Structural and biochemical study on the inhibitory activity of derivatives of 5-nitro-furan-2-carboxylic acid for RNase H function of HIV-1 reverse transcriptase

Article abstract of DOI:10.1016/j.bmc.2010.12.011

Rapid emergence of drug-resistant variants is one of the most serious problems in chemotherapy for HIV-1 infectious diseases. Inhibitors acting on a target not addressed by approved drugs are of great importance to suppress drug-resistant viruses. HIV-1 reverse transcriptase has two enzymatic functions, DNA polymerase and RNase H activities. The RNase H activity is an attractive target for a new class of antiviral drugs. On the basis of the hit chemicals found in our previous screening with 20,000 small molecular-weight compounds, we synthesized derivatives of 5-nitro-furan-2-carboxylic acid. Inhibition of RNase H enzymatic activity was measured in a biochemical assay with real-time monitoring of florescence emission from the digested RNA substrate. Several derivatives showed higher inhibitory activities that those of the hit chemicals. Modulation of the 5-nitro-furan-2-carboxylic moiety resulted in a drastic decrease in inhibitory potency. In contrast, many derivatives with modulation of other parts retained inhibitory activities to varying degrees. These findings suggest the binding mode of active derivatives, in which three oxygen atoms aligned in a straight form at the nitro-furan moiety are coordinated to two divalent metal ions located at RNase H reaction site. Hence, the nitro-furan-carboxylic moiety is one of the critical scaffolds for RNase H inhibition. Of note, the RNase H inhibitory potency of a derivative was improved by 18-fold compared with that of the original hit compound, and no significant cytotoxicity was observed for most of the derivatives showing inhibitory activity. Since there is still much room for modification of the compounds at the part opposite the nitro-furan moiety, further chemical conversion will lead to improvement of compound potency and specificity.

Full text of DOI:10.1016/j.bmc.2010.12.011

Bioorganic & Medicinal Chemistry 19 (2011) 816–825
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structural and biochemical study on the inhibitory activity of derivatives
of 5-nitro-furan-2-carboxylic acid for RNase H function of HIV-1
reverse transcriptase
Hiroshi Yanagita a, Emiko Urano b, Kishow Matsumoto a, Reiko Ichikawa b, Yoshihisa Takaesu a,
Masakazu Ogata a, Tsutomu Murakami b, Hongui Wu b,c, Joe Chiba c, Jun Komano b, Tyuji Hoshino a,
⇑
a Graduate School of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan
b AIDS Research Center, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku, Tokyo 162-8640, Japan
c Faculty of Industrial Science and Technology, Tokyo University of Science, Yamazaki 2641, Noda, Chiba 278-8510, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Rapid emergence of drug-resistant variants is one of the most serious problems in chemotherapy for
HIV-1 infectious diseases. Inhibitors acting on a target not addressed by approved drugs are of great
importance to suppress drug-resistant viruses. HIV-1 reverse transcriptase has two enzymatic functions,
DNA polymerase and RNase H activities. The RNase H activity is an attractive target for a new class of
antiviral drugs. On the basis of the hit chemicals found in our previous screening with 20,000 small
molecular-weight compounds, we synthesized derivatives of 5-nitro-furan-2-carboxylic acid. Inhibition
of RNase H enzymatic activity was measured in a biochemical assay with real-time monitoring of flores-
cence emission from the digested RNA substrate. Several derivatives showed higher inhibitory activities
that those of the hit chemicals. Modulation of the 5-nitro-furan-2-carboxylic moiety resulted in a drastic
decrease in inhibitory potency. In contrast, many derivatives with modulation of other parts retained
inhibitory activities to varying degrees. These findings suggest the binding mode of active derivatives,
in which three oxygen atoms aligned in a straight form at the nitro-furan moiety are coordinated to
two divalent metal ions located at RNase H reaction site. Hence, the nitro-furan-carboxylic moiety is
one of the critical scaffolds for RNase H inhibition. Of note, the RNase H inhibitory potency of a derivative
was improved by 18-fold compared with that of the original hit compound, and no significant cytotox-
icity was observed for most of the derivatives showing inhibitory activity. Since there is still much room
for modification of the compounds at the part opposite the nitro-furan moiety, further chemical conver-
sion will lead to improvement of compound potency and specificity.
Received 25 September 2010
Revised 2 December 2010
Accepted 3 December 2010
Available online 9 December 2010
Keywords:
Antiviral drugs
HIV-1 reverse transcriptase
RNase H enzymatic activity
Inhibitors
Dual metal chelation
Nitro-furan carboxylic acid
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
possible to take an inhibitor of RNase H activity with other ap-
proved drugs, the RNase H inhibition is expected to be one of the
A cocktail regimen of therapeutic agents known as highly active
antiretroviral therapy (HAART) showed a great advance in the
treatment of human immunodeficiency virus (HIV) infectious
diseases. The efficacy of this therapy is, however, limited by the
emergence of drug-resistant variants of HIV-1. Drug-resistant
viruses have become a serious issue in current HIV chemotherapy1
because HIV-infected disease inevitably requires long-term treat-
ment. One of the effective and practical measures to suppress drug
resistance is to produce new anti-HIV drugs that act on the target
not addressed by approved drugs. Drugs directly blocking the viral
enzymatic activity usually show a high therapeutic performance.
RNase H activity of reverse transcriptase (RT) is the enzymatic
activity of HIV-1 that no approved drugs still act on. Since it is
attractive targets for anti-HIV drugs.2
RT is a virally encoded enzyme of HIV-1. RT is a heterodimer of
p51 and p66 subunits and has enzymatic functions of DNA
polymerase and RNase H activity. That is, this enzyme converts sin-
gle-strand viral genomic RNA into double-strand DNA. The enzyme
also catalyzes the hydrolysis of RNA phosphodiester bonds of RNA
hybridized to DNA. Two spacely-separated active sites with the
same protein are responsible for these two enzymatic functions
respectively. Hence, RT-associated RNase H activity is one of the
attractive targets for developing a novel class of antiviral drugs.
Furthermore, dual inhibitory of RNase H activity and the activity
of RT-associated polymerase or HIV-1 integrase has been reported
because of the structural similarity of their catalytic sites.3–5
RNase H activity requires the presence of divalent metal cations
to be functionalized in catalysis of endo-nucleolytic phosphodies-
ter hydrolysis. Recent crystallographic studies have shown the
⇑
Corresponding author. Tel.: +81 43 290 2926; fax: +81 43 290 2925.
