Structure-guided design, synthesis, and evaluation of guanine-derived inhibitors of the eIF4E mRNA-cap interaction

Article abstract of DOI:10.1021/jm300037x

The eukaryotic initiation factor 4E (eIF4E) plays a central role in the initiation of gene translation and subsequent protein synthesis by binding the 5′ terminal mRNA cap structure. We designed and synthesized a series of novel compounds that display pot

Full text of DOI:10.1021/jm300037x

Article
pubs.acs.org/jmc
Structure-Guided Design, Synthesis, and Evaluation of Guanine-
Derived Inhibitors of the eIF4E mRNA−Cap Interaction†
Xiaoqi Chen,*,‡ David J. Kopecky,‡ Jeff Mihalic,‡ Shawn Jeffries,∥ Xiaoshan Min,§ Julie Heath,‡
Jeff Deignan,‡ SuJen Lai,‡ Zice Fu,‡ Cristiano Guimaraes,§ Shanling Shen,∥ Shyun Li,∥ Sheree Johnstone,§
Stephen Thibault,§ Haoda Xu,§ Mario Cardozo,§ Wang Shen,‡ Nigel Walker,§ Frank Kayser,‡
and Zhulun Wang*,§
‡Department of Chemistry Research & Discovery, §Department of Molecular Structure, ∥Department of Oncology, Amgen Inc., 1120
Veterans Boulevard, South San Francisco, California 94080, United States
ABSTRACT: The eukaryotic initiation factor 4E (eIF4E) plays a central role in the initiation of gene translation and subsequent
protein synthesis by binding the 5′ terminal mRNA cap structure. We designed and synthesized a series of novel compounds that
display potent binding affinity against eIF4E despite their lack of a ribose moiety, phosphate, and positive charge as present in
m7-GMP. The biochemical activity of compound 33 is 95 nM for eIF4E in an SPA binding assay. More importantly, the
compound has an IC50 of 2.5 μM for inhibiting cap-dependent mRNA translation in a rabbit reticular cell extract assay (RRL-
IVT). This series of potent, truncated analogues could serve as a promising new starting point toward the design of neutral eIF4E
inhibitors with physicochemical properties suitable for cellular activity assessment.
having highly structured 5′ UTR regions that generally inhibit
translation. The elevated level of eIF4E in tumor tissue
selectively facilitates translation of these messages while leaving
translation of bulk mRNAs unchanged. Activation of eIF4E lies
most downstream on the axis of growth factor signaling
through the PI3K−Akt−mTor pathway; signaling via this
network is frequently upregulated during human tumori-
genesis.16 Inhibition of cap-dependent translation by the
ectopic expression of the eIF4E repressor protein, 4E binding
protein 1, reduces breast cancer tumorgenicity and resistance to
apoptosis.17 Additionally, the development of eIF4E-specific
antisense oligonucleotides (ASOs) modified to enhance their
tissue stability and nuclease resistance required for systemic
anticancer therapy were reported. The impact of eIF4E
reduction in normal tissues was assessed. Despite reducing
eIF4E levels by 80% in mouse liver, eIF4E-specific ASO
administration did not affect body weight, organ weight, or liver
transaminase levels, thereby providing the first in vivo evidence
that cancer may be more susceptible to eIF4E inhibition than
normal tissues.18 The above research results generated renewed
interest in finding small molecule inhibitors of the eIF4E
mRNA-cap interaction suitable for probing the role of cap
dependent translation in cancer biology.
INTRODUCTION
■
Unregulated cell growth is the hallmark of aggressive metastatic
cancer.1 Traditional cytotoxic chemotherapy has attempted to
halt this growth by targeting the fundamental process of DNA
replication with limited success. A clinically unexplored avenue
for therapeutic intervention is the essential cellular process of
protein synthesis.2,3
Protein translation requires the coordinated action of
multiple cytoplasmic ribonucleic acid protein complexes and
is tightly regulated by mitogenic signal transduction pathways.4
Translation initiation is regarded as the rate-limiting step in
new protein synthesis. Initiation proceeds through the
recognition of methyl-7-guanosine (m7GpppN or cap) at the
5′ end of all nuclear encoded mRNAs by the cap binding
protein eIF4E. Once formed, the eIF4E/mRNA complex then
binds to the scaffolding protein eIF4G and the helicase eIF4A
forming the eIF4F complex, which is then recruited to
ribosomal subunits where translation ensues.5
eIF4E is overexpressed in a wide variety of malignant cell
lines and primary human tumors such as carcinomas of the
breast,6 colon,7 and head and neck,8 non-Hodgkin’s lympho-
mas,9 and chronic and acute M4/M5 myelogenous leukemias.10
mRNA transcripts that are most profoundly influenced by
activation of eIF4E typically include growth promoting factors
and proto-oncogenes such as cMyc, Ornithine decarboxylase,
Cyclin D1, and VEGF.11−15 These mRNAs are characterized by
Received: January 11, 2012
Published: March 29, 2012
© 2012 American Chemical Society
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Figure 1. m7-GTP binding to eIF4E (PDB code: 1IPC). (a) Ribbon representation of the m7-GTP binding in the cap-binding slot of eIF4E with
selected residues shown as sticks. Both eIF4E and m7-GTP are colored by element with carbon atoms in beige in the protein and green in m7-GTP,
oxygen atoms in red, nitrogen atoms in blue, and phosphorus atoms in orange. (b) Molecular surface representation of pockets I, II, and III in the
cap-binding site: (I) pronounced lipophilic pocket adjacent and perpendicular to the guanosine moiety formed by residues Phe48, Trp56, Leu60,
Pro100, Ser92, and Asp90; (II) small pocket formed by residues Arg112, Leu114, Val153, Asn155, Ala164, and Trp166; (III) phosphate binding
pocket.
As part of our efforts aimed at developing small molecule
inhibitors of eIF4E, a high-throughput screen (HTS) of our in-
house small molecule library was conducted. However, no bona
fide leads were identified from these screening efforts. Given
the attractiveness of the target and the availability of cocrystal
structures of eIF4E in complex with the cap analogues m7-
GDP, m7-GTP, and m7-GpppA as well as with m7-GpppA and
the 4E-BP1 fragment,19−23 we embarked on a structure based
design approach to identify novel small molecule eIF4E
inhibitors.
Analogues of the mRNA cap have been useful in studying
eIF4E function and cap-dependent translation. Previously,
several N7-substituted guanosine nucleoside and nucleotide
analogues were evaluated for their ability to inhibit eIF4E
binding to 7-methyl GTP.24−28 Further analysis of the eIF4E
cocrystal structures with cap analogues revealed that a unique
hydrophobic pocket presents itself adjacent to the guanosine
moiety. Our strategy was to first optimize the N7 position of
m7-GMP directed toward binding in this pocket and
subsequently investigate the other parts of these N7-alkyl
GMP analogues. We successfully discovered a series of 7-alkyl
guanosine nucleotide analogues as well as 7-alkyl guanosine
phosphonic acid analogues which demonstrated high binding
affinity for eIF4E. This report describes the structured-guided
medicinal chemistry efforts toward these novel eIF4E cap
binding antagonists as well as the cocrystal structures of these
compounds in complex with eIF4E.
backbone amide of Trp102. N1 and N2 are also engaged in
hydrogen bond contacts with Glu103. It appears that structural
changes in these regions would not be favorable, as it would
disrupt these key H-bond interactions. Furthermore, the
phosphate moiety also makes a large contribution to the
binding affinity of m7-GTP to the eIF4E protein. The
triphosphate moiety forms an extensive hydrogen-bonding
network through water molecules with Arg112, Arg157, and
Lys162. The ki of m7-GTP against eIF4E was 28 nM,
determined in the protein binding assay. Compared to the
case of m7-GTP, m7-GDP and m7-GMP’s binding affinities
against eIF4E were reduced by 39- and 407-fold, respectively.
While both the m7-guanosine and phosphate moieties make
important contributions for the binding of the cap analogues to
eIF4E, the ribose ring is positioned at the entrance of the cap-
binding side. The 3- and 4-hydroxy moieties on the ribose ring
point toward the solvent and do not participate in any specific
interactions with the protein. While analyzing the interactions
of m7-GTP analogues with eIF4E, we were intrigued by the
pronounced lipophilic pocket adjacent and perpendicular to the
guanosine moiety formed by Phe48, Leu60, Pro100, Ser92, and
Asp90 (pocket I, Figure 1b). We envisioned targeting this
pocket to increase the binding affinity of N7-modified GMP
analogues, thus compensating for the loss from β- and γ-
phosphate groups in regard to m7-GTP.
A number of analogues were synthesized by alkylation of
guanine monophosphate, as shown in Scheme 1. The SAR
results are summarized in Table 1.
In agreement with previous reports, the N7-benzyl analogue
2 showed similar binding affinity as m7-GMP 1. We were
delighted to find that when guanine monophosphate was
alkylated with a Cl-phenylpropyl group, a derivative with
RESULTS AND DISCUSSION
■
Various cocrystal structures of human eIF4E in complex with
cap analogues, including m7-GDP, m7-GTP, and m7-GpppA as
well as the ternary complex of eIF4E with m7-GpppA in the
presence of the 4E-BP1 fragment, have been reported.20−23 All
of the structures reveal that these cap analogues bind in a
narrow cap-binding slot on the protein’s concave surface. The
delocalized positively charged 7-methyl guanosine ring forms
cation-π interactions with Trp102 and Trp56 through being
sandwiched between these two conserved tryptophans (Figure
1a). Here, the delocalized positive charge makes a large
contribution to their binding affinity to eIF4E. The binding
potency of GpppG compared with m7-GpppG is 276-fold
lower against eIF4E. In addition, the carbonyl group of the
guanine engages in a hydrogen bond interaction with the
Scheme 1. General Synthesis of N7-Modified GMP
Analogues
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Table 1. SAR of N7-Substituted GMP Analogues
Figure 2. Cocrystal structure of eIF4E in complex with compound 9. (a) Binding mode of compound 9 in the cap-binding slot in eIF4E. The protein
is shown as a molecular surface representation. The selected residues involved in interactions with 9 are shown as sticks. The color code is the same
as that in Figure 1, except that the carbon atoms in compound 9 are colored in magenta. (b) Overlay of the cocrystal structures of compound 9 and
m7-GTP (PDB code: 1IPC) with eIF4E.
improved binding potency for eIF4E was obtained (7 vs 1).
Encouraged by this result we further probed the binding
pocket. Interestingly and quite unexpectedly, the Cl-phenox-
yethyl derivative 9 demonstrated significantly improved binding
potency for eIF4E, with an IC50 of ∼60 nM. This constitutes a
remarkable 200-fold increase in potency over m7-GMP and an
almost 100-fold increase over that of the unsubstituted
phenoxyethyl derivative 8. Similarly enhanced binding affinities
were observed for the 4-cyano and 4-bromo phenoxyethyl
derivatives 10 and 12, while the fluoro- and trifluorometyl
derivatives 11 and 13 only showed a moderate 20−30-fold
increase in affinity over the unsubstituted phenoxyethyl
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a
Scheme 2. General Synthesis of Deazaguanine Analogues
a
Conditions: (a) NaH, 4-Cl-PhOCH2CH2Br (for 24) or 4-BrPhOCH2CH2Br (for 25), MeCN, 55°C; (b) aq 1 M NaOH, 1,4-dioxane, reflux; (c) 7
N NH3 in MeOH, 170 °C; (d) SnCl4, β-D-ribofuranose 1-acetate 2,3,5-tribenzoate, MeNO2, 60°C; (e) NaOMe, MeOH, 23°C; (f) POCl3,
PO(MeO)3; 0 °C.
