Synthesis of novel benzosuberone derivatives using organophosphorus reagents and their antitumor activities

Article abstract of DOI:10.1515/znb-2012-0311

2-Arylidenebenzosuberones react with a Wittig-Horner reagent in the presence of sodium hydride as a base to give the novel dimethyl (4-(4-methoxyphenyl)-2-oxa-2,3,4,5,6,7-hexahydrobenzo-[6,7]cyclohepta[1,2-b] pyran-3-yl)phosphonate. On the other hand, 6,7-dihydrobenzo[6,7]cyclohepta-[1,2- b]pyran-2(5H)-ones were isolated from the reaction of 2-arylidenebenzosuberones with Wittig-Horner reagents using alcoholic sodium alkoxide. The reaction of 2-arylidenebenzosuberones with trialkyl phosphites affords the alkyl phosphonate derivatives. Tris(dialkylamino)phosphines react with 2-arylidenebenzosuberones to give the oxaphospholanoxide products. 2-Arylidenebenzosuberones react with Lawesson's reagent to yield the corresponding dimers. Some of the prepared products were screened for antitumor activity.

Full text of DOI:10.1515/znb-2012-0311

Synthesis of Novel Benzosuberone Derivatives using Organophosphorus
Reagents and their Antitumor Activities
Leila S. Boulos, Hoda A. Abdel-Malek, and Naglaa F. El-Sayed
Department of Organometallic and Organometalloid Chemistry, National Research Centre,
El-Behoos St., P. O. 12622, Dokki, Cairo, A. R. Egypt
Reprint requests to L. S. Boulos. E-mail: leilagoubran@yahoo.com
Z. Naturforsch. 2012, 67b, 243 – 252; received January 9, 2012
2-Arylidenebenzosuberones react with a Wittig–Horner reagent in the presence of sodium hy-
dride as a base to give the novel dimethyl (4-(4-methoxyphenyl)-2-oxa-2,3,4,5,6,7-hexahydrobenzo-
[6,7]cyclohepta[1,2-b]pyran-3-yl)phosphonate. On the other hand, 6,7-dihydrobenzo[6,7]cyclohepta-
[1,2-b]pyran-2(5H)-ones were isolated from the reaction of 2-arylidenebenzosuberones with Wittig–
Horner reagents using alcoholic sodium alkoxide. The reaction of 2-arylidenebenzosuberones with
trialkyl phosphites affords the alkyl phosphonate derivatives. Tris(dialkylamino)phosphines react
with 2-arylidenebenzosuberones to give the oxaphospholanoxide products. 2-Arylidenebenzosuber-
ones react with Lawesson’s reagent to yield the corresponding dimers. Some of the prepared products
were screened for antitumor activity.
Key words: Benzosuberone Derivatives, Organophosphorus Reagents, Antitumor Activity
Introduction
Results and Discussion
Chemistry
Benzosuberone derivatives are known as cytotoxic
and antitumor agents against L1210 murine leukemia
and HT2g cell lines [1 – 3], as potent inhibitors of tubu-
lin polymerization [4] and as blood platelet aggrega-
tion inhibitors [5]. This together with our interest in
organophosphorus chemistry [6 – 11] has encouraged
the synthesis of new organophosphorus compounds in-
corporating such structural units that may possibly lead
to further biological activity. The present study deals
with the reaction of 6-arylidene-6,7,8,9-tetrahydro-
5H-benzocyclohepten-5-ones 1 with Wittig-Horner
reagents 2, trialkyl phosphites 3, tris(dialkylamino)-
phosphines 4, and Lawesson’s reagent 5 (Scheme 1).
When benzosuberone 1a was treated with one
equivalent of trimethyl phosphonoacetate 2a in dry
xylene in the presence of sodium hydride at reflux
temperature for 10 h, compound 6a was obtained
(75 % yield) (Scheme 2). Compound 6a was formu-
lated as lactone 6a. Elemental and mass spectral anal-
ysis of 6a led to an empirical formula C23H25O6P.
The IR spectrum of 6a in KBr revealed of strong ab-
sorption bands at 1230 cm−1 (P=O, bonded) [12] and
at 1050 cm−1 (P-O-C) [12]. Moreover, the IR spec-
trum showed a carbonyl absorption band at 1726 cm−1
(CO, lactone). The spectrum of 6a lacked the carbonyl
absorption which is recorded [13] for 1a at 1660 cm−1
.
The 1H NMR spectrum of 6a gave signals at δ =
1.23 ppm (m, 2H, CH2), 1.79 ppm (m, 2H, CH2),
2.6 ppm (m, 2H, CH2). The two ortho-methine pro-
tons appeared as a pair of doublets at 2.7 ppm (dd,
2J
= 15 Hz, JHH = 7.5 Hz), and 5.6 ppm (dd, 3JHP
=
HP
1
10 Hz,JHH = 7.5 Hz). Moreover, the H NMR of 6a
exhibited signals at 3.4 ppm (s, 3H, C6H4-OCH3) and
3
3.5, 3.6 ppm (2d, 6H, J
= 11 Hz, PO(OCH3)2)
HP
for the two methoxy groups attached to the phospho-
rus and a multiplet at 6.9– 7.1 ppm (8H, aromatic).
The 31P NMR measurement of 6a supported the phos-
phonate structure. It exhibited a sharp signal at δ =
Scheme 1.
c
ꢀ 2012 Verlag der Zeitschrift fu¨r Naturforschung, Tu¨bingen · http://znaturforsch.com
Brought to you by | University of Georgia Libraries
Authenticated
Download Date | 5/30/15 9:19 AM

244
L. S. Boulos et al. · Novel Benzosuberone Derivatives
Scheme 2.
23.03 ppm [14]. The 13C NMR spectrum of 6a added idene-1-benzosuberones 1a and 1b were allowed to re-
good support for the proposed structure and revealed act with phosphonate 2b in an alcoholic sodium ethox-
the methine proton attached to the phosphorus as a ide solution, pyranones 7a and 7b were also isolated
doublet at 33.4 ppm with 1J = 168.8 Hz.
(Scheme 2).
