Synthesis of sugar-amino acid-nucleosides as potential glycosyltransferase inhibitors

Article abstract of DOI:10.1016/j.bmc.2010.11.044

Sugar-amino acid-nucleosides (SAAN) were synthesized to mimic glycosyl nucleotide donors based on the hypothesis that a basic amino acid may interact with carboxylate groups of the enzyme in a manner similar to the diphosphate metal ion complex. C-Glycoside analogues of the d-galactopyranose or l-arabinofuranose ring systems, and four amino acids (lysine, glutamine, tryptophan, and histidine), were chosen for this study. The targets were synthesized and tested against GlfT2, a galactofuranosyltransferase essential for cell wall galactan biosynthesis in Mycobacterium tuberculosis. The inhibition assay showed that analogues containing histidine and tryptophan are moderate inhibitors of GlfT2.

Full text of DOI:10.1016/j.bmc.2010.11.044

Bioorganic & Medicinal Chemistry 19 (2011) 58–66
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of sugar–amino acid–nucleosides as potential
glycosyltransferase inhibitors
Kannan Vembaiyan a, Jean A. Pearcey b, Milan Bhasin a, Todd L. Lowary b, Wei Zou a,
⇑
a Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada K1A 0R6
b Alberta Ingenuity Centre for Carbohydrate Science and Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2
a r t i c l e i n f o
a b s t r a c t
Article history:
Sugar–amino acid–nucleosides (SAAN) were synthesized to mimic glycosyl nucleotide donors based on
the hypothesis that a basic amino acid may interact with carboxylate groups of the enzyme in a manner
similar to the diphosphate metal ion complex. C-Glycoside analogues of the D-galactopyranose or L-arab-
inofuranose ring systems, and four amino acids (lysine, glutamine, tryptophan, and histidine), were cho-
sen for this study. The targets were synthesized and tested against GlfT2, a galactofuranosyltransferase
essential for cell wall galactan biosynthesis in Mycobacterium tuberculosis. The inhibition assay showed
that analogues containing histidine and tryptophan are moderate inhibitors of GlfT2.
Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
Received 21 October 2010
Revised 18 November 2010
Accepted 22 November 2010
Available online 25 November 2010
Keywords:
Synthesis
C-Glycoside
Amino acid
Nucleoside
Galactosyltransferase
Inhibitors
1. Introduction
many studies focused on replacing the diphosphate group with
non-charged entities including sulfone,7 amide,8 malonic and tar-
Glycosylation is an important biological process in all life forms
ranging from bacteria to humans.1 The majority of glycosyltransfer-
ases, the enzymes that make glycosidic bonds, involve sugar nucle-
otide donors, which all possess two negative charges presented
either by diphosphates such as in UDP-Glc, UDP-Gal, UDP-GlcNAc,
and GDP-Fuc or by one phosphate and one carboxylate in the case
of CMP-NeuAc.1,2 Structural analysis of enzyme–substrate com-
plexes in glycosyltransferases has revealed that these negative
charges often interact with enzyme motifs through a bivalent metal
ion (e.g., Mn2+ and Mg2+) bridge.3 Inhibitors of glycosyltransferases
are of therapeutic interest as potential anti-cancer agents, as well as
for treatment of bacterial disease.4 Consequently, numerous glyco-
syltransferase inhibitors have been designed and synthesized,5
which has provided insights to the essential role played by the
negative charges in sugar donors. In a systematic study using sialyl
C-glycosides with different numbers of phosphate groups as sialyl-
transferase inhibitors, Schmidt and co-workers found that the
monophosphate derivatives were weak inhibitors but derivatives
with two phosphate groups were much better inhibitors, likely
due to coordinated interaction.6 However, one of the drawbacks of
these charged inhibitors is their difficulty in being transported
across the cell membrane. In order to improve the bioavailability,
taric groups9 as coordinators of metal ions have been reported.
Unfortunately, most of these molecules display no significant activ-
ity against respective enzymes. We reasoned that replacement of
the diphosphate moiety in sugar nucleotide donors by a basic amino
acid may mimic the diphosphate–metal ion complex by direct inter-
action with the carboxylates in the catalytic site of the enzyme. In
addition, these molecules may have better bioavailability than
charged compounds.
In this report we describe the synthesis of C-a-D-galactopyrano-
syl–amino acid–uridine and C- /b- -arabinofuranosyl–amino acid–
a
L
uridine derivatives as a new type of molecular architecture for
glycosyltransferase inhibition. These compounds were then tested
as potential inhibitors of a key galactosyltransferase (GlfT2), which
is involved in cell wall galactan biosynthesis in Mycobacterium
tuberculosis, the organism that causes the disease tuberculosis.10,11
In addition, because M. tuberculosis infects alveolar macrophages
in the lungs, the membrane transport properties of these
compounds were investigated using macrophages.
2. Results and discussion
2.1. Synthesis of SAANs
Synthetic hybrids of sugar–amino acids (SAA)12 and peptide nu-
cleic acids (PNA)13 are well known, hybrid molecules consisting of
⇑
Corresponding author. Tel.: +1 613 991 0855; fax: +1 613 952 9092.
E-mail address: wei.zou@nrc-cnrc.gc.ca (W. Zou).
0968-0896/$ - see front matter Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.11.044

K. Vembaiyan et al. / Bioorg. Med. Chem. 19 (2011) 58–66
59
a sugar, an amino acid, and a nucleoside (SAAN) are another novel
class of structures yet to be fully explored chemically and biologi-
cally although various amino acid–nucleoside substructures pres-
ent in natural products.14 Because the amino acid is placed
between the sugar and the nucleoside, the synthesis of these hy-
brid molecules should be easily achieved by forming an amide
bond between the moieties. Four basic amino acids (lysine, gluta-
mine, tryptophan, and histidine), which are able to carry a positive
charge, were chosen to mimic the diphosphate metal ion complex.
The starting C-galactoside acid (1a)15 was prepared from the
previously reported allyl C-galactoside16 and 50-amino-uridine
derivative (4) was synthesized following known procedures.17
The C-glycoside moiety provides the necessary stability to resist
biodegradation. The SAAN can be assembled in two ways, by cou-
pling the C-glycoside and amino acid followed by coupling with
50-amino-uridine, or vice versa. We attempted both strategies
using lysine as the amino acid and obtained poor coupling yield be-
tween the sugar amino acid and 50-amino-uridine. In contrast, the
reversed coupling sequence, using an activated C-glycoside ester
for amide formation worked efficiently to give SAAN 5a–d in good
yield (Scheme 1). However, removal of O-benzyl groups by cata-
lytic hydrogenation [H2/Pd–C or Pd(OH)2] under the atmospheric
pressure was problematic with 5b and 5d. Incomplete de-O-benzy-
lation, particularly on the uridine moiety, was observed. Although
complete de-O-benzylation of 5a–c was achieved under the condi-
tions of H2 and 10% Pd–C in 10% HOAc–MeOH for 3–4 days, the
reduction of uridine C@C bond also occurred, resulting in an insep-
arable mixture of 6 and 7. The ratio of 6:7 varies depending on the
substrates (see Scheme 1). In the case of histidine derivative 5d,
complete removal of the O-benzyl groups required hydrogenation
at 50 psi, which led to complete reduction of the uridine C@C dou-
ble bond to afford 7d as single product. Apparently, the structure of
the amino acid side chain was able to affect significantly the cata-
lytic hydrogenation process.
Because the uridine double bond was reduced by catalytic
hydrogenation, we decided to avoid O-benzyl groups on both gal-
actose and ribose to circumvent the problematic catalytic hydroge-
nation. Thus, the isopropylidenated uridine amine derivative 918
and activated ester of per-O-acetylated C-galactosyl acid 1119 were
prepared for coupling with the four amino acids (lysine, glutamine,
tryptophan, and histidine). Amide bonds were formed first by con-
densation of the uridine amine with the amino acid, followed by
a
removal of N -protecting groups (Fmoc or Boc) to afford 10a–d,
which were in turn coupled with 11 to give SAAN 12a–d in
71–78% yield (see Scheme 2). Boc group deprotection of 8d with
TFA in CH2Cl2 also removed the isopropylidene acetal to give amide
10d, which was coupled successfully with 11. After global depro-
tection (TFA and then NaHCO3–MeOH) and purification on a
Bio-gel P-2 column, the desired C-galactosyl amino acid uridine
derivatives (6a–d) were obtained in good yield (58–66%). In addi-
tion, to understand the importance of the uridine C@C bond for
enzyme inhibition, 6a–d were subjected to catalytic hydrogenation
(H2/Pd–C in water) to afford uridine-reduced analogues 7a–d.
Although most galactosyltransferases use UDP-
natural substrate of GlfT2 is UDP- -Galf. Therefore, SAANs with
-C-galactofuranosyl moiety could be a better substrate mimic
than the Galp analogues. Because we had synthesized -C-arabino-
furanosides previously,16 we explored this system as a surrogate of
-galactofuranose. -Galactofuranose and -arabinofuranose differ
a-D-Galp, the
a-D
a
D
L
D
D
L
only in the side chain, appended to the ring at C4, with the latter
being one carbon shorter.
