U.S. patent application number 12/745670 was filed with the patent office on 2011-03-10 for method for preparing nanoparticles based on functional amphiphilic molecules or macromolecules, and the use thereof.
Invention is credited to Philippe Barthelemy, Florence Bouyer, Nathalie Campins, Michel Camplo, Bruno Chauffert.
Application Number | 20110059180 12/745670 |
Document ID | / |
Family ID | 39638675 |
Filed Date | 2011-03-10 |
United States Patent
Application |
20110059180 |
Kind Code |
A1 |
Barthelemy; Philippe ; et
al. |
March 10, 2011 |
Method for Preparing Nanoparticles Based on Functional Amphiphilic
Molecules or Macromolecules, and the Use Thereof
Abstract
The invention relates to a method for preparing nanoparticles
based on functional amphiphilic molecules or macromolecules,
optionally in the presence of at least one colipide, enabling the
encapsulation of therapeutic agents, especially anti-tumoral
agents, and the use thereof for the transport and vectorisation of
therapeutic agents, especially anti-tumoral agents.
Inventors: |
Barthelemy; Philippe;
(Merignac, FR) ; Camplo; Michel; (Marseille,
FR) ; Campins; Nathalie; (Valliguieres, FR) ;
Chauffert; Bruno; (Dijon, FR) ; Bouyer; Florence;
(Chenove, FR) |
Family ID: |
39638675 |
Appl. No.: |
12/745670 |
Filed: |
November 28, 2008 |
PCT Filed: |
November 28, 2008 |
PCT NO: |
PCT/FR08/01661 |
371 Date: |
November 24, 2010 |
Current U.S.
Class: |
424/490 ;
424/649; 514/492; 536/26.8; 536/27.23; 536/28.54; 977/773;
977/906 |
Current CPC
Class: |
A61K 31/706 20130101;
A61K 9/5123 20130101; A61K 47/26 20130101; C07H 19/06 20130101;
C07H 19/10 20130101; A61K 31/282 20130101; A61K 31/7072 20130101;
A61K 33/24 20130101; A61K 31/708 20130101; C07H 19/16 20130101;
A61P 35/00 20180101; A61K 9/4816 20130101; A61K 31/28 20130101;
A61K 31/7052 20130101 |
Class at
Publication: |
424/490 ;
424/649; 514/492; 536/26.8; 536/28.54; 536/27.23; 977/773;
977/906 |
International
Class: |
A61K 9/51 20060101
A61K009/51; A61K 33/24 20060101 A61K033/24; A61K 31/282 20060101
A61K031/282; C07H 19/10 20060101 C07H019/10; C07H 19/06 20060101
C07H019/06; C07H 19/16 20060101 C07H019/16; A61P 35/00 20060101
A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 30, 2007 |
FR |
07/08399 |
Claims
1. Process for encapsulating a therapeutic agent comprising the
stages consisting of: a) preparing a mixture of at least one
functional amphiphilic compound of formula (I) ##STR00027## in
which X represents an oxygen or sulphur atom or a methylene group,
B represents a purine or pyrimidine base such as uracil, adenine,
guanine, cytosine, thymine, hypoxanthine, or their derivatives, or
also a non-natural mono- or bi-cyclic heterocyclic base each ring
of which comprises 4 to 7 members, optionally substituted; L.sub.1
and L.sub.2, identical or different, represent hydrogen, an
oxycarbonyl --O--C(O)-- group, a thiocarbamate --O--C(S)--NH--
group, a carbonate --O--C(O)--O-- group, a carbamate
--O--C(O)--NH-- group, an oxygen atom, a phosphate group, a
phosphonate group or a heteroaryl group comprising 1 to 4 nitrogen
atoms, unsubstituted or substituted by a linear or branched,
saturated or unsaturated C.sub.2-C.sub.30 hydrocarbon chain, or
also, L.sub.1 and L.sub.2, together, form a ketal group of formula
##STR00028## or also L.sub.1 or L.sub.2 represents hydrogen, and
the other represents a hydroxy group or a heteroaryl group
comprising 1 to 4 nitrogen atoms, unsubstituted or substituted by a
linear or branched C.sub.2-C.sub.30 alkyl chain; R.sub.1 and
R.sub.2, identical or different, represent a linear or branched
C.sub.2-C.sub.30 hydrocarbon chain, preferably C.sub.6-C.sub.25, in
particular C.sub.8-C.sub.25, saturated or partially unsaturated,
optionally completely or partially fluorinated, unsubstituted or
substituted on the carbon at the end of the chain by a fluorine
atom or by a benzyl or naphthyl ester or ether, or a diacyl chain
in which each acyl chain is C.sub.2-C.sub.30, or a diacylglycerol,
sphingosine or ceramide group, or when L.sub.1 or L.sub.2
represents hydrogen, and the other represents a hydroxy group or a
heteroaryl group comprising 1 to 4 nitrogen atoms, R.sub.1 and
R.sub.2 do not exist; R.sub.3 represents a hydroxy, amino,
phosphate, phosphonate, phosphatidylcholine, O-alkyl
phosphatidylcholine, thiophosphate, phosphonium, NH.sub.2--R.sub.4,
NHR.sub.4R.sub.5 or NR.sub.4R.sub.5R.sub.6 group in which R.sub.4,
R.sub.5 and R.sub.6, identical or different, represent a hydrogen
atom or a linear or branched C.sub.1-C.sub.5 alkyl or hydroxyalkyl
chain, or a linear or branched C.sub.2-C.sub.30 alkyl chain
optionally substituted by a hydroxy group, or a cyclodextrin
radical, or a ##STR00029## radical, in which V represents an --O--,
--S--, or --NH-- bond, R.sub.7 represents H or CH.sub.3, and n=1 to
500, or a --(CH.sub.2).sub.n--V--R.sub.8 group, in which R.sub.8
represents a C.sub.2-C.sub.30 alkyl, and n=1 to 500, or a
heteroaryl group containing 1 to 4 nitrogen atoms, unsubstituted or
substituted by a C.sub.2-C.sub.30 alkyl, or by a
(CH.sub.2).sub.m--O--(CH.sub.2).sub.p--R.sub.9 group in which m=1
to 6 and p=0 to 10 and R.sub.9 represents a cyclic ketal group
containing 5 to 7 carbon atoms, unsubstituted or substituted by at
least one linear or branched C.sub.2-C.sub.30 alkyl or by a sterol
radical, or also R.sub.3 is bound by a covalent bond to another
substituent R.sub.3, identical or different, of another compound of
formula (I), identical or different, in order to form a compound of
formula (I) in the form of a dimer, and a therapeutic agent,
preferably an anti-neoplastic agent, b) subjecting said mixture to
repeated heating and freezing cycles, in order to obtain
nanoparticles containing said therapeutic agent, and c) recovering
the nanoparticles containing said therapeutic agent obtained in
this way.
2. Process according to claim 1, characterized in that, in formula
(I), X represents oxygen.
3. Process claim 1, wherein, in formula (I), B represents thymine
or adenine.
4. Process according to claim 1, wherein, in formula (I). X and B
are as defined in claim 1; L.sub.1 represents a phosphate group,
L.sub.2 represents hydrogen, R.sub.1 represents a C.sub.2-C.sub.30
alkyl group or a diacyl group in which each acyl chain is
C.sub.2-C.sub.30 and R.sub.3 is a hydroxy group; or L.sub.1 and
L.sub.2 represent an oxygen atom, R.sub.1 and R.sub.2 represent
hydrogen and R.sub.3 represents a triazole, tetrazole, pyrazole or
imidazole group substituted by a C.sub.2-C.sub.30 alkyl group, or
L.sub.1 represents a triazole, tetrazole, pyrazole or imidazole
group, L.sub.2 represents hydrogen, R.sub.1 represents a
C.sub.2-C.sub.30 alkyl group, and R.sub.3 is a hydroxy group, or
L.sub.1 represents a hydroxy group, L.sub.2 represents hydrogen and
R.sub.3 is a triazole group substituted by a C.sub.2-C.sub.30 alkyl
chain, optionally substituted by a hydroxy group, or R.sub.3 is a
group or a --(CH.sub.2)n-V--R.sub.8 group, in which V represents
--O-- or --NH-- and R.sub.8 represents a C.sub.2-C.sub.30 alkyl, or
L.sub.1 represents a hydroxy group, L.sub.2 represents hydrogen and
R.sub.3 is a triazole group substituted by a
(CH.sub.2).sub.m--O--(CH.sub.2).sub.p--R.sub.9 group in which m=1
to 6 and p=0 to 10 and R.sub.9 represents a cyclic ketal group
containing 5 to 7 carbon atoms, unsubstituted or substituted by at
least one linear or branched C.sub.2-C.sub.30 alkyl group or by a
sterol radical, or L.sub.1 represents a hydroxy group, L.sub.2
represents hydrogen and R.sub.3 is a triazole group substituted by
a (CH.sub.2).sub.m--O--(CH.sub.2).sub.p--R.sub.9 group in which m=1
to 6 and p=0 to 10 bound by a covalent bond to another identical
substituent R.sub.3, of another identical compound of formula (I),
in order to form a compound of formula (I) in the form of a dimer,
or L.sub.1 represents a phosphate group, L.sub.2 represents
hydrogen, R.sub.1 represents a diacyl chain in which each diacyl
chain is C.sub.2-C.sub.30, and R.sub.3 is a hydroxy group.
5. Process according to claim 1, wherein in formula (I): X, B,
L.sub.2, R.sub.1 and R.sub.2 are as defined in formula (I), and the
substituent L.sub.1 or the substituent R.sub.3 is constituted by or
comprises a triazole group, unsubstituted or substituted.
6. Process according to claim 1, further comprising the steps
consisting of: putting the compound of formula (I) in solution in
an organic solvent in order to form a lipid mixture, evaporating
said mixture in order to form a film, putting the desired quantity
of therapeutic agent, preferably an anti-neoplastic agent in
solution in distilled water, rehydrating the lipid film in the
solution of therapeutic agent, preferably an anti-neoplastic agent,
subjecting the mixture obtained in this way to a heating and
cooling cycle carried out 1 to 10 times.
7. Process according to claim 1, further comprising the step of
putting the compound of formula (I) in solution in an organic
solvent in the presence of a co-lipid.
8. Process according to claim 1, further comprising the step of
putting the compound of formula (I) t in solution in an organic
solvent in the presence of a co-lipid, wherein the co-lipid is
chosen from dioleylphosphatidylcholine (DOPC) and dioleyl
phosphatidyluridinephosphatidylcholine (DOUPC).
9. Process according to claim 1, wherein the therapeutic agent is
chosen from the platinum complexes or ruthenium capable of binding
to platinum complexes, or also the inorganic complexes without
platinum based on ruthenium II or III, titanium, gallium, cobalt,
iron or gold.
10. Process according to claim 1, wherein the therapeutic agent is
chosen from cis-platin, carboplatin, oxaliplatin, nedaplatin and
lobaplatin.
11. Nanoparticles capable of being obtained by the process
according to claim 1.