E-mail address: hoshino@faculty.chiba-u.jp (T. Hoshino).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.12.011

H. Yanagita et al. / Bioorg. Med. Chem. 19 (2011) 816–825
817
bimetal mode of divalent metal.6–8 The enzymatic active site con-
tains four carboxylate residues, creating an environment capable of
stabilizing two metal ions. Many RNase H inhibitors are assumed
to be bound to the catalytic center and interact with divalent metal
ions. That is, chelators of these metal ions are regarded as potential
inhibitors of the function of HIV-1 RNase H.
high inhibitory activity for RNase H. Parts A, B and C of the hit com-
pound shown in Figure 1a were converted into other chemical sub-
stitutes. Then compounds 1–53 were synthesized according to the
routes shown in the scheme of Figure 1b.
First, chemical modulation was performed for part A. Part A is
composed of large hydrophobic substitutes. Compounds 1–28
were prepared by nucleophilic substitution reaction of 5-nitro-
Several different scaffolds have been reported as inhibitors of
RNase H activity.9–13 Diketo acids have been well-known chemical
structure showing potent inhibitory for two divalent metal-related
enzymatic activity initiating endonucleolytic phosphodiester
hydrolysis.14 Therefore, diketo acid structure has served as a start-
ing point for the design and optimization of inhibitors of HIV-1
integrase or influenza endonuclease. Pyrimidinol is one of typical
derivatives bearing a scaffold called N-hydroxyimide,15 and it has
been reported to be a potent inhibitor of HIV-1 RNase H function,
acting through metal chelation at the active site. N-hydroxyimides
were firstly described as inhibitors of influenza endonuclease, but
they also show high potency in biochemical assays of HIV-1 RNase
H. An important feature in the structure of pyrimidinol is a
six-member ring having a polar atom alignment compatible with
diketo acids. The natural product b-thujaplicinol is another scaffold
and has been reported to be a highly potent inhibitor of HIV-1
RNase H activity.16 Using this chemical, the multiple inhibition of
HIV-1 enzymes such as HIV-1 integrase and HIV-1 RT-associated
polymerase has been investigated. The prominent feature in the
structure of b-thujaplicinol is a seven-member ring bonding with
two hydroxy groups and one carboxy group.
2-furoic acid with an
a-chloro carbonylate in the presence of
DMAP in DMF at 80 °C (eq. 1 of Fig. 1b). Second, chemical
modulation was performed for part B, which shows a large hydro-
philicity with a nitro-furan moiety. Compounds 29–45 were pre-
pared by experimental conditions similar to those used in the
reaction for converting part A. A substitution reaction was carried
out using carboxylic acids that contain a nitro group on the aro-
matic ring (eq. 2 of Fig. 1b). Third, the chemical structure of part
C was modulated. Part C is a region connecting parts A and B.
5-Nitro-2-furoic acid was treated with thionyl chloride to gener-
ate an acid chloride as an intermediate, followed by reaction of
the generated acid chloride with an
a-amino acid ester to pro-
duce compounds 46–51 (eq. 3 of Fig. 1b). Additionally, two com-
pounds 52 and 53 were prepared to modulate parts A and C with
keeping the ester bond. 5-Nitro-2-furoic acid was converted into
an acid chloride as an intermediate with thionyl chloride,
followed by the substitution reaction of nucleophilic reagents
(eq. 4 of Fig. 1b).
2.2. Evaluation of inhibitory activity
From an in vitro screening using 20,000 small molecular-weight
compounds, we found chemicals that blocked HIV-1 RT-associated
RNase H activity.17 Several analogues bearing the 5-nitro-furan-2-
carboxylic acid ester moiety were shown to work as retroviral
RNase H inhibitors. Two of the derivatives were capable of sup-
pressing HIV-1 replication in tissue culture. The distinguishable
feature in the structure of our 5-nitro-furan-2-carboxylic acid ester
is a five-member ring.
To date, no RNase H inhibitors have been approved for clinical
use. One of the problems in developing an RNase H active site
inhibitor is the absence of a deep pocket into which the inhibitors
can be bound.7,18 This makes it difficult to improve stable and spe-
cific binding of compounds to the RNase H catalytic site. Metal ions
at the active site, however, are a suitable aiming point for inhibitor
binding. A diketo acid inhibitor was shown to be bound in a metal-
dependent manner to the RNase H domain of RT.14,19 Pyrimidinol
analogues were designed to chelate the divalent metals of the
RNase H domain.6 b-Thujaplicinol also chelates the two metal ions
at the active site.7 Judging from the structural similarity to
pyrimidinol or b-thujaplicinol, derivatives bearing the 5-nitro-
furan-2-carboxylic acid ester moiety are assumed to chelate two
divalent metal ions as well.
Plasmids expressing p66 or p51 of RT with a hexahistidine tag
were prepared by cloning a DNA fragment encoding the HIV-1 RT
into the pQE-9 vector.17 The Escherichia coli strain BL21(DE3)pLysS
was transformed with the plasmids, and protein expression was
induced by treatment with 1 mM isopropyl b-D-thiogalactoside
for 3 h. For generating heterodimers, bacteria lysates expressing
p66 and p51 were mixed prior to purification. A gradient elution
was performed using HiTrap HP columns according to the manu-
facturer’s protocol. The yield of the purified protein was estimated
using bovine serum albumin as a standard, and the purification
was evaluated with Coomassie blue-stained SDS–polyacrylamide
gels. The concentration of HIV-1 RT was approximately 1 ꢀ 104
units/g, which was determined by a comparison of polymerase
activity with an RT standard.17
Alternatively, the expression plasmid vector RT69A, which was
kindly provided by Professor E. Arnold at Rutsgers University, was
used. This plasmid expresses a heterodimer of p66 and p51, which
was reported to produce RT crystals with a resolution below 2 Å in
X-ray diffraction analysis due to amino residue mutations of F160S
and C280S.20 The E. coli strain Rosetta transformed with the RT69
plasmid was incubated at 37 °C. Protein expression was induced
In this study, we examined chemical compounds for anti-HIV
drugs blocking RT-associated RNase H enzymatic activity. A variety
of compounds were synthesized on the basis of hit chemicals found
in in vitro screening in our previous study. Measurement of inhibi-
tory activity with a fluorescence-based assay and theoretical calcu-
lation using quantum mechanical (QM) and molecular mechanics
(MM) methods suggested the binding mode of the synthesized com-
pounds. The findings of the present study provide a strategy for
designing a chemical structure to enhance the inhibitory activity.
by adding 1 mM isopropyl b-D-thiogalactoside at an OD600 value
of 0.9 and completed by incubation for 3 h after induction. A cell
pellet corresponding to 1.5 L of culture was resuspended in
50 mM Tris–HCl at pH 8.0, 600 mM NaCl, and 1% Triton X-114.
