CA2199615A1 - Erythro-hydroxynonyladenine analogs with enhanced lipophilic and anti-ischemia traits - Google Patents

Abstract

An epoxy-silicate nanocomposite is prepared by dispersing an organically modified smectite-type clay in an epoxy resin together with diglycidyl ether of bisphenol-A (DGEBA), and curing in the presence of either nadic methyl anhydride (NMA), and/or benzyldimethyl amine (BDMA), and/or boron trifluoride monoethylamine (BTFA) at 100-200 ~C. Molecular dispersion of the layered silicate within the crosslinked epoxy matrix is obtained, with smectite layer spacings of 100.ANG. or more and good wetting of the silicate surface by the epoxy matrix. The curing reaction involves the functional groups of the alkylammonium ions located in the galleries of the organically modified clay, which participate in the crosslinking reaction and result in direct attachment of the polymer network to the molecularly dispersed silicate layers. The nanocomposite exhibits a broadened Tg at slightly higher temperature than the unmodified epoxy. The dynamic storage modulus of the nanocomposite was considerably higher in the glassy region and very much higher in the rubbery plateau region when compared to such modulus in the unmodified epoxy.

Description

~- W097128803 PCT~S96101990 "
E~'THRO-HYDROXYNONYLADENINE ANALOGS
WITH ENHA~r~ED LIPOP~ILIC AND ANTI-IS~MT~ TRAITS

This invent:ion is in the fields of chemistry and pharmacology, ancl relates to drugs that can inhibit an enzyme called adenosine d~min~e (ADA, also ~nown as adenosine aminohydrolase). ADA-inhibiting drugs can be used to reduce the 10 enzymatic degradat:ion of chemotherapeutic and anti-viral drugs, thereby increasincr the therapeutic utility of such drugs. As disclosed herein, ADA-inhibiting drugs can also be used to protect heart musc:le and brain tissue against damage caused by ischemia (inadequate blood Elow) or hypoxia (inadequate oxygen 15 supply), as occurs during stroke, cardiac arrest, heart attack, asphyxiation, and various ol:her crises.
The ~m~l ian enzyme called adenosine deaminase (ADA), which is designated E.C.3.5O4.4 under the international enzyme classification system, converts adenosine into inosine by 20 removing an amine group from the #6 carbon in the two-ring adenyl structure of adenosine. ADA can also degrade a number of other molecules, including several nucleoside analogs that are used in cancer chemotherapy or for anti-viral therapy. Since ADA
is known to reduce the therapeutic utility of various drugs used 25 to treat cancer and viral infections, a substantial amount of work has been done to develop drugs which function as ADA
inhibitors. The ADA inhibitor drugs can be used as adjuncts (i.e., as secondary agents to increase the effectiveness of a primary drug) to pI-olong the metabolic half-lives of therapeutic 30 drugs during cancel or anti-viral chemotherapy. ADA inhibitors can also be used to artificially create ADA deficiencies, which are of interest to some researchers.
A compound called erythro-hydroxynonyladenine (abbreviated as EHNA, usually pronounced as "eenahl') is a relatively mild ADA
35 inhibitor, and is of particular interest herein. EHNA is a stereoisomer with the following chemical structure, which shows the numbering of t;he carbon atoms in the nonyl "side chain~' (i.e., in the erythro-hydroxy-nonyl straight chain which is attached to the dou;ble-ringed adenyl group):

CA 0219961~ 1997-03-10 - W097/28803 PCT~S96/01990 ~'1 ~ ..

9 ~7~/

The "erythro-" prefix _ndicates a certain stereoisomeric arrangement of the atoms attached to the #2 and #3 carbon atoms in the nonyl side chain. Both the #2 and #3 carbon atoms are 10 chiral atoms (i.e., carbon atoms with four different groups attached to them, so that the spatial arrangement of the four groups will have either a dextrorotatory (or right, or +) or levorotatory (or sinister, or -) configuration, depending on how they rotate polarized light passing through an aqueous solution 15 of a purified stereoisomer. These rotations are abbreviated as D/L, R/S, or ~/-. Other purified steroeisomers having the same atoms as erythro compounds, but in a different stereoisomeric arrangement, are referred to as "threo-" compounds.
A "racemic" mixture of EHNA (i.e., a mixture containing 20 both D/R/~ and L/S/- isomers) was identified as an ADA inhibitor in Schaeffer and Schwender 1974. Subsequent reports, including ~astian et al 1981 and Baker and Hawkins 1~82, identified the (+)-2S,3R isomer (the erythro isomer) as the most potent ADA
inhibitor from among the various hydroxynonyladenine isomers.
Various analogs and derivatives of EHNA have been described in reports such as Harriman et al 1992. Those other analogs are not related to the EHNA analogs described herein.
EHNA apparently is metabolized and cleared from the mammalian bloodstream fairly rapidly (McConnell et al 1980;
30 Lambe and Nelson 1982). In addition, EHNA's activity as an ADA
inhibitor drug is not as strong as various other ADA inhibitor drugs, including deoxycoformycin (dCF, also known as Pentostatin). The so-called ~Ki~ value of dCF (i.e., the negative log value of a molar concentration of dCF required to 35 inactivate a standardized quantity of ADA) is very low, about 2.5 x lo-12, which indicates that dCF binds to ADA very tightly;
dCF is sometimes called a "suicide inhibitor," which indicates that the binding between dCF and ADA is ef~ectively irreversible, and neither molecule can be regenerated. This process of irrevl-rsible birlding is al~so referred to as ~poisoning" an erLzyme.
Because of its potency as an ADA inhibitor, dCF was tested - by several research teams to dete ;ne whether it can ~e used 5 therapeutically. Although dCF reportedly provided some beneficial activ~ y in cardiovascular models (e.g., Dorheim et al 1991), neuroprotection (e.g., Phillis and O'Regan 1989), and cancer therapy, i1_ was found to cause serious toxic side effects (e.g., O'Dwyer et: al 1986~. Therefore, attention subsequently lO returned to EHNA cmd various other milder or "softer" ADA
inhibitors, in th~ hope that the milder A~A inhibitors would have fewer side eifects and would be less toxic. The Ki value of (+)-EHNA is about 6 x 109, which indicates that EHNA binds to ADA a~out a thousand times less tightly than dCF.
This invention discloses a class of compounds in which a hydrogen atom coupled to one of the "far end" carbon atoms (i.e., the #8 or #9 car~on atoms) is replaced by a hydroxyl group, to create a #8 or #9 hydroxylated EHNA, or by various other types of moieties to create other #8 or #9 analogs 20 (including analogs which ar~ more soluble in lipids than the hydroxylated analogs, and which have shown better therapeutic utility against ischemia). As ~;scll~sed below, the analogs that are of interest h/-rein have both (1) a binding affinity ~or the ADA enzyme which Ls in the desired range, with a Ki value 25 between about 10 7 and about 10-10, and (2) additional properties which render them ,ubstantially more useful and beneficial than unmodified EHNA in protecting heart tissue and/or brain tissue against damage cau;ed by ischemia (inadequate blood flow) or hypoxia (inadequat:e oxygen supply), as occurs during stroke, 30 heart attack, carcl:iac arrest, asphyxiation, and various other types of crises or conditions.
The utility oi- ADA-inhibiting drugs in protecting heart muscle or brain tissue against ;~chem; c or hypoxic damage has not been widely re!c:ognized prior to this invention. Instead, 35 nearly all research on ADA inhibitors has focused on their potential ability, as adjuncts, to slow the degradation of anti-cancer or anti-viral drugs b~ the ADA enzyme, in order to increase the efficacy of such anti-cancer or anti-viral drugs.
However, as disclosed herein, various EHNA analogs have CA 0219961~ 1997-03-lo -- W097/28803 PCT~S96/01990 also been shown to provide substantial protection ~or the heart against ischemic or hypoxic damage, as would occur during a heart attack, cardiac arrest, or surgery reguiring cardiopulmonary bypass. It is also believed that at least some 5 of these analogs may also provide substantial protection ~or brain tissue against ischemic or hypoxic damage due to stro~e, cardiac arrest, asphyxiation, etc.
It should be noted that the ADA enzyme acts inside cells.
Despite the fact that this enzyme activity can affect the 10 quantity of adenosine released by a cell, which will react with adenosine receptors on other cells, the fact r~r~; n~ that the ADA enzyme, itself, functions almost exclusively inside cells.
Therefore, an ADA inhibitor drug must enter ~ -lian cells in order to function properly, and its efficacy will depend to a 15 large extent on how readily it can be taken into cells.
One object of this invention is to disclose a class of analogs of EHNA which have been modified at the #8 or #9 carbon atoms on the side chain, in a manner which substantially improves the therapeutic efficacy of these analogs against 20 ischemic or hypoxic damage to heart muscle or brain tissue, compared to either unmodified or hydroxylated EHNA.
Another object of this invention is to disclose a class of analogs of EHNA which have been modified at the ~8 or #9 carbon atoms on the side chain, in a manner which provides various 25 therapeutic advantages for these analogs while ret~;ning a binding affinity for the ADA enzyme which is in the desired range (preferably with a Ki value between about 107 and about 101~). This status as a relatively mild and reversible ADA
inhibitor allows such analogs to inhibit ADA activity at 30 therapeutically effective levels, without irreversibly inactivating (poisoning) the ADA enzyme and increasing the risk of toxic side effects.
Another object of this invention is to disclose synthetic reagents and methods that can be used to create 35 pharmacologically valuable analogs of EHNA which contain hydroxyl, halide, acid, ester, ether, amine, amide, imide, azide, nitrile, or other moieties at various controllable locations in the nonyl side chain, and particularly containing novel moieties coupled to the #8 or ~9 carbon atoms in the side -- W097/28803 PCT~S96101990 chain.
Another object of this invention is to disclose a new set o~ EHNA analogs which can be used to slow down the degradation by the ADA enzyme of certain types of anti-cancer, anti-viral, 5 or other therapeutic drugs.
These and ot:her objects of the invention will become more clear and apparent from the following summary, detailed description, and examples.

SUMM~ OF THE lN V l~:N'l lt lN
Analogs of erythro-hyclroxynonyladenine (EHNA) are disclosed which have been :modified by bonding various types of moieties to the #8 or #9 carbon atoms in the "side chain" portion of the 15 molecule (i.e., t.he 9-carbon erythro-hydroxynonyl straight chain portion, which i, attached to an adenosine ring structure).
Analogs of EHNA with various moieties coupled to the #8 or #9 positions on the :,ide chain. have been discovered to have new and unexpected value ~s therapeutic drugs, as described below.
In an early set of tests, one of the hydroxylated EHNA
analogs describecl below (designated herein as 9-OH-EHNA, where the hydroxyl moiety was coupled to the ~9 carbon atom on the EHNA side chain) was discovered to have a significant advantage in protecting hea.rt muscle against ischemic damage, compared to 25 unmodified EHNA. ~'his assay is described in Example 4.
Based on that. early fi].~ding, subsequent research on other newly-synthesized analogs o:E EHNA showed that several analogs had major therapeutic advantages, not just over unmodified EHNA, but also compared to the hydroxylated analogs. This subsequent 30 research, which used a some~Jhat different heart perfusion technique as described in Example 6, indicated that the 9-OH-EHNA analog did not perform substantially better than unmodified EHNA in most param,eters that were used to measure heart protection; howeve:r, by the time that was confirmed, it had been 35 discovered that several other EHNA analogs showed a marked improvement over e:ither the unmodified or the hydroxylated form of EHNA, and subsequent research was devoted to those other analogs while 9-OH-EHNA was disregarded and not tested further.
The analogs which have showed the best combinations of CA 0219961~ 1997-03-10 -- W097/28803 PCT~S96/0~990 traits in the research done to date are believed to be more lipophilic (i.e., more soluble in lipids and other fatty, non-polar fluids, and less soluble in water) than the hydroxylated analogs. These lipophilic analogs are disclosed below, along 5 with chemical methods for synthesizing them and any other desired analog of EHNA which has been modified at the ~8 or ~9 carbon atom of the side chain.
Any such analog which is synthesized as described herein can be screened, using assays as described below or otherwise lo known to those skilled in the art, to determine whether a particular analog has a desired combination of traits as described herein. These traits primarily involve (1) non-toxic potency as a mild, reversible ADA inhibitor; (2) a desirable level of lipophilicity, to increase cellular uptake; and (3) 15 therapeutic utility in reducing ischemic or hypoxic tissue damage, or in reducing ADA degradation of anti-cancer, anti-viral, or other drugs.

BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE 1 depicts a series of chemical reactions used to create 9l-hydroxy(+)-EHNA, designated as Compound ~10], an intermediate compound that was used to create other subsequent analogs of EHNA that are more lipophilic.
FIGURE 2 depicts the reactions that were used to create 8'-Z5 hydroxy(~)-EHNA, designated as Compound r23~.
FIGURE 3 depicts the reactions that were used to create 8',9'-dihydroxy(+)-EHNA, designated as Compound tl4J.
FIGURE 4 depicts the reactions that were used (see Example 5) to create analogs of EHNA that contained various non-hydroxy 30 moieties bonded to the ~9 carbon atom.
FIGURES 5, 6, AND 7 are bar graphs showing that 9-chloro-EHNA (compound ~29]) and 9-phthalimido-EHNA (compound ~27]
provided better protection for heart muscle against ischemic damage than either unmodified E~NA or 9-hydroxy-EHNA, in the 35 tests described in Example 6.

