Antitumor activities of some new 1,3,2-oxaza- and 1,3,2-diazaphosphorinanes against K562, MDA-MB-231, and HepG2 cells

Article abstract of DOI:10.1007/s00044-011-9737-7

New X-substituted 1,3,2-oxazaphosphorinanes, where X = NHC ₆H₅ (1), NHC₆H₄S(O) ₂NH₂-4 (2), NHC₆ H₄OCH₃-4 (3), NHC₆H₄NO₂-4 (4), OC₆H ₄CH₃-4 (5), NHC(O)C₆H₄NO ₂-4 (6), plus one X-substituted 1,3,2-diazaphosphorinane, where X = NHC₆H₄S(O)₂NH₂-4 (7), were synthesized and characterized by NMR, IR spectroscopy, and elemental analysis. The antitumor activities of these compounds, cyclophosphamide (CP), sulfanilamide (SA), and two X-substituted 5,5-dimethyl-1,3,2- diazaphosphorinanes, where X = NHC₆H₅ (8) OC ₆H₄CH₃-4 (9), were evaluated by cell culture on K562, MDA-MB-231, and HepG2 cell lines using MTT cell proliferation assay. The IC₅₀ values for CP and compounds 1-9 were in the range of 0.06 μM (for inhibition of HepG2 cells by compound 3) to 3.17 μM (for inhibition of HepG2 cells by compound 8). It was found that compounds 2 and 7 containing sulfonamide substituent and also SA itself are the best candidates for antitumor activity very close to CP. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9737-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:2185–2195
DOI 10.1007/s00044-011-9737-7
ORIGINAL RESEARCH
Antitumor activities of some new 1,3,2-oxaza- and 1,3,2-
diazaphosphorinanes against K562, MDA-MB-231, and HepG2
cells
•
Khodayar Gholivand Fatemeh Ghaziani Zahra Shariatinia
•
•
•
Nilufar Dorosti Manouchehr Mirshahi Sina Sarikhani
•
Received: 18 November 2010 / Accepted: 1 July 2011 / Published online: 20 July 2011
Ó Springer Science+Business Media, LLC 2011
Abstract New X-substituted 1,3,2-oxazaphosphorinanes,
where X = NHC6H5 (1), NHC6H4S(O)2NH2-4 (2), NHC6
H4OCH3-4 (3), NHC6H4NO2-4 (4), OC6H4CH3-4 (5),
NHC(O)C6H4NO2-4 (6), plus one X-substituted 1,3,2-di-
azaphosphorinane, where X = NHC6H4S(O)2NH2-4 (7),
were synthesized and characterized by NMR, IR spectros-
copy, and elemental analysis. The antitumor activities of
these compounds, cyclophosphamide (CP), sulfanilamide
(SA), and two X-substituted 5,5-dimethyl-1,3,2-diaza-
phosphorinanes, where X = NHC6H5 (8) OC6H4CH3-4 (9),
were evaluated by cell culture on K562, MDA-MB-231,
and HepG2 cell lines using MTT cell proliferation assay.
The IC50 values for CP and compounds 1–9 were in the
range of 0.06 lM (for inhibition of HepG2 cells by com-
pound 3) to 3.17 lM (for inhibition of HepG2 cells by
compound 8). It was found that compounds 2 and 7 con-
taining sulfonamide substituent and also SA itself are the
best candidates for antitumor activity very close to CP.
Introduction
Oxazaphosphorinanes are widely used as antineoplastic and
immunosuppressive agents that are characterized by a rel-
atively high oncologic specificity (Chen and Waxman,
1995). Cyclophosphamide (CP) prodrug is an oxazaphos-
phorinane that is activated by liver cytochrome P450
enzymes by a metabolic pathway that ultimately yields the
alkylating agent phosphoramide mustard (Schwartz and
Waxman, 2001). The parent compound is inactive in vitro
and in vivo and exerts its biologic activity through metab-
olites, mainly phosphoramide mustard, by hepatic micro-
somal enzymes (Huang et al., 2000). The alkylating
metabolite(s) can bind to a variety of molecules including
amino acids, proteins, and peptides, but the most important
binding site is DNA where cross-linking occurs (Murata
et al., 2004). CP is effective against a wide spectrum of
malignancies such as, leukemia, lymphoma, breast, lung,
prostate, and ovarian cancers (Moore 1991). In fact, CP is
one of the most extensively used chemotherapeutic agents
in the treatment of many types of cancer that has been the
focus of research efforts to understand its mode of action
and to develop analogs with improved function (Camerman
et al., 1983). The phosphorus-containing heterocycles such
as 1,3,2-oxazaphosphorinanes are also an essential part of
phosphorus chemistry indicating unique conformational
behavior (Bentrude et al., 1986; Setzer et al., 1989; Ben-
trude et al., 1988). A great number of studies have been
carried out on various monocyclic and fused bicyclic and
tricyclic 1,3,2-oxazaphosphorinane derivatives about their
synthesis, bioactivity, and conformational analyses (Vilja-
nen et al., 1998; Martinek et al., 2000; Bentrude et al.,
1984; Ludeman et al., 1986). Modifications of CP have led
to the design and synthesis of many cyclic analogs (Li et al.,
2003; Gholivand et al., 2005a, b; Ludeman et al., 1979) to
Keywords 1,3,2-Oxazaphosphorinane ꢀ
Antitumor activity ꢀ MTT assay ꢀ K562 ꢀ MDA-MB-231 ꢀ
HepG2
K. Gholivand (&) ꢀ F. Ghaziani ꢀ N. Dorosti
Department of Chemistry, Faculty of Basic Sciences,
Tarbiat Modares University, P.O. Box 14115-175, Tehran, Iran
e-mail: gholi_kh@modares.ac.ir
Z. Shariatinia
Department of Chemistry, Amirkabir University of Technology,
P.O. Box 159163-4311, Tehran, Iran
M. Mirshahi ꢀ S. Sarikhani
Department of Biochemistry, Faculty of Biology, Tarbiat
Modares University, P.O. Box 14115-175, Tehran, Iran
123

2186
Med Chem Res (2012) 21:2185–2195
Fig. 1 The structure of
cyclophosphamide (CP)
O
P
Fig. 2. A summary of the spectroscopic data of these
compounds is listed in Table 1. The phosphorus chemical
shift in CP is at the most downfield region (11.53 ppm)
compared to those of compounds 1–9. The phosphorus
atom in 4 (containing para-nitrophenyl moiety) is more
deshielded than the P atom in 6 (containing nitrobenzamide
moiety). In fact, among compounds 1–9, the d(31P) of 6
O
X
N
X = N(CH2CH2Cl)2
1
obtain relationships between structure and antitumor
activities. It has been shown that the steric and electronic
properties of the X substituent (Fig. 1) exert strong effects
on the conformational equilibrium of oxazaphosphorinane
ring (Martinek et al., 2000).
