PEG-SO3H catalyzed synthesis and cytotoxicity of α-aminophosphonates

Article abstract of DOI:10.1016/j.ejmech.2011.11.026

One pot three-component PEG-SO₃H catalyzed reaction of 4-(Pyridin-4-yl)benzaldehyde and triethyl phosphite with various primary amines afforded α-aminophosphonates with high yields by the Kabachnik-Field's reaction. These new structurally diver

Full text of DOI:10.1016/j.ejmech.2011.11.026

European Journal of Medicinal Chemistry 47 (2012) 553e559
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
PEG-SO3H catalyzed synthesis and cytotoxicity of a-aminophosphonates
C. Bhupendra Reddy a, K. Suresh Kumar a, M. Anil Kumar a, M. Veera Narayana Reddy a,
B. Satheesh Krishna a, M. Naveen b, M.K. Arunasree b, C. Suresh Reddy a, C. Naga Raju a,
,
C. Devendranath Reddy a
*
a Department of Chemistry, Sri Venkateswara University, Tirupati 517 502, India
b Institute of Lifesciences, University of Hyderabad Campus, Hyderabad 500 046, India
a r t i c l e i n f o
a b s t r a c t
Article history:
One pot three-component PEG-SO3H catalyzed reaction of 4-(Pyridin-4-yl)benzaldehyde and triethyl
Received 10 June 2011
Received in revised form
9 November 2011
Accepted 14 November 2011
Available online 20 November 2011
phosphite with various primary amines afforded
KabachnikeField’s reaction. These new structurally diversified set of
evaluated for their anti-tumor activity on human chronic myeloid leukemia cells (K 562), human colon
carcinoma cells (Colo 205) along with non-cancerous human embryonic kidney cells (HEK 293). They
showed moderate activity on both cancerous cells and non-cancerous cells.
Ó 2011 Elsevier Masson SAS. All rights reserved.
a
-aminophosphonates with high yields by the
-aminophosphonates (4aej) were
a
Keywords:
Poly ethylene glycol sulfonic acid (PEG-
SO3H)
4-(Pyridin-4-yl)benzaldehyde
Human chronic myeloid leukemia cells
(K 562)
Human colon carcinoma cells (Colo 205)
Human embryonic kidney cells (HEK 293)
1. Introduction
a-aminophosphonates could not improve the product yields.
Similarly many other reported methods were also not satisfactory
since they required toxic chemicals as reagents, complicated
experimental set-up, long reaction times and complicated workup
procedures. In the recent past, use of soluble polymer supported
a
-Aminophosphonates are an important class of bioactive
molecules. Their use as peptide mimics [1], enzyme inhibitors [2],
antibiotics [3] and anti HIV [4], anti cancer [5] and thrombotic [6]
agents has proved their vast potentiality both as direct drugs and
as drug precursors for several disease manifestations. Thus, their
significant role in life processes prompted the development of
many elegant synthetic approaches for them.
catalyst for the synthesis of a-aminophosphonates gained attention
due to the use of homogeneous reaction conditions [17e22].
However this procedure also could not afford the pure products in
satisfactory yields. Our continuous study to synthesize the target
specific less toxic and effective bioactive organophosphorus
compounds resulted in the development of an elegant one-pot
The chiral Brønsted acid catalyzed synthesis of
a-amino-
phosphonates had achieved through enantioselective hydro-
phosphonylation of imines, which is having the synthetic
importance [7]. Similarly the Lewis acid promoted nucleophilic
phosphite addition to imines is one of the most convenient
methods to obtain them [8e13]. The major drawback of reported
methodologies is deactivation of the lewis acid catalyst by water
released during the course of this reaction. Even the reported
catalysts such as In(OTf)3 [14], H3PW12O40 [15] and Na2CaP2O7
[16] used for the conversion of carbonyl compounds into
method for the synthesis of
a-aminophosphonates using poly
ethylene glycol sulfonic acid (PEG-SO3H) [23,24] as an efficient
recyclable catalyst under mild conditions.
In recent, some
viral drugs containing a pyridazine [25], amide moieties [26] and
anti-tumor drugs containing a chiral -amino carboxamide [27],
a-aminophosphonates were reported as anti-
a
benzthiazole moieties [28]. In this connection we had also reported
various biologically active organophosphorus compounds of
diverse classes [29e31] including a-aminophosphonates [32e35]
and some are identified as the potential anti cancer agents
[36,37]. Despite reasonable progress achieved in cancer chemo-
therapy, high toxicity and low specificity of the current medications
* Corresponding author. Tel.: þ918772289479; fax: þ918772289555.
E-mail address: profcdr@yahoo.co.in (C.D. Reddy).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.11.026

554
C.B. Reddy et al. / European Journal of Medicinal Chemistry 47 (2012) 553e559
N
Entry
4a
R
Entry
4f
R
O
P
O
P
OC2H5
OC2H5
H
C2H5O
C2H5O
H
Br
N
N
N
4b
4c
4d
4g
4h
4i
F
Cl
S
N
Br
N
N
S
N
(4c)
(4g)
Cl
Fig. 1. Cytotoxic a-aminophosphonates 4c and 4g.
NO
2
O N
2
S
N
are driving search for the development of more effective, less toxic
cancer drugs. In pursuit of this goal we continued our research and
accomplished synthesis of a series of new a-aminophosphonates
4e
4j
NO
2
(4aej) and studied their anti-tumor activity. We are successful in
identifying the newly synthesized compounds 4c & 4g as the
potential cancer cytotoxic compounds (Fig. 1). Further, this group of
compounds with an aminophosphonate pharmacophore may even
have broad range of bioactivity against many other bacterial and
viral diseases. Our survey revealed that no such work has been
reported on these compounds.