12. Solid nanoparticles, wherein they are constituted by a core
rich in therapeutic agent, preferably an anti-neoplastic agent,
surrounded by one or more lipid layers constituted by the compound
of formula (I), ##STR00030## in which X represents an oxygen or
sulphur atom, or a methylene group, B represents a purine or
pyrimidine base such as uracil, adenine, guanine, cytosine,
thymine, hypoxanthine, or their derivatives, or also a non-natural
mono- or bi-cyclic heterocyclic base each ring of which comprises 4
to 7 members, optionally substituted; L.sub.1 and L.sub.2,
identical or different, represent hydrogen, an oxycarbonyl
--O--C(O)-- group, a thiocarbamate --O--C(S)--NH-- group, a
carbonate --O--C(O)--O-- group, a carbamate --O--C(O)--NH-- group,
an oxygen atom, a phosphate group, a phosphonate group or a
heteroaryl group comprising 1 to 4 nitrogen atoms, unsubstituted or
substituted by a linear or branched, saturated or unsaturated,
C.sub.2-C.sub.30 hydrocarbon chain, or also, L.sub.1 and L.sub.2,
together, form a ketal group of formula ##STR00031## or also
L.sub.1 or L.sub.2 represents hydrogen, and the other represents a
hydroxy group or a heteroaryl group comprising 1 to 4 nitrogen
atoms, unsubstituted or substituted by a linear or branched
C.sub.2-C.sub.30 alkyl chain; R.sub.1 and R.sub.2, identical or
different, represent a linear or branched C.sub.2-C.sub.30
hydrocarbon chain, preferably C.sub.6-C.sub.25, in particular
C.sub.8-C.sub.25, saturated or partially unsaturated, optionally
completely or partially fluorinated, unsubstituted or substituted
on the carbon at the end of the chain by a fluorine atom or by a
benzyl or naphthyl ester or ether, or a diacyl chain in which each
acyl chain is C.sub.2-C.sub.30, or a diacylglycerol, sphingosine or
ceramide group, or when L.sub.1 or L.sub.2 represents hydrogen, and
the other represents a hydroxy group or a heteroaryl group
comprising 1 to 4 nitrogen atoms, R.sub.1 and R.sub.2 do not exist;
R.sub.3 represents a hydroxy, amino, phosphate, phosphonate,
phosphatidylcholine, O-alkyl phosphatidylcholine, thiophosphate,
phosphonium, NH.sub.2--R.sub.4, NHR.sub.4R.sub.5 or
NR.sub.4R.sub.5R.sub.6 group in which R.sub.4, R.sub.5 and R.sub.6,
identical or different, represent a hydrogen atom or a linear or
branched C.sub.1-C.sub.5 alkyl or hydroxyalkyl chain, or a linear
or branched C.sub.2-C.sub.30 alkyl chain optionally substituted by
a hydroxy group, or a cyclodextrin radical, or a ##STR00032##
radical, in which V represents an --O--, --S--, or --NH-- bond,
R.sub.7 represents H or CH.sub.3, and n=1 to 500, or a
--(CH.sub.2).sub.n--V--R.sub.8 group, in which R.sub.8 represents a
C.sub.2-C.sub.30 alkyl, and n=1 to 500, or a heteroaryl group
containing 1 to 4 nitrogen atoms, unsubstituted or substituted by a
C.sub.2-C.sub.30 alkyl, or by a
(CH.sub.2).sub.m--O--(CH.sub.2).sub.p--R.sub.9 group in which m=1
to 6 and p=0 to 10 and R.sub.9 represents a cyclic ketal group
containing 5 to 7 carbon atoms, unsubstituted or substituted by at
least one linear or branched C.sub.2-C.sub.30 alkyl or by a sterol
radical, or also R.sub.3 is bound by a covalent bond to another
substituent R.sub.3, identical or different, of another compound of
formula (I), identical or different, in order to form a compound of
formula (I) in the form of a dimer, with or without a co-lipid.
13. Compound of formula (I): ##STR00033## in which X represents an
oxygen or sulphur atom, B represents a purine or pyrimidine base
such as uracil, adenine, guanine, cytosine, thymine, hypoxanthine,
or their derivatives, or also a non-natural mono- or bi-cyclic
heterocyclic base each ring of which comprises 4 to 7 members,
optionally substituted; and L.sub.1, L.sub.2, R.sub.1, R.sub.2 and
R.sub.3 are chosen from one of the following definitions: L.sub.1
represents a phosphate group, L.sub.2 represents hydrogen, R.sub.1
represents a C.sub.2-C.sub.30 alkyl group or a diacyl group in
which each acyl chain is C.sub.2-C.sub.30, and R.sub.3 is a hydroxy
group; or L.sub.1 and L.sub.2 represent an oxygen atom, R.sub.1 and
R.sub.2 represent hydrogen and R.sub.3 represents a triazole,
tetrazole, pyrazole or imidazole group substituted by a
C.sub.2-C.sub.30 alkyl group, or L.sub.1 represents a triazole,
tetrazole, pyrazole or imidazole group, L.sub.2 represents
hydrogen, R.sub.1 represents a C.sub.2-C.sub.30 alkyl group, and
R.sub.3 is a hydroxy group, or L.sub.1 represents a hydroxy group,
L.sub.2 represents hydrogen and R.sub.3 is a triazole group
substituted by a C.sub.2-C.sub.30 alkyl chain, optionally
substituted by a hydroxy group, or R.sub.3 is a group or a
--(CH.sub.2)n-V--R.sub.8 group, in which V represents --O-- or
--NH-- and R.sub.8 represents a C.sub.2-C.sub.30 alkyl, or L.sub.1
represents a hydroxy group, L.sub.2 represents hydrogen and R.sub.3
is a triazole group substituted by a
(CH.sub.2).sub.m--O--(CH.sub.2).sub.p--R.sub.9 group in which m=1
to 6 and p=0 to 10 and R.sub.9 represents a cyclic ketal group
containing 5 to 7 carbon atoms, unsubstituted or substituted by at
least one linear or branched C.sub.2-C.sub.30 alkyl group or by a
sterol radical, or L.sub.1 represents a hydroxy group, L.sub.2
represents hydrogen and R.sub.3 is a triazole group substituted by
a (CH.sub.2).sub.m--O--(CH.sub.2).sub.p--R.sub.9 group in which m=1
to 6 and p=0 to 10 bound by a covalent bond to another identical
substituent R.sub.3 of another identical compound of formula (I),
in order to form a compound of formula (I) in the form of a dimer,
or L.sub.1 represents a phosphate group, L.sub.2 represents
hydrogen, R.sub.1 represents a diacyl chain in which each diacyl
chain is C.sub.2-C.sub.30, and R.sub.3 is a hydroxy group.
14. Compound of formula (I): ##STR00034## in which X, B, L.sub.2
and R.sub.2 are as defined in claim 1; L.sub.1 or R.sub.3 is
constituted by or comprises a triazole group, unsubstituted or
substituted.
15. Nanoparticle comprising a compound of formula (I) according to
claim 13 and a therapeutic agent.
16. Nanoparticle comprising a compound of formula (I) according to
claim 13 and a therapeutic agent, wherein the therapeutic agent is
an anti-neoplastic agent.
17. Nanoparticle according to claim 11, wherein the therapeutic
agent is chosen from cis-platin, carboplatin, oxaliplatin,
nedaplatin and lobaplatin.
18. (canceled)
19. (canceled)
20. (canceled)
21. (canceled)
22. Process according to claim 1, wherein in formula (I) X
represents oxygen and B represents thymine or adenine.
23. Nanoparticle according to claim 12, wherein the therapeutic
agent from is selected from the group consisting of cis-platin,
carboplatin, oxaliplatin, nedaplatin and lobaplatin.
24. Nanoparticle comprising a compound of formula (I) according to
claim 13 and a therapeutic agent, wherein the therapeutic agent is
an anti-neoplastic agent.
25. Nanoparticle comprising a compound of formula (I) according to
claim 13 and a therapeutic agent, wherein the therapeutic agent is
selected from the group consisting of cis-platin, carboplatin,
oxaliplatin, nedaplatin and lobaplatin.
26. A method for transportation, vectorization or intracellular
delivery of therapeutic agents, in particular of anti-neoplastic
agents, wherein the nanoparticles according to claim 11 are used as
for transportation, vectorization or delivery agents.
27. A method for transportation or vectorization of therapeutic
agents, in particular of anti-neoplastic agents, wherein the
nanoparticles according to claim 12 are used as for transportation
or vectorization agents.
28. A method for transportation or vectorization of therapeutic
agents, in particular of anti-neoplastic agents, wherein the
nanoparticles according to claim 13 are used as for transportation
or vectorization agents.
29. A method for transportation or vectorization of therapeutic
agents, wherein the nanoparticles according to claim 11 are used as
for transportation or vectorization agents and the therapeutic
agent is selected from the group consisting of cis-platin,
carboplatin, oxaliplatin, nedaplatin and lobaplatin.
29. A method of treating tumour diseases, wherein the nanoparticles
according to claim 11 are administered to a patient in need
thereof.
30. A method of treating tumour diseases, wherein the nanoparticles
comprising a compound of formula (I) according to claim 13 and an
anti-neoplastic agent are administered to a patient in need
thereof.
31. A method of treating tumour diseases, wherein the nanoparticles
according to claim 11 wherein the therapeutic agent is selected
from the group consisting of cis-platin, carboplatin, oxaliplatin,
nedaplatin and lobaplatin. are administered to a patient in need
thereof.
Description
[0001] The invention relates to a process for the preparation of
nanoparticles based on functional amphiphilic molecules or
macromolecules and the use thereof for the transport or
vectorization of therapeutic agents, in particular of
anti-neoplastic agents.
[0002] Among the anti-neoplastic agents, cis-platin is an
anti-neoplastic agent that is widely used, in particular for the
treatment of solid tumours. However, its use is limited by its
toxicity as well as the onset of an acquired resistance.
[0003] In order to overcome these drawbacks, different formulations
have been proposed in the prior art: for example, the U.S. Pat. No.
5,178,876 describes platinum derivatives in the form of hydrophobic
complexes intended for encapsulation in liposomes.
[0004] The U.S. Pat. No. 6,001,817 describes compositions
containing cis-platin and a vector comprising at least one
nucleoside or deoxynucleoside.
[0005] The U.S. Pat. No. 7,908,160 relates to cis-platin
derivatives bound to ligands, the activity of which is reversible
as a function of the bond to the ligand.
[0006] The application WO01/32139 describes compositions of
cis-platin encapsulated in lipophilic nanoparticles obtained by
repeated heating and freezing cycles, based on negatively charged
natural lipids, in particular dioleyl-phosphatidylserine. It is
indicated in this application that cis-platin forms, in water,
positively charged aggregates having a higher solubility than the
non-charged species, which allows their interaction with the
negatively charged lipid membranes and the reorganization of the
lipid membranes around the cis-platin aggregates.
[0007] However, there is still a need to solve the problems linked
to the vectorization of therapeutic agents, in particular the
anti-neoplastic agents.
[0008] In particular, a means is sought which allows transport of
the therapeutic agents (in particular cis-platin and/or its
derivatives) rapidly to the inside of the tumorous cells with a
high pharmacological activity, whilst preserving the healthy cells,
i.e. by reducing the neurological, renal, auditory, digestive, etc.
toxicity, by simultaneously limiting the phenomena of the
appearance of resistance to this therapeutic agent.
[0009] It is also sought to provide a vector having a sufficient
stability over time to avoid the early release of the therapeutic
agent and the drawbacks associated with the presence of the free
therapeutic agent in the biological medium, in particular in terms
of loss of activity and toxicity.
[0010] It has now been found that the use of nanoparticles formed
from functional amphiphilic molecules or macromolecules allows the
effective and rapid intracellular delivery of therapeutic agents
and exhibited improved stability properties, in particular at
37.degree. C., allowing a sustained vectorization of said
therapeutic agents over time.
[0011] By "therapeutic agents", is meant, for example, a natural or
synthetic molecule used for the prevention or treatment of a
pathology or the restoration of a biological function, in vitro or
in vivo, in particular in animals, including human beings, or also
on isolated cells, with the exception of nucleic acids or fragments
thereof.
[0012] Such a molecule can be chosen, for example, from the active
ingredients of medicaments, in particular from anti-neoplastic
agents such as, for example: [0013] platinum complexes, among which
there can in particular be mentioned cis-platin, carboplatin,
oxaliplatin, nedaplatin, lobaplatin, etc. or [0014] ruthenium
capable of binding to platinum complexes, or also [0015] inorganic
complexes without platinum based on ruthenium II and/or III,
titanium, for example titanocene dichloride, or gallium, for
example the gallium salts such as gallium nitrate, gallium
chloride, KP46, or [0016] iron derivatives, such as, for example,
ferrocenium salts, nucleoside analogues containing iron, iron (II)
complexes containing pyridyl-based pentadentate ligands, or [0017]
cobalt derivatives, such as, for example, hexacarbonyl-dicobalt
complexes, alkyne-cobalt complexes, Co (III) complex containing a
nitrogen mustard ligand, or [0018] gold derivatives such as, for
example, Auranofin, gold (I), (III) and (III) complexes,
aurothioglucose, etc.
