The present invention relates, in general, to prodrugs. In particular, the present invention relates to lipophilic, aminohydrolase-activated, anti-viral nucleoside prodrug compounds, pharmaceutical compositions containing these compounds, and methods of using these compounds.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates, in general, to prodrugs. In particular, the 
present invention relates to lipophilic anti-viral and anticancer 
nucleoside prodrugs activated by endogenous aminohydrolase enzymes. 
2. Background Information 
Nucleoside analogues are currently used as the primary mode of drug 
treatment for almost all viral diseases caused by human immunodeficiency 
virus (HIV) and the herpes virus family (herpes simplex I and II, 
varicella-zoster, cytomegalovirus, Epstein-Barr). This class of compounds 
is also useful for treating certain types of cancer. HIV causes AIDS, 
while the various members of the herpes family cause a range of problems 
(cancer, genital warts, shingles, herpes encephalitis). Many of these 
afflictions are fatal if not successfully treated. When the causative 
agent (virus or cancer cell) spreads to a sanctuary in the body (central 
nervous system (CNS), testes, eye, etc), drug treatment is often difficult 
because conventional active drugs have difficulty reaching the target 
organ site. Prodrugs designed to overcome these transport problems, 
especially to the CNS, and then be activated by endogenous aminohydrolases 
to active drugs, are described here. 
Acquired immune deficiency syndrome, or AIDS, is a fatal disease which has 
reached epidemic proportions among certain high risk groups. Several 
features of AIDS make therapy extremely difficult. The main target of the 
AIDS virus, now known as HIV, or human immunodeficiency virus, is the T4 
lymphocyte, a white blood cell that marshals the immune defenses. This 
depletion of T4 cells in AIDS causes a severe depression of the immune 
response, so that a compound which is to be effective against AIDS must 
modify virus effect without much help from host immunity. Furthermore, the 
virus also affects cells in the central nervous system (Berger, J. R. & 
Resnick, L. in AIDS. Modern Concepts and Therapeutic Challenges, Broder, 
S., Ed., M. Dekker, N. Y., 1987, pp 263-283; Snider, W. D. et al. Ann 
Neurol. 1983, 14, 403; Fauci, A. S. Science 1988, 239, 617; Price, R. W. 
et al; Science 1988, 239, 586; Lane, H. C. & Fauci, A. S. in AIDS. Modern 
Concepts and Therapeutic Challenges, Broder, S., Ed., M. Dekker, N.Y., 
1987, pp 185-203), where it is protected by the blood-brain barrier from 
compounds that might otherwise be effective against the virus (Mitsuya, Ho 
& Broder, S. Nature 1987, 325, 773; De Clercq, E. J. Med. Chem. 1986, 29, 
1561; Mitsuya, H. & Broder, S. Proc. Nat. Acad. Sci. USA 1986, 83, 1911). 
In infecting its host, the HIV binds to specific cell-surface receptor 
molecules. The virus penetrates the cell cytoplasm and sheds its protein 
coat, thereby baring its genetic material, a single strand of RNA. A viral 
enzyme, reverse transcriptase, accompanies the RNA. The virus, which is a 
retrovirus, reverse transcribes the RNA into DNA. Ultimately, some DNA 
copies of the HIV genome become integrated into the chromosomes of the 
host cell. 
This integrated viral genome, known as a provirus, may remain latent until 
the host cell is stimulated, such as by another infection. The proviral 
DNA is then transcribed into mRNA, which directs the synthesis of viral 
proteins. The provirus also gives rise to other RNA copies that will serve 
as the genetic material of viral progeny. The proteins and the genomic RNA 
congregate at the cell membrane and assemble to form new HIV particles, 
which then break off from the cell. Two HIV genes, tat and trs/art, appear 
to control this burst of replication, which destroys the cell. These genes 
code for small proteins that boost the transcription of proviral DNA and 
the synthesis of viral proteins. 
Several compounds have been shown to reduce the activity of reverse 
transcriptase in vitro. The reverse transcription is the step that is 
essential to viral replication and irrelevant to host cells. It has been 
found that HIV replication is considerably slower in the presence of 
compounds such as suramin, antimoniotungstate, phosphonoformate, and a 
class of nucleoside analogues known as dideoxynucleosides (ddN). 
Nucleoside analogues are a class of synthetic compounds that resemble the 
naturally occurring nucleosides, which are chemical precursors of DNA and 
RNA. A nucleoside comprises a single-or double-ring base linked to a 
five-carbon sugar molecule. An analogue differs from the 
naturally-occurring nucleoside in large or small features of the base or 
the sugar. An enzyme that normally acts on a nucleoside in the course of 
viral replication may also bind to the nucleoside analogue. Because the 
nucleoside and the analogue differ, however, binding to the analogue can 
incapacitate the enzymes, thereby disrupting a molecular process crucial 
to viral replication. 
Of the synthetic nucleoside analogues, dideoxyadenosine (ddA), 
dideoxyinosine (ddI) and dideoxycytidine (ddC), have been found to have 
potent in vitro activity against the human immunodeficiency virus (HIV) 
which causes AIDS. Additionally, dideoxycytidine has been found effective 
in vivo in treating patients with AIDS, and dideoxyinosine and 
dideoxyadenosine are currently being tested in vivo in patients with AIDS. 
The blood-brain-barrier protects the brain from potentially harmful 
materials in the systemic circulation. Unfortunately, this phenomenon can 
also exclude useful drugs. (Greig, N. Cancer Treat. Rev. 1987, 14, 1) 
Lipophilic, non-ionic, low molecular weight materials generally appear to 
have the best passive diffusion properties for CNS penetration. (Rall, D. 
P. & Zubrod, C. G. Annu. Rev. Pharmacol. 1962, 2, 109) It also has been 
reported that an active transport mechanism may play a role in BBB 
penetration of some nucleosides (Collins, J. M. et al. J. Pharmacol. Exp. 
Ther. 1988, 245, 466; Conford, E. M. & Oldendorf, W. H. Biochem. Biophys. 
Acta 1975, 394, 211) although the structural requirements necessary to 
make use of this possibility are not well defined. 
In an attempt to provide new antiviral and anticancer drugs by enhancing in 
vivo transport properties in general, and CNS transport properties in 
particular, the present invention specifically provides purine and 
pyrimidine nucleoside prodrugs. These compounds, because of their 
lipophilic, biologically stable character, can be transported after 
administration to disease-site sanctuaries and then converted by 
endogenous aminohydrolases to active anti-HIV, anti-herpes or anticancer 
drugs. It has been found that adenosine deaminase can convert certain 
6-substituted purine dideoxynucleoside prodrugs into antiretroviral (eg. 
HIV) active inosine and guanosine compounds. This general concept is also 
valid for prodrugs of certain other nucleosides and their analogues, 
including acyclic, purine-containing nucleosides which are antivirally 
active. The conversion of certain 4-substituted pyrimidin-2-one nucleoside 
prodrugs by cytidine deaminase to antiretroviral, antiviral and anticancer 
compounds is also possible. The prodrugs proposed, because of their need 
to be activated by an aminohydrolase, are, by definition, prodrugs of 
active inosine, guanosine, uridine, and thymidine analogues. 
SUMMARY OF THE INVENTION 
It is an object of this invention to provide synthetic nucleosides which 
are prodrugs, subject to activation by aminohydrolases. 
It is a specific object of this invention to provide synthetic nucleosides 
which have diffusion properties appropriate for CNS penetration. 
It is another object of the invention to provide synthetic lipophilic 
nucleosides. 
It is a further object of the invention to provide antiviral and anticancer 
pharmaceutical compositions. 
It is another object of the invention to provide a method of treating a 
patient infected with a virus or cancer. 
Further objects and advantages of the present invention will be clear from 
the description that follows.

DETAILED DESCRIPTION OF THE INVENTION 
Nucleosides and their analogues are known for their antiviral properties 
against retroviruses (eg. dideoxynucleosides vs. HIV) and herpes viruses 
(eg. acyclovir, DHPG, and PMEA vs. the herpes simplex family). This class 
of compounds also has activity as anticancer drugs (eg. IUDR). The in vivo 
biochemical pharmacology of a prodrug is important relative to whether an 
analogue has properties which are superior to the active parent compound. 
Important properties are activity, potency, and in vivo transport to an 
organ site which the parent compound reaches only with difficulty. 
Transport optimization is the property addressed through the design and 
synthesis of a selective set of 6-substituted purine-, and 4-substituted 
pyrimidine nucleoside analogues. The advantage of administering the 
proposed prodrugs, relative to their active parent compounds, is related 
to three factors. Prodrugs can be designed 1) to be more lipophilic than 
the parent compound which allows greater biological membrane penetration 
and therefore greater access to disease sanctuaries such as the CNS, 2) to 
have lower toxicity because the nucleoside prodrugs are poorer substrates 
for anabolic kinases. This allows the prodrug to be transported to its 
site of action (eg. CNS) in its inactive nucleoside form before 
aminohydrolase conversion and subsequent metabolic nucleotide formation, 
and 3) to allow the inactive prodrug to be converted to the active parent 
compound at its target site (eg. CNS) by endogenous enzymes (eg. 
aminohydrolases) which are abundant in the human body. 
In vitro testing experiments with certain nucleoside analogues can give 
misleading results relative to a new compound's clinical potential. These 
analogues are 6-substituted purine and 4-substituted pyrimidine 
nucleosides. This is because some of these compounds have the ability to 
be converted by aminohydrolase enzymes to active agents. Examples of this 
are experiments detailed below which show that Compounds 1f and 1i, which 
have only moderate anti-HIV activity in the ATH8 cell culture tests, can 
have activity abolished when the potent adenosine deaminase inhibitor, 
2'-deoxycoformycin (dCF) is added to the test system. Conversely, when 
extra adenosine deaminase (0.7 unit) was added to the test system, the 
activity of 1f was increased to 90% protection of ATH8 cells from the 
cytopathogenic effects of HIV. The manner in which the usual test 
procedure can be misleading relative to a new compound's clinical 
potential is related to the fact that the level of the aminohydrolase 
enzymes (eg. adenosine deaminase) in the cell culture test systems is very 
low relative to the levels in the human body. Therefore, compounds capable 
of being enzymatically hydrolyzed to an active drug, but which are 
converted slowly in vitro due to the low enzyme level, will appear to be 
poor candidate drugs. These same compounds, even though poor substrates 
for the aminohydrolases, would be hydrolyzed at an appropriate rate in 
patients to an active drug because of the large amount of enzyme present 
in mammals. The rate of hydrolysis would be determined by the prodrug 
structure which can be fine-tuned to allow for prodrug transport to the 
intended site (eg. CNS) and the controlled sustained or slow release of 
active material in the target organ by action of the endogenous 
aminohydrolase. 
Hansch and co-workers have pioneered the use of octanol-water partition 
coefficients (log P) to correlate compound structure with CNS penetration. 
(Hansch, C. et al. D. J. Med. Chem. 1967, 11, 1) This was the applicants 
starting point for maximizing the CNS anti-AIDS activity for the 2', 
3'-dideoxynucleoside series (Driscoll, J. et al. Vth Internat. Conf. on 
AIDS, Montreal, 1989, M.C.P. 107). 
The log P of AZT indicates that this compound is really neither lipophilic 
nor hydrophilic but partitions into octanol and pH 7.0 buffer almost 
equally. AZT, however, is one of the more lipophilic compounds 
investigated clinically, and it enters the CNS better than ddC (Collins, 
J. M. et al. J. Pharmacol. Exp. Ther. 1988, 245, 466; Broder, S. Medical 
Res. Rev. 1990, 10, 419). The failure of ddC to achieve significant CNS 
levels might be related to the more hydrophilic nature of this compound 
relative to AZT. Thus, ddN more lipophilic than AZT for anti-HIV 
evaluation were prepared. 
An uncertain aspect of designing lipophilic compounds is retention of 
anti-HIV activity after making the appropriate structural modifications, 
since the activity of dideoxynucleosides is critically dependent on a 
series of enzymatic events, any one of which might be adversely affected 
by a structural change. For this reason, the most minor structural changes 
possible--the addition of a methyl group (which should increase a log P 
value by ca. 0.5) (Leo, A. et al. Chem. Rev. 1971, 71, 525) at various 
purine positions were made. Purines rather than pyrimidines were modified 
initially, because purines are the more lipophilic class. Among the 
dideoxypurines, ddA was chosen to modify rather than ddI for the same 
reason, and a 2'-fluoro substituent was normally included for its acid 
stabilizing properties. (Marquez, V. E. et al. J. Med. Chem. 1990, 33, 
978) 
In the following embodiments, monophosphate, diphosphate, and triphosphate 
are defined as: 
##STR1## 
if n=0 then monophosphate, if n=1 then diphosphate, and 
if n=2 then triphosphate; 
wherein when the counter ion (M) is necessary the counter ion is an alkyl 
metal (more specifically, Na.sup.+ or K.sup.+), NH.sub.4.sup.+, or H. 
