Phosphorus-nitrogen compounds. Part 23: Syntheses, structural investigations, biological activities, and DNA interactions of new N/O spirocyclotriphosphazenes

Article abstract of DOI:10.1016/j.saa.2011.10.027

The Schiff base compounds (1 and 2) are synthesized by the condensation reactions of 2-furan-2-yl-methylamine with 2-hydroxy-3-methoxy- and 2-hydroxy-5-methoxy-benzaldehydes and reduced with NaBH₄ to give the new N/O-donor-type ligands (3 and 4

Full text of DOI:10.1016/j.saa.2011.10.027

Spectrochimica Acta Part A 86 (2012) 214–223
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Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Phosphorus–nitrogen compounds. Part 23: Syntheses, structural investigations,
biological activities, and DNA interactions of new N/O spirocyclotriphosphazenes
Nuran Asmafiliza, Zeynel Kılıc¸ a,∗, Zeliha Hayvalıa, Leyla Ac¸ ıkb, Tuncer Hökelekc, Hakan Dald,
Yag˘mur Önerb
a Department of Chemistry, Ankara University, 06100 Ankara, Turkey
b Department of Biology, Gazi University, 06500 Ankara, Turkey
c Department of Physics, Hacettepe University, 06800 Ankara, Turkey
d Department of Chemistry, Anadolu University, 26470 Eskis¸ehir, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
The Schiff base compounds (1 and 2) are synthesized by the condensation reactions of 2-furan-2-yl-
methylamine with 2-hydroxy-3-methoxy- and 2-hydroxy-5-methoxy-benzaldehydes and reduced with
NaBH4 to give the new N/O-donor-type ligands (3 and 4). The monospirocyclotriphosphazenes con-
taining 1,3,2-oxazaphosphorine rings (5 and 6) are prepared from the reactions of N3P3Cl6 with 3 and 4,
respectively. The reactions of 5 and 6 with excess pyrrolidine, morpholine, and 1,4-dioxa-8-azaspiro [4,5]
decane (DASD) produce tetrapyrrolidino (5a and 6a), morpholino (5b and 6b), and 1,4-dioxa-8-azaspiro
[4,5] deca (5c and 6c) spirocyclotriphosphazenes. The structural investigations of the compounds are
examined by 1H, 13C, 31P NMR, DEPT, HSQC, and HMBC techniques. The solid-state structures of 5, 5a, and
6 are determined using X-ray crystallography. The compounds 5a, 5b, 5c, 6a, 6b, and 6c are subjected to
antimicrobial activity against six patojen bacteria and two yeast strains. In addition, interactions between
these compounds and pBR322 plasmid DNA are presented by agarose gel electrophoresis.
© 2011 Elsevier B.V. All rights reserved.
Received 19 August 2011
Received in revised form 7 October 2011
Accepted 14 October 2011
Keywords:
N/O spirocyclotriphosphazenes
Syntheses
Spectroscopy
Crystal structure
DNA interactions
1. Introduction
nongeminal route to give ansa derivatives and/or (ii) in geminal
route to give spiro derivatives, (iii) the replacement of one chlorine
Organocyclophosphazenes (NPR2)n (n = 3, 4,. . .), all of which
contain phosphorus and nitrogen atoms bonded alternately, are
borderline in inorganic and organic chemistry and an important
family of inorganic ring systems [1–4]. A large number of the phosp-
hazene derivatives have been prepared by nucleophilic substitution
reactions on hexachlorocyclotriphosphazene, N3P3Cl6, introduc-
ing easily a wide variety of different mono [5,6] and difunctional
organic groups onto P atoms [7–9]. A scan of the literature shows
that the involvement of bifunctional ligands with the cyclophos-
phazene ring is considerably limited to monofunctional reagents.
The condensation reactions of bifunctional ligands with N3P3Cl6
may lead to spiro, ansa, bino, dispiro, diansa, spiro-ansa, spiro-
bino, and trispirocyclophosphazene derivatives [10–13]. On the
other hand, the next oligomer octachlorocyclotetraphosphazene,
N4P4Cl8, gives also similar products with bifunctional ligands
[14–16].
atom with one of the two functional groups of the ligand to produce
open-chain (dangling) compounds, (iv) intermolecular reactions
between Cl atoms on two different phosphazene rings to form
bridged (bino) phosphazene derivatives, and (v) intermolecular
condensation reactions to give cyclolinear or cyclomatrix poly-
mers [17,18]. Moreover, the distribution of these phosphazene
derivatives may depend on many factors, e.g. reaction time, solvent
polarities, temperature, size of the phosphazene ring and the prop-
erties of the bifunctional ligands [19]. It has been observed that,
when the reactions are carried out with N/O donor-type bidentate
ligands and N3P3Cl6, the major product is generally spiro derivative
in tetrahydrofuran [20].
Recently, phosphazene derivatives have drawn considerable
attention for the further design of highly selective anticancer
agents [21] and antimicrobial [22] reagents. Aziridine-crown sub-
stituted cyclotriphosphazene derivatives cleave the DNA and halt
the growth of cancer cells [23]. The Cu+2 complex of fully-phenoxy-
substituted star-branched cyclotetraphosphazene derivative is also
found to be active in the oxidative cleavage of DNA [24]. On
the other hand, cyclophosphazenes have found industrial appli-
cations such as in the production of ionic liquids [25], liquid
crystals [26], dendrimers having chiral ligands for asymmetric
catalysis [27], flame retardants [28–30], advanced elastomers
As known, there are five possible reaction pathways for the reac-
tions of N3P3Cl6 with bidentate ligands: both functional groups
of the ligand may replace with two chlorine atoms: (i) in cis
∗
Corresponding author. Tel.: +90 3122126720/1043; fax: +90 3122232395.
E-mail address: zkilic@science.ankara.edu.tr (Z. Kılıc¸ ).
1386-1425/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2011.10.027

N. Asmafiliz et al. / Spectrochimica Acta Part A 86 (2012) 214–223
215
N
CHO
OH
X
Compound
O
H2N
O
3-OCH3
5-OCH3
1
2
OH
MeOH
X
X
MeOH
NaBH4
Cl
Cl
N
P
X
O
N
H
P
N
O
O
N
THF
+
Cl
Cl
P
OH
Cl
Cl
N
N
P
Cl
Cl
Cl
X
N
P
P
X
Compound
N
Cl
3-OCH3
5-OCH3
3
4
X
Compound
3-OCH3
5-OCH3
5
6
THF
O
O
O
HN
HN
HN
X
O
X
X
N
O
N
O
O
N
O
O
O
P
O
P
P
O
O
N
N
N
N
N
N
P
N
P
O
N
N
N
N
N
N
N
N
P
P
P
P
N
N
N
N
N
N
O
O
O
O
O
X
Compound
X
Compound
X
Compound
3-OCH3
5-OCH3
5c
6c
3-OCH3
5-OCH3
5b
6b
3-OCH3
5-OCH3
5a
6a
Scheme 1. The reaction pathway for the phosphazene derivatives.
[31,32], rechargeable batteries [33,34], and biomedical materials
[35,36].
and heteronuclear multiple-bond correlation (HMBC) techniques.
