U.S. patent application number 11/989054 was filed with the patent office on 2009-10-15 for analogous compounds of 6-thioguanosine triphosphate, their use in medical fields and processes for their preparation.
Invention is credited to Sergio Baroni, Giancarlo Naccari.
Application Number | 20090258837 11/989054 |
Document ID | / |
Family ID | 38157753 |
Filed Date | 2009-10-15 |
United States Patent
Application |
20090258837 |
Kind Code |
A1 |
Naccari; Giancarlo ; et
al. |
October 15, 2009 |
Analogous compounds of 6-thioguanosine triphosphate, their use in
medical fields and processes for their preparation
Abstract
The invention relates to analogous compounds of 6-thioguanosine
triphosphate of general formula (I). A compound of the general
formula (I); wherein the dashed bond in the sugar moiety can be
either single or double and wherein R1, R2, R3, R4 or R5, equal or
different between each other, have general formula
-(Int).sub.m-Ter, wherein m is between 0 and 12 and Int and Ter are
Internal and Terminal building blocks, wherein Int is selected from
the group consisting of formula (II); and Ter is selected from the
group consisting of formula (III). And wherein X represents either
carbon or nitrogen atom within aromatic ring, Y represents either
oxygen or sulphur atom and an additional group Q, group Qi or
groups Qi (Qi indicates that the group or several groups may be
bound to any unsaturated moiety of the ring) are selected from the
group consisting of --OH, --COOH, --N(CH.sub.3).sub.2,
--N(CH.sub.2--CH.sub.3).sub.2|--CO--CH.sub.3, --CO--O--CH.sub.3,
--O--CH.sub.3, --S--CH.sub.3, --SO.sub.2--CH.sub.3, --CN,
--NO.sub.2 or -Halogen elements, and wherein R5 may be formula (IV)
and metal and ammonium salts thereof, wherein n is between O and 5,
or oxygen or phosphorus is partially or completely replaced by
nitrogen, sulphur, methyleno groups or their derivatives. The
invention also concerns the uses of the above mentioned compounds
in medical field and the process for their preparation.
##STR00001##
Inventors: |
Naccari; Giancarlo; (Monza,
IT) ; Baroni; Sergio; (Villa d' Adda, IT) |
Correspondence
Address: |
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
530 VIRGINIA ROAD, P.O. BOX 9133
CONCORD
MA
01742-9133
US
|
Family ID: |
38157753 |
Appl. No.: |
11/989054 |
Filed: |
July 24, 2006 |
PCT Filed: |
July 24, 2006 |
PCT NO: |
PCT/IE2006/000077 |
371 Date: |
June 6, 2008 |
Current U.S.
Class: |
514/48 ; 514/45;
536/26.26; 536/26.7; 536/27.21 |
Current CPC
Class: |
A61P 21/00 20180101;
A61P 17/00 20180101; C07H 19/16 20130101; A61P 1/00 20180101; A61P
1/16 20180101; A61P 37/06 20180101; A61P 37/00 20180101; A61P 17/16
20180101; C07H 19/20 20130101; A61P 17/06 20180101; A61P 35/00
20180101; A61P 13/12 20180101; A61P 7/06 20180101; A61P 9/00
20180101; A61P 1/04 20180101; A61P 21/04 20180101; A61P 7/00
20180101; A61P 19/02 20180101; A61P 29/00 20180101; A61P 25/00
20180101 |
Class at
Publication: |
514/48 ;
536/26.26; 536/27.21; 536/26.7; 514/45 |
International
Class: |
A61K 31/708 20060101
A61K031/708; C07H 19/20 20060101 C07H019/20; C07H 19/16 20060101
C07H019/16; A61P 37/06 20060101 A61P037/06; A61P 37/00 20060101
A61P037/00; A61P 17/00 20060101 A61P017/00; A61P 17/06 20060101
A61P017/06 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 22, 2005 |
IT |
RM2005A000391 |
Jun 14, 2006 |
IE |
2006/0448 |
Claims
1. A compound of the general formula (I): ##STR00207## wherein the
dashed bond in the sugar moiety can be either single or double and
wherein R1, R2, R3, R4 or R5, equal or different between each
other, have general formula -(Int).sub.m-Ter, wherein m is between
0 and 12 and Int and Ter are Internal and Terminal building blocks,
wherein Int is selected from the group consisting of ##STR00208##
and Ter is selected from the group consisting of ##STR00209##
##STR00210## ##STR00211## and wherein X represents either carbon or
nitrogen atom within aromatic ring, Y represents either oxygen or
sulphur atom and an additional group Q, group Qi or groups Qi (Qi
indicates that the group or several groups may be bound to any
unsaturated moiety of the ring) are selected from the group
consisting of --OH, --COOH, --N(CH.sub.3).sub.2,
--N(CH.sub.2--CH.sub.3).sub.2, --CO--CH.sub.3, --CO--O--CH.sub.3,
--O--CH.sub.3, --S--CH.sub.3, --SO.sub.2--CH.sub.3, --CN,
--NO.sub.2 or -halogen elements and wherein R5 may be ##STR00212##
and metal and ammonium salts thereof, wherein n is between 0 and 5,
or oxygen or phosphorus is partially or completely replaced by
nitrogen, sulphur, methyleno groups or their derivatives.
2. A compound according to claim 1 wherein Int is selected from the
group consisting of ##STR00213##
3. A compound according to claim 1 wherein Ter is selected from the
group consisting of ##STR00214## ##STR00215##
4. A compound according to claim 1 wherein said compounds are
labelled.
5. A compound according to claim 1 with the general formula (Ia):
##STR00216## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4 or R.sub.5,
equal or different between each other, have general formula
-(Int).sub.m-Ter, wherein m is between 0 and 12 and Int and Ter are
Internal and Terminal building blocks, wherein Int is selected from
the consisting of ##STR00217## and Ter is selected from the
consisting of ##STR00218## wherein an additional group Q, group Qi
or groups Qi (Qi indicates that the group or several groups may be
bound to any unsaturated moiety of the ring) are selected from the
group consisting of --OH, --COOH, --N(CH.sub.3).sub.2,
--N(CH.sub.2--CH.sub.3).sub.2 or -halogen elements
6. A compound according to claim 1, wherein sugar moiety of
compounds of formula (I) are selected from the group consisting of
the following sugar moieties or sugar-like moieties:
##STR00219##
7. A compound according to claim 1 wherein R.sub.3 or R.sub.4 are
selected from ##STR00220## and wherein Q is selected from --OH
(FAM) or --N(CH.sub.3).sub.2 (TAMRA).
8. A compound as claimed in claim 1 with the following structure:
##STR00221##
9. A compound as claimed in claim 1 with the following structure:
##STR00222##
10. A compound as claimed in claim 1 with the following structure:
##STR00223##
11. A compound as claimed in claim 1 with the following structure:
##STR00224##
12. A compound as claimed in claim 1 with the following structure:
##STR00225##
13. A compound as claimed in claim 1 with the following structure:
##STR00226##
14. A compound as claimed in claim 1 with the following structure:
##STR00227##
15. A compound as claimed in claim 1 with the following structure:
##STR00228##
16. A compound as claimed in claim 1 with the following structure:
##STR00229##
17. A compound as claimed in claim 1 with the following structure:
##STR00230##
18. A compound as claimed in claim 1 with the following structure:
##STR00231##
19. A compound according to claim 1 of the general formula (II):
##STR00232## wherein n=1, 2 or 3, m is between 0 and 5, Int is
selected from the group consisting of ##STR00233## and Ter is
selected from group the consisting of ##STR00234## ##STR00235##
wherein X represents either carbon or nitrogen atom within aromatic
ring, Y represents either oxygen or sulphur atom and an additional
group Q or groups Qi (i indicating the position of any unsaturated
moiety of the ring to which the group Q may be bound) are selected
from the group consisting of --CH.sub.3, --C(CH.sub.3).sub.3, --OH,
--COOH, --CO--CH.sub.3, --CO--O--CH.sub.3, --O--CH.sub.3,
--S--CH.sub.3, --SO.sub.2--CH.sub.3, --N(CH.sub.3).sub.2,
--N(CH.sub.2--CH.sub.3).sub.2, --CN, --NO.sub.2 or -halogen
elements.
20. A compound according to claim 19 wherein, Ter is selected from
the group consisting of ##STR00236##
21. A compound as claimed in claim 19 wherein n is 1-3.
22. A compound as claimed in claim 19 wherein n is 3.
23. A compound as claimed in claim 19 wherein n is 1.
24. A compound as claimed in claim 19 wherein n is 2.
25. A compound as claimed in claim 19 with the following general
formula: ##STR00237##
26. A compound as claimed in claim 19 with the following general
formula: ##STR00238##
27. A compound as claimed in claim 19 with the following general
formula: ##STR00239##
28. A compound as claimed in claim 19 with the following general
formula: ##STR00240##
29. A compound as claimed in claim 19 with the following general
formula: ##STR00241##
30. A compound as claimed in claim 19 with the following general
formula: ##STR00242##
31. A compound as claimed in claim 19 with the following general
formula: ##STR00243##
32. A compound as claimed in claim 19 with the following general
formula: ##STR00244##
33. A compound according to claim 1, wherein said compounds are
selected from the group consisting of 2',3'-EDA-6-Thio-GTP,
FAM-2',3'-EDA-6-Thio-GTP, TAMRA-2',3'-EDA-6-Thio-GTP,
Aspartate-2',3'-EDA-6-Thio-GTP, Glutamate-2',3'-EDA-6-Thio-GTP,
Threonine-2',3'-EDA-6-Thio-GTP, Serine-2',3'-EDA-6-Thio-GTP,
2',3',5',O-Triacetyl-N-2-(Acetyl-6''-aminohexyl)-guanosine,
2',3',5'-Triacetyl-N-2-(6''-thioacetamide-hexyl)-6-Thioguanosine,
N-2-(6''-thioacetamide-hexyl)-6-Thioguanosine,
N-2-(6''-Aminohexyl)-6-Thioguanosine,
N-2-(6''-guanidino-hexyl)-6-Thioguanosine,
N-2-(6''-Aminohexyl)-6-Thio-GMP,
N-2-(6''-guanidino-hexyl)-6-Thio-GMP,
N-2-(6''-Aminohexyl)-6-Thio-GTP,
N-2-(6''-guanidino-hexyl)-6-Thio-GTP,
N-2-(6''-Aspartate-hexyl)-6-Thioguanosine,
N-2-(6''-Glutamate-hexyl)-6-Thioguanosine,
N-2-(6''-Threonine-hexyl)-6-Thioguanosine,
N-2-(6''-Serine-hexyl)-6-Thioguanosine,
N-2-(6''-Aminobutyl)-6-Thio-GTP,
N-2-(6''-guanidino-butyl)-6-Thioguanosine,
N-2-(6''-Aspartate-butyl)-6-Thioguanosine,
N-2-(6''-Glutamate-butyl)-6-Thioguanosine,
N-2-(6''-Threonine-butyl)-6-Thioguanosine,
N-2-(6''-Serine-butyl)-6-Thioguanosine,
N-2-(6''-Aminopropyl)-6-Thioguanosine,
N-2-(6''-guanidino-propyl)-6-Thioguanosine,
N-2-(6''-Aspartate-propyl)-6-Thioguanosine,
N-2-(6''-Glutamate-propyl)-6-Thioguanosine,
N-2-(6''-Threonine-propyl)-6-Thioguanosine,
N-2-(6''-Serine-propyl)-6-Thioguanosine,
N-2-(6''-Amino-2-butene)-6-Thioguanosine,
N-2-(6''-guanidino-2-butene)-6-Thioguanosine,
N-2-(6''-Aspartate-2-butene)-6-Thioguanosine,
N-2-(6''-Glutamate-2-butene)-6-Thioguanosine,
N-2-(6''-Threonine-2-butene)-6-Thioguanosine,
N-2-(6''-Serine-2-butene)-6-Thioguanosine,
N-2-(6''-Amino-2-butyne)-6-Thioguanosine,
N-2-(6''-guanidino-2-butyne)-6-Thioguanosine,
N-2-(6''-Aspartate-2-butyne)-6-Thioguanosine,
N-2-(6''-Glutamate-2-butyne)-6-Thioguanosine,
N-2-(6''-Threonine-2-butyne)-6-Thioguanosine,
N-2-(6''-Serine-2-butyne)-6-Thioguanosine,
N-2-(6''-Amino-2,4-hexadiyne)-6-Thioguanosine,
N-2-(6''-guanidino-2,4-hexadiyne)-6-Thioguanosine,
N-2-(6''-Aspartate-2,4-hexadiyne)-6-Thioguanosine,
N-2-(6''-Glutamate-2,4-hexadiyne)-6-Thioguanosine,
N-2-(6''-Threonine-2,4-hexadiyne)-6-Thioguanosine,
N-2-(6''-Serine-2,4-hexadiyne)-6-Thioguanosine.
