Methods for treating adenosine kinase related conditions

Novel compounds which selectively inhibit adenosine kinase and methods of preparing adenosine kinase inhibitors are provided. Also provided are methods of treating various inflammatory conditions, including arthritis and SIRS, which may be ameliorated by increased local concentrations of adenosine using adenosine kinase inhibitors.

FIELD OF INVENTION 
This invention relates to the use of adenosine kinase inhibitors and 
specifically to purine, pyrrolo[2,3-d]pyrimidine and 
pyrazolo[3,4-d]pyrimidine nucleoside analogs having activity as adenosine 
kinase inhibitors. The invention also relates to the use of these and 
other adenosine kinase inhibitors in the treatment of inflammation, 
sepsis, septic shock, burns and diseases which can be regulated by 
increasing the local concentration of adenosine. 
BACKGROUND OF THE INVENTION 
Adenosine has been reported to have cardioprotective (Olafsson et al., 
Circulation, 1987, 76:1135-1145) and neuroprotective properties (Dragunow 
and Faull, Trends in Pharmacol. Sci., 1988, 9:193; Marangos, Medical 
Hypothesis, 1990, 32:45). It is reportedly released from cells in response 
to alterations in the supply of or demand for oxygen (Schrader, 
Circulation, 1990, 81:389-391), is said to be a potent vasodilator, and is 
believed to be involved in the metabolic regulation of blood flow (Berne, 
Circ. Res., 1980, 47:808-813). However, adenosine has a short half life 
(&lt;1 sec) in human blood (Moser, et al., Am. J. Physiol., 1989, 
256:C799-C806), and therefore high doses of adenosine would need to be 
administered continuously to achieve effective levels. Adenosine has been 
reported to exhibit negative inotropic, chronotropic and dromotropic 
effects (Belardinelli et al., Prog. in Cardiovasc. Diseases, 1989, 
32:73-97) and to cause coronary steal by preferentially dilating vessels 
in nonischemic regions. Consequently, high doses of adenosine are toxic 
and this toxicity severely limits its therapeutic potential. However, it 
is believed that by increasing adenosine concentration locally, i.e. at 
the target site within the target tissue, the beneficial effects of 
adenosine can be provided without the toxic systemic effects. Adenosine 
has been reported to be an endogenous modulator of inflammation by virtue 
of its effects on stimulated granulocyte function (Cronstein etal., J. 
Clin. Invest., 1986, 78:760-770) and on macrophage, lymphocyte and 
platelet function. Adenosine receptor agonists have been reported to be 
beneficial in an experimental model of inflammation (Schrier, et al., J. 
Immunol., 1990, 145:1874-1879). Adenosine and a related analog have been 
reported to inhibit in vitro production of the cytokine, tumor necrosis 
factor alpha (Parmely et al., FASEB Journal, 1991, 5:A 1602). 
Adenosine kinase is a cytosolic enzyme which catalyzes the phosphorylation 
of adenosine to AMP. Inhibition of adenosine kinase can potentially reduce 
the ability of the cell to utilize adenosine, leading to increased 
adenosine outside of the cell where it is pharmacologically active. 
However, the regulation of adenosine concentration is complex and involves 
other adenosine-metabolizing enzymes each with different kinetic 
properties and mechanisms of regulation. Adenosine can also be deaminated 
to inosine by adenosine deaminase (ADA) and condensed with L-homocysteine 
to S-adenosylhomocysteine (SAH) by SAH hydrolase. The role of each of 
these enzymes in modulating adenosine concentration is dependent on the 
prevailing physiological conditions, is tissue specific and is not well 
understood. 
A number of nucleosides including purine, pyrrolo[2,3-d]pyrimidine and 
pyrazolo[3,4-d]pyrimidine analogs have been evaluated for inhibition of 
adenosine kinase but were reported to have K.sub.i 's of greater than 800 
nM (Caldwell and Henderson, Cancer Chemother. Rep., 1971, 2:237-246; 
Miller et al., J. Biol. Chem., 1979, 254:2346-2352). A few compounds have 
been reported as potent inhibitors of adenosine kinase with Ki's of less 
than 100 nM. These are the purine nucleosides, 5'-amino-5'-deoxyadenosine 
(Miller et al., J. Biol. Chem., 1979, 254:2346-2352) and 
1,12-bis(adenosin-N6-yl)dodecane (Prescott et al., Nucleosides & 
Nucleotides, 1989, 8:297), and the pyrrolopyrimidine nucleosides, 
5-iodotubercidin (Henderson et al., Cancer Chemotherapy Rep. Part 2, 1972, 
3:71-85; Bontemps et al., Proc. Natl. Acad. Sci. USA, 1983, 80:2829-2833; 
Davies et al., Biochem. Pharmacol., 1986, 35:3021-3029)and 
5'-deoxy-5-iodotubercidin (Davies et al., Biochem. Pharmacol., 1984, 
33:347-355; Davies et al., Biochem. Pharmacol., 1986, 35:3021-3029). 
Some of these compounds have been used to evaluate whether adenosine kinase 
inhibition might lead to increased extracellular adenosine concentrations. 
In rat cardiomyocytes, inhibition of adenosine deaminase by 
2'-deoxycoformycin was reported to have no effect on adenosine release 
from the cells. In contrast, inhibition of ADA together with adenosine 
kinase by 5'-amino-5'-deoxyadenosine resulted in a 6-fold increase in 
adenosine release (Zoref-Shani et al., J. Mol. Cell. Cardiol., 1988, 
20:23-33). The effects of the adenosine kinase inhibitor alone were not 
reported. Similar results were reported in isolated guinea pig hearts; in 
these studies addition of 5'-amino-5'-deoxyadenosine to the perfusion 
medium, in the presence of EHNA to inhibit deamination, was reported to 
result in a 15-fold increase of adenosine release (Schrader, in Regulatory 
Function of Adenosine; (Berne et al.) eds. pp. 133-156, 1983). These 
effects were not apparent in the absence of ADA inhibition and other 
studies using isolated rat hearts perfused with 5-iodotubercidin alone, 
have reported no increase in perfusate adenosine concentration under 
normoxic conditions (Newby et al., Biochem. J., 1983, 214:317-323) or 
under hypoxic, anoxic or ischemic conditions (Achtenberg et al., Blochem, 
J., 1986, 235:13-17). In other studies, adenosine release has been 
measured in neuroblastoma cells in culture and compared with that of a 
variant deficient in adenosine kinase (AK-). The AK- cells used in this 
study were said to release adenosine at an accelerated rate; the 
concentration of adenosine in the growth medium was reported to be 
elevated compared to the normal cells (Green, J. Supramol. Structure, 
1980, 13:175-182). In rat and guinea pig brain slices, adenosine uptake 
was reportedly inhibited by the adenosine kinase inhibitors, 
5-iodotubercidin and 5'-deoxy-5-iodotubercidin (Davis et al., Biochem. 
Pharmacol., 1984, 33:347-355). However, inhibition of uptake and 
intracellular trapping via phosphorylation does not necessarily result in 
increased extracellular adenosine, since the adenosine could enter other 
metabolic pathways or the percentage of adenosine being phosphorylated 
could be insignificant compared to the total adenosine removed. 
The effects of adenosine and certain inhibitors of adenosine catabolism, 
including 5-iodotubericidin were evaluated in an experimental model in 
which dog hearts were subjected to ischemia and reperfusion; 
5-iodotubericidin was reported to have inconsistent effects (Wu, et al., 
Cytobios, 1987, 50:7-12). 
Although the adenosine kinase inhibitors, 5'-amino-5'-deoxyadenosine and 
5-iodotubercidin have been widely used in experimental models, the 
susceptibility of 5'-amino-5'-deoxyadenosine to deamination, and hence its 
potentially short half life, and the cytotoxicity of 5-iodotubercidin make 
their clinical utility limited and may limit interpretations based on 
these compounds. The pyrrolo[2,3-d]pyrimidines, 5-iodotubercidin and 
5'-deoxy-5-iodotubercidin have been reported to cause pronounced general 
flaccidity and much-reduced spontaneous locomotor activity in mice, 
interpreted to be skeletal muscle relaxation; to cause hypothermia in 
mice; and to decrease blood pressure and heart rate in anesthetized rats 
(Daves et al., Biochem. Pharmacol., 1984, 33:347-355; Daves et al., 
Blochem. Pharmacol., 1986, 35:3021-3029; U.S. Pat. No. 4,455,420). The 
skeletal muscle effects of these compounds have been poorly documented, 
while the other effects were considered significant toxicities. It is 
believed that studies using these compounds were curtailed due to these 
toxicities and also because of their limited availability. 
SUMMARY OF THE INVENTION 
The present invention is directed to novel uses of compounds which are 
potent and selective adenosine kinase inhibitors. 
Another aspect of the present invention is directed to the clinical use of 
adenosine kinase inhibitors as a method of increasing adenosine 
concentrations in biological systems. In vivo inhibition of adenosine 
kinase prevents phosphorylation of adenosine resulting in higher local 
concentrations of endogenous adenosine. As a result of the very short 
half-life of adenosine and very low quantities of adenosine in tissues, 
this effect is most pronounced in regions producing the most adenosine 
such as ischemic regions. Hence, the beneficial effects of adenosine are 
enhanced in a site and event specific manner and toxic systemic effects 
are reduced. 
In particular, in one preferred aspect, the present invention is directed 
to novel nucleoside analogs which comprise a 5'-modified ribose linked to 
a substituted purine, pyrrolo[2,3-d]pyrimidine, or 
pyrazolo[3,4-d]pyrimidine base. Certain preferred compounds within these 
groups possess potencies many times greater than previously described 
inhibitors of adenosine kinase. The compounds of the present invention 
possess advantages for pharmaceutical use such as enhanced pharmacological 
selectivity, efficacy, bioavailability, ease of manufacture and compound 
stability. 
The novel compounds of the present invention and other adenosine kinase 
inhibitors may be used clinically to treat medical conditions where an 
increased localized adenosine concentration is beneficial. Accordingly, 
the present invention is directed to the prophylactic and affirmative 
treatment of ischemic conditions such as myocardial infarction, angina, 
percutaneous transluminal coronary angiography (PTCA), stroke, other 
thrombotic and embolic conditions, neurological conditions such as 
seizures and psychosis, and other conditions benefited by enhanced 
adenosine levels such as inflammation, arthritis, autoimmune diseases, 
cardiac arrhythmias, ulcers and irritable bowel syndrome. 
In particular, the present invention is also directed to the prophylactic 
and affirmative treatment of sepsis, septicemia (including but not limited 
to endotoxemia), and various forms of septic shock (including but not 
limited to endotoxic shock.) For example, adenosine kinase inhibitors will 
be useful in the prophylactic or affirmative treatment of a localized or 
systemic inflammatory response to infection by one or more of several 
types of organisms, including bacteria (gram negative or gram positive), 
viruses (including retroviruses), mycobacteria, yeast, protozoa or 
parasites. 
Furthermore, the present invention is directed to the treatment of 
disorders in which vascular leakage is involved. In particular, the 
present invention is directed to the treatment of burn injury. 
DEFINITIONS 
In accordance with the present invention and as used herein, the following 
terms, are defined with the following meanings, unless explicitly stated 
otherwise. 
The term "hydrocarbyl" refers to an organic radical comprised of carbon 
chains to which hydrogen and other elements are attached. The term 
includes alkyl, alkenyl, alkynyl and aryl groups, groups which have a 
mixture of saturated and unsaturated bonds, carbocyclic rings and includes 
combinations of such groups. It may refer to straight-chain, 
branched-chain cyclic structures or combinations thereof. 
The term "aryl" refers to aromatic groups which have at least one ring 
having a conjugated pi electron system and includes carbocyclic aryl, 
heterocyclic aryl and biaryl groups, all of which may be optionally 
substituted. 
Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic 
ring are carbon atoms. Carbocyclic aryl groups include monocyclic 
carbocyclic aryl groups and optionally substituted naphthyl groups. 
The term "monocyclic carbocyclic aryl" refers to optionally substituted 
phenyl, being preferably phenyl or phenyl substituted by one to three 
substituents, such being advantageously lower alkyl, hydroxy, lower 
alkoxy, lower alkanoyloxy, halogen, cyano, trihalomethyl, lower acylamino 
or lower alkoxycarbonyl. 
"Optionally substituted naphthyl" refers to 1- or 2-naphthyl or 1- or 
2-naphthyl preferably substituted by lower alkyl, lower alkoxy or halogen. 
Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring 
atoms in the aromatic ring and the remainder of the ring atoms carbon 
atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and 
include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, 
pyrimidyl, pyrazinyl, imidazolyl, and the like, all optionally 
substituted. 
Optionally substituted furanyl represents 2- or 3-furanyl or 2- or 
3-furanyl preferably substituted by lower alkyl or halogen. 
Optionally substituted pyridyl represents 2-, 3- or 4-pyridyl or 2-, 3- or 
4-pyridyl preferably substituted by lower alkyl or halogen. 
Optionally substituted thienyl represents 2- or 3-thienyl, or 2- or 
3-thienyl preferably substituted by lower alkyl or halogen. 
The term "biaryl" represents phenyl substituted by carbocyclic aryl or 
heterocyclic aryl as defined herein, ortho, meta or para to the point of 
attachment of the phenyl ring, advantageously para; biaryl is also 
represented as the --C.sub.6 H.sub.4 --Ar substituent where Ar is aryl. 
The term "aralkyl" refers to an alkyl group substituted with an aryl group. 
Suitable aralkyl groups include benzyl, picolyl, and the like, and may be 
optionally substituted. 
The term "lower" referred to herein in connection with organic radicals or 
compounds respectively defines such with up to and including 7, preferably 
up to and including 4 and advantageously one or two carbon atoms. Such 
groups may be straight chain or branched. 
The terms (a)"alkyl amino", (b)"arylamino", and (c)"aralkylamino", 
respectively, refer to the groups --NRR' wherein respectively, (a) R is 
alkyl and R' is hydrogen or alkyl; (b) R is aryl and R' is hydrogen or 
aryl, and (c) R is aralkyl and R' is hydrogen or aralkyl. 
The term "acyl" refers to hydrocarbyl--C(O)-- or HC(O)--. 
The terms "acylamino" refers to RC(O)NR-- and (RC(O)).sub.2 N-- 
respectively, wherein each R is independently hydrogen or hydrocarbyl. 
The term ".alpha.-alkoxyalkylidene" refers to hydrocarbyl-O--CR (an 
orthoester) wherein R is hydrogen or hydrocarbyl. 
The term "hydrocarbyloxycarbonyloxy" refers to the group ROC(O)O-wherein R 
is hydrocarbyl. 
The term "lower carboalkoxymethyl" or "lower hydrocarbyloxycarbonymethyl" 
refers to hydrocarbyl--OC(O)CH.sub.2 -- with the hydrocarbyl group 
containing ten or less carbon atoms. 
The term "carbonyl" refers to --C(O)--. 
The term "carboxamide" or "carboxamido" refers to --CONR.sub.2 wherein each 
R is independently hydrogen or hydrocarbyl. 
The term "lower hydrocarbyl" refers to any hydrocarbyl group of ten or less 
carbon atoms. 
The term "alkyl" refers to saturated aliphatic groups including 
straight-chain, branched chain and cyclic groups. 
The term "alkenyl" refers to unsaturated hydrocarbyl groups which contain 
at least one carbon-carbon double bond and includes straight-chain, 
branched-chain and cyclic groups. 
The term "alkynyl" refers to unsaturated hydrocarbyl groups which contain 
at least one carbon-carbon triple bond and includes straight-chain, 
branched-chain and cyclic groups. 
The term "halogen" refers to fluorine, chlorine, bromine or iodine. 
The term "hydrocarbyioxycarbonylamino" refers to a urethane, 
hydrocarbyl--O--CONR-- wherein R is H or hydrocarbyl and wherein each 
hydrocarbyl is independently selected. 
The term "di(hydrocarbyloxycarbonyl)amino" refers to 
(hydrocarbyl--O--CO).sub.2 N-- wherein each hydrocarbyl is independently 
selected. 
The term "hydrocarbylamino" refers to --NRR' wherein R is hydrocarbyl and 
R' is independently selected hydrocarbyl or hydrogen. 
The term "mercapto" refers to SH or a tautomeric form, 
The term "methine" refers to 
##STR1## 
The term "methylene" refers to --CH.sub.2 --. 
The term "alkylene" refers to a divalent straight chain or branched chain 
saturated aliphatic radical. 
The term "oxy" refers to --O-- (oxygen). 
The term "thio" refers to --S-- (sulfur). 
The term "prodrug" as used herein refers to any compound that has less 
intrinsic activity than the "drug" but when administered to a biological 
system generates the "drug" substance either as a result of spontaneous 
chemical reaction or by enzyme catalyzed or metabolic reaction. Reference 
is made to various prodrugs such as acyl esters, carbonates, and 
urethanes, included herein as examples. The groups illustrated are 
exemplary, not exhaustive and one skilled in the art could prepare other 
known varieties of prodrugs. Such prodrugs of the compounds of Formula I, 
fall within the scope of the present invention. 
The term "pharmaceutically acceptable salt" includes salts of compounds of 
Formula I derived from the combination of a compound of this invention and 
an organic or inorganic acid. The compounds of Formula I are useful in 
both free base and salt form. In practice the use of salt form amounts to 
use of base form; both forms are within the scope of the present invention 
.

DETAILED DESCRIPTION OF THE INVENTION 
Novel Adenosine Kinase Inhibitors 
In one aspect, the present invention relates to the novel use of adenosine 
kinase inhibitors which comprise compounds of the general formula I. 
##STR2## 
wherein: 
(a) A is oxygen, methylene or sulfur; 
(b) B' is --(CH.sub.2).sub.n --B wherein n is 1, 2, 3 or 4 and B is 
hydrogen, alkyl, alkoxy, amino, alkylamino, acylamino, 
hydrocarbyloxycarbonylamino, mercapto, alkylthio, azido, cyano, halogen, 
or B' is alkenyl or alkynyl; 
(c) C.sub.1 and C.sub.2 are each independently hydrogen, acyl, 
hydrocarbyloxycarbonyl or taken together form a 5-membered ring wherein 
C.sub.1 is a single bond to C.sub.2 and C.sub.2 is carbonyl or 
.alpha.-alkoxyalkylidene; 
(d) X is --C(--D).dbd. and Y is --N.dbd. or --C(--E).dbd.; 
(e) D is hydrogen, halogen, alkyl, aryl, aralkyl, alkenyl, alkynyl, 
haloalkyl, cyano, cyanoaikyl, acyl, carboxamido, a carboxylic acid or 
carboxylic acid ester group, alkoxy, aryloxy, aralkyloxy, alkylthio, 
arylthio, aralkylthio, amino, alkylamino arylamino, aralkylamino, 
acylamino, or nitro; 
(f) E is hydrogen, halogen, alkyl, or alkylthio; 
(g) F is alkyl, aryl, aralkyl, halogen, amino, alkylamino, arylamino, 
aralkylamino, cyano, cyanoalkyl, alkoxy, aryloxy, aralkoxy, alkyithio, 
arylthio, aralkyithio; optionally substituted indolinyl or indolyl; 
pyrrolidinyl or piperazinyl; and 
(h) G is hydrogen, halogen, lower alkyl, lower alkoxy, lower alkylamino or 
lower alkylthio; and pharmaceutically acceptable salts thereof; with the 
proviso that: 
when A is oxygen and 
(i) X is --C(--D).dbd. and Y is --C(--E).dbd., then if B' is methyl, D is 
halogen, cyano or carboxamido, F is amino, then G is not hydrogen; or if D 
is hydrogen, then F is not amino; or 
(ii) X is --C(--D).dbd. and Y is --N.dbd., if B is hydrogen or halogen, D 
and G are hydrogen, then F is not amino; 
or when A is methylene, X is --C(--D).dbd., Y is --C(--E).dbd., B, D, E and 
G are hydrogen, then F is not amino. 
According to an alternative aspect of the present invention, novel 
adenosine kinase inhibitors are provided which have a 5'-group which 
comprises a hydroxyl or hydroxyl derivative. However, it is believed that 
due to their overall structures, those compounds which have a 5'-hydroxyl 
would not act as substrates for phosphorylation enzymes and, thus, would 
be unlikely to undergo 5'-phosphorylation or would be phosphorylated at an 
extremely slow rate. 
One preferred group of these adenosine kinase inhibitors comprise compounds 
of the formula: 
##STR3## 
wherein: 
(a) A is oxygen, methylene or sulfur; 
(b) B' is --(CH.sub.2).sub.n B wherein n is 1,2, 3 or 4 and B is hydroxy, 
acyloxy, hydrocarbyloxycarbonyloxy, or --OCONR.sub.2 wherein each R is 
independently hydrocarbyl; 
(c) C.sub.1 and C.sub.2 are each independently hydrogen, acyl, 
hydrocarbyloxycarbonyl or taken together form a 5-membered ring wherein 
C.sub.1 is a single bond to C.sub.2 and C.sub.2 is carbonyl or 
.alpha.-alkoxyalkylidene; 
(d) X is --C(--D).dbd. and Y is --N.dbd.; 
(e) D is halogen, aryl or aralkyl; 
(f) F is alkyl, aryl, aralkyl, halogen, amino, alkylamino, arylamino, 
aralkylamino, cyano, cyanoalkyl, alkoxy, aryloxy, aralkoxy, alkylthio, 
arylthio, aralkylthio, optionally substituted indolinyl or indolyl, 
pyrrolidinyl or piperazinyl; and 
(g) G is hydrogen, halogen, lower alkyl, lower alkoxy, or lower alkylthio; 
and pharmaceutically acceptable salts thereof; with the proviso that when 
A is oxygen and D is halogen, then F is not amino. 
Another preferred group of these adenosine kinase inhibitors comprise 
compounds of the formula: 
##STR4## 
wherein: 
(a) A is oxygen, methylene or sulfur; 
(b) B' is --(CH.sub.2).sub.n B wherein n is 1, 2, 3 or 4 and B is hydroxy, 
acyloxy, hydrocarbyloxycarbonyloxy, or --OCONR.sub.2 wherein each R is 
independently hydrocarbyl; 
(c) C.sub.1 and C.sub.2 are each independently hydrogen, acyl, 
hydrocarbyloxycarbonyl or taken together form a 5-membered ring wherein 
C.sub.1 is a single bond to C.sub.2 and C.sub.2 is carbonyl or 
.alpha.-alkoxyalkylidene; 
(d) X is --C(--D).dbd. and Y is --C(--E).dbd.; 
(e) D is aryl or aralkyl; 
(f) E is hydrogen, halogen, alkyl, or alkylthio; 
(g) F is alkyl, aryl, aralkyl, halogen, amino, alkylamino, arylamino, 
aralkylamino, cyano, cyanoalkyl, alkoxy, aryloxy, aralkyloxy, alkylthio, 
arylthio, aralkylthio, optionally substituted indolinyl or indolyl, 
pyrrolidinyl or piperazinyl; and 
(h) G is hydrogen, halogen, lower alkyl, bower alkoxy, or lower alkylthio; 
and pharmaceutically acceptable salts thereof; with the proviso that: when 
A is oxygen, D is oxadiazolyl, triazolyl or triazinyl, E and G are both 
hydrogen, then F is not amino. 
