Synthesis and antiproliferative activity of novel α-aminophosphonates

Article abstract of DOI:10.1248/cpb.c12-00676

A novel series of carbazole-based α-aminophosphonates were synthesized by three component coupling of 6-bromo-9-ethyl-9H-carbazole-3- carbaldehyde, amine and diethyl phosphite using polyethylene glycol (PEG-400) as a green reaction media. The antiproliferative activity of these molecules was evaluated against three cancer cell lines. Of these, compounds 4c, 4e and 4m were found to exhibit good antiproliferative activity against three cancer cells, A549, MCF-7, and NCI-N87.

Full text of DOI:10.1248/cpb.c12-00676

December 2012
Regular Article
Chem. Pharm. Bull. 60(12) 1531–1537 (2012)
1531
Synthesis and Antiproliferative Activity of Novel α-Aminophosphonates
,a
Anil Kumar Mungara,a Yong-Ki Park,b and Kap Duk Lee*
a Department of Nanomaterial Chemistry, Dongguk University; and b Department of Herbology, Dongguk University;
Gyeongju 780–714, South Korea.
Received July 30, 2012; accepted August 29, 2012; advance publication released online September 14, 2012
A novel series of carbazole-based α-aminophosphonates were synthesized by three component coupling
of 6-bromo-9-ethyl-9H-carbazole-3-carbaldehyde, amine and diethyl phosphite using polyethylene glycol
(PEG-400) as a green reaction media. The antiproliferative activity of these molecules was evaluated against
three cancer cell lines. Of these, compounds 4c, 4e and 4m were found to exhibit good antiproliferative activ-
ity against three cancer cells, A549, MCF-7, and NCI-N87.
Key words α-aminophosphonate; carbazole; antiproliferative activity; cancer cell
Organophosphorus compounds have been found a wide biologically important organophosphorus compounds,28–31)
variety of applications in the areas of agricultural, indus- we herein report the synthesis of a newly designed series of
trial, and medicinal chemistry due to their biological and carbazole-based α-aminophosphonates. The antiproliferative
physical properties as well as their utility as synthetic in- activity of these molecules was evaluated against three cancer
termediates.1–5) In recent years, a significant attention has cell lines A549 (human lung cancer), MCF-7 (human breast
been focused on the synthesis and biological evaluation of cancer), and NCI-N87 (human stomach cancer).
α-aminophosphonic acids and their phosphonate derivatives.
Since α-aminophosphonate derivatives are structural mim-
ics of α-amino acids, some of these compounds exhibit very
Results and Discussion
On the basis of our previous results28) polyethylene gly-
high potency in inhibiting the enzymes that are involved in col (PEG-400) was used for the one pot three component
the metabolism of the corresponding amino acids. Generally, synthesis of α-aminophosphonates. The carbazole-based
low mammalian toxicity of these compounds makes them at- α-aminophosphonates were synthesized by the coupling of
tractive for use in agriculture and medicine.6–9) Numerous 6-bromo-9-ethyl-9H-carbazole-3-carbaldehyde, amine, and
of them possess antifungal, pesticidal, herbicidal and plant diethyl phosphite in PEG-400. The reaction was complete in
growth regulatory activity and are of particular interest for 6–7h at 100°C temperature (Chart 1). After completion of the
agrochemistry.8–12) α-Aminophosphonates have also been reaction, cold water was added to the reaction mixture and the
found to act as enzyme inhibitors, haptens of catalytic anti- resulting precipitate was filtered and washed with water. The
biotics, inhibitors of serine hydrolases, uridine 5′-diphosphate solid product was then dried and purified by column chroma-
(UDP)-galactopyranose mutase, human immunodeficiency tography to give the pure α-aminophosphonate.
