Design, efficient synthesis, and anti-HIV activity of 4′-C-cyano- and 4′-C-ethynyl-2′-deoxy purine nucleosides

Article abstract of DOI:10.1081/NCN-120037508

Some 4′-C-ethynyl-2′-deoxy purine nucleosides showed the most potent anti-HIV activity among the series of 4′-C-substituted 2′-deoxynucleosides whose 4′-C-substituents were methyl, ethyl, ethynyl and so on. Our hypothesis is that the smaller the substitue

Full text of DOI:10.1081/NCN-120037508

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Design, Efficient Synthesis, and Anti‐HIV Activity
of 4′‐C‐Cyano‐ and 4′‐C‐Ethynyl‐2′‐deoxy Purine
Nucleosides
Satoru Kohgo Ph.D. a , Kohei Yamada a , Kenji Kitano a , Yuko Iwai a , Shinji Sakata a ,
Noriyuki Ashida a , Hiroyuki Hayakawa Ph.D. a , Daisuke Nameki b , Eiichi Kodama b ,
Masao Matsuoka b , Hiroaki Mitsuya c d & Hiroshi Ohrui e
a Chemistry and Pharmacology Laboratory , Biochemicals Division , Yamasa Corporation ,
10‐1, Araoicho 2‐Chome, Choshi, Chiba‐ken, 288‐0056, Japan
b Laboratory of Virus Immunology , Institute for Virus Research , Kyoto University , 53‐1
Kawaharacho‐Shogoin, Sakyo‐Ku, Kyoto, 606‐8507, Japan
c Department of Internal Medicine II , Kumamoto University School of Medicine , 1‐1‐1,
Honjo, Kumamoto, 860‐0811, Japan
d Experimental Retrovirology Section , Department of Developmental Therapeutics ,
Medicine Branch , National Cancer Institute , Bethesda, Maryland, 20892, USA
e Division of Life Science , Graduate School of Life Sciences , Tohoku University , 1‐1
Tsutsumidori‐Amamiyamachi, Aoba‐Ku, Sendai, 981‐8555, Japan
Published online: 07 Feb 2007.
To cite this article: Satoru Kohgo Ph.D. , Kohei Yamada , Kenji Kitano , Yuko Iwai , Shinji Sakata , Noriyuki Ashida ,
Hiroyuki Hayakawa Ph.D. , Daisuke Nameki , Eiichi Kodama , Masao Matsuoka , Hiroaki Mitsuya & Hiroshi Ohrui (2004)
Design, Efficient Synthesis, and Anti‐HIV Activity of 4′‐C‐Cyano‐ and 4′‐C‐Ethynyl‐2′‐deoxy Purine Nucleosides,
Nucleosides, Nucleotides and Nucleic Acids, 23:4, 671-690, DOI: 10.1081/NCN-120037508
To link to this article: http://dx.doi.org/10.1081/NCN-120037508
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 23, No. 4, pp. 671–690, 2004
Design, Efficient Synthesis, and Anti-HIV Activity of
4’-C-Cyano- and 4’-C-Ethynyl-2’-deoxy
Purine Nucleosides
Satoru Kohgo,1 Kohei Yamada,1 Kenji Kitano,1 Yuko Iwai,1
Shinji Sakata,1 Noriyuki Ashida,1 Hiroyuki Hayakawa,1,
*
Daisuke Nameki,2 Eiichi Kodama,2 Masao Matsuoka,2
Hiroaki Mitsuya,3,4 and Hiroshi Ohrui5
1Biochemicals Division, Yamasa Corporation, Chiba, Japan
2Laboratory of Virus Immunology, Institute for Virus Research,
Kyoto University, Kyoto, Japan
3Department of Internal Medicine II, Kumamoto University School
of Medicine, Kumamoto, Japan
4Experimental Retrovirology Section, Department of
Developmental Therapeutics, Medicine Branch,
National Cancer Institute, Bethesda, Maryland, USA
5Division of Life Science, Graduate School of Life Sciences,
Tohoku University, Sendai, Japan
ABSTRACT
Some 4’-C-ethynyl-2’-deoxy purine nucleosides showed the most potent anti-HIV
activity among the series of 4’-C-substituted 2’-deoxynucleosides whose 4’-C-
substituents were methyl, ethyl, ethynyl and so on. Our hypothesis is that the smaller
the substituent at the C-4’ position they have, the more acceptable biological activity
they show. Thus, 4’-C-cyano-2’-deoxy purine nucleosides, whose substituent is
smaller than the ethynyl group, will have more potent antiviral activity. To prove our
*
Correspondence: Hiroyuki Hayakawa, Ph.D., Chemistry and Pharmacology Laboratory, Bio-
chemicals Division, Yamasa Corporation, 10-1, Araoicho 2-Chome Choshi, Chiba-ken 288-0056,
Japan; Fax: +81-479-22-9821; E-mail: hayakawa@yamasa.com.
671
DOI: 10.1081/NCN-120037508
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com
Copyright D 2004 by Marcel Dekker, Inc.

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Kohgo et al.
hypothesis, we planned to develop an efficient synthesis of 4’-C-cyano-2’-deoxy
purine nucleosides (4’-CNdNs) and 4’-C-ethynyl-2’-deoxy purine nucleosides (4’-
EdNs). Consequently, we succeeded in developing an efficient synthesis of six 2’-
deoxy purine nucleosides bearing either a cyano or an ethynyl group at the C-4’
position of the sugar moiety from 2’-deoxyadenosine and 2,6-diaminopurine 2’-
deoxyriboside. Unfortunately, 4’-C-cyano derivatives showed lower activity against
HIV-1, and two 4’-C-ethynyl derivatives suggested high toxicity in vivo.
Key Words: NRTIs; Efficient synthesis; 4’-CNdNs; 4’-EdNs; Anti-HIV-1 agents.
INTRODUCTION
There are mainly two ways for the preparation of our 4’-C-substituted-2’-
deoxynucleosides (4’-SdNs). One is a condensation method and the other is
modification of nucleosides. During our exploration of novel nucleoside reverse
transcriptase inhibitors (NRTIs), we have prepared many 4’-SdNs by the use of
glycosidation reaction of 4-C-substituted sugar derivatives with nucleobases. This
method was effective to make various kinds of 4’-SdNs. However, this synthetic route
incurs some problems in making 4’-C-cyano-2’-deoxy purine nucleosides, because 4-C-
substituted sugars have low reactivity in glycosidation reaction, especially when its
substituent is an electron withdrawing group like a cyano group (data not shown).
These problems encouraged us to develop a preparation method of 4’-C-cyano-2’-deoxy
purine nucleosides from natural 2’-deoxynucleosides, which were so far difficult to be
synthesized by condensation of sugars with nucleobases. The synthetic routes of 4’-
SdNs from the corresponding nucleosides as starting materials were already reported.
Matsuda et al. synthesized various 4’-C-substituted pyrimidine nucleosides by
intramolecular radical cyclization reaction of 3’-O-diphenylvinylsilyl nucleosides with
their 4’-radical and then conversion of 4’-C-hydroxyethyl nucleosides.[1] O-Yang et al.
also reported an interesting article, which described the preparation of 4’-cyanothymi-
dine (4’-CNT) from thymidine via 5’-aldehyde.[2] Haraguchi et al. recently reported ring
opening reaction of 4’,5’-epoxy-nucleosides to make 4’-C-branched nucleosides.[3]
These methods were useful for the preparation of 4’-SdNs, but there were few cases of
the preparation of 4’-C-cyano purine nucleosides.
In this report, we describe the design and efficient synthesis of 4’-C-substituted 2’-
deoxy purine nucleosides bearing a cyano and an ethynyl group at the C-4’ position of
the sugar moiety from purine 2’-deoxynucleosides, and their anti-HIV activity. Further-
more, a preliminary toxicity test using 4’-C-ethynyl derivatives in vivo will be reported.
RESULTS AND DISCUSSIONS
From previous studies on structure-activity relationships of 4’-C-substituted
nucleosides,[1,2,4–10] it is expected that a smaller substituent at the C-4’ position will
give more acceptable biological activity against HIV-1. Actually, 4’-C-cyano-2’-
deoxycytidine showed highest activity compared to 4’-C-ethynyl and 4’-C-methyl
derivatives. From these results, we speculated that there was relationship between the

