Synthesis and antiviral activity of certain second generation methylenecyclopropane nucleosides

Article abstract of DOI:10.1016/j.bmc.2012.04.049

A second-generation series of substituted methylenecyclopropane nucleosides (MCPNs) has been synthesized and evaluated for antiviral activity against a panel of human herpesviruses, and for cytotoxicity. Although alkylated 2,6-diaminopurine analogs showed little antiviral activity, the compounds containing ether and thioether substituents at the 6-position of the purine did demonstrate potent and selective antiviral activity against several different human herpesviruses. In the 6-alkoxy series, antiviral activity depended on the length of the ether carbon chain, with the optimum chain length being about four carbon units long. For the corresponding thioethers, compounds containing secondary thioethers were more potent than those with primary thioethers.

Full text of DOI:10.1016/j.bmc.2012.04.049

Bioorganic & Medicinal Chemistry 20 (2012) 3710–3718
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antiviral activity of certain second generation
methylenecyclopropane nucleosides
John D. Williams a, , Atiyya R. Khan a, Emma A. Harden b, Caroll B. Hartline b, Geraldine M. Jefferson b,
⇑
Kathy A. Keith b, Mark N. Prichard b, Jiri Zemlicka c, Norton P. Peet a, Terry L. Bowlin a
a Microbiotix, Inc., One Innovation Drive, Worcester, MA 01605, USA
b Department of Pediatrics, University of Alabama School of Medicine, 1600 6th Avenue South, Birmingham, AL 53233, USA
c Department of Chemistry, Developmental Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, 110 E. Warren Avenue,
Detroit, MI 48201, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A
second-generation series of substituted methylenecyclopropane nucleosides (MCPNs) has been
Received 9 February 2012
Revised 17 April 2012
Accepted 25 April 2012
Available online 1 May 2012
synthesized and evaluated for antiviral activity against a panel of human herpesviruses, and for cytotox-
icity. Although alkylated 2,6-diaminopurine analogs showed little antiviral activity, the compounds
containing ether and thioether substituents at the 6-position of the purine did demonstrate potent and
selective antiviral activity against several different human herpesviruses. In the 6-alkoxy series, antiviral
activity depended on the length of the ether carbon chain, with the optimum chain length being about
four carbon units long. For the corresponding thioethers, compounds containing secondary thioethers
were more potent than those with primary thioethers.
Keywords:
Antiviral agents
Nucleoside analogs
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
several different human herpesviruses.10–13 A large series of
mono-hydroxymethyl (first-generation) MCPNs with varying sub-
The human herpesviruses (HHV) are a closely related family of
enveloped, double-stranded DNA viruses that cause a range of hu-
man diseases.1–3 There are eight distinct human herpesviruses,
each of which is thought to be responsible for a set of human ail-
ments, from oral and genital herpes (HHV-1 and HHV-2, respec-
tively) to Kaposi’s sarcoma (HHV-8). The human herpesviruses
are highly prevalent in both urban and rural populations, can
establish latent infections, and can have severe consequences,
especially for immunocompromised populations such as AIDS
patients, transplant recipients, neonates, and the elderly.
stituents at the purine 6-position has been synthesized (Fig. 2),
and several SAR trends have been elucidated within that series.14
O
N
O
N
H2N
O
O
N
N
N
N
NH
NH2
NH
NH2
HO
HO
O
O
valacyclovir
acyclovir
Although acyclovir4 and its valine ester prodrug, valacyclovir,5
(Fig. 1) have been extremely effective at treating herpes simplex
(HSV or HHV-1/2) and varicella zoster (VZV or HHV-3) viruses,
they are far less effective against the other members of the herpes-
virus family. Other drugs, such as ganciclovir,6 valganciclovir,7
cidofovir,8 and foscarnet9 have also been used as anti-herpesvirus
agents, particularly against human cytomegalovirus, but have rel-
atively narrow therapeutic ranges because of their inherent toxic-
ities. No drugs have been approved for use against HHV-6, HHV-7,
or HHV-8.
O
N
O
N
H2N
O
O
N
N
NH
NH2
NH
NH2
N
O
N
O
OH
ganciclovir
OH
valganciclovir
NH2
N
O
O
P
OH
OH
O
N
O
Methylenecyclopropane nucleosides (MCPNs) are a series of
acyclic nucleoside analogs that have showed promise against
HO
P
O
HO
HO
foscarnet
HO
cidofovir
⇑
Corresponding author.
E-mail address: jwilliams@microbiotix.com (J.D. Williams).
Figure 1. FDA-approved anti-herpes drugs
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.04.049

J. D. Williams et al. / Bioorg. Med. Chem. 20 (2012) 3710–3718
3711
First generation MCPNs
NH
corresponding first-generation compounds; given the limited
number of second-generation compounds, SAR trends are difficult
to establish. Herein we describe the synthesis of a range of second-
generation MCPNs, and the conclusions that can be drawn from the
different analogs.
O
N
XR
NH
NH2
N
N
N
N
HO
HO
NH2
N
2. Materials and methods
2.1. Chemistry
synguanol
X = NH, O, S
Second generation MCPNs
The chemistry used to synthesize the second-generation MCPNs
has been adapted from the previous work of Zemlicka.12,14 Thus,
we began by synthesizing a versatile intermediate (4a) that could
be scaled to multi-gram batches and used in the subsequent reac-
tions without resorting to repetitious procedures (Scheme 1). To
this end, commercially available 2-amino-5-chloropurine (2) was
treated with dibrominated diester 1 in a two-stage reaction with
base. In the initial low-temperature stage, the base is alkylated
by 1; after additional DBU is added, the temperature is raised,
and dehydrohalogenation produces a mixture of the protected
(Z)- and (E)-nucleosides 3a and 3b (1:1 mixture). Because it is
impractical to separate the isomers in large batches at this stage,
the subsequent saponification step was performed on the mixture.
The resulting diols 4a and 4b are readily separated by crystalliza-
tion, wherein the (Z)-isomer 4a was then used for subsequent
reactions.
Displacement of the 6-chloro group on the purine was readily
achieved with amines, alkoxides, or thiolates (Scheme 1). Treating
the intermediate 4a with an excess of amine in ethanol at 70 °C
provided the corresponding 2,6-diaminopurine analogs 5a–l in
88–46% yield. Using a solution of alkoxide in DMF at 70 °C, the cor-
responding 2-amino-6-alkoxypurines 6a–l were also synthesized
in 75–32% yield. Reaction of thiolates and 4a at elevated tempera-
tures gave complex mixtures of products containing large amounts
of a product containing two equivalents of thiol, as determined by
1H NMR and LC–MS (data not shown), presumably from a second
equivalent of thiolate adding to the double bond of the methylene-
cyclopropane moiety. Fortunately, we found that the thiolates
were sufficiently nucleophilic to displace the 6-chloro group on
the purine even at lower temperature; 4a was thus reacted at room
temperature with a series of thiolates, generated in situ from the
O
XR
N
N
N
N
NH
NH2
N
HO
HO
HO
HO
NH2
N
N
cyclopropavir
X = NH, O, S
Figure 2. First and second generation MCPNs. SAR is well-defined for first
generation compounds; SAR is little explored for second generation compounds.
Several first-generation MCPNs containing alkylamine, ether, and
thioether substituents at the 6-position of the purine were potent
inhibitors of HCMV (HHV-5), while the ether-containing analogs
were generally more potent against HHV-6 and HHV-8.15
The synthesis and subsequent development of these first-gener-
ation compounds have been complicated, however, by the pres-
ence of a chiral center in the molecule, which requires a more
complicated synthesis to execute with good enantiomeric excess.
A second generation of MCPNs that eliminate the chiral center by
adding a second hydroxymethyl group to the cyclopropyl ring
has been subsequently synthesized and tested for antiviral activ-
ity.10,12,16 Several of these second-generation MCPNs were found
to be potent inhibitors of several different human herpesviruses.
