Synthesis and anti-hepatitis B virus and anti-hepatitis C virus activities of 7-deazaneplanocin a analogues in vitro

Article abstract of DOI:10.1021/jm801418v

A series of 7-deazaneplanocin A (7-DNPA, 2) analogues were synthesized and evaluated for in vitro antiviral activity against HBV and HCV. The syntheses of target carbocyclic nucleosides were accomplished via a convergent procedure. 7-Substitutions were in

Full text of DOI:10.1021/jm801418v

206
J. Med. Chem. 2009, 52, 206–213
Synthesis and Anti-Hepatitis B Virus and Anti-Hepatitis C Virus Activities of
7-Deazaneplanocin A Analogues in Vitro
Hyo-Joong Kim,† Ashoke Sharon,† Chandralata Bal,† Jianing Wang,† Madhan Allu,† Zhuhui Huang,‡ Michael G. Murray,‡
Leda Bassit,§ Raymond F. Schinazi,§ Brent Korba,| and Chung K. Chu*,†
The UniVersity of Georgia College of Pharmacy, Athens, Georgia 30602, Southern Research Institute, Frederick, Maryland 21701, Emory
UniVersity School of Medicine/VA Medical Center, Atlanta, Georgia 30033, and Georgetown UniVersity Medical Center,
RockVille, Maryland 20850
ReceiVed NoVember 10, 2008
A series of 7-deazaneplanocin A (7-DNPA, 2) analogues were synthesized and evaluated for in vitro antiviral
activity against HBV and HCV. The syntheses of target carbocyclic nucleosides were accomplished via a
convergent procedure. 7-Substitutions were introduced by using 7-substituted-7-deaza heterocyclic base
precursors (F, Cl, Br, and I) or via substitution reactions after the synthesis of the carbocyclic nucleosides.
Among the synthesized compounds, 2, 13-15, 24, and 27 exhibited significant anti-HCV activity (EC50
ranged from 1.8 to 20.1 µM) and compounds 2, 15, 22, and 24 demonstrated moderate to potent anti-HBV
activity (EC50 ) 0.3-3.3 µM). In addition, compound 24 also showed activity against lamivudine- and
adefovir-associated HBV mutants.
Introduction
Neplanocin A (NPA,a Figure 1, 1), a fermentation-derived
carbocyclic nucleoside, exhibits a broad-spectrum antiviral
activity and may be associated with the inhibition of S-
adenosylhomocysteine hydrolase (AdoHcyase), a key enzyme
regulating biologically important transmethylation processes.1
However, the therapeutic utility of NPA has been limited
because of its cytotoxicity, which may be attributed mainly to
the phosphorylation of NPA at the 5′-position2 as well as the
lack of selective inhibition of AdoHcyase.1 The corresponding
Figure 1. Structures of neplanocin A (NPA) and 7-deazaneplanocin
A (7-DNPA).
NPA-triphosphate may interfere with the RNA synthesis of the
host-cell and be further metabolized to cytotoxic S-neplanocyl-
methionine.2 On the basis of these observations, numerous
efforts have been made to modify the structure of NPA to
produce the compounds with better therapeutic efficacy.3
Recently, our group has also been involved in discovering
biologically interesting carbocyclic nucleosides.4
Hepatitis B and hepatitis C virus (HBV and HCV, respec-
tively) belong to hepadnaViridae5 and flaViViridae6 families,
respectively. The infection of the two viruses accounts for major
proportions of hepatitis cases worldwide and is also strongly
associated with the development of cirrhosis and hepatocellular
carcinoma. Although important advances have been made in
the prevention of HBV infection, the current therapies remain
still unsatisfactory.7 Furthermore, there are no vaccines as well
as no effective therapeutic agents that are available for the
treatment of HCV. The current standard therapy for chronic
HCV infection is interferon R (or pegylated-interferon R) in
combination with ribavirin, which is inadequate because of the
low response rates as well as its side effects.8 Although a number
of inhibitors of HCV replication have recently been reported,
their therapeutic effectiveness and safety profile remain to be
demonstrated in clinical trials.9 As a part of our ongoing antiviral
drug discovery program, a number of carbocyclic nucleoside
analogues have been synthesized and evaluated for their
potential antiviral activity against HBV and HCV.10
Recently, we described the synthesis and antiviral activity
of 7-deazaneplanocin A (7-DNPA, Figure 1, 2) against ortho-
poxviruses (vaccinia and cowpox virus).4e In addition, the further
screening of the compound 2 revealed significant anti-HBV and
anti-HCV activity with low cytotoxicity. In view of these
interesting biological results, it was of interest to explore the
structure-activity relationships of 7-substituted-7-DNPA ana-
logues as potential antiviral agents. Herein, we report the
synthesis and anti-HBV and anti-HCV activities of 7-deazane-
planocin A analogues. The most potent compound against wild
type HBV was further evaluated against drug-resistant mutant
strains of HBV.
* To whom correspondence should be addressed. Phone: (706) 542-5379.
Fax: (706) 542-5381. E-mail: dchu@rx.uga.edu.
†
The University of Georgia College of Pharmacy.
Southern Research Institute.
Emory University School of Medicine/VA Medical Center.
Georgetown University Medical Center.
‡
§
|
a Abbreviations: HBV, hepatitis B virus; HCV, hepatitis C virus; NPA,
neplanocin A; 7-DNPA, 7-deazaneplanocin A; 2′-C-Me-C, 2′-C-Me-
cytosine; 2′-F-C-Me-C, 2′-F-C-Me-cytosine; 2′-C-Me-A, 2′-C-methylad-
enosine; RdRp, RNA dependent RNA polymerase; AdoHcyase, S-adeno-
sylhomocysteine hydrolase; DIAD, diisopropyl azodicarboxylate; TPP,
triphenylphosphine; PMB, p-methoxybenzyl; DDQ, 2,3-dichloro-5,6-dicy-
ano-1,6-benzoquinone; HMPA, hexamethylphosphoramide; TBAF, tetrabu-
tylammonium fluoride; DMEM, Dulbecco’s modified Eagle’s medium; FBS,
fetal bovine serum; RI, replication intermediates.
Results and Discussion
Chemistry. The modification of carbocyclic nucleosides
4-amino-7-(2,3-dihydroxy-4-hydroxymethyl-4-cyclopenten-1-
yl)-7H-pyrrolo[2,3-d]pyrimidine (7-DNPA, Figure 1, 2) was
investigated by synthesizing the corresponding 7-substituted-
7-DNPA analogues, as shown in Schemes 1-3. For the
10.1021/jm801418v CCC: $40.75
2009 American Chemical Society
Published on Web 12/15/2008

7-Deazaneplanocin A Analogues
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 207
Scheme 1a
a Reagent and conditions: (a) PPh3, DIAD, THF, room temp; (b) NH3, MeOH, 80 °C; (c) HCl, MeOH, THF, 50 °C.
Scheme 2a
Scheme 1 to produce the vinyl and acetylene derivatives from
the precursor of iodo compound 11 by a palladium catalyzed
reaction followed by ammonolysis. However, the acidic hy-
drolysis of the coupled products by HCl in MeOH and THF
gave a complex mixture that could not be separated. To avoid
the deprotection step in acidic conditions, a new key intermedi-
ate 19 (acetyl protected) was prepared from the cyclopentenol
intermediate 3 by alkylation with p-methoxybenzyl bromide and
deprotection of the trityl and acetonide groups followed by
acetylation and deprotection of p-methoxybenzyl (PMB) group
using 2,3-dichloro-5,6-dicyano-1,6-benzoquinone (DDQ). Sub-
sequently, the acetyl protected cyclopentenol intermediate 19
was treated with 7-iodo-7-deaza-6-chloropurine (7) under the
Mitsunobu conditions and provided the coupling product 20 in
54% yield. The iodo group of 20 was converted to the vinyl
group by the treatment of tetravinyltin in hexamethylphospora-
mide (HMPA) at 80 °C for 16 h to obtain 21 in 26% yield,
which was deprotected under basic conditions to give 7-vinyl-
7-DNPA 22. Compound 20 was reacted with trimethylsily-
lacetylene under palladium catalyzed conditions to give the TMS
protected acetylene substituted derivative 23 in 57% yield, which
was in turn converted to 7-acetylene-7-DNPA 24 by ammonolysis.
a Reagent and conditions: (a) Ac2O, pyridine, 60 °C; (b) HNO3, H2SO4,
CH2Cl2; (c) NH3, MeOH, 80 °C.
synthesis of 7-halogenated-7-DNPA analogues (Scheme 1), the
key intermediate 2,3-isopropylidenedioxy-4-trityloxymethyl-4-
cyclopenten-1-ol 3 was utilized as the starting material. The
cyclopentenol intermediate 3 was synthesized from D-ribose in
eight steps with 52% overall yield, as previously reported from
our group.4d The Mitsunobu coupling reaction of the cyclopen-
tenol intermediate 3 with 7-halogenated-7-deaza-6-chloropurines
4, 5, 6, and 7 provided corresponding coupling products as
protected carbocyclic nucleosides, which were aminated at
elevated temperature to give the amino derivatives 8, 9, 10, and
11 in 75-95% yield. The deprotection of the trityl and acetonide
groups of 8-11 was accomplished using hydrochloric acid in
a mixture of methanol and THF at 50 °C to yield the desired
carbocyclic nucleosides 12, 13, 14, and 15.
