N-phosphinyl ureas: Synthesis, characterization, X-ray structure, and in vitro evaluation of antitumor activity

Article abstract of DOI:10.1002/hc.20754

A new series of N-phosphinylureas 5b, 6a-7c was synthesized and characterized by ¹H, ¹³C, ³¹P NMR, IR, and elemental analysis. The three-dimensional structure of 5b has been determined by X-ray crystallography. The crystal structure revealed the existence of four independent molecules. All structures form two chains with different arrangements and connect to each other via hydrogen bonds to produce two-dimensional polymeric chains. The cytotoxicity of cyclophosphamide (a standard antitumor compound) and its nine analogues with formula R ¹C₆H₄ NHC(O)NHP(O)XCH₂C(R ²)₂ CH₂Y(X = Y = NH, R² = CH ₃, R¹ = H (5a), CH₃ (5b), NO₂ (5c), X = O, Y = NH, R² = H, R¹ = H (6a, CH₃ (6b), NO₂ (6c), and X = Y = O, R² = CH₃, R ¹ = H (7a), CH₃ (7b), NO₂ (7c)) as well as phenyl urea were evaluated in vitro against three human tumor cell lines K562, MDA-MB-231, and HepG2. The results showed that most of the compounds have significant activity against the selected cell lines. Also, HepG2 cells were more sensitive to all the tested compounds than other cell lines.

Full text of DOI:10.1002/hc.20754

Heteroatom Chemistry
Volume 23, Number 1, 2012
N-Phosphinyl Ureas: Synthesis,
Characterization, X-Ray Structure, and In Vitro
Evaluation of Antitumor Activity
Khodayar Gholivand,1 Nilufar Dorosti,1 Fatemeh Ghaziany,1
Manouchehr Mirshahi,2 and Sina Sarikhani2
1Department of Chemistry, Faculty of Science, Tarbiat Modares University, P.O. Box: 14115-175,
Tehran, Iran
2Department of Biochemistry, Faculty of Biology, Tarbiat Modares University, Tehran, Iran
Received 15 April 2011; revised 5 July 2011
INTRODUCTION
ABSTRACT: A new series of N-phosphinylureas
5b, 6a–7c was synthesized and characterized by
N-phosphinylureas are some important instances
of phosphoramidates that little attention has been
given to their biological properties [1,2] and struc-
tural studies [1,3]. These compounds can cause at-
tractive biological activities due to having urea and
peptide moieties. Recently, we evaluated the cy-
totoxic effects of several phosphoramidates with
the general formula RP(O)[NHCH2C(CH3)2CH2NH]
against human leukemia K562 [4]. Results re-
vealed that N-phosphinylureas are good candi-
dates for antitumor activity. On the other hand,
several studies are concentrated on the devel-
opment of six-membered P-heterocycles, owing
to their biological activities and unique confor-
mational and stereochemical aspects [5–7]. Cy-
clophosphamide is one of the phosphorus hete-
rocycles that is used in the treatment of human
cancers [8–11]. Nowadays, modifications in cy-
clophosphamides have led to the design and syn-
thesis of numerous cyclic analogues [12] to find
an antitumor agent with fewer side effects. In this
study, following our previous research, the novel
derivatives were synthesized and characterized by
1H, 13C, 31P NMR, and infrared (IR) spectroscopy
and elemental analysis. X-ray crystallography con-
firms the existence of four conformers in crystalline
lattice. Since few crystal structures of these types of
molecules were reported on [1,3], we believe that it
is the first example of N-phosphinylureas with four
1 H, 13C, 31P NMR, IR, and elemental analysis. The
three-dimensional structure of 5b has been determined
by X-ray crystallography. The crystal structure revealed
the existence of four independent molecules. All struc-
tures form two chains with different arrangements
and connect to each other via hydrogen bonds to pro-
duce two-dimensional polymeric chains. The cytotoxi-
city of cyclophosphamide (a standard antitumor com-
pound) and its nine analogues with formula R1C6 H
4
NHC(O)NHP(O)XCH2C(R2)2 CH2Y(X = Y = NH,
R2 = CH3, R1 = H (5a), CH3 (5b), NO2 (5c), X = O,
Y = NH, R2 = H, R1 = H (6a, CH3 (6b), NO2 (6c),
and X = Y = O, R2 = CH3, R1 = H (7a), CH3 (7b),
NO2 (7c)) as well as phenyl urea were evaluated in
vitro against three human tumor cell lines K562, MDA-
MB-231, and HepG2. The results showed that most
of the compounds have significant activity against
the selected cell lines. Also, HepG2 cells were more
sensitive to all the tested compounds than other cell
ꢀ
C
lines.
2011 Wiley Periodicals, Inc. Heteroatom Chem
23:74–83, 2012; View this article online at wileyonlineli-
brary.com. DOI 10.1002/hc.20754
Correspondence to: Khodayar Gholivand; e-mail: gholi kh@
modares.ac.ir.