DESCRIPTION OF T~E PREFERRED EMBODIMENTS
This invention describes analogs of EHNA in which the side chain (i.e., the straight chain erythro-hydroxynonyl portion, CA 02lss6l~ lgg7-o3-lo -W097/28803 PCT~S96/01990 which is attachecL to an ad,enyl ring structure) has been chemically modified by bonding certain types of chemical groups (moieties) to it. The preferred moieties provide the resulting analogs with certain pharmacological and therapeutic activities, ~ 5 which are substantially improved compared to unmodified EHNA.
In one embodiment of this invention, a hydroxyl group can be bonded to either the #8 or #9 car~on atom on the EHNA side chain, to generate hydroxylated analogs referred to herein as 8-OH-EHNA or 9-OH-EHNA (or a di-hydroxylated analog with hydroxyl 10 moieties bonded -to both car~on atoms). These hydroxylated analogs were synthesized as described in Examples l and 2, and were tested and discovered to have a slight but significant advantage, compar~d to unmcdified EHNA, in protecting heart muscle against ischemic damage, using a laboratory model with 15 intact perfused hearts taken from rats. The limited tests carried using the~;e hydroxylated analogs indicatecl that the 9-OH-EHNA analog waLs preferable to the 8-OH-EHNA analog.
Based on those initial f;n~;ngs, other analogs of EHNA were synthesized and tested in v~rious ways. Some of these analogs, 20 synthesized in the! laboratories of Prof. Elie Abl~ch~n~h at the University of Rhode Island, used a benzyl-protected precursor of the g-OH-EHNA analog (compo~md ~9] in the examples) as a starting reagent. Other analogs, synthesized in the laboratories of Cypros Pharmac~eutical Co~oration in Carlsbad, California, 25 used surplus quantities of the de-protected 9-OH-EHNA analog as a starting reagent. Research on these analogs showed that some of them had major therapeutic advantages over both unmodified EHNA, and over the 9-OH-EHNA analog as well.
The analogs ~hich have showed the best combinations of 30 traits in the research completed to date are believed to be more lipophilic (i.e., ~nore soluble in lipids and in other fatty, non-polar fluids) cmd less soluble in water than the hydroxylated 9-OH-~HNA analog. These analogs, which include 9-chloro-EHNA and 9-phthalimido-EHNA, are disclosed below.
35 Alternately, the chemical synthesis methods disclosed herein (and other methods known to those skilled in the art of chemical synthesis) can be used to create other analogs of EHNA which have been modified by the addition of nearly any type of moiety at the #8 or #9 carbon atom of the side chain. After synthesis, . .

CA 02l996l~ l997-03-l0 -= W097/28803 PCT~S96/01990 any such analog can be screened and tested, using the assays described herein or other assays known to those skilled in the art of biomedical testing, to determine whether any particular analog has a desired combination of traits.
Three traits are of primary interest herein. One such trait is, an E~NA analog intended for therapeutic use in humans should have suitable potency as a reversible ADA inhibitor, preferably with a binding affinity for ADA that provides a Ki value in the range of about 107 to about 101~. EHNA analogs with Ki values in 10 this desired range can inhibit the ADA enzyme reversibly, without permanently poisoning enzyme molecules, and without causing the types of toxic side effects that have been caused in some patients or test ~n;~l s by highly potent "suicide inhibitors" such as deoxycoformycin. The Ki value for any analog 15 of EHNA can be determined by assays such as the spectrophotometric assay described in Harriman et al 1992 (described in more detail in Example 3).
A second desirable trait for an EHNA analog intended for therapeutic use in humans involves a suitable level of lipid 20 solubility (also called lipophilicity). In general, lipophilic drugs tend to be taken into cells more readily and in greater quantities than hydrophilic drugs, because of two chemical factors. First, like droplets of oil in water, lipophilic drugs generate surface tension between themselves and water molecules;
25 they are not "comfortable" floating in the watery liquid that surrounds cells, and they seek configurations that minimize the area of their surface contact with water. This surface tension causes lipophilic molecules to attach and adhere to any lipophilic surfaces they encounter, including the surfaces of 30 cells, to minimize the area of their surface contact with water.
And second, since the membranes of mammalian cells are themselves made of lipid bilayers, molecules that are soluble in lipids tend to dissolve in and move into cell membranes. This is a major step in cellular uptake, and it does not require an 35 ac~ive ~ransport mechanism to help these drugs cross a cell membrane and enter a cell.
Both of these factors tend to promote and increase cellular intake of lipophilic drugs, compared to drugs which are hydrophilic and/or highly charged. This is assumed and believed CA 02lsg6l~ lgg7-o3-lo -- W097/28803 PCT~S96/01990 ~ . .
to be a potenti~lly important factor for ADA-inhibiting EHNA
analogs, since ~]~NA and its analogs act via molecular me~h~nl~m~
that occur inside cells.
However, the desired trait of high intake into cells does ~ 5 not increase in an unlimited manner as hydrophobicity increases;
extremely hydro~)hobic drugs can be difficult to a~min; ster to a patient via conventional routes such as injection or ingestion, and once inside t:he body, they often tend to sequester themselves in lipid vesicles or globules, or they tend to cling 10 to various membr2,nes, pla~1e deposits, or particulates, either in the intestines or inside blood vessels.
For these re;asons, a moderately high but not extreme level of lipophilicity (hydrophobicity) is often preferred for therapeutic drugs which must be taken inside cells in order to 15 be ~ully effective. AccordiLngly, now that it has been discovered that other, more lipophilic analogs provide better protection against ischemic damage than the hydroxylated 9-OH-EHNA analog, other EHNA analogs with other lipophilic moieties have been and will be synthesizled and te~ted, using the methods and assays 20 disclosed herein, to determine the optimal lipophilic values and moieties that pro~ide the ~est therapeutic benefits as described herein.
The lipophil:ic level of an EHNA analog with any candidate moiety can be assessed using a dual-solvent assay, such as the z5 widely used octanol-water partition assay. The partition coefficient is usually referred to as Po/W, where l'o/w" refers to oil and water; this value is usually referred to by a base 10 logarithm, comparable to pH values; a high log Po/W value indicates a high clegree of oil solubility, and a low (or 30 negative) value re~fers to a high degree of water solubility.
Since lipophilicity involves chemical reactions rather than complex biological functions, octanol-water partition coefficients can be estimated using commercially available computer software (such as t:he ACD/LogP software program, sold 35 by Advanced Chemistry Develc>pment, Inc. of Toronto, Canada).
This software was used to calculate the octanol-water partition coefficients listed in Table l. A description of the methods used to calculate and estimate partition coefficients, based on their chemical strllctures, is described in Bodor et al 1989.
_g_ CA 0219961~ 1997-03-lo -= W097/28803 PCT~S96/01990 Accordingly, without tying or limiting this invention to any particular theory, it is generally believed that EHNA
analogs which have a level of lipophilic solubility close to or greater than the chloro- or phthalimido- analogs described below 5 are likely to be preferable to hydroxylated analogs and other more hydrophilic (water-soluble) analogs, for protecting against ischemic or hypoxic damage or for increasing the half-lives of drugs that are degraded by the A~A enzyme.
The third primary desirable trait for an EHNA analog 10 intended for use as described herein involves therapeutic utility, either in m~m~l ian patients or in laboratory studies which provide good models of therapeutic utility against certain types of cell damage or drug degradation. Currently, the two primary and most urgent uses for the EHNA analogs described 15 herein are: (a) for reducing the amount of damage caused by ischemia or hypoxia in vulnerable tissues, especially in heart muscle or brain tissue; and (b) for prolonging the half-lives and increasing the therapeutic benefits of drugs that are being used to treat patients suffering from c~nc~, viral infection, 20 or other disease conditions, by reducing the rate of degradation of such drugs by the ADA enzyme. Various other uses may also be currently available, or they may be discovered in the future after these compounds are announced and made available to scientific and medical researchers.
This invention also discloses a method of synthesizing analogs of EHNA in which a moiety (such as a hydroxy group or halide atom) has been added to the side chain. This method comprises the following steps:
a. reacting an epoxide reagent having a desired chiral 30 orientation with an alkyl halide reagent having an unsaturated bond between two selected carbon atoms, under conditions which cause said reagents to create an unsaturated aliphatic compound comprising a first portion having a desired chiral orientation and a second portion having an unsaturated bond;
b. reacting the unsaturated aliphatic compound with at least one third reagent, under conditions which cause the third reagent to modify the unsaturated aliphatic compound by adding at least one hydroxyl group ~or other desired group or atom) to at least one of the carbon atoms involved in the unsaturated -- W097/28803 PCT~S96101990 bond, thereby creating a hydroxylated (or otherwise modified) saturated aliphatic compound, c. i~ a hydroxyl group was bonded to the EHNA side chain, the hydroxyl group can be replaced by or converted into a 5 dif~erent moiety, as discussed herein.
' When all o~ the init;al steps have been completed, any v additional proc~essiLng is carried out to complete the synthesis of the desired ,~nalog, such as removal of benzyl or other protective groups; such groups are commonly used during lO synthesis to prl-vent undesired reactions involving a protected constituent. The final de-protected analog is then puri~ied by any suitable means, such as chromatography, gel electrophoresis, or isoelectric ~ocusing.
The part}cu:Lar processing and puri~ication steps used to 15 create a specifi~ analog will depend on the exact molecular structure o~ the desired analog. Such steps are within the ordinary skill i.Tl the art, and various examples of suitable reagents and rea,ctions which can be used for such purposes are described below. - -In the Exam;ples and figures, each major starting reagent or intermediate is re~erred to by a bracketed number. For convenience, that brac~eted number is then used to refer to that compound in subsequent processing steps.
Example 1 (below) describes in detail the reagents and 25 reactions used to synthesize the EHNA analog which has a hydroxyl group bonded to the #9 carbon atom on the side chain.
This compound, referred to herein as 9-hydroxy-EHNA or as 9-OH-EHNA, is designai_ed as Comp~ound ~lO]. Its full chemical name is g-[2(S),9-dihydrc~xy-3(R)-nonyl]adenine, and its synthesis is 30 depicted in FIG. :L. The ~ull chemical name includes the term "dihydroxy" because this analog has two hydroxy groups on the side chain. One hydroxy group is attached to the ~2 chiral carbon atom, in the same "S" orientation that occurs in unmodified EHNA; t:he other hydroxy group was added to the #9 35 carbon atom, to create the 9-OH-EHNA analog.
Example 2 describes the reagents and reactions used to synthesize the EHNA analog which has a hydroxyl group bonded to the #8 carbon atom on the side chain. This compound, referred to herein as 8-hydroxy-EHNA or as 8-OH-EHNA, is designated as . .

CA 0219961~ 1997-03-10 - W097/28803 PCT~S96/01990 Compound [23]. Its full chemical namë is 9-[2(S),8-dihydroxy-3(R)-nonyl]adenine, and its synthesis is depicted in FIG. 2.
Example 2 also describes the synthesis of Compound ~14~, which is a di-hydroxylated EHNA analog with hydroxy groups added 5 to both the 8' and 9I carbon atoms (in addition to the st~n~rd hydroxy group on the #2 carbon atom). Its synthesis is depicted in FIG. 3.
All three of these hydroxylated analogs were shown to inhibit ADA activity in a reversible manner, with Ki values in 10 the desired range, as described in Example 3. They were then tested in certain types of in vitro tissue tests involving perfused hearts, as described in Example 4. In both the ADA
inhibition tests and the heart muscle tests, the 9-OH analog performed slightly better than either the 8-OH analog or the 15 8,9-dihydroxy analog. The 9-OH analog was the strongest binding agent of the three, with a Ki value of 3.8 x lO-9; the 8-OH
analog was the weakest, with a Ki value of 15.8 x lO-9, while the 8,9-dihydroxy analog had an intermediate strength, with a Ki value of 6.4 x 109. In addition, the 9-OH analog also displayed 20 a useful protective e~fect in the heart muscle reperfusion assays that was not shown at a significant level by the 8-OH
analog, involving a reduction of undesired muscle stiffness.
Accordingly, the 9-OH analog was identified as the preferred candidate, and it was used as a starting reagent for 25 synthesizing other analogs ~more precisely, a benzyl-protected precursor o~ the 9-OH analog, designated as Compound t9] in Example 1, was used; the benzyl group protec~ed the hydroxy group attached to the #2 carbon atom). If desired, either the 8-hydroxy or the 8,9-dihydroxy analogs could be used instead, to 30 create comparable lipophilic analogs with any desired moieties coupled to the #8 carbon atom instead of (or in addition to) the #g carbon atom, using the same general procedures and reagents described herein.
This general procedure, using hydroxylated EHNA analogs as 35 intermediates for synthesizing other analogs, can be used, if desired, to provide a general method for synthesizing any other desired type of EHNA analog, with any suitable type of moiety bonded to the side chain. However, since that is a relatively circuitous route which arose only because of the stepwise nature -- W O 97~28803 PCT~US96/01990 of the research described herein, synthetic chemists will recognize that ot:her, non-hydroxyl moieties can be added directly to the side chain, without having to go through hydroxylated int:ermediates, by using suitable alternate reagents 5 in one or more ol' the reactions that were used to create the hydroxyl analogs. Such more direct methods of synthesis will v likely provide better yields and require fewer purification steps, and will be generally be pre~erable to the indirect-via-hydroxyl method used during the initial research described l0 herein.
Nevertheless, the hydroxyl route should be recognized as a potentially useful route for synthesizing a large number of analogs that can be generat:ed by substituting or derivatizing hydroxyl groups, such as carboxylic acid groups, esters, and lS ethers, all of which can be created using techni~ues such as disclosed in the examples, or other t~hn~ ~ues known to those skilled in the art of chemical synthesis. In addition, hydroxide groups can be converted to numerous other groups by known method5. As o~e e~K~mple, a hydroxy~I group can be converted into 20 an azide group by reacting the hydroxyl with p-toluenesulfonyl chloride (TsCl) 1O create an O-tosyl group (abbreviated as OTs in the figures; t:osyl refers to toluenesul~onyl), then reacting the O-tosyl compotlnd with sodium azide (NaN3), which displaces the O-tosyl group and leaves an N3 group attached to the carbon Z5 chain. As a seconcl example, a hydroxyl group can be converted to a halide group (such as a chlorine, fluorine, bromine, or iodine atom) by methods such as in Example 5 relating to Compounds [28 and [29].
The analogs t:hat can be synthesized as described herein 30 include, but are not limited to, analogs in whiGh the ch2m,i~al moiety bonded to the #8 or ~9 carbon atom on the nonyl side chain consists of a halide; a nitrogen-containing moiety such as an amine, amide, azide, imide, or lactam; a carboxylic acid or salt thereof; or a moiety which is coupled to the #8 or #9 35 carbon atom via an ester or ether linkage. In order to be covered by the claims herein, any such analog must display the traits that can make such analogs therapeutically useful as disclosed herein (i.e., the resulting analog should have a Ki within the desired range of about 10-7 to about l0-1~; it must be CA 0219961~ 1997-03-10 - W097128803 PCT~S96/~l990 pharmacologically acceptable, and it must be therapeutically useful in protecting tissue against ischemic or hypoxic damage, or as an adjunct with one or more anti-cancer, anti-viral, or other drugs).
Epoxide [l] was synthesized as described in Abushanab et al 1984 and 1988. It controls the orientation of the substituents on the two chiral carbon atoms in the final EHNA analog, which are provided by the #3 and #4 carbons in the epoxide. To synthesize different stereoisomers of any of the EHNA analogs lO discussed herein, different epoxide stereoisomers having any desired chiral configuration can be used as the starting reagent.
The benzyl group ~-CH2C6H5) which was attached via an oxygen atom to the #3 carbon in the starting epoxide served as a 15 protective group for the oxygen atom. In the final step of synthesis of each of the hydroxylated EHN~ analogs, the benzyl group was displaced by hydrogen to create a hydroxyl group on the #2 carbon of the side chain. That #2 hydroxyl group is part of the normal EHNA molecule. If desired, that hydroxyl group can 20 be eliminated by using a starting epoxide without a protected oxygen atom, or it can be modified during synthesis to provide a halide, carboxylic, ester, ether, azide, or other group, as described above. If a moiety is desired at the ~l carbon atom in the final EHNA analog, it can be provided by using a starting 25 epoxide having the desired moiety or a precursor at the #4 carbon atom of the epoxide.
The synthesis reactions described herein also offer a method of derivatizing (i.e., bonding moieties to) the ~4, #5, #6, or ~7 carbon atoms on the nonyl side chain. In the synthetic 30 method used herein, those carbon atoms were provided by the reagent l-pentenylmagnesium bromide, which has a structure as shown in FIG. l in the reaction that converts epoxide [l] into compound [2~. The l-pentenyl notation indicates that the unsaturated double bond is positioned between the ~l and X2 35 carbon atoms in l-pentenylmagnesium bromide; those carbon atoms ultimately become the #8 and #9 carbon atoms in the EHNA analogs of this invention. The unsaturated carbon atoms in the double-bonded pentenyl compound became attachment points for hydrox~l groups during the reaction which converted compound [8~ into -- WO 97/28803 PCT/US96/Ul99O
compound [9~. Hydroxyl groups were added to both of the unsaturated carbons, and the compound having the hydroxyl moiety at the desired location was subse~uently purified. In an alternate approa,-h, the double bond supplied by the pentenyl 5 compound was converted into an epoxide intermediate, as shown in FIG. 2 in the re.~ction which generated compound [153.
Using either of these approaches, the location of a hydroxyl (or other desired) group on the side chain of an EHNA
analog can be controlled by using a pentenylmagnesium bromide 10 (or similar) compound having a double bond in any desired location. A 2-peni_enyl compound would have a double bond between its #2 and #3 carl~on atoms, which become the #8 and #7 carbon atoms in the fina:L EHNA analog. A 3-pentenyl reagent (having a double bond between its #3 and #4 carbon atoms) would generate 15 hydroxyl groups at:tached to the ~7 or #6 carbons in the EHNA
analog.
FIG. 2 also clepicts a halogenated analog, Compound ~21]. In Compound [21~, th;e halogen (chlorine) atom was su~stituted into the adenine ring structure. Although ~hat chlorine atom was 20 substituted by an amine group during the synthesis of compound ~22~, that particular reaction could be omitted if desired, so that the halogen ~loiety wou:Ld remain after removal of the benzyl protective group.
The method used to create the adenyl structure in the EHNA
25 analogs described herein offers a general method for making various changes in the aden:ine group. The adenyl structure was provided by supplying and then manipulating a heterocyclic compound, 5-amino-4,6-dichloropyrimidine (ADCP), which is shown in FIG. 1 in the reaction that generated compound [6]; this same 30 reagent was also used to generate compound r 20l shown in FIG. 2.
The ADCP was coupled to the side chain by displacing one of the chlorine atoms on the ADCP with an amine group that was coupled to the side chain. The five-member ring in the adenine structure was then closed by forming a carbon bond between two proximal 35 nitrogen atoms.
If desired, a:Lternate heterocyclic reagents could be used instead of ADCP, t:o create analogs of EHNA with modified adenine structures, either as moieties attached to one of the rings, or as differing atoms incorporated into either of the rings.