The 7-nitro analogues of 7-DNPA 18 was synthesized as
shown in Scheme 2. The acetylation of 7-DNPA 2 with acetic
anhydride in pyridine at 60 °C gave a mixture of 16a and 16b,
which were treated with fuming nitric acid and sulfuric acid in
methylene chloride to give the 7-nitro derivative 17 in good
yield. The removal of acetyl groups of 17 was accomplished
with saturated methanolic ammonia to give 7-nitro-7-DNPA 18
in 66% yield.
Additionally, the iodo group in 20 was used to the obtain
the cyano moiety by a palladium catalyzed reaction with
tributyltin cyanide to give 25 in moderate yield. The ammonoly-
sis reaction of 25 gave the desired product 7-cyano-7-DNPA
26 in 73% yield from 20. The 7-cyano group of 26 was further
converted to carboxamide analogues by the treatment with
hydrogen peroxide in ammonium hydroxide solution to yield a
7-carboxamide-7-DNPA 27 in quantitative yield.
Anti-HCV Activity. The anti-HCV activity and cytotoxicity
of synthesized nucleosides were evaluated in the HCV RNA
replicon assay system in Huh7 ET cells, as previous described,11
and the results are summarized in Table 1. Compounds were
initially assessed at a single concentration of 20 µM. Any
potentially active or cytotoxic compounds in the primary assay
were further evaluated in the dose-response assay. From these
studies we found that 6 out of 10 NPA analogues (2, 13-15,
24, and 27) exhibited significant anti-HCV activity with EC50
ranging from 1.8 to 20.1 µM. 7-DNPA 2 displayed anti-HCV
activity with an EC50 value of 2.5 µM without any cytotoxicity
at a concentration up to 100 µM (Table 1). This result was
confirmed independently in a separate anti-HCV evaluation, in
which the anti-HCV activity of 7-DNPA 2 is at least comparable
to, if not better than, the positive controls 2′-C-Me-cytosine (2′-
C-Me-C)9f and 2′-F-C-Me-cytosine (2′-F-C-Me-C)9g,h (Table 2).
The syntheses of vinyl, acetylene, and cyano substituted
derivatives of 7-DNPA, 22, 24, and 26, are illustrated in Scheme
3. During our first attempt, we used our strategy based on

208 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1
Kim et al.
Scheme 3a
a Reagents and conditions: (a) (i) NaH, p-methoxybenzyl chloride, DMF; (ii) HCl/MeOH/THF; (iii) Ac2O, pyridine; (iv) DDQ, CH2Cl2, H2O; (b) PPh3,
DIAD, THF, room temp, 7; (c) Bu3Sn(CHCH2), Pd(PPh3)4, Et3N, CuI, DMF; (d) NH3, MeOH, 80 °C; (e) TMSCt CH, Pd(PPh3)4, Et3N, CuI; (f) NH3,
MeOH, 80 °C; (g) Bu3SnCN, (PPh3)4Pd, ClCH2CH2Cl, reflux; (h) NH3, MeOH, 80 °C; (i) H2O2, NH4OH.
As the modifications at the 7-position of the 7-deaza analogues
of 2′-C-methyladenosine (2′-C-Me-A) exhibited enhanced anti-
HCV activity,9d it was of interest to explore the electrostatic
and steric requirements at the 7-position in the 7-DNPA
analogues. Electronegative substituents, such as 7-fluoro-12,
7-chloro-13, 7-bromo-14, and 7-iodo-15 analogues, were also
synthesized. In comparison to the parent compound 2, the fluoro
analogue 12 was devoid of anti-HCV activity. The anti-HCV
potency tends to be increased with decreasing electronegativity
as well as with increasing size of halogens at the 7-position
(13, EC50 ) 14.7 µM; 14, EC50 ) 16.7 µM; 15, EC50 ) 2.1
µM), while a concomitant increase in cytotoxicity was also
withdrawing groups have deleterious effects on the anti-HCV
activity in the 7-DNPA, which is clearly contradictory to the
observations of the 2′-C-methyl-7-substituted-7-deaza adenosine
analogues, in which electronegative substituents on the 7-posi-
tion appeared to be favored.9d These results suggest that the
two classes of compounds may have different mode of actions,
although the structures of heterocyclic moiety are same.
However, investigating the mode of action of 7-DNPAs is
beyond the scope of the current structure-activity relationships
studies and would be the future subject of study.
Anti-HBV Activity. The synthesized carbocyclic nucleosides
were also screened for their antiviral activity against wild type
HBV in vitro, and the results are summarized in Table 3. The
most potent compound was further evaluated against HBV drug-
resistant mutants. As shown in Table 3, compounds 2, 15, 22,
and 24 exhibited significant anti-HBV activity against wild-
type HBV with EC50 values of 0.60, 0.43, 3.3, and 0.32 µM,
respectively, in an extracellular virion HBV DNA assay.
7-DNPA 2 showed moderate cytotoxicity (IC50 ) 22 µM),
whereas the compounds 15, 22, and 24 did not show any
significant cytotoxicity up to 300 µM. The most potent
compound 24 (7-ethynyl derivative) was also evaluated against
observed (13, IC50 ) 50.1 µM; 14, IC50 ) 41.7 µM; 15, IC50
)
15.0 µM). Compounds with other substituents on the 7-position,
such as carboxamide 27, cyano 26, vinyl 22, and acetylene 24
derivatives, were also investigated, in which the 7-carboxamide
derivative 27 appeared to be the most active compound against
HCV (EC50 ) 1.8 µM); however, it was also the most cytotoxic
(IC50 ) 11.8 µM). The anti-HCV activity decreased significantly
when electron-withdrawing substituents (CN or NO2) were
present at the 7-position, such as the cyano-26 and nitro-18
derivatives. From these results, it appears that the electron-

7-Deazaneplanocin A Analogues
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 209
Table 1. Anti-HCV Activity of 7-DNPA Analogues Based on an HCV
RNA Replicon Assay and Cytotoxicity
Table 4. In Vitro Antiviral Activity against HBV Mutants Based on the
Intracellular HBV DNA Replication Assay
anti-HCV
activity,
EC50 (µM)
cytotoxicity,
compd
R
EC50 (µM)
IC50 (µM)
SI50
strain
WT
rtL180M
rtLM/rtMVa
rtM204I
anti-HBV activity
3TC
adefovir
2
H
F
Cl
Br
I
2.5
>100
112.7%
50.1
41.7
15.0
101.5%
99.7%
48.9
103.3%
11.8
>40
2.5
2.0
2.8
3.0
2.1
5.6
0.2
1.3
1.6
1.2
1.8
1.5
7.7
12a
13
14
9.5%
14.7
16.7
2.1
31.5
49.2%
20.1
0.5%
1.8
10.0
>100
>100
>100
0.3
3.4
2.5
7.1
15
rtM204V
rtN236T
18a
NO2
vinyl
ethynyl
CN
CONH2
22a
a rtLM/rtMV ) rtL180M/rtM204V double mutant.
24
2.4
26a
27
6.6
>66.7
(rtN236T). Again, the mode of action of the compound has not
been studied, but it will be the future subject of investigation.
In summary, to study the structure-activity relationships, a
series of novel 7-deazaneplanocin A analogues have been
synthesized and their anti-HCV and anti-HBV activity were
determined in vitro. Several synthesized compounds exhibited
significant anti-HCV activity, among which the most potent
analogue was the 7-unsubstituted compound 2 without any
cytotoxicity. The anti-HBV activities were also determined, from
which the ethynyl derivative 24 was found to be the most
significant against the wild-type, maintaining the potency against
drug-resistant HBV mutants. The present findings warrant future
investigation of the mechanism of action as well as additional
biological evaluation of these analogues.
2′-C-Me-A
0.15
>10.0
a Compounds assessed at a single-concentration of 20 (µM) with
percentage donating inhibition level compared to control cells.
Table 2. Additional Studies of Anti-HCV Activity and Cytoxicity of
7-DNPA 2 in the HCV RNA Replicon Huh7 Assay in Comparison to
the Known Agents (2′-F-C-Me-C and 2′-C-Me-C)
anti-HCV
activity, EC50
(µM)a
cytotoxicity,
EC90
CC50
compd
(µM)a
(µM)b
2
0.9
1.7
3.5
8.2
5.3
11
>50
>50
>50
2′-F-C-Me-C
2′-C-Me-C
a Effective concentrations required for reducing HCV level by 50% and
90% in 5 days. b Cytotoxicity concentration required for reducing the rRNA
levels by 50% in 5 days.