Contract grant sponsor: Research Office of Tarbiat Modares
University.
c
ꢀ
2011 Wiley Periodicals, Inc.
74

N-Phosphinyl Ureas: Synthesis, Characterization, X-ray Structure, and Evaluation of Antitumor Activitys 75
conformers. Furthermore, we would like to report
the final concentration of DMSO was 2%, the sol-
vent showed no activity in these assays at the level
that was used for screening. These compounds were
stored in a freeze prior to use. An MTT assay was
performed at 24, 48, and 72 h after adding all
compounds to assess cell viability. MTT was added
(20 μL of 5 mg/mL MTT in phosphate buffered saline
(PBS)), and the plates were incubated at 37◦C for
an additional 3–4 h. MTT can be reduced to a blue
formazan dye and thereby assesses cell viability by
measuring mitochondrial function. DMSO was then
added to dissolve the resultant crystals, and the opti-
cal absorbance was measured at 570 nm on an Eliza
reader. The results were compared with those of a
control reference plate fixed on the treatment day,
and the growth inhibition percentage was calculated
for each drug contact period. Each assay was set up
in triplicate wells and repeated one to three times.
Graph Pad PRISM version 5.0 [16] was used to fit
the data. Also, to study the degree of selectivity in
the cytotoxic activity of the compounds, separation
of lymphocyte was down according to the follow-
ing procedure [17]: Defibrinated or anticoagulant-
treated blood was diluted with an equal volume of
PBS and layered carefully over Ficoll–Paque PLUS
(without intermixing) in a centrifuge tube. After a
short centrifugation at room temperature (typically
at 400 gav for 30–40 min), lymphocytes, together with
monocytes and platelets, are harvested from the in-
terface between the Ficoll–Paque PLUS and sample
layers. This material was then centrifuged twice in a
balanced salt solution to wash the lymphocytes and
to remove the platelets.
on the structural modifications of phosphorus hete-
rocycles with the aim of examining their cytotoxic ef-
fects. However, the antitumor activity of cyclophos-
phamide, its nine analogues, and phenylurea were
determined against K562, MDA-MB-231, and HepG2
cell lines by applying the MTT colorimetric assay.
EXPERIMENTAL
Material and Methods
All reactions were carried out in an argon atmo-
sphere. All chemicals and solvents were purchased
from Merck (Tehran, Iran) and used without fur-
ther purification. 1H, 13C, and 31P NMR spectra were
recorded on a Bruker (Avance DRS) 500-MHz spec-
trometer. 1H and 13C chemical shifts were deter-
mined relative to TMS, 31P chemical shifts relative
to 85% H3PO4 as an external standard. IR spectra
were obtained using KBr pellets on a Shimadzu IR-
60 model spectrometer. Elemental analysis was per-
formed using a Heraeus CHN-O-RAPID apparatus.
Melting points were determined on an electrother-
mal apparatus.
X-Ray Measurements
X-ray data of compound 5b were collected on a
Bruker SMART 1000 CCD [13] single-crystal diffrac-
tometer with graphite monochromated Mo Kα radi-
˚
ation (k = 0.71073 A). The structure was refined with
SHELXL-97 [14] by full-matrix least-squares on F2.
The positions of hydrogen atoms were obtained from
the difference Fourier map. Routine Lorentz and po-
larization corrections were applied, and an absorp-
tion correction was performed using the SADABS
program for the title structure [15].
Synthesis
5,5-Dimethyl-2-(N-4-methylphenylureido)-1,3,2-
diazaphosphorinane-2-oxide (5b). A solution of 2,2-
dimethyl-1,3-diaminopropane (1.06 g, 10.4 mmol)
in dry diethylether (15 mL) was treated dropwise to
suspension of N-4-methylphenylureidophosphoryl
dichloride (3) (1.38 g, 5.2 mmol) in dry diethylether
(15 mL) with a 2:1 molar ratio and stirred at 0◦C.
After stirring for 5 h, the products were filtered off
and then washed with H2O.
In Vitro Cytotoxicity Assay
The cytotoxic activity of the synthesized compounds
was determined on three human cancer cell lines:
Human leukemia K562, Human breast MDA-MB-
231, and HepG2 hepatoma by MTT assay using cy-
clophosphamide as a conventional anticancer agent.
For a typical screening experiment, cells are inoc-
ulated into 96-well-microtiter plates in 100 μL at
plating densities ranging from 5000 to 8000 cells
per well depending on the doubling time of individ-
ual cell lines. After cell inoculation, the microtiter
plates are incubated at 37◦C, 5% CO2, 95% air, and
100% relative humidity for 24 h. After 24 h, expo-
nentially growing cells were exposed to various con-
centrations of several tested compounds. Although
all tested compounds were dissolved in DMSO and
Yield: 73%. mp 202–203◦C. Anal. Calcd. for
C13H21N4O2P (%): C, 52.70; H, 7.14; N, 18.91. Found:
C, 52.63; H, 7.20; N, 18.79. IR (KBr): νmax = 3185
(N H), 2925, 1686 (C O), 1600, 1545, 1507, 1441,
1325, 1256, 1180 (P O), 1106, 1043, 950, 910, 810,
1
761, 590 cm−1. H NMR (500.13 MHz, d6-DMSO):
δ = 0.79 (s, 3H, CH3), 1.03 (s, 3H, CH3), 2.21 (s, 3H,
CH3), 2.57 (ddd, 2 J(H, H) = 12.0 Hz, 3 J(H, H) =
3
2
5.25 Hz, J(P, H) = 24.31 Hz, 2H), 2.98 (dd, J(H,
H) = 12.0 Hz, 3 J(H, H) = 2.7 Hz, 2H), 4.65 (d, 2 J(P,
Heteroatom Chemistry DOI 10.1002/hc

76 Gholivand et al.
H) = 3.5 Hz, 2H, NHendocyclic), 7.25 (d, 3 J(H, H) = 8.2
Hz, 2Haromatic), 7.05 (d, 3 J(H, H) = 8.2 Hz, 2Haromatic),
(s), 129.15 (s), 131.19 (s), 136.39 (s), 152.71 (d, 2 J(P,
C) = 2.69 Hz). 31P NMR (202.46, d6-DMSO): δ =
−0.58 (b).
