Synthesis and antitumor activity of novel α-aminophosphonates from diterpenic dehydroabietylaminer

Article abstract of DOI:10.1002/hc.20471

A series of novel α-aminophosphonates were synthesized from diterpenic dehydroabietylamine, and their structures were characterized by IR, ¹H NMR, and ³¹P NMR spectroscopy. Their antitumor activities against SMMC7721 liver cancer cells were evaluated by the MTT method. Compounds 4 and 6 exhibited higher activities even at very low concentrations, and the inhibition ratios reached 75% and 79% at 0.1 μM, respectively. The inhibition ratio of compound 9 reached 99% after 72-h incubation. α-Aminophosphonates with a fluorine atom and a nitro group fused to the benzene ring exhibited higher activities.

Full text of DOI:10.1002/hc.20471

Heteroatom Chemistry
Volume 19, Number 5, 2008
Synthesis and Antitumor Activity of Novel
α-Aminophosphonates from Diterpenic
Dehydroabietylamine
Xiaoping Rao,1 Zhanqian Song,1 and Ling He2
1Institute of Chemical Industry of Forest Products, Chinese Academy of Forestry, Nanjing 210042,
People’s Republic of China
2Department of Pharmacology, China Pharmaceutical University, Nanjing 210009,
People’s Republic of China
Received 16 January 2008; revised 11 March 2008
proved as drugs by the U.S. Food and Drug Adminis-
ABSTRACT: A series of novel α-aminophosphonates
trator (FDA) from 1981 to 2002 were natural or nat-
were synthesized from diterpenic dehydroabiety-
ural product derived molecules [1]. Cancer is the pri-
lamine, and their structures were characterized by IR,
mary cause of death in most of the countries, and as
1H NMR, and 31P NMR spectroscopy. Their antitumor
a result there is a need to search for cancer-effective
activities against SMMC7721 liver cancer cells were
compounds [2]. During the past decade, many natu-
evaluated by the MTT method. Compounds 4 and 6
ral antitumor compounds such as taxol and topote-
exhibited higher activities even at very low concen-
can were isolated from natural sources and were
trations, and the inhibition ratios reached 75% and
clinically used [3]. However, the low content of ac-
79% at 0.1 µM, respectively. The inhibition ratio of
tive principles extracted from natural sources and
compound 9 reached 99% after 72-h incubation. α-
complex extract procedures compelled scientists to
Aminophosphonates with a fluorine atom and a nitro
search for other naturally abundant resources.
group fused to the benzene ring exhibited higher ac-
Abietane diterpenoids are widely distributed nat-
ꢀ
C
tivities. 2008 Wiley Periodicals, Inc. Heteroatom Chem
ural products, which exhibited a variety of industrial
uses [4]. In particular, the tricyclic diterpenoid abi-
etic acid is a highly interesting compound for its
19:512–516, 2008; Published online in Wiley InterScience
(www.interscience.wiley.com). DOI 10.1002/hc.20471
special structure and properties. It is the main com-
ponent of rosin, which has the yield of about 1.2
INTRODUCTION
million tonnes per year all over the world. Dehy-
droabietylamine is an abietane diterpenic amine de-
rived from abietic acid, which can be isolated from
an industrial product of Amine D. Its derivatives
exhibited a wide range of biological properties in-
cluding antibacterial, antifungal, and antipenetrant
activities [5,6]. Recently, antiinflammatory activities
for derivatives of dehydroabietylamine at N position
were also evaluated [7,8]. Encouraged by these re-
search results, dehydroabietylamine was chosen as
the raw material in screening for new potential an-
titumor compounds.
Natural products still play an important role in drug
discovery. About 30% of new chemical entities ap-
Correspondence to: Zhanqian Song; e-mail: rxping2001@163.
com.
Contract grant sponsor: Forestry Commonwealth Industry Spe-
cial Foundation of China.
Contract grant number: 200704008.
Contract grant sponsor: National Natural Science Foundation
of China.
Contract grant number: 30771690.
2008 Wiley Periodicals, Inc.
c
ꢀ
512

