3′-Oxo-, amino-, thio- and sulfone-acetic acid modified thymidines: Effect of increased acidity on ribonuclease A inhibition

Article abstract of DOI:10.1016/j.bmc.2013.05.047

A family of 3′-functionalized thymidines carrying XCH₂COOH (X = O, NH, S, SO₂) groups has been designed as inhibitors of RNase A. This is because it is possible to manipulate the overall acidity of this new class of nucleic 'acids' by changing X from oxygen to the SO₂ group in the series. It is also expected that the acyclic nature of the XCH ₂COOH group would provide enough flexibility to the -COOH group to have maximum interactions with the catalytic subsite P₁ of RNase A. As the -SO₂CH₂COOH substituted derivative showed better potency partially because of the increased acidity of the -COOH group, the inhibitory properties of both 5′-substituted and 3′,5′- disubstituted sulfone acetic acid modified thymidines were investigated. Two -SO₂CH₂COOH groups were incorporated with the expectation of targeting two phosphate binding sites simultaneously. Thus, 3′,5′-dideoxy-3′,5′-bis-S-[(carboxymethyl)sulfonyl] thymidine emerged as the best inhibitor in this series with a K_i value of 25 ± 2 μM.

Full text of DOI:10.1016/j.bmc.2013.05.047

Bioorganic & Medicinal Chemistry 21 (2013) 4634–4645
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Bioorganic & Medicinal Chemistry
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30-Oxo-, amino-, thio- and sulfone-acetic acid modified thymidines:
Effect of increased acidity on ribonuclease A inhibition
⇑
⇑
Dhrubajyoti Datta, Anirban Samanta , Swagata Dasgupta , Tanmaya Pathak
Department of Chemistry, Indian Institute of Technology Kharagpur, Kharagpur 721302, India
a r t i c l e i n f o
a b s t r a c t
A family of 30-functionalized thymidines carrying XCH2COOH (X = O, NH, S, SO2) groups has been
designed as inhibitors of RNase A. This is because it is possible to manipulate the overall acidity of this
new class of nucleic ‘acids’ by changing X from oxygen to the SO2 group in the series. It is also expected
that the acyclic nature of the XCH2COOH group would provide enough flexibility to the –COOH group to
have maximum interactions with the catalytic subsite P1 of RNase A. As the –SO2CH2COOH substituted
derivative showed better potency partially because of the increased acidity of the –COOH group, the
inhibitory properties of both 50-substituted and 30,50-disubstituted sulfone acetic acid modified thymi-
dines were investigated. Two –SO2CH2COOH groups were incorporated with the expectation of targeting
two phosphate binding sites simultaneously. Thus, 30,50-dideoxy-30,50-bis-S-[(carboxymethyl)sulfo-
Article history:
Received 22 April 2013
Revised 13 May 2013
Accepted 14 May 2013
Available online 3 June 2013
Keywords:
Ribonuclease A
Inhibitors
Acidic nucleosides
Kinetics
nyl]thymidine emerged as the best inhibitor in this series with a Ki value of 25
2 lM.
Ó 2013 Elsevier Ltd. All rights reserved.
Docking
1. Introduction
diester bond where n = 1) (Fig. 1). In all RNases the subsite P1 is
conserved, whereas subsites B1 and B2 on each side of P1 are
partially conserved.20 Nevertheless, B1 binds pyrimidines, while
B2 has a strong base preference for purines. Like all other
ribonucleases, the presence of the His12-Lys41-His119 catalytic
During the last two decades, the ribonuclease superfamily
members1 have generated significant biomedical interest as their
enzymatic activities2–5 are closely associated with some physiolog-
ical irregularities.6–8 The angiogenic and tumor-promoting
activities of angiogenin,9–11 the neurotoxic activities of eosino-
phil-derived neurotoxin (EDN)12,13 and the cytotoxic activities14
of bovine seminal ribonuclease15 are among the few potent biolog-
ical activities shown by the members of this family. Further studies
have shown that most of these pathological conditions are linked
to the ribonucleolytic activity of these proteins16 and hence, inhib-
itors of their enzymatic activity could be potent pharmaceutical
agents. Bovine pancreatic ribonuclease A (RNase A)16,17 is often
exploited as a model system to understand structure–function
relationships of members of this superfamily. All the members
are active against single-stranded RNA substrates and catalyze
RNA breakdown although they show various degrees of ligand rec-
ognition and discrimination.18,19 In RNase A, several subsites are
responsible in RNA binding which are conventionally termed as
P0. . .Pn, R0. . .Rn and B0. . .Bn corresponding to phosphate, ribose
and nucleobases of bound RNA, respectively (n indicates the posi-
tion of the group with respect to the cleaved phosphate phospho-
B1
Thr-45
Phe-120
Ser-123
NH2
N
O
P
O
O
O
P
B2
-O
P0
Lys-66
Gln-69
Asn-71
Glu-111
NH2
R1
P1
Lys-41
His-12
His-119
N
N
N
OH
O
O
N
O
P
B3
-O
O
NH2
Lys-1
N
N
N
R2
OH
O
O
N
O
-O
O
P2
R3
Lys-7
OH
O
Arg-10
⇑
Corresponding authors. Tel.: +91 3222 283306; fax: +91 3222 255303 (S.D.);
tel.: +91 3222 283342; fax: +91 3222 255303 (T.P.).
E-mail addresses: swagata@chem.iitkgp.ernet.in (S. Dasgupta), tpathak@chem.
iitkgp.ernet.in (T. Pathak).
Figure 1. Schematic representation of active clefts of RNase A where Bn, Rn, and Pn
are nucleobase-, ribose-, and phosphate-binding subsites, respectively, and labeled
with their constituent residues.
Current address: Division of Organic Chemistry, National Chemical Laboratory,
Pune 411008, India.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.05.047

D. Datta et al. / Bioorg. Med. Chem. 21 (2013) 4634–4645
4635
triad of P1 subsite in RNase A is essential for its ribonucleolytic
activity.16,21
attention to pyrimidine nucleosides modified with acyclic
functional groups expected to have minimum conformational
rigidity. Also, depending on the nature of ‘X’ in XCH2COOH, these
compounds are expected to have better binding through enhanced
electrostatic interactions.
The development of substrate mimics is a good strategy for the
search of a competitive type of inhibitor of an enzyme. Most of the
reported potent inhibitors and mechanism based inactivators22 of
RNase A are thus phosphate or pyrophosphate based nucleotide
molecules.20,23–25 The utility of these nucleotides as inhibitors is,
however, limited. The free phosphate groups are susceptible to
dephosphorylation by phosphatases which markedly diminish
their potency as the anionic phosphate groups plays an important
role in binding with the active site of RNase A.
Moreover, the polyionic nature of these inhibitors hinders their
transport across the cell membrane.26 To overcome these problems,
the replacement of the phosphate group by a carboxylic acid group
is considered as an alternative route. At physiological pH, inhibitors
carrying acidic groups would exist in the deprotonated form and
therefore interact electrostatically with the protonated histidine
groups present at the P1 subsite to perturb the protonating/deprot-
onating environment of this subsite.27,28 This in turn would affect
the ribonucleolytic activity of RNase A.29 Few modified mononucle-
osidic inhibitors containing carboxylic acid group are reported
where this acid group is linked through amide linkage30,31 or by
cyclic alkyl amino group28,32,33 with the backbone of pyrimidine
nucleosides. Modified dinucleosides where the phosphate group
is replaced with sulfonamide34 and amide group35 have also been
reported as inhibitors of RNase A. Again, nucleobases tethered with
polyphenols and bridged with a triazole moiety have been reported
to act as inhibitors of RNase A.36 Recently triazole pyrimidine
nucleosides have emerged as RNase A inhibitors.37
It is clearly evident from the earlier experiments carried out in
our laboratory that modified nucleosides functionalized with car-
boxylic group at different positions of the sugar residues as well
as dinucleosides having a carboxylic group attached to the internu-
cleoside linkage almost always perform as better inhibitors of
RNase A.35 We therefore selected a family of 30-functionalized
thymidines carrying XCH2COOH (X = O, NH, S, SO2) groups. We
argued that in this series we would be able to manipulate the
overall acidity of this new class of nucleic ‘acids’ carrying a
carboxylic group instead of phosphate by changing X from oxygen
to the SO2 group because it is known that in the series
MeSCH2COOH (pKa = 3.81), MeOCH2COOH (pKa = 3.55), MeSO2CH2-
COOH (pKa = 2.31) and MeNHCH2COOH (NH2-pKa = 10.2; COOH-
pKa = 2.36),38,39 acidity of the carboxylic group depends signifi-
cantly on the nature of X. Since the overall nature of the P1 site
is positively charged at physiological pH,28,29 a carboxylic group
with a pKa of 2.31 should be able to anchor more efficiently with
the P1 subsite of RNase A. This study is expected to establish that
even in the case of weak acids, the efficiency of inhibition of mod-
ified nucleosides would vary with the varying pKa values of the
acidic group attached to the sugar moiety of a nucleoside.
2. Results and discussion
2.1. Synthesis of inhibitors
For the designing of inhibitors the nucleobase thymine was left
unaltered for the recognition by the B1 site. The –XCH2COOH (X=,
O, N, S, SO2) functionalities were attached to the sugar ring of the
nucleoside. Thus, for the incorporation of the –OCH2COOH group
at the 30-position, N3 of 50-O-tritylthymidine was benzoylated fol-
lowing the literature procedure to synthesize compound 1.41,42
Compound 1 was reacted with bromoethylacetate in presence of
NaH in dry DMSO for 24 h at room temperature followed by base
hydrolysis to afford 30-O-(carboxymethyl)-50-O-tritylthymidine
243 in 55% overall yield. Compound 2 was treated with 80% TFA
in DCM for 3 h at room temperature to obtain compound 3 (T-
30OCH2COOH)44 in 69% yield (Scheme 1).
