Methods of treatment of viral infections using carbocyclic deoxyguanosine analogs

Method for prophylaxis and treatment of a viral infection characterized by the administration of a composition comprising a substantial molar excess of the D-stereoisomer of 2'CdG over the L-stereoisomer.

BACKGROUND OF THE INVENTION 
This invention relates to the use of the carbocyclic analogue of 
2'-deoxyguanosine (2'CdG) in the prophylaxis and treatment of infections 
by viruses including herpes simplex virus types I and II, cytomegalovirus, 
and hepatitis B virus. 
Among the many kinds of viruses that infect man, two types are especially 
important with regard to their ubiquity, clinical significance and 
economic impact: the herpes viruses and the hepatitis viruses. 
The six human herpesviruses, herpes simplex virus types 1 and 2 (HSV-1, 
HSV-2), varicella-zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr 
virus (EBV), and human herpes virus 6 (HHV 6), are widely disseminated in 
the population and are responsible for a broad spectrum of human diseases, 
ranging from minor annoyances such as cold sores to highly destructive 
infections of the central nervous system (encephalitis), and potentially 
fatal neonatal infections. Once infected, an individual may suffer lesions 
and recover, but the virus usually persists for the life of the individual 
in a state of latency in nerve or other cells and may periodically become 
reactivated to cause recurrent clinical lesions or other disease states. 
In addition, it has been long recognized that herpesvirus infections are 
considerably more severe in certain immunosuppressed patients, 
particularly those with depressed cell-mediated immunity such as cancer 
patients and organ transplant recipients, individuals with hereditary 
immune deficiencies, as well as individuals with acquired immune 
deficiency syndrome (AIDS). 
HSV-1 and HSV-2 manifest themselves in a number of clinical diseases, 
including infections of the central nervous system (encephalitis), skin, 
lips, and the genitalia. Oral herpes is caused generally by HSV-1 and 
genital herpes by HSV-2, but both viruses can cause either infection (and 
each can cause encephalitis). The estimated annual number of episodes of 
recurrent herpes labialis is over 100 million in the United States alone. 
It also has been estimated that over 1,000,000 new clinically reported 
cases of genital herpes occur each year in the United States, and that 
currently as many as 60 million cases per year of recurrent genital herpes 
may exist. In addition, a large number of individuals will excrete virus 
in the absence of clinical symptoms and thus constitute a silent but 
persistent reservoir for transmission of virus through sexual contacts. 
Human cytomegalovirus (CMV) infections are among the most common cause of 
congenital (human intrauterine and perinatal) infection in the world 
today, and represent a frequent and serious complication in 
immunosupressed inividuals. Congential CMV infections are associated with 
retinitis, hearing loss, birth defects, mental retardation, and death. In 
immunosuppressed adults, CMV is associated with serious infections of the 
eye such as chorioretinitis, as well as infections of other organs, 
including hepatitis, esophagitis, gastritis, pneumonitis and encephalitis. 
Hepatitis B virus (HBV) is the most prevalent of the highly contagious 
hepatitis viruses, infecting an estimated 200 million people worldwide. 
HBV is a major cause of acute and chronic hepatitis, cirrhosis, and 
primary hepatocellular carcinoma. Of the estimated 200,000-250,000 people 
in the United States who are infected annually, more than 100,000 require 
hospitalization and approximately 250 die of active disease each year. 
Between 6% and 10% of infected individuals become carriers of the virus. 
Approximately 25% of these carriers will develop cirrhosis and 
approximately 1% will develop hepatocellular carcinoma. 
Other than exclusion of virus-host contact, there are two types of defenses 
against viral infection: vaccination and chemotherapy. Vaccination has had 
some success in preventing or limiting certain viral infections. However, 
there are certain limitations associated with the use of vaccines. First, 
vaccination, with rare exception, is a prophylactic measure which is 
useful only prior to the onset of the disease. Second, factors such as the 
age of the individual, the presence of preexisting antibody, either 
passively acquired maternal antibody or antibody secondary to natural 
infection, and the site of injection may influence the effectiveness of 
some vaccines. In addition, vaccination involves the introduction into the 
body of material which can, in some instances, pose risks which result in 
adverse reactions in the individual. 
A number of nucleoside analogs have been proposed for chemotherapeutic 
treatment of viruses such as HSV, HBV and CMV. U.S. Pat. No. 4,396,623 
(Shealy et al.) refers to the use of certain carbocyclic analogs or uracil 
nucleosides for the treatment of various human and animal diseases caused 
by DNA viruses, such as Herpes simplex virus. U.S. Pat. No. 4,177,348 
(Shealy et al.) and U.S. Pat. No. 4,232,154 (Shealy et al.) refer to 
carbocyclic analogues of cytosine nucleosides and their activity against 
DNA viruses, such as herpes simplex virus Type 1. U.S. Pat. No. 4,543,255 
(Shealy et al.) and U.S. Pat. No. 4,728,736 (Shealy et al.) refer to 
carbocyclic analogues of purine 2'-deoxyribofuranosides and 
ribofuranosides, respectively, and their activity against DNA viruses, 
exemplified by herpes simplex virus Type 1 and Type 2. 
Of the nucleoside analogs available, acyclovir (acyloguanosine) is the 
agent currently indicated for the topical, oral or intravenous therapy of 
a number of clinical manifestations of HSV-1 and HSV-2 as it is a potent 
inhibitor of HSV replication. However, a number of toxic effects have been 
reported. Topical application of acyclovir can cause transient stinging, 
keratitis, follicular conjunctivitis and allergies when used to treat 
herpetic keratitis. Infusion may produce nephrotoxicity in patients 
receiving large doses due to deposition of drug crystals in renal tubules. 
Other toxic effects have been attributed to the high pH (alkalinity) 
required to keep acyclovir in solution. Furthermore, while the antiviral 
activity of acyclovir has been ascribed to its ability to be incorporated 
into the viral DNA, acyclovir induced-DNA chain incorporation and 
termination of cellular genes may lead to additional forms of toxicity, 
including chromosomal damage. 
Acyclovir has also been used against HBV although its use is disadvantaged 
by the same potential side effects described above. In addition, while it 
is effective during short term administration in reducing markers 
associated with HBV replication, such as plasma levels of HBV DNA 
polymerase, cessation of drug administration may result in the return to 
pretreatment level of virus replication. 
Other drugs currently used to treat HBV include adenosine arabinoside 
(ara-A) and adenine arabinoside monophosphate (ara-AMP, a form of the drug 
which allows it to be administered intramuscularly). These drugs are 
effective alone or in combination in decreasing levels of circulating HBV 
DNA polymerase activity in patients infected with HBV. However, complete 
inhibition of HBV may not result from these treatments, as DNA polymerase 
activity has been demonstrated to increase following cessation of drug 
therapy. Furthermore, both ara-A and ara-AMP are also associated with 
substantial toxicity. Untoward effects of these drugs commonly experienced 
by patients include nausea, anorexia, fatigue, diarrhea, vomiting, and 
reversible bone marrow suppression with thrombocytopenia. In addition, a 
peculiar neuromuscular pain syndrome that produces pain and cramping, most 
pronounced at the site of injection, and which may last for months 
following cessation of drug administration has been described. Payne, John 
A. "Chronic Hepatitis: Pathogenesis and Treatment," Disease a Month, 
March, pp. 117-59 (1988). 
Acyclovir has also been used for prophylaxis of CMV, but has been reported 
to be ineffective against established systemic CMV infections as well as 
against CMV retinitis. Broad spectrum antibiotics, corticosteroids, and 
antifungal agents have also been reported to be without therapeutic 
benefit against this disease. An antiviral agent, ganciclovir (dihydroxy 
propoxymethyl guanine) (DHPG) an acyclic nucleoside, has been reported to 
be effective against CMV retinitis but is of limited potency and is 
associated with dose-limiting toxicity. Its activity is described in 
Declercq et al., Antiviral Research Vol. 3, 17-24 (1983) and Vol. 4, 
119-133 (1984). 
The carbocyclic analogue of 
2'-deoxyguanosine(.+-.)-2-amino-1,9-dihydro-[(1.alpha.,3.beta.,4.alpha.)-3 
-hydroxy-4(hydroxymethyl)cyclopentyl]-6H-purin-6-one] (2'-CdG) has been 
reported to have in vitro antiviral activity against HSV-1 and HSV-2 
(Shealy et al., J. Med. Chem. 27:1416, 1987), human cytomegalovirus 
(Shannon et al., in Advances in Chemotherapy of AIDS, Diasio et al., eds., 
Pergamon Press, Inc., New York pp.75-95, 1990, as well as CMV (WO91/13549, 
1991)), and human hepatitis-B virus (Price et al., Proc. Natl. Acad. Sci. 
