Anchimerically Activated ProTides as Inhibitors of Cap-Dependent Translation and Inducers of Chemosensitization in Mantle Cell Lymphoma

Article abstract of DOI:10.1021/acs.jmedchem.7b00916

The cellular delivery of nucleotides through various pronucleotide strategies has expanded the utility of nucleosides as a therapeutic class. Although highly successful, the highly popular ProTide system relies on a four-step enzymatic and chemical process to liberate the corresponding monophosphate. To broaden the scope and reduce the number of steps required for monophosphate release, we have developed a strategy that depends on initial chemical activation by a sulfur atom of a methylthioalkyl protecting group, followed by enzymatic hydrolysis of the resulting phosphoramidate monoester. We have employed this ProTide strategy for intracellular delivery of a nucleotide antagonist of eIF4E in mantle cell lymphoma (MCL) cells. Furthermore, we demonstrated that chemical inhibition of cap-dependent translation results in suppression of c-Myc expression, increased p27 expression, and enhanced chemosensitization to doxorubicin, dexamethasone, and ibrutinib. In addition, the new ProTide strategy was shown to enhance oral bioavailability of the corresponding monoester phosphoramidate.

Full text of DOI:10.1021/acs.jmedchem.7b00916

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Article
Anchimerically Activated ProTides as Inhibitors of Cap-Dependent
Translation and Inducers of Chemosensitization in Mantle Cell Lymphoma
Aniekan Okon, Jingjing Han, Surendra Dawadi, Christos Demosthenous,
Courtney C. Aldrich, Mamta Gupta, and Carston R. Wagner
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00916 • Publication Date (Web): 31 Aug 2017
Downloaded from http://pubs.acs.org on September 3, 2017
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Anchimerically Activated ProTides as Inhibitors of Cap-dependent
Translation and Inducers of Chemosensitization
in Mantle Cell Lymphoma
‡
#
‡
#
‡
Aniekan Okon , JingJing Han , Surendra Dawadi , Christos Demostheneous , Courtney C. Aldrich ,
†
*
‡§*
Mamta Gupta, and Carston R. Wagner
*
Address correspondence to: wagne003@umn.edu and magupta@email.gwu.edu
‡
§
Departments of Medicinal Chemistry and Chemistry, University of Minnesota,
Minneapolis, Minnesota 55455, United States
#
Mayo Clinic, Division of Hematology, Department of InternalꢀMedicine Rochester, Minnesota 55905,
United States
†
Dept. of Biochemistry and Molecular Medicine, School of Medicine and Health Sciences, George
Washington University, GW Cancer Center, Washington DC, 20052, United States
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ABSTRACT: The cellular delivery of nucleotides through various pronucleotide strategies has
expanded the utility of nucleosides as a therapeutic class. Although highly successful, the highly popular
ProTide system relies on a fourꢀstep enzymatic and chemical process to liberate the corresponding
monophosphate. To broaden the scope and reduce the number of steps required for monophosphate
release, we have developed a strategy that depends on initial chemical activation by a sulfur atom of a
methylthioꢀalkylꢀprotecting group, followed by enzymatic hydrolysis of the resulting phosphoramidate
monoester. We have employed this proTide strategy for intracellular delivery of a nucleotide antagonist
of eIF4E in mantle cell lymphoma (MCL) cells. Furthermore, we demonstrated that chemical inhibition
of capꢀdependent translation results in suppression of cꢀMyc expression, increased p27 expression, and
enhanced chemosensitization to doxorubicin, dexamethasone, and ibrutinib. In addition, the new
ProTide strategy was shown to enhance oral bioavailability of the corresponding monoester
phosphoramidate.
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Keywords: ProTide, pronucleotide, eIF4E, phosphoramidate, cꢀMYC, p27, nucleoside, lymphoma
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Introduction
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Nucleoside and nucleoside analogs have proven to be important antiviral and antiꢀcancer agents.
As antiviral and antiꢀcancer agents, nucleoside and nucleoside analogs rely on conversion by cellular
kinases into the respective monoꢀ, diꢀ, and triꢀphosphate nucleoside metabolites, which could then
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interdict viral replication or cancer proliferation. A major drawback for nucleoside/nucleotideꢀbased
therapies lies in the activation pathway into the respective functional nucleotide metabolite(s).
Specifically, the first phosphorylation step by a nucleoside kinase has been shown to be inefficient for
1
many nucleoside analogs, because of the strict substrate specificity of nucleoside kinases.
One could propose a direct administration of nucleoside analog monophosphates as a means to
bypass the inefficient first phosphorylation step. However, such a strategy is impractical because of the
highly polar nature of a nucleotide, which renders it impermeable to the cell membrane. In addition,
nucleotides are metabolically unstable and are susceptible to dephosphorylation by cellular and plasma
phosphatases. To bypass the inefficient initial phosphorylation step, prodrugs of nucleoside
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, 2
monophosphates, referred to as ProTides have been developed.
Pronucleotides serve to mask the negatively charged phosphate backbone, thus ensuring passage
though the cell membrane. Once internalized, the nucleoside monophosphate is typically released by
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enzymatic and/or chemical processes. The unveiled monophosphate can ultimately undergo multiple
phosphorylation steps by cellular kinases to form the therapeutically active nucleotide(s). Kinase bypass
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strategies have been validated with a wide range of antiviral and anticancer nucleosides. Currently,
the most clinically successful ProTide strategy involves nucleoside prodrugs containing the aryloxy
5
amino acid phosphoramidate moiety (Fig. 1A). Activation of ProTides are initiated by esterase
hydrolysis of the amino acid ester, followed by spontaneous intramolecular nucleophilic attack at the
6
phosphorus center to eliminate the aryl moiety. The resulting cyclic intermediate is hydrolyzed by
water to form the amino acid monoester phosphoramidate, which is ultimately enzymatically
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hydrolyzed by the intracellular nucleoside phosphoramidase, Histidine Triad Nucleotide Binding
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Protein 1 (Hint1) to yield the corresponding nucleoside monophosphate (Fig. 1A).
Although the ProTide technology has been widely adapted for delivery of nucleotides in cells,
the first step of the activation of ProTides depends on the expression levels and stereospecific
preference of intracellular esterases. Typically, one of the phosphorous stereoisomers of a ProTide
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displays significantly more potent biological activity than the other, thus requiring the development of a
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stereospecific synthesis of the preferred isomer.
In addition, ProTides have been viewed as liver
targeting because the initial esterase hydrolysis occurs rapidly during first pass metabolism after oral
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, 11
dosing.
Consequently, ProTides may not be that effective for delivery of significant amounts of
monophosphate to tissues other than the liver. To further expand the utility of ProTides, we explored the
development of a new prodrug approach that incorporates a chemically releasable thiomethyl ethanol
moiety and Hint1 cleavable tryptamine phosphoramidate (Fig. 1C). This approach offers an alternative
to the ProTide approach, since the first activation step does not require enzymatic activation or
carboxylate attack at the chiral phosphorous. The resulting monoester phosphoramidate can then
undergo PꢀN bond hydrolysis by Hint1.
Amino acid nucleoside monoester phosphoramidates (Fig. 1B) have been shown to undergo
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2ꢀ17
intracellular uptake and to deliver antiviral and anticancer nucleoside monophosphates.
These
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monoester phosphoramidates were shown to be water soluble, nonꢀtoxic and cell permeable in vitro.
In particular, we have demonstrated that the monoester phosphoramidate, 4Eiꢀ1 (Fig. 2), is capable of
1
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undergoing cellular uptake and deliver the eIF4E selective antagonist, 7ꢀBnGMP. Treatment of tumor
cells with 4Eiꢀ1 has demonstrated that eIF4E is critical in epithelial to mesenchymal transition
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(EMT).
In addition, enhanced chemosensitization to gemcitabine and pemetrexed in mesothelioma,
14, 17
lung, and breast cancer cells has been shown to be dependent on eIF4E after treatment with 4Eiꢀ1.
Furthermore, 4Eiꢀ1 has been used to demonstrate that eIF4E is necessary for the induced proliferation
+
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ꢀ
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and expansion of both CD4 Foxp3 and CD4 Foxp3 Tꢀcells, as well as Tꢀcell subset identity. While
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Eiꢀ1 has been shown to be a useful probe of eIF4E function in cells, its low potency has hindered it
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from being used in vivo. The low potency of 4Eiꢀ1 is likely due to the low affinity of 7ꢀBnGMP for
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eIF4E (K = 0.8 ꢁM) and likely lower than expected cellular uptake of the zwitterionic monoester
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phosphoramidate. Consequently, we hypothesized that the development of a ProTide with greater
membrane permeability and incorporating a more potent eIF4E antagonist would afford an enhanced
chemical agonist of eIF4E.
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Results and Discussion
Design Strategy
In an effort to improve the membrane permeability of nucleoside phosphoramidate monoesters,
we chose to prepare nucleoside phosphoramidate diesters containing a methylthio alkyl moiety
conjugated to the phosphoramidate oxygen (Fig. 1C). Cieślak and coworkers had previously reported
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the use of methylthio alkyl protecting groups for the synthesis of oligonucleotides. Unmasking of the
phosphate oxygen is thought to proceed according to a sulfurꢀmediated intramolecular cycloꢀdeꢀ
esterification reaction. Although the halfꢀlife for the deprotection reactions were fast at high
ꢀ1
ꢀ1
temperatures (~ 115 s – 225 s at 55 °C), we reasoned that the halfꢀlife for deprotection of the
methylthio alkyl moieties would be considerably slower under physiological conditions and thus
sufficient to facilitate membrane permeability and intracellular uptake.
