U.S. patent application number 12/624734 was filed with the patent office on 2010-03-18 for phosphohalohydrins, process for the production thereof and use thereof.
This patent application is currently assigned to Institut National de la Sante et de la Recherche Medicale. Invention is credited to Christian Belmant, Marc Bonneville, Jean-Jacques Fournie, Marie-Alix Peyrat.
Application Number | 20100068179 12/624734 |
Document ID | / |
Family ID | 9530019 |
Filed Date | 2010-03-18 |
United States Patent
Application |
20100068179 |
Kind Code |
A1 |
Belmant; Christian ; et
al. |
March 18, 2010 |
Phosphohalohydrins, Process For The Production Thereof And Use
Thereof
Abstract
The invention provides compounds comprising at least one
phosphohalohydrin group of the formula: ##STR00001## where X is a
halogen selected from among I, Br, Cl, R1 is selected from among
--CH.sub.3 and --CH.sub.2--CH.sub.3, Cat+ is an organic or
inorganic cation, and n is an integer between 2 and 20, processes
for the production thereof and uses theroef, in particular
therapeutic uses and for activating primate T.gamma.9.delta.2
lymphocytes.
Inventors: |
Belmant; Christian;
(Six-Fours-les-Plages, FR) ; Fournie; Jean-Jacques;
(Corronsac, FR) ; Bonneville; Marc; (Vertou,
FR) ; Peyrat; Marie-Alix; (Saint-Sebastien Sur Loire,
FR) |
Correspondence
Address: |
SALIWANCHIK LLOYD & SALIWANCHIK;A PROFESSIONAL ASSOCIATION
PO Box 142950
GAINESVILLE
FL
32614
US
|
Assignee: |
Institut National de la Sante et de
la Recherche Medicale
Paris
FR
|
Family ID: |
9530019 |
Appl. No.: |
12/624734 |
Filed: |
November 24, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11512688 |
Aug 30, 2006 |
7625879 |
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12624734 |
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10682990 |
Oct 14, 2003 |
7109183 |
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11512688 |
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09786054 |
Mar 1, 2001 |
6660723 |
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PCT/FR99/02058 |
Aug 27, 1999 |
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10682990 |
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Current U.S.
Class: |
424/85.2 ;
514/106; 514/51; 536/26.14; 558/156 |
Current CPC
Class: |
C07H 19/10 20130101;
A61K 39/00 20130101; C12N 2501/23 20130101; A61P 31/18 20180101;
A61P 33/00 20180101; C12N 5/0636 20130101; A61P 35/00 20180101;
Y02A 50/412 20180101; A61K 45/06 20130101; A61P 31/00 20180101;
C07F 9/098 20130101; C12N 2501/999 20130101; Y02A 50/30 20180101;
A61K 31/66 20130101; A61P 37/04 20180101; A61K 2039/55511 20130101;
A61K 2035/124 20130101 |
Class at
Publication: |
424/85.2 ;
558/156; 536/26.14; 514/106; 514/51 |
International
Class: |
A61K 31/6615 20060101
A61K031/6615; C07F 9/09 20060101 C07F009/09; C07H 19/06 20060101
C07H019/06; A61K 31/7072 20060101 A61K031/7072; A61K 38/20 20060101
A61K038/20; A61P 35/00 20060101 A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 1, 1998 |
FR |
98/10913 |
Claims
1. A compound selected from the group consisting of: ##STR00039##
wherein X is a halogen; n is 2 to 20; Cat.sup.+ is a cation; R1 is
methyl or ethyl; and ##STR00040## where X is a halogen selected
from the group consisting of I, Br and Cl, R1 is methyl or ethyl,
Cat.sup.+ is a cation, n is an integer between 2 and 20, R3 is: a
halohydrin group of the formula: ##STR00041## an epoxide group of
the formula: ##STR00042## an alkene group of the formula:
##STR00043## m being an integer between 1 and 20.
2. The compound according to claim 1, wherein n is 2 and R1 is
methyl.
3. A method of treating a disease comprising administering to a
patient in need of treatment a composition comprising an amount of
a phosphohalohydrin compound according to claim 1 effective to
activate T.gamma.9.delta.2 lymphocytes
4. The method according to claim 3, wherein the method further
comprises administering an effective amount of IL-2 in combination
with said phosphohalohydrin compound.
5. A method of treating cancer in a patient comprising
administering to a patient in need thereof a composition comprising
an effective amount of a phosphohalohydrin compound according to
claim 1.
6. The method according to claim 5, wherein the method further
comprises administering an effective amount of IL-2 in combination
with said phosphohalohydrin compound.
7. A method of treating infectious diseases in a patient comprising
administering to a patient in need thereof a composition comprising
an effective amount of a phosphohalohydrin compound according to
claim 1.
8. The method according to claim 7, wherein the method further
comprises administering an effective amount of IL-2 in combination
with said phosphohalohydrin compound.
9. A process for the extracorporeal activation of T.gamma.9.delta.2
lymphocytes, comprising contacting T.gamma.9.delta.2 lymphocytes
with a phosphohalohydrin compound of the formula: ##STR00044##
wherein X is a halogen; n is 2 to 20; Cat.sup.+ is a cation; R1 is
methyl or ethyl; in an extracorporeal medium which contains
T.gamma.9.delta.2 lymphocytes, IL-2 and is compatible with T
lymphocyte growth to induce the proliferation of said
T.gamma.9.delta.2 lymphocytes.
10. A process for inducing the extracorporeal proliferation of
T.gamma.9.delta.2 lymphocytes, comprising contacting
T.gamma.9.delta.2 lymphocytes with a phosphohalohydrin compound of
the formula: ##STR00045## wherein X is a halogen; n is 2 to 20;
Cat.sup.+ is a cation; R1 is methyl or ethyl; and IL-2 in an
extracorporeal medium which contains T.gamma.9.delta.2 lymphocytes
and is compatible with T lymphocyte growth.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/512,688, filed Aug. 30, 2006, now U.S. Pat.
No. 7,625,879, which is a divisional of U.S. patent application
Ser. No. 10/682,990, filed Oct. 14, 2003, now U.S. Pat. No.
7,109,183, which is a divisional of U.S. patent application Ser.
No. 09/786,054, filed on Mar. 1, 2001, now U.S. Pat. No. 6,660,723,
which is the national stage application of PCT International
Application No. PCT/FR99/02058, filed on Aug. 27, 1999. The entire
contents of each of the above-identified applications are hereby
incorporated by reference.
[0002] This invention relates to novel phosphohalohydrins, to the
process for the production thereof and to the use thereof for
stimulating T.gamma.9.delta.2 lymphocytes bearing TCR receptors
with V.gamma.9 and V.delta.2 variable regions.
[0003] In healthy individuals, the T.gamma..delta. lymphocytes of
primates (humans, monkeys) present in the peripheral bloodstream
usually constitute from 1 to 5% of the lymphocytes in the blood and
play a role in the immune system. It has been demonstrated that
they recognize their antigenic ligands by direct interaction with
the antigen without presentation by molecules of the MHC by a
presenting cell. T.gamma.9.delta.2 lymphocytes (sometimes also
known as T.gamma.2.delta.2 lymphocytes) are T.gamma..delta.
lymphocytes bearing TCR receptors with V.gamma.9 and V.gamma.2
variable regions. They constitute the majority of T.gamma..delta.
lymphocytes in human blood.
[0004] When activated, T.gamma..delta. lymphocytes exercise a
strong cytotoxic activity which is unrestrained by the MHC and is
particularly effective in killing various types of cells, in
particular pathogenic cells. These may be cells infected by viruses
(".gamma..delta. T cell activation or anergy during infections: the
role of nonpeptidic TCR ligands and HLA class I molecules" Fabrizio
POCCIA et al, Journal of Leukocyte Biology, 62, 1997, p. 1-5), or
by other intracellular parasites, such as mycobacteria ("The
antituberculous Mycobacterium bovis BCG Vaccine is an attenuated
Mycobacterial producer of phosphorylated nonpeptidic Antigens for
human .gamma..delta. T cells" Patricia CONSTANT et al, Infection
and Immunity, vol. 63, no. 12, December 1995, p. 4628-4633); or by
protozoans ("Plasmodium falciparum stimuli for human .gamma..delta.
T Cells are related to phosphorylated Antigens of mycobacteria"
Charlotte BEHR et al, Infection and Immunity, Vol. 64, no. 8, 1996,
p. 2892-2896). They may also be cancer cells ("CD94/NKG2 inhibitory
receptor complex modulates both antiviral and antitumoral responses
of polyclonal phosphoantigen-reactive V.gamma.9 V.delta.2 T
lymphocytes" Fabrizio POCCIA et al, Journal of Immunology, 159, p.