In the preparation of the Araf SAANs, we initially explored the
use of O-acetyl protecting groups as was described above for the
Galp analogues. However, attempted 1-C-allylation using methyl
2,3,5-tri-O-acetyl-
-C- -arabinofuranoside exclusively. To overcome this problem,
benzyl ethers were used as the protecting groups. Subsequent
a-L-arabinofuranoside produced the undesired
a
L
1-C-allylation of methyl 2,3,5-tri-O-benzyl-a-L-arabinofuranoside
O
O
BnO
OBn
NH
NH
O
O
O
O
H N
N
H N
2
2
BnO
BocHN
O
O
HN
N
i, ii
OPfp
O
BnO
+
R
O
R
72-85%
BnO
OBn
OR
BnO
2a-d
OBn
3a-d
1a R = H
4
1b R = Pfp
AA
R
6:7
9:1
a
b
c
d
Lys
Gln
Trp
His
NH-Fmoc
i, iii
69-76%
O
BnO
BnO
OBn
NH
6:4
2
O
O
NH
O
BnO
HN
O
N
NH
7:3
O
N
O
R
H
BnO
OBn
5a-d
0:10
HN
N
iv
58-82%
HO
HO
HO
HO
OH
OH
O
O
O
O
NH
NH
O
O
OH
OH
HN
HN
O
O
N
N
NH
NH
+
O
O
O
R
O
R
HO
OH
HO
OH
7a-d
6a-d
for 6a and 7a: R = (CH ) NH
2 4
2
Scheme 1. Reagents and conditions: (i) DMAP/DMF, rt, 12 h; (ii) TFA/CH2Cl2, 0 °C–rt, 2 h; (iii) piperidine/DMF, rt, 10 min (for Lys); (iv) H2–Pd/C, 10% AcOH/MeOH, rt, 3–4
days.

60
K. Vembaiyan et al. / Bioorg. Med. Chem. 19 (2011) 58–66
O
O
NH
NH
O
AcO
AcO
O
OAc
O
H N
2
O
H N
N
O
2
HN
N
R'HN
i, ii
O
O
OH
R
+
R
69-80%
AcO
O
O
O
OR' OR'
8a-c R' = Fmoc
8d R' = Boc
OPfp
10a-c R', R' = Me C=
2
9
11
10d R', R' = H
AA
R
iii
a
b
c
d
Lys
Gln
Trp
His
AcO
AcO
71-78%
OAc
O
NH-Boc
O
O
NH
O
AcO
HN
NH
O
2
N
NH
O
O
R
OR' OR'
12a-c R', R' = Me C=
12d R', R' = H
iv, v
58-66%
2
N
H
6a-d
HN
N
vi
61-85%
7a-d
for 7a: R = (CH ) NH
2 4
2
Scheme 2. Reagents and conditions: (i) HOBt/EDC/DMF, rt, 6–12 h; (ii) piperidine, 10 min for 8a–c or TFA/CH2Cl2, 0 °C–rt, 2 h for 8d; (iii) DMAP/DMF, rt, 12 h; (iv) TFA:H2O
(9:1)/CH2Cl2, 0 °C–rt, 2 h; (v) NaHCO3/MeOH, 5 min; (vi) Pd/C, H2O, 6–12 h.
afforded a 1:1 isomeric mixture of C-arabinofuranosides, which
were inseparable by silica gel column chromatography. Routine
transformations were performed on known aldehyde16 to obtain
an activated ester 13 (see Supplementary data), which was also
an inseparable isomeric mixture. Because the preliminary inhibi-
tion screening of Galp analogues 6 and 7 showed that SAANs with
tryptophan and histidine were active, those two amino acids were
and glutamine analogues are very weak inhibitors. Similarly, the
C-arabinofuranosyl–amino acid–uridine derivatives were screened
at 2 mM against GlfT2 at pH 7.6 and 6.0. The tryptophan derivative
17c showed 30% inhibition at pH 7.6 whereas its isomer 16c had
only 8% inhibition. The histidine derivative 17d was also a better
inhibitor (37% inhibition) than its isomer 16d (14% inhibition) at
pH 7.6. All of the uridine-reduced analogues were not inhibitors
(<10% inhibition at 2 mM). Table 1 summarizes the inhibition re-
sults. Unexpectedly, when the assay was carried out at pH 6.0, only
16d was shown to be a weak inhibitor (24% inhibition) (data not
shown). This could be a result of poor substrate mimic due to the
charged amino acid, although a charged SAAN may be favored in
transition state.
Efficient catalysis by enzymes requires the specific binding of
substrates and the rapid release of the product(s). The substrate
binding by glycosyltransferases is usually specific but not neces-
sarily tight, with Km values in the micromolar to low millimolar
range. For example, kinetic characterization of GlfT2 using disac-
charide and trisaccharide acceptors showed Km values ranging
selected for coupling with uridine and the L-arabinofuranoside. The
products resulting from the coupling of 13 with either 10c or 10d
were separable by flash column chromatography and this provided
pure isomers 14c,d and 15c,d, respectively. Their configurations
were confirmed by 2D-NOESY experiments. Compounds 14c and
15c were treated with TFA to remove the isopropylidene acetal.
The resultant intermediates, as well as 14d and 15d, were sub-
jected to catalytic hydrogenation (H2/Pd–C in 10% HOAc–MeOH)
to produce products 16c,d and 17c,d (see Scheme 3). The final
products were purified on a Bio-gel P-2 column. A portion of the
above SAAN products was catalytically hydrogenated in water, to
give the corresponding uridine-reduced products, 18c,d and
19c,d. This process generated an additional eight
oside-based SAANs for screening against GlfT2.
L-C-arabinofuran-
from 204 lm to 1.7 mM, and tighter binding was observed for
the trisaccharide receptors.11 The IC50 obtained from inhibition
by 6d against GlfT2 in this study suggests a binding affinity
2.2. Inhibition evaluation
comparable to Km = 380 lM obtained from natural sugar donor
(UDP-Galf).20 Inhibition of the enzyme appears to require proper
interactions with the sugar, amino acid and uridine moieties
because analogues having the similar molecular architecture,
differing only in the side chain of the amino acid, did not effectively
inhibit the enzyme.
The ability of the compounds to inhibit GlfT2 was measured
based on a coupled spectrophotometric assay method developed
recently,20 where
(1?5)-b-
a
galactofuranosyl trisaccharide (b-
D
D-Galf-
D
-Galf-(1?6)-b- -Galf-Octyl) was used as the acceptor.
The C-galactosyl–amino acid–uridine analogues were initially
screened at 4 mM against GlfT2 at pH 7.6. Under these conditions,
tryptophan derivative 6c showed 75% inhibition and histidine
derivative 6d showed 79% inhibition. To determine the approxi-
mate inhibition potency of these compounds, further titration
The furanosyl analogues were only weak inhibitors of the
enzyme, but the results were consistent with what we observed
onpyranosideanalogues thatinhibitionactivitywas associated with
tryptophan and histidine in SAANs. In addition, the glycosyl config-
uration played a critical role. Only 17c and 17d appear to mimic the
sugar donor and therefore acted as the moderate inhibitors.
The interaction of glycosyltransferases with the donor is a mul-
tiple binding process; the sugar moiety, the nucleoside, and the
diphosphate all play critical roles. Sugar donor mimics resulting
experiments showed 6d had an IC50 of 332 lM. However, the ly-
sine 6a and glutamine 6b analogues showed only <12% inhibition
at 4 mM. These results indicate that the SAANs containing trypto-
phan and histidine are moderate inhibitors of GlfT2 and the lysine

K. Vembaiyan et al. / Bioorg. Med. Chem. 19 (2011) 58–66
61
O
BnO
BnO
O
O
NH
O
BnO
O
O
OPfp
O
O
HN
i
O
O
N
NH
BnO
NH
O
BnO
O
+
BnO
R
10c or 10d
HN
OBn
BnO
O
N
NH
BnO
O
OR' OR'
80-87%
13
R
14c R', R' = Me C=
14d R', R' = H
2
15c R', R' = Me C=
2
OR' OR'
15d R', R' = H
AA
Trp
His
R
ii,iii
ii,iii
59-62%
HO
O
57-64%
O
O
O
NH
O
O
HO
c
d
HO
NH
N
H
HN
O
O
O
N
O
NH
HO
HN
O
N
O
NH
R
HO
R
HO
OH OH
16c,d
HN
N
OH OH
17c,d
iv
iv
84-88%
81-82%
O
HO
O
NH
O
O
O
HO
O
HN
O
HO
O
NH
N
NH
O
O
HO
HN
R
HO
O
N
O
NH
HO
OH OH
R
18c,d
19c,d
OH OH
Scheme 3. Reagents and conditions: (i) DMAP/DMF, 6 h; (ii) TFA:H2O (9:1)/CH2Cl2, 0 °C–rt, 2 h for 10c; (iii) H2/Pd–C, AcOH/MeOH, rt, 24 h; (iv) H2/Pd–C, H2O, 6 h.
