Synthesis, X-ray crystal structure, DNA/protein binding and cytotoxicity studies of five α-aminophosphonate N-derivatives

Article abstract of DOI:10.1016/j.bioorg.2016.10.007

Five new α-aminophosphonates are synthesized and characterized by EA, FT-IR, ¹H NMR, ¹³C NMR, ³¹P NMR, ESI-MS and X-ray crystallography. The X-ray analyses reveal that the crystal structures of 1–5 are monoclinic or triclinic system with the space group P 21/c, P ? 1, P ? 1, P2(1)/c and P ? 1, respectively. All P atoms of 1–5 have tetrahedral geometries involving two O-ethyl groups, one C_α atom, and a double bond O atom. The binding interaction of five new α-aminophosphonate N-derivatives (1–5) with calf thymus(CT)-DNA have been investigated by UV–visible and fluorescence emission spectrometry. The apparent binding constant (Kapp) values follows the order: 1 (3.38 × 10⁵ M^?1) > 2 (3.04 × 10⁵ M^?1) > 4 (2.52 × 10⁵ M^?1) > 5 (2.32 × 10⁵ M^?1) > 3 (2.10 × 10⁵ M^?1), suggesting moderate intercalative binding mode between the compounds and DNA. In addition, fluorescence spectrometry of bovine serum albumin (BSA) with the compounds 1–5 showed that the quenching mechanism might be a static quenching procedure. For the compounds 1–5, the number of binding sites were about one for BSA and the binding constants follow the order: 1 (2.72 × 10⁴ M^?1) > 2 (2.27 × 10⁴ M^?1) > 4 (2.08 × 10⁴ M^?1) > 5 (1.79 × 10⁴ M^?1) > 3 (1.17 × 10⁴ M^?1). Moreover, the DNA cleavage abilities of 1 exhibit remarkable changes and the in vitro cytotoxicity of 1 on tumor cells lines (MCF-7, HepG2 and HT29) have been examined by MTT and shown antitumor effect on the tested cells.

Full text of DOI:10.1016/j.bioorg.2016.10.007

Accepted Manuscript
Synthesis, X-ray crystal structure, DNA/protein binding and cytotoxicity studies
of five α-aminophosphonate N-derivatives
Qingming Wang, Lei Yang, Hui Ding, Xuanrong Chen, Hua Wang, Xinhui Tang
PII:
S0045-2068(16)30229-2
DOI:
http://dx.doi.org/10.1016/j.bioorg.2016.10.007
YBIOO 1962
Reference:
Bioorganic Chemistry
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24 August 2016
12 October 2016
26 October 2016
Please cite this article as: Q. Wang, L. Yang, H. Ding, X. Chen, H. Wang, X. Tang, Synthesis, X-ray crystal structure,
DNA/protein binding and cytotoxicity studies of five α-aminophosphonate N-derivatives, Bioorganic Chemistry
(2016), doi: http://dx.doi.org/10.1016/j.bioorg.2016.10.007
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Title page
Synthesis, X-ray crystal structure, DNA/protein binding and cytotoxicity
studies of five α-aminophosphonate N-derivatives
Qingming Wang*a,b, Lei Yanga, Hui Dinga, Xuanrong Chena, Hua Wanga, Xinhui Tang*a
a School of Pharmacy, Jiangsu Provincial Key Laboratory of Coastal Wetland Bioresources and
Environmental Protection , Yancheng Teachers’ University, Yancheng, Jiangsu 224051, People’s
Republic of China, E-mail: wangqm@yctu.edu.cn (Wang Q) & xinhuitang@sina.com ( Tang X )
b State Key Laboratory of Coordination Chemistry, Nanjing University, Nanjing, 210093, People’s
Republic of China, E-mail: wangqm@yctu.edu.cn (Wang Q)
Corresponding author: Prof. Dr. Qingming Wang
School of Pharmacy, Jiangsu Provincial Key Laboratory of Coastal Wetland Bioresources and
Environmental Protection , Yancheng Teachers’ University, Yancheng, Jiangsu 224051, People’s
Republic of China,
Tel: 0515-88258905
Email: wangqm@yctu.edu.cn

Abstract
1
Five new α-aminophosphonates are synthesized and characterized by EA, FT-IR, H-NMR,
13C-NMR, 31P-NMR, ESI-MS and X-ray crystallography. The X-ray analyses reveal that the crystal
structures of 1-5 are monoclinic or triclinic system with the space group P 21/c, P-1, P-1, P2(1)/c
and P-1, respectively. All P atoms of 1-5 have tetrahedral geometries involving two O-ethyl
groups, one Cα atom, and a double bond O atom. The binding interaction of five new
α-aminophosphonate N-derivatives (1-5) with calf thymus(CT)-DNA have been investigated by
UV-visible and fluorescence emission spectrometry. The apparent binding constant (Kapp)values
follows the order: 1 ( 3.38×105 M-1 ) > 2 ( 3.04×105 M-1 ) > 4 ( 2.52×105 M-1 ) > 5 ( 2.32×105
M-1 )> 3 ( 2.10×105 M-1 ), suggesting moderate intercalative binding mode between the
compounds and DNA. In addition, fluorescence spectrometry of bovine serum albumin (BSA)
with the compounds 1-5 showed that the quenching mechanism might be a static quenching
procedure. For the compounds 1-5, the number of binding sites were about one for BSA and the
binding constants follow the order:1 (2.72×104 M-1) > 2 (2.27 × 104 M-1) > 4 (2.08 × 104 M-1) > 5
(1.79 ×104 M-1) > 3 (1.17 × 104 M-1). Moreover, the DNA cleavage abilities of 1 exhibit remarkable
changes and the in vitro cytotoxicity of 1 on tumor cells lines (MCF-7, HepG2 and HT29) have
been examined by MTT and shown antitumor effect on the tested cells.
Keywords: α-Aminophosphonate / DNA binding / Bovine Serum Albumin (BSA) binding /
Cytotoxicity