virus (HIV) protease, and antitumor agents.13–20) On the other
In this PEG mediated reaction, different kinds of substi-
hand, carbazole derivatives exhibit diverse biological activities tuted anilines carrying either electron donating or electron
such as antimalarial, antibacterial, anti-tuberculosis (TB), anti- withdrawing substituents and also benzylamine reacted well
HIV, anti-inflammatory, antihistaminic, and antitumor activi- to give the desired products in good yields and the results are
ties.21–24) Furthermore, carbazole-based oligomers or polymers summarized in Table 1. In this reaction PEG-400 not only
have also attracted much interest in materials science because acts as the solvent but also accelerates the imine formation
of their interesting electrical and optical properties.25–27)
and nucleophilic addition of phosphite to the imine by in-
Owing to their synthetic and biological importance, the creasing its electrophilicity through hydrogen bonding by its
chemistry of aminophosphonates and carbazole derivatives hydroxyl group with the imine nitrogen. The structures of all
1
has stimulated an increasing interest and the synthesis of the compounds were confirmed by IR, H-, 13C-, and 31P-NMR
carbazole-based aminophosphonates remains a great interest and mass spectroscopy. The IR spectra of compounds
in the field of medicinal chemistry.
4a–n showed the expected absorption bands at 3378–3269,
As part of our continued interest in developing new 1234–1231, and 973–963cm−1, which are attributed to N–H,
Chart 1. Synthesis of Carbazole-Based α-Aminophosphonates
The authors declare no conflict of interest.
*To whom correspondence should be addressed. e-mail: kdlee@dongguk.ac.kr
© 2012 The Pharmaceutical Society of Japan

1532
Vol. 60, No. 12
Table 1. Synthesis of Carbazole-Based α-Aminophosphonates Using PEG-400
Entry
Aldehyde
Amine
Product
Yield (%)a)
a
88
90
b
c
d
e
92
90
86
f
g
h
i
89
90
89
84
82
j

December 2012
1533
Table 1. Synthesis of Carbazole-Based α-Aminophosphonates Using PEG-400
Entry
k
Aldehyde
Amine
Product
Yield (%)a)
78
l
81
m
79
75
n
a) Isolated yields.
Table 2. Maximum Percentage of Inhibition for Compounds 4a–n at Doses of 5 and 10µM
Inhibition (%) of cell lines (mean S.E.M.)
MCF-7
Compound
A549
NCI-N87
5µM
10µM
5µM
10µM
5µM
10µM
4a
4b
4c
4d
4e
4f
42.19 0.21
21.09 0.29
63.76 0.02
50.45 0.21
56 0.05
36.85 0.32
36.91 0.05
68.64 0.24
51.65 0.56
56.72 0.42
22.35 0.22
16.99 4.54
17.65 0.50
4.34 0.13
11.23 1.17
8.4 0.51
0a)
0a)
27.17 0.31
5.23 0.54
44.13 0.22
27.91 0.19
76.67 0.14
21.32 0.93
0a)
35.72 0.11
25.5 1.72
55.84 0.98
46.66 0.59
85.93 0.14
26.61 0.77
8.27 0.57
13.25 1.11
40.72 0.74
13.88 1.01
5.09 0.34
16.49 0.54
89.86 1.39
0a)
0a)
0a)
26.15 2.62
31.78 2.12
0a)
0a)
39.04 1.01
48.92 3.01
15.71 0.77
8.61 0.05
4.69 1.20
4.69 0.85
10.35 0.37
1.31 0.56
29.71 0.16
71.47 0.32
1.29 0.31
0a)
0a)
4g
4h
4i
0a)
0a)
0a)
0a)
0a)
0a)
0a)
0a)
0a)
29.94 0.75
0a)
4j
4k
4l
0a)
0a)
0a)
38.64 0.50
84.05 0.56
61.36 0.46
0.85 0.73
25.58 3.10
0a)
3.46 0.57
61.81 0.97
0a)
2.31 0.63
53.23 0.45
0a)
4m
4n
a) The score zero shows no inhibition of cell proliferation.
