Method and compounds for aica riboside delivery and for lowering blood glucose

AICA riboside and prodrugs of AICA riboside are provided which lower blood glucose for the treatment of various pathologic conditions, including hypoglycemia, insulin deficiency, insulin resistance diabetes and Syndrome X. Prodrugs of AICA riboside provide AICA riboside in an orally bioavailable form. The use of adenosine kinase inhibition and ZMP enhancement for lowering blood glucose are also described.

FIELD OF THE INVENTION 
This invention generally relates to purine nucleosides, especially to 
1-.beta.-D-ribofuranosyl-5-amino-imidazole-4-carboxamide 
("5-amino-4-imidazolecarboxamide riboside" or "AICA riboside") prodrugs. 
It also relates to the preparation, use and administration of these 
compounds which, when introduced into the body, will metabolize into their 
active forms. This invention also relates to ischemic syndrome treatments, 
anticonvulsant therapeutic agents, methods and treatment of seizure and 
related disorders, and to lowering blood glucose and the treatment of 
blood glucose-related disorders including diabetes mellitus. 
BACKGROUND OF THE INVENTION 
The present invention is directed to compounds which act as prodrugs of 
AICA riboside and certain analogs of it. AICA riboside monophosphate is a 
naturally occurring intermediate in purine biosynthesis. AICA riboside is 
also naturally occurring and is now known to enable adenosine release from 
cells during net ATP catabolism. By virtue of its adenosine releasing 
abilities, AICA riboside has many therapeutic uses. However, we have 
discovered that AICA riboside does not cross the blood-brain barrier well 
and is inefficiently absorbed from the gastrointestinal tract; both 
characteristics decrease its full potential for use as a therapeutic 
agent. 
We have also discovered that AICA riboside, and AICA riboside pro-drugs and 
analogs can be used to lower blood glucose levels in animals, including 
rats, rabbits, dogs and man. These compounds are surprisingly efficacious 
for lowering blood sugar and are believed to be partially causing their 
effect by decreasing hepatic gluconeogenesis. These compounds will be 
useful for the treatment of animals for conditions including 
hyperglycemia, insulin resistance, insulin deficiency, diabetes mellitis, 
Syndrome X, to control the hyperglycemia and/or hyperlipidemia associated 
with total parenteral nutrition, or a combination of these effects. While 
AICA riboside does not have the enhanced bioavailability as described for 
those pro-drugs set forth herein as useful for penetrating the gut 
barrier, it may be nevertheless useful for the above conditions because 
AICA riboside itself will be present in amounts sufficient to reach the 
liver, as we have also discovered. AICA riboside monophosphate is 
implicated by our studies to be the causative agent and, accordingly, it 
and monophosphate forms of prodrug and analog compounds noted herein are 
within the scope of our invention. 
Adenosine, 9-.beta.-D-ribofuranosyladenine (the nucleoside of the purine 
adenine), belongs to the class of biochemicals termed purine nucleosides 
and is a key biochemical cell regulatory molecule, as described by Fox and 
Kelly in the Annual Reviews of Biochemistry, Vol. 47, p. 635, 1978. 
Adenosine interacts with a wide variety of cell types and is responsible 
for a myriad of biological effects. Adenosine serves a major role in brain 
as an inhibitory neuromodulator (see Snyder, S. H., Ann. Rev. Neural Sci. 
8:103-124 1985, Marangos, et al., NeuroSci and Biobehav. Rev. 9:421-430 
(1985), Dunwiddie, Int. Rev. Neurobiol., 27:63-130 (1985)). This action is 
mediated by ectocellular receptors (Londos et al., Regulatory Functions of 
Adenosine, pp. 17-32 (Berne et al., ed.) (1983)). Among the documented 
actions of adenosine on nervous tissue are the inhibition of neural firing 
(Phillis et al., Europ. J. Pharmacol., 30:125-129 (1975)) and of calcium 
dependent neurotransmitter release (Dunwiddie, 1985). Behaviorally, 
adenosine and its metabolically stable analogs have profound 
anticonvulsant and sedative effects (Dunwiddie et al., J. Pharmacol. and 
Exptl. Therapeut., 220:70-76 (1982); Radulovacki et al., J. Pharmacol. 
Exptl. Thera., 228:268-274 (1981)) that are effectively reversed by 
specific adenosine receptor antagonists. In fact, adenosine has been 
proposed to serve as a natural anticonvulsant, and agents that alter its 
extracellular levels are modulators of seizure activity (Dragunow et al., 
Epilepsia 26:480-487 (1985); Lee et al., Brain Res., 21:1650-164 (1984)). 
In addition, adenosine is a potent vasodilator, an inhibitor of immune 
cell function, an inhibitor of granulocyte oxygen free radical production, 
an anti-arrhythmic, and an inhibitory neuromodulator. Given its broad 
spectrum of biological activity, considerable effort has been aimed at 
establishing practical therapeutic uses for adenosine and its analogs. 
Since adenosine is thought to act at the level of the cell plasma membrane 
by binding to receptors anchored in the membrane, past work has included 
attempts to increase extra-cellular levels of adenosine by administering 
it into the blood stream. Unfortunately, because adenosine is toxic at 
concentrations that have to be administered to a patient to maintain an 
efficacious extracellular therapeutic level, the administration of 
adenosine alone is of limited therapeutic use. Further, adenosine 
receptors are subject to negative feedback control following exposure to 
adenosine, including down-regulation of the receptors. 
Other ways of achieving the effect of a high local extracellular level of 
adenosine exist and have also been studied. They include: a) interference 
with the uptake of adenosine with reagents that specifically block 
adenosine transport, as described by Paterson et al., in the Annals of the 
New York Academy of Sciences, Vol. 255, p. 402 (1975); b) prevention of 
the degradation of adenosine, as described by Carson and Seegmiller in The 
Journal of Clinical Investigation, Vol. 57, p. 274 (1976); and c) the use 
of analogs of adenosine constructed to bind to adenosine cell plasma 
membrane receptors. 
There are a large repertoire of chemicals that can inhibit the cellular 
uptake of adenosine. Some do so specifically, and are essentially 
competitive inhibitors of adenosine uptake, and others inhibit 
nonspecifically. P-nitrobenzylthioinosine and dipyridamole appear to be 
competitive inhibitors. A variety of other chemicals, including 
colchicine, phenethyalcohol and papaverine inhibit uptake nonspecifically. 
Extracellular levels of adenosine can be increased by the use of chemicals 
that inhibit enzymatic degradation of adenosine. Previous work has focused 
on identifying inhibitors of adenosine deaminase, which participates in 
the conversion of adenosine to inosine. Adenosine deaminase activity is 
inhibited by coformycin, 2'-deoxycoformycin, and 
erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride. 
A number of adenosine receptor agonists and antagonists have been generated 
having structural modifications in the purine ring, alterations in 
substituent groups attached to the purine ring, and modifications or 
alterations in the carbohydrate moiety. Halogenated adenosine derivatives 
appear to have been promising as agonists or antagonists and, as described 
by Wolff et al. in the Journal of Biological Chemistry, Vol. 252, p. 681, 
1977, exert biological effects in experimental systems similar to those 
caused by adenosine. Derivatives with N-6 or 5'-substitutions have also 
shown promise. 
Although all three techniques discussed above may have advantages over the 
use of adenosine alone, they have been found to have several 
disadvantages. The major disadvantages of these techniques are that they 
rely on chemicals that have adverse side effects, primarily due to the 
fact that they must be administered in doses that are toxic, and that they 
affect most cell types nonselectively. As described in Purine Metabolism 
in Man, (eds. De Baryn, Simmonds and Muller), Plenum Press, New York, 
1984, most cells in the body carry receptors for adenosine. Consequently 
the use of techniques that increase adenosine levels generally throughout 
the body can cause unwanted, dramatic changes in normal cellular 
physiology. In addition, adenosine deaminase inhibitors prevent the 
degradation of deoxyadenosine which is a potent immunotoxin. (Gruber et 
al., Ann. New York Acad. Sci. 451:315-318 (1985)). 
It will be appreciated that compounds which increase extracellular levels 
of adenosine or adenosine analogs at specific times during a pathologic 
event, without complex side effects, and which would permit increased 
adenosine levels to be selectively targeted to cells that would benefit 
most from them, would be of considerable therapeutic use. By way of 
example, such compounds would be especially useful in the prevention of, 
or response during, an ischemic event, such as heart attack or stroke, or 
other event involving an undesired restricted or decreased blood flow, 
such as atherosclerosis or skin flap surgery, for adenosine is a 
vasodilator and prevents the production of superoxide radicals by 
granulocytes. Such compounds would also be useful in the prophylactic or 
affirmative treatment of pathologic states involving increased cellular 
excitation, such as (1) seizures or epilepsy, (2) arrhythmias (3) 
inflammation due to, for example, arthritis, autoimmune disease, Adult 
Respiratory Distress Syndrome (ARDS), and granulocyte activation by 
complement from blood contact with artificial membranes as occurs during 
dialysis or with heart-lung machines. It would further be useful in the 
treatment of patients who might have chronic low adenosine such as those 
suffering from autism, cerebral palsy, insomnia and other neuropsychiatric 
symptoms, including schizophrenia. The compounds useful in the invention 
may be used to accomplish these ends. 
Clearly, there is a need for more effective anticonvulsant therapeutic 
compounds and strategies since most of the currently used antiseizure 
agents are toxic (e.g., dilantin), or are without efficacy in many 
patients. Adenosine releasing agents, which enhance adenosine levels 
during net ATP catabolism will be useful for the treatment of seizure 
disorders. 
Compounds which selectively increase extracellular adenosine will also be 
used in the prophylactic protection of cells in the hippocampus implicated 
in memory. The hippocampus has more adenosine and glutamate receptors than 
any other area of the brain. Accordingly, as described below, it is most 
sensitive to stroke or any condition of low blood flow to the brain. Some 
recent studies support the theory that Alzheimer's disease may result from 
chronic subclinical cerebral ischemia. The compounds of the invention will 
be used for the treatment and/or prevention of both overt stroke and 
Alzheimer's disease. 
It is now established that relatively short periods of brain ischemia (on 
the order of 2 to 8 minutes) set into motion a series of events that lead 
to an eventual death of selected neuronal populations in brain. This 
process is called delayed excitotoxicity and it is caused by the 
ischemia-induced release of the excitatory amino acid (EAA) 
neurotransmitters glutamate and aspartate. Within several days post-stroke 
the neurons in the brain are overstimulated by EAA's to the point of 
metabolic exhaustion and death. Because glutamate appears to be the major 
factor involved in post-stroke cell damage, the blockade of glutamate 
receptors in brain could be beneficial in stroke therapy. In animals, 
glutamate receptor blockers have been shown to be effective in alleviating 
or reversing stroke associated neural damage. These receptor blockers 
have, however, been shown to lack specificity and produce many undesirable 
side effects. Church, et al., "Excitatory Amino Acid Transmission," pp. 
115-118 (Alan R. Liss, Inc. 1987). 
Adenosine has been shown to be a potent inhibitor of glutamate release in 
brain. The CA-1 region of brain is selectively sensitive to post-stroke 
destruction. In studies, where observations were made at one, three and 
six days post-stroke the CA-1 area was shown to be progressively destroyed 
over time. However, where cyclohexyladenosine ("CHA") a global adenosine 
agonist, was given shortly after the stroke, the CA-1 area was markedly 
protected. (Daval et al., Brain Res. 491: 212-226 (1989).) That beneficial 
effect was also seen in the survival rate of the animals. Because of its 
global effect, however, CHA has non-specific side effects. For example it 
undesirably will lower blood pressure and could remove blood from the 
ischemic area, thereby causing further decreased blood flow. 
The compounds of the invention described and claimed herein not only show 
the beneficial adenosine release (glutamate inhibiting properties) but are 
both site and event specific, avoiding the unwanted global action of known 
adenosine agonists. These compounds will also be used in the treatment of 
neurodegenerative diseases related to the exaggerated action of excitatory 
amino acids, such as Parkinson's disease. 
Another area of medical importance is the treatment of neurological 
diseases or conditions arising from elevated levels of homocysteine (e.g., 
vitamin B12 deficiencies). The novel AICA riboside prodrugs of this 
invention may be used for such purposes as well. 
A further area of medical importance is the treatment of allergic diseases, 
which can be accomplished by either preventing mast cell activation, or by 
interfering with the mediators of allergic responses which are secreted by 
mast cells. Mast cell activation can be down-regulated by immunotherapy 
(allergy shots) or by mast cell stabilizers such as cromalyn sodium, 
corticosteroids and aminophylline. There are also therapeutic agents which 
interfere with the products of mast cells such as anti-histamines and 
adrenergic agents. The mechanism of action of mast cell stabilization is 
not clearly understood. In the case of aminophylline it is possible that 
it acts as an adenosine receptor antagonist. However, agents such as 
cromalyn sodium and the corticosteroids are not as well understood. 
It will be appreciated, therefore, that effective allergy treatment with 
compounds which will not show any of the side effects of the above noted 
compounds, such as drowsiness in the case of the anti-histamines, 
agitation in the case of adrenergic agents, and Cushing disease symptoms 
in the case of the corticosteroids would be of great significance and 
utility. In contrast to compounds useful in the present invention, the 
AICA riboside prodrugs, none of the three known mast cell stabilizers are 
known or believed to be metabolized in the cell to purine nucleoside 
triphosphates or purine nucleoside monophosphates. 
The use of AICA riboside and prodrugs of AICA riboside as antiviral agents 
and for increasing the antiviral activity of AZT is disclosed in 
commonly-assigned U.S. patent application Ser. No. 301,454, "Antivirals 
and Methods for Increasing the Antiviral Activity of AZT", filed Jan. 24, 
1989, the disclosure of which is incorporated herein by reference. 
Certain derivatives of AICA riboside have been prepared and used as 
intermediates in the synthesis of nucleosides such as adenosine or 
nucleoside analogs such as 3'-deoxy-thio-AICA riboside. See, e.g., U.S. 
Pat. No. 3,450,693 to Suzuki et al.; Miyoshi et al., Chem. Pharm. Bull. 
24(9): 2089-2093 (1976); Chambers et al., Nucleosides & Nucleotides 7(3): 
339-346 (1988); Srivastava, J. Org. Chem. 40(20): 2920-2924 (1975). 
Hyperglycemia has been reported to be associated with a poor prognosis for 
stroke. (Helgason, Stroke 19(8): 1049-1053 (1988). In addition, mild 
hypoglycemia induced by insulin treatment has been shown to improve 
survival and morbidity from experimentally induced infarct. (LeMay et al., 
Stroke 19(11): 1411-1419 (1988)). We believe that AICA riboside and the 
prodrugs of the present invention will be useful to help protect against 
ischemic injury to the central nervous system (CNS) at least partly by 
their ability to lower blood glucose. 
Hyperglycemia and related diabetic conditions are generally divided into 
"type I" or severe (typically insulin requiring) and "type II" or mild 
(typically controlled by oral hypoglycemic agents and/or diet and 
exercise). Type I diabetic patients have severe insulin deficiency with 
complications typically including hyperglycemia and ketoacidosis. Type II 
diabetic patients typically have milder insulin deficiency or decreased 
insulin sensitivity associated with hyperglycemia predominantly from 
accelerated hepatic gluconeogenesis. Both forms of diabetic conditions are 
associated with atherosclerosis and ischemic organ injury. 
Oral hypoglycemic agents that are currently available clinically include 
sulfonylureas (e.g., tolbutamide, tolazamide, acetohexamide, 
chlorpropamide, glyburide, glipizide) and biguanides (e.g. phenformin and 
metformin). The sulfonylurea class of drugs lower blood sugar acutely in 
man and experimental animals by causing insulin release but in long term 
studies, their activity appears to involve extra pancreatic effects. These 
drugs are active on potassium cation channels, but it is not known if this 
activity is related to their hypoglycemic effects. The sulfonyl-urea class 
of drugs are not ideal hypoglycemic agents for a variety of reasons; 
moreover, they have been associated with increased risk of cardiovascular 
disease and can be of insufficient efficacy for many Type II diabetes 
patients. 
The biguanide class of drugs reduce blood sugar by increasing peripheral 
utilization of glucose and by decreasing hepatic glucose production, both 
effects presumably caused by inhibiting oxidative phosphorylation. In 
addition, because of their inhibition of oxidative phosphorylation, the 
biguanides have been associated with fatal lactic acidosis and, for that 
reason, are at present not available clinically in the United States. 
