Synthesis of 4′-ethynyl-2′-deoxy-4′-thioribonucleosides and discovery of a highly potent and less toxic NRTI

Article abstract of DOI:10.1021/ml2001054

The synthesis of 4′-ethynyl-2′-deoxy-4′- thioribonucleosides was carried out utilizing an electrophilic glycosidation in which 4-ethynyl-4-thiofuranoid glycal 16 served as a glycosyl donor. Electrophilic glycosidation between 16 and the silylated nucleoba

Full text of DOI:10.1021/ml2001054

LETTER
pubs.acs.org/acsmedchemlett
Synthesis of 40-Ethynyl-20-deoxy-40-thioribonucleosides and
Discovery of a Highly Potent and Less Toxic NRTI
Kazuhiro Haraguchi,*,† Hisashi Shimada,† Keigo Kimura,† Genta Akutsu,† Hiromichi Tanaka,† Hiroshi Abe,‡
Takayuki Hamasaki,§ Masanori Baba,§ Elizabeth A. Gullen,|| Ginger E. Dutschman,||
Yung-Chi Cheng,|| and Jan Balzarini^
†School of Pharmacy, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
‡RIKEN Advanced Science Institute, Nanomedical Engineering Laboratory, 2-1, Hirosawa, Wako-shi, Saitama 351-0198, Japan
§Division of Antiviral Chemotherapy, Center for Chronic Viral Diseases, Graduate School of Medical and Dental Sciences,
Kagoshima University, Kagoshima 89-8544, Japan
Department of Pharmacology, School of Medicine, Yale University, New Haven, Connecticut 06520, United States
^Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
S Supporting Information
b
ABSTRACT: The synthesis of 40-ethynyl-20-deoxy-40-thioribonucleo-
sides was carried out utilizing an electrophilic glycosidation in which
4-ethynyl-4-thiofuranoid glycal 16 served as a glycosyl donor. Electro-
philic glycosidation between 16 and the silylated nucleobases (N4-
acetylcytosine, N6-benzoyladenine, and N2-acetyl-O6-diphenylcarba-
moylguanine) was carried out in the presence of N-iodosuccinimide
(NIS), leading to the exclusive formation of the desired β-anomers 29,
33, and 36. Anti-HIV studies demonstrated that these 40-thio nucleo-
sides were less cytotoxic to T-lymphocyte (i.e., MT-4 cells) than the
corresponding 40-ethynyl derivatives of 20-deoxycytidine (44), 20-deoxyadenosine (45), and 20-deoxyguanosine (46). Comparison
of the selectivity indices (SI) was made between 40-thionucleosides (32, 41, and 43) and the corresponding 40-oxygen analogues 44-
46 by using the reported CC50 and EC50 values. In the case of cytosine and adenine nucleosides, comparable SI values were obtained
as follows: 32 (545) and 44 (458); 41 (>230) and 45 (1630). In contrast, 40-ethynyl-20-deoxy-40-thioguanosine 43 was found to
possess a SI value of >18200, which is 20 times better than that of 46 (933).
KEYWORDS: 40-Thionucleosides, glycal, electrophilic glycosidation, anti-HIV-1 activity, nucleoside reverse transcriptase
inhibitors
ucleoside analogues are recognized as an important class of
biologically active compounds, especially as antiviral and
derivative 13 (Figure 3). The 40-acetoxy leaving group of 13
was introduced by diacetoxylation of the 40,50-anhydro derivative
14 with Pb(OAc)4. Compound 14 was prepared by a series of re-
actions initiated with NIS-mediated electrophilic glycosidation
between silylated thymine and TIPDS (1,1,3,3- tetraisopropyl-
disiloxane-1,3-diyl)-protected 4-thiofuranoid glycal 15.7 In the
present study, to enable a diverse set of nucleobases to be in-
troduced, the 4-thiofuranoid glycal 16 already substituted at the
4-position with the triethylsilylethynyl group was employed as a
glycosyl donor.
