Biologically active oligodeoxyribonucleotides, 5. 5'-end-substituted d(TGGGAG) possesses anti-human immunodeficiency virus type 1 activity by forming a G-quadruplex structure

Article abstract of DOI:10.1021/jm970658w

A series of hexadeoxyribonucleotides (6-mers), d(TGGGAG), substituted with a variety of aromatic groups at the 5'-end were synthesized and tested for anti-human immunodeficiency virus type 1 (HIV-1) activity. While unmodified d(TGGGAG) (31) had no anti-HI

Full text of DOI:10.1021/jm970658w

J . Med. Chem. 1998, 41, 3655-3663
3655
Biologica lly Active Oligod eoxyr ibon u cleotid es. 5.1 5′-En d -Su bstitu ted
d (TGGGAG) P ossesses An ti-Hu m a n Im m u n od eficien cy Vir u s Typ e 1 Activity by
F or m in g a G-Qu a d r u p lex Str u ctu r e
Hitoshi Hotoda,*,† Makoto Koizumi,† Rika Koga,† Masakatsu Kaneko,† Kenji Momota,‡ Toshinori Ohmine,‡
Hidehiko Furukawa,‡ Toshinori Agatsuma,‡ Takashi Nishigaki,‡ J unko Sone,§ Shinya Tsutsumi,§
Toshiyuki Kosaka,§ Koji Abe,§ Satoshi Kimura,|,# and Kaoru Shimada|,
Exploratory Chemistry Research Laboratories, Biological Research Laboratories, and Analytical and Metabolic Research
Laboratories, Sankyo Company, Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, J apan, and The Department of
Infectious Diseases, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku,
Tokyo 108, J apan
Received September 29, 1997
A series of hexadeoxyribonucleotides (6-mers), d(TGGGAG), substituted with a variety of
aromatic groups at the 5′-end were synthesized and tested for anti-human immunodeficiency
virus type 1 (HIV-1) activity. While unmodified d(TGGGAG) (31) had no anti-HIV-1 activity,
compound 23 with a 3,4-di(benzyloxy)benzyl (DBB) group at the 5′-end potently inhibited the
HIV-1IIIB-induced cytopathicity of MT-4 cells in vitro (IC50 ) 0.37 µM) without cytotoxicity up
to 40 µM. A thermal denaturation study on the 5′-end-substituted 6-mers by means of the
circular dichroism (CD) spectra demonstrated that the aromatic substituent attached at the
5′-end of the 6-mer strongly enhanced the formation of a parallel helical structure consisting
of four strands (quadruplex). On the contrary, compound 36, in which one of the guanosines
of 23 was replaced by a thymidine, did not form a quadruplex, thus exhibiting no anti-HIV-1
activity. Moreover, both compound 15, with a tert-butyldiphenylsilyl group solely at its 3′-
end, and compound 21, with a relatively small substituent, a benzyl group, at the 5′-end, formed
quadruplexes but had no anti-HIV-1 activity. These findings led us to the conclusion that
both the quadruplex structure and the aromatic substituent with adequate size at the 5′-end
are crucial for the interaction of the 5′-end-substituted 6-mers with the V3 loop as well as the
CD4 binding site on viral gp120, resulting in anti-HIV-1 activity.
In tr od u ction
thrombin-catalyzed fibrin-clot formation in vitro. Ojwang
et al.9 reported that a heptadecadeoxyribonucleotide (5′-
GTGGTGGGTGGGTGGGT-3′), which had single phos-
phorothioate internucleoside linkages at its 5′- and 3′-
ends (T30177, also known as AR177), inhibited HIV
integrase activity in HIV-infected cells. T30177 proved
to favor the formation of a compact, intramolecularly
folded structure dominated by two stacked guanosine-
quartets (G-quartets), resulting in resistance to
nucleases.9-11 Wyatt et al.12,13 reported that a phos-
phorothioate-type octadeoxyribonucleotide with a 5′-
TTGGGGTT-3′ sequence (ISIS5320, Chart 1) formed a
tetrameric structure arranged in a parallel helix stabi-
lized by G-quartets and bound to the cationic V3 loop
region of the viral envelope glycoprotein gp120. As a
result, both cell-to-cell transmission and virus-to-cell
transmission of HIV-1 were inhibited by submicromolar
concentrations of ISIS5320.
Since antisense oligodeoxyribonucleotides (AS-ODNs)
can interfere with transcription, mRNA processing, or
translation by the formation of a specific duplex with
the cognate mRNA, the development of these com-
pounds as potential therapeutic agents has received
increasing attention in recent years.2 There are a
number of reports regarding the biological activities of
both the AS-ODNs having natural-type oligodeoxyribo-
nucleotide (ODN) structures3 and the backbone-modified
AS-ODNs that are designed to be resistant to enzymatic
degradation4,5 or more effectively transported into living
cells than the natural-type counterpart.6,7
While the AS-ODN binds to the cognate mRNA, there
are some other ODNs that bind to specific proteins in
place of mRNAs. Bock et al.8 reported that a penta-
decadeoxyribonucleotide (15-mer) with a 5′-GGTTGGT-
GTGGTTGG-3′ sequence bound to thrombin to inhibit
For the last several years, we have studied the anti-
HIV-1 activity of ODNs with natural-type phosphodi-
ester backbone. First, the 15-mers complementary to
six sites in HIV-1 RNA were tested for anti-HIV-1
activity, and all 15-mers were found to be inactive in
our assay system. Unexpectedly, a synthetic precursor
of one of the six 15-mers, with a 4,4′-dimethoxytrityl
(DMTr) group at the 5′-end, complementary to the tat
second splicing acceptor region of HIV-1 (5′-TGGGAG-
GTGGGTCTG-3′) (1), was found to be the only active
* Author for correspondence. Fax: (81) 3-5436-8570. E-mail:
hotoda@shina.sankyo.co.jp.
† Exploratory Chemistry Research Laboratories, Sankyo Co., Ltd.
‡ Biological Research Laboratories, Sankyo Co., Ltd.
