Synthesis and evaluation of potential inhibitors of eIF4E cap binding to 7-methyl GTP

Article abstract of DOI:10.1016/j.bmcl.2005.01.080

Cap-dependent translation is initiated by the binding of eIF4E to capped mRNA (m⁷GpppN). We have prepared a small library of 7-methyl guanosine nucleoside and nucleotide analogs and evaluated their ability to inhibit eIF4E binding to 7-methyl GTP with a competitive eIF4E binding immunoassay. 5′-H-Phosphonate derivatives in which the 2′- and 3′-riboside hydroxyls were tethered together by an isopropylidene group were shown to be a new class of inhibitors of eIF4E binding to capped mRNA.

Full text of DOI:10.1016/j.bmcl.2005.01.080

Bioorganic& Medi ci nal Chemistry Letters 15 (2005) 2177–2180
Synthesis and evaluation of potential inhibitors of eIF4E
cap binding to 7-methyl GTP
a
Phalguni Ghosh, Chunkyung Park, Mark S. Peterson, Peter B. Bitterman,
a
b
b
b
Vitaly A. Polunovsky and Carston R. Wagner
a,
*
a
Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455, USA
b
Department of Medicine, University of Minnesota, Minneapolis, MN 55455, USA
Received 9 August 2004; revised 25 January 2005; accepted 27 January 2005
Available online 18 March 2005
7
Abstract—Cap-dependent translation is initiated by the binding of eIF4E to capped mRNA (m GpppN). We have prepared a small
library of 7-methyl guanosine nucleoside and nucleotide analogs and evaluated their ability to inhibit eIF4E binding to 7-methyl
0
0
0
GTP with a competitive eIF4E binding immunoassay. 5 -H-Phosphonate derivatives in which the 2 - and 3 -riboside hydroxyls were
tethered together by an isopropylidene group were shown to be a new class of inhibitors of eIF4E binding to capped mRNA.
ꢀ
2005 Elsevier Ltd. All rights reserved.
In eukaryotes, eukaryoticinitiation fa tc or 4E (eIF4E)
plays a key role in initiation of the translation process
prepared from guanosine monophosphate as previously
7
7
described. The synthesis of the m G derivatives 14–29 is
summarized in Scheme 1. Guanosine was first treated
with dimethoxy propane and toluene sulfonic ai cd
0
by recognition of the 5 terminal mRNA cap structure,
0
0
7
interaction between the 5 -cap of mRNAs and eIF4E
-methyl G(5 )ppp(5 )X (X = any nucleoside). A strong
0
0
0
(TsOH) to yield 2 ,3 -isopropylidene guanosine, 16.
Next, 16 was either alkylated with the appropriate alkyl
or aryl halide to yield compounds, 17–19, or treated
with diphenylphosphite and pyridine to afford the pro-
tected H-phosphonate of guanosine, 20. Compounds
21–24 were obtained from alkylation of 20 by a proce-
dure similar to that employed to synthesize compounds
17–19. Since removal of the isopropylidene from com-
pounds 21–24 resulted in significant depurination, com-
pound 14 was obtained by deprotection of compound 20
Dowex 50W resin, followed by alkylation of compound
13 with benzyl bromide. The amino acid phosphorami-
dates of compound 20 were prepared by oxidation with
1
is required for initiation of translation. Formation of
the eIF4E—cap mRNA complex is considered to be rate
limiting for translation initiation under most circum-
2
stances and its up-regulation is associated with cell
,4
3
growth, tumorgenicity and inhibition of apoptosis.
Recently, inhibition of cap-dependent translation by
the ectopic expression of the eIF4E repressor protein,
4
breast cancer tumorgenicity and resistance to apopto-
E binding protein 1 (4E-BP1), was shown to reduce
4
sis. These findings have renewed interest in the develop-
ment of pharmacological tools and assays capable of
modulating and monitoring translation initiation in gen-
5
,6
eral and eIF4E binding to capped RNA in particular.
timethylsilyl chloride (TMSCl) and I , followed by the
2
In this report, we describe the synthesis and evaluation
of the ability of a small library of 7-methyl G nucleoside
and nucleotide analogs to inhibit eIF4E binding to 7-
methyl GTP with an immunoassay.
addition of the carbomethoxy amino acid. The carbo-
methoxy group was removed by treatment with sodium
hydroxide, yielding compounds 25, 26, 28, and 29.
6
–8
Unlike previous assays that have monitored indirectly
eIF4E antagonism in the presence of accessory proteins,
we have successfully developed a high throughput pro-
cedure for the direct measurement of the inhibition of
A library of 29 members was assembled. The triphos-
phates 1–3 and nucleosides 7–9 were obtained from
commercial sources. The monophosphates 10–13 were
9
eIF4E binding capped mRNA in tissue extracts. Typi-
1
0
cally, eIF4E from pooled cell extracts was co-incu-
bated with variable concentrations of the potential
inhibitors in the presence of 7-methyl-GTP agarose
*
Corresponding author. Tel.: +1 612 625 2614; fax: +1 612 624
139; e-mail: wagne003@tc.umn.edu
0
0
960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.01.080