The bacterial cell membrane was disrupted by sonication. After
removing unnecessary disrupted fragments from the lysate by
centrifugation, the expressed protein was obtained from the super-
nant. Since the RT p51 subunit contains an N-terminal hexahisti-
dine tag, RT was purified by using a HiTrap Ni affinity column
with an elution buffer containing 500 mM imidazole. The eluted
protein fraction was dialyzed overnight against a buffer containing
50 mM Tris–HCl at pH 8.0 and 600 mM NaCl. The dialyzed RT was
incubated with HRV 3C protease for 24 h at 4 °C to cleave the hexa-
histidine tag attached to the N-terminus of the p51 subunit of the
heterodimer RT protein. The protein was again purified by Ni-NTA
according to the manufacturer’s recommendation to remove the
uncleaved protein and HRV 3C protease. The RT69 protein was dia-
2. Methods
2.1. Organic synthesis
Derivatives from a hit compound bearing 5-nitro-furan-2-car-
boxylic acid were synthesized to obtain chemical analogs showing

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H. Yanagita et al. / Bioorg. Med. Chem. 19 (2011) 816–825
a
b
O
O2N
CO2H
O
O
a
O2N
O
Cl
XR1R2R3
O
XR1R2R3
O
Compound 1-28
Yeild = 31-88 %
Ar-CO2H
O
Ar
O
a
XR1R2R3
O
Compound 28-45
Yeild = 25-82 %
R2
O
O
b, c
O
OR1
O2N
CO2H
N
O2N
H
O
Compound 46-51
Yeild = 10-63 %
O
b, d
R
O
O
O2N
Compound 52, 53
Yeild = 65-76 %
Conditions ; (a) 5-Nitro-2-furoic acid, DMAP, DMF, 80 ºC, 6 h
(b) SOCl2, CH2Cl2, reflux, 3 h (c) Amino acid ester, NEt3, CH2Cl2, 0 ºC-rt, 1 h
(d) , NEt , CH Cl , 0 ºC-rt, 1 h
Nucleophile
3
2
2
Figure 1. (a) Structure of a hit chemical showing RNase H inhibitory activity found in our previous in vitro screening. (b) Scheme for synthesis of the derivatives from the hit
chemical. Eqs. 1, 2 and 3 correspond to the reactions for modulating parts A, B and C in (a), respectively. Eq. 4 indicates the reaction modulating the moiety connected to the
ester bond at parts A and C.
lyzed against a buffer of 50 mM Tris–HCl at pH 7.5 and 200 mM
NaCl and was stored at ꢁ20 °C with adding 50% (v/v) glycerol.
The inhibitory activities of synthesized compounds were mea-
sured by an enzymatic assay in a manner similar to that in the previ-
ous studies.17,21,22 In short, a real-time monitoring assay was
applied. For substrate, two oligo-nucleotides were annealed at final
concentration of the compounds in these wells was 100 lM when
cells were cultured. (3) A sequence of wells with different com-
pound concentrations was prepared for the second, third and
fourth columns. The final concentrations of these columns were
50, 25 and 12.5 lM. The wells in the fifth column were used for
a control without adding any test compounds. (4) 100
l
L MT-4
concentrations of 2.5 and 0.25
lM. One was oligo-ribonucleotide
cells at a concentration of 4 ꢀ 105/mL or 100
lL 293T cells at a
50-GAUCUGAGCCUGGGAGCU-30 with 6-carboxy-fluoroscein (FAM)
conjugated at the 30 end, and the other was oligo-deoxyribonucleo-
tide 50-AGCTCCCAGGCTCAGATC-30 with black hole quencher (BHQ)
concentration of 2 ꢀ 105/mL was added to the respective wells.
The final concentration of DMSO in each well was 1%. (5) Cells
were incubated for 3 days at 37 °C with 5% CO2 atmosphere. (6)
conjugated at the 5’ end. Enzyme reaction with 100 ng RT, 0.025
oligo-ribonucleotide, and 0.25 M oligo-deoxyribonucleotide was
carried out in a volume of 10 L at 37 °C. Fluorescence at 488 nm
l
M
100
15
the cells were incubated for 1 h. (7) 100
l
l
L of supernant of the cultured medium was removed. Then
L MTT reagent for dye solution was added to each well and
L solution of solubiliza-
l
l
l
was monitored every 150 s using a multimode detector.
tion and stop mix was added, and the cells were incubated
overnight at 4 °C to sufficiently dissolve the dye. (8) Intensity of
2.3. Assessment of cytotoxicity
OD570/690 was measured by a spectrofluorometer, BIO-TEK
ELx808. Drug concentration showing 50% cell cytotoxicity was cal-
culated only if the viability of cells was below 50% in the presence
of compound at 100 lM.
Cytotoxicity of the synthesized compounds was tested by an
MTT assay with the celltiter 96 non-radioactive cell proliferation
assay system (Promega). Two cell lines, MT-4 and 293T, were used
in this assay. The cytotoxic assay was performed by the following
2.4. Theoretical computation
8-step procedure. (1) 100
with 10% FBS containing 2% DMSO was loaded in a 96-well plate,
and the outside of the wells was soaked with 100 L PBS to prevent
an edge effect. (2) 200 L RPM-1640 medium with 10% PBS and 2%
DMSO containing test compounds at a concentration of 200
was added to the wells in the first column of the plate. The final
lL RPM-1640 medium supplemented
A computational model of the target protein was constructed
from an X-ray crystal structure of the RNase H domain of HIV-1
RT: 3HYF.6 According to the results of the recent X-ray crystallo-
graphic studies on the complex of RNase H domain and chemical
compounds showing RNase H inhibitory activity,6–8 the RNase H
l
l
lM

H. Yanagita et al. / Bioorg. Med. Chem. 19 (2011) 816–825
819
domain contains two Mn2+ ions at the center of the active site.