P=O, P–O stretching vibrations, respectively. In 1H-NMR cancer), MCF-7 (human breast cancer) and NCI-N87 (human
spectra, doublet or doublet of doublet in the region of stomach cancer) by WST-1 cell proliferation assay. The re-
4.74–4.99ppm indicates the presence of methine proton. The sults are summarized in Table 2. As evident from the results
remaining proton signals are observed in the expected regions. obtained, all the aminophosphonates (4a–n) exhibited good to
In 13C-NMR spectra, doublet in the region of 55.3–59.5ppm moderate antiproliferative activity on A549 cells compare to
(1JP–C=150.2–154.8Hz) confirms the presence of methine MCF-7 and NCI-N87 cells. Especially, compounds 4c, 4d, 4e,
carbon which is directly attached to phosphorus. In 31P-NMR and 4m showed good antiproliferative activity on A549 cells.
spectra of all α-aminophosphonates 4a–n showed a signal in It was observed that the compounds 4a–n displayed lower ac-
the region of 22.83–25.11ppm. The high-resolution mass spec- tivity against MCF-7 cells. Except 4c, 4e, and 4m, the remain-
tral data for compounds 4a–n are provided in Experimental.
ing compounds showed zero or negligible antiproliferative
Antiproliferative Activity The antiproliferative activity activity on MCF-7 cells. In the case of NCI-N87 cells, com-
of the synthesized compounds 4a–n was evaluated in vitro pounds 4c, 4e, and 4m showed good antiproliferative activ-
against three different cancer cell lines A549 (human lung ity, and other compounds showed negligible antiproliferative

1534
Vol. 60, No. 12
antiproliferative activity of these compounds may perhaps be
attributed to the presence of relatively high electron density on
the exocyclic amine nitrogen atom which facilitates the bind-
ing ability of these compounds with the enzymes of cancer
cells thereby preventing the proliferation.
Furthermore, for compounds 4c, 4e, and 4m, the cell pro-
liferation assay was conducted in a concentration-dependent
(1–10 µM) manner (Fig. 1). The inhibition percentage of these
compounds at concentrations of 1, 2, 5, and 10µM is presented
in Table 3. It has been observed that the compounds 4c and
4e active against A549 cells and compound 4e active against
NCI-N87 cells even at concentration of 2µM. Compound 4m
exhibited the strong antiproliferative activity compared to 4c
and 4e against all cancer cells at higher concentration (10µM).
Conclusion
In summary, we have synthesized a novel series of carba-
zole-based α-amino phosphonates and subsequently evaluated
their antiproliferative activity. Among all the compounds, 4c,
4e and 4m were found to exhibit good anti-proliferative activ-
ity against all three cancer cells A549, MCF-7, and NCI-N87.
In this reaction, PEG-400 acts as an efficient “green” pro-
moter. The advantages of this method are simple and mild ex-
perimental conditions, avoiding hazardous solvents and toxic
organic reagents.
Experimental
The 6-bromo-9-ethyl-9H-carbazole-3-carbaldehyde, was
prepared by the ethylation32) followed by formylation33) and
bromination of carbazole.34) Melting points were determined
in an open capillaries using Electrothermal (IA 9100) digital
melting point apparatus and are uncorrected. IR spectra were
recorded on Bruker (Tensor 37) FT-IR spectrometer using
1
KBr pellets. H-, 13C-, and 31P-NMR spectra were recorded
on a VARIAN 200MHz instrument. Mass spectral data were
obtained from the Korea Basic Science Institute (Daegu) on
JEOL JMS-700 high resolution mass spectrometer.
Fig. 1. Dose-Dependent Antiproliferative Effect of 4c, 4e, and 4m on
Cancer Cells
General Experimental Procedure A mixture of 6-bromo-
9-ethyl-9H-carbazole-3-carbaldehyde (1mmol), amine (1
mmol), and diethyl phosphite (1.3mmol) was added to
PEG-400 (1g) and the mixture was heated to 100°C and
stirred for 6–7h. After completion of the reaction as moni-
Three cancer cells were treated with each compound at concentrations of 1,
2, 5, and 10µM for 24h, and measured the cell proliferation by WST-1 assay, as
described in Experimental. Results are represented as inhibition percentage of the
cell proliferation with the mean S.E.M. of three independent experiments.
