Synthesis, crystal structure, biological evaluation, electronic aspects of hydrogen bonds, and QSAR studies of some new N-(substituted phenylurea) diazaphosphore derivatives as anticancer agents

Article abstract of DOI:10.1007/s00044-016-1527-9

A new series of 2-[N-(R-phenylureido)]-1,3,2-diazaphosphore-2-oxide derivatives (R = CH₃, F, NO₂, CN) were synthesized and characterized by ³¹P, ¹H, ¹³C NMR and FT-IR spectral techniques. All the compounds were evaluated for their antibacterial activity against some Gram-positive, Gram-negative strains of bacteria, as well as for their cytotoxic effects on MCF-7, MDA-MB-231, PC-3, HeLa, and K562 human cell lines. In vitro activity results exhibited an important role for six-membered diaza ring in both assays as well as high effect of meta-methyl and ortho-fluoro substitutes on the aromatic ring against the studied human cell lines and B. subtilis bacteria, respectively. To understand the correlation between the anticancer activity and physicochemical properties of the synthesized compounds, the QSAR studies were carried out. Further, the crystal structure of compound 15 was investigated and revealed that the title derivative is composed of two symmetrically independent molecules in the solid state with anti configuration the C=O versus P=O. NBO and AIM analyses were performed to investigate electronic aspects of hydrogen bonding of the crystal cluster, which play an extremely important role in biochemical systems.

Full text of DOI:10.1007/s00044-016-1527-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1527-9
ORIGINAL RESEARCH
Synthesis, crystal structure, biological evaluation, electronic
aspects of hydrogen bonds, and QSAR studies of some new
N-(substituted phenylurea) diazaphosphore derivatives
as anticancer agents
Niloufar Dorosti1 Bahram Delfan2 Khodayar Gholivand3
Ali Asghar Ebrahimi Valmoozi3
•
•
•
Received: 1 October 2015 / Accepted: 4 February 2016
Ó Springer Science+Business Media New York 2016
Abstract A new series of 2-[N-(R-phenylureido)]-1,3,
2-diazaphosphore-2-oxide derivatives (R = CH3, F, NO2,
cluster, which play an extremely important role in bio-
chemical systems.
1
CN) were synthesized and characterized by 31P, H, 13C
NMR and FT-IR spectral techniques. All the compounds
were evaluated for their antibacterial activity against some
Gram-positive, Gram-negative strains of bacteria, as well
as for their cytotoxic effects on MCF-7, MDA-MB-231,
PC-3, HeLa, and K562 human cell lines. In vitro activity
results exhibited an important role for six-membered diaza
ring in both assays as well as high effect of meta-methyl
and ortho-fluoro substitutes on the aromatic ring against
the studied human cell lines and B. subtilis bacteria,
respectively. To understand the correlation between the
anticancer activity and physicochemical properties of the
synthesized compounds, the QSAR studies were carried
out. Further, the crystal structure of compound 15 was
investigated and revealed that the title derivative is com-
posed of two symmetrically independent molecules in the
solid state with anti configuration the C=O versus P=O.
NBO and AIM analyses were performed to investigate
electronic aspects of hydrogen bonding of the crystal
Keywords Diazaphosphore Á Anticancer Á Antibacterial Á
QSAR Á AIM Á NBO
Introduction
The increasing issue of drug resistance has prompted an
intensive search for new bioactive agents. Phosphor hete-
rocyclic compounds are attractive in this regard because
structural properties of these derivatives such as steric,
electronic, and conformational interactions allow them to
interact easily with the biopolymers of the living systems
(Hua et al., 2009; Venkatachalam et al., 2006; Mohe et al.,
2003). They inhibit acetylcholinesterase and butyryl-
cholinesterase enzymes (Elgorashi et al., 2006; Gholivand
et al., 2014) and, as such, are useful for inhibiting of cell
proliferation in the treatment of cancer (Voorde et al.,
2011; Monteil et al., 2014; Gholivand et al., 2010). In
addition, these compounds exhibit antimicrobial, anti-
malarial, and antiviral activities (Gholivand and Dorosti,
´
2013; Mara et al., 2011; Hockova et al., 2011). One rep-
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-016-1527-9) contains supplementary
material, which is available to authorized users.
resentative phosphor group is oxazaphosphorine family,
which is used as potent drugs in the treatment of human
cancers (Fig. 1a) (Li et al., 2003; Borch and Canute, 1991;
Ludeman et al., 1979). Modification of cyclophosphamide
(CP), one of oxazaphosphorinane derivatives, has led to
the synthesis of numerous phosphoramidate alkylating
agents (Venkatachalam et al., 2006; Li et al., 2003; Moon
et al., 1995) to find an antitumor agent that has fewer side
effects than drugs now present. In all the research, analogs
of CP have two important components including P ring
and the substituent on phosphor (Jiang et al., 2006; Sun
& Niloufar Dorosti
nilufardorosti@gmail.com
1
Department of Chemistry, Lorestan University,
Khoramabad 68135-465, Iran
2
Razi Herbal Medicines Research Center, Lorestan University
of Medical Sciences, Khoramabad, Iran
3
Department of Chemistry, Tarbiat Modares University,
P.O. Box 14115-175, Tehran, Iran
123

Med Chem Res
R1
P
Fig. 1 a Bioactive
oxazaphosphorines, b designed
diazaphosphore
O
H
N
H
N
NH
O
N
O
R2
P
A
B
R
N
NH
O
ClCH2CH2
13-34
R1 = H, R2 = CH2CH2Cl (Cyclophosphamide)
R
1 = CH2CH2Cl, R2 = H (Ifosfamide)
R1 = CH2CH2Cl, R2 = CH2CH2Cl (Trofosfamide)
(a)
(b)
et al., 2006). In our previous studies, we have designed CP
analogs based on the principle of conjugating the two
pharmacophores of the phosphoryl and urea, with antici-
pation that the obtained hybrids could possess powerful
biological activity (Gholivand et al., 2010, 2012). Our
findings highlighted the importance of –NHC(O)NHP(O)–
framework. In this paper, we describe the design and
synthesis of 22 compounds with the general skeleton of
determined relative to internal TMS, and 31P chemical
shifts, relative to 85 % H3PO4 as an external standard. IR
spectra were recorded on a Shimadzu model IR-60 spec-
trometer using KBr pellets. Melting points were obtained
with an electrothermal instrument.
General procedure for the synthesis
of diazaphosphorinanes 15, 17–19, and 22
RC6H5NHC(O)NHP(O)(NH)1(NH)2
(Fig. 1b).
New
desired derivatives 15, 17–19, 22, 24–34 were character-
1
To a suspension of related intermediates of 2–12 (6 mmol)
in dry diethyl ether (20 mL), 2, 2-dimethyl-1, 3-diamino-
propane (1.23 g, 12.0 mmol) at 0 °C was added with stir-
ring. After 5 h, the products were filtered off and washed
with H2O.
ized by IR, H, 13C, and 31P NMR. Cytotoxic activities of
22 compounds, phenylurea, and standard drug (CP) were
assayed against HeLa, PC-3, K562, MCF-7, and MDA-
MB-231 human cancer cell lines to elucidate the impor-
tance of replacement of the 5- and 6-membered diaza-
phosphore rings (A-ring) instead of oxazaphosphorinane
ring as well as the variation in the type of substituent and
their position on the aromatic ring (B-ring). In the fol-
lowing, the selected compounds were screened for their
antimicrobial activities. We also present quantitative
structure–activity relationship (QSAR) studies for valida-
tion of the observed pharmacological properties of the
investigated anticancer compounds and for determination
of the most important parameters controlling these prop-
erties. In addition, the crystal structure of compound 15
was investigated and employed as reference for quantum
mechanical (QM) calculations at the B3LYP level. It is
well known that the hydrogen bond plays a key role in the
chemistry of life, such as protein–ligand interactions and
enzymatic activity (Jeffrey and Saenger, 1991; Carletti
et al., 2010; Massiah et al., 2001). Moreover, the physic-
ochemical properties of compounds depend on the pres-
ence of intermolecular hydrogen bonds. Hence, the
electronic aspects of hydrogen bonds in the crystal struc-
ture of the compound 15 have been investigated by NBO
and AIM analyses.
5,5-Dimethyl-2-(N-4-fluoro-phenylureido)-1,3,2-diazaphos-
phorinane-2-oxide (15) Yield: 60 %, m.p. 198–199 °C.
1H NMR (500.13 MHz, d6-DMSO) d 0.78 (s, 3H, CH3), 1.03
(s, 3H, CH3), 2.57 (ddd, 2J(H, H) = 12.00 Hz, 3J(H,
H) = 5.30 Hz, 3J(P, H) = 24.4 Hz, 2H, CH), 2.98 (dd,
2J(H, H) = 8.4 Hz, 3J(H, H) = 3.65 Hz, 2H, CH), 4.67
(d,2(P, H) = 3.8 Hz, 2H, NHendocyclic), 7.08 (t, 3J(H,
3
H) = 8.85 Hz, 2H, Ar–H), 7.38 (dd, J(H, H) = 9.00 Hz,
3J(H, F) = 4.90 Hz, 2H, Ar–H), 7.68 (s, 1H, NHP), 9.37 (s,
1H, 4-F-C6H4NH) ppm; 13C NMR (d6-DMSO): d 23.31 (s,
CH3), 24.88 (s, CH3), 30.49 (d, J(P, C) = 4.2 Hz, CH2),
3
52.39 (d, 2J(P, C) = 1.40 Hz, CH2), 115.25 (d, 1J(F,
2
C) = 22.2 Hz, CH2), 119.8 (d, J(C, F) = 7.68 Hz), 135.7
2
(s), 153.49 (d, J(P, C) = 2.38 Hz), 156.4 (s); 31P NMR
(202.46 MHz, d6-DMSO) d 3.72 (m). IR (KBr, cm-1): 3215
(mN–H), 3112, 1690 (mC=O), 1614, 1569, 1468, 1446, 1310,
1337, 1220, 1178 (mP=O), 1090, 1046 (mP–N), 954 (mP–N),
864, 829, 766.
5,5-Dimethyl-2-(N-4-cyano-phenylureido)-1,3,2-diazaphos-
°
phorinane-2-oxide (17) Yield: 65 %, m.p. 192–193 C.
1H NMR (500.13 MHz, d6-DMSO) d 0.74 (s, 3H, CH3), 1.03
(s, 3H, CH3), 2.59 (ddd, 2J(H, H) = 11.95 Hz, 3J(H,
H) = 5.15 Hz, 3J(P, H) = 24.36 Hz, 2H, CH), 3.0 (d, 3J(H,
2
Experimental
H) = 11.95 Hz, 2H, CH), 4.7 (d, J(P, H) = 2.5 Hz, 2H,
NHendocyclic), 7.2 (d, 3J(H, H) = 8.7 Hz, 2H, Ar–H), 7.41 (d,
3J(H, H) = 8.7 Hz, 2H, Ar–H), 7.75 (d, 2J(P, H) = 3.65 Hz,
1H, NHP), 9.47 (s, 1H, 4-CN-C6H4NH) ppm; 13C NMR (d6-
Chemistry
1H, 13C, and 31P spectra were recorded on a Bruker Avance
DMSO): d 23.29 (s, CH3), 24.87 (s, CH3), 30.47 (d, J(P,
C) = 4.33 Hz), 52.38 (s), 119.64 (s), 125.48 (s), 128.61 (s),
3
1
DRX 500 spectrometer. H and 13C chemical shifts were
123