§ Analytical and Metabolic Research Laboratories, Sankyo Co., Ltd.
| The University of Tokyo.
# Current address: Department of Infection Control and Prevention,
The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113,
J apan.
Current address: Tokyo Senbai Hospital, 1-4-3 Mita, Minato-ku,
Tokyo 108, J apan.
S0022-2623(97)00658-4 CCC: $15.00 © 1998 American Chemical Society
Published on Web 08/15/1998

3656 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19
Hotoda et al.
Ch a r t 1
Sch em e 1a
a
This stands for 5′-phosphorylated nonanucleotide with a 5′-
GTGGGTCTG-3′ sequence bound to the 3′-end of 2 via a phos-
phodiester linkage.
a
Reagents: (a) TBDPSCl, imidazole, DMF; (b) (PhS)2P(O)O-H3-
N+C6H11, 2,4,6-triisopropylbenzenesulfonyl chloride, 1H-tetrazole,
pyridine.
compound of all the compounds tested.14 The following
study on the structure-activity relationship (SAR)
demonstrated that an aromatic group attached at the
5′-end strongly enhanced the anti-HIV-1 activity of the
15-mer.15 Recently, the 15-mer with a DMTr group at
its 5′-end proved to interfere with both viral adsorption
to the cell membrane and cell fusion (syncytium forma-
tion) by interacting with both the V3 loop and the CD4
binding site on the HIV envelope glycoprotein gp120,
instead of the so-called “antisense” fashion.16 A recent
study has also revealed that the 5′-region of the 5′-end-
substituted 15-mer containing hexadeoxyribonucleotide
(6-mer), 5′-TGGGAG-3′ (2), is important for its anti-
HIV-1 activity.17,18 ODN 2 also proved to exhibit anti-
HIV-1 activity by the same mechanism of action as the
5′-end-substituted 15-mer 1.18 This paper describes the
SAR of the 5′-end-substituted 6-mers and their confor-
mations in solution.
Sch em e 2a
a
Reagents: (a) Et3N, H2O, pyridine; (b) TFA, CHCl3; (c) 2,4,6-
Ch em istr y
triisopropylbenzenesulfonyl chloride, 3-nitro-1,2,4-triazole, pyri-
dine.
The 6-mers with a 5′-TGGGAG-3′ sequence and
attaching a tert-butyldiphenylsilyl (TBDPS) group at the
3′-end were synthesized by the liquid-phase phospho-
triester method. First, both 3′-O-TBDPS-guanosine
derivative 4 and 5′-O-TBDPS-thymidine derivative 6
were synthesized as shown in Scheme 1. Next, the
dimer unit of guanylyl-(3′f5′)-adenosine 9 was synthe-
sized according to Hata’s procedure as follows (Scheme
2).19,20 One of two P-S bonds of 7 was cleaved by
selective hydroxylation using Et3N-H2O-pyridine, fol-
lowed by condensation with the compound obtained by
the acidic treatment of 8 to give the dimer 9.
Scheme 3 illustrates the block condensation procedure
used to obtain the 6-mers with TBDPS groups. One of
two PhS groups at the 3′-end of 9 was selectively
removed using triethylammonium hypophosphate, fol-
lowed by condensation with the compound obtained by
the acidic treatment of 4 to give the fully protected
trimer 10. The above procedure was repeated using the
dimer unit 11 and thymidine monomers 13 or 6 to give
two types of protected hexamers (14a ,b). Deprotection
of 14a containing one TBDPS group was carried out as
follows. Selective hydroxylation of the P-S bonds of
14a using aqueous NaOH and the ammonolysis of the
protecting groups bound to the nucleoside bases followed
by the removal of the DMTr group attached at the 5′-
end using aqueous acetic acid led to the desired com-
pound 15 with a TBDPS group solely at its 3′-end.
Deprotection of 14b was carried out the same way as
deprotection of 14a , excluding treatment with aqueous
acetic acid, to give the desired compound 16 with two
TBDPS groups, one at each end. Compounds 36 and
37 were purchased from GENSET.
The rest of the 5′-end-substituted 6-mers were syn-
thesized according to the solid-phase phosphoramidite
method using an automatic DNA synthesizer by simple
modification of the nucleotide sequence described in a
previous paper.15
All compounds tested were purified using preparative
reverse-phase HPLC before being used in biological
assay. Negative ion LSI mass spectroscopy21 has proved
to be a good method for rapid confirmation of the
structures (see Supporting Information).
Resu lts a n d Discu ssion
To evaluate the anti-HIV-1 activity of the 5′-end-
substituted 6-mers along with an unmodified 6-mer,

Biologically Active Oligodeoxyribonucleotides
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19 3657
Sch em e 3a
a
Reagents: (a) TFA, CHCl3; (b) 3.3 M H3PO4, Et3N, pyridine; (c) 2,4,6-triisopropylbenzenesulfonyl chloride, 3-nitro-1,2,4-triazole, pyridine;
(d) (i) 0.1 N NaOH, H2O, pyridine, (ii) 28% aqueous NH4OH; (e) AcOH, H2O.
Ta ble 1. In Vitro Anti-HIV-1 Activity of 5′-End-Modified
6-mers. As shown in Table 1, the 6-mer substituted
with a trityl group (17) in place of a DMTr group had
d(TGGGAG)a
relatively potent anti-HIV-1 activity along with moder-
ate cytotoxicity like 2. Compounds 18 and 19, in which
two of three aromatic moieties of the tertiary alkyl
substituents were linked covalently, showed no cyto-
toxicity up to 40 µM but potent anti-HIV-1 activity.
Compound 20 with a secondary alkyl (benzhydryl) group
showed moderate anti-HIV-1 activity without cytotox-
icity, and compound 21 with a primary alkyl (benzyl)
group showed no anti-HIV-1 activity and no cytotoxicity.
These results demonstrate that reduction in the number
of phenyl groups of 17 resulted in a reduction of both
anti-HIV-1 activity and cytotoxicity. Next, the phenyl
group of 21 was further substituted with some aromatic
groups. The anti-HIV-1 activity of 22 with a benzyloxy
group as an extra substituent of 21 was moderate.