Other compounds which lower blood sugar have been described in the 
literature, but none of them is available clinically due to other 
toxicities. (See Sherratt, H. S. A., "Inhibition of Gluconeogenesis by 
Non-Hormonal Hypoglycaemic Compounds" in Short-Term Regulation of Liver 
Metabolism, pp. 199-277 (Hue, L. and Van de Werve, G., ed.s, 
Elsovier/North Holland Biomedical Press, 1981)). D-Ribose has been 
reported to cause hypoglycemia after oral or intravenous administration to 
experimental animals and humans and Foley (J. Clin. Invest. 37: 719-735 
(1958)) demonstrated an inhibition of phosphoglucomutase by 
ribose-5'-phosphate (formed intracellularly after ribose therapy). 
Although others have suggested that ribose lowers glucose via increased 
insulin release (Ishiwita et al., Endoncinol. Japan 25: 163-169 (1978)), 
the preponderance of evidence favors decreased glucose production over 
increased insulin release. 
Fructose diphosphatase has been suggested as an ideal target for new 
hypoglycemic agents, since it is one of two control steps in 
gluconeogenesis. (See, Sherratt, supra 1981) However, therapeutic agents 
which lower its activity are not presently clinically available. Fructose 
diphosphatase is inhibited by AMP and activated by ATP, being responsive 
to the cellular energy charge. Pyruvate carboxylase, the other major 
regulatory step in gluconeogenesis, is the first committed step towards 
glucose production and is regulated by the availability of acetyl CoA; 
however, its inhibition would result in interruption of mitochondrial 
function. 
The present invention is directed to purine prodrugs and analogs which 
exhibit and, in some cases improve upon, the positive biological effects 
of AICA riboside and other adenosine releasing compounds without the 
negative effects of systemic adenosine. The compounds herein defined may 
be used as prodrugs. The novel compounds typically exhibit one or more of 
the following improvements over AICA riboside: 1) more potent adenosine 
releasing effects; 2) increased half-lives; 3) increased brain 
penetration; 4) increased oral bioavailability; 5) increased myocardial 
targeting; 6) in some cases efficacy improvements over AICA riboside 
itself. 
The AICA riboside prodrugs of this invention may be used in treatment and 
prevention of a number of disorders, some of which already have been 
mentioned. 
SUMMARY OF THE INVENTION 
The present invention is directed to prodrugs of AICA riboside. We have 
surprisingly found that AICA riboside has very limited oral 
bioavailability. Accordingly, we have found that when AICA riboside is 
given orally, very little or none of it reaches the tissue(s) which 
comprise its site(s) of action. Among other factors, the present invention 
is based on our finding that oral administration of prodrugs of AICA 
riboside result in enhanced levels of AICA riboside in the blood and other 
tissues, as compared with oral administration of AICA riboside itself. Use 
of prodrugs of AICA riboside allow for delivery of therapeutically 
effective amounts of AICA riboside to the tissue(s) to be treated. 
AICA riboside has less than full gastrointestinal tract penetration and 
relatively low blood brain-barrier penetration. Derivatization of 
adenosine releasing agents, including AICA riboside, has been undertaken 
with the goals of increasing penetration of AICA riboside into the brain 
and through the gut by delivering it as a brain and/or gut permeable form 
that avoids first pass metabolism and, while reaching the target 
regenerates into the parent compound (a prodrug strategy). 
The present invention is directed to compounds which act as prodrugs of 
AICA riboside and their use as prodrugs in therapies as described below. 
These prodrug compounds comprise a modified AICA riboside having an AICA 
ribosyl moiety and at least one hydrocarbyloxycarbonyl or 
hydrocarbylcarbonyl moiety per equivalent weight of AICA ribosyl moiety. 
It has been found that AICA riboside may be chemically modified to yield an 
AICA riboside prodrug wherein one or more of the hydroxyl oxygens of the 
ribosyl moiety (i.e. 2'-, 3'- or 5'-) is substituted with a 
hydrocarbyloxycarbonyl or hydrocarbylcarbonyl moiety. 
These compounds function as prodrugs of AICA riboside and are better 
absorbed from the gastrointestinal system and are better able to cross the 
blood-brain barrier than AICA riboside itself. It is believed that the 
added ester side groups allow for improved absorption from the 
gastrointestinal system and decreased first pass metabolism, as well as in 
making more drug available for crossing the blood-brain barrier. As the 
prodrug molecule approaches or reaches the active site, intact modifying 
groups can be endogenously cleaved to regenerate AICA riboside. 
The prodrug compounds of the present invention are useful in treating a 
variety of clinical conditions where increasing extracellular levels of 
adenosine would be beneficial. Accordingly, the present invention is 
directed to the prophylactic and affirmative treatment of such conditions 
as stroke, Alzheimer's disease, homocysteineuria, skin flap and 
reconstructive surgery, post-ischemic syndrome and other seizure-related 
conditions, spinal cord ischemia, intraoperative ischemia especially 
during heart/lung bypass procedures, cardioplegia, diabetes mellitus, 
hyperglycemic conditions including that associated with total parenteral 
nutrition, and myocardial ischemia, including angina and infarct, using 
these prodrug compounds. These prodrugs are useful in treating other 
indications where AICA riboside has exhibited activity and where oral 
administration is preferred or would be advantageous. Thus, they are 
useful in delivering AICA riboside in an orally bioavailable form. This 
invention is also directed to pharmaceutical compositions comprising an 
effective amount of a prodrug compound of the present invention in a 
pharmaceutically acceptable carrier. 
Preferred prodrug compounds include those where at least one of the 
hydroxyl oxygens of the ribosyl moiety is substituted with a 
hydrocarbyloxycarbonyl or hydrocarbylcarbonyl moiety. One preferred class 
of compounds are those wherein at least one hydroxyl oxygen is substituted 
with a hydrocarbyloxycarbonyl moiety. One preferred class of prodrug 
compounds comprises compounds wherein either of the 3'- or 5'-hydroxyl 
oxygens, or both, of the ribosyl moiety is substituted with a 
hydrocarbyloxycarbonyl or hydrocarbylcarbonyl moiety. 
Compounds having a 5'-ester substituent constitute a preferred class of 
compounds due in part to the slower hydrolysis rates that have been 
observed in plasma, giving a longer half-life in the bloodstream and, 
thus, allowing less frequent dosing. 
Due to their enhanced oral bioavailability, preferred prodrug compounds 
include those substituted with 1 to 3 short chain acyl ester groups. In 
particular, compounds having a 5'-pivaloyl or isobutyryl substitution or 
having a 2',3',5'-triacetyl substitution have shown enhanced 
bioavailability when given orally. Also showing enhanced oral 
bioavailability are compounds having a 5'-butyryl or 3',5'-diacetyl 
substitutions. 
Another preferred group of compounds include those having a 
3'-hydrocarbyloxycarbonyl substitution, especially those having an 
isobutoxycarbonyl or neopentoxycarbonyl substitution. 
In one aspect, the present invention is directed to a class of novel 
prodrug compounds. In general, these compounds comprise a modified AICA 
riboside having an AICA ribosyl moiety and at least one 
hydrocarbyloxycarbonyl or hydrocarbylcarbonyl moiety, or combinations 
thereof, per equivalent weight of AICA ribosyl moiety, provided that said 
prodrug does not have three acetyl, propionyl or benzoyl moieties per 
equivalent weight of AICA ribosyl moiety and that it is not 
dibenzoyl-substituted or mono-acetyl substituted at the 5'-position of the 
ribosyl moiety. 
A particularly preferred group of compounds includes those mono- or di- 
substituted with a short chain acyl ester group. Such compounds include 
those having a 5'-acyl ester substitution or a 3',5'-diacyl substitution. 
Preferred diacyl substituted compounds include the 3',5'-diacetyl 
substituted compound and the 5'-n-butyryl substituted compound. Especially 
preferred compounds include those having either a 5'-pivalyl or 
5'-isobutyryl substitution. 
Another aspect of the present invention provides prodrugs of carbocyclic 
AICA riboside. 
Definitions 
As used herein, the following terms have the following meanings, unless 
expressly stated to the contrary: 
The term "alkyl" refers to saturated aliphatic groups, including straight, 
branched and carbocyclic groups. 
The term "alkenyl" refers to unsaturated alkyl groups having at least one 
double bond [e.g. CH.sub.3 CH.dbd.CH(CH.sub.2).sub.2 --] and includes both 
straight and branched-chain alkenyl groups. 
The term "alkynyl" refers to unsaturated groups having at least one triple 
bond [e.g. CH.sub.3 C.tbd.C(CH.sub.2).sub.2 --] and includes both straight 
chain and branched-chain groups. 
The term "aryl" refers to aromatic hydrocarbyl and heteroaromatic groups 
which have at least one aromatic ring. 
The term "alkylene" refers to straight and branched-chain alkylene groups 
which are biradicals, and includes, for example, groups such as ethylene, 
propylene, 2-methylpropylene 
##STR1## 
and the like. 
The term "hydrocarbyl" denotes an organic radical composed of carbon and 
hydrogen which may be aliphatic (including alkyl, alkenyl, and alkynyl 
groups and groups which have a mixture of saturated and unsaturated 
bonds), alicyclic (carbocyclic), aryl (aromatic) or combinations thereof; 
and may refer to straight-chained, branched-chain, or cyclic structures or 
to radicals having a combination thereof, as well as to radicals 
substituted with halogen atom(s) or heteroatoms, such as nitrogen, oxygen, 
and sulfur and their functional groups (such as amino, alkoxy, aryloxy, 
carboxyl, ester, amide, carbamate or lactone groups, and the like), which 
are commonly found in organic compounds and radicals. 
The term "hydrocarbyloxycarbonyl" refers to the group 
##STR2## 
wherein R' is a hydrocarbyl group. 
The term "hydrocarbylcarbonyl" refers to the group 
##STR3## 
wherein R' is a hydrocarbyl group. 
The term "ester" refers to a group having a 
##STR4## 
linkage, and includes both acyl ester groups and carbonate ester groups. 
The term "halo" or "halogen" refers to fluorine, chlorine, bromine and 
iodine. 
The term "carbonate ester" refers to the group 
##STR5## 
wherein R' is hydrocarbyl or to compounds having at least one such group. 
The term "acyl ester" refers to the group 
##STR6## 
wherein R' is hydrocarbyl or to compounds having at least one such group. 
The term "mixed ester" refers to compounds having at least one carbonate 
ester group and at least one acyl ester group or to compounds having 
combinations of different acyl ester or carbonate ester groups. 
In referring to AICA riboside and the AICA riboside prodrugs of the present 
invention, the following conventional numbering system for the rings is 
used: 
##STR7## 
The term "carbocyclic AICA riboside" refers to an analog of AICA riboside 
wherein the oxygen atom of the ribosyl ring has been replaced by a carbon 
atom. Accordingly, carbocyclic AICA riboside has the following structure 
and the following conventional number system for the rings, as noted, is 
used: 
##STR8## 
The term "prodrug" refers to compounds which are derivatives of a parent 
compound (such as AICA riboside) which have been derivativized to assist 
the parent compound in getting to the desired locus of action. The 
derivitized portion of the prodrug is cleaved (metabolized) or activated 
to give the parent compound either in transit or at the desired locus. 
Typically a prodrug may allow the parent compound to cross or better cross 
a biological barrier such as the gut epithelium or the blood-brain 
barrier, at which point it is cleaved to give the parent compound. 
The term "oral bioavailability" refers to the quantity of drug reaching the 
bloodstream after oral administration. Accordingly, an "orally 
bioavailable" drug is one which is well absorbed from the gut and reaches 
the blood stream when administered orally.

DETAILED DESCRIPTION OF THE INVENTION 
Preferred Prodrug Compounds 
Preferred prodrug compounds of the present invention comprise a modified 
AICA riboside having an AICA ribosyl moiety and at least one 
hydrocarbyloxycarbonyl or hydrocarbylcarbonyl moiety per equivalent weight 
of AICA ribosyl moiety. 
Preferred are AICA riboside prodrugs of the formula: 
##STR9## 
wherein X.sub.1, X.sub.2 and X.sub.3 are independently (a) hydrogen, (b) 
##STR10## 
wherein R.sub.1 is independently hydrocarbyl or independently mono- or 
dihydrocarbylamino and R.sub.2 is independently hydrocarbyl, or (c) two of 
X.sub.1, X.sub.2 and X.sub.3 taken together form a cyclic carbonate ring, 
provided that at least one of X.sub.1, X.sub.2 and X.sub.3 is not 
hydrogen. 
Since for many indications, it would be advantageous and preferred to 
administer these prodrugs orally, those prodrugs which exhibit enhanced 
oral bioavailability would offer a therapeutic advantage. Accordingly, 
prodrugs where one or more of X.sub.1, X.sub.2 and X.sub.3 comprises a 
short chain hydrocarbylcarbonyl group are preferred. In view of their 
enhanced bioavailability when given orally in either a liquid or solid 
(e.g., capsule) form, particularly preferred are those prodrugs where 
X.sub.1 is isobutyryl or pivaloyl and X.sub.2 and X.sub.3 are both 
hydrogen (compounds 10 and 11 of Table I) and where X.sub.1, X.sub.2 and 
X.sub.3 are acetyl (compound C1 of Table I). Also preferred are those 
prodrugs where X.sub.1 is n-butyryl and X.sub.2 and X.sub.3 are both 
hydrogen, and where X.sub.1 and X.sub.3 are both acetyl and X.sub.2 is 
hydrogen. Especially preferred are certain prodrug compounds which have 
been isolated in an advantageous crystalline form, in particular 
2',3',5'-triacetyl AICA riboside (Compound C1 of Table I), 3',5'-diacetyl 
AICA riboside (Compound 22 of Table I) and 3'-neopentoxycarbonyl (Compound 
17 of Table I). Moreover, in the acetyl-substituted prodrug compounds, the 
leaving groups comprise acetate which is advantageously relatively 
pharmacologically silent. 
Preferred Novel Prodrug Compounds 
The preferred novel prodrug compounds of the present invention include 
those having the following formula: 
##STR11## 
wherein X.sub.1, X.sub.2, and X.sub.3 are independently (a) hydrogen, or 
(b) 
##STR12## 
wherein R.sub.1 is independently hydrocarbyl preferably of from 1 to about 
24 carbon atoms or mono- or di-hydrocarbylamino, R.sub.2 is independently 
hydrocarbyl preferably of form 1 to about 24 carbon atoms or (c) two of 
X.sub.1, X.sub.2 and X.sub.3 taken together form a cyclic carbonate group; 
with the proviso that not all of X.sub.1, X.sub.2 and X.sub.3 are 
hydrogen, acetyl, propionyl or benzoyl, or if one of X.sub.1, X.sub.2 and 
X.sub.3 is hydrogen, the other two are not both benzoyl, or if X.sub.2 and 
X.sub.3 are hydrogen, then X.sub.1 is not acetyl. Preferred R.sub.1 and 
R.sub.2 groups include lower alkyl groups. One preferred class of lower 
alkyl groups are those having at least one secondary or tertiary carbon 
atom. Another preferred class of lower alkyl groups are those having up to 
about 6 carbon atoms and optionally having a secondary or tertiary carbon 
atom. Hydrocarbyl groups having more than 24 carbon atoms may be used and 
are considered to be within the scope of the present invention. 
Preferred compounds include those having one or two ester groups. 
Especially preferred are compounds having an ester group at either the 3'- 
or 5'-position or both positions of the ribosyl ring. 
One preferred class of compounds comprises carbonate esters. Particularly 
preferred carbonate esters include compounds wherein X.sub.1 or X.sub.3 is 
##STR13## 
especially preferred are such compounds where X.sub.2 is hydrogen. One 
such preferred group of compounds are those having a 3'-carbonate ester 
group. Especially preferred carbonate ester compounds, include those where 
X.sub.1 and X.sub.2 are both hydrogen and X.sub.3 is isobutoxycarbonyl 
(compound No. 1 of Table I) or neopentoxycarbonyl (Compound No. 17 of 
Table I). Other preferred 3'-substituted carbonate esters include 
compounds Nos. 4, 3, and 7 of Table I. 
One particularly preferred class of prodrug compounds include compounds 
which have enhanced water solubility. Such compounds are believed to 
exhibit enhanced bioavailability when given orally due to improved 
absorption from the gastrointestinal tract. For this reason, it is 
believed that prodrug compounds having one or two acyl ester groups are 
particularly advantageous. Especially preferred are short chain ester 
groups having less than about 6 carbon atoms. In particular, we have found 
compounds having an acyl ester at the 5' position or both the 3' and 5' 
positions of the ribosyl ring to be preferred. In particular, we have 
found compounds No. 10 (where X.sub.1 is isobutyryl and X.sub.2 and 
X.sub.3 are both hydrogen), and No. 11 (where X.sub.1 is pivalyl and 
X.sub.2 and X.sub.3 are both hydrogen) of Table I to be particularly 
preferred. Other preferred compounds include No. 31 (where X.sub.1 is 
n-butyryl and X.sub.2 and X.sub.3 are both hydrogen) and No. 22 (where 
X.sub.2 is hydrogen and X.sub.1 and X.sub.3 are both acetyl) of Table I. 