., -- WO 97/28803 PCT/US96/01990~
Cristalli et al 1988 and 1991 report that certain analogues of EHNA with modified adenine structures (such as a 3-deaza-EHNA
derivative) are active as ADA inhibitors. Such modifications to the adenyl structure could be incorporated into the analogs of 5 this invention, which have modified side Ch~; n~.
As mentioned above, all three of the hydroxylated EHNA
analogs which were tested for ADA inhibition (as described in Example 4) were shown to be active. The 9-hydroxy analog (compound [10]) was the strongest binding agent of the three, 10 with a Ki value of 3.8 x 10-9; the 8,9-dihydroxy analog (compound ~14]) was the weakest, with a Ki value of 15.8 x 10-9, while the 8-OH analog (compound ~23~) had an intermediate strength, with a Ki value of 6.4 x 10 9.
All three of these Ki values are within a desired range, 15 which covers about 10-7 to about 10-1~. At one end of the desired range, ADA inhibitors having Ki values lower than about 10-1~ run the risk of "poisoning" the enzyme by binding to it so tightly that the reaction is, for all practical purposes, irreversible.
At the other end of the desired range, ADA inhibitors having Ki 20 values higher than about 10-7 tend to be insufficiently potent to accomplish the desired level of ADA inhibition; they would need to be administered in relatively large quantities, and even in large quantities they might not be adequately potent.
The desired range of Ki values is relatively broad, since 25 candidate compounds can be administered to a patient in any desired quantity, by various routes. An analog having a Ki value in the range of about 10-9 should be a~mi n; -ctered in relatively low dosages, such as up to about 10 milligrams per kilogram of body weight per day if injected intravenously, and up to about 30 50 mg/kg/day if a~m~nictered orally. A less potent analog having a Ki value in the range of about 107 could be a~m; n;ctered in higher dosages, such as up to about 25 mg/kg/day if a~m; n; ~tered orally or injected in response to a major crisis, or up to 20 mg/kg/day if injected intravenously. Since the metabolic 35 problems caused by ADA deficiency tend to accumulate slowly, short-term dosages can be rather large.
After testing for ADA inhibition, the hydroxylated EHNA
analogs were tested for protection against ischemic damage to hearts, using procedures described in Example 4. Briefly, these ~CA 02l996l5 lss7-03-lo - W097/28803 PCT~S96/~1990 tests involved h.earts that were removed from laboratory rats, hooked up to perf.usion equipment and given electrical stimulation to su.stain the heartbeat, treated with the candidate drugs, subjected to a period of ischemia, and then reperfused, 5 to evaluate how well the hearts could recover their pumping functions. In these initial assays, 9-OH-EHNA provided a higher level o~ protection than wlmodi~ied EHNA in a particular parameter involving reduct:ion of unwanted muscle stiffness after ischemia. In subsequent assays using somewhat different heart lO preparations, described in Example 6, the advantages of 9-OH-EHNA were not as significant compared to unmodified EHNA;
however, other a:nalogs had been created by the time those sub~e~uent assays were car~-ied out, and the results of those other assays clearly indicated that the other preferred analogs 15 were substantial:Ly better than either 9-OH-EHNA or unmodified EHNA, in protect:i:ng hearts against ischemic or hypoxic damage.
Example 5 describes, aLnd Figure 4 depicts, the synthesis of several other an.~.logs, using the benzyl-protected precursor (compound t9l) o.E the 9-OH analog as a starting reagent. These 20 analogs include 1_wo relatively lipophilic analogs, referred to herein as 9-chloro-EHNA (Compound t29~3 and 9-phthalimido-EHNA
(Compound [26]). rrhese two analogs have shown the best therapeutic resul1_s observed to date, in protecting both heart muscle and brain 1:issue against ischemic damage.
Some additiollal analogs were also created by Cypros Pharmaceutical Col-poration, using the de-protected 9-OH-EHNA
analog as a start:ing reagent, since a quantity was still available after c:ompletion of the initial biological testing.
One such analog i.~; the silicon-contAi n; ng analog described in 30 Example 5 (compourld [33]). The silicon-containing moiety was chosen for two rezsons: (l) calculations indicated that it had a very high lipophil.icity, and could provide a potentially useful test compound to h.elp evaluate that factor; and (2) it could be added to the #9 atom in a de-protected 9-OH-EHNA molecule, 3s without disturbing the hydroxyl group on the #2 carbon atom of the side chain.
For convenie:nce, the Ki values and oil/water solubility values that were gathered or calculated on the final (deprotected) ana.logs listed in Examples l, 2, or ~ are compiled -~ W097/28803 PCT~S96/01990 in Table 1. In this table, these anaIogs are provided with simple names that indicate what type of modifying group was added to the side chain, and which carbon atom it was bonded to.
Complete chemical names are provided in Examples 1, 2, and 5, 5 correlated with bracketed compound numbers. It should be noted that the Ki values in Table 1 used extra-cellular ADA enzyme, and did not re~lect the apparent ability of lipophilic analogs to enter cells more readily and in greater quantities.

