Synthesis and biological evaluation of endocyclic 2′,3′-didehydro-2′,3′-dideoxymethanocarba adenosine

Article abstract of DOI:10.1081/NCN-120015724

This is the first report describing the synthesis and conformation of methanocarba nucleosides incorporating an endo (β-face) cyclopropyl at the 2′,3′ position of 2′,3′-didehydro-2′,3′-dideoxy carbocyclic nucleosides. These nucleoside isosteres have been shown to exist in a unique extreme eastern conformation. This prediction was confirmed by x-ray crystallography and high resolution NMR spectroscopy. As expected, the methanocarba adenosine compound was neither a substrate nor an inhibitor of adenosine deaminase. However, some of the compounds synthesized demonstrated moderate antiviral activity against HSV-1. The methanocarba adenosine and its triphosphate form were evaluated as inhibitors of HIV-1 reverse transcriptase.

Full text of DOI:10.1081/NCN-120015724

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SYNTHESIS AND BIOLOGICAL EVALUATION
OF ENDOCYCLIC 2′,3′-DIDEHYDRO- 2′,3′-
DIDEOXYMETHANOCARBA ADENOSINE
Danaè R. Quirk Dorr a & Robert Vince a
a Department of Medicinal Chemistry , College of Pharmacy , University of Minnesota , 308
Harvard Street S.E., Minneapolis, MN, 55455, U.S.A.
Published online: 17 Aug 2006.
To cite this article: Danaè R. Quirk Dorr & Robert Vince (2002) SYNTHESIS AND BIOLOGICAL EVALUATION OF ENDOCYCLIC
2′,3′-DIDEHYDRO- 2′,3′-DIDEOXYMETHANOCARBA ADENOSINE, Nucleosides, Nucleotides and Nucleic Acids, 21:10, 665-680,
DOI: 10.1081/NCN-120015724
To link to this article: http://dx.doi.org/10.1081/NCN-120015724
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 21, No. 10, pp. 665±680, 2002
SYNTHESIS AND BIOLOGICAL EVALUATION
OF ENDOCYCLIC 20,30-DIDEHYDRO-
20,30-DIDEOXYMETHANOCARBA ADENOSINE
Danae R. Quirk Dorr and Robert Vince*
Department of Medicinal Chemistry, College of Pharmacy,
University of Minnesota, 308 Harvard Street S. E.,
Minneapolis, MN 55455
ABSTRACT
This is the ®rst report describing the synthesis and conformation of
methanocarba nucleosides incorporating an endo (b-face) cyclopropyl at
the 20,30 position of 20,30-didehydro-20,30-dideoxy carbocyclic nucleosides.
These nucleoside isosteres have been shown to exist in a unique extreme
eastern conformation. This prediction was con®rmed by x-ray crystal-
lography and high resolution NMR spectroscopy. As expected, the
methanocarba adenosine compound was neither a substrate nor an
inhibitor of adenosine deaminase. However, some of the compounds
synthesized demonstrated moderate antiviral activity against HSV-1. The
methanocarba adenosine and its triphosphate form were evaluated as
inhibitors of HIV-1 reverse transcriptase.
INTRODUCTION
The concept of enzymatically stable nucleoside analogues led to the
synthesis of carbocyclic nucleosides. However, as a result of altered stereo-
electronic e€ects, carbocyclic nucleosides are generally less biologically
*Corresponding author. E-mail: vince001@maroon.tc.umn.edu
665
DOI: 10.1081/NCN-120015724
Copyright # 2002 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

