Note: Descriptions are shown in the official language in which they were submitted.
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IMPROVEMENTS TO ANALOGOUS COMPOUNDS OF 6-THIOGUANOSINE
TRIPHOSPHATE, THEIR USE IN MEDICAL FIELDS AND PROCESSES FOR THEIR
PREPARATION.
The present invention relates to analogous compounds of 6-thioguanosine
triphosphate, their
use in medical field and process for their preparation.
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).
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).
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).
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 Vong-lasting disease (Tiede, 2003).
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
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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-
xL gene
(Khoshnan, 2000; Noel,1996).
The steps through which an inhibition of apoptosis takes place, will be
synthesized as follows,
as shown in Figure 1:
-CD28 acts through its cytoplasmaic portion with a complex of "adaptor"
proteins and with a
molecule, named Vav (Frauwirth, 2002);
-Vav acts as guanosine nucleotide exchange factor (GEF) for another molecule
named Rac1
(Frauwirth, 2002);
-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);
-Activated Rac1, in its turn, leads to the activation of kinases (IKK) that
phosphorylate the NF-KB
inhibitory proteins (like I-KB alfa) (Marinari, 2002) through MEK
phosphorylation;
-Thus, NF-KB is not anymore retained in an inactive form in the cytosol but is
able to translocate
to nucleus where induces bcI-xL expression (Khoshnan, 2000);
-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 bcI-xL 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).
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
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processes, proliferation, and invasivity, since it stimulates alterations of
the gene expression, in
the present case of the gene bcf-xL, modulating the activity of transcription
factors, such as, in
the present case, NF-KB and STAT-3 (Van Aelst, 1997).
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.
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).
The key point is represented by the metabolite 6-thioguanosine triphosphate (6-
thioGTP) which
represents the real functional metabolite of the drug. Specifically, 6-thioGPT
binds directly to
Rac1 in place of GTP, thus blocking its activation. Such a block is highly
specific for Racl, 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.
The block of the activation of Rac1 would result in the block of NF-KB and
STAT-3, normally
induced by Rac1 itself, and thus in the block of the bcl-xL gene expression,
detected both at the
mRNA and protein levels. Thus, azathioprine, by modulating Racl activity
switches an anti-
apoptotic co-stimulatory signal, mediated by CD28, into a pro-apoptotic
signal.
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 Racl 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.
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.
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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.
It is therefore an object of the present invention to provide a class of
analogous compounds of
6-thioguanosine triphosphate of general formula (I):
R1
N N
< 1
N '~~
O N NH
R5 I
R2
R4 R3 (la)
wherein the dashed bond in the sugar moiety can be either single or double and
wherein Ri, R2,
R3, R4 or R5, equal or different between each other, have general formula -
(Int)m-Ter, wherein
m is between 0 and 12 and !nt and Ter are Internal and Terminal building
blocks, wherein Int is
selected from the group consisting of
-N-
-O- -s- -CHz -NH- CH -SOZ
3
-CH- -CH- -CH- -CH- -CH- -CH-
OH SH CH3 NHz COOH HZC-OH
-o- -S- CH NH 'H H -C-C I Qi
z
C QiY
N-OH - C(CHs)z
iNJ
Y
iX Q ~Qi~ -Y-
N N
H H H
X Q~X X CQ~ /Ns ~Qi N
~ ~Q~/
H
N-N NQ Qi I Q~ Qi I ~ X
N~Qi\ N N NQiN \ N
H
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and Ter is selected from the group consisting of
-H -CH3 \QI/
N
~ JN
-OH -SH N
O N
~
-COOH -NH2 No ~NNH TI a
\_j N
Y N
Di I QY \~ I Q\ ' f QY '1 Qi I I QN>
Y H
N\ N N' / \Qi ~ Qi Y _~ JJN Qi NH
\ Y N \ N
N
~i~ Q~> \iI Qi \iI QO O
1D O
O I Q~ D
O
\ O eQ~ O
~Qo :T" :::)~
O ~ o' O Q o a
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)
5 are selected from the group consisting of -OH, -COOH, -N(CH3)2, -N(CH2-
CH3)2, -CO-CH3,
-CO-O-CH3i -O-CH3, -S-CH3,-S02-CH3, , -CN, -NO2 or -Halogen elements.
Alternatively R5 may be
-O
I
HO P-O
I I
O
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.
In some embodiments (particularly, but not limited to those where R5 is HO-
[PO3]n,
and metal and ammonium salts thereof, wherein n is between 0 and 5, or oxygen
or phosphorus
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is partiafly or completely replaced by nitrogen, sulphur, methylene goups or
their derivatives)
TER may be selected from the group consisting of:
Br 32
\ S / ~ PICOOCH3 S H 3COOC\ S / N(CH
-CH N
~ CN
H H H S
N~N N~ HO N"
~ \ IN \ / D,\/,
HOOC H3COOC
S H
S H3COC S
-CH N ~CHZ ~ ~ ~ ~
(Q/
2 CH3
J/' H3COOC \ S/ SCH3
S/CH2 \ S/ N~ ~CH3
~//,N ,~~\
NC H3C CN
S CH CHa -CHZ~N~N.CH3 H3COOC S
/3 {
\ X N' '--' ~ /
3
N CH3 ~ ~N CH SO2CH3
S ~H3 H S
z
CH N N H3COOC
\N, C(CH3)
3 CH3 C(CH3)3
CH3 ~H3
~/N~ N N~
NII\% H3C CI ~N ~ ~N
CH3 C(CH3)3
Compounds of formula (I) can be labelled, particularly with R3 or R4 selected
from
Int-C=O Int-
~ ~ C=0
COOH HOOC
Q O Q or Q 0 Q
wherein Q is selected from -OH (FAM) or -N(CH3)2 (TAMRA).
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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:
H O H H O
~
H O H O
H H H OH H OH -
0-\O
Compounds of the invention may be of the general formula (Il) derived from
general formula (la)
and/or (I) where R, is [-SH] (it is understood that the Guanosine moiety may
undergo keto-enol
tautomeric shifts and so give rise to [=S]), R2 is [-H], R5 is [-(PO3),-OH]
and one of R3 and R4 is
[-OH] and the other of R3 and R4 is [-O-CO-NH-Intm Ter] (both versions are
provided in the
same mixture, i.e., [-O-CO-NH -Intrr, Ter]: will be the same, but a proportion
of the molecules
will have [-O-CO-NH- -Int,,,-Ter]: attached at R3 with R4 being OH, and
another proportion of the
molecules will have [-O-CO-NH-Intm-Ter]: attached at R4 with R3 being OH):
N N
<~ ~
O N N" NH
o 2
HO P-O O
n
O O
O=~
N-H
(nt)m
Ter
(II)
wherein n=1, 2 or 3, m is between 0 and 5, Int is selected from the group
consisting of
-N- ~
-Q- -s- -CHZ -NH- CH -SOZ
3
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-CH- -CH- -CH- -CH- -CH- -CH- I OH SH CH3 NH2 COOH HZC-OH i l
O S CH NH H H -C_C Q
z
N
-C- NJ' N V N-OH -C(CH3)z ~'~/~NI iN\/ ~
Y
\Q /x
N N
N N N~ X~
\Qi~x \Qi~x \Qi~x \Qiz N
N-N \
-Y-
~1Qi _(Qi Q OCW\ x
N N N--N H and Ter is selected from the consisting of
-H -CH3
N
-OH -SH \ ~J
N
N
-COOH -NHz No ~N~NH ( Q
v N
y Y N
~10 \Qi ~ i // N Q
N N
Qi~
\ i ~ Q~ ~ Qi *'CN ~- GI_N
Y Q~ Y Y H =
i I Qi i Y :,N\ QQNH
1\ N\ ~N\
y \ ~N Y N N
N
\~ I Q~ \~ I Q~ GQoi Q O~ Qi O
O O CO~
\ O~ Qi ~ \ Qi T c i ~
~ I\ O ~ 1 I\ O O
/ O O oo o
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
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unsaturated moiety of the ring to which the group Q may be bound) are selected
from the group
consisting of -CH3, -C(CH3)3i -OH, -COOH, -CO-CH3, -CO-O-CH3, -O-CH3, -S-CH3,-
SO2-CH3,
-N(CH3)2, -N(CH2-CH3)2i -CN, -NO2 or -Halogen elements.