Synthesis, X-ray crystal structure, DNA/protein binding, DNA cleavage
and cytotoxicity studies of five α-aminophosphonate N-derivatives
Qingming Wang*a,b, Lei Yanga, Hui Dinga, Xuanrong Chena, Hua Wanga, Xinhui Tang*a
a School of Pharmacy, Jiangsu Provincial Key Laboratory of Coastal Wetland Bioresources and
Environmental Protection , Yancheng Teachers’ University, Yancheng, Jiangsu 224051, People’
s Republic of China, E-mail: wangqm@yctu.edu.cn (Wang Q) & xinhuitang@sina.com ( Tang X )
b State Key Laboratory of Coordination Chemistry, Nanjing University, Nanjing, 210093, People’s
Republic of China, E-mail: wangqm@yctu.edu.cn (Wang Q)
Abbreviations:
MTT, 3-[4,5-Dimethylthiazo-2-yl]-2,5- diphenpyltetra-zolium bromide;
BSA, Bovine serum albumin;
CT-DNA, calf thymus-DNA;

Abstract
1
Five new α-aminophosphonates are synthesized and characterized by EA, FT-IR, H-NMR,
13C-NMR, 31P-NMR, ESI-MS and X-ray crystallography. The X-ray analyses reveal that the crystal
structures of 1-5 are monoclinic or triclinic system with the space group P 21/c, P-1, P-1, P2(1)/c
and P-1, respectively. All P atoms of 1-5 have tetrahedral geometries involving two O-ethyl
groups, one Cα atom, and a double bond O atom. The binding interaction of five new
α-aminophosphonate N-derivatives (1-5) with calf thymus(CT)-DNA have been investigated by
UV-visible and fluorescence emission spectrometry. The apparent binding constant (Kapp)values
follows the order: 1 ( 3.38×105 M-1 ) > 2 ( 3.04×105 M-1 ) > 4 ( 2.52×105 M-1 ) > 5 ( 2.32×105
M-1 )> 3 ( 2.10×105 M-1 ), suggesting moderate intercalative binding mode between the
compounds and DNA. In addition, fluorescence spectrometry of bovine serum albumin (BSA)
with the compounds 1-5 showed that the quenching mechanism might be a static quenching
procedure. For the compounds 1-5, the number of binding sites were about one for BSA and the
binding constants follow the order:1 (2.72×104 M-1) > 2 (2.27 × 104 M-1) > 4 (2.08 × 104 M-1) > 5
(1.79 ×104 M-1) > 3 (1.17 × 104 M-1). Moreover, the DNA cleavage abilities of 1 exhibit remarkable
changes and the in vitro cytotoxicity of 1 on tumor cells lines (MCF-7, HepG2 and HT29) have
been examined by MTT and shown antitumor effect on the tested cells.
Keywords: α-Aminophosphonate / DNA binding / Bovine Serum Albumin (BSA) binding /
Cytotoxicity
1 Introduction
Due to the structural similarity of α-aminophosphonates with α-amino acids, it is received an
increasing attention[1]. A considerable number of α-aminophosphonate derivatives are known

to be antiviral[2-3], antibacterial[4], anticancer[5-6], haptens of catalytic antibodies[7-8],
antibiotics and pharmacological agents[9-11] and herbicides[12]. The potential of
α-aminophosphonates such as a novel series of diphenyl 1-(arylamino)-1-(pyridin-3-
yl)ethylphosphonates, containing difluoro-methylenephosphonate group, 6-(phosphono-
difluoromethyl)-2-naphthoic acid and so on, acting as enzyme inhibitors pharmacological agents
has been established[13-19]. Moreover, our early research indicated that a-aminophosphonate
N-derivatives with the rigid structures can potently inhibit PTPs with some selectivity [20].
The field of anticancer metallodrugs is dominated by platinum based complex and the
mechanism of metallodrugs relies on direct DNA damage. DNA is the carrier of human genetic
information[21], the formation of human tumors has a close relationship with DNA[22], and
most of tumour was caused by the mutation of genetic[23]. The change of DNA structure were
detected in a variety of cancer cells, such as liver cancer[24], digestive system tumors[25] and
renal cell carcinoma[26], so DNA is always used as one of the most important targets for drug
design. While the toxic side of platinum based metallodrugs limited their for further use, so
many metal complexes and organic compounds with fewer side effects were investigated in
recent years for their tumor inhibiting properties. As early as 1961, Lerman discovered that
molecules with an aromatic structure could interact with DNA first, and this has been confirmed
by many molecules with such structures[27]. Therefore, α-aminophosphonate may also be
developed to interact with DNA.
Binding to plasma proteins is regarded as a reason for the α-aminophosphonate low general
toxicity. Bovine serum albumin is the most important and the most abundant carrier protein in
the plasma[28]. It plays an important role in the distribution and absorption of the drug[29-30].