34. A pharmaceutical composition comprising at least one of the
compounds as claimed in claim 1 as active principle and one or more
pharmaceutically acceptable co-adjuvants or excipients.
35. (canceled)
36. A method of preventing rejection of organ transplants or of
post-transplant nephropathy in an individual in need thereof
comprising administering to the individual a compound of claim
1.
37. The method of claim 36 further comprising administering
radiotherapy, corticosteroids or cytotoxic agents to the
individual.
38. A method of treating one or more pathologies in an individual
in need thereof, wherein the pathologies are selected from the
group consisting of inflammatory chronic intestinal diseases,
auto-immune enteropathy, active chronic hepatitis, rheumatoid
arthritis, Still's disease, systemic lupus erythematous, acquired
haemolytic anaemia, idiopathic thrombocytopenia, polyarthritis
nodosa, vasculitis, polyangitis, polymyositis, myasthenia gravis,
sarcoidosis, lipoid nephritis, multiple sclerosis, dermatomyositis,
pemphigus vulgaris, primary biliary cirrhosis, primary sclerosing
cholangitis, recurrent multiform erythema, chronic actinic
dermatitis, gangrenous hypoderm, ptyriasis rubra, Wegener's
granulomatosis, cutaneous vasculitis, atopic dermatitis, psoriasis,
pimply pemphigoid, comprising administering to the individual a
compound of claim 1.
39. The method of claim 38, wherein inflammatory chronic intestinal
diseases are selected from the group consisting of Crohn's disease,
ulcerous rectocolitis, indeterminate colitis.
40. The method of claim 38 further comprising administering
radiotherapy, corticosteroids or cytotoxic agents to the
individual.
41. A method for the treatment of cancer in an individual in need
thereof, comprising administering to the individual a compound of
claim 1.
42. A method of evaluating the binding properties of the compounds
of formula (I) by the RacI/Vav system wherein a compound of claim 1
is used in the RacI/Vav system.
43. (canceled)
44. The method of claim 36 wherein the organ transplants include
kidney, heart, lung, pancreas and liver transplantation.
45. A process for the preparation of a compound as claimed in claim
1 wherein the introduction of the --NH--R group at the 2 position
of guanosine ring comprises the following steps: a) protection of
the NH moiety of tri-O-acetyl-inosine; b) oxidative guanosine
ring-opening and O-deprotection; c) guanosine ring-closing and
introduction of a SH group at the 2 position of the guanosine ring
through the use of CS.sub.2; d) replacing the SH group at the 2
position with an amino-linker by using an excess of an aliphatic
diamine.
46. Process according to claim 45, comprising a further step e) of
protection of ribose OH groups and of the primary amine group by
acetylation.
47. Process according to claim 46, comprising a further step f) of
thiolation of C.dbd.O groups through the use of Lawesson's
reagent.
48-52. (canceled)
Description
[0001] The present invention relates to analogous compounds of
6-thioguanosine triphosphate, their use in medical field and
process for their preparation.
[0002] Particularly, the invention refers to the therapeutic use of
analogous compounds of 6-thioguanosine triphosphate for example as
immunosuppressant for the prevention of rejection of organ
transplants and of post-transplant nephropathy and in the treatment
of pathologies in which immune system is involved, such as, for
instance, inflammatory chronic intestinal diseases, such as Crohn's
disease, ulcerous rectocolitis, indeterminate colitis, or of
auto-immune enteropathy, active chronic hepatitis, rheumatoid
arthritis, Still's disease, systemic lupus erythematous, acquired
haemolytic anaemia, idiopathic thrombocytopenia, polyarthritis
nodosa, vasculitis, polyangitis, polymyositis, myasthenia gravis,
sarcoidosis, lipoid nephritis, multiple sclerosis, dermatomyositis,
pemphigus vulgaris, primary biliary cirrhosis, primary sclerosing
cholangitis, recurrent multiform erythema, chronic actinic
dermatitis, gangrenous hypoderm, ptyriasis rubra, Wegener's
granulomatosis, cutaneous vasculitis, atopic dermatitis, psoriasis,
pimply pemphigoid and, in general, in the immunosuppressive
treatment in addition to radiotherapy, corticosteroids and other
cytotoxic agents. The latter also involves immunosuppressive
therapy after organ transplantation (e.g. kidney, heart, lung,
pancreas and liver transplantation).
[0003] The cells involved in the inflammatory immune response are
able to survive at the inflammatory site, however, after completion
of such response, the majority of cells must "die" to maintain the
homeostasis of organism (Boise, 1995). Since the uncontrolled
lymphocyte proliferation may cause the development of inflammatory
chronic pathologies, the immune system controls the depletion of
activated lymphocytes by a process named apoptosis (programmed cell
death).
[0004] This would assume a particular importance for the immune
system of the mucosa, since the apoptosis resistance of lamina
propria cells can lead to a chronic inflammatory response at the
intestinal level (Tiede, 2003).
[0005] The activation of the mucosal immune system plays a key role
in the pathogenesis of Crohn's disease. Particularly,
pro-inflammatory cytokines produced by T lymphocytes and
macrophages, in particular interleukine-6 (IL-6) and
interleukine-12 (IL-12), may cause T lymphocytes resistance against
apoptosis, which in its turn provokes an intestinal accumulation of
lymphocytes and establishes a long-lasting disease (Tiede,
2003).
[0006] The lymphocytes activation starts with two signals: the
specific binding of antigens to the TCR (T cell receptor) and a
second co-stimulatory signal represented by transmembrane proteins,
such as CD28 (Maltzman, 2003). It has been shown that
co-stimulation with CD28 enhances in vitro survival of activated T
lymphocytes; in fact, CD28 induces an enhanced production of
interleukine-2 (IL-2) acting as extrinsic factor for T lymphocytes
survival, and the intrinsic ability of T lymphocytes to be
resistant against apoptosis (Boise, 1995 bis). This occurs since
CD28 action is associated with the expression of an anti-apoptotic
gene, named bcl-x.sub.L gene (Khoshnan, 2000; Noel, 1996).
[0007] The steps through which an inhibition of apoptosis takes
place, will be synthesized as follows, as shown in FIG. 1: [0008]
CD28 acts through its cytoplasmaic portion with a complex of
"adaptor" proteins and with a molecule, named Vav (Frauwirth,
2002); [0009] Vav acts as guanosine nucleotide exchange factor
(GEF) for another molecule named Rac1 (Frauwirth, 2002); [0010]
Rac1, a small GTPase, in such a way switches between an inactive
state bound to GDP and an active state bound to GTP (Frauwirth,
2002); [0011] Activated Rac1, in its turn, leads to the activation
of kinases (IKK) that phosphorylate the NF-.kappa.B inhibitory
proteins (like I-.kappa.B alfa) (Marinari, 2002) through MEK
phosphorylation; [0012] Thus, NF-.kappa.B is not anymore retained
in an inactive form in the cytosol but is able to translocate to
nucleus where induces bcl-x.sub.L expression (Khoshnan, 2000);
[0013] Further, activated Rac1 stimulates the activation of a
protein belonging to the STAT family (Signal Transducers and
Activators of Transcription), that is STAT-3, thus inducing its
translocation to the nucleus and the corresponding expression of
STAT-3 dependent genes (Faruqi, 2001). In particular, STAT-3
induces bcl-x.sub.L expression thus contributing to the resistance
against apoptosis and to the accumulation of T lymphocytes in the
inflamed mucosa during the course of Crohn's disease (Mudter,
2003). On the other hand, the study of intestinal T lymphocytes has
pointed out that STAT-3 is steadily activated in patients with
Crohn's disease, but not in healthy voluntaries (Lovato 2003).
[0014] Rac1, together with RhoA and Cdc42, belongs to the Rho
family which is a superfamily of small G protein characterized in
that they are able to bind guanosine nucleotides and to regulate
many cellular responses. They cycle between an inactive state, when
bound to GDP, to an active state with GTP in place of GDP. This
reaction is sustained upon guanosine nucleotide exchange factors
named GEFs (like Vav). The binding with GTP induces a
conformational change, which allows Rac1 and other GTPases to bind
to their effectors. The action of other proteins called
GTPase-activating protein (GAPs) stimulates the innate GTPase
activity of these small G proteins and causes them to turn back to
their GDP bound inactive state. Rac1, as in general all GTPase
belonging to the Rho family, plays an important role in the
mitogenesis processes, proliferation, and invasivity, since it
stimulates alterations of the gene expression, in the present case
of the gene bcl-x.sub.L, modulating the activity of transcription
factors, such as, in the present case, NF-.kappa.B and STAT-3 (Van
Aelst, 1997).
[0015] Azathioprine is regarded as "gold standard" of the
immunosuppressive therapy of Crohn's disease, also if the mechanism
of action of such active principle is still unknown. However, the
inhibition of the purine nucleotide biosynthesis with suppression
of DNA and RNA synthesis and downregulation of T and B lymphocytes
function (Tiede 2003), is assumed to be the main therapeutic
mechanism of azathioprine.
[0016] Recently, a new mechanism of action of azathioprine acting
at the T lymphocytes level has been shown. After the evidence that
azathioprine induces in vitro apoptosis of activated T lymphocytes
and that treatment with azathioprine causes apoptosis of both
circulating and lamina propria T lymphocytes of IBD patients, the
specific molecular mechanisms were pointed out (Tiede 2003).
[0017] The key point is represented by the metabolite
6-thioguanosine triphosphate (6-thioGTP) which represents the real
functional metabolite of the drug. Specifically, 6-thioGTP binds
directly to Rac1 in place of GTP, thus blocking its activation.
Such a block is highly specific for Rac1, as other GTPases
belonging to the same family are not inhibited by 6-thioGTP, and
this specificity suggests that the block would be correlated to the
structure of Rac1 protein. The fact that an accumulation of the
Rac1 guanosine nucleotide exchange factor vav was observed, is
consistent with a compensatory mechanism to achieve Rac1
activation.