Also included within the present invention are adenosine kinase inhibitors 
which comprise modified purine nucleosides of the formula: 
##STR5## 
wherein 
(a) A is oxygen, methylene or sulfur; 
(b) B' is --CH.sub.2 B wherein and B is amino, alkylamino, or acylamino; 
(c) C.sub.1 and C.sub.2 are each independently hydrogen, acyl, 
hydrocarbyloxycarbonyl or taken together form a 5-membered ring wherein 
C.sub.1 is a single bond to C.sub.2 and C.sub.2 is carbonyl or 
.alpha.-alkoxyalkylidene; 
(d) X is -N.dbd. and Y is --C(--E).dbd.; 
(e) E is hydrogen, halogen, alkyl, amino, alkylamino, azido, acylamino, 
alkoxy or alkylthio; 
(f) F is halogen, amino, alkylamino, arylamino, aralkylamino, cyanoalkyl, 
alkoxy, aryloxy, aralkoxy, alkylthio, arylthio, aralkylthio, alkyl, aryl, 
aralkyl, optionally substituted indolinyl or indolyl, pyrrolidinyl or 
piperazinyl; and 
(g) G is hydrogen, halogen, lower alkyl, lower alkoxy, or lower alkylthio 
and pharmaceutical acceptable salts thereof; with the proviso that: 
when A is oxygen, B is amino or hydrocarbylamino, E and G are hydrogen, 
then F is not amino. 
According to a further aspect of the present invention, novel adenosine 
kinase inhibitors are provided that comprise dimeric compounds of the 
formula: 
##STR6## 
wherein 
(a) A and A' are independently oxygen, methylene or sulfur; 
(b) B' and B" are independently --(CH.sub.2).sub.n B wherein n is 
independently 1, 2, 3 or 4 and B is independently hydrogen, hydroxy, 
alkyl, alkoxy, amino, alkylamino, acylamino, hydrocarbyloxycarbonylamino, 
mercapto, alkylthio, azido, or either or both of B' or B" is independently 
alkenyl or alkynyl; 
(c) C.sub.1 and C.sub.1' and C.sub.2 and C.sub.2' are each independently 
hydrogen, acyl, hydrocarbyloxycarbonyl, or C.sub.1 and C.sub.2 or C.sub.1' 
and C.sub.2 ' taken together form a 5-membered ring wherein C.sub.1 or 
C.sub.1' is a single bond to C.sub.2 or C.sub.2' and C.sub.2 or C.sub.2' 
is carbonyl or .alpha.-alkoxyalkylidene; 
(d) X and X' are each independently --C(--D).dbd. or --N.dbd.; and Y and Y' 
are each independently --N.dbd. or --C(--E).dbd., provided that either of 
X and Y or X' and Y' are not both --N.dbd.; 
(e) D is independently hydrogen, halogen, alkyl, aryl, aralkyl, alkenyl, 
alkynyl, haloalkyl, cyano, cyanoalkyl, acyl, carboxamido, a carboxylic 
acid or corresponding carboxylic acid ester group, alkoxy, aryloxy, 
aralkyloxy, alkylthio, arylthio, aralkylthio, amino, alkylamino, 
arylamino, aralkylamino acylamino or nitro; 
(f) E is independently hydrogen, halogen, alkyl, or alkylthio; 
(g) L is an optionally substituted piperazinyl divalent radical or 
--NH(ALKL)NH-- wherein ALKL is a divalent alkylene radical of 2 to 24 
carbon atoms; and 
(h) G and G' are each independently hydrogen, halogen, lower alkyl, lower 
alkoxy, or lower alkoxy; or pharmaceutically acceptable salts thereof; 
with the proviso that if B is OH, then X and X' are not both --N.dbd.. 
In general, preferred are compounds where G is hydrogen, halogen, alkyl or 
alkylthio. Especially preferred G groups include hydrogen. Preferred 
C.sub.1 and C.sub.2 groups include hydrogen and acetyl. 
Preferred E groups include hydrogen or halogen, especially preferred are 
compounds where E is hydrogen. 
Preferred are compounds where A is oxygen. 
Preferred are compounds where D is hydrogen, halogen, alkyl, aryl, aralkyl, 
alkenyl or alkynyl, cyano, cyanoalkyl, alkoxy, aryloxy, aralkoxy, 
alkylthio, arylthio, aralkylthio, amino, alkylamino, arylamino, 
aralkylamino, carboxamido, or hydrocarbyloxycarbonyl. Especially preferred 
D groups include hydrogen, halogen, alkyl, aryl, aralkyl, cyano, alkoxy, 
aryloxy, aralkoxy, alkenyl or alkynyl, more preferably hydrogen, halogen, 
aryl, cyano, alkoxy or aryloxy. A particularly preferred group of 
compounds include those wherein D is hydrogen, halogen or aryl. According 
to one preferred aspect, D is aryl such as heterocyclic aryl or monocyclic 
carbocyclic aryl, such as optionally substituted phenyl. 
Preferred compounds include those where B' is --(CH.sub.2).sub.n B, and n 
is 1 or 2, more preferably n is 1. B may preferably include hydrogen, 
halogen, alkyl, amino, alkylamino, alkoxy, mercapto, alkylthio, azido or 
cyano; more preferably B is hydrogen, halogen, lower alkyl, amino, lower 
alkylamino, azido or cyano. Particularly preferred B groups include 
hydrogen, amino or azido. Also preferred are compounds wherein B' is 
vinyl, ethynyl, or propargyl. 
Preferred F groups include halogen, amino, alkylamino, arylamino, 
aralkylamino, alkylthio, arylthio, alkyl, aryl or aralkyl, more preferably 
amino or arylamino. Especially, preferred F groups include optionally 
substituted anilino. 
A. Preferred Compounds 
The compounds of the present invention contain asymmetric carbon atoms and 
hence can exist as stereoisomers, both enantiomers and diastereomers. The 
individual preferred stereoisomers and mixtures thereof are considered to 
fall within the scope of the present invention. The compounds described by 
Formula I contain a 5-modified 1-.beta.-D-ribofuranosyl group and that 
isomer comprises a particularly preferred diastereomeric and enantiomeric 
form for compounds of the present invention. Aptly, the synthetic examples 
cited herein provide the most preferred isomer. It is evident that in 
addition to the sugar moiety, additional asymmetric carbons may be present 
in compounds of Formula I, being present in moieties B', C.sub.1 or 
C.sub.2 or the substituted heterocyclic purine, pyrrolo[2,3-d]pyrimidine 
or pyrazolo[3,4-d]pyrimidine ring. In this event, both of the resulting 
diastereomers are considered to fall within the scope of the present 
invention. 
It is noted that compounds of Formula I where B is hydroxy (i.e. a 
5'-hydroxyl moiety) are in many cases potent inhibitors of adenosine 
kinase. The use of compounds having Formula I wherein B' replaced by 
--CH.sub.2 OH, as adenosine kinase inhibitors are included in the scope of 
this invention. However, since some of these compounds may be 
phosphorylated in vivo and since the resulting 5'-phosphates may be toxic, 
mutagenic or teratogenic, 5'-hydroxy compounds which can serve as 
substrates for phosphorylation enzymes may not comprise preferred 
compounds for clinical or therapeutic use. An important aspect of the 
novel compounds of the present invention is that these preferred compounds 
are either non-phosphorylatable at the 5' position or are not substrates 
of enzymes that lead to phosphorylation. 
(i) Preferred Pyrazolo[3,4-d]pyrimidines 
Preferred adenosine kinase inhibitor compounds of the present invention 
include certain pyrazolo[3,4-d]pyrimidine compounds of Formulas I and II. 
Preferred pyrazolo[3,4-d]pyrimidine compounds of Formula I include those 
where G is hydrogen and A is oxygen. Preferred D groups include hydrogen, 
alkyl, aryl, aralkyl, cyano, alkoxy, aryloxy, aralkoxy, alkenyl or 
alkynyl, more preferably hydrogen, halogen, aryl, cyano, alkoxy or 
aryloxy, more particularly hydrogen, halogen or aryl. An especially 
preferred group of compounds includes those where D is aryl, especially 
heterocyclic aryl or monocyclic carbocyclic aryl, more preferably 
optionally substituted phenyl. Preferred B' groups include 
--(CH.sub.2).sub.n B wherein B is hydrogen, halogen, alkyl, amino, 
alkylamino, alkoxy, mercapto, alkylthio, azido or cyano; more preferably B 
is hydrogen, halogen, lower alkyl, amino, lower alkylamino, azido or 
cyano. Particularly preferred B groups include hydrogen, amino or azido. 
Preferably, n is 1 or 2, more preferably 1. Other preferred B' groups 
include vinyl and ethynyl. Preferred are compounds of Formula I wherein F 
is halogen, amino, alkylamino, arylamino, aralkylamino, alkylthio, 
arylthio, alkyl, aryl or aralkyl, more preferably amino or arylamino. 
Certain preferred compounds include F groups which comprise optionally 
substituted anilino. 
Examples of preferred pyrazolo[3,4-d]pyrimidine compounds include those 
noted as GP-1-515, GP-1-547, GP-1-560, GP-1-665, GP-1-666 GP-1-667, 
GP-1-695, GP-1-704, and GP-1-792. 
Preferred pyrazolo[3,4-d]pyrimidine compounds of Formula II include those 
where G is hydrogen and A is oxygen. Preferred D groups include aryl. 
Preferred aryl groups include heterocyclic carbocyclic aryl groups, 
especially optionally substituted phenyl. Preferred F groups include 
halogen, amino, alkylamino, arylamino, aralkylamino, alkylthio, arylthio, 
alkyl, aryl, or aralkyl, more preferably amino or arylamino. Certain 
preferred compounds of Formula II may include F groups which comprise 
optionally substituted anilino groups. 
(ii) Preferred Pyrrolo[2,3-d]pyrimidines 
Preferred adenosine kinase compounds of the present invention include 
pyrrolo[2,3-d]pyrimidine compounds of Formulas I and II. 
Preferred pyrrolo[2,3-d]pyrimidine compounds of Formula I include those 
wherein G is hydrogen. Preferred are compounds wherein E is hydrogen or 
halogen; more preferably E is hydrogen. Preferred are compounds where A is 
oxygen. Preferred compounds include those where D is hydrogen, halogen, 
alkyl, aryl, aralkyl, cyano, alkenyl or alkynyl, more preferably hydrogen, 
halogen or aryl. An especially preferred group of compounds includes those 
where D is aryl, especially heterocyclic aryl or monocyclic carbocyclic 
aryl, especially optionally substituted phenyl. Preferred B' groups 
include --(CH.sub.2).sub.n B wherein n is 1 or 2, preferably 1. 
Preferably, B is hydrogen, halogen, alkyl, amino, alkylamino, alkoxy, 
mercapto, alkylthio, azido or cyano, more preferably B is hydrogen, 
halogen, lower alkyl, amino, lower alkylamino, lower alkoxy, lower 
alkylthio, or azido, more particularly hydrogen, lower alkyl, amino, lower 
alkylamino, or azido. Especially preferred B groups include hydrogen, 
amino or azido. Other preferred B' groups include vinyl and ethynyl. 
Preferred pyrrolo[2,3-d]pyrimidine compounds of Formula I include those 
wherein F is halogen, amino, alkylamino, arylamino, aralkylamino, 
alkylthio, aralkylthio, alkyl, aryl or aralkyl, more preferably amino or 
arylamino. Certain preferred compounds include F groups which comprise 
optionally substituted anilino. Examples of preferred 
pyrrolo[2,3-d]pyrimidine compounds include those noted as GP-1-448, 
GP-1-606, GP-1-608, GP-1-639, GP-1-683, GP-1-684, GP-1-691, GP-1-711, 
GP-1-714, and GP-1-718. 
Preferred pyrrolo[2,3-d]pyrimidines of Formula II include those where G is 
hydrogen and A is oxygen. Preferably E is hydrogen or halogen, more 
preferably hydrogen. Preferred D groups include aryl. Preferred aryl 
groups include heterocyclic aryl groups and monocyclic carbocyclic aryl 
groups, especially optionally substituted phenyl. Preferred heterocyclic 
aryl groups include 2-furanyl, 2-thienyl and 3-thienyl. 
(iii) Preferred Purines 
Preferred purine compounds include those where G is hydrogen, halogen, 
lower alkyl or lower alkylthio, more preferably hydrogen. Preferred are 
compounds wherein A is oxygen. Preferred E groups include hydrogen, 
halogen or alkylthio. Preferred are compounds wherein B is amino. 
Preferred F groups include halogen, amino, alkylamino, arylamino, 
aralkylamino, alkylthio, arylthio, alkyl, aryl or aralkyl, more preferably 
amino or arylamino. 
(iv) Preferred Dimer Compounds 
Preferred dimeric compounds include those which comprise dimers of the 
above-described pyrazolo[3,4-d]pyrimidines, the pyrrolo[2,3-d]-pyrimidines 
and purines. These dimers may comprise monomeric units which are the same 
or different. 
SYNTHESIS OF PREFERRED COMPOUNDS 
A. General Synthetic Methods 
This invention is also directed to processes for preparing compounds of 
Formula I. Disclosed herein are general synthetic routes for preparing 
variously substituted purine nucleosides or pyrrolo[2,3-d]pyrimidine 
nucleosides, including a novel and improved synthesis of 
5'-deoxy-5-iodotubercidin; and pyrazolo[3,4-d]pyrimidine nucleosides of 
the present invention. 
A process for preparing 5'-azido, 5'-amino and 5'-deoxy analogs of 6- 
substituted-amino purine ribosides is depicted in FIG. 3. The protected 
azide (2a), prepared from 2',3'-O-isopropylideneinosine, is activated for 
nucleophilic attack at position six by chlorination with thionyl 
chloride/dimethylformamide. Other standard reagents may also be used to 
activate position six of compound (2) such as thionyl bromide, phosphorous 
oxychloride, triphenylphosphine dibromide-thiophenol-potassium 
permanganate or hexamethyldisilazane-ammonium sulfate. The chloride (3) or 
other activated intermediate (Br, RSO.sub.2, R.sub.3 SiO, etc.) is then 
reacted with ammonia or an appropriate amine such as aniline, piperazine 
or indoline in solvents such as water, alcohols, THF and the like. The 
resulting protected azide (4a) is deblocked using an aqueous acid such as 
50% formic acid, to provide the 6-substituted-amino 
5'-azido-5'-deoxyadenosine (5a). Reduction of the azide (5a) to the amine 
(6) is effected by catalytic hydrogenation with a catalyst such as 
platinum oxide, palladium on carbon and the like. For molecules containing 
other functional groups sensitive to hydrogenation, triphenylphosphine is 
used to selectively reduce the azide moiety to the amine. To prepare the 
N-acylamino (7a) and hydrocarbyloxycarbonylamino (7b) compounds, the azide 
(4a) is reduced to the amine and treated with an acyl anhydride or acyl 
chloride or alkyl chloroformate and deblocked to give (7a) or (7b) 
respectively. Analogous processes are used to prepare the 2- and 8- 
substituted analogs beginning with appropriately substituted 
intermediates. An alternative synthesis of 5'-amino and 
5'-hydrocarbylamino compounds comprises deblocking a 
2',3'-isopropylidene-5'-tosylate with aqueous acid and then reacting the 
deblocked tosylate with ammonia or a lower hydrocarbylamine. Further 
description of these procedures is set forth in the Examples. 
A similar process is used to prepare 5'-deoxy purine nucleosides. The 
appropriately substituted 5'-deoxy-2',3'-O-isopropylideneinosine (2b) is 
chlorinated or activated using other reagents described above, aminated to 
(4b) and subsequently deblocked to afford the 5'-deoxy nucleoside (5b). 
The overall process for preparing 5'-modified pyrrolo[2,3-d]pyrimidine 
riboside compounds of Formula I, is depicted in FIG. 6. A key step 
comprises the sodium salt glycosylation method (K. Ramasamy et al., 
Tetrahedron Letters, 1987, 28:5107) using the anion of a substituted 
4-chloropyrrolo[2,3-d]pyrimidine (18) and 
1-chloro-2,3-O-isopropylidene-5-O-tert-butyldimethylsilyl-.alpha.-D-ribofu 
ranoside (17). This method is also suitable for direct preparation of 
ribofuranosides wherein the 5-hydroxy group has been replaced with 
substituents such as hydrogen or azido or extended with additional carbons 
(FIG. 5). The azide sugars further provide for facile synthesis of 
5'-amino nucleosides by reductions of the azide function after 
ribosylation. An alternative to the sodium salt glycosylation method is a 
solid-liquid phase transfer reaction using the same substrates and 
potassium hydroxide in place of sodium hydride as described by Rosemeyer 
H., and Seela, F, Helvetica Chimica Acta, 1988, 71:1573. 
Preparation of the 5-substituted ribose analogs and homologs is outlined in 
FIGS. 4 and 5. The 5-substituted-5-deoxy ribose analogs (10) are prepared 
by tosylation of the protected ribose (8), displacement of the tosylate by 
appropriate nucleophiles and subsequent deblocking (Synder, J.; Serianni, 
A.; Carbohydrate Res., 1987, 163:169). The ribose homologs (FIG. 5) are 
prepared by oxidation of the protected ribose (8) to the aldehyde (11) 
(Moorman, A.; Borchaedt, R.; Nucleic Acid Chemistry-Part III, Townsend, 
L.; Tipson, R.; John Wiley & Sons, 1986). The aldehyde is homologated via 
the appropriate Wittig reagent to give the key intermediate protected 
vinyl sugar (12). The protected intermediate is deblocked to give the 
vinyl ribose homolog (16a) or reduced to (13) and then deblocked to give 
the saturated deoxy analog (16b). Alternatively, the vinylated 
intermediate (12) is hydroborated and oxidized affording the protected 
homologous ribose (14a) which is deblocked to the ribose homolog or 
converted to the azide (14b) via tosylation and displacement with azide. 
Deblocking of (14b) then affords the homologous azido ribose (16d). The 
protected 5-aldehyde (11) was also methylated to ultimately afford 
6-deoxy-D-allofuranose (16e). The various 5- substituted riboses are then 
converted to the corresponding 2,3-O-isopropylidine ketals (FIG. 6) which 
are chlorinated stereoselectively to 5-modified 
1-chloro-.alpha.-D-ribofuranosides (17) using carbon tetrachloride and 
hexamethylphosphorous triamide (Wilcox, C.; Otaski, R.; Tetrahedron Lett., 
1986, 27:1011). 
The preparation of various substituted 4-chloro-pyrrolo[2,3-d]pyrimidines 
is described in the Examples. The initial products from the ribosylation 
reactions, ribosyl protected 
5-substituted-4-chloropyrrolo[2,3-d]pyrimidine nucleosides and the 
corresponding deblocked compounds are versatile intermediates and comprise 
an aspect of the present invention. As examples, the 4-chloro substituent 
of (19) can be displaced by sulfur (such as thiourea or mercaptide anions) 
leading to thionated and hydrocarbylthio compounds. More importantly, 
displacement of the 4-chloro substituent by ammonia or amines leads to 
4-amino- and 4-arylaminopyrrolo[2,3-d]pyrimidine nucleosides. As further 
example, and an aspect of the present invention, an improved synthesis of 
the adenosine kinase inhibitor, 5'-deoxy-5-iodotubercidin is described. 
According to this novel method, coupling of the sodium salt of 
4-chloro-5-iodopyrrolo[2,3-d]pyrimidine with 
1-chloro-5-deoxy-2,3-isopropylidene-.alpha.-D-ribofuranoside (17, 
B'=CH.sub.3) in acetonitrile gives the protected 4-chloro compound. 
Amination of this product with ammonia, followed by deblocking affords 
5'-deoxy-5-iodotubercidin. 
Especially preferred intermediates are protected pyrrolo[2,3-d]pyrimidine 
nucleosides having a 4-chloro and a 5-iodo or bromo substituent. 
Another aspect of the present invention is directed to the use of 
arylboronic acids to prepare 4- and 5-arylated pyrrolo[2,3-d]pyrimidine 
bases and nucleosides from the corresponding 4- and 5-halogenated 
compounds. Thus, a halogenated nucleoside such as (19) or the 
corresponding base was heated with an arylboronic acid and a 
palladium-phosphine catalyst such as palladium 
tetrakis(triphenylphosphine) to prepare the analogous arylated compound by 
displacement of halogen. Various 4- and 5- arylated 
pyrrolo[2,3-d]pyrimidines also can be prepared using arylstannyl compounds 
in place of the arylboronic acids (Flynn, B.; Macolino, B.; Crisp, G. 
Nucleosides & Nucleotides, 1991, 10:763). Synthesis of 
5-arylpyrrolo[2,3-d]pyrimidines can also be effected by condensation of 
arylamino ketones and malononitrile to arylated pyrroles and subsequent 
ring closure to 5-arylpyrrolo[2,3-d]pyrimidines. (Taylor, E.; Hendess, R., 
J. Am. Chem. Soc., 1965, 87:1995). 
The various above-mentioned products of ribosylation reactions may be 
deblocked at appropriate points with aqueous acids such as 50% formic acid 
or trifiuoroacetic acid. Preparation of 5'-amino compounds consists of 
reducing an appropriate azide. The 5'-amides and urethanes are prepared 
analogously to those described previously for purine analogs. Further 
description of these procedures is set forth in the Examples. 
Still another aspect of this invention is the preparation of 5'-substituted 
pyrazolo[3,4-d]pyrimidine ribosides of Formula I as depicted in FIG. 7. 
Accordingly, a substituted pyrazolo[3,4-d]pyrimidine is ribosylated with 
an esterified 5-hydroxy, 5-azido or 5-deoxyribofuranoside in the presence 
of a Lewis acid such as boron trifluoride (Cottam, H., Petrie, C.; 
McKernan, P.; Goebel, R.; Dalley, N.; Davidson, R.; Robins, R.; Revankar, 
G.; J. Med. Chem., 1984, 27:1120). The 5-substituted sugar is prepared by 
esterification of the deblocked sugar (10a) to (10c) or (16a) to (16e) 
(See FIG. 6). Suitable esters include the acetate, benzoate, toluate, 
anisoate and the like. The substituted pyrazolo[3,4-d]pyrimidine base (22) 
may be prepared by a variety of procedures as illustrated in the Examples. 
Two general routes to the compounds of the present invention are described 
below. 
The first general route comprises coupling an esterified ribose (21), 
prepared from (10) or (16), with a 3-substituted 
pyrazolo[3,4-d]pyrimidin-4-one. After ribosylation the pyrimidone riboside 
(24a) may be activated by chlorination with thionyl 
chloride/dimethylformamide or other reagents previously described and then 
reacted with ammonia or an amine to provide a variety of substituted 
5'-modified N.sup.4 -substituted-amino-pyrazolo[3,4-d]pyrimidine 
nucleosides (24b). Examples of this aspect of the invention, 
3-iodopyrazolo[3,4-d]pyrimidone nucleosides, are prepared by nonaqueous 
diazotization-iodination of the 3-amino compounds using a nitrite ester 
such as isoamyl nitrite and methylene iodide. Previous attempts to 
diazotize the 3-aminopyrazolo[3,4-d]pyrimidones using aqueous nitrous acid 
gave only N-nitrosated pyrazolo[3,4-d]pyrimidin-3,4-diones (Cottam, H.; 
Petrie, C.; McKernan, P.; Goebel, R.; Dalley, N.; Davidson, R.; Robins, 
R.; Revankar, G.; , J. Med. Chem., 1984, 27:1119). Further modifications 
of (23) or (24) include reduction of the 5'-azido moiety to afford the 
5'-amino compounds or the 5'-amides and urethanes as described in FIG. 7. 
Ester prodrugs (C.sub.1 and C.sub.2) of various 5'-amino nucleosides are 
prepared by reduction of the 5'-azide esters (23) using previously 
described reagents. 
Various C-4 alkylated pyrazolo[3,4-d]pyrimidine nucleosides are prepared by 
reaction of the above mentioned suitably protected 
4-chloropyrazolo[3,4-d]pyrimidine nucleosides with carbanion nucleophiles. 
A specific catalyst for this alkylation reaction was found to be 
trimethylamine; these reactions either do not occur or proceed very slowly 
and in poor yield in the absence of trimethylamine. Suitable carbanions 
include those derived from diethyl malonate, ethyl cyanoacetate, 
malononitrile, nitromethane, cyanide salts and the like. This procedure is 
also used to prepare C-6 alkylated purine ribosides. The initial 
C-alkylated products were deblocked and optionally further modified by 
hydrolysis and decarboxylation to afford the desired products. 