[0019] Advantageously, the use of nanoparticles formed from
functional amphiphilic molecules or macromolecules of formula (I)
for encapsulating these compounds and ensuring their intracellular
delivery makes it possible to limit the phenomena of resistance to
these compounds.
[0020] The platinum complexes, in particular cis-platin, are
preferred therapeutic agents for the purposes of the invention.
[0021] Inorganic complexes based on ruthenium II and/or III, can
be, for example, the complexes called NAMI-A, RAPTA-C, KP1019. Such
non-platinum complexes are described in Ott I. and Gust R., Arch.
Pharm. Chem. Life Sci. 2007, 340, 117-126; Reedijk J., Curr Opin
Chem. Biol., 1999, 3, 236-40; Haimei Chen et al., J. Am. Chem.
Soc., 2003, 125, 173-186.
[0022] Nucleoside analogues containing iron are described in
Schlawe D. et al., Angew. Chem. Int. Ed., 2004, 1731-1734).
[0023] According to a first aspect the invention therefore relates
to a process for encapsulating a therapeutic agent, preferably an
anti-neoplastic agent, comprising the stages consisting of:
[0024] a) preparing a mixture of at least one functional
amphiphilic compound of formula (I)
##STR00001##
in which [0025] X represents an oxygen or sulphur atom or a
methylene group, [0026] B represents a purine or pyrimidine base
such as uracil, adenine, guanine, cytosine, thymine, hypoxanthine,
or their derivatives, or also a non-natural mono- or bi-cyclic
heterocyclic base each ring of which comprises 4 to 7 members,
optionally substituted; [0027] L.sub.1 and L.sub.2, identical or
different, represent hydrogen, an oxycarbonyl --O--C(O)-- group, a
thiocarbamate --O--C(S)--NH-- group, a carbonate --O--C(O)--O--
group, a carbamate --O--C(O)--NH-- group, an oxygen atom, a
phosphate group, a phosphonate group or a heteroaryl group
comprising 1 to 4 nitrogen atoms, unsubstituted or substituted by a
linear or branched, saturated or unsaturated C.sub.2-C.sub.30
hydrocarbon chain, [0028] or also, L.sub.1 and L.sub.2, together,
form a ketal group of formula
[0028] ##STR00002## [0029] or also L.sub.1 or L.sub.2 represents
hydrogen, and the other represents a hydroxy group or a heteroaryl
group comprising 1 to 4 nitrogen atoms, unsubstituted or
substituted by a linear or branched C.sub.2-C.sub.30 alkyl chain;
[0030] R.sub.1 and R.sub.2, identical or different, represent
[0031] a linear or branched C.sub.2-C.sub.30 hydrocarbon chain,
preferably C.sub.6-C.sub.25, in particular C.sub.8-C.sub.25,
saturated or partially unsaturated, optionally completely or
partially fluorinated, unsubstituted or substituted on the carbon
at the end of the chain by a fluorine atom or by a benzyl or
naphthyl ester or ether, or [0032] a diacyl chain in which each
acyl chain is C.sub.2-C.sub.30, or [0033] a diacylglycerol,
sphingosine or ceramide group, or [0034] when L.sub.1 or L.sub.2
represents hydrogen, and the other represents a hydroxy group or a
heteroaryl group comprising 1 to 4 nitrogen atoms, R.sub.1 and
R.sub.2 do not exist; [0035] R.sub.3 represents [0036] a hydroxy,
amino, phosphate, phosphonate, phosphatidylcholine, O-alkyl
phosphatidylcholine, thiophosphate, phosphonium, NH.sub.2--R.sub.4,
NHR.sub.4R.sub.5 or NR.sub.4R.sub.5R.sub.6 group in which R.sub.4,
R.sub.5 and R.sub.6, identical or different, represent a hydrogen
atom or a linear or branched C.sub.1-C.sub.5 alkyl chain or linear
or branched C.sub.1-C.sub.5 hydroxyalkyl, or [0037] a linear or
branched C.sub.2-C.sub.30 alkyl chain optionally substituted by a
hydroxy group, or [0038] a cyclodextrin radical, or [0039] a
[0039] ##STR00003## [0040] radical, in which V represents an --O--,
--S--, or --NH-- bond, R.sub.7 represents H or CH.sub.3, and n=1 to
500, or [0041] a --(CH.sub.2).sub.n--V--R.sub.8 group, in which
R.sub.8 represents a C.sub.2-C.sub.30 alkyl, and n=1 to 500, or
[0042] a heteroaryl group containing 1 to 4 nitrogen atoms,
unsubstituted or substituted by a C.sub.2-C.sub.30 alkyl, or by a
(CH.sub.2).sub.m--O--(CH.sub.2).sub.p--R.sub.9 group in which m=1
to 6 and p=0 to 10 and R.sub.9 represents a cyclic ketal group
containing 5 to 7 carbon atoms, unsubstituted or substituted by at
least one linear or branched C.sub.2-C.sub.30 alkyl or by a sterol
radical, or also [0043] R.sub.3 is bound by a covalent bond to
another substituent R.sub.3, identical or different, of another
compound of formula (I), identical or different, in order to form a
compound of formula (I) in the form of a dimer, and [0044] a
therapeutic agent, preferably an anti-neoplastic agent,
[0045] b) subjecting said mixture to repeated heating and freezing
cycles, so as to obtain nanoparticles containing said therapeutic
agent, and
[0046] c) recovering the nanoparticles containing said therapeutic
agent obtained in this way.
[0047] Advantageously, it has been found that the molecular and/or
macromolecular structures which constitute the compounds of formula
(I), comprising at least one ligand of the therapeutic agent
(nucleobase, nucleoside, modified nucleoside, nucleotides,
oligonucleotide, heterocycle, etc) represented by the substituent
B, and having an amphiphilic character due to the presence of at
least one hydrophilic part (phosphate, carboxylate, etc), and of at
least one hydrophobic part (hydrophobic segments which are
single-stranded, double-stranded and polar parts derived from
synthons of biological origin, etc.), making it possible to form
stable nanoparticles with the therapeutic agent.
[0048] By combining the amphiphilic properties of the compounds of
formula (I), the presence of ligands of the therapeutic agent
(active ingredient) in these compounds and any electrostatic
interactions between the therapeutic agents and these compounds,
the nanoparticles obtained in this way have a structure which
allows an effective and rapid intra-cell delivery of the
encapsulated active ingredients, in particular anti-neoplastic
agents.
[0049] Without wishing to restrict the invention to one theory, it
can be postulated that the intermolecular interactions of the
compounds of formula (I) lead to an increase in the cohesion forces
on the surface of the nanoparticles, which results in a greater
stability over time, under the conditions of use.
[0050] Advantageously, said nanoparticles also have a life span
compatible with their use as a vector for a therapeutic agent.
[0051] In formula (I) above, n is advantageously comprised between
1 and 500, preferably comprised between 1 and 100, in particular
comprised between 1 and 50, quite particularly comprised between 1
and 10.
[0052] By "linear or branched C.sub.1-C.sub.5 alkyl", is meant for
example a methyl, ethyl, propyl, i-propyl, n-butyl, i-butyl,
tert-butyl radical, preferably methyl or ethyl.
[0053] Also, in formula (I) above, the purine or pyrimidine base,
or the non-natural heterocyclic base can be substituted by at least
one substituent chosen, for example, from a halogen, an amino
group, a carboxy group, a carbonyl group, a carbonylamino group, a
hydroxy, azido, cyano, alkyl, cycloalkyl, perfluoroalkyl, alkyloxy
(for example, methoxy), oxycarbonyl, vinyl, ethynyl, propynyl, acyl
group etc.
[0054] By "non-natural heterocyclic base" is meant a base other
than uracil, adenine, guanine, cytosine, thymine or hypoxanthine,
which does not exist in nature.
[0055] By "heteroaryl group containing 1 to 4 nitrogen atoms", is
meant a monocyclic or bicyclic, aromatic or partially unsaturated,
carbocyclic group containing 5 to 12 atoms, interrupted by 1 to 4
nitrogen atoms, in particular the pyrazole, triazole, tetrazole or
imidazole groups.
[0056] For the preparation of the compounds of formula (I),
reference can be made to the application WO 2005/116043, which
describes different access routes to this type of compounds (see in
particular pp. 8-17 and examples).
[0057] The process according to the invention can comprise the
stages implemented under the following general conditions: [0058]
the compound of formula (I), is put in solution in an organic
solvent in order to form a lipid mixture, then, after removal,
evaporation is carried out in order to form a film; [0059] in
parallel, the desired quantity of therapeutic agent, preferably an
anti-neoplastic agent, is put in solution in distilled water;
[0060] the lipid film is rehydrated in the solution of therapeutic
agent, preferably an anti-neoplastic agent. A clear solution is
obtained by sonication and heating; [0061] the solution is cooled
down rapidly, for example by immersion in liquid nitrogen. This
heating/cooling cycle is preferably carried out 1 to 10 times, in
particular 5 to 10 times, in particular 10 times.
[0062] The solution obtained is centrifuged. The supernatant is
separated. After several centrifugations, the pellet is dried.
[0063] The organic solvent can be chosen, for example, from the
usual organic solvents in the field, such as, for example,
chloroform, an alcohol such as methanol or ethanol, etc.
[0064] The heating is carried out, preferably, to a temperature of
the order of 20.degree. C. to 80.degree. C., and the cooling to a
temperature of the order of -190.degree. C. (liquid nitrogen) to
0.degree. C. (ice). An appropriate heating/cooling cycle can, for
example, be 45.degree. C. for the heating and -78.degree. C. for
the cooling.
[0065] Preferably, the therapeutic agent is chosen from the
platinum complexes (cis-platin, carboplatinum, etc.), cis-platin
being particularly preferred, or ruthenium capable of binding to
platinum complexes, or also the abovementioned inorganic complexes
without platinum based on ruthenium II or III, titanium, gallium,
cobalt, iron or gold.
[0066] The molar ratio R of the compound of formula (I)/therapeutic
agent can be comprised, for example, between 0.01 and 50, in
particular R=0.2.
[0067] The nanoparticles obtained can be optionally extruded on
polycarbonate filter having, for example, a pore diameter of the
order of 100 or 200 nm.
[0068] In this way solid nanoparticles are obtained which are
constituted by a core rich in therapeutic agent (active ingredient)
surrounded by one or more lipid layers constituted by the
functional amphiphilic compound of formula (I) as defined above,
with or without co-lipid.
[0069] Said solid nanoparticles, constituted by a core rich in
therapeutic agent, preferably an anti-neoplastic agent, in
particular a platinum complex, surrounded by one or more lipid
layers constituted by the functional amphiphilic compound of
formula (I) as defined above, with or without co-lipid, represent a
subsequent subject of the invention.
[0070] In the case of a multi-layer surround, all the lipid layers
are constituted by the same lipids (compound of formula (I) with or
without co-lipid).
[0071] According to an aspect of the invention, at least one
co-lipid will be used in the lipid mixture, in addition to the
compound of formula (I).
[0072] By "co-lipid", is meant a compound used in combination with
the compound of formula (I), which contributes to the production of
the structure of the lipid layers(s) of the nanoparticle.
[0073] Preferably, a zwitterionic co-lipid will be used.
[0074] Said co-lipid can be, for example, chosen from
dioleylphosphatidylcholine (DOPC) or dioleylphosphatidyluridine
phosphatidylcholine (DOUPC), in combination with the compound of
formula (I) in order to form the lipid layer(s) of the
nanoparticle.