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673
biological activity and a parameter, namely, À DG0 values[11] between equatorial and
axial substituents on a cyclohexane rings (CN < F < C ꢀ CH < CH
CH2 < Me
Q Et <tert-Bu). On the other hand, we have already found that 4’-C-ethynyl-2’-deoxy
purine nucleosides exhibited potent anti-HIV activity and moderate cytotoxicity
compared to the corresponding pyrimidine derivatives.[6,7,10] Therefore, we were
interested in the synthesis and biological activity of 2’-deoxy purine nucleosides
bearing a cyano group at C-4’ position. However, the glycosidation reaction will have
drawbacks as described in the above introduction on synthetic problems. These
problems prompted us to develop a preparation method of 4’-C-substituted purine
nucleosides from the corresponding nucleosides, and this approach would enable us to
synthesize 4’-C-cyano purine nucleoside derivatives, which were so far difficult to be
synthesized by condensation of sugars with nucleobases.
Synthesis of 4’-C-cyano-2’-deoxy purine nucleosides starting from 2’-deoxyadeno-
sine and 2,6-diaminopurine 2’-deoxyriboside (dDAP) is shown in Schemes 1–3. To be-
gin with, 9-(2-deoxy-5-O-dimethoxytrityl-ribo-pentofuranosyl)-2,6-dibenzamidopurine
3a was prepared from dDAP 1 by way of N-benzoylation and 5’-O-dimethoxytritylation
(Scheme 1). Similarly, N6-benzoyl-2’-deoxy-5’-O-dimethoxytrityladenosine 3b, which
was commercially available from Yamasa Corporation, was also used as the starting
material for the synthesis of the target compounds.
The key intermediates 7a,b were obtained according to Scheme 2. In the process
for the synthesis of 3’-protected nucleosides 4b, we initially expected that protection of
the 3’-hydroxyl group as tert-butyldimethylsilyl ether (TBS) and the following
deprotection of the 5’-O-dimethoxytrityl group (DMTr) would give the compound 4b
in good yield. However, when deprotection of the dimethoxytrityl group with 80%
AcOH was tried at room temperature, the isolated yield of compound 4b was very low
because of acid sensitivity of N-acylated 2’-deoxyadenosine. On the contrary, when
2% p-toluenesulphonic acid (TsOH) in CHCl3–MeOH was utilized for removal of
DMTr group, this treatment gave 3’-TBS-derivative 4b in 84% yield. To give
4’-C-hydroxymethyl derivative 5b, compound 4b was oxidized to the corresponding
5’-carboaldehyde by Moffatt oxidation, and then the aldehyde was treated under aldol
reaction conditions with formaldehyde. The 4’-a-hydroxyl group of 5b was selectively
protected to give DMTr ether 6b in 67% yield. Compound 6b was converted to the
key intermediate 7b in 81% yield by tert-butyldimethylsilylation of the 5’-hydroxyl
group and the following deprotection of the 4’-hydroxymethyl group. These reactions
were also effective to prepare protected dDAP derivative 7a. The overall yields were
12.7% in 6 steps.
Scheme 1. Reagents and conditions: dDAP 1 was obtained by enzymatic base exchange reaction
from 2’-deoxyuridine. (a) 1. TMSCl, pyridine, 0°C, 30 min; 2. BzCl, pyridine, 0°C, 2 h; 3. NH4OH,
H2O, 0°C, 30 min; (b) DMTrCl, pyridine, rt., 3 h.

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Kohgo et al.
Scheme 2. Reagents and conditions: (a) 1. TBSCl, imidazole, DMF, rt., overnight; 2.
TsOH Á H2O, MeOH, CHCl3, 0°C, 15–30 min; (b) 1. EDC Á HCl, pyridine, TFA, toluene, DMSO,
rt., 2 h; 2. aq CH2O, 1N NaOH, dioxane, rt., 1–3 h; 3. NaBH4, EtOH, 0°C, 30 min; (c) DMTrCl,
Et3N, DMF, rt., 1 h, for 5a; DMTrCl Et3N, CH2Cl2, 0°C, 30 min, for 5b; (d) 1. TBSCl, imidazole,
DMF, rt., overnight; 2. TsOH Á H2O, MeOH, CHCl3, 0°C, 20–30 min.
Conversion of key intermediates 7a,b to the corresponding 4’-C-cyano derivatives
12a,b is shown in Scheme 3. 4’-C-Formyl derivatives 8a,b, which were obtained by
Moffatt oxidation of 4’-C-hydroxymethyl derivatives 7a,b, were derived to 4’-C-
aldoxime derivatives 9a,b by treatment with hydroxylamine hydrochloride in pyridine,
and were further dehydrated by methanesulfonyl chloride and triethylamine in CH2Cl2
Scheme 3. Reagents and conditions: (a) EDC Á HCl, pyridine, TFA, toluene, DMSO, rt., 1 h;
(b) NH2OH Á HCl, pyridine, rt., 30 min; (c) MsCl, Et3N, CH2Cl2, 0°C, 30 min; (d) aq MeNH2,
MeOH, rt., overnight, for 10a; NH4OH, MeOH, rt., overnight, for 10b; (e) TBAF, THF, rt., 15 min.

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675
Scheme 4. Reagents and conditions: (a) aq MeNH2, MeOH, rt., 46 h; (b) 1,1’-carbon-
yldiimidazole, MeCN, rt., 4 h; (c) TBAF, THF, rt., 10 min.
to give 4’-C-cyano derivatives 10a,b in good yields through 3 steps. These 4’-C-cyano
derivatives 10a and 10b were deblocked under aq MeNH2 or NH4OH in MeOH,
respectively, to give crude debenzoylated products 11a and 11b. Treatment of 11a and
11b with tetra-n-butylammonium fluoride (TBAF) in tetrahydrofuran (THF) gave 4’-C-
cyano-2’-deoxy-2,6-diaminopurine-ribonucleoside 12a and 2’-deoxyadenosine nucleo-
side 12b in 25% and 66% yield, respectively.
Next, an alternative route was investigated because the isolated yield of 12a was
very low (Scheme 4). The cyano group might be unstable under MeNH2 in MeOH for
debenzoylation. 4’-C-Aldoxime 9a, which was similarly derived from 7a, was
deblocked by treatment with aq MeNH2 in MeOH to give the debenzoylated aldoxime
derivative 13. Crude 13 was then dehydrated with 1,1’-carbonyldiimidazole in MeCN to
give 11a in 52% yield from 7a. In the case of compound 13 whose amino group is not
protected, when dehydration of oxime was performed with MsCl, sulfonylation of the
amino groups also occurred. Finally, desilylation of 11a gave the desired nucleosides
12a in 97% yield. The isolated yield of 12a from 7a was improved to 50% (as
compared to 22% yield in Scheme 3).
We have already reported the synthesis of 4’-C-ethynyl-2’-deoxyinosine and 4’-C-
ethynyl-2’-deoxyguanosine from the corresponding 2’-deoxyadenosine and 2,6-diami-
nopurine 2’-deoxyriboside derivatives by enzymatic deamination with adenosine
Scheme 5. Reagents and conditions: (a) adenosine deaminase, Tris-HCl buffer, 40°C, 1–2 h.

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Kohgo et al.
Scheme 6. Reagents and conditions: (a) EDC Á HCl, pyridine, TFA, toluene, DMSO, rt., 90 min–
3 h; (b) PPh3 = CHBr, THF, À40°C, 90 min–2 h; (c) tert-BuOK, THF, À40°C, 1–2 h; (d) TBAF,
THF, rt., 30 min; (e) aq MeNH2, MeOH, rt., overnight, for 17a; NH4OH, MeOH, rt., overnight,
for 17b.
deaminase.[6] This procedure was utilized to synthesize 4’-C-cyano-2’-deoxyguanosine
14a and 4’-C-cyano-2’-deoxyinosine 14b (Scheme 5). 4’-C-cyano nucleosides 12a and
12b were readily hydrolyzed to give 14a and 14b, respectively, by treatment with
adenosine deaminase in Tris-HCl buffer (pH 7.5).
The 4’-C-hydroxymethyl derivatives 7a,b are versatile intermediates for the
synthesis of various 4’-C-substituted nucleosides. Therefore, we also utilized these
intermediates 7a,b to synthesize 4’-C-ethynyl-2’-deoxy purine nucleosides 18a,b.
Scheme 6 shows the synthesis of 4’-C-ethynyl derivatives 18a,b from the key
intermediates 7a,b. Matsuda et al. synthesized 4’-C-ethynyl-2’-deoxycytidine by
treatment of the corresponding 4’-C-chlorovinyl derivative with n-BuLi in THF in
good yield.[5] However, conversion of N6-benzoyl-4’-C-chlorovinyl-2’-deoxyadenosine
to the corresponding 4’-C-ethynyl derivative 16b under this condition resulted in lower
isolated yield than that of 4’-C-ethynyl-2’-deoxycytidine derivative because of de-
composition of desired product. After investigating reaction conditions, we obtained
4’-C-ethynyl derivatives 16a,b by conversion of the 4’-C-formyl group of 8a,b to a
more reactive 4’-C-bromovinyl group with bromomethylenetriphenylphosphorane[12] in
Scheme 7. Reagents and conditions: (a) adenosine deaminase, Tris-HCl buffer, 40°C, 1–2 h.