2
7.58 (d, J(P, H) = 7.15 Hz, 1H, NHP), 9.22 (s, 1H,
PhNH). 13C NMR (125.77 MHz, d6-DMSO): δ = 20.22
(s, CH3), 23.29 (s, CH3), 24.84 (s, CH3), 30.46 (d, 3 J(P,
C) = 4.1 Hz), 52.32 (s), 118.16 (s), 129.09 (s), 130.75
(s), 136.75 (s), 153.33 (s). 31P NMR (202.46 MHz,
d6-DMSO): δ = 3.85 (m).
2-(N-4-nitrophenylureido)-1,3,2-oxazaphosphori-
nane-2-oxide (6c). Yield: 35%. mp = 204–205◦C.
Anal. Calcd. for C10H13N4O5P (%): C, 40.01; H, 4.36;
N, 18.66. Found: C, 40.09; H, 4.38; N, 18.61. IR
(KBr): νmax = 3300 (N H), 3080, 1704 (C O), 1610,
1550, 1499, 1343 (NO2), 1219, 1193 (P O), 1112,
1
1062, 1009, 935, 848, 815, 744, 650, 529 cm−1. H
General Procedure for the Synthesis of Oxazaphos-
phorinanes 6a–6c. To a suspension, intermediates
2–4 (5.2 mmol) in acetonitrile (20 mL), 3-amino-1-
propanol (0.78 g, 10.4 mmol) for 6a or title amino
alcohol (0.39 g, 5.2 mmol) and triethylamine (1.05 g,
10.4 mmol) for 6b and 6c at 0◦C were added with
stirring. The reaction mixture was stirred overnight
at room temperature; the solvent was then partially
evaporated off, was the oily residue dried after a few
days and washed with cooled water, and the precip-
itate filtered off and dried at room temperature.
2-(N-Phenylureido)-1,3,2-oxazaphosphorinane-2-
oxide (6a). Yield: 40%. mp 197–198◦C. Anal. Calcd.
for C10H14N3O3P (%): C, 47.06; H, 5.53; N, 16.46.
NMR (500.13 MHz, d6-DMSO): δ = 1.71 (d, 2 J(H,
H) = 14.25 Hz, 1H, CH2), 1.80 (m, 1H, CH2), 3.09
(m, 1H), 3.17 (m, 1H), 4.27 (m, 1H), 4.38 (m, 1H),
3
5.34 (d, J(H, H) = 3.1 Hz, 1H, NHendocyclic), 6.69 (s,
1H, NHP), 7.63 (d, 3 J(H, H) = 9.05 Hz, 2Haromatic),
7.93 (d, 3 J(H, H) = 9.05 Hz, 2Haromatic), 9.6 (s,
1H, PhNH). 13C NMR (125.77 MHz, d6-DMSO):
δ = 25.31 (d, 3 J(P, C) = 7.45 Hz), 40.27 (d, 2 J(P,
2
C) = 2.44 Hz), 68.69 (d, J(P, C) = 7.73 Hz), 112.33
(s), 116.88 (s), 117.78 (s), 126.28 (s), 141.53 (s),
2
145.42 (s), 152.53 (d, J(P, C) = 2.84 Hz). 31P NMR
(202.46 MHz, d6-DMSO): δ = –1.38 (m).
Found: C, 47.12; H, 5.56; N, 16.35. IR (KBr): νmax
=
General Procedure for the Synthesis of Dioxaphos-
phorinanes 7a–7c. These compounds were synthe-
sized from the reaction of (5.2 mmol) intermedi-
ates 2–4 suspended in 25 mL dichloromethane with
(0.53 g, 5.2 mmol) 2,2-dimethyl-1,3-propandiol and
(1.05 g, 10.4 mmol) triethylamine at 0◦C. The solu-
tion was stirred overnight at room temperature and
then evaporated in vacuum. The oily residue was
washed with cooled water and the precipitate was
filtered off and dried at room temperature.
3320 (N H), 3185, 1679 (C O), 1598, 1532, 1460,
1435, 1332, 1310, 1231 (P O), 1204, 1125, 1105,
1
1048, 982, 942, 848, 767, 737, 466 cm−1. H NMR
(500.13 MHz, d6-DMSO): δ = 1.69 (d, 2 J(H, H) =
12.65 Hz, 1H), 1.80 (m, 1H), 3.11 (m, 1H), 3.30
(m, 1H), 4.25 (m, 1H), 4.36 (m, 1H), 5.24 (s, 1H,
NHendocyclic), 6.98 (t, 3 J(H, H) = 6.8 Hz, 1Haromatic),
7.12 (t, 3 J(H, H) = 7.15 Hz, 2Haromatic), 7.38 (d, 3 J(H,
2
H) = 7.4 Hz, 2Haromatic), 7.97 (d, J(P, H) = 7.2 Hz,
1H, NHP), 8.97 (s, 1H, PhNH).13C NMR (125.77
5,5 - Dimethyl-2-(N-phenylureido)-1,3,2-dioxaph-
osphorinane-2-oxide (7a). Yield: 70%, mp = 232–
233◦C. Anal. Calcd. for C12H17N2O4P (%): C, 50.71;
H, 6.03; N, 9.86. Found: C, 50.85; H, 6.14; N, 9.75.