[0075] These compounds can play the role of co-lipid when they are
used in mixture with a compound of formula (I). Alternatively, they
can be included in formula (I), such as, for example,
dioleylphosphatidyluridinephosphatidylcholine (DOUPC). In this
case, they will either play the role of compound of formula (I) or,
in combination with another compound of formula (I), the role of
co-lipid.
[0076] According to a preferred aspect of the process, said lipid
mixture contains solely at least one compound of formula (I) and
does not contain co-lipid.
[0077] Preferably, the therapeutic agent will be used at a
concentration of the order of 0.1 ng/mL to 10 mg/mL in the aqueous
phase, so that the intracellular delivery of the active ingredient
is significant.
[0078] The preferred compounds of formula (I) are those in which X
represents oxygen.
[0079] The compounds of formula (I) in which B represents thymine
or adenine are also preferred compounds.
[0080] Among the compounds of formula (I) which are particularly
advantageous for the purposes of the invention, the compounds of
formula (I) can be mentioned in which: [0081] X and B are as
defined above; [0082] L.sub.1 represents a phosphate group, L.sub.2
represents hydrogen, R.sub.1 represents a C.sub.2-C.sub.30 alkyl
group or a diacyl group in which each acyl chain is
C.sub.2-C.sub.30, and R.sub.3 is a hydroxy group; or [0083] L.sub.1
and L.sub.2 represent an oxygen atom, R.sub.1 and R.sub.2 represent
hydrogen and R.sub.3 represents a triazole, tetrazole, pyrazole or
imidazole group substituted by a C.sub.2-C.sub.30 alkyl group, or
[0084] L.sub.1 represents a triazole, tetrazole, pyrazole or
imidazole group, L.sub.2 represents hydrogen, R.sub.1 represents a
C.sub.2-C.sub.30 alkyl group and R.sub.3 is a hydroxy group, or
[0085] L.sub.1 represents a hydroxy group, L.sub.2 represents
hydrogen and R.sub.3 is a triazole group substituted by a
C.sub.2-C.sub.30 alkyl chain, optionally substituted by a hydroxy
group, or R.sub.3 is a group or a --(CH.sub.2)n-V--R.sub.8 group,
in which V represents --O-- or --NH-- and R.sub.8 represents a
C.sub.2-C.sub.30 alkyl, or [0086] L.sub.1 represents a hydroxy
group, L.sub.2 represents hydrogen and R.sub.3 is a triazole group
substituted by a (CH.sub.2).sub.m--O--(CH.sub.2).sub.p--R.sub.9
group in which m=1 to 6 and p=0 to 10 and R.sub.9 represents a
cyclic ketal group containing 5 to 7 carbon atoms, unsubstituted or
substituted by at least one linear or branched C.sub.2-C.sub.30
alkyl group or by a sterol radical, or [0087] L.sub.1 represents a
hydroxy group, L.sub.2 represents hydrogen and R.sub.3 is a
triazole group substituted by a
(CH.sub.2).sub.m--O--(CH.sub.2).sub.p--R.sub.9 group in which m=1
to 6 and p=0 to 10 bound by covalent bond to another identical
substituent R.sub.3 of another identical compound of formula (I),
in order to form a compound of formula (I) in the form of a dimer,
or [0088] L.sub.1 represents a phosphate group, L.sub.2 represents
hydrogen R.sub.1 represents a diacyl chain in which each diacyl
chain is C.sub.2-C.sub.30, and R.sub.3 is a hydroxy group, which
are new compounds.
[0089] The compounds of formula (I) in which the substituent
L.sub.1 or the substituent R.sub.3 is constituted by or comprises a
triazole group, unsubstituted or substituted, are also preferred
new compounds for the purposes of the process according to the
invention.
[0090] The above compounds are new compounds which represent a
subject of the invention, as well as the nanoparticles comprising
these compounds and a therapeutic agent, in particular an
anti-neoplastic agent, in particular the platinum complexes (such
as, for example cis-platin, carboplatin, oxaliplatin, nedaplatin,
lobaplatin,), or ruthenium capable of binding to the platinum
complexes, or also the abovementioned inorganic complexes without
platinum based on ruthenium, titanium, gallium, cobalt, iron or
gold. Cis-platin is a preferred anti-neoplastic agent for the
purposes of the invention.
[0091] The compounds of formula (I) can comprise purine or
pyrimidine base derivatives having an anti-neoplastic activity,
such as, for example, aracytosine (AraC), 5-fluorouracil (5-FU),
Iododeoxyuridine (IdU), 2'-deoxy-2'-methylidenecytidine (DMDC) or
5-chloro-6-azido-5,6-dihydro-2'-deoxyuridine.
[0092] A subject of the invention is also the use of the
nanoparticles capable of being obtained by the process described
above, for the transport or vectorization of therapeutic agents, in
particular anti-neoplastic agents.
[0093] In particular, the invention relates to the use of the
nanoparticles capable of being obtained by the process described
above, for the intracellular delivery of therapeutic agents, in
particular anti-neoplastic agents.
[0094] The invention also relates to the use of the nanoparticles
capable of being obtained by the above process, for the preparation
of anti-neoplastic medicaments.
[0095] The invention also relates to the nanoparticles capable of
being obtained by the above process, for the treatment of tumour
diseases, in particular cancers, such as, for example, cancers of
the ovary, testicle, colon, cervix, lung, or adenosarcoma etc.
[0096] The invention is illustrated non-limitatively by the
examples below.
[0097] All of the starting products originate from suppliers of
chemical products (Aldrich, Alfa Aesar and Avanti Polar Lipid) and
are used without subsequent purification. The solvents were used
without additional distillation. The synthesized compounds were
characterized using standard spectroscopic analytical methods such
as NMR .sup.1H at 300.13 MHz, .sup.13C at 75.46 MHz and .sup.31P at
121.49 MHz) and mass spectroscopy (Characteristics). The chemical
shifts (.delta.) in NMR are expressed in ppm and relative to TMS.
The coupling constants J in NMR .sup.1H are expressed in Hz. Merck
RP-18 F254s plates were used for the thin layer chromatography
(TLC). SEPHADEX LH-20 (25-100 .mu.m) silica was used for the
purifications by quantitative chromatographies.
[0098] The examples below, entitled "Preparation" describe the
preparation of synthesis intermediates used for preparing the
compounds of formula (I). The preparation of the compounds of
formula (I) is then described in the synthesis examples entitled
"Example".
Preparation 1
5'-paratoluenesulphonylthymidine
##STR00004##
[0100] 2 g of thymidine (8.26 mmol) in 0.1 M solution is introduced
into anhydrous pyridine in a two-necked flask under an anhydrous
nitrogen atmosphere. The solution is then cooled down to 0.degree.
C. and 3.935 g of paratoluene sulphonic acid chloride (2.5
equivalents, 20.6 mmol) are added in small portions. The reaction
medium is left to return to ambient temperature, then stirred for
10 hours. The reaction is then stopped by the addition of 10 mL of
methanol, stirring is maintained for 30 min. 50 mL of
CH.sub.2Cl.sub.2 is added to the mixture then it is washed
successively with 20 mL of a 5% solution of NaHCO.sub.3, 20 mL of a
saturated solution of NaCl and 20 mL of a 5% solution of
NaHCO.sub.3. The solvent is eliminated under reduced pressure. The
expected compound is obtained pure by recrystallization from
methanol.
[0101] Rf: 0.47 (AcOEt/MeOH 9/1)
[0102] The yield is 75%.
[0103] NMR .sup.1H (300.13 MHz, DMSO d.sub.6): .delta. 1.77 (s, 3H,
CH.sub.3), .delta. 2.11 (m, 2H, CH.sub.2), .delta. 2.42 (s, 3H,
CH.sub.3), .delta. 3.52 (t, j=6 Hz, 4H, CH.sub.2), .delta. 4.18 (m,
1H, CH), .delta. 4.25 (m, 3H, CH, CH.sub.2), .delta. 5.42 (s, 1H,
OH), .delta. 6.15 (t, j=6 Hz, H, CH), .delta. 7.38 (s, 1H, CH),
.delta. 7.46 (s, 1H, CH), .delta. 7.49 (s, 1H, CH), .delta. 7.78
(s, 1H, CH), .delta. 7.81 (s, 1H, CH), .delta. 11.28 (s, 1H,
NH).
[0104] NMR .sup.13C (75.47 MHz, DMSO d.sub.6): .delta. 12.5
(CH.sub.3), .delta. 21.6 (CH.sub.3), .delta. 38.9 (CH.sub.2),
.delta. 70.4 (CH.sub.2), .delta. 70.6 (CH), .delta. 83.7 (CH),
.delta. 84.5 (CH), .delta. 110.3 (C), .delta. 128.1 (CH ar),
.delta. 130.6 (2 CH ar), .delta. 132.6 (C ar), .delta. 136.4 (C
ar), .delta. 145.6 (C), .delta. 150.8 (C.dbd.O), .delta. 164.1
(C.dbd.O).
[0105] High resolution MS [M+H].sup.+: 397.1
Preparation 2
2'-deoxy-5'-toluenesulphonyladenosine
##STR00005##
[0107] 2 g of 2'-deoxyadenosine (8 mmol) in a 0.1 M solution is
introduced into anhydrous pyridine in a two-necked flask under an
anhydrous nitrogen atmosphere. The solution is then cooled down to
0.degree. C. and 3.793 g of paratoluene sulphonic acid chloride
(2.5 equivalents, 20 mmol) are added by small portions. The
reaction medium is left to return to ambient temperature, then
stirred for 10 hours. The reaction is then stopped by the addition
of 10 mL of methanol, stirring is maintained for 30 min. 50 mL of
CH.sub.2Cl.sub.2 is added to the mixture then it is washed
successively with 20 mL of a 5% solution of NaHCO.sub.3, 20 mL of a
saturated solution of NaCl and 20 mL of a 5% solution of
NaHCO.sub.3. The solvent is eliminated under reduced pressure. The
expected compound is obtained pure by recrystallization from
methanol. 2.1 g of a white product is isolated in this way.
[0108] Rf: 0.37 (AcOEt/MeOH 9/1)
[0109] The yield is 63%.
Preparation 3
5'azido-5'-deoxythymidine
##STR00006##
[0111] 2 g of 5'-paratoluenesulphonylthymidine (5 mmol) as
described in Preparation 1 in a 0.1 M solution is introduced into
DMF in a two-necked flask provided with a condenser and under an
anhydrous nitrogen atmosphere. 1.3 g of sodium azide (4
equivalents, 20 mmol) is added. The solution is then stirred and
heated at 110.degree. C. for 10 hours. The mixture is cooled down
to ambient temperature. 50 mL of CH.sub.2Cl.sub.2 is added to the
mixture then it is washed successively with twice 15 mL of water
then with 15 mL of a saturated aqueous solution of NaCl. The
organic phase is dried over sodium sulphate then the solvent is
eliminated under reduced pressure. The expected compound is
obtained pure by recrystallization from methanol. 0.8 g of a white
solid is obtained in this way.
[0112] Rf: 0.47 (AcOEt/MeOH 9/1)
[0113] The yield is 60%.
[0114] MS [M+H].sup.+: 268.1
Preparation 4
5'-azido-5',2'-dideoxyadenosine
##STR00007##
[0116] 2 g of 2'-deoxy-5'-paratoluenesulphonyladenosine as
described in Preparation 2 (5 mmol) in a 0.1 M solution is
introduced into DMF in a two-necked flask provided of a condenser
and under an anhydrous nitrogen atmosphere. 1.3 g of sodium azide
(4 equivalents, 20 mmol) is added. The solution is then stirred and
heated at 110.degree. C. for 10 hours. The mixture is cooled down
to ambient temperature. 50 mL of CH.sub.2Cl.sub.2 is added to the
mixture then it is washed successively with twice 15 mL of water
then with 15 mL of a saturated aqueous solution of NaCl. The
organic phase is dried over sodium sulphate then the solvent is
eliminated under reduced pressure. The expected compound is
obtained pure by recrystallization from methanol. 0.8 g of a white
solid is obtained in this way.