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Table 1. Anti-HIV activity of 4’-C-ethynyl and 4’-C-cyano 2’-deoxy purine nucleosides.
Anti-HIV activitya
Compound no.
EC50 (mM)
CC50 (mM)
Selectivity index
4’-C-ethynyl dAdo 18b
4’-C-ethynyl dDAP 18a
4’-C-ethynyl dIno 19b
4’-C-ethynyl dGuo 19a
4’-C-cyano dAdo 12b
4’-C-cyano dDAP 12a
4’-C-cyano dIno 14b
4’-C-cyano dGuo 14a
3’-azidothymidine (AZT)
0.0098
0.00034
0.13
16
1633
2647
1054
933
0.9
137
0.0015
0.051
1.4
12
>0.034
23
235
>43
0.00079
0.051
451
0.000188
0.0032
>0.034
29.4
>181
9188
aAnti-HIV activity was determined by MTT assay.[13,14] MT-4 cells and HIV-1LAI
employed.
were
[15]
THF, and then the following dehydrobromination of bromoolefin 15a,b with tert-
BuOK. After removal of the TBS groups of 16a,b by treatment with TBAF in THF,
benzamide groups at the base moiety of 17a,b were cleaved by aq MeNH2 or NH4OH
in MeOH, respectively, to give 4’-C-ethynyl-2’-deoxy-2,6-diaminopurine 2’-deoxyribo-
side 18a and 2’-deoxyadenosine derivative 18b.
The preparation of 4’-C-ethynyl-2’-deoxyguanosine 19a and 4’-C-ethynyl-2’-
deoxyinosine 19b (Scheme 7) was already reported.[6]
Consequently, we were able to synthesize four 4’-C-cyano- and two 4’-C-ethynyl-
2’-deoxy- purine ribonucleosides by a more useful procedure using 2’-deoxyadenosine
and 2, 6-diaminopurine 2’-deoxyriboside as the starting materials.
Antiviral Evaluation
Table 1 shows a summary of anti-HIV activity of 4’-C-cyano-2’-deoxy purine nu-
cleosides together with that of 4’-C-ethynyl-2’-deoxy purine nucleosides. The structures
are summarized in Scheme 8.
4’-C-Ethynyl dAdo 18b, 4’-C-ethynyl dDAP 18a, and 4’-C-ethynyl dGuo 19a were
highly potent against HIV-1, with subnanomolar to nanomolar EC50s. 4’-C-Ethynyl
dIno 19b was only moderately active against the virus. It is noteworthy that both 4’-C-
Scheme 8. The structures of 4’-C-cyano- and 4’-C-ethynyl-2’-deoxy purine nucleosides.

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Kohgo et al.
Table 2. Anti-HIV activity of 4’-C-ethynyl and 4’-C-cyano 2’-deoxy purine nucleosides against
drug-resistant infectious clones.
Anti-HIV activitya, EC50 (mM)
Compound no.
Wild type (HXB2)
MDRb
M184Vc
4’-C-ethynyl dAdo 18b
4’-C-ethynyl dDAP 18a
4’-C-ethynyl dIno 19b
4’-C-ethynyl dGuo 19a
4’-C-cyano dAdo 12b
4’-C-cyano dIno 14b
3’-azidothymidine(AZT)
3’-thiacytidine (3TC)
0.008
0.0014
0.81
0.0062
0.001
0.51
0.047
0.0059
16.6
0.007
0.043
0.242
0.022
0.71
0.0048
0.083
0.296
15.3
0.008
2.28
6.06
0.01
1.1
>100
aAnti-HIV activity was determined by MAGI assay.[16-18] HeLa-CD4-LTR-b-gal cells were
employed.
bAn infectious molecular clone (HIV-1MDR) which contains five amino acid substitutions
(Ala62Val, Val75Leu, Phe77Leu, Phe116Tyr and Gln115Met) and shows a high level of resistance
against a variety of 2’,3’-dideoxy nucleoside analogues (AZT, ddI, ddC, d4T).[19]
cAn infectious molecular clone (HIV-1M184V) which contains an amino acid substitution (Met184-
Val) and shows a high level of resistance against 3TC.
ethynyl dDAP 18a and 4’-C-ethynyl dAdo 18b had a favorable toxicity profile, with
selectivity indices of 2647 and 1633, respectively. On the contrary, 4’-C-cyano dDAP
12a and 4’-C-cyano dGuo 14a showed remarkable cytotoxicity. Additionally, anti-HIV
activity of 4’-C-cyano dAdo 12b and 4’-C-cyano dIno 14b decreased in comparison to
that of 4’-C-ethynyl derivatives. Interestingly, 4’-C-cyano dIno 14b was as potent as 4’-
C-cyano dAdo 12b against HIV-1 in spite of showing the low activity of 4’-C-ethynyl
dIno 19b.
We finally summarized anti-HIV activity of 4’-CNdNs and 4’-EdNs against drug-
resistant infectious clones as shown in Table 2. The 4’-EdNs analogs (18a, 18b, and
19a) were active against the infectious clones tested, including HIV-1M184V. However,
the activity of 2’-deoxyinosine derivatives (14b and 19b) was low against, especially,
clone HIV-1M184V. Taken together, although both 4’-C-cyano and 4’-C-ethynyl de-
rivatives were active against HIV-1, the 4’-C-ethynyl substitution appears to be more
acceptable for activity against 3TC-resistant HIV-1M184V. On the other hand, we
have not explained any relationship between cytotoxicity and biological activity on 4’-
SdNs including both 4’-CNdNs and 4’-EdNs, but these biological data were already
reported.[6,7,10]
Preliminary Toxicity Test In Vivo
A preliminary toxicity test for three 4’-C-ethynyl derivatives (18a, 18b, and 19a)
was conducted, and the results are summarized in Table 3. Two of the three candidates
(18a and 19a) were toxic, but 4’-C-ethynyl dAdo 18b was not in mice.
We developed an efficient preparation method for purine 2’-deoxyribonucleo-
side derivatives bearing a cyano group at the C-4’ position from the corresponding

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679
Table 3. Preliminary toxicity test of three 4’-C-ethynyl-2’-deoxy purine nucleosides.a
Intravenous administration
Oral administration
Compound no.
Dose (mg/kg)
Mortality (%)
Dose (mg/kg)
Mortality (%)
4’-C-ethynyl dAdo 18b
100
10
3
0
100
10
3
0
0
0
0
0
1
0
1
0
4’-C-ethynyl dDAP 18a
4’-C-ethynyl dGuo 19a
100
10
3
100 (1 d)b
100 (2 d)b
0
100
10
3
100 (1 d)b
100 (2 d)b
100 (2 d)b
0
1
0
1
100
10
3
100 (1 d)b
100 (2 d)b
100 (4 d)b
0
100
10
3
100 (1 d)b
100 (4 d)b
100 (4 d)b
0
1
1
aSix-week-old ICR male mice were employed.
bNumbers in parentheses represent survival days of mice after administration.
2’-deoxynucleosides as the starting materials. This method was also applicable to
effective preparation of 4’-C-ethynyl-2’-deoxy purine nucleosides. The total yields of 4’-C-
substituted derivatives were improved (3.4 ꢁ 17%) compared to that of the method by
condensation of sugars with nucleobases (< 1%[6]) by the use of these routes. Sub-
sequently, it became easy for us to make derivatives bearing an electron withdrawing
substitutent like a cyano group.
We initially expected that the target derivatives having a smaller 4’-C-substituent
would lead to potent anti-HIV activity. However, we found that 4’-C-ethynyl deriva-
tives were superior to 4’-C-cyano derivatives in terms of anti-HIV activity. Addi-
tionally, it turned out that 4’-C-ethynyl dDAP 18a and dGuo 19a were toxic in the
preliminary toxicity test, but 4’-C-ethynyl dAdo 18b was not.
Further investigations to discover novel NRTIs are in progress in our laboratory.
EXPERIMENTALS
Chemistry
General Method for Chemistry. All melting points were determined on a
Yanagimoto MP-500D micro melting point apparatus and are uncorrected. The 1H-NMR
spectra were recorded on a JEOL JNM-GSX-400 spectrometer or a Bruker Avance 500
spectrometer in CDCl3 or DMSO-d6 as the solvent with tetramethylsilane (TMS) as
reference. UV spectra were recorded with a Shimazu UV-160A spectrophotometer.
Low-resolution mass spectra (MS) were taken on a JEOL JMS-AX500 spectrometer.
Merck precoated plates (Kieselgel 60F254) were used for thin-layer chromatography
(TLC) and spots were examined with ultraviolet light and sulfuric acid/anisaldehyde
solution. Merck Kieselgel 60 was used for column chromatography. Reversed-phase