Med Chem Res
138.35 (s), 153.39 (d, 2J(P, C) = 1.94 Hz). 31P NMR
(202.46 MHz, d6-DMSO) d 3.60 (m). IR (KBr, cm-1): 3295
(mN–H), 1709 (mC=O), 1595, 1527, 1467, 1409, 1314, 1257,
1187(mP=O), 1089, 1044 (mP–N), 850, 761, 733, 545.
General procedure for the synthesis of diazaphospholanes
24–34
These compounds were synthesized from the reaction of
(6.06 mmol) intermediates 2–12 suspended in 10 mL
dichloromethane with (0.73 g, 12.12 mmol) ethylenedi-
amine at 0 °C. The solution was stirred overnight at room
temperature and then evaporated in vacuum. The residue
was washed with cooled water, and the precipitate was
filtered off and dried at room temperature.
5,5-Dimethyl-2-(N-3-methyl-phenylureido)-1, 3, 2-diaza-
phosphorinane-2-oxide
(18) Yield:
53 %,
m.p.
163–164 °C. 1H NMR (500.13 MHz, d6-DMSO) d 0.79 (s,
3H, CH3), 1.03 (s, 3H, CH3), 2.18 (s, 3H, CH3), 2.56 (m,
2H, CH2), 2.98 (m, 2H, CH2), 4.74 (s, 2H, NH), 6.89 (m,
1H, Ar–H), 7.11 (m, 2H, Ar–H), 7.91 (d, 1H, NHP), 9.19
(s, 1H, 3-CH3-C6H4NH). 13C NMR (125.76 MHz, d6-
DMSO) d 17.79 (s,CH3), 23.36 (s, CH3), 24.89 (s), 30.54
2-(N-phenylureido)-1,3,2-diazaphospholane-2-oxide
°
(24) Yield: 50 %, m.p. 200–201 C. 1H NMR
3
3
(d, J(P, C) = 3.5 Hz), 52.21 (s), 119.93 (s), 122.26 (s),
(500.13 MHz, d6-DMSO) d 3.12 (d, J(P, H) = 5.05 Hz,
3
126.08 (s), 126.49 (s), 130.02 (s), 137.49 (s), 153.44 (d,
2J(P, C) = 2.54 Hz). 31P NMR (202.46 MHz, d6-DMSO) d
4.62 (m) ppm. IR (KBr, cm-1): 3320 (mN–H), 1680
(mC=O), 1616, 1588, 1558, 1455, 1326, 1193 (mP=O), 1099,
1038, 953, 861, 764, 607.
2H, CH2), 3.27 (d, J(P, H) = 7.1 Hz, 2H, CH2), 4.74 (d,
2J(P, H) = 12.5 Hz, 2H, NH), 6.95 (t, 3J(H, H) = 7.35 Hz,
1H, CH), 7.24 (t, 3J(H, H) = 8.8 Hz, 2H, CH, Ar–H), 7.36
(d, 3J(H, H) = 7.7 Hz, 2H, CH, Ar–H), 7.52 (s, 1H, NHP),
9.52 (s, 1H, C6H5NH). 13C NMR (125.76 MHz, d6-DMSO)
d 40.8 (d, 2J(P, C) = 11.52 Hz), 118.1 (s), 121.9 (s), 128.7
(s), 139.3 (s), 153.0 (d, 2J(P, C) = 2.3 Hz). 31P NMR
(202.46 MHz, d6-DMSO) d 23.54 (m). IR (KBr, cm-1):
3365 (mNH), 1695 (mC=O), 1600, 1540, 1482, 1443, 1409,
1309, 1285, 1213, 1155 (mP=O), 1103, 1083, 1057, 1027,
938, 847, 816, 776, 738, 689, 610, 557, 469.
5, 5-dimethyl-2-(N-3-fluoro-phenylureido)-1, 3, 2-diaza-
phosphorinane-2-oxide
(19) Yield:
60 %,
m.p.
198–199 °C. 1H NMR (500.13 MHz, d6-DMSO) d 0.78 (s,
3H, CH3), 1.03 (s, 3H, CH3), 2.58 (dd, 2J(H,
H) = 12.69 Hz, 3J(H, H) = 5.30 Hz, 2H, CH), 2.96 (d,
2J(H, H) = 11.4 Hz, 2H, CH), 4.69 (s, 2H, NHendocyclic),
7.08 (t, 3J(H, H) = 8.6 Hz, 2H, Ar–H), 7.37 (b, 2H, Ar–H),
2-(N-4-methyl-phenylureido)-1,3,2-diazaphospholane-2-
°
2
7.7 (d, 1H, J(P, H) = 5.18 Hz, NHP), 9.35 (s, 1H, 4-F-
C6H4NH) ppm; 13C NMR (d6-DMSO): d 23.28 (s, CH3),
24.87 (s, CH3), 30.46 (d, J(P, C) = 4.5 Hz, CH2), 52.38
oxide (25) Yield: 80 %, m.p. 189–190 C. 1H NMR
(500.13 MHz, d6-DMSO) d 2.20 (s, 3H, CH3), 3.12 (d,
3J(P, H) = 12.65 Hz, 2H, CH2), 3.26 (d, 3J(P,
H) = 7.4 Hz, 2H, CH2), 4.71 (d, 2J(P, H) = 12.35 Hz, 2H,
NHendocyclic), 7.04 (d, 3J(H, H) = 8.20 Hz, 2H, CH, Ar–H),
7.25 (d, 3J(H, H) = 8.20 Hz, 2H, CH, Ar–H), 7.50 (d, 2J(P,
H) = 6.00 Hz, 1H, NHP), 9.42 (s, 1H, 4-CH3-C6H4
NH).13C NMR (125.76 MHz, d6-DMSO) d 20.2 (s), 40.8
2
2
(s), 115.12 (s), 115.41 (s), 119.82 (d, J(C, F) = 7.7 Hz),
135.69 (d, 2J(P, C) = 1.97 HZ), 153.49 (d, 2J(P,
C) = 2.49 Hz); 31P NMR (202.46 MHz, d6-DMSO) d 3.8
(m). IR (KBr, cm-1): 3215 (mN–H), 1690 (mC=O), 1614,
1569, 1468, 1446, 1337, 1220, 1178(mP=O), 1090, 1046
(mP–N), 954 (mP–N), 864, 829, 766.
2
(d, J(P, C) = 11.53 Hz), 118.2 (s), 129.1 (s), 130.8 (s),
136.7 (s), 153.0 (d, 2J(P, C) = 2.454 Hz).31P NMR
(202.46 MHz, d6-DMSO) d 23.65 (m). IR (KBr, cm-1):
3385 (mN–H), 3255, 2900, 1694 (mC=O), 1599, 1536, 1490,
1404, 1281, 1211, 1159 (mP=O), 1059 (mP–N), 940 (mP–N),
809 (mP–N), 719, 464.
5,5-Dimethyl-2-(N-2-fluoro-phenylureido)-1,3,2-diazaphos-
phorinane-2-oxide (22) Yield: 50 %, m.p. 198-199 °C.
1H NMR (500.13 MHz, d6-DMSO) d 0.78 (s, 3H, CH3),
2
3
1.02 (s, 3H, CH3), 2.59 (ddd, J(H, H) = 12.4 Hz, J(H,
H) = 5.27 Hz, 3J(P, H) = 24.38 Hz, 2H, CH), 3.0 (d,
2J(H, H) = 12.0 Hz, 2H, CH), 4.69 (d, 2J(P,
H) = 3.28 Hz, 2H, NHendocyclic), 7.09 (t, 3J(H,
2-(N-4-fluoro-phenylureido)-1,3,2-diazaphospholane-2-ox-
°
ide (26) Yield: 70 %, m.p. 201–202 C. 1H NMR
3
3
H) = 8.76 Hz, 2H, Ar–H), 7.38 (dd, J(H, H) = 8.46 Hz,
3J(H, H) = 5.1 Hz, 2H, Ar–H), 7.67 (d, 2J(P, H) = 7.1 Hz,
(500.13 MHz, d6-DMSO) d 3.26 (d, J(P, H) = 12.0 Hz,
3
2H, CH2), 3.27 (d, J(P, H) = 7.8 Hz, 2H, CH2), 4.75 (d,
1H, NHP), 9.35 (s, 1H, 2-F-C6H4NH) ppm; 13C NMR (d6-
2J(P, H) = 12.3 Hz, 2H, NHendocyclic), 7.08 (t, 3J(H,
H) = 8. 0 Hz, 2H, Ar–H), 7.38 (m, 2H, Ar–H), 7.58 (s, 1H,
NHP), 9.55 (s, 1H, 4-F-C6H4NH). 13C NMR (125.76 MHz,
3
DMSO): d 23.34 (s, CH3), 24.94 (s, CH3), 30.5 (d, J(P,
C) = 4.5 Hz, CH2), 52.4 (s), 115.2 (s), 115.5 (s), 119.86
(d, 2J(C, F) = 7.8 Hz), 135.72 (s), 153.53 (d, 2J(P,
C) = 2.57 Hz); 31P NMR (202.46 MHz, d6-DMSO) d 3.79
(m). IR (KBr, cm-1): 3215 (mN–H), 1690 (mC=O), 1614,
1569, 1468, 1446, 1337, 1220, 1178(mP=O), 1090, 1046
(mP–N), 954 (mP–N), 864, 829, 766.
2
d6-DMSO) d 40.88 (d, J(P, C) = 11.55 Hz), 115.25 (d,
1J(C, F) = 22.2 Hz), 119.87 (d, 2J(P, C) = 7.6 Hz),
2
135.64 (s), 153.14 (d, J(P, C) = 1.89 Hz), 156.43 (s).31P
NMR (202.46 MHz, d6-DMSO) d 23.58 (m). IR (KBr,
cm-1): 3390(mN–H), 3250, 2955, 1701 (mC=O), 1612,
123

