Fluorinated methylenecyclopropane analogues of nucleosides. Synthesis and antiviral activity of (Z)- and (E)-9-{[(2-fluoromethyl-2-hydroxymethyl)-cyclopropylidene]methyl}adenine and -guanine

Article abstract of DOI:10.1016/j.bmc.2007.11.082

Synthesis and antiviral activity of the title fluoromethylenecyclopropane analogues 15a, 15b, 16a, and 16b is described. Methylenecyclopropane carboxylate was first transformed to 2,2-bis-hydroxymethylmethylenecyclopropane. Selective monoacetylation followed by introduction of fluorine gave 2-acetoxymethyl-2-fluoromethylmethylenecyclopropane as the key intermediate. The synthesis of analogues 15a, 15b, 16a, and 16b then followed alkylation-elimination procedure as described previously for other methylenecyclopropane analogues. The adenine Z-isomer 15a was found to be a potent inhibitor of Epstein-Barr virus (EBV) in vitro with EC₅₀/CC₅₀ (μM) 0.5/55.7. Compounds 15b, 16a, and 16b were also active but at higher concentrations, EC₅₀/CC₅₀ (μM) 3.2-7.5/53.6-64.1. Analogue 15a inhibited hepatitis C virus by virtue of its cytotoxicity and it moderately inhibited replication of the Towne strain of human cytomegalovirus (HCMV). The E-isomer 16a was a substrate for adenosine deaminase, whereas the Z-isomer 15a was not deaminated.

Full text of DOI:10.1016/j.bmc.2007.11.082

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 2148–2155
Fluorinated methylenecyclopropane analogues of nucleosides.
Synthesis and antiviral activity of (Z)- and
(E)-9-{[(2-fluoromethyl-2-hydroxymethyl)-
cyclopropylidene]methyl}adenine and -guanine
Chengwei Li,a Mark N. Prichard,b Brent E. Korba,c John C. Drachd and Jiri Zemlickaa,*
aDevelopmental Therapeutics Program, Barbara Ann Karmanos Cancer Institute,
Wayne State University School of Medicine, Detroit, MI 48201-1379, USA
bDepartment of Pediatrics, The University of Alabama School of Medicine, Birmingham, AL 35233, USA
cDivision of Molecular Biology and Immunology, Georgetown University Medical Center, Rockville, Maryland, MD 20850, USA
dDepartment of Biologic and Materials Sciences, School of Dentistry, University of Michigan, Ann Arbor, MI 48019-1078, USA
Received 19 October 2007; revised 30 November 2007; accepted 30 November 2007
Available online 5 December 2007
Abstract—Synthesis and antiviral activity of the title fluoromethylenecyclopropane analogues 15a, 15b, 16a, and 16b is described.
Methylenecyclopropane carboxylate was first transformed to 2,2-bis-hydroxymethylmethylenecyclopropane. Selective monoacetyla-
tion followed by introduction of fluorine gave 2-acetoxymethyl-2-fluoromethylmethylenecyclopropane as the key intermediate. The
synthesis of analogues 15a, 15b, 16a, and 16b then followed alkylation–elimination procedure as described previously for other
methylenecyclopropane analogues. The adenine Z-isomer 15a was found to be a potent inhibitor of Epstein–Barr virus (EBV)
in vitro with EC50/CC50 (lM) 0.5/55.7. Compounds 15b, 16a, and 16b were also active but at higher concentrations, EC50/CC50
(lM) 3.2–7.5/53.6–64.1. Analogue 15a inhibited hepatitis C virus by virtue of its cytotoxicity and it moderately inhibited replication
of the Towne strain of human cytomegalovirus (HCMV). The E-isomer 16a was a substrate for adenosine deaminase, whereas the
Z-isomer 15a was not deaminated.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
or pyrimidine Z- and E-2-fluoro analogues 5 and 6 were
effective against HCMV, EBV or varicella zoster virus
(VZV).7 Purine 3-fluoroanalogues 7, 8, 9, and 10 had
more narrow antiviral effects or they were less potent.8
This trend was also reflected in the bis(2,2-hydroxy-
methyl)-3-fluoro derivatives9 11 and 12.
The Z-methylenecyclopropane analogues of purine
nucleosides 1 and 2 are effective antiviral agents,
whereas the E-isomers 3 and 4 (Chart 1) are either inac-
tive or of limited potency.1–3 The guanine analogue 2b
(cyclopropavir) is currently under preclinical investiga-
tion as a possible drug against infections caused by hu-
man cytomegalovirus (HCMV).4,5 It is also effective
in vitro6 against Epstein–Barr virus (EBV) and human
herpes viruses HHV-6 and HHV-8. Structure–activity
relationship (SAR) studies have indicated that introduc-
tion of fluorine into the cyclopropane moiety of 1 and 3
can also provide new antiviral agents. Thus, purine and/
Fluorine can mimic both a hydrogen atom and a hydro-
xy group because of its small van der Waals radius and
polarity of the carbon–fluorine bond.10 Although all
possible monofluoromethylenecyclopropane analogues
(5–12) derived by replacement of hydrogens of the cyclo-
propane moiety were investigated,7–9 compounds having
the hydroxy group(s) replaced with fluorine have not
been described. Similar fluoro analogue of ganciclovir
13 exhibited activity11 against herpes simplex virus 1
(HSV-1). Because cyclopropavir 2b can be regarded as
a rigid bioisostere of anti-HCMV drug ganciclovir4 14
it was of interest to synthesize and investigate biological
activity of purine fluoromethylenecyclopropane ana-
logues 15a, 15b, 16a, and 16b.
Keywords: Methylenecyclopropanes; Nucleoside analogues; Alkyl-
ation–elimination; Methylenecyclopropane–methylenecyclobutane
rearrangement; Antiviral agents; Adenosine deaminase.
*
Corresponding author. Tel.: +1 313 578 4288; fax: +1 313 832
7294; e-mail: zemlicka@karmanos.org
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.11.082

C. Li et al. / Bioorg. Med. Chem. 16 (2008) 2148–2155
2149
Chart 1.