[0117] Rf: 0.37 (AcOEt/MeOH 9/1)
[0118] The yield is 60%.
[0119] High resolution MS [M+H].sup.+: calculated mass: 277.1161,
measured mass: 277.1157
Preparation 5
1-propargyloxyoctadecane
##STR00008##
[0121] 673 mg of propargylic alcohol (12 mmol) in a 0.5 M solution
is introduced into DMF in a clean and dry flask under an anhydrous
nitrogen atmosphere. The solution is then cooled down to 0.degree.
C. and 180 mg of sodium hydride (0.625 equivalent, 7.5 mmol) are
added by small portions. The reaction medium is left to return to
ambient temperature. 2 g of 1-bromo-octadecane (0.5 equivalent, 6
mmol) are added. Stirring is maintained for 5 hours. The reaction
is then stopped by the addition of 10 mL of methanol and stirring
is maintained for 30 min. 50 mL of CH.sub.2Cl.sub.2 is added to the
mixture then it is washed successively with twice 20 mL and 20 mL
of a saturated solution of NaCl. The organic phase is then dried
over Na.sub.2SO.sub.4 then the solvent is eliminated under reduced
pressure. The expected compound is obtained pure after separation
on a chromatographic column (hexane). 1.2 g of a white product is
isolated in this way.
[0122] Rf: 0.82 (Hexane)
[0123] The yield is 65%.
[0124] NMR .sup.1H (300.13 MHz, CDCl.sub.3): .delta. 0.90 (t, j=6
Hz, 3H, CH.sub.3), .delta. 1.28 (s, 30H, CH.sub.2), .delta. 1.61
(m, 2H, CH.sub.2), .delta. 2.43 (t, j=3 Hz, 1H, CH), .delta. 3.53
(t, j=6 Hz, 2H, CH.sub.2), .delta. 4.15 (d, j=3 Hz, 2H,
CH.sub.2).
[0125] NMR .sup.13C (75.47 MHz, CDCl.sub.3): NMR .sup.13C (75.47
MHz, CDCl.sub.3): .delta. 14.2 (CH.sub.2), .delta. 22.7 (CH.sub.2),
.delta. 26.1 (CH.sub.2), .delta. 29.4 (CH.sub.2), .delta. 29.5
(CH.sub.2), .delta. 29.6 (CH.sub.2), .delta. 32.0 (CH.sub.2),
.delta. 58.0 (CH.sub.2), .delta. 70.4 (CH.sub.2), .delta. 74.1
(CH), .delta. 80.1 (C).
Preparation 6
1,12-propargyloxydodecane
12-propargyloxydodecan-1-ol
[0126] 1 g of dodecan-1,12-diol (5 mmol) in a 0.5 M solution is
introduced into DMF in a clean and dry flask, under an anhydrous
nitrogen atmosphere. The solution is then cooled down to 0.degree.
C. and 360 mg of hydrogen hydride (3 equivalents, 15 mmol) is added
by small portions. The reaction medium is left to return to ambient
temperature. 1.49 g of propargyl bromide (2.5 equivalents, 12.5
mmol) is added. Stirring is maintained for 5 hours. The reaction is
then stopped by the addition of 10 mL of methanol, stirring is
maintained for 30 min. 50 mL of CH.sub.2Cl.sub.2 is added to the
mixture then it is washed successively with twice 20 mL and 20 mL
of a saturated solution of NaCl. The organic phase is then dried
over Na.sub.2SO.sub.4 then the solvent is eliminated under reduced
pressure. The products obtained are then separated on a
chromatographic column (Hex/ActEth 9/1). Two products are isolated,
namely 370 mg of a brown oil corresponding to
1,12-Propargyloxydodecane and 430 mg of a brown solid corresponding
to 12-propargyloxydodecan-1-ol.
1,12-propargyloxydodecane
##STR00009##
[0128] Rf: 0.53 (Hexane/AcOtEt 9/1)
[0129] The yield is 27%.
[0130] NMR .sup.1H (300.13 MHz, CDCl.sub.3): .delta. 1.31 (m, 16H,
CH.sub.2), .delta. 1.61 (m, 4H, CH.sub.2), .delta. 2.43 (t, j=3 Hz,
1H, CH), .delta. 3.52 (t, j=6 Hz, 4H, CH.sub.2), .delta. 4.15 (d,
j=3 Hz, 4H, CH.sub.2).
[0131] NMR .sup.13C (75.47 MHz, CDCl.sub.3): .delta. 26.1
(CH.sub.2), .delta. 29.4 (CH.sub.2), .delta. 29.5 (CH.sub.2),
.delta. 29.6 (CH.sub.2), .delta. 58.0 (CH.sub.2), .delta. 70.3
(CH.sub.2), .delta. 74.1 (CH), .delta. 80.1 (C).
12-propargyloxydodecan-1-ol
##STR00010##
[0133] Rf: 0.10 (Hexane/AcOtEt 9/1)
[0134] The yield is 36%.
[0135] NMR .sup.1H (300.13 MHz, CDCl.sub.3): .delta. 1.32 (m, 16H,
CH.sub.2), .delta. 1.59 (m, 4H, CH.sub.2), .delta. 2.43 (t, j=3 Hz,
2H, CH), .delta. 3.52 (t, j=6 Hz, 2H, CH.sub.2), .delta. 3.65 (t,
j=6 Hz, 2H, CH.sub.2), .delta. 4.15 (d, j=3 Hz, 2H, CH.sub.2).
[0136] NMR .sup.13C (75.47 MHz, CDCl.sub.3): .delta. 25.8
(CH.sub.2), .delta. 26.0 (CH.sub.2), .delta. 29.4 (2 CH.sub.2),
.delta. 29.5 (2 CH.sub.2), .delta. 29.6 (CH.sub.2), .delta. 32.7
(CH.sub.2), .delta. 57.9 (CH.sub.2), .delta. 62.7 (CH.sub.2),
.delta. 70.2 (CH.sub.2), .delta. 74.2 (CH), .delta. 79.9 (C).
Preparation 7
o-propargylcholesterol
##STR00011##
[0138] 500 mg of cholesterol (1.3 mmol) in a 0.5 M solution is
introduced into DMF in a clean and dry flask, under an anhydrous
nitrogen atmosphere. The solution is then cooled down to 0.degree.
C. and 47 mg of sodium hydride (1.5 equivalents, 2 mmol) is added
by small portions. The reaction medium is left to return to ambient
temperature. 238 mg of propargyl bromide (1.5 equivalents, 2 mmol)
is added. Stirring is maintained for 5 hours. The reaction is then
stopped by the addition of 10 mL of methanol and stirring is
maintained for 30 min. 50 mL of CH.sub.2Cl.sub.2 is added to the
mixture then it is washed successively with twice 20 mL of a
saturated solution of NaCl. The organic phase is then dried over
Na.sub.2SO.sub.4 then the solvent is eliminated under reduced
pressure. The expected compound is obtained after purification on a
chromatographic column (Hexane/AcOEt 8/2). 215 mg of a white
product are isolated in this way.
[0139] Rf: 0.83 (Hexane/AcOEt 8/2)
[0140] The yield is 39%.
EXAMPLE 1
Thymidine 3'-(1,2-dimyristoyl-sn-glycero-3-phosphate) (di
c14dT)
##STR00012##
[0142] 5'-O-(4,4'-dimethoxytrityl)-2'-deoxythymidine,
3'-[(2-cyano-ethyl)-N,N-diisopropyl)]phosphoramidite (0.500 g, 1
eq, 0.67 mmol), 1,2-dimyristoyl-sn-glycerol (0.447 g, 1.3 eq, 0.87
mmol) and a 0.45 M solution of tetrazole in acetonitrile (2 mL, 1.3
eq, 0.87 mmol) are dissolved in 4 mL of anhydrous acetonitrile
under nitrogen. The reaction medium is magnetically stirred for 24
hours at ambient temperature. The mixture is then oxidized by the
addition of 43 mL of a 0.02M solution of diiodine in
THF/Pyr/H.sub.2O. After 12 hours at ambient temperature, the
solvent is evaporated off under vacuum. The residue is dissolved in
8 mL of dichloromethane. Then, 0.2 mL of
1,5-diazabicyclo[5.4.0]undec-7-ene (DBU) (1.3 eq, 0.87 mmol) is
added to the reaction medium over 5 hours. The reaction medium is
washed with a 0.1 N solution of HCl then with a saturated solution
of Na.sub.2S.sub.2O.sub.7. The organic phase is concentrated under
vacuum. The compound is obtained after purification by flash
chromatography (381 mg) using an elution gradient (MeOH/DCM 9:1 to
1:1).
[0143] The yield is 69%.
[0144] Rf: 0.34 (DCM/MeOH 9:1)
[0145] NMR 1H (300 MHz CDCl3): .delta. in ppm 0.84 (t, 6H, J=6.92
Hz, 2*CH.sub.3), 1.21 (m, 40H, 20*CH.sub.2), 1.42 (dd, 4H, J1=8.45
Hz, J2=15.68 Hz, 2*CH2), 1.89 (s, 3H, Me), 2.30 (dd, 4H, J1=7.43
Hz, J2=15.92 Hz, 2*CH2), 2.83 (t, 2H, J=5.84, H2'), 3.84 (m, 1H,
H3'), 4.09-4.35 (m, 7H, 2*CH.sub.2 (glycerol), H4', H5'), 5.27 (s,
1H, CHglycerol), 6.22 (t, 1H, J=6.81 Hz, H1'), 7.61 (s, 1H,
Hbase).
[0146] NMR 13C (75 MHz, CDCl3): .delta.60 in ppm 19.29 (CH.sub.3),
23.71 (CH.sub.2), 26.57 (CH.sub.2), 28.73 (CH.sub.2), 32.76
(CH.sub.2), 37.85 (CH.sub.2), 48.90 (CH.sub.2), 166.15
(C.dbd.O).
[0147] NMR 31P (121 MHz, CDCl3): .delta. in ppm 0.61.
[0148] High Resolution Mass FAB- theoretical m/z=815.4823 observed
m/z=815.4794.
EXAMPLE 2
Thymidine 3'-(1,2-dipalmitoyl-sn-glycero-3-phosphate) (di
c16dT)
##STR00013##
[0150] 5'-O-(4,4'-dimethoxytrityl)-2'-deoxythymidine,
3'-[(2-cyano-ethyl)-N,N-diisopropyl)]phosphoramidite (0.500 g, 1
eq, 0.67 mmol), 1,2-dipalmitoyl-sn-glycerol (0.496 g, 1.3 eq, 0.87
mmol/solubilized in 3 mL of THF) and a 0.45 M solution of tetrazole
in acetonitrile (2 mL, 1.3 eq, 0.87 mmol) are dissolved in 3 mL of
anhydrous acetonitrile under nitrogen. The reaction medium is
magnetically stirred for 24 hours at ambient temperature and under
nitrogen. The mixture is then oxidized by the addition of 43 mL of
a 0.02M solution of diiodine in THF/Pyr/H.sub.2O. After 12 hours at
ambient temperature, the solvent is evaporated off under vacuum and
dried under P.sub.2O.sub.5 overnight using a pump. The residue is
dissolved in 8 mL of dichloromethane. Then, 0.2 mL of
1,5-diazabicyclo[5.4.0]undec-7-ene (DBU) (1.3 eq, 0.87 mmol) is
added to the reaction medium over 5 hours. The reaction medium is
washed with a 0.1 N solution of HCl then with a saturated solution
of Na.sub.2S.sub.2O.sub.3. The organic phase is concentrated under
vacuum. The compound is obtained after purification by flash
chromatography (180 mg) using an elution gradient (MeOH/DCM 98:2 to
1:1).