666
QUIRK DORR AND VINCE
Figure 1. Methanocarba adenosine isosteres. Note that although only one enantiomer is
depicted in each structure, all compounds and intermediates are racemic mixtures.
e€ective than their nucleoside counterparts.[1,2] In solution, the sugar moieties
of nucleosides exist in a rapid dynamic equilibrium between extreme confor-
mations as de®ned in the pseudorotational cycle.[3,4] In the solid state however,
only one of these conformations is present. Therefore, it is dicult to correlate
which conformation of a nucleoside is responsible for its biological activity.
It has been shown that substituting a bicyclo[3.1.0]hexane moiety for the
nucleoside glycone locks the ring pucker of the nucleoside analogue so that
the conformation is the same in both the solution and solid states.[5] The rigid
methanocarba nucleoside may then result in a stable nucleoside-receptor
interaction with the target enzyme. Synthesizing carbocyclic nucleoside
analogues incorporating rigid bicyclo[3.1.0]hexanes allows structure activity
relationships to be evaluated correlating the de®ned conformations with
speci®c enzyme activity.
Based on the exceptional antiviral activity of the 20,30-didehydro-20,30-
dideoxy carbocyclic nucleosides previously synthesized in our laboratory, we
prepared bicyclo[3.1.0]hexane nucleosides incorporating an endo cyclopropyl
at the 20,30-didehydro-20,30-dideoxy position.[6] Because both cyclopropyl and
vinyl groups demonstrate similar unsaturated properties, the exo 20,30-
methanocarba nucleosides have recently been synthesized.[7,8] X-ray crystal-
lography and high resolution NMR spectroscopy con®rmed that the endo
methanocarba adenosine 2 compound reported here (Fig. 1) exists in a
unique extreme eastern (C30-endo, when C30 supplants the oxygen of the
furanose) conformation. The methanocarba nucleosides were synthesized
and evaluated for activity against HSV-1 and HIV-1. The triphosphate
nucleotide analogue of compound 2 was also synthesized and tested as an
inhibitor of HIV-1 reverse transcriptase.
RESULTS
Chemical Synthesis
The basic strategy of synthesizing the methanocarba purine nucleoside
analogues involved two steps. The ®rst step was the synthesis of a functio-

ENDOCYCLIC METHANOCARBA ADENOSINE
667
nalized amino bicyclo[3.1.0]hexane moiety. The heterocylic purine was then
constructed from the amino bicyclohexane. The versatile lactam, 2-azabicy-
clo[2.2.1]hept-5-en-3-one, was utilized as the starting material because the
rigid bicyclo[2.2.1]heptene allowed for control of the stereochemistry of the
incoming substituent reactions.[9±11] This unsaturated lactam o€ered a cis
orientation for the hydroxymethyl and the heterocyclic functions. The endo
bicyclic template, 6, was constructed from the racemic lactam in four steps.
The lactam underwent acidic hydrolysis as previously described in the lite-
rature to yield the cis-1,3-cyclopentene system which was subsequently
esteri®ed and acetylated to give the blocked amino cyclopentene.[10] The
methyl ester was then reduced, and the corresponding alcohol was acetylated
to give compound 4.[10±13] Modi®ed Simmons-Smith reaction conditions
were employed for the diastereoselective delivery of the incoming methylene
function to generate the acetylated endo bicyclo[3.1.0]hexane, 5, in a quan-
titative yield as shown in Sch. 1.[14±16] The formation of the endo isomer was
consistent with the anticipated amide-directed cyclopropanation.[16] The
amide and ester moieties of compound 5 were removed by barium hydroxide
hydrolysis to the bicyclic amino alcohol, 6.
The endo bicyclo[3.1.0]hexane template, 6, and 5-amino-4,6-dichloro-
pyrimidine were condensed in re¯uxing n-butanol in the presence of tri-
ethylamine to yield the pyrimidinylamino compound, 7. Cyclization of
compound 7 was subsequently completed with triethyl orthoformate and
acid to give the corresponding purine. Any resulting formate ester was
removed by mild acid hydrolysis.[17] The halide of the 6-chloropurine, 1, was
displaced with ammonia to generate methanocarba adenosine, 2. When
compound 1 was heated in methanol, displacement of the chlorine from the
heterocyclic ring gave the methoxy derivative, 3.
The triphosphate derivative was synthesized from the methanocarba
adenosine isostere using a ``one pot'' procedure.[18] The methanocarba
Scheme 1. Synthesis of compounds 1, 2, and 3 using the endo-bicyclo [3.1.0]hexane.

668
QUIRK DORR AND VINCE
adenosine isostere, 2, was ®rst treated with phosphorous oxychloride to gene-
rate the phosphoric dichloride intermediate. Bis(tributylammonium) pyro-
phosphate was then added directly to the mixture and the triphosphate
derivative was isolated after the neutral mixture was eluted by a linear gradient
of aqueous triethylamine bicarbonate through an anion exchange column.
Bis(tributylammonium) pyrophosphate was generated by passing an aqueous
solution of tetrasodium pyrophosphate decahydrate through a cation
exchange column directly into a solution of tributylamine in ethanol.[19]
DISCUSSION
The regiostereochemical assignments for the conformations of the
1
adenosine isostere, 2, were con®rmed by high-resolution H NMR spectra
analysis, including COSY and NOSEY spectra. The x-ray crystal structure of
the adenosine isostere, 2, was also evaluated.
1
Two-Dimensional H NMR Spectra Analysis
The vicinal couplings between di€erent protons were identi®ed as cross
signals in the HOMO-COSY 2D spectra. The results of the COSY spectra
are summarized in Table 1. NOSEY spectral analysis provided de®nitive
Table 1. Cross Signals Observed in HOMO-COSY 2 D Spectra of the Methanocarba
Adenosine Isostere
H-40
CH2
H-20
H-30a
H-50
X
H-10
H-30b
X
H-60a
H-60b
H-40
CH2OH
H-20
H-30a
H-50
H-10
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
H-30b
H-60a
H-60b
X
X
X
X
X
X
X
X