In one embodiment, the present invention relates to a compound of formula 
(I) 
##STR2## 
wherein A is H or F; , B is H, monophosphate, diphosphate, or triphosphate 
wherein when the counter ion is necessary the counter ion is an alkyl 
metal (preferably, Na.sup.+ or K.sup.+), H, or NH.sub.4.sup.+ ; 
Y is H, NH.sub.2, or halogen (F, Cl, Br, I); 
X is NHR, NR.sub.2, NROR, halogen, SR, or OR' wherein R is H, C.sub.1 
-C.sub.16 alkyl (branched or unbranched), or (CH.sub.2).sub.1-8 Ar; and R' 
is C.sub.1 -C.sub.16 alkyl or (CH.sub.2).sub.1-8 Ar wherein Ar is 
unsubstituted phenyl, or phenyl substituted with C.sub.1 -C.sub.8 alkyl 
(preferably, unbranched) or OH; 
with the proviso that when A is H, X is not a halogen. 
Preferrably, A is F, B is H, Y is H or NH.sub.2, and X is a halogen 
(specifically, Cl) or NH(C.sub.1 -C.sub.8 alkyl) (specifically, 
NHCH.sub.3). 
In another embodiment, the present invention relates to a compound of 
formula (II) 
##STR3## 
wherein A is O or CH.sub.2 ; B is H, monophosphate, diphosphate, or 
triphosphate wherein when the counter ion is necessary the counter ion is 
an alkyl metal (preferably, Na.sup.+ or K.sup.+), H, or NH.sub.4.sup.+ ; 
Z is H, OH, or CH.sub.2 OH; 
Y is H, NH.sub.2, or halogen; 
X is NHR, NR.sub.2, NROR, halogen, SR, or OR' wherein R is H, C.sub.1 
-C.sub.16 alkyl (branched or unbranched), or (CH.sub.2).sub.1-8 Ar; and R' 
is C.sub.1 -C.sub.16 alkyl (preferably, unbranched) or (CH.sub.2).sub.1-8 
Ar wherein Ar is unsubstituted phenyl, phenyl substituted with C.sub.1 
-C.sub.8 alkyl, or phenyl substituted with OH; with the proviso that when 
B is H, X may not be NH.sub.2 or OH. 
Preferably, B is H, Z is CH.sub.2 OH, Y is NH.sub.2, and X is halogen 
(specifically, Cl) or NH(C.sub.1 -C.sub.8 alkyl) (specifically, 
NHCH.sub.3). 
In a further embodiment, the present invention relates to a compound of 
formula (III) 
##STR4## 
wherein B is H, monophosphate, diphosphate, or triphosphate wherein when 
the counter ion is necessary the counter ion is an alkyl metal 
(preferably, Na.sup.+ or K.sup.+), H, or NH.sub.4.sup.+ ; 
Y is H, NH.sub.2, or halogen; 
X is NHR, NR.sub.2, NROR, halogen, SR, or OR' wherein R is H, C.sub.1 
-C.sub.16 alkyl, or (CH.sub.2).sub.1-8 Ar; and R' is C.sub.1 -C.sub.16 
alkyl or (CH.sub.2).sub.1-8 Ar wherein Ar is unsubstituted phenyl, phenyl 
substituted with C.sub.1 -C.sub.8 alkyl, or phenyl substituted with OH. 
Preferably, B is H, Y is H, and X is halogen (specifically, Cl) or 
NH(C.sub.1 -C.sub.8 alkyl) (specifically, NHCH.sub.3). 
In yet another embodiment, the invention relates to a compound of formula 
(IV) 
##STR5## 
wherein B is H, monophosphate, diphosphate, or triphosphate wherein when 
the counter ion is necessary the counter ion is an alkyl metal 
(preferably, Na.sup.+ or K.sup.+), H, or NH.sub.4.sup.+ ; 
Z is H, OH, or CH.sub.2 OH; 
X is NHR, NR.sub.2, NROR, halogen, SR, or OR' wherein R is H, C.sub.1 
-C.sub.16 alkyl, or (CH.sub.2).sub.1-8 Ar; and R' is C.sub.1 -C.sub.16 
alkyl or (CH.sub.2).sub.1-8 Ar wherein Ar is unsubstituted phenyl, phenyl 
substituted with C.sub.1 -C.sub.8 alkyl, and phenyl substituted with OH. 
Preferably, B is H, X is halogen or NH(C.sub.1 -C.sub.8 alkyl) 
(specifically, X is Cl or NHCH.sub.3), and Z is H or CH.sub.2 OH. 
In a further embodiment, the present invention relates to a compound of 
formula (V) 
##STR6## 
wherein Z is H, OH, or CH.sub.2 OH; and X is NHR, NR.sub.2, NROR, halogen, 
SR, or OR' wherein R is H, C.sub.1 -C.sub.16 alkyl, or (CH.sub.2).sub.1-8 
Ar; and R' is C.sub.1 -C.sub.16 alkyl or (CH.sub.2).sub.1-8 Ar wherein Ar 
is unsubstituted phenyl, phenyl substituted with C.sub.1 -C.sub.8 alkyl, 
and phenyl substituted with OH. 
Preferably, X is halogen or NH(C.sub.1 -C.sub.8 alkyl) (specifically, X is 
Cl or NHCH.sub.3) and Z is H or CH.sub.2 OH. 
In another embodiment, the present invention relates to a compound of 
formula (VI) 
##STR7## 
wherein X is H, F, or N.sub.3 ; Y is H, monophosphate, diphosphate, or 
triphosphate wherein when the counter ion is necessary the counter ion is 
an alkyl metal (preferably, Na.sup.+ or K.sup.+), H, or NH.sub.4.sup.+ ; 
Z is H, (CH.sub.2).sub.0-5 CH.sub.3, or halogen; and 
R is H, OH, O(CH.sub.2).sub.0-5 CH.sub.3, C.sub.1 -C.sub.16 alkyl, or 
(CH.sub.2).sub.1-5 Ar; wherein Ar is unsubstituted phenyl, phenyl 
substituted with C.sub.1 -C.sub.8 alkyl, and phenyl substituted with OH. 
Preferably, X is N.sub.3, Y is H, R is C.sub.1 -C.sub.8 alkyl 
(specifically, CH.sub.3), and Z is C.sub.1 -C.sub.8 alkyl (specifically, 
CH.sub.3). 
In a further embodiment, the present invention relates to a compound of 
formula (VII) 
##STR8## 
wherein Y is H, monophosphate, diphosphate, or triphosphate wherein when 
the counter ion is necessary the counter ion is an alkyl metal 
(preferably, Na.sup.+ or K.sup.+), H, or NH.sub.4.sup.+ ; 
Z is H, (CH.sub.2).sub.0-5 CH.sub.3, or halogen; and 
R is H, OH, O(CH.sub.2).sub.0-5 CH.sub.3, C.sub.1 -C.sub.16 alkyl, or 
(CH.sub.2).sub.1-5 Ar; wherein Ar is unsubstituted phenyl, phenyl 
substituted with C.sub.1 -C.sub.8 alkyl, and phenyl substituted with OH. 
Preferably, Y is H, R is C.sub.1 -C.sub.8 alkyl (specifically, CH.sub.3), 
and Z is C.sub.1 -C.sub.8 alkyl (specifically, CH.sub.3). 
In another embodiment, the present invention relates to a compound of 
formula (VIII) 
##STR9## 
wherein X is H or F; Y is H, monophosphate, diphosphate, or triphosphate 
wherein when the counter ion is necessary the counter ion is an alkyl 
metal (preferably, Na.sup.+ or K.sup.+), H, or NH.sub.4.sup.+ ; 
Z is halogen or CH.dbd.CHBr; and 
R is H, OH, O(CH.sub.2).sub.0-5 CH.sub.3, C.sub.1 -C.sub.16 alkyl, or 
(CH.sub.2).sub.1-5 Ar; wherein Ar is unsubstituted phenyl, phenyl 
substituted with C.sub.1 -C.sub.8 alkyl, and phenyl substituted with OH. 
Preferably, Y is H, Z is halogen (specifically, I), and R is C.sub.1 
-C.sub.8 alkyl (specifically, CH.sub.3). 
In yet another embodiment, the present invention relates to antiviral 
pharmaceutical compositions comprising the above-described compounds in an 
antivirally effective amount and a pharmaceutically acceptable diluent, 
carrier, or excipient. 
Because the 2'-fluoro compounds of the present invention are stable in an 
acid environment such as is found in the human stomach, they can readily 
be formulated without the need for pH buffers into dosages suitable for 
oral administration, using a pharmaceutically acceptable carrier, diluent, 
or excipient, which are well known in the art. Such carriers may enable 
the compounds to be formulated as tablets, pills, capsules, liquids, gels, 
and the like, for oral ingestion by a patient to be treated for AIDS. 
The precise dosage amounts to be administered will be determined by routine 
experimentation. In general, however, the dosage amounts will be 
comparable to those already known from the experimental use of dideoxy 
adenosine, AZT, acyclovir, DHPG, oxetanocin, HPMPA, PMEA, and IUDR. 
Pharmaceutical compositions within the scope of the present invention 
include compositions wherein the active ingredient is contained in an 
effective amount to achieve its intended purpose. Determination of the 
effective amounts is well within the skill of the art. 
In addition to the nucleosides of the present invention, these 
pharmaceutical compositions may contain suitable pharmaceutically 
acceptable carriers comprising excipients and auxiliaries which facilitate 
processing of the active compounds into preparations which can be used 
pharmaceutically. The preparations may be formulated for oral 
administration, and are in the form of tablets, dragees, and capsules. 
Alternatively, the preparations may be administered rectally, such as in 
the form of suppositories. Alternatively, solutions may be prepared for 
oral or parenteral administration. The compositions of the present 
invention contain from about 0.1 to 99 percent, and preferably from about 
25 to 85 percent by weight of active ingredient, together with the 
excipient. 
The pharmaceutical compositions of the present invention are manufactured 
in a manner which is itself known, for example, by means of conventional 
mixing, granulating, dragee-making, dissolving, or lyophilizing processes. 
Thus, pharmaceutical preparations for oral use can be obtained by 
combining the active compounds with solid excipients, optionally grinding 
a resulting mixture, and processing the mixture of granules, after adding 
suitable auxiliaries, if desired, to obtain tablets or dragee cores. 
Suitable excipients are, in particular, fillers such as sugars, such as 
lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, 
for example, maize starch, wheat starch, rice starch, potato starch, 
gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose, 
sodium carboxymethylcellulose, and/or polyvinylpyrrolidone. If desired, 
disintegrating agents may be added, such as the cross-linked polyvinyl 
pyrrolidone, agar, or alginic acid or a salt thereof such as sodium 
alginate. Auxiliaries which can be used include flow-regulating agents and 
lubricants, such as silica, talc, stearic acid or salts thereof such as 
magnesium or calcium stearate, and/or polyethylene glycol. Dragee cores 
are provided with suitable coatings. For this purpose, concentrated sugar 
solutions may be used, which may optionally contain gum arabic, talc, 
polyvinyl pyrrolidone, polyethylene glycol, and/or titanium dioxide, 
lacquer solutions, and suitable organic solvents or solvent mixtures. 
Dyestuffs or pigments may be added to the tablets or dragee coatings for 
identification or in order to characterize different combinations of 
active compound doses. 
Other pharmaceutical preparations which can be used orally include push-fit 
capsules made of gelatin, as well as soft, sealed capsules made of gelatin 
and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can 
contain the active ingredients in a mixture with filler such as lactose, 
binders such as starches, and/or lubricants such as talc or magnesium 
stearate and, optionally, stabilizers. In soft capsules, the active 
compounds may be dissolved or suspended in suitable liquids, such as fatty 
oils, liquid paraffin, or liquid polyethylene glycols. In addition, 
stabilizers may be added. 
Possible pharmaceutical preparations which can be used rectally include, 
for example, suppositories, which consist of a combination of the active 
compounds with a suppository base. Suitable suppository bases are, for 
example, natural or synthetic triglycerides, paraffin hydrocarbons, 
polyethylene glycols or higher alkanols. In addition, it is also possible 
to use gelatin rectal capsules which consist of a combination of the 
active compounds with a base. Possible base materials include, for 
example, liquid triglycerides, polyethylene glycols, or paraffin 
hydrocarbons. 