Addition of another benzyloxy group to 22 gave 23 and
24, both of which showed very potent anti-HIV-1 activ-
ity. The anti-HIV-1 activity of 6-mers containing bi-
phenyl moieties (25 and 26) or fused aromatics (27 and
28) as substituents was moderate. A TBDPS group was
also found to be as effective as the 5′-substituent of the
6-mer (29). However, the 6-mer with a tert-butyldi-
methylsilyl (TBDMS) group (30) showed no anti-HIV-1
activity and potent cytotoxicity. As a result, compound
23 with a 3,4-di(benzyloxy)benzyl (DBB) group was the
most potent of all candidates listed in Table 1.
compd
R
IC50 (µΜ)b
CC50 (µΜ)c
2
17
18
19
20
21
22
23
24
25
26
27
28
29
30
(4-MeO-C6H4)2PhC
Ph3C
9-Ph-xanthen-9-yl
9-Ph-fluoren-9-yl
Ph2CH
1.0 ( 0.21
38
23 ( 2.4
0.80 ( 0.10
0.95 ( 0.18 >40
1.3 ( 0.16
4.3 ( 0.71
>40
>40
>40
>40
PhCH2
>40
4-(PhCH2O)-C6H4CH2
3,4-(PhCH2O)2-C6H3CH2
3,5-(PhCH2O)2-C6H3CH2
2-Ph-C6H4CH2
4-Ph-C6H4CH2
2-naphthyl-CH2
1-pyrenyl-CH2
tBu(Ph)2Si
1.9 ( 0.35
0.37 ( 0.060 >40
0.49 ( 0.064 >40
1.2 ( 0.19
2.8 ( 0.66
2.0 ( 0.44
1.8 ( 0.24
>40
>40
>40
>40
0.88 ( 0.070 >40
>40 1.2 ( 0.82
tBu(Me)2Si
a
Values represent the mean of duplicate or quadruplicate (
b
SD. The 50% inhibitory doses for the cytopathicities induced by
HIV-1IIIB were determined in vitro by MTT assay using MT-4 cells.
c The 50% cytotoxic doses were determined by MTT assay using
MT-4 cells.
d(TGGGAG), the 50% inhibitory concentrations (IC50)
for inhibiting the cytopathicity induced by HIV-1IIIB
were determined in vitro by MTT assay using MT-4
cells.22 The 50% cytotoxic concentrations (CC50) were
also determined using MT-4 cells. The 6-mer d(TGG-
GAG) with a DMTr group at its 5′-end (2) has been
found to possess potent anti-HIV-1 activity with moder-
ate cytotoxicity (Table 1).17,18 Consequently, 6-mers
with a variety of aromatic substituents were examined
initially to clarify the SAR for the 5′-end-substituted
Next, the effect of 5′-end substitution was assessed
by comparing it with 3′-end substitution. A TBDPS
group was used as the substituent because it was
relatively easy to introduce at the 3′-end of the 6-mer.
As shown in Table 2, it is obvious that 5′-end substitu-
tion is crucial for anti-HIV-1 activity. While compound
29 with a TBDPS group at its 5′-end possessed potent
anti-HIV-1 activity, the unmodified 6-mer (31) showed

3658 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19
Hotoda et al.
Ta ble 2. Modification Effect of a TBDPS Group on the in
that is, a helical structure consisting of four ODN
strands, stabilized by the formation of G-quartets.23,24
Accordingly, compound 39 with a sequence containing
five contiguous guanosines could form a G-quadruplex.25
In fact, the purification of 39 by preparative reverse-
phase HPLC was somewhat troublesome because a
major peak with a longer retention time than the peak
of monomeric 39 appeared. This additional peak was
thought to be attributable to the G-quadruplex formed
by self-assembly of 39 under HPLC conditions because
the peak was reproduced by HPLC analysis of the
fraction of monomeric 39 isolated by preparative HPLC.
Vitro Anti-HIV-1 Activity of d(TGGGAG)a
compd
R1
R2
IC50 (µM)b
>40
tBu(Ph)2Si >40
0.88 ( 0.070
6.0
CC50 (µM)c
31
15
29
16
H
H
H
>40
7.7
>40
>40
tBu(Ph)2Si
H
tBu(Ph)2Si tBu(Ph)2Si
To clarify the presence of the G-quadruplex structures
of the 6-mers in solution, the CD spectra of compounds
15, 21, 23, 29, 31-37, and 39 were measured. Figure
1A-G shows the CD spectra of these ODNs in 10 mM
PBS buffer (pH 7) at about 1 A260 unit/mL concentra-
tions (ca. 20 µM). As shown in Figure 1A, the spectrum
of 31, which was not substituted and had no anti-HIV-1
activity, had a positive Cotton effect at 253 nm. The
presence of the 253-nm band is consistent with the CD
spectrum expected by calculation using the nearest-
neighbor method.26 The molecular ellipticity for this
area decreased with the increase in temperature. How-
ever, the temperature dependency of the spectrum of
31 was not so drastic. The clear isosbestic points at 262
and 238 nm demonstrate a two-state transition probably
between disordered monomer and the monomer strand
ordered by intramolecular stacking interactions. On the
contrary, the spectra of 23 having a DBB group at the
5′-end was drastically changed as a function of temper-
ature (Figure 1B). While the spectra of 23 at above 50
°C were almost the same as those of 31, the spectra at
below 40 °C had a strong positive Cotton effect at about
264 nm, which is typical of CD spectra of parallel
quadruplexes with guanine-rich sequences.27 The shift
of the CD band as a function of temperature demon-
strates that 23 forms a parallel G-quadruplex stabilized
by G-quartets (Chart 2) at ambient temperature and
about 20 µM concentration, and the helix dissociates
gradually at above 50 °C. Compound 29 with a TBDPS
group at the 5′-end gave almost the same result as 23
(Figure 1C). Compound 15 having a TBDPS group at
the 3′-end without anti-HIV-1 activity also forms a
G-quadruplex; however, the G-quadruplex of 15 is less
stable than that of 29 (Figure 1D). It is presumed by
comparison of compounds 29 and 15 that the formation
of a G-quadruplex is not enough for anti-HIV-1 activity
and the aromatic substituent must be attached not at
the 3′-end but at the 5′-end. Another example is 21
having a relatively small substituent, a benzyl group,
at the 5′-end. Compound 21 having no anti-HIV-1
activity also forms a G-quadruplex (Figure 1E). This
result demonstrates that the 5′-substituents of the
G-quadruplex-forming ODNs must have enough size (or
suitable number of phenyl groups) as mentioned above.