1o TABLE 1 CHEMICAL DATA FOR VARIOUS EHNA ANALOGS
Compound Modifying Ki value Log P0/
number qroup x 109 (calcu~ated) 15 _ -- unmodified (+)-EHNA 6 Z.60 + 0.41 [10] 9-hydroxy-EHNA 3.8 + 0.4 0.59 + 0.41 [23] 8-hydroxy-EHNA 6.4 0.41 + 0.41 ~14] 8,9-dihydroxy-EHNA 15.8 + 0.4 -1.10 + 0.42 20 [25] 9-benzoyloxy-EHNA 0.2 3.50 + 0.41 [27] 9-phthalimido-EHNA 2.3 2.86 + 0.44 t29] 9-chloro-EHNA 3.7 2.28 + 0.41 ~31] 9-carboxymethyl-EHNA 5.0 0.95 + 0.41 t32] 8,9-unsaturated EHNA 2.5 2.06 + 0.41 25 ~33] 9-tert-BDPSi-EHNA ND 9.46 + 0.69 Example 6 describes the testing of various analogs to evaluate their ability to protect heart muscle against ischemia.
The results indicated that relatively lipophilic analogs 30 ~including 9-chloro-EHNA and 9-phthalimido-EHNA) provided substantially better protection against ischemic damage to heart muscle than either unmodified EHNA or hydroxylated EHNA.
Example 7 describes the results o~ cell culture tests to evaluate the ability o~ EHNA and several analogs both (1) to 35 enter human cells, and (2) inhibit ADA activity inside the cells. These tests did not stress the cells, or test any analogs against ischemic damage; instead, they evaluated the ability of various analogs to reach the intracellular enzyme molecules and inhibit their activity. These tests used both red blood cells 40 (which are easy to work with), and human astrocytoma cells (which are brain cells that can reproduce in cell culture; ~hese CA 02l996l5 lss7-03-lo - W097/28803 PCT~S96/01990 were used to provide an indication o~ whether EHNA analogs can help reduce ischemic damage in brain tissue). The results indicated that several EHNA analogs which are more lipophilic than unmodified EHNA or 9-OH-EHNA were substantially more potent 5 than unmodified EHNA or 9-OH-EHNA in inhibiting ADA activity inside cells, as indicated by lower IC50 values.
Example 8 describes the results of cell culture tests which used several different methods to generate ischemic damage in either brain cells or blood cells. some of these tests used lO toxins such as 2-deoxyglucose or sodium azide to interfere with respiration and g~ycolysisO Other tests used culture media containing no free oxygen, obtained by bubbling nitrogen gas rather than oxygen gas through the cell culture media. In all of these tests, the cells were subjected to a period of oxygen 15 deprivation (usually lasting several minutes), then the oxygen supply was reest;~blished. After a brief period to allow the cells to reestablish equilibrium, selected metabolic indicators were evaluated to determine how close the cells had come to regaining their proper metabolic rates. The results indicated 2 o that some EHNA analogs (especially the lipophilic analogs) can indeed protect brain cells against ischemic damage.
Example g describes several assays that can be used to test EHNA analogs to c~1antify their ability to protect intact m~m~ lian brain tissue against ischemia. Rather than usin~
25 isolated cultured brain cells, as in Example 7, these tests use intact slices of brain tissue, ~rom the hippocampal regions of sacri~iced rats. I'he hippocampal region is used because it is highly vulnerable to ischem;ic damage, and the use of intact hippocampal slices that can still generate brain waves in 30 response to electrical stimulation offers a better assurance of overall tissue ~unctioning than the metabolic rates of isolated cells. These tests are currently underway. Although the final results are not yet available, it is believed (based on protection levels ~provided in other tests, including perfused 35 heart tests and cu1tured brain cell tests) that at least some of the E~NA analogs c1escribed herein will provide a signi~icant and therapeutic reduct::ion in ischemic or hypoxic damage in brain tissue.
Analogs that show promising results in the hippocampal CA 0219961~ 1997-03-10 -- W097/28803 PCT~S96/01990 ~ .
slice tests described in Example 9 will be tested further, in ln vivo tests on intact ~n;~ls. These tests can use artery clamping, neck tourni~uets, or other methods to induce either local or global ischemia in the brains of test An;~ls, as 5 described in articles such as Nellgard and Wieloch 1992, Buchan and Pulsinelli l990, Michenfelder et al 1989, and Lanier et al 1988.
In summary, the examples, tables, and figures show that certain EHNA analogs described herein have a use~ul and lO previously unknown therapeutic benefit in protecting heart muscle and brain cells against ischemic damage. The benefits provided by the relatively lipophilic analogs exceed and surpass the benefits provided by unmodified EHNA or hydroxylated EHNA
analogs.
Included within the family of agents useful for the purposes described herein are any isomers (including "threo"
isomers), analogs, or salts of the compounds described herein, provided that such isomers, analogs, and salts are functionally effective as ADA inhibitors, are pharmacologically acceptable, 20 and are therapeutically effective in either reducing ischemic or hypoxic damage or in slowing the degradation of anti-cancer, anti-viral, or other drugs. The potency of any candidate isomer, analog, or salt in inhibiting ADA activity can be tested using methods such as described in Example 3. The therapeutic efficacy 25 of any candidate isomer, analog, or salt against ischemic or hypoxic damage can be tested using methods such as described in Examples 4, 6, and 7. The efficacy of any candidate isomer, analog, or salt in slowing the degradation of anti-cancer, anti-viral, or other drugs can be measured by methods known to those 30 skilled in the art, such as by administering an EHNA analog to animals (or humans) that have received the drug of interest, and after an appropriate period (which will usually be in the range of 2 to 24 hours later, depending on the drug), measuring the quantities of the drug that are present in the blood or tissue 35 of the test ~ ls (or humans, if blood tests are used), and comparing that quantity to the quantity of the same drug in animals or humans that have not been treated with an EHNA
analog.
The term 'Ipharmacologically acceptable" embraces those - WO 97/28803 PCT/US9~i/01990 characteristics ~hich make a drug suitable and practical for administration t:o humans; such compounds must be sufficiently chemically stable to have an adequate shelf life under reasonable storage conditions, and they must be physiologically 5 acceptable when introduced into the body by a suitable route of administration. ~.cceptable salts can include alkali metal salts as well as addition salts of free acids or free bases. Examples of acids which are widely used to form pharmacologically acceptable acid-addition salts include inorganic acids such as 10 hydrochloric acid, sulfuri< acid and phosphoric acid, and organic acids such as male:ic acid, succinic acid and citric acid. Alkali metal salts Ol- alkaline earth metal salts could include, for example, sodium, potassium, calcium or magnesium salts. All of these salts may be prepared by conventional means.
15 The nature of the salt is not critical, provided that it is non-toxic and does not sub~tantially interfere with the desired activity.
The term "analog" is used herein in the conventional pharmaceutical sense, to refer to a molecule that structurally 20 resembles a re~ere~nt molecule (EHNA, g-O~-EHNA, or 8-OH-EHNA, in this case) but wh:ich has been modified in a targeted and controlled manner to replace a specific substituent of the referent molecule with an alternate substituent, other than hydrogen (since replacement of the #9 hydroxyl group on 9-OH-25 EHNA with a hydrogen atom would give unmodified EHNA rather thana true analog of 9-OH-EHNA. A chemical analog reguires an "offspring" type of relationship, wherein an analog is created by chemical modification of a known compound (often called a parent or referent compound~. Accordingly, the hydroxylated 30 compounds [10~, [14~, and [23~ are analogs of EHNA, but EHNA is not regarded as an analog oi~ those hydroxylated compounds. A
substitution which converts a known molecule into a new analog may be inserted into or coupled to any location in the molecule, such as in one of the rings in the adenyl structure, in the 35 attached side chain, or in one of the pendant groups attached to the ring structure or side chain.
It should also be noted that analogs of 8-OH-EHNA or 9-OH-EHNA are covered ~y the claims herein only if they satisfy the re~uirements of pharmacological acceptability, ADA-inhibiting CA 0219961~ 1997-03-10 -= W097/28803 PCT~S96/Ol990~
e~ficacy, and therapeutic utility as'disclosed herein, and only if such analogs retain an ADA inhibiting potency with a Ki value which is within the desired range of about 107 to about l~10.
Administration of the compounds of this invention to humans 5 or ~n;m~l S can be by any t~chn;que capable of introducing the compounds into the bloodstream, including oral administration or via intravenous or intramuscular injections. The active compound is usually administered in a pharmaceutical formulation, such as in a liquid carrier for injection, or in a capsule, tablet, or lO liquid form for oral ingestion. Such formulations may comprise a mixture of one or more active compounds mixed with one or more pharmaceutically acceptable carriers or diluents. When lipophilic drugs are formulated for in~ection, they are usually mixed with water, a buf~er compound (such as a mixture of a lS carboxylic acid and a salt thereof), and an organic compound having a plurality of hydroxyl groups; propylene glycol, dextran compounds, and cyclodextrin compounds are often used for such purposes.
If desired, other therapeutic agents (such as anti-cancer 20 or anti-viral nucleoside analogs) may also be present in an injectable ingestible formulation which contains a suitable EHNA
analog as described herein. A mixture of an anti-cancer or anti-viral nucleoside analog, with an EHNA analog, can be very useful, since the EHNA analog can prolong the half-life and 25 efficacy of the nucleoside analog in the blood, by suppressing degradation of the nucleoside analog by AD~ enzymes.
The tests completed to date indicate that different analogs showed different efficacy levels in different types of cells or tissues. For example, although the differences between 9-chloro-30 EHNA and 9-phthalimido-EHNA in heart muscle tests were relatively slight, the phthalimido analog tended to show significantly better efficacy in cell culture tests on brain cells. Accordingly, it is anticipated that one type of analog may be preferred for protection of heart muscle, while a 35 different analog may be preferred for protection of brain tissue.
It should also be noted that the tissue-protecting efficacy of any analog will depend on a combination of factors, rather than on any ~actor in isolation. For example, as indicated in CA 02l996l~ lgg7-o3-lo -- W097/28803 PCT~S96/01990 - Table 1, unmodif'i'.ed EHNA Aas a log PO/~ coefficient that is roughly the same as for 9-~hloro-EHNA or 9-phthalimido-EHNA, while 9-hydroxy-F,HNA has a Ki value which is roughly the same as for 9-chloro-EHNA or 9-phthalimido-EHNA. However, neither 5 unmodified EHNA nor 9-hydroxy-EHNA have the combined desirable traits of a low ~;i value and a high Po/W value, as shown by 9-chloro-EHNA and 9~-phthalimido-EHNA, and neither unmodified EHNA
nor 9-hydroxy-EHNA show the same level of tissue protection shown by 9-chloro-EHNA and 9-phthalimido-EHNA. Accordingly, 10 unless and until other parameters are determined to be of even greater use in predicting protective utility in intact tissue tests or in vivo tests, a combination of low Ki value and high P~w value should be regarded as a better indicator than either trait considered by itself~ At the current time, based on the 15 tests completed to date, it: is believed that an analog should have both (a) a Ki value for adenosine d~;n~e inhibition which is less thi~n about 5 x 10'9, and (b) an octanol/water partition coefficient of at: least about 2. Neither unmodified EHNA nor any of ithe hydroxy~ated analogs created to date have 20 this combination of traits.
It was also ;noted that the 9-benzoyloxy-EHNA analog had the best combination of low Ki value and high PO/~ value out of all the analogs listed in Table 1. In the future, it will be tested in both cell cult:ure and intact tissue tests. In the assays 25 carried out to date, it was not tested, due to concerns that the benzoyloxy group would likely be cleaved off from the EHNA
molecule by various ~ ~lian enzymes, thereby converting it into 9-OH-EHNA, which has relatively low efficacy for tissue protection.
EXAMPLES
EXAME~IE 1: ~Y ~ S OF 9-o~-EENA ANALOG
This example describes how various intermediate and final compounds were synthesized. For convenience, bracketed numbers 35 which correspond to the subheadings below and to the callout numbers in the drawings are used to refer to each compound.
Unless otherwise indicated, all organic solutions were dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. Ratios of chromatography =-- = ~
CA 0219961~ 1997-03-lo - W097/28803 PCT~S96/01990 solvents are expressed in v/v. Silica gel (Davison, grade H, 230-425 mesh) suitable for flash column chromatography was purchased from Fisher Scientific. A Chromatotron (centrifugally accelerated, preparative thin-layer, radial chromatograph) Model 5 7924T was used to complete various separations. The 1.0 and 2.0 mm plates used were coated with silica gel PF254 containing CaS04 .
The molecular structures were confirmed in a number of ways. Elemental analyses (to compare measured values against 10 calculated values) were performed by MHW Laboratories (Phoenix, AZ). Melting points were determined on a Buchi 535 melting point apparatus, and 1H NMR spectra were recorded on a Varian EM-390, Bruker AM-300, or Bruker AM-500 spectrometer. Optical rotations were obtained with a Perkin Elmer Model 141 digital readout 15 polarimeter. All of these analyses provided data that were within the expected limits.

COMPOUND 1: (2S.3S)-3-~benzYloxY)-1,2-epoxYbutane The starting epoxide [1] was synthesized as described in 20 Abushanab et al 1984 and 1988. This epoxide determines the orientation of the substituents on the two chiral carbon atoms in the final EHNA analog, which are provided by the #3 and #4 carbons in the epoxide. To synthesize different stereoisomers of any of the EHNA analogs discussed herein, different epoxide 25 stereoisomers having any desired chiral configuration can be used as the starting reagent. A benzyl group attached to the #3 carbon atom via an oxygen atom was used to protect the oxygen atom during synthesis.

30 COMPOUND 2: (2S.3S~-2-O-Benzyl-2.3-non-8-en-diol A solution of epoxide tl] (2g, 11.24 mmol) in ether (50 mL) was added to a cold (-78~C) ether solution contA; ni ng 1-pentenylmagnesium bromide [(22.5 mmol), prepared by reacting magnesium (0.66g, 22.5 mmol) and 5-bromo-pentene (4.11g, 22.5 35 mmol)] and 0.1 mmol of lithium tetrachlorocuprate, while stirring was continued for 1 h. The reaction was quenched with a saturated solution of NH4Cl (100 mL) and extracted with ether.
Pure ~2] (2.67g, 96~) was obtained by silica gel column chromatography eluting with a mixture of ethyl acetate (EtOAc) - W097/28803 PCT~S96/01990 and hexane (5:95).

COMPOUND 3: (2s~3s~-2-o-Benz~l-3-o-tosvl-2~3-nona-8-en-diol To a stirrecL solution of the alcohol [2] (5.4g, 21.7 mmol) 5 in pyridine (10 mL) was added p-toluenesul~onyl chloride (TsCl;

4.5 g, 23.9 mmol) and stirring was continued for 12 h at room temperature (RT~. The mixtl~re was poured into water (100 mL) and extracted with CHzC12 (3 x 100 mL). The combined organic solutions were then washed with cold HCl (2x50 mL) and water (2 10 x 100 mL), dried ~MgSO4) and filtered. Removal of solvent left an oil, which was purified by silica gel chromatography eluting with EtOAc-hexane (1:50) to afford t3] (8.35g, 97~).

COMPOUND 4: (2S 3R)-3-Azido-2-O-benzyl-2-nona-8-en-ol Sodium azid~e (1.027g~ 15.8 mmol) was added to a stirred solution of [2] (4.0g, 13.2 mmol) in anhydrous DMF (20 mL).
After refluxing for 45 min, DMF was removed and pure r43 (2.61g, 96%) was obtained by silica gel column chromatography eluting with EtOAc-hexanes (5:95).
COMPOUND 5: (2S :3~R)-3-Amino-2-O-BenzYl-2-nona-8-en-01 To a stirred solution of lithium aluminum hydride (LAH, 0.25g, 5.2 mmol) in anhydrous ether (50 mL) was added, dropwise, a solution of azide [4] (1 g, 3. 66 mmol) in anhydrous ether (50 25 mL). The reaction mixture was then heated at reflux for 2 h, cooled to RT, ancl excess LA~ was decomposed by the care~ul successive dropwise addition o~ water (0.25 mL), 15% NAOH (0.24 mL), and water (0.5 mL). Filtration, drying and evaporation of the solvent gave a pure colorless liquid [5~ (1.06g, 96%).
COMPOUND 6: 5-Amino-6-chloro-4 r 2(S~-O-benzYl-3(R)-nona-8-enYl1aminoPYrimid~ine 5-Amino-4,6-dichloropyrimidine tADCP, 0.39g, 2.336 mmol), N-tributylamine ~n-Bu3N; 0.433g, 2.336 mmol) and [5~ (0.5907g, 35 2.336 mmol) in anhydrous pentanol (10 mL) was heated at reflux for 48 h under an N2 atmosphere. Pentanol and n-Bu3N were removed and the residue was chromatographed over silica gel (EtOAc-hexanes 1:10) to give [6] (0.65g, 74%).

- WO 97/28803 PCT/US96101990~
COMPOUND 7: 6-Chloro-9- r 2(S)-O-benzyl-3(R)-nona-8-en-enyl~purine An acidified (conc. HCl 0.3 mL) solution of [63 (0.58 g, 1.55 mmol) in triethyl orthoformate (TEOF; 15 mL) was stirred at RT for 24 h. The yellow oil obtained after removal of T~OF, was 5 purified by silica gel column chromatography eluting with EtOAc-hexanes (1:10) to provide ~7] (0.5 g, 85~).

COMPOUND 8: 9- r 2(S)-O-BenzYl-3(R)-nona-8-enylladenine Compound [7~ (0.3 g, 0.78 mmol) was dissolved in liquid 10 ammonia (15 mL) and heated at 90~C in a steel bomb for 24 h.
After cooling, excess ammonia was allowed to evaporate. The residue was taken up in CH2Cl2 (25 mL) and washed with water (10 mL). The organic layer was dried and pure r8] (0.245 g, 85~) was obtained as a white solid.
COMPOUND 9: 9-r2 (s) -O-BenzYl-9-hYdroXy-3(R)-nonYl~adenine To a solution of the olefin [8] (0.365g, 1 mmol) in dry tetrahydrofuran (THF; 1 mL), placed in a three-necked flask fitted with a condenser and a septum, was added a 1 M solution 20 of a diborane-THF complex (BH3.THF; 0.5 mL, 0.5 mmol) at 0~C.
The reaction was done under nitrogen atmosphere. The mixture was permitted to stir for additional hours at RT to continue the completion of the reaction. Water (0.05 mL) was added slowly and the mixture was allowed to stir at RT until hydrogen no longer 25 evolved. The flask was immersed in an ice bath and 3 molar NaOE
(0.17 mL) was rapidly added to the reaction mixture. The organoboronic acid inteL ?~i~te was oxidized by the slow addition of 30~ hydrogen peroxide (0.11 mL). The reaction mixture was then allowed to stir ~or 3 h at 50~C to ensure 30 completion of the oxidation. The mixture was brought to RT and NaCl was added to saturate the lower aqueous phase. The THF
phase was separated and dried (MgSO4). The crude compound obtained after solvent removal was purified by silica gel column chromatography eluting with EtOAc to provide 9 ~0.268g, 70%).
COMPOUND 10: 9- r 2fSl,9-dihYdroxY-3(R~-nonylladenine In order to remove the benzyl protective group, a solution of compound [9~ (0.227g, 0.593 mmol) in EtOH (25 mL) and cyclohexene (10 mL) was treated with ~0~ palladium hydroxide on .

-- WO 97/288(13 PCT/US96/01990 charcoal [Pd(OH~;,/C, often referred ~o as Pearlmann's reagent, 0.05g]. The resulting suspension was stirred at reflux for 12 h.
After cooling to RT, the mixture was filtered and the filtrate was concentrated at reduced pressure. The residue was - 5 chromatographed over silica gel (EtOAc-MeOH, 9:1) to give pure [10] (0.156g, 9C)~
This compound is the 9-hydroxy analog of EHNA, which was tested as described in Examples 4 and 6.

