Syntheses, spectroscopic properties, crystal structures, biological activities, and DNA interactions of heterocyclic amine substituted spiro-ansa-spiro- and spiro-bino-spiro-phosphazenes

Article abstract of DOI:10.1016/j.ica.2013.07.023

The heterocyclic amine e.g. pyrrolidine, piperidine, morpholine and 1, 4-dioxa-8-azaspiro[4, 5]decane (DASD) substituted spiro-ansa-spiro (sas) 5a-5d and spiro-bino-spiro (sbs) 6a-6d phosphazenes were prepared by the replacement reactions of the Cl-atoms

Full text of DOI:10.1016/j.ica.2013.07.023

Inorganica Chimica Acta 406 (2013) 160–170
Contents lists available at ScienceDirect
Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Syntheses, spectroscopic properties, crystal structures, biological
activities, and DNA interactions of heterocyclic amine substituted
spiro-ansa-spiro- and spiro-bino-spiro-phosphazenes
a
a
a
b
b
Selen Bilge Koçak a, , Serhat Koçog˘lu , Aytug˘ Okumußs , Zeynel Kılıç , Aslı Öztürk , Tuncer Hökelek ,
⇑
Yag˘mur Öner c, Leyla Açık c
a Department of Chemistry, Ankara University, 06100 Tandog˘an-Ankara, Turkey
b Department of Physics, Hacettepe University, 06800 Beytepe-Ankara, Turkey
c Department of Biology, Gazi University, 06500 Beßsevler-Ankara, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
The heterocyclic amine e.g. pyrrolidine, piperidine, morpholine and 1,4-dioxa-8-azaspiro[4,5]decane
(DASD) substituted spiro-ansa-spiro (sas) 5a–5d and spiro-bino-spiro (sbs) 6a–6d phosphazenes were pre-
pared by the replacement reactions of the Cl-atoms in 3 and 4 with heterocyclic amines in dry THF
(Scheme 1). All of the phosphazene derivatives were characterized by elemental analysis, FTIR, MS, 1D
1H, 13C and 31P NMR and DEPT and 2D HSQC techniques. The crystal structures of fully morpholine substi-
tuted sas 5c and sbs 6c phosphazenes were verified by X-ray diffraction analysis. The relationships
Received 17 January 2013
Received in revised form 21 June 2013
Accepted 14 July 2013
Available online 22 July 2013
Keywords:
between exocyclic OPN bond angles (a0) and dPOPN shifts, and the correlation of
(dP) or dPOPN shifts were presented. The phosphazene derivatives (3, 4, 5a–5d and 6a–6d) were sub-
D(P–N) values and
Spiro-ansa-spiro-phosphazenes
Spiro-bino-spiro-phosphazenes
Spectroscopy
Crystal structure
DNA interactions
D
jected to antimicrobial activity against six pathojen bacteria and two yeast strains. Fully pyrrolidine
substituted sbs 6a was found to be quite active against yeast strain Candida tropicalis. In addition, the nat-
ure of the interactions of these compounds with pBR322 plasmid DNA was investigated, and the results
displayed that partly substituted sas 3 and sbs 4 caused to cleave the DNA.
Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction
alized nucleophilic reagents, such as primary [9] and secondary
[10] amines, polyamines [11] and phenols [12]. Among these, the
Cyclophosphazenes are versatile inorganic ring systems com-
posed of a backbone that contains the repeating unit [NPR2]n
(n = 3, 4, 5, . . .) with trivalent nitrogen and pentavalent phosphorus
atoms and two organic, inorganic and organometallic side groups
(R), covalently linked to each phosphorus atom [1]. They have di-
verse applications as hydraulic fluids or lubricants [2], ionic liquids
[3], liquid crystalline materials [4], flame retardant additives to or-
ganic polymers [5], light-emitting diodes [6], electrolytes for lith-
ium-ion batteries [7], and monomers for inorganic high
molecular weight polymers [8]. The hexachlorocyclotriphospha-
zene (cyclic trimer, N3P3Cl6) has served as an important starting
compound in the field of phosphazene chemistry. One to all six
Cl-atoms in N3P3Cl6 can be substituted by the same or different
groups. Therefore, there is no limit for the number of PN/substitu-
ent combinations. A wide range of cyclotriphosphazene derivatives
with different side groups and with diverse application potential
has been prepared using the replacement of highly reactive Cl-
atoms at P-atoms in N3P3Cl6 by a variety of –NH or –OH function-
substitution reactions of N3P3Cl6 with difunctional nucleophiles
have received a lot of attention in recent two decades due to the
formation of numerous structural isomers and stereoisomers. The
reactions involving difunctional reagents with N3P3Cl6 have been
found to proceed via different routes. The interactions of one of
the Cl-atoms with one functional group of the nucleophile give
open-chain dangling precursors [13]. The replacement of two gem-
inal and two non-geminal Cl-atoms yields spiro- and ansa-substi-
tuted isomeric products, respectively [14]. The intermolecular
reactions between the heterofunctional dangling precursor with
a Cl-atom of the next molecule of N3P3Cl6 produce bino-architec-
ture composed of two chlorocyclotriphosphazene moieties bridged
with the respective difunctional reagent [15]. In general, the nature
of the products formed in these reactions is controlled by a variety
of factors such as the chain lengths of the reacting functional
groups, reaction temperature and the solvent polarities [16]. The
number of spiro-ansa-, spiro-bino- or ansa-bino-phosphazene deriv-
atives has been relatively limited in the literature [17–19]. On the
other hand, some of the cycloptrihosphazene derivatives substi-
tuted heterocyclic amines such as aziridine, pyrrolidine and mor-
pholine have revealed significant antimicrobial and antitumor
⇑
Corresponding author. Tel.: +90 312 2126720 1021; fax: +90 312 2232395.
E-mail address: sbilge@science.ankara.edu.tr (S. Bilge Koçak).
0020-1693/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ica.2013.07.023

S. Bilge Koçak et al. / Inorganica Chimica Acta 406 (2013) 160–170
161
NaBH
B O .10 H O
4 7 2
4
OH
NH
HO
HN
OH
HC
HO
CH
OH
Na
2
2
+
dry MeOH
dry MeOH
H N
2
NH
2
N
N
CHO
X
X
Dibenzo-diaza podand (2)
dry THF 2 K CO
Dibenzo-bis-imino podand (1)
P
N
N
4 XH
O
O
2
3
P
P
dry THF
N
N
N
2 XH Cl
2
O K
NH
K
O
Cl
Cl
Fully substituted sas phosphazene (5)
P
HN
N
P
N
Cl
Cl
P
O
N
O
N
X
N
P
N
P
N
P
K (2)
2
N
Cl
Cl
Cl
Cl
(5a) ; (6a)
N
Partly substituted sas phosphazene (3)
(5b) ; (6b)
(5c) ; (6c)
(5d) ; (6d)
N
Ar
dry THF
Cl
Cl
Cl
Cl
P
P
N
N
O
N
P
N
N
P
N
P
O
O
O
Cl
Cl
Cl
Cl
P
N
N
O
N
N
Partly substituted sbs phosphazene (4)
X
X
X
X
P
P
16 XH
N
N
N
P
N
P
dry THF
O
O
X
X
X
P
P
8 XH Cl
2
N
N
X
N
N
Fully substituted sbs phosphazene (6)
Scheme 1. The phosphazene derivatives obtained from the reactions of N3P3Cl6 with dibenzo- diaza podand, pyrrolidine, piperidine, morpholine and DASD.
activities and have been effective in changing the mobility of the
DNA [20–22]. Thus, it has been decided to introduce heterocyclic
amine groups to the partly substituted sas 3 and sbs 4 phosphaz-
enes with the aim of the investigations of their antimicrobial activ-
ities and DNA interactions.