Hence, two Mn2+ ions in the crystal structure were replaced with
Mg2+ ions. The protonation states of all the ionasable residues were
predicted by ProPKa program23 in the presence of two Mg2+ ions at
the active site. The prediction indicated deprotonation of Asp443,
Glu478, Asp498, and Asp549. Chemical structures of the active
compounds were built by using GaussView software, and geome-
try optimization was executed at the B3LYP/6-31G(d,p) level. Each
compound was manually placed at the active site of RNase H do-
main. A quantum mechanical and molecular mechanics (QM/
MM) approach was applied,24 utilizing the ONIOM method of
GAUSSIAN 03 program.25 In our preliminary computations, molecular
dynamics simulation hardly gave a reasonable binding conforma-
tion, because the RNase H domain contained divalent metal ions
at the active site and the parameterization would be insufficient
to reproduce the six-fold coordination of divalent metal ions. In
QM/MM calculation, Asp443, Glu478, Asp498, Asp549, His539,
compound, and two Mg2+ ions were set to the QM layer, and the
other residues were set to the MM layer. No water molecules were
included in the calculation model. Models including surrounding
water molecules had also been examined in our preliminary trials
of QM/MM calculation. Geometry optimization was hardly com-
pleted in the water-included models because of the difficulty in
meeting the convergence criteria, in which even slight forces on
atoms caused large displacements of the surrounding water mole-
cules. Molecular orbitals in the QM layer were calculated at the
B3LYP/6-31G(d,p) level and the universal force field was applied
to the atoms in the MM layer. Geometry optimization was exe-
cuted without any constraints to any atoms.
3. Results
Two hit chemicals, which were found in our previous in vitro
screening, contain a nitro-furan ring and amide group bonding to
Table 1
Structure and RNase H inhibitory activity of the derivatives modulated at part A
O
O
a
O2N
O
XR1R2R3
O
Cl
XR1R2R3
O
Compound 1-28
Yeild = 31-88%
Conditions ; (a) 5-Nitro-2-furoic acid, DMAP, DMF, 80 ºC, 6 h
Compound
X
O
N
R1, R2, R3
Yield (%)
64
IC50 (lM)
1
R1 = i-Pr
13.2
2
3
4
5
R
1 = H, R2 = t-Bu
58
48
43
48
18.0
5.8
8.2
R1 = H, R2 = CMe2Et
R1 = H, R2 = CMe2Ph
R1 = H, R2 = CMe2CH2Ph
4.3
R1 = H, R2 = CH2CH(CH2)3O
R1 = H, R2 =
6
7
8
64
59
38
4.2
5.5
6.1
R1 = H, R2 =
N
9
45
4.3
R1 = H, R2 =
10
11
12
13
N
N
R
1 = t-Bu, R2 = CH2Ph
82
74
82
88
0.9
R1 = t-Bu, R2 = CH2CH2Ph
R1 = t-Bu, R2 = CH2(CH2)2Ph
R1 = t-Bu, R2 = CH2CH2C(O)Ph
14.2
>50
>50
14
15
16
17
18
19
R
1 = t-Bu, R2 = CH2p-NO2C6H4
72
69
45
31
68
73
6.9
9.8
8.7
12.8
7.5
R1 = t-Bu, R2 = CH2m-NO2C6H4
R1 = t-Bu, R2 = CH2p-AcOC6H4
R1 = t-Bu, R2 = CH2o-AcOC6H4
R1 = t-Bu, R2 = CH2p-MeOC6H4
R1 = t-Bu, R2 = CH2p-BnOC6H4
>50
20
21
22
23
24
N
R
1 = t-Bu, R2 = CH2p-FC6H4
64
71
69
72
68
8.5
8.0
8.5
9.0
6.8
R1 = t-Bu, R2 = CH2p-CF3C6H4
R1 = t-Bu, R2 = CH2m-CF3C6H4
R1 = t-Bu, R2 = CH2o-CF3C6H4
R1 = t-Bu, R2 = CH22,3,4,5,6-F5C6
R1 = CH2CH(CH2)3O, R2 = CH2Ph
25
26
32
45
7.7
5.0
N
C
R1 = CH2CH(CH2)3O, R2 = CH2p-HOC6H4
27
28
R
1 = H, R2 = H, R3 = H
R1 = Me, R2 = Me, R3 = Me
56
49
7.1
21.9
Conditions: (a) 5-nitro-2-fuloric acid, DMAP, DMF, 80 °C, 6 h.

820
H. Yanagita et al. / Bioorg. Med. Chem. 19 (2011) 816–825
a hydrophobic substitute. The nitro-furan ring is connected to the
amide group through an ester linkage. Synthesis of these chemi-
cals was accomplished by coupling reaction of nitro-furoic acid
As shown in Table 1 and 28 analogues were synthesized by con-
verting the hydrophobic substitute at part A. In the in vitro assay
for inhibitory activity, most of the analogues exhibited a similar
degree of inhibitory potency to the hit chemical in Figure 1a, the
with a-chloro carbonylate containing of a hydrophobic substitute
and an amide group. Hence, three series of analogue compounds
were synthesized by the following strategy for modulating parts
A, B and C of Figure 1a: (A) conversion of the hydrophobic substi-
tute bound to the amide group to other substitutes, (B) conver-
sion of the nitro-furan ring to several other chemical structures,
and (C) conversion of the ester linkage to an amide bond.
The 50% inhibitory concentration (IC50) of the compounds for
HIV-1 RT-associated RNase H activity was determined from the
chemical concentration leading half the rate for substrate cleavage
reaction relative to the control. A real-time monitoring assay was
carried out to estimate IC50 of the synthesized compounds.