Experimental Section
Table 3. In Vitro Antiviral Activity Against Wild Type HBV and
Chemistry. Melting points were determined on a Mel-temp II
apparatus and are uncorrected. Nuclear magnetic resonance spectra
were recorded on a Varian Inova 500 spectrometer at 500 MHz
for 1H NMR and 125 MHz for 13C NMR with tetramethylsilane as
the internal standard. Chemical shifts (δ) are reported as s (singlet),
d (doublet), t (triplet), q (quartet), m (multiplet), or bs (broad
singlet). UV spectra were recorded on a Beckman DU-650
spectrophotometer. Optical rotations were measured on a Jasco DIP-
370 digital polarimeter. TLC was performed on Uniplates (silica
gel) purchased from Analtech Co. Column chromatography was
performed using either silica gel-60 (220-440 mesh) for flash
column chromatography or silica gel G (TLC grade, >440 mesh)
for vacuum column chromatography. Elemental analyses were
performed by Atlantic Microlab Inc., Norcross, GA.
Cytotoxicity of 7-DNPA Analogues
anti-HBV activity
virion,
HBV RI,
EC50 (µM)
cytotoxicity,
IC50 (µM)
compd
R
EC50 (µM)
2
H
F
Cl
Br
I
NO2
vinyl
acetylene
CN
CONH2
0.60
>10
>10
>10
0.43
>10
3.3
0.32
>10
>10
0.048
NDa
NDa
NDa
NDa
1.6
22
(-)-(1R, 2S, 3R)-4-Amino-5-fluoro-7-[2,3-(isopropylidene-
dioxy)-4-(trityloxymethyl)-4-cyclopenten-1-yl]-7H-pyrrolo[2,3-
d]pyrimidine (8). To a mixture of compound 3 (318 mg, 0.74
mmol), compound 4 (140 mg, 0.82 mmol), and Ph3P (388 mg, 1.48
mmol) in THF (30 mL) was added DIAD (299 mg, 1.48 mmol) at
0 °C. After being stirred for 30 min at room temperature, the mixture
was evaporated under reduced pressure and purified by column
chromatography on silica gel. The appropriate fraction was collected
and evaporated. The residue was treated with saturated methanolic
ammonia and heated to 80 °C in a steal bomb for 16 h. After
cooling, the mixture was purified via flash column chromatography
on silica gel (eluting up to 50% ethyl acetate in hexane) to give 8
(350 mg, 84%) as a white solid: mp 100-102 °C; [R]2D7 -25.33 (c
12
13
14
15
18
22
24
26
27
3TC
>300
>300
>300
>300
>300
>300
>300
>300
>300
>2000
NDa
10
1.8
NDa
NDa
0.15
a ND, not determined.
drug resistant HBV mutants (Table 4), in which compound 24
did not lose activity against all the lamivudine (3TC) and
adefovir associated HBV mutants, with an EC50 value compa-
rable to that of adefovir (ADV). It was also found that compound
24 confers a slight resistance (2-fold) to the ADV mutant
1
0.13, CHCl3); UV (CHCl3) λmax 283.0 nm; H NMR (500 MHz,
CDCl3) δ 8.33 (s, 1H), 7.4-7.5 (m, 6H), 7.2-7.35 (m, 9H), 6.48
(d, J ) 2.0 Hz, 1H), 5.99 (s, 1H), 5.88 (s, 1H), 5.37 (s, 2H), 5.18
(d, J ) 6.0 Hz, 1H), 4.54 (d, J ) 6.0 Hz, 1H), 4.01 (dt, J ) 15 and

210 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1
Kim et al.
2 Hz, 1H), 3.83 (d, J ) 15.5 Hz, 1H), 1.42 (s, 3H), 1.30 (s, 3H);
13C NMR (100 MHz, CDCl3) δ 155.51, 155.49, 153.20, 149.12,
145.84, 145.81, 144.14, 143.78, 141.71, 128.54, 128.09, 127.94,
127.20, 123.07, 112.48, 104.25, 103.99, 93.81, 93.66, 87.26, 84.99,
83.94, 77.24, 63.97, 61.48, 27.52, 26.10. HRMS ([M + H]+) calcd,
563.2453; found, 563.2458.
(10 mL), and water (1 mL) was heated to 50 °C for 16 h. It was
neutralized by NaHCO3 and purified by flash column chromatog-
raphy on silica gel (eluting up to 10% methanol in dichloromethane)
to give 12 (140 mg, 94%) as a white solid: mp 224-225 °C; [R]D26
-102.29 (c 0.14, MeOH); UV (H2O) λmax 282 nm (pH 2), 282 nm
1
(pH 7), 283 nm (pH 11); H NMR (500 MHz, DMSO-d6) δ 8.08
(s, 1H), 7.01 (d, J ) 2.5 Hz, 1H), 6.94 (br s, 2H), 5.59 (br s, 2H),
5.02 (d, J ) 6.5 Hz, 1H), 4.92 (d, J ) 6.5 Hz, 1H), 4.90 (t, J ) 5.5
Hz, 1H), 4.42 (t, J ) 6.0 Hz, 1H), 4.12 (m, 2H), 4.08 (m,, 1H);
13C NMR (100 MHz, DMSO-d6) δ 156.22, 156.20, 152.96, 150.52,
146.15, 146.12, 143.86, 141.44, 124.47, 104.73, 104.47, 92.57,
92.42, 77.34, 72.70, 63.71, 58.99. Anal. Calcd for
(C12H13FN4O3 ·0.2H2O).
(-)-(1R,2S,3R)-4-Amino-5-chloro-7-[2,3-(isopropylidenedioxy)-
4-(trityloxymethyl)-4-cyclopenten-1-yl]-7H-pyrrolo[2,3-d]pyri-
midine (9). To a mixture of 3 (193 mg, 0.45 mmol), 5 (110 mg,
0.59 mmol), and Ph3P (177 mg, 0.68 mmol) in THF (20 mL) was
added DIAD (0.13 mL, 0.68 mmol) at 0 °C. After being stirred for
30 min at room temperature, the mixture was evaporated under
reduced pressure and purified by silica gel column chromatography.
The isolated product was treated with saturated methanolic ammonia
and heated to 80 °C in a steal bomb for 16 h. After cooling, the
mixture was purified by flash column chromatography on silica gel
(eluting up to 50% ethyl acetate in hexane) to give 9 (205 mg,
79%) as a white solid: mp 94-95 °C; [R]2D8 -28.55 (c 0.20, CHCl3);
(-)-(1R,2S,3R)-4-Amino-5-chloro-7-[2,3-dihydroxy-4-hy-
droxymethyl-4-cyclopenten-1-yl]-7H-pyrrolo[2,3-d]pyrimidine
(13). A mixture of compound 9 (190 mg) and HCl (0.2 mL) in
MeOH (10 mL), THF (10 mL), and water (1 mL) was heated to
50 °C for 16 h. It was neutralized by NaHCO3 and purified by
flash column chromatography on silica gel (eluting up to 10%
methanol in dichloromethane) to give 13 (87 mg, 90%) as a white
solid: mp 236-237 °C; [R]D27 -140.51 (c 0.14, MeOH); UV (H2O)
1
UV (CHCl3) λmax 284.0 nm; H NMR (500 MHz, CDCl3) δ 8.34
(s, 1H), 7.4-7.5 (m, 6H), 7.2-7.35 (m, 9H), 6.68 (s, 1H), 5.99 (s,
1H), 5.85 (s, 1H), 5.57 (br s, 2H), 5.17 (d, J ) 5.5 Hz, 1H), 4.54
(d, J ) 5.5 Hz, 1H), 4.03 (m, 1H), 3.84 (d, J ) 16.0 Hz, 1H), 1.43
(s, 3H), 1.30 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 156.62,
153.20, 149.51, 149.02, 143.81, 128.58, 127.98, 127.25, 122.80,
118.67, 112.56, 103.10, 101.34, 87.35, 84.99, 83.98, 64.39, 61.54,
27.55, 26.13. HRMS ([M + H]+) calcd, 579.2157; found, 579.2163.
(-)-(1R,2S,3R)-4-Amino-5-bromo-7-[2,3-(isopropylidenedioxy)-
4-(trityloxymethyl)-4-cyclopenten-1-yl]-7H-pyrrolo[2,3-d]pyri-
midine (10). To a mixture of 3 (144 mg, 0.34 mmol), 6 (102 mg,
0.44 mmol), and Ph3P (134 mg, 0.51 mmol) in THF (20 mL) was
added DIAD (0.10 mL, 0.51 mmol) at 0 °C. After being stirred for
30 min at room temperature, the mixture was evaporated and
purified by column chromatography on silica gel. The appropriate
fraction was collected and evaporated. The residue was treated with
saturated methanolic ammonia and heated to 80 °C in a steal bomb
for 16 h. After cooling, the mixture was purified by flash column
chromatography on silica gel (eluting up to 50% ethyl acetate in
hexane) to give 10 (197 mg, 93%) as a white solid: mp 112-114
°C; [R]2D6 -39.47 (c 0.15, MeOH); UV (MeOH) λmax 284.0 nm; 1H
NMR (500 MHz, CDCl3) δ 8.34 (s, 1H), 7.4-7.5 (m, 6H), 7.2-7.35
(m, 9H), 6.75 (s, 1H), 5.99 (s, 1H), 5.85 (s, 1H), 5.61 (br s, 2H),
5.17 (d, J ) 5.5 Hz, 1H), 4.54 (d, J ) 6.0 Hz, 1H), 4.03 (d, J )
15.0 Hz, 1H), 3.85 (d, J ) 15.5 Hz, 1H), 1.43 (s, 3H), 1.30 (s,
3H); 13C NMR (125 MHz, CDCl3) δ 156.75, 153.01, 149.57,
149.51, 143.81, 128.58, 127.98, 127.26, 122.75, 121.14, 112.56,
103.10, 87.36, 86.71, 84.99, 83.97, 64.51, 61.55, 27.55, 26.13.