Next, the 5′-substituent was fixed with the most suitable
DBB group, and the sequence effect was examined.
Figure 1F shows the CD spectra for the ODNs truncated
gradually from the 3′-end of 23. While compounds 23,
32, and 33 having anti-HIV-1 activity proved to form
G-quadruplexes, compounds 34 and 35 without anti-
HIV-1 activity were too short to form G-quadruplexes.
Moreover, compound 36, in which one of the guanosines
a-c See legends of Table 1.
Ta ble 3. Sequence Effect on in Vitro Anti-HIV-1 Activity of
ODNs Having a 3,4-Di(benzyloxy)benzyl Groupa
compd
sequence
IC50 (µΜ)b
CC50 (µΜ)c
23
32
33
34
35
36
37
38
39
40
41
42
TGGGAG
TGGGA
TGGG
TGG
TG
TGTGAG
TTTTTT
TGGGG
TGGGGG
TTGGGG
TTGGG
TTGG
0.37 ( 0.060
1.9 ( 0.24
4.6 ( 0.56
>40
>40
>40
>70
>70
>100
>24
>100
>24
>24
>24
0.66 ( 0.036
0.26 ( 0.032
0.56 ( 0.15
>40
>40
>40
>40
>50
40
33 ( 2.4
a-c See legends of Table 1.
no anti-HIV-1 activity and no cytotoxicity up to 40 µM.
Compound 15 with a TBDPS group solely at its 3′-end
showed no anti-HIV-1 activity with moderate cytotox-
icity. The additional substitution of another TBDPS
group at the 3′-end of 29 reduced the anti-HIV-1 activity
(16).
The results obtained so far indicate that the 5′-end
substitution of the 6-mer is essential for anti-HIV-1
activity and the DBB group is the most effective
substituent of all tested. The remaining part of the SAR
study was to examine the sequence effect of the 6-mer.
Thus, the 5′-end substituent was fixed with a DBB
group, and the sequence of the ODN was changed.
First, the nucleotide sequence of 23 was truncated
gradually from the 3′-end toward the 5′-direction (32-
35). As shown in Table 3, it is obvious that the anti-
HIV-1 activity becomes less potent as the nucleotide
chain is decreased. Compound 33, with a TGGG
sequence, was found to contain the minimum structural
requirement for anti-HIV-1 activity in our assay system.
While the 4,4′-dimethoxytritylated ODNs with se-
quences TGGGAG, TGGGA, TGGG, and TGG possessed
moderate cytotoxicity,17,18 compounds 23 and 32-35
containing a DBB group in place of a DMTr group
showed no cytotoxicity up to 40 µM. Both compounds
36, in which one of the guanosines of 23 was replaced
by a thymidine, and 37, with a TTTTTT sequence, were
inactive. Compound 39, in which the adenosine of 23
was replaced by a guanosine, was found to be more
potent than the parent compound 23. The ODN con-
taining a TT sequence at its 5′-end was not appropriate
because of its tendency to exert moderate cytotoxicity
(41, 42). It is well-known that an ODN with a high
content of guanosine forms a G-quadruplex structure,

Biologically Active Oligodeoxyribonucleotides
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19 3659
F igu r e 1. CD spectra for the 5′-end-modified 6-mers. A-E, G: CD spectra for ca. 1 A260 unit/mL concentrations (ca. 20 µM) of
31 (A), 23 (B), 29 (C), 15 (D), 21 (E), and 39 (G) in 10 mM PBS buffer (pH 7) at 20 °C (black), 30 °C (blue), 40 °C (red), 50 °C
(green), 60 °C (pink), 70 °C (light blue), and 80 °C (light brown). F: CD spectra for ca. 1 A260 concentrations of 23 and 32-37 in
10 mM PBS buffer (pH 7) at ambient temperature. H: CD spectra for ca. 0.5 µM concentrations of G-quadruplex-forming ODNs;
(+), anti-HIV-1 active; (-), anti-HIV-1 inactive.
of 23 was replaced by a thymidine, did not form a
G-quadruplex, thus exhibiting no anti-HIV-1 activity.
Inactive compound 37 with a TTTTTT sequence did not
form a G-quadruplex, as was to be expected because of
the absence of guanosine. The above results suggest
the apparent relationship between G-quadruplex forma-
tion and anti-HIV-1 activity. The spectrum of 39 with
a 5′-TGGGGG-3′ sequence indicates that 39 shows a
very thermodynamically stable G-quadruplex (Figure
1G). This is attributable to the presence of five contigu-
ous guanosines as discussed above. The G-quadruplex
of 39 hardly dissociated even at 80 °C. Figure 1H shows
the CD spectra of G-quadruplex-forming ODNs at about
0.5 µM concentrations in PBS buffer at ambient tem-
perature. All the CD spectra still had a positive Cotton
effect at around 264 nm, demonstrating that these
ODNs form G-quadruplexes at the minimum concentra-
tions (about IC50 for 23) required for anti-HIV-1 activity.
Finally, the stability of 23 and 31 in human plasma
was assessed in vitro. As shown in Figure 2, an
apparent stabilization effect by the modification at the
5′-end of 31 was observed. The half-lives of 23 and 31
were roughly 1 h and 15 min, respectively. The nu-
clease resistance of 23 may be partially attributable to
the G-quadruplex structure containing G-quartet mo-
tifs.11

3660 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19
Hotoda et al.
coagulation, (iii) complement activation, and (iv) liver
and kidney toxicity, that are unrelated to their intended
antisense activity.30 Compound 23 with a natural-type
phosphodiester backbone might not have the drawbacks
of PS-ODNs.