Med Chem Res
3
1546, 1509, 1484, 1405, 1292, 1217, 1153 (mP=O), 1101,
1055 (mP–N), 939, 825, 783, 747, 708, 460.
2J(P, H) = 12.6 Hz, 2H, NHendocyclic), 6.76 (d, 1H, J(H,
3
H) = 4.28 Hz), 7.08 (t, J(H, H) = 8. 9 Hz, 2H, Ar–H),
3
7.38 (dd, J(H, H) = 8.9 Hz, J(H, F) = 4.9 Hz, 1H, Ar–
3
2-(N-4-nitro-phenylureido)-1,3,2-diazaphospholane-2-oxide
(27) Yield: 70 %, m.p. 195–196 °C. 1H NMR
H), 7.56 (b, 1H, NHP), 9.54 (s, 1H, 3-F-C6H4NH). 13C
NMR (125.76 MHz, d6-DMSO)
d
40.88 (d, 2J(P,
3
(500.13 MHz, d6-DMSO) d 3.16 (d, J(P, H) = 12.5 Hz,
C) = 11.43 Hz), 115.16 (s), 115.46 (s), 119.8 (s), 119.9 (s),
153.13 (s). 31P NMR (202.46 MHz, d6-DMSO) d 23.58
(m). IR (KBr, cm-1): 3390(mN–H), 3250, 2955, 1701
(mC=O), 1612, 1546, 1509, 1484, 1405, 1292, 1217, 1153
(mP=O), 1101, 1055 (mP–N), 939, 825, 783, 747, 708, 460.
2H, CH2), 3.29 (d, 3J(P, H) = 7.65 Hz, 2H, CH2), 4.83 (d,
2J(P, H) = 12.8 Hz, 2H, NHendocyclic), 7.62 (d, 3J(H,
H) = 9.05 Hz, 2H, Ar–H), 7.83 (s, 1H, NHP), 8.14 (d,
3J(H, H) = 9.00 Hz, 2H, Ar–H), 10.19 (s, 1H, 4-NO2-
C6H4NH). 13C NMR (125.76 MHz, d6-DMSO) d 40.8 (d,
2J(P, C) = 11.78 Hz), 117.6 (s), 125.1 (s), 141.3 (s), 145.7
(s), 152.8 (d, 2J(P, C) = 2.64 Hz).31P NMR (202.46 MHz,
d6-DMSO) d 23.30 (m). IR (KBr, cm-1): 3375 (mNH),
3265, 2960, 1705 (mC=O), 1549, 1489, 1407, 1328 (mNO2),
1299, 1155 (mP=O), 1059 (mP–N), 940 (mP–N), 847 (mP–N),
809, 732.
2-(N-3-nitro-phenylureido)-1,3,2-diazaphospholane-2-oxide
°
(31) Yield: 55 %, m.p. 187-188 C. 1H NMR
3
(500.13 MHz, d6-DMSO) d 3.17(d, J(P, H) = 12.2 Hz,
2H, CH2), 3.27 (d, 3J(P, H) = 7.91 Hz, 2H, CH2), 4.85 (d,
2J(P, H) = 12.7 Hz, 2H, NHendocyclic), 7.01 (t, 3J(H,
3
H) = 8.2 Hz, 1H, Ar–H), 7.22 (d, J(H, H) = 7.9 Hz, 1H,
3
Ar–H), 7.31 (d, J(H, H) = 7.5 Hz, 1H, Ar–H), 7.52 (s,
2-(N-4-cyano-phenylureido)-1,3,2-diazaphospholane-2-oxide
°
3
1H, PNH), 7.75 (d, J(H, H) = 8.0 Hz, 1H, Ar–H), 9.5 (s,
(28) Yield: 73 %, m.p. 192–193 C. 1H NMR
1H, 3-NO2-C6H4NH). 13C NMR (125.76 MHz, d6-DMSO)
d 40.9 (d, 2J(P, C) = 11.4 Hz), 123.55 (s), 123.75(s),
125.17(s), 134.1(s), 134.5(s), 138.3(s), 153.1(d, 2J(P,
C) = 2.35 Hz).31P NMR (202.46 MHz, d6-DMSO) d
23.53 (m). IR (KBr, cm-1): 3370(mN–H), 3135,
1700(mC=O), 1544, 1493, 1402, 1344, 1214 (mP=O), 1158,
1094, 1069, 905, 859, 836, 805, 732, 675, 610, 578, 547.
3
(500.13 MHz, d6-DMSO) d 3.13 (d, J(P, H) = 10.75 Hz,
2H, CH2), 3.27 (d, 3J(P, H) = 7.55 Hz, 2H, CH2), 4.76 (d,
2J(P, H) = 12.55 Hz, 2H, NHendocyclic), 7.28 (d, 3J(H,
3
H) = 8.8 Hz, 2H, Ar–H), 7.41 (d, J(H, H) = 8.8 Hz, 2H,
Ar–H), 7.62 (d, 2J(P, H) = 5.6 Hz, 1H, NHP), 9.65 (s, 1H,
4-CN-C6H4NH). 13C NMR (125.76 MHz, d6-DMSO) d
40.9 (d, 2J(P, C) = 11.56 Hz), 119.66 (s), 125.52(s),
128.61(s), 138.29(s), 153.03(d, 2J(P, C) = 2.6 Hz).31P
NMR (202.46 MHz, d6-DMSO) d 23.53 (m). IR (KBr,
cm-1): 3379(mN–H), 3140, 2918, 1706 (mC=O), 1592,
1525, 1485, 1413, 1365, 1297, 1217, 1157 (mP=O), 1106,
1054 (mP–N), 938, 772, 545, 488, 445.
2-(N-2-methyl-phenylureido)-1,3,2-diazaphospholane-2-oxide
°
(32) Yield: 73 %, m.p. 195-196 C. 1H NMR
(500.13 MHz, d6-DMSO) d 2.15 (s, 3H, CH3), 3.12 (d,
3J(P, H) = 12.13 Hz, 2H, CH2), 3.2 (d, 3J(P, H) = 7.3 Hz,
2H, CH2), 4.75 (d, 2J(P, H) = 12.35 Hz, 2H, NHendocyclic),
6.93 (t, 3J(H, H) = 7.33 Hz, 1H, Ar–H), 7.1 (d, 3J(H,
H) = 7.5 Hz, 1H, Ar–H), 7.18 (d, 3J(H, H) = 7.61 Hz,
1H, Ar–H), 7.31 (s, 1H, PNH), 7.52 (d,3J(H,
H) = 7.92 Hz,1H, Ar–H), 9.21 (s, 1H, 2-CH3-C6H4NH).
13C NMR (125.76 MHz, d6-DMSO) d 17.52(s), 40.92 (d,
2J(P, C) = 11.31 Hz), 119.87 (s), 122.3(s), 126.1 (s),
126.53(s), 130.02(s), 137.26 (s), 153.24(d, 2J(P,
C) = 2.2 Hz).31P NMR (202.46 MHz, d6-DMSO) d 24.12
(m). IR (KBr, cm-1): 3375(mN–H), 3150, 2895, 1701
(mC=O), 1693, 1612, 1588, 1537, 1490, 1452, 1408, 1290,
1209, 1165 (mP=O), 1106, 1048 (mP–N), 941, 850, 745,
618, 568, 476.
2-(N-3-methyl-phenylureido)-1,3,2-diazaphospholane-2-oxide
°
(29) Yield: 73 %, m.p. 195–196 C. 1H NMR
(500.13 MHz, d6-DMSO) d 2.17 (s, 3H, CH3), 3.12 (d,
3J(P, H) = 12.0 Hz, 2H, CH2), 3.23 (d, 3J(P,
H) = 7.95 Hz, 2H, CH2), 4.8 (d, 2J(P, H) = 12.45 Hz, 2H,
NHendocyclic), 6.89 (t, 3J(H, H) = 7.25 Hz, 1H, Ar–H), 7.09
(d, 3J(H, H) = 7.5 Hz, 1H, Ar–H), 7.11 (d, 3J(H,
H) = 7.65 Hz, 1H, Ar–H), 7.81 (s, 1H, PNH), 7.92
(d,3J(H, H) = 8.1 Hz,1H, Ar–H), 9.38 (s, 1H, 3-CH3-
C6H4NH). 13C NMR (125.76 MHz, d6-DMSO) d 17.73(s),
40.9 (d, 2J(P, C) = 11.44 Hz), 119.94 (s), 122.3(s),
2
126.09(s), 126.49(s), 130.04(s), 137.48 (s), 153.24(d, J(P,
C) = 2.4 Hz).31P NMR (202.46 MHz, d6-DMSO) d 24.1
(m). IR (KBr, cm-1): 3368(mN–H), 2957, 2894,
1694(mC = O), 1614, 1591, 1540, 1481, 1455, 1411, 1295,
1210, 1168 (mP=O), 1109, 1050(mP–N), 944, 854, 824, 796,
749, 622, 571, 480.
2-(N-2-fluoro-phenylureido)-1,3,2-diazaphospholane-2-oxide
°
(33) Yield: 70 %, m.p. 201–202 C. 1H NMR
3
(500.13 MHz, d6-DMSO) d 3.12 (d, J(P, H) = 12.88 Hz,
2H, CH2), 3.26 (d, 3J(P, H) = 9.89 Hz, 2H, CH2), 4.76 (d,
2J(P, H) = 12.61 Hz, 2H, NHendocyclic), 6.81 (d, 2J(H,
H) = 4.43 Hz, 1H, Ar–H), 7.08 (t, 3J(H, H) = 8.9 Hz, 2H,
2-(N-3-fluoro-phenylureido)-1,3,2-diazaphospholane-2-oxide
°
3
3
(30) Yield: 75 %, m.p. 201–202 C. 1H NMR
Ar–H), 7.39 (dd, J(H, H) = 8.96 Hz, J(H, F) = 4.9 Hz,
1H, Ar–H), 7.58 (s, 1H, NHP), 8.03 (b,1H, Ar–H), 9.54 (s,
1H, 2-F-C6H4NH). 13C NMR (125.76 MHz, d6-DMSO) d
3
(500.13 MHz, d6-DMSO) d 3.09 (d, J(P, H) = 10.11 Hz,
2H, CH2), 3.18 (d, J(P, H) = 5.4 Hz, 2H, CH2), 4.76 (d,
3
123