ENDOCYCLIC METHANOCARBA ADENOSINE
669
evidence for the regiochemistry assigned for the methanocarba andenosine
compound synthesized (Fig. 2). It is important to note that there are cross
signals between correlating H-10, H-20, H-40, and H-50 con®rming that these
four protons are all below the plane of the six-membered ring. This con®rms
that the cyclopropyl was added to form the endo bicyclic hexane.
The NOESY spectra also con®rms that a 60-Ha and 30-Hb interaction is
occurring. There are also interactions between 60-Hb and 10-H and between
30-Ha and 40-H.
Figure 2. NOSEY spectra of compound 2.

670
QUIRK DORR AND VINCE
X-Ray Crystallographic Analysis of Methanocarba Adenosine
The adenine rings in the crystal structure are stacked with an extensive
intermolecular H-bonding network among the neighboring methanocarba
adenosine molecules. It is suggested that intermolecular interactions in the
crystal do not have a major impact on the extent of puckering in the sugar
ring (Cmax). But, the interactions can tune the mode of puckering (P), the
glycosidic conformation (w), and the C20-CH2OH conformation (g) to
maximally optimize hydrogen bonding and stacking interactions (Fig. 3).
The pseudorotational value, P, was obtained from calculations based
on the crystal structure data obtained. If the pseudorotational concept is
applied to the bicyclo[3.1.0]hexane ring, the phase angle, P, is 92.5ꢀ. This
de®nes the pucker as C30-endo if the C30 is taken to be analogous to the
furanose O40. The pseudosugar is unique in that it is con®ned to the eastern
conformation. This is important because in natural nucleosides containing
riboses and deoxyriboses, P falls in the ranges of 0ꢀ±36ꢀ and 144ꢀ±180ꢀ. The
existence of these narrow ranges implies the existence of the true minimum
energy regions. The potential barriers near P ˆ 90ꢀ and 270ꢀ arise when the
molecule pseudorotates from one energy minimum to the other. It is
speculated that the pseudorotational pathway along the P ˆ 90ꢀ point is the
favored mode of north-south interconverion.[20] Therefore, this compound in
its uniqueness is an extremely useful tool for studying the conformational
preferences of adenosine metabolizing enzymes.
BIOLOGICAL EVALUATION
The endo methanocarba adenosine isostere, 2, was not recognized by
the adenosine deaminase enzyme. This result is in support of a previous study
Figure 3. The molecular structure of compound 2 as determined by X-ray crystallography.

ENDOCYCLIC METHANOCARBA ADENOSINE
671
demonstrating the conformational preference of adenosine deaminase[21]
.
Compounds were tested against a DNA virus, Herpes simplex virus, type 1
(strain F), in African green monkey kidney cells (VERO cell line). Both
compounds 2 and 3 were shown to inhibit HSV-1 with an IC50 of 40 mM.
Compound 2 showed no cytotoxicity and compound 3 showed only slight
cytotoxicity at 400 mM. The 6-chloropurine compound, 1, was the most
active with an IC50 ˆ 4.0 mM. As expected, compound 1 was also the most
cytoxic as cycotoxicity was observed at 40 mM. Compound 2 was evaluated
for antiviral activity against HIV-1 but was found to be inactive (the IC50
value was greater than 100 mM, AZT demonstrated an IC50 value of
0.003 mM). From this study, it can be postulated that the inactivity of this
compound could be a result of poor anabolism to the mono-, di-, or tri-
phosphate level or of the poor substrate properties of the 50-triphosphate
toward the viral encoded reverse transcriptase. Therefore, the methanocarba
nucleoside triphosphate of 2 was tested for HIV-1 reverse transcriptase
activity. Interestingly, the endo methanocarba nucleotide inhibited reverse
transcriptase with an IC50 of 15 mM. From this, it can be concluded that the
parent nucleoside form is probably not anabolized intracellularly to the tri-
phosphate form.
EXPERIMENTAL SECTION
Crystal Structure
The adenosine nucleoside analogue, 2, was recrystallized by evapora-
tion from methanol/water (10:1). All calculations were preformed with SGI
INDY R4400-SC and/or Pentium computers using the SHELXTL V5.10
suite of programs. A clear colorless 0.1460.1160.06 mm3 triangular crystal
was used for data collection on an automated Bruker SMART di€ractometer
platform equipped with a graphite monochromator. The frame time was 25
seconds and the detector distance was 4.95 cm. Lattice parameters were
determined from 20 re¯ections within y range 1.47ꢀ to 25.13ꢀ. The data
collection range of hkl was À 6 ꢁ h ꢁ 3, À 9 ꢁ k ꢁ 9, À 16 ꢁ 1 ꢁ 16 with
98.7% completeness to y ˆ 25.13ꢀ. The data collection was conducted using
MoKa radiation. A randomly oriented region of reciprocal space was sur-
veyed to the extent of 1.3 hemispheres and to a resolution of 0.84 A. Three
major sections of frames were collected with 0.30ꢀ steps in o at 3 di€erent j
settings and a detector position of À 28ꢀ in 2y. The intensity data were
corrected for absorption and decay.[22] Final cell constants were calculated
from the xyz centroids of 567 strong re¯ections from the actual data col-
lection after integration.

