(R)-(-)- and (S)-(+)-Synadenol: synthesis, absolute configuration, and enantioselectivity of antiviral effect.

Article abstract of DOI:10.1021/jm980323u

Synthesis of (R)-(-)- and (S)-(+)-synadenol (1a and 2a, 95-96% ee) is described. Racemic synadenol (1a + 2a) was deaminated with adenosine deaminase to give (R)-(-)-synadenol (1a) and (S)-(+)-hypoxanthine derivative 5. Acetylation of the latter compound g

Full text of DOI:10.1021/jm980323u

J . Med. Chem. 1998, 41, 5257-5264
5257
(
R)-(-)- a n d (S)-(+)-Syn a d en ol: Syn th esis, Absolu te Con figu r a tion , a n d
X
En a n tioselectivity of An tivir a l Effect
†
‡
‡
§
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3
Yao-Ling Qiu, Andrew Hempel, Norman Camerman, Arthur Camerman, Fiona Geiser, Roger G. Ptak,
3
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J ulie M. Breitenbach, Toshiko Kira, Ling Li, Elizabeth Gullen, Yung-Chi Cheng, J ohn C. Drach, and
J iri Zemlicka*,†
Department of Chemistry, Experimental and Clinical Chemotherapy Program, Barbara Ann Karmanos Cancer Institute,
Wayne State University School of Medicine, Detroit, Michigan 48201-1379, Biochemistry Department, University of Toronto,
Toronto, Ontario M5S 1A8, Canada, Ardono Research, Seattle, Washington 98105, Chiral Technologies, Inc., Exton,
Pennsylvania 19341, Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510-8066,
and Department of Biologic and Materials Science, School of Dentistry, University of Michigan, Ann Arbor,
Michigan 48019-1078
Received May 26, 1998
Synthesis of (R)-(-)- and (S)-(+)-synadenol (1a and 2a , 95-96% ee) is described. Racemic
synadenol (1a + 2a ) was deaminated with adenosine deaminase to give (R)-(-)-synadenol (1a )
and (S)-(+)-hypoxanthine derivative 5. Acetylation of the latter compound gave acetate 6.
Reaction with N,N-dimethylchloromethyleneammonium chloride led to 6-chloropurine derivative
7
. Ammonolysis furnished (S)-(+)-synadenol (2a ). Absolute configuration of 1a was established
by two methods: (i) synthesis from (R)-methylenecyclopropanecarboxylic acid (8) and (ii) X-ray
diffraction of a single crystal of (-)-synadenol hydrochloride. Racemic methylenecyclopropan-
ecarboxylic acid (10) was resolved by a modification of the described procedure. The
R-enantiomer 8 was converted to ethyl ester 13 which was brominated to give vicinal dibromides
1
4. Reduction with diisobutylaluminum hydride then furnished alcohol 15 which was acetylated
to the corresponding acetate 16. Alkylation-elimination procedure of adenine with 16 yielded
acetates 17 and 18. Deprotection with ammonia afforded a mixture of Z- and E-isomers 1a
and 19 of the R-configuration. Comparison with products 1a and 2a by chiral HPLC established
the R-configuration of (-)-synadenol (1a ). These results were confirmed by X-ray diffraction
of a single crystal of (-)-synadenol hydrochloride. The latter forms a pseudosymmetric dimer
with adenine-adenine base pairing in the lattice with the nucleobase in an anti-like
conformation. Enantiomers 1a and 2a exhibit varied enantioselectivity toward different viruses.
Both enantiomers are equipotent against human cytomegalovirus (HCMV) and varicella zoster
virus (VZV). The S-enantiomer 2a is somewhat more effective than R-enantiomer 1a in herpes
simplex virus 1 and 2 (HSV-1 and HSV-2) assays. By contrast, enantioselectivity of antiviral
effect is reversed in Epstein-Barr virus (EBV) and human immunodeficiency virus type 1 (HIV-
1
) assays where the R-enantiomer 1a is preferred. In these assays, the S-enantiomer 2a is
less effective (EBV) or devoid of activity (HIV-1).
In our previous reports1 we described a new series
of nucleoside analogues comprising a methylenecyclo-
propane function and exhibiting a strong antiviral effect
against a broad range of viruses. Of this group of
analogues, synadenol (1a + 2a ) and synguanol (1b +
-4
contain a single chiral center were obtained by chemical
1
-3
synthesis as racemic mixtures.
Resolution of the
racemates or synthesis of enantiopure biologically active
enantiomers is then a foremost task in further develop-
ment of these analogues as potential antiviral drugs.
Because methylenecyclopropane nucleoside mimics were
originally designed as analogues of the corresponding
2
b) are of particular importance because of their inhibi-
tion of replication of human and murine cytomegalovi-
rus (HCMV and MCMV) and Epstein-Barr virus (EBV)
as well as human herpes virus 6 (HHV-6). Synadenol
1a + 2a ) is also an agent against hepatitis B virus
HBV) and varicella zoster virus (VZV). Compounds 1a
1
allenes, a comparison of enantioselectivity of antiviral
effects in both series of analogues is also of interest. In
this communication, we report on synthesis, absolute
configuration, and antiviral activity of (R)- and (S)-
synadenol (1a and 2a ).
(
(
+
2a and 1b + 2b as well as related analogues which
X
Presented in part at the 13th International Roundtable: Nucleo-
sides, Nucleotides and Their Biological Applications, Montpellier,
France, Sept 6-10, 1998; Abstracts Poster 16; Nucleosides Nucleotides,
in press.
*
Send correspondence to this author at the Barbara Ann Karmanos
Cancer Institute, 110 E. Warren Ave., Detroit, MI 48201-1379.
Telephone: (313) 833-0715, ext. 2452 or 2262. Fax: (313) 832-7294.
E-mail: zemlicka@kci.wayne.edu.
†
Wayne State University.
University of Toronto.
Ardono Research.
Chiral Technologies, Inc.
University of Michigan.
Yale University.
‡
§
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0.1021/jm980323u CCC: $15.00 © 1998 American Chemical Society
Published on Web 12/01/1998

5
258 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 26
Qiu et al.
Sch em e 1a
F igu r e 1. Chiral HPLC of enantiomers 1a , 2a , 19, and 20.
Chiralpak AD column (10 µm, 4.6 × 250 mm, MeOH as eluent,
flow rate 1 mL/min, detection at 261 nm, attenuation 16). The
2
0-µL aliquots of the following solutions in MeOH were
injected: panel A, (R)-(-)-synadenol (1a ) from deamination,
.25 mg/mL; panel B, (S)-(+)-synadenol (2a ), 0.25 mg/mL;
0
panel C, (()-synadenol (1a + 2a ), 0.5 mg/mL; panel D, (R)-
synadenol (1a ) and R,E-isomer 19 (1.5:1), 0.5 mg/mL; panel
E, (()-E-isomers 19 + 20, 0.5 mg/mL; panel F, (()-synadenol
(1a + 2a ) and the (()-E-isomers 19 + 20, 1 mg/mL; panel G,
a
(
a) Adenosine deaminase, pH 7.5; (b) Ac2O, pyridine; (c)
+
-
[
Me2NdCHCl] Cl , CHCl3, 4; (d) NH3, MeOH, 4.
