Antineoplastic activity of fused nitrogen-phosphorus heterocycles and derived phosphonates

Article abstract of DOI:10.1007/s00706-013-0950-6

A variety of derivatives incorporating substituted heterocycle-phosphor motifs is described. Substituted N,P-heterocycles and derived phosphonates were produced efficiently in a tandem operation without intermediate isolation. The synthesis methodology is based on the reaction of dialkyl phosphites with Schiff base Kabachnik-Fields intermediates, which are generated in situ from 2-amino-4,6-di-tert-butylphenol and substituted benzaldehydes in dry THF/FeCl₃ (10 %) solution, to yield fused oxazole-2-phosphonates in moderate yield (≈55 %). The latter products could be also obtained in excellent yield (≥76 %) by directly applying the same P(III) reagents to the parent Schiff bases. On the other hand, oxazaphosphinine-2-amines were isolated in high yields (≈77 %) when the Schiff bases were allowed to react with hexaalkyltriamidophosphites at rt. More P-heterocycles and the derived phosphonates were also obtained when the same reagents were applied to another imino derivative derived from the aminophenol, 2-hydroxybenzaldehyde oxime. The synthesized scaffolds were biologically evaluated and found to possess potent anticancer activities. On the basis of bioassay data, the produced N,P-heterocycles exhibit remarkable antitumor activity against 17 tested human tumor cell lines, representing breast and prostate cancer and melanoma. Several phosphonates were found to possess specific anti-breast cancer activity (especially MDA-MB-435 cell lines) while others possess specific effects against melanoma (MI4 and SK-MEL-2 cancer cell lines). These findings form a foundation for further investigation in our continuing efforts to develop selective anticancer agents.

Full text of DOI:10.1007/s00706-013-0950-6

Monatsh Chem (2013) 144:1233–1242
DOI 10.1007/s00706-013-0950-6
ORIGINAL PAPER
Antineoplastic activity of fused nitrogen-phosphorus heterocycles
and derived phosphonates
•
Wafaa M. Abdou Reham F. Barghash
•
Rizk E. Khidre
Received: 29 November 2012 / Accepted: 19 February 2013 / Published online: 8 May 2013
Ó Springer-Verlag Wien 2013
Abstract A variety of derivatives incorporating substi-
tuted heterocycle-phosphor motifs is described. Substituted
N,P-heterocycles and derived phosphonates were produced
efficiently in a tandem operation without intermediate iso-
lation. The synthesis methodology is based on the reaction
of dialkyl phosphites with Schiff base Kabachnik–Fields
intermediates, which are generated in situ from 2-amino-
4,6-di-tert-butylphenol and substituted benzaldehydes in
dry THF/FeCl3 (10 %) solution, to yield fused oxazole-
2-phosphonates in moderate yield (&55 %). The latter
products could be also obtained in excellent yield (C76 %)
by directly applying the same P(III) reagents to the parent
Schiff bases. On the other hand, oxazaphosphinine-
2-amines were isolated in high yields (&77 %) when the
Schiff bases were allowed to react with hexaalk-
yltriamidophosphites at rt. More P-heterocycles and the
derived phosphonates were also obtained when the same
reagents were applied to another imino derivative derived
from the aminophenol, 2-hydroxybenzaldehyde oxime. The
synthesized scaffolds were biologically evaluated and
found to possess potent anticancer activities. On the basis of
bioassay data, the produced N,P-heterocycles exhibit
remarkable antitumor activity against 17 tested human
tumor cell lines, representing breast and prostate cancer and
melanoma. Several phosphonates were found to possess
specific anti-breast cancer activity (especially MDA-MB-
435 cell lines) while others possess specific effects against
melanoma (MI4 and SK-MEL-2 cancer cell lines). These
findings form a foundation for further investigation in our
continuing efforts to develop selective anticancer agents.
Keywords Oxazole-phosphor motifs Á
Oxazaphosphininones Á Multicomponent reaction Á
Kabachnik–Fields reaction Á Anticancer bioassay Á
QSAR
Introduction
Cancer is among the most critical health issues and con-
sidered the second leading cause of death worldwide, just
after the circulatory diseases. Despite the availability of
improved drugs, including targeted cancer therapies,
according to the World Health Organization (WHO) the
worldwide cancer burden is expected to increase by as
much as 50 % by the year 2020 unless further preventive
measures are put into practice [1–3]. Over the last two
decades, a continuous trend is observed toward the chem-
istry of P-heterocycles and derived phosphonates, largely,
because these compounds, especially those that contain
high nitrogen contents tend to have higher antitumor
activity [4–8].
The Kabachnik-reaction has been previously reported as
a facile method for the preparation of new aminophos-
phonates and N,P-heterocycles [9–11]. Consecutively,
several of these compounds were synthesized in our pre-
vious publications via the Kabachnik–Fields reaction
starting with amine derivatives, substituted benzaldehydes,
and nucleophilic P(III)-reagents. We have found that most
of these rings possess remarkable inhibitory effect on
carcinoma cell lines as well as they have anti-inflammatory
properties [12–16].
In sequel, we are herein investigating the Kabachnik–
Fields reaction further with an attempt to look for new
antineoplastic motifs. A series of new N,P-heterocycles and
their phosphonate derivatives were synthesized by the
W. M. Abdou (&) Á R. F. Barghash Á R. E. Khidre
Chemical Industries Division, National Research Centre,
Dokki, Cairo 12311, Egypt
e-mail: wabdou@intouch.com; wabdou@link.net
123

1234
W. M. Abdou et al.
Kabachnik–Fields reaction of 2-amino-4,6-di-tert-butyl-
phenol (2) with the appropriate aromatic aldehydes 5a or
5b, followed by addition of trialkyl phosphites (TAPs) 3a–
3c, dialkyl phosphites (DAPs) 4a–4c, or hexaalk-
yltriamidophosphites 9. The investigation also highlighted
the influence of replacing the Schiff base moiety with the
methylhydroxylamine motif (C=NOH) at the ortho position
with respect to the phenolic group in the behavior toward
the same phosphorus reagents. The quantitative structure-
anticancer activity relationship (QSR) is also described in
this paper.
Scheme 2
N
CH
+
Y
O
2
+
5a, 5b
P(OR)3 / HP(OR)2
OH
3a-3c / 4a-4c
7a, 7b
O
P
OR
OR
THF/FeCl3 (10%)
- H2
OH
6a-6f
Δ
N
H
Y
8a-8f
Results and discussion
The required 2-amino-4,6-di-tert-butylphenol (2) is not
commercially available, and was therefore synthesized by
aminolysis of the parent 3,5-di-tert-butyl-1,2-benzoqui-
none (1) according to the reported method [17, 18]
(Scheme 1). The substrate 2 could be used in situ without
further purification for the next step.