The synthesis of –NHCH2COOH was initiated by reacting
aminothymidine 445,46 with bromoethylacetate in presence of
diisopropylethylamine in dry DMF at room temperature to afford
30-N-(carboxymethyl)-50-O-tritylthymidine
5
in 92% yield.47
Bromoethylacetate (1 equiv) was added in portion (10 ꢀ 0.1 equiv)
over a period of 40 h to get the monoalkylated product as major
compound. Compound 5 was detritylated by 80% TFA in DCM for
3 h at room temperature to afford compound 7 (T-30NHCH2COOEt)
in 80% yield. Compound 5 was hydrolyzed by aq 1 M NaOH
solution to obtain compound 6 in 77% yield, which on acid
treatment afforded compound 8 (T-30NHCH2COOH) in 72% yield
(Scheme 2).
The preparation of thioglycolic acid modified thymidine re-
quired the reactions between the ‘up’-mesylated nucleoside 945
and the sodium salt of methyl 2-mercaptoacetate in DMF at
0 °C, which afforded compound 10 in 66% yield. Compound 10
on treatment with 80% TFA in DCM afforded the ester derivative
12 (T-30SCH2COOMe) in 79% yield. Another portion of compound
10 was hydrolyzed by 1 M NaOH in THF to afford compound 11 in
74% yield which was further converted to the free acid derivative
13 (T-30SCH2COOH) by 80% TFA in DCM. Compound 10 was trea-
ted with magnesium bis(monoperoxyphthalate)hexahydrate
(MMPP) in dry MeOH at room temperature to obtain compound
14 in 70% yield which on treatment with 80% TFA in DCM affor-
ded 15 (T-30SO2CH2COOMe) in 75% yield. Again, compound 14
was converted to 16 (T-30SO2CH2COOH) in 55% overall yield after
consecutive hydrolysis by lithium hydroxide and 80% TFA
(Scheme 3).
The crystal structure of 30-isonipecotic acid modified ara-uridine
with RNase A reveals that two inhibitors are bound to the periphe-
ral binding sites of RNase A in a different fashion.40 In one case, the
uracil base and the carboxylic acid group of 4-carboxypiperidine
moiety binds to the P0 and B1 subsite, respectively, while in the
other, the uracil base occupies the purine—preferring B2 subsite
but the 4-carboxypiperidine group points toward the solvent.
Similar phenomena are also observed from the crystal structures
of various cyclic alkylamino substituted 50-modified uridine or
thymidine nucleosides.32 It shows that the anchoring point of all
these inhibitors is the pyrimidine base which is bound at the B1
subsite but the 50-substituents are directed towards the solvent
rather than the active site residues. However, in all the
cases discussed above it has been observed that the cyclic nature
of the substituent generates a hindrance to the efficient binding
of these inhibitors to RNase A.32,40 Therefore, we turned our
Since sulfone containing compounds showed better efficiency as
inhibitors (see later) we decided to synthesize several related sulf-
onylated thymidines. For the incorporation of two thioglycolic acid
O
NBz
(i) BrCH2CO2Et,
O
N
NaH, DMSO
N
O
T
TrO
RO
rt, 24 h
O
O
NH
T =
(ii) 1M NaOH,
THF, rt,
O
HO
CO2H
O
overnight
1
2 R = Tr (55%)
3 R = H (69%)
80% TFA in
DCM, rt, 3 h
Scheme 1. Synthesis of T-30OCH2COOH 3.

4636
D. Datta et al. / Bioorg. Med. Chem. 21 (2013) 4634–4645
BrCH2CO2Et
DMF, (iPr)2NEt
rt, 40 h
1M NaOH
THF, rt, overnight
T
T
T
RO
RO
TrO
O
O
O
CO2Et
CO2H
HN
HN
NH2
4
5 R = Tr (92%)
7 R = H (80%)
80% TFA in
DCM, rt, 3 h
80% TFA in
DCM, rt, 3 h
6 R = Tr (77%)
8 R = H (72%)
Scheme 2. Synthesis of T-30NHCH2COOH 8.
2.1.1. Biophysical studies
HSCH2CO2Me
80% TFA in
DCM, rt, 3 h
NaH, DMF
OMs
O
T
T
T
The agarose gel-based assay was performed to get a qualitative
idea of the inhibitory activity of the synthesized compounds 3, 7,
8, 12, 13, 15, 16, 19–22, 24–27, 30 and 31 against RNase A. In the
agarose gel, the band in lane 1 showed maximum intensity due to
the presence of only tRNA. Due to the degradation of tRNA by
RNase A, the band in lane 2 was the least intense. Lanes 3, 4
and 5 of each gel contained tRNA and RNase A with increasing
concentrations of synthesized compounds 3, 7, 8, 12 and 13
[Fig. I in SI]. A significant difference in intensity between lanes
2 and 3 was observed for compounds 3, (T-30OCH2COOH) 8 (T-
30NHCH2COOH) and 13 (T-30SCH2COOH) and these differences
gradually increased from lane 3–5. It was evident that, the inten-
sity change was less prominent for the ester compounds, viz.; 7
(T-30NHCH2COOEt), 12 (T-30SCH2COOMe) 24 [T-30(up),50-bis-
SCH2COOMe], and 19 (T-30,50-bisSCH2COOMe). However, despite
being esters, compounds 15 (T-30SO2CH2COOMe), 20 (T-30,50-
bisSO2CH2COOMe), 25 [T-30(up),50-bisSO2CH2COOMe], and 30
(T-50SO2CH2COOMe) showed prominent intensity changes with
increase in inhibitor concentration [Fig. I and II in SI]. Similar
qualitative experiments have been performed for sulfone-acetic
acid modified thymidines viz. compound 16 (T-30SO2CH2COOH),
21 (T-30,50-bisSO2CH2COOH), 26 [T-30(up),50-bisSO2CH2COOH], 31
(T-50SO2CH2COOH) and sulfur-acetic acid modified thymidines
viz. 27 [T-30(up),50-bisSCH2COOH] and 22 (T-30,50-bisSCH2COOH)
[Fig. III in SI]. In Figures 2 and 3, representative images of the aga-
rose gel based electrophoresis assay have been given. From the
gel images, it was evident that sulfone-acetic acids always show
better inhibitory property than the other modified nucleosides
mentioned above.
TrO
HO
TrO
0 oC, 6 h
O
O
79%
66%
9
S
CO2Me
S
CO2Me
10
12
MMPP
MeOH
rt. 6 h
1M NaOH, THF
rt, overnight
74%
70%
T
TrO
T
O
TrO
O
20% TFA, DCM
rt, 0.5 h, 75%
CO2H
S
O2S
CO2Me
11
14
T
HO
O
80% TFA in
DCM, rt, 3 h
(i) LiOH.H2O, THF
62%
rt, 40 min
(ii) 20% TFA, DCM
rt, 0.5 h, 55%
O2S
CO2Me
HO
T
HO
O
T
O
15
CO2H
S
O2S
CO2H
13
16
Scheme 3. Synthesis of T-30SCH2COOH 13 and T-30SO2CH2COOH 16.
units, dithiothymidine 1748 was treated with ethylbromoacetate in
DCM in presence of TEA.49 Compound 18, thus obtained was con-
verted to its methyl ester analogue by a transesterification meth-
od50 to afford 19 (T-30,50-bisSCH2COOMe) in good yield.
Compound 19 was oxidized to the sulfone ester 20 (T-30,50-
bisSO2CH2COOMe) and thereafter the latter compound was
hydrolyzed under basic condition to afford the desired bis-acid
21 (T-30,50-bisSO2CH2COOH). The bis-ethylthioglycolate ester 18
was hydrolyzed by 1 M NaOH solution to obtain the corresponding
bis-thioglycolic acid 22 (T-30,50-bisSCH2COOH) in moderate yield
(Scheme 4).
After confirming the possibility of inhibition by the agarose gel
based assay, we performed the precipitation assay with com-
pounds 3, 7, 8, 12, 13, 15, 16, 19–22, 24–27, 30 and 31 against
RNase A. The reduction of the ribonucleolytic activity of these com-
pounds for RNase A (1.31 lM) at a fixed concentration (0.35 mM)
was compared. The activity of RNase A was reduced to a greater ex-
tent by the acids in comparison to the esters, following a range of
13–35% for acids (Fig. 4) and 3–18% for esters (see Fig. IV in SI).
Compound 3 (T-30OCH2COOH) exhibited 13% inhibition of ribonuc-
leolytic activity compared to 19% by 8 (T-30NHCH2COOH). Thiogly-
colic acid 13 (T-30SCH2COOH) exhibited a 17% reduction in the
ribonucleolytic activity. The 30-substituted sulfone acid 16 (T-
30SO2CH2COOH) showed better inhibitory properties (25% reduc-
tion) compared to the 50-analogue 31 (T-50SO2CH2COOH) (22%
reduction). The bis-thioglycolic acids 27 [T-30(up),50-bis-
SCH2COOH] and 22 (T-30,50-bisSCH2COOH) showed moderate po-
tency and were able to reduce the ribonucleolytic activity by 16%
and 18%, respectively, (Fig. 4).
Except sulfone-modified esters tested in this assay, other esters
like, 7 (T-30NHCH2COOEt), 12 (T-30SCH2CO2Me), 24 [T-30(up),50-
bisSCH2COOMe] and 19 (T-30,50-bisSCH2COOMe) exhibited very
low inhibitory activity (7%, 3%, 5% and 6%, respectively). Reduction
of ribonucleolytic property improved for sulfone-esters viz. 15 (T-
30SO2CH2COOMe), 25 [T-30(up),50-bisSO2CH2COOMe], 20 (T-30,50-
bisSO2CH2COOMe), and 30 (T-50SO2CH2COOMe) which showed
Ditosylate thymidine 2351 was treated with 2-mercaptoacetate
using similar reaction condition52 mentioned in Scheme 3 to obtain
the desired bis-modified 24 [T-30(up),50-bisSCH2COOMe] in high
yield. Thereafter 24 was oxidized by MMPP to generate bis-sulfone
ester 25 [T-30(up),50-bisSO2CH2COOMe], which on further hydroly-
sis afforded acid 26 [T-30(up),50-bisSO2CH2COOH]. Again, 24 was
hydrolyzed under basic condition with 1 M NaOH solution to ob-
tain the corresponding bis-thioglycolic acid derivative 27 [T-
30(up),50-bisSCH2COOH] in moderate yield (Scheme 5).