In some embodiments of the invention of formula (I), (Ia) and (II), Ter is
selected from the group
consisting of
Br \ S / ' /~ \ S / H3COOC
-CH '(N \ S / N(CH3)~
2 COOCH3 CN
H H H S
N N
\ /N Q Ho \ N /N
HOOC H3COOC
S NH
~ S H3COC S
U \ /N ~CH2
2 CF{3
\ S / ~CH2 0\/ S N~N ,CH3 H3COOC S ' SCH3
NC H3C CN
,CHZ N" ,CH3
3 GH3 CH3 H3GOOC S
N
N ' \ ~
CH3 \ ~N CH3 SO2CH3
CH
S 3 H S
-ICHz N N.N H3COOC
C(CH3)3 CH C(CH3)3
3
CH3 ~H3
N1 N~
N H3C I / Cl \ /N \ /N
CH3 C(CH3)3
According to some embodiments of the present invention the compounds of
formula (I) are the
compounds described below:
R~
N N
/
R O N N NH
5 I
K2
R4 R3 (la)
wherein RI, R2, R3, R4 or R5, equal or different between each other, have
general formula -
(Int)m Ter, wherein m is between 0 and 12 and lnt and Ter are Internal and
Terminal building
blocks, wherein Int is selected from the consisting of
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-CHz -NH- -N- Qi
-O- -S- CH -SOZ
3
-CH- -CH- -CH-
-CH- SH CH3 NHz -CH- -CH-
OH COOH HZC-OH
_ _ _ -C=C-
O s CH NH H H -C=C- I Q%
2
and Ter is selected from the consisting of
-H -CH3
N
-OH -SH ~ I \ ~J
N
-COOH -NH2
Q \ O ~ Q
5
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(CH3)2i -N(CH2-CH3)2 or -Halogen elements
In addition the sugar moiety of compounds of formula (I) can be selected from
the group
10 consisting of the following sugar moieties or sugar-like moieties:
H O H O O H O
H O H O
R H OH H OH -
~/
Compounds of formula (I), (Ia) and (II) can be labelled, particularly with R3
or R4 selected from
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Int- C=0 Int-
0
COOH HOOC
Q O Q or Q O Q
wherein Q is selected from -OH (FAM) or -N(CH3)2 (TAMRA).
According to some embodiments of the present invention the compounds of
formula (I), (Ia) and (II) are the compounds described below:
SM4410
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2',3'-EDA-6-Thio-GTP, ID: 05B-0
SH
O O O N ~ N
HOOPlO O-O O O N NNHZ
O O 60
O O
HN
\/ -O
NH2
m=0 Ter
-R1 -SH
n=3 1 2 3 Ter
-R5 --PO3 -PO3 -PO3 -OH
m=6 /ntl lnt2 /nt3 Int4 Int5 /nts Ter
-R3 0 -0- -C- -NH- -CH2 -CHz -NH- -H
11
-R4 0
m=0 Ter
-R3 0 -OH
-R4
m=0 Ter
-R2 -H
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FAM-2 ; 3'-EDA-6-Thio-GTP
SH
N LNNH2
q 8 ~ HO P~O-P~O~P~O N O p0 0O --~ I I O O
H ~O
H
N H
O
COOH
~ OH
O
HO
m=0 Ter
-RI -SH
n=3 1 2 3 Ter
-R5 -P03 -PO3 -POs -OH
m=0 Ter
-R2 -H
m=8 Intl Int2 Int3 Infd Int5 Infs Int, Int8 Ter
-R3 -0- -C- -NH- -CHZ -CHZ -NH- -C- ~ I ~
0 O O HO ~ ~O ~ OH
COOH
-R4
m=0 Ter
-R3o
-OH
R4
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TAMRA-2 ;3'-EDA-6-Thio-GTP
SH
O O O N 'N
HO'P-O-P-O-P-O
00 00 00 --~ I I N NHz
O
r O
H~o
O NH
COOH
_
O
-N
m=0 Ter
-R1 -SH
n=3 1 2 3 Ter
-R5 -P03 -P03 -F03 -OH
m=0 Ter
-R3 o
-OH
R4
m=8 Int, lnt2 Int3 lnt4 lnt5 Ints InG Int8 Ter
-R3 -O- -C- -NH- 1_0Hz -CH2 -NH- -C-
0 Q O N(CVi O N(CNz
COOH
R4
m=0 Ter
-R2 -H
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Aspartate-2',3'-EDA-6-Thio-GTP, ID: 05B-1
SH
O O 0,0 I
HO ~P~ ~O ~ ~O -- NN -N~ N
O O
HN
O NH
HZN
O
HO
m=0 Ter
-RI -SH
n=3 1 2 3 Ter
-R5 -F03 -PO3 -P03 -OH
m=9 lnt, Int2 lnt3 lnt4 lnt5 lnt6 Int, lnt$ lnt9 Ter
-R3 -0- -C -NH -CH -CH2 -NH -C- -CH- -CHa -COOH
o 0 0 NH2
-R4
m=0 Ter
-R2
m=0 Ter
-R3 o
-R4
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Glutamate-2;3'-EDA-6-Thio-GTP, ID: 05B-2
SH
0 N N
o q
HO P~ oO -oO -O N ~NNHZ
O O
~O
H
O NH
HZN O
OH
m=0 Ter
-R1 -SH
n=3 1 2 3 Ter
-R5 -P03 -P03 -P03 -OH
m=10 Int, lnt2 lnt3 Intq lnt5 lnts lnt7 lntg lntg lntlo Ter
-R3 o -O- -C -NH -CH -CHZ -NH- -C- -CH- -CH2 -CHz -COOH
0 NH2
-R4
m=0 Ter
-R2 -H
m=0 Ter
-R3 o -OH
-R4
Threonine-2;3'-EDA-6-Thio-GTP, ID: 05B-3
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SH
O O O N N
II II II I
HO oP- oO "
oO 0- N NNH2
O O
H
HN X-O
NH
HZN
OH
m=0 Ter
-RI -SH
n=3 1 2 3 Ter
-R5 -P03 -PO3 -F03 -OH
m=9 intl lnt2 /nt3 int4 /nt5 /nts Int, Int8 Int9 Ter
-R3 0 -O- -C- -NH- -CHz -CHZ -NH- -C- -CH- -CH- -CH3
O 0 NH 2 OH
-R4
m=0 Ter m=0 Ter
-R3 o -OH
-R2 -H -R4
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Serine-2', 3'-EDA-6-Thio-GTP
SH
O O O N N
HOOP- O O" 0 0~ 0 ~ CN I
NNHZ
O O Q -y\ y
~IOle/
H~ O
HN
NH
O
H2N OH
m=0 Ter
-RI -SH
n=3 1 2 3 Ter
-R5 -P03 -P03 -PO3 -OH
m=9 Intl lntZ lnt3 Int4 lnt5 lnts lnt7 Int8 lnt9 Ter
-R3 o -0- -C- -NH- -CH2 -CH2 -NH- -C- -CH- -CHZ -OH
0 0 NH2
-R4
m=0 Ter
-R3 o _OH
-R4
m=0 Ter
-R2 -H
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2', 3', 5', O-Triacetyl-N-2-(Acetyl-6"-am i nohexyl)-g uanosine
OH
O N N H
N N
O O ~N NN
I ~
H O
O~O O\r O
m=0 Ter m=2 Intl Int2 Ter
-OH -0- -C- -CH3
-RI -R5 0
m=2 Int, lnt2 Ter
-R3 e -O- -C- -CH3
-R4 0
m=8 Int, Int2 Int3 Int4 lnt5 lnt6 lnt7 Int8 er
-CH2 -CHz -CH2 -CHZ -CHz -CHZ -NH- -C- -CH3
-R2 0
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2',3',5'-Triacetyl-N-2-(6"-thioacetamide-hexyl)-6-Thioguanosine (V4) [TWI
107/7]
SH
O N ~N H