1
1
8
:1 mixture of 1a + 19 (panel D) and 1a (panel A); panel H,
:1 mixture of 1a + 19 (panel D) and 2a (panel B, attenuation
). The retention times (min, panel F) for each enantiomer
Syn th esis
1
Previously, in a preliminary experiment, we found
that synadenol (1a + 2a ) is a substrate for adenosine
deaminase from calf intestine. The reaction kinetics
indicated that after 48 h the deamination stopped at
approximately 50% conversion. This result suggested
that digestion with adenosine deaminase could possibly
be utilized for resolution of enantiomers. A similar
were as follows: 8.45 (19), 9.91 (20), 10.25 (2a ), and 10.99
(1a ).
5
approach was used before in our laboratory for resolu-
tion of the anti-HIV agent adenallene (3 + 4). In a
preparative experiment, deamination of (()-synadenol
(1a + 2a ) afforded the (-)-enantiomer 1a in 39% yield,
whereas the deaminated product, (+)-hypoxanthine
derivative 5, was obtained in 44-48% yield (Scheme 1).
Compound 5 was converted to (+)-synadenol (2a ) as
follows. Acetylation furnished the acetoxy derivative 6
in almost quantitative yield. Chlorination by N,N-
6
dimethylchloromethyleneammonium chloride in reflux-
ing CHCl3 gave 6-chloropurine derivative 7 (88%).
Ammonolysis with NH3 in methanol at 100 °C in an
autoclave afforded (+)-synadenol (2a ) in 96% yield. It
is interesting to note that similar conditions proved
5
destructive for an analogous allene derivative indicat-
ing a higher stability of the methylenecyclopropane
system. The optical purity of enantiomers 1a (95% ee)
and 2a (96% ee) was determined by chiral HLPC on a
Chiralpak AD column using methanol as an eluent
F igu r e 2. CD spectra of enantiomers of synadenol (1a + 2a )
and adenallene (3 + 4). For details see Experimental Section:
(
s) (R)-(-)-synadenol (1a ); (‚ ‚ ‚) (R)-(-)-adenallene (3); (- ‚ -
‚
- ‚) (S)-(+)-synadenol (2a ); (- - -) (S)-(+)-adenallene (4).
was observed7 although only in a limited number of
cases. The values of optical rotations of 1,3-disubstituted
methylenecyclopropane enantiomers are smaller than
those of the corresponding allenes. This was also
observed in our case because an absolute value of optical
rotation [R]D of (-)- and (+)-synadenol (1a and 2a ) is
120-123°, whereas that of (R)-(-)- and (S)-(+)-adenal-
(Figure 1, panels A-C).
Absolu te Con figu r a tion
Synadenol (1a + 2a ) and adenallene (3 + 4) have the
same type of chromophore (a double bond attached to
the adenine base), and the center of dissymmetry either
is a part of the chromophore (allene, 3 and 4) or is
located next to it (methylenecyclopropane, 1a and 2a ).
In fact, their UV spectra are almost superimposable. It
seemed then possible that enantiomers of the same sign
of optical rotation may have the same absolute config-
uration. Some support for such a reasoning is in the fact
that a correlation between the absolute configuration
of optically active allenes and methylenecyclopropanes
5
lene amounts to 179-181°. Similarity of CD spectra of
(R)-(-)- and (S)-(+)-adenallene (3 and 4) with (-)-
and (+)-synadenol (1a and 2a ) (Figure 2) is also in line
with the supposition that enantiomers with the same
sign of Cotton effect may have the same absolute
configuration. Enantiomers 1a , 2a , 3, and 4 display the
Cotton effects between 230 and 240 nm and a less-well-

(R)-(-)- and (S)-(+)-Synadenol
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 26 5259
Sch em e 2a
a
(a) KOH, MeOH/H2O; (b) 1. iBuOCOCl, NEt3, (R)-2-phenylglycinol, 2. separation; (c) 1 M H2SO4, THF; (d) HCl, EtOH; (e) Br2, CCl4;
(f) DIBALH, THF; (g) Ac2O, pyridine; (h) adenine, K2CO3, DMF, 4; (i) NH3/MeOH.
developed maximum at 260 nm (λmax in the UV spec-
trum). It is noteworthy that molar ellipticity at 230-
similar reaction of racemic 9 was recently described.3,8
Compounds 14 could have been successfully used for an
alkylation-elimination procedure with adenine. How-
1
2
40 nm is significantly higher in enantiomers of
adenallene 3 and 4 than synadenol 1a and 2a . This may
be related to the fact that the chromophore is directly a
part of the chiral axis in allenes 3 and 4, whereas in
the case of methylenecyclopropanes 1a and 2a it is
located next to the chiral center.
ever, to avoid any potential racemization of the chiral
center next to the carboxylate function, the latter was
reduced with diisobutylaluminum hydride (DIBALH) to
give hydroxymethyl derivatives 15 in 75% overall yield
based on four steps from amide 12. This procedure was
3
used previously in the synthesis of racemic synadenol
Further support for a tentative assignment of R-
configuration to the (-)-enantiomer 1a comes from the
(
1a + 2a ). Acetylation then furnished acetates 16 (96%).
3
5
The racemic counterparts of 16 were recently described.
Alkylation-elimination procedure with adenine3 af-
forded a mixture of acetates 17 and 18 in 86% yield.
Deprotection with methanolic ammonia giave (R)-syn-
adenol (1a ) and the corresponding E-isomer 19 in
quantitative yield, and the ratio of 1a :19 was 1.5:1 as
determined by chiral HPLC (Figure 1, panel D). No
racemization of 1a and 19 was observed.
biological data. We have previously shown that (S)-(+)-
adenallene is deaminated more readily than the (R)-
(-)-enantiomer but the enantioselectivity of their anti-
HIV effect is just the opposite. Because the same trend
of substrate activity toward adenosine deaminase and
anti-HIV potency was observed with (-)- and (+)-
synadenol (1a and 2a ) (Scheme 1; for HIV data see
Table 1), it seemed quite plausible that (-)-enantiomer
1
a has an R-configuration and (+)-enantiomer 2a an
Enantiomers 1a , 2a , 19, and 20 of the racemic E/Z-
8
1
S-configuration. Nevertheless, Cheng et al. reached
very recently an opposite conclusion which was based
on calculated conformational parameters of a series of
nucleoside analogues, and they assigned an S-configu-
ration to (-)-synadenol (1a ). These authors also resolved
racemic synadenol (1a + 2a ) on a chiral column, but
enantiomers 1a and 2a were characterized only by
optical rotations which are significantly lower ([R]D
isomeric mixture were all resolved on a Chiralpak AD
column (Figure 1, panels C, E, and F) which permitted
the determination of absolute configuraiton of (-)- and
(+)-synadenol (1a and 2a ) obtained from enzymic
deamination (Scheme 1) by a comparison with 1a
without separation of the Z- and E-isomers 1a and 19.