On the other hand, 50-O-tosylthymidine 2833 was treated with
the sodium salt of methyl 2-mercaptoacetate in dry DMF at room
temperature to afford compound 29 (T-50SCH2COOMe)53 in 76%
yield. Ester 29 was oxidized first and then hydrolyzed under basic
condition to obtain corresponding sulfone ester 30 (T-
50SO2CH2COOMe) and acid 31 (T-50SO2CH2COOH) (Scheme 6). It
should be noted that the yields of 21, 26, and 31 were too low un-
der routine hydrolysis conditions which were later improved by
applying trimethyltin hydroxide (TMTOH).54

D. Datta et al. / Bioorg. Med. Chem. 21 (2013) 4634–4645
4637
CO2Et
CO2Me
O
BrCH2CO2Et
DCM, TEA
rt, 0.4 h
nBu2SnO
MeOH, reflux, 12 h
T
T
T
S
HS
S
O
O
80%
70%
CO2Et
S
SH
17
CO2Me
S
19
18
1M NaOH,
THF, rt,
MMPP
MeOH
2 h, 61%
rt, 6h, 77%
CO2H
CO2H
CO2Me
O2S
Me3SnOH
T
T
DCE
T
O2S
S
O
O
70 oC, 6 h
O
75%
CO2H
CO2H
O2S
S
CO2Me
O2S
20
Scheme 4. Synthesis of T-30,50-bisSO2CH2COOH 21 and T-30,50-bisSCH2COOH 22.
21
22
CO2Me
S
CO2Me
O2S
HSCH2CO2Me
MMPP
MeOH
rt, 6 h
CO2Me
T
CO2Me
NaH, DMF, 0 oC
3 h
T
T
O2S
TsO
O
S
O
O
70%
76%
25
OTs
24
23
Me3SnOH
1M NaOH, THF
rt, 2 h, 60%
DCE, 70 oC
6 h, 75%
CO2H
CO2H
O2S
O2S
CO2H
T
CO2H
S
S
T
O
O
26
27
Scheme 5. Synthesis of T-30(up),50-bisSO2CH2COOH 26 and T-30(up),50-bisSCH2COOH 27.
CO2R
O2S
CO2Me
S
HSCH2CO2Me
MMPP
MeOH
rt, 6 h
NaH, DMF
T
T
T
0 oC, 0.8 h
O
TsO
O
O
76%
OH
28
OH
OH
29
Me3SnOH
30 R =Me (70%)
31 R = H (81%)
DCE
70 oC, 12 h
Scheme 6. Synthesis of T-50SO2CH2COOH 31.
Figure 2. Agarose gel for the inhibition of RNase A (2.0
l
M): (lane 1) tRNA; (lane 2)
Figure 3. Agarose gel for the inhibition of RNase A (2.0
l
M): (lane 1) tRNA; (lane 2)
tRNA and RNase A; (lanes 3–6) tRNA, RNase A and 21 (T-30,50-bisSO2CH2COOH)
tRNA and RNase A; (lanes 3–6) tRNA, RNase A and 26 [T-30(up),50-bisSO2CH2COOH]
(0.05, 0.10, 0.15 and 0.20 mM, respectively).
(0.05, 0.10, 0.15 and 0.20 mM, respectively).
18%, 12%, 15%, 14% inhibition, respectively [Fig. IV in SI]. Among
the ester and acids in this series, best results were shown by
bis-sulfone acids 26 [T-30(up),50-bisSO2CH2COOH] and 21 (T-30,50-
bisSO2CH2COOH) with almost equal potency (33% and 35%,
respectively). From the two qualitative experimental observations,
we could say that among the seventeen preliminarily compounds

4638
D. Datta et al. / Bioorg. Med. Chem. 21 (2013) 4634–4645
Figure 5. Lineweaver–Burk plots for inhibition of RNase
bisSO2CH2COOH) of 19.0 (N), 9.5 (j), 0.00 (ꢀ)
M, 20,30-cCMP concentrations
(0.77–0.42 mM) and RNase A concentration of 20.0 M.
A
by 21 (T-30,50-
l
l
the nature of the plots indicated reversible competitive inhibition
[Figs. V, VI and VII in SI].
Figure 4. Reduction of ribonucleolytic activity of RNase A (1.31 lM) in the presence
of acids (0.35 mM) by precipitation assay.
2.1.2. Docking studies
screened, acids have shown a better inhibitory activity compared
to the corresponding esters.
We performed docking experiment to get probable binding con-
formations of the inhibitors with RNase A. In docking, the thymine
base of 30-modified acid compounds 3 (T-30OCH2COOH), 8 (T-
30NHCH2COOH), 13 (T-30SCH2COOH) and 16 (T-30SO2CH2COOH)
has shown an improved interaction pattern with B1 subsite which
in turn, has been reflected in the observed inhibition constants also
[Figs. VIII and XI in SI]. In the docked pose, for compound 3 (Fig. 6),
the nucleobase occupies the active cleft of P1 subsite consisting of
His12, Lys41 and His119 (amino acid residues highlighted in cyan)
which moves towards the B1 subsite consisting of Val43, Asn44 and
Thr45 amino acid residues (highlighted in green) as the substitu-
tion is changed from oxygen to SO2 at the 30-position of the inhib-
itors (Fig. 6). Quite remarkably, it is found that for the sulfone-acid
16 (T-30SO2CH2COOH), there is appreciable inclination of the
nucleobase towards the B1 subsite (Fig. 6) which is likely due to
the effect of the sulfone group present at the 30-position which is
able to generate more H-bonding interactions. Eventually, H-bond-
ing interaction is observed for compound 16 with B1 site (Fig. 6 and
Tables II–IV in SI).
Binding of the acid groups of compound 3, 8 and 13 are dis-
tinctly different. In the docked conformation, the acid group of
compound 13 (T-30SCH2COOH) is within hydrogen bonding dis-
tance of the active site His12 and His119 residues while that of
compound 8 (T-30NHCH2COOH) is near His119 residue only. On
the other hand, the acid group of compound 3 (T-30OCH2COOH)
is bound to the Gln11 residue and observed to be away from the
active site His residues. From the docked structure it is noted that
the 30-hetero atom plays a vital role in binding. In compound 3, the
hydrogen bonding interaction of the 30-oxygen atom with His119
hinders binding of the acid group with the His residues. For com-
pound 8, the 30-NH is also hydrogen bonded with His119 and this
interaction favors the binding of the acid group with His119 but
not with His12. The 30-sulfur atom of compound 13 interacts with
Lys7 to facilitate the H-bonding interaction with His119 as well as
His12 which is the probable reason for slightly higher potency
compared to compound 3 and 8. Compound 16 consisting of ‘stron-
ger’ acid functionality, –SO2CH2COOH, that accommodates itself
into the active site due to proper nucleobase recognition and addi-
tional electrostatic interactions. These parameters make it superior
To determine the mode of inhibition and inhibition constants
(Ki) of compounds 3, 7, 8, 12, 13, 15, 16, 20–22, 25–27, 30 and
31 steady state kinetic experiments were performed. The inhibi-
tion constants of potent inhibitors of RNase A, predicted from qual-
itative assays, were determined from the Lineweaver–Burk plots
(SI). From the Ki values, it could be concluded that the thio and sul-
fone acetic acid modified thymidine compounds were more potent
than compound 8 (T-30NHCH2COOH) and 3 (T-30OCH2COOH). The
ester compounds 7 (T-30NHCH2COOEt) and 12 (T-30SCH2COOMe)
showed weak inhibition property against the enzyme. However,
sulfone-esters (15, 20, 25, and 30) exhibited slightly better inhibi-
tion with moderate Ki values (Table 1) which was in agreement
with the preliminary assays.
Finally, inhibition constants (Ki) for bis-sulfur and bis-sulfone
compounds were calculated. A representative Lineweaver–Burk
plot of compound 21 against RNase A has been depicted in Figure 5.
The Ki values mentioned in Table 1 reflect that the fact that the
thio-acids are much weaker inhibitors than sulfone and bis-sulfone
acids. Bis-sulfone acid 21 is the most potent inhibitor in this series.
This phenomenon can be explained as a result of greater H-bond-
ing ability of this acid, designed with two sulfone groups. Thus, it
is able to generate a larger H-bonding network in the proximity
of the active site of the enzyme. In all the cases mentioned above,
Table 1
Inhibition constants (Ki) of inhibitors
Inhibitors (acids)
Ki (
lM)
Inhibitors (esters)
Ki (lM)
a
0
3 (T3 OCH2COOH
)
267 11
0
0
8 (T3 NHCH2COOH
)
186
169
88
25
93
39
108
95
9
9
5
2
4
2
5
3
7 (T3 NHCH2COOEt
)
)
504 20
757 22
0
0
13 (T3 SCH2COOH
16 (T3 SO2CH2COOH
21 (T3 ,5 -bisSO2CH2COOH
22 (T3 ,5 -bisSCH2COOH
26 [T3 (up),5 -bisSO2CH2COOH
27 [T3 (up),5 -bisSCH2COOH
31 (T5 SO2CH2COOH
)
12 (T3 SCH2COOMe
15 (T3 SO2CH2COOMe
20 (T3 ,5 -bisSO2CH2COOMe)
18 (T3 ,5 -bisSCH2COOMe)
25 [T3 (up),5 -bisSO2CH2COOMe
24 [T3 (up),5 -bisSCH2COOMe]
30 (T5 SO2CH2COOMe
0
0
)
)
140
267
NDb
242
NDb
202
4
5
0
0
0
0
)
0
0
0
0
)
0
0
0
0
]
]
7
3
0
0
0
0
]
0
0
)
)
ND due to very poor response in agarose gel-based assay.
a
The ester T3’OCH2COOEt corresponding to the acid 3 not synthesized.
b
Kinetic experiments not done (ND) due to very poor response in agarose gel-
based assay.

D. Datta et al. / Bioorg. Med. Chem. 21 (2013) 4634–4645
4639
Figure 6. Docked poses of compound 3 (T-30OCH2COOH) (Ki = 267 11
l
M) and 16 (T-30SO2CH2COOH) (Ki = 88
5
l
M) with RNase A (1FS3) where movement of nucleobase
towards B1 site (amino acid residues colored in green) is observed.
than the oxo-, amino- and thio-glycolic acid modified nucleoside
inhibitors.
functionalities with different phosphate binding subsites (P0 and
P1), 21 elicits the best inhibitory property among the aforemen-
tioned molecules. In the docked pose of 26 with RNase A, dimin-
ished H-bonding interactions with the P1 subsite are noticeable
due to a conformational change in the 30-site of the inhibitor
(Fig. 7).