/
O N INN N~
I
H S
O-~ O O\r O
m=0 Ter
-R1 -SH
m=2 Int, lnt2 Ter m=2 Int, lntZ Ter
-0- -C- -CH3 -R3 e -0 -C- -CH3
-R5 O -R4 0
m=8 Intl lnt2 lnt3 lnt4 Int5 lnts lnt7 lnt8 Ter
-CH2 -CH2 -CHZ -CHz -CHZ -CHZ -NH- -C- -CH3
-R2 g
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N-2-(6"-thioacetam ide-hexyl)-6-Thioguanosine
SH
ON I N H
HO N Ni N
H S
OH OH
m=0 Ter m=0 Ter m=0 Ter
-R1 -SH -R5 -OH R43 e -OH
m=8 /nt, lnt2 lnt3 Int4 lnt5 lnt6 Int, lnt8 Ter
-R2 -CH2 -CH2 -CHF- -CH2 -CHZ -CHz -NH- -C- -CH3
S
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N-2-(6"-Aminohexyl)-6-Thioguanosine, ID: 05A-0
SH
N N
~ I
HO O N NN NHZ
H
OH OH
m=0 Ter
-R1 -SH
m=0 Ter
-R3 e -OH
-R4
m=6 Int, lnt2 lnt3 /nt4 int5 lnt6 Ter
m=0 Ter -R2 -CHZ -CHz _CH2 -CH2 -CHz -CHZ -NH2
-R5 -OH
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23
N-2-(6"-guanidino-hexyl)-6-Thioguanosine, ID: 05A-1
SH
N I N H
HO N Ni'~N NyNHZ
H NH
OH OH
m=0 Ter n=O Ter m=0 Ter
-RI -SH -R5 -OH -R4 e -OH
m=8 Int, lntZ Int3 Int4 Int5 Int6 lnt, IntB Ter
-CH2 -CHz -CH2 -CHi- -CHZ -CHZ -NH- -C- -NH2
-R2 NH
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24
N-2-(6"-Am inohexyl)-6-Thio-GM P
SH
O N N
II \
<
HO ~O O N I
Ni N NHz
o
--\~ y H
O
I~H----I OH
m=0 Ter n=1 1 Ter m=0 Ter
-R1 -sH -R5 -PO3 _OH -R4 e _OH
m=6 Intl lntz lnt3 lnt4 Int5 lnts Ter
-R2 -CHZ -CH2 -CHZ -CHZ -CHZ -CH2 -NH2
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N-2-(6"-guanidino-hexyl)-6-Thio-GMP, ID: 05A-2
SH
O N I N H
I I ~
HO ~'O N N N N~NHZ
6 --\~ H N H
OH OH
m=0 Ter n=1 1 Ter m=0 Ter
-R1 -SH -R5 -PO3 -OH -R4 e -OH
m=8 Intl lnt2 lnt3 lnt4 lnt5 Int6 lnt7 lnt$ er
-R2 -CHz -CH2 -CH2 -CH2 -CHZ -CH2 -NH- -C- -NH2
NH
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26
N-2-(6"-Aminohexyl)-6-Thio-GTP
SH
O 0 0 N INN
HO P~O'p,OP,O N ~ NHZ
N
Oo 00 00 --\ I I H
OH OH
m=0 Ter n=3 1 2 3 Ter
-R1 -SH -R5 -P03 -PO3 -P03 -OH
m=0 Ter
-R3 e -oH
-R4
m=6 Int, Int2 Int3 Int4 lnt5 Ints Ter
-R2 -CH2 -CH2 -CHZ -CHz -CHZ -CH2 -NH2
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27
N-2-(6"-guanidino-hexyl)-6-Thio-GTP, ID: 05A-3
SH
O O O <N N H
HOO-P~O O~O oO N I N1NH2
~ N N
~ e O --\O H OH OH
m=0 Ter n=3 1 2 3 Ter
-R1 -SH -R5 -P03 -P03 -P03 -OH
m=0 Ter
-R3 e -OH
-R4
m=8 int, lnt2 Int3 Int4 /nt5 Ints Int, IntB Ter
-R2 -CHZ -CHz -CH2 -CHZ -CHZ -CHz -NH- -C- -NH2
NH
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28
N-2-(6"-Aspartate-hexyl)-6-Thioguanosine
SH
N N H NHZ O
N ~ OH
HO--\ N IN ' 'v ' ~
H O
OH OH
m=0 Ter n=0 Ter m=0 Ter
-R1 -SH -R5 -OH -R4 e -OH
m=10 Int, Int2 Int3 Int4 Int5 Ints Int, Int8 Int9 Int,o Ter
-CHz -CHZ -CH2 -CHi -CH2 -CHZ -NH- -C- -CH- -COOH
-R2 O NH2 -CHz
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29
N-2-(6"-Glutamate-hexyl)-6-Thioguanosine
SH
N L N N H NH2
HO N i \ N~~OH
--\ N N
H O O
OH OH
m=0 Ter
-R1 -SH
n=0 Ter m=0 Ter
-R5 -OH -R4 -OH
=11 lnt, IntZ Int3 Int4 Int5 Infs InG lnf$ lnt9 lnflo Intl, Ter
-CH2 -CH2 -CHz -CHZ -CH2 -CHZ -NH- -C- -CH- -CHZ -CHa -COOH
-R2 0 NH2
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N-2-(6"-Threonine-hexyl)-6-Thioguanosine
SH
N e ~ N H NHz
HO N Ni Ny~,/
--\ H O IOH
OH OH
m=0 Ter n=0 Ter m=0 Ter
-R1 -SH -R5 -OH -R4 o -OH
=10 Int, lnt2 lnt3 1nt4 lnt5 Ints lnt, lnt8 Int9 lnt,o Ter
-CHZ -CHZ -CHZ -CHz -CHZ -CH2 -NH -C- -CH- -CH- -CH3
-R2 0 NH2 OH
N-2-(6"-Serine-hexyl)-6-Thioguanosine
SH
I ' N H NHZ
N i
HO--\~ O N N N~OH
H O
OH OH
m=0 Ter n=0 Ter m=0 Ter
-R1 -SH -R5 -OH -R3 e _OH
-R4
m=10 Int, IntZ lnt3 Int4 Int5 Int6 lnt7 Int8 Int9 Intlo Ter
-CH2 -CH2 -CH2 -CHZ -CH2 -CHz -NH- -C- -CH- -CH2 -OH
-R2 0 NHz
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31
N-2-(6"-Aminobutyl)-6-Thio-Guanosine, ID: 05C-0
SH
N N
HO N I Ni 'N~/NHz
OH OH
m=0 Ter n=0 Ter
-R1 -SH -R5 -OH
m=0 Ter
-R3 e
-OH
-R4
m=4 Infl Inf2 Inf3 Inf4 rer
-CHz -CHi--CH2 -CHZ -NH2
-R2
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2- N-2- (6"-guanidino-butyl)-6-Thioguanosine, ID: 05C-1
SH
N I N H
HO N N% Nu
H NH2
II
NH
OH OH
m=0 Ter n=0 Ter m=0 Ter
-R1 -SH -R5 -OH -R4 e -OH
m=6 Intl Int2 Int3 Int4 Int5 Ints Ter
-R2 -CHZ -CH2 -CHZ -CHz -NH- -C- -NHZ
NH
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33
2- N-2- (6"-guanidino-butyl)-6-Thio-GMP, ID: 05C-2
SH
O N ~ N H
HO'P'O I i uNH2
O N N II
H NH
Na+
OH OH
m=0 Ter n=1 I Ter m=0 Ter
-R1 -SH -R5 -P03 -OH -R4 e -OH
m=6 Inf, Int2 Int3 Int4 lnf5 -nt6 Ter
-CH2 -CH2 -CHZ -CHZ -NH- -C- -NH2
-R2 NH
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34
2- N-2- (6"-guanidino-butyl)-6-Thio-GTP, ID: 05C-3
SH
O 0 0 N I~N H
II II II 1
HOO-P~O 0"O O~O O \N Ni NuNH2
e 0 o H INI NH
3Na*
OH OH
m=0 Ter n=3 1 2 3 Ter
-Rl -SH -R5 -P03 -P03 -P03 -OH
m=0 Ter
-R3 e -OH
-R4
m=6 Int, IntZ Int3 Int4 lnt5 lnts Ter
-R2 -CHZ -CHa -CH2 -CHZ -NH- -C- -NH2
NH
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N-2-(6"-Aspartate-butyl)-6-Thioguanosine
SH
N AN H NHZ O
HO N N~/ ~~Nu'~/~OH