2.2. Physical measurements
Melting points were measured with a Gallenkamp apparatus
using a capillary tube. Microanalyses (C, H, N) were performed
using Leco CHNS-932 elemental analyzer by the Central Instru-
mental Analysis Lab., Faculty of Pharmacy, Ankara University. 1H
(400 MHz) and 13C (100 MHz) NMR, DEPT and HSQC spectra were
recorded employing a Varian Mercury 400 MHz FT spectrometer,
and 31P NMR spectra were obtained Bruker DPX FT-NMR
500 MHz spectrometer (SiMe4 was used as an internal standard
for 1H and 13C NMR, and external 85% H3PO4 for 31P NMR). IR spec-
tra were recorded on a Mattson 1000 FTIR spectrometer using KBr
pellets. ESI-MS and API-ES mass spectra were obtained on the Agi-
lent 1100 MSD spectrometer and Waters Micromass ZQ mass spec-
trometer, respectively.
The present paper reports herein: (i) The synthesis of fully het-
erocyclic amine substituted sas 5a–5d and sbs 6a–6d phosphaz-
enes (Scheme 1), (ii) The structure determinations of eight new
cyclotriphosphazene derivatives (5b, 5c and 6a–6d) by elemental
analysis; mass spectrometry (MS); fourier transform infrared
(FTIR); one-dimensional (1D) 1H, 13C and 31P NMR; distortionless
enhancement by polarization transfer (DEPT), and two-dimen-
sional (2D) heteronuclear single quantum correlation (HSQC) tech-
niques, (iii) The solid-state and molecular structures of fully
morpholine substituted sas 5c and sbs 6c phosphazenes, (iv) The
relationship between the dPOPN shifts and the exocyclic OPN bond
angles (a0), and the correlation of
D(P–N) values and D(dP) chem-
2.3. Single crystal X-ray structure determination
ical shift differences or dPOPN shifts, (v) The investigations of anti-
bacterial and antifungal activities of 3, 4, 5a–5d and 6a–6d, and (vi)
Interactions between all of the sas and sbs compounds and pBR322
plasmid DNA.
The detailed crystallographic data and structure refinement
parameters were summarized in Table 1 [23,24]. Selected bond
lengths and angles for fully morpholine substituted sas 5c and
sbs 6c phosphazenes are given in Table 1S. Crystallographic data
were recorded on an Enraf–Nonius CAD-4 (for 5c) and Bruker Kap-
2. Experimental
pa APEXII (for 6c) diffractometers using Mo Ka radiation
(k = 0.71073 Å) at T = 294 K (for 5c) and T = 100(2) K (for 6c).
Absorption corrections by psi-scan [25] (for 5c) and multi-scan
[26] (for 6c) were applied. Structures were solved by direct meth-
ods and refined by full-matrix least squares against F2 using all
data [27]. All non-H atoms were refined anisotropically. Aromatic,
methylene and methyl H atoms were positioned geometrically at
distances of 0.93 (CH), 0.97 (CH2) and 0.96 (CH3) (for 5c) and
0.95 (CH), 0.99 (CH2) and 0.98 (CH3) (for 6c) from the parent C
atoms; a riding model was used during the refinement process
and the Uiso(H) values were constrained to be 1.2 Ueq (for methine
and methylene carrier atoms) and 1.5 Ueq (for methyl carrier
atoms).
2.1. Materials used for synthesis
The solvents, salicylaldehyde, sodium borohydride, borax,
potassium carbonate, piperidine and morpholine were purchased
from Merck. Hexachlorocyclotriphosphazene, N3P3Cl6 (Fluka),
was purified by fractional crystallization from n-hexane. 1,2-
Diaminoethane, pyrrolidine and DASD were obtained from Fluka.
All reactions were monitored using thin-layer chromatography
(TLC) on Merck DC Alufolien Kiesegel 60 B254 sheets. Column chro-
matography was performed on Merck Kiesegel 60 (230–400 mesh
ATSM) silica gel.

162
S. Bilge Koçak et al. / Inorganica Chimica Acta 406 (2013) 160–170
Table 1
Crystallographic data for 5c and 6c.
(5c)
(6c)a
Empirical formula
Color/shape
Formula weight
C24H32N7O4P3ꢁC2H3N
Colorless/rod-shaped
616.53
C48H80N16O10P6ꢁ2C2H3N
Colorless/block
1309.21
T (K)
298(2)
100(2)
Radiation used, graphite monochromator
Mo K
a
(k = 0.71073 Å)
Mo Ka (k = 0.71073 Å)
Crystal system
Space group
a (Å)
P21/a
monoclinic
15.5465(18)
P21/c
monoclinic
15.8439(6)
b (Å)
11.0971(17)
10.0433(4)
c (Å)
18.2722(17)
21.0252(7)
a
(°)
90.00
90.00
b (°)
109.954(8)
107.223(2)
c
(°)
90.00
2963.1(6)
90.00
3195.6(2)
V (Å3)
Z
4
2
Absorption coefficient (mmꢀ1
)
0.248
1.382
0.237
1.361
Dcalc (Mg mꢀ3
)
Maximum crystal dimension (mm)
hmax (°)
0.25 ꢂ 0.30 ꢂ 0.35
0.32 ꢂ 0.38 ꢂ 0.48
26.29
28.44
Reflections measured
Range of h, k, l
Diffractometer/scan
5999
5605
ꢀ19 < h < 0, 0 < k < 13, ꢀ21 < l < 22
Enraf–Nonius Turbo CAD-4/non-profiled w
4739
ꢀ17 < h < 18, ꢀ11 < k < 11, ꢀ25 < l < 25
Bruker-Kappa APEXII CCD/ profiled
4921
u and w
Number of reflections with I > 2
r(I)
Corrections applied
lorentz-polarization
lorentz-polarization
Computer programs
SHELXS97, SHELXL97, ORTEP-3
SHELXS97, SHELXL97, ORTEP-3
Source of atomic scattering factors
Structure solution
Int. Table for X-ray Cryst., vol. IV, 1974
direct methods
Int. Table for X-ray Cryst., vol. IV, 1974
direct methods
Treatment of hydrogen atoms
No. of parameters var.
Goodness-of-fit (GOF)
R = ||Fo| ꢀ |Fc||/|Fo|
geometric calculations
372
geometric calculations
389
1.152
0.0562
0.1459
0.774
ꢀ0.746
1.015
0.0778
0.2056
1.597
ꢀ1.508
Rw
(D
D
/
/
q
q
)
)
(e Aꢀ3
)
max
(
(e Aꢀ3
)
min
a
The crystallographic data for 6c do not sufficiently fulfill the requirements of the checkCIF program, but the data of sbs 6c were used in this study for comparison with the
data (bond lengths and angles, hydrogen bondings, and conformation of the rings and Cremer–Pople parameters) of sas 5c.