reveals the most upfield shift (-2.16 ppm). The H NMR
spectra show different chemical shifts for the Haxial and
Hequatorial atoms of CH2 groups (Table 2). These diaste-
reotopic protons suggest a single chair conformation for
six-membered ring at room temperature and exclude flex-
ible boat forms in which pseudorotation would tend to
average the chemical shifts and coupling constants (Eliel
and Hutchins, 1969). This was confirmed by the X-ray
crystal structures of their analogs (Gholivand et al., 2005a,
b). The axial hydrogen atoms are at downfield relative to
their equatorial counterparts (Jackman and Sternhell,
1969). Furthermore, the axial H resonances are shifted
downfield for 1–6 as compared to those of 7–9 (Table 2).
3J(P,C)aliphatic coupling constants in these molecules present
To obtain compounds with comparative antitumor
activities against K562, MDA-MB-231, and HepG2 cell
lines to those of CP and to explore whether there are cor-
relations between the steric and electronic properties of the
substituents and inhibitory effect, a new series of 1,3,2-
oxazaphosphorinanes and 1,3,2-diazaphosphorinanes were
designed, synthesized, and characterized. The antitumor
activities of these compounds, CP and sulfanilamide (SA) to
inhibit the K562, MDA-MB-231, and HepG2 cells growth
have been evaluated in vitro using MTT cell proliferation
assay. Using spectroscopic data, IC50 and log P values, the
antitumor activities were compared and described.
2
greater values than J(P,C)aliphatic coupling constants.
Compound 5 revealed the coupling of phosphorus atoms
with carbonyl group (2J(P,C=O) = 6.8 Hz). IR spectra indi-
cate that m(P=O) varies from 1232 cm-1 in 3 to 1171 cm-1
in 8.
Results and discussion
The orientation of P=O double bond in axial or equa-
torial position depends on the changes in 31P chemical
shifts, proton chemical shift due to 1,3 diaxial interactions,
and 13C chemical shift arising from the shielding effect of
oxygen (Bentrude et al., 1986). Thus, it is suggested that
the P=O bond is in an axial position in compounds 1–6
Spectroscopic study
The structures of 1,3,2-oxazaphosphorinanes and 1,3,2-di-
azaphosphorinanes studied in this work are presented in
O
P
X = N(CH2CH2Cl)2
NHC6H5
(CP)
(1)
(2)
(3)
(4)
(5)
(6)
4-NHC6H4-SO2NH2
4-NH-C6H4OCH3
4-NH-C6H4NO2
4-O-C6H4CH3
X
N
H
O
4-NHC(O)C6H4-NO2
NH2(CH2)3OH
N(C2H5)3
X
P
X
P
O
P
NH2(CH2)3NH2
N(C2H5)3
NH2CH2C(CH3)2CH2NH2
O
N
H
O
N
H3C
N(C2H5)3
H
N
X
Cl
H
N
Cl
H
CH3
T0 = 0 0C,
Tfinal = R.T.
X = 4-NHC6H4-SO2NH2 (7)
Fig. 2 The preparation pathways for analogs 1–9
123
X = NHC6H5
(8)
4-O-C6H4CH3 (9)

Med Chem Res (2012) 21:2185–2195
2187
Table 1 Some spectroscopic data of cyclophosphamide (CP) and compounds 1–9
Compound
d(31P)/ppm
2J(P,Caliphatic-O)/Hz
2J(P,Cal-N)/Hz
3J(P,Cal)/Hz
3J(P,Car)/Hz
m
(P=O)/cm-1
Ref.
CP
1
11.53
2.41
6.9
7.04
7.2
7.0
7.3
7.7
7.7
–
2.8
3.1
2.5
–
6.3, 3.8
7.5
–
1223
1231
1222
1232
1223
1218
1229
1178
1171
1198
–
a
7.3
7.2
6.7
7.5
4.7
9.2
6.7
6.7
4.5
a
a
a
a
a
a
2
1.51
7.5
3
3.06
7.4
4
0.68
2.7
3.5
–
7.6
5
-0.87
-2.16
5.13
7.0
6
7.4
7
–
7.6
8
5.60
–
3.3
4.0
7.7
Gholivand et al. (2007b)
Gholivand et al. (2007b)
9
8.69
–
7.8
al aliphatic, ar aromatic
a
This work
while it places in an equatorial (or pseudo-equatorial)
position in 7–9. Therefore, the antibonding P–N orbital,
when the P=O bond is axial, is suitably aligned for stabi-
lizing overlap with a neighboring electron single pair on
N(1) or O(3), an interaction which is not available when
the P=O bond is equatorial. The interaction between the
P–N antibonding orbital and the lone pair of the oxygen has
been reported (Bentrude et al., 1991) to play a substantial
role in stabilization of chair conformation. It was shown
that the P=O bond in CP places in a pseudo-axial position
(Camerman and Camerman, 1973). Also, the crystal struc-
ture of a 1,3,2-benzoxazaphosphorinane in which the P=O
bond demonstrates a pseudo-axial orientation has been
reported (Gholivand et al., 2007a).
well as log D (computed at pH 7.4 by CSlog D software)
for CP, SA (sulfanilamide), and compounds 1–9 are pro-
vided in Table 3. The plots of IC50 (lM) versus compounds
obtained after exposure for 24, 48, and 72 h are shown in
Figs. 3, 4, and 5 exhibiting the increase of IC50 from 24 to
48 and 72 h in most of the cases. This means that the
antitumor activities are reduced due to further exposure to
the cancer cell lines. Thus, the main inhibition occurs
during 24 h. Also, the plots of mean IC50 (lM) against
concentration (mM) after 48 h exposure to K562 cancer
cells were presented in Figs. 6, 7, and 8 indicating the
enhancement of IC50 with increasing of the antitumor
concentration. This behavior was also observed for MDA-
MB-231 and HepG2 cell lines.