H IOI
OH OH
m=0 Ter n=0 Ter m=0 Ter
-RI -SH -R5 -OH -R3 e -OH
-R4
m=8 Int, Int2 lnt3 lnt4 lnt5 lnts Int, lnt8 Ter
-CHZ -CH2 -CHZ -CHZ -NH- "C- -CH- -CHZ -COOH
-R2 0 NH2
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36
N-2-(6"-Glutamate-butyl)-6-Thioguanosine
SH
IN ~ N N H NHZ
<N i
HO 0 N /N~~OH
H 0 O
OH OH
m=0 Ter n=0 Ter m=0 Ter
-R1 -SH -R5 -OH -R3 e -OH
-R4
m=9 Intl int2 /nt3 int4 /nt5 int6 int, Int8 int9 Ter
-CHi- -CHi- -CH2 -CHi- -NH- -C- -CH- -CHZ -CH2 -COOH
-R2 0 NH2
N-2-(6"-Threonine-butyl)-6-Thioguanosine
SH
N I~ N H NHz
HO ~N N" N-fy
--\~ H O OH
OH OH
m=0 Ter n=0 Ter m=0 Ter
-R1 -SH -R5 -OH _R4 e -OH
m=8 intl lnt2 int3 Int4 /nts lnts Int7 int8 Ter
-CH2 -CHz -CHi- -CHi- -NH- -C- -CH- -CH- -CH3
-R2 0 NH2 OH
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37
N-2-(6"-Serine-butyl)-6-Th ioguanosine
SH
/,N I N H NHZ
HO ~N NN/NY-~-OH
H 0
OH OH
m=0 Ter n=0 Ter m=0 Ter
-R1 -SH -R5 -OH -R3 e -OH
-R4
m=8 lnt, Int2 Int3 lnt4 int5 Ints Int7 Int8 Ter
-CHZ -CHZ -CHZ -CHZ -NH- -C- -CH- -CHZ -OH
-R2 0 NH2
N-2-(6"-Aminopropyl)-6-Thioguanosine
SH
/
\N I " N
HO O N N~N~U~NHZ
-\ H
OH OH
m=0 Ter n=0 Ter m=0 Ter
-RI -SH -R5 -OH -R3 e -OH
-R4
m=3 Int, Int2 Int3 Ter
-R2 -CHZ -CHZ -CH2 -NH2
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38
N-2-(6"-guanidino-propyl)-6-Thioguanosine
SH
</ N N NH
HO O N N~N~~N~NH2
H H
OH OH
m=0 Ter n=0 Ter m=0 Ter
-RI -SH -R5 -OH R4 -OH
m=5 Int, lnt2 lnt3 int4 lnt5 Ter
-CHZ -CHZ -CHZ -NH- -C- -NH2
-R2 NH
N-2-(6"-Aspartate-propyl)-6-Thioguanosine
SH
N I ~ O
HO--\~ I i O ~N N% N-_"~N-'~OH
H H NHZ O
OH OH
m=0 Ter n=0 Ter m=0 Ter
-R1 -SH -R5 -OH -R4e -OH
m=7 Intl lntZ lnt3 lnt4 Int5 Ints Int, Ter
-CHz -CHZ -CHZ -NH- -C- -CH- -CHz -COOH
-R2 0 11 NH2
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39
N-2-(6"-Glutamate-propyl)-6-Thioguanosine
SH
'N - N O O
\ i
HO--\ O N IN~N~~\'N OH
FI H NHZ
IOH IOH
m=0 Ter n=0 Ter m=0 Ter
-RI -SH -R5 -OH - R4 -OH
m=8 Int, Int2 Int3 lnt4 lnt5 lnt6 Int, IntB Ter
-CHZ -CHF- -CHZ -NH- =C- -CH- -CHF- -CHz -COOH
-R2 0 NH2
N-2-(6"-Threonine-propyl)-6-Thioguanosine
SH
N N O OH
HO O ~N I N
I I~
H H NH2
OH OH
m=0 Ter n=0 Ter m=0 Ter
-R1 -SH -R5 -OH -R3 e -OH
-R4
m=7 lnti lnt2 Int3 lnt4 lnt5 lnt6 lnt, Ter
-CHZ -CHF- -CHF- -NH- -C- -CH- -CH- -CH3
-R2 O NH2 OH
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N-2-(6"-Serine-propyl)-6-Thioguanosine
SH
N - N O
HO-- N
\ O ~ li H INHZ
OH OH
m=0 Ter n=0 Ter m=0 Ter
-R1 -SH -R5 -OH -R3 e -OH
-R4
m=7 Intl lnt2 lnt3 Int4 Int5 lnt6 Int, Ter
-CHZ -CHZ -CHz -NH- -C- -CH- -CHZ -OH
-R2 0 NH2
N-2-(6"-Amino-2-butene)-6-Thioguanosine
SH
\N \ N
f I
HO-\ I IN N~ N,,%\/NHZ
H
OH OH
m=0 Ter n=0 Ter m=0 Ter
-RI -SH -R5 -OH R4 -OH
m=3 Int, lnt2 lnt3 Ter
-CHZ -H=H- -CHz -NH2
-R2
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41
N-2-(6"-guanidino-2-butene)-6-Thioguanosine
SH
/,N I N H
HO 'N N% NyNHZ
41 NH
OH OH
m=0 Ter n=0 Ter m=0 Ter
-RI -SH -R5 -OH -R3 e -OH
-R4
m=5 Intl lnt2 Int3 Int4 int5 Ter
-CH~ -H=H -CH- -NH- -C11 - -NHZ
-R2 NH
N-2-(6"-Aspartate-2-butene)-6-Th ioguanosine
SH
N I N H NH2 O
HO N NN~~N Y ' 'OH
--\~ H O
OH OH
m=0 Ter n=O Ter m=0 Ter
-R1 -SH -R5 -OH RR4 -OH
m=7 Int1 Int2 lnt3 Int4 lnt5 lnts lnt7 Ter
-CH2 -H=H- -CHZ -NH- -C- -CH- -CH2 -COOH
-R2 0 NH2
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42
N-2-(6"-Glutamate-2-butene)-6-Thioguanosine
SH
,N I ~ N H NH2
HO-- 'N N~Ni'~% ~/NOH
H O O
~
OH OH
m=0 Ter n=0 Ter m=0 Ter
-R1 -SH -R5 -OH -R3 e -OH
-R4
m=8 Int, lnt2 lnt3 lnt4 lnt5 lnts lnt, IntB Ter
-CHZ -H=H- -CHz -NH- -C- -CH- -CHz -CHa -COOH
-R2 0 NH2
N-2-(6"-Th reon ine-2-butene)-6-Thioguanosine
SH
N I N H NH2
HO--\ N N N'_"~N II I
H O OH
OH OH
m=0 Ter n=0 Ter m=0 Ter
-RI -SH -R5 -OH R4 -OH
m=7 Intl lnt2 lnt3 Int4 lnt5 lnt6 lnt, Ter
-CH2 -H=H- -CHZ -NH- -C- -CH- -CH- -CH3
-R2 O NH2 OH
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N-2-(6"-Serine-2-butene)-6-Thioguanosine
SH
s,N I N H NH2
--~ I I ~" '
HO 'N I N N.~ l/OH
H 0
OH OH
m=0 Ter n=0 Ter m=0 Ter
-R1 -SH -R5 -OH -R3 e -OH
-R4
m=7 Intl Int2 Int3 Int4 Int5 Int6 Int7 Ter
-CHZ -H=N -CHZ -NH- -C- -CH- -CHT -OH
-R2 O NI
H2
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44
N-2-(6"-Amino-2-butyne)-6-Thioguanosine
SH
N ~N
~ IN N
/NH2
HO-- O N
\
H
OH OH
m=0 Ter n=0 Ter m=0 Ter
-R1 -SH -R5 -OH -R3 e -OH
-R4
m=3 Int, Int2 Int3 Ter
-CHZ -C-C- -CHZ -NH
2 -R2
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N-2-(6"-guanidino-2-butyne)-6-Thioguanosine
SH
N H
HO \N I NNNyNHZ
O I NH
H
OH OH
m=0 Ter n=O Ter m=0 Ter
-RI -SH -R5 -OH -R3 e -OH
-R4
m=5 Intl lnt2 lnt3 lnt4 lnt5 Ter
-R2 -CHZ -C=C- -CH2 -NH- -C- -NH2
NH
N-2-(6"-Aspartate-2-butyne)-6-Thioguanosine
SH
N ~ N H NHZ O
/
H
HO_- NIN N N~OH
~O~~ O
OH OH
m=0 Ter n=0 Ter m=0 Ter
-R1 -SH -R5 -OH -R3 e -OH
-R4
m=7 Intl int2 lnt3 Int4 Int5 Ints lnt7 Ter
-CHZ -C=C- -CHz -NH- -C - -CH- -CHZ -COOH
-R2 0 NH2
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46