This subtle change, affects the Ki value of 26, making it less po-
tent than 21. The proximity of the –COOH groups with the effective
side chain residues at the active site along with proper nucleobase
recognition are the most probable reasons for better activity of
these two sulfone-modified acids. Thus, docking results substanti-
ate the biological assays to a large extent.
Ester compounds, 7 and 12 interact with RNase A through the
thymine base but the 30-ester substituent is away from the P1
subsite. In spite of being esters, 15, 20, 25 and 30 perform as good
inhibitors of RNase A due to additional interactions between the
sulfone groups of the molecules and residues of the protein that
lie within H-bonding distance [Figs IX and X in SI]. For 50-modified
compounds 30 and 31, the 50-substitutions appear to move away
from the active site and interact weakly with the B1 subsite
[Fig. X in SI]. For 31, the nucleobase was away from the B1 subsite
due to a possible H-bonding interaction with Lys41.
Finally, for 21 (T-30,50-bisSO2CH2COOH) and 26 [T-30(up),50-bis-
SO2CH2COOH] docking studies reveal H-bonding interactions with
amino acid residues of the active site (P1) and other subsites (P0, P2,
B2) [Tables III–IV in SI]. Docked conformations of both 21 (T-30,50-
bisSO2CH2COOH) and 26 [T-30(up),50-bisSO2CH2COOH] depict
favorable alignment for interactions with the enzyme active site.
Here, due to proper recognition of two anchoring –SO2CH2COOH
After docking studies, rationalization of the experimentally ob-
tained inhibition constant (Ki) with docking parameters were
checked with the aid of PEARLS.55 During such analysis, the acids
viz. 3, 8, 13, 16, 21, 22, 26, 27, 31 and ester 15, which showed
considerably low Ki values, were chosen and their protein–ligand
merged PDB-format files were selected for total energy calcula-
tion of ligand–receptor interactions. The calculated protein–li-
Figure 7. Docked poses of compound 21 (T-30,50-bisSO2CH2COOH) and 26 [T-30(up),50-bisSO2CH2COOH] with RNase A (1FS3) where cyan, yellow and magenta colored amino
acid residues are of B1, P1 and other subsites respectively within H-bonding interaction.

4640
D. Datta et al. / Bioorg. Med. Chem. 21 (2013) 4634–4645
Table 2
mentally obtained data quite well which in turn, strengthen
our approach towards the design and synthesis of RNase A
inhibitors.
Theoretical total ligand–receptor interaction energy and experimentally obtained
inhibition constants for correlation
Inhibitor/ligand
Ki (lM)
Total ligand–receptor
interaction
energy (kcal/mol)
4. Materials and methods
3 (T-30OCH2COOH)
267
186
169
140
88
25
93
39
108
95
ꢁ5.65
ꢁ6.61
ꢁ5.84
ꢁ8.48
ꢁ8.23
ꢁ9.25
ꢁ6.13
ꢁ9.40
ꢁ6.10
ꢁ7.73
All reagents were commercially purchased. Column chromato-
graphic separations were done using silica gel (60–120 and 230–
400 mesh). Solvents were dried and distilled following standard
procedures. TLC was carried out on precoated plates (Merck silica
gel 60, f254) and the spots were visualized with UV light or by char-
ring the plates dipped in 5% H2SO4 in MeOH, 5% vanillin in MeOH
and 5% ninhydrin in n-butanol solutions. 1H NMR (400 MHz) and
13C NMR (100 MHz) spectra were recorded on Bruker NMR spec-
trometer unless stated otherwise. All 1H NMR in D2O were re-
corded using HPLC grade acetonitrile as internal standard. All 13C
NMR spectra in D2O were recorded using DMSO-d6 or HPLC grade
acetonitrile as internal standard. Chemical shifts are reported in
parts per million (ppm, d scale). DEPT experiments have been car-
ried out to identify the methylene carbons. Mass spectroscopy data
were obtained from Xevo G2QTof mass spectrometer in either ESI+
or ESIꢁ mode. Melting points were determined in open-end capil-
lary tubes and are uncorrected. Bovine pancreatic RNase A, yeast
tRNA, 20, 30-cCMP, 30-CMP and human serum albumin (HSA) were
purchased commercially. UV–Vis measurements were made using
a UV–Vis spectrophotometer (Model Lambda 25). Concentrations
of the solutions were estimated spectrophotometrically using the
8 (T-30-NHCH2COOH)
13 (T-30SCH2COOH)
15 (T-30SO2CH2COOMe)
16 (T-30SO2CH2COOH)
21 (T-30,50-bisSO2CH2COOH)
22 (T-30,50-bisSCH2COOH)
26 [T-30(up),50-bisSO2CH2COOH]
27 [T-30(up),50-bisSCH2COOH]
31 (T-50SO2CH2COOH)
20
15
10
5
R2 = 0.5353
0
following data:
268 = 8500 Mꢁ1 cm-1 (20, 30-cCMP).57
Purity of two most active compounds, 21 and 26 were analyzed
e
278.5 = 9800 Mꢁ1 cmꢁ1 (RNase A)56 and
0
100
200
300
400
e
Inhibition constant values (µM)
for purity on Chiralpak OJ-H equipped reverse phase column
(0.46 ꢀ 15 cm) having flow rate of 1.0 mL/min, k = 254 nm. The
mobile phase was a mixture of isopropanol and hexane. Applied
gradient was 40–60% isopropanol over 15 min using Chiralpak
OJ-H column. These compounds were more than 96% pure.
Figure 8. A correlation graph of ligand–receptor interaction and inhibition constant
values.
gand (here, RNase A and inhibitors of RNase A) interaction values
were obtained from the automated program PEARLS and the data
tabulated (Table 2). A correlation graph has been plotted with the
help of obtained data and corresponding inhibition constants
(Fig. 8). The linear nature of the curve suggests a distinct correla-
tion between theoretically and experimentally obtained data sets.
4.1. Chemistry
4.1.1. 30-Deoxy-30-O-[(carboxymethyl)oxo]-50-O-tritylthymidine
2
Compound 2 was synthesized according to the reported proce-
dure43 with the following modification. Compound 1 (0.43 g,
0.74 mmol) in dimethylsulfoxide (DMSO) (5 mL) was added to
the mixture of NaH (60% in dispersed in mineral oil, 0.036 g,
1 mmol) in DMSO (5 mL) at room temperature under N2 atmo-
sphere which was stirred for 1 h. Bromoethylacetate (0.14 mL,
1.10 mmol) was added to the mixture. After 12 h, another
0.14 mL (1.10 mmol) bromoethylacetate was added to the reaction
mixture and the mixture was stirred for 12 h. Satd aq NH4Cl solu-
tion was added to the reaction mixture and was partitioned and
washed with ethylacetate (EtOAc) (2 ꢀ 50 mL). Combined organic
layers were washed with brine, separated, dried over anhyd
Na2SO4 and filtered. The filtrate was evaporated to dryness under
reduced pressure. The crude residue was dissolved in a mixture
of aq NaOH solution (1 M, 7.5 mL) and tetrahydrofuran (THF)
(10 mL) and the solution was stirred overnight. Volatile matters
were removed under reduced pressure and the residue was neu-
tralized carefully with 1 M HCl. The solution was washed with
dichloromethane (3 ꢀ 50 mL). Combined organic layers were
washed with brine, separated, dried over anhyd Na2SO4 and fil-
tered. The filtrate was concentrated and the residue thus obtained,
was purified over a silica gel column to afford compound 2 (0.22 g,
55% in two steps). [Eluent: 0–10% of MeOH in CHCl3]. Hygroscopic
solid; 1H NMR (CDCl3, 25 °C, TMS): d = 1.15–1.18 ([CH3CH2]3N),
1.43 (s, 3H), 2.24–2.31 (m, 1H), 2.92–2.98 ([CH3CH2]3N), 3.30–
3.37 (m, 2H), 3.93 (br s, 1H), 4.45–4.50 (m, 3H), 6.30–6.33 (m,
3. Conclusion
Since nucleosides functionalized with acidic groups are known
to act as efficient inhibitors of P1 subsite of RNase A,32 we designed
a family of 30-functionalized thymidines carrying XCH2COOH
(X = O, NH, S, SO2) because in this series it would be possible to
manipulate the overall acidity of this new class of nucleic ‘acids’
by changing X from oxygen to the SO2 group. As expected, 30-
deoxy- and 50-deoxy-(30-oxy-, amino- and thio) acetic acid
modified pyrimidine nucleosides were identified as moderate
inhibitors of RNase A. The sulfone acetic acid modified thymidine
compounds, expected to be the most acidic nucleosides in this
series were found to be the more potent inhibitors of RNase A. A
bis-modified thymidine armed with two –SO2CH2COOH groups at
the 30 and 50 sites was found to be the most effective inhibitors
in the series with a Ki value 25
2 lM. It was observed in the dock-
ing studies that the –CH2COOH group of these compounds was
within H-bonding distance of the active site (P1) of the enzyme.
The acyclic nature of XCH2COOH also helped the carboxylic
function to easily reach the target site. Moreover, in the case
of –SO2CH2COOH functionalized thymidines, additional binding
patterns of the oxygen atom(s) of the SO2 group, makes these mol-
ecules more effective inhibitors. All these phenomena lead to suit-
able recognition of inhibitors, thereby enhanced their potency as
initially presumed. Extensive docking studies corroborated experi-

D. Datta et al. / Bioorg. Med. Chem. 21 (2013) 4634–4645
4641
1H), 7.16–7.25 (m, 9H), 7.33–7.35 (m, 6H), 7.48 ppm (s, 1H); 13C
NMR (CDCl3, 25 °C, TMS): d = 8.4 ([CH3CH2]3N), 12.6, 40.9 (CH2),
43.9 (CH2), 45.1 ([CH3CH2]3N), 63.8 (CH2), 71.6, 85.4, 85.8, 87.2
(C), 109.9 (C), 127.2, 127.9, 128.6, 133.9, 143.4 (C), 150.8 (C),
163.3 (C), 172.6 ppm (C); HRMS (ESI+), m/z calcd for (M+H)+
C31H31N2O7: 543.2131, found: 543.2139.