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Kohgo et al.
column chromatography was performed on Fuji Silysia DM1020T. Purity of all
compounds obtained was verified by TLC or high-performance liquid chromatography
1
(HPLC), and identity was verified by MS, H-NMR, and elementary analysis.
9-(2-Deoxy-ribo-pentofuranosyl)-2,6-dibenzamidopurine (2). 9-(2-Deoxy-ribo-
pentofuranosyl)-2,6-diaminopurine 1 (26.6 g, 100 mmol) was coevaporated with
pyridine and then it was suspended in pyridine (400 mL). Chlorotrimethylsilane (88.0
mL, 700 mmol) was added dropwise to the suspension at 0°C. After stirring for 30 min
at 0°C, the mixture was added dropwise benzoyl chloride (82.0 mL, 700 mmol) and
was stirred for 2 h. The reaction mixture was added ice-water. After stirring for 15 min,
the mixture was added conc. NH4OH and was stirred for 30 min. The mixture was
evaporated under reduced pressure. The residue was added AcOEt (600 mL) and
saturated NaHCO3 (600 mL) and was stirred for 1 h at 0°C. The precipitate formed was
collected by filtration to give 2 (36.2 g, 76.3 mmol, 76.3%).
1H-NMR (DMSO-d6) d 11.21, 11.00 (each 1H, s, NH), 8.62 (1H, s, H-8), 8.08–
7.51 (10H, m, aromatic), 6.42 (1H, t, H-1’, J = 6.5), 5.34 (1H, d, 3’-OH, J = 4.0), 4.94
(1H, t, 5’-OH, J = 6.0), 4.44 (1H, m, H-3’), 3.88 (1H, q, H-4’, J = 5.0), 3.62, 3.54 (each
1H, m, H-5’), 2.79, 2.34 (each 1H, m, H-2’). FABMS m/z: 475 (MH+). Anal. Found: C,
57.82; H, 4.80; N, 16.76. Calcd. for C24H22N6O5 Á 1.4H2O: C, 57.69; H, 5.00; N, 16.82.
9-(3-O-tert-Butyldimethylsilyl-2-deoxy-ribo-pentofuranosyl)-2,6-dibenzamido-
purine (4a). Compound 2 (36.2 g, 76.4 mmol) which was dried by coevaporation with
pyridine was dissolved in pyridine (300 mL). The solution was added dimethoxytrityl
chloride (37.6 g, 111 mmol) and stirred for 3 h at room temperature. After addition of
EtOH, the reaction mixture was evaporated. The residue was suspended to AcOEt and
water and the precipitate formed was removed by filtration. The organic layer was
washed successively water and brine. The organic layer was dried over Na2SO4 and was
evaporated under reduced pressure. The residue was purified by silica-gel column
chromatography (Eluent: AcOEt/n-hexane, 3:1–4:1–5:1) to give crude 3a (69.9 g),
which was used for the synthesis of 4a without further purification.
To a solution of crude 3a (69.9 g) in DMF (370 mL) was added imidazole (8.80 g,
129 mmol) and tert-butylchlorodimethylsilane (16.5 g, 109 mmol) and the solution was
stirred at room temperature overnight. After addition of water, the reaction mixture was
evaporated under reduced pressure. The residue was dissolved to AcOEt and washed
successively with water and brine. The organic layer was dried over Na2SO4 and
evaporated under reduced pressure.
The residue was dissolved in CHCl3 (510 mL) and was added dropwise
toluenesulfonic acid hydrate (14.6 g) in MeOH (220 mL) at 0°C. After addition, the
solution was stirred for 15 min at same temperature. The reaction mixture was
neutralized by addition of saturated NaHCO3. The organic layer was washed with
brine, dried over Na2SO4 and evaporated under reduced pressure. The residue was
dissolved to AcOEt and stirred for 30 min. The precipitate formed was collected by
filtration to give 4a (28.8 g, 48.9 mmol, 64.0%).
1H-NMR (CDCl3) d 9.30, 9.13 (each 1H, s, NH), 7.95 (1H, s, H-8), 8.06–7.45
(10H, m, aromatic), 6.29 (1H, dd, H-1’, J = 6.5, 8.0), 4.96 (1H, dd, 5’-OH, J = 4.5,
9.0), 4.85 (1H, m, H-3’), 4.04 (1H, m, H-4’), 3.99–3.83 (2H, m, H-5’), 3.10, 2.29 (each
1H, m, H-2’), 0.91 (9H, s, tert-Bu), 0.12, 0.10 (each 3H, s, Me). FABMS m/z: 589

ORDER
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681
(MH+). Anal. Found: C, 60.96; H, 6.14; N, 13.99. Calcd. for C30H36N6O5Si: C, 61.20;
H, 6.16; N, 14.27.
9-(3-O-tert-Butyldimethylsilyl-2-deoxy-4-C-hydroxymethyl-ribo-pentofurano-
syl)-2,6-dibenzamidopurine (5a). The solution of 4a (50.0 g, 85.0 mmol) in toluene
(190 mL) and DMSO (290 mL) was suspended 1-ethyl-3-(3-dimethylaminopropyl)car-
bodiimide hydrochloride (24.6 g, 128 mmol). Pyridine (6.90 mL) and trifluoroacetic
acid (3.30 mL) were added and stirred for 2 h at room temperature. The reaction
mixture was added AcOEt (750 mL) and ice-water (750 mL). The mixture was stirred
and precipitate formed was collected by filtration. The filtrate was washed successively
with water and brine and was dried over Na2SO4 and evaporated under reduced
pressure. This residue was combined with the precipitate to give crude aldehyde.
The crude aldehyde was dissolved in dioxane (240 mL) and added 37%
formaldehyde (45.0 mL) and 2 N NaOH (45.0 mL). After stirring for 3 h at room
temperature, the reaction mixture was neutralized by addition of AcOH. The mixture
was diluted with AcOEt and washed successively with water, saturated NaHCO3 and
brine. The solution was dried over Na2SO4 and evaporated under reduced pressure.
The residue was dissolved in EtOH (360 mL) and added NaBH4 (3.20 g, 85.0
mmol) at 0°C. After stirring for 30 min, the reaction mixture was neutralized by
addition of AcOH. The mixture was diluted with mixture of CHCl3 and MeOH and
washed successively with water and brine. The organic layer was dried over
Na2SO4, and evaporated under reduced pressure. After coevaporation of the residue
with AcOEt, it was dissolved to AcOEt (500 mL) and stirred at room temperature.
Precipitate formed was collected by filtration to give 5a (30.83 g, 49.83 mmol,
58.6%).
1H-NMR (DMSO-d6) d 11.20, 11.00 (each 1H, s, NH), 8.63 (1H, s, H-8), 8.08–
7.51 (10H, m, aromatic), 6.44 (1H, t, H-1’, J = 6.5), 4.90 (1H, t, OH, J = 5.5), 4.78
(1H, t, H-3’, J = 5.5), 4.46 (1H, t, OH, J = 5.5), 3.65–3.53 (4H, m, H-5’ and H-6’),
2.96, 2.43 (each 1H, m, H-2’), 0.89 (9H, s, tert-Bu), 0.10, 0.097 (each 3H, s, Me).
FABMS m/z: 619 (MH+). Anal. Found: C, 58.99; H, 6.14; N, 13.08. Calcd. for
C31H38N6O6Si Á 0.8H2O: C, 58.81; H, 6.30; N, 13.27.
9-(3-O-tert-Butyldimethylsilyl-2-deoxy-4-C-dimethoxytrityloxymethyl-ribo-pen-
tofuranosyl)-2,6-dibenzamidopurine (6a). To a solution of 5a (24.8 g, 40.0 mmol)
in DMF (200 mL) was added triethylamine (11.2 mL, 80.0 mmol) and dimethoxytrityl
chloride (20.3 g, 60.0 mmol) and stirred for 1 h at room temperature. The reaction
mixture was diluted with AcOEt and was washed with water. The organic layer
was dried over MgSO4 and evaporated. The residue was purified by silica-gel
column chromatography (Eluent: n-hexane/AcOEt, 2:1–1:1) to give 6a (22.2 g, 24.1
mmol, 60.3%).
1H-NMR (CDCl3) d 9.33, 9.23 (each 1H, s, NH), 8.07 (1H, s, H-8), 8.13–6.91
(23H, m, aromatic), 6.27 (1H, t, H-1’, J = 6.5), 5.14 (1H, dd, H-3’, J = 3.5, 6.0), 4.59
(1H, dd, 5’-OH, dd, J = 5.5, 8.5), 4.30 (1H, dd, H-5’a, J = 5.5, 12.5), 3.81 (1H, d, H-
5’b, J = 9.0, 12.5), 3.59 (1H, d, H-6’a, J = 10.5), 3.28 (1H, m, H-2’a), 3.17 (1H, d, H-
6’b, J = 11.0), 2.48 (1H, m, H-2’b), 0.84 (9H, s, tert-Bu), 0.09, 0.07 (each 3H, s, Me).
FABMS m/z: 921 (MH+). Anal. Found: C, 67.87; H, 6.35; N, 8.80. Calcd. for
C52H56N6O8Si: C, 67.80; H, 6.13; N, 9.12.