N
antitumor agents.1ꢀ3 Among their sugar-modified analogues, 40-
thionucleosides, in which the oxygen atom in the furanose ring is
replaced with a sulfur atom, have attracted much attention since
the discovery of the antiviral and antitumor activities of 40-
thiothymidine (1) and 20-deoxy-40-thiocytidine (2) (Figure 1).4
Also, it has been reported that 40-substituted thymidines such as
the 40-azido (3), 40-methoxy (4), 40-cyano (5), and 40-ethynyl (6)
derivatives exhibit potent anti-HIV activity.5
Having been stimulated by the above findings, we synthesized
the 40-substituted analogues 7ꢀ12 of 40-thiothymidine (Figure 2)
and found promising anti-HIV activity in the 40-azido (8), the 40-
cyano (11), and the 40-ethynyl (12) derivatives.6 This finding led
us to investigate the present study where synthesis of the 40-
ethynyl analogues having other nucleobases (cytosine, adenine,
and guanine) was carried out.
Our plan to introduce an ethynyl group in a tetrahydrothio-
phene ring is visualized in Scheme 1. Aldol reaction between A
and formaldehyde gives B, which is then converted to the O-silyl-
protected C. The formyl group of C is reacted with dimethyl
Received: April 22, 2011
Accepted: July 10, 2011
Published: July 20, 2011
In our previous study,6 the synthesis of 7ꢀ12 was accom-
plished through nucleophilic substitution of the 40-acetoxy
r
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Figure 1. Structures of compounds 1ꢀ6.
protecting the hydroxyl groups with the TIPDS group (yield of
28, 72%) and then the ethynyl group with a triethylsilyl group
(yield of 16, 90%).
With the glycosyl donor 16 in hand, electrophilic glycosida-
tion with a suitable nucleobase was examined. When 16 was
reacted with N4,O2-bis-trimethylsily-N4-acetylcytosine (1.5 equiv)
in the presence of NIS (1.5 equiv) in CH3CN/CH2Cl2 at room
temperature overnight, the desired β-anomer 29 of the glycosi-
dated product was formed as a single stereoisomer in 61% yield
(Figure 7).14 The depicted structure was confirmed by nuclear
Overhauser effect (NOE) experiment: H-6/H-20 (1%), H-6/
H-30 (5%), and H-6/CH2-50(0.2%). The observed exclusive for-
mation of 29 suggested that the presence of the ethynyl group at
the 4-position of 3,5-O-TIPDS-4-thiofuranoid glycal 15 does not
influence the β-face selectivity of the electrophilic glycosidation.7
The introduced iodine atom of 29 was removed by reaction
with Bu3SnH/Et3B at ꢀ70 ꢀC under an O2 atmosphere to give
30 in 94% yield. To circumvent the difficult chromatographic
separation of the free nucleoside and the side product derived
from the silyl-protecting group, 30 was converted to its
corresponding acetate 31 (99% isolated yield) by desilylation
with Bu4NF and subsequent acetylation in one pot. Finally,
31 was converted to the target 40-ethynyl-20-deoxy-40-thiocy-
tidine 32 (91% isolated yield) by treatment with K2CO3
in MeOH.
Figure 2. 40-Substituted 40-thiothymidines 7ꢀ12.
Figure 3. Structures of compounds 13ꢀ16.
1-diazo(2-oxopropyl)phosphonate8 to provide the ethynyl-sub-
stituted tetrahydrothiophene derivative D.
Compound 17 (Figure 4), which corresponds to the aldehyde
A of Scheme 1, was prepared from 2,3-O-isopropylidene-L-
lyxonolactone (18).9 Namely, by following the reported pro-
cedures,10 18 was converted to the dimesylate 19. Reaction of 19
with Na2S in DMF at 80 ꢀC led to the formation of the 1,4-
anhydro-4-thio-D-ribitol derivative 20 in 66% overall yield from
18. Compound 20 was desilylated with Bu4NF to give 21 in 81%
yield.11 Finally, oxidation of 21 with IBX (2-iodoxybenzoic acid)
in CH3CN provided the aldehyde 17 in 83% yield.12
Subsequent aldol reaction between 17 and 37% aqueous for-
maldehyde was carried out in 60% aqueous dioxane (room tem-
perature, overnight), and the resulting mixture was silylated with
TBSCl. In the presence of K2CO3, the aldols 22 and 23 (Figure 5)
were obtained in 21 and 13% yields, respectively, together
with the silyl enol ether 24 (16%). The yield of the desired
stereoisomer 22 was improved to 50% by using NaHCO3,
although the formation of 23 (18%) and 24 (14%) could not be
eliminated.