USA 86:8541, 1989). In addition, it has also shown in vivo antiviral 
activity against HSV-1 and HSV-2 (Shannon et al., in Proceedings of the 
American Society of Virology, Annual Meeting, 1985). 
SUMMARY OF THE INVENTION 
We have found that the balance of antiviral efficacy and toxicity risk 
associated with using 2'CdG antiviral agents is substantially improved by 
using the D-isomer of such agents. Accordingly, the invention features a 
method for preventing or treating a viral infection in a mammal 
characterized by administering an antiviral effective amount of a 
composition comprising a compound of the formula 
##STR1## 
wherein, the composition comprises a substantial molar excess of the 
D-stereoisomer of the compound over the L-stereoisomer of the compound; 
R.sup.1 is selected from the group consisting of, hydroxyl, and C.sub.1 
-C.sub.6 acyloxy; R.sup.2 is selected from the group consisting of 
hydroxyl, and C.sub.1 -C.sub.6 acyloxy; and R.sup.3 is oxygen bound 
through a double bond to carbon when R.sup.4 is hydrogen, or R.sup.3 is 
chosen from the group consisting of C.sub.1 -C.sub.6 alkoxy, amino, and 
halogen when R.sup.4 is bound to carbon 6 to form a double bond between 
the nitrogen of position 1 and the carbon of position 6 and R.sup.5 is 
amino. 
By "substantial molar excess" as used herein is meant that more than 70%, 
more preferably 80%, even more preferably 90%, and most preferably more 
that 95% of the compound in the composition is in the form of the 
D-isomer. By D-enantiomer is meant the enantiomer corresponding in 
configuration to DGuo. 
The term "carbocyclic analogue" as used herein refers to compounds which 
possess a cyclopentane ring in place of the tetrahydrofuran ring of the 
nucleoside compound. 
In preferred embodiments, the composition contains the compound 2'CdG. Also 
greatly preferred are the prodrugs of 2'CdG, that is, those compounds that 
are metabolized in vivo to 2'CdG. Examples of prodrugs would include, but 
not be limited to, such compounds as various O-acylated esters, the 
5'-O-phosphate, D-2'CdG analogues with a substituent at C-6 that is 
metabolized to D-2'CdG, and substituents such as alkoxy, halogen, 
methylthio, hydrogen, amino, or methylamino. 
The method of the invention is especially effective in the prevention and 
treatment of herpesviruses including HSV-1, HSV-2 and cytomegalovirus, as 
well as hepatitis viruses such as hepatitis B virus. 
Depending on the route of administration, which could normally be 
intravitreal injection (e.g., in treatment of cytomegalovirus induced 
chorioretinitis), topical, oral, intravenous or parenteral, compositions 
may be in the form of a solute, solid, semi-solid, liquid, oil, ingestible 
capsule or liposome or microencapsulated dosage form. In addition, the 
compounds may be present as the original compound or in the form of a 
pharmaceutically acceptable salt, and the compostion may include a 
pharmaceutically acceptable non-toxic carrier.

CHEMICAL SYNTHETIC SCHEME 
The enantiomers of 2'CdG and related compounds according to this invention 
may be prepared using the appropriate optically active starting materials. 
For example, the D and L forms may be prepared by an enzymatic method as 
described by Secrist et al. (J. Med. Chem. 30:746, 1987). Generally, in 
this method the racemic carbocyclic analogue of 2,6-diaminopurine 
2'deoxyribofuranoside (C-2,6-DAPR) is subjected to the action of 
commercially available adenosine deaminase for a short period at low 
concentration to yield D-2'-CdG and C-2,6-DAPdR. Then the unconverted 
C-L-2,6-DAPdR isolated from the filtrate may be converted to L-2'-CdG by 
increasing the concentration of adenosine deaminase, raising the 
temperature, and lengthening the reaction time. 
Alternatively, synthesis can involve schemes which use optically inactive 
starting materials and which introduce optical activity at later steps, 
using enzymes which operate preferentially on one stereoisomer such as 
Pseudomonas fluorescens lipase, pig lever esterase, pig pancreatic lipase, 
Mucoimieties lipase, or 5' nucleotidase from Crotalus atrax venom. After 
that reaction, chemical separations of starting materials from products 
will provide optically active fragments. 
A detailed description of one preferred method of preparing the enantiomers 
of 2'CdG is given below. 
General Methods 
Melting temperatures (mp) were determined in capillary tubes heated in a 
Mel-Temp apparatus. Ultraviolet spectra (UV) were recorded with a Cary 
Model 17 spectrophotometer, and absorption maxima are reported in 
nanometers: sh=shoulder. Solutions for ultraviolet determinations were 
prepared by diluting a 5-mL aliquot of a water solution of the carbocyclic 
analogue to 50 mL with 0.1 N hydrochloric acid, phosphate buffer (pH 7), 
or 0.1 N sodium hydroxide. Absorption maxima of these solutions are 
reported as being determined at pH 1,7, or 13, respectively. Extinction 
coefficients are given in parentheses. Mass spectra were determined at 70 
eV by the fast-atom-bombardment (FAB) method and with a Varian/MAT 311A 
spectrometer. Elemental analyses were performed by Atlantic Microlab., 
Inc., Atlanta, GA, and the Molecular Spectroscopy Section of Southern 
Research Institute. Thin-layer chromatography (TLC) was performed on 
plates of silica gel, and developed plates were examined with ultraviolet 
light. High-pressure liquid chromatography (HPLC) was performed with a 
Hewlett-Packard 1084B liquid chromatograph equipped with a 
variable-wavelength detector set at 280 nm, an automatic injector, and a 
C.sub.18 .mu.-Bondapak ODS 10-.mu.m column. Isolated specimens were 
dissolved in water at a concentration of 1 mg/mL. In order to determine 
accurately the progress of the deamination reactions with an ultraviolet 
detector, standards of racemic starting materials and products were run in 
order to calibrate peak areas. Unless indicated otherwise, aliquots of 
reaction solutions and most isolated specimens were assayed by using a 
gradient eluting solvent of water-acetonitrile (9:1--1:9 over 20 min); 
flow rate, 1 mL/min. Some of the aliquots and the analytical samples of 
D-2'-CdG, L-2'-CdG, and C-L-2,6-DAP-2'-dR were also assayed by using a 
gradient eluting solvent of 0.01 M NH.sub.4 H.sub.2 PO.sub.4 (pH 5.1)-MeOH 
(9:1--1:9 over 20 min). Optical rotations were measured with a 
Perkin-Elmer Model 141 polarimeter. Yield calculations are based upon 
conversion of one-half of the racemic starting materials. 
[1R,2S,3R,4R-(1.alpha.,2.beta.,3.beta.,4.alpha.)]-D-1,9-Dihydro-9-[2,3-dihy 
droxy-4-(hydroxymethyl)cyclopentyl]-6H-purin-6-one(D-C-Ino, 4a) and 
[1S,2R,3S,4S-(1.alpha.,2.beta.,3.beta.,4.alpha.)]-L-4-(6-Amino-9H-purin-9- 
yl)-2,3-dihydroxycyclopentanemethanol(L-C-Ado). 
A solution of 400 mg (1.51 mmol) of carbocyclic adenosine in 80 mL of hot 
water was cooled to room temperature before the addition of 100 .mu.L (250 
units) of Adenosine aminohydrolase (EC 3,5,4,4) (ADA from calf intestinal 
mucosa, purchased from Sigma Chemical Co., St. Louis, Mo. 63178; Product 
No. A-1030, Type VIII). After being stirred for 3 h at room temperature, 
the solution was boiled for 5 min., filtered through Celite, and examined 
by TLC and HPLC, which shows 52.3% carbocyclic inosine (0.79 mmol., 210 
mg), leaving 0.72 mmol, 200 mg of carbocyclic adenosine. The solution was 
applied to an ion-exchange column (diameter 1 cm) containing 3.3 mL (3 
equiv) of Amberlite IRA 400(OH.sup.-). The column was then eluted with 500 
mL of water to remove the adsorbed C-Ado (no further C-Ado was observed by 
TLC). Evaporation of this solution to dryness followed by 
recrystallization from water gave 143 mg (71.5%) of L-C-Ado: 
[.alpha.].sup.23 p+51.1.degree. (c 0.3, DMF); mp 208-210.degree. C. 