An alternative process for synthesis of 5'-azido- and 
5'-amino-5'-deoxypyrazolo[3,4-d]pyrimidine ribosides is also described. 
Accordingly, a substituted allopurinol riboside (24a) is protected by 
conversion to the 2',3'-isopropylidene derivative, tosylated and reacted 
with sodium azide in DMSO or DMF to form the azide. Activation of position 
four by chlorination with thionyl chloride/dimethylformamide or other 
reagents as described, followed by displacement of the activating group by 
ammonia or an amine results in a protected 5'-azido-5'-deoxy riboside. The 
azide is deblocked to afford (24b, B=N.sub.3) and subsequently reduced to 
the 5'-amino riboside using the previously described procedures. 
The second general route for preparation of substituted 
pyrazolo[3,4-d]pyrimidine nucleosides comprises coupling the esterified 
ribose (21) with various substituted 4-amino or 
4-hydrocarbylaminopyrazolo[3,4-d]pyrimidines. The resulting products are 
then further modified or deblocked to afford the desired compounds. The 
utility of this procedure is demonstrated in the Examples, by the 
preparation of 3-phenyl-4-(phenylamino)pyrazolo[3,4-d]pyrimidine 
5'-modified ribosides from 
3-phenyl-4-(phenylamino)pyrazolo[3,4-d]pyrimidine and various 5'-modified 
sugars. In another aspect of the present invention, halogenated 
pyrazolo[3,4]pyrimidine ribosides can be arylated using arylboronic acids 
and palladium catalysts as described for the pyrrolo[2,3-d]pyrimidines. 
Alternatively, the base can be boronated and then coupled with an aryl 
halide. Further description of these procedures is set forth in the 
Examples. 
B. Preferred Methods of Synthesis 
According to another aspect of the present invention, certain preferred 
methods of preparing the adenosine kinase inhibiting compounds of Formula 
I are provided. 
One preferred method of the present invention is a novel procedure for 
preparing C-6 alkylated purine nucleosides and C-4 alkylated 
pyrazolo[3,4-d]pyrimidine nucleosides from the 6-chloropurine and 
4-chloropyrazolo[3,4-d]pyrimidine nucleosides, respectively, using various 
carbanions (enolates, cyanide anion, etc.) and trimethylamine as a 
specific catalyst. Previous methodology for C-alkylation of 
6-chloropurines consisted of a multistep route involving alkylthiolation 
and oxidation to a sulfone followed by nucleophilic displacement with a 
carbanion (Yame, A.; Matsuda, A.; Veda, T.; Chem. Pharm. Bull. (Jap.), 
1980, 28:150). This multistep route can be accomplished in one step using 
the specific catalyst trimethylamine which reacts to form a quaternary 
salt and in turn is displaced by a carbanion in situ, regenerating 
trimethylamine. The reactions are specifically catalyzed by unhindered 
trialkylamines. 
Another preferred method of the present invention is a process for 
preparing arylated bases and nucleosides by reaction of a halogenated 
pyrrolo[2,3-d]pyrimidine or pyrazolo[3,4-d]pyrimidine with an aryl boronic 
acid in the presence of a palladium-phosphine catalyst. In this process, 
the halogen atom of a brominated or preferably, iodinated 
pyrrolo[2,3-d]pyrimidine or pyrazolo[3,4-d]pyrimidine base or nucleoside, 
is replaced by an aryl moiety such as phenyl, substituted phenyl or a 
heteroaryl moiety such as furanyl. A catalyst consisting of a metal such 
as palladium, complexed to an arylphosphine such as triphenylphosphine 
must be present as well as a base such as sodium carbonate. The resulting 
arylated nucleosides are important examples of the present invention and 
this method is shorter and more versatile than alternative syntheses of 
arylated nucleosides. 
Still another preferred method of the present invention is a process for 
preparing the previously unknown 3-iodo- and 
3-chloropyrazolo[3,4-d]pyrimidine nucleoside by nonaqueous diazotization 
of 3-aminopyrazolo[3,4-d]pyrimidine nucleosides. According to this 
invention, a suitably substituted 3-aminopyrazolo[3,4-d]pyrimidine 
nucleoside is diazotized by heating with an alkyl nitrite such as isoamyl 
nitrite in the presence of an iodine source (such as methylene iodide) 
resulting in replacement of the 3-amino moiety with an iodine atom. 
Alternatively, methylene iodide can be replaced by a chlorine source such 
as carbon tetrachloride resulting in replacement of the amino moiety by a 
chlorine atom. A previously reported attempt to effect replacement of the 
amino moiety in a 3-aminopyrazolo[3,4-d]pyrimidine riboside with other 
moleties using nitrous acid resulted only in replacement of the amino 
moiety by a hydroxyl group. The resulting 3-chloro- and particularly 
3-iodopyrazolo[3,4-d]pyrimidine nucleoside are an important subject of 
adenosine kinase inhibitors disclosed in the present invention. 
C. Preferred Intermediates 
According to a further aspect of the present invention, certain novel 
intermediates are provided which are useful in the synthesis of the 
adenosine kinase inhibitors of the present invention. 
(i) Intermediates for Pyrrolo[2,3-d]pyrimidines 
Certain intermediates useful in the preparation of certain preferred 
adenosine kinase inhibitors which comprise substituted 
pyrrolo[2,3-d]pyrimidine nucleosides include compounds of the formula: 
##STR7## 
wherein B' is lower alkyl or 1 to 3 carbon atoms optionally substituted 
with azido or hydroxy, or lower alkenyl of 1 to 3 carbon atoms; D is bromo 
or iodo, E is hydrogen, F is chloro, mercapto, arylamino and G is 
hydrogen. Certain especially preferred intermediates are set forth in FIG. 
8. 
These preferred intermediates include the following compounds: 
5-Bromo-4-chloro-7-(5-deoxy-1-.beta.-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidi 
ne; 
4-Chloro-5-iodo-7-(5-deoxy-1-.beta.-D-ribofuranosyl)pyrrolo-[2,3-d]pyrimidi 
ne; 
5-Iodo-7-(5-deoxy- 
1-.beta.-D-ribofuranosyl)pyrrolo[2,3-d]-pyrimidin-4(3H)-thione; 
5-Bromo-4-chloro-7-(5,6-dideoxy-1-.beta.-D-allofuranosyl)pyrrolo[2,3-d]pyri 
midine; 
4-Chloro-5-iodo-7-(5,6-dideoxy-1-.beta.-D-ribofuranosyl)pyrrolo-[2,3-d]pyri 
midine; 
5-Bromo-4-chloro-7-(5,6-dideoxy-5,6-didehydro-1-.beta.-D-allofuranosyl)pyrr 
olo[2,3-d]pyrimidine; 
4-Chloro-5-iodo-7-(5,6-dideoxy-5,6-didehydro-1-.beta.-D-allofuranosyl)pyrro 
lo[2,3-d]pyrimidine; 
4-Chloro-5-iodo-7-(5-azido-5-deoxy-1-.beta.-D-ribofuranosyl)pyrrolo[2,3-d]p 
yrimidine; 
5-Bromo-4-chloro-7-(5-azido-5-deoxy-1-.beta.-D-ribofuranosyl)pyrrolo[2,3-d] 
pyrimidine; 
4-Chloro-5-iodo-7-(6-azido-5,6-dideoxy-1-.beta.-D-allofuranosyl)pyrrolo[2,3 
-d]pyrimidine; 
5-Bromo-4-arylamino-7-(5-deoxy-1-.beta.-D-ribofuranosyl)pyrrolo[2,3-d]pyrim 
idine; 
5-Iodo-4-arylamino-7-(5-deoxy-1-.beta.-D-ribofuranosyl)pyrrolo[2,3-d]pyrimi 
dine; 
5-Iodo-4-arylamino-7-(1-.beta.-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine; 
and 
5-Bromo-4-arylamino-7-(1-.beta.-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine. 
In addition to being useful in the preparation of certain preferred 
adenosine kinase inhibitors, certain of these preferred intermediates 
exhibit activity as adenosine kinase inhibitors themselves. 
(ii) Intermediates for Pyrazolo[3,4-d]pyrimidines 
Certain intermediates useful in the preparation of certain preferred 
adenosine kinase inhibitor compounds comprise substituted 
pyrazolo[3,4-d]pyrimidines of the formula: 
##STR8## 
wherein Ar is an aryl group and F is halogen, preferably chloro. Preferred 
aryl groups include heterocyclic aryl groups and monocyclic carbocyclic 
aryl groups, including optionally substituted phenyl groups. These 
preferred intermediates include the following compounds: 
4-chloro-3-phenylpyrazolo[3,4-d]pyrimidine; 
4-chloro-3- (2-thienyl)pyrazolo[3,4-d]pyrimidine; 
4-chloro-3-(4-methoxyphenyl)pyrazolo[3,4-d]pyrimidine; and 
4-chloro-3-(4-chlorophenyl)pyrazolo[3,4-d]pyrimidine. 
UTILITY 
The adenosine kinase inhibitors of the present invention may be used in the 
treatment of a variety of clinical situations where increasing local 
levels of adenosine are beneficial. 
The compounds described herein and other adenosine kinase inhibitors are 
useful in treating conditions in which inflammatory processes are 
prevalent such as sepsis, arthritis, osteoarthritis, autoimmune disease, 
adult respiratory distress syndrome (ARDS), inflammatory bowel disease, 
necrotizing enterocolitis, chronic obstructive pulmonary disease (COPD), 
psoriasis, conjunctivitis, iridocyditis, myositis, cerebritis, meningitis, 
dermitis, renal inflammation, ischemia, reperfusion injury, peripheral 
vascular disease, atherosclerosis and other inflammatory disorders. 
Sepsis, septicemia and septic shock, which involve an inflammatory 
response to a variety of injuries such as burns, pancreatinitis and 
infection, for example, by gram negative or gram positive bacteria, may be 
treated with an adenosine kinase inhibitor, such as the adenosine kinase 
inhibitors described herein. 
To assist in understanding the present inventions and especially their 
properties and utilities, the results of a series of experiments are also 
included. These experiments demonstrated that a number of compounds 
described herein were potent inhibitors of a purified cardiac adenosine 
kinase with IC.sub.50 s of less than 1 .mu.M. Moreover, we have shown that 
these compounds are specific inhibitors of adenosine kinase with low 
affinity at the A1 adenosine receptor and no significant adenosine 
deaminase (ADA) inhibition (Example A). We have demonstrated that a number 
of these compounds are also inhibitors of adenosine kinase in intact cells 
(Example B). These compounds include pyrrolo[2,3-d]pyrimidine nucleosides 
modified at the 5'-position or at other positions such that it is less 
likely to serve as a substrate for phosphorylation enzymes and that, in 
contrast to 5-iodotubercidin (GP-1-202), these compounds are unlikely to 
be phosphorylated at the 5'-position, incorporated into nucleotides or 
DNA, which may cause toxicity to cells or animals. We have demonstrated 
that inhibition of the cardiac adenosine kinase was achieved in vivo 
following systemic administration or, in some cases, oral administration 
of these compounds. 
Selected compounds, such as GP-1-238, were also evaluated to determine the 
potential for toxic hemodynamic effects or hypothermia associated with 
administration of adenosine kinase inhibitors. No effects were observed in 
conscious animals on blood pressure, heart rate or temperature with doses 
of inhibitor greatly in excess of that required to inhibit the cardiac 
adenosine kinase (Example C). 
In other experimental models, the ability of selected adenosine kinase 
inhibitors (GP-1-272 and GP-1-456) to inhibit neutrophil adherence to 
endothelial cells, an inflammatory response mediated at the cellular level 
was evaluated (Example D). Certain adenosine kinase inhibitors were found 
to exhibit anti-inflammatory activity in animal models of inflammation. 
The ability of particular adenosine kinase inhibitors to improve survival 
in a mouse model of endotoxic shock, both when administered immediately 
after E. Coli LPS injection (Example E) and when administered 
prophylactically (Example F), supports the ability of adenosine kinase 
inhibitors to prevent and treat septic conditions, including endotoxemia 
and endotoxic shock. The efficacy of adenosine kinase inhibitors in 
treatment of sepsis is further demonstrated by the ability of particular 
adenosine kinase inhibitors to improve survival in another model of septic 
shock (Example G). 
The experiments described in Example H show that endotoxic mice treated 
with an adenosine kinase inhibitor have lower blood levels of tumor 
necrosis factor alpha (TNF-.alpha.) compared with placebo treated mice. 
Cytokines such as TNF-.alpha. have been suggested to be involved in many 
conditions including sepsis and septic shock (Zentella et al., Progress 
Clin. Biol. Research 367:9 (1990); Mathison et al., J. Clin. Invest. 
81:1925 (1988); Zanetti et al., J. Immunol. 148:1890 (1992); Creasey et 
al., Circ. Shock 33:82 (1991); Michie et al., N. Engl. J. Med. 318:1481 
(1988); Waage et al., Lancet 1(8529):355 (1987); Damas et al., Crit. Care 
Med. 17:975 (1989); Girardin et al., N. Engl. J. Med. 319:297 (1988)), 
other severe infectious diseases (Wakabayashi et al., J. Clin. Invest. 
87:1925 (1991)), adult respiratory distress syndrome (Miltar et al., 
Lancet 2(8665):712 (1989); Rinaldo et al., Clin. Chest Med. 11:621 (1990); 
Ferrai-Baliviera et al., Arch. Surg. 124(12):1400 (1989)), acquired immune 
deficiency syndrome (Folks et al., PNAS 86:2336 (1989)), reperfusion 
injury (Vedder et al., PNAS 87:2643 (1990)), and bone resorption diseases 
(Bertolini et al., Nature 319:516 (1986); Johnson et al., Endocrinology 
124(3):1424 (1989)), among others. Although definite correlation between 
such conditions and TNF-.alpha. has not been established (Eskandari et 
al., J. Immunol. 148:2724 (1992), the results of Example H may indicate a 
broader therapeutic role for adenosine kinase inhibitors, including the 
novel compounds disclosed herein. 
Additionally Example J describes the efficacy of GP-1-515 in the treatment 
of endotoxic shock in pigs, as the treated animals did not develop the 
hypoxemia, hypercapnia and acidosis exhibited in the control animals. 
An integral part of the inflammatory response involves an increase of 
vascular permeability to plasma proteins, herein termed "plasma leakage" 
or "vascular leakage". Such leakage occurs when there is a change of the 
barrier properties of the vasculature in a tissue, and may be due to 
contraction of activated endothelial cells from each other leading to 
formation of a pore or to partial destruction of the vessel by cells 
participating in an aggressive immune response. The suppression of 
vascular leakage by adenosine kinase inhibition is described in Example K. 
Therefore methods of the present invention may be useful in the treatment 
of conditions in which vascular leakage is present such as in inhalation 
injury, the treatment of burns both locally at the injury site and in 
other organs such as lung (ARDS) and gut, or other edema induced by 
sepsis, burns or trauma. 
Additional support for the use of adenosine kinase inhibitors in burn 
treatment is presented in Example L. Bacterial infection is a common 
occurrence during burn recovery. In a burn model, the use of GP-1-515 was 
shown to significantly reduce bacterial translocation after a severe burn 
or pancreatitis. 
FORMULATIONS 
Compounds of the invention are administered to the affected tissue at the 
rate of from 0.01 to 200 nmole/min/kg, preferably from 1 to 20 
nmol/min/kg. Such rates are easily maintained when these compounds are 
intravenously administered as discussed below. When other methods are used 
(e.g., oral administration), use of time-release preparations to control 
the rate of release of the active ingredient may be preferred. These 
compounds are administered in a dose of about 1 mg/kg/day to about 20 
mg/kg/day, preferably from about 3 mg/kg/day to about 8 mg/kg/day and most 
preferably about 5 mg/kg/day. 
For the purposes of this invention, the compounds of the invention may be 
administered by a variety of means including orally, parenterally, by 
inhalation spray, sublingually, topically, or rectally in formulations 
containing conventional non-toxic pharmaceutically acceptable carriers, 
adjuvants and vehicles. The term parenteral as used herein includes 
subcutaneous, intravenous, intramuscular, and intraarterial injections 
with a variety of infusion techniques. Intraarterial and intravenous 
injection as used herein includes administration through catheters. 
Preferred for certain indications are methods of administration which 
allow rapid access to the tissue or organ being treated, such as 
intravenous injections for the treatment of myocardial infarction. When an 
organ outside a body is being treated, perfusion is preferred. 
Pharmaceutical compositions containing the active ingredient may be in any 
form suitable for the intended method of administration. When used for 
oral use for example, tablets, troches, lozenges, aqueous or oil 
suspensions, dispersible powders or granules, emulsions, hard or soft 
capsules, syrups or elixirs may be prepared. Compositions intended for 
oral use may be prepared according to any method known to the art for the 
manufacture of pharmaceutical compositions and such compositions may 
contain one or more agents including those from the group consisting of 
sweetening agents, flavoring agents, coloring agents and preserving 
agents, in order to provide a palatable preparation. Tablets containing 
the active ingredient in admixture with non-toxic pharmaceutically 
acceptable excipient which are suitable for manufacture of tablets are 
acceptable. These excipients may be, for example, inert diluents, such as 
calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium 
phosphate; granulating and disintegrating agents, such as maize starch, or 
alginic acid; binding agents, such as starch, gelatin or acacia; and 
lubricating agents, such as magnesium stearate, stearic acid or talc. 
Tablets may be uncoated or may be coated by known techniques including 
microencapsulation to delay disintegration and adsorption in the 
gastrointestinal tract and thereby provide a sustained action over a 
longer period. For example, a time delay material such as glyceryl 
monostearate or glyceryl distearate alone or with a wax may be employed. 
Formulations for oral use may be also presented as hard gelatin capsules 
wherein the active ingredient is mixed with an inert solid diluent, for 
example calcium phosphate or kaolin, or as soft gelatin capsules wherein 
the active ingredient is mixed with water or an oil medium, such as peanut 
oil, liquid paraffin or olive oil. 
Aqueous suspensions of the invention contain the active materials in 
admixture with excipients suitable for the manufacture of aqueous 
suspensions. Such excipients include a suspending agent, such as sodium 
carboxymethylcellulose, methylcellulose, hydroxypropylmethyl celluose, 
sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and 
dispersing or wetting agents such as a naturally occurring phosphatide 
(e.g., lecithin), a condensation product of an alkylene oxide with a fatty 
acid (e.g., polyoxyethylene stearate), a condensation product of ethylene 
oxide with a long chain aliphatic alcohol (e.g., 
heptadeaethyleneoxycetanol), a condensation product of ethylene oxide with 
a partial ester derived from a fatty acid and a hexitol anhydride (e.g., 
polyoxyethylene sorbitan mono-oleate). The aqueous suspension may also 
contain one or more preservative such as ethyl of n-propyl 
p-hydroxybenzoate, one or more coloring agent, one or more flavoring agent 
and one or more sweetening agent, such as sucrose or saccharin. 
Oil suspensions may be formulated by suspending the active ingredient in a 
vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, 
or in a mineral oil such as liquid paraffin. The oral suspensions may 
contain a thickening agent, such as beeswax, hard paraffin or cetyl 
alcohol. Sweetening agents, such as those set forth above, and flavoring 
agents may be added to provide a palpable oral preparation. These 
compositions may be preserved by the addition of an antioxidant such as 
ascorbic acid. 
Dispersible powders and granules of the invention suitable for preparation 
of an aqueous suspension by the addition of water provide the active 
ingredient in admixture with a dispersing or wetting agent, a suspending 
agent, and one or more preservatives. Suitable dispersing or wetting 
agents and suspending agents are exemplified by those disclosed above. 
Additional excipients, for example sweetening, flavoring and coloring 
agents, may also be present. 
The pharmaceutical compositions of the invention may also be in the form of 
oil-in-water emulsions. The oily phase may be a vegetable oil, such as 
olive oil or arachis oil, a mineral oil, such as liquid paraffin, or a 
mixture of these. Suitable emulsifying agents include naturally-occurring 
gums, such as gum acacia and gum tragacanth, naturally occurring 
phosphatides, such as soybean lecithin, esters or partial esters derived 
from fatty acids and hexitol anhydrides, such as sorbitan mono-oleate, and 
condensation products of these partial esters with ethylene oxide, such as 
polyoxyethylene sorbitan mono-oleate. The emulsion may also contain 
sweetening and flavoring agents. 
Syrups and elixirs may be formulated with sweetening agents, such as 
glycerol, sorbitol or sucrose. Such formulations may also contain a 
demulcent, a preservative, a flavoring or a coloring agent. 
The pharmaceutical compositions of the invention may be in the form of a 
sterile injectable preparation, such as a sterile injectable aqueous or 
oleaginous suspension. This suspension may be formulated according to the 
known art using those suitable dispersing or wetting agents and suspending 
agents which have been mentioned above. The sterile injectable preparation 
may also be a sterile injectable solution or suspension in a non-toxic 
parenterally-acceptable diluent or solvent, such as a solution in 
1,3-butanediol or prepared as a lyophilized powder. Among the acceptable 
vehicles and solvents that may be employed are water, Ringer's solution 
and isotonic sodium chloride solution. In addition, sterile fixed oils may 
conventionally be employed as a solvent or suspending medium. For this 
purpose any bland fixed oil may be employed including synthetic mono- or 
diglycerides. In addition, fatty acids such as oleic acid may likewise be 
used in the preparation of injectables. 
The amount of active ingredient that may be combined with the carrier 
material to produce a single dosage form will vary depending upon the host 
treated and the particular mode of administration. For example, a 
time-release formulation intended for oral administration to humans may 
contain 20 to 200 .mu.moles of active material compounded with an 
appropriate and convenient amount of carrier material which may vary from 
about 5 to about 95% of the total compositions. It is preferred that 
pharmaceutical composition be prepared which provides easily measurable 
amounts for administration. For example, an aqueous solution intended for 
intravenous infusion should contain from about 20 to about 50 .mu.moles of 
the active ingredient per milliliter of solution in order that infusion of 
a suitable volume at a rate of about 30 mL/hr can occur. 
As noted above, formations of the present invention suitable for oral 
administration may be presented as discrete units such as capsules, 
cachets or tablets each containing a predetermined amount of the active 
ingredient; as a powder or granules; as a solution or a suspension in an 
aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a 
water-in-oil liquid emulsion. The active ingredient may also be 
administered as a bolus, electuary or paste. 
A tablet may be made by compression or molding, optionally with one or more 
accessory ingredients. Compressed tablets may be prepared by compressing 
in a suitable machine the active ingredient in a free-flowing form such as 
a powder or granules, optionally mixed with a binder (e.g., povidone, 
gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, 
preservative, disintegrant (e.g., sodium starch glycolate, cross-linked 
povidone, cross-linked sodium carboxymethyl cellulose) surface-active or 
dispersing agent. Molded tablets may be made by molding in a suitable 
machine a mixture of the powdered compound moistened with an inert liquid 
diluent. The tablets may optionally be coated or scored and may be 
formulated so as to provide slow or controlled release of the active 
ingredient therein using, for example, hydroxypropylmethyl cellulose in 
varying proportions to provide the desired release profile. Tablets may 
optionally be provided with an enteric coating, to provide release in 
parts of the gut other than the stomach. This is particularly advantageous 
with the compounds of formula (I) as such compounds are susceptible to 
acid hydrolysis. 
Formulations suitable for topical administration in the mouth include 
lozenges comprising the active ingredient in a flavored basis, usually 
sucrose and acacia or tragacanth; pastilles comprising the active 
ingredient in an inert basis such as gelatin and glycerin, or sucrose and 
acacia; and mouthwashes comprising the active ingredient in a suitable 
liquid carrier. 
Formulations for rectal administration may be presented as a suppository 
with a suitable base comprising for example cocoa butter or a salicylate. 
Formulations suitable for vaginal administration may be presented as 
pessaries, tampohs, creams, gels, pastes, foams or spray formulations 
containing in addition to the ddPN ingredient such carriers as are known 
in the art to be appropriate. 