The IR spectra of 6a–6f revealed the presence of NH
À1
ꢀ
around m ꢀ 3; 340 cm and the lack of stretching vibra-
tions at &3,450 cm-1 due to phenolic OH frequency (for
compounds 2 and 7: OH &3,455 cm-1). The phenolic
1
proton was not also displayed in the H NMR (CDCl3)
spectra of 6a–6f. Among other signals, the sp3–C– of the
oxazole ring appeared as a doublet (1JP–C = 138.5 Hz) at
d = 88.6 ppm in the 13C NMR (CDCl3) spectrum of 6a. A
plausible explanation for the mechanism of the reaction is
proposed in Scheme 2. According to the Kabachnik–Fields
reaction [9–11], the first step may involve the condensation
between the aldehyde and the amine in the presence of
FeCl3 and the formation of the intermediate Schiff bases 7a
and 7b, followed by the addition of the phosphorus
reagents 3a–3c or 4a–4c to produce 6a–6f via the inter-
mediates 8a–8f in a tandem extrusion of H2. We considered
that the thermal condition, coupled with the presence of an
excess of FeCl3 used for the coupling reaction, had pro-
moted the oxidation process. Furthermore, the tendency of
the intramolecular cyclization process was previously dis-
cussed for the transformation of 3,5-di-tert-butyl-2-
hydroxyphenylamino derivatives to the corresponding
benzoxazoles [17]. Nevertheless, the formation of dialkyl,
and not trialkyl adducts is acceptable since the presence of
acidic medium (FeCl3) causes the hydrolysis of TAPs to
DAP counterparts.
The Kabachnik–Fields reaction was carried out by
mixing the amine 2 with the aldehyde 5a or 5b and TAPs
3a–3c in THF/FeCl3 (10 %) solution at rt, followed by
heating under reflux for &12 h to yield the a-aminophos-
phonates 6a–6f (&55 %, Scheme 1). The oxazole ring
structure was determined to be 6 (Scheme 2) based on the
following: compatible elementary analysis and molecular
weight determinations (MS) were measured for all adducts.
Scheme 1
O
NH2
NH3 (aq) / MeOH
O
OH
1
2
O
O
Y
P(OR)3 / HP(OR)2
+
2
+
H
3a-3c / 4a-4c
5a, 5b
In favor of this mechanism, phosphonates 6a–6f
were independently synthesized in higher yield (C76 %)
by treating 2,4-di-tert-butyl-6-(4-fluorobenzylideneamino)
phenol (7a) or 2,4-di-tert-butyl-6-[4-(dimethylamino)benzy-
lideneamino]phenol (7b) with TAPs/DAPs in THF/FeCl3
(10 %) solution. The required Schiff bases 7a and 7b were
initially prepared from the condensation of the aldehydes 5
with the amine 2 in ethanol solution [17, 18] (Scheme 3).
Conversely, when the Schiff bases 7a and 7b were allowed
to react with an excess of hexaalkyltriamidophosphites 9a or
O
O
P(OR)2
THF/FeCl3 (10%)
Δ
/ - H2
N
H
Y
6a-6f
Y
R
Me
Y
R
6a 4-F-C6H4
6b 4-F-C6H4 Et
6c 4-F-C6H4 i-Pr
6d
6e
6f
4-Me2N-C6H4 Me
4-Me2N-C6H4 Et
4-Me2N-C6H4 i-Pr
123

Antineoplastic activity of fused nitrogen-phosphorus heterocycles
1235
reaction was observed between 14 and trialkyl phosphites,
even after heating the reaction mixture in toluene solution
under reflux for 48 h. On the other hand, the reaction
between dialkyl phosphites 4a–4c and 14 proceeded only
when a catalytic amount of TEA or benzoyl peroxide was
added to the reactants in absence of solvent (best yield was
obtained with benzoyl peroxide), and yielded the respective
isoxazol-3-ylphosphonates 16a–16c (&68 % yield) as it is
outlined in Scheme 5. Obviously, the phosphonates 16
resulted from the loss of a molecule of H2O from the
(hydroxyamino)methylphosphonate intermediates 15 ini-
tially formed.
Scheme 3
N
O
CH
Y
C2H5OH
Y
2
+
H
OH
5a, 5b
7a, 7b
Y = 4-F-C6H4; 4-(Me)2N-C6H4
Scheme 4
Finally, when the oxime 14 was stirred with an excess of
amidophosphites 9a or 9b in THF (or toluene) at rt for
12 h, oxaphospholes 18a and 18b were isolated in high
yields (&77 %). When 18a and 18b were treated with
H2O2 in THF solution, oxaphosphol-2-oxides 19a and 19b
(&77 %, dP = 12.7, 14.2 ppm) were formed as it was
predicted in Scheme 6.
OH
P(NR'2)3
THF
r.t.
P(NR'2)3
7a, 7b
+
N
H
Y
9a, 9b
10a-10d
O
O
H2O2
Satisfactory elementary analysis and molecular weight
determinations (MS) confirmed structures 18 and 19, 31P
NMR signals of 18a and 18b were found at d = 123.6 and
120.5 ppm. The 1H NMR spectrum (CDCl3) of 18a showed
-2NHR'2
O
N
PNR'2
PNR'2
only 11a,11c
Y
Y
N
12a, 12b
among others the NMe2 protons as a doublet (3JP–H
=
11a-11d
10.4 Hz) at d = 3.23 ppm. The 13C NMR (CDCl3) spec-
trum of 18a displayed the P–C=N signal at d = 143.6 ppm
(d, JP–C = 166.3 Hz).
11a,12a, Y = 4-F-C6H4; R' = Me; 11b, Y = 4-F-C6H4; R' = Et; 11c,
12b, Y = 4-(Me)2N-C6H4; R' = Me; 11d, Y = 4-(Me)2N-C6H4; R' = Et
1
Antitumor activity screening
9b in dry THF, the reactions went smoothly at rt (8 h) and
afforded the oxazaphosphinines 11a–11d in high yield
(&77 %). The synthesis route in Scheme 4 [19] shows that 11
was formed through the initial formation of the phosphini-
mines 10, followed by intramolecular cyclization reaction to
give the oxazaphosphinines 11 with concomitant elimination
of two moles of dialkylamine. The structures of 11a–11d were
Antitumor activity for the synthesized nitrogen-phosphorus
heterocycles and derived phosphonates 6a–6e, 12a, 12b,
Scheme 5
1
confirmed by H NMR, 13C NMR, 31P NMR, IR, MS spec-
CHO
troscopic data, and elemental analysis. Compounds 11a–11d
were quite stable at normal conditions, nevertheless, for the
pharmacological studies, 11a and 11c, as representative
examples, were oxidized to the oxazaphosphininone form 12a,
12b by boiling in THF containing hydrogen peroxide. Com-
pounds 12a, 12b showed 31P NMR (CHCl3) chemical shifts
around d = 11.8 and 13.6 ppm, confirming the presence of the
C–P–N linkage as an oxazaphosphininone moiety [20, 21].
This finding eliminates the formation of a phosphonate for
which a signal at d[17 ppm would be expected.
The investigation was next extended to obtain additional
substituted N,P-heterocycles and the corresponding phos-
phonate derivatives by applying the same P(III) reagents to
the oxime 14. The substrate 14 was obtained by treating
the parent 3,5-di-tert-butyl-2-hydroxybenzaldehyde (13)
with hydroxylamine hydrochloride [22]. Unfortunately, no
+ NH2OH·HCl
NOH
OH
OH
EtOH / Δ
14
13
O
neat
+
14
HP(OR)2
(PhCO)2O2
4a-4c
O
P(OR)2
O
NH
OH
OH
- H2O
- H2
N
P(OR)2
O
15a-15c
16a-16c
16a, R = Me; 16b, R = Et; 16c, R = i-Pr
123

1236
W. M. Abdou et al.
16a, 16b, 19a, and 19b at a dose of 10 lM utilizing sev-
enteen different human tumor cell lines representing breast
and prostate cancer and melanoma was carried out
according to the previously reported methods [23, 24].