Fig. 2. Structure of diethyl (aryl/heteroaryl amino)(4-(pyridine-4-yl)phenyl)methyl
phosphonates (4aej).
study the variations in cytotoxicity as a function of the chemical
structure (Fig. 2).
To establish optimization experimental conditions for the
preparation of 4aej, a typical reaction with aldehyde (1), aniline (2)
and phosphite (3), was carried out both by conventional as well as
by PEG-SO3H catalyzed condition in various solvents. In conven-
tional preparation in refluxing toluene even after 14e20 h of
reaction, only unsatisfactory product yield (70%) was observed.
When the same reaction was performed in the presence of PEG-
SO3H as catalyst at 40e50 ꢀC for the duration of 2 h 96% yield of the
product was obtained. Similarly the PEG-SO3H catalyzed synthesis
of 4a was carried out in various solvents like dichloromethane,
tetrahydrofuran, ethanol and toluene at various concentrations of
PEG-SO3H catalyst with 1.0, 0.5, 0.1 M ratios (Table 1). It was found
that toluene as a solvent with 0.1 M quantity of the PEG-SO3H
catalyst are ideal for the synthesis of compound 4a.
2. Results and discussion
2.1. Chemistry
The three-component reaction of 4-(Pyridin-4-yl)benzaldehyde
(1) and triethyl phosphite (3) with various aryl/heteroaryl
substituted primary amines (2) led to the formation of diethyl (aryl/
heteroaryl amino)(4-(pyridine-4-yl)phenyl)methyl phosphonates
(4aej) (Scheme 1). In the first step, the corresponding imine
intermediate (2laej) was formed from the aldehyde (1) and amines
(2aej) and in the second step subsequent addition of Phosphite (3)
to the C]N bond of the imine (2laej) occurs to from the corre-
sponding
a
-aminophosphonates (4aej). However, this one-pot
The same reaction for the synthesis of compound 4a with
various catalysts in toluene under different temperature conditions
was recorded and the analysis is presented in Table 2 as an effective
optimization for the selection of catalyst.
The advantage of PEG-SO3H as a catalyst is that it is water stable,
non-corrosive, environmentally benign, recyclable and works effi-
ciently with a wide variety of aryl/heteroaryl substrates. The
comparative experimental data on the preparation of all the title
compounds with conventional and PEG-SO3H catalyzed pathways
in toluene are given in Table 3.
KabachnikeFields reaction is not straightforward, because water
formed during imine formation in the first step hydrolyzes the
imine intermediate (2laej) and thus retards the forwarded process
of the reaction. This drawback is prevented or at least minimized by
the use of PEG-SO3H catalyst which traps the released water and
subsequently drive the reaction forward to the second step facili-
tating phosphite (3) addition to imine (2laej) to form
a-aminophosphonates.
Several members of structurally diversified set of
a-amino-
phosphonates (4aej) were designed with various aryl/heteroaryl
primary amine moieties substituted with different electron with-
The proposed structures for the synthesized compounds 4aej
were confirmed by IR, 1H, 13C, 31P and mass spectrometry and
elemental analyses. The IR spectra of 4aej showed the expected
absorption bands at 3350e3330 and 1255e1233 cmꢁ1 for theNH and
drawing and donating groups at the
a-carbon atom of 4aej. The
main aim in the design and synthesis of the title compounds is to
Scheme 1. Synthesis of Diethyl (aryl/heteroaryl amino)(4-(pyridine-4-yl)phenyl)methyl phosphonates (4aej).

C.B. Reddy et al. / European Journal of Medicinal Chemistry 47 (2012) 553e559
555
Table 1
Table 3
Optimization of catalyst at different concentrations with various solvents for the
Assessment of the synthesis of compounds 4aej with conventional and PEG-SO3H
preparation of 4a.
catalyzed methods.
Entry
Solvent
Yield (%)b
1.0a
Entry
R
Reaction time in h (yield in %) Melting
point (ꢀC)
0.5a
0.1a
Conventional
method
PEG-SO3H
Catalyzed
method
1
2
3
4
Dichloromethane
Tetrahydrofuran
Ethanol
60.0
65.0
70.0
95.0
45.0
55.0
60.0
93.0
20.0
40.0
55.0
95.0
Toluene
a
Moles of PEG-SO3H.
Yields were given as an average value from two independent consecutive
b
4a
4b
14.0 (75.0)
14.5 (80.0)
2.0 (96.0)
2.5 (93.0)
155e157
144e146
determinations.
P]O stretching vibrations [38]. In the 1H NMR spectra, the
compounds 4aej gave triplet at
couplingwiththeprotonatthe -carbonand insomecompoundsthe
NH proton appeared as a broad singlet at 5.67e5.04. The doublet of
doublet signal at 4.98e4.60 is for PeCH proton, the multiplet at
4.19e3.66 is for POCH2 proton [39]. In the 13C NMR the doublet at
68.6e52.8 is ascribed for the -carbon. The other carbons gave
d 5.78e5.02 due to its vicinal
a
F
d
d
Cl
d
d
a
chemical shifts in their expected region. 31P chemical shifts were
4c
15.5 (85.0)
3.5 (94.0)
163e165
observed as singlet at d 26.45e23.50 for all the compounds 4aej.
