Synthesis of ursolic phosphonate derivatives as potential anti-HIV agents

Article abstract of DOI:10.1080/10426500601088838

In order to search for new anti-tumor and anti-viral agents, a series of -aminophosphonate conjugates of 3-O - acetyl ursolic acid were prepared. Their biological activities as cytotoxic and anti-HIV agents were evaluated. The preliminary bioassays indicate that synthesized compounds 7a-j have anti-HIV activity (targeting HIV-1 gp120 and CD4) and no cytotoxicity on HT-29 cells (human colon adenocarcinoma cell line).

Full text of DOI:10.1080/10426500601088838

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Phosphorus, Sulfur, and Silicon
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Synthesis of Ursolic
Phosphonate Derivatives as
Potential Anti-HIV Agents
a
a
Sheng-Lou Deng , Isabelle Baglin , Mohammed
a
b
c
Nour , Oxana Flekhter , Claudio Vita & Christian
d
Cavé
a
Unité de Molécules d'Intérêt Biologique , UFR de
Pharmacie, Dijon, France
b
Institute of Organic Chemistry , Ufa Scientific
Center of the Russian Academy of Science, Ufa,
Russia
c
Department of Protein Engineering and Research ,
Centre d'Etude Atomique, Saclay, France
d
UMR CNRS BioCis 8076, Faculté de Pharmacie ,
Châtenay-Malabry, France
Published online: 19 Apr 2007.
To cite this article: Sheng-Lou Deng , Isabelle Baglin , Mohammed Nour , Oxana
Flekhter , Claudio Vita & Christian Cavé (2007) Synthesis of Ursolic Phosphonate
Derivatives as Potential Anti-HIV Agents, Phosphorus, Sulfur, and Silicon and the
Related Elements, 182:5, 951-967, DOI: 10.1080/10426500601088838
To link to this article: http://dx.doi.org/10.1080/10426500601088838
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Phosphorus, Sulfur, and Silicon, 182:951–967, 2007
Copyright © Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426500601088838
Synthesis of Ursolic Phosphonate Derivatives as
Potential Anti-HIV Agents
Sheng-Lou Deng
Isabelle Baglin
Mohammed Nour
Unit e´ de Mol e´ cules d’Int e´ r eˆ t Biologique, UFR de Pharmacie,
Dijon, France
Oxana Flekhter
Institute of Organic Chemistry, Ufa Scientific Center of the Russian
Academy of Science, Ufa, Russia
Claudio Vita
Department of Protein Engineering and Research, Centre d’Etude
Atomique, Saclay, France
Christian Cave´
UMR CNRS BioCis 8076, Facult e´ de Pharmacie,
Ch aˆ tenay-Malabry, France
In order to search for new anti-tumor and anti-viral agents, a series of α-
aminophosphonate conjugates of 3-O-β-acetyl ursolic acid were prepared. Their
biological activities as cytotoxic and anti-HIV agents were evaluated. The prelim-
inary bioassays indicate that synthesized compounds 7a–j have anti-HIV activity
(
targeting HIV-1 gp120 and CD4) and no cytotoxicity on HT-29 cells (human colon
adenocarcinoma cell line).
Keywords α-aminophosphonate; anti-HIV activity; conjugate; cytotoxicity; Ursolic acid
INTRODUCTION
Ursolic Acid (UA) 1 (Figure 1) is a pentacyclic triterpene, widely dis-
tributed in the vegetable kingdom in aglycone, glycosides, or acylated
Received August 26, 2006; accepted October 6, 2006.
We would like to thank Professor B. Chauffert for cooperation upon bioassays and the
Conseil R e´ gional de Bourgogne for financial support.
Address correspondence to Christian Cave, UMR CNRS BioCis 8076, Facult e´ de
Pharmacie, 5 Rue J.-B. Cl e´ ment, Ch aˆ tenay-Malabry, 92296 France. E-mail: christian.
cave@cep.u-psud.fr
951

9
52
S.-L. Deng et al.
FIGURE 1 Structure of UA.
form. In past decades, UA has attracted considerable interest owing
to its significant biological activities and promising clinical application
as a chemotherapeutic and chemopreventive agent. Indeed, UA and
its derivatives exhibit attractive pharmacological properties, including
1
2,3
anti-inflammatory activity and, anti-microbial activity and being an
4
inhibitor of mutagenesis in bacteria. It also shows a dose-dependent
cytotoxic activity in vivo through inhibiting implanted ascetic tumor
5
6
cells. UA is known as an anti-melanoma agent. It is obviously a po-
tent inhibitor of MCF-7 growth in the light of both cytostatic and cyto-
7
toxic activities. Moreover, UA is able to inhibit dimerisation of HIV-1
protease, although slight toxicity is observed. Some dicarboxylic acid
hemiesters at C-3 of ursolic acid also show anti-HIV activity. However,
8
−10
the action mechanism of these compounds is not clear.
Literature review reveals that only a few synthetic analogs of UA
have been reported for the purpose of cytotoxicity evaluation.11−14 Re-
sults from a more extensive investigation using a great number of
derivatives are needed, and criteria may be eventually established for
the design and synthesis of more effective terpenoid-derived chemother-
apeutic and chemopreventive agents. On the other hand, our recent
work upon the chemical modification of UA as glucoside at C-28 and
cinnamoyl esters at C-3 position showed that some of those derivatives
1
5
possessed cytotoxicity on HT-29 and anti-HIV activity. These excit-
ing discoveries prompted us to do more chemical modification of the
carboxyl group to meet the demand of bioactivity screening and the
Quantitative Structure-Activity Relationship (QSAR) study.
It is well known that α-aminophosphonic acids and their derivatives,
as analogs of natural α-amino acids, have attracted wide attention in
chemistry, medicine, and agricultural science. They play an important
role in living systems, as mimetic agents of the tetrahedral transi-
tion state of amide, esters, and peptides hydrolysis. α-Aminophosphonic
acid derivatives have been used as antibiotics, herbicides, anti-tumor
agents and enzyme inhibitors.1
6,17
In the past three decades, there has
been a growing interest in the chemistry of α-aminophosphonic acids