6009-6015; "Stimulation of .gamma..delta. T cells by
phosphoantigens" Jean-Jacques FOURNIE, Marc BONNEVILLE, Res.
Immunol., 66.sup.th FORUM IN IMMUNOLOGY, 147 p. 338-347).
[0005] It has been demonstrated that, in the event of a
mycobacterial infection, human T.gamma.9.delta.2 lymphocytes react
to four natural, nonpeptidic molecules of a phosphorylated
structure, known as phosphoantigens, which exhibit stimulation
activity at a concentration of 1 to 5 nM (nanomolar) (WO-95/20673
and "Stimulation of human .gamma..delta. T cells by nonpeptidic
Mycobacterial ligands" Patricia CONSTANT et al, Science, 264, p.
267-270).
[0006] These natural antigens have not been completely identified.
Certain authors have erroneously presented them as alkene
derivatives of pyrophosphate, in particular isopentenyl
pyrophosphate IPP (U.S. Pat. No. 5,639,653 and "Natural and
Synthetic nonpeptide antigens recognized by human .gamma..delta. T
cells", Yoshimasa TANAKA et al, Nature, 375, 1995, p. 155-158). It
has nonetheless now been demonstrated that none of these prenyl
pyrophosphates is active at a concentration of nanomolar magnitude.
The best results which have been obtained have been unable to
demonstrate activity at below 3 .mu.M for IPP and below 0.3 .mu.M
for dimethylallyl-UTP and 3-methyl-2-hexene pyrophosphate. The
minimum active concentration of these compounds is thus, at best,
of the order to 100 times higher than that of natural
phosphoantigens.
[0007] With regard to IPP, it should in particular be noted that
the most recent of the above-stated publications make the mistake
of deducing the structure of the isopentenyl radical solely on the
basis of mass spectrometry and the detection of a certain level of
bioactivity. Indeed, apart from the fact that the compound analyzed
in these publications was not purified and that a mass spectrum
cannot identify uncharged species, it may be demonstrated that
there are in fact several thousand different chemical structures
which may have the same molecular weight and be a substituent of
pyrophosphate in these molecules.
[0008] The fact that the minimum active concentration for IPP is
much higher (some 1000 times higher) and that the intensity of the
T.gamma.9.delta.2 lymphocyte responses obtained is much weaker than
that for natural phosphoantigens demonstrates that IPP is not one
of these natural phosphoantigens ("A novel nucleotide-containing
antigen for human blood .gamma..delta. T lymphocytes", Y. Poquet et
al, Eur. J. Immunol. 1996, 26, p. 2344-2349). This is moreover
confirmed by numerous other observations: IPP is not found in
sufficient concentration in mycobacterial extracts which stimulate
T.gamma.9.delta.2 lymphocytes; according to "High pH anion exchange
chromatographic analysis of phosphorylated compounds: application
to isolation and characterization of non peptide mycobacterial
antigens", Y. Poquet et al, Anal. Biochem, 243 no. 1, 1996, p.
119-126, IPP does not have the same chromatographic (HPAEC)
characteristics as natural phosphoantigens; IPP and other natural
isoprenoids are produced by all living cells, but these do not
stimulate T.gamma.9.delta.2 lymphocytes.
[0009] Moreover, it is known that substances having bioactivity of
the order of or greater than 1 .mu.M are only rarely compatible
with the economic constraints of operation on an industrial scale.
The synthetic phosphoantigens which have hitherto been proposed
thus cannot be processed on an industrial scale under acceptable
economic conditions.
[0010] Natural phosphoantigens, on the other hand, may only be
produced in very small quantities (WO 95/20673) and, since their
precise chemical structure has still not yet been established, they
cannot be synthesized. Economic industrial scale processing is thus
likewise out of the question, despite their demonstrated great
therapeutic worth.
[0011] The object of the invention is accordingly to provide novel
chemical compounds which activate T.gamma.9.delta.2 lymphocytes at
a minimum activation concentration of below 100 nM, in particular
of the order of 1 nM.
[0012] A further object of the invention is to provide compounds
which may be linked to a large number of organic groups, in
particular to natural or synthetic peptide groups, so as to permit
multifunctional compounds to be obtained.
[0013] A. further object of the invention is to provide such
compounds which may be synthesized simply, quantitatively and at
low cost, i.e. in a manner compatible with the economic constraints
of production on an industrial scale.
[0014] A further object of the invention is to provide an
advantageous synthetic pathway for these compounds.
[0015] A further object of the invention is to provide a process
for the production of the compounds according to the invention.
[0016] A further object of the invention is to suggest uses for the
compounds according to the invention as a T.gamma.9.delta.2
lymphocyte activator and in particular therapeutic uses of the
compounds according to the invention.
[0017] The invention accordingly provides compounds comprising at
least one phosphohalohydrin group of the formula:
##STR00002##
where X is a halogen selected from among iodine, bromine and
chlorine, [0018] R1 is selected from among --CH.sub.3 and
--CH.sub.2--Ch.sub.3, [0019] Cat.sup.+ represents one or more
organic or inorganic cation(s) (including the proton), which may be
identical or different in the same compound, [0020] and n is an
integer between, 2 and 20.
[0021] A compound according to the invention may in particular
comprise one or more phosphohalohydrin group(s) selected from among
the esters of the following groups (TUPA,C nomenclature) or among
the compounds formed from these groups: [0022]
3-(halomethyl)-3-butanol-1-yl diphosphate,
3-(halomethyl)-3-pentanol-1-yl diphosphate,
4-(halomethyl)-4-pentanol-1-yl diphosphate,
4-(halomethyl)-4-hexanol-1-yl diphosphate,
5-(halomethyl)-5-hexanol-1-yl diphosphate,
5-(halomethyl)-4-heptanol-1-yl diphosphate,
6-(halomethyl)-6-heptanol-1-yl diphosphate,
6-(halomethyl)-6-octanol-1-yl diphosphate,
7-(halomethyl)-7-octanol-1-yl diphosphate,
7-(halomethyl)-7-nonanol-1-yl diphosphate,
8-(halomethyl)-8-nonanol-1-yl diphosphate,
8-(halomethyl)-8-decanol-1-yl diphosphate,
9-(halomethyl)-9-decanol-1-yl diphosphate,
9-(halomethyl)-9-undecanol-1-yl diphosphate.
10-(halomethyl)-10-undecanol-1-yl diphosphate,
10-(halomethyl)-10-dodecanol-1-yl diphosphate,
11-(halomethyl)-11-dodecanol-1-yl diphosphate,
11-(halomethyl)-11-tridecanol-1-yl diphosphate, 12
-(halomethyl)-12-tridecanol-1-yl diphosphate,
12-(halomethyl)-12-tetradecanol-1-yl-diphosphate,
13-(halomethyl)-13-tetradecanol-1-yl-diphosphate,
13-(halomethyl)-13-pentadecanol-1-yl diphosphate,
14-(halomethyl)-14-pentadecanol-1-yl diphosphate,
14-(halomethyl)-14-hexadecanol-1-yl diphosphate,
15-(halomethyl)-15-hexadecanol-1-yl diphosphate,
15-(halomethyl)-15-heptadecanol-1-yl diphosphate,
16-(halomethyl)-16-heptadecanol-1-yl diphosphate,
16-(halomethyl)-16-octadecanol-1-yl diphosphate,
17-(halomethyl)-17-octadecanol-1-yl diphosphate,
17-(halomethyl)-17-nonadecanol-1-yl diphosphate,
18-(halomethyl)-18-nonadecanl-1-yl diphosphate,
18-(halomethyl)-18-eicosanol-1yl diphosphate,
19-(halomethyl)-19-eicosanol-1-yl diphosphate,
19-(halomethyl)-11-heneicosanol-1-yl
diphosphate,20-(halomethyl)-20-heneicosanol-1-yl diphosphate, 20
-(halomethyl)-20-docosanol-1-yl diphosphate,
21-(halomethyl)-21-docosanol-1-yl diphosphate,
21-(halomethyl)-21-tricosanol-1-yl diphosphate.
[0023] The phosphohalohydrin compounds in accordance with one of
the following formulae may be mentioned among the compounds
according to the invention:
##STR00003##
where R2 is an organic or inorganic substituent selected from among
the group comprising: [0024] substituents which do not prevent
formation of the halohydrin function
##STR00004##
[0024] starting from the alkene function
##STR00005##
and halogen X.sub.2 in the presence of water; [0025] and
substituents for which there is an R2--O--Y compound which is not
reactive towards the halohydrin function of the compound of the
formula:
##STR00006##
[0025] and selected such that R2--O--Y may react with the terminal
phosphate of this compound (3) in order to obtain the compound
(4).
[0026] Said compounds according to the invention are advantageously
characterized in that n=2 and R1 is CH.sub.3.