2. Results and discussion
21 but it led instead to a ring-expanded fluorocyclobu-
tane 22 as the only product in 87% yield. It is impor-
tant to note that this is a new synthesis of
2.1. Synthesis
methylenefluorocyclobutane skeleton. The parent com-
pound17 is accessible only by reaction of [1.1.1]propel-
lane with XeF2. Recently, ring expansion of prolinols
to fluoropiperidines effected by DAST was described.18
Nevertheless, the reaction course was not uniform and
ratio of five-membered to six-membered products was
about 2:3.
Methylenecyclopropane diol 17 was chosen as a conve-
nient starting material for synthesis of analogues 15
and 16 (Scheme 1). For the present purpose, compound
17 was obtained by an alternate approach. Methylene-
cyclopropane carboxylate12 18 was reduced using a less
than a stoichiometric amount of diisobutylaluminum
hydride (DIBALH). The intermediary aldehyde 19
was not isolated but it was subjected in situ to aldol
and crossed Cannizzaro reaction with formaldehyde
to give diol 17 in 64% yield. It should be noted that
this is a new synthesis of an important intermediate
for cyclopropavir (2b).4,13,14 Acetylation of 17 via the
corresponding cyclic orthoester13 gave monoacetate
20 in 78% yield. Reaction of 20 with diethylaminosul-
fur trifluoride (DAST)15,16 using pyridine in CH2Cl2
at ꢀ78 ꢁC did not give the expected fluoro derivative
The reaction is initiated by transformation of 20 by
DAST to intermediate 23 (Scheme 2). In the next step,
a non-classical19 cyclopropylmethyl carbocation 24 exist-
ing in equilibrium with cyclobutonium ion 25 reacts with
fluoride ion to give methylenefluorocyclobutane 22. The
reported solvolysis20 of methylenecyclopropylmethyl
chloride and deamination21 of methylenecyclopropylm-
ethylamine led also to methylenecyclobutanes in addition
to methylenecyclopropyl methyl derivatives.
Scheme 1. Reagents and conditions: (a) DIBALH, CH2Cl2, ꢀ78 ꢁC; (b) 1—37% CH2O, MeOH; 2—1 M HCl, D; (c) 1—MeC(OMe)3, cat. TSOH,
CH2Cl2; 2—NEt3; 3—80% AcOH; (d) DAST, pyridine, CH2Cl2, ꢀ78 ꢁC to rt; (e) MsCl, NEt3, CH2Cl2; (f) Bu4NF, THF; (g) PyridineÆHBr3, CH2Cl2,
0 ꢁC; (h) B–H, K2CO3, DMF, D; (i) K2CO3, MeOH–H2O (9:1), rt or 0 ꢁC; (j) 1—80% HCO2H, D; 2—NH3, MeOH, 0 ꢁC.

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C. Li et al. / Bioorg. Med. Chem. 16 (2008) 2148–2155
Scheme 2.
0
C3 shifts. The final confirmation of the Z- and E-iso-
It was then clear that avoiding formation of intermedi-
ary carbocation might lead to a successful synthesis of
methylenefluorocyclopropane 21. Therefore, monoace-
tate 20 was converted to methylsulfonate 26 (81%) using
methylsulfonyl chloride (MsCl) which, in turn, was
smoothly transformed to fluorocyclopropane 21 (72%)
using tetrabutylammonium fluoride (NBu4F) in THF.
Addition of bromine via pyridinium tribromide gave di-
bromo derivative 27 which was used for alkylation elim-
ination1–3 of nucleic acid heterocycles. The reaction of
27 with adenine gave the Z- and E-isomeric mixture
methylenecyclopropanes 28a in 65% yield. The yield of
28c obtained with 2-amino-6-chloropurine was lower
(46%). Deacetylation of 28a using K2CO3 in 90% aque-
ous methanol at room temperature furnished the target
analogues 15a and 16a after chromatographic separa-
tion in 49% and 43% yield, respectively. In a similar
fashion, deacetylation of intermediate 28c at 0 ꢁC affor-
ded the Z- and E-isomers 15c and 16c (46% and 54%).
Hydrolytic dechlorination of 15c and 16c using 80% for-
mic acid at 80 ꢁC provided guanine analogues 15b and
16b (84% and 91%).
meric assignment came from the NOE experiments per-
formed with adenine analogues 15a and 16a (Table 2).
In the Z-isomer 15a, the NOE enhancements were found
0
0
between the cis-arranged H1 and H3 protons as well as
between the H8 and protons of OH, CH2F, and CH2O
groups. By contrast, in the E-isomer 16a a strong
0
NOE interaction occurs between the cis-located H3
and H8. Also, the NOE enhancements were found be-
0
tween the H3 and OH, CH2F, and CH2O groups.