Most notably, the guanine analog, cyclopropavir, was found to be
a potent inhibitor of HCMV both in vitro and in vivo; it also has
moderate activity against EBV, HHV-6 and HHV-8. Cyclopropavir
is currently undergoing preclinical development as
a HCMV
therapeutic.17
To date, far fewer examples of second-generation MCPNs have
been synthesized and tested for antiviral activity than the
Cl
Cl
N
Cl
N
AcO
AcO
N
N
N
AcO
AcO
N
a
N
+
N
N
NH2
N
+
N
NH2
AcO
AcO
65%
yield
N
H
Br
NH2
3b
Br
3a
1
2
42%
b
yield (4a)
Cl
N
NRR'
N
Cl
N
N
N
N
N
N
N
N
N
c
HO
HO
HO
and
NH2
HO
HO
N
NH2
NH2
88-46%
yield
4b
HO
4a
5a-l
65-11%
yield
e
d
75-32%
yield
OR
N
SH
N
N
N
N
N
HO
HO
HO
HO
N
NH2
N
NH2
6a-l
7a-j
Scheme 1. Synthesis of 6-amino, 6-alkoxy, and 6-alkylthio MCPNs. Reagents and conditions: (a) DBU, DMF, 25–75 °C; (b) K2CO3, H2O/MeOH; (c) amine, EtOH, 70 °C; (d)
alcohol, NaH, DMF, 70 °C; (e) thiol, 1.0 M NaOH/H2O, DMF, 25 °C.

3712
J. D. Williams et al. / Bioorg. Med. Chem. 20 (2012) 3710–3718
corresponding thiols and aqueous sodium hydroxide, to provide a
series of 2-amino-6-alkylthiopurines 7a–j in 65–11% yield.
types 1 and 2 (HSV-1 and HSV-2), varicella zoster virus (VZV),
Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), two
variants of human herpes 6 (HHV-6A and HHV-6B), and human
herpesvirus 8 (HHV-8). Cytotoxicity was also determined for the
compounds in each of the cell lines used for the antiviral evalua-
tions using the same cell number and duration of compound expo-
sure. None of the compounds tested exhibited significant activity
against HSV-1, HSV-2. One of the new 2,6-diaminopurine analogs
(5d; see Table 1) demonstrated modest activity against HCMV,
but the spectrum of activity was quite limited, with little or no
activity against EBV, VZV, HHV-6, or HHV-8. This result was unex-
pected given the potent and relatively broad-spectrum antiviral
activity of the analogous first-generation MCPNs, which show
potent activity against all of these pathogens.
The 1H of all the target compounds, and the 13C NMR of repre-
sentative compounds 5b, 6a, and 7a corresponded very well with
the analogous 1st generation methylenecyclopropane nucleo-
sides,14 as well as the limited number of known 2nd generation
compounds.18 Interestingly, addition of the second hydroxymethyl
substituent in the methylenecyclopropane moiety resulted in some
signals which were much more indistinct in the 13C NMR spectra
than in the corresponding 1st generation mono-hydroxymethyl
compounds. Although the indistinct peaks complicated the identi-
fication of the compounds, we are unsure of the implication this
phenomenon has to the biological activity of the compounds.
2.2. Biological evaluation
In contrast to the diaminopurine analogs, compounds contain-
ing ether and thioether substituents (see Table 2 and Table 3) at
the 6-position had much broader antiviral profiles, very much like
the first-generation MCPNs. In particular, certain members of the
The methylenecyclopropane nucleosides were tested against a
range of human herpesviruses, including herpes simplex virus
Table 1
Antiviral activity and cytotoxicity for 6-alkylamino-substituted MCPN analogs
R
N
N
N
HO
HO
N
NH2
#
R
50% inhibitory concentrationa
(lM)
IC50
CC50 IC50
HFF EBV
(hybrid)
CC50 IC50
CC50 IC50
CC50
IC50
CC50
HSV-1
(CPE)
HSV-2
(CPE)
VZV
(CPE)
HCMV
(CPE)
Akata HHV-6A
(hybrid)
HSB- HHV-6B
MOLT- HHV-8
3
BCBL-
1
2
(hybrid)
(QPCR)
3.0
Cyclopropavir (CPV) >100
>100
>300
>100
>300
1.4
>300 45
>300 11
>20
>20
1.3
>20
8.0
>20
>20
67
DAPb
>300
>300
>300
>20
>20
>20
16
>20
>20
30
62
NH2
N
H
5a
>300
>300
>300
>300
>300
>300
265 >24
>300 >24
>24
>24
>20
>20
>20
67
n.d.c
n.d.c
n.d.c
n.d.c
>20
>20
>20
N
H
5b
5c
>300
>300
>20
>20
>300
>60
>300
>60
160
24
>300 >24
>300 >20
>24
>20
>20
>20
>20
N
H
5d
>60
>20
>20
>20
71
71.5
N
H
N
H
5e
5f
>300
>300
>300
>300
>300
>300
>60
>300 >24
>300 >24
>24
>24
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>300
n.d.c
n.d.c
N
H
5g
>60
>60
>60
>60
>300 >20
>20
>20
>20
>20
>20
63
71.7
N
H
5h
5i
>300
>300
>300
>300
>300
>60
>300
>60
>300 >24
>300 >24
>24
>24
>20
>20
>20
>20
n.d.c
>20
n.d.c
>20
>20
>20
N
H
N
H
n.d.c
19.9
n.d.c
n.d.c
n.d.c
n.d.c
>20
>20
>20
>20
N
N
5j
>300
>300
>300
>300
180
>300
>300
>300 >24
>300 >24
>24
>24
>20
>20
>20
>20
5k
>300
N
5l
>300
2.9
>300
2.8
>300
1.9
>300
>300 >24
>24
>20
>20
n.d.c
n.d.c
>20
>20
O
Acyclovir (ACV)
Ganciclovir (GCV)
>300
>300
2.1
a
b
c
Antiviral and cytotoxicity assays were performed in duplicate wells as described in the text.
DAP is 2,6-diaminopurine.
n.d. means value not determined.

J. D. Williams et al. / Bioorg. Med. Chem. 20 (2012) 3710–3718
3713
Table 2
Antiviral activity and cytotoxicity for 6-alkoxy-substituted MCPN analogs
R
N
N
N
N
HO
NH2
HO
#
R
50% inhibitory concentrationa
(lM)
IC50
VZV
(CPE)
>100
7.4
CC50 IC50
HFF EBV
(hybrid)
>300 45
171
CC50 IC50
CC50 IC50
CC50
IC50
CC50
HSV-1
(CPE)
HSV-2
CPE)
HCMV
(plaque)
Akata HHV-6A
(hybrid)
HSB- HHV-6B
MOLT- HHV-8
3
BCBL-
1
2
(hybrid)
(QPCR)
3.0
Cyclopropavir (CPV)
>100
>300
>100
>300
1.4
>20
>20
1.3
>20 8.0
>20 >20
>20
>20
>20
51
AMPb
2.8
1
>20
13
O
6a
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>100
2.9
1.6
1.1
1.1
1.6
0.5
3.9
29
>300 11.9
>300 8.9
>300 11.0
>300 1.6
259 0.5
>200 7.3
>300 13
>20
>20
>20
>20
>20
>20
>20
>20
18
>20 11.2
>20 6.6
>20 11.4
>20 3.2
>20 >20
>20 9.5
>20 11.5
>20
>20
>20
>20
>20
>20
>20
>20
16
>20
>20
>20
>20
>20
>20
>20
O
6b
O
6c
>300
>100
>60
>20
>20
>20
>20
>20
17
O
6d
>20
>20
>20
>20
O
6e
O
6f
n.d.c
>100
O
6g
O
6h
6i
>300
>300
>300
>300
>300
13.9
5.6
17
>300 1.4
>300 20
>20
>20
>20
>20
>20 8.9
>20 >20
>20
>20
19
>20
>20
O
CF3
O
>20
O
O
6j
>300
2.9
>300
2.8
53.0
1.9
45
>300 >20
>20
>20
>20 >20
>20
>20
>20
Acyclovir (ACV)
Ganciclovir (GCV)
>300
>300
2.1
a
b
c
Antiviral and cytotoxicity assays were performed in duplicate wells as described in the text.
AMP is 2-amino-6-methoxypurine.
n.d. means value not determined.