[0018] The block of the activation of Rac1 would result in the
block of NF-.kappa.B and STAT-3, normally induced by Rac1 itself,
and thus in the block of the bcl-x.sub.L gene expression, detected
both at the mRNA and protein levels. Thus, azathioprine, by
modulating Rac1 activity switches an anti-apoptotic co-stimulatory
signal, mediated by CD28, into a pro-apoptotic signal.
[0019] This new mechanism of action is able to explain the
well-known "delay" in the therapeutic effect of azathioprine, which
needs a long treatment time to elicit a clinical response in such a
way that benefits and clinical response were not observed earlier
than 4 months of therapy. This is due to the fact that 6-thioGPT
has 20-fold less affinity to Rac1 in comparison with GTP, which is
normally bound to Rac1. Therefore, the azathioprine treatment needs
the simultaneous and prolonged administration with high steroids
doses, having remarkable side effects such as osteoporosis,
diabetes, cataract.
[0020] In the light of the above it would be desirable to have at
disposal new immunosuppressive drugs eliciting a faster and more
efficacious therapeutic response in comparison with the already
known compounds.
[0021] According to the present invention, a new class of 6-thioGTP
analogous drugs able to inhibit Rac1 and characterized by greater
affinity to Rac1, an higher suppression of Rac1 activity and, thus
having a greater immunosuppressive power and action in comparison
with the delayed effect of azathioprine treatment and a best
therapeutic effect, was prepared.
[0022] It is therefore an object of the present invention to
provide a class of analogous compounds of 6-thioguanosine
triphosphate of general formula (I):
##STR00002##
wherein the dashed bond in the sugar moiety can be either single or
double and wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4 or R.sub.5,
equal or different between each other, have general formula
-(Int).sub.m-Ter, wherein m is between 0 and 12 and Int and Ter are
Internal and Terminal building blocks, wherein Int is selected from
the group consisting of
##STR00003##
and Ter is selected from the group consisting of
##STR00004## ##STR00005## [0023] wherein X represents either carbon
or nitrogen atom within aromatic ring, Y represents either oxygen
or sulphur atom and an additional group Q, group Qi or groups Qi
(Qi indicates that the group or several groups may be bound to any
unsaturated moiety of the ring) are selected from the group
consisting of --OH, --COOH, --N(CH.sub.3).sub.2,
--N(CH.sub.2--CH.sub.3).sub.2, --CO--CH.sub.3, --CO--O--CH.sub.3,
--O--CH.sub.3, --S--CH.sub.3, --SO.sub.2--CH.sub.3, --CN,
--NO.sub.2 or -Halogen elements. [0024] Alternatively R.sub.5 may
be
[0024] ##STR00006## [0025] and metal and ammonium salts thereof,
wherein n is between 0 and 5, or oxygen or phosphorus is partially
or completely replaced by nitrogen, sulphur, methylene groups or
their derivatives. [0026] In some embodiments (particularly, but
not limited to those where R.sub.5 is HO--[PO.sub.3].sub.n, and
metal and ammonium salts thereof, wherein n is between 0 and 5, or
oxygen or phosphorus is partially or completely replaced by
nitrogen, sulphur, methylene groups or their derivatives) TER may
be selected from the group consisting of:
##STR00007## ##STR00008##
[0027] Compounds of formula (I) can be labelled, particularly with
R.sub.3 or R.sub.4 selected from
##STR00009##
wherein Q is selected from --OH (FAM) or --N(CH.sub.3).sub.2
(TAMRA).
[0028] In addition the sugar moiety of compounds of formula (I) can
be selected from the group consisting of the following sugar
moieties or sugar-like moieties:
##STR00010##
[0029] Compounds of the invention may be of the general formula
(II) derived from general formula (Ia) and/or (I) where R.sub.1 is
[--SH] (it is understood that the Guanosine moiety may undergo
keto-enol tautomeric shifts and so give rise to [.dbd.S]), R.sub.2
is [--H], R.sub.5 is [--(PO.sub.3).sub.n--OH] and one of R.sub.3
and R.sub.4 is [--OH] and the other of R.sub.3 and R.sub.4 is
[--O--CO--NH-Int.sub.m-Ter] (both versions are provided in the same
mixture, i.e., [--O--CO--NH-Int.sub.m-Ter]: will be the same, but a
proportion of the molecules will have
[--O--CO--NH---Int.sub.m-Ter]: attached at R.sub.3 with R.sub.4
being OH, and another proportion of the molecules will have
[--O--CO--NH-Int.sub.m-Ter]: attached at R.sub.4 with R.sub.3 being
OH):
##STR00011##
wherein n=1, 2 or 3, m is between 0 and 5, Int is selected from the
group consisting of
##STR00012## ##STR00013##
and Ter is selected from the consisting of
##STR00014## ##STR00015##
wherein X represents either carbon or nitrogen atom within aromatic
ring, Y represents either oxygen or sulphur atom and an additional
group Q or groups Qi (i indicating the position of any unsaturated
moiety of the ring to which the group Q may be bound) are selected
from the group consisting of --CH.sub.3, --C(CH.sub.3).sub.3, --OH,
--COOH, --CO--CH.sub.3, --CO--O--CH.sub.3, --O--CH.sub.3,
--S--CH.sub.3, --SO.sub.2--CH.sub.3, --N(CH.sub.3).sub.2,
--N(CH.sub.2--CH.sub.3).sub.2, --CN, --NO.sub.2 or -Halogen
elements.
[0030] In some embodiments of the invention of formula (I), (Ia)
and (II), Ter is selected from the group consisting of
##STR00016## ##STR00017##
[0031] According to some embodiments of the present invention the
compounds of formula (I) are the compounds described below:
##STR00018##
wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4 or R.sub.5, equal or
different between each other, have general formula
-(Int).sub.m-Ter, wherein m is between 0 and 12 and Int and Ter are
Internal and Terminal building blocks, wherein Int is selected from
the consisting of
##STR00019##
and Ter is selected from the consisting of
##STR00020##
wherein an additional group Q, group Qi or groups Qi (Qi indicates
that the group or several groups may be bound to any unsaturated
moiety of the ring) are selected from the group consisting of --OH,
--COOH, --N(CH.sub.3).sub.2, --N(CH.sub.2--CH.sub.3).sub.2 or
-Halogen elements
[0032] In addition the sugar moiety of compounds of formula (I) can
be selected from the group consisting of the following sugar
moieties or sugar-like moieties:
##STR00021##
[0033] Compounds of formula (I), (Ia) and (II) can be labelled,
particularly with R.sub.3 or R.sub.4 selected from
##STR00022##
wherein Q is selected from --OH (FAM) or --N(CH.sub.3).sub.2
(TAMRA).
[0034] According to some embodiments of the present invention the
compounds of formula (I), (Ia) and (II) are the compounds described
below: [0035] SM4410
2',3'-EDA-6-Thio-GTP, ID: 05B-0
TABLE-US-00001 ##STR00023## [0036] m = 0 Ter --R1 --SH n = 3 1 2 3
Ter --R5 --PO.sub.3-- --PO.sub.3-- --PO.sub.3-- --OH m = 6
Int.sub.1 Int.sub.2 Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6 Ter
--R3 o --R4 --O-- ##STR00024## --NH-- --CH.sub.2-- --CH.sub.2--
--NH-- --H m = 0 Ter --R3 o --OH --R4 m = 0 Ter --R2 --H
FAM-2',3'-EDA-6-Thio-GTP
TABLE-US-00002 ##STR00025## [0037] m = 0 Ter --R1 --SH n = 3 1 2 3
Ter --R5 --PO.sub.3-- --PO.sub.3-- --PO.sub.3-- --OH m = 0 Ter --R2
--H m = 8 Int.sub.1 Int.sub.2 Int.sub.3 Int.sub.4 Int.sub.5
Int.sub.6 Int.sub.7 Int.sub.8 Ter --R3 o --R4 --O-- ##STR00026##
--NH-- --CH.sub.2-- --CH.sub.2-- --NH-- ##STR00027## ##STR00028##
##STR00029## m = 0 Ter --R3 o --OH R4
TAMRA-2',3'-EDA-6-Thio-GTP
TABLE-US-00003 ##STR00030## [0038] m = 0 Ter --R1 --SH n = 3 1 2 3
Ter --R5 --PO.sub.3-- --PO.sub.3-- --PO.sub.3-- --OH m = 0 Ter --R3
o --OH R4 m = 8 Int.sub.1 Int.sub.2 Int.sub.3 Int.sub.4 Int.sub.5
Int.sub.6 Int.sub.7 --R3 o --R4 --O-- ##STR00031## --NH--
--CH.sub.2-- --CH.sub.2-- --NH-- ##STR00032## m = 8 Int.sub.8 Ter
--R3 o --R4 ##STR00033## ##STR00034## m = 0 Ter --R2 --H
Aspartate-2,3'-EDA-6-Thio-GTP, ID: 05B-1
TABLE-US-00004 ##STR00035## [0039] m = 0 Ter --R1 --SH n = 3 1 2 3
Ter --R5 --PO.sub.3-- --PO.sub.3-- --PO.sub.3-- --OH m = 9
Int.sub.1 Int.sub.2 Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6
Int.sub.7 Int.sub.8 Int.sub.9 Ter --R3 o --R4 --O-- ##STR00036##
--NH-- --CH.sub.2-- --CH.sub.2-- --NH-- ##STR00037## ##STR00038##
--CH.sub.2-- --COOH m = 0 Ter --R2 m = 0 Ter --R3 o --R4
Glutamate-2',3'-EDA-6-Thio-GTP, ID: 05B-2
TABLE-US-00005 ##STR00039## [0040] m = 0 Ter --R1 --SH n = 3 1 2 3
Ter --R5 --PO.sub.3-- --PO.sub.3-- --PO.sub.3-- --OH m = 10
Int.sub.1 Int.sub.2 Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6