Substrates 7a and 14 were also biologically tested in a trial
to reflect the effect of introducing phosphor moiety. The
obtained results represent percentage growth of the tumor
cell lines treated with compounds under investigation rel-
ative to control cell.
The pharmacological results displayed in Table 1 show
that other than the substrates 7a and 14, all tested com-
pounds reflect remarkable antitumor activity against breast
(especially MDA-MB-23/ATCC cell lines) and prostate
cancer, whereas a moderate effect was observed on mela-
noma. On the other hand, only the fused phosphor-
heterocycles 12a, 12b, 19a, and 19b showed sensitivity
against melanoma. However, we considered that cell line
growth inhibition with[50 % at a concentration of 10 lM
usually seems to be a noticeable activity. Structural activity
relationship (SAR) correlation for the reported observa-
tions reveals that the presence of the fused phosphorus ring
usually associated with an enhancement in the antitumor
properties as indicated in our observations in Table 1.
Scheme 6
NHOH
14
P(NR'2)3
OH
THF
r.t.
- 2HNR'2
+
P(NR'2)3
9a, 9b
17a, 17b
Conclusion
NOH
NOH
Summarizing, previous [4–8] and present investigations
have shown that multicomponent reactions in a one-pot
synthesis are of significant value from both the synthetic,
chemical, and biological points of view. The convenience
and novelty of this work is reflected in its several
advantages, such as mild reaction conditions, short reac-
tion time, and easy workup procedure without the need to
chromatographic purification. The present study has also
NR'2
H2O2
PNR'2
P
O
O
O
18a, 18b
19a, 19b
18a,19a, R' = Me; 18b,19b, R' = Et
Table 1 Concentrations resulting in growth inhibition of 50 % (GI50/mg dm-3) of in vitro human tumor cell lines
Panel/cell line
Compounds
7a/14
6a
6b
6c
6d
6e
12a
12b
16a
16b
19a
19b
Breast cancer
McF7
[48
[48
[48
[48
[48
[48
[48
9.48
10.85
17.56
10.43
20.42
6.05
17.58
26.50
20.68
24.80
19.60
23.3
11.42
16.21
14.35
22.64
13.4
8.40
5.27
5.27
10.25
19.16
13.12
20.74
16.33
12.44
12.38
11.85
17.56
13.48
19.34
16.97
12.44
12.18
8.84
9.45
NCl/ADR-RES
MDA-MB-231/ATCC
HS578T
18.31
9.73
20.1
6.76
8.65
6.08
9.23
8.97
11.42
16.92
10.05
16.32
14.12
9.88
13.22
15.43
10.56
17.13
14.76
14.98
20.42
14.05
11.97
12.14
18.72
7.52
12.10
13.44
15.85
15.06
10.42
13.07
15.25
12.11
MDA-MB-435
BT-549
12.76
11.88
12.44
12.18
11.97
13.08
T-47D
30.95
Prostate cancer
PC-3
[48
[48
20.46
15.3
18.73
29.6
14.5
10.82
15.3
19.4
8.75
6.86
7.82
18.03
16.47
18.37
14.34
14.2
7.57
16.21
8.73
DU-145
[52
10.98
10.24
Melanoma
LOXIMVI
MALME-3M
MI4
[50
[50
[50
[50
[50
[50
[50
[50
30.15
14.33
17.25
28.56
16.35
18.54
8.64
33.41
14.74
15.72
27.03
14.38
11.44
13.44
20.51
33.42
28.61
40.23
30.35
30.36
14.22
19.23
33.26
34.51
14.73
5.64
34.11
16.43
7.66
9.38
10.36
12.57
15.78
16.96
17.76
10.16
12.16
8.30
8.66
24.52
31.31
27.52
26.47
30.03
22.34
23.25
23.66
24.52
31.31
27.52
26.47
30.32
37.4
13.24
11.75
12.15
14.23
26.61
12.42
10.54
9.52
15.16
12.55
12.95
14.92
24.21
12.06
11.88
9.52
15.72
13.43
14.09
14.16
10.46
11.62
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
10.33
14.35
11.44
8.64
10.44
15.75
13.64
8.21
21.22
24.77
20.17
22.21
27.61
123

Antineoplastic activity of fused nitrogen-phosphorus heterocycles
1237
demonstrated that although the initial step is the nucleo-
philic attack by the P(III) reagents at the azomethine-C
portion in the two C–N multiple bond substrates, the anils
7 and the oxime 14, the consequences of the initial step
varied markedly according to the type of both the func-
tional group and the type of the phosphorus reagent. In
parallel, it has also been noticed that some of these motifs
(e.g., compounds 6a and 6b) were found to possess spe-
cific anti-breast cancer activity (especially to MDA-MB-
435 cell lines) and others (e.g., compounds 6d and 6e)
possess specific effects against melanoma (MI4 and SK-
MEL-2 cell lines). Importantly, the introduction of an
oxazole-phosphor motif framework produced very potent
anticancer compounds. Finally, on the basis of bioassay
results, the produced N,P-heterocycles 12a and 12b
exhibited remarkable antitumor activity against the sev-
enteen tested human tumor cell lines representing breast
and prostate cancer and melanoma. They could be con-
sidered as lead molecules to be modified in order to
improve their anticancer activity. Furthermore, these
findings form the foundation for further investigation in
our continuing efforts to develop selective anticancer
agents.
(trimethyl (3a), triethyl (3b), or triisopropyl phosphite (3c),
6.2 mmol) in 10 cm3 of dry THF/FeCl3 (10 %) was heated
under reflux for 10–15 h. After completion of the reaction
(the evolved H2 gas was observed but not tested), 10 cm3
AcOEt was added to the mixture. The organic phase was
separated, washed with 20 cm3 distilled H2O, and dried
over anhydrous Na2SO4. Solvents were evaporated under
reduced pressure, and the residue therein was crystallized
from the proper solvent to give the corresponding phos-
phonates 6a–6f.
The products 6a–6f were obtained in analogy, almost in
the same yield, when DAPs [dimethyl (4a), diethyl (4b), or
diisopropyl phosphite (4c)] replaced the TAP counterpart
in the above reactions whereas experimental conditions,
stoichiometric amounts, and work up were inclusively
used.
