Phosphonate terminated PPH dendrimers: Influence of pendant alkyl chains on the in vitro anti-HIV-1 properties

Article abstract of DOI:10.1039/b908352a

The synthesis, characterization and in vitro anti-HIV activity of a series of generation one dendrimers having phosphonate groups with pendant alkyl chains are described. The influence of the lateral alkyl chains on the biological properties was correlate

Full text of DOI:10.1039/b908352a

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Phosphonate terminated PPH dendrimers: influence of pendant alkyl chains
on the in vitro anti-HIV-1 properties†
Alexandra Pe´rez-Anes,a,b,c Gre´gory Spataro,a,b Yannick Coppel,a,b Christiane Moog,d Muriel Blanzat,*c
Ce´dric-Olivier Turrin,*a,b Anne-Marie Caminade,a,b Isabelle Rico-Lattesc and Jean-Pierre Majorala,b
Received 27th April 2009, Accepted 2nd June 2009
First published as an Advance Article on the web 7th July 2009
DOI: 10.1039/b908352a
The synthesis, characterization and in vitro anti-HIV activity of a series of generation one dendrimers
having phosphonate groups with pendant alkyl chains are described. The influence of the lateral alkyl
chains on the biological properties was correlated to 1H-1H NOESY experiments.
sialic acid moieties, carboxylates, or galactose sulfates.9 The
Introduction
multivalency effects10,11 induced by dendrimeric structures upon
Although combination antiretroviral therapy has revolutionized
the ionic interactions are remarkably strengthened when den-
the management of HIV infection, the continuing HIV/AIDS
drimeric compounds are used, but to the best of our knowledge,
pandemic confirms the need for new antiretroviral drugs. Among
none of these multivalent systems have taken into account the
the new strategies currently under investigation, the early stages of
possible influence of lipophilic-lipophilic interaction that could
the virus replication involving the recognition between the virus
influence the anchorage of the inhibitors and thus its efficacy.
and host cells, and the subsequent fusion step, are often at the
This type of interaction is known to be determinant for numbers
center of the debate. CD4(+) lymphoid cells are a key target for
of supramolecular interactions, and we have thus assumed that
the HIV, and in this case the entry of the virus is initiated by
the multivalent interaction of polyanionic dendrimers with gp120
the formation of ternary gp120/CD4/co-receptors (namely CCR5
could be influenced by lipophilic interactions with the lipophilic
or CXCR4) complex1, followed by the anchoring of viral gp41
part of the V3 loop located on gp120. Actually, it has already
to the host cell. Recently FDA-approved Enfuvirtide2 and other
drug candidates (co-receptors antagonists, gp41 inhibitors) were
designed to disrupt this ternary complex.
been shown that a particular family of HIV-1 inhibitors (GalCer
analogs)12, that also prevent gp120-CD4 recognition, is sensitive
to lipophilic interactions.13 In fact, the hydrophobic long chain
grafted on these GalCer analogs, plays a key role in the interaction
with the hydrophobic portion of the V3 loop and therefore in the
enhancement of protein/ligand binding. In this respect, we present
herein the synthesis and preliminary anti-HIV-1 inhibitory activity
of a series of phosphorus-containing dendrimers, namely PPH
(Poly-(PhosphorHydrazone)) dendrimers, equipped with terminal
phosphonic acid moieties and lateral alkyl chains (Fig. 1). In order
to highlight the influence of the alkyl chain and according to
previous results14, the generation parameter was set to one.
Alternatively, anionic polymers have been used to inhibit HIV-1
infection by preventing virus-cell fusion through ionic interactions
with the V3 loop of gp120.3 Some of these polymers include
topically applied microbicides designed to prevent vaginal HIV-1
transmission, like BufferGel, a polyanionic carbopol containing
buffering gel (phase III trials).4 Multivalent effects5 are of great
importance for these charge-based interactions, and dendrimers
have naturally emerged as a new class of potential nanodevices for
antiviral strategies. Indeed, dendrimers have received constantly
increasing attention since the first synthesis was performed by
Vo¨gtle and co-workers.6 Actually, dendrimers offer a wide range
of surface function availability and a regular structure which is
tunable at will for the rational design of nanosized therapeutic
platforms.7 For instance, polysulfonated dendrimers based on
poly-L-lysine have given birth to Viva GelTM, a dendrimer-based
topical microbicide (phase I/II trials).8 Many other examples
deal with the use of polyanionic dendrimers terminated with
Fig. 1 A symbolic representation of phosphonic acid (red spheres) capped
dendrimers without (left) and with C3 (centre) and C10 (right) lateral alkyl
chains.
aLaboratoire de Chimie de Coordination du CNRS, UPR 8241, 205 route
de Narbonne, F-31077, Toulouse, France. E-mail: turrin@lcc-toulouse.fr;
Fax: +33(0)561 553 003; Tel: +33(0)561 333 134
bUniversite´ de Toulouse; UPS, INPT; LCC, F-31077, Toulouse, France
cLaboratoire des Interactions Mole´culaires et Re´activite´ Chimique et
Photochimique, CNRS UMR5623. Universite´ Paul Sabatier, 118 Route de
Narbonne, 31062, Toulouse cedex, (France). E-mail: blanzat@chimie.ups-
tlse.fr; Fax: +33 5 61 55 81 55
Results and discussion
Chemistry
dInstitut de Virologie, INSERM U544, Faculte´ de Me´decine, 3 rue Koeberle´,
F-67000, Strasbourg, France
We have designed series of phenols that can be easily grafted
onto the dichlorothiophosphorydrazone end groups which com-
pose one of the easily affordable electrophilic surface of PPH
† Electronic supplementary information (ESI) available: NMR spectra for
all compounds. See DOI: 10.1039/b908352a
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dendrimers.15 These phenols comprise dimethylphosphonate func-
tions with a pendant alkyl chain (scheme 1), and can be readily
transformed to the corresponding phosphonic acids after grafting
onto the dendrimer’s outer shell.16 Phenol 3 lacks lateral alkyl
chains and is easily obtained by coupling tyramine with the
deprotected P,P-dimethylphosphoacetic acid in the presence of
N,N¢-dicyclohexylcarbodiimide (DCC) and N-hydroxybenzo-
triazole (HOBt). Compound 3 is obtained with moderate yields
(46%) after purification by column chromatography, and shows on
1H NMR a typical doublet at 2.84 ppm with 2JPH = 21 Hz for the
activated methylene group, whereas the phosphorus atom of the
phosphonate resonates as a singlet at 25.6 ppm. The intermediary
carboxylic acid 2 is obtained by TFA-assisted deprotection of the
commercially available tert-butyl P,P-dimethylphosphonoacetate
(scheme 1), and the total deprotection is easily assessed on proton
NMR by disappearance of the signal corresponding to the terbutyl
group.
Scheme
2
Synthesis of dendrimers 3a-Gc¢1, 5a-Gc¢1 and 7a-Gc¢1
(a: 3, Cs2CO3/THF; b: 5, Cs2CO3/THF; c: 7, Cs2CO3/THF).
singlet at 68.4 ppm is rapidly observed, indicating the formation
of the intermediary –P(S)(OAr)Cl species on the surface of the
dendrimers, along with the progressive disappearance of the singlet
at 62.3 ppm corresponding to the starting P(S)Cl2 end groups. The
reaction goes to completion in one night at room temperature,
as confirmed by 31P NMR with the total disappearance of the
intermediary species at 68.4 ppm and the appearance of a new
singlet at 62.8 ppm for the terminal P(S)(OAr)2 functions. During
the course of the reaction the cyclotriphosphazene core and
the terminal phosphonate functions of the dendrimers remain
unaffected, and resonate at 8.4 ppm and 28.2 ppm respectively
on the 31P NMR spectra.
