Green synthesis of α-aminophosphonate derivatives on a solid supported TiO2-SiO2 catalyst and their anticancer activity

Article abstract of DOI:10.1002/ardp.201300214

Syntheses of a new series of biologically potent α-aminophosphonates were accomplished by one-pot Kabachnik-Fields reaction using TiO ₂-SiO₂ as solid supported catalyst under microwave irradiation conditions. The chemical structures of all the newly synthesized compounds were confirmed by analytical and spectral (IR, ¹H, ¹³C, ³¹P NMR, and mass) data. Their anticancer nature was evaluated by screening the in vitro activity on two human cancer cell lines, HeLa and SK-BR-3. Compounds 4i and 4o showed the best activity on these cancer cells even though the majority of the compounds, and particularly 4l and 4p, have good cytotoxic activity against them. TiO₂-SiO₂ was employed as solid supported catalyst for the solvent-free one-pot synthesis of α-aminophosphonates by Kabachnik-Fields reaction under microwave irradiation conditions. The newly synthesized compounds were assessed for in vitro anticancer activity against HeLa and SK-BR-3 cells. The majority of the compounds showed good cytotoxic activity.

Full text of DOI:10.1002/ardp.201300214

Arch. Pharm. Chem. Life Sci. 2013, 346, 667–676
667
Full Paper
Green Synthesis of a-Aminophosphonate Derivatives on a
Solid Supported TiO2–SiO2 Catalyst and Their Anticancer
Activity
Radha Rani Chinthaparthi1, Ira Bhatnagar2, Chandra Sekhar Reddy Gangireddy3,
Sundar Chereddy Syama1, and Suresh Reddy Cirandur1
1
Department of Chemistry, Sri Venkateswara University, Tirupati, Andra Pradesh, India
Laboratory of Nanotechnology, Centre for Cellular and Molecular Biology, Hyderabad, Andra Pradesh, India
Department of Chemistry, Sri Venkateswara Colleges, Tirupati, Andra Pradesh, India
2
3
Syntheses of a new series of biologically potent a-aminophosphonates were accomplished by one-pot
Kabachnik–Fields reaction using TiO2–SiO2 as solid supported catalyst under microwave irradiation
conditions. The chemical structures of all the newly synthesized compounds were confirmed by
analytical and spectral (IR, 1H, 13C, 31P NMR, and mass) data. Their anticancer nature was evaluated by
screening the in vitro activity on two human cancer cell lines, HeLa and SK-BR-3. Compounds 4i and 4o
showed the best activity on these cancer cells even though the majority of the compounds, and
particularly 4l and 4p, have good cytotoxic activity against them.
Keywords: a-Aminophosphonates / Anticancer activity / Green synthesis / HeLa and SK-BR-3 human cancer cells /
TiO2–SiO2
Received: June 5, 2013; Revised: June 27, 2013; Accepted: June 28, 2013
DOI 10.1002/ardp.201300214
Introduction
A number of synthetic methods [10–22] using various
Bronsted and Lewis acids, heteropoly acids, heterogeneous
catalysts, microwave irradiation (MWI), ultrasound irradia-
tion, catalyst-free, ionic liquid, and nanocatalysts have been
reported for a-aminophosphonates. Recently Kabachnik–
Fields reaction has been reviewed [23] well about the various
reactions conditions. Although significant advances have
been made in these syntheses, still some limitations such as
use of expensive catalyst, toxic solvents, longer reaction time,
elevated temperature, and low product yields are associated
with them. Therefore, the search continues for a better
synthetic procedure for a-aminophosphonates in terms of
operational simplicity, economic viability, and greater
selectivity. Green chemical synthetic approaches under
solvent-free conditions [24–26] with MWI [27–33] obviously
meet this objective.
In heterogeneous solid supported catalysis [34–37], the
titania–silica (TiO2–SiO2) is an important catalyst for
organic reactions due to advantages such as improving the
availability of the active sites, ease to handle as a benchtop
catalyst, inexpensive, commercial availability, and recyclable
due to stability even at a higher temperature. In pursuit
of our continued interest in the development of green
synthetic methods for the preparation of phosphonate
Synthesis and use of phosphonates have received great
attention during the last two decades. The a-functionalized
phosphonic acid esters serve as valuable intermediates in the
preparation of medicinal compounds and as synthetic
intermediates [1–6] in phosphonate chemistry for many
organic compounds and dyes. They also find use in laser
technology and as fluorescent materials for visualization of
biomolecules. Because of attractive biological activities of
a-aminophosphonates, which are the important a-function-
alized phosphonic acid esters, as anti-bacterial, anti-viral, and
anti-inflammatory agents [7] they have received much
attention and attracted special interest as peptide analogs
due to their structural similarity to a-amino acids. The
anticancer properties of a-aminophosphonate derivatives
have been recently reported [8, 9]. Cancer being the second
leading cause of death worldwide, there is a need for the
development of more effective anticancer agents.
Correspondence: Suresh Reddy Cirandur, Department of Chemistry, Sri
Venkateswara University, Tirupati 517 502, Andra Pradesh, India.
E-mail: csrsvu@gmail.com
Fax: þ91 877 2289555
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R. R. Chinthaparthi et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 667–676
O
∆
CHO
NH2
H
N
_
_
110 °
C 90 180
min
90
,
O
P
O
Catalyst
Neat
O
P
H
+
+
O
O
O
w
µ
_
_
O
2a
50 100 ° 5 10 min
C
,
4a
1
3
Scheme 1. Preparation of dimethyl ((4-methoxyphenyl)(naphthalen-2-ylamino)methyl)phosphonate (4a).
derivatives [38, 39], we accomplished solvent-free synthesis for
a new series of a-aminophosphonates via three-component
one-pot Kabachnik–Fields reaction using TiO2–SiO2 as sup-
ported catalyst under MWI (Scheme 1) and studied their
cytotoxic activity. We succeeded in identifying these newly
synthesized compounds as potential in vitro anticancer
compounds. Further, this group of compounds with an
aminophosphonate pharmacophore may even have broad
range of bioactivity against many other bacterial and viral
diseases.
catalyst also did not increase the product yield considerably
(Table 1, entry 22). Therefore, it was established that 5 mol% of
TiO2–SiO2 and 80 min of reaction time are necessary and
sufficient for the total completion of the model reaction to
obtain maximum product yield.
We have also examined the TiO2–SiO2 catalytic activity for
reusability. We reused the catalyst for five consecutive cycles
and observed no significant loss of catalytic activity in all
Table 1. Screening of catalyst quantity and reaction time on the
synthesis of dimethyl ((4-methoxyphenyl)(naphthalen-2-ylamino)-
methyl)phosphonate (4a).a)
Results and discussion
Chemistry
Conventional
Microwave
Reaction of naphthalen-2-amine (1), 4-methoxy benzaldehyde
(2a), and dimethyl phosphonate (3) to obtain the dimethyl ((4-
methoxyphenyl)(naphthalen-2-ylamino)methyl)phosphonate
(4a) under solvent-free conditions (Scheme 1) was run to
standardize the experimental conditions.