Int.sub.7 Int.sub.8 Int.sub.9 Int.sub.10 Ter --R3 o --R4 --O--
##STR00040## --NH-- --CH.sub.2-- --CH.sub.2-- --NH-- ##STR00041##
##STR00042## --CH.sub.2-- --CH.sub.2-- --COOH m = 0 Ter --R2 --H m
= 0 Ter --R3 o --OH --R4
Threonine-2',3'-EDA-6-Thio-GTP, ID: 05B-3
TABLE-US-00006 ##STR00043## [0041] m = 0 Ter --R1 --SH n = 3 1 2 3
Ter --R5 --PO.sub.3-- --PO.sub.3-- --PO.sub.3-- --OH m = 9
Int.sub.1 Int.sub.2 Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6
Int.sub.7 Int.sub.8 Int.sub.9 Ter --R3 o --R4 --O-- ##STR00044##
--NH-- --CH.sub.2-- --CH.sub.2-- --NH-- ##STR00045## ##STR00046##
##STR00047## --CH.sub.3 m = 0 Ter --R2 --H m = 0 Ter --R3 o --OH
--R4
Serine-2',3'-EDA-6-Thio-GTP
TABLE-US-00007 ##STR00048## [0042] m = 0 Ter --R1 --SH n = 3 1 2 3
Ter --R5 --PO.sub.3-- --PO.sub.3-- --PO.sub.3-- --OH m = 9
Int.sub.1 Int.sub.2 Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6
Int.sub.7 Int.sub.8 Int.sub.9 Ter --R3 o --R4 --O-- ##STR00049##
--NH-- --CH.sub.2-- --CH.sub.2-- --NH-- ##STR00050## ##STR00051##
--CH.sub.2-- --OH m = 0 Ter --R3 o --OH --R4 m = 0 Ter --R2 --H
2',3',5',O-Triacetyl-N-2-(Acetyl-6''-aminohexyl)-guanosine
TABLE-US-00008 ##STR00052## [0043] m = 0 Ter --R1 --OH m = 2
Int.sub.1 Int.sub.2 Ter --R5 --O-- ##STR00053## --CH.sub.3 m = 2
Int.sub.1 Int.sub.2 Ter --R3 e --R4 --O-- ##STR00054## --CH.sub.3 m
= 8 Int.sub.1 Int.sub.2 Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6
Int.sub.7 Int.sub.8 Ter --R2 --CH.sub.2-- --CH.sub.2-- --CH.sub.2--
--CH.sub.2-- --CH.sub.2-- --CH.sub.2-- --NH-- ##STR00055##
--CH.sub.3
2',3',5'-Triacetyl-N-2-(6''-thioacetamide-hexyl)-6-Thioguanosine
(V4) [TWI 107/7]
TABLE-US-00009 ##STR00056## [0044] m = 0 Ter --R1 --SH m = 2
Int.sub.1 Int.sub.2 Ter --R5 --O-- ##STR00057## --CH.sub.3 m = 2
Int.sub.1 Int.sub.2 Ter --R3 e --R4 --O-- ##STR00058## --CH.sub.3 m
= 8 Int.sub.1 Int.sub.2 Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6
Int.sub.7 Int.sub.8 Ter --R2 --CH.sub.2-- --CH.sub.2-- --CH.sub.2--
--CH.sub.2-- --CH.sub.2-- --CH.sub.2-- --NH-- ##STR00059##
--CH.sub.3
N-2-(6''-thioacetamide-hexyl)-6-Thioguanosine
TABLE-US-00010 ##STR00060## [0045] m = 0 Ter --R1 --SH m = 0 Ter
--R5 --OH m = 0 Ter --R3 e R4 --OH m = 8 Int.sub.1 Int.sub.2
Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6 Int.sub.7 Int.sub.8 Ter
--R2 --CH.sub.2-- --CH.sub.2-- --CH.sub.2-- --CH.sub.2--
--CH.sub.2-- --CH.sub.2-- --NH-- ##STR00061## --CH.sub.3
N-2-(6''-Aminohexyl)-6-Thioguanosine, ID: 05A-0
TABLE-US-00011 ##STR00062## [0046] m = 0 Ter --R1 --SH m = 0 Ter
--R3 e --OH --R4 m = 0 Ter --R5 --OH m = 6 Int.sub.1 Int.sub.2
Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6 Ter --R2 --CH.sub.2--
--CH.sub.2-- --CH.sub.2-- --CH.sub.2-- --CH.sub.2-- --CH.sub.2--
--NH.sub.2
N-2-(6''-guanidino-hexyl)-6-Thioguanosine, ID: 05A-1
TABLE-US-00012 ##STR00063## [0047] m = 0 Ter --R1 --SH n = 0 Ter
--R5 --OH m = 0 Ter --R3 e --OH --R4 m = 8 Int.sub.1 Int.sub.2
Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6 Int.sub.7 Int.sub.8 Ter
--R2 --CH.sub.2-- --CH.sub.2-- --CH.sub.2-- --CH.sub.2--
--CH.sub.2-- --CH.sub.2-- --NH-- ##STR00064## --NH.sub.2
N-2-(6''-Aminohexyl)-6-Thio-GMP
TABLE-US-00013 ##STR00065## [0048] m = 0 Ter --R1 --SH n = 1 1 Ter
--R5 --PO.sub.3-- --OH m = 0 Ter --R3 e --OH --R4 m = 6 Int.sub.1
Int.sub.2 Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6 Ter --R2
--CH.sub.2-- --CH.sub.2-- --CH.sub.2-- --CH.sub.2-- --CH.sub.2--
--CH.sub.2-- --NH.sub.2
N-2-(6''-guanidino-hexyl)-6-Thio-GMP, ID: 05A-2
TABLE-US-00014 ##STR00066## [0049] m = 0 Ter --R1 --SH n = 1 1 Ter
--R5 --PO.sub.3-- --OH m = 0 Ter --R3 e --OH --R4 m = 8 Int.sub.1
Int.sub.2 Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6 Int.sub.7
Int.sub.8 Ter --R2 --CH.sub.2-- --CH.sub.2-- --CH.sub.2--
--CH.sub.2-- --CH.sub.2-- --CH.sub.2-- --NH-- ##STR00067##
--NH.sub.2
N-2-(6''-Aminohexyl)-6-Thio-GTP
TABLE-US-00015 ##STR00068## [0050] m = 0 Ter --R1 --SH n = 3 1 2 3
Ter --R5 --PO.sub.3-- --PO.sub.3-- --PO.sub.3-- --OH m = 0 Ter --R3
e --OH --R4 m = 6 Int.sub.1 Int.sub.2 Int.sub.3 Int.sub.4 Int.sub.5
Int.sub.6 Ter --R2 --CH.sub.2-- --CH.sub.2-- --CH.sub.2--
--CH.sub.2-- --CH.sub.2-- --CH.sub.2-- --NH.sub.2
N-2-(6''-guanidino-hexyl)-6-Thio-GTP, ID: 05A-3
TABLE-US-00016 ##STR00069## [0051] m = 0 Ter --R1 --SH n = 3 1 2 3
Ter --R5 --PO.sub.3-- --PO.sub.3-- --PO.sub.3-- --OH m = 0 Ter --R3
e --OH --R4 m = 8 Int.sub.1 Int.sub.2 Int.sub.3 Int.sub.4 Int.sub.5
Int.sub.6 Int.sub.7 Int.sub.8 Ter --R2 --CH.sub.2-- --CH.sub.2--
--CH.sub.2-- --CH.sub.2-- --CH.sub.2-- --CH.sub.2-- --NH--
##STR00070## --NH.sub.2
N-2-(6''-Aspartate-hexyl)-6-Thioguanosine
TABLE-US-00017 ##STR00071## [0052] m = 0 Ter --R1 --SH n = 0 Ter
--R5 --OH m = 0 Ter --R3 e --OH --R4 m = 10 Int.sub.1 Int.sub.2
Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6 Int.sub.7 Int.sub.8
Int.sub.9 Int.sub.10 Ter --R2 --CH.sub.2-- --CH.sub.2--
--CH.sub.2-- --CH.sub.2-- --CH.sub.2-- --CH.sub.2-- --NH--
##STR00072## ##STR00073## --CH.sub.2-- --COOH
N-2-(6''-Glutamate-hexyl)-6-Thioguanosine
TABLE-US-00018 ##STR00074## [0053] m = 0 Ter --R1 --SH n = 0 Ter
--R5 --OH m = 0 Ter --R3 --OH e --R4 m = 11 Int.sub.1 Int.sub.2
Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6 Int.sub.7 Int.sub.8
Int.sub.9 Int.sub.10 Int.sub.11 Ter --R2 --CH.sub.2-- --CH.sub.2--
--CH.sub.2-- --CH.sub.2-- --CH.sub.2-- --CH.sub.2-- --NH--
##STR00075## ##STR00076## --CH.sub.2-- --CH.sub.2-- --COOH
N-2-(6''-Threonine-hexyl)-6-Thioguanosine
TABLE-US-00019 ##STR00077## [0054] m = 0 Ter --R1 --SH n = 0 Ter
--R5 --OH m = 0 Ter --R3 o --OH --R4 m = 10 Int.sub.1 Int.sub.2
Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6 Int.sub.7 Int.sub.8
Int.sub.9 Int.sub.10 Ter --R2 --CH.sub.2-- --CH.sub.2--
--CH.sub.2-- --CH.sub.2-- --CH.sub.2-- --CH.sub.2-- --NH--
##STR00078## ##STR00079## ##STR00080## --CH.sub.3
N-2-(6''-Serine-hexyl)-6-Thioguanosine
TABLE-US-00020 ##STR00081## [0055] m = 0 Ter --R1 --SH n = 0 Ter
--R5 --OH m = 0 Ter --R3 e --OH --R4 m = 10 Int.sub.1 Int.sub.2
Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6 Int.sub.7 Int.sub.8
Int.sub.9 Int.sub.10 Ter --R2 --CH.sub.2-- --CH.sub.2--
--CH.sub.2-- --CH.sub.2-- --CH.sub.2-- --CH.sub.2-- --NH--
##STR00082## ##STR00083## --CH.sub.2-- --OH
N-2-(6''-Aminobutyl)-6-Thio-Guanosine, ID: 05C-0
TABLE-US-00021 ##STR00084## [0056] m = 0 Ter --R1 --SH n = 0 Ter
--R5 --OH m = 0 Ter --R3 e --OH --R4 m = 4 Int.sub.1 Int.sub.2
Int.sub.3 Int.sub.4 Ter --R2 --CH.sub.2-- --CH.sub.2-- --CH.sub.2--
--CH.sub.2-- --NH.sub.2
2-N-2-(6''-guanidino-butyl)-6-Thioguanosine, ID: 05C-1
TABLE-US-00022 ##STR00085## [0057] m = 0 Ter --R1 --SH n = 0 Ter
--R5 --OH m = 0 Ter --R3 e --OH --R4 m = 6 Int.sub.1 Int.sub.2
Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6 Ter --R2 --CH.sub.2--
--CH.sub.2-- --CH.sub.2-- --CH.sub.2-- --NH-- ##STR00086##
--NH.sub.2
2-N-2-(6''-guanidino-butyl)-6-Thio-GMP, ID: 05C-2
TABLE-US-00023 ##STR00087## [0058] m = 0 Ter n = 1 1 Ter m = 0 Ter
--R1 --SH --R5 --PO.sub.3-- --OH --R3 e --R4 --OH m = 6 Int.sub.1
Int.sub.2 Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6 Ter --R2
--CH.sub.2-- --CH.sub.2-- --CH.sub.2-- --CH.sub.2-- --NH--
##STR00088## --NH.sub.2
2-N-2-(6''-guanidino-butyl)-6-Thio-GTP, ID: 05C-3
TABLE-US-00024 ##STR00089## [0059] m = 0 Ter n = 3 1 2 3 Ter m = 0
Ter --R1 --SH --R5 --PO.sub.3-- --PO.sub.3-- --PO.sub.3-- --OH --R3
e --R4 --OH m = 6 Int.sub.1 Int.sub.2 Int.sub.3 Int.sub.4 Int.sub.5
Int.sub.6 Ter --R2 --CH.sub.2-- --CH.sub.2-- --CH.sub.2--
--CH.sub.2-- --NH-- ##STR00090## --NH.sub.2
N-2-(6''-Aspartate-butyl)-6-Thioguanosine
TABLE-US-00025 ##STR00091## [0060] m = 0 Ter n = 0 Ter m = 0 Ter
--R1 --SH --R5 --OH --R3 e --R4 --OH m = 8 Int.sub.1 Int.sub.2
Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6 Int.sub.7 Int.sub.8 Ter
--R2 --CH.sub.2-- --CH.sub.2-- --CH.sub.2-- --CH.sub.2-- --NH--
##STR00092## ##STR00093## --CH.sub.2-- --COOH
N-2-(6''-Glutamate-butyl)-6-Thioguanosine
TABLE-US-00026 ##STR00094## [0061] m = 0 Ter n = 0 Ter m = 0 Ter
--R1 --SH --R5 --OH --R3 e --R4 --OH m = 9 Int.sub.1 Int.sub.2
Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6 Int.sub.7 Int.sub.8
Int.sub.9 Ter --R2 --CH.sub.2-- --CH.sub.2-- --CH.sub.2--
--CH.sub.2-- --NH-- ##STR00095## ##STR00096## --CH.sub.2--
--CH.sub.2-- --COOH
N-2-(6''-Threonine-butyl)-6-Thioguanosine
TABLE-US-00027 ##STR00097## [0062] m = 0 Ter n = 0 Ter m = 0 Ter
--R1 --SH --R5 --OH --R3 e --R4 --OH m = 8 Int.sub.1 Int.sub.2
Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6 Int.sub.7 Int.sub.8 Ter
--R2 --CH.sub.2-- --CH.sub.2-- --CH.sub.2-- --CH.sub.2-- --NH--
##STR00098## ##STR00099## ##STR00100## --CH.sub.3
N-2-(6''-Serine-butyl)-6-Thioguanosine
TABLE-US-00028 ##STR00101## [0063] m = 0 Ter n = 0 Ter m = 0 Ter
--R1 --SH --R5 --OH --R3 e --R4 --OH m = 8 Int.sub.1 Int.sub.2
Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6 Int.sub.7 Int.sub.8 Ter
--R2 --CH.sub.2-- --CH.sub.2-- --CH.sub.2-- --CH.sub.2-- --NH--
##STR00102## ##STR00103## --CH.sub.2-- --OH
N-2-(6''-Aminopropyl)-6-Thioguanosine
TABLE-US-00029 ##STR00104## [0064] m = 0 Ter n = 0 Ter m = 0 Ter
--R1 --SH --R5 --OH --R3 e --R4 --OH m = 0 Int.sub.1 Int.sub.2
Int.sub.3 Ter --R2 --CH.sub.2-- --CH.sub.2-- --CH.sub.2--
--NH.sub.2
N-2-(6''-guanidino-propyl)-6-Thioguanosine
TABLE-US-00030 ##STR00105## [0065] m = 0 Ter n = 0 Ter m = 0 Ter
--R1 --SH --R5 --OH --R3 e --R4 --OH m = 5 Int.sub.1 Int.sub.2
Int.sub.3 Int.sub.4 Int.sub.5 Ter --R2 --CH.sub.2-- --CH.sub.2--
--CH.sub.2-- --NH-- ##STR00106## --NH.sub.2
N-2-(6''-Aspartate-propyl)-6-Thioguanosine
TABLE-US-00031 ##STR00107## [0066] m = 0 Ter n = 0 Ter m = 0 Ter
--R1 --SH --R5 --OH --R3 e --R4 --OH m = 7 Int.sub.1 Int.sub.2
Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6 Int.sub.7 Ter --R2
--CH.sub.2-- --CH.sub.2-- --CH.sub.2-- --NH-- ##STR00108##
##STR00109## --CH.sub.2-- --COOH
N-2-(6''-Glutamate-propyl)-6-Thioguanosine
TABLE-US-00032 ##STR00110## [0067] m = 0 Ter n = 0 Ter m = 0 Ter
--R1 --SH --R5 --OH --R3 e --R4 --OH m = 8 Int.sub.1 Int.sub.2
Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6 Int.sub.7 Int.sub.8 Ter
--R2 --CH.sub.2-- --CH.sub.2-- --CH.sub.2-- --NH-- ##STR00111##
##STR00112## --CH.sub.2-- --CH.sub.2-- --COOH
N-2-(6''-Threonine-propyl)-6-Thioguanosine
TABLE-US-00033 ##STR00113## [0068] m = 0 Ter n = 0 Ter m = 0 Ter
--R1 --SH --R5 --OH --R3 e --R4 --OH m = 7 Int.sub.1 Int.sub.2
Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6 Int.sub.7 Ter --R2
--CH.sub.2-- --CH.sub.2-- --CH.sub.2-- --NH-- ##STR00114##
##STR00115## ##STR00116## --CH.sub.3
N-2-(6''-Serine-propyl)-6-Thioguanosine
TABLE-US-00034 ##STR00117## [0069] m = 0 Ter n = 0 Ter m = 0 Ter
--R1 --SH --R5 --OH --R3 e --R4 --OH m = 7 Int.sub.1 Int.sub.2
Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6 Int.sub.7 Ter --R2
--CH.sub.2-- --CH.sub.2-- --CH.sub.2-- --NH-- ##STR00118##
##STR00119## --CH.sub.2-- --OH
N-2-(6''-Amino-2-butene)-6-Thioguanosine
TABLE-US-00035 ##STR00120## [0070] m = 0 Ter n = 0 Ter m = 0 Ter
--R1 --SH --R5 --OH --R3 e --R4 --OH m = 3 Int.sub.1 Int.sub.2
Int.sub.3 Ter --R2 --CH.sub.2-- ##STR00121## --CH.sub.2--
--NH.sub.2
N-2-(6''-guanidino-2-butene)-6-Thioguanosine
TABLE-US-00036 ##STR00122## [0071] m = 0 Ter n = 0 Ter m = 0 Ter
--R1 --SH --R5 --OH --R3 e --R4 --OH m = 5 Int.sub.1 Int.sub.2
Int.sub.3 Int.sub.4 Int.sub.5 Ter --R2 --CH.sub.2-- ##STR00123##
--CH.sub.2-- --NH-- ##STR00124## --NH.sub.2
N-2-(6''-Aspartate-2-butene)-6-Thioguanosine
TABLE-US-00037 ##STR00125## [0072] m = 0 Ter n = 0 Ter m = 0 Ter
--R1 --SH --R5 --OH --R3 e --R4 --OH m = 7 Int.sub.1 Int.sub.2
Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6 Int.sub.7 Ter --R2
--CH.sub.2-- ##STR00126## --CH.sub.2-- --NH-- ##STR00127##
##STR00128## --CH.sub.2-- --COOH
N-2-(6''-Glutamate-2-butene)-6-Thioguanosine
TABLE-US-00038 ##STR00129## [0073] m = 0 Ter n = 0 Ter m = 0 Ter
--R1 --SH --R5 --OH --R3 e --OH --R4 m = 8 Int.sub.1 Int.sub.2
Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6 Int.sub.7 Int.sub.8 Ter
--R2 --CH.sub.2-- ##STR00130## --CH.sub.2-- --NH-- ##STR00131##
##STR00132## --CH.sub.2-- --CH.sub.2-- --COOH
N-2-(6''-Threonine-2-butene)-6-Thioguanosine
TABLE-US-00039 ##STR00133## [0074] m = 0 Ter n = 0 Ter m = 0 Ter
--R1 --SH --R5 --OH --R3 e --OH --R4 m = 7 Int.sub.1 Int.sub.2
Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6 Int.sub.7 Ter --R2
--CH.sub.2-- ##STR00134## --CH.sub.2-- --NH-- ##STR00135##
##STR00136## ##STR00137## --CH.sub.3
N-2-(6''-Serine-2-butene)-6-Thioguanosine
TABLE-US-00040 ##STR00138## [0075] m = 0 Ter n = 0 Ter m = 0 Ter
--R1 --SH --R5 --OH --R3 e --OH --R4 m = 7 Int.sub.1 Int.sub.2
Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6 Int.sub.7 Ter --R2
--CH.sub.2-- ##STR00139## --CH.sub.2-- --NH-- ##STR00140##
##STR00141## --CH.sub.2-- --OH
N-2-(6''-Amino-2-butyne)-6-Thioguanosine
TABLE-US-00041 ##STR00142## [0076] m = 0 Ter n = 0 Ter m = 0 Ter
--R1 --SH --R5 --OH --R3 e --OH --R4 m = 3 Int.sub.1 Int.sub.2
Int.sub.3 Ter --R2 --CH.sub.2-- --C.ident.C-- --CH.sub.2--
--NH.sub.2
N-2-(6''-guanidino-2-butyne)-6-Thioguanosine
TABLE-US-00042 ##STR00143## [0077] m = 0 Ter n = 0 Ter m = 0 Ter
--R1 --SH --R5 --OH --R3 e --OH --R4 m = 5 Int.sub.1 Int.sub.2
Int.sub.3 Int.sub.4 Int.sub.5 Ter --R2 --CH.sub.2-- --C.ident.C--
--CH.sub.2-- --NH-- ##STR00144## --NH.sub.2
N-2-(6''-Aspartate-2-butyne)-6-Thioguanosine
TABLE-US-00043 ##STR00145## [0078] M = 0 Ter n = 0 Ter m = 0 Ter
--R1 --SH --R5 --OH --R3 e --OH --R4 m = 7 Int.sub.1 Int.sub.2
Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6 Int.sub.7 Ter --R2
--CH.sub.2-- --C.ident.C-- --CH.sub.2-- --NH-- ##STR00146##
##STR00147## --CH.sub.2-- --COOH
N-2-(6''-Glutamate-2-butyne)-6-Thioguanosine
TABLE-US-00044 ##STR00148## [0079] m = 0 Ter n = 0 Ter m = 0 Ter
--R1 --SH --R5 --OH --R3 e --OH --R4 m = 8 Int.sub.1 Int.sub.2
Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6 Int.sub.7 Int.sub.8 Ter
--R2 --CH.sub.2-- --C.ident.C-- --CH.sub.2-- --NH-- ##STR00149##
##STR00150## --CH.sub.2-- --CH.sub.2-- --COOH
N-2-(6''-Threonine-2-butyne)-6-Thioguanosine
TABLE-US-00045 ##STR00151## [0080] m = 0 Ter n = 0 Ter m = 0 Ter
--R1 --SH --R5 --OH --R3 e --OH --R4 m = 7 Int.sub.1 Int.sub.2
Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6 Int.sub.7 Ter --R2
--CH.sub.2-- --C.ident.C-- --CH.sub.2-- --NH-- ##STR00152##
##STR00153## ##STR00154## --CH.sub.3
N-2-(6''-Serine-2-butyne)-6-Thioguanosine
TABLE-US-00046 ##STR00155## [0081] m = 0 Ter n = 0 Ter m = 0 Ter
--R1 --SH --R5 --OH --R3 e --OH --R4 m = 7 Int.sub.1 Int.sub.2
Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6 Int.sub.7 Ter --R2
--CH.sub.2-- --C.ident.C-- --CH.sub.2-- --NH-- ##STR00156##
##STR00157## --CH.sub.2-- --OH
N-2-(6''-Amino-2,4-hexadiyne)-6-Thioguanosine
TABLE-US-00047 ##STR00158## [0082] m = 0 Ter n = 0 Ter m = 0 Ter
--R1 --SH --R5 --OH --R3 e --OH --R4 m = 4 Int.sub.1 Int.sub.2
Int.sub.3 Int.sub.4 Ter --R2 --CH.sub.2-- --C.ident.C--
--C.ident.C-- --CH.sub.2-- --NH.sub.2
N-2-(6''-guanidino-2,4-hexadiyne)-6-Thioguanosine
TABLE-US-00048 ##STR00159## [0083] m = 0 Ter n = 0 Ter m = 0 Ter
--R1 --SH --R5 --OH --R3 e --OH --R4 m = 6 Int.sub.1 Int.sub.2
Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6 Ter --R2 --CH.sub.2--
--C.ident.C-- --C.ident.C-- --CH.sub.2-- --NH-- ##STR00160##
--NH.sub.2
N-2-(6''-Aspartate-2,4-hexadiyne)-6-Thioguanosine
TABLE-US-00049 ##STR00161## [0084] m = 0 Ter n = 0 Ter m = 0 Ter
--R1 --SH --R5 --OH --R3 e --OH --R4 m = 8 Int.sub.1 Int.sub.2
Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6 Int.sub.7 Int.sub.8 Ter
--R2 --CH.sub.2-- --C.ident.C-- --C.ident.C-- --CH.sub.2-- --NH--
##STR00162## ##STR00163## --CH.sub.2-- --COOH
N-2-(6''-Glutamate-2,4-hexadiyne)-6-Thioguanosine
TABLE-US-00050 ##STR00164## [0085] m = 0 Ter n = 0 Ter m = 0 Ter
--R1 --SH --R5 --OH --R3 e --OH --R4 m = 9 Int.sub.1 Int.sub.2
Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6 Int.sub.7 Int.sub.8
Int.sub.9 Ter --R2 --CH.sub.2-- --C.ident.C-- --C.ident.C--
--CH.sub.2-- --NH-- ##STR00165## ##STR00166## --CH.sub.2--
--CH.sub.2-- --COOH
N-2-(6''-Threonine-2,4-hexadiyne)-6-Thioguanosine
TABLE-US-00051 ##STR00167## [0086] m = 0 Ter n = 0 Ter m = 0 Ter
--R1 --SH --R5 --OH --R3 e --OH --R4 m = 8 Int.sub.1 Int.sub.2
Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6 Int.sub.7 Int.sub.8 Ter
--R2 --CH.sub.2-- --C.ident.C-- --C.ident.C-- --CH.sub.2-- --NH--
##STR00168## ##STR00169## ##STR00170## --CH.sub.3
N-2-(6''-Serine-2,4-hexadiyne)-6-Thioguanosine
TABLE-US-00052 ##STR00171## [0087] m = 0 Ter n = 0 Ter m = 0 Ter
--R1 --SH --R5 --OH --R3 e --OH --R4 m = 8 Int.sub.1 Int.sub.2
Int.sub.3 Int.sub.4 Int.sub.5 Int.sub.6 Int.sub.7 Int.sub.8 Ter
--R2 --CH.sub.2-- --C.ident.C-- --C.ident.C-- --CH.sub.2-- --NH--
##STR00172## ##STR00173## --CH.sub.2-- --OH
[0088] Further compounds of the invention are disclosed, with
reference titles indicated.