EX~MPIE 2: ~iYN~ ;SIS OF 8--O~I--EEINA AND 8 . g--DlnYl~KOXY ANAr~-S

COMPOUND 11: 6-Chloro-9-r2(S)-O-~enzYl-8.9-e~oxy-3 (R) -nona-8-enYllpurine To an ice cold solutic)n of the olefin C7~ (0.769g, 2 mmol) 15 in CH2Cl2 (15 mL) was added 85% m-chloroperbenzoic acid (0.488g, 2.4 mmol). After stirring t:he reaction mixture at RT overnight, it was diluted w:ith ether (50 mL~ and washed successively with saturated NaHC03 ~15 mL), 10~ NaHSO3 (15 mL), saturated NaHCO3 (15 mL), and brine and dried (MgSO4). The residue, obtained 20 after evaporation of ether, was purified by silica gel column chromatography e].uting with EtoAc-h~ane~ [1:10) to afford pure epoxide [11] (0.~'21g, 90%).

COMPOUND 12: 6-ch,]oro-9r2(s)-o-benzyl-8 9-dihydroxy-3(R) -nonyl l 25 Purine Compound ~113 (0.3g, 0.75 mmol), 5~ HCl04 ~2 mL), in acetonitrile (6 mI,) was stirred at RT for 2 h. The reaction mixture was neutralized with solid NaHCO3 and the mixture was filtered. The filtrate was diluted with CH2Cl2 (25 mL) and dried 30 over MgSO4. The re~sidue obtained after solvent evaporation was purified by silica gel column chromatography using EtOAc-hexanes (1:1) to provide diol [123 ~0.3g, 95%).

COMPOUND 13: 9- r 2fS~-O-Benzvl-8.9-dihYdroxY-3(R~-nonyl~adenine Compound [12l was obtained from ~11] according to the procedure described for the preparation of [83, in 90% yield.

COMPOUND 14: 9- r 2~';) 8~9-trihydroxY-3(R)-nonYlladenine Triol ~14] was prepared in 90~ yield by debenzylating [13]

. .

using the procedure described for ~10].
This compound is the 8,9-dihydroxy analog of EHNA, which was tested as described in Example 3. Although it was shown to be a moderately effective inhibitor of ADA activity, its potency 5 was lower than the 9-OH-EHNA analog, and it was not tested further.

COMPOUND 15: (2S~3S)-2-0-BenzYl-3.)-0-toSYl-8~ 9-e~PoXY - 2 . 3-nonanediol Epoxidation of [3] was carried out as described for compound [11]. After workup, the residue was chromatographed using hexane-EtOAc (7:3) to give the epoxide [15; (99%) as an oil.

15 COMPOUND 16: (2S~3S)-2-0-Benzvl-3-0-tosYl-2,3~8-nonenetriol A solution of aluminum hydride (30 mL, 15 mmol, 1 M
solution in THF), was added to epoxide [15~ (3.143g, 7.52 mmol) in dry ether (100 mL) at 0~C. The mixture was stirred at RT for 1 h and decomposed slowly by the addition of water (25 mL) and 20 the aqueous solution was extracted with ether (4 x 50 mL). The combined ether extracts were dried (MgSO4) and concentrated to furnish [16] (3g, 95%). An analytical sample was obtained by silica gel column chromatography with hexane-EtOAc (7:3) as eluent.
COMPOUND 17: (2S 3S)-2-0-Benzyl-3-0-tosYl-8-0-tetrahvdropyranyl-2,3,8-nonanetriol A solution of alcohol [16] (2.8g, 6.6667 mmol), and dihydropyran (1.68g, 13,33 mmol) in dry CHzCl2 (50 mL) 30 containing pyridinium p-toluenesulfonate (PPTS, 0.33g, 1.33 mmol) was stirred at RT for 4 h. The reaction mixture was diluted with ether and washed once with half saturated brine to remove the catalyst PPTS and dried (MgSO4). The residue obtained after evaporation of the solvent was purified by silica gel 35 column chromatography using hexane-EtOAc (95:5) as eluent, to af~ord ~17] ~3.3g, 99%).

COMPOUND 18: ~2S 3R)-3-Azido-2-0-benzyl-8-0-tetrahydropyranYl-2 8-nonanediol PCT~S96/~1990 Compound [18] was prepared from~ ~17] following the procedure described for the formation of [4]. The crude product, after column chromatography (hexane-EtOAc, ~5:5), gave pure ~18]
(8296) .
COMPOUND 19: (2S, 3R) -3-Amino-2-O--benzvl-8-O-tetrahYdro~vranY
2 -8 -nonanediol The azide t 18 3 was recluced by a procedure similar to that described ~or th/_ preparation of ~5], to afford the amine [19]
10 quantitatively.

COMPOUND Z0: 5--Amino--6--chl~ro--4 r2 fS)--O--benzyl--8--O--tetrahydropvranY L -3 tR) -2 8-dihydroxYnonyl~ aminopyrimidine Compound [20 ] was prepared ~rom 19, by following the 15 procedure described ~or the formation of [ 6 ] . The residue, obtained after so Lvent removal, was chromatographed over silica gel ( EtOAc-hexane; 1: 5 ) to give [ 2 0 ] ( 2 8 % ) .

COMPOUND 21: 6-Ch:Loro--9--~ 2 (S ) -O-benzvl-2 . 8--dihYdroxY-3 ~R) -2 0 nonYl ] t surine An acidifiecl (conc. HCl, 0.15 mL) solution of [20~ (0.3g, 0.63 mmol) in TEOF (15 mL) was stirred at RT for 24 h. The residue obtained, after removal of TEOF, was dissolved in absolute ethanol ~10 mL~, cont;3 ; n ; ng PPTS ( 0 . 025g) and refluxed 25 for 1 h. Solvent was then removed under reduced pressure and the crude product was purified by silica gel column chromatography, using EtOAc--hexane!s (3 :1) to give [21] (0.15g, 60~;) .

COMPOUND 2 2: 9 - r 2 ( S ) -O-Benz~1-2 8 -dihYdroxY--3 (R)--nonYl 1 adenine Amination of chloropur:ine derivative [21] was carried out as for the synthesis of [8] from [73. Crude product, obtained after workup, was purified by silica gel column chromatography, eluting with MeOH-EtOAc (5: 95? to give [22 ] (90%) .

3 5 COMPOUND 2 3: 9 - r 2 ( S ) . 8--Dihyclroxy- 3 ( R) -nonYl 1 adenine Diol [23] wa, prepared in 90g~ yield by debenzylating t22]
using the procedwre describ~d for [103.
This compound is the 8-hydroxy analog of EHNA which was tes~ed as described in Example 3. It was shown to inhibit ADA

- W097/28803 PCT~S96/01990 activity in the desired range, but its potency was lower than the 9-O~-EHNA analog, so it was not tested further.

EXAMPLE 3 ~ G FOR ADA INHIBITION
Compound [lO~ (the 9-hydroxy analog), compound [23] (the 8-hydroxy analog), and compound [14] (the 8,9-dihydroxy analog) were tested for ADA inhibition activity, using calf intestinal ADA (Type III, Sigma Chemical Company) measured at 30~C by direct spectrophotometric assays at 265 nm, as described in l0 Harriman et al 1992. These tests used extra-cellular enzyme preparations, and did not require any of the analogs to enter cells in order to reach the enzyme. The Ki values, listed in Table l, indicated that the 9-OH analog was more potent than either of the other hydroxylated analogs, since a smaller 15 quantity was required to achieve 50~ inactivation of a standardized quantity of the ADA enzyme. Since it was more potent, the 9-hydroxy analog was used as a starting compound for synthesizing other analogs.
Table l compiles ADA inhibition data and octanol-water 20 partitiion coefficients (an index of lipophilicity) for all of the final (deprotected) analogs listed in Examples l, 2, or 5.

EXAMPLE 4: TESTING OF 9--OH--EHNA FOR ~KU~ lON Af~AT~c:T
ISCEIEMIC nAMA~:~ TO TISSlJE
After synthesis of the 9-hydroxy and 8-hydroxy analogs of EHNA, samples were provided by the Applicant to Dr. Robert Rodgers of the Department of Pharmacology and Toxicology at the University of Rhode Island. There were sufficient quantities of 9-OH-EHNA, while quantities of 8-O~-EHNA were smaller.
30 Accordingly, most tests used 9-OH-EHNA and compared it to unmodified EHNA and to disulfiram, an unrelated compound that is known to have certain protective anti-ischemic effects in cardiovascular tissue.
The tests carried out by Dr. Rodgers used a widely-used 35 protocol known as a "working heart" preparation. These tests involved removing intact hearts from sacrificed lab ~nim~-s (male Sprague-Dawley rats were used), and perfusing the hearts with liquids containing controlled quantities (or deficits) of oxygen and glucose for fixed periods of time. The procedures used in these experiments are describëd in detail in Davidoff and Rodgers, HYpertension 15: 633-642 (1990), with certain minor modifications. The lef't atrium was filled at 15 cm H2O pressure, and the left vent:ricle ejected into a buffer-filled column 5 against a pressure which e~uated to 72 mm Hg, except during ischemic periods. The perfusate was Krebs-Henseleit buffer with HCO3 (25 mM), Cai' (2.2 mM), and glucose (10 mM). When gassed with 95% ~2 and 5~ CO2, the p~ of the perfusate was 7.4 + 0.2.
Perfusate and ambient temperatures were held at 37~c, and the 10 hearts were allowed to beat spontaneously.
After perfusion began, the isolated hearts were allowed to stabilize for 10 minutes, then they were treated for 10 minutes with one of the test drugs (unmodified EHNA, 9-OH-EHNA, or disulfiram) or buf'fered saline cont~ining either dilute ethyl 15 alcohol tused to increase the solubility of EHNA or 9-hydroxy-EHNA) or dilute dimethyl su:Lfoxide (used to increase the so~ubility of disulfiram).
Following stabilization and treatment, the hearts were subjected to simulated ischemia for 20 minutes; no oxygen was 20 added to the perfusate during this period. When the ischemic period ended, oxygen was again added to the perfusion buffer, and the following parameters were measured over a period of 10 minutes:

LVPP - :L,eft ventricular pulse pressures ~time-dependent pre~sures, calculated as peak pressure minus diastolic pre~3sure, in mm Hg, millimeters of mercury column) LVEDP - :Left ventricular end diastolic pressure (i.e, time-dependen1: pressures as the ventricle relaxed during diastolic filling, in mm Hg) CFR - coronary flow rate (mL/min) HR - sporltaneous h~eartbeat rate (beats/min) ECG - electrocardiogram (surface potential, in mV) The results indicated that both EHNA and 9-hydroxy-EHNA
reduced the incide~ce of fibrillation; the difference ~etween them was not significant. Both EHNA and 9-OH-EHN~ also caused moderate increases in both L~PP and coronary flow rate after CA 0219961~ 1997-03-lo - W097/28803 PCT~S96/01990 ischemia; again, their effects were not different from each other at a significant level.
The most important difference observed between EHNA and s-oH-EHNA in these initial tests appeared in measurements of 5 LVEDP (left ventricular end diastolic pressure). This parameter indicates whether the muscles of the left ventricular wall are able to relax promptly following contraction. Prompt relaxation is essential, since it allows the heart's pumping chambers to fill with blood during diastolic relaxation, between lO contractions. A high LVEDP level is very undesirable, since it indicates that the heart muscle has become stiffened by ischemic damage and no longer has sufficient flexibility and elasticity to properly fill the pumping chambers with blood during diastolic relaxation. In these tests, 9-OH-EHNA provided a 15 higher level of protection against muscle stiffening than unmodified EHNA.
Testing of the 8-OH-EHNA analog in these perfused heart preparations was limited, due to the small quantity that was available. However, those limited tests indicated that 8-OH-EHNA
20 analog was not as potent as 9-OH-EHNA in reducing left ventricular stiffness and LVEDP.
Because 9-OH-EHNA was more potent as an A~A inhibitor than 8-OH-EHNA, and because 9-OH-EHNA appeared to have an additional use~ul effect in reducing heart muscle stiffness, subse~uent 25 research used 9-OH-EHNA, compound [lO], or its benzyl-protected precursor, compound [9], as starting compounds for synthesizing other analogs.
The methods used by Prof. Rodgers were slightly different in some respects from the heart muscle tests described in 30 Example 6, which were carried out subsequently at a contract research firm called Coromed, Inc. (Troy, New York). In the tests carried out by Coromed, under contract to the Applicant, Cypros Pharmaceutical Corporation, 9-OH-EHNA showed little or no improvement over unmodified EHNA in protecting heart muscle, and 35 in some tests it did not perform as well as EHNA. However, by then, other more lipophilic analogs (including 9-chloro-EHNA and 9-phthalimido-EHNA) had been synthesized and were being tested.
As described in Example 6, those other analogs showed major advan~ages in protecting heart muscle against ischemia, compared - WO 97/:28803 PCT/US96101990 ~ to either EHNA or 9-OH-EHNA. Accordingly, subsequent research has been devoted to those other analogs, while 9-OH-EHN~ and 8-OH-EHNA are not :being actively tested ~urther.
.

EXAMPIE 5: ~YNl~SIS OF OIHER ANALOGS OF EHNA
This exampl,e and Fig. 4 depict the synthesis of several additional analogs of EHNA. Except as noted, the synthetic reactions described below used the benzyl-protected compound [9]
(described in ~xample l) as the starting reagen~. Elemental and l0 NMR analyses con:Eirmed that: each compound was created in analytically pure form, except as noted for compounds [26] and [28]; these were intermediates rather than completed analogs, and they were no1 fully purified.
The final (de-protecte!d) analogs (Compounds ~25]/ [273, 15 [29], r3l]~ and li:3~]) described herein were tested for inhibition of the ADA enzym.e, using the procedures descri~ed in Example 3, and a]:L were found to have a potency in the desired range, indicating that they can inhibit the ADA enzyme without irreversib~y poisoning it. These Ki values are provided in Table 20 l.