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Kohgo et al.
9-(3,5-Di-O-tert-Butyldimethylsilyl-2-deoxy-4-C-hydroxymethyl-ribo-pentofura-
nosyl)-2,6-dibenzamidopurine (7a). To a solution of 6a (29.9 g, 32.5 mmol) in DMF
(100 mL) was added imidazole (3.30 g, 48.8 mmol ) and tert-butylchlorodimethylsilane
(5.90 g, 39.0 mmol) and stirred at room temperature overnight. The reaction mixture
was diluted with AcOEt and was washed with water. The organic layer was dried over
MgSO4 and evaporated.
The residue was dissolved to CHCl3 (620 mL) and was added dropwise TsOH
hydrate (6.20 g) in MeOH (190 mL) at 0°C. After addition, the solution was stirred for
20 min at same temperature. The reaction mixture was neutralized by addition of
saturated NaHCO3. The organic layer was dried over MgSO4 and evaporated. The
residue was purified by silica-gel column chromatography (Eluent: n-hexane/AcOEt,
3:1) to give 7a (17.5 g, 23.9 mmol, 73.5%).
1H-NMR (CDCl3) d 9.14, 9.05 (each 1H, s, NH), 8.13 (1H, s, H-8), 8.02–7.53
(10H, m, aromatic), 6.45 (1H, t, H-1’, J = 6.5), 4.99 (1H, dd, H-3’, J = 4.5, 6.5), 3.91–
3.74 (4H, m, H-5’ and H-6’), 3.12, 2.54 (each 1H, m, H-2’), 2.70 (1H, dd, 6’-OH,
J = 5.0, 8.5), 0.93, 0.86 (each 9H, s, tert-Bu), 0.17, 0.15, 0.02, 0.01 (each 3H, s, Me).
FABMS m/z: 733 (MH+). Anal. Found: C, 60.51; H, 7.19; N, 11.37. Calcd. for
C37H52N6O6Si2: C, 60.63; H, 7.15; N, 11.46.
9-(3,5-Di-O-tert-Butyldimethylsilyl-4-C-cyano-2-deoxy-ribo-pentofuranosyl)-2,6-
dibenzamidopurine (10a). The solution of 7a (2.00 g, 2.73 mmol) in toluene
(6.00 mL) and DMSO (12.0 mL) was suspended 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide hydrochloride (1.57 g, 8.19 mmol). The mixture was added pyridine
(0.22 mL) and trifluoroacetic acid (0.11 mL) and was stirred for 1 h at room tem-
perature. The reaction mixture was diluted with AcOEt and washed with water. The
organic layer was dried over MgSO4 and evaporated to give crude aldehyde 8a.
To a solution of crude aldehyde 8a in pyridine (20.0 mL) was added hydrox-
ylamine hydrochloride (0.28 g, 4.03 mmol) and stirred for 30 min at room temperature.
The reaction mixture was evaporated and the residue was partitioned between AcOEt
and water. The organic layer was dried over MgSO4 and evaporated to give crude
oxime 9a.
To a solution of crude oxime 9a in CH2Cl2 (20.0 mL) was added triethylamine
(0.76 mL, 5.45 mmol) and methanesulfonyl chloride (0.32 mL, 4.13 mmol) at 0°C
and stirred for 30 min at same temperature. The reaction mixture was diluted with
CHCl3 and washed with saturated NaHCO3. The organic layer was dried over MgSO4
and evaporated. The residue was purified by silica-gel column chromatography (Eluent:
n-Hexane/AcOEt, 1:1) to give 10a (1.77 g, 2.43 mmol, 89.0%).
1H-NMR (CDCl3) d 9.54, 9.29 (each 1H, s, NH), 7.97 (1H, s, H-8), 8.03–7.49
(10H, m, aromatic), 6.43 (1H, t, H-1’, J = 6.5), 5.21 (1H, t, H-3’, J = 5.5), 4.22, 3.99
(each 1H, d, H-5’, J = 11.0), 3.42, 2.53 (each 1H, m, H-2’), 0.97, 0.86 (each 9H, s, tert-
Bu), 0.24, 0.17, 0.054, 0.051 (each 3H, s, Me). FABMS m/z: 728 (MH+). Anal. Found:
C, 57.87; H, 6.97; N, 12.78. Calcd. for C37H49N7O5Si2 Á 2H2O: C, 58.17; H, 6.99;
N, 12.83.
9-(4-C-Cyano-2-deoxy-ribo-pentofuranosyl)-2,6-diaminopurine (12a). The
mixture of 10a (1.00 g, 1.37 mmol) in MeOH (10.0 mL) and 40% MeNH2 (10.0 mL)

ORDER
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4’-C-Substituted 2’-Deoxy Purine Nucleosides
683
was stirred at room temperature overnight. Precipitate formed was collected by filtra-
tion to give crude debenzoylated product 11a (0.25 g).
To a solution of crude 11a (0.25 g) in THF (9.00 mL) was added tetra-n-
butylammonium fluoride (1M solution in THF, 1.00 mL, 1.00 mmol) and stirred for
15 min at room temperature. The reaction mixture was evaporated and the residue
was purified by silica-gel column chromatography (Eluent: AcOEt/MeOH,10:1).
The residue was triturated with 2-propanol to give 12a (0.10 g, 0.34 mmol, 24.8%
from 10a).
1H-NMR (DMSO-d6) d 7.93 (1H, s, H-8), 6.77 (2H, s, NH2), 6.34 (1H, t, H-1’,
J = 7.0), 6.28 (1H, d, 3’-OH, J = 4.5), 5.87 (1H, t, 5’-OH, J = 6.5), 5.85 (2H, s, NH2),
4.64 (1H, q, H-3’, J = 4.5), 3.78 (1H, dd, H-5’a, J = 6.0, 12.0), 3.65 (1H, dd, H-5’b,
J = 6.5, 12.0), 2.87, 2.37 (each 1H, m, H-2’). FABMS m/z: 292 (MH+). Anal. Found: C,
43.18; H, 4.62; N, 31.80. Calcd. for C11H13N7O3 Á 0.8H2O: C, 43.22; H, 4.81; N, 32.07.
Improved Route for the Synthesis of 9-(4-C-Cyano-2-deoxy-ribo-pentofurano-
syl)-2,6-diaminopurine (12a) from 7a. The solution of 7a (1.00 g, 1.40 mmol) in
toluene (3.00 mL) and DMSO (6.00 mL) was suspended 1-ethyl-3-(3-dimethylamino-
propyl)carbodiimide hydrochloride (0.79 g, 4.10 mmol). Pyridine (0.11 mL) and
trifluoroacetic acid (60.0 mL) were added and stirred for 90 min at room temperature.
The reaction mixture was diluted with AcOEt and washed with water. The organic
layer was dried over MgSO4 and evaporated to give crude aldehyde 8a.
To a solution of crude aldehyde 8a in pyridine (10.0 mL) was added hydrox-
ylamine hydrochloride (0.19 g, 2.70 mmol) and stirred for 1 h at room temperature.
The reaction mixture was evaporated and the residue was partitioned between AcOEt
and water. The organic layer was dried over MgSO4 and evaporated to give crude
oxime 9a.
The mixture of crude oxime 9a in MeOH (10.0 mL) and 40% MeNH3 (10.0 mL)
was stirred for 46 h at room temperature. Precipitate formed was collected by filtaration
to give crude debenzoylated 4’-C-oxime derivative 13 (0.61 g).
To a suspension of crude 13 (0.61 g) in MeCN (5 mL) was added 1,1’-
carbonyldiimidazole (0.40 g, 2.50 mmol) and stirred for 4 h at room temperature. The
reaction mixture was diluted with AcOEt and washed successively with water and
brine. The organic layer was dried over MgSO4 and evaporated under reduced pressure.
The residue was purified by silica-gel column chromatography (Eluent: CHCl3/MeOH,
100:1–50:1) to give 11a (0.38 g, 0.73 mmol, 52.1% from 9a).
1H-NMR (CDCl3) d 7.62 (1H, s, H-8), 6.32 (1H, t, H-1’, J = 6.25), 5.35 (2H, s,
NH2), 4.93 (1H, t, H-3’, J = 5.75), 4.66 (2H, s, NH2), 4.00, 3.87 (each 1H, d, H-5’,
J = 11.0), 3.08, 2.50 (each 1H, m, H-2’), 0.96, 0.89 (each 9H, s, tert-Bu), 0.20, 0.17,
0.086, 0.046 (each 3H, s, Me). FABMS m/z: 520 (MH+). Anal. Found: C, 52.50; H,
7.84; N, 18.39. Calcd. for C23H41N7O3Si2 Á 0.4H2O: C, 52.42; H, 7.99; N, 18.60.
To a solution of 11a (0.272 g, 0.523 mmol) in THF (4.80 mL) was added tetra-n-
butylammonium fluoride (1M solution in THF, 1.10 mL, 1.10 mmol) and stirred for 10
min at room temperature. The reaction mixture was evaporated and the residue was
purified by silica-gel column chromatography (Eluent: AcOEt/MeOH,10:1–5:1) to give
12a (0.147 mg, 0.506 mmol, 96.7%). Total isolated yield of 12a from 7a by this
procedure was 50.4%.