IC50 value of which was 16.5 lM. Inhibitory activity was retained
with conversion of the amide bond to an ester linkage and
substitution of the hydrophobic region with iso-propyl (1). A set
of derivatives containing two methyl groups and one hydrophobic
substitute such as t-butyl, pentyl or phenyl bound to an alkyl car-
bon connecting to the amide group was surveyed (2–5). Substitu-
tion of tetrahydrofuran was also tested (6). These derivatives had
similar potencies. In particular, tetrahydrofuran substitution (6) in-
creased compound potency by approximately fourfold from the hit
chemical. The effect of introduction of a phenyl group was exam-
ined in two forms: one is through connection with cyclo-carbons
Table 2
Structure and RNase H inhibitory activity of the derivatives modulated at part B
O
O
a
Ar
O
XR1R2R3
Cl
XR1R2R3
O
Compound 28-45
Yeild = 25-82%
Conditions ; (a) Ar-CO2H, DMAP, DMF, 80 ºC, 6 h
Compound
X
Ar-CO2H
R1, R2, R3
Yield (%)
IC50 (lM)
29
30
31
32
N
o-Nitro-C6H4CO2H
m-Nitro-C6H4CO2H
p-Nitro-C6H4CO2H
3,5-Dinitro-C6H3CO2H
R1 = H, R2 = t-Bu
R1 = H, R2 = t-Bu
R1 = H, R2 = t-Bu
R1 = t-Bu, R2 = CH2Ph
57
60
49
34
>50
>50
>50
>50
O
CO2H
33
34
R1 = t-Bu, R2 = CH2Ph
R1 = H, R2 = t-Bu
67
30
>50
>50
N
N
O
Br
CO2H
O
O
35
36
R1 = H, R2 = t-Bu
R1 = H, R2 = t-Bu
43
25
>50
>50
CO2H
Ph
O
CO2H
N
NO2
O
37
R1 =H, R2 = t-Bu
82
>50
CO2H
O2N
O2N
N
38
39
R1 = H, R2 = t-Bu
R1 =H, R2 = t-Bu
61
46
>50
>50
O
CO2H
O
CO2H
S
S
CO2H
CO2H
O2N
O2N
40
41
R1 = H, R2 = t-Bu
61
32
2.8
5.8
R1 = H, R2 = CMe2Et
N
S
S
R1 = CMe2Et, R2 = CH2p-NO2C6H
R1 = CMe2Et, R2 = CH2m-NO2C6H4
58
64
33.5
25.8
O2N
O2N
CO2H
CO2H
42
43
S
S
CO2H
CO2H
O2N
O2N
44
45
R1 = H, R2 = H, R3 =H
56
48
9.5
5.7
C
R1 = Me, R2 = Me, R3 =Me
Conditions: (a) Ar-CO2H, DMAP, DMF, 80 °C, 6 h.

H. Yanagita et al. / Bioorg. Med. Chem. 19 (2011) 816–825
821
Table 3
(7–9) and the other with an alkyl chain (10–13). A highly potent
Structure and RNase H inhibitory activity of the derivatives modulated at part C
compound (10) was found in the analogues in which a benzyl
group with a t-butyl substitute was introduced. This derivative
(10) showed an 18-fold increment of RNase H inhibitory activity
from the original hit chemical. In contrast, no inhibitory activity
was observed for the analogues with a phenyl group introduced
via more than two chain atoms of carbon or oxygen (12 and 13).
The effect of introduction of a phenyl group via an alkyl chain
was further examined by connecting some polar functional groups,
nitro, acetyl, methoxy and phenoxy, to the phenyl (14–19). These
derivatives showed inhibitory activities similar to that of the hit
chemical, while no activity was observed only for the connection
of phenoxy (19), which was a considerably large substitute. The
introduction of fluoride was examined with the phenyl group
linked to an amide bond via an alkyl chain (20–24). These deriva-
tives exhibited similar degrees of inhibitory activity, but no highly
potent compound was found with fluoride introduction. The intro-
duction of tetrahydrofuran with a benzyl or hydroxy benzyl group
(25 and 26) was examined. Both compounds showed inhibitory
activity similar to that of the hit chemical. The amide group was
converted to a carbonyl group (27 and 28). A similar degree of
inhibitory activity was maintained with conversion of the amide
bond to an acetyl group, while conversion to oxy t-butyl decreased
the inhibitory potency.
As shown in Table 2 and 17 analogues were synthesized by con-
verting the nitro-furan ring at part B into other ring structures.
Most of the analogues lost inhibitory potency for RNase H enzy-
matic activity or had significantly reduced inhibitory activity. Con-
version of the furan ring into benzene (29–32) resulted in loss of
compound potency, regardless of the location of the nitro group
at the orto-, meta- or para-position. The derivative containing
two nitro groups (32) also showed no inhibitory activity. A series
of derivatives with modification of the nitro group (33–36) exhib-
ited no inhibitory activity. Removal of the nitro group (33),
replacement by bromide (34), replacement by carbonyl phenyl
(35), or removal of even hydrogen (36) resulted in complete loss
of compound potency. Substitution of the nitro group by nitro-ben-
zene (37–39) resulted in no inhibitory activity regardless of the po-
sition of the nitro group bonding to benzene. Substitution of the
furan ring with thiophene (40–45) was surveyed. Some derivatives
were more potent than the hit chemical. An analog derived from
compound 3 with conversion of the furan ring into thiophene
(40) showed high inhibitory activity.
R2
O
OR1
a, b
O
O
O2N
CO2H
N
H
O2N
O
Compound 46-51
Yeild = 10-63%
Conditions ; (a) SOCl2, CH2Cl2, reflux, 3 h,
(b) Aminoacidester, NEt3, CH2Cl2, 0 ºC-rt, 1 h
Compound
R1, R2
Yield (%)
IC50 (lM)
46
47
48
49
50
51
R1 = Me, R2 = H
R1 = Et, R2 = Me
R
10
70
45
60
63
61
>50
>50
>50
>50
>50
>50
1 = Et, R2 = CH2i-Pr
R1 = Et, R2 = CH2Ph
R1 = Et, R2 = CH2p-OHC6H4
R1 = Et, R2 = Ph
Conditions: (a) SOCl2, CH2Cl2, reflux, 3 h; (b) amino acid ester, NEt3, CH2Cl2, 0 °C to
rt, 1 h.
that a nitro-furan core has little cytotoxicity for MT-4 and 293T
cells and that the scaffold tested in this study is favorable from a
cytotoxic viewpoint. It is notable that there is some degree of
difference in concentrations of compounds showing noticeable
cytotoxicity between MT-4 and 293T cells.
4. Discussion
Two hit chemicals found in our previous in vitro screening bear
an ester linkage at the connection of the nitro-furan group and the
hydrophobic moiety.17 All of the compounds showing RNase H
inhibitory activity in Tables 1 and 2 have this ester linkage. Table
3 clearly indicates that conversion of the ester linkage into an
amide bond results in loss of inhibitory potency. The amide bond
is likely to form a planar configuration. Hence, a straight form is
favorable for the amide linkage of nitro-furan and the hydrophobic
moiety. If a compound has a straight form, the side part of the com-
pound will collide with the inside wall of the binding pocket of the
RNase H domain. Accordingly, it will be difficult for the compound
to combine with the binding pocket. This suggests that an amide
bond adjacent to the nitro-furan group is unfavorable for RNase
H inhibitors.