We next turned our attention to the synthesis of the adenine
and guanine nucleosides. Under similar reaction conditions for the
electrophilic glycosidation of N4-acetylcytosine, bis-trimethylsilyl-
N6-benzoyladenine was reacted with 16. In this reaction, the
target nucleoside 33 could be obtained in 48% isolated yield as a
single stereoisomer together with its regioisomers 34 (12%) and
35 (13%) (Figure 8).15 The depicted structures of 33ꢀ35 were
determined on the basis of comprehensive NMR studies includ-
ing NOE, heteronuclear multiple quantum coherence, and
heteronuclear multiple bond correlation experiments.16 A similar
regiochemical outcome was also observed in the glycosidation of
N2-acetyl-O6-diphenylcarbamoylguanine,17 where three isomeric
nucleosides 36ꢀ38 were isolated in 25, 12, and 29% yields, re-
spectively (Figure 9).
The N9-glycosidated products 33 and 36 were successfully
converted to 40-ethynyl-40-thio-20-deoxyadenosine 41 and the
respective guanosine nucleoside 43 by three steps as follows: (1)
Bu3SnH/Et3B/PhMe, ꢀ70 ꢀC (yield of 39, 88%; yield of 42,
72%), (2) Bu4NF/Ac2O/THF (yield of 40, quant.), and (3)
K2CO3/MeOH (yield of 41, 82%; yield of 43, 63% from 42 for
two steps) (Figure 10).
The formyl group of 22 was converted to an ethynyl group
through its reaction with dimethyl 1-diazo(2-oxopropyl)pho-
sphonate in MeOH in the presence of K2CO3. Upon reacting the
crude product with Bu4NF, the 4-ethynyl derivative 25 was
isolated in 73% yield from 22.
Compound 25 was transformed to 4-thiofuranoid glycal 26 by
reaction with tert-BuLi (4 equiv) at ꢀ70 ꢀC in THF (Figure 6).13
This reaction furnished the glycal 26 in 61% yield along with the
ring-opened sulfide 27 (9%) and the starting material 25 (11%).
The actual glycosyl donor 16 was prepared from 26 by first
The anti-HIV-1 activities of 32, 41, and 43 were evaluated, and
the results are summarized in Table 1.18,19 To compare the antiviral
activity and cytotoxicity with the corresponding 40-oxygen count-
erparts, reported biological data20,21 of 40-ethynyl derivatives of 20-
deoxycytidine 44, 20-deoxyadenosine 45, and 20-deoxyguanosine
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LETTER
Scheme 1. Introduction of an Ethynyl Group on a Tetrahydrothiophene Ring
Figure 4. Structures of compounds 17ꢀ21.
Figure 5. Structures of compounds 22ꢀ25.
derivatives also exhibited antiviral activity against herpes
simplex virus and vaccinia virus without measurable cytotoxicity
to the host cells up to 100 μM (entries 1ꢀ3). These potencies are
at least 100 times less than that of ganciclovir, but their selectivity
indices against HSV-1 and HSV-2 were >50ꢀ100 for 32 and 43.
However, it is noteworthy that all compounds synthesized in this
study suppressed the replication of the thymidine kinase-deffi-
cient (TKꢀ) HSV-1 KOS strain at an almost equal potency as
wild-type HSV-1. In contrast, the potency of ganciclovir against
HSV-1 TKꢀ strain (1 μM) is 100-fold weaker as compared
to wild-type HIV-1. These data are somewhat surprising
but interesting and may suggest that the antiherpesvurus
activity of 32, 41, and 43 is independent of the activation
(phosphorylation) by the virus-encoded thymidine kinase. This
may, in turn, point to another mechanism of antihepetic action of
these compounds.