Ursolic Phosphonate Derivatives
953
with an emphasis on their synthesis and diverse potential biological
significance in metabolic processes of life.1 Herein we are interested
in the introduction of α-aminophosphonates to the parent structure
of UA. Conjugation of these two entities may improve bioactivities
and minimize to some extent the frequently encountered problems
such as bioavailability, solubility, and pharmacokinetic asssociated with
UA derivatives. In the present article, a variety of new phosphorus-
containing conjugates of this triterpenoid were therefore synthesized
and biologically evaluated as cytotoxic agents on HT-29 cells (human
colon adenocarcinoma cell line) and anti-HIV agents.
8
The preliminary bioassays reveal that such compounds have some
anti-HIV activity and no cytotoxicity on HT-29 cells.
RESULTS AND DISCUSSION
Synthesis of α-Aminophosphonates
A variety of N-blocked α-aminophosphonates were prepared in good to
excellent yields by a one-pot three-component (aldehydes, benzyl carbo-
mate, and triphenyl phosphite) condensation reaction according to the
1
9
literature. Ammonium hydrobromides 3 were sequentially obtained
in quantitative yields after deprotection with a solution of hydrobro-
mide in acetic acid. The subsequent neutrolization was carried out by
treatment with triethylamine (>3 equivalents) in THF. The filtration
and evaporation under reduced pressure resulted in free amines 4 as
oils with yields of 50–75% (Scheme 1). Without further purification, the
obtained α-aminophosphonates (4) were then dissolved in anhydrous
THF and used directly in the next step.
◦
SCHEME 1 Reagents and conditions: (a) AcOH, 80–85 C, 2 h; (b) HBr/AcOH,
3 2 2
RT, 2 h; (c) NEt /CH Cl .

9
54
S.-L. Deng et al.
SCHEME 2 Reagents and conditions: (a) Ac
2 3
SOCl , 60–75 C, 5 h; (c) 4, NEt /THF, 0 C, 0.5 h; RT, 5 h.
2
O, DMAP, pyridine, RT, 2 h; (b)
◦
◦
Synthesis and Structural Characterization of Ursolic
Phosphonates 7a–j
To prepare the designed α-aminophosphonate conjugates of UA, a
straightforward approach was first attempted, which involved a one-
step condensation of UA or acetylated UA 5 with amines 4 (1 equiv.)
in the presence of DiCyclohexylCarbodiimide (DCC) (1.2 equiv.) and
4
-Dimethylamino-Pyridine (DMAP) in THF (or dichloromethane). Un-
fortunately, the reaction did not run to the desired product, presumably
due to the hindrance of both reactants. To facilitate the coupling reac-
tion, UA 1 was then transformed into acyl chloride. However, protection
of hydroxyl group at 3 position was accordingly required prior to cou-
pling reaction. Thus, acetylation of UA was conducted first according
to the literature.20 It should be indicated that improving the yield of
the desired product 5 required one equivalent of DMAP when a large
excess of acetic acid anhydride was used. It should be noted that the
side reactions were increased by heating or strong light irradiation.
Conversion of 3β-acetoxy-urs-12-ene-28-oic acid 5 to acid chloride 6
◦
was accomplished when treated with excess thionyl chloride at 60–75 C

Ursolic Phosphonate Derivatives
955
TABLE I 31P NMR Data of Compounds 7a–j (δ, ppm)
Compds
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
Major isomer 17.19 19.71 11.20 19.25 14.89 10.85 14.23 13.75 13.64 14.51
Minor isomer
d.e. (%)
—
0
19.24 12.16 19.72 15.86 11.91 15.09 14.70 14.50 13.43
10 13 12 24 14 25 22 11
3
for 5 h under nitrogen atmosphere, which was slightly different from the
21
procedure reported in the literature. The excess thionyl chloride was
removed by distillation under reduced pressure. Complete removal of
thionyl chloride required further distillation on high vacuum (oil pump)
at r.t. for another 1 h. The obtained acid chloride was then used directly
in the next step.
The coupling reaction of acid chloride 6 with α-aminophosphonates
was routinely performed in the presence of excess triethylamine on
an ice bath. It should be indicated that both reactants should be well
dissolved in anhydrous THF prior to a coupling reaction. This could
improve the yield of the product. Purification of the products by flash
column chromatography gave the expected compounds 7a–j as oils
in 48–82% yields, which crystallized after standing for 3 days at r.t.
(
Scheme 2).
Products 7a–j were characterized by spectroscopic ( H NMR, 31P
1
NMR, C NMR, IR) and microanalytical methods. 31P NMR data of
13
compounds 7a–j are summarized in Table I. For each of compounds 7b–j,
3
1
the P NMR spectrum showed two peaks with chemical shift difference
ranging from 0.47 to 1.06 ppm, indicating the existence of diastereoiso-
1
mers. The corresponding H NMR spectrum also implied the existence
of isomers by appearance of two doublets of doublets at 4.70–6.80 ppm
corresponding to CH moiety adjacent to a phosphorus atom and two
broad single peaks corresponding to the unsaturated CH residue at
C-12 position. The diastereoisomeric excess was in the range 3–25%.
Biological Activity
The preliminary bioassay of compounds 7a–j was conducted on an HT-
2
9 colon cancer cell line at the highest concentration (500 µM). The
results showed that these new phosphonate derivatives of UA had no
cytotoxicity.
Our previous study upon global anti-HIV activity revealed that some
1
5
of the UA derivatives (Figure 2) had good activity. For example, urso-
lic glucopyranosyl ester (9) and 3-O-3-(3-pyridyl)-prop-2-enoyl ursolic

9
56
S.-L. Deng et al.
Compounds
R1
R2
Compounds
R1
R2
UA
8
H
H
-OH
13
OH
OH
O
β D Glc(OAc)4 14
9
H
O
O
β
β
D
D
Glc
15
O
β D Glc
1
1
1
0
1
2
Glc(OAc)4 16
17
Ac
H
OH
OH
OH
FIGURE 2 Structures of the reported compounds.
acid (13) were potent anti-HIV candidates with EC50 at 4 µM and 7.5
µMµ, respectively (Table II). Both displayed higher activity than UA
(
10 µM). These results prompted us to examine the anti-HIV prop-
erty of compounds 7a–j. It is noteworthy that these compounds showed
specific anti-HIV activity in some degree (Table III). They proved to
be entry inhibitors capable of inhibiting CD4-gp120 interaction. How-
ever, the HIV-1 entry is a multiple-step process ([a] interaction of the
viral envelope gp120 glycoprotein with the host cell receptor CD4;[b]
interaction of gp120-CD4 complex with CCR5 or CXCR4 chemokine re-
ceptors; [c] insertion of the envelope fusion domain into the host cell
membrane; and [d] fusion of the virus’ cell membrane); each of these
steps may represent a potential target of our new compounds. In order to
search the real target of our derivatives, assay inhibiting HIV-1 entry in