Table 1
even without charge, the hydrophilic sugar moiety is still a signif-
icant barrier for cell membrane crossing.
Inhibition of GlfT2 activity by SAANs at pH 7.6
Compound
Inhibition% at 4 mM
Compound
Inhibition% at 2 mM
In conclusion, we report here a novel type of potential galactosyl-
transferase inhibitor by replacing the diphosphate moiety in sugar
donor with a basic amino acid. Analogues containing tryptophan
and histidine showed moderate inhibition against mycobacterial
GlfT2, which we suggest may arise by mimicking the interaction
between the diphosphate metal ion complex with the carboxylate
residues present in the active site of the enzyme. However, the poor
bioavailability of these compounds suggests that these molecules
will likely only be useful as biological tools instead of therapeutics.
6a
6b
6c
6d
7a
7b
7c
7d
11
<10
75
16c
16d
17c
17d
18c
18d
19c
19d
<10
14
30
79, IC50 = 332
lM
37
<10
<10
53
<10
<10
<10
<10
18
from the replacement of the diphosphate with proper amino acids
could not only provide similar interaction with enzyme carboxyl-
ate but also should not disrupt other interactions between enzyme
and sugar moiety and nucleoside. The inhibition observed reflects
the sum of these three interactions. Based on the results described
here we propose that histidine and tryptophan are two amino acids
that are likely able to mimic the carboxylate–diphosphate metal
ion interaction. However, it should be noted that validation of this
hypothesis requires further study.
3. Experimental
3.1. General methods
1H and 13C NMR spectra were recorded at 400 MHz and
100 MHz, respectively, on a Varian instrument at 293 K. Chemical
shifts are given in ppm downfield to the signal of internal TMS,
and were assigned on the basis of 2D 1H–1H COSY and 1H–13
C
chemical-shift correlated experiments. In order to maintain consis-
tency in NMR assignments carbon and proton numberings of the
sugar were used despite the compounds being named otherwise.
For high resolution mass spectroscopic analysis, samples were
mixed with Agilent ES tuning mix for internal mass calibration
and infused into an AB/MDS-Sciex (Concord, ON) QSTAR mass
2.3. Cross-cell membrane ability
The cross-cell membrane ability of molecules correlates to their
bioavailability. Therefore, we examined the cross-membrane per-
meability of 6a–d using the mouse macrophage cell line J774A.1
(ATCC TIB-67). A known TB drug, ethambutol, which inhibits arabi-
nosyltransferases, was used as a positive control in the study. Mac-
rophage cells were cultured with SAAN-containing medium
spectrometer at a flow-rate of 4 lL/min. All chemicals were pur-
chased from Aldrich Co. and used without further purification.
(100 lM) for 2 min, 4.5 h, and 22 h. After removal of medium the
3.2. Spectrophotometric inhibition assays
cells were washed with PBS, sonicated and centrifuged through a
3 kDa filter to obtain sample solutions. The solutions were
analyzed by multiple reaction monitoring (MRM) through MRM
chromatograms and MS/MS analysis. Although stable in the med-
ium as indicated by MRM and MS/MS, the permeability of these
SAANs was, unfortunately, negligible under these conditions while
ethambutol permeated reasonably well. The results suggest that
GlfT2 inhibition assays were performed in 384-array microtitre
plate wells, which contained 100 mM MOPS, pH 7.6, 50 mM KCl,
20 mM MgCl2, 1.1 mM NADH, 3.5 mM PEP, 3.75 U pyruvate kinase
(PK, EC 2.7.1.40), and 16.8 U lactate dehydrogenase (LDH, EC
1.1.1.27). MOPS at pH 7.6, was added as a 20-fold stock solution.
KCl, MgCl2, and b-D-Galf-(1?5)-b-D-Galf-(1?6)-b-D-Galf-Octyl

62
K. Vembaiyan et al. / Bioorg. Med. Chem. 19 (2011) 58–66
acceptor were dissolved in de-ionized distilled water (MQ). All
other assay components were prepared in 100 mM MOPS, pH 7.6,
with the exception of PEP, which, owing to its acidity, was buffered
in 250 mM MOPS, pH 7.6. Stock solutions of NADH, PEP, PK, and
LDH were made fresh on the day of use and all solutions were
stored on ice. The final concentration of the compound was
2 mM. Test master mixes were prepared for 40 assays and stored
on ice, then used for 10 test assays at a time. Likewise blanks were
prepared for 12 reactions and 2 were used per run. Two com-
pounds in triplicate and one set of controls in triplicate were in-
cluded in each run. Reactions were initiated with the addition of
room temperature, and the mixture was stirred overnight. After
completion of reaction, the solvent was evaporated under reduced
pressure. The residue was diluted with water and extracted with
EtOAc (three times). The combined extracts were sequentially
washed with saturated NH4Cl solution, brine, dried (Na2SO4) and
concentrated under vacuum. The residue was purified by flash col-
umn chromatography to give pure compounds 12a–d in 71–78%
yield.
3.6. General procedure for deprotection of isopropylidene and
acetate groups
0.5 lg GlfT2 diluted in 100 mM MOPS. Closest duplicates of test
and control reaction velocities were corrected by subtracting the
mean blank velocity per run, and were expressed as a percentage
of the mean control velocity.
A solution of 90% trifluoroacetic acid in water (1 mL) was added
dropwise to a solution of the compound 12a–c in CH2Cl2 at 0 °C,
and the mixture was stirred for 2 h at room temperature. After
completion of the reaction, the solvent and TFA were evaporated
under vacuum. The residue was dissolved in methanol and passed
through a pad of NaHCO3 to deprotect the acetates. Similarly, the
adduct 12d was also deacetylated by passage through a pad of
NaHCO3. The filtrate was concentrated and the residue was loaded
onto a column of Bio-gel P-2 (2 ꢀ 100 cm) and eluted with water.
The fractions containing the product were pooled and lyophilized
to afford 6a–d in 58–66% yield.
Addition of 2.5
tures initiated each activity and inhibition test, bringing the final
volume to 20 L. Reactions were incubated at 37 °C and monitored
lL of diluted GlfT2 to the compound assay mix-
l
at 340 nm at 10–15 s intervals using a Spectra Max 340PC micro-
plate reader. Data were acquired using SoftmaxÒPRO software
operated in the Michaelis–Menten Protocol kinetic mode.
3.3. Permeability assay
J774 macrophage cells were grown in Dulbecco’s modified med-
ium (DMEM) which contained 10% FBS (fetal bovine serum), Hepes
buffer and NEAA (non-essential amino acids). The cells were grown
in 10 cm plates at 37 °C in a 5% CO2/air incubator. Cells were pas-
saged when 80–90% confluency was observed. The cells were
washed with 10 mL of HBSS (Hank’s balanced salt solution) to re-
move non-adherent cells. The cells were harvested using a rubber
cell scrapper. These cells were split into three plates with approx-
imately 2 million cells in each plate. The cell count was performed
using hemocytometer.
3.7. General procedure for hydrogenation of the adducts 6a–d
To a solution of adducts 6a–d (1 mmol) in water was added 10%
Pd/C (0.1 mmol) and stirred under hydrogen atmosphere for 6–
12 h. The reaction mixture was then filtered and washed with
water and the aqueous solution was lyophilized to give the re-
duced analogues 7a–d in 61–85% yield.
e
3.8. 50-N-(N -Boc-lysinamido)-50-deoxy-20,30-O-isoporopylidene-
uridine (10a)
J774 macrophage cells were seeded into Multiwell 24-well tissue
culture plate. The cells were grown at 37 °C till confluency (4 days).
The cell count was 1.7 ꢀ 105 for each well. Solutions (1 mM) of
Yield 79%; syrup; [
a
]
ꢁ13 (c 1.6, CHCl3); 1H NMR (CDCl3,
D
400 MHz) d 1.35 (s, 3H), 1.38–1.60 (m, 17H), 1.84 (br s, 1H),
3.09–3.18 (m, 2H), 3.32–3.40 (m, 1H), 3.50–3.58 (m, 1H), 3.65–
3.76 (m, 1H), 4.15–4.21 (m, 1H), 4.70 (br s, 1H), 4.77 (dd, 1H,
J = 5.6, 5.2 Hz), 5.09 (d, 1H, J = 5.2 Hz), 5.44 (s, 1H), 5.73 (d, 1H,
J = 8.0 Hz), 7.24 (d, 1H, J = 8.0 Hz), 7.47 (br s, 1H); 13C NMR (CDCl3,
100 MHz) d 22.9, 25.6, 27.4, 28.6 (2C), 29.8, 29.9, 34.5, 40.3, 40.6,
55.3, 79.5, 81.0, 83.9, 85.5, 96.2, 103.1, 115.0, 143.4, 150.5, 156.4,
163.3, 175.9; HRMS: calcd for C23H38N5O8 [M+H]+ 512.2720, found
512.2722.