The structure–activity relationships (SAR) is base on
this definition that the physico-chemical properties that
affect the biological activities of a molecule are of three
major types: electronic, steric, and hydrophobic and other
factors, such as hydrogen bonding, polarizability, dipole
moment, topology, and steric effects of substitutions play
less important roles (Hansch and Leo, 1995). The SAR
studies were performed on phosphoramidate compounds
and in their investigations, they have reported linear rela-
tionships between log(1/IC50) and log P, d(31P) and para-
substituent constants (Ghadimi et al., 2008, 2009). In the
following sections, the effects of several parameters on the
SAR are discussed.
Structure–activity relationships
As indicated in Fig. 2, the rationale design in this study is
the replacement of X = N(CH2CH2Cl)2 group in CP by
para-substituted aniline, benzoyl, or phenol moieties in
compounds 1–9. Also, one of the endocyclic NH groups
was replaced with its para-element oxygen atom in 7–9.
The effects of these structural changes on the conforma-
tional and biological properties are of our interest.
The antiproliferative activities of CP analogs 1–9 were
compared in three types of human cancer cell lines by
means of a colorimetric microculture assay technique
(MTT assay), and the corresponding mean IC50 values are
reported in Table 3. All the derivatives showed potent
antitumor activities against three cell lines in micromolar
concentrations. The cells were exposed to various con-
centrations of the compounds for 24, 48, and 72 h, and the
inhibition percentages of cell growth (I) were measured for
various times after exposure. The IC50 values (lM) after
48 h of incubation for three cell lines and also the log
P values (which is a measure of molecular lipophilicity) as
Effect of sulfonamide substitution
In evaluation of the activities of the heterocyclic ring
compounds, we found that sulfanilamide is also an anti-
cancer agent with the average IC50 value of *0.12 lM in
three cell lines. It should be mentioned that the inclusion
of this amide on a phosphorus atom as in compounds
2 and 7 can increase the cytotoxicity against cancer cells.
123

2188
Med Chem Res (2012) 21:2185–2195
Table 2 The C, H, and Y chemical shifts of cyclophosphamide (CP) and compounds 1–9
d(CH),
d(CH),
d(CH),
dYF
(JPF
dYX (JPE
)
dNHY
dHC
(JPD
dHD (JPC
)
dHA
(JPB
dHB
(JPA
Compound
2J(P,C)al
2J(P,C)al
3J(P,C)al
)
(JPNH
)
)
)
)
40.55 (2.8)
40.67 (3.1)
40.57 (2.5)
67.25 (6.8)
67.95 (7.0)
68.25 (7.2)
25.50 (6.3)
26.30 (7.5)
26.10 (7.5)
1.69–1.72 (m)
4.65 (3.6)
3.14 (m) 3.06 (m) 4.17 (m) 4.14 (m) CP
1.80 (m) 1.60 (m) 4.98 (5.5)
3.11 (m)
3.11 (m)
4.11 (2.5) 4.20 (m)
4.10 (m) 4.21 (m)
1
2
1.79 (s) 1.57
(13.6)
1.74 (m) 1.55
(14.0)
1.81 (m) 1.62 (s)
5.19 (–)
40.66 (s)
67.76 (7.0)
26.30 (7.4)
4.88 (4.2)
3.1 (m)
4.10 (m) 4.18 (m)
3
40.47 (2.7)
40.67 (3.5)
40.48 (s)
68.49 (7.3)
69.70 (7.7)
69.30 (7.7)
25.95 (7.6)
25.80 (7.0)
25.59 (7.4)
5.37 (m)
3.13 (m)
3.12(m)
3.23
4.13 (m) 4.27 (m)
4.31 (m)
4
5
6
1.82 (m) 1.61 (m) 5.48 (m)
1.86 (m) 1.67
(13.9)
1.41–1.61 (m)
5.36 (s)
3.11
(22.0)
4.33 (m)
41.71 (s)
–
–
–
26.75 (7.6)
27.03 (7.7)
26.25 (6.8)
4.46 (s)
2.99–3.06 (m)
3.02 (m)
7
8
9
41.86 (3.3)
42.04 (4.0)
1.50 (m) 1.40 (m) 4.26 (m)
1.52 (m) 1.46 (m) 4.78 (m)
3.03 (m)
al aliphatic
O
HC
HA
HD
1
P
2
6
X
N
H
5
YF
4
O
3
HB
YE
CP, 1-6
X
HC
HA
4
HD
1
P
2
6
O
N
H
3
5
YF
N
HB
H
YE
7-9
Diazaphosphorinanes 8 and 9 are much less toxic among
these compounds. Hence, it could be concluded that 1,3,2-
diazaphosphorinanes are often less toxic than 1,3,2-
oxazaphosphorinanes as this is seen from the IC50 values in
Table 3. The reason may be the easier opening of the ali-
phatic oxazaphosphorinane ring and P–O bond cleavage
than the diazaphosphorinane ring and P–N bond breaking.
A comparison of the IC50 values discloses that molecules 2
and 7 have much efficient activities than other diazaphos-
phorinanes. These higher activities are in consistent with
their log P values which are greater for these compounds. It
is very interesting that the IC50 values of molecules 2 and 7
are nearly the same that is most probably due to the pres-
ence of sulfonamide (SA) moiety with its close anticancer
activity to CP in these derivatives.
Electronic effects of substituents
The chemical reactivity is dependent on one or both of two
factors: steric and electronic effects (Hansch et al., 1963).
A comparison of analogs 1, 3, and 4 indicates that due to
the electronic effects, the phosphorus atom in 3 is the most
positive one. The substituents at para position of the phe-
nyl ring in compounds 1, 3, and 4 are H, OCH3, and NO2
123

Med Chem Res (2012) 21:2185–2195
2189
Table 3 The in vitro cytotoxic activity (IC50, lM) of the compounds
1–9 toward human tumor cell lines after 48 h and their log P, log D
values
Compound Mean IC50 Mean IC50
K562
Mean IC50 log D log P
MDA-MB-231 HepG2
CP
SA
1
0.15
0.20
0.55
0.15
0.20
0.89
0.71
0.67
0.19
3.11
1.44
0.09
0.09
0.83
0.17
0.23
0.09
0.87
0.27
0.11
2.04
0.23
0.24
0.08
0.60
0.14
0.06
0.15
0.59
0.57
0.13
3.17
2.20
0.05
-0.38
0.16
1.12
1.47
0.29
1.88
2
-0.70
3
0.46 -0.71
0.50 -0.53
4
Fig. 5 The plot of IC50 (lM) against compounds to inhibit HepG2
cells
5
0.28
0.76
6
0.52 -0.45
7
-1.00
1.56
compounds to inhibit K562 cells exhibit the same trend,
i.e., reduction from 4 to 1 and 3 (Table 3). Similarly, d(31P)
is more positive in compound 7 (containing electron-
withdrawing p-NH2SO2 moiety) than in compound 8
(containing neutral H atom). The IC50 values of 8 are
greater than those of 7 against all three K562, MDA-MB-
231, and HepG2 cell lines. Figure 9 represents the plot of
log (1/IC50) against d(31P) for compounds CP and 1, 4–7
exhibiting a nearly linear relationship. This linear correla-
tion diagram is not obtained for all compounds 1–9 that
shows the inhibition mechanism is dependent to several
parameters and one of them is d(31P).