Table 2. eIF4E Binding Affinity Comparison of N7-Modified Guanosine 5-Monophosphate vs N9-Modified Deazaguanine 5-
Monophosphate Analogues
compound 8. More importantly, compounds 9, 10, and 12
were about 30-fold more effective inhibitors of cap dependent
mRNA translation in a rabbit reticular lysate in vitro translation
assay (RRL-IVT) than m7-GMP .
phenoxyethyl linker. The rest of the molecule adopts a binding
mode similar to that of m7-GTP (Figure 2b). Overall, there are
no significant movements within the protein upon the binding
of the Cl-phenoxy ethyl group. We noticed that the side chain
of Ser92 adopts an alternative conformation to accommodate
the binding of the Cl-atom. In addition, the side chains of
Asp90, Trp56, and Met101 also shift to accommodate the
phenyl group. The Glu103 side chain shows some shift as well
but still maintains good H-bond interactions with the amino-
guanosine moiety. Subsequent to our work, Brown et al.
reported a similar effort on generating guanosine derivatives for
targeting eIF4E activity.29 While our GMP analogues target the
pocket I behind Trp56 (Figure 2), both their 7-benzyl-GMP
(Bn7GMP) and 7-(p-fluorobenzyl)-GMP (Fbn7GMP) com-
The cocrystal structure of eIF4E complexed with 9 was
determined to a resolution of 2.7 Å. The structure revealed that
9 maintains the same binding mode as that of m7-GTP in the
cap-binding slot with the p-chlorophenoxy ethyl group fitting
pocket I almost perfectly, in both shape and size (Figure 2a).
The chlorophenyl moiety makes numerous favorable van der
Waals interactions with residues including Val153, Asp90,
Phe48, Leu60, and Pro100 lining pocket I. The Cl atom is in
close van der Waals contact with Phe48, Leu60, and Ser92, but
there are no specific interactions with the oxygen atom of the
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pounds make an induced fit to pocket III around Trp102
(Figure 1).
26 (Table 3) had little or no effect on its binding affinity for
eIF4E, consistent with previous observations.21
Apparently, the para-Cl atom prevents the phenyl ring from
sampling other smaller pockets, further stabilizing the ligand
binding. It was found that the chlorine atom can also be
replaced by a bromo or a cyano group while retaining the
binding potency. Disubstitution of the aryl ring was not
tolerated. The chlorophenoxy ethyl group seems to fit the
binding pocket very well. Simple replacement of the linker
oxygen atom with a carbon atom resulted in a 100-fold loss of
activity in the SPA assay. Thermodynamic analysis indicated
that there is a 0.5 kcal/mol energy difference between the
global energy minimum of compound 9 in water and its bound
conformation.30 However, for compound 7 there is a 3.0 kcal/
mol difference between its global energy minimum in water and
the bound conformation, indicating a larger penalty upon
binding for this compound. This may explain the potency
difference between compounds 7 and 9.
Table 3. eIF4E Binding Affinity Data for N7-Modified
Guanosine 5-Monophosphate Analogues
The eIF4E binding potency relies to a significant extent on
the delocalized positive charge of the guanine moiety, mainly
due to favorable cation-π interactions with Trp56 and Trp102
and a decrease in overall negative molecular charge, which likely
reduces the desolvation penalty upon binding. However, a
highly charged molecule is generally incompatible with cell
permeability. After establishing the ideal side chain at the N7
position, we moved on to investigate 9-deazaguanosine
analogues to possibly eliminate the positive charge from the
guanosine ring. The 9-deazaguanosine nucleoside was synthe-
sized according to literature procedures shown in Scheme 2.31
2,4-Dichloro-5H-pyrrolo[3,2-d]pyrimidine 17 was alkylated
with 1-(2-bromoethoxy)-4-chlorobenzene for compound 24
or 1-(2-bromoethoxy)-4-bromobenzene for compound 25 to
yield ideal side-chain substituted pyrrolopyrimidine 18. The
dichloro group of 2,4-dichloropyrrolopyrimidin 18 was
selectively hydrolyzed by treatment with NaOH to furnish
compound 19, which was then animated at the 2 position of 20.
The ribose moiety 21 was installed by a SnCl4 catalyzed
glycoside formation to give compound 22. The benzoyl
protecting groups of 22 were removed using NaOMe to give
23, the 5-hydroxymethyl moiety of which was then
phosphorylated to give compound 24. Compound 25 was
synthesized similarly.
It was found that the 9-deazaguanosine monophosphate 24
had 100-fold reduced binding affinity against eIF4E as
compared to the 7-alkylated guanosine analogue 9 (Table 2).
This was also true for the corresponding p-bromophenoxy ethyl
derivatives 10 and 25. The affinity differences between the 9-
deazaguarnine and 7-methylguanine were consistent with those
previously reported for GpppG compared with m7-GpppG,
which was 276-fold weaker against eIF4E.18 This SAR trend
clearly indicated the importance of the positive charge for the
formation of the cation-π interactions with Trp102 and Trp56.
However, the 9-deazaguanosine monophosphates are still in the
same affinity range as m7-GMP, validating our predictions that
neutral analogues with appropriate features might be designed
to replace the positively charged guanosine core.25
Inspection of the three-dimensional structure of the complex
of 9 bound to eIF4E revealed that the ribose ring only provided
a conformationally restricted connection linking the phosphate
group with the guanine ring in a “folded-back” orientation. As
previously noted, the two hydroxyl groups on the 3- and 4-
positions of the ribose ring extend into the solvent accessible
region without being engaged in any specific interaction with
the eIF4E protein. The phosphate moiety on the 5′-position of
the ribose was positioned approximately 4.2 Å away from the 8
position of the guanine (Figure 3). We reasoned that it should
be possible to use an alternate linker extending from the 8-
position of the guanine as a means of connecting the phosphate
group with the guanine ring. Because deletion of the ribose
would also decrease hydrophilicity, this modification might be
anticipated to improve membrane permeability in those
compounds. We sought to substitute the phosphate group
with potential bioisosteric equivalents, such as a phosphonic
acid or a carboxylic acid. Such groups would ideally be placed in
the optimal position to interact with the residues Arg112,
Arg157, and Lys162.
The designed compound 33 was synthesized as illustrated in
Scheme 3. Since it had previously been found by us as well as
others24 that 2-methyl amine GMP analogues were about 2-fold
more potent than 2-amino GMP analogues (data not shown),
the final analogues were designed bearing a 2-methylamino
group. The diamino compound 27 was synthesized via the
published procedure.32,33 The more basic amino group of the
diamino compound 27 was first acetylated with 2-(4-
chlorophenoxy)acetyl chloride to furnish compound 28,
which was subjected to borane reduction to generate the
secondary amine 29. The secondary amine 29 was then
coupled with 4-bromobenzoic acid to furnish compound 30.
The ring closure was attempted with trimethylsilyl poly-
phosphanic acid treatment. However, the reaction only gave
20% yield of cyclization product 31. We found that the ring
closure can be accomplished more efficiently by treatment with
t-BuONa to give 31 in 52% yield. . Palladium mediated reaction
of 31 smoothly transferred the bromo group of 31 to a diethyl
phosphonate 32. The removal of one or both ethyl groups of
Next, we turned our attention toward designing analogues
that would probe the role of the ribose moiety. The ribose ring
is positioned at the entrance of the cap-binding site without
participating in any specific interactions with the protein.
Notably, the 2′- and 3′-hydroxyl groups of the ribose ring
extend into a solvent accessible region. Indeed, we found that
the incorporation of an isopropylidene group as in compound
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Figure 3. (a) Distance of phosphate group in relation to the 8 position of guanine in compound 9 bound to eIF4E; (b) possible design of 8-
substituted N7-(chlorophenoxyethyl)guanine analogues. L: linker. FG: COOH, PO(OH)2, SO2NH2, etc.
32 was accomplished by stirring the material with TMSBr in
CH2Cl2 to yield final products 33 and 34.
tolerated. Indeed, compound 39 was found to have similar
affinity to compound 33. Furthermore, the difluoro analogue
42 was 4-fold more active than the phosphonic acid 33. While it
is possible that the fluoro atoms are picking up additional
hydrogen bonding interactions, it is more likely that these
atoms simply increase the acidity of the phosphonic acid, thus
strengthening its salt bridge interaction with Arg157 and
Lys162. Discouragingly, the less charged carboxylic acid
analogue 46 showed much reduced activity when compared
with compound 33.
Compound 33 is remarkably selective for eIF4E, with no
significant off-target activities found at concentrations up to 10
μM in Upstate kinase panel and CEREP receptor panel screens.
Unfortunately, however, compound 33 had compromised cell
permeability with a Paap of 1.1 × 10−6 cm/s (Table 4) in the
PAMPA assay.36 This is very likely due to the large polar
surface area (PSA) contributed by the phosphonic acid moiety
(calculated PSA for 33: 142 Å2) and its dianionic nature at
physiological pH. This was confirmed by the diethyl
phosphonate analogue 32, which was significantly more
permeable. Among the analogues, only the monoethyl
phosphonate 34 and the carboxylic acid analogue 46 showed
some affinity for eIF4E, as measured in the SPA assay (1.4 and
3.14 μM, respectively). Just like the mono phosphonic acid
analogue 34, the carboxylic acid analogue 46 showed limited
cellular permeability.
We determined the binding mode of compound 33 with an
X-ray cocrystal structure at a resolution of 2.9 Å. This revealed
that 33 adopts a binding mode similar to that of 9 (Figure 4a).
While preserving the same H-bond interactions to the
backbone amide of Trp120 and to the side chains of Glu103,
the plane of the guanosine core is skewed about 10° in
comparison with that of compound 9 (Figure 4b). The Cl-
phenyl side-chain still occupies the binding pocket I very well,
forming numerous favorable van der Waals interactions with
the protein. The 8-phenyl moiety occupies the space between
the guanosine and α-phosphate of the m7-GTP positions,
which is less solvent exposed when compared to the ribose ring.
Similar to the ribose, the phenyl group makes no specific
interactions with eIF4E. The phosphonic acid moiety is
positioned between the α- and β- phosphate groups of m7-
GTP and forms direct H-bond interactions with Arg157 and
Lys162. Due to the limitation of the diffraction resolution, very
few water molecules were observed in the cocrystal structure.
Thus, the water mediated H-bonds with Arg112 are not seen
here. Overall, there are no significant conformational changes in
the protein except for a slight shift of the side chain of Glu103,
whose movement seems to be in concert with the guanosine
ring rotation.
