþ 413 (16.1%), 215
(38.3%), 177 (90.9%), 135 (100%), 187 (9%), 158 (6%), 202 (14%), 135
(100%), 74 (11%).
3.1.8.1.4. 9-Methoxy-7-oxo-7H-furo[3,2-g]chromen-4-sulfonic
acid furan-2-yl-methylene amide (9d) (C19H12BrNO6S, 462.38, 85.7,
236–238). Anal. Calcd.: C, 49.36; H, 2.62; N, 3.03; S, 6.94. Found: C,
Cells were plated in 96-well plates at a density of 104 cells/well
in 190
were added to the wells. All dilutions were made into 50% DMSO
and were added to the wells (10 L of appropriate dilutions in 50%
DMSO to 190
media containing 104 cells) to make final
concentrations of 5, 10, 50 and 100 g compound/mL with a final
concentration of 2.5% DMSO in 200 L media in each well. The
200 L volume was used, because in earlier assays with 100 L of
mL media. At 24 h, appropriate dilutions of test compounds
49.35; H, 2.59; N, 2.99; S, 6.92. IR (cmꢀ1, KBr): 1731 (C]O
a
-
m
pyrone), 1397, 1169 (SO2–N), 1677 (C]N). MS: Mþ 462 (9%), 295
(64%), 253 (90%), 215 (40%), 205 (55%), 161 (100%).
mL
m
3.1.8.1.5. 9-Methoxy-7-oxo-7H-furo[3,2-g]chromen-4-sulfonica-
cid-4-methoxybenzylidine-amide (9e) (C17H11NO7S, 373.33, 72.6,
260–263). Anal. Calcd.: C, 54.69; H, 2.97; N, 3.75; S, 8.59 . Found: C,
m
m
m
media in the wells, there was significant evaporation especially of
wells in the outside rows of the plates. (The outside rows were not
used in subsequent assays.) The plates were incubated for 72 h and
were read with a plate reader (Synergy HT Multi-Detection
Microplate Reader with Gen5 software, BioTek Instruments,
Winooski, VT). The Cell titer 96 Aqueous One Solution Reagent
(Promega Corporation, Madison, WI) was then added to the wells
54.67; H, 2.92; N, 3.73; S, 8.54. IR (cmꢀ1, KBr): 1740 (C]O
a-
pyrone), 1395, 1172 (SO2–N), 1670 (C]N). MS: Mþ 373 (2.5%), 295
(92%), 215 (100%), 202 (25%), 188 (28%), 160 (38%).
3.1.9. Preparation of 7-oxo-furochromen sulphonic acid imide
derivatives (10a–c)
To
a
solution of xanthotoxin-4-sulfonyl hydrazide (3.14 g,
(20 mL/well) and the plates were read again after 3 h. Each
0.01 mol) in acetic acid, the appropriate anhydrides, namely,
phthalic anhydride, pyridine anhydride (0.01 mol) or benzen dia-
nhydride (0.005 mol) was added . The mixture was refluxed for 6–
8 h, poured onto cold water; the formed precipitate was filtered,
washed with water and crystallized from glacial acetic acid to give
the cyclic imide dione derivatives (10a–c).
concentration of the test compound and controls with and without
DMSO was tested in triplicate and the percent viability in the wells
with test compounds was calculated by comparing absorbance to
that of the control wells containing 2.5% DMSO.
The Cell Titer 96 Aqueous One Solution Reagent contains a novel
tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carbo-
methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt;
MTS] and an electron coupling reagent (phenazine ethosulfate;
PES). The MTS tetrazolium compound is reduced by cells to
a colored formazan product which is soluble in culture medium, so
that it can be read by a plate reader with no other steps after
addition of the reagent. Since some of the test compounds were
highly colored, affecting the color of the medium in the wells even
before the reagent was added, the plates were read immediately
before addition of the reagent to the wells and the absorbance
before addition of reagent was subtracted from the absorbance
recorded 3 h after addition to determine the absorbance attributed
to reduction of the MTS by viable cells.
3.1.9.1. Microanalytical and spectral data of compounds 10a–c (Mol.
For., M. wt., yield % and m.p. ꢁC)
3.1.9.1.1. 9-Methoxy-7-oxo-7H-furo[3,2-g]chromen-4-sulfonica-
cid(1,3-dioxo-1,3-dihydro-isoindol-2-yl) (10a) (C20H12N2O8S, 440.39,
70.45, 220–222). Anal. Calcd.: C, 54.54; H, 2.75; N, 6.36; S, 7.28.
Found: C, 54.50; H, 2.71; N, 6.32; S, 7.25. IR (cmꢀ1, KBr): 1765 (C]O
a
-pyrone), 1746, 1773 (C]O imide), 1370, 1150 (SO2–N). MS: Mþ
440 (2.91%), 294 (4.04%), 279 (3.51%), 247 (9.09%), 216 (6.72%) and
63 (100%).
3.1.9.1.2. 9-Methoxy-7-oxo-7H-furo[3,2-g]chromen-4-sulfonica-
cid(5,7-dioxo-5,7-dihydro-pyrrolo[3,4-b]pyridin-6-yl)-amide (10b)
(C19H11N3O8S, 441.37, 72.72, >250). Anal. Calcd.: C, 51.70; H, 2.51; N,
9.52; S, 7.26. Found: C, 51.66; H, 2.49; N, 9.50; S, 7.25. IR (cmꢀ1, KBr):
References
1750 (C]O a-pyrone), 1740, 1772 (C]O imide), 1370, 1165 (SO2–N).
MS: Mþ 440 (2.91%), 294 (4.04%), 279 (3.51%), 247 (9.09%), 216
(6.72%) and 63 (100%).
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3.1.9.1.3. 2,6-Bis-(9-methoxy-7-oxo-7H-furo[3,2-g]chromen-4-
sulfonicacid)-1,3,5,7-tetraoxo-3,5,6,7-tetrahydro-1H-pyrrolo
[3,4-
b]isoindol-2-yl)-amide (10c) (C34H18N4O16S, 802.64, 72.5,227–
230). Anal. Calcd.: C, 50.87; H, 2.26; N, 6.98; S, 7.99. Found: C,
50.85; H, 2.25; N, 6.96; S, 7.97. IR (cmꢀ1, KBr): 1740 (C]O
a-pyrone),
1745, 1776 (C]O imide), 1372, 1160 (SO2–N). MS: Mþ 802 (6%), 477
[8] F.D. Popp, W. Kirsch, J. Org. Chem. 26 (1961) 3858.
(8%), 276 (84%),173 (42%), 78 (74%), 62 (100%).
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3.2. In vitro antitumor screening
The ten compounds (Xanthotoxin, 2d, 2g, 3c, 4a, 5b, 5c, 7, 9d
and 10a) were tested for their ability to inhibit the growth of the
HeLa cervical cancer cell line (obtained from ATCC, Cat. No. CCL-2)
and the (MCF-7) breast cancer cell line (ATCC, Cat. No. HTB-22). The
cells were grown in Eagle’s Minimal Essential medium with Earle’s
salts and 2 mM
L-glutamine, that was further modified to contain
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