[0027] The compounds according to the invention advantageously
additionally comprise at least one group selected from among the
group comprising nucleoside derivatives, oligonucleotides, nucleic
acids (RNA, DNA); amino acids, peptides, proteins, monosaccharides,
oligosaccharides, polysaccharides, fatty acids, simple lipids,
complex lipids, folic acid, tetrahydrofolic acid, phosphoric acids,
inositol, vitamins, co-enzymes, flavonoids, aldehydes, epoxides and
halohydrins.
[0028] In particular, the invention provides the phosphohalohydrin
compounds of the formula (4) above in which R2 is selected from
among the group comprising nucleoside derivatives,
oligonucleotides, nucleic acids (RNA, DNA), amino acids, peptides,
proteins, monosaccharides, oligosaccharides, polysaccharides, fatty
acids, simple lipids, complex lipids, folic acid, tetrahydrofolic
acid, phosphoric acids, inositol, vitamins, co-enzymes, flavonoids,
phosphohalohydrins of the formula (1), aldehydes, epoxides and
halohydrins.
[0029] The invention also provides compounds, the structure of
which incorporates two or more groups of the formula (1), which may
he identical or different, for example monomers, polymers,
oligomers or dendrimers, or more generally molecules with two or
more phosphorylated branches of the formula (1).
[0030] It should be noted that the compounds according to the
invention are esters (monoesters or diesters) of phosphoric acid
(this term encompassing those acids in which phosphorus is in
oxidation state V, namely orthophosphoric acid, pyrophosphoric
acid, metaphosphoric acid, triphosphoric acid, other polyphosphorie
acids).
[0031] The invention provides a process for the production of the
compounds according to the invention. According to the invention,
halogen X.sub.2 is reacted in the presence of water with a starting
compound comprising at least one phosphorylated alkene group of the
formula:
##STR00007##
[0032] Advantageously and according to the invention, a salt formed
from said starting compound is reacted in an aqueous or
aqueous/alcoholic medium, at neutral pH, at a temperature of below
30.degree. C., by mixing with an aqueous solution of the halogen
X.sub.2. Advantageously and according to the invention, the
reaction is performed at atmospheric temperature at a temperature
of between 0.degree. C. and 25.degree. C.
[0033] The starting compounds may themselves be obtained from
alcohol:
##STR00008##
[0034] Advantageously and according to the invention, the starting
compound is a salt of the formula:
##STR00009##
[0035] The pyrophosphohalohydrin compound of the formula (2) is
then obtained.
[0036] An example of a complete reaction scheme for obtaining the
compound (2) from the alcohol (9) is given below:
##STR00010##
where TsCl is tosyl chloride, [0037] 4-DMAP is
4-dimethylaminopyridine, [0038] Bu.sub.4 N+ is tetrabutylammonium,
[0039] (Bu.sub.4N+).sub.3 HP.sub.2O.sub.7 is tris(tetra
n-butylammonium) hydrogenpyrophosphate,. [0040] PP represents the
pyrophosphate group.
[0041] The reactions which allow compound (6) to be obtained from
the alcohol (9) may be performed as described by: DAVISSON V. J. et
al. "Phosphorylation of Isoprenoid Alcohols" J. Org. Chem 1986, 51,
4768-4779, et DAVISSON V. J. et al. "Synthesis of Allylic and
Homoallylic Isoprenoid Pyrophosphates" Methods in Enzymology, 1984,
110, 130-144.
[0042] Advantageously and according to the invention, the starting
compound is a salt of the formula:
##STR00011##
[0043] The triphosphohalohydrin compound of the formula (3) is then
obtained.
[0044] An example of a complete reaction scheme for obtaining the
compound (3) from the alcohol (9) is given below:
##STR00012##
where PPP is the triphosphate group,
[0045] (Bu .sub.4N.sup.+).sub.4HP.sub.3O.sub.10 is
tetrakis(tetra-n-butylammonium) hydrogentriphosphate.
[0046] The compound (10) is obtained from the alcohol (9) as stated
above. The reaction which allows compound (7) to be obtained from
the compound (10) may be performed under conditions similar to
those described in the publications by DAVISSON V. J. et al or in
DAVISSON V. J. et al "Synthesis of Nucleotide 5'-Diphosphates from
(5'-O-Tosyl Nucleosides" J. Org. Chem. 1987, 52, 1794-1801.
[0047] Advantageously and according to the invention, in a first
variant which allows a compound according to the invention of the
formula (4) to be obtained, the above-stated production process
according to the invention (reaction of X.sub.2 in the presence of
water with a phosphorylated alkene function) may be performed by
using a salt of the following formula as the starting compound:
##STR00013##
where R2 is an organic or inorganic substituent of a nature such as
not to prevent formation of the halohydrin function
##STR00014##
starting from the alkene function
##STR00015##
and halogen X.sub.2 in the presence of water.
[0048] The starting compound (8) may itself be prepared in
accordance with one of the following reaction schemes:
##STR00016##
where Ts is tosyl.
[0049] The compound (7) may be obtained as stated above starting
from the alcohol (9) and the inter mediate compound (10). The
reaction which allows compound (8) to be obtained from the compound
(7) may be performed under conditions similar to those described in
the publications by DAVISSON V. J. et al. This scheme may be used
when R2-O-Ts is commercially available.
##STR00017##
[0050] The intermediate compound (10) may be obtained as stated
above starting from the alcohol (9). The reaction which allows
compound (8) to be obtained from the compound (7) may be performed
under conditions similar to those described in the publications by
DAVISSON V. J. et al. This scheme may be used when R2-O-PPP is
commercially available.
##STR00018##
where DMF is dimethylformamide, MeOH is methanol.
[0051] This reaction scheme 3 may be performed under conditions
similar to those described in D. G. KNORRE et al "General method
for the synthesis of ATP gamma derivatives" Febs letters, 1976, 70,
105-108.
[0052] This reaction scheme 3 cannot be used when R2 comprises a
carbodiimide-reactive function (carboxylate, triphosphate etc.). It
is, however, advantageous when R2-O-PPP is commercially
available.
[0053] In the specific case where R2- is itself a halohydrin group
of the formula:
##STR00019##
the following reaction scheme may be used:
##STR00020##
[0054] This compound (4') is a particular instance of the compound
according to the invention of the formula (4).
[0055] It should be noted that, in all these reactions,
acetonitrile may be replaced by any other aprotic dipolar solvent
(dimethylformamide DMF, dimethyl sulfoxide DMSO etc.).
[0056] It should be noted that when preparing compounds (2), (3)
and (4') and in the event that n.noteq.2, the intermediate compound
(10) may also be replaced by the chloride or bromide compound of
the formula:
##STR00021##
where A is chlorine or bromine.
[0057] The alcohols (9) are commercially available or may readily
be obtained by a well known Grigmard reaction between an alkenyl
organomagnesium compound and formaldehyde or ethylene oxide.
[0058] In a second variant usable in certain cases, a compound of
the formula (4) could be prepared by reacting the triphosphate
compound according to the invention of the formula (3) starting
from a salt soluble in an organic medium, such as a Bu.sub.4N+
salt, in a subsequent stage with a compound R2--O--Y where --O--Y
is a leaving group and R2 is an organic or inorganic substituent
selected such that R2--O--Y is capable of forming, by reaction with
the compound (3), the compound according to the invention of the
formula:
##STR00022##
[0059] In order to be capable of forming the compound of the
formula (4) in this manner, the compound R2--O--Y must in
particular not be reactive towards the halohydrin function:
##STR00023##
Moreover, R2--O--Y must react with the terminal phosphate of
compound (3) to form the compound (4).
[0060] The reaction of the compound of the formula (3) with
R2--O--Y is a nucleophilic substitution. This reaction is in
particular possible and advantageous for R2 selected from among the
group comprising alkyls and alkenes. Y is selected such that
R2--O--Y may give rise to the compound (4) by nucleophilic
substitution. Y is selected, for example, from among tosyl, brosyl
and triflyl.
[0061] A compound according to the invention may accordingly be
difunctional or multifunctional. The phosphohalohydrin function(s)
bring(s) about a specific desired antigenic property towards
T.gamma.9.delta.2 lymphocytes, and R2 or the other functional
groups of the compound may exhibit other, in particular
therapeutic, properties.
[0062] In the case of a compound according to the invention having
two or more phosphohalohydrin groups of the formula (1), it is
sufficient either to start from a starting compound having the
corresponding number of phosphorylated alkene groups of the formula
(5) and the corresponding chemical structure, or to use the
compound of the formula (3) and to react it with an intermediate
compound R2--O--Y having the corresponding number of --O--Y
functions.
