Synthesis and biological evaluation of LNA phosphoramidates

Article abstract of DOI:10.1080/15257770701571834

The synthesis of LNA phosphoramidates is presented. The LNA phosphoramidates were evaluated for their ability to inhibit cell proliferation of the human prostate cancer cell line 15PC3. A number of the LNA phosphoramidates showed cell proliferation inhibition determined by the MTS assay. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770701571834

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Synthesis and Biological Evaluation of
LNA Phosphoramidates
Jacob Jensen a , Gitte Sjøgren a , Jens Bo Hansen a , Christoph
Rosenbohm a & Troels Koch a
a Santaris Pharma A/S, Hørsholm, Denmark
Version of record first published: 16 Dec 2010.
To cite this article: Jacob Jensen, Gitte Sjøgren, Jens Bo Hansen, Christoph Rosenbohm & Troels Koch
(2008): Synthesis and Biological Evaluation of LNA Phosphoramidates, Nucleosides, Nucleotides and
Nucleic Acids, 27:1, 37-42
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Nucleosides, Nucleotides, and Nucleic Acids, 27:37–42, 2008
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701571834
SYNTHESIS AND BIOLOGICAL EVALUATION OF LNA
PHOSPHORAMIDATES
Jacob Jensen, Gitte Sjøgren, Jens Bo Hansen, Christoph Rosenbohm, and
Tro el s Ko ch
Santaris Pharma A/S, Hørsholm, Denmark
The synthesis of LNA phosphoramidates is presented. The LNA phosphoramidates were
2
evaluated for their ability to inhibit cell proliferation of the human prostate cancer cell line 15PC3.
A number of the LNA phosphoramidates showed cell proliferation inhibition determined by the MTS
assay.
Keywords LNA; phosphoramidates; proliferation
Nucleoside analogues are an important class of drugs that have found
widespread use, in particular as anti-viral agents. Well-known examples
include AZT, which revolutionized AIDS therapy, and the highly efficacios
anti-cancer agent gemcitabine.[1]
The mode of action of the majority of nucleoside and nucleotide drugs is
based on chain termination; i.e., they are analogues of naturally occurring
deoxynucleotides required for the synthesis of viral DNA and hence com-
pete with natural deoxynucleotides for incorporation into the growing viral
DNA chain. However, unlike the natural deoxynucleotide substrates they
often lack the 3ꢁ-hydroxyl group on the deoxyribose moiety. As a result, fol-
lowing their incorporation chain elongation is terminated. For nucleosides
such as zidovudine (AZT) or didanosine (ddI) to be incorporated into viral
DNA, they must be converted to the active 5ꢁ-triphosphates within the cell.
Since therapeutically relevant nucleosides are synthetic analogues of the
natural congeners they usually have poor affinity for kinases and monophos-
phates. Consequently they are often introduced chemically.[2] However,
usually monophosphotylation is not adequate for a putative drug candidate
since the phosphate is readily hydrolysed intracellularly. Moreover, the
hydrophilicity of the monophosphate generally blocks spontaneous cellular
Received 27 March 2007; accepted 29 June 2007.
Address correspondence to Christoph Rosenbohm, Santaris Pharma A/S, 3 Bøge Alle, DK-2970,
Hørsholm, Denmark. E-mail: cr@santaris.com
37