CP
When 1a was allowed to react with one equiva-
A possible explanation for the reaction course of 1a
lent of trimethyl phosphonoacetate 2a, in a methano- with Wittig–Horner reagent 2a in the presence of
lic sodium methoxide solution, adduct 7a was isolated sodium hydride as base is shown in Scheme 2. Initial
in 80 % yield (Scheme 2). The structure 7a was es- attack of trimethyl phosphonacetatete 2a on the most
1
tablished from its elemental analysis and its IR, H, reactive center of 1a gave the intermediate A. Elimi-
13C NMR and mass spectral data (cf. Experimental nation of one molecule of alcohol followed by cycliza-
Section). Similarly, when 1b was allowed to react with tion result in the formation of the phosphonate deriva-
trimethyl phosphoacetate 2a in a methanolic sodium tive 6a. Compounds 7a,b presumably are also formed
methoxide solution, 7b was obtained (78% yield) via intermediate A. Under the influence of the base
(Scheme 2, cf. Experimental Section). When 2-benzyl- present in the reaction medium, elimination of dialkyl
Brought to you by | University of Georgia Libraries
Authenticated
Download Date | 5/30/15 9:19 AM

L. S. Boulos et al. · Novel Benzosuberone Derivatives
245
Scheme 3.
phosphite together with the loss of one molecule of al- (H3CO)2P), 3.3 (dd, 2JHP = 18.7 Hz, JHH = 7.5 Hz, 1H,
cohol and cyclization after a suitable proton transfer CH-P), 3.5 (dd, 3JHP = 10.7 Hz, JHH = 7.5 Hz, CH-CH-
gives the final products 7a and/or 7b (Scheme 2). The P), 1.7, 2.5, 2.7 (m, 6H, 3CH2) and 6.7 – 7.3 ppm (m,
dialkyl phosphite was detected in the water layer by 8H, aromatic). The 31P NMR measurement of 8a sup-
the development of a violet color on addition of 3,5- ported the phosphonate structure. It exhibited a sharp
dinitrobenzoic acid [15]. Moreover, when 6a was al- signal at δ = 23.10 ppm. The 13C NMR spectrum
lowed to react with sodium methoxide in an alcoholic of 8a gave signals at δ = 25.4, 29.3, 32.6 (3CH2),
1
2
solution under reflux for 6 h, compound 7a was ob- 31.6 (CH-P=O, JCP = 100 Hz), 39.2 (CH-C=O, JCP
tained (60 % yield). The dialkyl phosphite was again = 21.72 Hz), 52.1 (C6H4-OCH3), 54.6 (PO(OCH3)2),
detected in the water layer.
157 (C-OCH3), and 205.6 ppm (C=O). The mass spec-
Furthermore, this study was extended to include the trum of 8a showed the molecular ion peak at m/z =
386 (30 %), and the peak for [M–P(O)(OCH3)2]+ at
m/z = 278 (90%). Similarly, 2-benzylidene-1-benzo-
behavior of benzosuberone 1a towards trimethyl phos-
phite 3a to determine the preferential site of attack. We
have found that the reaction of 1a with 3a was success- suberone (1a) reacts with an excess of triisopropyl
fully completed (TLC) by using an excess of phosphite phosphite 3b as solvent to give 8b (70 % yield). The
◦
structure of 8b is derived from its spectral data (cf.
Experimental Section). When benzosuberone 1b re-
as solvent and heating the mixture for 4 h at 105 C.
Separation of the product mixture yielded phospho-
nate 8a (75 % yield) (Scheme 3). Compound 8a was acted with an excess of (3b) as solvent, product 8c
was obtained (65 % yield). Methyl phosphonate 8c has
chromatographically pure and shows a sharp melting
point. The assigned methylphosphonate structure 8a
been identified on the basis of its elemental analy-
1
is based on correct microanalysis and IR, 1H, 13C, sis and its IR, H, 13C, 31P NMR and mass spectral
31P NMR and mass spectral data.
data (Scheme 3, cf. Experimental Section). Worthy to
mention is that only one isomer of phosphonate 6a
and compound 8 were isolated, which are assumed to
have a cis configuration. The assigned cis configura-
tion for these products, although not established with
certainty, is supported by an inspection of Newman
The IR spectrum of 8a revealed the presence of the
carbonyl absorption band at 1660 cm−1. In addition,
it exhibited intense bands corresponding to the P=O
(1256 cm−1) and P-O-alkyl (1082 cm−1) stretching
1
vibrations [12]. The H NMR spectrum (in DMSO)
1
projections [16] as well as by the H NMR chemi-
of the adduct showed signals at δ = 3.8 ppm (s, 3H,
3
cal shifts and coupling constants of the two ortho me-
H3CO-C6H4), 3.41, 3.46 (2d, JHP = 11.2 Hz, 6H,
Brought to you by | University of Georgia Libraries
Authenticated
Download Date | 5/30/15 9:19 AM

246
L. S. Boulos et al. · Novel Benzosuberone Derivatives
Scheme 4.
thine protons attached to phosphorus. The observed for the dimethyl amino group, a singlet at 3.8 (3H,
coupling constants of (JH H = JH Ha , ca. 6 – 7.5 Hz) C6H4-OCH3), and multiplets at 1.8, 2.3, 2.8 ppm
a
b
b
indicate a cis configuration, rather than a trans config- (3CH2). The aromatic protons appeared as multiplets
uration which would give rise to larger coupling con- at δ = 6.9 – 7.9 ppm (8H). The 31P NMR spectrum
stants (9 – 15 Hz).
of 9a gave one signal at δ = 43.7 ppm that sup-
ported the cyclic oxaphospholanoxide [14, 18]. Simi-
larly, tris(diethylamino)phosphine (4b) reacts with 1a
to give 9b (70 % yield). When 2-benzylidene-1-benzo-
suberone 1b reacted with 4a and 4b, the corresponding
oxaphospholoxide derivatives 9c and 9d were obtained
(Scheme 4).
We have found that 1a reacted with tris(dimeth-
ylamino)phosphine (4a) in refluxing toluene to give
a chromatographically pure adduct formulated as 9a
(Scheme 4). The structure elucidation of product 9a is
based on the following evidences. Elemental analysis
and molecular weight determination (MS) of 9a sup-
ported the molecular formula C21H24NO3P (369.39).