Recently, Lacoste et al.31 reported that an acridine
moiety attached at the 5′-end of triplex-forming ODN
stabilized the triplex by intercalation of the acridine ring
at the triplex/duplex junction. Bates et al.32 successfully
used a psoralen moiety as a 5′-end substituent of triplex-
forming ODN to form triplex-directed photoadduct. As
to the stability of the quadruplex, Wolfe et al.33 reported
that a cholesteryl substituent attached at the 3′-
terminal phosphodiester bond of the phosphorothioate
20-mer, 5′-TGGGGCTTACCTTGCGAACA-3′, stabilized
the quadruplex structure. In our present study, thermal
denaturation of the 5′-end-substituted 6-mers measured
by CD spectra indicated that the aromatic substituents
strongly enhanced the formation of G-quadruplex struc-
tures. In both cases, hydrophobic interactions of the
substituents might have contributed to the stability of
the structure.
F igu r e 2. Stability of 23 (9) and 31 (b) in 93% human
plasma. Initial concentration of ODN: 20 µg/mL; n ) 3; mean
( SD. ODNs 23 and 31 were incubated with human plasma,
and these reaction mixtures were analyzed by reverse-phase
HPLC at 0, 0.25, 0.5, 1, 2, and 4 h after initial mixing to
quantify the amount of ODNs remaining.
Ch a r t 2. Parallel G-Quadruplex Structure (A) and
G-Quartet (B)
All ODNs with anti-HIV-1 activity in the present
study form G-quadruplexes. In addition, compound 36,
in which one of the guanosines of 23 was simply
replaced by a thymidine, did not form a G-quadruplex,
thus exhibiting no anti-HIV-1 activity. These results
suggest that the G-quadruplex structures are the ulti-
mate active species. On the other hand, both compound
15 with a 3′-end substituent and compound 21 with a
relatively small 5′-end substituent, a benzyl group, form
G-quadruplexes but have no anti-HIV-1 activity. These
findings led us to the conclusion that both the G-
quadruplex structure and the aromatic substituent with
adequate size at the 5′-end were crucial for anti-HIV-1
activity. The mechanism for anti-HIV-1 activity of 5′-
end-substituted 6-mers has been elucidated as the
interference of both cell-to-cell transmission and virus-
to-cell transmission of HIV-1 by interaction of the
6-mers with both the V3 loop and the CD4 binding site
on viral gp120.17,18 We presume that both the G-
quadruplex-forming ODN backbone and the cluster of
5′-end substituents might have contributed to the
interaction of the 6-mers with viral gp120. At present,
the results of the CD experiment are the only clues for
determining the fundamental conformation of 23 which
possesses potent anti-HIV-1 activity. Further experi-
ments involving NMR analysis are currently underway
in an attempt to gain insight into the structural and
conformational requirements of this series of com-
pounds.
Con clu sion
Our previous efforts in both synthetic and biological
studies on 5′-end-substituted ODNs with anti-HIV-1
activity have led to the identification of non-antisense
lead compound 2 with a 5′-TGGGAG-3′ sequence.17,18,28
The present study dealt with the optimization of both
the 5′-end substituent and the sequence of the ODN. A
3,4-di(benzyloxy)benzyl (DBB) group was found to be
the best substituent of all tested with respect to both
potent anti-HIV-1 activity and low cytotoxicity. In
terms of optimal sequence, compound 39 with a 5′-
TGGGGG-3′ sequence was perhaps the best on the basis
of the anti-HIV-1 activity. Fujihashi et al.29 reported
that oligodeoxyriboguanylic acids with a chain length
of more than three nucleotides and their oligoribogua-
nylic acid counterparts both inhibited cytopathicity
induced by HIV-1 in vitro. Since these oligonucleotides
containing contiguous guanosines have a tendency to
form tetrameric aggregates, the manipulation of these
ODNs involving 39 on a large scale was expected to be
troublesome. Therefore, we concluded that compound
23, which showed a simple profile on HPLC analysis,
was superior to 39 on the basis of both potent anti-
HIV-1 activity and ease of manipulation.
Exp er im en ta l Section
Ch em ist r y. 1H NMR spectra were recorded on a J EOL
J NM-EX 270 spectrometer (270 MHz) with tetramethylsilane
as an internal standard. UV absorption spectra were recorded
on a HITACHI U-3210 spectrophotometer. CD spectra were
recorded on a J ASCO J -500C spectrometer. Negative ion LSI
mass spectra were recorded on a VG 70-4SE using 3-nitroben-
zyl alcohol as a matrix. TLC was done on Merck Kieselgel 60
F
254 precoated plates. Column chromatography was performed
with Merck Kieselgel 60 (70-230 mesh). HPLC was per-
formed on a HITACHI 655A-11 liquid chromatograph equipped
with an L-5000 LC controller, an L-3000 photodiode array
detector, and a D-2500 chromato-integrator.
Compounds 17-35 and 38-42 were synthesized using a
Cyclone Plus DNA synthesizer equipped with additional
In vitro anti-HIV-1 activity of 23 has proved to be
almost the same as that of ISIS5320 (IC50 ) 0.3 µM), a
phosphorothioate (PS) octamer.12 PS-ODNs have ap-
peared to possess a number of biological effects, i.e., (i)
nonspecific binding to protein, (ii) prolonged blood

Biologically Active Oligodeoxyribonucleotides
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19 3661
reagents, 5′-end-substituted thymidine derivatives, which were
synthesized according to the previous method.15 Compounds
36 and 37 were purchased from GENSET. Compounds 15 and
16 were synthesized on the basis of Hata’s procedure.19 Thus,
intermediates 3, 7, 8, 11, and 13 were synthesized according
to the literature.20 Intermediate 5 was also synthesized
according to the literature.15 Syntheses of other intermediates
(4, 6, 9, 10, 12, and 14a ,b) and desired compounds 15 and 16
were performed as follows.