2.3. Antiviral activity
Compounds 15a, 15b, 16a, and 16b were tested against
the following viruses: herpes simplex virus 1 and 2
(HSV-1 and HSV-2), human cytomegalovirus (HCMV,
Towne and AD 169 strains), varicella zoster virus
(VZV), Epstein–Barr virus (EBV), human immunodefi-
ciency virus (HIV-1), hepatitis B and C virus (HBV
and HCV). They were all effective against EBV in Akata
cells using a DNA hybridization assay.22 The adenine
analogue 15a was the most potent (Table 3) and least
cytotoxic. It was more effective than cyclopropavir
(2b). The E-isomer 16a and guanine derivatives 15b
and 16b were less effective than 15a. Interestingly, a
somewhat similar anti-EBV activity pattern was found
with Z- and E-isomers of fluoroanalogues7 5a, 5b, 6a,
and 6b which can be regarded as lower homologues of
2.2. The Z- and E-isomeric assignment
As in previous cases of methylenecyclopropane ana-
logues,2,4 the NMR spectroscopy was indispensable to
confirm the Z- and E-isomeric structure of analogues
15a, 15b, 16a, and 16b. The chemical shift patterns of
relevant protons parallel those of analogues 2a, 2b, 4a,
1
Table 2. The NOE enhancements of relevant 1H NMR signals of (Z)-
and (E)-2-fluoromethyl-2-hydroxymethylmethylenecyclopropanes 15a
and 16a
and 4b (Table 1). Thus, the H NMR signals of OH
and H8 of the Z-isomers 15a and 15b are more deshield-
ed than those of the E-isomers 16a and 16b, whereas an
0
opposite pattern was found in the alkene H1 signals. In
0
the 13C NMR spectra, the cyclopropane C4 of the Z-iso-
NH2
6
NH2
6
mers 15a and 15b is located at a lower field than in the
E-isomers 16a and 16b in contrast to the corresponding
5
4
5
N
N
1'
H
N
N
8
8
N
N
H
H
H
F
2
H
2
H
H
3'
4'
4
N
N
Table 1. Comparison of chemical shifts (d) of the relevant 1H and 13
C
1'
4'
OH
2'
OH
3'
H
H
NMR signals of the (Z)- and (E)-2, 2-bis(hydroxymethyl)- and 2-
fluoromethyl-2-hydroxymethylmethylenecyclopropanes 2a, 4a, 2b, 4b,
15a, 16a, 15b, and 16b
15a
16a
F
H
Compound
Hirr
d
Hobs
d
NOE (%)
Compounda
Isomer
OH
H8
C3
0
H1
0
0
C4
0
0
0
0
15a
H1
H3
7.45
1.54
H3
1.54
7.45
8.57
5.37
8.57
8.57
1.83
2.17
4.0
2a
4a
Z
E
Z
E
Z
E
Z
E
5.07
4.76
4.99
4.76
5.37
5.02
5.31
5.01
7.37
7.48
7.07
7.21
7.45
7.52
7.16
7.26
8.82
8.49
8.41
8.03
8.57
8.49
8.15
8.04
11.7
14.4
11.5
14.3
12.0
15.0
11.9
14.8
31.4
29.7
31.3
29.5
29.4
27.6
29.2
27.5
H1
OH
H8
5.37
8.57
H8
OH
H8
H8
2b
4b
1.71
3.16
3.84
CH2F
CH2O
4.44–4.70
3.48–3.80
15a
16a
15b
16b
0
16a
H3
OH
1.76
5.02
4.47
3.46
H8
8.49
7.52
7.52
7.52
2.75
1.42
1.34
1.87
0
0
0
H1
H1
H1
CH2F
CH2O
a CD3SOCD3 as solvent. For numbering of signals, see Table 2. Values
for 2a, 4a, 2b, and 4b were taken from Ref. 4.

C. Li et al. / Bioorg. Med. Chem. 16 (2008) 2148–2155
2151
Table 3. Inhibition of replication of EBV with fluoromethyl methyl-
enecyclopropane nucleoside analoguesa
viruses. The E-isomer 15b was a moderate substrate
for adenosine deaminase, whereas Z-isomer 15a was
not deaminated.
Compound
EC50/CC50 (lM)b
Selectivity index
2b
5a
0.22/>46c
6.8/>213
8.0/>199
167/>209d
29.1/>199e
0.5/55.7
209
>31.3
>24.9
>1.25
>6.8
111
4. Experimental
5b
6a
6b
4.1. General methods
15a
15b
16a
16b
7.5/59.7
3.4/53.6
3.2/64.1
8
The UV spectra were measured in ethanol and NMR
spectra were determined at 300 or 400 MHz (1H), 75
or 100 MHz (13C), and 376 MHz (19F) in CD3SOCD3
unless stated otherwise. For 19F NMR, CFCl3 was used
as a reference. Mass spectra were determined in elec-
tron-impact (EI-MS) or electrospray ionization (ESI,
methanol–NaCl) mode. Thin-layer chromatography
(TLC) was performed on Analtech aluminum foils
coated with silica gel F254.
15.8
20
a Akata cells, DNA hybridization assay. For details see 4. Acyclovir as
a control had EC50 1.7 lM.
b Results for analogues 5a–6b were taken from Ref. 7 (DNA hybrid-
ization assay in Daudi cells).
c Data from Ref. 22.
d EC50 2.3 lM in viral capsid immunofluorescence (VCA) ELISA and
3.6 lM in H-1 cells (DNA hybridization).
e EC50 < 0.32 lM in VCA ELISA.
4.2. 2,2-Bis(hydroxymethyl)methylenecyclopropane (17)
15a, 15b, 16a, and 16b. However, an exact comparison is
not possible because of the differences in assays. In the
series of fluoroanalogues 7–12 only adenine Z-isomer8
9a was effective against EBV. It is likely that the mech-
anism of anti-EBV action of analogues 15a, 15b, 16a,
and 16b includes their phosphorylation to triphosphates
which then inhibit the viral DNA polymerase as sug-
gested for other fluorinated methylenecyclopropane
analogues.7,8
A solution of DIBALH in hexane (1 M, 26 mL, 26 mmol)
was added dropwise to ethyl methylenecyclopropane car-
boxylate12 18 (4.12 g, 32.7 mmol) in dichloromethane at
ꢀ78 ꢁC with stirring. The stirring was continued for 1 h.
TLC (hexane–AcOEt, 4:1) indicated the presence of alde-
hyde 19 as the major product accompanied by minor
amounts of the faster moving starting ester 18 and slower
moving methylenecyclopropylmethanol. The reaction
was quenched with saturated aqueous NH4Cl (100 mL).