activity. Among all the aminophosphonates, compounds 4c, tored by TLC, the mixture was diluted with cold water
4e, and 4m were found to exhibit good antiproliferative activi- and the resulting precipitate was filtered and washed with
ty against all three cell lines A549, MCF-7, and NCI-N87. The water. The residue was then dried and purified by column
Table 3. Maximum Percentage of Inhibition for 4c, 4e, and 4m at Doses of 1, 2, 5, and 10µM
Inhibition (%) of cell proliferation (mean S.E.M.)
Cancer cell
A549
Compound
1µM
2µM
5µM
10µM
4c
49.15 0.24
58.32 0.88
12.43 0.53
13.09 1.87
30.23 4.32
0a)
55.25 0.26
61.04 0.77
13.71 0.05
18.89 1.95
33.74 0.97
0a)
63.76 0.02
56.00 0.05
71.47 0.32
26.15 2.62
39.04 1.01
25.58 3.10
44.13 0.22
76.67 0.14
53.23 0.45
68.64 0.24
56.72 0.42
84.05 0.56
31.78 2.12
48.92 3.01
61.81 0.97
55.84 0.98
85.93 0.14
89.86 1.39
4e
4m
4c
MCF-7
4e
4m
4c
NCI-N87
28.38 1.14
49.68 0.42
0a)
34.30 0.56
64.22 0.82
4.21 0.37
4e
4m
a) The score zero shows no inhibition of cell proliferation.

December 2012
1535
chromatography over silica gel (ethyl acetate–hexane) to 1H), 6.34 (dd, J=8.6, 2.8Hz, 1H), 6.57 (d, J=3.0Hz, 1H), 7.17
give the pure α-aminophosphonate. In the case of 4m, the (d, J=8.6Hz, 1H), 7.23–7.27 (m, 1H), 7.36 (d, J=8.6Hz, 1H),
product was gummy therefore it was extracted with ethyl 7.50–7.60 (m, 2H), 8.09–8.17 (m, 2H); 13C-NMR (CDCl3) δ:
acetate and then, washed with water, and dried over sodium 13.7, 16.3 (d, JP–C=6.0Hz), 23.0, 37.7, 56.1 (d, JP–C=151.7Hz),
sulfate. Removal of the solvent followed by purification on 63.2, 108.9, 109.9, 111.6, 112.8, 116.4, 119.8, 119.9, 122.1,
column chromatography over silica gel afforded the pure 123.2, 124.3, 126.1, 128.4, 132.5, 138.2, 138.8, 140.0, 145.6,
α-aminophosphonate.
145.9; 31P-NMR (CDCl3) δ: 23.92; HR-MS (EI) m/z: Calcd for
Entry 4a: White solid; mp 175–176°C; IR (KBr) cm−1: 3307, C26H29Br2N2O3P: 606.0283 (M+), Found: 606.0281
1
2976, 1601, 1234, 1020, 964, 754; H-NMR (CDCl3) δ: 1.08
Entry 4f: Pale yellow solid; mp 147–148°C; IR (KBr)
(t, J=7.0Hz, 3H), 1.30 (t, J=7.0Hz, 3H), 1.40 (t, J=7.0Hz, cm−1: 3293, 2979, 1509, 1232, 1025, 969, 793; 1H-NMR
3H), 3.57–3.73 (m, 1H), 3.83–3.98 (m, 1H), 4.09–4.17 (m, 2H), (CDCl3) δ: 1.08 (t, J=7.0Hz, 3H), 1.31 (t, J=7.2Hz, 3H),