Ursolic Phosphonate Derivatives
957
TABLE II Anti-HIV Activity of the Reported compounds
Compounds
UA
8
9
10
11
12
13
14
15
16
17
a
c
4
d
d
c
c
c
c
EC50 µM
Inhibition %
10
24
30
9
7.5
11
b
13
16
2
7
13
0
16
11
aGlobal anti-HIV activity; EC50: the agent concentration that inhibited viral
replication in H9 cell by 50%.
b
c
−4
HIV entry inhibitor activity (10 M).
Low activity.
No activity.
d
lymphocyte cells was performed. This experiment was intended to tar-
get gp120, more specifically, the binding site between gp120 and CD4.
The ability of compounds 7a–j to inhibit CD4M33-F1 binding to gp120
was tested in florescence polarization assay. In this test, a small and
fully functionalized CD4 mimic CD4M33 was engineered, which binds
the recombinants gp120, gp40 proteins, and HIV-1 particles with CD4-
like affinity. A florescent-labelled CD4M33 derivative was also synthe-
sized and used as florescence tracer; additionally, a competition assay
was developed to characterize different gp120 ligands.
In contrast to the reported results in Table II, compounds 7a–j
showed a comparable anti-HIV activity. Three of them (7a, 7b, and
−
4
7
c) exhibited higher inhibition activity (18, 17, and 20 at 10 M)
than the rest of the others except UA. It was found that the varia-
tion of substituents at α position of phosphoryl group did affect anti-
HIV activity in some degree. Based on the results in Table III, it
can be concluded that electron-donating substituents generally lead
to higher activity than the electron-withdrawing counterparts. On the
other hand, EC50 of compounds 8∼17 in Table II implies that free
hydroxyl at 3 position and hydroxyl or carboxylic acid at 28 posi-
tion are important for their activity. Therefore, keeping hydroxyl moi-
ety at 3 position intact may improve bioactivity of UA derivatives.
In this connection, removal of acetyl at 3 position and hydrolysis
of phosphonate of compounds 7a–j will be our task in the next
program.
−
4
TABLE III HIV Entry Inhibitor Activity (10 M)
Compounds
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
Inhibition (%)
18
17
20
0
11
6
0
5
12
2

9
58
S.-L. Deng et al.
In conclusion, we have synthesized a series of α-aminophosphonate
conjugates of 3-O-β-acetyl UA. All of the prepared compounds have
been biologically evaluated. The bioassay results reveal that these com-
pounds have no cytotoxicity on HT-29 cells. Interestingly, these new
derivatives of UA exhibit specific anti-HIV activity. They prove to be
an HIV-1 entry inhibitor by inhibiting gp120-CD4 interaction. These
results suggest that our UA derivatives may be potential candidates to
inhibit HIV infection. Nevertheless, with these results in hand, we still
cannot find the target of our compounds. To understand the role and
the mechanism of action of these UA derivatives, further work needs
to be done in the future.
EXPERIMENTAL
Reagents and solvents were purchased from commercial sources and
used without further purification unless indicated otherwise. Tetrahy-
drofuran was dried over sodium and distilled before use. Thionyl chlo-
ride and triethylamine were freshly distilled before use. Melting points
1
13
31
were determined with a Kofler bench and uncorrected. H, C, P NMR
spectra were recorded on a BRUKER AC-P300 instrument. Elemental
analyses were carried out on a Yanaco MT-3 instrument. All spectra
were recorded in CDCl3 or DMSO-d6; TMS was used as an internal
1
standard for H NMR, and 85% phosphoric acid (H3PO4) was used as
an external standard for 31P NMR spectrum; chemical shifts (δ) were
expressed in ppm. Analytical TLC was performed on Merck silica gel
60F254 pre-coated plates. Column chromatography was performed us-
ing commercially obtained silica gel H 40. All reactions involving air-
or water-sensitive compounds were routinely conducted under nitrogen
atmosphere.
General Procedure for the Preparation of
Diphenyl-α-Aminophosphonates Hydrobromides (3)
To a flask containing N-benzyloxycarbonyl-diphenyl-α-aminophos-
phonates 2 (0.01 mol) was added a solution of HBr in AcOH (25 mL,
3
0%). The mixture was stirred at r.t. for 2 h. When evolution of bub-
bles stopped, excess HBr and solvent were subsequently removed by
distillation under reduced pressure. The residue was washed with di-
ethyl ether (30 mL), producing a white solid. Further filtration and
washing with diethyl ether yielded white powder. The spectral data
of all known products are in accordance with that reported in the
literature.