The mixture was stirred for 6 h, the aqueous layer was ex-
tracted with ether (2 · 100 mL), the combined organic
phase was dried (MgSO4), and it was concentrated to
about 10 mL by distillation at <45 ꢁC at an atmospheric
pressure. A mixture of this product, aqueous formalde-
hyde (37%, 65 mL, 0.8 mol), and KOH (18.3 g,
0.33 mmol) in methanol (60 mL) was stirred for 5 days
at room temperature. Methanol was removed in vacuo
and the aqueous portion was extracted with ethyl acetate
(10 · 100 mL). The organic phase was dried (MgSO4) and
concentrated. The precipitated paraformaldehyde was fil-
tered off using a short silica gel column which was then
washed with AcOEt–hexanes (4:1). The solvents were
evaporated and the residue was refluxed in 1 M HCl
(5 mL) for 2 h. The volatile components were evaporated
and the crude product was chromatographed on a silica
gel column using AcOEt–hexanes (1:1) to give diol 17
(1.88 g, 64% based on DIBALH) as a yellow oil. TLC
(AcOEt–hexanes, 2:1) and 1H NMR spectrum were iden-
tical with those of authentic samples.4,13
Compound 15a also inhibited HCV in Huh7 AVA5
cells23 (replicon assay) with EC50/CC50 (lM) 6.5/11
using 20-methylcytidine as a control (EC50/CC50 1.8/
>300) but the antiviral activity was poorly separated
from cytotoxicity. Compound 15a moderately inhibited
the replication of HCMV Towne strain but not AD169
strain (plaque reduction assay) in human foreskin fibro-
blast (HFF) cells with EC50/CC50 (lM) 46/>100, ganci-
clovir (14) as a control exhibited EC50/CC50 2.5/>100.
No significant activity against the rest of tested viruses
was detected.
2.4. Adenosine deaminase (ADA)
Adenine analogues 15a and 16a were investigated as
substrates for adenosine deaminase. In agreement with
the general trend in the series of methylenecyclopropane
analogues,1,2 the E-isomer 16a was a moderate substrate
and it was deaminated after 28 h, whereas the Z-isomer
15a was resistant to deamination.
4.3. 2-Acetoxymethyl-2-hydroxymethyl-1-methylenecy-
clopropane (20)
3. Conclusion
A mixture of diol 17 (1.80 g, 15.8 mmol), trimethyl ort-
hoacetate (2.9 g, 23.7 mmol), and p-toluenesulfonic acid
(2 mg) in CH2Cl2 (20 mL) was stirred for 1 h at room
temperature. The reaction was quenched with Et3N
(0.1 mL) and solvent was evaporated. The residue was
dissolved in 80% acetic acid (5 mL) and the solution
was allowed to stand at room temperature for 30 min
whereupon it was diluted with dichloromethane
(200 mL). The organic phase was washed with saturated
Fluoromethylenecyclopropane analogues 15a, 15b, 16a,
and 16b were synthesized and evaluated for antiviral
activity. All analogues were inhibitors of replication of
EBV in Akata cells with adenine derivative 15a being
the most potent with EC50/CC50 (lM) 0.5/55.7. Against
HCMV, only compound 15a had a moderate effect
whereas its potency against HCV was offset by cytotox-
icity. No activity was observed against other tested

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C. Li et al. / Bioorg. Med. Chem. 16 (2008) 2148–2155
NaHCO3 (2 · 200 mL, caution!) and water (2 · 200 mL).
It was dried (MgSO4) and the solvent was removed to
give product 20 (1.87 g, 78%) as a colorless oil. 1H
NMR (CDCl3) d 5.52 (t, 1H, J = 3.1 Hz), 5.43 (t, 1H,
J = 1.8 Hz, CH2@), 4.15, 4.10 (AB, 2H, J = 11.6 Hz,
CH2OAc), 3.56, 3.51 (AB, 2H, J = 11.6 Hz, CH2OH),
2.09 (s, 3H, CH3), 1.25 (t, 2H, J = 2.4 Hz, H3). 13C
NMR 171.9 (C@O), 134.9 (C@), 105.2 (CH2@), 66.7,
65.2 (CH2O), 26.2 (C2), 21.2 (CH3), 13.8 (C3). ESI-MS
179 (84.8, M+Na), 157 (26.6, M+H), 97 (100.0). Anal.
Calcd for C8H12O3·0.25 H2O: C, 59.80; H, 7.84. Found:
C, 59.74; H, 7.73.
7 mmol) in THF (100 mL) under N2 at room tempera-
ture. The stirring was continued for 6 h, the mixture
was diluted with ether (200 mL), the organic phase
was washed with saturated NaHCO3 (2 · 200 mL),
water (2 · 200 mL), and it was dried (MgSO4). The sol-
vent was removed by distillation at an atmospheric pres-
sure. The crude product was chromatographed on a
silica gel column using 1-pentane–ether (15:1) to give
compound 21 (0.80 g, 72%) as a colorless oil. 1H
NMR (CDCl3) d 5.57 (t, 1H, J = 2.4 Hz), 5.49 (poorly
resolved dd, 1H, CH2@), 4.43, 4.40 and 4.30, 4.28
(2AB, 2H, JH,F = 48.8 Hz, JAB = 9.8 Hz, CH2F), 4.17,
4.10 (AB, 2H, JAB = 12.0 Hz, CH2OAc), 2.09 (s, 3H,
CH3), 1.38 (m, 2H, H3). 13C NMR 171.2 (C@O), 133.2
(C@), 106.3 (CH2@), 85.5 (d, J = 172.3 Hz, CH2F),
65.7 (CH2OAc), 24.3 (d, J = 23.1 Hz, C2), 21.2 (CH3),
14.0 (d, J = 6.7 Hz, C3). 19F NMR ꢀ216.52 (poorly re-
solved tt, J = 48.8, 2, 6 Hz). EI-MS 138 (16.7, MꢀHF),
116 (23.8, MꢀCH2CO), 97 (100.0). HRMS calcd for
C8H10O2 (MꢀHF) 138.0681. Found 138.0687.
4.4. 3-Acetoxymethyl-3-fluoro-1-methylenecyclobutane
(22)
DAST (0.16 mL, 0.81 mmol) was added dropwise to a
stirred solution of acetate 20 (75 mg, 0.48 mmol) and
pyridine (0.16 mL, 2 mmol) in CH2Cl2 (20 mL) at
ꢀ78 ꢁC. The temperature was allowed to rise, the
solvent was evaporated, and the crude product was
chromatographed on a silica gel column using hex-
anes–ether (4:1) to give compound 22 (70 mg, 87%) as
a colorless oil. 1H NMR (CDCl3) d 4.97 (m, 2H,
CH2@), 4.26 (d, 2H, J = 22.8 Hz, CH2O), 3.05 (dt, 2H,
J = 19.0, 2.9 Hz), 2.84 (m, 2H, H2, H4), 2.11 (s, 3H,
CH3). 13C NMR 171.1 (C@O), 136.9 (d, J = 15.7 Hz,
C@), 109.7 (d, J = 8.2 Hz, CH2@), 91.0 (d,
J = 216.4 Hz, C3), 66.1 (d, J = 23.1 Hz, CH2O), 41.7
(d, J = 23.1 Hz, C2, C4), 21.0 (CH3). 19F NMR
ꢀ149.32 (m). EI-MS 138 (34.5, MꢀHF), 116 (22.6,
MꢀCH2CO), 97 (100.0). HRMS calcd for C8H10O2
(MꢀHF) 138.0681. Found: 138.0682. Anal. Calcd for
C8H11FO2: C, 60.75; H, 7.01. Found: C, 61.02; H, 7.07.