4.31 (q, J=7.0Hz, 2H), 4.93 (d, J=23.6Hz, 1H), 6.63–6.70 (m, 1.40 (t, J=7.2Hz, 3H), 3.54–3.74 (m, 1H), 3.82–3.98 (m, 1H),
3H), 7.05–7.13 (m, 2H), 7.23–7.28 (m, 2H), 7.36 (d, J=8.6Hz, 4.06–4.20 (m, 2H), 4.31 (q, J=7.2Hz, 2H), 4.85 (d, J=23.4Hz,
1H), 7.50–7.64 (m, 2H), 8.12–8.19 (m, 2H);13C-NMR (CDCl3) 1H), 6.54–6.61 (m, 2H), 6.74–6.83 (m, 2H), 7.22–7.27 (m, 1H),
δ: 13.7, 16.3, 37.7, 56.1 (d, JP–C=150.2Hz), 63.1, 108.9, 109.9, 7.36 (d, J=8.6Hz, 1H), 7.50–7.61 (m, 2H), 8.10–8.18 (m, 2H);
111.6, 113.9, 118.3, 119.9, 123.2, 124.3, 126.1, 126.5, 128.3, 13C-NMR (CDCl3) δ: 13.7, 16.3, 37.7, 56.8 (d, JP–C=151.7Hz),
129.1, 138.8, 139.9, 146.3, 146.6; 31P-NMR (CDCl3) δ: 24.20; 63.2, 108.9, 109.9, 111.6, 114.8, 115.3, 115.8, 119.9, 122.0,
high resolution (HR)-MS (electron ionization (EI)) m/z: Calcd 123.2, 124.3, 126.1, 128.4, 138.8, 140.0, 142.6, 142.9, 153.8,
for C25H28BrN2O3P: 514.1021 (M+), Found: 514.1020.
158.5; 31P-NMR (CDCl3) δ: 24.09; HR-MS (EI) m/z: Calcd for
Entry 4b: White solid; mp 115–116°C; IR (KBr) cm−1: 3296, C25H27BrFN2O3P: 532.0927 (M+), Found: 532.0927.
1
2978, 1616, 1232, 1024, 965, 797; H-NMR (CDCl3) δ: 1.08 (t,
Entry 4g: White solid; mp 138–139°C; IR (KBr) cm−1: 3292,
1
J=7.0Hz, 3H), 1.30 (t, J=7.0Hz, 3H), 1.39 (t, J=7.2Hz, 3H), 2978, 1598, 1232, 1024, 967, 795; H-NMR (CDCl3) δ: 1.08 (t,
2.15 (s, 3H), 3.58–3.75 (m, 1H), 3.83–3.99 (m, 1H), 4.09–4.17 J=7.2Hz, 3H), 1.31 (t, J=7.0Hz, 3H), 1.40 (t, J=7.0Hz, 3H),
(m, 2H), 4.29 (q, J=7.2Hz, 2H), 4.91 (d, J=23.8Hz, 1H), 3.53–3.72 (m, 1H), 3.82–3.98 (m, 1H), 4.06–4.22 (m, 2H),
6.57 (d, J=8.4Hz, 2H), 6.90 (d, J=8.6Hz, 2H), 7.22–7.26 4.30 (q, J=7.2Hz, 2H), 4.87 (d, J=23.6Hz, 1H), 6.53–6.61 (m,
(m, 1H), 7.35 (d, J=8.4Hz, 1H), 7.49–7.63 (m, 2H), 8.12–8.18 2H), 6.99–7.06 (m, 2H), 7.23–7.27 (m, 1H), 7.36 (d, J=8.6Hz,
(m, 2H); 13C-NMR (CDCl3) δ: 13.7, 16.3, 20.3, 37.7, 56.4 (d, 1H), 7.50–7.60 (m, 2H), 8.01–8.16 (m, 2H); 13C-NMR (CDCl3)
JP–C=151.7Hz), 63.1, 108.8, 109.8, 111.6, 114.0, 119.9, 122.0, δ: 13.7, 16.4, 37.7, 56.2 (d, JP–C=151.7Hz), 63.2, 108.9, 109.9,
123.2, 124.3, 126.1, 126.7, 127.5, 128.3, 129.6, 138.8, 139.9, 115.0, 115.6, 119.8, 120.0, 122.0, 122.8, 123.2, 124.2, 126.1,
143.9, 144.2; 31P-NMR (CDCl3) δ: 24.36; HR-MS (EI) m/z: 128.4, 128.9, 138.7, 139.9, 144.9, 145.2; 31P-NMR (CDCl3) δ:
Calcd for C26H30BrN2O3P: 528.1177 (M+), Found: 528.1181.