Med Chem Res
40.89 (d, 2J(P, C) = 11.59 Hz), 114.94 (s), 115.16 (s),
three times. The IC50 value is defined as compound con-
centration that inhibits cell growth by 50 %. Also, to study
the degree of selectivity in the cytotoxic activity of the
compounds, separation of lymphocyte was down according
to the following procedure (Bøyum, 1976): 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
2
2
115.45 (s), 119.86 (d, J(F, C) = 7.8 Hz), 135.66 (d, J(F,
C) = 2.3 Hz), 153.16 (d, 2J(P, C) = 2.7 Hz). 31P NMR
(202.46 MHz, d6-DMSO) d 23.66 (m). IR (KBr, cm-1):
3390(mN–H), 3250, 2955, 1701 (mC=O), 1612, 1546, 1509,
1484, 1405, 1292, 1217, 1153 (mP=O), 1101, 1055 (mP–N),
939, 825, 783, 747, 708, 460.
2-(N-2-nitro-phenylureido)-1,3,2-diazaphospholane-2-oxide
°
(34) Yield: 60 %, m.p. 187–188 C. 1H NMR
g
av for 30–40 min), lymphocytes, together with monocytes
3
(500.13 MHz, d6-DMSO) d 3.14(d, J(P, H) = 12.6 Hz,
2H, CH2), 3.25 (d, J(P, H) = 8.9 Hz, 2H, CH2), 4.7 (d,
and platelets, are harvested from the interface 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.
3
2J(P, H) = 12.7 Hz, 2H, NHendocyclic), 7.2 (t, 3J(H,
3
H) = 7.9 Hz, 1H, Ar–H), 7.65 (t, J(H, H) = 8.0 Hz, 1H,
3
Ar–H), 8.02 (d, J(H, H) = 8.15 Hz, 1H, Ar–H), 8.2 (d,
3J(H, H) = 8.4 Hz, 1H, Ar–H), 8.39 (s, 1H, PNH), 10.3 (s,
1H, 2-NO2-C6H4NH). 13C NMR (125.76 MHz, d6-DMSO)
d 40.9 (d, 2J(P, C) = 11.52 Hz), 122.65 (s), 123.32(s),
125.12(s), 134.01(s), 134.5(s), 138.5(s), 153.06(d, 2J(P,
C) = 2.5 Hz, d).31P NMR (202.46 MHz, d6-DMSO) d
22.94 (m). IR (KBr, cm-1): 3334(mN–H), 3135, 2935,
1715(mC=O), 1609, 1586, 1483, 1433, 1344, 1278, 1209,
1172 (mP=O), 937, 864, 742.
Antibacterial evaluation assay
The cup-plate agar method (Barry, 1977) was used to
determine the antibacterial activity of compounds 13–34
against three Gram-positive bacteria, namely S. aureus
(ATCC 25923), B. subtilis (ATCC 12711), and B. cereus
(ATCC 11778), three Gram-negative bacteria, viz. E. coli
(ATCC 25922), P. aeruginosa (ATCC 27853), and Proteus
vulgaris (NCIMB 8066). The compounds dissolved in
dimethyl sulfoxide (DMSO) at a concentration of 6000 lg/
cm3 were used. Then, the solutions of the tested com-
pounds were placed on the well of the media inoculated
with the microorganisms. DMSO was used as a solvent
control. Gentamicin and tetracycline were used as refer-
ence antibacterial drugs. The diameter of the growth inhi-
bition zone was read after 24 h of incubation at 35 °C.
These compounds were further examined by the broth
dilution method to determine their MIC (minimal inhibi-
tory concentration) (Vincent and Vincent, 1944). Minimal
inhibitory concentrations were read after 24 h of incuba-
tion at 35 °C.
Crystal structure determination
Crystals suitable for X-ray crystallography were obtained
from ethanol at room temperature. X-ray data of compound
15 were collected on a Bruker SMART 1000 CCD (Bruker,
1998) area detector with graphite monochromated Mo Ka
˚
radiation (k = 0.71073 A). The structure was refined with
SHELXL-97 (Sheldrick, 2008) by full-matrix least-squares
on F2. The positions of hydrogen atoms were obtained
from the difference Fourier map. An absorption correction
was performed using the SADABS program for the titled
structure.
Cytotoxicity assay
Computational details
The cytotoxic activity of the synthesized compounds was
determined on five human cancer cell lines: cervical cancer
(HeLa), breast cancer (MCF-7 and MDA-MB-231), pros-
tate cancer (PC-3), and leukemia (K562) by MTT assay.
The cells suspended in the corresponding culture medium
were inoculated in 96-well microtiter plates at a density of
2000–8000 cells per well and incubated for 24 h at 37 °C
in a humidified atmosphere with 95 % air and 5 % CO2.
An equal volume of additional medium containing either
the serial dilutions of the test compounds, positive control
(cyclophosphamide), or negative control (2 %DMSO) was
added to the desired final concentrations, and the microtiter
plates were further incubated for 24, 48, and 72 h. Each
assay was set up in triplicate wells and repeated one to
The solid-state structures were used as the starting point for
density functional theory (DFT) calculations in the gas
phase and fully optimized at the B3LYP/6–31 ? G* level.
Whereas the X-ray crystallography cannot determine
accurately the position of the H atoms, the optimization of
H atom positions was performed to investigate the hydro-
gen bond characters in solid-state structures. To achieve
this goal, the solid-state structure of a conformer was
modeled as clusters in which the target molecule is sur-
rounded by two neighboring conformers and a similar
molecule (Fig. 5). The positions of H atoms were opti-
mized for model clusters, while other atoms were kept
frozen during the optimization. Such computational justi-
fications have also been used to describe well the geometry
123

Med Chem Res
and electronic aspects of X-ray structures (Mirzaei et al.,
2006; Esrafili et al., 2007). The NBO and AIM analyses
have been performed to compare the electronic features of
the gas-phase structures of the compound with those of
model clusters at the B3LYP/6–311 ? G** level. The
hydrogen-bonding energies have been calculated on the
basis of the energy difference between the hydrogen-bon-
ded tetramer and its fragments, as represented in the
equation EHB(1) = [Etetramer - (Efrag1 ? Efrag2)]/2, where
fragment 1 is composed of three monomers (conformers A,
B, and B0 on the left in Fig. 5) and fragment 2 is the right
principal component analysis (PCA) was utilized to find
the relationship between the dependent and independent
variables, reducing the set of independent variables (Pinto
et al., 2001). The toxicities of 22 compounds, CP, and
phenylurea were expressed in terms of IC50, which is
defined as necessary molar concentration of compound
causing 50 % inhibition against human cell lines. All
obtained IC50 values are usually converted into the nega-
tive logarithm of IC50 in QSAR study (log (1/IC50)).
molecule (conformer A0). The [Etetramer - (Efrag1
?
Results and discussion
Spectroscopic study
E
frag2)] term corresponds to the energies of the two sym-
metry-related contacts between the two pairs of molecules,
and the value is therefore divided by 2. Also, other
hydrogen-bonding energies have been calculated on the
Sixteen new derivatives of diazaphosphore with the general
formula RC6H4NHC(O)NHP(O)R1R2 were synthesized
(Scheme 1) (Kirsanov, 1954; Kirsanov and Zhmurova,
basis of the equation
EHB(2), (3) = [Etetramer -
(Efrag1 ? Efrag2)], where fragment 1 is composed of three
monomers (conformers A, A0, and B in EHB(2); conformers
A, A0, and B0 in EHB(3)) and fragment 2 is the conformer B0
in EHB(2) and conformer B in EHB(3), respectively. The
hydrogen-bonding energies were then corrected for basis
set superposition error (BSSE) using the counterpoise
method (Boys and Bernardi, 1970). All quantum chemical
calculations have been carried out using the GAUSSIAN 03
package (Frisch et al., 2003) on a Pentium 4 (2.8 GHz CPU
and 1024 MB RAM) work station.
1
1956) and characterized by 31P, 13C, and H NMR and IR
spectroscopy. Comparison of the d(31P) data in Table 1
shows that chemical shift of compounds bearing nitro
group is at the most upfield region rather than those of
other groups (CH3, F, and CN). For instance, derivative 32
(2-CH3) shows 31P chemical shift value 24.12 ppm,
whereas derivatives 33 (2-F) and 34 (2-NO2) exhibit 23.66
and 22.94 ppm, respectively. The 31P NMR spectra of
compounds 13–34 also show a chemical shift in the range
from 2.86 (in 23) to 24.12 (in 19) ppm. 31P nuclei in
compounds 13–23 are shielded relative to those of other
Statistical analysis
1
compounds. Inspection of H and 13C NMR spectra of the
In order to identify the effect of descriptors on the mole-
cule’s anticancer activity, QSAR studies were performed
using the model described (Hansch and Fujita, 1964). The
stepwise multiple linear regression (MLR) procedure,
which is a common method used in QSAR studies, was
used for selection of the descriptors using SPSS 16.0. The
electronic and structural descriptors were obtained by both
the quantum chemical calculations. The electronic
descriptors included the highest occupied and lowest
unoccupied molecular orbital (EHOMO and ELUMO), after
highest occupied and next lowest unoccupied molecular
orbital (EH-1 and EL?1), the energy difference between the
LUMO and HOMO (DEL-H), electrophilicity (w) (Parr
et al., 1999), polarizability (PL), and the net atomic charges
(Q). Hydrophobic coefficient (log P), dipole moment (l),
molecular volume (Mv), and molecular total energy (TE)
were the structural descriptors. EH, EL, EH-1, EL?1, w, PL,
Q, l, Mv, and TE (low optimized energy) values were
obtained from the DFT results (Sharma et al., 2012). The
logarithm of partition coefficient (log P) was calculated by
the Viswanadhan’s fragmentation method (Viswanadhan,
1989). In this study, only the variables that contain the
necessary information for modeling were used. The
new derivatives 15, 17–19, and 22 reveals two separate
signals for Haxial and Hequatorial and carbons of two methyl
groups. Also, it is observed from Table 1 two different
signals of Haxial and Hequatorial for compounds 24–33. The
3J(PNCH) coupling constant from 10.11 to 12.88 is related
to the Hequatorial atom, and for Haxial atom, the coupling
with phosphorus atom is from 5.4 to 9.89 ppm. This led us
to conclude that the synthesized compounds are toward one
chair conformation (Denmark et al., 1999) that adopt with
3
X-ray crystallography for 15. The small values of J(H, P)
indicate that the relevant dihedral angles are close to
orthogonal. In the case of compound 15, the torsional
angles of P–N–C–Haxial and P–N–C–Hequatorial are 68 and
173, respectively, obtained from X-ray crystallography.
Considering the Karplus equation (Breitmaier and Voelter,
1990), the Hequatorial proton has the largest coupling with
phosphorus atom. In these heterocycles, the highest value
coupling constant is 24.76 and 12.88 Hz for diazaphos-
phorinane 20 and diazaphospholane 33. In addition to the
d(31P), 2J(PNH)endocyclic and 2J(P,C)endocyclic values in
compounds 24–34 are larger than those of compounds 13–
1
23. The H NMR spectrum of 24–34 exhibits a doublet
signal for the two equivalent amino protons (of the five-
123

Med Chem Res
Scheme 1 Preparation pathway
for the synthesis of compounds
13–34
a
PCl
NH COOC H
+
Cl P(O)NCO
5
2
2
5
2
1
b
H
N
H
N
O
Cl
Cl
2-12
P
R
O
c
d
H
N
H
N
O
H
H
N
OH
N
A
N
CH3
N
H
P
P
HN
B
B
R
R
N
A
O
CH3
H
O
13-23
24-34
Reagents and conditions:
(a) 1,2-dicholoroethan
(b) Anilin derivatives, 0 0
C
(c) 2,2-dimethyl-1,3-diaminopropane, 0 0 C, diethyl ether
(d) ethylendiamine, triethylamine, 0 0 C, dicholoromethane
A-ring
B-ring
4-Hi
A-ring
B-ring
4-H
Comp.
13
Comp.
diazaphosphorinane
diazaphosphorinane
diazaphosphorinane
diazaphosphorinane
diazaphosphorinane
diazaphosphorinane
diazaphosphorinane
diazaphosphorinane
diazaphosphorinane
diazaphosphorinane
diazaphosphorinane
diazaphospholane
diazaphospholane
diazaphospholane
diazaphospholane
diazaphospholane
diazaphospholane
diazaphospholane
diazaphospholane
diazaphospholane
diazaphospholane
diazaphospholane
24
25
26
27
28
29
30
31
32
33
34
ii
4-CH3
4-CH3
4-F
14
4-F
15
i
4-NO2
4-CN
3-CH3
3-F
4-NO2
4-CN
3-CH3
3-F
16
17
18
19
iii
3-NO2
3-NO2
2-CH3
2-F
20
iii
2-CH3
2-F
21
22
iii
2-NO2
2-NO2
23
For compounds 13, 14, 16, 20, 21, and 23 see the following references: iGholivand and Dorosti., 2011; iiGholivand
et al., 2012; iiiGholivand et al., 2010.
2
membered ring) with a high value for phosphorus–hydro-
gen coupling constant about 12.8 Hz and a doublet signal
with 2J(PNH) about 6.0 Hz for the amidic proton. 2J(PNH)
coupling constants of endocyclic amino protons in com-
pounds containing six-membered (13–34) are in the range
2.5–3.8 Hz. The major reduction in the J(PNH) coupling
constant from 12.8 (in 27) to 3.8 (in 15) is due to the
increase in ring size because the five-membered ring has
2
2
high ring strain. Similar to the J(PNH) value, J(P, C)
endocyclic was observed for diazaphospholanes 24–34 (about
123

