Aminoacyl-tRNA synthetase inhibitors as potent and synergistic immunosuppressants

Article abstract of DOI:10.1021/jm8000746

The aminoacyl-tRNA synthetase family of enzymes is the target of many antibacterials and inhibitors of eukaryotic hyperproliferation. In screening analogues of 5′-O-(N-L-aminoacyl)-sulfamoyladenosine containing all 20 proteinogenic amino acids, we found t

Full text of DOI:10.1021/jm8000746

3020
J. Med. Chem. 2008, 51, 3020–3029
Aminoacyl-tRNA Synthetase Inhibitors as Potent and Synergistic Immunosuppressants
Pieter Van de Vijver,† Tomasz Ostrowski,‡ Brian Sproat,§ Jozef Goebels, Omer Rutgeerts, Arthur Van Aerschot,†
Mark Waer, and Piet Herdewijn*,†
Laboratory for Medicinal Chemistry, Rega Institute for Medical Research, Catholic UniVersity of LeuVen, Minderbroedersstraat 10,
B-3000 LeuVen, Belgium, and Laboratory for Experimental Transplantation, Campus Gasthuisberg, Catholic UniVersity of LeuVen,
B-3000 LeuVen, Belgium
ReceiVed January 28, 2008
The aminoacyl-tRNA synthetase family of enzymes is the target of many antibacterials and inhibitors of
eukaryotic hyperproliferation. In screening analogues of 5′-O-(N-L-aminoacyl)-sulfamoyladenosine containing
all 20 proteinogenic amino acids, we found these compounds to have potent immunosuppressive activity.
Also, we found that combinations of these compounds inhibited the immune response synergistically. Based
on these data, analogues with modifications at the aminoacyl and ribose moieties were designed and evaluated,
and several of these showed high immunosuppressive potency, with one compound having an IC50 of 80
nM, when tested in a cellular mixed lymphocyte reaction assay. Apart from showing the potential of
aminoacyl-tRNA synthetase inhibitors as immunosuppressants, the current study also provides arguments
for careful evaluation of the immunosuppressive activity of developmental antibacterials that target these
enzymes.
Introduction
Aminoacyl-tRNA synthetases (aaRSsa) are the enzymes that
catalyze the transfer of amino acids to their cognate tRNA. They
play a pivotal role in protein biosynthesis and are necessary
for growth and survival of all cells. Consequently, inhibition
of these enzymes is an attractive target for cell-growth inhibition,
both as antibacterials and as inhibitors of cell (hyper)proliferation
in humans.1–7 Indeed, an inhibitor of IleRS, mupirocin (1), is
in clinical use for the topical treatment of infections caused by
Gram-positive bacteria and several more are in clinical develop-
ment.1
The largest group of aaRS inhibitors (aaRSi) are the reaction-
intermediate mimics. These compounds are analogues of aa-
AMP (2), which is the natural reaction intermediate of these
enzymes. Mupirocin belongs to this group, as do the 20 5′-O-
(N-L-aminoacyl)-sulfamoyladenosines (aaSA, 3, Figure 1). The
latter are commercially available and show Ki values in the low
nanomolar range against the corresponding isolated aaRSs.8–12
Because the aaSA compounds differ only little from the natural
reaction intermediates, they do not show noticeable selectivity
toward either bacterial or eukaryotic aaRSs. This is a conse-
quence of the use of the same general reaction intermediate by
Figure 1. Structures of the IleRS inhibitor mupirocin (1), the universal
aaRSs of all kingdoms of life, despite extensive divergence in
the structure and amino acid sequence of these enzymes.
Nevertheless, researchers at Cubist Pharmaceuticals have been
aaRS reaction intermediate aa-Amp (2), and the universal aaRS inhibitor
aaSA (3).
able to develop analogues of IleSA with extensive modifications
at the nucleobase moiety that inhibit bacterial IleRS at nano-
molar concentrations and that show good selectivity with respect
to the human homologue. Also, they showed that one of the
analogues, CB-432 (4) was active in a systemic mouse protec-
tion test with Streptococcus pyogenes, thereby validating the
systemic potential of this class of compounds. Unfortunately,
clinical development of this compound was halted as a
consequence of low bioavailability, caused by high serum
albumin binding (Figure 2).4,13
* To whom correspondence should be addressed. Tel.: 32(0)16337387.
Fax: 32(0)16337340. E-mail: piet.herdewijn@rega.kuleuven.be.
†
Laboratory for Medicinal Chemistry, Catholic University of Leuven.
Institute of Bioorganic Chemistry, Polish Academy of Sciences, ul.
‡
Noskowskiego 12/14, 61-704 Poznan, Poland.
§
Integrated DNA Technologies BVBA, B-3000 Leuven, Belgium.
Laboratory for Experimental Transplantation, Catholic University of
Leuven.
a Abbreviations: aa, amino acid; aaRS, aminoacyl-tRNA synthetases;
aaRSi, aminoacyl-tRNA synthetase inhibitors; aaSA (where aa can be
replaced by any amino acid 3-letter code), 5′-O-(N-aminoacyl)-sulfamoy-
ladenosine; Boc, tert-butoxycarbonyl; DBU, 1,8-diazabicyclo [5.4.0]undec-
7-ene; DMAc, N,N-dimethylacetamide; -OSu, N-hydroxysuccinimide ester;
MLR, mixed lymphocytes reaction; rt, room temperature; TBDMS, tert-
butyldimethylsilyl; TFA, trifluoroacetic acid; TIPDS, 1,1,3,3-tetraisopro-
pyldisiloxane-1,3-diyl.
Resistance and resistance development are essential aspects
of current antimicrobial development, and it was recently found
that mupirocin and REP8839 (5), a selective inhibitor of
bacterial MetRS, act synergistically in preventing resistance
10.1021/jm8000746 CCC: $40.75
2008 American Chemical Society
Published on Web 04/26/2008

ImmunosuppressiVe aaRS Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10 3021
Figure 2. Structures of relevant aminoacyl-tRNA synthase inhibitors. Antibacterial IleRS inhibitor CB-432 (4), antibacterial MetRS inhibitor
REP8839 (5), antipsoriatic 5′-deoxy-adenosine 5′-N-(N-L-aminoacyl)-sulfamide (6), antitumoral IleRS inhibitor reveromycin A (7), and borrelidin
(8), which is an inhibitor of ThrRS and has antiangiogenetic properties.
Figure 4. Structures of aminoacyl-modified analogues 27-32 and the
amino acids these are derived from (34-38).
recent years. For instance, Cubist Pharmaceuticals has patented
several aaRS inhibitors for the treatment of psoriasis, a common
hyperproliferative skin disease.6 The compounds described in
this patent are 19 aminoacyl analogues of 5′-deoxy-adenosine
Figure 3. Structures of new analogues modified at the sugar moiety.
5′-N-(N-L-aminoacyl)-sulfamide (6), which are structurally very
similar to the aaSA compounds. Another well-characterized
development, though neither synergism or subadditivity was
found with respect to the MIC values. A fixed combination of
mupirocin and REP8839 for the treatment of topical infections
is currently in clinical development.114 It was also found that
mutations leading to resistance against REP8839 come with
significant fitness costs. The latter makes antibiotic restriction
or antibiotic rotation a feasible option to control antibiotic
resistance against these agents.
inhibitor of a human aaRS is reveromycin A (7). Reveromycin
A, an inhibitor of human IleRS, was discovered in a screening
effort to identify compounds that can cause morphology
reversion of srcts-NRK cells. It was also found to be an inhibitor
of the epidermal growth factor-dependent responses of mouse
epidermal cells and to have antitumoral effects against human
ovarian carcinoma BG-1.15 Finally, reveromycin A also induces
apoptosis in bone osteoclasts at concentrations far lower than
those needed for the inhibition of other cell lines. Woo et al.
show that this selectivity is a consequence of the acid microen-
vironment of these cells, which greatly improves the uptake of
The effects of aaRS inhibitors on human cells have been
studied less extensively. Nevertheless, several aaRS inhibitors
with potential application in humans have been described in

3022 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10
Scheme 1. Synthesis of aaSA Analogues of Figure 4a
Van de VijVer et al.