Catalyst
(10 mol%)
Temp Time Yieldb) Temp Yieldb)
Entry
(°C) (min)
(%)
(°C)
(%)
1
2
3
4
5
6
7
8
AlCl3
ZrOCl2 · 8H2O
InF3
110
110
110
110
110
110
110
110
110
110
110
110
110
110
110
110
110
110
90
140
120
110
120
120
130
100
150
120
180
100
160
130
120
150
100
100
90
53
58
63
45
48
52
60
49
55
50
56
53
61
59
70
68
67
80
55
75
63
81
–
70
70
70
70
70
70
70
70
70
70
70
70
70
70
70
70
70
70
70
70
70
70
65
70
76
60
58
67
73
62
62
62
69
69
81
82
84
81
80
97
97
96
83
97
94
64
In order to establish optimum conditions for Kabachnik–
Fields reaction under both conventional and MWI conditions
(at 210 W power), the model reaction was run using various
catalysts (Table 1) in 10 mol% under solvent-free conditions.
All the reactions were carried out at 90–110 and 50–100°C for
90–180 and 5–10 min under conventional and MWI con-
ditions, respectively. When the model reaction was run under
conventional conditions, the product yield was very low even
after prolonged reaction time. However, the same reaction
under MWI conditions afforded high product yields (Table 1,
entries 1–18). This observation motivated us to search for a
suitable catalyst under MWI conditions. We found that the
TiO2–SiO2 worked as a better catalyst among all the catalysts
used at 70°C (Table 1, entry 18) under these conditions. We
also found that the yields were significantly affected by the
amount of TiO2–SiO2 loaded. Most excitingly, the reaction
progressed very smoothly and gave 4a in 97% yield when
5 mol% TiO2–SiO2 was used (Table 1, entry 19). Interestingly
we could find no drastic change in the product yield when the
reaction time was decreased from 10 to 5 min (Table 1, entry
20) at 5 mol% concentration of the catalyst. But, when loaded
with 3 mol% of TiO2–SiO2 catalyst, the reaction remained
incomplete (Table 1, entry 21) and use of excess amount of
ZnBr2
ZnCl2
ZnO
ZnCl2–SiO2
MnCl2 · 4H2O
Yb(OAc)3 · H2O
CuBr
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
NbCl5
K-10
Al2O3
NiCl2 · 6H2O
Amberlyst-15
PS/GaCl3
PS/AlCl3
TiO2–SiO2
TiO2–SiO2 (5 mol%)
TiO2–SiO2 (5 mol%)
TiO2–SiO2 (3 mol%)
TiO2–SiO2 (15 mol%)
TiO2–SiO2 (5 mol%)
TiO2–SiO2 (5 mol%)
90
90
90
90
90
90
90
–
–
100
50
–
–
–
a)
Reaction conditions: naphthalen-2-amine (1), 4-methoxybenz-
aldehyde (2a), and dimethyl phosphonate (3) in 1:1:1 ratio. All the
microwave irradiation reactions were run for 10 and 5 min for
entries 1–19 and 20–24, respectively. All the entries were treated
under neat (solvent-free) conditions in both methods.
b)
Isolated yield.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 667–676
a-Aminophosphonates as Potential Anticancer Drugs
669
Table 2. Effect of the solvent and temperature on the synthesis of
dimethyl ((4-methoxyphenyl)(naphthalen-2-ylamino)methyl)phos-
phonate (4a).a)
Conventional
Microwave
Temp
(°C)
Yieldb) Temp Yieldb)
Entry
Solvent
(%)
(°C)
(%)
1
2
3
4
5
6
7
8
9
10
Toluene
Carbontetrachloride
Chloroform
Dichloromethane
Tetrahydrofuran
Ethanol
Methanol
Acetonitrile
Dimethylsulfoxide
Solvent-free
110
75
60
60
65
75
70
80
150
90
57
55
51
52
56
61
65
76
80
75
100
70
70
70
70
70
70
100
100
70
65
61
59
55
63
73
78
82
86
96
Figure 1. Reusability of TiO2–SiO2 catalyst for the model reaction in
consecutive cycles.
a)
Reaction conditions: naphthalen-2-amine (1), 4-methoxybenz-
aldehyde (2a), and dimethyl phosphonate (3) in 1:1:1 ratio. All the
reactions under conventional and microwave (at 210 and 245 W)
conditions were run at 90 and 5 min reaction time, respectively.
these reactions; the corresponding product was obtained in
95%, 93%, 91%, 90%, and 88% yields (Fig. 1). These data qualify
the catalyst for reusability.
b)
Isolated yield.
Suitable microwave power required for the model reaction
was also studied by using variable MW energy and it was
concluded that 210 W MW power output and 70°C were
sufficient for the accomplishment of maximum conversion of
reactants to the product 4a. Increasing to 245 W or decreasing
to 140 W MW energy, no more appreciable increment in the
product yield (Table 1, entries 23 and 24, respectively) was
observed.
The effect of solvent and temperature on this model
reaction was investigated for both conventional and MWI
conditions with 5 mol% of TiO2–SiO2 catalyst using different
solvents at varying temperatures (Table 2, entries 1–9) for 90
and 5 min, respectively. The results indicated that the
reaction efficiency was also affected by the solvent. In non-
polar solvent, lower product yields (Table 2, entries 1–3), and,
in polar solvent, better yields were obtained (Table 2, entries
6–9). The polar solvents undergo dipole rotation when
exposed to microwaves and generate heat energy, which
results in product yield at a faster rate. However, the best
result was obtained under solvent-free conditions (Table 2,
entry 10). This motivates us to synthesize the target molecules
under MWI and solvent-free conditions. We found that
the reaction proceeded smoothly in much less reaction time
(3–5 min) at lower reaction temperature (70°C) leading to
higher product yields (85–97%).
After finding TiO2–SiO2 as the best catalyst system for the
model reaction, we applied this methodology successfully for
a series of aldehydes (2b–l) and amines (Scheme 2) and
achieved excellent yields (Table 3). The aromatic aldehydes
(2a–h) reacted at a faster rate irrespective of the nature of
substrates (electron withdrawing or donating) on them. On
the other hand, the product yields were lower with aliphatic
aldehydes (2i–l), although the reactions went smoothly.