The compounds were not significantly inhibitory against
other viruses, including parainfluenza virus, reovirus-1, Sind-
bis virus, Coxsackie virus B4, Punta Toro virus in Vero cell
cultures, VSV and RSV in HeLa cell cultures, feline corona
virus (FIPV) and feline herpesvirus in CrFK cell cultures,
and influenza virus A (H1N1, H3N2) and B in MDCK cell
cultures.
In conclusion, we have developed a novel synthetic approach
to 40-ethynyl-20-deoxy-40-thioribonucleosides on the basis of
electrophilic glycosidation utilizing 4-ethynyl-4-thiofuranoid gly-
cal 16 as a glycosyl donor. The synthesis of 16 was initiated with
the β-face-selective aldol reaction of 1,4-anhydro-2,3-O-isopro-
pylidene-4-thio-D-ribitol 5-aldehyde 17 with formaldehyde. The
aldol 22 was reacted with dimethyl 1-diazo(2-oxopropyl)pho-
sphonate to provide the ethynyl-substituted tetrahydrothiophene
derivative 25. 4-Ethynyl-4-thiofuranoid glycal 26 was obtained by
the reaction of 25 with tert-BuLi. The actual glycosyl donor 16
Figure 6. Structures of compounds 26ꢀ28.
46 are also included in Table 1. As can be seen in entry 1, 40-ethynyl-
20-deoxy-40-thiocytidine 32 exhibited a 10 times lower inhibitory
activity than that of the corresponding deoxycytidine derivative 44.
However, because 32 was less toxic to MT-4 cells, the SI value (545)
of 32 was found to be comparable to that of 44 (458). In the case of
adenine nucleosides as shown in entry 2, a similar trend was seen in
terms of EC50 and CC50 values. In contrast, 40-ethynyl-20-deoxy-40-
thioguanosine 43 was found to be a highly promising anti-HIV agent
(entry 3). Indeed, 43 exhibited comparable antiviral activity (EC50:
0.0055 μM for 43 vs 0.0015 μM for 46) and did not show any
cytotoxicity to MT-4 cells up to 100 μM in contrast to the highly
toxic 20-deoxyguanosine derivative 46 (CC50: 1.4 μM). The
promising guanine nucleoside 43 possesses a SI value of >18200,
which is 20 times better than that of 40-ethynyl-20-deoxyguanosine
46 (SI 933).
With the above promising anti-HIV-1 activity in hand, next,
the 40-substituted 20-deoxy-40-thioribonucleosides 32, 41, and
43 were also evaluated for their inhibitory activity against a
series of other viruses including HSV-1 strain KOS, HSV-2
strain G, TKꢀ HSV-1 strain KOS resistant to ACV, and vaccinia
virus Lederle strain, and the results were summarized in
Table 2.22 Antiviral data of ganciclovir are also included as a
reference compound. As can be seen, these nucleoside
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LETTER
was prepared by silyl-protection of the hydroxyl and ethynyl
groups of 26.
The glycosidation between 16 and the silylated nucleobase
(N4-acetylcytosine, N6-benzoyladenine, and N2-acetyl-O6-diphenyl-
carbamoylguanine) proceeded with facial selectivity and β-anomers
29, 33, and 36 of the glycosidated products could be
obtained exclusively. These glycosides were efficiently trans-
formed into the 40-ethynyl derivatives of 20-deoxy-40-thiocy-
tidine (32), -adenosine (41), and -guanosine (43). It is
noteworthy that these novel nucleoside analogues synthe-
sized in this study were found to be less cytotoxic to MT-4
cells as compared to the corresponding 20-deoxycytidine
(44), 20-deoxyadenosine (45), and 20-deoxyguanosine (46)
derivatives. By comparison with the reported SI value of 40-
ethynyl-20-deoxyguanosine 46, it was found that the SI for
the 20-deoxy-40-thioguanosine derivative 43 has a 20-fold
better value (>18200) than that of 20-deoxyguanosine coun-
terpart (933). These facts suggest that replacement of the
furanose oxygen with sulfur atom is a promising approach for the
development of less cytotoxic antiviral nucleosides. We are
currently investigating the mechanism of the promising biologi-
cal profile of these compounds.