Formations suitable for parenteral administration include aqueous and 
non-aqueous isotonic sterile injection solutions which may contain 
anti-oxidants, buffers, bacteriostats and solutes which render the 
formation isotonic with the blood of the intended recipient; and aqueous 
and non-aqueous sterile suspensions which may include suspending agents 
and thickening agents. The formulations may be presented in unit-dose or 
multi-dose sealed containers, for example, ampoules and vials, and may be 
stored in a freeze-dried (lyophilized) condition requiring only the 
addition of the sterile liquid carrier, for example water for injections, 
immediately prior to use. Extemporaneous injection solutions and 
suspensions may be prepared from sterile powders, granules and tablets of 
the kind previously described. 
Preferred unit dosage formulations are those containing a daily dose or 
unit, daily sub-dose, or an appropriate fraction thereof, of an adenosine 
kinase inhibitor compound. 
It will be understood, however, that the specific dose level for any 
particular patient will depend on a variety of factors including the 
activity of the specific compound employed; the age, body weight, general 
health, sex and diet of the individual being treated; the time and route 
of administration; the rate of excretion; other drugs which have 
previously been administered; and the severity of the particular disease 
undergoing therapy, as is well understood by those skilled in the art. 
Examples of use of the method of the invention includes the following. It 
will be understood that these examples are exemplary and that the method 
of the invention is not limited solely to these examples. 
The method may be used following thrombolysis for coronary occlusion. The 
compound would be given as a sterile injectable preparation with water or 
isotonic sodium chloride as the solvent. The solution can be administered 
intravenously or directly into the coronary artery at the time of left 
heart catheterization or into a carotid artery. The rate of administration 
could vary from 1 to 20 nmole/min/kg with, for example, an infusion volume 
of 30 mL/hr. Duration of therapy would typically be about 96 hours. 
Capsules comprising adenosine kinase inhibitors suitable for oral 
administration according to the methods of the present invention may be 
prepared as follows: (1) for a 10,000 capsule preparation: 1500 g of 
adenosine kinase inhibitor is blended with other ingredients (as described 
above) and filled into capsules which are suitable for administration 
depending on dose, from about 4 capsules per day (1 per 6 hours) to about 
8 capsules per day (2 capsules per 6 hours), to an adult human. 
The compounds of this invention and their preparation can be understood 
further by the examples which illustrate some of the processes by which 
these compounds are prepared. These examples should not however be 
construed as specifically limiting the invention and variations of the 
invention, now known or later developed, are considered to fall within the 
scope of the present invention as hereinafter claimed. 
EXAMPLES 
EXAMPLE 1 
Preparation of 5'-Azido-5'-deoxy-2', 3'-O-(1-methylethylidene) inosine 
This material was prepared by tosylation of 2', 
3'-(1-methylethylidene)inosine and subsequent reaction with sodium azide 
in DMSO as described by Hampton, A.; J. Org. Chem., 1968, 11:1220. 
EXAMPLE 2 
Preparation of 
9-[5-Azido-5-deoxy-2,3-O-(1-methylethylidene)-1-.beta.-D-ribofuranosyl]-6- 
chloropurine 
A solution of the azide (Example 1) (12.01 g) in dry CH.sub.2 Cl.sub.2 (500 
mL) was added, during 1 hr, to a warm solution of SOCl.sub.2 (8.1 mL) and 
DMF (4.05 mL,) in CH.sub.2 Cl.sub.2 (50 mL). The resulting solution was 
refluxed for 6 hrs and cooled and added to a cold aqueous solution of 
KHCO.sub.3. The layers were separated and the organic layer was washed 
with cold aqueous K.sub.2 CO.sub.3, water(2.times.), dried and 
concentrated. The residue was redissolved in CH.sub.2 Cl.sub.2 and 
filtered through silica. Evaporation under vacuum gave 11.3 g of the title 
compound as a pale yellow oil. 
EXAMPLE 3 
General Procedure for the Preparation of N.sup.6 
-Substituted-5'-azido-5'-deoxy-2',3'-O-(1-methylethylidene)adenosines 
To a solution of chloride (Example 2) (1 mmole/5 mL) in EtCH or n-BuOH was 
added amine (1.3 equivalents) and Et.sub.3 N (1.8 equivalents). The 
solution was heated to reflux for 12-24 hours. The mixture was evaporated 
under vacuum, dissolved in methylene chloride and washed. The organic 
solution was dried, concentrated and used directly in the next step or 
chromatographed on silica gel. 
EXAMPLE 4 TO 14 
General Procedure for the Preparation of N.sup.6 
-Substituted-5'-azido-5'-deoxyadenosines 
The N.sup.6 -substituted isopropylidene azide (Example 3) (1.0 g) was 
dissolved in formic acid (10-20 mL) and diluted with an equal volume of 
water. After 12-48 hr, the mixture was evaporated under vacuum, 
coevaporated with water (3.times.) then EtOH (2.times.). The residue was 
crystallized from water, alcohol or mixtures. The compounds in Table I 
(Examples 4-14) were prepared by this procedure: 
TABLE I 
______________________________________ 
GP-1-# 
Example F m.p. (.degree.C.) 
______________________________________ 
266 4 .O slashed.NH 121-126.degree. 
-- 5 CH.sub.3 NH 180.degree. (d) 
317 6 4-Cl.O slashed.NH 
209-210.degree. 
299 7 .O slashed.CH.sub.2 CH.sub.2 NH 
144-146.degree. 
337 8 N-indolinyl 157-159.degree. 
346 9 4-(HOCH.sub.2 CH.sub.2).O slashed.NH 
147-151.degree. 
-- 10 --NH(CH.sub.2).sub.12 NH-(dimer) 
foam 
385 11 N-indolyl.sup.1 186-188.degree. 
391 12 N-(5-bromoindolinyl) 
94-196.degree. 
421 13 N-(5-methoxyindolinyl) 
184-185.degree. 
557 14 1,4-piperazinyl 198-204.degree. 
______________________________________ 
.sup.1 Prepared from indole and sodium hydride in DMF. 
EXAMPLES 15-20 
General Procedure for the Preparation of N.sup.6 
-Substituted-5'-amino-5'-deoxyadenosines and Hydrochloride Salts 
A solution of the azide in EtOH or MeOH containing 10% Pd-C (25-50% weight 
of azide) was hydrogenated at 25 psi for 4-8 hours. The mixture was 
filtered, the catalyst rinsed with solvent and the filtrate evaporated. 
The residue was recrystallized to give the free base or converted to the 
salt. The hydrochloride salt was prepared by adding the base to ETCH, 
adding dry ethanolic HCl, warming and then chilling to crystallize out the 
salt (in some cases Et.sub.2 O was added). The compounds below were 
prepared by this procedure: 
TABLE II 
______________________________________ 
GP-1-# Example F m.p. .degree.C. (salt) 
______________________________________ 
272 15 CH.sub.3 NH 170-2.degree. (HCO.sub.2 H) 
286 16 .O slashed.NH 169-173.degree. (HCl) 
328 17 .O slashed.CH.sub.2 CH.sub.2 NH 
130.degree. (d) (HCl) 
345 18 N-indolinyl 202-203.degree. (HCl) 
373 19 --HN(CH.sub.2).sub.12 NH-(dimer) 
151-153.degree. (HCl) 
565 20 1,4-piperazinyl 140-145.degree. (HCl) 
______________________________________ 
EXAMPLE 21 
Preparation of 5'-deoxy-2',3'-O-(1-methylethylidene)inosine 
A solution of 5'-deoxy-5'-iodo-2',3'-O-(1-methylethylidene)inosine (5.45 g) 
in 80 mL of methanol containing triethylamine (2 g) and 10% palladium on 
charcoal (737 mg) was hydrogenated for 2 hr under 50 psi H.sub.2. The 
reaction mixture was filtered, concentrated and allowed to crystallize. 
The product was dried under vacuum to give 2.45 g (82% yield) of the title 
compound. 
EXAMPLE 22 
Preparation of 
6-Chloro-9-[5-deoxy-2,3-O-(1-methylethylidene)-1-.beta.-D-ribofuranosyl]pu 
rine 
A solution of the blocked 5'-deoxyinosine (1.4 g, 4.8 mmol), 
tetraethylammonium chloride (1.9 g, 11.5 mmol), diethylaniline (1.2 mL, 
7.2 mmol) and phosphorous oxychloride (3.35 mL, 36 mmol) in CH.sub.3 CN 
(24 mL) was refluxed for 10 minutes then evaporated. The residue was 
dissolved in CH.sub.2 Cl.sub.2, washed with water, KHCO.sub.3 solution, 
water and dried. The solution was filtered and evaporated to give 860 mg 
(65% yield) of title compound as a yellow oil. 
EXAMPLE 23 
General Procedure for Preparation of N.sup.6 substituted 5'-deoxyadenosines 
The above identified compounds were prepared using the procedures described 
in Example 3 and Examples 4-14. 
The compound listed in Table III were prepared by this procedure. 
TABLE III 
______________________________________ 
GP-1-# EXAMPLE F m.p. (.degree.C.) 
______________________________________ 
595 23 1,4-piperazinyl 
220-225.degree. 
______________________________________ 
EXAMPLE 24 
Preparation of 
8-Bromo-2',3'-O-(1-methylidene)-5'-O-(4-methylbenzenesulfonyl)adenosine 
The above-identified compound may be prepared as described: Ikshara, M.; 
Kaneko, M.; Sagi, M.; Tetrahedron, 1970, 26:5757. 
EXAMPLE 25 
Preparation of N.sup.6 
-Formyl-8-bromo-2',3'-O-(1-methylethylidene)-5'-O-(4-methylbenzenesulfonyl 
)adenosine 
To acetic-formic anhydride (prepared by stirring 25 mL of acetic anhydride 
and 12.5 mL of formic acid for 15 minutes at 45.degree. C.) at 0.degree. 
C., was added the tosylate (Example 24) (4.0 g, 7.30 mmol). The solution 
was allowed to warm to 22.degree. C. and stirred for 48 hours. The 
reaction mixture was evaporated, chased 2.times. with toluene and the 
residue dissolved in CHCl.sub.3. Filtration and evaporation of the 
filtrate and crystallization of the residue from ethanol gave 4.0 g (96%) 
of the title compound. 
EXAMPLE 26 
Preparation of 5'-Deoxy-5',8-diazido-2',3'-O-(1-methylethylidene)adenosine 
To a 75.degree. C. slurry of NaN.sub.3 (2.23 g, 34.3 mmol) in 
dimethylsulfoxide (35 mL) was added the formyl tosylate (Example 25) (4.00 
g, 6.9 mmoles). The reaction temperature was held at 75.degree. C. for one 
hour then cooled to 25.degree. C. The mixture was poured into stirring 
H.sub.2 O (90 mL) and slurried for ten minutes. The solid was collected by 
filtration, rinsed with H.sub.2 O (3.times.), cold ethanol, and then 
dried. The crude product was dissolved in CHCl.sub.3, filtered through 
silica gel and the filtrate evaporated to give the N.sup.6 -formyl 
derivative 1.20 g; m.p. 107.degree.-110.degree. C. 
The N.sup.6 -formyl derivative was deformylated by slurrying in MeOH, 
adding saturated methanolic ammonia (80 mL) and warming until homogenous. 
After 15 minutes the solution was evaporated, the residue recrystallized 
from EtOH and dried to give the title compound; 0.900 g (60% yield); m.p. 
166.degree.-168.degree. C. 
EXAMPLE 27 
Preparation of 5'-Deoxy-5',8-diazidoadenosine 
The isopropylidene diazide of Example 26 (1.00 g, 3.15 mmol) was deblocked 
as described under Example 4 and recrystallized from H.sub.2 O; 760 mg 
(85% yield); m.p. 128.degree.-130.degree.. 
EXAMPLE 28 
Preparation of 5'-Deoxy-5'-8-diaminoadenosine Formate 
The diazide of Example 27 (0.660 g, 2.0 mmol) was hydrogenated as described 
under Example 15 and recrystallized from EtOH to give, after drying, 400 
mg of the free base (61% yield). This material was further purified by 
conversion to the formate salt (HCO.sub.2 H/EtOH/Et.sub.2 O); m.p. 
98.degree. C.(d). 
EXAMPLE 29 
Preparation of 5'-Deoxy-5'-formylaminoadenosine 
To cold (5.degree. C.) acetic-formic anhydride (10 mL acetic anhydride and 
5 mL formic acid) was added 
5'-amino-5'-deoxy-2',3'-O-(1-methylethylidene)adenosine (670 mg, 2.0 
mmol). The solution was stirred for 24 hours then evaporated, and 
coevaporated with toluene (2.times.) then EtOH. The residual foam was 
dissolved in methanolic ammonia containing CH.sub.2 Cl.sub.2 and stirred 
overnight. TLC indicated the initial product was converted to a more polar 
product. The solution was evaporated, the residue dissolved in CHCl.sub.3 
with 3% MeOH and filtered through silica. Evaporation of the filtrate gave 
520 mg of a white foam. This material (500 mg) was deblocked with 
HCO.sub.2 H as described for Example 4 and recrystallized from 
EtOH-H.sub.2 O to give the title compound: yield 0.35 g (55%); m.p. 
212.degree.-213.degree. C. 
EXAMPLE 30 
Preparation of 
6-(N-Indolinyl)-9-[2,3-O-(1-methylethylidene)-1-.beta.-D-ribofuranosyl)]pu 
rine 
A mixture of the isopropylidene of 6-chloropurine riboside (11.5 g, 0.035 
mol), indoline (5.13 mL, 0.046 mole) and triethylamine (8.82 mL, 0.063 
mole) in n-butanol (60 mL) was stirred and heated to reflux for 24 hours. 
The reaction was cooled and the solid collected by filtration, rinsed with 
EtOH and dried to give the title compound: 10.70 g (75% yield); m.p. 
119.degree.-125.degree. C. 
EXAMPLE 31 
Preparation of 
6-(N-Indolinyl)-9-[2,3-O-(1-methylethylidene)-5-O-(4-methylbenzenesulfonyl 
)-1-.beta.-D-ribofuranosyl)]purine 
To a cold (0.degree. C.) solution of the alcohol (Example 30) (6.0 g, 0.015 
mol) in dry pyridine (40 mL) was added with stirring, p-toluenesulfonyl 
chloride (6.96 g, 0.36 mol). The solution was sealed and stored at 
0.degree.-10.degree. C. for 72 hours then poured into cold H.sub.2 O (30 
mL). The solid was collected by filtration and rinsed 3.times. with 
H.sub.2 O. After drying at 25.degree. C. under vacuum, the title compound 
was obtained: 6.85 g (83% yield); m.p. 195.degree. C. (d). 
EXAMPLE 32 
Preparation of 
6-(N-Indolinyl)-9-[5-O-(4-methylbenzenesulfonyl)-1-.beta.-D-ribofuranosyl) 
]purine 
A slurry of the protected tosylate (Example 31) (5.80 g, 10.0 mmole) in 
hydrochloric acid (23 mL) and EtOH (255 mL) was heated until homogenous 
then refluxed for 15 minutes. The solution was cooled in an ice bath and 
neutralized. The solid was collected by filtration, washed with H.sub.2 O, 
EtOH then MeOH. After drying, 3.38 g (65% yield) of the title compound 
were obtained; m.p. 112.degree. C.(d). 
EXAMPLE 33 
Preparation of 
6-(N-Indolinyl)-9-(5-methylamino-5-deoxy-1-.beta.-D-ribofuranosyl)purine 
Hydrochloride 
To 40% aqueous methylamine (40 mL) was added the tosylate (Example 32) (2.0 
g, 3.8 mmol) and sufficient MeOH to give a clear solution. The solution 
was stirred for one week then concentrated. The residue was coevaporated 
3.times. with MeOH then recrystallized from MeOH to give the free base, 
0.310 g (21% yield). A portion of this material was converted to the 
hydrochloride salt, m.p. 170.degree.-172.degree.. 
EXAMPLE 34 
Preparation of 4-Chloro-7H-pyrrolo[2,3-d]pyrimidine 
The above-identified compound was prepared as described: Davoll, J.; J. 
Chem. Soc., 1960, 131. 
EXAMPLE 35 
Preparation of 5-Bromo-4-chloro-7H-pyrrolo[2,3-d]pyrimidine 
The above identified compound was prepared as described: Hinshaw, B.; 
Gerster, J.; Robins, R.; Townsend, L.; J. Heterocyclic Chem., 1969, 215. 
EXAMPLE 36 
Preparation of 4-Chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine 
The above-identified compound was prepared as described: Pudlo, J.; 
Nassiri, M.; Kern, E.; Wartiny, L.; Drach, J.; Townsend, L.; J. Med. 
Chem., 1990, 33, 1984. 
EXAMPLE 37 
Preparation of 4-Chloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidine 
The above-identified compounds was prepared as described: Pudlo, J.; 
Nassir, M.; Kern, E.; Wotring, L.; Drach, J.; Townsend, L.; J. Med. Chem., 
1990, 33, 1984. 
EXAMPLE 38 
Preparation of 4-Chloro-2-methylthio-7H-pyrrolo[2,3-d]pyrimidine 
The above-identified compound was prepared as described: Noel, C., Robins, 
R.; J. Heterocyclic Chem., 1964, 1, 34. 
EXAMPLE 39 
Preparation of 2-Amino-4-Chloro-7H-pyrrolo[2,3-d]pyrimidine 
The above-identified compound was prepared as described: Pudlo, J.; 
Nassiri, M.; Kern, E.; Wotring, L.; Drach, J.; Townsend, L.; J. Med. 
Chem., 1990, 33, 1984. 
EXAMPLE 40 
Preparation of 2-Amino-4-chloro-7H-pyrrolo[2,3-d]pyrimidine 
The above-identified compound was prepared as described. Seela, F.; Stiker, 
H.; Driller, H.; Binding, N.; Liebigs Ann. Chem., 1987, 15. 
EXAMPLE 41 
Preparation of 4-Chloro-5-methylthio-7H-pyrrolo[2,3-d]pyrimidine 
A solution of 5-bromo-4-chloro-7H-pyrrolo[2,3-d]pyrimidine (Example 35) 
(2.53 g, 10 mmol) in THF (30 mL) was cooled to -78.degree. C. and a 
solution of n-butyl lithium (12 mL of 2.3M solution, 25 mmol) was added 
keeping the reaction temperature below -72.degree. C. The reaction mixture 
was stirred at -78.degree. C. for 45 min, a solution of methyl disulfide 
(0.95 mL, 10 mmol) in tetrahydrofuran (10 mL) was added over a period of 
30 minutes maintaining the temperature below -72.degree. C. The reaction 
mixture was stirred at -78.degree. C. for 2.5 hours then allowed to warm 
to room temperature. A saturated solution of ammonium chloride (40 mL) was 
added to the reaction with stirring. The organic layer was separated, the 
aqueous layer extracted with ethyl acetate (2.times.40 mL) and the 
combined organic extracts were dried, filtered and evaporated to obtain a 
pale yellow solid which was crystallized from EtOH: yield 1.65 g; (70%) 
m.p. 166.degree.-167.degree. C. 
EXAMPLE 42 
Preparation of 4-Chloro-5-cyano-7H-pyrrolo[2,3-d]pyrimidine 
N-Butyllithium in hexane (2.31M, 6.1 mL, 14.0 mmol) was added dropwise to a 
solution of 4-chloro-5-bromopyrrolopyrimidine (Example 35) (1.481 g, 6.37 
mmol) in 65 mL THF at -78.degree. C. and the resulting light yellow 
suspension stirred at this temperature for 1 hour. A cold (-78.degree. C.) 
solution of p-tolylsulfonylcyanide (2.08 g, 11.5 mmol) in 35 mL THF was 
added dropwise and stirred at this temperature for 1 hour. Aqueous 
NH.sub.4 Cl was added and the resulting solution was diluted with 100 mL 
CH.sub.2 Cl.sub.2. Washed the organic layer with water, brine, dried and 
evaporated to provide a tan solid (1.3 g) which appeared to be a 1:1 
mixture of the title nitrile and 4-chloropyrrolo-pyrimidine by .sup.1 H 
NMR. This material was recrystallized from 25 mL ethanol to provide 435 mg 
(38%) of the title nitrile as a tan solid: m.p. 300.degree. C. 
This compound may also be prepared as described: Tollman et al., J. Amer. 
Chem. Soc., 1969, 91:2102. 
EXAMPLE 43 
Preparation of 4-Chloro-5-ethoxycarbonyl-7H-pyrrole[2,3-d]pyrimidine 
A solution of 5-bromo-4-chloropyrrolo[2,3-d]pyrimidine (Example 35) (232 
mg; 1 mmol) in THF (5 mL) was cooled to -78.degree. C. and a solution of 
n-butyl lithium (1.3 mL of 2.31M) was added keeping the temperature below 
-72.degree. C. After stirring the reaction mixture at -78.degree. C. for 
45 minutes, a solution of ethyl chloroformate (0.15 mL) in THF (2 mL) was 
added slowly, maintaining the reaction temperature below -72.degree. C. 
The reaction mixture was stirred at -78.degree. C. for 2 hours then 
allowed to warm to room temperature. A saturated solution of NH.sub.4 Cl 
(20 mL) was added to the reaction mixture. The organic layer was separated 
and the aqueous layer was extracted with ethyl acetate (2.times.25 mL). 
The combined organic extracts were dried and evaporated to a white solid: 
yield 210 mg (92%): m.p. 140.degree.-141.degree. C. 
EXAMPLE 44 
Preparation of 
5-O-[(1,1-Dimethylethyl)dimethylsilyl]-2,3-O-(1-methylethylidene)-D-ribofu 
ranose 
The above-identified compound was prepared as described: H. Rosemeyer, H.; 
Seela, Helv. Chim. Acta., 1988, 71:1573. 
EXAMPLE 45 
Preparation of 5-Deoxy-D-ribofuranose 
The above-identified compound was prepared as described: Snyder J.; 
Serianni, A.; Carbohydrate Research, 1987, 163:169. 
EXAMPLE 46 
Preparation of 5-O-Methyl-D-ribofuranose 
The above-identified compound was prepared as described: Snyder, J.; 
Serianni, A.; Carbohydrate Research, 1987, 163:169. 
EXAMPLE 47 
Preparation of 
1-O-Methyl-2,3-O-(1-methylethylidene)-5-O-(4-methylbenzenesulfonyl)-D-ribo 
furanoside 
The above-identified compound was prepared as described: Snyder, J.; 
Serianni, A.; Carbohydrate Research, 1987, 163:169. 
EXAMPLE 48 
Preparation of 5-Deoxy-2,3-O-(1-methylethylidene)-D-ribofuranose 
5-Deoxy-D-ribofuranose (8 g, 60 mmole) was dissolved in DMF (25 mL) and to 
the solution was added dimethoxypropane (10 mL) and p-toluenesulfonic acid 
(150 mg). The reaction was stirred overnight then neutralized with 
OH.sup.- resin. The mixture was filtered, concentrated and the residue 
chromatographed on silica gel. Collected like fractions and evaporated to 
yield 4.1 g (39% yield) of viscous liquid. 
EXAMPLE 49 
Preparation of 5-O-Methyl-2,3-O-(1-methylethylidene)-D-ribofuranose 
To a solution of 5-O-methyl-D-ribofuranose (6.0 g, 36 mmole) in dry DMF (25 
mL), 2,2-dimethoxypropane (25 mL) and p-toluenesulfonic acid (250 mg) were 
added and the solution was stirred at room temperature for 20 hours, 
evaporated and chromatographed over silica. Collected and evaporated like 
fractions to give the title compound as an oily product, yield: 5.0 g 
(68%). 