Br
2.2. Pharmacology
The cytotoxicity of synthesized compounds 4aej was evaluated
in vitro against human chronic myeloid leukemia cells (K 562) and
human colon carcinoma cells (Colo 205) after 24 h exposure and
their IC50 values were determined from a graph of cell viability
4d
4e
14.0 (79.0)
15.5 (82.0)
2.5 (91.0)
3.0 (95.0)
175e177
153e155
Cl
NO
2
measured over a range of concentrations between 1 and 100 mM.
Each data point was the average of two determinations that in all
cases differed by 8e10% or less. These results are summarized in
Table 4. Initially, the IC50 was determined at a broad range of
NO
concentrations specifically 1, 10 & 100
range of concentrations specifically 2, 4, 8, 10, 16, 32 & 64
m
M and then at a narrow
2
mM of the
title compounds against the cell lines. For this data, a line graph was
plotted between concentrations (X-axis) versus % inhibition (Y-axis)
and then an intersection drawn at 50% inhibition on Y-axis and then
correlated to the concentration value on X-axis. Finally that
4f
15.5 (70.0)
17.0 (76.0)
17.5 (72.0)
3.5 (91.0)
4.0 (87.0)
4.5 (89.0)
148e150
150e152
159e161
N
N
concentration value is considered as IC50 in
are summarized in Table 4.
mM and these results
4g
4h
From the data it is revealed that all compounds exhibited
different range of significant cytotoxic activities varying from
35
cytotoxicity data compounds 4c and 4g with 3-bromo aniline and
3-aminopyridine moieties at the -carbon atom showed highest
activity against the K 562 cell lines with (IC50 10.0 and 10.0
mM to 5 mM due to structural differences. As evident from the
S
N
a
mM
respectively). Similarly the same compounds 4c and 4g exhibit high
S
N
156e158
170e172
4i
4j
18.0 (70.0)
20.0 (71.0)
5.0 (82.0)
6.0 (85.0)
Table 2
Optimization of catalyst at different temperatures with various catalysts for the
preparation of 4a.a
Yield (%)b
O N
2
Entry
Catalyst
Time (h)
S
N
rt
50 ꢀC
1
2
3
4
5
6
7
8
9
Bismuth chloride
Ferric chloride
Silica sulfuric acid
Silica supported Tantalum chloride
Triphenyl phosphine
6
2
5
22
12
24
6
1.5
1.5
2
e
92
92
87
92
86
61
42
59
91
98
20
87
92
e
cytotxicity against Colo 205 cell lines with IC50 5.0 and 8.0
mM
respectively. The 4-methylamino pyridine derivative (4f) also has
very low IC50 data. Their bioactivity data is presented in the
graphical representation also (Fig. 3).
b-Cyclodextrin
64
38
59
60
89
Sulfamic acid
p-Toulenesulfonic acid
Cellulose Sulfuric acid
PEG-Sulfuric acid
The Percentage inhibition of growth in Human chronic myeloid
leukemia cells (K 562), Human colon carcinoma cells (Colo 205)
and Human embryonic kidney cells (HEK 293) following treatment
10
a
Reaction conditions: All reactions were carried in toluene with 1 mmol of
with by the compounds 4aej were measured at 1
mM and 10 mM
catalyst.
b
concentrations which were the mean values determined from two
Isolated yield.

556
C.B. Reddy et al. / European Journal of Medicinal Chemistry 47 (2012) 553e559
Table 4
In vitro cytotoxic activity of title compounds (4aej) against K 562 and Colo 205 cell
lines.a
Entry
IC50
(m
M)b
K 562
Colo 205
4a
4b
4c
4d
4e
4f
4g
4h
4i
>100.0
22.0
10.0
19.0
20.0
32.0
10.0
24.0
35.0
31.0
18.0
10.0
5.0
12.0
14.0
10.0
8.0
10.0
15.0
10.0
4j
a
IC50 values were determined from growth inhibition curves (1e100
The reported values are the average of two determinations that in all cases
m
M).
b
differed by 8e10 % or less.
120
100
80
K562
Colo-205
IC50
(µM)
60
40
20
Fig. 4. Diethyl (phenyl amino)(4-(pyridine-4-yl)phenyl)methyl phosphonate.
3. Conclusion
In conclusion, this work describes an efficient one-pot synthesis
of
a-aminophosphonates in excellent yields by the reaction of
aldehyde, amine and tri ethyl phosphite in the presence of PEG-
SO3H as catalyst. The advantages of this method are mild reaction
conditions, use of eco-friendly reusable catalyst, easy workup and
high product yields. Being an effective and green procedure, this
0
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
Compounds (4a-j)
Fig. 3. IC50 values of the title compounds (4aej) against K 562 and Colo 205.
may be the method of choice for commercial production of
a-
aminophosphonates. Further two of these compounds, 4c and 4g
exhibited good cytotoxicity on K562 and Colo 205 cancerous cells.
The structure activity relationship studies analyzed for the title
compounds were strengthened the results obtained for the
compounds 4c, 4g as promising cytotoxic agents. The relation for
this had established by the designing itself by the presence of 4-
(Pyridin-4-yl)phenyl group as a fixed bioactive moiety in all the
title compounds. In addition to this incorporation of active 3-
bromophenyl and pyridine-3-methylene electron withdrawing
independent experiments with duplicates in each other and the
reported values are the average of two determinations that in all
cases differed by 7e10% or less.