Therefore, the study of the interaction mechanism between drug and bovine serum albumin can
provide the basis of theory for the design and development of drugs. Serum albumin contains
many kinds of ligands[31], which can interact with various endogenous and exogenous
compounds[32-33], so serum albumin can play a role in the storage and transportation of some
drugs or some other biological activities[34]. When smaller molecules interact with serum
albumin in the form of covalent bond, the serum albumin conformation will be changed[35-36],
which could provide valuable information and data to the disease prevention, diagnosis, curative
effect.
Here, we intend to report the preparation of five new α-aminophosphonate N-derivatives
(Scheme 1) and their DNA and BSA binding, cytotoxicity and preliminary apoptotic.
2 Results and discussion
2.1 Synthesis of α-aminophosphonate N-derivatives 1-5
A powerful and direct method for construction of P–C–N bonds is Pudovik reaction [37-39]. It
could form an asymmetric center in α-position to the phosphorus atom if there are two different
substituents in C-terminal. In this paper, five new α-aminophosphonate N-derivatives were
prepared, which exhibited characteristic either the rigid structure with one
α-aminoalkylphosphonate fragment.
2.2 Characterization of α-aminophosphonate N-derivatives 1–5
The elemental analysis data for 1–5 were consistent with the compositions of desired products.
All the compounds showed absorption bands of infrared in the region 3148–3244, 2983–2988,
1221–1228 and 970–975 cm-1 for stretch vibrations of N–H, Cα–H, P=O and P–Cα, respectively

[20].
The 31P-NMR spectroscopy is the most precise method and a very convenient tool for
determining the structure of the organophosphorus compounds. Chemical shifts of 31P NMR
spectra in the α-aminophosphonic acids are in range of 15–25 ppm referenced to 85% H3PO4 [37].
The 31P NMR spectra of all the compounds show one single peak. The 31P NMR spectra of 1-5
have signals with the chemical shifts of 20.447-22.573 ppm. The values agree with other diester
aminophosphonates, such as diethyl (1R)-1-{[(carbobenzyloxy]amino}ethylphosphonate (31P
NMR, DMSO-d6 25.89 ppm) [40], p-[N-Methyl(dietoxy- phosphonyl)(2-furyl)]toluidine (31P NMR
at 101 MHz, CDCl3 21.13 ppm) [41], it is also agree with our early results [20].
2.3 Crystal structures of 1-5.
In order to comprehensively understand the structural features of 1-5, the crystals in several
solvents or mix-solvents were attempted to grow. Suitable crystals for x-ray single crystal
diffraction were obtained from ethanol (1-4) and ethanol / H2O (5). Crystallographic data and
hydrogen bonds are listed in Table 1 & Table S1(see supporting information). Also the X-ray
structures of the α-aminophosphonates N-derivatives 1–5 are shown in Figs. 1-5.
Table 1 here
The crystal structures of 1-5 are monoclinic or triclinic system with the space group P 21/c, P-1,
P-1, P2(1)/c and P-1, respectively. Seen from Figs. 1-5, all P atoms have tetrahedral geometries
involving two O-ethyl groups, one Cα atom, and a double bond O atom. In the structures, Cα
atoms are chiral centres, which are responsible for the existence of optically activity. The C-P and
P=O bond lengths are almost comparable with the similar structures [20]. There are a lot of weak
interactions, such as O-H···O, N-H···O, C-H···O, C-H···π and N-O···π, resulting in stabilization in the

structures.
Figs. 1-5 here.
2.4 DNA-binding studies.
It is reported that one of an effective method in examining the binding mode of DNA with the
metal complex was electronic absorption spectroscopy[42]. The intercalative binding usually
results in a large hypochromism or hyperchromic and red shift due to the interaction between
the base pairs of DNA and aromatic chromophores. Absorption spectral changes of 1 in the
absence (dash line) and presence (solid line) of CT-DNA are shown in Fig. 6. The addition of
CT-DNA to 1 solution results in hyperchromic and no bathochromic shift. It is suggested the
association interation between 1 and DNA is intercalation[43]. The binding constant between 1
and CT-DNA was calculated according to the equation (1) & (2), is 1.69 × 104 M-1. The binding
constants Kb for compounds 2-5 were also calculated and were shown in Table 2. From Table 2,
we can see that the similar binding constants Kb for compounds 1-5.
Table 2 here
Fig. 6 here
To further clarifying the binding of 1-5, fluorescence spectrum was a sensitive test method and
was used to analyze and verify the form of interaction between compounds 1-5 and DNA.
Ethidium Bromide emits intense fluorescent light in the presence of DNA due to its strong
intercalation between the adjacent DNA base pairs[44], which could be quenched by the
addition of another molecule. The relative binding of 1 to CT - DNA was studied with an EB
bound CT - DNA solution in 5 mM Tris - HCl / 50 mM NaCl buffer ( pH = 7.20 ) and it was shown