4.7. (Z,E)-1-Acetoxymethyl-1-fluoromethyl-2-bromo-2-
bromomethylcyclopropane (27)
A mixture of pyridinium tribromide (2.12 g, 6.6 mmol)
and compound 21 (0.7 g (4.4 mmol)) in CH2Cl2 (20 mL)
was stirred at 0 ꢁC for 1 h. The solid portion was filtered
off and it was washed with CH2Cl2 (5 mL). The filtrate
was diluted with ether (100 mL), the organic phase was
washed with saturated Na2S2O3 (2 · 100 mL) and water
(2 · 100 mL), and it was dried with Na2SO4. The solvents
were evaporated and the residue was chromatographed
on a silica gel column using AcOEt–hexanes (1:10) to give
1
product 27 (1.12 g, 80%) as a colorless oil. H NMR
(CDCl3) d 4.80–4.20 (cluster of m, 6H, CH2F, CH2Br,
CH2OAc), 2.08, 2.07 (2s, 3H, CH3), 1.46, 1.32 (2m, 2H,
H3). 13C NMR 170.9 (C@O), 86.8, 81.6 (2d,
J = 173.8 Hz, CH2F), 67.9 (d, J = 1.5 Hz), 61.7 (d,
J = 1.5 Hz, CH2OAc), 42.1 (d, J = 4.5 Hz), 42.0 (d,
J = 4.3 Hz, CH2Br), 41.3, 41.2 (C1), 33.1 (d,
J = 23.1 Hz), 32.8 (d, J = 20.9 Hz, C2), 26.5, 26.3 (2d,
J = 6.7 Hz, C3), 21.09, 21.06 (CH3). 19F NMR ꢀ219.24
(dt, J = 48.9, 6.2 Hz), ꢀ219.92 (tt, 47.4, 9.0, 4.1 Hz).
ESI-MS 339, 341, 343 (53.3, 100.0, 51.8, M+Na). Anal.
Calcd for C8H11Br2FO2: C, 30.22; H, 3.49. Found: C,
30.61; H, 3.50.
4.5. 2-Acetoxymethyl-2-methylsulfonyloxymethylmethyl-
enecyclopropane (26)
Methylsulfonyl chloride (0.90 mL 11.5 mmol) was added
dropwise with stirring and external ice cooling to a solu-
tion of acetate 20 (1.80 g, 11.5 mmol) and triethylamine
(3.3 mL, 23 mmol) in CH2Cl2 (20 mL). The stirring was
continued for 1 h, the mixture was diluted with ether
(150 mL), the organic phase was washed with water
(100 mL), saturated NaHCO3 (2 · 100 mL), water
(2 · 100 mL), and it was dried with MgSO4. The solvent
was evaporated to give compound 26 (2.2 g, 81%) as a col-
1
orless oil. H NMR (CDCl3) d 5.59 (t, 1H, J = 3.1 Hz),
4.8. (Z,E)-9-[(2-Acetoxymethyl-2-fluoromethylcyclopro-
pylidene)methyl]adenine (28a)
5.51 (t, 1H, J = 1.8 Hz, CH2@), 4.20, 4.16 (AB, 2H,
J = 10.5 Hz), 4.14, 4.05 (AB, 2H, J = 11.6 Hz, CH2O),
3.01 (s, 3H, CH3SO2), 2.07 (s, 3H, CH3CO), 1.42 (t, 2H,
J = 1.8 Hz, H3). 13C NMR 171.1 (C@O), 132.9 (C@),
107.0 (CH2@), 72.1 (CH2OMs), 65.5 (CH2OAc), 37.8
(CH3SO2), 23.1 (C2), 21.1 (CH3 of AcO), 14.7 (C3). ESI-
MS (MeOH+LiCl) 241 (M+Li, 100.0), 475 (2M+Li,
48.8). Anal. Calcd for C9H14O5S·H2O: C, 42.85; H.