##STR00174## ##STR00175## ##STR00176## ##STR00177## ##STR00178##
##STR00179## ##STR00180## ##STR00181## ##STR00182##
[0089] Compounds according to the present invention, can be
advantageously used in medical field; therefore another object of
the present invention are pharmaceutical composition comprising at
least one of the compounds of the above mentioned formula (I) as
active principle and one or more pharmaceutically acceptable
co-adjuvants or excipients, that are known to those skilled in the
art and currently in use in the pharmaceutical technology.
[0090] It is an object of the invention to provide for compounds
for the preparation of an immunosuppressive drug. Its uses and
therapeutical and medical uses thereof. for It is an object of the
present invention to provide the compounds and compositions of the
invention, and their uses, in the following methods of treatment,
and/or therapy; the prevention of rejection of organ transplants
(e.g. kidney, heart, lung, pancreas, liver transplantation) and of
post-transplant nephropathy and in the treatment of pathologies in
which immune system is involved, such as, for instance,
inflammatory chronic intestinal diseases, such as Crohn's disease,
ulcerous rectocolitis, indeterminate colitis, or auto-immune
enteropathy, active chronic hepatitis, rheumatoid arthritis,
Still's disease, systemic lupus erythematous, acquired haemolytic
anaemia, idiopathic thrombocytopenia, polyarthritis nodosa,
vasculitis, polyangitis, polymyositis, myasthenia gravis,
sarcoidosis, lipoid nephritis, multiple sclerosis, dermatomyositis,
pemphigus vulgaris, primary biliary cirrhosis, primary sclerosing
cholangitis, recurrent multiform erythema, chronic actinic
dermatitis, gangrenous hypoderm, ptyriasis rubra, Wegener's
granulomatosis, cutaneous vasculitis, atopic dermatitis, psoriasis,
pimply pemphigoid and, in general, in the immunosuppressive
treatment in addition to radiotherapy, corticosteroids and other
cytotoxic agents. In addition, the compounds according to the
present invention can be advantageously used for the preparation of
a medicament for the treatment of cancer.
[0091] The present invention further relates to the use of labelled
compounds of formula (I), particularly with R.sub.3 or R.sub.4
selected from
##STR00183##
wherein Q is selected from --OH (FAM) or --N(CH.sub.3).sub.2
(TAMRA) as probes for the evaluation of the binding properties of
the compounds of formula (I) by the RacI/Vav system.
[0092] According to a further aspect, the present invention refers
to a process for the preparation of compounds of formula (I), (Ia)
and (II), wherein the introduction of the --NH--R group at the 2
position of guanosine ring comprises the following steps:
a) protection of the NH moiety of tri-O-acetyl-inosine; b)
oxidative guanosine ring-opening and O-deprotection; c) guanosine
ring-closing and introduction of a SH group at the 2 position of
the guanosine ring through the use of CS.sub.2; d) replacing the SH
group at the 2 position with an amino-linker by using an excess of
an aliphatic diamine. The process may further comprise an
additional step e) of protection of ribose OH groups and of the
primary amine group by acetylation, and, moreover, an additional
step f) of thiolation of C.dbd.O groups through the use of
Lawesson's reagent.
[0093] The present invention will be now described, for
illustrative but not limitative purposes, according to its
preferred embodiments, with particular reference to the Figures of
the enclosed drawings.
DESCRIPTION OF DRAWINGS
[0094] FIG. 1 shows the scheme of the mechanism of action of the
apoptosis inhibition and of the action of azathioprine.
[0095] FIG. 2 shows the apoptosis induction in human CD4+ T
Lymphocytes by V1=BMB20=EDA-6-Thio-GTP, V2=TWI
35/1=N-2-(6''-Aminohexyl)-guanosine, V3=TWI
71/2=2',3',5',o-Triacetyl-N-2-(Acetyl-6''-aminohexyl)-guanosine,
V4=TWI
107/7=2',3',5'-Triacetyl-N-2-(6''-thioacetamide-hexyl)-6-Thioguano-
sine, V5=BMB=TAMRA-EDA-6-Thio-GTP after 3 days.
[0096] FIG. 3 shows the apoptosis induction in human CD4+ T
Lymphocytes by V1=BMB20=EDA-6-Thio-GTP, V2=TWI
35/1=N-2-(6''-Aminohexyl)-guanosine, V3=TWI
71/2=2',3',5',o-Triacetyl-N-2-(Acetyl-6''-aminohexyl)-guanosine,
V4=TWI
107/7=2',3',5'-Triacetyl-N-2-(6''-thioacetamide-hexyl)-6-Thioguano-
sine, V5=BMB=TAMRA-EDA-6-Thio-GTP after 4 days.
[0097] FIG. 4 shows the apoptosis induction in human CD4+ T
Lymphocytes by V1=BMB20=EDA-6-Thio-GTP, V2=TWI
35/1=N-2-(6''-Aminohexyl)-guanosine, V3=TWI
71/2=2',3',5',o-Triacetyl-N-2-(Acetyl-6''-aminohexyl)-guanosine,
V4=TWI
107/7=2',3',5'-Triacetyl-N-2-(6''-thioacetamide-hexyl)-6-Thioguano-
sine, V5=BMB=TAMRA-EDA-6-Thio-GTP after 5 days.
[0098] FIG. 5: Induction of apoptosis. Buffy stands for "buffy
coat" which is the fraction of blood obtainable by centrifugation
and containing leukocytes and platelets. In this case buffy coats
were used to isolate monocytes for the experiments. "Buffy 1 vom
08/03/05" means "Buffy coat 1 of Mar. 8, 2005". V1 and V3 have been
tested 4 times in four independent experiments, while V2 and V5
were tested twice in two separate experiments. For instance: V1 was
tested twice on Aug. 3, 2005 (where Aug. 3, 2004 is written this is
in error and should read 08/03/2005) and twice on 22/04/2005
[0099] FIG. 6: Results of biological activity of compounds compiled
in FIG. 5.
[0100] FIG. 7: list of further compounds of the invention, as well
as providing key to some of the shorthand used in identifying some
of the molecules. It will be noted that some compounds have
different enantiomeric forms, and the representations in FIG. 7 may
show an alternative enantiomeric form than that discussed in the
text.
[0101] FIG. 8: Caspase-graph: Luminescence value of untreated cells
was defined as 100% Caspase activity. Due to their specific
luminescence value the caspase activity of treated cells were
calculated accordingly. Induction of Caspase
activity.sub.x[%]=Caspase activity.sub.x[%]-Caspase
activity.sub.untreated[%]. Means.+-.SEM (Standart Error of the
Mean=Standard deviation/radical(n)) of three different experiments
are presented. Summary of this graph: 6-thio-GTP was able to induce
caspase activity (as positive control). 05B-0 was also very
effective. 05B-1 was less effective than 05B-0 in agreement with
the previous data on AnnexinV/PI-staining. Concerning Group-D
derivatives 06D-13, 06D-14 and to a lesser extent 06D-22 were
promising.
EXAMPLE 1
Process for the Preparation of 2-substituted-6-thio-guanosine
Nucleotides
Preparation of 2',3',5'-Tri-O-acetyl-1-[(2-methoxyethoxy)methyl]
inosine
[0102] (Kohyoma et al., 2003)
##STR00184##
[0103] Inosine-2',3',5'-triacetate (4 g, 10.2 mmol) was dissolved
in 100 ml dichloromethane and the solution was treated with 1.4 ml
(12.2 mmol) 2-methoxyethoxymethylchloride at 0.degree. C. in the
presence of diisopropylethylamine as a supportive base. After 1
hour the reaction was quenched with water. The solution was stirred
for 30 min and chloroform was added afterwards.
[0104] The aqueous layer was extracted with chloroform and the
combined and washed organic layers were concentrated to dryness.
The reaction yielded 3.93 g (8.2 mmol, 80%) of
2',3',5'-Tri-O-acetyl-1-[(2-methoxyethoxy)methyl] inosine after
chromatographic purification (silica gel, EtOAc-MeOH, 50:1).
Preparation of 5-Amino-1-O-ribofuranosylimidazole-4-carboxamide
(Kohyoma et al., 2003)
##STR00185##
[0106] Aqueous ammonia solution (28%, 20 ml) was added to a
solution of 2',3',5'-Tri-O-acetyl-1-[(2-methoxyethoxy)methyl]
inosine (3.5 g, 7.3 mmol) in 50 ml methanol. The reaction mixture
was stirred for 1 hour at room temperature and concentrated,
yielding the deprotected nucleoside (2.44 g, 6.9 mmol, 95%). This
product was used in the next step without further purification.
[0107] The nucleoside (2.44 g, 6.9 mmol) was refluxed with 50 ml
aqueous sodium hydroxide (0.2 M) for 1 hour, cooled to room
temperature, neutralized with HCl (6 M) and evaporated to dryness.
The residue was dissolved in ethanol, filtered from insoluble
material and concentrated to dryness. Purification of the crude
product by column chromatography (silica gel, CHCl.sub.3:MeOH, 3:1)
gave 1.32 g (5.11 mmol, 70%) of
5-Amino-1-.beta.-ribofuranosylimidazole-4-carboxamide.