Moreover, the molecular and solid-state structures of 5, 5a, and 6
have been established by X-ray diffraction techniques.
As a particular interest in our ongoing studies about N/O
spirocyclic phosphazene derivatives, we report here in detail: (i)
the synthesis of new N-(furan-2-yl-methyl)-3 and 5-methoxy-
2-hydroxybenzylamines (3 and 4), (ii) the preparation of new
tetrachloro (5 and 6), tetrapyrrolidino (5a and 6a), tetramorpholino
(5b and 6b), and tetra(1,4-dioxa-8-azaspiro [4,5] deca) (5c and
6c) monospirocyclotriphosphazenes (Scheme 1), (iii) investiga-
tions of antibacterial and antifungal activity of 5a, 5b, 5c, 6a,
6b, and 6c, and (iv) interactions between these compounds and
pBR322 plasmid DNA examined by agarose gel electrophoresis.
The determination of the structures of the compounds have been
made using elemental analyses, mass spectrometry (MS), Fourier
transform (FTIR), one-dimensional (1D) 1H, 13C, and 31P NMR,
distortionless enhancement by polarization transfer (DEPT), two-
dimensional (2D) heteronuclear single quantum coherence (HSQC),
2. Experimental
2.1. General methods
Hexachlorocyclotriphosphazatriene (Aldrich), 2-hydroxy-3-
methoxy- and 2-hydroxy-5-methoxy-benzaldehydes (Aldrich),
2-furan-2-yl-methylamine (Acros Organics), pyrrolidine (Fluka),
morpholine (Fluka), and 1,4-dioxa-8-azaspiro [4,5] decane (Fluka)
were purchased and used without further purification. All reac-
tions were monitored using thin-layer chromatography in different
solvents and chromatographed using silica gel. All experiments
were carried out in an argon atmosphere. The 1H, 13C, and 31P
NMR, DEPT, HSQC, and HMBC spectra were recorded on a Bruker

216
N. Asmafiliz et al. / Spectrochimica Acta Part A 86 (2012) 214–223
DPX FT-NMR (500 MHz) spectrometer (SiMe4 as internal and 85%
H3PO4 as external standards). The spectrometer was equipped
with a 5 mm PABBO BB inverse-gradient probe. Standard Bruker
pulse programs were used [37]. The IR spectra were recorded
on a Mattson 1000 FTIR spectrometer in KBr disks and were
reported in cm−1 units. APIES mass spectrometric analyses were
performed on an AGILENT 1100 MSD spectrometer. The melting
points were measured on a Gallenkamp apparatus using a capillary
tube. Antimicrobial susceptibility testing was performed by the
agar-well diffusion method (Section S1 Supplementary Material).
The DNA binding abilities were examined using agarose gel
electrophoresis (Section S2 Supplementary Material).
(C–H arom.), 2972; 2875 (C–H aliph.), 1236; 1174 (P N), 564; 527
(PCl). APIES-MS (based on 35Cl, Ir %): m/z = 507 ([MH]+, 26%).
2.2.5. 4ꢀ,4ꢀ,6ꢀ,6ꢀ-Tetrachloro-3-(2-furanylmethyl)-6-(methoxy)-
3H,4H-spiro{[1,3,2-benzoxazaphosphinine]-2,2ꢀ-
[1,3,5,2,4,6]triazatriphosphinine]}
(6)
The work-up procedure was similar to that of compound 5,
using 4 (1.70 g, 7.30 mmol), N3P3Cl6 (2.54 g, 7.30 mmol) and tri-
ethylamine (2.05 mL). Yield: 2.68 g (72%). mp: 96 ◦C. Anal. cal. for
C13H13N4O3P3Cl4; C: 30.74; H: 2.58; N: 11.03. Found; C: 30.80; H:
2.62; N: 10.90%. FTIR (KBr, cm−1): ꢀ 3117; 3074 (C–H arom.), 2934;
2839 (C–H aliph.), 1242; 1186 (P N), 581; 519 (PCl). APIES-MS
(based on 35Cl, Ir %): m/z = 507 ([MH]+, 90%).
2.2. Preparation of compounds
2-{(E)-[(2-furanylmethyl)imino]methyl}-6-(methoxy)phenol
(1) was obtained from the reaction of 3-methoxy-2-hydroxy-
benzaldehyde with 2-furan-2-yl-methylamine according to the
methods reported in the literature [38].
2.2.6. 4ꢀ,4ꢀ,6ꢀ,6ꢀ-Tetra(1-pyrrolidinyl)-3-(2-furanylmethyl)-8-
(methoxy)-3H,4H-spiro{[1,3,2-benzoxazaphosphinine]-2,2ꢀ-
[1,3,5,2,4,6]triazatriphosphinine]}
(5a)
A solution of compound 5 (0.90 g, 1.77 mmol) and triethylamine
(1.00 mL) in dry THF (150 mL) was added slowly to a solution
of pyrrolidine (1.74 mL, 21.00 mmol) with stirring and refluxing
for 30 h. The oily product was purified by column chromatogra-
phy using benzene-THF (2:1) as an eluent and was crystallized
from acetonitrile. Yield: 0.67 g (59%). mp: 113 ◦C. Anal. cal. for
C29H45N8O3P3; C: 53.87; H: 7.01; N: 17.33. Found; C: 53.97; H:
6.86; N: 17.20%. FTIR (KBr, cm−1): ꢀ 3098; 3058 (C–H arom.), 2964;
2852 (C–H aliph.), 1224; 1199 (P N). APIES-MS (based on 35Cl, Ir
%): m/z = 647 ([M]+, 100%).
2.2.1.
2-{(E)-[(2-furanylmethyl)imino]methyl}-4-(methoxy)phenol (2)
A
solution of 5-methoxy-2-hydroxy-benzaldehyde (2.28 g,
15.00 mmol) in dry methanol (50 mL) was added to 1.45 g of 2-
furan-2-yl-methylamine (15.00 mmol) in dry methanol (50 mL).
The mixture was refluxed for 3 h and then the product was crys-
tallized from methanol. Yield: 2.90 g (84%). mp: 77 ◦C. Anal. cal. for
C13H13NO3; C: 67.52; H: 5.67; N: 6.06. Found; C: 67.49; H: 5.63;
N 6.08%. FTIR (KBr, cm−1): ꢀ 3126 (O–H), 3091; 3065 (C–H arom.),
2967; 2880 (C–H aliph.).