Figure 7. Structures of compounds 29ꢀ32.
Figure 8. Structures of compounds 33ꢀ35 (R = CtCSiEt3).
Figure 9. Structures of compounds 36ꢀ38 (R = CtCSiEt3).
Figure 10. Structures of compounds 39ꢀ43.
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Table 1. Inhibitory Effect of 40-Ethynyl-20-deoxy-40-thioribonucleosides (32, 41, and 43) and Its Oxygen Analogues (44ꢀ46) on
HIV-1 in MT-4 Cells
entry
compd
EC50 (μM)a
CC50(μM)b
SIc
545
compd
EC50 (μM)d
CC50(μM)d
SI c
1
2
3
32
41
43
0.011 ( 0.001
0.087 ( 0.017
6.0 ( 1.2
>20
44
45
46
0.0048 ( 0.001
0.0098 ( 0.0043
0.0015 ( 0.0003
2.2 ( 1.0
16 ( 7.9
458
1630
933
>230
0.0055 ( 0.0016
>100
>18200
1.4 ( 0.16
a Inhibitory concentration required to achieve 50% protection of MT-4 cells against the cytopathic effect of HIV-1. b Cytotoxic concentration required to
reduce the viability of mock-infected MT-4 cells by 50%. c SI = CC50/EC50 d Data taken from refs 20 and 21.
Table 2. Inhibitory Effect of 40-Ethynyl-20-deoxy-40-thioribonucleosides (32, 41, and 43) against Herpes Simplex Virus and
Vaccinia Virus in HEL Cell Culturesa
EC50 (μM)b
herpes simplex virus-1
(KOS)
herpes simplex virus-2
(G)
vaccinia
virus
herpes simplex virus-1 (KOS, TKꢀ
ACV)
minimum cytotoxic concentrationc
entry compd
(μM)
1
2
3
4
32
41
2 ( 0
7 ( 5
3 ( 1
0.01
1.5 ( 0.5
4 ( 0
6.5 ( 2.5
52 ( 6.0
27 ( 7.0
100
2.5 ( 0.5
8 ( 4.0
4.5 ( 2.5
1
>100
>100
>100
>100
43
1.5 ( 0.5
ganciclovir
0.01
a Data derived from two independent experiments. b Required to reduce virus-induced cytopathogenicity by 50%. c Required to cause a microscopically
detectable alternation of normal cell morphology.
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b
full characterization for compounds 16ꢀ17 and 20ꢀ43. This material
is available free of charge via the Internet at http://pubs.acs.org.
(4) For a review, see Yokoyama, M. Synthesis and biological activity
of thionucleosides. Synthesis 2000, 1637–1655.
’ AUTHOR INFORMATION
(5) For a review, seeHayakawa, H.; Kohgo, S.; Kitano, K.; Ashida, N.;
Kodama, E.; Mitsuya, H.; Ohrui, H. Potential of 40-substituted nucleosides for
the treatment of HIV-1. Antiviral Chem. Chemother. 2004, 15, 169–187.
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Corresponding Author
*Tel: 81-3-3784-8187. Fax: 81-3-3784-8252. E-mail: harakazu@
pharm.showa-u.acjp.
Funding Sources
C.-Y.C. is a Fellow for the National Foundation for Cancer
Research (NFCR). Financial support from the Japan Society for
the Promotion of Science (KAKENHI No. 21590123 to K.H.),
the NIH (#AI-38204 to Y.-C.C.), and the K.U. Leuven (GOA
No. 10/014 to J.B.) are gratefully acknowledged.
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We are also grateful to Y. Odanaka and M. Matsubayashi
(Center for Instrumental Analysis, Showa University) for tech-
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LETTER
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