Synthesis and Antitumor Activity of Novel α-Aminophosphonates from Diterpenic Dehydroabietylamine 513
As phosphorus analogs of α-amino acids and
δ/ppm, 400 MHz): 7.69–6.82 (18H, Ph-H); 4.82 (1H,
N CH ); 3.38 (2H, N CH2 ); 2.88 (1H, CH(Me)2);
2.28–1.72 (10H, CH2 );1.70 (1H, >CH ); 1.22–1.09
(12H, CH3); 31P NMR (DAc; δ/ppm, 400 MHz): 5.46.
2: C40H48NO4P; mp 223.6◦C; yield 27.5%; IR
(cm−1): 3434 ( NH); 2930 ( CH3ꢁ CH2); 1219 (P O);
their esters, α-aminophosphonates are widely stud-
ied as biologically active substances. They exhibit an-
tibacterial, anticancer, insecticidal, hypoglycemic,
herbicidal properties and are investigated as start-
ing materials for the synthesis of phosphonopeptides
[9–12]. α-Functionalized organic phosphonates are
valuable medical compounds and synthetic interme-
diates. However, the biological activities of tricyclic
diterpenic modified phosphonates have seldom been
studied.
In this report, we describe the synthesis and
antitumor activity of synthesized diterpenic de-
hydroabietylamine modified α-aminophosphonates
against SMMC7721 liver cancer cell, and prelimi-
nary structure–activity relationships of these com-
pounds were also investigated.
1
1094 (C N); 913(Ar-H); H NMR (DAc; δ/ppm, 400
MHz): 7.18–6.84 (17H, Ph-H); 4.72 (1H, N CH );
3.84 (3H, OCH3); 3.38 (2H, N CH2–); 2.82 (1H,
CH(Me)2); 2.28–2.06 (10H, CH2–); 1.70 (1H,
>CH ); 1.22–0.98 (12H, CH3); 31P NMR (DAc;
δ/ppm, 400 MHz): 5.88.
3: C39H45ClNO3P; mp 230.5◦C, yield 30.7%; IR
(cm−1): 3458 ( NH); 2929 ( CH3ꢁ CH2); 1218 (P O);
1
1093 (C N); 910 (Ar-H); H NMR (DAc; δ/ppm, 400
MHz): 7.48–6.86 (17H, Ph-H); 4.80 (1H, N CH );
3.40 (2H, N CH2 ); 2.82 (1H, CH(Me)2); 2.29–2.06
(10H, CH2 ); 1.71 (1H, >CH ); 1.20–1.00 (12H,
CH3); 31P NMR (DAc; δ/ppm, 400 MHz): 4.92.
4: C39H45FNO3P; mp 225.6◦C, yield 18.9%; IR
RESULTS AND DISCUSSION
Synthesis
(cm−1): 3437 ( NH); 2931 ( CH3ꢁ CH2); 1220 (P O);
1
1094 (C N); 910 (Ar-H); H NMR (DAc; δ/ppm, 400
MHz): 7.07–6.83 (17H, Ph-H); 4.85 (1H, N CH );
3.40 (2H, N CH2 ); 2.89 (1H, CH(Me)2); 2.84–2.06
(10H, CH2 ); 1.76 (1H, >CH ); 1.71–0.99 (12H,
CH3); 31P NMR (DAc; δ/ppm, 400 MHz): 5.22.
5: C39H45FNO3P; mp 234.6◦C, yield 19.7%;
IR(cm−1); 3421 ( NH); 2932 ( CH3ꢁ CH2); 1223
(P O); 1097 (C N); 913 (Ar-H); 1H NMR (DAc;
δ/ppm, 400 MHz): 7.51–6.84 (17H, Ph-H); 4.87(1H,
N CH ); 3.47 (2H, N CH2 ); 2.90 (1H, CH(Me)2);
2.84–2.06 (10H, CH2 ); 1.63 (1H, >CH ); 1.58–
1.01 (12H, CH3); 31P NMR (DAc; δ/ppm, 400 MHz):
4.63.
Novel α-aminophosphonates were synthesized from
diterpenic dehydroabietylamine by two steps of re-
actions through imines intermediates (Fig. 1). Al-
though high steric hindrance of tricycle structure to
amine group, dehydroabietylamine and substituted
benzaldehyde afforded good yields of imines without
catalysts [13]. The addition of phosphite to imines
was proved to be very difficult because of high steric
hindrance from two reaction components. Although
we obtained the diterpenic α-aminophosphonates
easily in the toluene solution, the yields were rel-
atively low to 20%–30% for benzyl phosphonates,
ethyl phosphonates give higher yields due to less
steric hindrance than benzene [14].
6: C40H45F3NO3P; mp 237.8◦C, yield 20.5%; IR
(cm−1): 3428 ( NH); 2931 ( CH3ꢁ CH2); 1217 (P O);
1
1: C39H46NO3P; mp 229.4◦C, yield 22.8%; IR
(cm−1): 3429 ( NH); 2926 ( CH3ꢁ CH2); 1220 (P O);
1096 (C N); 832(Ar-H); 1H NMR (DAc (CH3COOD);
1094 (C N); 911 (Ar-H); H NMR (DAc; δ/ppm, 400
MHz): 7.87–6.84 (17H, Ph-H); 4.99 (1H, N CH );
3.52 (2H, N CH2 ); 2.90 (1H, CH(Me)2); 2.84–2.06
FIGURE 1 Synthetic scheme of α-aminophosphonates.
Heteroatom Chemistry DOI 10.1002/hc