38
J. Jensen et al.
FIGURE 1 The four LNA nucleosides: β-d-LNA-adenine (1); β-d-LNA-thymine (2); α-l-LNA-thymine
(3); β-d-xylo-LNA-adenine (4).
uptake of nucleotides. To solve this problem lipophilic monophosphate
prodrugs such as simple phosphate esters (Adefovir dipivoxil), CycloSal,[3]
SATE,[4] and phosphoramidates[5,6] have been prepared. Due to the many
promising prospects of nucleotide prodrugs research within the field is
continuously growing.
Recent years have seen the development of several LNA (locked nucleic
acid) nucleosides but with a single exception[7] they have only been used as
building blocks for oligonucleotides. LNA nucleosides as therapeutic agents
in their own right remain an essentially unexplored area.
Herein we wish to report the synthesis of a series of lipophilic LNA
prodrug analogues and their properties as inhibitors of cell proliferation.[8]
The prodrug strategy was based on the phosphoramidate approach devel-
oped by McGuigan and co-workers.[9,10]
RESULTS AND DISCUSSION
To explore the bicyclic chemical space present in LNA four phos-
phoramidates were investigated: two purine and two pyrimidine bicyclic
nucleosides in both the α-l and β-d configuration along with a β-d-xylo-
adenosine derivative (Figure 1).[11,12]
The phosphoramidate moity was prepared according to the method by
McGuigan and co-workers[6] and linked to the various bicyclic nucleosides
as shown in Scheme 1.
SCHEME 1 Reagents and conditions: (a) POCl3, Et3N, Et2O, −78◦C, 5–12 hours; (b) Et3N, l-alanine
methyl- or benzyl- ester hydrochloride, DCM, −78◦C to rt, 2–5 hours; (c) LNA nucleoside 1-4, tBuMgCl,
Pyr/MeCN 2:1, 2 days (low to moderates yields).

Synthesis of LNA Phosphoramidates
39
FIGURE 2 LNA phosphoramidates: A, adenine; T, thymine; PhCl, p-chlorophenyl.
Synthesis of phosphochloridate species 6 was accomplished according
to the procedures published by McGuigan and co-workers.[10] The aromatic
moiety was treated with phosphorusoxychloride at −78◦C to give phospho-
rdichloridates 5 followed by treatment with l-alanine methyl or benzyl ester
hydrochloride at −78◦C to give phosphochloridates 6.
The reactions were monitiored closely by LC-MS to prevent the forma-
tion of sideproducts. After concentration the residue was suspended in ether
and filtered. The filtrate was concentrated in vacuo and stored as a 0.2 M so-
lution in THF. The three selected phosphochloridates (X = H and R = Me;
X = H and R = Bn; X = Cl and R = Me) have previously been reported to be
among the most biologically active nucleoside derivatives within this class.[6]
The three different phosphochloridates were reacted under Grignard
conditions[6] with four different LNA nucleosides (1–4, Figure 1) to give
12 LNA phosphoramidates as depicted in Figure 2.
The four LNA nucleosides 1–4, LNA phosphoramidates 8–19, and
gemcitabine (as positive control) were tested for their ability to inhibit
the proliferation of the human prostate cancer cell line 15PC3 as de-
tected by the MTS assay. Only a few of these showed activity in the assay
(Figure 3). The four parent LNA nucleosides 1–4 showed no inhibition
in this assay. Compound 9 was the most potent compound and showed
a clear dose–response relationship, with an IC50 value below 100 µM
after incubating for 72 hours. Gemcitabine (2ꢁ-deoxy-2ꢁ,2ꢁ-difluorocytidine
hydrochloride) also showed a clear dose–response profile, with an IC50
value below 0.1 µM after incubating for 72 hours. Compound 9 reached an

40
J. Jensen et al.
120
100
80
60
40
20
0
24 h
48 h
72 h
96 h
8
9
10
15
GEM
Treatment
FIGURE 3 MTS assay of selected LNA phosphoramidates and gemcitabine (GEM, positive control)
normalized to Mock (blank).
inhibition level comparable to gemcitabine but at approximately 100 times
the concentration. The different dose- and time-response profiles between
9 and gemcitabine could be a punitive indication that the mechanisms of
action are different.
When the four active compounds (8, 9, 10, and 15) were compared
it was clear that the adenine-containing nucleoside was more potent than
the thymine nucleoside (compound 9 versus 15) Moreover, the benzylester
alanine amino acid susbtituent gave more potent compounds than the
corresponding methylesters (compound 8 versus 9).
Interestingly, none of the isomers with unnatural stereochemistry
(α-l-LNA and xylo-LNA) showed any activity. The MTS assay did not give
any mechanistic insight into the mode of action of the phosphoramidate
derivatives.
In conclusion, we have developed a route for the synthesis of LNA phos-
phoramidates. Twelve phosphoramidates based on four LNA nucleosides
and three phosphoramidite building blocks resulted in the identification of
one compound with a significant effect on the growth of 15PC3 cancer cells.
Based on the obtained data further work with LNA-based bicyclic nucleoside
prodrugs is under way and will be reported in due course.
EXPERIMENTAL
General Procedure for the Synthesis of LNA Phosphoramidates
(1R,3R,4R,7S)Phenyl-[benzyloxy-l-alaninyl]-[(-3-(adenine-9-yl)-7-hydroxy-
2,5-dioxabicyclo[2:2:1]heptane-1-yl)methyl] phosphate (9):(1S,3R,4R,7S)-
3-(adenine-9-yl)-7-hydroxy-1-hydroxymethyl-2,5-dioxabicyclo[2:2:1]heptane