Dimethyl 5,7-di-tert-butyl-2-(4-fluorophenyl)-2,3-
dihydrobenzo[d]oxazol-2-ylphosphonate
(6a, C23H31FNO4P)
Colorless crystals; m.p.: 119 °C (cyclohexane); yield: 1.4 g
ꢀ
(53 %); IR (KBr): m = 3,350w (NH), 1,240 (P=O), 1,164
1
(P–O–C) cm-1; H NMR (500.7 MHz, CDCl3): d = 1.35,
1.40 [2 s, 2 9 9H, 2(Me3C)], 3.70 (d, JP–H = 11.6 Hz,
3
6H, (MeO)2P), 6.23 (d, JH–H = 4.2 Hz, 1H, H(4)-Ar), 6.93
(d, JH–H = 4.2 Hz, 1H, H(6)-Ar), 7.40, 7.70 (2 m, 4H, H–
Ar), 8.78 (br s, 1H, HN) ppm; 13C NMR (125.4 MHz,
Experimental
CDCl3): d = 163.5, 149.0, 148.7, 134.2, 132.2, 128.5,
Melting points were determined with an open capillary
tube on an Electrothermal (variable heater) melting point
apparatus and are corrected. IR spectra were recorded on a
Perkin Elmer 297 grating IR spectrophotometer using KBr
pellets. NMR spectra were measured with a JEOL E.C.A-
1
120.8, 117.3, 116.7, 112.0 (C–Ar), 88.6 (d, JP–C
=
2
138.5 Hz, C–P), 54.6 (d, JP–C = 14.8 Hz, (MeO)2P),
37.7, 30.5 (2CMe3), 31.1, 29.6 (2Me3C) ppm; 31P NMR
(200.7 MHz, CDCl3): d = 27.6 ppm; MS (EI, 70 eV): m/z
(%) = 434 (35) [M?-1], 414 (23) [M?-20, (H ? F)],
305 (100) [M?-129, (H ? F ? P(O)(OMe)2)], 191 (84),
109 (66) [P(O)(OMe)2)], 77 (80).
1
500 MHz (13C: 125 MHz, H: 500 MHz, 31P: 200 MHz)
spectrometer. 31P NMR spectra were recorded with H3PO4
1
(85 %) as an external reference. H and 13C NMR spectra
were measured using SiMe4 as an internal reference in
CDCl3 or DMSO-d6. Chemical shifts (d) are given in ppm.
Mass spectrometry was performed on a JEOL JMS-AX 500
spectrometer. The appropriate precautions in handling
moisture-sensitive compounds were considered. Solvents
were dried using standard techniques. Thin-layer chroma-
tography (TLC) used Merck 0.2 mm silica gel 60 F254
aluminum plates. Elemental analyses were carried out at
the Microanalysis Laboratory, Cairo University, Cairo,
Egypt, using Elemental C, H, N analyzer Vario EL III
Germany. Their values agreed favorably with the calcu-
lated ones.
Diethyl 5,7-di-tert-butyl-2-(4-fluorophenyl)-2,3-
dihydrobenzo[d]oxazol-2-ylphosphonate
(6b, C25H35FNO4P)
Colorless crystals; m.p.: 114 °C (pentane); yield: 1.5 g
ꢀ
(54 %); IR (KBr): m = 3,345w (NH), 1,245 (P=O), 1,125
1
(P–O–C) cm-1; H NMR (500.7 MHz, CDCl3): d = 1.29
(dt, JH–H = 6.6 Hz, 4JP–H = 4.8 Hz, 6H, (MeCO)2P), 1.37,
1.44 [2 s, 2 9 9H, 2(Me3C)], 4.22 (dq, JH–H = 6.6,
3JP–H = 5.7 Hz, 4H, (H2CO)2P), 6.35 (d, JH–H = 4.2 Hz,
1H, H(4)-Ar), 6.96 (d, JH–H = 4.2 Hz, 1H, H(6)-Ar), 7.36,
7.82 (2 m, 4H, H–Ar), 8.91 (br s, 1H, HN) ppm; 13C NMR
(125.4 MHz, CDCl3): d = 163.9, 149.2, 148.6, 134.0,
132.3, 128.2, 119.4, 117.5, 116.4, 112.2 (C–Ar), 90.2 (d,
1JP–C = 136 Hz, C–P), 63.2 (d, 2JP–C = 14.8 Hz,
(CH2O)2P), 38.7, 30.5 (2CMe3), 31.2, 29.4 (2Me3C), 16.2
General procedure for the one-pot preparation
of phosphonates 6a–6f
3
(d, JP–C = 7.8 Hz, MeCOP) ppm; 31P NMR (200.7 MHz,
A stirred mixture of 0.8 g of aldehyde 5a, 5b (6 mmol),
1.3 g of amine 2 (6 mmol), and trialkyl phosphite
CDCl3): d = 27.9 ppm; MS (EI, 70 eV): m/z (%) = 462
(23) [M?-1], 442 (53) [M?-20, (H ? F)], 305 (100)
123

1238
W. M. Abdou et al.
[M?-157, (H ? F ? P(O)(OEt)2)], 191 (90), 138 (62)
ppm; 13C NMR (125.4 MHz, CDCl3): d = 151.2, 149.6,
148.2, 133.7, 132.6, 127.0, 117.5, 117.2, 113.0, 112.4
[P(O)(OEt)2)], 77 (78).
1
(C–Ar), 102.1 (d, JP–C = 140 Hz, C–P), 63.2 (d,
Diisopropyl 5,7-di-tert-butyl-2-(4-fluorophenyl)-2,3-dihy-
drobenzo[d]oxazol-2-ylphosphonate (6c, C27H39FNO4P)
Colorless crystals; m.p.: 123 °C (cyclohexane); yield: 1.7 g
2JP–C = 14.8 Hz, (CH2O)2P), 40.4 (Me2N), 39.7, 30.5
3
(2CMe3), 32.5, 29.2 (2Me3C), 16.8 (d, JP–C = 6.8 Hz,
MeCOP) ppm; 31P NMR (200.7 MHz, CDCl3):
d = 23.8 ppm; MS (EI, 70 eV): m/z (%) = 487 (17)
[M?-1], 442 (22) [M?-45, (H ? NMe2)], 305 (100)
[M?-182, (H ? NMe2 ? P(O)(OEt)2)], 191 (74), 137
(48) [P(O)(OEt)2], 77 (83).
ꢀ
(58 %); IR (KBr): m = 3,336w (NH), 1,242 (P=O), 1,064
1
(P–O–C) cm-1; H NMR (500.7 MHz, CDCl3): d = 1.25
(dd, JH–H = 6.5 Hz, JP–H = 5.1 Hz, 12H, (Me2CO)2P),
4
1.35, 1.40 [2 s, 2 9 9H, 2(Me3C)], 4.28 (dsept,
3
JH–H = 6.5, JP–H = 5.8 Hz, 2H, (HCO)2P), 6.26 (d,
JH–H = 4.2 Hz, 1H, H(4)-Ar), 6.86 (d, JH–H = 4.2 Hz,
1H, H(6)-Ar), 7.34, 7.76 (2 m, 4H, H–Ar), 8.88 (br s, 1H,
HN) ppm; 13C NMR (125.4 MHz, CDCl3): d = 164.0,
150.2, 148.3, 133.8, 132.0, 128.0, 119.1, 117.7, 116.3,
Diisopropyl 5,7-di-tert-butyl-2-[4-(dimethylamino)phenyl]-
2,3-dihydrobenzo[d]oxazol-2-yl-phosphonate
(6f, C29H45N2O4P)
Straw yellow crystals; m.p.: 140 °C (MeCN); yield: 1.6 g
1
ꢀ
112.7 (C–Ar), 103.5 (d, JP–C = 135.5 Hz, C–P), 69.4 (d,
(52 %); IR (KBr): m = 3,356w (NH), 1,236 (P=O), 1,144
2JP–C = 9.5 Hz, (CHO)2P), 39.7, 30.5 (2CMe3), 31.1, 29.6
(2Me3C), 23.7 (d, 3JP–C = 7.9 Hz, Me2CO) ppm; 31P NMR
(200.7 MHz, CDCl3): d = 31.2 ppm; MS (EI, 70 eV): m/z
(%) = 490 (28) [M?-1], 470 (35) [M?-20, (H ? F)],
305 (100) [M?-185, (H ? F ? P(O)(OC3H7)2)], 191 (92),
165 (55) [P(O)(OC3H7)2)], 77 (85).