N-2-(6"-Glutamate-2-butyne)-6-Thioguanosine
SH
H NH2
~N ~ N ~
/ I N\~~OH
HO O N N/
--\~ I I H 0 0
OH OH
m=0 Ter I n=0 Ter I I m=0 Ter
-RI -SH -R5 -OH -R3 e -OH
-R4
m=8 Int, Int2 Inf3 Int4 lnt5 Ints Inf, Int8 Ter
-CH2 -CC- -CHa -NH -C- -CH- -CH2 -CH2 -COOH
-R2 0 NH2
N-2-(6"-Threonine-2-butyne)-6-Thioguanosine
SH
N j N H NH2
HO Y1'
/ N N~N N
I 0 OH
H
OH OH
m=0 Ter I n=0 Ter m=0 Ter
-R1 -SH -R5 -OH -R3 e -OH
-R4
m=7 Int, Int2 Int3 Int4 Int5 Ints Int, Ter
-CHZ -C=C- -CH2 -NH- -C- -CH- -CH- -CH3
-R2 0 NH2 OH
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47
N-2-(6"-Serine-2-butyne)-6-Thioguanosine
SH
H NH2
< N ( N;r,~
N/-_N~OH
HO O N
--\ H O
OH OH
m=0 Ter n=O Ter m=0 Ter
-R1 -SH -R5 -OH R4 -OH
m=7 Intl Inf2 Inf3 /nf4 Int5 /nfs Int, Ter
-CHZ -CC- -CHZ -NH- -C- -CH- -CHZ -OH
-R2 0 NH2
N-2-(6"-Amino-2,4-hexadiyne)-6-Thioguanosine
SH
~ NH2
\N \ N -
HO--, O N eI NN/
H
OH OH
m=0 Ter n=0 Ter m=0 Ter
-R1 -SH -R5 -OH -R3 e -OH
-R4
m=4 Intl Int2 Int3 Int4 Ter
-CH- -CC- -CC- -CH- -NHZ
-R2
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48
N-2-(6"-guanidino-2,4-hexadiyne)-6-Thioguanosine
SH
H
<N N N~NH2
HO-- O N ~ - -
I NH
\~ '~/ N H
IO~H~IO/H
m=0 Ter n=O Ter m=0 Ter
-R1 -SH -R5 -OH -R3 e -OH
-R4
m=6 Intl lnt2 lnt3 lnt4 lnt5 lnt6 Ter
-CH2 -C=C- -CC -CHZ -NH- -C- -NH2
-R2 NH
N-2-(6"-Aspartate-2,4-hexadiyne)-6-Thioguanosine
SH
NI ~ N - - li NHZ 0
/ ~I
HO O N %~ ~ - OH
N N 0
H
OH OH
m=0 Ter n=O Ter m=0 Ter
-R1 -SH -R5 -OH R4 -OH
m=8 Int, lnt2 lnt3 lnt4 lnt5 lnts Int, lnt8 Ter
-CHZ -CC- -CC -CH- -NH- -C- -CH- -CHZ -COOH
-R2 0 NH2
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49
N-2-(6"-Glutamate-2,4-hexadiyne)-6-Thioguanosine
SH
NI N - - H NHZ
OH
HO O N Ni \ - -
N\~I~
N O O
H
OH OH
m=0 Ter n=0 Ter m=0 Ter
-R1 -SH -R5 -OH -R4 -OH
m=9 Intl Int2 Int3 Int4 Int5 Ints Int, lnt8 Int9 Ter
-CHZ -C=C- -CC -CHZ -NH- -C- -CH- -CHz -CHZ -COOH
-R2 0 NH2
N-2-(6"-Threonine-2,4-hexadiyne)-6-Thioguanosine
SH
N j , N H NH2
HO N ~~ N\ ~ /
' '
O - -
--~~ N H O OH
OH OH
m=0 Ter n=0 Ter m=0 Ter
-R1 -SH -R5 -OH R4 -OH
m=8 Intl int2 Int3 /nt4 Int5 Int6 /nt, Int8 Ter
-CHZ -C=C- -C=C -CHZ -NH- -C- -CH- -CH- -CH3
-R2 0 NH2 OH
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N-2-(6"-Serine-2,4-hexadiyne)-6-Thioguanosine
SH
N H NHZ
~ /OH
s ~ N' N\" "
--~ ~ I I 0
H
HO N NJ~. ~
OH OH
m=0 Ter n=0 Ter m=0 Ter
-R1 -SH -R5 -OH R4 -OH
m=8 Int, IntZ Int3 Int4 Int5 Int6 int7 Int8 Ter
-CHZ -C=C -C=C- -CHZ -NH- -C- -CH- -CHz -OH
-R2 0 NH2
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51
Further compounds of the invention are disclosed, with reference titles
indicated.
06D-1
S
<~ ~ N
0- O' O- O N NNH2
HO-P-O-~-O--O
O 0 0
O 0
ONH
Br / '
S
06D-2
N
O' O O N IN/'NH2
O 0 O
O 0
O~NH
d
NI
H
06D-3
/~ I N
I- I O 'N N NH2
HO-P-O-P-O--FO
O 0 0
O 0
ONH
~ ~
S
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52
06D-4
I N
- I - I p N ~NH2
HO-P-O-P-O-P-O
O 0 0
O p
O==~
NH
\N
06D-5
/~ N
OI 'N~ ~NHz
HO-P-O-P-O-PO
O 0 0
O O
O=~NH
N
H3C ~
CH3
06D-6
N
O I N N NHZ
HO-P-O-P-O-P-O
0 0 0
O O
ONH
ci
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53
06D-7
N
/ ~
I I I O N NHZ
HO-P-O-P-O-P-O
O 0 0
O 0
O=<
NH
~ '
C~ ~ CH3
06D-8
N
T O ? O N NHZ
/ ~
HO-P-O-P-O~-P-O
11 O 0 0
O. O
O~NH
H s C N 'H
06D-9
/ N
I O O N N"LNHZ
HO-P-O-P-O~-P-O
11 O 0 0
O O
ONH
j \N
HO N-'
H
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54
06D-10
S
/~ N
O' I " I p \N 'tNH2
HO-P-O-P-O-P-O
11 O 0 0
O O
O
NH
H3COOC
S
06D-11
/~ I N
i O t O 'N ~NHZ
-O-P-O
HO-P-O-P
11
O 0 0
O, O
ONH
CN
06D-12
N
O 1 p N ~NH2
HO-P-O-P-O-P-O
11
O 0 0
O O
ONH
/ S
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06D-13
N
/~ I~
I 1 I O N NHZ
HO-P-O-P-O-P-O
11
O 0 10~
O O
ONH
N
I
CH3
06D-14
S
</I N
Q O 0 N ~NHZ
HO- IP-O-P-O-P-O
O 0 0
O O
ONH
N
N--CH3
CH3
06D-1 5
X N
I- I Q O N ~NH2
HO-P-O-P-O-P--O
0 0 0
11
O O
ONH
= ~N~
C(CH3)3 N H
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06D-16
/N I N
I i ~ O 'N N"~NH2
HO-P-O-P-O-P-O
11 O 0 0
l',~w
O, O
HOOC ONH
N
N
I
H
06D-17
S
/f ' N
O' O" q O 'N N/'NHZ
HO-P-O-P--O-P-O 11
O 0 0
O O
ONH
COOCH3
06D-18
S
_ N
_ \~
I Q O N NNHZ
HO-P-O-P-O-P-O
O 0 0
O O
ONH
s COOCH3
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06D-19
N
O' ~- ~ p N N"'NH2
HO-P-O-P-O-P
11 O 0 0
O O
ONH
N~N0CCH3
06D-20
S
</ I N
O- O' O- N Ni'NHZ
HO-P-p-P--p-P-O
O 0 0
O O
O~NH
N,CH3
N
CH3
06D-21
I N
O- O- O- p N Ni~NH2
HO-P-O-P-O-P-O
O 0 0
O O
pNH
H3C N~N~CH3
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06D-22
S
<'I "
O- O- ~- N NH2
HO-P-O-P-O-P-O
O 0 0
O O
O.NH
/ S
H3C
06D-23
S
<~ "
~ O Q p NXNi:~:~'NHZ
HO-P-O-P-O- IP-O
11
O 0 0
O O
ONH
NC
CH3S s COOCH3
06D-24
"
_ p_ <~ ( I I ~ " ~NHz
HO-P-O-P-O-P-O
0 0 0
O O
O=1(
NH
N
C(C'HA
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06D-25
X N
I t I O N ~NHZ
HO-P-O-P-O-PO
11
O O O
O p
ONH
s COCH3
06D-26
N
4 I I O N ~NHZ
HO-P-O-P-O-P-O
11
O O O
O O
O=x-NH
CN
1:.