HRMS ([M + H]+) calcd, 623.1652; found, 623.1657.
1
λmax 285 nm (pH 2), 285 nm (pH 7), 283 nm (pH 11); H NMR
(400 MHz, DMSO-d6) δ 8.07 (s, 1H), 7.21 (s, 1H), 6.90 (br s,
2H), 5.56 (d, J ) 1.2 Hz, 1H), 5.52 (br s, 1H), 5.01 (d, J ) 7.2 Hz,
1H), 4.89 (d, J ) 6.0 Hz, 1H), 4.85 (t, J ) 5.6 Hz, 1H), 4.39 (t, J
) 5.6 Hz, 1H), 4.1-4.15 (m, 3H); 13C NMR (100 MHz, DMSO-
d6) δ 157.16, 152.87, 150.74, 149.33, 124.29, 119.56, 102.04,
100.19, 77.39, 72.74, 64.32, 59.00. Anal. Calcd for (C12H13ClN4O3).
(-)-(1R,2S,3R)-4-Amino-5-bromo-7-[2,3-dihydroxy-4-hy-
droxymethyl-4-cyclopenten-1-yl]-7H-pyrrolo[2,3-d]pyrimidine
(14). A mixture of 10 (100 mg) and HCl (0.1 mL) in MeOH (5
mL), THF (5 mL), and water (0.5 mL) was heated to 50 °C for
16 h. It was neutralized by NaHCO3 and purified by flash column
chromatography on silica gel (eluting up to 10% methanol in
dichloromethane) to give 14 (49 mg, 90%) as a white solid: mp
242-243 °C; [R]D26 -142.06 (c 0.21, MeOH); UV (H2O) λmax 285
nm (pH 2), 283 nm (pH 7), 284 nm (pH 11); 1H NMR (400 MHz,
DMSO-d6) δ 8.08 (s, 1H), 7.26 (s, 1H), 6.70 (br s, 2H), 5.56 (br s,
1H), 5.53 (br s, 1H), 5.02 (d, J ) 6.4 Hz, 1H), 4.90 (d, J ) 6.4 Hz,
1H), 4.86 (t, J ) 5.6 Hz, 1H), 4.39 (t, J ) 6.4 Hz, 1H), 4.0-4.15
(m, 3H); 13C NMR (100 MHz, DMSO-d6) δ 157.34, 152.68, 150.79,
149.81, 124.28, 122.10, 101.38, 86.02, 77.42, 72.75, 64.47, 59.00.
Anal. Calcd for (C12H13BrN4O3 ·0.8H2O).
(-)-(1R,2S,3R)-4-Amino-5-iodo-7-[2,3-dihydroxy-4-hydroxym-
ethyl-4-cyclopenten-1-yl]-7H-pyrrolo[2,3-d]pyrimidine (15). A
mixture of 11 (130 mg) and HCl (0.2 mL) in MeOH (10 mL), THF
(10 mL), and water (1 mL) was heated to 50 °C for 16 h. It was
neutralized by NaHCO3 and purified by flash column chromatog-
raphy on silica gel (eluting up to 10% methanol in dichloromethane)
and recrystallized with ethanol to give 15 (65 mg, 87%) as a white
solid: mp 230-231 °C; [R]2D6 -98.93 (c 0.12, MeOH); UV (H2O)
(-)-(1R,2S,3R)-4-Amino-5-iodo-7-[2,3-(isopropylidenedioxy)-
4-(trityloxymethyl)-4-cyclopenten-1-yl]-7H-pyrrolo[2,3-d]pyri-
midine (11). To a mixture of 3 (380 mg, 0.89 mmol), 7 (310 mg,
1.11 mmol), and Ph3P (467 mg, 1.78 mmol) in THF (40 mL) was
added DIAD (360 mg, 1.78 mmol) at 0 °C. After being stirred for
30 min at room temperature, the mixture was evaporated and
purified by column chromatography on silica gel to give a white
solid (580 mg). A portion of solid (550 mg) was treated with
saturated methanolic ammonia and heated to 80 °C for 16 h. After
cooling, the mixture was purified by flash column chromatography
on silica gel (eluting up to 50% ethyl acetate in hexane) to give 11
(545 mg, 94%) as a white solid: mp 114-116 °C; [R]2D7 -34.68 (c
1
λmax 290 nm (pH 2), 286 nm (pH 7), 285 nm (pH 11); H NMR
(400 MHz, DMSO-d6) δ 8.08 (s, 1H), 7.28 (s, 1H), 6.60 (br s,
2H), 5.56 (d, J ) 1.2 Hz, 1H), 5.51 (br s, 1H), 5.00 (d, J ) 6.8 Hz,
1H), 4.89 (d, J ) 6.0 Hz, 1H), 4.85 (t, J ) 5.6 Hz, 1H), 4.39 (t, J
) 5.6 Hz, 1H), 4.0-4.15 (m, 3H); 13C NMR (100 MHz, DMSO-
d6) δ 157.59, 152.19, 150.74, 150.42, 127.34, 124.37, 103.60, 77.48,
72.75, 64.56, 59.00, 50.91. Anal. Calcd for (C12H13IN4O3 ·
0.15C2H5OH).
1
0.34, CHCl3); UV (CHCl3) λmax 287.0 nm; H NMR (500 MHz,
(-)-(1R,2S,3R)-4-Acetylamino-5-nitro-7-[2,3-diacetoxy-4-ac-
etoxymethyl-4-cyclopenten-1-yl]-7H-pyrrolo[2,3-d]pyrimidine (17).
A mixture of 2 (183 mg, 0.70 mmol) and acetic anhydride (1 mL)
in pyridine (8 mL) was heated to 60 °C for 2 days. The mixture
was evaporated and purified by column chromatography on silica
gel to give mono- and diacetyl derivatives 16a and 16b (260 mg)
as a white solid. To a mixture of 16a and 16b (260 mg) in
methylene chloride (30 mL) were added H2SO4 (0.5 mL) and HNO3
(fuming, 0.5 mL) (prepared from the addition of H2SO4 into HNO3)
at 0 °C. The mixture was stirred at 0 °C for 15 min and neutralized
by NaHCO3 (excess) and MeOH (5 mL). It was filtered through
Celite and washed with ethyl acetate. The combined filtrate was
CDCl3) δ 8.34 (s, 1H), 7.4-7.5 (m, 6H), 7.2-7.35 (m, 9H), 6.84
(s, 1H), 6.00 (s, 1H), 5.85 (s, 1H), 5.71 (br s, 2H), 5.18 (d, J ) 5.5
Hz, 1H), 4.54 (d, J ) 6.0 Hz, 1H), 4.03 (d, J ) 15.0 Hz, 1H), 3.85
(d, J ) 16.0 Hz, 1H), 1.44 (s, 3H), 1.30 (s, 3H); 13C NMR (125
MHz, CDCl3) δ 156.93, 152.63, 150.16, 149.56, 143.82, 128.59,
127.98, 128.00, 127.26, 126.41, 122.77, 112.55, 104.59, 87.31,
85.03, 83.98, 64.64, 61.58, 49.36, 27.56, 26.14. HRMS ([M + H]+)
calcd, 671.1514; found, 671.1519.
(-)-(1R,2S,3R)-4-Amino-5-fluoro-7-[2,3-dihydroxy-4-hydroxym-
ethyl-4-cyclopenten-1-yl]-7H-pyrrolo[2,3-d]pyrimidine (12). A
mixture of 8 (300 mg) and HCl (0.2 mL) in MeOH (10 mL), THF

7-Deazaneplanocin A Analogues
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 211
purified by flash column chromatography on a silica gel to give 17
(225 mg, 68%) as a light-yellow solid: mp 66-67 °C; [R]2D7 -141.94
(-)-(1R,2S,3R)-4-Chloro-5-ethenyl-7-[2,3-diacetoxy-4-ace-
toxymethyl-4-cyclopenten-1-yl]-7H-pyrrolo[2,3-d]pyrimidine (21).