IR (KBr): νmax = 3380 (N H), 3250, 3140, 1700
(C O), 1600, 1541, 1487, 1443, 1310, 1244 (P O),
3
MHz, d6-DMSO): δ = 25.43 (d, J(P, C) = 7.3 Hz),
40.32 (s), 68.5 (d, 2 J(P, C) = 7.6 Hz), 118.33 (s),
2
122.36 (s), 128.68 (s), 138.97 (s), 152.77 (d, J(P, C)
= 2.69 Hz). 31P NMR (202.46 MHz, d6-DMSO): δ =
−0.64 (m).
1
2-(N-4-methylphenylureido)-1,3,2-oxazaphosph-
orinane-2-oxide (6b). Yield: 40%. mp = 196–197◦C.
Anal. Calcd. for C11H16N3O3P (%): C, 49.07; H,
5.99; N, 15.61. Found: C, 49.03; H, 6.05; N,15.65.
IR (KBr): νmax = 3310 (N H), 3170, 1680 (C O),
1596, 1527, 1458, 1238 (P O), 1201, 1113, 1050,
981, 926, 845, 451 cm−1. 1H NMR (500.13 MHz,
d6-DMSO): δ = 1.70 (d, 2 J(H, H) = 13.4 Hz, 1H,
CH2), 1.81 (m, 1H, CH2), 2.23 (s, 3H, CH3), 3.08
(m, 1H), 3.29 (m, 1H), 4.23 (m, 1H), 4.35 (m, 1H),
1046, 1010, 983, 750, 482 cm−1. H NMR (500.13
MHz, d6-DMSO): δ = 0.89 (s, 3H, CH3), 1.14 (s, 3H,
CH3), 3.97 (dd, 2 J(H, H) = 10.45 Hz, 3 J(P, H) =
2
3
18.91 Hz, 2H), 4.29 (dd, J(H, H) = 10.35 Hz, J(P,
H) = 3.95 Hz, 2H), 7.015 (t, 3 J(H, H) = 7.15 Hz,
1Haromatic), 7.28 (t, 3 J(H, H) = 7.65 Hz, 2Haromatic), 7.38
(d, 3 J(H, H) = 7.9 Hz, 2Haromatic), 8.38 (d, 2 J(P, H)
= 8.3 Hz, 1H, NHP), 8.74 (s, 1H, PhNH). 13C NMR
(125.77 MHz, d6-DMSO): δ = 19.82 (s), 21.22 (s),
31.58 (d, 3 J(P, C) = 6.7 Hz), 77.40 (d, 2 J(P, C) = 7.07
Hz), 118.70 (s), 122.80 (s), 138.48 (s), 152.50 (d, 2 J(P,
C) = 3.53 Hz). 31P NMR (202.46 MHz, d6-DMSO):
δ = −8.95 (m).
3
5.19 (s, 1H, NHendocyclic), 7.06 (d, J(H, H) = 8.1 Hz,
2Haromatic), 7.25 (d, 3 J(H, H) = 8.2 Hz, 2Haromatic),
7.9 (d, 2 J(PNH) = 7.9 Hz, 1H, NHP), 8.87 (s, 1H,
PhNH). 13C NMR (125.77 MHz, d6-DMSO): δ =
20.25 (s), 25.4 (d, 3 J(P, C) = 7.4 Hz), 40.26 (d, 2 J(P,
C) = 2.9 Hz), 68.4 (d, 2 J(P, C) = 7.61 Hz), 118.39
5,5 - Dimethyl - 2 - (N - 4 - methylphenylureido)-
1,3,2-dioxaphosphorinane-2-oxide (7b). Yield: 65%,
mp = 209–210◦C. Anal. Calcd. for C13H19N2O4P (%):
Heteroatom Chemistry DOI 10.1002/hc

N-Phosphinyl Ureas: Synthesis, Characterization, X-ray Structure, and Evaluation of Antitumor Activitys 77
2
3
C, 52.36; H, 6.42; N, 9.39. Found: C, 52.27; H, 6.42;
N, 9.45. IR (KBr): νmax = 3380 (N H), 3250, 1700
(C O), 1600, 1541, 1487, 1443, 1310, 1244 (P O),
Hz, 2H), 4.29 (dd, J(H, H) = 10.50 Hz, J(P, H) =
3
4.30 Hz, 2H), 7.65 (d, J(H, H) = 9.2 Hz, 2Haromatic),
3
2
8.19 (d, J(H, H) = 9.2 Hz, 2Haromatic), 8.63 (d, J(P,
H) = 9.1 Hz, 1H, NHP), 9.38 (s, 1H, PhNH). 13C
NMR (125.77 MHz, d6-DMSO): δ = 19.78 (s), 21.19
1
1046, 1010, 983, 750, 462 cm−1. H NMR (500.13
MHz, d6-DMSO): δ = 0.89 (s, 3H, CH3), 1.14 (s, 3H,
2
3
2
CH3), 2.23 (s, 3H, CH3), 3.97 (dd, J(H, H) = 10.45
(s), 31.65 (d, J(P, C) = 6.97 Hz), 77.57 (d, J(P, C)
= 6.88 Hz), 118.18 (s), 125.0 (s), 141.88 (s), 145.00
(s), 151.99 (d, 2 J(P, C) = 3.34 Hz). 31P NMR (202.46
MHz, d6-DMSO): δ = −9.68 (m).