1H), 2.30–2.37 (m, 1H), 3.39–3.51 (m, 3H), 3.81–3.95 (m, 3H),
4.20 (q, J = 14.4 Hz, 2H), 6.12–6.15 (m, 1H), 7.46 (s, 1H), 9.12 ppm
(br s, 1H); 13C NMR (CDCl3, 25 °C, TMS): d = 12.5, 14.1, 38.5
(CH2), 49.0 (CH2), 57.2, 61.2 (CH2), 62.4 (CH2), 85.2, 86.0, 110.7
(C), 136.5, 150.3 (C), 163.9 (C), 172.3 ppm (C); HRMS (ESI+), m/z
calcd for (M+H)+ C14H22N3O6: 328.1508, found: 328.1517.
4.1.2. 30-Deoxy-30-O-[(carboxymethyl)oxo]thymidine
4.1.6. 30-Deoxy-30-N-[(carboxymethyl)amino)]thymidine
(T-30NHCH2COOH) 8
(T-30OCH2COOH) 3
Compound 2 (0.25 g, 0.46 mmol) was stirred with trifluoroace-
tic acid (TFA) in DCM (80%, 5 mL) at room temperature. After 3 h,
the volatile matters were evaporated to dryness under reduced
pressure and residual liquid was co-evaporated with toluene
(2 ꢀ 5 mL). The residue, thus obtained was dissolved in MeOH
(2–3 mL), loaded onto the silica gel column, and purified to obtain
compound 3 (0.96 g, 69%) [Eluent: 5–20% of MeOH in CHCl3].
Hygroscopic solid; 1H NMR (D2O, 25 °C): d = 1.92 (s, 3H), 2.38–
2.40 (m, 2H), 3.74–3.86 (m, 2H), 4.02 (s, 1H), 4.45–4.51 (m, 3H),
6.30 (t, J = 6.4 Hz, 1H), 7.70 ppm (s, 1H); 13C NMR (D2O, 25 °C):
d = 13.9, 40.4 (CH2), 45.6 (CH2), 62.6 (CH2), 71.8, 87.3, 88.1, 111.7
(C), 137.2, 152.8 (C), 166.1 (C), 174.8 ppm (C); HRMS (ESI+), m/z
calcd for (M+Na)+ C12H16N2O7Na: 323.0856, found: 323.0861.
Compound 6 (0.40 g, 0.74 mmol) was converted to compound 8
(0.16 g, 72%) following the method described for compound 3
[Eluent: 5–20% of MeOH in CHCl3]. Hygroscopic solid; 1H NMR
(DMSO-d6, 25 °C): d = 1.77 (s, 3H), 2.12–2.17 (m, 2H), 3.25 (s,
2H), 3.41–3.42 (m, 1H), 3.54–3.65 (m, 2H), 3.80–3.81 (m, 1H),
6.15 (t, J = 6.4 Hz, 1H), 7.73 (s, 1H), 11.28 ppm (br s, 1H); 13C
NMR (DMSO-d6, 25 °C): d = 12.7, 36.9 (CH2), 49.1 (CH2), 57.7, 62.0
(CH2), 84.1, 84.6, 109.7 (C), 136.6, 150.9 (C), 164.2 (C), 174.5 ppm
(C); HRMS (ESI+), m/z calcd for (M+H)+ C12H18N3O6: 300.1195,
found: 300.1190.
4.1.7. 30-Deoxy-30-S-[(methoxycarbonyl)methylthio]-50-O-
tritylthymidine 10
To a suspension of NaH (60% in dispersed in mineral oil, 0.19 g,
8 mmol) in DMF (10 mL), methyl 2-mercaptoacetate (0.64 mL,
7 mmol) was added dropwisely at 0 °C under argon. The suspen-
sion was allowed to warm at room temperature while the solu-
tion turned yellow. The reaction mixture was cooled to 0 °C and
compound 945 (1.12 g, 2 mmol) in DMF (5 mL) was added to it.
After 6 h, Satd aq NH4Cl solution was added to the reaction mix-
ture and was partitioned and washed with EtOAc (2 ꢀ 50 mL).
Combined organic layers were washed with brine, separated,
dried over anhyd Na2SO4 and filtered. The filtrate was removed
under reduced pressure and the residue was purified over a silica
gel column to afford compound 10 (0.74 g, 66%) [Eluent: 50–60%
of EtOAc in pet ether]. White solid; mp 60–63 °C; 1H NMR (CDCl3,
25 °C, TMS): d = 1.44 (s, 3H), 2.41–2.49 (m, 1H), 2.57–2.63 (m,
1H), 3.17 (s, 2H), 3.34–3.42 (m, 1H), 3.59–3.68 (m, 4H), 3.84–
3.88 (m, 1H), 3.90–3.98 (m, 1H), 6.19–6.21 (m, 1H), 7.27–7.34
(m, 9H), 7.38–7.43 (m, 6H), 7.70 (s, 1H), 8.25 ppm (s, 1H); 13C
NMR (CDCl3, 25 °C, TMS): d = 11.9, 32.9 (CH2), 40.1 (CH2), 41.3,
52.6, 62.2 (CH2), 84.6, 84.9, 87.2(C), 110.9 (C), 127.4, 127.9,
128.8, 135.4, 143.2 (C), 150.2 (C), 163.8 (C), 170.2 ppm (C); HRMS
(ESI+), m/z calcd for (M+Na)+ C32H32N2O6SNa: 595.1879, found:
595.1887.
4.1.3. 30-Deoxy-30-N-[(ethoxycarbonyl)methylamino]-50-O-
tritylthymidine 5
To a suspension of compound 4 (1.22 g, 2.52 mmol) in DMF
(30 mL), diisopropylethylamine (1.32 mL, 7.56 mmol) was added
under N2 atmosphere at room temperature. Bromoethylacetate
was added in portions (0.1 ꢀ 10 equiv) to the reaction mixture over
a period of 40 h.47 Satd aq NaHCO3 solution was added to the reac-
tion mixture and the aq solution was washed with EtOAc
(2 ꢀ 50 mL). Combined organic layers were washed with brine,
dried over anhyd Na2SO4 and filtered. The filtrate was removed un-
der reduced pressure and the residue was purified over a silica gel
column to afford compound 5 (1.32 g, 92%) [Eluent: 50–60% of
EtOAc in pet ether]. White solid; mp 121–124 °C; 1H NMR (CDCl3,
25 °C, TMS): d = 1.24 (t, J = 7.2 Hz, 3H), 1.50 (s, 3H), 2.26–2.31 (m,
2H), 3.30–3.34 (m, 3H), 3.49–3.52 (m, 1H), 3.56–3.61 (m, 1H),
3.91–3.92 (m, 1H), 4.13–4.19 (m, 1H), 6.27 (t, J = 6.0 Hz, 1H),
7.24–7.33 (m, 9H), 7.42–7.44 (m, 6H), 7.59 ppm (s, 1H); 13C NMR
(CDCl3, 25 °C, TMS): d = 11.9, 14.1, 38.9 (CH2), 48.9 (CH2), 57.5,
61.0 (CH2), 63.6 (CH2), 84.6, 84.8, 87.3(C), 110.8 (C), 127.3, 127.9,
128.6, 135.5, 143.3 (C), 150.3 (C), 163.9 (C), 171.9 ppm (C); HRMS
(ESI+), m/z calcd for (M+H)+ C33H36N3O6: 570.2604, found:
570.2615.
4.1.8. 30-Deoxy-30-S-[(carboxymethyl)thio]-50-O-tritylthymidine
11
4.1.4. 30-Deoxy-30-N-[(carboxymethyl)amino]-50-O-
tritylthymidine 6
Compound 10 (0.30 g, 0.52 mmol) was converted to compound
11 (0.22 g, 74%) following the method described for compound 2
[Eluent: 0–8% of MeOH in CHCl3]. White solid; mp 185 °C
(decompose); 1H NMR (CDCl3, 25 °C, TMS): d = 1.43 (s, 3H),
2.47–2.54 (m, 1H), 2.60–2.66 (m, 1H), 3.19 (s, 2H), 3.37–3.41
(m, 1H), 3.59–3.62 (m, 1H), 3.81–3.85 (m, 1H), 3.95–3.97 (m,
1H), 6.12–6.14 (m, 1H), 7.24–7.33 (m, 9H), 7.42–7.43 (m, 6H),
7.75 (s, 1H), 9.58 ppm (br s, 1H); 13C NMR (CDCl3, 25 °C, TMS):
d = 11.9, 33.3 (CH2), 40.4 (CH2), 41.6, 62.2 (CH2), 85.0, 85.1, 87.4
(C), 110.7 (C), 127.5, 128.1, 128.7, 136.2, 143.2 (C), 150.4 (C),
165.0 (C), 174.2 ppm (C); HRMS (ESI+), m/z calcd for (M+H)+
C31H31N2O6S: 559.1903, found: 559.1911.
Compound 5 (0.76 g, 1.33 mmol) was converted to compound 6
(0.53 g, 77%) following the method described for compound 2 [Elu-
ent: 2–10% of MeOH in CHCl3]. White solid; mp 162–165 °C; 1H
NMR (DMSO-d6, 25 °C): d = 1.45 (s, 3H), 2.24 (br s, 2H), 3.18–3.27
(m, 4H), 3.56–3.57 (m, 1H), 3.94 (br s, 1H), 6.20 (t, J = 6.0 Hz, 1H),
7.25–7.28 (m, 3H), 7.31–7.35 (m, 6H), 7.39–7.50 (m, 6H), 7.50 (s,
1H), 11.35 ppm (br s, 1H); 13C NMR (DMSO-d6, 25 °C): d = 12.2,
36.9 (CH2), 49.7 (CH2), 58.0, 64.8 (CH2), 83.1, 84.2, 86.7(C), 109.9
(C), 127.5, 128.4, 128.7, 136.1, 143.9 (C), 150.8 (C), 164.1 (C),
173.0 ppm (C); HRMS (ESI+), m/z calcd for (M+Na)+ C31H31N3O6Na:
564.2111, found: 564.2118.