SAANs and ethambutol were filtered through a sterile 0.22 lM
Millex-GV filter. Each compound was added to three wells to obtain
a final concentration of 100 M with 1 mL of total volume. The com-
l
pounds and cells were incubated for 2 min, 4.5 h and 22 h before the
medium was removed and the cells were washed with 2 mL PBS
buffer three times. The cells become detached from the plate after
washing with PBS. These cells were centrifuged for 10 min at 300g
in a Centra CL3R Thermo IEC centrifuge and the cell pellet was
washed again with PBS twice (2 mL) and centrifuged. The cells were
then sonicated for 4 min with 40% power output. The cell lysate was
then centrifuged through a 3 kDa MWCO Amicon centrifugal filter
unit for 30 min at 3570g. The filtrate was collected and analyzed
by mass spectrometer.
3.9. 50-N-Glutaminamido-50-deoxy-20,30-O-isopropylidene-
uridine (10b)
Yield 80%; syrup; [a]
D +15 (c 0.5, H2O); 1H NMR (D2O, 400 MHz)
d 1.41 (s, 3H), 1.61 (s, 3H), 1.86–1.98 (m, 2H), 2.33 (t, J = 7.6 Hz,
2H), 3.48 (t, 1H, J = 6.8 Hz), 3.52 (dd, 1H, J = 5.6, 14.4 Hz), 3.65
(dd, 1H, J = 7.2, 14.4 Hz), 4.30 (m, 1H), 4.88 (dd, 1H, J = 4.4,
6.4 Hz), 5.25 (dd, 1H, J = 2.4, 6.4 Hz), 5.81 (d, 1H, J = 2.4 Hz), 5.87
(d, 1H, J = 8.0 Hz), 7.66 (d, 1H, J = 8.0 Hz); 13C NMR (D2O,
100 MHz) d 24.5, 26.1, 30.0, 31.2, 40.8, 54.1, 81.4, 83.9, 84.6,
93.9, 102.2, 115.3, 144.3, 152.0, 167.5, 176.6, 178.4; HRMS: calcd
for C17H26N5O7 [M+H]+ 412.1832, found 412.1864.
3.4. General procedure for the coupling of amino acids with
uridine amine 9 followed by Fmoc deprotection
To a solution of the amino acid 8a–d (1 mmol) in DMF were
added HOBt (1 mmol), EDC (1 mmol) and uridine amine (1 mmol)
and stirred for 6–12 h at room temperature. After completion of
reaction, piperidine (5 mmol) was added and stirred for 10 min.
The solvent was removed under reduced pressure and the residue
was purified by flash column chromatography to afford the corre-
sponding amides 10a–d in 69–80% yield.
3.10. 50-N-Tryptophanamido-50-deoxy-20,30-O-isopropylidene-
uridine (10c)
3.5. General procedure for the coupling of amides 10a–d with 11
Yield 69%; syrup; [
a
]
ꢁ39 (c 1, CHCl3); 1H NMR (CDCl3,
D
400 MHz) d 1.31 (s, 3H), 1.53 (s, 3H), 2.93 (dd, 1H, J = 8.4,
14.4 Hz), 3.31 (dd, 1H, J = 8.4, 14.4 Hz), 3.46–3.52 (m, 1H), 3.62–
3.79 (m, 2H), 4.08–4.13 (m, 1H), 4.64 (dd, J = 4.8, 6.0 Hz, 1H),
A solution of amide 10a–d (1 mmol) in DMF was added to a
solution of 11 (1 mmol) and catalytic amount of DMAP in DMF at

K. Vembaiyan et al. / Bioorg. Med. Chem. 19 (2011) 58–66
63
4.91 (d, 1H, J = 6.4 Hz), 5.41 (s, 1H), 5.62 (d, 1H, J = 8.0 Hz), 7.01–
7.18 (m, 4H), 7.32 (d, 1H, J = 8.0 Hz), 7.52 (br s, 1H), 7.61 (d, 1H,
J = 8.0 Hz), 8.63 (br s, 1H); 13C NMR (CDCl3, 100 MHz) d 25.6,
27.4, 30.9, 40.7, 55.8, 81.2, 84.0, 85.6, 95.5, 102.8, 111.5, 114.9,
119.1, 119.6, 122.3, 123.6, 127.7, 136.4, 136.6, 143.2, 150.3,
163.7, 175.5; HRMS: calcd for C23H28N5O6 [M+H]+ 470.2039, found
470.2054.
1H, J = 8.0 Hz), 7.04–7.17 (m, 4H), 7.29 (d, 1H, J = 8.0 Hz), 7.62 (d,
1H, J = 7.6 Hz), 8.77 (s, 1H); 13C NMR (D2O, 100 MHz) d 20.90,
20.92, 20.94, 25.6, 27.4, 28.7, 34.6, 41.2, 54.4, 61.2, 67.3, 68.1,
69.3, 69.5, 80.9, 83.7, 85.2, 96.6, 102.9, 110.5, 111.5, 114.9, 119.0,
119.7, 122.3, 123.7, 127.7, 136.4, 143.6, 150.5, 163.8, 169.6,
170.0, 170.2, 170.3, 170.9, 172.3; HRMS: calcd for C39H48N5O16
[M+H]+ 842.3096, found 842.3129.
a
3.11. 50-N-Hisditinamido-50-deoxy-uridine (10d)
3.15. 50-N-[N -(2,6-Anhydro-3,4,5,7-tetra-O-acetyl-
D
-glycero-
L-
gluco-heptit-1-yl-acetamido)-histidinamido]-50-deoxy-uridine
Yield 70%; syrup; [
a]
+2 (c 1, CHCl3); 1H NMR (CD3OD,
(12d)
D
400 MHz) d 2.76 (dd, 1H, J = 7.2, 14.4 Hz), 2.96 (dd, 1H, J = 6.0,
14.8 Hz), 3.25–3.28 (m, 1H), 3.42–3.52 (m, 2H), 3.57 (dd, 1H,
J = 6.4, 6.8 Hz), 3.90–3.96 (m, 2H), 4.15 (dd, 1H, J = 4.4 Hz), 5.67
(d, 1H, J = 4.4 Hz), 5.69 (s, 1H), 6.84 (s, 1H), 7.56 (s, 1H), 7.59 (d,
1H, J = 8.4 Hz); 13C NMR (CD3OD, 100 MHz) d 33.4, 41.7, 56.2,
72.0, 74.3, 83.5, 92.4, 102.8, 117.7, 134.8, 136.2, 143.2, 152.1,
165.9, 176.6; HRMS: calcd for C15H21N6O6 [M+H]+ 381.1522, found
381.1512.
Yield 77%; syrup; [
a
]
+33 (c 1, CH3OH); 1H NMR (CDCl3,
D
400 MHz) d 2.06 (s, 3H), 2.08 (s, 3H), 2.09 (s, 3H), 2.19 (s, 3H),
2.69 (dd, 1H, J = 5.6, 15.6 Hz), 2.79 (dd, 1H, J = 9.2, 15.2 Hz), 3.06
(d, 1H, J = 8.4, 15.2 Hz), 3.18 (dd, 1H, J = 6.0, 14.8 Hz), 3.50–3.62
(m, 2H), 4.02–4.08 (m, 2H), 4.17 (dd, 1H, J = 4.8, 11.6 Hz), 4.22–
4.38 (m, 3H), 4.59–4.68 (m, 2H), 5.24–5.34 (m, 2H), 5.44–5.48
(m, 1H), 5.77 (d, 1H, J = 4.4 Hz), 5.90 (d, 1H, J = 8.0 Hz), 7.11 (s,
1H), 7.62 (d, 1H, J = 8.0 Hz), 8.03 (s, 1H); 13C NMR (CDCl3,
100 MHz) d 20.1, 20.17, 20.22, 20.3, 28.1, 33.7, 40.9, 53.8, 62.6,
68.0, 68.2, 69.9, 70.5, 71.9, 72.2, 73.2, 81.9, 90.8, 102.4, 117.0,
131.4, 135.2, 142.4, 151.6, 166.2, 172.1, 172.7, 172.9, 173.2,
173.4, 173.7; HRMS: calcd for C31H41N6O16 [M+H]+ 753.2579,
found 753.2566.