(P–O–C) cm-1; H NMR (500.7 MHz, CDCl3): d = 1.22
1
4
(dd, JH–H = 6.8 Hz, JP–H = 5.8 Hz, 12H, (Me2CHO)2P),
1.33, 1.51 (2 s, 2 9 9H, 2(Me3C)), 3.09 (s, 6H, Me2N),
3
4.35 (dsept, JH–H = 6.8, JP–H = 4.6 Hz, 2H, (HCO)2P),
6.25 (d, JH–H = 4.2 Hz, 1H, H(4)-Ar), 6.95 (d,
JH–H = 4.2 Hz, 1H, H(6)-Ar), 7.27, 7.69 (2 m, 4H, H–
Ar), 8.94 (br s, 1H, HN) ppm; 13C NMR (125.4 MHz,
CDCl3): d = 150.2, 149.4, 148.0, 134.0, 132.5, 127.8,
117.5, 116.4, 114.0, 112.5 (C–Ar), 103.5 (d,
1JP–C = 136.5 Hz, C–P), 69.5 (d, 2JP–C = 8.5 Hz,
(CHO)2P), 40.4 (Me2N), 38.7, 29.5 (2CMe3), 32.2, 29.0
(2Me3C), 23.5 (d, 3JP–C = 7.9 Hz, Me2CO) ppm; 31P NMR
(200.7 MHz, CDCl3): d = 25.8 ppm; MS (EI, 70 eV): m/z
(%) = 515 (15) [M?-1], 470 (47) [M?-45,
(H ? NMe2)], 305 (100) [M?-210, (H ? N(Me)2 ?
P(O)(OC3H7)2)], 191 (72), 165 (47) [P(O)(OC3H7)2], 77
(69).
Dimethyl 5,7-di-tert-butyl-2-[4-(dimethylamino)phenyl]-
2,3-dihydrobenzo[d]oxazol-2-ylphosphonate
(6d, C25H37N2O4P)
Straw yellow needles; m.p.: 133 °C (CH2Cl2); yield: 1.5 g
ꢀ
(55 %); IR (KBr): m = 3,334w (NH), 1,248 (P=O), 1,155
1
(P–O–C) cm-1; H NMR (500.7 MHz, CDCl3): d = 1.35,
1.47 [2 s, 2 9 9H, 2(Me3C)], 2.93 (s, 6H, (Me)2N), 3.78
3
(d, JP–H = 12.3 Hz, 6H, (MeO)2P), 6.28 (d, JH–H
=
4.2 Hz, 1H, H(4)-Ar), 6.95 (d, JH–H = 4.2 Hz, 1H, H(6)-
Ar), 7.28, 7.48 (2 m, 4H, H–Ar), 9.07 (br s, 1H, HN) ppm;
13C NMR (125.4 MHz, CDCl3): d = 150.2, 149.0, 148.7,
134.0, 132.6, 127.2, 118.2, 117.9, 113.7, 112.1 (C–Ar),
Preparation of the Schiff bases 7a and 7b
1
2
95.6 (d, JP–C = 129.5 Hz, C–P), 54.6 (d, JP–C
=
14.8 Hz, (MeO)2P), 40.4 (Me2N), 37.5, 30.5 (2CMe3),
32.4, 29.5 (2Me3C) ppm; 31P NMR (200.7 MHz, CDCl3):
d = 24.6 ppm; MS (EI, 70 eV): m/z (%) = 459 (18)
[M?-1], 414 (67) [M?-45, (H ? NMe2)], 305 (100)
[M?-154, (H ? NMe2 ? P(O)(OMe)2)], 191 (87), 109
(72) [P(O)(OMe)2], 77 (85).
According to Refs. [17, 18], a mixture of 8.0 g of 2-amino-
4,6-di-tert-butylphenol (2, 36.2 mmol) and the aldehyde 5a
or 5b (36.3 mmol) in 50 cm3 ethanol was refluxed for 3 h.
The product mixture was further concentrated to its half,
followed by filtration. The collected material was crystal-
lized from ethanol to give 2,4-di-tert-butyl-6-(4-
fluorobenzylideneamino)phenol (7a, 10 g, 84 %) or 2,4-di-
tert-butyl-6-[4-(dimethylamino)benzylideneamino]phenol
(7b, 11.2 g, 87 %).
Diethyl 5,7-di-tert-butyl-2-[4-(dimethylamino)phenyl]-2,3-
dihydrobenzo[d]oxazol-2-ylphosphonate
(6e, C27H41N2O4P)
Straw yellow needles; m.p.: 127 °C (CH2Cl2); yield: 1.6 g
ꢀ
(56 %); IR (KBr): m = 3,340w (NH), 1,235 (P=O), 1,110
Synthesis of 6a–6f by reaction of 7a, 7b with dialkyl
phosphites 4a–4c
1
(P–O–C) cm-1; H NMR (500.7 MHz, CDCl3): d = 1.29
(dt, JH–H = 6.6 Hz, 4JP–H = 4.8 Hz, 6H, (MeCO)2P), 1.31,
1.47 [2 s, 2 9 9H, 2(Me3C)], 2.86 (s, 6H, (Me)2N), 4.17
A mixture of 0.8 g of the Schiff bases 7a, 7b (2.2 mmol)
and dimethyl (4a), diethyl (4b), or diisopropyl phosphite
(4c) (2.5 mmol) in 15 cm3 THF containing 10 % FeCl3
was heated under reflux for 10–15 h (TLC). After the usual
3
(dq, JH–H = 6.6, JP–H = 6.2 Hz, 4H, (H2CO)2P), 6.22 (d,
JH–H = 4.2 Hz, 1H, H(4)-Ar), 6.78 (d, JH–H = 4.2 Hz, 1H,
H(6)-Ar), 7.28, 7.69 (2 m, 4H, H–Ar), 8.83 (br s, 1H, HN)
123

Antineoplastic activity of fused nitrogen-phosphorus heterocycles
1239
6,8-Di-tert-butyl-3-[4-(dimethylamino)phenyl]-N,N-dime-
working up, as it is described in the general procedure, and
crystallization from the proper solvent, phosphonates 6a–6f
were isolated: 6a: 727 mg (76 %); 6b: 795 mg (78 %); 6c:
864 mg (80 %); 6d: 779 mg (77 %); 6e: 827 mg (77 %);
6f: 863 mg (76 %). Compounds 6a–6f were characterized
by m.p., mixed m.p., and comparable IR spectra with the
material previously obtained.