Ursolic Phosphonate Derivatives
959
Diphenyl[1-(1-amino)methyl]phosphonate
2
2
Hydrobromide (3a)
◦
1
R = H, yield: 96%; m.p. 134–136 C; H NMR (DMSO-d6): δ-3.89 (d, 2H,
2
3
+
JP-H = 17.96 Hz, CH2), 6.74-7.46 (m, 10H, Harom), 8.81 (brs, 3H, NH3);
1
P NMR (DMSO-d6): δ = 14.6.
Diphenyl [1-(1-amino)butyl]phosphonate Hydrobromide (3b)
◦
1
R = n-Pr, yield: 97%; mp 195–197 C; H NMR (DMSO-d6): δ = 0.93 (t,
3
4
H, CH3), 1.50–1.65 (m, 2H, CH2CH3), 1.85–2.03 (m, 2H, CH2CH2CH3),
+
31
.20 (m, 1H, CH), 7.10–7.49 (m, 10H, Harom), 8.84 (brs, 3H, NH3);
P
NMR (DMSO-d6): δ = 15.83.
Diphenyl [1-(1-amino)methyl-p-methoxyphenyl]phosphonate
2
3
Hydrobromide (3c)
◦
1
R = p-MeOPh, yield: 98%; mp 193.5 C; H NMR (DMSO-d6): δ = 3.77
2
(
s, 3H, OCH3), 5.58 (d, 1H, JP-H = 18.08 Hz, CH), 6.91–7.64 (m, 14H,
+
31
Harom), 9.37 (brs, 3H, NH3); P NMR (DMSO-d6): δ = 12.29.
Diphenyl [1-(1-amino)pentyl]phosphonate Hydrobromide (3d)
◦
−1
R = n-Bu, yield: 98%; mp 172.5 C; IR (ν cm ): 3403, 2958, 1559, 1488,
1
1
234, 1206, 1175, 1159, 936, 764; H NMR (DMSO-d6): δ 0.87 (t, 3H,
3
CH3, JH H = 7.20 Hz), 1.30–1.58 (m, 4H), 1.92–2.05 (m, 2H), 4.20 (m,
+
13
1
H, CH), 7.22–7.45 (m, 10H, Harom), 8.89 (brs, 3H, NH3); C NMR
2
(
4
DMSO-d6): δ = 13.57 (s), 21.67 (s), 27.02 (d, JP-C = 9.0 Hz), 27.62 (s),
1
3
3
6.36 (d, JP-C = 156 Hz), 120.50 (d, JP-C = 4.1 Hz), 120.57 (d, JP-C
2
31
=
5.3 Hz), 125.77 (s), 130.02 (s), 149.30 (d, JP-C = 10.1 Hz); P NMR
(
DMSO-d6): δ 15.73.
Diphenyl [1-(1-amino)benzyl]phosphonate
2
3
Hydrobromide (3e)
◦
1
R = Ph, yield: 99%; mp 195–196 C; H NMR (DMSO-d6): δ = 5.65 (d,
2
1
H, JP-H = 18.42 Hz, CH), 6.86–7.68 (m, 15H, Harom), 9.46 (br.s, 3H,
+
31
NH3); P NMR (DMSO-d6): δ = 12.22.
Diphenyl [1-(1-amino)methyl-p-tolyl]phosphonate
2
3
Hydrobromide (3f)
◦
1
R = p-MePh, yield: 99%; mp 196–198 C; H NMR (DMSO-d6): δ = 2.34
2
(
s, 3H, CH3), 5.59 (d, 1H, JP-H = 18.82 Hz, CH), 6.91–7.59 (m, 14H,
+
31
Harom), 9.41 (br.s, 3H, NH3); P NMR (DMSO-d6): δ = 12.10.

9
60
S.-L. Deng et al.
Diphenyl [1-(1-amino)methyl-p-chlorophenyl]phosphonate
2
3
Hydrobromide (3g)
◦
1
R = p-ClPh, yield: 98%; mp 191.4 C; H NMR (DMSO-d6): δ = 5.70 (d,
2
3
1
H, CH, JP-H = 18.6 Hz), 6.92 (d, 2H, JH-H = 8.10 Hz), 7.19–7.97 (m,
31
+
1
2H, Harom), 9.53 (br.s, 3H, NH3); P NMR (DMSO-d6): δ = 10.48.
Diphenyl [1-(1-amino)methyl-o-chloropheny]phosphonate
2
3
Hydrobromide (3h)
◦
1
R = o-ClPh, yield: 99%; mp 203.1 C; H NMR (DMSO-d6): δ = 5.74 (d,
2
1
H, CH, JH-P = 18.3 Hz), 6.96–7.74 (m, 14H, Harom), 9.53 (br.s, 3H,
+
31
NH3); P NMR (DMSO-d6): δ = 11.51.
Diphenyl [1-(1-amino)methyl-2,
-dichlorophenyl]phosphonate Hydrobromide (3i)
4
◦
−1
R = 2,4-Cl2Ph, yield: 99%; mp 196.9 C; IR (ν cm ): 3402, 3101, 2973,
1
2
798, 2570, 1589, 1505, 1490, 1268, 1206, 1174, 959, 936, 760; H NMR
2
3
(
8
(
DMSO-d6) : δ = 5.72 (d, 1H, CH, JH P = 18.6 Hz), 6.97 (d, 2H, JH H =
3
.40 Hz), 7.18-7.45 (m, 8H, Harom), 7.69 (d, 1H, JH H = 10.50 Hz), 7.83
3
+
13
s, 1H), 7.95 (d, 1H, JH H = 8.40 Hz), 9.49 (br.s, 3H, NH3); C NMR:
2 2
δ = 149.27 (d, JP C = 8.5 Hz), 149.21 (d, JP C = 11.0 Hz), 135.25 (d,
3
2
JP C = 3.3 Hz), 134.52 (d, JP C = 7.5 Hz), 131.17, 130.14 (s), 130.00
3
(
1
=
s), 129.61, 128.19, 127.46 (d, JP C = 4.4 Hz) , 125.99 (s), 125.83 (s),
3
3
1
20.32 (d, JP C = 4.5 Hz), 119.88 (d, JP C = 4.3 Hz), 47.18 (d, JP
C
3
1
158 Hz); P NMR (DMSO-d6): δ = 10.15.
Diphenyl [1-(1-amino) methyl-o-nitrophenyl]phosphonate
Hydrobromide (3j)
◦
−1
R = o-NO2Ph, yield: 96%; mp 191.4 C; IR (ν cm ): 3423, 2990, 2550,
1
1
589, 1540, 1489, 1347, 1264, 1205, 1175, 1150, 961, 941, 774; H NMR
2
3
(
DMSO-d6): δ = 6.14 (d, 1H, CH, JH H = 19.8 Hz), 6.94 (d, 2H, JH H =
8
.29 Hz), 7.13–7.43 (m, 8H, Harom), 7.74–8.20 (m, 4H), 9.43 (brs, 3H,
+
13
1
3
NH3); C NMR: δ = 46.25 (d, JP C = 158 Hz), 119.96 (d, JP C =
3
4
.3 Hz), 120.27 (d, JP C = 4.3 Hz), 125.07, 125.12, 125.87, 125.99,
2
1
29.72, 129.99 (s), 130.09 (s), 131.04, 134.25, 149.17 (d, JP C = 9.9
2
31
Hz), 149.25 (d, JP C = 9.9 Hz); P NMR (DMSO-d6): δ = 11.00.
General Procedure for the Preparation
of Diphenyl-α-Aminophosphonates (4)
To a suspension of diphenyl-α-aminophosphonate hydrobromide 3
(
0.25 mmol) in anhydrous dichloromethane (20 mL) was added triethy-
24
lamine (0.3 mL, 2.15 mmol) dropwise at r.t. The reaction mixture was