Although the solubility properties of PPH dendrimers are
closely connected to the nature of the outer-shell functions, it
is usually possible to separate them from excess of reagents by
simple washing procedures involving a suitable mixture of solvents
in which the dendrimers are scarcely soluble and the impurities
or excess reactants are soluble. In the present case, phosphonate
containing phenols 3, 5 and 7 do not show significant differences
in their solubility properties compared to the parent dendrimers
3a-Gc¢1, 5a-Gc¢1 and 7a-Gc¢1, which were consequently purified by
flash column chromatography. These compounds were univocally
characterized by 31P, 1H and 13C NMR techniques, including two
dimension experiments. As expected for PPH dendrimers, no clear
information could be obtained from mass spectrometry analysis.17
Phosphonic acid terminated dendrimers 3b-Gc¢1, 5b-Gc¢1
and 7b-Gc¢1 are obtained from the corresponding phos-
phonate terminated dendrimers 3a-Gc¢1, 5a-Gc¢1 and 7a-Gc¢1
via silylation/methanolysis of the phosphonate end groups
(scheme 3) as previously described for aminomethylene
bis(dimethylphosphonate) capped dendrimers.18 The resulting
compounds are scarcely soluble in water and poorly soluble in
dimethylsulfoxide or methanol. The 31P NMR spectroscopy allows
us to observe the complete transformation of the phosphonate
surface by the total disappearance of the singlets at 25.4, 28.2 and
28.1 ppm and the disappearance of singlets at 16.9, 23.2, 22.8 ppm
for 3b-Gc¢1, 5b-Gc¢1 and 7b-Gc¢1 respectively. The latter are readily
transformed into the corresponding monosodium salts 3c-Gc¢1,
5c-Gc¢1 and 7c-Gc¢1 by addition of the stoichiometric amount of
sodium hydroxide in water and subsequent lyophilisation. During
this step, the signals corresponding to the terminal phosphonic
acids are all moderately shielded to ca. 13 ppm on the 31P NMR
spectra. As previously observed14,19, proton spectra obtained from
diluted water solutions are not well resolved (although sufficient
Scheme 1 Synthesis of phenols 3, 5 and 7 [a: TFA (20% in CH2Cl2); b:
tyramine, DCC, HOBt, c: HNa, IC3H7, cat. crown 15C5; d: HNa, IC10H21,
cat. crown 15C5].
Phenols 5 and 7 are obtained with moderate yields in three
steps (34% and 36% respectively). The alkyl chain is grafted on 1
by deprotonation of the activated methylene with sodium hydride
and reaction of the resulting carbanion with 1-iodopropane or
1-iododecane to afford 4 and 6 respectively (scheme 1). After
tertbutyl ester removal with TFA, the phenols 5 and 7 are
obtained by acylation of tyramine with a DCC/HOBt procedure.
No stereochemical control is induced during this procedure,
racemate species are thus obtained for compounds 4 to 7, and
the diastereotopical methoxy groups of the phosphonate present
a typical set of two doublets located around 3.7 ppm (3JHP
=
1
10 Hz) on H spectra and a typical set of two doublets located
around 53 ppm (2JPC = 7 Hz) on the 13C spectra. The influence of
the alkyl chain is observed on the 31P NMR spectra of compounds
5 and 7 on which the chemical shift of the phosphorus atom of the
phosphonate group is displaced ca. 2.5 ppm upfield compared to
compound 3.
The phosphonate terminated dendrimers are obtained by
nucleophilic substitution of the twelve terminal chlorine atoms
located on the surface of a first generation dendrimer Gc1 in the
presence of the corresponding phenols and cesium carbonate as a
base (scheme 2). The chain length has no influence on the course
of the reactions, which can be monitored by 31P NMR directly
from the reaction mixture. Typically, in the case of 5a-Gc¢1, a
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the alkyl chains (figure 2, right). Additionally, one should notice
that the signal of the terminal methyl group is relatively shielded,
d(1H) = 0.6 ppm, instead of ca. 0.9 ppm for a terminal methyl
group of a free alkyl chain, which is in agreement with shielding
effects due to the dendrimer’s interior.
In the case of 7c-Gc¢1, a clear correlation between the aromatic
rings of the zeroth and the first layer can be observed (data not
shown), indicating that these moieties are located within a 0.5 nm
distance range, which is almost half of the expected distance.
This observation is consistent with previous experiments run on
PPH dendrimers equipped with water soluble end groups20, and
produces a drastic compaction of the structure. The terminal
methyl group of the lateral alkyl chain is identified as a broad
singlet centered at 0.49 ppm on the 1H spectrum, and the methylene
groups resonate as a non-resolved multiplet (0.84–1.00) ppm.
Again, a strong shielding effect is observed on the terminal methyl
group of the alkyl chain, causing it to bury into the interior of the
dendrimer. The 2D NOESY spectrum shows a clear correlation
between the methylene groups of the alkyl chain and the aromatic
systems located on the zeroth and on the first generation, and an
unambigous correlation between the alkyl chains (terminal methyl
group and methylene groups) and the signals of the aromatic
systems located at 6.70, 7.40 and 7.45 ppm and attributed to
Scheme 3 Synthesis of dendrimers 3b,c-Gc¢1, 5b,c-Gc¢1 and 7b,c-Gc¢1
(i: BrTMS, MeOH; j: NaOH).
1
to perfom 2D NOESY H-1H), due to a lack of mobility of the
interior of the dendrimers, but this phenomenon was surprisingly
less pronounced in the case of 7-Gc¢1. In all cases, addition
of a co-solvent (acetone or acetonitrile) allowed us to observe
fully resolved signals and all compounds could thus be fully
characterized by multi-nucleus NMR.
1
2D NOESY H-1H experiments were performed with mixing
2
3
=
time of 100 ms to prevent spin-diffusion phenomenon that can
impair the interpretation of NOESY spectra for macromolecules.
This technique allows the observation of 2D mapped correlations
the C0 -H, C0 -H and CH N moieties respectively (figure 3 left).
Another interaction between the alkyl chain and the signals at 6.90
and 7.05 ppm corresponding to the OC6H4 groups located on the
first generation is also evidenced, while in the 2.4–3.6 ppm region,
the terminal methyl group correlates with the N(CH3) groups at
2.95 ppm. The fact that the aromatic rings of the zeroth layer
correlate with the aromatic rings of the first layer supports the
idea that the extremities of the lateral C10 alkyl chains are burried
inside lipophilic pockets located within the contracted dendrimer
structure. This hypothesis is consistent with the observation of
shortened distances between the different moieties of the structure.
In addition, the fact that resolution of the spectra obtained in water
is much better in the case of 7c-Gc¢1 than in the case of 5c-Gc¢1 can
˚
that traduce through space connectivities in a 5 A distance range.
It allowed us to observe for dendrimers 5c-Gc¢1 and 7c-Gc¢1
an unambiguous interaction between the terminal methyl group
and the methylene groups of the lateral alkyl chains with specific
regions of the interior of the dendritic skeleton. In the case of
5c-Gc¢1, the CH3-(CH2)n entity appears on proton spectra as a
set of overlapped singlets from 0.60 ppm (CH3) to 0.89 ppm
((CH2)n) and the 2D NOESY spectrum evidences a weak NOE
correlation (figure 2, left) between the CH3-(CH2)n entities and
aromatic protons centered on 6.8 ppm belonging to the OC6H4
groups located on the first generation, whereas no correlation can
be detected between the N(CH3) group located at 3.20 ppm and
Fig. 2 Selected regions of the NOESY 1H-1H spectrum obtained for
5c-Gc¢1.
Fig. 3 Selected regions of the NOESY 1H-1H spectrum obtained for
7c-Gc¢1.
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be explained by the mutual interaction of the alkyl chain with the
dendrimer internal structure, which provides lipophilic pockets to
the alkyl chains and is reciprocally “solvated” by the latter.
The collected NMR data indicate an expected segregation
between the lipophilic and hydrophilic domains of the dendrimers
in water solutions, proving that the lateral alkyl chains of the
terminal phosphonic acid monosodium salt functions interact
with the dendrimer structure. In the case of 7c-Gc¢1 strong
interactions of the C10 alkyl chains with the interior of the PPH
dendrimer structure can be assumed, whereas for 5c-Gc¢1 the
interaction of the C3 alkyl chains with the dendrimer’s interior
is assumed to be weaker.
inhibitory effect was found for the dendrimer 5c-Gc¢1, grafted with
the short C3 alkyl chain. This dendrimer inhibits HIV-1 replication
on CEM-SS cells at 1.5 ¥ 10-6 mol L-1. It is noteworthy that these
values were determined by the viral reverse transcriptase titration,
representative of the viral activity. To confirm these results, the
three dendrimers were then evaluated on MT-4 cells, another
T4-lymphoblastoid cell line that is very sensitive to the HIV
infection (figure 5).