Cytotoxicity
The anticancer activity of test compounds 4a–p was
investigated on HeLa (human cervical cancer) and SK-BR-3
(human breast adenocarcinoma) cells. It was determined by
measuring the number of live cells after 24 h of treatment
(MTT assay); their IC50 values are presented [40] in Table 4. The
results showed that majority of title compounds (4a–4d, 4f,
O
H
-
T
NH2
R
iO2 SiO2 (5 mol% )
′
R
H
C O
N
+
solvent-free
+
R
P
O
H
O
O
′
w
µ
R
P
_
min
O
O
70 ° 3 5
C
,
Scheme 2. Solvent-free microwave synthesis
of a-aminophosphonates (4a–l) on solid
supported TiO2–SiO2 catalyst.
_
_
1
2a l
4a l
3
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670
R. R. Chinthaparthi et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 667–676
Table 3. Synthesis of a-aminophosphonates (4a–p) on TiO2–SiO2 catalyst by MWI via Scheme 2.a)
a)
Reaction conditions: naphthalen-2-amine/2-aminofluorene, various aldehydes, and dimethyl phosphonate on 5 mol% TiO2–SiO2
catalyst at 210 W.
b)
Isolated yields.
4g, 4i–4m, 4o, and 4p) possessed good anti-proliferative
activity against the two types of cancer cells. Among them, the
compound 4o has the highest cytotoxicity with the IC50 at
concentrations of 0.95 ꢀ 0.06 and 1.21 ꢀ 0.09 mg/mL for
HeLa and SK-BR-3 cells, respectively. Almost equal levels of
activity were observed for 4i, 4l, and 4p. From these
observations one may conclude that the dimethyl (1-((9H-
fluorescent microscope for both HeLa and SK-BR-3 cells (Figs. 2
and 3, respectively). The control group that is without test
compound showed normal healthy and intact nuclei without
any cytological abnormalities (Fig. 2A and C for HeLa cells and
Figs. 3A and C for SK-BR-3 cells).
The cells treated with highly active test compound 4o for
24 h showed obvious morphological changes such as
destruction of cellular membrane, chromatin fragmentation,
and appearance of apoptotic bodies as granules in the culture
media (Fig. 2B). These light microscopy results were consistent
with those of fluorescence microscopy using Hoechst 33342
stain for control and treated cells, where most of the treated
cells exhibited the symptoms of apoptosis but the damage was
severe in some HeLa cells prior to cell membrane blebbing
fluoren-2-yl)amino)butyl)phosphonate (4o) is
a potential
pharmacophore showing higher cytotoxic activity against
the two types of cancerous cells.
Microscopic observations
The cytomorphological abnormalities accrued by the effect of
test compounds were observed under a phase-contrast and
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Arch. Pharm. Chem. Life Sci. 2013, 346, 667–676
a-Aminophosphonates as Potential Anticancer Drugs
671
Table 4. Cytotoxic activitya) of test compounds 4a–r against
HeLa and SK-BR-3 cells.
(inset of Fig. 2B). Hoechst staining revealed typical horseshoe-
shaped nuclei of the HeLa cells, which indicates early
apoptosis leading to deformed nuclear cytoplasmic regularity
followed by margination of chromatin (Fig. 2D).
IC50 in mg/mLb)
When observed for the SK-BR-3 cells similar results were
obtained but the damage to the cells was less compared to
HeLa cells even at the IC50 concentration of the ideal
compound, 4o. Interestingly, the SK-BR-3 cells appeared
circular with the enlargement of cell membrane in almost
all the treated cells (Fig. 3B). This implies that the cell damage
is not as quick as in the case of HeLa cells. The bright,
condensed and segregated chromatin was identified in the
nuclei of the treated cells. The typical characteristic nuclear
deformities such as karyorrhexis and picknosis are well
evident in the treated SK-BR-3 cells (Fig. 3D).
Compound
HeLa
SK-BR-3
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
20.05 ꢀ 1.26
22.16 ꢀ 1.38
29.70 ꢀ 1.35
79.29 ꢀ 3.53
>100
13.52 ꢀ 0.89
12.76 ꢀ 0.86
>100
1.18 ꢀ 0.35
6.16 ꢀ 0.19
8.34 ꢀ 0.24
2.10 ꢀ 0.85
26.80 ꢀ 1.15
>100
0.95 ꢀ 0.06
1.45 ꢀ 0.08
13.65 ꢀ 0.55
3.57 ꢀ 0.33
32.26 ꢀ 1.52
35.12 ꢀ 1.75
32.78 ꢀ 1.81
68.37 ꢀ 3.12
>100
19.45 ꢀ 0.91
11.29 ꢀ 0.81
>100
2.52 ꢀ 0.15
5.41 ꢀ 0.16
7.89 ꢀ 0.22
3.37 ꢀ 0.95
29.37 ꢀ 1.29
>100
1.21 ꢀ 0.09
2.79 ꢀ 0.11
9.73 ꢀ 0.42
2.83 ꢀ 0.11
Structure–activity relationship (SAR)
An organophosphorus compound with a general structure (I)
is biologically active [41–43]. Its phosphorylating ability on the
target molecules depends on the strength of the P–X bond.
A weak P–X bond makes it a good phosphorylating agent
because of the more electrophilic nature of phosphorus that
can facilitate its nucleophilic attack of electron-rich centers of
enzymes and proteins. The ease and effectiveness of phospho-
rus substitution are critically governed by nature, steric size,
structure, and configuration of groups attached to phospho-
rus [44, 45]. Even though, the mechanism of phosphorylation
is not clearly proved, this hypothesis opened a new concept for
the design of novel organophosphorus compounds that could
regulate the normal metabolic biochemical processes in living
organisms (Scheme 3).
Etoposide
Camptothecin
a)
Exponentially growing cells were treated with different
concentrations of test compounds for 24 h and cell growth
inhibition was analyzed through MTT assay.
b)
IC50 is defined as the concentration that results in a 50%
decrease in cell number as compared with that of the control in
the absence of an inhibitor. The values represent the mean ꢀ SD
of five individual observations.
Figure 2. Light and Hoechst stained micro-
graphs of normal and treated HeLa cells. Left-
hand panel (A and C) represents the untreated/
normal HeLa cells, and the right-hand panel
represents the HeLa cells (B and D) treated
with IC50 concentration (0.95 mg/mL) of the
ideal test compound 4o.
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672
R. R. Chinthaparthi et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 667–676
Figure 3. Light and Hoechst stained micro-
graphs of normal and treated SK-BR-3 cells.
Left-hand panel (A and C) represents the
untreated/normal SK-BR-3 cells, and the right-
hand panel represents the SK-BR-3 cells (B
and D) treated with IC50 concentration
(1.21 mg/mL) of the ideal test compound 4o.