The structure of the new products 9b – d was
The IR spectrum, in KBr, exhibited an intense band deduced from their spectral data (cf. Experimental
at 1240 cm−1 corresponding to the P=O absorp- Section). These compounds were formed presum-
tion, and two bands at 1320 cm−1 and 860 cm−1 ably through nucleophilic attack of the phosphite-
due to the absorption of P-N(CH3)2 [17]. Moreover, phosphorus on the most reactive center of 1 leading
the IR spectrum of the oxaphosphole-2-oxide 9a re- to the dipolar adduct A, which undergoes ring clo-
vealed the absence of the carbonyl absorption which sure giving structure B. The latter, due to its struc-
is recorded for 1a at 1660 cm−1. The 1H NMR tural features, could transform into the most sta-
spectrum of 9a showed signals at δ = 3.5 (d, 1H, ble form 9 through rapid hydrolysis (by the pres-
2JHP = 18.5 Hz) for the methine proton attached ence of unavoidable moisture) to give intermediate C,
3
to the phosphorus, 2.8 (d, 6H, JHP = 11.10 Hz) which undergoes further decomposition yielding com-
Brought to you by | University of Georgia Libraries
Authenticated
Download Date | 5/30/15 9:19 AM

L. S. Boulos et al. · Novel Benzosuberone Derivatives
247
Scheme 5.
pound 9 [8, 18, 19]. Similar results have been reported cant therapeutic potential [1 – 3]. Based on these con-
previously [8, 19]. siderations, six of the newly synthesized compounds
Furthermore, this study was extended to include the were screened for their in vitro cytotoxic and growth
behavior of 2-benzyliene-1-benzosuberones 1a, b to- inhibitory activities against human breast adenocar-
ward Lawesson’s reagent (5). We have found that 1a cinoma cells (MCF 7) and/or human hepatocellular
reacted with half a molar equivalent of 5 in reflux- carcinoma (HEPG2). The cytotoxicity of the tested
ing toluene for 1 h to give the dimeric product 10a extracts was measured against MCF 7 cells and/or
(65 % yield). Similarly, when 1b reacted with half HEPG2 cells using the MTT Cell Viability Assay [22].
an equivalent of 5 in refluxing toluene, the dimeric According to the American National Cancer Institute
product 10b was obtained (60 % yield). The structures guidelines [23] drugs with IC50 < 30 are active. The
of 10a and 10b were confirmed by analytical and spec- cytotoxicity of the extracts was tested in the Cancer
tral data (Scheme 5, cf. Experimental Section).
Biology laboratory, Center of Excellence for Advanced
The dimeric compounds 10a, b were formed Sciences, National Research Center.
through the straightforward thionation of carbonyl
group of 1a, b to yield the thione monomer which im-
mediately underwent cyclization with another thione
monomer [2+4] to yield the corresponding thione
dimers 10a and 10b (Scheme 5) [20]. It is wor-
thy to note that when 1a and 1b react with phos-
phorus pentasulfide (P4S10) in refluxing toluene, the
dimeric compounds 10a and 10b were also obtained
(Scheme 5).
Treatment of MCF 7 cells with sample 7a led to a
high inhibition in the cell proliferation as concluded
by the low IC50 value of 13.32 µg mL−1 (Fig. 1). On
the other side, the other tested samples 6a, 8a, 9b,
Pharmacological evaluation
Cancer diseases are a serious threat to health and
development of mankind, and searching for effec-
tive anti-cancer agents remain actual. Considerable
progress has been made in recent years in the field
of drug development against different types of can-
cer. Moreover, chemotherapy is a major approach for
both localized and metastasized cancers [21], and ben-
zosuberone derivatives have proved to have a signifi-
Fig. 1. Effect of compound 7a on MCF 7 tumor cell lines
(breast cancer).
Brought to you by | University of Georgia Libraries
Authenticated
Download Date | 5/30/15 9:19 AM

248
L. S. Boulos et al. · Novel Benzosuberone Derivatives
Table 1. Effect of the tested compounds on HEPG2 tumor
cell lines (liver cancer).
Reaction of trimethyl phosphonoacetate 2a with 6-(4-meth-
oxybenzylidene)-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-
one 1a in the presence of sodium hydride
Compound
IC50 (µgmL−1
)
Paclitaxel
7a
0.517
27.89
28.56
29.27
Trimethyl phosphonoacetate 2a (0.18 g, 1 mmol) was dis-
solved in dry xylene (25 mL), and sodium hydride (0.024 g,
1 mmol) was added carefully with stirring. Then the aryli-
dene suberone 1a (0.27 g, 1 mmol) was added, and the mix-
ture was refluxed for 10 h. After evaporation of the volatile
material under reduced pressure, the residue was subjected
to silica gel column chromatography to give compound 6a.
9a
10b
10a, and 10b revealed a non-cytotoxic effect against
of MCF 7 cells. Paclitaxel, a known anti-cancer drug,
was used as a positive control, and its IC50 was
0.452 µg mL−1 against MCF 7 cells.
As shown in Table 1, the treatment of HEPG2 cells
with 7a, 9a, and 10b led to a high inhibition in the
cell proliferation as concluded by the low IC50 val-
ues of 27.89, 28.56, and 29.27 µg mL−1, respectively,
whereas the other samples 6a, 8a and 10a revealed a
non-cytotoxic effect against HEPG2 cells. Paclitaxel, a
known anti-cancer drug, was used as a positive control
and its IC50 was 0.517 µg mL−1 against HEPG2 cells.
Dimethyl-(4-(4-methoxyphenyl)-2-oxo-2,3,4,5,6,7-hexa-
hydrobenzo[6,7]cyclohepta[1,2-b]pyran-3-yl)phosphonate
(6a)
Eluent: petroleum ether-ethyl acetate (50/50, v/v). Prod-
uct 6a was separated as colorless crystals, yield 75 %. –
◦
M. p. 216 – 217 C. – IR (KBr): ν = 1050 (P-O-C), 1230
(P=O, bonded), 1726 (C=O, lactone) cm−1. – 1H NMR
(500.14 MHz, CDCl3): δ = 1.23 (m, 2 H, CH2), 1.79 (m,
2 H, CH2), 2.6 (m, 2 H, CH2), 2.7 (dd, 2JHP = 15 Hz, JHH
=
7.5 Hz, CH-P=O), 3.4 (s, 3 H, OCH3-C6H4), 3.5, 3.6 (2 d,
3
6 H,3JHP = 11 Hz, P=O (OCH3)2), 5.6 (dd, JHP = 10 Hz,
Conclusion
JHH = 7.5 Hz , CH-C6H4-OCH3), 6.9 – 7.1 (m, 8H, Harom). –
13C NMR (125.76 MHz, CDCl3): δ = 20.0, 22.01, 32.12 (3
The reaction of 2-benzylidene-1-benzosuberones
1 with Wittig–Horner reagents, trialkyl phosphites,
tris(dialkylamino)phosphines and Lawesson’s reagent
leads to different products, depending on the reac-
tion conditions as well as on the stability of the addi-
tion products. As a result pyran phosphonates, cyclo-
heptapyranone, phosphonates benzocycloheptenes and
oxaphospholoxide derivatives with anticipated biolog-
ical activity could be obtained.