672
QUIRK DORR AND VINCE
Biological Experimental
Adenosine Deaminase Assay
Adenosine deaminase (150-200 units/mg protein), originally isolated
from calf intestinal mucosa, was obtained from Sigma Chemical Company,
St. Louis, Missouri. Adenosine deaminase solution was prepared by dilution
into potassium phosphate bu€er (0.05M, pH 7.4) to the concentration of
approximately 100 units/mL. The enzymatic reaction was preformed in 1 mL
potassium phosphate bu€er containing 40 mM compound and 50 milliunits of
adenosine deaminase solution. The assay was monitored by following the
decrease in absorbance at 265 nm (37ꢀC) for 15 min. If there was no change in
absorbance within 15 min, an additional enzyme (50 milliunits) was added
and the reaction was continued overnight. A decrease in UV absorbance
indicated that the test compound was deaminated and thus a substrate for
adenosine deaminase. The inhibitory e€ects of the test compounds against
adenosine deaminase were evaluated by comparing the rate of deamination
of adenosine with or without the presence of test compound. If deamination
rate was delayed by the coexistance of the test compound, the compound was
considered an inhibitor of adenosine deaminase.
Antiviral Assays
HSV-1
The in vitro antiviral assays were carried out by virus induced cyto-
pathogenic e€ects (CPE) inhibition studies. Con¯uent monolayers of African
green monkey kidney cells (VERO cell lines) grown in minimal essential
media supplemented with 5% fetal calf serum in COSTAR 96-well microtiter
plates were infected with an inoculum of HSV-1, strain F. Final concentra-
tions of 100 mg/mL followed by ten fold serial dilutions of test compounds
were added to the wells (100 mg/mL to 0.1 mg/mL). Virus controls contained
no test compounds. Other wells remained free of virus to determine the drug
toxicity e€ects against the VERO cells. Acyclovir was used as the positive
control. The 96-well plates were incubated for 3 days at 37ꢀC in a humidi®ed
atmosphere with 5% CO2 until the maximum CPEwere observed in the virus
control cultures. The cell monolayers were microscopically examined for
virus induced CPEand for drug cytotoxicity. The IC
value is the con-
50
centration of the test compound the decreased the total number of plaques
50% to that present in the control wells with no test compound.
HIV-1
The HIV inhibitory assay of the compounds were evaluated by micro-
titer anti-HIV assays with CEM-SS cells or fresh human peripheral blood