[0151] The yield is 24%.
[0152] Rf: 0.3 (DCM/MeOH 8:2)
[0153] NMR 1H (300 MHz, CDCl3): .delta. in ppm 0.88 (t, 6H, J=6.9
Hz, 2*CH.sub.3), 1.25 (m, 48H, 24*CH.sub.2), 1.42 (dd, 4H, J1=8.4
Hz, J2=15.6 Hz, 2*CH2), 1.90 (s, 3H, Me), 2.33 (m, 4H, 2*CH.sub.2),
2.83 (t, 2H, J=5.6 Hz, H.sub.2'), 3.84 (m, 1H, H3'), 4.09-4.35 (m,
7H, 2*CH.sub.2 (glycerol), H.sub.4', H.sub.5'), 5.27 (s, 1H, CH
glycerol), 6.21 (t, 1H, J=6.7 Hz, H1'), 7.54 (s, 1H, H base).
[0154] NMR 1H (CDCl3): .delta. in ppm 12.4 (CH.sub.3 base), 14.1
(CH.sub.3 chain), 19.6 (CH.sub.2), 19.7 (CH.sub.2), 22.6
(CH.sub.2), 24.8 (CH.sub.2), 29.1-29.6 (CH.sub.2), 31.9 (CH.sub.2),
33.9 (CH.sub.2), 34.1 (CH.sub.2), 61.5 (CH.sub.2), 61.7 (CH.sub.2),
62.5 (CH.sub.2), 62.6 (CH.sub.2), 66.1 (CH.sub.2), 66.2 (CH.sub.2),
69.1 (CH), 78.8 (CH), 85.5 (CH), 86.1 (CH), 111.3 (C base), 136.8
(CH base), 150.5 (C.dbd.O base), 164.1 (C.dbd.O base), 173.0
(C.dbd.O chain), 173.5 (C.dbd.O chain).
[0155] NMR 31P (121 MHz, CDCl3): .delta. in ppm 2.1.
[0156] Mass ESI-: theoretical m/z=872.5 observed m/z=871.3.
EXAMPLE 3
5'-(4-Hexadecyloxymethyl-[1,2,3]triazol-1-yl)-5',2'dideoxythymidine
##STR00014##
[0158] 200 mg of 5'-azido-5'-deoxythymidine as described in
Preparation 3 (0.75 mmol) and 231 mg of 1-propargyloxyoctadecane as
described in the Preparation 5 (1 equivalent) in a 0.1 M solution
are introduced into a mixture of THF and water (1/1) in a flask.
Then, the following are added successively: 30 mg of sodium
ascorbate (0.2 equivalents, 0.15 mmol) and 12 mg of copper sulphate
(0.1 equivalent, 0.075 mmol). The reaction medium is stirred and
heated at 60.degree. C. for 5 hours. The mixture is then cooled
down to ambient temperature. The reaction medium is immediately
adsorbed on silica and the solvent eliminated by evaporation. 180
mg of a white solid are obtained after chromatography on a silica
column (AcOEt/MeOH 8/2).
[0159] Rf: 0.72 (AcOEt/MeOH 8/2)
[0160] The yield is 42%.
[0161] High resolution MS [M+H].sup.+: calculated mass: 576.4125,
measured mass: 576.4120
EXAMPLE 4
5'-(4-Hexadecyloxymethyl-[1,2,3]triazol-1-yl)-5',2'-dideoxyadenosine
##STR00015##
[0163] 200 mg of 5'-azido-5',2'-dideoxyadenosine as described in
Preparation 4 (0.72 mmol) and 223 mg of 1-propargyloxyoctadecane as
described in Preparation 5 (1 equivalent) in a 0.1 M solution are
introduced in a mixture of THF and water (1/1) in a flask. Then,
the following are added successively: 30 mg of sodium ascorbate
(0.2 equivalents, 0.15 mmol) and 12 mg of copper sulphate (0.1
equivalent, 0.075 mmol). The reaction medium is stirred and heated
at 60.degree. C. for 5 hours, then the mixture is cooled down to
ambient temperature. The reaction medium is immediately adsorbed on
silica and the solvent eliminated by evaporation. 150 mg of a white
solid are obtained after column chromatography (AcOEt/MeOH
8/2).
[0164] Rf: 0.65 (AcOEt/MeOH 8/2)
[0165] The yield is 35%.
[0166] NMR .sup.1H (300.13 MHz, CDCl.sub.3): .delta. 0.89 (t, j=6
Hz, 3H, CH.sub.3), .delta. 1.26 (m, 30H, CH.sub.2), .delta. 1.55
(m, 2H, CH.sub.2), .delta. 2.54 (m, 1H, CH.sub.2), .delta. 3.06 (m,
1H, CH.sub.2), .delta. 3.45 (t, j=6 Hz, 2H, CH.sub.2), .delta. 4.50
(m, 4H, CH.sub.2, CH), .delta. 4.89 (m, ???), .delta. 5.88 (s, 2H,
NH.sub.2), .delta. 6.40 (t, j=6 Hz, 1H, CH), .delta. 7.42 (s, 1H,
CH), .delta. 7.81 (s, 1H, CH), .delta. 8.35 (s, 1H, CH).
[0167] High resolution MS [M+H].sup.+: calculated mass: 585.4241,
measured mass: 585.4254
EXAMPLE 5
5'-(4-(1-hydroxy-hexyl)-[1,2,3]triazol-1-yl)-5',2'dideoxythymidine
##STR00016##
[0169] 215 mg of 5'-azido-5'-deoxythymidine as described in
Preparation 3 (0.8 mmol) and 101 mg of the racemic mixture of
oct-1-yn-3-ol (1 equivalent) in a 0.1 M solution in a THF/water
mixture (1/1) are introduced into a flask. Then, the following are
added successively: 31.5 mg of sodium ascorbate (0.2 equivalent,
0.15 mmol) and 13 mg of copper sulphate (0.1 equivalent, 0.075
mmol). The reaction medium is stirred and heated at 60.degree. C.
for 5 hours. The mixture is then cooled down to ambient
temperature. The reaction medium is then immediately adsorbed on
silica and the solvent eliminated by evaporation. The compound is
obtained pure by column chromatography (AcEt/MeOH 85/15). 255 mg of
a white solid is obtained.
[0170] Rf: 0.48 (AcOEt/MeOH 85/15)
[0171] The yield is 78%.
[0172] NMR .sup.1H (300.13 MHz, DMSO d.sub.6): .delta. 0.83 (t, j=6
Hz, 3H, CH.sub.3), .delta. 1.24 (m, 6H, CH.sub.2), .delta. 1.68 (m,
2H, CH.sub.2), .delta. 1.81 (s, 3H, CH.sub.3), .delta. 4.06 (m, 1H,
CH), .delta. 4.27 (m, 1H, CH), .delta. 4.62 (m, 3H, CH.sub.2, CH),
.delta. 5.2 (d, j=6 Hz, 1H, OH), .delta. 5.5 (s, 1H, OH), .delta.
6.17 (t, j=6 Hz, 1H, CH), .delta. 7.37 (s, 1H, CH), .delta. 7.89
(s, 1H, CH).
[0173] NMR .sup.13C (75.47 MHz, DMSO d.sub.6): .delta. 12.5
(CH.sub.3), .delta. 14.4 (CH.sub.3), .delta. 22.6 (CH.sub.2),
.delta. 25.1, .delta. 31.7 (CH.sub.2), .delta. 38.4 (CH.sub.2),
.delta. 51.6 (CH.sub.2--N), .delta. 66.0 (CH--O), .delta. 71.3 (C),
.delta. 84.4, .delta. 84.5, .delta. 110.3 (.dbd.C--N), .delta.
122.8, .delta. 136.4, .delta. 150.9, .delta. 152.4, .delta.
164.1.
[0174] High resolution MS [M+Na].sup.+: calculated mass: 416.1910,
measured mass: 416.1897
EXAMPLE 6
5'-(4-(hexyl)-[1,2,3]triazol-1-yl)-5',2'dideoxythymidine
##STR00017##
[0176] 200 mg of 5'-azido-5'-deoxythymidine as described in
Preparation 3 (0.75 mmol) and 82.65 mg of non-1-yne (1 equivalent)
in a 0.1 M solution are introduced into a THF water mixture (1/1)
in a flask. The following are added successively: 30 mg of sodium
ascorbate (0.2 equivalent, 0.15 mmol) and 12 mg of copper sulphate
(0.1 equivalent, 0.075 mmol). The reaction medium is stirred and
heated at 60.degree. C. for 5 hours. The mixture is then cooled
down to ambient temperature. The reaction medium is immediately
adsorbed on silica and the solvent eliminated by evaporation. The
compound is obtained pure by column chromatography (AcOEt/MeOH
8/2). 130 mg of a white solid is obtained.
[0177] Rf: 0.62 (AcOEt/MeOH 8/2)
[0178] The yield is 46%.
[0179] High resolution MS [M+H].sup.+: calculated mass: 576.4125,
measured mass: 576.4120
EXAMPLE 7
5'-(4-(heptyl)-[1,2,3]triazol-1-yl)-5',2'dideoxythymidine
##STR00018##
[0181] 200 mg of 5'-azido-5'-deoxythymidine as described in
Preparation 3 (0.75 mmol) and 93 mg of non-1-yne (1 equivalent) in
a 0.1 M solution are introduced into a THF water mixture (1/1) in a
flask. The following are added successively: 30 mg of sodium
ascorbate (0.2 equivalent, 0.15 mmol) and 12 mg of copper sulphate
(0.1 equivalent, 0.075 mmol). The reaction medium is stirred and
heated at 60.degree. C. for 5 hours. The mixture is then cooled
down to ambient temperature. The reaction medium is immediately
adsorbed on silica and the solvent eliminated by evaporation. The
compound is obtained pure by column chromatography (AcOEt/MeOH
85/15). 120 mg of a white solid is obtained.
[0182] Rf: (AcOEt/MeOH 85/15)
[0183] The yield is 41%.
[0184] NMR .sup.1H (300.13 MHz, CDCl.sub.3): .delta. 0.83 (t, j=6
Hz, 3H, CH.sub.3), .delta. 1.25 (m, 8H, CH.sub.2), .delta. 1.55 (m,
2H, CH.sub.2), .delta. 1.79 (s, 3H, CH.sub.3), .delta. 2.10 (t, j=6
Hz, 2H, CH.sub.2), .delta. 2.61 (t, j=6 Hz, 2H, CH.sub.2), .delta.
4.06 (s, 1H, CH), .delta. 4.27 (s, 1H, CH), .delta. 4.59 (m, 2H,
CH.sub.2), .delta. 5.5 (s, 1H, OH), .delta. 6.16 (t, j=6 Hz, 1H,
CH), .delta. 7.29 (s, 1H, CH), .delta. 7.82 (s, 1H, CH), .delta.
11.31 (s, 1H, NH).
[0185] NMR .sup.13C (75.47 MHz, DMSO d.sub.6): .delta. 12.6
(CH.sub.3), .delta. 14.4 (CH.sub.3), .delta. 22.5 (CH.sub.2),
.delta. 25.4 (CH.sub.2), .delta. 28.9 (CH.sub.2), .delta. 29.0
(CH.sub.2), .delta. 29.4 (CH.sub.2), .delta. 31.7 (CH.sub.2),
.delta. 38.4 (CH.sub.2), .delta. 51.5 (CH.sub.2), .delta. 71.1
(CH), .delta. 84.4 (CH), .delta. 110 (C), .delta. 123.1 (CH),
.delta. 136.5 (CH), .delta. 147.4 (C), .delta. 150.9 (C.dbd.O),
.delta. 164.1 (C.dbd.O).