It is worthwhile to note that all these compounds have no
significant cytotoxicity on non-cancerous HEK 293 cells. This study
thus discovered a new family of diethyl (substituted amino)
(4-(pyridine-4-yl)phenyl)methyl phosphonates (4aej) that have
significant target specific cytotoxicity on some cancer cells (Table 5).
groups at nitrogen atom of the
linked to phosphorus atom through the bioactive
a
-aminophosphonate which is
-carbon. These
Table 5
a
Percent inhibition of growth of in three cancer cell lines caused by 4aej 1
10 M concentration.
mM and
two compounds 4c & 4g were also supported by the significant
properties like logp (5.72 & 4.55), polaraizability and hydrophobic
nature (41.80 & 42.16), total energy (92.94e133.76 & 96.25e138.15)
and also sterically feasible ones with molecular weights (475.31 &
411.43) which satisfy the requirements of a potential drug and
possible to have precise interaction with the biological systems
also. Finally it is concluded that the results provided a foundation
for the design and development of some more structurally diver-
m
Entry
% of inhibitionb
K 562
Colo 205
1.0
HEK293a
1.0
m
M
10.0
m
M
m
M
10.0
m
M
1.0
mM
10.0 mM
4a
4b
4c
4d
4e
4f
4g
4h
4i
0.0
0.0
26.2
53.8
24.9
25.8
15.7
48.4
24.0
15.7
18.9
18.1
44.5
31.3
35.0
39.5
14.0
27.6
37.1
36.6
34.4
31.0
47.0
71.6
45.1
41.1
53.4
60.1
47.9
41.0
48.1
0.0
0.2
1.0
1.2
0.0
0.5
3.1
1.0
2.5
0.0
0.0
1.2
2.3
3.4
1.2
1.9
3.6
2.5
2.5
0.3
26.0
41.7
14.3
0.0
0.0
26.0
0.0
sified a-aminophosphonates as potential cytotoxic agents.
4. Experimental
0.0
0.9
4.1. General
4j
a
HEK 293 is taken as non-cancerous cell line.
b
Chemicals were procured from SigmaeAldrich and used as such
without further purification. All solvents used for the spectroscopic
The reported values are the average of two determinations that in all cases
differed by 7e10% or less.

C.B. Reddy et al. / European Journal of Medicinal Chemistry 47 (2012) 553e559
557
and other physical studies were reagent grade and were further
purified by literature methods [40]. Diethyl phosphite was purified
by vacuum distillation. All solvents were freshly distilled prior to
use. Melting points were determined using a calibrated ther-
mometer by Guna Digital Melting Point apparatus. They were
expressed in degrees centigrade (ꢀC) and are uncorrected. IR
spectra of samples were recorded as potassium bromide on
a Bruker Vector 21 FTeIR spectrophotometer. Absorptions were
reported in wave numbers (cmꢁ1). 1H, 13C and 31P NMR spectra
were recorded as solutions in DMSO-d6 on a Bruker AMX 500 MHz
spectrometer operating at 400 MHz for 1H, 100 MHz for 13C and
161.9 MHz for 31P NMR. The 1H and 13C chemical shifts were
expressed in parts per million (ppm) with reference to tetrame-
thylsilane (TMS) as an internal standard and 31P chemical shifts to
85% H3PO4 as an external standard. LCMS mass spectra were
recorded on a Jeol SX 102 DA/600 Mass spectrometer. Elemental
analysis was performed on Thermo Finnigan Insturment.
(C-2a’), 68.5 (d,
J
¼
150.5 Hz, C-2), 63.3 (d,
J
¼
6.1
Hz, eOeCH2eCH3), 16.2 (d, J ¼ 5.8 Hz, eOeCH2eCH3). 31P NMR
(161.9 MHz, DMSO-d6)
Calcd for C22H25N2O3P (%): C, 66.66; H, 6.36; N, 7.07. Found (%):
66.42; H, 6.21; N, 6.96.
d
: 24.73. LCMS, (m/z): 397 [Mþ1]þ. Anal.
4.3.2. Diethyl (3-chloro-4-fluorophenylamino)(4-(pyridine-4-yl)
phenyl)methylphosphonate (4b)
Yield : 93%. mp: 144e146 ꢀC. IR (KBr) cmꢁ1: 3350 (NeH), 1255
(P]O). 1H NMR (400 MHz, DMSO-d6)
d
: 8.58 (d, 2H, J ¼ 8.1, AreH),
7.92e7.45 (m, 8H, AreH), 6.91e6.80 (m, 1H, AreH), 5.67 (br s, 1H,
NH), 4.90 (dd, 1H, J ¼ 22.8, 8.5 Hz, CHP), 4.17e4.07 (m, 2H, OCH2),
3.99e3.87 (m, 2H, OCH2), 1.29 (t, 3H, J ¼ 6.9 Hz, CH3), 1.16 (t, 3H,
J ¼ 6.9 Hz, CH3). 13C NMR (100 MHz, DMSO-d6)
d
: 149.9 (C-70 & C-90),
147.7 (C-3a’), 147.4 (C-1a’), 147.3 (C-50), 137.6 (C-10), 136.6 (C-40),
130.4 (C-5a’), 128.4 (C-20 & C-120), 127.2 (C-30 & C-110), 123.0 (C-60
&
C-100), 121.3 (C-4a’), 116.5 (C-6a’), 112.2 (C-2a’), 63.3
(d, J ¼ 7.2 Hz, eOeCH2eCH3), 56.3 (d, J ¼ 150.5 Hz, C-2), 16.1
4.2. Synthesis
(d, J ¼ 5.9 Hz, eOeCH2eCH3). 31P NMR (161.9 MHz, DMSO-d6)
d:
24.56. LCMS, (m/z): 450 [Mþ2]þ, 449 [Mþ1]þ. Anal. Calcd for
C22H23ClFN2O3P (%): C, 58.87; H, 5.16; N, 6.24. Found (%): C, 58.68;
H, 5.02; N, 6.17.