8
-0.31 -0.03
9
0.22
0.12
CP cyclophosphamide, SA sulfanilamide
Lipophilicity effect
As known, the lipophilicity of a compound plays a pivotal
role during its penetration into the cells and enhances the
activity. The log P values of the compounds 1–9 were
calculated using Hyperchem 7.0 software (Table 3). Typ-
ically, partition coefficients in the order of 100–1000 (log
P = 2–3) are required for efficient passive diffusion pro-
cess (Taylor, 1996). The results demonstrate that com-
pounds 2 and 7 both of them containing sulfonamide
moiety as well as SA itself have the maximum lipophilicity
values (*1.5–2.0). The log D values of SA and com-
pounds 2, 7 are the most negative ones. Thus, the log D
values cannot be measure of toxicity, and the log P values
are more informative and applicable. Considering this
matter plus the IC50 values, it may be suggested that
compounds 2 and 7 as well as SA are the best candidates
for anticancer activity very close to CP. It is noteworthy
that the high log P value of 1.47 for sulfonamide (SA)
confirms that presence of this substituent in compounds 2
and 7 cause their cytotoxic effects. In fact, it has been
indicated that sulfonamide derivatives possess many
biological activities such as anticancer, antibacterial,
hypoglycemic, diuretic, anti-carbonic anhydrase, and anti-
thyroid (Ghorab et al., 2009; Abbate, et al., 2004).
Figure 10 presents the plot of log (1/IC50) against log P for
Fig. 3 The plot of IC50 (lM) against compounds to inhibit K562
cells
Fig. 4 The plot of IC50 (lM) against compounds to inhibit MDA-
MB-231 cells
groups that are neutral, electron-donating, and electron-
withdrawing groups, respectively. Results indicate that the
d(31P) values appear at more down field from 4 to 1 and 3
that means a more electron donating group gives a more
positive phosphorus atom. The IC50 values of these
123

2190
Med Chem Res (2012) 21:2185–2195
Fig. 6 The plot of IC50 (lM)
against concentration (lM) to
inhibit K562 cells for CP, SA,
and compounds 1–3
Antitumor
1
80.00
60.00
40.00
20.00
0.00
2
3
CP
SA
.01 .05 .19 .75 2.50 4.15 5.00 5.50 5.81 8.25 8.25 9.43 10.00 10.30 13.99 14.55 15.00 16.49 20.00
Concentration/mM
100.00
80.00
60.00
40.00
20.00
0.00
Antitumor
4
5
6
.05
.19
.32
.75
.82
1.32 1.82 2.50 4.54 5.00 5.50 5.81 5.98 9.13 9.18 10.00 15.42 15.57 31.99 47.25
Concentration/mM
Fig. 7 The plot of IC50 (lM) against concentration (lM) to inhibit K562 cells for compounds 4–6
compounds CP, SA and 1, 2, 4, 6, 7 which is a nearly linear
diagram. Similar to the electronic effect, the linear
behavior between lipophilicity and inhibition potency is
not obtained for all compounds 1–9. This means that the
inhibition mechanism does not just depend on log P, but
several parameters can affect it.
Material and methods
All of the chemicals and solvents for syntheses were
prepared from Fluka and Merck companies. The K562,
MDA-MB-231, and HepG2 cell lines were purchased from
Pasteur Institute of Iran (Tehran, Iran). Melting points were
1
In summary, results of bioassay demonstrate that
attachment of sulfonamide to the phosphorus atom in 1,3,2-
oxaza-, 1,3,2-diazaphosphorinanes 1–9 does lead to potent
and selective antitumor activities against the studied K562,
MDA-MB-231, and HepG2 cell lines in compounds 2 and
7. Moreover, the electronic features of the substituents,
lipophilicity as well as conformational properties can affect
the inhibition potencies of CP and its analogs.
determined on an electrothermal apparatus. H, 13C, and
31P NMR spectra were recorded on a Bruker (Avance
DRS) 500 spectrometer. H, 13C, and 31P chemical shifts
1
were determined relative to TMS and 85% H3PO4,
respectively, as external standards. IR spectra (KBr pellets)
were obtained with a Shimadzu, IR-60 model spectrometer.
Elemental analysis was performed using a Heraeus CHN-
O-RAPID instrument.
123

Med Chem Res (2012) 21:2185–2195
2191
100.00
Antitumor
7
8
9
80.00
60.00
40.00
20.00
0.00
.05 .11 .14 .19 .25 .26 .32 .35 .53 .60 .75 .82 1.00 1.30 1.32 1.50 1.58 1.82 2.50 2.51 5.81
Concentration/mM
Fig. 8 The plot of IC50 (lM) against concentration (lM) to inhibit K562 cells for compounds 7–9
Fig. 10 The plot of log (1/IC50) against log P for compounds CP, SA,
and 1, 2, 4, 6, 7
Fig. 9 The plot of log (1/IC50) against d(31P) for compounds CP, 1,
and 4–7
phosphoramidic dichloride (Cates and Lemke, 1974), and
4-tolyl-dichlorophosphate (Tolkimth, 1959) in dry CCl4, a
mixture of 10 mmol of related diamine (propane-1,3-dia-
mine, 2,2-dimethylpropylene diamine and 3-aminopropane-
1-ol) plus 10 mmol triethylamine was added dropwise at
about 0°C and the reaction mixture stirred for 10 h. Then
the precipitate was filtered and washed with distilled water
and dried.