diastereomers: 1H NMR (CDCl3) δ 9.02 and 9.00 (2s, 1H), 8.24
and 8.22 (2s, 1H), 8.14 (m, 1H), 8.05 (m, 1H), 7.65-7.15 (m,
39H), 6.81 (m, 4H), 6.40 (m, 1H), 6.18-6.04 (m, 1H), 5.57 and
5.43 (2m, 1H), 5.32 (m, 1H), 4.73-4.46 (m, 2H), 4.33-4.18 (m,
2H), 3.72 (s, 6H), 3.48-3.32 (m, 2H), 3.00-2.80 (m, 4H), 2.60-
2.38 (m, 1H), 1.18 (m, 3H); HRMS (FAB) m/z 1616.3867 (M +
Na)+
∆
) -0.4.
ppm
[S-P h en yl 5′-O-(4,4′-d im eth oxytr ityl)-N2-p r op ion yl-O6-
(d ip h en ylca r ba m oyl)gu a n osin e-3′-O-p h osp h or oth ioyl]-
(3′f5′)-[S-p h en yl N6-b en zoyla d en osin e-3′-O-p h osp h o-
r o t h io y l]-(3′f5′)-[3′-O -(t er t -b u t y ld im e t h y ls ily l)-N 2-
propionyl-O6-(diphenylcarbamoyl)guanosine](10). Compound
9 (250 mg, 0.17 mmol) was dissolved in 5 M H3PO2-pyridine
(10.2 mL) and triethylamine (5.1 mL), and the solution was
stirred at 40 °C. After 1 h, the mixture was diluted with CHCl3
(100 mL) and washed with 1 M triethylammonium bicarbonate
(TEAB) (pH 8, 3 × 100 mL), and the washings were extracted
with CHCl3 (100 mL). The organic layers were combined,
dried over MgSO4, and then evaporated to give a residue
containing a phosphodiester component. This residue and 4
(151 mg, 0.142 mmol) were subjected to the reactions described
in the synthesis of 9 to give 10 (150 mg, 50%). Compound 10
is a mixture of diastereomers: 1H NMR (CDCl3) δ 8.97 (m,
1H), 8.72-8.53 (m, 1H), 8.23 (m, 1H), 8.16-7.98 (m, 4H), 7.70-
7.04 (m, 54H), 6.80 (m, 4H), 6.40 (m, 2H), 6.13 (m, 1H), 5.41
(m, 1H), 5.25 (m, 1H), 4.68-3.92 (m, 8H), 3.74 (s, 3H), 3.72 (s,
3H), 3.47-3.27 (m, 2H), 3.05-2.12 (m, 10H), 1.28-0.70 (m,
15H).
[S-P h en yl 5′-O-(4,4′-d im eth oxytr ityl)-N2-p r op ion yl-O6-
(d ip h en ylca r ba m oyl)gu a n osin e-3′-O-p h osp h or oth ioyl]-
(3′f5′)-[S-p h en yl N2-p r op ion yl-O6-(d ip h en ylca r ba m oyl)-
gu a n osin e-3′-O-p h osp h or oth ioyl]-(3′f5′)-[S-p h en yl N2-
p r o p io n y l-O 6 -(d ip h e n y lc a r b a m o y l)g u a n o s in e -3′-O -
ph osph or oth ioyl]-(3′f5′)-[S-ph en yl N6-ben zoyladen osin e-
3′-O-p h osp h or oth ioyl]-(3′f5′)-[3′-O-(ter t-bu tyld im eth yl-
silyl)-N2-p r op ion yl-O6-(d ip h en ylca r b a m oyl)gu a n osin e]
(12). Compounds 10 (159 mg, 0.071 mmol) and 11 (139 mg,
0.085 mmol) were subjected to the reactions described in the
synthesis of 10 to give 12 (87.6 mg, 37%). Compound 12 is a
mixture of diastereomers: 1H NMR (CDCl3) δ 8.92-8.57 (m,
2H), 8.20-7.95 (m, 6H), 7.70-6.90 (m, 87H), 6.74 (m, 4H),
6.45-6.07 (m, 5H), 5.29 (m, 4H), 4.60-3.90 (m, 14H), 3.68 (br
s, 6H), 3.45-3.22 (m, 2H), 2.85-2.20 (m, 18H), 1.30-0.70 (m,
21H).
3′-O-(ter t-Bu tyld im eth ylsilyl)-5′-O-(4,4′-d im eth oxytr i-
tyl)-N2-p r op ion yl-O6-(d ip h en ylca r ba m oyl)gu a n osin e (4).
Alcohol 3 (164 mg, 0.2 mmol) and imidazole (27 mg, 0.4 mmol)
were dissolved in DMF (1 mL). To the solution was added
tert-butyldimethylsilyl chloride (78 µL, 0.3 mmol), and the total
mixture was stirred at room temperature. After 42 h, 5%
aqueous NaHCO3 (50 mL) was added, and the resulting
mixture was extracted with EtOAc (50 mL). The organic layer
was washed with 5% aqueous NaHCO3 (50 mL) and dried over
MgSO4. The solvent was removed by evaporation, and the
residue was eluted on a silica gel column (20 g) with 1%
MeOH-CH2Cl2 to give 4 (151 mg, 71%). Rotational isomers
were observed: 1H NMR (CDCl3) δ 7.96 and 7.81 (2s, 1H),
7.63-6.68 (m, 33H), 4.52 (br s, 1H), 4.22 (br s, 1H), 3.79 and
3.72 (2s, 6H), 3.07 (m, 2H), 2.80 (m, 2H), 2.43 (m, 2H), 1.19 (t,
J ) 7.3 Hz, 3H), 1.07 (s, 9H); HRMS (FAB) m/z 1059.4476 (M
+ H)+
∆
) 0.