Thus, Figure 1 (panel G) shows that addition of (-)-
synadenol (1a ) obtained from enzymic deamination of
racemate 1a + 2a to a mixture of Z- and E-isomers 1a
and 19 of the R-configuration obtained by enantiose-
lective synthesis from (R)-methylenecyclopropanecar-
boxylic acid (8) (Scheme 2) caused only an increase of
the peak corresponding to the R-enantiomer 1a as
compared with panel D (Figure 1). By contrast, addition
of (+)-synadenol (2a ) resulted in a clear separation of
the three peaks of 1a , 2a , and 19 (Figure 1, panel H).
Therefore, the R-configuration must be assigned to the
(26.3°) than those described herein ([R]D (120-123°).
The absolute configuration of (-)- and (+)-synadenol
1a and 2a ) was determined by two methods: (A)
(
correlation of (-)-synadenol (1a ) with (R)-methylenecy-
clopropanecarboxylic acid9 (8) and (B) single-crystal
X-ray diffraction of hydrochloride of 1a .
A. Cor r ela t ion of (-)-Syn a d en ol (1a ) w it h (R)-
Meth ylen ecyclop r op a n eca r boxylic Acid (8). The
1
0
known ethyl methylenecyclopropanecarboxylate (9)
was hydrolyzed with KOH in aqueous methanol to give
the corresponding racemic acid 10 in 98% yield (Scheme
(-)-enantiomer 1a . It is also worthy of note that
alkylation agent 16 and the corresponding diastereoi-
somers derived from (S)-methylenecyclopropanecar-
). The reported procedure9 using K2CO3 in aqueous
2
9
boxylic acid can be used for the synthesis of enan-
methanol proved unsuitable for this purpose. Compound
0 was then converted to the R,S- and R,R-diastereoi-
tiopure (R)- and (S)-methylenecyclopropane analogues
of nucleobases other than adenine by the procedures
described for the corresponding racemic compounds.1
1
someric amides of (R)-phenylglycinol 11 and 12 which
-3
were then resolved by a column chromatography on
9
silica gel by a modification of the described procedure
The configuration of (R)-methylenecyclopropanecar-
9
in 39% and 41% yield, respectively. Acid hydrolysis of
boxylic acid (8) was previously determined by HPLC
the diastereoisomer 12 gave quantitatively (R)-methyl-
enecyclopropanecarboxylic acid (8) which was readily
esterified to afford ester 13. Adddition of bromine
provided 1S,2S- and 1S,2R-diastereoisomers 14. A
elution profiles of diastereoisomeric (R)-phenylglycinol
derivatives 11 and 12 which were compared with
patterns of similar amides derived from structurally
unrelated compounds. In addition, optical rotations of
9

5
260 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 26
Qiu et al.
in solution (DMSO) was also indicated by the NMR
1
spectra of the racemic compound 1a + 2a. The adenine-
adenine dimer displaying an unusual base pairing
observed in the lattice of the hydrochloride of 1a is of
interest. A dimeric structure involving a different set
of hydrogen bonds was also observed in crystals of (R)-
5
(
-)-adenallene (3).
Our results have unambiguously shown that a recent
8
assignment of the S-configuration of (-)-synadenol (1a )
is in error. The present investigation also provides
conclusive support for the original configurational as-
signment of (R)-methylenecyclopropanecarboxylic acid9
(8).
An tivir a l Activity
Antiviral activity of (R)-(-)- and (S)-(+)-synadenol (1a
and 2a ) was investigated with seven different viruses:
human cytomegalovirus (HCMV), herpes simplex virus
F igu r e 3. ORTEP-3 view of the two independent (-)-synadnol
hydrochloride molecules showing 50% probability displacement
ellipsoids and hydrogen-bonded A and B molecules forming
dimers.
1
and 2 (HSV-1 and HSV-2), Epstein-Barr virus (EBV),
varicella zoster virus (VZV), human immunodeficiency
virus 1 (HIV-1), and hepatitis B virus (HBV) (Table 1).
Most surprisingly, both enantiomers were virtually
equipotent as inhibitors of replication of HCMV virus
in plaque and yield reduction assay (EC50 2.4-2.9 and
EC90 0.5-2.0 µM, respectively). The same phenomenon
was observed in VZV-infected cell culture (EC50 1.5 µM
for both enantiomers). In HBV assay the S-enantiomer
2a was somewhat more active (EC50 9 µM) than the
R-enantiomer 1a (EC50 15.7 µM). (()-Synadenol (1a +
8
, 11, 12, and the S-enantiomer of 8 were not reported.
Therefore, the configuration of (-)-synadenol (1a ) was
independently confirmed by X-ray diffraction of a single
crystal.
B. Cr ysta llogr a p h ic Resu lts. The absolute config-
uration was determined by refinement of the Flack
parameter for (-)-synadenol hydrochloride, which showed
that (-)-synadenol (1a ) has the R-absolute configuration
1
2a ) is only a moderate anti-HSV agent, but some
1
1
according to Cahn, Ingold, and Prelog notation. The
two independent molecules of (-)-synadenol hydrochlo-
ride which constitute the crystallographic asymmetric
unit are presented in Figure 3. Formation of the
hydrochloride has occurred by protonation at N1 of
adenine, the position also favored in adenine nucleo-
enantioselectivity was observed. Thus, the (S)-(+)-
enantiomer 2a with EC50 8.8 (ELISA), 16, and 35 µM
(Vero) is a somewhat better inhibitor of replication of
both HSV-1 and HSV-2 than the (R)-(-)-enantiomer 1a
(EC50 38 (ELISA) and >50 µM in Vero cell culture). In
EBV/H-1 assay the R-enantiomer 1a was more potent
(EC50 0.09 µM) than S-enantiomer 2a (EC50 0.62 µM).
However, it is clear that both enantiomers are effective
inhibitors of EBV. Addition of coformycin, an inhibitor
of adenosine and AMP deaminase, did not influence the
anti-EBV potency of 1a and 2a . This result indicated
that neither of the enantiomers is subject to intracel-
lular enzymic deamination. The (S)-(+)-synhypoxanthol
(5) was inactive. Although (()-synadenol (1a + 2a ) is
1
2
sides. The hydroxyl group of the B molecule is disor-
dered; there are three partial occupancy positions for
the O16 oxygen atom (occupancies refined to 0.48, 0.24,
and 0.33 for O16b,c,d, respectively), and consequently
two hydrogen atoms, one at O16 and one at C15, could
not be reliably positioned from the difference map. Apart
from this, the geometrical parameters (bonds and
angles) of both molecules are in good agreement with
commonly accepted values. Structural details are avail-
able as Supporting Information. The conformations of
the two crystallographically independent molecules
differ in the side chain. The cyclopropyl ring of the A
molecule is coplanar with the plane of the adenine rings
system plus N10 and C11, as opposed to the B molecule
where the angle between normals to these planes is
1
only a moderate inhibitor of HIV-1, the (R)-(-)-enan-
tiomer 1a showed an EC50 value of 11 µM, whereas the
(S)-(+)-enantiomer 2a was inactive. A similar pattern
of activity was found in adenallene (3 + 4) which is a
more potent anti-HIV agent than synadenol (1a + 2a ),
where the (R)-(-)-enantiomer 3 is significantly more
5
effective than the (S)-(+)-enantiomer 4.