ORDER
REPRINTS
684
Kohgo et al.
4’-C-Cyano-2-deoxyguanosine (14a). To a solution of 12a (70.0 mg, 0.24 mmol)
in 50 mM Tris-HCl buffer (pH, 7.50, 13.3 mL) was added adenosine deaminase (0.13
mL, 58.5 units) and stirred for 1 h at 40°C. The precipitate formed was collected by
filtaration and recrystallized from water to give 14a (56.0 mg, 0.19 mmol, 79.2%).
1H-NMR (DMSO-d6) d 10.68 (1H, s, NH), 7.93 (1H, s, H-8), 6.53 (2H, s, NH2),
6.29 (1H, t, H-1’, J = 6.8), 6.27 (1H, d, 3’-OH, J = 4.9), 5.73 (1H, t, 5’-OH, J = 5.8),
4.60 (1H, dd, H-3’, J = 4.9, 10.8), 3.75 (1H, dd, H-5’a, J = 5.8, 11.7), 3.64 (1H, dd, H-
5’b, J = 5.9, 11.7), 2.80, 2.40 (each 1H, m, H-2’). FABMS m/z: 293 (MH+). Anal.
Found: C, 41.54; H, 4.37; N, 26.58. Calcd. for C11H12N6O4 Á 0.6H2O: C, 41.41; H,
4.17; N, 26.34.
N6-Benzoyl-3’-O-tert-butyldimethylsilyl-2’-deoxyadenosine (4b). To a solution
of 3b (2.00 g, 3.04 mmol) in DMF (6.00 mL) was added imidazole (0.83 g, 12.2 mmol)
and tert-butylchlorodimethylsilane (0.92 g, 6.10 mmol) and the solution was stirred at
room temperature overnight. After addition of MeOH, the reaction mixture was diluted
with AcOEt and washed with water. The organic layer was dried over MgSO4 and
evaporated under reduced pressure.
The residue was dissolved in CHCl3 (70.0 mL) and added dropwise toluenesulfonic
acid hydrate (0.60 g) in MeOH (30.0 mL) at 0°C. After addition, the solution was
stirred for 30 min at same temperature. The reaction mixture was neutralized by addi-
tion of saturated NaHCO3. The organic layer was dried over MgSO4 and evaporated
under reduced pressure. The residue was purified by silica-gel column chromatography
(Eluent: AcOEt/n-hexane, 3:1) to give 4b (1.20 g, 2.56 mmol, 84.2%).
1H-NMR (CDCl3) d 9.02 (1H, s, NH), 8.79, 8.10 (each 1H, s, H-2 and H-8), 8.03–
7.52 (5H, m, aromatic), 6.37 (1H, dd, H-1’, J = 5.5, 9.5), 5.78 (1H, dd, 5’-OH, J = 1.5,
11.5), 4.74 (1H, d, H-3’, J = 6.5), 4.17 (1H, s, H-4’), 4.00–3.74 (2H, m, H-5’), 3.07, 2.27
(each 1H, m, H-2’), 0.94 (9H, s, t-Bu), 0.13 (6H, s, Me). FABMS m/z: 470 (MH+). Anal.
Found: C, 58.45; H, 6.56; N, 14.76. Calcd. for C23H31N5O4Si: C, 58.83; H, 6.65; N, 14.91.
N6-Benzoyl-3’-O-tert-butyldimethylsilyl-2’-deoxy-4’-C-hydroxymethyladenosine
(5b). The solution of 4b (2.55 g, 5.43 mmol) in toluene (10.0 mL) and DMSO (15.0
mL) was suspended 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(3.12 g, 16.3 mmol). The mixture was added pyridine (0.41 mL) and trifluoroacetic
acid (0.21 mL) and stirred for 2 h at room temperature. The reaction mixture was
diluted with AcOEt and washed with water. The organic layer was dried over MgSO4
and evaporated under reduced pressure.
The residue was dissolved in dioxane (15.0 mL) and added 37% aqueous
formaldehyde solution (2.86 mL) and 2 N sodium hydroxide (2.86 mL). After stirring
for 1 h at room temperature, the reaction mixture was neutralized by addition of AcOH,
diluted with AcOEt and washed with water. The organic layer was dried over MgSO4
and evaporated under reduce pressure.
The residue was dissolved in EtOH (25.0 mL) and added NaBH4 (0.21 g, 5.55
mmol) at 0°C. After stirring for 30 min, the reaction mixture was neutralized by
addition of AcOH. The mixture was diluted with AcOEt and washed with water. The
organic layer was dried over MgSO4 and evaporated under reduced pressure. The
residue was purified by silica-gel column chromatography (Eluent: AcOEt/n-hexane,
3:1–4:1–5:1) to give 5b (1.68 g, 3.36 mmol, 61.9%).

ORDER
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4’-C-Substituted 2’-Deoxy Purine Nucleosides
685
1H-NMR (CDCl3) d 9.03 (1H, s, NH), 8.79, 8.08 (each 1H, s, H-2 and H-8), 8.03–
7.52 (5H, m, aromatic), 6.45 (1H, dd, H-1’, J = 6.0, 9.0), 5.61 (1H, dd, OH, J = 2.0,
11.0), 4.94 (1H, dd, H-3’, J = 1.5, 6.0), 3.87–3.66 (4H, m, H-5’ and H-6’), 3.24 (1H, m,
H-2’a), 2.68 (1H, dd, OH, J = 4.0, 9.0), 2.37 (1H, m, H-2’b), 0.96 (9H, s, tert-Bu), 0.19,
0.18 (each 3H, s, Me). FABMS m/z: 500 (MH+). Anal. Found: C, 55.41; H, 6.50; N,
13.32. Calcd. for C24H33N5O5Si Á 1.1H2O: C, 60.63; H, 7.15; N, 11.46.
N6-Benzoyl-3’-O-tert-butyldimethylsilyl-2’-deoxy-4’-C-dimethoxytrityloxy-
methyladenosine (6b). To a solution of 5b (0.84 g, 1.68 mmol) in CH2Cl2 (17.0 mL)
was added triethylamine (0.47 mL, 3.37 mmol) and dimethoxytrityl chloride (0.85 g,
2.51 mmol) and stirred for 30 min at 0°C. After addition of MeOH, the reaction
mixture was diluted with CHCl3 and washed with saturated NaHCO3. The organic layer
was dried over MgSO4 and evaporated under reduced pressure. The residue was
purified by silica-gel column chromatography (Eluent: n-hexane/AcOEt, 2:1–1:1) to
give 6b (0.91 g, 1.13 mmol, 67.3%).
1H-NMR (CDCl3) d 9.17 (1H, s, NH), 8.96, 8.24 (each 1H, s, H-2 and H-8), 8.23–
6.94 (18H, m, aromatic), 6.41 (1H, dd, H-1’, J = 6.0, 8.5), 5.44 (1H, dd, 5’-OH,
J = 2.5, 11.0), 4.84 (1H, dd, H-3’, J = 1.5, 5.5), 4.43 (1H, dd, H-5’a, J = 3.0, 12.5),
3.92 (6H, s, OMe), 3.78 (1H, t, H-5’b, J = 12.0), 3.67 (1H, d, H-6’a, J = 11.0), 3.30
(1H, m, H-2’a), 3.20 (1H, d, H-6’b, J = 11.0), 2.39 (1H, m, H-2’b), 0.90 (9H, s, tert-
Bu), 0.13, 0.11, (each 3H, s, Me). FABMS m/z: 802 (MH+). Anal. Found: C, 67.02; H,
6.52; N, 8.55. Calcd. for C45H51N5O7Si: C, 67.39; H, 6.41; N, 8.73.
N6-Benzoyl-3’,5’-di-O-tert-butyldimethylsilyl-2’-deoxy-4’-C-hydroxymethylade-
nosine (7b). To a solution of 6b (1.61 g, 2.01 mmol) in DMF (8.00 mL) was added
imidazole (0.41 g, 6.02 mmol) and tert-butylchlorodimethylsilane (0.45 g, 2.99 mmol)
and stirred at room temperature overnight. After addition of MeOH, the reaction
mixture was diluted with AcOEt and washed with water. The organic layer was dried
over MgSO4 and evaporated.
The residue was dissolved to CHCl3 (70.0 mL) and added dropwise 1% TsOH
hydrate in MeOH (30.0 mL) at 0°C. After addition, the solution was stirred for 30 min
at same temperature. The reaction mixture was neutralized by addition of saturated
NaHCO3. The organic layer was dried over MgSO4 and evaporated under reduced
pressure. The residue was purified by silica-gel column chromatography (Eluent: n-
hexane/AcOEt, 3:1) to give 7b (1.00 g, 1.63mmol, 81.1%).
1H-NMR (CDCl3) d 9.04 (1H, s, NH), 8.81, 8.28 (each 1H, s, H-2 and H-8), 8.04–
7.52 (5H, m, aromatic), 6.52 (1H, t, H-1’, J = 6.5), 5.78, 4.88 (1H, dd, H-3’, J = 4.5,
6.5), 3.89–3.73 (4H, m, H-5’ and H-6’), 3.04, 2.58 (each 1H, m, H-2’), 2.48 (1H, dd, 6’-
OH, J = 5.5, 8.5) 0.95, 0.89 (each 9H, s, tert-Bu), 0.16, 0.06 (each 6H, s, Me). FABMS
m/z: 614 (MH+). Anal. Found: C, 58.33; H, 7.80; N, 11.14. Calcd. for C30H47N5O5Si2:
C, 58.69; H, 7.72; N, 11.41.
N6-Benzoyl-3’,5’-di-O-tert-butyldimethylsilyl-4’-C-cyano-2’-deoxyadenosine
(10b). The solution of 7b (1.00 g, 1.63 mmol) in toluene (3.00 mL) and DMSO (6.00
mL) was suspended 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(0.94 g, 4.90 mmol). Pyridine (0.13 mL) and trifluoroacetic acid (62.8 mL) were
added and stirred for 1 h at room temperature. The reaction mixture was diluted with