(racemic C-Ado, 244 C.degree.); HPLC 98.67%; TLC, homogeneous in (3:1 
CHCl.sub.3 -MaOH) containing 5% acetic acid. Anal. (C.sub.11 H.sub.15 
N.sub.5 O.sub.3 +).25H.sub.2 O) C, H, N. 
The ion-exchange column was eluted with 200 mL of 3 N acetic acid. 
Evaporation of the eluate followed by recrystallization from water gave 
90.7 mg (43.2%) of chromatographically pure D-carbocyclic inosine, which 
was 100% pure by HPLC, mp 240.degree. C. with shrinking from 237.degree. 
C. (lt.mp (racemic) 225-227.degree. C., 235.degree. C.), 
[.alpha.].sup.23.sub.D -48.9 (C 0.2, DMF). Anal. (C.sub.11 H.sub.14 
N.sub.4 O.sub.4) C, H, N. 
[1R,3S,4R-(1.alpha.,3.beta.,4.alpha.)]-D-2-Amino-1,9-dihydro-9-[3-hydroxy-4 
-(hydroxymethyl)cyclopentyl]-6H-purin-6-one(D-2'-CdG) 
Racemic carbocyclic 2,6-diaminopurine 2'-deoxyribofuranoside (350 mg, 1.32 
mmol) was dissolved in 70 mL of 0.05 M phosphate buffer (pH 7.4) at 
50.degree. C. The solution was cooled to room temperature, ADA[250 .mu.L 
containing 625 units (0.5 unit/.mu.mol of (.+-.)-C-2,6-DAPdR)] was added 
in one portion, and the progress of the reaction was monitored by HPLC. 
The deamination reaction had essentially stoped within 2 h, and the HPLC 
data indicated that the reaction solution contained approximately equal 
amounts of D-2'-CdG and L-2,6-diaminopurine 2'deoxyribofuranoside 
(C-L-2,6DAPdR). The reaction solution was heated at 100.degree. C. for 3 
min. to deactivate the enzyme, the mixture was filtered through Celite to 
remove agglutinated protein, and the filtrate was refrigerated when 
crystals began to form. D-2'-CdG was filtered off, washed with cold water, 
and dried in vacuo at 78.degree. C.; yield, 80 mg (45%); mp 
244-247.degree. C. (inserted at 100.degree. C., 3.degree. C./min). 
Additional D-2'-CdG crystallized when the filtrate (including water 
washings) from the first crop was concentrated in vacuo to a final volume 
of 8 mL: yield after drying in vacuo at 78.degree. C. for 2 h, 30 mg (17%, 
2 crops); mp 244-246.degree. C. (inserted at 100.degree. C., 3.degree. 
C./min). The analytical sample was obtained by combining the three crops 
of D-2'-CdG and recrystallizing (twice) from water: mp 242-245.degree. C. 
(inserted at 100.degree. C., 3.degree. C./min); HPLC, t.sub.R =6.3 min 
(99.8%); TLC, 1 spot (5:2:3 BuOH-HOAc-H.sub.2 O and4:1 2-propanol-1 M 
NH.sub.4 OAc); MS (FAB), m/e266 (M+1): UV.sub.max 254 nm (11800) and 279 
(8000) at pH 1,253 (13000 and 270-275 sh at pH 7,255-260 sh and 268 
(11300) at pH 13; [.alpha.].sup.23.sub.546 +5.5.degree., 
[.alpha.].sup.23.sub.576 +4.80.degree., [.alpha.].sup.23.sub.D 
+4.9.+-.0.1.degree. (c 1.0, 0.1 N NaOH). Anal. (C.sub.11 H.sub.15 N.sub.5 
O.sub.3 .cndot.1.5H.sub.2 O) C, H, N. 
[1S,3R,4S-(1.alpha.,3.beta.,4.alpha.)]-L-2-Amino-1,9-dihydro-9-[3-hydroxy-4 
-(hydroxymethyl)cyclopentyl]-6H-purin-6-one (L-2'CdG) 
A solution of 40 mg (0.15 mmol) of L-C-2,6-DAPdR and 240 .mu.L of ADA (600 
units, 4 units/.mu.mol of 2b) in 80 mL of phosphate buffer (pH 7.5) was 
stirred at room temperature (18-22.degree. C.) for 20 h. The deamination 
reaction was monitored by using the ammonium dihydrogen phosphate gradient 
eluting system. After 20 h, the ratio of L-C-2,6-DAPdR to L-2'CdG was 
about 2:1, and the reaction solution was then stirred at 37.degree. C., 
HPLC indicated that the relative amounts of L-2'CdG and L-C-2,6-DAPdR to 
in the reaction soluton were 98% and 0.4%, respectively. The reaction 
mixture was heated at 100.degree. C. for 3 min to deactivate the enzyme, 
the mixture was filtered through Celite to remove suspended protein, and 
the filtrate and water washings were combined and concentrated in vacuo to 
a volume of 7 mL. After crystals began to form, the mixture was 
refrigerated. The white crystalline product was filtered away, washed with 
cold water, and dried in vacuo at 78.degree. C.; yield, 28 mg (70%); mp 
238-241.degree. C. (inserted at 135.degree. C., 3.degree. C./min). 
Recrystallization of this specimen from 2.2 mL of water afforded the 
analytical sample of L-2'CdG recovery, 26 mg (93%); mp 243-246.degree. C. 
(inserted at 100.degree. C., 3.degree. C./min); HPLC, t.sub.R =6.3 min 
(99.6%); TLC, 1 spot (5:2:3 BuOH-HOAc-H.sub.2 O and 4:1 2-propanol-1 M 
NH.sub.4 OAc); MS (FAB), m/e 266 (M+1); UV.sub.max 254 nm (11900), 279 
(8100) at pH 1, 253 (12800) and 270-275 sh at pH7, 255-260 sh and 268 
(11200) at pH 13; [.alpha.].sup.16.sub.546 -6.0.degree., 
[.alpha.].sup.18.sub.578 -5.5.degree., [.alpha.].sup.16.sub.D -5.2.degree. 
(c 1.0, 0.1 N NaOH). Anal. (C.sub.11 H.sub.15 N.sub.5 O.sub.3 
.cndot.1.5H.sub.2 O) C, H, N. 
[1S,2R,4S-(1.alpha.,2.beta.,4.alpha.)]-L-4-(2,6-Diamino-9H-purin-9-yl)-2-hy 
droxycyclopentanemethanol (L-C-2,6-DAPdR) 
The filtrate from the isolation of D-2'-CdG was shown by HPLC analysis to 
contain L-C-2,6DAPdR and D-2'CdG in a ratio of about 85:15. The solution 
was diluted to 75 mL with water and was stirred for 1.5 h with 5 mL of an 
anion-exchange resin (Dowex 1-X8, OH form). HPLC analysis of the 
supernatant solution indicated that all of the remaining D-2'CdG had been 
absorbed on the resin. The mixture was filtered, and the filtrate 
(combined with the water washings) was chromatographed on a column that 
contained 30 mL of a cation-exchange resin (Bio-Rad AG 50W-X4, H.sup.+ 
form). The column was washed thoroughly with water and was then eluted 
with 1 N aqueous ammonia. Product-containing fractions were identified by 
UV analysis of the column effluent and were concentrated in vacuo to 
crystalline L-C-2,6-DAPdR: yield, 100 mg (57%); HPLC, 99%. 
Additional L-C-2,6DAPdR was obtained by extracting the Dowex 1-X8 resin 
used above with three 25-mL portions of boiling water: weight, 15 mg (9%); 
HPLC, 99.3%. The analytical sample was obtained as a white, crystalline 
solid by recrystallizing the combined crops of L-C-2,6DAPdR: recovery, 90 
mg (78%); mp 210-213.degree. C. dec (inserted at 100.degree. C., 3.degree. 