2.2.7. 4ꢀ,4ꢀ,6ꢀ,6ꢀ-Tetra(1-pyrrolidinyl)-3-(2-furanylmethyl)-6-
(methoxy)-3H,4H-spiro{[1,3,2-benzoxazaphosphinine]-2,2ꢀ-
[1,3,5,2,4,6]triazatriphosphinine]}
2.2.2. 2-{[(2-Furanylmethyl)amino]methyl}-6-(methoxy)phenol
(3)
(6a)
A
solution of compound 1 (3.46 g, 15.00 mmol) in dry
The work-up procedure was similar to that of compound 5a,
using 6 (1.08 g, 2.13 mmol), pyrrolidine (2.09 mL, 26.00 mmol) and
triethylamine (1.20 mL). Yield: 0.85 g (62%). mp: 145 ◦C. Anal. cal.
for C29H45N8O3P3; C: 53.87; H: 7.01; N: 17.33. Found; C: 53.97; H:
6.86; N: 17.18%. FTIR (KBr, cm−1): ꢀ 3097; 3056 (C–H arom.), 2964;
2859 (C–H aliph.), 1220; 1186 (P N). APIES-MS (based on 35Cl, Ir
%): m/z = 647 ([M]+, 100%).
methanol (100 mL) was added to the excess sodium borohy-
dride (2.28 g, 60.00 mmol). The crude product was extracted with
dichloromethane (150 mL, three times). Yield: 0.86 g (86%). mp:
67 ◦C. Anal. cal. for C13H15NO3; C: 66.94; H: 6.48; N: 6.00. Found;
C: 67.13; H: 6.45; N: 5.94%. FTIR (KBr, cm−1): ꢀ 3301 (N–H), 3124
(O–H), 3106; 3046 (C–H arom.), 2996; 2935 (C–H aliph.).
2.2.8. 4ꢀ,4ꢀ,6ꢀ,6ꢀ-Tetra(4-morpholinyl)-3-(2-furanylmethyl)-8-
(methoxy)-3H,4H-spiro{[1,3,2-benzoxazaphosphinine]-2,2ꢀ-
[1,3,5,2,4,6]triazatriphosphinine]}
2.2.3. 2-{[(2-Furanylmethyl)amino]methyl}-4-(methoxy)phenol
(4)
A
solution of compound 2 (3.46 g, 15.00 mmol) in dry
methanol (100 mL) was added to the excess sodium borohy-
dride (2.28 g, 60.00 mmol). The crude product was extracted with
dichloromethane (150 mL, three times). Yield: 0.90 g (90%). mp:
55 ◦C. Anal. cal. for C13H15NO3; C: 66.94; H: 6.48; N: 6.00. Found;
C: 66.86; H: 6.29; N: 6.17%. FTIR (KBr, cm−1): ꢀ 3289 (N–H), 3124
(O–H), 3091; 3065 (C–H arom.), 2960; 2890 (C–H aliph.).
(5b)
To a THF (75 mL) solution of 5 (1.00 g, 1.96 mmol) and triethy-
lamine (1.10 mL) was added 2.05 mL of morpholine (24.00 mmol)
in THF (75 mL) and the mixture was refluxed for 27 h. The solvent
was evaporated and the crude product was purified by column
chromatography with benzene-THF (1:1). Yield: 0.81 g (58%). mp:
160 ◦C. Anal. cal. for C29H45N8O7P3; C: 49.01; H: 6.34; N: 15.77.
Found; C: 48.88; H: 6.13; N: 15.73%. FTIR (KBr, cm−1): ꢀ 3091; 3071
(C–H arom.), 2960; 2842 (C–H aliph.), 1255; 1185 (P N). APIES-MS
(based on 35Cl, Ir %): m/z = 711 ([M]+, 100%).
2.2.4. 4ꢀ,4ꢀ,6ꢀ,6ꢀ-Tetrachloro-3-(2-furanylmethyl)-8-(methoxy)-
3H,4H-spiro{[1,3,2-benzoxazaphosphinine]-2,2ꢀ-
[1,3,5,2,4,6]triazatriphosphinine]}
(5)
A solution of 3 (1.00 g, 4.29 mmol) in THF (150 mL) and triethy-
lamine (1.21 mL) was added to a stirred solution of N3P3Cl6 (1.49 g,
4.29 mmol) in THF (50 mL) at room temperature. The mixture
was stirred for 25 h and the precipitated triethylaminehydrochlo-
ride was filtered off. The solvent was evaporated and the product
purified by column chromatography with benzene. The powdery
product was crystallized from acetonitrile. Yield: 1.71 g (78%). mp:
125 ◦C. Anal. cal. for C13H13N4O3P3Cl4; C: 30.74; H: 2.58; N: 11.03.
Found; C: 30.70; H: 2.45; N: 11.07%. FTIR (KBr, cm−1): ꢀ 3092; 3076
2.2.9. 4ꢀ,4ꢀ,6ꢀ,6ꢀ-Tetra(4-morpholinyl)-3-(2-furanylmethyl)-6-
(methoxy)-3H,4H-spiro{[1,3,2-benzoxazaphosphinine]-2,2ꢀ-
[1,3,5,2,4,6]triazatriphosphinine]}
(6b)
The work-up procedure was similar to that of compound 5b,
using 6 (1.20 g, 2.36 mmol), morpholine (2.47 mL, 28.00 mmol) and
triethylamine (1.33 mL). Yield: 0.90 g (54%). mp: 157 ◦C. Anal. cal.
for C29H45N8O7P3; C: 49.01; H: 6.34; N: 15.77. Found; C: 49.31; H:
6.30; N: 15.56%. FTIR (KBr, cm−1): ꢀ 3093; 3075 (C–H arom.), 2962;

N. Asmafiliz et al. / Spectrochimica Acta Part A 86 (2012) 214–223
217
2848 (C–H aliph.), 1255; 1187 (P N). APIES-MS (based on 35Cl, Ir
process and the Uiso(H) values were constrained to 1.2Ueq(carrier
atom) for CH and CH2 and 1.5Ueq(carrier atom) for CH3 groups.
%): m/z = 711 ([M]+, 100%).
2.2.10. 4ꢀ,4ꢀ,6ꢀ,6ꢀ-Tetra(1,4,7-dioxazonan-7-yl)-3-(2-
furanylmethyl)-8-(methoxy)-3H,4H-spiro{[1,3,2-
3. Results and discussion
benzoxazaphosphinine]-2,2ꢀ-[1,3,5,2,4,6]triazatriphosphinine]}
(5c)
3.1. Synthesis
The new N/O donor-type bidentate ligands (3 and 4) have been
prepared by the reduction of the corresponding Schiff bases (1
and 2) with NaBH4. The reactions of equal amounts of N3P3Cl6
with the N/O bifunctional ligands (3 and 4) produce N-furan-2-
yl-methyl substituted monospirocyclotriphosphazenes (5 and 6)
in THF. Scheme 1 illustrates the reaction pathway of N3P3Cl6 with
the ligands for providing a better understanding of the nucleophilic
replacement reactions. All the reactions of N3P3Cl6 with the biden-
tate ligands (3 and 4) appear to be regioselective because only the
spirocyclic arrangements are favored. The reactions of 5 and 6 with
excess pyrrolidine, morpholine, and DASD give tetrapyrrolidino (5a
and 6a), morpholino (5b and 6b), and 1,4-dioxa-8-azaspiro [4,5]
deca (5c and 6c) spirocyclotriphosphazenes, respectively.
Data from the microanalyses, FTIR, APIES-MS and NMR are con-
sistent with the proposed structures of the compounds. While the
mass spectra of 5a, 5b, 5c, 6a, 6b, and 6c showed the molecular ion
(M+) peaks, the protonated molecular ion (MH+) peaks appear in
the spectra of 5 and 6.