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AcOEt and washed with water. The organic layer was dried over MgSO4 and
evaporated to give crude aldehyde 8b.
To a solution of crude aldehyde 8b in pyridine (10.0 mL) was added hydroxylamine
hydrochloride (0.17 g, 2.45 mmol) and stirred for 30 min at room temperature. The
reaction mixture was evaporated and the residue was partitioned between AcOEt and
water. The organic layer was dried over MgSO4 and evaporated to give crude oxime 9b.
To a solution of crude oxime 9b in CH2Cl2 (10.0 mL) was added triethylamine
(0.45 mL, 3.23 mmol) and methanesulfonyl chloride (0.19 mL, 2.45 mmol) at 0°C and
stirred for 30 min at same temperature. The reaction mixture was diluted with CHCl3
and washed with saturated NaHCO3. The organic layer was dried over MgSO4 and
evaporated. The residue was purified by silica-gel column chromatography (Eluent:
CHCl3/MeOH, 100:1) to give 10b (0.87 g, 1.41 mmol, 87.7%).
1H-NMR (CDCl3) d 9.10 (1H, s, NH), 8.79, 8.14 (each 1H, s, H-2 and H-8), 8.04–
7.52 (5H, m, aromatic), 6.52 (1H, dd, H-1’, J = 6.0, 6.5), 5.02 (1H, t, H-3’, J = 6.0), 4.05,
3.89 (each 1H, d, H-5’, J = 11), 3.22, 2.62 (each 1H, m, H-2’), 0.98, 0.87 (each 9H, s, tert-
Bu), 0.21, 0.19, 0.08, 0.03 (each 3H, s, Me). FABMS m/z: 609 (MH+). Anal. Found: C,
58.64; H, 7.38; N, 13.60. Calcd. for C30H44N6O4Si2 Á 0.2H2O: C, 58.83; H, 7.31; N, 13.72.
4’-C-Cyano-2’-deoxyadenosine (12b). The mixture of 10b (0.70 g, 1.15 mmol)
in MeOH (10.5 mL) and conc. NH4OH (3.50 mL) was stirred at room temperature
overnight. Precipitate formed was collected by filtration to give crude debenzoylated
product 11b (0.50 g).
To a solution of crude 11b (0.50 g) in THF (7.80 mL) was added tetra-n-
butylammonium fluoride (1 M solution of THF, 2.18 mL, 2.18 mmol) and stirred
for 15 min at room temperature. The reaction mixture was evaporated and the residue
was purified by silica-gel column chromatography (Eluent: CHCl3/MeOH, 20:1–10:1).
The residue was recrystallized from water to give 12b (0.21 g, 0.76 mmol, 66.1%
from 10b).
1H-NMR (DMSO-d6) d 8.34 (1H, s, H-8), 8.16 (1H, s, H-2), 7.36 (2H, s, NH2), 6.52
(1H, t, H-1’, J = 6.5), 6.32 (1H, d, 3’-OH, J = 5.0), 5.83 (1H, t, 5’-OH, J = 6.0), 4.74 (1H,
dd, H-3’, J = 5.5, 11.5), 3.82 (1H, dd, H-5’a, J = 5.0, 11.5), 3.66 (1H, dd, H-5’b, J = 6.0,
12), 3.01, 2.47 (each 1H, m, H-2’). FABMS m/z: 277 (MH+). Anal. Found: C, 45.90; H,
4.42; N, 29.12. Calcd. for C11H12N6O3 Á 0.6H2O: C, 46.02; H, 4.63; N, 29.28.
4’-C-Cyano-2’-deoxyinosine (14b). To a solution of 12b (0.15 g, 0.54 mmol) in
Tris-HCl buffer (pH, 7.50, 30.0 mL) was added adenosine deaminase (0.30 mL, 135
units) and stirred for 2 h at 40°C. The reaction mixture was concentrated and purified
reverse phase column chromatography (0–2.5% EtOH). The residue was recrystalized
from water to give 14b (90.0 mg, 0.32 mmol, 59.3%).
1H-NMR (DMSO-d6) d 12.46 (1H, s, NH), 8.32 (1H, s, H-8), 8.10 (1H, s, H-2), 6.49
(1H, t, H-1’, J = 6.5), 6.36 (1H, d, 3’-OH, J = 5.0), 5.76 (1H, t, 5’-OH, J = 6.0), 4.69 (1H,
dd, H-3’, J = 5.5, 11.5), 3.78 (1H, dd, H-5’a, J = 6.0, 12.0), 3.66 (1H, dd, H-5’b, J = 6.0,
12.0), 2.92, 2.49 (each 1H, m, H-2’). FABMS m/z: 278 (MH+). Anal. Found: C, 44.91; H,
4.06; N, 24.06. Calcd. for C11H11N5O4 Á 1H2O: C, 44.75; H, 4.44; N, 23.72.
9-(3,5-Di-O-tert-Butyldimethylsilyl-2-deoxy-4-C-ethynyl-ribo-pentofuranosyl)-
2,6-dibenzamidopurine (16a). The solution of 7a (2.00 g, 2.73 mmol) in toluene