a Reagents and conditions: (a) N-Boc amino acid N-hydroxysuccinimide ester, DBU, DMF, 77-86%; (b) TFA/H2O (5:2 v/v), 37-61%; (c) N-Boc,N-
Bzl-L-phenylalanine N-hydroxysuccinimide ester, DBU, DMF, 52%; (d) (i) TFA/H2O (5:2 v/v); (ii) Et3N·3HF, THF, 43%.
reveromycin A.16 A third well-characterized inhibitor of both
bacterial and eukaryotic cell growth is the macrolide-polyketide
Borrelidin (8).17 This compound targets ThrRS and has anti-
angiogenetic properties, though recent findings suggest that
ThrRS inhibition is not the only mode of action of this
compound.18 In addition and in contrast to the other compounds
mentioned here, borrelidin is a noncompetitive inhibitor with
respect to both the amino acid and ATP.
lic acid, 35) are substrates for ProRS and L-R-aminobutyric acid
(36) and L-norvaline (37) are substrates for ValRS and
MetRS.20–22 L-Norvaline is a known substrate for LeuRS and
IleRS as well.2021 L-γ-Methallylglycine is a substrate of PheRS
and LeuRS. Furthermore, L-azetidine-2-carboxylic acid, L-
thioproline, and DL-γ-methallylglycine (38) are bactericidal
against Escherichia coli.23
For the synthesis of all compounds, the general synthetic
approach devised by Castro-Pichel et al. was used.24
Analogue Design and Synthesis
In preparing aaSA analogues containing nonproteinogenic
amino acids (Scheme 1), appropriately, 2′,3′-O-protected ad-
enosine was sulfamoylated using fresh, in situ generated
sulfamoylchloride in N,N-dimethylacetamide (DMAc).2526 The
resulting sulfamate 39/44 was coupled with N-Boc protected,
N-hydroxysuccinimide activated amino acid in the presence of
DBU, after which the protecting groups were removed to give
the target compounds.24 Initially, 2′,3′-O-isopropylidene was
used to protect the secondary hydroxyls of adenosine, as in the
original synthesis.24 We used this procedure for compounds
27-31, but found the starting product 39 and 40-43 to be prone
to cyclonucleoside formation, resulting in low yields.27 There-
fore, we switched to O-tert-butyldimethylsilyl (TBDMS) protec-
tion for the synthesis of the N-benzyl-L-phenylalanyl congener
32, based on the synthesis of 5′-O-(N-salicyl)-sulfamoyladenos-
ine by Ferreras et al.28 This, however, made a second depro-
tection step necessary, as aqueous TFA alone did not completely
remove TBDMS protection. Hence, following acid treatment,
the residue was treated with Et3N·3HF in THF to give
compound 32.
For the synthesis of the anhydrohexitol analogues 16-21
(Scheme 2) and 2′-deoxy-2′-fluoro-ꢀ-D-ribose-analogues 22-26
(Scheme 3), we started from the unprotected nucleoside
analogues, 1,5-anhydro-2-(adenin-9-yl)-2,3-dideoxy-D-arabino-
hexitol (46). and 2′-deoxy-2′-fluoroadenosine (57), respec-
tively.29,30 Both were first monomethoxytritylated (MMTr) at
the primary hydroxyl and subsequently O-TBDMS protected
at the secondary hydroxyl, after which the MMTr was removed.
For the synthesis of the altritol compounds (Scheme 4), we
started from 1,5-anhydro-2-deoxy-2-(adenin-9-yl)-D-altro-hexitol
(65) and used 1,1,3,3-tetraisopropyldisiloxane-1,3-diyl for pro-
We wanted to investigate the potential use of aaRSi as
immunosuppressants. Many antibacterial aaRS inhibitors have
been characterized, both of natural and synthetic origin. In
contrast, nothing is really known about the immunosuppressive
activity of aaRS inhibitors, and it may be reasoned that inhibition
of tRNA synthetases might lead to the inhibition of protein
synthesis in mammalian cells and, thus, may have an immu-
nosuppressive effect. Therefore, we first subjected all 20
analogues of 3 to an immunosuppressive screen. Based on these
initial data, we then designed, synthesized, and evaluated
analogues.
In 3, four moieties can be distinguished, with respectively
an aminoacyl, a linker, a sugar, and a nucleobase part. Analogues
with modifications at all of these positions have already been
made by other researchers, with most attention concentrated on
the nucleobase and linker part. Therefore, we focused on sugar
and aminoacyl modifications. For the sugar moiety, two six-
membered sugar analogues were used, altritol (9-15) and
anhydrohexitol (16-21). Both of these lack an anomeric carbon,
making the compounds more stable toward enzymatic hydroly-
sis. We also prepared analogues containing 2′-deoxy-2′-fluoro-
D-ribose (22-26) instead of the normal D-ribose sugar.
For the aminoacyl-modified analogues 27-32, we replaced
the amino acid moiety with nonproteinogenic amino acids that
are either substrates of aaRS or inhibitors thereof. The pheny-
lalanyl analogue N-benzyl-L-phenylalanine (33) is a substrate
for PheRS.19 Surprisingly, this is the case for both the L- and
the D-enantiomers of this compound, although we only prepared
analogues containing the L-enantiomer. Similarly, L-azetidine-
2-carboxylic acid (34) and thioproline (L-4-thiazolidinecarboxy-

ImmunosuppressiVe aaRS Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10 3023
Scheme 2. Synthesis of the Anhydrohexitol Analogues of Figure 3a
a Reagents and conditions: (a) MMTrCl, pyr, 93%; (b) TBDMSCl, imidazole, DMF, 68%; (c) TsOH in MeOH/CH2Cl2 (2:8 v/v), 81%; (d) H2NSO2Cl,
CH3CN/DMAc, 69%; (e) N-Boc L-amino acid N-hydroxysuccinimide ester, DBU, DMF, 80-96%; (f) TFA/H2O (5:2 v/v), 57-87%.
Scheme 3. Synthesis of the 2′-Deoxy-2′-fluoro-ꢀ-D-ribose
raphy and size exclusion chromatography, subsequently. Before
use, the compounds were further purified by reverse phase
HPLC.
Analogues of Figure 3a
Biological Evaluation
a. Immunosuppressive Properties of the 20 aaSA Com-
pounds. All 20 aaSA analogues were screened for their
immunosuppressive potential (Table 1), by determining their
ability to inhibit the allogeneic mixed lymphocyte reaction
(MLR), a laboratory test which is clinically used as a predictive
in vitro test of in vivo transplant rejection.32 Interestingly, all
20 compounds showed activity in the 0.1-10 µM range. For
reference purposes, we also subjected mupirocin to this test and
this compound showed no immunosuppressive effect up to the
highest concentration used (10 µM).
As we expected that the simultaneous inhibition of multiple
aminoacyl-tRNA synthetases would lead to an increase in the
biological effect, we then evaluated the effect of combinations
of aaSA compounds with other aaSA compounds, or with the
clinically used immunosuppressants rapamycin (Rapa) or cy-
closporin A (CsA). Rapamycin is an inhibitor of mTOR
(mammalian target of rapamycin), while cyclosporine A is a
calcineurin inhibitor. In practice, this was done by comparing
the MLR inhibiting effect of compounds alone with the effect
of compounds together at the same concentration. Testing for
statistical significance was done by 2 × 2 multifactorial analysis
of variance (ANOVA).33 The results of these tests are sum-
marized in Table 2; interestingly, all combinations of aaSA
compounds showed synergism. In contrast, no statistically
significant interaction was found between aaSA compounds and
rapamycin. With cyclosporine A, subadditivity was found in
all but one of the combinations tested. A possible explanation
for the antagonistic effect seen between aaRS inhibitors and
CsA would be that aaRS inhibition limits the synthesis of
proteins that are necessary for the action of this immunosup-
a Reagents and conditions: (a) (i) MMTrCl, Et3N, pyr; (ii) TBDMSCl,
imidazole, DMF; (iii) TsOH in MeOH/CH2Cl2 (2:8 v/v), 67%; (b)
H2NSO2Cl, CH3CN/DMAc, 88%; (c) N-Boc L-amino acid N-hydroxysuc-
cinimide ester, DBU, DMF, 61-85%; (d) TFA/H2O (5:2 v/v), 34-57%.
tection of the secondary hydroxyls.31 The remainder of the
synthesis of these sugar analogues was similar to the synthesis
of aaSA analogue 32 (Scheme 1).