Med Chem Res
Table 1 Spectroscopic data of compounds 13–34
Comp. d31P (ppm) 2J(P,H)exo (HZ) 2J(P,H)endo (HZ) 3J(PNCH) (HZ)
2J(P,C)exo (HZ) 2J(P,C)endo (HZ) 3J(P,C)endo (HZ) mP=O mC=O
13a
14b
15
16a
17
18
19
20c
21c
22
23c
24
25
26
27
28
29
30
31
32
33
34
3.78
3.85
6.76
7.15
–
2.7
3.5
3.8
–
24.2
24.1
24.4
24.3
24.36
m
2.3
–
1.76
1.4
4.3
4.1
4.2
4.53
4.33
3.5
–
1170 1677
1180 1686
1178 1690
1196 1699
1187 1709
1193 1680
1178 1690
1176 1699
1190 1680
1178 1690
1164 1702
1155 1695
1159 1694
1153 1701
1155 1705
1157 1706
1168 1694
1153 1701
1165 1701
1156 1701
1153 1701
1172 1715
3.72
2.38
–
3.23
7.59
3.65
–
3.6
2.5
–
1.94
2.54
2.49
2.54
–
–
4.62
–
3.8
5.18
–
–
m
4.5
1.42
–
3.41
3.4
–
24.76
21.9
m
4.33
3.5
4.5
4.62
–
4.62
–
3.79
7.1
–
3.28
2.57
–
–
2.86
23.1
23.54
23.65
23.58
23.30
23.53
24.1
0.0
6.0
0.0
0.0
5.6
0.0
–
12.5
12.85(eq), 7.1(ax) 2.3
12.65(eq), 7.4(ax) 2.45
11.52
11.53
11.55
11.78
11.56
11.44
11.43
11.4
12.35
12.30
12.8
–
12.0(eq), 7.8(ax)
1.89
–
12.85(eq), 7.65(ax) 2.64
10.75(eq), 7.55(ax) 2.6
12.0(eq), 7.95(ax) 2.4
–
12.55
12.45
12.6
–
–
23.58
23.53
24.12
23.66
22.94
10.11(eq), 5.4(ax)
–
–
–
12.7
12.2(eq), 7.91(ax) 2.35
12.13(eq), 7.3(ax) 2.2
12.88(eq), 9.89(ax) 2.7
–
–
12.35
12.6
11.31
11.59
11.52
–
0.0
0.0
–
12.7
12.6(eq), 8.9(ax)
2.5
–
For compounds 13, 14, 16, 20, 21, and 23, see the following references: aGholivand and Dorosti, 2011; bGholivand et al., 2012; cGholivand et al.,
2010
11.51 Hz), which are larger than the values of diazaphos-
phorinanes 13–23. 3J(P, C) coupling constant was obtained
for compounds 13–24 (3.5–4.62 Hz).
with the surrounding angles around the P atoms in the
range of 101.92 (9)°–116.36 (10)° and 101.84 (9)°–116.14
(9)° for molecules 15a and 15b, respectively. The P–N
distances are significantly shorter than the related
endocyclic
X-ray crystallography
P–NC(O)NHP(O) bond distance (Table 3). All of these bonds
˚
are in the range 1.6218(18)–1.6985(18) A and thus are
Single crystals of compound 15 were obtained by slow
evaporation of the solvent at room temperature. Compound
crystallizes in the monoclinic crystal system with space
group P21/n, and the crystal system contains two inde-
pendent molecules (15a and 15b). The crystallographic
data, bond lengths, and angles are listed in Tables 2 and 3,
respectively. Molecular structures are shown in Fig. 2. The
molecules are significantly different as manifested by their
torsion angles. For instance, the O1P1N1C1, O1AP1A-
N1AC1A, and O1P1N2C3, O1AP1AN2AC3A torsion
angles are -162.26°, 160.29° and 165.49°, -164.53°,
respectively. The P=O distances in molecules 15a and 15b
significantly shorter than a typical P–N single bond
˚
(1.77 A) (Roy et al., 2006). The P=O bonds in molecules
15a and 15b are in an equatorial position of the related 1,
3-diazaphosphorinane rings. The endocyclic nitrogen
atoms in compounds 15a and 15b are distorted from pla-
narity. The sum of angles around these atoms in 15a is
353.15° and 353.69° for N(1) and N(2) atoms, and in 15b,
is 351.39° and 352.72° for N(7) and N(8) atoms. The sum
of the surrounding angles for all the exocyclic nitrogen
atoms is almost 360°; therefore, the environment of the N
atoms is practically planar. As shown in Fig. 3, the car-
boxyl and phosphoryl groups of ligands are involved in
intermolecular N–H…O hydrogen bonding and form two
types of robust hydrogen bond synthons, namely R22 (8)
I and R66(28) II. These intermolecular interactions con-
nected the various components into a 2D network. There
are also intramolecular P = O…H-NPh hydrogen bonds in
˚
are 1.4818 and 1.4823 A and are larger than the normal
˚
P=O bond length (1.45 A) (Corbridge 1995). These values
are comparable to those reported for phosphoramidates
(Gholivand and Dorosti 2011; Gholivand et al. 2009). The
P atoms have slightly distorted tetrahedral configurations
123

Med Chem Res
Table 2 Crystallographic data for compound 15
Compound
15
Empirical formula
Formula weight
Temperature
C12H18F N4O2P
300.27
120(2) K
˚
0.71073 A
Wavelength
Crystal system
Space group
Monoclinic
P 21/n
˚
Unit cell dimensions
a = 17.3891(16) A a = 90°
˚
b = 5.7264(5) A b = 9.635(2)°
˚
c = 28.892(3) A c = 90°
3
˚
Volume
2836.4(4) A
Z
8
Density (calculated)
Absorption coefficient
F(000)
1.406 Mg/m3
0.213 mm-1
1264
0.30 9 0.30 9 0.05 mm3
Crystal size
Theta range for data collection
Index ranges
1.43–28.99°
-23 B h B 23, -7 B k B 7, -39 B l B 39
26921
Reflections collected
Independent reflections
Completeness to theta = 28.99°
Absorption correction
Max. and min. transmission
Refinement method
Data/restraints/parameters
Goodness of fit on F2
Final R indices
7536 [R(int) = 0.0557]
99.9 %
Semi-empirical from equivalents
0.984 and 0.939
Full-matrix least-squares on F2
7536/0/365
1.001
R1 = 0.0483, wR2 = 0.1073
R1 = 0.0896, wR2 = 0.1208
-3
R indices (all data)
Largest diff. peak and hole
˚
0.781 and -0.430 e A
both conformers. In addition to numerous strong hydrogen
34) with –NHC(O)NHP(O)– functional group, were eval-
uated by MTT assay against five human cancer cell lines:
cervical cancer (HeLa), breast cancer (MCF-7 and MDA-
MB-231), prostate cancer (PC-3), and leukemia (K562),
with cyclophosphamide (CP) as the positive control. Their
IC50 values are listed in Table 4. The plots of IC50 values
for all of the compounds are also shown in Fig. 4. As
shown in Table 4, most of the compounds displayed higher
activity than phenyl urea against the selected cell lines and
even preferable cytotoxic activities than the commercial
anticancer drug CP with slightly different capacity, which
was consistent with our previous studies (Gholivand et al.,
2010, 2012). On the basis of our previous work, –N1
HC(O)N2HP(O)– scaffold was assayed as the optimum
structure and the side chain of at P-position would be the
key moiety to be explored. Thus, we attempted to replace
the substituent on the phenyl ring attached to N1H group
with electron-donating and electron-withdrawing groups
which are usually considered to play a key role in cytotoxic
bonds, intermolecular interactions in 15 include weaker
C9A-H9AA…F1Ai
(i = 1/2 - x,
-1/2 ? y,
-1/
2 - z) interaction with C…F distance of 3.146(2). There
are also C–H…p interactions [H… centroid dis-
˚
tance = 2.82 (2), 2.72(2) A] that occur between C1-H1B,
C11AH11B, and Cg2 [Cg2i (i = -x, 2 - y, -z), Cg2ii
(ii = x, y, z)] as well as [H… centroid distance = 2.96 (2),
˚
2.96 (2) A] that occur between C1A–H1AA, C11H11A,
and Cg4 [Cg4i (i = 1 - x, 1 - y, -z), Cg4ii (ii = x,
1 ? y, z)].
Cytotoxic activity
The goal of this study was to gain further insights into the
structural requirements for the anticancer activity of
diazaphosphore derivatives. Hence, the in vitro antitumor
activities of the 22 synthesized compounds, namely
diazaphosphorinane (13–23) and diazaphospholane (24–
123

Med Chem Res
Table 3 Optimized and experimental geometries of compound 15
Parameters
Experimental
B3LYP/6-31 ? G*
B3LYP/6-311 ? G**
˚
Bond lengths (A)
P(1)–N(2)
1.6218 (18)
1.6315 (18)
1.6985(18)
1.6268 (18)
1.6299 (17)
1.6948 (18)
1.671
1.670
1.719
1.671
1.670
1.719
1.666
1.665
1.715
1.665
1.665
1.715
P(1)–N(1)
P(1)–N(3)
P(1A)–N(2A)
P(1A)–N(1A)
P(1A)–N(3A)
Bond angles (°)
O(1)–P(1)–N(2)
113.71 (9)
116.36 (10)
107.89 (9)
113.87 (9)
116.14 (9)
107.81 (9)
114.292
117.282
107.740
114.291
117.287
107.740
114.292
117.372
107.621
114.290
117.378
107.621
O(1)–P(1)–N(1)
O(1)–P(1)–N(3)
O(1A)–P(1A)–N(2A)
O(1A)–P(1A)–N(1A)
O(1A)–P(1A)–N(3A)
Torsion angles
O(1)–P(1)–N(1)–C(1)
O(1)–P(1)–N(2)–C(3)
N(3)–P(1)–N(1)–C(1)
N(3)–P(1)–N(2)–C(3)
O(1A)–P(1A)–N(1A)–C(1A)
O(1A)–P(1A)–N(2A)–C(3A)
N(3A)–P(1A)–N(1A)–C(1A)
N(3A)–P(1A)–N(2A)–C(3A)
-162.26 (14)
165.49 (15)
77.93 (17)
-159.366
163.487
80.139
-157.488
161.872
82.134
-72.80 (18)
160.29 (15)
-164.53 (15)
-80.00 (17)
73.73 (17)
-74.409
159.375
-163.499
-80.128
74.397
-76.214
157.467
-161.859
-82.155
76.228
Fig. 2 Molecular structure of
15 with its atom (50 %
probability level) labeling
scheme
via drug–target interactions. When H unit was replaced
with methyl, fluoro, and nitro substituent, data showed that
electron-donating substituents revealed better activities
than electron-withdrawing substituents such as 18 (3-CH3,
IC50, 1.33 lM), 19 (3-F, IC50, 4.75 lM), and 20 (3-NO2,
IC50, 8.58 lM) against MCF-7 cells. Similarly, 18 (3-CH3,
IC50, 2.25 lM), 21 (2-CH3, IC50, 2.66 lM), and 29 (3-
CH3, IC50, 0.88 lM) exhibited higher activities than the
other substituents such as 16 (4-NO2, IC50, 6.70 lM), 26
(4-F, IC50, 5.41 lM), and 31 (3-CN, IC50, 8.97 lM)
123