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(6.00 mL) and DMSO (12.0 mL) was suspended 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide hydrochloride (1.57 g, 8.19 mmol). Pyridine (0.22 mL) and trifluoroacetic
acid (0.11 mL) were added and stirred for 90 min at room temperature. The reaction
mixture was diluted with AcOEt and washed with water. The organic layer was dried
over MgSO4 and evaporated to give crude aldehyde 8a.
To a suspension of bromomethyltriphenylphosphonium bromide (2.40 g, 5.50
mmol) in THF (32.0 mL) was added potassium tert-butoxide (0.93 g, 8.29 mmol) at
À40°C and stirred for 90 min at same temperature to prepare bromomethylene
triphenylphosphorane. The solution was added crude aldehyde 8a in THF (32.0 mL)
and stirred for 2 h at À40°C. The reaction mixture was added saturated NH4Cl and
stirred. The organic layer was dried over MgSO4 and evaporated under reduced
pressure. The residue was purified by silica-gel column chromatography (Eluent: n-
hexane/AcOEt, 3:1–2:1–1:1) to give crude bromoethene 15a (1.96 g).
To a solution of crude 15a (1.96 g) in THF (50.0 mL) was added potassium tert-
butoxide (0.91 g, 8.11 mmol) at À40°C and the solution was stirred for 1 h at same
temperature. After addition of saturated NH4Cl, the mixture was stirred at room
temperature. The organic layer was dried over MgSO4 and evaporated under reduced
pressure. The residue was purified by silica-gel column chromatography (Eluent: n-
hexane/AcOEt, 2:1) to give 16a (1.21 g, 1.66 mmol, 60.8% from 7a).
1H-NMR (CDCl3) d 9.38, 9.37 (each 1H, s, NH), 8.19 (1H, s, H-8), 8.04–7.49
(10H, m, aromatic), 6.54 (1H, dd, H-1’, J = 4.5, 7.0), 4.84 (1H, t, H-3’, J = 6.5), 4.03,
3.83 (each 1H, d, H-5’, J = 11.0), 2.86, 2.66 (each 1H, m, H-2’), 2.55 (1H, s, ethynyl),
0.94, 0.90 (each 9H, s, tert-Bu), 0.14, 0.13, 0.08, 0.06 (each 3H, s, Me). FABMS m/z:
727 (MH+). Anal. Found: C, 61.91; H, 6.94; N, 11.21. Calcd. for C38H50N6O5-
Si2 Á 0.5H2O: C, 62.01; H, 6.98; N, 11.42.
9-(2-Deoxy-4-C-ethynyl-ribo-pentofuranosyl)-2,6-diaminopurine (18a). To a
solution of 16a (1.24 g, 1.71 mmol) in THF (30.0 mL) was added tetra-n-
butylammonium fluoride (1 M solution of THF, 4.30 mL, 4.30 mmol) and stirred for
30 min at room temperature. The reaction mixture was evaporated and the residue was
purified by silica-gel column chromatography (Eluent: CHCl3/MeOH, 20:1) to give 17a
(0.70 g, 1.40 mmol).
The mixture of 17a (0.65 g, 1.30 mmol) in MeOH (15.0 mL) and 40% MeNH2
aqueous solution (30.0 mL) was stirred at room temperature overnight. The reaction
mixture was evaporated and the residue was purified by silica-gel column chroma-
tography (Eluent: CHCl3/MeOH, 10:1). The residue was recrystallized from water to
give 18a (0.33 g, 1.14 mmol, 87.7%), whose structure was confirmed by comparing
spectroscopic data of 18a with these of previously reported 9-(2-deoxy-4-C-ethynyl-
ribo-pentofuranosyl)-2,6-diaminopurine.[6]
N6-Benzoyl-3’,5’-di-O-tert-butyldimethylsilyl-4’-C-ethynyl-2’-deoxyadenosine
(16b). The solution of 7b (1.80 g, 2.93 mmol) in toluene (6.00 mL) and DMSO (12.0
mL) was suspended 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(1.69 g, 8.82 mmol). Pyridine (0.24 mL) and trifluoroacetic acid (0.12 mL) were
added and stirred for 3 h at room temperature. The reaction mixture was diluted with
AcOEt and washed with water. The organic layer was dried over MgSO4 and evap-
orated to give crude aldehyde 8b.

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Kohgo et al.
To a suspension of bromomethyltriphenylphosphonium bromide (2.56 g, 5.87
mmol) in THF (35.0 mL) was added potassium tert-butoxide (1.00 g, 8.91 mmol) at
À40°C and stirred for 2 h at same temperature to prepare bromomethylene
triphenylphosphorane. The solution was added crude aldehyde 8b in THF (35.0 mL)
and stirred for 2 h at À40°C. The reaction mixture was added saturated NH4Cl and
stirred. The organic layer was dried over MgSO4 and evaporated under reduced
pressure. The residue was purified by silica-gel column chromatography (Eluent: n-
hexane/AcOEt, 3:1–2:1–1:1) to give crude bromoethene 15b.
To a solution of crude 15b in THF (70.0 mL) was added potassium tert-butoxide
(1.00 g, 8.91 mmol) at À40°C and the solution was stirred for 2 h at same temperature.
After addition of saturated NH4Cl, the mixture was stirred at room temperature. The
organic layer was dried over MgSO4 and evaporated under reduced pressure. The
residue was purified by silica-gel column chromatography (Eluent: n-hexane/AcOEt,
2:1–1:1) to give 16b (1.21 g, 1.99 mmol, 67.9% from 7b).
1H-NMR (CDCl3) d 8.99 (1H, s, NHBz), 8.81, 8.30 (each 1H, s, H-2 and H-8),
8.04–7.51 (5H, m, aromatic), 6.54 (1H, dd, H-1’, J = 4.9, 7.3), 4.83 (1H, t, H-3’,
J = 6.8), 3.97 (1H, d, H-5’a, J = 11.2), 3.81 (1H, d, H-5’b, J = 11.2), 2.79, 2.68 (each
1H, m, H-2’), 2.57 (1H, s, ethynyl), 0.94, 0.89 (each 9H, s, tert-Bu), 0.14, 0.13, 0.08,
0.04 (each, 3H, s, Me). FABMS m/z: 608 (MH+). Anal. Found: C, 60.25; H, 7.50; N,
11.10. Calcd. for C31H45N5O4Si2 Á 0.5H2O: C, 60.36; H, 7.52; N, 11.35.
2’-Deoxy-4’-C-ethynyladenosine (18b). To a solution of 16b (0.118 g, 0.235
mmol) in THF (3.2 mL) was added tetra-n-butylammonium fluoride (1 M solution of
THF, 0.7 mL, 0.7 mmol) and stirred for 30 min at room temperature. The reaction
mixture was evaporated and the residue was purified by silica-gel column chro-
matography (Eluent: CHCl3/MeOH, 20:1) to give crude 17b (0.122 g).
The mixture of 17b (0.122 g) in MeOH (2.1 mL) and conc. NH4OH (0.7 mL) was
stirred at room temperature overnight. The reaction mixture was evaporated and the
residue was purified by silica-gel column chromatography (Eluent: CHCl3/MeOH,
20:1–10:1). The residue was recrystallized from water to give 18b (0.057 g, 0.207
mmol, 88.1% from 16b), whose structure was confirmed by comparing spectroscopic
data of 18b with these of previously reported 2’-deoxy-4’-C-ethynyladenosine.[6]
Antiviral Evaluation
Antiviral Agents. 3’-Azido-3’-deoxythymidine (AZT or zidovudine), 2’,3’-
dideoxyinosine (ddI or didanosine), and 2’,3’-dideoxycytidine (ddC or zalcitabine)
were purchased from Sigma (St. Louis, MO). (-)-2’,3’-Dideoxy-3’-thiacytidine (3TC or
lamivudine) was a kind gift from Dr. R. F. Schinazi (Atlanta, GA). A series of 4’-
position substituted nucleosides were designed and synthesized as described by us.
Determination of Drug Susceptibility of HIV-1. The inhibitory effects of test
compounds on HIV-1 replication were monitored by the inhibition of virally induced
cytopathicity in MT-4 cells. Briefly, MT-4 cells were suspended at 105 cells/mL and
exposed to HIV-1LAI at 100 50% tissue culture infectious doses (TCID50s).
Immediately after viral exposure, the cell suspension (104 cells in 100 mL) was
brought into each well of a 96-well flat microtiter culture plate (Costar, Cambridge,

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Mass.) containing various concentrations of test compounds. After incubation for 5
days, the number of viable cells was determined by the MTT [3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide] method as previously described.[13,14]
The sensitivity of infectious clones to various RTIs was determined in the
multinuclear activation of the galactosidase indicator (MAGI) assay,[17] with some
modifications using the viral preparations titrated as previously described.[18] Briefly,
target cells (HeLa CD4-LTR/b-gal; 104/well) were plated in 96-well flat microtiter
culture plates. On the following day, the medium was aspirated and the cells were
inoculated with HIV-1 clones (70 MAGI units/well, which gave 70 blue cells after 48 h
of incubation) and cultured in the presence of various concentrations of drug in fresh
medium. Forty-eight hours after viral exposure, all blue cells in each well were
counted. The cytotoxicity of the compound was determined by the MTT method as
previously described.[13] All experiments were performed in triplicate.
Preliminary Toxicity Test for Mice. Six-week-old, random-bred, Swiss albino
ICR male mice, (Jcl:ICR) were purchased from Clea Japan. The drugs were dissolved
in saline and administered once to the mice either orally (p.o.) or intravenously (i.v.).
The mice were observed twice daily for 7 days for their death.
ACKNOWLEDGMENT
The authors are grateful to Dr. K. Kodama of Yamasa Corporation for his
encouragement throughout this work.
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Received January 5, 2004
Accepted February 23, 2004

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