1
2.9(6)°. In the crystal, the molecules are stacked along
The tested viruses can then be divided into three
groups according to their response toward enantiomers
1a and 2a : (i) no enantioselectivity (HCMV, VZV), (ii)
moderate enantioselectivity favoring the (S)-(+)-enan-
tiomer 2a (HSV-1, HSV-2, HBV), and (iii) enantioselec-
tive preference for the (R)-(-)-enantiomer 1a (EBV,
HIV-1).
Many antiviral nucleoside analogues are essentially
prodrugs which have to be converted to effective agents
(triphosphates) inhibiting the respective viral DNA
polymerase or reverse transcriptase. Multiple enzymes
(nucleoside and nucleotide kinases, nucleotidases, etc.)
involved in the activation process (phosphorylation) can
the b-axis direction and bound together by a three-
dimensional hydrogen bond network. The two crystal-
lographically independent molecules are paired by two
symmetrical hydrogen bonds between N7 and the amino
N10 group of the adenines; the dimers are bonded to
other dimers through interactions involving N1, N10,
and the chloride ions and through hydroxy groups
(Table 2).
The X-ray diffraction studies also showed that the
conformation of the base (adenine) is anti in both the
hydrochloride of 1a (Figure 3) and the free base (Sup-
porting Information, Figure 1). The same conformation

(R)-(-)- and (S)-(+)-Synadenol
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 26 5261
Ta ble 1. Antiviral Activity of (()-Synadenol (1a + 2a ), R-(-)-Enantiomer 1a , and S-(+)-Enantiomer 2a
antiviral activity, 50% inhibitory concentration (EC50, µM)a
HSV-1
compd
HCMV HFF
BSC-1b
Vero
HSV-2 Vero
EBV H-1
HIV-1 CEM-SS
17e
HBV 2.215
VZV HEL
c
d
d
d
1
1
2
a + 2a
2.1 (1.3)
26
38
28
>50
16
59
>50
35
0.2
17
15.7
9
1.5
1.5
1.5
f
c
0.09g
e
a
2.9 (2.0)
11
f
c
h,i
>100e
a
2.4 (0.5)
8.8
0.63
5
j
3.5j
k
k
j
0.003l
m
k
control
7.4
9
25
1.4
3
a
For details of antiviral assays, see ref 1. ELISA. c Plaque reduction. d Yield reduction (EC90, µM). e The HIV-1 assay in MT-4 cells
b
8
f
g
showed a similar pattern of antiviral effect: EC50 26 µM (1a + 2a ), 13 µM (1a ), and 50 µM (2a ). Optical purity 95-96%. EC50 0.13 µM
in the presence of 10 µM coformycin. The same value was obtained in the presence of 10 µM coformycin. EC50 of (S)-hypoxanthine
derivative 5 was >50 µM. j Ganciclovir. Acyclovir. l AZT. m Zalcitabine (ddC).
h
i
k
Ta ble 2. Hydrogen Bonds (Å)a
donor acceptor D-H
N1A H1A
N1B H1B
N10A H10A N7B
N10A H10B Cl1a
N10B H10C N7A
N10B H10D Cl2
O16A H16A O16Ab
Adenosine deaminase from calf intestine (type II, Sigma
Chemical Co., St. Louis, MO; 30 mg, 45 units) was added, and
the mixture was stirred at room temperature. The progress
H
H‚‚‚A
D‚‚‚A
angle (deg)
Cl2a
Cl1
0.81(6) 2.29(6) 3.057(3)
0.88(5) 2.15(5) 3.014(4)
0.80(6) 2.28(6) 3.028(5)
0.79(6) 2.39(6) 3.106(4)
0.94(4) 2.03(5) 2.934(5)
0.91(5) 2.25(5) 3.131(4)
1.11(9) 1.83(9) 2.793(5)
157(5)
165(4)
157(5)
152(5)
162(4)
162(4)
141(7)
2 2
of the reaction was followed by TLC (CH Cl -MeOH, 9:1). The
deamination stopped after 72 h, and the ratio of (1a + 2a ):5
was 7:3 as determined by UV spectrophotometry of the eluted
spots. Addition of another two portions of enzyme (30 mg every
2
4 h) did not improve this ratio. The mixture was lyophilized,
and the resultant product was sonicated with several portions
of CH Cl -MeOH (1:1, first 100 mL and then 40 mL) until no
2
2
a
UV absorption was detectable in the solvent. The filtrate and
washings were combined and evaporated. The residue was
adsorbed on silica gel (4 g) and loaded on a column made of
Translation symmetry codes: a ) 1/2+x, -1/2+y, z; b ) 1/2-
x, -1/2+y, -z.
the same material. Chromatography using CH
2 2
Cl -MeOH (9:
each exhibit a different enantioselectivity toward nu-
cleoside analogues.13 For example, enantioselectivity (or
lack of it) of carbovir and zalcitabine (ddC) is determined
1
) as an eluent afforded (-)-synadenol (1a ) (135 mg, 66.5%),
25
[R]
D
-59.1° (c 0.04, MeOH), and elution with CH
2
Cl
2
-MeOH
2
5
(4:1) gave (+)-synhypoxanthol (5) (85 mg), [R]
D
91.8° (c 0.1,
by enzymes involved in the phosphorylation path-
MeOH).