COMPOUND 24: 9-r9--Benzovloxv-2(S)-O-benzYl-3(R)-nonYl1adenine This analog, which has benzoyloxy groups coupled to the #2 carbon and a benz~loxy group at the #9 carbon atom, was prepared 25 by adding n,n-dii.~;opropylazo-dicarboxylate (DIAD, 202 mg, l mmol) to a stirrecl solution o~ compound [9~ (314 mg, 0.82 mmol), benzoic acid (BzOF[, 122 mg, l mmol), and triphenyl phosphine (PPh3, 262 mg, l ~mol) in THF (5 ml). The mixture was stirred at room temperature for 24 hr and precipitated triphenyl phosphine 30 oxide was filtered, out. The filtrate was concentrated and the residue was chroma.tographed on silica gel using ethyl acetate (EtOAc) to provide. [24], which was further purified by preparative thin layer chromatography (TLC) using EtOAc and methanol (MeOH) at 9:l. Yie:Ld was 250 mg (63%).
COMPOUND 25: 9-~9-Benzoylox~-2~S)-hYdroxY-3(R)-nonyl7adenine This alcohol, which has a benzoyloxy group coupled to the ~9 carbon atom, was created by treating compound [24~ (200 mg) in ethanol (EtOH, 18 ml) and cyclohexene (6 ml) with 20%

= palladium hydroxide on charcoal (PdOH2/C, 0.15 g). The suspension was stirred at reflux for 12 hours. After cooling to room temperature, the mixture was filtered and the filtrate was concentrated at reduced pressure. The residue was 5 chromatographed over silica (EtOAc and MeOH at 9:1) to give pure [25~, 130 mg (80%).

COMPOUND 26: 9- r 2tS)-O-benzYl-9-phthalimido-3(R3-nonylladenine Compound [26~ was prepared ~rom compound t9] in 87% yield 10 in the same way as compound ~24], except that phthalimide (1 mmol, 147 mg) was used in place of benzoic acid. Analytically pure compound could not be obtained as it always contained traces of triphenyl phosphine oxide and dihydro-DIAD. It was used as a rea~ent in the following step, to create compound 15 [26].

COMPOUND 27: 5-r2 (s) -hYdroxy-9-~hthalimido-3(R)-nonylladenine This alcohol was created in 85% yield, with ~26] as the starting reagent (255 mg, 0.5 mmol) using the same palladium on 20 charcoal (PdOH2/C) catalytic procedure used to create t25].

co~ou~v 28: 9-~2(S)-O-benzYl-9-chloro-3(~)-nonvl~adenine Analog [28~, with a benzyl ring on the ~2 carbon atom and a chlorine atom coupled to the #9 carbon atom, was created by 25 adding PPh3 (400 mg, 1.5 mmol) to a stirred solution of [9] (500 mg, 1.3 mmol) and NaHCO3 (50 mg) in anhydrous CCl4 (5 ml). The mixture was treated at reflux for 12 hours, then filtered, and the filtrate was concentrated. The residue was chromatographed over silica gel (EtOAc) to provide 380 mg (75%). Analytically 30 pure compound could not be obtained, since it contained traces of triphenyl phosphine oxide. It was used as a reagent in the following step, to create compound ~29~.

COMPOUND 29: 9- r 9 -chloro-2fS)-hYdroxy-3(R)-nonylladenine This analog, with a halide moiety at the #9 carbon and an alcohol group at the #2 carbon atom, was prepared in 82% yield using ~28] as the starting reagent and using the same palladium on charcoal (PdOH2/C) catalytic procedure used to create [25]~
Other halide analogs with fluorine, bromine, or iodine --- W097/28803 PCT~S96/01990 - atoms can be created, if desired, by proper selection of reagents containing such at:oms. For example, the synthetic procedure described for Compound ~28] could be modified by using CBr4 or CI4 instead of CCl4. As another example, a 9-fluoro-~HNA
5 analog could be produced by reacting compound ~9] (the benzyl-protected 9-oH-E~aNA analog) with the well known fluorination agent diethylamin~sulfur trifluoride (DAST).

COMPOUND 30: MethYl-7rR)-adenine-9- Yl) -8(5)-O-benzyl-nonoate Analog t30], with an ester group at the #9 carbon and a benzyl group at 1-he #2 car~on, was created by adding pyridinium dichromate (PDC, 2.755 g, 7.3 mmol) to a solution of ~9] (1.369 g, 3.4 mmol) in climethyl formamide (DMF, 2 ml). The mixture was stirred at room t~amperature for 24 hours, then diluted with 15 ethyl acetate ancl passed through a mixture of silica gel and Na2SO4 (l:l) to give the corresponding acid (220 mg, 15.7%
yield).

COMPOUND 31: Meth~rl- 7 (R~ - adenine- 9-Yl) - 8 (S) - hydroxy-nonoate Analog [31~, with an ester group at the ~9 carbon and a hydroxy group at 1:he ~2 carbon, was created in 82% yield using the same palladiuTn on charcoal (PdOH2/C) catalytic procedure used to create [25]~ using the benzyl analog [30~ as the starting reagent. Ester 31 can also be referred to as 9-[2(s)-25 hydroxy-9-carboxy~1ethyl-3(R)-nonyl]adenine.

COMPOUND 32: 9-~2~S)-hYdroxy-3(R)-non-8-envl3adenine Unsaturated analog [32J was created by using the unsaturated benzyl-protected analog ~7], shown in FIG. 3 and 30 described in Example l, as the starting reagent. Compound [7]
(200 mg, 0.55 mmol) in toluene (l0 ml) was cooled with dry ice/acetone and a~onia was bubbled through the solution until the volume of the mixture reached 40 ml. Sodium metal was added in portions with vigorous slirring until the mixture was 35 neutralized with NH4Cl and methanol and evaporated to dryness.
The compound was then extracted with CH2Cl2 and the extracts were dried over Na2SO4 and evaporated. The product was purified by preparative thin layer chromatography using ethyl acetate to give a 70% yield of [32].

CA 02lss6l~ lgg7-o3-lo -- W097/28803 PCT~S96/01990 ComPound 33: 9-r9-tert-Butvldiphenvlsilyloxy-2rs)-hvdroxy-3(R) nonYlladenine An additional analog that deserves note was synthesized by Cypros Pharmaceutical Corporation, using de-protected 9-OH-EHNA
5 as a starting reagent, since a surplus of that compound was available after completion o~ the initial biological testing of the 9-OH-EHNA. The silicon-contA;ning moiety was chosen for two reasons: (1) calculations indicated that it had a very high level of lipophilicity (with a log Po/W value in the range of 9, lo compared to 2 or 3 for the chloro and phthalimido analogs) and could provide a potentially useful compound to help evaluate high-level lipophilicity; and, (2) this moiety could be added to the ~9 atom in a de-protected 9-OH-EHNA molecule without disturbing the unprotected hydroxyl group on the #2 carbon atom 15 of the side chain.
Accordingly, compound ~10~ (30 mg, 0.102 mmol) was dissolved in 0.5 ml of DMF and added to a solution of 50 ~l of diisopropylethylamine and 15 mg of dimethylaminopyridine cont~;~e~ in l ml of CH2Cl2. To this solution was added 40 mg 20 (0.14 mmol) of t-butylchlorodiphenyl-silane and the mixture was stirred at room temperature for 16 hours. The solvent was evaporated and the product was purified by chromatography over silica gel (10~ CH30H/CHCl3) to give 26 mg (48%) of 9-t-BDPSi-EHNA
[compound 333.

EXAMPl,E 6: PROTECTION OF HEART M~SCLE ~ TN.~T Ist~MTA
Several of the analogs described in Example 5 were tested to evaluate their ability to protect heart muscle against ischemia, using a so-called "Langendorf heart preparation". Briefly, male 30 Sprague-Dawley rats weighing between 250 and 350 grams were anaesthetized with sodium heparin and sacrificed with CO2. ~he heart was rapidly excised via thoracotomy and placed in physiological salt solution (PSS~ until contraction ceased, The heart was then mounted via the aortic root to a cannula and 35 retrogradely perfused with PSS containing (in mM): NaCl (118), KCl (4.7), CaCl2 (2.2), KH2PO4 (1.18), MgSO4 (1.17), NaHCO3 (25), dextrose (11) at 80 mm Hg at 37 degrees C. The perfusion so~ution was aerated with 95~ ~2/5~ CO2 to maintain pH at 7.~. Hearts were allowed to e~uilibrate for 15 min, during which time a balloon-.
CA 02l996l~ lgg7-o3-lo - W097/28803 PCT~S96/01990 tipped catheter was introduced into the lumen of the left ventricle via a small incision in the left atrium. The catheter was connected to a pressure transducer and was used to measure left ventricular hemodynamic performance, i.e. left ventricular 5 systolic pressure (LVSP), l.eft ventricular end-diastolic pressure (LVEDP), left ventricular cleveloped pressure (LVDP), +dP/dt~X
(the m~ximllm rate at which pressure developed in the left ventricle during each contraction), -dP/dt~X (the ~;mll~ rate at which left ventr~ ular pressure declined following each 10 contraction), and heart rate. Following placement of the balloon-tipped catheter, ~the pulmonary artery was c~nnlllated to collect coronary effluenl ~or measu.rement of coronary flow.
At the conc~Lusion of the sta~ilization period, measurements of left ventricul.lr hemodynamic performance, heart rate and 15 coronary flow were made. The hearts were then perfused for 10 minutes with PSS cont~;n;ng vehicle, 9-hydroxy-EHNA, 9-chloro-EHNA, or 9-phthal:imido-EHNA., and measurements were repeated.
Global ischemia was produced by clamping the aortic c~nn~la, and measurements of t:hese parameters were made at 5-minute intervals.
20 After 35 min of g~Lobal ischemia, the hearts were reperfused with PSS for 20 min at: a pressure of 80 mm Hg. Measurements were again taken at 5 minute intervals during the reperfusion period.
The results, in Tables 2-4 and Figures 5-7, indicated that the more lipophil.iLc analogs provided substantially better 25 protection against: ischemic damage to heart muscle than either unmodified EHNA or 9-OH-EHN~.

EX~MPIE '7: OEI L C~l~TURE ~ESTS, UN~-l~;~SED CET.T~
In a first set of cell culture tests, EHNA and its analogs 30 were evaluated for their ability to inhibit ADA activity in two different types of cells: human red blood cells (which are relatively easy tc, work Wit]l), and human astrocytoma cells (which are brain cells that can reproduce in cell culture; these were used to provide an indication of whether EHNA analogs can help 35 reduce ischemic damage in brain tissue).
A first set of tests was carried out, using ~Inormoxic~
conditions (i.e., the cells had not been stressed by hypoxia or by simulated ischemia, using deoxyglucose or sodium azide), to determine an IC50 value for EHNA and several analogs. The IC~o o ~ ~ ~ -1 ~1 X
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~s ~ ~ c s_ s_ ~ ~, c c s_ E ~ E F E E. O, -~1 1) 0 Il~ O '~ o In o . -39-PCTtUS96/01990 - WO 97/28~03 - value indicates the concentration of drug which was required to inhibit half of the ADA activity in the cells; this value reflects both the ability of a drug to enter cells and reach the ADA enzyme, and the potency of a drug in binding to and 5 inhibiting the ADA enzyme inside the cells. A low IC50 value indicates that a drug is a potent ADA inhibitor and can enter cells readily.
To carry out these tests, cell populations were preincubated with EHNA or an EHNA analog, in varying concentrations, for 1 10 hour. The cells were then incubated for 30 minutes with 10 uM 5-iodotubercidin, which inhibits the activity of a different enzyme called adenosine kinase, which adds phosphate groups to adenosine. This step ensured that adenosine levels would not be altered by a phosphorylation pathway which can consume adenosine lS in intact cells. The cells were then ;ncl~hAted for 30 minutes with 100 uM radiolabelled adenosine. After 30 minutes, concentrations of radiolabelled inosine and hypoxanthine (the molecules that are created when the ADA enzyme degrades adenosine~ were measured in cell medium after separation using 20 cellulose thin layer chromatography. Several concentrations of EHNA or an EHNA analog were used in each set of tests, and an ICso for each compound was calculated based on the dose-response curve for that compound.
The results, in Table 5, indicate that 9-chloro-EHNA was 25 substantially more potent than unmodified EHNA or 9-OH-EHNA in inhibiting ADA activity inside red blood cells cells (as indicated by lower inhibitory concentration values), and that 9-phthalimido-EHNA was substantially more potent than unmodified EHNA or 9-OH-EHNA in inhibiting ADA ac~ivity inside brain 30 (astrocytoma) cells. IC50 ~alues in this table are averages followed by stAn~A~d deviations.

_ .

-- W097/28803 PCT~S96/01990 T~TRTTIcl~r OF ADA A~llVl-l-Y IN CELL CULTURE TESTS
Cell tY~e/compouncl IC50 fUM) Red Blood Cells EHNA 1.200 + 0.70 9-OH-EHNA ~cpcl. 10] 2.100 + 0.90 9-chloro-EHNA ~cpd. 29] 0.220 + 0.14 Astrocytoma cells EHNA 3.0 + 2.5 9-OH-EHNA ~10~ 3.5 1 0.8 9-chloro-EHN~. [cpd. 29] 4.8 + 1.9 9-phthal imidc,--EHNA [cpd. 273 1.1 + O.9 8,9-unsaturat.c!d-EHNA [cpd. 32~ 2.5 1 0.8 EXAMPLE 8: (']3LL CnLTURE TESTS FOR ISCHEMIC ~~ ON
In a second se!t of cell culture tests, cells were stressed by either of two meithods which simulate hypoxic or ischemic damage. In these te;sts, a population of astrocytoma cells con~i ni nq radiola~elled A~P was incubated with EHNA or an analog 20 for 60 minutes. Then, to dis~upt glycolysis and respiration, 2-deoxyglucose or sodium azide (5.5 mM final concentration for either toxin) was added, and the cells were in~l~h~ted for 60 minutes. After ;~c~h~tion, the cultures were tested to determine how much radiolabelled adenosine they released into medium.
25 Adenosine release .is a norma] and proper metabolic function of cells during is~e~ or hypoxia, and the quantity of adenosine released by EHNA-t:reated or analog-treated cells was compared to the quantity of ad~2nosine released by control cells, which had been identically stressed by the same toxin without any treatment 30 by EHNA or an analog. The results, in Tables 6 and 7, are expressed in percentages of adenosine release by treated cells, compared to untreat/2d control. cells.