[0063] The invention also in particular relates to the novel
phosphohalohydrin .beta. ester compounds of the formula:
##STR00024##
where X is a halogen selected from among iodine, bromine and
chlorine, [0064] R1 is selected from among --CH.sub.3 and
--CH.sub.2--CH.sub.3, [0065] Cat.sup.+ represents one or more
organic or inorganic cation(s) (including the proton), which may be
identical or different in the same compound, [0066] n is an integer
between 2 and 20, [0067] R3- is selected from among: [0068] a
halohydrin group of the formula (12), [0069] an epoxide group of
the formula:
[0069] ##STR00025## [0070] an alkene group of the formula:
[0070] ##STR00026## [0071] m being an integer between 1 and 20.
[0072] These compounds (14) are obtained by firstly performing the
following initial step:
##STR00027##
[0073] Then, the symmetrical diphosphodihalohydrin compound (14a)
is obtained as follows:
##STR00028##
[0074] The asymmetrical .alpha.,.beta. phosphodiester
halohydrin/alkene compound (14b) is obtained as follows:
##STR00029##
[0075] The asymmetrical .alpha.,.beta. phosphodiester
halohydrin/epoxide compound (14c) is obtained as follows:
##STR00030##
[0076] The invention also provides uses of the compounds according
to the invention, in particular the compounds of the formula (2),
as primate T.gamma.9.delta.2 lymphocyte activators, in particular
to activate proliferation and/or cytotoxic activity and/or
production of mediator substance(s) for primate T.gamma.9.delta.2
lymphocytes with TCR receptors comprising V.gamma.9 and V.delta.2
variable regions.
[0077] The invention also provides uses of the compounds according
to the invention for the treatment of cells sensitive to primate
T.gamma.9.delta.2 lymphocytes in a natural or artificial medium
which may contain T.gamma.9.delta.2 lymphocytes, in which medium
said cells may be brought into contact with T.gamma.9.delta.2
lymphocytes and which is compatible with the compounds according to
the invention (i.e. which is not likely to cause the breakdown
thereof, at least under certain treatment conditions).
[0078] A "cell sensitive to T.gamma.9.delta.2 lymphocytes" is taken
to mean any cell subject to the induced effector activity of the
T.gamma.9.delta.2 lymphocytes: cell death (cell destruction by
T.gamma.9.delta.2 lymphocytes); reception of cytokine released by
T.gamma.9.delta.2 lymphocytes (TNF-.alpha., INF-.gamma. . . . );
possibly cellular proliferation induced by T.gamma.9.delta.2
lymphocytes.
[0079] The invention accordingly provides a T.gamma.9.delta.2
lymphocyte activation process, in particular a process for
activating the proliferation of T.gamma.9.delta.2 lymphocytes
and/or the cytotoxic activity of T.gamma.9.delta.2 lymphocytes
and/or the production of mediator substance(s) by T.gamma.9.delta.2
lymphocytes, in which process these T.gamma.9.delta.2 lymphocytes
are brought into contact with at least one compound according to
the invention in a medium which contains T.gamma.9.delta.2
lymphocytes and is compatible with T lymphocyte growth.
Advantageously and according to the invention, interleukin, in
particular interleukin-2 (IL-2), is introduced into the medium in a
proportion suitable to bring about lymphocyte growth in this
medium. Indeed, the presence of the lymphocyte growth factor IL-2
is essential to achieve proliferation of the T lymphocytes, among
which only the T.gamma.9.delta.2 lymphocytes have been activated by
a compound according to the invention. This growth factor must thus
be present in the medium for those uses in which proliferation of
T.gamma.9.delta.2 lymphocytes is desired. This lymphocyte growth
factor may pre-exist in the natural state or be induced or
introduced into the medium, simultaneously or not with the
incorporation of the compound according to the invention, in the
same therapeutic composition or not. Nevertheless, in certain uses
in which activation without proliferation of the T.gamma.9.delta.2
lymphocytes is desired (for example induced cytotoxicity), the
presence of this growth factor is not helpful.
[0080] More specifically, the invention provides uses of the
compounds according to the invention for therapeutic purposes for
the curative or preventive treatment of pathological conditions
producing cells sensitive to primate T.gamma.9.delta.2 lymphocytes
in a medium which may contain these T.gamma.9.delta.2 lymphocytes
and in which these cells may be brought into contact with the
T.gamma.9.delta.2 lymphocytes.
[0081] Advantageously and according to the invention, at least one
compound according to the invention is used at a concentration in
the medium which brings about activation of polyclonal
proliferation of T.gamma.9.delta.2 lymphocytes. This medium may be
selected from among human blood, non-human primate blood, human
blood extracts, non-human primate blood extracts.
[0082] Said medium may be extracorporeal, said activation process
according to the invention then being an extracorporeal cellular
treatment, in particular applicable in a laboratory, for studying
T.gamma.9.delta.2 lymphocytes or the properties thereof, or for
diagnostic purposes. The invention also provides a composition for
extracorporeal (ex vivo) diagnostics, wherein it comprises at least
one compound according to the invention.
[0083] Said medium may also be intracorporeal, activation of the
T.gamma.2.delta.2 lymphocytes then being of therapeutic use.
[0084] More particularly, said medium is the peripheral bloodstream
of a primate. The invention accordingly in particular provides a
process for the activation of T.gamma.9.delta.2 lymphocytes in the
peripheral bloodstream of a primate, in particular humans, into
which is administered at least one compound according to the
invention in a quantity suitable for activating T.gamma.9.delta.2
lymphocytes. At least one compound according to the invention is
thus administered by a general route, in particular parenterally
into the peripheral bloodstream.
[0085] Said medium may also comprise a cellular site to be treated
and at least one compound according to the invention is
administered directly in contact with the cellular site to be
treated (topical administration).
[0086] The invention accordingly in particular provides therapeutic
uses of the compounds according to the invention for treating
pathological conditions in primates, said conditions belonging to
the group comprising cancers, infectious diseases, in particular
mycobacterial infections (leprosy, tuberculosis etc.); parasitic
conditions (malaria etc.); pathological immunodeficiency syndromes
(AIDS etc.). According to the invention, a therapeutic composition
is administered which is suitable for releasing into the peripheral
bloodstream and/or at a cellular site to be treated a quantity of
at least one compound according to the invention capable of
activating T.gamma.9.delta.2 lymphocytes. Indeed, it has been
demonstrated in general terms in the above-stated prior art that a
composition having the property of activating T.gamma.9.delta.2
lymphocytes may advantageously be used for treating these
pathological conditions.
[0087] As is conventional, throughout the text the terms "therapy"
or "therapeutic" encompass not only curative treatment or care, but
also preventive treatment (prophylaxis), such as vaccination,
together with intracorporeal diagnostics (administration for
diagnostic purposes). Indeed, by permitting activation of
T.gamma.9.delta.2 lymphocytes, the invention allows
immunostimulatory treatments which may be advantageous not only
prophylactically by preventing the development of pathogenic cells
sensitive to T.gamma.9.delta.2 lymphocytes, but also curatively by
inducing destruction of pathogenic cells sensitive to
T.gamma.9.delta.2 lymphocytes.
[0088] The invention accordingly provides a therapeutic composition
comprising at least one compound according to the invention. More
specifically, the invention relates to a therapeutic composition
comprising a quantity capable of being administered to a primate,
in particular in contact with the peripheral bloodstream or
topically, of at least one compound according to the invention, in
particular for the preventive or curative treatment of the
above-stated pathological conditions. A composition according to
the invention may be an immunostimulant composition or a vaccine,
the compounds according to the invention being antigens which
activate T.gamma.9.delta.2 lymphocytes.
[0089] Advantageously and according to the invention, the
therapeutic composition is characterized in that it moreover
comprises a proportion of interleukin, in particular interleukin-2,
suitable for bringing about lymphocyte growth in the medium into
which it is to be administered.
[0090] A therapeutic composition according to the invention may be
prepared in a dosage form capable of being administered by a
general route, in particular parenterally directly into the
peripheral bloodstream of a primate, with at least one compound
according to the invention in a quantity suitable to activate
T.gamma.9.delta.2 lymphocytes and one or more appropriate
excipient(s). Given the very low active concentration of the
compounds according to the invention (of the order of 0.1 to 10
nM), such administration may be made without risk of toxicity.
[0091] A therapeutic composition according to the invention may
also be prepared in a dosage form appropriate for topical
administration, directly in contact with the cells sensitive to
T.gamma.9.delta.2 lymphocytes.
[0092] The dosage form of a therapeutic composition according to
the invention is produced in accordance with the selected route of
administration using conventional pharmaceutical formulation
methods. The quantity and concentration of compound(s) according to
the invention and the dosage are determined by reference to known
chemotherapeutic methods for the diseases to be treated, taking
account of the bioactivity of the compounds according to the
invention towards T.gamma.9.delta.2 lymphocytes, the individual to
be treated, the disease in question and the desired biological
effects.