A mixture of 20 (550 mg. 1.03 mmol), tri-n-butylvinyltin (0.9 mL,
3.0 mmol), CuI (39 mg, 0.206 mmol), triethylamine (0.29 mL, 2.0
mol), and (PPh3)4Pd (119 mg, 0.10 mmol) in DMF (10 mL) was
stirred at 60 °C for 8 h. The mixture was poured into water (10
mL) and extracted with ethyl acetate (100 mL). The organic layer
was dried over Na2SO4, evaporated under reduced pressure, and
purified by flash column chromatography on a silica gel (eluting
up to 30% ethyl acetate in hexane) to yield 21 (310 mg, 69%) as
a light-yellow sticky liquid: [R]2D7 -142.24 (c 0.21, CHCl3); UV
(CHCl3) λmax 243 nm; 1H NMR (400 MHz, CDCl3) δ 8.60 (s, 1H),
7.35 (s, 1H), 7.22 (ddd, J ) 17.6, 12, 0.8 Hz, 1H), 6.15 (m, 1H),
6.05 (m, 1H), 5.97 (dd, J ) 6, 1.2 Hz, 1H), 5.45 (t, J ) 5.6 Hz,
1H), 5.29 (dd, J ) 10.8, 1.2 Hz, 1H), 4.76 (m, 2H), 2.14 (s, 3H),
2.13 (s, 3H), 2.00 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 170.35,
170.26, 169.74, 152.54, 151.79, 150.78, 140.97, 130.72, 126.91,
121.71, 115.83, 115.29, 114.75, 75.85, 73.00, 61.60, 60.45, 20.75,
20.65, 20.46. HR-MS calcd for (M + H)+ 434.1113, found
434.1110.
1
(c 0.19, CHCl3); UV (CHCl3) λmax 347, 279 nm; H NMR (500
MHz, CDCl3) δ 10.69 (br s, 1H), 8.74 (s, 1H), 8.17 (s, 1H), 6.16
(m, 1H), 6.06 (m, 1H), 5.97 (m, 1H), 5.44 (t, J ) 6.0 Hz, 1H),
4.78 (m, 2H), 2.53 (s, 3H), 2.15 (s, 3H), 2.14 (s, 3H), 2.00 (s, 3H);
13C NMR (100 MHz, CDCl3) δ 170.29, 170.11, 169.67, 169.56,
153.95, 151.16, 151.12, 142.86, 128.91, 128.73, 128.05, 98.34,
75.84, 73.04, 62.91, 60.25, 26.21, 20.72, 20.61, 20.38. Anal. Calcd
for (C20H21N5O9).
(-)-(1R,2S,3R)-4-Amino-5-nitro-7-[2,3-dihydroxy-4-hydroxym-
ethyl-4-cyclopenten-1-yl]-7H-pyrrolo[2,3-d]pyrimidine (18). A
mixture of 17 (215 mg) in saturated methanolic ammonia was
heated to 80 °C for 16 h in a steal bomb. After cooling, the mixture
was purified by flash column chromatography on silica gel (eluting
up to 10% methanol in dichloromethane) to give 18 (92 mg, 66%)
as a yellow solid: mp 245-246 °C; [R]2D3 -195.08 (c 0.28, DMSO);
UV (H2O) λmax 339, 261 nm (pH 2), 371, 276 nm (pH 7), 371, 275
nm (pH 11); 1H NMR (400 MHz, DMSO-d6) δ 8.43 (s, 1H), 8.23
(s, 1H), 7.80 (br s, 1H), 7.20 (br s, 1H), 5.64 (d, J ) 2.0 Hz, 1H),
5.59 (m, 1H), 5.13 (d, J ) 6.8 Hz, 1H), 4.98 (d, J ) 6.0 Hz, 1H),
4.92 (t, J ) 5.6 Hz, 1H), 4.41 (t, J ) 6.0 Hz, 1H), 4.22 (q, J ) 6.4
Hz, 1H), 4.10 (br s, 2H); 13C NMR (100 MHz, CDCl3) δ 156.98,
154.49, 152.02, 150.65, 128.98, 128.33, 123.08, 95.44, 77.41, 72.76,
65.69, 59.04. Anal. Calcd for (C12H13N5O5 ·0.4H2O).
(+)-(1S,2S,3R)-2,3-Diacetoxy-4-acetoxymethyl-4-cyclopenten-
1-ol (19). To a solution of compound 3 (6.00 g, 14.0 mmol) in
DMF (60 mL) was added a 60% dispersion of sodium hydride (1.12
g, 28 mmol) at 0 °C. After 10 min, the mixture was treated with
4-methoxybenzyl chloride (2.1 mL, 15.4 mmol) and stirred at room
temperature for 3 h. It was poured into water (300 mL) and extracted
with ethyl ether (100 mL × 2). The combined ether layer was dried
(MgSO4) and evaporated. The resulting solid was dissolved in
MeOH (150 mL), THF (150 mL), and water (15 mL) and treated
with concentrated HCl (0.5 mL). The mixture was heated to 55-60
°C for 4 h and neutralized by NaHCO3. After evaporation, the
mixture was purified by vacuum column chromatography on a silica
gel to give the triol compound (2.61 g, 70%) as a white solid. Part
of the triol (101 mg, 0.38 mmol) and acetic anhydride (0.25 mL)
in pyridine (2 mL) and methylene chloride (2 mL) was stirred at
room temperature overnight. The mixture was evaporated and
purified by flash column chromatography on a silica gel. The
appropriate fraction was collected and evaporated and dissolved in
CH2Cl2/H2O (10 mL/0.5 mL). It was treated with DDQ (260 mg,
1.14 mmol) and stirred at room temperature for 3 h. The mixture
was mixed with water (20 mL) and extracted with methylene
chloride. The combined organic layer was dried over Na2SO4 and
purified by flash column chromatography on a silica gel to give a
reddish sticky liquid. It was dissolved in chloroform and the
insoluble solid was filtered off and the filtrate was evaporated to
give 19 (80 mg, 78%) as a white solid: mp 47 °C; [R]2D4 +24.95 (c
0.45, CHCl3); 1H NMR (400 MHz, CDCl3) δ 6.14 (s, 1H), 5.68 (d,
J ) 6 Hz, 1H), 5.29 (t, J ) 5.6 Hz, 1H), 4.69 (br s, 3H), 2.13 (s,
3H), 2.10 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 170.45, 170.06,
139.84, 133.36, 73.14, 72.43, 72.00, 60.15, 20.74, 20.71, 20.63.
Anal. Calcd for (C12H16O7).
(-)-(1R,2S,3R)-4-Amino-5-ethenyl-7-[2,3-dihydroxy-4-hy-
droxymethyl-4-cyclopenten-1-yl]-7H-pyrrolo[2,3-d]pyrimidine
(22). A mixture of 21 (290 mg), methanol (5 mL), dioxane (3 mL),
and liquid ammonia (∼15 mL) was heated to 80 °C for 16 h in a
steel bomb. After cooling, the mixture was purified by flash column
chromatography on silica gel (eluting up to 10% methanol in
dichloromethane) to give 22 (106 mg, 55%) as a light-yellow solid:
mp 168-170 °C; [R]D26 -187.84 (c 0.11, MeOH); UV (H2O) λmax
1
294 nm (pH 2), 287 nm (pH 7), 287 nm (pH 11); H NMR (400
MHz, DMSO-d6) δ 8.03 (s, 1H), 7.32 (s, 1H), 7.09 (dd, J ) 17.6,
10.8 Hz, 1H), 6.65 (br s, 2H), 5.5-5.6 (m, 3H), 5.0-5.1 (m, 2H),
4.85-4.95 (m, 2H), 4.40 (t, J ) 5.6 Hz, 1H), 4.05-4.15 (m, 3H);
13C NMR (100 MHz, DMSO-d6) δ 157.99, 151.77, 151.02, 150.32,
129.63, 124.78, 119.07, 113.93, 112.68, 100.86, 77.62, 72.76, 63.97,
59.02. Anal. Calcd for (C14H16N4O3 ·0.15H2O).
(-)-(1R,2S,3R)-4-Chloro-5-trimethylsilanylethynyl-7-[2,3-di-
acetoxy-4-acetoxymethyl-4-cyclopenten-1-yl]-7H-pyrrolo[2,3-
d]pyrimidine (23). A mixture of 20 (350 mg. 0.66 mmol),
trimethylsilylacetylene (0.186 mL, 1.32 mmol), CuI (13 mg, 0.066
mmol), triethylamine (0.184 mL, 1.32 mol), and (PPh3)4Pd (38 mg,
0.033 mmol) in DMF (7 mL) was stirred at 60 °C for 3 h. The
mixture was poured into water and extracted with ethyl acetate.