EXAMPLE 50 
5-Azido-5-deoxy-1-O-methyl-2,3-O-(1-methylethylidene)-D-ribofuranoside 
A mixture of 
1-O-methyl-2,3-O-(1-methylethylidene)-5-O-(4-methylbenzenesulfonyl)-D-ribo 
furanoside (8.0 g, 22 mmol), DMF (40 mL) and NaN.sub.3 (4.0 g, 62 mmol) was 
heated at 80.degree. C. for 12 hours. The solvent was evaporated and the 
residue chromatographed. The faster moving fractions were pooled and 
evaporated to obtain 4.8 g (94% yield) of a syrupy product. 
EXAMPLE 51 
Preparation of 5-Azido-5-deoxy-D-ribofuranose 
A solution of 
5-azido-5-deoxy-1-O-methyl-2,3-O-(1-methylethylidene)-D-ribofuranoside 
(4.6 g, 20 mmol) in 0.1% H.sub.2 SO.sub.4 (300 mL) was refluxed for 3 
hours. The acid was neutralized with OH.sup.- resin. Filtered and washed 
the resin with ethanol. The filtrate was evaporated to give a syrupy 
residue; .sup.1 H and .sup.13 C NMR confirmed the product identity as a 
mixture of .alpha. and .beta. anomers. 
EXAMPLE 52 
Preparation of 
5-Azido-5-deoxy-2,3-O-(1-methyl-ethylidene)-.beta.-D-ribofuranose 
The crude 5-azido-5-deoxyribose (Example 51) was dissolved in DMF (10 mL) 
and treated with 2,2-dimethoxypropane (10 mL) and p-toluenesulfonic acid 
(100 mg). The solution was stirred at room temperature for 20 hours then 
evaporated. The residue was chromatographed. The appropriate fractions 
were pooled and evaporated to obtain the title compound, yield 2.4 g (56% 
yield). 
EXAMPLE 53 
Preparation of 
1-O-Methyl-2,3-O-(1-methylethylidene)-D-pentodialdo-1,4-furanoside 
The above-identified compound was prepared as described: Moorman, A., 
Borchardt, R.; Nucleic Acid Chemistry-Part III, Ed. Towsend, L., Tipson, 
R.; John Wiley and Sons, N.Y.; 1986, pages 38-41. 
EXAMPLE 54 
Preparation of 5-Benzoyl-D-allofuranose 
The sugar aldehyde from Example 53 (100 mmol) was dissolved in THF and 
treated with methyl magnesium bromide (100 mmol). After 2 hours of 
stirring at room temperature, a saturated solution of ammonium chloride in 
water (180 mL) was added. The organic layer was separated and the aqueous 
layer was extracted with ether (2.times.100 mL). The combined organic 
layers were dried and evaporated to obtain an oily product whose NMR was 
consistent with methyl-6-deoxy-2,3-isopropylidene-D-allofuranoside. The 
crude product was dissolved in pyridine (50 mL) and treated with benzoic 
anhydride (120 mmole). After stirring for 18 hours, methanol (2 mL) was 
added and the reaction mixture was evaporated. The residue was dissolved 
in ethyl acetate (300 mL) and washed with water, saturated bicarbonate 
solution, and brine. The organic layer was dried and evaporated to obtain 
a glassy product which was purified by chromatography. Identity of the 
product was confirmed by IR and NMR spectroscopy. The intermediate 
protected sugar was heated with aqueous sulfuric acid solution (0.01N in 
water, 300 mL) to 80.degree. C. for 2 hours and neutralized with resin. 
The aqueous layer was separated and evaporated to obtain the title 
compound as a sticky mass. The product was confirmed by NMR and used in 
the next step without further purification. 
EXAMPLE 55 
Preparation of 5-Benzoyl-6-deoxy-2,3-O-(1-methylethylidene)-D-allofuranose 
The benzoylated sugar (Example 54) was dissolved in a mixture of dry DMF 
(20 mL), 2,2-dimethoxypropane (20 mL) and p-toluenesulfonic acid (200 mg) 
and stirred at room temperature. After 2 hours the reaction mixture was 
neutralized by strongly basic ion exchange resin and the resin removed by 
filtration and washed. The combined washings and filtrate were evaporated 
and the residue was purified by chromatography. The pure product obtained 
was a glassy solid. 
EXAMPLE 56 
Preparation of 
5,6-Dideoxy-5,6-didehydro-1-O-methyl-2,3-O-(1-methylethyldene)-D-allofuran 
oside 
To a suspension of potassium-tert-butoxide (9.36 g) in ether (300 mL), 
methyl triphenylphosphonium bromide (29.6 g) was added over a 5 minute 
period. The yellow colored solution was stirred for 11/2 hours then a 
solution of methyl-2,3-isopropylidene-D-pentodialdo-1,4-furanoside (8.0 
g), Example 53, in ether (75 mL) was added over 5 minute period. The 
reaction mixture was stirred overnight at room temperature. The solid 
material that formed was removed by filtration and washed repeatedly with 
ether. The combined washings and filtrate were evaporated and the residue 
purified by chromatography to obtain 6.5 g of product as an oil; TLC 
Rf=0.5 (Silica gel, 97:3 hexane: EtOAc). 
EXAMPLE 57 
Preparation of 
5,6-Dideoxy-5,6-didehydro-2,3-O-(1-methylethylidene)-D-allofuranose 
A mixture of 5,6-dideoxy-5,6 
didehydro-1-O-methyl-2,3-O-(1-methylethylidene)-D-allofuranose (Example 
56) (2.0 g), and aqueous H.sub.2 SO.sub.4 (0.1%, 50 mL) was heated to 
90.degree. for 3 hours. The pH of the solution was adjusted to 7.5 with 1N 
NaOH solution, and evaporated to dryness. The residue was evaporated with 
DMF (2.times.20 mL) and the resulting semi solid product was slurried in 
methanol (25 mL). The undissolved solid was removed by filtration and 
washed with methanol (2.times.20 mL). The combined washings and the 
filtrate were evaporated to dryness and the resulting residue was 
dissolved in a mixture of dry DMF (10 mL), 2,2-dimethoxypropane (6 mL) and 
p-toluenesulfonic acid (50 mg). After stirring for 2 hours at room 
temperature the acid was neutralized with ion exchange resin and the resin 
was removed by filtration. Evaporation of the filtrate gave a product 
which was purified by chromatography over silica gel. 
EXAMPLE 58 
Preparation of 
5,6-Dideoxy-1-O-methyl-2,3-O-(1-methylethylidene)-D-allofuranoside 
A solution of the vinylic sugar (6.2 g), Example 56, in methanol (55 mL) 
was hydrogenated using 10% platinum on carbon as catalyst at 80 psi for 
100 hours. The catalyst was filtered and washed with methanol. The 
combined washings and filtrate were evaporated to obtain a colorless oil. 
Yield: quantitative. 
EXAMPLE 59 
Preparation of 5,6-Dideoxy-2,3-O-(1-methylethylidene)-D-allofuranose 
A mixture of 
5,6-dideoxy-1-methyl-2,3-O-(1-methylethylidene)-D-allofuranoside (5.8 
g)(Example 58), and water (160 mL) containing 0.16 mL of conc. H.sub.2 
SO.sub.4 was heated to 90.degree. C. for 3 hours. The pH of the reaction 
mixture was adjusted to 7.5 with 1N NaOH solution and evaporated to 
dryness. The residue was coevaporated with DMF (2.times.50 mL) then 
dissolved in methanol (25 mL) and filtered. The filtrate was evaporated, 
dissolved in a mixture of dry DMF (10 mL), 2,2-dimethoxypropane (10 mL) 
and potoluenesulfonic acid (200 mg) and stirred for 2 hours. The acid was 
neutralized with basic ion exchange resin and the resin filtered. The 
filtrate was evaporated and the residue chromatographed to obtain the 
title product; yield: 4.3 g.; TLC, Rf=0.6 (Silica gel, 2:1 hexane: EtOAc). 
EXAMPLE 60 
Preparation of 
5-deoxy-1-O-methyl-2,3-O-(1-methylethylidene)-D-allofuranoside 
To a solution of the vinylic sugar Example 56 (4.1 g) in THF (20 mL), 
borane:THF solution (10.65 mL of 1M solution in THF) was added over 10 
minutes. After stirring 2 hours the reaction vessel was immersed in a 
cooling bath (ice-water) and NaOH (8 mL of 3M solution) was added with 
stirring. After 15 minutes a solution of H.sub.2 O.sub.2 (30% aq., 4 mL) 
was added dropwise, and stirring was continued for 15 minutes. The flask 
was maintained at 55.degree. C. for 30 minutes and cooled. The contents 
were extracted with methylene chloride (3.times.150 mL) and the organic 
layer was dried. The solvent was evaporated and the residue was 
chromatographed. The minor product, Rf=0.7, (10%) was identified as 
6-deoxy-1-methyl-2,3-O-(1-methylethylidene)-D-allofuranoside. The slower 
moving major product (90%), Rf=0.3 was identified by proton NMR to be the 
title compound; yield 3.7 g; Rf=0.3 (silica gel, 2:1 hexane:EtOAc). 
EXAMPLE 61 
Preparation of 
6-O-(t-Butyldimethylsilyl)-2,3-O-(1-methylethylidene)-D-allofuranose 
This compound may be prepared by t-butyldimethylsilylation of the 6-hydroxy 
sugar, using t-butyldimethylsilyl chloride and imidazole in DMF. 
EXAMPLE 62 
Preparation of 
5-deoxy-1-methyl-2,3-O-(1-methylethylidene)-6-p-toluenesulfonyl-D-allofura 
noside 
To an ice-cold solution of the hydroxy sugar Example 60, (3.69 g) in 
pyridine (25 mL), p-toluenesulfonyl chloride (3.7 g) was added. The 
reaction mixture was stirred and allowed to warm to room temperature. 
Unreacted p-toluenesulfonylchloride was quenched by adding 1 mL of 
methanol and the volatile components were evaporated. The residue was 
evaporated with DMF (2.times.20 mL), then dissolved in ethyl acetate (350 
mL). The solution was washed and the organic layer was dried. Evaporation 
gave a residue which was chromatographed. Proton NMR indicated the product 
to be a mixture of .alpha. and .beta. anomers. 
EXAMPLE 63 
Preparation of 
6-azido-5,6-dideoxy-1-O-methyl-2,3-O-(1-methylethylidene)-D-allofuranoside 
A mixture of the tosyl sugar, Example 62, (4.0 g), dry DMF (20 mL) and 
sodium azide (1.5 g) was heated at 100.degree. C. for 24 hours. The 
solvent was evaporated and the residue was dissolved in ethyl acetate (200 
mL) and washed with water. The organic layer was dried and evaporated to 
obtain a colorless oil which was sufficiently pure by TLC and NMR for use 
in the next reaction; yield 2.09 g; Rf=0.35 (silica gel, 93:7 
hexane:EtOAc). 
EXAMPLE 64 
Preparation of 
6-azido-5,6-dideoxy-2,3-O-(1-methylethylidene)-D-allofuranose 
A mixture of the azido sugar Example 63 (2.8 g), and aqueous sulfuric acid 
solution (100 mL of 0.1% by volume) was heated to 90.degree. C. for 31/2 
hours. The pH of the reaction mixture was adjusted to 7.5 with 1N NaOH and 
evaporated to dryness. The residue was coevaporated with DMF (2.times.20 
mL) and treated with methanol (25 mL). The insoluble solids were removed 
by filtration and washed with methanol (2.times.20 mL). The combined 
filtrates were evaporated to dryness. The oily product was dissolved in a 
mixture of dry DMF (10 mL), 2,2-dimethoxypropane (6 mL) and p-toluene 
sulfonic acid (50 mg) and stirred for 2 hours at room temperature. The 
solvents were evaporated and the residue was chromatographed. Fractions 
containing the main product were combined and evaporated to obtain the 
title compound as a colorless oil. Yield was 1.89 g. 
EXAMPLES 65-81 
General procedure for the preparation of 5'-substituted-4- 
chloropyrrolo[2,3-d]pyrimidine-7-(1-.beta.-D-ribosides) 
A solution of the 5-substituted (H, OCH.sub.3, N.sub.3 or TBDMS-O-) 
5-deoxyisopropylideneribose (1 eq) in CCl.sub.4 (1.4 eq) and THF was 
cooled to -78.degree. C. Hexamethylphosphorous triamide (1.2 eq) was added 
dropwise and the reaction mixture stirred for 2 hours at -78.degree. C. 
This solution of 1-.alpha.-chloro sugar was used directly in the next 
step. 
To a slurry or solution of the substituted 4-chloropyrrolo[2,3-d]pyrimidine 
(1.4 eq corresponding to the sugar) in DMF, was added in four portions, 
NaH (1.4 eq) over 10 minutes. The solution was stirred 30 minutes then the 
above solution of chlorosugar (-25.degree. C.) was added and the reaction 
was stirred for 24 hours. The mixture was concentrated, diluted with 
EtOAc, filtered and the filtrate concentrated under vacuum. The residue 
was chromatographed. The appropriate fractions were collected and 
evaporated to yield the protected nucleoside. 
The protected nucleoside was deblocked by dissolving in 90% trifiuoroacetic 
acid and stirring for 2 hours. The solvent was evaporated and chased with 
methanol (3.times.). The product was crystallized from EtOH. 
The compounds in Table IV (Examples 65-81) were prepared by this procedure: 
TABLE IV 
______________________________________ 
GP-1-# EXAMPLE B' D G m.p. (.degree.C.) 
______________________________________ 
475 65 CH.sub.2 OH 
I H 183-181.degree. 
-- 66 CH.sub.2 N.sub.3 
I NH.sub.2 
203-205.degree. 
406 67 CH.sub.2 OH 
Br H &gt;230.degree. 
448 68 CH.sub.3 I H 180-181.degree. 
449 69 CH.sub.3 CH.sub.3 
H 155-157.degree. 
462 70 CH.sub.2 OCH.sub.3 
CH.sub.3 
H 142-144.degree. 
460 71 CH.sub.2 OCH.sub.3 
I H 179-180.degree. 
464 72 CH.sub.2 OCH.sub.3 
H H 122-124.degree. 
692 73 CH.sub.2 CH.sub.3 
Br H 163-165.degree. 
690 74 CH.sub.2 CH.sub.3 
I H 181-183.degree. 
529 75 CH.sub.2 N.sub.3 
I H 203-205.degree. 
554 76 CH.sub.3 Br H 174-175.degree. 
555 77 CH.sub.3 H CH.sub.3 S 
140-142.degree. 
569 78 CH.sub.3 SCH.sub.3 
H 147-148.degree. 
605 79 CH.sub.2 N.sub.3 
Br H 156-158.degree. 
-- 80 CH.sub.2 CH.sub.2 N.sub.3 
I H foam 
713 81 CH.dbd.CH.sub.2 
I H 183-185.degree. 
______________________________________ 
EXAMPLES 82-83 
Preparation of 
4-Amino-7-(5-amino-5-deoxy-1-.beta.-D-ribofuranosyl)-5-halopyrrolo[2,3-d]p 
yrimidines 
A mixture of 
4-chloro-5-iodo-7-(5-azido-5-deoxy-1-.beta.-D-ribofuranosyl)pyrrolo[2,3-d] 
pyrimidine (Example 75 or 79) (500 mg), triphenylphosphine (550 mg) and 
pyridine (6 mL) was stirred at room temperature for 24 hours. Pyridine was 
evaporated and the residue triturated with ether. The residual semi-solid 
was treated with ammonium hydroxide (5 mL). Ethanol was added to cause 
complete dissolution of the compound. After stirring for 5 hours at room 
temperature the mixture was evaporated and the residue was triturated with 
water (10 mL). The insoluble material was filtered and the pH of the 
filtrate was adjusted to 5.5 with dilute HCl. The solution was refiltered 
and lyophilized to obtain a hygroscopic solid, whose NMR was compatible 
with the structure. 
The above hygroscopic solid was dissolved in methanol, saturated with dry 
ammonia at -15.degree. C., then heated in a steel bomb at 80.degree. C. 
for 24 hours. The bomb was cooled and opened. The solution was evaporated 
and the residue was dissolved in water, charcoaled and filtered. The 
filtrate was lyophilized to obtain the title compound as a hygroscopic 
solid. The compounds listed in Table V were obtained by this procedure. 
TABLE V 
______________________________________ 
GP-1-# EXAMPLE D F m.p. (.degree.C.) 
______________________________________ 
550 82 I H 166-206.degree. 
649 83 Br H 217-219.degree. 
______________________________________ 
EXAMPLE 84 
Preparation of 
4-Amino-5-iodo-7-(5-acetylamino-5-deoxy-1-.beta.-D-ribofuranosyl)pyrrolo[2 
,3-d]pyrimidine 
To an ice cold solution of the 5'-amino compound from Example 54 (50 mg), 
in pyridine (5 mL), acetic anhydride (0.5 mL) was added. The reaction 
mixture was allowed to warm to room temperature over 1 hour. The flask was 
reimmersed into the cooling bath and 15 mL of methanol was added to 
neutralize unreacted acetic anhydride. The solvent was evaporated under 
reduced pressure and the residue purified by chromatography to give the 
above-identified product, m.p. 160.degree.-163.degree. C. 
EXAMPLES 85 TO 113B 
General Procedure for the Preparation of N.sup.4 
-Substituted-4-aminopyrrolo[2,3-d]pyrimidine Nucleosides 
A suspension of the substituted 4-Cl-pyrrolo[2,3-d]pyrimidine nucleoside (1 
eq) in EtOH containing the amine (3 eq) and triethylamine (5 eq) was added 
to a small stainless steel bomb (in the case of diamines a 25% excess of 
chloride was used). The bomb was heated overnight (70.degree.-120.degree. 
C.), cooled, opened and the reaction mixture evaporated. 
The product was crystallized from ethanol or ethyl acetate. The compounds 
in Table VI (Examples 85 to 113B) were prepared by this procedure: 
TABLE VI 
__________________________________________________________________________ 
GP-1-# 
Example 
B' D F G m.p. (.degree.C.) 
__________________________________________________________________________ 
334 85 CH.sub.2 OH 
H NH.sub.2 
H 249-250.degree. 
394 86 CH.sub.2 OH 
H N-indolinyl 
H Foam 
393 87 CH.sub.2 OH 
H N-prolinyl 
H Foam 
296 88 CH.sub.2 OH 
H cyclopentyl-NH 
H Foam 
376 89 CH.sub.2 OH 
Br NH.sub.2 
H Foam 
321 90 CH.sub.2 OH 
H NH.O slashed. 
H Foam 
476 91 CH.sub.2 OH 
I N-Indolinyl 
H 185-188.degree. 
456 92 CH.sub.3 
I NH.sub.2 
H 245-246.degree. 
470 93 CH.sub.3 
I N-indolinyl 
H 188-190.degree. 
457 94 CH.sub.3 
I CH.sub.3 NH 
H 226-228.degree. 
485 95 CH.sub.3 
I N.sub.3 H 213-214.degree. 
498 96 CH.sub.3 
CH.sub.3 
NH.sub.2 
H 212-214.degree. 
461 97 CH.sub.3 
CH.sub.3 
N-indolinyl 
H 171-173.degree. 
463 98 CH2OCH.sub.3 
I NH.sub.2 
H 216-218.degree. 
465 99 CH.sub.2 OCH.sub.3 
I CH.sub.3 NH 
H 188-189.degree. 
474 100 CH.sub.2 OCH.sub.3 
H N-indolinyl 
H 205-208.degree. 
480 101 CH.sub.2 OCH.sub.3 
H CH.sub.3 NH 
H 163-164.degree. 
513 102 CH.sub.3 
H N-piperazinyl 
H 216-219.degree. 
499 103 CH.sub.3 
I N,N'-piperazinyl 
H 220-223.degree. 
500 104 CH.sub.3 
I --NH(CH.sub.2).sub.6 NH--.sup.1 
H 227-229.degree. 
512 105 CH.sub.3 
I --NH(CH.sub.2).sub.2 NH--.sup.1 
H &gt;230.degree. 
559 106 CH.sub.3 
I NH.sub.2 
CH.sub.3 S 
200-202.degree. 
561 107 CH.sub.3 
H 1,4-piperazinyl.sup.2 
H foam 
606 108 CH.sub.2 N.sub.3 
Br NH.sub.2 
H 182-184.degree. 
639 109 CH.sub.3 
I NH.O slashed. 
H &gt;230.degree. 
581 110 CH.sub.3 
CO.sub.2 Et 
NH.sub.2 
H 162-168.degree. 
681 111 CH.sub.2 OH 
I NH.O slashed. 
H 224-225.degree. 
680 112 CH.sub.2 OH 
I NH(4-Cl.O slashed.) 
H 234-235.degree. 
689 113 CH.sub.2 OH 
I NH(4-CH.sub.3 O-.O slashed.) 
H 212-214.degree. 
711 113A CH.sub.2 CH.sub.2 N.sub.3 
I NH.sub.2 
H 151-153.degree. 
714 113B CH.dbd.CH.sub.2 
I NH.sub.2 
H 224-226.degree. 
__________________________________________________________________________ 
.sup.1 Dimers having two pyrrolo[2,3d]pyrimidine riboside moieties linked 
by the listed diamine. 
.sup.2 Dimer with purine riboside. 
EXAMPLE 114 
Preparation of 
5-Iodo-7-(5-deoxy-1-.beta.-D-ribofuransyl)pyrrolo[2,3-d]pyrimidin-4(3H)-th 
ione 
A solution of 
4-chloro-5-iodo-7-[5-deoxy-1-.beta.-D-ribofuranosyl]pyrrolo[2,3-d]pyrimidi 
ne (Example 68) (250 mg, 0.60 mmol) and thiourea (250 mg) in absolute, EtOH 
was refluxed for 16 hours. The solvent was evaporated and the residue was 
triturated with water (10 mL). The solid was collected by filtration, 
washed with water and dried in air: Yield 200 mg (81%); m.p. 
161.degree.-163.degree. C. 
EXAMPLES 115 TO 120 
General Procedure for S-Alkylation of 
5-Iodo-7-(5-deoxy-1-6-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidin-4(3H)-thione 
To a solution of 5-iodo-7-(5-deoxy-1-.beta.-D-ribofuranosyl) 
pyrrolo[2,3-d]pyrimidine-4-thione (Example 114) (50 mg) in concentrated 
NH.sub.4 OH (10 mL), the appropriate alkylating agent (e.g. methyl iodide, 
alkyl or substituted benzyl bromide) was added and the mixture stirred at 
room temperature for 20 hours. Volatile material was evaporated and the 
residue triturated with ether. To the residue, water (5 mL) was added and 
the solid was collected by filtration. 
The products obtained by this procedure (Examples 115 to 120) are listed in 
Table VII: 
TABLE VI 
______________________________________ 
GP-1-# Example B' D F m.p. (.degree.C.) 
______________________________________ 
482 115 CH.sub.3 
I SCH.sub.3 212-233.degree. 
493 116 CH.sub.3 
I SCH.sub.2 CH.dbd.CH.sub.2 
192-193.degree. 
494 117 CH.sub.3 
I SCH.sub.2 .O slashed.NO.sub.2 (4) 
224-226.degree. 
502 118 CH.sub.3 
I SC.sub.4 H.sub.9 
186-187.degree. 
503 119 CH.sub.3 
I SCH.sub.2 .O slashed. 
212-213.degree. 
511 120 CH.sub.2 OH 
I SCH.sub.3 214-150.degree. 
______________________________________ 
EXAMPLE 121 
Preparation of 
4-Phenyl-7-(1-.beta.-D-ribofuranosyl)-pyrrolo[2,3d]pyrimidine 
To a solution of 
4-chloro-7-[2,3-O-(1-methylethylidene)-1-.beta.-D-ribofuranosyl]pyrrolo[2, 
3-d]pyrimidine (200 mg) and phenylboronic acid (250 mg) in diglyme (10 mL) 
was added palladium-tetrakis-triphenylphosphine (30 mg), followed by 
aqueous Na.sub.2 CO.sub.3 solution (0.2 mL of 2M solution). The reaction 
mixture was heated to 90.degree. C. for 6 hours. The solvent was 
evaporated and the residue was purified by HPLC. The purified intermediate 
was treated with 2 mL of trifluoroacetic acid (80%) and stirred for 15 
minutes then evaporated and the residue crystallized from ethanol; yield 
20 mg, m.p. 163.degree.-164.degree. C. 