s COOCH3
06D-27
S
N
O- O- Q O 'N N~'NHZ
HO-P-O-P-O-P-O
O O O
O, O
ONH
CH3
H3C NiN
1
CH3
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06D-28
S
/~ N
O Q- Q 'N N/'~NH2
HO-P-O-P-O-P-O
O 0 0
O O
NH
O
H3C
/
S COOCH3
06D-29
N
O NI ~NH2
HO-~ IP-O-P-O-P-O
O 0 0
O Q
O==(
NH
N
Br ~
(''(CH3)3
06D-30
S
/~ I N
O' O- 'N Ni'NHZ
HO-P-O-P-p-P-O
0 0 0
O O
O~NH
CH3
H3C N~N
i
CH3
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06D-31
N
/ IN/
O- O N 'NHZ
HO-P-O-O--[?--O
O 0 ~O~
O O
O~NH
~ N~
C(CH3)3 ~N CH3
06D-32
S
~ I N
O' O- O N Ni'NH2
HO-P-O-P-O-P-O
O 0 IOI
O O
pNH
CN
(CH 3)2N $ COOCH3
06D-33
S
N
I I I O NI NtNH2
HO-P-O-P-O-P-O
O 0 0
O O
O
NH
H3CO2S
Z:C
S COOCH3
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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.
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.
The present invention further relates to the use of labelled compounds of
formula (I), particularly
with R3 or R4 selected from
_ I nt- C=0
Int-
~ C=0
I / I
COON HOOC Q o Q or Q o a
wherein Q is selected from -OH (FAM) or -N(CH3)2 (TAMRA) as probes for the
evaluation of
the binding properties of the compounds of formula (I) by the RaclNav system.
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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 0-deprotection;
c) guanosine ring-closing and introduction of a SH group at the 2 position of
the guanosine ring
through the use of CS2;
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=O groups through the use of Lawesson's reagent.
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.
Descripton of Drawings
Figure 1 shows the scheme of the mechanism of action of the apoptosis
inhibition and of the
action of azathioprine.
Figure 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-Thioguanosine, V5 = BMB = TAMRA-EDA-6-Thio-GTPafter 3
days.
Figure 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-Thioguanosine, V5 = BMB = TAMRA-EDA-6-Thio-GTPafter 4
days.
Figure 4 shows the apoptosis induction in human CD4+ T Lymphocytes by
V1=BMB20= EDA-6-
Thio-GTP, V2= TWI 3511= 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-Thioguanosine, V5 = BMB = TAMRA-EDA-6-Thio-GTPafter 5
days.
Figure 5: Induction of apoptosis. Buffy stands for "buffy coat" which is the
fraction of blood
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64
obtainable by centrifugation and containing leukocytes and platelets.ln this
case buffy
coats were used to isolate monocytes for the experiments. "Buffy 1 vom
08/03/05" means
"Buffy coat 1 of march 8th, 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: VI
was tested twice on 08/03/2005 (where 08/03/2004 is written this is in error
and should read
08/03/2005) and twice on 22/04/2005
Figure 6: Results of biological activity of compounds compiled in Figure 5.
Figure 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 Figure 7 may show an
alternative
enantiomeric form than that discussed in the text.
Figure 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 activityx [%]=Caspase
activityX[%] -
Caspase aCtivityuntreated[%]= 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/Pl-
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
(Kohyoma et al., 2003)
0
o ~
~/ N~J H \N N~O~O
ACO--~~N N Ac0 O N Ns
2-Methoxyethoxymethylchloride
OAc OAc O C OAc OAc
Inosine-2",3",5"-triacetate (4g, 10.2mmol) was dissolved in 100 ml
dichloromethane and the
solution was treated with 1,4 ml (12.2 mmol) 2-methoxyethoxymethylchloride at
0 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.
The aqueous layer was extracted with chloroform and the combined and washed
organic layers
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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-1-0-ribofuranosylimidazole-4-carboxamide (Kohyoma et
al.,
5 2003)
~
0
-0 0
NH2
<N ~ e\\O (/N I N NHz
N \
c-~~oy 0.2 N NaOH aq. Ho O N
reflux,lh ~
OAc OAc )1.
OH OH
Aqueous ammonia solution (28%, 20 ml) was added to a solution of 2',3',5'-Tri-
O-acetyl-1-[(2-
10 m ethoxyethoxy) m ethyl] 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.
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 HCI (6 M) and
evaporated to dryness.
15 The residue was dissolved in ethanol, filtered from insoluble material and
concentrated to
dryness. Purification of the crude product by column chromatography (silica
gel, CHC13:MeOH,
3:1) gave 1,32 g(5.11 mmol, 70%) of 5-Amino-l-(3-ribofuranosylimidazole-4-
carboxamide.
Preparation of 2-Mercaptoinosine (Imai et a1.,1971)
0 O
20 <N NH2 ~N I NH
~
HO 0 N 'NHz Phenylisothiocyanate HO O N N SH
~ Pyridine, reflux, 2h
OH OH OH OH
The 5-Amino-l-R-ribofuranosyiimidazole-4-carboxamide (1,3 g, 5 mmol) was
dissolved in
25 pyridine and 18m1 (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 C and concentrated under vacuum. Methanol
was then
30 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.
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According to an alternative method of preparation, compound-Amino-l-(i-
ribofuranosylimidazole-4-carboxamid (1,3 g, 5 mmol) was added to a solution of
sodium
hydroxide (1 g, 25 mmol) in 20m1 of methanol at 30 C. Carbon disulfide (1,9 g,
25 mmol) was
added, and the solution was heated in an autoclave at 180 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
0
N O
< I ' "
N~ NH
~ O C
HO--\O N N SH Hydrogen peroxide HO N I SOaNa
OH OH
OH OH
O O
NH N
~i~ NIH
< ~ NH2
HO
õ N I j~ N SONa 1,6-Diaminohexane HON N N
~I\_"'.I/y 155 C H
OH OH OH OH
2-Mercaptoinosine (1 g, 3 mmol) was dissolved in 100 ml water and cooled to 0
C. A clear
solution was obtained after ultrasonic irradiation. Hydrogen peroxide (1 mi, 9
mmol) was added
within 20 min under vigorous stirring. After 1 hour of stirring at 0 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 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 aI.,1999)
0 0
N I NH " I NH
HO O N Nj \N "HZ AcO O ~N Ni \N NHAc
~ H acetic anhydride ~ H
OH OH RT OAc OAc
A solution of nucleoside (220 mg, 0.575 mmol) in 20 ml dry pyridine was
stirred with Imi
(10mmol) 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
CHCI3 (10 mi)
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and CH3OH (2 ml). This solution was loaded on silica gel and eluted with CHCI3
/ CH3OH (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
o s
/N AN N I NH H
\ 5 Ac0 /N NNHAc AcO O N N~N N
H Lawesson's reagent H s
OAc OAc $0 C OAc OAc
Dioxane (200m1) 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 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, CHC13), resulted in
2',3',5'-triacetyl-N-
2-(6"-thioacetamido-hexyl)-6-thioguanosine (2,92 g, 5,02mmol) in 55% yield.