Purification of the final compounds was found to be rather
difficult, probably because of their highly polar, zwitterionic
nature. Therefore, they were purified by silica gel chromatog-

3024 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10
Scheme 4. Synthesis of the Altritol Analogues of Figure 3a
Van de VijVer et al.
a Reagents and conditions: (a) MMTrCl, pyr; (b) TIPDSCl2, pyr, 80% over steps a and b; (c) TsOH in MeOH/CH2Cl2 (2:8, v/v), 71%; (d) H2NSO2Cl,
CH3CN/DMAc, 71%; (e) N-Boc L-aminoacid N-hydroxysuccinimide ester, DBU, DMF, 90%; (f) (i) TFA/H2O (5:2, v/v); (ii) Et3N·3HF, THF, 40-71%.
Table 1. Immunosuppressive Activity of aaSA Compounds
tested, we found synergism. Interestingly, synergism was also
found when combining 32 with PheSA. This was surprising, as
both compounds were expected to target PheRS, and this result
suggests a more complex mode of action for 32 than inhibition
of PheRS alone. As we did not have access to enzymatic assays
to evaluate our compounds against isolated aaRS, it is difficult
to prove the direct relationship between aaRS inhibition and
the inhibitory effects observed with these compounds against
lymphocytes. Based on the data available in this article, the
overall growth-inhibitory activity of single compounds may be
the result of synergistic inhibition of multiple aaRS.
a
cmpd
MLR IC50 (µM)
cmpd
MLR IC50 (µM)
AsnSA
CysSA
MetSA
TrpSA
ProSA
GlnSA
GlySA
PheSA
LysSA
ArgSA
0.3
0.4
0.5
0.5
0.7
0.8
0.8
1.0
1.0
1.0
HisSA
LeuSA
AlaSA
AspSA
SerSA
GluSA
ThrSA
IleSA
1.0
1.1
1.7
2.4
4.8
4.9
5.2
5.6
5.6
5.6
TyrSA
ValSA
a MLR IC50 values for the analogues of aaSA.
Conclusions and Discussion
pressant: this may either be the direct molecular target of CsA
or a protein downstream of the target.
Though lacking in whole-cell antibacterial activity, several
of the compounds reported in this article show a pronounced
effect on the growth of stimulated immune cells, at micromolar
and submicromolar concentrations. We also show that combin-
ing multiple compounds that target aaRS leads to a significant
synergistic effect with respect to the immunosuppressive activity.
Finally, the potent immunosuppressive effects found with these
agents provide sufficient arguments for the screening of
developmental antibacterials that target aaRS enzymes for their
immunosuppressive effects in humans, because even slight
immunosuppressive effects may hamper the use of these
compounds as systemic antibiotics in humans.
b. Immunosuppressive Properties of the Modified
aaSA Compounds. For each of the modified aaSA compounds,
the IC50 in the MLR was determined (Table 3). The compounds
were active in the same concentration range as the parent
compounds. Replacement of the sugar moiety by anhydrohexitol
(16-21) was found to decrease the activity. In contrast,
replacement of the sugar by altritol (9-14) or 2′-deoxy-2′-
fluoro-ribose (22-26) gave similar or slightly better activity.
The aminoacyl-modified analogues (27-32) also showed good
immunosuppressive activity. Notable in this series is the
N-benzyl-L-Phe containing compound 32, which was found to
have an IC50 of 200 nM. To evaluate the modularity of our
modifications, we prepared a more extensively modified com-
pound combining the altritol sugar replacement with the
N-benzyl-L-Phe at the aminoacyl position. This compound (15)
inhibited the MLR with an IC50 of 80 nM, which is comparable
to the MLR-IC50 of cyclosporin A (74 ( 8 nM).32
Experimental Section
Chemistry. General Information. Reagents and solvents were
from commercial suppliers (Acros, Sigma-Aldrich, Bachem, Nova-
biochem) and used as provided, unless indicated otherwise. DMF
and THF were analytical grade and were stored over 4 Å molecular
sieves. All other solvents used for reactions were analytical grade
and used as provided. Reactions were carried out in oven-dried
We also evaluated several combinations of modified aaSA
analogues with aaSA analogues (Table 4): in all 3 combinations

ImmunosuppressiVe aaRS Inhibitors
Table 2. Combination Studies 1a
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10 3025
cmpd 1 (µM)
individual (%)
cmpd 2 (µM)
individual (%)
combined (%)
difference (%)
P value
PheSA (0.5)
LysSA (1.0)
LeuSA (0.5)
TrpSA (0.5)
ProSA (0.5)
TrpSA (0.1)
TrpSA (0.1)
AlaSA (0.1)
AlaSA (0.5)
HisSA (0.1)
HisSA (0.1)
PheSA (0.1)
PheSA (0.5)
LeuSA (0.1)
LeuSA (0.1)
ProSA (0.1)
10.8 ( 7.4
67.0 ( 6.5
28.4 ( 6.3
27.6 ( 8.6
18.6 ( 5.0
2.3 ( 22.8
2.3 ( 22.8
6.5 ( 8.2
4.9 ( 13.7
12.4 ( 6.1
11.1 ( 25.9
10.7 ( 9.6
53.4 ( 17.8
11.4 ( 11.0
16.0 ( 18.6
-4.1 ( 8.0
TrpSA (0.5)
ProSA (0.1)
MetSA (0.5)
MetSA (0.5)
LeuSA (0.5)
CsA (0.01)
Rapa (0.0004)
CsA (0.06)
Rapa (0.0008)
CsA (0.06)
Rapa (0.0004)
CsA (0.08)
Rapa (0.0004)
CsA (0.06)
Rapa (0.0008)
CsA (0.06)
27.6 ( 8.6
-2.7 ( 9.8
16.3 ( 6.9
28.4 ( 6.9
16.3 ( 6.3
32 ( 6.9
13.1 ( 13.5
65.7 ( 5.9
32.5 ( 6.5
65.7 ( 5.9
13.1 ( 13.5
75.2 ( 6.0
13.1 ( 13.5
65.7 ( 5.9
32.5 ( 6.5
65.7 ( 5.9
90.9 ( 7.1
87.0 ( 7.1
94.5 ( 7.0
96.9 ( 7.1
92.5 ( 6.8
-4.6 ( 28.6
-4.7 ( 11.3
36.3 ( 9.9
55.8 ( 15.4
54.4 ( 7.7
6.8 ( 19.2
62.1 ( 3.7
93.1 ( 21.2
32.4 ( 10.9
26.5 ( 10.1
37.2 ( 8.2
52.5b
22.7b
49.8b
40.9b
57.6b
-38.9
-20.1
-35.9b
18.4
<0.0001
0.0105
<0.0001
0.0001
<0.0001
0.0611
0.7412
0.0018
0.2121
0.0043
0.3836
0.0057
0.1703
0.0014
0.0562
0.0101
-24.1b
-17.4
-23.8b
26.6
-44.7b
-22
24.4b
a MLR inhibititory activity of combinations of aaSA compounds with aaSA compounds and with CsA/Rapa. For each combination, the meaningful
difference with lowest P value is reported. Activities are % inhibition ( SD. Differences are differences between observed effect of combinations and sum
of activities of individual compounds. b Indicates significant differences (P < 0.05).