3
2
Hz, J(P, H) = 18.91 Hz, 2H), 4.29 (dd, J(H, H) =
10.40 Hz, 3 J(P, H) = 4.40 Hz, 2H), 7.08 (d, 3 J(H,
3
H) = 8.2 Hz, 2Haromatic), 7.26 (d, J(H, H) = 8.3 Hz,
2Haromatic), 8.34 (d, 2 J(P, H) = 9.35 Hz, 1H, NHP), 8.64
(s, 1H, PhNH). 13C NMR (125.77 MHz, d6-DMSO): δ
= 19.83 (s), 20.28 (s), 21.22 (s), 31.58 (d, 3 J(P, C)
RESULTS AND DISCUSSION
2
= 7.11 Hz), 77.39 (d, J(P, C) = 6.8 Hz), 118.77 (s),
Synthetic and Spectroscopic Aspects
2
129.17 (s), 131.73 (s), 135.91 (s), 152.03 (d, J(P, C)
= 2.88 Hz). 31P NMR (202.46 MHz, d6-DMSO): δ =
−8.85 (m).
As indicated in Scheme 1, compounds 5b and
6a–7c were prepared from phosphorus pentachlo-
ride as a starting material, which was treated
with ethyl carbamate in ethylene chloride to give
dichloroisocyanatophosphine oxide (1) [18]. Then,
treatment of aniline derivatives with 1 afforded
RC6H4NHC(O)NHP(O)Cl2 (R = H (2), CH3 (3), NO2
(4)) [19]. Afterward, derivatives 5b and 6a–7c were
obtained from the reaction of intermediates 2–4 with
the corresponding diamine, amino alcohol, and diol
in the presence of an HCl scavenger.
5,5 - Dimethyl - 2 - (N - 4-nitrophenylureido)-1,3,2-
dioxaphosphorinane-2-oxide (7c). Yield: 60%, mp =
233–235◦C. Anal. Calcd. for C12H16N3O6P (%): C,
43.78; H, 4.89; N, 12.76. Found: C, 43.69; H, 4.75;
N, 12.88. IR (KBr): νmax = 3350 (N H), 3065, 1700
(C O), 1611, 1552, 1476, 1339 (NO2), 1261(P O),
1064, 1012, 943, 856, 499 cm−1. H NMR (500.13
1
MHz, d6-DMSO): δ = 0.89 (s, 3H, CH3), 1.15 (s, 3H,
CH3), 3.99 (dd, 2 J(H, H) = 10.55 Hz, 3 J(P, H) = 19.21
O
N
P
H
H
N
R
Y
X
X = NH, Y = O, R = H (6a)
X = Y = O, R = CH3 (7a)
O
R
O
H
H
Cl
Cl
N
N
P
O
(2)
+ C6H5NH2
O
P
H
H
Cl
Cl
−C2H5Cl, 2HCl
N
N
+ 4−CH3C6H4NH2
(3)
PCl5 + NH2COOC2H5
Cl2P(O)NCO
O
(1)
H3C
+ 4−NO2C6H4NH2
O
H
N
H
O
P
H
H
N
Cl
Cl
R
P
Y
N
N
X
O
R
O
H3C
(4)
O2N
X = Y = NH, R = CH3 (5b)
X = NH, Y = O, R = H (6b)
X = Y = O, R = CH3 (7b)
O
P
X = NH, Y = O, R = H (6c)
X = Y = O, R = CH3 (7c)
H
H
N
N
R
Y
X
O
R
O2N
SCHEME 1
Heteroatom Chemistry DOI 10.1002/hc

78 Gholivand et al.
TABLE 1 Some Spectroscopic Data of Compounds 5a–7c
δ (31P) 3J(PNCH)
3J(POCH)
(Hz)
2J(P,H)exocycl i c 2J(P,C)exocycl i c 3J(P,C)endocyclic
Compound (ppm)
(Hz)
(Hz)
(Hz)
(Hz)
ν(P O) ν(C O)
5aa
5b
5ca
6a
6b
6c
7a
3.78
3.85
3.23
−0.64
−0.54
−1.38
−8.95
−8.85
−9.68
24.21
24.01
24.47
m
b
b
–
–
–
–
–
–
m
b
b
6.76
7.15
7.59
7.2
7.9
9.0
8.3
9.35
9.1
–
–
–
2.69
2.69
2.84
3.53
2.88
3.34
4.3
4.1
4.53
7.3
7.4
7.45
6.7
7.11
6.97
1184
1180
1200
1231
1238
1240
1244
1244
1261
1677
1686
1699
1679
1680
1704
1700
1700
1700
18.91(eq), 3.95(ax)
18.91(eq), 4.4(ax)
19.21(eq), 4.3(ax)
7b
7c
aFor compounds 5a and 5c, see Ref. [1].