23.87; HR-MS (EI) m/z: Calcd for C25H27BrClN2O3P: 548.0631
Entry 4c: Pale yellow solid; mp 182–183°C; IR (KBr) cm−1: (M+), Found: 548.0631.
1
3297, 2958, 1615, 1233, 1025, 966, 796; H-NMR (CDCl3) δ:
Entry 4h: Light grey solid; mp 137–139°C; IR (KBr) cm−1:
1
1.04–1.14 (m, 9H), 1.29 (t, J=7.2Hz, 3H), 1.40 (t, J=7.2Hz, 3292, 2978, 1594, 1232, 1024, 969, 796; H-NMR (CDCl3) δ:
3H), 2.62–2.83 (m, 1H), 3.54–3.73 (m, 1H), 3.82–3.98 (m, 1H), 1.08 (t, J=7.0Hz, 3H), 1.31 (t, J=7.0Hz, 3H), 1.40 (t, J=7.2Hz,
4.07–4.13 (m, 2H), 4.31 (q, J=7.2Hz, 2H), 4.90 (d, J=23.6Hz, 3H), 3.52–3.72 (m, 1H), 3.82–4.01 (m, 1H), 4.07–4.21 (m,
1H), 6.59 (d, J=8.4Hz, 2H), 6.96 (d, J=8.4Hz, 2H), 7.23–7.27 2H), 4.31 (q, J=7.0Hz, 2H), 4.87 (d, J=23.4Hz, 1H), 6.53
(m, 1H), 7.36 (d, J=8.4Hz, 1H), 7.52 (dd, J=8.4, 2.0Hz, 1H), (d, J=7.6Hz, 2H), 7.16 (t, J=7.4Hz, 2H), 7.24–7.28 (m, 1H),
7.59–7.65 (m, 1H), 8.12–8.19 (m, 2H); 13C-NMR (CDCl3) δ: 7.37 (d, J=8.4Hz, 1H), 7.50–7.60 (m, 2H), 8.10–8.18 (m, 2H);
13.7, 16.4, 24.1, 33.0, 37.7, 56.4 (d, JP–C=150.2Hz), 63.1, 108.8, 13C-NMR (CDCl3) δ: 13.7, 16.3, 37.7, 56.1 (d, JP–C=151.7Hz),
109.9, 111.6, 113.8, 119.9, 122.0, 123.2, 124.4, 126.2, 127.0, 63.3, 108.9, 109.9, 111.6, 115.5, 119.8, 122.1, 123.2, 124.2,
128.3, 138.7, 139.9, 144.3, 144.6; 31P-NMR (CDCl3) δ: 24.30; 126.0, 128.4, 131.8, 138.8, 140.0, 145.3, 145.6; 31P-NMR
HR-MS (EI) m/z: Calcd for C28H34BrN2O3P: 556.1490 (M+), (CDCl3) δ: 23.83; HR-MS (EI) m/z: Calcd for C25H27Br2N2O3P:
Found: 556.1489.
592.0126 (M+), Found: 592.0125.