ENDOCYCLIC METHANOCARBA ADENOSINE
673
mononuclear cells (PBMCs), which quantify the ability of a compound to
inhibit HIV-induced cell killing or HIV replication.[23] AZT was used as
the positive control. Quanti®cation was performed with the tetrazolium dye
2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2M-tetrazolium-5-carboxanilide
(XTT), which is metabolized to a colored formazan product by the viable
cells.[24]
HIV-1 Teverse Transcriptase
Puri®ed recombinant HIV-1 reverse transcriptase was obtained from the
University of Alabama at Birmingham, Center for AIDS Research, Gene
Expression Core Facility (supported in part by the NIH Centers for AIDS
Research program grant P30 AI27767). For the study of RNA transcription,
HIV-1 reverse transcriptase activity was measured in 50-mL reactions con-
taining 50 mM Tris (pH 8.0), 50 mM KCl, 10 mM MgCl2, 4mM b-mercapto-
ethanol, 3% glycerol, 1 mg/mL bovine serum albumin, 3.33 mg/mL of primed
16S rRNA from E. coli, 10 mM dTTP, 10 mM dGTP, 10 mM dCTP, and
0.25 mM [33P] dATP (the Km concentration). The primer was annealed to the
template at a ratio of 3 to 1 as described previously.[25] After incubation, the
DNA in each sample was precipitated onto glass ®ber ®lters using a 5%
trichloroacetic acid solution containing 10 mM pyrophosphate. These ®lters
were batch washed and counted for radioactivity.[26] Assays were done in
duplicate. An initial range ®nding experiment was ®rst conducted. The data
from this experiment was then used to narrow the concentration range for
each compound, which was then tested in smaller increments. The experi-
ments were repeated in duplicate and these two experiments were used to
calculate the average IC50 values.
Chemical Experimental
General Synthesis
All chemicals and solvents were reagent grade unless otherwise speci-
®ed. The chemicals and solvents used were obtained from Aldrich Chemical
Company, Milwaukee, WI. All intermediates and ®nal compounds were
racemic mixtures. Analytical thin layer chromatography (TLC) was per-
formed on 0.25 mm layers of Merck silica gel 60 F254 on glass-backed plates.
The plates were visualized by viewing under UV light and by exposure
to iodine vapor unless otherwise indicated. Column chromatography was
performed on Merck silica gel 60 A, 230±400 mesh. Chromatography on
silica involved dissolving the crude material in a suitable solvent, treatment of

674
QUIRK DORR AND VINCE
the solution with silica gel, followed by evaporation of the solvent and then
drying of the silica invacuo. The dried compounds containing silica were then
loaded onto a slurry packed column of silica gel and eluted with the solvent
system listed. Then, the fractions containing pure product (as indicated by
TLC) were combined and concentrated under reduced pressure. Evaporation
under reduced pressure refers to the use of a rotary evaporator using
aspirator pressure. Evaporation in vacuo was carried out with a rotary eva-
porator under 0.2 mm Hg of pressure at a temperature below 30ꢀC. The
melting points were determined in open capillary tubes heated in a Mel-Temp
II melting point apparatus and are uncorrected. Routine mass spectra, ele-
1
mental analysis, H, and 31P NMR spectra were recorded using standard
methods. Positive-ion fast-atom bombardment mass spectra (FABMS) were
obtained on a VG 7070E-HF spectrometer. MBNA was used as the sample
matrix and ionization was e€ected by a beam of xenon atoms. The peaks
‡
listed are those from the molecular ion, designated M , and fragments that
can be assigned as plus or minus relative to the molecular ion. The elemental
1
analyses were preformed by M-H-W Laboratories in Phoenix, AZ. The H
and 31P NMR spectra were obtained on Varian Unity VAC-200 or VAC-300
with an automatic sample changer or on a Varian Inova, VI-500 in the sol-
vent listed and are given in parts per million (d). The peak multiplicities are
abbreviated as follows: broad, br; singlet, s; doublet, d; triplet, t; and mul-
tiplet, m.
;Æ)-cis-endo-Acetic acid 4-acetylamino-bicyclo[3.1.0]hex-2-yl methyl
ester ;5). Compound 4 (2.04 g, 10.4 mmol) was dissolved in anhydrous
methylene chloride (50 mL) and cooled to 0 ꢀC under N2 (g) as diethylzinc
(100 mL, 1 M in hexanes) was carefully added. The clear yellow solution was
stirred for 30 min. Diiodomethane (8.91 mL, 111 mmol) was diluted with
methylene chloride (40 mL) and then added dropwise to the above cold
diethylzinc/4 solution. The yellow and white mixture continued stirring
as the reaction mixture warmed to room temperature overnight (19 h).
Saturated sodium chloride (40 mL) and 3N hydrochloric acid (80 mL) were
added to the reaction over 15 min. Methylene chloride (100 mL) and satu-
rated sodium sul®te (40 mL) were then added to the clear yellow mixture. The
aqueous layer was removed and washed with chloroform (100 mL). The
chloroform layer and the methylene chloride layer were combined and
washed with saturated sodium bicarbonate (200 mL). The orange organic
layer was removed and dried over anhydrous magnesium sulfate and con-
centrated to a yellow oil, 5 (2.20 g, 10.4 mmol, 100%): Rf value 0.778 (iso-
propanol : ammonium hydroxide : water À 7:1:2); 1H NMR (200 MHz,
dimethyl-d6 sulfoxide): d 7.74±7.78 (1H, d, NH), 4.23±4.33 (1H, m, H-40),
3.81±4.00 (2H, m, OCH2), 2.32±2.43 (1H, m, H-20), 1.99 (3H, s, CH3CO2),
1.76 (3H, s, NCOCH3), 1.64±1.79 (1H, m, H-30a), 1.34±1.43 (1H, m, H-50),