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EXAMPLE 2: Process for the preparation of analogous of ribose-modified 6-thio-
guanosine-
triphosphate.
Preparation of 6-Thio-Guanosine-Triphosphate (Ludwig, 1981)
s s
<"~~ POCI3, (HNBu3)2H2P207 o 0 0 ~" I NH
HO N N NH O0~'+ HO OOP, Ni
Z 00 00 00 O~ N NHZ
OHr-vfOH 3Na OH OH
Under argon atmosphere, 6-thio-guanosine (1 g, 3.34 mmol) was dissolved in 6
mi
trimethylphosphate. The solution was cooled to 0 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 POCI3 was removed under vacuum within ten minutes.
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, I mmol, 30%).
Preparation of 2'/3'-EDA-6-Thio-Guanosine-triphosphate
s s
N
0 0 0 N NH O O O / AN HO 'F' O-?-O~?~O N I N" NH HOO,P~O O~O O~O O 'N NIiZ
0 Oo Oo O Z O O O
carbonyidimidazole,
-~-
e ethlyene diamine 3 Na 0 0
3 Na OH OH oe'. I I
H
HN
NH2
The dry tributylammonium salt of compound (0.5 mmol) was treated with 500 mg
carbonyidiimidazole in 25 ml dimethylformamide. The resulting mixture was
stirred for 6 hours at
0 C, brought to room temperature and 0,2 ml methanol and subsequently 0,3 ml
ethylene
diamine were added.
The resulting precipitate was centrifuged down and dissoived in water. The
solution was
adjusted to pH 2, in order to decompose the resuiting intermediate
phosphoramidate at the
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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
s s
O O O N O O O N NH
HO'P'OsP'O-P-O NI HO'P-O1P-O'P" O N~~
00 90 e0 N~NHZ ~ 00 00 ~-
3Na N NHZ
oO TAMRA-NHS-Ester s Na 001 1 100mM sodium borate I I
H ~o pH 8.5, RT H ~o
H N HN
NH2 NH
TAMRA
The N-hydroxy-succinimide ester of TAMRA (1 mg, 2 lamol) was dissolved in
200p1 of dry
dimethylformamide and added to a solution of I mg (3 tamo4) of 2'/3'-EDA-6-
Thio-Guanosine-
Triphosphate in 500pI of 100mM 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 pmol) 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)
s
N SH
AcO N AN
CN~N H
O N N N~ I ~ N
H S HO- N N N
OAc OAc H ~
9 CH3OH / NH3 OH OH 10
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)
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SH
N'N H SH
i I
HO O N NN~N~ (N ' sN
H S HO O N NN/~~/~/NHz
5J oH OH NH3 ~ H
OH OH
10 11
A solution of nucleoside 10 (0.1 g, 0.22 mmol) in 5 ml NH3/H20 (30%) was
stirred for 90 minutes
at 80 C. The solvent was removed in vacuo and the residue was dissolved in
water (2 m1). N-2-
10 (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)
15 SH
SH
\N
~ N ~
NH2 < I N
HO N N N~/~/\/ I
o H HO N N JN/\/N~NHz
I}--{I Triazole-l-carboxamldlne HCI H NH
OH OH I~===I/
OH OH
11 12
20 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-l-
carboxamidine
hydrochloride at room temperature in the presence of 39 pl (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
25 (silica gel RP 18, linear gradient from 100% H20 to 100% ACN) gave 0.92 g
(0.21 mmol, 90%)
of N-2-(6"-Guanidino-hexyl)-6-Thioguanosine (12).
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71
Preparation of N-2-(6"-Aminohexyl)-6-Thioguanosine-5'-monophosphate (13)
SH
N - N S
NO 0 N Ni \N/\/~/~~tJ~/ /N NH H
H ISI HOl
P~ \ Ni
OH OH POCIg Oo ~oy H s
0 C Na' ~I--{
OH OH
9
s SH
~
N
0 ~N~ H 0
~ I
11 HO0 O\ /N N~ N~ ~/ HO~p-O 0\/ N N~ N
Nao H ISI NH3 % l--/
OH OH Na IOH IOH
13
Under argon atmosphere, nucleoside 9 (0.3 g, 0.6 mmol) was dissolved in 3 ml
trimethyiphosphate. The solution was cooled to 0 C and treated with 0.3 ml of
Lutidine.
After 10 minutes, 0.15 ml (1.1 mmol) phosphorous oxychloride was carefully
added. After 1
hour, the excess of POCI3 was removed in vacuo within ten minutes.
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 NH3/H20
(30%) and
stirred for 90 minutes at 80 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)
SH
O N \ N SH
11 0 N ~
HOIP'~O N N N~/~/\/NHz II / N H
00 H Ho'P30 N NUHHz
~ FI-~{ Triazole-l-carboxamfdine HCI Op ~o ~ I~
Na OH OH Nal I-.I/ H NH
OH OH
13 14
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-l-carboxamidine hydrochloride at room
temperature in the
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presence of 18 pI (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% H20
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-273'-EDA-6-Thio-Guanosine-Triphosphate (4)
SH
S
O 0 O N '
O O O N NH Hp-?~ IPI lP~ '
I I O~ O/ ON N NH
HOOP~po~0 O~B o N NJ~NHZ 0o 00 oB 0 2
0 0
00
3Na' 0 0
3 Na O 0 0 Boc-Aspartate-tBu-NHS-Ester
I I 100mM sodium borate
pH 8.5, RT H 30 H N
H o
H NNH
> O
'NHZ
HZN
Ho 0
1 4
The N-hydroxy-succinimide ester of Boc/tBu protected Aspartate (2.8 mg, 7pmol)
was
dissolved in 200 pl of dry DMF and added to a solution of 1 (5 mg, 7 pmol) in
500 lal 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
pmol) after workup of the reaction mixture by reversed phase HPLC.
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Preparation of Glutamate-2'/3'-EDA-6-Thio-Guanosine-Triphosphate (5)
S
O O O 'N
O O O NH Hp F' 'P~ ip~
HO P~O~/P~O~P~O N ~ ~ p 0~ o~ O~ n_ N IN NHZ
0 0 N
N NHZ
0 0 00 ~ll---~~ly
3Na' O O
3 Na 0 0 Boc-Glutamate-tBu-NHS-Ester
100mM sodium borate N \>=O
pH 8.5, RT HN
HN 1-O O NH
CNHZ HZN 0
OH
5
The N-hydroxy-succinimide ester of Boc/tBu-protected Glutamate (2.9 mg, 7
pmol) was
dissolved in 200p1 of dry DMF and added to a solution of 1 (5 mg, 7pmol) in
500 lal 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 pmol) after workup of the reaction mixture by reversed phase HPLC.
Preparation of Threonine-2'/3'-EDA-6-Thio-Guanosine-Triphosphate (6)
SH
O O O ,N INj
HO P~ iP~ lP~ ' ~JJ~~
O O O !N A 00000 000 O~N N NHZ
HOOP,O O, O - O O '!J NNHZ
0 0 90 ~I'~ 3Na' O O
~I/
3 Na i Boc-Threonine-NHS-Este H HN /-O
100mM sodium borate
H >-O pH 8.5, RT
HN O NH
NH2 H:N)~
OH
6
The N-hydroxy-succinimide ester of Boc-protected Threonine (2.3 mg, 7 pmol)
was dissolved in
200 pi of dry DMF and added to a solution of 1 (5 mg, 7 pmol) in 500 iai 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 pmol) 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+ T Lymphocytes.
Used substances:
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= Azathioprine
= 6-Mercaptopurine
= 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-
Thioguanosine
= V5 = BMB = TAMRA-EDA-6-Thio-GTP
V3 and V4 were not soluble in water, therefore V3 was reconstituted with
ethanol and V4 was
reconstituted with methanol.