Although many of the anti-HIV compounds of the invention are designed to be 
administered orally because of their stability in low pH environments such 
as in gastric juices, pharmaceutical preparations may be prepared for 
parenteral administration, especially for antiviral and anticancer 
prodrugs. Suitable formulation for parenteral administration include 
aqueous solutions of the prodrugs in water-soluble form. Additionally, 
suspensions of the compounds may be prepared as appropriate oily injection 
suspensions. Suitable lipophilic solvents or vehicles include fatty oils 
such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate 
or triglycerides. Aqueous injection suspensions may contain substances 
which increase the viscosity of the suspension, such as sodium 
carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the 
suspension may also contain stabilizers. 
In a further embodiment, the present invention relates to a method of 
treating a patient infected with a virus comprising administering to the 
patient an amount of the above-described compounds sufficient to effect 
said treatment. Preferably, the therapeutically effective amount is 
sufficient to inhibit viral replication in cells infected with a 
retrovirus or is sufficient to inhibit the infectivity of a retrovirus. 
Once the prodrugs are converted by endogenous aminohydrolase enzymes to 
active compounds, the mechanism of action is as previously established 
(inhibition of retrovirus reverse transcriptase, inhibition of viral DNA 
polymerase after herpes-induced thymidine kinase activation or 
incorporation into cancer cell DNA). 
The present invention is described in further detail in the following 
non-limiting examples. 
EXAMPLES 
The following protocols and experimental details are referenced in the 
examples that follow: 
Melting points were taken on a Mel-Temp II apparatus and were uncorrected. 
UV spectra were recorded on a Beckman model 34 spectrophotometer or 
on-the-fly during HPLC analysis on a Perkin-Elmer LC-235 array 
spectrophotometer. Optical rotations were recorded on a Perkin-Elmer 241 
polarimeter at the sodium D line. Infrared spectra were recorded on a 
Perkin-Elmer 727B spectrophotometer. Proton and .sup.13 C NMR spectra were 
run on a Variant XL-200 spectrometer at 200 and 50 MHz, respectively. 
Chemical shifts are given in ppm relative to TMS and are referenced 
against the solvent in which the samples were run. Analytical and 
preparative TLC analyses were performed on Uniplate GHLF silica gel 
(Analtech, 250 and 1000 microns, respectively). Column chromatography was 
accomplished with Kieselgel 60 (mesh size 230-400). Reverse phase 
purification was performed either on C.sub.18 disposable extractions 
columns (J. T. Baker, 6 ml) or at medium pressure on bonded phase C.sub.18 
silica gel. Moisture sensitive reactions were run under argon in flasks 
previously dried at 110.degree. C. Ether and THF were distilled from 
sodium/benzophenone ketyl. All other solvents came from Sure Seal bottles 
purchased from Aldrich. Silylation reagents were premixed in 1 ml vials 
and purchased from Alltech. 
Positive ion fast atom bombardment mass spectra were obtained on a VG 7070E 
mass spectrometer operated at an accelerating voltage of 6 kV and a 
resolution of 2000. Glycerol was used as the sample matrix and ionization 
was effected by a beam of xenon atoms derived by charge-exchange 
neutralization of a 1.0-1.2 mA beam of xenon ions accelerated through 
8.0-9.2 kV. Spectra were acquired under the control of a VG 11/250 J.sup.+ 
data system at a scan speed of 10 s/decade, and the background due to the 
glycerol matrix was automatically subtracted. Accurate mass measurements 
of the protonated molecular ions (MH.sup.+) for compounds 1d and 1i (FIG. 
2) were obtained by a limited mass range voltage scan at a resolution of 
3000 with repetitive data accumulation and averaging under computer 
control. The MH.sup.+ peak was then mass measured with the appropriate 
data system software using selected glycerol peaks within the mass range 
as internal references. 
A 50 .mu.l aliquot of a 10 mg/ml suspension of adenosine deaminase 
(adenosine aminohydrolase, EC 3.5.4.4) from calf intestinal mucosa was 
centrifuged at 600.times.g for 3 min. The residue remaining after removal 
of the (NH.sub.4).sub.2 SO.sub.4 supernatant was dissolved in 0.5 ml pH 
7.4 tris(hydroxymethyl)-aminomethane (Tris) buffer to give an enzyme 
solution of 1 mg/ml (274 units/ml). 2'-Deoxycoformycin (dCF), 2', 
3'-dideoxy-2'-fluoroarabinosyladenine (2'-F-dd-ara-A, 1a) and 2', 
3'-dideoxy-2'-fluoroarabinosylhypoxanthine (2'-F-dd-ara-I, 1b) were 
obtained from the Pharmaceutical Resources Branch, NCI. A pH 7.4 buffer 
was prepared by adjusting 0.01M Tris to pH 7.4 with 0.5N HCl, The 1.0 HCl 
, HPLC-grade acetonitrile, monobasic potassium phosphate, sodium hydroxide 
and ultrapure Tris were all commercially available and were used without 
further purification. 
Enzymatic Deamination of Adenosine Analogues. Solutions (1 mM) of 
dideoxynucleoside substrates (ddA, 1a, 1f, 1g, 1i and 21) (FIG. 2) were 
prepared in 0.01M pH 7.4 Tris buffer, while 1 mM and 2 .mu.M solutions of 
the adenosine deaminase inhibitor, 2'-deoxycoformycin, were prepared in 
distilled water. A 50 .mu.L aliquot of substrate solution was diluted with 
0.95 ml 0.01M Tris buffer in a 1.5-mL Eppendorf tube and equilibrated at 
37.degree. C. in a Dubnoff metabolic shaking incubator. Reaction was 
initiated by addition of 2.5 .mu.L of adenosine deaminase solution (0.7 
unit). At specified time intervals, a 50 .mu.L aliquot of the reaction 
mixture was withdrawn and quenched by mixing with 0.45 mL cold 2 .mu.M 
2'-deoxycoformycin. This diluted sample was ultrafiltered to remove enzyme 
in a Centrifree Micropartition unit by centrifugation at 600.times.g at 
4.degree. C. in a high speed refrigerated centrifuge. The decrease in 
substrate concentration was monitored by HPLC analysis of the resultant 
ultrafiltrate (see below). The ability of 2'-deoxycoformycin to inhibit 
deamination under the above conditions was evaluated by adding 2 .mu.L of 
1.0 mM inhibitor to diluted substrate solutions at the time of 
equilibration. Reaction, sampling and analysis were carried out as above. 
In additional experiments, 4-6 concentrations of each substrate (ddA, 1a, 
1f, 1i) were reacted with ADA (0.01, 0.03 or 0.4 units depending on 
substrate) at pH 7.4 and 37.degree. C. Initial hydrolysis rates for each 
substrate concentration were measured through linear least-squares curve 
fitting of the concentration versus time data for reaction of the first 
10% of substrate. K.sub.M and v.sub.max values for each substrate were 
then determined from a graphical lineweaver-Burke plot of these initial 
rates (FIG. 1). 
HPLC Analysis of Dideoxypurine Nucleosides. 
Concentrations of dideoxynucleosides were measured by the HPLC analysis of 
100 .mu.L aliquots of ultrafiltered samples. A 4.6.times.250 mm 5-.mu.m 
Ultrasphere-ODS column, protected by a Waters guard column packed with 
37-50 .mu.m Vydac 201SC, was eluted with 10-20% CH.sub.3 CN in 0.01M pH 
7.0 phosphate buffer at a flow rate of 1.0 mL/min. Dideoxynucleosides and 
deamination products were detected at the appropriate wavelength of 
maximum absorption with a Gilson 116 variable wavelength detector. Peak 
identity was determined from coincidence of retention times with standards 
and by comparison of on-the-fly UV spectra obtained with a Perkin-Elmer 
LC-235 diode array detector. Peak areas and peak heights were measured 
simultaneously on a Spectra-Physics SP4200 computing integrator. For 
kinetic studies, this data was plotted as a function of time and, where 
possible, fitted to a first order decomposition using Graph-Pad, a 
commercial non-linear least squares, curve fitting program. 
Measurement of Octanol-Water Partition Coefficients. 
n-Octanol-water partition coefficients (P) were determined by a microscale 
shake-flask procedure, (Ford, H. et al Abstracts of Papers, 200th National 
Meeting of the American Chemical Society, Washington, D.C.; American 
Chemical Society: Washington, D.C., 1990; CARB 13) which was modification 
of an earlier method (Nahum, A. & Horvath, C. J. Chromatogr. 1980, 192, 
315). A 20 .mu.L aliquot of a 0.5 mg/mL DMSO solution of the 
dideoxynucleoside was dissolved in 1.0 mL of octanol-saturated, pH 7.0, 
0.01M potassium phosphate buffer. This was mixed thoroughly with 1.0 ml of 
buffer-saturated n-octanol in a 2-mL Mixxor apparatus at 
24.degree.-26.degree. C. and then allowed to stand for 15 min. The phases 
were separated, centrifuged at 600.times.g for 5 min, and the relative 
concentration of sample in each phase determined by HPLC analysis of a 50 
.mu.l aliquot. The partition coefficient was calculated by dividing the 
absolute area of the appropriate integrated peak from the octanol phase by 
that of the buffer phase (Table I). 
HIV Cytopathic Effect Inhibition Assay. 
The HIV cytopathic effect inhibition assay was performed as previously 
described. (Mitsuya, H. & Broder, S. Proc. Nat. Acad. Sci. USA 1986, 83, 
1911) Briefly, 200,000 target CD4.sup.+ ATH8 cells were exposed to 
cell-free HIV-1/III.sub.B at a dose of 43 (or 1087 for compound 1g) 
TCID.sub.50 (50% tissue culture infectious dose) for 1 h, resuspended in 2 
ml of fresh culture media containing interleukin 2, and cultured at 
37.degree. C. with or without test compounds in 5% CO.sub.2 -containing 
humidified air. On day 8 in culture, the viable cells were counted by 
using the dye exclusion method. All compounds, including inactives, were 
evaluated a minimum of two times in separate experiments. Data reported in 
Table II are from representative tests. The % protection against the virus 
was determined by the following formula: 100 times [(the number of viable 
cells exposed to HIV-1 and cultured in the presence of the compound minus 
the number of viable cells exposed to HIV-1 and cultured in the absence of 
compound) divided by (the number of viable cells cultured alone minus the 
number of viable cells exposed to HIV-1 and cultured in the absence of the 
compound)]. The % toxicity of a compound on the target cells was 
determined by the following formula: 100 times [1 minus (the number of 
total viable cells cultured in the presence of the compound divided by the 
number of total viable cells cultured alone)]. Calculated percentages 
equal to or less than zero are expressed as 0%. 
See FIGS. 2-5 for synthetic schemes. 
See Table V for the elemental analyses of new compounds. 
EXAMPLE 1 
Chemistry 
9-(3', 
5'-di-O-benzoyl-2'-deoxy-2'-fluoro-.beta.-D-arabinofuranosyl)-9-H-purine 
(3a). 
Purine (250 mg, 2.08 mmol) was suspended in dry acetonitrile (7 ml, argon 
atmosphere) and treated with BSTFA (1 ml vial, Pierce) at room 
temperature. After 30 min the homogeneous solution was evaporated to 
dryness in vacuo. The resulting yellow oil was placed under an argon 
atmosphere and compound 2b (807 mg, 1.90 mmol), dissolved in dry CH.sub.2 
Cl.sub.2 (10 ml), was added to the trimethylsilyl purine via syringe. 
After removing the solvent on the rotoevaporator (40.degree. C.) the neat 
mixture was heated to 100.degree. C. and rotated under vacuum for 45 min. 
After cooling to room temperature the resulting syrup was dissolved in 
CH.sub.2 Cl.sub.2, filtered and concentrated. Purification on silica gel 
(1% MeOH/CH.sub.2 Cl.sub.2 -3% MeOH/CH.sub.2 Cl.sub.2) afforded 564.2 mg 
(63.9%) of a 3:1 mixture of isomers 3a and 3b as a foam along with a minor 
amount (&lt;5%) of 3c. .sup.1 H NMR of the major component 3a (CDCl.sub.3): 
.delta.9.19 (s, H2 or H6); 9.01 (s, H2 or H6); 8.40 (d, J=2.8 Hz, H8); 
8.0-8.2 (m, aromatic); 7.3-7.7 (m, aromatic); 6.75 (dd, J=2.7 and 12.4 Hz, 
H1'); 5.80 (dd, J=2.7 Hz and 16.3 Hz, H3'); 5.38 (dd, J=2.7 and 49.8 Hz, 
H2'); 4.82 (d, H5', H5"); 4.62 (q, H4'). The mixture was carried through 
the next step. 