2-amino-6-alkoxypurine analog series 6a–j demonstrated broad
antiviral activity, especially against EBV, HCMV, and HHV-6b, with
some analogs possessing activity moderate against VZV and HHV-8
as well; the antiviral activity of these compounds was similar to
that of the corresponding 1st generation analogs. Antiviral activity
generally varied with the size of the ether carbon chain; with the
optimal size for broad activity being about 4 or 5 carbons in length
(6d and 6e, respectively), although any analog with less than
6-carbon ether functionalities (e.g., 6a, 6b, 6c, etc.) also had good
activity. Inclusion of a heteroatom in the ether unit (i.e., 6i) de-
creased the activity relative to the corresponding hydrocarbon
(6d), but the incorporation of halogens (i.e., 6h) was largely toler-
ated. The benzyl ether 6j, however, was much less active than the
other analogs.
The thioether series (7a–i) was somewhat less potent in the
antiviral assays than the corresponding ethers, but several com-
pounds did possess notable antiviral potency, especially against
EBV. The most potent compound among the thioether analogs
was the isopropylthio analog 7c, which had the best activity
against HCMV, and the cyclopentylthio analog 7f, which had the
highest activity against EBV. Interestingly, both of these com-
pounds are synthesized from secondary thiols, whereas the com-
pounds synthesized from primary thiols (e.g., 7b and 7d) had
somewhat lower activity against HCMV. Because of the more lim-
ited commercial availability of secondary thiols, however, we were
unable to determine whether this was a general phenomenon, or
merely a coincidence. Compared to the 1st generation compounds,
the 6-alkylthio analogs in the current study were somewhat less
potent against HCMV and HHV-6, but much more potent against
EBV
3. Conclusion
In conclusion, we have synthesized a range of second-genera-
tion methylenecyclopropane nucleosides designed to test the anti-
viral activity and antiviral spectrum for the series. The compounds
were synthesized by nucleophilic displacement of a versatile
2-amino-6-chloropurine intermediate that could be further elabo-
rated into the desired target compounds. The 2-amino-6-alkyl-
amino analogs were generally much less active then their
corresponding 1st generation counterparts. The 2-amino-6-alkyl-
thio analogs generally had potent anti-EBV activity and moderate
activity against HHV-6A and HHV-6B. The most potent compounds
were the 2-amino-6-alkoxypurine analogs; these compounds
demonstrated potent and selective antiviral activity against several
different human herpesviruses, including HCMV, EBV, and HHV-6B.
Additional studies evaluating the suitability of the ether series for
additional development are underway, and will be reported in the
future.
4. Experimental section
4.1. Biology
4.1.1. Cells and viruses
Human foreskin fibroblast (HFF) cells were prepared from
human foreskin tissue obtained from the University of Alabama
at Birmingham tissue procurement facility with approval from its
IRB. The E-377 strain of HSV-1 and the MS strain of HSV-2 were
a gift of Jack Hill (Burroughs Wellcome). The AD169 strain of HCMV
and the Ellen strain of VZV were obtained from the American Type

3714
J. D. Williams et al. / Bioorg. Med. Chem. 20 (2012) 3710–3718
Table 3
Antiviral activity and cytotoxicity for 6-alkylthio-substituted MCPN analogs
R
N
N
N
N
HO
NH2
HO
#
R
50% inhibitory concentrationa
(
l
M)
CC50 IC50
HSB- HHV-6B
IC50
VZV
(CPE)
>100
>60
CC50 IC50
HFF EBV
(hybrid)
>300
CC50 IC50
CC50
IC50
CC50
HSV-1
(CPE)
HSV-2
(CPE)
HCMV
(plaque)
Akata HHV-6A
(hybrid)
MOLT- HHV-8
BCBL-
1
2
(hybrid)
3
(QPCR)
>20
Cyclopropavir (CPV)
>100
>300
>100
1.4
45
>20
1.3
>20
8.0
>20
3.0
>20
7a
>300
>300
>50
>300 11.4
>300 >20
>20
>100
>100 11.6
>20
>20
S
7b
>300
>300
>300
>300
>300
>60
42
>20
>20
>20
>20
63
98
94
89
60
14.3
7.5
>20
>20
>20
n.d.b
>20
>20
>20
>20
>20
>20
>20
>20
S
7c
>300
>300
>300
4.0
>50
31
>300 3.1
>300 2.5
>300 2.3
70
S
7d
>300
>50
75
7.0
S
n.d.b
S
7e
7f
>20
>300
>300
>300
>50
>300 0.9
>20
35
91
9.9
>20
>20
>20
S
S
S
S
7g
7h
>300
>300
>300
>300
>300
>60
>50
20
>300 7.1
>300 >20
>20
>20
>20
>20
60
60
5.4
>20
>20
>20
>20
>20
>20
11.0
7i
>300
2.9
>300
2.8
>60
1.9
>50
>300 3.1
>20
88
>100 >20
>20
>20
>20
Acyclovir (ACV)
Ganciclovir (GCV)
>300
>300
2.1
a
Antiviral and cytotoxicity assays were performed in duplicate wells as described in the text.
n.d. means value not determined.
b
Culture Collection (ATCC) (Manassas, VA). The phenotype of these
isolates as well as the genotype of relevant genes was reported pre-
viously.19 Epstein-Barr virus (EBV)-infected Akata cells were a gift
from John Sixbey (Louisiana State University, Baton Rouge). HSB-2
cells and the GS strain of human herpesvirus 6 variant A (HHV-6A),
as well as BCBL1 cells infected with HHV-8, were obtained from the
AIDS Research and Reference Reagent Program, Division of AIDS,
NIAID, NIH. The Z29 strain of the B variant of human herpesvirus
6 (HHV-6B) and MOLT-3 cells were obtained from Scott Schmid
at the Centers for Disease Control and Prevention, Atlanta, GA.
Antiviral activity against HCMV was determined in plaque
reduction assays by methods reported previously.20 Briefly, mono-
layers of HFF cells in 6-well plates were prepared 2 days prior to
infection which were then infected with virus suspensions
sufficient to yield 30 plaques per well. Compound dilutions were
then prepared in MEM with 2% FBS and were supplemented with
standard concentrations of L-glutamine, penicillin, gentamycin,
and pooled human gamma globulin. After a 1 h adsorption period,
compound solutions were added and the infected monolayers were
incubated for 8 days. Cell monolayers were then stained with neu-
tral red and plaques were enumerated using a stereomicroscope.
The experimental data were used to interpolate EC50 values by
standard methods.
4.1.2. Evaluation of antiviral activity against HSV-1, HSV-2,
HCMV, and VZV
Antiviral activity was evaluated in primary CPE reduction as-
says. Briefly, compounds were diluted in 96-well plates containing
monolayers of HFF cells; the cells were then infected at a multiplic-
ity of infection of approximately 0.01 PFU/cell for HSV-1 or HSV-2.
Approximately 100 infected cells were used to initiate infection
with VZV. Infected monolayers were incubated in a humidified
5% CO2 incubator for 3 days for HSV-1 and HSV-2, 7 days for VZV,
and 14 days for CMV. After the incubation period, the media was
aspirated and the cells stained with a 0.1% crystal violet in a forma-
lin solution for 4 h. The stain was removed and the monolayers
were rinsed with water until all excess stain was removed. The
plates were allowed to dry for 24 h and the absorbance at
595 nm was used to calculate compound concentrations that re-
duced viral CPE by 50% (EC50). The same drug concentrations used
to determine efficacy were also used on uninfected cells in each
assay to determine the cytotoxicity of each experimental com-
pound. The drug concentration sufficient to reduce cell number
by 50% as estimated by crystal violet staining was used to deter-
mine cytotoxicity (CC50).
4.1.3. Evaluation of activity against EBV
Determination of antiviral activity against the Akata strain of
EBV was described previously.21 Briefly, Akata cells were passaged
routinely in RPMI 1640 supplemented with 10% FBS and standard
concentrations of L-glutamine, penicillin, and gentamicin. Com-
pounds were diluted directly in growth media in 96-well round
bottom assay plates. Wells containing compound dilutions were
then seeded with latently infected Akata cells that were induced
to undergo a productive infection by the addition of a goat anti-
human immunoglobulin G antibody. After 72 h, the DNA in the
cultures was denatured and transferred onto a positively charged
nylon membrane and viral DNA was quantified using a digoxi-
genin-labeled probe and a monoclonal antibody specific for the
hapten. Compound concentrations sufficient to reduce viral DNA
by 50% were used to calculate EC50 values. Assays for cytotoxicity
were prepared in the same manner as those for the antiviral assay
but with uninfected cells. Cells were incubated with drug for seven
days using the same conditions as the antiviral assay and 50 lL of

J. D. Williams et al. / Bioorg. Med. Chem. 20 (2012) 3710–3718
3715
CellTiter-Glo was added to each well. Luminescence was quantified
on a luminometer and concentrations of compounds sufficient to
reduce cell numbers by 50% (CC50) were calculated from the data.