Phosphorus–hydrogen and phosphorus–carbon
coupling constants and δ(31P) data of compounds
5a–7c are summarized in Table 1. The 31P NMR
spectra of these compounds reveal the substituent
effect on the δ(31P) so that the phosphorus atom in
derivatives containing NO2 shows a high upfield shift
compared to the CH3 group. Substitution of the NH
group in a diazaphosphorinane ring with an oxy-
gen atom causes extremely great changes in δ(31P),
X-Ray Crystallography
Colorless crystals of 5b suitable for X-ray diffraction
analysis were grown from an ethanol/chloroform
mixture after slow evaporation at room temperature.
Compound crystallizes in the triclinic crystal system
with space group P¯ı. The crystal data and the details
of the X-ray analysis are presented in Table 2, and
selected bond lengths and angles are summarized in
Table S1 (in the Supporting Information). Molecu-
lar structures (ORTEP view) and hydrogen-bonding
chains are shown in Figs. 1 and 2, respectively. Com-
pound contains four conformers 5b (1), 5b (2), 5b
(3), and 5b (4) in the solid state due to different spa-
tial orientations of the four conformers relative to
each other that cause different torsion angles (see
Table S1). Four types of hydrogen bonds are
established among these conformers. Hydrogen
bonding data of these structures are presented
in Table 3. There are intramolecular P O· · ·H–
NPh, intermolecular C O· · ·NHP, C O· · ·NH, and
P O· · ·NH hydrogen bonds. Each pair conformer
(5b (1), 5b (3), and 5b (2), 5b (4)) produces a
centrosymmetric dimer via C(O)· · ·H–N hydrogen
bonds. These conformers create two types of chains
with different arrangements in the crystal lattice of
5b. Linking of these chains by hydrogen bonding
leads to the formation of a two-dimensional poly-
meric chain in the crystal lattice.
2 J(PNHexocyclic), J(PNHendocyclic), and J(P, H) in ox-
aza and dioxaphosphorinanes. The 31P NMR spectra
for dioxaphosphorinanes 7a–7c demonstrate large
shifts to the upper field compared to those of their
related oxaza and diazaphosphorinanes. The 1H and
13C NMR spectra of molecules 5a–5c and 7a–7c
show two separate signals for CH3 groups on di-
aza and dioxaphosphorinane rings. The Hequatorial and
Haxial atoms of CH2 groups in these compounds ap-
2
3
1
pear at different chemical shifts in H NMR spec-
3
3
tra. As shown in Table 1, J(PNCH) and J(POCH)
coupling constants for Hequatorial are about 24.5 and
19.0 Hz. The Haxial atom has the largest coupling
with the phosphor atom from 3.95 to 4.40 Hz
in dioxaphosphorinanes 7a–7c. Furthermore, the
2 J(PNHendocyclic) coupling constant in oxazaphospho-
rinanes decreases to zero, whereas it ranges from
2.7 to 3.5 Hz for diazaphosphorinanes 5a–5c. It
can be deduced that the oxygen atom decreases
ring strain and hence diminishes the 2 J(PNHendocyclic
)
The mean P O distances fall in the range of
coupling constant. Besides, 2 J(PNHexocyclic) and 2 J(P,
Cexocyclic) coupling constants in dioxaphosphorinans
are larger than oxaza and diazaphosphorinanes,
respectively. The IR spectra for these compounds
show the absorption stretching band values in the
range of 1261–1180 cm−1 and 1704–1677 cm−1 for
P O and C O groups, respectively. Higher val-
ues of stretching absorption bands are observed
for the compounds with electron-withdrawing
substituents.
˚
1.4803–1.4845 A, which are slightly longer than
˚
the normal P O bond length (1.45 A). The phos-
phoryl oxygen atoms (O(2), O(22), O(23), and
O(24)) occupy a pseudoaxial position, and the 4-
methylphenylureido groups adopt a pseudoequato-
rial orientation. In all of the compounds, the P–Namide
bond length is longer than the P–Namine bond lengths
(Table 3), because of the electrostatic interaction
of the Namide lone pair with the π∗ CO. All of these
Heteroatom Chemistry DOI 10.1002/hc

N-Phosphinyl Ureas: Synthesis, Characterization, X-ray Structure, and Evaluation of Antitumor Activitys 79
TABLE 2 Crystallographic Data and Structure Refinement
rinane 7a–c and phenyl urea were assessed in
vitro against three human tumor cell lines, that is,
leukemia human cell line (K562), human breast cell
line (MDA-MB-231), and hepatocellular carcinoma
cell line (HepG2); and the cyclophosphamide (CP)
was used as a control. The results are summarized
in Table 4. The plots of IC50 values for all of the
compounds are shown in Fig. 3. Cell viability was
analyzed using the MTT colorimetric assay [24].
As shown in Table 4, the IC50 values of all the N-
phosphinylureas, except derivative 7a, were in the
range of 153–202 nM in K562 cells. Similarly, in
MDA-MB-231 and HepG2 cell lines, the IC50 values
for all the compounds were found in the ranges of
57–260 and 63–339 nM, respectively (Table 4).