The compounds 4a–p in general are more active than the
compound 4o in particular, hence the necessary steric and
electronic configuration in structure (II) that can arrest the
abnormal cell metabolic disorders and maintain normal
function by phosphorylating cancer cell components
(Scheme 4).
solvent-free conditions using TiO2–SiO2 as a solid supported
catalyst with MWI in one-pot multi-component Kabachnik–
Fields reaction. In this procedure the amount of waste is
minimized and atom efficiency is increased in each organic
transformation, qualifying it as the best method for the
synthesis of a-aminophosphonates. All the newly synthesized
a-aminophosphonates were screened for their in vitro antican-
cer activities on HeLa and SK-BR-3 cells and the IC50 values
obtained were compared against those of standard drugs.
Majority of them have good to excellent anticancer activity. The
results further signify that these compounds can be developed
as potential pharmacological leads to combat cancer.
Conclusions
We have successfully synthesized a series of new a-amino-
phosphonates from easily available starting materials under
( )
O S
( )
O S
R1
R2
Experimental
R1
R2
P
P
X
Y
X
Y
Z
Z
Chemistry
Materials and methods
(I)
R1 R2
=
= alkoxy, aryloxy, alkyl, aryl groups
=
H, C, O, S or halogens
All the chemicals purchased from Sigma–Aldrich (Hyderabad,
India), Merck (Mumbai, India), and Lancaster Chemical (Mumbai,
India) were used as such without further purification. The
solvents used for spectroscopic and other physical studies were of
analytical grade and were further purified employing the
reported methods. All the reactions and purity of products were
monitored by thin layer chromatography (TLC) using aluminum
=
X
Y
Z
= sp2
hybridized carbon
Scheme 3. Biologically active pharmacophore unit of organophos-
phorus compounds.
O
O
O
e
M
AChE
O
MeO
HN
P
OMe
OMe
O
OMe
P
P
+
MeOH
AChE
O
H
+
′
R
′
R
AChE-OH
HN
R
′
R
HN
R
(II)
R
(Phosphorylated organism)
Scheme 4. Hypothetical mechanistic presentation of bioactivity of the title compounds (4a–p).
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Arch. Pharm. Chem. Life Sci. 2013, 346, 667–676
a-Aminophosphonates as Potential Anticancer Drugs
673
3
3
plates coated with silica gel (Merck) using 3:7 of ethyl acetate
and hexane as mobile phase. Melting points were determined
using a calibrated thermometer by Guna Digital melting point
apparatus. IR Prestige-21, Fourier transform infrared (FT-IR)
spectrometer using KBr optics. 1H, 13C, and 31P NMR spectra were
recorded in CDCl3 on a Varian 400 MHz NMR spectrometer
operating at 400 MHz for 1H NMR, 100 MHz for 13C NMR, and
161.89 MHz for 31P NMR at Pusan National University, Pusan,
Republic of Korea, and referenced to TMS (1H and 13C) and 85%
(3H, d, JH–P ¼ 10.4 Hz, P–OCH3), 3.72 (3H, d, JH–P ¼ 10.4 Hz,
2
3
P–OCH3), 6.51–6.54 (1H, dd, JH–P ¼ 8.4 Hz and JH–H ¼ 2.0 Hz,
P–CH), 6.74 (1H, m, NH), 7.93–6.92 (11H, m, Ar–H). 13C NMR
(100.56 MHz, CDCl3) d: 105.5 (C-1), 148.7 (C-2), 119.4 (C-3), 130.8 (C-4),
129.2 (C-5), 122.3 (C-6), 127.1 (C-7), 126.2 (C-8), 134.2 (C-9), 129.2 (C-10),
71.2 (C-12), 131.2 (C-13), 130.1 (C-14 and C-28), 115.6 (C-15 and 17),
160.5 (C-16), 53.6 (P–OCH3), 53.9 (P–OCH3), 55.4 (Ar–OCH3). 31P NMR
(161.89, MHz, CDCl3) d: 21.07. EI-MS (m/z, %): 371 (Mþ•). Anal. calcd.
for C20H21NO4P: C, 64.68; H, 5.97; N, 3.77; Found: C, 64.63; H, 5.92; N,
3.73.
H3PO4 (
31P). Mass spectra were recorded at Pukyong National
University, Busan, Republic of Korea on a Jeol JMS-700 mass
spectrometer. Elemental analyses were performed on a Thermo
Finnigan instrument at the University of Hyderabad, Hyderabad,
India.
Dimethyl ((4-ethoxyphenyl)(naphthalen-2-ylamino)methyl)-
phosphonate (4b): Brown solid, mp: 186–188°C. IR (KBr)
(nmax cmꢁ1): 3323 (N–H), 2956 and 2848 (C–Haromatic), 1221
1
–
(P O), 757 (P–Caliphatic). H NMR (400 MHz, CDCl3) d: 3.45 (2H, q,
–
3
Synthetic procedure for the model reaction
Ar–OCH2), d: 1.56 (3H, t, JH–P ¼ 10.4 Hz, OCH2CH3), 3.55 (3H, d,
3JH–P ¼ 10.4 Hz, P–OCH3), 3.75 (3H, d, JH–P ¼ 10.4 Hz, P–OCH3),
3
Method 1: Microwave irradiation (MWI) method: A two-
necked round bottom (RB) flask, which contains a mixture of
naphthalen-2-amine (1, 0.716 g, 0.005 mol), 4-methoxybenzalde-
hyde (2a, 0.61 mL, 0.005 mol), dimethyl phosphonate (3, 0.46 mL,
0.005 mol), and various catalysts with 10 mol% without any
solvent, with an air condenser and a thermo probe was exposed to
MWI using a CATA-4R – Scientific Microwave oven at various
temperatures shown in Table 1 with 140, 210, and 245 W at
ambient pressure. While irradiating with MWI the reaction
mixture was stirred continually to maintain the irradiating field
homogeneously throughout the reaction mixture. The reaction
was stopped as indicated by TLC after 3–10 min. Then the crude
products were obtained by separation of the catalyst by filtration
followed by evaporation of the filtrate. The crude products were
further purified by column chromatography on 60–120 mesh
silica gel using ethyl acetate/hexane (1:3) as eluent, and the solvent
was evaporated in a rotary evaporator. The residue was recrystal-
lized from ethyl acetate to afford pure 4a (Table 1; Scheme 1).