On this cell line, the antiviral efficiency is directly monitored
by the inhibition of the virus-induced cytopathicity. These in
vitro assays allowed us to confirm the better inhibitory activity
of the phosphonated dendrimer equipped with a C3 alkyl chain.
Actually, IC50 were evaluated respectively at 1.0 ¥ 10-5 and 3.5 ¥
10-5 mol L-1 for 3c-Gc¢1 and 7c-Gc¢1, and 1.5 ¥ 10-6 mol L-1 for
5c-Gc¢1 (figure 5). The fact that the dendrimer 5c-Gc¢1 is 10 fold
more active than dendrimers 3c-Gc¢1 (no alkyl chain) and 7c-Gc¢1
(C10 alkyl chains) can be partially rationalized in the light
of the NOESY 1H-1H experiments. Actually, one can assume
that according to the hypothesis that the C10 alkyl chains of
7c-Gc¢1 are strongly interacting with the contracted interior of
the dendrimer structure, these chains are not available for any
further interaction with external partners. In this regard, the fact
that compound having a major structural difference like 3c-Gc¢1
and 7c-Gc¢1 present the same inhibitory activity is not surprising
if one considers that the C10 alkyl chains are irreversibly buried
in the PPH skeleton. On the contrary, one can assume that the
C3 alkyl chains of 5c-Gc¢1 are more available to interact with the
lipophilic regions of the V3 loop, responsible for the increased
antiviral activity.
Anti-HIV-1 assays
The inhibitory effects of dendrimers 3c-Gc¢1, 5c-Gc¢1 and 7c-Gc¢1
against HIV-1 were evaluated in vitro on two different types of
cell lines, namely CEM-SS and MT-4 cells, according to published
procedures.21–23 The percentage of inhibition on both cell lines
are plotted in figure 4 and 5. Remarkably, none of these three
dendrimers present a cytotoxicity on both cell lines for the whole
range of concentrations (1 ¥ 10-7 to 1 ¥ 10-4 mol L-1). These
results are therefore a guarantee that the measured IC50 effectively
correspond to an antiviral activity.
Conclusions
To summarize, we have developed an efficient strategy to syn-
thesize phosphonic acid capped dendrimers equipped with alkyl
chains in the vicinity of the surface functions. This strategy is
adaptable to a wide range of other surface functions to produce
functionalized phosphonic acid capped dendrimers. The three
dendrimers were all found to be non-toxic at concentrations up to
1 ¥ 10-4 mol L-1. The inhibitory assays performed both on CEM-SS
and MT4 cells indicate that the length of the alkyl chain influences
the efficiency of these inhibitors. Actually, a comparable activity
was evaluated for both 3c-Gc¢1 and 7c-Gc¢1 having respectively
no alkyl chain and C10 alkyl chains grafted on a positions of
the phosphonic acid terminations, whereas the inhibitory activity
5c-Gc¢1 having a C3 alkyl chain was found to be 10 fold better.
2D NOESY experiments performed on these compounds in water
solutions indicated that the alkyl chains of 7c-Gc¢1 could be more
tightly buried in the internal dendrimer skeleton. The availability
of the alkyl chains for external interaction was then assumed to be
a key parameter in the design of such anti HIV inhibitors.
Fig. 4 Inhibition of HIV-1 infection of CEM-SS cells by dendrimers
3c-Gc¢1, 5c-Gc¢1 and 7c-Gc¢1.
Experimental
Fig. 5 Inhibition of HIV-1 infection of MT4 cells by dendrimers 3c-Gc¢1,
5c-Gc¢1 and 7c-Gc¢1.
Chemistry
The phosphonated dendrimers 3c-Gc¢1 and 7c-Gc¢1 were found
to inhibit HIV replication on CEM-SS cells in the same range of
concentration with IC50 values respectively at 2.5 ¥ 10-5 and 1.6 ¥
10-5 mol L-1 (figure 4). On the contrary, a significant increase in the
All manipulations were carried out using standard high-vacuum
and dry-argon techniques. Chemicals were used as received and
solvents were dried and distilled by routine procedures.24 1H, 13C,
19F and 31P NMR spectra were recorded at 25 ◦C with Bruker
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AC 200, AV 300, DPX 300, AV 400 or AV500 spectrometers. The
following abbreviations were used in reporting spectra: s = singlet,
d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet
of doublets. References for NMR chemical shifts are 85% H3PO4
for 31P NMR and SiMe4 for 1H and 13C NMR. The attribution of
13C NMR signals was done using Jmod, 2D 1H-13C HSQC, 1H-13C
cesium carbonate (410 mg, 1.260 mmol). The mixture was
stirred at RT for 12 h, centrifuged, filtered and evaporated. The
resulting residue was purified by silica-gel flash chromatography
(acetone/MeOH/H2O, 49:49:2) to give 3a-Gc¢1 as a white solid
1
(240 mg, 71%). 31P{ H} NMR (CDCl3, 121.5 MHz): d = 8.4 (s,
1
=
N3P3), 25.4 (s, PO3Me2), 62.8 (s, P S) ppm; H NMR (CDCl3,
1
3
HMBC and H-31P HMQC, Broad Band or CW 31P decoupling
300.1 MHz): d = 2.76 (t, JHH = 7.2 Hz, 24H, ArCH2), 2.83
2
3
experiments when necessary. Mass spectrometry was recorded on
a Finniganmat TSQ 7000. The numbering schemes used for NMR
are depicted in Fig. 6. Compound Gc1 was prepared according to
published procedures.25,26
(d, JPH = 20.7 Hz, 24H, PCH2), 3.24 (d, JPH = 10.2 Hz, 18H,
3 3
NCH3), 3.43 (q, JHH = JHH = 6.6 Hz, 24H, NHCH2), 3.72 (d,
3JPH = 11.1 Hz, 72H, OCH3), 6.99–7.05 (m, 24H, C0 H and NH),
2
2
3
3
7.09 (br s, 48H, C1 H and C1 H), 7.61 (m, 12H, C0 H), 7.64 (m,
6H, N CH) ppm; 13C{ H} NMR (CDCl3, 75.5 MHz): d = 33.0
1
=
(d, 2JPC = 12.2 Hz, NCH3), 34.2 (d, 1JPC = 131.9 Hz, PCH2), 34.8
(s, ArCH2), 41.1 (s, NHCH2), 53.2 (d, 2JPC = 6.6 Hz, OCH3), 121.3
2
2
3
3
4
(s, C0 ), 121.4 (s, C1 ), 128.3 (s, C0 ), 129.8 (s, C1 ), 132.2 (s, C0 ),
136.1 (s, C1 ), 138.7 (d, 4JPC = 14.2 Hz, N CH), 149.1 (d, JPC
=
4
2
=
7.0 Hz, C1 ), 151.2 (s, C0 ), 163.9 (d, 2JPC = 4.3 Hz, CONH) ppm.
1
1
Fig. 6 Numbering scheme for dendrimers 3-Gc¢1, 5-Gc¢1 and 7-Gc¢1.
3b-Gc¢1. To a vigorously stirred solution of 3a-Gc¢1 (100 mg,
0.021 mmol) in a mixture of dry acetonitrile and dicloromethane
(1:1, 2 mL) bromotrimethylsilane (74 mL, 0.570 mmol) was added
at 0 ◦C. The mixture was stirred for 12 h at RT, and then evaporated
to dryness under reduced pressure. The crude residue was washed
twice with methanol (10 mL) for 1 h at RT and dried under
reduced pressure. The resulting white solid was washed with water,
methanol and Et2O to afford 3b-Gc¢1 as a white solid (89 mg, 94%).