2.4. Antibacterial activity
were prepared and immediately applied to plasmid DNA. Plasmid
DNA aliquots were incubated in the presence of increasing concen-
trations of the compounds ranging from 625 to 5000 M at the
37 °C for 24 h. The aliquots of the compound-DNA mixtures
(10 L) were mixed with the buffer (0.1% bromophenol blue, 0.1%
xylene cyanol) and loaded onto the 1% agarose gel. Electrophoresis
was carried out under TAE (tris-acetate-EDTA) buffer for 3 h at
80 V. The gel was stained with ethidium bromide (0.5 lg/mL),
viewed with a transilluminator (BioDoc Analyzer, Biometra), and
The agar well-diffusion method [28] was used to test the anti-
microbial activities of the compounds against reference bacterial
strains Staphylococcus aureus ATCC 25923 (G+), Bacillus subtilis
ATCC 6633 (G+), Bacillus cereus NRRL-B-3711 (G+), Enterococcus
faecalis ATCC 292112 (G+), Pseudomonas aeruginosa ATCC 27853
(Gꢀ), and Escherichia coli ATCC 25922 (Gꢀ). In addition, Candida
albicans ATCC 10231 andCandida tropicalis ATCC 13803 were used
for antifungal activities. Gram positive and gram negative bacteria
were grown on Nutrient Broth agar plates and incubated at 37 °C
for 24 h, and adjusted to a final concentration of 108 cfu/mL by
diluting fresh cultures and compared to McFarland density. The
yeast strains were grown in Sabouraud dextrose agar medium
and incubated at 30 °C for 72 h. The medium was prepared, mixed
with culture suspension, and poured into plates. The wells with a
6.0 mm diameter were made. The test compound was added to
the well. After incubation, the diameter of inhibition zone was
l
l
the image was captured by a video-camera as a TIFF file.
2.6. Syntheses
Dibenzo-bis-imino-podand {N,N0-bis(salicylidene)-1,2-etanedi-
amine} (1) and dibenzo-diaza-podand {bis[N,N’-(2-hydroxyben-
zyl)]1,2-diaminoetane} (2) were synthesized according to the
published procedure [30]. The partly substituted sas phosphazene
{8,8-dichloro-18,19-dihydro-6k5,8k5,10k5-6,10-nitrilo-16H,21H
[1,3,5,7,2,4,6]tetrazatriphosphonino[2,1-b:6,7-b0]bis[1,3,2]ben-
zoxazaphosphorine} (3) and sbs phosphazene {3,300-ethane-1,
2-diylbis[40,40,60,60-tetrachloro-3,4-dihydrospiro[1,3,2-benzoxaza-
phosphorine-2,20k5-[4k5,6k5][1,3,5,2,4,6]triazatriphosphorine]]}
(4) were prepared using a method in which dibenzo-diaza-podand
(2) and N3P3Cl6 are refluxed in dry THF according to the published
procedure by our research group [31]. Fully pyrrolidine and DASD
substituted sas phosphazene derivatives {meso-18,19-dihydro-8,8-
dipyrrolidine-1-yl-6k5,8k5,10k5-6,10-nitrilo-16H,21H[1,3,5,7,2,4,6]
tetrazatriphosphecino[2,1-b:6,7-b0]bis[1,3,2]benzoxazaphosph-
orine} (5a) and {meso-18,19-dihydro-8,8-di(1,4-dioxa-8-azaspiro
[4,5]decane)-1-yl-6k5,8k5,10k5-6,10-nitrilo-16H,21H[1,3,5,7,2,4,6]
measured in millimeters. Ampisilin (10
col (30 g/mL) were used as standard antibacterial agents. Ketocon-
azole (50 g/mL) was used as an antifungal control. All the
lg/mL) and Chlorampheni-
l
l
experiments were repeated three times.
2.5. DNA cleavage activity
The interactions between starting compound (N3P3Cl6), pyrrol-
idine, morpholine, piperidine, DASD, dibenzo-diaza podand (2),
sas 3, 5a-5d and sbs 4, 6a-6d phosphazenes and pBR322 plasmid
DNA were studied by agarose gel electrophoresis [29]. These com-
pounds were dissolved in DMSO. The solutions of the compounds

S. Bilge Koçak et al. / Inorganica Chimica Acta 406 (2013) 160–170
163
tetrazatriphosphecino[2,1-b:6,7-b0]bis[1,3,2]benzoxazaphospho-
rine} (5d) were synthesized according to the our published proce-
dure [32].
1)], crystallized from CH3CN. Yield: 0.72 g (70%). mp: 232 °C. Anal.
Calc. for C56H96N16O2P6: C, 55.53; H, 7.99; N, 18.50. Found: C,
55.79; H, 7.51; N, 18.60%. IR (KBr, m
(cmꢀ1)): 3028 (C–H arom.),
2929–2817 (C–H aliph.), 1589 (C@C), 1180 (P@N). ESI-MS (Ir%):
m/z 620 {[(piperidine)4P3N3OArCH2NCH2CH3]+, 4}, 606 {[(piperi-
dine)4P3N3OArCH2NCH3]+, 100}.
2.6.1. Preparation of fully substituted sas phosphazenes (5b and 5c)
Compounds (5b and 5c) were prepared by similar methods;
therefore, the experimental procedure of the preparation is only
described in detail for the first case.
2.6.2.3. 3,300-Ethane-1,2-diyllbis[3,4-dihydro-40,40,60,60-tetramorpho-
line-1-yl(spiro[1,3,2-benzoxazaphosphorine-2,20k5-[4k5,6k5] [1,3,5,2,
4,6]triazatriphosphorine]] (6c). Compound (6c) was prepared from
morpholine (1.18 g, 13.6 mmol) and 4 (0.7 g, 0.85 mmol) (60 h),
column chromatography [silica gel (10 g), toluene/THF (1/1)], crys-
tallized from CH3CN. Yield: 0.64 g (69%). mp: >350 °C. Anal. Calc.
for C48H80N16O10P6: C, 46.98; H, 6.57; N, 18.26. Found: C, 46.66;
2.6.1.1. meso-18,19-Dihydro-8,8-dipiperidine-1-yl-6k5,8k5,10k5-6,10-
nitrilo-16H,21H[1,3,5,7,2,4,6]tetrazatriphosphecino[2,1-b:6,7-b0] bis[1,3,
2]benzoxazaphosphorine (5b). A solution of piperidine (0.72 g,
8.48 mmol) in 50 mL of dry THF was slowly added, over 0.5 h to
a stirred solution of 3 (1.0 g, 2.10 mmol) in 200 mL of dry THF at
ambient temperature with argon being passed over the reaction
mixture. The mixture was stirred for 34 h at room temperature,
and followed by TLC indicating no starting material remaining.
The precipitated piperidine hydrochloride was filtered off, and
the solvent was evaporated at reduced pressure. The crude product
was subjected to column chromatography [silica gel 60 (70–230
mesh) (15 g) as adsorbent and toluene/THF (4/1) as the eluent]
and crystallized from CH3CN. Yield: 0.80 g (66%). mp: 218 °C. Anal.
Calc. for C26H36N7O2P3: C, 54.64; H, 6.35; N, 17.15. Found: C, 54.79;
H, 6.54; N, 17.74%. IR (KBr, m
(cmꢀ1)): 3055 (C–H arom.), 2958–
2843 (C–H aliph.), 1589 (C@C), 1198 (P@N). API-ES (Ir%): m/z
1227 {[MH]+, 43}, 614 {[(morpholine)4P3N3OArCH2NCH3]+, 12}.
2.6.2.4. 3,300-Ethane-1,2-diyllbis[3,4-dihydro-40,40,60,60-tetra(1,4-diox-
a-8-azaspiro[4,5]decane)-1-yl(spiro[1,3,2-benzoxazaphosphorine-2
,20k5-[4k5,6k5][1,3,5,2,4,6]triazatriphosphorine]]
(6d). Compound
H, 6.28; N, 17.07%. IR (KBr, m
(cmꢀ1)): 3041 (C–H arom.), 2931–2829
(6d) was prepared from DASD (1.95 g, 13.6 mmol) and 4 (0.7 g,
0.85 mmol) (60 h), column chromatography [silica gel (10 g), ben-
zene/THF (1/1)], crystallized from CH3CN. Yield: 1.02 g (72%). mp:
>350 °C. Anal. Calc. for C72H112N16O18P6: C, 51.61; H, 6.74; N, 13.37.