[0093] Advantageously and according to the invention, in the case
of a bioactive compound at a concentration of between 1 nM and 10
nM, the quantity of compound(s) according to the invention
administered by a general route is between 0.1 .mu.g and 100 .mu.g,
in particular between 1 .mu.g and 10 .mu.g, per kilogram of patient
body weight.
[0094] It has moreover been demonstrated in vitro that the
compounds according to the invention exhibit no general toxicity
ever at concentrations of up to 100 .mu.M, i.e. of the order of
10.sup.5 times the bioactive concentration. Furthermore, it is
known that the biochemical class of molecules to which the
compounds according to the invention belong (phosphoesters)
comprises a family of metabolic compounds found in any living cell.
The compounds according to the invention thus exhibit no toxic
effects other than those induced by the bioactivity thereof upon
T.gamma.9.delta.2 lymphocytes.
[0095] Moreover, certain compounds according to the invention have
a sufficiently low molecular weight (in particular below 500) to be
compatible with the elimination thereof via the kidneys and
urine.
[0096] One example formulation of an injectable therapeutic
composition according to the invention for a primate weighing 1 kg
is as follows: 5 .mu.g of 3-(iodomethyl)-3-butanol-1-yl diphosphate
(IHPP) diluted in 0.5 ml of sterile phosphate buffer at pH 7 and
adjusted to 37.degree. C.
[0097] In this manner, 5 .mu.g of IHPP (compound of the formula
(2)) are administered per 1 kg of animal body weight, corresponding
to a concentration in the circulating blood such as to be greater
than the bioactive concentration of IHPP (a concentration of 10 nM
of IHPP corresponding, to approx. 5 ng/ml).
[0098] It should be noted that the majority of the excipients or
other conventionally used phalinaceutically acceptable additives
are chemically compatible with the compounds according to the
invention.
[0099] A therapeutic composition according to the invention may
also advantageously comprise one or more other active
ingredient(s), in particular to bring about a synergistic effect.
In particular, a compound according to the invention may act as a
vaccine adjuvant. The therapeutic vaccine composition according to
the invention then comprises a known vaccine composition to which
is added a quantity of compound(s) according to the invention
capable of activating the T.gamma.y9.delta.2 lymphocytes which will
not only be able to exert their anti-infective activity directly,
but will also be able to activate the T lymphocytes which effect
the conventional vaccine response.
[0100] A therapeutic composition according to the invention may
also itself incorporate primate T.gamma.9.delta.2 lymphocytes in a
culture in a medium compatible with T lymphocyte growth. It may
then be used for treating primates, or more generally vertebrates
with which administration of primate T.gamma.9.delta.2 lymphocytes
may be performed under conditions of immune compatibility towards
said primate T.gamma.9.delta.2 lymphocytes. Such a composition
according to the invention may be administered by a general route,
or even by a topical route, in contact with target pathogenic
cells, sensitive to said primate T.gamma.9.delta.2 lymphocytes.
[0101] The invention also provides the use of at least one compound
according to the invention for the production of a therapeutic
composition according to the invention. More particularly, the
invention relates to the use of at least one compound according to
the invention for the production of a therapeutic composition
intended for the preventive or curative treatment of a pathological
condition of humans or vertebrates which produces cells sensitive
to primate T.gamma.9.delta.2 lymphocytes, in particular a
pathological condition selected from the group comprising cancers,
infections diseases, parasitic conditions and pathological
immunodeficiency syndromes. To this end, the invention also
provides the use of at least one compound according to the
invention for the production of a therapeutic composition intended
to be administered, in particular in contact with the peripheral
bloodstream or by a topical route, to a primate, in particular to
humans, for the preventive or curative treatment of a pathological
condition as mentioned above.
[0102] The invention also provides a process for the production of
a composition, in particular a therapeutic composition, according
to the invention having the characteristic of activating
T.gamma.9.delta.2 lymphocytes, in which process at least one
compound according to the invention is used.
[0103] The invention also relates to a process for the production
of a therapeutic composition intended for the preventive or
curative treatment of a pathological condition of humans or
vertebrates which produces cells sensitive to primate
T.gamma.9.delta.2 lymphocytes, in which process at least one
compound according to the invention is used. The invention in
particular is relates to a process for the production of a
therapeutic composition intended to be administered, in particular
in contact with the peripheral bloodstream or by a topical route,
to a primate, for the preventive or curative treatment of a
pathological condition which produces cells sensitive to
T.gamma.9.delta.2 lymphocytes, in particular a pathological
condition belonging to the group stated above, in which process at
least one compound according to the invention is used.
[0104] Advantageously and according to the invention, in a
production process according to the invention, at least one
compound according to the invention is brought into contact with a
medium which contains primate T.gamma.9.delta.2 lymphocytes, and is
compatible with T lymphocyte growth, in a quantity suitable to
activate these T.gamma.9.delta.2 lymphocytes in this medium.
Advantageously and according to the invention, said medium
comprises a substance selected from among primate blood and primate
blood extracts. A therapeutic composition containing activated
T.gamma.9.delta.2 lymphocytes is then obtained so allowing a
cellular therapeutic approach to be performed.
[0105] It should be noted that the compounds according to the
invention are halogenated and, for this reason alone, cannot
correspond to natural phosphoantigens, in particular to the
molecules known as Tubag1, Tubag2, Tubag3 and Tubag4 obtained as
described in WO 95/20673. It is in any event possible to
demonstrate for example that these natural phosphoantigens are
broken down by the bromine water used for the chemical production
of the phosphobromohydrins according to the invention. The
compounds according to the invention are thus not natural antigens,
but are synthetic antigens which activate T.gamma.9.delta.2
lymphocytes at concentrations of the same order and with an
efficiency similar to or even greater than that of natural
antigens.
[0106] It should also be noted that, contrary to the prior art as
illustrated by U.S. Pat. No. 5,639,653, which considered that the
presence of an alkyl or alkene group was essential to activate
human T.gamma.9.delta.2 lymphocytes, the inventors have observed
that by destroying the alkene bond with the addition of halogen, an
element absent from natural biological compounds, the
T.gamma.9.delta.2 lymphocytes are activated extremely strongly and
at very low concentration. In particular, it may be observed that
the effect may even exceed that of phosphoantigens of natural
origin.
[0107] Further features, objects and advantages of the invention
will be evident from the following Examples, which are provided in
non-limitative manner merely for purposes of explanation, and from
the figures:
[0108] FIG. 1 is a graph showing the results obtained in Example
10,
[0109] FIG. 2 is a graph showing the results obtained in Example
11,
EXAMPLE 1
Production of 3-(bromomethyl)-3-butanol-1-yl diphosphate
(BrHPP)
[0110] Preparation of 3-methyl-3-butene-1-yl tosylate (isopentenyl
tosylate):
[0111] 2.32 mmol (442 mg) of tosyl chloride and 2.55 mmol (312 mg)
of 4-(N,N-dimethylamino)pyridine are introduced while stirring with
a magnetic stirrer into 5 ml of anhydrous dichloromethane in a
glass reaction vessel which is equipped for handling under an inert
atmosphere and has been carefully dried. 2.32 mmol (200 mg) of
isopentenol dissolved in approx. 1 ml of dichloromethane are slowly
added to this mixture through a septum using a syringe. The
reaction is monitored by thin-layer chromatography on silica gel 60
F-254; eluent: pentane/ethyl acetate 85/15 vol./vol.; R.sub.f
(product)=0.4 and R.sub.f (TsCl)=0.5). After approx. 3 hours'
stirring under a nitrogen atmosphere, the reaction mixture is
diluted in a large volume of hexane (approx. 100 ml), resulting in
the immediate formation of a white precipitate. The mixture is then
filtered and the filtrate concentrated by evaporation under reduced
pressure. The solution is then diluted with diethyl ether and
refiltered. Once the solvent has evaporated, a yellowish oil is
obtained. The product is purified by preparative chromatography
through a silica column (silica gel 60; eluent: pentane/ethyl
acetate 85/15). In this manner, 1.98 mmol (475 mg) of
3-methyl-3-butene-1-yl tosylate (85% isolated yield) are obtained.
The compound (colorless oil) is stored at +4.degree. C. in an
anhydrous medium.
Preparation of tris(tetra-n-butylammonium)
hydrogenpyrophosphate:
[0112] 4.5 mmol (1 g) of dihydrogenpyrophosphate disodium salt
(Na.sub.2H.sub.2P.sub.2O.sub.7) are dissolved in 10 ml of cold
deionized water which has previously been adjusted to pH 9 with a
10 mM ammonia solution. The solution is passed through a column
containing 19 milliequivalents (4 g) of DOWEX.RTM. 50-WX8-200
cationic resin (H.sup.+ form). The acid solution is eluted with
15-20 ml of cold deionized water at pH 9. The collected solution is
immediately titrated at pH 7.3 using a 40% aqueous solution of
tetra-n-butylammonium hydroxide (Bu.sub.4NOH). After freeze-drying,
4 g of tetra-n-butylammonium salt are obtained as a hygroscopic
white solid. The salt is dissolved in 10 ml of anhydrous
acetonitrile. The solution is subsequently filtered, then dried by
evaporation of the solvent under reduced pressure in successive
stages. In this manner, a solution of tris(tetra-n-butylammoniurn)
hydrogenpyrophosphate is obtained with a purity of 98% (result
deduced from analysis by ionic chromatography (HPAEC)). The volume
is adjusted to achieve a salt concentration of between 0.5 and 1 M.