2
C, 3 CH2), 21.4 (CH-C6H4-OCH3, JCP = 17.5 Hz), 33.4
(1JCP = 168.8 Hz, CH-P=O), 50.8 (C6H4-OCH3), 52.5 (2
C, P=O(OCH3)2), 125.4 (C=C-O), 125.8 – 137.0 (aromatic
C-H), 140.9 (C=C-O), 164.0 (C-OCH3), 165.0 (C=O). –
31P NMR: δ = 23.03. – MS (EI, 70 eV):m/z = 428 [M]+. –
Anal. for C23H25O6P (428.41): calcd. C 64.48, H 5.88,
P 7.23; found C 64.53, H 6.1, P 7.30.
Reaction of trimethyl phosphonoacetate 2a with 6-(4-meth-
oxybenzylidene)-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-
one 1a in the presence of sodium methoxide
Experimental Section
A solution of sodium methoxide (0.054 g, 1 mmol) in
absolute methanol (30 mL) was treated with an equimolar
amount of trimethyl phosphonoacetate 2a (0.18 g, 1 mmol).
Then 2-arylidenebenzosuberone 1a (0.27 g, 1 mmol) was
added, and the reaction mixture was refluxed for 12 h (TLC).
The mixture was poured on a small amount of water, ex-
tracted with ethyl acetate, and dried, and the extracts were
evaporated under reduced pressure. The residue was sub-
jected to silica gel column chromatography to give com-
pound 7a. When compound 6a was reacted with sodium
methoxide in alcoholic solution under reflux for 6 h, com-
pound 7a was obtained in 60 % yield.
Melting points were determined in open glass capillaries
using an Electrothermal IA 9100 series digital melting point
apparatus (Electorthermal, Essex, UK), and IR spectra were
measured as KBr pellets with a Perkin-Elmer spectropho-
tometer model 157. The 1H and 13C NMR spectra were
recorded in CDCl3 or [D6]DMSO as solvents on a Joel 500
(500/125 MHz) spectrometer. The chemical shifts δ (in ppm)
are given relative to TMS as internal reference. The 31P NMR
spectra were taken with a Varian CFT-20 spectrometer (vs.
external 85 % H3PO4 as standard). The mass spectra were
recorded at 70 eV with a Kratos MS equipment or a Var-
ian MAT311A spectrometer. Elemental analyses were per-
formed using an Elementar Vario E1 instrument. The re-
ported yields correspond to pure isolated materials obtained
by column chromatography on silica gel 60 (Merck). 2-benz-
ylidene-1-benzosuberones 1a and 1b were prepared accord-
ing to a reported method [24].
4-(4-Methoxyphenyl)-6,7-dihydrobenzo[6,7]cyclohepta[1,2-
b]pyran-2(5H)-one (7a)
Eluent: petroleum ether-ethyl acetate (95/5, v/v). Prod-
uct 7a was obtained as yellow crystals, yield 80 %. – M. p.
Brought to you by | University of Georgia Libraries
Authenticated
Download Date | 5/30/15 9:19 AM

L. S. Boulos et al. · Novel Benzosuberone Derivatives
249
◦
148 – 149 ◦C. – IR (KBr):v = 1627 (C=O) cm−1. – 1H NMR fluxed for 4 h at 105 C (TLC). After evaporation of the
(500.14 MHz, CDCl3): δ = 2.0, 2.5, 2.8 (m, 6 H, 3CH2), volatile material under reduced pressure, the residu◦e was
3.8 (s, 3 H, OCH3), 7.2 (s, 1 H, CH), 7.4 – 7.8 (m, 8H, washed several times with petroleum ether (60 – 80 C) to
Harom). – 13C NMR (125.76 MHz, CDCl3): δ = 24.7, 32.4, give compound 8a.
38.7 (3 CH2), 55.0 (OCH3), 102.0 (CH-C=O), 114.0 – 130.0
(aromatic C-H), 136.0 (O-C=C), 158.0 (C=C-C6H4-OCH3),
Dimethyl-(6,7,8,9-tetrahydro-5-oxo-5H-benzocyclohepten-
6-yl)(4-methoxyphenyl)methyl-phosphonate (8a)
160.0 (C-OCH3), 161.0 (C=O). – MS (EI, 70 eV): m/z = 303
[M–16]+. – Anal. for C21H18O3(318.37): calcd. C 79.22,
Solvent of crystallization: petroleum ether-ethyl acetate.
H 5.70; found C 79.49, H 5.92.
Product 8a was obtained as colorless crystals, yield 75 %. –
◦
M. p. 202 – 203 C. – IR (KBr):v = 1082 (P-O-alkyl), 1256
Reaction of trimethyl phosphonoacetate 2a with 6-benzylid-
(P=O), 1660 (C=O) cm−1. – 1H NMR (500.14 MHz,
ene-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-one 1b in the
2
DMSO): δ = 1.7, 2.5, 2.7 (m, 6 H, 3 CH2), 3.3 (dd, JHP
=
=
=
presence of sodium methoxide
3
18.7 Hz, JHH = 7.5 Hz, 1 H, CH-P), 3.4, 3.46 (2 d, JHP
3
A solution of sodium methoxide (1 mmol) in absolute
methanol (30 mL) was treated with an equimolar amount of
trimethyl phosphonoacetate 2a (0.18 g, 1 mmol). Then 1b
(0.24 g, 1 mmol) was added, and the resulting reaction mix-
ture was refluxed for 10 h (TLC). Then it was poured on
a small amount of water, extracted with ethyl acetate, and
dried, and the extracts were evaporated under reduced pres-
sure. The residue was subjected to silica gel column chro-
matography to afford compound 7b.