Especially preferred are 3'-neopentoxycarbonyl-AICA riboside (Compound 17 
of Table I), 3',5'-diacetyl-AICA riboside (Compound No. 22) which have 
been isolated in an advantageous crystalline form; and, with respect to 
3',5'-diacetyl AICA riboside, its 3'- and 5'-leaving groups comprise 
acetate (which is relatively pharmacologically silent). 
Preparation of Preferred Compounds 
The preferred carbonate ester and acyl ester compounds of the present 
invention may be conveniently prepared according to the following reaction 
scheme: 
##STR14## 
wherein X.sub.1, X.sub.2, X.sub.3, R.sub.1, and R.sub.2, are as defined in 
conjunction with formula (I). 
Reaction (1) is carried out by combining II, AICA riboside, and III, the 
appropriate acid chloride, acid anhydride or chloroformate, in solvents. 
The acid chloride may be conveniently prepared by conventional procedures 
such as reaction of the corresponding acid with thionyl chloride. Some 
acid chlorides and acid anhydrides are commercially available. Many 
chloroformates are commercially available; also, the chloroformates may be 
conveniently prepared by conventional procedures known to those skilled in 
the art by the reaction of phosgene with the appropriate alcohol. Reaction 
(1) is conducted at a temperature of from about -10.degree. C. to about 
5.degree. C., preferably from about -5.degree. C. to about 0.degree. C. 
and is generally complete within about 2 to about 4 hours. For ease of 
handling, the reaction is carried out in solvents. Suitable solvents 
include dimethylformamide (DMF), pyridine, methylene chloride and the 
like. For convenience, the reaction is carried out at ambient pressure. 
The reaction product(s) are isolated by conventional procedures such as 
column chromatography, crystallization and the like. As may be 
appreciated, the reaction may result in a mixture of products, mono, di, 
and tri-esters at the 2'-, 3'- and/or 5'-positions of the ribosyl moiety. 
The product esters may be separated by conventional procedures such as 
thin layer chromatography (TLC), high pressure liquid chromatography 
(HPLC), column chromatography, crystallization, and the like which are 
well known to those skilled in the art. 
The 5'-monoesters may be conveniently prepared according to the following 
reaction scheme to give an intermediate blocked at the 2' and 3' 
positions: 
##STR15## 
wherein X.sub.1a is 
##STR16## 
and DbAg is a deblocking agent. 
Reaction (2) is conducted by combining II, IV, V and VI. Although the 
reactants may be combined in any order, it may be preferred to add II to a 
mixture of IV, V and VI. The reaction is carried out at a temperature of 
about 10.degree. C. to about 25.degree. C., preferably from about 
15.degree. C. to about 25.degree. C. and is generally complete within 
about 45 minutes. Intermediate VI is isolated by conventional procedures. 
Reaction (3) is the reaction of intermediate VII with the appropriate acid 
chloride, acid anhydride or chloroformate and is carried out as described 
in connection with Reaction (1). 
Reaction (4) is an optional step to remove, if desired, the cyclic blocking 
group from the 2' and 3' positions. It is carried out by reacting with IX, 
the appropriate deblocking agent. Suitable deblocking agents include 
H.sup.+ resin in water/acetone, tetraethyl-ammonium fluoride/THF, acetic 
acid/water, formic acid/water and the like. Such deblocking reactions are 
conventional and well known to those skilled in the art. 
Mixed ester compounds may be conveniently prepared by first reacting AICA 
riboside with the appropriate acid chloride or acid anhydride according to 
Reaction (1) to add the acyl ester group and then reacting the acyl 
ester-substituted compound with the appropriate chloroformate according to 
Reaction (1) to obtain the mixed ester. Alternatively, mixed ester 
compounds may be prepared by first converting AICA riboside to a monoacyl 
ester according to Reaction (1) or Reaction (2) and then reacting the 
purified monoacylated product with the appropriate chloroformate according 
to Reaction (1). In addition, some mixed esters are prepared by first 
converting AICA riboside to a mono-alkoxy-carbonate according to Reaction 
(1) or (2) and then reacting the purified carbonate ester with an 
appropriate acid chloride or acid anhydride according to Reaction (1). 
Carbocyclic AICA Riboside Compounds 
We have discovered that carbocyclic AICA riboside has adenosine releasing 
agent properties. Thus, in another aspect, the present invention also 
provides a novel class of prodrugs of carbocyclic AICA riboside, and the 
use of carbocyclic AICA riboside and its prodrugs as adenosine releasing 
agents. These prodrugs are also useful as antiviral agents. 
These prodrug compounds comprise a modified carbocyclic AICA riboside 
having a carbocyclic AICA ribosyl moiety which comprises an AICA moiety 
and a cyclopentyl moiety (where a carbon has replaced the oxygen in the 
ribosyl ring), and at least one hydrocarbyloxycarbonyl or 
hydrocarbylcarbonyl moiety per equivalent weight of carbocyclic AICA 
ribosyl moiety. 
The carbocyclic AICA riboside and its prodrug compounds are useful in 
treating a variety of clinical conditions where increasing extracellular 
levels and release of adenosine would be beneficial. This invention is 
also directed to pharmaceutical compositions comprising an effective 
amount of carbocyclic AICA riboside or a prodrug compound thereof in a 
pharmaceutically acceptable carrier. 
These carbocyclic AICA riboside prodrugs may be conveniently prepared by 
methods similar to those used in the preparation of the AICA riboside 
prodrugs described herein, substituting carbocyclic AICA riboside for AICA 
riboside as a starting material. 
Utility 
As noted previously, the prodrug compounds of the present invention are 
useful in treating a variety of clinical conditions where increased 
extracellular levels (and/or release) of adenosine are beneficial. 
In particular, these compounds are useful in stroke therapy, either by 
prophylactic treatment or by treatment soon after the cerebral vascular 
event. These compounds are useful in mitigating the effects of other 
post-ischemic syndromes in the central nervous system, including the brain 
and spinal chord. 
It is now clear that relatively short periods of brain ischemia set into 
motion a series of events that lead to an eventual death of selected 
neural populations in brain caused by the ischemia induced overproduction 
of the EAA neurotransmitters. Thrombolytic therapy of stroke is therefore 
not sufficient to protect against the ensuing neurologic damage after the 
occlusion is removed. 
Since EAA's appear to be the major factor involved in post-stroke cell 
damage, blockade of EAA release in brain with adenosine might be 
beneficial in stroke therapy. Known glutamate receptor blockers have 
however been shown to lack specificity and produce many undesirable side 
effects, and the undesirable effects of adenosine administration have been 
noted. However, low doses of adenosine or an adenosine agonist with a high 
A.sub.1 to A.sub.2 affinity ratio or co-administration of a centrally 
acting agonist with an antagonist that cannot enter the central nervous 
system might avoid cardiovascular side effects and bind the A.sub.1 
receptors in the hippocampal regions thereby preventing or reducing EAA 
release. 
AICA-riboside has been shown to protect against cellular degeneration that 
results after experimentally induced brain ischemia in two different 
animal model systems. The claimed prodrugs, by delivering AICA riboside 
should provide similar efficacy. In a gerbil model employing 5 minutes of 
global ischemia followed by reperfusion, AICA-riboside prevented the 
degeneration of hippocampal CA-1 cells, which in the control animals (non 
AICA-riboside treated) were virtually completely destroyed. Both 
intracerebroventricular (ICV) and IP administration of AICA riboside was 
effective in the gerbil model. In addition to the gerbil, two different 
rat models of focal ischemia were also used to evaluate AICA-riboside. One 
model employed partial reperfusion and the second total reperfusion. Both 
protocols showed a highly significant reduction in infarct size when a 800 
mg/kg dose of AICA-riboside was given IP. 
These compounds are also useful in treating other ischemic conditions, 
particularly those involving myocardial ischemia such as heart attacks and 
angina pectoris. 
During a heart attack, adenosine is normally released and assists in 
maintaining the patency of ischemic vessels through vasodilation and 
inhibition of granulocyte free radical production and concomitant 
microvascular plugging, as described below. The prodrug compounds of the 
present invention enhance adenosine release and, therefore enhance the 
normal protective effect of adenosine during such an ischemic event. 
Adenosine levels are not altered significantly throughout the patient 
because alterations of adenosine production only occur in areas of, and at 
the time of, net ATP use and because adenosine is rapidly degraded. Thus, 
there will be a localized increased level of extracellular adenosine 
instead of a systemic or generalized adenosine enhancement. 
Since many of the damaging events during ischemia occur rapidly, the 
prodrugs of the present invention should be present at the earliest 
possible moment. Accordingly, prophylactic use of these prodrugs may slow 
or interrupt the damaging process early enough to prevent any permanent 
damage. For example, the increased microvascular blood flow from 
vasodilation and decreased white cell sticking could maintain 
microvascular patency as well as in a sense wash away clots, clot 
promoting matter, or other deleterious agents from the proximal 
atherosclerotic regions. 
In addition, since the prodrugs of the present invention when taken 
prophylactically would enhance adenosine release during an ischemic event, 
a heart attack patient undergoing such treatment would have a greater 
chance of not dying of a sudden arrhythmia before entry to the hospital. 
Such a prophylactic therapeutic regimen would protect the microvascular 
system and allow a longer time frame in which to institute thrombolytic 
therapy. 
Moreover, the prodrugs of the present invention will also be useful in 
combination with thrombolytic agents such tissue plasminogen activator, 
streptokinase, and the like and also with other agents which are either 
free radical scavengers or agents which prevent the production of free 
radicals. 
The prodrugs of the present invention are useful in treating reduced blood 
flow caused by myocardial arrhythmia. Prophylactic treatment with AICA 
riboside has been shown to result in decreased numbers of premature 
ventricular depolarizations and ventricular tachycardia episodes in 
animals and, more recently, decreased fatal ventricular fibrillation. 
In addition, the prodrugs of the present invention may be useful in 
treating other conditions in which administration of AICA riboside has 
been beneficial. These include conditions such as treatment of autoimmune 
and inflammatory diseases and neurodegenerative diseases, conditions 
potentially associated with chronically low adenosine (including autism, 
insomnia, cerebral palsy, schizophrenia and other neuropsychiatric 
conditions), conditions associated with hyperglycemia (including diabetes 
mellitus and/or resulting from total parenteral nutrition), allergic 
conditions (especially by preventing the release of pharmacologically 
active substances by mast cells), and viral conditions, especially those 
associated with the human immunodeficiency disease. 
However, as previously noted, AICA riboside is inefficiently absorbed from 
the gut and is poor in crossing the blood-brain barrier to penetrate the 
affected foci in the brain. 
The advantageous features of more efficient absorption from the GI tract 
and better crossing of the blood brain barrier of the prodrug compounds of 
the present invention should give them increased efficacity and improved 
therapeutic effect as compared to AICA-riboside itself. 
In addition the prodrug compounds of the present invention are useful as 
anticonvulsants and in preventing seizures in individuals with epilepsy 
including patients with homocysteineuria. 
Both AICA riboside and some of these prodrug compounds have been shown to 
be active in preventing homocysteine-induced seizures in laboratory 
animals. 
In addition AICA riboside and the prodrugs of the present invention should 
be efficacious in reducing ischemic injury to the CNS. The enhanced 
localized adenosine should cause local vasodilation, decreased granulocyte 
activation and trapping, and decreased glutamate release and excitatory 
neurotoxicity. The mild hypoglycemia resulting from administration of 
these compounds is also protective. 
We have demonstrated that AICA riboside can cause hypoglycemia in rats, 
rabbits, dogs and man. This hypoglycemic effect may contribute to the 
anti-ischemic properties of the molecule. Ribose is known to lower blood 
glucose in several animal species, including man, in relatively high doses 
(on the order of about 1000 mg/kg) by an unknown mechanism, possibly by 
inhibition of phosphoglucomutase. Accordingly, in theory, AICA riboside 
could cause hypoglycemia at extremely high doses (approximately 3 gm/kg) 
due to its ability to deliver ribose phosphate to cells. Other means of 
increasing intracellular ribose such as treatment with ribose itself or 
other nucleosides and prodrugs and analogs of them which can be 
metabolized to yield ribose-1-phosphate (which is converted 
ribose-5-phosphate) or ribose-5-phosphate may also lower blood sugar. 
Surprisingly, we have found AICA riboside to lower blood glucose in 
rabbits at doses an order of magnitude lower than those effective for 
ribose, as low as about 200 mg/kg (see Table VI). At higher doses of AICA 
riboside, hypoglycemic seizures and death were induced in rabbits and 
mice. We have found rats to also be sensitive to the hypoglycemic effects 
of AICA riboside. Initial studies were performed using non-fasted rats 
(Sprague Dawley) and mice (Swiss-Webster). Decreases in plasma glucose 
levels ranging from 30-40% were seen with a dose of 750 mg/Kg in rats at 1 
hour after administration. In non-fasted mice the decrease of glucose 
levels at this dose was greater, on the order of 30-50%. In both cases the 
hypoglycemia was statistically significant at the p&lt;0.01 level. Fasted 
rodents have been shown to be more sensitive to the hypoglycemic effects 
of AICA riboside than fed rodents (See FIG. 7). Fasting the animals for 
2-16 hours before drug administration significantly increased the 
hypoglycemic effect of AICA riboside. In fasted mice, AICA riboside is 
potent in lowering blood glucose. (See e.g., FIG. 6). In fasted mice AICA 
riboside may cause seizures at doses of 500 mg/Kg and above. In fasted 
mice (2 hours) a dose of 500 mg/Kg of AICA riboside reduced plasma glucose 
levels for 2-3 hours. In fasted mice (16 hours) a dose of 250 mg/Kg 
reduced glucose levels by 50% (p&lt;0.01) and 60% at doses of 500 and 750 
mg/Kg (p&lt;0.01). Fasting therefore appears to potentiate the hypoglycemic 
effect of AICA riboside. To date, of the species tested, humans have been 
found to be the species most sensitive to the hypoglycemic effects of AICA 
riboside; doses of 25 mg/kg given intravenously over 30 minutes have 
caused significant reductions in serum glucose. (See FIG. 8). In rats made 
hyperglycemic by treatment with 50 mg/Kg streptozocin I.V, the 
hypoglycemic effect of AICA riboside was found to be more pronounced than 
in normal animals. Doses of AICA riboside as low as 100 mg/Kg I.P. 
resulted in marked decreases in plasma glucose that approached euglycemic 
levels. Thus, it appears that the potency of AICA riboside as a 
hypoglycemic agent may be potentiated in a diabetic state. We have also 
shown that tolerance to the chronic administration of AICA riboside does 
not develop. Repeated administration of the drug for 6 days continues to 
yield a significant hypoglycemic response. 
This leads to investigation of the mechanism of AICA riboside induced 
hypoglycemia. It appears that adenosine is not involved in the mechanisms 
of AICA riboside induced hypoglycemia, since adenosine and adenosine 
receptor agonists such as cyclohexyladenosine (CHA) and N-ethylcarboxamide 
adenosine (NECA) do not result in hypoglycemia but in fact produce a 
profound hyperglycemia (200-300% increases in plasma glucose, p&lt;0.01) when 
administered I.P. in either rats or mice. This adenosine induced 
hyperglycemia is reversed by adenosine receptor antagonists (theophylline 
and sulphophenyltheophylline). Additionally, the AICA riboside-induced 
hypoglycemia is not blocked by theophylline. It is therefore concluded 
that the AICA riboside effect on plasma glucose is not mediated by 
adenosine. In rats, high doses of AICA riboside suppress blood insulin 
levels even at early time points after administration of the dose. 
However, human subjects exhibit significant elevations of serum insulin 
levels with administration of AICA riboside which precede the drop in 
blood glucose levels. (See FIG. 13). 
A dose of 750 mg/Kg in mice was shown to increase liver glycogen by 55% 
(p&lt;0.01) suggesting that the drug inhibits liver glycogenolysis or 
activates glycogen synthesis and, thereby, contributes to the lowered 
plasma glucose levels. Rats exhibit an elevation of liver glycogen and of 
serum lactate and pyruvate; however, the lactate to pyruvate ratio is not 
changed. The lactate elevation suggests an interruption in 
gluconeogenesis, while the lack of change in the lactate to pyruvate ratio 
suggests no effect on mitochondrial function. (See FIGS. 9 and 10A to 
10D). The glucose lowering effects of AICA riboside are related temporally 
to the generation and maintenance of liver ZMP levels in rats. (See FIGS. 