4.1.5. 30-Deoxy-30-N-[(ethoxycarbonyl)methylamino]thymidine
(T-30NHCH2COOEt) 7
4.1.9. 30-Deoxy-30-S-[(methoxycarbonyl)methylthio]thymidine
(T-30SCH2COOMe) 12
Compound 5 (0.30 g, 0.53 mmol) was converted to compound 7
(0.14 g, 80%) following the method described for compound 3 [Elu-
ent: 0–3% of MeOH in CHCl3]. Hygroscopic solid; 1H NMR (CDCl3,
25 °C, TMS): d = 1.27 (t, J = 7.2 Hz, 3H), 1.89 (s, 3H), 2.18–2.25 (m,
Compound 10 (0.46 g, 0.8 mmol) was converted to compound
12 (0.21 g, 79%) following the method described for compound 3
[Eluent: 0–4% of MeOH in CHCl3]. Hygroscopic solid; 1H NMR
(CDCl3, 25 °C, TMS): d = 1.86 (s, 3H), 2.36–2.43 (m, 1H), 2.54–2.56

4642
D. Datta et al. / Bioorg. Med. Chem. 21 (2013) 4634–4645
(m, 1H), 3.34 (s, 2H), 3.59–3.65 (m, 1H), 3.73 (s, 3H), 3.78–3.89 (m,
2H), 3.97–4.00 (m, 1H), 6.09 (br s, 1H), 7.65 (s, 1H), 9.69 ppm (s,
1H); 13C NMR (CDCl3, 25 °C, TMS): d = 12.4, 33.2 (CH2), 39.5
(CH2), 41.1, 52.8, 60.9 (CH2), 85.3, 85.9, 110.6 (C), 136.6, 150.5
(C), 164.3 (C), 170.9 ppm (C); HRMS (ESI+), m/z calcd for (M+H)+
C13H19N2O6S: 331.0964, found: 331.0973.
and the solution thus obtained, was washed with EtOAc
(3 ꢀ 15 mL). Combined organic layers were separated, dried over
anhyd Na2SO4 and filtered. The filtrate was concentrated under re-
duced pressure. Crude solid thus obtained, was converted to com-
pound 16 (0.032 g, 55%) following the method described for
compound 15. White solid; mp 210–215 °C; 1H NMR (DMSO-d6,
25 °C): d = 1.76 (s, 3H), 2.34–2.42 (m, 1H), 2.67–2.72 (m, 1H),
3.55–3.58 (m, 1H), 3.71–3.74 (m, 1H), 4.25–4.28 (m, 1H), 4.39–
4.49 (m, 3H), 6.08–6.12 (m, 1H), 7.72 (s, 1H), 11.33 ppm (s, 1H);
13C NMR (DMSO-d6, 25 °C): d = 12.6, 32.1 (CH2), 56.7 (CH2), 61.7,
62.8 (CH2), 78.2, 84.3, 110.1 (C), 136.3, 150.8 (C), 164.1 (C),
164.6 ppm (C); HRMS (ESI+), m/z calcd for (M+H)+ C12H16N2O8S:
349.0706, found: 349.0719.
4.1.10. 30-Deoxy-30-S-[(carboxymethyl)thio]thymidine
(T-30SCH2COOH) 13
Compound 11 (0.22 g, 0.39 mmol) was converted to compound
13 (0.08 g, 62%) following the method described for compound 3
[Eluent: 5–20% of MeOH in CHCl3]. Hygroscopic solid; 1H NMR
(DMSO-d6, 25 °C): d = 1.76 (s, 3H), 2.22–2.29 (m, 1H), 2.34–2.41
(m, 1H), 3.27 (s, 2H), 3.51–3.63 (m, 1H), 3.67–3.70 (m, 1H), 3.78–
3.80 (m, 1H), 6.04 (t, J = 5.6 Hz, 1H), 7.84 ppm (s, 1H); 13C NMR
(DMSO-d6, 25 °C): d = 12.6, 35.5 (CH2), 38.9 (CH2), 41.6, 61.1
(CH2), 84.0, 86.0, 109.4 (C), 136.7, 150.8 (C), 164.2 (C), 173.9 ppm
(C); HRMS (ESI+), m/z calcd for (M+Na)+ C12H16N2O6S: 339.0627,
found: 339.0626.
4.1.14. 30,50-Dideoxy-30,50-bis-S-[(ethoxycarbonyl)
methylthio]thymidine 18
To a solution of compound 1748 (0.4 g, 1.45 mmol) in dry DCM
(5 mL), TEA (0.48 mL, 3.5 mmol) was added at 0 °C. To the result-
ing mixture, ethylbromoacetate (0.41 mL, 3.5 mmol) was added
slowly. The reaction mixture was quenched with cold water after
0.4 h and the aq layer was washed with DCM (2 ꢀ 20 mL). Organ-
ic layers were separated, dried over anhyd CaCl2, filtered and the
filtrate was concentrated under reduced pressure. Crude product
thus obtained, was purified over column to afford compound 18
(0.39 g, 70%) [Eluent: 40–60% of EtOAc in pet ether]. Transparent yel-
low gum; 1H NMR (200 MHz, CDCl3, 25 °C, TMS): d = 1.25–1.33 (m,
6H), 1.95 (s, 3H), 2.45–2.52 (m, 2H), 3.00–3.24 (m, 2H), 3.25–3.42 (m,
4H), 3.46–3.58 (m, 1H), 4.01–4.09 (m, 1H), 4.14–4.26 (m, 4H) 6.15–
6.21 (m, 1H), 7.43 (s, 1H), 9.26 ppm (s, 1H); 13C NMR (50 MHz, CDCl3,
25 °C, TMS): d = 12.7, 14.3, 33.6 (CH2), 34.6 (CH2), 34.7 (CH2), 40.0
(CH2), 44.9, 61.7(CH2), 61.9(CH2), 84.3, 84.6, 111.5 (C), 135.6, 150.4(C),
163.9 (C), 170.1 (C), 170.3 ppm (C); HRMS (ESI+), m/z calcd for
(M+Na)+ C18H26N2O7S2Na: 469.1079, found: 469.1097.
4.1.11. 30-Deoxy-30-S-[(methoxycarbonyl)methylsulfonyl]-50-O-
tritylthymidine 14
MMPP (0.65 g, 1.32 mmol) was added to a solution of 10 (0.25 g,
0.44 mmol) in anhyd MeOH and the mixture was stirred at room
temperature and N2 atmosphere. After 8 h, this reaction mixture
was concentrated, treated with Satd aq NaHCO3 and the solution
was washed with EtOAc (2 ꢀ 25 mL). Organic layers were sepa-
rated, dried over anhyd Na2SO4, filtered and the filtrate was con-
centrated under reduced pressure. The residue thus obtained was
purified over silica gel column to afford compound 14 (0.2 g,
76%) [Eluent: 70% of EtOAc in pet ether].White solid; mp 108–
112 °C; 1H NMR (CDCl3, 25 °C, TMS): d = 1.47 (s, 3H), 2.55–2.59
(m, 1H), 3.00–3.06 (m, 1H), 3.45–3.48 (m, 1H), 3.62–3.65 (m,
1H), 3.76 (s, 3H), 3.96 (d, J = 14.8 Hz, 1H), 4.08 (d, J = 15.2 Hz,
1H), 4.63–4.66(m, 2H), 6.25–6.28 (m, 1H), 7.25–7.47(m, 15H),
7.56 (s, 1H). 9.29 ppm (br s, 1H); 13C NMR (CDCl3, 25 °C, TMS):
d = 12.0, 34.1 (CH2), 53.7, 56.9 (CH2), 60.8, 64.0 (CH2), 77.6, 85.2,
87.8 (C), 111.7 (C), 127.7, 128.3, 128.8, 135.3, 143.3 (C), 150.3
(C), 163.3 (C), 164.0 ppm (C); HRMS (ESI+), m/z calcd for (M+Na)+
C32H32N2O8S: 627.1777, found: 627.1770.
4.1.15. 30,50-Dideoxy-30,50-bis-S-
[(methoxycarbonyl)methylthio]thymidine (T-30,50-
bisSCH2COOMe) 19
A mixture of compound 18 (0.13 g, 0.29 mmol) and dibutyltin
oxide (0.014 g, 0.058 mmol) in MeOH was heated under reflux
for 12 h. After completion of reaction (TLC), MeOH was removed
under reduced pressure to obtain a crude mass which was purified
over silica gel column to obtain 19 (0.1 g, 80%) [Eluent: 50–80% of
EtOAc in pet ether]. Colorless gum; 1H NMR (CDCl3, 25 °C, TMS):
d = 1.92 (s, 3H), 2.43–2.47 (m, 2H), 3.02 (dd, J = 5.2, 14.4 Hz, 1H),
3.15 (dd, J = 3.6, 14.4 Hz, 1H), 3.28–3.38 (m, 4H), 3.45–3.52 (m,
1H), 3.71 (s, 3H), 3.73 (s, 3H), 3.99–4.04 (m, 1H), 6.13–6.16 (m,
1H), 7.38 (s, 1H), 9.47 ppm (br s, 1H); 13C NMR (CDCl3, 25 °C,
TMS): d = 12.7, 33.3 (CH2), 34.3 (CH2), 34.7 (CH2), 39.9 (CH2),
45.0, 52.7, 52.9, 84.3, 84.7, 111.5 (C), 135.6, 150.5 (C), 164.1 (C),
170.6 (C), 170.7 ppm (C); HRMS (ESI+), m/z calcd for (M+H)+
C16H23N2O7S2: 419.0947, found: 419.0939.