Synthesis (general procedure)
To a solution of 10 mmol of corresponding phospho-
ramidic dichlorides (N-4-phenylsulfonylphosphoramidic
dichloride, N-4-nitrophenylphosphoramidic dichloride,
N-nitrobenzoylphosphoramidic dichloride (Amirkhanov et al.,
1997), N-phenylphoramidic dichloride, 4-methoxyphenyl
123

2192
Med Chem Res (2012) 21:2185–2195
2J(PNH) = 10.0 Hz, 1H, NHexocyclic). 13C NMR (d6-DMSO):
d 26.32 (d, 3J(P,C) = 7.35 Hz, 1C, CH2), 40.66 (s), 55.15(s),
2-(N-phenyl)-1,3,2-oxazaphosphorinane-2-oxide
2
C6H5NHP(O)[NHCH2CH2CH2O]
67.8 (d, J(P,C) = 6.98 Hz, 1C, CH2), 114.08(s), 118.67(d,
(1)
3J(P,C) = 6.7 Hz, 2C, CH), 135.07 (s), 153.28 (s). 31P{1H}
NMR (d6-DMSO): d 3.06 (s). IR (KBr), t (cm-1): 3275
(NH), 1510, 1457, 1278, 1232 (P=O), 1209, 1178, 1127,
1030, 977, 947 (P–N), 816 (P–N), 760, 587, 492.
Yield: 89%, m.p. 185°C. Anal. Calc. for C9H13N2O2P: C,
50.95%; H, 6.18%; N, 13.20%. Found: C, 50.94%; H, 6.19%;
N, 13.25%. 1H NMR (d6-DMSO): d 1.54–1.58 (m, CH2), 1.77
(m, 1H, CH2), 3.21 (m, 1H, CH2), 4.11 (m, 1H, CH2),),
4.19 (m, 1H, CH2), 4.98 (m, 1H, NHcyclic), 6.79 (t,
2-(N-4-Nitrophenyl)-1,3,2-oxazaphosphorinane-
2-oxide
3
3J(H,H) = 7.3 Hz, 1H), 7.04 (d, J(H,H) = 7.8 Hz, 2H, CH),
7.15 (m, 2H), 7.51 (d, 2J(P,NH) = 10.65 Hz 1H, NHsulf). 13
C
NMR (d6-DMSO): d 26.30 (d, 3J(P,C) = 7.53 Hz, 1C, CH2),
40.68 (d, J(P,C) = 3.08 Hz, 1C, CH2–N), 67.98 (d,
4-NO2C6H4NHP(O)[NHCH2CH2CH2O]
(4)
2
3
2J(P,C) = 7.0 Hz, 1C, CH2–O), 117.23 (d, J(P,C) = 7.3 Hz,
Yield: 67%. Anal. Calc. for C10H16N3O4P: C,43.96%;
CH–Ar), 119.80 (s), 128.64 (s), 145.07 (s). 31P NMR (d6-
DMSO): d 2.41 (s). IR (KBr), t (cm-1): 3235 (NH), 3090,
1601, 1497, 1429, 1371, 1338, 1284, 1231 (P=O), 1202, 1125,
1034, 998, 933 (P–N), 863(P–N), 747, 691, 617, 497.
H,5.90%; N,15.38%;. Found: C, 43.99%; H,5.95%; N,
15.39%. 1H NMR (d6-DMSO): d = 1.62 (d, J(P,H)
3
=
12.6 Hz, 1H, CH2), 1.80 (m, 1H, CH2), 3.12 (m, 2H, N–
CH2), 4.12 (m, 1H, O–CH2), 4.26 (m, 1H, O–CH2), 5.38
3
(m, 1H, NHcyclic), 7.18 (d, J(H,H) = 9.0 Hz, 2H, Ar–H),
3
8.10 (d, J(H,H) = 9.0 Hz, 2H, Ar–H), 8.50 (s, 1H, NH).
2-(N-4-phenylsulfonyl)-1,3,2-oxazaphosphorinane-
2-oxidez
3
13C NMR (d6-DMSO): d = 25.95(d, J(P,C) = 7.6 Hz, 1C,
CH2), 40.47 (d, J(P,C) = 2.7 Hz, 1C, N–CH2), 68.49(d,
2
3
2J(P,C) = 7.3 Hz, 1C, O–CH2), 116.57 (d, 2C, J(P,C)
=
4-NH2SO2C6H4NHP(O)[NHCH2CH2CH2O]
(2)
7.47 Hz, CH), 125.2 (s), 139.81 (s), 149.48 (s). 31P{1H}
NMR (d6-DMSO): d = 0.68 (s). IR (KBr), t (cm-1): 3335,
3170, 2870, 1598, 1517, 1477, 1340, 1254, 1223 (P=O),
1100, 1039, 985, 928 (P–N), 843, 782, 745, 541, 479.
Yield: 49%. Anal. Calc. for C9H14N3O4PS: C, 37.11%;
H, 4.84%; N, 14.43%. Found: C, 37.21%; H, 4.89%; N,
14.35%. 1H NMR (d6-DMSO): d 1.60 (d, J(H,H)
13.7 Hz, 1H, CH2), 1.78(b, 1H, CH2), 3.095 (b, 2H, CH2),
3
=
3
3
4.12 (t, J(H,H) = 10.9 Hz, 1H, CH2), 4.23(d, J(H,H)
2-(4-Methyl-phenoy)-1,3,2-oxazaphosphorinane-
2-oxide
=
2
10.4 Hz, 1H, CH2), 5.19 (d, J(P,NH) = 4.8 Hz, 1H, NHa-
3
mine), 7.07 (s, 2H, NHsulf), 7.14 (d, J(H,H) = 8.0 Hz, 2H,
3
CH), 7.63 (d, J(H,H) = 8.0 Hz, 2H), 8.05 (d, J(P,NH)
2
4-CH3C6H4OP(O)[NHCH2CH2CH2O]
=
(5)
10.5 Hz 1H, NHamine). 13C NMR (d6-DMSO): d 26.15 (d,
3J(P,C) = 7.47 Hz, 1C, CH2), 40.58 (d, J(P,C) = 2.54 Hz,
2
Yield: 47%, m.p. 84–85°C. Anal. Calc. for C10H14NO3P:
C,54.32%; H,7.46%; N,5.76%;. Found: C, 54.31%; H,
7.43%; N, 5.73%. 1H NMR (d6-DMSO): d = 1.61 (m, 1H,
CH2), 1.83 (m, 1H, CH2), 2.26 (s, 3H, CH3), 3.11 (m, 2H, N–
CH2), 4.31 (m, 2H, O–CH2), 5.29 (m, 1H, NH), 7.09 (d,
1C, CH2), 68.29 (d, 2J(P,C) = 7.2 Hz, 1C, CH2), 116.50 (d,
3J(P,C) = 7.2 Hz, 2C, CH), 126.83 (s), 135.09 (s), 145.59
(s). 31P{1H} NMR (d6-DMSO): d 1.51 (s). IR (KBr), t
(cm-1): 3340 (NH), 3210 (NH), 2940, 1597, 1500, 1467,
1319(s), 1222 (P=O), 1202, 1156 (SO2), 1094, 1044, 982,
932 (P–N), 830 (P–N), 751, 590, 540.