C./min); HPLC,.sup.26 t.sub.R =7.5 min (99.7%); TLC, 1 spot (5:2:3 
BuOH-HOAc-H.sub.2 O and 4:1 2-propanol-1 M NH.sub.4 OAc); MS (FAB), m/e 
265 (M+1); UV.sub.max 217 nm (22300), 253 (9500), and 291 (9900) at pH 
1,215 (29000) and 245-250 sh, 255 (8200), and 280 (10500) at pH 7 and 13; 
[.alpha.].sup.23.sub.546 -5.9.degree., [.alpha.].sup.23.sub.578 
-5.5.degree., [.alpha.].sup.23.sub.D -4.8.degree. (c 1.0, H.sub.2 O). 
Anal. (C.sub.11 H.sub.16 N.sub.6 O.sub.2 .cndot.0.25H.sub.2 O) C, H, N. 
Results 
The antiviral activity of a number of guanine derivatives including 
acyclovir and related acyclic compounds has been attributed to their 
phosphorylation, a reaction that is believed to be catalyzed by a 
virus-encoded nucleoside kinase. For example, in viruses which produce 
viral thymidine kinase, such as HSV, acyclovir is phosphorylated to the 
monophosphate form which is then converted by cellular kinases to 
acyclovir triphosphate. The ability of this triphosphate form to interfere 
with the viral DNA polymerase is believed to be a source of antiviral 
activity of this compound. (Fyfe et al., J. Biol. Chem. 252:8721, 1978; 
Chu et al., J. Heterocycl. Chem. 23:289, 1986; De Clercq et al., in: 
Progress in Medicinal Chemistry, Ellis et al., eds., Vol. 23, pp. 187-218, 
Elsevier, Amsterdam, 1986). The structure of 2'CdG is substantially 
different from the 9-acyclic derivatives of guanine, and it is by no means 
certain that the presence of a virus-encoded kinase is essential to 2'-CdG 
activity. However, viral kinase, when present, appears to be involved in 
2'-CdG activity. We therefore examined the interaction of racemic 2'CdG 
and its D- and L-enantiomers with HSV thymidine kinase. 
Interaction of D-2'CdG, L-2'CdG, and D,L-2'CdG with HSV Thymidine Kinase 
HSV thymidine kinase (HSV TK) was isolated from from HELA (BU25) cells 
infected with the SW148 strain of HSV-1 at a multiplicity of infection of 
10 CCID.sub.50 (1 CCID.sub.50 =the number of virions required to infect 
50% of the cells) as described by Balzarini et al. (Mol. Pharmacol. 
37:395, 1992). Both enantiomers of 2'CdG were tritiated by Moravek 
Biochemicals (Brea, Calif.) to yield [8-.sup.3 H]-D-2'CdG (250 mCi/mmole) 
and [8-.sup.3 H]-L-2'CdG (900 mCi/mmole). The samples had an initial 
radiopurity of greater than 99%. [Methyl-.sup.3 H]-thymidine (70-85 
Ci/mmole) was obtained from Amerxham Corp. (Arlington Heights, Ill.). 
9-Benzyl-9-deazaguanine was synthesized in our laboratories. Pyruvate 
kinase, dGuo, dCyd and mycophenolic acid were obtained from Sigma Chemical 
Co. (St. Louis, Mo.). HeLa (BU25) cells, a line deficient in thymidine 
kinase, were provided by Dr. Y.-C. Cheng (Yale University) and were 
maintained in Eagle's minimal essential medium supplemented with bovine 
calf serum. The S148 strain of HSV-1 was maintained in H.Ep.-2 cells as 
described previously (Bennet et al. Biochem. Pharmacol. 48:1515, 1990). 
The purified HSV TK was incubated with various concentrations of 
radiolableled nucleoside. The HSV TK was incubated for 1 hour at 
37.degree. in 50 .mu.l volumes containing 5.0 mM ATP, 5 mM MgCl.sub.2, 9 
mM potassium fluoride, 5 mM phosphoenol pyruvate, 2.8 .mu.g of pyruvate 
kinase, 10 mM .beta.-mercaptoenthanol, and various concentrations of 
unlabeled and labeled nucleosides (1, 1.25, 1.5, 2, 2.5, 3.33, 5, 6.66, 8, 
10, 12.5, 15, 20, 33.3, 50, or 100 .mu.M D-2'CdG; 1, 1.25, 1.5, 2, 2.5, 
3.33, 5, 6.66, 8, 10, 12.5, 15, 20, 33.3, 50, or 100 .mu.M L-2'CdG; 0.1, 
0.125, 0.15, 0.2, 0.25, 0.33, 0.4, 0.5, 0.66, 0.8, or 1.0 .mu.M 
thymidine). The reactions were terminated by spotting onto DE-81 discs 
(Whatman Lab Sales, Hillsboro, Ore.), after which the disks were washed 
with 100% ethanol. The radioactivity remaining on the disk (nucleoside 
monophosphate) was determined by transferring the disk to a vial, adding 
Scinti-Verse (Fisher Scientifi co., St. Louis, Mo.) and counting in a 
Packard Tri-Carb liquid scintillation spectrometer (Packard, Cowners 
Grove, Ill.). 
Lineweaver-Burke plots for both enantiomers demonstrated non-linear 
kinetics at the higher concentrations (FIG. 1). The lines were 
extrapolated from the linear portions of the double reciprocal plots to 
obtain K.sub.m values. Table 1 presents a summary of the kinetic constants 
for the enantiomers of 2'CdG and also for thymidine. The K.sub.m and 
V.sub.max values for L-2'CdG did not differ significantly from those for 
D-2'CdG. The K.sub.m values for the 2'CdG enantiomers were much higher 
than that for dThd and the V.sub.max values were about 50% greater than 
that for dThd. 
TABLE 1 
______________________________________ 
Compound K.sub.m (.mu.M) 
V.sub.max (nmole/hour/mg protein) 
______________________________________ 
dThd 0.31 8.7 
D-2'CdG 20.8 12.6 
L-2'DdG 28.6 13.4 
______________________________________ 
To measure inhibition by the different forms of 2'CdG, thymidine kinase was 
incubated with [.sup.3 H]-thymidine alone or in the presence of D-2'CdG, 
L-2'CdG, or D,L-2'CdG. The phosphorylation of thymidine was determined by 
spotting the reaction mixture onto DE-81 filters, washing away unreacted 
substrate with ethanol, and determining the radioactivity ([.sup.3 H]TMP) 
remaining on the filters. 
Both enantiomers of 2'CdG competitively inhibit the phosphorylation of 
thymidine by the partially purified HSV-1 TK (FIG. 2). The K.sub.i 's were 
determined from a plot of the slope of each line versus the concentration 
of the competitor (D-, L, or D,L-2'CdG) and are summarized below: 
TABLE 2 
______________________________________ 
Compound 
K.sub.i 
______________________________________ 
D-2'CdG 2.1 .mu.M 
L-2'CdG 3.4 .mu.M 
D,L-2'CdG 3.0 .mu.M 
dGuo 10.5 .mu.M 
______________________________________ 
These data demonstrate that each of the enantiomers as well as the racemic 
mixture of 2'CdG are substrates for HSV thymidine kinase and competitively 
inhibit the phosphorylation of thymidine by HSV thymidine kinase at 
similar levels. 
Phosphorylation of D-, L, and D,L-2'CdG 
The assays discussed above were accomplished by the disk method which 
measures total radioactivity retained on the disk. Because of the 
unexpected finding that the L-enantiomer of 2'CdG was as good a substrate 
as the D-enantiomer, it was desirable to examine nucleotide formation by 
another method. 
HSV thymidine kinase activity was measured in 200 .mu.l volumes containing 
HSV-TK extract obtained as described previously (Belzarini et al., supra), 
5.0 mM ATP, 5 mM MgCl.sub.2, 9 mM potassium fluoride, 11.2 .mu.g 
phosphoenol pyruvate, 5 .mu.g of pyruvate kinase, 10 mM 
.beta.-mercaptoethanol and either 50 .mu.M [.sub.3 H]-D-, L, or no drug 
for 60 minutes at 37.degree. C. The reaction was stopped with perchloric 
acid (PCA), neutralized with potassium bicarbonate, and the reaction 
products were analyzed by SAX HPLC. The UV absorption spectra of selected 
peaks were determined with a rapid spectral detector (LKB, Gaithersburg, 
Md.). The monophosphate and triphosphate derivatives were detected by 
absorbance at 254 nm. The data (FIG. 3) demonstrates that L-2'CdG is 
phosphorylated primarily to the monophosphate form (L-2'CdG-MP) whereas 
the major phosphorylated derivative of D-2'CdG is the triphosphate 
(D-2'CdG-TP). The amount of monophosphate derived from L-2'CdG was about 
the same as the amount of triphosphate derived from D-2'CdG, and confirmed 
the results of the disk assay. From these results, one would expect that 
the racemate would yield approximately equal amounts of monophosphate and 
triphosphate, and this was, in fact, the case. 