Table 3. Immunosuppressive Activity of Modified aaSA Compounds
(39). To an ice-cooled solution of 2′,3′-O-isopropylidene-adenosine
(8.00 g, 26.0 mmol, 1.0 equiv) in N,N-dimethylacetamide (40 mL)
was added an ice-cooled, freshly prepared 2.0 M solution of
sulfamoyl chloride in CH3CN (30 mL, 2.3 equiv).34 After stirring
for 1 h at rt, the reaction mixture was cooled to 0 °C and the reaction
was quenched by the subsequent additions of Et3N (15 mL) and
MeOH (50 mL). The volatiles were removed in vacuo and the oily
residue was taken up into EtOAc (500 mL), extracted with aqueous
NaHCO3 5% (2 × 150 mL), and brine (2 × 100 mL). Next, the
organic layer was dried over anhydrous Na2SO4, filtered, and
evaporated to give 7.77 g of 39 (21.2 mmol, 82%). Spectral data
(1H NMR, 13C NMR, HRMS) were as reported by Heacock et al.11
2′,3′-O-Bis(tert-butyldimethylsilyl)-5′-O-sulfamoyladenosine (44).
Following the sulfamoylation procedure used for the preparation
of 39, 2′,3′-O-bis(tert-butyldimethylsilyl)adenosine (10.00 g, 20.2
mmol, 1.0 equiv) was converted to 11.40 g of 44 (19.8 mmol,
98%).35 NMR spectra (1H NMR, 13C NMR) were as reported by
Ferreras et al.28 HRMS for C22H43N6O6SSi2 [M + H]+ calcd,
575.2503; found, 575.2498.
L-(N-Boc-azetidine)-2-carboxylic Acid N-Hydroxysuccinimide
Ester (76). The amino acid L-azetidine-2-carboxylic acid (0.25 g,
2.47 mmol, 1.0 equiv) was dissolved in aqueous Na2CO3 solution
(10% w/w, 10 mL) and a solution of ditert-butyl dicarbonate (0.70
g, 3.21 mmol, 1.3 equiv) in dioxane (5 mL) was added. The mixture
was stirred overnight, H2O was added (15 mL), and the mixture
was extracted with Et2O (2 × 30 mL). The aqueous layer was
cooled (0 °C), acidified with concd HCl to pH ∼ 2, and extracted
with EtOAc (3 × 50 mL). The organic layers were dried (anhydrous
Na2SO4), filtered, and evaporated. The oily residue (0.50 g) obtained
this way was used in the second step without further purification.
The residue was dissolved in DMF (15 mL), N-hydroxysuccinimide
was added (345 mg, 3.0 mmol, 1.2 equiv) and the solution cooled
to 0 °C. N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydro-
chloride (671 mg, 3.50 mmol, 1.4 equiv) was added, and the mixture
was allowed to come to rt overnight. The volatiles were removed
in vacuo, and the residue was taken up into EtOAc (100 mL). The
mixture was washed with HCl 2 N (2 × 100 mL), NaHCO3 satd
(100 mL), brine (100 mL), and dried over anhydrous Na2SO4. After
filtering, the volatiles were removed in vacuo, yielding 615 mg of
76 (2.06 mmol, 85%). 1H NMR (DMSO-d6): δ 5.02 (dd, 1H, 2-H,
J ) 5.4 Hz, 9.4 Hz), 3.88 (t, 2H, 4-H2, J ) 7.6 Hz), 2.83 (s, 4H,
OSu-H2), 2.78-2.58 (m, 1H, 3-HA), 2.30-2.10 (m, 1H, 3-HB), 1.37
(s, 9H, Boc-C(CH3)3). 13C NMR (DMSO-d6): δ 170.1, 167.6, 79.8,
58.0, 47.1, 27.8, 25.5, 20.4. HRMS for C13H19N2O6 [M + H]+
calcd, 299.1243; found, 299.1248.
a
cmpd
MLR IC50 (µM)
cmpd
MLR IC50 (µM)
9
0.3
1
21
22
23
24
25
26
27
28
29
30
31
32
0.6
0.4
0.45
0.4
0.3
0.45
0.6
0.55
5.5
4.7
0.55
0.2
10
11
12
13
14
15
16
17
18
19
20
5.2
0.75
1.2
0.6
0.08
5.5
6.8
5.5
2.8
6.7
a MLR IC50 values for the modified analogues of aaSA.
glassware under a nitrogen atmosphere and stirred at room
temperature, unless indicated otherwise.
1H and 13C NMR spectra of the compounds were recorded on a
200 MHz Varian Gemini spectrometer or a Bruker UltraShield
Avance 300 MHz spectrometer. Spectra were recorded in DMSO-
d6, D2O, or CDCl3. The chemical shifts are expressed as δ values
in parts per million (ppm), using the residual solvent peaks
(chloroform: 1H, 7.26 ppm, 13C, 77.36 ppm; DMSO: 1H, 2.50 ppm,
13C, 39.60 ppm; HOD: 1H, 4.79 ppm) as a reference. For 13C
spectra in D2O, ∼0.1% dioxane (67.19 ppm) was added as a
reference. Coupling constants are given in Hertz (Hz). The peak
patterns are indicated by the following abbreviations: bs ) broad
singlet, d ) doublet, m ) multiple, q ) quadruplet, s ) singlet,
and t ) triplet. High resolution mass spectra were recorded on a
quadrupole time-of-flight mass spectrometer (Q-Tof-2, Micromass,
Manchester, U.K.) equipped with a standard ESI interface; samples
were infused in 2-propanol/H2O (1:1, v/v) at 3 µL·min-1
.
For TLC, precoated aluminum sheets were used (Merck, Silica
gel 60 F254). The spots were visualized by UV light. Column
chromatography was performed on ICN silica gel 60A 60-200.
For size exclusion chromatography, a 2 × 30 cm column of
Sephadex LH-20 was used as the solid phase and MeOH/H2O (7:
3, v/v) as the eluent. Preparative HPLC was done using a Waters
Xbridge Prep. C18 (19 × 150 mm) column connected to a Waters
1525 binary HPLC pump and a Waters 2487 Dual Absorbance
Preparative Detector. Method HPLC1: solvent A ) H2O, solvent
B ) CH3CN. Method HPLC2: solvent A ) triethylammonium
bicarbonate 50 mM, CH3CN 1%, H2O 99%; solvent B ) triethy-
lammonium bicarbonate 50 mM, H2O 1%, CH3CN 99%. Eluent
compositions are expressed as v/v.
2′,3′-O-Isopropylidene-5′-O-[N-(L-(N-Boc-azetidine)-2-carbon-
yl)-sulfamoyl]adenosine (Et3N Salt; 40). To a solution of 39 (350
mg, 0.91 mmol, 1.0 equiv) and 76 (300 mg, 1.01 mmol, 1.1 equiv) in
DMF (10 mL) was added DBU (277 mg, 1.82 mmol, 2 equiv). When
TLC analysis (Et3N 1%, MeOH 5%, CH2Cl2) showed complete
Synthesis of Analogues of 5′-O-(N-Aminoacyl)-sulfamoylad-
enosine Containing Nonproteinogenic Amino Acids (27-32).