Preparation of 2-Mercaptoinosine (Imai et al., 1971)
##STR00186##
[0109] The 5-Amino-1-.beta.-ribofuranosylimidazole-4-carboxamide
(1.3 g, 5 mmol) was dissolved in pyridine and 18 ml (15 mmol)
phenyl-isothiocyanate were added slowly. The reaction mixture was
refluxed for 2 hours under argon atmosphere. The solution was
cooled to room temperature, the precipitate collected by filtration
and washed with diethyl ether. The obtained pyridinium salt of the
product was dissolved in aqueous sodium hydroxide (15%, 40 ml). The
solution was heated 30 minutes at 60.degree. C. and concentrated
under vacuum. Methanol was then added and the solution was kept in
the refrigerator overnight. After 20 hours the precipitated
colourless prisms were collected by filtration to give 1.2 g (80%)
2-Mercaptoinosine.
[0110] According to an alternative method of preparation,
compound-Amino-1-.beta.-ribofuranosylimidazole-4-carboxamide (1.3
g, 5 mmol) was added to a solution of sodium hydroxide (1 g, 25
mmol) in 20 ml of methanol at 30.degree. C. Carbon disulfide (1.9
g, 25 mmol) was added, and the solution was heated in an autoclave
at 180.degree. C. for 3 hours. The mixture was cooled to room
temperature, the precipitate was filtered off, washed with cold
methanol and recrystallized from water to yield 1.1 g (3.8 mmol,
75%) 2-Mercaptoinosine.
Preparation of N-2-(6''-aminohexyl)-Guanosine
##STR00187##
[0112] 2-Mercaptoinosine (1 g, 3 mmol) was dissolved in 100 ml
water and cooled to 0.degree. C. A clear solution was obtained
after ultrasonic irradiation. Hydrogen peroxide (1 ml, 9 mmol) was
added within 20 min under vigorous stirring. After 1 hour of
stirring at 0.degree. C., HPLC chromatography indicated that the
starting compound has been completely oxidized to the sodium salt
of Inosine-2-sulfonic acid. Without further purification, the
resulting solution was treated with an excess of 1,6-diaminohexane
(20 g, 200 mmol). The mixture was refluxed for 2.5 hours at
155.degree. C. The excess of 1,6-diaminohexane was removed by
vacuum distillation to give an orange residue.
N-2-(6''-Aminohexyl)-guanosine (0.85 g, 2.2 mmol, 50%) was obtained
after purification of the crude product by column chromatography
(silica gel RP-18, linear gradient from 100% water to 100%
methanol). Preparation of
2',3',5'-triacetyl-N-2-(6''-acetamide-hexyl)-guanosine (Ostermann
et al., 1999)
##STR00188##
[0113] A solution of nucleoside (220 mg, 0.575 mmol) in 20 ml dry
pyridine was stirred with 1 ml (10 mmol) acetic anhydride at room
temperature for 15 hours under argon atmosphere. The solvent was
removed under vacuum and the residue dissolved in a mixture of
CHCl.sub.3 (10 ml) and CH.sub.3OH (2 ml). This solution was loaded
on silica gel and eluted with CHCl.sub.3/CH.sub.3OH (5:1) to give
284.7 mg (0.52 mmol, 90%) of fully protected nucleoside.
Preparation of
2',3',5'-triacetyl-N-2-(6''-thioacetamido-hexyl)-6-thioguanosine
##STR00189##
[0115] Dioxane (200 ml) was added to the fully protected nucleoside
(5.03 g, 9.13 mmol). After addition of 8 g (19.8 mmol) of
Lawesson's reagent the suspension was vigorously stirred for 2
hours at 80.degree. C. The initially opaque reaction mixture became
clear after 10 minutes. The solution was cooled at room temperature
and the solvent was evaporated by vacuum distillation. Purification
of the raw product by column chromatography (silica gel,
CHCl.sub.3), resulted in
2',3',5'-triacetyl-N-2-(6''-thioacetamido-hexyl)-6-thioguanosine
(2.92 g, 5.02 mmol) in 55% yield.
EXAMPLE 2
Process for the Preparation of Analogous of ribose-modified
6-thio-guanosine-triphosphate
Preparation of 6-Thio-Guanosine-Triphosphate (Ludwig, 1981)
##STR00190##
[0117] Under argon atmosphere, 6-thio-guanosine (1 g, 3.34 mmol)
was dissolved in 6 ml trimethylphosphate. The solution was cooled
to 0.degree. C. and 1.3 ml of Lutidine were added. After 10
minutes, 0.4 ml (4.4 mmol) phosphorous oxychloride was carefully
added to the solution. After 1 hour the excess of POCl.sub.3 was
removed under vacuum within ten minutes.
[0118] The solution of the initially formed intermediate
dichlorophosphate was then treated with a solution of tri-n-butyl
ammonium pyrophosphate (17 ml, 100 mM) in dimethylformamide. After
2 minutes, the reaction was quenched by adding 100 ml of 0.25 M
triethylammonium bicarbonate buffer. Purification by ion exchange
chromatography gave 6-Thio-Guanosine-Triphosphate (0.5 g, 1 mmol,
30%).
Preparation of 2'/3'-EDA-6-Thio-Guanosine-triphosphate
##STR00191##
[0120] The dry tributylammonium salt of compound (0.5 mmol) was
treated with 500 mg carbonyldiimidazole in 25 ml dimethylformamide.
The resulting mixture was stirred for 6 hours at 0.degree. C.,
brought to room temperature and 0.2 ml methanol and subsequently
0.3 ml ethylene diamine were added.
[0121] The resulting precipitate was centrifuged down and dissolved
in water. The solution was adjusted to pH 2, in order to decompose
the resulting intermediate phosphoramidate at the triphosphate
moiety. After 18 hours, pH was adjusted to 7.5 and the solvent was
subsequently removed under reduced pressure. Purification of the
crude product by ion exchange chromatography gave 116 mg (0.4 mmol,
80%) 2'/3'-EDA-6-Thio-Guanosine-triphosphate.
Preparation of TAMRA-2'/3'-EDA-6-Thio-Guanosine-Triphosphate
##STR00192##
[0123] The N-hydroxy-succinimide ester of TAMRA (1 mg, 2 .mu.mol)
was dissolved in 200 .mu.l of dry dimethylformamide and added to a
solution of 1 mg (3 .mu.mol) of
2'/3'-EDA-6-Thio-Guanosine-Triphosphate in 500 .mu.l of 100 mM
sodium borate buffer (pH 8.5) at room temperature. After 2 hours,
the reaction mixture was quenched with methanol. The TAMRA labelled
product was obtained in 70% yield (2.4 mg, 1.4 .mu.mol) after
workup of the reaction mixture by reversed phase HPLC.
EXAMPLE 3
Synthesis of 2-Substituted 6-Thio-Guanosine nucleotides
Preparation of N-2-(6''-Thioacetamido-hexyl)-6-Thioguanosine
(10)
##STR00193##
[0125] A, 7 M solution of ammonia in methanol (70 ml) was added to
the fully protected nucleoside 9 (1.15 g, 1.97 mmol). The solution
was stirred at room temperature for 20 hours. The solvent was
removed by distillation to give an off white residue. The crude
N-2-(6''-thioacetamido-hexyl)-6-Thioguanosine (10) (0.85 g, 1.8
mmol, 95%) was used without further purification.
Preparation of N-2-(6''-Aminohexyl)-6-Thioguanosine (11)
##STR00194##
[0127] A solution of nucleoside 10 (0.1 g, 0.22 mmol) in 5 ml
NH.sub.3/H.sub.2O (30%) was stirred for 90 minutes at 80.degree. C.
The solvent was removed in vacuo and the residue was dissolved in
water (2 ml). N-2-(6''-Aminohexyl)-6-Thioguanosine (11) (0.80 g,
2.0 mmol, 91%) was obtained after purification of the crude product
by column chromatography (silica gel RP-18, linear gradient from
100% water to 100% methanol).
Preparation of N-2-(6''-Guanidino-hexyl)-6-Thioguanosine (12)
##STR00195##
[0129] N-2-(6''-Aminohexyl)-6-Thioguanosine (11) (0.091 g, 0.23
mmol) was dissolved in 1 ml DMF and the resulting solution was
treated with 0.34 g (0.23 mmol) Triazole-1-carboxamidine
hydrochloride at room temperature in the presence of 39 .mu.l (0.23
mmol) N-Ethyl-diisopropylamine acting, as a supportive base. After
2 hours, the reaction mixture was evaporated to dryness.
Purification of the crude product by reversed phase chromatography
(silica gel RP 18, linear gradient from 100% H.sub.2O to 100% ACN)
gave 0.92 g (0.21 mmol, 90%) of
N-2-(6''-Guanidino-hexyl)-6-Thioguanosine (12).
Preparation of
N-2-(6''-Aminohexyl)-6-Thioguanosine-5'-monophosphate (13)
##STR00196##
[0131] Under argon atmosphere, nucleoside 9 (0.3 g, 0.6 mmol) was
dissolved in 3 ml trimethylphosphate. The solution was cooled to
0.degree. C. and treated with 0.3 ml of Lutidine.
[0132] After 10 minutes, 0.15 ml (1.1 mmol) phosphorous oxychloride
was carefully added. After 1 hour, the excess of POCl.sub.3 was
removed in vacuo within ten minutes.
[0133] The solution was quenched by adding 100 ml of 0.25 M (pH
7.5) triethylammonium bicarbonate buffer. Purification by ion
exchange chromatography gave
N-2-(6''-thioacetamido-hexyl)-6-Thioguanosine-5'-monophosphate. The
product was dissolved in 5 ml NH.sub.3/H.sub.2O (30%) and stirred
for 90 minutes at 80.degree. C. The solvent was subsequently
removed in vacuo.
N-2-(6''-Aminohexyl)-6-Thioguanosine-5'-monophosphate (13) (0.17 g,
0.36 mmol, 60%) was obtained after purification of the crude
product by ion exchange chromatography.
Preparation of
N-2-(6''-Guanidino-hexyl)-6-Thioguanosine-5'-monophosphate (14)
##STR00197##
[0135] N-2-(6''-Aminohexyl)-6-Thioguanosine-5'-monophosphate (13)
(0.053 g, 0.11 mmol) was dissolved in a mixture of 0.8 ml water and
0.5 ml DMF. The solution was subsequently treated with 0.16 g (0.11
mmol) Triazole-1-carboxamidine hydrochloride at room temperature in
the presence of 18 .mu.l (0.11 mmol) N-Ethyl-diisopropylamine,
acting as a supportive base. After 16 hours, the reaction mixture
was concentrated to dryness. Purification of the crude product by
reversed phase chromatography (silica gel RP 18, linear gradient
from 100% H.sub.2O to 100% ACN) gave 0.037 g (0.072 mmol, 65%) of
N-2-(6''-Guanidino-hexyl)-6-Thioguanosine-5'-monophosphate
(14).
EXAMPLE 4
Synthesis of Ribose-Modified 6-Thio-GTP Analogs
Preparation of Aspartate-2'/3'-EDA-6-Thio-Guanosine-Triphosphate
(4)
##STR00198##
[0137] The N-hydroxy-succinimide ester of Boc/tBu protected
Aspartate (2.8 mg, 7 .mu.mol) was dissolved in 200 .mu.l of dry DMF
and added to a solution of 1 (5 mg, 7 .mu.mol) in 500 .mu.l of 100
mM sodium borate buffer (pH 8.5) at room temperature. After 16
hours, the reaction mixture was quenched with methanol. The
Aspartate derivative 4 was obtained in 70% yield (3.6 mg, 4.9
.mu.mol) after workup of the reaction mixture by reversed phase
HPLC.