9-(2'-deoxy-2'-fluoro-.beta.-D-arabinofuranosyl)-9-H-purine (4a). 
A 3:1 mixture of 3a and 3b (563 mg, 1.21 mmol) was placed in a pressure 
bottle and treated with a solution of ammonia saturated methanol (10 ml). 
The mixture was kept at 10 .degree. C. for 14 h. Argon was bubbled through 
the solution for 5 min and the methanol was removed. The resulting gum was 
taken up in water and washed with CH.sub.2 Cl.sub.2 (3.times.). The 
aqueous layer was freeze dried and purified first by silica gel flash 
chromatography (FC) (5% MeOH/CH.sub.2 Cl.sub.2 eluant) and subsequently by 
reverse phase chromatography (C.sub.18, linear gradient of H.sub.2 O-10% 
MeOH/H.sub.2 O) affording 285 mg of the diol as a powder (92%) in the same 
isomer ratio (3:1). .sup.1 H NMR (DMSO-d.sub.6) for the major component 
4a: .delta.9.22 (s, H2 or H6); 8.98 (s, H2 or H6); 8.75 (d, J=1.8 Hz, H8); 
6.58 (dd, J=4.9 and 13.0 Hz,H1'); 5.30 (dt, J=4.4 Hz and 52.6 Hz, H2'); 
4.49 (dt, J=4.4 and 19.0 Hz, H3'); 3.89 (q, J=4 9 Hz,H4'); 3.68 (m, H5', 
H5") This was also carried through to the next step. Anal. (C.sub.10 
H.sub.11 N.sub.4 O.sub.3 F. 0.2 H.sub.2 O ) C, H, N, F. 
9-(5'-O-tert-butyldimethylsilyl-2'-deoxy-2'-fluoro-.beta.-D-arabinofuranosy 
l)-9-H-purine (5). 
A mixture of 4a and 4b (285 mg, 1.12 mmol) was dissolved in dry 
dimethylformamide (DMF, 4 ml), treated with 2 ml of a premixed solution of 
TBDMS-Cl/imidazole in DMF (Alltech) at room temperature and stirred for 15 
min. Water was added and the aqueous mixture was extracted with ethyl 
acetate (3.times.). The combined organic layers were washed with brine 
(3.times.), dried (Na.sub.2 SO.sub.4) and concentrated. Silica gel FC (1:1 
EtOAc/PE eluant) afforded a clean separation of the N-9 and N-7 purine 
isomers. The desired .beta.-N-9 compound, 5, was obtained as an oil in 
62%. .sup.1 H NMR (CDCl.sub.3): .delta.9.15 (s, H2 or H6); 8.98 (s, H2 or 
H6); 8.42 (d, J=2.4 Hz, H8); 6.63 (dd, J=4.0 and 16.7 Hz, H1'); 5.16 (ddd, 
J=3.0, 3.9 and 51.8 Hz, H2'); 4.73 (dm, J=18.1 Hz, H3'); 4.06 (q, J=4.7 
Hz, H4'); 3.91 (m, H5', H5"); 0.91 (s, 9H, t-butyl); 0.10 (s, 6H, 
Si--(CH.sub.3).sub.2). 
9-(5'-O-tert-butyldimethylsilyl)-2'3'-dideoxy-2'-fluoro-.beta.-D-arabinofur 
anosyl)-9-H-purine (7). 
Method A. Compound 5 (54 mg, 0.147 mmol) and CS.sub.2 (111 mg, 1.47 mmol) 
were dissolved in dry DMF and cooled to 0.degree. C. Sodium hydride (80% 
suspension, 10 mg) was added and the mixture was stirred at the same 
temperature for 30 min. Methyl iodide (208 mg, 1.47 mmol) was added via 
syringe and the solution was stirred for an additional 30 min. Water was 
added and the mixture was extracted with ether (3.times.). The combined 
organic layers were washed with brine, dried (Na.sub.2 SO.sub.4) and 
concentrated. Following purification on silica gel, the 3'-O-xanthate, 6, 
was obtained in 64% yield based on recovered starting material. This 
compound (30 mg, 0.066 mmol) was dissolved in toluene (3 ml), a trace of 
AIBN was added followed by 0.1 ml of tri-n-butyltin hydride. The solution 
was heated to 90.degree. C. for 10 min and the solvent was evaporated. 
Silica FC (2% MeOH/CH.sub.2 Cl.sub.2) afforded 15 mg (65%) of the 3'-deoxy 
derivative 7 as a powder. Compound 6 .sup.1 H NMR (CDCl.sub.3): 
.delta.9.16 (s, 1H); 8.98 (s, 1H); 8.42 (d, J=3.1 Hz, H8); 6.64 (dd, J=3.0 
and 21.5 Hz, H1'); 6.25 (dd, J=2.6 and 16.0 Hz, H3'); 5.28 (dd, J=3.0 and 
49.8 Hz, H2'); 4.31 (q, J=4.0 Hz, H4'); 3 98 (m, H5', H5"); 2.62 (s, 3H); 
0.91 (s, 9H); 0.11 (s, 6H). Compound 7: .delta.9.15 (s, 1H); 8.96 (s, 1H); 
8.48 (d, J=2.5 Hz, H8); 6.42 (dd, 3.5 and 16.7 Hz, H1'); 5.32 (dq, 
J.sub.2'F =53.5 Hz, H2'); 4.30 (m, H4'); 3.83 (d, H5'H5"); 2.59 (m, H3'); 
2.44 (m, H3"); 0.92 (s, 9H); 0.11 (s, 3H); 0.103 (s, 3H). Compound 7 was 
used as such for the next experiment. 
Method B. A mixture of t-butyl nitrite (1.12 g, 0.011 mmol) and dry THF (8 
ml) was heated to reflux under an atmosphere of argon. The flask was 
illuminated with a 200 watt unfrosted bulb and compound 16 (200 mg, 0.55 
mmol) dissolved in 2 ml of THF was added over 15 min via syringe. After 1 
h at reflux TLC analysis showed disappearance of starting material and 
appearance of a major less polar spot, along with several very minor 
compounds. The solvent was evaporated and the crude residue was purified 
by silica flash chromatography. The major spot was isolated and determined 
by NMR analysis to be identical with compound 7 prepared by method A. 
Compound 7 was used as such for the next experiment. 
9- (2',3'-dideoxy-2'-fluoro-.beta.-D-arabinofuranosyl)-9-H-purine (1c). 
Compound 7 (105 mg, 0.298 mmol) was dissolved in 80% acetic acid and heated 
to 90.degree. C. for 35 min. The acid was removed in vacuo and the 
resulting syrup was eluted through a short silica column (5% MeOH/CH.sub.2 
Cl.sub.2). The nucleoside obtained was recrystallized from acetone: ether 
affording needles (top 93.degree.-95.degree. C.) of pure 1c. Yield of the 
hydrolysis was 88%. .sup.1 H NMR (CD.sub.3 COCD.sub.3): .delta.9.08 
(s,1H); 8.90 (s, 1H); 8.66 (d, J=2.3 Hz,H8); 6.56 (dd, J=3.8 and 15.9 Hz, 
H1'); 5.52 (dm, J.sub.2'F =54.4 Hz, H2');4.38 (m, H4');3.81 (m, H5', H5"); 
2.78 (dddd, H3'); 2.45 (dddd, H3"). .sup.13 C NMR (CD.sub.3 COCD.sub.3): 
153.2, 148.9, 145.9, 92.2, 85.2, 79.3, 64.2, 33.5. FAB-MS m/z (rel 
intensity), 239 (MH.sup.+, 85), 121 bH.sub.2.sup.+, 100). UV (H.sub.2 O) 
.lambda..sub.max 261, 205 nm. [.alpha.].sub.D.sup.25 =+38.1.degree. (c 
1.8, MeOH). Anal. (C.sub.10 H.sub.11 FN.sub.4 O.sub.2) C, H, N, F. 
2-methyl-9-(3',5'-di-O-benzoyl-2'-deoxy-2'-fluoro-.beta.-D-arabinofuranosyl 
) adenine (8a). 
2-methyladenine hemisulfate (250 mg, 1.26 mmol) was per-trimethylsilylated 
under argon at room temperature (acetonitrile, BSTFA) and the solvent was 
evaporated in vacuo. The resulting yellow oil was placed under an argon 
atmosphere and compound 2b (500 mg, 1.18 mmol) dissolved in dry CH.sub.2 
Cl.sub.2 (8 ml) was added via syringe. The solvent was removed on the 
rotoevaporator (40.degree. C.), the water bath was replaced with an oil 
bath (100.degree. C.) and rotation under vacuum was continued at this 
temperature for 45 min. The brown syrup was dissolved in CH.sub.2 
Cl.sub.2, filtered and concentrated. Purification on silica gel (1% 
MeOH/CH.sub.2 Cl.sub.2 -3% MeOH/CH.sub.2 Cl.sub.2) afforded a mixture of 
isomers (71%), the desired .beta.-N-9 nucleoside 8a being 55% of the 
mixture. A pure sample of 8a was obtained as a foam by repeated silica gel 
FC. .sup.1 H NMR (CDCl.sub.3): .delta.7.3-8.2 (m, aromatics, H8); 6.59 
(dd, J=2.8 and 22.9 Hz, H1'); 5.88 (br s, NH.sub.2); 5.76 (dd, J=2.7 and 
17.2 Hz, H3'); 5.33 (dd, J=2.7 and 50.1 Hz, H2'); 4.79 (d, H5', H5"); 4.55 
(q, J=4.1 Hz, H4'); 2.59 (s, 2-Me). At this point, it was more expedient 
to carry the mixture through the next step. 
2-methyl-9-(2'-deoxy-2'-fluoro-.beta.-D-arabinofuranosyl)adenine (9). 
The mixture of 8a and 8b (490 mg, 1 mmol) was placed in a pressure bottle 
along with 10 ml of ammonia saturated methanol. The solution was allowed 
to stand at 5.degree. C. for 6 h whereupon argon was bubbled through the 
solution. The reaction was worked up as per compound 4. Purification (FC, 
5%-10% MeOH/CH.sub.2 Cl.sub.2 eluant) yielded 288 mg (90%) of a 4:1 
mixture of 9a and 9b. A pure sample of 9a was obtained as an oil by 
preparative silica gel TLC (15% MeOH/CH.sub.2 Cl.sub.2). Compound 9a 
.sup.1 H NMR (D20): .delta.8.12 (d, J=2.1 Hz, H8); 7.22 (br s, NH.sub.2); 
6.35 (dd, J=4.6 and 14.6 Hz, H1'); 5.16 (dt, J=4.2 and 52.9 Hz, H2'); 4.43 
(dt, J=4.0 and 19.0 Hz, H3'); 3.81 (q, J=4.6 Hz, H4'); 3.64 (br m, H5', 
H5"); 2.38 (s, 2-CH.sub.3). .sup.13 C NMR (DMSO): 161.6, 155.6, 149.95, 
139.0, 116.4, 95.4, 83.4, 81.2, 72.6, 60.4, 25.4. UV (MeOH) 
.lambda..sub.max 260, 212 nm. [.alpha.].sub.D.sup.25 : +31.1.degree. (c 7, 
MeOH). The mixture was carried through to the next step. 
2-methyl-9- (5 '-O-(tert-butyldimethylsilyl) 
-2'-deoxy-2'-fluoro-.beta.-D-arabinofuranosyl)adenine(10). 
The 4:1 mixture of 9a and 9b (63 mg, 0.22 mmol) was dissolved in dry DMF 
and treated with premixed silylating agent (1 ml) as described for 
compound 4. After standard workup and purification on silica gel (1% 
MeOH/CH.sub.2 Cl.sub.2) a clean separation of 5' protected nucleoside 
derivatives was effected, the major product being the desired compound 10 
(68 mg, 77%) which was obtained as an oil. .sup.1 H NMR (CDCl.sub.3): 
.delta.8.03 (d, J=2.8 Hz, H8); 6.56 (dd, J=3.5 and 19.2 Hz, H1'); 5.73 (br 
s, NH.sub.2); 5.08 (ddd, J=2.2, 3.3 and 51.9 Hz, H2'); 4.67 (ddd, J=2.3, 
3.7 and 17.0 Hz, H3'); 4.06 (m, H4'); 3.88 (m, H5', H5"); 2.57 (s, 
2-CH.sub.2); 0.89 (s, 6H, t-butyl); 0.08 (s, 3H, Si--CH.sub.3); 0.07 (s, 
3H, Si--CH.sub.3). Anal. (C.sub.17 H.sub.28 N.sub.5 O.sub.3 FSi 
.0.5H.sub.2 O ) C, H, N, (C+0.5). 