ENDOCYCLIC METHANOCARBA ADENOSINE
675
1.20±1.31 (1H, m, H-10), 0.560±0.730 (1H, m, H-30b), 0.459±0.522 (1H, m, H-
60a), 0.158±0.263 (1H, m, H-60b). Anal. calcd. for C11H17NO3: C, 62.54; H,
8.11; N, 6.63. Found: C, 62.39; H, 8.24; N, 6.61.
;Æ)-cis-endo-;4-Amino-bicyclo[3.1.0]hex-2-yl)methanol ;6). Compound
5 (2.16 g, 10.2 mmol) was re¯uxed with aqueous barium hydroxide (0.5N,
250 mL) overnight (17.5 h), cooled to 0ꢀC and carefully neutralized with
carbon dioxide (g). The white precipitate was removed by ®ltration and the
clear ®ltrate was concentrated to dryness. The dry solid was triturated with
water (2650 mL) and the water mixture was ®ltered through Celite 545. The
yellow ®ltrate was passed through an anion (-OH) exchange column
(Amberlite IRA-400, 15-50 wet mesh, activated from the chloride form by
washing with 1N sodium hydroxide). The clear solution was concentrated
in vacuo (37 ꢀC, 0.2 mm Hg) to a white solid. Absolute ethanol (150mL) was
added, and the white precipitate was removed by ®ltration. The clear ®ltrate
was concentrated to an oil, 6 (1.31 g, 10.3 mmol, 100%): mp (dec) 93±95ꢀC;
Rf value 0.416 (isopropanol : ammonium hydroxide : water, 7:1:2); 1H NMR
(200 MHz, dimethyl-d6 sulfoxide): d 6.41 (1H, b, NH-a, D2O exchangeable),
4.33 (1H, b, OH, D2O exchangeable), 4.03 (1H, b, NH-b, D2O exchangeable),
3.30±3.43 (1H, m, H-40), 3.23±3.26 (2H, m, CH2OH), 2.03±2.13 (1H, m, H-
20), 1.51±1.65 (1H, m, H-30a), 1.31±1.14 (2H, m, H-10 and H-50), 0.468±0.300
(2H, m, H-30b and H-60a), 0.016±0.086 (1H, m, H-60b). MS (FAB) 127.19,
‡
(M‡H) .
;Æ)-cis-endo-{4-[;5-Amino-6-chloro-pyrimidin-4-yl)amino]-bicyclo[3.1.0]-
hex-2-yl}methanol ;7). Compound 6 (400 mg, 3.14 mmol) was combined
with 5-amino-4,6-dichloropyrimidine (781 mg, 4.40 mmol) in 1-butanol
(50 mL). Triethylamine (3.12 mL) was added and the mixture was re¯uxed for
45 h. The brown mixture was concentrated invacuo to an orange residue and
puri®ed by ¯ash column chromatography (hexanes/ethyl acetate À 1:1, 1:2,
1:3, 1:4, 1:5 and 1:6) to a ¯u€y white solid, 7 (601 mg, 2.36 mmol, 75.2%): mp
1
176±178 ꢀC; Rf value 0.241 (ethyl acetate); H NMR (200 MHz, dimethyl-d6
sulfoxide): d 7.71 (1H, s, H-2), 6.58±6.65 (1H, d, NH, D2O exchangeable),
5.09 (2H, s, NH2, D2O exchangeable), 4.59±4.62 (1H, m, H-40), 4.44±4.50
(1H, t, OH, D2O exchangeable), 3.30±3.36 (2H, m, CH2OH), 2.20±2.32 (1H,
m, H-20), 1.95±1.81 (1H, m, H-30a), 1.36±1.62 (1H, m, H-50), 1.27±1.34 (1H,
m, H-10), 0.633±0.801 (1H, m, H-30b), 0.483±0.545 (1H, m, H-60a), 0.152±
0.256 (1H, m, H-60b). Anal. calcd. for C11H15N4OCl: C, 51.87; H, 5.94; N,
22.0. Found: C, 52.10; H, 5.97; N, 22.21.
;Æ)-cis-endo-[4-;6-Chloro-9H-purin-9-yl)bicyclo[3.1.0]hex-2-yl]-methanol
;1). Compound 7 (519 mg, 2.04 mmol) was mixed with triethyl ortho-
formate (13 mL) and hydrochloric acid (12N, 0.50 mL). The clear yellow