2. A solution of 20% of TFA in dichloromethane (15 mL)
was dropped on tert-butyl-2-dimethylphosphonoacetate (100 mg,
0.472 mmol) and the reaction mixture was stirred at RT for 1.5 h.
The residue was co-evaporated with ethyl acetate so that remaining
traces of TFA were removed upon evaporation to dryness. This
sequence was repeated until total disappearance of the signal of
TFA in 19F NMR to afford 2 as a white powder (75 mg, 100%).
The product was used in the next step without further purification.
1
31P{ H} NMR (DMSO-d6, 121.5 MHz): d = 8.4 (s, N3P3), 16.9 (s,
1
=
PO3H2), 62.5 (s, P S) ppm; H NMR (DMSO-d6, 200.1 MHz):
d = 2.50–2.70 (m, 48H, ArCH2 and PCH2), 3.10–3.30 (m, 42H,
1
31P{ H} NMR (CDCl3, 121.5 MHz): d = 22.7 ppm (s, PO3Me2);
2
2
3
NCH3 and NHCH2), 6.95–7.25 (m, 60H, C0 H, C1 H and C1 H),
1H NMR (CDCl3, 300.1 MHz): d = 3.14 (d, 2JPH = 22.2 Hz, 2H,
3
3
1
7.61 (s, 6H, CH N), 7.63 (d, JHH = 8.4 Hz, C0 H) ppm; 13C{ H}
=
1
PCH2), 3.89 (d, 3JPH = 11.4 Hz, 6H, OCH3) ppm; 13C{ H} NMR
NMR (DMSO-d6, 62.9 MHz): d = 32.8 (br s, NCH3), 34.3 (s,
(CDCl3, 75.5 MHz): d = 32.8 (d, 1JPC = 136.5 Hz, PCH2), 53.5 (d,
ArCH2), 37.2 (d, 1JPC = 132.0 Hz, PCH2), 120.7 (s, C1 and C0 ),
2
2
2JPC = 6.5 Hz, OCH3), 167.4 (d, 2JPC = 5.6 Hz, COOH) ppm.
3
3
4
4
128.2 (s, C0 ), 129.9 (s, C1 ), 132.0 (s, C0 ), 136.6 (s, C1 ), 139.0 (m,
1
1
3. To a vigorously stirred mixture of 2 (117 mg, 0.700 mmol)
and HOBt (107 mg, 1.400 mmol) in dry DMF (5 mL) was added
DCC (163 mg, 1.400 mmol) at 0 ◦C. The mixture was stirred at RT
for 1h, then a solution of tyramine (197 mg, 1.400 mmol) in dry
DMF (1 mL) was added dropwise at 0 ◦C. The resulting mixture
was stirred at RT for 12h and filtered. After solvent removal
(lyophilization), the resulting dark red residue was diluted in
chloroform (50 mL) and washed with an aqueous solution of HCl
(0.1 M, 3 ¥ 10 mL) and with brine (10 mL). The organic phase was
separated, dried over Na2SO4, filtered and concentrated to dryness
under reduced pressure. The residue was purified by silica-gel flash
chromatography (DCM/MeOH, 95:5 then acetone/MeOH, 95:5)
=
N CH), 148.4 (m, C1 and C0 ), 165.4 (s, CONH) ppm.
3c-Gc¢1. The sodium monosalt form was obtained by adding
aqueous sodium hydroxide (0.1023 N, 2.58 mL) to a suspension of
3b-Gc¢1 (100 mg, 0.023 mmol) in water (5 mL) at 0 ◦C. The solution
was filtered on micropore (1.2 mm) and lyophilized to give 3c-Gc¢1
1
as a white powder (110 mg, 90%). 31P { H} NMR (D2O/CD3CN,
3:1, 121.5 MHz): d = 9.7 (s, N3P3), 13.8 (s, PO3HNa), 64.0 (s,
1
=
P S) ppm; H NMR (D2O/CD3CN, 3:1, 300.1 MHz): d = 2.86
2
(d, JPH = 21.0 Hz, 24H, PCH), 2.97 (m, 24H, ArCH2), 3.49 (d,
3JPH = 9.0 Hz, 18H, NCH3), 3.55 (m, 24H, NHCH2), 7.13 (d,
3JHH = 6.0 Hz, 12H, C0 H), 7.31 (d, 3JHH = 7.3 Hz, 24H, C1 H),
2
2
3
7.43 (d, 3JHH = 9.0 Hz, 24 Hz, C1 H), 7.85 (d, 3JHH = 9.0 Hz, 12H,
1
to give 3 as a white solid (92 mg, 46%). 31P{ H} NMR (CDCl3,
C0 H), 8.02 (s, 6H, N CH) ppm; 13C { H} NMR (D2O/CD3CN,
3
1
121.5 MHz): d = 25.6 ppm (s, PO3Me2); 1H NMR (CDCl3,
=
3
3
3:1, 75.5 MHz): d = 32.9 (d, JPC = 11.7 Hz, N-CH3), 34.3 (s,
300.1 MHz): d = 2.71 (t, JHH = 6.9 Hz, 2H, ArCH2), 2.84 (d,
ArCH2), 38.2 (d, 1JPC = 117.9 Hz, PCH), 41.2 (s, NHCH2), 121.3
2JPH = 21.0 Hz, 2H, PCH2), 3.46 (q, JHH = JHH = 6.6 Hz, 2H,
3
3
2
2
3
3
4
3
3
(s, C1 ), 121.6 (s, C0 ), 128.9 (s, C0 ), 130.3 (s, C1 ), 132.7 (s, C0 ),
NHCH2), 3.74 (d, JPH = 11.4 Hz, 6H, OCH3), 6.77 (d, JHH
=
4
1
1
8.4 Hz, 2H, C2H), 6.81 (m, 1H, NH), 7.00 (d, JHH = 8.4 Hz,
3
=
137.1 (s, C1 ), 140.0 (s, N CH), 148.9 (s, C1 ), 150.7 (s, C0 ), 171.0
(s, CONH) ppm.
2H, C3H) ppm; 13C{ H} NMR (CDCl3, 75.5 MHz): d = 34.1 (d,
1
1JPC = 132.1 Hz, PCH2), 34.4 (s, ArCH2), 41.5 (s, NHCH2), 53.3
(d, 2JPC = 6.0 Hz, OCH3), 115.5 (s, C2), 129.6 (s, C4), 129.7 (s, C3),
155.4 (s, C1), 163.9 (d, 2JPC = 4.5 Hz, CONH) ppm; CI-MS: m/z =
305 [M + NH4]+.
4a. To a s◦uspension of NaH (24 mg, 1.000 mmol) in DMF
(2.5 mL) at 0 C, was added dropwise a solution of tert-butyl-2-
dimethylphosphonoacetate (224 mg, 1.000 mmol) in DMF (2 mL).
The mixture was stirred for 1 h. Then, a solution of 1-iodopropane
(0.195 mL, 2.000 mmol) and crown 15C5 (20 mL, 0.100 mmol)◦in
DMF (1.0 mL) was dropped. The reaction was stirred at 50 C
3a-Gc¢1. To a solution of Gc1 (96 mg, 0.053 mmol) in THF
(3 mL) were added the phenol 3 (200 mg, 0.690 mmol) and
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overnight. It was then cooled at RT, diluted in a saturated aqueous
solution of NH4Cl and extracted with Et2O (2 ¥ 50 mL). The
organic phase was washed with water (2 ¥ 50 mL) and brine
(50 mL) and then it was dried over Na2SO4, filtered and evaporated
to dryness under reduced pressure to afford 4a as a yellow oil
(173 mg, 65%). The product was used in the next step without
(s, C2), 129.7 (s, C3 and C4), 155.3 (s, C1), 167.4 (d, 2JPC = 2.3 Hz,
CONH) ppm; CI-MS: m/z = 361 [M + NH4]+.