11.2 Hz, 6 H, PO(OCH3)2, 3.5 (dd, JHP = 10.7 Hz, JHH
7.5 Hz, CH-CH-P), 3.8 (s, 3 H, OCH3), 6.7 – 7.3 (m, 8H,
Harom). – 13C NMR (125.76 MHz, DMSO): δ = 25.4,
1
29.3, 32.6 (3 CH2), 31.6 (CH-P=O, JCP= 100 Hz), 39.2
(CH-C=O, 2JCP =21.72 Hz), 52.1 (C6H4-OCH3), 54.6 (PO-
(OCH3)2), 126.0 – 130.0 (aromatic C-H), 157 (C-OCH3),
205.6 (C=O). – 31P NMR: δ = 23.1. – MS (EI, 70 eV):
m/z = 386 [M–2]+. – Anal. for C21H25O5P (388.39): calcd.
C 64.94, H 6.49, P 7.97; found C 64.83, H 6.50, P 7.82.
4-Phenyl-6,7-dihydrobenzo[6,7]cyclohepta[1,2-b]pyran-
2(5H)-one (7b)
Reaction of triisopropyl phosphite (3b) with 1a
Excess triisopropyl phosphite (3b) was added to 1a
(0.27 g, 1 mmol) without solvent. The reaction mixture was
refluxed for 4 h. After evaporation of the volatile material un-
der reduced pressure, the residue was subjected to silica gel
column chromatography to give product 8b.
Eluent: petroleum ether-ethyl acetate (95/5, v/v). Prod-
uct 7b was obtained as pale-yellow crystals, yield 78 %. –
M. p. 136 – 137 ◦C. – IR (KBr):v = 1620 (C=O) cm−1. –
1H NMR (500.14 MHz, CDCl3): δ = 2.0, 2.6, 2.8 (m, 6 H, 3
CH2), 7.2 (s, 1 H, CH), 7.3 – 7.5 (m, 9 Harom). – 13C NMR
(125.76 MHz, CDCl3): δ = 24.7, 33.4, 39.1 (3 CH2),
102.1 (CH-C=O), 126.0 – 137.0 (aromatic C-H), 137.13 (O-
C=C), 159.0 (C=C-C6H5), 162.0 (C=O). – MS (EI, 70 eV):
m/z = 273 [M–16]+. – Anal. for C20H16O2(288.34): calcd.
C 83.31, H 5.59; found C 83.69, H 5.97.
Diisopropyl-(6,7,8,9-tetrahydro-5-oxo-5H-benzocyclo-
hepten-6-yl)(4-methoxyphenyl)methyl phosphonate (8b)
Eluent: petroleum ether-ethyl acetate (80/20, v/v). Prod-
uct 8b was obteined as colorless crystals, yield 70 %. – M. p.
210 – 211 ◦C. – IR (KBr): v = 1080 (P-O-alkyl), 1252 (P=O),
1
1667 (C=O) cm−1. – H NMR (500.14 MHz, DMSO): δ =
General procedure for the reaction of triethyl phosphonoac-
etate (2b) with 1a,1b
1.3 (m, 12 H, 4 CH3), 1.6, 1.9, 2.9 (m, 6 H, 3 CH2), 3.1 (dd,
2JHP = 19.3 Hz, JHH = 7.5 Hz, 1H, CH-P), 3.3 (dd, JHP
=
3
Sodium ethoxide (0.068 g, 1 mmol) in absolute alcohol
(30 mL) was added to a solution of an equimolar amount of
triethyl phosphonoacetate (2b) (0.22 g, 1 mmol), and then 1a
or 1b (1 mmol) was added. The resulting mixture was re-
fluxed for 4 h, then poured on a small amount of water, ex-
tracted with ethyl acetate, and dried, and the extracts were
evaporated under reduced pressure. The residue was sub-
jected to silica gel column chromatography. Elution of the
column with petroleum ether (60 – 80 ◦C)-ethyl acetate (95/5,
v/v) to give products 7a, 7b (mixed melting points and com-
parative IR spectra with authentic samples).
10.70 Hz, JHH = 7.5 Hz, 1 H, CH-CH-P), 3.8 (s, 3 H, OCH3),
4.6, 4.8 (2 m, 2 CH, isopropyl), 7.1 – 7.3 (m, 8 H, Harom). –
13C NMR (125.76 MHz, DMSO): δ = 24.2 (4 CH3, iso-
2
propyl), 25.5, 29.8, 31.1 (3 CH2), 35.0 (CH-C=O, JCP
=
1
35 Hz), 44.2 (CH-P=O, JCP = 98 Hz), 55.8 (OCH3), 72.0
2
(CH-(CH3)2), JCP = 32.4 Hz), 114.2 – 141.9 (aromatic C-
H), 159.5 (C-OCH3), 205.0 (C=O). – 31P NMR δ = 23.2. –
MS (EI, 70 eV): m/z = 444 [M]+. – Anal. for C25H33O5P
(444.50): calcd. C 67.55, H 7.48, P 6.97; found C 67.50,
H 7.50, P 7.20.
Reaction of triisopropyl phosphite (3b) with 1b
Reaction of trimethyl phosphite (3a) with 1a
Excess trimethyl phosphite (3a) was added to 1a (0.27 g,
Excess triisopropyl phosphite (3b) was added to 1b
1 mmol) without solvent, and the reaction mixture was re- (0.24 g, 0.001 mol) without solvent. The reaction mixture
Brought to you by | University of Georgia Libraries
Authenticated
Download Date | 5/30/15 9:19 AM

250
L. S. Boulos et al. · Novel Benzosuberone Derivatives
was refluxed for 4 h. After evaporation of the volatile ma- 194 ◦C. – IR (KBr): v = 863, 1242 (P-N(C2H5)2), 1328
terial under reduced pressure, the residue was subjected to (P=O) cm−1. – 1H NMR (500.14 MHz, CDCl3): δ = 1.1
silica gel column chromatography to give compound 8c.