Med Chem Res
Fig. 3 A view of the robust N–
H…O hydrogen bond synthons,
R22(8) I and R66(28) II, that link
the molecules forming a two-
dimensional network in 15 (the
dashed lines show donor–
acceptor distances of hydrogen
bonds)
against MDA-MB-231 cells. For this reason, synthesis of
methyl analogs of this class compounds seems to be ben-
eficial for anticancer drug design. Likewise, the above-
mentioned structure–activity relationship was also found in
the other titled cell lines. It is noteworthy that replacement
of H with the selected groups at 4-position of aromatic ring
did not follow a clear trend against the studied cell lines. In
an attempt to explore the effects of substituent insertion in
different positions of phenyl ring on the activity, the H in
aromatic ring was replaced with 4-, 3-, and 2-methyl, flu-
orine, and nitro to afford the corresponding compounds
(14–16, 25–27), (18–20, 29–31), and (21–23, 32–34),
respectively. Table 4 demonstrates that the newly synthe-
sized methyl analogs exhibited antitumor activity on the
studied cell lines with the average activity order of
3-CH3 [ 2-CH3 [ 4-CH3 such as 29 (3-CH3, IC50,
1.74 lM), 32 (2-CH3, IC50, 6.08 lM), and 25 (4-CH3,
IC50, 13.90 lM) against PC-3 cells, whereas cell lines from
our panel respond to nitro and fluoro analogs significantly
differently. Finally, replacement of the six-membered ring
in 13–23 with a five-membered ring 24–34 resulted in an
interesting structure–activity correlation in two carcinoma
cells, HeLa and MCF-7. For instance, fluorine analogs (15;
4-F, IC50, 3.94 lM, 19; 3-F, IC50, 4.75 lM, and 22; 2-F,
IC50, 3.14 lM) in six-membered ring displayed slightly
higher average potency in comparison with these analogs
in five-membered ring (26; 4-F, IC50, 16.64 lM, 30; 3-F,
IC50, 16.06 lM, and 33; 2-F, IC50, 3.2 lM) against MCF-
7. Also, among modified analogs, ortho substituent in
diazaphosphorinane ring (21; 2-CH3, 2.13 lM, 22; 2-F,
3.14 lM, 23; 2-NO2, 5.64 lM) showed higher anticancer
activity than similar counterpart in diazaphospholane ring
(32; 2-CH3, 6.69 lM, 33; 2-F, 3.2 lM, 34; 2-NO2,
17.57 lM) against MCF-7, suggesting that the ring size
plays a significant role for activity. It can be explained by
reduction of the electron donation to the phosphorus atom
in five-membered ring compared to six-membered ring. In
contrast, as shown in Table 1, inhibitory activities of meta
and para positions to the titled cancer cell lines were dif-
ferent. For example, phosphorinane derivatives 16 (4-NO2)
and 18 (3-CH3) showed an IC50 value 8.92 and 1.33 lM
against MCF-7 cell line, whereas phospholanes 27 (4-NO2)
and 29 (3-CH3) exhibited IC50 values of 1.28 and 0.72 lM,
respectively. These differences may result from a number
of factors, in addition to the interaction with the target
involved in the cytostatic/cytotoxic response. These factors
include number of cell cycles during the incubation period,
drug uptake, efflux, and metabolism. On the other hand,
few differences among cell lines would suggest that some
non-specific cytotoxic effects are responsible for the
activity of the tested compounds. To study the degree of
selectivity in the cytotoxic activity of the compounds,
assays using lymphocyte isolation from whole human
blood were carried out on some representative compounds
such as 14, 20, and 33, which showed relatively high
activity in tumor cells. The assay showed that survival
values were 97 % for these compounds.
Antibacterial activity
The in vitro antibacterial activity of the synthesized
derivatives 13–34 was tested against three Gram-positive
123

Med Chem Res
Table 4 In vitro antiproliferative activity of derivatives 13–34, cyclophosphamide (CP), and phenylurea
H
N
H
N
O
NH
P
A
R
B
NH
O
Compound
A-ring
B-ring
ICa50(lM) SD
HeLa
MCF-7
K562
PC-3
MDA-MB-231
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
a
Diazaphosphorinane
Diazaphosphorinane
Diazaphosphorinane
Diazaphosphorinane
Diazaphosphorinane
Diazaphosphorinane
Diazaphosphorinane
Diazaphosphorinane
Diazaphosphorinane
Diazaphosphorinane
Diazaphosphorinane
Diazaphospholane
Diazaphospholane
Diazaphospholane
Diazaphospholane
Diazaphospholane
Diazaphospholane
Diazaphospholane
Diazaphospholane
Diazaphospholane
Diazaphospholane
Diazaphospholane
Phenyl ureaa
4-H
3.33 0.35
6.51 0.92
3.05 0.19
–
6.2 0.71
4.75 0.15
4.14 0.27
8.17 0.65
7.23 0.39
11.46 1.70
1.64 0.02
9.75 1.85
–
29.66 2.41
5.06 0.11
3.45 0.32
2.82 0.18
2.68 0.31
1.10 0.27
7.31 0.49
3.89 0.23
2.24 0.17
4.01 0.12
3.95 0.44
30.08 3.51
13.95 2.98
18.38 3.52
2.59 0.55
1.69 0. 29
1.74 0.27
4.09 0.89
6.54 0.41
6.08 0.39
–
4.13 0.24
2.96 0.09
5.29 0.28
6.70 0.91
0.49 0.05
2.25 0.72
3.03 0.74
5.25 0.25
2.66 0.83
4.77 0.33
4.69 0.06
0.68 0.03
5.59 0.20
5.41 0.53
2.01 0.04
2.84 0.38
0.88 0.07
–
4-CH3
4-F
2.32 0.03
3.94 0.27
8.92 0.58
5.52 0.97
1.33 0.10
4.75 0.64
8.58 1.51
2.13 0.77
3.14 0.67
5.64 0.22
8.10 0.55
9.67 0.39
16.64 1.34
1.28 0.25
1.69 0.99
0.72 0.02
16.06 2.91
5.75 0.38
6.69 0.71
3.20 0.03
17.57 3.00
7.79 0.92
39.64 3.94
4-NO2
4-CN
3-CH3
3-F
4.80 0.82
1.52 0.06
3.50 0.42
5.56 0.21
2.10 0.36
17.70 2.89
3.32 0.01
6.42 0.13
–
3-NO2
2-CH3
2-F
3.84 0.63
4.50 0.73
2.82 0.07
–
2-NO2
4-H
4-CH3
4-F
–
4.25 0.26
2.90 0.79
3.07 0.06
1.35 0.31
–
4.67 0.27
–
4-NO2
4-CN
3-CH3
3-F
–
3.00 0.16
5.82 0.59
–
3-NO2
2-CH3
2-F
30.02 4.37
5.38 0.44
–
8.97 0.11
1.40 0.01
1.65 0.16
8.94 0.88
17.6 2.31
2.70 0.33
4.05 0.22
–
2-NO2
–
20.65 3.80
16.07 1.06
41.23 5.31
4.44 0.39
15.07 2.21
13.39 2.91
10.22 0.48
7.09 0.29
Cyclophosphamidea
IC50 is in terms of mM
Fig. 4 IC50 values of
compounds 13–34 against
HeLa, MCF-7, K562, PC-3, and
MDA-MB-231
123

Med Chem Res
Table 5 Diameter of inhibition zone and MIC valuesa of compounds 13–34 against selected microorganisms
Com.
A-ring
B-ring Gram-positive bacteria
Gram-negative bacteria
E. coli P. aeruginosa
S. aureus
B. cereus
B. subtilis
P. vulgaris
Za MICa
Z
MIC
Z
MIC
Z
MIC
Z
MIC
Z
MIC
13
Diazaphosphorinane 4-H
12 385
–
7
–
6
[2500
11 1000
10 1000
7
[2500
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
22
25
–
14
Diazaphosphorinane 4-CH3 11 1000
Diazaphosphorinane 4-F
Diazaphosphorinane 4-NO2 11 600
Diazaphosphorinane 4-CN 12 [2500
Diazaphosphorinane 3-CH3 10 1200
[2500
11 500
20
–
[2500
–
15
10 [2500 16 [2500
17 [2500
14 300
–
–
–
–
16
6
8
–
–
9
–
–
1200
11 1200
11
–
1200
–
17
[2500
17 [2500
12 600
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
18
–
–
–
–
19
Diazaphosphorinane 3-F
11 [2500
–
16 833
–
–
–
20
Diazaphosphorinane 3-NO2
–
–
[2500
18 [2500
11 [2500
15 25
–
–
–
21
Diazaphosphorinane 2-CH3 10 [2500
–
–
–
–
–
22
Diazaphosphorinane 2-F
9
–
–
–
–
–
–
–
–
8
[2500
–
–
–
23
Diazaphosphorinane 2-NO2
–
11 [2500
–
–
–
–
–
–
8
–
–
–
–
24
Diazaphospholane
Diazaphospholane
Diazaphospholane
Diazaphospholane
Diazaphospholane
Diazaphospholane
Diazaphospholane
Diazaphospholane
Diazaphospholane
Diazaphospholane
Diazaphospholane
4-H
–
–
–
–
–
–
–
7
8
–
8
–
–
–
–
–
–
25
4-CH3
4-F
–
–
–
–
–
–
26
–
–
–
–
–
–
27
4-NO2
4-CN
3-CH3
3-F
–
–
–
–
–
–
28
–
–
–
–
–
–
29
–
–
[2000
–
–
–
30
–
[2500
[2500
–
12 [1000
10 [2500
–
–
–
31
3-NO2
[2500
–
–
–
32
2-CH3 10 [2500
7
[2500
–
–
–
33
2-F
9
8
[2500
[1000
[2500
–
13 [2000
–
–
–
34
2-NO2
–
–
–
–
–
Gentamicin
Tetracycline
a
23 [500
33 0.025
27 125
33 125
26 125
25
125
[0.05
125
0.025
40 [0.025 36 [0.025 33 [0.025 31
The values indicate the diameters in mm for the zone of growth inhibition (GIZ) and minimal inhibitory concentration (MIC) in lg/ml
observed after 24 h of incubation at 35 °C. Error values are within 1 mm. Moderately active (8–13); highly active ([14)
–, not active
bacteria, viz. S. aureus (ATCC 25923), B. subtilis (ATCC
12711), and B. cereus (ATCC 11778), and three Gram-
negative bacteria, viz. E. coli (ATCC 25922), P. aerugi-
nosa (ATCC 27853), and Proteus vulgaris (NCIMB 8066),
using the cup test method and minimum inhibitory con-
centration (MIC) experiments. While carrying out
antibacterial activity, gentamicin and tetracycline were
used as reference compounds and MIC were determined in
terms of lg/ml (Table 5). Twenty-two heterocyclic com-
pounds with six- and five-membered rings of phosphor
were prepared and evaluated. At first, the objective was to
identify the effect of A-ring size on activity. A perusal of
the results from Table 5 reveals that diazaphosphorinanes
(13–23) with six rings showed higher activity toward the
tested Gram-positive bacteria than diazaphospholane (24–
34) with five rings. For example, 19 with six rings (3-F,
MIC; 833 lg/ml) exhibited potent activity against B. sub-
tilis than 30 with similar structure attached to five rings
(3-F, MIC; [ 1000 lg/ml). The second goal was to iden-
tify substitution effect on activity against the selected
microorganisms. Among the synthesized analogs at para
position, the most potent compound against S. aureus and
B. subtilis was 16. It has NO2 group attached, whereas
when CH3, F, and CN group substituents were attached to
ring, the same as in case of 17 (4-CN), inhibition decreased
(Table 5). Further, if NO2 group is attached to meta and
ortho positions, activity decreased as observed in 20 and
23, respectively. Reduced activity was observed when CH3
group was attached at the titled positions as in compounds
18 and 21, whereas substitution of (3-F, 2-F) group to the
phenyl group of 19 and 22 compounds increased inhibitory
activity against B. subtilis. The most valuable finding is the
inhibition of B. subtilis growth by compound 22 (MIC,
25 lg/ml) having electron-withdrawing group (F) at ortho
position. The MIC values of compounds against certain
bacterial strains indicate that B. subtilis were more
123