The solution is stored at -20.degree. C. in an anhydrous
medium.
Preparation of 3-methyl-3-butene-1-yl diphosphate (isopentenyl
pyrophosphate):
[0113] 2.5 ml of a solution of tris(tetra-n-butylammonium)
hydrogenpyrophosphate at a concentration of 0.7 M (1.75 mmol) in
anhydrous acetonitrile are introduced into a glass reactor which
has been dried carefully. The reactor is cooled with an ice bath,
then 0.70 mmol (168 mg) of 3-methyl-3-butene-1-yl tosylate
dissolved in a minimum quantity of acetonitrile (0.5-1 M) are added
using a syringe, while stirring with a magnetic stirrer. Once the
tosylate has been introduced, the ice bath is removed, then the
reaction is allowed to continue at room temperature with stirring.
The progress of the reaction is then monitored by ionic
chromatography (HPAEC). After approx. 3 hours, the solvent is
evaporated under reduced pressure and the reaction medium
redissolved in 3 ml of a 98/2 (vol./vol.) water/2-propanol mixture.
The solution is passed through a column containing 19
milliequivalents (4 g) of DOWEX.RTM. 50-WX8-200 cationic resin
(NH.sub.4.sup.+form), then eluted with 10 ml of the water (pH
9)/2-propanol 98/2 (vol./vol.) mixture. After freeze-drying, a
white solid containing the crude product is collected.
Purification:
[0114] The pyrophosphate and the traces of ammonium monophosphate
are separated from the medium by coprecipitation in the presence of
ammonium hydrogencarbonate. The crude product obtained in the
preceding stage is dissolved in 4 ml of 0.1 M ammonium
hydrogencarbonate, which is transferred into a 25 ml centrifuge
tube, The solution is then treated with 10 ml of a 1/1 (vol./vol.)
acetonitrile/2-propanol mixture by shaking the mixture vigorously
(vortex) for a few minutes until a white precipitate has formed.
The tube is then centrifuged at 2000 rpm at 10.degree. C. for 5
minutes. The supernatant, into which the organic salts have been
extracted, is stored at +4.degree. C. The procedure is repeated by
redissolving the precipitate in 3 ml of 0.1 M ammonium
hydrogencarbonate, to which 7 ml of the acetonitrile/2-propanol
mixture are added. The two supernatants are combined and the
solvent evaporated under a vacuum. An oily liquid is obtained which
is stored at +4.degree. C.,
[0115] The ammonium tosylate is separated from the reaction medium
by extraction with the 1/1 (vol./vol.) chloroform/methanol solvent.
The oily liquid from the preceding stage is dissolved in 4 ml of
water at pH 9 and treated with 1 ml of this solvent by a
conventional extraction procedure which is repeated 3 times. Any
traces of solvent are then removed from the aqueous phase by
evaporation under reduced pressure at 30.degree. C. On the basis of
analysis by ionic chromatography (HPAEC), an 83% yield of
3-methyl-3-butene-1-yl diphosphate (0.58 mmol, 172 mg) is obtained.
The solution is stored at -20.degree. C.
[0116] The product is then purified in accordance with requirements
by anion exchange chromatography through 360 mg to 10 g Sep-Pak
Accell Plus QMA (Waters.RTM.) cartridges eluted in succession
respectively by 20 mM, 40 mM, 100 mM then 200 mM aqueous ammonium
hydrogencarbonate solutions, with the eluted fractions being
monitored by chromatography (HPAEC). The fractions corresponding to
the purified. product are combined and then freeze-dried.
Preparation of 3-(bromomethyl)-3-butanol-1-yl diphosphate:
[0117] 0.34 mmol (100 mg) of isopentenyl pyrophosphate (ammonium
salt) dissolved in 2 ml of neutral pH deionized water are treated
under a fume hood at room temperature with 1.9 ml (0.34 mmol) of
bromine in a saturated aqueous solution (0.18 M). The bromine
solution is added gradually and preferably to a cold solution of
the ammonium salt, with the mixture being stirred periodically
until the bromine water has become colorless. In the event that
bromine is added in a slight excess (persistent yellow color), the
solution is transferred into a glass flask and then exposed to
reduced pressure (rotary evaporator) for a few minutes at a
temperature of 30.degree. C. until the color disappears. the
product 3-(bromomethyl)-3-butanol-1-yl diphosphate is produced
quantitatively (0.33 mmol, 130 mg); this result being deduced from
analysis by ionic chromatography (HPAEC). For the purpose of
performing biological testing, the resultant aqueous solutions are
filtered and then neutralized by being passed through a cationic
resin column. The bromide ions may be removed from the solution
using a DIONEX.RTM. apparatus comprising an OnGuard.RTM.-Ag
cartridge attached to an OnGuard.RTM.-H cartridge. This apparatus
allows selective retention of the halide ions from the solution.
For the purpose of performing biological testing, the aqueous
solutions of the product are sterilized by filtration through a 0.2
.mu.m filter and stored (preferably with a neutral to slightly
acidic pH) at -20.degree. C. In the case of testing performed in
vivo, the solutions are passed beforehand through a DOWEX.RTM.
50-WX8-200 cationic resin column (Na.sup.+ form) eluted by two
column volumes of deionized water.
EXAMPLE 2
Production of 3-(iodomethyl)-3-butanol-1-yl diphosphate (IHPP)
Preparation of Iodized Water:
[0118] A solution of iodized water of the order of 0.5 to 1 mM is
prepared by extended sonication (approx. 15 minutes) of a few
crystals of iodine in a solution of deionized water and filtration.
For tests involving larger quantities, solutions having a higher
iodine concentration may be obtained by adding a small proportion
of alcohol to the initial aqueous solution. The iodized water is
then titrated with sodium thiosulfate using starch solution as a
color indicator.
Preparation of 3-(iodomethyl)-3-butanol-1-yl diphosphate:
[0119] One micromole (1 ml of a milliniolar solution) of
isopentenyl pyrophosphate prepared according to Example 1 in the
form of the ammonium salt in an aqueous or aqueous/alcohol medium
of neutral pH is treated at room temperature by addition of one
micromole of iodine in an aqueous solution (1.43 ml of 0.7 mM
iodized water). The solution is left for 30 minutes at room
temperature, then for 30 minutes at +4.degree. C. while
periodically being stirred vigorously. Once the iodized water has
become colorless, the product 3-(iodomethyl)-3-butanol-1-yl
diphosphate is produced quantitatively (1 micromole in approx. 2.5
ml). The solution is then treated as in Example. 1 for the
performance of biological testing and/or for the performance of in
vivo testing and stored at -20.degree. C.
EXAMPLE 3
Production of 3-(chloromethyl)-3-butanol-1-yl diphosphate
(ClHPP)
[0120] Preparation of chlorine water:
[0121] The solution of chlorine water is prepared by bubbling
gaseous chlorine through a solution of deionized water. The
solution is then titrated with sodium thiosuifate in the presence
of excess potassium iodide and using starch solution as a color
indicator.
Preparation of 3-(chloromethyl)-3-butanol-1-yl diphosphate:
[0122] One micromole (1 ml of a millimolar solution) of isopentenyl
pyrophosphate prepared according to Example 1 in the form of the
ammonium salt in an aqueous or aqueous/alcohol medium of neutral pH
is treated at room temperature by addition of one micromole of
chlorine in an aqueous solution (72 .mu.l of 14 mM chlorine water).
After 30 minutes at room temperature with periodic stirring, the
product 3-(chloromethyl)-3-butanol-1-yl diphosphate is produced
quantitatively (in this case 1 micromole in approx. 1.1 ml). The
solution is then treated as in Example 1 for the performance of
biological testing and/or for the performance of in vivo testing
and stored at -20.degree. C.