(C–H aliph.), 1583 (C@C), 1184 (P@N). ESI-MS (Ir%): m/z 572
{[MH]+, 92}.
Found: C, 51.50; H, 6.79; N, 13.27%. IR (KBr, m
(cmꢀ1)): 3030 (C–H
2.6.1.2.
meso-18,19-Dihydro-8,8-dimorpholine-1-yl-6k5,8k5,10k5-
6,10-nitrilo-16H,21H[1,3,5,7,2,4,6]tetrazatriphosphecino[2,1-b:6,7-b0]
bis[1,3,2]benzoxazaphosphorine (5c). Compound (5c) was prepared
from morpholine (0.37 g, 4.20 mmol) and 3 (0.5 g, 1.05 mmol)
(36 h), column chromatography [silica gel (10 g), benzene/THF (1/
1)], crystallized from CH3CN. Yield: 0.45 g (70%). mp: 135 °C. Anal.
Calc. for C24H32N7O4P3: C, 50.09; H, 5.60; N, 17.04. Found: C, 50.16;
arom.), 2956–2844 (C–H aliph.), 1591 (C@C), 1180 (P@N). ESI-MS
(Ir%): m/z 838 {[(DASD)4P3N3OArCH2NCH3]+, 100}.
3. Results and discussion
H, 5.81; N, 16.97%. IR (KBr,
m
(cmꢀ1)): 3071 (C–H arom.), 2960–2850
3.1. Syntheses
(C–H aliph.), 1585 (C@C), 1185 (P@N). API-ES (Ir%): m/z 576 {[MH]+,
100}, 490 {[M-morpholine]+, 3}.
The reaction of N3P3Cl6 with K2(2) was investigated by our
group and two kinds of architectures, namely, spiro-ansa-spiro
(sas) (3) and spiro-bino-spiro (sbs) (4) skeletons were obtained
(Scheme 1). This reaction was carried out using two different ways
of driving with or without triethylamine and different yields of sas
3 and sbs 4 phosphazenes were obtained. The yield of sbs 3 (50%)
was higher than that of sas 4 (30%) in the absence of triethylamine
and on a 1:2 M ratio reaction of N3P3Cl6 and K2(2). Whereas, the
higher yield of sas (65%) and the lower yield of sbs (20%) were ob-
tained in the presence of triethylamine as an HCl acceptor and in a
1:1 M ratio. This may be caused by the facilitate the nucleophilic
attack of N–H nitrogen to P-atom with the formation of intermo-
lecular hydrogen bonding involving triethylamine and N–H hydro-
gen. Fully substituted phosphazenes (sas 5a–5d and sbs 6a–6d)
were prepared by the reaction of partly substituted phosphazenes
(sas 3 and sbs 4) with excess heterocyclic amines (pyrrolidine,
piperidine, morpholine and DASD) in dry THF.
In the literature, only spiro phosphazenes were obtained from
the reaction of N3P3Cl6 with ethane-1,2-diamine [33], therefore
sas (3 and 5a–5d) and sbs (4 and 6a–6d) are the first examples
of the ansa and bino structures having ethane-1,2-diamine precur-
sors, respectively. The P–N bonds of the seven membered ansa
rings of 3, 5a, 5c and 5d have cis configuration according to the
crystallographic data [34]. Analogously, the cis configuration is ex-
pected for sas 5b. The nucleophilic substitution reaction of K2(2)
with N3P3Cl6 is of importance in providing tetra-substituted
phosphorus atoms which are centers of chirality. As a result of
the reaction, each sas compound (3, 5a–5d) contains 2 equiv chiral
P-atoms in seven membered ansa ring. Therefore, they have R and S
configurations (meso form).
2.6.2. Preparation of fully substituted sbs phosphazenes (6a–6d)
Compounds (6a–6d) were prepared by similar methods; there-
fore, the experimental procedure of the preparation is only de-
scribed in detail for the first case.
2.6.2.1.
3,300-Ethane-1,2-diyllbis[3,4-dihydro-40,40,60,60-tetrapyrroli-
dine-1-yl(spiro[1,3,2-benzoxazaphosphorine-2,20k5-[4k5,6k5] [1,3,5,
2,4,6]triazatriphosphorine]] (6a). A solution of pyrrolidine (0.97 g,
13.6 mmol) in 50 mL of dry THF was slowly added, over 0.5 h to
a stirred solution of 4 (0.7 g, 0.85 mmol) in 100 mL of dry THF at
ambient temperature with argon being passed over the reaction
mixture. The mixture was stirred for 65 h at room temperature,
and followed by TLC indicating no starting material remaining.
The precipitated pyrrolidine hydrochloride was filtered off, and
the solvent was evaporated at reduced pressure. The crude product
was subjected to column chromatography [silica gel 60 (70–230
mesh) (20 g) as adsorbent and toluene/THF (1/4) as the eluent]
and crystallized from n-hexane. Yield: 0.69 g (74%). mp: 164 °C.
Anal. Calc. for C48H80N16O2P6: C, 52.50; H, 7.34; N, 20.39. Found:
C, 52.73; H, 6.92; N, 19.80%. IR (KBr, m
(cmꢀ1)): 3043 (C–H arom.),
2960–2827 (C–H aliph.), 1587 (C@C), 1178 (P@N). API-ES (Ir%):
m/z 1099 {[MH]+, 100}, 547 {[(pyrrolidine)4P3N3OArCH2NCH3]+, 23}.
2.6.2.2.
3,300-Ethane-1,2-diyllbis[3,4-dihydro-40,40,60,60-tetrapiperi-
dine-1-yl(spiro[1,3,2-benzoxazaphosphorine-2,20k5-[4k5,6k5] [1,3,5,
2,4,6]triazatriphosphorine]] (6b). Compound (6b) was prepared
from piperidine (1.16 g, 13.6 mmol) and 4 (0.7 g, 0.85 mmol)
(46 h), column chromatography [silica gel (10 g), benzene/THF (3/

164
S. Bilge Koçak et al. / Inorganica Chimica Acta 406 (2013) 160–170
3
3.2. FTIR spectroscopy
6a–6d) give multiplet and doublet (ca. JPH = 11 Hz) signals at
d = 3.12–3.54 ppm, respectively. In the 1H NMR spectra of sas 5a–
5d, the two substituents bonded to the same P-atom show two
groups of NCH2 signals (3.12 and 3.27 ppm for 5a, 3.14 and
3.20 ppm for 5b, 3.21 and 3.25 ppm for 5c, 3.32 and 3.38 ppm for
5d) with small separations, indicating that the two geminal sub-
stituents are not equivalent to each other (Table 3, see a schematic
representation of sas-structure). The ArCH2 benzylic protons of sas
derivatives are separated from each other at ca. 3.90 and 4.50 ppm
as two peak groups. In addition, the ArCH2 benzylic protons give
rise to doublet with three bond coupling to the P-atom (3JPH) of
ca. 15 Hz for sbs derivatives indicating that the ArCH2 protons
are equivalent. The four different proton signals of the aromatic
rings for the phosphazene derivatives are two sets of doublets
and triplets.