The mixture of 5 (0.62 g, 1.22 mmol), triethylamine (0.69 mL)
and DASD (1.88 mL, 14.64 mmol) in THF (150 mL) was refluxed for
30 h. The product was purified by column chromatography with
benzene-THF (1:1). Yield: 0.69 g (60%). mp: 181–183 ◦C. Anal. cal.
for C41H61N8O11P3; C: 52.67; H: 6.58; N: 11.99. Found; C: 52.47; H:
6.53; N: 11.78%. FTIR (KBr, cm−1): ꢀ 3071; 3056 (C–H arom.), 2954;
2844 (C–H aliph.), 1205; 1156 (P N). APIES-MS (based on 35Cl, Ir
%): m/z = 935 ([M]+, 100%).
2.2.11. 4ꢀ,4ꢀ,6ꢀ,6ꢀ-Tetra(1,4,7-dioxazonan-7-yl)-3-(2-
furanylmethyl)-6-(methoxy)-3H,4H-spiro{[1,3,2-
benzoxazaphosphinine]-2,2ꢀ-[1,3,5,2,4,6]triazatriphosphinine]}
(6c)
The work-up procedure was similar to that of compound 5c,
using 6 (0.75 g, 1.48 mmol), DASD (2.27 mL, 18.00 mmol) and tri-
ethylamine (0.83 mL). Yield: 0.87 g (63%). mp: 168 ◦C. Anal. cal. for
C
41H61N8O11P3; C: 52.67; H: 6.58; N: 11.99. Found; C: 52.44; H:
6.53; N: 12.03%. FTIR (KBr, cm−1): ꢀ 3091; 3075 (C–H arom.), 2954;
2846 (C–H aliph.), 1215; 1174 (P N). APIES-MS (based on 35Cl, Ir
%): m/z = 935 ([M]+, 100%).
3.2. NMR and FTIR spectroscopy
The 31P NMR spectral data of all the compounds refer to the
spiro structures. The spin systems of the phosphazenes are inter-
preted as AX2 for 5, 5b, 5c, 6, 6b, and 6c, and AB2 for 5a and 6a,
give rise to one triplet and one doublet in the 31P NMR spectra.
These findings are consistent with the two kinds of P atoms present
2.3. X-ray crystallography
The suitable crystals of compounds 5, 5a, and 6 were crystal-
lized from acetonitrile at room temperature. The crystallographic
data are given in Table 1. Crystallographic data were recorded on a
Bruker Kappa APEXII CCD area-detector diffractometer using Mo K␣
2
in the cyclophosphazene ring. The coupling constants, JPP, are in
the range of 47.5–58.4 Hz. The chemical shift values, ␦P(OArN), of
aminophosphazenes (5a–6c) are larger than those of chloro deriva-
tives (5 and 6, Table 2), and they are consistent with the findings of
N/O donor-type spirocyclotriphosphazenes [41].
˚
radiation (ꢁ=0.71073 A) at T = 100(2) K for 5 and 6 and at T = 294(2) K
for 5a. Absorption corrections by multi-scan [39] were applied.
Structureswere solved by direct methods and refined by full-matrix
least squares against F2 using all data [40]. All non-H atoms were
refined anisotropically. H atom positions were calculated geometri-
In all the spirocyclotriphosphazenes, the 13C and 1H NMR sig-
nals are assigned on the basis of chemical shifts, multiplicities,
and coupling constants (Tables 3 and 4). The assignments are
made undoubtedly by the HSQC and HMBC (Fig. S1 Supplementary
˚
˚
˚
cally at distances of 0.93 A (CH), 0.97 A (CH2) and 0.96 A (CH3) from
the parent C atoms; a riding model was used during the refinement
Table 1
Crystallographic data for 5, 5a, and 6.
5
5a
6
Empirical formula
Fw
C13H13N4O3P3Cl4
508.01
C29H45N8O3P3
646.64
C13H13N4O3P3Cl4
507.98
Crystal system
Space group
a (Å)
triclinic
P-1
triclinic
P-1
triclinic
P-1
8.0555(2)
9.1964(2)
13.3201(3)
92.392(2)
98.199(3)
93.540(2)
973.55(4)
2
9.9991(8)
10.9588(9)
15.4961(12)
94.439(3)
103.033(3)
99.123(3)
1622.0(2)
2
7.7561(2)
8.8500(2)
14.6474(3)
97.107(3)
96.818(3)
93.389(2)
987.86(4)
2
b (Å)
c (Å)
˛ (◦)
ˇ (◦)
ꢂ (◦)
V (Å3)
Z
ꢃ (cm−1
)
0.878 (Mo K␣
1.733
)
0.228 (Mo K␣
1.324
)
0.866 (Mo K␣
1.708
)
ꢄ (calcd) (g cm−1
)
Number of reflections total
Number of reflections unique
17371
4886
28,194
8022
29,132
4914
Rint
2ꢅmax (◦)
0.0431
56.90
0.0403
56.80
0.0780
56.64
Tmin/Tmax
0.8630/0.9146
245
0.0235
0.9265/0.9453
389
0.0570
0.7515/0.8811
245
0.0365
Number of parameters
R[F2 > 2ꢆ(F2)]
wR
0.0643
0.1338
0.0929

218
N. Asmafiliz et al. / Spectrochimica Acta Part A 86 (2012) 214–223
Table 2
31P NMR (decoupled) spectral data of the compounds (ı are reported in ppm; J values in Hz].a
Compound
Spin system
ı (ppm)
2JPP
P(OArN)
PCl2
P(NR)2
5
6
AX2
AX2
AB2
AB2
AX2
AX2
AX2
AX2
5.82
5.25
23.98
23.87
–
–
58.4
57.5
47.5
49.1
48.5
49.3
49.1
49.5
5a
6a
5b
6b
5c
6c
17.88
17.57
16.62
16.27
16.16
15.70
–
–
–
–
–
–
19.36
19.09
22.04
21.75
21.71
21.40
a
31P NMR measurements in CDCl3 solutions at 293 K.
Material). As examples, the HSQC and HMBC spectra of 6a are illus-
trated in Fig. S1 (Supplementary Material). The expected carbon
signals are assigned from the 13C NMR spectra of all the phosp-
hazenes. The average ␦-shift values of C7, C8, NCH2 (pyrr), NCH2
(mor), and NCH2 (DASD) carbons are 48.50, 44.36, 46.10, 44.32,
and 42.53 ppm, respectively. Thus, the aromatic and NCH2 carbon
signals of the spirocyclotriphosphazenes are verified by the HSQC
and HMBC spectra (Fig. S1 Supplementary Material). The charac-
teristic OCH3 carbon peaks of all the phosphazenes are observed
as singlets at ca. 55.98 ppm. In the 13C NMR spectra of pyrrolidino
(5a and 6a), morpholino (5b and 6b), and DASD (5c and 6c) sub-
stituted monospirocyclotriphosphazenes, the geminal substituents
show two groups of NCH2CH2 (pyrr and DASD), OCH2 (mor and
DASD), and OCO (DASD) signals with small separations, indicating
that the two geminal groups are not equivalent to each other (Fig.