(t, 6 H, 2 CH3, ethyl), 1.7, 1.9, 2.1 (m, 6 H, 3 CH2),
3
2
2.8 (q, JHP = 10 Hz, 4 H, 2 CH2, ethyl), 3.2 (d, JHP
=
Diisopropyl((5-oxo-6,7,8,9-tetrahydro-5H-benzocyclo-
hepten-6-yl)(phenyl)methyl)phosphonate (8c)
18.5 Hz, 1 H, CH), 3.8 (s, 3 H, OCH3), 6.9 – 7.8 (m, 8 H,
Harom). – 13C NMR (125.76 MHz, CDCl3): δ = 14.1 (2
CH3, ethyl), 25.9, 30.1, 35.7 (3 CH2), 39.0 (2 CH2, ethyl),
Eluent: petroleum ether-ethyl acetate (80/20, v/v). Prod-
uct 8c was obtained as colorless crystals, yield 65 %. – M. p.
190 – 191 ◦C. – IR (KBr): v = 1081 (P-O-alkyl), 1250 (P=O),
1
48.8 (CH, JCP = 107.30 Hz), 55.3 (OCH3), 107.8 (C=C-
CH), 114.0 – 130.0 (aromatic C-H), 158.0 (C-OCH3), 184.0
(C-O). – 31P NMR: δ = 42.5. – MS (EI, 70 eV): m/z = 397
[M]+. – Anal. for C23H28NO3P (397.45): calcd. C 69.51,
H 7.10, N 3.52, P 7.79; found C 69.47, H 7.17, N 3.5,
P 7.70.
1
1669 (C=O) cm−1. – H NMR (500.14 MHz, CDCl3): δ =
1.3 (m, 12 H, 4 CH3), 1.6, 2, 2.9 (m, 6 H, 3 CH2), 3.2 (dd,
3
2JHP = 18.3 Hz, JHH = 7.5 Hz, 1 H, CH-P), 3.4 (dd, JHP
=
11.20 Hz, JHH = 7.5 Hz, 1 H, CH-CH-P), 4.7, 4.8 (2 m,
2 CH, isopropyl), 7.2 – 7.3 (m, 9 H, Harom). – 13C NMR
(125.76 MHz, CDCl3): δ = 24.7 (4 CH3, isopropyl), 26.2,
28.02, 35.1 (3 CH2), 31.0 (1JCP = 99.0 Hz, CH-P=O), 36.6
(CH-C=O, 2JCP = 20.0 Hz), 72.6 (CH-(CH3)2), 114.1 – 140
(aromatic C-H). – 31P NMR: δ = 23.4. – MS (EI, 70 eV):
m/z = 399 [M–Me]+. – Anal. for C24H31O4P (414.47):
calcd. C 69.55, H 7.54, P 7.47; found C 69.54, H 7.60, P 7.1.
2-(Dimethylamino)-3-phenyl-3,4,5,6-tetrahydro-2H-benzo-
[3,4]cyclohepta[1,2-d][1,2]oxaphosphole-2-oxide (9c)
Eluent: petroleum ether-acetone (85/15, v/v). Product 9c
was obtained as colorless needles, yield 60 %. – M. p. 143 –
144 ◦C. – IR (KBr): v = 862, 1240 (P-N(CH3)2), 1323 (P=O)
cm−1. – 1H NMR (500.14 MHz, CDCl3): δ = 2.2, 2.3, 2.33
3
(m, 6 H, 3 CH2), 2.8 (d, JHP = 10.8 Hz, 6 H, 2 CH3), 3.8
General procedure for the reaction of 2-arylidenebenzo-
suberones 1a,b with tris(dialkylamino)phosphines 4a,b
2
(d, JHP = 18.2 Hz, 1 H, CH), 7.2 – 7.8 (m, 9 H, Harom). –
13C NMR (125.76 MHz, CDCl3): δ = 23.9, 27.1, 35.8 (3
CH2), 43.7 (2 C, N(CH3)2), 48.1 (CH), 115.0 – 131.0 (aro-
matic C-H), 108.0 (C=C-CH), 184.0 (C-O). – 31P NMR:
δ = 43.7. – MS (EI, 70 eV): m/z = 339 [M]+. – Anal.
for C20H22NO2P (339.37): calcd. C 70.78, H 6.53, N 4.13,
P 9.13; found C 70.80, H 6.56, N 4.1, P 9.1.
Tris(dialkylamino)phosphine 4 (1 mmol) was added to a
solution of compound 1 (1 mmol) in dry toluene (30 mL),
and the reaction mixture was refluxed for 7 h (TLC). After
evaporation of the volatile material under reduced pressure,
the residue was subjected to silica gel column chromatogra-
phy to give the products 9a – d.
2-(Diethylamino)-3-phenyl-3,4,5,6-tetrahydro-2H-benzo-
[3,4]cyclohepta[1,2-d][1,2]oxaphosphole-2-oxide (9d)
2-(Dimethylamino)-3-(4-methoxyphenyl)-3,4,5,6-tetra-
hydro-2H-benzo[3,4]cyclohepta[1,2-d][1,2]oxaphosphole-
2-oxide (9a)
Eluent: petroleum ether-acetone (90:10, v/v). Product 9d
was obtained as a colorless powder, yield 70 %. – M. p. 163 –
164 ◦C. – IR (KBr): v = 862, 1242 (P-N(C2H5)2), 1323
(P=O) cm−1. – 1H NMR (500.14 MHz, CDCl3): δ = 1.4 (t,
6 H, 2 CH3, ethyl), 1.7, 1.8, 2.2 (m, 6 H, 3 CH2), 3.0 (q, 4 H,
2 CH2, ethyl), 3.5 (d, 2JHP = 17.9 Hz, 1 H, CH), 6.7 – 7.1 (m,
9 H, Harom). – 13C NMR (125.76 MHz, CDCl3): δ = 14 (2
CH3, ethyl), 26, 30.5, 35.5 (3 CH2), 38.8 (2 CH2, ethyl), 48.5
(CH), 107.1 (C=C-CH), 114.0 – 130.0 (aromatic C-H), 185.0
(C-O). – 31P NMR: δ = 42.5. – MS (EI, 70 eV): m/z = 367
[M]+. – Anal. for C22H26NO2P (367.42): calcd. C 71.92,
H 7.13, N 3.81, P 8.43; found C 71.90, H 7.18, N 3.14, P 8.4.