4.1.12. 30-Deoxy-30-S-[(methoxycarbonyl)
methylsulfonyl]thymidine (T-30SO2CH2COOMe) 15
Compound 14 (0.1 g, 0.17 mmol) was stirred with TFA in DCM
(20%, 15 mL) at room temperature. After 0.5 h, the volatile matters
were evaporated to dryness under reduced pressure and residual
liquid was co-evaporated with the mixture of toluene and carbon
tetrachloride. The residue was triturated well with chilled diethyl-
ether (Et2O) to obtain solid compound 15 (0.059 g, 93%). White so-
lid; mp 195–198 °C; 1H NMR (DMSO-d6, 25 °C): d = 1.78 (s, 3H),
2.38–2.46 (m, 1H), 2.69–2.74 (m, 1H), 3.58–3.61 (m, 1H), 3.73–
3.76 (m, 4H), 4.26–4.28 (m, 1H), 4.43(br s, 1H), 4.62–4.66 (m,
2H), 6.11–6.15 (m, 1H), 7.74 (s, 1H), 11.36 ppm (s, 1H); 13C NMR
(DMSO-d6, 25 °C): d = 12.6, 32.1 (CH2), 53.3, 56.2 (CH2), 61.9, 62.7
(CH2), 78.2, 84.3, 110.1 (C), 136.3, 150.8 (C), 163.8 (C), 164.1 ppm
(C); HRMS (ESI+), m/z calcd for (M+H)+ C13H18N2O8S: 363.0862,
found: 363.0867.
4.1.16. 30,50-Dideoxy-30,50-bis-S-[(methoxycarbonyl)
methylsulfonyl]cthymidine (T-30,50-bisSO2CH2COOMe) 20
Compound 19 (0.6 g, 1.24 mmol) was converted to compound
20 (0.53 g, 77%) following the method described for compound
14. [Eluent: 6% of MeOH in dichloromethane].White solid; mp
76–80 °C; 1H NMR (DMSO-d6, 25 °C): d = 1.78 (s, 3H), 2.59–2.67
(m, 1H), 2.72–2.78 (m, 1H), 3.64–3.73 (m, 4H), 3.77 (s, 3H),
4.07–4.13 (m, 1H), 4.41 (s, 2H), 4.43–4.48 (m, 1H), 4.61–4.71
(m, 2H), 4.75–4.78 (m, 1H), 6.08–6.11 (m, 1H), 7.56 (s, 1H),
11.39 ppm (s, 1H); 13C NMR (DMSO-d6, 25 °C): d = 12.6, 31.2
(CH2), 53.3, 53.6, 57.0 (CH2), 57.7 (CH2), 58.7 (CH2), 64.4, 72.3,
86.4, 110.5 (C), 137.3, 150.9 (C), 163.7 (C), 164.1 (C), 164.3 ppm
(C); HRMS (ESI+), m/z calcd for (M+H)+ C16H23N2O11S2:
483.0743, found: 483.0757.
4.1.13. 30-Deoxy-30-S-[(carboxymethyl)sulfonyl]thymidine
(T-30SO2CH2COOH) 16
LiOHꢂH2O (0.03 g, 0.68 mmol) and water (4 mL) were added to a
solution of compound 14 (0.1 g, 0.17 mmol) in THF (16 mL). The
reaction mixture was stirred for 1 h and volatile matters were re-
moved under reduced pressure. Solid NaHSO4ꢂH2O (0.1 g) was
added in portions to the residue and the mixture was stirred until
the pH reached 7. The residue was diluted with water (10 mL)

D. Datta et al. / Bioorg. Med. Chem. 21 (2013) 4634–4645
4643
4.1.17. 30,50-Dideoxy-30,50-bis-S-[(carboxymethyl)sulfonyl]
thymidine (T-30,50-bisSO2CH2COOH) 21
25 °C): d = 12.8, 31.9 (CH2), 53.1, 53.4, 54.3 (CH2), 57.8 (CH2),
58.6 (CH2), 62.2, 72.1, 83.2, 110.3 (C), 136.1, 150.7 (C), 163.5 (C),
164.0 ppm (2 C); HRMS (ESIꢁ), m/z calcd for (MꢁH)ꢁ
C16H21N2O11S2: 481.0587, found: 481.0564.
Compound 20 (0.05 g, 0.11 mmol) and trimethyltin hydroxide
(TMTOH) (0.098 g, 0.55 mmol) dissolved in 1,2-dichloroethane
(DCE) was heated at 70 °C for 6 h. After completion of reaction,
volatile matters were evaporated under reduced pressure and
the crude mass thus obtained, was purified over silica gel column
to obtain compound 21 (0.035 g, 75%) [Eluent: 30–80% of MeOH
in chloroform]. Yellowish white hygroscopic solid; 1H NMR
(D2O, 25 °C, water suppressed): d = 1.91 (s, 3H), 2.73–2.81 (m,
1H), 2.92–2.99 (m, 1H), 3.86–3.89 (m, 1H), 4.09–4.23 (m, 5H),
4.56–4.62 (m, 1H), 4.99–5.04 (m, 1H), 6.16–6.19 (m, 1H),
7.54 ppm (s, 1H); 13C NMR (DMSO-d6, 25 °C): d = 12.6, 31.3
(CH2), 57.1 (CH2), 57.9 (CH2), 58.7 (CH2), 64.5, 72.3, 86.4, 110.6
(C), 137.3, 150.8 (C), 163.1 (C), 163.6 (C), 164.3 ppm (C); HRMS
(ESIꢁ), m/z calcd for (MꢁH)ꢁ C14H17N2O11S2: 453.0274, found:
453.0252.
4.1.21. 1-[30,50-Bis-S-(carboxymethyl)-20-deoxy-30,50-disulfonyl-
b-D
-threo-pentofuranosyl]thymine [T-30(up),50-bisSO2CH2COOH]
26
Compound 25 (0.05 g, 0.11 mmol) was converted to compound
26 (0.035 g, 75%) following the method described for compound 21
[Eluent: 30–80% of MeOH in chloroform]. Yellow hygroscopic so-
lid; 1H NMR (D2O, 25 °C, water suppressed): d = 1.92 (s, 3H),
2.54–2.60 (m, 1H), 3.03–3.11 (m, 1H), 4.05–4.25 (m, 5H), 4.41–
4.47 (m, 1H), 4.58–4.60 (m, 1H), 4.93–4.98 (m, 1H), 6.25–6.28
(m, 1H), 7.70 ppm (s, 1H); 13C NMR (DMSO-d6): d 12.7, 32.0
(CH2), 54.5 (CH2), 57.9 (CH2), 58.7 (CH2), 62.3, 72.1, 83.2, 110.3
(C), 136.0, 150.7 (C), 162.9 (C), 163.4 (C), 164.0 ppm (C); HRMS
(ESIꢁ), m/z calcd for (MꢁH)ꢁ C14H17N2O11S2: 453.0274, found:
453.0296.
4.1.18. 30,50-Dideoxy-30,50-bis-S-[(carboxymethyl)thio]
thymidine (T-30,50-bisSCH2COOH) 22
4.1.22. 1-[30,50-Bis-S-(carboxymethyl)-20-deoxy-30,50-dithio-b-
threo-pentofuranosyl]thymine [T-30(up),50-bisSCH2COOH] 27
D-
Compound 18 (0.091 g, 0.22 mmol) was dissolved in a mixture
of aq NaOH solution (1 M, 2.5 mL) and THF (5 mL) and this solu-
tion was stirred at room temperature for 2 h. Volatile matters
were removed under reduced pressure and the solution was neu-
tralized with 1 M HCl solution. The resulting solution was evapo-
rated to dryness under reduced pressure and the crude mass thus
obtained, was purified over silica gel column to obtain compound
22 (0.048 g, 61%) [Eluent: 5–80% of MeOH in chloroform]. Hygro-
scopic yellowish white solid; 1H NMR (D2O, 25 °C): d = 1.90 (s,
3H), 2.49–2.55 (m, 1H), 2.61–2.66 (m, 1H), 2.95 (dd, J = 6.4,
14.4 Hz, 1H), 3.10 (dd, J = 3.6, 14.4 Hz, 1H), 3.30–3.35 (m, 4H),
3.47–3.54 (m, 1H), 4.10–4.15 (m, 1H), 6.20–6.23 (m, 1H),
7.59 ppm (s, 1H); 13C NMR (D2O, 25 °C): d = 12.2, 35.0 (CH2),
36.3 (CH2), 37.4 (CH2), 38.5 (CH2), 45.3, 84.6, 85.4, 112.2 (C),
138.2, 152.3 (C), 167.1 (C), 177.3 (C), 177.6 ppm (C); HRMS
(ESI+), m/z calcd for (M+Na)+ C14H18N2O7S2Na: 413.0453, found:
413.0457.
Compound 24 (0.1 g, 0.24 mmol) was converted to compound 27
(0.056 g, 60%) following the method described for compound 22
[Eluent: 5–80% of MeOH in chloroform]. Yellow solid; mp 115–
120 °C; 1H NMR (D2O, 25 °C): d = 2.20 (s, 3H), 2.36–2.44 (m, 1H),
2.99–3.06 (m, 1H), 3.15–3.17 (m, 2H), 3.38–3.44 (m, 4H), 3.83–
3.87 (m, 1H), 4.60–4.65 (m, 1H), 6.24–6.27 (m, 1H), 7.96 ppm (s,
1H); 13C NMR (D2O, 25 °C): d = 11.8, 33.4 (CH2), 37.1 (CH2), 37.4
(CH2), 38.1 (CH2), 45.7, 81.2, 84.8, 111.2 (C), 137.9, 151.9 (C), 166.8
(C), 177.6 (C), 177.9 ppm (C); HRMS (ESI+), m/z calcd for (M+Na)+
C14H18N2O7S2Na: 413.0453, found: 413.0462.
4.1.23. 50-Deoxy-50-S-[(methoxycarbonyl)methylthio]thymidine
29
Compound 2833 (1.5 g, 3.8 mmol) was converted to compound
2953 (0.95 g, 76%) following the method described for compound
10. [Eluent: 0–3% of MeOH in CHCl3]. White solid; mp 117–
120 °C; 1H NMR (DMSO-d6, 25 °C): d = 1.78 (s, 3H), 2.01–2.06 (m,
1H), 2.16–2.21 (m, 1H), 2.80–2.91 (m, 2H), 3.41 (q, J = 20.0 Hz,
2H), 3.61 (s, 3H), 3.81–3.85 (m, 1H), 4.13–4.16 (m, 1H), 5.35 (d,
J = 4.4 Hz, 1H), 6.12–6.16 (m, 1H), 7.45 (s, 1H), 11.29 ppm (br s,
1H); 13C NMR (DMSO-d6, 25 °C): d = 12.6, 33.7 (CH2), 34.7 (CH2),
38.5 (CH2), 52.5, 72.9, 84.2, 85.7, 110.2 (C), 136.5, 150.9 (C),
164.1 (C), 170.9 ppm (C); HRMS (ESI+), m/z calcd for (M+Na)+
C13H18N2O6SNa: 353.0783, found: 353.0784.