Synthesis of LNA Phosphoramidates
41
(125 mg, 0.45 mmol) was concentrated from anhydrous pyridine
twice, dried under vacuum over P2O5 for 2 hours, and dissolved in
a mixture of pyridine and acetonitrile (2:1; 5 mL). To the solution
was added tBuMgCl (1 M in THF, 0.5 mL, 0.5 mmol) and the
solution was stirred at room temperate under argon for 15 minutes.
Phenyl-[benzyloxy-l-alaninyl]phosphorchloridate (0.2
M
in THF,
4.5 mL, 0.9 mmol) was added and the solution was stirred under argon for
t
24 hours. After this time BuMgCl (1 M in THF, 0.5 mL, 0.5 mmol) was
added followed by the phenyl-[benzyloxy-l-alaninyl]phosphorchloridate
(0.2 M in THF, 4.5 mL, 0.9 mmol). After an additional 24 hours the solution
was concentrated in vacuo and the residue redissolved in CH2Cl2 (25 mL).
The CH2Cl2 solution was washed with aqueous HCl (1 N, 25 mL), brine (2 ×
25 mL), dried (MgSO4), filtered, and concentrated in vacuo to give a yellow
gum. Purification by dry column vacuum chromatography (DCVC)[13]
,
eluted with MeOH in CH2Cl2 0–10%, v/v gave phosphoramidate 9 (13 mg)
as a colorless solid.
LC-MS (ES; M+H), found 597.2 (calculated 597.18)
31P NMR (400 MHz, CDCl3) δP: 4.53 (double peak)
1H NMR (400 MHz, DMSO-d6) δH: 8.21 (1H, s, H2); 8.15 (1H, s, H8);
7.39–7.11 (10H, m, 2 × Ph); 6.12 (1H, m, CHCH3); 5.91 (1H, s, H1ꢁ);
5.06 (2H, d, J 5.5 Hz); 4.48–4.32 (4H, m, H5ꢁ and Ph-CH2); 3.94 (1H, d,
3
3
3J HH 8, H2ꢁ/H3ꢁ); 3.75 (1H, d, J HH 8, H2ꢁ/H3ꢁ); 1.27 (3H, d, J HH 7,
CH3)
MTS Assay
15PC3 cells were seeded to a density of 12,000 cells per well in white
96-well plate (Nunc 136101) in DMEM the day prior to transfection. The
next day, cells were washed once in prewarmed OptiMEM followed by
addition of 72 µL OptiMEM containing 5 µg/mL Lipofectamine2000 (In
Vitrogen). Cells were incubated for 7 minutes before adding 18 µL LNA
phosphoramidate diluted in OptiMEM. The final LNA phosphoramidate
concentration ranged from 0.1 nM to 500 nM. After 4 hours of treatment,
cells were washed in OptiMEM and 100 µL serum containing DMEM was
added. Following treatment with the LNA phosphoramidate compound,
cells were allowed to recover for the period indicated, viable cells were mea-
sured by adding 20 µL of the tetrazolium compound [3-(4,5-dimethyl-2-yl)-
5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt;
MTS] and an electron coupling reagent (phenazine ethosulfate; PES)
(CellTiter 96 AQueous One Solution Cell Proliferation Assay, Promega) per
100 µL DMEM. Viable cells were measured at 490 nm in a Powerwave
(Biotek Instruments). Growth rates (ꢀOD/h) were plotted against the
concentration of the LNA phosphoramidate.

42
J. Jensen et al.
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