Eluent: petroleum ether-acetone (80/20, v/v). Product 9a
was obtained as colorless crystals, yield 60 %. – M. p. 148 –
149 ◦C. – IR (KBr): v = 860, 1320 (P-N(CH3)2), 1240 (P=O)
1
cm−1. – H NMR (500.14 MHz, CDCl3): δ = 1.8, 2.3, 2.8
(m, 6 H, 3 CH2), 2.8 (d, 3JHP = 11.10 Hz, 6 H, 2 CH3), 3.5
(d, 2JHP = 18.5 Hz, 1 H, CH), 3.8 (s, 3 H, OCH3), 6.9 – 7.8
(m, 8 H, Harom). – 13C NMR (125.76 MHz, CDCl3): δ =
24.1, 27.6, 35.9 (3CH2), 43.7 (2C, N (CH3)2), 48.0 (CH),
50.8 (OCH3), 108.0 (C=C-CH), 114.0 – 130.3 (aromatic C-
H), 158.2 (C-OCH3) 185.0 (C-O). – 31P NMR: δ = 43.7. –
MS (EI, 70 eV): m/z = 369 [M]+. – Anal. for C21H24NO3P
(369.39): calcd. C 68.28, H 6.55, N 3.79, P 8.39; found
C 68.2, H 6.6, N 4.1, P 8.3.
Reaction of Lawesson’s reagent (5) with 1a
A mixture of 2-arylidene-benzosuberone 1a (0.27 g,
1 mmol) and Lawesson’s reagent (5) (0.2 g, 0.5 mmol) was
refluxed for 1 h in (30 mL) of dry toluene. The volatile ma-
terial was evaporated under reduced pressure and the residue
2-(Diethylamino)-3-(4-methoxyphenyl)-3,4,5,6-tetrahydro-
2H-benzo[3,4]cyclohepta[1,2-d][1,2]oxaphosphole-2-
oxide (9b)
Eluent: petroleum ether-acetone (90/10, v/v). Product 9b subjected to silica gel column chromatography to give prod-
was obtained as colorless crystals, yield 70 %. – M. p. 193 – uct 10a.
Brought to you by | University of Georgia Libraries
Authenticated
Download Date | 5/30/15 9:19 AM

L. S. Boulos et al. · Novel Benzosuberone Derivatives
Dimeric compound 10a
251
volatile material was evaporated under reduced pressure. The
residue was subjected to silica gel column chromatography.
The dimeric compound 10b was separated using petroleum
ether-ethyl acetate (95/5, v/v) as an eluent (mixed melting
point and comparative IR spectrum).
Eluent: Petroleum ether-ethyl acetate (95/5, v/v). Prod-
uct 10a was obtained as yellow crystals; yield 65 %. – M. p.
202 – 203 ◦C. – IR (KBr): v =1210 (C=S) cm−1. – 1H NMR
(500.14 MHz, CDCl3): δ = 1.7, 2.3, 2.6 (m, 12 H, 6 CH2),
3.8 (2 s, 6 H, 2 OCH3), 4.9 (s, CH-C6H4-OCH3), 5.6 (s, 1 H,
S-CH-C6H4-OCH3), 6.9 – 7.7 (m, 16 H, Harom). – 13C NMR
(125.76 MHz, CDCl3): δ = 23.2 – 35.9 (6 C, 6 CH2), 43.3
(CH-C6H4-OCH3), 49.2 (S-CH-C6H4-OCH3), 53.2 (2 C, 2
OCH3), 73.0 (C6H4-C-C-C=S), 114.0 – 142.9 (aromatic C-
H), 241.0 (C=S). – MS (EI, 70 eV): m/z = 468 [M–(C6H4-
OCH3-CH)]+. – Anal. for C38H36O2S2 (588.82): calcd.
C 77.51, H 6.16, S 10.89; found C 77.55, H 6.2, S 11.
Pharmacological activity
Material and methods
Chemicals: All cell culture material was obtained from
Cambrex BioScience (Copenhagen, Denmark). All chemi-
cals were from Sigma/Aldrich, USA, unless otherwise indi-
cated. All experiments were repeated three times.
Cell culture: Cells were routinely cultured in DMEM
(Dulbeco’s Modified Eagle’s Medium), which was supple-
mented with 10 % fetal bovine serum (FBS), 2 mM L-
glutamine, containing 100 units/mL penicillin G sodium, 100
units mL−1 streptomycin sulfate and 250 mg mL−1 ampho-
Reaction of Lawesson’s reagent (5) with 1b
A mixture of (0.24 g, 1 mmol) of 2-arylidenebenzosuber-
one 1b and (0.20 g, 0.5 mmol) of Lawesson’s reagent (5) was
refluxed for 1 h in 30 mL of dry toluene. The solvent was
evaporated under reduced pressure. The residue was washed
several times with acetone to give product 10b.
◦
tericin B. Cells were maintained at sub-confluence at 37 C
in humidified air containing 5 % CO2.
In vitro cytotoxicity assay: Cells (0.5×105 cells per well),
in serum-free media, were plated in a flat bottom 96-well
microplate, and treated with 20 µL of diff◦erent concentra-
tions of the tested extract for 48 h at 37 C in a humidi-
fied 5 % CO2 atmosphere. After incubation, media were re-
moved, and 40 µL MTT solution per well was added and in-
cubated for an additional 4 h. MTT crystals were solubilized
by adding 180 µL of acidified isopropanol per well, and the
plate was shaken at r. t., followed by photometric determina-
tion of the absorbance at 570 nm using a microplate ELISA
reader. Triplicate repeats were performed for each concen-
tration, and the average was calculated. Data were expressed
as the percentage of relative viability compared with the un-
treated cells and the vehicle control, with cytotoxicity indi-
cated by < 100 % relative viability.