4.1.19. 1-[30,50-Bis-S-(methoxycarbonyl)methyl-20-deoxy-30,50-
dithio-b-D
-threo-pentofuranosyl]thymine [T-30(up),50-
bisSCH2COOMe] 24
Compound 2349 (1.1 g, 2 mmol) was converted to compound 24
(0.64 g, 76%) following the method described for compound 10.
[Eluent: 40–60% of EtOAc in pet ether]. Transparent gum; 1H
NMR (CDCl3, 25 °C, TMS): d = 1.86 (s, 3H), 2.03–2.09 (m, 1H),
2.77–2.84 (m, 1H), 2.94–3.04 (m, 2H), 3.21–3.35 (m, 4H), 3.64–
3.67 (s, 7H), 4.30–4.35 (m, 1H), 6.02–6.05 (m, 1H), 7.51 (s, 1H),
9.92 ppm (s, 1H); 13C NMR (CDCl3, 25 °C, TMS): d = 12.4, 33.6
(CH2), 33.7(CH2), 33.8 (CH2), 39.3 (CH2), 46.2, 52.4, 52.6, 81.3,
84.1, 110.8 (C), 135.6, 150.6 (C), 164.1 (C), 170.2 (C), 170.6 ppm
(C); HRMS (ESI+), m/z calcd for (M+Na)+ C16H22N2O7S2Na:
441.0766, found: 444.0757.
4.1.24. 50-Deoxy-50-S-
[(methoxycarbonyl)methylsulfonyl]thymidine
(T-50SO2CH2COOMe) 30
Compound 29 (0.9 g, 2.75 mmol) was converted to compound
30 (0.77 g, 70%) following the method described for compound
14. [Eluent: 2–8% of MeOH in CHCl3]. White solid; mp 195–
200 °C; 1H NMR (DMSO-d6, 25 °C): d = 1.78 (s, 3H), 2.04–2.09 (m,
1H), 2.22–2.29 (m, 1H), 3.60–3.85 (m, 4H), 3.77–3.85 (m, 1H),
4.09–4.11 (m, 1H), 4.24–4.27 (m, 1H), 4.31–4.39 (m, 2H), 5.52–
5.53 (m, 1H), 6.15–6.19 (m, 1H), 7.50 (s, 1H), 11.32 ppm (s, 1H);
13C NMR (DMSO-d6, 25 °C): d = 12.4, 37.7 (CH2), 53.1, 56.6 (CH2),
58.4 (CH2), 73.2, 80.3, 85.1, 110.2 (C), 136.9, 150.8 (C), 163.9 (C),
164.1 ppm (C); HRMS (ESI+), m/z calcd for (M+H)+ C13H19N2O8S:
363.0862, found: 363.0882.
4.1.20. 1-[30,50-Bis-S-(methoxycarbonyl)methyl-20-deoxy-30,50-
disulfonyl-b-D
-threo-pentofuranosyl]thymine [T-30(up),50-
bisSO2CH2COOMe] 25
Compound 24 (0.6 g, 1.24 mmol) was converted to compound
25 (0.48 g, 70%) following the method described for compound
14 [Eluent: 6% of MeOH in dichloromethane].White solid; mp
97–100 °C. 1H NMR (DMSO-d6, 25 °C): d = 1.76 (s, 3H), 2.41–2.46
(m, 1H), 2.77–2.85 (m, 1H), 3.66 (s, 3H), 3.72 (s, 3H), 3.86–3.89
(m, 1H), 4.12–4.19 (m, 1H), 4.37–4.51 (m, 3H), 4.55 (d,
J = 14.8 Hz, 1H), 4.72 (d, J = 15.2 Hz, 1H), 4.76–4.80 (m, 1H), 6.15–
6.18 (m, 1H), 7.54 (s, 1H), 11.42 ppm (s, 1H); 13C NMR (DMSO-d6,
4.1.25. 50-Deoxy-50-S-[(carboxymethyl)sulfonyl]thymidine (T-
50SO2CH2COOH) 31
Compound 30 (0.032 g, 0.09 mmol) was converted to com-
pound 31 (0.025 g, 81%) following the method described for com-

4644
D. Datta et al. / Bioorg. Med. Chem. 21 (2013) 4634–4645
pound 21. [Eluent: 20–80% of MeOH in CHCl3].White hygroscopic
solid; 1H NMR (DMSO-d6, 25 °C): d = 1.21 (s, 3H), 2.02–2.15 (m,
2H), 3.54–3.57 (m, 2H), 3.71–3.75 (m, 1H), 4.02–4.08 (m, 1H),
4.13–4.16 (m, 1H), 4.23–4.24 (m, 1H), 5.66 (br s, 1H), 6.13–6.17
(m, 1H), 7.65 (s, 1H), 11.30 ppm (br s, 1H); 13C NMR (DMSO-d6,
25 °C): d = 11.9, 37.8 (CH2), 54.5 (CH2), 61.8 (CH2), 72.9, 80.7,
84.5, 109.9 (C), 136.3, 150.5 (C), 163.8 (C), 164.0 ppm (C);. HRMS
(ESI+), m/z calcd for (M+H)+ C12H17N2O8S: 349.0706, found:
349.0699.
aver–Burk plot. For the Lineweaver–Burk plot the reciprocal of
initial velocity was plotted against the reciprocal of substrate con-
centration at a constant inhibitor concentration according to the
following equation:
ꢀ
ꢁ
ꢀ
ꢁ
1
v
Km
Vmax
½Iꢃ
1
1
½Iꢃ
¼
1 þ
þ
1 þ
Ki ½Sꢃ Vmax
Ki
Here
v is the initial velocity, [S] the substrate concentration, [I]
the inhibitor concentration, Km the Michaelis constant, Ki the inhi-
bition constant, and Vmax the maximum velocity. The kinetics
experiments were performed with two fixed inhibitor concentra-
tions and another in absence of inhibitor with varying substrate
(20,30-cCMP) concentrations. The slopes from the double reciprocal
plot were again plotted against the corresponding inhibitor con-
centrations to get inhibition constants (Ki).
4.2. Biophysical studies
4.2.1. Agarose gel electrophoresis assay
(i) Inhibition of RNase A was assayed qualitatively by the deg-
radation of tRNA in an agarose gel. In this method, 20 lL
of RNase A (1.51
(0.14 mM) and 60
l
l
M) was mixed with 20 (0.07 mM), 40
L (0.20 mM) of compounds 3, 7, 8, 12,
5. Docking studies
13, 15, 19, 20, 24, 25 and 30 to a final volume of 100
and the resulting solutions incubated for 3 h. 20 L aliquots
of the incubated mixtures were then mixed with 20 L of
tRNA solution (5.0 mg/mL tRNA, freshly dissolved in RNase
free water) and incubated for another 30 min. Then 10
of sample buffer (containing 10% glycerol and 0.025% bro-
mophenol blue) was added to this mixture and 15 L from
each solution was taken and loaded into a 1.1% agarose
gel. The gel was run using 0.04 M Tris–Acetic acid–EDTA
(TAE) buffer (pH 8.0). The residual tRNA was visualized by
ethidium bromide staining under UV light.
lL
The crystal structures of the protein 1FS3 (PDB entry for RNase
A) was downloaded from the Protein Data Bank.59 Water mole-
cules and other ions present in the crystal structures were sub-
tracted to prepare the protein PDB file for docking. The 3D
structures of compounds 3, 7, 8, 12, 13, 15, 16, 20–22, 25–27,
30 and 31 were generated in Sybyl6.92 (Tripos Inc., St. Louis,
USA) and their energy-minimized conformations were obtained
with the help of the MMFF94 force field using MMFF94 charges
with a gradient of 0.005 kcal/mol by 1000 iterations with all other
default parameters. The FlexX software as part of the Sybyl suite
was used for docking of the ligands to the protein. The ranking
of the generated solutions was performed using a scoring func-
l
l
lL
l
(ii) Inhibition of RNase A by compounds 16, 21, 22, 26, 27 and
31 were assayed qualitatively by the degradation of tRNA
tion that estimates the free binding energy (DG) of the protein–li-
individually. In this method, 20
was mixed with 20 (0.06 mM), 40 (0.12 mM), 60
(0.18 mM) and 80 L (0.24 mM) of compounds 16, 22, 26
and 31 separately to a final volume of 100 L and the result-
ing solutions were incubated for 3 h. Again, 20 L of RNase A
(2.00 M) was mixed with 20 (0.05 mM), 40 (0.10 mM), 60
(0.15 mM) and 80 (0.20 mM) L of compounds 21 and 26
separately to a final volume of 100 L. Solutions were incu-
lL of RNase A (1.26 lM)
gand complex considering various types of molecular interactions
as described in Rarey et al.60 Each docked conformation is looked
upon as a ‘suggestion’ of how the ligand may bind with the pro-
tein. PyMol61 was used for visualization of the docked
conformations.
lL
l
l
l
l
l
Acknowledgments
l
bated for 3 h. After running the gel, residual tRNA was visu-
alized after ethidium bromide staining under UV light.
Authors thank the Council for Scientific and Industrial Research,
New Delhi for fellowships, Department of Chemistry and Central
Research Facility, IIT Kharagpur for instrumental facilities. The
authors are also thankful to Mr. Sk Mohammad Aziz for HPLC
analysis.
4.3.1. Precipitation assay
Inhibition of the ribonucleolytic activity of RNase A was quanti-
fied by the precipitation assay as described by Bond58 In this meth-
Supplementary data
od 10
(0.35 mM) individually to a final volume of 100
for 2 h at 37 °C. 20 L of the resulting solutions from the incubated
mixtures were then mixed with 40 L of tRNA (5 mg/mL tRNA
freshly dissolved in RNase A free water), 40 L of Tris–HCl buffer
of pH 7.5 containing 5 mM EDTA and 0.5 mg/mL HSA. After incuba-
tion of the reaction mixture at 25 °C for 30 min, 200 L of ice-cold
l
L of RNase A (1.31
lM) was mixed with 40
lL of compounds
lL and incubated
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.05.047.
l
l
l
References and notes
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