6.39. Found: C 42.97; H, 6.40.
A mixture of dibromide 27 (400 mg, 1.26 mmol), adenine
(170 mg, 1.26 mmol), and K2CO3 (1.8 g, 12.6 mmol) in
DMF (25 mL) was stirred for 5 h at 110–115 ꢁC. After
cooling, solids were filtered off and they were washed with
DMF (5 mL). The filtrate was concentrated in vacuo and
the residue was chromatographed on a silica gel column
using CH2Cl2–methanol (200:5) to give compound 28a
(240 mg, 65%) as a white solid. The Z/E ratio was 1:1 as
determined by 1H NMR, mp 189–196 ꢁC. UV kmax
277 nm (e 8400), 263 (e 11,800), 227 (e 24,900). 1H NMR
d 8.50, 8.34 (1H, 2s, 1H, H8), 8.19, 8.18 (2s, 1H, H2),
4.6. 2-Acetoxymethyl-2-(fluoromethyl)methylenecyclo-
propane (21)
0
A solution of Bu4NF (1 M, 35 mL, 35 mmol) in THF
was added with stirring to compound 26 (1.65 g,
7.58 (t, J = 2.5 Hz), 7.51 (m, 1H, H1 ), 7.39 (bs, 2H,
NH2), 4.79, 4.65 and 4.62, 4.49 (2AB, JH,F = 49.0 Hz,

C. Li et al. / Bioorg. Med. Chem. 16 (2008) 2148–2155
2153
JAB = 10.1 Hz), 4.46 (d, 2H, J = 49.2 Hz, CH2F), 4.40,
4.13 and 4.20, 4.14 (2AB, 2H, J = 11.7 Hz, CH2OAc),
2.06, 1.91 (2s, 3H, CH3), 1.95, 1.70 (2m, 2H, H3). 13C
NMR 171.1, 170.6 (C@O), 156.8 (C6), 153.9 (C2), 149.0,
148.9 (C4), 138.0 (C8), 119.1 (C5), 115.6 (d, J = 8.0 Hz),
at 110–115 ꢁC. After cooling, the solid portion was fil-
tered off and it was washed with DMF (5 mL). Filtrate
was concentrated in vacuo and the residue was chro-
matographed on a silica gel column using CH2Cl2–
methanol (200:1) to give product 28c (220 mg, 46%) as
a white solid. The Z/E ratio was 1:1 as determined by
1H NMR, mp 153–170 ꢁC. UV kmax 311 nm (e 7900),
231 (e 29,900). 1H NMR d 8.45, 8.25 (2s, 1H, H8),
0
0
115.3 (d, J = 7.0 Hz, C2 ), 113.1, 112.9 ðC1 ), 86.4 (d,
J = 169.4 Hz), 85.5 (d, J = 169.2 Hz, CH2F), 65.8, 65.2
(CH2OAc), 26.6 (d, J = 23.2 Hz), 24.6 (d, J = 23.2 Hz,
0
0
C4 ), 21.4, 21.1 (CH3), 15.9 (d, J = 6.8 Hz), 12.8 (d,
0
7.42 (d, J = 2.4 Hz), 7.34 (bs, 1H, H1 ), 7.06, 7.05 (2bs,
J = 7.1 Hz, C3 ). 19F NMR ꢀ215.10, ꢀ214.94 (2 over-
2H, NH2), 4.76–4.33 (cluster of m, 4H, CH2F,
CH2OAc), 2.05, 1.89 (2s, 3H, CH3), 1.94 (poorly re-
lapped t, J = 48.2 Hz). ESI-MS 292 (100.0, M+H), 314
(44.4, M+Na). Anal. Calcd for C13H14FN5O2: C, 53.60;
H, 4.84; N, 24.04. Found: C, 53.51; H, 4.89; N, 23.87.
solved t), 1.70 (bs, 2H, H3 ). 13C NMR 171.0, 170.7
0
(C@O), 160.8 (C6), 153.3, 153.2 (C2), 150.4 (C4), 140.5,
140.3 (C8), 123.78, 123.75 (C5), 116.7 (d, J = 8.2 Hz),
0
0
4.9. (Z)-9-[(2-Fluoromethyl-2-hydroxymethylcyclopropy-
lidene)methyl]adenine (15a) and (E)-9-[(2-Fluoromethyl-
2-hydroxymethylcyclopropylidene)methyl]adenine (16a)
116.6 (d, J = 9.7 Hz, C2 ), 112.8, 112.6 (C1 ), 86.2 (d,
J = 168.6 Hz), 85.5 (d, J = 170.1 Hz, CH2F), 65.7, 65.2
0
(CH2OAc), 26.6, 24.7 (2d, J = 23.1 Hz, C4 ), 21.4, 21.1
0
(CH3), 16.1, 12.9 (2d, J = 6.7 Hz, C3 ). 19F NMR
A mixture of compound 28a (220 mg, 0.76 mmol) and
K2CO3 (200 mg, 1.45 mmol) in methanol–water (9:1,
20 mL) was stirred for 1 h at room temperature. The sol-
vent was evaporated and the residue was chromato-
ꢀ214.99 (2 overlapped dt). ESI-MS 191 (100.0), 326,
328 (6.5, 2.0, M+H), 348, 350 (5.9, 2.0, M+Na). Anal.
Calcd for C13H13ClFN5O2: C, 47.94; H, 4.02; N,
21.50. Found: C, 47.93; H, 4.08; N, 21.23.
graphed on
a silica gel column using CH2Cl2–
methanol (20:1) to give the Z-isomer 15a (93 mg,
49%), followed by E-isomer 16a (80 mg, 43%).
4.11. (Z)-2-Amino-6-chloro-9-[(2-hydroxymethyl-2-fluo-
romethylcyclopropylidene)methyl]-purine (15c) and (E)-2-
Amino-6-chloro-9-[(2-hydroxymethyl-2-fluoromethyl-
cyclopropylidene)methyl]purine (16c)
Z-Isomer 15a. Mp 234–236 ꢁC. UV kmax 278 nm (e 7700),
261 (e 10,700), 227 (e 22,800). 1H NMR d 8.57 (s, 1H, H8),
0
8.17 (s, 1H, H2), 7.45 (s, 1H, H1 ), 7.37 (bs, 2H, NH2), 5.37
A mixture of compound 28c (210 mg, 0.65 mmol) and
K2CO3 (178 mg, 1.30 mmol) in methanol–water (9:1,
30 mL) was stirred for 1 h at 0 ꢁC. The solvent was evap-
orated and the residue was chromatographed on a silica
gel column using CH2Cl2 - methanol (100: 3) to give the
Z-isomer 15c (85 mg, 46%), followed by E-isomer 16c
(100 mg, 54%).
(t, 1H, J = 5.4 Hz, OH), 4.70, 4.56 and 4.58, 4.44 (two
partly overlapped AB, 1H, JH,F = 47.8 Hz, JAB = 8.8 Hz,
CH2F), 3.80 (dd, 1H, J = 10.4, 4.8 Hz), 3.48 (dd, 1H,
J = 11.6, 5.6 Hz, CH2OH), 1.54 (m, 2H, H3 ). 13C NMR
0
156.7 (C6), 153.8 (C2), 148.7 (C4), 138.0 (C8), 119.1 (C5),
0
0
116.2 (d, J = 9.0 Hz, C2 ), 112.2 (C1 ), 85.5 (d,
J = 168.6 Hz, CH2F), 62.6 (CH2OH), 29.4 (d, J =
23.1 Hz, C4 ), 12.0 (d, J = 7.5 Hz, C3 ). 19F NMR
ꢀ216.38 (t, J = 48.2 Hz). ESI-MS 250 (100.0, M+H),
272 (13.7, M+Na). Anal. Calcd for C11H12FN5O: C,
53.01; H, 4.85; N, 28.10. Found: C, 52.99; H, 4.82; N,
27.81.