5a-Gc¢1. To a solution of Gc1 (167 mg, 0.091 mmol) in THF
(3 mL) were added the phenol 5 (400 mg, 1.210 mmol) and cesium
carbonate (720 mg, 2.200 mmol). The mixture was stirred for 12 h
at RT, centrifuged, filtered and evaporated. The resulting residue
was purified by silica-gel flash chromatography (DCM/MeOH
80:20 then acetone/MeOH, 95:5) to give 5a-Gc¢1 as a white solid
1
further purification. 31P{ H} NMR (CDCl3, 121.5 MHz): d =
1
26.3 (s, PO3Me2) ppm; H NMR (CDCl3, 300.1 MHz): d = 0.87
(t, 3JHH = 6.6 Hz, 3H, CH2CH3), 1.20–1.40 (m, 2H, CH3CH2), 1.42
(s, 9H, C(CH3)3), 1.60–2.00 (m, 2H, CHCHxHy), 2.85–2.95 (m, 1H,
PCH), 3.71 (d, 3JPH = 4.5 Hz, 3H, OCH3), 3.75 (d, 3JPH = 4.5 Hz,
1
(240 mg, 71%). 31P{ H} NMR (CDCl3, 121.5 MHz): d = 8.4 (s,
1
=
N3P3), 28.2 (s, PO3Me2), 62.9 (s, P S) ppm; H NMR (CDCl3,
300.1 MHz): d = 0.86 (t, 3JHH = 7.2 Hz, 36H, CH2CH3), 1.10–1.40
(m, 24H, CH3CH2), 1.60–2.00 (m, 24H, CHCHxHy), 2.61–2.82 (m,
36H, ArCH2 and PCH), 3.22 (d, 3JPH = 10.2 Hz, 18H, NCH3), 3.43
(br s, 24H, NHCH2), 3.68 (d, 3JPH = 8.7 Hz, 36H, OCH3), 3.72 (d,
3JPH = 8.1 Hz, 36H, OCH3), 6.76–6.98 (br s, 12H, NH), 6.98 (d,
1
3H, OCH3) ppm; 13C{ H} NMR (CDCl3, 75.5 MHz): d = 13.4 (s,
3
CH2CH3), 21.3 (d, JPC = 15.4 Hz, CHCH2), 27.7 (s, C(CH3)3),
2
1
28.7 (d, JPC = 5.4 Hz, CHCH2CH2), 44.6 (d, JPC = 131.8 Hz,
2
2
PCH), 53.5 (d, JPC = 7.0 Hz, OCH3), 53.7 (d, JPC = 6.6 Hz,
OCH3), 82.0 (s, C(CH3)3), 170.7 (d, 3JPC = 4.3 Hz, COO) ppm.
2JHH = 7.8 Hz, C0 H), 7.08 (br s, 48H, C1 H and C1 H), 7.50–7.64
2
2
3
(m, 18H, C0 H and N CH) ppm; 13C NMR (CDCl3, 75.5 MHz):
3
4b. A solution of 20% of TFA in dichloromethane (15 mL)
was dropped on 4a (120 mg, 0.450 mmol) and the reaction mixture
was stirred at RT for 1.5 h. The residue was co-evaporated with
ethyl acetate so that remaining traces of TFA were removed upon
evaporation to dryness. This sequence was repeated until total
disappearance of the signal of TFA in 19F NMR to afford 4b as a
white powder (95 mg, 100%). The product was used in the next step
=
d = 13.7 (s, CH2CH3), 21.3 (d, 3JPC = 15.1 Hz, CHCH2CH2), 29.2
(d, 2JPC = 3.8 Hz, CHCH2), 33.0 (d, 2JPC = 12.1 Hz, NCH3), 34.8
(s, ArCH2), 41.0 (s, NHCH2), 45.8 (d, 1JPC = 130.6 Hz, PCH), 53.0
2
2
(br s, OCH3), 53.4 (br s, OCH3), 121.3 (s, C1 and C0 ), 128.3 (s,
3
3
4
4
3
C0 ), 129.8 (s, C1 ), 132.2 (s, C0 ), 136.1 (s, C1 ), 138.7 (d, JPC
=
2
1
1
=
14.3 Hz, N CH), 149.1 (d, JPC = 6.8 Hz, C1 ), 151.2 (s, C0 ),
1
without further purification. 31P{ H} NMR (CDCl3, 121.5 MHz):
167.5 (d, 2JPC = 2.3 Hz, CONH) ppm.
d = 25.3 (s, PO3Me2) ppm; 1H NMR (CDCl3, 300.1 MHz): d = 0.96
(t, 3JHH = 5.4 Hz, 3H, CH2CH3), 1.30–1.55 (m, 2H, CHCH2CH2),
5b-Gc¢1. To a vigorously stirred solution of 5a-Gc¢1 (100 mg,
0.018 mmol) in a mixture of dry acetonitrile and dicloromethane
(1:1,◦2 mL) bromotrimethylsilane (67 mL, 0.520 mmol) was added
at 0 C. The mixture was stirred for 12 h at RT, and then dried
under reduced pressure. The crude residue was washed twice with
methanol (2 ¥ 10 mL) for 1 h at RT and evaporated to dryness
under reduced pressure. The resulting white solid was washed once
with water, once with methanol and once again with Et2O to afford
1.75–2.05 (m, 2H, CHCHxHy), 3.13 (ddd, 2JPH = 11.1 Hz, 3JHHx
=
8.1 Hz, 3JHHy = 3.0 Hz, 1H, PCH), 3.86 (2 d, 3JPH = 5.4 Hz, 3H,
3
OCH3), 3.89 (d, JPH = 5.4 Hz, 3H, OCH3) ppm, (COOH could
1
not be detected); 13C{ H} NMR (CDCl3, 75.5 MHz): d = 13.3
2
3
(s, CH2CH3), 21.3 (d, JPC = 11.2 Hz, CHCH2), 28.5 (d, JPC
4.0 Hz, CH3CH2), 44.5 (d, 1JPC = 100.8 Hz, PCH), 54.2 (d, 2JPC
=
=
=
2
2
5.3 Hz, OCH3), 54.4 (d, JPC = 5.1 Hz, OCH3), 174.3 (d, JPC
1
5b-Gc¢1 as a white solid (83 mg, 92%). 31P{ H} NMR (CD3OD,
2.9 Hz, COO) ppm.
=
121.5 MHz): d = 9.3 (s, N3P3), 23.2 (s, PO3H2), 62.8 (s, P S)
5. To a vigorously stirred mixture of 4b (147 mg, 0.7 mmol),
HOBt (107 mg, 1.4 mmol) and dry DMF (5 mL) was added
DCC (163 mg, 1.4 mmol) at 0 ◦C. The mixture was stirred at
RT for 1 h, then, a solution of tyramine (197 mg, 1.4 mmol)
ppm; 1H NMR (CD3OD, 300.1 MHz): d = 0.76 (t, 3JHH = 6.9 Hz,
36H, CH2CH3), 1.07–1.24 (m, 24H, CH3CH2), 1.40–1.90 (m, 24H,
CHCHxHy), 2.64 (br s, 36H, ArCH2 and PCH), 3.19–3.29 (m, 42H,
2
2
NCH3 and NHCH2), 7.02 (m, 36H, C0 H and C1 H), 7.15 (d,
◦
in dry DMF (1 mL) was added dropwise at 0 C. The resulting
3JHH = 7.2 Hz, 48H, C1 H), 7.63 (d, 3JHH = 6.3 Hz, 12H, C0 H),
3
3
mixture was stirred at RT for 12 h and filtered. After solvent
removal (lyophilisation), the resulting dark red residue was diluted
in chloroform (50 mL) and washed with HClaq (0.1 M, 3 ¥
10 mL) and brine (10 mL). The organic phase was separated,
dried over Na2SO4, filtered and concentrated to dryness under
reduced pressure. The residue was purified by silica-gel flash
chromatography (DCM/MeOH, 95:5 then acetone) to afford 5 as
7.73–8.01 (m, 18H, N CH and NH) ppm; 13C NMR (DMSO-
=
3
d6, 75.5 MHz): d = 14.1 (s, CH2CH3), 21.3 (d, JPC = 15.9 Hz,
2
CH3CH2), 29.3 (br s, CHCH2), 33.4 (d, JPC = 11.3 Hz, NCH3),
34.8 (s, ArCH2), 41.2 (s, NH-CH2), 46.9 (d, 1JPC = 129.1 Hz, PCH),
2
2
3
3
121.1 (s, C1 ), 121.4 (s, C0 ), 128.7 (s, C0 ), 130.4 (s, C1 ), 132.7 (s,
4
4
2
1
=
C0 ), 137.2 (s, C1 ), 140.4 (s, N CH), 148.8 (d, JPC = 6.8 Hz, C1 ),
150.9 (s, C0 ), 169.1 (d, 2JPC = 4.5 Hz, CONH) ppm.