HSV extracts were also incubated with 10 .mu.M of either [.sup.3 H]-D- or 
L-2'Cdg, as described above, to measure the metabolism of these 
entantiomers. After 2 hours the reaction was stopped with perchloric acid 
(PCA), neutralized with potassium bicarbonate, and the acid-soluble 
metabolites were separated using SAX HPLC as described above. The results 
are summarized below.: 
TABLE 3 
______________________________________ 
DPM 
Compound nucleoside 
mono di tri total 
______________________________________ 
D-2'CdG 38555 2138 2427 85073 128000 
L-2'CdG 40944 115000 2050 410 158000 
______________________________________ 
The in vivo metabolism of D- and L-2'CdG was also measured in 
HSV-[TK+]-infected, HSV-[TK-]-infected and mock infected H.Ep.-2 cells 
(human epidermal carcinoma cells, ATCC CCL23). Uninfected and infected 
cells were incubated in fresh medium containing 2 .mu.Ci/ml of either 
[.sup.3 H]-D- or L-2'CdG, as described above, to measure the metabolism of 
these entantiomers. After 8 hours the cells were harvested and washed free 
of medium with PBS, and extracts were prepared with cold 0.5 N perchloric 
acid for 0.5 hr. The extract was then neutralized with potassium 
bicarbonate and the resulting precipitate was removed by centrifugation. 
The supernatant was lyophilized to dryness and the acid-soluble 
metabolites were separated using SAX HPLC as described above. 
In H.Ep.-2 cells infected with HSV-1 the principal soluble metabolite of 
D-2'CdG was the triphosphate, and the principal metabolite of L-2'CdG was 
the monophosphate (FIG. 4). While these findings are similar to those 
obtained with the isolated viral kinase, they differ from the resulted 
with the isolated enzyme in that relatively more di- and triphosphate were 
formed from L-2'CdG. In uninfected CEM cells, the total amounts of 
phosphates formed from either enantiomer were much less than in infected 
cells, and again the predominant metabolite of L-2'CdG was the 
monophosphate, whereas the predominant metabolite of D-2'CdG was the 
triphosphate. The total amount of phosphates of L-2'CdG formed in these 
cells was about twice that formed from D-2'CdG (FIG. 5); thus L-2'CdG 
appears to be superior to D-2'CdG as a substrate from one or more cellular 
phosphorylating enzymes. 
Identity of the Cellular Enzyme(s) Responsible for the Phsophorylation of 
the Enantiomers of 2'CdG 
The cellular enzymes most likely to catalyze the phosphorylation of 2'CdG 
to the monophosphate form are dCyd kinase, which appears to the principal 
enzyme responsible for the phosphorylation of dGuo in mammalian cells 
(Sarup et al., Biochemistry 26:590, 1987; Hurley et al., J. Biol. Chem. 
258:15021, 1983), and 5'-nucleotidase, which has been shown to catalyze 
phosphorylation of several nucleoside analogs including 
2',3'dideoxyinosine, carbovir, and acyclo derivatives of guanine (Keller 
et al., J. Biol. Chem. 260:8664, 1985; Bondoc et al., Biochemistry 
29:9839, 1990; Johnson et al., Mol. Pharmalcol. 36:291, 1989). To 
determine if dCyd kinase was involved, studies were carried out in which 
cells were indubated with D- or L-2'CdG and dCyd, and the formation of 
phosphates was monitored by HPLC. To determine if 5'nucleotidase was 
involved cells were incubated with D- or L-2'CdG or mycophenolic acid, 
which has been shown to increase the activity of this enzyme by producing 
a buildup of IMP (Ahluwalia et al., Biochem. Biophys. Res. Communs. 
171;1297, 1990; Hartman et al. Mol. Pharmac. 40:118, 1991). As shown in 
FIG. 6, the presence of dCyd markedly decreased the amount of phosphates 
of both D- or L-2'CdG; the reduction, calculated from the radioactivity in 
the peaks, was 80-90% for both enantiomers. The presence of mycophenolic 
acid produced an increase in the phosphates of D- or L-2'CdG of 2.5-fold 
and 1.4-fold, respectively. These results indicate that dCyd kinase may be 
the principal enzyme responsible for catalyzing the initial 
phosphorylation of both enantiomers, although the participation of 
5'-nucleotidase as well as other, as yet unidentified enzymes, is not 
excluded. The fact that the presence of mycophenolic acid increases the 
phosphorylation of both enantiomers (FIG. 6) would suggest the involvement 
of 5'-nucleotidase. However, an alternative interpretaion of these data 
would be that mycophenolic acid, by lowering the concentrations of guanine 
nucleotides, decreases the normal feedback control of dCyd kinase. 
It is likely that the enzyme responsible for the phosphorylation of the 
D-2'CdG-MP is human guanylate kinase that co-purifies with the HSV TK. 
Alternatively, the HSV TK also contains a monophosphate kinase and it is 
possible that this enzyme is responsible for the phosphorylation of 
D-2'CdG-MP. In turn, D-2'CdG-DP could be phosphorylated by either human 
nucleoside diphosphate kinase, which co-purifies with the HSV-TK, or the 
pyruvate kinase which is a component of the assay. 
Comparison of dGuo and 2'CdG as Substrates for HSV-1 TK 
It was of interest to compare 2'CdG and its natural counterpart, dGuo, as 
substrates for the viral kinase. since preparation of the viral enzyme was 
only partially purified, it was necessary to take into account the 
possible presence of interfereing enzymes. The kinase preparation was in 
fact found to contain some purine nucleoside phosphorylase (PNP) activity. 
Therefore, we added 9-benzyl-9-deazaguanine (BzDAG), a potent inhibitor of 
PNP, to the incubation mixture. The phosphorylation of 2'CdG proceeded at 
a rate much higher than that for dGuo (FIG. 7). Similar experiments 
performed in the absence of BzDAG yielded about the same results with 
D-2'CdG and showed, at best, a slight decrease in the rate of 
phosphorylation of dGuo. Thus, although PNP activity was present in the 
enzyme preparation, it did not have a significant effect on the 
phosphorylation of dGuo. Although it has been reported that dGuo is a poor 
substrate for this enzyme (Fyfe et al., J. Biol. Chem., 253:8721, 1978), 
it is unexpected that the replacement of the 4'-O atom by a methylene 
group would improve substrate activity so markedly. 
As was previously discussed, the inhibitory forms of many antiviral 
nucleosides are the triphosphates, and the target is the virus-coded DNA 
polymerase. Activity of nucleoside analogs for viral inhibition may 
therefore be determined by the potency of the triphosphate forms as 
inhibitors of the viral polymerase. Therefore, we analyzed the effects of 
D-2'CdG on the viral and host DNA polymerases. 
Incorporation of [.sup.3 H]-D-2'CdG into HSV and Host DNA 
Mock-infected and HSV-infected cells tretated with the desired label were 
collected by centrifugation. The cell pellet was resuspended in 0.5 ml of 
10 mM Tris, pH 8.0, 40 mM EDTA, 0.5% sodium dodecyl sulfate, 200 .mu.g/ml 
proteinase K, and the mixture was incubated at 37.degree. overnight. One 
hundred microliters of each sample wre mixed with 5.0 ml of a CsCl 
solution, such that the final concentration of the CsCl was 1.75 g/ml. 
Host DNA labeled with [.sup.14 -C]dThd was included in each sample as an 
internal control. The samples were centrifuged to equilibrium at 
20.degree. (30,000 rpm for 72 hr, with a Ti 70.1 Beckman rotor). 
Twenty-five microliter fractions were collected from the top of each 
gradient, and the DNA in each fraction was precipitated onto glass fiber 
filters with a 5% trichloroacetic acid solution containing 10 mM 
pyrophosphate. These filters were washed three times with this solution 
followed by two washes with 95% ethanol and dried. The radioactivity in 
the acid-insoluble portion in every fourth fraction of each gradient was 
then determined and plotted in relation to its internal standard, the 
.sup.14 C-labeled host DNA. 