The FTIR spectra of sas (5b and 5c) and sbs (6a–6d) phospha-
zene derivatives display strong stretching bands at 1198–
1180 cmꢀ1 related to P@N bonds of the phosphazene rings. The
aromatic C–H stretching bands are observed at around 3071–
3030 cmꢀ1. The partly substituted sas 3 and sbs 4 give the stretch-
ing bands of PCl2 bonds at 546 and 575 cmꢀ1, respectively. These
bands disappear in the IR spectra of the fully heterocyclic amine
substituted phosphazenes.
3.3. MS spectrometry
The fragments were observed under electrospray ionization
(ESI-MS) (for 5b, 6b and 6d) and atmospheric pressure ionization
with electrospray (API–ES) (for 5c, 6a and 6c) mass spectrometry
conditions. The compounds (5b, 5c, 6a and 6c) give protonated
molecular ions [MH]+ which are the parent or the second peaks
in relative intensity. Notably, the MS spectra of 6a–6d show [(hete-
reocyclic amine)4P3N3OArCH2NCH3]+ ions. These peaks are ob-
served as parent peaks for 6b and 6d. In the unimolecular
decomposition of the phosphazenes, the major fragmentation
pathway involves the initial cleavage of the C–N bonds in the N–
CH2–CH2–N precursor for sbs (6a–6d) and the exocyclic P–N bond
with the loss of hetereocyclic amine groups for sas (5a, 5c and 5d).
All of the expected carbon peaks are interpreted from the 13C
NMR spectra of the compounds. The most reliable evidence coming
from the substitution of all the Cl-atoms of partly substituted sas 3
and sbs 4 phosphazenes is that the carbon signals of heterocyclic
amine substituents are observed in the 13C NMR spectra of the
3
compounds. The values of the three-bond coupling constants JPC
are observed for NCH2CH2 carbons of sas (5a and 5b) and sbs (6a
and 6b) phosphazenes (3JPC ca. 8.2 Hz) and OCH2 carbons of fully
morpholine substituted derivatives (5c and 6c) (3JPC ca. 7.8 Hz).
2
In addition, the values of the two-bond coupling constants, JPC
,
are estimated at 3.1, 7.0 and 6.0 Hz for bino-bridge NCH2 carbons
of partly (4), and fully piperidine (6b) and morpholine (6c) substi-
3.4. NMR spectroscopy
tuted phosphazene derivatives. The couplings, 2JPC1, 3JPC2, and 3JPC6
,
The proton-decoupled 31P NMR spectra of the fully substituted
phosphazenes exhibit an AB2 spin system containing two magnet-
ically equiv 31P NMR nuclei in the phosphazene rings, indicating
the heterocyclic amine substituents replace all of the Cl-atoms in
compounds 3 and 4 (Table 2). The 31P NMR spectra of fully hetero-
cyclic amine substituted sas 5b and 5c do not show a typical five-
line resonance pattern consisting of a doublet and a triplet and the
dP shifts overlap centering at ca. 24 ppm. The 31P NMR spectra of
6b–6d give two sets of peaks around d = 16 and 22 ppm in a dou-
blet–triplet pattern because of their sbs structures, which are as-
signed to the two OPN and four XPX phosphorus atoms.
are observed as triplets for the sas phosphazenes and doublets for
the sbs phosphazenes. The triplets observed for the sas phosphaz-
enes may be due to the second-order effects which have previously
been observed [35], and the JPC coupling constants between the
external transitions of the triplets are estimated. The peaks of non-
protonated C atoms disappear in the DEPT spectra, as compared
with the 1H decoupled 13C NMR spectra (Fig. 1S).
3.5. X-ray crystallography
The molecular and solid state structures of 5c and 6c along with
the atom-numbering schemes are depicted in Figs. 2 and 3, respec-
tively. The asymmetric unit of 5c contains one uncoordinated CH3-
CN and a phosphazene derivative containing a sas structure
together with two morpholino substituents (Fig. 2S). The symmet-
ric phosphazene derivative with a sbs structure and eight morpho-
lino substituents contains 0.5 mol of 6c and 1 mol of CH3CN in its
asymmetric unit (Fig. 3S). As can be seen from the packing dia-
grams of 5c and 6c, the CH3CN molecules occupy the cavities
[36]. Dipole–dipole and van der Waals interactions are effective
in the molecular packing. There are intra- and intermolecular C–
Hꢁ ꢁ ꢁN hydrogen bonds in 5c, while 6c has intramolecular C–
Hꢁ ꢁ ꢁN and intermolecular C–Hꢁ ꢁ ꢁO hydrogen bonds (Table 2S).
31
Whereas, the P NMR spectrum of 6a gives an AB2A0B02 spin sys-
tem, which is characterized by a small shift in the phosphorus-
phosphorus coupling constants (2JPP).
The 1H and 13C NMR assignments of the compounds are sum-
marized in Table 3. The interpretations are made unambiguously
by HSQC and DEPT spectra. The HSQC spectrum of 5c is depicted
in Fig. 1, as an example. The aliphatic regions of the 1H NMR spec-
tra of the sas compounds are very complex since all of the aliphatic
protons are diastereotopic. In the fully heterocyclic amine substi-
tuted derivatives (5a, 5b and 5d) and (6a, 6b and 6d), the NCH2
proton signals of the heterocyclic amines are easily distinguished
from those of the ansa and bino rings by the HSQC spectra of these
compounds. The NCH2 protons of sas (3 and 5a–5d) and sbs (4 and
The findings in the IR spectra of 5c and 6c (mCHꢁ ꢁ ꢁN stretching band
for 5c at 3442 cmꢀ1 and mCHꢁ ꢁ ꢁN and
mCHꢁ ꢁ ꢁO stretching bands for 6c
at 3446 cmꢀ1) support X-ray crystallographic data.
Table 2
31P NMR Data (CDCl3) of 3, 4, 5a–5d and 6a–6d (d in ppm, J in Hz).
The phosphazene rings of 5c and 6c are in twisted and flat-
2JPP
tened-boat conformations, respectively [Fig. 4Sa;
u2 = 90.4(2)°,
Compound
Spin system
dClPCl
dXPX
dOPN
2JAX:70.1
2JAB:53.5
h2 = 53.5(2)° (P1/N1/P2/N2/P3/N3) (for 5c) and Fig. 5Sa;
(3)
A2X
AB2
AB2
AB2
AB2
AX2
AB2A0B02
PX:29.35
–
–
PA:19.59
PB:24.10
u
2 = ꢀ168.3(8)°, h2 = 110.7(8)°6 (for 6c)] having total puckering
(5a)
(5b)
(5c)
(5d)
(4)
PA:20.15
ꢃ23.69
ꢃ23.90
ꢃ23.50
–
amplitudes [37], QT, of 0.421(2) Å for 5c and QT of 0.170(3) Å for
6c. In 5c, the seven-membered ansa ring (N1/P1/N6/C23/C24/N7/
P2) and the six-membered spiro rings (P1/N6/C9/C10/C15/O3)
and (P2/N7/C16/C17/C22/O4) are in twisted, boat and twisted con-
PX:25.10
–
PA:6.56
PA:17.47
2JAX:56.1
(6a)
PB:18.98
0
2JAB:47.3
2
0
0
0
P
B :18.96
PA :17.42
JA B :44.5
2JAB:46.0
2JAB:47.0
2JAB:47.3
formations, respectively [Fig. 4Sb; QT = 1.133(3) Å,
h2 = 12.0(1)° (N1/P1/N6/C23/C24/N7/P2), QT = 0.552(2) Å, u2
162.9(3)°, h2 = 92.7(3)° (P1/N6/C9/C10/C15/O3), QT = 2.478(2) Å,
2 = 148.1(3)°, h2 = 21.1(1)° (P2/N7/C16/C17/C22/O4)]. In 6c, the
u2 = 98.9(4)°,
(6b)
(6c)
(6d)
AB2
AB2
AB2
–
–
–
PB:22.49
PB:21.62
PB:21.58
PA:16.68
PA:16.53
PA:16.12
=
u

Table 3
1H and 13C NMR Data (CDCl3) of 3, 4, 5a–5d and 6a–6d (d in ppm, J in Hz, s: singlet, d: doublet, t: triplet, q: quartet and m: multiplet peak).