S2 Supplementary Material). The same situation is also observed
for NCH2 carbons (except 5b, 6b, and 6c). The couplings 3JPC1 2JPC2
,
(except 6c), 3JPC3, and 3JPC9 of the monospirocyclotriphosphazenes
2
are observed as doublets. In addition, the JPC2 values of 6a and
2
6b are larger than those of 5a, 5b, and 5c. Additionally, the JPC8
values are assigned in the range of 4.0–5.3 Hz, except morpholino
derivatives (5b and 6b) (Table 3).
Afterwards, the 1H NMR spectra of the compounds are
evaluated. The benzylic H7 protons give rise to doublets for
monospirocyclotriphosphazenes, except 6b and 6c, indicating that
these protons are equivalent to each other. The geminal H8 pro-
tons of all the phosphazenes also give doublets, and the 3JPH values
are between 4.6 and 13.8 Hz. The characteristic OCH3 peaks of the
Table 3
13C NMR (decoupled) spectral data for the phosphazene derivatives. (ı are reported in ppm and J values in Hz].
5
6
4
10
11
3
1
7
2
X
9
12
8
O
N
O
P
N
N
Compound
5
6
5a
6a
5b
6b
5c
6c
C1
C2
C3
125.00
3JPC = 7.3
149.81
2JPC = 5.2
149.30
3JPC = 6.9
124.34
112.32
118.16
48.20
124.14
3JPC = 7.1
149.73
2JPC = 5.1
119.50
3JPC = 8.5
114.08
156.01
111.70
48.49
125.94
3JPC = 7.8
149.39
2JPC = 6.3
152.51
3JPC = 8.2
122.26
111.61
118.90
48.46
124.52
3JPC = 7.8
152.40
2JPC = 9.4
119.24
3JPC = 7.7
113.32
154.66
111.83
48.82
126.12
3JPC = 7.7
149.29
2JPC = 6.1
151.51
3JPC = 7.0
122.77
111.21
118.40
48.40
124.58
3JPC = 8.0
151.36
2JPC = 8.4
119.06
3JPC = 7.7
113.54
155.04
111.95
48.54
125.81
3JPC = 7.7
149.45
2JPC = 6.2
151.81
3JPC = 8.2
122.28
111.58
118.43
48.40
124.79
3JPC = 7.8
151.63
119.30
3JPC = 7.6
113.47
154.82
111.67
48.72
C4
C5
C6
C7
2JPC = 1.7
44.33
2JPC = 2.0
44.38
C8
C9
44.63
44.68
44.64
43.37
44.44
44.43
2JPC = 5.2
139.47
3JPC = 7.9
109.18
110.44
142.91
56.46
2JPC = 5.3
143.51
3JPC = 8.1
109.28
110.48
142.96
55.71
2JPC = 4.4
140.67
3JPC = 8.3
108.24
110.15
142.37
56.52
2JPC = 4.0
145.96
3JPC = 7.5
107.92
110.20
141.86
55.72
2JPC = 4.7
141.26
3JPC = 8.1
108.37
110.11
142.22
56.27
2JPC = 4.6
145.32
3JPC = 8.0
108.44
110.13
142.26
55.63
140.67
3JPC = 8.3
108.44
110.23
142.31
55.89
67.19
67.21
–
145.12
3JPC = 7.7
108.53
110.24
142.37
55.62
67.15
67.23
–
C10
C11
C12
OCH3
OCH2
–
–
–
–
64.15
64.19
64.20
64.28
N–CH2–CH2
–
–
25.58
3JPC = 8.4
26.62
3JPC = 8.3
46.04
46.13
–
26.32
3JPC = 8.0
26.38
3JPC = 8.1
46.04
46.18
–
35.52
35.55
3JPC = 6.7
35.60
3JPC = 6.4
35.56
3JPC = 6.9
42.54
3JPC = 6.5
42.42
NCH2
OCO
–
–
–
–
44.64
–
44.00
–
42.63
107.62
107.86
107.59
107.77

Table 4
1H NMR spectral data for 2–6, 5a–5c, and 6a–6c [s: singlet, d: doublet, dd: doublet of doublets, m: multiplet, and bp: broad peak].a
Compound[ı(ppm), J(Hz)]
2
3
4
5
6
5a
6a
5b
6b
5c
6c
H3
H4
6.92 (d, 1H)
3JH3–H4 = 9.0
6.95 (dd, 1H)
3JH3–H4 = 9.0
4JH4–H6 = 2.6
–
–
6.81 (d, 1H)
3JH3–H4 = 8.8
6.76 (dd, 1H)
3JH3–H4 = 8.8
4JH4–H6 = 2.9
–
–
6.99 (d, 1H)
3JH3–H4 = 8.9
6.77 (dd, 1H)
3JH3–H4 = 8.9
4JH4–H6 = 2.5
–
–
6.81 (d, 1H)
3JH3–H4 = 7.9
6.67(d, 1H)
3JH3–H4 = 7.9
4JH4–H6 = 2.9
–
–
6.82 (d, 1H)
3JH3–H4 = 8.8
6.70 (dd, 1H)
3JH3–H4 = 8.8
4JH4–H6 = 2.5
–
–
6.89 (d, 1H)
3JH3–H4 = 8.8
6.70 (dd, 1H)
3JH3–H4 = 8.8
4JH4–H6 = 2.8
–
6.85 (dd, 1H)
3JH4–H5 = 8.1
4JH4–H6 = 1.4
6.78 (dd, 1H)
3JH4–H5 = 8.1
3JH5–H6 = 7.6
6.68 (dd, 1H)
3JH5–H6 = 7.6
4JH4–H6 = 1.4
3.87 (s, 2H)
6.88 (d, 1H)
6.75(d, 1H)
6.81(d, 1H)
6.77 (d, 1H)
3JH4–H5 = 8.2
3JH4–H5 = 8.0
3JH4–H5 = 8.1
3JH4–H5 = 8.5
H5
H6
7.01 (dd, 1H)
3JH4–H5 = 8.2
3JH5–H6 = 7.8
6.65 (d, 1H)
3JH5–H6 = 7.8
6.85(dd, 1H)
3JH4–H5 = 8.0
3JH5–H6 = 7.7
6.56 (d, 1H)
3JH5–H6 = 7.7
6.93 (dd, 1H)
3JH4–H5 = 8.1
3JH5–H6 = 7.7
6.60 (d, 1H)
3JH5–H6 = 7.7
6.88 (dd, 1H)
3JH4–H5 = 8.5
3JH5–H6 = 7.8
6.59 (d, 1H)
3JH5–H6 = 7.8
6.80 (d, 1H)
6.58 (d, 1H)
6.57 (d, 1H)
6.51(d, 1H)
6.54 (d, 1H)
6.53 (d, 1H)
4JH4–H6 = 2.6
4JH4–H6 = 2.9
4JH4–H6 = 2.5
4JH4–H6 = 2.9
4JH4–H6 = 2.5
4JH4–H6 = 2.8
H7
H8
8.35 (s, 1H)