EXAMPLES 122 TO 124 
General Procedure for the Preparation of 4-Amino- and 
4-Arylamino-5-aryl-7-(1-.beta.-D-ribofuranosyl)pyrrolo-[2,3-d]pyrimidines 
To a stirred mixture of the 4-amino- or 
4-arylamino-5-iodopyrrolo[2,3-d]pyrimidine riboside (or corresponding 
hydroxyl protected compound) (0.1 mmol), and Pd(PPh.sub.3).sub.4 (10 mg, 
0.01 mmol) in diglyme was added a solution of the arylboronic acid (0.4 
mmol) in EtOH and 0.4 mL of aqueous 2M Na.sub.2 CO.sub.3. The lo mixture 
was heated to 100.degree. C. and the reaction monitored by TLC. After the 
reaction was complete, the cooled mixture was filtered and concentrated 
under vacuum. The residue was chromatographed over silica or by HPLC. 
The compounds in Table VIII may be prepared by this procedure: 
TABLE VIII 
______________________________________ 
GP-1-# Example D F m.p. (.degree.C.) 
______________________________________ 
-- 122 .O slashed. 
.O slashed.NH 
-- 
-- 123 .O slashed. 
NH.sub.2 
-- 
718 124 2-furanyl .O slashed.NH 
foam 
______________________________________ 
EXAMPLES 125-126 
General Procedure for the Preparation of 4-Amino- and 
4-Arylamino-5-aryl-7-(5-deoxy-1-.beta.-d-ribofuranosyl)pyrrolo[2,3-d]pyrim 
idines 
The above-identified compounds were prepared as described in Example 
122-124 from the 4-amino- or 
4-arylamino-5-iodo-7-(5-deoxy-1-.beta.-D-ribofuranosyl)pyrrolo[2,3-d]pyrim 
idine and an arylboronic acid. 
The compounds in Table IX were prepared by this procedure: 
TABLE IX 
______________________________________ 
GP-1-# Example B' D F G m.p. (.degree.C.) 
______________________________________ 
684 125 CH.sub.3 
.O slashed. 
NH.sub.2 
H 106-109 
683 126 CH.sub.3 
.O slashed. 
NH-.O slashed. 
H 207-208 
______________________________________ 
EXAMPLE 127 
Preparation of 
4-Chloro-5-iodo-7-(6-deoxy-1-.beta.-D-allofuranosyl)pyrrolo[2,3-d]pyrimidi 
ne 
The above-identified compound was prepared according to the general 
procedure used for Examples 65-81; m.p. 211.degree.-213.degree. C. 
EXAMPLE 128 
Preparation of 
4-Amino-5-iodo-7-(6-deoxy-1-.beta.-D-allofuranosyl)pyrrolo[2,3-d]pyrimidin 
The 4-chloro compound, Example 127, was heated in a steel bomb with 
methanolic ammonia at 120.degree. C. for 12 hours followed by the usual 
work up (see Example 85). A white crystalline product was obtained; m.p. 
206.degree.-208.degree. C. 
EXAMPLES 129-130 
General Procedure for the Preparation of 
4-Amino-5-halo-7-(5,6-dideoxy-1-.beta.-D-allofuranosyl)pyrrolo[2,3-d]pyrim 
idine 
The above-described compounds were prepared by the general procedures for 
Examples 65 to 81. 
The compounds obtained by this procedure are listed in Table X. 
TABLE X 
______________________________________ 
GP-1-# Example B' D F G m.p. (.degree.C.) 
______________________________________ 
693 129 CH.sub.2 CH.sub.3 
Br NH.sub.2 
H 229-230 
691 130 CH.sub.2 CH.sub.3 
I NH.sub.2 
H 233-234 
______________________________________ 
EXAMPLE 131 
Preparation of 4-Amino-3-bromopyrazolo[3,4-d]pyrimidine 
The above-identified compound was prepared as described: Leonova, T.; 
Yashunskii, V.; Khim. Get. Soed., 1982, 982. 
EXAMPLE 132 
Preparation of 4-Amino-3-(cyanomethyl)pyrazolo[3,4-d]pyrimidine 
The above-identified compound was prepared as described: Carboni, R.,; 
Coffman, D.,; Howard, E.; J.Am. Chem. Soc., 1958 80:2838. 
EXAMPLE 133 
Preparation of 4-Amino-3-cyanopyrazolo[3,4-d]pyrimidine 
The above-identified compound was prepared as described: Taylor, E.; 
Abul-Hsan, A.; J. Org. Chem., 1966, 31:342. 
EXAMPLE 134 
Preparation of 4-Amino-3-phenylpyrazolo[3,4-d]pyrimidine 
The above-identified compound was prepared from trimethyl orthobenzoate as 
described: Kobayashi, S.; Chem. Pharm. Bull. (Jap.) 1973, 21:941. 
EXAMPLES 135-139 
General Procedure for the Preparation of Aryl Thiomorpholides 
A mixture of the aromatic carboxaldehyde (0.1 mole), sulfur (4.8 g, 0.15 
mole) and morpholine (18 mL, 0.15 mole) was heated at 180.degree. C. for 
3-5 hours then cooled and diluted with H.sub.2 O. The solid was collected 
by filtration or, if oily, extracted with CH.sub.2 Cl.sub.2, dried and 
concentrated. The crude product was recrystallized or chromatographed over 
silica. 
The compounds in Table XI were prepared by this procedure: 
TABLE XI 
______________________________________ 
Example Aryl m.p. (.degree.C.) 
______________________________________ 
135 4-CH.sub.3 O.O slashed. 
95-98.degree. 
136 4-Cl.O slashed. 
137-140.degree. 
137 2-Br.O slashed. 
-- 
138 2-thienyl 
75-77.degree. 
3-thienyl 
84-87.degree. 
139 3-CH.sub.3 O.O slashed. 
134-139.degree. 
______________________________________ 
EXAMPLES 140-144 
General Procedure for the Preparation of 5-Amino-3-aryl-4-cyanopyrazoles 
The above-identified compounds were prepared from the corresponding aryl 
thiomorpholides (Examples 135-139) following the general procedure 
described: Tominaga, Y.,; et al.; J. Heterocyclic Chem., 1990, 27:647. 
The compounds listed in Table XII were prepared by this procedure: 
TABLE XII 
______________________________________ 
Example Aryl m.p. (.degree.C.) 
______________________________________ 
140 4-CH.sub.3 O.O slashed. 
155-160.degree. 
141 4-Cl.O slashed. 
218-222.degree. 
142 2-Br.O slashed. 
-- 
143 2-thienyl 
260-265.degree. 
144 3-thienyl 
229-231.degree. 
______________________________________ 
EXAMPLES 145-148 
General Procedure for the Preparation of 
5-Amino-3-aryl-4-carboxamidopyrazoles 
The above compounds were obtained from the corresponding cyano compounds 
(Example 140-144) following the general procedure described: Kobayashi, 
S.; Chem. Pharm. Bull. (Jap.), 1973, 21:941. 
The compounds listed in Table XIII were prepared by this procedure: 
TABLE XIII 
______________________________________ 
Example Aryl m.p. (.degree.C.) 
______________________________________ 
145 .O slashed. 
203-205.degree. 
146 4-CH.sub.3 O.O slashed. 
-- 
147 4-Cl.O slashed. 
210-215.degree. 
148 2-Br.O slashed. 
-- 
______________________________________ 
EXAMPLE 149-154 
General Procedure for the Preparation of 
4-Amino-3-arylpyrazolo[3,4-d]pyrimidines 
A mixture of the 5-amino-3-aryl-4-cyanopyrazole and formamide (5 mL/g) was 
refluxed (190.degree.-200.degree. C.) for 4 hours. The cooled mixture was 
diluted with H.sub.2 O and the solid collected by filtration. The crude 
products were used directly for subsequent steps or purified by 
recrystallization. The compounds listed in Table XIV were prepared by this 
procedure. 
TABLE XIV 
______________________________________ 
Example Aryl m.p. (.degree.C.) 
______________________________________ 
149 .O slashed. &gt;220 
150 4-CH.sub.3 O.O slashed. 
&gt;220 
151 4-Cl.O slashed. 
&gt;220 
152 2-Br.O slashed. 
&gt;220 
153 2-Thienyl &gt;220 
154 3-Thienyl &gt;283 (dec.) 
______________________________________ 
EXAMPLES 155-158 
General Procedure for the Preparation of 
3-Arylpyrazolo[3,4-d]pyrimidin-4-ones from 
5-Amino-3-aryl-4-carboxamidopyrazoles 
A mixture of the 5-amino-3-aryl-4-carboxamidopyrazole and formamide (5 
mL/g) was refluxed at 190.degree.-200.degree. C. for 2 hours, cooled and 
diluted with H.sub.2 O. The solid was collected by filtration and dried. 
Further purification was effected by dissolving the compound in dilute 
sodium hydroxide, followed by charcoal treatment and precipitation with 
acetic acid. 
The compounds listed in Table XV were prepared by this procedure: 
TABLE XV 
______________________________________ 
Example Aryl m.p. (.degree.C.) 
______________________________________ 
155 .O slashed. 
&gt;200.degree. 
156 4-CH.sub.3 O.O slashed. 
&gt;220.degree. 
157 4-Cl.O slashed. 
&gt;220.degree. 
158 2-Thienyl 
&gt;220.degree. 
______________________________________ 
EXAMPLES 159-160 
General Procedure for Preparation of 3-Arylpyrazolo-[3,4d]pyrimidin-4-ones 
from 4-Amino-3-aryl-pyrazolo[3,4-d]pyrimidines 
To a slurry of the 3-aryl-4-aminopyrazolo[3,4-d]pyrimidine (25 mmoles) in 
175 mL of 9% HCl at 0.degree. to 5.degree. C., was added, over 45 minutes, 
an aqueous solution of sodium nitrite (15.0 g in 30 mL). The mixture was 
allowed to warm to room temperature and solid sodium nitrite (5.0 g) was 
added. After 15 minutes the mixture was cautiously heated to boiling 
(foaming!), then cooled. The product was collected by filtration, rinsed 
with H.sub.2 O and dried at 50.degree. C. under vacuum. 
The compounds listed in Table XVI were prepared by this procedure: 
TABLE XVI 
______________________________________ 
Example Aryl m.p. (.degree.C.) 
______________________________________ 
159 .O slashed. 
&gt;220.degree. 
160 2-thienyl 
&gt;220.degree. 
______________________________________ 
EXAMPLES 161-163 
General Procedure for the Preparation of N.sup.4 -Aryl and N.sup.4 -Alkyl 
Substituted 4-amino-3-aryl-pyrazolo[3,4-d]pyrimidines 
A mixture of the 3-arylpyrazolo[3,4-d]pyrimidin-4-one (15 mmoles), 
POCl.sub.3 (18 mL, 195 mmoles) and diethylaniline (5 mL, 31 mmoles) was 
refluxed for 4 hours then concentrated. The residue was decomposed by 
addition of ice and extracted (4.times.) with 3:1 ether-ethyl acetate. The 
combined organic extracts were washed with water and dried. The solution 
was concentrated and the crude 4- chloro-3-arylpyrazolo[3,4-d]pyrimidine 
(50-70% yield) was added to a solution of amine (2.2 equivalents) in EtOH 
(25 mL/mmole chloro compound). The mixture was refluxed for 30 minutes 
then cooled and the product collected by filtration and rinsed with ETCH. 
Recrystallization gave the title compounds. 
The compounds listed in Table XVII were prepared by this procedure: 
TABLE XVII 
______________________________________ 
Example 3-Aryl 4-Arylamino 
m.p. (.degree.C.) 
______________________________________ 
161 .O slashed. .O slashed. 
229-232.degree. 
162 .O slashed. 4-Cl.O slashed.NH 
232-233.degree. 
163 .O slashed. 4-CH.sub.3 O.O slashed.NH 
218-220.degree. 
______________________________________ 
EXAMPLE 164 
Preparation of 3-Bromopyrazolo[3,4-d]pyrimidin-4-one 
The above-identified compound was prepared as described: Chu, I.; Lynch, 
B.; J. Med. Chem., 1975, 18:161. 
EXAMPLE 165 
Preparation of 
3-Bromo-1-(2,3,5-O-tribenzoyl-1-.beta.-D-ribofuranosyl)pyrazolo[3,4-d]pyri 
midin-4-one 
The above-identified compound was prepared as described: Cottam, H.; 
Petrie, C.; McKernan, P.; Goebel, R.; Dailey, N.; Davidson, R.; Robins, 
R.; Revankar, G.; J. Med. Chem., 1984, 27:1120. 
EXAMPLE 166 
Preparation of 
3-Substituted-4-chloro-1-(2,3,5,-O-tribenzoyl-1-.beta.-D-ribofuranoyl)pyra 
zolo[3,4-d]pyrimidin-4-ones 
The above-identified compounds may be prepared from the corresponding 
pyrazolo[3,4-d]-pyrimidones by a procedure analogous to the one described 
in Example 2. 
EXAMPLES 167 TO 169 
General Procedure for Preparation of 3-Substituted 4-amino- and 
4-(arylamino)-1-(1-.beta.-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidines 
To a slurry of the 3-substituted-4-chloropyrazolo[3,4-d]pyrimidine 
nucleoside tribenzoate (1.0 eq) (Example 166) in a mixture of EtOH and 
THF, was added ethanolic ammonia or the amine (1.5 eq) and Et.sub.3 N (3.5 
eq). The reaction rapidly became homogenous and after 0.5-12 hours, was 
evaporated. The residue was dissolved in CH.sub.2 Cl.sub.2, washed with 
aqueous K.sub.2 CO.sub.3 then H.sub.2 O and the solution dried. After 
evaporation, the residue was recrystallized or chromatographed. The 
resulting tribenzoate of the title compound was deblocked by stirring in 
methanolic NaOMe. The mixture was neutralized with amberlite IR-120(+) 
resin, filtered and evaporated. The residue was recrystallized to give the 
title compounds. 
Examples 167 to 169 listed in Table XVIII were prepared by this procedure: 
TABLE XVIII 
______________________________________ 
GP-1-# Example 3- 4- m.p. (.degree.C.) 
______________________________________ 
596 167 I NH.sub.2 
180-185.degree. 
469 168 Br N-indolinyl 
195-196.degree. 
536 169 CH.sub.3 
NH.sub.2 
241-242.degree. 
______________________________________ 
EXAMPLE 170 
Preparation of 
4-(N-Indolinyl)-1-(1-.beta.-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine 
A solution of the bromide (Example 168) (351 mg, 0.78 mmol) in methanol 
containing 300 mg 10% Pd-C and Raney Ni was hydrogenated at 40 psi. The 
mixture was filtered, the filtrate concentrated and the product collected 
by filtration to give the title compound: 120 mg (42%); m.p. 
215.degree.-219.degree.. 
EXAMPLE 171 
Preparation of 
3-Bromo-1-[2,3-O-(1-methylethylidene)-1-.beta.-D-ribofuranosyl]pyrazolo[3, 
4-d]pyrimidin-4-one 
Crude 3-bromoallopurinol riboside (prepared from 33.0 g of tribenzoate and 
NaOMe/MeOH (Example 83) was added to a 5.degree. C. solution of 1M 
ethanolic HCl (6.5 mL) and dimethoxypropane (20 mL) in 1.1 L of acetone. 
The mixture was stirred 45 minutes. Na.sub.2 CO.sub.3 (5.0 g) and 
concentrated NH.sub.4 OH (5 mL) were added and the mixture pH reached 6-7. 
The reaction was filtered and evaporated. The residual solid was dissolved 
in 300 mL of boiling EtOH and the solution concentrated. The solution was 
chilled overnight and the solid collected by filtration. After drying 
(50.degree. C.), 16.7 g (86%) of the title compound were obtained; m.p. 
221+.congruent.224.degree. C. 
EXAMPLE 172 
Preparation of 
3-Bromo-1-[2,3-O-(1-methylethylidene)-5-O-(4-methylbenzenesulfonyl)-1-.bet 
a.-D-ribofuransyl]pyrazolo[3,4-d]pyrimidin-4-one 
To a solution of the isopropylidene alcohol (Example 171) (3.0 g, 7.74 
mmol) in pyridine (18 mL) at 0.degree. C. was added p-toluenesulfonyl 
chloride (1.77 g, 9.30 mmol). The reaction was held at 0.degree. C. for 3 
hours then poured into 160 mL of cold H.sub.2 O. The H.sub.2 O was 
decanted and the residue dissolved in CH.sub.2 Cl.sub.2. The CH.sub.2 
Cl.sub.2 solution was washed with 0.5N H.sub.2 SO.sub.4, 5% aqueous 
K.sub.2 CO.sub.3 and dried. After evaporation, 4.03 g (96% yield) of the 
title compound were obtained as a foam. 
EXAMPLE 173 
Preparation of 
1-[5-Azido-5-deoxy-2,3-O-(1-methylethylidene)-1-.beta.-D-ribofuranosyl]-3- 
bromopyrazolo[3,4-d]pyrimidin-4-one 
To a solution of NaN.sub.3 (7.69 g, 0.12 moles) in DMSO (70 mL) was added 
the tosylate (Example 172) (16.0 g, 0.03 mol). The solution was rapidly 
heated to 80.degree. C. and stirred for 45 minutes. After cooling, the 
reaction mixture was added to H.sub.2 O (600 mL). The mixture was 
extracted and the combined extracts were washed with water, dilute brine, 
dried and concentrated to give 11.0 g of a white foam. TLC indicated a 
mixture of three products in the approximate ratio of 1:2:1. The middle 
spot was subsequently determined to be the desired azide. 
The mixture was purified by chromatography. The fractions containing the 
desired product were combined and evaporated to give the title compound; 
5.90 g (48% yield), m.p. 168.degree. C. (d). 
EXAMPLE 174 
Preparation of 
3-Amino-1-[2,3-O-(1-methylethylidene)-1-.beta.-D-ribofuranosyl]pyazolo[3,4 
-d]pyrimidin-4-one 
A mixture of bromide (Example 171) (2.35 g, 6.1 mmol) CuCl (88 mg) and Cu 
(101 mg) in MeOH (45 mL) was placed in a bomb and saturated with ammonia. 
The bomb was sealed and heated to 110.degree. C. for 10 hours. After 
cooling, the bomb was opened, the contents filtered and the filtrate 
evaporated. The residue was chromatographed. The appropriate fractions 
were combined and evaporated to yield the title compound as a solid; 2.1 g 
(98% yield); m.p. 142.degree.-144.degree. C. 
EXAMPLE 175 
Preparation of 
3-Iodo-1-[2,3-O-(1-methylethylidene)-1-.beta.-D-ribofuranosyl]pyrazolo[3,4 
-d]pyrimidin-4-one 
A mixture of the amine (Example 174) (2.58 g, 8.0 mmole), isoamyl nitrite 
(30 mL), methylene iodide (20 mL) and CH.sub.3 CN was refluxed for 10 
minutes. The cooled mixture was evaporated and chromatographed. 
Appropriate similar fractions were combined and evaporated to give the 
title compound: 1.08 g (66% yield); m.p. &gt;220.degree. C. 
EXAMPLE 176 
Preparation of 3-Iodo-1-[2,3-O-(1-methylethylidene)-5-O-(4- 
methylbenzenesulfonyl)-1-.beta.-D-ribofuranosyl]pyrazolo-[3,4-d]pyrimidin- 
4-one 
The above identified compound was prepared by a procedure analogous to the 
one described for Example 172. 
EXAMPLE 177 
Preparation of 
3-Iodo-1-[5-azido-5-deoxy-2,3-O-(1-methylethylidene)-1-.beta.-D-ribofurano 
syl]pyrazolo-[3,4-d]pyrimidin-4-one 
The above identified compound was prepared by a procedure analogous to the 
one described for Example 173 in 45% yield; m.p. 203.degree. C.(d). 
EXAMPLE 178 
Preparation of 
3-Halo-4-chloro-1-[5-azido-5-deoxy-2,3-O-(1-methylethylidene)-1-.beta.-D-r 
ibofuransyl)]pyrazolo[3,4-d]pyrimidine 
The above identified compounds were prepared by a procedure analogous to 
the one described for Example 2 from the pyrimidin-4-one (Example 173 or 
177). The title compounds were obtained as unstable yellow oils and used 
immediately in the next step. 
EXAMPLES 179 TO 181 
General Procedure for the Preparation of 4-Amine and 4- 
hydrocarbyl-amino-1-(5-azido-5-deoxy-1-.beta.-D-ribofuranosyl)pyrazolo[3,4 
d]pyrimidines 
To a solution of the chloro azide (Example 178) (1 eq) in 1:1 THF-EtOH (10% 
w/v), was added the amine (1.2-2.0 eq) and excess Et.sub.3 N (for the 
4-amine compounds the solution was saturated with NH.sub.3 gas). The 
resulting solution was stirred for 2-24 hours. 
The reactions using amines were worked up in the following manner. The 
reaction mixture was evaporated, the residue dissolved in CH.sub.2 
Cl.sub.2 and the solution washed with aqueous NaHCO.sub.3, H.sub.2 O and 
dried. Concentration of the CH.sub.2 Cl.sub.2 solution and chromatography 
of the residue gave the purified isopropylidene N4-substituted compounds. 
The isopropylidene 4-amino compounds were isolated by evaporating the 
reaction mixture and recrystallizing the residue from EtOH. 
The isopropylidene compounds were deblocked using the procedure described 
under Example 3. 
The compounds in Table XIX (Examples 179 to 181) were prepared by this 
procedure: 
TABLE XIX 
______________________________________ 
GP-1-# Example D E m.p. (.degree.C.) 
______________________________________ 
507 179 Br NH.sub.2 
169-170.degree. 
501 180 Br N-indolinyl 
133-138.degree. 
-- 181 I NH.sub.2 
193-195.degree. 
______________________________________ 
EXAMPLES 182 TO 185 
General Procedure for the Preparation of 4-Amino- and 
4-Substituted-amino-1-(5-amino-5-deoxy-1-.beta.-D-ribofuranosyl)-3-halopyr 
azolo[3,4-d]pyrimidines and Their Hydrochloride Salts 
A solution of the azide (Examples 179 to 181) (1.0 equivalent) and 
triphenylphosphine (1.5 equivalents) in pyridine (5 mL/g of azide) was 
stirred for 2 hours. To the reaction mixture was added NH.sub.4 OH (1.25 
mL/g of azide) and the solution stirred overnight. The solution was 
evaporated, slurried in Et.sub.2 O, filtered and the insoluble residue 
dried. The resulting solid was recrystallized or converted to its HCl salt 
and crystallized to give the title compounds. 
The compounds in Table XX (Examples 182-185) were prepared by this 
procedure: 
TABLE XX 
______________________________________ 
m.p. (.degree.C.) 
GP-1-# 
Example D E (HCl salt) 
______________________________________ 
515 182 Br NH.sub.2 &gt;230.degree. 
516 183 Br N-indolinyl 
170.degree. (broad) 
547 184 I NH.sub.2 188 
658 185 Br 1,4-piperazinyl.sup.1 
195.degree. (broad) 
______________________________________ 
.sup.1 a dimer 
EXAMPLE 186 
Preparation of 1,2,3-O-Triacetyl-5-deoxy-D-ribofuranoside 
The above identified compound was prepared as described: Snyder, J.; 
Serianni, A.; Carbohydrate Research, 1987, 163:169. 