2
3
6.54–6.59 (1H, dd, JH–P ¼ 8.4 Hz, P–CH and JH–H ¼ 2.0 Hz,
NH–CH), 6.58 (1H, m, NH), 7.91–6.90 (11H, m, Ar–H). 13C NMR
(100.56 MHz, CDCl3) d: 105.2 (C-1), 148.3 (C-2), 119.5 (C-3), 130.6 (C-
4), 129.4 (C-5), 122.7 (C-6), 127.3 (C-7), 126.5 (C-8), 134.6 (C-9), 129.5
(C-10), 71.1 (C-12), 131.6 (C-13), 130.5 (C-14 and C-28), 115.3 (C-15
and 17), 160.6 (C-16), 53.3 (P–OCH3), 53.5 (P–OCH3), 65.6 (Ar–OCH2),
15.2 (OCH2CH3). 31P NMR (161.89, MHz, CDCl3) d: 21.34. EI-MS (m/z,
%): 385 (Mþ•). Anal. calcd. for C21H24NO4P: C, 65.45; H, 6.28; N,
3.63; Found: C, 65.39; H, 6.22; N, 3.58.
Dimethyl ((naphthalen-2-ylamino)(p-tolyl)methyl)phospho-
nate (4c): Yellowish solid, mp: 189–181°C. IR (KBr) (nmax cmꢁ1):
–
3317 (N–H), 2965 and 2861 (C–Haromatic), 1258 (P O), 752
–
(P–Caliphatic). 1H NMR (400 MHz, CDCl3) d: 2.18 (3H, s, Ar–CH3),
3.52 (3H, d, 3JH–P ¼ 10.4 Hz, P–OCH3), 3.74 (3H, d, 3JH–P ¼ 10.4 Hz,
2
3
P–OCH3), 6.54–6.59 (1H, dd, JH–P ¼ 8.4 Hz and JH–H ¼ 2.0 Hz,
P–CH), 6.12 (1H, m, NH), 7.91–7.16 (11H, m, Ar–H). 13C NMR
(100.56 MHz, CDCl3) d: 105.8 (C-1), 148.5 (C-2), 119.5 (C-3), 130.7
(C-4), 129.3 (C-5), 122.6 (C-6), 127.3 (C-7), 126.0 (C-8), 134.0 (C-9),
129.6 (C-10), 71.5 (C-12), 131.8 (C-13), 130.5 (C-14 and C-28), 115.1
(C-15 and 17), 160.6 (C-16), 53.3 (P–OCH3), 53.5 (P–OCH3), 25.6
(Ar–CH3). 31P NMR (161.89, MHz, CDCl3) d: 22.13. EI-MS (m/z, %):
355 (Mþ•). Anal. calcd. for C20H22NO3P: C, 67.60; H, 6.24; N, 3.94;
Found: C, 67.54; H, 6.18; N, 3.90.
Method 2: Conventional (heating) method: The reactants
were heated to reflux in an oil bath at 90–110°C for 90–180 min
and kept stirring, and the workup of the products was done as in
the MWI method.
Synthesis of a-aminophosphonates (4a–p): As model reac-
tion the amine (1a/1b, 0.005 mol), various aldehydes (2b–p,
0.005 mol), and dimethyl phosphonate (3, 0.46 mL, 0.005 mol)
were mixed thoroughly with 5 mol% TiO2–SiO2 in a RB flask and
exposed to MWI at 210 W and 70°C under ambient pressure
(Scheme 2). TLC showed the reaction completion in 3–6 min. The
product separation and purification were done according to MWI
method. Pure products were obtained by recrystallization from
ethyl acetate (Table 3). The structures of all the newly synthesized
Dimethyl ((4-hydroxyphenyl)(naphthalen-2-ylamino)methyl)-
phosphonate (4d): Pale yellow solid, mp: 209–211°C. IR (KBr)
(nmax cmꢁ1): 3317 (N–H), 2968 and 2878 (C–Haromatic), 1239 (P O),
–
–
751 (P–Caliphatic). 1H NMR (400 MHz, CDCl3) d: 10.27 (3H, s, Ar–OH),
3
3
3.54 (3H, d, JH–P ¼ 10.4 Hz, P–OCH3), 3.72 (3H, d, JH–P ¼ 10.4 Hz,
2
3
P–OCH3), 6.51–6.58 (1H, dd, JH–P ¼ 8.4 Hz and JH–H ¼ 2.0 Hz,
P–CH), 6.33 (1H, m, NH), 7.90–6.96 (11H, m, Ar–H). 13C NMR
(100.56 MHz, CDCl3) d: 105.2 (C-1), 148.6 (C-2), 119.8 (C-3), 130.4 (C-4),
129.5 (C-5), 122.3 (C-6), 127.5 (C-7), 126.6 (C-8), 134.5 (C-9), 129.8 (C-10),
71.3 (C-12), 131.4 (C-13), 130.9 (C-14 and C-28), 115.5 (C-15 and 17),
160.8 (C-16), 53.5 (P–OCH3), 53.9 (P–OCH3). 31P NMR (161.89, MHz,
CDCl3) d: 22.75. Anal. calcd. for C19H20NO4P: C, 63.86; H, 5.64; N,
3.92; Found: C, 63.81; H, 5.58; N, 3.87.
title compounds were characterized by IR, 1H NMR, 13C NMR, 31
NMR, mass spectral, and elemental analysis.
P
Physical and spectral characterization of the title
compounds (4a–p)
Dimethyl ((4-methoxyphenyl)(naphthalen-2-ylamino)methyl)-
Dimethyl ((naphthalen-2-ylamino)(4-nitrophenyl)methyl)-
phosphonate (4a): Brown solid, mp: 162–164°C. IR (KBr) (nmax
phosphonate (4e): Yellow solid, mp: 168–170°C. IR (KBr)
cmꢁ1): 3304 (N–H), 2955 and 2866 (C–Haromatic), 1257 (P O), 761
(nmax cmꢁ1): 3321 (N–H), 2960 and 2869 (C–Haromatic), 1229
–
–
(P–Caliphatic). 1H NMR (400 MHz, CDCl3) d: 3.78 (3H, s, Ar–OCH3), 3.56
(P O), 1532 and 1347 (N–O), 755 (P–Caliphatic). 1H NMR (400 MHz,
–
–
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

674
R. R. Chinthaparthi et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 667–676
3
CDCl3) d: 3. 28 (3H, d, JH–P ¼ 10.4 Hz, P–OCH3), 3.47 (3H, d,
Ar–H). 13C NMR (100.56 MHz, CDCl3) d: 105.2 (C-1), 148.3 (C-2),
119.5 (C-3), 130.1 (C-4), 130.3 (C-5), 122.4 (C-6), 125.5 (C-7), 126.3
(C-8), 134.5 (C-9), 128.7 (C-10), 66.7 (C-12), 26.2 (C-13), 16.1 (C-14).
31P NMR (161.7, MHz, CDCl3) d: 21.62. EI-MS (m/z, %): 293 (Mþ•).
Anal. calcd. for C15H20NO3P: C, 61.43; H, 6.87; N, 4.78; Found: C,
61.37; H, 6.81; N, 4.73.