way.1
4,15
Nevertheless, target viral polymerases (e.g.,
The obtained (-)-synadenol (1a ) (135 mg, 0.62 mmol) was
subjected to a second round of deamination (60 mg, 90 units
of enzyme) under the conditions described above. TLC showed
a ratio of (1a + 2a ):5 as 7:3 after 40 h. Prolonged reaction
time did not improve the conversion. Workup followed by
chromatography as described above gave optically enriched
HSV-1 and HBV) can also respond differently toward
enantiomeric triphosphates.16,17
(()-Synadenol (1a + 2a ) is unique in this aspect of
antiviral activity since its enantioselectivity is com-
pletely lost with HCMV, VZV, and, to a large extent,
HBV, HSV-1, and HSV-2, but it is reversed in the case
of HIV-1 and EBV (Table 1). To the best of our
knowledge, such varied pattern of enantioselectivity has
not been observed with any broad-spectrum antiviral
agent. Currently, it is assumed that triphosphates of
enantiomers 1a and/or 2a are the actual inhibitors of
viral DNA polymerases. The loss of enantioselectivity
toward HCMV, VZV, and HBV indicates a little differ-
ence in the activation capability of enantiomers 1a and
2
5
(-)-synadenol (1a ), 95 mg, 47.5%, [R]
D
-112.0° (c 0.095,
MeOH), and (+)-synhypoxanthol (5), 40 mg, [R]25
MeOH). The combined portions of product 5 (125 mg) were
rechromatographed on silica gel using CH Cl -MeOH (4:1) to
89.0° (c 0.1,
D
2
2
afford optically pure (+)-synhypoxanthol (5), 96 mg, 47.8%,
2
5
[
°
R]
D
112.5° (c 0.08, MeOH), as a white solid: mp 235-240
C; UV (EtOH) λmax 228 nm (ꢀ 31 300); IR (KBr) 3450 (OH),
1600 and 1670-1690 (purine ring and olefin), 1040 cm-1
1
2
(cyclopropane ring); H NMR (DMSO-d
6
) δ 1.21 (ddd, 1 H, J
3
4
2
)
8.4 Hz, J trans ) 5.4 Hz, J ) 1.8 Hz) and 1.48 (td, 1 H, J )
3
4
3
J
cis ) 8.7 Hz, J ) 1.5 Hz, H3′), 2.12 (dqd, 1 H, J cis ) 9.0 Hz,
3 4
3
J
trans ) J ) 5.4 Hz, J ) 1.8 Hz, H4′), 3.28 (dt, overlapped
2 3
2
a and inhibition of the target polymerases by the
with H
2
O, 1 H, J ) 11.1 Hz, J ) 7.2 Hz) and 3.72 (dd, 1 H,
respective triphosphates. The differences between enan-
tiomers are more accentuated in the case of HBV, HSV-
2
3
3
J ) 10.8 Hz, J ) 5.1 Hz, H5′), 5.09 (t, 1 H, J ) 4.8 Hz, OH),
4
7
.33 (d, 1 H, J ) 1.8 Hz, H1′), 8.07 (s, 1 H, H
), 12.41 (s, 1 H, NH); 13C NMR 6.67 (C3′), 19.62 (C4′), 63.11
(C5′), 110.50 (C1′), 117.58 (C2′), 124.18 (C ), 137.56 (C ), 146.61
); EI-MS 217 (M - H, 26.3), 200
O, 58.6), 187 (40.5), 159 (11.7), 148 (16.1), 135
hypoxanthine - H, 100.0). Anal. (C10 ‚0.6H O) C, H,
2
), 8.69 (s, 1 H,
1
, HSV-2, and, particularly, HIV-1 and EBV (Table 1).
H
8
These results suggest that stereorecognition patterns
of enzymes responsible for activation (phosphorylation)
or the respective viral DNA polymerases toward enan-
tiomers 1a and 2a could vary significantly with the type
of virus involved. The mechanism of action of methyl-
enecyclopropane analogues 1a and 2a may then vary
not only with the class of virus involved (HCMV, VZV,
and HBV vs HIV) but also within the same class
5
8
(
(
(
C
2
), 147.19 (C
4 6
), 156.97 (C
M - H
2
H
10
N
4
O
2
2
N.
In another experiment (()-synadenol (1a + 2a ) (450 mg,
2
.07 mmol) furnished optically enriched (-)-enantiomer 1a
(200 mg, 44.4%) and (+)-synhypoxanthol (5) (200 mg, 44.4%).
The combined portions of optically enriched adenine analogue
1
a (280 mg, 1.29 mmol) from both experiments were incubated
(HCMV, HSV-1, HSV-2, VZV, or EBV). These intriguing
with adenosine deaminase (100 mg, 150 units) in phosphate
buffer (250 mL) as described above. The ratios of 1a :5 were
mechanistic possibilities remain to be fully explored.
9
4:6 after 24 h and 93:7 after 48 h. Workup and purification
Exp er im en ta l Section
as described above gave optically pure 1a (250 mg, 38.5%
Gen er a l Meth od s. See ref 1. The circular dichroism (CD)
spectra were determined using J ASCO J -600 CD spectrometer
overall yield based on starting material 1a + 2a , 650 mg, 2.99
5
25
mmol), [R]
D
-120.0° (c 0.075, MeOH), and optically enriched
25
in 0.05 M Na
-)-Syn a d en ol (1a ) a n d (+)-Syn h yp oxa n th ol (5). A
suspension of (()-synadenol (1a + 2a ; 200 mg, 0.92 mmol) in
.05 M Na HPO (pH 7.5, 100 mL) was briefly sonicated.
2
HPO
4
(pH 7.0).
hypoxanthine analogue 5 (20 mg, 7.1%), [R]
D
60.6° (c 0.11,
(
MeOH). Optical purity of 1a was 95% as determined by chiral
HPLC (Figure 1, panel A): mp 237-238 °C; UV (EtOH) λmax
277 nm (shoulder, ꢀ 8 800), 260 (ꢀ 12 300), 227 (ꢀ 26 900); IR,
0
2
4

5
262 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 26
Qiu et al.
1H NMR, and EI-MS were identical to the racemic material1
filtrate and washings were concentrated, and the crude product
1
1
a + 2a . Anal. (C10 O), C, H, N.
O-Acet yl-(+)-syn h yp oxa n t h ol (6). A mixture of (+)-
synhypoxanthol (5) (131 mg, 0.6 mmol) and acetic anhydride
1.0 mL, 10.6 mmol) in pyridine (10 mL) was stirred at room
H
11
N
5
was distilled to give compound 9 (36.60 g, 85.8%). The H NMR
spectrum was identical to that reported.10
(()-Meth ylen ecyclop r op a n eca r boxylic Acid (10). Po-
tassium hydroxide (9.82 g, 175 mmol) was added to a mixture
of ester 9 (12.61 g, 0.1 mol) in MeOH-H O (4:1, 120 mL) with
2
stirring and ice cooling. The stirring was continued at room
temperature for 18 h. Volatile components were removed in
vacuo, and the residue was dissolved in water (60 mL). The
pH was adjusted to 2.0 by a cautious addition of 4 M HCl with
stirring and ice cooling. The resultant solution was extracted
(
temperature for 16 h. Volatile components were removed in
vacuo, and the residue was passed through a short silica gel
column using CH Cl -MeOH (92:8) as an eluent to give
2 2
25
acetate 6 (155 mg, 99%): mp 234-237 °C; [R]
D
+108.6° (c
0
3
.084, MeOH); UV (EtOH) λmax 227 nm (ꢀ 27 300); IR (KBr)
460 and 3140 (NH), 1735 (ester), 1695 and 1595 (hypoxan-
-
1
1
with CH
(Na SO
98.3%) as a colorless oil: H NMR (CDCl
2
Cl
2
(10 × 100 mL). The combined extracts were dried
thine ring and olefin), 1040 cm (cyclopropane); H NMR
2
3
4
2
4
) and evaporated to give carboxylic acid 10 (9.64 g,
(DMSO-d
6
) δ 1.38 (ddd, 1 H, J ) 11.7 Hz, J trans ) 5.4 Hz, J
1
2
2
3
4
3
) δ 1.72 (ddt, 1 H, J
)
1.8 Hz) and 1.63 (td, 1 H, J ) J cis ) 8.9 Hz, J ) 1.5 Hz,
3 3
3
4
2
) 9.0 Hz, J cis ) 8.3 Hz, J ) 2.4 Hz) and 1.89 (ddt, 1 H, J )
H
3′), 1.88 (s, 3 H, CH
3
), 2.32 (dqd, 1 H, J cis ) 9.0 Hz, J trans
)
3
4
3
3
4
2
9.3 Hz, J trans ) 4.8 Hz, J ) 2.4 Hz, CH
2
), 2.27 (ddt, 1 H, J cis
J ) 5.4 Hz, J ) 1.8 Hz, H4′), 3.93 (dd, 1 H, J ) 11.3 Hz,
3
4
3
2
3
) 8.3 Hz, J trans ) 4.8 Hz, J ) 2.1 Hz, CH), 5.55-5.59 (m, 2
H, J ) 2.1 Hz, dCH ), 9.5 (bs, 1 H, CO H).