Synthesis of 2′,3′-O-Isopropylidene-5′-O-sulfamoyladenosine

3026 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10
Table 4. Combination Experiments with Modified Analogues of aaSAa
Van de VijVer et al.
cmpd 1 (µM)
individual (%)
cmpd 2 (µM)
individual (%)
combined (%)
difference (%)
P value
9 (0.5)
32 (0.1)
32 (0.1)
68.0 ( 8.2
31.1 ( 10.1
31.1 ( 10.1
MetSA(0.5)
PheSA(0.5)
TrpSA (0.5)
1.0 ( 9.8
-1.8
-8.6
86.2 ( 9.0
92.0 ( 12.7
91.6 ( 12.4
17.2
0.0935
0.001
0.0009
( 9.5
( 9.2
62.7b
69.1b
a MLR inhibititory activity of combinations of modified analogues of aaSA with aaSA. For each combination, the meaningful difference with lowest P
value is reported. Activities are % inhibition ( SD. Differences are differences between observed effect of combinations and sum of activities of individual
compounds. b Indicates significant differences (P < 0.05).
conversion of 39 (6 h), the reaction mixture was concentrated in vacuo,
and the residue was purified by silica gel LC (Et3N 1%, MeOH 5% in
CH2Cl2), giving 473 mg (0.71 mmol, 77%) of 40. 1H NMR (DMSO-
d6): δ 8.39 (s, 1H, 8-H), 8.15 (s, 1H, 2-H), 6.15 (d, 1H, 1′-H, J ) 3.0
Hz), 5.36 (m, 1H, 2′-H), 5.03 (d, 1H, 3′-H, J ) 5.2 Hz), 4.38 (m, 1H,
4′-H), 4.18 (dd, 1H, azetidine-2-H, J ) 4.4 Hz, 8.8 Hz), 4.00 (d, 2H,
5′-H2, J ) 4.4 Hz), 3.88-3.44 (m, 2H, azetidine-4-H2), 2.98 (q, 6H,
Et3NH+-H2, J ) 7.2 Hz), 2.30 (m, 1H, azetidine-3-HA), 1.79 (m, 1H,
azetidine-3-HB), 1.54 (s, 3H, isopropylidene-H3), 1.42-0.90 (m, 21H,
isopropylidene-H3, Boc-C(CH3)3, Et3NH+-H3). 13C NMR (DMSO-
d6): δ 175.6, 156.3, 155.4, 153.0, 149.3, 139.6, 119.0, 113.2, 89.3,
83.9, 83.6, 81.8, 78.0, 67.0, 64.5, 46.9, 45.7, 28.3, 27.1, 25.2, 20.7,
9.0. HRMS for C22H32N7O9S [M + H]+ calcd, 570.1982; found,
570.1983.
5′-O-[N-(L-Azetidine-2-carbonyl)-sulfamoyl]adenosine (27). Com-
pound 40 (440 mg, 0.66 mmol, 1.0 equiv) was dissolved in TFA/
H2O (4 mL, 5:2, v/v) and the reaction mixture was stirred for 2 h,
after which the volatiles were evaporated, coevaporated twice with
EtOH, and once with EtOH + 1 mL Et3N, to neutralize any
remaining acid. The residue was purified by silica gel LC (Et3N
1%, MeOH 20 f 50% in CH2Cl2) and by size exclusion
chromatography, giving 172 mg (0.40 mmol, 61%) of 27 as a white
solid after lyophilization from H2O.
oil was purified by silica column chromatography (0.5% Et3N,
MeOH 1f3% in CH2Cl2) to give 4.13 g of 48 (6.34 mmol, 68%).
1H NMR (DMSO-d6/CDCl3 1:1): δ 8.30 (s, 1H, 8-H), 8.13 (s, 1H,
2-H), 7.50-7.06 (m, 14 H, aryl-H, Ade-NH2), 6.81 (d, 2H, aryl-H,
J ) 9.2 Hz), 4.81 (bs, 1H, 2′-H), 4.38 (d, 1H, 1′-HA, J ) 14.2 Hz),
4.02 (dd, 1H, 1′-HB, J ) 2.0 Hz, 11.7 Hz), 3.73 (s, 3H, MMTr-
OCH3), 3.78-3.44 (m, 2H, 4′-H, 5′-H), 3.22 (dd, 1H, 6′-HA, J )
5.8 Hz, 12.4 Hz), 3.07 (dd, 1H, 6′-HB, J ) 5.1 Hz, 9.9 Hz), 2.48
(obscured by solvent peak, 3′-HA), 1.93 (app. dt, 1H, 3′-HB, J )
4.0 Hz, 11.4 Hz), 0.56 (s, 9H, Si-(CH3)3), -0.26 (s, 3H, Si-CH3),
-0.43 (s, 3H, Si-CH3).
13C NMR (DMSO-d6/CDCl3, 1:1 v/v): δ 158.3, 156.1, 152.4,
149.5, 144.4, 139.2, 135.2, 130.2, 128.2, 127.6, 126.7, 118.7, 113.0,
85.6, 81.6, 68.4, 63.1, 54.9, 50.1, 36.4, 25.3, 17.2, -4.6, -5.6.
HRMS for C37H46N5O4Si [M + H]+ calcd, 652.3319; found,
652.3301.
2-(Adenin-9-yl)-1,5-anhydro-4-O-tert-butyldimethylsilyl-2,3-
dideoxy-D-arabino-hexitol (49). Compound 48 (4.13 g, 6.34 mmol)
was added to an ice-cooled solution of 2% p-toluenesulfonic acid
monohydrate in MeOH/CH2Cl2 (2:8, v/v). After one hour, TLC
(CH2Cl2/MeOH 9:1 v/v) indicated complete conversion, and the
reaction was quenched by the addition of NaHCO3. Silica gel (20 g)
was added and the volatiles were removed under reduced pressure.
The adsorbed residue was purified by silica gel chromatography
(CH2Cl2/MeOH 9:1 v/v) giving 49 (1.95 g, 5.14 mmol, 81%).
1H NMR (DMSO-d6): δ 8.30 (s, 1H, 8-H), 8.15 (s, 1H, 2-H),
7.24 (s, 2H, Ade-NH2), 4.80 (bs, 1H, 2′-H), 4.72 (t, 1H, 6′-OH, J
) 5.7 Hz), 4.21 (d, 1H, 1′-HA, J ) 12.4 Hz), 3.91 (dd, 1H, 1′-HB,
J ) 2.6 Hz, 14.1 Hz), 3.85-3.45 (m, 3H, 6′-H2, 4′-H), 3.26 (m,
1H, 5′-H), 2.39 (d, 1H, 3′-HA, J ) 13.6 Hz), 1.96 (m, 1H, 3′-HB),
0.79 (s, 9H, Si-C(CH3)3), -0.07 (s, 3H, Si-CH3), -0.13 (s, 3H,
Si-CH3). 13C NMR (DMSO-d6): δ 156.2, 152.6, 149.6, 139.8, 118.5,
82.7, 68.1, 62.8, 60.1, 50.1, 36.2, 25.6, 17.6, -4.7, -5.1. HRMS
for C17H30N5O3Si [M + H]+ calcd, 380.2118; found, 380.2119.
2-(Adenin-9-yl)-1,5-anhydro-6-O-sulfamoyl-4-O-tert-butyldim-
ethylsilyl-2,3-dideoxy-D-arabino-hexitol (50). Following the proce-
dure used for the synthesis of 39, compound 49 (1.0 g, 2.63 mmol,
1.0 equiv) was sulfamoylated to give 50 (0.83 g, 1.80 mmol, 69%).
1H NMR (DMSO-d6): δ 8.20 (s, 1H, 8-H), 8.15 (s, 1H, 2-H), 7.54
(bs, 2H, sulfamoyl-NH2), 7.24 (bs, 2H, Ade-NH2), 4.81 (bs, 1H,
2′-H), 4.40-4.10 (m, 3H, 6′-H2, 1′-HA), 3.97 (m, 1H, 1′-HB),
3.75-3.35 (m, 2H, 4′-H, 5′-H), 2.5 (3′-HA, hidden under DMSO
signal), 2.02 (m, 1H, 3′-HB), 0.80 (s, 9H, Si-C(CH3)3), -0.04 (s,
3H, Si-CH3), -0.09 (s, 3H, Si-CH3). 13C NMR (DMSO-d6): δ
156.3, 152.7, 149.6, 139.5, 118.6, 79.5, 68.1, 67.9, 63.2, 50.0, 36.0,
25.6, 17.6, -4.5, -5.1. HRMS for C17H29N6O5SSi [M - H]- calcd,
457.1689; found, 457.1690.