Ursolic Phosphonate Derivatives
961
then kept stirring for 3 h. The precipitate was filtered off, and the sol-
vent was evaporated under reduced pressure. The resulting colorless
sticky oil was used directly without further purification.
Preparation of 3β-Acetoxy-urs-12-ene-28-oic Acid (5)
To a 50-mL flask was added UA (0.1 g, 0.22 mmol), pyridine (10
mL), acetic acid anhydride (2 mL, 36.67 mmol), and DMAP (0.027 g,
0
.22 mmol). The mixture was kept stirring at r.t. under nitrogen for 2
h. Ground ice was then added. After the ice dissolved completely, the
mixture was extracted with methylene chloride (15 mL × 3). The ex-
tracted organic layers were combined and washed with water (20 mL ×
5
), dried over sodium sulfate, and followed by filtration and evaporation
on a rotavapor. The residue was allowed to evaporate with dry toluene
20 mL) under reduced pressure. After repetition of the addition and
(
evaporation of toluene (4 times), the crude pale powder was obtained.
Recrystallization from ethyl acetate/hexane (1/6, v/v) afforded a white
solid in 95% yield. m.p. 289-290 C. (Lit. , m.p. 289–290 C).
◦
22
◦
Preparation of 3-β-Acetoxy-urs-12-ene- 28-oyl Chloride (6)
To a 10-mL flask was added 3β-acetoxy-urs-12-ene-28-oic acid (43 mg,
0
.086 mmol) and freshly distilled thionyl chloride (2 mL). The reaction
◦
was warmed to 60–75 C and kept stirring for about 5 h. The solution
was then concentrated by distillation under reduced pressure at 60 C,
◦
followed by evaporation on vacuum (oil pump) for another 1 h. The
obtained acid chloride was dissolved in anhydrous THF and used im-
mediately in the next reaction.
General Procedure for the Synthesis of 3β-Acetoxy-ursolic
Phosphonate Derivatives (7a–j)
To a solution of α-aminophosphonates 4 (0.2 mmol) in anhydrous THF
(
10 mL) was added triethylamine (0.2 mL). The solution was then cooled
with stirring on an ice bath. A solution of acid chloride 6 (0.1 mmol) in
anhydrous THF (5 mL) was added dropwise over 10 min. After com-
◦
pletion of the addition, the reaction was allowed to stir at 0 C for 0.5
h and at r.t. for another 5 h. The precipitate was then filtered off, and
the solvent was removed on a rotavapor. The residue was subject to col-
umn chromatography using a mixture of ethyl acetate/hexane (1/4, by
volume) as an eluent. The fractions (Rf = 0.3–0.4) were collected. The
subsequent evaporation gave pure products 7a–j as oils. After standing
for 3 days, the products solidified as a yellow powder.

9
62
S.-L. Deng et al.
Diphenyl [(N-3β-acetoxyurs-12-en-28-oyl)aminomethyl]
Phosphonate (7a)
◦
−1
Yield: 75%; mp 48–50 C; IR (ν cm ): 3395, 2922, 1737, 1715, 1528,
1
1
244, 1027; H NMR (CDCl3): δ = 0.65–1.95 (m, 43H, parent structure
3
of UA unless otherwise indicated), 1.80 (d, 1H, JH H = 9.62 Hz, H-18),
2
2
1
.98 (s, 3H, CH3C=O), 3.51 (dddd, Ha, JHa-Hb = 15.90 Hz, JP−Ha = 11.9
3
Hz, JHa-H = 3.67 Hz, CHaHbP), 4.22–4.30 (m, 1Hb, CHaHbP), 4.40 (m,
3
1
7
1
3
1
H, H-3), 5.25 (m, 1H, H-12), 6.29 (t, 1H, JH H = 3.87 Hz, NH), 7.10–
.30 (m, 10H, Harom); 13C NMR (CDCl3): δ = 14.8, 15.9, 17.0, 17.1,
7.4, 21.5, 21.7, 23.7, 23.9, 24.5, 28.1, 28.4, 29.4, 30.1, 32.9, 37.0, 37.1,
8.1, 38.6, 38.9, 39.4, 39.8, 42.6, 47.8, 48.4, 53.4, 55.5, 81.5 (C3), 116.1,
20.8, 126.9 (C12), 130.1, 139.1 (C13), 156.2, 171.9 (O=C-(CH3)), 181.5
3
1
(
C28); P NMR(CDCl3): δ = 17.19; Analysis of compound C45H62NO6P
calcd.: C, 72.65; H, 8.40; found: C, 72.44; H, 8.60.
Diphenyl [(N-3β-Acetoxyurs-12-en-28-oyl)
(
Aminomethyl-n-propyl)] Phosphonate (7b)
◦
−1
Yield: 80%; mp 63–65 C; IR (ν cm ): 3371, 2928, 1737, 1700,
1
1
595, 1489, 1244, 1024; H NMR (CDCl3): δ = 0.69–1.96 (m, 50H,
CH2CH2CH3 + parent structure of UA unless otherwise indicated), 1.97
3
(
s, 3H, CH3C=O), 2.08 (d, 1H, JH H = 10.74 Hz, H-18), 4.40 (m, 1H,
H-3), 4.78–4.84 (m, 1H, CHP), 5.26 (m, 1H, H-12), 5.97 and 6.17 (d, 1H,
3
13
JH H = 9.42 Hz, NH), 6.73–7.25 (m, 10H, Harom); C NMR (CDCl3):
δ = 14.1, 14.5, 15.9, 17.1, 17.3, 17.5, 17.8, 18.1, 21.5, 21.8, 23.5, 23.6,
2
4
1
7.3, 28.4, 30.1, 31.2, 37.2, 38.0, 38.6, 38.9, 39.1, 39.9, 40.0, 42.8, 42.9,
7.8, 48.7, 48.9, 55.6, 81.5 (C3), 115.8, 120.8, 126.8 (C12), 129.9, 138.1,
3
1
39.0 (C13), 156.2, 171.9 (O=C-(CH3)), 181.5 (C28); P NMR (CDCl3):
δ = 19.24, 19.71; Analysis of compound C48H68NO6P calcd.: C, 73.34;
H, 8.72, found: C, 73.69; H, 8.34.
Dipheny[(N-3β-Acetoxyurs-12-en-28-oyl)
Aminomethyl-p-methoxyphenyl)]Phosphona te (7c)
◦
−1
Yield: 75%; mp 127–128 C; IR (ν cm ): 3392, 2926, 1737, 1700, 1592,
1
1
513, 1247, 1030; H NMR (CDCl3): δ = 0.68–1.97 (m, 43H, parent
structure of UA unless otherwise indicated), 2.07 (s, 3H, CH3C=O), 2.15
3
(
d, 1H, JH H = 10.80 Hz, H-18), 3.80 and 3.81 (s, 3H, OCH3,), 4.48 (m,
3
1
8
H, H-3), 5.29 and 5.41 (m, 1H, H-12), 5.75 and 5.93 (dd, 1H, JH H =
2
.18 Hz, JP-H = 19.45 Hz, CHP), 6.60 (br s, 1H, NH), 6.64–7.42 (m, 14H,
1
3
Harom); C NMR (CDCl3): δ = 14.1, 15.2, 15.6, 16.7, 17.0, 17.3, 21.1, 21.3,
2
4
1
2.7, 23.0, 23.5, 28.1, 29.7, 36.6, 36.8, 37.1, 37.6, 38.8, 39.4, 39.5, 42.3,
2.4, 47.9, 48.1, 55.2, 80.8 (C3), 114.2, 115.4, 120.3, 126.6 (C12), 129.5,
38.1, 137.9 (C13), 155.9, 171.1 (O=C (CH3)), 181.5 (C28); P NMR
3
1