With the discovery of the phosphonic acid compound 33, we
carried out more detailed SAR studies of its analogues. The
homologated phosphonic acid 39 was prepared starting from
compound 35.34 The more basic amine was alkylated with 2-(4-
chlorophenoxy)acetaldehyde through a reductive amination
reaction to give compound 36, which was then coupled with
diethyl 4-(chlorocarbonyl)benzylphosphonate to generate
compound 37. The ring closure was achieved by heating
compound 37 at reflux in 40% NaOMe in MeOH. The
phosphonate ester was partially hydrolyzed to monomethyl
ester 38 during the reaction. Deesterification was completed by
treatment with TMSBr to furnish compound 39. The difluoro
substituted phosphonic acid 42 was similarly generated after
acylation with 4-((diethoxyphosphoryl)difluoromethyl)benzoic
acid35 to give compound 40. The carboxylic acid analogue 46
was generated by the same reaction sequence using methyl 4-
(chlorocarbonyl)benzoate.
CONCLUSION
■
In summary, we designed and synthesized a novel series of high
affinity and selective eIF4E inhibitors starting from m7-GMP.
Attaching a 4-phenylphosphonic acid moiety to the 8-position
of the guanine ring gave a potent eIF4E inhibitor eliminating
the ribose moiety, phosphate group, and positive charge. The
presence of the ribose moiety was shown to be unnecessary for
maintaining high levels of inhibitory activity. The biochemical
activity of compound 33 is 95 nM for eIF4E in an SPA binding
assay. More importantly, the compound has an IC50 of 2.5 μM
for inhibiting cap-dependent mRNA translation in a rabbit
reticular cell extract assay (RRL-IVT). Unfortunately, the
compound displayed limited cellular permeability hindering its
further evaluation in cellular assays. Further optimization or the
potential use of prodrug strategies might be useful in increasing
the cellular permeability of this series to achieve cellular activity
and enable future testing in vivo. This series of potent,
truncated analogues could serve as a promising new starting
On the basis of the structural observation that the
phosphonic acid moiety of compound 33 is situated between
the α- and β-phosphates of m7-GTP, we envisioned that
extending the phosphonic acid out by one carbon should be
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a
Scheme 3. Synthesis of 8-Substituted N7-(Chlorophenoxyethyl) Guanine Analogues
a
Conditions: (a) 2-(4-chlorophenoxy)acetyl chloride, K2CO3, CH2Cl2, 66% yield; (b) BH3, THF, 47% yield; (c) 4-bromobenzoic acid, HOBT,
DMAP, EDC, DMF, 80% yield; (d) t-BuONa in i-PrOH, reflux, 52% yield; (e) Pd(OAc)2, dicyclohexylmethylamine, diethyl phosphonate, 91%
yield; (f) TMSBr, CH2Cl2, 42% yield for 33 and 92% yield for 34; (g) NaBH(OAc)3, 2-(4-chlorophenoxy)acetaldehyde, CH2Cl2, 32% yield; (h)
Hunig’s base, DMAP, diethyl 4-(chlorocarbonyl)benzylphosphonate, CH2Cl2; (i) 40% NaOMe/MeOH, 90° C, 15−66% yield; (j) TMSBr, DMF,
4.5% yield; (k) 40% MeNH2, 160 °C, microwave, 25−68% yield; (l) Et3N, HATU, 4-((diethoxyphosphoryl)difluoromethyl)benzoic acid; (m)
Hunig’s base, DMAP, methyl 4-(chlorocarbonyl)benzoate; (n) LiOH, MeOH, 100 °C, 74% yield.
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Figure 4. Binding of phosphonic acid analogue 33 in eIF4E. (a) Compound 33 in the cap-binding site of eIF4E. The protein is shown in molecular
surface representation with selected residues involved in ligand recognition shown as sticks. The color scheme is the same as that in Figure 1 except
that carbon atoms are colored in cyan. (b) Overlay of the cocrystal structures of compound 33 (in cyan) and 9 (in magenta) with eIF4E.
A: Analytical Agilent Eclipse Plus C18 5 μm column, 4.6 mm × 150
mm, gradient elution of 10% MeCN in water to 100% MeCN in water
over a 12 min period, where the CH3CN and water contains 0.1%
TFA. HPLC method B: Analytical Chromolith SpeedRod RP18
column (Merck, Darmstadt, Germany), 4.6 mm × 150 mm, gradient
elution of 0% MeCN in water to 20% MeCN in water over a 4 min
period, where the CH3CN and water contains 0.1% TFA. HPLC
method C: CAPCELL PAK C18, 4.6 mm × 75 mm, 3 μm column
(Shiseido, Tokyo, Japan), gradient elution of 0% MeCN in water to
50% MeCN in water over a 4 min period, where the CH3CN and
water contains 0.1% TFA. HPLC method D: Analytical Agilent Eclipse
Plus C18 5 μm column, 4.6 mm × 150 mm, gradient elution of 10%
MeCN in water to 100% MeCN in water over a 7.5 min period, where
the CH3CN and water contains 0.1% TFA. HPLC method E:
CAPCELL PAK C18, 4.6 mm × 75 mm, 3 μm column (Shiseido,
Tokyo, Japan), gradient elution of 0% MeCN in water to 100% MeCN
in water over a 15 min period, where the CH3CN and water contains
0.1% TFA. HPLC method F: Agilent SB-C8 column, gradient elution
of 0% MeCN in water to 50% MeCN in water over a 1 min period,
where the CH3CN and water contains 0.1% TFA.
Table 4. eIF4E Binding Affinity Data for 8-Substituted N7-
(Chlorophenoxyethyl) Guanine Analogues
Synthetic Procedures and Characterization for Com-
pounds. 7-(Benzyl)guanosine-5′-monophosphate (2). To a
slurry of guanosine-5′-monophosphate disodium salt hydrate
(401 mg, 0.98 mmol, 1 equiv) in DMSO (5 mL) was added
benzyl bromide (600 μL, 4.94 mmol, 5 equiv). The resulting
white slurry was stirred at room temperature for 27 h; during
this time, all solids dissolved. The reaction mixture was purified
directly by reverse phase prep HPLC (Prep C18 OBD 10 μm
column (Waters, Milford, MA), gradient elution of pure water
to 15% MeCN in water over a 30 min period, where both
solvents contain 0.1% TFA). After discarding mixed fractions,
61 mg (11%, 0.11 mmol) of the trifluoroactetate salt of 2 was
1
isolated as a white solid: H NMR (400 MHz, DMSO-d6): δ
9.85 (s, 1 H), 7.52−7.58 (m, 2 H), 7.31−7.44 (m, 3 H), 5.85
(d, J = 3.3 Hz, 1 H), 5.58 (s, 2 H), 4.42−4.48 (m, 1 H), 4.11−
4.23 (m, 3 H), 3.95−4.03 (m, 1 H); MS (LRES in negative
mode) calcd for C17H19N5O8P [M − H]− 452.1, found 452.0;
HPLC method A tR = 3.25 min, purity 98%.
point toward the design of neutral eIF4E inhibitors with
physicochemical properties suitable for cellular activity.
7-(3-Chlorobenzyl)guanosine-5′-monophosphate (3). To a slurry
of guanosine-5′-monophosphate disodium salt hydrate (302 mg, 0.74
mmol, 1 equiv) in DMSO (3.6 mL) was added 3-chlorobenzyl
bromide (350 μL, 2.59 mmol, 3.5 equiv). The resulting white slurry
was stirred at room temperature for 22 h; during this time all solids
dissolved. The reaction mixture was purified directly by reverse phase
prep HPLC (Prep C18 OBD 10 μm column (Waters, Milford, MA),
gradient elution of 5% MeCN in water to 30% MeCN in water over a
30 min period, where both solvents contain 0.1% TFA). After
discarding mixed fractions, 61 mg (14%, 0.10 mmol) of the
trifluoroactetate salt of 3 was isolated as a white solid: 1H NMR
(400 MHz, DMSO-d6): δ 9.70 (s, 1 H), 7.62 (s, 1 H), 7.46−7.51 (m, 1
H), 7.39−7.44 (m, 2 H), 5.85 (d, J = 3.1 Hz, 1 H), 5.57 (s, 2 H),
EXPERIMENTAL SECTION
■
General Experimental. Reagents and solvents used below were
obtained from commercial sources and when required were purified.
1H NMR spectra were obtained on a Varian Gemini 400 or 500 MHz
NMR spectrometer. Electron ionization (EI) mass spectra were
recorded on a Hewlett-Packard 5989A mass spectrometer. Electro-
spray ionization (ESI) mass spectrometry analysis was performed
using a Hewlett-Packard 1100 MSD electrospray mass spectrometer
using the HP1 100 HPLC for sample delivery. Combustion analyses
were performed by Atlantic Microlab Inc. (Norcross, GA). Air and/or
moisture sensitive reactions were carried out under N2 using flame-
dried glassware and standard syringe/septa techniques. HPLC method
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4.43−4.49 (m, 1 H), 4.12−4.23 (m, 3 H), 3.98−4.07 (m, 1 H); MS
(LRES in negative mode) calcd for C17H18ClN5O8P [M − H]− 486.1,
found 486.1; HRMS: theoretical 490.0889, observed 490.0886; HPLC
method A tR = 4.13 min, purity 98%.
theoretical 516.1046, observed 516.1052; HPLC method C, tR = 3.09
min, purity 98%.
7-(Phenoxyethyl)guanosine-5′-monophosphate (8). To a slurry
of guanosine-5′-monophosphate disodium salt hydrate (500 mg, 1.20
mmol, 1 equiv) in DMSO (12 mL) was added phenyl-2-bromoethyl
ether (1.20 g, 6.0 mmol, 5 equiv). The resulting white slurry was
stirred at room temperature for 3 days and then heated to 50 °C for an
additional 21 h; during the heating period, all solids dissolved. A
portion of the reaction mixture was purified directly by reverse phase
prep HPLC (Prep C18 OBD 10 μm column (Waters, Milford, MA),
gradient elution of 2% MeCN in water to 20% MeCN in water over a
30 min period, where both solvents contain 0.1% TFA). After
discarding mixed fractions, 3.2 mg (0.4%, 0.005 mmol) of the
7-(2-Chlorobenzyl)guanosine-5′-monophosphate (4). To a slurry
of guanosine-5′-monophosphate disodium salt hydrate (521 mg, 1280
μmol, 1 equiv) in DMSO (6 mL) was added 2-chlorobenzyl bromide
(498 μL, 3839 μmol, 3 equiv). The resulting white slurry was stirred at
room temperature for 2 days; during this time all solids dissolved. The
reaction mixture was filtered and purified directly by reverse phase
prep HPLC (Prep C18 OBD 10 μm column (Waters, Milford, MA),
gradient elution of 0 to 30% MeCN in water over a 30 min period,
where both solvents contain 0.1% TFA). After discarding mixed
fractions, 110 mg (18%, 0.23 mmol) of the trifluoroactetate salt of 4
1
trifluoroactetate salt of 8 was isolated as a white solid: H NMR (400
1
was isolated as a white solid: H NMR (400 MHz, DMSO-d6) δ ppm
MHz, DMSO-d6): δ 9.55 (s, 1 H), 7.23−7.31 (m, 2 H), 6.88−6.96 (m,
3 H), 5.89 (d, J = 3.9 Hz, 1 H), 5.70−5.79 (m, 1 H), 4.73−4.81 (m, 2
H), 4.44−4.51 (m, 1 H), 4.42 (t, J = 5.3 Hz, 2 H), 4.13−4.21 (m, 3
H), 3.99−4.07 (m, 2 H); MS (LRES in negative mode) calcd for
C18H21N5O9P [M − H]− 482.1, found 482.0; HRMS: theoretical
486.1384, observed 486.1387; HPLC method A, tR = 3.96 min, purity
93%.