2-(Adenin-9-yl)-1,5-anhydro-4-O-tert-butyldimethylsilyl-6-O-[N-
(N-Boc-L-alanyl)sulfamoyl]-2,3-dideoxy-D-arabino-hexitol (51). To
a solution of compound 50 (200 mg, 0.44 mmol, 1.0 equiv) and
N-Boc-L-alanine N-hydroxysuccinimide ester (187 mg, 0.65 mmol,
1.5 equiv) in dry DMF (5 mL) was added DBU (165 mg, 1.09
mmol, 2.5 equiv). The reaction mixture was stirred at rt until
complete conversion (2 h) of the starting compound (TLC, HOAc
1%, MeOH 5% in CH2Cl2). Next the volatiles were evaporated
and the oily residue was purified by silica gel column chromatog-
raphy (HOAc 1%, MeOH 5% in CH2Cl2) giving the title compound
51 as a white solid (252 mg, 0.40 mmol, 91%). 1H NMR (DMSO-
d6): δ 10.6 (bs, 1H, R-NH) 8.22 (s, 1H, 8-H), 8.15 (s, 1H, 2-H),
7.25 (bs, 2H, Ade-NH2), 6.76 (bs, 1H, sulfamoyl-NH), 4.80 (bs,
2′-H), 4.40-4.10 (m, 3H, 1’HA, 6′-H2,), 4.00-3.75 (m, 2 H, 1′-
1H NMR (D2O) δ 8.35 (s, 1H, 8-H), 8.17 (s, 1H, 2-H), 6.08 (d,
1H, 1′-H, J ) 4.4 Hz), 4.89 (t, 1H, azetidine-2-H, J ) 8.7 Hz), 4.73
(m, 1H, 2′-H), 4.56-4.34 (m, 4H, 3′-H, 4′-H, 5′-H2), 4.18-4.03 (m,
1H, azetidine-4-HA), 3.97-3.82 (m, 1H, azetidine-4-HB), 2.88-2.71
(m, 1H, azetidine-3-HA), 2.61-2.44 (m, 1H, azetidine-3-HA). 13C NMR
(D2O + dioxane) δ 175.0, 156.1, 153.4, 149.5, 140.3, 119.2, 88.0,
82.8, 74.6, 70.7, 69.0, 61.0, 43.9, 24.1. HRMS for C14H20N7O7S [M
+ H]+ calcd, 430.1145; found, 430.1136.
The synthesis and spectroscopic data of other analogues of 5′-
O-(N-aminoacyl)-sulfamoyladenosine containing nonproteinogenic
amino acids (28-32) can be found in the Supporting Information.
Synthesis of the Anhydrohexitol Derivatives (16-21). 2-
(Adenin-9-yl)-1,5-anhydro-6-O-monomethoxytrityl-2,3-dideoxy-D-
arabino-hexitol (47). A suspension of 2-(adenin-9-yl)-1,5-anhydro-
2,3-dideoxy-D-arabino-hexitol (2.64 g, 9.95 mmol, 1.0 equiv; 46)
and 4-monomethoxytrityl chloride (3.25 g, 10.5 mmol, 1.05 equiv)
in dry pyridine (220 mL) was allowed to react for 2 days at rt.29
Next, the resulting solution was concentrated under reduced pressure
and coevaporated three times with toluene. The residue was purified
by column chromatography using 0.5% Et3N and MeOH 1 f 3%
in CH2Cl2 as the eluent to give 47 (4.99 g, 93%) as a white foam.
1H NMR (DMSO-d6/CDCl3 1:1): δ 8.33 (s, 1H, 8-H), 8.14 (s, 1H,
2-H), 7.50-7.00 (m, 14H, aryl-H, Ade-NH2), 6.83 (d, 2H, aryl-H,
J ) 8.8 Hz), 4.86 (d, 1H, 4′-OH, J ) 4.0 Hz), 4.79 (bs, 1H, 2′-H),
4.32 (d, 1H, 1′-HA, J ) 12.0 Hz), 3.94 (d, 1H, 1′-HB, J ) 10.6
Hz), 3.78-3.50 (m, MMTr-OCH3, 4′-H), 3.50-3.07 (m, partly
obscured by residual H2O peak, 5′-H, 6′-H2), 2.41 (m, 1H, 3′-HA),
1.88 (m, 1H, 3′-HB). 13C NMR (DMSO-d6): δ 158.3, 156.3, 152.7,
149.6, 144.8, 142.1, 139.6, 135.4, 130.3, 128.3, 128.0, 126.9, 118.7,
113.3, 85.5, 81.4, 68.3, 63.0, 61.1, 55.1, 50.4, 36.3. HRMS for
C31H32N5O4 [M + H]+ calcd, 538.2454; found, 538.2432.
2-(Adenin-9-yl)-1,5-anhydro-6-O-monomethoxytrityl-4-O-tert-
butyldimethylsilyl-2,3-dideoxy-D-arabino-hexitol (48). A solution
of 47 (4.99 g, 9.28 mmol, 1.0 equiv) in DMF (∼100 mL) was
reacted with TBDMSCl (3.00 g, 19.9 mmol, 2.1 equiv) in the
presence of imidazole (1.40 g, 20.6 mmol, 2.2 equiv) at rt over
weekend. The resulting yellow solution was concentrated under
reduced pressure and coevaporated (3×) with toluene. The residual

ImmunosuppressiVe aaRS Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10 3027
HB, R-H), 3.70-3.50 (m, partially obscured by H2O peak, 5′-H,
4′-H), 2.44 (m, partially obscured by DMSO-d6 peak, 3′-HA),
2.10-1.90 (m, 1H, 3′-HB), 1.36 (s, 9H (Boc-C-(CH3)3), 1.19 (d,
3H, ꢀ-H3, J ) 7.4 Hz), 0.78 (s, 9H, Si-C(CH3)3), -0.07 (s, 3H,
Si-CH3), -0.12 (s, 3H, Si- CH3). 13C NMR (DMSO-d6): δ 173.1,
156.2, 155.2, 152.6, 149.6, 139.7, 118.5, 79.5, 78.2, 69.8, 68.1,
63.3, 50.8, 50.0, 36.1, 28.3, 25.3 and 25.6, 17.6, 8.7, -4.6, -5.1.
HRMS for C25H44N7O8SSi [M + H]+ calcd, 630.2741; found,
630.2740.
5′-O-[N-(N-Boc-L-alanyl)sulfamoyl]-3′-O-(tert-butyldimethylsi-
lyl)-2′-deoxy-2′-fluoroadenosine Et3N Salt (60). Following the
procedure used for the synthesis of 51, compound 59 (185 mg,
0.40 mmol, 1.0 equiv) and N-Boc-L-alanine N-hydroxysuccinimide
ester (115 mg, 0.40 mmol, 1.0 equiv) were reacted with DBU (91
mg, 0.6 mmol, 1.5 equiv). The product isolated by column chro-
matography was dried under vacuum to afford 179 mg of solid 60
(0.24 mmol, 61%). 1H NMR (DMSO-d6): δ 8.40 (s, 1H, 8-H), 8.15
(s, 1H, 2-H), 7.36 (s, 2H, Ade-NH2), 6.24 (dd, 1H, 1′-H, J ) 16.2
Hz, 4.4 Hz), 6.06 (d, 1H, Ala-NH, J ) 7.2 Hz), 5.62 (ddd, 1H,
2′-H, J ) 52.8 Hz), 4.76-4.64 (m, 1H, 3′-H), 4.19-3.96 (m, 3H,
4′-H, 5′-H2), 3.71 (m, 1H, R-H), 3.08 (q, 6H, Et3NH+-H2, J ) 11.0
Hz), 1.35 (s, 9H, Boc-C(CH3)3), 1.16 (t, 9H, Et3NH+-H3), 1.17 (d,
3H, ꢀ-H3), 0.90 (s, 9H, Si-C(CH3)3), 0.14 and 0.12 (2 × s, 6H,
Si(CH3)2). 13C NMR (DMSO-d6): δ 177.3, 156.4, 155.1, 153.4,
149.6, 139.9, 119.2, 92.5 (J ) 192.3 Hz), 85.3 (J ) 32.0 Hz), 82.7,
78.1, 71.1 (J ) 15.4 Hz), 66.9, 52.0, 46.1, 28.5, 25.9, 19.9, 18.1,
8.9, -4.9. HRMS for C24H40N7O8FSSi [M + H]+ calcd, 634.2490;
found, 634.2497.