As shown in Table 1, no drastic change was observed in inhibi-
tory activity when the hydrophobic moiety connecting to carbonyl
carbon was substituted by various kinds of chemical groups. This
indicates that the substituted region has little interaction with
the RNase H domain, suggesting that the substituted region will
be located outside the binding pocket and exposed to the solvent.
This indicates that a strategy for increasing inhibitory activity is for
the position of the substitute to be closer to the nitro-furan group
or instead more distant from it. The former conversion may make
the aromatic ring or hydrophobic substitute interact with the tar-
get protein inside the binding pocket. The latter will make the sub-
stitute interact with a neighboring hollow site outside the binding
pocket.
As shown in Table 3 and six analogues were synthesized by
exchanging the ester bond at part C. Ester linkage is disadvanta-
geous for medicine because esterase digests the linkage and the
drug concentration in a body is rapidly decreased. The ester linkage
was replaced by an amide group (46–51). None of the derivatives
showed noticeable inhibitory activity.
As shown in Table 4 and two analogues were synthesized by
converting the region connected to the ester bond at parts A and
C. Both derivatives (52 and 53) increased compound potency
6–8-fold from the hit chemical. It is interesting to note that the dis-
tance from the ring part of the substituted moiety to the ester bond
is small compared to a series of derivatives listed in Table 1.
In the measurement of cytotoxicity using MT-4 cells, almost all
chemical compounds showed no cytotoxicity at a concentration of
100
Table 5. In the measurement using 293T cells, many compounds
showed no noticeable cytotoxicity at a concentration of 100 M,
while seven compounds, 11, 15, 21, 22, 23, 24 and 52 showed
cytotoxicity in which CC50 ranged from 36 to 83 M. Compounds
lM, except for compounds 21, 23, 52 and 53 as shown in
Table 2 shows that conversion of the nitro-furan group into
other chemical structures drastically decreases the inhibitory
activity except for nitro-thiophen. This means that a nitro-furan
core is indispensable for inhibitory potency induced by analogues
of the hit chemical in Figure 1a. The characteristic property of ni-
tro-furan is large electric polarity. Oxygen atoms are negatively
charged. These oxygen atoms will be coordinated to divalent me-
tal ions at the RNase H active site. Accordingly, enhancing the
l
l
showing cytotoxicity contain several fluoride atoms (21–24). Cyto-
toxicity was also observed in a compound bearing a nitro-benzyl
group (15 and 52), which is a chemical structure known as a cause
for genotoxicity. Overall, the assessment of cytotoxicity suggested

822
H. Yanagita et al. / Bioorg. Med. Chem. 19 (2011) 816–825
Table 4
Structure and RNase H inhibitory activity of the derivatives modulated at the moiety connected to the ester bond in parts A and C
O
a, b
O
R
O
O2N
CO2H
O
O2N
Compound 52,53
Yeild = 65-76 %
Conditions ; (a) SOCl2, CH2Cl2, reflux, 3 h,
(b) Nucleophile, NEt3, CH2Cl2, 0 ºC-rt, 1 h
Compound
Nucleophile
R
Yield (%)
76
IC50
2.1
(lM)
NO2
NO2
52
R = (CH2)2p-NO2C6H4
HO
Me
Me
NO2
N
N
R =
53
O
O
65
2.7
HO
O
O
Conditions: (a) SOCl2, CH2Cl2, reflux, 3 h; (b) nucleophile, NEt3, OH2Ol2, 0 °C to rt, 1 h.
Table 5
Evaluation of cytotoxicity of the synthesized compounds
Compound
CC50
(l
M): MT-4
CC50
(lM): 293T
Compound
CC50
(l
M): MT-4
CC50 (lM): 293T
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
—
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
67
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
—
—
—
—
—
—
—
—
—
—
—
>100
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
>100
—
—
—
—
—
—
—
—
>100
>100
>100
>100
>100
—
>100
71
>100
95
>100
>100
>100
>100
>100
>100
78
>100
>100
>100
—
>100
36
68
65
83
>100
>100
>100
>100
46
47
48
49
50
51
52
53
—
—
—
—
—
—
28
86
—
—
—
—
—
—
36
>100
— means that the measurement for cytotoxicity was not performed because the compound shows no or little inhibitory activity.
coordination force to metal ions is one strategy for increasing
inhibitory activity.
coordination force to the active site becomes strong with change
from Mg and Mn. A single metal ion is observed in most metal-
bound crystal structures. Double coordination of divalent metal
ions was observed only in 1RTD28 at that time point. Accordingly,
it had been controversial how many metal ions were required at
the RNase H reaction site to exert its enzymatic activity.18,29 A the-
oretical study by De Vivo et al. suggested that the presence of two
divalent metal ions is essential for RNase H activity and that two
metal ions act cooperatively with facilitating nucleophilic binding
of a substrate and stabilizing the transition state for the enzymatic
reaction.30 De Vivo et al. also suggested that there is a difference in
role of those two metal ions. One of the ions is stably bound to the
RNase H of HIV-1 exerts its enzymatic activity by incorporating
divalent metal ions at the reaction site.26,27 In April 2009, there
were 119 entries for the crystal structure of HIV-1 reverse trans-
criptase in Protein Data Bank. We surveyed the number of divalent
metal ions observed at the RNase H domain through all of these
119 crystal structures. As shown in Table S1 in Supplementary
data, no metal ion was observed in many of the crystal structures.
The presence of Mg2+ ions was detected in 16 crystal structures
and coordination of Mn2+ ions was observed in five structures.
Mn2+ ion is often used in protein crystallization, because the

H. Yanagita et al. / Bioorg. Med. Chem. 19 (2011) 816–825
823
RNase H active site with an ideal octahedral coordination through
the reaction, while the other is slightly irregular with changing the
coordination bond. This theoretical finding strongly suggests dou-
ble coordination of divalent metal ions at the RNase H domain, but
the number of coordinated metal ions was still not clearly deter-
mined when an RNase H inhibitor was bound to the active site.