514 Rao, Song, and He
TABLE
2 Inhibition Ratios of Compounds against
(10H, CH2 ); 1.73 (1H, >CH ); 1.20–1.01 (12H,
CH3); 31P NMR (DAc; δ/ppm, 400 MHz): 4.29.
7: C39H45N2O6P; mp 271.5◦C, yield 22.3%; IR
(cm−1): 3408 ( NH); 2956 ( CH3ꢁ CH2); 1261 (P O);
SMMC7721 Liver Cancer Cells at Different Concentrations
Inhibition
Ratio (%)
Inhibition
Ratio (%)
Concentration
1
1095 (C N); 916 (Ar-H); H NMR (DAc; δ/ppm, 400
Compounds
(µM)
24 h
72 h
MHz): 8.59–6.92 (17H, Ph-H); 5.02 (1H, N CH );
3.54 (2H, N CH2 ); 2.95 (1H, CH(Me)2); 2.83–2.06
(10H, CH2 ); 1.84 (1H, >CH ); 1.57–1.07 (12H,
CH3); 31P NMR (DAc; δ/ppm, 400 MHz): 9.56.
Control
1
0
6.79
15.08
5.70
12.26
0.00
0
2.12
29.49
38.03
40.38
0.00
10
100
10
100
10
2
8: C39H45ClNO4P; mp 248.8◦C, yield 20.3%; IR
(cm−1): 3428 ( NH); 2931 ( CH3ꢁ CH2); 1217 (P O);
3
1
1094 (C N); 911 (Ar-H); H NMR (DAc; δ/ppm, 400
100
10
100
10
100
10
100
10
100
10
100
10
10.94
6.25
41.40
33.07
5.45
10.70
55.21
20.30
57.81
5.53
41.41
3.67
19.04
40.19
40.19
17.50
40.38
9.81
66.46
46.79
75.00
38.03
99.00
2.76
5
MHz): 7.67–6.84 (17H, Ph-H); 4.86 (1H, N CH );
3.41 (2H, N CH2 ); 2.89 (1H, CH(Me)2); 2.82–2.06
(10H, CH2 ); 1.84 (1H, >CH ); 1.57–1.00 (12H,
CH3); 31P NMR (DAc; δ/ppm, 400 MHz): 4.93.
6
7
9: C31H45N2O6P; mp 244.4◦C, yield 66.2%; IR
(cm−1): 3394 ( NH); 2948 ( CH3ꢁ CH2); 1234 (P O);
8
1
1060 (C N); 827 (Ar-H); H NMR (DAc; δ/ppm, 400
9
MHz): 8.49–6.89 (17H, Ph-H); 5.08 (1H, N CH );
3.82 (2H, N CH2 ); 2.89 (1H, CH(Me)2); 2.85–2.06
(10H, CH2 ); 1.78 (1H, >CH ); 1.76–1.05 (12H,
CH3); 31P NMR (DAc; δ/ppm, 400 MHz): 8.58.
10
100
25.26
29.70
10: C35H45ClNO4P; mp 235.2◦C, yield 63.5%; IR
(cm−1): 3398 ( NH); 2944 ( CH3ꢁ CH2); 1237 (P O);
1
1064 (C N); 822 (Ar-H); H NMR (DAc; δ/ppm, 400
tive. It has been found from Table 1 that the growth
of SMMC7721 liver cancer cells was strongly inhib-
ited by compounds 4 and 6, even at very low con-
centration of 0.1 µM, after the 48-h incubation, the
inhibition ratios of compounds 4 and 6 reached 75%
and 79%, respectively. The dose-dependent response
of these two compounds to the tumor cells was also
monitored; it was found that when the concentra-
tion of the drugs was changed, the inhibition ratios
were nearly 75% at different concentrations, and the
increase in the concentration has no obvious effects
on the inhibition ratios after the 48-h incubation.
The inhibition ratios of other compounds
against SMMC7721 liver cancer cells were evaluated
at the concentrations of 10 and 100 µM at 24 and
72 h, respectively; the data of inhibition ratios are
listed in Table 2. It was found that compounds 7,
8, and 9 exhibited high activities when at 100 µM
after the 72-h incubation, and the inhibition ratios
reached 66.46%, 75.00%, and 99.00%, respectively.
Most compounds exhibited relatively low activity
after the 24-h incubation. The dose-dependent re-
sponse indicated that most of the compounds exhib-
ited higher activity when at higher concentrations;
however, compound 6 is the exception. Compared
with the data of inhibition ratios of compound 6 at
24, 48, and 72 h, it exhibited highest activity when
incubated for 48 h.
MHz): 7.52–6.89 (17H, Ph-H); 4.83 (1H, N CH );
3.85 (2H, N CH2 ); 2.89 (1H, CH(Me)2); 2.82–2.06
(10H, CH2 ); 1.84 (1H, >CH ); 1.57–1.00 (12H,
CH3); 31P NMR (DAc; δ/ppm, 400 MHz): 9.26.
Biological Activity
Compounds 4 and 6 were treated with SMMC7721
liver cancer cells at different concentrations from 0.1
to 100 µM. The data of inhibition ratios are listed
in Table 1. Usually, when the concentration of the
compound solution is 1 µmol/L, the inhibition ra-
tio of the compound to cancer growth is more than
50%, the compound is considered as strongly effec-
TABLE 1 Inhibition Ratios of Compounds 4 and 6 against
SMMC7721 Liver Cancer Cells at Different Concentrations
Concentration
Optical
Density
Inhibition
Ratio(%)
Compounds
(µM)
Control
4
1.74 0.05
0.44 0.07
0.42 0.04
0.43 0.05
0.37 0.05
0.40 0.06
0.41 0.08
0.47 0.08
0.38 0.03
0
0.1
1.0
10
100
0.1
1.0
10
75.00
76.00
76.00
77.00
79.00
76.00
73.00
78.00
6
On the basis of the screening results,
we made the preliminary conclusions. Some
100
Heteroatom Chemistry DOI 10.1002/hc