4.2.2. (Z)-2-Amino-6-chloro-9-{[2,2-bis(acetoxymethyl)-
cyclopropylidene]methyl}purine (3a) and (E)-2-Amino-6-
chloro-9-{[2,2-bis(acetoxymethyl)cyclopropylidene]methyl}-
purine (3b)
4.1.4. Assessment of activity against HHV-6
To a stirred solution of 1,1-bis(acetoxymethyl)-2-bromo-2-
(bromomethyl)cyclopropane (1, 329 g, 0.92 mol) and 2-amino-6-
chloropurine (2, 136 g, 0.80 mol) in anhydrous DMF (2.0 L) cooled
to 0 °C was added DBU (139 mL, 0.92 mol). The solution was al-
lowed to warm to room temperature over 3 days with moderate
stirring. Additional DBU (146 mL, 0.92 mol) was then added, and
the solution was heated to 75 °C, and stirred at this temperature
for 16 h. The reaction was poured into a mixture of H2O (5.0 L),
ice (1 kg), 85% phosphoric acid (280 mL), and EtOAc (6.0 L). The
mixture was agitated, then separated. The organic layer was
washed with 5% aqueous NaCl (5.0 L), saturated aqueous NaCl
(2.5 L), then evaporated to a volume of 1.0 L and refrigerated for
24 h. The resulting precipitate was filtered, washed with 1:1 hex/
EtOAc (250 mL), and dried to yield 189 g (65%) of 3a and 3b
(ꢀ1:1 mixture) as light-colored powder: 1H NMR (CDCl3) d 8.43
(s, 1H), 8.32 (s, 1H), 7.40 (s, 1H), 7.30 (s, 1H), 7.06 (s, 2H), 7.02 (s,
2H), 4.28 (d, 4H), 4.06–4.15 (m, 4H), 2.04 (s, 6H), 1.94 (s, 6H),
1.89 (d, 2H), 1.64 (s, 2H).
Compounds were evaluated by methods reported previously.20
Briefly, stock solutions were serially diluted in cell culture media in
96-well plates. Uninfected HSB-2 or Molt-3 cells were then seeded
into 96 well plates and the infection was initiated with HHV-6A in-
fected HSB-2 cells, or HHV-6B infected Molt-3 cells. Cultures were
incubated for 7 days at 37 °C. Infected cells were then lysed in
denaturation buffer (4.5 M NaCl, 1.2 M NaOH) and the lysate was
applied to a positively charged nylon membrane to immobilize
the DNA. A digoxygenin-labeled probe specific for viral DNA was
used in a hybridization assay to quantify the accumulation of viral
DNA and EC50 values were calculated from the experimental data.
Cytotoxicity was determined in uninfected cells by the same meth-
ods used for Akata cells.
4.1.5. Activity against HHV-8
Compounds were evaluated by methods reported previously.20
Briefly, BCBL1 cells were maintained in growth medium consisting
of RPMI 1640 supplemented with 10% FBS, penicillin, gentamycin,
and L-glutamine. Compounds were diluted in triplicate wells of a
4.2.3. (Z)-2-Amino-6-chloro-9-{[2,2-bis(hydroxymethyl)cyclo-
propylidene]methyl}purine (4a)
96-well plate and cells that had been induced to undergo a lytic
infection with phorbol 12-myristate 13-acetate were added to
the assay plates. Cultures were incubated for 7 days at 37 °C in a
humidified CO2 incubator. Total DNA was prepared with a Wizard
SV 96-well purification kit (Promega), and viral DNA was quanti-
fied by quantitative PCR using forward primer 50-TTC CCC AGA
TAC ACG ACA GAA TC-30, reverse primer 50-CGG AGC GCA GGC
TAC CT-30, and probe 50-(6-carboxyfluorescein)-CCT ACG TGT TCG
TCG AC-(6-carboxytetramethylrhodamine)-30. Compound concen-
trations sufficient to reduce viral DNA accumulation by 50%
(EC50s) were calculated by standard methods. Cytotoxicity was
determined with CellTiter-Glo by methods used for Akata cells.
To a stirred solution of (Z)-2-amino-6-chloro-9-{[2,2-bis(acet-
oxymethyl)cyclopropylidene]methyl}purine and (E)-2-amino-6-
chloro-9-{[2,2-bis(acetoxymethyl)cyclopropylidene]methyl}purine
(1:1 3a:3b, 193 g, 0.53 mol) in MeOH (6.5 L) and H2O (720 mL)
was added potassium carbonate (75.5 g, 0.54 mol). The suspen-
sion was stirred at room temperature for 30 min, then AcOH
(62 mL, 1.1 mol) was added in one portion, and the mixture stir-
red at room temperature for 16 h. The resulting precipitate was
filtered and rinsed with MeOH (500 mL) to provide the unde-
sired
E isomer 4b. The filtrate was evaporated, and EtOH
(2.5 L) was added to the residue. The EtOH was then evaporated
and the residue triturated in CHCl3 (2.2 L) and MeOH (250 mL).
The mixture was stirred at room temperature for 16 h, and the
resulting solids filtered, rinsed with 10% MeOH/CHCl3, and dried
to yield 62.9 g (42%) of 4a (containing ꢀ8% of 4b) as a pale tan
powder. An additional 1.1 g of pure 4a was obtained upon
concentration of the filtrate: 1H NMR (DMSO-d6) d 1.34 (s, 2H),
3.47–3.66 (m, 4H), 5.04 (t, 2H), 7.03 (s, 2H), 7.18 (s, 1H), 8.81
(s, 1H).
4.2. Chemistry
4.2.1. General procedures
1,1-Bis(acetoxymethyl)-2-bromo-2-(bromomethyl)cyclopro-
pane (1, Scheme 1) was obtained according to literature proce-
dures;12 all other reagents and solvents were obtained from
commercial sources and used without additional purification.
Synthesis of (Z)-2-amino-6-chloro-9-{[2,2-bis(acetoxymethyl)cycl-
opropylidene]methyl}purine and (E)-2-amino-6-chloro-9-{[2,2-
bis(acetoxymethyl)cyclopropylidene]methyl}purine (3a/b). and
(Z)-2-amino-6-chloro-9-{[2,2-bis(hydroxymethyl)cyclopropylid-
ene]methyl}purine (4a) were carried out as large-scale modifica-
tions of the literature procedure12 as described below. Evaporation
of solvents was accomplished under reduced pressure (40–
60 mmHg), at less than 40 °C, unless otherwise noted. Chromatogra-
phy solvent systems are expressed in v/v ratios or as % vol. Melting
points were taken on EZ-Melt automated melting point apparatus
(Stanford Research Systems, Inc.) in manual mode, and are uncor-
rected. Thin-layer chromatography was performed on silica gel
GHLF plates from Analtech (Newark, DE). Chromatograms were
visualized under UV light at 254 nm. 1H NMR spectra were obtained
at 300 MHz on a Bruker DPX300 spectrometer. Chemical shift
values for 1H were determined relative to an internal tetramethylsil-
ane standard (0.00 ppm). High-resolution mass spectrometry
(HRMS) was performed at the Scripps Institute mass spectrometry
facility (La Jolla, CA). Analytical HPLC was performed at CreaGen
Biosciences (Woburn, MA). All compounds were found to be
P95% pure by analytical HPLC, except for 5a (90%), 5b (90%) and
5g (89%).
4.2.4. General method A; 2-amino-6-alkylaminopurine analogs
To
a solution of (Z)-2-amino-6-chloro-9-{[2,2-bis(hydroxy-
methyl)cyclopropylidene]methyl}purine (4a; 100 mg, 0.35 mmol)
in absolute EtOH (3 mL) was added the respective amine (0.5 mL,
excess). The mixture was heated to 85 °C for 2–48 h, then cooled
and evaporated under vacuum. The crude mixture was then puri-
fied by flash chromatography on silica gel with 0–8% MeOH/CHCl3.