in Fig.7. As shown in Fig.7, the emission intensity at 580nm (510 nm excitation) quenched
gradually upon addition of 1 in the Tris - HCl ( pH=7.20 ) solution of fixed EB - DNA concentration,
indicating that the form of interaction between 1 and DNA. Meanwhile, the fluorescence spectra
of EB - DNA showed that 1 can effectively compete and substitute EB[45]. The Kapp for 1 is 4.21 ×
104 M-1. Fluorescence titration results indicated that the derivative molecule has some
interaction with CT-DNA. The binding mode needs to be further explored. From Table 2, we
could find that the calculated apparent binding constant values (Kapp) follows the order: 1 ( 3.38
×105 M-1 ) > 2 ( 3.04×105 M-1 ) > 4 ( 2.52×105 M-1 ) > 5 ( 2.32×105 M-1 )> 3 ( 2.10×105 M-1 ),
which is consistent with the results obtained Kb values by UV spectroscopy. The calculated
binding constant is less than 107 M-1, indicating that the bonding effect between 1-5 and DNA
was medium[46-47]. Compounds 1-5 show medium potency between DNA and the substitute
groups and their positions slightly influence the inhibitory effects. Compared 1-4, we can see
that compound 3 with o-NO2 moiety reduces potency. Compared 5 with other four compounds,
substituent Cl atom at 4-position of aniline or aldehyde have the differences effect on DNA. The
results indicate that by rational design, α-aminophosphonate compounds are possibly developed
to bind to DNA.
Fig. 7 here
2.5 MTT assay
MTT assay is a colorimetric assay based on the conversion of the yellow tetrazolium salt to
purple formazan crystals by metabolically active cells[4]. The cytotoxic effects of 1 on the
viability of several human cancer cell lines ( MCF-7, HepG2 and HT29 ) were examined by MTT

assay. A variety of tumor cells were treated with 1 and incubated for 48 h at increasing
concentration, respectively. As shown in Fig 8, we observe that 1 was cytotoxic to all tested
tumor cells and inhibited the growth of cells in a dose-dependent manner. The IC50 values of 1
on MCF-7, HepG2 and HT29 were 83 ± 8 μM, 89 ± 7μM, 92 ± 6 μM. Cisplatin was taken as a
reference drug, and the IC50 values of cisplatin on MCF-7, HepG2 and HT29 were 9 ± 0.9 μM, 21±
2.7 μM, 13 ± 1.6 μM.
Fig. 8 here
2.6 Protein binding studies.
The interactions between BSA and chemicals have attracted increasing research interest in
recent years, since BSA is one of the most important proteins in the plasma, and has many
physiological functions in drug transport and drug metabolism[48]. From Fig. 9, the fluorescence
emission of BSA was reduced regularly with the increasing the concentration of 1 (there is no
fluorescent signal was found when compound 1 was excited at 280 nm), which confirmed that
the interaction between 1 and BSA has occurred.
The fluorescence quenching between 1 and BSA was described by the Stern–Volmer
equation, F0/F = 1+ Kqτ0[Q] = 1+ KSV[Q]. Where F0 and F represent the fluorescence intensities in
the absence and in the presence of quencher 1, kq is the quenching rate constant, τ0 the average
life-time of the biomolecule without quencher (about 10-8 s). KSV is the quenching constant and
[Q] is the concentration of quencher. KSV can be obtained from a slope by the plot of F0/F vs. [Q]
as shown in Table 3. The kq values ( 1012 M-1 s-1 ) of 1-5 are higher than the maximum scatter
collision-quenching constant, indicating the existence of static quenching mechanism[49].

Fig. 9 here
The binding constant and stoichiometry of BSA and 1 were estimated through the static
quenching equation [50].
F0  F
lg
 lg KA  nlg[Q]
F
where F and F0 are current and initial fluorescence intensities, respectively. Q is the quencher,
KA is the binding constant and n stands for the stoichiometry of BSA and 1. The n and KA can be
calculated by the slope and the intercept of the line of log(F0 - F)/F vs. log[Q] (Fig.10). Table 3
shows that 1 exhibits higher binding constants for BSA than 2-5, and the KA and n values follow
the order:1 (2.72×104 M-1, 1.03) > 2 (2.27 × 104 M-1, 0.95) > 4 (2.08 × 104 M-1, 0.79) > 5 (1.79 ×
104 M-1, 0.82) > 3 (1.17 × 104 M-1, 0.78). As expected, the values of n are indicated that 1:1
complex was formed between BSA and quencher.
Fig. 10 here
Table 3 here
3 Conclusions
Five new α-aminophosphonate N-derivatives (1-5) have been synthesized and characterized.
The structures of compounds 1-5 were determined by X-ray crystal analysis. The partial
intercalation between 1-5 and CT-DNA has been confirmed by using absorption and emission
spectral methods and medium binding strength follow the order: 1 > 2 > 4 > 5 > 3. Further, the
ability to bind to BSA also has been explored, and the quenching mechanisms of BSA with 1-5
were static procedures. The in vitro cytotoxicity of 1 on tumor cells lines (MCF-7, HepG2 and
HT29 ) have been assessed by MTT and the results indicated that 1 showed antitumor effect on
the tested cells.