To verify that the radioactivity associated with both the host and viral 
DNA peaks was due to the incorporation of [.sup.3 H]-D-2'CdG, the DNA 
samples were pooled, dialyzed against water to remove the CsCl, 
lyophilized, and resuspended in 50 .mu.l of DNase I in 50 mM glycine, pH 
9.0. After incubation for 2 hr at 37.degree. C., 50 .mu.l of 100 units/ml 
concentrations of both phosphodiesterase I and alkaline phosphatase in 50 
mM glycine, pH 9.0 were added to each sample, and the reaction was stopped 
by boiling for 2 min, the precipitated porteins were removed by 
centrifugation, and the samples were analyzed by reverse phase HPLC for 
the appearance of radiolabeled nucleoside. To determine whether the 
[.sup.3 H]-D-2'CdG was incorporated into internal or terminal positions, 
the DNA after the lyophilization step was digested by the sequential 
action of micrococcal nuclease (50 units/ml micrococcal nuclease, 5 
.mu.g/ml pentostatin, 5 mM CaCl.sub.2, 1 mM Tris, pH 9.5, for 2 hr at 
37.degree. C.) and spleen phosphodiesterase (three consecutive additions 
of 75 .mu.g/ml spleen phosphodiesterase, 1 mM EDTA, in the same buffer at 
pH 7.0, for 1 hr each time at 37.degree. C.), as described by Pelling et 
al. (Virology 109:323, 1981). These enzymes specifically cleave RNA and 
DNA to generate 3'-monophosphates of all internally located nucleotices 
and the nucleoside of any 3'-terminal nucleotides. After digestion, the 
samples were analyzed by strong anion exchange HPLC, to separate 
monophosphates from nucleosides. 
The incorporation of [.sup.3 H]-D-2'CdG into the DNA of cells infected with 
HSV was determined at various times after infection to optimize conditions 
for the incorporation of D-2'CdG into either HSV or host DNA. When 
HSV-infected cells were incubated for 9 hr with 8 .mu.M [.sup.3 
H]-D-2'CdG, starting at thte time of infection (FIG. 8), label was found 
only in host DNA. Very little incorporation of [.sup.3 H]-D-2'CdG into DNA 
was observed in cells that were not infected with virus. These results 
indicated that [.sup.3 H]-D-2'CdG-TP formed from the sequential action of 
the HSV TK and host nucleotide kinases was then utilized by a host DNA 
polymerase for DNA synthesis. When HSV-1-infected cells were treated for 5 
hr with 1 .mu.M [.sup.3 H]-D-2'CdG, starting 4 hr after infection (FIG. 
2), label was found primarily in viral DNA. The small amount oflabel in 
the host DNA is presumably due to the near-total inhibition of host DNA 
synthesis by 4 hr of the viral infection. The lack of label in viral DNA 
incubated with [.sup.3 H]-D-2'CdG from 0 to 9 hr of virus infection was 
probably due to effective inhibition of viral replication by drug 
treatment (8 .mu.M 2'CdG is 10 times the concentration required to inhibit 
viral replication by 50%). Although [.sup.3 H]-D-2'CdG is undoubtedly 
incorporated into viral DNA under these conditions, the amount of 
incorporation is below the level of detection. 
The density of viral DNA labeled with 1.0 .mu.M [.sup.3 H]-D-2'CdG was the 
same as that seen with [.sup.3 H]Thd-labeled viral DNA. However, if the 
concentration of [.sup.3 H]-D-2'CdG was increased to 8 .mu.M, the density 
of the labeled DNA was decreased, so that the viral DNA banded between the 
host and viral DNA markers. The reason for this shift in the binding in 
the CsCl gradient is not known. However viral DNA obtained from 
HSV-infected cells treated with 8 .mu.M [.sup.3 H]-D-2'CdG sedimented in 
either neutral or alkaline sucrose gradients, prepared according to 
standard methods (McGuirt et al., Antimicrob. Agents Chemother. 25:507, 
1984; Balzarini et al., Mol. Pharmacol. 37:402, 1990), in a manner similar 
to that of viral DNA labeled with [.sup.3 H]dThd, indicating that the 
viral DNA containing 2'CdG was the same size as viral DNA from untreated 
cells. 
The nucleosides of the radioactive viral and host DNAs were then analyzed 
using reverse phase HPLC as described above. All of the radioactivity in 
the DNA eluted from the reverse phase column with authentic 2'CdG, 
confirming that the radioactivity in the DNA was due to the incorporation 
of 2'CdG. The viral and host DNA labeled with [.sup.3 H]-D-2'CdG were also 
degraded with micrococcal nuclease and spleen phosphodiesterase I, to 
generate 3'-monophosphates of all internally located nucleotides and the 
nucleosides of any terminal nucleotides. The nucleosides and nucleotides 
were then analyzed by strong anion exchange chromatography. Most of the 
radioactivity in both the viral and host DNA was converted to the 3' 
monophosphate of D-2'CdG-Mp, indicating that D-2'CdG was incorporated into 
internal linkages by the HSV DNA polymerase and the host polymerase 
responsible for incorporation. The percentage of D-2'CdG incorporated into 
internal linkages in the DNA chain was the same as that seen in similar 
experiments using [.sup.3 H]dThd to label the DNA. 
Inhibition of purified HSV and host DNA polymerases by 2'CdG-TP. Because 
D-2'CdG was incorporated into both HSV and host DNA, the interaction of 
2'CdG-TP with the HSV and host DNA polymerases was studied. 
HSV DNA polymerase was purified from H.Ep.-2 cells infected with HSV-1. 
Approximately 4.times.10.sup.8 cells were infected with virus at a 
multiplicity of infection of 10 CCID.sub.50 /cell. After a 1 hr adsorption 
period, the unattached virus was waxhed from the cells and the cultures 
were returned to the incubator at 37.degree. C. for another 8 hr. The 
cells were collected by centrifugation and mixed with a 0.3 M potassium 
phosphate buffer, pH 7.5, containing 0.3% Triton X-100 and 10% glycerol. 
The HSV DNA polymerase was purified approximately 288-fold from this crude 
extract, as described by Derse et al. (J. Biol. Chem. 257:10251, 1982). 
The specific activity of the HSV DNA polymerase was 4140 units/mg protein. 
Human DNA polymerases, .alpha., .beta., and .gamma., were purified from 
5.0 ml of packed K562 cells grown in cell culture as previously described 
(Parker et al., J. Biol. Chem. 266:1754, 1991). The specific activities of 
DNA polymerases .alpha., .beta., and .gamma. used in these studies were 
approximately 34, 1600, and 8 units/mg protein, respectively. One unit of 
enzyme activity is defined as the amount of enzyme needed to incorporate 1 
nmol of [.sup.3 H]dGTP into acid-precipitable material per hour at 
37.degree. C., using gapped DNA as a template. HSV DNA polymerase activity 
was measured in 50 .mu.l volumes containing 50 mM Tris, pH 8.0, 3 mM 
MgCl.sub.2, 0.5 mM dithiothreitol, 0.2 mg/ml bovine serum albumin, 0.2 M 
KCl, 12.5 .mu.g/ml gapped DNA, 10 .mu.M [.sup.3 H]dGTP (12 Ci/mmol), and 
100 .mu.M each of dATP, dCTP, and TTP. DNA polymerase a activity was 
measured in 50 .mu.l volumes containing 50 mM Tris, pH 8.0, 1 mg/ml bovine 
serum albumin, 10 mM MgCl.sub.2, 1 mM dithiothreitol, 100 .mu.g/ml gapped 
duplex DNA, 10 .mu.M [.sup.3 H]dGTP (1 Ci/mmol), and 50 .mu.M of each 
dATP, dCTP and TTP. DNA polymerase .beta. and .gamma. activities were 
measured as described for DNA polymerase .alpha., except that the 
reactions included 100 mM KCl. After incubation for the desired time, the 
DNA in each sample was precipitated onto glass fiber filters using a 5% 
trichloroacetic acid solution containing 10 mM pyrophosphate. The filters 
were then washed three times with this solution followed by two washes 
with 95% ethanol, dried, and counted for radioactivity. 
2'CdG-TP was a potent competitive inhibitor of the incorporation of [.sup.3 
H]dGTP into DNA by both the HSV DNA polymerase and the host DNA polymerase 
.alpha. (FIG. 9). The average K.sub.i of D-2'CdG-TP against HSV DNA 
polymerase was 0.35 .mu.M, and with DNA polymerase .alpha. it was 0.95 
.mu.M. The K.sub.i /K.sub.m ratio for both DNA polymerases was 
approximately 1, indicating that the affinity of 2'CdG-TP for the active 
site of these enzymes was similar to that of dGTP. 