Product-containing fractions were pooled and evaporated, and the
resulting solids recrystallized from hot CH3CN to provide the
desired product.
4.2.4.1.
(Z)-2-Amino-6-methylamino-9-{[2,2-bis(hydroxy-
Intermediate
methyl)cyclopropylidene]methyl}purine (5a).
4a was treated with a solution of methylamine in THF according
to method A for 16 h to provide 66 mg (68%) of 5a as a fluffy white
powder: mp 231–233 °C; Rf 0.09 (10% MeOH/CHCl3); 1H NMR
(DMSO-d6) d 8.37 (s, 1H), 7.3–7.1 (br s, 1H), 7.15 (s, 2H), 5.89 (s,
2H), 4.98 (t, 2H), 3.55 (ddd, 4H), 2.88 (br s, 3H), 1.29 (d, 2H);
HRMS (ESI) m/z calcd for C12H17N6O2 [M+H]+ 277.1407, found
277.1405.

3716
J. D. Williams et al. / Bioorg. Med. Chem. 20 (2012) 3710–3718
4.2.4.2.
(Z)-2-Amino-6-(1-propylamino)-9-{[2,2-bis(hydroxy-
Intermediate
line solid: mp 183–184 °C; Rf 0.50 (10% MeOH/CHCl3); 1H NMR
(DMSO-d6) d 8.40 (s, 1H), 7.16 (s, 1H), 6.92 (d, 1H), 5.85 (s, 2H),
5.00 (t, 2H), 4.07 (br s, 1H), 3.64 (dd, 2H), 3.51 (dd, 2H), 1.83–
1.72 (dd, 4H), 1.60 (d, 1H), 1.36–1.27 (m, 7H); HRMS (ESI) m/z calcd
for C17H25N6O2 [M+H]+ 345.2033, found 345.2031.
methyl)cyclopropylidene]methyl}purine (5b).
4a was treated with 1-aminopropane according to method A for
48 h to provide 80 mg (75%) of 5b as a fluffy white powder: mp
190–192 °C; Rf 0.18 (10% MeOH/CHCl3); 1H NMR (DMSO-d6) d
8.39 (s, 1H), 7.16 (s, 2H), 5.86 (s, 2H), 5.00 (t, 2H), 3.66 (dd, 2H),
3.62 (dd, 2H), 3.33 (m, 2H under H2O), 1.57 (q, 2H), 1.30 (s, 2H),
0.88 (t, 3H); 13C NMR (DMSO-d6) d 160.52, 155.00, 149.51,
134.15, 116.00, 112.91, 111.49/109.65, 65–60 (indistinct multi-
plet), 30.62, 25–20 (indistinct multiplet), 13–8 (indistinct multi-
plet); HRMS (ESI) m/z calcd for C14H21N6O2 [M+H]+ 305.1720,
found 305.1720.
4.2.4.9.
(Z)-2-Amino-6-(benzylamino)-9-{[2,2-bis(hydroxy-
Intermediate
methyl)cyclopropylidene]methyl}purine (5i).
4a was treated with benzylamine according to method A for 16 h
to provide 58 mg (46%) of 5i as a white powder: mp 175–176 °C;
Rf 0.30 (10% MeOH/CHCl3); 1H NMR (DMSO-d6) d 8.40 (s, 1H),
7.9–7.7 (br s, 1H), 7.33–7.15 (m, 6H), 5.7–5.8 (br s, 2H), 4.97 (t,
2H), 4.8–4.5 (br s, 2H), 3.62 (dd, 2H), 3.50 (dd, 2H), 1.28 (d, 2H);
HRMS (ESI) m/z calcd for C18H21N6O2 [M+H]+ 353.1720, found
353.1719.
4.2.4.3.
(Z)-2-Amino-6-(2-propylamino)-9-{[2,2-bis(hydroxy-
Intermediate
methyl)cyclopropylidene]methyl}purine (5c).
4a was treated with 2-aminopropane according to method A for
16 h to provide 66 mg (61%) of 5c as a white powder: mp 163–
165 °C; Rf 0.36 (10% MeOH/CHCl3); 1H NMR (DMSO-d6) d 8.40 (s,
1H), 7.16 (s, 1H), 6.95 (d, 1H), 5.86 (s, 2H), 5.00 (t, 2H), 4.35 (br
s, 1H), 3.64 (dd, 2H), 3.52 (dd, 2H), 1.30 (s, 2H), 1.17 (d, 6H); HRMS
(ESI) m/z calcd for C14H21N6O2 [M+H]+ 305.1720, found 305.1722.
4.2.4.10.
(Z)-2-Amino-6-dimethylamino-9-{[2,2-bis(hydroxy-
Intermediate
methyl)cyclopropylidene]methyl}purine (5j).
4a was treated with dimethylamine according to method A for
48 h to provide 82 mg (80%) of 5j as a white powder: mp 179–
181 °C; Rf 0.32 (10% MeOH/CHCl3); 1H NMR (DMSO-d6) d 8.46 (s,
1H), 7.19 (s, 1H), 5.90 (s, 2H), 4.99 (t, 2H), 3.65 (dd, 2H), 3.50
(dd, 2H), 3.35 (s, 6H, overlapping H2O peak), 1.30 (t, 2H); HRMS
(ESI) m/z calcd for C13H19N6O2 [M+H]+ 291.1564, found 291.1563.
4.2.4.4. (Z)-2-Amino-6-cyclopropylamino-9-{[2,2-bis(hydroxy-
methyl)cyclopropylidene]methyl}purine (5d).
Intermediate
4a was treated with cyclopropylamine according to method A for
16 h to provide 80 mg (76%) of 5d as a white powder: mp 172–
175 °C; Rf 0.21 (10% MeOH/CHCl3); 1H NMR (DMSO-d6) d 8.39 (s,
1H), 7.36 (s, 1H), 7.15 (s, 1H), 5.91 (s, 2H), 4.98 (s, 2H), 3.61 (d,
2H), 3.50 (d, 2H), 3.02 (s, 1H), 1.4–1.2 m, 2H), 0.7–0.5 (m, 4H);
HRMS (ESI) m/z calcd for C14H19N6O2 [M+H]+ 303.1564, found
303.1566.
4.2.4.11. (Z)-2-Amino-6-(pyrrolidin-1-yl)-9-{[2,2-bis(hydroxy-
methyl)cyclopropylidene]methyl}purine (5k).
Intermediate
4a was treated with pyrrolidine according to method A for 16 h to
provide 60 mg (54%) of 5k as a white powder: mp 217–218 °C; Rf
0.34 (10% MeOH/CHCl3); 1H NMR (DMSO-d6) d 8.43 (s, 1H), 7.18
(s, 1H), 5.88 (s, 2H), 5.00 (t, 2H), 3.95 (br s, 2H), 3.67–3.63 (m,
3H), 3.55–3.49 (m, 3H), 1.90 (br s, 4H), 1.30 (s, 2H); HRMS (ESI)
m/z calcd for C15H21N6O2 [M+H]+ 317.1720, found 317.1719.
4.2.4.5.
(Z)-2-Amino-6-(1-butylamino)-9-{[2,2-bis(hydroxy-
Intermediate
methyl)cyclopropylidene]methyl}purine (5e).
4a was treated with 1-aminobutane according to method A for
48 h to provide 62 mg (55%) of 5e as a fluffy white powder: mp
206–209 °C; Rf 0.19 (10% MeOH/CHCl3); 1H NMR (DMSO-d6) d
8.39 (s, 1H), 7.16 (br s, 2H), 5.86 (br s, 2H), 5.00 (t, 2H), 3.64 (dd,
2H), 3.52 (dd, 2H), 3.41 (br s, 2H), 1.57–1.50 (m, 2H), 1.36–1.30
(m, 4H), 0.90 (t, 3H); HRMS (ESI) m/z calcd for C15H23N6O2
[M+H]+ 319.1877, found 319.1874.