Med Chem Res
sensitive to the toxicity of the synthesized compounds than
other microorganisms. In addition, only derivatives 13, 14,
and 16 showed negligible activities against E. coli and P.
aeruginosa as compared to reference drugs.
surrounded by the same conformer (A0) and two neighbors
(B, B0) (Fig. 5). The electronic parameters of the hydrogen-
bonded clusters of compound 15 were calculated byAIM and
NBO methods. The results of AIM and NBO analyses for
H-optimized clusters are presented in Table 6. The bond
lengths in optimized clusters are equal to those obtained for
X-ray structures (Table S1), except for C–H and N–H bonds,
since the optimizations have only been performed for H atom
positions. Also, the donor–acceptor distances for hydrogen
bonds in model clusters are equal to the experimental values.
NBO analysis reveals [LP(OC) ? r*(N–H)] and
[Lp(OP) ? r*(N–H)] interactions among the subunits
within the clusters. This electronic delocalization leads to the
weakening of the N–H bond. This is in good agreement with
the decrease in the q value at the bcp of the N–H bond from
the monomers to their corresponding clusters (Table 6 and
S1). Moreover, the D–H–A angles in the optimized clusters
are more linear than those in the solid-state structures. These
indicate that the hydrogen bonds in the optimized clusters are
stronger than those in the X-ray structures. It has been pre-
viously explained that the charge density at the bcp of
DH…A increased when the donor–acceptor distance
shortens. The results of AIM analysis show that q value at
bcp of intramolecular hydrogen bond A(NH)…(OP)-
NBO and AIM analyses
Fully optimized conformers in the gas phase
The geometries of conformers 15a and 15b were separately
optimized as single molecules in vacuum in order to
compare the structural and energy aspects of the two
conformers in the gas phase. The optimized geometries
were confirmed to be the minimum energy point with no
imaginary vibrations. The P=O, P–N, C–N, and N–H bond
lengths, charge densities, and vibrational data have been
represented in Table S1 for two conformers. In both of the
conformers, the weak intramolecular CH…OC hydrogen
bond between the ortho-proton of the 4-FC6H4NH and the
carbonyl O atom creates a six-membered ring via the O–C–
N–C–C–H bond paths. This intramolecular contact stabi-
lizes a gauche configuration between the C=O bond and the
ortho-proton of 4-fluoroaniline. Furthermore, intramolec-
ular 4-FC6H4N-H…O=P hydrogen bond creates a six-
membered ring via the O–P–N–C–N–H bond paths. Based
on the results of DFT calculations, we found that the
change in basis set used for geometry optimizations has no
significant effect on the measure of structural parameters
and the two optimized conformers convert to a unique
structure. Hence, only one molecular structure was selected
to obtain electronic properties by AIM and NBO analyses.
The NBO analysis reveals a weak electronic delocalization
between the lone pair of the carbonyl oxygen, Lp(OC), and
the vacant r*(C-Hortho) orbital, whereas Lp(OP) and the
vacant r*(N–H) orbital strengthen NH…OP hydrogen
bond between the proton of the 4-FC6H4NH and the
phosphoryl O atom. Stabilization energies E2 of 4.7 and
8.2 kJ mol-1 were obtained for the Lp(OC) ? r*(C–
A (0.0362 e A-3) is larger in magnitude than that calculated
˚
for the intermolecular hydrogen bonds, i.e., A(NH)…(OP)-
-3
-3
0
B (0.0277 e A ), B (NH)…(OP)A (0.0221 e A ), and A0
˚
˚
(NH)…(OC)A (0.0266 e A-3). The smaller q values at the
˚
bcp of the N–H bond confirm the presence of the stronger
interaction in intramolecular hydrogen bond in comparison
with intermolecular hydrogen bonds. This is in good agree-
ment with stabilizing energies E(2) for the title electronic
delocalization in the model cluster. The stabilizing energies
E(2) of 29.8 and 25.1 kJ mol-1 have been calculated for
LP(OP) ? r*(N–H) electron density transfer in (OP)B
0
0
…HA–NA and (OP)A…HB –NB hydrogen bonds, while it is
41.1 kJ mol-1 in the electronic delocalization LP(OC) ?
r*(N–H) in the case of (OC)A …HA´–NA´. Furthermore, the
stabilizing energy E2 of 45.6 kJ mol-1 has been calculated
for LP(OP) ? r*(N–H) in (OP)A …HA–NA hydrogen bond.
This is in agreement with the values of distance for these
H
ortho) interaction in conformers 15a and 15b, respectively,
whereas those of Lp(OP) ? r*(N–H) interaction were
calculated as 21.1 and 40.0 kJ mol-1. At the same level,
AIM analysis reveals charge density (q) values at the bcp
of the CH…OC and NH…OP contacts in 0.0177 and
˚
hydrogen bonds in NA–HA…OA–PA (2.722(2)A), (OP)B…-
˚
˚
0
0
HA–NA (2.933(2)A), (OP)A…HB –NB (3.040(2)A), (OC)A
0.0321 e A-3. The q value at the ring critical point (rcp)
…HA´ –NA´ (2.969(2)A) models. Besides, the hydrogen-
˚
˚
for the O–C–N–C–C–H and O–P–N–C–N–H six-mem-
bered rings (Fig. S1) is 0.0114 and 0.0138 e A-3. This
˚
bonding energy in the (OP)B …HA–NA and (OP)A…HBNB
models (-9.7 and -7.1 kJ mol-1) is smaller than the value
calculated for (OC)A …HA´–NA´ (-39.9 kJ mol-1) model. It
should be noted that the stabilizing energies E(2) differ from
the hydrogen-bonding energy. The hydrogen-bonding
energy is related to the sum of the total attractive and
repulsive forces between two hydrogen-bonded fragments,
and E2 refers to the stabilization energy of electronic delo-
calization between the donor–acceptor orbital.
values confirm the formation of weak and strong interac-
tions for CH…OC and NH…OP contacts.
Electronic parameters of the hydrogen-bonded clusters
For the compound 15, the conformers were modeled as
hydrogen-bonded clusters in which the target molecule (A) is
123

Med Chem Res
were identified in MCF-7 model. The electronic and
structural descriptors were obtained by quantum chemical
calculations (Table 7). An optimal QSAR equation based
on experimental data (d, log P, and Mr) shown in Table 2
(eq. a), as well as eq. b is produced by replacement of
experimental variables with the descriptors derived from
the DFT calculations. Afterward, PCA method was used to
reduce the independent variables (Pillai et al., 2005). The
variable selection in PCA was performed by using the
Fisher’s weights approach (Molfetta et al., 2005), and the
results are summarized as the following Eqs. (1, 2).
PC1 ¼ 0:346 Eh þ 0:330El þ 0:338Elþ1 þ 0:301EhÀ1
À 0:326W þ 0:063Qp À 0:303QN2 À 0:236QC
À 0:187PLP¼O À 0:117 PLðNfÀÀgHÞ1
À 0:269PLðNfÀÀgHÞ2 À 0:213MV þ 0:235TE
ð1Þ
PC2 ¼ 0:001 Eh À 0:005 El À 0:034 Elþ1 À 0:054 EhÀ1
À 0:033W þ 0:514Qp À 0:212 QN2 À 0:362 QC
À 0:437 PLP¼O þ 0:224 PLðNfÀÀgHÞ1
Fig. 5 Model hydrogen-bonded cluster for DFT calculations, in
which the target molecule (conformer A) is in the center. A similar
model was considered for the cluster in which conformer B in the
center is the target molecule
þ 0:178 PLðNfÀÀgHÞ2 þ 0:299 MV À 0:311 TE
ð2Þ
The main variables were found from the principle scores of
the normalized eigenvalue of the two principal compo-
nents. The results showed that the first and second factor
PC on the total variance were 46.7 and 18.3 %, respec-
tively. Also, from the above equations, it was deduced that
QSAR analysis
In order to understand the observed pharmacological
properties of the investigated analogs and determine the
crucial factors governing these activities, QSAR studies
were undertaken. The stepwise MLR procedure was used
for model selection, which is a common method used in
QSAR studies. Therefore, experimental IC50 values of 22
tested compounds with the general formula of RC6H5
(NH)1C(O)(NH)2P(O)R1R2 (13–34), phenylurea, and CP
were employed for constructing QSAR models against five
data sets corresponding to the five tested cell lines, i.e.,
MDA-MB-231, PC-3, MCF-7, K562, and HeLa. For each
data set, compounds with inactive cytotoxic activity were
excluded from the analysis. For example, compound 30
was identified as an outlier for the MDA-MB-231 model
and was subjected to removal, whereas none of the outliers
the electronic parameters EHOMO, ELuMO, W, QP, QC, QN2
,
PLP=O, PLN–H, and TE predominated over those related to
structural parameter (Mv).
MDA-MB-231 model
The MLR was performed using these thirteen descriptors
and equations are shown in Table S2. This method led to an
optimal QSAR Eq. 3:
Logð1=IC50Þ ¼ 14:350EHÀ1 þ 0:006W À 1:078QP
þ 77:247QC þ 13:463PLðNfÀÀgHÞ1
À 28:317PLðNfÀÀgHÞ2 À 83:257
ð3Þ
Table 6 Hydrogen bond data for the X-ray structure (values in brackets), model cluster (at B3LYP/6-311?G**), charge densities (from AIM
analysis), and delocalization energy (from NBO)
D–H…A
d(N–H)
d(H…O)
d(N…O)
\(NHO)
q(a.u.)
SE
DEHB
bcp1
bcp2
N(2A)–H(2NA)…O(1)i
N(2)–H(2N)…O(1A)ii
N(3)–H(3 N)…O(2)iii
N(4)–H(4 N)…O(1)ii
[0.860]1.024
[1.00]1.024
[0.92]2.969
[0.82]2.722
[2.20]2.02
[1.94]1.91
[2.05]1.94
[2.01]1.82
3.040(2)
2.933(2)
2.969(2)
2.722(2)
[168.0]176.3
[171.0]173.3
[175.0]175.9
[145.0]144.9
0.3166
0.3172
0.3134
0.3235
0.0221
0.0277
0.0264
0.0362
[25.2]
[29.8]
[41.1]
[45.6]
[-7.1]
[-9.7]
[-39.9]
–
Symmetry codes: (i) x, y - 1, z; (ii) x, y, z; (iii) -x, -y ? 1, -z. SE is the stabilizing energy E(2) (in kJ mol-1) for the [LP(OC) ? r*(N–H)]
and [Lp(OP) ? r*(N–H)] electronic delocalization. The binding energy is in kJ mol-1 for hydrogen bonds
q is the calculated charge density at the bcp of the D–H bond (bcp1) and H…A contact (bcp2) in the cluster
123