X
X
H
6
H
4
X
N
X
X
X
X
X
X
5
2
X
X
P
H
6
P
P
H
H
N
P
N
5
N
P
N
N
P
N
P
1
3
H
O
O
4
X
O
O
O
X
X
X
P
1
H
P
3
N
2
N
N
N
N
X
N
N
H
O
N
O
N
Schemetic representative
Schemetic representative
structure of sas
structure of sbs
(3)
(5a)
(5b)
(5c)
(5d)
(4)
(6a)
(6b)
(6c)
(6d)
H
NCH2CH2CH2
NCH2CH2
–
–
–
1.67 (m,4H)
1.59 (m,8H)
–
–
–
–
–
–
1.67 (m,16H)
1.53 (m,32H)
–
–
–
1.86 (m,8H)
1.62 (m,2H)
1.77 (m,6H)
DASD
3.32 (m,4H)
3.38 (m,4H)
ansa
3.32 (m,32H)
1.67 (m,32H)
NCH2
pyrrolidine
3.23 (m,4H)
3.27 (m,4H)
ansa
piperidine
3.13 (m,4H)
3.20 (m,4H)
ansa
morpholine
3.21 (m,4H)
3.25 (m,4H)
ansa
pyrrolidine
3.17 (m,32H)
piperidine
3.09 (m,32H)
morpholine
3.10 (m,32H)
DASD
3.21 (m,32H)
ansa
bino
bino
bino
bino
bino
3.30 (m,2H)
3.54 (m,2H)
–
3.12 (m,2H)
3.43 (m,2H)
–
3.14 (m,2H)
3.44 (m,2H)
–
3.14 (m,2H)
3.45 (m,2H)
3.71 (m,8H)
3.15 (m,2H)
3.44 (m,2H)
3.99 (s,4H)
4.00 (s,4H)
3.84 (q,2H)
4.48 (d,2H)
3JPH = 14.8
3.34 (d,4H)
3JPH = 14.0
–
3.32 (d,4H)
3JPH = 10.6
–
3.31 (d,4H)
3JPH = 10.4
–
3.28 (d,4H)
3JPH = 9.9
3.66 (m,32H)
3.26 (d,4H)
3JPH = 10.0
3.93 (s,16H)
3.96 (s,16H)
4.35 (d,4H)
3JPH = 14.4
OCH2
ArCH2N
3.94 (m,2H)
3JPH = 15.0
4.56 (m,2H)
3JPH = 15.1
3.84 (d,2H)
3JPH = 15.0
4.47 (d,2H)
3JPH = 15.0
3.85 (q,2H)
4.49 (d,2H)
3JPH = 16.3
3.84 (q,2H)
2JHH = 15.0
3JPH = 11.0
4.47 (d,2H)
2JHH = 15.0
3JPH = 10.2
7.04 (d,2H)
7.01 (t,2H)
7.21 (t,2H)
7.03 (d,2H)
8.7
4.38 (d,4H)
4.33 (d,4H)
4.37 (d,4H)
4.38 (d,4H)
3JPH = 15.5
3JPH = 14.3
3JPH = 14.8
3JPH = 14.4
ArH
H3
7.09–7.29
7.09–7.29
7.09–7.29
7.03 (d,2H)
7.00 (t,2H)
7.18 (t,2H)
7.02 (d,2H)
8.4
7.05 (d,2H)
7.00 (t,2H)
7.20 (t,2H)
7.03 (d,2H)
8.7
7.02 (d,2H)
6.96 (t,2H)
7.16 (t,2H)
7.01 (d,2H)
8.5
6.96–7.09
6.96–7.09
7.28 (t,2H)
6.96–7.09
6.94–6.84
6.94–6.84
7.13 (t,2H)
6.94–6.84
6.90–6.86
6.91 (t,2H)
7.14 (t,2H)
6.90–6.86
7.09–6.96
7.09–6.96
7.19 (t,2H)
6.87–7.09
6.87–7.09
7.12 (t,2H)
H4
H5
H6
7.09–7.29
6.88 (d,2H)
6.87–7.09
3J3–4
3J4–5
3J5–6
*
*
*
*
*
*
*
7.3
8.0
7.7
8.2
7.6
8.5
7.3
8.1
7.6
7.9
C
NCH2CH2CH2
–
–
–
25.0
25.1
–
–
–
–
–
–
25.0
25.2
–
–
NCH2CH2
NCH2
26.3
26.2
35.3
26.36
26.3
35.76
35.82
3JPC = 6.3
26.4
3JPC = 5.9
26.4
3JPC = 5.7
35.6
3JPC = 9.3
26.37
3JPC = 7.9
26.4
3JPC = 5.9
pyrrolidine
46.1
3JPC = 7.1
piperidine
45.1
3JPC = 4.9
DASD
42.6
3JPC = 8.7
pyrrolidine
46.0
3JPC = 8.0
piperidine
45.3
morpholine
42.8
42.7
morpholine
44.6
42.7
DASD
42.8
42.9
2JPC = 3.8
46.3
45.2
42.8
46.2
45.4
2JPC = 4.0
ansa
ansa
ansa
ansa
ansa
bino
bino
bino
bino
bino
52.0
53.6
51.5
51.7
53.5
47.3
2JPC = 3.1
–
47.6
47.6
2JPC = 7.0
–
47.1
46.9
2JPC = 6.0
67.14
3JPC = 8.5
67.18
3JPC = 8.1
–
OCH2
OCO
–
–
–
–
–
–
65.4
64.2
64.3
–
–
64.4
3JPC = 6.6
65.2
3JPC = 6.7
–
107.6
–
–
107.7
(continued on next page)

166
S. Bilge Koçak et al. / Inorganica Chimica Acta 406 (2013) 160–170
six-membered ring (P1/O1/C1/C6/C7/N4) is in twisted form
[Fig. 5Sb; QT = 0.527(3) Å, 2 = 123.5(5)°, h2 = 91.3(5)°]. The bicy-
u
clic system made up of phosphazene and ansa rings of 5c is in a
sofa conformation (Fig. 4Sc). Each ring is V-shaped, with the non-
planar two halves (P2/N2/P3/N3/P1) and (P1/N6/C23/C24/N7/P2).
The sums of the bond angles around the atoms N6 and N7
[345.1(2)° and 347.6(2)°] show changes in hybridizations of the
atoms from trigonal planar towards a pyramidal for 5c [38]. The
distortions of the N-atoms especially may depend on the confor-
mations of phosphazene and spiro rings (Fig. 4Sb). Since the com-
pounds (5a–5d) are fluxional in solution, the distortion may also
be purely caused by packing effects in the solid state.