4.2.1. Synthesis of Diethyl (phenyl amino)(4-(pyridine-4-yl)phenyl)
methyl phosphonate (4a)
In a 50 mL round bottom flask a mixture of 4-(Pyridin-4-yl)
benzaldehyde (1) (0.0025 mol, 0.46 g), aniline (2) (0.0025 mol,
0.23 mL) and triethyl phosphite (3) (0.0030 mol, 0.52 mL) were
taken in toluene (10 mL) as solvent and stirred for 15 min in the
presence of PEG-SO3H (0.1 mol) for a duration of 2 h at 40e50 ꢀC.
The reaction progress was monitored with Thin Layer Chromatog-
raphy by using ethyl acetate, hexane mixture (1:9) as mobile phase.
After completion of the reaction the reaction mixture was allowed
to settle for 15 min the catalyst was separated by filtration washed
with toluene and dried, for its reuse. The solvent from filtrate was
removed in rotaevaporator. The crude compound obtained was
purified by column chromatography using neutral silica gel as an
absorbent, ethyl acetate, hexane mixture (3:7) as an eluant. Finally
the collected product is recrystallized from hot ethanol to get the
pure diethyl(phenylamino) (4-(pyridine-4-yl)phenyl)methyl phos-
phonate 4a (Yield 96%; mp: 155e157 ꢀC) (Fig. 4). The same proce-
dure is followed for the synthesis of the remaining title compounds
4bej.
4.3.3. Diethyl (3-bromophenylamino)(4-(pyridine-4-yl)phenyl)
methylphosphonate (4c)
Yield : 94%. mp: 163e165 ꢀC. IR (KBr) cmꢁ1: 3202 (NeH), 1247
(P]O). 1H NMR (400 MHz, DMSO-d6)
d
: 8.66 (d, 2H, J ¼ 8.1 Hz,
AreH), 7.84e7.50 (m, 6H, AreH), 6.94 (d, 1H, J ¼ 8.0 Hz, AreH), 6.82
(d, 1H, J ¼ 7.9 Hz, AreH), 6.78e6.49 (m, 2H, AreH), 5.02 (t, 1H,
J ¼ 8.2 Hz, NH), 4.79 (dd, 1H, J ¼ 24.5, 9.1 Hz, CHP), 4.14e4.03 (m,
2H, OCH2), 3.99e3.85 (m, 1H, OCH2), 3.76e3.66 (m, 1H, OCH2), 1.32
(t, 3H, J ¼ 7.05 Hz, CH3), 1.16 (t, 3H, J ¼ 7.06 Hz, CH3). 13C NMR
(100 MHz, DMSO-d6) d
: 149.9 (C-70 & C-90), 147.7 (C-3a0), 147.4
(C-1a0), 147.3 (C-50), 137.6 (C-10), 136.6 (C-40), 130.4 (C-5a’), 128.4
(C-20 & C-120), 127.2 (C-30 & C-110), 123.0 (C-60 & C-100), 121.3 (C-4a’),
116.5 (C-3a’), 112.2 (C-2a’), 63.3 (d, J ¼ 6.1 Hz, eOeCH2eCH3), 56.2
(d, J ¼ 150.5 Hz, C-2), 16.2 (d, J ¼ 5.9 Hz, eOeCH2eCH3). 31P NMR
(161.9 MHz, DMSO-d6)
d
: 23.68. LCMS, (m/z): 476 [Mþ2]þ, 475
[Mþ1]þ. Anal. Calcd for C22H24BrN2O3P (%): C, 55.59; H, 5.09; N,
5.89. Found (%): C, 54.97; H, 5.03; N, 5.82.
4.2.2. Preparation of PEG-SO3H
4.3.4. Diethyl (4-chloro-3-nitrophenylamino)(4-(pyridine-4-yl)
phenyl)methylphosphonate (4d)
Chlorosulfonic acid (10 mmol) was added to a solution of
PEG-6000 (1 mmol) in CH2Cl2 (10 ml) at ꢀC and the resulting
solution was stirred at room temperature overnight. Then, the
solution was concentrated under reduced presence and ether
(25 ml) was added to it. The resulting precipitate was filtered and
washed with ether (10 ml) three times to afford PEG-SO3H as
a gummy solid [41,42]. After its first use, it was again successfully
reused for three times.
Yield : 91%. mp: 175e177 ꢀC. IR (KBr): 3339 (N-H), 1238 (P]O).
1H NMR (400 MHz, DMSO-d6)
d
: 8.63 (d, 2H, J ¼ 8.0 Hz, AreH),
7.80e6.33 (m, 9H, Ar-H), 5.04 (br s, 1H, NH), 4.60 (dd, 1H, J ¼ 22.8,
7.8 Hz, CHP), 4.12e4.10 (m, 2H, OeCH2), 4.03e3.99 (m, 1H, OCH2),
3.60 (m, 1H, OCH2), 1.41 (t, 3H, J ¼ 7.03 Hz, CH3), 1.18 (t, 3H,
J ¼ 7.0 Hz, CH3). 13C NMR (100 MHz, DMSO-d6)
d
: 149.9 (C-70 & C-90),
147.7 (C-3a’), 147.4 (C-1a’), 147.3 (C-50), 137.6 (C-10), 136.6 (C-40),
130.4 (C-5a’), 128.4 (C-20 & C-120), 127.2 (C-30 & C-110), 123.0 (C-60
4.3. Spectral data of the synthesized compounds (4aej)
&
C-100), 121.3 (C-4a’), 116.5 (C-6a’), 112.2 (C-2a’), 63.3
(d, J ¼ 6.9 Hz, eOeCH2eCH3), 55.8 (d, J ¼ 152.2 Hz, C-2), 16.2
4.3.1. Diethyl (phenyl amino)(4-(pyridine-4-yl)phenyl)
methylphosphonate (4a)
(d, J ¼ 6.8 Hz, eOeCH2eCH3). 31P NMR (161.9 MHz, DMSO-d6)
d:
26.45. LCMS, (m/z): 477 [Mþ2]þ, 476 [Mþ1]þ. Anal. Calcd for
C22H23ClN3O5P (%): C, 55.53; H, 4.87; N, 8.83. Found (%): 55.35; H,
4.65; N, 8.68.