Med Chem Res
123

Med Chem Res
n ¼ 16; R2 ¼ 0:939; Ra2dj ¼ 0:885; F ¼ 17:497; Sreg
¼ 0:103
compound with lower net charge QN2 and higher molecular
orbital EH is indicative of higher potency against MCF-7
cell line. The interrelationships between the variables and
the correlation matrix results are presented in Table S3.
The high and low interrelationships were observed between
QN2 and PLC=O (r = -0.840), and QN2 and EH
(r = -0.325), respectively. The high interrelationship
between QN2 and PLC=O (r = -0.840) shows that PLC=O
controls the effect of net charge of the N–H nitrogen QN2
on the inhibition of the titled cell line. Diazaphosphorinane
with fluorine substituent at ortho position 22
(PLC=O = -1.476) and phenylurea 36 (PLC=O = -1.451)
are in order of the highest and the lowest inhibitory
potentials against MCF-7 cell line.
In this equation, the inhibitory potency of MDA-MB-231
cell line is influenced mainly by the electronic parameters.
Qc with the coefficient values of ?77.247 has the highest
contribution to log (1/IC50) rather than the other electronic
parameters such as the net charge of P=O phosphorus
(-1.078). The positive coefficient associated with the
parameter Qc suggests that increases in the negative charge
or electron density at the C=O carbon may be favorable to
higher antitumor activity against MDA-MB-231 cell line.
The correlation matrix was used to determine the interre-
lationship between the independent variables (Table 8).
The high interrelationship observed between QC descriptor
and W (r = ?0.721) showed that electrophilicity proper-
ties of the studied derivatives affect the inhibition of human
MDA-MB-231. Compounds 16 (W = 0.205) and 36
(W = 0.062) are in order of the highest and the lowest
inhibitory potentials.
PC-3 and HeLa models
Similar results were found in both PC-3 and HeLa models
in which the electronic descriptors were governing the
cytotoxic activity. The QSAR equation of the PC-3 model
is shown below:
MCF-7 model
Logð1=IC50Þ ¼ À27:559ELþ1 À 8:847EHÀ1 þ 26:613W
þ 343:388QN2 þ 8:79QC þ 1:586l
Besides net charge of the N–H nitrogen QN2, EH properties
of compounds are highly involved in cytotoxic activity
against the MCF-7 cell line as shown by the QSAR
equation below:
À 0:004 PLðP¼OÞ À 335:95
ð5Þ
n ¼ 11; R2 ¼ 0:965; Ra2dj ¼ 0:883; F ¼ 11:814; Sreg
¼ 0:034
Logð1=IC50Þ ¼ 10:13EH À 2:702EHÀ1 À 10:201QN2
À 5:699l þ 0:019PLðP¼OÞ þ 0:001PLðC¼OÞ
þ 1:705
In Eq. (5), QN2 representing the net charge of the N–H
nitrogen seems to be the most important descriptor as
deduced from its highest regression coefficient value of
343.388. High colinearity (r [ 0.5) was observed between
different parameters (Table S4). The high interrelationship
was observed between QN2 and W (r = -0.841), and low
interrelationship was observed between this descriptor and
PLP=O (r = 0.087). From the correlation matrix, it was
observed that the electrophilicity (W) was found to be most
effective in describing the anticancer activity of the
ð4Þ
n ¼ 11; R2 ¼ 0:939; Ra2dj ¼ 0:885; F ¼ 17:497; Sreg
¼ 0:103
QN2 and EH with the coefficient values of -10.201 and
10.130 have the highest contribution to log (1/IC50) rather
than the structural parameters. The negative and positive
signs of QN2 and EH in log (1/IC50) disclose that the
Table 8 Correlation matrix for log (1/IC50) and selected parameters in Eq. 3
Selected variables
MDA-MB-231
EH-1
W
QP
QC
PL(N–H)1
PL(N–H)2
Mv
EH-1
W
1
-0.814
0.283
1
QP
-0.215
0.721
0.115
0.635
0.511
1
QC
-0.640
-0.201
-0.509
-0.422
-0.675
0.089
0.140
0.258
1
PL(N–H)1
PL(N–H)2
Mv
-0.166
0.318
0.175
1
0.650
0.452
1
0.587
1
123

Med Chem Res
synthesized compounds. As shown in Table 7, compounds
16 and 36 with electrophilicity (W) values 0.205 and 0.062
have the highest and the lowest inhibitory potentials
against the titled cell line.
1. QSAR studies demonstrated that cytotoxic activities of
compounds 13–34, CP, and phenylurea against the
investigated cell lines were influenced by the elec-
tronic descriptors. This indicates that such distinct
chemical properties are crucial for the cytotoxic
activity against particular cell lines.
The QSAR equation of the HeLa model is shown below:
Logð1=IC50Þ ¼ 10:13EH À 6:661EHÀ1 þ 261:821QN2
þ 50:015l þ 0:012PLðP¼OÞ
2. QSAR studies also emphasize that higher probability
of electrophilic attack at the nitrogen atom number 2
may be favorable to the biological activity compared
to the nitrogen atom number 1. This is in agreement
with the values of the electron density (q) for nitrogen
À 0:007PLðC¼OÞ À 155:829
ð6Þ
n ¼ 11; R2 ¼ 0:86; Ra2dj ¼ 0:651; F ¼ 4:105; Sreg ¼ 0:095
-3
˚
atom in two amidic (8.383 eA
) and aniline
Similarly the model described by Eq. 6 showed that the
most effective variable in the inhibition of HeLa cell line by
the tested derivatives was QN2, with the coefficient value of
261.821. The interrelationships between the variables and
the correlation matrix results are presented in Table S5. A
high interrelationship was observed between QN2 and EH
(r = -0.758). Compounds 14 (EH = -0.2105) and 35
(EH = -0.268) are in order of the highest and the lowest
inhibitory potentials. The positive coefficients of QN2 at
Eqs. 5 and 6 indicate that the increase in the negative charge
or electron density at the N–H nitrogen may be favorable to
higher antitumor activity.
(8.174 eA-3) bonds obtained by AIM analysis. This
may explain much interaction N1 with neighbor groups
compared to the N2 atom. NBO analysis reveals an
[Lp(N) r*(C=O)] interaction with the stabilizing
energies E2 of 56.99 and 32.39 kcal mol-1 for lone
pair N1 and N2, respectively. This electronic delocal-
ization leads to reduction in the (q) value for nitrogen
atom from N2–H bond to N1–H bond.
˚
3. Net charge of the N–H nitrogen (QN2) seemed to be
important descriptor for the cytotoxic activity against
MCF-7, PC-3, and HeLa cell lines, whereas net charge
of the C=O carbon (Qc) and EL?1 were shown to be
crucial for MDA-MB-231 and K562, respectively.
With respect to importance of the electronic descrip-
tors against the studied cell lines, it could be hypoth-
esized that the mechanism of cytotoxic activity of
diazaphosphore compounds may be similar.
K562 model
Distinct from other cell lines, the cytotoxic activity of
compounds against the K562 cell line is dependent on the
next lowest unoccupied molecular orbital shown in the
equation below:
4. Experimental and theoretical data demonstrate the
importance of six-membered ring for cytotoxicity.
This might be explained by the electronic nature of
phosphor atom.
Logð1=IC50Þ ¼ 22:06ELþ1 À 3:1EHÀ1 þ 7:877QN2
þ 3:078l þ 0:004PLðP¼OÞ þ 0:281
ð7Þ
n ¼ 12; R2 ¼ 0:941; Ra2dj ¼ 0:869; F ¼ 13:193; Sreg
Validation of QSAR
¼ 0:006
In each cell lines, a set of compounds was used as a
training set for QSAR modeling. The remaining com-
pounds were adopted as a test set for validating the QSAR
model. Considering the balance of the QSAR quality and
the number of employed quantum chemical descriptors, an
optimal equation was achieved for the selected compounds
in the training set by MLR analysis for the studied cell lines
(Table S7). The developed QSAR models were cross-val-
idated with q2 value (q2 [ 0.5) obtained by leave-one-out
(LOO) method. The value of q2 more than 0.5 indicates
that the model developed is a valid one. According to the
recommendations of Golbraikh and Tropsha, the only way
to estimate the true predictive power of a model is to test
their ability to predict accurately the biological activities of
compounds. As the observed and predicted values are close
It can be seen that the EL?1 descriptor is responsible for
prediction of the cytotoxic activity of K562 cell line as
deduced from its high regression coefficient value of 22.06.
The positive sign of EL?1 in log (1/IC50) discloses that the
compound with higher molecular orbital (EL?1) may be
conducive to the biological activity of these diazaphos-
phore compounds. The correlation matrix of different
descriptors used in the QSAR models is shown in Table S6.
The high interrelationship was observed between EL?1 and
EH-1 (r = -0.553), and low interrelationship was
observed for QN2 (r = -0.01). Then, compound 21 having
high EH-1 value (-0.240) displayed high anticancer
activity. Similarly, compound 27 having low EH-1 value
(-0.277) exhibited low anticancer potential. Obtained
results from the above data can be summarized as follows:
123

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123

Med Chem Res
Bøyum
A
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Conclusion
In this study, new sixteen diazaphosphore derivatives,
differing in the nature of the aliphatic and aromatic rings,
were synthesized and investigated for their biological
activity. Novel anticancer agents 18 and 29 with methyl
group at meta position of aromatic ring exhibited higher
cytotoxic activity against most of tested cell lines than
other derivatives which may be due to size, steric, and
electronic properties of the substituent. The models
obtained through QSAR study gave a better prediction
capability of anticancer activity against the studied human
cell lines. Electronic parameters such as QN2, QC, and
EL?1 were major factors responsible for positively affect-
ing the anticancer activity of the selected diazaphosphore
derivatives. As evident from the experimental and QSAR
data, the presence of six-membered ring of diazaphosphore
is essential for higher antitumor activity. Besides, impor-
tant of the nitrogen atom number 2 at anticancer activity
compared with the nitrogen atom number 1 expressed by
result of NBO and AIM analysis. Comparison of the results
of antibacterial studies exhibited further emphasis on the
importance of six-membered diaza ring in addition to the
requirement of substitutions in the aromatic ring.
Supplementary data
Crystallographic data for structure 15 have been deposited
with the Cambridge Crystallographic Data Centre as sup-
plementary publication Nos. CCDC 1428625 (C12H18F1N4
O2P). Copies of the data may be obtained free of charge upon
request from CCDC, 12 Union Road, Cambridge CB2 1EZ,
UK (fax: ?44 1223 336033; E-mail: deposit@ccdc.-
cam.ac.uk or www: http://www.ccdc.cam.ac.uk
Gholivand K, Dorosti
N (2013) Some new compounds with
P(E)NHC(O) (E = lone pair, O, S) linkage: synthesis, spectro-
scopic, crystal structures, theoretical studies, and antimicrobial
evaluation. Monatsh Chem 144:1417–1425
Acknowledgments The financial support of this work provided by
the Research Council of Tarbiat Modares University and the Razi
Herbal Medicines Research Centre of Lorestan University of Medical
Sciences is gratefully acknowledged.
Gholivand K, Shariatinia Z, Mahdieh S, Farzaneh Daeepour M,
Farshidnasab N, Mahzouni HR, Taheri N, Amiri S, Ansar S
(2009) Structural diversity in phosphoramidate’s chemistry:
syntheses, spectroscopic and X-ray crystallography studies.
Polyhedron 28:307–321
Gholivand K, Dorosti N, Shariatinia Z, Ghaziany F, Sarikhani S,
Mirshahi M (2010) Cyclophosphamide analogues: synthesis,
spectroscopic study, and antitumor activity of diazaphosphori-
nanes. Med Chem Res 21:2185–2195
Gholivand K, Dorosti N, Ghaziany F, Mirshahi M, Sarikhani S (2012)
N-Phosphinyl ureas: synthesis, characterization, X-ray structure,
and in vitro evaluation of antitumor activity. Heteroatom Chem.
23:74–83
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