9.65 (s, 1 H), 7.52 (dd, J = 8.0, 1.37 Hz, 1 H), 7.38 (dt, J = 7.6, 1.96
Hz, 1 H), 7.31 (dt, J = 7.6, 1.17 Hz, 1 H), 7.22 (dd, J = 7.8, 1.56 Hz, 1
H), 5.89 (d, J = 3.1 Hz, 1 H), 5.70 (s, 2 H), 4.49−4.58 (m, 1 H), 4.21
(t, J = 5.1 Hz, 1 H), 4.09−4.18 (m, 2 H), 3.92−4.05 (m, 1 H); MS
(LRES in negative mode) calcd for C17H18ClN5O8P [M − H]− 486.1,
found 486.1; HRMS: theoretical 490.0889, observed 490.0892, HPLC
method B tR = 2.11 min, purity 100%.
7-(4-Chlorobenzyl)guanosine-5′-monophosphate (5). To a slurry
of guanosine-5′-monophosphate disodium salt hydrate (250 mg, 0.61
mmol, 1 equiv) in DMSO (3.1 mL) was added 4-chlorobenzyl
bromide (625 mg, 2.95 mmol, 4.8 equiv). The resulting white slurry
was stirred at room temperature for 22 h; during this time all solids
dissolved. The reaction mixture was purified directly by reverse phase
prep HPLC (Prep C18 OBD 10 μm column (Waters, Milford, MA),
gradient elution of 2% MeCN in water to 20% MeCN in water over a
30 min period, where both solvents contain 0.1% TFA). After
discarding mixed fractions, 139 mg (38%, 0.23 mmol) of the
trifluoroactetate salt of 5 was isolated as a white solid: 1H NMR
(400 MHz, DMSO-d6): δ 9.94 (s, 1 H), 7.58 (d, J = 8.4 Hz, 2 H), 7.39
(d, J = 8.6 Hz, 2 H), 5.84 (d, J = 3.1 Hz, 1 H), 5.57 (s, 2 H), 4.41−4.47
(m, 1 H), 4.10−4.24 (m, 3 H), 3.93−4.01 (m, 1 H); MS (LRES in
negative mode) calcd for C17H18ClN5O8P [M − H]− 486.1, found
486.1; HRMS: theoretical 490.0889, observed 490.0893; HPLC
method A, tR = 4.21 min, purity 97%.
7-(4-Chlorophenethyl)guanosine-5′-monophosphate (6). To a
slurry of guanosine-5′-monophosphate disodium salt hydrate (299 mg,
0.73 mmol, 1 equiv) in DMSO (4.5 mL) was added 4-chlorophenethyl
bromide (550 μL, 3.67 mmol, 5.0 equiv). The resulting white slurry
was heated at 50 °C for 26 h; during this time all solids dissolved. The
reaction mixture was purified directly by reverse phase prep HPLC
(Prep C18 OBD 10 μm column (Waters, Milford, MA), gradient
elution of 5% MeCN in water to 30% MeCN in water over a 30 min
period, where both solvents contain 0.1% TFA). Heavily mixed
fractions were discarded, and the remaining impure product was
purified again by reverse phase prep HPLC (Prep C18 OBD 10 μm
column (Waters, Milford, MA), gradient elution of 10% MeCN in
water to 25% MeCN in water over a 30 min period, where both
solvents contain 0.1% TFA). After discarding mixed fractions, 3.2 mg
(1%, 0.005 mmol) of the trifluoroactetate salt of 6 was isolated as a
white solid: 1H NMR (400 MHz, DMSO-d6): δ 9.94 (s, 1 H), 7.58 (d,
J = 8.4 Hz, 2 H), 7.39 (d, J = 8.6 Hz, 2 H), 5.84 (d, J = 3.1 Hz, 1 H),
5.57 (s, 2 H), 4.41−4.47 (m, 1 H), 4.10−4.24 (m, 3 H), 3.93−4.01
(m, 1 H); MS (LRES in negative mode) calcd for C17H18ClN5O8P [M
− H]− 486.1, found 486.1; HPLC method A, tR = 4.43 min, purity
90%.
7-(4-(Chlorophenyl)propyl)guanosine-5′-monophosphate (7).
The compound was prepared according to the same procedure used
in preparing compound 2. Purification by reverse-phase preparative
HPLC (SB C8, 30 mm × 250 mm, 5 μm column; Agilent, Santa Clara,
CA) (eluent: 0.1% TFA in acetonitrile/water, gradient 2% to 30% over
25 min) to provide the trifluoroactetate salt of 7 as a white solid (79.3
mg, 13% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 9.56 (1 H, s),
7.24−7.28 (2 H, m), 7.17−7.22 (2 H, m), 5.81 (1 H, d, J = 3.1 Hz),
4.36−4.44 (3 H, m), 4.11−4.22 (3 H, m), 3.99 (1 H, m), 2.61− 2.68
(2 H, m), 2.18−2.22 (2 H, m); MS (ESI) 514.2 [M − 2H]−; HRMS:
7-(4-Chlorophenoxyethyl)guanosine-5′-monophosphate (9). To
a slurry of guanosine-5′-monophosphate disodium salt hydrate (380
mg, 0.93 mmol, 1 equiv) in DMSO (4.3 mL) was added 4-
chlorophenyl-2-bromoethyl ether (879 mg, 3.73 mmol, 4 equiv). The
resulting white slurry was heated 55 °C for 3.5 days; during this time
all solids dissolved. The reaction mixture was purified directly by
reverse phase prep HPLC (Prep C18 OBD 10 μm column (Waters,
Milford, MA), gradient elution of 10% MeCN in water to 30% MeCN
in water over a 30 min period, where both solvents contain 0.1%
TFA). After discarding mixed fractions, 23.1 mg (4%, 0.037 mmol) of
1
the trifluoroactetate salt of 9 was isolated as a white solid: H NMR
(400 MHz, DMSO-d6): δ 9.65 (s, 1 H), 7.47 (br s, 1 H), 7.26 (d, J =
8.6 Hz, 2 H), 6.93 (d, J = 9.0 Hz, 2 H), 5.86 (d, J = 3.9 Hz, 1 H), 4.77
(br s, 2 H), 4.47 (t, J = 3.9 Hz, 1 H), 4.40 (t, J = 5.1 Hz, 2 H), 4.20 (t, J
= 4.7 Hz, 1 H), 4.09−4.18 (m, 2 H), 3.95−4.05 (m, 2 H); 13C NMR
(125 MHz, DMSO-d6): δ 157.2, 156.5, 154.1, 150.0, 137.4, 129.9,
125.5, 117.0, 107.6, 89.9, 84.5, 74.7, 70.1, 66.1, 64.5, 48.8; MS (HRES
in negative mode) calcd for C18H20ClN5O9P [M − H]− 516.1, found
516.1. HRMS: theoretical 516.0693, observed 516.0695; HPLC
method A, tR = 3.88 min, purity 95%.
7-(4-Bromophenoxyethyl)guanosine-5′-monophosphate (10).
To a slurry of guanosine-5′-monophosphate disodium salt hydrate
(329 mg, 0.81 mmol, 1 equiv) in DMSO (4.3 mL) was added 4-
bromophenyl-2-bromoethyl ether (816 mg, 2.91 mmol, 3.6 equiv).
The resulting white slurry was heated 55 °C for 4.5 days; during this
time all solids dissolved. The reaction mixture was purified directly by
reverse phase prep HPLC (Prep C18 OBD 10 μm column (Waters,
Milford, MA), gradient elution of 10% MeCN in water to 30% MeCN
in water over a 30 min period, where both solvents contain 0.1%
TFA). After discarding mixed fractions, 13.7 mg (2.5%, 0.02 mmol) of
1
the trifluoroactetate salt of 10 was isolated as a white solid: H NMR
(400 MHz, DMSO-d6): δ 9.76 (s, 1 H), 7.42 (d, J = 8.8 Hz, 2 H), 6.92
(d, J = 8.9 Hz, 2 H), 5.87 (d, J = 3.7 Hz, 1 H), 4.74−4.82 (m, 2 H),
4.48 (t, J = 4.1 Hz, 1 H), 4.38−4.45 (m, 2 H), 4.20 (t, J = 4.6 Hz, 1 H),
4.09−4.17 (m, 2 H), 3.93−4.04 (m, 2 H); MS (LRES in negative
mode) calcd for C18H20BrN5O9P [M − H]− 560.0, found 560.0;
HRMS: theoretical 562.0333, observed 562.0323; HPLC method A, tR
= 4.64 min, purity 96%.
7-(4-Fluorophenoxyethyl)guanosine-5′-monophosphate (11). To
a slurry of guanosine-5′-monophosphate disodium salt hydrate (331
mg, 0.813 mmol, 1 equiv) in DMSO (4.3 mL) was added 1-(2-
bromoethoxy)-4-fluorobenzene (641 mg, 2.93 mmol, 3.6 equiv). The
resulting white slurry was heated at 55 °C for 3 days; during this time
all solids dissolved and the reaction turned yellow. The reaction
mixture was purified directly by reverse phase prep HPLC (Prep C18
OBD 10 μm column (Waters, Milford, MA), gradient elution of 10 to
30% MeCN in water over a 30 min period, where both solvents
contain 0.1% TFA). Heavily mixed fractions were discarded, and the
remaining impure product was purified again by reverse phase prep
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HPLC (Prep C18 OBD 10 μm column (Waters, Milford, MA),
gradient elution of 2% MeCN in water to 20% MeCN in water over a
30 min period, where both solvents contain 0.1% TFA). After
discarding mixed fractions, 2 mg (0.4%, 0.003 mmol) of the
hydrate (551 mg, 1.35 mmol, 1 equiv) in DMSO (4.5 mL) was added
2-chloro-N-(4-chlorophenyl)acetamide (828 mg, 4.06 mmol). The
resulting white slurry was stirred at 55 °C for 2 days; during this time
most of the solids dissolved. The reaction mixture was filtered and
purified directly by reverse phase prep HPLC (Prep C18 OBD 10 μm
column (Waters, Milford, MA), gradient elution of 17 to 27% MeCN
in water over a 30 min period, where both solvents contain 0.1%
TFA). After discarding mixed fractions, 5 mg (0.7%, 0.008 mmol) of
1
trifluoroactetate salt of 11 was isolated as a white solid: H NMR
(400 MHz, DMSO-d6) δ ppm 9.79 (s, 1 H), 7.00−7.14 (m, 2 H),
6.89−6.99 (m, 2 H), 5.86 (d, J = 3.9 Hz, 1 H), 4.77 (t, J = 5.09 Hz, 2
H), 4.46−4.51 (m, 1 H), 4.40 (t, J = 5.3 Hz, 2 H), 4.19−4.27 (m, 1
H), 4.13 (d, J = 2.74 Hz, 2 H), 3.97 (d, J = 5.9 Hz, 1 H); MS (LRES in
negative mode) calcd for C18H21FN5O9P [M − H]− 500.1, found
500.0; HRMS: theoretical 502.1134, observed 502.1132, HPLC
method A, tR = 7.52 min, purity 96%.