2 2
J ) 7.4 Hz) and 4.14 (dd, 1 H, J ) 11.4 Hz, J ) 6.9 Hz,
4
4
H
1
2
1
2
5′), 7.37 (q, 1 H, J ) 1.8 Hz, H1′), 8.06 (s, 1 H, H ), 8.36 (s,
1
3
N-[(R)-1′-P h en yl-2′-h yd r oxyet h yl]-(1S)-m et h ylen ecy-
clop r op a n eca r boxa m id e (11) a n d N-[(R)-1′-P h en yl-2′-
h yd r oxyet h yl]-(1R)-m et h ylen ecyclop r op a n eca r b oxa m -
H, H
8
), 12.40 (bs, 1 H, NH); C NMR 7.79 (C3′), 15.89 (CH
3
),
),
0.89 (C4′), 65.87 (C5′), 111.56 (C1′), 117.03 (C2′), 124.19 (C
37.65 (C ), 146.68 (C ), 147.42 (C ), 156.99 (C
5
8
2
4
6
), 170.53 (CO,
9
ide (12). The described procedure was followed with carboxylic
acetate); EI-MS 260 (M, 26.1), 218 (M - Ac + H, 5.4), 201 (M
OAc, 66.5), 174 (15.6), 137 (hypoxanthine + H, 100.0);
HRMS calcd for C12 M 260.0909, found M 260.0906.
Anal. (C12 ) C, H, N.
+)-(Z)-6-Ch lor o-9-((2-(acetoxym eth yl)cyclopr opyliden -
e)m eth yl)p u r in e (7). N,N-Dimethylaminochloromethylene-
ammonium chloride in CHCl (0.2 M, 3.5 mL, 8.7 mmol) was
added to a suspension of acetate 6 (150 mg, 0.58 mmol) in
CHCl (15 mL). The mixture was refluxed for 1 h. The solvent
was evaporated, and the residue was chromatographed on a
silica gel column using CH Cl -MeOH (96:4) to give product
(141 mg, 87.8%): mp 122-125 °C; [R]
MeOH); UV (EtOH) λmax 260 nm (shoulder, ꢀ 8 200), 228 (ꢀ
8 500); IR (KBr) 1730 (ester), 1590 and 1563 (purine ring and
acid 10 (9.63 g, 98.2 mmol), triethylamine (30.1 mL, 0.216 mol),
isobutyl chloroformate (12.7 mL, 98.2 mmol), and (R)-phenyl-
glycinol (13.47 g, 98.2 mmol) in THF (400 mL), but the
separation of diastereoisomers 11 and 12 was modified.
-
12 4 3
H N O
12 4 3
H N O
(
Several chromatographic runs on silica gel columns using CH
Cl -THF (95:5 f 85:15) followed by CH Cl -MeOH (95:5 f
:1) afforded the R,R-diastereoisomer 12 (8.78 g, 41.2%) and
2
-
2
2
2
3
9
the R,S-diastereoisomer 11 (8.34 g, 39.4%) as white solids.
3
The reported9 combined yield of both diastereoisomers was
5
8%.
2
2
2
3
S,R-Isom er 11: mp 133-136 °C; [R]
D
-149.4° (c 0.87,
3 4
2
5
7
D
93.9° (c 0.095,
1
2
EtOH); H NMR (CDCl
3
) δ 1.65 (tt, 1 H, J ) J cis ) 9.0 Hz, J
2
3
4
)
2.4 Hz) and 1.75 (ddt, 1 H, J ) 9.0 Hz, J trans ) 5.0 Hz, J
2.4 Hz, CH
trans ) 4.8 Hz, J ) 2.4 Hz, CH of cyclopropane), 2.52 (bs, 1
2
3
)
2
of cyclopropane), 2.21 (ddt, 1 H, J cis ) 8.7 Hz,
-
1
1
olefin), 1030 cm (cyclopropane); H NMR (DMSO-d
6
) δ 1.40
3
4
J
2
3
4
(
ddd, 1 H, J ) 9.2 Hz, J trans ) 5.1 Hz, J ) 2.0 Hz) and 1.73
2
3
H, OH), 3.87 (dd, 1 H, J ) 11.4 Hz, J ) 5.2 Hz) and 3.92 (dd,
1
2
3
4
(
td, 1 H, J ) J cis ) 9.0 Hz, J ) 1.9 Hz, H3′), 2.04 (s, 3 H,
2
3
3
H, J ) 11.4 Hz, J ) 5.2 Hz, CH
2
O), 5.07 (dt, 1 H, J ) 5.7
), 5.55-5.59 (m, 2 H, J ) 2.1 Hz,
), 6.26 (bs, 1 H, NH), 7.26-7.42 (m, 5 H, C ).
-139.0° (c 0.81,
2
3
CH
3
), 2.26-2.37 (m, 1 H, H4′), 3.77 (dd, 1 H, J ) 11.4 Hz, J
3
Hz, J ) 5.2 Hz, CHC
dCH
R,R-Isom er 12: mp 137-139 °C; [R]
6 5
H
2
3
)
7
and H
8.7 Hz) and 4.53 (dd, 1 H, J ) 11.4 Hz, J ) 5.7 Hz, H5′),
.55 (q, 1 H, J ) 1.8 Hz, H1′), 8.74 and 8.76 (2s, 1 H each, H
2
6 5
H
4
2
2
5
1
3
D
8
); C NMR 7.46 (C3′), 15.98 (CH
3
), 20.80 (C4′), 66.36
1
2
3
4
EtOH); H NMR (CDCl
3
) δ 1.63 (tt, 1 H, J ) J cis ) 9.0 Hz, J
(
(
C
C
5′), 111.11 (C1′), 116.25 (C2′), 131.41 (C
5 8
), 142.32 (C ), 150.32
2
3
4
)
2.4 Hz) and 1.77 (ddt, 1 H, J ) 9.0 Hz, J trans ) 4.7 Hz, J
2.4 Hz, CH
trans ) 4.8 Hz, J ) 2.4 Hz, CH of cyclopropane), 2.32-2.49
4
), 151.18 (C ), 152.44 (C ), 170.56 (CO, acetate); EI-MS 280
2
6
3
)
2
of cyclopropane), 2.19 (ddt, 1 H, J cis ) 10.8 Hz,
and 278 (M, 7.3, 22.0), 238 and 236 (M - Ac + H, 2.6, 7.0),
21 and 219 (M - OAc, 36.2, 81.1), 207 (8.9), 183 (8.4), 157
and 155 (6-chloropurine + H, 29.4, 85.9), 82 (63.0), 43 (Ac,
3
4
J
2
2
3
(
bs, 1 H, OH), 3.85 (dd, 1 H, J ) 11.4 Hz, J ) 4.2 Hz) and
2
3
3
.91 (dd, 1 H, J ) 11.4 Hz, J ) 5.4 Hz, CH
2
O), 5.07 (dt, 1 H,
), 5.57-5.62 (m, 2 H, dCH ),
.27 (bs, 1 H, NH), 7.28-7.42 (m, 5 H, C ).