a
3.12. 50-N-[N -(2,6-Anhydro-3,4,5,7-tetra-O-acetyl-
D
-glycero-
L
-
e
gluco-heptit-1-yl-acetamido)-N -Boc-lysinamido]-50-deoxy-20,30-
O-isopropylidene-uridine (12a)
Yield 71%; syrup; [
a
]
D
ꢁ28 (c 1.2, CHCl3); 1H NMR (CDCl3,
400 MHz) d 1.33–1.64 (m, 20H), 1.87–1.98 (m, 1H), 1.99–2.12 (m,
12H), 2.42 (dd, 1H, J = 4.0, 14.8 Hz), 2.54 (dd, 1H, J = 10.0,
14.8 Hz), 3.04–3.18 (m, 1H), 3.50–3.70 (m, 2H), 4.08–4.14 (m,
2H), 4.20–4.26 (m, 1H), 4.33–4.49 (m, 2H), 4.61–4.78 (m, 2H),
4.89 (dd, 1H, J = 2.0, 6.8 Hz), 5.16 (dd, 1H, J = 2.0, 6.8 Hz), 5.19 (s,
2H), 5.28 (s, 1H), 5.41 (s, 1H), 5.72 (d, 1H, J = 8.0 Hz), 6.87 (d, 1H,
J = 8.4 Hz), 7.04 (s, 1H), 7.19 (d, 1H, J = 8.0 Hz); 13C NMR (CDCl3,
100 MHz) d 20.8, 20.90, 20.91, 21.0, 22.4, 25.4, 27.4, 28.6, 29.7,
31.5, 35.9, 39.4, 40.2, 53.3, 60.6, 66.6, 67.8, 68.5, 68.6, 70.3, 79.7,
80.0, 83.9, 85.2, 97.1, 103.3, 115.1, 144.0, 151.1, 156.8, 163.3,
169.4, 169.7, 169.8, 170.0, 170.9, 172.4; HRMS: calcd for
a
3.16. 50-N-[N -(2,6-Anhydro-
D
-glycero-
L
-gluco-heptit-1-yl-aceta-
mido)-lysinamido]-50-deoxy-uridine (6a)
Yield 66%; solid; [a]
D +9 (c 1.6, H2O); 1H NMR (D2O, 400 MHz) d
1.33–1.50 (m, 2H), 1.53–1.83 (m, 4H), 2.66 (dd, 1H, J = 4.4,
15.2 Hz), 2.74 (dd, 1H, J = 10.4, 15.2 Hz) 2.94 (dd, 2H, J = 7.6,
15.2 Hz), 3.52–3.63 (m, 2H), 3.66–3.79 (m, 3H), 3.88 (dd, 1H,
J = 5.6, 5.6 Hz), 3.98–4.14 (m, 4H), 4.28 (dd, 1H, J = 6.0, 6.0 Hz),
4.33–4.40 (m, 1H), 4.45–4.53 (m, 1H), 5.84 (d, 1H, J = 5.2 Hz),
5.87 (d, 1H, J = 8.0 Hz), 7.61 (d, 1H, J = 8.0 Hz); 13C NMR (D2O,
100 MHz) d 22.2, 26.6, 30.7, 32.5, 39.3, 41.0, 54.2, 61.2, 67.7,
69.0, 69.8, 70.9, 72.8, 73.1, 73.3, 81.8, 90.4, 102.7, 141.7, 155.7,
C
39H58N5O18 [M+H]+ 884.3776, found 884.3788.
a
3.13. 5-N-[N -(2,6-Anhydro-3,4,5,7-tetra-O-acetyl-
D
-glycero-
L
-
161.1, 174.0, 174.4; HRMS: calcd for
576.2516, found 576.2509.
C
23H38N5O12 [M+H]+
gluco-heptit-1-yl-acetamido)-glutamido]-5-deoxy-2,3-O-isopro-
pylidene-uridine (12b)
a
3.17. 50-N-[N -(2,6-Anhydro-
D
-glycero-
L
-gluco-heptit-1-yl-aceta-
Yield 76%; syrup; [
a]
D
+26 (c 1, CHCl3); 1H NMR (CDCl3,
mido)-glutamido]-50-deoxy-uridine (6b)
400 MHz) d 1.34 (s, 3H), 1.53 (s, 3H), 1.83–2.16 (m, 13H), 2.22–
2.55 (m, 4H), 2.69 (dd, 1H, J = 10.0, 14.8 Hz), 3.44–3.50 (m, 1H),
3.69–3.76 (m, 1H), 4.07–4.29 (m, 4H), 4.53 (dd, 1H, J = 8.0,
8.4 Hz), 4.64–4.74 (m, 1H), 4.85 (dd, 1H, J = 6.0 Hz), 5.07–5.43 (m,
5H), 5.72 (d, 1H, J = 8.0 Hz), 6.62 (s, 1H), 7.05 (s, 1H), 7.21 (d, 1H,
J = 8.0 Hz), 7.46 (d, 1H, J = 8.0 Hz), 7.51 (s, 1H); 13C NMR (CDCl3,
100 MHz) d 20.7, 20.73, 20.77, 20.8, 25.3, 27.3, 29.2, 32.2, 34.6,
40.3, 52.7, 60.9, 67.0, 67.8, 67.9, 69.1, 69.4, 80.4, 84.1, 85.4, 96.3,
102.8, 114.7, 143.8, 150.7, 163.7, 169.8, 169.9, 170.0, 170.1,
170.7, 171.8; HRMS: calcd for C33H46N5O17 [M+H]+ 784.2888,
found 784.2876.
Yield 65%; solid; [a]
D
+6 (c 1, H2O); 1H NMR (D2O, 400 MHz) d
1.96–2.04 (m, 1H), 2.06–2.16 (m, 1H), 2.38 (t, 2H, J = 7.6 Hz), 2.67
(dd, 1H, J = 8.4, 15.2 Hz), 2.75 (dd, 1H, J = 10.4, 15.2 Hz), 3.53–
3.91 (m, 7H), 3.99–4.16 (m, 3H), 4.30–4.39 (m, 2H), 4.44–4.54
(m, 1H), 5.83 (d, 1H, J = 5.6 Hz), 5.89 (d, 1H, J = 8.0 Hz), 7.64 (d,
1H, J = 8.0 Hz); 13C NMR (D2O, 100 MHz) d 27.1, 30.4, 31.2, 32.5,
41.0, 53.7, 61.4, 67.7, 69.0, 69.8, 70.8, 72.8, 73.1, 81.8, 90.5, 102.7,
141.8, 160.8, 163.3, 173.8, 174.0, 178.0; HRMS: calcd for
C
22H34N5O13 [M+H]+ 576.2153, found 576.2136.
a
3.18. 50-N-[N -(2,6-Anhydro-
D
-glycero-
L
-gluco-heptit-1-yl-aceta-
a
3.14. 50-N-[N -(2,6-Anhydro-3,4,5,7-tetra-O-acetyl-
D
-glycero-
L
-
mido)-tryptophanamido]-50-deoxy-uridine (6c)
gluco-heptit-1-yl-acetamido)-tryptophanamido]-50-deoxy-20,30-
O-isopropylidene-uridine (12c)
Yield 61%; solid; [a]
D +12 (c 0.5, H2O); 1H NMR (D2O, 400 MHz)
d 2.61 (dd, 2H, J = 4.2, 14.0 Hz), 2.72 (dd, 1H, J = 10.4, 15.2 Hz),
3.15–3.33 (m, 3H), 3.45–3.56 (m, 2H), 3.68 (d, 2H, J = 6.0 Hz),
3.71–3.77 (m, 2H), 3.79–3.88 (m, 2H), 3.97–4.06 (m, 2H), 4.43–
4.51 (m, 1H), 4.68 (dd, 1H, J = 6.0, 6.4 Hz), 5.51 (d, 1H, J = 4.0 Hz),
5.65 (d, 1H, J = 8.0 Hz), 7.07–7.26 (m, 4H), 7.42 (d, 1H, J = 8.0 Hz),
7.63 (d, 1H, J = 8.0 Hz); 13C NMR (D2O, 100 MHz) d 27.2, 32.3,
40.2, 55.2, 61.0, 67.7, 68.9, 69.7, 69.8, 72.6, 73.1, 73.2, 81.6, 90.1,
102.3, 108.9, 111.7, 118.5, 119.5, 122.2, 124.6, 126.9, 136.2,
Yield 78%; syrup; [
a
]
+23 (c 2.2, CHCl3); 1H NMR (CDCl3,
D
400 MHz) d 1.30 (s, 3H), 1.50 (s, 3H), 1.96 (s, 3H), 1.99 (s, 3H),
2.02 (s, 3H), 2.10 (s, 3H), 2.51 (dd, 1H, J = 4.8, 15.2 Hz), 2.61 (dd,
1H, J = 9.2, 15.6 Hz), 3.23 (d, 2H, J = 6.8 Hz), 3.38–3.56 (m, 2H),
4.03–4.18 (m, 3H), 4.36 (dd, 1H, J = 5.2 Hz), 4.69–4.74 (m, 1H),
4.76–4.88 (m, 1H), 5.14 (dd, 1H, J = 2.8, 8.8 Hz), 5.22–5.32 (m,
2H), 5.36 (s, 1H), 5.65 (d, 1H, J = 8.0 Hz), 6.90 (br s, 1H), 7.01 (d,

64
K. Vembaiyan et al. / Bioorg. Med. Chem. 19 (2011) 58–66
141.4, 151.4, 166.2, 173.6, 173.8; HRMS: calcd for C28H36N5O12
[M+H]+ 634.2360, found 634.2326.