thyl-2H-benzo[e][1,4,2]oxazaphosphinin-2-amine
(11c, C25H36N3OP)
Yellow crystals; m.p.: 159 °C (CHCl3); yield: 748 mg
-1
ꢀ
(80 %); IR (KBr): m = 1,334, 856 (P–NMe2) cm
;
1H
NMR (500.7 MHz, CDCl3): d = 1.42, 1.48 (2 s, 2 9 9H,
3
2(Me3C)), 2.96 (s, 6H, Me2N-Ar), 3.38 (d, JP–H
=
11.4 Hz, 6H, Me2N–P), 6.33 (d, JH–H = 4.2 Hz, 1H,
H(4)-Ar), 6.99 (d, JH–H = 4.2 Hz, 1H, H(6)-Ar), 7.31,
7.53 (2 m, 4H, H–Ar) ppm; 13C NMR (125.4 MHz,
Synthesis of 11a and 11b by reaction of the Schiff bases
7a, 7b with hexaalkyltriamidophosphites 9a and 9b
CDCl3): d = 151.1, 149.5, 143.0, 142.5, 140.2, 129.6,
1
127.0, 120.3, 118.5, 113.2 (C–Ar), 154.1 (d, JP–C
=
The amidophosphite 9a (R’ = Me) or 9b (R’ = Et)
(2.3 mmol) in 4 cm3 dry THF was added dropwise to 0.8 g
7a, 7b (2.2 mmol) in 15 cm3 THF and the reaction mixture
was stirred at rt for 8 h (TLC). The solvent was evaporated
under vacuum, the residue was collected, washed with light
petroleum, and crystallized from the proper solvent to give
compounds 11a–11d.
143.3 Hz, C–P), 39.9 (s, 6H, Me2N), 37.5 (d,
2JP–C = 22.2 Hz, Me2N–P), 39.6, 30.9 (2CMe3), 30.5,
28.9 (2Me3C) ppm; 31P NMR (200.7 MHz, CDCl3):
d = 123.6 ppm; MS (EI, 70 eV): m/z (%) = 425 (13)
[M?], 381 (22) [M?-44, NMe2], 306 (100) [M?-119,
(NMe2 ? PNMe2)], 191 (77), 77 (92).
6,8-Di-tert-butyl-3-[4-(dimethylamino)phenyl]-N,N-di-
ethyl-2H-benzo[e][1,4,2]oxazaphosphinin-2-amine
(11d, C27H40N3OP)
6,8-Di-tert-butyl-3-(4-fluorophenyl)-N,N-dimethyl-2H-
benzo[e][1,4,2]oxazaphosphinin-2-amine
(11a, C23H30FN2OP)
Straw yellow crystals; m.p.: 145 °C (MeCN); yield:
Yellow crystals; m.p.: 147 °C (n-hexane); yield: 563 mg
(78 %); IR (KBr): m = 1,320, 863 (P–NMe2) cm
-1
;
1H
ꢀ
697 mg (75 %); IR (KBr): m = 1,344, 836 (P–NEt2)
ꢀ
cm-1
;
1H NMR (500.7 MHz, CDCl3): d = 0.89 (dt,
NMR (500.7 MHz, CDCl3): d = 1.30, 1.36 [2 s, 2 9 9H,
2(Me3C)], 2.89 (d, JP–H = 10.8 Hz, 6H, Me2N–P), 6.25
4
3
JH–H = 6.2, JP–H = 3.5 Hz, 6H, MeCN–P), 1.30, 1.36
[2 s, 2 9 9H, 2(Me3C)], 2.96 (s, 6H, Me2N-Ar), 3.21 (dq,
3
JH–H = 6.2, JP–H = 5.8 Hz, 4H, H2CN–P), 6.23 (d,
(d, JH–H = 4.2 Hz, 1H, H(4)-Ar), 6.97 (d, JH–H = 4.2 Hz,
1H, H(6)-Ar), 7.33, 7.64 (2 m, 4H, H–Ar) ppm; 13C NMR
(125.4 MHz, CDCl3): d = 163.0, 148.8, 144.1, 142.5,
140.0, 132.4, 127.9, 120.9, 118.9, 116.2 (C–Ar), 154.1
JH–H = 4.2 Hz, 1H, H(4)-Ar), 6.93 (d, JH–H = 4.2 Hz,
1H, H(6)-Ar), 7.00, 7.49 (2 m, 4H, H–Ar) ppm; 13C NMR
(125.4 MHz, CDCl3): d = 153.0, 149.5, 141.8, 141.1,
140.5, 129.0, 126.4, 120.3, 117.5, 112.5 (C–Ar), 155.7
1
2
(d, JP–C = 166.3 Hz, C–P), 38.0 (d, JP–C = 25.2 Hz,
Me2N–P), 37.6, 30.5 (2CMe3), 31.3, 29.9 (2Me3C) ppm;
31P NMR (200.7 MHz, CDCl3): d = 121.2 ppm; MS (EI,
70 eV): m/z (%) = 400 (21) [M?], 381 (28) [M?-19, F],
306 (100) [M?-94, (F ? PNMe2)], 191 (52), 77 (83).
1
2
(d, JP–C = 163.7 Hz, C–P), 41.4 (d, JP–C = 25.7 Hz,
CH2–N–P), 39.9 (s, Me2N–P), 37.6, 30.9 (2CMe3), 31.5,
3
30.3 (2Me3C), 15.8 (d, JP–C = 8.8 Hz, MeCN–P) ppm;
31P NMR (200.7 MHz, CDCl3): d = 118.4 ppm; MS (EI,
70 eV): m/z (%) = 453 (20) [M?], 409 (17) [M?-44,
NMe2], 306 (100) [M?-147, (NMe2 ? PNEt2)], 191 (62),
77 (95).
6,8-Di-tert-butyl-N,N-diethyl-3-(4-fluorophenyl)-2H-
benzo[e][1,4,2]oxazaphosphinin-2-amine
(11b, C25H34FN2OP)
Yellow crystals; m.p.: 137 °C (CH2Cl2); yield: 678 mg
-1
(72 %); IR (KBr): m = 1,328, 848 (P–NEt2) cm
;
1H
ꢀ
Synthesis of 12a and 12b by oxidation of 11a and 11c
NMR (500.7 MHz, CDCl3): d = 0.98 (dt, JH–H = 6.8,
4JP–H = 4.6 Hz, 6H, Me2CN–P), 1.35, 1.42 [2 s, 2 9 9H,
2(Me3C)], 3.97 (dq, JH–H = 6.8, JP–H = 5.7 Hz, 2H,
H2CN–P), 6.24 (d, JH–H = 4.2 Hz, 1H, H(4)-Ar), 6.88 (d,
JH–H = 4.2 Hz, 1H, H(6)-Ar), 7.38, 7.88 (2 m, 4H, H–Ar)
ppm; 13C NMR (125.4 MHz, CDCl3): d = 163.5, 149.2,
142.5, 141.9, 140.3, 132.8, 127.1, 121.4, 118.5, 116.0 (C–
When the compounds 11a and 11c were treated with H2O2
in THF solution at rt, the oxazaphosphininones 12a and
12b were formed after 1 h.