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The recent discovery of N ꢀ(pꢀchlorophenoxyethyl)guanosineꢀ5´ꢀmonophosphate (7ꢀClꢀPhꢀ
2
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EthylꢀGMP) (Fig. 2) as an inhibitor of eIF4E, presents an attractive substrate to develop a more potent
eIF4E ProTide inhibitor compared to 4Eiꢀ1. 7ꢀClꢀPhꢀEthylꢀGMP was found to have a greater than 200ꢀ
2
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fold affinity for eIF4E compared to 7ꢀBnGMP. Nevertheless, 7ꢀClꢀPhꢀEthylꢀGMP, was found to
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possess no cellular activity, likely due to its low cellular permeability. Therefore, we chose to prepare
tryptamine methylthio alkyl 7ꢀClꢀPhꢀEthylꢀGMP ProTides to enhance the cellular potency of 7ꢀClꢀPhꢀ
EthylꢀGMP. This ProTide strategy adds to our previous work, which demonstrated that tryptamine
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nucleoside phosphoramidates are excellent substrates for Hint1 and thus nucleoside monophosphate
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intracellular release.
Aberrant eIF4E activity has been shown to cause malignant transformations in a variety of cells,
and has been found to be a driver of tumorigenesis, disease progression, and poor survival rates in
certain carcinomas of the breast, head and neck, acute and chronic myelogenous leukemias, colon, and
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nonꢀHodgkin’s lymphomas.
Consistent with these findings, several studies have shown that
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interdiction of eIF4E activity reduces tumorigenesis and resistance to apoptosis in certain carcinomas.
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eIF4E is highly expressed in 76% of diffuse large B cell lymphomas, which correlates with poor
prognosis. In particular, patients with recurrent/refractory mantle cell lymphoma (MCL) have been
shown to have significant failureꢀfree survival (FFS) and overall survival (OS) upon modulation of
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eIF4E activity.
These results were consistent with finding that shRNA targeted eIF4E expression
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resulted in an ~80% reduction in colony formation in cancer cells. Given the dependence of MCL on
eIF4E, we chose to determine the uptake and biological activity of our new antiꢀeIF4E ProTide
approach in MCL cells.
Chemistry
For preparation of our target compounds 6aꢀc, we envisioned introduction of the methylthio
alkyl moieties via 5´–O phosphonation using phosphonating agents such as 1aꢀc. (Scheme 1A) Such
phosphonating agents were synthesized by treating the appropriate methylthio alcohol with diphenyl
phosphite followed by aqueous workup and dichloromethane (DCM) washes. The phosphonating agents
1
were then used without further purification, after being judged to be of sufficient purity by H NMR and
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P NMR. The alkylating agent 2 was prepared by alkylation of pꢀchlorophenol with dibromoethane in
7
the presence of sodium hydroxide. Alkylation of 2´,3´ꢀOꢀTBS guanosine with 2 provided the N
alkylated product 4 in 30% yield. With the guanosine derivative 4 in hand, phosphonation of 5´ꢀO was
performed by treatment with phosphonating agents (1aꢀc) in the presence of pivaloyl chloride. The
resulting Hꢀphosphonate product was not isolated due to its propensity to degrade during silica column
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chromatography. Hence, we attempted a oneꢀpot approach to install the tryptamine by employing
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AthertonꢀTodd oxidation conditions. Reaction of the Hꢀphosphonate intermediate with five
equivalents of tryptamine under stringent anhydrous conditions gave only a 1:1 mixture of the desired
tryptamine phosphoramidates 5aꢀc and the phosphodiester side product. (Scheme 1B) We reasoned that
the oxygen source likely resulted from formation of a pivalyl acylꢀphospho mixed anhydride formed the
AthertonꢀTodd oxidation in the presence of pivalic acid, followed by nucleophilic attack at the carbonyl
center (Scheme 1B). Removal of pivalic acid by aqueous workup prior to AthertonꢀTodd oxidation was
found to be beneficial and afforded the desired phosphoramidate 5aꢀc, without significant amounts of
the phosphodiester side products. Subsequent 2´,3´ꢀO desilylation afforded the tryptamine
phosphoramidate diester 6aꢀc.
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In Vitro Stability of Methylthio Alkyl Nucleoside Phosphoramidates
With the synthesized compounds in hand, we next examined the deprotection kinetics of the
methylthio alkyl moieties in an aqueous environment. The compounds 6aꢀc were incubated in HEPES
buffer (pH 7.4, 37 °C) and the progress of the deprotection monitored by reverse phase high
performance liquid chromatography (RPꢀHPLC) (Fig. 3). The ethyl and butyl derivatives, 6a and 6c
underwent significant deprotection under neutral conditions with halfꢀlives (t ) of 8.5 ± 0.9 h and 10.4
½
±
1.1 h, respectively (Table 1). In contrast, deprotection of the propyl derivative, 6b, was found to have a
halfꢀlife of greater than 60 h. The slow deprotection observed for 6b under neutral aqueous conditions is
likely due to the highly unfavorable ability of the 3ꢀ(methylthio)propyl moiety to form the necessary
3
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fourꢀmembered ring sulfonium species.
Given the stability of the protecting groups under neutral
conditions, we then assessed the kinetics of deprotection under acidic condition (HEPES buffer pH 2,
37 °C). Similar to neutral conditions, compounds 6a and 6c exhibited deprotection halfꢀlives of 5.0 ±
0
.4 h and 13.0 ± 2.5 h, respectively (Table 1).
Given the moderate stability of 6a under both neutral and acid conditions, we further evaluated
its halfꢀlife in rat plasma. Incubation of 6a with rat plasma at physiological pH and 37 °C produced
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reasonable deprotection of the 2ꢀ(methylthio)ethyl moiety, with a halfꢀlife of 7.2 ± 1.2 h, which is
similar to the 8.5 h ± 0.9 h halfꢀlife observed at pH 7.2. (Table 1) Consequently, we chose to determine
the ability of 6a to undergo intracellular uptake and delivery of 7ꢀClꢀPhꢀEthylꢀGMP.
Intracellular Uptake of 6a.
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Mantle cell lymphoma cells Granta 519, Mino, and Z138 were treated with 6a and assessed after
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2 hours for cellular uptake and formation of the corresponding monophosphate metabolite, using
tandem liquid chromatography/mass spectrometry (LCꢀMS/MS). Upon cellular uptake of 6a, we found
that 6a undergoes chemical deprotection to form the corresponding monoester phosphoramidate
metabolite 7, which is ultimately hydrolyzed by cellular HINT1 to form the monophosphate metabolite
(Figure 4). LCꢀMS/MS analyses showed substantial accumulation of 7ꢀClꢀPheꢀEthyl GMP in Mino
cells (217.7 ± 29.6 pmol per 5 million cells) and Z138 cells (272 ± 14.2 pmol per 5 million cells). The
observed amount of monophosphate metabolite in these cells was significantly greater than that
observed after exposure to monoester phosphoramidate derivative 7 (Table 2). Furthermore, the
observed amount of monophosphate (per 5 million cells) was significantly lower in Granta 519 cells ꢀ
1
0.3 ± 0.92 pmol (Table 2). Consistent with the significantly higher accumulation of monophosphate
metabolite in Mino and Z138, we observed that these cell lines also had significantly higher
intracellular amounts of monoester phosphoramidate 7 (Table 2). Taken together, 6a increased the
amount of 7ꢀClꢀPheꢀEthyl GMP in MCL cells relative to the corresponding phosphoramidate monoester
7, from 520ꢀfold (Z138 cells) to 1.9ꢀfold (Granta 519 cells) (Table 1). It is not clear why within this set
of MCL cells the delivery efficiency for 7ꢀClꢀPheꢀEthyl GMP varies by over 250ꢀfold. In relation to the
mechanism of cellular uptake and release of the nucleotide, it is likely that intracellular release of 7ꢀClꢀ
PheꢀEthyl GMP proceeds via Route A (Fig. 5). Although, we cannot rule out some degree of uptake via
extracellular activation, followed by uptake of the corresponding phosphoramidate monoester (7)
(Route B in Fig. 5), the low intracellular amounts of 7ꢀClꢀPheꢀEthyl GMP observed in all the cell lines
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after incubation with monoester phosphoramidate 7, suggests that the cellular uptake is dominated by
Route A (Fig. 5) and not Route B (Fig. 5).
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Biological Activity of 6a.
With the confirmed enhanced intracellular delivery of 7ꢀClꢀPheꢀEthylꢀGMP by 6a over 7, we set
out to assess the inhibition of eIF4E activity in MCL cells treated with 6a. First, disruption of the
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interaction between mRNA 5´ cap and eIF4E was assessed by a 7ꢀmethylguanosine 5´ꢀtriphosphate (m
GTP) pullꢀdown assay on lysates generated from MCL cells exposed to 6a. The binding of free eIF4E
was clearly blocked for cells treated with 6a, compared to untreated MCL cells (Fig. 6). These results
are consistent with the results of our intracellular uptake and metabolism studies demonstrating delivery
of 7ꢀClꢀPheꢀEthylꢀGMP.
3
3
Due to the implication of eIF4E hyperactivation as a driver of lymphomagenesis , we then
assessed if 6a was able to inhibit proliferation and reduce survival in MCL cells. MCL proliferation, as
a measure of thymidine incorporation, was significantly reduced in a dose dependent manner by 6a
(IC = 50 ꢁM, Fig. 6B), with complete inhibition of proliferation observed at 200 ꢁM. Surprisingly,
5
0
with the exception of Z138 cells, survival of MCL cells was not significantly affected by 6a (IC > 200
5
0
ꢁ
M) when compared to untreated control (Fig. 6C). The observed decrease in cellular proliferation after
exposure to 6a may result from reduced translation of proꢀproliferative mRNA transcripts.