2-methyl-9-(5'-O-(tert-butyldimethylsilyl) 
-3'-O-methoxythiocarbonyl-2'-deoxy-2 
'-fluoro-.beta.-D-arabinofuranosyl)adenine (11). 
Compound 10 (60 mg, 0.15 mmol) was dissolved in dry DMF, the solution was 
heated to 80.degree. C. and 1,1-thiocarbonyldiimidazole (107 mg, 0.604 
mmol) was added at once. The mixture was stirred at 80.degree. C. for 1 h 
and the solvent was removed in vacuo. The brown gum was dissolved in 
anhydrous methanol (5 ml) and this solution was refluxed for 30 min. After 
removing the solvent the mixture was purified by silica FC (80% EtOAc/PE 
eluant). Compound 11 was obtained as a powder in 70% yield (48 mg) for the 
two steps. .sup.1 H NMR (CDCl.sub.3): .delta.8.03 (d, J=3.3 Hz, H8); 6.47 
(dd, J=2.9 and 22.3 Hz, H1'); 5.93 (dd, J=2.7 and 16.0 Hz, H3'); 5.63 (br 
s, NH.sub.2); 5.19 (dd, J=3.0 and 49.7 Hz, H2') 4.23 (m, H4') 4.10 (s, 
3H, OCH.sub.3); 4.04-3.87 (m, H5', H5"); 2.57 (s, 3H, 2-CH.sub.3); 0.91 
(s, 9H, t-butyl); 0.10 (s, 6H, Si (CH.sub.3).sub.2). The compound was used 
without further purification in the following step. 
2-Methyl-9-(5'-O-(tert-butyldimethylsilyl)-2',3'-dideoxy-2'-fluoroarabinofu 
ranosyl)adenine (12). 
Compound 11 (48 mg, 0.102 mmol) was dissolved in dry toluene and AIBN 
(trace) was added followed by tri-n-butyltin hydride (0.1 ml). The 
solution was heated to 90.degree. C. for 10 min. The solvent was 
evaporated and the mixture was purified by silica FC (2% MeOH/CH.sub.2 
Cl.sub.2). Compound 12 was obtained as a white solid in 80% yield (31.1 
mg). .sup.1 H NMR (CDCl.sub.3): .delta.8.05 (d, J=2.9 Hz, H8); 6.28 (dd, 
J=3.2 and 19.0 Hz, H1'); 5.50 (br s, NH.sub.2); 5.23 (dm, J.sub.2'F =53.6 
Hz, H2'); 4.25 (m, H4'); 3.82 (m, H5', H5"); 2.57(s, 2-CH.sub.3); 0.92 (s, 
9H, t-butyl); 0.10 (s, 3H, Si--CH.sub.3); 0.09 (s, 3H, Si--CH.sub.3). The 
compound was used directly in the next step. 
2-methyl-9-(2', 3'-dideoxy-2'-fluoro-.beta.-D-arabinofuranosyl)adenine 
(1d). 
Compound 12 (31.1 mg, 0.082 mmol) was dissolved in 4 ml of acetic acid. To 
this solution was added 1 ml of water with stirring and the flask was 
heated to 90.degree. C. for 1 h. The acid was removed in vacuo and the 
resulting gum purified first by silica FC (5% MeOH/CH.sub.2 Cl.sub.2) and 
then by reverse phase C.sub.18 chromatography eluting with a gradient of 
water10% MeOH/H.sub.2 O. The desired nucleoside was recrystallized from 
acetone affording needles (mp 237.degree.-239.degree. C.) in 87% yield (19 
mg). .sup.1 H NMR (CD.sub.3 COCD.sub.3): .delta.8.08 (d, J=2.3 Hz, H8); 
6.48 (br s, NH.sub.2); 6.32 (dd, J=3.9 and 16.6 Hz, H1'); 5.42 (dm, 
J.sub.2'F =54.5 Hz, H2'); 4.57 (m, H4'); 4.29 (m, H5', H 5"); 2.64 (m, 
H3'); 2.42 (m, H3"); 2.41 (s, 2-CH.sub.3). .sup.13 C NMR (D.sub.2 O): 
165.2, 157.4, 151.5, 143.0, 118.4, 93.7, 87.3, 80.5, 65.8, 34.7, 26.4. 
FAB-MS m/z (rel intensity), 268 (MH.sup.+, 100), 150 (bH.sub.2.sup.+, 53); 
accurate mass m/z 268.1180 (MH.sup.+, calcd 268.1210). UV (H.sub.2 O) 
.lambda..sub.max 260, 208 nm. [.alpha.].sub.D.sup.25 :+46.1.degree. (c 
3.1, H.sub.2 O). Anal. (C.sub.11 H.sub.14 O.sub.2 FN.sub.5) C, H, N. 
8-Methyl-9-(2',3'-dideoxy-2'-fluoro-.beta.-D-arabinofuranosyl)adenine (1h). 
A solution of diisopropylamine (260 mg, 2.72 mmol) in dry THF (3 ml) was 
cooled to -78.degree. C. n-Butyl lithium (1.35 ml of a 1M solution) was 
added dropwise via syringe and the solution was stirred at the same 
temperature for 15 min. Compound 16 (Marquez, V. E. et al. J. Med. Chem. 
1990, 33, 978) (200 mg 0.544 mmol) dissolved in THF (3.5 ml) was next 
added under a positive pressure of argon and the solution was stirred for 
an additional 20 min. Methyl iodide (0.9 ml, 1.36 mmol) was added and 
after 15 min at -78.degree. C. the reaction was quenched with acetic acid. 
The solution was warmed to room temperature and diluted with ether. The 
organic layer was washed sequentially with water, saturated NaHCO.sub.3 
solution and brine, dried over Na.sub.2 SO.sub.4 and evaporated. After 
purification on silica gel, a mixture of the desired 8-methyl derivative 
and several minor byproducts was isolated (134 mg). This was treated with 
80% acetic acid/water at 90.degree. C. for 40 min and the acid was 
evaporated. Purification by prep TLC (silica, 10% CH.sub.2 OH/CH.sub.2 
Cl.sub.2, eluant) followed by semi-preparative HPLC (C.sub.18 silica, 15% 
acetonitrile/water eluant) afforded pure 1h (30 mg) which recrystallized 
from acetone as a white solid (mp 208.degree.-209.degree. C.) in 22% 
overall yield. .sup.1 H NMR (D.sub.2 O): .delta.7.86 (s, H 2); 6.08 (dd, 
J=3.7 and 20.2 Hz, H1'); 5.21 (dm, J.sub.2'F =54.5 Hz, H2'); 4.24 (m, 
H4'); 3.76 (dd, J=2.5 and 12.4 Hz, H5'); 3.63 (dd, J=4.8 and 12.4 Hz, 
H5"); 2.60 (m, H3'); 2.42 (s, 8-Me); 2.16 (m, H3"). .sup.13 C NMR (D.sub.2 
O): 156.6, 153.9, 153.8, 152.1, 119.3, 94.5, 89.2, 80.4, 65.5, 35.0, 17.2. 
FAB-MS m/z (rel intensity), 268 (MH.sup.+, 100), 150 (bH.sub.2.sup.+, 35). 
UV (D.sub.2 O) .lambda..sub.max 261, 209 nm. [.alpha.].sub.D25 
=+37.degree. (c 2.0, MeOH). Anal. (C.sub.11 H.sub.14 FN.sub.5 O.sub.2) C, 
H, N, F. 
2-Methyl-N.sup.6 
-benzoyl-9-(2'-deoxy-5'-O-(tert-butyldimethylsilyl-2'-fluoro-.beta.-D-arab 
inofuranosyl)adenine (13). 
Benzoyl chloride (0.5 ml, 1.57 mmol) was added to a solution of compound 10 
(58 mg, 0.157 mmol) in dry pyridine (3 ml) previously cooled to 0.degree. 
C. After 2 h at this temperature the reaction was quenched with saturated 
sodium bicarbonate solution and the resulting mixture was extracted with 
ethyl acetate (3.times.). The combined organic layers were washed with 
water and brine, dried (Na.sub.2 SO.sub.4) and concentrated to a gum. The 
crude dibenzoate was dissolved in 4 ml of pyridine:methanol:water 
(65:30:5), cooled to 0.degree. C. and treated with 2 ml of 2N NaOH 
solution. After 30 min at this temperature solid NH.sub.4 Cl was added 
followed by water and the aqueous layer was extracted with ethyl acetate 
as described above. Chromatography on silical gel afforded 58 mg (78%) of 
the monobenzoate 13 as an oil. 1H NMR (CDCl.sub.3): .delta.8.95 (br s, 
NH); 8.20 (d, J=2.7 Hz, H8); 8.04 and 7.55 (m, aromatics); 6.59 (dd, J=3.6 
and 19.0 Hz, H1'); 5.11 (ddd, J.sub.2'F =51.6 Hz, H2'); 4.67 (dm, 
J.sub.3'F =17.8 Hz, H3'); 4.03 (m, H4' ); 3.87 (m, H5', H5"); 2.75 (s, 
2-CH.sub.3); 0.90 (s, 9H, t-butyl); 0.09 (s, 6H, Si--(CH.sub.3).sub.2). 
This compound was used directly in the next step. 
2,N.sup.6 -dimethyl-N.sup.6 
-benzoyl-9-(5'-O-(tert-butyldimethylsilyl)-3'-O-xanthyl-2'deoxy-2'-fluoro- 
.beta.-D-arabinofuranosyl)adenine (14). 
A solution of compound 13 (80 mg, 0.17 mmol) and carbon disulfide (129 mg, 
1.7 mmol) in dry DMF (4 ml) was cooled to 0.degree. C. and sodium hydride 
(11 mg of an 80% slurry in mineral oil) was added at once. The bright red 
mixture was stirred at 0.degree. C. for 30 min. Methyl iodide (241 mg, 1.7 
mmol) was added via syringe and the resulting yellow solution was stirred 
for an additional 20 min. Water was added and the solution was extracted 
with ether (3.times.). The combined ether layers were washed with brine 
(3.times.), dried (Na.sub.2 SO.sub.4) and concentrated. Silica FC yielded 
63.8 mg (65.4%) of the desired N-methyl-3'-xanthate as an oil along with 
20% of a by product believed to be the N1-methylated compound. .sup.1 H 
NMR (CDCl.sub.3): .delta.8.12 (d, J=3.1 Hz, H8); 7.1- 7.5 (m, aromatics); 
6.49 (dd, J=2.9 and 22.0 Hz, H1'); 6.22 (dd, J=2.7 and 16.5 Hz, H3'); 5.20 
(dd, J=3.1 and 49.8 Hz, H2'); 4.25 (m, H4'); 3.95 (m, H5', H5"); 3.79 (s, 
N--CH.sub.3); 2.62 (s, S--CH.sub.3); 2.47 (s, 2-CH.sub.3); 0.90 (s, 
t-butyl); 0.09 (s, Si--(CH.sub.3)2). The compound was used as such for the 
next step. 
2,N.sup.6 -Dimethyl-N.sup.6 
-benzoyl-9-(5'-O-(tert-butyldimethylsilyl)-2',3'-dideoxy-2'-fluoro-.beta.- 
D-arabinofuranosyl)adenine (15). 
A toluene solution (3 ml) of compound 14 (63 mg, 0.109 mmol) containing 
AIBN (trace) and tri-n-butyltin hydride (95 mg, 0.327 mmol) was heated to 
90.degree. C. for 10 min. The solvent was evaporated and the mixture 
purified on silica gel (2% MeOH/CH.sub.2 Cl.sub.2) affording 45 mg (86.6%) 
of the 3' deoxy product 15 as an oil. .sup.1 H NMR (CDCl.sub.3): 
.delta.8.15 (d, J=2.7 Hz, H8); 7.1-7.5 (m, aromatics); 6.28 (dd, J=3.2 and 
18.4 Hz, H1'); 5.22 (dm, J.sub.2'F =53.6 Hz, H2'); 4.23 (m, H4'); 3.79 (d, 
H5', H5"); 3.77 (s, N--CH.sub.3); 2.3-2.6 (m, H3', H3"); 2.44 (s, 
2-CH.sub.3); 0.89 (s, t-butyl); 0.07 (s, Si(CH.sub.3).sub.2). This 
compound was used directly in the following final reaction. 