From the latter half of 2009, crystal structures on the complex
of the RNase H domain and its inhibitor were successively reported
from three different research groups.6–8 All of the crystal structures
in the reports showed the presence of two metal ions at the active
site. One of the divalent metal ions was held deep inside the bind-
ing pocket of the RT RNase H domain with making coordination
bonds to three carboxyl groups of Asp443, Glu478 and Asp498.
The other was fixed with making coordination bonds to two car-
boxyl groups of Asp443 and Asp549. The distance between two
metal ions was about 4 Å. Every inhibitor in crystal structures
was revealed to have a similar binding mode. That is, inhibitors
are stabilized with forming coordination bonds to both metal
ions.
charged atoms being aligned in a straight form. This negatively
charged region was strongly bound to two divalent metal ions.
The oxygen atom located at the center of the straightly aligned po-
lar atoms was positioned between the two metal ions. Ethoxy or
cyclo-pentane groups are attached to the side opposite the nega-
tively charged region on the hetero-rings. These groups were also
shown to stick out from the binding pocket and to be exposed to
the solvent in the crystal structures; PDB codes: 3LP0 and 3LP1.
The above three X-ray crystal analyses have revealed that the
RNase H active site holds two divalent metal ions and that an
RNase H inhibitor is bound to the active site with forming coordi-
nation bonds to these metal ions. Accordingly, it is highly probable
that the chemical compounds showing RNase H inhibitory activity
examined in this study are also coordinated to two divalent metal
ions. These compounds have a structural core of nitro-furan and
bear an ester linkage at the site opposite the nitro group on the fur-
an ring. Hence, negatively charged oxygen atoms of the nitro
group, furan, and carbonyl group are aligned in a straight form.
This negatively charged region will be attached to the divalent me-
tal ions.
In order to examine whether the binding mode described in the
above paragraph is stable or not, theoretical calculation with
QM/MM method was performed. Geometry of the QM region was
optimized in the B3LYP/6-31G(d,p) level and that for the MM region
was optimized by molecular mechanics approach with the univer-
sal force field. All of the atoms were allowed to move freely during
geometry optimization. The X-ray crystal structure; PDB code:
3HYF,6 was used for the computational model for the RNase H do-
main. The initial atom coordinates before geometry optimization
were set to the same as those of the crystal structure. The inhibitor,
two Mg2+ ions, and side chains of Asp443, Glu478, Asp498, Asp549
and His 539 were assigned to the atoms in the QM region. QM/MM
calculations were executed using three kinds of chemical com-
pounds; 3, 7 and 10. The dashed lines in Table 1 separate the com-
pounds into several groups in terms of chemical structure, and
compounds; 3, 7 and 10, bear a typical chemical structure of each
group. The inhibitory activity of these compounds is high and the
mass-weight is relatively low compared to other chemicals in the
same group. Therefore, these compounds will be a basis for our next
study. In every optimized structure, the nitro-furan group was
shown to be stably bound to two Mg2+ ions as shown in Figure 2.
The oxygen atom on the furan ring is also oriented toward the diva-
lent metals, while the coordination force seems weak. Carbonyl
oxygen at the ester linkage connecting to the furan ring is strongly
coordinated to Mg2+ ion. Therefore, a large ring-shaped configura-
tion of –O–C–N–O–Mg–Mg–O–C–C– is formed. In compound 3,
the inter-atomic distance of the two Mg2+ ions is 4.4 Å. The distance
between the nitro-oxygen atom and one Mg2+ ion is 2.3 Å, and that
between carbonyl oxygen and the other Mg2+ ion is 2.2 Å. The pen-
tyl group positioned at the opposite side of the compound sticks out
from the binding pocket. Hence, there is much room for improve-
ment in this hydrophobic region. The oxygen atom at the amide
bond has a noticeable interaction with the hydroxyl group of
Ser499 of the RNase H domain. In compound 7, the distance be-
tween the two Mg2+ ions is 3.8 Å. The distance between the nitro-
oxygen and one Mg2+ ion is 2.0 Å, and that between carbonyl oxy-
gen and the other Mg2+ ion is 2.1 Å. The polar atoms composing
the amide bond have an interaction with the RNase H domain. In
compound 10, the distance between the two Mg2+ ions is 4.3 Å.
The distance between nitro-oxygen and one Mg2+ ion is 2.1 Å, and
that between carbonyl oxygen and the other Mg2+ ion is 2.3 Å.
The oxygen atom at the amide bond has an interaction with
Ser499 of the RNase H domain, while the large hydrophobic moiety
at the side part is positioned outside the binding pocket. The QM/
MM calculations confirmed that all of the compounds; 3, 7 and
10, can be stably bound to the active site of the RNase H domain
In the X-ray crystallographic study by Kirschberg et al.6 the crys-
tallization was archived using the isolated RNase H domain. The
inhibitor was a kind of pyrimidinol carboxylic acid derivative. This
compound bears a pyrimidine ring connected to one carboxyl group
and two hydroxyl groups. This chemical structure shows a signifi-
cant feature of negatively charged functional groups being aligned
in a straight form. This negatively charged region is strongly
attached to two divalent metal ions. An oxygen atom located at
the center of the straightly aligned polar atoms is positioned be-
tween the two metal ions. In their study, many chemical structures
were examined by modulating the substitute located at the position
opposite the straightly aligned negatively charged atoms on the
pyrimidine ring. IC50 values of the derivatives were ranged from
0.1 to 70 lM. In their crystal structure; PDB code: 3HYF, the inhibi-
tor has a dichlorobenzyl group as a substitute at the opposite region
on the pyrimidine ring. This region was demonstrated to stick out
from the binding pocket and to be exposed to the solvent.
An X-ray crystallographic study by Himmel et al.7 was per-
formed using a protein expressed by RT69A vector. The protein
consists of the RT p66 subunit containing F160S and C280S muta-
tions and the p51 subunit containing C280S mutation and trun-
cated at residue 428. The apo-crystal without an inhibitor using
this protein was reported to give a high-resolution X-ray diffrac-
tion.20 The X-ray analysis by Himmel et al. is the first report for
co-crystallization of whole RT and an RNase H inhibitor. The inhib-
itor was b-thujaplicinol, which shows a high level of RNase H
inhibitory activity.16 b-Thujaplicinol has a unique chemical struc-
ture, in which two hydroxyl groups and one carbonyl group are
connected to the adjacent three carbon atoms on a seven-member
ring. Those negatively charged oxygen atoms are also aligned in a
straight form in a manner similar to that for pyrimidinol carboxylic
acids. These negatively charged atoms are strongly coordinated to
two divalent metal ions. All of the charged atoms are located on
one side of the seven-member ring and the oxygen atom posi-
tioned at the center of the three aligned negatively charged atoms
is coordinated to both metal ions. An alkyl chain is connected to
the region opposite to the three negatively charged atoms on the
seven-member ring in b-thujaplicinol. In the X-ray crystal struc-
ture; PDB code: 3IG1, this alkyl chain was demonstrated to be lo-
cated outside the binding pocket and exposed to the solvent.