4 Experimental
4.1 general methods
All reagents and solvents were purchased commercially and used without further purification.
Deionized water was used for all chemical experiments and double-distilled water was used to
prepare buffer solutions as well as biological evaluation. pH values of buffer solutions were
measured using a PHS-3TC pH meter. All reactions were magnetically stirred. Elemental analyses
(EAs) were carried out with a VARI-EL elemental analyzer. IR spectra (4,000–400 cm-1) were
1
recorded using a Shimadzu Fourier transform IR-8300 spectrometer in KBr disks. H-NMR and
13C-NMR spectra were recorded on a Bruker DRX-400 spectrometer with CCl3D as the internal
standard. 31P NMR spectra were recorded with the same spectrometer at 121.49 MHz with
H3PO4 (85%) as external standard. Electrospray ionization mass spectra (ESI-MS, negative mode)
were recorded with a Triple TOFTM 5600+ system in methanol/water solution. Fluorometric
experiments were carried out on a Cary Eclipse Spectrophotometer. The X-ray data of crystal 1
were collected by ω - scan method on a SMART 1K CCD area detector single-crystal
diffractometer that uses graphite mono-chromated Mo Kα radiation ( λ = 0.71073 Å ). The
structure was solved by direct methods and refined by least squares procedures on F2 using
SHELX-97 package.
4.2 Synthesis of α-aminophosphonates 1-5
Target α-aminophosphonates 1-5 were prepared by the method analogous to our reported
procedure[20]. According to scheme 1, α-aminophosphonates 1-5 were prepared by a two-step
procedure: (1) equimolar amounts (0.02 mol) of amine and salicylaldehyde were mixed in a 20

mL of ethanol solution and refluxed for 2 h. After the reacting mixture were cooled to room
temperature, the generated Schiff bases were crystallized from solution ( Yield: 1a 82%, 2a 92%,
3a 90%, 4a 88%, , 5a 76% ); (2) a 10-20 mL of ethanol solution containing 20 mmol of diethyl
phosphonate was added dropwise into the stirred solution of an equimolar amount of Schiff
base compounds in a 30 mL of ethanol. And then, the mixture was refluxed for 14-16 h with
constant stirring. The solvent was removed by a rotary evaporator. The oily residue was dissolved
in 10–20 mL of ethanol, block crystal were obtained from ethanol (1-4) and ethanol / H2O (5).
4.2.1 Diethyl (4-chlorophenylamino)(2-methoxyphenyl)methylphosphonate ( 1 )
Yield: 2.681 g, 35%. Yellow crystals were obtained from ethanol. EAs: calcd / found ( % ) for
C18H23ClNO4P ( 1 ): C 56.33 / 56.30, H 6.04 / 5.99, N 3.65 / 3.65. IR ( cm-1 ): 3242 ν( O - H, N - H ),
2988 ν ( Cα - H ), 1226 ν ( P = O ), 1043 and 1022 ν ( P - O - C ), 975 ( C - P ). 31P NMR ( D2O / CDCl3,
1
ppm ): δ 22.494. H NMR (400 MHz, CDCl3) δ 7.35 (dd, J = 8.8, 2.3 Hz, 2H), 7.04 (d, J = 8.9 Hz, 2H),
6.87 (d, J = 8.5 Hz, 2H), 6.52 (d, J = 8.7 Hz, 2H), 4.65 (d, J = 23.7 Hz, 1H), 4.15 – 4.07 (m, 2H), 3.97 –
3.88 (m, 1H), 3.78 (s, 3H), 3.72 – 3.64 (m, 1H), 1.29 (t, J = 7.1 Hz, 3H), 1.13 (t, J = 7.1 Hz, 3H). 13C
NMR( CDCl3, ppm ): δ 159.418, 159.389, 144.866, 144.722, 128.993, 127.081, 123.132, 115.132,
114.138, 63.426, 63.356, 63.256, 63.188, 56.293, 55.237, 54.776, 16.475, 16.418, 16.290, 16.233.
Exact mass for 1: 383.11, ESI-MS: [ M - H ]-, found: 382.15.
4.2.2 Diethyl (4-chlorophenylamino)(4-methoxyphenyl)methylphosphonate ( 2 )
Yield: 3.217 g, 42%. Yellow crystals were obtained from ethanol. EAs: calcd / found ( % ) for
C18H23ClNO4P ( 2 ): C 56.33 / 56.35, H 6.04 / 5.92, N 3.65 / 3.64. IR ( cm-1 ): 3244 ν( O - H, N - H ),
2986 ν ( Cα - H ), 1222 ν ( P = O ), 1048 and 1026 ν ( P - O - C ), 975 ( C - P ). 31P NMR ( D2O / CDCl3,
1
ppm ): δ 22.537. H NMR (400 MHz, CDCl3) δ 7.35 (dd, J = 8.8, 2.3 Hz, 2H), 7.04 (d, J = 8.8 Hz, 2H),