Z-Isomer 15c. Mp 206–208 ꢁC. UV kmax 310 nm (e 7000),
0
0
1
232 (e 28,600). H NMR d 8.53 (s, 1H, H8), 7.26 (s, 1H,
0
H1 ), 7.04 (2H, bs, NH2), 5.33 (t, 1H, J = 5.2 Hz, OH),
4.69, 4.53 and 4.57, 4.41 (2AB, 2H, JH,F = 48.1 Hz,
JAB = 9.8 Hz, CH2F), 3.80 (dd, 1H, J = 11.2, 4.8 Hz),
3.44 (dd, 1H, J = 11.2, 5.6 Hz, CH2O), 1.54 (m, 2H,
13
0
H3 ). C NMR 160.8 (C6), 153.1 (C2), 150.4 (C4), 140.1
E-Isomer 16a. Mp 251–253 ꢁC. UV kmax 277 nm (e
1
0
0
7800), 262 (e 11,000), 226 (e 24,200). H NMR d 8.49
(C8), 123.7 (C5), 117.1 (d, J = 8.2 Hz, C2 ), 111.7 (C1 ),
85.4 (d, J = 167.9 Hz, CH2F), 62.6 (CH2OH), 29.4 (d,
0
(s, 1H, H8), 8.16 (s, 1H, H2), 7.52 (s, 1H, H1 ), 7.37
(bs, 2H, NH2), 5.02 (t, 1H, J = 5.6 Hz, OH), 4.47 (d,
2H, J = 47.8 Hz, CH2F), 3.54 (dd, 1H, J = 11.0,
6.2 Hz), 3.46 (dd, 1H, J = 11.2, 5.8 Hz, CH2O), 1.76
J = 23.1 Hz, C4 ), 12.1 (d, J = 6.7 Hz, C3 ). 19F NMR
ꢀ216.29 (t, J = 48.0 Hz). ESI-MS (MeOH+KOAc) 123
(100.0), 284, 286 (M+H, 11.0, 2.7), 322, 324 (18.2, 6.9,
M+K) 605, 607 (14.9, 11.0, 2M+K). Anal. Calcd for
C11H11ClFN5O: C, 46.57; H, 3.91; N, 24.69. Found: C,
46.54; H, 3.91; N, 24.45.
0
0
(m, 2H, H3 ). 13C NMR 156.7 (C6), 153.8 (C2), 149.0
0
0
(C4), 137.9 (C8), 119.1 (C5), 117.1 (d, J = 9.0, C2 ),
0
112.0 (C1 ), 85.1 (d, J = 168.6 Hz, CH2F), 62.5
0
(CH2O), 27.6 (d, J = 23.1 Hz, C4 ), 15.0 (d, J = 7.5 Hz,
0
C3 ). 19F NMR ꢀ215.42 (poorly resolved dt, J = 48.8,
E-Isomer 16c. Mp 216 ꢁC (decomp). UV kmax 311 nm (e
1
3.0 Hz). ESI-MS 250 (100.0, M+H), 272 (29.8,
M+Na). Anal. Calcd for C11H12FN5O: C, 53.01; H,
4.85; N, 28.10. Found: C, 53.25; H, 4.89; N, 28.18.
6700), 232 (e 27,000). H NMR d 8.45 (s, 1H, H8), 7.36
0
(poorly resolved d, 1H, H1 ), 7.04 (2H, bs, NH2), 5.04
(poorly resolved t, 1H, OH), 4.52, 4.39 (two poorly re-
solved ddd, 2H, JH,F = 48.6 Hz, CH2F), 3.54–3.52,
13
3.46–3.44, (2m, 2H, CH2O), 1.76 (m, 2H, H3 ). C NMR
0
4.10. (Z,E)-2-Amino-6-chloro-9-[(2-acetoxymethyl-2-flu-
oromethylcyclopropylidene)methyl]purine (28c)
160.8 (C6), 153.3 (C2), 150.4 (C4), 140.2 (C8), 123.7 (C5),
0
0
118.1 (d, J = 9.7 Hz, C2 ), 111.6 (C1 ), 85.0 (d, J =
168.6 Hz, CH2F), 62.4 (CH2OH), 27.8 (d, J = 23.9 Hz,
A mixture of dibromide 27 (470 mg, 1.48 mmol), 2-ami-
no-6-chloropurine (256 mg, 1.48 mmol), and K2CO3
(2.08 g, 15 mmol) in DMF (25 mL) was stirred for 5 h
C4 ), 15.1 (d, J = 6.7 Hz, C3 ). 19F NMR ꢀ215.47 (t,
0
0
J = 48.2 Hz). ESI-MS (MeOH+KOAc) 123 (100.0), 322,

2154
C. Li et al. / Bioorg. Med. Chem. 16 (2008) 2148–2155
324 (22.9, 8.0, M+K), 605, 607 (6.0, 4.8, 2M+K). Anal.
Calcd for C11H11ClFN5O·0.5H2O: C, 45.13; H, 4.13;
N, 23.93. Found: C, 45.28; H, 4.14; N, 23.57.
4.15. Antiviral assays
The antiviral assays, with the exception of EBV and
HCV, were described previously.7
4.12. (Z)-9-[(2-hydroxymethyl-2-fluoromethylcyclopropy-
lidene)methyl]guanine (15b)
4.15.1. EBV DNA hybridization assay.22 Akata cells were
maintained in RPMI 1640 (Mediatech, Inc., Herndon,
VA) supplemented with 10% fetal bovine serum (Hy-
clone, Logan, Utah), L-glutamine, penicillin, and genta-
micin at 37 ꢁC in a humidified 5% CO2 atmosphere.