Ursolic Phosphonate Derivatives
963
(
7
CDCl3): δ = 11.20, 12.16. Analysis of compound C52H68NO7P calcd.: C,
3.47; H, 8.06, found: C, 73.38; H, 8.22.
Diphenyl [(N-3β-acetoxyurs-12-en-28-oyl)
aminomethyl-n-butyl)] Phosphonate (7d)
(
◦
−1
Yield: 82%; mp 87–89 C; IR (ν cm ): 3369, 2925, 1737, 1715,
1
1
624, 1518, 1242, 1027; H NMR (CDCl3): δ = 0.70–1.97 (m, 52H,
CH2CH2CH2CH3 + parent structure of UA unless otherwise indicated),
3
2
.05 (s, 3H, CH3CO), 2.15 (d, 1H, JH H = 11.10 Hz, H-18), 4.48 (m, 1H,
H-3), 4.84–4.94 (m, 1H, CHP), 5.34 (m, 1H, H-12), 6.04 and 6.25 (d, 1H,
3
13
JH H = 9.54 Hz, NH), 7.11–7.36 (m, 10H, Harom); C NMR (CDCl3):
δ = 12.7, 12.8, 13.2, 14.4, 14.5, 15.7, 16.0, 16.4, 16.8, 17.1, 18.2, 18.4,
2
3
4
1
0.0, 20.1, 20.3, 22.0, 22.3, 22.5, 23.7, 26.8, 27.0, 28.7, 29.8, 29.9, 32.0,
5.8, 36.2, 36.6, 37.3, 37.7, 38.5, 38.6, 41.4, 41.5, 41.6, 46.4, 46.5, 47.4,
7.5, 52.3, 52.8, 54.2, 59.5, 80.0 (C3), 114.4, 119.0, 119.4, 119.5, 119.7,
24.0, 124.4, 125.0 (C12), 128.5, 128.6, 128.8, 137.7(C13), 155.2, 170.4
31
(
O=C (CH3)), 178.2 (C28); P NMR (CDCl3): δ = 19.25, 19.72; Analy-
sis of compound C49H70NO6P calcd.: C, 73.56; H, 8.82; found: C, 73.49;
H, 8.91.
Diphenyl[(N-3β-Acetoxyurs-12-en-28-oyl)
(
aminomethylphenyl)]Phosphonate (7e)
◦
−1
Yield: 70%; mp 98–101 C; IR (ν cm ): 3416, 2924, 1736, 1715, 1595,
1
1
497, 1244, 1024; H NMR (CDCl3): δ = 0.59–2.03 (m, 43H, parent
structure of UA unless otherwise indicated), 2.11 (s, 3H, CH3CO), 2.22
3
(
d, 1H, JH H = 10.28 Hz, H-18), 4.55 (m, 1H, H-3), 5.35 and 5.46 (m,
3
2
1
H, H-12), 5.92 and 6.06 (dd, 1H, JH H = 9.42 Hz, JP-H = 19.80 Hz,
13
CHP), 6.64–7.87 (m, 15H, Harom), 7.95 (br s, 1H, NH); C NMR (CDCl3):
δ = 15.6, 16.0, 16.7, 16.9, 17.0, 17.3, 17.4, 17.6, 18.3, 21.4, 21.5, 21.7,
2
4
1
3.5, 23.6, 28.1, 28.4, 29.7, 30.1, 36.7, 37.0, 37.2, 38.0, 38.1, 39.7, 39.8,
0.0, 42.6, 42.8, 47.8, 48.4, 48.6, 48.8, 55.6, 81.6 (C3), 115.8, 120.6,
20.8, 121.0, 129.4 (C12), 129.9, 130.3, 138.2 (C13), 139.2, 156.3, 172.0
31
(
O=C-(CH3)), 181.5 (C28); P NMR (CDCl3): δ = 14.89, 15.86; Analysis
of compound C51H66NO6P calcd.: C, 74.70; H, 8.11; found: C, 74.89; H,
.23.
8
Diphenyl [(N-3β-Acetoxyurs-12-en-28-oyl)
aminomethyl-p-tolyl)] Phosphonate (7f)
(
◦
−1
Yield: 78%; mp 112–114 C; IR (ν cm ): 3412, 2925, 1737, 1715, 1596,
1
1
500, 1243, 1026; H NMR (CDCl3): δ = 0.56–2.04 (m, 43H, parent
structure of UA unless otherwise indicated), 2.14 (s, 3H, CH3CO), 2.22
3
(
d, 1H, JH H = 10.90 Hz, H-18), 2.42 (s, 3H, CH3), 4.54 (m, 1H, H-3),