The average endocyclic PN bond lengths of 5c and 6c are
1.591(2) Å (for 5c) and 1.588(3) Å (for 6c), respectively, which
are considerably shorter than the average exocyclic PN bonds
[1.636(2) Å for 5c and 1.644(3) Å for 6c], indicating that the endo-
cyclic PN bonds have double bond character. The value of the endo-
cyclic P1N1P2 angle of 5c [112.8(1)°] is highly smaller than those
of P2N2P3 [117.4(1)°] and P1N3P3 [122.8(1)°], and the correspond-
ing value of the starting dichloro sas 3 [114.1(3)°] [39]. The endo-
cyclic NPN angles of 5c and 6c are considerably narrowed with
respect to the corresponding value in the ‘‘standard’’ compound
N3P3Cl6 [118.3(2)°]. Moreover, all the endocyclic PNP angles of 5c
are noticeably narrowed, except P1N3P3 angle but, the endocyclic
PNP angles of 6c are slightly expanded with respect to correspond-
ing value of N3P3Cl6 [121.4(3)°] [40].
3.6. The relationship between 31P NMR spectroscopy and X-ray
crystallography
A number of relationships gained from 31P NMR spectra and X-
ray crystallographic results is observed using the exocyclic OPN
bond angles (a0) and the bond lengths (a, a0, b and b0) of the phos-
phazene rings (Scheme 2, Table 4). These include (i) a0 versus dPOPN
shifts (Fig. 4A) and (ii) the electron density transfer parameters
D
(P–N) [
D
(a–b): the difference between the bond lengths of two
(dP) and
adjacent P–N bonds] and their relationships with both
D
dPOPN shifts (Fig. 4B). The variations in the bond angles and dP-
shifts of the phosphazenes depend on the electronic and steric fac-
tors. It is found that relatively small changes in a0 bond angles are
shown to cause large changes in dPOPN shifts according to Fig. 4A.
Moreover, the correlations between dPOPN shifts and a0 bond angles
for the fully heterocyclic amine (Fig. 4Aa) and partly (Fig. 4Ab)
substituted sas, sbs and spiro [41–45] phosphazene derivatives
show contrasting trends. On the other hand,
measure of the electron releasing and withdrawing capacities of
the substituents bonded to phosphazene ring [46] and the (P–
D(P–N) indicates the
D
N) values are calculated from the equations given in Scheme 2.
When the substituents bonding to the phosphazene ring withdraw
electrons from the phosphazene ring,
D(P–N) values are getting in-
creases. In addition, the shortening of the exocyclic P–N bonds is to
be a measure of the electron withdrawing capacities of the substit-
uents, hence, which is likely to be a measure of the negative hyper-
conjugation. The electron releasing capacities of the substituents
for 3, 5a, 5c and 5d are in the following order: DASD > pyrroli-
dine > morpholine > Cl. A similar situation is observed for partly
(I) and fully morpholino (6c) substituted sbs compounds. The
homologous partly and fully heterocyclic amine substituted
spiro-phosphazenes given in Scheme 2 can also be compared with
each other in Fig. 4. Based on the electron releasing capacities of
the morpholine substituents in the sas 5c and sbs 6c skeletons,
the electrons are slightly transferred from morpholine groups to
the phopsphazene ring in 5c.
Consequently, the origin of the PN bonds in the cyclophospha-
zene derivatives is still not clearly understood. A number of
attempts have been made to explain the origin of the PN bonds

S. Bilge Koçak et al. / Inorganica Chimica Acta 406 (2013) 160–170
167
Fig. 1. HSQC spectrum (a) for the aliphatic region and (b) aromatic region of 5c (a:ansa, m:morpholine).
using the island [47] and the negative hyperconjugation models
[48]. In the negative hyperconjugation model, which is the newest
one, the electronic structures and the PN bonds of phosphazenes
are investigated using natural bond orbital (NBO) and topological
electron density analyses. The ionic bonding and negative hyper-
conjugation alternatives for phosphazene derivatives are mutually
exclusive. While the electron releasing groups bonding to the P-
atoms decrease the negative hyperconjugation of the PN bonds,
the electron withdrawing groups increase the multiple-bond char-
acter of the PN bonds. Therefore, the narrowing and broadening of
the endocyclic NPN and PNP angles, and the shortening of the PN
bonds of the phosphazenes may significantly depend on the con-
formation and electron releasing and withdrawing capacities of
the substituents, the steric hindrances of the exocyclic groups,
the spiro rings bonded to the P-atoms and the negative
hyperconjugation.
3.7. Antimicrobial activity
Fig. 2. ORTEP-3 drawing of 5c with the atom-numbering scheme. Displacement
ellipsoids are drawn at the 30% probability level.
The starting compound (N3P3Cl6), pyrrolidine, piperidine, mor-
pholine, DASD and dibenzo-diaza podand (2) were subjected to
antimicrobial activity against the bacteria and yeast strains tested
for phosphazene derivatives (3, 4, 5a–5d and 6a–6d) in this study.
N3P3Cl6 exhibits weak antibacterial activity against B. subtilis ATCC
6633 (G+), B. cereus NRRL-B-3711 (G+) and E. coli ATCC 25922 (Gꢀ),
piperidine to B. cereus NRRL-B-3711 (G+), morpholine to P. aerugin-
osa ATCC 27853 (Gꢀ), and DASD to B. subtilis ATCC 6633 (G+) and B.
cereus NRRL-B-3711 (G+). The results were given in Table 3S for
comparison to the cyclotriphosphazene derivatives. Phosphazene
derivatives 3, 4, 5a–5d and 6a–6d exhibit no antibacterial activity
even if they have been used at 5000 lM. The hetereocyclic amines
and starting compounds do not show antifungal activity. However,
compound 6a displays good activity (15.0 0.0) against yeast
strain C. tropicalis compared with that of Ketoconazole
(29.0 0.0) (Table 3S). The activity for 6a may be attributed to
the presence of both the sbs architecture and pyrrolidinyl side
groups.
3.8. Interactions of DNA with the compounds
The interactions of N3P3Cl6, pyrrolidine, piperidine, morpholine,
Fig. 3. ORTEP-3 drawing of 6c with the atom-numbering scheme. Displacement
ellipsoids are drawn at the 30% probability level.
DASD, 2 and cyclotriphosphazene derivatives (3, 4, 5a–5d and

168
S. Bilge Koçak et al. / Inorganica Chimica Acta 406 (2013) 160–170
X
No
3
X
X
Cl
b
b
P
Δ(δP) = δPX2 ─ δPOPN
N
5a
5c
5d
N
N
a + a′
Δ(P-N) = ──── ─ b
2
a'
a'
O
O
P
a
P
a
α '
α '
N
N
O
N
N
N
O
O
X
X
X
X
X
n
0
No
6c
P
P
b
Δ(δP) = δPX2 ─ δPOPN
N
P
N
N
P
N
P
a'
N
O
O
O
X
X
X
a + a′
b + b′
α '
P
a
Δ(P-N)= ──── ─ ────
N
N
X
N
N
b'
Cl
1
I
2
2
n
Ar
Y
X
No
II
H
H
Cl
Δ(δP) = δPX2 ─ δPOPN
Y
O
a + a′
b + b′
Me
Δ(P-N)= ──── ─ ────
Ar
N
N
α '
α '
O
O
Cl
Cl
III
IV
2
2
N
P
a
P
a'
a
a'
N
N
N
N
b
b'
b
b'
p-OMe
o-OMe
Cl
Cl
Cl
Cl
X
X
X
P
P
P
P
O
X
N
V
N
N
VI
O
Scheme 2. The exocyclic OPN bond angles (a0) and bond lengths (a, a0, b and b0) on the formulae of the sas, sbs and spiro-phosphazenes.
Table 4
Exoxcyclic OPN bond angles (a0), bond lengths (a, a0, b and b0), dP–shifts,
D
(P–N) and D
(dP) values for the compounds [dP in ppm, a0 angles in (°)].