3
5
1
2
00.0); HRMS calcd for C12
78.0568. Anal. (C12 11ClN
+)-Syn a d en ol (2a ). A mixture of compound 7 (130 mg,
.47 mmol) in methanolic ammonia (20%, 70 mL) was heated
H
4 2
11 ClN
4
O
2
M 278.05705, found
3
3
J ) 6.6 Hz, J ) 5.1 Hz, CHC
6
H
5
2
H
O ) C, H, Cl, N.
6
6 5
H
(
(
1R)-(-)-Meth ylen ecyclop r op a n eca r boxylic Acid (8).
0
9
The described procedure was modified as follows. The R,R-
diastereoisomer 12 (8.68 g, 39.95 mmol) in THF (150 mL) and
in a stainless steel bomb at 100 °C for 18 h. After cooling,
the contents were evaporated. The residue was chromato-
graphed on silica gel using CH Cl -MeOH (9:1 f 85:15) to
2 2
give (S)-(+)-synadenol (2a ; 91 mg, 90%) after drying at <0.01
mmHg and 100 °C for 2 h. The optical purity of 2a was 96%
as determined by chiral HPLC (Figure 1, panel B): mp 233-
2 4
aqueous H SO (1 M, 150 mL) was refluxed for 48 h. After
cooling, the mixture was evaporated to about half of the
original volume. Saturated aqueous NaCl (80 mL) was added
to the residue and extracted with ether (300 mL, then 4 × 80
mL). The extraction was monitored by TLC in hexane-ethyl
2
1
8
35 °C (modification change between 213 and 230 °C); [R]25
D
acetate (1:1.5). The combined extracts were dried (Na
2 4
SO ) and
23.0° (c 0.073, MeOH); UV (EtOH) λmax 277 nm (shoulder, ꢀ
evaporated leaving a yellow oil of compound 8 (3.92 g, 100%),
1
25
600), 261 (ꢀ 12 200), 227 (ꢀ 26 700); IR, H NMR, and MS
[R]
-17.0° (c 1.0, EtOH).
D
1
were identical to the racemic compound 1a + 2a . Anal.
Eth yl (1R)-(-)-Meth ylen ecyclopr opan ecar boxylate (13).
The (R)-carboxylic acid 8 (3.92 g, 39.95 mmol) was stirred at
room temperature in ethanolic HCl (1 M, 60 mL) for 24 h.
Water (60 mL) was added, and the mixture was extracted with
pentane (8 × 80 mL). The combined extracts were washed with
(C
10
H
11
N
5
O) C, H, N.
Eth yl (()-Meth ylen ecyclop r op a n eca r boxyla te (9). Eth-
anol (2.0 mL) was added to a mixture of ethyl 2-bromo-2-
1
0
methylcyclopropanecarboxylate (70.00 g, 0.34 mol), ether (250
mL), and sodium hydride (50% in mineral oil, 32.64 g, 0.68
mol) with stirring at room temperature. Within a few minutes,
exothermic reaction started resulting in a vigorous refluxing
of ether. Another portion of ethanol (2.0 mL) was added after
3
water, saturated aqueous NaHCO , water, and brine (80 mL
each). The extraction was monitored by TLC in hexane-ethyl
acetate (19:1). After drying (Na SO ) the solvents were distilled
2
4
off at an atmospheric pressure, and the crude ester 13 was
used directly in the next step.
1
h, and the reaction mixture was refluxed for 16 h. After the
mixture had cooled, pentane (150 mL) was added and the
mixture was filtered through a Celite pad. The insoluble
portion was washed with pentane (3 × 50 mL). The combined
E t h yl (2R)-2-Br om o-2-(b r om om et h yl)-(1S)-cyclop r o-
p a n eca r boxyla te a n d Eth yl (2S)-2-Br om o-2-(br om om -
eth yl)-(1S)-cyclop r op a n eca r boxyla te (14). Bromine (2.25

(R)-(-)- and (S)-(+)-Synadenol
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 26 5263
mL, 43.67 mmol) was added to a solution of (R)-ester 13 (39.95
mmol) from the preceding experiment in CCl (80 mL) over
including Friedel pairs; 2θmax ) 52°; h ) -25:25, k ) -5:6, l
) -27:28.
4
1
5 min at 0 °C with stirring. After 0.5 h, TLC (hexane-ethyl
Str u ctu r e Solu tion , Absolu te Con figu r a tion , a n d Re-
fin em en t. The structure was solved by direct methods using
the program19 SHELXS-97 and refined using SHELXL-97.
A total of 3132 reflections with I > 2σ(I) were included in the
refinement. All hydrogen atoms (except one at O16b and one
at C15b of the B molecule) were found from the difference map.
All the atoms were refined (non-hydrogen atoms anisotropi-
cally and hydrogen isotropically; 416 parameters). Final R )
0.0439. The highest peak in the final difference map was 0.17
acetate, 9:1) showed a complete reaction. Volatile components
20
were removed in vacuo to afford a yellow oil of compound 14
(
11.02 g) which was used in the next step. The 1H NMR
1
spectrum was identical to that of the racemic mixture except
for the E/Z-ratio which was 1:9. Chiral HPLC on Chiralpak
AD in hexane-2-propanol (9:1, 1 mL/min, detection at 220 nm)
showed no detectable amount of the corresponding 1R,2S- and
1
R,2R-diastereoisomers. The retention times (min) of all four
1
3
3
diastereoisomers were as follows: 4.34 (1S,2S), 4.47 (1R,2S),
.65 (1S,2R), and 6.02 (1R,2R).
2R)-2-Br om o-2-(br om om eth yl)-(1S)-(h yd r oxym eth yl)-
cyclop r op a n e a n d (2S)-2-Br om o-2-(br om om eth yl)-(1S)-
h yd r oxym eth yl)cyclop r op a n e (15). The procedure de-
e/Å , lowest -0.18 e/Å . The absolute configuration was
21
5
established by refinement of the Flack parameter: x ) -0.03-
(
0
8). The discrepancy factor for the inverted structure was R )
(
.0451, and the Flack parameter was 0.97(8).