Latently infected cells were induced to undergo a lytic
infection by adding a F(ab0)2 fragment of goat anti-hu-
man IgG antibody (MP Biomedicals, Aurora, OH). Total
DNA from the cells was purified and genome copy
number was quantified by Real-Time PCR. The primers
50-CGG AAG CCC TCT GGA CTT C-30 and 50-CCC
TGT TTA TCC GAT GGA ATG-30 were used with the
fluorescent probe, 6FAM-TGT ACA CGC ACG AGA
AAT GCG CC-TAMRA corresponding to coordinates
155,959–155,981 in the EBV genome (Applied Biosys-
tems). The PCR was performed in an optical 96-well plate
using an ABI 7300 Real-Time PCR system. The PCR con-
tained 900 nM primers, 200 nM probe, 12.5 lL Taqman
Universal Master Mix (Applied Biosystems, Foster City,
CA), and 5 lL target DNA in a final volume of 25 lL.
Each sample was run in duplicate and EC50 values were
calculated by standard methods.
A solution of the Z-isomer 15c (85 mg, 0.3 mmol) in for-
mic acid (80%, 15 mL) was heated at 80 ꢁC for 4 h. The
solvent was removed and the crude product was dissolved
in methanolic NH3 (20%, 30 mL) at 0 ꢁC with stirring
which was continued for 5 h. The volatile components
were evaporated and methanol was evaporated from the
residue (three times). The resultant solid was washed with
methanol (5 mL) to give product 15b (67 mg, 84%) as a
white solid, mp > 280 ꢁC. UV kmax 273 nm (e 10,200),
1
230 (e 25,200). H NMR d 10.68 (s, 1H, CONH), 8.15
0
(s, 1H, H8), 7.16 (s, 1H, H1 ), 6.54 (bs, 2H, NH2), 5.31
(t, 1H, J = 5.0 Hz, OH), 4.64–4.42 (two overlapped AB,
CH2F), 3.74 (poorly resolved dd, 1H), 3.46 (dd, 2H,
13
J = 11.2, 4.8 Hz, CH2O), 1.49 (m, 2H, H3 ). C NMR
0
157.3 (C6), 154.7 (C2), 150.4 (C4), 134.5 (C8), 116.9 (C5),
0
0
115.7 (d, J = 7.1 Hz, C2 ), 112.0 (C1 ), 85.3 (d,
J = 168.6 Hz, CH2F), 62.4 (CH2O), 29.2 (d, J = 23.1 Hz,
C4 ), 11.9 (d, J = 6.7 Hz, C3 ). 19F NMR ꢀ216.48 (t,
J = 47.9 Hz). ESI-MS 266 (100.0, M+H), 288 (48.2,
M+Na). Anal. Calcd for C11H12FN5O2·0.2H2O: C,
49.14; H, 4.65; N, 26.04. Found: C, 49.13; H, 4.54; N,
25.83.
0
0
4.15.2. HCV studies. Antiviral activity of test compounds
was assessed in the stably expressing HCV replicon cell
line, AVA5 (subgenomic CON1, genotype 1b)23, main-
tained at sub-confluent cultures on 96-well plates as previ-
ously described.24 Antiviral activity was determined by
blot hybridization analysis of intracellular HCV RNA
and cytotoxicity was assessed by neutral red dye uptake
after 3 days of treatment. Compounds were added each
day in fresh medium. Intracellular RNA levels and cyto-
toxicity were assessed 24 h after the last dose of
compound.
4.13. (E)-9-[(2-hydroxymethyl-2-fluoromethylcyclopropy-
lidene)methyl]guanine (16b)
The procedure described for the Z-isomer 15b was fol-
lowed with E-isomer 16c (128 mg, 0.45 mmol). The prod-
uct was recrystallized from methanol (20 mL) to give
compound 16b (109 mg, 91%) as a white solid,
mp > 300 ꢁC. UV kmax 272 nm (e 10,200), 229 (e 26,700).
1H NMR (d) 10.70 (s, 1H, CONH), 8.04 (s, 1H, H8),
0
7.26 (s, 1H, H1 ), 6.53 (bs, 2H, NH2), 5.01 (t, 1H,
J = 5.8 Hz, OH), 4.52, 4.47 and 4.40, 4.35 (2AB, 2H,
JH,F = 48.1 Hz, JAB = 9.8, 8.8 Hz, CH2F), 3.51, 3.43
(2dd, 2H, J = 11.0, 5.6 Hz, CH2O), 1.71 (m, 2H,
Acknowledgments
13
0
H3 ). C NMR 157.4 (C6), 154.6 (C2), 150.6 (C4), 134.4
We thank L.M. Hryhorczuk from the Central Instrumen-
tation Facility of the Department of Chemistry, Wayne
State University, for mass spectra. The work described
herein was supported by Grant RO1-CA32779 from the
National Cancer Institute and contract NO1-AI-30049
from the National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Bethesda, MD,
USA. We also thank a reviewer for Ref. 18.
0
0
(C8), 117.0 (C5), 116.7 (d, J = 8.9 Hz, C2 ), 111.9 (C1 ),
85.1 (d, J = 167.6 Hz, CH2F), 62.4 (CH2OH), 27.5 (d,
J = 23.0 Hz, C4 ), 14.8 (d, J = 6.7 Hz, C3 ). 19F NMR
ꢀ215.38 (t, J = 48.9 Hz). ESI-MS 266 (100.0, M+H),
288 (73.2, M+Na). Anal. Calcd for C11H12FN5O2·
0.2H2O: C, 49.14; H, 4, 65; N, 26.04. Found: C, 49.35;
H, 4.57; N, 25.96.
0
0
4.14. Adenosine deaminase (ADA) assay7
References and notes
The Z- and E-isomers 15a and 16a (4.2–4.4 lmol) were
incubated with ADA from calf intestine (Worthington,
Lakewood, NJ, USA, 1.5 U/mL) in 0.05 M Na2HPO4
(pH 7.5, 1.2 mL) with magnetic stirring at room temper-
ature. Aliquots were withdrawn, they were diluted with
the buffer (0.2 mL/10 mL), and the UV spectra were re-
corded. The UV maximum of 16a at 260 nm completely
disappeared after 28 h, whereas the spectrum of 15a was
unchanged (UVmax 260 nm).
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