2-(Adenin-9-yl)-1,5-anhydro-6-O-[N-(L-alanyl)sulfamoyl]-
2,3-dideoxy-D-arabino-hexitol (16). A solution of compound 51
(242 mg, 0.38 mmol, 1.0 equiv) in TFA/H2O (4 mL, 5:2, v/v) was
stirred for 5 h at rt. Next, the volatiles were removed by rotary
evaporation and coevaporated (EtOH, 3×) to give a white foam.
This foam was further purified by silica gel chromatography (HOAc
1%, MeOH 30% in CH2Cl2), and the fractions containing 16 were
collected and evaporated to dryness. The residue was further purified
by size exclusion column chromatography (2×, 30% H2O in
MeOH). Finally, lyophilization from H2O yielded 135 mg (0.32
mmol, 87%) of pure 16.
5′-O-[N-(L-Alanyl)sulfamoyl]-2′-deoxy-2’fluoroadenosine (22).
Following the procedure used for the synthesis of compound 16,
compound 60 (194 mg, 0.26 mmol, 1.0 equiv) was deprotected to
1H NMR (D2O): δ 8.38 (s, 1H, 8-H), 8.13 (s, 1H, 2-H), 4.83
(bs, 1H, 2′-H), 4.47-4.34 (m, 3H, 1′-HA, 6′-H2), 4.12 (dd, 1H, 6′-
HB, J ) 2.6 Hz, 13.1 Hz), 3.88 (q, 1H, R-H, J ) 7.2 Hz), 3.79-3.66
(m, 2H, 5′-H, 4′-H), 2.55-2.45 (m, 1H, 3′-HA), 2.14-2.03 (m, 1H,
3′-HB), 1.51 (d, 3H, ꢀ-H3, J ) 7.2 Hz). 13C NMR (D2O + dioxane):
δ 177.2, 156.0, 152.9, 149.3, 141.7, 118.6, 80.1, 69.1, 69.0, 61.6,
52.1, 51.4, 35.5, 17.2. HRMS for C14H20N7O6S [M - H]- calcd,
414.1196; found, 414.1187.
1
give 22 as a white solid (51 mg, 46% yield). H NMR (DMSO-
d6): δ 8.36 (s, 1H, 8-H), 8.16 (s, 1H, 2-H), 7.35 (s, 2H, Ade-NH2),
6.25 (dd, 1H, 1′-H, J ) 17.6, 2.4 Hz), 5.47 (ddd, 1H, 2′-H, J )
53.4 Hz), 4.61-4.37 (m, 1H, 3′-H), 4.27-4.05 (m, 3H, 4′-H, 5′-
H2), 3.49 (q, 1H, R-H, J ) 11.1 Hz), 1.30 (d, 3H, ꢀ-H3, J ) 10.5
Hz). 13C NMR (DMSO-d6): δ 173.6, 156.3, 153.0, 149.2, 139.5,
119.1, 93.4 (J ) 186.19 Hz), 85.7 (J ) 32.0 Hz), 81.3, 69.1 (J )
16.7 Hz), 67.2, 50.9, 17.3. HRMS for C13H18N7O6FS [M + H]+
calcd, 420.1101; found, 420.1101.
The synthesis and spectroscopic data of the anhydrohexitol
derivatives 17-21 can be found in the Supporting Information.
Synthesis of the 2′-Deoxy-2′-fluoroadenosine derivatives (22-26).
3′-O-(tert-Butyldimethylsilyl)-2′-deoxy-2′-fluoroadenosine (58). 2′-
Deoxy-2′-fluoroadenosine (57, 1.16 g, 4.31 mmol, 1.0 equiv) was
coevaporated with anhydrous pyridine (3×) and then dissolved in
anhydrous pyridine (50 mL).30 Triethylamine (567 mg, 5.60 mmol,
1.3 equiv) was added to the solution followed by monomethoxytrityl
chloride (2.26 g, 7.32 mmol, 1.7 equiv). After stirring overnight at rt,
the volatiles were evaporated. The residue was dissolved in DMF (50
mL) and tert-butyldimethylsilyl chloride (974 mg, 6.46 mmol, 1.5
equiv) and imidazole (733 mg, 10.77 mmol, 2.5 equiv) were added.
The mixture was stirred at rt for 1 day and the volatiles were
evaporated. Finally, the residue was treated with a 2% solution of
p-toluenesulfonic acid monohydrate in MeOH/CH2Cl2 (150 mL, 2:8,
v/v) at 0 °C for 2 h. The solution was then diluted with CH2Cl2 (400
mL) and the reaction was quenched with 5% aq NaHCO3 (150 mL).
The organic layer was separated and the aqueous layer was extracted
with CH2Cl2 (3 × 100 ml). The organic layers were combined, dried
(Na2SO4), filtered, and evaporated. The residue was further purified
by silical gel chromatography (MeOH 2 f 6% in CH2Cl2) to give 58
as a white solid (1.12 g, 67% yield). 1H NMR (DMSO-d6): δ 8.38 (s,
1H, 8-H), 8.15 (s, 1H, 2-H), 7.39 (s, 2H, Ade-NH2), 6.23 (dd, 1H,
1′-H, J ) 16.6, 3.4 Hz), 5.56 (ddd, 1H, 2′-H, J ) 52.4 Hz), 5.30 (t,
1H, 5′-OH), 4.67-4.77 (m, 1H, 3′-H), 3.99 (m, 1H, 4′-H), 3.42-3.78
(m, 2H, 5′-H2), 0.91 (s, 9H, C(CH3)3), 0.14 and 0.12 (2 s, 6H,
Si(CH3)2). 13C NMR (DMSO-d6): δ 156.4, 153.0, 149.2, 140.0, 119.3,
92.5 (J ) 189.2 Hz), 85.9 (J ) 32.0 Hz), 84.9, 70.2 (J ) 15.3 Hz),
60.4, 25.8, 18.0, -4.8, -5.0. HRMS for C16H26N5O3FSi [M + H]+
calcd, 384.1867; found, 384.1870.
The synthesis and spectroscopic data of the 2′-deoxy-2′-fluoro-
adenosine derivatives 23-26 can be found in the Supporting
Information.
Synthesis of the Altritol Derivatives (9-15). 2′-(Adenin-9-yl)-
1′,5′-anhydro-2-deoxy-6′-O-monomethoxytrityl-3′,4′-O-(1,1,3,3-tet-
raisopropyldisiloxane-1,3-diyl)-D-altro-hexitol (67). To a suspension
of 2-(adenin-9-yl)-1,5-anhydro-2-deoxy-D-altro-hexitol (65, 206 mg,
0.73 mmol, 1.0 equiv) in dry pyridine (15 mL) was added
monomethoxytrityl chloride (226 mg, 0.73 mmol, 1.0 equiv) and
the resulting mixture was stirred overnight.31 TLC (CH2Cl2/MeOH,
9:1 v/v) indicated complete conversion of the starting material 65,
and 1,3-dichloro-1,1,3,3-tetrasiopropyldisiloxane (0.25 mL, 0.80
mmol, 1.1 equiv) was added and the resulting solution was stirred
for 3 days until TLC (CH2Cl2/MeOH, 9:1, v/v) indicated complete
conversion of the monomethoxytritylated intermediate 66. The
volatiles were removed under reduced pressure and the resulting
viscous oil was purified using column chromatography (MeOH
0f6%, Et3N 1% in CH2Cl2) to yield pure 67 (465 mg, 0.58 mmol,
80%).
1H NMR (200 MHz, CDCl3): δ 8.58 (s, 1H, 8-H), 8.34 (s, 1H,
2-H), 7.52-7.43 (m, 4H, Ar-H), 7.40-7.14 (m, 10H, Ar-H), 4.75
(s, 1H, 2′-H), 4.43 (d, 1H, 1′-HA, J ) 12.4 Hz), 4.24 (d, 1H, 1′-
HB, J ) 12.8 Hz), 4.18-4.05 (m, 2H, 3′-H, 4′-H), 3.93-3.82 (m,
1H, 5′-H), 3.79 (s, 3H, MMTr-O-CH3), 3.46 (dd, 1H, 6′-HA, J )
9.8 Hz, 1.8 Hz), 3.29 (dd, 1H, 6′-HB, J ) 4.1 Hz, 9.9 Hz),
1.22-0.65 (m, 28H, TIPDS-CH(CH3)2).