2,N.sup.6 
-Dimethyl-9-(2',3'-dideoxy-2'-fluoro-.beta.-D-arabinofuranosyl)adenine 
(1e). 
Compound 15 (45 mg, 0.957 mmol) was treated with a saturated solution of 
ammonia in methanol in a pressure bottle at room temperature. After 4 h 
argon was bubbled through the solution and the methanol was evaporated. 
The resulting oil (15a) was dissolved in THF (3 ml) and treated with 
tetrabutylammonium fluoride (0.1 ml of a 1.0M solution in THF). After 5 
min at 25.degree. C. the solvent was evaporated and the oil was purified 
by silica prep TLC (10% MeOH/CH.sub.2 Cl.sub.2) and reverse phase C.sub.18 
chromatography (gradient of water-20% MeOH/H.sub.2 O ). The desired 
nucleoside 1e was recovered as a glass in 78% (20 mg) over the two steps. 
.sup.1 H NMR (CD.sub.3 COCD.sub.3): .delta.8.05 (d, J=2.2 Hz, H8); 6.75 
(br s, N.sub.H); 6.32 (dd, J=3.9 and 16.5 Hz, H1'); 5.42 (dm, J.sub.2'F 
=54.6 Hz, H2'); 4.63 (br s, OH); 4.29 (m, H4'); 3.75 (m, H5', H5"); 3.11 
(br s, N--CH.sub.3); 2.3-2.8 (2 dddd, H3', H3"); 2.43 (s, 2-CH.sub.3). 
.sup.13 C NMR (CD.sub.3 COCD.sub.3): 162.8, 156.1, 155.9, 139.6, 92.3, 
85.6, 78.9, 64.2, 33.4, 29.4, 26.0. FAB-MS m/z (rel intensity), 282 
(MH.sup.+, 100), 164 (bH.sub.2.sup.+, 64). UV (H.sub.2 O) .lambda..sub.max 
267, 212 nm. [.alpha.].sub.D.sup.25 : +37.8.degree. (c 6, H.sub.2 O). 
Anal. (C.sub.12 H.sub.16 N.sub.5 O.sub.2 F.O.8H.sub.2 O) C,H,N (N-0.7). 
6-Amino-9-[5-O-tert-butyldimethylsilyl)-2,3-dideoxy-2-fluoro-.beta.-D-threo 
-pento-furanosyl]-9H-purine (16). 
Compound 16 was prepared as previously described (Marquez, V. E. et al. J. 
Med. Chem. 1990, 33, 978). 
6-Chloro-9-(2',3'-dideoxy-2-fluoro-.beta.-D-arabinofuranosyl)purine (1i). 
Compound 16 (216 mg, 0.59 mmol) was suspended in dry CCl.sub.4 (7 ml) and 
1.34 ml (11.8 mmol) of freshly distilled t-butyl nitrite was added. The 
mixture was heated to 80.degree. C. and illuminated with a 200 Watt 
unfrosted bulb maintained one inch from the reaction flask. After 12 h the 
reagents and solvent were evaporated in vacuo. The residue was dissolved 
in 10% CH.sub.3 OH/CH.sub.2 Cl.sub.2, a small amount of celite was added 
and the solution was filtered through sodium sulfate and evaporated. 
Chromatography on silica gel (CH.sub.2 Cl.sub.2 then 2% CH.sub.3 
OH/CH.sub.2 Cl.sub.2, eluant) afforded 85 mg (37%) of the 
5'-protected-6-chloro derivative. This was treated with 80% acetic acid at 
85.degree. C. for 30 min. The acid was evaporated and the residue was 
purified on a C.sub.18 silica extraction column (Baker, 2.5% CH.sub.3 
OH/H.sub.2 O eluant) yielding 37 mg (62%) of the target nucleoside 1i. An 
analytically pure sample was obtained by a second chromatography on an 
Altex C.sub.18 HPLC column employing 20% CH.sub.3 CN/H.sub.2 O. .sup.1 H 
NMR (CD.sub.3 COCD.sub.3): .delta.8.78 (d, J=2.2 Hz, H8); 8.72 (s, H2); 
6.55 (dd, J=3.4 and 15.0 Hz, H1'); 5.59 (dm J.sub.2'F =54.3 Hz, H2'); 4.38 
(m, H4'); 3.82 (br AB, H5', 5"); 2.72 (m, H3'); 2.46 (m, H3"). .sup.13 C 
NMR (D.sub.2 O): .delta.151.9, 151.1, 150.2, 146.4, 91.5, 78.5, 63.3, 
32.3. FAB-MS m/z (rel intensity), 273 (MH.sup.+, .sup.35 Cl, 100), 239 
(MH--Cl+H, 29, 155 (bH.sub.2.sup.+,.sup.35 Cl, 70); accurate mass m/z 
273.0561 (MH.sup.+ calcd 273.0555). UV (CH.sub.3 OH): .lambda..sub.max 
260, 252 (inflection), 208. [.alpha.].sub.D.sup.25 =+55.7 (C 1.4). Anal. 
(C.sub.10 H.sub.10 N.sub.4 O.sub.2 ClF) C, H, N. 
N.sup.6 
-Benzoyl-9-(5'-O-(tert-butyldimethylsilyl)-2,3'-dideoxy-2'-fluoro-.beta.-D 
-arabinofuranosyl) adenine (17). 
Compound 16 (200 mg, 0.545 mmol) was treated under identical conditions as 
in the preparation of compound 13. After workup and purification by silica 
FC (2% MeOH/CH.sub.Cl.sub.2) 206 mg (77.8%) of the monobenzoate 17 was 
obtained as an oil along with approximately 10% of the dibenzoyl 
intermediate. .sup.1 H NMR(CDCl.sub.3): 9.11 (br s, NH); 8.79 (s, H2); 
8.35 (d, J=2.6 Hz, H8); 6.40 (dd, J=3.4 and 17.7 Hz, H1'); 5.31 (dm, 
J.sub.2'F =53.5 Hz, H2'); 4.32 (m, H4'); 3.85 (d, H5', H5"); 2.57 (m, 
H3'); 2.43 (m, H3"); 0.92 (s, t-butyl); 0.11 (s, Si--CH.sub.3); 0.10 (s, 
Si--CH.sub.3). This compound was carried to the next step. 
N.sup.6 -Methyl-N.sup.6 -benzoyl-9-(5'-O-(tert-butyldimethylsilyl)-2',340 
-dideoxy-2'-fluoro-.beta.-D-arabinofuranosyl)adenine (18). 
This was prepared in an identical manner to compound 14. After workup and 
purification, 100 mg (47%) of the desired N.sup.6 methyl derivative 18 was 
recovered as an oil along with 38 mg (18%) of what was most likely the 
N1-methyl derivative. .sup.1 H NMR (CDCl.sub.3): .delta.8.51 (s, H2); 8.32 
(d, J=2.6 Hz, H8); 7.1-7.5 (m, aromatics); 6.31 (dd, J=3.4 and 17.4 Hz, 
H1'); 5.25 (dm, J.sub.2'F =53.7 Hz, H2'); 4.26 (m,H4'); 3.80 (s, 
N--CH.sub.3); 3.79 (m, H5', H5"); 2.53 (m, H3'); 2.39 (m, H3"); 0.90 (s, 
9H, t-butyl); 0.10 (s, 6 H, Si--(CH.sub.3).sub.2). This compound was used 
directly in the next step. 
N.sup.6 -Methyl-9-(2'3'-dideoxy-2'-fluoro-.beta.-D-arabinofuranosyl)adenine 
(1f). 
Compound 18 (100 mg, 0.206 mmol) was debenzoylated as per the other 
benzoates (ammonia saturated methanol, pressure bottle, 0.degree. C. for 4 
h then room temperature for 2 h) affording after typical workup and FC on 
silica the N.sup.6 -methyl-5'-O-protected derivative 19 in &gt;90% yield. 
This was dissolved in 80% glacial acetic acid and heated to 90.degree. C. 
for 20 min. After evaporation of the acid and preparative TLC on silica 
(10% MeOH/CH.sub.2 Cl.sub.2) 45 mg (82% for two steps) of the desired 
nucleoside if was obtained as a glass. Compound 19 .sup.1 H NMR 
(CDCl.sub.3): .delta.8.38 (s, H2); 8.07 (d J=2.7 Hz, H8); 6.29 (dd, J=3.2 
and 18.4 Hz, H1'); 6.01 (br s, NH); 5.24 (dm, J.sub.2'F =53.7 Hz, H2'); 
4.25 (m, H4'); 3.81 (d, H5', H5"); 3.19 (br d, J=4.6 Hz, N--CH.sub.3); 
2.53 (m, H3'); 2.36 (m, H3"); 0.91 (s, t-butyl); 0.09 (s, Si--CH.sub.3); 
0.08 (s, Si--CH.sub.3). Compound 1f .sup.1 H NMR (D.sub.2 O): .delta.8.25 
(br s, H2); 8.18 (d J=2.3 Hz, H8); 7.01 (br s, NH); 6.38 (dd, J=3.8 and 
16.5 Hz, H1'); 5.42 (dm, J.sub.2'F =54.4 Hz, H2'); 4.59 (br s, OH): 4.31 
(m, H4'); 3.78 (br m, H5', H5"); 3.11 (br s, N--CH.sub.3); 2.30-2.83 (2 
dddd, H3', H3"). .sup.13 C NMR (D.sub.2 O): 156.8, 154.5, 148.9, 142.4, 
120.0, 93.5, 87.1, 80.5, 65.9, 34.8, 29.7. FAB-MS m/z 268 (MH.sup.+, 100), 
150 (bH2.sup.+, 30). UV (H.sub.2 O ): 265, 211 nm. [.alpha.].sub.D.sup.25 
: +56.8.degree. (c 1.9, MeOH ). Anal. (C.sub.11 H.sub.14 O.sub.2 
FN.sub.5.0.7 H.sub.2 O) C, H, N. 
N.sup.6 
-benzoyl-9-(2',3'-dideoxy-2'-fluoro-.beta.-D-arabinofuranosyl)adenine 
(1g). 
Compound 17 (49 mg, 0.104 mmol) was hydrolyzed with 80% acetic acid at 
90.degree. C. for 30 min. After evaporation of the acid and 
recrystallization from ether/CH.sub.2 Cl.sub.2, the desired nucleoside 1g 
was obtained in 68% yield (23.4 mg), mp 187.degree.-189.degree. C. .sup.1 
H NMR (CD.sub.3 COCD.sub.3): .delta.9.97 (br s, NH); 8.65 (s, H2); 8.51 
(d, J=2.3 Hz, H8); 6.52 (dd, J=3.8 and 16.1 Hz, H1'); 5.52 (dm, J.sub.2'F 
=54.4 Hz, H2'); 4.36 (m, H4'); 4.30 (t, J=6.0 Hz, OH); 3.80 (m, H5', H5"); 
2.3-2.9 (2 dddd, H3', H3"). .sup.13 C NMR (CD.sub.3 COCD.sub.3): 168.9, 
152.3, 151.9, 149.5, 144.5, 133.7, 133.0, 129.2, 128.4, 123.5, 91.8, 85.3, 
78.7, 63.6, 32.6. FAB-MS m/z (rel intensity), 358 (MH.sup.+, 100), 240 
(bH.sub.2.sup.+, 61), 105 (93). UV (MeOH) .lambda..sub.max 278, 231 (sh), 
205 nm. [.alpha.].sub.D.sup.25 : 31.7.degree. (c 1.7, MeOH). Anal. 
(C.sub.17 H.sub.15 O.sub.3 FN.sub.5. 0.2 H.sub.2 O) C, H, N, F. 
9-(2',3'-Dideoxy-2'-fluoro-.beta.-D-arabinofuranosyl)adenine-1-oxide (21). 
Compound 1a (225 mg, 0.89 mmol) was dissolved in 8 ml of acetic acid and 
1.5 ml of 30% solution of hydrogen peroxide was added. The mixture was 
stirred at room temperature for 5 days, the flask was cooled to 0.degree. 