- W097/28803 PCT~S96/01990 = TABLE 6 ADENOSINE ~R~.~A.~ BY ASTRO~YlOI~ CELLS DURING SIMULATED HYPOXIA
fSTRESSED BY DEOXYGLUCOSE) 5 Protective druq Percent of control values Untreated = 100~ (baseline~
EHNA 339 + 25 9-OH-EHNA [10] 289 + 12 9-chloro-E~NA [cpd. 29~ 375 + 25 10 9-phthalimido-EHNA ~cpd. 27] 559 + 19 8,9-unsaturated-EHNA [cpd. 32~ 300 + 48 9-butyldiphenylsilyloxy-EHNA [33] 244 + 21 ADENOSINE RRr~ ~E BY AsTRG~Y-~ ~.~ CELLS
IN N~ r ~X I C CONDITIONS AND SIM~LATED ~Y~VXlA

Protective dru~ (uM: conditions) Percent of control values 20 Untreated = 100% (baseline) EHNA
0.01 normoxic 9g + 10 0.1 normoxic 92 + 8 0.01 stressed 139 + 7 0.1 stressed 253 + 21 9-phthalimido-EHNA [cpd. 27]
O.01 normoxic 102 + 11 0.1 normoxic 111 + 13 0.01 stressed 242 + 9 0.1 stressed 425 + 40 8,9-unsaturated-EHNA ~cpd. 32]
0.01 normoxic 115 + 22 0.1 normoxic 119 + 12 0.01 stressed 159 ~ 11 0.1 stressed 225 + 31 These results indicate that the 9-chloro and 9-phthalimido CA 02l996l5 l997-03-lO

- ~ W097/28803 PCT~S96/01990 - EHNA analogs provided better adenosine release duri.ng hypoxic damage, in brain c:ell cultures, than unmodified E~NA.
In addition, 1:he absence of any significant effects in cells that were culturecL under normal oxygen ('1normoxic") conditions is 5 important, because it indicates that EHNA does not disrupt the normal metabolic aLctivities of cells that are not being stressed.
It only becomes act:ive in ce-ls that are being stressed.
In a third set: of cell culture tests, astrocytoma cells were subjected to actual hypoxia eOr 2 hours, in an anaerobic chamber 10 while nitrogen gas (rather than oxygen) was bubbled through the cell culture mediu~. A~ter the hypoxic period release of radiolabelled adenc~ine into the culture medium (TABLE XXX).

ADENOSINE R~'r;~A.~E BY BR~IN OEIIS AFT~R 2 HOURS ~v~OXlA

Protective druq (uM~ Percent of control value Untreated = lOO~ (baseline) EHNA
0.05 257 + 34 0.5 769 + 67 9-phthalimido-EHNA
0.05 386 + 51 0.5 866 + 83 These results :indicate that EHNA and its 9-phtha7imido analog elevate adenosine release in both simulated and unsimulated hypoxia" and that the phthalimido analog o~fers a higher level of pro1:ection. This ability to help sustain a normal 30 metabolic function, in brain cells, in the face of ischemic insult indicates thLzlt the phthalimido analog can help protect brain tissue against: ischemic damage.

- EXAMP~E 9: ADDITIONAL ASSAYS FOR PROTECTION OF BRAIN TISSUE
Various assays are currently underway to quantify the ability of several EHNA analogs to reduce hypoxic or ischemic damage in intact brain tissue" as distinct from cultured cells.
These assays are being sponsored and funded by the Applicant, Cypros Pharmaceutical Corporat:ion, and are being carried out at the UCLA Medical Center in Los Angeles by an independent investigator. Based upon the results o~ other tests already completed ~including the heart tests and the cultured brain cell tests), it is anticipated that at least some of the EHNA analogs 5 will display a substantial ability to reduce hypoxic or ischemic damage in intact brain tissue. Accordingly, this example describes experimental protocols that can be used to carry out such evaluative tests, using intact tissue slices in relatively simple, rapid and inexpensive tests.
The in vitro assays currently being carried out use intact slices of hippoc~mr~l tissue from the brains of sacrificed rats, for two reasons. First, tissue from the hippocampal region of the brain is known to be highly vulnerable to ischemic damage. And second, tests using perfused intact tissue slices are often 15 preferred in neurological research, since intact cohesive tissue often provides a better model of in vivo cell and tissue behavior than either broken cell fragments, or isolated cells cultured under artificial conditions (especially when such cells are cancerous or have been genetically altered to increase their 20 replication rates in artificial cell culture conditions).
If properly perfused, so-called "CAl" neurons in hipro~ ~-1 tissue slices will generate electrophysiological responses (comparable to brain waves in living ~ c) which can be measured quickly and easily, using a device comparable to an 25 electroencephalograph, for several hours. Accordingly, a candidate neuroprotective drug can be tested to find out whether it can prolong, restore, or otherwise increase the ability of neurons in perfused hippocampal tissue slices to generate electrical spikes having normal and desirable amplitudes and 30 ~re~uencies ~as distinct from seizure-inducing convulsant drugs, which induce spikes having abnormal amplitudes or frequencies).
If a candidate drug allows hippocampal slices to regain or prolong their normal electrophysio}ogical responses despite ischemic insult, this provides strong and direct evidence that 35 the candidate drug does ; n~ have substantial protective activity against ischemic damage to brain tissue. Such tests are widely used and accepted, and are described in various references such as Whittingham et al 1984, and Schuur and Rigor 1992.
The protocols described below are described in more detail ~ _ CA 02199615 19s7-03-1o -- W097/28803 PCT~S96/01990 - in Wallis et al 1992. Briefly, Sprague-Dawley rats are anesthetized with halothane, then decapitated. The brain is quickly removed and placed in cold artificial cere~rospinal fluid (CSF) ~or one minute. This f~uid co~tains (in mM) NaCl, 126: KCl, 5 4.0; KH2PO4, l.4; MgSO4, l.3: CaClz, 2.4: Na~CO3, 26; and glucose, 4.0, with a pH o~ 7.4, saturated with a gas mixture of 95% ~2 and ~ 5~ CO2. The chillecl brain tissue is then sliced to provide hippo~mpAl tissue slices, which are placed in recording wells with the temperature of the surrounding bath thermostatically l0 controlled to 34~C.
One hour afte:r placement of slices into the recording wells, the orthodromic CAL population spike (PS) is measured for each tissue slice. This output, which is an indicator of cell function, is elici1:ed as a response to electrical stimulation 15 using a twisted bipolar electrode placed over the CA3 Schaffer collatera~s. Respon,es are recorded using tungsten electrodes inserted into the p~rr~; dA 1 layer of CAl. Current strengths (in the stimulating electrodes) and e}ectrode depth (for the measuring electrodes) are adjusted to obtain r~Y;mAl amplitude of 20 the CAl spikes. on]y slices showing an orthodromic CAl PS of 3 mV
or greater on initial ass~c ~t are used for further testing.
In control sa~les, tissue slices are submerged in artificial CSF fluicl that dose not contain any E~NA analogs or other neuroprotecti~re drugs. Test samples are treated 25 identically, but th~e CSF fluid contains a known c~n~ntration of EHNA, or an EHNA analog as described herein.
The assays involve subjecting hippoc~mr~l tissue slices to hypoxic conditions f'or limited periods of time, and then measuring the ability of the CAl cells to respond to electrical 30 stimulation after that period of hypoxia. To initiate hypoxia, paired hippocampal slices (i.e., two tissue slices from the same ~;m~l ) are placed in two recording wells, and the perfusion fluid to both wells is changed to artificial CSF cont~;n;ng no free oxygen; the CSF fluid is saturated with 95~ N2 (instead of 35 ~2) and 5~ CO2.
one slice in e<~ch pair aclditionally receives exposure to EHNA or an EHNA analog, beginning 30 minutes prior to the onset of hypoxia, and continued until 15 minutes after the termination of hypoxia. The per:iod of hypoxic deprivation is variable for - dif~erent slices; during hypoxia, each control tissue slice (i.e., each slice that had not been treated by EHNA or an EHNA
analog) is monitored to ensure that a "hypoxic injury potential"
(HIP; see Fairchild et al ~988) is still present.
The hypoxic deprivation for paired slices is terminated by adding oxygen to the perfusion medium 5 minutes after the disappearance of the HIP in the untreated slice. The two paired slices are then monitored for an additional 90 minutes, and brain wave amplitudes are recorded. Final CA1 orthodromic PS amplitude 10 i5 then compared to the original CA1 orthodromic PS amplitude which was measured prior to treatment. Antidromic PS amplitude is also assessed before hypoxia, and after 90 minutes of recovery.
In some tests, hippocAm~l tissue slices are given electrical stimulation every 30 seconds throughout the entire 15 perfusion period, including the hypoxic period. In these tests, ongoing periodic stimulation imposes additional metabolic demands r which a~yldvates the excitotoxic injury and provides an even more rigorous test~
The data gathered from stimulated slices, which are 20 monitored for mean PS recoveries throughout hypoxia, are analyzed using Student's t-test. Data from unstimulated slices, which are assessed for CAl orthodromic and antidromic PS amplitude only at the beg; nn ing and end of experiments, are analyzed using the Wilcoxon rank-sum test.
Due to the design of these test protocols, the CAl injury produced by hypoxia in this assay is severe, and usually quite reliable, in unmedicated, unstimulated slices, and it provides a useful baseline for comparative purposes.
As noted above, these assays have not yet been completed, 30 but they are currently underway, and they are expected to show that at least some of the EHNA analogs descri~ed herein offer substantially improved levels of neuroprotection against ischemia, compared to unmodified EHNA or hydroxylated EHNA. Any specific EHNA analog which substantially reduces hypoxic or 35 ischemic damage in these relatively simple and inexpensive in vitro tests can then be tested in intact Ani~ls, using in vivo studies as described in articles such as Nellgard and ~ieloch 1992 (surgically-induced ischemia in rats), Buchan and Pulsinelli 1990 (surgical ischemia in gerbils), Michenfelder et al 1989 - (surgical ischemia in dogs), and Lanier et al 1988 (neck tourniquets on pr:Lmates), or using other protocols known to those skilled in the art:
Thus, there h~s been shown and described a new class of EHNA
analogs which are useful for reducing ischemic or hypoxic damage and for slowing the metabolic degradation of useful anti-cancer and anti-viral drugs. Also disclosed herein are methods o~
synthesizing these~ compounds, and methods of using these compounds to treat patien~s in need of such treatment. Although 10 this invention has been exemplified for purposes of illustration and description by reference to certain specific embodiments, it will be apparent to those skilled in the art that various modifications and alterations of the illustrated examples are possible. Any such changes which derive directly from the 1~ teachings herein, and which clo not depart from the spirit and scope of the invention, are ~le -~ to be covered by this invention .

R~k~CES
Abll~h~n~h, E.,, et al, "Practical Enantiospecific Synthesis o~ (+~-erythro-9-(2-Hydroxy-3-nonyl)~n;ne," Tetrahedron Lett.
25: 3841 (1984) Ab~7~h~n~h, E., et al, "The ~h~ ;~try o~ L-Ascorbic and D-isoascorbic Acids: :L. The Preparation of Chiral Butanetriols 25 and -tetrols" J. orc~. Chem. 53: 2598-2602 (1988) Bastian, G., ~!t: al, "Adenosine Deaminase Inhibitors:
Conversion of a Sinc~le Chiral Synthon into erythro- and threo-9-(2-hydroxy-3-nonyl)ad~;n~," J. Med. Chem. 24:1383-1385 (1981) Baker, D.C., an,d Hawkins, ~.D., "Synthesis of Inhibitors of 30 Adenosine De -in~ce: A Total ~;ynthesis of erythro-3-(Adenin-9-yl)-2-nonanol and Its Isomers from Chiral Precursors," J. or~.
Chem. 47: 2179-2184 (1982) Bodor, N., et al., "A New Method for the Estimation of - Partition Coe~ficients," J. Am. Chem. Soc. 111: 3783-3786 (1989) Buchan, A. and Pulsinelli, W.A., "Hypothermia, but not the NMDA Antagonist MK-801, attenuates the neuronal damage in gerbils subjected to transient global ischemia," J. Neuroscience 10: 311-316 (19g~) Cristalli, G., et al, "Adenosine De~m;n~se Inhibitors:

CA 0219961~ 1997-03-lo ~- W097/28803 PCT~S96101990 ~

= Synthesis and Biologica~ Activity of Deaza Analogues of erythro-9-(2-Hydroxy-3-nonyl)adenine," J. Med. Chem. 31: 390-397 (1988) Cristalli, G., et al, "Adenosine D~m;n~e Inhibitors:
Synthesis and Structure-Activity Relationships of Imidazole 5 Analogues of erythro-9-(2-Hydroxy-3nonyl)adenine," J. Med. Chem.
34: 1187-1192 (1991) Davidoff, A.J., and Rodgers, R.L., ~Insulin, thyroid hormone, and heart ~unction of diabetic spontaneously hypertensive rat," Hypertension 15: 633-642 (1990) Dorheim, T.A., et al, "Enhanced interstitial fluid adenosine attenuates myocardial stunning," SurqerY 110: 136-145 (1991) ~ arriman, G.C.B., et al, "Adenosine Deaminase Inhibitors:
Synthesis and Biological Evaluation of Cl' and Nor-Cl' Derivatives of (~)-erythro-9-(2(S)-Hydroxy-3(R)-nonyl)adenine,"
15 J. Med. Chem. 35: 4180-4184 (1992) Lambe, C.U., and Nelson, D.J., "Pharmacokinetics of Inhibition of Adenosine D~;n~e by erythro-9-(2-hydroxy-3-nonyl)adenine in CBA Mice," Biochem. Pharmacol. 31: 535-539 (1982) Lanier, W., et al, "The effects of dizocilpine maleate (MK-801), an antagonist of the NMDA receptor, on neurological recovery and histopathology following complete cerebral ischemia in primates," Anesthes. Rev. lS: 36-37 (1988) McConnell, W.R., et al, "Metabolism and Disposition of 25 erythro-9-(2-Hydroxy-3-nonyl)r14C]adenine in the Rhesus Monkey,"
Druq Metab. Disp. 8: 5-7 (1980) Michenfelder, J., et al, "Evaluation of the glutamate antagonist dizocilpine maleate (MK-801) on neurological outcome in a canine model of complete cerebral ischemia: correlation with 30 hippocampal histopathology," Brain Research 481: 228-234 (1989) Nellgard, B. and Wieloch, T., "Postischemic blockage of AMPA
but not NMDA receptors mitigates neuronal damage in the rat brain following transient severe cerebral ischemia," J Cereb Blood Flow Metab 12: 2-11 (1992) O'Dwyer, P.J., et al, "Association of severe fatal infections and treatment with Pentostatin," Cancer Treat. ~eP.
70: 1117-1120 (1986) Phillis, J.W. and O'Regan, M.H., "Deoxycoformycin antagonizes ischemia-induced neuronal degeneration," Brain Res.