Protocol:
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/mI Penicillin/ Streptomycin + 3mM L-Glutamin) for
3, 4 or 5
days with coated antibodies to CD3 (0,04 pg/mi) and soluble CD28 antibodies
(PharMingen; I
~g/ ml) plus IL-2 (R & D Systems, Wiesbaden, Germany; 40 U/mI). 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 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 106 cells per milliliter. Annexin V FITC
and propidium iodide
were added (5 lal of each per 105 cells). Samples were gentiy mixed and
incubated for 15
minutes at room temperature in the dark before FACS analysis.
Results:
= 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.
= Induction of apoptosis = (Rate of apoptotic cells after indicated treatment)
- (Rate of
apoptosis of untreated cells)
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= V1, V2 and V3 were tested in 4 independent experiments. V4 and V5 were
tested in 2
independent experiments.
Conclusion:
Our first results showed, that VI and V5 were able to induce apoptosis in
CD3/CD28
5 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 (Figures 2-4).
Example 5
Preparation of 2'/3'-Methylenoaminocarbamate derivatives of 6-Thio-Guanosine-
triphosphate
S 0 O O N NH
O O O N I NH HO~P'O 0~O ~O CN I NNH2
,~
HO P" o~P~0~1P~0 N O O 80
Ni NH o
00 00 00 0 carbonyldimldazole I I
R=CHZ=NHy Et3N 3 Na
O+ ,
3 Na OH OH o=c H ~O
HN
'CHy
R
1 2
The dry tributylammonium salt of 6-Thio-GTP 1(0.2mmol) was treated with 200 mg
carbonyl-
diimidazole in 4 ml dimethylformamide. The resulting mixture was stirred for 6
hours at 00 C,
brought to room temperature and, subsequently, 80 ial 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
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2'/3'-Methylenoaminocarbamate-6-Thio-Guanosine-triphosphate derivative 2a
s
I
O O O N NH
HO P-oO oO O 'IJ N" NHZ
3 Na O O
H O
HN\
CH6S./-
2a
Reaction of 3-thienyl-methylamine (226 mg, 2 mmol) with I according to the
general procedure
yielded 2a (0.58 mmol, 29 %) after purification by ion exchange chromatography
and
subsequent reversed phase HPLC.
2'/3'-M ethylen oam in ocarbam ate-6-Th io-G uan osi ne-tri phosphate
derivative 2b
s
O O O N NH
N NHZ
HO'P-O~P:00h1w ~P~
Na
H ~-O
HN~
cH2
N 2b
~N
Reaction of (1,5-Dimethyl-lH-pyrazol-3-yl)methylamine (250 mg, 2 mmol) with I
according to
the general procedure yielded 2b (0.66 mmol, 33 %) after purification by ion
exchange
chromatography and subsequent reversed phase HPLC.
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Example 7
Preparation of 2'/3'-Carbamate derivatives of 6-Thio-Guanosine-triphosphate
s s
O O O /N &NNH2 O O O /N &NNH2
HOOP- O O~O O" O O 'N HOO-P~O O~O "O O 'N O O --\ O O 80
carbonyldlmldazole
O R-NHZ, KHMDS O+
3Na OH OH 3Na I I
o c
H O
HN\
R
1 3
The dry tributylammonium salt of compound 1(0.2mmol) was treated with 200 mg
carbonyl-
diimidazole in 4 ml dimethylformamide. The resulting mixture was stirred for 6
hours at 00 C,
brought to room temperature and, subsequently, 80 pi methanol were added.
After 10 minutes,
2 mmol of the appropriate amine and 2 ml of I M potassium hexamethyidisilazide
(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
In relation to figures 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.
BO was the
strongest candidate drug for apoptosis induction. Two issues should be
considered in these
results.
= 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.
= 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,
BO
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and some of the D compunds were able to induce apoptosis suggesting that they
are
candidate drugs.
In addition, an alternative method was performed for screening of group-D
derivatives (figure 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:
= It is an easier protocol. Therefore there are fewer possibilities for
individual errors.
= 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.
= The measurement is done in duplicate. In this way there is an internal
control.
The Caspase-Glo 317 Assay (PromegaTM) was used. This assay is based on a
caspase
dependent luminescent signal. Protocol: CD4+ 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/mI Penicillin/ Streptomycin + 3mM L-Glutamin) for
3 days with
coated antibodies to CD3 (0,04 Ng/mI) and soluble CD28 antibodies
(PharMingenTM; I pg/ mi)
plus IL-2 (R & D SystemsTM, Wiesbaden, Germany; 40 U/mi) in 96-well plates.
Cells were
treated with different group-D derivatives or were left untrated. At day three
of culture Caspase-
3/7 Assay was performed. 25 pl of Capase-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
pi of each probe were transfered 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 (figure
8)
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Example 8
2'13'-Carbamate-6-Thio-Guanosine-triphosphate derivative 3a
s
O O O SN I NH
HO ~P~ oo ~pC~ "o--\ O 'N N" 'NHy
3 Na I I
H
H N
N
N'J
3a
Reaction of 3-aminopyrimidine (190 mg, 2 mmol) with I according to the general
procedure
yielded 3a (0.076 mmol, 3.8 %) after purification by ion exchange
chromatography and
subsequent reversed phase HPLC.
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Example 9
2'/3'-Carbamate-6-Thio-Guanosine-triphosphate derivative 3b
5 s
O O O N NH
HO ~P 00 ~~O ,O- O NNHZ
3 Na I I
10 ~ '>=O
H HN\
CHp
--N
No
(
3b
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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BIBLIOGRAPHY
-Boise LH et al. Receptors that regulate T-cell susceptibility to apoptotic
cell death. Ann N Y
Acad Sci 1995; 766:70-80.
-Tiede I. et al. CD28-dependent Rac1 activation is the molecular target of
azathioprine in
primary human CD4+ T lymphocytes. J Clin Invest 2003; 111: 1133-1145.
-Maltzman JS et al. Azathioprine: old drug, new action. J Clin Invest 2003;
111: 1122-1124.
-Boise LH et al. CD28 costimulation can promote T cell survival by enhancing
the expression of
bcl-xL . Immunity 1995; 3: 87-98.
-Khoshnan A. et al. The NF-KB cascade is important in bcl-xL expression and
for the anti-
apoptotic effects of CD28 receptor in primary human CD4+ T lymphocytes. J
Immunol 2000;
165: 1743-1754.
-Noel PJ et al. CD28 costimulation prevents celi death during primary T cell
activation. J
Immmunol 1996; 157: 636-642.
-Frauwirth KA et al. Activation and inhibition of lymphocytes by
costimulation. J Clin Invest
2002; 109: 295-299.
-Marinari B et al. Vav cooperates with CD28 to induce NF-KB activation via a
pathway involving
Rac1 an mitogen-activated kinase kinase 1. Eur J Immunol 2002; 32: 447-456.
-Faruqi TR et al. Rac1 mediates STAT-3 activation by autocrine IL-6. PNAS
2001; 98: 9014-
9019.
-Mudter J and Neurath MF. The role of signal transducers and activators of
transcription in T
inflammatory bowel diseases. IBD 2003; 9: 332-337.
-Lovato P et al. Constitutive STAT-3 activation in intestinal T cells from
patients with Crohn's
disease. J Biol Chem 2003; 278: 16777-16781.
-Van Aelst L et al. Rho GTPases and signaling networks. Genes & Development
1997; 11:
2295-2322.
-Kohyoma et al.(2003) A facile synthesis of AICAR from inosine. Synthesis
17:2639.
-Imai et al. (1971) Synthesis of compounds related to inosine 5'-phosphate and
their flavor
enhancing activity. IV 2-substituted inosine lo'-phosphates. Chem. Pharm.
Bull. 19:576.
CA 02615980 2008-01-18
WO 2007/010515 PCT/IE2006/000077
82
-Ostermann et al (1999), New N-2-labelled fluorescent derivates of guanosine
nucleotides and
their interaction with GTP-binding proteins. Nucleosides & Nucleotides 18:245.
-Ludwig (1981) Acta Biochim. Acad. Sci.Hung. 16:131