C. and 10% palladium on carbon was added. After stirring for 30 min, the 
mixture was filtered through celite and concentrated. The crude syrup was 
purified by flash chromatography on C.sub.18 silica, eluting with a 
gradient of water-5% MeOH/H.sub.2 O, affording 132 mg of product. This was 
crystallized from 95% EtOH affording 101 mg (42%) as needles, mp, 
dec&gt;230.degree. C. .sup.1 H NMR (DMSO-d.sub.6 : .delta.8.56 (s, H2); 8.45 
(d, J=2.0 Hz, H8); 6.30 (dd, J=3.9 and 15.1Hz, H1'); 5.43 (dm, J.sub.2'F 
=54.6 Hz, H2'; 5.04 (t, J=5.8 Hz, 5'-OH); 4.17 (m, H4'); 3.61 (br s, H5', 
H5"); 2.54 (m, H3'); 2.24 (m, H3"). .sup.13 C NMR (DMSO-d.sub.6): 148.3, 
143.4, 142.5, 141.4, 117.9, 91.5, 83.5, 78.0, 62.7, 32.2. FAB-MS m/z (rel 
intensity), 270 (MH.sup.+, 48), 254 (MH--O.sup.+, 100), 152 
(bH.sub.2.sup.+, 16), 136 (bH.sub.2 --O.sup.+, 62). UV .lambda.max 
(H.sub.2 O): 232, 263. [.alpha.].sub.D.sup.25 : +12.2.degree. (c=1.0, 
MeOH). Anal. (C.sub.10 H.sub.12 N.sub.5 O.sub.2 F) C, H, N, F. 
The compounds of interest were prepared via a common carbohydrate 
precursor, 1,3,5-tri-O-benzoyl-2'-fluoro-.alpha.-D-ribofuranose 2a. (Tann, 
C. H. et al. J. Org. Chem., 1985, 50, 3644) Anomeric bromination with 30% 
HBr/acetic acid followed by condensation of the resulting sugar 2b with 
the appropriate pertrimethylsilylated base afforded the protected 
nucleosides 3 and 8 as intermediates in the synthesis of target compounds 
1c, 1d and 1e (Scheme 1). Fusion of 2b with purine gave mainly 3a along 
with minor percentages of the .beta.-N-7 and .alpha.-N-9 nucleosides, 3b 
and 3c. The analogous coupling of 2-methyladenine gave 8a as the major 
product along with 25% of the .beta.-N-9 compound 8b and very minor 
amounts of N-7 and N-6 alkylated bases. 
Ammonolysis of the acyl groups with ammonia-saturated methanol in a 
pressure bottle at 4.degree. C. afforded the 2'-fluoro-2'-deoxy 
nucleosides 4a and 9a in high yield. 
The stereochemistries of the major products were assigned by NMR coupling 
constants based on precedent (Herdewijn, P. et al. J. Med. Chem. 1987, 30, 
2131) and the regiochemistries (N9 versus N-7) by UV (Albert, A. in 
"Synthetic Procedures in Nucleic Acid Chemistry", W W Zorbach and R. S. 
Tipson, eds. 1973, Chapter 2) 71 and one dimensional nOe difference (ID 
nOe) spectra. In general, .sup.3 J.sub.1'2' in .beta.-nucleosides with a 
2'-ara substituent is in the range of 3-4 Hz whereas the 
.alpha.-nucleosides show extremely small or no coupling between the 1'- 
and 2'- protons. Also characteristic of .beta.-D-2'-ara-fluoro nucleosides 
of the purine series is the small but finite coupling (1-3 Hz) of the 
2'-fluorine atom to H8 of the base (Herwewijn, P. et al Nucleosides and 
Nucleotides, 1989, 8, 65), .sup.5 J.sub.8,F =2.5 Hz in these compounds). 
Distinguishing the N-7 and N-9 regioisomers of the 2-methyladenine 
derivatives 9a and 9b was based on the significant difference in the UV 
maxima of alkyl substituted adenines. (Albert, A. in "Synthetic Procedures 
in Nucleic Acid Chemistry", W. W. Zorbach and R. S. Tipson, eds. 1973, 
Chapter 2) This correlation was used previously to distinguish the N-7 and 
N-9 isomers of compound 1a (Marquez, V. E. et al. J. Med. Chem. 1990, 33, 
978) Since the methyl group at the 2-position has little effect on the 
position of the UV maxima, compound 9a whose UV maximum was centered at 
260 nm was assigned the N-9 structure. In the case where unsubstituted 
purine is the base (compounds 4a and 4b), the assignment of regiochemistry 
based on a similar argument is ambiguous (maxima of 4a and 4b are 261 and 
264 nm, respectively). The .sup.1 H NMR spectrum of 4a was virtually 
identical to 4b except for a subtle difference in the chemical shifts of 
the H1' protons. When H1' of 4b was irradiated in a 1D nOe experiment, 
positive enhancements were observed at H6 and H8 in addition to the 
expected enhancement at H2'. When a similar experiment was performed on 
compound 4a, a positive nOe was observed on only one base proton (H8). 
These data suggest the regiochemistries of 4a and 4b to be N-9 and N-7, 
respectively. This qualitative assessment was confirmed by conversion of 
the known 5'-protected adenosine derivative 16 to the corresponding 
nebularine analogue 7 by reductive deamination (Nair, V. & Richardson, S. 
G. J. Org. Chem. 1980, 45, 3969) (Scheme 2). 
Selective 5'-protection of compounds 4a and 9a yielded the alcohols 5and 
10, while monobenzoylation (Jones, R. A. in "Oligonucleotide Synthesis", 
M. Gait ed 1984 chapter 2) of 10 gave 13, all in high yield (Scheme 1). 
Reductive deoxygenation of the 3'-hydroxyl groups of compounds 5 and 10 to 
produce 7 and 12 proceeded without incident through the methyl xanthate 6 
and the methoxythiocarbonyl derivative (Sanghvi, Y. S. et al. Nucleosides 
and Nucleotides, 1987, 6, 761), 11, respectively. Acid catalyzed 
deprotection of 7 and 12 gave the corresponding targets, 1c and 1d. 
Preparation of the xanthate of 13 produced the N.sup.6 -methylated 
product, 14, the precursor of nucleoside 1e. Radical deoxygenation of 14 
and sequential deblocking of the resulting product 15, via 15a, afforded 
the desired 2,N.sup.6 -dimethyladenine analogue, 1e. 
Compounds 1f, 1g and 1h were prepared from the known 
5'-O-(t-butyl-dimethylsilyl)-2',3'-dideoxy nucleoside, 16 (Marquez, V. E. 
et al. J. Med. Chem. 1990, 33, 978) (Scheme 2). Dibenzoylation of 16 
followed by NaOH cleavage (Jones, R. A. in "Oligonucleotide Synthesis", M. 
Gait, ed., 1984, chapter 2) to the monobenzoyl derivative and desilyation 
afforded the N.sup.6 -benzoyl nucleoside 1g. Compound if was prepared from 
17 via methylation and sequential deblocking of the N-and O-protecting 
groups as for 1e. Direct lithiation (Hayakawa, H. et al. J. Heterocyclic 
Chem., 1989, 16, 189) of the 8-position of 16, followed by quenching with 
methyl iodide and deprotection, gave the 8-substituted derivative 1h in 
low to moderate yield. A significant amount of the olefin, 20, was formed 
from base-catalyzed elimination of hydrofluoric acid. Loss of the 
absorption for H1' and the appearance of an olefinic multiplet at 5.58 ppm 
which couples strongly to the H3' methylene in the .sup.1 H NMR provided 
evidence of structure 20. 
The 6-chloro derivative 1i was prepared via compound 16 by the method of 
Nair (Nair, V. & Richardson, S. G. J. Org. Chem. 1980, 45, 3969) (Scheme 
2). Direct replacement of the 6-amino group with chlorine proceeds in 
modest but acceptable yield (ca. 50%). Compound 1a was oxidized to the 
1-oxide, 21 by the action of hydrogen peroxide in acetic acid. 
EXAMPLE 2 
Partition Coefficients 
Molecules with a 100-fold range of lipophilicities were designed within the 
2'-fluoropurine ddN series (Table I). Octanol/pH 7.0 buffer partition 
coefficients were determined using a newly developed microscale method. 
The N.sup.6 -benzoyl compound, 1g, was the most lipophilic compound 
produced in this series with an octanol/pH 7.0 buffer partition 
coefficient (P) of 5.4 (log P 0.73). The 1-oxide of ddA, 21, was the most 
hydrophilic compound synthesized with a P of 0.04 (log P -1.38). 
Lipophilicities of these two compounds are five times greater and 27 times 
less, respectively, than AZT. The other compounds synthesized had 
intermediate P values. The addition of a methyl group normally increases 
the log P value of a compound by about 0.5. (Craig, P. J. Med. Chem. 1971, 
14, 680) In the dideoxynucleoside series, however, the lipophilicity 
constant (pi value) for a methyl group proved to be somewhat less, 0.3-0.4 
(Table I). 
TABLE I 
______________________________________ 
Octantol-Water Partition Coefficients and Chromatographic 
Properties of 2',3'-Dideoxy Nucleosides 
NPLC Mobile .lambda..sub.max.sup.c 
Compound 
Log P.sup.a Phase.sup.b (nm) 
______________________________________ 
21 -1.38 .+-. 0.06 
B 231,260 
ddC -1.33 .+-. 0.01 
A 271 
ddI -1.24 .+-. 0.03 
C 249 
1b -1.21 .+-. 0.02 
C 247 
Ic -0.40 .+-. 0.01 
D 261 
ddA -0.29 .+-. 0.01 
D 260 
1a -0.18 .+-. 0.01 
D 259 
AZT 0.05 .+-. 0.01 
F 266 
1h 0.10 .+-. 0.01 
D 260 
1d 0.12 .+-. 0.02 
D 260 
1f 0.27 .+-. 0.01 
E 265 
1i 0.32 .+-. 0.02 
F 264 
1e 0.64 .+-. 0.01 
F 265 
1g 0.70 .+-. 0.02 
F 280 
______________________________________ 
.sup.a Mean .+-. standard deviation of three independent determinations 
.sup.b The following mobile phases were used at 1.0 ml/min with a 4.6 
.times. 250 mm 5.mu.m 
Ultrasphere ODS column: A) 4%, B) 7%, C) 10%, D) 12%, E) 15% or F) 20% 
CH.sub.3 CN in pH 7.0, 0.01M phosphate buffer. ALL dideoxynucleosides had 
a retention time of 4-9 min under the above conditions. 
.sup.c Wavelength determined onthe-fly in HPLC mobile phase. 
EXAMPLE 3 
Anti-HIV Activity in vitro 
The various monomethyl analogues (1d, 1f, 1h) were prepared in an attempt 
to produce an increase in lipophilicity without adversely affecting the 
anti-HIV activity of the parent compound, 1a. The N6-methyl analogue (1f) 
(Driscoll, J. et al. Vth Internat. Conf. on AIDS, Montreal, 1989, M.C.P. 
107) was the only monomethyl compound with any in vitro activity (under 
the in vitro test conditions), and that activity was reduced relative to 
1a (Table II). Activity with the corresponding non-fluorinated compound 
had been reported earlier by Chu and co-workers (Chu, C. K. et al. Proc. 
197th Am. Chem. Soc. Meeting, Miami, 1989, Medicinal Chemistry, 89), and 
the activity of 1f against HIV in peripheral blood mononuclear cells was 
recently reported by the same group (Chu, C. K. et al. J. Med. Chem. 1990, 
33, 1553). Compound 1e is the 2-methyl analogue of 1f. As with 1d, the 
methyl group in the 2-position abolished activity. Substitution at N.sup.6 
with a benzoyl group (1g) resulted in the preservation of modest activity. 
Generation of the 1-oxide (21) of the parent compound, 1a, or removal of 
the 6-amino group to produce the nebularine analogue, 1c, abolished 
activity. The 6-chloro analogue (1i) however, provided ca. 50% protection 
to HIV-infected ATH8 cells. This finding, as well as the activity found 
for 1f, demonstrated that the 6-substituted compounds were perhaps being 
converted to an active metabolite. A reasonable explanation was that 
adenosine deaminase (ADA) catalyzed the hydrolysis of these compounds to 
the known active inosine analogue, 1b, (Scheme 3). The ATH8 cells and the 
incubation medium (which contains 15% fetal calf serum) contain some ADA 
which can act on the ddN during the seven day in vitro anti-HIV test. For 
this reason, it was decided to quantitate how rapidly the N6-methyl 
compound, 1f, was converted to 1b by ADA, and determine if this property 
could be used to advantage with prodrugs of ddI, ddG, and their 2'-fluoro 
analogues. 