3JH–P ¼ 10.4 Hz, P–OCH3), 6.25–6.51 (1H, dd, JH–P ¼ 8.4 Hz and
2
3JH–H ¼ 2.0 Hz, P–CH), 6.53 (1H, m, NH), 7.84–7.32 (11H, m, Ar–H).
13C NMR (100.56 MHz, CDCl3) d: 105.4 (C-1), 148.7 (C-2), 119.9 (C-3),
129.4 (C-4), 129.6 (C-5), 121.7 (C-6), 127.6 (C-7), 126.5 (C-8), 134.4
(C-9), 129.9 (C-10), 70.8 (C-12), 143.3 (C-13), 130.1 (C-14 and C-28),
125.3 (C-15 and 17), 153.6 (C-16), 53.7 (P–OCH3), 53.8 (P–OCH3). 31
P
NMR (161.89, MHz, CDCl3) d: 21.35. EI-MS (m/z, %): 386 (Mþ•). Anal.
calcd. for C19H19N2O5P: C, 59.07; H, 4.96; N, 7.25; Found: C, 59.01;
H, 4.91; N, 7.21.
Dimethyl (1-(naphthalen-2-ylamino)butyl)phosphonate (4j):
Pale yellow solid, mp: 153–155°C. IR (KBr) (nmax cmꢁ1): 3322 (N–H),
2963 and 2861 (C–Haromatic), 1241 (P O), 751 (P–Caliphatic). 1H NMR
–
–
(400 MHz, CDCl3) d: 1.38–1.51 (4H, m, 2 ꢂ CH2), 1.12 (3H, t, CH3),
Dimethyl ((4-fluorophenyl)(naphthalen-2-ylamino)methyl)-
phosphonate (4f): Pale yellow solid, mp: 183–185°C. IR (KBr)
3.55 (3H, d, 3JH–P ¼ 10.4 Hz, P–OCH3), 3.67 (3H, d, 3JH–P ¼ 10.4 Hz,
2
3
P–OCH3), 5.61–5.66 (1H, dd, JH–P ¼ 8.4 Hz and JH–H ¼ 2.0 Hz,
P–CH), 6.36 (1H, m, NH), 7.68–7.18 (7H, m, Ar–H). 13C NMR
(100.56 MHz, CDCl3) d: 105.6 (C-1), 146.6 (C-2), 119.8 (C-3), 130.4 (C-
4), 130.6 (C-5), 122.2 (C-6), 125.8 (C-7), 126.7 (C-8), 134.8 (C-9), 128.9
(C-10), 66.3 (C-12), 27.3 (C-13), 20.5 (C-14), 14.2 (C-15). 31P NMR
(161.7, MHz, CDCl3) d: 21.52. EI-MS (m/z, %): 307 (Mþ•). Anal. calcd.
for C16H22NO3P: C, 62.53; H, 7.22; N, 4.56; Found: C, 62.46; H, 7.15;
N, 4.51.
(nmax cmꢁ1): 3323 (N–H), 2960 and 2872 (C–Haromatic), 1235 (P O),
–
–
755 (P–Caliphatic). 1H NMR (400 MHz, CDCl3) d: 3.56 (3H, d,
3
3JH–P ¼ 10.4 Hz, P–OCH3), 3.71 (3H, d, JH–P ¼ 10.4 Hz, P–OCH3),
2
3
6.51–6.58 (1H, dd, JH–P ¼ 8.4 Hz and JH–H ¼ 2.0 Hz, P–CH), 6.42
(1H, m, NH), 7.95–7.36 (11H, m, Ar–H). 13C NMR (100.56 MHz,
CDCl3) d: 105.3 (C-1), 148.3 (C-2), 119.1 (C-3), 130.5 (C-4), 129.6 (C-5),
122.2 (C-6), 127.6 (C-7), 126.3 (C-8), 134.4 (C-9), 129.2 (C-10), 71.9
(C-12), 135.5 (C-13), 130.8 (C-14 and C-28), 119.3 (C-15 and 17), 161.7
(C-16), 53.7 (P–OCH3), 53.9 (P–OCH3). 31P NMR (161.89, MHz, CDCl3)
d: 21.35. EI-MS (m/z, %): 359 (Mþ•). Anal. calcd. for C19H19FNO3P: C,
63.51; H, 5.33; N, 3.90; Found: C, 63.45; H, 5.28; N, 3.85.
Dimethyl (3-methyl-1-(naphthalen-2-ylamino)butyl)phos-
phonate (4k): Pale yellow solid, mp: 161–163°C. IR (KBr)
(nmax cmꢁ1): 3324 (N–H), 2965 and 2862 (C–Haromatic), 1246
–
Dimethyl ((4-chlorophenyl)(naphthalen-2-ylamino)methyl)-
(P O), 754 (P–Caliphatic). 1H NMR (400 MHz, CDCl3) d: 1.28–1.59
–
3
(6H, m, 3 ꢂ CH2), 1.05 (3H, d, CH3), 3.55 (3H, d, JH–P ¼ 10.4 Hz,
phosphonate (4g): Pale yellow solid, mp: 173–175°C. IR (KBr)
P–OCH3), 3.65 (3H, d, 3JH–P ¼ 10.4 Hz, P–OCH3), 5.45–5.54 (1H, dd,
(nmax cmꢁ1): 3334 (N–H), 2969 and 2865 (C–Haromatic), 1237 (P O),
–
–
3
2JH–P ¼ 8.4 Hz and JH–H ¼ 2.0 Hz, P–CH), 6.43 (1H, m, NH),
757 (P–Caliphatic). 1H NMR (400 MHz, CDCl3) d: 3.51 (3H, d,
7.61–7.20 (7H, m, Ar–H). 31P NMR (161.7, MHz, CDCl3) d: 21.54.
EI-MS (m/z, %): 321 (Mþ•). Anal. calcd. for C17H24NO3P: C, 63.54; H,
7.53; N, 4.36; Found: C, 63.45; H, 7.46; N, 4.31.
3
3JH–P ¼ 10.4 Hz, P–OCH3), 3.68 (3H, d, JH–P ¼ 10.4 Hz, P–OCH3),
2
3
6.53–6.59 (1H, dd, JH–P ¼ 8.4 Hz and JH–H ¼ 2.0 Hz, P–CH), 6.35
(1H, m, NH), 7.90–7.32 (11H, m, Ar–H). 13C NMR (100.56 MHz,
CDCl3) d: 105.4 (C-1), 148.7 (C-2), 119.6 (C-3), 130.3 (C-4), 130.6 (C-5),
122.8 (C-6), 125.7 (C-7), 126.7 (C-8), 134.7 (C-9), 128.4 (C-10), 70.8
(C-12), 135.1 (C-13), 131.2 (C-14 and C-28), 125.4 (C-15 and 17), 136.5
(C-16), 53.3 (P-OCH3), 53.7 (P-OCH3). 31P NMR (161.7, MHz, CDCl3) d:
21.43. EI-MS (m/z, %): 375 (Mþ•). Anal. calcd. for C19H19ClNO3P: C,
60.73; H, 5.10; N, 3.73; Found: C, 60.68; H, 5.02; N, 3.65.