13C NMR (CDCl3): δ 158.6, 155.5, 153.1, 150.5, 144.7, 140.52,
135.8, 130.5, 128.5, 127.8, 126.8, 117.6, 113.0, 86.0, 76.3, 69.5,
67.9, 64.2, 62.7, 55.8, 55.1, 17.2, 14.0, 12.8. HRMS for C43-
H58N5O6Si2 [M + H]+: calcd, 796.3926; found, 796.3930.
2-(Adenin-9-yl)-1,5-anhydro-2-deoxy-3,4-O-(1,1,3,3-tetraisopro-
pyldisiloxane-1,3-diyl)-D-altro-hexitol (68). To an ice-cooled solution
of 2% p-toluenesulfonic acid monohydrate in MeOH/CH2Cl2 (200
mL, v/v) was added 3.80 g (4.77 mmol, 1.0 equiv) of 67. After 1 h
at 0 °C, TLC (CH2Cl2/MeOH, 9:1, v/v) indicated complete
conversion of 67 to a lower Rf compound. The reaction was
quenched by the addition of solid NaHCO3 and the reaction mixture
was adsorbed onto 10 g of silica by rotary evaporation and subjected
to column chromatography with gradient elution (MeOH 0 f 10%
in CH2Cl2) to give solid 68 (2.15 g, 4.10 mmol, 86%).
3′-O-(tert-Butyldimethylsilyl)-2′-deoxy-2′-fluoro-5′-O-sulfamoy-
ladenosine (59). Following the procedure used for the synthesis of
39, compound 58 (505 mg, 1.32 mmol, 1.0 equiv) was sulfamoy-
1
lated to give 59 (537 mg, 1.16 mmol, 88%). H NMR (DMSO-
d6): δ 8.30 (s, 1H, 8-H), 8.15 (s, 1H, 2-H), 7.62 (bs, 2H, SO2NH2),
7.39 (bs, 2H, Ade-NH2), 6.29 (dd, 1H, 1′-H, J ) 18.8 Hz, 2.2 Hz),
5.61 (ddd, 1H, 2′-H, J ) 55.0 Hz), 4.98-4.83 (m, 1H, 3′-H),
4.34-4.17 (m, 3H, 4′-H, 5′-H2), 0.91 (s, 9H, Si-C(CH3)3), 0.17
and 0.15 (2 × s, 6H, Si(CH3)2). 13C NMR (DMSO-d6): δ 156.4,
153.0, 149.1, 139.9, 119.3, 92.3 (J ) 189.2 Hz), 86.2 (J ) 33.6
Hz), 80.4, 70.2 (J ) 15.3 Hz), 67.8, 25.6, 17.8, 0.2. HRMS for
C16H27N6O5FSSi [M + H]+ calcd, 463.1595; found, 463.1591.

3028 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10
Van de VijVer et al.
1H NMR (DMSO-d6): δ 8.32 (s, 1H, 8-H), 8.12 (s, 1H, 2-H),
7.26 (bs, Ade-NH2), 4.85-4.72 (m, 2H, 3′-H, 6′-OH), 4.68-4.60
(m, 1H, 2′-H), 4.24-4.08 (m, 2H, 1′-H2), 4.10-3.92 (m, 1H, 4′-
H), 3.80-3.55 (m, 3H, 6′-H2, 5′-H), 1.20-0.75 (m, 28H, TIPDS-
CH(CH3)2).
culture plates (TTP, Switzerland). After five days, cells were pulsed
with 1 µCi of methyl-3H thymidine (MP Biomedicals, U.S.A.),
harvested 18 h later on glass filter paper and counted. Proliferation
values were expressed as counts per minute (cpm) and converted
to % inhibition with respect to a blank MLR test (identical but
without added compound). The IC50 was determined from a graph
with at least four points, each derived from the mean of 3-6
experiments.
Statistics. Basic data analysis was done in Microsoft Excel
2003 (Microsoft Corporation, Redmond, WA). Two-factorial
ANOVA was done using Graphpad Prism 4.03 (Graphpad
Software, San Diego, CA). Where applicable, data are repre-
sented as the mean ( SD. P values <0.05 were considered
statistically significant.
13C NMR (DMSO-d6): δ 156.2, 152.7, 149.9, 139.7, 118.5, 77.5,
69.5, 67.9, 63.3, 59.9, 55.0, 17.1, 13.1, 12.7, 12.4. HRMS for
C23H42N5O5Si2 [M+H]+ calcd, 524.2724; found, 524.2726.
2-(Adenin-9-yl)-1,5-anhydro-2-deoxy-6-O-sulfamoyl-3,4-O-(1,1,3,3-
tetraisopropyldisiloxane-1,3-diyl)-D-altro-hexitol (69). Following the
procedure used for the synthesis of 39, compound 68 (1.50 g, 2.86
mmol, 1.0 equiv) was sulfamoylated to give 69 (1.24 g, 2.06 mmol,
72%) as a white solid.
1H NMR (DMSO-d6): δ 8.23 (s, 1H, 8-H), 8.13 (s, 1H, 2-H),
4.86 (bs, 1H, 3′-H), 4.65 (bs, 1H, 2′-H), 4.40-4.14 (m, 4H, 6′-H2,
1′-H2), 3.96-3.78 (m, 2H, 4′-H, 5′-H), 1.25-0.75 (m, 28H, 4 ×
TIPDS-CH (CH3)2).
Acknowledgment. We thank Luc Baudemprez for as-
sistance in NMR recording, Roger Busson for help with
interpretation of NMR data and helpful discussions, and Jef
Rozenski and Stephanie Vandenwaeyenberg for recording of
MS spectra.
13C NMR (DMSO-d6): δ 156.3, 152.8, 149.9, 139.3, 118.5, 74.4,
68.9, 68.1, 67.9, 63.4, 55.1, 12.4, 12.7, 13.3, 17.1. HRMS for
C23H42N6O7SSi2 [M + H]+ calcd, 603.2452; found, 603.2458.
2-(Adenin-9-yl)-1,5-anhydro-2-deoxy-6-O-[(N-Boc-L-alanyl)sul-
famoyl]-3,4-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-D-altro-
hexitol (70). Following the procedure used for the synthesis of 51,
compound 69 (300 mg, 0.50 mmol, 1.0 equiv) was reacted with
N-Boc-L-alanine N-hydroxysuccinimide ester (280 mg, 0.98 mmol,
2.0 equiv) and DBU (114 mg, 1.05 mmol, 2.1 equiv) to give 349
mg of 70 (0.45 mmol, 90%).
Supporting Information Available: Synthetic procedures and
analytical data for the preparation of compounds 10-15, 17-21,
23-26, and 28-32 and for the corresponding intermediate
compounds. HPLC purity data of final compounds. This material
is available free of charge via the Internet at http://pubs.acs.org.
1H NMR (CD3OD) δ 8.47 (s, 1H, 8-H), 8.21 (s, 1H, 2-H),
4.90-4.70 (partly obscured by D2O peak, 2H, 2′-H, 3′-H),
4.63-4.48 (m, 2H, 6′-H2), 4.47-4.26 (m, 2H, 1′-H2), 4.16-3.75
(m, 3H, R-H, 4′-H, 5′-H), 1.30-0.76 (m, 40H, 4 × -CH(CH3)2,
Boc-(CH3)3, Ala-CH3). 13C NMR (CD3OD): δ 153.8, 153.2, 152.0,
142.0, 119.5, 75.7, 72.6, 70.5, 68.9, 64.8, 57.3, 28.7, 18.0, 17.6,
15.1, 14.4, 14.1. HRMS for C33H56N7O10SSi2 [M + H]+ calcd,
774.3348; found, 774.3334.
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