11A and 11B). In studies employing rabbit liver, ZMP, but not AICA 
riboside, has been found to inhibit fructose diphosphatase ("FDPase") with 
a K.sub.1 of about 40 .mu.M (see FIG. 12). 
Inhibition of pyruvate carboxylase, PEP carboxykinase or oxidative 
phosphorylation can interrupt mitochondrial function. However, AICA 
riboside does not interrupt mitochondrial function; the mild lactate 
build-up which occurs after administration of AICA riboside is not 
associated with a change in redox potential or a reduction in ATP pools. 
The mild lactate build up represents a minor interruption of the Cori 
cycle and should not, in itself, have detrimental effects. Doses of AICA 
riboside substantially higher than those causing elevation in lactate 
levels have been shown safe in toxicological studies. 
In vivo, AICA riboside may be phosphorylated by adenosine kinase to give 
AICA riboside-5'-monophosphate ("ZMP"). We believe that inhibition of 
fructose diphosphatase probably results from ZMP binding to the AMP 
inhibitory site of the enzyme. Thus, the enzyme falsely interprets a 
reduction in energy charge by the build-up in "false AMP", namely ZMP. ZMP 
can also cause an elevation of AMP levels (AMP is also an inhibitor of 
FDPase) via its inhibition of AMP deaminase. We have found that fructose 
diphosphatase may be inhibited using other ZMP analogs, including 
carbocyclic AICA riboside monophosphate. (See FIG. 16). Also useful are 
agents which cause or result in a build-up in ZMP (AICA ribotide). These 
agents include precursors in the de novo purine synthesis pathway or 
nucleosides, bases or prodrugs of such precursors. (See Lehninger, 
Biochemistry, p. 569 (1970)). Endogeneous ZMP levels may also be increased 
by agents which directly or indirectly inhibit AICA ribotide 
transformylase (thereby inhibiting folate metabolism), the enzyme which 
converts ZMP to 5'-formamido-imidazole-4-carboxamide ribonucleotide 
(precursor to inosinic acid). Accordingly, blood glucose levels may be 
decreased by administering agents which increase ZMP, either by increasing 
its synthesis or decreasing its conversion by AICA ribotide 
transformylase. Increased ZMP levels result in decreased FDPase activity 
and thus lower blood glucose levels. Thus, concomitant administration of 
one of these prodrugs of AICA riboside With an inhibitor of AICA ribotide 
transformylase may give enhanced hypoglycemic effects. Administration of 
any of the de novo purine synthesis intermediates (after the first 
committed step for purine synthesis, or their nucleosides or bases or 
their prodrugs, may similarly result in lowered blood glucose levels as 
mediated by ZMP. In addition, we have shown that 
5-amino-1-.beta.-D-ribofuranosyl-1,2,3-triazole-4-carboxamide, a purine 
nucleoside analog of AICA riboside lowers blood glucose in mice. AMP 
deaminase inhibitors can also be used to raise AMP concentration and 
inhibit FDPase, and thereby treat hyperglycemic conditions such as 
diabetes mellitis. 
In summary, AICA riboside lowers blood glucose levels by at least three 
mechanisms. In man, AICA riboside causes (1) an early elevation of serum 
insulin levels, probably due to increased pancreatic release of insulin. 
AICA riboside administration causes (2) increased glycogen storage related 
to increased synthesis and/or decreased breakdown of glycogen. The 
glycogen storage effects of AICA riboside are probably only partially 
contributory to its hypoglycemic effects, since surprisingly, in glycogen 
depleted states such as prolonged fasting, profound hypoglycemia is 
induced more readily, i.e. at lower AICA riboside doses. Finally, AICA 
riboside appears to lower blood glucose (3) by inhibiting gluconeogenesis 
at the level of fructose diphosphatase; an ideal mechanism of therapy for 
type II diabetic patients who exhibit accelerated hepatic gluconeogluesis. 
Control of gluconeogenesis by inhibition of fructose diphosphatase is a 
preferred site of action in the gluconeogenesis pathway because fructose 
diphosphatase is specific for the synthesis of glucose; several other 
enzymes are used in both the synthesis and degradation pathways of 
glucose. Additionally, inhibition of fructose diphosphatase does not 
interfere with mitochondrial function. The combined effects from AICA 
riboside treatment, namely, increased insulin release, direct inhibition 
of gluconeogenesis and decreased glycogen utilization provide a profound, 
yet safe, reduction in blood glucose levels. 
Due to the chronic nature of therapies for hypoglycemia and related 
diabetic conditions, and especially in the case of type II diabetic 
patients, therapeutic agents which may be administered orally are 
preferred. In another aspect of the present invention, we have found 
prodrugs of AICA riboside useful for increased delivery of the drug to the 
pancreas and liver after oral administration. Although AICA riboside 
itself is not well absorbed when given orally, administration of prodrugs 
of AICA riboside of the present invention, including acyl and carbonate 
esters, result in enhanced levels of serum AICA riboside and heart and 
liver ZMP. We have shown that oral administration of some of the AICA 
riboside prodrugs of the present invention produced hypoglycemia; whereas 
due to its low oral bioavailability, oral administration of AICA riboside, 
did not produce detectable hypoglycemia. (See FIG. 14) 
As noted, the AICA riboside prodrugs of the present invention are useful in 
therapies for diabetes and related conditions. In addition, AICA riboside 
and these prodrugs are useful as a supplement to total parenteral 
nutrition as an agent to control hyperglycemia and/or hyperlipidemia. 
As noted previously we have found that adenosine releasing agents such as 
AICA riboside prevent or reduce (ischemic) injury associated with 
atherosclerosis. Since ischemic injury to heart, brain, eyes, kidneys, 
skin and nerves constitute significant long term complications associated 
with both type I and type II diabetes, the anti-ischemic properties of 
AICA riboside, its prodrugs and related analogs, will thereby confer added 
therapeutic benefits to diabetic patients. In addition, we have found that 
AICA riboside lowers serum triglycerides in streptozotocin-induced 
diabetes in rats. (See FIG. 17). Elevated triglycerides are associated 
with accelerated atherosclerosis and their normalization in those with 
diabetic conditions by the present invention should provide additional 
therapeutic utility in the treatment of diabetes. 
S-AICA ribosyl homocysteine is formed from AICA riboside and homocysteine 
utilizing the enzyme S-adenosyl homocysteine hydrolase. This compound is a 
prodrug of AICA riboside. In addition, we have found AICA riboside is a 
weak inhibitor of adenosine kinase and that S-AICA riboside homocysteine 
is more potent. Inhibition of adenosine kinase by either AICA riboside or 
S-AICA ribosyl homocysteine can lead to increased adenosine release from 
cells undergoing net ATP catabolism. 
During our study of these prodrugs we observed a surprising decrease in AMP 
concentrations in conjunction with no change in ATP concentration which 
would be caused by either an inhibition of adenosine kinase or an 
activation of AMP-5'-nucleotidase, or a combination of those effects. AMP 
5'-nucleotidase is a highly regulated enzyme which has a regulatory 
protein which, when bound to the enzyme, inhibits its activity. AICA 
riboside or a metabolite thereof may activate AMP 5'-nucleotidase by 
preventing the binding of the regulatory protein to the enzyme. 
This work on AICA riboside and its prodrugs has demonstrated that adenosine 
kinase is a potential site of action for the adenosine releasing agents. 
We have expanded this work on AICA riboside and S-AICA riboside 
homocysteine to examine other known adenosine kinase inhibitors as 
adenosine releasing agents. The flux (substrate made into product per unit 
of time), through adenosine kinase under physiological conditions is low; 
substrate metabolism is low partly due to low substrate availability. 
However, during net ATP catabolism, substrate availability (i.e. adenosine 
concentration) dramatically increases. In cell culture and in the rat 
heart ischemia models, we have found that inhibition of adenosine kinase 
by 5-iodotubercidin or by 5'-amino-5'-deoxyadenosine resulted in increased 
adenosine concentrations during net ATP breakdown. When 
5'-amino-5'deoxyadenosine was administered to rats, it did not produce any 
significant changes in blood pressure or heart rate (i.e. no 
adenosine-mediated effects), yet in mice it was observed to prevent 
HTL-induced seizure, with the antiseizure effect being blocked by 
coadministration of the centrally acting adenosine antagonist 
theophylline. 5'-Amino-5'deoxyadenosine was also effective in blocking 
pentylene-tetrazole (PTZ) induced seizures. (See FIG. 15). 
Furthermore, AICA riboside has now been shown to be generated from ZMP 
during ischemia. The localized dephosphorylation of ZMP in a region of net 
ATP catabolism results in selectively high concentrations of AICA riboside 
and, therefore, ischemic selective effects of the molecule. For example, 
adenosine kinase and adenosine deaminase (ADA) would be only slightly 
inhibited by a dose of up to 500 mg/kg AICA riboside, but during tissue 
ischemia, the localized build-up of AICA riboside should cause increased 
inhibition of adenosine kinase and ADA. This ischemia-specific effect 
would avoid the deleterious effects of systemic ADA inhibition. 
As noted, these AICA riboside prodrugs are useful as antiviral agents. They 
may be used to treat viral infections such as that caused by HIV. These 
prodrugs may also be used in combination with other antiviral agents, and 
when used in combination, may result in enhanced antiviral activity 
allowing lower doses and, thus, potentially decreased side effects 
resulting from those other antiviral agents. 
Description of Preferred Embodiments 
We have identified a series of prodrugs of AICA riboside having 
advantageous therapeutic properties. The structures of some of these 
prodrug compounds are depicted in Table I. Additional compounds proposed 
to be useful as prodrug are depicted in Tables II and III. These prodrug 
compounds should improve penetration of the blood-brain barrier in 
comparison with AICA riboside itself because of their longer plasma 
half-life. 
A prodrug of AICA riboside with the assigned structure: 
3'-isobutoxycarbonyl-AICA riboside which appears as Compound 1 of Table 1 
("5-amino-3'-(2-methyl-1-propoxycarbonyl)-1-.beta.-D-ribofuranosyl-imidazo 
le-4-carboxamide" or "Prodrug A") has been found to be particularly good 
for prolonging the half-life of AICA riboside and in penetrating the gut 
barrier. It has improved anticonvulsant activity against HTL-induced 
seizures when compared to AICA riboside. (FIG. 1). 
The ability of this compound to enhance adenosine production in an ischemic 
heart model has also been demonstrated (FIG. 2). Prodrug A was about 30% 
more potent on a molar basis than AICA riboside. The detection of 
substantial phosphorylated derivative of AICA riboside (ZMP) following 
administration of this compound further demonstrated that Prodrug A was in 
fact being cleaved to AICA riboside because that cleavage is necessary for 
the intracellular phosphorylation of AICA riboside to ZMP to occur. 
Surprisingly, Prodrug A also led to less AICA riboside and, therefore, 
less ZMP accumulation in the heart than an equimolar dose of AICA riboside 
and yet had more adenosine production indicating it may have intrinsic 
(analog) activity. 
To further evaluate the intrinsic activity of Prodrug A, we have 
synthesized and tested a series of 3'-hydrocarbyloxycarbonyl derivatives 
of AICA riboside. As seen in FIG. 19, with an increasing number of carbon 
atoms in the side chain of the 3'-carbonate ester group, there is 
increased potency until four carbons are reached. Compounds having side 
chains in the carbonate ester of more than 4 carbon atoms (i.e. 5 and 
above) exhibited decreased activity in this experiment. We believe that 
these studies demonstrate the important and specific nature of this 
portion of the molecule for adenosine releasing activity. 
In summary, 3'-isobutoxycarbonyl-AICA riboside has demonstrated improved 
enhancement of adenosine production as compared with the AICA riboside 
itself. It has an increased half-life as evidenced by the fact that it is 
cleared and phosphorylated more slowly than AICA riboside. Also, the 
maximum therapeutic effect of the compound appears to be greater than AICA 
riboside on a molar basis. This compound, furthermore, exhibits 
anti-seizure activity in the homocysteine-induced seizure model and 
increases adenosine production in the myocardial ischemia model. This 
compound also crosses the gut better than AICA riboside, as there is 5 
times more ZMP accumulation in the liver after an equimolar gavage. 
To assist in understanding the present invention, the following examples 
follow, which include the results of a series of experiments. The 
following examples relating to this invention are illustrative and should 
not, of course, be construed as specifically limiting the invention. 
Moreover, such variations of the invention, now known or later developed, 
which would be within the preview of one skilled in the art are to be 
considered to fall within the scope of the present invention hereinafter 
claimed. 
EXAMPLE 1 
Preparation of Carbonate Esters of AICA Riboside 
Carbonate esters of AICA riboside are prepared according to the following 
procedure: 
A 70 mmol portion of AICA riboside is suspended in a mixture of 50/ml N, 
N-dimethylformamide and 50/ml pyridine and then cooled in an ice-salt 
bath. To the resulting mixture the appropriate chloroformate (94 mmol, a 
20 percent excess) is added under anhydrous conditions over a period of 
about 15 to 30 minutes with constant stirring. The ice salt bath is 
removed. The reaction mixture is allowed to warm to room temperature over 
about 1 to 2 hours. The progress of the reaction is monitored by TLC on 
silica gel, eluting with 6:1 methylene chloride:methanol, Disappearance of 
AICA riboside indicates completion of the reaction. The solvents are 
removed by evaporation under high vacuum (bath temperature less than 
40.degree. C.). The residue is chromatographed on a silica gel column 
packed with methylene chloride and is eluted initially with methylene 
chloride and then with methylene chloride: methanol 95:5. Fractions 
showing identical (TLC) patterns are pooled and then the eluate is 
evaporated to give a foam. The foam is dried overnight under high vacuum 
at room temperature. 
The yield of the product carbonate esters is about 45 to 65%. Although the 
primary product is the 3'-carbonate ester, other product esters are 
formed. 
EXAMPLE 2 
Preparation of 3'-Isobutoxycarbonyl AICA Riboside 
A solution of AICA riboside (18.06 g, 70 mmol) in a mixture of pyridine 
(50/ml) and N,N-dimethylformamide (50/ml) was cooled in an ice-salt 
mixture. To it was added an isobutyl chloroformate (11.47 g, 94 mmol) 
slowly over a period of 30 minutes with constant stirring. The initial red 
color of the reaction turned pale yellow in about 40 minutes. Stirring was 
continued for 2 hours at the end of which TLC on silica gel, eluting with 
methylene chloride: methanol 9:1 (Rf=0.3), indicated completion of the 
reaction. Methanol (2 ml) was added to neutralize unreacted reagents. The 
solvents from the reaction mixture were removed by evaporation under high 
vacuum (bath temperature approximately 40.degree. C.). The sticky mass 
remaining was chromatographed over a silica gel column packed in a 9:1 
methylene chloride: methanol mixture. The column was eluted with the same 
mixture and several fractions were collected. Fractions showing identical 
TLC spots were pooled and evaporated to obtain an off-white foam. The 
product isolated from the foam had the assigned structure, based on the 
nmr spectrum: 3'-isobutyloxycarbonyl-AICA riboside. Yield 8.5/g; mp 
71.degree.-73.degree. (not a sharp mp). 
IR (nujol): 1725 cm.sup.-1 (--OCO.sub.2 CH.sub.2 CH(CH.sub.3).sub.2). 
NMR(DMSO-d.sub.6), .delta. ppm; 0.9 [d, 6H (CH.sub.3).sub.2 ], 1.9 (m, 1H, 
CH of isobutyl side chain), 3.6 (m, 2H, 5'-CH.sub.2), 3.9 (d, 2H, CH.sub.2 
of isobutyl side chain), 4.1(m, 1H, 4'-CH), 4.6(1, 1H, 2'-CH), 5.01(dd, 
1H, 3'-CH), 5.45-5.55(m, 2H, 1'-CH and 5'-OH), 5.92(d, 1H, 2-OH), 
6.02(br.s, 2H, 5-NH.sub.2), 6.6-6.9(br. d, 2H, 4-CONH.sub.2), 7.35 (S, 1H, 
2-CH). 
The spectra of this compound was compared with that of its parent compound, 
AICA riboside and showed that 3-CH (which appears at 4.05 ppm in AICA 
riboside), had shifted down field by 1 ppm due to a substitution on the 
oxygen attached to the same carbon atom, while the positions of all the 
other protons remained unchanged for the most part, thus confirming the 
substitution to be on 3'-C. 