34, 35
The transcription factor cꢀMyc is a known promoter of cell proliferation
and its
3
6
overexpression correlates with a negative prognosis in a variety of lymphomas. Specifically, previous
studies have shown that overexpression of cꢀMyc positively cooperates with eIF4E and other
3
7ꢀ39
components of the eIF4F complex to drive translation in lymphomas.
eIF4F is a protein complex
consisting of eIF4E, eIF4G and RNA helicase eIF4A, of which eIF4E is the limiting component. To
assess if translation of cꢀMyc in MCL was governed by eIF4E, we determined if eIF4E physically
associates with cꢀMyc mRNA. RNA immunoprecipitation with eIF4E suggests that eIF4E does
physically associate with cꢀMyc transcripts in MCL and that such association is much higher when
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compared to eIF4E association with the proꢀsurvival Bclꢀ2 transcript (Supporting Information Figure
S1). This result suggests that eIF4E might be directly regulating translation of cꢀMyc in MCL. Indeed
MCL cell lines Mino and Granta, showed a dose dependent reduction in the expression levels of cꢀMyc
and a dose dependent increase in p27 levels upon exposure to 6a, with only a negligible effect on
expression of Bclꢀ2 and Mclꢀ1 (Fig. 6D). Furthermore, the observed reduction in the expression levels
of cꢀMyc appears to be strictly due to reduced translation since the levels of cꢀMyc mRNA remain
unchanged in Mino cells exposed to 6a, compared to untreated control (Supporting Information Figure
S2). The observed dose dependent buildꢀup of p27 in response to 6a is interesting since it suggests
protection against loss of p27, which is a hallmark in MCL. Reduced p27 expression correlates with
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poor overall survival rates and prognosis in MCL patients. p27 is an inhibitor of cyclinꢀdependent
4
0ꢀ42
kinase (CDK) and cyclin D1 is overexpressed in MCL.
Thus, the result suggests that 6a causes an
accumulation of p27, which could counter the abnormally high levels of cyclin D1 and elicit an antiꢀ
proliferative phenotype in MCL cells.
Mantle cell lymphoma is an aggressive Bꢀcell lymphoma, largely resistant to traditional
chemotherapy. Consistent with the effect of eIF4E antagonism on cꢀMyc, previous studies have reported
4
3
that EꢀꢁMyc/eIF4E lymphomas are resistant to doxorubicin as a single anticancer therapy. However,
the observed chemoresistance in EꢀꢁMyc/eIF4E lymphoma mice was reversed when doxorubicin
43
therapy was coupled with attenuated eIF4F activity. Thus, we next assessed if chemoresistance in
MCL cells can be reversed upon treatment with 6a. Mino cells showed no reduction in proliferation
when treated with doxorubicin alone compared to untreated control (Fig. 7A). However, treatment with
6
a alone showed significant reduction in proliferation, which was further reduced when Mino cells were
exposed to both doxorubicin and 6a. The observed chemosensitization was significant (p = 0.05) when
compared to untreated control, doxorubicin treated cells, or cells treated with 6a alone (Fig. 7A).
Surprisingly, there was no significant impact on survival of Mino cells with the doxorubicin/6a
combination treatment compared to untreated control, doxorubicin treated cells, or 6a treated cells. We
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speculate that the observed resistance to apoptosis could possibly be a result of proꢀsurvival signaling
by Bclꢀ2, whose levels were relatively unchanged in Mino cells treated with 6a (Fig. 6D).
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To assess if concomitant inhibition of eIF4E and downregulation of Bclꢀ2 is advantageous for
reducing survival in Mino cells, we treated Mino cells with 6a and dexamethasone. A previous study
had shown that dexamethasone induced down regulation of Bclꢀ2 expression in acute lymphoblastic
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leukemia. Combination treatment of Mino cells with 6a and dexamethasone produced results that
were similar to that observed in doxorubicin/6a treated cells (p = 0.05). We observed an enhanced antiꢀ
proliferative response with dexamethasone/6a combination treatment when compared to untreated
control, dexamethasone alone, or 6a alone in Mino cells (Fig. 7B). However, survival of Mino cells was
little changed when compared to cells treated with 6a alone or dexamethasone alone (Fig. 7B). This
result suggests the involvement of other molecular processes as drivers of Mino cell survival. However,
we do not rule out the possibility that Mino cells are resistant to dexamethasone induced apoptosis or
that dexamethasone does not induce down regulation of proꢀsurvival Bclꢀ2 protein in Mino cells.
To further probe if downregulation of Bclꢀ2 and inhibition of eIF4E are sufficient to reduce
survival in Mino cells, we elected to disrupt Bꢀcell receptor (BCR) signaling through inactivation of
Bruton tyrosine kinase (BTK). BCR signaling has been shown to be involved in activation of NFꢀκB
4
5, 46
47
pathway in MCL.
NFꢀκB is a transcription factor that regulates expression of Bclꢀ2 , which is
46
overexpressed in MCL cells and primary tumors. Recently, therapeutic inhibition of BCR/BTK
pathway has entered the clinic for treatment of patients with recurrent MCL. Ibrutinib is a potent
inhibitor of BTK and despite its approval as a single agent; many patients still acquire resistance to
48
ibrutinib. We next assessed if combination treatment of 6a and ibrutinib could reverse
chemoresistance in MCL cells. Mino cells were treated with ibrutinib (25 ꢁM) in the presence or
absence of 6a, and proliferation and survival were analyzed. Mino cells showed no reduction in
proliferation or survival when treated with ibrutinib alone compared to untreated control (Fig. 7C).
However, treatment with 6a alone showed significant reduction in proliferation (p = 0.05), which was
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further reduced when exposed to both ibrutinib and 6a. Interestingly, the combination treatment also
had a significant effect (p=0.05) on the survival of Mino cells (Fig. 7C). We also evaluated the effect of
ibrutinib alone or in combination with 6a on cꢀMyc protein by western blotting. Interestingly both
ibrutinib and 6a reduced the level of cꢀMyc, but the combination treatment had a much more profound
effect on the downregulation of cꢀMyc (Fig. 8). Altogether, these results suggest that chemoresistance in
MCL cells could be reversed through a combination of eIF4E inhibition and attenuation of BCR
signaling. This is consistent with the previous report by Wu and coworkers, who showed that
simultaneous inhibition of BTK activity and MNK mediated phosphorylation of eIF4E elicited a strong
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apoptotic response in hematologic malignancies.
Cap dependent translation initiation complex is negatively regulated by phosphorylated 4EBP1
protein. Several studies have shown that 4EBP1 is hyperphosphorylated in lymphoma patient samples
50, 51
and correlates with poor prognosis.
Next, we sought to determine if the combination of 6a and
ibrutinib could reduce phosphorylation of 4EBP1 at both serine (Ser 65) and threonine sites (Thr
5
2
37/46). Phosphorylation of 4EBP1 at Thr37/46 is regulated by mTOR and does not prevent its binding
53
to eIF4E, but primes 4EBP1 for subsequent phosphorylation at Ser 65. Compound 6a alone did not
have much effect on 4EBP1 phosphorylation at either site. However, ibrutinib alone was able to
decrease 4EBP1 phosphorylation at Ser 65 without any change in phosphorylation at Thr 37/46.
Interestingly the combination treatment reduced 4EPBP1 phosphorylation at only the Ser 65 site (Fig.
8).
In vivo properties of 6a.
To assess the potential of our proTide approach to enhance the bioavailability of the parent nucleoside
phosphoramidate, we carried out pharmacokinetic (PK) studies of 6a and 7 in female SpragueꢀDawley
rats (pharmacokinetic parameters of 6a and 7 are summarized in Table 3.) After intravenous (i.v.)
administration (2.5 mg/kg), 6a exhibited biexponential kinetics with a rapid distribution phase followed
by a slow terminal elimination phase resulting in an extremely large steady state volume of distribution
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–
1
–1
(
V = 205 ± 45) (Supporting Information Figure S3). The high clearance of 6a (1.8 ± 0.2 L·min ·kg )
d
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indicates extraꢀhepatic metabolism since the clearance vastly exceeds rat hepatic blood flow (0.055
ꢀ1
L·min ) and is consistent with nonenzymatic cleavage of the methylthioꢀalkylꢀprotecting group to
release 7. The extensive tissue distribution of 6a compensates for the rapid clearance resulting in a
respectable halfꢀlife (t ) of 79 minutes. The C of 6a and 7 following oral (p.o.) dosing (25 mg/kg) of
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½
max
6
a were 5.0 ng/mL and 6.0 ng/mL, respectively, which were achieved at 30 minutes. The oral
bioavailability (F) of 6a was 23% based on the combined AUC0ꢀ8h values of 6a and 7, which
underestimates F since the levels of 6a and 7 remained steady and did not decline at 8 hours. The parent
drug 7ꢀClꢀPheꢀEthylꢀGMP was not detected in the plasma after both oral and i.v. doses of 6a, which
further confirms the prodrug design strategy as phosphoramidate cleavage of 7 by Hint1 is expected to
primarily occur in tissues. Given the highly polar (cationic) nature of the nucleoside, it is probable that a
higher level of oral bioavailability and enhanced in vivo life time will be observed with more nonꢀpolar
5
4
nucleosides. To evaluate the impact of the methylthioꢀalkylꢀprotecting group in 6a, we also performed
analogous PK studies with phosphoramidate monoester 7, which exhibited substantially lower oral
bioavailability (F = <1%) affirming the importance of the methylthioꢀalkylꢀprotecting group. The
volume of distribution and clearance of 7 were 3ꢀ and 6ꢀfold lower than 6a, respectively resulting a
nearly 2ꢀfold reduced terminal elimination rate constant and corresponding 2ꢀfold longer halfꢀlife. The
–1
–
oral exposure of 7 (AUC0ꢀ8h = 1.9 µg·min·mL ) was nearly identical to 6a (AUC
= 1.7 µg·min·mL
0ꢀ8h
1
) because the 6ꢀfold reduced clearance of 7 relative to 6a was nearly perfectly offset by the 6ꢀfold
lower bioavailability of 7. Direct i.v administration of 7 to rats provided substantially higher levels of
–
this compound enabling detection of the parent drug 7ꢀClꢀPheꢀEthylꢀGMP (AUC0ꢀ8h = 13.1 µg·min·mL
1
, C5min = 583 ng/mL) in plasma. Collectively, these results suggest that the prodrug strategy might offer
sufficient protection from first pass hepatic extraction, thus making it possible to achieve systemic
circulation of 2ꢀ(methylthio)ethyl and phosphoramidate monoester containing proTides. Such feasibility
could be especially attractive for the delivery of both pronucleotides to organs other than the liver.