1
1
a white solid (122 mg, 53%). 31P{ H} NMR (CDCl3, 121.5 MHz):
d = 28.3 ppm (s, PO3Me2) ppm; 1H NMR (CDCl3, 300.1 MHz):
5c-Gc¢1. The sodium monosalt form was obtained by adding
aqueous sodium hydroxide (0.1023 N, 2.58 mL) to a suspension of
5b-Gc¢1 (100 mg, 0.021 mmol) in water (3 mL) at 0 ◦C. The solution
was filtered on micropore (1.2 mm) and lyophilized to give 5c-Gc¢1
3
d = 0.91 (t, JHH= 7.2 Hz, 3H, CH2CH3), 1.20–1.50 (m, 2H,
CH3CH2), 1.70–1.95 (m, 2H, CHCHxHy), 2.66–2.78 (m, 2H, PCH
3
3
and ArCH2), 3.51 (q, JHH = JHH = 6.6 Hz, 2H, NHCH2), 3.75
3
3
1
(d, JPH = 10.5 Hz, 3H, OCH3), 3.76 (d, JPH= 10.5 Hz, 3H,
as a white powder (110 mg, 91%). 31P{ H} NMR (D2O/CD3CN,
3
OCH3), 6.30 (br s, 1H, NH), 6.58 (s, 1H, OH), 6.78 (d, JHH
=
3:1, 121.5 MHz): d = 9.9 (s, N3P3), 17.4 (s, PO3HNa), 63.5 (s,
8.1 Hz, 2H, C2H), 7.05 (d, 3JHH = 8.4 Hz, 2H, C3H) ppm; 13C{ H}
P S) ppm; H NMR (D2O/CD3CN, 3:1, 300.1 MHz): d = 1.09
1
1
=
NMR (CDCl3, 75.5 MHz): d = 13.7 (s, CH2CH3), 21.3 (d, 3JPC
=
(t, 3JHH = 6.0 Hz, 36H, CH2CH3), 1.35–1.61 (m, 24H, CH3CH2),
14.3 Hz, CHCH2CH2), 29.2 (d, 2JPC = 4.5 Hz, CHCH2CH2), 34.4
(s, ArCH2), 41.3 (s, NHCH2), 45.9 (d, 1JPC = 130.6 Hz, PCH), 53.3
1.85–2.08 (m, 24H, CHCHxHy), 2.59–2.71 (m, 12H, PCH), 3.01–
3
3.12 (m, 24H, ArCH2), 3.55 (d, JPH = 12.0 Hz, 18H, NCH3),
2
2
2
(d, JPC = 7.6 Hz, OCH3), 53.6 (d, JPC = 6.8 Hz, OCH3), 115.5
3496 | Org. Biomol. Chem., 2009, 7, 3491–3498
3.65–3.76 (m, 24H, NHCH2), 7.20 (d, 3JHH = 9.0 Hz, 12H, C0 H),
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7.37 (d, 3JHH = 6.0 Hz, 24H, C1 H), 7.49 (d, 3JHH = 9.0 Hz, 24H,
10 mL) and brine (10 mL). The organic phase was separated,
dried over Na2SO4, filtered and concentrated to dryness under
reduced pressure. The residue was purified by silica-gel flash
chromatography (DCM/MeOH, 95:5 than acetone) to afford 2c as
2
3
3
3
=
C1 H), 7.90 (d, JHH = 6.0 Hz, 12H, C0 H), 8.09 (s, 6H, N CH)
ppm; 13C NMR (D2O/CD3CN, 3:1, 100.6 MHz): d = 13.3 (s,
2
CH2CH3), 21.6 (s, CHCH2), 31.3 (s, CH3CH2), 32.8 (d, JPC
22.4 Hz, NCH3), 34.0 (s, ArCH2), 40.3 (s, NHCH2), 49.9 (d, 1JPC
=
=
1
a white solid (185 mg, 62%). 31P{ H} NMR (CDCl3, 121.5 MHz):
2
2
3
121.1 Hz, PCH), 120.8 (br s, C0 and C1 ), 128.4 (s, C0 ), 130.0 (s,
d = 28.4 (PO3Me2) ppm; 1H NMR (CDCl3, 300.1 MHz): d = 0.89
(t, 3JHH = 6.6 Hz, 3H, CH2CH3), 1.26 (br s, 16H, CH2), 1.70–1.90
(m, 2H, CHCHxHy), 2.68 (ddd, 2JPH = 14.7 Hz, 3JHHx = 10.5 Hz,
3
4
1
1
=
C1 ), 136.5 (s, C1 ), 139.5 (s, N CH), 148.5 (s, C1 ), 150.8 (s, C0 ),
4
174.0 (s, CONH) ppm (C0 could not be detected).
3
3JHHy = 4.2 Hz, 1H, PCH), 2.75 (t, JHH = 6.9 Hz, 2H, ArCH2),
6a. To a suspension of NaH (24 mg, 1 mmol) in DMF
(2.5 mL) at 0 ◦C, we added dropwise a solution of tert-butyl-P,P-
dimethyphosphonoacetate (224 mg, 1 mmol) in DMF (2 mL). The
mixture was stirred during 1 h. Then, a solution of 1-iododecane
(0.426 mL, 2 mmol) and crown 15C5 (20 mL, 0.1 mmol) in DMF
(1.0 mL) was dropped. The reaction was stirred at 50 ◦C overnight.
It was then cooled at RT, diluted in a saturated aqueous solution
of NH4Cl and extracted with Et2O (2 ¥ 50 mL). The organic phase
was washed with water (2 ¥ 50 mL) and brine (50 mL), dried
over Na2SO4, filtered and evaporated to dryness under reduced
pressure to afford 6a as a yellow oil (219 mg, 60%). The product
3
3
3
3.51 (q, JHH = JHH = 6.6 Hz, 2H, NHCH2), 3.74 (d, JPH
=
11.1 Hz, 3H, OCH3), 3.77 (3JPH = 11.1 Hz, 3H, OCH3), 6.32 (m,
1H, NH), 6.78 (d, 3JHH = 8.4 Hz, 2H, C2H), 7.04 (d, 3JHH = 8.4 Hz,
2H, C3H) ppm; 13C{ H} NMR (CDCl3, 75.5 MHz): d = 14.1 (s,
1
2
CH2CH3), 22.7 (s, CH2), 27.2 (d, JPC = 5,3 Hz, CH2), 28.1 (d,
3JPC = 15.1 Hz, CH2), 29.2–31.9 (m, CH2), 34.7 (s, ArCH2), 41.3
(s, NHCH2) 46.1 (d, 1JPC = 130.6 Hz, PCH), 53.3 (d, 2JPC = 6.8 Hz,
OCH3), 53.4 (d, 2JPC = 6.8 Hz, OCH3), 115.5 (s, C2), 129.7 (s, C3),
130.0 (s, C4), 155.0 (s, C1), 167.3 (d, 2JPC = 2.3 Hz, CONH) ppm.
CI-MS: m/z = 445 [M + NH4]+.