9
64
S.-L. Deng et al.
3
5
.36 and 5.49 (m, 1H, H-12), 5.85 and 6.02 (dd, 1H, JH H = 9.46 Hz,
2
3
JP-H = 20.29 Hz, CHP), 6.69–7.44 (m, 14H, Harom), 7.70 (d, 1H, JH H =
.34 Hz, NH); 1 C NMR (CDCl3): δ = 14.1, 14.6, 15.2, 15.6, 15.7, 16.0,
0.0, 20.1, 20.3, 22.1, 23.7, 26.7, 27.0, 28.7, 29.7, 31.8, 35.6, 35.7, 36.6,
7.7, 38.2, 38.4, 38.6, 41.2, 41.4, 46.4, 47.2, 50.3, 53.1, 54.1, 59.5, 79.9
3
9
2
3
(
1
C3), 114.4, 119.0, 119.3, 119.4, 119.5, 124.1, 124.3, 125.0, 127.9 (C12),
28.0, 128.4, 128.5, 128.6, 128.7, 129.3, 138.0, 137.9 (C13), 149.0, 149.2,
3
1
1
55.3, 170.2 (O=C-(CH3)), 176.2, 176.4, 181.5 (C28); P NMR (CDCl3):
δ = 10.85, 11.91. Analysis of compound C52H68NO6P calcd.; C, 74.88;
H, 8.22, found: C, 74.50; H, 8.51.
Diphenyl [(N-3β-Acetoxyurs-12-en-28-oyl)
(
aminomethyl-p-chlorophenyl)] Phosphonate (7g)
◦
−1
Yield: 67%; mp 116–118 C; IR (ν cm ): 3383, 2925, 1733, 1715, 1669,
1
1
594, 1490, 1243, 1088; H NMR (CDCl3): δ = 0.66–2.01 (m, 43H, parent
structure of UA unless otherwise indicated), 2.07 (s, 3H, CH3C=O), 2.13
3
(
d, 1H, JH H = 10.56 Hz, H-18), 4.47 (m, 1H, H-3), 5.26 and 5.46 (m, 1H,
3
2
H-12), 6.33 and 6.72 (dd, 1H, JH H = 8.76 Hz, JP-H = 22.23 Hz, CHP),
3
13
6
.62–7.48 (m, 14H, Harom), 7.72 (d, 1H, JH H = 8.46 Hz, NH); C NMR
(
CDCl3): δ = 14.1, 15.7, 15.9, 16.0, 20.0, 20.1, 20.3, 22.1, 25.9, 27.0, 28.7,
2
1
1
9.7, 31.8, 35.7, 36.6, 38.3, 38.4, 41.4, 46.4, 47.1, 53.1, 54.1, 80.2 (C3),
14.4, 119.1, 119.4, 119.5, 125.5, 127.7, 127.9 (C12), 128.1, 128.5, 128.7,
3
1
29.0, 136.7, 137.9 (C13), 154.8, 170.6 (O=C-(CH3)), 178.9 (C28);
P
NMR (CDCl3): δ = 14.23, 15.09. Analysis of compound C51H65ClNO6P
calcd.: C, 71.69; H, 7.67, found: C, 71.57; H, 7.40.
Diphenyl [(N-3β-Acetoxyurs-12-en-28-oyl)
(
aminomethyl-o-chlorophenyl)] Phosphonate (7h)
◦
−1
Yield: 61%; mp 125–127 C; IR (ν cm ): 3409, 2925, 1737, 1718, 1656,
1
1
595, 1490, 1242, 1069; H NMR (CDCl3): δ = 0.61–2.06 (m, 43H, parent
structure of UA unless otherwise indicated), 2.09 (s, 3H, CH3C=O), 2.16
3
(
d, 1H, JH H = 10.68 Hz, H-18), 4.51 (m, 1H, H-3), 5.31 and 5.44 (m, 1H,
3
2
H-12), 5.76 and 5.98 (dd, 1H, JH H = 9.40 Hz, JP−H = 20.25 Hz, CHP),
1
3
6
1
3
1
1
.64–7.45 (m, 14H, Harom), 7.78 (br s, 1H, NH); C NMR (CDCl3): δ =
4.1, 15.7, 15.9, 16.0, 20.0, 20.1, 20.3, 22.1, 25.9, 27.0, 28.7, 29.7, 31.8,
5.7, 36.6, 38.3, 38.4, 41.4, 46.4, 47.1, 53.1, 54.1, 80.2 (C3), 114.4, 119.1,
19.4, 119.5, 125.5, 127.7, 127.9 (C12), 128.1, 128.5, 128.7, 129.0, 136.7,
3
1
37.9 (C13), 154.8, 170.6 (O=C-(CH3)), 178.9 (C28); P NMR (CDCl3):
δ = 13.75, 14.70. Analysis of compound C51H65ClNO6P calcd.: C, 71.69;
H, 7.67; found: C, 71.66; H, 7.33.