Entry 4d: Pale yellow solid; mp 155–156°C; IR (KBr)
Entry 4i: Light brown solid; mp 206–207°C; IR (KBr)
cm−1: 3298, 2979, 1511, 1233, 1025, 967, 795; 1H-NMR cm−1: 3378, 3197, 2979, 1515, 1231, 1026, 963, 796; H-NMR
(CDCl3) δ: 1.09 (t, J=7.0Hz, 3H), 1.30 (t, J=7.0Hz, 3H), (DMSO-d6) δ: 0.99 (t, J=7.0Hz, 3H), 1.18–1.30 (m, 6H),
1.40 (t, J=7.2Hz, 3H), 3.56–3.76 (m, 4H), 3.83–3.99 (m, 1H), 3.56–3.69 (m, 1H), 3.78–3.90 (m, 1H), 4.01–4.15 (m, 2H), 4.39
4.06–4.21 (m, 2H), 4.30 (q, J=7.2Hz, 2H), 4.86 (d, J=23.4Hz, (q, J=7.0Hz, 2H), 4.98 (dd, J=24.1, 10.8Hz, 1H), 5.67–5.76 (m,
1H), 6.58–6.70 (m, 4H), 7.23–7.27 (m, 1H), 7.36 (d, J=8.4Hz, 1H), 6.44 (d, J=8.8Hz, 2H), 6.67 (d, J=8.8Hz, 2H), 7.53–7.66
1H), 7.52 (dd, J=8.6, 2.0Hz, 1H), 7.57–7.63 (m, 1H), 8.10–8.19 (m, 4H), 8.25–8.30 (m, 2H), 8.45 (s, 1H); 13C-NMR (DMSO-
(m, 2H); 13C-NMR (CDCl3) δ: 13.7, 16.3, 37.7, 55.5, 57.0 (d, d6) δ: 13.6, 16.2 (d, JP–C=9.1Hz), 37.1, 55.3 (d, JP–C=153.2Hz),
JP–C=150.2Hz), 63.1, 108.8, 109.9, 111.6, 114.7, 115.2, 119.9, 62.2 (dd, JP–C=15.1, 6.0Hz), 108.9, 110.7, 111.2, 115.1, 115.3,
122.0, 123.2, 124.3, 126.2, 126.7, 128.3, 138.8, 139.9, 140.3, 120.7, 122.5, 123.8, 127.0, 127.9, 138.4, 139.3, 139.5, 139.8,
140.6, 152.6; 31P-NMR (CDCl3) δ: 24.36; HR-MS (EI) m/z: 149.1; 31P-NMR (CDCl3) δ: 24.39; HR-MS (EI) m/z: Calcd for
1
Calcd for C26H30BrN2O4P: 544.1127 (M+), Found: 544.1123.
C25H28BrN2O4P: 530.0970 (M+), Found: 530.0973.
Entry 4e: Pale yellow solid; mp 167–168°C; IR (KBr) cm−1:
Entry 4j: Yellow solid; mp 148–149°C; IR (KBr) cm−1:
1
1
3292, 2977, 1600, 1233, 1024, 966, 794; H-NMR (CDCl3) δ: 3269, 2978, 1597, 1231, 1023, 973, 796; H-NMR (CDCl3) δ:
1.08 (t, J=7.0Hz, 3H), 1.31 (t, J=7.0Hz, 3H), 1.40 (t, J=7.4Hz, 1.09 (t, J=7.0Hz, 3H), 1.22–1.43 (m, 6H), 3.51–3.71 (m, 1H),
3H), 2.23 (s, 3H), 3.53–3.73 (m, 1H), 3.85–3.98 (m, 1H), 3.83–3.99 (m, 1H), 4.07–4.22 (m, 2H), 4.30 (q, J=7.2Hz,
4.05–4.21 (m, 2H), 4.31 (q, J=7.2Hz, 2H), 4.87 (d, J=23.8Hz, 2H), 4.99 (dd, J=23.5, 7.2Hz, 1H), 6.06 (t, J=8.2Hz, 1H),

1536
Vol. 60, No. 12
6.61–6.68 (m, 2H), 7.20–7.25 (m, 1H), 7.39 (d, J=8.4Hz, 1H), serum (FBS), 100U/mL of penicillin and 100µg/mL of strep-
7.48–7.60 (m, 2H), 7.96–8.04 (m, 3H), 8.13 (t, J=2.2Hz, 1H); tomycin at 37°C in a humidified incubator with an atmosphere
13C-NMR (CDCl3) δ: 13.7, 16.4, 37.7, 55.5 (d, JP–C=154.8Hz), containing 5% CO2.