1
was used in the next step without further purification. 31P{ H}
7a-Gc¢1. To a solution of Gc1 (150 mg, 0.081 mmol) in THF
(3 mL) were added the phenol 7 (200 mg, 0.69 mmol) and cesium
carbonate (457 mg, 1.06 mmol). The mixture was stirred for 12 h
at RT, centrifuged, filtered and evaporated. The resulting residue
was purified by silica-gel flash chromatography (DCM/MeOH,
80:20 then acetone/MeOH, 95:5) to give 7a-Gc¢1 as a white solid
NMR (CDCl3, 81.0 MHz): d = 29.7 (s, PO3Me2) ppm; 1H NMR
(CDCl3, 200.1 MHz): d = 0.80 (t, 3JHH = 7.1 Hz, 3H, CH2CH3),
1.18 (br s, 16H, CH2), 1.60–2.00 (m, 2H, CHCHxHy), 2.78 (ddd,
2JPH = 22.0 Hz, 3JHHx = 11.0 Hz, 3JHHy = 4.0 Hz, 1H, PCH), 3.68
(d, 3JPH = 10.0 Hz, 3H, OCH3), 3.74 (d, 3JPH = 10.0 Hz, 3H, OCH3)
1
(407 mg, 77%). 31P{ H} NMR (CDCl3, 121.5 MHz): d = 8.4 (s,
1
ppm; 13C{ H} NMR (CDCl3, 50.3 MHz): d = 14.1 (s, CH2CH3),
1
2
=
N3P3), 28.1 (s, PO3Me2), 63.0 ppm (s, P S); H NMR (CDCl3,
22.6 (s, CH2), 26.9 (d, JPC = 4.5 Hz, CH2), 27.9 (s, C(CH3)3),
300.1 MHz): d = 0.87 (t, 3JHH = 6.6 Hz, 36H, CH2CH3), 1.25 (br
28.3 (d, 3JPC = 15.5 Hz, CH2), 29.0–31.9 (m, CH2), 46.1 (d, 1JPC
=
=
2
2
s, 192H, CH2), 1.60–2.10 (m, 24H, CHCHxHy), 2.73–2.79 (m, 36H,
127.0 Hz, PCH), 53.1 (d, JPC = 7.5 Hz, OCH3), 53.1 (d, JPC
3
7.5 Hz, OCH3), 81.7 (s, C(CH3)3), 168.1 (d, 3JPC = 2.5 Hz, COO)
ArCH2 and PCH), 3.24 (d, JPH = 10.2 Hz, 18H, NCH3), 3.35–
3
3.52 (m, 24H, NHCH2), 3.71 (d, JPH = 10.8 Hz, 18H, OCH3),
ppm.
3
3.75 (d, JPH = 9.6 Hz, 36H, OCH3), 6.90 (br s, 12H, NH), 7.1
(d, 3JHH = 7.1 Hz, 12H, C0 H), 7.10 (br s, 48 H, C1 H and C1 H),
2
2
3
6b. A solution of 20% of TFA in dichloromethane (15 mL)
was dropped on 6a (164 mg, 0.450 mmol) and the reaction mixture
was stirred at RT for 1.5 h. The residue was co-evaporated with
ethyl acetate so that remaining traces of TFA were removed upon
evaporation to dryness. This sequence was repeated until total
disappearance of the signal of TFA in 19F NMR to afford 6b as a
white powder (75 mg, 100%). The product was used in the next step
7.64 (m, 18H, C0 H and N CH) ppm; 13C{ H} NMR (CDCl3,
3
1
=
75.5 MHz): d = 14.1 (s, CH2CH3), 22.7 (s, CH2), 27.1 (s, CH2),
28.2 (d, 3JPC = 14.3 Hz, CH2), 29.2–31.8 (s, CH2), 32.9 (d, 2JPC
=
=
11.9 Hz, NCH3), 34.9 (s, ArCH2), 41.1 (s, NHCH2), 45.9 (d, 1JPC
129.4 Hz, PCH), 53.2 (d, 2JPC = 38.6 Hz, OCH3), 121.81 (s, C12 and
2
3
4
4
3
C0 ), 128.2 (s, C1 ), 132.1 (s, C0 ), 136.1 (s, C1 ), 138.7 (d, JPC
=
1
without further purification. 31P{ H} NMR (CDCl3, 81.0 MHz):
2
1
1
=
14.0 Hz, N CH), 149.1 (d, JPC = 27.0 Hz, C1 ), 151.2 (s, C0 ),
1
d = 29.9 (s, PO3Me2) ppm; H NMR (CDCl3, 200.1 MHz): d =
167.6 (s, CONH) ppm.
3
0.86 (t, JHH = 7.1 Hz, 3H, CH2CH3), 1.23 (br s, 16H, CH2),
7b-Gc¢1. To a vigorously stirred solution of 7a-Gc¢1 (100 mg,
0.018 mmol) in a mixture of dry acetonitrile and dicloromethane
(1:1, 2 mL) bromotrimethylsilane (67 mL, 0.520 mmol) was added
at 0 ◦C. The mixture was stirred at RT for 12 h and then evaporated
to dryness under reduced pressure. The crude residue was washed
twice with methanol (10 mL) for 1 h at RT and evaporated to
dryness under reduced pressure. The resulting white solid was
washed with water, methanol and with Et2O to afford 2f as a white
1.60–2.05 (m, 2H, CHCHxHy), 2.98 (ddd, 2JPH = 24.0 Hz, 3JHHx
=
12.0 Hz, 3JHHy = 3.4 Hz, 1H, PCH), 3.79 (d, 3JPH = 10.0 Hz, 3H,
OCH3), 3.83 (d, 3JPH = 10.0 Hz, 3H, OCH3), 9.13 (s, 1H, COOH)
1
ppm; 13C{ H} NMR (CDCl3, 50.3 MHz): d = 14.1 (s, CH2CH3),
22.6 (s, CH2), 26.9 (d, 2JPC = 5.1 Hz, CH2), 28.3 (d, 3JPC = 19.5 Hz,
1
CH2), 29.0–31.9 (m, CH2), 45.1 (d, JPC = 130.0 Hz, PCH), 53.4
2
2
(d, JPC = 7.0 Hz, OCH3), 53.1 (d, JPC = 7.0 Hz, OCH3), 171.3
(d, 3JPC = 2.0 Hz, COO) ppm.
1
solid (83 mg, 92%). 31P{ H} NMR (CD3OD, 161.9 MHz): d = 9.3
1
=
7. To a vigorously stirred mixture of 6b (215 mg, 0.7 mmol),
HOBt (107 mg, 1.4 mmol) and dry DMF (5 mL) was added
DCC (163 mg, 1.4 mmol) at 0 ◦C. The mixture was stirred at
RT for 1 h, then, a solution of tyramine (197 mg, 1.4 mmol)
(s, N3P3), 22.8 (s, PO3H2), 62.8 (s, P S) ppm; H NMR (CD3OD,
400.1 MHz): d = 0.94 (br s, 36H, CH2CH3), 1.33 (br s, 192H,
CH2), 1.70–2.10 (m, 24H, CHCHxHy), 2.82 (br s, 36H, ArCH2,
3
PCH), 3.29 (d, JPH = 7.2 Hz, 18H, NCH3), 3.52 (br s, 24H,
◦
2
2
3
in dry DMF (1 mL) was added dropwise at 0 C. The resulting
NHCH2), 6.95 (br s, 12H, C0 H), 7.17 (m, 48H, C1 H and C1 H),
3
13
1
=
mixture was stirred at RT for 12 h and filtered. After solvent
removal (lyophilisation), the resulting dark red residue was diluted
in chloroform (50 mL) and was washed with HClaq (0.1 M, 3 ¥
7.70 (m, 12H, C0 H), 7.8 (s, 6H, N CH) ppm; C{ H} NMR
(CD3OD, 100.6 MHz): d = 13.3 (s, CH2CH3), 22.4 (s, CH2), 26.9
(s, CH2), 28.0 (d, 2JPC = 16.2 Hz, CH2), 29.0–31.8 (m, CH2), 32.6 (d,
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2JPC = 10.8, NCH3), 34.5 (s, ArCH2), 40.8 (s, NHCH2), 46.5 (d,
Notes and references
2
2
3
1JPC = 26.8 Hz, PCH), 121.1 (s, C1 ), 121.3 (s, C0 ), 128.2 (s, C0 ),
1 E. A. Berger, P. M. Murphy and J. M. Farber, Annu. Rev. Immunol.,
1999, 17, 657.
2 T. Matthews, M. Salgo, M. Greenberg, J. Chung, R. DeMasi and D.
Bolognesi, Nature Rev. Drug Discov., 2004, 3, 215–225.