From the biological results of compounds 5a–
c, 6a–c, and 7a–c, it is found that substituent on
the phenyl ring significantly influences the activity
against the title cell lines. Derivatives such as 6b
(the 4-CH3 group) and 6c (the 4-NO2 group) showed
higher anticancer activity (168 and 180 nm against
K562 and 57, 60 against MDA-MB-231, respectively)
than an unsubstituted molecule 6a (198 nm against
K562 and 260 nm against MDA-MB-231). Also, the
activity of these molecules against the HepG2 cell
line showed that replacement of H with NO2 af-
fords the more potent derivatives. The compound
5c showed an IC50 value of 73 nM. In contrast,
5a and 5b exhibited IC50 values of 94 and 97 nM,
respectively.
In an attempt to explore the influence of incorpo-
rating a heteroatom in the moiety of phosphorinane
on the activity, the NH in 5a–c was replaced with
O to afford the corresponding compounds 6a–c and
7a–c. Biological results indicated that introduction
of an O atom led to an increase in activity against the
HepG2 cell line. For example, dioxaphosphorinane
7a showed an IC50 value of 67 nM, whereas oxaza-
phosphorinane 6a and diazaphosphorinane 5a ex-
hibited IC50 values of 78 and 94 nM against HepG2,
respectively. In contrast, as shown in Table 4, re-
placement of NH with oxygen resulted in a de-
crease in K562 inhibitory activity, so that the IC50
value decreased from 179 (in compound 5a) to
198 nM (in compound 6a) and compound 7a was
nonactive.
Results for 5b
Empirical Formula
C13H21N4O2P1
296.31
Formula weight
Temperature (K)
120(2)
˚
Wavelength (A)
0.71073
Crystal system, space group
Unit cell dimensions
Triclinic, P¯ı
˚
a (A)
11.9433(10)
15.6496(13)
18.3524(16)
111.866(3)
108.349(2)
91.766(2)
2978.6(4)
8, 1.322
0.192
1264
˚
b (A)
˚
c (A)
α (◦)
β (◦)
γ (◦)
3
˚
V (A )
Z, Calculated density (Mg m−3
)
Absorption coefficient (mm−1
F(000)
)
Crystal size (mm3)
0.55 × 0.40 × 0.20
ꢀ Range for data collection (◦)
1.82–27.00
Index ranges
−15 ≤ h ≤ 15, −19 ≤ k
≤ 19, −23 ≤ l ≤ 23
28,818
Reflections collected
Independent reflections
Completeness to θ = 27.00◦
Absorption correction
12,934 [R(int) = 0.0410]
99.4%
Semiempirical from
equivalents
Maximum and minimum
transmission
Refinement method
0.970 and 0.914
Full-matrix least
squares on F2
12934/0/733
1.007
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [for 6883
reflections with I > 2σ(I )]
R indices (all data)
R1 = 0.0581, wR2 =
0.1003
R1 = 0.1068, wR2 =
0.1120
Larges−t 3difference peak and hole 0.1068 and 0.1120
˚
(e A
)
˚
bonds are in the range of 1.608(2)–1.639(2) A and
thus are significantly shorter than a typical P–N sin-
˚
gle bond (1.77 A) [20]. The phosphorus atoms have
a slightly distorted tetrahedral configuration with
the angles in the range of 102.66(12)–116.01(12)◦
(in 1), 102.25(12)–115.58(12)◦ (in 2), 102.06(12)–
115.50(12)◦ (in 3), and 105.52(12)–116.55(12)◦(in 4).
It is noteworthy, similar to most of the phosphorami-
dates, that the P O and C O bonds are in antiposi-
tion with respect to each other [21–23].
Table 4 demonstrates that the derivatives 5b–
c, 6b–c, and 7b–c exhibited antitumor activity on
the MDA-MB-231 cell line with the average activ-
ity order of 6b–c < 5b–c < 7b–c. The only excep-
tions were unsubstituted molecules 5a, 6a, and 7a,
which showed that the presence of dioxaphospho-
rinane ring (7a) afforded the more potent deriva-
tive of diaza (5a) and oxazaphosphorinane (6a),
respectively.
Biological Activity
Preliminary antitumor activities of all the synthe-
sized compounds, namely, diazaphosphorinane 5a–
c, oxazaphosphorinane 6a–c, and dioxaphospho-
Heteroatom Chemistry DOI 10.1002/hc

80 Gholivand et al.
FIGURE 1 Thermal ellipsoids (at the 50% probability level) of four conformers.
TABLE 3 Data of the Hydrogen Bonds in the Crystalline Compound
D H· · ·A
d(D H)
d(H· · ·A)
d(D· · ·A)
<(DHA)
N(1) H(1N)· · ·O(2)#1
0.85
0.85
0.79
0.91
0.85
0.96
0.82
0.88
0.89
0.91
0.78
0.86
0.83
0.84
1.91
2.01
2.11
2.53
1.89
1.77
1.94
2.05
2.04
1.96
2.09
2.03
2.03
2.58
2.685(3)
2.844(3)
2.893(3)
3.362(3)
2.675(3)
2.669(3)
2.698(3)
2.927(3)
2.930(3)
2.870(3)
2.859(3)
2.883(3)
2.853(3)
3.370(3)
152
167
176
152
153
155
154
172
178
178
173
172
169
156
N(2) H(2N)· · ·O(13)#2
N(3) H(3N)· · ·O(24)#1
N(4) H(4N)· · ·O(1)#2
N(12) H(12N)· · ·O(22)#1
N(13) H(13N)· · ·O(23)#1
N(14) H(14N)· · ·O(24)#1
N(22) H(22N)· · ·O(14)#3
N(23) H(23N)· · ·O(1)#2
N(24) H(24N)· · ·O(12)#3
N(32) H(32N)· · ·O(2)#1
N(33) H(33N)· · ·O(22)#1
N(34) H(34N)· · ·O(23)#4
N(44) H(44N)· · ·O(14)#3
Furthermore, the insertion of a phosphorinane
ring at the terminal position of phenyl urea led
to formation of compounds 5a, 6a, and 7a, with
better cytotoxic activity compared to the parent
molecule in HepG2 cells (Table 4). All novel syn-
thesized compounds also exhibit an enhanced ac-
tivity at nanomolar concentrations compared to the
reported urea derivatives against the title cell lines
[9,25–27]. These results indicate that the presence
of C(O)NHP(O) moiety and phosphorinane ring
may be increased the antiproliferative activities of
the new compounds.