6,8-Di-tert-butyl-3-(4-fluorophenyl)-N,N-dimethyl-2H-ben-
zo[e][1,4,2]oxazaphosphininone-2-amine
(12a, C23H30FN2O2P)
1
Ar), 155.7 (d, JP–C = 160.6 Hz, C–P), 41.4 (d,
2JP–C = 25.7 Hz, CH2–N–P), 38.7, 30.6 (2CMe3), 32.2,
Straw yellow crystals; m.p.: 167 °C (EtOH); yield: 713 mg
3
ꢀ
(78 %); IR (KBr): m = 1,262 (P=O), 1,338, 874 (P–NMe2)
29.2 (2Me3C), 15.2 (d, JP–C = 8.8 Hz, MeCN–P) ppm;
cm-1; 1H NMR (500.7 MHz, CDCl3): d = 1.30, 1.36 [2 s,
2 9 9H, 2(Me3C)], 3.11 (d, JP–H = 10.4 Hz, 6H, Me2N–
31P NMR (200.7 MHz, CDCl3): d = 123.1 ppm; MS (EI,
70 eV): m/z (%) = 428 (13) [M?], 409 (28) [M?-19, F],
306 (100) [M?-122, (F ? PNEt2)], 191 (52), 77 (83).
3
P), 6.35 (d, JH–H = 4.2 Hz, 1H, H(4)-Ar), 6.83 (d,
123

1240
W. M. Abdou et al.
1
JH–H = 4.2 Hz, 1H, H(6)-Ar) ppm; 31P NMR (200.7 MHz,
CDCl3): d = 11.8 ppm; MS (EI, 70 eV): m/z (%) = 416
(24) [M?], 397 (33) [M?-19, F], 306 (100) [M?-110,
(F ? P(O)NMe2)], 191 (57), 77 (80).
(P–O–C) cm-1; H NMR (500.7 MHz, CDCl3): d = 1.31
(dt, JH–H = 6.8 Hz, 4JP–H = 5.8 Hz, 6H, (MeCO)2P), 1.35,
1.53 [2 s, 2 9 9H, 2(Me3C)], 4.24 (dq, JH–H = 6.8,
3JP–H = 5.8 Hz, 4H, (H2CO)2P), 6.26 (d, JH–H = 4.2 Hz,
1H, H(4)-Ar), 6.87 (d, JH–H = 4.2 Hz, 1H, H(6)-Ar) ppm;
13C NMR (125.4 MHz, CDCl3): d = 153.0, 143.7, 135.8,
6,8-Di-tert-butyl-3-[4-(dimethylamino)phenyl]-N,N-dime-
thyl-2H-benzo[e][1,4,2]oxazaphosphininone-2-amine
(12b, C25H36N3O2P)
1
128.2, 122.7, 118.9 (C–Ar), 135.9 (d, JP–C = 168 Hz, C–
2
P), 62.2 [d, JP–C = 14.8 Hz, (CH2O)2P)], 36.2, 34.5
Straw yellow crystals; m.p.: 177 °C (EtOH); yield: 776 mg
3
(2CMe3), 30.8, 29.4 (2Me3C), 15.8 (d, JP–C = 6.8 Hz,
ꢀ
(80 %); IR (KBr): m = 1,255 (P=O), 1,325, 867 (P–NMe2)
(MeCO)2P) ppm; 31P NMR (200.7 MHz, CDCl3):
d = 25.7 ppm; MS (EI, 70 eV): m/z (%) = 367 (45)
[M?], 230 (100) [M?-137, (P(O)(OEt)2)], 191 (70), 138
(64), 77 (84).
cm-1; 1H NMR (500.7 MHz, CDCl3): d = 1.29, 1.39 [2 s,
2 9 9H, 2(Me3C)], 2.93 (s, 6H, (Me)2N), 3.25 (d,
3JP–H = 10.4 Hz, 6H, Me2N–P), 6.22 (d, JH–H = 4.2 Hz,
1H, H(4)-Ar), 6.93 (d, JH–H = 4.2 Hz, 1H, H(6)-Ar), 6.94
(d, JH–H = 4.2 Hz, 1H, H(6)-Ar), 7.00, 7.49 (m, 4H, H–Ar)
ppm; 31P NMR (200.7 MHz, CDCl3): d = 13.6 ppm; MS
(EI, 70 eV): m/z (%) = 441 (11) [M?], 397 (48) [M?-44,
NMe2], 306 (100) [M?-135, (NMe2 ? P(O)NMe2)], 191
(62), 77 (82).
Diisopropyl 5,7-di-tert-butylbenzo[d]isoxazol-3-ylphos-
phonate (16c, C21H34NO4P)
Colorless crystals, m.p.: 155 °C (CHCl3); yield: 884 mg
-1
;
ꢀ
(70 %); IR (KBr): m = 1,265 (P=O), 1,165 (P–O–C) cm
1H NMR (500.7 MHz, CDCl3): d = 1.21 (dd,
JH–H = 6.6 Hz, JP–H = 3.8 Hz, 2 9 6H, (Me2CHO)2P),
4
Synthesis of the phosphonate derivatives 16a–16c
by reaction of the oxime 14 with dialkyl phosphites
4a–4c
1.35, 1.53 [2 s, 2 9 9H, 2(Me3C)], 4.43 (dsept,
3
JH–H = 6.6, JP–H = 7.2 Hz, 2H, (HC(Me)2O)2P), 6.31
(d, JH–H = 4.2 Hz, 1H, H(6)-Ar), 6.97 (d, JH–H = 4.2 Hz,
1H, H(6)-Ar) ppm; 13C NMR (125.4 MHz, CDCl3):
d = 154.5, 142.4, 134.8, 126.8, 121.2, 117.8 (C–Ar),
To a mixture of 0.8 g of 3,5-di-tert-butyl-2-hydro-
xybenzaldehyde oxime (14, 3.2 mmol) and dimethyl (4a),
diethyl (4b), or diisopropyl phosphite (4c) (3.3 mmol), a
catalytic amount of benzoylperoxide was added. The
reaction mixture was further refluxed for 12–15 h (evolu-
tion of H2 gas was observed but not tested). After the usual
working up, as it is described in the general procedure, and
crystallization from the proper solvent the phosphonates
16a–16c were isolated.
1
2
137.5 (d, JP–C = 192 Hz, C–P), 70.8 (d, JP–C = 8.5 Hz,
(CHO)2P), 37.2, 35.0 (2CMe3), 32.4, 28.6 (2Me3C), 23.4
3
(d, JP–C = 7.9 Hz, Me2CO) ppm; 31P NMR (200.7 MHz,
CDCl3): d = 26.5 ppm; MS (EI, 70 eV): m/z (%) = 395
(48) [M?], 230 (100) [M?-165, (P(O)(OC3H7)2)], 191
(66), 165 (58), 77 (87).
Synthesis of 18a and 18b by reaction of the oxime 14
with hexaalkyltriamidophosphites 9a and 9b
Dimethyl 5,7-di-tert-butylbenzo[d]isoxazol-3-ylphosphonate
(16a, C17H26NO4P)
Colorless crystals; m.p.: 141 °C (CH2Cl2); yield: 737 mg
The aminophosphine 9a (R’=Me) or 9b (R’=Et)
(3.3 mmol) in 4 cm3 dry THF was added dropwise to 0.8 g
14 (3.2 mmol) in 15 cm3 of the same solvent, and the
reaction mixture was stirred at rt for 10 h (TLC). The
solvent was evaporated under reduced pressure; the residue
was collected, washed with light petroleum, and crystal-
lized from the proper solvent to give compounds 18a and
18b.