EXAMPLE 8
5'-(4-(2,2-ditridecyl-[1,3]dioxolan-4-ylmethoxymethyl)-[1,2,3]triazol-1-yl-
)-5',2'dideoxythymidine
##STR00019##
[0187] 264 mg of 5'-azido-5'-deoxythymidine as described in
Preparation 3 (1 mmol) and 500 mg of
4-Prop-2-ynyloxymethyl-2,2-ditridecyl-[1,3]dioxolane (1 equivalent)
in a 0.1 M solution are introduced into a THF water mixture (1/1)
in a flask. The following are added successively: 39.5 mg of sodium
ascorbate (0.2 equivalent, 0.2 mmol) and 16 mg of copper sulphate
(0.1 equivalent, 0.1 mmol). The reaction medium is stirred and
heated at 60.degree. C. for 5 hours. The mixture is then cooled
down to ambient temperature. The reaction medium is then
immediately adsorbed on silica and the solvent eliminated by
evaporation. The compound is obtained pure by column chromatography
(AcEt/MeOH 8/2). 550 mg of a white solid are obtained.
[0188] The yield is 72%.
[0189] High resolution MS [M+H].sup.+: calculated mass: 774.5745,
measured mass: 774.5739
EXAMPLE 9
5'-(4-(2,2-ditridecyl-[1,3]dioxolan-4-ylbutoxymethyl)-[1,2,3]triazol-1-yl)-
-5',2'dideoxythymidine
##STR00020##
[0191] 195 mg of 5'-azido-5'-deoxythymidine as described in
Preparation 3 (0.73 mmol) and 400 mg of
4-(4-Prop-2-ynyloxy-butyl)-2,2-ditridecyl-[1,3]dioxolane (1
equivalent) in a 0.1 M solution are introduced into a THF water
mixture (1/1) in a flask. The following are added successively: 30
mg of sodium ascorbate (0.2 equivalent, 0.15 mmol) and 12 mg of
copper sulphate (0.1 equivalent, 0.073 mmol). The reaction medium
is stirred and heated at 60.degree. C. for 5 hours. The mixture is
then cooled down to ambient temperature. The reaction medium is
immediately adsorbed on silica and the solvent eliminated by
evaporation. The compound is obtained pure by column chromatography
(AcOEt/MeOH 9/1). 180 mg of a white solid is obtained.
[0192] Rf: 0.43 (AcOEt/MeOH 9/1)
[0193] The yield is 30%.
EXAMPLE 10
5'-(4-((O-cholesteryl)-methyl)-[1,2,3]triazol-1-yl)-5',2'dideoxythymidine
##STR00021##
[0195] 170 mg of 5'-azido-5'-deoxythymidine as described in
Preparation 3 (0.63 mmol) and 270 mg of o-propargylcholesterol as
described in Preparation 7 (1 equivalent) in a 0.1 M solution are
introduced into a THF water mixture (1/1) in a flask. The following
are added successively: 20 mg of sodium ascorbate (0.2 equivalent,
0.13 mmol) and 10 mg of copper sulphate (0.1 equivalent, 0.063
mmol). The reaction medium is stirred and heated at 60.degree. C.
for 5 hours. The mixture is cooled down to ambient temperature. The
reaction medium is immediately adsorbed on silica and the solvent
eliminated by evaporation. The compound is obtained pure by column
chromatography (AcOEt/MeOH 8/2). 260 mg of a white solid is
obtained.
[0196] Rf: 0.57 (AcOEt/MeOH 8/2)
[0197] The yield is 59%.
[0198] MS [M+H].sup.+: 692.3
EXAMPLE 11
1,12-bis-[5'-(4-(methyl)-[1,2,3]triazol-1-yl)-5',2'dideoxythymidine]-oxydo-
decane
##STR00022##
[0200] 100 mg of 5'-azido-5'-deoxythymidine as described in
Preparation 3 (0.375 mmol) and 52 mg of 1,12-dipropargyloxydodecane
prepared from the compound described in Preparation 6 (0.5
equivalent) in a 0.1 M solution are introduced into a THF water
mixture (1/1) in a flask. The following are added successively: 15
mg of sodium ascorbate (0.2 equivalent, 0.075 mmol) and 6 mg of
copper sulphate (0.1 equivalent, 0.0375 mmol). The reaction medium
is stirred and heated at 60.degree. C. for 5 hours. The mixture is
then cooled down to ambient temperature. The reaction medium is
immediately adsorbed on silica and the solvent eliminated by
evaporation. The compound is obtained pure by column chromatography
(AcOEt/MeOH 8/2). 90 mg of a white solid is obtained.
[0201] The yield is 59%.
[0202] NMR .sup.1H (300.13 MHz, MeOH d.sub.4): .delta. 1.28 (m,
16H, CH.sub.2), .delta. 0.83 (m, 4H, CH.sub.2), .delta. 1.89 (s,
6H, CH.sub.3), .delta. 2.17 (s, 2H, CH.sub.2), .delta. 2.25 (m, 4H,
CH.sub.2), .delta. 3.51 (t, j=6 Hz, 4H, CH.sub.2), .delta. 4.18 (m,
2H, CH) .delta. 4.42 (m, 2H, OH) .delta. 4.58 (s, 4H, CH.sub.2),
.delta. 4.76 (qd, j=6 Hz, 4H, CH.sub.2), .delta. 6.21 (t, j=6 Hz,
2H, CH), .delta. 7.23 (s, 2H, CH), .delta. 7.99 (s, 2H, CH).
EXAMPLE 12
5'-(4-(1(R)-hydroxy-hexyl)-[1,2,3]triazol-1-yl)-5',2'dideoxythymidine
##STR00023##
[0204] 215 mg of 5'-azido-5'-deoxythymidine as described in
Preparation 3 (0.8 mmol) and 101.5 mg of (R) oct-1-yn-3-ol (1
equivalent) in a 0.1 M solution are introduced into a THF water
mixture (1/1) in a flask. The following are added successively:
31.5 mg of sodium ascorbate (0.2 equivalent, 0.15 mmol) and 13 mg
of copper sulphate (0.1 equivalent, 0.075 mmol). The reaction
medium is stirred and heated at 60.degree. C. for 5 hours. The
mixture is then cooled down to ambient temperature. The reaction
medium is then immediately adsorbed on silica and the solvent
eliminated by evaporation. The compound is obtained pure by column
chromatography (AcEt/MeOH 9/1). 240 mg of a white solid is
obtained.
[0205] Rf: 0.48 (AcEt/MeOH 9/1)
[0206] The yield is 76%.
[0207] High resolution MS [M+H].sup.+: calculated mass: 576.4125,
measured mass: 576.4120
EXAMPLE 13
5'-(4-(1(S)-hydroxy-hexyl)-[1,2,3]triazol-1-yl)-5',2'dideoxythymidine
##STR00024##
[0209] 215 mg of 5'-azido-5'-deoxythymidine as described in
Preparation 3 (0.8 mmol) and 101.5 mg of (S) oct-1-yn-3-ol (1
equivalent) in a 0.1 M solution are introduced into a THF water
mixture (1/1) in a flask. The following are added successively:
31.5 mg of sodium ascorbate (0.2 equivalent, 0.15 mmol) and 13 mg
of copper sulphate (0.1 equivalent, 0.075 mmol). The reaction
medium is stirred and heated at 60.degree. C. for 5 hours. The
mixture is then cooled down to ambient temperature. The reaction
medium is then immediately adsorbed on silica and the solvent
eliminated by evaporation. The compound is obtained pure by column
chromatography (AcOEt/MeOH 85/15). 255 mg of a white solid are
obtained.
[0210] Rf: 0.48 (AcOEt/MeOH 85/15)
[0211] The yield is 78%.
[0212] High resolution MS [M+H].sup.+: calculated mass: 576.4125,
measured mass: 576.4120
EXAMPLE 14
5'-(4-(1-hydroxy-hexyl)-[1,2,3]triazol-1-yl)-5',2'dideoxyadenosine
##STR00025##
[0214] 200 mg of 5'-azido-5'-deoxythymidine as described in
Preparation 3 (0.75 mmol) and 95 mg of the racemic mixture of
oct-1-yn-3-ol (1 equivalent) in a 0.1 M solution are introduced
into a THF water mixture (1/1) in a flask. The following are added
successively: 30 mg of sodium ascorbate (0.2 equivalent, 0.15 mmol)
and 12 mg of copper sulphate (0.1 equivalent, 0.075 mmol). The
reaction medium is stirred and heated at 60.degree. C. for 5 hours.
The mixture is then cooled down to ambient temperature. The
reaction medium is immediately adsorbed on silica and the solvent
eliminated by evaporation. The compound is obtained pure by column
chromatography (AcOEt/MeOH 8/2). 240 mg of a white solid is
obtained.
[0215] Rf: 0.47 (AcOEt/MeOH 8/2)
[0216] The yield is 80%.
[0217] High resolution MS [M+H].sup.+: calculated mass: 576.4125,
measured mass: 576.4120
EXAMPLE 15
Preparation of the Nanoparticles
[0218] The compound thymidine
3'-(1,2-dipalmitoyl-sn-glycero-3-phosphate) (di C16 dT) prepared in
Example 2 was used as a compound of formula (I) and
dioleylphosphatidylcholine (DOPC) as a co-lipid.
[0219] 1) Preparation of the Stock Solutions
[0220] a) Preparation of the Cis-Platin Solution:
[0221] 15 mg of cis-platin are solubilized in 10 mL of milli-Q
water (5 mM). This suspension is stirred for 1 min (vortex), then
incubated at 37.degree. C. for 24 h.
[0222] b) Preparation of the Lipid Solutions:
[0223] Solution A: 20 mg of diC16dT are solubilized in 2 mL of
chloroform (10 mg/mL). This sample is stored at -20.degree. C.
[0224] Solution B: DOPC: solution at 20 mg/mL in chloroform, stored
at -20.degree. C.
[0225] 2) Preparation of the Lipid Formulations
[0226] 52.3 .mu.L of solution A are mixed with 23.6 .mu.L of
solution B in a 2 mL Eppendorf.RTM. tube. These volumes correspond
to a molar ratio of 1/1.
[0227] The chloroform is evaporated off under nitrogen in order to
obtain a homogeneous lipid film.
[0228] 3) Preparation of the Nanoparticles
[0229] 1.2 mL of the cis-platin solution pre-incubated at
37.degree. C. are used to rehydrate the lipid film prepared
beforehand. The mixture is incubated at 37.degree. C. for 30 min. A
series of 10 heating (water bath at 45.degree. C.) and freezing
(dry ice/methanol -78.degree. C.) cycles is carried out.
[0230] 4) Washing and Recovery of the Nanoparticles
[0231] Once the series of 10 cycles is completed, the tube is
centrifuged at 2100 rpm at 4.degree. C. for 5 min. After
elimination of the supernatant the pellet is re-suspended in 1 mL
of milli-Q water. A second centrifugation is carried out (2100 rpm
at 4.degree. C. for 5 min), then the supernatant is removed and the
pellet is dried.
[0232] The concentration (ICP/Optical) of the pellet re-suspended
in 1 mL of milli-Q water is 2.844 mM equivalent in cis-platin
(852.2 mg/L). This concentration corresponds to 47.4% of the
cis-platin initially used.
EXAMPLE 16
Preparation of the Nanocapsules
[0233] The compound thymidine
3'-(1,2-dipalmitoyl-sn-glycero-3-phosphate) (di C16 dT) prepared in
Example 2 is used as a compound of formula (I) and
dioleylphosphatidylcholine (DOPC) as a co-lipid.
[0234] 1) Preparation of the Stock Solutions
[0235] a) Preparation of the Cis-Platin Solution:
[0236] 15 mg of cis-platin are solubilized in 10 mL of milli-Q
water (5 mM). This suspension is stirred for 1 min (vortex), then
incubated at 37.degree. C. for 24 h, stirring from time to
time.