4.2.4.12. (Z)-2-Amino-6-(morpholin-4-yl)-9-{[2,2-bis(hydroxy-
methyl)cyclopropylidene]methyl}purine (5l).
Intermediate
4a was treated with morpholine according to method A for 16 h
to provide 60 mg (51%) 5l as light brown micro-crystalline solid:
mp 214–215 °C; Rf 0.38 (10% MeOH/CHCl3); 1H NMR (DMSO-d6)
d 8.50 (s, 1H), 7.19 (s, 1H), 6.01 (s, 2H), 4.98 (t, 2H), 4.09 (s, 4H),
3.69–3.62 (m, 6H), 3.54–3.48 (m, 2H), 1.30 (s, 2H); HRMS (ESI)
m/z calcd for C15H21N6O3 [M+H]+ 333.1670, found 333.1672.
4.2.4.6.
(Z)-2-Amino-6-isobutylamino-9-{[2,2-bis(hydroxy-
Intermediate
methyl)cyclopropylidene]methyl}purine (5f).
4.2.5. General method B; 2-amino-6-alkoxypurine analogs
A solution of the respective alcohol (1 mL) in DMF (1 mL) was
treated with sodium hydride (67 mg, 1.67 mmol, 4.2 eq) at room
temperature and stirred for 30 minutes. (Z)-2-amino-6-chloro-9-
4a was treated with isobutylamine according to method A for
48 h to provide 62 mg (55%) of 5f as a fluffy white powder: mp
190–192 °C; Rf 0.18 (10% MeOH/CHCl3); 1H NMR (DMSO-d6) d
8.40 (s, 1H), 7.22 (br s, 1H), 7.17 (s, 1H), 5.85 (s, 2H), 5.00 (t, 2H),
3.67 (dd, 2H), 3.52 (dd, 2H), 3.4–3.1 (br s, 2H), 1.93 (quint, 1H),
1.30 (s, 2H), 0.88 (d, 6H); HRMS (ESI) m/z calcd for C15H23N6O2
[M+H]+ 319.1877, found 319.1877.
{[2,2-bis(hydroxymethyl)cyclopropylidene]methyl}purine
(4a;
100 mg, 0.35 mmol) was added in one portion, and the resulting
mixture was heated to 70 °C for 4–48 h. The reaction was then
cooled to room temperature, and glacial acetic acid (200 lL) was
added. The crude mixture was adsorbed onto silica gel (12 g),
and suction was applied for 16 h. The product was eluted with
20% MeOH/CH2Cl2, and evaporated to provide an oil that was fur-
ther purified by flash chromatography on silica gel with 0–10%
MeOH/CHCl3. Product-containing fractions were pooled and evap-
orated, and the resulting solids recrystallized from hot CH3CN to
provide the desired product.
4.2.4.7. (Z)-2-Amino-6-(cyclopentylamino)-9-{[2,2-bis(hydroxy-
methyl)cyclopropylidene]methyl}purine (5g).
Intermediate
4a was treated with cyclopentylamine according to method A for
16 h to provide 101 mg (88%) of 5g as an off-white solid: mp
110–113 °C; Rf 0.35 (10% MeOH/CHCl3); 1H NMR (DMSO-d6) d
8.39 (s, 1H), 7.15 (t, 1H), 7.08 (d, 1H), 5.84 (s, 2H), 4.98 (t, 2H),
4.6–4.4 (s, 1H), 3.62 (dd, 2H), 3.50 (dd, 2H), 2.0–1.8 (m, 2H), 1.8–
1.6 (m, 2H), 1.6–1.4 (m, 4H), 1.28 (d, 2H); HRMS (ESI) m/z calcd
for C16H23N6O2 [M+H]+ 331.1877, found 331.1877.
4.2.5.1. (Z)-2-Amino-6-ethoxy-9-{[2,2-bis(hydroxymethyl)cyclo-
propylidene]methyl}purine (6a).
Intermediate 4a was trea-
ted with ethanol according to method B for 48 h to provide
45 mg (44%) of 6a as an off-white powder: mp 182–187 °C; Rf
0.19 (5% MeOH/CHCl3); 1H NMR (DMSO-d6) d 8.59 (s, 1H), 7.21
(s, 1H), 6.51 (s, 2H), 5.02 (t, 2H), 4.47 (q, 2H), 3.67 (dd, 2H),
3.52(dd, 2H), 1.4–1.33 (m, 5H); 13C NMR (DMSO-d6) d 160.35,
4.2.4.8. (Z)-2-Amino-6-(cyclohexylamino)-9-{[2,2-bis(hydroxy-
methyl)cyclopropylidene]methyl}purine (5h).
Intermediate
4a was treated with cyclohexylamine according to method A for
16 h to provide 83 mg (68%) of 5h as a pale-yellow chunky crystal-

J. D. Williams et al. / Bioorg. Med. Chem. 20 (2012) 3710–3718
3717
160.11, 152.51, 136.51, 116.99, 113.42, 110.26, 62.17, 61.67, 30.62,
NMR (DMSO-d6) d 8.69 (s, 1H), 7.22 (s, 1H), 6.75 (s, 2H), 5.17 (q,
14.58, 10.99; HRMS (ESI) m/z calcd for
C
13H18N5O3 [M+H]+
2H), 5.05 (s, 2H), 3.68 (d, 2H), 3.52 (d, 2H), 1.35 (s, 2H); HRMS
292.1404, found 292.1403.
(ESI) m/z calcd for C
13H15F3N5O3 [M+H]+ 346.1121, found
346.1121.
4.2.5.2.
(Z)-2-Amino-6-(1-propyloxy)-9-{[2,2-bis(hydroxy-
Intermediate
methyl)cyclopropylidene]methyl}purine (6b).
4.2.5.9. (Z)-2-Amino-6-(2-methoxyethoxy)-9-{[2,2-bis(hydroxy-
methyl)cyclopropylidene]methyl}purine (6i). Intermediate
4a was treated with 1-propanol according to method B for 24 h to
provide 55 mg (51%) of 6b as a white powder: mp 195–202 °C; Rf
0.17 (5% MeOH/CHCl3); 1H NMR (DMSO-d6) d 8.58 (s 1H), 7.2 (s,
1H), 6.48 (s, 2H), 5.00 (t, 2H), 4.37 (t, 2H), 3.66 (dd, 2H), 3.51 (dd,
2H), 1.77 (q, 2H), 1.33 (s, 2H), 0.97 (t, 3H); HRMS (ESI) m/z calcd
for C14H20N5O3 [M+H]+ 306.1561, found 306.1561.
4a was treated with 2-methoxyethanol according to method B
for 24 h to provide 86 mg (75%) of 6i as an off-white powder: mp
210–213 °C; Rf 0.20 (5% MeOH/CHCl3); 1H NMR (300 MHz-DMSO-
d6) d 8.59 (s, 1H), 7.20 (s, 1H), 6.53 (s, 2H), 5.01 (s, 2H), 4.54 (s,
2H), 3.8–3.6 (m, 4H), 3.6–3.4 (m, 2H), 3.31 (s, 3H, overlaps H2O
peak), 1.33 (s, 2H); HRMS (ESI) m/z calcd for C14H20N5O4 [M+H]+
322.1510, found 322.1508.
4.2.5.3.
(Z)-2-Amino-6-allyloxy-9-{[2,2-bis(hydroxy-
Intermediate
methyl)cyclopropylidene]methyl}purine (6c).
4a was treated with allyl alcohol according to method B for 24 h
to provide 60 mg (46%) of 6c as a white powder: mp 203–206 °C;
Rf 0.20 (5% MeOH/CHCl3); 1H NMR (DMSO-d6) d 8.61 (s, 1H), 7.21
(s, 1H), 6.53 (s, 2H), 6.17–6.06 (m, 1H), 5.42 (d, 1H), 5.28 (d, 1H),
5.02 (t, 2H), 4.97 (d, 2H), 3.67 (dd, 2H), 3.54 (dd, 2H), 1.34 (s,
2H); HRMS (ESI) m/z calcd for C14H18N5O3 [M+H]+ 304.1404, found
304.1404.
4.2.5.10.
(Z)-2-Amino-6-benzyloxy-9-{[2,2-bis(hydroxy-
Intermediate
methyl)cyclopropylidene]methyl}purine (6j).