-1
;
ꢀ
(68 %); IR (KBr): m = 1,262 (P=O), 1,180 (P–O–C) cm
1H NMR (500.7 MHz, CDCl3): d = 1.34, 1.57 [2 s,
2 9 9H, 2(Me3C)], 3.88 (d, JP–H = 12.6 Hz, 6H,
3
(MeO)2P), 6.33 (d, JH–H = 4.2 Hz, 1H, H(4)-Ar), 6.97
(d, JH–H = 4.2 Hz, 1H, H(6)-Ar) ppm; 13C NMR
(125.4 MHz, CDCl3): d = 153.5, 143.4, 135.2, 127.8,
1
123.2, 119.6 (C–Ar), 134.2 (d, JP–C = 172 Hz, C–P),
2
5,7-Di-tert-butyl-2-(dimethylamino)benzo[d][1,2]oxaphos-
phol-3(2H)-oxime (18a, C17H27N2O2P)
52.8 (d, JP–C = 14.8 Hz, (MeO)2P), 36.8, 34.5 (2CMe3),
31.4, 29.6 (2 Me3C) ppm; 31P NMR (200.7 MHz, CDCl3):
d = 26.2 ppm; MS (EI, 70 eV): m/z (%) = 339 (42) [M?],
230 (100) [M?-109, (P(O)(OMe)2)], 191 (73), 109 (65),
77 (80).
Yellow crystals; m.p.: 214 °C (EtOH); yield: 773 mg
ꢀ
(75 %); IR (KBr): m = 3,335 (NOH), 1,320, 863 (P–NMe2)
1
cm-1; H NMR (500.7 MHz, DMSO-d6): d = 1.25, 1.37
[2 s, 2 9 9H, 2(Me3C)], 3.23 (d, JP–H = 10.4 Hz, 6H,
3
Diethyl 5,7-di-tert-butylbenzo[d]isoxazol-3-ylphosphonate
(16b, C19H30NO4P)
Me2N–P), 6.93 (d, JH–H = 4.2 Hz, 1H, H(4)-Ar), 6.93
(d, JH–H = 4.2 Hz, 1H, H(6)-Ar), 10.55 (br s, 1H, HON)
ppm; 13C NMR (125.4 MHz, DMSO-d6): d = 150.9,
140.4, 137.6, 128.9, 126.2, 122.5 (C–Ar), 143.6 (d,
Colorless crystals; m.p.: 127 °C (cyclohexane); yield:
ꢀ
763 mg (65 %); IR (KBr): m = 1,275 (P=O), 1,155
123

Antineoplastic activity of fused nitrogen-phosphorus heterocycles
1241
1JP–C = 166.3 Hz, C–P), 37.5 (d, 2JP–C = 22.2 Hz, Me2N–
P), 34.9, 34.4 (2CMe3), 31.4, 30.6 (2Me3C) ppm; 31P NMR
(200.7 MHz, DMSO-d6): d = 123.6 ppm; MS (EI, 70 eV):
m/z (%) = 322 (14) [M?], 321 (18) [M?-1], 291 (48)
[M?-31, NOH], 216 (100) [M?-106, (NOH ? PNMe2)],
77 (83).
d = 14.2 ppm; MS (EI, 70 eV): m/z (%) = 366 (18) [M?],
365 (34) [M?-1], 335 (40) [M?-31, NOH], 77 (84).
Antitumor activity screening
Antitumor potency of the new phosphorus compounds
6a-6e, 12a, 12b, 16a, 16b, 19a, and 19b in addition to the
substrates 7a and 14 was tested at a dose of 10 lM utilizing
17 different human tumor cell lines, representing breast and
prostate cancer and melanoma using adriamycin as a ref-
erence standard according to the reported methods [23, 24].
The human tumor cell lines of the cancer screening panel
are grown in RPMI 1640 medium containing 5 % fetal
bovine serum and 2 mm3 glutamine.
5,7-Di-tert-butyl-2-(diethylamino)benzo[d][1,2]oxaphos-
phol-3(2H)-oxime (18b, C19H31N2O2P)
Yellow crystals; m.p.: 188 °C (EtOH); yield: 873 mg
ꢀ
(78 %); IR (KBr): m = 3,345 (NOH), 1,317, 874 (P–NEt2)
cm-1
;
1H NMR (500.7 MHz, DMSO-d6): d = 0.99 (dt,
JH–H = 6.4 Hz, JP–H = 4.2 Hz, 6H, Me2CN–P), 1.25,
4
3
1.37 [2 s, 2 9 9H, 2(Me3C)], 3.62 (q, JP–H = 6.4 Hz,
4H, H2CN–P), 6.41 (d, JH–H = 4.2 Hz, 1H, H(4)-Ar), 6.93
(d, JH–H = 4.2 Hz, 1H, H(6)-Ar), 10.82 (br s, 1H, HON)
ppm; 13C NMR (125.4 MHz, DMSO-d6): d = 152.8,
143.4, 137.2, 128.5, 125.2, 122.3 (C–Ar), 146.3 (d,
1JP–C = 178 Hz, C–P), 41.4 (d, 2JP–C = 19.7 Hz,
(CH2)2N–P), 35.2, 32.4 (2CMe3), 30.9, 28.6 (2Me3C),
Percentage growth inhibition (GI) is calculated as
[(Ti-Tz)/(C-Tz)] 9 100 for concentration for which
Ti C Tz and [(Ti-Tz)/Tz)] 9 100 for concentration for
which Ti \ Tz. Growth inhibition of 50 % (GI50) is cal-
culated from [(Ti-Tz)/(C-Tz)] 9 100 = 50, which is the
tested compound concentration resulting in a 50 % reduc-
tion in the net protein increase (as measured in SRB
staining) in control cells during the compound incubation.
Table 1 displays the observed percentage growth of each
cell line treated with a certain tested compound relative to
control cell line experiments.
3
15.6 (d, JP–C = 8.8 Hz, Me2N–P) ppm; 31P NMR
(200.7 MHz, DMSO-d6): d = 120.5 ppm; MS (EI,
70 eV): m/z (%) = 350 (18) [M?], 349 (28) [M?-1],
319 (54) [M?-31, NOH], 216 (100) [M?-134,
(NOH ? PNEt2)], 77 (80).
Acknowledgments We thank NYU Cancer Institute, NY, USA for
carrying out the pharmacological screening.
Synthesis of 19a and 19b by oxidation of 18a and 18b
When the compounds 18a, 18b were treated with H2O2 in
THF solution at rt, the amidates 19a, 19b were formed after
1 h.
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ꢀ
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Yellow crystals; m.p.: 198 °C (EtOH); yield: 874 mg
ꢀ
(78 %); IR (KBr): m = 3,340 (NOH), 1,252 (P=O), 1,330,
1
872 (P–NEt2) cm-1; H NMR (500.7 MHz, DMSO-d6):
4
d = 1.11 (dt, JH–H = 6.4 Hz, JP–H = 4.2 Hz, 6H,
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1H, H(4)-Ar), 6.93 (d, JH–H = 4.2 Hz, 1H, H(6)-Ar), 10.40
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123