~- ~ W097/28803 PCT~S96/Ol990 - Bull. 22: 537-40 (1989) Schaeffer, H:.J. and Schwender, C.F., "Enzyme Inhibitors XXVI: Bridging Hydrophobic and Hydrophilic Regions on Adenosine Deaminase with scme 9-(2-Hydroxy-3-alkyl)adenines," J. Med. Chem.
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Claims (24)

1. A pharmaceutical agent consisting of a chemically modified form of erythro-hydroxynonyladenine with a molecular structure as follows:
wherein at least one of R1 and R2 comprises a chemical moiety, other than a hydrogen atom or hydroxyl group, which makes the chemically modified form of erythro-hydroxynonyladenine:
(a) pharmacologically acceptable and non-toxic when administered to humans and laboratory animals;
(b) capable of entering human cells and inhibiting intracellular enzymatic activity of adenosine deaminase in a reversible manner, in cell culture tests, more effectively than either erythro-hydroxynonyladenine or 9-[2(S),9-dihydroxy-3(R)-nonyl]adenine;
(c) therapeutically effective in reducing hypoxic and ischemic damage in at least one type of mammalian tissue, selected from the group consisting of heart muscle and brain tissue, more effectively than either erythro-hydroxynonyladenine or 9-[2(S),9-dihydroxy-3(R)-nonyl]adenine.

2. The pharmaceutical agent of Claim 1, wherein the chemically modified form of erythro-hydroxynonyladenine has a Ki value for adenosine deaminase inhibition in the range of about 10 -7 to about 10 -10.

3. The pharmaceutical agent of Claim 1, wherein the chemically modified form of erythro-hydroxynonyladenine has:
(a) a Ki value for adenosine deaminase inhibition which is less than about 5 x 10 -9; and, (b) an octanol/water partition coefficient of at least about 2.

4. The pharmaceutical agent of Claim 1, wherein at least one of R1 and R2 is selected from the group consisting of:
a. halide atoms;
b. halide-containing groups which allow the chemically modified form of erythro-hydroxynonyladenine to have a Ki value for adenosine deaminase inhibition which remains within the range of about 10 -7 to about 10 -10;
c. nitrogen-containing groups which allow the chemically modified form of erythro-hydroxynonyladenine to have a Ki value for adenosine deaminase inhibition which remains within the range of about 10 -7 to about 10 -10;
d. ester-linked groups which allow the chemically modified form of erythro-hydroxynonyladenine to have a Ki value for adenosine deaminase inhibition which remains within the range of about 10 -7 to about 10 -10, and, e. ether-linked groups which allow the chemically modified form of erythro-hydroxynonyladenine to have a Ki value for adenosine deaminase inhibition which remains within the range of about 10 -7 to about 10 -10.

5. A pharmaceutical agent consisting of a chemically modified form of erythro-hydroxynonyladenine, comprising an adenyl ring structure coupled to a nonyl side chain, wherein the side chain has been modified by bonding at least one chemical moiety other than a hydroxyl moiety to the side chain, wherein the chemically modified from of erythro-hydroxynonyladenine:
(a) is pharmacologically acceptable and non-toxic when administered to humans and laboratory animals;
(b) is capable of entering human cells and inhibiting intracellular enzymatic activity of adenosine deaminase in a reversible manner, in cell culture tests, more effectively than either erythro-hydroxynonyladenine or 9-[2(S),9-dihydroxy-3(R)-nonyl]adenine;
(c) is therapeutically effective in reducing hypoxic and ischemic damage in at least one type of mammalian tissue, selected from the group consisting of heart muscle and brain tissue, more effectively than either erythro-hydroxynonyladenine or 9-[2(S),9-dihydroxy-3(R)-nonyl]adenine.

6. The pharmaceutical agent of Claim 1, wherein the chemically modified form of erythro-hydroxynonyladenine has:
(a) a Ki value for adenosine deaminase binding which is less than about 5 x 10-9; and, (b) an octanol/water partition coefficient of at least about 2.

7. The pharmaceutical agent of Claim 1, wherein the chemical moiety is coupled to a carbon atom on the side chain which is designated as a #9 carbon atom, using conventional terminology.

8. The pharmaceutical agent of Claim 1, wherein the chemical moiety is coupled to a carbon atom on the side chain which is designated as a #8 carbon atom, using conventional terminology.

9. The pharmaceutical agent of Claim 1, wherein the chemical moiety is selected from the group consisting of:
a. halide atoms;
b. halide-containing groups which allow the chemically modified form of erythro-hydroxynonyladenine to have a Ki value for adenosine deaminase inhibition which remains within the range of about 10-7 to about 10-10;
c. nitrogen-containing groups which allow the chemically modified form of erythro-hydroxynonyladenine to have a Ki value for adenosine deaminase inhibition which remains within the range of about 10-7 to about 10-10;
d. ester-linked groups which allow the chemically modified form of erythro-hydroxynonyladenine to have a Ki value for adenosine deaminase inhibition which remains within the range of about 10-7 to about 10-10; and, e. ether-linked groups which allow the chemically modified form of erythro-hydroxynonyladenine to have a Ki value for adenosine deaminase inhibition which remains within the range of about 10-7 to about 10-10.

10. A method of inhibiting adenosine deaminase activity in a mammalian patient in need thereof, comprising administering to the patient a therapeutically effective quantity of a chemically modified form of erythro-hydroxynonyladenine with a molecular structure as follows:

wherein at least one of R1 and R2 comprises a chemical moiety, other than a hydrogen atom or hydroxyl group, which makes the chemically modified form of erythro-hydroxynonyladenine:
(a) pharmacologically acceptable and non-toxic when administered to humans and laboratory animals;
(b) capable of entering human cells and inhibiting intracellular enzymatic activity of adenosine deaminase in a reversible manner, in cell culture tests, more effectively than either erythro-hydroxynonyladenine or 9-[2(S),9-dihydroxy-3 (R) -nonyl]adenine;
(c) therapeutically effective in reducing hypoxic and ischemic damage in at least one type of mammalian tissue, selected from the group consisting of heart muscle and brain tissue, more effectively than either erythro-hydroxynonyladenine or 9-[2(S),9-dihydroxy-3(R)-nonyl]adenine.

11. The method of Claim 10, wherein the patient is receiving a useful therapeutic drug that is degraded by adenosine deaminase in the absence of an adenosine deaminase inhibitor.

12. The method of Claim 10, wherein the patient is suffering ischemic or hypoxic heart damage.

13. The method of Claim 10, wherein the patient is suffering ischemic or hypoxic brain damage.

14. The method of Claim 10, wherein the chemically modified form of erythro-hydroxynonyladenine has:
(a) a Ki value for adenosine deaminase inhibition which is less than about 5 x 10-9; and, (b) an octanol/water partition coefficient of at least about 2.

15. The method of Claim 10, wherein at least one of R1 and R2 is selected from the group consisting of:
a. halide atoms;
b. halide-containing groups which allow the chemically modified form of erythro-hydroxynonyladenine to have a Ki value for adenosine deaminase inhibition which remains within the range of about 10-7 to about 10-10;
c. nitrogen-containing groups which allow the chemically modified form of erythro-hydroxynonyladenine to have a Ki value for adenosine deaminase inhibition which remains within the range of about 10-7 to about 10-10;
d. ester-linked groups which allow the chemically modified form of erythro-hydroxynonyladenine to have a Ki value for adenosine deaminase inhibition which remains within the range of about 10-7 to about 10-10; and, e. ether-linked groups which allow the chemically modified form of erythro-hydroxynonyladenine to have a Ki value for adenosine deaminase inhibition which remains within the range of about 10-7 to about 10-10.

16. A method of inhibiting adenosine deaminase activity in a mammalian patient in need thereof, comprising administering to the patient a chemically modified form of erythro-hydroxynonyladenine comprising an adenyl ring structure coupled to a nonyl side chain, wherein the side chain has been modified by bonding at least one chemical moiety other than a hydroxyl moiety to the side chain, wherein the chemically modified form of erythro-hydroxynonyladenine:
(a) is pharmacologically acceptable and non-toxic when administered to humans and laboratory animals;
(b) is capable of entering human cells and inhibiting intracellular enzymatic activity of adenosine deaminase in a reversible manner, in cell culture tests, more effectively than either erythro-hydroxynonyladenine or 9-[2(S),9-dihydroxy-3(R)-nonyl]adenine;
(c) is therapeutically effective in reducing hypoxic and ischemic damage in at least one type of mammalian tissue, selected from the group consisting of heart muscle and brain tissue, more effectively than either erythro-hydroxynonyladenine or 9-[2(S),9-dihydroxy-3(R)-nonyl]adenine.

17. The method of Claim 16, wherein the chemically modified form of erythro-hydroxynonyladenine has:
(a) a Ki value for adenosine deaminase inhibition which is less than about 5 x 10-9; and, (b) an octanol/water partition coefficient of at least about 2.

18. The method of Claim 16, wherein the chemical moiety is selected from the group consisting of:
a . halide atoms;
b . halide-containing groups which allow the chemically modified form of erythro-hydroxynonyladenine to have a Ki value for adenosine deaminase inhibition which remains within the range of about 10-7 to about 10-10;
c . nitrogen-containing groups which allow the chemically modified form of erythro-hydroxynonyladenine to have a Ki value for adenosine deaminase inhibition which remains within the range of about 10-7 to about 10-10;
d. ester-linked groups which allow the chemically modified form of erythro-hydroxynonyladenine to have a Ki value for adenosine deaminase inhibition which remains within the range of about 10-7 to about 10-10; and, e. ether-linked groups which allow the chemically modified form of erythro-hydroxynonyladenine to have a Ki value for adenosine deaminase inhibition which remains within the range of about 10-7 to about 10-10.

19. The use of a chemically modified form of erythro-hydroxynonyladenine in preparing a medicament for reducing adenosine deaminase activity in mammalian patients, wherein the chemically modified form of erythro-hydroxynonyladenine has a molecular structure as follows:

wherein at least one of R1 and R2 comprises a chemical moiety, other than a hydrogen atom or hydroxyl group, which makes the chemically modified form of erythro-hydroxynonyladenine:
(a) pharmacologically acceptable and non-toxic when administered to humans and laboratory animals;
(b) capable of entering human cells and inhibiting intracellular enzymatic activity of adenosine deaminase in a reversible manner, in cell culture tests, more effectively than either erythro-hydroxynonyladenine or 9-[2(S),9-dihydroxy-3(R)-nonyl]adenine;
(c) therapeutically effective in reducing hypoxic and ischemic damage in at least one type of mammalian tissue, selected from the group consisting of heart muscle and brain tissue, more effectively than either erythro-hydroxynonyladenine or 9-[2(S),9-dihydroxy-3(R)-nonyl]adenine.

20. The method of Claim 19, wherein the chemically modified form of erythro-hydroxynonyladenine has:
(a) a Ki value for adenosine deaminase inhibition which is less than about 5 x 10 -9; and, (b) an octanol/water partition coefficient of at least about 2.

21. The method of Claim 19, wherein at least one of R1 and R2 is selected from the group consisting of:
a. halide atoms;
b. halide-containing groups which allow the chemically modified form of erythro-hydroxynonyladenine to have a Ki value for adenosine deaminase inhibition which remains within the range of about 10 -7 to about 10 -10;
c. nitrogen-containing groups which allow the chemically modified form of erythro-hydroxynonyladenine to have a Ki value for adenosine deaminase inhibition which remains within the range of about 10 -7 to about 10 -10;
d. ester-linked groups which allow the chemically modified form of erythro-hydroxynonyladenine to have a Ki value for adenosine deaminase inhibition which remains within the range of about 10 -7 to about 10 -10; and, e. ether-linked groups which allow the chemically modified form of erythro-hydroxynonyladenine to have a Ki value for adenosine deaminase inhibition which remains within the range of about 10-7 to about; 10-10.

22. The use of a chemically modified form of erythro-hydroxynonyladenine in preparing a medicament for reducing adenosine deaminase activity in mammalian patients, wherein the chemically modified form of erythro-hydroxynonyladenine comprises an adenyl ring structure coupled to a nonyl side chain wherein the side chain has been modified by bonding at least one chemical moiety, other than a hydroxyl moiety, to the side chain, wherein the chemically modified form of erythro-hydroxynonyladenine:
(a) is pharmacologically acceptable and non-toxic when administered to humans and laboratory animals;
(b) is capable of entering human cells and inhibiting intracellular enzymatic activity of adenosine deaminase in a reversible manner, in cell culture tests, more effectively than either erythro-hydroxynonyladenine or 9-[2(S),9-dihydroxy-3(R)-nonyl]adenine;
(c) is therapeutitically effective in reducing hypoxic and ischemic damage in at least one type of mammalian tissue, selected from the group consisting of heart muscle and brain tissue, more effectively than either erythro-hydoxynonyladenine or 9-[2(S),9-dihydroxy-3(R)-nonyl]adenine.

23. The method of Claim 22, wherein the chemically modified form of erythro-hydroxynonyladenine has:
(a) a Ki value for adenosine deaminase inhibition which is less than about 5 x 10-9; and, (b) an octanol/water partition coefficient of at least about 2.

24. The method of Claim 22, wherein the chemical moiety is selected from the group consisting of:
a. halide atoms;
b. halide-containing groups which allow the chemically modified form of erythro-hydroxynonyladenine to have a Ki value for adenosine deaminase inhibition which remains within the range of about 10-7 to about 10-10;
c. nitrogen-containing groups which allow the chemically modified form of erythro-hydroxynonyladenine to have a Ki value for adenosine deaminase inhibition which remains within the range of about 10-7 to about 10-10;
d. ester-linked groups which allow the chemically modified form of erythro-hydroxynonyladenine to have a Ki value for adenosine deaminase inhibition which remains within the range of about 10-7 to about 10-10; and, e. ether-linked groups which allow the chemically modified form of erythro-hydroxynonyladenine to have a Ki value for adenosine deaminase inhibition which remains within the range of about 10-7 to about 10-10.