EXAMPLE 197 
Preparation of 1,2,3-O-Triacetyl-5-azido-5-deoxy-D-ribofuranoside 
To a cooled solution of 5-azido-5-deoxyribose (6.2 g, 0.035 mole) (Example 
51) in 10 mL of pyridine was added acetic anhydride (18 mL) and the 
mixture stirred for 24 hours. The mixture was concentrated, the residue 
dissolved in CH.sub.2 Cl.sub.2 and the solution washed with 5% 
NaHCO.sub.3. The organic layer was then washed with 0.5N H.sub.2 SO.sub.4, 
dried and evaporated. The residue was dissolved in CH.sub.2 Cl.sub.2, 
filtered through a plug of silica gel and the filtrate concentrated to 
afford the title compound, 9.0 g (98% yield) as a semisolid mixture of 
.alpha. and .beta. isomers. 
EXAMPLES 188-203 
General Procedure for the Preparation of 
5'-Substituted-3,4-disubstituted-pyrazolo[3,4-d]pyrimidine Nucleosides 
To a slurry of the 3,4-disubstituted pyrazolo[3,4-d]pyrimidine (5.0 mmol) 
in nitromethane, nitroethane or benzonitrile, was added the acyl-protected 
ribose (5.0-7.0 mmoles). To the stirred mixture was added BF.sub.3 
-Et.sub.2 O (7.0 mmoles) and the mixture was refluxed for 90 minutes, then 
cooled and evaporated. If a 5'-deoxy derivative was used, Et.sub.3 N was 
added prior to the evaporation of the solvent. 
The residue was taken up in CH.sub.2 Cl.sub.2, filtered and 
chromatographed. Later fractions contained the N-2 isomer. Fractions 
containing the desired N-1 isomer were combined and evaporated to yield 
the title compounds as foams. 
The compounds in Table XXI (Examples 188-203) were prepared by this 
procedure: 
TABLE XXI 
______________________________________ 
Example B' D E m.p. (.degree.C.) 
______________________________________ 
188 .O slashed.CO.sub.2 CH.sub.2 
CN NH.sub.2 
foam 
189 .O slashed.CO.sub.2 CH.sub.2 
CH.sub.2 CN 
NH.sub.2 
foam 
190 .O slashed.CO.sub.2 CH.sub.2 
.O slashed. 
NH.O slashed. 
foam 
191 CH.sub.2 N.sub.3 
Br NH.sub.2 
foam 
192 CH.sub.2 N.sub.3 
CN NH.sub.2 
foam 
193 CH.sub.2 N.sub.3 
CH.sub.2 CN 
NH.sub.2 
foam 
194 CH.sub.2 N.sub.3 
.O slashed. 
NH.sub.2 
foam 
195 CH.sub.2 N.sub.3 
4-Cl.O slashed. 
NH.sub.2 
foam 
196 CH.sub.2 N.sub.3 
4-CH.sub.3 O.O slashed. 
NH.sub.2 
foam 
197 CH.sub.2 N.sub.3 
2-thienyl NH.sub.2 
foam 
198 CH.sub.3 .O slashed. 
NH.sub.2 
foam 
199 CH.sub.3 4-CH.sub.3 O.O slashed. 
NH.sub.2 
foam 
200 CH.sub.3 4-Cl.O slashed. 
NH.sub.2 
foam 
201 CH.sub.3 2-thienyl NH.sub.2 
foam 
202 CH.sub.3 3-thienyl NH.sub.2 
foam 
203 CH.sub.3 .O slashed. 
NH.O slashed. 
foam 
______________________________________ 
EXAMPLE 204 
General Procedure for the Preparation of 3-Substituted 
1-(5-azido-5-deoxy-2,3-O-diacetyl-1-.beta.-D-ribo-furanosyl)-4-chloropyraz 
olo[3,4-d]pyrimidines,5'-Deoxy Analogs and Protected 5'-Hydroxy Analogs 
The above identified compounds were prepared from the 
pyrazolo[3,4-d]pyrimidone esters by a procedure analogous to the one 
described in Example 2 and were used immediately in the next step. 
EXAMPLES 205-221 
General Procedure for the Preparation of 
3,4-Disubstituted-1-(5-azido-5-deoxy-1-.beta.-D-ribofuranosyl))pyrazolo-[3 
,4-d]pyrimidines, 5'-Deoxy Analogs and 5'-Hydroxy Analogs 
The above identified compounds were prepared from the diestem by a 
procedure analogous to the one described in Example 167-169. Methanolic 
NH.sub.3 (method A) or NaOMe (method B) was used to deblock the 
acyl-protected nucleosides (Examples 188-204). In the case of the 
cyano-substituted compounds, these methods led to different products by 
further reaction of the cyano group. The title compounds were isolated by 
conventional techniques. 
The compounds listed in Table XXII (Examples 205-221) were prepared by this 
procedure. 
TABLE XXII 
______________________________________ 
Example 
GP-1-# B' D F m.p. (.degree.C.) 
______________________________________ 
205 612 CH.sub.2 OH 
CH.sub.2 CN 
NH.sub.2 A 
220.degree. (dec) 
206 613 CH.sub.2 OH 
CH.sub.2 C(.dbd.NH)OCH.sub.3 
NH.sub.2 B 
75.degree.(dec) 
207 695 CH.sub.2 OH 
.O slashed. 
NH.O slashed. B 
220-224.degree. 
208 507 CH.sub.2 N.sub.3 
Br NH.sub.2 B 
172.degree. (d) 
209 623 CH.sub.2 N.sub.3 
C(.dbd.NH)NH.sub.2 
NH.sub.2 A 
203-206.degree. 
210 624 CH.sub.2 N.sub.3 
CH.sub.2 CN 
NH.sub.2 A 
153-156.degree. 
211 641 CH.sub.2 N.sub.3 
.O slashed. 
NH.sub.2 B 
203-205.degree. 
212 662 CH.sub.2 N.sub.3 
4-Cl.O slashed. 
NH.sub.2 B 
175-177.degree. 
213 666 CH.sub.2 N.sub.3 
4-CH.sub.3 O.O slashed. 
NH.sub.2 B 
153-155.degree. 
214 654 CH.sub.2 N.sub.3 
2-Thienyl NH.sub.2 B 
180-181.degree. 
215 667 CH.sub.2 N.sub.3 
.O slashed. 
NH.O slashed. B 
120-125.degree. 
216 663 CH.sub.3 
.O slashed. 
NH.sub.2 B 
223-224.degree. 
217 678 CH.sub.3 
4-Cl.O slashed. 
NH.sub.2 B 
130-133.degree. 
218 679 CH.sub.3 
4-CH.sub.3 O.O slashed. 
NH.sub.2 B 
175-176.degree. 
219 664 CH.sub.3 
2-Thienyl NH.sub.2 B 
174-175.degree. 
220 685 CH.sub.3 
3-Thienyl NH.sub.2 B 
153-154.degree. 
221 665 CH.sub.3 
.O slashed. 
NH.O slashed. B 
207-208.degree. 
______________________________________ 
EXAMPLES 222-229 
General Procedure for the Preparation of 4-Amino- and 
4-Arylamino-3-substituted-1-(5-amino-5-deoxy-1-.beta.-D-ribofuranosyl)pyra 
zolo[3,4-d]pyrimidines and Their Salts 
The above-identified compounds were prepared from the 5'-azides (Examples 
205-221) by catalytic hydrogenation as described in Examples 15-20 (method 
A) or triphenylphosphine followed by ammonium hydroxide as described in 
Examples 82-83 (method B). The salts were prepared by standard methods. 
The compounds listed in Table XXIII were be prepared by these methods: 
TABLE XXIII 
______________________________________ 
GP-1-# 
Example 3- 4- Method 
Salt m.p. (.degree.C.) 
______________________________________ 
515 222 Br NH.sub.2 
B HCl &gt;230.degree. 
614 223 H NH.sub.2 
A HBr 160.degree. (d) 
625 224 CH.sub.2 CN 
NH.sub.2 
B -- 175.degree. (d) 
642 225 .O slashed. 
NH.sub.2 
A HCl 218-219.degree. 
682 226 2-thienyl 
NH.sub.2 
B HCl &gt;220.degree. 
694 227 4-CH.sub.2 O.O slashed. 
NH.sub.2 
A -- 222-225.degree. 
701 228 4-Cl.O slashed. 
NH.sub.2 
B HCl 189-194.degree. 
704 229 .O slashed. 
NH.O slashed. 
A CF.sub.3 CO.sub.2 H 
185-190.degree. 
______________________________________ 
EXAMPLES 230-231 
General Procedure for the Preparation of 4-Amino- and 
4-Arylamino-1-(5-amino-2,3-O-diacetyl-5-deoxy-1-.beta.-D-ribofuranosyl)-3- 
substituted pyrazolo[3,4-d]pyrimidines 
A slurry of 10% Pd-C in a solution (MeOH or EtOH with THF, dioxane or 
EtOAc) of the 5'-azido-2',3'-diacetate nucleoside (Examples 191-198) was 
hydrogenated in a Parr shaker at 40 psi. After disappearance of the 
starting material (TLC), the mixture was filtered and concentrated. The 
residual product was purified by recrystallization or HPLC. 
The compounds listed in Table XXIV were prepared by this method: 
TABLE XXIV 
______________________________________ 
GP-1-# Example D F C1, C2 
m.p (.degree.C.) 
______________________________________ 
-- 230 Br NH.sub.2 
OAc -- 
-- 231 .O slashed. 
NH.O slashed. 
OAc -- 
______________________________________ 
EXAMPLES 232-233 
General Procedure for the Preparation of 
3-Substituted-4(1,1-dicarboethoxyalkyl)-1-(2,3,5-O-tribenzoyl-1-.beta.-D-r 
ibofuranosyl)pyrazolo[3,4d]pyrimidines 
To a solution of the diethyl(alkyl)malonate (0.10 mol) in DMF (100 mL) was 
added 80% NaH in mineral oil (0.125 mol). After stirring for 10 minutes, a 
solution of the 
3-substituted-4-chloro-1-(2,3,5-tribenzoyl-1-.beta.-D-ribofuranosyl)pyrazo 
lo[3,4-d]pyrimidine (0.09 mol) (Example 178) in DMF (75 mL) was added 
dropwise. The solution was cooled and anhydrous trimethylamine was bubbled 
into the solution for 4 minutes. The solution was stirred for 3 hours at 
room temperature then quenched with dilute acetic acid. The mixture was 
extracted with ether-ethyl acetate (9:1) and the organic extract dried and 
concentrated. The residue was chromatographed and the appropriate 
fractions combined and evaporated to yield the title compounds. The 
identity of the compounds were confirmed by NMR. 
The compounds listed in Table XXV were prepared by this procedure: 
TABLE XXV 
______________________________________ 
Example D F m.p. (.degree.C.) 
______________________________________ 
232 Br CH(CO.sub.2 C.sub.2 H.sub.5).sub.2 
foam 
233 Br C.O slashed.(CO.sub.2 C.sub.2 H.sub.5).sub.2 
foam 
______________________________________ 
EXAMPLES 234-235 
General Procedure for Preparation of 4-Alkyl, 4-Arylalkyl- and 
3,4-disubstituted-1-(1-.beta.-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidines 
The diester (Examples 232-233) was dissolved in aqueous ethanolic sodium 
hydroxide and heated. The solution was neutralized with acetic acid, 
evaporated, extracted with hot ethanol and the extract then evaporated and 
recrystallized or chromatographed. The appropriate fractions were combined 
and evaporated to yield the title compounds. 
The compounds described in Table XXVI were prepared by the procedure: 
TABLE XXVI 
______________________________________ 
GP-1-# Example D E m.p. (.degree.C.) 
______________________________________ 
719 234 Br CH.sub.3 
204-205.degree. 
-- 235 Br CH.sub.2 .O slashed. 
-- 
______________________________________ 
EXAMPLES 286-237 
General Procedure for Preparation of 
3-Substituted-4-(1,1-dicarboethoxyalkyl)-1-(5-azido-5-deoxy-2,3-O-diacetyl 
-1-.beta.-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidines 
The above identified compounds were prepared by a procedure analogous to 
the one described in Examples 232-233 using the 
3-substituted-(5-azido-5-deoxy-2,3-O-diacetyl-1-.beta.-D-ribofuranosyl)-4- 
chloropyrazolo[3,4-d]pyrimidine. 
The compounds listed in Table XXVII were prepared by this procedure: 
TABLE XXVII 
______________________________________ 
Example D F m.p. (.degree.C.) 
______________________________________ 
236 Br C.O slashed.(CO.sub.2 C.sub.2 H.sub.5).sub.2 
-- 
237 Br CH(CO.sub.2 C.sub.2 H.sub.5).sub.2 
-- 
______________________________________ 
EXAMPLES 238-239 
General Procedure for Preparation of 4-Alkyl-, 4-Phenylalkyl- and 
4-Substituted-3-Substituted-1-(5-azido-5-deoxy-1-.beta.-D-ribofuranosyl) 
pyrazolo[3,4-d]-pyrimidines 
The above identified compounds were prepared by a procedure analogous; to 
the one described in Examples 234-235 from the 5'-azide esters described 
in Examples 236-237. 
The following compounds listed in Table XXVIII were prepared by this 
procedure: 
TABLE XXVIII 
______________________________________ 
GP-1-# Example D F m.p. (.degree.C.) 
______________________________________ 
-- 238 Br CH.sub.3 
-- 
-- 239 Br CH.sub.2 .O slashed. 
-- 
______________________________________ 
EXAMPLES 240-241 
General Procedure for the Preparation of 4-Alkyl-4-Phenylalkyl- and 
3,4-Disubstituted-1-(5-amino-5-deoxy-1-.beta.-D-ribofuranosyl)pyrazolo[3,4 
-d]pyrimidines 
The above-identified compounds were prepared by reduction of the 5-azido 
ribosides listed in Examples 238-239 by catalytic hydrogenation as 
described in Examples 15-20 or by treatment with triphenylphosphine and 
ammonium hydroxide as described in Examples 182-185. 
The compounds listed in Table XXIX were prepared by this procedure: 
TABLE XXIX 
______________________________________ 
GP-1-# Example D F m.p. (.degree.C.) 
______________________________________ 
-- 240 Br CH.sub.3 
-- 
-- 241 Br CH.sub.2 .O slashed. 
-- 
______________________________________ 
EXAMPLES 242-243 
General Procedures for the Preparation of 
3-Substituted-1-(5-deoxy-2,3-O-diacetyl-1-.beta.-D-ribofuranosyl)-4-(1,1-d 
icarboethoxyalkyl)pyrazolo[3,4-d]pyrimidines 
The above-identified compounds were prepared by a procedure analogous to 
the one described in Examples 232-233 using 
3-substituted-4-chloro-1-(5-deoxy-2,3-O-diacetyl-1-.beta.-D-ribofuranosyl) 
pyrazolo[3, 4-d]pyrimidines (Examples 198-203). 
The compounds listed in Table XXX were prepared by this procedure: 
TABLE XXX 
______________________________________ 
GP-1-# Example D F m.p. (.degree.C) 
______________________________________ 
-- 242 Br CH(CO.sub.2 C.sub.2 H.sub.5).sub.2 
-- 
-- 243 Br C(CO.sub.2 C.sub.2 H.sub.5).sub.2 CH.O slashed. 
-- 
______________________________________ 
EXAMPLES 244-245 
General Procedure for the Preparation of 4-Alkyl-, 4-Phenylalkyl- or 
4-Substituted-3-substituted-1-(5-deoxy-1-.beta.-D-ribofuranosyl)pyrazolo[3 
,4-d]pyrimidines 
The above-identified compounds were prepared from the esters (Examples 
242-243) using the procedure described in Examples 234-235. 
The compounds listed in Table XXXI were prepared by this method: 
TABLE XXXI 
______________________________________ 
GP-1-# Example D F m.p. (.degree.C.) 
______________________________________ 
-- 244 Br CH.sub.2 CH.sub.2 .O slashed. 
-- 
-- 245 Br CH.sub.2 .O slashed. 
-- 
______________________________________ 
By following the procedures described in the Detailed Description of the 
Invention and Examples 1 to 245 and using the appropriate starting 
materials and reagents, the following compounds were made: 
4-Amino-7-(5-deoxy-1-.beta.-D-ribofuranosyl)-5-vinylpyrrolo[2,3-d]pyrimidin 
e; 
4-Amino-5-ethynyl-7-(5-deoxy-1-.beta.-D-ribofuranosyl)pyrrolo[2,3-d]pyrimid 
ine; 
5-(2-Chlorophenyl)-7-(5-deoxy-1-.beta.-D-ribofuranosyl)-4-phenylaminopyrrol 
o[2,3-d]pyrimidine; 
5-(3-Chlorophenyl)-7-(5-deoxy-1-.beta.-D-ribofuranosyl)-4-phenylaminopyrrol 
o[2,3-d]pyrimidine; 
5-(4-Chlorophenyl)-7-(5-deoxy-1-.beta.-D-ribofuranosyl)-4-phenylaminopyrrol 
o[2,3-d]pyrimidine; 
5-(2-Methoxyphenyl)-7-(5-deoxy-1-.beta.-D-ribofuranosyl)-4-phenylaminopyrro 
lo[2,3-d]pyrimidine; 
5-(4-Methoxyphenyl)-7-(5-deoxy-1-.beta.-D-ribofuranosyl)-4-phenylaminopyrro 
lo[2,3-d]pyrimidine; 
5-(2-Furanyl)-7-(5-deoxy-1-.beta.-D-ribofuranosyl) 
-4-phenylaminopyrrolo[2,3-d]pyrimidine; 
7-(5-Deoxy-1-.beta.-D-ribofuranosyl)-4-phenylamino-5-(2-pyridyl)pyrrolo[2,3 
-d]pyrimidine; 
7-(5-Deoxy-1-.beta.-D-ribofuranosyl)-4-phenylamino-5-(4-pyridyl)pyrrolo[2,3 
-d]pyrimidine; 
7-(5-Deoxy-1-.beta.-D-ribofuranosyl)-5-phenyl-4-(4-pyridylamino)pyrrolo[2,3 
-d]pyrimidine; 
7-(5-Deoxy-1-.beta.-D-ribofuranosyl)-5-phenyl-4-(2-pyridylamino)-pyrrolo[2, 
3-d]pyrimidine; 
7-(5-Deoxy-1-.beta.-D-ribofuranosyl)-5-phenyl-4-(1-piperazinyl)-pyrrolo[2,3 
-d]pyrimidine; 
4-(2-Chlorophenyl)-7-(5-deoxy- 
1-.beta.-D-ribofuranosyl)-5-phenylpyrrolo[2,3-d]pyrimidine; 
4-(3-Chlorophenyl)-7-(5-deoxy-1-.beta.-D-ribofuranosyl)-5-phenylpyrrolo[2,3 
-d]pyrimidine; 
7-(5-Deoxy-1-.beta.-D-ribofuranosyl)-5-phenyl-4-(2-thiazolyl-amino)pyrrolo[ 
2,3-d]pyrimidine; 
4-Cyclohexylamino-7-(5-deoxy-1-.beta.-D-ribofuranosyl)-5-phenylpyrrolo[2,3- 
d]pyrimidine; 
7-(5-Deoxy-1-.beta.- 
D-ribofuranosyl)-5-phenyl-4-phenylthiopyrrolo[2,3-d]pyrimidine; 
4-Benzyl-7-(5-deoxy-1-.beta.-D-ribofuranosyl)-5-phenylpyrrolo[2,3-d]pyrimid 
ine;; 
7-(5-Deoxy-1-.beta.-D-ribofuranosyl)-4-ethynyl-5-phenylpyrrolo[2,3- 
d]pyrimidine; 
7-(5-Deoxy-1-.beta.-D-ribofuranosyl)-4-methyl-5-phenylpyrrolo[2,3-d]pyrimid 
ine; 
4-Benzyl-7-(5-deoxy-1-.beta.-D-ribofuranosyl)-5-iodopyrrolo[2,3-d]pyrimidin 
e; 
7-(5-Deoxy-1.beta.-D-ribofuranosyl)-5-iodo-4-methylpyrrolo[2,3-d]pyrimidine 
7-(5-Deoxy-1-.beta.-D-ribofuranosyl)-5-phenyl-4-phenylaminopyrrolo[2,3-d]py 
rimidine; 
4-Amino-5-phenyl-7-(1-.beta.-D-ribofuranosyl)pyrrolo[2,3-d]-pyrimidine; 
4-Amino-7-(5-deoxy-5-mercapto-1-.beta.-D-ribofuranosyl)-5-iodopyrrolo[2,3-d 
]pyrimidine; 
7-(5-Deoxy-5-mercapto-1-.beta.-D-ribofuranosyl)-5-iodo-4-phenylaminopyrrolo 
[2,3-d]pyrimidine; 
7-(5-Deoxy-5-mercapto-1-.beta.-D-ribofuranosyl)-5-phenyl-4-phenylaminopyrro 
lo[2,3-d]pyrimidine; 
7-(5-Amino-5-deoxy-1-.beta.-D-ribofuranosyl)-5-phenyl-4-phenylaminopyrrolo[ 
2,3-d]pyrimidine; 
7-(5,6-Dideoxy-1-.beta.-D-allofuranosyl)-5-iodo-4-phenytamino-pyrrolo[2,3-d 
]pyrimidine; 
7-(5,6-Dideoxy-1-.beta.-D-allofuranosyl)-5-phenyl-4-phenylaminopyrrolo[2,3- 
d]pyrimidine; 
4-Amino-7-(5,6-dideoxy-1-.beta.-D-allofuranosyl)-5-phenylpyrrolo[2,3-d]pyri 
midine; 
4-Amino-7-(5-deoxy-5-fluoro-1-.beta.-D-ribofuranosyl)-5-iodopyrrolo[2,3-d]p 
yrimidine; 
4-Amino-7-(5-deoxy-5-chloro-1-.beta.-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidi 
ne; 
7-(5-Deoxy-5-fluoro-1-.beta.-D-ribofuranosyl)-5-phenyl-4-phenylaminopyrrolo 
[2,3-d]pyrimidine; 
4-Amino-7-(6-azido-5,6-dideoxy-1-.beta.-D-alIofuranosyl)-5-iodopyrrolo[2,3- 
d]pyrimidine; 
7-(6-Azido-5,6-dideoxy-1-.beta.-D-allofuranosyl)-5-phenyl-4-phenylaminopyrr 
olo[2,3-d]pyrimidine; 
4-Amino-7-(6-amino-5,6-dideoxy-1-.beta.-D-allofuranosyl)-5-iodopyrrolo[2,3- 
d]pyrimidine; 
7-(6-Amino-5,6-dideoxy-1-.beta.-D-allofuranosyl)-5-phenyl-4-phenylaminopyrr 
olo[2,3-d]pyrimidine; 
5-(2-Methoxyphenyl)-7-[1-.beta.-D-ribofuranosyl]-4-phenylaminopyrrolo[2,3-d 
]pyrimidine; 
4-Amino-5-bromo-7-(5,6-didehydro-5,6-dideoxy-1-.beta.-D-allofuranosyl)pyrro 
lo[2,3-d]pyrimidine; 
7-(5,6-Didehydro-5,6-dideoxy-1-.beta.-D-allofuranosyl)-5-phenyl-4-phenylami 
nopyrrolo[2,3-d]pyrimidine; 
4-Amino-1-(5-amino-5-deoxy-1-.beta.-D-ribofuranosyl)-3-methoxypyrazolo[3,4- 
d]pyrimidine; 
4-Amino-1-(5-amino-5-deoxy-1-.beta.-D-ribofuranosyl)-3-phenoxypyrazolo[3,4- 
d]pyrimidine; 
4-Amino-1-(5-amino-5-deoxy-1-.beta.-D-ribofuranosyl)-3-phenylthiopyrazolo[3 
,4-d]pyrimidine; 
4-Amino-1-(5-amino-5-deoxy-1-.beta.-D-ribofuranosyl)-3-methylthiopyrazolo[3 
,4-d]pyrimidine; 
4-Amino-1-(5-amino-5-deoxy-1-.beta.-D-ribofuranosyl)-3-chloropyrazolo[3,4-d 
]pyrimidine; 
4-Amino-1-(5-amino-5-deoxy-1-.beta.-D-ribofuranosyl)-3-cyclopropylpyrazolo[ 
3,4-d]pyrimidine; 
4-Amino-1-(5-amino-5-deoxy-1-.beta.-D-ribofuranosyl)-3-dimethylaminopyrazol 
o[3,4-d]pyrimidine; 
4-Amino-1-(5-amino-5-deoxy-1-.beta.-D-ribofuranosyl)-3-fluoropyrazolo[3,4-d 
]pyrimidine; 
4-Amino-1-(5-amino-5-deoxy-1-.beta.-D-ribofuranosyl)-3-(3-pyridyl)pyrazolo[ 
3,4-d]pyrimidine; 
1-(5-Amino-5-deoxy-1-.beta.-D-ribofuranosyl)-4-(3-chlorophenyl)-3-(4-methox 
yphenyl)pyrazolo[3,4-d]pyrimidine; 
1-(5-Amino-5-deoxy-1-.beta.-D-ribofuranosyl)-4-(4-chlorophenyl)-3-(4-methox 
yphenyl)pyrazolo[3,4-d]pyrimidine; 
1-(5-Amino-5-deoxy-1-.beta.-D-ribofuranosyl)-4-(4-ethoxyphenyl)-3-(4-methox 
yphenyl)pyrazolo[3,4-d]pyrimidine; 
1-(5-Amino-5-deoxy-1-.beta.-D-ribofuranosyl)-4-(3-carboxamidophenylamino)-3 
-(4-methoxyphenyl)pyrazolo[3,4-d]pyrimidine; 
1-(5-Amino-5-Deoxy-1-.beta.-D-ribofuranosyl)-4-(2-furanyl)-3-(4-methoxyphen 
yl)pyrazolo[3,4-d]pyrimidine; 
1-(5-Deoxy-1-.beta.-D-ribofuranosyl)-3-(4-methoxyphenyl)-4-(phenylamino)pyr 
azolo[3,4-d]pyrimidine; 
1-(5-Deoxy-1-.beta.-D-ribofuranosyl)-3-(3-methoxyphenyl)-4-(phenylamino)pyr 
azolo[3,4-d]pyrimidine; 
1-(5-Deoxy-1-.beta.-D-ribofuranosyl)-3-(2-pyridyl)-4-(phenylamino)pyrazolo[ 
3,4-d]pyrimidine; 
1-(5-Deoxy-1-.beta.-D-ribofuranosyl)-3-(4-methoxyphenyl)-4-(4-pyridylamino) 
pyrazolo[3,4-d]pyrimidine; 
1-(5-Deoxy-1-.beta.-D-ribofuranosyl)-3-(3-methoxyphenyl)-4-(4-pyridylamino) 
pyrazolo[3,4-d]pyrimidine; 
1-(5-Deoxy-1-.beta.-D-ribofuranosyl)-3-(2-pyridyl)-4-(4-pyridylamino)pyrazo 
lo[3,4-d]pyrimidine; 
1-(5-deoxy-1-.beta.-D-ribofuranosyl)-3-(4-methoxyphenyl)-4-(2-methoxyphenyl 
amino)pyrazolo[3,4-d]pyrimidine; 
1-(5-Deoxy-1-.beta.-D-ribofuranosyl)-3-(3-methoxyphenyl)-4-(2-methoxyphenyl 
amino)pyrazolo[3,4-d]pyrimidine; 
1-(5-Deoxy-1-.beta.-D-ribofuranosyl)-3-(4-pyridyl)-4-(2-methoxyphenylamino) 
pyrazolo[3,4-d]pyrimidine; 
1-(5-Deoxy-1-.beta.-D-ribofuranosyl)-3-(4-methoxyphenyl)-4-(2-imidazolylami 
no)pyrazolo[3,4-d]pyrimidine; 
1-(5-Deoxy-1-.beta.-D-ribofuranosyl)-3-(3-methoxyphenyl)-4-(2-imidazolylami 
no)pyrazolo[3,4-d]pyrimidine; 
1-(5-Deoxy- 
1-.beta.-D-ribofuranosyl)-3-(2-pyridyl)-4-(2-imidazolylamino)pyrazolo[3,4- 
d]pyrimidine; 
1-(5-Deoxy-1-.beta.-D-ribofuranosyl)-3-(2-pyrazinyl)-4-phenylaminopyrazolo[ 
3,4-d]pyrimidine; 
1-(5-Deoxy-1-.beta.-D-ribofuranosyl)-3-(2-pyrazinyl)-4-(N-indolinyl)pyrazol 
o[3,4-d]pyrimidine; 
1-(5,6-Dideoxy-1-.beta.-D-allofuranosyl)-3-phenyl-4-phenylaminopyrazolo[3,4 
-d]pyrimidine; 
4-Amino-1-(5,6-dideoxy-1-.beta.-D-allofuranosyl)-3-iodopyrazolo[3,4-d]pyrim 
idine; 
1-(5-Deoxy-1-.beta.-D-ribofuranosyl)-3-phenyl-4-phenylthiopyrazolo[3,4-d]py 
rimidine; 
1-(5-Amino-5-deoxy-1-.beta.-D-ribopuranosyl)-3-bromo-4-methylpyrazolo[3,4-d 
]pyrimidine; 
1-(5-Amino-5-deoxy-1-.beta.-D-ribofuranosyl)-4-methyl-3-iodopyrazolo[3,4-d] 
pyrimidine; 
7-(5-Deoxy-1-.beta.-D-ribofuranosyl)-5-iodo-4-methylpyrrolo[2,3-d]pyrimidin 
e; 
4-Methyl-3-phenyl-1-(1-.beta.-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine; 
1 
-(5-Deoxy-1-.beta.-D-ribofuranosyl)-3-phenyl-4-(phenylmethyl)pyrazolo[3,4- 
d]pyrimidine; and 
7-(5-Amino-5-deoxy-1-.beta.-D-ribofuranosyl)-5-bromo-4-chloropyrrolo[2,3-d] 
pyrimidine(GP-1-608). 