Dimeric compound 10b
Solvent of crystallization: benzene. Product 10b was
obtained as colorless crystals; yield 60 %. – M. p. 230 –
232 ◦C. – IR (KBr): v =1212 (C=S) cm−1. – 1H NMR
(500.14 MHz, CDCl3): δ = 1.7, 2.3, 2.8 (m, 12 H, 6 CH2), 4.9
(s, 1 H, CH-C6H4-OCH3), 5.7 (s, 1 H, S-CH-C6H4-OCH3),
6.8 – 7.6 (m, 18 H, Harom). – 13C NMR (125.76 MHz,
CDCl3): δ = 23.0 – 35.7 (6 C, 6 CH2), 42.9 (CH-C6H5), 48.9
(S-CH-C6H5), 73.2 (C6H5-C-C-C=S), 113.9 – 140.1 (aro-
matic C-H), 241.1 (C=S). – MS (EI, 70 eV): m/z = 406
[M–(C6H5-CH-S)]+. – Anal. for C36H32S2(528.77): calcd.
C 81.77, H 6.10, S 12.13; found C 81.9, H 6.22, S 12.19.
Reaction of 1a with phosphorus pentasulfide
Calculation: The percentage of relative viability was cal-
culated using the following equation: [absorbance of treated
cells/absorbance of control cells)] X 100.
A mixture of 1a (0.27g, 1 mmol) and P4S10 (0.02 g,
0.5 mmol) was refluxed for 2 h in 30 mL of dry toluene.
A yellow powder precipitated after the evaporation of the
volatile material. The solid material was collected and crys-
tallized from methanol to give 10a (mixed melting point and
comparative IR spectrum).
Then the half-maximum inhibitory concentration (IC50
)
was calculated from the equation of the dose response curve.
Acknowledgement
We are grateful to Dr. A. Abd El-Fatah, the Cancer
Biology labratory, the Center of Excellence for Advanced
Sciences, and the National Research Center, Dokki, Cairo,
Egypt.
Reaction of 1b with phosphorus pentasulfide
A mixture of 1b (0.24 g, 1 mmol) and P4S10 (0.02 g,
0.5 mmol) was refluxed for 2 h in 30 mL of dry toluene. The
[1] J. R. Dimmock, G. A. Zello, E. O. Oloo, J. W. Quail,
H. B. Kraatz, P. Perjesi, F. Aradi, K. Taka´cs-Nova´k,
T. M. Allen, C. L. Santos, J. Balzarini, E. De Clercq,
J. P. Stables, J. Med. Chem. 2002, 45, 3103 –
3111.
[2] A. Afonso, J. M. Kelly, J. Weinstein, R. L. Wolin, S. B.
Rosenblum, US patent 6218401, 2001; Chem. Abstr.
2001, 134, 295746.
[3] B. Joseph, D. Alagille, J. Y. Me´rour, S. Le´once, Chem.
Pharm. Bull. 2000, 48, 1872 – 1876.
Brought to you by | University of Georgia Libraries
Authenticated
Download Date | 5/30/15 9:19 AM

252
[4] M. Sriram, J. J. Hall, N. C. Grohman, T. E. Strecker,
T. Wooton, A. Franken, M. L. Trawick, K. G. Pinney,
Bioorg. Med. Chem. 2008, 16, 8161 – 8171.
[5] T. Nakayama, H. Takashi, JP 03167178 A2, 1991;
Chem. Abstr. 1991, 115, 256204.
L. S. Boulos et al. · Novel Benzosuberone Derivatives
(Eds.: M. Grayson, E. J. Griffith), Interscience Pub-
lishers, John Wiley
pp. 227 – 458.
& Sons, New York 1968,
[15] B. C. Saunders, B. P. Stark, Tetrahedron 1958, 4, 169 –
170.
[6] M. H. N. Arsanious, S. S. Maigali, L. S. Boulos, Phos-
phorus, Sulfur and Silicon and the Related Elem. 2010,
185, 57 – 67.
[7] L. S. Boulos, M. H. N. Arsanious, E. F. Ewies, Phos-
phorus, Sulfur and Silicon and the Related Elem. 2009,
184, 275 – 290.
[8] M. H. N. Arsanious, N. K. EL-Din, L. S. Boulos, Phos-
phorus, Sulfur and Silicon and the Related Elem. 2009,
184, 2813 – 2826.
[9] L. S. Boulos, M. H. N. Arsanious, E. F. Ewies,
F. Ramzy, Z. Naturforsch. 2008, 63b, 1211 – 1218.
[10] L. S. Boulos, M. H. N. Arsanious, E. M. A. Yakout,
Monatsh. Chem. 2007, 138, 979 – 984.
[16] J. March, Advanced Organic Chemistry, Reactions,
Mechanisms, and Structures, John Wiley & Sons, New
York, 1985, chap. 4, pp. 120.
[17] N. B. Colthup, L. H. Daly, S. E. Wiberley, Introduction
to Infrared and Raman Spectroscopy, Academic Press,
New York, 1964.
¨
[18] E. Ohler, E. Zbiral, Liebigs Ann. Chem. 1991, 229 –
236.
[19] A. A. El-Kateb, L. S. Boulos, H. A. Abd El-Malek,
Phosphorus, Sulfur and Silicon and the Related Elem.
1993, 83, 105 – 110.
[20] M. I. Hegab, Phosphorus, Sulfur and Silicon and the
Related Elem. 2000, 166, 137 – 148.
[11] L. S. Boulos, M. H. N Arsanious, Monatsh. Chem.
2006, 137, 1177 – 1184.
[12] L. J. Bellamy, The Infrared Spectra of Complex
Molecules, John Wiley and Sons, New York, 1964,
p. 311.
[13] V. Peesapat, K. Anuradha, P. Sreelakshmi, Synth. Com-
mun. 1999, 29, 4381 – 4395.
[14] M. M. Crutchfield, C. H. Dungan, J. H. Letcher,
V. Mark, J. R. Van Wazer, 31P Nuclear Magnetic
Resonance, Topics in Phosphorus Chemistry, Vol. 5,
[21] Y. J. Surh, Nat. Rev. Cancer 2003, 3, 768 – 780.
[22] M. B. Hansen, S. E. Nielsen, K. Berg, J. Immunol.
Methods 1989, 119, 23 – 210.
[23] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. Mc
Mahon, D. Vistica, J. T. Warren, H. Bokesch, S. Ken-
ney, M. R. Boyd, J. Natl. Cancer Inst. 1990, 82, 1107 –
1112.
[24] N. R. El-Rayyes, N. H. Bahtiti, J. Heterocycl. Chem.
1989, 26, 209 – 214.
Brought to you by | University of Georgia Libraries
Authenticated
Download Date | 5/30/15 9:19 AM