ppm
S,S′-Dip h en yl 5′-O-(ter t-Bu tyld ip h en ylsilyl)th ym id in e-
3′-O-p h osp h or od ith ioa te (6). Alcohol 5 (48 mg, 0.1 mmol),
cyclohexylammonium S,S′-diphenylphosphorodithioate (76 mg,
0.2 mmol), and 1H-tetrazole (14 mg, 0.2 mmol) were coevapo-
rated with pyridine, and the residue was dissolved in pyridine
(1 mL). To the solution was added 2,4,6-triisopropylbenzene-
sulfonyl chloride (60 mg, 0.2 mmol), and the total mixture was
stirred at room temperature. After 1 h, the reaction mixture
was diluted with EtOAc (50 mL), and the solution was washed
with 5% aqueous NaHCO3 (2 × 50 mL) followed by drying over
MgSO4. The solvent was removed by evaporation, and the
residue was eluted on a silica gel column (5 g) with 3% MeOH-
CH2Cl2 to give 6 (62 mg, 91%): 1H NMR (CDCl3) δ 8.05 (br s,
1H), 7.67-7.30 (m, 21H), 6.33 (dd, J ) 5.2, 9.3 Hz, 1H), 5.34
(dd, J ) 5.8, 9.5 Hz, 1H), 4.02 (m, 1H), 3.90 (dd, J ) 2.1, 11.7
Hz, 1H), 3.81 (dd, J ) 2.1, 11.7 Hz, 1H), 2.39 (dd, J ) 5.3,
13.9 Hz, 1H), 2.20 (m, 1H), 1.54 (s, 3H), 1.09 (s, 9H); HRMS
(FAB) m/z 745.1972 (M + H)+ ∆ppm ) -2.6. Anal. (C38H41N2O6-
S2PSi‚0.5H2O) C, H, N, S, P.
[S-P h en yl 5′-O-(4,4′-d im eth oxytr ityl)-N2-p r op ion yl-O6-
(d ip h en ylca r ba m oyl)gu a n osin e-3′-O-p h osp h or oth ioyl]-
(3′f5′)-[S,S′-d ip h en yl N6-b en zoyla d en osin e-3′-O-p h os-
ph or odith ioate] (9). S,S′-Diphenyl 5′-O-(4,4′-dimethoxytrityl)-
N6-benzoyladenosine-3′-O-phosphorodithioate (8) (856 mg, 1
mmol) was dissolved in CHCl3 (100 mL) and then stirred in
an ice-water bath. To the solution was added trifluoroacetic
acid (2 mL), and the mixture was stirred for 10 min. The
mixture was neutralized by pyridine (3 mL), washed with 5%
aqueous NaHCO3 (2 × 50 mL), dried over MgSO4, and then
evaporated to give a residue containing a 5′-hydroxyl compo-
nent. Separately, S,S′-diphenyl 5′-O-(4,4′-dimethoxytrityl)-N2-
propionyl-O6-(diphenylcarbamoyl)guanosine-3′-O-phosphoro-
dithioate (7) (1.225 g, 1.2 mmol) was treated with pyridine (12
mL), triethylamine (12 mL), and H2O (6 mL) in an ice-water
bath for 5 min. The temperature was raised to room temper-
ature, and the reaction was continued for 1 h. The solution
was repeatedly coevaporated with pyridine, combined with the
above 5′-hydroxyl component and 3-nitro-1,2,4-triazole (342
mg, 3 mmol), coevaporated with pyridine to dryness, and
dissolved in pyridine (10 mL). To the solution was added 2,4,6-
triisopropylbenzenesulfonyl chloride (606 mg, 2 mmol), and the
total mixture was stirred at room temperature for 1 h. The
mixture was diluted with CH2Cl2 (100 mL) and then washed
with 5% aqueous NaHCO3 (2 × 100 mL), and the washings
were extracted with CH2Cl2 (50 mL). The organic layers were
combined, dried over MgSO4, and then evaporated. Chroma-
tography (70-230 mesh silica gel, MeOH-CH2Cl2, 2:98)
provided 9 (1.227 g, 84%). Compound 9 is a mixture of
[S-P h en yl 5′-O-(4,4′-d im eth oxytr ityl)th ym id in e-3′-O-
p h osp h or oth ioyl]-(3′f5′)-[S-p h en yl
N2-p r op ion yl-O6-
(d ip h en ylca r ba m oyl)gu a n osin e-3′-O-p h osp h or oth ioyl]-
(3′f5′)-[S-phenylN2-propionyl-O6-(diphenylcarbamoyl)guanosine-
3′-O-p h osp h or oth ioyl]-(3′f5′)-[S-p h en yl N2-p r op ion yl-O6-
(d ip h en ylca r ba m oyl)gu a n osin e-3′-O-p h osp h or oth ioyl]-
(3′f5′)-[S-phenylN6-benzoyladenosine-3′-O-phosphorothioyl]-
(3′f5′)-[3′-O-(ter t-b u t yld im et h ylsilyl)-N2-p r op ion yl-O6-
(d ip h en ylca r ba m oyl)gu a n osin e] (14a ). Compounds 12 (43
mg, 0.013 mmol) and 13 (20 mg, 0.026 mmol) were subjected
to the reactions described in the synthesis of 10 to give 14a
(19.4 mg, 40%). Compound 14a is a mixture of diastereo-
mers: 1H NMR (CDCl3) δ 9.15-8.62 (m, 3H), 8.20-7.90 (m,
6H), 7.73-6.90 (m, 92H), 6.82 (m, 4H), 6.45-6.05 (m, 6H),
5.40-5.12 (m, 5H), 4.60-3.90 (m, 17H), 3.74 (m, 6H), 3.47-
3.12 (m, 2H), 2.85-2.20 (m, 20H), 1.45-0.70 (m, 24H).