The organic layer was evaporated and purified by flash column
chromatography on a silica gel (eluting up to 20% ethyl acetate in
hexane) to yield 23 (190 mg, 57%) as a light-yellow solid: mp
56-57 °C; [R]2D5 -156.83 (c 0.22, CHCl3); UV (CHCl3) λmax 310,
235 nm; 1H NMR (500 MHz, CDCl3) δ 8.63 (s, 1H), 7.42 (s, 1H),
6.10 (br s, 1H), 6.02 (br s, 1H), 5.94 (d, J ) 6 Hz, 1H), 5.40 (t, J
) 6 Hz, 1H), 4.74 (s, 2H), 2.133 (s, 3H), 2.128 (s, 3H), 1.99 (s,
3H), 0.27 (s, 9H); 13C NMR (100 MHz, CDCl3) δ 170.61, 170.46,
169.88, 153.85, 151.98, 151.09, 141.68, 130.46, 130.31, 117.46,
99.17, 98.93, 96.03, 76.14, 73.27, 62.25, 60.66, 21.02, 20.92, 20.70,
0.00. Anal. Calcd for (C23H26ClN3O6Si).
(-)-(1R,2S,3R)-4-Amino-5-ethynyl-7-[2,3-dihydroxy-4-hy-
droxymethyl-4-cyclopenten-1-yl]-7H-pyrrolo[2,3-d]pyrimidine
(24). A mixture of 23 (180 mg), methanol (3 mL), dioxane (3 mL),
and liquid ammonia (∼10 mL) was heated to 80 °C for 16 h in a
steel bomb. After cooling, the mixture was purified by flash column
chromatography on silica gel (eluting up to 10% methanol in
dichloromethane) to give 24 (75 mg, 73%) as a light-yellow solid:
mp 126-128 °C; [R]D26 -194.42 (c 0.10, MeOH); UV (H2O) λmax
(-)-(1R,2S,3R)-4-Chloro-5-iodo-7-[2,3-diacetoxy-4-acetoxym-
ethyl-4-cyclopenten-1-yl]-7H-pyrrolo[2,3-d]pyrimidine (20). A
mixture of 19 (470 mg, 1.72 mmol), 7 (290 mg, 1.03 mmol), and
Ph3P (1.35 g, 5.16 mmol) was taken in THF (30 mL), and DIAD
(1.04 g, 5.16 mmol) was added at 0 °C to the reaction mixture.
The reaction mixture was stirred for 1 h at room temperature and
then concentrated under reduced pressure. The crude was further
purified by flash column chromatography on silica gel (eluting up
to 30% ethyl acetate in hexane) to yield 20 (500 mg, 54%) as a
light-yellow solid: mp 49 °C; [R]2D6 -126.83 (c 0.25, CHCl3); UV
1
287 nm (pH 2), 281 nm (pH 7), 279 nm (pH 11); H NMR (400
MHz, CD3OD) δ 8.12 (s, 1H), 7.37 (s, 1H), 5.80 (m, 1H), 5.63
(m, 1H), 4.58 (d, J ) 5.6 Hz, 1H), 4.29 (m, 2H), 4.19 (t, J ) 5.2
Hz, 1H), 3.71 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ 157.92,
153.08, 150.83, 149.84, 127.71, 124.17, 102.84, 93.52, 83.20, 78.08,
77.41, 72.77, 64.58, 58.99. Anal. Calcd for (C14H14N4O3 ·0.3H2O).
(-)-(1R,2S,3R)-4-Amino-5-cyano-7-[2,3-dihydroxy-4-hydroxym-
ethyl-4-cyclopenten-1-yl]-7H-pyrrolo[2,3-d]pyrimidine (26). A
mixture of Bu3SnCN (499 mg, 1.58 mmol) and Pd(PPh3)4 (91 mg,
0.079 mmol) in dichloroethane (15 mL) was heated to reflux for
1
(CHCl3) λmax 308, 269, 240 nm; H NMR (400 MHz, CDCl3) δ
8.62 (s, 1H), 7.35 (s, 1H), 6.11 (m, 1H), 6.03 (m, 1H), 5.95 (dd, J
) 5.6, 0.8 Hz, 1H), 5.42 (t, J ) 5.6 Hz, 1H), 4.75 (m, 2H), 2.14
(s, 3H), 2.13 (s, 3H), 2.00 (s, 3H). Anal. Calcd for (C18H17ClIN3O6).

212 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1
Kim et al.
30 min. To this mixture 20 (425 mg, 0.79 mmol) in dichloromethane
(10 mL) was added and refluxed overnight. The mixture was
evaporated and purified by flash column chromatography on a silica
gel to give a crude oily compound 25. A mixture of crude oily
compound was saturated with methanolic ammonia and was heated
to 80 °C for 16 h in a steal bomb. After cooling, the mixture was
purified by flash column chromatography on a silica gel (eluting
up to 10% methanol in dichloromethane) to give 26 (100 mg, 44%)
as a white solid: mp 178-180 °C; [R]2D7 -161.12 (c 0.13, MeOH);
UV (H2O) λmax 277 nm (pH 2), 278 nm (pH 7), 278 nm (pH 11);
1H NMR (500 MHz, CD3OD) δ 8.26 (s, 1H), 7.95 (s, 1H), 5.86
(m, 1H), 5.73 (m, 1H), 4.64 (d, J ) 5.5 Hz, 1H), 4.3-4.4 (m, 2H),
4.26 (t, J ) 5.5 Hz, 1H); 13C NMR (125 MHz, DMSO-d6) δ 157.45,
153.84, 151.45, 150.71, 132.96, 123.62, 116.01, 101.84, 82.41,
77.43, 72.78, 65.32, 59.03. Anal. Calcd for (C13H13N5O3 ·0.6H2O).
(-)-(1R,2S,3R)-4-Amino-7-[2,3-dihydroxy-4-hydroxymethyl-
4-cyclopenten-1-yl]-7H-pyrrolo[2,3-d]pyrimidine-5-carboxam-
ide (27). A mixture of 26 (50 mg) and 30% H2O2 (0.4 mL) in
NH4OH (4 mL) was stirred at room temperature for 1 h. The
mixture was evaporated and the residue was dispersed in MeOH/
EtOH (2 mL/ 4 mL) and filtered to give 27 (47 mg, 92%) as a
light-yellow solid: mp 260-261 °C; [R]2D7 -134.41 (c 0.20, H2O);
UV (H2O) λmax 280 nm (pH 2), 282 nm (pH 7), 283 nm (pH 11);
1H NMR (400 MHz, CD3OD) δ 8.10 (s, 1H), 7.87 (s, 1H), 5.86
(m, 1H), 5.67 (m, 1H), 4.63 (d, J ) 5.6 Hz, 1H), 4.26 - 4.40 (m,
2H), 4.19 (t, J ) 5.2 Hz, 1H); 13C NMR (125 MHz, DMSO-d6) δ
167.58, 158.21, 152.10, 150.64, 150.17, 125.44, 124.31, 110.43,
101.51, 77.88, 72.91, 64.71, 58.79. Anal. Calcd for
(C13H15N5O4 ·0.6H2O).
Virology. Anti-HCV Assay Using 2′-C-Me-A as Positive
Control. The subgenomic HCV replicon cell line, ET, was obtained
from Dr. Ralf Bartenschlager and ReBLikon GmbH. The cell line
harbors a dicistronic self-replicating HCV RNA replicon with a
firefly luciferase gene and three cell culture-adaptive mutations.
Measurement of the activity of the luciferase reporter is directly
proportional to the replication of the RNA replicon in the
experimental conditions used. Antiviral and cytotoxicity assays were
performed as described previously11 with minor modifications. For
the marginally active compounds, we were unable to calculate the
exact EC50 values. This is the reason that we expressed them as
“percentage of level of inhibition to control cells at 20 µM”.12
Briefly, the ET cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM), supplemented with 2 mM L-glutamine, 1×
nonessential amino acids, 10% fetal bovine serum (FBS), and
0.25mg/mL G418 (Invitrogen, Carlsbad, CA). The cells were seeded
in 96-well tissue culture plates dedicated for analysis of antiviral
activity or cell numbers (cytotoxicity) at a density of 5000/well in
100 µL of DMEM without G418 overnight. Serial dilutions of
compounds were prepared in DMEM and added to the appropriate
wells. Following 3 days of incubation, the cells were processed to
assess the replicon-derived luciferase activity with the Steady-Glo
luciferase assay system (Promega, Madison, WI) according to
manufacturer’s instruction. Cytotoxic effect of the compound on
the viability of the replicon cells was determined using a tetrazo-
lium-based CytoTox-1 cell proliferation assay (Promega). Each data
point represents an average of four replicates in cell culture to derive
applicable EC50 (concentration of compound inhibiting luciferase
activity by 50%), IC50 (concentration of compound decreasing cell
viability by 50%), and SI50 (selective index, EC50/IC50) values.
A Further Anti-HCV Assay Using 2′-C-Me-C and 2′-F-C-
Me-C as Positive Controls. Subgenomic HCV replicon RNA-
containing Huh-7 cells (clone B, kindly provided by Dr. C. M. Rice)
were seeded in a 96-well plate at 3000 cells/well, and the
compounds were added in duplicate at two different dilutions
immediately after seeding. Following 5 days of incubation (37 °C,
5% CO2), total cellular RNA was isolated by using RNeasy 96-kit,
Qiagen. Replicon RNA and an internal control (TaqMan rRNA
contro reagents, Applied Biosystems) were amplified in a single
step multiplex real time RT-PCR assay.13 The antiviral effectiveness
of the compounds was calculated by subtracting the threshold RT-
PCR cycle of the test compound from the threshold RT-PCR cycle
of the no-drug control (∆CtHCV). A ∆Ct of 3.3 equals a 1 log
reduction (equal to 90% less starting material) in replicon RNA
levels. The cytotoxicity of the compounds was also calculated by
using the ∆Ct rRNA values. Two compounds (2′-C-Me-C and 2′-
F-C-Me-C) were used as positive controls. To determine EC50, EC90,
and CC50 values, ∆Ct values were first converted into fraction of
starting material by using CalcuSyn computer software (Biosoft,
Ferguson, MO).