3
3J(H,H) = 8.3 Hz, 2H, Ar–H), 7.15 (d, J(H,H) = 8.3 Hz,
2H). 13C NMR (d6-DMSO): d = 20.21 (s, 1C, CH3), 25.85
(d, 3J(P,C) = 7.04 Hz 1C, CH2), 40.68 (d, 2J(P,C) = 3.5 Hz,
1C, CH2), 69.73 (d, 2J(P,C) = 7.66 Hz, 2C, CH2), 119.75 (d,
3JP,C = 4.67 Hz, 2C, CH), 129.92 (s, 2C, CH), 133.26 (s,1C,
C), 148.70(d, 2J(P,C) = 6.78 Hz, 1C, C). 31P{1H} NMR (d6-
DMSO): d = -0.87 (s). IR (KBr), t (cm-1): 3230, 2925,
1737, 1437, 1257, 1218 (P=O), 1166, 990, 932, 910 (P–N),
869 (P–N), 818, 763, 682, 634, 479.
2-(N-4-methoxy-phenyl)-1,3,2-oxazaphosphorinane-
2-oxide
4-CH3OC6H4NHP(O)[NHCH2CH2CH2O]
(3)
Yield: 70%, m.p. 185–186°C. Anal. Calc. for
C10H15N2O3P: C, 49.59%; H, 6.24%; N, 11.57%. Found: C,
49.59%; H, 6.27%; N, 11.55%. 1H NMR (d6-DMSO): d 1.55
(d, 2J(H,H) = 13.5 Hz, 1H, CH), 1.74 (m, 1H, CH2), 3.03 (m,
2H, CH2), 3.65(s, OCH3), 4.14 (m, 2H, CH2–O), 4.88 (d,
2J(P,NH) = 4.2 Hz, 1H, NH), 6.75 (d, 3J(H,H) = 8.2 Hz, 2H,
2-(N-4-Nitrobenzueil)-1,3,2-oxazaphosphorinane-
2-oxide
(4-NO2)C6H4CONHP(O)[NHCH2CH2CH2O]
3
(6)
Ar–H), 6.97 (d, J(H,H) = 8.2 Hz, 2H, Ar–H), 7.25 (d,
123

Med Chem Res (2012) 21:2185–2195
2193
between the Ficoll-Paque PLUS and sample layers. This
material is then centrifuged twice in balanced salt solution to
wash the lymphocytes and to remove the platelets.
Yield: 75%, m.p. 219°C. Anal. Calc. for C10H12N3O5P:
C,42.11%; H,4.24%; N,14.73%;. Found: C, 42.07%;
H,4.33%; N,14.68%. 1H NMR (d6-DMSO): d 1.67 (d,
3
2J(H,H) = 13.9 Hz, 1H), 1.86 (m, 1H), 3.11 (dm, J(P,H)
=
In vitro antitumor assays
22.0 Hz, 1H), 3.23 (t, 1H), 4.33 (m, 2 H), 5.36 (s, 1 H, NHa-
3
mine), 8.15 (d, J(H,H) = 8.2 Hz, 2 H), 8.30 (d, J(H,H)
3
=
8.2 Hz, 2 H), 9.85 (s, 1 H, NHamide). 13C NMR (d6-DMSO): d
For evaluation of antiproliferative activity of CP and
compounds 1–9, three types of cells, i.e. K562 cells
(5 9 103 cells/well), MDA-MB-231 cells (2.5 9 103 cells/
well), and HepG2 cells (3 9 103 cells/well), were plated in
96-multiwell microtiter plate for 24 h before treatment
with the test compounds to allow attachment of cell to the
wall of the plate. Cells were maintained in RPMI medium
(Gibco BRL) supplemented with 10% fetal bovine serum,
2% L-glutamine (Gibco BRL), penicillin (100 U/ml), and
streptomycin (100 lg/ml) at 37°C in an atmosphere
humidified with 5% CO2 and 95% air. Cells were seeded in
96-well cell culture plates and treated on the second day
with the candidate drugs.
3
2
25.59 (d, J(P,C) = 7.4 Hz), 40.48 (s), 69.30 (d, J(P,C)
=
7.7 Hz), 123.38 (s), 129.54 (s), 139.00 (d, 3J(P,C) = 9.2 Hz),
149.47(s),167.13(s).31P{1H}NMR(d6-DMSO):d = -2.16
(s). IR (KBr, cm-1): 3230 (NH), 3105 (NH), 2870, 1686
(C=O), 1515, 1465, 1343, 1260, 1229(P=O), 1202, 1100, 988,
880 (P–N), 852 (P–N), 715, 498.
2-(N-4-phenylsulfonyl)-1,3,2-diazaphosphorinane-
2-oxide
4-NH2SO2C6H4NHP(O)[NHCH2CH2CH2NH]
(7)
Test compounds stock solutions were prepared by dis-
solving of each compound in DMSO and then aliquots of
stock solutions further diluted with sterile RPMI to the
required concentration, such that the total DMSO concen-
tration did not exceed 2%. At this concentration, DMSO was
found to be nontoxic to the cells tested. As a control, a
solution containing 2% DMSO in RPMI was used. Various
concentrations of compounds 1–9 were added to each well in
triplicate at 37°C with 5% CO2 in the incubator for 24, 48,
and 72 h. After incubation for the indicated times, the cell
number was estimated by the MTT assay as described in the
literature (Mosmann, 1983). After treatment, 20 ll of
aqueous MTT solution (5 mg/ml) was added and incubated
for 3–4 h at 37°C in an atmosphere humidified with 5% CO2
and 95% air. After incubation, the medium/MTT mixtures
were removed, and formazan crystals were dissolved in
200 ll DMSO/well. Optical densities at 540 nm were
measured with an ELIZA plate reader (Labsystems Multi-
scan, MS). The IC50 values of compounds 1–9, CP, and
sulfonamide (SA) for 50% cell death were measured after
48 h of incubation. Statistical analyses were performed
using GraphPad prism software, version 5 (GraphPad soft-
ware) so that using nonlinear regression modeling, one IC50
value was calculated from each set of triplicate wells.