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Conclusions
Kinase bypass strategies have proven to be an effective means for delivering otherwise charged
2, 55
nucleotides in cells.
The US Food and Drug Administration (FDA) approval of the phosphoramidate
ProTide, sofosbuvir, for the curative treatment of HCV is a testament to the effectiveness/utility of
bypass strategies to deliver clinically relevant nucleotide analogs into cells. Phosphoramidate diester
ProTides, such as in sofosbuvir, mask the negative charge of the intermediate phosphoramidate
monoester, thus enhancing their cellular uptake and potential for monophosphate delivery. Here, we
have described the design and development of a ProTide strategy, which incorporates a chemically
cleaved 2ꢀ(methythio)ethyl moiety and an enzymatically cleaved tryptamine phosphoramidate as
phosphate protecting groups. The incorporation of 2ꢀ(methylthio)ethyl as a phosphate protecting group
eliminates the need for carboxyesteraseꢀdependent activation. Furthermore, the new strategy was
successfully utilized to generate 6a, which exhibited efficient in vitro and inꢀcell activation to produce
the parental nucleotide, 7ꢀClꢀPhꢀEthylꢀGMP, a potent inhibitor of eIF4E in capꢀdependent translation. In
vitro, 6a was found to inhibit MCL cellular proliferation, without significantly affecting cell survival. It
is possible that the inhibition of eIF4E activity induces cell cycle arrest similar to the effect observed
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3, 56
with mTOR inhibitors rapamycin and PP242 in lymphoma.
Work on this hypothesis is on going in
our laboratory and will be reported in due time. The combination of doxorubicin, dexamethasone, or
ibrutinib with 6a demonstrated the ability to reverse chemoresistance observed in Mino cells that were
treated with doxorubicin, dexamethasone, or ibrutinib alone. Taken together, these results demonstrate
that 2ꢀ(methylthio)ethyl phosphoramidate ProTides are capable of enhancing cellular delivery of
monoester nucleoside phosphoramidates and the delivery of the corresponding nucleoside
monophosphate. In addition, we have demonstrated that pharmacological abrogation of eIF4E:mRNA 5´
ꢀcap interaction could be an attractive anticancer strategy in mantle cell lymphomas. Most importantly,
this work has established 6a as a chemical tool, which is specific to eIF4E, for use in studying
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Journal of Medicinal Chemistry
translational control in biological systems where aberrant capꢀdependent protein translation leads to a
disease phenotype.
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6
Experimental Section
All chemicals and reagents were obtained from commercial sources and were used without further
purification. Anhydrous N,NꢀDimethylformamide (DMF) was obtained from a dry solvent purification
system (MBraun) and dispensed under argon. Dexamethasone, doxorubicin, and ibrutinib were
purchased from Sigma Pharmaceuticals. Antibodies for eIF4E, eIF4G, cꢀMyc, p27, p4EBP1, and Bclꢀ2
were purchased from Cell Signaling Technologies. Antibody to Actin was purchased from Santa Cruz
0
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0
(Dallas, TX, USA). All reactions were performed under an atmosphere of dry nitrogen unless otherwise
noted. All silica gel chromatography and preparative reverse phase purification was performed on a
Teledyne Isco CombiFlash Rf system, using Redisep Rf high performance gold silica gel columns (for
normal phase purifications) and Redisep Rf high performance gold C18 columns (for reverse phase
purifications). Reverse phase purifications were with water and acetonitrile. Lyophilization of
compounds after reverse phase purification was performed on a FreeZone 12 Plus freeze dry system
(
Labconco). All analytical HPLC analyses were performed on a Beckman Coulter System Gold
instrument using a Haisil 100 C18 column (4.6 mm X 250 mm, 5 ꢁm, Higgins Analytical Inc.); eluting
with a gradient of 10% acetonitrile in phosphate buffer (50 mM, pH 7.4) to 90% acetonitrile in
phosphate buffer over 24 minutes at a flow rate of 1.0 mL/min. The purity of all test compounds was
determined with RPꢀHPLC and all test compounds were ≥95 % pure. All Nuclear Magnetic Resonance
spectra were obtained on a Brucker Avance III HD 500 MHz spectrometer or on an Agilent/Varian 600
MHz spectrometer at ambient temperature. Chemical shifts were recorded in parts per million using the
solvent peaks as internal references. All highꢀresolution mass spectroscopy (HRMS) were performed on
a LTQ Orbitrap Velos instrument (Thermo Scientific) in positiveꢀion mode.
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General Procedure for the Synthesis of Methylthio Alkyl HꢁPhosphonates Triethylammonium Salts 1a-c.
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Diphenyl phosphite (1 equiv) and anhydrous pyridine (10 mL) were added to an ovenꢀdried round
bottom flask and purged/evacuated extensively with N under vacuum. To the mixture was added the
2
appropriate methythio alcohol and stirred for two hours at ambient temperature. Triethylamine/water
(
1:1, 6 mL) was added and the reaction was stirred for three hours at ambient temperature. The reaction
was concentrated in vacuo at ambient temperature. The resulting oily residue was dissolved with water
10 mL) and washed with DCM (5 X 10 mL). The aqueous layer was concentrated in vacuo without
heating, dried overnight on high vacuum, and used without further purification after being judged to be
0
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0
(
1
31
of sufficient purity by NMR spectroscopy ( HNMR and PNMR).
Triethylammonium 2ꢁ(methylthio)ethyl phosphonate (1a). The title compound was prepared from 2ꢀ
(Methylthio)ethanol (0.76 mL, 8.74 mmol) using the general procedure for synthesis of methylthio alkyl
1
Hꢀphosphonate triethylammonium salts. The compound was obtained as a clear oil. H NMR (500
MHz, DMSOꢀd ) δ 1.18 (t, J = 7.5 Hz, 6H), 2.08 (s, 3H), 2.66 (t, J = 7.0 Hz, 2H), 3.04 (q, J = 7.0 Hz,
6
1
3
4H), 3.87 (q, J = 7.0 Hz, 2H), 6.64 (d, JPH = 619.5 Hz, 1H), 10.32 (br s, 1H). C NMR (125 MHz,
31
DMSOꢀd ) δ 8.4, 14.9, 33.7 (d, JCꢀP = 6.3 Hz), 45.3, 62.2 (d, JCꢀP = 4.6). P NMR (202 MHz, DMSOꢀ
6
d6) δ 2.28.
Triethylammonium 3ꢁ(methylthio)propyl phosphonate (1b). The title compound was prepared from 3ꢀ
(Methylthio)ꢀ1ꢀpropanol (0.90 mL, 8.75 mmol) using the general procedure for synthesis of methylthio
1
alkyl Hꢀphosphonate triethylammonium salts. The compound was obtained as a clear oil. H NMR (500
MHz, DMSOꢀd ) δ 1.17 (t, J = 7.5 Hz, 5H), 1.79 (quint, J = 7.0, 2H), 2.04 (s, 3H), ~2.50 (obscured by
6
13
solvent peak), 3.04 (m, 4H), 3.83 (q, J = 6.5 Hz, 2H), 6.61 (d, JPH = 625.0 Hz, 1H), 10.2 (br s, 1H). C
3
1
NMR (125 MHz, DMSOꢀd ) δ 8.4, 14.6, 29.5, 29.7 (d, J = 6.4 Hz), 45.4, 61.9 (d, JCꢀP = 4.6). P
6
CꢀP
NMR (202 MHz, DMSOꢀd ) δ 2.69.
6
Triethylammonium 4ꢁ(methylthio)butyl phosphonate (1c). The title compound was prepared from 4ꢀ
(Methylthio)ꢀ1ꢀbutanol (1.06 mL, 8.74 mmol) using the general procedure for synthesis of methylthio
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1
alkyl Hꢀphosphonate triethylammonium salts. The compound was obtained as a clear oil. H NMR (500
1
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
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6
MHz, DMSOꢀd ) δ 1.18 (t, J = 7.5 Hz, 5H), 1.59 (m, 4H), 2.03 (s, 3H), 2.47 (t, J = 7.0 Hz, 2H), 3.04 (q,
6
1
3
J = 7.0 Hz, 4H), 3.74 (q, J = 6.0 Hz, 2H), 6.59 (d, JPH = 614.5 Hz, 1H), 10.23 (br s, 1H). C NMR (125
31
MHz, DMSOꢀd ) δ 8.5, 14.6, 25.0, 29.3 (d, J = 6.8 Hz), 32.8, 45.4, 62.6 (d, J = 5.0). P NMR (202
6
CꢀP
MHz, DMSOꢀd ) δ 2.58.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
1
ꢁ(2ꢁbromoethoxy)ꢁ4ꢁchlorobenzene (2). 4ꢀchlorophenol (6.43 g, 50 mmol) and 22.5% NaOH solution
(18.5 mL) were added to a round bottom flask at ambient temperature. To the mixture was added 1,2ꢀ
dibromoethane (17.2 mL, 200 mmol) and the reaction was refluxed overnight. The reaction extracted
with DCM (3X) and the organic extract was washed with H O, dried over Na SO , filtered and
2
2
4
concentrated in vacuo. The crude product was purified by silica gel flash column chromatography,
1
eluting with petroleum ether/EtOAc (0 – 5% pet. ether) to give 2 as a clear oil (9.47 g, 80% yield). H
NMR (500 MHz, CDCl ) δ 3.64 (t, J = 6.0 Hz, 2H), 4.28 (t, J = 6.0 Hz, 2H), 6.87 (d, J = 9.0 Hz, 2H),
3
1
3
7
.27 (d, J = 9.0 Hz, 2H). C NMR (125 MHz, CDCl ) δ 29.0, 68.3, 116.2, 126.5, 129.6, 156.8. HRMSꢀ
3
+
+
ESI (m/z) calcd [M + H] for C H BrClO: 234.9520. Found: 234.9526.