63.6 (dd, JP–C=20.5, 6.1Hz), 109.0, 109.8, 111.6, 112. 3, 119.7,
Cell Proliferation Assay The antiproliferative activity of
122.2, 123.0, 124.0, 125.1, 126.0, 128.4, 138.7, 140.0, 152.1, all compounds was measured by the WST-1 Cell Proliferation
152.4; 31P-NMR (CDCl3) δ: 22.83; HR-MS (EI) m/z: Calcd for Assay Kit (Cayman Chemical Co., Ann Arbor, MI, U.S.A.).
C25H27BrN3O5P: 559.0872 (M+), Found: 559.0870.
The cells were seeded at 1×105 cells/well in 24-well flat-bot-
Entry 4k: Pale yellow solid; mp 116–117°C; IR (KBr) cm−1: tom culture plates, and incubated with compounds at various
3277, 2979, 2213, 1606, 1233, 1023, 969, 797; 1H-NMR (CDCl3) concentrations for 24h. The WST-1 reagent (10µL) was added
δ: 1.09 (t, J=7.2Hz, 3H), 1.25–1.43 (m, 6H), 3.51–3.71 (m, to each well and further incubated for 2h at 37°C in a CO2 in-
1H), 3.83–3.98 (m, 1H), 4.09–4.21 (m, 2H), 4.31 (q, J=7.2Hz, cubator. The plate was then gently mixed on an orbital shaker
2H), 4.94 (dd, J=23.7, 7.0Hz, 1H), 5.77 (t, J=7.8Hz, 1H), 6.65 for 1min to ensure homogenous distribution of color. The
(d, J=9.0Hz, 2H), 7.22 (d, J=8.6Hz, 1H), 7.29–7.40 (m, 3H), absorbance was read at 450nm using an automated microplate
7.48–7.59 (m, 2H), 8.04 (d, J=1.8Hz, 1H), 8.12 (d, J=1.8Hz, reader (SpectraMAX 340; Molecular Devices, Sunnyvale, CA,
1H); 13C-NMR (CDCl3) δ: 13.7, 16.3 (d, JP–C=4.5Hz), 37.6, U.S.A.).
55.3 (d, JP–C=151.7Hz), 63.4 (dd, JP–C=14.4, 6.0Hz), 99.6,
Statistical Analysis All statistical analyses were per-
108.9, 109.7, 111.4, 113.4, 119.7, 120.1, 122.1, 123.0, 124.0, formed using GraphPad Prism 5.0 for the Macintosh
125.3, 126.0, 128.2, 133.4, 138.6, 139.9, 150.0, 150.3; 31P-NMR (GraphPad Software, San Diego, CA, U.S.A.). Data signify
(CDCl3) δ: 23.13; HR-MS (EI) m/z: Calcd for C26H27BrN3O3P: the means plus or minus the standard error of mean (means
539.0973 (M+), Found: 539.0976.
S.E.M.) of three samples and are representative of three inde-
Entry 4l: White solid; mp 185–186°C; IR (KBr) cm−1: pendent experiments.
1
3286, 2980, 1611, 1232, 1026, 969, 796; H-NMR (CDCl3) δ:
1.10 (t, J=7.0Hz, 3H), 1.26–1.43 (m, 6H), 3.54–3.74 (m, 1H),
Acknowledgement We are thankful to the Dongguk
3.84–4.00 (m, 1H), 4.10–4.34 (m, 4H), 4.89 (d, J=23.8Hz, University, Gyeongju, South Korea for providing financial
1H), 6.66 (dd, J=8.7, 2.8Hz, 1H), 7.04–7.23 (m, 3H), 7.37 support.
(d, J=8.4Hz, 1H), 7.46–7.59 (m, 2H), 7.99–8.14 (m, 2H),
13C-NMR (CDCl3) δ: 13.7, 16.3, 37.7, 55.9 (d, JP–C=153.2Hz),
References
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1
3297, 2977, 1605, 1233, 1024, 968, 799; H-NMR (CDCl3) δ:
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