1
the trifluoroactetate salt of 15 was isolated as a white solid: H NMR
(400 MHz, DMSO-d6) δ ppm 11.35 (s, 1 H), 9.99 (s, 1 H), 7.60 (d, J
= 9.0 Hz, 2 H), 7.33 (d, J = 9.0 Hz, 2 H), 5.90 (d, J = 3.1 Hz, 1 H),
5.43 (d, J = 16.0 Hz, 1 H), 5.33 (d, J = 16.4 Hz, 1 H), 4.38−4.52 (m, 1
H), 4.20−4.27 (m, 1 H), 4.09−4.18 (m, 2 H), 3.91 (dd, J = 9.6, 4.9
Hz, 1 H); MS (LRES in positive mode) calcd for C18H20ClN6O9P [M
+ H]+ 531.1, found 531.0; HPLC method A, tR = 6.06 min, purity
85%.
7-N-(5-Chloro-2-methylbenzofuranyl)guanosine-5′-monophos-
phate (16). The compound was prepared according to the same
procedure used in preparing compound 2. Purification by reverse-
phase preparative HPLC (SB C8, 30 mm × 250 mm, 5 μm column;
Agilent, Santa Clara, CA) (eluent: 0.1% TFA in acetonitrile/water,
gradient 5% to 40% over 25 min) to provide 44 mg (6.4%) of the
trifluoroactetate salt of 16 as a white solid: 1H NMR (500 MHz,
DMSO-d6) δ ppm 3.93−4.00 (m, 1 H), 4.11−4.15 (m, 1 H), 4.15−
4.21 (m, 1 H), 4.26 (t, J = 5.3 Hz, 1 H), 4.47 (t, J = 3.7 Hz, 1 H),
5.78−5.92 (m, 3 H), 7.08 (s, 1 H), 7.31 (dd, J = 8.9, 2.1 Hz, 1 H), 7.57
(d, J = 8.9 Hz, 1 H), 7.67 (d, J = 1.8 Hz, 1 H), 9.95 (s, 1 H); MS
(LRES in negative mode), found 526.0 [M − 2H]−; HPLC method E,
tR = 11.3 min, purity 95%.
2,4-Dichloro-5-(2-(4-chlorophenoxy)ethyl)-5H-pyrrolo[3,2-d]-
pyrimidine (18). Sodium hydride (60% in mineral oil) (0.28 mL, 6.7
mmol) was added to an acetonitrile solution (75 mL) containing 2,4-
dichloro-5H-pyrrolo[3,2-d]pyrimidine (1.000 g, 5.3 mmol). After
stirring for 20 min at room temperature, 4-chlorophenyl 2-bromoethyl
ether (1.5 g, 6.4 mmol) was added, and the resulting mixture was
stirred overnight at 55 °C. The solution was then concentrated and
purified on silica, eluting with 10−100% hexane/ethyl acetate gradient
to give 1.7 g (94% yield) of 2,4-dichloro-5-(2-(4-chlorophenoxy)-
ethyl)-5H-pyrrolo[3,2-d]pyrimidine. LCMS-ESI (POS), m/z, M + 1:
Found 342, Calculated 342.
2-Chloro-5-(2-(4-chlorophenoxy)ethyl)-5H-pyrrolo[3,2-d]-
pyrimidin-4-ol (19). Aqueous 1 M sodium hydroxide (6 mL, 6 mmol)
was added to a dioxane solution (60 mL) containing 2,4-dichloro-5-(2-
(4-chlorophenoxy)ethyl)-5H-pyrrolo[3,2-d]pyrimidine (2.13 g, 6
mmol). The resulting mixture was stirred at reflux for 3 h and then
cooled to room temperature and neutralized with acetic acid. After the
mixture was cooled on ice, a precipitate formed and was collected
(1.98 g, 99% yield). This material was used in the next step without
further purification. LCMS-ESI (NEG), m/z, M − 1: Found 322,
Calculated 322.
2-Amino-5-(2-(4-chlorophenoxy)ethyl)-5H-pyrrolo[3,2-d]-
pyrimidin-4-ol (20). 2-Chloro-5-(2-(4-chlorophenoxy)ethyl)-5H-
pyrrolo[3,2-d]pyrimidin-4-ol (2 g, 6 mmol) was suspended in a
methanolic ammonia solution (7 N, 50 mL), sealed in a Parr steel
reactor, and heated at 170 °C overnight. The reaction was cooled to
room temperature and purified on silica gel, eluting with a
dichloromethane/methanol gradient (0−20%). Similar fractions were
pooled and concentrated to give 1.56 g (84% yield) of 2-amino-5-(2-
(4-chlorophenoxy)ethyl)-5H-pyrrolo[3,2-d]pyrimidin-4-ol: LCMS-ESI
(POS), m/z, M + 1: Found 305, Calculated 305.
(2S,3S,4R,5R)-2-(2-Amino-5-(2-(4-chlorophenoxy)ethyl)-4-hy-
droxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-5-(benzoyloxymethyl)-
tetrahydrofuran-3,4-diyl Dibenzoate (22). Tin(IV) chloride (0.19
mL, 1.6 mmol) was added to a nitromethane (20 mL) solution
containing β-D-ribofuranose 1-acetate 2,3,5-tribenzoate (0.35 g, 0.69
mmol) and 2-amino-5-(2-(4-chlorophenoxy)ethyl)-5H-pyrrolo[3,2-
d]pyrimidin-4-ol (0.18 g, 0.58 mmol). The resulting mixture was
stirred for 3 h at 60 °C. Excess solvent was then removed using
reduced pressure, and the remaining residue was purified on silica,
eluting with a dichloromethane/methanol gradient (0−20%), giving
7-(4-Cyanophenoxyethyl)guanosine-5′-monophosphate (12). To
a slurry of guanosine-5′-monophosphate disodium salt hydrate (0.53 g,
1.3 mmol, 1 equiv) in DMSO (4.5 mL) was added 4-(2-
bromoethoxy)benzonitrile (0.88 g, 3.9 mmol, 3 equiv). The resulting
white slurry was stirred at 60 °C for 28 h; during this time all solids
dissolved. The reaction mixture was filtered and purified directly by
reverse phase prep HPLC (Prep C18 OBD 10 μm column (Waters,
Milford, MA), gradient elution of 10−30% MeCN in water over a 30
min period, where both solvents contain 0.1% TFA). The lyophilized
impure product was suspended in water and then fully dissolved by
adding 2 equiv of 1 N NaOH to pH = 8. The solution was then
acidified to pH = 4 by adding 1 equiv of 1 N HCl. The solution was
extracted with EtOAc and n-butanol to remove impurities, and then it
was lyophilzed to afford 12 was as a white solid: 1H NMR (400 MHz,
DMSO-d6) δ ppm 10.11 (s, 1 H), 7.70 (d, J = 8.6 Hz, 2 H), 7.12 (d, J
= 8.6 Hz, 2 H), 5.84 (d, J = 3.5 Hz, 1 H), 4.83 (s, 2 H), 4.53 (dt, J =
13.7, 4.11 Hz, 3 H), 4.28 (t, J = 4.3 Hz, 1 H), 4.09 (s, 2 H), 3.89 (br. s,
1 H); MS (LRES in negative mode) calcd for C19H20N6O9P [M −
H]− 507.1, found 507.1; HRMS: theoretical 509.1180, observed
502.1179, HPLC method A, tR= 4.33 min, purity 100%.
7-(4-Trifluoromethylphenoxyethyl)guanosine-5′-monophos-
phate (13). To a slurry of guanosine-5′-monophosphate disodium salt
hydrate (0.53 g, 1.3 mmol, 1 equiv) in DMSO (4.5 mL) was added 1-
(2-bromoethoxy)-4-(trifluoromethyl)benzene (1.1 g, 3.9 mmol, 3
equiv). The resulting white slurry was stirred at 55 °C for 3 days;
during this time all solids dissolved. The reaction mixture was purified
directly by reverse phase prep HPLC (Prep C18 OBD 10 μm column
(Waters, Milford, MA), gradient elution of 10 to 30% MeCN in water
over a 30 min period, where both solvents contain 0.1% TFA). After
discarding mixed fractions, 2 mg (0.02%, 0.03 mmol) of the
1
trifluoroactetate salt of 13 was isolated as a white solid: H NMR
(400 MHz, DMSO-d6) δ ppm 9.79 (s, 1 H), 7.61 (d, J = 9.0 Hz, 2 H),
7.12 (d, J = 8.2 Hz, 2 H), 5.86 (d, J = 3.5 Hz, 1 H), 4.82 (t, J = 4.7 Hz,
2 H), 4.45−4.62 (m, 3 H), 4.23 (t, J = 4.7 Hz, 1 H), 4.14 (s, 2 H),
3.93−4.03 (m, 1 H); MS (LRES in positive mode) calcd for
C19H21F3N5O9P [M + H]+ 552.1, found 552.2; HPLC method D, tR =
2.52 min, purity 81%.
7-(3-Fluoro-4-Chlorophenoxyethyl)guanosine-5′-monophos-
phate (14). To a slurry of guanosine-5′-monophosphate disodium salt
hydrate (527 mg, 1.29 mmol, 1 equiv) in DMSO (4.5 mL) was added
4-(2-bromoethoxy)-1-chloro-2-fluorobenzene (1.3 g, 5.2 mmol, 4
equiv). The resulting white slurry was stirred at 55 °C for 4 days;
during this time all solids dissolved and starting material still remained.
The reaction mixture was filtered and purified directly by reverse phase
prep HPLC (Prep C18 OBD 10 μm column (Waters, Milford, MA),
gradient elution of 10 to 30% MeCN in water over a 30 min period,
where both solvents contain 0.1% TFA). After discarding mixed
fractions, 12 mg (1.7%, 0.02 mmol) of the trifluoroactetate salt of 14
1
was isolated as a white solid: H NMR (400 MHz, DMSO-d6) δ ppm
9.70 (s, 1 H), 7.36 (t, J = 8.8 Hz, 2 H), 7.02 (dd, J = 11.4, 2.7 Hz, 1
H), 6.76 (ddd, J = 8.8, 2.7, 1.0 Hz, 1 H), 5.80 (d, J = 3.5 Hz, 1 H), 4.72
(t, J = 4.9 Hz, 2 H), 4.41−4.47 (m, 1 H), 4.34−4.41 (m, 2 H), 4.16 (t,
J = 4.7 Hz, 1 H), 3.99−4.12 (m, 2 H), 3.87−3.94 (m, 1 H); MS (LRES
in negative mode) calcd for C18H19Cl FN5O9P [M − H]− 534.1, found
534.1; HRMS: theoretical 528.0682, observed 528.0675, HPLC
method A, tR = 6.71 min, purity 91%.