An tivir a l Assa ys. Assays used for biological evaluation of
(
(
()-synadenol (1a + 2a ), (R)-(-)-synadenol (1a ), and (S)-(+)-
scribed for the preparation of a mixture of all four diastereo-
isomers of ethyl 2-bromo-2-(bromomethyl)cyclopropanecar-
1
synadenol (2a ) were described in detail previously. The
following virus-infected cultures were employed: human cy-
tomegalovirus (HCMV) in human foreskin fibroblasts (HFF;
plaque and yield reduction assay), herpes simplex virus 1
(HSV-1) in BSC-1 cells (ELISA) and in Vero cells (plaque
reduction), herpes simplex virus 2 (HSV-2) in Vero cells
3
boxylate was followed with 1S,2R- and 1S,2S-diastereoiso-
mers 14. Chromatography on a silica gel column using
hexane-ethyl acetate (4:1 f 1.5:1) as eluents gave compound
1
1
5 (7.31 g, 75% overall yield based on amide 12). The H NMR
3
spectrum was identical to that reported previously except for
the diastereoisomeric composition.
(plaque reduction), Epstein-Barr virus (EBV) in H-1 cells,
human immunodeficiency virus 1 (HIV-1) in CEM-SS cells,
and hepatitis B virus (HBV) in 2.2.15 cell culture. The varicella
zoster virus (VZV) was assayed by growth inhibition of human
embryonic lung (HEL) cells. The results are summarized in
Table 1.
(
2R)-2-Br om o-2-(br om om eth yl)-(1S)-(a cetoxym eth yl)-
cyclop r op a n e a n d (2S)-2-Br om o-2-(br om om eth yl)-(1S)-
a cetoxym eth yl)cyclop r op a n e (16). Again, the described
(
3
procedure was followed. Acetylation of 15 (7.31 g, 29.97 mmol)
gave acetate 16 (8.23 g, 96% yield) which was used as such in
VZV Gr ow th In h ibition Assa y. Confluent HEL cells in
1
the next step. The H NMR spectrum was identical to that
1
2-well plates were inoculated with 30-40 pfu/well of virus.
3
reported except for the diastereoisomeric composition.
After 1-h adsorption at 37 °C, the inoculum was replaced with
medium containing 10% FBS and tested compounds at various
concentrations. Each dosage was duplicated. The infected cells
were harvested at 72-h postinfection. The cells were lysed by
(
R),(Z)-9-((2-(Acetoxym eth yl)cyclopr opyliden e)m eth yl)-
a d en in e (17) a n d (R),(E)-9-((2-(Acetoxym eth yl)cyclop r o-
p ylid en e)m eth yl)a d en in e (18). The alkylation-elimination
3
procedure performed with adenine (43 mg, 0.32 mmol) and
“
freeze-thaw”, and the lysate was treated with protease K and
2
-bromo-2-(bromomethyl)-(1S)-(acetoxymethyl)cyclopropanes
RNase. The DNA was then subjected to slot-blot assay as
described previously. The viral DNA was detected by hy-
bridization with P-labeled DNA fragment of VZV thymidine
(16; 45 mg, 0.16 mmol) in DMF (5 mL) in the presence of K
2
-
22
CO
3
(221 mg, 1.60 mmol) at 100 °C for 24 h afforded the title
32
compounds 17 and 18 (35 mg, 85.8% yield) after column
chromatography in CH Cl
EtOH) λmax 277 nm (shoulder, ꢀ 7 600), 261 (ꢀ 11 200), 226 (ꢀ
kinase (TK) gene. Autoradiographic results were quantitated
by personal densitometer SI (Molecular Dynamics, Sunnyvale,
CA). The same membrane was stripped and rehybridized with
human b-actin gene fragment. The viral DNA amount was
subsequently normalized by VZV DNA/human actin DNA. The
concentration of the tested compounds, which inhibited 50%
of viral DNA synthesis compared to untreated control, was
2
2
-MeOH (95:5); mp 160-174 °C; UV
(
1
9 300), 197 (ꢀ 7 600). This product was used directly in the
next step.
(
R)-(-)-Syn a d en ol (1a ) a n d R,E-Isom er 19. The mixture
of 17 and 18 (30 mg, 0.11 mmol) was stirred in methanolic
ammonia (20%, 10 mL) at room temperature for 16 h. Volatile
components were evaporated, and the residue was dried at
defined as EC50
.
<
0.01 mmHg at 78 °C for 1 h leaving a mixture of (R)-(-)-
Ack n ow led gm en t. We thank the Central Instru-
mentation Facility, Department of Chemistry, Wayne
State University (Dr. Robin J . Hood, Director), for mass
spectra. The work described herein was supported by
U.S. Public Health Service Research Grants RO1-
CA32779 (J .Z.) from the National Cancer Institute and
RO1-CA44358 (Y.-C.C.), U19-Al31718, and RO1-Al33332
synadenol (1a ) and the E-isomer 19 as a white solid (25 mg,
1
1
00%). The H NMR spectra were identical to those of the
1
respective racemic compounds. The chiral HPLC (Figure 1,
panel D) showed the ratio of 1a :19 as 1.5:1. The ee values for
1
a and 19 were 98.4% and 98.5%, respectively.
Cr ysta l Da ta a n d Da ta Collection . For an X-ray diffrac-
tion study (-)-synadenol (1a ; 95% ee) was further enriched
by exhaustive deamination. (-)-Synadenol (1a ) obtained as
described above (65 mg, 0.3 mmol) was incubated with
adenosine deaminase in phosphate buffer (60 mL). The
recovered 1a had 99.2% ee as determined by chiral HPLC.
Crystals of (-)-synadenol hydrochloride for X-ray diffraction
were obtained by slow crystallization of an ethanol solution
of approximately equal molar amounts of (-)-synadenol and
hydrochloric acid. The specimen chosen for preliminary study
and data collection was a colorless thin needle of dimensions
(J .C.D.), and RO1-Al40392 (A.C.) from the National
Institute of Allergy and Infectious Diseases, National
Institutes of Health, Bethesda, MD, and by the National
Cancer Institute of Canada with funds from the Cana-
dian Cancer Society (N.C.).
Su p p or tin g In for m a tion Ava ila ble: Crystallographic
data of (R)-(-)-synadenol (1a ) (hydrochloride and free base)
and Figure 1 (14 pages); tables of observed and calculated
structure factors (20 pages). Ordering information is given on
any current masthead page.
1
.00 × 0.075 × 0.080 mm. Unit cell parameters were deter-
mined from refinement of all reflections: a ) 20.986(1), b )
4
0
.951(0), c ) 22.845(1) Å; â ) 99.30(0)°; Mo KR radiation λ )
.71073 Å; F(000) ) 1056; room temperature; space group C2;
Refer en ces
3
3
V ) 2342 Å ; Z ) 8; D(calcd) ) 1.439 g/cm . Nonius Kappa
CCD diffractometer and Kappa CCD server software were
employed. Processing and data reduction were carried out
using the program Denzo-SMN. No absorption corrections
were necessary. A total of 4015 reflections were measured,
(
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