Su et al. also provided X-ray crystal structures on the complex
of full-length HIV-1 RT and chemical compounds showing RNase
H inhibitory activity.8 The chemical compounds have a structural
basis of pyridine hetero-rings to which one hydroxyl group and
one carbonyl group are connected. IC50 values of those pyridine
heteroring-based derivatives were reported to be 0.1–0.2
lM. The
chemical structure also shows a common feature of negatively

824
H. Yanagita et al. / Bioorg. Med. Chem. 19 (2011) 816–825
There exists a polar residue, Ser499, deep inside at this space on the
RNase H domain. This residue would have little influence on the
function of RNase H. Therefore, one of the designs to improve inhib-
itory activity is to modify the compound to bear some polar func-
tional group that can interact with Ser499. Substitution of ether
oxygen with nitrogen or carbon atom to enable the incorporation
of a polar functional group is one of the possible conversions of
our derivatives to enhance binding affinity to the RNase H domain.
The difficulty in developing an RNase H inhibitor for practical
use lies in the specificity and toxicity. In spite of much effort to en-
hance the inhibitory potency, the 50% inhibitory concentrations of
many compounds reported so far are still in the order of sub-micro
molar and they often lack sufficient specificity for HIV-1 RT-associ-
ated RNase H activity. Most problematically, they sometime dis-
play cytotoxicity to mammalian cells. Many previous compounds
have shown little inhibitory activity in an in vitro cell culture rep-
lication assay. The derivatives synthesized in this work have a scaf-
fold different from that of the previously reported inhibitors. It was
shown in our previous study17 that the inhibitory potency of the
hit chemical was highly specific to RNase H of retrovirus and the
hit chemical further displayed an inhibitory activity in a cell cul-
ture replication assay. The present study showed that our deriva-
tives had little cytotoxicity and that the chemical conversion at a
part other than the 5-nitro-furan-2-carboxylic moiety increased
the inhibitory activity. Moreover, there is still much room for mod-
ulation of the chemical structure. Accordingly, the analogues bear-
ing the scaffold addressed in this study are good candidates for
anti-HIV-1 drugs acting on RT-associated RNase H.
5. Summary
RNase H activity of reverse transcriptase is an attractive target of
an antiviral agent for HIV-1 that is not yet addressed by currently
approved drugs. A series of chemical compounds were synthesized
on the basis of a hit chemical found in our previous in vitro screen-
ing. Inhibition of RNase H enzymatic activity was measured in a
biochemical assay with a real-time fluorescence monitoring tech-
nique. Conversion of the nitro-furan group into other chemical
structures drastically decreased the inhibitory activity except for
nitro-thiophene. This means that the structural basis of nitro-furan
is indispensable for inhibitory activity induced by analogues of the
hit chemical. No notable change was observed in inhibitory potency
when the hydrophobic moiety located at the opposite part of nitro-
furan was modulated. This indicates that the modulated region has
little interaction with the RNase H domain. Theoretical calculation
with QM/MM method suggested the binding mode of the synthe-
sized compounds to RNase H reaction active site. The characteristic
property of the nitro-furan group is large electric polarity. Since
oxygen atoms are negatively charged, these oxygen atoms will be
strongly coordinated to divalent metal ions of the active site. The
findings obtained in this work will be informative for designing po-
tent inhibitors of RNase H enzymatic activity.
Figure 2. Optimized binding structures of the active compound to the RNase H
domain, obtained by QM/MM calculation. (a), (b) and (c) correspond to compounds
(3), (7) and (10), respectively. Chemical structures of the compounds are shown at
the right top. Inhibitor compound and several polar residues are shown in stick
representation. Two Mg2+ ions are denoted by spheres. Inter-atomic distances are
shown in units of Å.
Acknowledgments
This work was supported by a Health and Labor Science
Research Grant for Research on Publicly Essential Drugs and Med-
ical Devices from the Ministry of Health and Labor of Japan. A part
of this work was also supported by a Grant-in-Aid for Scientific Re-
search (C) from Japan Society for the Promotion of Science (JSPS).
Theoretical calculations were performed at the Research Center
for Computational Science, Okazaki, Japan and at the Information
Technology Center of the University of Tokyo and also by the
high-performance computer system at Institute for Media Informa-
tion Technology in Chiba University.
with coordinating to two divalent metal ions, while the interaction
of other moiety is moderate.
Close observation of the optimized geometry by QM/MM calcu-
lation indicates that there is some space between the RNase H do-
main and inhibitory compounds around the ether oxygen at the
ester linkage. This suggests that a few water molecules occupy
the space when the compounds are bound to the RNase H domain.

H. Yanagita et al. / Bioorg. Med. Chem. 19 (2011) 816–825
825
16. Budihas, S. R.; Gorshkova, I.; Gaidamakov, S.; Wamiru, A.; Bona, M. K.; Parniak,
M. A.; Crouch, R. J.; McMahon, J. B.; Beutler, J. A.; Le Grice, S. F. Nucleic Acids Res.
2005, 33, 1249.
17. Fuji, H.; Urano, E.; Futahashi, Y.; Hamatake, M.; Tatsumi, J.; Hoshino, T.;
Morikawa, Y.; Yamamoto, N.; Komano, J. J. Med. Chem. 2009, 52, 1380.
18. Davies II, J. F.; Hostomska, Z.; Hostomsky, Z.; Jordan, S. R.; Matthews, D. A.
Science 1991, 252, 88.
19. Klumpp, K.; hang, J. Q.; Rajendran, S.; Yang, Y.; Derosier, A.; Wong Kai In, P.;
Overton, H.; Parkes, K. E.; Cammack, N.; Martin, J. A. Nucleic Acids Res. 2003, 31,
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.12.011.
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