4a was treated with benzyl alcohol according to method B for
24 h to provide 55 mg (44%) of 6j as a white powder: mp 182–
187 °C; Rf 0.19 (5% MeOH/CHCl3); 1H NMR (DMSO-d6) d 8.61 (s,
1H), 7.52 (d, 2H), 7.44–7.36 (m, 3H), 7.22 (s, 1H), 6.60 (s, 2H),
5.52 (s, 2H), 5.02 (t, 2H), 3.67 (dd, 2H), 3.52 (dd, 2H), 1.34 (s,
2H); HRMS (ESI) m/z calcd for C18H20N5O3 [M+H]+ 354.1561, found
354.1561.
4.2.5.4.
(Z)-2-Amino-6-(1-butyloxy)-9-{[2,2-bis(hydroxy-
Intermediate
methyl)cyclopropylidene]methyl}purine (6d).
4a was treated with 1-butanol according to method B for 4 h to
provide 66 mg (58%) of 6d as an off-white microcrystalline solid:
mp 210–212 °C; Rf 0.17 (5% MeOH/CHCl3); 1H NMR (DMSO-d6) d
8.58 (s, 1H), 7.20 (s, 1H), 6.48 (s, 2H), 5.01 (t, 2H), 4.42 (t, 2H),
3.66 (dd, 2H), 3.51 (dd, 2H), 1.73 (quint, 2H), 1.46–1.40 (m, 2H),
1.32 (s, 2H), 0.94 (t, 3H); HRMS (ESI) m/z calcd for C15H22N5O3
[M+H]+ 320.1717, found 320.1717.
4.2.6. General method C; 2-amino-6-thioalkylpurine analogs
To
a solution of (Z)-2-amino-6-chloro-9-{[2,2-bis(hydroxy-
methyl)cyclopropylidene]methyl}purine (4a; 100 mg, 0.35 mmol)
and the respective thiol in DMF (1 mL) was added 1.0 M aq. NaOH,
(1.0 mL, 1.0 mmol). The reaction mixture was stirred at room tem-
perature for 0.5–24 h, then glacial AcOH (200 lL) was added in one
portion. The crude mixture was adsorbed onto silica gel (12 g), and
suction was applied for 16 h. The product was eluted with 20%
MeOH/CH2Cl2, and evaporated to provide an oil that was further
purified by flash chromatography on silica gel with 0–10%
MeOH/CHCl3. Product-containing fractions were pooled and evap-
orated, and the resulting solids recrystallized from hot CH3CN/H2O
to provide the desired product.
4.2.5.5.
(Z)-2-Amino-6-(1-pentyloxy)-9-{[2,2-bis(hydroxy-
Intermediate
methyl)cyclopropylidene]methyl}purine (6e).
4a was treated with 1-pentanol according to method B for
30 min to provide 73 mg (62%) of 6e as a white powder: mp
148–149 °C; Rf 0.32 (10% MeOH/CHCl3); 1H NMR (DMSO-d6) d
8.58 (s, 1H), 7.20 (s, 1H), 6.47 (s, 2H), 5.00 (t, 2H), 4.41 (t, 2H),
3.67 (dd, 2H), 3.51 (dd, 2H), 1.76 (t, 2H), 1.37–1.33 (m, 6H), 0.90
(t, 3H); HRMS (ESI) m/z calcd for C16H24N5O3 [M+H]+ 334.1874,
found 334.1876.
4.2.6.1.
(Z)-2-Amino-6-methylthio-9-{[2,2-bis
(hydroxy-
methyl)cyclopropylidene]methyl}purine (7a).
Intermediate
4a was treated with sodium thiomethoxide (145 mg, 2.1 mmol)
according to a modification of method C in which no sodium
hydroxide was added. The reaction proceeded for 30 min to pro-
vide 66 mg (58%) of 7a as an off-white powder: mp 146–147 °C;
Rf 0.28 (10% MeOH/CHCl3); 1H NMR (DMSO-d6) d 8.65 (s, 1H),
7.20 (s, 1H), 6.59 (s, 2H), 4.99 (t, 2H), 3.65 (dd, 2H), 3.51 (dd,
2H), 2.58 (s, 3H), 1.34 (s, 2H); 13C NMR (DMSO-d6) d 160.16,
159.85, 149.11, 138.81, 123.843, 117.56, 111.26, 62.12, 30.69,
4.2.5.6.
(Z)-2-Amino-6-(1-hexyloxy)-9-{[2,2-bis(hydroxy-
Intermediate
methyl)cyclopropylidene]methyl}purine (6f).
4a was treated with 1-hexanol according to method B for 4 h to
provide 56 mg (45%) of 6f as a white powder: mp 142–143 °C; Rf
0.20 (5% MeOH / CHCl3); 1H NMR (DMSO-d6) d 8.58 (s, 1H), 7.20
(s, 1H), 6.48 (s, 2H), 5.00 (t, 2H), 4.41 (t, 2H), 3.66 (dd, 2H), 3.51
(dd, 2H), 1.75 (t, 2H), 1.5–1.3 (m, 8H), 0.88 (t, 3H); HRMS (ESI)
m/z calcd for C17H26N5O3 [M+H]+ 348.2030, found 348.2031.
11.79, 9.91; HRMS (ESI) m/z calcd for
C
12H16N5O2S [M+H]+
294.1019, found 294.1018.
4.2.5.7.
(Z)-2-Amino-6-(1-octyloxy)-9-{[2,2-bis(hydroxy-
Intermediate
4.2.6.2.
(Z)-2-Amino-6-(1-propylthio)-9-{[2,2-bis(hydroxy-
Intermediate
methyl)cyclopropylidene]methyl}purine (6g).
methyl)cyclopropylidene]methyl}purine (7b).
4a was treated with 1-octanol according to method B for 4 h to
provide 67 mg (50%) of 6g as a white powder: mp 148–150 °C; Rf
0.20 (5% MeOH/CHCl3); 1H NMR (DMSO-d6) d 8.58 (s, 1H), 7.20
(s, 1H), 6.48 (s, 2H), 5.00 (t, 2H), 4.40 (t, 2H), 3.66 (dd, 2H), 3.51
(dd, 2H), 1.75 (t, 2H), 1.5–1.3 (m, 12H), 0.86 (t, 3H); HRMS (ESI)
m/z calcd for C19H30N5O3 [M+H]+ 376.2343, found 376.2343.
4a was treated with 1-propanethiol (0.13 mL, 1.4 mmol) according
to method C for 30 min to provide 66 mg (58%) of 7b as an off-
white powder: mp 212–214 °C; Rf 0.25 (10% MeOH/CHCl3); 1H
NMR (DMSO-d6) d 8.65 (s, 1H), 7.20 (s, 1H), 6.57 (s, 2H), 5.00 (t,
2H), 3.67 (dd, 2H), 3.51 (dd, 2H), 3.28 (t, 2H), 1.69 (q, 2H), 1.34
(s, 2H), 1.00 (t, 3H); HRMS (ESI) m/z calcd for C14H20N5O2S
[M+H]+ 322.1332, found 322.1331.
4.2.5.8.
bis(hydroxymethyl)cyclopropylidene]methyl}purine
(6h). Intermediate 4a was treated with 2,2,2,-trifluoroethanol
(Z)-2-Amino-6-(2,2,2-trifluoroethoxy)-9-{[2,2-
4.2.6.3.
(Z)-2-Amino-6-(2-propylthio)-9-{[2,2-bis(hydroxy-
Intermediate
methyl)cyclopropylidene]methyl}purine (7c).
according to method B for 24 h to provide 50 mg (41%) of 6h as a
4a was treated 2-butanethiol (0.13 mL, 1.4 mmol) according to
method C for 30 min to provide 60 mg (65%) of 7c as an off-white
white powder: mp 182–192 °C; Rf 0.20 (5% MeOH/CHCl3); 1H

3718
J. D. Williams et al. / Bioorg. Med. Chem. 20 (2012) 3710–3718
powder: mp 163–183 °C; Rf 0.25 (10% MeOH/CHCl3); 1H NMR
(DMSO-d6) d 8.65 (s, 1H), 7.20 (s, 1H), 6.58 (s, 2H), 5.01 (t, 2H),
4.28 (quint, 1H), 3.67 (dd, 2H), 3.50 (dd, 2H), 1.39 (d, 6H), 1.33
(s, 2H); HRMS (ESI) m/z calcd for C14H20N5O2S [M+H]+ 322.1332,
found 322.1330.