Yield: 69%, m.p. 193.5°C. Anal. Calc. for C9H15N4O3PS:
C, 37.24%; H, 5.21%; N, 19.30%. Found: C, 37.21%; H,
1
5.18%; N, 19.25%. H NMR (d6-DMSO): d 1.54 (s, 2 H,
CH2), 3.04 (d, 3J(P,H) = 24.9 Hz, 2H, CH), 3.15 (s, 2H, CH),
4.46 (s, 2 H, NHamine), 6.80 (b, 2H, NHsulf), 7.19 (d,
3J(H,H) = 8.4 Hz, 2H), 7.58 (d, 3J(H,H) = 8.4 Hz, 2H), 7.66
(d, 2J(P,NH) = 9.8 Hz 1H, NHamide). 13C NMR (d6-DMSO):
d 26.78 (d, 3J(P,C) = 7.6 Hz, 1C, CH2), 41.71 (s), 116.45 (d,
3J(P,C) = 6.7 Hz, 2C, CH), 126.49 (s), 134.21 (s), 146.63 (s).
31P{1H} NMR (d6-DMSO): d 5.13 (s). IR (KBr), t (cm-1):
3320 (N–H), 3195(N–H), 2930, 1588, 1490, 1468, 1332,
1305, 1178 (P=O), 1143 (SO2), 1086, 918 (P–N), 825 (P–N),
536.
Cell culture and growth inhibition assay
In order to study the degree of selectivity of the cytotoxic
activity of the compounds under investigation, assays using
healthy cells (Human Peripheral Blood Mononuclear Cells)
were carried out on some compounds. The compounds
selected were those that showed activity in tumor cells.
Isolation and culture of human peripheral blood
mononuclear cells lymphocytes
Acknowledgments We wish to thank the Research Office of
Tarbiat Modares University for the financial support of this work.
Separation of lymphocyte was done according to the fol-
lowing procedure (Noble et al., 1968): defibrinated or anti-
coagulant-treated blood is diluted with an equal volume of
phosphate buffered saline (PBS) and layered carefully over
Ficoll-Paque PLUS (without intermixing) in a centrifuge
tube. After a short centrifugation at room temperature (typ-
ically at 400 rpm for 30–40 min), lymphocytes together with
monocytes and platelets are harvested from the interface
References
Abbate F, Casini A, Owa T, Scozzafava A, Supuran CT (2004)
Carbonic anhydrase inhibitors: E7070, a sulfonamide anticancer
agent, potently inhibits cytosolic isozymes I and II, and
123

2194
Med Chem Res (2012) 21:2185–2195
transmembrane, tumor-associated isozyme IX. Bioorg Med
Chem Lett 14:217–223. doi:10.1016/j.bmcl.2003.09.062
Gholivand K, Mojahed F, Mohamadi L, Bijanzadeh HR (2007a)
Conformation of 2-Oxo-2-dimethylamino-1,3,2-k5-benzoxaza-
phosphorinane: X-ray, NMR, and ab initio studies. Phosphorus
Sulfur Silicon 182:631–638. doi:10.1080/10426500601046901
Gholivand K, Pourayoubi M, Shariatinia Z (2007b) 2,3J(P, X)
[X = H, C] coupling constants dependency to the ring size,
hybridization and substituents in new diazaphospholes and
diazaphosphorinanes, NMR and X-ray crystallography studies.
Polyhedron 26:837–844. doi:10.1016/j.poly.2006.09.092
Ghorab MM, Ragab FA, Hamed MM (2009) Design, synthesis and
anticancer evaluation of novel tetrahydroquinoline derivatives
containing sulfonamide moiety. Eur J Med Chem 44:4211–4217.
doi:10.1016/j.ejmech.2009.05.017
Amirkhanov VM, Ovchynnikov VA, Glowiak T, Kozlowski H (1997)
Crystal and molecular structures of N,N0-diphenyl-N00-trichlor-
oacetyl-phosphorictriamide and N,N0-tetraethyl-N00-benzoylpho-
sphorictriamide. The effect of various substituents on the
structural parameters of the [C(O)N(H)P(O)] moiety. Z Natur-
forsch 52B:1331–1336
Bentrude WG, Day RO, Holmes JM, Quin GS, Setzer WN, Sopchik
AE, Holmes RR (1984) Conformations of saturated six-mem-
bered phosphorus heterocycles. X-ray crystallographic and
proton NMR study of cis-2-oxo-2-(dimethylamino)-5-phenyl-1,
3, 2-oxazaphosphorinane, a cyclophosphamide-like molecule in a
twist conformation. J Am Chem Soc 106:106–111. doi:10.1021/
ja00313a022
Hansch C, Leo A (1995) Exploring QSAR. ACS Professional
References Book, Washington, DC
Bentrude WG, Setzer WN, Sopchik AE, Bajwa GS, Burright DD,
Hutchinson JP (1986) Conformations of saturated six-mem-
bered-ring phosphorus heterocycles related to cyclophospha-
mide. NMR, X-ray, and infrared studies of 2-methoxy-2-oxo-
1,3,2-oxazaphosphorinane and 2-thio-1,3,2-oxazaphosphorinane.
J Am Chem Soc 108:6669–6675. doi:10.1021/ja00281a037
Bentrude WG, Setzer WN, Sopchik AE, Chandrasekaran S, Ashby
MT (1988) Conformations of saturated six-membered ring
phosphorus heterocycles. 2-Aryl-1,3,2-.lambda.5-oxazaphospho-
rinanes. J Am Chem Soc 110:7119–7127. doi:10.1021/ja00229a
027
Bentrude WG, Setzer WN, Ramli E, Khan M, Sopchik AE (1991)
Anomeric-like substituent effects on the chair–chair conforma-
tional equilibrium of the 2-oxo-1,3,2-oxazaphosphorinane ring
system. J Org Chem 56:6127–6131. doi:10.1021/jo00021a031
Camerman N, Camerman A (1973) Cyclophosphamide structure.