8
8
2
´,3´ꢁdiꢁOꢁTBDMSꢁguanosine (3). To an ovenꢀdried round bottom flask was added guanosine (3.0 g,
0.6 mmol), imidazole (5.8 g, 84.7 mmol), and DMF (57.0 mL). The reaction vessel was
1
purged/evacuated extensively N , TBDMSCl (6.40 g, 42.4 mmol) was added and the reaction was
2
stirred overnight at room temperature. The reaction was quenched/diluted with water (30 mL) and
extracted with DCM (3X). The organic extract was washed with sat. aq. NH Cl (1X), water (2X), brine
4
(
1X), dried over MgSO , filtered, and concentrated in vacuo. To the resulting residue was added 80%
4
AcOH (86.0 mL) and stirred at 60 °C for 12 hours. Acetic acid was removed under vacuum and
coevaporated with EtOH. The resulting residue was purified by silica gel flash chromatography, eluting
CHCl /MeOH (0 to 20% MeOH) to give the title compound as a white solid (4.01 g, 74% yield over
3
1
two steps). H NMR (500 MHz, DMSOꢀd ) δ ꢀ0.31 (s, 3H), ꢀ0.09 (s, 3H), 0.10 (s, 3H), 0.11 (s, 3H),
6
0
.73 (s, 9H), 0.91 (s, 9H), 3.53 – 3.57 (m, 1H), 3.64 – 3.68 (m, 1H), 3.91 (t, J = 4.0 Hz, 1H), 4.23 (d, J
17
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Journal of Medicinal Chemistry
Page 18 of 44
=
4.5 Hz, 1H), 4.68 (dd, J = 4.5 Hz and 7.0 Hz, 1H), 5.21 (t, J = 5.5 Hz, 1H), 5.73 (d, J = 7.0 Hz, 1H),
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
3
6
.44 (s, 2H), 7.97 (s, 1H), 10.6 (br s, 1H). C NMR (125 MHz, DMSOꢀd ) δ ꢀ5.5, ꢀ4.78, ꢀ4.76, ꢀ4.7,
6
+
17.6, 17.8, 25.5, 25.7, 61.2, 73.1, 75.0, 85.7, 86.7, 116.7, 135.7, 151.4, 153.6, 156.6. HRMSꢀESI (m/z)
+
calcd [M + H] for C H N O Si : 512.2719. Found: 512.2725.
2
2
41
5
5
2
2
ꢀaminoꢀ9ꢀ((2R,3R,4R,5R)ꢀ3,4ꢀbis((tertꢀbutyldimethylsilyl)oxy)ꢀ5ꢀ(hydroxymethyl)tetrahydrofuranꢀ2ꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
yl)ꢀ7ꢀ(2ꢀ(4ꢀchlorophenoxy)ethyl)ꢀ6ꢀoxoꢀ6,9ꢀdihydroꢀ1Hꢀpurinꢀ7ꢀium (4). To an ovenꢀdried round
bottom flask was added 3 (1 g, 1.95 mmol) and dry DMF (20 mL). The solution was purged/evacuated
extensively with N under vacuum. To the solution was added 2 (2.02 g, 8.60 mmol) and the reaction
2
was stirred for 72 h at 70 °C. The mixture was concentrated in vacuo and the resulting residue was
purified by silica gel flash column chromatography, eluting with CHCl /MeOH (+10% NH OH) –
3
4
1
gradient 0 – 20% to give 4 as slight yellow solid (0.444 g, 34% yield). H NMR (500 MHz, DMSOꢀd )
6
δ ꢀ0.26 (s, 3H), ꢀ0.09 (s, 3H), 0.08 (s, 3H), 0.11 (s, 3H), 0.75 (s, 9H), 0.90 (s, 9H), 3.59 – 3.61 (m, 1H),
3
.76 – 3.79 (m, 1H), 4.03 (d, J = 3.0 Hz, 1H), 4.27 (m, 1H), 4.37 – 4.44 (m, 2H), 4.81 (t, J = 5.0 Hz,
2H), 4.85 (t, J = 5.5 Hz, 1H), 5.55 (s, 2H), 5.82 (d, J = 6.0 Hz, 1H), 5.90 (br s, 1H), 6.95 (d, J = 9.0 Hz,
13
2
H), 7.31 (d, J = 9.0 Hz, 2H), 9.21 (s, 1H). C NMR (125 MHz, DMSOꢀd ) δ ꢀ5.4, ꢀ5.1, ꢀ4.9, ꢀ4.6, 17.5,
6
1
7.7, 25.5, 25.7, 47.7, 60.6, 65.9, 72.1, 74.4, 87.1, 88.9, 107.9, 116.3, 124.8, 129.2, 133.2, 149.4, 156.6.
+
+
+
HRMSꢀESI (m/z) calcd [M] for C H ClN O Si : 666.2904. Found: 666.2885.
30
49
5
6
2
General Procedure for Preparation of Methylthio Alkyl Tryptamine Nucleoside Diester
Phosphoramidates. Compound 4 (1 equiv) and the appropriate Hꢀphosphonate 1aꢀc (1.5 equiv) were
added to an ovenꢀdried round bottom flask and dried overnight on the high vacuum. To the flask was
added anhydrous pyridine and the mixture was purged/evacuated extensively with N under vacuum. To
2
the mixture was added PivCl (2.75 equiv) in a dropwise manner and the reaction was stirred for seven
hours at ambient temperature. The reaction was quenched over sat. aq. NaHCO and extracted three
3
times with DCM. The organic extract was dried over Na SO , filtered, and concentrated in vacuo at
2
4
ambient temperature and dried for an hour on the high vacuum. The dried residue was dissolved with
18
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Page 19 of 44
Journal of Medicinal Chemistry
anhydrous pyridine and the reaction vessel was purged/evacuated extensively with N under vacuum. To
2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the solution was added Et N (2 equiv). Tryptamine (4.0 equiv, dissolve in dry pyridine) and CCl (1.5
3
4
equiv) were added simultaneously to the reaction mixture and stirred for 25 min at ambient temperature.
The reaction was diluted with MeOH and concentrated in vacuo at ambient temperature. The resulting
residue was purified by silica gel flash column chromatography, eluting with CHCl /MeOH (+10%
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
NH OH) – gradient 0 – 20% to give semiꢀpurified products 5aꢀc as offꢀwhite solids. The products tend
4
to coelute with unreacted nucleoside 4, hence all fractions with product were pooled and advanced to
the desilylation step.
General Procedure for Desilylation. To a solution of semiꢀpurified 5 (1.0 equiv) in dry THF at 0 °C was
added 1 M TBAF (3.0 equiv) dropwise until reaction became cloudy. The reaction was removed from
the ice bath and stirred for five minutes at ambient temperature. The reaction was diluted with MeOH
and concentrated in vacuo at ambient temperature. The resulting residue was purified by silica gel flash
column chromatography, eluting with CHCl /MeOH (+10% NH OH) – gradient 0 – 20% to give 6 as
3
4
slight yellow solid. Reverse phase flash column purification afforded the products as white powders
after lyophilization.
9
ꢀ((2R,3R,4R,5R)ꢀ5ꢀ(((((2ꢀ(1Hꢀindolꢀ3ꢀyl)ethyl)amino)(2ꢀ(methylthio)ethoxy)phosphoryl)oxy)methyl)ꢀ
,4ꢀbis((tertꢀbutyldimethylsilyl)oxy)tetrahydrofuranꢀ2ꢀyl)ꢀ2ꢀaminoꢀ7ꢀ(2ꢀ(4ꢀchlorophenoxy)ethyl)ꢀ6ꢀ
3
oxoꢀ6,9ꢀdihydroꢀ1Hꢀpurinꢀ7ꢀium (5a). The title compound was prepared from 4 (734 mg, 1.10 mmol),
PivCl (373 ꢁL, 3.02 mmol), and 1a (424 mg, 1.65 mmol) in anhydrous pyridine (17.4 mL). The second
step of the synthesis was performed with CCl (160 ꢁL, 1.65 mmol), tryptamine (705 mg, 4.40 mmol),
4
Et N (0.31 mL, 2.20 mmol). The title compound was obtained as an offꢀwhite solid after silica gel flash
3
column chromatography. The semiꢀpurified product was advanced to the next step.
9
ꢀ((2R,3R,4R,5R)ꢀ5ꢀ(((((2ꢀ(1Hꢀindolꢀ3ꢀyl)ethyl)amino)(3ꢀ
(methylthio)propoxy)phosphoryl)oxy)methyl)ꢀ3,4ꢀbis((tertꢀbutyldimethylsilyl)oxy)tetrahydrofuranꢀ2ꢀ
yl)ꢀ2ꢀaminoꢀ7ꢀ(2ꢀ(4ꢀchlorophenoxy)ethyl)ꢀ6ꢀoxoꢀ6,9ꢀdihydroꢀ1Hꢀpurinꢀ7ꢀium (5b). The title compound
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Journal of Medicinal Chemistry
Page 20 of 44
was prepared from 4 (101 mg, 0.151 mmol), PivCl (52 ꢁL, 0.416 mmol), and 1b (62 mg, 0.227 mmol)
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
in anhydrous pyridine (2.3 mL). The second step of the synthesis was performed with CCl (22 ꢁL,
4
0.227 mmol), tryptamine (97 mg, 0.604 mmol), Et N (42 ꢁL, 0.302 mmol). The title compound was
3
obtained as an offꢀwhite solid after silica gel flash column chromatography and the semiꢀpurified
product was advanced to the next step.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
9
ꢀ((2R,3R,4R,5R)ꢀ5ꢀ(((((2ꢀ(1Hꢀindolꢀ3ꢀyl)ethyl)amino)(4ꢀ(methylthio)butoxy)phosphoryl)oxy)methyl)ꢀ
,4ꢀbis((tertꢀbutyldimethylsilyl)oxy)tetrahydrofuranꢀ2ꢀyl)ꢀ2ꢀaminoꢀ7ꢀ(2ꢀ(4ꢀchlorophenoxy)ethyl)ꢀ6ꢀ
3
oxoꢀ6,9ꢀdihydroꢀ1Hꢀpurinꢀ7ꢀium (5c). The title compound was prepared from 4 (150 mg, 0.225 mmol),
PivCl (76 ꢁL, 0.619 mmol), and 1c (91.5 mg, 0.337 mmol) in anhydrous pyridine (3.6 mL). The second
step of the synthesis was performed with CCl (52 ꢁL, 0.337 mmol), tryptamine (144 mg, 0.900 mmol),
4
Et N (63 ꢁL, 0.450 mmol). The title compound was obtained as an offꢀwhite solid after silica gel flash
3
column chromatography and the semiꢀpurified product was advanced to the next step.