[0237] b) Preparation of the Lipid Solutions:
[0238] Solution A: 20 mg of diC16dT are solubilized in 2 mL of
dichloromethane (10 mg/mL). The solution is stored at -20.degree.
C.
[0239] Solution B: DOPC: solution at 20 mg/mL in dichloromethane,
stored at -20.degree. C.
[0240] 2) Preparation of the Lipid Formulations
[0241] 52.3 .mu.L of solution A are mixed with 47.2 .mu.L of
solution B in a 2 mL Eppendorf.RTM. tube. These volumes correspond
to a molar ratio of 1/1.
[0242] The dichloromethane is evaporated off with compressed
nitrogen in order to obtain a homogeneous lipid film.
[0243] 3) Preparation of the Nanoparticles
[0244] 1.2 mL of the cis-platin solution (5 mM) are incubated
overnight at ambient temperature with the lipid film prepared
beforehand, without stirring. A series of 10 heating (water bath at
45.degree. C.) and freezing (dry ice/ethanol -78.degree. C.) cycles
is carried out.
[0245] 4) Washing and Recovery of the Nanoparticles
[0246] Once the series of 10 cycles is completed, the suspension is
stirred and placed in a glass haemolysis tube, then subjected to
sonication for 5 min. After sonication, the suspension is
centrifuged at 1000 rpm/2.5 min/20.degree. C. in order to remove
the large capsules which are to be found in the pellet which will
be eliminated. The supernatant is again centrifuged at 10,000 rpm/5
min/20.degree. C. The nanoparticles are to be found in the pellet.
The latter is resuspended in 1 mL of milli-Q water and a second
centrifugation is carried out. The pellet is suspended in 1 mL and
subjected to an Inductively Coupled Plasma (ICP) Optical assay.
EXAMPLE 17
Stability Test
[0247] The nanoparticles prepared according to the protocol of
Example 16 are assayed by ICP optical spectrometry (the measured
value corresponds to the total concentration). The suspension of
the nanoparticles is aliquoted into 5 Eppendorf.RTM. tubes (150
.mu.L). The latter are incubated at 37.degree. C. under stirring
(300 rpm) for different periods of time (0, 2.5, 5, 10 and 24
hours).
[0248] At a given time (x), the tube is centrifuged at 14,000
rpm/10 min/20.degree. C. and 50 .mu.L of supernatant (recovered
carefully so as not to resuspend the pellet) are assayed.
[0249] Nanoparticles based on
1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS) with
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) as a co-lipid are
prepared according to the same protocol as a comparison.
[0250] The percentage of release of cis-platin is calculated
according to the following equation:
% of cis-platin released=Cx-C0/Ct-C0
Cx: concentration found at a given time (x). C0: concentration
found in the supernatant before incubation. CT: total concentration
found without incubation and without centrifugation.
[0251] The curve of the release of cis-platin as a function of the
incubation time is represented in FIG. 1. The nanoparticles of
Example 16 are represented by the symbol --O-- and the
nanoparticles based on DOPC/DOPS by the symbol
[0252] The results show that the half life (incubation time
necessary to release 50% of the cis-platin) is greater than 24 h
for the nanoparticles according to the invention, whereas it is of
the order of 6.5 h for the nanoparticles based on DOPC/DOPS.
EXAMPLE 18
Measurement of the Size of the Nanoparticles
[0253] The nanoparticles prepared according to the protocol of
Example 16 were analyzed with a MALVERN zetasizer.
[0254] The nanoparticles are suspended in 2 mL of milli-Q water
(volume necessary for the size measurement). The concentration is
approximately 0.5 mM (the suspension must be cloudy in order to
diffuse the light). Single-use cuvettes (1 cm/1 cm) are used for
the measurement.
[0255] The results show that more than 95% of the nanoparticles
have a size between 100 and 250 nm with a polydispersity of
0.228.
EXAMPLE 19
Assay of Intracellular Cis-Platin
[0256] Protocol
[0257] IGROV1 cells (ovarian adenocarcinoma line) at 80% confluence
(dish 10 cm in diameter) are treated with 100 .mu.M of free
cis-platin or cis-platin encapsulated in the nanoparticles of
Example 16 for 2, 4 or 6 h. On completion of this treatment two
washings with PBS are carried out. The cells are treated with
trypsin and resuspended in PBS. Two washings of the cell
suspensions with PBS are carried out (centrifugation 1000 rpm/1
min). The cells are suspended in 1 mL of PBS and counted.
[0258] ICP Optical Assay
[0259] 10.sup.6 cells are lysed with 500 .mu.L of the cell lysis
solution (lysis buffer from SIGMA). The volume is topped up to 5 mL
with milli-Q water with 1% HNO.sub.3 acid.
[0260] Results
[0261] The results are represented in FIG. 2, which shows the
concentration of cis-platin released after cell lysis as a function
of time, corresponding to the concentration of cis-platin
internalized in the treated cells.
[0262] For each period of time, the hatched column (on the left)
corresponds to the free cis-platin and the dotted column (on the
right) to the nanoparticles containing cis-platin.
[0263] The results show that the internalization of cis-platin is
clearly more effective in the presence of the nanoparticles. For
example, under identical conditions (10.sup.6 cells, 100 .mu.M, 2
h) 0.5 nanomole of cis-platin is internalized in the case of the
free cis-platin whereas the internalization is 4.5 times greater in
the case of the nanoparticles (2.3 nanomoles).
EXAMPLE 20
Study of the Cytotoxic Effects of the Nanoparticles Prepared
According to the Invention
[0264] The nanoparticles were prepared according to the process
described in Example 15. Compound C20dT of formula
##STR00026##
[0265] in which n=18,
[0266] described in Nathalie Campins et al., New J. Chem 2007, 31,
1928-1934 is used as a compound of formula (I).
[0267] Protocol
[0268] The test implemented uses the human cancer cell line HCT8
(colorectal adenocarcinoma), known for its intrinsic resistance to
platinum derivatives.
[0269] The protocol is as follows: [0270] On D3, the cells are
implanted in 96-well plates at a density of 50,000 cells per well.
[0271] On D0, the cells are treated by the formulation test, either
for a short exposure period (30 min) in physiological serum, or for
a long exposure period (72 h) in culture medium. The range of
concentrations produced comprises the points
0-0.4-0.8-1.5-3-6.25-12.5-25-50 and 100 mg/L. Negative controls
(untreated cells) and positive controls (cis-platin) were carried
out simultaneously. [0272] On D3, the culture medium is changed,
and the 72 h treatment stopped. [0273] On D6, the protocol is
completed, and the cells remaining at the bottom of the well are
stained with crystal violet.
[0274] Expression of the Results
[0275] The survival of the cells was determined, and the overall
percentage of death (or cytotoxicity) obtained at the different
test formulation concentrations was compared with that obtained
with the negative controls (untreated cells). The cytotoxicity
results were presented in the form of dose-effect curves
characterized by their inhibitory concentration 50 (IC.sub.50),
i.e. the concentration at which 50% dead cells are observed.
[0276] Preliminary Results IC.sub.50 at 30 Minutes
[0277] The results are given in Table 1 below.
TABLE-US-00001 TABLE 1 Molar ratio of the Compound (I) Co-lipid
mixture IC.sub.50 (mg/L) None -- Not determined (Cis-platin alone)
IC.sub.50 > at 100 mg/L C20dT DOPC DOPC/C20dT 33 mg/L) 1:1
[0278] The results show that the nanoparticles according to the
invention, containing the compound of formula (I) C20dT, exhibit in
this test, a cytotoxicity at 30 min expressed by the IC.sub.50 at a
dose significantly less than that of the cis-platin used alone.
EXAMPLE 21
Study of the Cytotoxic Effects of the Nanoparticles Prepared
According to the Invention
[0279] The following were used as compounds of formula (I): [0280]
diC16dT (Thymidine 3'-(1,2-dipalmitoyl-sn-glycero-3-phosphate) of
Example 2 and a co-lipid (DOPC), or [0281]
5'-(4-(1(R)-hydroxy-hexyl)-[1,2,3]triazol-1-yl)-5',2'dideoxythymidine
of Example 12, but without adding a co-lipid.
[0282] The nanoparticles were prepared according to the process
described in Example 15, but with a single heating/cooling cycle
for the compound of Example 12.
[0283] Protocol
[0284] 1000 to 5000 IGROV1 cells are incubated in 100 .mu.L of
medium with serum per well (96-well plate). After 24 h the medium
is removed and the cells are treated for 1 to 3 days with the
formulations according to the invention and/or free cis-Pt (at the
desired concentrations). The cells are incubated at 37.degree. C.
in 200 .mu.L of medium with serum. The cell viability is revealed
by an MTS colorimetric assay at the end of the treatment.
[0285] Expression of the Results
[0286] The survival of the cells was determined, and the overall
percentage of death (or cytotoxicity) obtained at the different
concentrations of formulations tested was compared with that
obtained with the negative controls (untreated cells). The
cytotoxicity results were presented in the form of dose-effect
curves characterized by their inhibitory concentration 50
(IC.sub.50), i.e. the concentration at which 50% dead cells are
observed.
[0287] The results are reported in Table 2 below, in which the IC50
is expressed either in .mu.M, or in mg/L of cis-platin (incubation
time: 24 h)
TABLE-US-00002 TABLE 2 IC.sub.50 IC.sub.50 compound (I) Co-lipid.
Expressed in mg/L Expressed in .mu.M None -- 9.48 31.6 (Cis-platin
alone) Example 2 DOPC 0.48 1.6 Example 12 -- 0.95 3.16
[0288] The results show that the nanoparticles according to the
invention, containing the compounds of formula (I), prepared in the
presence or absence of co-lipid, exhibit in this test, a
cytotoxicity at 24 h expressed by the IC.sub.50 at a dose
significantly less than that of the cis-platin used alone.
EXAMPLE 22
Study of the Cytotoxic Effects of the Nanoparticles Prepared
According to the Invention
[0289] Protocol
[0290] a/ Preparation and Treatment of the Cells:
[0291] 2500 cells (IGROV1, SKOV3, ovarian adenocarcinoma lines) per
well are incubated in 100 .mu.L of the medium with serum in a
96-well plate. After 24 hours the medium is aspirated and the cells
are treated with free cis-platin or cis-platin encapsulated in the
nanoparticles of Example 16 in 100 .mu.L of the medium without
serum at different concentrations (500, 100, 10, 1, 0.1, 0.01,
0.001 .mu.M). After treatment for 24 hours, the medium is removed
and the cells are washed twice with 100 .mu.L of PBS then incubated
with 100 .mu.L of the medium with serum.
[0292] b/ Revelation of the Toxicity:
[0293] 48 hours after the two washings, the cell viability is
revealed by adding 20 .mu.L of MTS. The absorbance at 490 nm is
measured after incubation for 2 to 4 hours at 37.degree. C. The
absorbance is proportional to the cell viability.
[0294] c) Results:
[0295] The results are represented in FIG. 3, which shows the
concentration necessary to obtain 50% cell death (IC50) with free
cis-platin (column A) or the nanoparticles containing cis-platin
(column B).
[0296] FIG. 3A relates to the cell line IGROV1 and FIG. 3B relates
to the cell line SKOV3.
[0297] The results show that the nanoparticles of Example 16
containing cis-platin are more effective than the free cis-platin
in the two cell lines, IGROV1 (sensitive to cis-platin) and SKOV3
(resistant to cis-platin).
[0298] On the line IGROV1, 50% cell death is obtained with 0.27
.mu.M of nanoparticles containing cis-platin whereas it is
necessary to use 2.41 .mu.M of free cis-platin in order to obtain
this result. On the line SKOV3, 50% cell death is obtained with
0.54 .mu.M of nanoparticles containing cis-platin as against 4.3
.mu.M of free cis-platin.
[0299] The nanoparticles containing cis-platin are respectively 9
and 8 times more effective than the free cis-platin on the lines
IGROV1 and SKOV3.
* * * * *