6.87 (d, J = 8.5 Hz, 2H), 6.54 – 6.50 (m, 2H), 4.65 (d, J = 23.7 Hz, 1H), 4.17 – 4.05 (m, 2H), 3.97 – 3.88
(m, 1H), 3.78 (s, 3H), 3.73 – 3.63 (m, 1H), 2.92 (d, J = 28.7 Hz, 1H), 1.29 (t, J = 7.1 Hz, 3H), 1.13 (t, J =
7.1 Hz, 3H). 13C NMR( CDCl3, ppm ): δ 159.423, 159.394, 144.878, 144.729, 129.002, 127.080,
127.056, 123.127, 115.119, 114.145, 63.448, 63.378, 63.213, 56.273, 55.245, 54.757, 16.474,
16.418, 16.289, 16.232. Exact mass for 2: 383.11, ESI-MS: [ M - H ]-, found: 382.12.
4.2.3 Diethyl (4-chlorophenylamino)(2-nitrophenyl)methylphosphonate( 3 )
Yield: 2.627 g, 33%. Yellow crystals were obtained from ethanol. EAs: calcd / found ( % ) for
C18H23ClNO4P ( 3 ): C 51.20 / 51.23, H 5.06 / 4.99, N 7.02 / 7.05. IR ( cm-1 ): 3243 ν( O - H, N - H ),
2984 ν ( Cα - H ), 1226 ν ( P = O ), 1045 and 1026 ν ( P - O - C ), 973 ( C - P ). 31P NMR ( D2O / CDCl3,
1
ppm ): δ 20.447. H NMR (400 MHz, CDCl3) δ 8.21 (d, J = 8.5 Hz, 2H), 7.63 (dd, J = 8.8, 2.2 Hz, 2H),
7.06 (dd, J = 8.7, 1.6 Hz, 2H), 6.46 (d, J = 8.9 Hz, 2H), 4.80 (d, J = 25.0 Hz, 1H), 4.20 – 4.10 (m, 2H), 4.07
– 3.99 (m, 1H), 3.87 (dd, J = 15.0, 7.9 Hz, 1H), 1.31 (t, J = 7.1 Hz, 3H), 1.18 (t, J = 7.1 Hz, 3H). 13C
NMR( CDCl3, ppm ): δ 147.718, 147.679, 144.217, 144.072, 143.522, 143.488, 129.254, 128.621,
128.571, 123.869, 123.844, 114.982, 63.844, 63.775, 63.662, 63.594, 56.869, 55.393, 16.461,
16.405, 16.289, 16.232. Exact mass for 3: 398.08, ESI-MS: [ M - H ]-, found: 397.08.
4.2.4 Diethyl (4-chlorophenylamino)(4-nitrophenyl)methylphosphonate ( 4 )
Yield: 3.582 g, 45%. Yellow crystals were obtained from ethanol. EAs: calcd / found ( % ) for
C18H23ClNO4P ( 4 ): C 51.20 / 51.18, H 5.06 / 5.12, N 7.02 / 7.02. IR ( cm-1 ): 3183 ν( O - H, N - H ),
2986 ν ( Cα - H ), 1221 ν ( P = O ), 1052 and 1028 ν ( P - O - C ), 970 ( C - P ). 31P NMR ( D2O / CDCl3,
1
ppm ): δ 21.836. H NMR (400 MHz, CDCl3) δ 7.91 (d, J = 8.8 Hz, 2H), 7.44 (dd, J = 8.8, 2.2 Hz, 2H),
7.26 (dd, J = 8.3, 1.5 Hz, 2H), 6.67 (d, J = 8.7 Hz, 2H), 3.80 (d, J = 25.0 Hz, 1H), 4.11 – 4.24 (m, 2H), 3.87
(dd, J = 15.0, 7.9 Hz, 1H), 1.28 (t, J = 7.3 Hz, 3H), 1.14 (t, J = 7.3 Hz, 3H). 13C NMR( CDCl3, ppm ): δ