7-(N-(4-Chlorophenyl)acetamido)guanosine-5′-monophosphate
(15). To a slurry of guanosine-5′-monophosphate disodium salt
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0.11 g (26% yield) of pure product: LCMS-ESI (POS), m/z, M + 1:
Found 749, Calculated 749.
flask equipped with a mechanical stirrer was added potassium
carbonate (34.3 g, 569 mmol). To this solution was added 4-
chlorophenoxyacetyl chloride (44.4 mL, 285 mmol), dropwise and
with stirring. The reaction needed occasional cooling in an ice bath to
avoid overheating. When LCMS analysis indicated complete reaction,
400 mL of water was added. The resulting precipitate was filtered off.
The solid was thoroughly washed with water and dried under reduced
pressure to give 51.5 g of the first batch of compound. The aqueous
layer was filtered again and washed with water to give 9.25 g of a
second batch of material. The solid was vacuum-dried to give N-(4-
amino-2-(methylamino)-6-oxo-1,6-dihydropyrimidin-5-yl)-2-(4-
chlorophenoxy)acetamide (60.4 g, 65.6% yield).
6-Amino-5-(2-(4-chlorophenoxy)ethylamino)-2-(methylamino)-
pyrimidin-4(3H)-one (29). A 1 M solution of BH3·THF in THF (77
mL, 77 mmol) was added to a THF (100 mL) solution containing N-
(4-amino-2-(methylamino)-6-oxo-1,6-dihydropyrimidin-5-yl)-2-(4-
chlorophenoxy)acetamide (6.20 g, 19 mmol). The resulting mixture
was stirred for 5 min at 23 °C and then at reflux for 1 h, at which point
LCMS indicated the reaction was completed. To the reaction mixture
was added methanol (5 mL) and 4 M HCL in dioxane (∼5 mL)
slowly. A white precipitate formed. The suspension was heated for 10
h at reflux and then cooled to room temperature and concentrated.
THF was then added to the solid, and the suspension was filtered to
give the 6-amino-5-(2-(4-chlorophenoxy)ethylamino)-2-
(methylamino)pyrimidin-4(3H)-one (3.5 g, 54% yield).
N-(4-Amino-2-(methylamino)-6-oxo-1,6-dihydropyrimidin-5-yl)-
4-bromo-N-(2-(4-chlorophenoxy)ethyl)benzamide (30). To a sol-
ution of p-bromobenzoic acid (0.358 g, 1.78 mmol) in DMF (18 mL)
was added HATU (1.02 g, 2.67 mmol) and N,N-diisopropylethyl-
amine (1.24 mL, 7.12 mmol). The reaction mixture was stirred for half
an hour before 6-amino-5-(2-(4-chlorophenoxy)ethylamino)-2-
(methylamino)pyrimidin-4(3H)-one (0.607 g, 1.96 mmol) was
added to the mixture. The combined reaction mixture was stirred at
room temperature overnight. It was purified on silica gel, eluting with a
dichloromethane/methanol gradient (0−20%). Similar fractions were
pooled and concentrated to give N-(4-amino-2-(methylamino)-6-oxo-
1,6-dihydropyrimidin-5-yl)-4-bromo-N-(2-(4-chlorophenoxy)ethyl)-
benzamide (0.666 g, 75.9% yield).
8-(4-Bromophenyl)-7-(2-(4-chlorophenoxy)ethyl)-2-(methylami-
no)-1H-purin-6(7H)-one (31). To a solution of N-(4-amino-2-
(methylamino)-6-oxo-1,6-dihydropyrimidin-5-yl)-4-bromo-N-(2-(4-
chlorophenoxy)ethyl)benzamide (0.666 g, 1.35 mmol) in iPrOH (10
mL) dried over molecular sieves was added sodium tert-butoxide
(0.649 g, 6.76 mmol). The reaction mixture was stirred at 100 °C
overnight. Next the reaction mixture was acidified with acetic acid
(0.34 mL, 5.9 mmol) and concentrated to give 8-(4-bromophenyl)-7-
(2-(4-chlorophenoxy)ethyl)-2-(methylamino)-1H-purin-6(7H)-one
(0.334 g, 52.1% yield).
(2S,3R,4S,5R)-2-(5-(2-(4-Chlorophenoxy)ethyl)-4-hydroxy-2-
(methylamino)-5H-pyrrolo[3, 2-d]pyrimidin-7-yl)-5-
(hydroxymethyl)tetrahydro-3,4-furandiol (23). Sodium methoxide
(0.081 g, 1.5 mmol) was added to a methanol solution (10 mL)
containing (2S,3S,4R,5R)-2-(2-amino-5-(2-(4-chlorophenoxy)ethyl)-
4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-5-(benzoyloxymethyl)-
tetrahydrofuran-3,4-diyl dibenzoate (0.11 g, 0.15 mmol). The resulting
mixture was stirred overnight at room temperature and then purified
by reverse phase HPLC (5−70% water/acetonitrile gradient with 0.1%
TFA). Desired fractions were pooled and lyophilized to give 40 mg
(61% yield) of (2S,3R,4S,5R)-2-(2-amino-5-(2-(4-chlorophenoxy)-
ethyl)-4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-5-
(hydroxymethyl)tetrahydrofuran-3,4-diol: LCMS-ESI (POS), m/z, M
+ 1: Found 437, Calculated 437.
((2R,3S,4R,5S)-5-(5-(2-(4-Chlorophenoxy)ethyl)-4-hydroxy-2-
amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-3,4-dihydroxytetrahydro-2-
furanyl)methyl Dihydrogen Phosphate (24). Phosphorus oxychloride
(29 μL, 309 μmol) and trimethylphosphate (12 μL, 103 μmol) were
premixed at 0 °C for 10 min and then added to (2S,3R,4S,5R)-2-(2-
amino-5-(2-(4-chlorophenoxy)ethyl)-4-hydroxy-5H-pyrrolo[3,2-d]-
pyrimidin-7-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (0.045 g,
103 μmol). The mixture was stirred for 4 h and then diluted with
water and purified directly via preparative HPLC (5−50% water/
acetonitrile gradient with 0.1% TFA). Similar fractions were pooled
and lyophilized, yielding 12 mg (23% yield) of final product: 1H NMR
(500 MHz, DMSO-d6) δ ppm 3.81 −3.89 (m, 1 H), 3.89−4.06 (m, 4
H), 4.28 (t, J = 5.49 Hz, 2 H), 4.56−4.64 (m, 2 H), 4.66 (d, J = 7.32
Hz, 1 H), 6.96 (d, J = 9.16 Hz, 2 H), 7.31 (d, J = 9.16 Hz, 2 H), 7.42
(s, 1 H), 7.69−7.97 (m, 2 H). LCMS-ESI (POS), m/z, M + 1: Found
517, Calculated 517.
((3aS,4S,6S,6aS)-6-(2-amino-7-(2-(4-chlorophenoxy)ethyl)-6-oxo-
1H-purin-7-ium-9(6H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]-
dioxol-4-yl)methyl Phosphate (26). The 2′,3′-O-isopropylidienegua-
nosine (Sigma-Aldrich) (400 mg, 1.24 μmol) and 4-chlorophenyl 2-
bromoethyl ether (Sigma-Aldrich) (874 mg, 3712 μmol) were
dissolved in 3 mL of DMSO and heated at 60° for 3 days. The
reaction was diluted in ethyl acetate and extracted three times with
water. The product-containing aqueous layer was lyophilized to a clear
yellowish oil which contained DMSO and product. Purification of the
residue by preparatory RP-HPLC (10−30% acetonitrile, water, 0.1%
TFA, gradient elution) over 30 min using a SunfireTM Prep C18 OBD
column, 10 μm, 30 mm × 150 mm (Waters, Milford, MA) at 30 mL/
min provided the alkylated product as a white solid after lyophilization
to give 2-amino-7-(2-(4-chlorophenoxy)ethyl)-9-((3aR,4R,6R,6aR)-6-
(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-
6-oxo-6,9-dihydro-1H-purin-7-ium (156 mg, 26%). LCMS, MS (neg
ion) 477 [M − H]-Placed trimethyl phosphate (1.0 mL, 8686 μmol)
at 0°, then POCl3 was added (100 μL, 1085 μmol), followed by the 2-
amino-7-(2-(4-chlorophenoxy)ethyl)-9-((3aR,4R,6R,6aR)-6-(hydroxy-
methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-6-oxo-6,9-
dihydro-1H-purin-7-ium (52 mg, 109 μmol). Then the reaction was
warmed to room temperature and stirred for 30 min. It was recooled
to 0° and quenched with a small amount of NaHCO3 (saturated) until
pH ∼ 7. Purification of the solution by preparatory RP-HPLC (10 to
40% acetonitrile, water, 0.1% TFA, gradient elution) over 30 min using
SunfireTM Prep C18 OBD column, 10 μm, 30 mm × 150 mm
(Waters, Milford, MA) at 30 mL/min provided the phosphate product
Diethyl 4-(7-(2-(4-Chlorophenoxy)ethyl)-6-hydroxy-2-(methyla-
mino)-7H-purin-8-yl)phenylphosphonate (32). To the starting
material in a 25 mL sealed tube was added EtOH, palladium(II)
acetate (0.95 mg, 4.2 μmol), diethyl phosphite (6.5 μL, 51 μmol), and
N,N-dicyclohexylmethylamine (13 μL, 63 μmol) under Ar. The
mixture was purged with Ar for 10 min, and triphenyl phosphine (2.0
μL, 8.4 μmol) was added. The reaction mixture was stirred at 100 °C
overnight. The solvent was removed under vacuum. The crude
product was purified by chromatoraphy on silica gel, eluting with 0−
10% MeOH−CH2Cl2 to give diethyl 4-(7-(2-(4-chlorophenoxy)-
ethyl)-6-hydroxy-2-(methylamino)-7H-purin-8-yl)phenylphosphonate
1
(31 mg, 49%) as a fluffy white powder after lyophilization. H NMR
1
(17 mg, 76% yield). H NMR (500 MHz, chloroform-d) δ ppm 1.37
(500 MHz, DMSO-d6) δ ppm 1.33 (s, 3 H), 1.52 (s, 3 H), 3.97 (dt, J =
11.55, 5.84 Hz, 1 H), 4.05−4.20 (m, 1 H), 4.43 (dq, J = 11.62, 5.67
Hz, 2 H), 4.52 (br s, 1 H), 4.77 (br s, 2 H), 5.11 (dd, J = 6.11, 2.45 Hz,
1 H), 5.32 (d, J = 6.36 Hz, 1 H), 6.20 (s, 1 H), 6.99 (m, J = 8.80 Hz, 2
H), 7.32 (m, J = 8.80 Hz, 2 H), 9.45 (s, 1 H). LCMS, MS (neg ion)
[M − 2H]− C21H24ClN5O9P calc 556.1, found 556.1, HRMS:
(t, J = 7.09 Hz, 6 H), 2.97 (br s, 3 H), 4.02−4.26 (m, 4 H), 4.43 (t, J =
5.01 Hz, 2 H), 4.72 (t, J = 5.01 Hz, 2 H), 6.56−6.78 (m, 2 H), 7.06−
7.23 (m, 2 H), 7.83−8.07 (m, 5 H).