9
ꢀ((2R,3R,4S,5R)ꢀ5ꢀ(((((2ꢀ(1Hꢀindolꢀ3ꢀyl)ethyl)amino)(2ꢀ(methylthio)ethoxy)phosphoryl)oxy)methyl)ꢀ
3,4ꢀdihydroxytetrahydrofuranꢀ2ꢀyl)ꢀ2ꢀaminoꢀ7ꢀ(2ꢀ(4ꢀchlorophenoxy)ethyl)ꢀ6ꢀoxoꢀ6,9ꢀdihydroꢀ1Hꢀ
purinꢀ7ꢀium (6a). The title compound was prepared using 5a (402.8 mg, 0.418 mmol), 1 M TBAF (1.25
mL, 1.25 mmol), and dry THF (22 mL). The product was obtained as a white powder after reverse
1
phase flash column chromatography (0.152 g, 19% yield over two steps). H NMR (500 MHz, DMSOꢀ
d6) δ 2.02 (d, J = 17.5 Hz, 3H), 2.65 (q, J = 6.5 Hz, 2H), 2.83 (q, J = 6.0 Hz, 2H), 3.05 (m, 2H), 3.88 –
3.99 (m, 2H), 4.05 – 4.29 (m, 4H), 4.40 – 4.44 (m, 2H), 4.55 (br s, 1H), 4.66 – 4.77 (m, 2H), 5.19 (dq, J
=
–
13.5, 7.0 Hz, 1H), 5.46 (br s, 1H), 5.81 (br s, 2H), 5.86 (d, J = 4.5 Hz, 1H), 6.91 – 6.96 (m, 3H), 7.02
7.06 (m, 1H), 7.13 (d, J = 11.0 Hz, 1H), 7.26 – 7.33 (m, 3H), 7.45 – 7.49 (m, 1H), 9.09 (d, J = 7.0 Hz,
1
3
1H), 10.8 (s, 1H). C NMR (125 MHz, DMSOꢀd ) δ 14.8, 14.9, 27.6, 33.1, 41.7, 47.5, 64.2, 65.9, 69.9,
6
7
1
3.2, 83.2, 88.7, 107.8, 111.4, 111.5, 116.4, 118.1 (d, J = 3.9 Hz), 118.2 (d, J = 3.4 Hz), 120.9, 122.7,
31
24.8, 127.1, 129.3, 132.5, 136.2, 149.8, 156.6, 162.7, 163.7. P NMR (202 MHz, DMSOꢀd ) δ 10.45
6
+
+
+
and 10.31. HRMSꢀESI (m/z) calcd [M] for C H ClN O PS : 734.1923. Found: 734.1908.
31
38
7
8
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Journal of Medicinal Chemistry
9
ꢀ((2R,3R,4S,5R)ꢀ5ꢀ(((((2ꢀ(1Hꢀindolꢀ3ꢀyl)ethyl)amino)(3ꢀ
methylthio)propoxy)phosphoryl)oxy)methyl)ꢀ3,4ꢀdihydroxytetrahydrofuranꢀ2ꢀyl)ꢀ2ꢀaminoꢀ7ꢀ(2ꢀ(4ꢀ
chlorophenoxy)ethyl)ꢀ6ꢀoxoꢀ6,9ꢀdihydroꢀ1Hꢀpurinꢀ7ꢀium (6b). The title compound was prepared using
b (66.1 mg, 0.058 mmol), 1 M TBAF (203 ꢁL, 0.203 mmol), and dry THF (3.7 mL). The product was
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
5
obtained as a white powder after reverse phase flash column chromatography (15.3 mg, 9% yield over
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
two steps). H NMR (500 MHz, DMSOꢀd ) δ 1.77 (sex, J = 14.5, 6.5 Hz, 2H), 1.98 (d, J = 11.5 Hz,
6
3
H), 2.45 (dt, J = 12.0, 7.5 Hz, 2H), 2.82 (q, J = 7.0 Hz, 2H), 2.97 – 3.06 (m, 2H), 3.84 – 3.94 (m, 2H),
4.04 – 4.28 (m, 4H), 4.37 – 4.44 (m, 2H), 4.54 (d, J = 4.0 Hz, 1H), 4.66 – 4.77 (m, 2H), 5.15 (dq, J =
1
4.0, 7.0 Hz, 1H), 5.44 (t, J = 4.5 Hz, 1H), 5.75 (d, J = 5.0 Hz, 1H), 5.81 (br s, 1H), 5.86 (d, J = 4.5 Hz,
H), 6.94 (m, 3H), 7.04 (m, 1H), 7.12 (dd, J = 10.0, 1.5 Hz, 1H), 7.27 (dd, J = 9.0, 6.0 Hz, 2H), 7.32
1
1
3
(dd, J = 8.0, 3.5 Hz, 1H), 7.46 (dd, J = 12.5, 8.0 Hz, 1H), 9.09 (d, J = 6.0 Hz, 1H), 10.79 (br s, 1H). C
NMR (150 MHz, DMSOꢀd ) δ 14.5, 27.7, 29.3 (d, J = 2.9 Hz), 29.35 (d, J = 6.6 Hz), 41.7 (d, J = 4.4
6
Hz), 47.5, 64.1, 65.2, 65.8, 69.9, 73.2, 83.2, 88.7, 107.8, 111.4, 111.5, 116.4 (d, J = 3.5 Hz), 118.1, 118.2
(d, J = 4.2 Hz), 120.9, 122.7, 124.8, 127.1 (d, J = 3.3 Hz), 129.3, 132.5, 136.2, (d, J = 1.8 Hz), 149.9,
31
+
1
56.6 (d, J = 2.4 Hz), 162.4, 163.4. P NMR (202 MHz, DMSOꢀd ) δ 10.55 and 10.39. HRMSꢀESI
6
+
+
(m/z) calcd [M] for C H ClN O PS : 748.2080. Found: 748.2058.
32 40 7 8
9
ꢀ((2R,3R,4S,5R)ꢀ5ꢀ(((((2ꢀ(1Hꢀindolꢀ3ꢀyl)ethyl)amino)(4ꢀ(methylthio)butoxy)phosphoryl)oxy)methyl)ꢀ
,4ꢀdihydroxytetrahydrofuranꢀ2ꢀyl)ꢀ2ꢀaminoꢀ7ꢀ(2ꢀ(4ꢀchlorophenoxy)ethyl)ꢀ6ꢀoxoꢀ6,9ꢀdihydroꢀ1Hꢀ
purinꢀ7ꢀium (6c). The title compound was prepared using 5c (15.2 mg, 0.0153 mmol), 1 M TBAF (46
3
ꢁ
L, 0.046 mmol), and dry THF (1 mL). The product was obtained as a white powder after reverse phase
1
flash column chromatography (8.2 mg, 10% yield over two steps). H NMR (600 MHz, DMSOꢀd ) δ
6
1.50 ꢀ1.60 (m, 4H), 1.94 – 1.99 (m, 3H), 2.35 – 2.43 (m, 2H), 2.77 – 2.84 (m, 2H), 3.01 – 3.03 (m, 2H),
3
.78 – 3.83 (m, 3H), 4.03 – 4.24 (m, 3H), 4.35 – 4.43 (m, 3H), 4.54 (m, 1H), 4.68 – 4.76 (m, 2H), 5.71
(br s, 2H), 5.86 (s, 1H), 6.90 – 6.96 (m, 2H), 7.01 – 7.05 (m, 1H), 7.11 – 7.14 (m, 1H), 7.24 – 7.33 (m,
1
3
2
H), 7.42 – 7.48 (m, 2H), 9.08 (d, J = 7.8 Hz, 1H), 10.80 (br s, 1H). C NMR (151 MHz, DMSOꢀd ) δ
6
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1
4.5, 24.6, 27.6, 28.9, 32.7, 41.7, 47.4, 64.9, 65.0, 65.9, 73.3, 107.9, 111.3, 111.5, 116.4, 118.1, 120.8,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
1
1
22.7, 124.8, 127.1, 129.3, 132.1, 136.16, 136.17, 149.9, 156.6, 162.7, 163.7. P NMR (202 MHz,
+
+
+
CD OD) δ 10.89 and 10.77. HRMSꢀESI (m/z) calcd [M] for C H ClN O PS : 762.2236. Found:
3
33 42
7
8
7
62.2220.
9
ꢁ((2R,3R,4S,5R)ꢁ5ꢁ(((((2ꢁ(1Hꢁindolꢁ3ꢁyl)ethyl)amino)(hydroxy)phosphoryl)oxy)methyl)ꢁ3,4ꢁ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
dihydroxytetrahydrofuranꢁ2ꢁyl)ꢁ2ꢁaminoꢁ7ꢁ(2ꢁ(4ꢁchlorophenoxy)ethyl)ꢁ6ꢁoxoꢁ6,9ꢁdihydroꢁ1Hꢁpurinꢁ7ꢁ
ium (7). Compound 6a (74.2 mg, 0.101 mmol) was dissolved with a mixture of MeOH/MeCN (1:1, 15
mL) and the to the mixture was added 10 mM HEPES buffer (340 mL, pH 7.2). The resulting
suspension was incubated for 30 hours at 37 °C. Water was removed in vacuo and the resulting residue
was purified by silica gel flash column chromatography, eluting first with solvent A (20% MeOH in
CHCl ) then with solvent B (CHCl /MeOH/H O/NH OH – 5:3:0.5:0.005). The fractions containing the
3
3
2
4
title compound were pooled and concentrated in vacuo and the resulting residue was purified by reverse
phase C18 flash column chromatography to give product as a white fluffy solid (40.6 mg, 61% yield).