[S-P h en yl 5′-O-(ter t-bu tyld im eth ylsilyl)th ym id in e-3′-
O-p h osp h or oth ioyl]-(3′f5′)-[S-p h en yl N2-p r op ion yl-O6-
(d ip h en ylca r ba m oyl)gu a n osin e-3′-O-p h osp h or oth ioyl]-
(3′f5′)-[S-phenylN2-propionyl-O6-(diphenylcarbamoyl)guanosine-
3′-O-p h osp h or oth ioyl]-(3′f5′)-[S-p h en yl N2-p r op ion yl-O6-
(d ip h en ylca r ba m oyl)gu a n osin e-3′-O-p h osp h or oth ioyl]-
(3′f5′)-[S-phenylN6-benzoyladenosine-3′-O-phosphorothioyl]-
(3′f5′)-[3′-O-(ter t-b u t yld im et h ylsilyl)-N2-p r op ion yl-O6-
(d ip h en ylca r ba m oyl)gu a n osin e] (14b). Compounds 12 (43
mg, 0.013 mmol) and 6 (20 mg, 0.03 mmol) were subjected to
the reactions described in the synthesis of 10 to give 14b (13.3
mg, 28%). Compound 14b is a mixture of diastereomers: 1H
NMR (CDCl3) δ 9.20-8.60 (m, 3H), 8.20-7.93 (m, 6H), 7.70-

3662 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19
Hotoda et al.
6.90 (m, 93H), 6.45-6.05 (m, 6H), 5.40-5.12 (m, 5H), 4.60-
3.54 (m, 19H), 2.90-1.93 (m, 20H), 1.45-0.70 (m, 33H).
Tr ieth yla m m on iu m Sa lt of Th ym id in e-3′-O-p h osp h o-
r yl-(3′f5′)-gu a n osin e-3′-O-p h osp h or yl-(3′f5′)-gu a n osin e-
3′-O-phosphoryl-(3′f5′)-guanosine-3′-O-phosphoryl-(3′f5′)-adeno-
sine-3′-O-phosphoryl-(3′f5′)-3′-O-(tert-butyldimethylsilyl)guanosine
(15). Compound 14a (10 mg), pyridine (1 mL) and 0.2 N
aqueous NaOH (1 mL) were stirred in an ice-water bath for
30 min. The mixture was applied on a column of Dowex
50Wx2 (φ1 × 5 cm, pyridinium form), followed by elution with
pyridine-H2O (1:1, v/v, 20 mL). The eluent was evaporated,
and the residue was dissolved in 28% aqueous NH4OH (20
mL). The resulting solution was stirred at room temperature
for 18 h and then at 50 °C for 3 h. The solution was evaporated
to dryness, and the residue was dissolved in AcOH-H2O (4:1,
v/v, 10 mL), stirred at room temperature for 15 min, evapo-
rated to dryness, and then dissolved in 0.1 M triethylammo-
nium acetate (TEAA) (pH 7, 2 mL). The solution was subjected
to preparative HPLC (Inertsil Prep-ODS, φ20 × 250 mm; 0.1
M TEAA, pH 7, 20f50% CH3CN/30 min, 9 mL/min, 254 nm),
and the fraction that eluted at 19.7 min was collected. The
solvent was removed by evaporation, and the residue was
dissolved in H2O (20 mL) and then lyophilized to give the
triethylammonium salt of 15 (46.7 A260 units).
42 (Table 4), PAGE for compounds 17, 20, 21, 23, 28-30, 32-
35, and 38 (Figure 3), and HPLC profile for 39 (Figure 4) (3
pages). Ordering information is given on any current mast-
head page.
Refer en ces
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Tr ieth ylam m on iu m Salt of 5′-O-(ter t-Bu tyldim eth ylsilyl)-
t h y m id in e -3′-O -p h o s p h o r y l-(3′f5′)-g u a n o s in e -3′-O -
p h osp h or yl-(3′f5′)-gu a n osin e-3′-O-p h osp h or yl-(3′f5′)-
gu a n osin e-3′-O-p h osp h or yl-(3′f5′)-a d en osin e-3′-O-p h os-
p h or yl-(3′f5′)-3′-O-(ter t-b u t yld im et h ylsilyl)gu a n osin e
(16). Compound 14b (13 mg) was subjected to the reaction
described in the synthesis of 15 except for treatment with
aqueous AcOH. The crude product was subjected to reversed-
phase open column chromatography (Preparative C18 125,
55-105 µm, Waters; A: 50 mM TEAB, pH 7, 5% CH3CN; B:
50 mM TEAB, pH 7, 60% CH3CN, linear gradient; 150 drops/
fraction, 254 nm), and the main fraction (fractions 30 and 31)
was collected. The solvent was removed by evaporation, and
the residue was dissolved in H2O (20 mL) and then lyophilized
to give the triethylammonium salt of 16 (48.1 A260 units).
The structures of all compounds tested (15-42) were
confirmed by means of negative ion LSI mass spectroscopy.
Biology. Mea su r em en t of Sta bility in Hu m a n P la sm a .
To each solution of 30 µg of ODNs 23 and 31 in 100 µL of PBS
buffer was added 1.4 mL of human plasma at 37 °C; 100 µL of
the reaction mixture was taken into 100 µL of lysis buffer
(Perkin-Elmer) at 0, 0.25, 0.5, 1, 2, and 4 h after the initial
mixing. To the sampling mixture were added 70 µL of PBS
buffer (pH 7.4), 10 µL of Tris-HCl (pH 8), and 10 µL of 25 mg/
mL proteinase K (Perkin-Elmer). After 30 min, the reaction
mixture was washed with 300 µL of phenol-CHCl3-isoamyl
alcohol (25:24:1, v/v/v) and with 300 µL of CHCl3. The
resulting mixture was analyzed by reverse-phase HPLC (Wa-
kopak WS-DNA, Wako Co., Ltd. J apan, 4.6 × 150 mm; 0.1 M
TEAA (pH 7.0)-CH3CN (74.5:25.5, v/v), 1 mL/min, 260 nm).
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medium and infected with 100 TCID50 HIV-1 for 1 h. The cells
were then washed with RPMI-1640 medium, resuspended in
the same medium, and distributed in 96-well plates containing
serial dilutions of modified oligonucleotides. Each well con-
tained 2.5 × 104 cells in 200 µL of medium. At day 6, cell
viability was assessed by the MTT method. The concentra-
tions of the compounds giving 50% inhibition of HIV-induced
cytopathic effect (IC50) were determined from the dose-
response curves.
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Su p p or tin g In for m a tion Ava ila ble: Table of negative
ion LSI MS and retention times of HPLC for compounds 15-

Biologically Active Oligodeoxyribonucleotides
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19 3663
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