Yield: 96%. mp: 155e157 ꢀC. IR (KBr) cmꢁ1: 3330 (NeH), 1235
(P]O). 1H NMR (400 MHz, DMSO-d6)
d
: 8.62 (d, 2H, J ¼ 8.0 Hz,
AreH), 7.78e7.35 (m, 6H, AreH), 7.25 (s, 1H, AreH), 6.95e6.75 (m,
3H, AreH), 6.45 (d, 1H, J ¼ 8.3 Hz, AreH), 5.22 (t, 1H, J ¼ 8.3 Hz, NH),
4.69 (dd, 1H, J ¼ 24.9, 7.55 Hz, CHP), 4.17e4.09 (m, 2H, OCH2),
4.01e3.92 (m, 1H, OCH2), 3.78e3.70 (m, 1H, OCH2), 1.37 (t, 3H,
J ¼ 6.7 Hz, CH3), 1.11 (t, 3H, J ¼ 6.79 Hz, CH3). 13C NMR (100 MHz,
4.3.5. Diethyl (3-nitrophenylamino)(4-(pyridine-4-yl)phenyl)
methylphosphonate (4e)
Yield : 95%. mp: 153e155 ꢀC. IR (KBr) cmꢁ1: 3340 (NeH), 1237
(P]O). 1H NMR (400 MHz, DMSO-d6)
AreH), 7.64e7.55 (m, 4H, AreH), 7.50 (d, 2H, J ¼ 5.9 Hz, AreH),
6.90e6.43 (m, 4H, AreH), 5.10 (br s, 1H, NH), 4.70 (dd, 1H, J ¼ 21.8,
9.6 Hz, CHP), 4.18e4.13 (m, 2H, OCH2), 4.01e3.98 (m, 1H, OCH2),
d
: 8.65 (d, 2H, J ¼ 5.7 Hz,
DMSO-d6)
d
: 149.9 (C-70 & C-90), 147.4 (C-1a’), 147.4 (C-50), 137.6
(C-10), 136.6 (C-40), 130.4 (C-3a’ & C-5a’), 128.4 (C-20 & C-120), 127.2
(C-30 & C-110), 123.0 (C-60 & C-100), 121.3 (C-4a’), 116.5 (C-6a’), 112.2

558
C.B. Reddy et al. / European Journal of Medicinal Chemistry 47 (2012) 553e559
3.80 (m, 1H, OCH2), 1.31 (t, 3H, J ¼ 7.03 Hz, CH3), 1.16 (3H, t,
1H, J ¼ 23.8, 8.3 Hz, CHP), 4.16e3.64(m, 4H, OCH2), 1.63 (t, 3H,
J ¼ 7.0 Hz, CH3), 1.33 (t, 3H, J ¼ 7.0 Hz, CH3). 13C NMR (100 MHz,
J ¼ 7.0 Hz, CH3). 13C NMR (100 MHz, DMSO-d6)
d
: 149.9 (C-70 & C-90),
147.7 (C-6a’), 147.4 (C-1a’), 147.3 (C-50), 137.6 (C-10), 136.6 (C-40),
130.4 (C-5a’), 128.4 (C-20 & C-120), 127.2 (C-30 & C-110), 123.0 (C-60 &
C-100), 121.3 (C-4a’), 116.5 (C-3a’), 112.2 (C-2a’), 63.3 (d, J ¼ 6.3
Hz, eOeCH2eCH3), 56.7 (d, J ¼ 151.5 Hz C-2), 16.2 (d, J ¼ 6.9
DMSO-d6) d
: 172.3 (C-1a’), 152.9 (C-8a’), 149.4 (C-70 & C-90), 147.1
(C-50), 136.5 (C-10), 139.8 (C-40), 129.9 (C-3a’), 127.3 (C-20 & C-120),
127.0 (C-30 & C-110), 125.6 (C-6a’), 124.7 (C-5a’), 121.1 (C-4a’), 120.5
(C-60 & C-100), 119.6 (C-7a’), 62.5 (d, J ¼ 5.8 Hz, eOeCH2eCH3), 56.7
(d, J ¼ 151.6 Hz, C-2), 16.3 (d, J ¼ 6.4 Hz, eOeCH2eCH3). 31P NMR
Hz, eOeCH2eCH3). 31P NMR (161.9 MHz, DMSO-d6)
d: 25.30. LCMS,
(m/z): 442 [Mþ1]þ. Anal. Calcd for C22H24N3O5P (%): C, 59.86; H,
(161.9 MHz, DMSO-d6)
Calcd for C23H24N3O3PS (%): C, 60.92; H, 5.33; N, 9.27. Found (%): C,
60.24; H, 5.27; N, 9.16.
d
: 23.58. LCMS, (m/z): 454 [Mþ1]þ. Anal.