1
H NMR (500 MHz, DMSOꢀd ) δ 2.73 ꢀ 2.77 (m, 2H), 2.97 ꢀ 2.99 (m, 2H), 3.80 (m, 1H), 3.97 (m, 1H),
6
4
1
.08 (br s, 1H), 4.25 (br s, 1H), 4.40 – 4.44 (m, 3H), 4.76 (br s, 2H), 5.75 (br s, 1H), 5.88 (d, J = 3.5 Hz,
H), 5.93 – 5.99 (m, 1H), 6.10 (br, 1H), 6.85 – 6.88 (m, 3H), 6.98 – 7.01 (m, 1H), 7.06 (br s, 1H), 7.18
1
3
(d, J = 8.5 Hz), 7.28 – 7.30 (m, 1H), 7.42 (d, J = 8.0 Hz, 1H), 9.99 (br s, 1H), 10.79 (br s, 1H). C NMR
(151 MHz, DMSOꢀd ) δ 28.0, 42.7, 47.2, 62.5, 65.9, 70.4, 75.1, 84.7, 88.5, 107.3, 111.2, 112.6, 116.27,
6
31
116.31, 116.36, 117.9, 118.2, 120.6, 122.4, 124.6, 127.3, 129.1, 136.2, 150.0, 156.7, 189.3. P NMR
+
+
+
(202 MHz, DMSOꢀd ) δ 6.30. HRMSꢀESI (m/z) calcd [M] for C H ClN O P : 660.1733. Found:
6 28 32 7 8
6
60.1724.
Methylthio alky deprotection studies.
An aliquot (400 ꢁL) of either ProTides 6a or 6c (80 ꢁM in 20 mM HEPES buffer, pH 7.2 or 2.0)
was subjected to RPꢀHPLC (t = 0) and the remaining solution was incubated at 37 °C. Subsequent RPꢀ
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HPLC analyses on aliquots of the solution was performed at t = 1, 4, 6,12, 18, and 24 hours. The same
analyses were performed for compound 6b at t = 1, 3, 6, 11, 17, 24, and 60 hours. The halfꢀlife of
deprotection was determined by plotting the area under the curve (AUC) against time.
1
2
3
4
5
6
7
8
9
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1
1
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57
Plasma Stability Study with 6a.
Stability of ProTide 6a in plasma was performed with pooled female Sprague Dawley rat plasma
0
1
2
3
4
5
6
7
8
9
0
1
2
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9
0
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7
8
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0
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7
8
9
0
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6
7
8
9
0
(BioChemed, Winchester, VA) in triplicates. Briefly, rat plasma (500 ꢁL) was preꢀincubated at 37 °C for
two minutes. The plasma was then spiked with 6a so that the final concentration was 150 ꢁM. Aliquots
(100 ꢁL) of the reaction were drawn at different time points (t = 1, 2, 4, 6, and 9 hours) for RPꢀHPLC
analysis. To the aliquots were added MeCN (100 ꢁL) and the mixture was spun at 13,300 x g for 10
minutes to separate the precipitated proteins. The supernatant was removed and filtered through a 0.2
ꢁm nylon filter and then subjected to RPꢀHPLC analysis. The stability of 6a was determined by
calculating the halfꢀlive from curves generated by plotting AUCs of remaining parent compound against
time.
In vitro cellular uptake.
Human Mantle cell lymphoma cell lines Mino, Granta, and Z138 were purchased from ATCC
(Manassas, VA, USA). The cells were cultured in Roswell Park Memorial Institute medium (RPMI)
ꢀ1
ꢀ1
supplemented with 10% fetal bovine serum (FBS), 100 U mL penicillin, 100 ꢁg mL streptomycin,
and ꢀglutamine at 37 °C and 5% CO . MCL cells (five million) were incubated with either 6a (150 ꢁM)
L
2
or 7 (150 ꢁM) for 12 hours at 37 °C. Cells were rinsed with iceꢀcold phosphate–buffered saline (PBS)
upon removal of ProTide–containing media. Cells were harvested and cell pellet was obtained by
centrifugation. The resulting cell pellet was resuspended in MeOH/NH OAc (v/v = 60%: 40%), spiked
4
with 80 fmol each of internal standards 11 and 13 (Supporting Information Table S1), and subjected to
three freeze – thaw cycles in liquid nitrogen. The resulting mixture was centrifuged at max speed
(13500 rpm, 4 °C) and the supernatant was removed. The supernatant was filtered through a 0.2 ꢁm
nylon filter and the filtrate was dried in a speedꢀvac. The resulting residue was resuspended with 100 ꢁL
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HEPES buffer (20 mM, pH 7.2) and analyzed by LCꢀMS/MS. LCꢀMS/MS method: Analytical Agilent
Zorbax SBꢀC18 5 ꢁm column 5.0 mm X 150 mm. Buffer A – water (+0.1% formic acid); Buffer B:
MeCN (+0.1% formic acid). Analysis was performed on a gradient of 98% Buffer A (0 to 2 min), then
to 82% Buffer A (2 to 18 min), then to 5% Buffer A (18 to 20 min), and isocratic 5% Buffer A (20 to 23
min), followed by a linear gradient to 98% Buffer A (23 to 25 min), and finally isocratic 98% Buffer A
1
2
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4
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6
7
8
9
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1
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1
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0
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0
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0
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0
1
2
3
4
5
6
7
8
9
0
(25 to 40 min). All LCꢀMS/MS analyses were performed at a flow rate of 15.0 ꢁL/min. Standards for 7ꢀ
58
ClꢀPhꢀEthylꢀGMP (see notes for synthesis) and phosphoramidate 7 were prepared and spiked with 80
fmol of either internal standard 11 (for phosphoramidate 7 standards) or internal standard 13 (for 7ꢀClꢀ
PhꢀEthyl_GMP standards). All standards were prepared so that the ratio of analyte to internal standard
(in fmol) was 1:50, 1:10, 1:2, 1:1, and 2:1. A standard curve was generated by plotting the observed
internal standard to analyte ratios against the expected internal standard to analyte ratios. The amount of
analyte in each sample was determined from the following equation.
slope x AUCanalyte
Analyte amount
=
AUCinternal standard
Amountinternal standard
Cap affinity assay.
ꢀMethyl guanosine triphosphateꢀSepharose 4B (m7GTP) beads were purchased from GE
7
Healthcare (Buckinghamshire, HP7 9NA UK). Briefly eight to ten million MCL cells treated with 6a
were washed with iceꢀcold 1X PBS followed by lysis with cap binding buffer, and cap affinity assay
29
was performed as described before.
In vivo properties of 6a and 7 in SpragueꢀDawley rats.
All animal care, housing, and laboratory procedures were approved by the University of
Minnesota Institutional Animal Care and Use Committee (IACUC, protocol #1509A33023). The animal
care and housing was maintained by University of Minnesota Research Animal Resources (RAR) and
veterinarian staff.
The single dose pharmacokinetic studies were performed with rats by monitoring plasma time
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Journal of Medicinal Chemistry
course of 7 and 6a after administration via oral (p.o.) and intravenous (i.v.) routes. A group of three
female SpragueꢀDawley rats (250ꢀ274 g, Envigo, Indianapolis, IN) was used first in a p.o study
followed by an i.v. study after a threeꢀday washout period. All rats were received with inꢀdwelling dual
femoral/jugular vein catheters, which were used for blood withdrawing and i.v. dosing, respectively. 10
mg/mL solutions of compounds were prepared in 0.5% aqueous sodium carboxymethylcellulose (CMC)
with 5% DMSO and rats were given a single p.o. or i.v. dose of 25 or 2.5 mg/kg by oral gavage or
injection respectively. Blood samples (0.15 mL) were drawn at various times post dose (p.o.: 0, 15, 30,
45, 60, 120, 240, 360, and 480 min; i.v.: 0, 5, 10, 15, 30, 45, 60, 120, 240, and 480 min) and collected
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6
0
1
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0
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7
8
9
0
in vacutainer tubes containing 3.6 mg K EDTA and transported on ice. They were centrifuged at 3000 g
2
for 5 min and plasma was harvested and stored at –80 ºC until analyzed. Plasma (20 ꢁL) was proteinꢀ
precipitated with acetonitrile (20 ꢁL) containing a mixture of corresponding internal standards (100 nM
each, Supporting Information Table S1), centrifuged, and the supernatant was analyzed by LCꢀMS/MS
(ShimadzuꢀAB Sciex Qtrap 5500).
Reverseꢀphase LC was performed on a Kinetix C18 column (50 mm × 2.1 mm, 2.6 µm particle
size; Phenomenex, Torrance, CA). Mobile phase A was 0.1% aqueous formic acid while mobile phase
B was 0.1% formic acid in acetonitrile. Initial conditions were 5% B from 0 to 0.5 min, after which the
%
B was increased to 95% from 0.5 to 3 min. The column was washed in 95% B for 2 min, returned to
% over 0.2 min, and allowed to reꢀequilibrate for 2.8 min in 5% B to provide a total run time of 8 min.
The flow rate was 0.5 mL/min and the column oven was maintained at 40 °C. The injection volume was
0 µL. All analytes were analyzed by mass spectrometry in positive ionization mode by Multiple
5
1
Reaction Monitoring (MRM). To determine the optimum MRM settings (Supporting Information Table
S1), each analyte was infused at a concentration of 10 µM (in 1:1 water: acetonitrile containing 0.1%
formic acid) onto the MS by a syringe pump at a flow of 10 µL/min.