Ursolic Phosphonate Derivatives
965
Diphenyl[(N-3β-Acetoxyurs-12-en-28-oyl)
(
aminomethyl-2,4-dichlorophenyl)]phosphornate (7i)
◦
−1
Yield: 57%; mp 142–145 C; IR (ν cm ): 3405, 2924, 1737, 1718, 1654,
1
1
589, 1488, 1247, 1071; H NMR (CDCl3): δ = 0.65–2.04 (m, 42H, parent
structure of UA unless otherwise indicated), 2.07 (s, 3H, CH3C=O), 2.22
3
(
d, 1H, JH H = 10.35 Hz, H-18), 4.50 (m, 1H, H-3), 5.26 and 5.44 (m,
3
2
1
H, H-12), 6.26 and 6.64 (dd, 1H, JH H = 7.57 Hz, JP-H = 14.26 Hz,
13
CHP), 6.60–7.59 (m, 14H, Harom), 7.82 (br s, 1H, NH); C NMR (CDCl3):
δ = 14.1, 14.9, 15.2, 16.2, 16.7, 17.0, 17.1, 21.0, 21.3, 22.7, 23.1, 23.3,
2
5
1
1
1
7
8.0, 29.7, 36.7, 37.6, 38.2, 38.4, 38.8, 39.3, 39.6, 42.3, 42.4, 42.7, 48.2,
3.9, 55.0, 55.2, 80.8 (C3), 119.7, 119.9, 120.0, 120.4, 120.5, 125.2, 125.4,
26.5 (C12), 129.5, 129.6, 129.7, 129.9, 138.1 (C13), 148.0, 150.0, 150.2,
56.3, 171.1 (O=C-(CH3)), 179.8, 179.9 (C28); P NMR (CDCl3): δ =
3.64, 14.50. Analysis of compound C51H64Cl2NO6P calcd.: C, 68.91; H,
.26; found: C, 69.21; H, 7.17.
3
1
Diphenyl[(N-3β-Acetoxyurs-12-en-28-oyl)
aminomethyl-o-nitrophenyl)]phosphonate (7j)
(
◦
−1
Yield: 48%; mp 112–114 C; IR (ν cm ): 3419, 2924, 1733, 1716, 1654,
1
1
540, 1488, 1245, 1070; H NMR (CDCl3): δ = 0.65–2.02 (m, 43H, parent
structure of UA unless otherwise indicated), 2.03 (s, 3H, CH3C=O), 2.15
3
(
d, 1H, JH H = 9.12 Hz, H-18), 4.45 (m, 1H, H-3), 5.23 and 5.46 (m,
3
2
1
H, H-12), 6.64 and 6.90 (dd, 1H, JH H = 6.60 Hz, JP-H = 18.84 Hz,
13
CHP), 6.54–8.07 (m, 14H, Harom), 8.04 (brs, 1H, NH); C NMR (CDCl3):
δ = 14.1, 15.2, 15.3, 16.4, 16.7, 21.3, 22.7, 24.4, 28.0, 29.3, 29.7, 30.7,
3
7
1
1
0.8, 31.9, 36.7, 37.6, 39.0, 39.3, 39.6, 47.3, 48.3, 53.9, 55.2, 59.5, 73.8,
9.7 (C3), 98.5, 111.9, 113.9, 114.4, 119.8, 120.0, 120.4, 120.7, 125.1,
29.5 (C12), 129.8, 138.0 (C13), 170.9 (O=C-(CH3)), 172.7, 176.2, 176.4,
3
1
81.5 (C28); P NMR (CDCl3): δ = 13.43, 14.51. Analysis of compound
C51H65N2O8P calcd.: C, 70.81; H, 7.57, found: C, 70.87; H, 7.28.
Cell and Drug Cytotoxicity Assay
The HT-29 colon cancer cell line originated from a colic adenocarcinoma
in an untreated patient and was obtained from American Type Culture
Collection (ATCC) (Rockville, MD). The culture medium was a mixture
of Ham F10 medium (Bio Whittaker, Ververs, Belgium) and 10% fetal
calf serum (Boerhinger Ingelheim, Gagny, France). Cells were grown
◦
as monolayers in a controlled atmosphere (37 C, 5% CO2) in Ham’s
2
5
F-10 medium supplemented with 10% fetal calf serum. All test com-
pounds were solubilized in DMSO and were tested twice at different
concentrations.

9
66
S.-L. Deng et al.
4
HT29 cells (2 × 10 per well) were seeded in 96-well culture plates
2
5
and cultured for two days before treatment. The prepared compounds
were dissolved immediately before use in a mixture of DMSO and ab-
solute EtOH (1:1, v/v), then diluted in serum-free Ham’s F-10 medium.
Final concentration of DMSO and EtOH, which did not exceed 1%, did
not affect cell survival. Cells were treated for 3 h with drugs alone. After
treatment, cells were washed twice with Ham’s F-10 and cultured again
for 7 days in a drug-free culture medium. Cell survival was measured
by the crystal violet colorimetric assay. In brief, cells were rinsed with
phosphate buffered saline, and then surviving adherent cells were fixed
for 5 min by pure ethanol. After drying, cells were stained by crystal
violet (5g/L in distilled water). Dye in excess was flushed off using tap
water. Cell-fixed dye was eluted by 33% acetic acid.
Fluorescence Polarization Assay Specific to the CD4 Binding
26
Site of HIV-1 Glycoproteins
Anisotropy measurements in the Beacon 2000 analyzer are quite repro-
ducible, but require 200 µL total volume and 20 min equilibration time
after each determination. Therefore, it is rather consuming in terms
of material and time. The assay was thus performed in 384-well mi-
croplates a method that uses a 10-fold smaller total volume and allows
multiple determinations in a short time. The fluorescence anisotropy of
CD4M33-Fl was evaluated in quadruplicate experiments, with 16 two-
fold dilutions of 200 nM of gp120. CD4M33-Fl was tested at different
concentrations (0.1, 0.5, 1.0, and 10 nM) to minimize the use of gp120. At
0
.1 nM, the intensity of fluorescence was very weak (the signal-to-noise
ratio was less than 10), while 1 nM was found to be the best compromise
between an acceptable signal-to-noise ratio and the use of gp120.
Measurements were made in an Lev J. Leytes analyst (LJL Biosys-
tems, Sunnyvale, CA) microplate reader, using the fluorescence polar-
ization detection mode, 485 nm excitation, and 530 nm emission filters,
and an additional 505 nm dichroic filter to limit blank response. A 1.2-G
parameter was used. Samples were in 10 mM sodium phosphate buffer,
pH 7.0, containing 135 mM NaCl and 0.05% Tween 20. The binding as-
say was performed in a final volume of 20 mL (titration experiments) or
2
1 mL (competitive experiments) in a 384-well small-volume black mi-
crotitration plate by using 1 nM fluorescein–CD4M33. Titration assays
were performed in quadruplicate by the addition of 10 mL of fluores-
cein ligand (1 nM final concn.) to 10 mL of gp120 (two-fold dilutions in
1
6 wells and starting at 200 nM). Competition assays were performed
in triplicate by mixing 7.0 mL of competing unlabelled ligand, 7.0 mL
of fluorescein-labelled ligand (1 nM final concn.), and 7.0 mL of gp120

Ursolic Phosphonate Derivatives
967
(
4
12.5 nM final concn.). Fluorescence anisotropy was determined after
0 min equilibration at 25 C.
◦
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