67.7, 68.9, 69.8, 70.3, 70.7, 72.7, 73.1, 81.2, 88.1, 117.3, 131.4,
136.1, 151.6, 166.1, 173.4, 173.8; HRMS: calcd for C23H36N6O12
[M+H]+ 587.2313, found 587.2304.
a
3.19. 50-N-[N -(2,6-Anhydro-
D
-glycero-
L
-gluco-heptit-1-yl-aceta-
a
mido)-histidinamido]-50-deoxy-uridine (6d)
3.24. 50-N-[N -(2,5-Anhydro-3,4,5-tri-O-benzyl-
L-glucit-1-yl-acet-
amido)-tryptophanamido]-50-deoxy-uridine (14c)
Yield 58%; solid; [a]
D
+44 (c 1, H2O); 1H NMR (D2O 400 MHz) d
2.59 (dd, 1H, J = 4.0, 15.2 Hz), 2.70 (dd, 1H, J = 10.8, 15.2 Hz), 3.01
(dd, 1H, J = 7.6, 15.2 Hz), 3.08 (dd, 1H, J = 7.2, 15.2 Hz), 3.51 (dd,
2H, J = 4.0, 6.4 Hz), 3.63–3.70 (m, 2H), 3.73 (dd, 1H, J = 3.2 Hz),
3.83 (dd, J = 6.0, 6.8 Hz, 1H), 3.95–4.05 (m, 4H), 4.27 (dd, 1H,
J = 4.8, 5.2 Hz), 4.39–4.47 (m, 1H), 4.59 (dd, 1H, J = 6.8, 7.6 Hz),
5.76 (d, 1H, J = 4.8 Hz), 5.88 (d, 1H, J = 8.0 Hz), 6.98 (s, 1H), 7.60
(d, 1H, J = 8.0 Hz), 7.76 (s, 1H); 13C NMR (D2O, 100 MHz) d 28.7,
32.3, 40.8, 54.3, 61.0, 67.7, 68.9, 69.8, 70.5, 72.7, 73.1, 73.2, 81.9,
90.5, 102.4, 117.4, 131.5, 135.8, 142.3, 151.7, 166.4, 173.3, 173.8;
HRMS: calcd for C23H33N6O12 [M+H]+ 585.2156, found 585.2147.
Yield 87%; syrup; [
a]
ꢁ5 (c 2, CHCl3); 1H NMR (CDCl3,
D
400 MHz) d 1.25 (s, 3H), 1.46 (s, 3H), 2.44 (dd, 1H, J = 3.6,
J = 14.8 Hz), 2.65 (dd, 1H, J = 9.6, J = 15.2 Hz), 3.14 (dd, 1H, J = 6.8,
J = 14.8 Hz), 3.20–3.33 (m, 2H), 3.44–3.60 (m, 3H), 3.80 (s, 1H),
3.94 (s, 1H), 3.98–4.02 (m, 1H), 4.30–4.55 (m, 9H), 4.68 (d, 1H,
J = 6.8 Hz), 4.74 (m, 1H), 5.16 (s, 1H), 5.56 (d, 1H, J = Hz), 6.79 (br
s, 1H), 6.85 (d, 1H, J = 8.0 Hz), 6.94–7.32 (m, 19H), 7.53 (d, 1H,
J = 7.6 Hz), 8.34 (s, 1H); 13C NMR (D2O, 100 MHz) d 25.6, 27.4,
28.3, 40.5, 41.1, 54.3, 70.4, 71.9, 72.0, 73.6, 79.9, 80.5, 82.4, 83.7,
84.8, 84.9, 87.1, 95.9, 102.9, 110.2, 111.3, 114.9, 119.2, 119.5,
122.0, 124.2, 127.7, 127.9, 128.10, 128.12, 128.14, 128.2, 128.65,
128.66, 128.70, 136.2, 137.6, 137.7, 138.1, 143.2, 150.5, 163.5,
170.8, 172.6; HRMS: calcd for C51H56N5O11 [M+H]+ 914.3976,
found 914.3942.
a
3.20. 50-N-[N -(2,6-Anhydro-
D
-glycero-
L
-gluco-heptit-1-yl-aceta-
mido)-lysinamido]-50-deoxy-60,70-dihydro-uridine (7a)
Yield 82%; solid; [a]
D +3 (c 0.5, H2O); 1H NMR (D2O, 400 MHz) d
a
1.35–1.53 (m, 2H), 1.62–1.88 (m, 4H), 2.62–2.81 (m, 4H), 2.92–3.03
(m, 2H), 3.42–3.59 (m, 5H), 3.66–3.80 (m, 4H), 3.87 (dd, 1H, J = 5.2,
5.6 Hz), 3.98–4.09 (m, 5H), 4.25–4.34 (m, 2H), 4.46–4.54 (m, 1H),
5.81 (d, 1H, J = 6.4 Hz); 13C NMR (D2O, 100 MHz) d 22.2, 26.7,
30.7, 32.5, 36.9, 37.7, 39.4, 41.2, 54.1, 61.2, 67.7, 68.9, 69.8, 70.2,
71.1, 72.8, 73.3, 81.2, 88.1, 155.8, 161.4, 174.1, 174.4; HRMS: calcd
for C23H40O12 [M+H]+ 578.2673, found 578.2650.
3.25. 50-N-[N -(2,5-Anhydro-3,4,5-tri-O-benzyl-
L-glucit-1-yl-acet-
amido)-histidinamido]-50-deoxy-uridine (14d)
Yield 80%; syrup; [
a]
ꢁ16 (c 2, CHCl3); 1H NMR (CD3OD,
D
400 MHz) d 2.47 (dd, 1H, J = 4.0, 14.0 Hz), 2.60 (dd, 1H, J = 9.2,
14.0 Hz), 2.95 (dd, 1H, J = 8.8, 14.8 Hz), 3.13 (dd, 1H, J = 4.8,
14.8 Hz), 3.40–3.54 (m, 4H), 3.87–3.99 (m, 4H), 4.12–4.23 (m,
2H), 4.33–4.51 (m, 7H), 4.62 (dd, 1H, J = 6.4, 6.8 Hz), 5.66–5.74
(m, 2H), 6.99 (s, 1H), 7.16–7.30 (m, 15H), 7.56 (d, 1H, J = 8.0 Hz),
7.82 (s, 1H); 13C NMR (CD3OD, 100 MHz) d 29.7, 40.9, 42.1, 54.8,
71.3, 72.1, 72.8, 72.9, 74.3, 74.6, 81.6, 83.5, 83.8, 85.8, 88.1, 92.3,
103.2, 118.6, 128.83, 128.86, 128.9, 129.0, 129.1, 129.2, 129.4,
129.5, 133.5, 135.9, 139.1, 139.2, 139.4, 143.4, 152.4, 166.0,
173.0, 173.5; HRMS: calcd for C43H49N6O11 [M+H]+ 825.3459,
found 825.3449.
a
3.21. 50-N-[N -(2,6-Anhydro-
D
-glycero-
L
-gluco-heptit-1-yl-aceta-
mido)-glutamido]-50-deoxy-60,70-dihydro-uridine (7b)
Yield 83%; solid; [a]
D
+3 (c 2, H2O); 1H NMR (D2O, 400 MHz) d
1.93 (s, 3H), 1.98 (m, 1H), 2.06–2.18 (m, 1H), 2.39 (t, 2H,
J = 7.6 Hz), 2.63–2.80 (m, 4H), 3.44–3.57 (m, 4H), 3.64–3.80 (m,
3H), 3.88 (dd, 1H, J = 5.2 Hz), 3.99–4.09 (m, 4H), 4.27–4.36 (m,
2H), 4.46–4.54 (m, 1H), 5.81 (d, 1H, J = 6.0 Hz); 13C NMR (D2O,
100 MHz) d 23.4, 27.2, 30.2, 31.2, 32.5, 36.9, 41.1, 53.6, 61.1,
67.7, 68.9, 69.8, 70.3, 70.9, 72.8, 81.2, 88.1, 160.9, 163.7, 173.8,
174.0, 178.0; HRMS: calcd for C22H36N5O13 [M+H]+ 578.2309,
found 578.2295.
a
3.26. 50-N-[N -(2,5-Anhydro-3,4,5-tri-O-benzyl-
L
-mannit-1-yl-
acetamido)-tryptophanamido]-50-deoxy-uridine (15c)
Yield: 87%; syrup; [
a]
ꢁ11 (c 1.3, CHCl3); 1H NMR (CDCl3,
D
400 MHz) d 1.26 (s, 3H), 1.46 (s, 3H), 2.50 (dd, 1H, J = 4.4,
14.8 Hz), 2.62 (dd, 1H, J = 8.4, 14.8 Hz), 3.10–3.53 (m, 6H), 3.87
(s, 1H), 3.98–4.04 (m, 1H), 4.26–4.51 (m, 8H), 4.68 (d, 1H,
J = 6.8 Hz), 4.76–4.82 (m, 1H), 5.31 (s, 1H), 5.56 (d, 1H, J = 8.0 Hz),
6.82 (d, 1H, J = 7.6 Hz), 6.91 (d, 1H, J = 8.0 Hz), 6.95–7.32 (m,
19H), 7.55 (d, 1H, J = 8.0 Hz), 8.52 (s, 1H); 13C NMR (D2O,
100 MHz) d 25.6, 27.4, 28.1, 36.8, 41.0, 54.2, 70.3, 71.7, 71.8,
73.4, 77.9, 80.7, 82.8, 83.1, 83.2, 83.8, 84.9, 95.0, 102.9, 110.4,
111.5, 115.0, 119.0, 119.6, 122.2, 123.7, 127.9, 127.8, 127.94,
127.97, 28.05, 128.08, 128.11, 128.2, 128.61, 128.67, 128.7, 136.3,
137.7, 137.8, 138.1, 143.0, 150.4, 163.7, 171.6, 172.5; HRMS: calcd
for C51H56N5O11 [M+H]+ 914.3976, found 914.3962.