4.78 (s, 2H)
3.83 (s, 2H)
3.94 (s, 2H)
4.27 (d, 2H)
3JPH = 11.8
4.29 (d, 2H)
3JPH = 13.8
4.26 (d, 2H)
3JPH = 3.5
4.20 (d, 2H)
3JPH = 13.8
4.24 (d, 2H)
3JPH = 8.4
4.18 (d, 2H)
3JPH = 14.6
4.26 (d, 2H)
3JPH = 7.3
4.24 (d, 2H)
3JPH = 2.4
4.20 (s, 2H)
4.18 (d,
4.17 (s, 2H)
2H)3JPH = 2.5
4.21 (d, 2H)
3JPH = 8.3
4.00 (s, 2H)
4.29 (d, 2H)
4.21 (d, 2H)
4.27 (d, 2H)
4.20 (d, 2H)
3JPH = 5.7
3JPH = 7.5
3JPH = 5.2
3JPH = 4.6
H10
H11
H12
6.30 (m, 1H)
3JH10–H11 = 3.3
4JH10–H12 = 1.9
6.38 (m, 1H)
3JH10–H11 = 3.3
3JH11–H12 = 3.2
7.40 (m, 1H)
3JH11–H12 = 3.2
4JH10–H12 = 1.9
3.79 (s, 3H)
–
6.27 (m, 1H)
3JH10–H11 = 3.1
4JH10–H12 = 1.8
6.36 (m, 1H)
3JH10–H11 = 3.1
3JH11–H12 = 2.5
7.40 (m, 1H)
3JH11–H12 = 2.5
4JH10–H12 = 1.8
3.90 (s, 3H)
–
6.23 (d, 1H)
6.35 (m, 1H)
3JH10–H11 = 3.2
4JH10–H12 = 1.8
6.35 (m, 1H)
3JH10–H11 = 3.2
3JH11–H12 = 2.7
7.40 (m, 1H)
3JH11–H12 = 2.7
4JH10–H12 = 1.8
3.88 (s, 3H)
–
6.37 (m, 1H)
6.37 (m, 1H)
7.41 (m, 1H)
6.31 (m, 1H)
6.28 (m, 1H)
6.30 (m, 1H)
7.35 (m, 1H)
6.31 (m, 1H)
6.28 (m, 1H)
3JH10–H11 = 3.1
4JH10–H12 = 1.7
6.32 (m, 1H)
3JH10–H11 = 3.1
3JH11–H12 = 2.8
7.35 (m, 1H)
3JH11–H12 = 2.8
4JH10–H12 = 1.7
3.72 (s, 3H)
6.31 (m, 1H)
6.31 (m, 1H)
7.39 (m, 1H)
6.32 (m, 1H)
3JH10–H11 = 3.2
3JH10–H12 = 1.5
6.33 (m, 1H)
3JH10–H11 = 3.2
3JH11–H12 = 2.8
7.39 (m, 1H)
3JH11–H12 = 2.8
3JH10–H12 = 1.5
3.74 (s, 3H)
3JH10–H11 = 3.1
3JH10–H11 = 3.1
6.36 (m, 1H)
3JH10–H11 = 3.1
3JH11–H12 = 2.0
7.41 (m, 1H)
3JH11–H12 = 2.0
6.31 (m, 1H)
6.34 (m, 1H)
3JH10–H11 = 3.1
3JH11–H12 = 1.9
7.38 (m, 1H)
3JH11–H12 = 1.9
3JH11–H12 = 1.2
7.35 (m, 1H)
3JH11–H12 = 1.2
OCH3
OCH2
3.76 (s, 3H)
–
3.76 (s, 3H)
–
3.79 (s, 3H)
–
3.73 (s, 3H)
–
3.82 (s, 3H)
3.65 (t, 8H)
3JHH = 8.9
3.69 (t, 8H)
3JHH = 8.4
–
3.82 (s, 3H)
3.96 (s, 8H)
3.62 (t, 8H)
3.96 (s, 8H)
3.97 (s, 8H)
3JHH = 8.5 3.65 (t, 3.97 (s, 8H)
8H) 3JHH = 9.1
N–CH2–CH2
NCH2
–
–
–
–
–
–
–
–
–
1.77 (t, 8H)
3JHH = 6.9
1.76 (t, 8H)
3JHH = 6.7
–
1.63–1.70
(m, 16H)
1.65 (t, 8H)
3JHH = 5.6
1.69 (t, 8H)
3JHH = 5.6
3.23–3.28
(m, 16H)
1.80 (t, 8H)
1.82 (t, 8H)
3JHH = 6.1
3JHH = 7.6
–
3.13 (m,8H)
3.19 (m,8H)
3.10 (m,8H)
3.18 (m,8H)
3.12–3.26
(m, 16H)
3.07–3.19
(m, 16H)
3.06–3.40
(m, 16H)
OH
12.85 (bp, 1H)
a
Numberings of protons are given in Table 3.

220
N. Asmafiliz et al. / Spectrochimica Acta Part A 86 (2012) 214–223
Fig. 1. ORTEP-3 [48] drawing of 5 with the atom-numbering scheme. Displacement
Fig. 2. ORTEP-3 [48] drawing of 5a with the atom-numbering scheme. Displacement
ellipsoids are drawn at the 50% probability level.
ellipsoids are drawn at the 50% probability level.
Table 5
ligands (2, 3 and 4) and all the phosphazenes are observed as sin-
glets at ca. 3.79 ppm. In the 1H NMR spectra of pyrrolidino (5a and
6a), morpholino (5b and 6b), and DASD (5c and 6c) substituted
monospirocyclotriphosphazenes, the geminal substituents show
two groups of NCH2 signals with small separations, as it is observed
in the 13C NMR spectra. The same situation is also observed for
NCH2CH2 (pyrr and DASD) and OCH2 (mor and DASD) protons (Fig.
S2 Supplementary Material). The average values of JHH and JHH
for benzene rings are 8.2 Hz and 2.5 Hz, respectively. In addition,
the 3JHH values of furan rings of all the compounds are very smaller
than those of benzene rings (Table 4).
The FTIR spectra of the ligands (3 and 4) and all the phos-
phazenes show two medium intensity absorption bands at
3117–3071 cm−1 and 3076–3046 cm−1 attributed to asymmetric
and symmetric stretching vibrations of the Ar-H protons. In addi-
tion, all the phosphazenes exhibit intense bands between 1255
and 1156 cm−1 related to ꢀP–N bonds of the phosphazene rings
[42]. The characteristic ꢀNH stretching bands of N-alkyl(or aryl)-
o-hydroxybenzylamines (3301 cm−1 for 3 and 3289 cm−1 for 4)
disappear in the FTIR spectra of monospirocyclotriphosphazenes.
Selected bond lenghts (Å) and angles (◦) for 5, 5a, and 6.