Molecular structure of 4-ketocyclophosphamide. J Am Chem
Soc 95:5038–5041. doi:10.1021/ja00796a042
Hansch C, Muir RM, Fujita T, Maloney PP, Geiger F, Streich M
(1963) The correlation of biological activity of plant growth
regulators and chloromycetin derivatives with Hammett con-
stants and partition coefficients. J Am Chem Soc 85:2817–2824.
doi:10.1021/ja00901a033
Huang Z, Raychowdhury MK, Waxman DJ (2000) Impact of liver
P450 reductase suppression on cyclophosphamide activation,
pharmacokinetics and antitumoral activity in cytochrome P450-
based cancer gene therapy model. Cancer Gene Ther 7:1034–1042
Jackman LM, Sternhell S (1969) Nuclear magnetic resonance
spectroscopy in organic applications of chemistry. Pergamon
Press, London
Li Z, Han J, Jiang Y, Browne P, Knox RJ, Hu
L (2003)
Nitrobenzocyclophosphamides as potential prodrugs for biore-
ductive activation: synthesis, stability, enzymatic reduction, and
antiproliferative activity in cell culture. Bioorg Med Chem
11:4171–4178. doi:10.1016/S0968-0896(03)00459-0
Ludeman SM, Zon G, Egan W (1979) Synthesis and antitumor
activity of cyclophosphamide analogues. 2.1 Preparation, hydro-
lytic studies, and anticancer screening of 5-bromocyclophospha-
mide, 3,5-dehydrocyclophosphamide, and related systems. J
Med Chem 22:151–158. doi:10.1021/jm00188a006
Camerman A, Smith HW, Camerman N (1983) Activated cyclophos-
phamide anticancer drugs: molecular structures of cis- and trans-
4-hydroperoxyisophosphamides. J Med Chem 26:679–683. doi:
10.1021/jm00359a
Cates LA, Lemke TL (1974) Phosphorus-nitrogen compounds XVIII:
hydrazides and thiosemicarbazides. J Pharm Sci 63:1736–1739.
doi:10.1002/jps.2600631114
Chen G, Waxman DJ (1995) Identification of glutamine S-transferase
as a determinant of 4-hydroxycyclophosphamide resistance in
human breast cancer cells. Biochem Pharmacol 49:1691–1701
Eliel EL, Hutchins RO (1969) Conformational analysis. XVIII. 1,3-
Dithianes. Conformational preferences of alkyl substituents and
the chair-boat energy difference. J Am Chem Soc 91:2703–2715.
doi:10.1021/ja01038a050
Ludeman SM, Boyd VL, Regan JB, Gallo KA, Zon G, Ishii AK
(1986) Synthesis and antitumor activity of cyclophosphamide
analogs. 4. Preparation, kinetic studies, and anticancer screening
of phenylketophosphamide and similar compounds related to the
cyclophosphamide metabolite aldophosphamide. J Med Chem
29:716–727
´
¨¨
¨ ¨
Martinek T, Forro E, Gu¨nther G, Sillanpaa R, Fulop F (2000)
Synthesis and conformational study of 1,3,2-oxazaphosphori-
no[4,3-a]isoquinolines: a new ring system. J Org Chem 65:316–
321. doi:10.1021/jo991047?
Ghadimi S, Ebrahimi Valmoozi AS (2009) Lipophilicity, electronic,
steric and topological effects of some phosphoramidates on
acethylcholinesterase inhibitory property. J Iran Chem Soc 6:
838–848
Ghadimi S, Ebrahimi Valmoozi AS, Pourayoubi M, Samani KA
(2008) Structure-activity study of phosphoramido acid esters as
acetylcholinesterasf inhibitors. J Enzyme Inhib Med Chem 23:
556–561
Moore MJ (1991) Clinical pharmacokinetics of cyclophosphamide.
Clin Pharmacokinet 20:194–208
Mosmann T (1983) Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays. J
Immunol Methods 65:55–63. doi:10.1016/0022-1759(83)90303-4
Murata M, Suzuki T, Midorikawa K, Oikawa S, Kawanishi S (2004)
Oxidative DNA damage induced by a hydroperoxide derivative
of cyclophosphamide. Free Radic Biol Med 37:793–802
Noble PB, Cutts JH, Carroll AK (1968) Ficoll flotation for the
separation of blood leukocyte types. Blood 31:66–73
Schwartz PS, Waxman DJ (2001) Cyclophosphamide induces caspase
9-dependent apoptosis in 9L tumor cells. Mol Pharmacol
60:1268–1279
Gholivand K, Shariatinia Z, Pourayoubi M, Farshadian S (2005a)
Syntheses and spectroscopic study of some new diazaphosp-
holes and diazaphosphorinanes; crystal structure of
.
Z
Naturforsch 60b:
4-F-C6H4C(O)N(H)P(O)[NHC6H4NH]
1021–1026
Gholivand K, Pourayoubi M, Farshadian S, Molani S, Shariatinia Z
(2005b) Synthesis and crystal structure of 5,5-dimethyl-2-(p-
methylanilino)-2-oxo-1,3,2-diazaphosphorinane. Anal Sci 21:x55.
doi:10.2116/analscix.21.x55
Setzer WN, Black BG, Hovanes BA (1989) Conformational analy-
sis of 1,3,2-oxazaphospholanes derived from ephedrine and
pseudoephedrine. J Org Chem 54:1709–1713. doi:10.1021/
jo00268a038
123

Med Chem Res (2012) 21:2185–2195
2195
¨
¨ ¨
Taylor MD (1996) Improved passive oral drug delivery via prodrugs.
Adv Drug Deliv Rev 19:131–148. doi:10.1016/0169-409X(95)
00104-F
Tolkimth H (1959) Electron group polarizability and molecular
properties of organophosphorus compounds. Ann NY Acad Sci
79:189–232. doi:10.1111/j.1749-6632.1959.tb42781.x
Viljanen T, Tahtinen P, Pihlaja K, Fulop AF (1998) Conformational
study of some saturated 2-[bis(2-chloroethyl)amino]-1,3,2-ben-
zoxazaphosphorinane 2-oxides. J Org Chem 63:618–627. doi:
10.1021/jo971490p
Software for Windows (2000) GraphPad Software Inc., San Diego,
CA. http://www.graphpad.com
123