Ethyl Hydrogen (4-(7-(2-(4-Chlorophenoxy)ethyl)-6-hydroxy-2-
(methylamino)-7H-purin-8-yl)phenyl)phosphonate (34). To diethyl
4-(7-(2-(4-chlorophenoxy)ethyl)-6-hydroxy-2-(methylamino)-7H-
purin-8-yl)phenylphosphonate (20 mg, 37.6 μmol) in a 10 mL flask
was added CH2Cl2 and bromotrimethylsilane (30 μL, 224 μmol). The
solution was stirred at 30 °C over 24 h. The solvent was removed
under vacuum, and the residue was purified by reverse phase HPLC to
theoretical 558.1157, observed 558.1157, HPLC method A, tR
=
5.06 min, purity 99%.
N-(4-Amino-2-(methylamino)-6-oxo-1,6-dihydropyrimidin-5-yl)-
2-(4-chlorophenoxy)acetamide (28). To 5,6-diamino-2-
(methylamino)pyrimidin-4(3H)-one (44.15 g, 285 mmol) in a 1 L
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give ethyl hydrogen (4-(7-(2-(4-chlorophenoxy)ethyl)-6-hydroxy-2-
(methylamino)-7H-purin-8-yl)phenyl)phosphonate (8.0 mg, 42%
yield). H NMR (500 MHz, DMSO-d6) δ ppm 1.31 (t, J = 8.0 Hz,
3 H), 2.81 (d, J = 4.65 Hz, 3 H), 4.31 (t, J = 5.26 Hz, 2 H), 4.51 (q, J =
8.0 Hz, 2 H), 4.63 (t, J = 5.14 Hz, 2 H), 6.06 (d, J = 4.65 Hz, 1 H),
6.77 (m, J = 9.05 Hz, 2 H), 7.25 (m, J = 8.80 Hz, 2 H), 7.79−8.03 (m,
4 H), 10.90 (s, 1 H). HRMS: theoretical 472.1387, observed 472.1385;
HPLC method F, tR = 0.657 min, purity 97%.
the resulting residue was purified by reverse phase HPLC using a C-8
Agilent column, eluting with a 10−90% water/acetonitrile gradient
with 0.1% TFA. Desired fractions were pooled and concentrated
(0.101 g, 66% yield). LCMS-ESI (POS), m/z, M + H2O: Found 615,
Calculated 615.
1
(4-(7-(2-(4-Chlorophenoxy)ethyl)-2-(methylamino)-6-oxo-6,7-di-
hydro-1H-purin-8-yl)benzyl)phosphonic Acid (39). Bromotrimethyl-
silane (0.188 mL, 1.42 mmol) was added to a DMF solution
containing methyl hydrogen 4-(2-(benzylthio)-7-(2-(4-
chlorophenoxy)ethyl)-6-oxo-6,7-dihydro-1H-purin-8-yl)-
benzylphosphonate (0.170 g, 0.285 mmol). The resulting mixture was
stirred overnight at room temperature. Next, a few drops of methanol
were added, and the resulting mixture was purified directly by reverse
phase HPLC using a C-8 Agilent column, eluting with a 10−90%
water/acetonitrile gradient with 0.1% TFA. Desired fractions were
pooled and lyophilized to give 0.143 g (86% yield) of pure product.
LCMS-ESI (POS), m/z, M + 1: Found 583, Calculated 583.
An aqueous 40% methyl amine solution (2 mL) containing 4-(2-
(benzylthio)-7-(2-(4-chlorophenoxy)ethyl)-6-oxo-6,7-dihydro-1H-
purin-8-yl)benzylphosphonic acid (5 mg, 9 umol) was sealed in a tube
and heated in a microwave at 160 °C for 12 h. The reaction was then
concentrated to dryness, and the resulting residue was purified by
reverse phase HPLC using a C-8 Agilent column, eluting with a 5−
50% water/acetonitrile gradient with 0.1% TFA. Desired fractions
were pooled and lyophilized to give 3 mg (68% yield) of pure product.
1H NMR (400 MHz, MeOH) δ ppm 2.86 (s, 3 H), 4.34 (t, J = 4.99
4-(7-(2-(4-Chlorophenoxy)ethyl)-6-hydroxy-2-(methylamino)-7H-
purin-8-yl)phenylphosphonic Acid (33). To diethyl 4-(7-(2-(4-
chlorophenoxy)ethyl)-6-hydroxy-2-(methylamino)-7H-purin-8-yl)-
phenylphosphonate (17 mg, 32 μmol) in a 10 mL flask was added
CH2Cl2 and bromotrimethylsilane (30 μL, 224 μmol). The solution
was stirred at 30 °C over 3 days. The solvent was removed under
vacuum, and the residue was purified by reverse phase HPLC to give
4-(7-(2-(4-chlorophenoxy)ethyl)-6-hydroxy-2-(methylamino)-7H-
purin-8-yl)phenylphosphonic acid (14 mg, 92% yield). 1H NMR (500
MHz, DMSO-d6) δ ppm 2.81 (d, J = 4.65 Hz, 3 H), 4.31 (t, J = 5.26
Hz, 2 H), 4.63 (t, J = 5.14 Hz, 2 H), 6.06 (d, J = 4.65 Hz, 1 H), 6.77
(m, J = 9.05 Hz, 2 H), 7.25 (m, J = 8.80 Hz, 2 H), 7.79−8.03 (m, 4
H), 10.90 (s, 1 H). HRMS: theoretical 444.1074, observed 444.1073;
HPLC method F, Agilent SB-C8 column, 10−50% H2O−CH3CN, tR
= 0.457 min, purity 98%.
6-Amino-2-(benzylthio)-5-(2-(4-chlorophenoxy)ethylamino)-
pyrimidin-4(3H)-one (36). 5,6-Diamino-2-(benzylthio)pyrimidin-
4(3H)-one (7.09 g, 28.6 mmol) and 2-(4-chlorophenoxy)acetaldehyde
(4.87 g, 28.5 mmol) were combined with methylene chloride (80.00
mL, 1225 mmol) and stirred at room temperature for 60 min. The
reaction was cooled to room temperature. Next, sodium triacetox-
yborohydride (18.18 g, 85.8 mmol) was added. The reaction was
stirred at room temperature overnight. The reaction was quenched
with water. The reaction was diluted with dichloromethane and water
until all solids were dissolved. The aqueous layer was extracted with
dichloromethane three times. The organics were combined and
washed with brine. The chloromethane layer was filtered through
Na2SO4 and concentrated in vacuum. The crude material was purified
on silica gel eluting with a dichloromethane/methanol gradient (0−
20%) to give 6-amino-2-(benzylthio)-5-(2-(4-chlorophenoxy)-
ethylamino)pyrimidin-4(3H)-one (3.72 g, 32.3% yield).
Diethyl 4-((4-Amino-2-(benzylthio)-6-oxo-1,6-dihydropyrimidin-
5-yl)(2-(4-chlorophenoxy)ethyl)carbamoyl)benzylphosphonate
(37). Triethyl phosphite (6.07 mL, 35.4 mmol) was added to a toluene
solution containing p-(bromomethyl)benzoic acid (6.92 g, 32.2
mmol). The resulting mixture was stirred overnight at 111 °C. The
solvent was then removed using reduced pressure, and the remaining
residue was used in the next step without purification. LCMS-ESI
(NEG), m/z, M − 1: Found 271, Calculated 271.
DMF (5 drops) was added to a DCM solution containing oxalyl
chloride (0.305 mL, 3.44 mmol) and 4-((diethoxyphosphoryl)-
methyl)benzoic acid (0.468 g, 1.72 mmol). The resulting mixture
was stirred for 3 h at room temperature. The mixture was then
concentrated and used in the next step without purification.
Hunig’s base (0.300 mL, 1.72 mmol) and dimethylaminopyridine
(0.210 g, 1.72 mmol) were added to a DCM (40 mL) solution
containing 6-amino-2-(benzylthio)-5-(2-(4-chlorophenoxy)-
ethylamino)pyrimidin-4(3H)-one (0.693 g, 1.72 mmol) and diethyl
4-(chlorocarbonyl)benzylphosphonate (0.500 g, 1.72 mmol) . The
resulting mixture was stirred overnight at 23 °C. The solution was then
washed with water, dried over sodium sulfate, and concentrated. This
material was used in the next step without further purification. LCMS-
ESI (POS), m/z, M + 1: Found 657, Calculated 657.
Methyl Hydrogen 4-(2-(Benzylthio)-7-(2-(4-chlorophenoxy)ethyl)-
6-oxo-6,7-dihydro-1H-purin-8-yl)benzylphosphonate (38). Diethyl
4-((4-amino-2-(benzylthio)-6-oxo-1,6-dihydropyrimidin-5-yl)(2-(4-
chlorophenoxy)ethyl)carbamoyl)benzylphosphonate (0.170 g, 0.259
mmol) was suspended in 6 mL of a 40% NaOMe/MeOH solution and
heated to 90 °C for 1 h. After cooling to room temperature,
concentrated HCl was added until the solution was acidic. Excess
solvent was then removed, and the remaining solid was washed
repeatedly with absolute ethanol. The filtrate was concentrated, and
Hz, 2 H), 4.65−4.72 (m, 2 H), 6.58−6.70 (m, 2 H), 7.01−7.15 (m, 2
H), 7.49 (d, J = 6.46 Hz, 2 H), 7.69 (d, J = 8.02 Hz, 2 H); LCMS-ESI
(POS), m/z, M + 1: Found 490, Calculated 490. HPLC method F,
20−95% H2O−CH3CN, tR = 0.227 min, purity: 97%.
Methyl Hydrogen ((4-(7-(2-(4-Chlorophenoxy)ethyl)-2-(methyla-
mino)-6-oxo-6,7-dihydro-1H-purin-8-yl)phenyl)difluoromethyl)-
phosphonate (41). Diethyl bromodifluoromethylphosphonate (0.877
mL, 4.94 mmol) was added to a dry DMF solution containing
cadmium (0.0704 mL, 5.41 mmol) (under nitrogen). The resulting
mixture was stirred at room temperature for 2 h and then filtered into
a flask containing copper(I) chloride (0.097 mL, 3.46 mmol) and
benzyl 4-iodobenzoate (1.00 g, 2.96 mmol) (under nitrogen). The
resulting mixture was stirred overnight at room temperature. The
reaction was then purified on silica, eluting with a hexane/ethyl acetate
gradient (0−100%). Desired fractions were pooled and concentrated
to give 1.06 g (90.8% yield) of pure product. LCMS-ESI (POS), m/z,
M + 1: Found 399, Calculated 399.
10% palladium on carbon (0.290 g, 2.70 mmol) was added to an
ethanol solution containing benzyl 4-((diethoxyphosphoryl)-
difluoromethyl)benzoate (1.6 g, 2.68 mmol). The suspension was
then placed under an atmosphere of hydrogen (1 atm) and stirred
vigorously for 4 h. Next, the solution was filtered though Celite and
concentrated. This material was used in the next step without further
purification (0.83 g, 100% crude yield). LCMS-ESI (NEG), m/z, M −
1: Found 307, Calculated 307.