Although nmr of the product of Example 2 indicated that it was at least 80% 
of the 3'-isobutoxycarbonate ester (Compound 1 of Table I), HPLC analysis 
showed several peaks. The fractions corresponding to each peak were 
collected and analyzed on HPLC. Each peak also showed the presence of two 
major products, designated A and B. One of them (product A) was determined 
to be AICA riboside and the other (product B) was isolated in small 
quantities and characterized as AICA riboside-2', 3'-cyclic carbonate 
based on its nmr and mass spectral data. NMR(DMSO-d.sub.6) .delta. ppm; 
3.6-3.7(m, 2H, 5'-CH.sub.2), 4.3 (g, 1H, 4'-CH), 5.35 (m, 1H, 3'-CH), 5.6 
(m, 1H, 2'-CH), 5.2-6.7 (br, 1H, 5'-OH), 5.8-6.0 (br, 2H, 5-NH.sub.2), 6.1 
(d, 1H, 1'-CH), 6.7-6.95(br, d, 2H, 4-CONH.sub.2), 7.45 (S, 1H, 2-CH). 
Mass spec, (FAB) M.sup.+, 284; M.sup.+1 285, M.sup.+2 286. These data 
confirmed the structure of the compound (product B) to be 2'3'-cyclic 
carbonate of AICA riboside. A preferred method of synthesis of this 
compound is set forth in Example 3 below. 
EXAMPLE 3 
Preparation of AICA Riboside 2', 3'-Cyclic Carbonate 
To a suspension of AICA-riboside (5.16/g, 20 mmol) in pyridine (50/ml), 
p-nitrophenyl chloroformate 92.5/g, 25 mmol) was added in one lot and 
stirred at room temperature for 5 days at the end of which TLC on silica 
gel, eluting with methylene chloride: methanol, (6:1 Rf=0.4), indicated 
completion of the reaction. Pyridine from the reaction mixture was removed 
by evaporation. The residue was chromatographed over a silica gel column, 
eluting with methylene chloride:methanol (9:1). The fractions which showed 
identical TLC were pooled and evaporated to obtain a foam (yield, 4.0 g). 
This product was identical to AICA riboside-2',3'-cyclic carbonate, 
isolated as one of the by-products from the synthesis described in Example 
2 and characterized by nmr and mass spectral analysis. 
EXAMPLE 3A 
Preparation of AICA Riboside 2',3'-Cyclic Carbonate 
In a 100 ml round bottom flask fitted with a vacuum adapter 5.0 g of 
3'-isobutoxy carbonyl AICA-riboside was taken and immersed in a preheated 
oil bath (bath temperature 100.degree.-110.degree. C.) while vacuum was 
applied gradually. After about 45 minutes of heating under vacuum, the 
product was cooled and crystallized from hot methanol. The colorless 
crystalline product was collected by filtration and dried under vacuum. 
The product was found to be identical to AICA-riboside-2',3'-cyclic 
carbonate, isolated as one of the byproducts from an earlier procedure 
(Example 2) as characterized by TLC and other spectroscopic data 
comparison. Yield was 3.1 g of a solid melting point 66.degree.-68.degree. 
C. 
EXAMPLE 4 
Preparation of 5'-Acetyl AICA Riboside 
(A) Preparation of 2',3'-Isopropylidene AICA Riboside: 
To a mixture of dry HCl gas (9.0 g) dissolved in dry acetone (115 ml) and 
absolute ethanol (138 ml), AICA-riboside (12.9 g), was added. The mixture 
was stirred at room temperature for two hours. Completion of the reaction 
was monitored by TLC. The reaction mixture was stirred an additional two 
hours at room temperature at which time TLC indicated that the reaction 
was complete. The reaction mixture was poured slowly into an ice-cold 
mixture of ammonium hydroxide (18 ml) and water (168 ml). The pH of the 
solution was adjusted to about 8 by adding a few ml of ammonium hydroxide. 
The reaction was concentrated to 100 ml. The ammonium chloride precipitate 
was removed by filtration. The filtrate was concentrated again to 
precipitate additional ammonium chloride. After filtering, the filtrate 
was evaporated to dryness. The residue was extracted three times with 200 
ml aliquots of methylene chloride. Evaporation of methylene chloride gave 
a foam which was characterized by nmr spectroscopy to be the product 
2',3'-isopropylidene AICA riboside which was used in the following 
reaction without further purification. 
(B) To a solution of 2',3'-isopropylidene AICA riboside in 25 ml dry 
pyridine cooled in an ice-salt mixture, 10 ml acetic anhydride was added 
dropwise with stirring; the mixture was warmed to room temperature over a 
period of two hours. The reaction was shown to be complete by TLC (9:1 
methylene chloride: methanol). The solvents were removed from the reaction 
mixture by evaporation. The residue was coevaporated twice with two 25 ml 
aliquots of N, N-dimethylformamide. That product was treated with 100 ml 
of 80% acetic acid for twenty-four hours. Completion of the reaction was 
indicated by TLC on silica gel eluting with 6:1 methylene 
chloride:methanol. Water and acetic acid were removed by evaporation under 
reduced pressure. The residue was coevaporated four times with 100 ml 
aliquots of water to remove the acetic acid. The residue was crystallized 
from 25 ml 1:1 ethanol:water. The crystalline product was collected by 
filtration, washed with water and dried under vacuum to give 3.0 g of the 
above-identified product, melting point 165.degree.-166.degree. C. 
IR(nujol); 1745 cm.sup.-1 (--OCOCH.sub.3). NMR (DMSO-d.sub.6), .delta. ppm: 
2.0 (S,3H, COCH.sub.3), 4.0-4.1 (m, 2H, 5'-CH.sub.2), 4.1-4.4 (m, 3H, 
2'-CH, 3'-CH, 4'-CH), 5.3 (d, 1H, 1'-CH), 5.4-5.6 (m, 2H, 3'-OH, 4'-OH), 
5.7-5.9 (br, 2H, 5-NH.sub.2), 6.6-7.0 (br. d, 2H, CONH.sub.2), 7.3 (S, 1H, 
2-CH). 
EXAMPLE 4A 
Preparation of 2',3'-Isopropylidene AICA Riboside 
To a solution of 230 ml acetone, 275 ml ethanol and 58 ml of 9.5M HCl in 
ethanol, 26 g AICA riboside were added. The resulting mixture was stirred 
for 35 minutes. The reaction mixture was added to a solution of 500 ml ice 
and 75 ml ammonium hydroxide (14N). The solution was concentrated to 100 
ml; then 300 ml n-butanol and 100 ml water were added. The organic phase 
was separated and washed with 50 ml water. The combined aqueous phases 
were extracted with 100 ml n-butanol. The combined organic (n-butanol) 
phases were concentrated to give a white foam. The foam was dissolved in 
75 ml ethanol and left in the freezer. The crystalline product was 
collected by filtration, washed with ethanol and dried under vacuum to 
give 19.9 g of the above-identified product, melting point 
184.degree.-185.degree. C. (literature: 185.degree.-186.degree. C.). 
Reference: Srivastava et al., J. Med. Chem. 18:1237 (1975). 
EXAMPLE 5 
Preparation of 5'-Alkoxycarbonyl-AICA Riboside Derivatives 
Four different 5'-alkoxycarbonyl-AICA riboside derivatives were made 
according to the following general procedure using the appropriate 
starting materials: 
To an ice-cold solution of 10 mmol 2',3'-isopropylidene-AICA riboside in 40 
ml pyridine, a solution of 15 mmol of the appropriate alkylchloroformate 
in 10 ml methylene chloride was added over a period of about 15 minutes. 
The cooling bath was removed and the reaction mixture was stirred for 
about four hours. At the end of that time period, thin layer 
chromatography (TLC) with silica gel, eluting with methylene 
chloride:methanol 9:1, indicated that the reaction was complete. The 
solvent was removed by evaporation under high vacuum. The residue was 
coevaporated with DMF (2.times.20 ml). The product was dissolved in about 
100 ml methylene chloride and extracted with water (2.times.100 ml). The 
organic layer was dried over sodium sulfate and evaporated to obtain a 
syrup-like product which was carried into the following step for 
deblocking of isopropylidene group. 
The above product was dissolved in 60 ml of 50% formic acid and then heated 
at 65.degree. C. for two hours. At the end of that time, TLC on silica gel 
eluting with methylene chloride: methanol 6:1, indicated that the reaction 
was complete. Water and formic acid were removed by evaporation under high 
vacuum. The residue was co-evaporated with water (2.times.25 ml) and 
ethanol (2.times.25 ml). The product was chromatographed over a silica gel 
column, eluting with methylene chloride: methanol, 9:1. Effluents 
containing fast-moving products were rejected. Effluents containing the 
major product were pooled and evaporated to obtain a glassy product which 
was dried under high vacuum. The yields of each of the products made 
according to this procedure and their physical data are summarized below. 
A. 5'-Ethoxycarbonyl AICA-Riboside 
Yield about 35%. 
IR (KBr) cm.sup.-1 : 3000-4000 (broad peaks, OH, NH.sub.2, CONH.sub.2), 
1730 (0-C00-Et), 1660 (CONH.sub.2) 
.sup.1 H-NMR(DMSO-d.sub.6) .delta.ppm: 1.2(t, 3H, CH.sub.3 of ethyl), 
3.9-4.1 (m, 2H, 5'-CH.sub.2), 4.1-4.25 (q, 2H, --OCH.sub.2 of ethyl side 
chain), 4.25-4.4 (m, 3H, 2'-CH, 3'-CH, and 4'-CH), 5.45 (d, 1H, 1'-CH), 
5.45-5.6 (2d, 2H, 2'-OH and 3'-OH), 5.8-9.0 (br.5, 2H, 5-NH.sub.2), 
6.6-6.9 (br.d, 2H, CONH.sub.2), and 7.25 (S, 1H, 2-CH). 
5'-Isobutoxycarbonyl-AICA Riboside 
Yield 40% 
IR(KBr) cm.sup.-1 : 3000-4000 cm.sup.-1 (broad peaks, OH, NH.sub.2, 
CONH.sub.2, etc. ) 
.sup.1 H-NMR (DMSO-d.sub.6), .delta.ppm, 0.8-0.9 (d, 6H, 2CH.sub.3 of 
isobutyl side chain), 1.8-2.0 (m, 1H, CH of isobutyl side chain), 3.8-3.9 
(d, 2H, CH.sub.2 of isobutyl side chain), 4.0-4.1 (m, 2H, 5'-CH.sub.2), 
4.1-4.4 (m, 3H, 2'-CH, 3'-CH, and 4'-CH), 5.4 (d, 1H, 1'-CH), 5.5-5.1 (2d, 
2H, 2'-OH and 3'-OH), 5.8-5.9 (br.s, 2H, 5-NH.sub.2), 6.6-6.9 (br.d, 2H, 
COHN.sub.2) and 7.25 (5, 1H, 2-CH). 
C. 5-Neopentoxycarbonyl-AICA-Riboside: 
Yield: 35% 
.sup.1 H-NMR (DMSO-d.sub.6), .delta.ppm: 0.8 (S, 9H, 3CH.sub.3), 3.8 (5, 
2H, -CH.sub.2 O--of neopentoxy side chain), 4.0-4.1 (m, 2H, 3'-CH and 
4'-CH), 4.2-4.45 (m, 3H, 2'-CH and 5'-CH.sub.2), 5.5 (d, 1H, 1'-CH), 
5.3-5.7 (m, 2H, 2'-OH and 3'-OH), 5.8-5.9 (br.s, 2H, 5-NH.sub.2), 6.6-7.0 
(br.d, 2H, CONH.sub.2), and 7.3 (5, 1H, 2-CH). 
D. 5'-Cyclopentyloxycarbonyl-AICA-Riboside 
Yield: 38% 
.sup.1 H-NMR (DMSO-d.sub.6), .delta.ppm: 1.3-2(br, m, 8H, 4 CH.sub.2 groups 
of the cyclopentane ring), 3.9-4.2 (m, 2H, 3'-CH and 4'-CH), 4.2-4.4 (m, 
3H, 2'-CH and 5'-CH), 4.9-5.1 (m, 1H, CH--O of cyclopentane ring), 5.4 (d, 
1H, 1'-CH), 5.45-5.65 (2d, 2H, 2'-OH and 3'-OH), 5.9 (br.s, 2H, 
5-NH.sub.2), 6.6-6.9 (br.d, 2H, CONH.sub.2), 7.3 (5H, 2'-CH). 
EXAMPLE 6 
Preparation of 2'3'5'-Tri-O-N-Butyryl-AICA-Riboside 
This compound was prepared according to the method described by for the 
preparation of 2', 3', 5'-tri-o-acetyl-AICA-riboside. (Reference: Suzuki 
and Kumashiro, U.S. Pat. No. 3,450,693; Chem. Abstr. 71:81698Z (1969)). 
The yield was 70% and the product had a melting point of 
97.degree.-100.degree. C. 
IR (KBr) cm.sup.-1 : 3200-3400 (NH.sub.2, CONH.sub.2), 1720-1745 
(OCOCH.sub.2 CH.sub.2 CH.sub.3), 1650 (CONH.sub.2). 
.sup.1 H-NMR (DMSO-d.sub.6), .delta.ppm: 0.7-1.0 (m, 9H, CH.sub.3), 1.3-1.7 
(m, 6H, --CH.sub.2 --), 2.1-2.6 (m, 6H, --COCH.sub.2 --), 4.2 (br.s, 3H, 
5'-CH.sub.2 and 4'-CH), 5.3-5.4 (m, 1H, 3'-CH) 5.6 (t, 1H, 2'-CH), 5.9 (d, 
1H, 1'-CH) 6.0 (bros, 2H, NH.sub.2), 6.8 (br.d, 2H, CONH.sub.2) 7.4 (S, 
1H, 2-CH) 
EXAMPLE 7 
Preparation of 2',3',5'-Tri-O-Succinyl-AICA-Riboside 
A mixture of 5.2 g AICA riboside and 12.0 g succinic anhydride dissolved in 
a mixture of 30 ml DMF and 30 ml pyridine was stirred at 40.degree. C. for 
18 hours and then evaporated to dryness under high vacuum. The residue was 
dissolved in water and applied to a 150 ml column of amberlite IRC-120 
(H+). The column was washed with water and then eluted with 1N ammonium 
hydroxide. The eluate was evaporated to dryness under vacuum. The residue 
was dissolved in water and applied to a 30 ml column of Dowex-2 (formate). 
The column was washed with water and eluted with formic acid. The formic 
acid eluate was concentrated and then lyophilized to yield 0.55 g of the 
product (approximately 5% yield). 
.sup.1 H NMR (D.sub.2 O), .delta.ppm: 2.4-2.8 (m, 12H, --CH.sub.2 --), 4.4 
(m, 2H, 5'-CH.sub.2), 4.6 (m, 1H, 4'-CH), 5.2 (m, 1H, 3'-CH), 5.4 (m, 1H, 
2'-CH), 5.8 (d, 1H, 1'-CH), 7.9 (S, 1H, 2-CH). 
EXAMPLE 8 
Preparation of 3',5'-Diacetyl-AICA-Riboside 
To a solution of 10.0 g 2',3',5'-tri-o-acetyl-AICA-riboside in 150 ml 
pyridine, 3.0 g hydroxylamine hydrochloride was added. The reaction 
mixture was stirred at room temperature; progress of the reaction was 
monitored using TLC (silica gel, methylene chloride: methanol 9:1) After 
three days of stirring, an additional 2.0 g of hydroxylamine hydrochloride 
was added. The reaction mixture was stirred for an additional three days. 
At the end of that time, 80% of the starting material had disappeared, 
according to TLC. Acetone (10 ml) was added to the reaction mixture to 
neutralize unreacted hydroxylamine hydrochloride; the resulting mixture 
was stirred for an additional five hours. Solvent was removed by 
evaporation under reduced pressure. The residue was coevaporated with 
N,N-dimethylformamide (DMF) (2.times.100 ml). The product obtained was 
chromatographed over a silica gel column which was packed in, and eluted 
with, a 9:1 mixture of methylene chloride: methanol. Fractions showing 
homogeneous spots at R.sub.f =about 0.5 were pooled and then evaporated. 
The residue was crystallized from ethyl acetate to give a light pink 
crystalline product whose H.sup.1 -NMR indicated that it was a mixture 
2',5'-diacetyl-AICA-riboside and 3',5'-diacetyl-AICA-riboside in a ratio 
of about 1:5. A second crystallization from ethyl acetate gave a product 
which was about 96% isomerically pure 3',5'-diacetyl-AICA-riboside, having 
a melting point of 132.degree.-134.degree. C. About 3.0 g of product was 
obtained (about 35% yield). 