5.48; N, 9.52. Found (%): C, 59.66; H, 5.23; N, 9.39.
4.3.6. Diethyl (pyridine-4-ylamino)(4-(pyridine-4-yl)phenyl)
methylphosphonate(4f)
4.3.10. Diethyl(6-nitrobenzo[d]thiazol-2-ylamino)(4-(pyridine-4-
yl)phenyl)methyl phosphonate (4j)
Yield : 91%. mp: 148e150 ꢀC. IR (KBr) cmꢁ1: 3340 (NeH), 1245
(P]O). 1H NMR (400 MHz, DMSO-d6)
d
: 8.68 (d, 2H, J ¼ 8.1 Hz,
Yield : 85%. mp: 170e172 ꢀC. IR (KBr) cmꢁ1: 3341 (NeH), 1239
AreH), 8.51 (d, 2H, J ¼ 8.2 Hz, AreH), 7.68e7.12 (m, 8H, AreH), 5.78
(t, 1H, J ¼ 8.1 Hz, NH), 4.94 (dd, 1H, J ¼ 23.9, 8.5 Hz, CHP), 4.14e3.65
(m, 4H, OCH2), 1.68 (t, 3H, J ¼ 7.1 Hz, CH3), 1.29 (t, 3H, J ¼ 7.1 Hz,
(P]O). 1H NMR (400 MHz, DMSO-d6)
d
: 8.62 (d, 2H, J ¼ 8.2 Hz,
AreH), 8.35e7.18 (m, 9H, AreH), 5.78 (t, 1H, J ¼ 8.0 Hz, NH), 4.98
(dd, 1H, J ¼ 22.9, 8.1 Hz, CHP), 4.10e3.66 (m, 4H, OCH2), 1.60 (t, 3H,
J ¼ 7.0 Hz, CH3), 1.35 (t, 3H, J ¼ 7.0 Hz, CH3). 13C NMR (100 MHz,
CH3). 13C NMR (100 MHz, DMSO-d6) : 149.9 (C-70 & C-90), 148.4
d
(C-6a’ & 5a’), 147.4 (C-1a’), 147.4 (C-50), 137.6 (C-10), 136.6 (C-40),
128.4 (C-20 & C-120), 127.2 (C-30 & C-110), 123.0 (C-60 & C-100), 121.3
(C-4a’), 112.2 (C-2a’), 63.3 (d, J ¼ 5.8 Hz, eOeCH2eCH3), 56.8
(d, J ¼ 151.6 Hz, C-2), 16.2 (d, J ¼ 6.4 Hz, eOeCH2eCH3). 31P NMR
DMSO-d6) d
: 173.9 (C-1a0), 159.6 (C-8a0), 149.7 (C-70 & C-90), 147.3
(C-50), 144.7 (C-5a0), 140.2 (C-40), 136.7 (C-10), 130.8 (C-3a0), 127.7
(C-20 & C-120), 127.4 (C-30 & C-110), 121.6 (C-6a0), 119.7 (C-4a0), 120.2
(C-60 & C-100), 117.7 (C-7a’), 62.4 (d, J ¼ 5.8 Hz, eOeCH2eCH3), 55.6
(d, J ¼ 151.6 Hz, C-2), 16.1 (d, J ¼ 6.4 Hz, eOeCH2eCH3). 31P NMR
(161.9 MHz, DMSO-d6)
Calcd for C21H24N3O3P (%): C, 63.57; H, 6.09; N, 10.57. Found (%): C,
62.87; H, 6.02; N, 10.45.
d
: 23.50. LCMS, (m/z): [Mþ1]þ 398. Anal.
(161.9 MHz, DMSO-d6)
Calcd for C23H23N4O5PS (%): C, 55.42; H, 4.65; N, 11.24. Found (%): C,
54.81; H, 4.59; N, 11.12.
d
: 24.62. LCMS, (m/z): 499 [Mþ1]þ. Anal.
4.3.7. Diethyl(pyridine-4-ylmethylamino)(4-(pyridine-4-yl)phenyl)
methylphosphonate (4g)
4.4. Biological assay
Yield : 87%. mp: 150e152 ꢀC. IR (KBr) cmꢁ1: 3330 (NeH), 1233
(P]O). 1H NMR (400 MHz, DMSO-d6)
d
: 8.66 (d, 2H, J ¼ 8.0 Hz,
4.4.1. Cell lines and culture conditions
AreH), 7.67e7.43 (m, 6H, AreH), 7.26 (d, 2H, J ¼ 7.1 Hz, AreH), 6.91
(d, 2H, J ¼ 6.96 Hz, AreH), 5.34 (t, 1H, J ¼ 8.0 Hz, NH), 4.85 (dd, 1H,
J ¼ 23.1, 8.6 Hz, CHP), 4.72e4.68 (m, 2H, OCH2), 4.19e4.14 (m, 2H,
CH2), 4.05e3.99 (m, 1H, OCH2), 3.80e3.75 (m, 1H, OCH2), 1.17 (t, 3H,
J ¼ 7.04 Hz, CH3), 1.31(t, 3H, J ¼ 7.0 Hz, CH3). 13C NMR (100 MHz,
The title compounds 4aej were evaluated for cytotoxic activity
against human chronic myeloid leukemia cells, K 562, human colon
carcinoma cells, Colo 205 and human embryonic kidney cells, HEK
293. They were procured from National Center for Cell Sciences,
Pune, India. All cells were grown in RPMI-1640 supplemented with
10% heat inactivated fetal bovine serum (FBS), 100 IU/mL penicillin,
100 mg/mL streptomycin and 2 mm-glutamine. Cultures were
maintained in a humidified atmosphere with 5% CO2 at 37 ꢀC. The
cells were subcultured twice each week, seeding at a density of
about 2 ꢂ 103 cells/mL. Before the analysis of the compounds, cells
were washed with PBS and fresh medium was added. For final
analysis, exponentially growing cells were collected and resus-
pended in fresh culture medium with 10% FBS.