5
5a
6
P1–N1
P1–N3
P1–N4
P1–O1
P2–N1
P2–N2
P3–N2
P3–N3
N1–P1–N3
N1–P2–N2
N2–P3–N3
N4–P1–O1
P1–N1–P2
P2–N2–P3
P1–N3–P3
N5–P2–N6
N7–P3–N8
1.605(1)
1.592(1)
1.613(3)
1.587(1)
1.565(1)
1.589(1)
1.579(1)
1.574(1)
1.583(2)
1.577(2)
1.641(2)
1.621(2)
1.600(2)
1.589(2)
1.590(2)
1.606(2)
1.604(2)
1.594(2)
1.631(2)
1.583(2)
1.557(2)
1.587(2)
1.584(2)
1.567(2)
3
4
116.2(1)
116.7(1)
115.8(1)
116.4(1)
100.5(1)
122.1(1)
123.6(1)
122.2(1)
100.6(1)
101.4(2)
114.8(1)
118.8(1)
120.5(1)
101.7(1)
123.1(1)
119.5(1)
121.5(2)
–
119.4(1)
120.3(1)
102.7(1)
123.7(1)
118.8(1)
122.7(1)
–
–
–
In the phosphazene rings of 5, 5a, and 6, the endocyclic
P–N bond lengths are in the ranges of 1.565(1)–1.605(1) A (for
5), 1.577(2)–1.606(2) A (for 5a), and 1.557(2)–1.604(2) A (for
6) and there are regular variations with the distances from
P1: P1–N1 ≈ P1–N3 > P2–N2 ≈ P3–N2 > P2–N1 ≈ P3–N3. In addition,
˚
As expected, ꢀPCl
and ꢀPCl
bands emerge at 564 and
2(asym)
2(sym)
˚
˚
527 cm−1 for 5, and 581 and 519 cm−1 for 6.
3.3. X-ray structures of 5, 5a, and 6.
The molecular and solid-state structures of 5, 5a, and 6 along
with the atom-numbering schemes, are depicted in Figs. 1–3,
respectively. The selected bond lengths and angles are listed in
Table 5 and hydrogen-bond data are given in Table 6. The phos-
phazene rings of 5 and 6 are nearly planar (Fig. S3a Supplementary
Material); ϕ2 = −22.6(0.8)◦, ꢅ2 = 99.6(0.8)◦, Fig. S5a (Supplemen-
tary Material); ϕ2 = −147.8(5.7)◦, ꢅ2 = 15.7(1.5)◦, while that of 5a
is not planar, and it is in twisted-boat conformation (Fig. S4a
Supplementary Material); ϕ2 = −153.0(0.6)◦, ꢅ2 = 123.2(0.5)◦ hav-
˚
˚
ing total puckering amplitudes QT of 0.067(1) A (for 5), 0.178(2) A
˚
(for 5a) and 0.050(1) A (for 6) [43]. In tetrachloro monospirocy-
clotriphosphazene (5), the six-membered ring P1/O1/C1/C6/C7/N4
is in boat conformation (Fig. S3b Supplementary Material);
◦
QT = 1.221(3) A, ϕ2 = 128.3(1) , ꢅ2 = 77.0(1)◦, and in 5a and 6, the
˚
six-membered rings, P1/O1/C1/C6/C7/N4, are in twisted con-
˚
formations (Fig. S4b Supplementary Material); QT = 0.705(2) A,
ϕ2 = 44.9(3)◦, ꢅ2 = 35.5(2)◦ an◦d Fig. S5b (Supplementary Material);
Fig. 3. ORTEP-3 [48] drawing of 6 with the atom-numbering scheme.Displacement
ellipsoids are drawn at the 50% probability level.
QT = 1.471(4) A, ϕ2 = −66.7(1) , ꢅ2 = 122.1(1)◦.
˚

N. Asmafiliz et al. / Spectrochimica Acta Part A 86 (2012) 214–223
221
Table 6
Hydrogen-bond geometries (Å,◦) for 5, 5a, and 6.
D-H. . .A
D-H
H. . .A
D. . .A
D-H. . .A
5
5a
C8–H8A...N3
C8–H8A...N3
C12–H12...O3i
C8–H8B...N3
0.97
0.97
0.93
0.97
2.61
2.63
2.51
2.72
3.092(4)
3.114(4)
3.291(4)
3.144(3)
111
110
142
107
6
Symmetry code: (i) x + 1, y, z.
the average endocyclic P–N bond lengths in phosphazene rings are
1.584(1) and 1.591(2), and 1.582(2) A, which are shorter than the
On the other hand, compounds 5, 5a, and 6 have intramolecular
C–H. . .N hydrogen bonds (Table 6) between furanyl-CH2 and nitro-
gen atoms of phosphazene rings (Figs. 1–3), indicating that the CH2
protons of these compounds have asidic properties. In compound
5a, intermolecular C–H. . .O hydrogen bonds link the molecules
into a two-dimensional network. The ␲ . . . ␲ contacts between
the benzene rings, Cg1· · ·Cg1i [for 5, (i) 2 − x, −y, −z], Cg1· · ·Cg1ii
[for 5a, (ii) 1 − x, 2 − y, 1 − z] and Cg1· · ·Cg1iii [for 6, (iii) 2 − x, 1 − y,
1 − z] [where Cg1 is the centroid of the ring (C1–C6)] with the
˚
exocyclic P–N bonds of 5, 5a, and 6, respectively (Table 5). It is note-
worthy that, the P–N bonds are thought to be the most intriguing
bonds in chemistry. In recent years, natural bond orbital (NBO) and
topological electron density analyses have been used to investi-
gate the electronic structures of the phosphazenes [44]. The two
kinds of phosphazene bonding alternatives are suggested, namely
negative hyperconjugation [44,45] and ionic bonding, which are
scrutinized using NBO. The estimations imply that the ionic com-
ponent is the dominant feature. However, these two alternatives
are not mutually exclusive and in fact, they are both important. The
major donor-acceptor interactions (NBO overlaps) are depicted in
Fig. 4 for N3P3Cl6, taken from reference 44. In addition, the pres-
ence of the negative hyperconjugation strongly contribute to the
multiple-bond character, and the electron withdrawing or releas-
ing substituents bonding to the P atoms increase or decrease the
negative hyperconjugation of the exocyclic and endocyclic P–N
bonds. Consequently, the shortenings of the endo- and exocyclic
P–N bonds in 5, 5a, and 6 could be explained by the negative hyper-
conjugation concept.
In monospirocyclic phosphazenes (5, 5a, and 6), the endocylic
N1–P1–N3 (˛) angles are considerably narrowed, while the exo-
cyclic O1–P1–N4 (˛ꢀ) angles are hardly changed with respect to
the corresponding values in the “standard” compound, N3P3Cl6
(Table 5). In N3P3Cl6, the ˛ and ˛ꢀ angles are 118.3(2) and 101.2(1)◦,
respectively [46]. It is suggested that the negative hyperconjugation
and substituent-dependent charge at the P centers play an impor-
tant role in the variations of exocyclic and endocyclic angles. The
charge separations between the P and N atoms in the phosphazene
rings differ significantly depending on the electron withdrawing or
releasing capacities of the substituents bonded to the P atoms [47].
The narrowing of the endocyclic NPN (˛) angles in 5, 5a, and 6 could
also be explained by the negative hyperconjugation concept.