IR (KBr) cm.sup.-1 : 3200-3460 (broad peaks, OH, NH.sub.2, CONH.sub.2, 
etc.); 1760, 1740 (3'-OCOCH.sub.3 and 5'-OCOCH.sub.3), 1660 (CONH.sub.2). 
.sup.1 H-NMR (DMSO-d.sub.6), .delta.ppm: 2.0-2.1 (2s, 6H, 3'-OCOCH.sub.3 
and 5'-OCOCH.sub.3), 4.25 (br.s, 2H, 5'-CH.sub.2), 4.6 (q, 1H, 2'-CH), 5.1 
(m, 1H, 4'-CH), 5.5 (d, 1H, 2'-OH), 5.9 (m, 3H, 5-NH.sub.2 and 1'-CH), 6.8 
(br.d, 2H, --CONH.sub.2), 7.35 (s, 1H, 2-CH). 
EXAMPLE 9 
Preparation of AICA-Riboside-5'-N,N-Diethylsuccinamate 
To a solution of 2.98 g 2',3'-isopropylidene AICA riboside, 1.73 g 
N,N-diethylsuccinamic acid and 1.2 g 4-dimethylaminopyridine in 25 ml of 
DMF which was cooled in a dry ice-methanol bath, 2.26 g 
N,N-dicyclohexylcarbodiimide was added in one lot. The reaction mixture 
was stirred and allowed to warm to room temperature. The reaction was 
complete after about 18 hours, as evidenced by the disappearance of most 
of the starting material as determined by TLC. The by product 
cyclohexylurea that separated as a white solid was removed by filtration 
and then washed with DMF (2.times.10 ml). The filtrate and DMF washings 
were combined and then concentrated under high vacuum. The residue was 
chromatographed over a silica gel column, eluting with methylene chloride: 
methanol, 19:1. The fractions showing the major spot (TLC R.sub.f 
.about.0.6) were pooled and evaporated to obtain a syrupy product which 
was taken to the following step to remove the isopropylidene blocking 
group. 
The syrupy product was dissolved in 25 ml of a 60% formic acid solution. 
The resulting mixture was stirred at room temperature for 48 hours. At the 
end of that time TLC indicated hat the reaction was complete. The reaction 
mixture was concentrated under high vacuum to give a thick syrup. The 
syrupy residue was coevaporated with water (2.times.20 ml) and ethanol 
(2.times.25 ml). The product was crystallized from 25 ml ethanol:water 
(9:1) to give 900 mg of the above-identified compound, melting point 
180.degree.-181.degree. C. 
IR(KBr)cm.sup.-1 : 3000-4000 (NH.sub.2, OH, etc.), 1725, 
##STR17## 
1610-1650 (, CONH.sub.2 and CON(CH.sub.2 CH.sub.3).sub.2). 
.sup.1 H-NMR (DMSO-d.sub.6), .delta.ppm: 0.9-1.15 (2t, 6H, 2CH.sub.3 of the 
two ethyl groups), 2.5 (m, 4H, --CO--CH.sub.2 CH.sub.2 --CO--), 3.1-3.4 
(m, 4H, --H.sub.2C C--N--CH.sub.2 --), 4.0 (m, 2H, 5'-CH.sub.2), 4.15-4.35 
(m, 3H, 2'-CH, 3'-CH and 4'-CH), 5.35 (d, 1H, 1'-CH), 5.5 (2d, 2H, 2'-OH 
and 3'-OH) 5.8 (br, 2H, 5-NH.sub.2), 6.6-6.9 (br.d, 2H, CONH.sub.2), and 
7.3 (s, 1H, 2-CH). 
EXAMPLE 10 
Preparation of 3'-Neopentoxy Carbonyl-AICA Riboside 
The above-identified compound was prepared according to the procedure 
described in Example 2 for the preparation of 3'-isobutoxycarbonyl-AICA 
riboside, substituting neopentyl chloroformate for isobutylchloroformate. 
In this preparation, the product was crystallized from hot water to give 
8.1 g (yield about 30%) of the above-identified compound as a crystalline 
solid, melting point 119.degree.-121.degree.C. 
.sup.1 H-NMR (DMSO-d.sub.6), .delta.ppm: 0.8-1 (m, 9H, 3CH.sub.3 groups of 
the neopentyl side chain), 3.6 (m, 2H, 5'-CH.sub.2), 3.8 (s, 2H, 
--CH.sub.2 O-- of the neopentyl side chain), 4.1 (m, 1H, 2'-CH), 3.6 (q, 
1H, 4'-CH), 5.05 (d, 1H, 3'-CH), 5.5 (m, 2H, 2'-OH and 5'-OH), 5.9 (d, 1H, 
1'-CH), 6.05 (br.s, 2H, 5-NH.sub.2), 6.6-6.9 (br.d, 2H, CONH.sub.2), and 
7.3 (S, 1H, 2-CH). 
EXAMPLE 11 
Preparation of 2',5'-Di-O-Acetyl-3'-Neopentoxycarbonyl-AICA Riboside 
To an ice-cold solution of 1.85 g 3'-neopentoxy-carbonyl-AICA-riboside in 
25 ml pyridine, 0.25 ml acetic anhydride was added slowly. The resulting 
mixture was stirred at room temperature for about three hours. TLC (on 
silica gel, using methylene chloride:methanol 9:1) of the reaction mixture 
indicated that the reaction was complete. A 0.5 ml aliquot of methanol was 
added to the reaction mixture which was then evaporated under high vacuum 
to give a syrupy residue. The residue was coevaporated with DMF 
(2.times.10 ml). The resulting product was dissolved in 100 ml methylene 
chloride and extracted twice with 25 ml of 5% aqueous sodium bicarbonate 
solution. The organic layer was dried over anhydrous sodium sulfate and 
then evaporated. The residue was crystallized from hot ethyl acetate to 
give 1.7 g. of the above-identified compound, melting point 
160.degree.-161.degree.C. 
IR(KBr)cm.sup.1 : 3000-4000 (broad peaks, NH.sub.2, CONH.sub.2), 1740-1775 
(--OCOCH.sub.3 and O-COO-neopentyl). 
.sup.1 H-NMR (DMSO-d.sub.6), .delta.ppm: 0.9 (5, 9H, t-butyl), 2.05 (2s, 
6H, 2'-COCH.sub.3 and 3'-COCH.sub.3), 3.8-3.95 (q, 2H, 5'-CH.sub.2), 
4.2-4.4 (m, 3H, 4'-CH and --CH.sub.2 -- of neopentyl side chain), 5.25 (m, 
1H, 3'-CH), 5.65 (t, 1H, 2'-CH), 5.9 (d, 1H, 1'-CH), 6.0 (br.s, 2H, 
5-NH.sub.2), 6.7-6.9 (br.d, 2H, 4-CONH.sub.2) and 7.4 (s, 1H, 2-CH). 
EXAMPLE 12 
Preparation of 5'-O-Acetyl-3'-Isobutoxycarbonyl-AICA-Riboside and 
5'-O-Acetyl-2'-Isobutoxycarbonyl-AICA Riboside 
To an ice-cold solution of 4.0 g 5'-O-Acetyl-AICA Riboside (the product of 
Example 4) in 10 ml pyridine and 10 ml DMF, a solution of 2.6 g 
isobutylchloroformate in 10 ml methylene chloride was added over a period 
of about 30 minutes. The reaction mixture was allowed to warm to room 
temperature over about three hours at the end of which TLC (silica gel 
with methylene chloride:methanol 9:1) indicated that the reaction was 
complete. A 1 ml aliquot of methanol was added to the reaction mixture and 
the solvents were removed under high vacuum. The residue was coevaporated 
with DMF (2.times.10 ml) and chromatographed on a silica gel column, 
eluting with methylene chloride:methanol (9:1). The fractions having 
identical TLC patterns were pooled and evaporated (under high vacuum) to 
give a colorless glassy product. The glassy product was dried under high 
vacuum to give 2.3 g product. The .sup.1 H-NMR of this product indicated 
that it was a mixture of 5'-O-acetyl-3'-isobutoxycarbonyl-AICA riboside 
and 5'-O-acetyl-2'-isobutoxycarbonyl-AICA riboside in a ratio of about 2:1 
based on a comparison of the areas under the peaks for the aromatic 
proton. 
IR (KBr) cm.sup.-1 : 3020, 3240-3500 (OH, NH.sub.2, CONH.sub.2 etc.) 
.sup.1 H-NMR (DMSO-d.sub.6), .delta.ppm: 0.8-1.0 (2d, 6H, 2CH.sub.3 groups 
of isobutyl side chain), 1.8-2.0 (m, 1H, CH of the isobutyl side chain), 
2.05 (2s, 3H, COCH.sub.3), 3.85-3.95 (2d, 2H, CH.sub.2 --O-- of the 
isobutyl side chain), 7.3 (s, 1H, 2-CH of the 
5'-O-acetyl-3'-isobutoxycarbonyl-AICA riboside molecule) and 7.4 (s, 1H, 
2-CH of the 5'-O-acetyl-2'-isobutoxycarbonyl-AICA riboside molecule). The 
ratio of those last listed peaks represented the relative percent of 
5'-O-acetyl-3'-isobutoxycarbonyl-AICA riboside and 
5'-O-acetyl-2'-isobutoxycarbonyl-AICA riboside in the composition as a 
whole as about 66% and 33%, respectively. 
EXAMPLE 13 
Preparation of 5-N,N-Dimethylaminomethylene-AICA Riboside 
A mixture of 10.0 g 2',3',5'-tri-O-acetyl-AICA riboside, 50 ml DMF and 15 
ml N,N-dimethylformamide dimethyl acetyl was stirred at room temperature 
for about 18 hours. The solvent and unreacted reagent were removed by 
evaporation under reduced pressure. The residue was dried under high 
vacuum for 12 hours at 40.degree. C. to give a syrupy residue. The residue 
was dissolved in 30 ml dry cyclohexylamine. The resulting mixture was 
stirred overnight. The solvent was removed by evaporation under reduced 
pressure to give a gum. The gum was crystallized from ethanol to give 4.2 
g of the above-identified compound as white crystals, melting point 
173.degree.-175.degree. C. 
.sup.1 H-NMR (MeOH-d.sub.4), .delta.ppm: 3.0-3.05 (2s, 6H, 
N(CH.sub.3).sub.2), 3.75 (m, 2H, 5'-CH.sub.2), 4.0 (q, 1H, 4'-CH), 4.2 (t, 
1H, 3'-CH), 4.35 (t, 1H, 2'-CH), 5.8 (d, 1H, 1'-CH), 7.7 (s, 1H, 2-CH), 
and 8.25 (s, 1H, 5-N.dbd.CH--N ). 
EXAMPLE 14 
Preparation of AICA Riboside-5'-N-Butylcarbamate 
To a solution of 2.6 g AICA riboside dissolved in 20 ml DMF, 5.0 g 
n-butylisocyanate was added in portions over 72 hours. The reaction 
mixture was evaporated to dryness under vacuum. The residue was applied to 
a 350 ml silica gel column, prepared with methylene chloride: methanol 
(10:1) and eluted with methylene chloride methanol (9:1). One hundred 
milliliter fractions were collected. Fractions 26 to 30 (which contained 
the desired product) were pooled and evaporated to dryness to give 0.6 g 
of the above-identified product (yield about 16%). 
.sup.1 H-NMR (DMSO-d.sub.6), .delta.ppm: 0.9 (t, 3H, CH.sub.3 --), 1.1-1.5 
(m, H, --CH.sub.2 --CH.sub.2 --), 3.0 (q, 2H, CH.sub.2 --NH), 4.0 (br.s, 
2H, 5'-CH.sub.2), 4.1-4.4 (m, 3H, 2'-CH, 3'-CH and 4'-CH), 5.3-5.5 (m, 2H, 
2'-OH and 3'-OH), 5.8 (br.s, 1H, 1'-CH), 6.0 (br.s, 2H, NH.sub.2), 6.8 
(br.d, 2H, CONH.sub.2), 7.3 (s, 1H, 2-CH). 
EXAMPLE 15 
Preparation of AICA Riboside-5'-T-Butylcarbamate 
The above-identified compound was prepared according to the procedure 
described in Example 14 substituting t-butylisocyanate for 
n-butylisocyanate in the reaction mixture. The above-identified compound 
was isolated by chromatography using a silica gel column to give a yield 
of approximately 8%. 
.sup.1 H-NMR (DMSO-d.sub.6), .delta.ppm: 1.1 (S, 9H (CH.sub.3).sub.3 
--C--), 4.0 (m, 3H, 4'-CH and 5'-CH.sub.2), 4.1 (m, 1H, 2'-CH), 4.3 (m, 
1H, 3'-CH), 5.2-5.5 (br.m, 3H, 2'-OH, 3'-OH, and 1'-CH), 5.8 (br.s, 2H, 
NH.sub.2), 5.9 (br.s, 1H, CONH.sub.2), 6.3 (br.d, 2H, CONH.sub.2), 7.3 (5, 
1H, 2-CH). 
EXAMPLE 16 
Preparation of 5-AMINO 
-2,3'5'-Tri-O-Acetyl-1-.beta.-D-Ribofuranosylimidazole-4-Carboxamide 
("AICA Riboside-Triacetate) 
To a well-stirred, ice-cooled suspension of 50.0 g AICA riboside in 500 ml 
pyridine, 72 ml acetic anhydride was added over a period of 15 minutes. 
The cooling bath was removed; stirring of the mixture was continued for 
four hours during which a clear solution formed. TLC of a small aliquot 
drawn and evaporated indicated that the reaction was complete. The 
reaction vessel was cooled in ice and treated with 5 ml methanol. Pyridine 
was removed by evaporation under high vacuum. The residue was 
co-evaporated with N,N-dimethyl-formamide (3.times.150 ml). The 
tan-colored viscous product obtained was dissolved in ethanol. The 
resulting mixture was seeded with a few crystals of 2',3',5'-triacetyl 
AICA riboside. The crystalline product formed after 24 hours and was 
collected by filtration, washed with ice cold ethanol and dried under 
vacuum (40.degree. C.) to give 65.0 g of the above-identified product, 
melting point 128.degree.-130.degree. C. 
The filtrate and washings were combined and evaporated down to about 50 ml 
and seeded with 2',3',5'-triacetyl AICA riboside crystals to obtain an 
additional 7.0 g of product. Thus, giving a total yield of 72.0 g. 
EXAMPLE 17 
Preparation of 
5-Amino-5'-Isobutyryl-1-.beta.-D-Ribofuranosylimidazole-4-Carboxamide 
("5'-Isobutyryl-AICA-Riboside") 
To an ice-cooled solution of 14.9 g 2',3'-isopropylidene-AICA riboside and 
6.1 g 4-N,N-dimethylamino-pyridine in 150 ml N,N-dimethylformamide, 8.69 g 
isobutyric anhydride (10% excess) was added over a period of 15 minutes. 
The cooling bath was removed and the reaction mixture stirred at room 
temperature overnight. TLC of a small aliquot of the reaction mixture 
which was drawn and worked up indicated that the reaction was complete. 
The reaction mixture was treated with 5 ml methanol and evaporated under 
reduced pressure. The syrupy product so obtained was treated with 100 ml 
of 60% formic acid and allowed to stand at room temperature for 48 hours. 
Water and formic acid were removed by evaporation under reduced pressure. 
The residue was chromatographed using a silica gel column with methylene 
chloride:methanol (9:1) as the solvent phase. The solvent was evaporated 
to give a syrupy product which was then stirred in hot toluene. The 
toluene was decanted. The residue was ground with hexane. The solid 
product formed and was collected by filtration, washed with hexane and 
dried under vacuum to give 10.2 g of the above-identified product. 
IR(KBr)cm.sup.1, 3500-2800 (OH,NH.sub.2), 1710 
##STR18## 
1650 (CONH.sub.2). NMR (DMSO-d.sub.6), .delta.ppm. 0.9-1.1 (2d,6H, 
2CH.sub.3), 2.5-2.65 (m, 1H, C--CH), 4.0 (m, 2H, 5'-CH.sub.2), 4.15-4.3 
(m, 3H, 2'-CH, 3'-CH, and 4'-CH) 5.35(d,1H, 1'-CH),5.4-5.6(2d, 2H, 2'-OH 
and 3'OH) 5 8 (br.s, 2H, NH.sub.2), 6.6-6.95(br.d, 2H, CONH.sub.2), and 
7.3 (S, 7H, 2-CH). 