Compound
(a)
(a0)
(b)
(b0)
D
(P–N)
(
a0
)
dPOPN
dPX2
D(dP)
(3)
1.588(5)
1.579(5)
1.589(3)
1.584(3)
1.5811(18)
1.5888(19)
1.590(5)
1.577(5)
1.584(3)
1.594(5)
1.575(5)
1.600(3)
1.589(4)
1.594(2)
1.592(3)
1.602(5)
1.621(6)
1.577(3)
1.567(3)
1.5890(19)
1.5775(19)
1.586(4)
1.585(4)
1.571(4)
1.589(5)
1.582(6)
1.583(3)
1.597(4)
1.604(2)
1.607(3)
1.564(5)
1.621(6)
1.697(3)
1.600(3)
–
–
–
–
–
–
–
–
0.0310
104.0(3)
104.4(2)
19.59
29.35
9.76
3.95
0.30
0.25
ꢀ0.0210
ꢀ0.0260
ꢀ0.0245
ꢀ0.0209
ꢀ0.0206
ꢀ0.0700
ꢀ0.0230
ꢀ0.0230
0.0235
0.0185
0.0255
0.0265
0.0370
(5a)
(5c)
(5d)
101.55(16)
104.44(15)
101.33(9)
105.17(9)
102.2(2)
20.15
23.9
24.10
24.2
1.6059(18)
1.6037(18)
1.595(5)
1.604(4)
1.601(4)
1.563(5)
1.566(5)
1.563(3)
1.569(3)
1.567(2)
1.563(3)
23.25
23.50
102.6(2)
(6c)
(I)
1.600(3)
1.573(6)
1.554(6)
1.569(3)
1.564(3)
1.557(2)
1.554(3)
100.96(16)
103.81(17)
102.65(15)
102.97(13)
100.3
16.53
6.78
21.62
25.09
5.09
18.31
(II)
3.90
5.15
5.25
1.60
24.73
26.32
23.87
24.5
20.83
21.17
18.62
24.00
(III)
(IV)
(V)
102.7(1)
101.03
0.0410
26.7
(VI)
1.577(2)
1.583(2)
1.606(2)
1.600(2)
ꢀ0.023
100.5(1)
17.88
19.36
1.48
6a–6d) with supercoiled pBR322 DNA were investigated using the
agarose gel electrophoresis. Fig. 5 gives the electrophoretograms
applying to have incubated mixtures of pBR322 plasmid DNA
The phosphazene derivatives 5d, 6b and 6c cause a decrease in
the mobility and intensity of form I DNA, whereas that of the form
II band remains essentially unchanged. When pBR322 plasmid
DNA is interacted with 5b and 5c, there is a slight decrease in
intensity of form I, and the mobility and intensity of form II remain
unchanged. In case of 5a, 6a and 6d, there is a slight decrease in
mobility of form I at two higher concentrations. The linear band
is also observed for 6a in addition to decrease in mobility. The
partly substituted phosphazene derivatives (3 and 4) are found
to be more damaging to DNA. Compounds 3 and 4 increase in
mobility and intensity of form II DNA. The ClPCl angle of partly
substituted sas 3 phosphazene (99.6°) is close to the ClPtCl angle
and varying concentrations (5000–625 lM) of the compounds.
Lane P applies to the untreated pBR322 plasmid DNA (control
DNA), showing the major supercoiled circular form I and minor
singly nicked relaxed circular form II of the plasmid DNA. Lanes
1–4 apply to pBR322 plasmid DNA incubated with the compounds
ranging from 5000 to 625 lM. Pyrrolidine, morpholine and 2 have
no effect on plasmid DNA. Piperidine and DASD cause only a very
small decrease in the mobility of form I DNA. However, N3P3Cl6
causes significant conformational change.

S. Bilge Koçak et al. / Inorganica Chimica Acta 406 (2013) 160–170
169
35
25
20
15
10
5
5c
5c
30
25
20
15
10
5
5d
5d
5c
V
5d
5d
(b)
(a)
5a
3
3
5a
III
II
I
5a
3
3
3
VI
I
VI
IV
6c
6c
(a)
δ
3
I
I
IV
III
6c
VI
δ
5a
II
V
(b)
5d
δ
I
I
0
5d
5c
III
IV
II
-5
(a) Δ(P-N):δ POPN
(b) Δ(P-N):Δ(δ P)
V
-10
0
99
100
101
102
103
104
105
106
-0.10 -0.08 -0.06 -0.04 -0.02 0.00
0.02
0.04
0.06
0.08
OPN (α')
(A)
Δ((PB-N))
Fig. 4. The relationship between (A) exocyclic OPN bond angles (
a
’) and dPOPN shifts for (a) the fully heterocyclic amine and (b) partly substituted sas, sbs and spiro-
(dP) values for the partly and fully heterocyclic amine substituted sas, sbs and spiro-phosphazenes.
phosphazenes and (B) (a) (P–N) and dPOPN shifts and (b) (P–N) and D
D
D
Fig. 5. Gel electrophoretic mobility of pBR322 plasmid DNA, when incubated with different concentrations of N3P3Cl6, pyrrolidine, piperidine, morpholine, DASD, 2–4, 5a–5d
and 6a–6d. Lane P applies untreated pBR322 plasmid DNA. Concentrations (in M) are as follows: lanes 1 to 4 apply to plasmid DNA interacted with decreasing
l
concentrations of the compounds: lane 1: 5000; lane 2: 2500; lane 3 1250; lane 4: 625. The top and the bottom bands correspond to form II (single nicked open circular), form
I (covalently closed circular or supercoiled) and form III (linear) plasmids, respectively.

170
S. Bilge Koçak et al. / Inorganica Chimica Acta 406 (2013) 160–170
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4. Conclusions
The syntheses and characterizations of fully heterocyclic amine
substituted sas 5a–5b and sbs 6a–6b phosphazenes were de-
scribed. The partly substituted sas 3 and sbs 4 phosphazenes re-
acted with excess heterocyclic amines in THF to produce the
fully substituted sas 5a–5d and sbs 6a–6d phosphazenes, respec-
tively. All the compounds were characterized by one and two
dimensional NMR techniques, where the fully morpholine
substituted sas 5c and sbs 6c phosphazenes were characterized
crystallographically. In sas compounds, there are two stereogenic
P-atoms. They have meso form. Compound 6a is active against
yeast strain C. tropicalis. Interactions between the phosphazenes
and pBR322 plasmid DNA show that the partly substituted sas 3
and sbs 4 phosphazenes are effective in changing the mobility
and intensity of form II DNA.
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The authors would like to express their deep sense of gratitude
and heartiest thanks to Ankara University Scientific Research Pro-
jects (BAP) (Grant No. 10B4240008), Hacettepe University Scien-
tific Research Unit (Grant No. 02 02 602 002) and State Planing
Organization (grant No.1998K121480) for financial support of this
work.
Appendix A. Supplementary material
[32] S. Bilge, Sß. Demiriz, A. Okumußs, Z. Kılıç, B. Tercan, T. Hökelek, O. Büyükgüngör,
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The crystal packing diagrams, ring conformations and hydro-
gen-bond geometries for sas (5c) and sbs (6c) phosphazenes, and
antimicrobial activities of N3P3Cl6 and heterocyclic amines. CCDC
917208 and 917209 contains the supplementary crystallographic
data for these compounds 5c and 6c. These data can be obtained
free of charge from The Cambridge Crystallographic Data Centre
via www.ccdc.cam.ac.uk/data_request/cif. Supplementary data
associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.ica.2013.07.023.
(2007) 121.
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