2178
P.Ghosh et al./ Bioorg.Med.Chem.Lett.15 (2005) 2177–2180
O
N
O
N
O
N
R
R
N
N
N
N
N
N
NH
NH2
NH
NH2
NH
NH2
O
P
H
O
P
H
HO
-O
O
-O
Na+
O
O
O
O
Et NH+
3
O
O
O
O
OH OH
17-19
R = CH3, C2H5, -CH2C6H5,
CH2C10H8
21-24
14
(iii)
(v)
(vi)
O
N
O
O
O
N
N
N
NH
NH2
N
N
N
NH
NH2
NH
NH2
(ii)
NH
NH2
(iv)
O
N
O
N
N
N
N
-O
-O
Na+
O
HO
P
H
O
HO
P
O
O
O
O
(i)
+
Et NH H
3
OH OH
OH OH
O
O
O
O
1
6
20
13
O
N
O
N
N
N
NH
NH
NH2
O
P
O-
N
O
P
O-
NH2
(viii)
N
O
(vii)
R
MeO2C
N
H
O
N
O
O
H
+
O
O
+
O
O
Et NH
Et NH
3
3
25-29
R = glycine, alanine,
phenylalanine, tryptophan
Scheme 1. Reagents and conditions: (i) GuanosineÆxH
Et N, 0.5 h; silica column, CHCl –MeOH–H O (5:2:0.25) + 1% NH
iv) 20, Dowex 50W · 4 resign, H O, 8 h, rt, silica column, CHCl
CH Br–DMF, rt, 7–24 h; (vi) C CH Br, DMF, rt, 48 h; 0.5 mL DMSO, rt, 24 h, 15%; (vii) (a) TMSCl, Py, I
rt, 20–24 h; (viii) (vii); 1 N NaOH, rt, 2 h.
2
O, 2,2-dimethoxy propane, TsOH, acetone, 63%; (ii) diphenylphosphite, Py, rt, 6 h; H
OH, 44%; (iii) RI, R = CH , C , C CH , C10 CH Br–DMF, rt, 18–24 h;
–MeOH–H O (5:2:0.25) + 1% NH OH, 58%; (v) RI, R = CH , C , or
, Et N, (AA)-OMeÆHCl, argon,
2
O,
3
3
2
4
3
2
H
5
6
H
5
2
H
8
2
(
2
3
2
4
3
2 5
H
C
6
H
5
2
6
H
5
2
2
3
beads. After thorough washing to remove unbound pro-
teins, bound eIF4E was assayed by eluting the beads
with 7-methyl-GTP followed by spot immunoblot anal-
ysis (Fig. 1a). As can be seen for the representative
inhibitor compound 24, the optical densities determined
by densitometry were plotted and the IC50 values ex-
tracted (Fig. 1b, Table 1).
placement of the 7-methyl guanosine cation in a deep
hydrophobicpo kc et oc mposed of three tryptophans,
0
0
while the 2 - and 3 -hydroxyl groups are solvent
exposed. None of the nucleosides, compounds 4–9,
exhibited binding affinity for eIF4E, establishing that
effective binding by a 7-methyl GTP analog is likely
0
dependent on having at least one 5 -phosphate group.
Indeed, binding could be recovered when the 7-methyl
G analog, 9 (IC50 = >400 lM), was converted to the
respective monophosphate, 10 (IC50 = 50 lM). Increas-
ing the stericbulk at N-7 by substitution with either
ethyl, allyl, or benzyl groups reduced inhibitor potency
The IC50 for 7-methyl GTP (1) was similar to the
previously reported K value for inhibition of in vitro
translation (5 vs 4.39 lM) (Table 1). While loss of
the 7-methyl group reduced the potency by greater than
i
8
8
7
0-fold, no loss of potency was observed for the
0
from 2- to 4-fold. This is in contrast to K values
i
previously reporting a 3.5-fold increase in potency for
-methyl 2 -deoxy GTP. This is consistent with the
9
Figure 1. eIF4E binding assay for 24.