HBV Antiviral Assays. HBV antiviral assays were conducted
as previous described.14 Briefly, confluent cultures of 2.2.15 cells
were maintained on 96-well flat-bottomed tissue culture plates
(confluence in this culture system is required for active, high levels
of HBV replication equivalent to that observed in chronically
infected individuals). Cultures were treated with nine consecutive
daily doses of the test compounds. HBV DNA levels were assessed
by quantitative blot hybridization 24 h after the last treatment.
Cytotoxicity was assessed by uptake of neutral red dye 24 h
following the last treatment.
Activity Against Drug-Resistant HBV Mutants. Activity
against lamivudine-resistant and adefovir-resistant HBV mutants
was performed in a 5-day assay using a transient transfection
method as previously described.15 Antiviral activity was determined
by quantitative Southern blot hybridization of intracellular HBV
DNA replication intermediates (HBV RI) following 4 days of drug
treatment.
Acknowledgment. This research was supported by the U.S.
Public Health Service Grants U19-AI 056540 (C.K.C.), 5R37-
AI-41980 (R.F.S.), 5P30-AI-50409 (R.F.S.) and by Contracts
N01-AI-30047 (Z.H. and M.G.M.) and NO1-AI-30046 (B.K.)
from the National Institute of Allergy and Infectious Diseases
and the Department of Veterans Affairs (R.F.S.).
Supporting Information Available: Elemental analysis data for
the final compounds and few key intermediates. This material is
available free of charge via the Internet at http://pubs.acs.org.
References
(1) (a) Yaginuma, A.; Muto, N.; Tsujino, M.; Sudate, Y.; Hayashi, M.;
Otani, M. Studies on neplanocin A, new antitumor antibiotic. I.
Producting organism, isolation and characterization. J. Antibiot. 1981,
34, 359. (b) Borchart, R. T.; Keller, B. T.; Patel-Thombre, U.;
Neplanocin, A. A potent inhibitor of S-adenosylhomocysteine hydro-
lase and of vaccinia virus multiplication in mouse L-929 cells. J. Biol.
Chem. 1984, 259, 4353–4358. (c) De Clercq, E. Antiviral and
antimetabolic activities of neplanocins. Antimicrob. Agents Chemother.
1985, 28, 84–89.
(2) Turner, M. A.; Yang, X.; Yin, D.; Kuczera, K.; Borchardt, R. T.;
Howell, P. L. Structure and function of S-adenosylhomocysteine
therapy. Cell Biochem. Biophys. 2000, 33, 101–125.
(3) (a) Borcherding, D. R.; Scholtz, S. A.; Borchart, R. T. Synthesis of
analogues of neplanocin A: utilization of optically active dihydroxy-
clopentenones derived from carbohydrates. J. Org. Chem. 1987, 52,
5457. (b) Borcherding, D. R.; Narayanan, S.; Hasobe, M.; McKee,
J. G.; Keller, B. T.; Borchardt, R. T. Potential inhibitors of S-
adenosylmethionine-dependent methyltransferases. 11. Molecular dis-
sections of neplanocin A as potential inhibitors of S-adenosylho-
mocysteine hydrolase. J. Med. Chem. 1988, 31, 1729–1738. (c) Tseng,
C. K. H.; Marquez, V. E.; Fuller, R. W.; Goldstein, B. M.; Haines,
D. R.; McPherson, H.; Parsons, J. L.; Shannon, W. M.; Arnett, G.;
Hollingshead, M.; Driscoll, J. S. Synthesis of 3-deazaneplanocin A, a
powerful inhibitor of S-adenosylhomocysteine hydrolase with potent
and selective in vitro and in vivo antiviral activities. J. Med. Chem.
1989, 32, 1442–1446. (d) De Clercq, E.; Cools, M.; Balzarini, J.;
Marquez, V. E.; Borcherding, D. R.; Borchardt, R. T.; Drach, J. C.;
Kitaoka, S.; Konno, T. Broad-spectrum antiviral activities of nepl-
anocin A, 3-deazaneplanocin A, and their 5′-nor derivatives. Antimi-
crob. Agents Chemother. 1989, 33, 1291–1297. (e) Wolfe, M. S.; Lee,
Y.; Bartlett, W. J.; Borcherding, D. R.; Borchardt, R. T. 4′-Modified
analogues of aristeromycin and neplanocin A: synthesis and inhibitory
activity toward S-adenosyl-L-homocysteine hydrolase. J. Med. Chem.
1992, 35, 1782–1791, and references cited therein. (f) Shuto, S.; Obara,
T.; Saito, Y.; Andrei, G.; Snoeck, R.; De Clercq, E.; Matsuda, A. New
neplanoin analogues. 6. Synthesis and potent antiviral activity of 6′-
homoneplanocin A. J. Med. Chem. 1996, 39, 2392–2399. (g) Shuto,
S.; Minakawa, N.; Niizuma, S.; Kim, H.-S.; Wataya, Y.; Matsuda, A.

7-Deazaneplanocin A Analogues
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 213
New neplanocin analogues. 12 Alternative synthesis and antimalarial
effect of (6′R)-6′-C-methylneplanocin A, a potent adoHcy hydrolase
inhibitor. J. Med. Chem. 2002, 45, 748–751, and references cited
therein. (h) Jeong, L. S.; Yoo, S. J.; Lee, K. M.; Koo, M. J.; Choi,
W. J.; Kim, H. O.; Moon, H. R.; Lee, M. Y.; Park, J. G.; Lee, S. K.;
Chun, M. W. Design, synthesis, and biological evaluation of fluor-
oneneplanocin A as the novel mechanism-based inhibitor of S-
adenosylhomocysteine hydrolase. J. Med. Chem. 2003, 46, 201–203.
(4) (a) Song, G. Y.; Paul, V.; Choo, H.; Morrey, J.; Sidwell, R. W.;
Schinazi, R. F.; Chu, C. K. Enantiomeric synthesis of D- and
L-cyclopentenyl nucleosides and their antiviral activity against HIV
and West Nile virus. J. Med. Chem. 2001, 44, 3985–3993. (b) Jin,
Y. H.; Liu, P.; Wang, J.; Baker, R.; Huggins, J.; Chu, C. K. Practical
synthesis of D- and L-2-cyclopentenone and their utility for the
synthesis of carbocyclic antiviral nucleosides against orthopox viruses
(smallpox, monkeypox, and cowpox virus). J. Org. Chem. 2003, 68,
9012–9018. (c) Chu, C. K.; Jin, Y. H.; Baker, R. O.; Huggins, J.
Antiviral activity of cyclopentenyl nucleosides against orthopox viruses
(smallpox, monkepox, and cowpox). Bioorg. Med. Chem. Lett. 2003,
13, 9–12. (d) Cho, J. H.; Bernard, D. L.; Sidwell, R. W.; Kern, E. R.;
Chu, C. K. Synthesis of cyclopentenyl carbocyclic nucleosides as
potential antiviral agents against orthopoxviruses and SARS. J. Med.
Chem. 2006, 49, 1140–1148. (e) Arumugham, B.; Kim, H.; Prichard,
M. N.; Kern, E. R.; Chu, C. K. Synthesis and antiviral activity of
7-deazaneplanocin A against orthopoxviruses (vaccinia and cowpox
virus). Bioorg. Med. Chem. Lett. 2006, 16, 285–287. (f) Rao, J. R.;
Schinazi, R. F.; Chu, C. K. Enantioselective synthesis and antiviral
activity of purine and pyrimidine cyclopentenyl C-nucleosides. Bioorg.
Med. Chem. 2007, 15, 839–846.
(5) Koshy, R.; Caselmann, W. H. Hepatitis B Virus: Molecular Mechanism
in Disease and NoVel Strategies for Therapy; Imperial College Press:
London, 1997.
(6) Tan, S. L.; Pause, A.; Shi, Y.; Sonenberg, N. Hepatitis C therapeutics:
current status and emerging strategies. Nat. ReV. Drug. DiscoVery 2002,
1, 867–881.
(7) (a) Buti, M.; Esteban, R. Drugs in development for hepatitis B. Drugs
2006, 65, 1451–1460. (b) Zoulim, F. Mechanism of viral persistence
and resistance to nucleoside and nucleotide analogs in chronic hepatitis
B virus infection. AntiViral Res. 2004, 64, 1–15. (c) Ganem, D.; Prince,
A. M. Hepatitis B virus infection. Natural history and clinical
consequences. N. Engl. J. Med 2004, 350, 1118–1129.