EXAMPLE 18 
Preparation of 
5-AMINO-5'-Pivaloyl-1-.beta.-D-Ribofuranosylimidazole-4-Carboxamide 
("5'-Pivaloyl-AICA Riboside") 
To an ice-cooled solution of 14.9 g 2',3'-isopropylidene-AICA riboside in 
N,N-dimethylformamide, 10.23 g pivalic anhydride and 6.1 g 
4'N,N-dimethyl-aminopyridine were added in sequence. The cooling bath was 
removed and the reaction mixture as stirred at room temperature for 24 
hours. TLC of a small aliquot of the reaction mixture drawn and worked up 
indicated that the reaction was complete. The reaction mixture was treated 
with 5 ml methanol and evaporated to dryness under high vacuum. The 
residue so obtained was treated with 100 ml of 60% formic acid and then 
allowed to stand at room temperature for 48 hours. Formic acid and water 
were removed by evaporation under reduced pressure. The resulting residue 
was chromatographed on a silica gel column using methylene 
chloride:methanol (9:1) as the solvent phase. The syrupy product obtained 
after evaporation of the solvent was stirred in hot toluene. The toluene 
was decanted and the product was ground with 150 ml hexane. The solid 
product formed and was collected by filtration, washed and hexane and 
dried under vacuum to give 11.5 g of the above-identified product. 
IR(KBr) cm.sup.1, 3500-2900 (OH,NH.sub.2), 1720 
##STR19## 
1645 (CONH.sub.2). 
NMR(DMSO-d.sub.6), .delta.ppm, 1.15 (s, 9H, 3CH.sub.3), 3.95-4.05 (m, 2H, 
5'-CH.sub.2), 4.15-4.3 (m, 3H, 2'-CH, 3'-CH, and 4'-CH), 5.35 and 5.55 
(2d, 2H, 2'OH, and 3'-OH), 5.48 (d, 1H, 1'-CH), 5.75-5.9 (br.s, 2H, 
5-NH.sub.2), 6.6-6.9 (br.d, 2H, CONH.sub.2) 7.25 (s,1H, 2-CH). 
EXAMPLE 19 
Preparation of 
5-AMINO-5'-n-Butyryl-1-.beta.-Ribofuranosylimidazole-4-Carboxamide 
("5'-n-Butyryl-AICA Riboside") 
To an ice-cooled solution of 12.2 g 2', 3'-isopropylidene AICA riboside in 
a mixture of 50 ml N,N-dimethylformamide and 50 ml pyridine, 5.0 ml 
n-butyric anhydride was added over a period of 10 minutes. The cooling 
bath was removed and the reaction mixture was stirred for 20 hours. TLC of 
a small aliquot of the reaction mixture drawn and evaporated indicated 
that the reaction was complete. The reaction mixture was treated with 5 ml 
methanol and evaporated under high vacuum. The residue was coevaporated 
twice with 50 ml N,N-dimethylformamide. The resulting product was 
dissolved in 120 ml of 60% formic acid and then allowed to stand at room 
temperature for 48 hours. Water and formic acid were removed by 
evaporation under high vacuum. The residue was chromatographed on a silica 
gel column using methylene chloride:methanol (9:1) as the solvent phase. 
The sticky product obtained after evaporation of solvent was triturated 
with hot toluene. The toluene was decanted. The product was ground with 
hexane. The amorphous powder which formed was collected by filtration, 
washed with hexane, and dried under high vacuum to give 2.7 g of the 
above-identified product. 
IR(KBr)cm.sup.-1, 3500-2900 (OH, NH.sub.2, etc ), 1695 
##STR20## 
NMR(DM50-d.sub.6), .delta.ppm. 0.8-0.95(t, 3H, CH.sub.3), 1.5(m, 2H, 
--CH.sub.2 attached to CH.sub.3), 2.18-2.2(t, 2H, 
##STR21## 
3.95-4.1(m, 2H, 5'-CH.sub.2), 4.15-4.35(m, 3H, 2'-CH, 3'-CH, and 4'-CH), 
5.35(d, 1H, 1'-CH), 5.4-5.6 (2d, 2H, 2'-OH and 3'-OH) 5.75-5.9(br.s, 2H, 
5-NH.sub.2), 6.6-6.9(br.d, 2H, CONH.sub.2), 7.3(s, 1H, 2-CH). 
By using the procedures described in Examples 1 to 19 and in the Detailed 
Description of the Invention, and using the appropriate starting materials 
and reagents the compounds listed in Table I were prepared. Also, by using 
the procedures described in Examples 1 to 19 and in the Detailed 
Description of the Invention, the compounds listed in Tables II and III 
are prepared. 
EXAMPLE A 
Activity in Inhibiting HTL-Induced Seizures 
Compounds were tested for their activities in inhibiting HTL-induced 
seizures in rats. 
Animals used were male Swiss Webster mice weighing 21-30 grams (Charles 
River Breeding Labs, Wilmington, Mass.). All animals were adapted to the 
laboratory for at least 5 days prior to use. 
All solutions to be injected were prepared as a single injection cocktail 
at a concentration such that 1 ml per 100 g of body weight yielded the 
desired dose. The solutions were compounded as follows: Homocysteine 
Thiolactone--HCl (HTL-HCl) (Sigma Chemical Company, St. Louis, Mo.) was 
dissolved in distilled water and the pH adjusted to 6.7 with NaOH. 
Pentylenetetrazol (PTZ) was dissolved in 0.9% saline. Prodrug compounds or 
AICA riboside (Sigma) when used alone was dissolved in distilled water. 
All solutions containing Mioflazine (Janssen Pharmaceuticals) were 
prepared at a final DMSO concentration of 10-15% as were the Dipyridamole 
(Sigma) solutions. N-ethyl-carboxamide adenosine, NECA (Sigma) and 
Flunitrazepam (Hoffman La Roche) injections were prepared in a final 
ethanol concentration of 0.2%. In all cases carrier control solutions of 
carrier were injected that were matched for both tonicity and solvents to 
the test solutions. All test and control solutions were injected via a 
bolus, I.P., using a 27 gauge needle. HTL and PTZ were injected 
subcutaneously in the upper back of the animal. 
Animals were preinjected with either control solution containing only 
carrier or test solution containing candidate compound (prodrug or AICA 
riboside) and carrier in groups of 6-8 per test solution or control. The 
seizure inducing composition solution was injected at a specific time 
interval thereafter (ranging from 15 minutes to several hours, most 
experiments utilized a 30 minute interval). After injection of the seizure 
inducing composition animals were isolated in separate cages and observed 
for the onset of a seizure. In most experiments animals were scored as 
being fully protected from a seizure if they failed to seize for a period 
2-3 hours following homocysteine thiolactone (HTL) injections (carrier 
control latency about 20 minutes) and 1 hour after PTZ administration 
(carrier control seizure latency of 4 minutes). Seizures noted were either 
clonic or clonic-tonic in nature and varied in severity from forelimb 
clonus to full tonic extension of hind limbs and forelimbs. In all 
experiments the seizure latency was also noted as was the mortality rate 
in animals having seizures. The overt character of both the PTZ and HTL 
seizures were quite similar, although the latency of the former was 
markedly shorter. 
Results of testing one of the compounds of the present invention, 
3'-isobutoxycarbonyl AICA riboside (Prodrug A, Compound 1 of Table I), and 
AICA riboside for prevention of HTL induced seizures are shown in FIG. 1. 
EXAMPLE B 
Adenosine, AICA Riboside and ZMP Levels in Ischemic Heart Tissue 
Prodrug compounds of the present invention and AICA riboside were tested 
for their activity in enhancing the production of adenosine and increasing 
production of AICA riboside from ZMP in ischemic heart tissue in rats. 
Samples of heart tissue after ischemia were analyzed for nucleoside and 
nucleotide levels. Samples were measured for adenosine, AICA riboside and 
ZMP concentrations by HPLC. 
A comparison of adenosine production induced by saline, AICA riboside and 
Prodrug A (Compound 1 of Table I) is shown in FIG. 2. The fall in ZMP and 
quantitatively equivalent rise in AICA riboside level is shown in FIG. 3. 
The enhancement of adenosine production by Prodrug A as compared with an 
equimolar dose of AICA riboside without a corresponding high AICA-riboside 
level is tabulated in Table IV. 
EXAMPLE C 
Activity in Protection Against Ischemic Injury in Skin Flap 
Compounds were tested for their activity in protecting against ischemic 
injury in a skin flap model in rats. 
Animals were pretreated with AICA riboside or AICA riboside plus adenosine 
deaminase (ADA) 45 minutes before surgery or, as a positive control, 
superoxide dismutase (SOD) was used at the time of surgery. A skin flap 
was raised on the abdomen of a rat for 6 hours and then sewn down. The 
percent viability of the flaps was evaluated at 3 days post-surgery. 
Results are tabulated in Table V. 
Animals treated with AICA riboside showed an increase in skin flap 
viability (compared with controls) which was statistically significant 
according to the Fisher Exact Test (p&lt;0.05). This effect was not as 
pronounced in the presence of ADA, supporting the importance of 
adenosine's protective role in this setting. 
EXAMPLE D 
Enhancement of Adenosine Release by Lymphoblasts 
Prodrug compounds of the present invention and AICA riboside were tested 
for their activity in increasing adenosine release in cell culture. 
With regard to the enhanced in vitro release of adenosine, a human splenic 
lymphoblast cell line (WI-L2) was used to demonstrate the effect of AICA 
riboside and prodrugs of the present inventions of the cell line have been 
described and properties of the cell line have been described by 
Hershfield et al. in Science, Vol. 197, p. 1284, 1977. The cell line was 
maintained in RPMI 1640 cell culture media supplemented with 10% fetal 
calf serum and 2 mM glutamine and equimolar concentrations of prodrug or 
AICA riboside and grown for 36 hours in an atmosphere of 5% carbon dioxide 
in air. Fetal bovine serum contains purines and purine metabolizing 
enzymes; however, and to establish the effect of AICA riboside or prodrug 
during 2-deoxyglucose exposure, the WI-L2 cells were incubated in RPMI 
1640 glucose-deficient medium supplemented with 10% heat-inactivated, 
dialyzed fetal bovine serum, 2 mM glutamine, and 1 .mu.M deoxycoformycin. 
Catabolism of cellular ATP stores was stimulated by adding 2-deoxyglucose 
to a final concentration of 10 mM. At sixty minutes, the amount of 
adenosine released by the cells into the supernatant was determined by 
mixing 30 microliters of chilled 4.4N perchloric acid with 300 microliters 
of supernatant and centrifuging the mixtures at 500.times.G for 10 minutes 
at 4.degree. C. Each resulting supernatant was neutralized with 660 
microliters of a solution containing 2.4 grams of tri-n-octylamine 
(Alamine 336) (General Mills) in 12.5 milliliters of 
1,1,2-trichloro-1,2,2-trifluoroethane (Freon-113) solvent as described by 
Khym in Clinical Chemistry, Vol. 21, p. 1245, 1975. Following 
centrifugation at 1500.times.G for 3 minutes at 4.degree. C., the aqueous 
phase is removed and frozen at -20.degree. C. until assayed for adenosine 
and inosine. Adenosine was evaluated isocratically on a C-18 
micro-Bondapak reverse phase column equilibrated with 4 millimolar 
potassium phosphate, pH 3.4:acetonitrile 60% in water (95:5 v/v) buffer. 
Adenosine elutes at 8-10 minutes and its identity was confirmed by its 
sensitivity to adenosine deaminase and by spiking with adenosine 
standards. Continuous monitoring was performed by absorbance at 254 and 
280 nm. Peaks were quantitated by comparison with high pressure liquid 
chromatography analysis of suitable standards. 
FIG. 4 shows the effect of 36 hour pretreatment with AICA riboside or 
Prodrug A on enhancement of adenosine release from lymphoblasts. 
EXAMPLE E 
Enhanced Oral Bioavailability 
AICA riboside was administered to Sprague-Dawley rats at a dose of 250 
mg/kg or 500 mg/kg, prodrug compounds of the present invention were 
administered at an equal molar dose. 
At 15, 30, 60 and 120 minutes after gavage, the animals were sacrificed. 
The tissues were obtained and frozen immediately for nucleoside and 
nucleotide analysis. The tissue samples obtained were liver, heart, brain 
and whole blood. After initial freezing in liquid nitrogen, the tissue 
samples were extracted with trichloroacetic acid and neutralized with 
alamine freon. The tissue samples were evaluated by HPLC on a Whatman 
Partsil-10 (SAX) column for nucleosides and bases as described in Example 
6. 
In two separate experiments, at a dose equimolar to the dose of AICA 
riboside used, 3'-isobutoxycarbonyl AICA riboside ("Prodrug A") exhibited 
increased oral bioavailability as evidenced by an increase in ZMP levels 
in liver, whole blood and heart. Tests were run with 8 rats. 
FIGS. 5(a) and (b) show ZMP concentrations in rat liver at doses of a molar 
equivalent of 250 mg/kg and 500 mg/kg AICA riboside. 
EXAMPLE F 
Effect of Length of Side Chain of 3'-Carbonate Esters of AICA Riboside on 
Adenosine Levels 
Adenosine and AMP levels resulting from the administration of equimolar 
amounts of AICA riboside or some 3'-carbonate esters of AICA riboside 
having side chain lengths of 2 to 6 carbon atoms were studied using the 
ischemic rat heart model of Example B. 
After one hour following administration of AICA riboside (500 mg/kg) or the 
molar equivalent of carbonate ester, the hearts were excised and incubated 
at 37.degree. C. for one hour as described in Example B. Tissue adenosine 
and, for some of the carbonate esters, AMP levels were measured in the 
ischemic hearts by HPLC. Values are means .+-. S.E.M. Results are shown in 
FIGS. 19A and 19B. 
EXAMPLE G 
Glucose Levels in Fasted Mice 
Male mice (Swiss-Webster) were injected IP with the indicated treatment. 
The mice were fasted for 120 minutes before treatment. Ribose and AICA 
riboside were formulated in saline. Glucose levels were measured on serum 
(heparinized blood centrifuged at 10,000.times.g for ten minutes). Glucose 
levels were measured one hour after AICA riboside administration. Results 
are reported in FIG. 6. 
EXAMPLE H 
Effect of AICA Riboside on Plasma Glucose Levels in Fasted and Non-Fasted 
Rats 
Fasted rats were fasted for 16 hours before administration of AICA 
riboside. Rats were given a 750 mg/kg dose of AICA riboside IP (8 animals 
per group). Forty minutes later blood samples were taken. Blood was 
centrifuged at 10,000 x/g for 10 minutes and then analyzed for glucose 
levels using the hexokinase procedure (See Example K). Results are shown 
in FIG. 7. 
EXAMPLE H-2 
Effect of AICA Riboside on Blood Glucose in Rabbits 
Rabbits (New Zealand White) were given IV doses of AICA riboside, either 2 
ml/kg (100 mg/kg) or 4 ml/kg (200 mg/kg). Blood was obtained by 
venipuncture before AICA riboside administration and two hours 
post-administration. Blood glucose concentrations before administration 
and two hours post-administration were measured by the hexokinase 
procedure (see Example K). The percent change (+/-) from pre-dose values 
are reported. Both dosage levels decreased blood glucose levels as 
compared with pre-AICA riboside administration levels. Results are 
tabulated in Table VI. 
EXAMPLE I 
Effect of AICA Riboside on Plasma Glucose Levels in Humans 
Healthy male volunteers were given a 30-minute intravenous infusion of AICA 
riboside at doses of 25 mg/kg, 50 mg/kg or 100 mg/kg. 
Plasma glucose levels were monitored over a four hour period during and 
following the 30 minute infusion. Plasma glucose was measured by clinical 
chemistry autoanalyzer. The onset of the serum glucose lowering effect was 
evident by the end of the AICA riboside infusion period and plasma glucose 
reached a nadir approximately 30 minutes after the AICA riboside infusion 
was stopped. Recovery to euglycemic levels was complete by about three 
hours. Results are shown in FIG. 8. 
EXAMPLE J 
Effect of AICA Riboside on Liver Glycogen in Mice 
Swiss Webster mice were treated with saline (as a control) or 750 mg/kg of 
AICA riboside administered intraperitoneally, six animals per group. The 
mice were sacrificed one hour post-administration. The livers were removed 
and extracted. Liver glycogen was determined by the method of Dubois, et 
al., Anal. Chem. 28:350-356 (1956). Glucose was measured by the hexokinase 
method (See Example K). Results are reported in FIG. 9. 
EXAMPLE K 
Effects of AICA Riboside on Blood Lactate and Pyruvate in Rats 
AICA riboside (100, 250 or 500 mg/kg) or saline (as a control) was 
administered intraperitoneally to rats. Sixty minutes after AICA riboside 
administration, the rats were sacrificed by cervical dislocation. Blood 
glucose was determined spectrophotometrically measuring O.D. at 340 nm by 
the hexokinase method using the glucose SR Reagent (Medical Analysis 
Systems, Inc.) Blood lactate and pyruvate were determined 
spectrophotometrically measuring O.D. at 340 nm using lactate 
dehydrogenase in the presence of excess NAD or NADH, respectively. Values 
were expressed as mean +S.E.M. Results are reported in FIGS. 10A to 10D. 