DMSO-d6) d
: 149.5 (C-70 & C-90), 147.4 (C-50), 146.6 (C-3a’), 145.1
(C-5a’), 140.6 (C-40), 134.7 (C-2a’), 135.4 (C-7a’), 132.6 (C-10), 127.3
(C-20 & C-120), 127.1 (C-30 & C-110), 122.3 (C-6a’), 120.8 (C-60 & C-100),
63.2 (d, J ¼ 5.8 Hz, eOeCH2eCH3), 53.2 (C-1a’), 52.8 (d, J ¼ 151.6 Hz,
C-2), 16.3 (d, J ¼ 6.4 Hz, eOeCH2eCH3). 31P NMR (161.9 MHz,
DMSO-d6)
d
: 24.89. LCMS, (m/z): 412 [Mþ1]þ. Anal. Calcd for
C22H26N3O3P (%): C, 64.22; H, 6.37; N, 10.21. Found (%): C, 63.51; H,
6.29; N, 10.09.
4.3.8. Diethyl(thiazol-2-ylamino)(4-(pyridine-4-yl)phenyl)
methylphosphonate (4h)
4.4.2. MTT assay
Cell viability was assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay as described by Mos-
mann [43]. All the cells (5 ꢂ 103 cells/well) were seeded to 96-well
Yield : 89%. mp: 159e161 ꢀC. IR (KBr) cmꢁ1: 3346 (NeH), 1242
(P]O). 1H NMR (400 MHz, DMSO-d6)
d: 8.60 (d, 2H, J ¼ 8.1 Hz,
AreH), 8.26 (s, 1H, AreH), 7.78e7.54 (m, 5H, AreH), 7.41 (d, 1H,
J ¼ 6.5 Hz, AreH), 6.79 (d,1H, J ¼ 6.5, AreH), 5.58 (br s,1H, NH), 4.96
(dd, 1H, J ¼ 29.0, 13.0 Hz, CHP), 4.04e3.92 (m, 2H, OCH2), 3.52e3.41
(m, 2H, OCH2), 1.31 (t, 3H, J ¼ 6.9 Hz, CH3), 1.19 (t, 3H, J ¼ 6.9 Hz,
culture plate and cultured with or without compounds at 1
m
M and
L. After
L of MTT (5 mg/mL in
PBS) was added to the fresh medium. After 2 h incubation at 37 ꢀC,
100 L of DMSO was added to each well and plates were agitated
for 1 min. The colored precipitate formed was dissolved in 100 L of
10
mM concentration for 24 h in a final volume of 200 m
treatment, the medium was removed and 20
m
CH3). 13C NMR (100 MHz, DMSO-d6)
d
: 162.8 (C-1a’),149.7 (C-70 & C-
m
90), 147.3 (C-50), 139.5 (C-40), 136.8 (C-4a’), 136.4 (C-10), 127.4 (C-20 &
C-120), 127.1 (C-30 & C-110), 120.8 (C-60 & C-100), 112.2 (C-3a’), 68.6
(d, J ¼ 151.6 Hz, C-2), 62.8 (d, J ¼ 5.8 Hz, eOeCH2eCH3), 16.4 (d,
m
DMSO and the absorbance was read at 570 nm on a multi-well plate
reader (Victor3, Perkin Elmer). The Percent inhibition of prolifera-
tion of the title compounds were calculated with respect to the
control. The concentration that inhibited the cell growth by 50%
(IC50) was determined from cell survival plots and the detailed
procedure for the determination of IC50 values was followed by the
graphical method as explained here. The IC50 was preliminarily
J ¼ 6.4 Hz, eOeCH2eCH3). 31P NMR (161.9 MHz, DMSO-d6)
d: 25.63.
LCMS, (m/z): 404 [Mþ1]þ. Anal. Calcd for C19H22N3O3PS (%): C,
56.57; H, 5.50; N, 10.42. Found (%): C, 55.94; H, 5.44; N, 10.31.
4.3.9. Diethyl(benzo[d]thiazol-2-ylamino)(4-(pyridine-4-yl)phenyl)
methyl phosphonate (4i)
determined at a broad range of concentrations viz., 1, 10 & 100
and then at a narrow range of concentrations viz., 2, 4, 8,10,16, 32 &
64 M of the title compounds against the cell lines. For this data,
a line graph was plotted between concentrations (X-axis) versus %
mM
Yield: 82%. mp: 156e158 ꢀC; IR (KBr) cmꢁ1: 3337 (NeH), 1235
(P]O). 1H NMR (400 MHz, DMSO-d6)
AreH), 8.20e7.15 (10H, m, AreH), 5.75 (t, 1H, J ¼ 8.1, NH), 4.90 (dd,
d
: 8.64 (d, 2H, J ¼ 8.2 Hz,
m

C.B. Reddy et al. / European Journal of Medicinal Chemistry 47 (2012) 553e559
559
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on Y-axis and then correlated to the concentration value on X-axis.
Finally that concentration value is considered as IC50 in mM and the
same procedure is applied for the evaluation of MTT assay of all the
compounds.
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