3 M. Moulard, H. Lortat-Jacob, I. Mondor, G. Roca, R. Wyatt, J.
Sodroski, L. Zhao, W. Olson, P. D. Kwong and Q. J. Sattentau, J.
Virol., 2000, 74, 1948–1960.
4 J. Weber, K. Desai and J. Darbyshire, PLoS Med., 2005, 2, e142.
5 A. Mulder, J. Huskens and D. N. Reinhoudt, Org. Biomol. Chem., 2004,
2, 3409–3424.
129.7 (s, C1 ), 132.7 (s, C0 ), 136.5 (s, C1 ), 138.9 (d, 3JPC = 12.7 Hz,
3
4
4
2
1
1
=
N CH), 149.2 (d, JPC = 7.1 Hz, C1 ), 151.0 (s, C0 ), 170.2 (s,
CONH) ppm.
7c-Gc¢1. The sodium monosalt form was obtained by adding
aqueous sodium hydroxide (0.1023 N, 2.58 mL) to a suspension
of 7b-Gc¢1 (100 mg, 0.017 mmol) in water (3 mL) at 0 ◦C. The
solution was filtered on micropore (1.2 mm) and lyophilized to
6 E. Buhleier, F. Wehner and F. Vo¨gtle, Synthesis, 1978, 78, 155–158.
7 O. Rolland, C. O. Turrin, A. M. Caminade and J. P. Majoral, New J.
Chem., 2009, DOI: 10.1039/b901054h.
8 T. D. McCarthy, P. Karellas, S. A. Henderson, M. Giannis, D. F.
O’Keefe, G. Heery, J. R. A. Paull, B. R. Matthews and G. Holan,
Mol. Pharm., 2005, 2, 312–318.
afford 7c-Gc¢1 as a white powder (110 mg, 95%). d = 9.1 (s, N3P3),
1
=
16.5 (s, PO3HNa), 64.0 (s, P S) ppm; H NMR (D2O/CD3CN,
3
3:1, 500.3 MHz): d = 0.92 (t, JHH = 6.0 Hz, 36H, CH2CH3),
1.13–1.61 (m, 192H, CH2), 1.90 (br s, 24H, CHCH2), 2.62 (br s,
12H, PCH), 3.01 (m, 24H, ArCH2), 3.45–3.80 (m, 42H, NCH3
9 A. R. Borges and C. L. Schengrund, Current Drug Targets - Infectious
Disorders, 2005, 5, 247–254.
3
2
and NHCH2), 7.11 (d, JHH = 7.0 Hz, 12H, C0 H), 7.30 (br s,
24H, C1 H), 7.42 (d, 3JHH = 8.0 Hz, 24H, C1 H), 7.83 (d, 3JHH
=
2
3
10 M. Mammen, S. K. Choi and G. M. Whitesides, Angew. Chem., Int.
Ed., 1998, 37, 2754–2794.
8.0 Hz, 12H, C0 H), 7.95 (s, 6H, N CH) ppm; 13C{ H} NMR
(D2O/CD3CN, 3:1, 125.8 MHz): d = 14.0 (s, CH2CH3), 22.7 (s,
CH2), 28.8 (d, 2JPC = 13.9 Hz, CH2), 29.2–29.8 (m, CH2), 33.0 (d,
3JPC = 7.3 Hz, NCH3), 34.4 (s, CH2), 40.5 (s, NHCH2), 50.2 (d,
3
1
=
11 P. I. Kitov and D. R. Bundle, J. Am. Chem. Soc., 2003, 125, 16271–
16284.
12 I. Rico-Lattes, M. F. Gouzy, C. Andre´-Barre`s, B. Guidetti and A.
Lattes, New J. Chem., 1998, 451–457.
13 J. Fantini, D. Hammache, O. Delezay, N. Yahi, C. Andre´-Barre`s, I.
Rico-Lattes and A. Lattes, J. Biol. Chem., 1997, 272, 7245–7252.
14 M. Blanzat, C. O. Turrin, A. M. Aubertin, C. Couturier-Vidal, A. M.
Caminade, J. P. Majoral, I. Rico-Lattes and A. Lattes, ChemBioChem,
2005, 6, 2207–2213.
2JPC = 115.9 Hz, PCH), 121.3 (s, C02 and C1 ), 128.4 (s, C4 ), 141.6
2
1
4
1
1
(s, C0 ), 149.0 (s, C1 ), 151.3 (s, C0 ), 175.1 (s, CONH) ppm.
Antiviral assays
15 A. M. Caminade, C. O. Turrin, R. Laurent, A. Maraval and J. P.
Majoral, Curr. Org. Chem., 2006, 10, 2333–2355.
16 L. Griffe, M. Poupot, P. Marchand, A. Maraval, C. O. Turrin, O.
Rolland, P. Me´tivier, G. Bacquet, J. J. Fournie´, A. M. Caminade, R.
Poupot and J. P. Majoral, Angew. Chem., Int. Ed., 2007, 46, 2523–
2526.
CEM-SS and MT4 cells were cultured in RPMI medium supple-
mented with 10% fetal calf serum (heated at 56 C for 30 min).
◦
CEM-SS cells were infected with HIV-1 LAI and the production
of virus was evaluated after five days, by measuring the reverse
transcriptase (RT) which expresses the presence of HIV in the
supernatant culture medium. RT inhibition percentage, providing
IC50 values (concentration of drug at which virus production
is inhibited by 50%), was determined in comparison with the
non-treated cells. MT4 cells were infected with HIV-1 IIIB. The
determination of the antiviral activity of the tested compounds
was based on a reduction of HIV-1-induced pathogenicity, the
metabolic activity of the cells being measured by the ability of
mitochondrial dehydrogenases to reduce 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan. The
produced quantity of formazan (characterized by OD540) is
directly proportional to the number of living cells and to the IC50.
17 J. C. Blais, C. O. Turrin, A. M. Caminade and J. P. Majoral, Anal.
Chem., 2000, 72, 5097–5105.
18 O. Rolland, L. Griffe, M. Poupot, A. Maraval, A. Ouali, Y. Coppel, J.
J. Fournie´, G. Bacquet, C. O. Turrin, A. M. Caminade, J. P. Majoral
and R. Poupot, Chem.–Eur. J., 2008, 14, 4836–4850.
19 M. Blanzat, C. O. Turrin, E. Perez, I. Rico-Lattes, A. M. Caminade
and J. P. Majoral, Chem. Commun., 2002, 1864–1865.
20 J. Leclaire, R. Dagiral, S. Fery-Forgues, Y. Coppel, B. Donnadieu, A.
M. Caminade and J. P. Majoral, J. Am. Chem. Soc, 2005, 127, 15762–
15770.
21 C. Moog, A. Wick, P. L. Ber, A. Kirn and A.-M. Aubertin, Antivir.
Res., 1994, 24, 275.
22 R. Pauwels, J. Balzarini, M. Baba, R. Snoeck, D. Schols, P.
Herdewijn, J. Desmyter and E. D. Clercq, J. Virol. Methods, 1988, 20,
309.
23 V. Peytou, R. Condom, N. Patino, R. Guedj, A.-M. Aubertin, N. Gelus,
C. Bailly, R. Terreux and D. Cabrol-Bass, J. Med. Chem., 1999, 42,
4042–4053.
Acknowledgements
24 D. D. Perrin and W. L. F. Armarego, Purification of Laboratory
Chemicals, Third Edition, Oxford, 1988.
The European Community is acknowledged for funding support
(A. P.-A., Marie Curie EST “Nanotool” and G. S., FSE-Fonds
Social Europe´en). The ANRS is acknowledged for molecular
screening.
25 N. Launay, A. M. Caminade, R. Lahana and J. P. Majoral, Angew.
Chem., Int. Ed. Engl., 1994, 33, 1589–1592.
26 N. Launay, A. M. Caminade and J. P. Majoral, J. Organomet. Chem.,
1997, 529, 51–58.
3498 | Org. Biomol. Chem., 2009, 7, 3491–3498
This journal is
The Royal Society of Chemistry 2009
©