Under similar incubation conditions, the concentration of D-2'CdG-TP 
required to inhibit the incorporation of 1 .mu.M [.sup.3 H]dGTP into the 
DNA by 50% was 2 .mu.M with DNA polymerase .alpha., but was 2200 and 300 
.mu.M for DNA polymerase .beta. and .gamma., respectively (Table 4). The 
L-enantiomer of 2'CdG-TP also inhibited DNA polymerase .beta. and .gamma. 
at concentrations similar to or less than that required for inhibition by 
the D-enantiomer. 
TABLE 4 
______________________________________ 
IC.sub.50 
D-2'CdG-TP 
L-2'CdG-TP 
.mu.M 
______________________________________ 
HSV DNA polymerase 
1.5 460 
DNA polymerase .alpha. 2.0 470 
DNA polymerase .beta. 2200 1400 
DNA polymerase .gamma. 300 215 
______________________________________ 
Antiviral Activity of D-; L- and D,L-2'CdG 
The antiviral activities of the racemate and each 2'CdG enantiomer were 
studied. 
EXAMPLE 1 
D-2'-CdG and L-2'-CdG were tested, alongside D,L-2'-CdG, against both HSV-1 
(strain 377, TK+; obtained from Dr. Earl Kern, University of Alabama) and 
HSV-2 (strain MS, TK+; also obtained from Dr. Earl Kern). In these tests 
(Table 5), Ara-A and acyclovir (ACV) were the positive control drugs. In 
four tests vs. HSV-1, the antiviral activity (VR) and potency (MIC.sub.50) 
of D-2'-CdG were approximately the same as those of racemic 2-CdG. 
L-2'-CdG was only modestly active. 
D,L-2'-CdG and D-2'-CdG were more effective than was the positive control 
drug, Ara-A. D-2'-CdG, (D,L)-2'CdG, and ACV were comparable in antiviral 
activity, but the two forms of 2'CdG were 5-10 times more potent than was 
ACV. 
These compounds were tested against HSV-2(TK.sup.+). The VR of ACV was 
somewhat higher than the VR values of D-2'-CdG and D,L-2'-CdG, but the 
values of MIC.sub.50 of the two forms of 2'-CdG were lower. In addition, 
experiments comparing the antiviral activity D-2'-CdG with acyclovir 
against HSV-1 in human foreskin fibroblasts have shown an even greater 
potency, in favor of D-2'-CdG. 
TABLE 5 
______________________________________ 
Activity of Enantiomers of 2'-CdG 
Against Herpes Simplex Virus Types 1 and 2 
HSV-1 HSV-2 
Mic.sub.50 Mic.sub.50 
Compound VR (.mu.g/ml) VR (.mu.g/ml) 
______________________________________ 
D-2'-CdG 4.8-6.3 0.1-0.3 3.8 0.7 
D,L-2'-CdG 5.3-7.0 0.2-0.3 3.7 2.6 
L-2'-CdG 0.8-2.4 39-257 0 -- 
Ara-A 1.6-2.5 10-34 1.3 50 
Acyclovir 5.7-6.5 1.5-2.9 4.5 5.3 
______________________________________ 
EXAMPLE 2 
The D- and L-enantiomers of 2'-CdG were prepared from racemic C-2,6-DAPR by 
an enzymatic method as described above Secrist et al. (J. Med. Chem. 
30:746, 1987). The effectiveness of racemic-2'-CdG and of D-2'-CdG in 
reducing CMV yields in MRC5 cell monolayer cultures was determined by a 
plaque assay with the results set forth in tables 6 and 7. 
TABLE 6 
______________________________________ 
THE EFFECT OF THE CARBOCYCLIC ANALOGUE OF 2' 
DEOXYGUANOSINE [(.+-.-2'-CdG] ON CMV YIELDS IN MRC5 
CELL MONOLAYER CULTURES 
Reduction (%) of Drug Cytotox- 
CPE by CdG at CMV Yield icity (Gross 
(+-2'-CdG.sup.1a Time of Harvest (log.sub.10 CCID.sub.50 /mL) Morphology 
) 
______________________________________ 
32 .mu.g/mL 
-- -- toxic 
10 100 0 sl. toxicity 
3.2 100 1.0 v. sl. toxicity 
1.0 95 3.6 0 
0.32 20 4.5 0 
0.1 0 5.0 0 
0.032 0 5.4 0 
0 (Virus -- 5.5 -- 
Control) 
______________________________________ 
.sup.a Drug treatment began immediately following the virus adsorption 
period (1 1/2 hr). 
TABLE 7 
______________________________________ 
THE EFFECT OF THE CARBOCYCLIC ANALOGUE OF D-2'- 
DEOXYGUANOSINE [D-2'-CdG] ON CMV YIELDS IN MRC5 
CELL MONOLAYER CULTURES 
Reduction (%) of 
CPE by D-2'CdG 
CMV Yield Drug Cytotox- 
at Time of Virus log.sub.10 
icity (Gross 
D-2'CdG.sup.a Harvest PFU/mL PFU/mL Morphology) 
______________________________________ 
320 .mu.M 
-- -- -- toxic 
100 50 0 slight toxicity 
32 50 0 -- v. sl. toxicity 
10 25 2 .times. 10.sup.1 1.3 0 
3.2 10 8.4 .times. 10.sup.2 2.9 0 
1.0 10 5.4 .times. 10.sup.3 3.7 0 
0.32 0 1.4 .times. 10.sup.4 4.1 0 
0.1 0 3.8 .times. 10.sup.4 4.6 0 
0 (Virus -- 1.1 .times. 10.sup.5 5.1 -- 
Controls) 
______________________________________ 
.sup.a Drug treatment began immediately following the 1 1/2 hour virus 
adsorption period. 
The MIC.sub.50 of D-2'-CdG (&lt;0.029 .mu.L/mL) was about half (or less) of 
the MIC.sub.50 of D,L-2'-CdG (0.069 .mu.g/mL) measured under the same 
conditions in MRC5 cells. 
EXAMPLE 3 
The in vitro activity of D-2'-CdG was also compared with the current 
investigational drug of choice for human CMV, DHPG. These experiments were 
carried out in human foreskin fibroblast cells. The ED.sub.50 value 
(median dose at which 50% antiviral efficacy is observed) was 0.06 
.mu.g/ml for D-2'-CdG and 0.20 for DHPG, demonstrating that that the 
antiviral potency of D-2'-CdG is more than 3 times that of DHPG. 
EXAMPLE 4 
New Zealand white rabbits were found to develop reproducible clinical 
symptoms of infection within 3-4 days after intravitreal inoculation with 
human CMV. The symptoms included vitritis, iritis, retinal pathology 
(micro hemorrhages and focal necrosis of the retinal surface), corneal 
stromal haze and neovascularization, and corneal endothelial pigmented 
precipitates. Administration of D-2'-CdG by intravitreal injection, i.e., 
by injection directly within the vitreous humor (100 .mu.g in 100 .mu.l of 
sterile water) at 48-hour intervals beginning at day 4 was effective in 
reducing the develpment of retinal pathology and in reducing the severity 
of the vitritis and iritis as xompared to rabbits receiving a placebo. It 
was found that in most cases treatment with D-2'-CdG was clinically better 
than treatment with ganciclovir (DHPG) under the same conditions. The 
intravitreal route offers some significant advantages for intraocular 
infections including: 1) delivery of maximal concentrations of drug to the 
desired site of action; 2) increased potential for sustained release 
(i.e., longer half-life, T.sub.1/2) of the drug; and 3) reduced potential 
for systemic toxicity. 
EXAMPLE 5 
2'-CdG was tested at three concentrations (50 ng/ml, 500 ng/ml, and 5 
ug/ml) against the HBV-producing, human liver cell line, 2.2.15 (Sells et 
al., J. Virol. 62:2836, 1989). A racemic (D,L) preparation, as well as the 
purified D and L isomers were tested. For these analyses compounds were 
tested in duplicate at all concentrations. Ara-AMP and ddG 
(2'3'-dideoxyguanosine) were used as HBV inhibitory control compounds. 