148.908, 148.003, 145.112, 144.876, 144.002, 143.978, 131.234, 129.221, 128.674, 124.345,
123.244, 115.482, 63.776, 63.549, 63.362, 63.288, 55.692, 55.421, 16.645, 16.612, 16.544,
16.532. Exact mass for 4: 398.08, ESI-MS: [ M - H ]-, found: 397.09.
4.2.5 Diethyl (4-nitrophenylamino)(4-chlorophenyl)methylphosphonate ( 5 )
Yield: 3.821 g, 48%. Yellow crystals were obtained from ethanol. EAs: calcd / found ( % ) for
C18H23ClNO4P ( 5 ): C 51.20 / 51.22, H 5.06 / 5.02, N 7.02 / 7.04. IR ( cm-1 ): 3242 ν( O–H, N–H ),
2983 ν ( Cα–H ), 1228 ν ( P=O ), 1042 and 1026 ν ( P–O–C ), 972 ( C–P ). 31P NMR ( D2O / CDCl3,
ppm ): δ 20.600. 1H NMR (400 MHz, CDCl3) δ 8.04 – 7.97 (m, 2H), 7.40 (dd, J = 8.6, 1.9 Hz, 2H), 7.34
(dd, J = 8.6, 2.7 Hz, 2H), 6.57 (dd, J = 9.2, 2.4 Hz, 2H), 4.79 (dd, J = 24.2, 2.6 Hz, 1H), 4.19 – 4.08 (m,
2H), 3.97 (ddd, J = 17.3, 10.8, 5.0 Hz, 1H), 3.79 – 3.68 (m, 1H), 1.31 (td, J = 7.1, 1.3 Hz, 3H), 1.16 (t, J =
7.1 Hz, 3H). 13C NMR( CDCl3, ppm ): δ 151.528, 139.315, 139.223, 134.490, 134.454, 133.107,
129.156, 126.103, 112.464, 64.069, 63.997, 63.700, 63.666, 55.719, 55.666, 54.207, 54.155,
16.454, 16.398, 16.259,16.203. Exact mass for 5: 398.08, ESI-MS: [ M - H ]-, found: 397.04.
4.3 X-ray Crystallography
All the data of the compounds 1-5 were collected on a Bruker Smart Apex II diffractometer
equipped with 1K CCD instrument by using a graphite monochromator utilizing Mo-Kα radiation
(λ = 0.71073 Å) at room temperature. Cell parameters were determined using SMART software
[51]. Data reduction and corrections were performed using SAINTPlus. Absorption corrections
were made via SADABS [52]. The structures were solved by direct methods with the program
SHELXS-97 and refined by full-matrix least-squares methods on all F2 data with SHELXL-97 [53].
The non-H atoms were refined anisotropically. Hydrogen atoms attached C were added
theoretically and treated as riding on the concerned atoms. The final cycle of full-matrix

least-squares refinement was based on observed reflections and variable parameters.
4.4 DNA binding study
UV absorption and fluorescence spectrometry have been employed to investigate the
interaction between the compounds 1-5 and CT-DNA( 5 mM Tris-HCl / 50 mM NaCl, pH
7.20 ). The buffered solution of CT-DNA has been added successively to a fixed amount of
the complex. By monitoring the change in the absorption intensity of the spectral bands
after incubated each time for 5 min, the intrinsic equilibrium binding constant (Kb) and the
binding site size (s) of 1-5 to CT-DNA were obtained by regression analysis using the
equation:[54]
2
(b (b2  2Kb Ct[DNA]/ s)1/2
)
a  f

(1)
(2)
b  f
2KbCt
b 1 KbCt Kb[DNA]/ 2s
Where εa is the absorb intensity observed at a given DNA concentration, εf is the absorb
intensity of free complex in solution, εb is the absorb intensity of complex when fully bound to
DNA (it is assumed that when further addition of DNA does not change the absorb intensity), Kb
is the equilibrium binding constant, Ct is the total DNA concentration, [DNA] is the DNA
concentration, and s is the binding site size. Both Kb and s are obtained from the best fit line. The
non-linear least-squares analyses were done through the use of Origin Lab, version 7.5.
The relative binding capacity of 1-5 to CT-DNA have been examined by fluorescence using
ethidium bromide (EB) as fluorescence probe. The samples were excited at 510 nm, and the
emission range was recorded at 500 - 700 nm. The fluorescence intensities were recorded with
increasing amounts of the added 1-5 concentrations.
Acknowledgments

This work were financially supported by the National Natural Science Foundation for
Young Scientists of china(21301150, 21571154), the Natural Science Foundation of the
Jiangsu Higher Education Institutions of China (13KJB150037, 14KJB150027), the
Post-Doctoral Foundation of Jiangsu Provincial (1501032B), the six Taleng Peak Project in
Jiangsu Province ( SWYY-063 ), the practice innovation training program projects for the
Jiangsu College students (201310324014Z, 201610324029Y, 201410324053), the Natural
Science Foundation of Yancheng Teachers’ University (14YCKL006) and sponsored by Qing
Lan Project of Jiangsu Provincial.
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Captions for Tables, Figs and Scheme
Table 1 Crystallographic data for 1 - 5
Table 2 Absorption spectral properties of 1-5 bound to CT-DNA
Table 3 The quenching constants (KSV), binding constants (KA), binding sites (n) of the BSA-1-5
Fig. 1 X-ray structure of the 1 with a 30% probability of thermal ellipsoids.
Fig. 2 X-ray structure of the 2 with a 30% probability of thermal ellipsoids.
Fig. 3 X-ray structure of the 3 with a 30% probability of thermal ellipsoids.
Fig. 4 X-ray structure of the 4 with a 30% probability of thermal ellipsoids.
Fig.5 X-ray structure of the 5 with a 30% probability of thermal ellipsoids.
Fig.6 Absorption spectra of 1 ( 10 μM ) in the absence (dash line) and presence (solid line) of
increasing amounts of CT-DNA (40, 80, 120, 160, 200, 240 μM) in 5 mM Tris-HCl/50 mM NaCl
buffer (pH = 7.20 ). The arrow shows the absorbance changes on increasing DNA concentration.
Inset: Plot of (εa-εf)/(εb-εf) versus [DNA] for the titration of DNA to 1.
Fig.7 Emission spectra of EB-CT-DNA in the absence (dash line) and in the presence (other lines)
of 1 with increasing amounts (10, 20, 80, 160, 240, 320, 400, 480 and 560 μM). Inset: plot of F0 /
F versus [Compound 1] showing linearity.
Fig. 8 Cell viability of MCF-7, HepG2 and HT29 cells treated with 1 at 0, 10, 50, 100, 300 and
500 μM for 48 h.