EXAMPLE 4
Production of 3-(bromomethyl)-3-butanol-1-yl triphosphate
(BrHPPP)
[0123] Preparation of tetrakis(tetra-n-butylammonium)
hydrogentriphosphate:
[0124] 2.1 mmol (1 g) of tripolyphosphate hexahydrate pentasodium
salt (Na.sub.5P.sub.3O.sub.10.6H.sub.2O) are dissolved in 10 ml of
cold deionized water which has previously been adjusted to pH 9
with a 10 mM ammonia solution. The solution is passed through a
column containing 21 milliequivalents (4.4 g) of DOWEX.RTM. 50-WX8
cationic resin (H.sup.+form). The acid solution is eluted with
20-25 ml of cold deionized water at pH 9. The collected solution is
immediately titrated at pH 7.0 using a 40% aqueous solution of
tetra-n-butylammonium hydroxide (Bu.sub.4NOH). After freeze-drying,
2.5 g of tetra-n-butylammonium salt are obtained as a hygroscopic
white solid. The salt is dissolved in 10 ml of anhydrous
acetonitrile. The solution is subsequently filtered, then dried by
evaporation of the solvent under reduced pressure in successive
stages. In this manner, a solution of
tetrakis(tetra-n-butylammonium) hydrogentriphosphate with a purity
of 95% is obtained (result deduced from analysis by ionic
chromatography (HPAEC)). The volume is adjusted to achieve a salt
concentration of between 0.5 and 1 M. The solution is stored at
-20.degree. C. in an anhydrous medium.
Preparation of 3-methyl-3-butene-1-yl triphosphate (isopentenyl
triphosphate):
[0125] Using the procedure described for the preparation of
3-methyl-3-butene-1-yl diphosphate (Example 1), 2 mmol of a molar
solution of tetrakis(tetra-n-butylammonium) hydrogentriphosphate
are reacted under a nitrogen atmosphere with 1 mmol (240 mg) of
3-methyl-3-butene-1-yl tosylate prepared according to Example 1 in
4 ml of anhydrous acetonitrile for 24 hours. By using a
precipitation/extraction purification procedure similar to that
applied to 3-methyl-3-butene-1-yl diphosphate, on the basis of
analysis by ionic chromatography (HPAEC), a yield of 74% of
3-methyl-3-butene-1-yl triphosphate (0.74 mmol, 292 mg) is
obtained. Phosphohalohydrin compounds according to the invention
are prepared for the purpose of biological testing by using a
fraction of the product obtained at this stage which is purified by
HPAEC through an IonPac.RTM. AS11 column, with two or more
chromatographic passes being combined. In this manner, approx. 2 ml
of an aqueous millimolar solution of neutral pH of
3-methyl-3-butene-1-yl triphosphate are prepared in the form of an
ammonium salt.
Preparation of 3-(bromomethyl)-3-butanol-1-yl triphosphate:
[0126] 300 nmol (300 .mu.l of a millimolar solution) of isopentenyl
triphosphate are treated at room temperature by addition of 300
nmol of bromine in a saturated aqueous solution (1.7 .mu.l of 180
mM bromine water). The color of the bromine water disappears
virtually instantaneously. Once the mixture has been stirred and
the bromine has become colorless (virtually instantaneously), the
product 3-(bromomethyl)-3-butanol-1-yl triphosphate is produced
quantitatively (300 .mu.l of a millimolar solution). The solution
is then treated as in Example 1 for the performance of biological
testing and/or for the performance of in vivo testing and stored at
-20.degree. C.
EXAMPLE 5
Production of 3-(iodomethyl)-3-butanol-1-yl diphosphate (IHPPP)
[0127] 300 nmol (300 .mu.l of a millimolar solution) of isopentenyl
triphosphate prepared according to Example 4 are treated in an
aqueous or aqueous/alcohol medium of neutral pH by addition of 429
.mu.l of 0.7 mM iodized water prepared according to
[0128] Example 2. The solution is left for 30 minutes at room
temperature while periodically being stirred vigorously. Once the
iodized water has become colorless, the product
3-(iodomethyl)-3-butanol-1-yl triphosphate is produced
quantitatively (729 .mu.l of a 411 .mu.M solution). The solution is
then treated as in Example 1 for the performance of biological
testing and/or for the performance of in vivo testing and stored at
-20.degree. C.
EXAMPLE 6
Production of .alpha.,.gamma. di-[3-(bromomethyl)-3-butanol-1-yl]
triphosphate (diBrHTP)
[0129] Preparation of .alpha.,.gamma. di-[3-methyl-3-butene-1-yl]
triphosphate:
[0130] Using the procedure described for the preparation of
3-methyl-3-butene-1-yl diphosphate (Example 1), 0.5 mmol of a molar
solution of tetrakis(tetra-n-butylammonium) hydrogentriphosphate
(prepared according to Example 4) are reacted under a nitrogen
atmosphere with 1 mmol (240 mg) of 3-methyl3-butene-1-yl tosylate
(prepared according to Example 1) in 4 ml of anhydrous acetonitrile
for 24 hours. By using a precipitation/extraction purification
procedure similar to that applied to 3-methyl-3-butene-1-yl
diphosphate, on the basis of analysis by ionic chromatography
(HPAEC), a yield of 81% of .alpha.,.gamma.
di-[3-methyl-3-butene-1-yl] triphosphate (0.4 mmol, 178 mg) is
obtained. Phosphohalohydrin compounds according to the invention
are prepared for the purpose of biological testing by using a
fraction of the product obtained at this stage which is purified by
HPAEC through an IonPac.RTM. AS11 column, with two or more
chromatographic passes being combined. Before each chromatographic
pass and in order to improve isolation of the product, the fraction
to be purified is treated enzymatically with alkaline phosphatase
in order to break down the isopentenyl triphosphate which is a
secondary product of the reaction. In this manner, approx. 1 ml of
an aqueous millimolar solution of neutral pH of .alpha.,.gamma.
di-[3-methyl-3-butene-1-yl] triphosphate are prepared in the form
of an ammonium salt,
Preparation of .alpha.,.gamma. di-[3-(bromomethyl)-3-butanol-1-yl]
triphosphate:
[0131] 250 nmol (250 .mu.l of a millimolar solution) of
.alpha.,.gamma. di-[3-methyl-3-butene-1-yl] triphosphate are
treated at room temperature by addition of 250 nmol of bromine in a
saturated aqueous solution (1.4 .mu.l of 180 mM bromine water).
Once the mixture has been stirred and the bromine has become
colorless (virtually instantaneously), the product .alpha.,.gamma.
di-[3-(bromomethyl)-3-butanol-1-yl] triphosphate is produced
quantitatively (approx. 250 .mu.l of a millimolar solution). The
solution is then treated as in Example 1 for the performance of
biological testing and/or for the performance of in vivo testing
and stored at -20.degree. C.
EXAMPLE 7
Production of .alpha.,.gamma. di-[3-(iodomethyl)-3-butanol-1-yl]
triphosphate (diIHTP)
[0132] 250 nmol (250 .mu.l of a millimolar solution) of
.alpha.,.gamma. di-[3-methyl-3-butene-1-yl] triphosphate prepared
according to Example 6 are treated in an aqueous or aqueous/alcohol
medium of neutral pH by addition of 358 .mu.l of 0.7 mM iodized
water prepared according to Example 2. The solution is left for 30
minutes at room temperature while periodically being stirred
vigorously. Once the iodized water has become colorless, the
product .alpha.,.gamma. di-[3-(iodomethyl)-3-butanol-1-yl]
triphosphate is produced quantitatively (608 .mu.l of a 411 .mu.M
solution). The solution is then treated as in Example 1 for the
performance of biological testing and/or for the performance of in
vivo testing and stored at -20.degree. C.
EXAMPLE 8
Production of uridine 5'-triphosphate
.gamma.-[3-(iodomethyl)-3-butanol-1-yl]
[0133] Preparation of uridine 5'-triphosphate
.gamma.-[3-methyl-3-butene-1-yl]:
[0134] This product is prepared in accordance with the procedure
described by KNORRE D. C. et al. "General Method for the synthesis
of ATP Gamma-derivatives" Febs Letters, 1976, 70-1, 105-108,
starting from 40 .mu.mol of uridine 5'-triphosphate (UTP)
(triethylammonium salt) in the presence of an excess of
isopentenol. Phosphohalohydrin compounds according to the invention
are prepared for the purpose of biological testing by purifying a
fraction of the product obtained by HPAEC through an IonPac.RTM.
AS11 column, with two or more chromatographic passes being
combined. Before each chromatographic pass and in order to improve
isolation of the product, the fraction to be purified is treated
enzymatically with alkaline phosphatase in order to break down the
secondary products (UDP and UMP) and unreacted UTP. In this manner,
approx. 500 .mu.l of an aqueous 300 .mu.M solution of neutral pH of
uridine 5'-triphosphate .gamma.-[3-methyl-3-butene-1-yl] are
prepared in the form of an ammonium salt.
Preparation of uridine 5'-triphosphate
.gamma.-[3-(iodomethyl)-3-butanol-1-yl]:
[0135] 75 nmol (250 .mu.l of a 300 .mu.M solution) of uridine
5'-triphosphate, .gamma.y-[3-methyl-3-butene-1-yl] in ammonium salt
form are treated in an aqueous medium of neutral pH by addition of
108 .mu.l of 0.7 mM iodized water prepared according to Example 2.