Triethylamine (0.045 mL, 0.32 mmol) was added to a DMF (100
mL) solution containing 4-((diethoxyphosphoryl)difluoromethyl)-
benzoic acid (0.100 g, 0.32 mmol) and 6-amino-5-(2-(4-
chlorophenoxy)ethylamino)-2-(methylamino)pyrimidin-4(3H)-one
(0.20 g, 0.65 mmol). The resulting mixture was stirred for 6 h at 23
°C. The solution was then partitioned with water/ethyl acetate, and
the organic layer was dried over sodium sulfate and concentrated. This
material was then suspended in a 25% NaOMe/MeOH solution and
was heated at 90 °C for 1 h. Next, concentrated HCl was added until
the pH was 1 and the solvent was removed. The resulting solid was
washed with absolute ethanol, and the filtrate was collected and
concentrated. This material was used in the next step without further
purification. LCMS-ESI (POS), m/z, M + 1: Found 540, Calculated
540.
((4-(3-(2-(4-Chlorophenoxy)ethyl)-6-(methylamino)-4-oxo-4,5-di-
hydro-3H-imidazo[4,5-c]pyridin-2-yl)phenyl)difluoromethyl)-
phosphonic Acid (42). Bromotrimethylsilane (73 μL, 556 μmol) was
added to a DMF (1 mL) solution containing methyl hydrogen (4-(7-
(2-(4-chlorophenoxy)ethyl)-2-(methylamino)-6-oxo-6,7-dihydro-1H-
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purin-8-yl)phenyl)difluoromethylphosphonate (30 mg, 56 μmol). The
resulting mixture was stirred for 2 h at 60 °C. The mixture was then
directly injected on reverse phase HPLC using a C-8 Agilent column
eluting with a 5−50% water/acetonitrile gradient with 0.1% TFA.
Desired fractions were pooled and lyophilized to give 1.3 mg (4.5%
yield) of pure product. 1H NMR (500 MHz, MeOH) δ ppm 2.99 (s, 3
H), 4.48 (t, J = 5.01 Hz, 2 H), 4.84 (t, J = 5.01 Hz, 2 H), 6.71−6.89
(m, 2 H), 7.16−7.29 (m, 2 H), 7.94 (s, 4 H). LCMS-ESI (POS), m/z,
M + 1: Found 526, Calculated 526, Agilent SB-C8 column, 10−50%
H2O−CH3CN, HPLC method F, tR = 0.422 min, purity 100%.
Methyl 4-(2-(Benzylthio)-7-(2-(4-chlorophenoxy)ethyl)-6-oxo-6,7-
dihydro-1H-purin-8-yl)benzoate (44). N,N-Dimethylpyridin-4-amine
(0.121 g, 0.993 mmol) and N-ethyl-N-isopropylpropan-2-amine (0.128
g, 0.993 mmol) were added to a DCM (40 mL) solution containing 6-
amino-2-(benzylthio)-5-(2-(4-chlorophenoxy)ethylamino)pyrimidin-
4(3H)-one (0.400 g, 0.993 mmol) and methyl 4-(chlorocarbonyl)-
benzoate (0.197 g, 0.993 mmol). The resulting mixture was stirred for
3 h at 23 °C. The solution was then partitioned with water, dried over
sodium sulfate, and concentrated. The remaining residue was
suspended in a 40% NaOMe/MeOH solution and was heated at 90
°C for 1 h. The reaction was then cooled to room temperature, and
concentrated HCl was added until the pH was 1. Excess solvent was
removed, and the resulting solid was washed with absolute ethanol.
The filtrate was concentrated, and the remaining residue was purified
on silica, eluting with DCM/MeOH stepwise gradient (0−20%).
Desired fractions were pooled and concentrated (0.0831 g, 15% yield).
LCMS-ESI (POS), m/z, M + 1: Found 547, Calculated 547.
diffraction data sets were collected at the synchrotron
beamlines 501/502 at Advanced Light Source (ALS) in
Berkeley, and they were processed with the programs
MOSFLM38 and SCALA in the CCP4 program suite.39 The
structures were solved by the molecular replacement method
MOLREP40 using a previously published eIF4E structure (PDB
code: 1IPB) as a search model. Model building and refinement
were carried out in QUANTA (Accelrys, San Diego), COOT,41
and REFMAC42 in CCP4.39 All structural figures were prepared
using Pymol (http://www.pymol.org).
In Vitro IC50 Determination: SPA Assay. Human eIF4E protein
(NP_001959) was produced as an N-terminal FLAG-6xHis fusion
protein in E. coli and purified by m7-GTP-sepharose affinity
chromatography (GE Healthcare). Protein was eluted with 100 μM
m7-GDP and dialyzed extensively into storage buffer. For binding
analysis, eIF4E protein (50 nM final) was biotinylated and bound to
streptaviden coated scintillation proximity assay beads (GE Health-
care) in the presence of 3H-m7-GTP (1 mCi/mL, Moravek
Biochemicals) in binding buffer (20 mM HEPES, pH 7.4, 50 mM
KCL, 1 mM DTT, and 0.5 mM EDTA). To determine competitive
inhibition of eIF4E binding, compounds were added in an 11 point, 3-
fold dilution dose series, and loss of the radioactive signal was
monitored by scintillation counting. Assays were performed in
triplicate, and IC50 values were calculated by nonlinear regression
curve fitting using GraphPad Prism analysis software (GraphPad
Software).
In Vitro IC50 Determination: Rabbit Reticulocyte Lysate In Vitro
Translation Assay. Inhibition of cap dependent translation was
measured in vitro by utilization of the pRenilla-HCV-Firefly (pRIF)
luciferase reporter mRNA. The pRIF reporter construct was generated
by insertion of renilla and firefly luciferase cDNAs into multiple
cloning sites of the pIRES vector (Clonetech) using standard
techniques. The HCV internal ribosome entry site (bases 14−383)
was cloned into Mlu-1, Sal-1 restriction sites located between renilla
and firefly cDNAs. For in vitro transcription, the pRIF plasmid was
linearized by digestion with Bgl-II and m7-GTP capped, poly
adenylated mRNA was transcribed with T7 polymerase according to
manufacturer’s guidelines (Ambion, AM1345). In vitro translations
were performed in 20 μL reactions containing 5 μL of rabbit
reticulocyte lysate (Promega), 100 mM potassium acetate, 1.4 mM
magnesium chloride, 10 μM amino acids, 20 units of RNasin
(Promega), and 20 ng of pRIF mRNA. eIF4E inhibitor compounds
were added in an 11 point, 3-fold dilution dose series with a final
DMSO concentration of 1%. Reactions were incubated at 30 °C for 90
min prior to determination of renilla and firefly luciferase activities
(Promega, Dual-Glo E2920). Assays were performed in triplicate, and
IC50 values were calculated by nonlinear regression curve fitting using
GraphPad Prism analysis software (GraphPad Software).
4-(2-(Benzylthio)-7-(2-(4-chlorophenoxy)ethyl)-6-oxo-6,7-dihy-
dro-1H-purin-8-yl)benzoic Acid (45). Lithium hydroxide (4 mg, 0.15
mmol) was added to a water/MeOH (1:1) (30 mL) solution
containing methyl 4-(2-(benzylthio)-7-(2-(4-chlorophenoxy)ethyl)-6-
oxo-6,7-dihydro-1H-purin-8-yl)benzoate (0.0831 g, 0.15 mmol). The
resulting mixture was stirred for 2 h at 100 °C. The material was then
acidified and concentrated. The residue was washed with absolute
ethanol, and the filtrated was concentrated. This material was used in
the next step without purification (0.60 g, 74% crude yield). LCMS-
ESI (POS), m/z, M + 1: Found 533, Calculated 533.
4-(7-(2-(4-Chlorophenoxy)ethyl)-2-(methylamino)-6-oxo-6,7-di-
hydro-1H-purin-8-yl)benzoic acid (46). An aqueous methylamine
40% wt solution (2 mL) and 4-(2-(benzylthio)-7-(2-(4-
chlorophenoxy)ethyl)-6-oxo-6,7-dihydro-1H-purin-8-yl)benzoic acid
(0.025 g, 0.047 mmol) were sealed in a tube and heated in a
microwave at 160 °C for 12 h. The reaction was then concentrated to
dryness, and the resulting residue was purified by reverse phase HPLC
using a C-8 Agilent column eluting with a 5−50% water/acetonitrile
gradient with 0.1% TFA. The desired fractions were pooled and
1
lyophilized to give 5.19 mg (25% yield) of pure product. H NMR
(400 MHz, MeOH) δ ppm 2.87 (s, 3 H), 4.33 (s, 2 H), 4.71 (s, 2 H),
6.62 (m, J = 9.00 Hz, 2 H), 7.06 (m, J = 9.19 Hz, 2 H), 7.83 (m, J =
8.41 Hz, 2 H), 8.11 (m, J = 8.41 Hz, 2 H); LCMS-ESI (POS), m/z, M
+ 1: Found 440, Calculated 440. Agilent SB-C8 column, 20−95%
H2O−CH3CN, HPLC method F, tR = 0.286 min, purity 100%.
Biological Characterization of Compounds. Protein Produc-
tion and Crystallography. Recombinant eIF4E protein for
crystallographic studies was prepared as previously re-
ported.20,37 In brief, the full length human eIF4E was cloned
into vector pET101 with an N-terminal FLAG 6xHis tag
followed by an rTEV cleavage site. The protein was expressed
in E. coli and purified through a m7-GTP-sepharose column.
The purified protein which contained 100 μM m7-GTP was
then concentrated to about 7 mg/mL in 20 mM Hepes, pH 7.6,
100 mM KCl, 1 mM DTT, 0.1 mM EDTA for crystallization.
The m7-GTP-bound eIF4e protein was crystallized by the
hanging drop vapor diffusion method at 16 °C with a 1:1 ratio
of protein solution to reservoir solution of 17−20% PEG-3350
and 0.1−0.4 M Na formate. The compound was introduced to
the crystals by a soaking method in a solution of 32% PEG-
3350, 0.4 M Na formate, 10% glycerol with powder compound.
Harvested crystals were transferred to the mother liquor with
30% glycerol and then flash frozen in liquid nitrogen. The X-ray
ASSOCIATED CONTENT
■
Accession Codes
†Coordinates of the crystal structures have been deposited with
the RCSB Protein Data Bank with the accession codes of 4DT6
and 4DUM.
AUTHOR INFORMATION
Corresponding Author
*Phone: (650) 244-2596 (X.C.) and (650) 244-2446 (Z.W.).
E-mail: xiaoqic@amgen.com and zwang@amgen.com.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Larry McGee for critical reading of the manuscript.
The Advanced Light Source is supported by the Director,
Office of Science, Office of Basic Sciences, Materials Sciences
Division, of the U.S. Department of Energy under Contract No.
3849
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Journal of Medicinal Chemistry
Article
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