˚
˚
centroid–centroid distances of 3.532(1) A (for 5), 3.939(2) A (for 5a),
˚
and 3.899(1) A (for 6). In compound 5a, there is also a ␲ . . . ␲ con-
tact between the furan rings, Cg2· · ·Cg2iv [(iv) −x, 1 − y, 1 − z, where
Cg2 is the centroid of the ring (O2/C9–C12)] with centroid–centroid
˚
distance of 3.534(2) A.
3.4. Antibacterial activity
The pyrrolidine and the morpholine derivatives, and some of the
organic compounds containing furan side groups have widespread
structural features of natural and synthetic biologically designed
active molecules, and they can be used for pharmaceutical and
medicinal purposes [43–51]. The herbicidal, plant growth regula-
tory, fungicidal, anti-microbial, anti-inflammatory, and anti-cancer
activities of these heterocycles have been known in the literature
[52–55]. So, furan, pyrrolidine, and morpholine are especially cho-
sen as the substituents in this study.
The antibacterial activity of 5a, 5b, 5c, 6a, 6b, and 6c are
determined against eight different microorganisms [Staphylococ-
cus aureus ATCC 25923 (G+), Pseudomonas aeruginosa ATCC 27853
(G−), Escherichia coli ATCC 25922 (G−), Bacillus subtilis ATCC 6633
(G+), Bacillus cereus NRRL-B-3711 (G+), and Enterococcus faecalis
ATCC 292112 (G+), Candida albicans ATCC 10231 and Candida tropi-
calis ATCC 13803]. All the compounds with 5000 ␮M and 10,000 ␮M
concentrations exhibit no antimicrobial activity. Moreover,
ꢀ
Fig. 4. Major NBO overlaps in [N3P3Cl6]: (a) Nlp2 → ó*PCl, (b) Nlp1 → ó*PN , and (c) Nlp1 → ó*PN
.

222
N. Asmafiliz et al. / Spectrochimica Acta Part A 86 (2012) 214–223
Fig. 5. Agarose gel electrophoresis of pBR322 plasmid DNA with the compounds 5a, 5b, 5c, 6a, 6b, and 6c of different concentrations. Lane 1: untreated plasmid DNA as a
control, lanes 2–6: plasmid DNA treated with different concentrations of compound ranging from (5000, 2500, 1250, 625, and 125 ␮M).
pyrrolidine, morpholine, DASD, and 2-furan-2-yl-methylamine,
which are the substituents of the N/O spirocyclotriphosphazenes,
are tested against the same bacteria and fungi. However, no activ-
ities have been observed against the tested bacteria and fungi. The
reason for that could be protective outer membrane of microorgan-
isms. All of the antimicrobial agents demonstrate selective toxicity
towards the bacteria and fungi. Antibiotics or any antimicrobial
agent may inhibit protein, DNA, and folic acid syntheses. The resis-
tance to antibiotics or the compounds may attribute to the inability
of the molecules to enter into the cell or after entering export
from the cell. In addition, the resistance of microorganisms may
be depend on the production of an enzyme by the cell to inactivate
the compounds.
supercoiled plasmid DNA decreases with the increasing concentra-
tions of 5b. Moreover, the effect of 6b is maximum at the highest
concentration of the compound. When the DNA cleavage assay for
the compound 5c is examined, the mobility of Form I decreases with
the decreasing concentrations of the compound. The intensity of
Form II diminishes with the decreasing concentrations of 5c (lanes
2–6). It can be seen that Form I of supercoiled plasmid DNA dimin-
ishes gradually with decreasing concentrations of 5c, whereas the
intensity of Form III increases. It appears that the compound cleaves
the supercoiled Form I DNA to linear Form III. In the case of 6c, the
mobility of the supercoiled DNA slightly decreases with the increas-
ing concentrations of the compound. It is clearly understood that
compound 6c also causes the conformational changes on pBR322
plasmid DNA by cleaving supercoiled DNA to linear Form III, as
compound 5c.
3.5. Interactions of DNA with the compounds
Consequently, the activities of the compounds are considered to
be associated with their bindings with nucleotides in DNA, in which
they cause some changes in DNA conformations or damage to DNA.
Binding modes are possible intercalatives as all of the compounds
are aromatic and also have hydrogen bond donors/acceptors [58].
DNA is a very valuable target for the actions of many anticancer
agents. Therefore, it is very important to find out whether the com-
pound can bind to DNA, and the binding may suppress its function.
So, DNA targeted compounds offer opportunity for development of
new anticancer agents. The results obtained in this study could pro-
vide the important scientific knowledge for scientists interesting in
this field.
Agarose gel electrophoresis is used to demonstrate the confor-
mational change and damage caused to plasmid DNA, due to their
bindings with the compounds. When the plasmid DNA loads on
the gel in an electric field, the plasmid DNA will migrate towards
the positive electrode. The plasmid DNA is found in three different
forms, supercoiled circular form I, singly nicked relaxed circular
form II, and doubly nicked linear form III. Generally, the untreated
plasmid DNA migrates on the gel with two DNA bands, which form
I migrating faster and form II migrating slower. Form III takes place
in the middle when plasmid DNA restricted with the enzyme or
damage. Interactions with compounds may cause conformational
changes on the plamid DNA, and in mobility of DNA through gel
[56,57]. Therefore, gel electrophoresis has been carried out using
agarose gel in order to investigate the abilities of 5a, 5b, 5c, 6a,
6b, and 6c in effecting the plasmid DNA pBR322 DNA. Fig. 5 shows
the gel electrophoresis of plasmid pBR322 DNA after 24 h incuba-
tion in the presence of the compounds. The concentrations of the
compounds are 5000, 2500, 1250, 625, and 125 ␮M. It is observed
that all the compounds exhibit the effect in more or less, dose
dependent manner. In addition, compounds 5c and 6c exhibit dif-
ferent effects of all the concentrations on plasmid DNA than the
other tested compounds. In the case of 5a, the mobilities of the
bands of supercoiled DNA decrease slightly at concentration of
5000 ␮M. The intensities of these bands also decrease. In all the
concentrations, compound 6a has considerable effects on super-
coiled plasmid DNA, but the maximum effect is observed at the
concentration of 5000 ␮M. It is noteworthy that the effect of 5b is
highly noticable in three high concentrations. The intensity of the
4. Conclusions
The spirocyclotriphosphazene derivatives containing 1,3,2-
oxazaphosphorine rings are very limited in the literature. In
this study, new tetrachloro (5 and 6), tetrapyrrolidino (5a
and 6a), tetramorpholino (5b and 6b), and tetra(1,4-dioxa-8-
azaspiro[4,5]deca) (5c and 6c) monospirocyclotriphosphazenes
have been obtained. The molecular and solid-state structures of 5,
5a, and 6 are determined by X-ray crystallography, and the phos-
phazene rings of 5 and 6 are nearly in planar and the other one
is in twisted-boat conformations. In addition, the compounds 5a,
5b, 5c, 6a, 6b, and 6c have no antimicrobial activities. Interac-
tions between these compounds and pBR322 plasmid DNA show
that the compounds are effective in changing the mobility of
the DNA.

N. Asmafiliz et al. / Spectrochimica Acta Part A 86 (2012) 214–223
223
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