EXAMPLE L 
Effect of AICA Riboside on Blood Glucose Levels in Conjunction with Liver 
ZMP Levels 
The association of AICA riboside-induced reduction in blood glucose with 
hepatic ZMP in the mouse was investigated. 
AICA riboside (either 100 or 500 mg/kg) was administered intravenously by 
tail vein injection to mice which had been fasted for four hours. At a 
time of 2, 5, 10, 30 or 120 minutes post-administration, the mice were 
sacrificed by cervical dislocation. Blood glucose was measured 
spectrophotometrically, measuring O.D. at 340 nm, by the hexokinase method 
using the glucose SR Reagent (Medical Analysis Systems, Inc.). Portions of 
liver (0.2 to 0.3 g) were freeze clamped in situ, homogenized and, 
following centrifugation, the neutralized supernatant was analyzed by 
ion-exchange HPLC. Values were expressed as means +S.E.M. Results are 
reported in Table 11. 
EXAMPLE M 
Inhibition of Fructose-1,6-Diphosphatase 
Inhibition of fructose-1,6-diphosphatase from rabbit liver (Sigma) by AICA 
riboside monophosphate (ZMP) and AMP was measured according to the assay 
technique described in Methods in Enzymology 90:352-357 (1982). Results 
are reported in FIG. 12. 
EXAMPLE N 
Plasma Insulin Levels in Humans after AICA Riboside Administration 
Human (male) volunteers were given a 15-minute intravenous infusion of 50 
mg/kg AICA riboside. Plasma concentrations of immunoreactive insulin was 
determined by RIA during and following the administration for about 4 
hours. (RIA kit, hersham Clinical). Results are reported in FIG. 13. 
EXAMPLE O 
Hypoglycemic Effects of AICA Riboside Prodrugs 
Male mice (Swiss Webster) were given equimolar amounts of either saline, 
AICA riboside, or compounds Cl or 10 of Table I orally. Blood glucose 
levels were measured one hour after administration by the glucose strip 
method (Chemstrip B.G.). Results are reported in FIG. 14. 
EXAMPLE P 
Effects of Inhibition of Adenosine Kinase on PTZ-Induced Seizures 
Swiss Webster mice were given the indicated dose (100, 200 or 400 mg/kg) of 
the adenosine kinase inhibitor, 5'-amino-5'-deoxyadenosine, or saline (as 
a control) intraperitoneally. One hour later the animals were given a 75 
mg/kg dose of pentylenetetrazole (PTZ) and the seizure frequency observed. 
Results are reported in FIG. 15. 
EXAMPLE Q 
Inhibition of Fructose 1,6-Diphosphatase by ZMP and Carbocyclic ZMP 
The indicated concentrations (250 .mu.m) of ZMP and carbocyclic ZMP were 
incorporated into the fructose 1,6-diphosphatase assay (see Example M), 
and the resulting activity was determined. Activity was expressed as a 
velocity (rate of conversion of substrate). Results are reported in FIG. 
16. 
EXAMPLE R 
Effect of Chronic AICA Riboside Treatment on Triglyceride Levels in 
Diabetic Rats 
Rats were made diabetic by treatment with streptozocin (50 mg/kg IV) and 
then treated with either saline or AICA riboside (500 mg/kg, twice a day) 
for 22 days. Plasma triglyceride levels were analyzed 18 hours after the 
last injection of AICA riboside using the Sigma Procedure #334 Assay Kit 
(coupled assay employing lipase, glycerokinase, pyruvate kinase and 
lactate dehydrogenase) which measures decreases in OD.sub.340 over time 
(NADH disappearance). 
EXAMPLE S 
Effect of Oral Administration of AICA Riboside or an AICA Riboside Prodrug 
on Plasma AICA Riboside Levels 
Plasma concentrations of AICA riboside in dogs were determined by HPLC 
following oral administration of 50 mg/kg AICA riboside and the (molar) 
equivalent amount of 50 mg/kg AICA riboside or one of two prodrugs, 
compounds 10 and 17 of Table I. The compounds were administered in 
solution in PEG 400:water (1:1). Results are shown in FIG. 18. A different 
prodrug. Compound 22 of Table I, was administered in solid form in a 
capsule (50 mg/kg). Results are shown in FIG. 20. Plasma concentration of 
AICA riboside was determined according to: Dixon, R., et al., "AICA 
riboside: Direct quantitation in ultrafiltrate of plasma by HPLC," Res. 
Commun. Chem. Path. Pharm., in press (1989). 
EXAMPLE T 
Effect of AICA Riboside on Serum Glucose Levels in Diabetic Mice 
Mice were made diabetic by low dose Streptozotocin treatment (40 mg/kg/day 
for 5 days followed by a 10-day incubation period). These diabetic mice, 
11 per group, were treated with the indicated dose of AICA riboside or 
saline in an IP bolus of 1 ml/100 g body weight. The animals were 
exsanguinated 1.5 hours post-administration (of AICA riboside or saline). 
The plasma was isolated by centrifugation and analyzed for glucose by the 
hexokinase/glucose-6-phosphate dehydrogenase spectrophotometric method 
(see Example K). Normoglycemic levels were determined from saline-treated 
nondiabetic mice by the same protocol. Values were expressed as mean .+-. 
sem. Results are reported in FIG. 21. 
EXAMPLE U 
Effect of Chronic AICA Riboside Treatment on Blood Glucose and Water Intake 
in Diabetic Rats 
Rats made diabetic with Streptozotocin (60 mg/kg, 5 days post-treatment), 9 
per group, were treated twice daily with 500 mg/kg AICA riboside or with 
physiological saline via injection IP, except for days 6, 13 and 20 when a 
single 750 mg/kg dose was administered and except for days 7, 14 and 21 
when no treatment was given. Blood was drawn by tail bleeds, two hours 
post-injection, on the days indicated, analyzed for glucose using 
Chemstrip bG glucose reagent strips and an Accuchek II blood glucose 
monitor (both from Boehringer Manheim). Data was calculated as percent of 
pretreated levels and expressed as mean .+-. sem. Results are reported in 
FIG. 22. 
The water intake from these rats was measured by determining the amount of 
water lost from the individual cage water bottles each day. Values are 
expressed as a cumulative mean .+-. sem. Results are reported in FIG. 23. 
EXAMPLE V 
Effects of Chronic AICA Riboside Treatment on Hepatic Fructose 
1,6-Diphosphatase (FDPase) Activity in Diabetic Rats 
Rats made diabetic with Streptozotocin (60 mg/kg, 5 days post treatment) 
were treated with either 2.times.500 mg/kg/day AICA riboside or 0.9% 
saline twice a day for three weeks. Eighteen hours after the last 
treatment the livers were removed. Livers from the treated rats and from 
native rats were homogenized in 20 .mu.M potassium phosphate buffer (pH 
7.4) with 100 .mu.M EDTA and 100 .mu.M dithiothreitol and then centrifuged 
for 20 minutes at 45,000.times.g. FDPase activity was assayed both in this 
native form and following passage of the supernatant over a sephadex G25 
column by the method of Marcus et al (Methods in Enzymology 90:352-356 
(1982)). Protein concentrations were determined by the Bradford method 
(Anal. Biochem. 72:248 (1976); Anal. Biochem. 86:142 (1978)). Enzyme 
activity per mg protein was expressed as mean .+-. sem. Results are shown 
in FIG. 24. 
EXAMPLE W 
Determination of IC50 for Inhibition of FDPase by ZMP 
Liver samples (from the indicated species) were homogenized in 20 mM 
potassium phosphate buffer (pH 7.4) with 100 .mu.M EDTA and 100 .mu.M 
dithiothreitol. The liver homogenates were centrifuged at 45,000.times./g. 
The supernatants were passed over a sephadex G25 column and assayed for 
FDPase activity by the method of Marcus et al (Methods in Enzymology 
90:352-356 (1982)) in the presence of ZMP over a range of concentration 
and in the absence of ZMP. The IC50 value was defined as the concentration 
of ZMP, which inhibited 50 percent of the baseline FDPase activity under 
the assay conditions. Results are shown in Table VII. 
EXAMPLE X 
Effect of AICA Riboside on Hepatic Fructose 1,6-Diphosphate Levels in Mouse 
Mice which had been fasted for 6 hours were given either an IP injection of 
500 mg/kg AICA riboside or 0.9% saline. The animals were sacrificed 1.5 
hours post-injection; their livers were removed and extracted into iced 
perchloric acid. The extracts were neutralized and analyzed for fructose 
1,6-diphosphate by the method of Shrinivas et al (Biochem J. 262: 721-725 
(1989)). Results are shown in FIG. 25. 
EXAMPLE Y 
Comparison of Glyburide (Oral) and AICA Riboside (IP) on Blood Glucose 
Levels in Fasted and Non-Fasted Mice 
Fed or 24-hour fasted mice were treated either with vehicle, 5 mg/kg 
glyburide (an oral hyperglycemic agent) administered orally or 600 mg/kg 
AICA riboside administered IP. Either three hours post-administration or 
at the time of hypoglycemic seizure, whichever came first, blood was 
taken. The serum was isolated by centrifugation and analyzed for glucose 
levels by the hexokinase/glucose-6-phosphate dehydrogenase 
spectrophotometric assay (see Example K). Drug group values were expressed 
as percent of vehicle levels. Results are shown in FIG. 26. 
EXAMPLE Z 
Effect of AICA Riboside on Serum Glucose Levels in Diabetic Rats 
Sprague-Dawley rats (200 g each) made diabetic with Streptozotocin (55 
mg/kg, 4 days post-treatment) were anesthetized with diethyl ether. After 
anesthetizing, an incision was made and a cannula of silastic medical 
grade tubing (0.020 inch I.D/0.037 inch O.D.) was inserted into the right 
jugular 5 mm rostral to the clavicle and extended 20 mm toward the heart. 
The cannula was anchored and exteriorized through the back of the neck, 
filled with heparized saline (500 U/ml) and tied off. The following day, 
the rats were given an IP injection of either 750 mg/kg AICA riboside or 
physiological saline. Serial blood draws were made via the cannula. Serum 
was isolated by centrifugation and was analyzed for glucose by the 
hexokinase/glucose-6-phosphate dehydrogenase method (see Example K). 
Results are reported in FIG. 27. 
EXAMPLE AA 
Evaluation of Oral Bioavailability of AICA Riboside Prodrugs 
The bioavailability of AICA riboside after oral administration of either 
AICA riboside or one of the AICA riboside prodrugs of the present 
invention was evaluated in dogs. Plasma concentrations of AICA riboside 
and intact prodrug were measured using HPLC. (See Dixon, R., et al., Res. 
Commun. Chem. Path. Pharm. 65:405-408 (1989)). Bioavailability of AICA 
riboside was evaluated in terms of the time required to reach maximum 
plasma concentration (Tmax), the maximum concentration achieved (Cmax) and 
the area under the plasma concentration-time curve (AUC) from time zero to 
the last measurable plasma concentration. Absolute bioavailability (F %) 
was calculated by dividing the AUC for AICA riboside following oral 
administration of the prodrug (or AICA riboside itself) by the AUC 
following intravenous administration of an equivalent amount of AICA 
riboside (100% bioavailability). Results are tabulated in Table VIII. 
TABLE I 
__________________________________________________________________________ 
Compounds of the formula: 
##STR22## 
Compound 
X.sub.1 X.sub.2 X.sub.3 
__________________________________________________________________________ 
1 H H 
##STR23## 
2 
##STR24## 
##STR25## H 
3 
##STR26## H H 
4 H H 
##STR27## 
5 
##STR28## H H 
6 
##STR29## 
##STR30## H 
7 H H 
##STR31## 
8 H 
##STR32## 
##STR33## 
9 H 
##STR34## H 
10 
##STR35## H H 
11 
##STR36## H H 
12 
##STR37## H H 
13 H H 
##STR38## 
14 H 
##STR39## 
15 H 
##STR40## H 
16 
##STR41## H H 
17 H H 
##STR42## 
18 H H 
##STR43## 
19 
##STR44## H H 
20 
##STR45## H H 
21 
##STR46## 
##STR47## 
22 
##STR48## H 
##STR49## 
23 
##STR50## 
##STR51## 
##STR52## 
24 
##STR53## H 
##STR54## 
25 
##STR55## 
##STR56## H 
26 
##STR57## 
##STR58## 
##STR59## 
27 
##STR60## H H 
28 
##STR61## H H 
29 
##STR62## H H 
30 
##STR63## 
##STR64## 
##STR65## 
31 
##STR66## H H 
32 
##STR67## 
##STR68## 
##STR69## 
33 
##STR70## H H 
C1 
##STR71## 
##STR72## 
##STR73## 
C2 
##STR74## 
##STR75## 
##STR76## 
C3 
##STR77## H H 
C4 
##STR78## 
##STR79## 
##STR80## 
__________________________________________________________________________ 
TABLE II 
______________________________________ 
Compounds of the formula: 
##STR81## 
Compound X.sub.1 
______________________________________ 
34 
##STR82## 
35 
##STR83## 
36 
##STR84## 
37 
##STR85## 
38 
##STR86## 
39 
##STR87## 
40 
##STR88## 
41 
##STR89## 
42 
##STR90## 
43 
##STR91## 
44 
##STR92## 
______________________________________ 
TABLE III 
__________________________________________________________________________ 
Compounds of the formula 
##STR93## 
Compound 
X.sub.1 X.sub.2 
X.sub.3 
__________________________________________________________________________ 
45 
##STR94## H H 
46 H H 
##STR95## 
47 
##STR96## H H 
48 H H 
##STR97## 
49 
##STR98## H H 
50 H H 
##STR99## 
51 
##STR100## H H 
52 H H 
##STR101## 
53 
##STR102## H H 
54 H H 
##STR103## 
__________________________________________________________________________ 
TABLE IV 
______________________________________ 
Tissue Concentration (nMoles/g) 
AICA 
Treatment Adenosine Riboside ZMP 
______________________________________ 
Control (Saline) 
272 .+-. 31 0 0 
AICA-riboside 
409 .+-. 60 774 .+-. 73 
385 .+-. 15 
Prodrug A 553 .+-. 46 592 .+-. 55 
161 .+-. 9 
______________________________________ 
TABLE V 
______________________________________ 
Protection against Ischemic Injury in Skin Flap 
Number 
Treatment Evaluated 
% Viable 
______________________________________ 
Control 24 33 
AICA riboside 8 75 
AICA riboside + ADA 
7 43 
SOD 18 68 
______________________________________ 
TABLE VI 
______________________________________ 
RABBIT PRE-DOSE 2H POST-DOSE 
CHANGE(+/-) 
______________________________________ 
AICA Riboside - 2 ml(100 mg/kg) - Blood Glucose - mg % 
1 111 87 -24 
2 117 89 -28 
3 113 89 -24 
4 112 122 +10 
5 118 96 -22 
6 121 98 -23 
7 117 106 -11 
8 152 99 -53 
9 118 93 -25 
10 113* 215* +102* 
Mean 120 98 -22 
AICA Riboside - 4 ml(200 mg)/kg 
Blood Glucose - mg % 
1 114 100 -14 
2 108 32** -76 
3 121 79 -42 
4 125 56 -69 
5 121 12 -59 
Mean 118 66 -52 
______________________________________ 
*Values considered outliers due to hemolysis of 2h sample were not 
included in mean. 
**Rabbit convulsed and was sacrificed. 
TABLE VII 
______________________________________ 
IC50 for Inhibition 
of FDPase by ZMD 
Species IC50 (.mu.M) 
______________________________________ 
Dog 90 
Gerbil 50 
Guinea Pig 
12 
Human 48 
Mouse 150 
Rabbit 41 
Rat 375 
______________________________________ 
TABLE VIII 
______________________________________ 
BIOAVAILABILITY 
Tmax 
Compound (hr) Cmax(ug/ml) 
AUC(.mu.g .multidot. hr/ml) 
F(%) 
______________________________________ 
AICA riboside (i.v.) 
-- -- 8.3 100 
AICA riboside (oral) 
0.75 1.1 0.9 11 
CI*(2',3',5'-Triacetyl- 
1.5 2.2 3.2 39 
AICA riboside 
10*(5'-isobutyryl-AICA 
0.75 5.0 4.9 60 
riboside 
11*(5'-pivalyl-AICA 
1.0 3.2 3.7 44 
riboside 
______________________________________ 
*Compounds from Table I