Analyte and internal standard peak areas were calculated (MultiQuant, version 2.0.2). Analyte
peak areas were normalized to the corresponding internal standard peak areas and the analyte
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Page 26 of 44
concentrations were determined using an appropriate standard curve for each compound. The standard
solutions were prepared by serial dilution in rat plasma and treated similarly as for samples described
above. PK parameters were calculated from concentrationꢀtime profiles by nonꢀcompartamental analysis
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
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6
(Phoenix WinNonLin, version 6.3, Pharsight Corporation). Oral bioavailability (%F) was calculated
individually for each rat using the dose normalized p.o. and i.v. area under the curve (AUC).
Thymidine incorporation assay.
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9
0
MCL lymphoma cell lines were treated with 6a for 48 hours and then thymidine was added for
5
9
another 16 hours. Proliferation was assessed by thymidine incorporation assay as described earlier.
ProTide 6a was added everyday.
Annexin V/PI flow cytometry assay for survival.
MCL cell lines were treated with 6a for 48 hours and inhibition of survival was assessed by
59
AnnexinV/PI staining using flow cytometry, as described before. ProTide 6a was added everyday.
RNAꢀImmunoprecipitation (RNAꢀIP).
RNAꢀIP was performed on Granta and Mino cells with Magna RIP RNAꢀBinding Protein
60
Immunoꢀprecipitation Kit (Millipore, Billerica, MA, USA) as described earlier.
Western blotting. MCL cell lines were treated with 6a for 48 hours and protein quantification was
performed. Western blotting was performed as described before using eIF4E, eIF4G, actin, cꢀMyc, and
60
Bclꢀ2 antibodies.
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website.
1
13
31
HNMR, CNMR, PNMR, RNAꢀIP data (PDF)
Molecular Formula Strings data (CSV)
Corresponding Author
*
Eꢀmail: magupta@gwu.edu
Eꢀmail: wagne003@umn.edu
*
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Journal of Medicinal Chemistry
Funding Sources
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This work is supported by ASH Bridge Grant Award from American Society of Hematology and the
University of Minnesota Foundation; this work is also supported in part through start up package from
George Washington University. Mass spectrometry was carried out with the assistance of Dr. Bruce A.
Witthuhn in the Analytical Biochemistry Shared Resource of the Masonic Cancer Center, University of
Minnesota, funded in part by Cancer Center Support Grant CAꢀ77598. We also gratefully acknowledge
financial support from the National Institutes of Health Grant AI070219 (to C.C.A.).
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Notes
7ꢀClꢀPhꢀEthylꢀGMP was synthesized according to ref 48; purified by ion exchanged with Dowex50X8
to remove triethylammonium salt and finally reverse phase C18 column.
ACKNOWLEDGMENT
We thank Xun Ming for his technical expertise with the LCꢀMS/MS analyses.
ABBREVIATIONS
DMF, N,Nꢀdimethylformamaide; TBAF, Tetraꢀnꢀbutylammonium fluoride; TBDMSCl, tertꢀ
Butyldimethylsilyl chloride; PivCl, Pivaloyl chloride; eIF4E, eukaryotic translation initiation factor 4E;
eIF4G, eukaryotic translation initiation factor 4E; eIF4A, eukaryotic translation initiation factor 4A.
REFERENCES
1
.
Jordheim, L. P.; Durantel, D.; Zoulim, F.; Dumontet, C. Advances in the development of
nucleoside and nucleotide analogues for cancer and viral diseases. Nat. Rev. Drug Discov. 2013, 12,
47ꢀ464.
4
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Page 28 of 44
2
.
Pradere, U.; GarnierꢀAmblard, E. C.; Coats, S. J.; Amblard, F.; Schinazi, R. F. Synthesis of
nucleoside phosphate and phosphonate prodrugs. Chem. Rev. 2014, 114, 9154ꢀ9218.
3. Wagner, C. R.; Iyer, V. V.; McIntee, E. J. ProTides: toward the in vivo delivery of antiviral and
anticancer nucleotides. Med. Res. Rev. 2000, 20, 417ꢀ451.
Meier, C. Proꢀnucleotides ꢀ recent advances in the design of efficient tools for the delivery of
biologically active nucleoside monophosphates. Synlett 1998, 233ꢀ242.
McGuigan, C.; Pathirana, R. N.; Balzarini, J.; De Clercq, E. Intracellular delivery of bioactive
AZT nucleotides by aryl phosphate derivatives of AZT. J. Med. Chem. 1993, 36, 1048ꢀ1052.
Saboulard, D.; Naesens, L.; Cahard, D.; Salgado, A.; Pathirana, R.; Velazquez, S.; McGuigan,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
.
6
.
C.; De Clercq, E.; Balzarini, J. Characterization of the activation pathway of phosphoramidate triester
prodrugs of stavudine and zidovudine. Mol. Pharmacol. 1999, 56, 693ꢀ704.
7
.
Murakami, E.; Tolstykh, T.; Bao, H.; Niu, C.; Steuer, H. M.; Bao, D.; Chang, W.; Espiritu, C.;
Bansal, S.; Lam, A. M.; Otto, M. J.; Sofia, M. J.; Furman, P. A. Mechanism of activation of PSIꢀ7851
and its diastereoisomer PSIꢀ7977. J. Biol. Chem. 2010, 285, 34337ꢀ34347.
8
.
Cho, A.; Zhang, L.; Xu, J.; Lee, R.; Butler, T.; Metobo, S.; Aktoudianakis, V.; Lew, W.; Ye, H.;
Clarke, M.; Doerffler, E.; Byun, D.; Wang, T.; Babusis, D.; Carey, A. C.; German, P.; Sauer, D.; Zhong,
W.; Rossi, S.; Fenaux, M.; McHutchison, J. G.; Perry, J.; Feng, J.; Ray, A. S.; Kim, C. U. Discovery of
the first Cꢀnucleoside HCV polymerase inhibitor (GSꢀ6620) with demonstrated antiviral response in
HCV infected patients. J. Med. Chem. 2014, 57, 1812ꢀ1825.
9
.
Zhou, L.; Zhang, H. W.; Tao, S.; Bassit, L.; Whitaker, T.; McBrayer, T. R.; Ehteshami, M.;
Amiralaei, S.; Pradere, U.; Cho, J. H.; Amblard, F.; Bobeck, D.; Detorio, M.; Coats, S. J.; Schinazi, R.
F. βꢀDꢀ2'ꢀCꢀMethylꢀ2,6ꢀdiaminopurine ribonucleoside phosphoramidates are potent and selective
inhibitors of hepatitis C Virus (HCV) and are bioconverted intracellularly to bioactive 2,6ꢀ
diaminopurine and guanosine 5'ꢀtriphosphate forms. J. Med. Chem. 2015, 58, 3445ꢀ3458.
28
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Page 29 of 44
Journal of Medicinal Chemistry
1
0.
Sofia, M. J.; Bao, D.; Chang, W.; Du, J.; Nagarathnam, D.; Rachakonda, S.; Reddy, P. G.; Ross,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
B. S.; Wang, P.; Zhang, H. R.; Bansal, S.; Espiritu, C.; Keilman, M.; Lam, A. M.; Steuer, H. M.; Niu,
C.; Otto, M. J.; Furman, P. A. Discovery of a βꢀdꢀ2'ꢀdeoxyꢀ2'ꢀαꢀfluoroꢀ2'ꢀβꢀCꢀmethyluridine nucleotide
prodrug (PSIꢀ7977) for the treatment of hepatitis C virus. J. Med. Chem. 2010, 53, 7202ꢀ7218.
1
1.
McGuigan, C.; Gilles, A.; Madela, K.; Aljarah, M.; Holl, S.; Jones, S.; Vernachio, J.; Hutchins,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
J.; Ames, B.; Bryant, K. D.; Gorovits, E.; Ganguly, B.; Hunley, D.; Hall, A.; Kolykhalov, A.; Liu, Y.;
Muhammad, J.; Raja, N.; Walters, R.; Wang, J.; Chamberlain, S.; Henson, G. Phosphoramidate
ProTides of 2'ꢀCꢀmethylguanosine as highly potent inhibitors of hepatitis C virus. Study of their in vitro
and in vivo properties. J. Med. Chem. 2010, 53, 4949ꢀ4957.
1
2.
Wagner, C. R.; Chang, S. L.; Griesgraber, G. W.; Song, H.; McIntee, E. J.; Zimmerman, C. L.
Antiviral nucleoside drug delivery via amino acid phosphoramidates. Nucleosides Nucleotides 1999, 18,
9
1
13ꢀ919.
3. Chang, S.; Griesgraber, G. W.; Southern, P. J.; Wagner, C. R. Amino acid phosphoramidate
monoesters of 3'ꢀazidoꢀ3'ꢀdeoxythymidine: relationship between antiviral potency and intracellular
metabolism. J. Med. Chem. 2001, 44, 223ꢀ231.
1
4.
Li, S.; Jia, Y.; Jacobson, B.; McCauley, J.; Kratzke, R.; Bitterman, P. B.; Wagner, C. R.
Treatment of breast and lung cancer cells with a Nꢀ7 benzyl guanosine monophosphate tryptamine
phosphoramidate ProTide (4Eiꢀ1) results in chemosensitization to gemcitabine and induced eIF4E
proteasomal degradation. Mol. Pharm. 2013, 10, 523ꢀ531.
1
5.
Wagner, C. R.; McIntee, E. J.; Schinazi, R. F.; Abraham, T. W. Aromatic aminoꢀacid
phosphoramidate diesters and triesters of 3´ꢀazidoꢀ3´ꢀdeoxythymidine (AZT) are nontoxic inhibitors of
HIVꢀ1 replication. Bioorganic & Medicinal Chemistry Letters 1995, 5, 1819ꢀ1824.
1
6.
Ghosh, B.; Benyumov, A. O.; Ghosh, P.; Jia, Y.; Avdulov, S.; Dahlberg, P. S.; Peterson, M.;
Smith, K.; Polunovsky, V. A.; Bitterman, P. B.; Wagner, C. R. Nontoxic chemical interdiction of the
29
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