The solution is left for 20 minutes at room temperature while
periodically being stirred vigorously. Once the iodized water has
become colorless, the product uridine 5'-triphosphate
.gamma.-[3-(iodomethyl)-3-butanol-1-yl] is produced quantitatively
(approx. 360 .mu.l of a 200 .mu.M solution). The solution is then
treated as in Example 1 for the performance of biological testing
and/or for the performance of in vivo testing and stored at
-20.degree. C.
EXAMPLE 9
Production of .alpha.,.beta. di[3-bromomethyl-3-butanol-1-yl]
diphosphate
[0136] This product is prepared in accordance with a procedure
analogous to that described in Example 6, with
tetrakis(tetra-n-butylammonium) hydrogentriphosphate being replaced
by tris(tetra-n-butylammonium) hydrogenpyrophosphate (prepared
according to Example 1) for the preparation of the intermediate
compound: .alpha.,.beta. di-[3-methyl-3-butene-1-yl]
diphosphate.
EXAMPLE 10
Measurement of Antigenic Activity by Stimulation of the
Proliferation of T.gamma.9.delta.2 Lymphocytes in a Culture
[0137] 20 microliters of the aqueous solution of the compound
according to the invention adjusted to the final concentration
specified in the test are added to an in vitro culture of 10.sup.6
total T lymphocytes in 1 ml, separated from the blood of a healthy
adult human donor and initially containing 1-5% of
T.gamma.9.delta.2 lymphocytes, in an adequate culture medium (RPMI
1640+10% of inactivated human serum and 50 U/ml of human
interleukin-2 (hIL-2)). After culturing for 4 days, 50 U of dIL-2
are added per milliliter of culture medium. After 8 days, the cells
are counted, collected, washed with phosphate buffer, and the
T.gamma.9.delta.2 type cells are detected in the culture by
labeling with conventional commercial reagents (fluorescein-labeled
monoclonal antibodies) and the proportion thereof determined by
flux cytometry analysis. The parameter measured is either the
change in the proportion of or the increase in the number of
T.gamma.9.delta.2 cells in cultures in the presence of the compound
according to the invention in comparison with cultures not
containing a compound according to the invention. The results of
this testing are represented by plotting curves of these values
(y-axis in FIG. 1) as a function of concentration on a logarithmic
scale of the compound according to the invention placed in the
culture (x-axis in FIG. 1).
[0138] FIG. 1 shows the results obtained with the compounds
according to the invention obtained in Examples 1 (BrHPP) and 2
(IHPP), with the dotted line being a negative control (value
obtained in the absence of the compound according to the
invention).
[0139] Table 1 below shows the ED50 values, the effective dose at
50% of the maximum polyclonal lymphocyte amplification effect
obtained as stated above with various compounds according to the
invention.
TABLE-US-00001 TABLE I MOLECULE ED 50% Name Abbreviation Structure
nM isopentenyl pyrophosphate IPP ##STR00031## 3000
3-(chloromethyl)- 3-butanol-1-yl diphosphate ClHPP ##STR00032## 100
3-(bromomethyl)- 3-butanol-1-yl diphosphate BrHPP ##STR00033## 10
3-(iodomethyl)-3- butanol-1-yl diphosphate IHPP ##STR00034## 3
3-(bromomethyl)- 3-butanol-1-yl triphosphate BrHPPP ##STR00035##
120 3-(iodomethyl)-3- 3-butanol-1-yl triphosphate IHPPP
##STR00036## 70 .alpha., .gamma. di-3- (bromomethyl)-3-
butanol-1-yl triphosohate di-BrHTP ##STR00037## 3000 .alpha.,
.gamma. di-3- (iodomethyl)-3- butanol-1-yl triphosphate di-IHTP
##STR00038## 7000
EXAMPLE 11
Measurement of Antigenic Activity by Stimulation of Induced
Cytotoxicity
[0140] The specific cytotoxic activity of a T.gamma.9.delta.2
lymphocyte clone measured in accordance with the induced
cytotoxicity test is compared, said activity being stimulated with
decreasing concentrations of the phosphoantigen Tubag3 obtained as
described by WO 95/20673 (curve represented by black diamonds in
FIG. 2), of the compound BrHPP according to the invention obtained
in Example 1 (curve represented by black squares in FIG. 2), of the
compound IHPP according to the invention obtained in Example 2
(curve represented by white, circles in FIG. 2) and of isopentenyl
pyrophosphate IPP (curve represented by black triangles in FIG.
2).
[0141] It may be noted that the compounds according to the
invention are active at a concentration of the order of 10 nM,
which is of the same order as that of the natural phosphoantigen
Tubag3, whereas the prior art compound IPP is active at a
concentration of the order of 3 .mu.M, or 300 times higher.
EXAMPLE 12
Toxicity of BrHPP (Sodium Salt)
[0142] Eight 30 g mice received an intravenous injection (caudal
vein) of 300 .mu.l of PBS buffer containing 1 mg of BrHPP (sodium
salt). No sign of shock or pyrogenicity is observed: 8 mice are
still alive after 30 days; no significant variation in the weight
of the mice is recorded during the stady. Toxicity is thus less
than 12.5% for a dose of 33.4 mg of BrHPP (Na.sup.+) per kilogram
of animal body weight.
EXAMPLE 13
Pyrogenicity of BrHPP (Sodium Salt)
[0143] This test is performed in accordance with a protocol
specified by standards ISO-10993-11 (1993) and USP XXIII. The test
involves assessing the febrile reaction in rabbits after
intravenous administration of BrHPP (sodium salt) at 400 .mu.g/ml
(i.e. 10 ml of extract per kilogram) to three rabbits. Temperature
is taken every 30 minutes for 3 hours starting 30 minutes after the
injection.
[0144] Only one rabbit exhibited an increase in temperature of
greater than 0.5.degree. C. (minimum threshold). On injection of
the 400 .mu.g/ml solution, the rabbits exhibited a side-effect
taking the form of muscle tremors. Another rabbit injected with the
same solution diluted 1:40, namely to 10 .mu.g/ml, in 19 minutes
exhibited no reaction to the injection.
EXAMPLE 14
In vivo Bioactivity of BrHPP (Sodium Salt) in the Macaque
[0145] These tests are performed on monkeys of the species Macaca
fascicularis (males weight 3-4 kg) anaesthetized with 15 mg/kg
Zoletil (zolazepam).RTM./0.01 mg/kg atropine. The batches of BrHPP
(in NA.sup.+ form) are subiected to prior purity testing (>99%),
quantified and have been tested in accordance with the pyrogenicity
tests of Example 12. The animals are injected by rapid intravenous
injection (2-5 minutes into the saphenous vein) with 0.1 mg/kg of
BrHPP (i.e. 0.3-0.4 mg per animal) diluted in 10 ml of Ringer
lactate, Each animal also receives a subcutaneous injection of
90,000 units of interleukin-2 (IL-2). These injections are
performed once daily for four days. Control animals receive only
the subcutaneous injection of IL-2 once daily for four days.
[0146] The proportion of T.gamma.9.delta.2 lymphocytes in total
lymphocytes in the various animals is compared in the following
three situations: [0147] in the animal's bloodstream, [0148] in a
culture of total lymphocytes taken from the animal receiving only
BrHPP, [0149] in a culture of total lymphocytes taken from the
animal receiving BrHPP+IL-2.
[0150] The results are shown in Table II below:
TABLE-US-00002 TABLE II Reactivity ex vivo of T.gamma.9.delta.2
lymphocytes (% after 10 days' culturing in vitro) % of
T.gamma.9.delta.2 lymphocytes Amplification Amplification Animals
in the blood in vivo by 40 nM by 40 nM injected: (7 days after
injection) BrHPP BrHPP + IL-2 Controls or IL-2 ~1%* [0.8-1.4] 1%
~8% [4.7-12] only BrHPP only ~1% 1% ~11% [10-12] BrHPP only, ~1% 1%
40.2% twice (1 injection + 1 booster) BrHPP + IL-2 7% [5-10] 10%
50.0%
It may be noted that the single or repeated intravenous injection
of BrHPP alone preactivates the monkey's T.gamma.9.delta.2
lymphocytes, but does not stimulate the growth thereof in the
animal. The injection of BrHPP alone brings about preacfivation of
the T.gamma.9.delta.2 lymphocytes, which is in particular
manifested by expression of the growth factor IL-2 from the surface
of the receptor cells. In contrast, intravenous injection of BrHPP
in the presence of subcutaneous IL-2 preactivates these same cells
and brings about the growth thereof in vivo. Injection of the
growth factor IL-2 alone brings about no specific response in the
T.gamma.9.delta.2 lymphocytes.
* * * * *