TABLE II 
______________________________________ 
Effect of 2'-Deoxycoformycin on the Anti-HIV Activity of 
2',3'-Dideoxyadenosine Analogues in ATH-8 Cells.sup.a 
Com- dCF Concentration 
pound (5 .mu.m) 
.mu.M % Protection 
% Toxicity 
______________________________________ 
1a - 0,20,50 0,70,84 0,0,0 
+ 0,20,50 0,68,48 11,17,54 
1f - 0,5,10,20,50,100 
0,13,24,32,37,27 
0,0,0,0,6,44 
+ 0,5,10,20,50,100 
0,1,2,0,0,0 
11,3,7,0,46,70 
1g - 0,5,20,50,100 
0,8,30,29,21 
0,2,0,0,8 
1i - 0,10,20,50,100 
0,48,57,53,35 
0,5,26,46,52 
+ 0,10,20,50,100 
3,15,11,14,17 
14,49,58,56,72 
ddA - 10 100 0 
+ 10 86 22 
ddI - 20 100 0 
______________________________________ 
.sup.a ATH8 cells were exposed to HIV1/III.sub.B per cell for 1 hr and 
cultured in the presence of various concentrations of each compound. On 
day 8, the total viable cells were counted. Orders of numbers in the 
column for concentrations correspond to the orders of numbers in other 
columns. 
EXAMPLE 4 
Adenosine Deaminase 
Kinetic data already available indicated that the deamination of N6-methyl 
adenosine riboside analogues was slow. (Chassy, B. M. & Suhadolnik, R. J. 
J. Biol. Chem. 1967, 2.42, 3655; Baer, H. P. et al. Arch. Biochem. 
Biophys. 1968, 123, 172) Reaction of the N.sup.6 -methyl analogue, 1f, 
with ADA as the isolated enzyme (0.7 U/mL) at 37.degree. C. showed that a 
hydrolysis reaction occurred at a rate substantial enough to be easily 
quantified (FIG. 1, t.sub.1/2 =3.0 h), but which was 135 times slower than 
1a (FIG. 1, inset). It was also established that the corresponding inosine 
analogue, 1b, was the product formed, and that the rate of formation of 1b 
corresponded to the rate of disappearance of 1f (FIG. 1). Dideoxyadenosine 
and its 2'-fluoro analogue, 1a, were deaminated at much faster rates 
(t.sub.1/2 =5 and 80 sec, respectively) under these conditions (FIG. 1, 
inset). As expected (Frederiksen, S. & Rasmussen, A. H. Cancer. Res. 1967, 
27, 385; Williamson, J. & Scott-Finnigan, T. J. Antimicrob. Agents 
Chemother. 1978, 13, 735), no deamination was observed under the same 
conditions with the N-oxide, 21. Similarly, the N.sup.6 -benzoyl compound, 
1g, was unaffected by ADA. 
Because of the ubiquitous nature of ADA in vivo, it occurred to us that the 
ADA-catalyzed hydrolysis of 6-substituted dideoxypurine nucleoside 
analogues might possibly be used to advantage in anti-AIDS therapy in 
general, and in CNS therapy, in particular. The ADA reaction might be of 
general utility if the proper hydrolysis rate could be achieved for a 
compound which was converted into an active material, eg. 1b. In addition, 
if a compound could be designed which was catabolized slowly enough by ADA 
in the peripheral circulation to allow transport into the CNS, but fast 
enough by ADA in the CNS to provide therapeutic concentrations of an 
active, more hydrophilic inosine analogue, then a drug delivery system 
could be available which provided enhanced CNS prodrug entry with reduced 
therapeutic drug exit. This would be a variation on the CNS "locked-in" 
effect of very polar molecules which is a part of the dihydropyridine CNS 
prodrug system developed by Bodor and coworkers (Pop, E. et al. J. Med. 
Chem. 1989, 32, 1774) and is important since it is generally thought that 
compounds which enter the CNS easily also exit easily (Palomino, E. et al 
J. Med. Chem. 1990, 33, 258). The reported values for ADA in the CNS are 
somewhat variable, but it appears clear that certain CNS diseases, 
especially meningeal tuberculosis, greatly increase ADA activity relative 
to normal controls. (Hankiewicz, J. & Lesniak, M. Enzymologia 1972, 43, 
385; Piras, M. A. & Gakis, C. Enzyme 1972/73, 14, 311; Malan, C. et al J. 
Trop. Med. Hygiene 1984, 87, 33; Norstrand, I. F. et al. Enzyme 1984, 32, 
20) Whether AIDS causes a similar effect on ADA CNS levels is, however, 
presently unknown. 
Since it is reported that ADA hydrolyzes a number of groups other than the 
amino function in the 6-position of purine nucleosides (Chassy, B. M. & 
Suhadolnik, R. J. J. Biol. Chem. 1967, 242, 3655; Baer, H. P. et al. Arch. 
Biochem. Biophys. 1968, 123, 172; Simon, L. N. et al. Biochemistry 1970, 
9, 573; Maguire, M. H. & Sim, M. K. Eur. J. Biochem. 1971, 23, 22), the 
6-chloro derivative, 1i, also appeared to be an attractive target, since 
the pi value for an aromatic chlorine is +0.71. This should result in a 
compound, 1i, with a predicted log P value of 0.31 based on 1c. Table I 
shows that this is the case. Another reason for the interest in 1i is the 
recently demonstrated anti-HIV activity of the non-fluorosugar analogues 
of the 2',3'-dideoxy-6-halopurines in multiple CD4+ cell systems, 
including the ATH8 system (Shirasaka, T. et al Proc. Nat. Acad. Sci. 
(USA), in press). Because of the reproducible, albeit unspectacular, 
activity found for 1i and 1f (Tabe II), it was decided to examine the role 
of ADA by determining the enzyme kinetics for the hydrolysis of several 
active 2'-fluoro analogues (1a, 1f and 1i, Scheme 3) relative to the 
non-fluorinated dideoxynucleoside, ddA. 
As seen in Table III, there are significant variations in both the binding 
affinities (K.sub.M) and the maximum reaction velocities (v.sub.max) among 
the 2'-fluoro analogues and between ddA. None of the 2'-fluoro 
dideoxynucleosides bound as tightly or reacted as rapidly with ADA as the 
parent compound, ddA. Therefore, large differences thus exist in the 
measured relative rates of ADA hydrolysis determined at 50 .mu.M substrate 
for these compounds as compared to ddA. Both the 6-chloro (1i) and 
6-methylamino (1f) analogues are hydrolyzed much more slowly (ca. 1700 and 
2500 times slower, respectively) than ddA. While if was bound to ADA about 
70 times tighter than 1i, its v.sub.max was 50 times slower resulting in 
the measured relative rates being fairly similar. Compound 1a was 
hydrolyzed 17 times slower than ddA, which compares with a value of ca. 10 
times slower determined previously under slightly different conditions. 
(Masood, R. et al Mol. Pharmacol. 1990, 37, 590) Relative rates were 
measured using 50 .mu.M substrate (Table III), since this concentration 
produced an anti-HIV protective effect with the compounds shown in Table 
II. Preliminary experiments indicate that the ADA level in media alone (no 
ATH8 cells) is more than 1000 times lower than the 0.7 U/mL concentration 
used in our isolated enzyme experiments. ADA is also present in a number 
of cell lines, (Cooney, D. A. et al Biochem. Pharmacol. 1987, 36, 1765) 
including ATH8 cells. (Masood, R. et al Mol. Pharmacol. 1990, 57, 590) 
Additional studies are underway to quantitate the effects of media and 
cellular ADA on ddN analogues, and will be reported at a future time. 
In order to further establish the importance of ADA in the anti-HIV 
experiments, the effect of the powerful ADA inhibitor, 2'-deoxycoformycin 
(dCF), was evaluated. dCF affected the compounds in this study in 
different ways when added to the in vitro anti-HIV test system (Table II). 
dCF alone (5 .mu.M), did not produce significant toxicity or protection of 
ATH8 cells against the effects of HIV-1. Similarly, there were no 
important changes in the protection afforded by ddA and its 2' fluoro 
analogue, 1a, in the presence of dCF. Toxicity, however, appeared to be 
potentiated in each instance. The ddA/dCF anti-HIV results are consistent 
with reported data (Cooney, D. A. et al Biochem. Pharmacol. 1987, 36, 
1765). The effects observed were quite different when dCF was used in 
combination with the N.sup.6 -methyl (1f) and 6-chloro (1i) compounds. In 
these cases, protection was either abolished or greatly decreased relative 
to experiments conducted in the absence of dCF (Table II). This is 
qualitatively consistent with the isolated enzyme data shown in FIG. 1 and 
Table III. 
As a consequence of the above results, studies were conducted to evaluate 
the effect of augmenting the HIV/ATH8 test system with additional ADA. In 
these experiments (Table IV), the combination of 10 .mu.M if (N.sup.6 
-methyl analogue) and ADA (0.7 U/mL) gave 90% protection, whereas 1f, 
alone, at the same concentration gave only 13% protection. Compound 1a 
with supplementary ADA under the same conditions did not change its 
activity. ADA by itself neither influenced the viability nor protected 
infected ATH8 cells. These data suggest that 1f and 1i are prodrug forms 
of 2'-F-dd-ara-I (1b), which require hydrolysis by ADA as a necessary 
first step in their activation. 
TABLE III 
______________________________________ 
Adenosine Deamimase/Dideoxynucleoside Kinetic Parameters.sup.a 
.nu.max Measured 
K.sub.M (.mu.mol/ Relative 
Substrate (Molar) min/U) Rate.sup.b 
______________________________________ 
ddA 1.4 .times. 10.sup.-5 
1.32 100 
2'-F-dd-ara-A (1a) 
3.3 .times. 10.sup.-4 
2.3 .times. 10.sup.-1 
6.0 
6-Cl-2'-F-dd-ara-P (1i) 
7.5 .times. 10.sup.-3 
2.6 .times. 10.sup.-2 
0.06 
N.sup.6 --CH.sub.3 -2'-F-dd-ara-A 
1.1 .times. 10.sup.-4 
5.5 .times. 10.sup.-4 
0.04 
(1f) 
______________________________________ 
.sup.a pH 7.4 and 37.degree. C. 
.sup.b Measured at 50 .mu.M substrate concentration 
TABLE IV 
______________________________________ 
Effect of Added Adenosine Deaminase (ADA) or 2'- 
Deoxycoformycin (dCF) on the Anti-HIV Activity of 10 .mu.M 
Compound 1f. 
Additive % Protection.sup.a 
______________________________________ 
None (10 .mu.M 1f only) 
13% 
dCF (2 .mu.M) &lt;10% 
ADA (0.7 U/ml) 90% 
______________________________________ 
.sup.a Protection of ATHS cells from the cytopathogenic effects of 
HIV.sub.IIIB. 
TABLE V 
______________________________________ 
Elemental Analyses 
Compound Theory Found 
______________________________________ 
1c C: 50.42 50.57 
H: 4.62 4.70 
N: 23.53 23.49 
F: 7.98 8.03 
1d C: 49.43 49.54 
H: 5.24 5.24 
N: 26.22 26.15 
1e 0.8 H.sub.2 O 
C: 48.75 48.83 
H: 5.96 5.84 
N: 23.70 22.97 
1f 0.7 H.sub.2 O 
C: 47.14 47.08 
H: 5.71 5.42 
N: 25.00 24.63 
1g 0.2 H.sub.2 O 
C: 56.51 56.21 
H: 4.43 4.52 
N: 19.39 19.38 
F: 5.26 5.47 
1h C: 49.44 49.56 
H: 5.24 5.30 
N: 26.22 26.20 
F: 7.12 7.19 
1i C: 44.12 43.84 
H: 3.67 3.65 
N: 20.58 20.36 
4a 0.2 H.sub.2 O 
C: 46.58 46.84 
H: 4.43 4.75 
N: 21.74 21.54 
F: 7.38 6.96 
5 0.5 H.sub.2 O C: 50.93 50.55 
H: 6.90 6.73 
N: 14.85 15.15 
10 0.5 H.sub.2 O 
C: 50.25 49.93 
H: 7.14 7.24 
N: 17.24 17.70 
21 H.sub.2 O C: 41.81 41.67 
H: 4.88 4.92 
N: 24.39 24.11 
F: 6.62 6.73 
______________________________________ 
All publications mentioned hereinabove are hereby incorporated in their 
entirety by reference. 
While the foregoing invention has been described in some detail for 
purposes of clarity and understanding, it will be appreciated by one 
skilled in the art from a reading of this disclosure that various changes 
in form and detail can be made (for example, BO-- or YO-- of Formulas 
I-VIII can be Ph--O--C--O--) without departing from the true scope of the 
invention and appended claims.