a
3.22. 50-N-[N -(2,6-Anhydro-
D
-glycero-
L
-gluco-heptit-1-yl-aceta-
mido)-tryptophanamido]-50-deoxy-60,70-dihydro-uridine (7c)
Yield 61%; solid; [a]
D +5 (c 0.6, H2O); 1H NMR (D2O, 400 MHz) d
2.42–250 (m, 2H), 2.55–3.01 (m, 4H), 3.15–3.34 (m, 3H), 3.37–3.83
(m, 8H), 3.92–4.05 (m, 2H), 4.48 (m, 1H), 4.68 (m, 2H), 5.58 (d, 1H,
J = 6.0 Hz), 7.15–7.30 (m, 3H), 7.50 (d, 1H, J = 7.6 Hz), 7.67 (d, 1H,
J = 8.0 Hz); 13C NMR (D2O, 100 MHz) d 27.4, 30.1, 36.1, 40.1, 55.0,
61.0, 67.7, 68.9, 69.7, 69.8, 70.1, 72.6, 73.9, 78.4, 80.7, 87.9,
109.1, 112.0, 118.6, 119.5, 122.2, 124.6, 127.0, 136.3, 154.3,
173.5, 173.6, 173.8; HRMS: calcd for
636.2517, found 636.2546.
C
28H38N5O12 [M+H]+
a
3.27. 50-N-[N -(2,5-Anhydro-3,4,5-tri-O-benzyl-
L-mannit-1-yl-
a
3.23. 50-N-[N -(2,6-Anhydro-
D
-glycero-
L
-gluco-heptit-1-yl-aceta-
acetamido)-histidinamido]-50-deoxy-uridine (15d)
mido)-histidinamido]-50-deoxy-60,70-dihydro-uridine (7d)
Yield 80%; syrup; [
a]
ꢁ2 (c 2.5, CH3OH); 1H NMR (CD3OD,
D
Yield 85%; solid; [
a]
D
+8 (c 1, H2O); 1H NMR (D2O, 400 MHz) d
400 MHz) d 2.43 (dd, 1H, J = 4.0, 14.5 Hz), 2.68 (dd, 1H, J = 9.2,
14.0 Hz), 2.88 (dd, J = 8.8, 14.8 Hz), 2.96 (s, 1H), 3.11 (dd, 1H, J
= 4.0, 14.8 Hz), 3.34–3.54 (m, 4H), 3.86–3.99 (m, 4H), 4.10–4.14
(m, 1H), 4.25–4.49 (m, 6H), 4.62 (dd, 1H, J = 4.8, 5.2 Hz), 5.72 (d,
1H, J = 8.0 Hz), 5.76 (d, 1H, J = 4.4 Hz), 6.83 (s, 1H), 7.13–7.30 (m,
15H), 7.35 (d, 1H, J = 8.0 Hz), 7.46–7.51 (m, 2H); 13C NMR (CD3OD,
100 MHz) d 30.1, 37.1, 41.8, 42.1, 50.0, 52.5, 55.2, 71.5, 71.8, 72.4,
2.59 (dd, 1H, J = 4.0, 14.8 Hz), 2.68–2.77 (m, 3H), 3.00 (dd, 1H, J
= 7.6, 15.2 Hz), 3.08 (dd, 1H, J = 6.8, 14.8 Hz), 3.39–3.51 (m, 5H),
3.64–3.78 (m, 3H), 3.86 (dd, J = 6.0, 6.0 Hz, 1H), 3.90–4.05 (m,
4H), 4.19 (dd, 1H, J = 5.2, 6.0 Hz), 4.40–4.48 (m, 1H), 4.60 (dd, 1H,
J = 7.2 Hz), 5.77 (d, 1H, J = 6.4 Hz), 6.98 (s, 1H), 7.70 (s, 1H); 13C
NMR (D2O, 100 MHz) d 29.1, 30.2, 32.3, 36.9, 40.9, 54.4, 61.0,

K. Vembaiyan et al. / Bioorg. Med. Chem. 19 (2011) 58–66
65
a
72.7, 74.3, 74.7, 79.8, 83.7, 84.0, 84.1, 84.3, 92.0, 103.5, 118.6,
3.32. 50-N-[N -(2,5-Anhydro-
L
-glucit-1-yl-acetamido)-tryptopha-
128.1, 128.82, 128.88, 128.9, 129.0, 129.2, 129.3, 129.50, 129.52,
133.6, 135.8, 129.9, 136.4, 139.2, 139.3, 139.4, 143.0, 145.4,
152.9, 166.7, 173.9, 174.0; HRMS: calcd for C43H49N6O11 [M+H]+
825.3459, found 825.3436.
namido]-50-deoxy-60,70-dihydro-uridine (18c)
Yield 88%; solid; [
a]
D ꢁ51 (c 0.6, H2O); 1H NMR (D2O, 400 MHz)
d 2.51 (dd, 2H, J = 6.4, 10.8 Hz), 2.66 (dd, 2H, J = 2.4, 5.2 Hz), 2.76–
2.85 (m, 1H), 2.97 (m, 1H), 3.22–3.30 (m, 3H), 3.44–3.55 (m, 2H),
3.59 (dd, 1H, J = 6.4, 6.8 Hz), 3.64–3.82 (m, 4H), 3.88–3.92 (m,
1H), 3.97 (dd, 1H, J = 6.4, 6.0 Hz), 4.06 (dd, 1H, J = 6.0, 6.0 Hz),
4.12–4.18 (m, 1H), 4.68 (dd, 1H, J = 6.4, 6.0 Hz), 5.58 (d, 1H,
J = 6.4 Hz), 7.17–7.22 (m, 1H), 7.21–7.29 (m, 2H), 7.52 (d, 1H,
J = 8.0 Hz), 7.70 (d, 1H, J = 7.6 Hz); 13C NMR (D2O, 100 MHz) d
27.3, 30.1, 36.2, 39.2, 40.2, 55.0, 61.2, 69.7, 70.0, 76.5, 79.1, 80.1,
80.9, 82.7, 87.9, 109.0, 112.0, 118.6, 119.6, 122.2, 124.6, 126.9,
136.3, 154.4, 172.8, 173.7, 173.9; HRMS: calcd for C27H36N5O11
[M+H]+ 606.2411, found 606.2377.
a
3.28. 50-N-[N -(2,5-Anhydro-
L-glucit-1-yl-acetamido)-tryptopha-
namido]-50-deoxy-uridine (16c)
Yield 59%; solid; [
a
]
D ꢁ29 (c 0.5, H2O); 1H NMR (D2O, 400 MHz)
d 2.67 (dd, 2H, J = 5.6, 8.0 Hz), 3.26 (dd, 2H, J = 3.2, 6.8 Hz), 3.35 (dd,
1H, J = 3.2, 14.8 Hz), 3.50–3.57 (m, 2H), 3.70 (ddd, 2H, J = 3.6, 8.0,
12.0 Hz), 3.80 (dd, 1H, J = 4.4, 4.8 Hz), 3.86–3.95 (m, 2H), 3.96
(dd, 1H, J = 6.0, 6.4 Hz), 4.06 (dd, 1H, J = 6.0, 6.4 Hz), 4.12–4.18
(m, 1H), 4.67 (dd, 1H, J = 6.4, 7.2 Hz), 5.54 (d, 1H, J = 4.8 Hz), 5.70
(d, 1H, J = 8.0 Hz), 7.13–7.23 (m, 2H), 7.27 (s, 1H), 7.44 (d, 1H,
J = 8.0 Hz), 7.66 (d, 1H, J = 7.2 Hz); 13C NMR (D2O, 100 MHz) d
27.1, 39.2, 40.2, 55.1, 61.2, 69.7, 73.2, 76.5, 79.1, 80.1, 81.6, 82.7,
90.2, 102.3, 108.9, 111.8, 118.5, 119.5, 122.2, 124.6, 126.9, 136.3,
141.5, 154.1, 172.9, 173.5, 174.0; HRMS: calcd for C27H33N5O11Na
[M+Na]+ 626.2074, found 626.2086.