Fig.9 Fluorescence emission spectra of the BSA (1.0 μM) system in the absence and presence of
complex 1 (0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75μM, respectively). Inset: the plot of F0/F versus 1
concentration.
Fig. 10 Plot of log (F0 - F)/F vs. log[Q] for BSA in the presence of compounds 1-5
Scheme 1 The synthesis of the rigid α-aminophosphonate N-derivatives 1-5 in two-step by
Pudovik reaction.

Table 1
Table 1 Crystallographic data for 1 - 5
1
2
3
4
5
C18H23ClNO4
P
Empirical formula
C18H23ClNO4P C17H20ClN2O5P C17H20ClN2O5P C17H20ClN2O5P
CCDC
1405033
383.79
296(2) K
0.71073 A
monoclinic
P 21/c
1405040
383.79
296(2) K
0.71073 A
Triclinic
P-1
1405043
398.77
1405035
398.77
296(2) K
0.71073 A
Monoclinic
C2
1405037
398.77
Formula weight
Temperature
Wavelength
Crystal system
space group
296(2) K
0.71073 A
Triclinic
P-1
296(2) K
0.71073 A
Triclinic
P-1
9.1255(3)
15.1556(5)
14.4507(5
90
8.8782(3)
8.9732(3)
13.2136(5)
81.446(2)
73.770(2)
83.748(2)
996.96(6)
2
8.1644(2)
11.6975(3)
11.7798(4)
106.796(2)
109.576(2)
100.364(2)
965.83(5)
2
22.3212 (2)
8.1823(9)
21.6247(2)
90
7.7916(2)
10.6084(3)
12.7751(4)
107.2550(10)
92.8790(10)
101.7190(10)
980.48(5)
2
a(Ǻ)
b(Ǻ)
c(Ǻ)
α(°)
97.445(2)
90
98(3)
β(°)
γ(°)
V(Ǻ3)
Z
90
1981.72(11)
4
3903.9(7)
8
Dcalc(g cm-3)
1.286
1.278
1.371
1.357
1.351
808
404
416
1664
416
F(000)
Goodness of fit
Reflections collect.
Reflections unique
Rint
1.091
1.047
1.044
1.259
1.051
12853
16516
15925
14955
15412
3412
4562
4329
4153
4434
0.0270
0.0524
0.1389
0.0652
0.1530
97.50%
0.0221
0.0623
,0.1769
0.0710,
0.1940
98.30%
0.0239
0.0297
0.0912
0.2378
0.1093,
0.2647
98.10%
0.0231
0.0925
0.0725
R1, wR2[I> 2(I)]
R1,wR2(all data)
0.2745
0.2051
0.1111,
0.3009
0.0855,
0.2296
98.30%
98.60%
Completeness (%)
Largest diff. peak
(e Ǻ3) and hole
0.379, -0.318 0.383, -0.525
1.221, -0.777
0.664, -0.751
0.456, -0.499
-10 ≤ h ≤ 10
-16 ≤ k ≤ 18
-17 ≤ l ≤ 16
-11 ≤ h ≤ 11
-11 ≤ k ≤ 11
-17 ≤ l ≤ 17
-10 ≤ h ≤ 10
-15 ≤ k ≤ 15
-13 ≤ l ≤ 15
-15 ≤ h ≤ 15
-11 ≤ k ≤ 11
-23 ≤ l ≤ 23
-9 ≤ h ≤ 10
-13 ≤ k ≤ 13
-16 ≤ l ≤ 16
Limiting indices

Table 2
Table 2 Absorption spectral properties of 1-5 bound to CT-DNA
Complex λmax/nm
Change in absorbance
Kb(M-1)
Kapp(M-1)
1
2
3
4
5
278
280
278
276
278
Hyperchromism
Hyperchromism
Hyperchromism
Hyperchromism
Hyperchromism
1.69 × 104
1.52 × 104
1.05 × 104
1.26 × 104
1.16 × 104
3.38×105
3.04×105
2.10×105
2.52×105
2.32×105

Table 3
Table 3 The quenching constants (KSV), binding constants (KA), binding sites (n) of the BSA-1-5
System
KSV×105(M-1)
Kq×1013(M-1) KA×104(M-1)
n
BSA-1
BSA-2
BSA-3
BSA-4
BSA-5
2.58
1.97
1.26
1.62
1.44
2.58
1.97
1.26
1.62
1.44
2.71
2.27
1.17
2.08
1.79
1.03
0.95
0.79
0.82
0.78

Scheme 1
Scheme 1 The synthesis of the rigid α-aminophosphonate N-derivatives 1-5 in two-step by
Pudovik reaction.

Fig. 1
Fig. 1 X-ray structure of the 1 with a 30% probability of thermal ellipsoids.

Fig. 2
Fig. 2 X-ray structure of the 2 with a 30% probability of thermal ellipsoids.