676
QUIRK DORR AND VINCE
solution was then stirred overnight (14 h). The reaction mixture was con-
centrated and hydrochloric acid (0.5 N, 12 mL) was added. After the yellow
solution was stirred at room temperature for 1 h, it was neutralized to pH 7.2
with aqueous sodium hydroxide (1N, 27 mL) and concentrated to a thick
white mixture. The mixture was puri®ed by ¯ash column chromatography
(5% methanol/methylene chloride) to a dark brown residue (646 mg) and
recrystallized (ethyl acetate) to yield 1 as a white solid (378 mg, 1.43 mmol,
70.1%): mp 129±130ꢀC; Rf value 0.459 (10% methanol/methylene chloride);
1H NMR (200 MHz, dimethyl-d6 sulfoxide): d 8.82 (1H, s, H-2), 8.77 (1H, s,
H-8), 5.15±5.20 (1H, m, H-40), 4.55±4.60 (1H, b, OH, D2O exchangeable),
3.70±3.46 (2H, d, CH2OH), 2.38±2.48 (1H, m, H-20), 2.17±2.28 (1H, m, H-
30a), 1.77±1.81 (1H, m, H-50), 1.52±1.58 (1H, m, H-10) 1.31±1.43 (1H, m, H-
30b), 0.936±0.984 (1H, m, H-60a), 0.461±0.431 (1H, m, H-60b). Anal. calcd.
for C12H13N4OCl: C, 54.44; H, 4.95; N, 21.17; Cl, 13.39. Found: C, 54.16; H,
5.06; N, 21.49; Cl, 13.61.
;Æ)-cis-endo-[4-;6-Amino-9H-purin-9-yl)bicyclo[3.1.0]hex-2-yl]-methanol
;2). Compound 1 (707 mg, 2.67 mmol) was placed in a bomb. Excess liquid
ammonia was added. The bomb was sealed and heated at 75ꢀC for 42 h. The
bomb was then cooled, and the ammonia was evaporated. The remaining
white solid was puri®ed by ¯ash column chromatography (methanol/
methylene chloride, 0%±15%) to yield 2 as a ®ne white powder (611 mg,
2.49 mmol, 93.2%): mp 123±124 ꢀC; Rf value 0.478 (20% methanol/methy-
1
lene chloride); H NMR (500 MHz, dimethyl-d6 sulfoxide): d8.25 (1H, s, H-
2), 8.12 (1H, s, H-8), 7.18 (2H, s, NH2-C6, D2O exchangeable), 5.01±5.06
(1H, m, H-40), 4.54 (1H, t, OH, D2O exchangeable), 3.39±3.40 (2H, m,
CH2OH), 2.40±2.45 (1H, m, H-20), 2.09±2.14 (1H, m, H-30a), 1.71±1.75 (1H,
m, H-50), 1.48±1.52 (1H, m, H-10), 1.24±1.31 (1H, m, H-30b), 0.934±0.960
(1H, m, H-60a), 0.391±0.433 (1H, m, H-60b). Anal. calcd. for C12H15N5O: C,
58.76; H, 6.16; N, 28.55. Found: C, 58.63; H, 6.17; N, 28.62.
;Æ)-cis-endo-[4-;6-Methoxy-9H-purin-9-yl)bicyclo[3.1.0]hex-2-yl]-metha-
nol ;3). Compound 1 (537 mg, 2.11 mmol) was mixed with triethyl ortho-
formate (10 mL) and hydrochloric acid (12N, 0.50 mL) and stirred overnight
(14 h). When dilute hydrochloric acid (0.5N, 12 mL) was added to the white
reaction mixture, it turned to a clear yellow solution. After the solution
stirred at room temperature for 1 h, sodium hydroxide (1N, 32 mL) was
added until the pH of the solution was slightly basic. Methanol (10 mL) was
added, and the reaction mixture was concentrated invacuo to a residue. The
residue was puri®ed by ¯ash column chromatography (10% methanol/
chloroform) and recrystallized (ethyl acetate) to yield 3 (357 mg, 1.46 mmol,
69.3%): mp 163±164 ꢀC; Rf value 0.404 (10% methanol/methylene chloride);