Ara-AMP is a widely used compound in antiviral research and has been 
demonstrated to have activity against HBV in chronically infected patients 
(Alexander et al. Brit. Ned. J. 292:915, 1986). The nucleoside analog, ddG 
was used as an additional control compound since ddG has activity against 
duck hepatitis B virus in chronically infected Peking ducks (Lee et al., 
Antimicrob. Agents Chemother. 33:336, 1989) and 2'-CdG is also a 
deoxyguanosine analog. 
2.2.15 cells were seeded in 6 well culture plates and grown to confluence 
over a 10 day period in medium with 5% FBS. Test compounds were then added 
daily for a continuous 10 day period in medium with 1% dialyzed FBS. This 
reduced serum level does not affect HBV replication in confluent cultures 
of 2.2.15 cells and helps to eliminate uncontrolled variations of 
endogenous low molecular weight compounds (such as nucleosides) present in 
FBS. Compounds were tested, in duplicate cultures, at 3 concentrations 
covering a 100-fold range. Culture medium (changed daily during the 
treatment period) was collected and stored for analysis of extracellular 
(virion) HBV DNA from days 0, 3, 6, and 10 of the treatment period. This 
allowed for an analysis of the level of HBV virion production during 
discrete 24 hour intervals and reduced the potential for the buildup of 
toxic metabolites derived from the test compounds. Treated cells were 
lysed following the 10th day of treatment for the analysis of 
intracellular HBV genomic forms. HBV DNA was analyzed in a quantitative 
manner for (i) overall levels (both extracellular and intracellular DNA) 
and (ii) the relative rate of HBV replication (intracellular DNA only). 
HBV DNA levels were measured by comparison to known amounts of HBV DNA 
standards applied to every nitrocellulose filter (gel or slot blot) using 
an AMBIS Beta Scanner. Standard curves, generated by multiple analyses, 
were used to correlate CPM measurements with relative levels of target HBV 
DNA. The levels of HBV DNA in each of three classes of intracellular viral 
genomic forms were individually quantitated: integrated HBV DNA, episomal 
monomeric genomes and HBV DNA replication intermediates (RI). Integrated 
HBV DNA was used to normalize the relative amounts of DNA in each lane 
because the levels of this class of HBV DNA would be expected to remain 
constant on a per cell basis. The levels of the monomeric HBV genomes and 
RI were used as an indicator of the relative level of HBV replication. 
Levels of extracellular, or virion HBV DNA, in the culture medium and the 
levels of intracellular HBV DNA (Table 9), varied no more than 3-fold in 
the untreated control cell cultures. Ara-.mu.MP and ddG were effective in 
reducing the levels of HBV DNA production and replication. The doses used 
were previously determined to be nontoxic (by measurement of cell growth 
rates) to 2.2.15. 
The racemic (D,L) mixture of 2'-CdG was a potent inhibitor of HBV 
replication in 2.2.15 cells (Table 8). In comparison to ddG, 2'-CdG 
exhibited approximately the same level of inhibition of HBV replication at 
a 100-fold lower concentration. The minimal inhibitory concentration of 
approximately 50 ng/ml was approximately 10-fold higher than that 
originally reported for the racemic mixture on these same cells (Price et 
al., PNAS 86:806, 1989). The D isomer was as active an inhibitor of HBV 
replication as the racemic mixture. The L isomer did not exhibit any 
significant anti-HBV activity at the concentrations tested. 
No obvious toxicity was observed for any of the three compound 
preparations. Toxicity in these experiments was not quantitatively 
assessed. Toxicity was based upon the physical appearance of the cells 
(e.g. vacuolization, granulation, morphological changes, detachment of 
cells). 
TABLE 8 
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Effect of test compounds on HBV production in 2.2.15 cell cultures. 
INTRACELLULAR HBV DNA 
(PG/UG CELL DNA) HBV DNA IN CULTURE MEDIUM 
(PG/ML) 
WELL 
TREATMENT INTEG. 
MONO. 
RI DAY 0 
DAY 3 
DAY 6 
DAY 10 
__________________________________________________________________________ 
25A UNTREATED CELLS 
1.4 3.0 53 50 50 46 40 
25B " 1.2 2.0 50 55 100 42 45 
25C ARA-AMP @ 300 UG/ML 1.3 0.1 0.2 100 25 0 0 
25D " 1.1 0.1 0.3 95 30 0 0 
25E DDG @ 100 uM 1.6 0.2 0.1 49 35 8 0 
25F " 1.7 0.1 0.1 58 41 2 0 
25G D,L 2-CdG @ 5 ug/ml 1.6 1.7 0.1 88 24 0.4 0 
25H " 1.0 1.9 0.3 87 16 0.3 0 
25I D,L 2-CdG @ 500 ng/ml 1.4 1.8 0.4 89 41 8 0.1 
25J " 1.5 2.0 1 90 55 17 0.2 
25K D,L 2-CdG @ 50 ng/ml 1.3 2.1 12 120 88 55 11 
25L " 1.2 1.4 18 95 75 77 6 
25M D, 2-CdG @ 5 ug/ml 1.6 2.0 0.3 100 18 3 0 
25N " 0.7 2.0 0.1 120 21 6 0 
25O D, 2-CdG @ 500 ng/ml 1.0 1.9 1 80 36 15 1 
25P " 1.6 1.8 1 90 43 14 0.4 
25Q D, 2CdG @ 50 ng/ml 1.6 1.3 16 100 110 38 6 
25R " 1.3 2.1 20 77 70 40 13 
25S L, 2-CdG @ 5 ug/ml 1.5 2.0 97 50 67 77 90 
25T " 0.9 1.1 86 88 110 120 120 
25U L, 2-CdG @ 500 ng/ml 1.4 1.9 82 90 63 78 97 
25V " 1.5 1.9 99 110 83 56 91 
25W L, 2-CdG @ 50 ng/ml 1.3 1.4 90 48 47 90 90 
25X " 0.8 1.1 76 67 83 63 61 
__________________________________________________________________________ 
@ Analysis of intracellular HBV DNA was 24 hours following the 10th day o 
treatment. 
These data demonstrate that the D-isomer of 2'-CdG is considerably more 
potent against human CMV HSV-1 and HSV-2 in comparison to the D,L-mixture 
of 2'-CdG and is at least equally as potent as the racemic mixture against 
HBV, whereas the L-isomer demonstrated very little, if any, activity. 
However, we have also shown that, unexpectedly, D- and L-2'CdG are equally 
effective competitive inhibitors of the phosphorylation of thymidine by 
the virus-specific kinase, and do not differ in activity as substrates for 
the initial phosphorylation by the viral nucleoside kinase. Thus, while 
the difference in their effectiveness as antiviral agents appears to 
associated with the dramatic differences in the further phosphorylation of 
their monophosphates and in the polymerase-inhibitory activity of their 
triphosphates, it is apparent that the L-isomer is not totally inert in 
the cell. The total amount of L-2'CdG-monophosphate formed was greated 
than that of D-2'CdG-triphosphate. Although, it has been reported that 
very little if any short term cytotoxicity is associated with the 
L-isomer, long term effects L-2'CdG-monophosphate accumulation the host 
cells cannot be ruled out. 
Thus, use of the purified D-isomer of 2'CdG as well as analogues and 
prodrugs of this compound in the treatment and prophylaxis of a number of 
viral infections provides the advantage of increased efficacy at a reduced 
dosage when compared to presently available agents and methods. 
The composition can be administered at a dosage of between 10.sup.-5 and 10 
mg/m.sup.2 /dose. The therapeutically antiviral effective amount of the 
compositions to be used in accordance with this invention to provide 
prophylaxis and treatment for individuals infected with, or at risk of 
being infected, can be optimized by methods known in the art. A preferred 
range is between 10.sup.-4 and 10.sup.-1 mg/m.sup.2 /dose. Dose frequency 
can be determined by measuring half-life according to standard techniques 
and preferably will be 1-3 times daily, although less frequent 
administration (every other day or even weekly) may have a positive 
effect. Treatment is continued until no further clinical improvement is 
observed (e.g., by viral load or other clinical measures), and preferably 
longer. 
While we have hereinbefore presented a number of embodiments of this 
invention, it is apparent that our basic construction can be altered to 
provide other embodiments which utilize the processes of this invention. 
Therefore, it will be appreciated that the scope of this invention is to 
be defined by the claims appended hereto rather than the specific 
embodiments which are presented by way of example.