Dimethyl (1-(naphthalen-2-ylamino)pentyl)phosphonate
(4l): Pale yellow solid, mp: 173–175°C. IR (KBr) (nmax cmꢁ1):
–
3323 (N–H), 2963 and 2859 (C–Haromatic), 1251 (P O), 756
–
(P–Caliphatic). 1H NMR (400 MHz, CDCl3) d: 1.45–1.53 (2H, m,
CHCH2), 1.72 (1H, m, CH2CH), 1.21 (6H, d, (CH3)2), 3.51 (3H, d,
3
3JH–P ¼ 10.4 Hz, P–OCH3), 3.63 (3H, d, JH–P ¼ 10.4 Hz, P–OCH3),
Dimethyl ((4-bromophenyl)(naphthalen-2-ylamino)methyl)-
2
3
5.46–5.58 (1H, dd, JH–P ¼ 8.4 Hz and JH–H ¼ 2.0 Hz, P–CH), 6.48
(1H, m, NH), 7.64–7.19 (7H, m, Ar–H). 31P NMR (161.7, MHz, CDCl3)
d: 22.45. EI-MS (m/z, %): 321 (Mþ•). Anal. calcd. for C17H24NO3P: C,
63.54; H, 7.53; N, 4.36; Found: C, 63.49; H, 7.47; N, 4.30.
phosphonate (4h): Brown solid, mp: 206–208°C. IR (KBr)
(nmax cmꢁ1): 3325 (N–H), 2968 and 2863 (C–Haromatic), 1234
(P O), 751 (P–Caliphatic). 1H NMR (400 MHz, CDCl3) d: 3.56 (3H,
–
–
d, 3JH–P ¼ 10.4 Hz, P–OCH3), 3.62 (3H, d, 3JH–P ¼ 10.4 Hz, P–OCH3),
2
3
6.55–6.61 (1H, dd, JH–P ¼ 8.4 Hz and JH–H ¼ 2.0 Hz, P–CH), 6.36
(1H, m, NH), 7.96–7.30 (11H, m, Ar–H). 13C NMR (100.56 MHz,
CDCl3) d: 105.8 (C-1), 148.9 (C-2), 119.2 (C-3), 130.6 (C-4), 130.4 (C-5),
122.2 (C-6), 125.3 (C-7), 126.9 (C-8), 134.2 (C-9), 128.1 (C-10), 70.1
(C-12), 135.9 (C-13), 131.5 (C-14 and C-28), 134.2 (C-15 and 17), 126.5
(C-16), 53.4 (P–OCH3), 53.8 (P–OCH3). 31P NMR (161.7, MHz, CDCl3)
d: 21.65. EI-MS (m/z, %): 419 (Mþ•). Anal. calcd. for C19H19BrNO3P: C,
54.30; H, 4.56; N, 3.33; Found: C, 54.23; H, 4.51; N, 3.25.
Dimethyl (((9H-fluoren-2-yl)amino)(4-ethoxyphenyl)methyl)-
phosphonate (4m): Brown solid, mp: 185–187°C. IR (KBr)
(nmax cmꢁ1): 3315 (N–H), 2965 and 2865 (C–Haromatic), 1255 (P O),
–
–
752 (P–Caliphatic). 1H NMR (400 MHz, CDCl3) d: 3.82 (3H, q, Ar–OCH2),
d: 1.29 (3H, t, OCH2CH3), 3.51 (3H, d, 3JH–P ¼ 10.4 Hz, P–OCH3), 3.81
(3H, d, 3JH–P ¼ 10.4 Hz, P–OCH3), 3.68 (2H, s, Ar–CH2–Ar), 6.59–6.65
2
3
(1H, dd, JH–P ¼ 8.4 Hz and JH–H ¼ 2.0 Hz), 6.56 (1H, m, NH),
7.29–7.73 (11H, m, Ar–H). 13C NMR (100.56 MHz, CDCl3) d: 114.5
(C-1), 150.7 (C-2), 108.1 (C-3), 122.7 (C-4), 122.3 (C-5), 128.2 (C-6), 127.7
(C-7), 127.1 (C-8), 36.9 (C-9), 145.7 (C-10), 128.8 (C-11), 137.9 (C-12),
140.1 (C-13), 56.8 (C-15), 130.5 (C-16), 126.5 (C-17 and C-21), 114.7
(C-18 and C-20), 158.9 (C-19), 55.3 (C-23), 52.6 (P–OCH3), 53.5
(P–OCH3). 31P NMR (161.89, MHz, CDCl3) d: 22.07. EI-MS (m/z, %): 423
(Mþ•). Anal. calcd. for C24H26NO4P: C, 68.07; H, 6.19; N, 3.31; Found:
C, 68.01; H, 6.12; N, 3.26.
Dimethyl (1-(naphthalen-2-ylamino)propyl)phosphonate
(4i): Pale yellow solid, mp: 145–147°C. IR (KBr) (nmax cmꢁ1):
–
3312 (N–H), 2954 and 2867 (C–Haromatic), 1233 (P O), 752
–
(P–Caliphatic). 1H NMR (400 MHz, CDCl3) d: 1.38 (2H, m, CHCH2),
3
1.11 (3H, t, CH2CH3), 3.53 (3H, d, JH–P ¼ 10.4 Hz, P–OCH3), 3.66
(3H, d, 3JH–P ¼ 10.4 Hz, P–OCH3), 5.64–5.71 (1H, dd, 2JH–P ¼ 8.4 Hz
3
and JH–H ¼ 2.0 Hz, P–CH), 6.31 (1H, m, NH), 7.52–7.15 (7H, m,
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2013, 346, 667–676
a-Aminophosphonates as Potential Anticancer Drugs
675
Dimethyl (((9H-fluoren-2-yl)amino)(4-nitrophenyl)methyl)-
medium at a final density of 2 ꢂ 104 cells/well, in 96-well
microtiter plates under identical conditions. After overnight
incubation, the cells were treated with different concentrations of
test compounds (0.1–100 mg/mL) or DMSO (carrier solvent) in a
final volume of 200 mL with three replicates each. After 24 h,
10 mL of MTT (5 mg/ mL) was added to each well and the plate
was incubated at 37°C in the dark for 4 h. Then the media along
with MTT was removed and the formazan crystals were solubilized
by adding DMSO (100 mL/well). Finally, the reduction of MTT was
quantified by reading the absorbance at 570 nm using GENios1
microplate reader (Tecan Austria GmbH, Austria). The effects of
the test compounds on cell viability were evaluated using
untreated cells added with DMSO as control. The data were
subjected to linear regression analysis and the regression lines
were plotted for the best straight-line fit. The IC50 (the
concentration at which 50% of the cells are dead) concentrations
were calculated using the respective regression equation.