P.Ghosh et al./ Bioorg.Med.Chem.Lett.15 (2005) 2177–2180
2179
a
Table 1. Inhibition data for 7-methyl guanosine nucleoside and nucleotide analogs
O
N
R1
N
NH
R2
N
N
R O
4
O
R3
O
O
R5 R5
b
No.
R
1
R
2
R
3
R
4
R
5
IC50 (lM)
ꢀ
ꢀ
+
1
2
3
4
5
6
–CH
—
3
–H
–H
–H
–CH
–CH
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
(–P(@O(–O Na )
2
2
2
)
)
)
3
3
3
–H
–H
5
>400
4
+
(–P(@O(–O Na )
0
ꢀ
+
–CH
—
3
(2 -deoxy)
–H
–H
(–P(@O(–O Na )
–H
–H
3
3
–H
–H
–H
–H
–H
–H
>400
>400
>400
>400
>400
>400
50
—
–CH
3
–H
—
p-CH
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
13
3
–PhSH–
–H
–H
c
7
–CH
–CH
–CH
–CH
3
3
3
3
d
8
–H
–H
9
ꢀ
+
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
–P(@O)(O Na )
2
2
2
2
–H
–H
ꢀ
+
2
–C H
5
–P(@O)(O Na )
100
220
ꢀ
+
–CH
–CH
—
2
(CH@CH)
H
–P(@O)(O Na )
–H
–H
ꢀ
+
2
C
6
5
–P(@O)(O Na )
220
130
ꢀ
ꢀ
+
–P(H)(@O)(O Na )
–P(H)(@O)(O Na )
–H
–H
+
–CH
—
2
C
6
H
5
200
–H
–H
–H
–C(CH
–C(CH
–C(CH
–C(CH
–C(CH
–C(CH
–C(CH
–C(CH
–C(CH
–C(CH
–C(CH
–C(CH
–C(CH
–C(CH
3
3
3
3
3
3
3
3
3
3
3
3
3
3
)
)
)
)
)
)
)
)
)
)
)
)
)
)
2
2
2
2
2
2
2
2
2
2
2
2
2
2
>400
>400
>400
>400
180
–CH
3
2
–C H
5
–CH
—
2
C
6
H
5
–H
ꢀ
+
NH )
+
–P(H)(@O)(O Et
–P(H)(@O)(O Na )
–P(H)(@O)(O Na )
–P(H)(@O)(O Na )
3
ꢀ
ꢀ
ꢀ
ꢀ
–CH
3
55
45
+
2
–C H
5
+
–CH
–CH
—
2
C
6
H
5
85
70
+
2
C
10
H
8
–P(H)(@O)(O Na )
ꢀ
+
ꢀ
+
–P(@O)(O Na )NHCH
2
C(@O)O Na
>400
400
ꢀ
+
ꢀ
+
—
—
–P(@O)(O Na )NH(CH
2
)
2
C(@O)O Na
ꢀ
ꢀ
ꢀ
+
–P(@O)(O Na )NHCH(CH
2
C
6
H
5
-(C(@O)OCH
3
ꢀ
>400
>400
400
+
+
—
—
–P(@O)(O Na )NHCH(CH
2
-3-indolyl.)C(@O)O Na
+
ꢀ
+
–P(@O)(O Na )NHCH (4-benzoyl)C(@O)O Na
2
a
b
c
1
31
All compounds were characterized by H, C, and P NMR and HRMS.
The IC50 (in lM) is the concentration necessary to inhibit 7-Me-GTP binding to eIF4E by 50%. Percent variance was within 20%.
-Methylinosine.
7
d
7-Methyl-6-thioguanosine.
7
-benzyl GMP relative to 7-methyl GMP when assayed
able H-phosphonates by constraining the ring with an
isopropylidene group. While this modification had little
effect on the activity of the H-phosphonate of guanosine
(Table 2, 14 vs. 20), the potency of the isopropylidene 23
was enhanced over 2-fold when compared to that for the
unconstrained H-phosphonate, 15. Replacement of the
7-benzyl group with either methyl or ethyl moieties
further enhanced inhibitory potency by approximately
2-fold. Surprisingly, replacement of 7-benzyl with
7-naphthyl modestly enhanced binding affinity. The
requirement for incorporating the H-phosphonate
group was demonstrated by observing little or no activ-
ity for the corresponding nucleosides. Because phospho-
ramidates of nucleosides have been shown to be stable
intracellularly, we proposed that they might serve as
potential di- and tri-phosphate mimics, since they are
dianions under physiological conditions. Nevertheless,
none of the phosphoramidates (25–29) of compound
20 exhibited inhibitory activity.
as inhibitors of in vitro translation. This discrepancy
may stem from differences in the conformation of eIF4E
bound to 7-methyl GTP resin and when actively
1
1
engaged in translation.
0
Since nucleoside 5 -monophosphates are not stable in
vivo and are not cell permeable, we prepared H-phos-
phonate analogs, 14 and 15, as potential phosphate
mimics. No significant difference in the binding affinity
of 7-benzyl GMP (Table 1, 13) an 7-benzyl G H-phos-
phonate (15) was observed, thus confirming the ability
1
2
0
of this substitution to replace 5 -monophosphate. Sur-
prisingly, the H-phosphonate of guanosine (Table 1,
1
4) exhibited nearly 2-fold greater potency than 7-benzyl
0
0
G H-phosphonate (Table 1, 15). Since the 2 - and 3 -hy-
droxyl groups of 7-methyl GTP do not interact with
eIF4E, we chose to examine the role of the ribose ring
conformation on the binding of potentially cell perme-