Preparation of Glutamate-2'/3'-EDA-6-Thio-Guanosine-Triphosphate
(5)
##STR00199##
[0139] The N-hydroxy-succinimide ester of Boc/tBu-protected
Glutamate (2.9 mg, 7 .mu.mol) was dissolved in 200 .mu.l of dry DMF
and added to a solution of 1 (5 mg, 7 .mu.mol) in 500 .mu.l of 100
mM sodium borate buffer (pH 8.5) at room temperature. After 16
hours, the reaction mixture was quenched with methanol. The
Glutamate labeled product 5 was obtained in 70% yield (3.7 mg, 4.9
.mu.mol) after workup of the reaction mixture by reversed phase
HPLC.
Preparation of Threonine-2'/3'-EDA-6-Thio-Guanosine-Triphosphate
(6)
##STR00200##
[0141] The N-hydroxy-succinimide ester of Boc-protected Threonine
(2.3 mg, 7 .mu.mol) was dissolved in 200 .mu.l of dry DMF and added
to a solution of 1 (5 mg, 7 .mu.mol) in 500 .mu.l of 100 mM sodium
borate buffer (pH 8.5) at room temperature. After 16 hours, the
reaction mixture was quenched with methanol. The Threonine
derivative 6 was obtained in 70% yield (3.4 mg, 4.9 .mu.mol) after
workup of the reaction mixture by reversed phase HPLC.
EXAMPLE 5
Analysis of the Ability of 5 New Synthesized 6-Thio-GTP-Derivatives
to Induce Apoptosis in Human CD4.sup.+ T Lymphocytes
Used Substances:
[0142] Azathioprine [0143] 6-Mercaptopurine [0144]
V1=BMB20=EDA-6-Thio-GTP [0145] V2=TWI
35/1=N-2-(6''-Aminohexyl)-guanosine [0146] V3=TWI
71/2=2',3',5',o-Triacetyl-N-2-(Acetyl-6''-aminohexyl)-guanosine
[0147] V4=TWI
107/7=2',3',5'-Triacetyl-N-2-(6''-thioacetamide-hexyl)-6-Thioguano-
sine [0148] V5=BMB=TAMRA-EDA-6-Thio-GTP
[0149] V3 and V4 were not soluble in water, therefore V3 was
reconstituted with ethanol and V4 was reconstituted with
methanol.
Protocol:
[0150] Human peripheral blood mononuclear cells (PBMC) from 4
buffycoats were isolated using Ficoll-Hypaque gradients. PBMC were
further purified using CD4 monoclonal antibodies attached to
immunomagnetic microbeads according to the protocol provided by the
manufacturer (Miltenyi Biotec). T lymphocytes were stimulated in
complete RPMI-1640 medium (RPMI-1640+10% FCS+100 U/ml
Penicillin/Streptomycin+3 mM L-Glutamin) for 3, 4 or 5 days with
coated antibodies to CD3 (0.04 .mu.g/ml) and soluble CD28
antibodies (PharMingen; 1 .quadrature.g/ml) plus IL-2 (R & D
Systems, Wiesbaden, Germany; 40 U/ml). Azathioprine,
6-Mercaptopurine, V1, V2, V3, V4 or V5 were added to the T cell
cultures at day 0 at a final concentration of 5 .mu.M. To determine
induction of apoptosis in these T lymphocytes, cells were analyzed
by FACS. For FACS analysis, apoptotic cells were detected by
staining with annexin V and propidium iodide using the Annexin V
FITC Apoptosis Detection Kit I (PharMingen). In brief, T cells were
washed twice in PBS, and the pellet was resuspended in annexin V
binding buffer (PharMingen) at a concentration of 10.sup.6 cells
per milliliter. Annexin V FITC and propidium iodide were added (5
.mu.l of each per 10.sup.5 cells), Samples were gently mixed and
incubated for 15 minutes at room temperature in the dark before
FACS analysis.
Results:
[0151] Annexin-positive, propidium iodide-negative cells (black
bars) present the rate of early apoptotic cells. Annexin-positive,
propidium iodide-positive cells (white bars) present late apoptotic
or necrotic cells. [0152] Induction of apoptosis=(Rate of apoptotic
cells after indicated treatment)-(Rate of apoptosis of untreated
cells) [0153] V1, V2 and V3 were tested in 4 independent
experiments. V4 and V5 were tested in 2 independent
experiments.
CONCLUSION
[0154] Our first results showed, that V1 and V5 were able to induce
apoptosis in CD3/CD28 costimulated T Lymphocytes. V2, V3 and V4
were not able to induce apoptosis. Comparing V1 and V5 mediated
induction of apoptosis with azathioprine or 6-mercaptopurine
mediated induction of apoptosis, V1 and even V5 mediated effects
seemed to be more pronounced and appeared earlier (FIGS. 2-4).
EXAMPLE 5
Preparation of 2'/3'-Methylenoaminocarbamate derivatives of
6-Thio-Guanosine-triphosphate
##STR00201##
[0156] The dry tributylammonium salt of 6-Thio-GTP 1 (0.2 mmol) was
treated with 200 mg carbonyl-diimidazole in 4 ml dimethylformamide.
The resulting mixture was stirred for 6 hours at 0.degree. C.,
brought to room temperature and, subsequently, 80 .mu.l of methanol
were added. After 10 minutes, 2 mmol of the appropriate
methyleno-amine and 2 ml of triethylamine were also added to the
reaction mixture. The solution was stirred overnight at room
temperature and the solvent was then removed under reduced
pressure. The residue was taken up in 30 ml water and the mixture
was adjusted to pH 1, in order to decompose the resulting
intermediate phosphoramidate at the triphosphate moiety. After 20
minutes, the solution was adjusted to pH 7.5, the precipitate
filtered off and the solvent was removed in vacuo. The resulting
crude product was purified by ion exchange chromatography and
subsequently by reversed phase HPLC.
EXAMPLE 6
2'/3'-Methylenoaminocarbamate-6-Thio-Guanosine-triphosphate
derivative 2a
##STR00202##
[0158] Reaction of 3-thienyl-methylamine (226 mg, 2 mmol) with 1
according to the general procedure yielded 2a (0.58 mmol, 29%)
after purification by ion exchange chromatography and subsequent
reversed phase HPLC.
2'/3'-Methylenoaminocarbamate-6-Thio-Guanosine-triphosphate
Derivative 2b
##STR00203##
[0160] Reaction of (1,5-Dimethyl-1H-pyrazol-3-yl)methylamine (250
mg, 2 mmol) with 1 according to the general procedure yielded 2b
(0.66 mmol, 33%) after purification by ion exchange chromatography
and subsequent reversed phase HPLC.
EXAMPLE 7
Preparation of 2'/3'-Carbamate Derivatives of
6-Thio-Guanosine-triphosphate
##STR00204##
[0162] The dry tributylammonium salt of compound 1 (0.2 mmol) was
treated with 200 mg carbonyl-diimidazole in 4 ml dimethylformamide.
The resulting mixture was stirred for 6 hours at 0.degree. C.,
brought to room temperature and, subsequently, 80 .mu.l methanol
were added. After 10 minutes, 2 mmol of the appropriate amine and 2
ml of 1 M potassium hexamethyldisilazide (KHMDS) in THF were
carefully added to the solution. The solution was stirred at room
temperature for 1 hour and the solvent was then removed under
reduced pressure. The residue was taken up in 30 ml water and the
mixture was adjusted to pH 1, in order to decompose the resulting
intermediate phosphoramidate at the triphosphate moiety. After 20
minutes, the solution was adjusted to pH 7.5, the precipitate
filtered off and the solvent was removed in vacuo. The resulting
crude product was purified by ion exchange chromatography and
subsequently by reversed phase HPLC.
EXAMPLE 8
[0163] In relation to FIGS. 5 and 6, an overview of at least three
experimental data sets on apoptosis is shown below. Here, some of
the D compounds were able to induce apoptosis. B0 was the strongest
candidate drug for apoptosis induction. Two issues should be
considered in these results. [0164] Negative induction of apoptosis
means that there were more apoptotic cells in the untreated group
than in the treated group. This phenomenon may appear from time to
time and may be explained by a kind of statistical variance. [0165]
The drug 6-Thio-GTP on average was not able to induce apoptosis in
these experiments. Generally 6-Thio-GTP should be a positive
control to induce apoptosis in T cells. In these experiments the
positive control did not work very well. This might be explained by
the fact that the experiments are often preformed with primary T
cells, which are freshly isolated from blood of different donors.
It is well known, that some people are not sensitive for
azathioprine therapy. In this way, T cells of some donors might be
resistant against 6-Thio-GTP induced apoptosis. In any case,
however, B0 and some of the D compounds were able to induce
apoptosis suggesting that they are candidate drugs.
[0166] In addition, an alternative method was performed for
screening of group-D derivatives (FIG. 8). It was decided to
analyse the activity of caspase-3/7 in T cells, which were treated
with group-D derivatives. As compared to AnnexinV/PI staining this
new method might have some advantages: [0167] It is an easier
protocol. Therefore there are fewer possibilities for individual
errors. [0168] Increased activity of caspase-3 is very specific for
apoptosis. Therefore this method is very sensitive for the
detection of apoptosis. There is no interfering influence of
necrotic cells. [0169] The measurement is done in duplicate. In
this way there is an internal control.
[0170] The Caspase-Glo 3/7 Assay (Promega.TM.) was used. This assay
is based on a caspase dependent luminescent signal. Protocol:
CD4.sup.+ T cells were isolated from human blood by magnetic beads
(Dynal). T lymphocytes were stimulated in complete RPMI-1640 medium
(RPMI-1640+10% FCS+100 U/ml Penicillin/Streptomycin+3 mM
L-Glutamin) for 3 days with coated antibodies to CD3 (0.04
.mu.g/ml) and soluble CD28 antibodies (PharMingen.TM.; 1 .mu.g/ml)
plus IL-2 (R & D Systems.TM., Wiesbaden, Germany; 40 U/ml) in
96-well plates. Cells were treated with different group-D
derivatives or were left untreated. At day three of culture
Caspase-3/7 Assay was performed. 25 .mu.l of Caspase-Glo 3/7
reagent was added to each well. Probes were mixed gently for 2
minutes and incubated at room temperature for 30 minutes. Finally
100 .mu.l of each probe were transferred to a white walled 96-well
luminometer plate and analysed in a plate-reading luminometer. The
added reagent contains a specific substrate of caspase-3 and
caspase-7. Cleavage of this substrate by activated caspase-3
results in luciferase reaction. Luminescence is proportional to the
amount of caspase activity present (FIG. 8)
EXAMPLE 8
2'/3'-Carbamate-6-Thio-Guanosine-triphosphate derivative 3a
##STR00205##
[0172] Reaction of 3-aminopyrimidine (190 mg, 2 mmol) with 1
according to the general procedure yielded 3a (0.076 mmol, 3.8%)
after purification by ion exchange chromatography and subsequent
reversed phase HPLC.
EXAMPLE 9
2'/3'-Carbamate-6-Thio-Guanosine-triphosphate derivative 3b
##STR00206##
[0174] Reaction of 3-(tert.-Butyl)-1-methyl-1H-pyrazol-5-amine (306
mg, 2 mmol) with 1 according to the general procedure yielded 3b
(0.11 mmol, 5.5%) after purification by ion exchange chromatography
and subsequent reversed phase HPLC.
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