EXAMPLE A 
A Method of Measuring the Inhibition of Adenosine Kinase Activity 
Inhibition of enzyme activity was determined using a 0.1 mL assay mixture 
containing 50 mM Tris-maleate, pH 7.0, 0.1% (w/v) BSA, 1 mM ATP, 1 mM 
MgCl.sub.2, 0.5 .mu.M [U-.sup.14 C]adenosine (500 mCi/mmol) and 0.1 .mu.g 
of purified pig heart adenosine kinase. Different concentrations of the 
test compounds were incubated in the assay mixture for 20 min. at 
37.degree. C. From each reaction mixture, 20 .mu.l portions were removed 
and spotted on 2 cm.sup.2 pieces of Whatman DE81 filter paper. The papers 
were then washed to remove [.sup.14 C]adenosine in 1 mM ammonium formate 
followed by deionized water and finally 95% ethanol. The papers were 
dried, and [.sup.14 C]AMP measured by scintillation counting. Activities 
were determined from the amount of [.sup.14 ]LAMP formed. 
A1 receptor binding affinity was determined using 0.5 mL mixture containing 
50 mM Tris HCl, pH 7.4, 1 nM [.sup.3 H]cyclohexyladenosine (CHA) and 0.5 
mg of neuronal membrane incubated with different concentrations of the 
test compound for 60 min at 37.degree. C. The reaction was stopped and 
unbound [.sup.3 H]CHA removed by rapid filtration through Whatman GF/B 
filters. The filter papers, were then solubilized and bound [.sup.3 H]CHA 
determined by scintillation counting. 
Inhibition of adenosine deaminase activity was determined 
spectrophotometrically using a 1 mL assay mixture containing 50 mM 
potassium phosphate, pH 7.0, 1 mM ADP, 2.5 mM alpha-ketoglutarate, 15 
units glutamic dehydrogenase, 0.125 mM NADH, 80 .mu.M adenosine and 0.002 
units of calf intestinal mucosa adenosine deaminase. Different 
concentrations of the test compounds were incubated in the assay mixture 
for 10 min at 37.degree. C. The reaction was monitored continuously for 
oxidation of NADH from the change in absorbance at 340 nm. 
Illustrative of the invention, the compounds designated GP-1-515, GP-1-608, 
GP-1-683, GP-1-695, GP-1-718, GP-1-704, GP-1-665, and GP-1-667, were found 
to have an IC.sub.50 of less than 10 nM in the adenosine kinase inhibition 
assay. The compound GP-1-515 was found to be much less potent in the A1 
receptor assay and in the adenosine deaminase inhibition assay, having an 
IC.sub.50 greater than 100 .mu.M in the A1 receptor assay and an IC.sub.50 
greater than 1000 .mu.M in the adenosine deaminase inhibition assay. 
EXAMPLE B 
Adenosine Kinase Inhibition in Intact Cells 
Inhibition of adenosine kinase in intact cells was determined from the 
amount of incorporation of radioisotope from adenosine into the adenylates 
(AMP, ADP and ATP) in the presence of adenosine deaminase inhibition. 
Capillary endothelial cells from bovine heart were incubated for 60 min. 
with 20 .mu.M 2'-deoxycoformycin, a potent adenosine deaminase inhibitor. 
Different concentrations of the test compounds were then added to the 
cells and incubated for 15 min. after which 5 .mu.M [.sup.3 H]adenosine 
was added and the cells incubated for a further 15 min. The media was then 
discarded and the cells were treated with 50 .mu.l 0.4M perchloric acid, 
centrifuged and the supernalants neutralized with 100 .mu.l alanine: freon 
(1:4). Radioisotope-labeled adenylates were separated by TLC on PEI 
cellulose plates developed in methanol:water (1:1) and incorporation of 
.sup.3 H determined by scintillation counting. 
Illustrative of the invention, the compounds designated GP-1-515, GP-1-683 
and GP-1-665 were shown to have an IC.sub.50 of 9 nM, 73 nM and 4.5 nM, 
respectively, in the adenosine kinase inhibition assay in intact cells. 
EXAMPLE C 
Effect on Adenosine Kinase Inhibition on Acute I.V. Hemodynamics in the Rat 
The ability of the adenosine kinase inhibitor GP-1-238 to show effects on 
blood pressure, heart rate or body temperature was compared in 
anesthetized and conscious rats. Sprague Dawley rats were anesthetized 
with pentobarbital and catheterized in the jugular vein and carotid 
artery. GP-1-238 (0.1-5 mg/kg/min) was infused intravenously in stepwise 
increments (0.2 mL/min.times.5 minutes). The experiments in conscious rats 
were conducted in the same manner after rats had been catheterized and 
allowed to recover for 2 days following surgery. In conscious rats, in 
contrast to anesthetized animals, no hemodynamic effects were seen at 
doses which completely inhibited adenosine kinase in vivo See FIG. 1. 
EXAMPLE D 
Inhibition of Neutrophil Adherence to Fibroblasts or Endothelial Cells 
The ability of an adenosine kinase inhibitor to affect neutrophil adherence 
to fibroblasts and endothelial cells was evaluated in a cell culture 
model. Cultures of human dermal fibroblasts or human umbilical vein 
endothelial cells were washed and then incubated for 2 hours at 37.degree. 
C. in a 5% CO.sub.2 atmosphere in fresh medium containing different 
concentrations of the adenosine kinase inhibitors GP-1-272 and GP-1-456. 
These incubations were carried out in the presence of fMLP-stimulated 
human neutrophils isolated from whole blood (1.25.times.106/mL) with or 
without adenosine deaminase (0.125 U/mL). At the end of the incubation, 
the medium was removed and the monolayers of fibroblasts or endothelial 
cells and adherent neutrophils were fixed by addition of formaldehyde 
(3.7%) and, after washing to remove non-adherent neutrophils, adherent 
neutrophils were stained with Weigart's hematoxylin and counted under a 
light microscope. The results depicted in FIG. 2 show that the adenosine 
kinase inhibitors GP-1-272 and GP-1-456 inhibit neutrophil adhesion to 
endothelial cells and that this inhibition is reversed by adenosine 
deaminase treatment. 
EXAMPLE E 
Improved Survival in Endotoxemia in Adensoine Kinase Inhibitor-Treated Mice 
An adenosine kinase inhibitor (GP-1-515) was used to increase endogenous 
adenosine production in vivo. FIG. 9 shows the results of an experiment in 
which Balb/C mice received an intravenous injection of 900 ug of E. coli 
LPS (Sigma Chemical Co., St Louis, Mo.) followed immediately by an 
intravenous injection of an adenosine kinase inhibitor (GP-1-515) or 
carrier (10 animals per group). The adenosine kinase inhibitor-treated 
mice showed a dose-dependent increase in survival over that observed in 
the placebo group (p=0.015). Animals receiving 200 mg/kg i.p. of the 
adenosine receptor antagonist 8-p-sulphophenyltheophylline (8SPT) 30 
minutes prior to the injection of LPS and drug completely inhibited the 
protective effect of GP-1-515. 
EXAMPLE F 
Adenosine Kinase Inhibitor as Prophylactic Treatment for Endotoxemia 
As shown in Example E, intravenous treatment with an adenosine kinase 
inhibitor improves survival of mice if administered immediately after 
intravenous injection with LPS. In the following experiments, it was shown 
that an adenosine kinase inhibitor (GP-1-515) protects animals against 
endotoxemia if administered prophylactically. In these experiments, 25-30 
gram male Balb/C mice received oral GP-1-515 (5 mg/kg in water) or vehicle 
by gavage. Six hours later, the animals received an intravenous injection 
of 700 ug of E. coli LPS (Sigma Chemical Co.). In an experiment involving 
10 animals per group, 50% of mice in the adenosine kinase 
inhibitor-treated group survived for 2 days, while none of animals in the 
vehicle treated group survived for that time period (data not shown). 
EXAMPLE G 
Efficacy of an Adensoine Kinase Inhibitor in Model of Chronic Sepsis, Cecal 
Ligation and Puncture 
Cecal ligation and puncture (CLP) is a model of bacterial peritonitis and 
septic shock which mimics systemic infections in humans. In these 
experiments, male CD rats were fasted overnight and treated orally with 
either GP-1-515 (5 mg/kg) or vehicle by gavage. Two hours later, animals 
were anesthetized with ether and the anterior abdominal wall was shaved. A 
midline incision was made and the cecum was exteriorized and ligated with 
3-0 silk suture near the ilial-cecal junction without causing bowel 
obstruction. The cecum was punctured twice on the anti-mesenteric side 
using a yellow tip disposable pipette tip (Fisher, Tustin, Calif.) and 
squeezed to ensure patency. The cecum was placed back in the abdominal 
cavity and the peritoneal wall was closed with 5-0 nylon sutures. The skin 
was closed with 9 mm stainless steel wound clips. The animals were 
resuscitated with a subcutaneous injection of 2 mL of sterile saline. No 
antibiotics were administered. The results of the experiments are 
presented in FIG. 10, which shows that the adenosine kinase inhibitor, 
GP-1-515, improved survival in this model of septic shock. 
EXAMPLE H 
Inhibition of TNF-Alpha Production by an Adenosine Kinase Inhibitor 
In these experiments, plasma TNF-.alpha. levels were measured in 
GP-1-515and placebo-treated mice (8 animals per group). Balb/C mice 
received an intravenous injection of E. coli LPS (900 ug/animal) (Sigma 
Chemical Co.) followed immediately by a second injection with 0.1 mg/kg of 
GP-1-515 or vehicle. Some animals were pretreated with an intraperitoneal 
injection of the adenosine receptor antagonist 8SPT. One hour later, blood 
was obtained from ether anesthetized animals by intracardiac puncture 
using a heparinized syringe. Blood samples were chilled on ice, 
centrifuged at 5000 rpm in a microcentrifuge for 5 minutes, and the plasma 
removed. TNF-.alpha. levels in the plasma were assayed by ELISA according 
to the instructions provided by the manufacturer (Endogen, Cambridge, 
Mass.). As shown in FIG. 11, the adenosine kinase inhibitor, GP-1-515, 
significantly decreased plasma TNF-.alpha. levels (p&lt;0.01). The decrease 
was prevented in animals pre-treated with 200 mg/kg i.p. of the adenosine 
receptor antagonist 8SPT 30 minutes prior to injection with LPS and 
GP-1-515. 
EXAMPLE I 
Effects on the Adenosine Kinase Inhibitor GP-1-456, in a Model of 
Inflammation 
The adenosine kinase inhibitor, GP-1-456, was examined for 
anti-inflammatory activity in adjuvant arthritis in rats. Lewis rats 
received a single subcutaneous tail injection of 0.75 mg Mycobacterium 
butyricum mixed in paraffin oil on day 0. The rats were treated with 3 
mg/kg GP-1-456 p.o., or a vehicle control from day 1 through day 19. Hind 
paw volume was measured by Mercury displacement. On day 19, total mean 
hind paw edema, in mL, was 1.41.+-.0.21 for treated rats versus 
2.55.+-.0.20 for untreated rats (p&lt;0.05). This model demonstrated 
significant reduction of hind paw edema in adjuvant arthritis in animals 
treated with the adenosine kinase inhibitor, GP-1-456. 
EXAMPLE J 
Endotoxic Shock in Pigs 
Male or female Yucatan miniature pigs (20-27 kg) were pre-medicated with 
xylazine (550 mg i.m.), ketamine (150 mg i.m.), and atropine (1 mg i.m.). 
The animals were intubated, administered 10 mg/kg of pentobarbital 
intravenously, and covered with a blanket to maintain normal body 
temperature. A pentobarbital infusion was maintained at 10 mg/kg/hr. The 
pigs were ventilated with 30% O.sub.2 using a Harvard large animal 
respirator at 7-10 breaths/rain in order to achieve a blood pH of 
approximately 7.45-7.50. A bolus of 4 mg of Pancuronium bromide was given 
to inhibit muscle contraction in response to the cautery and the animals 
were hydrated with a continuous infusion of saline at 60-80 cc/hr. A left 
thoracotomy was performed in the third intercostal space and the pulmonary 
artery, carotid artery, external jugular vein were cannulated with PE-190 
tubing. A line was also placed in the left atrium and a catheter tipped 
micromanometer was inserted into the left ventricle via an apical stab 
wound. A transit-time flow probe was placed on the pulmonary artery. 
Pressure readings were obtained using a Statham pressure transducer 
connected to a Gould strip chart recorder. Heparinized arterial blood 
samples for blood gases were obtained through the carotid arterial 
catheter. Drugs and endotoxin were infused through the external jugular 
catheter. 
After the animals had stabilized, a continuous infusion of GP-1-515 (0.3 
.mu.g/kg/m in) was started, followed 30 min later by a slow infusion of E. 
Coli 0111:B4 lipopolysaccharide (LPS) (Sigma Chemical Co., St. Louis, Mo.) 
over 30 min. The animals continued to receive GP-1-515 or vehicle for 4 
additional hours and were ventilated with a fixed tidal volume and 
FiO.sub.2. The most prominent findings pertained to gas exchange and 
pulmonary function, with marked protection observed in the 
GP-1-515-treated animals. Control animals developed severe hypoxemia, 
hypercapnia, and acidosis. However, the GP-1-515-treated animals had 
minimal changes in blood gases (see FIG. 12a,b,c for pH, pO.sub.2, and 
pCO.sub.2 data). Blood pressures were similar in the treated and control 
groups, although the control group,, required a significantly higher heart 
rate to maintain this pressure (see FIG. 12d,e for blood pressure and 
heart rate data). Other hemodynamic parameters, including pulmonary flow 
and pulmonary vascular resistance, were either not different or modestly 
improved compared to untreated animals. 4/7pigs in the control group died 
(several of whom had frank pulmonary edema) compared to 1/7in the 
GP-1-515-treated group. Upon completion of the study, the surviving 
animals were euthanized with an intravenous bolus of pentobarbital. 
EXAMPLE K 
Suppression of Vascular Leakage by Adenosine Kinase Inhibitor 
Leakage of plasma into inflamed skin was measured using Evans' Blue (Sigma 
Chemical Co., St. Louis, Mo.), which binds irreversibly to albumin. Evans' 
Blue and bovine serum albumin (Sigma) were mixed (20 and 40 mg/mL PBS, 
respectively) and incubated for 15 min at room temperature in order to 
form a conjugate. The mixture was then filtered through a 0.45 .mu.m pore 
syringe filter and immediately used. Male SA rats (150-200 g, Simonsen, 
Gilroy, Calif.) that had been shaved the previous day received GP-1-792 
(4-amino-1-(5-amino-5-deoxy-2,3-di-O-acetyl-1-.beta.-D-ribofuranosyl)-3-br 
omopyrazolo[3,4-d]pyrimidine, an orally bioavailable pro-drug of GP-1-515) 
or vehicle by gavage 1 hr prior to induction of skin lesions. The rats 
were then briefly anesthetized with halothane and dorsal skin injection 
sites (up to 4/rat) were stamped with a 12 mm test tube inked with a felt 
pad. Every site received 100 .mu.l of an inflammatory agent or PBS 
injected intradermally into the middle of the circular mark. Inflammatory 
agents included carrageenan (1%) and histamine (10.sup.-3 M) (Sigma). Rats 
were then returned to their cages. Two hrs later, animals were again 
anesthetized and 0.5 mL blood was obtained through orbital plexus bleed. 
Following sacrifice of the rats, the injection sites were excised along 
the marked lines, and subcutaneous muscle and fat layers were removed from 
the skin biopsies. Each skin biopsy was weighed and cut in four, and its 
Evans' Blue content was extracted overnight by incubation in 1 mL 
N,N-dimethylformamide (Aldrich, Milwaukee, Wis.). The absorbance at 650 nm 
of plasma samples and skin extracts was read in a plate reader, and the 
plasma content of each skin piece was calculated and expressed as .mu.l 
plasma/g tissue. 
Carrageenan and histamine induced plasma leakage in rat skin, since the 
plasma content was significantly higher in skin sites injected with these 
agents compared to PBS-injected sites. Treatment with GP-1-792 (5 mg/kg 
p.o. 1 hr prior to experiment) significantly inhibited carrageenan- and 
histamine-induced plasma leakage by 47.+-.5 and 51.+-.5%, respectively 
(FIG. 14). Since histamine acts directly on endothelial cells, the 
protective effect of GP-1-792 can be mediated by an effect on endothelial 
cells in addition to a decrease in neutrophil accumulation in the tissue 
in this model as occurs with carrageenan. 
EXAMPLE L 
Effect on an Adenosine Kinase Inhibitor in a Burn Model 
Female CF-1, non-Swiss, non-albino mice (age 8-12 weeks) (Charles River 
Laboratory, Wilmington, Mass.) were anesthetized with a 1-2 minute 
exposure to methoxyflurane (Abbott Labs, Chicago, Ill.). The shaved dorsum 
was burned by a 6-second exposure to steam using a 3.5.times.5.5 cm 
template to produce a 32% total body surface area full thickness burn. 
Animals were resuscitated with 1 mL of normal saline i.p., followed by 0.1 
mg/kg i.p./injection of GP-1-515 or an equal amount of vehicle. Animals 
also received an i.p. injection of morphine sulfate (15 mg/kg). The 
animals were administered a second injection of GP-1-515 (0.1 mg/kg) or 
vehicle i.p. 6 hours after the burn. Animals were fasted for 4 hours 
before the burn and for 24 hours thereafter. After 48 hours, the animals 
were sacrificed by cervical dislocation and the abdomen was opened using 
aseptic techniques. Mesenteric lymph nodes were harvested and weighed in 
sterile bags, homogenized in Trypticate Soy Broth (BBL, Becton Dickinson 
Microbiology System, Cockeysville, Md.). Ser. dilutions of the homogenate 
were plated on head-brain-infusion agar plates (BBL, Becton Dickinson) at 
37.degree. C. and the presence or absence of bacterial colonies was 
determined 48 hours later. In experiments involving approximately 120 
animals, 52% of control mice had positive mesenteric lymph node cultures 
compared to 32% of GP-1-515-treated mice (p&lt;0.05). Hence bacterial 
translocation after a severe burn was significantly decreased by GP-1-515.