2180
P.Ghosh et al./ Bioorg.Med.Chem.Lett.15 (2005) 2177–2180
In conclusion, a new assay for direct binding to eIF4E
was developed and used to screen a small library of 29
assess their relative activity. Cell lysate generated from
T3/4E cells using a freeze–thaw lysis method was
3
prepared in advance and aliquots stored at ꢀ70 ꢁC. Each
sample required 20 lL of packed 7-methyl GTP agarose
beads (Amersham Biosciences) pre-equilibrated with
freeze–thaw lysis buffer. Beads were placed into micro-
centrifuge tubes and 7-methyl GTP was added to form a
set of standards. Test compounds were added in parallel to
other sets of tubes. Freeze–thaw lysis buffer was added to
the tubes to bring the volume in each tube to 150 lL. At
the start of the binding step 50 lL of 3T3/4E cell lysate at
a concentration of 3 mg/mL was added to each tube,
which was placed in a horizontal rotating platform to mix
for 2 h at 4 ꢁC. Following the binding step, tubes were
centrifuged to form a pellet, which was washed with
7
-methyl guanosine nucleoside and nucleotide analogs.
0
Constrained 5 -H-phosphonate derivatives were shown
to be a new class of potential cell permeable inhibitors
of eIF4E binding to cap mRNA and thus regulators
of cap-dependent translation. Results of a cell based
translational screen are underway and will be reported
in due course.
Acknowledgements
This work was partially supported by a grant from NIH-
NCI (U01-CA91220).
0.5 mL of freeze–thaw lysis buffer and centrifuged. The
wash step was repeated once. Bound eIF4E was eluted
from the beads by the addition of 250 lL of elution buffer
(
150 mM KCl, 25 mM Tris pH 7.5, and 100 lM 7-methyl
GTP). The beads were centrifuged to form a pellet and
00 lL of each eluate was removed and spotted onto a
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2
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NFD milk in TTBS buffer and probed with mouse anti-
eIF4E (1:500, B D Biosciences) for 1 h. The blot was
washed three times for 5 min each and then incubated with
HRP conjugated Goat anti mouse IgG (1:2000, Sigma
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film using ECL reagents (Amersham Biosciences). Densi-
tometricanalysis was performed by s ca nning and quanti-
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(Bio-Rad). Values were expressed in arbitrary optical
density (OD) units.
2
10, 268.
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7
8
9
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compounds were tested in triplicate in each run and
compared to the reference compound, 7 methyl GTP, to
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