ENDOCYCLIC METHANOCARBA ADENOSINE
677
1H NMR (300 MHz, dimethyl-d6 sulfoxide): d 8.50 (1H, s, H-2), 8.49 (1H, s,
H-8), 5.09±5.17 (1H, m, H-40), 4.53±4.56 (1H, t, OH, D2O exchangeable),
3.99±4.06 (3H, s, CH3), 3.38±3.49 (2H, m, CH2OH), 2.38±2.40 (1H, m, H-20),
2.11±2.21 (1H, m, H-30a), 1.72±1.80 (1H, m, H-50), 1.48±1.56 (1H, m, H-10),
1.15±1.41 (1H, m, H-30b), 0.928±0.971 (1H, m, H-60a), 0.380±0.452 (1H, m,
H-60b). Anal. calcd. for C13H16N4O2: C, 59.99; H, 6.20; N, 21.52. Found: C,
60.06; H, 6.34; N, 21.95.
;Æ)-cis-endo-P-{[4-;6-Amino-9H-purin-9-yl)bicyclo[3.1.0]hex-2-yl]
methyl} ester triphosphoric acid. Compound 2 (100 mg, 0.407 mmol) was
suspended in dry trimethylphosphate (4 mL) and cooled to 0 ꢀC. Phosphorus
oxychloride (0.100 mL, 1.05 mmol, 166 mg) was then added and the cool
mixture continued stirring at 0 ꢀC for two hours. The reaction mixture
was then stirred at room temperature for 2 h and recooled 0 ꢀC. Bis
(tributylammonium) pyrophosphate (1.11 g, 2.04 mmol), and tributylamine
(3.11 mg, 4.12 mL) in dimethylformamide (6 mL) was added to the above
reaction mixture. After the mixture stirred 0ꢀC for 15 min, it was poured into
ice water (200 mL). This neutral solution was stored at 4 ꢀC overnight, con-
centrated invacuo and lypholized to an oil. Ether (26125 mL) was added to
the oil to extract some of the remaining trimethyl phosphate. The residue was
concentrated and dissolved in water (20 mL) and appÀlied to a 4 6 40 cm
column containing DEAE Sephadex A-25 (25 g), HCO3 form. The elution
was performed by a linear gradient of water (1 L) to 0.75 M triethylamine
bicarbonate (1.5 L). The fractions containing the triphosphate compound
were concentrated and lyophilized. The solid was evaporated several times
with ethanol (46100 mL) to remove the triethylammonium bicarbonate, and
then lyophilized to yield the triphosphate of compound 2 as a clear, colorless
¯u€y solid (227 mg crude, UV: 52.6% with A ˆ 0.44054, e ˆ 15.46103,
1
119 mg, 0.246 mmol, 60.4% actual); H NMR (300 MHz, dimethyl-d6 sulf-
oxide): d 8.29 (1H, s, H-2), 8.13 (1H, s, H-8), 7.17 (2H, s, NH2-C6, D2O
exchangeable), 5.06 (1H, m, H-40), 3.39 (2H, m, CH2OH), 2.49 (1H, m, H-20),
2.08 (1H, m, H-30a), 1.75 (1H, m, H-50), 1.50 (1H, m, H-10), 1.35±1.43 (1H, m,
H-30b), 0.846-0.905 (1H, m, H-60a), 0.414±0.433 (1H, m, H-60b). 31P NMR
(300 MHz, 1.6 mL Hepes Bu€er, pH7 containing 0.5 mL deuterium oxide and
0.05 mL EDTA, 150mg/mL): d À 21.65 (Pb), À 10.24 (Pa), À 6.57 (Pg). MS
‡
(FAB) 486.0, (M‡H) ; 484.2, (M À H)À .
ACKNOWLEDGMENTS
The adenosine deaminase assay and the in vitro antiviral assays were
performed by Jay Brownell in our laboratory at the University of Minnesota.
The testing against HIV-1 was performed by Southern Research Institute in
Frederick, Maryland. The assay for HIV reverse transcriptase enzyme

678
QUIRK DORR AND VINCE
recognition was performed by Lucile White at Southern Research Institute in
Birmingham, Alabama. Data collection and structure solution were con-
ducted by Victor Young and William W. Brennessel at the X-Ray Crystal-
lographic Laboratory, 160 Kolto€ Hall, Chemistry Department, University
of Minnesota. Some of the intermediates were also synthesized by Lakshmi
Akella in our laboratory at the University of Minnesota.
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Received April 5, 2002
Accepted July 2, 2002