(8) (a) Heathcote, E.; Shiffman, M.; Cooksley, W.; Dusheiko, G. M.; Lee,
S. S.; Balart, L.; Reindollar, R.; Reddy, R. K.; Wright, T. L.; Lin, A.;
Hoffman, J.; De Pamphilis, J. Peginterferon alfa-2a in patients with
chronic hepatitis C and cirrhosis. N. Engl. J. Med. 2000, 343, 1673–
1680. (b) Manns, M.; McHutchison, J.; Gordon, S. C.; Rustgi, V. K.;
Shiffman, M.; Reindollar, R.; Goodman, Z. D.; Koury, K.; Lin, M.;
Albrecht, J. K. Peginterferon alfa-2b plus ribavirin compared with
interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis
C: a randomized trial. Lancet 2001, 358, 958–965.
(9) (a) Hugle, T.; Cerny, A. Current therapy and new molecular approaches
to antiviral treatment and prevention of hepatitis C. ReV. Med. Virol
2003, 13, 361–371. (b) Gordon, C. P.; Keller, P. A. Control of hepatitis
C: a medicinal chemistry perspective. J. Med. Chem. 2005, 48, 1–20,
and references cited therein. (c) Eldrup, A. B.; Allerson, C. R.; Bennett,
C. F.; Bera, S.; Bhat, B.; Bhat, N.; Bosserman, M. R.; Brooks, J.;
Carroll, S. S.; Cook, P. D.; Getty, K. L.; MacCoss, M.; McMasters,
D. R.; Olsen, D. B.; Prakash, T. P.; Prhavc, M.; Song, Q.; Tomassini,
J. E.; Xia, J. Structure activity relationship of purine ribonucleosides
for inhibition of hepatitis C virus RNA-dependent RNA polymerase.
J. Med. Chem. 2004, 47, 2283–2295. (d) Eldrup, A. B.; Prhavc, M.;
Brooks, J.; Bhat, B.; Prakash, T. P.; Song, Q.; Bera, S.; Bhat, N.;
Dande, P.; Cook, P. D.; Bennett, C. F.; Carroll, S. S.; Ball, R. G.;
Bosserman, M.; Burlein, C.; Colwell, L. F.; Fay, J. F.; Flores, O. A.;
Getty, K.; Lafemina, R. L.; Leone, J.; MacCoss, M.; McMasters, D. R.;
Tomassini, T. E.; Von Langen, D.; Wolanski, B.; Olsen, D. B.
Structure-relationship of heterobase-modified 2′-C-methyl ribonucleo-
sides as inhibitors of hepatitis C virus RNA replication. J. Med. Chem.
2004, 47, 5284–5297. (e) Wang, P.; Hollecker, L.; Pankiewicz, K. W.;
Patterson, S. E.; Whitaker, T.; McBrayer, T. R.; Tharnish, P. M.;
Sidwell, R. W.; Stuyver, L. J.; Otto, M. J.; Schinazi, R. F.; Watanabe,
K. A. Synthesis of N3,5′-cyclo-4-(ꢀ-D-ribofuranosyl)-Vic-triazolo[4,5-
b]pyridine-5-one, a novel compound with anti-hepatitis C virus activity.
J. Med. Chem. 2004, 47, 6100–6103. (f) Pierra, C.; Amador, A.;
Benzaria, S.; Cretton-Scott, E.; D’Amours, M.; Mao, J.; Mathieu, S.;
Moussa, A.; Bridges, E. G.; Standring, D. N.; Sommadossi, J. P.;
Storer, R.; Gosselin, G. Synthesis and pharmacokinetics of valopic-
itabine (NM283), an efficient prodrug of the potent anti-HCV agent
2′-C-methylcytidine. J. Med. Chem. 2006, 49, 6614–6620. (g) Clark,
J. L.; Hollecker, L.; Mason, J. C.; Stuyver, L. J.; Tharnish, P. M.;
Lostia, S.; McBrayer, T. R.; Schinazi, R. F.; Watanabe, K. A.; Otto,
M. J.; Furman, P. A.; Stec, W. J.; Patterson, S. E.; Pankiewicz, K. W.
Design, synthesis, and antiviral activity of 2′-deoxy-2′-fluoro-2′-C-
methylcytidine, a potent inhibitor of hepatitis C virus replication.
J. Med. Chem. 2005, 48, 5504–5508. (h) Murakami, E.; Bao, H.;
Ramesh, M.; McBrayer, T. R.; Whitaker, T.; Micolochick Steuer,
H. M.; Schinazi, R. F.; Stuyver, L. J.; Obikhod, A.; Otto, M. J.;
Furman, P. A. Mechanism of activation of beta-D-2′-deoxy-2′-fluoro-
2′-c-methylcytidine and inhibition of hepatitis C virus NS5B RNA
polymerase. Antimicrob. Agents Chemother. 2007, 51, 503–509.
(10) (a) Chu, C. K.; Ma, T.; Shanmuganathan, K.; Wang, C.; Xiang, Y.;
Pai, S. B.; Yao, G. Q.; Sommadossi, J. P.; Cheng, Y. C. Use of 2′-
fluoro-5-methyl-ꢀ-L-arabinofuranosyluracil as a novel antiviral agent
for hepatitis B virus and Epstein-Barr virus. Antimicrob. Agents
Chemother. 1995, 39, 979–981. (b) Rajagopalan, P.; Chu, C. K.;
Kobra,B. E.; Sommadossi, J. P. Selective Inhibition of Human
Immunodeficiency Virus and Hepatitis B Virus by L-ꢀ-Enantiomers
of Dioxolane-Purines. Presented at the 33rd Interscience Conference
on Antimicrobial Agents and Chemotherapy, New Orleans, LA, Oct
17-20, 1993; Abstract 222427. (c) Lee, K.; Choi, Y.; Gullen, E.;
Schlueter-Wirtz, S.; Schinazi, R. F.; Cheng, Y. C.; Chu, C. K. Synthesis
and anti-HIV and anti-HBV activities of 2′-fluoro-2′,3′-unsaturated
L-nucleosides. J. Med. Chem. 1999, 42, 1320–1328. (d) Wang, P.;
Gullen, B.; Newton, M. G.; Cheng, Y. C.; Schinazi, R. F.; Chu, C. K.
Asymmetric synthesis and antiviral activities of L-carbocylic 2′,3′-
didehydro-2′,3′-dideoxy and 2′,3′-dideoxy nucleosides. J. Med. Chem.
1999, 42, 3390–3399. (e) Chu, C. K.; Ma, L.; Olgen, S.; Pierra, C.;
Du, J.; Gumina, G.; Gullen, E.; Cheng, Y. C.; Schinazi, R. F. Synthesis
and antiviral activity of oxaselenolane nucleosides. J. Med. Chem.
2000, 43, 3906–3912.
(11) (a) Ma, S.; Boerner, J. E.; Yip, C. T.; Weidmann, B.; Ryder, N. S.;
Cooreman, M. P.; Lin, K. NIM811, a cyclophilin inhibitor, exhibits
potent in vitro activity against hepatitis C virus alone or in combination
with alpha interferon. Antimicrob. Agents Chemother. 2006, 50, 2976–
2982. (b) Hao, W.; Herlihy, K. J.; Zhang, N. J.; Fuhrman, S. A.; Doan,
C.; Patick, A. K.; Duggal, R. Development of a novel dicistronic
reporter-selectable hepatitis C virus replicon suitable for high-
throughput inhibitor screening. Antimicrob. Agents Chemother. 2007,
51, 95–102.
(12) Koch, U.; Attenni, B.; Malancona, S.; Colarusso, S.; Conte, I.; Filippo,
M.; Harper, S.; Pacini, B.; Giomini, C.; Thomas, S.; Incitti, I.; Tomei,
L.; Di Francesco, R.; Altamura, S.; Matassa, V. G.; Narjes, F. 2-(2-
Thienyl)-5,6-dihydroxy-4-carboxypyrimidines as inhibitors of the
hepatitis C virus NS5B polymerase: discovery, SAR, modeling, and
mutagenesis. J. Med. Chem. 2006, 49, 1693–1705.
(13) Stuyver, L. J.; Whitaker, T.; McBrayer, T. R.; Hernandez-Santiago,
B. I.; Lostia, S.; Tharnish, P. M.; Ramesh, M.; Chu, C. K.; Jordan,
R.; Shi, J.; Rachakonda, S.; Watanabe, K. A.; Otto, M. J.; Schinazi,
R. F. Ribonucleoside analogue that blocks replication of bovine viral
diarrhea and hepatitis C viruses in culture. Antimicrob. Agents
Chemother. 2003, 47, 244–254.
(14) Korba, B. E.; Gerin, J. L. Use of a standardized cell culture assay to
determine activities of nucleoside analogs against hepatitis B virus
replication. AntiViral Res. 1992, 19, 55–70.
(15) Iyer, R. P.; Jin, Y.; Roland, A.; Morrey, J. D.; Mounir, S.; Korba, B.
Phosphorothioate di- and tri-nucleotides as a novel class of anti-HBV
agents. AntiViral Agents Chemother. 2004, 48, 2199–2205.
JM801418V