phosphonate (4n): Yellow solid, mp: 186–188°C. IR (KBr)
(nmax cmꢁ1): 3332 (N–H), 2964 and 2863 (C–Haromatic), 1260
1
–
(P O), 754 (P–Caliphatic). H NMR (400 MHz, CDCl3) d: 3.55 (3H, d,
–
3JH–P ¼ 10.4 Hz, P–OCH3), 3.74 (3H, d, JH–P ¼ 10.4 Hz, P–OCH3),
3
2
3.78 (2H, s, Ar–CH2–Ar), 6.65–6.72 (1H, dd, JH–P ¼ 8.4 Hz and
3JH–H ¼ 2.0 Hz), 6.65 (1H, m, NH), 7.24–7.74 (11H, m, Ar–H). 13C
NMR (100.56 MHz, CDCl3) d: 114.7 (C-1), 150.5 (C-2), 108.3 (C-3),
121.6 (C-4), 122.2 (C-5), 127.9 (C-6), 128.4 (C-7), 126.7 (C-8), 37.1
(C-9), 145.1 (C-10), 129.3 (C-11), 138.7 (C-12), 140.8 (C-13), 56.5
(C-15), 138.3 (C-16), 128.2 (C-17 and C-21), 125.7 (C-18 and C-20),
151.7 (C-19), 52.5 (P-OCH3), 53.6 (P-OCH3). 31P NMR (161.7, MHz,
CDCl3) d: 21.16. EI-MS (m/z, %): 424 (Mþ•). Anal. calcd. for
C22H21N2O5P: C, 62.26; H, 4.99; N, 6.60; Found: C, 62.20; H, 4.91; N,
6.51.
The altered morphology of exposed cells (1 ꢂ 104 cells/well) at
the respective IC50 concentrations of the most potential
compound was studied after 24 h using a phase contrast
microscope (DMI6000B, Leica Microsystems, Wetzlar, Germany).
Subsequently, the cells were Hoechst stained to observe the
nuclear/chromosomal condensation that occurred due to the
treatment of the highly active test compound. For staining, 96-
well cell culture plates were used to culture the cells (1 ꢂ 104
cells/well) in three replicates to treat with the best test compound.
Then the cells were incubated at 37°C overnight and the media
was removed to wash the cells twice with phosphate buffered
saline (PBS) and fixed with 4% paraformaldehyde in PBS for 1 day
at 4°C. Further, the cells were stained with 1 mg/mL of the
fluorescent DNA-binding dye, Bisbenzimide Hoechst 33342 stain,
and incubated for 20 min at room temperature to reveal nuclear
condensation/aggregation due to the effect of the test compound.
The Hoechst-stained cells were visualized and photographed
under fluorescence microscope (CTR 6000; Leica Microsystems).
Dimethyl (1-((9H-fluoren-2-yl)amino)butyl)phosphonate (4o):
Brown solid, mp: 134–136°C. IR (KBr) (nmax cmꢁ1): 3313 (N–H), 2965
and 2865 (C–Haromatic), 1255 (P O), 759 (P–Caliphatic). 1H NMR
–
–
(400 MHz, CDCl3) d: 3.54 (3H, d, 3JH–P ¼ 10.4 Hz, P–OCH3), 3.76 (3H,
d, 3JH–P ¼ 10.4 Hz, P–OCH3), 3.52 (2H, s, Ar–CH2–Ar), 6.63–6.66 (1H,
dd, 2JH–P ¼ 8.4 Hz and 3JH–H ¼ 2.0 Hz), 5.79 (1H, m, NH), 7.23–7.67
(7H, m, Ar–H), 1.34–1.49 (4H, m, 2 ꢂ CH2), 1.12 (3H, t, CH3). 31P NMR
(161.7, MHz, CDCl3) d: 21.62. EI-MS (m/z, %): 345 (Mþ•). Anal. calcd. for
C19H24NO3P: C, 66.07; H, 7.00; N, 4.06; Found: C, 66.00; H, 6.92; N,
4.01.
Dimethyl (1-((9H-fluoren-2-yl)amino)pentyl)phosphonate (4p):
Pale yellow solid, mp: 140–142°C. IR (KBr) (nmax cmꢁ1): 3314 (N–H),
1
–
2964 and 2857 (C–Haromatic), 1257 (P O), 755 (P–Caliphatic). H NMR
–
(400 MHz, CDCl3) d: 3. 47 (3H, d, 3JH–P ¼ 10.4 Hz, P–OCH3), 3.70 (3H, d,
3JH–P ¼ 10.4 Hz, P–OCH3), 3.51 (2H, s, Ar–CH2–Ar), 6.65–6.69 (1H, dd,
2JH–P ¼ 8.4 Hz and 3JH–H ¼ 2.0 Hz), 5.54 (1H, m, NH), 7.27–7.56 (7H, m,
Ar–H), 1.31–1.54 (6H, m, 3 ꢂ CH2), 1.06 (3H, t, CH3). 31P NMR
(161.7, MHz, CDCl3) d: 21.32. EI-MS (m/z, %): 359 (Mþ•). Anal. calcd. for
C20H26NO3P: C, 66.84; H, 7.29; N, 3.90; Found: C, 66.76; H, 7.21; N, 3.83.
The authors express their grateful thanks to Prof. C. Devendranath
Reddy, Department of Chemistry, Sri Venkateswara University,
Tirupati, for his helpful discussions and DRDO, New Delhi, for
providing financial assistance (ERIP/ER/1103894M/01/1346).
Pharmacology
Cell lines
Human cervical cancer cell line (HeLa) and human breast
adenocarcinoma cell line (SK-BR-3) were obtained from
American Type Culture Collection (Manassas, VA, USA). Dulbecco’s
modified Eagle’s medium (DMEM) was purchased from BioWhit-
taker1, and Roswell Park Memorial Institute (RPMI) medium, fetal
bovine serum (FBS), and other cell culture materials were
purchased from Sigma1 (USA). Cells were cultured either in
DMEM (HeLa) or in RPMI (SK-BR-3) media supplemented with 10%
v/v heat-inactivated fetal bovine serum (FBS), 100 units/mL
penicillin, and 100 mg/ mL streptomycin. Cells were maintained
in culture at 37°C in an atmosphere of 5% CO2.
The authors have declared no conflict of interest.
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