4.2.6.9.
(Z)-2-Amino-6-benzylthio-9-{[2,2-bis(hydroxy-
Intermediate
methyl)cyclopropylidene]methyl}purine (7i).
4a was treated with benzyl mercaptan (0.17 mL, 1.4 mmol) accord-
ing to method C for 30 min to provide 74 mg (56%) of 7i as a white
powder: mp 202–204 °C; Rf 0.26 (10% MeOH/CHCl3); 1H NMR
(DMSO-d6) d 8.65 (s, 1H), 7.48 (d, 2H), 7.33–7.20 (m, 4H), 6.69 (s,
2H), 4.98 (t, 2H), 4.56 (s, 2H), 3.66 (dd, 2H), 3.50 (dd, 2H), 1.34
(s, 2H); HRMS (ESI) m/z calcd for C18H20N5O2S [M+H]+ 370.1332,
found 370.1333.
4.2.6.4.
(Z)-2-Amino-6-(1-butylthio)-9-{[2,2-bis(hydroxy-
Intermediate
methyl)cyclopropylidene]methyl}purine (7d).
4a was treated with 1-butanethiol (0.15 mL, 1.4 mmol) according
to method C for 30 min to provide 32 mg (13%) of 7d as an off-
white powder: mp 185–195 °C; Rf 0.23 (10%MeOH/CHCl3); 1H MR
(DMSO-d6) d 8.64 (s, 1H), 7.20 (s, 1H), 6.55 (s, 2H), 4.99 (t, 2H),
3.67 (dd, 2H), 3.53 (dd, 2H), 3.27 (t, 2H, overlapping H2O peak),
1.65 (quint, 2H), 1.42 (quint, 2H), 1.33 (s, 2H), 0.91 (t, 3H); HRMS
(ESI) m/z calcd for C15H22N5O2S [M+H]+ 336.1489, found 336.1489.
Acknowledgments
This work was supported by National Institutes of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID) con-
tracts N01-AI-30049, and HHSN2722011000010C (M.N.P) and
Grant 1R43AI082799.
4.2.6.5.
(Z)-2-Amino-6-isobutylthio-9-{[2,2-bis(hydroxy-
Intermediate
methyl)cyclopropylidene]methyl}purine (7e).
Supplementary data
4a was treated with 2-methyl-1-propanethiol (0.15 mL, 1.4 mmol)
according to method C for 30 min to provide 26 mg (11%) of 7e as
an off-white powder: mp 174–177 °C; Rf 0.28 (10% MeOH/CHCl3);
1H NMR (DMSO-d6) d 8.64 (s, 1H), 7.19 (s, 1H), 6.54 (s, 2H), 4.99 (t,
2H), 4.18 (q, 1H), 3.67 (dd, 2H), 3.50 (dd, 2H), 1.69 (quint, 2H),
1.38–1.33 (m, 5H), 0.99 (t, 3H); HRMS (ESI) m/z calcd for
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.04.049.
References and notes
C
15H22N5O2S [M+H]+ 336.1489, found 336.1488.
1. Human Herpesviruses: Biology, Therapy, and Immunoprophylaxis; Arvin, A.,
Campadelli-Fiume, G., Mocarski, E., Moore, P. S., Roizman, B., Whitley, R.,
Yamanishi, K., Eds.; Cambridge University Press: Cambridge, 2007.
2. The Human Herpesviruses; Roizman, B., Whitley, R. J., Lopez, C., Eds.; Raven
Press: New York, 1993.
3. Pellett, P. E.; Roizman, B. The Family Herpesviridae: A Brief Introduction. In
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A., Roizman, B., Straus, S. E., Eds., 5th ed.; Lippincott Williams & Wilkins:
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4. O’Brien, J. J.; Campoli-Richards, D. M. Drugs 1989, 37, 233.
5. Beutner, K. R. Antiviral Res. 1995, 28, 281.
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4.2.6.6.
(Z)-2-Amino-6-cyclopentylthio-9-{[2,2-bis(hydroxy-
Intermediate
methyl)cyclopropylidene]methyl}purine (7f).
4a was treated with cyclopentanethiol (0.15 mL, 1.4 mmol) accord-
ing to method C for 30 min to provide 80 mg (65%) of 7f as a white
powder: mp 188–190 °C; Rf 0.24 (10% MeOH/CHCl3); 1H NMR
(DMSO-d6) d 8.64 (s, 1H), 7.20 (s, 1H), 6.55 (s, 2H), 4.99 (t, 2H),
4.29 (t, 1H), 3.67 (dd, 2H), 3.50 (dd, 2H), 2.4–2.1 (m, 2H), 1.9–1.5
(m, 6H), 1.34 (s, 2H); HRMS (ESI) m/z calcd for C16H22N5O2S
[M+H]+ 348.1489, found 348.1489.
9. Wagstaff, A. J.; Bryson, H. M. Drugs 1994, 48, 199.
10. Zemlicka, J. Methylenecyclopropane Analogues of Nucleosides as Anti-herpes
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11. Qiu, Y. L.; Ksebati, M. B.; Ptak, R. G.; Fan, B. Y.; Breitenbach, J. M.; Lin, J. S.;
Cheng, Y. C.; Kern, E. R.; Drach, J. C.; Zemlicka, J. J. Med. Chem. 1998, 41, 10.
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Zhou, S.; Zemlicka, J.; Prichard, M. N. Antimicrob. Agents Chemother. 2005, 49,
1039.
14. Chen, X.; Kern, E. R.; Drach, J. C.; Gullen, E.; Cheng, Y. C.; Zemlicka, J. J. Med.
Chem. 2003, 46, 1531.
15. Kushner, N. L.; Williams, S. L.; Hartline, C. B.; Harden, E. A.; Bidanset, D. J.; Chen,
X.; Zemlicka, J.; Kern, E. R. Nucleosides, Nucleotides Nucleic Acids 2003, 22, 2105.
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Antimicrob. Agents Chemother. 2004, 48, 4745.
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4.2.6.7.
(Z)-2-Amino-6-(1-hexylthio)-9-{[2,2-bis(hydroxy-
Intermediate
methyl)cyclopropylidene]methyl}purine (7g).
4a was treated with 1-hexanethiol (0.20 mL, 1.4 mmol) according
to method C for 30 min to provide 48 mg (55%) of 7g as an off-
white powder: mp 148–150 °C; Rf 0.23 (10% MeOH/CHCl3); 1H
NMR (DMSO-d6) d 8.65 (s, 1H), 7.20 (s, 1H), 6.55 (s, 2H), 5.00 (t,
2H), 3.67 (dd, 2H); 3.51 (dd, 2H), 3.28 (t, 2H), 1.66 (quint, 2H),
1.5–1.2 (m, 8H), 0.87 (t, 3H); HRMS (ESI) m/z calcd for C17H26N5O2S
[M+H]+ 364.1802, found 364.1800.
4.2.6.8.
(Z)-2-Amino-6-(1-octylthio)-9-{[2,2-bis(hydroxy-
Intermediate
methyl)cyclopropylidene]methyl}purine (7h).
4a was treated with 1-octanethiol (0.26 mL, 1.4 mmol) according
to method C for 30 min to provide 89 mg (32%) of 7h as a white
powder: mp 136–137 °C; Rf 0.28 (10% MeOH/CHCl3); 1H NMR
(DMSO-d6) d 8.64 (s, 1H), 7.20 (s, 1H), 6.55 (s, 2H), 4.99 (t, 2H),
3.67 (dd, 2H), 3.50 (dd, 2H), 3.27 (t, 2H, overlapping H2O peak),
1.66 (quint, 2H), 1.5–1.2 (m, 12H), 0.85 (t, 3H); HRMS (ESI) m/z
calcd for C19H30N5O2S [M+H]+ 392.2115, found 392.2116.
21. Prichard, M. N.; Daily, S. L.; Jefferson, G. M.; Perry, A. L.; Kern, E. R. J. Virol.
Methods 2007, 144, 86.