Comparison of potencies of the tested molecules
and conventional anticancer agent, cyclophos-
phamide, demonstrate that all of the derivatives
were more potent than the standard antitumor
Heteroatom Chemistry DOI 10.1002/hc

N-Phosphinyl Ureas: Synthesis, Characterization, X-ray Structure, and Evaluation of Antitumor Activitys 81
FIGURE 2 A two-dimensional polymeric chain produced by strong and weak hydrogen bonds in the crystalline lattice of
compound 5b.
compound in both the HepG2 and K562 tests, ex-
cept molecule 7a, which was inactive toward k562
cells. Also, most of the compounds 5a–7c showed an
enhanced activity compared to cyclophosphamide
against MDA-MB-231 cells. Since hydrogen bonds
play a key role in biology processes [28,29], these re-
sults suggest that an increase in the hydrogen bonds
of new derivatives toward cyclophosphamide may
be beneficial for drug-target interactions. Also, the
present study reveals that HepG2 cells were more
sensitive to all the tested compounds than other cell
lines.
On the other hand, although the lipophilicity
plays an important role during the penetration of
compounds into the cells and increases the activ-
FIGURE 3 IC50 values of compounds 5a–7c, CP and phenyl
urea against K562, MDA-MB-231, and HepG2.
ity [30], Table 4 demonstrates that there were no
direct correlations between the anticancer activ-
ity and the lipophilicity of the tested molecules.
SUPPORTING INFORMATION
This may suggest that the antitumor activity de-
Supporting Information is available from the
pends on other properties, such as the electronic
corresponding author (gholi kh@modares.ac.ir.) on
request.
nature of the substituent, a reduction in poten-
tial [31], an increase in the number, and the kind
of heteroatom in the phosphorinane ring in these
molecules.
SUPPLEMENTARY MATERIAL
Finally, to study the degree of selectivity in
the cytotoxic activity of the compounds, assays us-
ing lymphocyte isolation from whole human blood
were carried out on some representative com-
pounds such as 5a, 6b, and 7a, which showed
relatively high activity in tumor cells. The assay
showed that survival values were 98–100% for these
compounds.
CCDC 817643 contains the supplementary crys-
tallographic data for 5b (C13H21N4O2P1). These
data can be obtained free of charge via
http://www.ccdc.cam.ac.uk/conts/retrieving.html, or
from the Cambridge Crystallographic Data Center,
12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44)
1223-336-033; or e-mail: deposit@ccdc.cam.ac.uk.
Heteroatom Chemistry DOI 10.1002/hc

82 Gholivand et al.
TABLE 4 Cytotoxicity Activity of Compounds in K562, MDA-MB-231 and HepG2 Cell Cultures
O
P
X
R1-C6H4NHC(O)NH
R2
Y
R2
Cell Line, (IC50(nM))a
Compound
R1
R2
X
Y
K562
MDA-MB-231
HepG2
log Pb
5a
H
CH3
CH3
CH3
H
H
H
CH3
CH3
CH3
NH
NH
NH
O
O
O
O
O
O
NH
NH
NH
NH
NH
NH
O
179 2.1
157 2.0
171 3.2
198 5.9
168 1.2
180 3.1
NAd
173 1.5
202 7.3
NA
118 7.6
75 2.5
78 1.4
260 18.6
57 1.9
60 2.9
100 1.4
94 1.5
86 5.4
97 5.5
94 4.6
94 5.9
97 6.5
73 1.1
78 3.3
82 2.4
69 1.3
67 1.1
70 6.7
63 1.0
130 6.2
339 25.7
0.77
1.19
0.73
−0.19
0.23
−0.23
2.3
2.76
0.82
0.75
0.86
5b
4-CH3
4-NO2
H
4-CH3
4-NO2
H
5cc
6a
6b
6c
7a
7b
4-CH3
4-NO2
O
O
7c
CPe
Phenyl urea
153 2.8
aDose required to inhibit cell growth by 50%. Values are the means of at least three independent determinations.
bLog P values of the synthesized compounds were calculated using ChemDraw Ultra 8.0 calculation software.
cFor compound5c, see [4].
dNA: not active (IC50 > 400 nM).
eCyclophosphamide.
[11] Petitclerc, E’.; Deschesnes, R. G.; Coˆte´, M.-F.;
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ACKNOWLEDGMENTS
We thank Miss. M. Shojai for the financial support
of this work.
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