Synthesis and Antitumor Activity of Novel α-Aminophosphonates from Diterpenic Dehydroabietylamine 515
α-aminophosphonates from diterpenic dehy-
were added in the concentration gradient. The fi-
nal concentrations were maintained at l00, l0, l, and
0.1 µM, respectively. Control was added the same
amount of solvent. The plates were maintained at 37◦
in a humidified 5% CO2, and incubated for 48 h, then
20 µL MTT solution was added, and the plate was in-
cubated for additional 4 h. Supernatant from each
well was taken out carefully, and l50 mL of DMSO
was added to each well and was subjected to vibra-
tion on an ELISA spectrophotometer at 570 nm. In-
hibition ratios were calculated by using the following
equation:
droabietylamine exhibited high activity against
SMMC7721 liver cancer cells. They exhibited time-
and dose-dependent activities. The compounds
containing a fluorine atom and a nitro group in
the benzene ring exhibited high activities; this kind
of compounds exhibited higher activity at 48 h
of bioassay. The different substituted group has
prodigious diversity of antitumor activity; changing
the group is possible to improve their activity. The
title compound is a kind of lead compounds of
antitumor agent that warrants further investigation.
A0 − A1
Inhibiton ratio =
× 100%
A0
EXPERIMENTAL
General
where A0 represents total absorbance of the tumor
cell control and A1 represents the absorbance of the
drug treatment group.
Melting points were determined with XT5 melt-
ing point apparatus, and the temperature was
uncorrected. Infrared spectra were obtained by us-
ing the KBr method on a Bio-Rad FTS-185 IR spec-
trophotometer. 1H NMR and 31P NMR spectra were
recorded on DPX a Bruker AVANCE spectrometer
(CDCl3 as the solvent of imines and DAc as the sol-
vent of α-aminophosphonates).
CONCLUSIONS
Novel α-aminophosphonates were synthesized from
natural product of diterpenic dehydroabietylamine.
Their antitumor activities against SMMC7721 liver
cancer cells were evaluated by the MTT method.
They exhibited time- and dose-dependent activi-
ties to SMMC7721 liver cancer cells. Compounds
4 and 6 exhibited high activities even at very
low concentrations. The inhibition ratio of com-
pound 9 reached 99% after the 72-h incubation.
α-Aminophosphonates with a fluorine atom and a
nitro group fused to the benzene ring exhibited high
activities. Further investigations will be required for
a more detailed biological evaluation of these agents.
Synthesis
Synthetic Procedure for Imine Intermediates.
Dehydroabietylamine (10 mmol) was dissolved in
20 mL ethanol, 10 mmol of substituted benzalde-
hyde was added to the mixture and heated to reflux
for 2 h, and the resulted solids were crystallized from
ethanol. The crystals were collected and dried under
vacuum.
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heated to 120◦C and was maintained for 6 h. Then
the mixture was cooled to room temperature, and the
solids were crystallized from acetic acid. The crys-
tals were collected and dried under vacuum. Diethyl
phosphonates were synthesized at the same condi-
tions without using solvents.
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