a
3.33. 50-N-[N -(2,5-Anhydro-
L
-mannit-1-yl-acetamido)-histidin-
amido]-50-deoxy-60,70-dihydro-uridine (18d)
Yield 84%; solid; [
a]
D ꢁ12 (c 1.5, H2O); 1H NMR (D2O, 400 MHz)
d 2.59 (d, 2H, J = 6.8 Hz), 2.70–2.78 (m, 2H), 3.02 (dd, 1H, J = 7.6,
14.8 Hz), 3.14 (dd, 1H, J = 6.0, 14.8 Hz), 3.42–3.52 (m, 4H), 3.68
(dd, 1H, J = 5.6, 12.8 Hz), 3.72–3.83 (m, 3H), 3.92–4.07 (m, 4H),
4.22 (dd, 1H, 1H, J = 6.0, 5.6 Hz), 4.28–4.32 (m, 1H), 4.63 (dd, 1H,
J = 5.2, 6.0 Hz), 5.77 (d, 1H, J = 6.0 Hz), 7.02 (s, 1H), 7.80 (s, 1H);
13C NMR (D2O, 100 MHz) d 28.8, 30.2, 35.7, 36.9, 40.9, 54.0, 61.7,
70.3, 70.8, 77.5, 77.9, 78.1, 81.2, 85.0, 88.2, 117.1, 132.8, 136.0,
154.6, 173.4, 173.5, 173.8; HRMS: calcd for C22H33N6O11 [M+H]+
557.2207, found 585.2189.
a
3.29. 50-N-[N -(2,5-Anhydro-
L
-glucit-1-yl-acetamido)-histidin-
amido]-50-deoxy-uridine (16d)
Yield 62%; solid; 1H NMR (D2O, 400 MHz) d 2.59 (d, 2H,
J = 6.4 Hz), 2.74 (dd, 1H, J = 6.8, 12.4 Hz), 3.06 (dd, 1H, J = 7.2,
J = 14.8 Hz), 3.12 (dd, 1H, J = 6.0, 15.2 Hz), 3.46 (dd, 2H, J = 6.0,
6.8 Hz), 3.55 (d, 1H, J = 4.0 Hz), 3.64–3.81 (m, 3H), 3.91–4.07 (m,
4H), 4.21 (dd, 1H, J = 5.6 Hz), 4.30 (dd, 1H, J = 4.8, 4.8 Hz), 4.62
(dd, 1H, J = 6.4, 7.6 Hz), 5.77 (d, 1H, J = 4.4 Hz), 5.89 (d, 1H,
J = 8.0 Hz), 6.99 (s, 1H), 7.60 (d, 1H, J = 8.0 Hz), 7.72 (d, 1H,
J = 4.4 Hz); 13C NMR (D2O, 100 MHz) d 30.2, 35.7, 40.7, 54.2, 61.7,
70.3, 70.8, 73.2, 77.5, 78.1, 81.9, 85.0, 88.2, 90.5, 102.5, 117.9,
136.1, 142.4, 154.6, 166.5, 173.5, 173.6; HRMS: calcd for
a
3.34. 50-N-[N -(2,5-Anhydro-
L
-mannit-1-yl-acetamido)-trypto-
phanamido]-50-deoxy-60,70-dihydro-uridine (19c)
Yield 82%; solid; [
a]
D ꢁ23 (c 0.7, H2O); 1H NMR (D2O, 400 MHz)
d 2.54 (t, 2H, J = 6.4 Hz), 2.60 (d, 2H, J = 6.8 Hz), 2.84–2.92 (m, 1H),
3.03 (m, 1H), 3.18–3.37 (m, 3H), 3.47–3.56 (m, 2H), 3.63–3.84 (m,
6H), 4.01–4.07 (m, 3H), 4.28–4.32 (m, 1H), 4.72 (dd, 1H, J = 7.2,
7.6 Hz), 5.61 (d, 1H, J = 6.0 Hz), 7.19–7.29 (m, 3H), 7.52 (d, 1H,
J = 7.6 Hz), 7.70 (d, 1H, J = 8.0 Hz); 13C NMR (D2O, 100 MHz) d
27.3, 30.2, 35.7, 36.3, 40.3, 54.9, 61.7, 69.9, 70.1, 77.5, 77.9, 78.1,
80.9, 85.0, 88.0, 109.0, 112.0, 118.6, 119.6, 122.2, 124.6, 126.9,
136.3, 154.4, 173.3, 173.7, 173.9; HRMS: calcd for C27H36N5O11
[M+H]+ 606.2411, found 606.2468.
C
22H31N6O11 [M+H]+ 555.2051, found 555.2114.
a
3.30. 50-N-[N -(2,5-Anhydro-
L
-mannit-1-yl-acetamido)-trypto-
phanamido]-50-deoxy-uridine (17c)
Yield: 57%; solid; [
a]
D ꢁ18 (c 1, H2O); 1H NMR (D2O, 400 MHz) d
2.60 (d, 2H, J = 6.8 Hz), 3.19–3.42 (m, 4H), 3.48–3.90 (m, 8H), 4.01–
4.08 (m, 2H), 4.28–4.32 (m, 1H), 4.69 (dd, 1H, J = 6.8, 8.0 Hz), 5.57
(d, 1H, J = 4.0 Hz), 5.72 (d, 1H, J = 8.0 Hz), 7.13–7.29 (m, 4H), 7.45
(d, 1H, J = 8.0 Hz), 7.76 (d, 1H, J = 7.6 Hz); 13C NMR (D2O,
100 MHz) d 27.1, 35.7, 40.3, 55.0, 61.6, 69.8, 73.2, 77.5, 77.9,
78.1, 81.6, 85.0, 90.3, 102.4, 108.9, 111.8, 118.5, 119.5, 122.2,
124.6, 126.9, 136.2, 141.5, 152.4, 172.2, 173.4, 174.0; HRMS: calcd
for C27H34N5O11 [M+H]+ 604.2255, found 604.2280.
a
3.35. 50-N-[N -(2,5-Anhydro-
L
-mannit-1-yl-acetamido)-histidin-
amido]-50-deoxy-60,70-dihydro-uridine (19d)
Yield 81%; solid; [
a]
D ꢁ38 (c 1.5, H2O); 1H NMR (D2O, 400 MHz)
d 2.58–2.79 (m, 4H), 3.02 (dd, 1H, J = 7.6, 14.4 Hz), 3.11 (dd, 1H, J
= 6.8, 14.8 Hz), 3.42–3.52 (m, 4H), 3.66 (dd, 1H, J = 5.6, 12.8 Hz),
3.73 (dd, 1H, J = 3.2, 12.4 Hz), 3.92–3.98 (m, 4H), 4.02–4.7 (m,
1H), 4.20 (dd, 1H, J = 5.2, 5.6 Hz), 4.30 (dd, 1H, J = 6.0, 5.6 Hz),
4.63 (dd, 1H, J = 6.8, 7.2 Hz), 5.77 (d, 1H, J = 6.4 Hz), 7.01 (s, 1H),
7.74 (s, 1H); 13C NMR (D2O, 100 MHz) d 28.9, 30.2, 36.9, 39.2,
40.9, 54.2, 61.3, 70.2, 70.7, 76.5, 79.0, 80.0, 81.2, 82.7, 88.1, 117.7,
133.7, 136.1, 154.6, 172.9, 173.5, 173.8; HRMS: calcd for
a
3.31. 50-N-[N -(2,5-Anhydro-
L-mannit-1-yl-acetamido)-histidin-
amido]-50-deoxy-uridine (17d)
Yield 64%; solid; 1H NMR (D2O, 400 MHz) d 2.58–2.78 (m, 2H),
3.05 (dd, 1H, J = 7.2, J = 12.4 Hz), 3.14 (dd, 1H, J = 6.4, 14.8 Hz),
3.44–3.50 (m, 2H), 3.55 (d, 1H, J = 4.8 Hz), 3.67 (dd, 1H, J = 6.8,
13.6 Hz), 3.74 (dd, 1H, J = 3.6, 12.4 Hz), 3.86–4.08 (m, 6H), 4.20
(dd, 1H, J = 5.2, 5.6 Hz), 4.28–4.32 (m, 1H, J = 4.8, 5.2 Hz), 4.61
(dd, 1H, J = 7.2 Hz), 5.77 (d, 1H, J = 4.4 Hz), 5.88 (d, 1H, J = 8.0 Hz),
7.01 (s, 1H), 7.62 (d, 1H, J = 8.0 Hz), 7.87 (s, 1H); 13C NMR (D2O,
100 MHz) d 30.2, 39.2, 40.8, 53.9, 61.3, 70.3, 70.5, 76.5, 79.0, 80.0,
82.0, 82.7, 90.6, 102.4, 117.7, 128.9, 136.2, 142.4, 151.7, 166.3,
173.0, 173.8; HRMS: calcd for C22H31N6O11 [M+H]+ 555.2051,
found 555.2068.
C
22H33N6O11 [M+H]+ 557.2207, found 557.2190.
Acknowledgements
This is NRC-CNRC Publication No. 50009. We thank Dr. Jianjun
Li, Mr. Ken Chan and Mr. Jacek Stupak (IBS/NRC) for MS analysis
and Dr. Wangxue Chen and Mr. Harvey Miller (IBS/NRC) for the
assistance in macrophage growth. Support from the Alberta Inge-
nuity Centre for Carbohydrate Science and NSERC is gratefully
acknowledged.

66
K. Vembaiyan et al. / Bioorg. Med. Chem. 19 (2011) 58–66
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